SYNTHESIS, CHARACTERIZATION AND EVALUATION OF PAMAM
DENDRIMER-GOLD COMPLEX AS AN ANION RECOGNITION
MATERIAL
MUHAMMAD NOR FAZLI BIN ABD MALEK
A Project Report Submitted in Partial Fulfillment of the
requirements for the Award of the degree of
Master of Science (Chemistry)
Faculty of Science
Universiti Teknologi Malaysia
SEPTEMBER 2009
iv
This report writing is dedicated to my beloved parent
Abdul Malek Mohd Yusop & Jemilah Ahmad and my family members,
to my adorable supervisor, Prof Dr Salasiah Endud, and also
to my fellow friends.
Thanks for everything…
v
ACKNOWLEDGEMENT
My most appreciation is dedicated to Allah the Almighty with His concern to
give consent for me completing the postgraduate research project on time.
As the person who has been raising me up to who I am now, I would never
utter even a word to describe my everlasting love towards my father, Abd Malek Bin
Mohd Yusop, and my mother Jemilah Bt Ahmad.
Thank you for being the
wonderful parents on earth!
Special thanks to Prof. Dr Salasiah Endud who handled the supplements
process with care and attention to detail and also having the vision to see the project
report before it existed and jump in with her own, to make sure every detail was in
place to make the project report a success.
There is a saying goes that’s what friends are for. I wish fabulous
appreciations to all my friends. The sharing of idea through teamwork among us has
developed honestly for the sake of learning. Thanks, guys for always being there for
me. In addition, my appreciation also goes to all staff at Chemistry Department
especially the lab assistants and all staff in Institute Ibnu Sina.
I would also like to thank to my friends, Chin and Azizi for helping me in this
research, for their support and also valuable knowledge for me in carrying out the
laboratory work.
In the spirit of knowledge, I hope to provide useful inputs and remarkable
insights for the readers in my research area.
vi
ABSTRACT
Dendrimers are highly branched, monodisperse macromolecules and this field of
study has increased rapidly from the time they were discovered about twenty years
ago. In this study, PAMAM dendrimer was successfully synthesized by a divergent
synthesis route using the reagent excess method starting from ethylenediamine
(EDA) followed by consecutive Michael addition and ester amidation reaction.
Methanol was used as solvent and three dendrimer generations were prepared: G 1.0,
G 2.0 and G 3.0. For preparation of PAMAM-thiol functionalized gold nanoparticle
in-situ reduction of gold from Au3+ to Au0 was achieved through the reaction of
nanogold-thiol functionalized PAMAM dendrimer with sodium borohydrate
(NaBH4). The PAMAM dendrimer-gold complex gave light purple solution and was
characterized by 1H nucleus magnetic resonance (NMR), Fourier transform infrared
(FT-IR), and ultraviolet-visible (UV-Vis) spectroscopies. Analysis of the UV-Vis
spectral analysis of the PAMAM dendrimers showed that the wavelength maximum,
λmax significantly shifted from 330.15 nm to 517.28 nm with the addition of goldthiol nanoparticles due to binding of the thiol functional group to gold particles.
Anion recognition ability of the PAMAM-thiol functionalized gold nanoparticle has
been studied by treating the PAMAM dendrimer-gold complex with nitrate ion.
Based on the UV-Vis spectra, the wavelength maximum of Au(III) was shifted from
526.98 nm to higher wavelength upon binding of the nitrate anion to the surfaces of
gold-thiol nanoparticles which the process involved excitation of the electrons from π
→ π*.
vii
ABSTRAK
Dendrimer adalah makromolekul ekasebar dengan struktur bercabang-cabang dan
bidang kajian ini telah berkembang pesat sejak penemuannya hampir dua puluh
tahun yang lalu. Dalam kajian ini, dendrimer PAMAM telah berjaya disintesis
dengan menggunakan kaedah sintesis divergen dengan menggunakan kaedah reagen
berlebihan bermula dengan etilenadiamina (EDA) diikuti tindak balas penambahan
Michael dan amidasi ester secara berturutan. Metanol telah digunakan sebagai
pelarut dan tiga generasi dendrimer telah disediakan iaitu G 1.0, G 2.0 dan G 3.0.
Bagi penyediaan PAMAM-tiol berfungsikan nanopartikel emas penurunan in-situ
emas dari Au3+ kepada Au0 telah dijalankan melalui tindak balas dendrimer PAMAM
berfungsikan nanoemas-tiol dengan natrium borohidrat (NaBH4). Kompleks
dendrimer PAMAM-emas tersebut menghasilkan larutan berwarna ungu muda dan
telah dicirikan menggunakan spektroskopi Resonan Magnet Nuklear 1H (RMN),
inframerah transformasi Fourier (FT-IR) dan ultralembayung-nampak. Analisis
spectrum ultralembayung-nampak dendrimer PAMAM menunjukkan panjang
gelombang maksimum, λmax secara signifikan telah beranjak dari 330.15 nm kepada
517.28 nm dengan penambahan partikel nanoemas-tiol akibat penambatan kumpulan
berfungsi tiol oleh partikel emas. Sifat pengenalpastian anion PAMAM berfungsikan
partikel nanoemas-tiol telah dikaji melalui tindak balas kompleks dendrimer
PAMAM-emas dengan ion nitrat. Berdasarkan spektrum UV-vis, panjang gelombang
maksimum Au(III) didapati beranjak dari 526.98 nm kepada panjang gelombang
yang lebih tinggi apabila terjadi penambatan ion nitrat pada permukaan nanopartikel
emas-tiol yang mana proses tersebut melibatkan pengujaan elektron dari orbital π →
π*.
viii
TABLE OF CONTENTS
CHAPTER
I
TITLE
PAGE
SUPERVISOR VERIFICATION
ii
DECLARATION
iii
DEDICATION
iv
ACKNOWLEDGEMENTS
v
ABSTRACT
vi
ABSTRAK
vii
TABLE OF CONTENTS
viii
LIST OF TABLES
xi
LIST OF FIGURES
xii
LIST OF ABBREVIATIONS
xv
LIST OF APPENDICES
xvi
INTRODUCTION
1.1 Background of the Study
1
1.2 Problem Statement
5
1.3 Objective of the Study
7
1.4 Scope of the Study
7
1.5 Outline of the Study
8
ix
II
LITERATURE REVIEW
2.1 Introduction
10
2.2 Synthesis of Dendrimer
11
2.3 Poly(amidoamine) (PAMAM) Dendrimer
16
2.4 Synthesis usingEthylenediamine as Core
17
2.5 Characterization
20
2.5.1 Fourier Transformed Infrared (FTIR) Spectroscopy
20
2.5.2 UV-Vis Spectroscopy
21
1
13
23
2.6 Application of PAMAM Dendrimer
23
2.7 PAMAM-thiol functionalized gold nanoparticles
25
2.5.3 H and C Nuclear magnetic resonance (NMR)
III
EXPERIMENTAL
3.1 Chemicals and Materials
28
3.2 Preparation of Poly(amidoamine) (PAMAM) Dendrimer
28
3.3 Synthesis of Dendrimer Gold Nanoparticles
31
3.4 Synthesis of PAMAM Dendrimer Encapsulated Gold
Nanoparticles
3.5 Anion Recognition Test
IV
31
32
RESULTS AND DISCUSSION
4.1 Preparation of PAMAM Dendrimer
33
4.2 Characterization of PAMAM Dendrimer
41
4.2.1 Fourier Transformed Infrared (FTIR) Spectroscopy
41
4.2.2 Nucleus Magnetic Resonance (NMR) Spectroscopy
44
4.3 PAMAM-thiol functionalized gold nanoparticles
48
4.4 Preparation of PAMAM-gold nanocomposite
50
4.5 Characterization of PAMAM-thiol functionalized gold
x
nanoparticles
4.6 Anion Recognition Test
V
51
54
CONCLUSION AND RECOMMENDATIONS
5.1 Conclusion
57
5.2 Recommendations
58
REFERENCES
59
APPENDICES
69
xi
LIST OF TABLES
TABLE NO
TITLE
PAGE
2.1
Band position of dendrimer spectrum
21
2.2
UV absorption peak of PAMAM SH–Au samples
22
3.1
Stoichiometry of reactants in preparation of PAMAM.
30
4.1
Main peaks that obtained from FTIR spectrum for PAMAM G
0.5 and G 1.0
4.2
4.3
41
Assignment of 1H NMR for PAMAM dendrimers of half
generation and full generation
47
UV absorption peak of PAMAM -thiol functionalized gold
52
nanoparticles samples.
