ISSN 1870-249X
APRIL - JUNE - 2015
J. Mex. Chem. Soc. 2015 59 (2)
Pages 75-171
Quarterly publication
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ISSN 1870-249X
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Ta b l e o f C o n t e n t s
Articles
75-82
Intermolecular Lennard-Jones (22-11) Potential Energy
Surface in Dimer of N8 Cubane Cluster
Jamshid Najafpour*, Mehran Aghaie and Forouzan Zonouzi
83-92
Synthesis and Characterization of K2Ln2/3Ta2O7·nH2O (Ln=
La, Pr, Nd), Layered Tantalates Photocatalysts for Water
Splitting
Hoover Valencia-Sanchez*, Heriberto Pfeiffer, Dwight
Acosta, Alicia Negron-Mendoza and Gustavo Tavizon
93-98
Simultaneous Determination of Acetaminophen, Pamabrom and Pyrilamine Maleate in Pharmaceutical Formulations Using Stability Indicating HPLC Assay Method
Atif Saleem, Shahid Anwar, Talib Hussain, Rehan Ahmad,
Ghulam Mustafa and Muhammad Ashfaq*
99-104
ZnO-Photocatalyzed Oxidative Transformation of Diphenylamine. Synergism by TiO2, V2O5, CeO2 and ZnS
Chockalingam Karunakaran* and Swaminathan
Karuthapandian
105-118
Role of Different Transporting Systems in the Secretion
of Alkaloids by Hairy Roots of Catharanthus roseus (L)
G. Don
Juan Luis Monribot-Villanueva, Eliel Ruiz-May, Rosa
María Galaz-Ávalos, Dayakar Badri and Víctor Manuel
Loyola-Vargas*
*The asterisk indicates the name of the author to whom inquires about the paper should be addressed
Ta b l e o f C o n t e n t s
119-129
Reaction Parameters for Controlled Sonosynthesis of
Gold Nanoparticles
Alma Laura González-Mendoza and Lourdes I. Cabrera-Lara*
130-136
Phenylboronic Acid/CuSO4 as an Efficient Catalyst for
the Synthesis of 1,4-Disubstituted-1,2,3-Triazoles from
Terminal Acetylenes and Alkyl Azides
José Emilio de la Cerda-Pedro, Susana Rojas-Lima, Rosa
Santillan and Heraclio López-Ruiz*
137-142
Synthesis of New Dicoumarol Based Zinc Compounds
and their Invitro Antimicrobial Studies
Sadia Rehman* and Muhammad Ikram*
143-150
Separation of Micro-Macrocomponent Systems: 149Pm
– Nd, 161Tb-Gd, 166Ho-Dy and 177Lu-Yb by Extraction
Chromatography
F. Monroy-Guzman and E. Jaime Salinas
151-160
Synthesis of fluorescent oligo(p-phenyleneethynylene)
(OPE3) via Sonogashira reactions
Mariana Flores-Jarillo, Francisco Ayala-Mata, Gerardo
Zepeda-Vallejo, Rosa Ángeles Vázquez-García, Gabriel
Ramos-Ortiz, Miguel Ángel Méndez-Rojas, Oscar Rodolfo
Suárez-Castilloa and Alejandro Alvarez-Hernández*
161-171
Enantioselective Synthesis of Isoxazolecarboxamides and
their Fungicidal Activity
Mirosław Gucma, W. Marek Gołębiewski and Alicja K.
Michalczyk
Article
J. Mex. Chem. Soc. 2015, 59(2), 75-82
© 2015, Sociedad Química de México
ISSN 1870-249X
Intermolecular Lennard-Jones (22-11)Potential Energy Surface
in Dimer of N8 Cubane Cluster
Jamshid Najafpour,1* Mehran Aghaie2 and Forouzan Zonouzi1
1 Department
of Chemistry, Faculty of Science, Yadegar-e-Imam Khomeini (RAH) Shahre Rey Branch, Islamic Azad
University, Tehran, P.O. Box: 18155-144, Iran.
2 Faculty of Chemistry, North Tehran Branch, Islamic Azad University, Tehran, Iran.
Received June 16th, 2014; Accepted January 16th, 2015
Abstract. We have calculated the intermolecular potential energy surface (IPES) of the dimer of cubic N8 cluster using ab initio and the
density functional theory (DFT) calculations. The ab initio (HF/321G(d)) and DFT (B3LYP/6-31G(d) and aug-cc-pVDZ) calculations
were performed for two relative orientations of N8-N8 system as a
function of separation distance between the centers of cubic N8 clusters. In this research, the IPES, U(r), of the N8-N8 system is studied,
where the edge of N8 approaches to face or edge of the other considered N8. Then, the Lennard-Jones (12-6) and (22-11) adjustable parameters are fitted to the computed interaction energies for edge-face
and edge-edge orientations. In this research for the first time, the IPESs proportionated to the Lennard-Jones (22-11) potential are derived
that are compatible with the computed IPES curves. Assuming a set of
Lennard-Jones parameters, the second virial coefficients are obtained
for the N8-N8 complex at a temperature range of 298 to 1000 K. Both
the corrected and uncorrected basis set superposition error (BSSE)
results are presented confirming the significance of including BSSE
corrections.
Key words: Nitrogen cluster; IPES; Lennard-Jones (22-11) potential;
second virial coefficient; BSSE; ab initio; DFT
Resumen. Se calculó la superficie de energía potencial intermolecular
(SEPI) para el dímero del cúmulo N8 cúbico usando cálculos ab initio
y teoría de funcionales de la densidad (TFD). Los cálculos ab initio
(HF/3-21G(d)) y TFD (B3LYP/6-31G(d) se realizaron para dos orientaciones relativas del sistema N8-N8 como función de la distancia de
separación entre los centros de los cúmulos N8 cúbicos. En esta investigación se estudió la SEPI, U(r), del sistema N8-N8 donde la arista de
un N8 se aproxima a la cara o la arista del otro N8 considerado. Entonces, los parámetros ajustables de Lennard-Jones (12-6) y (22-11) se
ajustan a las energías de interacción calculadas para las orientaciones
arista-cara y arista-arista. En este trabajo, por primera vez, las SEPIs
proporcionadas para el potencial de Lennard-Jones (22-11) se derivan
de manera que sean compatibles con las curvas SEPI calculadas. Asumiendo un conjunto de parámetros de Lennard-Jones, los segundos
coeficientes del virial se obtienen para el complejo N8-N8 en el rango
de temperaturas 298 a 1000 K. Se presentan resultados corregidos y
sin corregir para error de superposición de bases (ESB), confirmándose la relevancia de incluir correcciones ESB.
Palabras clave: cúmulos de nitrógeno, SEPI, potencial Lennard-Jones (22-11), segundos coeficientes del virial, ESB, ab initio; TFD
Introduction
an important role in determining the properties of HEDMs
such as homo-polyatomic nitrogen clusters. These interactions
are conclusive in determining the thermodynamic properties of
molecular solids and liquids, while their magnitude are normally one or two orders of magnitude weaker than covalent
bonds [30]. Quantum mechanical approaches have been widely employed to drive intermolecular interaction potential.
Those approaches, which are sometimes practically difficult to
implement, can be used to extract detailed information about
the potential energy surface (PES). However, the quality of derived PES substantially depends on the applied computational
level. In addition, the basis set superposition error (BSSE)
should be corrected, because it has an important effect on the
calculated interaction potential [31,32].
In this work, the IPES, U(r), of the dimer of cubic N8 cluster is investigated. The Lennard-Jones potential is used to determine the IPES components. The thermal decomposition and
detonation of cubic N8 has been simulated from 2030 to 4642
K by molecular dynamics [33]. Therefore in this research, the
second virial coefficients for the dimer of cubic N8 are estimated computationally from 298 to 1000 K before simulation of
thermal decomposition.
A group of high energy density materials (HEDMs) are homo-polyatomic nitrogen clusters [1-2]. Although numerous
nitrogen clusters are studied theoretically [3-14], only a few of
them are known experimentally [15-22]. This stems from the
fact that the triple bond of N2 molecule has higher stability
compared to single or double bond species in other polynitrogen compounds.
N8 cubane is a hypothetical compound of interest for theoretical studies. However, there are speculations that it could
be a moderately stable compound [10,23-26]. It is isoelectronic with the corresponding carbon cubane (C8H8) compound,
which has been experimentally shown to be stable [27,28].
Whereas (C8H8) has been known experimentally for many
years [29], cubic N8 has never been observed experimentally.
Besides, cubic N8 is a candidate for HEDMs, with a high ratio
of the energy release to the specific weight; since its energy
content relative to the dissociation products (four N2 molecules) is extremely high [10].
Intermolecular potential energy surface (IPES), or van der
Waals interactions (intermolecular interaction potentials) has
76
J. Mex. Chem. Soc. 2015, 59(2)
Jamshid Najafpour, et al.
Methods and Computations
tal energy as a function of the system geometry is minimized,
an error appears due to differences between the long range energies from the unmixed basis sets and the short range energies
resulting from the mixed basis sets. The BSSE was computed
based on the Boys and Bernardi approach [31]. The IPES,
U(r), is analyzed for two interacting systems (i.e. A and B;
monomers) forming a supermolecule (i.e. AB; dimer) [32,44].
In principle, we can simply give the interaction energy of two
molecules A and B at a distance r, as
The equilibrium geometry of a single cubic N8 molecule was
obtained at the HF/3-21G(d) level of theory. We modeled the
cubic N8 dimer first by fixing the nearest nitrogen-nitrogen,
N-N, distance while the two monomers were left to rotate freely to obtain the most stable geometry. With several initial
choices of reciprocal orientations and approaching the monomers from the far side, the edge-face and face-face orientations
were found as the minimum-energy conformations (Fig. 1).
Since the cubic N8 molecule does not have a central atom, subsequently the nearest and farthest N-N distance were sampled
in deceasing and increasing steps with a step size of 0.1 Å from
minimum energy conformer to 2 and to 14 Å, respectively.
During the scan, the individual N8 molecular structures were
allowed to entirely relax. We optimized all geometries at the
framework of Hartree-Fock and DFT using the three-parameter Becke’s exchange [34,35] and Lee-Yang-Parr’s correlation
non-local functional [36], usually known as B3LYP. We used
the split valence type basis sets with polarized d function (321G(d) and 6-31G(d)) for electronic structure description of
N8 molecule and the interaction energy between N8 dimer with
HF and B3LYP methods, respectively. Also we used an augmented correlation consistent, polarized valance, double zeta
basis set (aug-cc-pVDZ) [37] for electronic structure description of N8 molecule and the interaction energy between N8 dimer with B3LYP method. Subsequently, we examined all
optimized geometries by re-optimizing them at the MP2
[38,39]/6-31G(d) [40-42] level in addition to re-computation
of the second order derivatives of energy at the same level.
Good agreement was found between both computational levels. To ensure the local minimum nature of all these species, all
optimized geometries were undergone frequency analysis. All
computations were performed using Spartan’10 suite of programs [43].
In cubic N8 dimer, the calculated interaction energy is sensitive to the BSSE if a finite basis set is used. As the interacting
nitrogen atoms of N8 dimer approach each other, their basis
functions start overlapping. Each monomer borrows basis
functions from other species then the basis set is “effectively”
increased and the calculated energy is improved. When the to-
U(r) = EAB(r) – EA – EB(1)
The total energies of the two discrete molecules EA and EB
and the total energy of the interacting system EAB are taken
from the solutions of Schrödinger’s equation. The IPES, U(r),
is estimated by Roothaan’s algebraic procedure as
α β
U (AB) = EAB
(AB)-EAα (A)-EBβ (B) (2)
EXσ (Y) is the total energy of the Y system at the X geometry
as calculated with the s basis set, where α, β and α  β illustrate the basis sets applied to compute A, B, and AB systems,
respectively.
In the counterpoise approach (CP), it is proposed to do the
calculation of the energies of the separate components with the
complete basis set of both monomers applied in the calculation
of the supermolecule energy
α β
U (AB)CP = EAB
(AB)-EAα β (A)-EBα β (B) (3)
In this manner, the three terms on the right hand side of
Eq. (3) are calculated using the same basis set. The BSSE is
known as the difference between the IPESs computed by Eqs.
(2) and (3)
δ BSSE = U (AB)CP -U (AB) (4)
U (AB)CP is a consistent method for correction of the
IPES, U, with α  β . For AB, A and B systems, α  β is a
balanced basis set. The magnitude of these parameters are
(a) Edge-Face orientation
(b) Edge-Edge orientation
Fig. 1. Two different orientations of N8 dimer.
77
Intermolecular Lennard-Jones (22-11) Potential Energy Surface in Dimer of N8 Cubane Cluster
determined by the second virial coefficient, B2, that calculated
using the IPES, U(r), (Equation (5)) [45],
B2 = 2π N A
∫
∞
0
⎧⎪
⎡ U ( r ) ⎤⎫⎪
⎥⎬ r 2 dr (5)
⎨1− exp ⎢−
⎢⎣ RT ⎥⎦⎭⎪
⎩⎪
where r is the distance separation of two components, T is temperature and NA is the Avogadro constant.
Results and Discussion
The ab initio and DFT methods are applied for exploring bond
lengths, bond angles and dihedral angles (Table 1). A good
agreement was found for the optimized geometrical parameters between B3LYP/6-31G(d) and B3LYP/aug-cc-pVDZ levels of theory.
For determining the IPES, U(r), of the cubic N8 dimer, the
approaching of monomers from the far side based on the edgeface and face-face orientations were done. Two different orientations of N8 dimer are shown in Fig. 1. Thermodynamic
properties of N8 and N8 dimer clusters in two different faceface and edge-face orientations at the cited computational levels are listed in Table 2. The obtained thermodynamic properties
for edge-face orientation show good agreement between the
both 6-31G(d) and aug-cc-pVDZ basis sets with B3LYP method, but with aug-cc-pVDZ basis set the edge-edge orientation
was not obtained.
Fig. 2 and Fig. 3 show the calculated IPES, U(r), as a
function of distance separation between the centers of N8-N8
system in the two different orientations using HF/3-21G and
B3LYP/6-31G(d) levels, respectively. Fig. 4 shows the calculated IPES, U(r), as a function of distance separation between
(a) Edge-Face orientation
(b) Edge-Edge orientation
Fig. 2. The BSSE corrected (CP) and uncorrected (NCP) potentials of
N8 dimer for (a) Edge-Face and (b) Edge-Edge orientations using RHF/3-21G(d) level and comparison with Lenard-Jones (12-6) potential.
Table 1. The set of optimized geometry parameters of N8 cluster.
Values
Optimized geometry parameters
HF/3-21G(d)
B3LYP/6-31G(d)
B3LYP/aug-cc-pVDZ
N-N bond length
1.561 Å
1.521 Å
1.522 Å
NNN bond angle
90.00˚
90.00˚
90.00˚
NNNN dihedral angle
0.00˚
90.00˚
0.00˚
90.00˚
0.00˚
90.00˚
Table 2. Thermodynamic properties of optimized N8 and N8 dimer clusters at 298.15 K and 1.00 atm.
Cluster
N8
N8 dimer (Edge-Face)
Level of theory
E0 (au)
ZPE (kJ/mol) Hth (kJ/mol) Sth (J/mol K) Cv (J/mol.K) Hth-TSth (kJ/mol)
HF/3-21G
-432.468727
102.7165
114.9938
278.1022
59.2031
32.0777
B3LYP/6-31G(d)
-437.428470
90.5353
104.1746
283.7048
71.4168
19.5880
B3LYP/aug-cc-pVDZ -437.502149
91.0127
104.5836
283.4026
70.8652
20.0871
HF/3-21G
-864.941938
207.7040
229.7346
378.6261
142.5478
116.8473
B3LYP/6-31G(d)
-874.861102
182.4899
207.2814
390.5892
167.5787
90.8272
B3LYP/aug-cc-pVDZ -875.006390
183.2978
207.9857
390.4852
166.7697
91.5626
-864.941349
207.7070
229.7420
378.3436
142.5822
116.9389
-874.861158
183.0752
207.8362
390.2179
167.2825
91.4928
N8 dimer (Edge-Edge) HF/3-21G
B3LYP/6-31G(d)
78
J. Mex. Chem. Soc. 2015, 59(2)
the centers of the N8-N8 in the edge-face orientation using
B3LYP/aug-cc-pVDZ. In Figures 2 to 4, the calculated IPES
are compared with Lenard-Jones (12-6) potential with the
BSSE corrected (CP) and uncorrected (NCP). The difference
of Lenard-Jones (12-6) with computed data is very large.
Therefore, this is a clear sign that Lenard-Jones (12-6) potential is “not” a proper function to fit the calculated data for N8N8 supermolecule. The IPESs, U(r), strongly depend on the
BSSE corrections. The IPESs corrected for the BSSE are energetically much lower than the potential energy calculated without the BSSE correction. In particular, the BSSE correction is
important for calculating the IPESs with small basis sets.
It is seen from comparison of edge-face and face-face orientations in Figures 2 and 3 that the orientations and separation
distances between the centers of N8-N8 clusters are fairly effective on the position of minimum, depth and width of the
calculated potential well of the IPES curves, especially at
HF/3-21G(d) level (Fig. 2). Figures 2 and 3 show that the
edge-face orientation has the IPES curve with the largest value
of De (De = - Eint(Re) = - Emin)2, at the HF/3-21G(d) level of
theory; whereas, at the B3LYP/6-31G(d) level of theory, both
of orientations have relatively similar potential energy curves.
Also based on the position of the minimum point (Re) of the
potential energy curves, the computed IPESs can be further
compared. These quantities are very sensitive to the values of
Jamshid Najafpour, et al.
Fig. 4. The BSSE corrected (CP) and uncorrected (NCP) potentials of
N8 dimer with Edge-Face orientation using B3LYP/aug-cc-pVDZ
level and comparison with Lenard-Jones (12-6) potential.
the separation distance of N8-N8 and different orientations
of N8 monomers to each other that is applied in the computational methods. In Table 3, the values of Re and De are tabulated for the edge-face and edge-edge orientations. The potential
well depths of the calculated IPESs, without the BSSE correction at RHF/3-21G(d) level are 2.5 to 3 times more than the
BSSE corrected potential, while these ratios for B3LYP/6-31G(d) are 2 to 2.5 times more than the BSSE corrected
potential and also for B3LYP/aug-cc-pVDZ level is less than 2
times more than the BSSE corrected potential.
In this work, the IPES, U(r), for the cubic N8 dimer system, was fitted to the Lennard-Jones (12-6) model (Equation
(6)), which can be expressed as
⎡⎛ ⎞12 ⎛ ⎞6 ⎤
σ
σ
U ( r ) = 4ε ⎢⎜ ⎟ − ⎜ ⎟ ⎥ (6)
⎢⎣⎝ r ⎠ ⎝ r ⎠ ⎥⎦
(a) Edge-Face orientation
(b) Edge-Edge orientation
Fig. 3. The BSSE corrected (CP) and uncorrected (NCP) potentials of
N8 dimer (a) Edge-Face and (b) Edge-Edge orientations using B3LYP/6-31G(d) level and comparison with Lenard-Jones (12-6) potential.
where r is the separation distance between the centers of N8N8, s is the size parameter and e is the depth of Lennard-Jones
function. We fitted the adjustable parameters s and e for two
different orientations with the ab initio HF/3-21G(d) and
B3LYP/6-31G(d) interaction energies, whereas these parameters are obtained for edge-face orientation at B3LYP/aug-ccpVDZ level. In Table 3, the values of the adjustable IPES
parameters are tabulated for various orientations. Also, the fitted curves of the calculated IPESs to the Lennard-Jones (12-6)
potential with the BSSE corrected (CP) and uncorrected
(NCP), are shown in Figures 2, 3 and 4. The fitted IPESs to the
Lennard-Jones (12-6) potential are not completely compatible
with the curves calculated ISEPs at HF/3-21G(d), B3LYP/6-31G(d) and B3LYP/aug-cc-pVDZ levels. In addition, in
this research, we demonstrated that it is possible to obtain a
IPES, U(r), for the cubic N8 dimer by quantum mechanical
calculations fitted to the Lennard-Jones (22-11) potential,
(Equation (7)), which can be expressed as
⎡⎛ ⎞22 ⎛ ⎞11 ⎤
σ
σ
U ( r ) = 4ε ⎢⎜ ⎟ − ⎜ ⎟ ⎥ (7)
⎢⎣⎝ r ⎠ ⎝ r ⎠ ⎥⎦
79
Intermolecular Lennard-Jones (22-11) Potential Energy Surface in Dimer of N8 Cubane Cluster
where the adjustable parameters e and s in Equation (7) are the
same as the Lennard-Jones (12-6) model are tabulated in Table
3. The calculated IPES curves at HF/3-21G(d) and B3LYP/6-31G(d) levels are fitted to the Lennard-Jones (22-11) potential with the BSSE corrected (CP) and uncorrected (NCP)
potential energy surfaces, as shown in Figures 5, 6 and 7. The
IPESs fitted to the Lennard-Jones (22-11) potential are completely compatible with the curves of the calculated ISPs. Also,
from the known U(r) formula, the second virial coefficients,
B2, are estimated for the cubic N8 dimer system with the BSSE
corrected (CP) and uncorrected (NCP) by quantum mechanical
calculations. We calculated the second virial coefficients with
the Lennard-Jones (12-6) and (22-11) potentials using Equation (5) and then tabulated the results for a range of temperatures from 298 to 1000 K in Table 4. However the equation (5)
is valid if the systems is “spherical” (like an atom) but for this
research we assumed N8 molecule is almost spherical. In Figures 8 and 9, the computed values of the second virial coefficients for selected temperatures (Table 4) are plotted.
We considered the BSSE corrections to yield the following
systematic results. The obtained second virial coefficients (B2)
with the Lenard-Jones (12-6) potential in the range of 298 to
1000 K with the BSSE corrected (CP) and uncorrected (NCP)
potential energy surface are negatives, i.e. the attractive forces
are dominated in the all range of 298 to 1000 K. Negative and
positive second virial coefficients (B2) imply dominance of attractive and repulsive forces, respectively between N8 clusters.
When we use the Lennard-Jones (22-11) potential, the obtained
second virial coefficients in the range of 298 to 1000 K with the
BSSE uncorrected (NCP) are negative, i.e. the attractive forces
are dominated in the all range of 298 to 1000 K. While B2 computed with the BSSE corrected (CP) potential energy surface
are varied from negative (approximately for a range of temperatures from 298 to 600 K) to positive (approximately for a
range of temperatures from 700 to 1000 K) values.
Conclusion
In this research the calculated IPESs were systematically studied
for cubic N8 dimer at the edge-face and edge-edge orientations
applying both Hartree-Fock and DFT methods. The edge-face
and edge-edge orientations were scanned at the HF/3-21G(d)
and B3LYP/6-31G(d) levels but the edge-face is the only orientation that was obtained at B3LYP/aug-cc-pVDZ level. It
(a) Edge-Face orientation
(b) Edge-Edge orientation
Fig. 5. The BSSE corrected (CP) and uncorrected (NCP) potentials of
N8 dimer for (a) Edge-Face and (b) Edge-Edge orientations using RHF/3-21G(d) level and comparison with Lenard-Jones (22-11) potential.
was demonstrated that the BSSE corrections significantly affect the quality of calculated potentials. From this survey several significant conclusions about applying the current
theoretical methods can be extracted to generate the IPESs.
Although in this study, we do not apply the long-range intermolecular interactions by DFT methods with dispersion-corrected and meta-hybrid functional and also MP2 methods with
correlation energy for weakly bound systems (in van der Waals
interactions), the results yield attractive potentials for the cubic
N8 dimer system. The IPESs fitted to the Lennard-Jones (12-6)
potential are not completely compatible with the curves of calculated IPESs at HF/3-21‫‏‬G(d), B3LYP/6-31G(d) and B3LYP/
aug-cc-pVDZ levels, while the IPESs fitted to the Lennard-Jones
Table 3. Adjustable parameters (ε = De and σ) fitted to the Lenard-Jones potential and Re for the two different orientations.
Adjustable parameters
RHF/3-21G
Different orientations
B3LYP/6-31G(d)
B3LYP/aug-cc-pVDZ
ε (kJ/mol)
σ (Å)
Re (Å)
ε (kJ/mol)
σ (Å)
Re (Å)
ε (kJ/mol)
σ (Å)
Re (Å)
11.87
4.60
5.00
12.07
4.49
4.81
6.82
4.65
5.00
Edge-Face (CP)
4.15
4.88
5.20
5.42
4.65
4.93
4.13
4.72
5.05
Edge-Edge (NCP)
10.53
4.60
5.18
12.04
4.50
4.84
-
-
-
Edge-Edge (CP)
4.07
4.90
5.27
5.18
4.64
4.97
-
-
-
Edge-Face (NCP)
80
J. Mex. Chem. Soc. 2015, 59(2)
Jamshid Najafpour, et al.
(a) Edge-Face orientation with NCP
(a) Edge-Face orientation
(b) Edge-Face orientation with CP
(b) Edge-Edge orientation
Fig. 6. The BSSE corrected (CP) and uncorrected (NCP) potentials
of N8 dimer (a) Edge-Face and (b) Edge-Edge orientations using
B3LYP/6-31G(d) level and comparison with Lenard-Jones (22-11) potential.
(c) Edge-Edge orientation with NCP
Fig. 7. The BSSE corrected (CP) and uncorrected (NCP) potentials of
N8 dimer with Edge-Face orientation using B3LYP/aug-cc-pVDZ
level and comparison with Lenard-Jones (22-11) potential.
(d) Edge-Edge orientation with CP
Fig. 8. Comparison between the computational data of the second virial
coefficients for selected temperatures of N8 dimer (Edge-Face orientation (a, b) and Edge-Edge orientation (c, d)) with BSSE corrected (CP)
and uncorrected (NCP) potentials using B3LYP/6-31G(d) level.
81
Intermolecular Lennard-Jones (22-11) Potential Energy Surface in Dimer of N8 Cubane Cluster
(a) NCP
(b) CP
Fig. 9. Comparison between the computational data of the second virial coefficients for selected temperatures of N8 dimer (Edge-Face orientation) with BSSE corrected (CP) and uncorrected (NCP) potentials using B3LYP/aug-cc-pVDZ level.
Table 4. Second virial coefficients from the theoretical potential for selected temperatures
B2 (cm3/mol)
RHF/3-21G
Edge-Face
Lenard-Jones (12-6)
Edge-Edge
Lenard-Jones (22-11)
Lenard-Jones (12-6)
Lenard-Jones (22-11)
T (K)
NCP
CP
NCP
CP
NCP
CP
NCP
CP
298
-11391.95
-916.78
-4722.26
-238.21
-7270.67
-895.93
-2928.11
-228.84
400
-4155.73
-545.76
-1591.67
-98.50
-2981.58
-534.23
-1096.84
-92.98
500
-2299.15
-370.93
-813.37
-34.79
-1743.82
-362.95
-586.11
-30.71
600
-1522.67
-268.29
-496.77
1.76
-1190.33
-262.12
-364.18
5.12
700
-1112.25
-201.01
-333.37
25.28
-884.94
-195.93
-244.61
28.21
800
-862.61
-153.64
-235.97
41.58
-693.60
-149.27
-171.17
44.24
900
-696.09
-118.56
-172.12
53.50
-563.22
-114.69
-121.96
55.96
1000
-577.63
-91.59
-127.36
62.54
-468.98
-88.09
-86.88
64.86
298
-11334.90
-1321.00
-4715.88
-412.43
-11295.59
-1198.62
-4696.98
-364.66
400
-4060.20
-754.80
-1563.24
-191.42
-4057.25
-691.45
-1560.89
-167.92
500
-2226.62
-509.40
-792.94
-98.99
-2227.96
-467.96
-792.64
-84.24
600
-1467.51
-371.75
-482.71
-48.46
-1469.47
-341.48
-482.77
-38.05
700
-1068.90
-284.08
-323.64
-16.90
-1070.78
-260.45
-323.72
-9.04
800
-827.52
-223.51
-229.24
4.55
-829.20
-204.26
-229.29
10.77
B3LYP/6-31G(d)
900
-667.04
-179.25
-167.57
20.02
-668.51
-163.08
-167.55
25.09
1000
-553.16
-145.54
-124.44
31.66
-554.44
-131.67
-124.36
35.88
298
-2176.77
-822.28
-758.66
-212.76
400
-1161.29
-489.72
-349.23
-87.61
500
-763.88
-332.81
-194.88
-30.46
600
-553.17
-240.63
-115.28
2.34
700
-423.61
-180.19
-67.37
23.46
800
-336.23
-137.62
-35.61
38.11
900
-273.44
-106.08
-13.11
48.81
1000
-226.23
-81.84
3.59
56.94
B3LYP/aug-cc-pVDZ
82
J. Mex. Chem. Soc. 2015, 59(2)
(22-11) potential are completely compatible with the curves of
the calculated IPESs.
Our calculated Lennard-Jones (22-11) potential with the
BSSE corrected (CP) from 298 ∼ 600 K show that the attractive forces are dominated and from 700 ∼ 1000 K relatively
repulsive forces are dominated.
These conclusions can be useful for subsequent studies on
thermal decomposition and detonation of the cubic N8 as a
high energy density material.
Acknowledgement
The authors are grateful to Dr. S. Shahbazian and Dr. C.
Foroutan-Nejad for their helpful comments and valuable suggestions that helped the authors to improve the quality of this
communication.
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Article
J. Mex. Chem. Soc. 2015, 59(2), 83-92
© 2015, Sociedad Química de México
ISSN 1870-249X
Synthesis and Characterization of K2Ln2/3Ta2O7·nH2O (Ln= La, Pr, Nd),
Layered Tantalates Photocatalysts for Water Splitting.
Hoover Valencia-Sanchez,1,5* Heriberto Pfeiffer,2 Dwight Acosta,3 Alicia Negron-Mendoza4
and Gustavo Tavizon1
1 Facultad de Química.
Instituto de Investigaciones en Materiales.
3 Instituto de Física.
4 Instituto de Ciencias Nucleares; Universidad Nacional Autónoma de México. Ciudad Universitaria, C.P. 04510, México D.F.
5 Permanent address: Escuela de Química, Universidad Tecnológica de Pereira, Carrera 27 10-02 Los Alamos, C. P. 660003,
Pereira-Colombia, Tel. (+57)63137242, hvalencia@utp.edu.co
2 Received July 7th, 2014; Accepted January 27th, 2015
Abstract. Three compounds of the K2Ln2/3Ta2O7 (Ln = La, Nd, Pr)
cation-deficient Ruddlesden-Popper series were prepared by the
Pechini (polymeric complex) method. The crystal structures of
the hydrated form of these compounds were determined by Rietveld
analysis of the X-ray powder diffraction data and High Resolution
Transmission Electron Microscopy (HRTEM). The samples were also
analyzed to determine specific area (BET), degree of hydration
(TGA), and photocatalytic activity for hydrogen evolution from water
and aqueous methanol solution.
Key words: Photocatalyst, Water Splitting, Tantalates, Ruddlesden-Popper, Hydrogen production.
Resumen. Tres compuestos del tipo K2Ln2/3Ta2O7 (Ln = La, Nd, Pr)
de la serie de Ruddlesden-Popper deficientes de cationes fueron
preparados por el método de Pechini (complejo polimerizable). Las
estructuras cristalinas de los compuestos hidratados fueron determinadas por análisis de Rietveld de los patrones de difracción de rayos
X y mediante microscopia electrónica de alta resolución (HRTEM).
Las muestras también fueron analizadas para determinar su área superficial (BET); grado de hidratación (TGA), y actividad fotocatalítica en la producción de hidrógeno a partir de agua y de soluciones
metanol/agua.
Palabras Clave: Fotocatalizadores, Disociación del agua, Tantalatos,
Ruddlesden-Popper, Producción de Hidrógeno.
Introduction
splitting activity[9],[10],[16],[17], the interlayer hydration
plays a key role because the intercalated water molecules are
accessible to the active sites responsible for the photocatalytic reaction. The number of publications on the photocatalytic
activity of layered tantalates has increased in recent years,
such as Kudo et al.[18] reported. These new semiconductors
are laminar compounds exhibiting a layered structure in
which perovskite blocks consisting of TaO6 octahedra are
thought to be responsible of such photoactivity. One of them,
the NaTaO3 has the highest quantum yield in the photocatalytic reaction that splits water into H2 and O2. This compound
was studied using different lanthanide doping such as: La, Pr,
Nd, Sm, Gd, Tb and Dy[19]. NaTaO3 presents a quantum
yield of 56% at 270 nm when it is doped with lanthanum and
Ni-loading (NaTaO3:La (2%) NiO (0,2 % w))[20],[21]. On
the other hand, the undoped compound, NaTaO3:NiO (0,05
%w), presented a yield of 28% at 270 nm (surface specific
area of 0,52 m2/g) as reported Kato et al.[22]. The significant
decrease in particle size and a topography in which terraces
predominate separating active sites for oxidation and reduction are thought to be the main responsible factors of such
phenomenon[23].
With the aim of investigating the synthetic route and the
main crystal features of hydrated layered tantalates and to explore the effect of the partially filled 4f shell cations (Pr, Nd)
Tantalum-oxide based compounds crystallizing in the Ruddlesden-Popper (RP) structure[1] have recently attracted
much attention due to their ion-exchange[2]–[5] (96 and intercalation[6]–[8] properties and as photocatalysts for hydrogen generation from water splitting[9]–[11]. Additionally,
high proton conductivity has been recently reported[12] in
randomly oriented grains of H2SrTa2O7 and SrTa2O6. In their
anhydrous form, the K2[Ln3/2Ta2O7] RP tantalates correspond
to the formula A’2[An-1BnO3n+1], where A’ is often an alkali
metal, A is a rare earth metal, B is a transition metal, and n
defines the number of BO6 octahedra forming perovskite layers[13]. In this structure there are two A’ interlayer cations
per formula unit interconnecting the n perovskite sheets.
Most of the compounds of the RP series with B=Ti and/or Ta
crystallize in the space group (SG) I4/mmm (No. 139), which
consists of perovskite blocks separated by rock-salt type A’O
blocks. This structure produces a staggered arrangement in
which two contiguous perovskite blocks are shifted by
(a+b)/2 in the crystal cell. During intercalation of water into
the RP structure, the water molecules form new layers inside
the rock-salt type block[14], and a structural change from the
I4/mmm to the P4/mmm (No. 123) SG occurs[15]. As reported for several layered tantalates with photocatalytic water
84
J. Mex. Chem. Soc. 2015, 59(2)
Hoover Valencia-Sanchez, et al.
on photocatalytic activity for hydrogen evolution of the RP
tantalates, a study of the K2Ln3/2Ta2O7 (Ln = La, Pr, Nd) series of compounds is presented. These compounds were synthesized via the Pechini method (PC) that results in good
crystallinity of the samples and higher or equal specific areas
than the synthesized by the solid-state reaction method.
These characteristics are important in the heterogeneous photocatalytic process of these systems. The compounds synthesized in this work are lanthanide-deficient (1/3 per formula
unit) and, as reported for the RbLnTa2O7 tantalate[24], the
role played by these lanthanides in the neighborhood of the
TaO6 octahedra cannot be considered as that of “spectator” in
the photocatalytic activity behavior of these layered perovskite tantalates.
Results and Discussion
XRD patterns of the hydrated phase (HP) of K2La2/3Ta2O7
(LaN2), K2Pr2/3Ta2O7 (PrN2), and K2Nd2/3Ta2O7 (NdN2)
are shown in Fig. 1. In the Table 1, a list of reflections that
correspond to diffraction pattern of the Fig 1 is presented.
The reflections marked with (*) in Table 1, contribute to
the total intensity and the observed reflection corresponds
to the sum of several reflections. For example, the first reflection should be assigned to the 003, 010 and 011 contributions. The second one results from the 013, 110, 111 and
014 reflections. The formation of the PR phases were also
supported by HRTEM (see below). Likewise, the wide
character of the main reflections observed in these patterns
must be associated with the small size of crystallites obtained by the Pechini method of synthesis. In this synthetic
route, an important element to consider refers to the calcination temperature because once this exceeds 900°C, undesired tungsten-bronze type compounds appear in the XRD
patterns[25]. Several authors have referred to the appearance of tungsten bronzes, obtained by the solid-state reaction method[26]–[28].
To determine the number of hydrating water molecules
in samples, dynamic thermogravimetric analysis (TGA) was
performed and the compounds were heated under airflow
from 25°C to 1000 °C (10 °C/min). To consider only the water molecules intercalated into the crystal structure, without
taken into account the water molecules on the crystal surface,
the weight-loss was considered from 100°C. According to
these thermogravimetric analyses, the number of hydration
water molecules for LaN2, PrN2 and NdN2 is 2.5, 1.8 and
0.83, respectively. A plot of this thermal behavior is presented in Fig. 2. As observed in this plot, the number of intercalated water molecules in these layered structure compounds
is different for each lanthanide, decreasing in the order
LaN2>PrN2>NdN2.
As previously reported for alkaline laminar tantalates (except Li[10],[16],[17],[27], Cs and Rb[29]), these compounds
exhibit spontaneous water intercalation when exposed to air
under room temperature[27],[28],[30]. For the anhydrous
Fig. 1. XRD patterns of the HP of a) K2La2/3Ta2O7 (LaN2); b)
K2Pr2/3Ta2O7 (PrN2) and d) K2Nd2/3Ta2O7 (NdN2), obtained at room
temperature. The hkl Miller indices are indicated above the main reflections. The peak width is associated to the loss of crystallinity and
very small size of crystallites. No additional reflections as those assigned to tungsten-bronze phases were detected.
phase of K2La2/3Ta2O7, Crosnier-Lopez et al[27] determined
an I4/mmm space group with a = 3.9679Å and c = 22.0807Å
crystal cell parameters. Water intercalation of this compound
is extremely rapid and one structural transition was observed
from the body centered (I4/mmm) to a primitive lattice (P4/
mmm) in the hydrate form of this compound,
K2La2/3Ta2O7·2H2O. For this compound the cell parameters
are a = 3.9427Å and c = 12.887Å[27]. As occurs in other
laminar compounds of the Ruddlesden-Popper series as
K2Nd2Ti3O10, water intercalation produces a change in the
symmetry due to an (a+b)/2 sliding of the central slab relative to the adjacent slabs[31]. The SG for all the LnN2 (Ln =
La, Pr and Nd) hydrated phases is P4/mmm and the diffraction patterns are presented in Fig. 1.
Crystal structure of K2La2/3Ta2O7 has been previously
reported by Crosnier-Lopez et al[27] through Rietveld refinements of XRD data. In the present work, Rietveld refinement
of the hydrated form of this phase is presented, and this study
has been extended to include the Pr and Nd cations in the
crystal unit cell (see Fig. 3). For the Rietveld analysis the P4/
mmm SG was attempted[4], and the water molecule position
was the 2e Wyckoff site (0, 1/2, 1/2; 1/2, 0, 1/2); however, its
occupancy was not refined and the different water stoichiometries of the compounds correspond to those estimated from
thermal analysis. However, the values of the isotropic thermal parameters for Ow (oxygen atom of the water molecule)
are acceptable for LaN2, PrN2 and NdN2, when considering
the interlayer distance in which those molecules are located.
Site-occupancy refinements of Ta and O were also omitted
because of the low volatility (and very low solubility in water) of the metal-oxide, and stability of the oxidation state of
Ta. Considering the ab plane as that formed by the O(2) at the
4i Wyckoff position, Ta rises from this plane and the TaO(2)-Ta angle is 163.7° (LaN2); 163.56°(PrN2) and
85
Synthesis and Characterization of K2Ln2/3Ta2O7·nH2O (Ln= La, Pr, Nd), Layered Tantalates Photocatalysts for Water Splitting
Table 1. Reflectioins for the diffraction pattern to K2La2/3Ta2O7, K2Pr2/3Ta2O7 and K2Nd2/3Ta2O7 compounds of Fig 1.
K2La2/3Ta2O7
K2Pr2/3Ta2O7
K2Nd2/3Ta2O7
Miller Indices (HKL)
2θ (Degrees)
Distance (Å)
2θ (Degrees)
Distance (Å)
2θ (Degrees)
Distance (Å)
003
19.481
4.5529
19.419
4.5673
21.471
4.1353
010*
22.608
3.9298
22.520
3.9449
22.283
3.9864
011
23.538
3.7766
23.448
3.7909
23.421
3.7953
013*
30.014
2.9749
29.905
2.9855
31.138
2.8700
110*
32.187
2.7788
32.060
2.7895
31.718
2.8188
111
32.865
2.7230
32.736
2.7334
32.549
2.7488
014
34.777
2.5776
34.653
2.5865
36.683
2.4479
020*
46.162
1.9649
45.975
1.9725
45.469
1.9932
007
46.504
1.9512
46.349
1.9574
51.525
1.7723
021
46.665
1.9449
46.476
1.9523
46.086
1.9680
023
50.552
1.8041
50.351
1.8108
50.810
1.7955
116*
51.880
1.7610
51.689
1.7670
55.036
1.6672
017
52.305
1.7477
52.120
1.7534
56.805
1.6194
123*
56.046
1.6395
55.817
1.6457
56.136
1.6371
117
57.682
1.5969
57.469
1.6023
61.783
1.5004
026*
62.379
1.4874
62.133
1.4927
64.931
1.4350
127*
72.297
1.3059
72.002
1.3105
75.594
1.2569
011*
73.319
1.2902
73.044
1.2943
81.127
1.1845
033*
75.454
1.2589
75.119
1.2633
75.018
1.2651
130*
76.611
1.2427
76.264
1.2475
75.331
1.2606
*Reflections that contribute more intensity in the Fig 1.
Fig. 2.Thermogravimetric analysis of the K2Ln2/3Ta2O7·nH2O (Ln =
La, Pr, Nd) samples under air flow. The number of intercalated water
molecules was calculated from the weight loss from 100°C (dashed
line). The first derivatives of the TGA curves are shown in the plot
inset.
164.66°(NdN2). In the LnN2 series, the Ta-(O2)-Ta angle remains almost constant, independent of the H2O/Ta ratio. For
the cell parameters of the rare earth tantalates (see Table 1)
there is no correlation between the lanthanide ionic radii
(1.36, 1.31 and 1.27Å for La3+, Pr3+ and Nd3+, respectively,
all them in coordination 12[32]) and the values of the a and c
Fig. 3. Crystal unit cell of the HP of K2Ln2/3Ta2O7 (Ln=La, PrandNd).
This figure shows the P4/mmm SG in which the water molecules
(only the oxygen atom in red is shown) have been intercalated in the
original I4/mmm crystal cell. The interlayer spaces of the intercalated
water were evaluated from Rietveld refinements.
86
J. Mex. Chem. Soc. 2015, 59(2)
Hoover Valencia-Sanchez, et al.
Table 2. Atomic and thermal parameters obtained from Rietveld refinements of the XRD data on K2La2/3Ta2O7·2.5H2O, K2Pr2/3Ta2O7·1.8H2O,
and K2Nd2/3Ta2O7·0.8H2O at room temperature. In these structural refinements, the SG is P4/mmm (No. 123); O(w) represent the oxygen atoms
of the water molecules, and the K and Ln site occupancies were fixed to the stoichiometric values.
Compound
Atom
Site
occupancy
x
y
z
100Uiso
(Å2)
Wyckoff
Symmetry
K2La2/3Ta2O7.2.5H2Oa
K(2)
0.833
1/2
1/2
0.33674
0.01000
2h
K(1)
0.333
1/2
1/2
0
0.05491
1c
La
0.667
·
K2Pr2/3Ta2O7 1.8H2O
b
K2Nd2/3Ta2O7·0.8H2Oc
aa
1/2
1/2
0
0.05491
1c
Ta
0
0
0.15061
0.02806
2g
O(1)
0
0
0
0.01000
1a
O(2)
0
1/2
0.1300
0.01000
4i
O(3)
0
0
0.28876
0.01000
2g
O(w)
0
1/2
1/2
0.07700
2e
K(1)
0.333
1/2
1/2
0
0.0100
1c
K(2)
0.833
1/2
1/2
0.32820
0.0100
2h
Pr
0.667
1/2
1/2
0
0.0100
1c
Ta
0
0
0.15080
0.0100
2g
O(1)
0
0
0
0.0100
1a
O(2)
0
1/2
0.1300
0.0100
4i
O(3)
0
0
0.28852
0.0100
2g
O(w)
0
1/2
1/2
0.0100
2e
K(1)
0.333
1/2
1/2
0
0.19222
1c
K(2)
0.833
1/2
1/2
0.34998
0.02500
2h
Nd
0.667
1/2
1/2
0
0.19222
1c
Ta
0
0
0.14740
0.08297
2g
O(1)
0
0
0
0.02500
1a
O(2)
0
1/2
0.12569
0.02500
4i
O(3)
0
0
0.29862
0.02500
2g
O(w)
0
1/2
1/2
0.02500
2e
χ2
= 3.9297 Å, c = 13.6587 Å, Rwp = 5.77, = 2.97.
= 3.9449 Å, c = 13.7019 Å, Rwp = 6.42, χ2 = 4.276.
ca = 3.9864 Å, c = 12.4058 Å, R
2
wp = 3.21, χ = 6.757.
ba
parameters with the water content in the crystal structure of
the HPs. Comparing the water content to LnN2 compounds,
the larger water content corresponds to the shorter distance
between the slice of the TaO6 octahedra (see Fig. 3). Hydrated systems that exhibit the configuration of adjacent perovskite blocks with alkaline or alkaline-earth metals in the
rock salt block have been reviewed by Lehtimäki et al[14].
According to this study, the main factors affecting the water
intercalation are: the size of cations in the rock-salt block, the
oxygen content of compounds, and the valence of the transition metal in the structure.
An additional aspect of the LnN2 crystal structure,
which is defective in 1/3 of the Ln3+ ion per unit formula, is
that we have not observed ordering of vacancies that could
be shown as additional reflections in the XRD pattern. This
is probably due to the particle small size of the hydrated
samples and the width of reflections. For K2La2/3Ta2O7,
Crosnier-Lopez et al[27] observed by XRD and HRTEM,
that the atomic vacancies are restricted to 9-coordinated K
sites. As observed the diffraction patterns of the HP of LnN2
(see Fig. 1), all of them exhibit the characteristic broad reflections associated to the presence of very small crystallites in samples. Using the Scherrer equation to evaluate the
average size of the crystallite, we obtain 4 nm. By Scanning
Electron Microscopy (not shown), the average size of particles is about 50 nm.
The HRTEM images of LaN2 (Fig. 5) reveal the good
crystallinity of the tetragonal phase, this confirms the fact
that a single phase can be obtained by the synthetic route of
this work. Most of the HRTEM micrographs exhibit patterns corresponding to the (110), (100), (111), (010), (011),
(014) and (013) crystallographic planes, with interplanar
spacings of approximately 2.82, 3.69, 2.68, 4.12, 3.67, 2.64
and 2.99Å, respectively. The incidence of these planes re-
Synthesis and Characterization of K2Ln2/3Ta2O7·nH2O (Ln= La, Pr, Nd), Layered Tantalates Photocatalysts for Water Splitting
87
Fig. 4.Rietveld refinement plots for the HP of K2Ln2/3Ta2O7, a) Ln=La, b) Ln=Pr, and c) Ln=Nd; in these systems a P4/mmm SG was used and
the intercalated water molecules between the TaO6 octahedral slices was modeled as O(w), see Table 1.
veals a predominant preferential orientation[27] of crystals
in the LaN2 phase. These orientations agree well with the
intense reflections observed in the DRX pattern. By HRTEM, the existence of an infrequent plane was also found,
the (002) plane with 6.12Å (see Fig. 5a zone I, Fig. 5e zone
I). Based on the goodness-of-fit parameters (Rp, Rwp, Rexp
and χ2) of the structural refinements and the HRTEM images, the synthesis of the HPs of K2Ln2/3Ta2O7 was confirmed.
The adsorption-desorption isotherms
To determine the specific surface area of the HP of LnN2, the
BET method was used and the graphical results are presented
in the Fig. 6. Adsorption isotherms of K2Ln2/3Ta2O7 can be associated to the type II of the IUPAC classification, which represent an unrestricted monolayer-multilayer adsorption[33].