4.4
UV-vis absorption peak of PAMAM-thiol functionalized gold
nanoparticles and reaction with nitrate ion samples.
55
xii
LIST OF FIGURES
FIGURE NO
TITLE
PAGE
1.1
Generation 1.0 of PAMAM dendrimer with ethylenediamine
3
core and amine surface group
1.2
Divergent procedures for macromolecular construction
4
1.3
Flowchart of the study
8
2.1
Convergent method in synthesizing dendrimer
11
2.2
Divergent method in synthesizing dendrimer
12
2.3
Various types of coordinated dendrimers
14
2.4
Typical PAMAM construction via a divergent process
18
2.5
A route to unsymmetrical PAMAMs derived from an
ethylenediamine core
2.6
FT-IR spectra of G3.0 PAMAM dendrimer
2.7
UV–vis spectral change of PAMAM-SH–Au aqueous
suspension at 20 °C
2.7
1
19
20
22
H NMR spectra of the (1) G3.0 PAMAM dendrimer and (2)
thiol-terminated G3.0 PAMAM dendrimer. (3) The peak
assignment
23
2.8
Example of metal ion located at central core of dendrimer
26
3.1
Reaction of half generation of PAMAM dendrimer
29
3.2
Reaction of full half generation of PAMAM dendrimer
30
4.1
Synthesis of PAMAM dendrimer
34
4.2
Defective structure of the PAMAM dendrimer A) missing
arm, B) dimmers, C) Intramolecular cyclization.
35
xiii
4.3
The structures of PAMAM dendrimer (a) G 0.5 (b) G 1.0 (c)
G 1.5 (d) G 2.0 (e) G 2.5 (f) G 3.0.
4.4
Rubbery product due to oxidation of the full generation of
PAMAM dendrimer
4.5
40
The comparison spectrum between half generation (ester
terminated) and full generation of amine-terminated
4.6
39
42
FTIR spectrum of PAMAM dendrimer (G 0.5, G1.0, G1.5,
G2.0, G2.5 and G3.0)
43
4.7
Formation of amide bonding
44
4.8
Half branch of PAMAM dendrimer of (a) G0.5 (b) G 1.0 (c)
G1.5 (d) G2.0 (e) G2.5 and (f) G3.0
4.9
Reaction (a) and proposed mechanism (b) of PAMAM-thiol
functionalized gold nanoparticles.
4.10
49
Violet solution of PAMAM-thiol functionalized gold
nanoparticles.
4.11
46
49
Purple-red solution of PAMAM-gold nanocomposite
solution
50
4.12
Formation of PAMAM-gold nanocomposite
51
4.13
The crystal field diagram to show the occurring of electronic
transition in d9 octahedral system.
9
4.14
The change in d orbital energy label in gold
4.15
UV-vis spectrum for PAMAM dendrimer, PAMAM + gold
and PAMAM + thiol gold.
4.16
52
53
Proposed mechanism for the PAMAM dendrimer-gold
complex
4.17
51
52
UV-vis absorption spectrum of PAMAM-thiol functionalized
gold nanoparticles and reaction with ammonium nitrate
samples.
4.18
Binding between S, Au and NO3- in formation of PAMAM
dendrimer-gold complex.
53
54
xiv
4.19
Mechanism of anion recognition by PAMAM dendrimergold complex
54
xv
LIST OF ABBREVIATIONS
FTIR
-
Fourierr transform infrared
EDA
-
Ethylenediamine
TEM
-
Transmission electron microscopy
UV-vis
-
Ultraviolet-visible
Au
-
Aurum
PAMAM
-
Poly(amidoamide)
λmax
-
Maximum wavelength
SH
-
Thiol group
NMR
-
Nuclear magnetic resonance
NaBH4
-
Sodium Boro Hydrate
S
-
Sulfur
-
Nitrate ion
NO3
-
xvi
LIST OF APPENDICES
APPENDICES
TITLE
PAGE
A
1
H NMR Spectra for G 0.5 PAMAM Dendrimer
69
B
1
H NMR Spectra for G 1.0 PAMAM Dendrimer
70
C
1
H NMR Spectra for G 1.5 PAMAM Dendrimer
71
D
1
H NMR Spectra for G 2.0 PAMAM Dendrimer
72
E
1
H NMR Spectra for G 2.5 PAMAM Dendrimer
73
F
1
H NMR Spectra for G 3.0 PAMAM Dendrimer
74
G
Infrared Spectra of PAMAM dendrimer G 0.5
75
H
Infrared Spectra of PAMAM dendrimer G 1.0
76
I
Infrared Spectra of PAMAM dendrimer G 1.5
77
J
Infrared Spectra of PAMAM dendrimer G 2.0
78
K
Infrared Spectra of PAMAM dendrimer G 2.5
79
L
Infrared Spectra of PAMAM dendrimer G 3.0
80
CHAPTER 1
INTRODUCTION
1.1
Background of Study
Since the pioneering work of well-defined, three-dimensional structural order
macromolecules by Vögtle [1], Tomalia [2,3], and Newkome [4], interest in dendrimers
and hyperbranched polymers has been increasing at an amazing rate.
The study of these polymers expands to all areas including theory, synthesis,
characterization of structures, and properties, and investigations of potential applications.
In the beginning the research on dendrimers focused on the synthesis, characterization,
and properties of perfect dendrimers of higher generations.
For the synthesis of
dendrimers constructed by step-by-step sequences, two fundamentally different
strategies, the divergent approach (from the inside out) [2,4] and convergent approach
(from the outside in) [5], were employed. In either way, dendrimers can be prepared with
high regularity and controlled molecular weights, and the macromolecules consist of a
polyfunctional central core covalently linked to layers of repeating units (generations)
and a number of terminal groups (Figure 1.1). These units are interdependent and create
a unique molecular shape, leading to intrinsic properties such as high solubilities and low
viscosity.
Dendrimers free-shaped synthetic macromolecule has garnered a great deal of
scientific interest due to their unique molecular nanostructure. Used in a variety of
2
scientific applications, the use of dendrimers is now widely regarded as a safer, more
precise, and more effective way to practice medicine [6].
Nanocomposites are materials that are created by introducing nanoparticulates
(often referred to as filler) into a macroscopic sample material (often referred to as the
matrix).
This is part of the growing field of nanotechnology.
After adding
nanoparticulates to the matrix material, the resulting nanocomposite may exhibit
drastically enhanced properties.
For example, adding carbon nanotubes tends to
drastically add to the electrical and thermal conductivity. Other kinds of nanoparticulates
may result in enhanced optical properties, dielectric properties or mechanical properties
such as stiffness and strength.
In general, the nanosubstance is dispersed into the matrix during processing. The
percentage by weight (called mass fraction) of the nanoparticulates introduced can
remain very low (on the order of 0.5% to 5%) due to the incredibly low filler percolation
threshold, especially for the most commonly used non-spherical, high aspect ratio fillers
(e.g. nanometer-thin platelets, such as clays, or nanometer -diameter cylinders, such as
carbon nanotubes). Dendrimers are known for their three-dimensional, monodispersed,
highly branched, macromolecular nanoscopic architecture with a number of reactive end
groups [7]. Commercially available PAMAM (poly(amidoamine)) dendrimer prepared
by the divergent growth approach of Tomalia et al. are one of the most widely used
dendrimer scaffolds in biology. These macromolecules have uniform size and monodispersed. Furthermore, high structural and chemical homogeneity of dendrimer might
facilitate quality control of their drug conjugates in production.
3
NH2
H2N
O
O
NH
HN
N
N
NH
HN
O
O
NH2
H2N
Figure 1.1: Generation 1.0 of PAMAM dendrimer with ethylenediamine
core and amine surface group.
The divergent route to dendrimer synthesis is based on the construction of a
molecular superstructure starting with a focal point or core and progressing outward to
the periphery, as illustrated in Figure 1.2. Dendrimers are built in layers, or generations,
upon a defined core that possesses a specific number of active sites, to which the
successive tiers are, for the most part, covalently attached. The number of active sites on
the core determines their n-directionality and limits the number of building blocks that
can be added to form the next generation. This trend is repeated (iterative synthesis) as
the reactive sites on the periphery of the previous generation are revealed for the
assembly of the next generational growth layer.
4
Figure 1.2: Divergent procedures for macromolecular construction.
Assuming that the monomer’s functional group(s), steric hindrance, and active
site accessibility do not interfere with the construction of ideal dendrimers, the divergent
process permits the exponential growth of free active sites per generation. Perfect growth
is only achieved when each active site is occupied by a building block (monomer)
denoting the next tier; otherwise, imperfect structural assembly results leading to internal
termini and variable internal void regions, which starts to resemble a hyperbranched
motif normally derived by a random one-step procedure.
If these imperfections or
‘‘branching defects’’ occur early in the generational growth, they can have serious
repercussions on the overall micellar properties of the resulting dendrimer.
5
1.2
Problem Statement
The study of anion has been a critical part in the most recent research in the area
host-guest chemistry [8]. In order to differentiate a target anion from other, the host
molecule must be carefully designed, considering not only the structural complementary
interaction between the ion-molecule pair, but also interaction with solvent molecules.
The host molecules for anion recognition comprise at least one interaction site that
improves the selective interaction for the target anion and overcomes the salvation energy
of the target anion in the aqueous phase. Among this host molecules, dendrimers are of
considerable interest as anion recognition material because they can provide dedicated
single-point interactions with the capability to further modify the host molecular structure
with a number of reactive end groups as well as possess internal cavities [2]. These
characteristics, along with water solubility, are some of the features that make them
attractive for environmental remediations [9].
Dendrimers that are functionalized with transition metals in the core can
potentially mimic properties of enzymes, their efficient natural counterparts (e.g.
cytochrome P-450), whereas the peripheral-functionalized systems is proposed to provide
ideal building blocks for the development of high-capacity, selective and recyclable
ligands for the recovery of anions.
One of the major problems related to the preparation of dendrimer is to modify
the surface of the molecules [10]. The difficult part is to protect the active site in the
dendrimer. It is known that the difference functional group at end terminal of the
dendrimer gave difference characteristics. Hydrophilic functional group that is attached
to end terminal will make the dendrimer soluble in water meanwhile, hydrophobic
functional group given the ability to soluble in organic solvent. Recently, dendrimers
have been used in medical application as a censoring device by using gold encapsulated
at intermolecules cavities of the dendrimer. For example, detection of α-1-fetoprotein
6
(AFP) has been designed based on antibody functionalized core shell nanocomposite
particles [11].
By introducing gold at the peripheral of a dendrimer molecule, sophisticated
artificial receptors exhibiting specific anion recognition can be obtained. Molecular
recognition moieties attached at the peripheries of dendrimers may act as exo-receptors
for analytes. In previous studies researchers have developed metallocene as hosts for
recognition of various anions [9].
The presence of –NH groups within the dendritic structure was established to be
important for anion recognition [12-14]. In this research, PAMAM dendrimer-gold
complex was prepared by attaching gold-thiol nanoparticles to the periphery of PAMAM
dendrimer by the divergent method with the aim to prevent its aggregation and improve
the solubility in water. PAMAM encapsulated gold nanoparticles was prepared only as a
comparison. Gold was chosen in this study because of its nanoparticles size and high
sensitivity to UV-Vis detection even in a small amount and soluble in most inorganic
solvents. The capability of a methanol soluble dendrimer to encapsulate and transport
PAMAM-gold thiol derivatives selected both as model compounds and for their potential
anion recognition properties e.g. nitrate will be investigated. The anion recognition
studies will be conducted by UV-Vis spectroscopy. The recognition of anions is deemed
possible as a result of electrostatic interaction between the gold third linkage of the
dendrimer and the anion.
7
1.3
Objective of the Study
1. To synthesize and characterize Poly(amidoamine) (PAMAM) dendrimers with
various generation number (G = 0.5,1.0,1.5,2.0,2.5 and 3.0)
2. To synthesize and characterize PAMAM dendrimer-gold complex.
3. To synthesize PAMAM encapsulated gold nanoparticles for comparison with
PAMAM dendrimer-gold complex.
4. To study the ability of PAMAM dendrimer-gold complex to bind with nitrate
ion using UV-Vis spectroscopy.
1.4
Scope of the Study
The scope of this study includes the synthesis of PAMAM dendrimer via Michael
Addition reaction by divergent method. PAMAM dendrimer-gold complex was
synthesized by addition of gold-thiol nanoparticles into the full generation of PAMAM
dendrimer. PAMAM encapsulated gold nanoparticles was synthesized by reduction of
tetrachloroauric acid (HAuCl4) by using reducing agent, sodium borohydrate (NaBH4).
The generation number of PAMAM dendrimer was determined by using several
characterization techniques such as Fourier Transform Infrared (FTIR) Spectroscopy and
1
H Nuclear Magnetic Resonance (NMR) Spectroscopy. After addition of gold-thiol
nanoparticles, the chemical properties of the PAMAM dendrimer gold-thiol
functionalized complex was characterized using Ultra Violet-Visible (UV-vis)
Spectroscopy.
8
1.5
Outline of the Study
This dissertation illustrates the information concerning the synthesis and
characterization of PAMAM based gold nanocomposites. Chapter 1 elucidates the
research background and the important strategies to respond the current issue. Chapter 2
presents the literature review regarding this project where it contains some background
information about the whole research done. Chapter 3 describes the research
methodology with the characterization techniques used in this research as shown in the
flowchart in Figure 1.3. Chapter 4 explains the results and discussion of the PAMAM
dendrimers obtained their and its characterization. Finally, chapter 5 summarizes the
results obtained with recommendation for future work.
9
Ethylene Diamine
Methyl Acrylate
Synthesis of PAMAM Dendrimer
Characterization of PAMAM
FTIR and NMR
Gold-thiol Nanoparticles
Synthesis of PAMAM dendrimer-Gold
Complex
Characterization of PAMAM dendrimer-Gold
Complex
UV-vis Spectroscopy
Anion Recognition Test
Figure 1.3: Flowchart of the Research Methodology
CHAPTER 2
LITERATURE REVIEW
2.1
Introduction
Dendrimers are repeatedly branched molecules. The huge number of papers on
dendritic architectures such as dendrimers, dendronized, hyperbranched and brushpolymers has generated a vast variety of inconsistent terms and definitions making a clear
and concise unfolding of this topic highly difficult.
Dendritic molecules are repeatedly branched species that are characterized by
their structure perfection. The latter is based on the evaluation of both symmetry and
polydispersity. The area of dendritic molecules can roughly be divided into the lowmolecular weight and the high-molecular weight species. The first category includes
dendrimers and dendrons whereas the second encompasses dendronized polymers,
hyperbranched polymers, and brush-polymers (also called bottle-brushes).
There are over fifty families of dendrimers with their own unique properties [13].
The solubility of dendrimers is strongly influenced by the nature of surface group. A
dendrimers can be designed to be soluble in polar solvents by terminating with
hydrophilic groups at its surface and nonpolar solvents by having hydrophobic end
groups. For example, from previous study, HAuCl4 in aqueous solution was extracted to
11
toluene or chloroform using a hydrophobically modified poly(amidoamine) dendrimer
[14].
Therefore there are many possible applications that could be done on the different
specific properties provided by the dendrimers. It has reported that research on
dendrimers can be used in catalysis [15], delivery of drug, light harvesting properties
[16], used as low dielectric materials [17], as templates for the growth of single-wall
carbon nanotubes [18] and for sensing purpose [19].
2.2
Synthesis of Dendrimer
The synthesis of dendrimers is more closely related to “organic chemistry” rather
than “traditional polymer synthesis” regarding with the requirement for a number of
synthetic processes and procedures including repeating purification and exact
characterization. Two different synthetic strategies, a convergent [15], (Figure 2.1) and a
divergent growth approach [16], (Figure 2.2), are generally employed to construct
dendritic frameworks.
Figure 2.1: Convergent method in synthesizing dendrimer
12
The convergent methods were developed as a response to the weaknesses of the
divergent synthesis [18]. In the convergent approach, the dendrimer is constructed
stepwise, starting from the end groups and progressing inwards. When the growing
branched polymeric arms, called dendrons, are large enough, they are attached to a
multifunctional core molecule. The convergent growth method has several advantages. It
is relatively easy to purify the desired product and the occurrence of defects in the final
structure is minimised. It becomes possible to introduce subtle engineering into the
dendritic structure by precise placement of functional groups at the periphery of the
macromolecule. The convergent approach does not allow the formation of high
generations because steric problems occur in the reactions of the dendrons and the core
molecule
Figure 2.2: Divergent method in synthesizing dendrimer
In the divergent methods, dendrimer grows outwards from a multifunctional core
molecule. The core molecule reacts with monomer molecules containing one reactive and
two dormant groups giving the first generation dendrimer. Then the new periphery of the
molecule is activated for reactions with more monomers. The process is repeated for
several generations and a dendrimer is built layer after layer. The divergent approach is
13
successful for the production of large quantities of dendrimers. Problems occur from side
reactions and incomplete reactions of the end groups that lead to structure defects. To
prevent side reactions and to force reactions to completion large excess of reagents is
required.It causes some difficulties in the purification of the final product.