For most of the compounds at low P/Po (~0.25-0.35) ratio, the
Fig. 5. HRTEM micrographs series of samples a) LaN2; b) LaN2; c) LaN2; d) PrN2; e) PrN2; f) NdN2, g) NdN2, and h) NdN2. For all
the samples interplanar distances and crystallographic information have been derivedafter FT (Fourier Transform analysis) and displayed
in the corresponding insets. Even though samples look with high levels of crystallinity, faulted structures (dislocations and stacking faults)
can be observed everywhere in all the samples. These faulted structures might be relatedwith the insertion of ions into the materials during
the synthesis processes.
88
J. Mex. Chem. Soc. 2015, 59(2)
Hoover Valencia-Sanchez, et al.
stage of complete monolayer coverage can be observed (see
Fig. 6). The most relevant feature of this isotherm is that they
did not exhibit any limiting adsorption at high P/Po ratio. This
behavior can be caused by the existence of non-rigid aggregates of plate-like particles of slit-shaped pores[34]. The BET
surface areas of the LnN2 compounds of the present work are
3.0 m2/g, 3.9 m2/g, and 5.4 m2/g for the HPs of K2Pr2/3Ta2O7,
K2La2/3Ta2O7, and K2Nd2/3Ta2O7, respectively. These values
are in the order reported for K2La2/3Ta2O7 (3.7 m2/g), obtained
by the solid state reaction method[9]. This behavior can be directly associated to the kind of cation (alkaline or alkaline-earth
metals).
Absorbance spectra
To obtain the absorbance spectra of the K2Ln2/3Ta2O7 compounds, these were scanned in the wavelength range from 250
nm to 700 nm, using the DRS technique[35],[36]. As observed
in the absorption spectra of the Fig. 7, for PrN2 and NdN2,
redshifted absorption appear with maxima at 380 nm (Pr) and
585 nm (Nd). The band gaps values were calculated with the
Kubelka-Munk function, and the values obtained are: 3.70 eV,
3.6 eV, and 3.5 eV for LaN2, PrN2, and NdN2, respectively.
These absorption signals are associated with internal transitions in the localized 4f states[37], even though they are expected to be sharp peaks. This graph (Fig 7), and electronic
structure calculations, support[38] the incomplete localization
of the Ln (Pr and Nd) 4f electrons and thus its participation in
the chemical bonding via orbital hybridization in the Ln-O-Ta
system, while the interlayer cations (K) have a negligible effect on the electronic structure of layered Ln-tantalates. As expected from previous considerations, in the MLnTa2O7 (M=Cs,
Rb, Na and H; Ln=La, Pr, Nd and Sm) series, a Dion-Jacobson
type system, the experimental activity in water photolysis decreases in the sequence Nd>Sm>La>Pr; the low activity in the
Fig. 6. Adsorption-desorption isotherms for LnN2 (Ln = La, Pr, Nd).
From this plot, the estimated values of the specific surface area are 3.9
(LaN2), 3.0 (PrN2) and 5.4(NdN2) m2/g.
Fig. 7. UV-Vis spectrafrom DRS for the samples of this work, LaN2,
PrN2 and NdN2. As can be observed from the absorption spectra of
PrN2 and NdN2, there is redshifted absorption whose maxima appear
at 380 nm (Pr) and 585 nm (Nd).
Pr system is explained by their acting as a trapping center of
electronic holes[29].
Photocatalytic activity
The photocatalytic activity of the LnN2 compounds is
presented in the graph (Fig. 8). The photocatalytic reaction
was carried out in an inner irradiation quartz cell. The catalyst was dispersed in aqueous methanol solution (14% vol/
vol), using deionized water, then it was irradiated by a high
pressure Hg lamp (400W). Amounts of evolved hydrogen
gas were analyzed by gas chromatography. The highest activity was achieved when the catalysts were used in aqueous
methanol mixture (see Table 3), except to PrN2. On the other hand, as the larger surface area corresponds to NdN2; it
is considered that the smaller particle size increases the activity for water splitting. Comparing the most active powder
(NdN2), the activity was higher by twice than that when
LaN2 and PrN2 were used in an aqueous methanol solution
(97.5 μmol H2/g.h). When the reaction was tried in pure water (see Table 4), the NdN2 compound also had higher activity than PrN2 and LaN2; although the NdN2 and LaN2
activity decreased in pure water, while that of PrN2 remained low. Therefore, the high photocatalytic activity of
the NdN2 sample should be associated with the high specific surface area of this sample. In an analogous system,
RbLnTa2O7 (Ln = La, Pr, Nd, Sm), Machida et al.[39]
showed that the Nd system also exhibited the highest activity. The explanation of this activity is associated to the electronic structure change that comes out from the unfilled 4f
levels of the lanthanide. The Ln-O-Ta hybridization affects
the position of both; the conduction band and the valence
89
Synthesis and Characterization of K2Ln2/3Ta2O7·nH2O (Ln= La, Pr, Nd), Layered Tantalates Photocatalysts for Water Splitting
Fig. 8. Photocatalytic activity for water splitting of the LaN2, PrN2
and NdN2 compounds (deionized water, pH=7); Hydrogen evolution from 14% (vol/vol) methanol/water solution (right-side scale),
and Hydrogen evolution from deionized water (left-side scale) with
NiOx (0.5 wt %) as cocatalyst.
band edges, as well as their density of states (DOS)[38].
Electronic structure calculations show[37]–[39] that the unoccupied 4f (Ln = La) levels lay at the bottom of the con-
duction band, whereas the occupied 4f levels become
lowered as the number of 4f electrons increases. Finally,
these levels overlap with O-2p band for Ln = Nd and
Sm[37]. In this way, the presence of empty Ln-4f bands is a
possible reason for the low level of the conduction band
edge. This empty 4f bands are not necessarily manifested as
a drop in the band gap values, but these states can act as
‘stepping stones’ for the electrons to jump to the conduction
band[38]. Previous results on the photocatalytic activity of
the layered tantalates RbLnTa2O7 (Ln = La, Pr, Nd, and Sm)
[24],[39], with different cation arrangement between two
contiguous perovskite slabs, account for the H2 evolution
over the catalysts; 6, 4.2, and 47 for La, Pr, and Nd, respectively (all in units of mmol·h-1 for 1 g of catalyst). As a
matter of comparison, when the NiOx loading was performed, the photocatalytic activity was notably improved,
as reported by Shimizu et al. [9]. When NiOx was loaded
onto the H2La2/3Ta2O7 and K2La2/3Ta2O7 surfaces, the hydrogen evolution increased by about ten times in the first
compound (from 146 to 940 mmol·h-1). In the present work,
the hydrogen evolution on the NiOx-LaN2 sample increased
from 2.9 to 26.6 μmol h-1, and it was the highest increment
observed in the photocatalytic activity.
In order to explore the NiOx-cocatalyst effect on the H2
production of the LnN2 samples, NiOx-loaded LnN2 compounds were performed. The H2 evolution rates of the LaN2
and NdN2 samples were significantly increased by the addi-
Table 3. Photocatalytic activity obtained in a 14 % methanol/water solution.
K2La2/3Ta2O7 in 14% methanol/water
solution
K2Pr2/3Ta2O7 in 14% methanol/water
solution
K2Nd2/3Ta2O7 in 14% methanol/water
solution
Time (min)
H2 (μmol/g)
Time (min)
H2 (μmol/g)
Time (min)
H2 (μmol/g)
0
43
83
120
168
201
208
248
271
295
0
2.38
4.59
7.53
27.19
52.34
55.80
75.91
92.17
113.91
0
75
135
165
195
255
285
0
2.14
4.44
7.16
9.41
11.75
14.09
0
25
37
51
68
82
99
117
135
153
169
189
0
12.7
30.59
60.35
87.67
110.21
135.61
172.07
212.13
253.88
284.07
315.78
Table 4. Photocatalytic activity obtained in water deionized.
K2La2/3Ta2O7 in water
K2Pr2/3Ta2O7 in water
K2Nd2/3Ta2O7 in water
Time (min)
H2 (μmol/g)
Time (min)
H2 (μmol/g)
Time (min)
H2 (μmol/g)
0
62
124
185
243
299
362
0
2.54
5.00
7.29
10.37
13.41
17.61
0
33
58
90
132
167
191
209
0
2.90
6.64
11.45
17.07
22.34
27.57
31.53
0
55
105
180
237
295
344
0
7.27
19.73
38.32
50.77
64.77
71.97
90
J. Mex. Chem. Soc. 2015, 59(2)
Hoover Valencia-Sanchez, et al.
tion of NiOx (0.5 wt %) as cocatalyst (Fig. 8, Table 5). In
contrast to this behavior, the NiOx loading of the PrN2 sample resulted in a small decrease of the activity. However, the
increase of NiOx-LaN2 activity was higher than the other
compounds (from 2.92 µmol H2/g.h to 26.63 µmol H2/g.h).
The higher activity of the NiOx-loaded compounds is due to
the type of heterojunction formed at the semiconductor interface[40]. When NiOx and LnN2 are brought into contact,
their Fermi levels align, due to the charge transfer phenomenon. Therefore, under illumination, the LnN2 compounds
produce electron diffusion across the depletion region to the
NiOx for H2 evolution. When the NiOx cocatalyst is pretreated under H2 reduction conditions, and then a subsequent O2 oxidation, a NiO/Ni double layer structure is
produced, and the electron transference to the photocatalyst
active sites is facilitated.
Conclusions
By the polymeric complex method, we successfully synthesized isostructural layered perovskite tantalates which correspond to the hydrated phases of K2Ln2/3TaO7 (Ln=La, Pr,
Nd). These compounds are 1/3 Ln-deficient Ruddlesden-Popper systems of layered tantalates. Rietveld structural refinements and HRTEM images confirm the synthesis of
Ln-deficient double-perovskite tantalates. The K2Ln3/2Ta2O7
(Ln = La, Pr, Nd) systems have a large ability to intercalate
water molecules. This has been experimentally proved to be
important in water splitting. According to the absorption-desorption isotherms, the higher surface area was obtained in
the NdN2 compound. The DRS plot shows maxima absorption at 380 nm (Pr) and 585 nm (Nd), and this seems to depend on the nature of the lanthanide (La, Pr, Nd) in the crystal
structure. The higher photocatalytic activity was observed in
the NdN2 compound and it increased when NiOx cocatalyst
was loaded on the surface of powders. Finally, the H2 evolution was higher when the reaction was carried out in an aqueous methanol mixture, due to the sacrificial reagent effect
(except to PrN2). Layered lanthanide tantalates, particularly
the Nd layered tantalate, are suitable compounds to reach
high photocatalytic activity in water splitting. Through the
experiments of this work, the role played by the Ln-deficien-
cies in the crystal structure is not clear and further experiments are needed in this direction.
Experimental
Preparation of samples
Samples with the general formula K2Ln2/3Ta2O7, with Ln =
La, Pr, and Nd, were prepared by the Pechini method[41]–
[43] in which nitrates of the respective lanthanides
(La(NO3)3.6H2O, Nd(NO3)3.6H2O and Pr(NO3)3.6H2O (all
of them Aldrich, 99.9%); tantalum chloride (TaCl5, Aldrich,
99.99%); and potassium carbonate (K2CO3, Aldrich,
99.995%) were dissolved in methanol in the cationic proportion 2:2/3:2. An excess, 100 % of K2CO3, was added to
compensate for the volatility of the oxide form at high temperature. Then, the temperature was increased to 80°C and
ethylene glycol was added to facilitate the solubility of
salts. The solution was fully translucent and free of precipitates and suspended particles. At this step and with vigorous
stirring, citric acid was added to enhance the solubility and
form a condensation polymer until the solution became a
viscous brown gel. These gels were slowly heated in a
high-alumina crucible to 450°C for two hours, then black,
highly porous, solids were formed. These solids were then
finely ground for a subsequent calcination at 850°C for 48
hours. Subsequently, the compounds were suspended in water with stirring for approximately 30 minutes to dissolve
the potassium excess and obtain the hydrated phases. The
resulting HPs of the lanthanide tantalates were K2La2/3Ta2O7
(LaN2) (white powders); K2Nd2/3Ta2O7 (NdN2) (pale-purple powders) and K2Pr2/3Ta2O7 (PrN2) (pale-yellow powders). As previously reported by Crosnier-Lopez[27] for
K2La2/3Ta2O7, the Ln-tantalates undergo rapid hydration
when the samples are stored under room temperature conditions. Therefore, the crystal structure, X-ray diffraction
(XRD), High Resolution Transmission Electron Microscopy (HRTEM)), specific surface area, thermal analysis, and
photocatalytic activity characterization of the samples of
this work, correspond to the hydrated form of the lanthanide
tantalates, K2Ln2/3Ta2O7 (LnN2) (Ln = La, Pr, Nd).
NiOx-Loaded catalysts were prepared by impregnation of
Table 5. Photocatalytic activity of the compounds loaded with NiOx over water deionized.
K2La2/3Ta2O7/NiOx in water
K2Pr2/3Ta2O7/NiOx in water
K2Nd2/3Ta2O7/NiOx in water
Time (minutes)
H2 (μmol/g)
Time (minutes)
H2 (μmol/g)
Time (minutes)
H2 (μmol/g)
0
36
67
96
124
153
185
227
0
10.54
24.63
41.13
54.49
68.58
85.97
100.76
0
27
53
88
121
150
180
212
0
2.73
5.34
9.54
13.88
18.25
22.71
26.84
0
31
63
95
117
150
177
207
0
5.70
15.95
23.46
30.32
41.55
53.32
64.67
Synthesis and Characterization of K2Ln2/3Ta2O7·nH2O (Ln= La, Pr, Nd), Layered Tantalates Photocatalysts for Water Splitting
the samples (2g) with an aqueous solution of Ni(NO3)2.6H2O
(Aldrich, 99.99%). After stirring for 30 minutes, the water
was evaporated in an oven. The powders were dried at
80°Cfor 4 h and then calcined at 350°C for 1 h, and then at
450°C for 2 h in air. Subsequently, the catalysts were reduced in a H2 atmosphere at 500 °C for 2 h, and oxidized in
O2 atmosphere at 200°C for 1 h to produce NiO/Ni clusters
(NiOx).
Characterization
XRD patterns of powders were gathered using a Siemens
D-5000 diffractometer in a Bragg-Brentano geometry configuration (CuKα1 radiation, λ = 1.5406 Å), in air at room temperature (RT), with the operating conditions of 35kV and 35 mA.
For the three hydrated compounds, the scanning angular range
was 6°≤2θ≤90° with a step scan of 0.02°/10 seconds. Rietveld
analyses were performed using the General Structure Analysis
System[44] (GSAS Package) code with the graphical user interface EXPGUI[45]. After full hydration of the samples by
continuous washing with deionized water, thermogravimetric
analyses (TGA) were performed using a TGA Q50 (TA Instruments) with a heating rate of 10°C/min in airflow, from RT to
1000°C.
HRTEM analyses of hydrated samples were performed
using a JEOL 2010-TEM/STEM microscope operated at 200
kV. The samples were softly ground to obtain fine powders
and then dispersed in deionized water using an ultrasonic
bath. A drop of this suspension was placed on a carbon-coated copper grid. Crystallographic information for the compounds was derived from the HRTEM micrographs and
studied and analyzed using the commercial Digital Micrograph computing programme[46]. The electronic spectra
(diffuse reflectance) were measured on a Cary 5E UV-VisNIR spectrophotometer in the 250-700 nm range. To determine the surface area, N2 adsorption-desorption isotherms
were obtained using a Bel Japan Minisorp-II instrument. All
the samples were previously degassed at 200°C under vacuum conditions for 4 hours. The photocatalytic activity was
measured by hydrogen evolution with an inner irradiation
cell made of quartz. The catalyst (1 g) was dispersed in aqueous methanol solution (14%) and deionized water by magnetic stirring and was irradiated by a high pressure Hg lamp
(400W). Amounts of evolved gases were analyzed by gas
chromatography (Varian Aerograph 1400, Ar carrier, stainless steel column 40/60 mesh) through a gas sampler (3 cm3)
which was directly connected to the reaction system to avoid
any contamination from air.
Acknowledgements
The authors thank M in C. Cecilia Salcedo-Luna (USAI-FQUNAM) for her help in the XRD experiments. This work was
partially supported by the PAPIIT projects IN- 118710 and IN214313. HV-S gratefully acknowledges the financial support
from DGAPA (UNAM).
91
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Article
J. Mex. Chem. Soc. 2015, 59(2), 93-98
© 2015, Sociedad Química de México
ISSN 1870-249X
Simultaneous Determination of Acetaminophen, Pamabrom and
Pyrilamine Maleate in Pharmaceutical Formulations Using Stability
Indicating HPLC Assay Method
1,3
Atif Saleem, 1Shahid Anwar, 1Talib Hussain, 2Rehan Ahmad, 3Ghulam Mustafa and 3Muhammad Ashfaq*
1 Department of Chemistry, Minhaj University, Lahore-54000, Pakistan
CCL Pharmaceuticals (Pvt.) Ltd, Lahore-54000, Pakistan
3 Department of Chemistry, University of Gujrat, H.H. Campus, Gujrat-50700, Pakistan
* Author for Correspondence, e-mail: m.ashfaq@uog.edu.pk Ph# +92333-4043110
2 Received May 15th, 2014; Accepted February 17th, 2015
Abstract. A simple, specific and accurate stability indicating RPHPLC method was developed for the determination of acetaminophen, pamabrom and pyrilamine maleate simultaneously in
pharmaceutical dosage forms. Successful separation of all the components was enacted within 10 min using C18 column with mobile
phase of methanol and acidified water (pH 1.8) in the ratio of (27: 73
v/v respectively). Flow rate of the mobile phase was 1.5 mL/min
with detection at 300 nm. The method was validated in accordance
with ICH guidelines. Response was a linear function of concentration over the range of 50- 150 μg/mL for acetaminophen, 2.5-7.5 μg/
mL for pamabrom and 1.5-4.5 μg/mL for pyrilamine maleate. The
method resulted in excellent separation of all the analytes along with
their stress induced degradation products with acceptable peak tailing and good resolution. It is therefore can be applied successfully
for simultaneous determination of acetaminophen, pamabrom and
pyrilamine maleate in pharmaceutical formulations and their stability studies.
Key words: RP-HPLC, Acetaminophen, Pamabrom, Pyrilamine,
ICH guidelines.
Resumen. Se ha desarrollado un procedimiento simple y específico
procedimiento para la determinación de acetaminofeno, pamabrom y
maleato de pirilamina en formulaciones farmacéuticas por HPLC en
fase inversa. La separación de todos los componentes se obtuvo en 10
min utilizando una columna C18 y la fase móvil compuesta por metanol y solución acuosa a pH 1.8 (con ácido sulfúrico) en relación 27:
73 v/v. La velocidad del flujo en la columna fue de 1.5 mL/min y la
detección espectrofotométrica en 300 nm. El método fue validado siguiendo los criterios ICH. La respuesta fue lineal en el intervalo de
concentraciones 50- 150 μg/mL para acetaminofeno, 2.5-7.5 μg/mL
para pamabrom y 1.5-4.5 μg/mL para maleato de pirilamina. El procedimiento permitió la separación de los tres analitos y de sus productos de degradación inducidos en condiciones de estrés con aceptable
simetría de picos y resolución cromatográfica. Es por ello que este
procedimiento puede ser empleado para la determinación de acetaminofeno, pamabrom y maleato de pirilamina en formulaciones farmacéuticas y en estudios de su estabilidad.
Palabras clave: Cromatografía de líquidos de alta resolución en
fase inversa (RP-HPLC), Acetaminofoeno, Pamabrom, Pirilamina,
criterios ICH (Conferencia Internacional sobre Armonización de los
Requisitos Técnicos para el Registro de Productos Farmacéuticos
para Uso Humano).
Introduction
is an antihistamine used to reduce the allergic conditions and
reduce symptoms of cold. [4]
The combination of three active ingredients acetaminophen, pamabrom and pyrilamine is used for the treatment of
symptoms of mild to moderate premenstrual syndrome in addition to additive effects as analgesic as wells as antihistamine
and mild diuretic effects.[5] This combination is available in
different strengths in different dosage form with various brand
names. Most common are the tablet dosage form having
strength of 500mg, 25mg and 15mg respectively of Acetaminophen, Pamabrom and Pyrilamine.
Literature review did not provide any analytical method
for the simultaneous determination of above three components.
Individual search of the three components regarding their analytical methods revealed a flood of HPLC methods for acetaminophen either individually or in combination with other
Acetaminophen commonly called Paracetamol is chemically
designated as 4-hydroxyacetanalide (Fig. 1). It shows both analgesic and antipyretic properties and is available from the
open market without prescription as well as through prescription. Different dosage forms of paracetamol such as tablet,
capsules, drops etc. are available in the open market and its
different combinations with other drugs have been enlisted in
pharmacopeias. [1-2] Pamabrom (Fig. 2) is a common diuretic
existed as 1:1 mixture of 8-Bromo-3,7-dihydro-1,3-dimethyl-1H-purine-2,6-dione and 2-amino-2-methyl-1-propanol, where
8-Bromo-3,7-dihydro-1,3-dimethyl-1-H-purine-2,6-dione is the
active diuretic agent. [3] Pyrilamine maleate (Fig. 3) having
the chemical name 1,2-Ethanediamine, N-[(4-methoxyphenyl)
methyl]-N’, N’-dimethyl-N-2-pyridinyl-,(Z)-2-butenedioate (1:1)
94
J. Mex. Chem. Soc. 2015, 59(2)
active ingredients. [6-17] The search of the other two components viz. pamabrom and pyrilamine resulted in only few analytical methods. [18-21]
The fixed dose combination containing acetaminophen,
pamabrom and pyrilamine maleate is available in the market in
many countries yet no official pharmacopoeia has adopted that
combination. Literature review also resulted in failure of any
reported HPLC method for the simultaneous determination of
these three drugs in fixed dose combination. Therefore efforts
were done to develop and validate RP-HPLC method for simultaneous determination of these drugs and their stress induced degradation products in pharmaceutical formulations.
The described method is able to separate all three drugs from
the stress induced degradation within 10 min, so it can be used
for stability studies also.
Results and discussion
Method Development and Optimization
The main objective of this research work was to achieve the
best conditions for separation of acetaminophen, pamabrom
and pyrilamine maleate simultaneously in their fixed dose
combination.
Initially various mobile and stationary phases were tried to
accomplish the best separation conditions and resolution be-
Atif Saleem, et al.
tween acetaminophen, pamabrom and pyrilamine maleate. For
the initial trial, Hypersil BDS C8 column was chosen with mobile phase consisting of 1 M Potassium dihydrogen phosphate
and acetonitrile in different ratios (50 : 50, 40 : 60, 30 : 70, 20
: 80). Under all these condition elution of acetaminophen occurred only and there was no separation between pamabrom
and pyrilamine maleate.
Then stationary phase was switched to Cyano column using same ratios of 1 M Potassium dihydrogen phosphate and
acetonitrile as they were used with Hypersil BDS C8 column.
Here also the elution of acetaminophen occurred with good
peak shape but remaining two peaks remain merged with each
other. pH of 1 M Potassium dihydrogen phosphate was varied
from 2 - 7 in order to separate the merged components but all
attempts remained fruitless.
Further trials were carried out using Promosil C18 column
but result was the same as for other two columns. Then mobile
phase was changed from buffer to acidified water and methanol was used instead of acetonitrile with different concentrations (50:50, 40:60, 30:70, 20:80) and with different pH values
of acidified water (1.5 - 4.5). The trick works here and all the
three components were eluted. At last, mobile phase of acidified water (pH 1.8) and methanol in the ratio of 73: 27 was
selected that provides best separation conditions for the mentioned three components along with C18 column. Under these
conditions tailing of all the components was less than 1.5 with
retention times of 2.0, 2.8 and 7.5 minutes for acetaminophen,
pamabrom and pyrilamine maleate at a flow rate of 1.5 ml per
minute.
Analytical method validation
Fig. 1. Chemical Structure of Acetaminophen
Fig. 2. Chemical Structure of Pamabrom
Fig. 3. Chemical Structure of Pyrilamine Maleate
The developed analytical method was validated using ICH
guidelines. [22] Linearity, accuracy, precision, robustness,
specificity, stability of solutions and limit of detection and
quantitation were performed.
Linear calibration plots of the proposed method were obtained over concentration ranges of 50-150 mg/mL (50, 60, 70,
80, 90, 100, 110, 120, 130, 140 and 150 µg/mL) for acetaminophen, 2.5-7.5 mg/mL for pamabrom (2.5, 3.0, 3.5, 4.0, 4.5,
5.0, 5.5, 6.0, 6.5, 7.0 and 7.5 µg/mL) and 1.5-4.5 mg/mL for
pyrilamine maleate (1.5, 1.8, 2.1, 2.4, 2.7, 3.0, 3.3, 3.6, 3.9,
4.2, 4.5).The linear regression equation for acetaminophen was
Y= 12039X + 401076 with correlation coefficient of 0.9993.
For pamabrom, it was Y= 1246.9X + 30914 with correlation
coefficient of 0.9993 and for pyrilamine maleate it was Y=
1579.5X + 38151 with correlation coefficient of 0.9995. The
graphics of linearity are shown in Figs. 4-6.
The limit of detection (LOD) and quantitation (LOQ) were
determined by making serials of dilutions. LOD was found to
be 0.23 µg/mL for acetaminophen, 0.09 µg/mL for pamabrom
and 0.26 µg/mL for pyrilamine maleate respectively (signal to
noise ratio of 3: 1). LOQ was found to be 0.77 µg/mL for acetaminophen, 0.30 µg/mL for pamabrom and 0.87 µg/mL pyrilamine maleate respectively (signal to noise ratio of 10: 1).
Accuracy of the method was evaluated in triplicates using
standard addition technique at three concentration levels i.e.
95
Simultaneous Determination of Acetaminophen, Pamabrom and Pyrilamine Maleate in Pharmaceutical Formulations
80 %, 100 % and 120 % of target test concentration (100 mg/
mL of acetaminophen, 5 mg/mL of pamabrom & 3 mg/mL of
pyrilamine maleate). Percentage recoveries along with standard deviation and relative standard deviations for each analyte are given in Table 1. Recovery studies showed the method
to be highly accurate and suitable for intended use.
For intra-day precision, three set of concentrations of acetaminophen, pamabrom and pyrilamine maleate were tested
six times within the same day. For intermediate precision, two
different analysts from the same Laboratory tested the same
three concentrations six times. Relative standard deviation
(RSD %) of the peak area was then calculated. The results of
intra-day and inter-day precision are presented in Table 2.
For carrying out the robustness, very small changes were
carried out in mobile phase composition, flow rate and pH of
acidified water. The results (Assay, tailing factor, theoretical
plates and resolution) showed that slight variations in chromatographic conditions had negligible effect on the chromatographic
parameters. All the chromatographic parameters remained within the acceptable criteria as described earlier. It was thus concluded that the method is robust for the intended use.
Specificity of the developed method was evaluated by applying different stress conditions (acid, base, oxidation, thermal, humidity and photolytic) to acetaminophen, pamabrom
and pyrilamine maleate in combination form. From the result
of forced degradation studies, it is clear that all the three components remain intact under heat stress and humid conditions.
In acidic conditions, acetaminophen and pyrilamine maleate
were degraded up to 4 % whereas no degradation was observed
for pamabrom. Basic stress caused the degradation of acetaminophen and pyrilamine up to 12.5 and 13.8 % respectively.
Remarkable degradation was observed in case of pamabrom
and pyrilamine maleate in oxidative conditions, where pamabrom and pyrilamine maleate were degraded up to 10.6 % and
Fig. 4. Linearity graph for acetaminophen
Fig. 5. Linearity graph for pamabrom
Fig. 6. Linearity graph for pyrilamine maleate
Table 1. Accuracy of the Proposed HPLC Method
Drugs
Spiked Concentration (µg mL-1)
Recovery (%)
SD
RSD (%)
Acetaminophen
80.0
102.7
0.28
0.27
100.0
100.8
0.09
0.09
120.0
98.3
0.15
0.15
4.0
99.3
0.39
0.39
5.0
101.7
0.37
0.36
6.0
101.3
0.21
0.21
2.4
101.6
0.46
0.45
3.0
100.1
0.41
0.41
3.6
103.1
0.18
0.17
Pamabrom
Pyrilamine
Table 2. Intra-Day and Intermediate Precision of the Proposed HPLC Method
Ingredient
n
Repeatability
± RSD (%)
Intermediate Precision
± RSD (%)
Acetaminophen
18
0.06
0.13
Pamabrom
18
0.56
0.71
Pyrilamine
18
0.88
1.13
96
J. Mex. Chem. Soc. 2015, 59(2)
Atif Saleem, et al.
In addition to the percentage degradation of each drug, a
number of degradation products (impurities) were produced
under acidic, oxidative and photolytic stress conditions. The
stress induced degradation products (impurities) were unique
to acidic (7.93, 9.47 min), oxidative (3.82, 6.16, and 10.82
min) and photolytic (5.10, 6.09, 7.17, and 8.45) stress conditions. The data is shown in Table 3.
Application of the Method
Fig. 7. Chromatogram of acetaminophen, pamabrom and pyrilamine
maleate standard
Application of the method was checked by analyzing the acetaminophen, pamabrom and pyrilamine maleate in commercially available pharmaceutical products. The results are provided
in Table 4 which showed high percentage recoveries and low
RSD (%) values for both analytes.
Experimental
Chemicals and Reagents
Fig. 8. Chromatogram of acetaminophen, pamabrom and pyrilamine
maleate under basic stress
Reference standards of acetaminophen, pamabrom and pyrilamine maleate with stated purity of 98.81, 99.12 and 99.20 %
respectively were obtained from CCL Pharmaceuticals (Lahore, Pakistan). Femistar tablet claimed to contain 500 mg per
tablets of acetaminophen, 25 mg per tablets of pamabrom and
15 mg per tablet of pyrilamine maleate were used in this study.
Methanol (HPLC grade), sulphuric Acid, hydrochloric acid,
sodium hydroxide and hydrogen peroxide (analytical reagent
grade) were of Fluka and were purchased from their local
agent in Lahore, Pakistan. Mobile phase was filtered using
0.45 mm nylon filters by Millipore (USA).
Equipment and Chromatographic Conditions
Fig. 9. Chromatogram of acetaminophen, pamabrom and pyrilamine
maleate under oxidative stress
HPLC apparatus consisted of Shimadzu LC-20A system (Kyoto, Japan) with auto sampler and UV detector set at 300 nm.
BDS Promosil C18 column (250 × 4.6 mm, 5 mm particle size)
was used for carrying out all the experimental work. Acidified
water (pH 1.8) and methanol in the ratio of (73: 27 v/v respectively) were used as mobile phase at a flow rate of 1.5 mL/min.
All chromatographic experiments were performed at room
temperature (250 C± 20 C).
Preparation of Mobile Phase
Fig. 10. Chromatogram of acetaminophen, pamabrom and pyrilamine
maleate under acidic stress
44 % respectively. The amount degraded was calculated by
subtracting the recovered amount in each stress condition from
the recovered amount of un-stressed samples. The representative chromatograms under all the stress conditions are shown
in Figs. s (7-10).
Mobile phase was prepared by mixing acidified water (pH 1.8)
and methanol in the ratio of (73: 27 v/v respectively). Acidified
water was prepared using sulphuric acid and then dilution to
set pH 1.8 with water.
Preparation of Standard Solution
500 mg acetaminophen, 25 mg of pamabrom and 15 mg of
pyrilamine maleate were accurately weighed in 100 ml flask,
dissolved and then diluted with mobile phase. This solution
was then further diluted to get the desired concentrations.
97
Simultaneous Determination of Acetaminophen, Pamabrom and Pyrilamine Maleate in Pharmaceutical Formulations
Table 3. Stress Testing Results of Acetaminophen, Pamabrom, Pyrilamine Maleate
Nature of stress
Recovery of Acetaminophen ± RSD
(%)
Recovery of Pamabrom ± RSD
(%)
Recovery of Pyrilamine ± RSD
(%)
Heat stress
100.60 ± 0.22
98.59 ± 0.22
97.41 ± 2.14
1N HCl
95.97 ± 0.08
99.62 ± 0.84
95.47 ± 1.31
1N NaOH
87.41 ± 0.15
100.42 ± 0.29
86.22 ± 0.69
10 % H2O2
97.99 ± 0.03
89.40 ± 0.44
56.04 ± 2.22
Humidity & Light
100.49 ± 0.06
98.94 ± 0.77
99.66 ± 1.08
Table 4. Assay results of acetaminophen, pamabrom and pyrilamine maleate in commercial tablets
Product
Femistar
Ingredient
Label Value
(mg per Tablet)
Found
(mg)
Recovery ± RSD
(%)
Acetaminophen
500
502.0
100.4 ± 0.21
Pamabrom
25
24.96
99.84 ± 0.75
Pyrilamine
15
15.04
101.33 ± 0.88
* n = Average of 10 determinations
Linearity
Linear calibration plots of the proposed method were obtained
by analyzing eleven solutions over concentration ranges of 50150 mg/mL (50, 60, 70, 80, 90, 100, 110, 120, 130, 140 and 150
µg/mL) for acetaminophen, 2.5-7.5 mg/mL for pamabrom (2.5,
3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5 µg/mL) and 1.54.5 mg/mL for pyrilamine maleate (1.5, 1.8, 2.1, 2.4, 2.7, 3.0,
3.3, 3.6, 3.9, 4.2 and 4.5 µg/mL). Each solution was prepared
in triplicate. The acceptance criterion was the value of correlation coefficient which should be close to 1.
Accuracy
Accuracy of the method was evaluated in triplicates using standard addition technique at three concentration levels i.e. 80 %,
100 % and 120 % of target test concentration (100 mg/mL of acetaminophen, 5 mg/mL of pamabrom & 3 mg/mL of pyrilamine
maleate). The recoveries of analytes were then calculated. Generally acceptable values for accuracy ranges from 95-105 %.
Precision
For intra-day precision, three set of concentrations of acetaminophen, pamabrom and pyrilamine maleate were tested six
times within the same day. From these replicates, percentage
RSD was calculated. For intermediate precision, two different
analysts from the same Laboratory tested the same three concentrations six times. RSD was then calculated from those.
RSD less than 2 % is generally accepted for precision so this
criterion was set as acceptable for this study.
Specificity
To demonstrate the stability indicating properties of the proposed method, accelerated degradation studies were performed on acetaminophen, pamabrom and pyrilamine in
tablet dosage form by applying different stress conditions.
The stress conditions employed include light & humidity exposure, heat (60 oC), acid (1N HCl), base (1N NaOH), and
Oxidative (10% H2O2) stress. The monitoring time was 24
hours for acid (1N HCl), base (1N NaOH), oxidative (10%
H2O2), light exposure and humidity stress and 60 minutes of
heat stress (60 oC ).
Robustness
For carrying out the robustness, very small changes were carried out in mobile phase composition, flow rate and pH of acidified water. The effect of these small changes on retention
time, tailing factor, resolution and number of theoretical plates
of each analyte was then assessed. Tailing factors less than 1.5,
theoretical plates greater than 2000 and resolution greater than
1.5 was acceptable criteria for the proposed method.
Solution stability and Mobile Phase stability
To check the stability of all the three active components of this
ternary combination in solution form, the solution of these
components was placed in tight containers at room temperature for 48 hours and their stability was checked after each 12
hours period. Mobile phase stability was also checked by using
12-48 hour old mobile phase for the preparation of analyte
solution and then calculating the recovery of the active components in that mobile phase solution.
Application of the method in tablets
20 tablets were weighed and the average weight was calculated. These tablets were then ground to fine powder. Weight of
the powder equivalent to mean weight of one tablet of Femistar
(composition 500 mg acetaminophen, 25 mg of pamabrom and
15 mg of pyrilamine maleate per tablet) was diluted to 100 mL
98
J. Mex. Chem. Soc. 2015, 59(2)
with mobile phase. 2 ml of this solution was then diluted to 100
mL with mobile phase to obtain concentration equal to 100 mg/
mL of acetaminophen, 5 mg/mL of pamabrom and 3 mg/mL of
pyrilamine maleate. Percentage recovery of each analyte was
then calculated using the developed method. Percentage recovery from 95-105 % was considered as acceptable for this study.
Conclusions
A simple, sensitive, isocratic and accurate reverse phase HPLC
method has been described for simultaneous determination of
acetaminophen, pyrilamine maleate and pamabrom in pharmaceutical formulations. The proposed method was validated by
testing its linearity, accuracy, and precision, limits of detection
and quantitation and specificity. The method was good enough
to separate the peaks of active pharmaceutical ingredients
(APIs) from the degradation products (produced during forced
degradation studies). It is also clear from the chromatograms
that both the active ingredient peaks in all the stress conditions
were free from any sort of degradation impurities. All these
convince us to conclude that the method can be successfully
used for any sort of stability and validation studies.
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Article
J. Mex. Chem. Soc. 2015, 59(2), 99-104
© 2015, Sociedad Química de México
ISSN 1870-249X
ZnO-Photocatalyzed Oxidative Transformation of Diphenylamine.
Synergism by TiO2, V2O5, CeO2 and ZnS
Chockalingam Karunakaran* and Swaminathan Karuthapandian1
* Corresponding author: Prof. Dr. C. Karunakaran, CSIR Emeritus Scientist, Department of Chemistry, Annamalai University,
Annamalainagar 608002, India
1 Present address: Department of Chemistry, VHNSN College, Virudhunagar 626001, Tamilnadu, India
Received September 24th, 2014; Accepted February 17th, 2015
Abstract. Diphenylamine (DPA) undergoes photocatalytic transformation on nanoparticulate ZnO surface yielding N-phenyl-p-benzoquinonimine (PBQ). The reaction rate increases with the increase of
(i) DPA-concentration, (ii) ZnO-loading, (iii) airflow rate and (iv)
light intensity. The formation of PBQ is larger on using UV-C light
instead of UV-A light. The photocatalyst is reusable. The mechanism
of the photocatalytic transformation has been discussed with a suitable kinetic law. Nanoparticulate TiO2, V2O5, CeO2 and ZnS enhance
the ZnO-photocatalyzed PBQ formation indicating interparticle
charge transfer in semiconductor mixtures.
Key words: Photocatalysis; Semiconductor; Kinetic law; Interparticle charge transfer; N-phenyl-p-benzoquinonimine
Resumen. La Difenil amina (DPA) se transforma en N-fenil-p-benzoquinonimina (PBQ) a través de un proceso fotocatalítico en la superficie de ZnO nanoestructurado. La velocidad de reacción aumenta con
(i) la concentración DPA, (ii) la cantidad de ZnO, (iii) el flujo de aire
y, (iv) la intensidad de la luz. La tasa de de formación de PBQ es
mayor si se usa iluminación con UV-C comparada con UV-A. El fotocatalizador se puede usar de nuevo. El mecanismo de la conversión
fotocatalítica se discute en términos de una ley cinética apropiada.
La presencia de TiO2, V2O5, CeO2 y ZnS nanoestructurados incrementa la formación fotocatalítica de PBQ en ZnO nanoestructurado,
indicando que hay transferencia de carga entre las partículas de los
diferentes materiales semiconductores.
Palabras clave: Fotocatalísis; Semiconductor; Ley cinética; Transferencia de carga entre partículas; N-fenil-p-benzoquinonimina.
Introduction
tantly, it is a promising photocatalyst for environmental remediation [12] and is believed to be an alternative to TiO2 as both
semiconductors possess approximately the same band gap.
Furthermore, their conduction band (CB) energy levels and
their valence band (VB) edges are not significantly different.
In addition, there are reports that the photocatalytic activity of
ZnO is better than that of TiO2 [13]. ZnO has also been employed as photocatalyst for organic transformation [1]. Hence
it is of interest to study the photocatalytic transformation of
DPA using nanocrystalline ZnO. It is possible to improve the
photocatalytic activity by enhancing the lifetime of the charge
carriers in semiconductor nanocrystals. This may be achieved
through interparticle charge transfer (IPCT). If a mixture of
two nanoparticulate semiconductors is employed as a photocatalyst for degradation of organic pollutants enhanced photocatalytic activity is observed [14, 15]. This has been explained
invoking IPCT. However, such enhancement is not realized in
the photocatalytic oxidation of iodide ion suggesting the IPCT
to be slower than hole-transfer to iodide ion [16]. Hence it is of
interest to study photocatalytic organic transformation using
nanoparticulate semiconductor mixture as photocatalyst. Here
we report enhanced photocatalytic organic transformation of
DPA on mixing nanoparticulate TiO2 or V2O5 or CeO2 or ZnS
with ZnO nanocrystals. Systematic study on ZnO-photocatalyzed transformation of DPA is necessary to obtain the kinetic
law governing the reaction. The UV light (ca. 5%) present in
Heterogeneous photocatalysis provides a promising method to
realize green chemical process in organic synthesis and application of semiconductor-photocatalysis in selective organic
transformations has been highlighted by Lang et al [1], Palmisano et al [2], Shiraishi and Hirai [3], etc. Selective photocatalytic (i) oxidation of alcohols to aldehydes by TiO2 [1, 3, 4],
CdS [5] and Pd@CdS [6] and to ketones by TiO2 [2] and Au/
TiO2 [1], benzene to phenol by Au/TiO2 [1], amines to imines
by TiO2 [1], Pt/TiO2 [3] and Au/TiO2 [1], (ii) oxidative dehydrogenation of 1,2,3,4-tetrahydroquinoline to 3,4-dihydroquinoline by TiO2 [1] and (iii) reduction of nitrobenzene to
aniline by Au/TiO2 [1], nitrophenol to aminophenol by N-TiO2
[2] and CdS [5], nitrotoluene to aminotoluene by N-TiO2 [2],
nitroaniline to phenylenediamine by N-TiO2 [2] and CdS [5]
and nitrobenzaldehyde to aminobenzaldehyde by N-TiO2 [2]
have been reported. Diphenylamine (DPA) is used in post-harvest treatment of apple and pear [7] and photosensitized oxidation of DPA has been studied; cyanoanthracenes [8] and
benzophenone [9] are some of the photosensitizers employed.
The unsensitized photoconversion of DPA into N-phenyl-p-benzoquinonimine (PBQ) is slow [10]. Nanocrystalline
ZnO is a unique functional oxide because of its dual semiconducting and piezoelectric properties. It has a wide range of applications in optics, optoelectronics, sensors, actuators, energy
and biomedical sciences, and spintronics [11]. More impor-
100
J. Mex. Chem. Soc. 2015, 59(2)
the solar spectrum could photoexcite ZnO and hence the reaction has also been studied using natural sunlight.
Experimental
Materials and measurements
ZnO, TiO2, CeO2, V2O5 and ZnS (Merck) were used as supplied and their specific surface areas, obtained by BET method, are 12.2, 14.7, 11.0, 16.1 and 7.7 m2 g-1, respectively. The
mean particle sizes (t) of ZnO, TiO2, CeO2, V2O5 and ZnS,
obtained using the formula t = 6/ρS, where ρ is the material
density and S is the specific surface area, are 87, 104, 76, 111
and 190 nm, respectively. The secondary particle size distributions occurring because of agglomeration in alcohol have been
measured using particle sizer Horiba LA-910 or Malvern
3600E (focal length 100 mm, beam length 2.0 mm). The obtained powder X-ray diffractograms of ZnO, TiO2, CeO2 and
ZnS show the crystalline structures as hexagonal wurtzite,
tetragonal anatase, cubic fluorite and cubic zinc blende, respectively [17]. The UV-visible diffuse reflectance spectra of
the materials were obtained using a Shimadzu UV-2600 spectrophotometer with ISR-2600 integrating sphere attachment.
The Kubelka-Munk plots provide the band gaps of ZnO, TiO2,
CeO2 and ZnS as 3.15, 3.18, 2.89 and 3.57 eV, respectively
[17]. Potassium tris(oxalato)ferrate(III), K3[Fe(C2O4)3].3H2O,
was prepared by standard method [18]. DPA, AR (Merck) was
used as received. Commercial ethanol was purified by distillation with calcium oxide.
UV light-induced transformation
The UV light induced reaction on ZnO was carried out in a
multilamp photoreactor fitted with eight 8 W mercury UV
lamps (Sankyo Denki, Japan) of wavelength 365 nm, shielded
by highly polished anodized aluminum reflector. Four cooling
fans at the bottom of the reactor dissipate the generated heat.
The reaction vessel was a borosilicate glass tube of 15-mm
inner diameter and was placed at the center of the reactor. The
UV light-induced reaction was also studied with a micro-photoreactor fixed with a 6 W 254 nm low-pressure mercury lamp
and a 6 W 365 nm mercury lamp. Quartz and borosilicate glass
tubes were employed as reaction vessels for 254 and 365 nm
lamps, respectively. The light intensity (25.2 μeinstein L-1 s-1
unless otherwise mentioned) was determined by ferrioxalate
actinometry. The volume of the reaction solution was always
maintained as 25 mL in the multilamp photoreactor and 10 mL
in the micro-photoreactor. Air was bubbled through the ethanolic solution of DPA (5 mmol L-1 unless otherwise stated) to
keep the catalyst powder (1.0 g unless otherwise given) under
suspension and at constant motion. The airflow rate (7.8 mL s-1
unless otherwise stated) was measured by soap bubble method.
The UV-visible spectra were recorded using a Hitachi U-2001
UV-visible spectrophotometer. The solution was diluted
5-times to decrease the absorbance to the Beer-Lambert law
Chockalingam Karunakaran and Swaminathan Karuthapandian
limit. The PBQ formed was estimated from its absorbance at
450 nm.
Sunlight-induced transformation
The sunlight-induced reaction on ZnO was carried out under
clear sky in summer (March-July) at 11.30 am - 12.30 pm. The
sunlight intensity (W m-2) was measured using a Global pyranometer (Industrial Meters, Bombay). The solar irradiance
(einstein L-1 s-1) was also measured by ferrioxalate actinometry. The measured 440 W m-2 corresponds to 22 μeinstein L-1
s-1. Ethanolic solutions of DPA of required concentration (5.0
mmol L-1 unless otherwise stated) were prepared afresh for
each set of experiments and taken in wide cylindrical glass
vessels of uniform diameter. The entire bottom of the vessel
(11.36 cm2 unless otherwise mentioned) was covered by ZnO
powder (1.0 g). Air was bubbled (4.6 mL s-1 unless otherwise
stated) with a micro-pump without disturbing the catalyst bed.
The volume of DPA solution was 25 mL and the loss of solvent
due to evaporation was compensated periodically. PBQ formed
was estimated spectrophotometrically.
Results and Discussion
Photo-induced oxidative transformation on ZnO
The UV light-induced oxidative transformation of DPA on
ZnO surface in ethanol was performed in the presence of air
using a multilamp photoreactor equipped with UV lamps of
wavelength 365 nm. The UV-visible spectra of the DPA solution, recorded at different illumination time, reveal the formation of PBQ (λmax= 450 nm). Fig. 1 ([DPA]0 = 25 mmol L-1)
displays the time spectra. The illuminated solution does not
show electron paramagnetic resonance (EPR) signal indicating
the absence of formation of diphenylnitroxide. Furthermore,
thin layer chromatographic analysis reveals formation of a single product. The illuminated DPA solution was evaporated after the separation of ZnO and the solid was dissolved in
chloroform to develop the chromatogram on a silica gel
G-coated plate using benzene as eluent. The PBQ formed was
estimated from the absorbance at 450 nm using its molar extinction coefficient [19, 20]. The linear increase of [PBQ] with
illumination time (not shown) provides the initial rate of PBQ
formation and the rates are reproducible to ±6%. The uncatalyzed photooxidation of DAP is slow [10] and the rate of PBQ
formation on ZnO was obtained by measuring the rates of PBQ
formation in the presence and absence of ZnO. The oxidative
transformation of DPA into BPQ on ZnO surface takes place
under natural sunlight also. The results obtained are similar to
those with UV light. Measurement of the solar irradiance (W
m-2) shows fluctuation of sunlight intensity during the experiment even under clear sky. Hence, the solar experiments at different reaction conditions were carried out in a set to maintain
the quantity of sunlight incident on unit area the same. This
makes possible to compare the solar results. A pair of solar
ZnO-Photocatalyzed Oxidative Transformation of Diphenylamine. Synergism by TiO2, V2O5, CeO2 and ZnS
experiments carried out simultaneously under identical reaction conditions yields results within ±6% and this is so on different days. The effect of operational parameters on the
solar-induced oxidative transformation was investigated by
carrying out the given set of experiments simultaneously and
the results presented in each figure represent identical sunlight
intensity. The rate of PBQ formation was obtained by illuminating the DPA solution on ZnO bed for 60 min. Fig. 2 shows
the enhancement of PBQ formation on ZnO with [DPA]. The
observed enhancement corresponds to Langmuir-Hinshelwood
kinetics with respect to [DPA]. The rate of surface reaction
with UV light increases with loading of ZnO in the DPA solution and the rate reach a limit at high ZnO-loading. The results
are presented in Fig. 3. Measurement of the rate of PBQ formation on ZnO at different airflow rates shows enhancement of
the surface reaction by oxygen and the rate dependence on the
airflow rate conforms to the Langmuir-Hinshelwood kinetic
law. Fig. 4 presents the results. The PBQ formation on ZnO
was also determined without bubbling air but the solution was
not deoxygenated. The dissolved oxygen itself brings in the
light-induced surface reaction. However, the reaction is slow.