In both step-by-step synthetic approaches quantitative coupling reactions are
required to construct high generation dendrimers. A host of dendrimers have been
presented in the literatures: polyamidoamine (PAMAM), poly(propyl imine)(DAB-dendrNH2), polyethers, polyesters, poly(ester amides), poly(ether amides), polyalkanes,
polyphenylenes, poly(phenylacetylenes), polysilanes, phosphorus dendrimers and others
[20-24]. In addition to these covalently linked dendrimers, various types of coordinated
dendrimers have also been reported [25]. Typical examples used are shown in Figure 2.3
14
DAB-dendrimer-NH2
Freéchet’s Dendron
N
=
CH2CH2CONHCH2CH2N
Z
=
NH2
R
=
H2
C
n
PAMAM Dendrimer
Figure 2.3: Various types of coordinated dendrimers
For the sake of the rapid growth of exploration of dendrimers, the development of
more efficient synthetic processes circumventing the laborious and time-consuming steps
of activation or protection of monomers, condensation reactions, and purification by
chromatographic separations is highly desirable. Several methods to reduce the number
of synthetic steps and to obtain the desired dendrimer in high yields have been
demonstrated; a double-stage convergent growth approach [26] a hypercore or branched
15
monomer approach [27,28] double-exponential dendrimer growth [29], and orthogonal
coupling strategies [30].
Tomalia et al. [31] initially reported in the literature the synthesis of
polyamidoamine dendrimers, which were generated from a three-directional core (e.g.,
ammonia) and possessed ½ N-branching centers as well as amide connectivity. Each
generation was iteratively constructed by the exhaustive Michael-type addition of methyl
acrylate to the amine termini (e.g., for an ammonia core) to generate a b-aminoacid ester
by amidation with excess ethylenediamine to produce the new, branched polyamine 16c.
This general procedure was repeated to create the higher generations (e.g., 16e). Similar
dendrimers were prepared by employing related cores, such as ethylenediamine as well as
aminoalcohols and other functionalized groups, such as amino and thiol moieties [30].
This procedure is applicable to most primary amines, resulting in the ½ Nbranching motif and has been commercialized based predominately on an
ethylenediamine core resulting in the most readily available dendritic [PAMAM]
architecture to-date. Other stable and practical frameworks have been considered [33-35].
In order to realize a high degree of synthetic perfection at each step (or a quest for
monodispersity) in the intermediates and products, the potential synthetic problems
associated with amidations using esters, such as intramolecular cyclization (lactam
formation), retro-Michael reactions [36], incomplete addition, and intermolecular
coupling have to be minimized; thus large excesses of the diamine, maintaining reaction
temperatures (<80 °C), and avoiding aqueous solvents are critical to optimize the
conversion at each branching termini.
This simple two-step procedure was noteworthy by allowing the preparation of
high molecular weight dendritic polymers possessing a repetitive, fractal-branched
infrastructure. It is important to note that even with optimized conditions, defects
produced by these undesired reactions can be, for the most part, suppressed but not totally
16
circumvented. An ESI-MS study (reported in 1999) on the G4 PAMAM indicated that the
analyzed sample possessed a structural purity of <8% ; this may bear out the statement
‘‘.the excess EDA required to make 95% or greater purity at generations higher than 4.0
becomes prohibitive experimentally’’ [37].
Although these dendritic structures derived from commercial sources possess low
structural ideality at G > 4, Baker et al. addressed the question “if these commercial
PAMAMs possess both generational and skeletal disparity due to the divergent synthetic
methodology, how many terminal amine groups reside on the proposed spheriodal
surface?”. Their conclusions, based on the G5 PAMAM used in their engineered
nanodevices, were derived from acetylation studies from which it was concluded that the
model G5 PAMAM had a ‘‘practical number of terminal amino groups’’ of 110
(calculated by NMR and potentiometric acidebase titration [38] vs. the theoretical
number of 128). The use of capillary electrophoresis added further support to the
assessment of the nanoplatforms for novel medical applications [39].
2.3
Poly(amidoamine) Dendrimer (PAMAM)
Poly(amidoamine) (PAMAM) dendrimers are the first complete dendrimer family
to be synthesized by the divergent method starting from ammonia or ethylenediamine
initiator core reagents. They are constructed using two-step reiterative reaction sequence
consisting of (a) double Michael addition of methyl acrylate to a primary amino group
followed by (b) amidation of the resulting carbomethoxy intermediated with a large of
ethylenediamine.
The related PAMAM-type dendrons have been conveniently and efficiently
synthesized on a solid support, and the products possessed good homogeneity [49]. This
solid phase procedure demonstrated that peptides and drugs can also be attached directly
17
onto dendrimer lattices or bound via a linker to its periphery. The G0.5 PAMAMs were
synthesized and capillary zone electrophoresis was used to separate the different
generations as well as for the characterization of specific generations [40 and 42].
Particularly, the hyperbranched PAMAM or the ‘‘dendrimer equivalent’’ has been
reported [41 and 44] and shown to possess a Mn of ca. 2000 and a polydispersity of 2
[45].
2.3.1
Synthesis using Ethylenediamine as Core
The synthesis of polyamidoamine dendrimers, which were generated from a
three-directional core (e.g., ammonia) and possessed ½ N-branching centers as well as
amide connectivity (Figure 2.4). Each generation was iteratively constructed by the
exhaustive Michael-type addition of methyl acrylate to the amine termini (e.g., for an
ammonia core, 2.4a) to generate a b-aminoacid ester (e.g., 2.4b), followed by amidation
with excess ethylenediamine to produce the new, branched polyamine 16c.
This general procedure was repeated to create the higher generations (e.g., 2.4e).
Similar dendrimers were prepared by employing related cores, such as ethylenediamine
as well as aminoalcohols and other functionalized groups, such as amino and thiol
moieties [46]. This procedure is applicable to most primary amines, resulting in the ½ Nbranching motif and has been commercialized based predominately on an
ethylenediamine core resulting in the most readily available dendritic [PAMAM]
architecture to-date.
18
b
NH2
CO2Me
NH3
a
N
O
H2N
CO2Me
NH2
3
N
HN
c
CO2Me
O
NH
N
CO2Me
HN
2
3
d
O
O
NH
N
NH
HN
HN
HN
NH2
O
e
2
2 3
Figure 2.4: Typical PAMAM construction via a divergent process
The ‘‘genealogically directed’’ synthetic nature of the PAMAM preparative
protocol was elaborated by Dvornic and Tomalia [47]. This protocol was essentially
comprised of an ‘‘excess monomer method’’ facilitating the isolation of dendritic
intermediates (i.e., generations) without excessive loss due to potential side reactions that
may occur with the reagents that were not intended to be structurally incorporated. Thus,
true molecular genealogy of this series can be examined from generation to generation by
electrospray mass spectroscopy. These authors [48-52] further published a treatise
describing the use of PAMAMs, as well as the concept of other dendritic systems, to
branched macromolecular architectures.
Unsymmetrical PAMAM-like dendrimers (Figure 2.4 (d)).have been crafted by a
divergent strategy whereby after the focal site was t-BOC-protected, the typical
sequential growth was terminated at the desired generation by capping with iso-
19
butylamine e the focal group was deprotected to generate a new starting point (Figure 2.5
(c)) for elaborating the other direction [53]. This procedure also gave rise to the
formation of the PAMAM-like dendron series
1.EDA
TFA/CH2Cl
2.MA
3.repeat 1,2,1
a
1.MA
c
2.EDA
1.MA
Figure 2.5: A route to unsymmetrical PAMAMs derived from an ethylenediamine core
[58].
20
2.4
Characterization
2.4.1
Fourier Transform Infrared (FT-IR) Spectroscopy
FT-IR was used to identify functional groups in the dendrimer that may determine
the formation of either ester terminated (half generation) or amide-terminated (fullgeneration) of PAMAM dendrimers. From the previous study [54], FT-IR spectrum
shows peaks at several wavenumbers like those shown in Figure 2.6.
Figure 2.6: FT-IR spectra of G3.0 PAMAM dendrimer [55]
Fig. 2.5 shows the FT-IR spectra between 4000 and 500 cm−1 of the dendrimer.
The band positions and their assignments are listed in Table 2.1. The band at 2574 cm−1
is due to the -SH group for thiol-terminated dendrimer, the bands positions of amides I
and II (1644 and 1555 cm−1) of dendrimer seems to be invariant, while the amides A and
B shift to higher regions (3425 and 3078 cm−1) after thiol modification. The strong
absorbance band at 1736 cm−1 from ester group of methyl mercaptoacetate disappears for
G 3.0-SH.
21
Table 2.1: Band position of dendrimer spectrum
Assignment
Amide A
Amide B
CH2 antisymmetric stretching
CH2 symmetric stretching
-SH
-COOAmide I
Amide II
CH2 scissoring
CH2 scissoring
CH2 wagging + amide III
2.4.2
Band position (cm-1)
3284
3075
2936
2865
1644
1556
1463
1438
1359
UV-Vis Spectroscopy
The effect of dendrimer generation on the optical properties of the suspensions
can be conveniently studied by UV–vis spectroscopy. The formation of nanogold
composite was demonstrated by the wavelength broadening and shifting at 520 nm as
shown in Figure 2.7. The peak shifts to higher wavelength for the lower generation
dendrimer, as shown in Table 2.2. The size of assembled nanogold composite decreased
with increasing dendrimer generation from 2 to 5.
22
Figure 2.7: UV–vis spectral change of PAMAM-SH–Au aqueous suspension at
20 °C.[53]
Table 2.2: UV absorption peak of PAMAM SH–Au samples
Sample
Peak (nm)
G2.0 Au
520
G2.0 SH–Au
558
G3.0 SH–Au
548
G4.0 SH–Au
542
G5.0 SH–Au
531
23
2.4.3
H1 and C13 Nuclear magnetic resonance (NMR)
Proton 1H NMR is a powerful tool to analyze the quality and purity of dendrimers
although it cannot determine the molecular distribution of PAMAM species [3]. The
spectrum that obtained was shown like Figure 2.8.
Figure 2.8: 1H NMR spectra of the (1) G3.0 PAMAM dendrimer and (2) thiol-terminated
G3.0 PAMAM dendrimer. (3) The peak assignment.[55]
2.5
Application of PAMAM Dendrimer
Dendrimers have been tested in preclinical studies as contrast agents for
magnetic resonance imaging [56]. Magnetic resonance imaging (MRI) is a diagnostic
method producing anatomical images of organs and blood vessels. Placing a patient in a
generated, defined, inhomogeneous magnetic field results in the nuclear resonance signal
of water, this is assigned to its place of origin and converted into pictures. Addition of
contrast agents (paramagnetic metal cations) improves sensitivity and specificity of the
method.
24
There are attempts to use dendrimers in the targeted delivery of drugs and other
therapeutic agents. Drug molecules can be loaded both in the interior of the dendrimers as
well as attached to the surface groups. Sialylated dendrimers, called sialodendrimers,
have been shown to be potent inhibitors of the haemagglutination of human erythrocytes
by influenza viruses. The first step in the infection of a cell by influenza virus is the
attachment of the virion to the cell membrane. The attachment occurs through the
interaction of a virus receptor haemagglutinin with sialic acid groups presented on the
surface of the cell [57]. Sialodendrimers bind to haemagglutinin and thus prevent the
attachment of the virus to cells. They can be useful therapeutic agents in the prevention of
bacterial and viral infections.
The combination of high surface area and high solubility makes dendrimers useful
as nanoscale catalysts [58]. They combine the advantages of homogenous and
heterogeneous catalysts. Homogenous catalysts are effective due to a good accessibility
of active sites but they are often difficult to separate from the reaction stream.
Heterogeneous catalysts are easy to separate from the reaction mixture but the kinetics of
the reaction is limited by mass transport. Dendrimers have a multifunctional surface and
all catalytic sites are always exposed towards the reaction mixture. They can be recovered
from the reaction mixture by easy ultrafiltration methods. The first example of a catalytic
dendrimer was described by the group of van Koten [59]. They terminated soluble
polycarbosilane dendrimers in diamino arylnickel (II) complexes. Such dendrimers can
be used in addition reactions of polyhaloalkanes.
An alternative application of dendrimers that has gained some attention is based
on nanostructures which can find use in environment friendly industrial processes.
Dendrimers can encapsulate insoluble materials, such as metals, and transport them into a
solvent within their interior. Cooper and co-workers [60] synthesised fluorinated
dendrimers which are soluble in supercritical CO2 and can be used to extract strongly
hydrophilic compounds from water into liquid CO2. This may help develop technologies
in which hazardous organic solvents are replaced by liquid CO2.
25
2.6
PAMAM-thiol functionalized gold nanoparticles
Composite materials (or composites for short) are engineered materials made
from two or more constituent materials with significantly different physical or chemical
properties which remain separate and distinct on a macroscopic level within the finished
structure. The physical properties of composite materials are generally not isotropic
(independent of direction of applied force) in nature, but rather are typically orthotropic
(different depending on the direction of the applied force or load).
Nanocomposites are materials that are created by introducing nanoparticulates
(often referred as filler) into a matrix material [61]. After adding nanoparticulates to the
matrix material, the resulting nanocomposites may exhibit an enhanced properties
whether optical properties, dielectric properties or mechanical properties such as stiffness
and strength.
The incorporation of metal component utilizing non-covalent interaction at the
exterior surface of or embedded within dendrimer framework is attractive strategy for the
design of applicable dendrimers. Based on a number of established coordination
chemistry, a variety of organometallic units as a core, junction units, modification motif
on the periphery, and repeat unit throughout the dendrimer are incorporated into the
structures. Among them the dendrimers with metals as repeat units are constructed by a
strategy called “complexes as metal/complexes as ligand”, utilizing rigid organic
molecules with two or three metal-binding sites, which are subsequently bridged with the
complementary metal component.
Balzani and colleagues [45] studied in details the construction of dendrimers
based on polypyridine-transition metal complexes). For such dendrimer syntheses, key
components are 2,20-bipyridine (bpy) as terminal ligand, 2,3-bis(2-pyridyl)pyrazine (2,3dpp) as bridging ligand and its methylate form. A typical example was shown in Figure
26
2.9 (Mc, MI, and Mp were metal ions located at central core, interior, and periphery).
Terpyridine and tetra (2-pyridyl)pyradine derivatives are also useful scaffold for the
construction of metallodendrimers [18]. The most striking characteristic of these
metallodendrimers is that each building block has intrinsic properties and that different
ligands-metal component or organic units can be placed into the specific sites in
dendrimers.
Figure 2.9 : Example of metal ion located at central core of dendrimer [82]
So far, metals such as Fe, Cu, Zn, Ni, Au, Co, Pd/Pt, Os/Ru, Rh, and Ge have
been incorporated as branching units or periphery of dendrimers. Some of these
metallodendrimers are widely studied as mimics of biological redox potential, sensors,
catalysts, new materials for energy conversion, and organic semiconductors.
27
Poly(amidoamine) (PAMAM) dendrimers, polymer molecules in the size range of
1-15 nm, have been successfully used as stabilizers and templates for inorganic
nanoclusters in aqueous or methanol solution In this approach, precursor ions are
accumulated within the dendrimer molecule due to electrostatic attraction or coordination
to its amine groups. For higher generation dendrimers, chemical reactions on these
precursor ions lead to the formation of inorganic colloids that are located inside
individual dendrimers [61].
CHAPTER 3
EXPERIMENTAL
3.1
Chemicals and Materials
Poly(amidoamine) (PAMAM) dendrimer (G 1.0, G 2.0 and G 3.0) were
prepared according to the previous literatures [1-4]. Dodecanethiol functionalized
gold nanoparticles (AuSHCH2(CH2)10CH3) was purchased from Aldrich Co.
Methanol that is used as solvent was obtained from Merck Co.
3.2
Preparation of Poly(amidoamine) (PAMAM) Dendrimer
There were two stages in preparing an EDA-core PAMAM dendrimer. First
stage is the addition of primary amine, ethylenediamine (EDA) to methyl acrylate
and followed by the amidation of the formed multiester with EDA. The repetition of
these two-stage procedures leads to higher generation of dendrimers.
STAGE 1: Synthesis of Ester-terminated dendrimer (Half Generation)
The synthesis of half generation of PAMAM dendrimers is illustrated in
Figure 3.1. The reaction mixtures were prepared in two parts. Part A consists of
ethylenediamine (EDA) dissolve in methanol and part B consists of methyl acrylate
(MA) in methanol. The mol ratio of these two materials (EDA and MA) is about 1:8.
29
These two part were then mixed together to initiate the reaction. Solution of part A
was added dropwise while stirring into the solution of part B at room temperature (27
ºC). The mixture was continuously stirred for 72 hours and was kept at room
temperature.
Then, the excess reactant, methyl acrylate and solvent were removed by
evaporation using a rotary evaporator at 70 ºC.