PBQ formation on ZnO with UV light was examined at different light intensities (I). The reaction was performed with two,
four and eight lamps and the angles sustained by the adjacent
lamps are 180°, 90° and 45°, respectively. The dependence of
the surface reaction rate on photon flux is shown in Fig. 5.
PBQ is not formed in the dark. Study of the PBQ formation on
ZnO with UV-A and UV-C light, employing a 6 W 365 nm
mercury lamp (I = 18.1 μeinstein L-1 s-1) and a 6 W 254 nm
low-pressure mercury lamp (I = 5.22 μeinstein L-1 s-1), separately in the micro-photoreactor under identical conditions
shows that UV-C light is more efficient than UV-A light in inducing the organic transformation on ZnO. The PBQ formation with UV-A and UV-C light are 11.2 and 44.2 nmol L-1 s-1,
respectively. The solar PBQ formation on ZnO increases linearly with the apparent area of ZnO-bed. Fig. 6 displays the
results. ZnO retains its photocatalytic efficiency on usage. Reuse of ZnO shows sustainable light-induced PBQ formation.
Singlet oxygen quencher azide ion (5 mmol L-1) does not inhibit the PBQ formation revealing the absence of involvement
of singlet oxygen in the light-induced organic transformation
on ZnO. This is in agreement with the literature; Fox and Chen
[21] excluded the possibility of singlet oxygen in the TiO2-photocatalyzed olefin-to-carbonyl oxidative cleavage.
101
Fig. 1. PBQ formation in presence of ZnO under UV light: the
UV-visible spectra of DPA solution at different illumination time
(5-times diluted).
Fig. 2. Variation of ZnO-photocatalyzed PBQ formation rate with
[DPA].
Mechanism
Band gap illumination of ZnO creates electron-hole pairs,
electrons in the CB and holes in the VB. Recombination of the
electron-hole pair is very fast, takes place in picosecond-time
scale, and for an effective photocatalysis the reactants are to be
adsorbed on the surface of ZnO. The hole is likely to pick up
an electron from the surface adsorbed DPA molecule to form
diphenylamine radical-cation (Ph2NH•+). The oxygen molecule adsorbed on the ZnO surface takes up the CB electron.
Fig. 3. Dependence of photocatalyzed PBQ formation on ZnO-loading.
102
J. Mex. Chem. Soc. 2015, 59(2)
Chockalingam Karunakaran and Swaminathan Karuthapandian
The superoxide radical-anion formed probably reacts with diphenylamine radical-cation to afford PBQ.
ZnO + hν → h+(VB) + e‒(CB)
Ph2NH(ads) + h+(VB) → Ph2NH•+
O2(ads) + e‒(CB) → O2•‒
Ph2NH•+
+ O2•‒ →
N
O
+ H2O
Kinetic law
Fig. 4. ZnO-photocatalyzed PBQ formation at different airflow rates.
Fig. 5. Effect of photon flux on ZnO-photocatalyzed PBQ formation.
Kinetic analysis is possible with the results obtained using artificial UV light. The heterogeneous photocatalytic reaction
taking place in a continuously stirred tank reactor (CSTR) conforms to the kinetic law [22]:
rate of PBQ formation on ZnO = kK1K2SIC[DPA]γ/(1 +
K1[DPA]) (1 + K2γ)
where K1 and K2 are the adsorption coefficients of DPA
and O2 on illuminated ZnO surface, k is the specific rate of
oxidation of DPA on ZnO surface, γ is the airflow rate, S is the
specific surface area of ZnO, C is the amount of ZnO suspended per litre and I is the photon flux. The obtained data fit to the
Langmuir-Hinshelwood kinetic profile, drawn using a computer program [22] (Figs. 2 and 4). The linear double reciprocal
plots of surface reaction rate versus [DPA] and airflow rate
(not shown) also confirm the Langmuir-Hinshelwood kinetic
law. The data-fit provides the adsorption coefficients K1 and K2
as 140 L mol-1 and 0.064 mL-1 s, respectively, and the specific
reaction rate k as 28 μmol L m-2 einstein-1. However, the rate of
PBQ formation on ZnO surface fails to increase linearly with
the catalyst-loading. This is because of the high catalyst loading. At high catalyst loading, the surface area of the catalyst
exposed to illumination does not correspond to the weight of
the catalyst. The amount of ZnO used is beyond the critical
amount corresponding to the volume of the reaction solution
and the reaction vessel; the whole amount of ZnO is not exposed to light. The photoinduced transformation lacks linear
dependence on illumination intensity; less than first power dependence of surface-photocatalysis rate on light intensity at
high photon flux is well known [23].
Synergism by TiO2, V2O5, CeO2 and ZnS
Fig. 6. Variation of the photocatalyzed PBQ formation rate with ZnObed area .
In coupled semiconductors, also known as semiconductor
composites, vectorial transfer of photoproduced charge carriers from one semiconductor to another is possible. This charge
separation enhances the photocatalytic efficiency and examples for coupled semiconductors are many [24]. In coupled
semiconductors, both the semiconductors coexist in the same
particle and the charge separation occurs within the particle.
But what we observe here is enhanced photoinduced transformation of DPA to PBQ on mixing nanoparticulate ZnS or TiO2
or CeO2 or V2O5 with ZnO nanoparticles. Fig. 7a presents the
ZnO-Photocatalyzed Oxidative Transformation of Diphenylamine. Synergism by TiO2, V2O5, CeO2 and ZnS
103
Fig. 7 (a). Enhanced PBQ formation on mixing TiO2 or V2O5 or CeO2 or ZnS with ZnO, (b) Secondary particle size distributions of ZnO, CeO2,
ZnS, TiO2 and V2O5 measured by particle size analyzer.
enhancement of photocatalytic formation of PBQ by ZnO
mixed with ZnS or TiO2 or CeO2 or V2O5 illuminated by UV
light - the two nanoparticulate semiconductors are in suspension and at constant motion. This observed enhanced photocatalytic transformation is likely due to interparticle charge
transfer. Nanoparticles in suspension aggregate [25]. Fig. 7b
displays the particle size distributions of ZnO, ZnS, TiO2,
CeO2 and V2O5 nanoparticles in suspension.
Examination of Fig. 7b in conjunction with the determined
particle sizes shows aggregation of the nanoparticles. As observed in individual nanoparticulate semiconductor suspension, aggregation is likely in nanoparticulate semiconductor
mixtures under suspension and both the semiconductor
nanoparticles are likely to be present in the aggregates. Charge
transfer between ZnO and ZnS or TiO2 or CeO2 or V2O5
nanoparticles is likely to occur when both the semiconductors
are under band gap-illumination and in contact with each other.
Electron from CB of a semiconductor may move to another if
the latter is of lower energy and so is the hole from VB. The CB
electron of ZnO is less cathodic than those of ZnS [26] and
CeO2 [27] and more cathodic than those of V2O5 and TiO2 [28].
This enables transfer of CB electron of ZnS and CeO2 to the
CB of ZnO (in ZnO-ZnS and ZnO-CeO2 mixtures) and migration of CB electron of ZnO to the CB of TiO2 and V2O5 (in
ZnO-TiO2 and ZnO-V2O5 mixtures). Similarly, the VB of ZnO
is more anodic than those of ZnS and CeO2 and less anodic
than those of TiO2 and V2O5. This favors hole-transfer from the
VB of ZnO to those of ZnS and CeO2 (in ZnO-ZnS and ZnOCeO2 mixtures) and hole-migration from VB of TiO2 and V2O5
to that of ZnO (in ZnO-TiO2 and ZnO-V2O5 mixtures). This
interparticle charge transfer enhances the photocatalytic transformation. The energy difference between the CB electrons of
the two semiconductors is the driving force for the interparticle
electron separation and the free energy change is given by –ΔG
= e(E(CBSC1) – E(CBSC2) [29]. In terms of redox chemistry, the
CB and VB refer to the reduced and oxidized states in the semiconductor. In TiO2, CeO2,V2O5 and ZnO or ZnS the CB electrons refer to the reduced forms of Ti4+ (i.e., Ti3+), Ce4+ (i.e.,
Ce3+), V4+ (i.e., V3+) and Zn2+ (i.e., Zn+), respectively. Similarly, the VB hole corresponds to the oxidized forms of the respective O2- (i.e., O-) or S2- (i.e., S-). The interparticle
charge-transfer, the transfer of electron from the CB of ZnS or
CeO2 to CB of ZnO refers to the electron jump from Zn+ of
ZnS or Ce3+ to Zn2+ of ZnO. The electron jump from CB of
ZnO to those of TiO2 and V2O5 corresponds to electron migration from Zn+ of ZnO to V5+ or Ti4+. The hole-transfer from the
VB of ZnO to those of ZnS and CeO2 refers to the electron-jump
from O2- of CeO2 or S2- of ZnS to O- of ZnO. Similarly, the
hole-jump from the VB of TiO2 and V2O5 implies electron
movement from O2- of ZnO to O- of TiO2 and V2O5. The possibility of cross-electron–hole combination, the transfer of
electron from the CB of one semiconductor (SC1) to the VB of
the other (SC2) is very remote; the very low population of the
excited states renders the electron transfer between two excited
states highly improbable. A possible reason for not observing
the maximum photocatalytic transformation at 50% wt. composition of the semiconductor mixtures is the densities and particle sizes of the semiconductors and also the aggregation.
Conclusions
DPA on light-induced oxidative transformation on ZnO surface
affords BPQ. The BPQ formation on ZnO increases with [DPA]
and airflow rate and conforms to Langmuir-Hinshelwood kinetic law. The PBQ formation on ZnO is larger with UV-C light
than with UV-A light. ZnO mixed with ZnS or TiO2 or CeO2 or
V2O5 yields more PBQ than by the individual semiconductor
and this is likely to be due to interparticle charge transfer.
Acknowledgement
Prof. C. Karunakaran is thankful to the Council of Scientific
and Industrial Research (CSIR), New Delhi for the Emeritus
Scientist Scheme [21(0887)/12/EMR-II].
104
J. Mex. Chem. Soc. 2015, 59(2)
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Article
J. Mex. Chem. Soc. 2015, 59(2), 105-118
© 2015, Sociedad Química de México
ISSN 1870-249X
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by Hairy Roots of Catharanthus roseus (L) G. Don
Juan Luis Monribot-Villanueva,1,3 Eliel Ruiz-May,1,3 Rosa María Galaz-Ávalos,1 Dayakar Badri2 and
Víctor Manuel Loyola-Vargas*1
1 Unidad
de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán. Calle 43 No. 130,
Col. Chuburná de Hidalgo, Mérida, Yucatán, México. vmloyola@cicy.mx Tel.: 52-999-9428330
2 Center for Rhizosphere Biology, Colorado State University, Fort Collins, CO 80523, USA.
3 Current address: Red de Estudios Moleculares Avanzados, Cluster Biomimic®, Instituto de Ecología A.C., Carretera Antigua
a Coatepec No. 351, Congregación el Haya, CP 91070, Xalapa, Veracruz, México.
Received October 27th, 2014; Accepted February 17th, 2015
Abstract. Knowledge on the biosynthetic pathways of the monoterpene alkaloids is enormous, but little is known about their mechanism
of transporting system from the plant cell. There is not concrete evidence confirming the role of ABC transporters in the secretion of
monoterpene indole alkaloids (MIAs) in Catharanthus roseus. Therefore, in order to determine the role of different transporting systems
involved in the MIAs translocation, we employed a pharmacological
approach by using transport inhibitors such as, KCN, Na3VO4, quinidine and glibenclamide in hairy root cultures of C. roseus. It was
found that the accumulation of ATP drastically decreased in the presence of KCN or 100 µM acetylsalicylic acid (ASA)/100 µM KCN.
The treatment with the inhibitors KCN and glibenclamide in the presence of ASA significantly increased the ajmalicine secretion compared to the control. The secretion of serpentine was undetected
during the first 24 h in all the samples. Treatment with the inhibitors
quinidine and glibenclamide provoked a significant reduction of serpentine secretion in the hairy roots compared to the control. Based on
our results, we found evidence that ABC transporters might participate in the secretion of MIAs by C. roseus hairy roots.
Key words: ABC transporters; alkaloids; Catharanthus roseus; exudation; inhibitors.
Resumen. El conocimiento de las rutas de biosíntesis de los alcaloides
monoterpénindólicos (AMIs) es amplio, pero prácticamente no se conoce nada sobre los mecanismos de transporte que se requieren para
que dichas rutas funcionen adecuadamente. Puesto que sólo se ha descubierto uno de estos transportadores, perteneciente a la familia de
transportadores ABC, en la investigación reportada en este artículo presentamos evidencia de la participación de tales transportadores utilizando un acercamiento farmacológico mediante el uso de los inhibidores
KCN, Na3VO4, quinidina y glibenclamida durante la secreción de
AMIs en raíces transformadas de Catharanthus roseus bajo condiciones de inducción y no inducción. La acumulación de ATP disminuye
drásticamente en presencia de KCN o 100 µM ácido acetil salicílico
(AAS)/100 µM KCN. El tratamiento con los inhibidores KCN y glibenclamida, en presencia de AAS, aumenta significativamente la secreción
de ajmalicina, comparada con el testigo. La secreción de serpentina fue
indetectable durante las primeras 24 h en todas las muestras. Las raíces
en el tratamiento con los inhibidores quinidina y glibenclamida mostraron una significativa disminución en la secreción de serpentina comparada con el testigo. Nuestros resultados muestran evidencia de que los
transportadores ABC pueden estar participando en la secreción de
AMIs por las raíces transformadas de C. roseus.
Palabras clave: alcaloides; Catharanthus roseus; inhibidores; transportadores ABC; secreción.
Introduction
A pioneer study reported the transport of vindoline and
ajmalicine by a specific proton-antiporter system [5]. Followed
by this report, another study demonstrated that the transport of
vindoline is carried out by an energy-dependent transporter by
using C. roseus protoplasts [6], besides an ion-trap mechanism
that could contribute to the vacuolar uptake of these endogenous alkaloids [7]. Recently, it was demonstrated that vacuolar
transport of MIAs is mediated by a proton-driven antiport and
not by an ion-trap mechanism or ABC transporters [8]. However, the expression of the CjMDR1 gene, a transmembrane
ABC transporter from Coptis japonica, in C. roseus cells, produced an increase in the accumulation of ajmalicine and tetrahydroalstonine, alkaloids from C. roseus but not of berberine,
Catharanthus roseus synthesizes more than 140 secologanin-derived monoterpene indole alkaloids (MIAs) [1], of the
approximately 2,000 known compounds, and some of them
such as ajmalicine, vincamine and reserpine (peripheral vasodilators), ajmaline (antiarrhythmic), vinblastine and vincristine
(anticancer), and yohimbine (pro-erectile) possess important
pharmacological effects [2] widely used in medicine. The biosynthesis of these compounds involves a very complex pathway and also requires the participation of different cell types
and different organelles within the cell [3, 4]. These pathways
require of a very precise transport system.
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J. Mex. Chem. Soc. 2015, 59(2)
the main substrate of the CjMDR1 transporter [9]. Recently, the screening of the Plant Medicinal Genomics Resource
database allowed to El-Guizani et al. [10] to identify 16 ABC
transporters partial sequences in C. roseus.
The alkaloids biosynthetic pathway can be regulated at
the cellular and at the molecular level [11], which includes
their transport through the membranes of different organelles
and different tissues. The De Luca’s laboratory has found
that vindoline is accumulated inside the leaf cells, meanwhile
catharanthine accumulates in leaf wax exudates [12]. This
separation is mediated by a catharanthine transporter
(CrTPT2) that is present in the epidermis of young leaves
[13]. The secretion of alkaloids to the leaves surface may be
mediated by unique transporters to MIA-producing plant
species. A strong candidate could be the transporters involved in the cuticle assembly, which are of the kind of ABC
transporters.
Previously, it was reported the accumulation and secretion
of MIA by treatment with different elicitors (methyl jasmonate, acetyl salicylic acid and nitric oxide) in in vitro tissue cultures of C. roseus, such as hairy roots [14, 15], cell suspensions
[16] and tumor lines [17]. In our laboratory, we observed that
the hairy roots of C. roseus elicited with methyl jasmonate
(MeJA) showed a differential secretion of ajmalicine, serpentine, ajmaline and catharanthine compared to the controls [15].
This treatment also increased the accumulation of H2O2 during
the first 48 h [18].
It has been demonstrated the involvement of ABC transporters in the root secretion of phytochemicals [19-22]. In
agreement with these studies, recently it has been showed a
tight regulation in the export and accumulation of defense phytochemicals in the rhizosphere [23, 24]. In addition, the ABC
transporter, GmPDR12 expression was induced in response to
salicylic acid and MeJA [19]. Differential expression of ABC
transporters in Arabidoposis thaliana root tissues was observed in response to nitric oxide, salicylic acid and MeJA
[22]. Based on these observations, one can hypothesize that
ABC transporters might be involved in transporting MIAs
from C. roseus cell cultures.
Better understanding about the transport mechanism of
secondary metabolites and their regulatory networks would
provide the development of novel methods to engineer C. roseus plants for commercial applications. In the present study,
we aimed to identify the type of transporters for MIAs by employing pharmacological approach using C. roseus hairy root
cultures.
Juan Luis Monribot-Villanueva et al.
Effect of inhibitors in plant growth and ATP accumulation
To determine the effect of inhibitors on hairy roots growth, we
treated the hairy roots with 100 mm of all inhibitors independently and dry weight (DW) was analyzed. We did not observe any difference in the dry weight after 48 h of all inhibitors
treatment but significant reduction on the DW of hairy roots
was observed after 72 hours only in 100 µM ASA treatment
(Fig. 1A).
We also analyzed the ATP levels in response to the inhibitors. The accumulation of ATP drastically decreased (close to
zero) in the presence of KCN or 100 µM ASA/100 µM KCN
(Fig. 1B) and the condition was lasted for the next 60 h. The
treatment with 100 µM ASA alone slightly decreased the accumulation of ATP during the first 36 h and slowly the ATP levels
were increased to 70 mg ATP g-1 fresh weight (FW) (Fig. 1B),
while the control remains around 40 mg ATP g-1 FW after an
increase during the first 36 h. High significant differences were
observed in the levels of ATP between the treatments of 100
mM ASA and KCN (Fig. 1B).
Results
To test whether the secretion of alkaloids is facilitated by ABC
transporters, we employed a pharmacological approach by using inhibitors of different transporting systems. If the secretion
of MIAs is mediated by ABC transporters, ATP must play a
central role and inhibition of ATP production must modify the
exudation process.
Fig. 1. Treatments effect on hairy roots growth (A) and ATP accumulation (B). Hairy roots were treated as indicated in “Materials and
Methods”, control ( ) or presence of ASA ( ), KCN ( ) and ASA/
KCN ( ). Error bars represent ± SE (n = 3). Asterisk represents statistical significance of mean differences at a given time by Turke´s
test (*, P ≤ 0.05; **, P ≤ 0.01). The experiment was performed three
times.
Role of Different Transporting Systems in the Secretion of Alkaloids by Hairy Roots of Catharanthus roseus (L) G. Don
Effect of different transporting systems
inhibitors in ajmalicine secretion by hairy roots
of C. roseus.
Ajmalicine was the more abundant MIA identified in the culture medium of hairy roots. The secretion of this alkaloid was
significantly increased after 48 h of the treatment with 100 µM
ASA (Fig. 2A) and the level of ajmalicine was two-fold more
(7.75 mg L-1) than the control (3.06 mg L-1) after 72 h of treatment. In the presence of inhibitors alone (KCN, orthovanadate,
quinidine and glibenclamide), we did not observe any significant changes in ajmalicine secretion compared to control. But,
we observed differential changes in ajmalicine secretion when
hairy roots treated simultaneously with the inhibitor and ASA.
For instance, treatment with the inhibitors, KCN (Fig. 2A) and
glibenclamide (Fig. 2D) in the presence of ASA significantly
increased the ajmalicine secretion compared to control. Unlikely, the inhibitors orthovanadate (Fig. 2B) and quinidine
107
(Fig. 2C) in the presence of ASA did not show any significant
changes in ajmalicine secretion.
Effect of inhibitors in serpentine secretion
by hairy roots of C. roseus
The secretion of serpentine was undetected during the first 24
h in all the samples (Figs. 3A-3D). In the control samples there
was a gradual increase in the secretion of serpentine (0.8 mg
L-1) at 48 h (Figs. 3A-3D). In the presence of ASA, the serpentine secretion was increased to 2.0 mg L-1 after 24 h and stayed
high till 72 h period of the study (Fig. 3A). When treated with
the inhibitors alone we observed different trends: KCN did not
show significant change compared to control (Fig. 3A) and unlikely orthovanadate showed significant increase in serpentine
secretion compared to control at 48 h (Fig. 3B). On the contrary, the inhibitors quinidine and glibenclamide showed sig-
Fig. 2. Secretion of ajmalicine on hairy roots treated with ASA and inhibitors. A) Ajmalicine secretion on treatments with ASA ( ), KCN ( ),
ASA/KCN ( ) ; B) ASA ( ), orthovanadate ( ), ASA/orthovanadate ( ); C) ASA ( ), quinidine ( ), ASA/quinidine ( ) ; D) ASA ( ),
glibenclamide ( ), ASA/ glibenclamide ( ). Each treatments were correlated with respective control ( ). Error bars represent ± SE (n = 3).
Asterisk represents statistical significance of mean differences at a given time by Turke´s test (*, P ≤ 0.05; **, P ≤ 0.01). The experiment was
performed three times.
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J. Mex. Chem. Soc. 2015, 59(2)
Juan Luis Monribot-Villanueva et al.
nificant reduction of serpentine secretion compared to control
(Figs. 3C-3D). Interestingly, the treatment with quinidine in
the presence of ASA also showed significant reduction in serpentine secretion at 72 h (Fig. 3C) but the presence of ASA
with other inhibitors showed significant increase in serpentine
secretion (Figs. 3A-3D).
Effect of inhibitors in catharanthine secretion
by hairy roots of C. roseus
Catharanthine secretion did not show significant difference between the treatments with 100 µM ASA and the control
(Fig. 4A-4D). However, the treatment with 100 µM KCN
alone, significantly increased the secretion of catharanthine at
24 h (Fig. 4A). Furthermore, the treatment of hairy roots with
100 µM ASA/100 µM KCN significantly increased catharanthine secretion in the culture media in comparison with other treatments and control (Fig. 4A). But, the treatment of hairy
roots with other inhibitors (orthovanadate, quinidine and glibenclamide) in the presence and absence of ASA did not show
any significant changes in catharanthine secretion compared to
control (Fig. 4B-4D).
Non-target profile of secreted compounds
by hairy roots of C. roseus in the presence of
inhibitors
The treatment of C. roseus hairy roots with 100 µM ASA increased the secretion of several unidentified compounds (Table
1; peaks 1, 4, 9 and 21) along the 72 h of observation (Tables
1, 3, 5, 7). After 12 h of treatment with 100 µM KCN, it was
observed the decrease of the peaks 2, 6 and 13 (Table 1); same
for peaks 1, 2 and 13 at 24 h of treatment (Table 3). The treatment with ASA/KCN reduced the secretion of compounds 2, 5
and 13 during the first 12 h of treatment. The inhibition was
still observed after 48 h of treatment for peaks 5 and 13. The
inhibitory effect of the KCN, alone or in combination with
ASA disappeared after 48 h of treatment (Table 5). Several
peaks, such as 1, 4, 6, 9 and 12 increased their secretion in response to the presence of ASA or ASA/KCN along the period
of study (Tables 1, 3, 5, 7).
Under the treatment with orthovanadate the absence of
peak 6 and the decrease of peak 13 were observed in comparison to the treatment with 100 µM ASA after 12 h (Table 1). The
reduction of peaks 6 and 21, and the absence of the peak 18
were observed with the treatment of orthovanadate after 48 h
Table 1. Catharanthus roseus hairy roots alkaloids profile secreted evaluated at the sampling times of 12 hours after treatments. Treatments
with ASA, KCN, ASA/KCN, orthovanadate, ASA/ orthovanadate. Error bars represent ± SE (n = 3). The experiment was performed three times.
Peak
Treatment (high of the peak, mAU)
Control
*ASA
KCN
ASA/KCN
Orthovanadate
ASA/Orthovanada-te
1
371.55±52.26
492.20±182.42
552.45±128.80
490.57±136.11
438.01±89.78
390.27±93.93
2
356.88±123.69
305.31±155.08
42.55±42.55
105.82±105.82
247.17±143.02
541.90±273.70
3
0
0
42.22±42.22
0
52.13±52.13
29.94±29.94
4
319.91±15.55
384.81±24.01
407.61±39.25
490.35±34.14
433.43±17.81
348.31±23.68
5
245.60±106.30
0
233.13±134.86
0
100.71±70.49
276.34±138.18
6
111.42±65.73
319.93±27.79
0
397.56±12.68
0
87.34±87.34
7
0
0
93.51±55.16
0
134.91±45.88
0
8
0
0
0
0
0
36.45±36.45
9
448.57±46.19
723.43±108.30
707.77±152.77
861.18±119.17
679.22±74.18
1133.01±190.35
10
45.715±15.38
83.11±3.89
84.34±36.02
0
0
101.74±5.21
11
344.50±31.91
272.88±33.78
285.75±48.95
293.51±34.52
268.06±14.23
269.01±12.57
12
0
16.32±16.32
394.94±133.20
641.90±39.18
38.038±14.09
63.41±31.75
13
165.85±27.62
150.15±19.87
0
20.18±20.18
105.05±10.03
66.79±33.50
14
232.88±11.43
257.59±61.65
179.88±39.10
229.59±38.33
335.81±26.54
339.28±12.20
15
0
0
34.14±25.10
127.75±16.52
0
0
16
10.59±10.59
0
49.80±29.01
28.79±28.79
0
0
17
0
0
0
0
51.39±30.38
67.08±17.36
18
11.71±11.71
0
48.20±29.21
224.06±28.87
0
8.16±8.16
19
21.85±13.60
0
91.52±45.43
0
31.85±18.40
46.17±4.39
20
0
0
0
0
20.96±20.96
0
21
0
0
25.09±25.09
14.40±14.40
0
0
22
43.78±14.59
28.63±14.34
20.38±11.77
39.71±2.04
28.79±9.73
36.36±0.07
*ASA = acetyl salicylic acid; Ort. = orthovanadate; Qui. = quinidine; Gli. = glibenclamide
109
Role of Different Transporting Systems in the Secretion of Alkaloids by Hairy Roots of Catharanthus roseus (L) G. Don
(Table 5). The peaks 1, 4, 6, 8 and 21 were also reduced after
72 h of treatment (Table 7). Peak 4 incremented with the ASA
treatment. Under the treatment with ASA/orthovanadate
through the 72 h, the peaks 1, 4, 13 and 21 were reduced (Table
7). On the other hand, the peaks 2, 6, 8 and 9 were increased
(Table 7).
The decrease of peaks 4, 6 and 14 were observed through
the treatment with quinidine after 12 h (Table 2), as well as the
absence of peak 1. After 48 h of treatment, peaks 4, 13 and 19
decreased (Table 6). From 24 to 72 h the absence of peaks 1, 2,
3 and the decrement of peak 21 was observed (Tables 4, 6, 8).
When the hairy roots were treated with ASA/quinidine, some
compounds such as, peaks 1, 2 and 3, were not detected (Tables 2, 4, 6, 8). However, other peaks, such as 4, 6, 11 and 20
increased after 72 h of treatment with ASA/quinidine (Table
8). It is important to indicate that most of these peaks increased
with the treatment of 100 µM ASA in comparison with the respective control.
Several peaks decreased with the treatment of 100 µM
glibenclamide. Peaks 1, 2, 6, 9, 13, 14, and 19 decreased after
12 h of treatment (Table 2). Peaks 2 and 21 were absence after
72 h of treatment (Table 8). On the other hand, several peaks
increased with the treatment of ASA/glibenclamide, such as
the case for peaks 2, 4, 6, 9 and 14 after 12 h of treatment (Tables 2, 4, 6, 8). Peaks 2, 3, 4 and 7, even after 72 h of treatment,
are higher than the controls (Table 8).
The secretion of unknown compounds by C. roseus hairy
roots did not change after the treatment with NO (data no
showed). Indeed, we observed that the treatments with NO/
KCN, NO/orthovanadate, NO/quinidine and NO/glibenclamide did not result in an inhibitory effect of the compounds
secretion when comparing with the respectively controls.
HPLC analysis revealed clear differences in the pattern of
secretion of the alkaloids from hairy roots treated with ASA
and ABC transporters inhibitors (Tables 1-8). Using the principal components analysis (PC) we were able to determinate that
the different treatments segregated from the control sample
and they were clearly grouped along the first PC axis (Fig. 5).
Interesting, at 72 h the treatments that include ASA make a
clear group, except the treatment ASA/KCN. Moreover, the
treatments with quinidine and glibenclamide were closed correlated with DMSO as control. On the other hand, a close
corre­lation was observed between KCN and orthovanadate but
not with the respective control (Fig. 5)
Table 2. Catharanthus roseus hairy roots alkaloids profile secreted evaluated at the sampling times of 12 hours after treatments. Treatments with
ASA, quinidine, ASA/quinidine, glibencamine, ASA/glibencamine. Error bars represent ± SE (n = 3). The experiment was performed three times.
Peak
Treatment (high of the peak, mAU) (12 h)
Control
*ASA
Quinidine
ASA/Quinidine
Glibencamine
ASA/Glibenca-mine
1
501.01±81.85
605.01±176.69
0
0
215.85±84.31
409.94±21.94
2
131.88±131.88
119.79±119.79
0
0
220.44±88.86
658.87±7.17
3
0
0
42.79±42.79
0
0
0
4
338.41±18.25
390.59±39.10
315.35±37.28
269.37±21.59
260.18±34.54
500.19±5.05
5
0
0
188.93±98.89
116.34±116.34
0
0
6
306.23±22.13
398.73±45.40
193.80±71.11
280.33±141.61
180.64±25.60
474.92±9.15
7
0
0
0
0
0
0
8
0
0
0
0
0
0
9
375.55±16.61
668.00±70.58
433.86±46.20
646.36±91.09
267.06±41.08
878.16±34.10
10
37.31±21.68
106.54±27.41
103.48±17.70
121.54±6.45
53.09±6.69
160.28±6.05
11
299.21±18.17
323.74±15.47
345.19±36.95
234.91±32.56
198.75±27.68
378.31±7.17
12
0
0
0
43.78±27.96
28.52±10.43
0
13
155.35±10.72
226.90±26.95
175.96±27.56
80.00±44.95
66.37±10.54
285.09±6.12
14
255.69±18.02
357.22±34.24
309.80±49.64
266.35±31.10
248.86±49.87
424.83±13.71
15
0
0
0
0
0
0
16
0
76.15±18.39
30.50±18.52
0
0
60.70±8.79
17
0
0
0
0
0
8.84±8.84
18
0
44.60±4.34
12.69±12.69
0
0
40.27±0.82
19
8.84±8.84
83.45±21.78
51.99±30.02
0
21.70±21.70
63.32±1.93
20
0
0
0
0
0
0
21
0
27.83±27.83
15.65±15.65
0
0
0
22
43.46±2.52
40.89±0.83
30.34±10.20
26.55±13.53
17.84±10.30
43.03±1.46
*ASA = acetyl salicylic acid; Ort. = orthovanadate; Qui. = quinidine; Gli. = glibenclamide
110
J. Mex. Chem. Soc. 2015, 59(2)
Juan Luis Monribot-Villanueva et al.
Table 3. Catharanthus roseus hairy roots alkaloids profile secreted evaluated at the sampling times of 24 hours after treatments. Treatments
with ASA, KCN, ASA/KCN, orthovanadate, ASA/ orthovanadate. Error bars represent ± SE (n = 3). The experiment was performed three times.
Treatment (high of the peak, mAU)
Peak
Control
*ASA
KCN
ASA/KCN
Orthovanadate
ASA/Orthovanada-te
1
407.02±252.58
453.46±64.12
409.99±142.23
469.22±44.41
582.88±189.06
344.46±55.52
2
480.29±240.97
729.11±116.62
347.41±203.20
832.59±145.93
558.49±293.02
636.63±204.33
3
0
0
69.41±69.41
104.83±104.83
0
117.21±117.21
4
412.59±52.22
555.03±27.93
464.20±71.33
797.42±44.64
682.51±86.60
757.68±145.08
5
46.43±46.43
0
67.23±67.23
0
0
0
6
362.84±52.18
359.18±71.76
408.39±45.42
689.72±40.11
396.99±38.91
291.78±17.35
7
0
0
0
0
0
0
8
0
0
0
147.80±78.31
0
0
9
551.47±52.14
857.88±30.71
879.37±59.82
1569.78±280.01
815.48±139.51
1029.20±367.83
10
41.15±41.15
75.016±37.52
99.05±49.69
54.52±54.52
78.79±39.61
0
11
462.63±28.24
404.16±53.52
403.78±15.13
555.72±50.31
489.59±27.49
353.96±45.47
12
0
0
539.76±69.82
1003.30±14.36
16.85±16.85
41.22±26.40
13
252.49±50.59
257.64±12.77
0
0
248.76±34.54
154.96±20.09
14
341.04±38.94
241.49±23.43
276.25±28.00
318.45±18.04
663.67±37.60
264.48±41.40
15
0
0
38.54±21.74
140.81±18.45
0
0
16
45.62±22.91
72.80±12.34
71.55±9.08
98.62±30.11
16.37±16.37
33.05±18.35
17
8.82±8.82
0
0
13.94±13.94
12.36±12.36
13.22±13.22
18
43.20±8.14
27.16±27.16
49.39±24.71
345.39±44.03
21.01±11.23
17.44±17.44
19
61.84±17.93
126.59±26.94
106.92±33.92
0
47.93±13.20
43.34±21.75
20
0
17.10±17.10
0
19.59±19.59
0
0
21
0
81.67±45.50
0
38.76±38.76
0
30.40±30.40
22
46.13±23.67
45.47±1.98
55.19±8.95
55.41±3.43
56.62±6.64
30.43±16.90
*ASA = acetyl salicylic acid; Ort. = orthovanadate; Qui. = quinidine; Gli. = glibenclamide
Discussion
In order to enhance and manipulate secondary metabolites mediated defense responses, it is necessary to understand the biosynthesis, regulation and transporting mechanism of these
compounds. In the present study, we made an attempt to identify the different transporting systems to translocate the indole
alkaloids by employing pharmacological approach with the
help of inhibitors.
The secretion of secondary metabolites from the plant
cells is often reported to be an energy-dependent transport [5,
25]. On the other hand, recent studies demonstrated that ABC
transporters are involved in the transport of some secondary
metabolites from plant root cells [13, 20, 26-28].
If the secretion of MIAs is mediated by ABC transporters,
ATP must play a central role and inhibition of ATP production
must modify the secretion process. We used potassium cyanide
an inhibitor of ATP synthesis, [29], Sodium orthovanadate is
an inhibitor of the membranal ATPases [30, 31] and also inhibits all kinds of ABC transporters [32, 33], quinidine and glibenclamide are potassium channel blockers [32]. Besides these,
there are other type of sulfonylurea receptor inhibitors that are
also able to inhibit the function of some ABC transporters [34].
In our study, the treatment of C. roseus hairy roots with
KCN inhibited the accumulation of ATP, both in the presence
or absence of an elicitor ASA (Fig. 1B) and it suggests that the
inhibitor KCN clearly reducing the ATP levels in the cell. Shitan et al. [35] demonstrated that the addition of KCN inhibited
the levels of berberine uptake by Cjmdr1-injected oocytes.
Furthermore, Loyola-Vargas et al. [20] reported that KCN inhibited the roots-secretion of phytochemicals in Arabidopsis
thaliana plants. Based on these observations, KCN is considered to be a good inhibitor and reduce ATP levels in the cell
and it is worth to analyze the secretion levels MIA by supplementing KCN in hairy roots to determine the ABC transporters
role in MIAs transport.
In A. thaliana P-type H+-ATPase isoforms found in membranes [36] and P-glycoproteins are strongly inhibited by vanadate [33, 37]. In our case, both potassium cyanide and
sodium orthovanadate inhibited the alkaloid secretion of several peaks, specially numbers 2, 5 and 13 by C. roseus hairy roots
(Tables 1, 3).
Orthovanadate is generally accepted as a strong inhibitor
of ABC transporters [33, 37], and their inhibitory effect were
observed on ajmalicine secretion after 48 h of treatment (Fig.
2B) and serpentine secretion after 24 h (Fig. 3B). In Lycoper-
111
Role of Different Transporting Systems in the Secretion of Alkaloids by Hairy Roots of Catharanthus roseus (L) G. Don
Table 4. Catharanthus roseus hairy roots alkaloids profile secreted evaluated at the sampling times of 24 hours after treatments. Treatments with
ASA, quinidine, ASA/quinidine, glibenclamide, ASA/glibenclamide. Error bars represent ± SE (n = 3). The experiment was performed three times.
Peak
Treatment (high of the peak, mAU)
Control
*ASA
Quinidine
ASA/Quinidine
Glibencamine
ASA/Glibenca-mine
1
692.66±176.13
616.35±57.59
0
0
267.25±9.58
441.74±37.82
2
231.34±231.34
914.35±30.02
0
0
596.54±75.76
983.09±234.55
3
0
0
0
456.27±37.67
0
139.45±139.45
4
506.64±59.78
748.93±42.33
428.41±40.43
284.62±16.30
497.36±61.72
688.58±26.07
5
0
0
258.87±19.27
530.12±39.55
0
0
6
461.79±66.32
671.72±50.88
464.05±57.40
0
360.93±42.97
462.79±11.28
7
0
0
0
0
0
0
8
0
0
0
0
0
0
9
526.26±70.10
985.98±111.29
667.18±32.37
784.75±22.52
586.26±10.12
1060.64±70.15
10
162.77±20.16
102.18±51.92
137.76±20.43
71.06±38.44
116.44±12.32
145.82±14.20
11
519.50±44.18
585.44±43.54
622.17±77.38
333.28±30.57
372.20±31.89
459.87±35.76
12
0
0
0
18.49±18.49
20.55±20.55
0
13
328.22±38.27
355.38±4.63
283.98±39.45
164.20±29.52
187.35±35.80
299.39±54.33
14
505.19±77.61
369.22±17.26
638.42±73.02
199.39±26.19
476.50±86.23
337.66±84.81
15
0
0
0
96.63±33.22
0
0
16
115.16±17.22
79.05±41.01
59.78±33.02
0
24.99±13.52
80.39±41.72
17
0
0
0
0
22.26±11.56
0
18
43.13±22.19
72.66±2.36
60.03±16.80
16.20±16.20
13.50±13.50
24.54±24.54
19
183.73±47.13
117.12±1.74
125.80±49.53
69.45±35.08
78.54±25.03
99.45±49.91
20
0
52.33±3.38
0
0
0
42.21±23.87
21
93.87±56.86
168.11±28.13
105.06±20.89
69.99±14.86
0
26.53±26.53
22
52.93±6.91
55.78±7.99
57.08±2.41
39.13±0.180
49.12±6.45
441.74±3.06
*ASA = acetyl salicylic acid; Ort. = orthovanadate; Qui. = quinidine; Gli. = glibenclamide
sicon peruvianum cell cultures, orthovanadate causes moderate alkalinization after it is added [38], suggesting a possible
link between proton fluxes across the plasma membrane and
the secretion of compounds into the rhizosphere. However, alkalinization did not occur, since the pH value of the medium,
during the culture period varied from 5.75 to 6.34, for all the
treatments including the controls. These data suggest that the
secretion process is ATP-dependent and that either a primary
or secondary active transporter could be involved in the secretion of phytochemicals into the culture medium by C. roseus
hairy roots.
When C. roseus hairy roots were cultured in MS medium
we found that the conductivity of the medium was between 2.1
and 3 mSi for the treatments. These data suggest that it is more
probable that the secretion of phytochemicals occurs through a
primary transporter. Nevertheless, we cannot discard the possibility that in the microenvironment of the apoplast, around the
cell wall, the concentration of protons is enough to drive the
efflux of phytochemicals. Another possibility is that the secreted phytochemicals are stored in the vacuole; if this is the case,
it is possible that the secretion of the compounds is mediated
by active secondary transport systems that require the V-AT-
Pase and vacuolar pyrophosphatase for maintenance of a proton gradient across the tonoplast.
Another two compounds which inhibit the activity of various transporters were examined by addition to the medium at
the concentration of 100 mM of each inhibitor. Quinidine and
glibenclamide are inhibitors of channel blockers and of ABC
transporters [39, 40] that function as a drug efflux pump in human cancer cells [41]. In plants, it is well documented the role
of ABC transporters in translocating the secondary metabolites
by using inhibitors. For instance, berberine uptake by a vacuolar P-glycoprotein is inhibited by nifedipine and quinidine in a
dose-dependent manner [42]. Verapamil, nifedipine and glibenclamide also inhibit berberine uptake in Cjmdr1-injected
oocytes [35]. It has also been shown that an ABC-type efflux-transporter is functioning in Thalictrum minus suspension
cultures [43]. Geisler et al. [44] by using MDR/PGP inhibitors
like cyclosporin A and verapamil inhibited the efflux of auxin
in PGP 1 transformed yeast.
As shown in tables 1-8, and figures 2C, 3C and 3D the
secretion of some unidentified phytochemicals as well as
ajmalicine and serpentine produced by C. roseus hairy roots
was inhibited by these compounds, suggesting that a primary
transporter might be involved in their exudation into the medi-
112
J. Mex. Chem. Soc. 2015, 59(2)
Juan Luis Monribot-Villanueva et al.
Table 5. Catharanthus roseus hairy roots alkaloids profile secreted evaluated at the sampling times of 48 hours after treatments. Treatments
with ASA, KCN, ASA/KCN, orthovanadate, ASA/ orthovanadate. Error bars represent ± SE (n = 3). The experiment was performed three times.
Peak
Treatment (high of the peak, mAU)
Control
*ASA
KCN
ASA/KCN
Orthovanadate
ASA/Orthovana-date
1
1056.03±723.93
676.89±12.59
238.63±20.00
629.23±126.21
392.68±23.07
480.44±49.43
2
442.80±442.80
770.35±34.84
260.86±131.21
694.81±389.70
865.68±88.93
851.28±64.75
3
791.28±5.82
505.26±8.71
260.66±22.76
755.24±110.04
496.22±27.04
589.49±72.94
4
0
1367.47±121.89
615.75±54.73
1792.33±184.61
859.85±50.50
858.29±68.40
5
0
0
0
0
0
0
6
673.59±34.90
646.66±47.55
337.76±43.18
895.17±174.73
412.33±27.06
431.56±36.15
7
225.30±225.30
0
0
329.21±329.21
0
0
8
0
185.81±94.78
0
177.46±177.46
0
0
9
911.28±149.87
798.39±105.29
647.85±56.33
1423.62±242.50
899.45±16.60
974.85±93.84
10
0
34.52±34.52
122.78±19.36
122.54±67.83
44.04±44.04
0
11
605.32±10.10
755.78±57.93
363.19±57.85
944.66±172.82
479.11±25.15
518.88±57.03
12
220.22±109.26
43.72±21.87
266.07±39.85
584.19±91.51
87.83±6.87
48.24±24.14
13
325.60±325.60
339.35±33.76
131.46±66.20
154.27±87.21
204.99±22.57
197.83±9.82
14
661.08±118.92
113.13±4.82
577.13±91.23
427.27±116.09
137.36±20.26
109.19±11.93
15
0
152.07±10.23
13.82±13.82
158.79±82.10
118.62±5.53
183.44±36.67
16
36.48±36.48
111.34±13.78
110.31±20.52
171.92±17.72
46.84±26.44
27.34±27.34
17
0
0
0
0
62.10±34.73
0
18
80.28±15.85
246.17±18.20
0
662.90±45.77
0
0
19
65.17±5.41
0
200.32±51.79
0
182.97±11.00
185.38±11.88
20
0
72.90±6.70
43.70±23.84
89.07±8.46
76.13±16.73
32.75±17.30
21
0
287.47±57.78
49.19±49.19
382.86±149.82
86.88±5.87
96.23±9.44
22
36.08±6.00
65.22±4.98
48.55±5.87
108.99±7.99
46.09±2.40
51.74±9.48
*ASA = acetyl salicylic acid; Ort. = orthovanadate; Qui. = quinidine; Gli. = glibenclamide
um. Only the secretion of a few compounds is inhibited by
each inhibitor, suggesting that the inhibition process is very
selective and that different transporters are involved in the secretion of the different secondary metabolites by the hairy
roots. However, in some cases unrelated compounds can be
moved by the same transporters, like yeast pdr5p which is able
to transport flavonoids, indole alkaloids and taxol [45].
In our research the secretion of MIAs by hairy roots treated
with the different inhibitors in presence of ASA did not followed the same pattern for all the alkaloids, suggesting the presence of more than one MIAs transport mechanism. For instance,
ajmalicine was the most abundant alkaloid secreted by the treatment with ASA/KCN after 72 h (Fig. 2A). However, at the same
time the treatment with ASA/orthovanadate significantly decreased the secretion of ajmalicine when compared with the
treatment with ASA (Fig. 2B). A similar effect was observed for
the secretion of serpentine with the treatments with ASA/KCN
and ASA/quinidine after 72 h of treatment (Figs. 3A and 3C).
HPLC analysis reveals the existence of 22 main peaks
present in the C. roseus hairy roots exudates. The peaks were
enumerated from 1 to 22 following the elution time. The peaks
3, 11 and 12 corresponded to serpentine, ajmalicine and catharanthine respectively. Among the unknown peaks, the secre-
tion of twelve of them (1, 2, 4, 6, 8, 9, 13, 14, 15, 18, 19 and
21) were inhibited or reduced when the hairy roots was treated
with KCN, orthovanadate, quinidine or glibenclamide (Tables
1-8). However, in many of the cases the treatments with KCN,
orthovanadate, quinidine or glibenclamide in presence of
ASA, induced again the secretion of these compounds (Tables
1-8), suggesting that the increase in the amount of alkaloids
produced by the ASA induction is higher than inhibition of the
transport produced by the inhibitor. Also could be possible
that the ASA induced the presence or additional transporters,
which in turn can increase the secretion of the alkaloids. In
transgenic plant cell suspension cultures of N. tabacum, carrying PRD5 genes from yeast, it as has been shown that those
genes can be used to stimulate the secretion of secondary metabolites [46].
The increase in secretion of some peaks (Tables 1-8) observed at 100 µM quinidine could be the result of a plasmatic
membrane leak because of the strong inhibition of the transport system; however, this explanation appears improbable
since there is a clear inhibition of several other peaks. It is
possible that the increase in secretion of these peaks may be an
indirect effect of the inhibitor since some transporters, such as
P-glycoprotein and members of the multidrug-resistance-relat-
113
Role of Different Transporting Systems in the Secretion of Alkaloids by Hairy Roots of Catharanthus roseus (L) G. Don
Table 6. Catharanthus roseus hairy roots alkaloids profile secreted evaluated at the sampling times of 48 hours after treatments. Treatments with
ASA, quinidine, ASA/quinidine, glibenclamide, ASA/glibenclamide. Error bars represent ± SE (n = 3). The experiment was performed three times.