Generation 0.5
Figure 3.1: Synthesis of half generation of PAMAM dendrimer
STAGE 2: Synthesis of amine-terminated dendrimer (Full Generation).
The reaction mixtures were also prepared in two parts. Part one, primary
generation of PAMAM (A) prepared from stage 1 was dissolved in methanol and
part two consists of ethylenediamine (EDA) in methanol. Next, the multiester
solution in part one was gradually added to the stirred EDA solution from part two
until the addition was completed. Then, the mixture was stirred and allowed to react
for 72 hours at room temperature (Figure 3.2).
The excess methanol was removed by evaporation process in a rotary
evaporator at 70 ºC. Next, an excess EDA was removed using an azeotropic mixture
of toluene and methanol (9:1 v/v) and the remaining toluene was then removed by
azeotropic distillation with methanol. Finally, the excess methanol was removed by
evaporation process in a rotary evaporator.
30
Generation 0.5
Generation 1.0
Figure 3.2: Synthesis of full half generation of PAMAM dendrimer
Table 3.1 shows the stoichiometry of reactants used in preparation of
PAMAM dendrimer. The moles of reactants are important in order to ensure that the
reaction is complete.
Table 3.1 : Stoichiometry of reactants in preparation of PAMAM.
Generation
of PAMAM
Molecular
Weight (gmol1
EDA
)
M.A
0.5
404
0.1
0.4
1.0
516
0.3
-
0.076 (0.5)
1.5
1204
-
0.4559
0.0569 (1.0)
2.0
1428
0.276
-
0.034 (1.5)
2.5
2804
-
0.272
0.017 (2.0)
3.0
3252
0.1553
-
0.0097 (2.5)
*G = generation
Mole
PAMAM (G)*
31
3.3
Synthesis of PAMAM-thiol functionalized gold nanoparticles
Gold-thiol solution and 1 wt.% dendrimer aqueous solutions were mixed at
the ratio of [PAMAM]/[Au] = 100/1 (mol/mol) and incubated for 5 hours at room
temperature to form stable aqueous solutions. A control experiment was also
conducted by using PAMAM encapsulated gold nanoparticles.
3.4
Synthesis of PAMAM Dendrimer Encapsulated Gold Nanoparticles
Dendrimer-gold nanocomposites were prepared by in situ reduction of
HAuCl4 in the presence of PAMAM dendrimers being synthesized. First, 50 mL of a
0.01 M aqueous solution of HAuCl4.3H2O (0.1969g) was mixed with 50 mL of
solution either a first or a second full-generation (G1 or G2), amine-terminated
PAMAM dendrimers. The reacting mixture was stirred vigorously during the
addition of HAuCl4.3H2O. The molar ratio of Au to the number of primary amine
groups of the dendrimers was kept constant at ~ 1:11. The pale yellow solution
obtained was stirred for another 20 minutes to provide ample time for HAuCl4 to
react with PAMAM dendrimers.
In the next stage, the above solution was reduced by slow addition of freshly
prepared 0.01 M aqueous solution of NaBH4 and the resulting Au(0) was formed.
The reaction was left at room temperature for another one hour with vigorous stirring
to complete the reaction. The initially pale yellow solution would immediately turn
to brown and finally to purple-red solution. The colloidal solution is expected to
remain stable at room temperature for several weeks and does not agglomerate. The
colloidal nanocomposite was precipitated by slow addition of non-solvent,
tetrahydrofuran (THF) into the solution. Finally, the nanocomposites obtained were
centrifuged to separate them from the liquid phase.
32
3.5
Anion Recognition Test
Full generation of PAMAM was mixed with saturated solution of ammonium
nitrate (NH4NO3) and stirred for 24 hours. The solution mixture was then
characterized by using UV-vis Spectrocopy.
CHAPTER 4
RESULTS AND DISCUSSION
4.1
Preparation of PAMAM Dendrimer
PAMAM dendrimers are generally known as monodispersed, highly
branched and globular assembles. Therefore, synthesis and analysis of the dendrimer
mixtures are complicated problems not only because of the large number of different
regular structural units, but also because of numerous possible “structural errors”
[62] present in the polymeric mixtures.
In this study, PAMAM dendrimers were synthesized using divergent method.
Ethylenediamine (EDA) as core unit and methyl acrylate (MA) as building blockwas
mixed in methanol (MeOH) that acts as a solvent. This mixture was stirred for at
least 3 days to complete the reaction. The overall reaction in the preparation of
PAMAM dendrimer is shown in Figure 4.1.
34
O
O
OMe
MeO
NH2
Methyl Acrylate
N
H2 N
N
Ethylenediamine
PAMAM G 0.5
MeO
OMe
O
O
Ethylenediamine
NH2
H2N
O
O
NH
HN
N
N
NH
PAMAM G 1.0
HN
O
O
NH2
H2N
Figure 4.1: Synthesis of route to PAMAM dendrimer
As discussed in section 1.1, there is a possibility for the dendrimer to have
incomplete structure, which is also known as “missing arms” or defective branched.
In cases where the reaction was not complete, the possibility of ‘missing arms’ in the
dendrimer was quite high. Figure 4.2 shows the possible defective structure of
PAMAM dendrimer that could be obtained in this study.
35
H
N
C
H2
NH2
designates
NH2
O
H2N
H
N
N
A
NH2
H2N
H2N
NH2
H2N
O
O
H
N
N
N
N
N
H
N
B
NH2
H2N
NH2
H2N
O
NH
N
N
H2N
C
NH
O
Figure 4.2: Defective structure of PAMAM dendrimer with A) missing arm, B)
dimers, C) Intramolecular cyclization.
36
O
O
OMe
MeO
N
N
OMe
MeO
O
O
PAMAM G 0.5
(a)
NH2
H2N
O
O
NH
HN
N
N
PAMAM G 1.0
(b)
HN
O
NH
O
NH2
H2N
[continue on next page]
37
MeO
OMe
O
N
O
OMe
O
MeO
O
O
N
NH
O
HN
N
N
NH
O
PAMAM G 1.5
(c)
HN
MeO
O
OMe
O
N
O
O
N
O
MeO
OMe
NH2
H 2N
HN
NH
NH2
O
H 2N
N
O
HN
NH
O
O
O
N
NH
O
HN
N
N
NH
O
HN
HN
O
N
PAMAM G 2.0
(d)
O
O
NH
O
N
O
H 2N
NH2
HN
NH
NH2
H 2N
[continue on next page]
PAMAM G 2.5
(e)
38
[continue on next page]
PAMAM G 3.0
(f)
39
Figure 4.3: Generation of PAMAM dendrimer (a) G 0.5 (b) G 1.0 (c) G 1.5 (d) G
2.0 (e) G 2.5 and (f) G 3.0
40
Theoretically, the structures for the different generation of PAMAM
dendrimers are shown in Figure 4.3. For each half generation of PAMAM dendrimer
(Figure 4.3 (a),(c) and (d)), the terminal group is ester (-COOCH3). Meanwhile, for
full generation (Figure 4.3 (b),(d) and (e)), the terminal group is amine (-NH2) due to
the bonding between EDA and MA by formation of an amide bond.
During preparation of full generation dendrimer, the formation of rubbery
yellowish product was obtained as shown in Figure 4.4. The rubbery form was very
stable and difficult to handle because it did not dissolved in any solvent. It is
suggested that the product was formed due to intramolecular cyclization of the
terminal amine groups. Formation of this rubbery compound can be avoided by
keeping the PAMAM dendrimer in an oxygen-free atmosphere or placed under
flowing N2 gas.
Figure 4.4: Rubbery product due to intramolecular cyclization of the
PAMAM dendrimer
41
4.2
Characterization of PAMAM Dendrimer
4.2.1
Fourier Transformed Infrared (FTIR) Spectroscopy
The most useful aspect of infrared spectroscopy is its ability to identify
functional groups in the dendrimer that could determine the formation of either ester
terminated (half generation) or amide-terminated (full-generation) of PAMAM
dendrimers. Table 4.1 shows the main peaks obtained from the FTIR spectra for all
generations of PAMAM dendrimers.