Treatment (high of the peak, mAU)
Peak
Control
*ASA
Quinidine
ASA/Quinidine
Glibencamine
ASA/Glibenca-mine
1
691.68±52.35
547.78±108.46
0
0
503.73±70.02
214.70±115.96
2
0
555.58±102.05
0
0
94.61±94.61
546.65±75.25
3
196.50±196.50
470.30±98.22
0
0
0
483.34±52.53
4
389.02±196.92
1158.99±347.51
831.36±129.92
992.69±76.26
570.23±39.65
1099.23±158.58
5
0
0
0
0
0
0
6
493.54±30.80
624.14±179.41
445.71±71.46
441.01±14.62
261.76±19.96
594.07±117.11
7
0
0
0
0
0
0
8
0
122.94±122.94
0
0
0
0
9
417.14±8.89
612.36±64.37
312.63±33.06
478.42±42.56
345.04±18.73
639.13±36.45
10
121.65±12.45
31.11±31.11
159.61±23.58
90.08±45.04
108.10±7.69
53.63±53.63
11
445.35±45.90
688.08±208.08
557.51±94.39
443.16±85.25
353.17±32.70
667.86±136.27
12
49.33±4.10
42.74±21.47
0
34.07±27.82
65.31±4.31
30.74±30.74
13
175.16±6.42
260.61±26.38
223.10±31.63
161.26±20.67
160.75±16.89
236.98±39.22
14
357.00±25.51
201.55±64.14
449.59±91.12
147.89±47.69
451.75±94.61
385.85±47.57
15
0
33.74±33.74
0
21.64±21.64
0
0
16
0
109.25±14.38
85.16±12.32
21.80±21.80
85.40±15.73
0
17
0
0
0
0
0
0
18
0
0
0
0
0
171.75±18.68
19
44.02±1.69
219.72±36.94
228.05±35.89
119.54±45.80
144.27±44.08
47.98±24.14
20
0
33.31±18.47
0
0
28.94±28.94
52.83±4.91
21
0
252.95±78.70
173.95±46.19
120.23±28.99
0
158.69±37.27
22
38.04±1.89
50.28±3.06
39.73±19.96
36.65±3.91
40.57±4.22
64.35±8.04
*ASA = acetyl salicylic acid; Ort. = orthovanadate; Qui. = quinidine; Gli. = glibenclamide
ed protein (MRP) subfamily in animal cells are able to efflux
anticancer drugs from the cytosol [47]).
Glibenclamide inhibits the potassium channels and some
ATP transporters in animal cells [34]. When added to hairy
roots cultures inhibited the secretion of several peaks, including serpentine (Fig. 3; Tables 1-8). Recently it has been shown
that a Crmdr1 is constitutively expressed in the root of C. roseus plants and that may be involved in the transport and accumulation of secondary metabolites [48].
On the other hand, glibenclamide, in the presence of NaCl,
inhibits the growth of the roots of the mutant Atmrp5-2 grown
in NaCl alone [49]. This inhibition is reversed by diazoxide, a
known K+- channel opener that reverses the inhibitory effects
of sulfonylureas in animal cells. However, it cannot discard the
possibility of an indirect effect of this compound with MATE
transporters or the possible indirect effect on vacuolar, pH-dependent transport [50].
Understanding the biosynthetic pathway of these compounds is
important, indeed the understanding of the transporting mechanisms of these compounds would be a novel area to engineer the
plants to enhance the secretion of these compounds to increase
the value of secondary metabolites for plant to defend biotic and
abiotic stresses that lead to plant health and production [51].
Conclusions
Inhibition assays
Taken together, the data presented here provides evidence that
ABC transporters could be involved in the secretion of MIAs.
Fourteen-day-old hairy roots were washed with water twice
and transferred in to liquid Gamborg B5 media supplemented
Experimental section
Plant material and growth conditions
Catharanthus roseus hairy roots were obtained through genetic transformation of roots with Agrobacterium rhizogenes
strain 1855 bearing plasmid pBI 121.1 [52]. The hairy roots
were maintained by sub-culturing every 15 days using halfstrength Gamborg B5 medium [53] supplemented with 3%
(w/v) of sucrose. The cultures were kept on orbital shakers at
100 rpm in the dark at 25 ± 2 ºC.
114
J. Mex. Chem. Soc. 2015, 59(2)
Juan Luis Monribot-Villanueva et al.
Table 7. Catharanthus roseus hairy roots alkaloids profile secreted evaluated at the sampling times of 72 hours after treatments. Treatments
with ASA, KCN, ASA/KCN, orthovanadate, ASA/ orthovanadate. Error bars represent ± SE (n = 3). The experiment was performed three times.
Peak
Treatment (high of the peak, mAU)
Control
*ASA
KCN
ASA/KCN
Orthovanadate
ASA/Orthovana-date
1
628.98±25.84
709.96±70.08
242.50±28.44
867.76±103.97
287.82±28.88
171.84±92.18
2
0
665.92±99.13
402.12±34.84
1260.39±70.82
521.45±66.25
645.56±78.70
3
219.35±110.31
540.53±68.93
304.78±28.79
878.72±41.75
416.73±61.58
486.54±65.83
4
633.96±81.43
1687.73±152.86
841.08±89.40
2373.26±80.45
1089.15±153.67
1046.22±186.55
5
0
0
0
0
0
0
6
334.66±51.10
776.86±98.43
389.99±32.03
922.68±82.44
457.43±25.40
397.92±59.98
7
0
0
0
1262.90±52.03
0
0
8
0
493.06±190.55
0
0
0
308.45±74.21
9
391.11±35.91
196.29±98.73
646.05±117.43
770.57±103.21
619.82±71.33
469.69±53.45
10
36.06±36.06
76.09±38.19
84.66±43.88
241.55±5.60
162.41±15.80
0
11
453.70±72.88
1063.15±155.91
506.38±60.05
967.67±415.91
634.38±62.25
632.12±121.75
12
12.47±12.47
0
165.95±27.73
526.57±95.67
112.13±6.612
0
13
180.13±29.75
296.46±15.01
166.53±8.68
324.67±204.80
268.98±30.47
180.84±26.68
14
401.37±24.26
252.37±79.68
723.12±145.54
602.44±301.52
876.81±175.23
105.57±14.46
15
0
63.69±63.69
0
0
0
153.22±23.73
16
91.28±10.40
0
121.46±12.91
39.31±39.31
54.70±28.18
44.57±22.60
17
0
76.53±15.16
0
293.21±19.28
117.89±19.29
30.84±30.84
18
180.12±18.44
0
263.13±21.18
370.18±89.90
325.18±53.32
196.18±34.88
19
0
150.66±13.11
0
32.44±32.44
0
0
20
0
99.25±18.48
67.45±24.44
22.46±22.46
21.72±21.72
24.74±24.74
21
109.84±8.79
412.27±80.16
141.97±18.56
752.82±90.72
183.21±4.83
182.93±48.19
22
20.99±17.14
62.79±7.65
64.93±8.04
282.81±131.64
81.68±5.04
51.66±26.33
*ASA = acetyl salicylic acid; Ort. = orthovanadate; Qui. = quinidine; Gli. = glibenclamide
with 3% (w/v) of sucrose, elicitors and/or inhibitors (100 µM
potassium cyanide, KCN; 100 µM sodium orthovanadate, Na3VO4; 100 µM quinidine or 100 µM glibenclamide). KCN and
Na3VO4 were dissolved in water while quinidine and glibenclamide were dissolved in DMSO. The elicitors used in this
study were acetylsalicylic acid (ASA; 100 µM) and sodium
nitroprusside (Na2[Fe(CN)5NO]; 10 µM) as nitric oxide (NO)
donor. Both elicitors were dissolved in water. The experiment
was conducted with 13 treatments in different combinations of
inhibitors and elicitors and their respective controls. The hairy
roots were exposed to ASA and collected the tissues at 12, 24,
48 and 72 hours and to NO for only 12 hours. In each sampling-time, the plant material was weighed and alkaloids were
extracted from both the culture media and the hairy roots.
Fresh (FW) and dry weight (DW) determination
After 12, 24, 48 and 72 h of elicitation with 10, 100 and 250
µM of ASA, hairy roots were collected and weighed for FW
determination. For DW determination, the roots were frozen at
-80°C and freeze-dried. After total elimination of water was
achieved, the lyophilized roots were weighted. Each sample
was done by triplicate. The experiment was repeated three
times with triplicate.
ATP determination by high performance liquid
chromatography (HPLC)
ATP was extracted from the mitochondrial sample as previously reported by Yang et al. [54]. The HPLC method reported by
Liu et al. [55] was followed. Briefly, sample from isolated mitochondria were chromatographed by gradient elution on a 4.6
mm x 150 mm reverse phase, Zorbax Eclipse XDB, 5 µm particle size C18 column (Agilent Technology). The chromatographic system (Agilent series 1200) consists of quaternary
G1311A pumps connected to a G1329A automatic sample injector. The injected samples (20 µL) were detected at 254 nm
with Gold 168 diode array detector G1315B (Agilent technology). The mobile phase A consisted 60 mM K2HPO4 and 40
mM KH2PO4 dissolved in HPLC quality water and adjusted to
pH 7.0 with 100 mM KOH, while mobile phase B consisted of
100% acetonitrile. HPLC separation was achieved using continuous gradient elution. ATP in the samples were identified by
comparison with the retention time of the standards, while the
115
Role of Different Transporting Systems in the Secretion of Alkaloids by Hairy Roots of Catharanthus roseus (L) G. Don
Table 8. Catharanthus roseus hairy roots alkaloids profile secreted evaluated at the sampling times of 72 hours after treatments. Treatments with
ASA, quinidine, ASA/quinidine, glibenclamide, ASA/glibenclamide. Error bars represent ± SE (n = 3). The experiment was performed three times.
Peak
Treatment (high of the peak, mAU)
Control
*ASA
Quinidine
ASA/Quinidine
Glibencamine
ASA/Glibencami-ne
1
446.22±33.36
373.56±15.13
0
0
422.98±123.58
0
2
150.58±77.28
564.19±94.13
0
0
0
974.26±119.75
3
0
386.95±63.37
0
877.35±190.85
0
540.65±69.32
4
446.47±52.78
1275.70±123.44
696.21±127.48
1049.32±260.05
496.19±116.57
1477.51±171.79
5
0
0
0
0
0
0
6
238.36±32.90
739.43±124.51
453.16±79.02
919.76±133.79
265.11±65.88
600.86±78.45
7
0
0
0
268.38±268.38
0
442.24±124.27
8
31.86±15.93
51.85±51.85
0
0
35.46±21.58
0
9
133.60±13.19
217.74±9.43
131.32±13.27
208.12±39.20
143.07±49.80
268.34±29.15
10
76.74±10.12
0
91.95±19.71
133.80±23.59
71.77±18.98
16.44±16.44
11
281.47±58.09
717.62±41.26
367.02±63.62
828.34±228.27
257.64±67.34
617.23±105.12
12
39.27±5.95
15.07±15.07
0
176.33±143.64
62.86±2.48
0
13
133.13±19.00
114.22±57.38
125.78±28.84
121.27±63.13
67.99±34.39
0
14
87.64±9.54
147.36±73.71
183.29±42.33
293.44±26.52
163.81±75.13
0
15
48.26±4.53
0
0
0
21.95±21.95
0
16
66.71±9.46
0
30.47±30.47
57.31±57.31
48.24±24.33
0
17
0
41.53±20.77
30.56±30.56
0
0
18
132.62±18.05
73.15±20.67
89.50±44.83
153.00±20.88
0
33.24±2.66
19
0
0
0
0
66.43±34.91
0
20
0
46.60±23.51
0
327.76±327.76
0
0
21
31.08±31.08
231.36±36.59
52.31±26.15
161.33±88.06
0
70.83±9.11
22
17.15±17.15
56.11±8.80
23.75±11.89
41.98±5.93
30.55±15.77
54.58±1.59
*ASA = acetyl salicylic acid; Ort. = orthovanadate; Qui. = quinidine; Gli. = glibenclamide
concentrations of ATP were determined using the external
standard method. Data were expressed as means of three replicate determinations.
Extraction of alkaloids from culture medium
To examine the secreted phytochemicals from C. roseus hairy
roots, liquid media samples from in vitro-grown Catharanthus
hairy roots were collected (final volume of 100 mL), filtered
through a nylon syringe filter of pore size 0.45 µm (Life Sciences Cat. PN 4612 or Nalgene cat. 195-2520) to remove any
cellular debris, and concentrated by freeze-drying (Labconco)
to remove water. The concentrate was dissolved in 5 mL 2.5%
(v/v) sulphuric acid and extracted as described by Monforte et
al., [56]. The final concentrate was dissolved in 500 µL of absolute methanol (Fisher Scientific Co.) and analyzed by HPLC.
The same procedure was followed for each treatment.
HPLC analysis of alkaloids from the exudates
Compounds from roots and media were chromatographed by
gradient elution on a 4.6 mm x 150 mm reverse phase, Zorbax
Eclipse XDB, 5-µm particle size C8 column (Agilent technology). The chromatographic system (Agilent series 1100) consists of two G1312A pumps connected to a G1328A manual
sample injector. The injected samples (20 µL) were detected at
280 nm with a variable UV-vis detector G1365B (Agilent technology). The mobile phase consisted of acetonitrile: 10 mM
(NH4)2HPO4 (43:57) a flow rate of 1.5 mL min-1. The solution
was filtered through a 0.22 µm nylon filter and degassed under
vacuum.
Retention times and peak heights of commercially purchased ajmaline, serpentine, vincamine, vindoline, ajmalicine
and catharanthine (Sigma Chemical Co.), were run in HPLC to
determine the possible presence and concentration of compounds in the root exudates and tissues.
Statistical Analysis
Each experiment was conducted with triplicate. The statistical
analysis was performed by one-way ANOVA analysis, taking
P ≤ 0.05 and P ≤ 0.01 (Tukey’s test) as significant and highly
significant, respectively. Principal components analysis (PCA)
was performed using the average of the peak areas of the chro-
116
J. Mex. Chem. Soc. 2015, 59(2)
Juan Luis Monribot-Villanueva et al.
Fig. 4. Secretion of catharanthine on hairy roots treated with ASA and
inhibitors. Figure description as Fig. 2.
matogram of each treatment (see Figs. 4 and 5) to identify the
most important variables in the secretion of compounds. Matrix was constructed with Pearson’s correlation coefficients
[57], using the program MVSP Version 3.13q Copyright©
1985-2008 Kovach Computing Service (http://www.kovcomp.
com). The first three axes account for >69% of total variation,
giving a clear idea of the structure underlying the quantitative
variables analyzed.
Acknowledgements
We acknowledge the research funds provided by Conacyt
(Grant No. 157014).
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inhibitors. Figure description as Fig. 2.
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Article
J. Mex. Chem. Soc. 2015, 59(2), 119-129
© 2015, Sociedad Química de México
ISSN 1870-249X
Reaction Parameters for Controlled Sonosynthesis of Gold Nanoparticles
Alma Laura González-Mendoza1 and Lourdes I. Cabrera-Lara*2,3
1 Facultad
de Química, Universidad Autónoma del Estado de México, Paseo Colón esq. Paseo Tollocan S/N; C.P. 50120 Toluca
de Lerdo, Edo. México, Mexico.
2 Chemistry Institute, Universidad Nacional Autónoma de México
3 Centro Conjunto de Investigación en Química Sustentable UAEMéx-UNAM, Km 14.5 Carr Toluca-Atlacomulco, Camus
UAEMéx “El Rosedal”, San Cayetano-Toluca, C.P. 50200, Edo. México, Mexico
Received November 14th, 2014; Accepted February 19th, 2015
Abstract. The synthesis of gold nanoparticles by sonochemical technique has been previously performed with excellent results. The synthesis has been carried out in the presence of citric acid, a strong
reducing agent, which allows the nucleation and growth of gold
nanoparticles, at the same time that controls particle size. In this work,
we report the use of sodium tartrate as a mild reducing agent that allows a better understanding of the effect of the reaction parameters
during gold nanoparticle synthesis. A conventional sonication bath
(37 kHz) was used for the sonochemical synthesis. This work focuses
on the reaction temperature effect and the effect of sodium tartrate
concentration. It was confirmed that particle size, and particle morphology is dependent of these two reaction parameters. Equally, colloidal stabilization was related to reaction temperature and sodium
tartrate concentration. It was also determined that Ostwald ripening
takes place during sonochemical reaction under our conditions, allowing us to understand the mechanism that takes place during synthesis. Gold nanoparticles with main particle size of 17 nm were
achieved by this method.
Key words: Gold colloidal suspension; nanoparticles; sonosynthesis;
sodium tartrate.
Resumen. La síntesis de nanopartículas de oro por el método de sonosíntesis ha sido previamente realizada con excelentes resultados.
La síntesis se ha llevado a cabo en presencia de ácido cítrico, un agente redactor fuerte, el cual permite la nucleación y crecimiento de nanopartículas de oro, al tiempo que controla el tamaño de partícula. En
este trabajo, se describe el empleo de tartrato de sodio como un agente redactor suave que permite dilucidar el efecto de los parámetros de
reacción durante la síntesis de nanopartículas de oro. Un baño de ultrasonido convencional (37 kHz) fue utilizado para la síntesis sonoquímica. Este trabajo se enfoca en el efecto de la temperatura de
reacción y concentración de tartrato de sodio. Se confirmó que el tamaño y morfología de las nanopartículas está en función de estos dos
parámetros de reacción. De igual forma, la estabilización de la suspensión coloidal depende de la temperatura de reacción y de la concentración de tartrato de sodio. Se determinó que el fenómeno de
maduración de Ostwald ocurre durante la reacción sonoquímica bajo
nuestras condiciones, permitiendo comprender el mecanismo que
ocurre durante la síntesis. Se lograron obtener nanopartículas de oro
con un tamaño promedio de 17 nm por este método.
Palabras clave: Suspensión coloidal de oro; nanopartículas; sonosíntesis; tartrato de sodio.
Abbreviations
properties such as high catalytic activities, or interesting optical properties.[2] Therefore, the potentialities of nanoparticles
relay on careful control of particle size, particle distribution,
and stability.[3] Accordingly, considerable effort has been focused on the development of synthetic techniques for tailoring
metal nanoparticles’ shape, size and distribution.[1, 4]
In the past few decades, gold colloids have been the subject of great interest. Their uniformity and stability, as well as
size-related electronic, magnetic, and optical characteristics,
make them promising in the fields of catalysis, imaging, nanophotonics, nanomagnetic, nanoelectronic devices, biosensors,
chemical sensors, and drug delivery, among others.[5-11]
Stabilization of the nanoparticles against coalescence into
large aggregates is however prerequisite for their remarkable
properties to be exploited in a variety of applications,[12]
particularly the strong surface plasmon resonance (SPR) absorption.[8, 10, 11] For these applications, maintaining the stability of colloidal gold suspensions is paramount, and this is
achieved by the adsorption of organic molecules with functional groups that bind to the gold nanoparticles (Au NPs) surface
Surface plasmon resonance: SRP; gold nanoparticles: Au NPs;
for example (exempli gratia): e.g.; kelvin: K; atmospheres: atm;
trisodium citrate dehydrate: TCD; sodium dibasic tartrate:
SDBT; millimolar: mM; minutes: min; milliliters: mL; revolutions per minute: RPM; hour: h; temperature: T; time: t; Fourier
transformed infrared: FT-IR; X-ray diffraction: XRD; watts: W;
ultraviolet-visible: UV/vis; nanometers: nm; atomic force microscopy: AFM;dynamic light scattering: DLS; ultra high resolution scanning electron microscopy: UHR SEM; kilovolts: kV;
thermogravimetric analysis: TGA; centimeters: cm; approximately (circa): ca.; millimeter of mercury: mmHg; differential
scanning calorimetry: DSC; lmax; polydispersity index: PDI;
hydrodynamic diameter: Dh; standard deviation:s; figure: Fig.
Introduction
Nanosized noble metal particles, because of their high surfaceto-bulk ratio and quantum-size effects,[1] display many novel
120
J. Mex. Chem. Soc. 2015, 59(2)
(e.g. carboxylic, phosphate, sulfhydryl, amino groups, etc.),
which depends on the preparative conditions of Au NPs.
[6, 10, 12]
As shown in the literature, many studies focus on the development of methods for the synthesis of Au NPs, which include photochemical, and controlled chemical reduction,
microwave assisted heating, laser ablation, annealing from
high-temperature solutions, metal evaporation, and sonochemical reduction.[13-15]
The sonochemical reduction has received much attention
in recent years for Au NPs synthesis,[15-17] due to the low
cost and effectiveness of the procedure.[18] The reaction
routes induced by acoustic cavitation in solution (the formation, growth and implosive collapse of micro bubbles or gas
cavities within a liquid),[17] provide extreme conditions of
transient high temperature and high pressure estimated to be
over 5000 K and 1000 atm respectively, cooling rates in excess
of 1010 K s-1, shock wave generation, and water molecules
dissociation into primary hydrogen radicals (H•) and hydroxyl
radicals (•OH).[15, 18-26] This method allows the simple and
effective preparation of fine powders on a nanometer scale and
with homogeneous particle size distribution.[17]
It is reported that a number of factors influence cavitation
efficiency, which in turn affects the chemical and physical
properties of the products. The dissolved gas, ultrasonic power
and frequency, temperature of the bulk solution, and solvent
are all important factors that control the yield and properties of
the synthesized materials, such as particle’s crystallinity.[2,
16-19, 21, 22, 27-32]
An ultrasonic horn delivers from 10 to 100 watts of acoustic energy. Hence, the ultrasonic power output must be calibrated by calorimetry, a critical parameter commonly
overlooked. The use of ultrasonic cleaning baths can be considered as an alternative. Ultrasonic cleaning baths have a
power density that corresponds to a small percentage of that
generated by an ultrasonic horn. The use of cleaning baths in
sonochemistry is limited, considering that fully homogeneous
particle size and morphology is not always reached. This is due
to the physical effects of ultrasound over nucleation and growing processes.[33]
In the literature has been reported the formation of gold
nanoparticles with different shapes and sizes (e.g., nanoprisms,
nanodumbbells, spherical and triangular nanoparticles) by ultrasonic-assisted reduction of a gold precursor in an aqueous
media in the sole presence of alcohol in solution.[18]
The size of gold particles depended strongly on the rate of
gold (III) reduction, suggesting that this rate affects the initial
nucleation of the gold particles.[21, 34] The rates of gold (III)
reduction are strongly influenced by the cavitation phenomenon, hence dependent on reaction parameters. The size of the
gold particles is correlated to the initial rate of gold (III) reduction, where the higher the rate of reduction, the smaller the
particles.[21]
The sonochemical reduction of AuCl4- to Au(0) has been
examined as a function of the concentration of various surface-active solutes.[21] It was found that the efficiency of reduction of AuCl4- in the presence of the surfactants such as
Alma Laura González-Mendoza and Lourdes I. Cabrera-Lara
sodium dodecyl sulfate,[11] chitosane,[14] amines, fatty acids,
ammonium salts,[11] or octaethylene glycol monodecyl ether
is related to the concentration of the surfactant in solution.[21,
35] Sonochemical formation of Au NPs with a narrow size distribution was also achieved with polyethylene(40)glycol
monostearate, polyoxyethylene-sorbitan monolaurate, or polyvinylpyrrolidone.[13, 35] Stabilizing ligands also confine the
growth in the nanometer regime and prevent agglomeration.
The use of capping agents commonly produces spherical particles due to the low surface energy associated with such particles.[36]
Among the common stabilizing ligands, trisodium citrate
dihydrate (TCD) is used as both a reducing agent of AuCl4- and
as a stabilizer of the gold nanoparticles, where citrate ions bind
physically at gold surfaces and stabilize the suspension (Fig.
1a).[37] Au NPs can be synthesized using TDC at room temperature under vigorous stirring for a couple of hours.[38]
However, particle size distribution and morphology is not uniform. Both parameters can be improved by increasing the reaction temperature, or by varying gold (III) concentration and
TCD concentration. Still, during the sonochemical generation
of Au NPs using TCD, these have the tendency to aggregate in
short period of time.[39]
Sodium dibasic tartrate (SDBT) is an organic compound
that resembles to TCD, and can also be used as a reducing
agent for gold precursors, and as a stabilizer for Au NPs in a
milder way (Fig. 1b). The application of ultrasound in a reaction media with SDBT present can promote the increase of the
reaction kinetics, allowing a better control over the rate of gold
(III) reduction, hence in Au NPs size and morphology. With
these motivations, in this study, we report the sonosynthesis of
Au NPs using SDBT, the effect of its concentration, reaction
temperature and reaction time on the formation of Au NPs in
the presence of constant ultrasonic power.[19]
In this work, the sonochemical synthesis of gold NPs was
performed based on the use of SDBT as the promoter with a
commercially-available low-frequency ultrasound cleaner
bath (37 kHz).
Experimental
Sonochemical synthesis of Au nanoparticles
For the sonosynthesis of Au NPs, three reaction parameters
were studied: sodium tartrate dibasic (SDBT) concentration,
reaction time and reaction temperature. The concentrations of
SDBT (Fluka, ≥98.0%) used for this work were: 5 mM, 10
mM, and 15 mM. The synthesis was performed at different
Fig. 1 (a) Trisodium citrate; (b) sodium dibasic tartrate.
121
Reaction Parameters for Controlled Sonosynthesis of Gold Nanoparticles
Table 1. Reaction conditions used for the sonosynthesis of Au NPs.
[SDBT] = concentration of SDBT; T = temperature; t = reaction time.
Sample code
[STDB] (mM)
T (°C)
t (min)
M5a
5
30
60
M5b
5
40
60
M5c
5
50
60
M5c’
5
50
120
M10a
10
30
60
M10b
10
40
60
M10c
10
50
60
M10c’
10
50
120
M15a
15
30
60
M15b
15
40
60
M15c
15
50
60
M15c’
15
50
120
temperatures: 30°C, 40°C, and 50°C. The reaction times under
study were 60 min and 120 min.
In a round bottom flask, 10 mL of a 1 mM solution of
HAuCl4 (Aldrich, 99.99%) was mixed with 10 mL of the
SDBT solution. The round bottom flask was placed at the center of the sonication bath (Elmasonic S30), which water bath
temperature was adjusted. The system was isolated from any
light source. The reaction solution was bubbled with nitrogen
gas during 20 minutes, after which the flask was sealed. After
the sonochemical reaction was finished, the Au NPs suspension was concentrated by removing the excess of water. The
Au NPs were removed by centrifugation at 3000 RMP for 30
min. The supernatant was discarded and the precipitated solid
was washed with isopropanol. The Au NPs were resuspended
in 5 mL of water and left for dialysis during 72 h. Table 1 presents the parameters used in each reaction.
Characterization techniques
To determine the concentration of non-reacted Au(III), the colorimetric method using NaBr salt was employed. For this
method, 0.15 mL of a 2.4 M solution of NaBr was added to 1.5
mL aliquot of the sample to be studied. The wavelength of
maximal absorption is found at ca. 382 nm.[15, 27, 40]
The Fourier transform infrared (FTIR) spectra were performed in a FTIR spectrometer Perkin Elmer Spectrum BX
sweeping the energy region between 4,000 and 500 cm-1. The
measurement resolution is of 2 cm-1. X-ray diffraction (XRD)
data were collected using a monocrystal Bruker Apex-Duo diffractometer with a 3-circle goniometer for charge-coupled device detector using a micro source apex II copper radiation (Cu
Ka) Incoatec ImS 30 W. The collection strategy used was as
follows: exposure time 600 s, with a Phi scanning from 180°
placing the detector in six different positions, with a
2Theta:Omega ratio 2:1 from -12°:174° to -72°:144° with a
difference of 12:6 degrees between each position to cover the
diffraction angles in the range of 0° to 83°. The data were pro-
cessed by the suite APEX2, using software XRD2 Eval. The
ultraviolet-visible (UV-Vis) spectra were performed in a Jasco
V-670 spectrometer, recording the spectral region between 300
and 800 nm.
An atomic force microscope (AFM) was used in order to
determine particle size of the synthetic product of reaction
M10c by tapping mode. The AFM employed was an Asylum
Research model FMP-3D Origin. The silicon AFM tips were
used, also provided by Asylum Research, model AC 24OTS-R3
(f = 45 – 95 KHz)with a tip radium of 9±2 nm. Zones of 2 mm
x 2 mm, were measured in the presence of air. The number of
scan lines were 426, scan rate was 0.25 Hz
Dynamic Light Scattering (DLS) measurements were performed on a ZETASIZER NANO ZS from Marvin Instruments. The energy source was a laser which emits a green
light, and the angle between the sample and detector is 173°.
DLS measurements were carried out on the tartrate-stabilized
Au NPs at a temperature of 25°C.
Ultra high resolution scanning electron microscopy (UHR
SEM) analysis was performed on a FEI Dual Beam Helios
Nanolab 600 instrument operated at an accelerating voltage of
5 kV. Samples for SEM and analysis were prepared by placing
a drop of diluted NP suspensions on carbon-coated copper
grids, allowing the solvent to dry before the analysis was carried out. Particles morphology was studied, and particle size
was determined from the measurement of 200 particles.
The thermogravimetric analyses (TGA) were performed
using a Seiko TG/ATD 320 U, SSC 5200 equipment. The analyses were carried out from an initial temperature of 20°C to a
final temperature of 550°C with an increasing temperature gradient of 10°C min-1 in the presence of air with a flow rate of 100
mL min-1 to allow the elimination of residues from the sample.
Results and discussion
The sonochemical synthesis of Au NPs has been well-documented, in which Au(0) is generated from the reduction of
Au(III) (HAuCl4) in aqueous solution by radicals of H• (from
H2O) followed by a number of Au(0) that nucleate and grow
into gold NPs (Aun). The sonochemical method relies on anaerobic environment, due to the intervening reaction between
free oxygen and H• radical.[41] For this reason, the HAuCl4 salt
aqueous solution was placed under N2(g) atmosphere for 20 min
after which the sonochemical reaction took place.
The stabilizing agent under study, SDBT, has two carboxylic groups that can coordinate to gold nanoparticle’s surface, and
can also provide stability in aqueous media. The initial clear
yellow solution changed to a red or purple color upon ultrasonic
irradiation, depending of the reaction conditions used (Table 1).
Concentration of SDBT was varied, in order to study if particle size and optical properties were dependent on this parameter. The concentrations selected were 5, 10 and 15 mM. Reaction
kinetics was controlled by adjusting the reaction temperature.
Reaction time was also studied to determine the optimum time
in which most of the gold precursor was consumed. The reaction times selected were 60 and 120 minutes.
122
J. Mex. Chem. Soc. 2015, 59(2)
Fig. 2. FTIR spectra of (a) SDBT and (b) Au NPs prepared in the
present work in the presence of SDBT.
FTIR was employed to determine if SDBT was adsorbed to
the surface of Au NPs. The spectrum for SDBT was generated
for comparison (Fig 2a). The signal at 1617 cm-1 corresponds
to C=O asymmetric stretching due to carbonyl group. On the
other hand, the band at 1410 cm-1 corresponds to the symmetric vibration of the C=O group. The broad absorption band occurring around 3440 cm-1 is characteristic of O–H bending,
revealing the presence of hydroxyl groups. The signal that corresponds to the C–H stretch is found at 2970 cm-1. Fig. 2b
shows FTIR spectrum of Au NPs with SDBT. It can be observed the O–H stretching at around 3300 cm-1. The C=O
stretching due to carbonyl group is observed around 1720 cm1. The O–H out of plane bending is seen around 1000 cm-1. The
C–O stretching vibration is observed around 1236 cm-1. The
C–H stretching is also observed around 1103 cm-1. This suggests that SDBT adsorbs onto the surface of Au NPs through
its carboxylic groups.[42]
Alma Laura González-Mendoza and Lourdes I. Cabrera-Lara
Fig. 4 (a) TGA curve and (b) DSC plot of Au NPs prepared by sonosynthesis. The analyses were carried in the presence of air with a flow
rate of 100 mL min-1.
The Au NPs generated by this method are highly crystalline, as was confirmed by XRD (Fig. 3). The data were generated by a monocrystal Bruker Apex-Duo, by using a Cu source,
since a powder diffractometer was not available for this work.
Hence, for these cases, the calculation of crystallite size using
Scherrer’s formula was not performed. The X-ray diffractograms were very similar, reason why only one is shown. The
characteristic peaks at 38.2°, 44.4°, 64.7°, 77.7° and 81.8° are
assigned to the (111), (200), (220), (311) and (222) reflections
of face centered cubic unit cell, which are typical for Au particles (JCPDS card no. 4-784).
TGA was performed for all cases after dialysis. The results
were very similar, reason why we only show one thermogram.
Fig. 4 shows that the Au NPs start losing mass at about 100°C
(7% mass), which corresponds to the loss of water present on
the surface of Au NPs. Water loss begins almost as soon as
heating is initiated and a gradual sloping TG loss curve is observed.
The second weight loss (ca. 8%) observed within the region of 250–400 °C is attributed to the decomposition of the
SDBT absorbed to the Au surface. This is in agreement with
the boiling point of SDBT, which is 399.3°C at 760 mmHg.
[43] The TGA study shows that the weight loss occurs gradually, but more rapidly around the boiling point of the ligand.
Differential scanning calorimetry (DSC) analysis shows two
exothermic temperatures. The first one, a relatively broad DSC
exotherm at 34°C corresponds to slow and gradual water loss.
The second one at 368°C corresponds to an exothermic reaction, which may be due to the formation of gaseous products
from SDBT.[44]
Effect of tartrate concentration during the sonochemical
reaction for the generation of Au NPs
Fig. 3. XRD diffractogram of Au NPs sonochemically synthesized.
All the sonochemical reactions for this study were performed
at 30°C during 60 minutes. SDBT concentration was varied,
Reaction Parameters for Controlled Sonosynthesis of Gold Nanoparticles
using 5 mM, 10 mM and 15 mM solutions (samples M5a,
M10a, and M15a, respectively). SDBT is very similar to trisodium citrate, a weak base that has several roles in the formation
of gold nanoparticles. It is a reducing agent, and its ligands
protect the recently formed nanoparticle. However, citrate also
changes the solution’s pH as its concentration varies.[45] It has
been reported that the reactivity of gold complexes changes
with pH values.[45] Hence, it is of importance to study the response of the reaction towards the change in concentration of
SDBT.
The effect of pH on the distribution of Au(III) complex
ions has been studied by other groups,[46, 47] which have
pointed out that low pH values facilitate the formation of well
dispersed Au NPs, whereas high pH values lead to the formation of large ensembles and large Au aggregates.
At pH > 6, the predominant species is AuCl(OH)3-, and at
pH > 10, Au(OH)4- is the predominant one, where both species
are difficult to reduce. With the variation of the pH of the system, both Au(III) complexes as well as SDBT can markedly
change their reactivity, inducing influence in the reaction pathways and rates.[48] It is the control of hydrolysis to tune the
speciation of [AuClx(OH)4-x]- that subsequently influences Au
nanoparticle’s size.[49]
The pH of the reaction solutions was measured (X mM
SDBT and 1 mM HAuCl4, X = 5, 10, 15), obtaining the values
of pH 5.92 for M5a, pH 6.12 for the reaction mixture of M10a
and pH 6.30 for M15a. Hence, it was assumed that AuCl2(OH)2- and AuCl(OH)-3 ions participate in the reactions.
[50] The species AuCl2(OH)2- is easily reduced (probably
present in M5a), which is an advantage for the synthesis of Au
NPs, since the nucleation process is faster than the growth process, allowing the generation of finer Au colloids.[47] On the
other hand, AuCl3(OH)- could be mostly present for M10a and
M15a. This species may possibly reduce the reaction rate to
achieve Au NPs, and might as well generate particles of higher
dimensions.
All final suspensions showed a purple color (Fig 5a) at the
end of each reaction. It was first determined the concentration
of non-reacted Au(III) by addition of NaBr. A maximum absorbance appears at 380 nm.[15] The M10a reaction showed the
lowest concentration of Au(III) was present at the end of its
reaction (0.19 mM Au3+) when compared M5a (0.21 mM
Au3+) and M15a (0.20 mM Au3+). In all the reactions, ca. 80%
of the initial concentration of Au(III) was consumed in order to
form Au NPs.
It has been reported that Au NPs with a diameter smaller
than 25 nm show a strong absorption band due to surface plasmon resonance (SPR) ca. 520 nm.[18, 51] UV-Vis spectra for
all cases showed the SPR absorption at wavelengths red shifted (Fig 5b). For M5a, SPR was located at lmax = 540 nm, for
M10a lmax = 539 nm, and for M15a lmax = 541 nm. The shifting of the SPR absorption peak when compared to what has
been reported, is an indication that particle size is greater than
25 nm.
Interestingly, at higher SDBT concentrations (15 mM),
absorbance values decreased with respect to 10 mM. The concentration of free Au(III) is lower than the concentration found
123
Fig. 5 (a) Image of the Au colloidal suspensions using different SDBT
concentrations (M5a, M10a, and M15a) at T = 30°C during 60 minutes. (b) UV-Vis spectra of Au NPs colloidal suspensions synthesized
with different SDBT concentrations (M5a, M10a, and M15a) at T =
30°C during 60 minutes.
for the reaction performed with 5 mM of SDBT. It can be
thought that the amount of Au(0) is greater for the reaction
M10a. However, it appears that these particles are more aggregated than particles generated at M15a. At this concentration,
the amount of carboxylic groups available is the one responsible to form these aggregates, which would explain the decrease
in absorbance. The pH value of the reaction media could also
allow the formation of aggregates.
It was also noticed in every case that the curve was not
symmetrical. The SPR peak wavelength depends directly on
the size and shape of the nanoparticles.[18, 52] As the light can
no longer polarize the nanoparticles homogeneously, when the
average diameter of gold nanoparticles is greater than 20 nm,
retardation effects of the electromagnetic field across the particle cause the red shift and broadening of the SPR with increasing particle size.[18, 53] As mentioned by other groups, this
implies that size distribution is broad or particles are aggregated. From the three concentrations studied, the reaction performed at 10 mM showed a higher absorbance, which implies
that higher concentration of Au nanoparticles were produced,
which is in agreement with the NaBr colorimetric technique.
From the DLS measurements, it was possible to determine
their hydrodynamic size. For M5a, particle size was predominantly of 62 nm with a polydispersity index (PDI) of 0.588.
For M10a, hydrodynamic particle size was ca. 50 nm (PDI =
0.277). For M15a, hydrodynamic particle size was ca. 45 nm
(PDI = 0.553). As it can be observed, as SDBT concentration
increases, particle size decreases (Table 2).
SDBT acts as a growth inhibitor that occupies active sites
at the surface of gold nanoparticles. The diffusion of gold ions
to the active sites is hindered by the SDBT molecules, not allowing the gold nanoparticles to grow more. Hence, as there is
more amount of SDBT present in the reaction suspension, the
124
J. Mex. Chem. Soc. 2015, 59(2)
Alma Laura González-Mendoza and Lourdes I. Cabrera-Lara
Table 2. Particle size determined by DLS of Au NPs generated using
different concentrations of SDBT (M5a, M10a, and M15a).
Sample
[STDB] (mM)
Dh (nm)
PDI
M5a
5
62
0.588
M10a
10
50
0.277
M15a
15
45
0.553
smaller the particle size will be [54]. However the PDI is high
for M5a and M15a. UV-Vis spectra interpretation is in agreement with DLS data, i.e. particle size distribution is broad.
The role of pH in these experiments is subtle. Species AuCl(OH)3- appears to be predominant for the reaction carried
out with 15 mM of SDBT (pH 6.30). AuCl(OH)3- is more difficult to reduce than AuCl2(OH)2- species, which may be predominant for the reaction performed with 5 mM of SDBT (pH
5.92), explaining why UV-Vis absorbance of the former one
was lower than that for 5 mM. However, at 5 mM there is not
enough SDBT to promote the reduction of Au(III). For the reaction performed with 10 mM of SDBT, both species AuCl2(OH)2- and AuCl(OH)3- could be present (pH 6.12), but the
amount of SDBT appears to be enough to promote the reduction of all AuCl2(OH)2- present.
From the experimental results generated in this section,
that SDBT concentration of 10 mM was the one that was used
for the rest of the experiments, it was the one that gave a better hydrodynamic diameter (Dh) with respect to the rest of the
results.
Effect of temperature during the sonochemical reaction
for the generation of Au NPs
The sonochemical reaction was performed at three different
temperatures: 30°C (M10a), 40°C (M10b), and 50°C (M10c)
during 60 min, using a SDBT concentration of 10 mM. Optical
differences were observed (Fig. 6a). The final color of the colloidal suspension of the reactions carried out at 30 and 40°C
(M10a and M10b) were purple, while the color of the colloidal
suspension for the reaction performed at 50°C (M10c) slowly
turned during the reaction from purple to red. (For the time vs
lmax absorbance curves constructed for reactions M5b, M10b
and M15b, with T = 40°C during 60 min, please refer to supplementary information).
M10c showed the lowest concentration of Au(III) at the
end of the reaction (0.12 mM Au3+) when compared to M10a
(0.18 mM Au3+) and M10b (0.18 mM Au3+). It is at the highest
temperature that ca. 90% of the initial concentration of Au(III)
was consumed in order to form Au NPs.
In fig. 6b, the UV-Vis spectra of Au NPs generated at different reaction temperatures, all in aqueous suspension are presented. It can be observed that as the reaction temperature
increases the Au NPs SPR peak is blue shifting (lM10a = 558
nm; lM10b = 544 nm; lM10c = 527 nm). For M10a and M10b,
the absorption spectra show broad and unsymmetrical SPR
peaks, which indicate that NPs size distribution is broad and
Fig. 6 (a) Image of the Au colloidal suspensions using [SDBT] = 10
mM, at different temperatures (M10a, M10b, and M10c) during 60
minutes. (b) UV-Vis spectra of Au NPs colloidal suspensions synthesized with [SDBT] = 10 mM, at different temperatures (M10a, M10b,
and M10c) during 60 minutes.
that probably they are aggregated. For M10c, the SPR peak in
this case is narrow and very symmetrical. Hence particle size is
homogeneous and particles are very well dispersed
The M10c reaction was followed by UV-Vis spectroscopy.
An UV-Vis spectrum was recorded every 10 minutes in order
to study its optical behavior (supplementary information). For
the first 10 minute reaction aliquot, a lmax = 546 nm corresponding to Au SPR was observed (the dispersion had a purple
color). As the reaction continued, the lmax had a blue shifting.
At the end of the reaction, lmax registered was at 527 nm (the
dispersion had a red color).
In order to study the evolution of the particle size during
the M10c reaction, we decided to analyze particle size by AFM
using the tapping mode. Aliquots were taken at different reaction times: 10, 20, and 30 minutes. A drop of the reaction suspension was placed on a TEM copper grid. The sample was
allowed to dry at room temperature and then it was analyzed
by AMF. Figure 7 shows the images corresponding to the AFM
analysis and the particle size distribution built for each case.
Micrographies are shown as phase images, in which different
densities are observed in the studied zone (2 mm x 2 mm). The
darker tone corresponds to material of higher densities, in our
case, Au NPs. As it can be observed after a 10 min reaction
time (Fig. 7a), particles show aggregation, and a broad particle
distribution, which covers from 30 nm to 85 nm, with an average particle size of 48 nm. At this point of the reaction, the reaction suspension had a purple color. Particle size was
measured for M10c after a reaction time of 20 min (Fig. 7b).
Average particle size was of 49 nm with a standard deviation
(s) of 7 nm. No significant change is observed, however, particle size distribution is narrower. The aliquot taken after a reaction time of 30 min showed an average particle size of 27 nm
(Fig. 7c). It can also be observed in the image, that particles are
Reaction Parameters for Controlled Sonosynthesis of Gold Nanoparticles
125
Fig. 7 (a) AFM microscography for M10c 10 min reaction and particle size distribution diagram; (b) AFM microscography for M10c 20 min
reaction and particle size distribution diagram; (a) AFM microscography for M10c 30 min reaction and particle size distribution diagram.
aggregated. Particle distribution diagram takes in account this
aggregates. At the end of the reaction, the NPs Au suspension
had a red color.
AFM images confirm what was observed by UV-vis spectroscopy. As M10c reaction takes place, particle size evolves,
from aggregates with an average size of 48 nm to well dis-
persed NP, with an average size of 17 nm at the end of the reaction. It can also be observed that particle size distribution
narrows as the reaction time increases.
Hydrodynamic size differences were observed in DLS
measurements. From this technique, the three reactions showed
a high value for PDI. For the experiment M10a, the Dh was of
126
J. Mex. Chem. Soc. 2015, 59(2)
ca. 50 nm (PDI = 0.277). For the reaction M10b, Dh was calculated to be of ca. 27 nm with a PDI of 0.556. In the case of
the reaction M10c, hydrodynamic size was slightly reduced,
with an average size of ca. 25 nm (PDI = 0.149). As it can be
observed, as temperature increases, particle size decreases.[54]
The spectroscopical difference among the reactions performed at different temperatures can be explained based in this
parameter. At T = 50°C, the reaction kinetics is increased. At
50°C, the reaction starts generating relatively big Au NPs (ca.
> 30 nm). Under less energetic conditions, the evolution process of inhomogeneous particle size takes place, which is
known as Ostwald ripening. However, in this case, the extra
amount of energy allows that under this ultrasonic frequency, a
higher number of Au(0) are available due to particle collision
(i.e. particle erosion),[55] which will result in smaller Au NPs
than when the reaction is carried out at 30 or 40°C. This is also
observed optically, since the colloidal color changes during the
reaction from purple to red, an indicative that particle size has
changed.
The effect of temperature might not only be reflected on
the reaction kinetics, but also the reaction mixture pH. As the
temperature increases, pH value decreases,[56] allowing a predominance for the AuCl2(OH)2- species, which is easier to reduce than AuCl(OH)3-.
Alma Laura González-Mendoza and Lourdes I. Cabrera-Lara
minutes of reaction time. However, the reaction M10c has
higher absorbance values, not reaching a plateau at the end of
the reaction time.
Hence, the reaction time for the synthesis performed at
50°C was increased to 120 min (M10c’) (Fig. 9a). It can be
noticed, that after 80 minutes, the reaction has reached a maximum absorbance value after 80 minutes.
The same kinetic study was performed for the reactions
using a SDBT concentration of 5 and 15 mM (M5c’ and
M15c’, respectively). Fig. 9a shows the kinetic curves for a
reaction time of 120 min. It is obvious that the reaction does
not proceed via the same path as for M10c’. The kinetics is
slower in both cases, and the amount of product in both reactions does not increase after a 60 min reaction.[56]
Fig. 9b shows the UV-Vis spectra generated after a 60 min
time reaction for 5 m M (M5c), 10 mM (M10c) and 15 mM
(M15c) SDBT concentrations. When comparing the final
Effect of reaction time during for the generation
of Au NPs
The study of the effect of reaction time for the generation of Au
NPs under different temperatures was also performed.
For the three temperatures a kinetic study was performed
during the 60 minute reaction. For these studies, an aliquot of
the reaction mixture was taken every 10 minutes and its UVVis spectrum was generated. Curves of time vs. lmax absorbance were constructed (Fig. 8).
As it can be observed, the reactions M10a and M10b have
reached an almost constant absorbance value at 0.5 after 60
Fig. 8. Time vs lmax absorbance curves constructed for reactions
M10a, M10b and M10c, with a SDBT 10 mM during 60 min.
Fig. 9. (a) Time vs lmax absorbance curves constructed for the reactions M5c’, M10c’ and M15c’, with T = 50°C during 120 min; (b)
UV-Vis spectra for the reactions performed with [SDBT] = 5 mM, 10
mM and 15 mM, with T = 50°C at trxn = 60 min (M5c, M10c and
M15c, respectively).
127
Reaction Parameters for Controlled Sonosynthesis of Gold Nanoparticles
Table 3. Au(III) concentration at the end of the reaction for T = 30, 40 and 50°C , in function of temperature.
T = 30°C
[STDB] (mM)
[Au(III)] (mM) λAu (nm)
T = 40°C
T = 50°C
Dh (nm)
[Au(III)] (mM)
λAu (nm)
Dh (nm)
[Au(III)] (mM)
λAu (nm)
Dh (nm)
5
0.21
540
62
0.19
550
44
0.19
551
22
10
0.19
539
50
0.19
544
27
0.21
527
25
15
0.20
541
45
0.20
546
35
0.15
536
32
Fig. 10. UHR SEM micrographies generated for reactions M5c (a), M10c (c), M15c (e, g), with their corresponding size distribution graphs (b,
d, and f, respectively).
128
J. Mex. Chem. Soc. 2015, 59(2)
concentration of Au(III) at 30°C for all the reactions with the
final Au(III) concentrations at 50°C, it is appreciated that
the amount of Au(III) present has decreased, but not in a significant amount for the reactions performed at 5 mM and 15 mM
of SDBT (Table 3).
UHR SEM micrographies were generated for these reactions. As it can be observed, NPs synthesized from M5c have
a great particle size distribution (Fig. 10a,b). Their morphology is not homogeneous, and they are found forming aggregates. Au NPs synthesized from M10c are very well dispersed,
and present an excellent Gaussian behavior (Fig. 10c,d). They
present a semispherical morphology, a mean particle size of 17
nm with a standard deviation (s) of 5 nm. The NPs generated
from M15c are also semispherical and had a mean particle size
of 19 nm (s = 5 mn) (Fig. 10e,f). However, they formed aggregates (Fig. 10g). A closer look to the samples shows a great
amount of organic surrounding the surface of the Au NP. This
explains the UV-Vis results and Dh values. The hydrodynamic
diameters of prepared gold nanoparticles are slightly larger
than the mean diameter determined from UHR SEM images.
This discrepancy can be accounted for by considering the
thickness of the surfactant layers adsorbed on the surface of
the Au NPs.[41]
As it can be noticed, the reaction performed at 50°C during
60 min in the presence of [SDBT] = 10 mM, generated gold
nanoparticles with an average particle size of 17 nm and a Dh
of 25 nm.
Conclusions
Au NPs were generated by sonochemical synthesis using a
conventional ultrasonic bath. In order to achieve particles with
very narrow particle distribution with a homogeneous morphology, reaction parameters such as ligand concentration, reaction temperature, pH value, and reaction time are important
to consider. In this work, we were able to achieve Au NPs with
a particle size ca. 17 nm with a s = 5 nm by using a ligand
concentration (SDBT) of 10 mM, a reaction temperature of
50°C, during a reaction time of 60 min, with a an initial solution pH value of 6.12, without adjusting it during the reaction
synthesis.
Acknowledgements
The authors will like to acknowledge the financial support of
PAPIIT UNAM with the project IIB200113-RR260113. UHR
SEM was performed at IPICyT, at the division of advanced
materials by Ph.D. Gladis Labrada Delgado. The authors want
to acknowledge M. Sc. Lizbeth Triana, M. Sc. Alejandra
Núñez, Ph. D. Diego Martínez, M. Sc. Melina Tapia Tapia, and
Ph. D. Marisol Reyes for their support in the characterization
of the material here described. The authors are also thankful to
Professor Jorge Tiburcio Baez from Centro de Investigación y
Alma Laura González-Mendoza and Lourdes I. Cabrera-Lara
de Estudios Avanzados del Instituto Politécnico Nacional, for
his support in the DLS measurement during the development
of this work.
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Supplementary information
S.I. 2. Time vs λmax absorbance curves constructed for reactions M5b,
M10b and M15b, with T = 40°C during 60 min.
S.I. 2. UV-Vis spectra recorded for M10c every 10 min during a 60
min reaction.
Article
J. Mex. Chem. Soc. 2015, 59(2), 130-136
© 2015, Sociedad Química de México
ISSN 1870-249X
Phenylboronic Acid/CuSO4 as an Efficient Catalyst for the Synthesis of
1,4-Disubstituted-1,2,3-Triazoles from Terminal Acetylenes and Alkyl Azides
José Emilio de la Cerda-Pedro,1 Susana Rojas-Lima,1 Rosa Santillan2 and Heraclio López-Ruiz1*
1 Área Académica
de Química (AAQ), Universidad Autónoma del Estado de Hidalgo (UAEH), Ciudad Universitaria, Carretera
Pachuca-Tulancingo Km 4.5, C.P 42184 Mineral de la Reforma, Hidalgo, México. Dirección: *corresponding author:
heraclio@uaeh.edu.mx
2 Departamento de Química, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, A.P. 14-740,
Avenida IPN 2508, Esquina Ticoman, C.P. 07000 México, D.F., México..
Received January 27th, 2015; Accepted March 9th, 2015
Abstract. The synthesis of 1,4-disubstituted-1,2,3-triazoles from alkyl azides and terminal alkynes at room temperature and under microwave heating was attained using Cu(I), generated in-situ from
copper(II) sulfate and phenylboronic acid, as catalyst. Twelve new
triazoles were obtained in moderate to good yields (53-98%), and the
products were obtained by crystallization from the mixture reaction
without further purification.
Key words: triazoles, phenylboronic acid, CuSO4, Microwave irradiation.
Resumen. Se describe la síntesis de 1,2,3-triazoles-1,4-disustituidos
a partir de alquilazidas y alquinos terminales a temperatura ambiente
y calentamiento por microondas empleando Cu(I) generado in-situ a
partir de sulfato de cobre(II) y ácido fenilborónico, como catalizador.
Bajo estas condiciones se prepararon doce triazoles nuevos en rendimientos de moderados a buenos (53-98%), que cristalizan de la mezcla de reacción sin purificación adicional.
Palabras clave: triazoles, ácido fenilborónico, CuSO4, irradiación de
microondas.
Introduction
Results and discussion
The Cu(I)-catalyzed azide-alkyne cycloaddition reaction
(CuAAC) has become the preferred synthetic route to
1,4-disubstituted 1,2,3-triazoles, some of which have interesting biological properties and/or applications in drug design
[1-3]. Because of the thermodynamic instability of Cu(I),
Cu(II) is usually reduced in situ, by the addition of reducing
agents such as sodium ascorbate [4], glucose in the presence of
Fehling’s reagent [5], or NaN3 [6], and NaCN [7]. In general,
longer reaction times are required when the Cu(II)/ascorbate
process is used, therefore we believe there is still potential for
further development in this reaction.
The readily available phenylboronic acid [8] is stable to
heat, air, and moisture, making it an attractive and valuable
precursor of phenol by its oxidation with CuSO4 [9-10]. Thus,
stirring phenylboronic acid in a solvent, followed by addition
of CuSO4 produces Cu(I). We employed this reaction to develop an efficient, synthesis of 1,4-disubstituted-1,2,3-triazoles
from alkyl acetylenes that can be effected at room temperature,
or more rapidly upon microwave irradiation.
The assistance of aryl boronic acid in the synthesis of triazoles by this route using Cu-catalysts in H2O and other solvents has been reported earlier [6, 11]. However, Cu(I)
generated in this manner has not been used to catalyze the formation of 1,4-disubstituted-1,2,3-triazoles from alkyl azides
and mono-substituted acetylenes therefore we describe herein
the application of this methodology to the synthesis of new six
new triazole derivatives.
Initially, studies were carried out using phenyl acetylene and
benzyl azide in the presence of varying amounts of CuSO4
and PhB(OH)2 at room temperature. Therefore, a 1:1 mixture
of H2O/iPrOH was chosen as the solvent, and several experiments were carried out (see table 1). The data (Table 1) clearly
show that the best conditions for the formation of 3a involved
the use of 10 mol % CuSO4 and 20 mol % PhB(OH)2 (Table 1,
entry 5). We also evaluated the solvent effect, although previous studies from our laboratory have documented the advantage of a H2O/iPrOH (1:1) solvent mixture [7,12]. In addition,
the influence of different bases was investigated finding
that the highest yields were obtained using a molar excess of
Et3N. Under his conditions, the cycloaddition reaction is favored by minimizing the formation of by products, also it prevents degradation of Cu(I) by oxidation or switching and helps
to solubilize the copper in the reaction medium [13].
After optimization of the conditions, the scope of the reaction in regard to alkyne and azide structures was explored by
reacting various azides with terminal alkynes in the presence of
CuSO4 (10 mol%) and PhB(OH)2 (20 mol%). The results are
summarized in Table 3. It is obvious from these data that a wide
variety of azides possessing different functional groups are tolerated. The reaction also showed considerable tolerance for
substituents in the phenyl alkynes (Table 3). Both electron donating and electron withdrawing substituents in the phenyl
alkynes led to the desired products in high yields with little
differences in reaction times. In contrast, both the reaction of
Phenylboronic Acid/CuSO4 as an Efficient Catalyst for the Synthesis of 1,4-Disubstituted-1,2,3-Triazoles from Terminal Acetylenes...
Scheme 1. Search for optimal conditions.
Scheme 2. Synthesis of compound 3c and bistriazole 4.
Table 1. Catalyst screening for the synthesis of 3a.
Conditions
Yield
(%)b
Entry
CuSO4
(mol%)
PhB(OH)2
(mol%)
1
10
10
NRc
2
10
5
14b
3
10
7
29 b
4
10
10
54 b
5
10
20
85 b
6
10
-----
NR
a
All reactions were carried out using 0.75 mmol of benzyl azide,
0.75 mmol of phenylacetylene and 1 mL of Et3N in 2 mL of iPrOH/H2O (1:1) at room temperature during 5 h.
b
isolated yield after purification via flash chromatography.
c
without Et3N
Fig. 1. X-Ray structure of compounds 3f and 4.
phenylacetylene and 1-chloro-4-ethynylbenzene with 5-azido-2-(2-methoxyphenyl)benzoxazole gave modest yields of the
expected products 3g and 3h respectively, in spite of increased
reactions times (Table 3, entries 7-8). However, when reactions
with benzyl azide and 4-ethynylbenzonitrile were carried out
using catalytic CuSO4 in the presence of phenylboronic acid, a
mixture of triazole and bistriazole was obtained in agreement
with the observations of Burgess [14] and Cuevas-Yañez [15]
(Scheme 2). We rationalized the formation of this by-product
on the basis of the reducing power of PhB(OH)2 at room temperature. The structures of most of the 1,2,3-triazoles was supported by the usual spectroscopic data. In contrast, the structures
of triazole 3h and bistriazole 4 were unequivocally established
by X-ray crystallography (Fig. 1) [16].
Microwave irradiation has revolutionized modern organic
synthesis due the energy saving, shortened reaction times, and
the formation of fewer side products [17]. Accordingly, the
132
J. Mex. Chem. Soc. 2015, 59(2)
methodology already described above was explored under microwave irradiation. Thus, the same reactions between azides
and terminal alkynes using Cu(I) generated with CuSO4 and
phenylboronic acid were run in water/iPrOH (1:1) using microwave irradiation at 100 W and 125 oC (Table 2). The data
clearly show that the best conditions for the formation of 3a
involved the use of CuSO4 (10 mol%) and PhB(OH)2 (20
mol%) at 100 W and 125 oC for 10 min (Table 2, entry 3). Increasing the reaction time to 30 min led to a decrease in yield.
With these optimized reaction conditions in hand, the scope
and generality of this protocol was examined employing benzyl azide and various aromatic azides with terminal alkynes
(Table 3). When we used benzyl azide with different phenylalkynes the reaction took only 10 min (Table 3, entries 1-6).
However, the reaction of phenylacetylene, 1-chloro-4-ethynylbenzene and 4-ethynylanisole with 5-azido-2-(2-methoxyphenyl)benzoxazole required 15 min to give good product yields
(Table 3, entries 7-9). In contrast to the room temperature results described above, no diyne or bis-triazole formation was
observed under these conditions.
The role of phenylboronic acid is to reduce copper(II) sulfate to Cu(I), which is responsible for catalyzing the reaction,
obtaining only the 1,4-disubstituted-1,2,3-triazoles in excellent yields. A possible mechanism for the catalytic CuSO4/
PhB(OH)2 reaction is depicted in Scheme 3 [18].
The structure of most 1,2,3-triazoles was supported by the
usual spectroscopic data and by X-ray crystallography.
José Emilio de la Cerda-Pedro et al.
Table 2. Catalyst screening for the synthesis of 3a in MW irradiation.a
Conditions
Time
(min.)
Yield
(%)b
--
10
NR
5
10
10
89
10
20
10
97
10
--
10
53
--
20
10
NR
10
20
30
96
Entry
CuSO4
(mol%)
PhB(OH)2
(mol%)
1
--
2
3
4
5
6
a
All reactions were carried out using 0.75 mmol of benzyl azide,
0.75 mmol of phenylacetylene and 1 mL of Et3N in 2 mL of iPrOH:H2O (1:1) at 100W and 125oC.
b
isolated yields purified by crystallization.
Conclusions
In summary, a very efficient and straightforward procedure for
the regioselective synthesis of alkyl and aryl-1,2,3-triazoles
has been developed using commercially available CuSO4 (10
mol%) and PhB(OH)2 (20 mol%), at room temperature or at
125 oC by microwave irradiation at 100 W. Microwave irradiation dramatically decreases reaction time from hours to minutes with excellent yields. 1,2,3-Triazoles thus produced often
crystallize from the reaction mixture and do not require any
further purification.
Experimental
Unless otherwise noted, materials were obtained from commercial suppliers and used without further purification. Thin
Fig. 2. X-Ray structure of compounds 3h and 3j.
Scheme 3. Postulated Mechanism.
Phenylboronic Acid/CuSO4 as an Efficient Catalyst for the Synthesis of 1,4-Disubstituted-1,2,3-Triazoles from Terminal Acetylenes...
Table 3: Synthesis of 1,4-disubstituted-1,2,3-triazoles from alkyl or aryl azides and terminal alkynes at room temperature or using MW.
Entry
Yield (%)c
Product
N
N
Time (min.)
r.t.b
MW
r.t.
97
85
10
300
129-131
[7]
96
93
10
300
181-182
[7]
89
78
10
300
144-146
[7]
93
88
10
280
144-146
[7]
97
97
10
240
117-119
[7]
93
75
10
300
102-104
[7]
N
1
3a
N
N
2
m.p. (°C)
[Ref.]
MWa
N
3b
NH2
N
N
3
N
3c
CN
N
4
N
N
3d
OCH3
N
N
5
N
3e
N
N
N
N
6
3f
Cl
134
J. Mex. Chem. Soc. 2015, 59(2)
Entry
José Emilio de la Cerda-Pedro et al.
Yield (%)c
Product
N
O
8
a
N
9
3i
N
3j
N N
N
11
91
53
15
24h
211-213
[7]
93
85
15
24h
164-166
[7]
73
67
15
24h
222-223
[7]
98
95
10
300
127-128
[12]
98
-----
15
-----
251-253
N N
N
OMe
r.t.
OCH3
O
10
MW
N N
N
OMe
r.t.
Cl
3h
O
m.p. (°C)
[Ref.]
MW
N N
N
OMe
Time (min.)
b
F
N N
N
O
3k
7
O
6'
12
5'
2
4'
3'
2'
5
N
1'
OMe
7'
6
8a
N
N N
4´´
5´´
3a 4
17´´
O
N
16´´
O2N
O
6´´
14´´
7´´
12´´
8´´
9´´
10´´
11´´
3l
All reactions were carried out using 0.75 mmol of benzyl azide, 0.75 mmol of phenylacetylene and 1 mL of Et3N in 2 mL of iPr-OH/H2O
(1:1) at 100W and 125oC.
b
All reactions were carried out using 0.75 mmol of benzyl azide, 0.75 mmol of phenylacetylene and 1 mL of Et3N in 2 mL of iPr-OH/H2O
(1:1) at room temperature.
c
isolated yields.
a
layer chromatography (TLC) was performed on glass plates
coated with silica gel 60 F254 and visualized by UV (254 nm).
Flash column chromatography was performed using Merck silica gel (230-240 mesh). Melting points were measured in open
capillary tubes on a Büchi Melting Point B-540 apparatus and
have not been corrected. 1H and 13C NMR spectra were recorded on Varian VNMRS 400 (400 and 100 MHz) spectrometer.
Chemical shifts (δ) are indicated in ppm downfield from inter-
nal TMS used as a reference; coupling constants (J) are given
in Hz. IR spectra were measured on a Perkin Elmer GX FT-IR.
High resolution mass spectra were obtained with an Agilent
G1969A spectrometer. All microwave irradiation experiments
were carried out in a CEM-Discover mono-mode microwave
apparatus, operating at a frequency of 2.45 GHz with continuous irradiation power from 0 to 100 W utilizing the standard
absorbance level of 300 W maximum power.
Phenylboronic Acid/CuSO4 as an Efficient Catalyst for the Synthesis of 1,4-Disubstituted-1,2,3-Triazoles from Terminal Acetylenes...
General procedure for the preparation of 1,2,3-triazoles at
room temperature
In a 50 mL round-bottomed flask containing a magnetic stirring bar was placed 1 equiv. of azide in 2 mL of a mixture of 1
mL of H2O and 1 mL of isopropanol as solvent, followed by the
addition of 1 equiv. of alkyne, 0.10 equiv. of CuSO4, 0.20
equiv. of phenylboronic acid, and 1.0 mL of Et3N. The resulting solution was stirred at room temperature for 5-24 h, followed by extraction with EtOAc (3 x 25 mL). The collected
organic layers were dried with MgSO4 and the solvent was removed under vacuum to give the corresponding triazole. After
removal of ethyl acetate, the crude residue was purified by column chromatography (EtOAc/hexane) to afford 1,2,3-triazoles.
General procedure for the preparation of 1,2,3-triazoles
by microwave irradiation
In a MW tube equipped with a magnetic stirrer was placed 1
equiv. of azide in 2 mL of a mixture of 1 mL of H2O and 1 mL
of isopropanol as solvent, followed by the addition of 1 equiv.
of alkyne, CuSO4 (10 mol%), phenylboronic acid (20 mol%),
and 1.0 mL of Et3N. This tube was sealed, and the content was
subjected to focused microwave irradiation at 100 W for 10 or
15 min at 125 oC. Then the reaction mixture was diluted with
ethyl acetate and washed with a saturated solution of NH4Cl,
the organic phase was collected, dried with MgSO4, filtered
and the solvent was vacuum removed. The crude product was
purified by crystallization to afford 1,2,3-triazoles.
Compound 3a, 3b, 3c, 3d, 3e, 3f, 3g, 3h, 3i, 3j and 3k are
known, and their spectra are consistent with the literature data
(Table 3).
2-(2-methoxyphenyl)-5-(4-((2-(5-nitrobenzoxazol-2-yl)
phenoxy)methyl)-1H-1,2,3-triazol-1-yl)benzoxazole (3l). White
solid: mp 251-253 oC; yield: 0.11 g (98%). IR (KBr); ν max
2925, 1603, 1532, 1494, 1263, 1352, 1021, 746 cm-1; 1H
NMR (400 MHz, DMSO-d6) δ 9.01 (s, 1H, H-5”), 8.65 (d,
1H, J = 2.4 Hz, H-14”), 8.30 (m, 2H, H-4, H-6), 8.07 (m, 2H,
H-16”, H-17”), 7.97 (m, 2H, H-7, H-10”), 7.62 (m, 3H, H-6’,
H-8”, H-11”), 7.29 (d, 1H, J = 8.4 Hz, H-3’), 7.21 (t, 1H, J =
7.5 Hz, H-5’), 7.15 (td, 1H, J = 8.0 Hz, J = 2.5 Hz, H-9”),
5.73 (s, 2H, H-6”), 3.93 (s, 3H, H-7’); 13C NMR (DMSO-d6,
100 MHz) δ 163.5 (C-2), 162.7 (C-13”), 157.3 (C-7”), 157.1
(C-2’), 156.1 (C-17a”), 150.4 (C-8a), 150.0 (C-13a”), 144.4
(C-4”), 142.4 (C-3a), 141.5 (C-6’), 137.7 (C-11”), 133.6 (C4’), 132.4 (C-9”), 129.7 (C-5), 123.1 (C-6), 121.7 (C-16”),
121.5 (C-10”), 121.0 (C-5”), 120.3 (C-15”), 119.9 (C-4),
119.1 (C-3’), 117.3 (C-8”), 116.6 (C-14”), 115.2 (C-17”),
111.5 (C-7), 110.6 (C-12”), 109.2 (C-1’), 72.6 (C-6”), 56.1
(C-7’); HRMS (ESI) calcd for C30H20N6O6 (M + H)+
561.1444, found 561.1521.
Acknowledgements
We are indebted to Dr. Joseph M. Muchowski for friendly,
helpful discussions and to CONACyT for financial support
(grant CB-2012-01-182415). One of the authors (J. E. de la
Cerda-Pedro) thanks CONACyT for a scholarship (239377).
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136
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Crystallographic Data Centre as a Supplementary Publication
Numbers, CCDC 1007167 No. for 3f, CCDC 1007170 No. for 4,
CCDC 1007168 No. for 3h, and CCDC 1007169 No for 3j. Copy
of the data can be obtained, free of charge, on application to
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Article
J. Mex. Chem. Soc. 2015, 59(2), 137-142
© 2015, Sociedad Química de México
ISSN 1870-249X
Synthesis of New Dicoumarol Based Zinc Compounds and their Invitro
Antimicrobial Studies
Sadia Rehman*1,a, Muhammad Ikram*,a and Fazle Subhan1
1 Department of Chemistry, Abdul Wali Khan University Mardan, Pakistan. E-mail: sadia@awkum.edu.pk
ikram@awkum.edu.pk
a Equal Contributors
* Received January 9th, 2015; Accepted March 18th, 2015
Abstract. The dicoumarol derivatives were reacted with Zn (II)
salt yielding the complexes (1-10) where metal centre was seen to
be coordinated with dicoumarols through hydroxyl and carbonyl
sites of attachments. All the synthesized compounds were studied
spectroscopically using 1H, 13C{1H}-NMR, infrared spectroscopic
method, and analytically using ES(+,-)-MS, elemental analyses and
conductance studies. The combined NMR and mass spectral data
suggested the attachment of two ligands to the zinc (II) centre.
Hydroxyl site is deprotonated and take part in charge neutralization of metal center. The synthesized zinc based dicoumarol compounds were screened for antimicrobial activities against Gram
negative bacteria Escherichia coli, Salmonella typhus, Agrobacterium
tumefaciens, Erwinia carotovora, Pseudomonas aeruginosa, Klebsiella
pneumoniae, Gram positive bacteria Staphylococcus aureus, Bacillus
subtilis, Bacillus atrophaeus and fungal Strain Candida albicans. All
the compounds shown exceptional antimicrobial and antifungal
activities.
Key words: Dicoumarols, zinc compounds, spectral analysis, antimicrobial, antifungal activities
Resumen: Derivados del dicoumarol se hicieron reaccionar con sales de
Zn (II) para formar los complejos (1-10), en donde el centro metálico se
encuentra coordinado con la moléculas de dicumarol a través de los grupos hidroxilos y carbonilos. Todos los compuestos sintetizados fueron
estudiados espectroscópicamente, utilizando: RMN de 1H, 13C{1H}, espectroscopia de infrarrojo; y analíticamente, utilizando: espectrometría
de masas con ionización por electroespray ES(+,-)-MS, análisis elemental
y conductancia. Los datos de RMN y de espectrometría de masas sugieren que existen dos ligandos unidos al centro de Zn (II). Los grupos hidroxilo se encuentran desprotonados y contribuyen a la neutralización de
la carga del metal central. Los compuestos de dicoumarol base Zinc, sintetizados en este trabajo, fueron evaluados en su actividad antimicrobiana
con bacterias Gram Negativas: Escherichia coli, Salmonella typhus,
Agrobacterium tumefaciens, Erwinia carotovora, Pseudomonas
aeruginosa, Klebsiella pneumoniae; bacterias Gram positivas:
Staphylococcus aureus, Bacillus subtilis, Bacillus atrophaeus y la
cepa fungica de Candida albicans. Todos los compuestos mostraron
actividades excepcionales, tanto antimicrobianas como anti fúngicas.
Palabras clave: Dicoumarol; compuestos de zinc; análisis espectroscópico; actividad antimicrobiana; actividad antifúngica
Introduction
proteins[6]. For example epigallocatechin-3-gallate (EGCG) is
an excellent remedy for neuronal diseases. Structurally it is
comprised of coumarin unit that neutralizes iron (III) safeguarding the cell from the oxidative stress of iron (III) [7].
Since the discovery of metal ion role inside body the metal
based chemistry is becoming attractive field for the scientific
community. Metal ions like zinc, copper, iron, and cobalt are
crucial for the proper functioning of cells inside the living
matrix, and any disruption can lead to serious neuropsychiatric
diseases such as Alzheimer’s, Menke’s, Wilson’s and Parkinson’s diseases, Friedreich’s ataxia and Hallervorden–Spatz
syndrome [8].
Carbonic anhydrase (CAs) is one of the important enzymes involved in the conversion of carbon dioxide to carbonate and proton, a very simple but essential step to overcome the
carbon dioxide concentration inside cells. This process is slow
without the presence of a suitable metallic catalyst [9]. CAs
evolved independently at least five times, with five genetically
distinct enzyme families known to date: the α-, β-, γ-, δ-, and
ζ-CAs [9-15]. All of them are metalloenzymes with their own
distinctions. The α-, β-, and δ- CAs use Zn(II) ions at the active
Zinc is one of the essential metal ion found in all forms of life
and is involved in many regulatory activities inside body which
includes growth and development of the body, normal brain
functioning and gene expressions. An average adult human being has around 3 g of zinc in the body. It also play vital role in
preventing many diseases like pneumonia, malaria, common
cold and cancers etc [1,2].
Zinc if combined with coumarin derivatives may function
as good inhibitor of many enzymes, cure diseases by inhibiting
the activities of many pathogenic microbes and recently tested
for the anticancer activities. Through coordination of dicoumarols with zinc (II) ion a library of compounds can be synthesized with new type of zinc binding groups (ZBG’s). Though
the ZBG of hydroxamic acid and other heterocyclic ZBG’s
were also synthesized but the present library of ZBG’s provide
insight into the natural product based zinc (II) binders [2-5].
Coumarins can act as effective drugs in treating many diseases like cancer, neuronal diseases, AIDS, etc because they
are regarded as good chelator for the iron in haem or inside
138
J. Mex. Chem. Soc. 2015, 59(2)
site, the γ-CAs are probably Fe(II) enzymes (but they are also
active when bounded to Zn(II) or Co(II) ions), whereas the
ζ-class uses Cd(II) or Zn(II) to perform the physiologic reaction catalysis [13-16].
Metallo thioneins, the ZIP and ZnT families of proteins
distribute the zinc ions for specific activities. Therefore zinc is
very essential metal ion either as mobile or chelatible and
much attention was paid by the scientists all round the world.
The role of zinc can be seen in many zinc enriched tissues of
hippo campus, pancreas, and most important prostate [17]. Intra cellular zinc trafficking therefore is attracting much attention, hence our work aimed to synthesize the coordination
complex of biologically important coumarins and use them for
the in vitro antimicrobial studies [18]. The active dicomarols
reported earlier were used for the synthesis of zinc based derivatives [19].
2. Results and discussion
2.1 Spectral analyses
Dicoumarols derived from the condensation of different aldehydes with 4-hydroxycoumarin were reacted with
[Zn{N(SiMe3)2}2] [21] to get the tetrahedral complexes in
1:2 molar metal to ligand ratio. These complexes were characterized unambiguously using different spectroscopic and
analytical techniques. All the ligands were found to be anionic in nature and coordinating to the zinc center through
lactons and hydroxyl site of attachments [19].
The high resolution ES+-MS analyses revealed the specific molecular ion peaks with reasonable abundance, exceptions
were seen for the 4 and 10 metal complexes. The reason may
be due to the unstable nature of these two complexes in solution formation. The compositions of all the zinc based metal
complexes (1-10) of dicoumarols were also confirmed by elemental analyses.
Further structural studies was done using techniques like
1H and 13C{1H} NMR along with infrared spectral studies.
where R = aldehydes with different substitutions
Fig. 1. Proposed structure of Zinc (II) complexes of dicoumarols
Sadia Rehman et al.
Multinuclear NMR and infrared studies confirm the formulation revealed from elemental analyses and MS spectral studies.
The resonances caused by the N(SiMe3)2 were replaced by dicoumarols. The deprotonation of strong hydrogen bonded phenols is therefore very easily attained. In previous studies such
deprotonation was obtained with sodium metals or sodium methoxide which is also confirmed by us in our sodium work in
the field of the same ligands [20]. The unambiguous and successful deprotonation along with successful approach to break
the strong intramolecular hydrogen bonds is therefore one of
beneficial aspects of the [Zn{N(SiMe3)2}2]. The CH proton
found at the linking site of the two coumarin groups was found
to be resonating at 5.3-5.9 ppm depending upon the variation
in the aromatic aldehydes [19]. All the complexes show multiplates in the region of 6-9 ppm, assigned to the resonances
caused by aromatic protons. The hydroxyl resonance, observed at 11-17 ppm in the free ligands, was found completely
absent in the 1H-NMR spectra of all the zinc complexes. The
13C{1H}-NMR also support the 1H-NMR spectral analyses,
the hydroxyl based carbon and lactone carbonyl resonances
were found low field compared to the neat ligand (∆δ = ~30
ppm). Therefore all the observations were found completely in
line with the complexation behavior for the dicoumarol ligands
coordinating through these two sites of attachments. In an impure NMR spectra of the samples show a singlet at 0 ppm
which was assigned to the HN(SiMe3)2. On purification such
resonances were completely removed. The infrared spectra
also established the attachment of dicoumarol ligands through
lactone and phenolic sites. The vibration caused by hydroxyl
group was completely diminished by the complexation to zinc
centre whereas in other cases it is broadened to a very large
extent compared to the neat ligand. The lactonic stretch was
found misplaced by ∆υ = 40-70 cm-1, suggesting its participation in coordination. The general structure of the produced
complexes is shown in Fig. 1.
2.2 Antimicrobial activities
All the synthesized zinc complexes were subjected to their antimicrobial activities against selected pathogenic Gram positive bacteria, Gram negative bacteria and a fungal strain. The
activities of all the synthesized compounds were compared
with a standard drug already used for stopping the pathogenic
activities of tested microorganisms. As may be depicted from
table 1, all the compounds were found much more active
against the Bacillus atrophaeus except 6. By comparing these
results with the bare dicoumarol ligands (as given in table 2)
[19] it can be concluded that metal complexation make the ligands much more active. The reason for the enhanced activities may be due to the increase in hydrophobicity in complexes.
The results for the antimicrobial activities of these compounds
against Bacillus subtilis and Bacillus atrophaeus are very
close. Therefore we can conclude that all the zinc based metal
complexes of dicoumarols are showing potential antimicrobial
activities against Bacillus species of Gram positive bacteria.
On the other hand all the zinc based metal complexes of dicoumarol were found moderately active against Gram negative
139
Synthesis of New Dicoumarol Based Zinc Compounds and their Invitro Antimicrobial Studies
bacteria viz Klebsiella pneumoniae, Salmonella typhus, Pseudomonas aeruginosa, and Escherichia coli and Gram positive
bacteria like Staphylococcus aureus. Strong antimicrobial activities were observed against Agrobacterium tumefaciens,
Erwinia carotovora, and Candida albican. All the compounds
were found much more active than the bare ligands. These
results show that zinc which is also essential metal for many
body functions can also be used as potential antimicrobial therapeutic agent for decreasing or completely vanishing the functions of the microbes in the form of metal complex of
dicoumarol ligand.
Compound 1 was found more active than the parent dicoumarol ligand against all the tested microbes except Candida albican and Erwinia carotovora, compound 2 was more
active than the dicoumarol ligand except the Pseudomonas
aeruginosa, similar activities were seen for all the compounds
from 3-10. Compounds like 3, 5, 7, 8, & 9 were found much
active against Bacillus atrophaeus than the standard drug
erythromycin. Compounds 2 & 10 showed similar activities
against Bacillus atrophaeus which are aslo comparable to the
drug. Compound 2 was found much active than the standard
drug in inhibiting the activities of Bacillus subtilis. None of the
metal based compound was found active against the Klebsiella
pneumoniae, Salmonella typhus, Pseudomonas aeruginosa,
Escherichia coli, Erwinia carotovora, and Staphylococcus aureus. Except compound 1, 4 and 6 all the tested compounds
showed greater activities against the Candida albican as compared to the standard drug. Similarly all the tested compounds
reveal greater activities against Agrobacterium tumefaciens
except the compound 6.
Table 1. In Vitro antimicrobial activities of zinc complexes dicoumarols against different animal and plant pathogens
Compounds
Bacillus Bacillus Klebsiella Salmonella Pseudomonas Escherichia Staphylococcus Candida Agrobacterium Erwinia
atrophaeus subtilis pneumoniae
typhus
aeruginosa
coli
aureus
albican
tumefaciens carotovora
(mm)
(mm)
(mm)
(mm)
(mm)
(mm)
(mm)
(mm)
(mm)
(mm)
1
24
24
16
16
---
15
16
---
22
---
2
25
28
19
3
27
22
12
12
---
24
15
22
26
15
13
21
14
22
23
24
16
4
22
22
5
26
22
16
---
16
16
17
---
22
15
09
13
22
25
19
24
22
14
6
19
15
7
29
20
11
---
20
24
21
19
13
15
---
12
20
22
19
21
26
15
8
29
22
---
---
21
25
21
25
24
18
9
27
25
14
20
20
19
21
23
27
22
10
25
20
20
10
20
20
21
24
26
16
Standard
25
26
29
42
36
38
34
16
15
26
Gram positive bacteria: Bacillus atrophaeus, Bacillus subtilis, Staphylococcus aureus, standard used was erythromycin in 6 µM
Gram negative bacteria: Klebsiella pneumoniae, Salmonella typhus, Pseudomonas aeruginosa, Escherichia coli, Agrobacterium tumefaciens,
Erwinia carotovora, standard used was ciprofloxacin in 6 µM
Fungal Strain: Candida albican, standard used was clothrimazol in 6 µM
Table 2. In Vitro antimicrobial activities of dicoumarols against different animal and plant pathogens*
Compounds
Bacillus Bacillus Klebsiella Salmonella Pseudomonas Escherichia Staphylococcus Candida Agrobacterium Erwinia
atrophaeus subtilis pneumoniae
typhus
aeruginosa
coli
aureus
albican
tumefaciens carotovora
(mm)
(mm)
(mm)
(mm)
(mm)
(mm)
(mm)
(mm)
(mm)
(mm)
L1
25
22
---
---
20
14
16
22
22
15
L2
12
09
09
06
09
---
16
12
10
10
L3
26
22
---
---
16
---
09
15
20
---
L4
17
17
---
---
18
12
__
12
16
11
L5
15
12
---
11
15
10
13
30
20
---
L6
13
15
---
---
18
---
13
19
27
13
L7
21
22
---
12
18
12
11
20
24
17
L8
16
20
11
---
20
09
15
20
20
---
L9
21
21
12
10
19
19
09
21
20
15
L10
22
25
---
---
15
12
12
---
28
11
Standard
25
26
29
42
36
38
34
16
15
26
[19] Rehman, S.; Ikram, M.; Baker, R. J.; Zubair, M.; Azad, E.; Min, S.; Riaz, K.; Mok, K. H.; Rehman, S.-U-.; Chem. Cent. J., 2013, 7, 68.
140
J. Mex. Chem. Soc. 2015, 59(2)
By comparison of the activities of dicoumarols and their
zinc based metal complexes it becomes clear that the zinc complexation make the ligands much active against the pathogenic
microbes.
3. Conclusion
Zinc metal complexes of the dicoumarols were synthesized insitu by reacting [Zn{N(SiMe3)2}2] with the ligands in THF as
a medium. All the metal complexes (1-10) were assigned geometries using various spectro-analytical techniques. In vitro
antimicrobial activities revealed that all the metal complexes
(1-10) except 6 are more active against Gram positive bacteria
and Candida albican, whereas moderate activities were observed against Gram negative bacteria. The most important
aspect of these metal complexes is the presence of nontoxic
and bioregulatory zinc ion, which can make them very useful
as effective pharmaceutical drug.
4. Experimental
4.1 Materials and methods
All chemicals, buffers and solvents used were of analytical
grade. Benzaldehyde, 4-nitrobenzaldehyde, 4-chlorobenzaldehyde, and N,N-dimethyl-4-benzaldehyde were obtained from
Fluka whereas 3-pyridinecarboxaldehyde, 3-indolecarboxaldehyde, 4-methoxybenzaldehyde, vaniline, 2-hydroxynaphthaldehyde, salicyldehyde, dimethylsilazane and butyllithium
were obtained from Sigma Aldrich and were used as such without further purification. Zinc chloride was obtained from fluka,
and solvents were obtained from local suppliers of Sigma Aldrich, Merck or Fluka and were distilled at least twice before
use. Unless otherwise stated, all reactions were carried out under a dinitrogen atmosphere.
4.2 Instrumentation
Elemental analyses were carried out on Varian Elementar II.
Melting points were recorded on a Gallenkamp apparatus. IR
spectra were recorded using Shimadzu FTIR Spectrophotometer Prestige-21. 1H-NMR were measured with Bruker DPX
400MHz (400.23 MHz) whereas, 13C{1H}NMR were recorded
on Bruker AV 400MHz (150.9 MHz) spectrometers in CD3OD
at room temperature. Chemical shifts are reported in ppm and
standardized by observing signals for residual protons. Molar
conductance of the solutions of the metal complexes was determined with a conductivity meter type HI-8333. All measurements were carried out at room temperature with freshly
prepared solutions. Mass spectra were recorded on a LCT Orthogonal Acceleration TOF Electrospray mass spectrometer.
4.3 Antimicrobial activity
Sadia Rehman et al.
About 2.8 g/L nutrient agar and nutrient broth were prepared in
deionized water and kept in autoclave set at 1.5 Pounds pressure for about 15 min. The nutrient agar media were poured
aseptically into sterilized petri dishes in laminar flow under
inert atmosphere. The petri dishes were kept in inverted position for about 24 hrs at 37 oC. Bacterial cultures were adjusted
to 0.5 McFarland turbidity standards and Candida albican was
adjusted to 108 cfu/ml. Sterile filter paper of diameter 6mm
was used for bacterial strains whereas its thickness ranged upto
13 mm for fungal strains. These filter papers were in the form
of discs and were seeded with 0.5 McFarland and 106 cfu/ml
cultures of bacteria and fungi respectively. Solutions (0.5 mM)
of the synthesized compounds were applied to the prepared
discs and incubated for 18 hr at 37 oC. Subsequent measurements of the zone of activity were carried out [21].
4.4 Synthesis of dicoumarols
Synthesis of dicoumarols has been described by Sadia et al.
2013 [19]. The sequence of codes L1-L10 used in this manuscript is following the sequence as described earlier by us [19].
4.5 Synthesis of zinc compounds
Zinc derivatives of the coumarin ligands [19] were prepared by
following the same procedure. [Zn{N(SiMe3)2}2] was synthesized according to the literature procedure [22]. 10 mmol of
butyllithium in n-hexane was added to the 50 mmol dimethyl
silazane dissolved in degassed dry diethyl ether and the reaction mixture stirred for one hour at 0 oC in completely inert
atmosphere. 20 mmol of synthesized yellow liquid Li{N(SiMe3)2}2 was added to the 10mmol ethereal solution of zinc
chloride and the mixture stirred under argon for one hour.
White floating powder of [Zn{N(SiMe3)2}2] was obtained after purifying the sample by washing and recrystallizing from
diethyl ether.
10 mmol of [Zn{N(SiMe3)2}2] was added to 5 cm3 THF
and stirred at 0 oC, 5 mmol of dicoumarol ligand dissolved in
minimum amount of THF was added to this solution and the
mixture stirred for 4-5 hour at room temperature. After the formation of powder, the mixture was filtered and washed many
times with diethyl ether and THF and dried in vacou.
4.5.1 Bis[3,3’-(1H-indole-3-ylmethanediyl-4-hydroxy-2Hchromen-2-one)]zinc (II) (1)
IR: 3500(bd), 2980(w), 2612(w), 1689(s), 1636(w), 1584(w),
1518(w), 1483(s), 1417(s), 1260(s), 1241(w), 1222(w),
1107(w), 1073(s), 1025(w), 900(w), 848(s), 791(w), 749(w),
695(w), 660(w) cm-1, 1H-NMR (400.23 MHz, CD3OD, 303k):
δ = 6.93 (s, 1H, methyl), 7.21 (m, 1H), 7.22 (m, 1H), 7.23 (dd,
JHH = 8.77 Hz, 1H), 7.25 (m, 1H), 7.48 (dd, JHH = 8.71 Hz,
1H), 7.78 (s, 1H), 7.80 (s, 1H) ppm, 13C{1H}-NMR (150.9
MHz, CD3OD, 303k): δ = 67.03 (CH, pyrrole), 105 (C, pyrrole), 112-138 (CH, aromatic), 155 (C, phenolic), 164 and 165
(C=O, lactone) ppm, Elemental Analysis (C54H36N2O12Zn),
Calc. C: 66.84%, H: 3.74%, N: 2.89%, Zn: 6.74%, Exp. C:
Synthesis of New Dicoumarol Based Zinc Compounds and their Invitro Antimicrobial Studies
66.44%, H: 3.69%, N: 2.90%, Zn: 6.34%, EI-MS: m/z (%)
968.1554 (100%) [C54H36N2O12Zn +].
4.5.2 Bis[(3,3’-(4-chlorophenyl)methanediyl-4-hydroxy-2Hchromen-2-one)]zinc(II) (2)
IR: 3420(bd), 2924(w), 2890(w), 1703(w), 1649(s), 1605(s),
1524(s), 1411(s), 1347(s), 1277(w), 1182(s), 1107(s), 1045(s),
921(s), 894(s), 831(s), 797(s), 758(s), 742(s), 712(s), 699(s),
669(s) cm-1, 1H-NMR (400.23 MHz, CD3OD, 303k): δ = 5.9
(s, 1H, methyl), 7.18-8.0 (aromatic protons) ppm, 13C{1H}NMR (150.9 MHz, CD3OD, 303k): δ = 103 (CH, methyl),
114.8-132 (Aromatic carbons), 152 (C, Chloro), 154 (C, Phenolic), 162 & 164 (C, lactone) ppm. Elemental Analysis
(C50H28Cl2O12Zn), Calc. C: 62.75%, H: 2.95%, Zn: 6.83%,
Exp. C: 62.14%, H: 3.09%, Zn: 6.91%, EI-MS: not observed.
4.5.3 Bis[(3,3’-(4-hydroxyphenyl)methanediyl-4-hydroxy2H-chromen-2-one)]zinc(II) (3)
IR: 3700(bd), 2980(bd), 2800(bd), 1704(s), 1649(s), 1605(s),
1526(s), 1469(s), 1414(s), 1245(s), 1182(s), 1107(s), 1042(s),
921(s), 892(s), 831(w), 799(w), 758(s), 743(s), 712(s), 669(s)
cm-1, 1H-NMR (400.23 MHz, CD3OD, 303k): δ = 5.34 (s, 1H,
methyl), 6.63-7.81 (m, aromatic protons) ppm, 13C{1H}-NMR
(150.9 MHz, CD3OD, 303k): δ = 104.6 (CH, methyl), 109-142
(aromatic carbons), 164 (C, lactone) ppm, Elemental Analysis
(C50H30O14Zn), Calc. C: 65.26%, H: 3.29%, Zn: 7.11%, Exp.
C: 65.80%, H: 5.10%, Zn: 8.89%. EI-MS: m/z (%) 918.0921
(40%) [C50H30O14Zn +].
4.5.4 Bis[(3,3’-(4-nitrophenyl)methanediyl-4-hydroxy-2Hchromen-2-one)]zinc(II) (4)
IR: 3420(bd), 2924(w), 2890(w), 1703(w), 1649(s), 1605(s),
1524(s), 1411(s), 1347(s), 1277(w), 1182(s), 1107(s), 1045(s),
921(s), 894(s), 831(s), 797(s), 758(s), 742(s), 712(s), 699(s),
669(s) cm-1, 1H-NMR (400.23 MHz, CD3OD, 303k): δ = 5.9
(s, 1H, methyl), 7.18-8.0 (aromatic protons) ppm, 13C{1H}NMR (150.9 MHz, CD3OD, 303k): δ = 103 (CH, methyl),
114.8-132 (Aromatic carbons), 152 (C, nitro), 154 (C, Phenolic), 162 & 164 (C, lactone) ppm. Elemental Analysis (C50H28N2O16Zn), Calc. C: 61.39%, H: 2.89%, N: 2.86 %, Zn:
6.69%, Exp. C: 63.01%, H: 2.11%, N: 2.89 %, Zn: 5.93%, EIMS: m/z (%) 976.0724 (5%) [C50H28N2O16Zn +].
4.5.5 Bis[3,3’-[(3-methoxy-4-hydroxyphenyl)methanediyl-4hydroxy-2H-chromen-2-one)]zinc(II) (5)
IR: 3300(bd), 2980(bd), 1704(s), 1649(s), 1605(s), 1526(s),
1469(s), 1414(s), 1245(s), 1182(s), 1107(s), 1042(s), 921(s),
892(s), 831(w), 799(w), 758(s), 743(s), 712(s), 669(s) cm-1,
1H-NMR (400.23 MHz, CD OD, 303k): δ = 2.51 (br, s, 2H,
3
methyl), 6.55-6.64 (m, 1H), 7.19 – 7.31 (m, 2H), 7.50 (t, 3JHH
= 7.78 Hz, 1H), 7.82 (d, 3JHH = 8.03Hz, 1H) ppm, = Elemental
Analysis (C52H34O16Zn), Calc. C: 63.72%, H: 3.50%, Zn:
6.67%, Exp. C: 62.23%, H: 4.10%, Zn: 6.89%. EI-MS: m/z
(%) 978.1132 (23 %) [C52H34O16Zn +].
4.5.6 Bis[3,3’-(pyridin-3-ylmethanediyl-4-hydroxy-2Hchromen-2-one)]zinc(II) (6)
141
IR: 3700(bd), 2980(bd), 2800(bd), 1704(s), 1649(s), 1605(s),
1526(s), 1469(s), 1414(s), 1245(s), 1182(s), 1107(s), 1042(s),
921(s), 892(s), 831(w), 799(w), 758(s), 743(s), 712(s), 669(s)
cm-1, 1H-NMR (400.23 MHz, CD3OD, 303k): δ = 5.34 (s, 1H,
methyl), 6.63-7.81 (m, aromatic protons) ppm, 13C{1H}-NMR
(150.9 MHz, CD3OD, 303k): δ = 104.6 (CH, methyl), 109-142
(aromatic carbons), 164 (C, lactone) ppm, Elemental Analysis
(C48H28N2O12Zn), Calc. C: 62.40%, H: 3.17%, N: 3.15%, Zn:
7.35%, Exp. C: 62.43%, H: 3.79%, N: 3.98%, Zn: 7.06%, EIMS: m/z (%) 888.0928 (100%) [C48H28N2O12Zn +].
4.5.7 Bis[3,3’-[(4-methoxyphenyl)methanediyl-4-hydroxy2H-chromen-2-one)]zinc(II) (7)
IR: 2980(w), 1622(s), 1597(s), 1577(s), 1556(s), 1521(s),
1481(s), 1452(s), 1402(s), 1340(s), 1271(s), 1242(s), 1209(s),
1153(w), 1105(s), 1072(s), 1024(s), 979(s), 947(s), 881(s),
815(s), 765(s), 750(s), 717(s), 688(s), 677(s), 610(w), 526(s)
cm-1, 13C{1H}-NMR (150.9 MHz, CD3OD, 303k): δ = 55.57
(CH, N-CH3), 103.73 (CH, methyl), 114.85-146.99 (aromatic
region), 152.34 (C, phenolic), 164.82 & 167.82 (C, lactone)
ppm, Elemental Analysis (C52H34O14Zn), Calc. C: 65.87%, H:
3.61%, Zn: 6.90%, Exp. C: 66.32%, H: 3.83%, Zn: 7.03%, EIMS: m/z (%)946.1234 (7%) [C52H34O14Zn +].