Table 4.1: The main peaks of FTIR spectra for all generations of PAMAM
dendrimer
Functional
Group
C=O (ester)
Wavenumber (cm-1)
Generation
G 0.5
1743
G 1.0
-
G 1.5
1732
G 2.0
1732
G 2.5
1735
G 3.0
1727
C=O (amide)
-
1649
1646
1656
1650
1644
N-H (amine)
3453
3354
3296
3290
3372
3407
C-N
-
1028
-
1031
-
1025
C-O
1202
-
1199
-
1203
-
Figure. 4.5 shows a comparison of the FT-IR spectra of the half and full
generation of PAMAM dendrimer. The spectra exhibit the peaks of the functional
groups that exist in two types of PAMAM dendrimers, which are ester-terminated
groups and amide-terminated groups. The main peak is the carbonyl group with
different end-substitutes on C=O. For the ester-terminated PAMAM groups, the
absorption of carbonyl ester group (-COOCH3) is shown at around 1730 cm-1 which
is due to the formation of ester. It should be noted that the FTIR spectrum of the halfgeneration of PAMAM dendrimer, shows this ester band, whereas the band is not
observed for the full-generation dendrimer.
42
Half Generation
Full Generation
Figure 4.5: The comparison spectra between half generation (ester terminated) and
full generation of amine-terminated dendrimer.
Meanwhile, the carbonyl groups of amides absorbed at particularly low
frequency around 1640 - 1650 cm-1. This is because the dipolar resonance structure
places part of the pi electron between carbon and nitrogen (-CO-NH-), leaving less
than a full C=O double bond.
43
G 3.0
G 2.5
G 2.0
G 1.5
G 1.0
G 0.5
Figure 4.6: FTIR spectra of PAMAM dendrimers (G 0.5, G1.0, G1.5, G2.0,
G2.5 and G3.0)
Figure 4.6 shows the FTIR spectra of various generations of dendrimer
prepared in this study. Based on previous study [60], full generation dendrimer
contains primary amines as terminal groups. There are two spikes for the primary
amine in the N-H stretching region about 3300 cm-1. But according to the FTIR
spectra shown in Figure 4.6, most of full generations have two spikes of primary
amine stretching bands mixed with broadening of OH band in the range of 32003400 cm-1. This phenomenon is due to the methanol used stabilizing the PAMAM
dendrimer which was not totally removed in the synthesis. As a result, intense broad
band is observed in the FTIR spectra.
From the spectra, the peaks of ester band occurred at PAMAM G 2.0 and
PAMAM G 3.0. These peaks are not supposed to be observed if the ester group has
been reacted with EDA and formed amide bonding as shown in the reaction scheme
44
in Figure 4.7. However, it is suggested that ‘missing arms’ of amide were formed
because this reaction did not go to completion.
NH2
O
N
H
+
OCH3
H2N
- CH3OH
Amide bond
O
NH2
N
H
N
H
Figure 4.7: Formation of amide bonding
4.2.2
1
H Nuclear Magnetic Resonance (NMR) Spectroscopy
Proton 1H NMR is a powerful tool to analyze the quality and purity of
dendrimers although it cannot determine the molecular distribution of PAMAM
species. Figure 4.8 shows the half branch of PAMAM dendrimer of generation (a)
G0.5 (b) G 1.0 (c) G1.5 (d) G2.0 (e) G2.5 and (f) G3.0. Table 4.2 below shows the
summary of the proton assignment from the 1H NMR spectra for half and full
generation numbers of PAMAM dendrimers, respectively.
45
2
1
3
2
N
NH
4
7
1
4
5
N
OMe
NH2
6
3
O
5
O
8
N
N
(b)
(a)
O
3
4
1
5
7
N
N
OMe
2
NH
6
9
8
O
10
N
(c)
O
6
7
4
8
10
N
N
NH
1
NH2
3
9
12
2
11
13
N
5
NH
O
(d)
[continue on next page]
46
O
8
9
6
10
12
N
N
NH
7
NH
1
2
N
5
OMe
11
14
3
4
13
O
15
O
N
(e)
O
13
15
N
9
N
10
NH
3
NH
N
8
NH
4
7
16
18
O
NH2
1
5
14
17
N
2
11
12
6
O
(f)
Figure 4.8: Half branch of PAMAM dendrimer of (a) G 0.5 (b) G 1.0 (c) G 1.5 (d)
G 2.0 (e) G 2.5 and (f) G 3.0.
47
Table 4.2: Assignment of 1H NMR for PAMAM dendrimers of half generation and
full generation.
Assignment of
Proton
1
Intergral*
(δ ppm)
3H s
2
3
4
1&2
2H t
2Ht
2Hs
2H mix multiplet
3
4
5
6
7
1
2H mix multiplet
1H broad
2H broad
2H triplet
2H broad triplet
3H s
2&6
3&7
4&9
5
8
1
2H t
2H quartet
2H t
2H quintet
Not detected
2H triplet
3.902
2.709
3.014
2.768
2.8-2.9
~ 3.4
~ 8.41
~ 2.6
~3.0
~2.7
3.74
~ 2.6
2.8-2.9
~2.7
~3.3
2
3
4&9
5 & 10
6 & 11
7
8
12 & 13
1
2H quintet
2H quartet
1H t
2H t
2H t
2H
2H quartet
2H t
3H s
G 2.5
(Appendix E)
2, 6 & 12
3, 9, 13, 14 & 15
4 & 10
5 & 11
1
2H t
2H t
2H quartet
1H m
2H m
2
6.8
5.2
G 3.0
(Appendix F)
2
3
5, 9, 19
6, 11, 16
2H quintet
2H t
2H t
2H quartet
7.2
6.8
Not detected
5.6
Generation
G 0.5
(Appendix A)
G 1.0
(Appendix B)
G 1.5
(Appendix C)
G 2.0
(Appendix D)
*Multiplicity
s = singlet
t = triplet
m = multiplet
14.4
5.6
6.4
6.0
6.4
4.8
4.8
5.6
5.6
48
In the 1H NMR spectra (see Appendices A-F), the appearance of a peak at δ
3.9 ppm, is in the range of chemical shift for ester group. So, it can be concluded that
amine group has been replaced by acrylate (ester group) in the compound.
Meanwhile, for the full generation, the peak at δ 3.9 (-OCH3) disappeared meaning
that the ester group has been replaced by ethylenediamine (-NH2).
However, in the synthesis of PAMAM dendrimers, there are always present
‘trailing’ generation(s) as well as dimers and real PAMAM molecules always have
missing arms and have molecular loops [3, 6, 16]. These defects result from the four
main types of side reactions during the synthesis namely, are a retro-Micheal reaction
giving rise to asymmetrical structures due to the missing arms, dimers may form in
the amidation step, intramolecular cyclization (molecular loops) may also occur
during the same step as ethylenediamine is a bifunctional reactant and the residual
ethylenediamine (EDA) acts as a new core to initiate a trailing generation [63].
4.3
PAMAM-thiol functionalized gold nanoparticles
A PAMAM-thiol functionalized gold nanoparticle was successfully
synthesized by using in situ method. Nanogold-thiol particles was added into
PAMAM dendrimer and stirred for 60 minutes to complete the reaction as shown in
Figure 4.9(a). The proposed route to the formation PAMAM was shown
schematically in Figure 4.9(b). This step was done in order to bind gold nano
particles to dendrimer end terminal groups. Violet solution was obtained from this
addition (Figure 4.10) and gave evidence to support the formation of stable
PAMAM-thiol functionalized gold nanocomposite solution.
49
PAMAM
HS
NH2
Au
PAMAM
N
H
S
Au
(a)
+
PAMAM
Thiol-gold
nanoparticles
(b)
PAMAM-thiol functionalized
gold nanoparticles
Figure 4.9: Reaction (a) and proposed mechanism (b) of PAMAM-thiol
functionalized gold nanoparticles.
Figure 4.10: Violet solution of PAMAM-thiol functionalized gold
nanoparticles.
50
4.4
Preparation of PAMAM-gold nanocomposite
Preparation of gold nanocomposites were done using the first and second full
generation of PAMAM dendrimers having about more than 50% of their pheripheral
amine at the end of the PAMAM dendrimers. A two-step process to functionalize
gold with PAMAM dendrimer was used. Firstly, an aqueous gold solution (AuCl4-)
was mixed with PAMAM dendrimer solution (Gn) and then followed by reduction
process with 0.01 M NaBH4 in aqueous solution.
The pale yellow solution was vigorously stirred for 20 minutes to provide
enough time for HAuCl4 to react with dendrimer. Next, 0.01 M NaBH4 was slowly added
to this solution with stirring to reduce the Au(III) to zerovalent, Au(0). The purple-red
color of solution (Figure 4.11) formed after reduction aqueous solution of NaBH4
provide supports the formation of stable gold nanocomposite solutions [64]. The
formation of PAMAM dendrimer encapsulated with gold nanoparticles is shown
schematically in Figure 4.12.