4.5.8 Bis[3,3’-(phenylmethanediyl-4-hydroxy-2H-chromen2-one)]zinc(II) (8)
IR, : 3400(bd), 3026(w), 2980(w), 1598(s), 1516(s), 1446(s),
1405(s), 1366(s), 1356(w), 1277(w), 1250(w), 1211(w),
1107(s), 1084(s), 1058(w), 937(w), 837(s), 757(s), 727(s),
693(s) cm-1, 1H-NMR (400.23 MHz, CD3OD, 303k): δ = 6.988.11 (aromatic protons) ppm, 13C{1H}-NMR (150.9 MHz,
CD3OD, 303k): δ = 103.9 (CH, methyl), 111.9-141.89 (aromatic), 166.7 & 168 (C=O, lactone) ppm, Elemental Analysis
(C50H30O12Zn), Calc. C: 67.61%, H: 3.40 %, Zn: 7.36%, Exp.
C: 67.21%, H: 3.76 %, Zn: 7.99 %, EI-MS: m/z (%) 886.1023
(60%) [C50H30O12Zn +].
4.5.9 Bis[(3,3’- (4-N,N-dimethylaminophenyl)methanediyl4-hydroxy-2H-chromen-2-one)]zinc(II) (9)
IR: 3414(bd), 3242(w), 2980(w), 1622(s), 1597(s), 1577(s),
1556(s), 1521(s), 1481(s), 1452(s), 1402(s), 1340(s), 1271(s),
1242(s), 1209(s), 1153(w), 1105(s), 1072(s), 1024(s), 979(s),
947(s), 881(s), 815(s), 765 (s), 750 (s), 717 (s), 688 (s), 677 (s),
610 (w), 526 (s) cm-1, 13C{1H}-NMR (150.9 MHz, CD3OD,
303k): δ = 55.57 (CH, N-CH3), 103.73 (CH, methyl), 114.85146.99 (aromatic region), 152.34 (C, C-N-(CH3)2), 164.82 &
167.82 (C, lactone) ppm, Elemental Analysis (C54H40N2O12Zn),
Calc. C: 66.57%, H: 4.14%, N: 2.88%, Zn: 6.71, Exp. C:
66.57%, H: 4.14%, N: 2.88%, Zn: 6.71, EI-MS: m/z
(%)972.1867 (100%) [C54H40N2O12Zn +].
4.5.10 Bis[(3,3’- (2-hydroxy-1,2-dihydronaphthalen-1-yl-4hydroxy-2H-chromen-2-one)]zinc(II) (10)
IR: 2980(w), 1622(s), 1597(s), 1577(s), 1556(s), 1521(s),
1481(s), 1452(s), 1402(s), 1340(s), 1271(s), 1242(s), 1209(s),
1153(w), 1105(s), 1072(s), 1024(s), 979(s), 947(s), 881(s),
815(s), 765(s), 750(s), 717(s), 688(s), 677(s), 610(w), 526(s)
142
J. Mex. Chem. Soc. 2015, 59(2)
cm-1, 13C{1H}-NMR (150.9 MHz, CD3OD, 303k): 103.73
(CH, methyl), 114.85-146.99 (aromatic region), 152.34 (CH,
phenolic), 164.82 & 167.82 (C, lactone) ppm, Elemental Analysis (C58H34O14Zn), Calc. C: 68.28%, H: 3.36%, Zn: 6.41%,
Exp. C: 68.31%, H: 3.87%, Zn: 6.12%. EI-MS: m/z
(%)1018.1234 (10%) [C58H34O14Zn +].
Conflict of interest
The author declares no conflict of interest.
Author’s contribution
Both the authors M. Ikram and S. Rehman equally contributed
to the content of this manuscript and equal first authors.
Acknowledgment
The authors are gratefully acknowledged to the Higher Education Commission (HEC) Pakistan for providing financial
assistance.
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Article
J. Mex. Chem. Soc. 2015, 59(2), 143-150
© 2015, Sociedad Química de México
ISSN 1870-249X
Separation of Micro-Macrocomponent Systems: 149Pm – Nd, 161Tb-Gd,
166Ho-Dy and 177Lu-Yb by Extraction Chromatography
F. Monroy-Guzman and E. Jaime Salinas
Instituto Nacional de Investigaciones Nucleares, Carretera México-Toluca S/N, La Marquesa Ocoyoacac, 52750
Edo. de México, México. fabiola.monroy@inin.gob.mx
Received February 20th, 2015; Accepted April 4th, 2015
Abstract. 149Pm, 161Tb, 166Ho, and 177Lu have advisable nuclear properties to be used in radiotherapy. They can be produced by neutron
irradiation of a lanthanide target followed by β- decay and a posterior
radiochemical separation of micro-amounts of daughter radionuclide
from macro-amounts of the parent target. In order to accomplish the
radiochemical separation of micro-macro systems: Nd/149Pm,
Gd/161Tb, Dy/166Ho and Yb/177Lu, this work proposes the use of an
extraction chromatographic with Ln SPS resin. Distribution coefficients and separation factors were determined and established the separation conditions of these micro-macro systems.
Key words: separation, extraction chromatography, 149Pm, 161Tb,
166Ho, 177Lu
Resumen. 149Pm, 166Ho, 161Tb y 177Lu poseen propiedades nucleares
apropiadas para ser utilizados en medicina nuclear. Estos radiolantánidos pueden ser producidos por irradiación de neutrones del blanco,
seguido de un decaimento β- y posteriormente la separación radioquímica de macro-cantidades de los blancos de las micro-cantidades de
los radionuclides hijos producidos por decaimento. Con el fin de establecer la metodología de separación radioquímica de los sistemas micro-macro: Nd/149Pm, Gd/161Tb, Dy/166Ho y Yb/177Lu, se determinaron
los coeficientes de distribución y factores de separación de estos sistemas en la resina Ln SPS. Se presentan las condiciones de separación
de estos sistemas.
Palabras clave: separación, cromatografía extractiva, 149Pm, 161Tb,
166Ho, 177Lu
1. Introduction
ecarboxylic acids, 2-ethylhexanoic, 2-methyl-cyclohexanecarboxylic, 2,2,3-trimetylbutanoic, 2-butyl-2-ethylhexanoic, versatic,
amines, etc. [10-12]. Nevertheless solvent extraction poses
environmental problems because a large amount of organic
solvent is inevitably used, due to the number of stages for solvent extraction cascades or batteries increases with the rise in
purity of each individual rare earth produced. Further purification of micro-macro amounts of rare earths into the high ninety-nines purity are usually carried out by ion exchange and/or
chromatographic techniques [1,5,9,10,13-15]. Ion exchange
resins with alpha-hydroxyisobutyric acid (α–HIBA) have been
currently used for lanthanides separation: however these procedures are either slow or give only partial separation or are
unsuitable at high levels of activity [3,7,9, 12, 16,17]. For example, 166Ho has been separated from mg quantities of Dy target by a Aminex-A5 cation exchange and 0.085 M α–HIBA at
pH = 4.3 as eluent, and purified with a cation exchange column
from HCl solutions. This separation was achieved around 2 h,
with a radiochemical yield of 95% and a radionuclide purity
166Ho of 99.9 % [1,9]. 177Lu from Yb target has been performed
by Na(Hg) amalgam from Cl−/CH3COO− electrolytes, followed by a final cation exchange purification. The cementation
separation process provides a decontamination factor of
Yb(III) of 104, the cation exchange purification adding a decontamination factor of >102 and is performed within 4–5 h.
The radiochemical yield of this process is about 75% [6]. In the
case of 161Tb, an isolation of 80 to 90% of 161Tb from massive
160Gd targets with a decontamination factor from gadolinium
>105 has been achieved using a cation exchange resin Aminex
Radioactive lanthanides such as 149Pm, 161Tb, 166Ho and 177Lu,
have a great potential in radiotherapy because they are beta or
Auger-electron emitters with just enough gammas to enable
imaging, with half-lives long enough to allow preparation and
distribution of the radiopharmaceuticals, and can be prepared
at high specific activities (carrier-free) [1,2].
Carrier-free radiolanthanides of high specific activity can
be produced in nuclear reactors via neutron irradiation of massive lanthanide targets (>1 mg), as described in the reaction 1,
followed by a radiochemical separation of the daughter radionuclide ( AZ++11 Ln ,< 1µg) from the macro-amounts of the parent
target ( AZ Ln )[3-9].
A
Z
−
−
A+1
Ln + 01 n → A+1Z Ln ⎯β⎯→ A+1
Ln ⎯β⎯→ Z+2
Ln Z+1
(1)
The difficulty in separating micro from macro-amounts of
irradiated target are particularly arduous because lanthanides
show great similarities in their chemical properties due to lanthanide contraction: the atomic size or ionic radii of tripositive
lanthanide ions show a steady and gradual decrease with the
increase in atomic number from La to Lu. A lot of works on
lanthanides separation by liquid-liquid extraction have been
reported and examined for improving the extraction and separation of individual rare earths, using as primary extractants:
di(2-ethylhexyl)orthophosphoric acid (HDEHP), tributyl phosphate (TBP), carboxylic acids (naphthenic acids: n-hexanoic,
n-octanoic, 3-cyclohexylpropanoic, iso-nonanoic, cyclohexan-
144
J. Mex. Chem. Soc. 2015, 59(2)
A6 eluted with α–HIBA at ph 4.5 and purified with a Bio-Rad
AG 50W-X8 cation exchanger to produce a 0.04 M HCl solution [3].
The extraction chromatography column is becoming
widely advantageous in the separation of very similar ions
such as lanthanides because it couples the favorable selectivity
features of the organic compounds used in liquid-liquid extraction, with the multistage character of a chromatographic
process [18]. In addition, extraction chromatography is a fast,
efficient technique and generates less waste than other separation technique. The organic phosphorus compounds that are
most frequently used as stationary phases in the lanthanides
separation are HDEHP, TBP, Diphenyl(dialkylcarbamoylmethyl) phosphine oxides, etc., however the best separation
factors (> 2.5) between adjacent rare earths are obtained with
HDEHP as stationary phase fixed in Kiselguhr, Corvic
(poly(vinyl chloride-vinyl acetate)copolymer), Kel-F (polychlorotrifluoroethane), Zorbax-SIL,(porous silica), PMS
(polymethysilane) or Celite® (diatomaceous earth) and as mobile phases solutions containing acids (HNO3, HCl, HClO4)
[1,5, 13,19-22]. In general, the separations of all the rare earth
elements at a tracer level on HDEHP-solid support are satisfactory by gradient elution; however some difficulties have
been reported in the separation of consecutive lanthanides such
as: Nd-Pm, Dy-Ho and Yb-Lu depending on the type of support used and the column length [20-22]. Ketring performed a
very neat separation of Nd/Pm, Dy/Ho and Yb/Lu using the Ln
spec resin (50-100 μm) from Eichrom, formed by HDEHP (40
% by weight) supported on an inert polymeric absorbent (60 %
by weight), nervertheless the precise conditions of these separations have not been reported [5].
The objective of this work was to establish a radiochemical method to separate the carrier free radiolanthanides: 149Pm,
161Tb, 166Ho and 177Lu from irradiated natural targets (Nd, Gd,
Dy and Yb), using an extraction chromatographic resin. Horwitz reported distribution coefficients (Kd) versus nitric acid
only for Pm, Gd, Tb and Lu using HDEHP on a hydrophobic
support, thus distribution coefficients for Nd, Dy, Ho and Yb
were determined in this work as well as those of Pm, Gd, Tb
and Lu under our experimental conditions and compared with
the values reported by Hotwitz [19].
2. Results
2.1. Distribution Coefficients of Nd, Pm, Gd, Tb, Dy, Ho,
Yb and Lu
Figure 1 shows the Kd values of Nd, Pm, Gd, Tb, Dy, Ho, Yb
and Lu as a function of HNO3 concentration in Ln SPS Eichrom
resin. The Kd values decrease with an increase in HNO3 solution concentration and a diminution of the lanthanide atomic
number at a fixed HNO3 concentration. For example, Dysprosium (Z = 66) at 3 mol/L HNO3 has a Kd value of 26 cm3/g
while Holmium (Z = 67), Yterbium ( Z = 70) and Lutetium ( Z
= 71), at the same HNO3 concentration, present Kd values of
37, 310 and 707 cm3/g, respectively. Thus, the parent lantha-
F. Monroy-Guzman and E. Jaime Salinas
Fig. 1. Effect of HNO3 concentration on the distribution coefficients
of Nd, Pm, Gd, Tb, Dy, Ho, Yb and Lu in Ln SPS Eichrom resin.
Table 1. Mathematical behavior of the Kd values of Nd, Pm, Gd, Tb,
Dy, Ho, Yb and Lu in Ln SPS Eichrom resin as a function of [HNO3].
Element
Mathemathical expression
Nd
log Kd = 2.283 – 2.919 log [HNO3] + 2.999 log[HNO3]2
Pm
log Kd = 2.894 – 3.559 log [HNO3] + 3.629 log[HNO3]2
Gd
log Kd = 1.208 - 2.558 log [HNO3] + 2.856 log[HNO3]2
Tb
log Kd = 1.905 - 2.928 log [HNO3] + 2.488 log[HNO3]2
Dy
log Kd = 2.227 - 2.673 log [HNO3] + 2.048 log [HNO3]2
Ho
log Kd = 2.525 - 2.865log [HNO3] + 1.766 log [HNO3]2
Yb
log Kd = 5.621 – 9.159 log [HNO3] + 5.433 log [HNO3]2
Lu
log Kd = 6.240 – 9.822 log [HNO3] + 5.729 log [HNO3]2
nide of the parent/daughter pairs: Nd/Pm, Gd/Tb, Dy/Ho and
Yb/Lu, will be eluted at first during the chromatographic separation process. The Kd values of Pm, Gd, Tb and Lu are in
good agreement with those reported by Hotwitz [19].
Table 1 presents the mathematical equations that express
the behavior of the Kd values of Nd, Pm, Gd, Tb, Dy, Ho, Yb
and Lu shown in Figure 1, which present an parabolic behavior.
2.2. Separation factors (α) of pairs: Nd/Pm, Gd/Tb, Dy/Ho
and Yb/Lu
The separation factors (α) of the Nd/Pm, Gd/Tb, Dy/Ho and
Yb/Lu pairs were calculated from their Kd values by using the
mathematical expressions depicted in Table 1. The α values
obtained then were plotted as a function of HNO3 concentration (Figure 2) and their respective mathematical equations are
shown in Table 2.
The α value determines the separation performance of the
pairs. When α is equal to one, the Kd values of both elements
are identical and they are not able be separated. So, the greater
the α value, the more efficient the separation and the higher purity of the radioisotope isolated is obtained. Additionally, the α
Separation of Micro-Macrocomponent Systems: 149Pm – Nd, 161Tb-Gd, 166Ho-Dy and 177Lu-Yb by Extraction Chromatography
145
Table 3. Separation factors (α) tested to define the separation conditions of parent/daughter pair.
α
PAIR
[HNO3] mol/L
3.5-3.0
Pm/Nd
0.18 - 0.25
5.7-5.5
Tb/Gd
0.7 - 0.80
1.9-1.7
Ho/Dy
1.25 - 1.75
2.3-2.2
Lu/Yb
3.0 - 3.5
select the separation conditions for each parent/daughter pair
are shown in Table 3.
2.4 Separation of the Nd/Pm, Gd/Tb, Dy/Ho and Yb/Lu
pairs
Fig. 2. Separation factors (α) of the Nd/Pm, Gd/Tb, Dy/Ho and Yb/Lu
pairs in Ln SPS Eichrom resin as a function of [HNO3].
Table 2. Mathematical behaviour of the separation factors (α) of the
Nd/Pm, Gd/Tb, Dy/Ho and Yb/Lu pairs in Ln SPS Eichrom resin as
a function of [HNO3].
Α
Mathemathical expression
Pm/Nd
α = -6.9 + 154.3log [HNO3] – 822.1log[HNO3]2 +
1939.4log[HNO3]3 - 2160.1log [HNO3]4 + 931.4log
[HNO3]5
Tb/Gd
α = 5.92e-([HNO3]/1.92) + 1.55
Ho/Dy
α = 1.79e-([HNO3]/3.36) + 0.65
Lu/Yb
α = 2.7e-([HNO3]/1.96) + 1.78
values of the pairs studied decrease with the increase of the
HNO3 concentration. It follows that the slopes of αDaughter/Parent
functions define if α decreases rapidly (αPm/Nd) or slowly (αLu/
Yb). The separation factors of the pair Tb/Gd are the highest
and those for Ho/Dy lower. Tb and Gd separation have a better
resolution that that of Ho/Dy or Lu/Yb.
2.3 Selection of separation conditions of the Nd/Pm, Gd/
Tb, Dy/Ho and Yb/Lu pairs
In principle, it is feasible to separate the Nd/Pm, Gd/Tb, Dy/
Ho and Yb/Lu pairs if α value (See Figure 2) is greater than 1.
Therefore, the best separation performances are essentially obtained with high αDaughter/Parent values (>>1); however it is also
important to consider the HNO3 volume used to elute lanthanides, which is closely dependent on the Kd values. Distribution coefficients higher than 200 impose the use of large
volumes of eluent and also require more time to achieve an
efficient separation. For this reason, the separation conditions
of the Nd/Pm, Gd/Tb, Dy/Ho and Yb/Lu pairs were selected
when higher separation factor (α) values at Kd values lower
than 200 were found. HNO3 concentration ranges chosen to
The separation tests of the parent/daughter pairs at HNO3 concentrations stated in Table 3, were determined following the
same methodology described in section 4.1. The separation
efficiencies of the parent/daughter pairs and the radionuclide
purity of daughters obtained are shown in Table 4. Highest separation efficiencies and radionuclide purities of more than
99.9% were selected as the optimal separation conditions of
each parent/daughter pair. Thus, the separation conditions selected for each pair are shown bolded in Table 4 and the respective elution profiles in Figure 3. Gadolinium and Terbium
were separated with an efficiency of 100% using 22 mL of 0.8
mol/L HNO3 and 18mL of 3 mol/L HNO3 to recover Gd and
161Tb respectively, with a 100% radionuclide purity for 161Tb.
In the case of Neodymium and Promethium, the use of 106 mL
and 30 mL of 0.18 and 1.5 mol/L HNO3, respectively, is required to achieve a separation efficiency of 98.4 % and a 149Pm
radionuclide purity of 99.9%. Dysprosium and Holmium separation was attained with an efficiency of 100% by using 50 mL
and 35 mL of 1.4 and 3 mol/L HNO3 respectively, and 100%
pure166Ho was obtained. Finally, Ytterbium and Lutetium were
separated with an efficiency of 89.7% by using 165 mL of 3.4
mol/L HNO3 to obtain Ytterbium and 55 mL of 8 mol/L HNO3
to recover the 177Lu at a 99.9% radionuclide purity. In order to
get a 177Lu radionuclide purity higher than 99.9%, the first 6
mL of the 177Lu eluate were removed (See Figure 3).
3. Discussion
The production of carrier-free 149Pm, 161Tb, 166Ho and 177Lu is
based on the neutron irradiation of 148Nd, 160Gd, 164Dy and
176Yb enriched targets in nuclear reactors, followed by a radiochemical separation of the 149Pm, 161Tb, 166Ho and 177Lu from
the macro-amounts of the neodymium, gadolinium, dysprosium and ytterbium targets [3-9]. However, Nd, Gd, Dy and Yb
natural targets were irradiated in this work, producing the nuclear reactions listed in table 5. The radioisotopes used to follow the separation processes of Nd/Pm, Gd/Tb, Dy/Ho and Yb/
Lu pairs and to determine the distribution coefficients of these
146
J. Mex. Chem. Soc. 2015, 59(2)
F. Monroy-Guzman and E. Jaime Salinas
Table 4. Separation efficiencies of parent/daughter pairs and radiochemical purities of the daughters obtained at different concentrations of
HNO3.
Pair
[HNO3] mol/L
recovery parent
[HNO3] mol/L
recovery daughter
Separation efficiency
(%)
Radionuclide purity of
daughter (%)
Gd/Tb
0.70
3.00
65.4
95.2
Gd/Tb
0.80
3.0
100
100
Nd/Pm
0.18
1.5
98.4
99.9
Nd/Pm
0.20
0.5
63.1
24.8
Nd/Pm
0.21
2.0
88.3
99.8
Nd/Pm
0.22
1.0
66.6
100
Nd/Pm
0.24
1.0
97.1
99.3
Nd/Pm
0.25
5.0
73.9
32.7
Dy/Ho
1.40
1.4
100
100
Dy/Ho
1.40
3.0
98.9
99.7
Dy/Ho
1.50
1.5
99.7
100
Yb/Lu
3.00
3.0
53.1
97.6
Yb/Lu
3.30
7.0
63.8
97.2
Yb/Lu
3.40
3.4
72.36
98.50
Yb/Lu
3.40
8.0
89.72
99.9
Yb/Lu
3.50
6.0
96.11
83.80
Yb/Lu
3.50
8.0
71.2
90.3
161
Tb
161
Gd
0
HNO
3
10
20
0.18 mol/L HNO3
149
Nd
149
Pm
30
40
0
50
20
40
60
HNO
3
166
Ho
Eluated activity (a.u.)
0
10
20
30
40
50
60
70
120
140
8 mol/L HNO3
177
Yb
Eluated activity (a.u.)
Dy
100
3.4 mol/L HNO3
0.15 mol/L
3 mol/L
1.4 mol/L
166
80
HNO3 (mL)
HNO3 (mL)
0.15 mol/L
1.5 mol/L HNO3
Eluated activity (a.u.)
Eluated activity (a.u.)
HNO
3
0.15 mol/L
3 mol/L HNO3
0.8 mol/L HNO3
0.15 mol/L
80
0
HNO3 (mL)
50
Lu
100
HNO3 mL
Fig. 3. Elution curve of the Gd/ Tb, Nd/Pm, Dy/Ho and Yb/Lu separation from Ln SPS resin.
Figure 3.
150
177
Lu
200
Separation of Micro-Macrocomponent Systems: 149Pm – Nd, 161Tb-Gd, 166Ho-Dy and 177Lu-Yb by Extraction Chromatography
147
Table 5. Nuclear reactions of the neodymium, gadolinium, dysprosium and ytterbium target irradiation.
Target
Nuclear reaction
142Nd
27.13 %
142
Nd (n, γ) 143Nd (stable)
143Nd
12.18%
143
Nd (n, γ) 144Nd (stable)
144Nd
145Nd
23.8%
144Nd
(n, γ) 145Nd (stable)
8.3 %
145Nd
(n, γ) 146Nd (stable)
146
Nd 17.19 %
148Nd
150
146Nd
5.72%
Nd 5.64 %
152Gd
148Nd
150Nd
(n,
(n, γ) 147Nd(11d)ª147Pm(2.2y)ª147Sm (stable)
(n, γ) 149Nd(1.7h) ª149Pm*(2.2d)ª149Sm (stable)
γ) 151Nd(12.4min)ª151Pm(1.2d)ª151Sm(93y)ª151Eu
0.2 %
152Gd
(n, γ) 153Gd(241.6d)ª153Eu (stable)
154
154Gd
155
155
Gd (n, γ) 156Gd(stable)
156
156
Gd (n, γ) 157Gd(stable)
157
Gd (n, γ) 158Gd(stable)
Gd 2.18 %
Gd 14.8 %
Gd 20.4 %
157Gd
20.4 %
158
Gd 24.8 %
160
Gd 21.86%
156
Dy 0.052 %
158
160
156
(n, γ) 155Gd(stable)
Gd (n, γ) 159Gd(18.6h) ª149Tb(stable)
(n, γ) 161Gd(3.7min)ª161Tb*(17.6h)ª161Dy (stable)
Dy (n, γ) 157 Dy(8.2h) ª 157Tb(71y)ª157Gd (stable)
Dy 0.09 %
158Dy
Dy 2.298 %
161
162
158
160Gd
Dy 18.8 %
(n, γ) 159 Dy(134d)ª159Tb (stable)
160Dy
(n, γ) 161 Dy (stable)
161Dy
(n, γ) 162 Dy (stable)
Dy 25.53 %
162
Dy (n, γ) 163 Dy (stable)
163
Dy 24.97 %
163
Dy (n, γ) 164 Dy (stable)
164
Dy 28.18 %
165
Dy
164
Dy (n, γ) 165 Dy(1.3min)ª165Ho(stable)
165
Dy (n, γ) 166 Dy(81.6h)ª166Ho*(26.7h)ª166Er (stable)
168Yb
0.14 %
170Yb
3.03 %
170Yb
171Yb
14.3 %
171
168Yb
172
16.13 %
174Yb
31.84 %
176
Yb 12.73 %
(n, γ) 169Yb(32d)ª169Tm (stable)
173
174Yb
176Yb
(n,
lanthanides, were selected in accordance with the following
criteria:
(A) For Neodymium/Promethium: Nd-147 (11 d), Nd-149
(1.7 h) and Nd-151 (12.4 min) are produced after 3 h of irradiation; Nd-151 and Nd-149 disappear after a decay time of 14 h.
Thus Kd determinations and Nd/Pm separation process were
performed with Nd-147. Pm can be evaluated with any radioisotopes produced by decay (Pm-147, 149 and 151); however
the best option is Pm -151 at 340 keV with a relative intensity
of 100% because the low relative intensity of gamma-rays
emitted by Pm-147 and Pm-149 (<0.01%).
(B) For Gadolinium/Terbium: target irradiation for 15
minutes produces Gd-153 (241.6 d), Gd-159 (18.6 h) and Gd-
(n, γ) 171Yb (stable)
Yb (n, γ) 172Yb (stable)
172Yb
Yb 21.8 %
173Yb
(stable)
(n, γ) 173Yb (stable)
Yb (n, γ) 174Yb (stable)
(n, γ) 175Yb(4.2d)ª175Lu (stable)
γ) 177Yb(1.9h)ª177Lu*(6.7d)ª177Hf
(stable)
161(3.2 min). The latter disappears after 30 min and Gd-153
requires longer counting times for its half-life, therefore Gd159 was chosen and measured at 363 keV (100%). Only one
terbium radioisotope is produced by the Gd irradiation and its
decay: Tb-161.
(C) For Dysprosium/Holmium: Dy-157 (8.2 h), Dy-159
(134 d), Dy-165(1.25 min) and Dy-166 are produced during
irradiation of dysprosium target. Dy-165 decays at 10 minutes,
Dy-159 activity produced is very low and requires long counting time, while the 82 keV gamma-ray of Dy-166 can interfered with the gamma-rays emitted by Ho-166, therefore,
Dy-157 was selected and counted at 326 keV (100%). Ho-166
is the only one holmium radioisotope generated.
148
J. Mex. Chem. Soc. 2015, 59(2)
F. Monroy-Guzman and E. Jaime Salinas
(D) For Ytterbium/Lutetium: ytterbium radioisotopes produced are: Yb-169 (32 d), Yb-175 (4.2 d) and Yb-177 (1.9 h)
and after 7 d decay to generate Lu-177, Yb-177 disappears and
Yb-175 decays considerably. Lu-177 is the only one lutetium
radioisotope generated in the irradiation-decay process.
Therefore Yb-169 and Lu-177 were selected to follow Yb/Lu
separation process.
One of the mechanisms postulated for the extraction adduct between a lanthanide ion and HDEHP (reaction 2) can be
divided into two steps: the formation of extractable species in
aqueous phase (reaction 3), and its partition between the two
phases (reaction 4), considering that HDEHP exists as a dimmer [18]:
Ln 3+ + 3(HDEHP)2 ↔ Ln[H(DEHP)2 ]3 + 3H +
⎡Ln[H(DEHP) ⎤ ⎡H + ⎤3
2 ⎦ 3 ⎣
⎦
⎣
K=
(2)
3
⎡(HDEHP) ⎤ ⎡Ln 3+ ⎤
2 ⎦ ⎣
⎦
⎣
Ln 3+ + 3(HDEHP)2 ↔ Ln[H(DEHP)2 ]3 + 3H +
⎡Ln ⎡H(DEHP) ⎤ ⎤ ⎡H + ⎤3
2 ⎦3 ⎦ ⎣
⎦
⎣ ⎣
Kc =
(3)
3
⎡⎣(HDEHP) ⎤⎦ ⎡Ln 3+ ⎤
2 ⎣
⎦
Ln[H(DEHP)2 ]3 ↔ Ln[H(DEHP)2 ]3 P=
Ln[H(DEHP)2 ]3
Ln[H(DEHP)2 ]3
(4)
(barred symbols denote the organic phase and Ln lanthanides
(III)).
The distribution ratio of lanthanides can be given as:
D=
⎡Ln⎤
⎡Ln[H(DEHP) ] ⎤
2 3 ⎦
⎣ ⎦
⎣
=
(5)
⎡⎣Ln⎤⎦ ⎡Ln 3+ ⎤ + ⎡Ln[H(DEHP) ] ⎤
⎣
2 3 ⎦
⎣
⎦
Thus, the two essential factors determining the magnitude
of distribution coefficient are: complex formation and the partition between the two phases. Extraction is greater for that
lanthanide which more readily forms an extractable complex
can be then expressed as a function of the partition coefficient
(P) and the stability constant of the complex (Kc), substituting
the constants into the above equation yields the following simplified form:
log Kd = log Z – 3log [H+] (6)
In accordance with expression 6, the lanthanides’s distribution coefficients follow a linear function form, where the
constant and the linear coefficient correspond to logZ, where Z
represents the sum of P, Kc and HDEHP concentration, consid-
ering that HDHEP is constant, and -3log[H+] (equation 6) respectively. However, the mathematical expressions of
lanthanides’s Kd, shown in Table 1, correspond to quadratic
functions where the constant and the linear coefficient agree to
logZ and -3log[H+] of the equation 6; while the quadratic coefficient constants, positive for all lanthanides, indicate that
the Ln[H(DEPH)2]3 complex dissociates in Ln3+ and HDEHP
at high HNO3 and thus Kd values decrease. It is important to
note that these Kd values reach to a minimum even if the acid
concentration is very high, because the full complex dissociation is never reached.
On the other hand, in the case of Yb and Lu, the logP values and the quadratic coefficient constants are approximately
double that for the rest of lanthanides, while their linear coefficient constants are three times greater than that obtained for the
other lanthanides.
The extractability of HDEHP is highly dependent upon
the acidity of the aqueous phase, and the distribution coefficients of lanthanides substantially increase with Z and the concentration of the nitric acid (See Figure 1) because the changes
in the stability constant with Z. Larger stability constant for the
complex Ln[H(DEHP)2]3, results in a larger concentration of
extractable species in the aqueous phase and consequently a
larger distribution coefficient. Therefore, the order of elution
of consecutive lanthanides is proportional to Z: Nd is eluted
first Pm, Gd first Tb, etc. (see Figure 3). This order favors the
separation between micro-amounts of the daughter radioisotope from the macro-amounts of lanthanide target because the
chromatographic column retains the micro-component while
the macro-component is in the meantime eluted away. However, when a stationary phase is loaded with macro-amounts of
retained elements, variations of its retention features may often
occur. Distribution coefficients usually are referred to the extraction of micro or even tracer amounts of metals and it is
known that the behaviour of many extractants at such metal
levels can be quite different from that shown at high organic
phase loading conditions [18]. Thus, the distribution coefficients of Nd, Pm, Gd, Tb, Dy, Ho, Yb and Lu were determined
under the same conditions used in the separation of pairs: Nd/
Pm, Gd/Tb, Dy/Ho and Yb/Lu. These values are in accordance
with those reported by Horwitz et. al. for Pm, Gd, Tb and Lu,
using undiluted HDEHP on Celite at 50°C [19].
Distribution coefficients are “suitable” for chromatographic separations of lanthanides not only when they result in
an advantageous separation factor for the neighbor lanthanides, but also when they are not so low or high so as to prevent the separation of elements or their recovery into acceptable
eluent volumes [18]. Therefore, the values of the distribution
coefficients selected to desorb the daughter radioisotopes:
149Pm, 161Tb and 166Ho were less than 20 in order to recover it
at the smallest possible volume. In the case of 177Lu, the minimum value of Kd is about 100 as shown in Figure 1, for this
reason its eluate volume is higher than 50 mL.
As regards the mathematical function of alpha (See Table
2), it is important to note that: Exponential and polynomial
models were applied to alpha data in order to find the mathematical function that gives the best approximation to our re-
Separation of Micro-Macrocomponent Systems: 149Pm – Nd, 161Tb-Gd, 166Ho-Dy and 177Lu-Yb by Extraction Chromatography
sults. All alpha data fit with the exponential model presented
a SSR > 0.999, however a best approximation of Nd/Pm data
(SSR=1) were obtained in the polynomial model. This difference could be explained in terms of lanthanide contraction,
which causes a decrease in atomic and ionic size from La to
Lu. Therefore, the chemical properties of Nd and Pm (light
lanthanides) in aqueous solution vary slightly to those of Dy
to Lu (heavy lanthanides).
4. Conclusions
The method proposed in this work to separate micro-amounts of
149Pm, 161Tb, 166Ho and 177Lu, produced via a neutron capture
Ln , from macfollowed by β- decay: AZ Ln ( n,γ ) A+1Z Ln β − ,γ A+1
Z+1
ro-amounts of the irradiated target (Nd, Gd, Dy or Yb) is based
on extraction chromatographic using Ln SPS resin and HNO3
solutions as mobile phase.
The distribution coefficients of lanthanides in Ln SPS resin decrease with an increase in HNO3 solution concentration
and a diminution of the lanthanide atomic number at a fixed
HNO3 concentration. Thus, the parent lanthanides of the parent/daughter pairs: Nd/Pm, Gd/Tb, Dy/Ho and Yb/Lu are eluted at first during the chromatographic separation process.
The best separation performances of the parent/daughter
pairs: Nd/Pm, Gd/Tb, Dy/Ho and Yb/Lu were obtained with
high αDaughter/Parent values (>>1) at Kd values lower than 200,
using the following conditions: for Neodymium and Promethium use 0.18 and 1.5 M HNO3, for Gadolinium and Terbium
use 0.8 and 3 M HNO3, for Dysprosium and Holmium use 1.5
M HNO3 and for Ytterbium and Lutetium use 3.4 and 8 M
HNO3 respectively.
(
)
5. Experimental
The distribution coefficients (Kd) of Nd, Pm, Gd, Tb, Dy, Ho,
Yb and Lu in Ln SPS Eichrom resin from Eichrom Darien
were determined for dynamic conditions by radiotracer technique using 147Nd, 149Pm, 159Gd, 161Tb, 157Dy, 166Ho, 169Yb and
177Lu (See Table 6) .
Radiolanthanides were produced by irradiation of 10 mg
Nd2(NO)3, Gd2(NO)3, Dy2(NO)3 and Yb2(NO)3 in the TRIGA
MARK III Reactor of the National Institute of the Nuclear Research (ININ) in Mexico, to a neutron fluence rate of 1.6 x1012
n cm-2s 1. Neodymium and dysprosium salts were irradiated for
149
3 h, ytterbium for 20 min and gadolinium for 15 min. 147Nd,
159Gd, 157Dy and 169Yb were produced by neutron irradiation
of the nitrate salts, while 151Pm, 161Tb, 166Ho and 177Lu were
formed from radioactive nitrate salt decay after 14 h, 3 min, 16
h and 7 d respectively. The radioactive nitrate salts were dissolved in 300 µL of 0.15 mol/L HNO3, to generate 0.098 mol/L
solutions with a specific activity of 0.148 kBq/µL.
The Kd measurements were determined using glass chromatographic columns (12 mm × 70 mm) loaded with 2 g of Ln
spec resin (50-100 µm) from Eichrom Industries of Darien, IL
(USA) previously preconditioned with 0.15 mol/L HNO3. The
Ln spec resin is comprised of a solution of di(2-ethylhexyl)
orthophosphoric acid (HDEHP) (40% by weight) loaded onto
the inert polymeric absorbent (60% by weight) Amberchrom™
CG-71.
All experiments were performed in the elution mode process. Radiolanthanide solutions (~100 μL) were loaded on the
column which was then eluted with the selected medium,
HNO3 solutions at different concentrations. Elution profiles
were obtained by collecting each fraction measured under a
coaxial gamma detector HPGe (Canberra 7229P) connected to
a PC-multichannel analyzer (ACCUSSPECT-A, Canberra).
Gamma-ray spectra were analyzed using the gamma software
for “Genie 2000” Canberra Acquisition and Analysis with
fixed geometry and varied counting time between 200 and 500
seconds. Table 6 shows the corresponding photopeaks used to
calculate the Kd values for 147Nd, 151Pm, 159Gd, 161Tb, 157Dy,
166Ho, 169Yb and 177Lu [23].
The Kd values were calculated using the relation:
⎡ V − v
max
m
Kd = ⎢
⎢
vm
⎣
⎤ ⎛ ⎞
⎥*⎜ v m ⎟ (7)
⎥ ⎜⎝ v s ⎟⎠
⎦
where Vmax is the eluate volume to peak maximum or retention
volume, vm is the void volume or the volume of the mobile
phase, vs is the volume of the stationary phase.
The Kd values were used to determine the separation conditions of the Parent/Daughter pairs (Nd/Pm, Gb/Tb, Dy/Ho
and Yb/Lu) by calculating the separation factor or selectivity
(α), which is defined as the ratio of the distribution coefficients
of two adsorbed solutes measured under the same conditions:
α=
Kd D
Kd P
(8)
Table 6. Gamma energies of radiolanthanides used in Kd determination.
Parent isotope
Energy (KeV)
Half-life
Daugther isotope
Energy (KeV)
Half-life
147Nd
531.00
11.06 d
151Pm
340.08
1.18 d
18.56 h
161Tb
74.60
6.91 d
80.57
1.11 d
208.36
6.71 d
159Gd
363.56
157Dy
326.40
8.00 h
166Ho
169Yb
197.70
32.03 d
177Lu
150
J. Mex. Chem. Soc. 2015, 59(2)
where KdP and KdD are the Kd values of the parent and daughter element respectively.
The separation factor can be visualized as the distance between the apices of the two chromatographic peaks. High α
values indicate good separation power.
Acknowledgements
This work was supported by the CONACYT-SALUD-2004-C01-001. The authors indebted to the technical
staff of reactor Triga Mark III (Mexico): Maximiano Hernández, Wenceslao Nava, Margarito Alva, Braulio Ortega, Edgar
Herrera and Roberto Raya.
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Article
J. Mex. Chem. Soc. 2015, 59(2), 151-160
© 2015, Sociedad Química de México
ISSN 1870-249X
Synthesis of Fluorescent oligo(p-phenyleneethynylene) (OPE3)
via Sonogashira Reactions
Mariana Flores-Jarillo,1 Francisco Ayala-Mata,2 Gerardo Zepeda-Vallejo,2 Rosa Ángeles Vázquez-García,3
Gabriel Ramos-Ortiz,4 Miguel Ángel Méndez-Rojas,5 Oscar Rodolfo Suárez-Castillo,1 and Alejandro
Alvarez-Hernández1*
1 Área Académica
de Química, Universidad Autónoma del Estado de Hidalgo, Carr. Pachuca Tulancingo Km 4.5, Pachuca,
Hidalgo 42184, México
2 Departamento de Química Orgánica, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional,
Prol. Carpio y Plan de Ayala s/n, 11340 México, D.F., México
3 Área Académica de Ciencias de la Tierra y Materiales, Universidad Autónoma del Estado de Hidalgo, Carr. Pachuca
Tulancingo km 4.5, Pachuca, Hidalgo 42184, México
4 Centro de Investigaciones en Óptica. A.P. 1-948, León, México
5 Departamento de Química y Biología, Universidad de las Américas-Puebla, Ex-Hda de Sta. Catarina Mártir, A. P. 100,
Cholula 72820, Puebla, México
* Corresponding author: Alejandro Alvarez-Hernández, email: alvarez@uaeh.edu.mx, alalvareher@yahoo.com
Received February 16th, 2015; Accepted May 28th, 2015
Abstract. Sonogashira reactions of 4-(2,5-diiodobenzoyl)morpholine
and 4-(5-bromo-2-iodobenzoyl)morpholine with arylacetylenes catalyzed by Pd2(dba)3 in DMSO allowed preparation of fluorescent oligo(p-phenyleneethynylene)s (OPE3) with fluorescence quantum
yields up to 0.87. DMSO proved to be very efficient for this double
Sonogashira coupling in which other solvents failed.
Key words: Sonogashira coupling, oligo(p-phenyleneethynylene)s,
OPE3, fluorescence
Resumen. Las reacciones de Sonogashira de la 4-(2,5-diiodobenzoil)
morfolina y la 4-(5-bromo-2-iodobenzoil)morfolina con arilacetilenos catalizadas por Pd2(dba)3 en DMSO permitieron preparar oligo(p-fenilenetinilenos) (OPE3) fluorescentes con rendimientos
cuánticos de hasta 0.87. El DMSO demostró ser muy eficiente para
este doble acoplamiento de Sonogashira en el cual otros disolventes
no fueron adecuados.
Palabras clave: Acoplamiento de Sonogashira, oligo(p-fenilenetinilenos), (OPE3), fluorescencia
Introduction
polyphenylenethynylenes [13]. Substituents on the terminal
aryl groups allow tuning of the optical properties and the morpholine amide group at the central aryl unit was designed to
confer solubility to phenyleneethynylenes in organic solvents.
The synthesis of pseudo symmetrical compounds was envisaged as a double Sonogashira coupling of diiodoamide 2 with
two equivalents of an arylacetylene 3. (Scheme 1)
Thus, synthesis of the required diiodoarene 2 started with
iodination of commercial 2-iodobenzoic acid 4 with iodine/
NaIO4 [14] in sulfuric acid that led to diiodo acid 5, which was
converted to 2 by reaction with thionyl chloride followed by
addition of morpholine and catalytic DMAP (Scheme 2).
Coupling of diiodoarene 2 with two equivalents of 4-ethynylanisole (3a) to produce 1a was chosen as model reaction to
establish a protocol for preparation of a set of related OPE3s 1.
The Sonogashira [1] coupling reaction and its variants [2] have
found widespread use in the synthesis of oligomeric arylenethynylene materials [3] These conjugated materials incorporate
aryl, heteroaryl [4] and organometallic [5] units joined by acetylene links and display remarkable electronic and optical properties [6], such as semiconduction and fluorescence, for which
they have been used to construct molecular wires [7], chemosensors [8], imaging materials [9] and the emitting layer in
light emitting devices [10]. Short oligomers [11] and small
molecules [12] of precise structure can retain some of the electronic and optical properties of their polymeric counterparts
and are more easily manipulated and prepared. Thus, in this
work a series of oligo(p-phenyleneethynylene)s OPE3s (Fig.
1) was prepared in search for highly fluorescent molecules of
low molecular weight for future adaptation and application as
biological probes. Herewith the synthesis of these compounds
by double Sonogashira couplings in DMSO and their optical
properties are described.
At the onset of this work, OPE3 compounds of type 1
were designed as low molecular weight models of fluorescent
Fig. 1. Structure of oligo(p-phenyleneethynylene)s (OPE3)
152
J. Mex. Chem. Soc. 2015, 59(2)
Mariana Flores-Jarillo et al.
Scheme 1
Scheme 2
Scheme 3
Unexpectedly, the reaction proved to be problematic under
standard Sonogashira conditions (PdCl2, CuI and triphenylphosphine) in toluene, THF and triethylamine (Scheme 3).
Thus, it was decided to study the possible effect of other
solvents in this Sonogashira coupling. Thus, coupling of 2 and
3a was carried out using several organic solvents of different
polarity [15] (Scheme 4) and Pd2(dba)3 [16] was used as the
source of catalyst. Results are summarized in Table 1. Reaction
in Et3N (entry 1) gave a mixture of OPE3 1a and monoalkyne
6a, whose structure was elucidated by HMBC and X-ray diffraction studies. Reactions in toluene (entry 2) and THF (entry
3) were sluggish. Coupling in anhydrous DMF (entry 4) gave
products 1a (double coupling) and 6a (monocoupling) in almost equal yields. Remarkably, reaction in anhydrous DMSO
(entry 5) proceeded in 1 h and gave exclusively the desired
double-coupled product 1a in good yield. Couplings in acetonitrile (entry 6) and nitromethane (entry 7) yielded mixtures of
mono (minor) and double coupling (major) products. Ulven
and coworkers [17] have reported the beneficial effect of addition of water to TMEDA used as solvent in a case of a problematic Sonogashira coupling. Accordingly, the use of a 9:1
TMEDA-water mixture was tested (entry 8). However, this
reaction gave a mixture of coupling products 1a, 6a and 7a in
low yield. Thus, of the solvents studied only DMSO was effective for the model double Sonogashira coupling.
It was explored if adventitious water in DMSO represented any problem in these Sonogashira couplings since this sol-
Table 1. Effect of the solvent in the model Sonogashira coupling of diiodoarene 2 and 3a according to Scheme 4.
Entry
Solventa
Polarityb
1ª(%)
6a(%)
7a(%)
2(%)
1
TEA
32.1
56
24
0
0
2
dry toluene
33.9
0
0
0
d
3
dry THF
37.4
d
d
0
d
4
DMF
43.2
48
34
0
0
5
DMSO
45.1
81
0
0
0
6
CH3CN
45.6
73
8
0
0
7
CH3NO2
46.3
52
21
0
0
8
9:1 TMEDA-H2O
--
48
(14)e
e
25
a Anhydrous
solvents (DMF 0.150% H2O; DMSO 0.013% H2O, by Karl Fisher determination) were used as received. TEA, THF and toluene
were dried according to standard procedures.
b Polarity values of solvents is based in E (30) scale. Ref. 15.
T
c Yields refer to isolated products. Identity of the coupling product 6a is supported by HMBC and X-ray diffraction.
d Compound detected by TLC
e Inseparable 2:1 mixture (NMR) of isomers 6a and 7a isolated in 14% yield.
153
Synthesis of Fluorescent oligo(p-phenyleneethynylene) (OPE3) via Sonogashira Reactions
Scheme 4
Scheme 5
vent is hygroscopic and also known for remarkable changes of
its properties in the presence of water [18]. Consequently,
model experiments in DMSO containing 5, 33 and 67% of water were carried out (Scheme 5) and their results are shown in
Table 2. While coupling of 2 and 3a in commercial “anhydrous
DMSO” (0.013% H2O) gave 81% of 1a (Table 2, entry 1) the
same reaction in DMSO containing 5% water led to a 10%
improved yield (entry 2; both coupling reactions in dry and wet
DMSO were repeated at least three times). The reaction in
DMSO containing 33% water led to a significant decrease in
yield of 1a and formation of monocoupled 6a as the major
product (entry 3). Reaction in 33% DMSO - 67% H2O (entry
4) led to reactant solubility problems and gave almost equal
amounts of mono (6a) and double (1a) coupled products. In
addition, Sonogashira couplings with other arylacetylenes
were tested in 5:95 water-DMSO (entries 5-10). The results
show that couplings using arylacetylenes substituted with electron donating groups OCH3 (entry 1 vs. entry 2) and Me2N
(entry 5 vs. entry 6) improved their chemical yield by about
10% in 5% water-DMSO vs. reaction in dry DMSO. However,
water had little effect in chemical yield of coupling reactions
with arylacetylenes substituted with electron withdrawing
groups such as CN (entries 7 and 8), NO2 (entries 9 and 10)
and methylketone (entries 11 and 12).
The observed solvent effect in these coupling reactions
could be due to coordination of DMSO with Pd. The coordinating ability of DMSO towards Pd is known [19] and it has been
associated to keep catalytically active Pd° in solution, thus
avoiding formation of black palladium which is inactive as catalyst. Other examples of Pd catalyzed alkynylation reactions in
Table 2. Effect of DMSO containing different amounts of water in the Sonogashira reactions of 2 and arylalkynes according to Scheme 5.
Entry
Alkyne
R
Water
[%]
1
[%]
6
[%]
Total yield [%]
1
3a
4-MeO-C6H4
0*
1a
81
6a
0
81
2
3a
4-MeO-C6H4
5
1a
91
6a
0
91
3
3a
4-MeO-C6H4
33
1a
24
6a
64
88
4
3a
4-MeO-C6H4
67
1a
47
6a
42
89
5
3f
4-Me2N-C6H4
0
1f
83
6f
0
83
6
3f
4-Me2N-C6H4
5
1f
94
6f
0
94
7
3g
4-NC-C6H4
0
1g
80
6g
0
80
8
3g
4-NC-C6H4
5
1g
79
6g
0
79
9
3h
4-O2N-C6H4
0
1h
80
6h
0
80
10
3h
4-O2N-C6H4
5
1h
78
6h
0
78
11
3i
4-MeC(O)-C6H4
0
1i
71
6i
0
71
12
3i
4-MeC(O)-C6H4
5
1i
67
6i
0
67
* “Dry” DMSO contained 0.013% water, as determined by Karl-Fischer titration.
154
J. Mex. Chem. Soc. 2015, 59(2)
Mariana Flores-Jarillo et al.
DMSO are known [20] Studies of some other reactions involving the use of Pd and DMSO show the active role of this solvent
in catalysis [21]. Studies of solvent dependence in cases of problematic Sonogashira couplings are rather limited [2d, 22] as
compared to the more common practice of an exhaustive ligand/
catalyst search, in spite of a more practical change of solvent.
With reaction conditions established, a series of OPE3
products 1a-i were prepared in good yields (Scheme 6, Table
3). Despite the known acceleration of Sonogashira cross-couplings with alkynes substituted with electron withdrawing
groups [23] couplings in DMSO worked equally well within 1
h with aryl acetylenes substituted either with strong electron
Scheme 6
Table 3. Synthesis of OPE3s 1 by Sonogashira couplings in dry
DMSO via Scheme 6.
Entry
Alkyne
R
Product
Yield [%]
1
3a
4-MeO(C6H4)
1a
81
2
3b
C6H5
1b
60
donating (entry 5), electron withdrawing groups (entries 6-8)
or heterocyclic acetylenes (entries 3 and 4). Compounds 1a-i
were soluble in common organic solvents. 1H NMR spectra of
all these compounds indicated high mobility of the morpholine
moiety in solution.
At this stage it was decided to prepare OPE3s substituted
with both electron donating groups and electron withdrawing
groups in a push-pull electronic architecture, a common feature
found in optoelectronic materials [3-5]. Thus, it was required to
sequentially install different arylacetylene groups in dihaloarene
9 by means of a regioselective Sonogashira coupling at the more
reactive C-I bond [1, 2d, 24]. The required dihaloarene 9 was in
turn prepared by bromination [25] of acid 4 followed by treatment with thionyl chloride and morpholine (Scheme 7).
Sonogashira reactions of 9 in DMSO with aryl acetylenes
3a,f-h were carried out for preparation of mono coupled products 10a,f-h. (Scheme 8, Table 4). These reactions produced
the desired mono coupled products 10a,f-h along with minor
amounts of the undesired double coupled product 1a,f-h. Sonogashira coupling of 9 with arylacetylenes substituted with electron donating groups OCH3 (3a, entry 1) and NMe2 (3f, entry
2) clearly gave better results in terms of yield and selectivity
than those observed using arylacetylenes bearing electron
withdrawing groups CN (3g, entry 3) and NO2 (3h, entry 4).
Installation of the second arylacetylene group at the C-Br
bond of compounds 10a,f-h proved more difficult than expected,
but eventually was accomplished using reaction conditions reported by Buchwald [26] to afford compounds 1j-p. (Scheme 9)
3
3c
3-Piridyl
1c
86
4
3d
3-Thiophenyl
1d
90
5
3e
4-Me(C6H4)
1e
70
6
3f
4-Me2N(C6H4)
1f
83
Entry
Alkyne
R1
9(%)
10
%
1
%
1
3a
4-MeO-C6H4
0
10a
73
1a
15
3f
4-Me2NC6H4
0
10f
72
1f
10
7
3g
4-NC(C6H4)
1g
80
8
3h
4-O2N(C6H4)
1h
80
9
3i
4-MeC(O)(C6H4)
1i
71
Table 4. Regioselective Sonogashira cross coupling at the C-I bond in
compound 10 via Scheme 8.
2
Reaction conditions: 5% Pd2(dba)3, 6% PPh3, 3% CuI, iPr2NH 2.5
eq, DMSO, 45°C, N2 atm, 1h.
Scheme 7
Scheme 8
3
3g
4-NC-C6H4
20
10g
43
1g
22
4
3h
4-O2N-C6H4
16
10h
42
1h
20
155
Synthesis of Fluorescent oligo(p-phenyleneethynylene) (OPE3) via Sonogashira Reactions
Scheme 9
The low reactivity of substrates 10a and 10f (see Table 5) can
be explained by the enhanced electron density at the C-Br bond
due to the strong electron donating groups (OMe and NMe2)
on the arylacetylene unit which likely made worse the Pd(0)
oxidative addition step of the coupling reaction [1]. By contrast, bromoarene 10h bearing a nitro substituted arylacetylene
(entry 7) gave compound 1p in higher yield.
With compounds 1a-p at hand, their photoluminescence
properties were studied and results are shown in Table 6. Several compounds including the parent phenyleneethynylene 1b,
methyl substituted 1e, and heterocyclic 3-pyridyl 1c and
3-thiophenyl analogs 1d display almost identical UV maxima
absorbance. The rest of the compounds show bathochromic
(red) shifts. All compounds are UV active and their ε values
indicate intense absorption bands due to π-π* transitions.
Compounds 1h, n, p containing one or two NO2 groups and the
diketone compound 1i did not show fluorescence. All other
compounds emit fluorescence in the 352-505 nm (violet to
green) range. Compounds 1l and 1o, substituted with the electron withdrawing keto group, show modest fluorescence (entries 11 and 14).
Compounds with equal terminal aryl groups are listed in
entries 1 through 9. Among them, compounds 1f with electron
donating NMe2 substituents (entry 6) and 1g with electron
withdrawing CN substituents (entry 7) displayed high quantum fluorescence yields, which were measured by Brouwer´s
method [27] On the other hand, compounds with push-pull architecture (entries 10-15) show intramolecular charge transfer,
as evidenced by large Stokes shifts which are associated to fluorescence quenching [28] However, cyano substituted compounds 1g (entry 7), 1k (entry 10) and 1m (entry 12) show
high quantum fluorescence yields; the cyano group is a common feature of molecular wires [13b]. Similar OPE3s with
high quantum yields are known [29]
In summary, we have prepared a series of soluble, fluorescent oligo phenyleneethynylenes OPE3, some of which display high quantum yields. Additionally, it was found that
Sonogashira couplings proceeded much better in DMSO without need for other additives, the chemical yields were good and
reactions took place within 1 h at 40-45°C. Couplings in
DMSO containing 5% water gave a small increment of the
yield of reactions of 2 with arylacetylenes substituted with
electron donating groups.
Acknowledgments
Financial support from Conacyt-México (grants 61247 and
221360 and a predoctoral fellowship to MFJ) is gratefully acknowledged.
Experimental
General
Commercial reagents were used without further purification.
Toluene was distilled from Na and stored over activated 4 Å
molecular sieves under N2. THF was refluxed in the presence
of triphenylphosphine then distilled and stored over activated
4-Å molecular sieves under N2. Reactions were monitored by
TLC on Merck silica gel F254 plates and spots were visualized
by a UV lamp at 254 and 365 nm. Column flash chromatography was performed using Whatman silica gel 60 (230-400
mesh). 1H and 13C NMR spectra were recorded on a Varian
VNMR System 400 MHz spectrometer using tetramethylsilane (TMS δ=0.0 ppm) and CDCl3 (δ = 77.16 ppm) as internal
standards; bs means broad signal, app d means apparent doublet in 1H NMR spectra. Infrared spectra were measured on a
Perkin-Elmer FT-IR Spectrum GX spectrometer. High resolution mass spectra were recorded in a Jeol Gcmate mass spectrometer using EI (70 eV) as ionization mode. UV spectra were
recorded on a Perkin-Elmer Lambda XLS spectrometer using
quartz cells. For UV and fluorescence determinations spectros-
Table 5. Preparation of push-pull OPE3s 1j-p via Scheme 9.
Entry
Arene 10
R1
Alkyne 3
1
10a
4-MeO-C6H4
3g
2
10a
4-MeO-C6H4
3h
3
10a
4-MeO-C6H4
3i
4
10f
4-Me2N-C6H4
3g
5
10f
4-Me2N-C6H4
3h
6
10f
4-Me2N-C6H4
3i
7
10h
4-O2N-C6H4
3a
R2
Product
Yield [%]
4-NC-C6H4
1j
80
4-O2N-C6H4
1k
0
4-MeC(O)-C6H4
1l
55
4-NC-C6H4
1m
36
4-O2N-C6H4
1n
35
4-MeC(O)-C6H4
1o
35
4-MeO-C6H4
1p
65
156
J. Mex. Chem. Soc. 2015, 59(2)
Mariana Flores-Jarillo et al.
Table 6. Optical characterization of OPE3 compounds 1a-o depicted in Schemes 6 and 9.
Entry data
a
λmaxa
(nm)
OPE3
R2
R1
1
1a
MeO
MeO
338
2
1b
H
H
325
3-pyr
326
λmxb
(nm)
Stokes
Shift
(nm)
Φc
5.50
389
51
0.475
5.56
375
50
0.103
5.57
352
26
0.121
ε(104)
cm-1 M-1
3
4
1d
3-thioph
3-thioph
326
4.54
380
54
0.077
5
1e
Me
Me
330
6.93
380
50
0.178
6
1f
Me2N
Me2N
385
6.58
441
56
0.792
7
1g
CN
CN
338
6.71
369
31
0.708
-
0
-
0
1c
3-pyr
d
e
8
1h
NO2
NO2
359
5.30
nd
9
1i
MeC(O)
MeC(O)
344
6.99
nd
10
1j
CN
OMe
341
5.52
406
65
0.874
11
1l
COMe
OMe
345
5.19
416
71
0.130
12
1m
CN
NMe2
388
4.65
495
157
0.786
13
1n
NO2
NMe2
328
3.09
nd
-
0
14
1o
COMe
NMe2
384
3.48
505
121
0.494
15
1p
OMe
NO2
364
4.63
nd
-
0
-6
UV absorption (10 M solution in CHCl3).
Fluorescence emission (10-6 M solution in CHCl3) with UV excitation at 10 nm below each maximum absorption.
c
Quantum fluorescence yield determined using quinine sulfate / H2SO4 as standard. Ref. 27
d
3-piridyl
e
3-thiophenyl
nd
No fluorescence emission was detected.
b
copy quality CHCl3 was used. Fluorescence spectra were recorded at room temperature on a Perkin Elmer LS 55
spectrofluorometer. Fluorescence quantum yields φ in CHCl3
are relative to quinine sulfate 1N in H2SO4 [27]. Melting points
were measured using a Büchi Melting Point B-540 apparatus
and are uncorrected.
4-(2,5-Diiodobenzoyl)morpholine (2) Iodination of
commercial 2-iodobenzoic acid 4 leading to 2,5-diiodobenzoic
acid (5) was carried out according to a reported method [14].
Thus, in a 100 mL round bottom flask containing 30 mL of
concentrated sulfuric acid were added 1.20 g of iodine and
0.34 g of NaIO4 at room temperature under vigorous magnetic
stirring. When the solids dissolved it was added 2.48 g of 2-iodobenzoic acid 4 and the mixture was left stirring at room temperature (23°C) for 48 h. Then, the crude reaction mixture was
poured on ice and the pink solid was filtered off. The solid was
dissolved in ethyl acetate and washed with a saturated sodium
thiosulfate solution. The organic layer was separated and dried
with anhydrous sodium sulfate, filtered and concentrated in
vacuo to afford 2.62 g (70%) of 5 as a white powder. Then,
1.68 g (4.5 mmol) of carboxylic acid 5 was suspended in 8 mL
of thionyl chloride and heated to reflux for 2 h under N2 and
allowed to cool to room temperature. Excess of thionyl chloride was removed in vacuo and the reaction mixture was diluted with 10 mL of anhydrous toluene. Catalytic DMAP and
morpholine (1.6 mL, 18.0 mmol) were added under stirring at
room temperature. After completion of the reaction (15 min),
indicated by TLC analysis, the mixture was washed with 3%
HCl solution, then with saturated NaHCO3 solution and extracted with ethyl acetate. The organic layer was dried with
anhydrous Na2SO4 and concentrated to yield a yellow syrup
that precipitated upon addition of hexane. Recrystallization
from toluene-hexane afforded 1.40 g of 2 (70%) as an offwhite powder, mp 145-146 °C; IR (KBr): λ 1600 (νN-C=O),
1700 (νC=O) cm–1; 1H NMR (400 MHz, CDCl3): δ 7.53 (d, J=
8.34, 1H), 7.50 (d, J= 1.99, 1H), 7.39 (dd, J=8.33, J=2.04, 1H),
3.9-3.1 (bs, 8H); 13C NMR (100 MHz, CDCl3): δ 167.8, 143.8,
140.9, 139.5, 135.9, 94.2, 91.8, 66.8, 66.6, 47.4, 42.1; HRMS
(EI) m/z calcd. for C11H11I2NO2: 442.8880, found: 442.8886.
General procedure for double Sonogashira cross-coupling
reactions of 4-(2,5-diiodobenzoyl)morpholine 2 with
arylacetylenes 3a-i in DMSO
A mixture of 4-(2,5-diiodobenzoyl)morpholine 2 (177.2 mg,
0.40 mmol), alkynes 3a-i (0.84 mmol), Pd2(dba)3 (10.4 mg, 10
μmol), CuI (2.3 mg, 12 μmol), triphenylphosphine (6.3 mg,
24 μmol) and dry DMSO (5 mL) under N2, was degassed. iPr2NH (140 μL, 1 mmol) was added and then the mixture was
heated at 45 °C (temperature of the external bath) under vigorous stirring. TLC analysis showed completion of the reaction
after 1 h. After aqueous work-up and extraction with ethyl acetate the residue was adsorbed on silica gel and purified by
flash chromatography (elution with hexane/EtOAc gradient).
Synthesis of Fluorescent oligo(p-phenyleneethynylene) (OPE3) via Sonogashira Reactions
Evaporation of solvent gave a solid residue which was triturated with hexane/acetone to afford the desired pure product 1a-i.
The same procedure was followed for those reactions carried out in DMSO containing 5%, 33% and 67% of water (v/v)
shown in Table 3.
2,5-Bis((4-ethoxyphenyl)ethynyl)morpholinebenzamide (1a) Obtained in 81% yield from the reaction of
2,5-diiodo-N-morpholinebenzamide (2) with 4-ethynylanisole
(3a) as a white powder, mp 149-151 °C. IR (KBr,): λ 1604 (νN-1 1
C=O), 1633 (νC=O), 2213 (νCsp-Csp) cm ; H NMR (400 MHz,
CDCl3): δ 7.50-7.41 (m, 7H), 6.89 (app d, 4H), 3.90-3.25 (bs,
m, 8H), 3.84 (s, 6H); 13C NMR (100 MHz, CDCl3): δ 168.3,
160.2, 160.1, 138.5, 133.32, 133.26, 132.0, 131.8, 129.6,
124.0, 119.9, 114.9, 114.7, 114.3, 114.2, 95.0, 92.4, 87.3, 85.6,
67.1, 67.0, 55.50, 55.48, 47.5, 42.3; HRMS (EI) m/z calcd. for
C29H25NO4: 451.1784, found: 451.1765.
2,5-Bis(phenylethynyl)morpholinebenzamide (1b) Prepared in 60% yield by the reaction of 2,5-diiodo-N-morpholinebenzamide (2) with phenylacetylene (3b) as yellow syrup
that solidifies upon standing. IR (film): λ 1599 (νN-C=O), 1639
(νC=O), 2216 (νCsp-Csp) cm-1; 1H NMR (400 MHz, CDCl3): δ
7.55-7.47 (m, 7H), 7.38-7.34 (m, 6H), 3.95-3.20 (bs, m, 8H);
13C NMR (100 MHz, CDCl ): δ 168.0, 138.7, 132.2, 132.0,
3
131.8, 131.7, 129.7, 129.1, 128.9, 128.6, 128.5, 124.0, 122.7,
122.5, 119.8, 94.9, 92.4, 88.3, 86.6, 67.0, 66.9, 47.5, 42.2;
HRMS (EI) m/z calcd. for C27H21NO2: 391.1572, found:
391.1589.
2,5-Bis(3-pyridinylethynyl)morpholinebenzamide (1c)
Prepared in 86% yield by the reaction 2,5-diiodo-N-morpholinebenzamide (2) with 3-ethynylpyridine (3c) as an off-white
powder, mp 157.7-158.2 °C. IR (KBr): λ 1613 (νC=O), 2211
(νCsp-Csp) cm-1 ; 1H NMR (400 MHz, CDCl3): δ 8.70 (bs, app d,
2H), 7.80 (app t, 3H), 7.61-7.51 (m, 4H), 7.33 (bs, s, 2H), 3.903.25 (bs, m, 8H); 13C NMR (100 MHz, CDCl3): δ 167.6, 152.4,
152.3, 149.4, 149.2, 139.0, 138.6, 138.5, 132.5, 132.1, 129.8,
123.8, 119.7, 123.25, 123.34, 91.5, 91.3, 89.6, 89.2, 67.0, 66.9,
47.5, 42.3; HRMS (EI) m/z calcd. for C25H19N3O2: 393.1477,
found: 393.1482.
2,5-Bis(3-thiophenylethynyl)morpholinebenzamide
(1d) Prepared in 90% yield by the reaction 2,5-diiodo-N-morpholinebenzamide (2) with 3-ethynylthiophene (3d) as a brown
powder, mp 156.0-156.5 °C. IR (KBr): λ 1633 (νC=O), 2207
(νCsp-Csp) cm-1; 1H NMR (400 MHz, CDCl3): δ 7.55 (dd, J=2.97
Hz, J= 1.18 Hz, 1H), 7.53 (dd, J=2.98 Hz, J= 1.17 Hz, 1H),
7.51-7.49 (m, 2H), 7.49-7.48 (app d, 1H), 7.33 (t, J= 2.93, 1H),
7.32 (t, J= 2.93, 1H) , 7.19 (dd, J=5.02 Hz, J= 1.18 Hz, 1H),
7.16 (dd, J=4.97 Hz, J= 1.18 Hz, 1H), 3.90-3.25 (bs, m, 8H);
13C NMR (100 MHz, CDCl ): δ 167.9, 138.5, 132.0, 131.8,
3
129.7, 129.6, 129.53, 129.46, 129.3, 125.6, 123.8, 121.7,
121.5, 119.6, 89.9, 87.8, 87.5, 86.0, 66.9, 66.8, 47.3, 42.1;
HRMS (EI) m/z calcd. for C23H17NO2S2: 403.0701, found:
403.0706.
2,5-Bis(p-tolylethynyl)morpholinebenzamide (1e) Prepared in 70% yield by the reaction 2,5-diiodo-N-morpholinebenzamide (2) with 4-methylphenylacetylene (3e) as a white
powder, mp 179.9-180.8 °C. IR (KBr): λ 1630 (νC=O), 2213
(νCsp-Csp) cm-1; 1H NMR (400 MHz, CDCl3): δ 7.52-7.48 (m,
157
3H), 7.42 (app d, 2H), 7.38 (app d, 2H), 7.17 (app d, 4H), 3.953.20 (bs, m, 8H), 2.38 (s, 6H); 13C NMR (100 MHz, CDCl3): δ
168.0, 139.2, 139.0, 138.5, 132.0, 131.8, 131.5, 131.5, 129.6,
129.3, 129.2, 123.9, 119.7, 119.5, 119.3, 95.0, 92.5, 87.7, 85.9,
66.9, 66.8, 47.3, 42.1; HRMS (EI) m/z calcd. for C29H25NO2:
419.1885, found: 419.1890.
2,5-Bis((4-(dimethylamino)phenyl)ethynyl)morpholinebenzamide (1f) Prepared in 83% yield by the reaction
2,5-diiodo-N-morpholinebenzamide (2) with 4-ethynyl-N,N-dimethylaniline (3f) as a yellow powder, mp 209.4-210.2 °C. IR
(KBr): λ 1607(nN-C=O), 1630 (νC=O), 2204 (νCsp-Csp) cm-1; 1H
NMR (400 MHz, CDCl3): δ 7.45 (s, 3H), 7.41-7.34 (m, 4H),
6.68-6.62 (m, 4H), 3.92-3.20 (bs, m, 8H), 3.00 (s, 12H); 13C
NMR (100 MHz, CDCl3): δ 168.6, 150.5, 150.4, 138.1, 133.0,
132.9, 131.7, 131.6, 129.4, 124.0, 119.9, 111.91, 111.89, 109.5,
109.2, 96.3, 93.6, 86.9, 85.2, 67.1, 67.0, 47.5, 42.2, 40.33,
40.31; HRMS (EI) m/z calcd. for C31H31N3O2: 477.2416,
found: 477.2432.
2,5-Bis((4-cyanophenyl)ethynyl)morpholinebenzamide (1g) Prepared in 80% by the reaction 2,5-diiodo-N-morpholinebenzamide (2) with 4-ethynylbenzonitrile
(3g) as a yellow powder, mp 228.0-230.0 °C (dec.) IR (KBr):
λ 1602 (nN-C=O), 1640 (νC=O), 2217 (νCsp-Csp), 2227 (νCsp-N)
cm-1; 1H NMR (400 MHz, CDCl3): δ 7.68 (app dd, 4H), 7.647.54 (m, 7H), 3.9-3.25 (bs, m, 8H); 13C NMR (100 MHz,
CDCl3): δ 167.3, 139.1, 132.5, 132.3, 132.3, 132.2, 132.1,
129.9, 127.3, 127.0, 123.7, 119.6, 118.3, 118.2, 112.4, 112.1,
93.1, 92.0, 90.9, 90.2, 66.9, 66.8, 47.4, 42.2; HRMS (EI) m/z
calcd. for C29H19N3O2: 441.1477, found: 441.1488.
2,5-Bis((4-nitrophenyl)ethynyl)morpholinebenzamide
(1h) Prepared in 80% yield by the reaction 2,5-diiodo-N-morpholinebenzamide (2) with 1-ethynyl-4-nitrobenzene (3h) as a
yellow powder, mp 204.8-205.7 °C. IR (KBr): λ 1347 and
1519 (νNO2), 1593 (νN-C=O), 1638 (νC=O), 2218 (νCsp-Csp), cm-1;
1H NMR (400 MHz, CDCl ): δ 8.26 (app d, 4H), 7.71-7.55 (m,
3
7H), 3.90-3.25 (bs, m, 8H); 13C NMR (100 MHz, CDCl3): δ
167.4, 147.6, 147.4, 139.3, 132.7, 132.6, 132.5, 132.3, 130.0,
129.4, 129.0, 124.0, 123.8, 119.7, 93.0, 92.9, 91.1, 90.8, 67.0,
66.9, 47.5, 42.3; HRMS (EI) m/z calcd. for C27H19N3O6:
481.1274, found: 481.1280.
2,5-Bis((4-acetophenyl)ethynyl)morpholinebenzamide
(1i) Prepared in 71% yield by the reaction 2,5-diiodo-N-morpholinebenzamide (2) with 4-ethynylacetophenone (3i) as a
green-yellow powder, mp 194.1-196.0 °C. IR (KBr): 1600 (νNC=O), 1630 (νC=O), 1677 (νC=O), 1690 (νC=O), 2208 (νCsp-Csp)
cm-1; 1H NMR (400 MHz, CDCl3): δ 7.95 (app d, 4H), 7.637.52 (m, 7H), 3.90-3.25 (bs, m, 8H), 2.61 (s, 6H); 13C NNMR
(100 MHz, CDCl3): δ 197.4, 197.3, 167.7139.1, 136.9, 136.7,
132.5, 132.2, 131.9, 131.9, 129.9, 128.6, 128.5, 127.4, 127.1,
123.8, 119.8, 94.2, 91.8, 91.2, 89.5, 67.0, 66.9, 47.5, 42.3,
26.8; HRMS (EI) m/z calcd. for C31H25NO4: 475.1784, found:
475.1800.
4-(5-Bromo-2-iodobenzoyl)morpholine (9) Bromination of commercial 2-iodobenzoic acid 4 leading to 5-bromo-2-iodobenzoic acid (8) was carried out according to a
reported method [25] Thus, in a 100 mL round bottom flask
containing 30 mL of concentrated sulfuric acid at 60 °C, was
158
J. Mex. Chem. Soc. 2015, 59(2)
added 2-iodobenzoic acid 4 (3.72 g, 15 mmol), then maintaining this temperature three portions of solid NBS (6 mmol)
were added every 15 min, for a total of 3.2 g (18 mmol) of
NBS. After the last addition, the mixture was left stirring for 1
h at 60°C, then left to cool to room temperature and poured
over crushed ice. The pink solid was filtered off and dissolved
in ethyl acetate and washed with a concentrated solution of
sodium thiosulfate. The organic portion was dried with anhydrous Na2CO3, filtered and concentrated in vacuo to afford
3.58 g (73%) of the carboxylic acid 8 as a white solid. Then,
1.471 g (4.5 mmol) of carboxylic acid 8 was suspended in 8
mL of thionyl chloride and heated to reflux for 2 h under N2
and allowed to cool to room temperature. Excess of thionyl
chloride was removed in vacuo and the reaction mixture was
diluted with 10 mL of anhydrous toluene. Catalytic DMAP and
morpholine (1.6 mL, 18.0 mmol) were added under stirring at
room temperature. After completion of the reaction (15 min),
indicated by TLC analysis, the mixture was washed with 3%
HCl solution, then with saturated NaHCO3 and extracted
with ethyl acetate. The organic layer was dried with anhydrous Na2SO4 and concentrated to yield a yellow syrup that
precipitated upon addition of hexane. Recrystallization from
toluene-hexane afforded 1.301 g of 10 (73%) as a white powder, mp 148 °C; IR (KBr) λ 1620 (νN-C=O) cm-1, 1H NMR (400
MHz, CDCl3): δ 7.68 (d, J= 8.44, 1H), 7.34 (d, J= 2.31, 1H),
7.22 (dd, J=8.43, J=2.35, 1H), 3.9-3.1 (bs, 8H); 13C NMR (100
MHz, CDCl3): δ167.92, 143.60, 140.76, 133.67, 130.15,
123.09, 90.55, 66.76, 66.62, 47.31, 42.12; HRMS (EI) m/z calcd. for C11H11BrINO2: 394.9018, found: 394.9020
General procedure for C-I regioselective Sonogashira
cross-coupling reactions of 9 with arylacetylenes. A mixture
of 9 (158.4 mg, 0.40 mmol), alkynes 3a,f-h (0.44 mmol),
Pd2(dba)3 (10.4 mg, 10 μmol), CuI (2.3 mg, 12 μmol), triphenylphosphine (6.3 mg, 24 mmol) and DMSO (5 mL) under N2,
was degassed. iPr2NH (140 μL, 1 mmol) was added and then
the mixture was heated at 45 °C (temperature of external bath)
under vigorous stirring for 1 h as TLC analysis indicated completion of the reaction. Aqueous work-up and extraction gave
the crude reaction mixture which was adsorbed on silica gel
and after flash chromatography (elution with hexane/EtOAc
gradient) the solid residue was washed with hexane/acetone to
afford the desired pure products 10a, f-h.
5-Bromo-2-((4-methoxyphenyl)ethynyl)morpholinebenzamide (10a) Prepared in 73% yield by the reaction of
5-bromo-2-iodo-N-morpholinebenzamide (9) with 4-ethynylanisole (6a) as a yellow powder, mp 138-139 °C. IR (KBr): λ
1600 (νN-C=O), 1692 (νC=O), 2212 (νCsp- Csp) cm-1; 1H NMR
(400 MHz, CDCl3): δ 7.52-7.47 (m, 2H), 7.44-7.37 (m, 3H),
6.88 (app d, 2H), 3.90-3.25 (bs, m, 8H), 3.84 (s, 3H); 13C NMR
(100 MHz, CDCl3): δ 167.2, 160.1, 139.7, 133.2, 133.1, 132.2,
129.7, 122.5, 119.5, 114.24, 114.19, 94.4, 84.6, 66.8, 66.7,
55.4, 47.3, 42.1; HRMS (EI) m/z calcd. for C20H18BrNO3:
399.0470, found: 399.0472.
5-Bromo-2-((4-(dimethylamino)phenyl)ethynyl)morpholinebenzamide (10f) Prepared in 72% yield by the reaction of 5-bromo-2-iodo-N-morpholinebenzamide (9) with
4-ethynyl-N,N-dimethylaniline (3f) as a yellow powder, mp
Mariana Flores-Jarillo et al.
123-124 °C. IR (KBr): λ 1604 (νN-C=O), 1688(νC=O), 2204
(νCsp- Csp) cm-1; 1H NMR (400 MHz, CDCl3): δ 7.49-7.45 (m,
2H), 7.38-7.32 (m, 3H), 6.64 (app d, 2H), 3.90-3.25 (bs, m,
8H), 3.00 (s, 6H); 13C NMR (100 MHz, CDCl3): d167.4, 150.4,
139.4, 133.0, 132.8, 132.1, 129.7, 121.7, 120.1, 111.7, 108.6,
96.0, 84.0, 66.8, 66.7, 47.3, 42.1, 40.1; HRMS (EI) m/z calcd.
for C21H21BrN2O2: 412.0786, found: 412.0770.
5-Bromo-2-((4-cyanophenyl)ethynyl)morpholine­
benzamide (10g) Prepared in 43% yield by the reaction of
5-bromo-2-iodo-N-morpholinebenzamide (9) and 4-ethynylbenzonitrile (3g) as a yellow powder, mp 237-238 °C (dec). IR
(KBr): λ 1602 (νN-C=O), 1687 (νC=O), 2224 (νCsp-N) cm-1; 1H
NMR (400 MHz, CDCl3): δ 7.66 (app d, 2H), 7.58-7.54 (m,
3H), 7.51 (app d, 1H), 7.44(dd, J=8.2 Hz J= 0.26 Hz, 1H),
3.90-3.25 (bs, m, 8H); 13C NMR (100 MHz, CDCl3): δ 166.9,
140.4, 133.8, 132.6, 132.4, 132.2, 130.0, 127.2, 124.2, 118.4,
118.3, 112.5, 92.2, 89.9, 67.0, 66.9, 47.5, 42.3; HRMS (EI) m/z
calcd. for C20H15BrN2O2: 394.0317, found: 394.0306.
5-Bromo-2-((4-nitrophenyl)ethynyl)morpholinebenzamide (10h) Prepared in 42% yield by the reaction of 5-bromo-2-iodo-N-morpholinebenzamide (9) with 1-ethynyl-4-nitrobenzene (3h) as a yellow powder, mp 229-230 °C (dec). IR
(KBr): λ 1347 and 1528 (νNO2), 1600 (νN-C=O), 1691 (νC=O),
2217 (νCsp- Csp) cm-1; 1H NMR (400 MHz, CDCl3): δ 8.23 (app
d, 2H), 7.62 (app d, 2H), 7.56 (dd, J=8.30 Hz J= 2.01 Hz, 1H),
7.52 (d, J= 1.97 Hz, 1H), 7.45 (d, J= 8.27 Hz, 1H), 3.90-3.25
(bs, m, 8H); 13C NMR (100 MHz, CDCl3): δ 166.7, 147.4,
140.3, 133.7, 132.5, 132.3, 129.8, 129.0, 124.2, 123.8, 118.0,
91.8, 90.6, 66.8, 66.8, 47.4, 42.2; HRMS (EI) m/z calcd. for
C19H15BrN2O4: 414.0215, found: 414.0227.
General procedure for Sonogashira cross-coupling reactions used to prepare 1j-p. Compounds 10a, 10f and 10h
were subjected to Sonogashira couplings at the C-Br bond with
arylacetylenes following conditions reported by Buchwald.26
Thus, a mixture of the bromoarene 10a,f-h (0.40 mmol, 1.0
equiv), arylalkynes 3a,g-1 (0.44 mmol, 1.1 equiv), PdCl2(CH3CN)2 (3.1 mg, 0.03 equiv, 12 mmol), XPhos (11.4 mg,
24 mmol, 0.06 equiv), Cs2CO3 (338.9 mg, 1.04 mmol, 2.6
equiv) and CH3CN (6 mL) under N2, was degassed. Then, the
mixture was heated at 75 °C (temperature of external bath) under vigorous stirring for 12 h. After this time, TLC analysis of
the reaction mixture indicated completion of the reaction.
Aqueous work-up and extraction with ethyl acetate (3 × 15
mL) gave the crude reaction mixture which was dried over anhydrous Na2CO3, filtered, and finally adsorbed on silica gel.
After flash chromatography (elution with hexane/EtOAc gradient) the solid residue was washed with hexane/acetone to
afford the desired pure products 1j-p.
5-((4-Cyanophenyl)ethynyl)-2-((4-methoxyphenyl)
ethynyl)morpholinebenzamide (1j) Prepared in 80% yield
by the reaction of 10a with 4-ethynylbenzonitrile (3g) as a yellow powder, mp 188.4-188.8 °C (dec). IR (KBr): λ 1605 (νN-1 1
C=O), 1625 (νC=O), 2205 (νCsp-Csp), 2225 (νCsp-N) cm ; H NMR
(400 MHz, CDCl3): δ 7.66 (app d, 2H), 7.60 (app d, 2H), 7.547.52 (m, 3H), 7.44 (app d, 2H), 6.90 (app d, 2H), 3.90-3.25 (bs,
m, 8H), 3.85 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 167.8,
160.2, 138.5, 133.2, 132.1, 131.98, 132.02, 132.0, 129.9,
Synthesis of Fluorescent oligo(p-phenyleneethynylene) (OPE3) via Sonogashira Reactions
127.6, 122.3, 121.1, 118.4, 114.2, 114.2, 111.9, 95.7, 92.4,
90.2, 85.2, 66.9, 66.8, 55.4, 47.3, 42.1; HRMS (EI) m/z calcd.
for C29H22N2O3: 446.1631, found: 446.1627.
5-((4-Acetophenyl)ethynyl)-2((4-methoxyphenyl)
ethynyl)morpholinebenzamide (1l) Prepared in 55% yield
from the reaction of 10a with 4-ethynylacetophenone (3i) as a
yellow powder, mp 182.0-182.5 °C. IR (KBr): λ 1605 (νN-C=O),
1627 (νC=O), 1677 (νC=O), 2208 (νCsp-Csp) cm-1; 1H NMR (400
MHz, CDCl3): δ 7.96 (app d, 2H), 7.60 (app d, 2H), 7.53 (s,
3H), 7.44 (app d, 2H), 6.90 (app d, 2H), 3.90-3.25 (bs, m, 8H),
3.85 (s, 3H), 2.63 (s, 3H); 13C NMR (100 MHz, CDCl3): δ
197.2, 167.9, 160.2, 138.5, 136.4, 133.2, 132.0, 131.9, 131.7,
129.8, 128.3, 127.5, 122.8, 120.8, 114.3, 114.2, 95.5, 91.4,
91.2, 85.3, 66.9, 66.8, 55.3, 47.3, 42.1, 26.7; HRMS (EI) m/z
calcd. for C30H25NO4: 463.1784, found: 463.1804.
5-((4-Cyanophenyl)ethynyl)-2-((4-(dimethylamino)
phenyl)ethynyl)morpholine benzamide (1m) Prepared in
36% yield by the reaction of 10f with 4-ethynylbenzonitrile
(3g) as a yellow powder, mp 210.7-212.1 °C. IR (KBr): λ 1593
(νN-C=O), 1633 (νC=O), 2213 (νCsp-Csp), 2223 (νCsp-N) cm-1; 1H
NMR (400 MHz, CDCl3): δ 7.65 (app d, 2H), 7.59 (app d, 2H),
7.50 (s, 3H), 7.36 (d app, 2H), 6.65 (app d, 2H), 3.95-3.20 (bs,
m, 8H), 3.01 (s, 6H); 13C NMR (100 MHz, CDCl3): δ 168.0,
150.5, 138.1, 132.9, 132.09, 132.07, 132.0, 131.7, 130.0,
127.7, 121.8, 121.6, 118.5, 111.7, 111.7h, 108.5, 97.5, 92.7,
89.9, 84.8, 66.9, 66.8, 47.3, 42.1, 40.1; HRMS (EI) m/z calcd.
for C30H25N3O2: 459.1947, found: 459.1968.
2-((4-(Dimethylamino)phenyl)ethynyl)-5-((4-nitrophenyl)ethynyl)morpholine benzamide (1n) Prepared in 35%
yield by the reaction of 10f with 1-ethynyl-4-nitro benzene
(3h) as a red powder, mp 227.3 - 227.9 °C. IR (KBr): λ 1339
and 1512 (νNO2), 1589 (νN-C=O), 1624 (νC=O), 2205 (νCsp-Csp)
cm-1; 1H NMR (400 MHz, CDCl3): δ 8.21 (app d, 2H), 7.64
(app d, 2H), 7.53-7.49 (m, 3H), 7.36 (app d, 2H), 6.64 (app d,
2H), 3.95-3.20 (bs, m, 8H), 3.00 (s, 6H); 13C NMR (100 MHz,
CDCl3): δ 167.9, 150.5, 147.1, 138.2, 132.9, 132.3, 132.0,
131.7, 130.0, 129.7, 123.7, 122.0, 121.4, 111.7, 108.5, 97.6,
93.6, 89.7, 84.8, 66.9, 66.8, 47.4, 42.1, 40.1; HRMS (EI) m/z
calcd. for C29H25N3O4: 479.1845, found: 479.1832.
5-((4-Acetophenyl)ethynyl)-2-((4-(dimethylamino)
phenyl)ethynyl)morpholine benzamide (1o) Prepared in
35% yield by the reaction of 10f with 4-ethynylacetophenone
(3i) as a yellow powder, mp 214.8-215.3 °C. IR (KBr): λ 1592
(νN-C=O), 1641 (νC=O), 1677 (νC=O), 2199 (νCsp-Csp) cm-1; 1H
NMR (400 MHz, CDCl3): δ 7.94 (app d, 2H), 7.59 (app d, 2H),
7.52-7.47 (m, 3H), 7.35 (app d, 2H), 6.64 (app d, 2H), 3.953.20 (bs, m, 8H), 2.99 (s, 6H), 2.60 (s, 3H); 13C NMR (100
MHz, CDCl3): δ 197.3, 168.0, 150.5, 138.1, 136.4, 132.9,
131.9, 131.7, 131.6, 129.9, 128.3, 127.6, 122.0, 121.4, 111.7,
108.6, 97.2, 91.7, 90.9, 84.8, 66.9, 66.8, 47.3, 42.1, 40.1, 26.7;
HRMS (EI) m/z calcd. for C31H28N2O3: 476.2100, found:
476.2130.
5-((4-Methoxyphenyl)ethynyl)-2-((4-nitrophenyl)
ethynyl)morpholinebenzamide (1p) Prepared in 65% yield
by the reaction of 10h and 4-ethynylanisole (3a) as a yellow
powder, mp 194.3-194.5 °C. IR (KBr): λ 1340 y 1513 (νNO2),
1590 (νN-C=O), 1636 (νC=O), 2209 (νCsp-Csp), cm-1; 1H NMR
159
(400 MHz, CDCl3): δ 8.24 (app d, 2H), 7.63 (app d, 2H), 7.587.50 (m, 3H), 7.47 (app d, 2H), 6.90 (app d, 2H), 3.90-3.25 (bs,
m, 8H), 3.84 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 167.8,
160.3, 147.4, 139.1, 133.4, 132.6, 132.4, 131.9, 129.5, 129.4,
125.6, 124.0, 118.2, 114.5, 114.3, 93.5, 92.4, 91.7, 87.0, 67.1,
67.0, 55.5, 47.5, 42.3; HRMS (EI) m/z calcd. for C28H22N2O5:
466.1529, found: 466.1512.
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Article
J. Mex. Chem. Soc. 2015, 59(2), 161-171
© 2015, Sociedad Química de México
ISSN 1870-249X
Enantioselective Synthesis of Isoxazolecarboxamides and their Fungicidal
Activity
Mirosław Gucma, W. Marek Gołębiewski and Alicja K. Michalczyk
Institute of Industrial Organic Chemistry Annopol 6, 03-236 Warsaw, Poland. Telephone: +(48) 22-8111231; golebiewski@ipo.
waw.pl
Received February 10th, 2015; Accepted June 10th, 2015
Abstract. A series of new 3-substituted isoxazolecarboxamides have
been prepared from aldehydes. The key step was a 1,3-dipolar cycloaddition reaction of nitrile oxides to a,b-unsaturated esters and amides. The cycloadditions to amides were mediated by chiral ligands
and several products displayed excellent enantioselectivities. Some of
the title compounds exhibited good fungicidal activities against Alternaria alternata, Botrytis cinerea, Fusarium culmorum, Phytophthora
cactorum, and Rhizoctonia solani strains.
Key words: cycloaddition, isoxazole derivatives, regioselectivity, enantioselectivity, fungicides.
Resumen. Una serie de nuevas isoxazolcarboxamidas 3-sustituidas se prepararon a partir de aldehídos. El paso clave fue la reacción de cicloadición
dipolar-1,3 de óxidos de nitrilo con ésteres o amidas a,b-insaturados. Las
cicloadiciones con amidas fueron llevadas a cabo en presencia de ligantes
quirales, y varios productos mostraron excelentes enantioselectividades.
Algunos de los compuestos preparados mostraron buena actividad fungicida contra las cepas de Alternaria alternata, Botrytis cinerea, Fusarium
culmorum, Phytophthora cactorum y Rhizoctonia solani.
Palabras clave: cicloadición, derivados de isoxazol, regioselectividad, enantioselectividad, fungicidas.
Introduction
Results and Discussion
Biological activity of carboxamides is known for a long time.
Fungicidal activity of some amides (pyridinecarboxamides
and benzamides) results from disrupting the succinate dehydrogenase complex in the respiratory electron transport chain
[1,2a], inhibition of rybosomic RNA synthesis (acylalanines
such as metalaxyl, oxazolidinones such as oxadixyl) [2a] and
targeting cellulose synthases [2b]. Herbicidal activity is presumably due to inhibiting phytoenone desaturase, enzyme involved in biosynthesis of carotenoids [3]. Simple derivatives
of isoxazole show also biological activity: 3-hydroxy-5-methylisoxazole disturbs RNA metabolism of fungi [4]. Fast appearance of resistance in pathogenic organisms and climatic
changes result in a continuous quest for new plant protective
agents which would exhibit a selective activity against pests
and apropriate durability in the environment. There is a growing interest in agrochemistry to use pure optical isomers since
the desired biological activity occurs generally only in one of
the enantiomers. Application of single enantiomers induced by
legislative, environmental and commercial factors brings several benefits, such as a decrease of environmental pollution,
elimination of useless or even detrimental activity of the undesired antipode and reduced costs of raw materials, labor, and
effluent treatment. Those facts induced us to synthesize several 3-aryl- and 3-alkylisoxazolecarboxamides showing fungicidal activity [5,6]. In continuation of these studies we have
prepared a number of new 3-aryl (alkyl)isoxazolecarboxamides and examined their activity against Alternaria alternate,
Botrytis cinerea, Rhizoctonia solani, Fusarium culmorum, and
Phytophthora cactorum fungal strains.
The title compounds have been prepared by two methods. Following the first method, fifteen 3-arylisoxazole-(3-aryl-2-isoxazoline-)-5-carboxamides 6-8 were synthesized from
arylaldehydes 1 (Scheme 1, Table 1) via carboxylates 4. Then
three 3-t-butyl-2-isoxazoline-5-carboxamides 9-11 were similarly obtained (Fig. 1). In the second method, fourteen 3-arylcarboxamides 14-15 were prepared by enantioselective
1,3-dipolar cycloadditon reaction of benzonitrile oxides 12a-c
and unsaturated amides 13a-h (Scheme 2).
Preparation of isoxazolecarboxamides 6a-f and
8a-i
The isoxazolecarboxamides listed at Scheme 1 and in Table 1
were prepared in a few steps starting from aldehydes which
were oximated with hydroxylamine. E-configuration of the oximes was established based on chemical shift values of HC=N
proton in 1H NMR spectra above 8.0 ppm [7-9]. The next step
was chlorination of oximes with NCS in DMF [10]. The key
step was a 1,3-dipolar cycloaddition reaction of ethyl acrylate
or a,b-unsaturated amides and nitrile oxides generated in situ
in the presence of triethylamine (Huisgen method) [11] or on a
basic Amberlyst A-21 column in the case of the cycloaddition
reaction of a,b-unsaturated amides 13a-h, which lead to cycloadducts 14-15 [12] (Scheme 2). The reaction with ethyl acrylate
showed
high
regioselectivity
and
only
3-aryl-2-isoxazoline-5-carboxylates 4 were isolated. Some
3-aryl-2-isoxazolinecarboxylates were dehydrated to give the
162
J. Mex. Chem. Soc. 2015, 59(2)
Mirosław Gucma et al.
corresponding isoxazoles 7 using N-bromosuccinimide bromination, followed by potassium acetate-promoted dehydrobromination [13]. The title amides were synthesized by reaction of
acid chlorides prepared by saponification of the corresponding
esters, reaction of the obtained acids with oxalyl chloride, followed by acylation of the aromatic, heterocyclic or alkyl
amines in the presence of tertiary amines (Method A1 and A2,
see experimental part). In the case of weakly nucleophilic aromatic amines, the amidation process was carried out by activation of amines with n-butyllithium in diethyl ether (Method
B1, see experimental part) or by formation of lithium amides
with t-butyllithium (Method B2, see experimental part) to
avoid formation of side products due to degradation of the acid
chlorides [14] and to increase yields of the amides.
Fig. 1
2-Isoxazolinecarboxamides 9-11 (Fig. 1) were similarly prepared starting from trimethylacetaldehyde via a cycloaddition of the corresponding nitrile oxide with ethyl acrylate and
acylation of an amine with the prepared 2-isoxazoline-5-carboxylic acid chloride (a description is provided in the experimental part).
Enantioselective cycloaddition reactions of
nitrile oxides to amides
In another approach to isoxazolinecarboxamides, we examined the cycloadditon reaction of benzonitrile oxides to acrylamides with application of chiral ligands (+)-(4,6-benzylidene)
Scheme 1
163
Enantioselective synthesis of isoxazolecarboxamides and their fungicidal activity
Table 1. Synthesized amides 6-8
Compd
No.
NR
6a
NHCH2CH2CH2Br
R1
R2
R3
R4
R5
Yield
[%]
Method
Cl
Cl
H
H
Cl
15
A1
Cl
Cl
H
H
Cl
38
A2
Cl
Cl
H
H
Cl
75
A2
Cl
Cl
H
H
Cl
27
B2
Cl
Cl
H
H
Cl
44
B2
F
H
F
F
H
42
B2
H
H
CF3
H
H
95
A1
H
H
CF3
H
H
30
A2
H
H
CF3
H
H
32
B2
H
H
CF3
H
H
14
B2
H
H
CF3
H
H
49
B2
H
H
CF3
H
H
44
A2
H
H
CF3
H
H
70
A2
H
H
CF3
H
H
77
B1
H
H
CF3
H
H
78
A2
Br
6b
NH
Br
6c
NH
F3CO
6d
NH
Br
F
NH
6e
N
Cl
F
Cl
F
NH
6f
F
N
Cl
F
CH(CH3)2
8a
N
8b
NH
CH(CH3)2
NH
Cl
8c
NO2
Cl
NH
8d
NO2
Cl
NH
8e
8f
NH
8i
N
NH CH2
8g
8h
CF3
O2N
O
O
H
N
N
O
164
J. Mex. Chem. Soc. 2015, 59(2)
Mirosław Gucma et al.
methyl-a-D-glucopyranoside (A), 1,2:5,6-di-O-isopropylidene- a-D-glucofuranose (B), 1,2:3,4-di-O-isopropylidene-a-D-galactopyranose
(C),
and
R-(+)-1,1’-bi-2-naphthol (D) (Fig. 2). We have tested before
application of chiral complexes to control regio- and enantioselectivity of the dipolar cycloaddition reaction of nitrile
oxides to crotonamides and cinnamides [15] as well as to unsaturated esters [16]. In this approach we describe the cycloadditions of benzonitrile oxides 12a-c to substituted
acrylamides 13a-h mediated by new complexes of chiral ligands A-D and Lewis acids, especially lanthanides (Table 2).
Structure-reactivity relationship
The reactivity of aromatic amides as dipolarofiles in the cycloaddition reaction of nitrile oxides depends on the nature of
the substituent and its position on the aromatic ring of the am-
Fig. 2.
Scheme 2.