Figure 4.11: Purple-red solution of PAMAM-gold nanocomposite solution
51
PAMAM Dendrimer
Encapsulated Au
nanoparticles
PAMAM Dendrimer
Figure 4.12: Formation of PAMAM-gold nanocomposite
4.5
Characterization of PAMAM-thiol functionalized gold nanoparticles
The formation of the dendrimer-thiol functionalized gold nanoparticles was
characterized by solution UV-Vis spectroscopy. This technique is useful because
gold is a d9 transition metal in Periodic Table. The configuration of electron at
ground state is t2g6eg3 changed to t2g5eg4 after being excited as shown in Figure 4.13
Ground State
Excited State
Figure 4.13: The crystal field diagram to show the occurring of electronic
transition in d9 octahedral system.
Therefore, the splitting energy ∆o or Dq of d9 are directly determined from
the maximum absorption wavelength of the respective spectrum. The observation
suggests that two possible transition processes with small different transition energy
level take place [63]. The change occurred due to the unsymmetrical occupying of
electron in d-orbital makes three degenerated orbitals in t2g state and two degenerated
orbital in eg state are no longer degenerated [49]. The energy level of the orbitals
split to various state labels as shown in Figure 4.14.
52
dy2-x2
dy2-x2
dy2-x2
dz 2
dxy
dz2
dz2
dxy
dxy
dyz
dxz
dxz
dyz
dxz
dyz
Figure 4.14: The change in d9 orbital energy label in gold.
Table 4.3 shows the λmax obtained from the spectra for PAMAM, PAMAMgold and PAMAM-thiol-gold. Figure 4.15 shows UV-Vis spectra of PAMAM
dendrimer, PAMAM + gold and PAMAM + thiol gold.
Table 4.3: UV absorption peak of PAMAM-thiol functionalized gold
nanoparticles samples.
Sample
Peaks (nm)
PAMAM
330.15
PAMAM – Gold
547.85
PAMAM – thiol - Gold
517.28
53
Figure 4.15: UV-vis spectrum for PAMAM dendrimer, PAMAM + gold and
PAMAM + thiol gold.
From the Table 4.4, there was shifting of the absorption peaks after
incorporated gold species in the PAMAM dendrimer. The maximum wavelength for
PAMAM-gold was shown by λmax, 547.85 nm. The excitation energy of shifting
obtained was lower than the shifting obtained for λmax of PAMAM-gold-thiol which
is at 517.28 nm.
The binding between thiol and gold in PAMAM-gold-thiol gave a lower λmax
than that of PAMAM-gold. Protonation of gold due to bonding between PAMAM
dendrimer and gold-thiol nanoparticles gave a lower λmax. The lone pair in no longer
available to interact with π cloud of thiol. A shift to shorter wavelength is called blue
shift [64].
On the other hand, reducing oxidation number of gold from Au(+3) to Au(0)
gave a higher shifting of λmax. This is because of addition of lone pair in gold [24].
This lone pair is available to interact with the π cloud of thiol. A shift to longer
wavelength is called red shift.
54
It is suggested that there are two possible steps for the formation of
dendrimer–gold complex: the first step is that the gold nanoparticle is surrounded by
several thiol-terminated dendrimers to form self-assembly monolayer (SAM); the
second step is that dendrimer-mediated networks are formed, each gold nanoparticle
may associate with more than one dendrimer molecule and, likewise, each dendrimer
molecule may bind to more than one gold nanoparticle, the result being a bridging
aggregation leading to controlled organization as shown in Figure 4.16.
Au-SH
PAMAM
Figure 4.16: Proposed mechanism for the dendrimer-mediated self-assembly
of gold nanoparticles.
4.5
Anion Recognition Test
PAMAM-thiol functionalized gold nanoparticles produced was reacted with
nitrate ion (NO3-) to test its potential ability as an anion recognition material. The
idea is a cationic ion will bind to gold from the dendrimer. From the result, there was
peak shifting of gold (III) to high wavelength as shown in Table 4.4.
55
Table 4.4: UV-vis absorption peak of PAMAM-thiol functionalized gold
nanoparticles and reaction with nitrate ion samples.
Sample
Peak (nm)
PAMAM + Goldthiol
517.28
PAMAM + Goldthiol + NO3-
526.98
From the spectra obtained (Figure 4.17), it was suggested that gold ions bind
to nitrate as shown by shifting of the peak to higher wavelength. It shows that the
binding that formed will reduce the energy of the nanocomposite or specifically gold
thiol bond due to bonding between thiol and nitrate as shown in Figure 4.18.
Figure 4.17: UV absorption spectrum of PAMAM-thiol functionalized gold
nanoparticles and reaction with ammonium nitrate samples.
56
S
Au
+
NO3-
S
Au
NO3
PAMAM
Figure 4.18: Binding between S, Au and NO3- in formation of PAMAM
dendrimer-gold complex.
The cationic characteristic of gold attracted nitrate ion to form electrostatic
interaction between them. Nitrate which is known as high field ligand makes the
bonding between gold and nitrate become weaker and gave a higher wavelength in
the UV-vis spectrum [56]. The proposed interaction of PAMAM-thiol functionalized
gold nanoparticles with nitrate ion is shown in Figure 4.19.
NO3-
PAMAM
Figure 4.19: Proposed interaction of nitrate ion with PAMAM-thiol functionalized
gold nanoparticles
CHAPTER 5
CONCLUSIONS AND RECOMMENDATIONS
5.1
Conclusions
PAMAM dendrimer-gold complexes were successfully synthesized via
Micheal addition and followed by amidation of ester. Three generation of dendrimers
were prepared which are G1.0, G2.0 and G3.0. The full generation of PAMAM
dendrimer was then used as a matrix for the preparation of dendrimer-gold
nanocomposites in solution via in-situ reduction of dodecanethiol functionalized gold
nanoparticles with sodium borohydrate (NaBH4) to produce Au(0). In recognition of
anion, there was red shifting of the wavelength λmax.
From FT-IR results, dendrimers of generation 2.0 and 3.0 exhibit defective
structures due to ‘missing arm’ as a result of incomplete synthesis. This conclusion is
supported by NMR data of the products obtained for G0.5 until G3 dendrimers.
PAMAM dendrimer is easily oxidized and this is the reason why some of the
generations PAMAM dendrimers obtained were in the gel or rubbery form.
.
Successful synthesis of PAMAM dendrimer-gold complex was confirmed by
UV-vis spectroscopy. The peak at around 520 nm shows the appearance of gold (III)
in the dendrimer. Peaks for PAMAM encapsulated gold nanoparticles shifted to
lower wavelength compared to PAMAM-gold-thiol complex were different due to
bonding between gold and the thiol functional group.
58
The interaction between the nitrate ion and gold-thiol was indicated by
shifting of λmax in the UV-vis spectrum for gold-thiol to higher upon addition of
nitrate ion wavelength. It is suggested that the bonding is electrostatic attraction
between cation gold and nitrate ion.
5.2
Recommendations
In this study, the characterization of dendrimer was carried out using UV-vis
spectroscopy. In order to validify the result obtained, it is suggested that other
characterization method such as cyclic voltammetry is employed as comparison.
In order to determine higher generation numbers of PAMAM dendrimers, it
is recommended a study on the molecular weight of PAMAM is done by using gel
permeation chromatography (GPC). This is important because all the dendrimers are
colorless and the generation number cannot be differentiated based solely on the
color.
Besides that, the ability of the PAMAM-gold thiol to binding with different
anions such as sulfate or phosphate can also be studied and compared with nitrate
ion.
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APPENDIX A
1
H NMR Spectra for G 0.5 PAMAM Dendrimer
70
APPENDIX B
1
H NMR Spectra for G 1.0 PAMAM Dendrimer
71
APPENDIX C
1
H NMR Spectra for G 1.5 PAMAM Dendrimer
72
APPENDIX D
1
H NMR Spectra for G 2.0 PAMAM Dendrimer
73
APPENDIX E
1
H NMR Spectra for G 2.5 PAMAM Dendrimer
74
APPENDIX F
1
H NMR Spectra for G 3.0 PAMAM Dendrimer
75
APPENDIX G
Infrared Spectra of PAMAM dendrimer G 0.5
76
APPENDIX H
Infrared Spectra of PAMAM dendrimer G 1.0
77
APPENDIX I
Infrared Spectra of PAMAM dendrimer G 1.5
78
APPENDIX J
Infrared Spectra of PAMAM dendrimer G 2.0
79
APPENDIX K
Infrared Spectra of PAMAM dendrimer G 2.5
80
APPENDIX L
Infrared Spectra of PAMAM dendrimer G 3.0