Table 2. Enantioselective nitrile oxide cycloaddition reactions to amides 13a–h
Entry
14,15
R
R1
R2
Chiral catalyst
Yielda
(%)
14/15b
14
Reg.5
% ee
15
Reg.4
% ee
1
a
CF3
H
C6H5
-
30
100/0
-
-
2
b
CF3
H
C6H4-4-sec-Bu
-
40
100/0
-
-
3c
c
CF3
H
C6H4-4-OMe
-
71
100/0
-
-
4
c
CF3
H
C6H4-4-OMe
Yb(OTf)3-C
53
100/0
-
-
5
d
CF3
Me
C6H4-4-OMe
Yb(OTf)3-B
96
54/46
0.1
93.0
6
e
CF3
Me
C6H4-4-sec-Bu
Yb(OTf)3-A
43
2/1
2.0
18.0
7
f
CF3
Me
C6H4-2-OMe
Yb2O3-A
61
41/59
99.9
0.0
8
f
CF3
Me
C6H4-2-OMe
AlCl3-A
21
20/80
7.4
0.0
9
f
CF3
Me
C6H4-2-OMe
RuCl3-A
31
38/62
-
0.6
10
f
CF3
Me
C6H4-2-OMe
YbF3-A
54
37/63
5.0
0.4
11
f
CF3
Me
C6H4-2-OMe
La(OTf)3-A
80
70/30
2.4
0.6
12
g
i-Pr
Me
C6H4-4-OMe
-
29
20/1
-
-
13
h
H
Me
C6H4-2-OMe
Yb(OTf)3-D
65
1/1
96.0
5.0
14
h
H
Me
C6H4-2-OMe
Yb(OTf)3-A
85
1/1
87.0
0.6
15
h
H
Me
C6H4-2-OMe
CsF-C
60
1/1
81
0.1
16
-
CF3
Me
C6H4-3-OMe
-
0
-
-
-
-
-
-
17
a Isolated
CF3
Me
C6H4-4-CF3
0
yield of amides 14 and 15, b Regioisomer-5(14)/regioisomer-4(15), c Reaction described in [15]
Enantioselective synthesis of isoxazolecarboxamides and their fungicidal activity
ide fragment (R2, Scheme 2). Amides with electron donating
substituents (EDG) on the aromatic ring such as methoxy and
sec-butyl in the ortho or para position are the most reactive;
meta substituted amide (Table 2, entry 16) was unreactive. On
the other hand amides with electron withdrawing substituents
(EWG) on the aromatic ring, such as CF3, did not react in the
cycloaddition reactions as well.
Reactions of acrylamides 13a-c afforded as expected only
5-substituted regioisomers 14a-c. The rest of the cycloadditions gave mixtures of 4- and 5-substituted regioisomers (Table 2, entries 5-15). Good regioselectivity was observed in the
uncatalyzed reaction of 4-isopropylbenzonitrile oxide (Table
2, entry 12), where regioisomer-5 was favored (20:1), and in
the reaction mediated by a complex of aluminum chloride-carbohydrate A, where regioisomer-4 was favored (4:1) (entry 8).
Excellent enantioselectivities were achieved in reactions
mediated by complexes of ytterbium triflate with carbohydrates A, B and binaphthol D (Table 2, entries 5, 13, 14). Very
good enantioselectivities were also observed in cycloadditions
mediated by systems Yb2O3-A and CsF-C (Table 2, entries 7
and 15). The observed enantioselectivity of the reaction lead-
165
ing to (4S,5S)-5-carbamoyl derivatives could be explained by
binding of the amides to the chiral catalytic complex of e.g.
ytterbium triflate with R-BINOL, followed by a preferential
attack of nitrile oxide from lower si-face of the dipolarophile
opposite to the chirally twisted R-BINOL-Yb(OTf)3 complex
affording isoxazolines of (4S,5S) configuration (Scheme 3).
This direction of enantioselectivity was found also for cesium
fluoride-carbohydrate C system.
On the other hand, the opposite chiral induction indicated
by opposite elution order of enantiomers of the same compound, from the chiral column and opposite sign of optical rotation, was observed in the reaction mediated by the ytterbium
triflate-carbohydrate A complex. The observed enantioselectivity could be explained by a preferred attack of the nitrile
oxide from upper re-face of the dipolarophile opposite to the
ligand alpha-1,2-substituents affording isoxazolines of (4R,5R)
configuration (Scheme 4). This chirality was observed also for
the catalytic systems Yb2O3-carbohydrate A.
Absolute configuration of the carboxamides was established
via Li-Selectride reduction to the known isoxazoline methanol
derivative [15,18].
Scheme 3
Scheme 4
Biological activity
The biological activity of the compounds 6b-15h against several fungal strains was examined. Preliminary assays showed
high fungistatic potency of cycloadducts 14d and 14f. Cycloadduct 14f (S,S enantiomer) showed 100% growth retardation against Alternaria alternate, Botrytis cinerea, Fusarium
culmorum, Phytophtora cactorum Rhizoctonia solani and was
the most active of all the tested compounds (Table 3). The reference compound (chlorothalonil) showed smaller 38% and
88% activities against these strains. The presented data show
the importance of the optical purity of the screened compounds
since R,R antipode of 14f was significantly less active. Similarly S,S-rich enantiomer of 14d exhibited much higher biological activity than a racemic mixture.
Analyzing structure-activity relationship (SAR) some regularities were found. Compounds with a stronger electron-with-
drawing character (EWG) of a C-3 aryl substituent (CF3) and
electron-donating character (EDG) of an amide group (OMe)
were the most active amides, 14d (S,S) and 14f (S,S). Stronger
EWG character of F atoms compared to Cl substituents at the
C-3 moiety was reflected by a higher antifungal activity of 6f
compared to 6e. Presence of a weaker EDG at the amide function at 14e (sec-Bu) lowered the biological activity. A similar
negative effect exerted by the EDG (or hydrogen atom) at the
C-3 aryl group (i.e., compounds 14g and 14h).
The antifungal activity can be correlated with lipophilicity of the compounds measured by the logarithm of octanol-water partition coefficient logP [19]. Although no simple
dependence between calculated clogP and the biological activity was found, the optimal range of clogP as a measure of
lipophilicity was observed and for the most potent antifungal
amides clogP fell in the range 3.5-3.3 (Table 3).
We have not examined the mechanism of action of the
new antifungal compounds. However, it can be tentatively
166
J. Mex. Chem. Soc. 2015, 59(2)
Mirosław Gucma et al.
assumed that described here derivatives interact with fungal
wall enzymes as was recently demonstrated for the other
carboxylic acid amides. [2b]
lectivity and regioselectivity for some systems. By the
appropriate choice of the chiral catalysts, both enantiomers
of 3-aryl-4(5)-methyl-2-isoxazolinecarboxylates can be obtained. The use of single enantiomers is highly advisable as
pure or enriched enantiomers exhibiting often a much higher
fungicidal activity than racemic mixtures (compounds 14d
and 14f, Table 3). The type and position of the substituent on
the aromatic ring of the amide fragment of the dipolarophile
has a decisive influence on the reactivity in the 1,3-dipolar
cycloaddition reaction of nitrile oxides. We are continuing
research to diminish the amount of chiral Lewis acid from
equimolar to catalytic quantities.
Conclusion
We have applied new chiral complexes of carbohydrates A,
B, C, and R-binaphthol, with inorganic salts of metals belonging to several groups of elements, especially lanthanides
to study the 1,3-dipolar cycloaddition reaction of nitrile oxides and substituted acrylamides achieving high enantioseTable 3. Fungicidal activity of compounds 6b-15h at 200 μg/mL.a
Fungicidal activity
Comp.
No.
cLogP
(+/-) 0.75
Alternaria
alternata
Boritis cinerea
Fusarium
culmorum
Phytophtora
cactorum
Rhizoctonia
solani
6b
4.23
-
3
5
7
9
6c
5.22
-
4
3
5
18
6d
6.24
-
5
8
19
23
6e
4.56
-
0
0
2
0
6f
4.30
-
23
27
67
19
8a
2.43
-
3
7
9
5
8d
3.81
-
0
5
8
18
8e
5.02
-
17
7
5
4
8f
2.34
-
20
5
23
7
8g
2.34
-
5
3
7
8
8h
2.50
-
6
7
4
5
8i
1.11
-
9
3
8
7
9
4.89
-
30
9
63
9
10
5.19
-
18
-
21
-
11
2.88
-
0
0
0
0
14a RS
2.95
-
0.0
12.9
4.2
10
14b RS
4.82
-
44
50
30
85
14c RS
2.96
58
0
19
48.3
38
14c RR
2.96
0.8
0
0.3
1.0
30
14d SS
3.46
100
100
78
100
78
14d RS
3.46
0.8
71
0.0
40
38
14e RR
5.31
6
47
25
0
3.9
15e RS
5.31
0
47
7
-
11
14f SS
3.34
100
100
100
100
100
14f RS
3.34
46
0.0
17
38
7
14f RR
3.34
51
33
12.5
12.5
44
15f RS
3.34
-
0.0
17
0.0
0.0
14g RS
4.22
6
31
9
5
7.8
14h RR
2.77
45
33
15
-
47
14h SS
2.77
0
53
41
-
55
15h RR
4.22
41
53
48
-
80
Chlorothalonilb
2.88
-
80.0
38.0
61.0
88.0
aPercentage
of linear growth inhibition.
bReference
compound.
Enantioselective synthesis of isoxazolecarboxamides and their fungicidal activity
Experimental
Reagent grade chemicals were used without further purification
unless otherwise noted. Elemental analyses were performed at the
Microanalysis Laboratory of Institute of Organic Chemistry, Polish Academy of Sciences, Warsaw. Spectra were obtained as follows: IR spectra on JASCO FTIR-420 spectrometer, 1H and 13C
NMR spectra on Varian 500 UNITY plus-500 and Varian 200
UNITY plus 200 spectrometers in deuterated chloroform using
TMS as internal standard, and EI mass spectra on AMD M-40. In
13C NMR spectra, signals of fluorine-substituted carbon atoms
and some alpha carbon atoms were not observed because of strong
19F–13C coupling. In order to further characterize these compounds, 19F spectra were recorded. Flash chromatography was
carried out using silica gel S 230-400 mesh (Merck) and hexanes-ethyl acetate mixtures as eluents. Hydroximinoyl acid chlorides were prepared from the corresponding aryl aldehyde oximes
and NCS in DMF [10]. The enantiomeric excess of the separated
regioisomers was determined by HPLC analysis (AD-H column).
LogP was calculated using ACD/CNMR Predictor v.12 computer
program of Advanced Chemistry Development (ACD/Labs), Toronto, Canada.
General procedure for the 1,3-dipolar cycloaddition
reactions to obtain adducts 4a-f and 9-11.
A solution of chlorooxime (13 mmol) in anhydrous toluene (15
mL) was added dropwise over 30 min to a stirred mixture of anhydrous toluene (60 mL), anhydrous Et3N (6 mL), MgSO4 (2 g),
and ethyl acrylate (8 mL, 80 mmol). The reaction mixture was
stirred overnight at room temperature, diluted with toluene (50
mL), washed with water (5 x 50 ml), and evaporated in vacuo.
General procedure for the synthesis of isoxazoles 7.
The flask was charged with CCl4 (50 mL), 3-(4-trifluoromethylphenyl)-5-ethoxycarbonyl-2-isoxazoline (4) (2.6 mmol) and a
pinch of azoisobutylnitrile. NBS (5 mmole) was added portionwise with stirring over 0.5 h. The reaction was carried out for 5 h
at reflux. After cooling to room temperature (rt), a mixture of CH3COOH (1.65 mL, 27.5 mmol) and CH3COOK (4 g, 40.8 mmol)
was added, the reaction was continued for 70 min at reflux. The
reaction progress was monitored by TLC. Then, after cooling, the
reaction mixture was poured into ice water, containing NaOH
(4.86 g, 121.5 mmol) and stirred for 5 minutes. Dichloromethane
(50 mL) was added, the mixture was washed with water (3 x 50
mL). The organic phase was dried over MgSO4. After filtration
and evaporation the product was purified by dissolving in dichloromethane and precipitation with hexanes.
General procedure for the synthesis of amides 6a, 8a and
11 with tertiary amines (Method A1)
A solution of an amine derivative (1.2 mmol) in anhydrous dichloromethane (10 mL) was added with stirring to an acid chloride prepared from compounds 5 or 7 followed by anhydrous
167
triethyl amine (4 mL, 30.0 mmol). The solution was stirred for 1
h at 0 °C. Water (10 mL) was added, the organic layer was washed
with 3% hydrochloric acid solution and water, and was dried over
magnesium sulfate. A crude amide obtained after evaporation of
the solvent was purified by crystallization.
General procedure for the synthesis of amides 6b, 6c, 8b,
8f, 8g and 8i with tertiary amines (Method A2).
A solution of an aniline derivative (1.2 mmol) in anhydrous
toluene (10 mL) was added with stirring to an acid chloride
followed by anhydrous triethyl amine (4 mL, 30.0 mmol). The
solution was stirred under reflux for 1 h and overnight at rt.
Water (10 mL) was added, the organic layer was washed with
3% hydrochloric acid solution and water, and was dried over
magnesium sulfate. A crude amide obtained after evaporation
of the solvent was purified by crystallization.
General procedure for the synthesis of amide 8h with
n-butyl lithium (Method B1)
A 2.5 M solution of n-BuLi in hexanes (0.2 mL, 0.5 mmol) was
added dropwise to a stirred solution of 4-aminopyridine derivative (0.4 mmol) in anhydrous diethyl ether at -78 °C. Stirring
was continued for 1 h and a solution of acid chloride (0.3
mmol) in anhydrous diethyl ether (or HMPA) was added dropwise. The mixture was stirred for 2 h at -78 °C and for 0.5 h at
0 °C. The reaction was quenched with ammonium chloride
solution, product was extracted with methylene chloride and
purified by flash chromatography.
General procedure for the synthesis of amides 6d, 6e, 6f,
8c, 8d, 8e, 9 and 10 with tert-butyl lithium (Method B2)
A 1.7 M solution of tert-BuLi in hexanes (0.2 mL, 0.5 mmol)
was added dropwise to a stirred solution of 4-aminopyridine
derivative (0.4 mmol) in anhydrous diethyl ether at 0 °C. Stirring was continued for 1 h and a solution of acid chloride (0.3
mmol) in anhydrous diethyl ether (or HMPA) was added dropwise. The mixture was stirred for 5 h at 0 °C and overnight at
rt. The reaction was quenched with ammonium chloride solution, the product was extracted with methylene chloride and
purified by flash chromatography.
General procedure for the synthesis of dipolarophile
amides 13a-h
A solution of an aniline derivative (1.2 mmol) in anhydrous
toluene (or anhydrous dichloromethane) (10 mL) was added
with stirring to an acid chloride (1.0 mmol) followed by an
anhydrous triethylamine (30 mmol) at rt. The obtained solution
was stirred under reflux for 1 h and overnight at rt. Water (10
mL) was added, the organic layer was separated, washed with
3% hydrochloric acid solution and water, and was dried over
magnesium sulfate. Product was extracted with dichloromethane and purified by flash chromatography.
168
J. Mex. Chem. Soc. 2015, 59(2)
General procedure for the enantioselective cycloaddition
to carboxamides 14a, 14b, 14c-h, and 15c-h.
A mixture of carbohydrate A (1.0 mmol) and Yb(OTf)3 (1.0
mmol) in dry dichloromethane was stirred at rt for 30 min.
Dipolarophile (1 mmol) was added dropwise followed by a
solution of dipole in the same solvent generated by passing a
hydroximinoyl chloride solution through a column of Amberlyst A-21 over 20–30 min. The solution was stirred at rt for
ca. 20 h, and water was added to quench the reaction followed
by the usual work-up. The crude product was purified by flash
column chromatography over silica gel and the enantiomeric
excess of the separated regioisomers was determined by HPLC
analysis (AD-H column).
N-(3-Bromopropyl)-3-(2,3,6-trichlorophenyl)-4,5-dihydro-1,2-oxazole-5-carboxamide (6a). A greenish oil. 1H
NMR (CDCl3, 200 MHz) d 7.65 (m, 3H, NH, H-4”, H-5”),
5.26-5.18 (m, 1H, H-5), 3.76-3.36 (m, 6H, H-4, -CH2Br,
-CH2NH], 2.11 (sept. J = 6.5 Hz, 2H, BrCH2CH2CH2NH).
Anal. Calcd for C13H12 BrCl3N2O2: C 37.67: H 2.92. Found: C
37.98; H .2.97.
N-(2-Bromophenyl)-3-(2,3,6-trichlorophenyl)-4,5-dihydroisoxazole-5-carboxamide (6b). A colorless glass. IR
(KBr) νmax 3357, 3080, 2920, 2840, 1699, 1650, 1591, 1521,
1438, 1400, 1304, 1180, 1150, 1040, 1020, 950, 870, 817, 735
cm-1. 1H NMR (CDCl3, 200 MHz) d 9.16 (s, 1H, NH), 8.33
(dd, J = 8.3; 1.6 Hz, 1H, H-3”), 7.58 (dd, J = 8.2; 1.6 Hz, 1H,
H-6”), 7.50 (d, J = 8.8 Hz, 1H, H-4’), 7.36 (td, J = 8.3; 1.6 Hz,
1H, H-4”), 7.33 (d, J = 8.8 Hz, 1H, H-3’), 7.04 (td, J = 8.3; 1.6
Hz, 1H, H-5”), 5.38 (dd, J = 11.0; 5.3 Hz, 1H, H-5), 3.74 (d, J
= 11.0 Hz, 1H, H-4), 3.69 (d, J = 5.3 Hz, 1H, H-4). Anal. Calcd
for C16H10 BrCl3N2O2: C 42.85: H 2.25. Found: C 42.63; H
.2.56.
N-(3-Bromophenyl)-3-(2,3,6-trichlorophenyl)-4,5-dihydroisoxazole-5-carboxamide (6c). A greenish pulp. IR
(KBr) νmax 3480, 3373, 3080, 2920, 1685, 1620, 1591, 1530,
1480, 1450, 1400, 1303, 1280, 1180, 1140, 1070, 1040, 990,
870, 820, 774, 680 cm-1. 1H NMR (CDCl3, 200 MHz) d 8.54
(s, 1H, NH), 7.89 (t, J = 2.0 Hz, 1H, H-2“), 7.51 (d, J = 8.7 Hz,
1H, H-4‘), 7.34 (d, J = 8.7 Hz, 1H, H-5’), 7.24 (d, J = 8.1 Hz,
1H, H-4”), 7.00 (td, J = 8.1; 0.7 Hz, 1H, H-5”), 6.86 (dm, J =
8.1 Hz, 1H, H-6”), 5.34 (dd, J = 10.9; 5.5 Hz, 1H, H-5), 3.73
(d, J = 10.9 Hz, 1H, H-4), 3.68 (d, J = 5.5 Hz, 1H, H-4). 13C
NMR (CDCl3, 50 MHz) d 168.92, 155.70, 138.06, 132.33,
130.60, 128.91, 128.31, 123.23, 118.74, 79.34 (C-5), 42.21 (C4). Anal. Calcd for C16H10 BrCl3N2O2: C 42.85: H 2.25. Found:
C 42.68; H .2.48.
3-(2,3,6-Trichlorophenyl)-4,5-dihydroisoxazole5-carboxylic acid 2-trifluorometoxy-4-bromophenylamide
(6d). A greenish wax. IR (KBr) νmax 3395, 3120, 2960, 2928,
2850, 1702, 1640, 1519, 1519, 1440, 1400, 1303, 1250, 1211,
1190, 1150, 1080, 1044, 941, 879, 818, 753, 730, 670 cm-1. 1H
NMR (CDCl3, 200 MHz) d 8.94 (s, 1H, NH), 8.33 (d, J = 9.2
Hz, 1H, H-6”), 7.94 (d, J = 8.8 Hz, 1H, H-5’), 7.53-7.43 (m,
3H, H-4’, H-3”, H-5”), 5.35 (dd, J = 10.8; 5.8 Hz, 1H, H-5),
3.72 (d, J = 10.8 Hz, 1H, H-4), 3.68 (d, J = 5.8 Hz, 1H, H-4).
13C NMR (CDCl , 50 MHz) d 168.95, 155.33, 138.81, 133.32,
3
Mirosław Gucma et al.
132.91, 132.44, 132.15, 131.83, 130.74, 128.93, 128.78,
128.72, 124.73, 124.05, 122.78, 122.07, 117.793, 116.94,
79.17, 41.91. ESI MS m/z (rel. int.) 532 [M+] (95). Anal. Calcd
for C17H9 BrCl3F3N2O3: C 38.34: H 1.70. Found: C 38.09; H
.1.56.
N-(5-Chloro-2,3,6-trifluoropyridin-4-yl)-3-(2,3,6-trichlorophenyl)-4,5-dihydroisoxazole-5-carboxamide (6e). A
white-brownish semisolid. IR (KBr) νmax 3490, 3350, 3256,
3080, 2925, 2850, 1715, 1623, 1500, 1474, 1440, 1400, 1320,
1270, 1240, 1203, 1182, 1150, 1100, 1067, 1042, 1021, 869,
818, 760, 703, 675 cm-1. 1H NMR (CDCl3, 200 MHz) d 8.81
(s, 1H, NH), 7.53 (d, J = 8.7 Hz, 1H, H-4’), 7.35 (d, J = 8.7 Hz,
1H, H-5’), 5.46 (dd, J = 10.8; 5.5 Hz, 1H, H-5), 3.77 (d, J =
10.8 Hz, 1H, H-4a), 3.73 (d, J = 5.5 Hz, 1H, H-4b). ESI MS
m/z calcd for C15H6N3O2F3Cl4Na: 479.9064. Found: 479.9026.
Anal. Calcd for C15H6 Cl4F3N3O3: C 39.25; H 1.32. Found: C
39.09; H .1.46.
N-(2,6-Difluoro-3,5-dichloropyridin-4-yl)-3-(2,4,5-trifluorophenyl)-4,5-dihydroisoxazole-5-carboxamide (6f). A
white-brownish semisolid. IR (KBr) νmax 3360, 3280, 3080,
2915, 2850, 1709, 1630, 1597, 1540, 1512, 1485, 1437, 1409,
1373, 1260, 1230, 1191, 1140, 1060, 1040, 1005, 916, 890,
804, 781, 735 cm-1. 1H NMR (CDCl3, 200 MHz) d: 8.59 (1H,
NH), 7.72 (m, 1H, H-6’), 7.05 (td, J = 10.2; 6.3 Hz, 1H, H-3’),
5.36 (t, J = 8.4 Hz, 1H, H-5), 3.87 (d, J = 8.4 Hz, 1H, H-4a),
3.86 (d, J = 8.4 Hz, 1H, H-4b). ESI-MS m/z calcd for C15H6O2N3F5Cl2Na: 447.9655. Found: 447.9655. Anal. Calcd for C15H6
Cl2F5N3O2: C 42.28; H 1.42. Found: C 42.07; H .1.59.
N,N-Diisopropyl-3-(4-trifluoromethylphenyl)-4,5-dihydroisoxazole-5-carboxamide (8a). A colorless glass. IR
(KBr) νmax 3440, 3121, 2974, 2910, 1645, 1478, 1437, 1380,
1323, 1175, 1120, 1063, 1040, 1020, 990, 951, 920, 846, 820,
760, 690 cm-1. 1H NMR (CDCl3, 200 MHz) d 7.95 (d, J = 8.2
Hz, 1H, H-3’, H-5’), 7.75 (d, J = 8.2 Hz, 2H, H-2’, H-6’), 4.11
(m, 1H, CH, CH(CH3)2), 3.63 (m, 1H, CH, CH(CH3)2), 1.54
(d, J = 6.2 Hz, 6H, HC(CH3)2), 1.31 (d, J = 6.2 Hz, 6H,
HC(CH3)2). 13C NMR (CDCl3, 50 MHz) d 167.27 (C=O),
161.33 (C-3), 157.94 (C-5), 132.66 (C-1‘), 131.97 (m, C-4‘),
127.40 (2C, C-2‘, C-6‘), 126.23 (q, J = 3.6 Hz, 2C, C-3‘, C-5‘),
104.10 (C-4), 50.93 (HC(CH3)3), 47.07 (HC(CH3)3), 21.17
(2C, CH, HC(CH3)2), 20.39 (2C, HC(CH3)2). 19F NMR
(CDCl3, 471 MHz) d -63.35 (s, 3F, F3C-Ar). ESI MS m/z calcd
for C17H19O2N2F3Na: 363.1296. Found: 363.1262.
N-Cyclohexyl-3-(4-trifluoromethylphenyl)isoxazole-5-carboxamide (8b). A yellowish semisolid. IR (KBr)
νmax 3460, 3318, 3287, 3160, 2936, 2854, 1650, 1536, 1521,
1450, 1438, 1326, 1283, 1260, 1240, 1181, 1135, 1115, 1095,
1070, 1020, 950, 832, 782, 761, 670 cm-1. 1H NMR (CDCl3,
200 MHz) d 7.96 (d, J = 8.4 Hz, 2H, H-5’, H-3’), 7.75 (d, J =
8.4 Hz, 2H, H-6’, H-2’), 7.28 (s, 1H, H-4), 6.53 (s, 1H, NH),
3.99 (m, 1H, -CH, NHCH(CH)2, H-1”), 2.07-1.08 (m, 10H,
-CH2, H-2”, H-3”, H-4”, H-5”, H-6”). Anal. Calcd for C17H17F3N2O2: C 60.35; H 5.06. Found: C 60.07; H .5.27.
N-(4-Chloro-3-nitrofenyl)-3-(4-trifluoromethylphenyl)isoxazole-5-carboxamide (8c). A white-brownish solid. mp. 217-219 oC. IR (KBr) νmax 3416, 3100, 2920, 2880,
1696, 1615, 1595, 1534, 1480, 1438, 1350, 1325, 1245,
Enantioselective synthesis of isoxazolecarboxamides and their fungicidal activity
1175, 1132, 1067,1019, 950, 850, 828, 755, 680 cm-1. 1H
NMR (CDCl3,, 200 MHz) d 8.48 (s, 1H, NH), 8.40 (d, J =
2.6 Hz, 1H, H-2”), 7.99 (d, J = 8.1 Hz, 2H, H-5’, H-3’), 7.83
(dd, J = 8.7; 2.6 Hz, 1H, H-6”), 7.79 (d, J = 8.1 Hz, 2H,
H-6’, H-2’), 7.59 (d, J = 8.7 Hz, 1H, H-5”), 7.44 (s, 1H,
H-4). Anal. Calcd for C17H9 ClF3N3O4: C 49.59; H 2.20.
Found: C 49.41; H .2.38.
N-(2-Chloro-5-nitrofenyl)-3-(4-trifluoromethylphenyl)
isoxazole-5-carboxamide (8d). A white semisolid. IR (KBr)
νmax 3376, 3300, 3150, 2925, 2850, 1703, 1678, 1618, 1595,
1536, 1460, 1440, 1421, 1348, 1327, 1275, 1241, 1163, 1128,
1069,1018, 950, 870, 848, 740 cm-1. 1H NMR (CDCl3, 200
MHz) d 9.44 (d, J = 2.5 Hz, 1H, H-6”), 8.97 (s, 1H, NH), 8.05
(dd, J = 8.8; 2.5 Hz, 1H, H-4”), 8.01 (d, J = 8.2 Hz, 2H, H-5’,
H-3’), 7.79 (d, J = 8.2 Hz, 2H, H-6’, H-2’), 7.66 (d, J = 8.8 Hz,
1H, H-3”), 7.47 (s, 1H, H-4). Anal. Calcd for C16H10 BrCl3N2O2:
C 42.85; H 2.25. Found: C 42.99; H .2.41.
N-(2-Chloro-4-trifluoromethyl-6-nitrophenyl)-3-(4-(trifluoromethylphenyl)isoxazole-5-carboxamide
(8e).
A
white-brownish semisolid. IR (KBr) νmax 3444, 3280, 3080,
2920, 1687, 1618, 1549, 1510, 1440, 1400, 1360, 1326, 1270,
1240, 1170, 1133, 1071,1020, 950, 900, 830, 751, 680 cm-1. 1H
NMR (CDCl3, 200 MHz) d 9.01 (s, 1H, NH), 8.25 (d, J = 2.0 Hz,
1H, H-5”), 8.06 (d, J = 2.0 Hz, 1H, H-3”), 7.99 (d, J = 8.3 Hz,
2H, H-3’, H-5’), 7.79 (d, J = 8.3 Hz, 1H, H-2’, H-6’), 7.45 (s,
1H, H-4). Anal. Calcd for C18H8 ClF6N3O4: C 45.07; H 1.68.
Found: C 45.31; H .1.51.
N-(Pyridin-4-yl)-3-(4-trifluoromethylphenyl)isoxazole-5-carboxamide (8f). A grey wax. Mp. 247-249 oC. IR
(KBr) νmax 3418, 3360, 3120, 2940, 2830, 1686, 1591, 1514,
1440, 1417, 1334, 1293, 1240, 1170, 1114, 1071, 1020, 1000,
950, 890, 822, 750 cm-1. 1H NMR (CDCl3, 200 MHz) d 8.63
(m, 2H, H-3”, H-5”), 8.49 (s, 1H, -NH), 7.99 (d, J = 7.7 Hz,
2H, H-3’, H-5’), 7.78 (d, J = 7.7 Hz, 2H, H-2’, H-6’), 7.68
(m, 2H, H-2”, H-6”), 7.47 (s, 1H, H-4). 13C NMR (CDCl3,
50 MHz) d 163.75 (C=O), 161.71 (C-3), 154.55 (C-5), 150.46
(2C, C-2“, C-6“), 144.63 (C-4“), 131.41 (C-1‘), 130.68 (q, J
= 31.6 Hz, C-4‘), 127.61 (2C, C-2‘, C-6‘), 126.17 (q, J = 4.0
Hz, 2C, C-3‘, C-5‘), 114.28 (2C, C-3“, C-5“), 106.53 (C-4).
19F NMR (CDC , 471 MHz) d -61.85 (s, 3F, F C-Ar). Anal.
l3
3
Calcd for C16H10 F3N3O2: C 57.66; H 3.02. Found: C 57.49;
H .2.89.
N-(Furan-2-yl-methyl)-3-(4-trifluoromethylphenyl)isoxazole-5-carboxamide (8g). A white-brownish solid: mp.
188-190 oC. IR (KBr) νmax 3437, 3296, 3052, 2937, 1670,
1622, 1538, 1525, 1502, 1435, 1418, 1384, 1332, 1297, 1269,
1253, 1197, 1122, 1076, 1066, 1032, 1019, 997, 953, 937, 924,
848, 771, 747, 697 cm-1. 1H NMR (CDCl3, 200 MHz) d 7.95
(d, J = 8.1 Hz, 2H, H-5’, H-3’), 7.75 (d, J = 8.1 Hz, 2H, H-6’,
H-2’), 7.41 (dd, J = 10.0; 1.8 Hz, 1H, -OCH=), 7.29 (s, 1H,
H-4), 6.92 (s, 1H, NH), 6.36 (m, 2H, C=CH-CH=C), 4.67 (d, J
= 5.6 Hz, 2H, CH2, CH2NH). 13C NMR (CDCl3, 50 MHz) d
164.26 (C=O), 162.48, 155.55, 150.02, 142.96, 127.51 (s, 2C,
C-2’, C-6’), 126.5 (q, J = 3.6 Hz, 2C, C-3’, C-5’), 110.83,
108.58, 105.63, 36.67. 19F NMR (CDCl3, 471 MHz) d -63.39
(s, 3F, F3C-Ar). ESI-MS m/z calcd for C16H11O3N2F3Na:
359.0620. Found: 359.0625.
169
4-Benzyloxazolidinone-2-3-(4-trifluoromethylphenyl)
isoxazole-5-carboxamide (8h). It was obtained as a
white-brownish solid from 4-benzyloxazolidinone-2 derivative [17]: mp: 156-159 oC. IR (KBr) νmax 3122, 3040, 3000,
1802, 1777, 1690, 1571, 1490, 1456, 1434, 1389, 1357, 1323,
1244, 1210, 1168, 1120, 1063, 1020, 952, 843, 770, 720, 701,
695 cm-1. 1H NMR (CDCl3) d 7.98 (d, J = 8.1 Hz, 2H, H-5’,
H-3’), 7.76 (d, J = 8.1 Hz, 2H, H-6’, H-2’), 7.37 (s, 1H, H-4),
7.29 (m, 5H, H-2”, H-3”, H-4”, H-5”, H-6”), 4.90 (m, 1H,
H-11, CH, CH2CHCH2), 4.37 (d, J = 10.8 Hz, 1H, H-10a, CH2,
OCH2CH), 4.34 (d, J = 7.5 Hz, 1H, H-10b, CH2, OCH2CH),
3.47 (dd, J = 13.4; 3.5 Hz, H-12b, CH2, ArCH2CH), 2.95 (dd,
J = 13.4; 9.2 Hz, 1H, H-12a). 13C NMR (CDCl3, 50.3 MHz) d
161.81 (C=O), 161.67, 156.64, 152.19, 134.65, 131.60, 129.63,
129.34, 127.88, 127.51 (s, 2C, C-2’, C-6’), 126.3 (q, J = 3.6
Hz, 2C, C-3’, C-5’), 107.73, 67.23, 56.27, 37.68. 19F NMR
(CDCl3, 471 MHz) d -63.32 (s, 3F, F3C-Ar). ESI MS m/z calcd
for C21H15O4N2F3Na: 439.0882. Found: 439.0860.
Pyrrolidine-3-(4-trifluoromethylphenyl)isoxazole5-carboxamide (8i). A white-brownish solid: mp. 164-166 oC.
IR (KBr) νmax 3116, 2977, 2887, 1626, 1595, 1467, 1438,
1413, 1383, 1326, 1320, 1228, 1157, 1125, 1114, 1064, 1016,
950, 933, 884, 850, 756, 694 cm-1. 1H NMR (CDCl3) d 7.97 (d,
J = 8.6 Hz, 2H, H-5’, H-3’), 7.75 (d, J = 8.6 Hz, 2H, H-2’,
H-6’), 7.25 (s, 1H, H-4), 3.95 (t, J = 6.6 Hz, 2H, H-5a, H-8a),
3.70 (t, J = 6.6 Hz, 2H, H-5a, H-8b), 2.02 (m, 4H, H-6, H-7).
13C NMR (CDCl , 50 MHz) d 166.27, 161.47, 155.56, 131.98,
3
131.84, 127.37 (s, 2C, C-2’, C-6’), 126.2 (q, J = 3.6 Hz, 2C,
C-3’, C-5’), 106.68, 47.98, 47.44, 26.51, 23.84. 19F NMR
(CDCl3, 471 MHz) d -63.39 (s, 3F, F3C-Ar). ESI MS m/z Calcd
for C15H13O2N2F3Na: 333.0827. Found: 333.0828.
N-(4-Trifluoromethyl-2-chloro-6-nitrophenyl)-3-(tertbutyl)-4,5-dihydroisoxazole-5-carboxamide (9). A greyish
semisolid by method B2, 12%. 1H NMR (CDCl3, 200 MHz) d
9.14 (s, 1H, NH), 8.14 (d, J = 1.4 Hz, 1H, H-5’), 7.97 (d, J =
1.4 Hz, 1H, H-3’), 5.13 (dd, J = 9.7; 6.5 Hz, 1H, H-5), 3.40 (d,
J = 9.7 Hz, 1H, H-4), 3.39 (d, J = 6.5 Hz, 1H, H-4), 1.26 (s, 9H,
C(CH3)3)). Anal. Calcd for C15H15 ClF3N3O4: C 45.76; H 3.84.
Found: C 46.00; H .3.61.
N-(4-Trifluoromethyl-2,6-dichlorophenyl)-3-(tertbutyl)-4,5-dihydroisoxazole-5-carboxamide (10). A yellowish solid, method B2, 15%: mp. 98-103 oC. IR (KBr) νmax
3437, 2970, 1691, 1497, 1391, 1323, 1170, 1133, 880, 813 cm1. 1H NMR (CDCl , 200 MHz) d 8.44 (s, 1H, NH), 7.65 (s, 2H,
3
H-3’, H-5’), 5.17 (t, J = 7.9 Hz, 1H, H-5), 3.42 (d, J = 7.9 Hz,
2H, H-4) 1.24 (s, 9H, (CH3)3C). 13C NMR (CDCl3, 50 MHz) d
169.92 (C=O), 167.28 (C-3), 134.54 (C-1’), 134.22 (C-4’),
131.50 (2C, C-2’, C-6’), 130.82, 125.68 (C-3’), 125.60 (C-5’),
78.22 (C-5), 39.62 (C-4), 33.23 (C(CH3)3), 28.06 (3C,
(CH3)3C). 19F NMR (CDCl3, 471 MHz) d -63.39 (3F, F3C-Ar).
Anal. Calcd for C15H15Cl2F3N2O2: C 47.02; H 3.95. Found: C
47.32; H 4.02.
N,N-Diisopropyl-3-(tert-butyl)-4,5-dihydroisoxazole5-carboxamide (11). A greyish semisolid, method A1, 98%.
IR (KBr) νmax 2969, 1647, 1446, 1368, 1300, 1212, 1160,
1136, 1043, 874, 818, 755 cm-1. 1H NMR (CDCl3, 200 MHz)
d 5.11 (dd, J = 10.9; 8.8 Hz, 1H, H-5), 4.30 (sept., J = 6.6 Hz,
170
J. Mex. Chem. Soc. 2015, 59(2)
1H), 3.80 (dd, J = 16.9; 10.9 Hz, 1H, H-4), 3.47 (sept., J = 6.7
Hz, 1H, CH(CH3)2), 3.44 (m, J = 6.7 Hz, 1H, CH(CH3)2), 2.92
(dd, J = 16.9; 10.9 Hz, 1H, H-4), 1.47 (d, J = 6.7 Hz, 6H,
CH(CH3)2), 1.41 (d, J = 6.7 Hz, 3H, HCCH3) 1.40 (d, J = 6.7
Hz, 3H, HCCH3), 1.23 (s, 9H, C(CH3)3). EI MS m/z (rel. int.)
254 [M+] (9), 224 (M+ - 2xCH3, 5), 126 (M+ - (O=C-N(CH(CH3)2), 50). Anal. Calcd for C14H26N2O2: C 66.10; H 10.30.
Found: C 66.40; H 10.11.
N-Phenylacrylamide (13a). A yellowish semisolid, 35 %.
IR (neat) νmax 3430, 3307, 3295, 3200, 3144, 3100, 3060,
1666, 1640, 1607, 1552, 1497, 1443, 1408, 1333, 1298, 1254,
1202, 1067, 986, 960, 940, 900, 840, 800, 755, 688 cm-1. 1H
NMR (CDCl3, 200 MHz) d 7.85 (s, 1H, HNC=O), 7,59 (d, J =
7.8 Hz, 2H, H-2‘, H-6‘), 7.30 (m, 2H, H-3‘, H-5‘), 7,11 (t, J =
7.4 Hz, 1H, H-4‘), 6.42 (dd, J = 16.8; 2.1 Hz, 1H, H-3a,
H2C=CC=O), 6.30 (dd, J = 16.8; 9.4 Hz, 1H, H-3b,
H2C=CC=O), 5.73 (dd, J = 9.4; 2.1 Hz, 1H, -C=CHC=O). EI
MS m/z (rel. int.) 147 [M+], 93 (HNC6H5 + H), 77 (C6H5), 55
(O=CCH=CH2). Anal. Calcd for C9H9NO: C 73.45; H 6.16.
Found: C 73.1; H .6.11.
Carboxamides 13b and 13d were described [15].
N-(4-sec-Butylphenyl)acrylamide (13c). A yellowish
semisolid, 30 %. 1H NMR (CDCl3, 500 MHz) d 7.51 (d, J =
8.3 Hz, 2H, H-2‘, H-6‘), 7.12 (d, J = 8.3 Hz, 2H, H-3‘,
H-5‘), 6.40 (d, J = 17.0 Hz 1H, H-3a, H2C=CC=O), 6.27
(dd, J = 17.0; 10.5 Hz, 1H, H-3b, H2C=CC=O), 5.70 (dd, J
= 10.5; 1.5 Hz, 1H, H-2, C=CHC=O), 2.58 (m, J = 7.0 Hz,
1H, H3CCHCH2), 1.58 (quint., J = 7.0 Hz, 2H, H3CH2CCH), 1.20 (d, J = 7.0 Hz, 3H, H3CCH), 0.81 (t, J = 7.0
Hz, 3H, H3CCH2). HR ESI MS m/z Calcd for C13H17NONa:
226.1208. Found: 226.1214. Anal. Calcd for C13H17NO: C
76.81; H 8.43. Found: C 76.50: H .8.21.
N-(3-Methoxyphenyl)crotonamide (13g). A yellowish
wax, 12 %, method A2. IR (KBr) νmax 3420, 3285, 2960, 2910,
2830, 1665, 1605, 1524, 1485, 1450, 1434, 1320, 1271, 1214,
1150, 1035, 960, 870, 830, 777, 750, 720, 680 cm-1. 1H NMR
(200 MHz, CDCl3) d 9.33 (s, 1H, NH), 7.40-6.65 (m, 5H, H-2‘,
H-4‘, H-5‘, H-6‘, H3C-CH=C), 5.94 (d, J = 15.2 Hz, 1H, CH,
O=CHC=C), 3.81 (s, 3H, H3CO), 1.82 (d, J = 6.8 Hz, 3H,
H3C-CH=C). Anal. Calcd for C11H13NO2: C 69.09: H 6.85.
Found: C 69.19: H .6.61.
N-(4-Trifluoromethylphenyl)crotonamide (13h). A yellowish semisolid, 10 %, method A2. 1H NMR (CDCl3, 200
MHz) d 7.70 (m, 1H, -NH), 7.69 (d, J = 8.0 Hz, 2H, H-5‘,
H-3‘), 7.56 (d, J = 8.0 Hz, 2H, H-2‘, H-6‘), 7.03 (m, J = 14.9;
6.5 Hz, 1H, H-3, H3C-CH=C), 5.97 (d, J = 14.9 Hz, 1H, H-2,
O=CHC=C), 1.91 (d, J = 6.5 Hz, 3H, H3C-CH=C). Anal. Calcd for C11H10 F3NO: C 57.64: H 4.40. Found: C 57.89: H .4.57.
N-Phenyl-3-(4-trifluoromethylphenyl)-4,5-dihydroisoxazole-5-carboxamide (14a).
A yellowish semisolid, 30 %. 1H NMR (CDCl3, 200 MHz)
d 8.48 (s, 1H, HN-C=O), 7.81 (d, J = 8.2 Hz, 2H, H-2‘, H-6‘),
7.69 (d, J = 8.2 Hz, 2H, H-3‘, H-5‘), 7.57 (d, J = 8.0; 1.7 Hz,
2H, H-2“, H-6“), 7.34 (td, J = 8.0; 1.7 Hz, 2H, H-3“, H-5“),
7.15 (td, J = 8.0; 1.7 Hz, 1H, H-4“), 5.32 (dd, J = 10.9; 6.4 Hz
1H, H-5), 3.82 (d, J = 6.2 Hz, 1H, H-4a), 3.79 (d, J = 10.9 Hz,
1H, H-4b). EI-MS m/z (%) 334 (M+), 315 (M+-F), 214 [M+-
Mirosław Gucma et al.
C=ONHC6H5], 145 (F3CC6H4), 93 (M+-HNC6H5), 77 (C6H5).
Anal. Calcd for C17H13F3N2O2: C 61.08; H 3.92. Found: C
60.80: H .4.17.
N - ( 4 - s e c - B u t y l p h e n y l ) - 3 - ( 4 trifluoromethylphenyl)-4,5-dihydroisoxazole-5-carboxamide (14b). A colorless glass, 40 %. 1H NMR (CDCl3, 200 MHz)
d 8.48 (s, 1H, HNC=O), 7.78 (d, J = 8.0 Hz, 2H, H-2‘, H-6‘),
7.67 (d, J = 8.0 Hz, 2H, H-3‘, H-5‘), 7.48 (d, J = 8.3 Hz, 2H,
H-2“, H-6“), 7.14 (d, J = 8.3 Hz, 2H, H-3“, H-5“), 5.30 (dd, J =
11.8; 5.2 Hz 1H, H-5), 3.83 (dd, J = 17.3; 5.2 Hz, 1H, H-4a),
3.73 (dd, J = 17.3; 11.8 Hz, 1H, H-4b), 2.56 (quint., J = 7.0 Hz,
1H, H3CCHCH2), 1.58 (m, 2H, H3CCH2CH), 1.20 (d, J = 7.0
Hz, 3H, H3CCH), 0.79 (t, J = 7.5 Hz, 3H, H3CCH2). HR ESI
MS m/z Calcd for C21H21F3N2O2Na: 413.1453. Found:
413.1434. Anal. Calcd for C21H21F3N2O2: C 64.61; H 5.42.
Found: C 64.78; H 5.37.
Compounds 14c-g and 15c-g were described [15].
N-(2-Methoxyphenyl)-4-methyl-3-phenyl-4,5-dihydroisoxazole-5-carboxamide (14h). A colorless glass. {[α]D
– 45o, (c 0.9. in acetone) [87.0% ee, (R,R) rich]}, {[α]D + 40o,
(c 0.76 in acetone) [96.0% ee, (S,S) rich]}. IR (KBr) νmax
3398, 3380, 3080, 3040, 2910, 2860, 1687, 1603, 1536, 1490,
1463, 1440, 1328, 1295, 1254, 1115, 1024, 874, 760, 743,
697 cm-1. 1H NMR (CDCl3, 200 MHz) d 9.16 (s, 1H, NH),
8.4-6.8 (m, 9H, H-6’, H-2’, H-5’, H-3’, H-4‘, H-6”, H-4”,
H-5”, H-3”), 4.84 (d, J = 3.4 Hz, 1H, H-5), 4.18 (dq, J = 7.3;
3.4 Hz, 1H, H-4), 3.88 (s, 3H, H3CO), 1.48 (d, J = 7.3 Hz, 3H,
H3CCH). HR ESI MS m/z Calcd for C18H18N2O3Na:
333.1215. Found: 333.1219.
N-(2-Methoxyphenyl)-5-methyl-3-phenyl-4,5-dihydroisoxazole-4-carboxamide (15h). A colorless glass. IR
(KBr) νmax 3446, 3080, 3005, 2980, 2910, 2840, 1687, 1600,
1533, 1490, 1460, 1440, 1380, 1340, 1290, 1255, 1220, 1180,
1120, 1024, 920, 950, 805, 757, 697 cm-1. 1H NMR (CDCl3,
200 MHz) d 8.3-6.8 (m, 9H, H-6“, H-5’, H-4‘, H-3’, H-2’, H-6’,
H-5”, H-4”, H-3”), 5.19 (qd, J = 6.4; 4.4 Hz, 1H, H-5), 4.09 (d,
J = 4.4 Hz, 1H, H-4), 3.72 (s, 3H, H3CO), 1.48 (d, J = 6.4 Hz,
3H, H3CCH). HR ESI MS m/z Calcd for C18H18N2O3Na:
333.1215. Found: 333.1214.
Fungicidal testing
The compounds were screened for fungicidal activity in vitro
test carried out for Fusarium culmorum Sacc., Phytophthora
cactorum Schroek, Alternaria alternata Keissl.(Fr.), Rhizoctonia solani Kuhn, Botrytis cinerea Pers. Ex Fr, which involved
determination of mycelial growth retardation in potato-glucose
agar (PGA). Stock solutions of test chemicals in acetone were
added to agar medium to give a concentration of 200 μg mL-1
and dispersed into Petri dishes. Four discs containing test fungus were placed at intervals on the surface of the solidified
agar and the dishes were then inoculated for 4-8 days depending on the growth rate of the control samples, after which fungal growth was compared with that in untreated control
samples. The fungicidal activity was expressed as the percentage of plant infection compared to that on the control. The results of the screening are given in Table 3.
Enantioselective synthesis of isoxazolecarboxamides and their fungicidal activity
Acknowledgment
This work was supported in part by the Polish Ministry of
Science and Higher Education Research (Grant
429/E-142/S/2010), which is gratefully acknowledged.
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