Monitoring the competence of a new ketotetrahydrocarbazole based fluorosensor under
homogeneous, micro-heterogeneous and serum albumin
environments
Amrit Krishna Mitraa, Abhishek Saub, Subhas Chandra Berab, Suchandra Chakrabortyc,
Chandan Sahac, Samita Basub,*
a Department of Chemistry, Government General Degree College, Singur, Hooghly, West
Bengal, Pin:712409, India
b Chemical Sciences Division, Saha Institute of Nuclear Physics, Kolkata 700064, India
c Department of Clinical and Experimental Pharmacology, School of Tropical Medicine,
Kolkata 700073, India
*Corresponding Author:
E-mail address: samita.basu@saha.ac.in
Telephone: +91-33-2337-5345, Fax: +91-33-2337-4637
1
Supporting Informations
Supporting Information S1:
Table 1: Physical properties and empirical parameters of solvents
Solvents Dielectric ET(30)
π*
α
β
SP
SdP
SA
SB
Constant
BZ
2.28
34.3
0.59
0
0.1
0.793
0.270
0
0.124
DOX
2.21
36
0.55
0
0.37
0.737
0.312
0
0.444
EtAc
6.02
38.1
0.45
0
0.45
0.656
0.603
0
0.542
ACN
37.5
45.6
0.75
0.19
0.31
0.645
0.974
0.044
0.286
DMF
36.7
43.2
0.88
0
0.69
0.759
0.977
0.031
0.613
DMSO
46.6
45.1
1
0
0.76
0.830
1
0.072
0.647
HOH
78.54
63.1
1.09
1.17
0.47
0.681
0.997
1.062
0.025
MeOH
32.6
55.4
0.6
0.98
0.66
0.608
0.904
0.605
0.545
EtOH
22.4
51.9
0.54
0.86
0.75
0.633
0.783
0.4
0.658
BuOH
18.2
49.7
0.47
0.84
0.84
0.674
0.655
0.341
0.809
HxOH
13.3
48.8
0.4
0.8
0.84
0.698
0.552
0.315
0.879
OcOH
10.3
48.1
0.4
0.77
0.81
0.713
0.454
0.299
0.923
DcOH
8.1
47.7
0.45
0.7
0.82
0.722
0.383
0.259
0.912
DdOH
6.5
47.5
-----
-----
-----
-----
-----
-----
-----
π* represents polarity/polarizability of the solvent, hydrogen bond donor acidity is represented as α
and β represents hydrogen bond acceptor basicity. Other parameters are its acidity (SA), Basicity
(SB), polarizability (SP) and dipolarity (SdP).
2
Supporting Information S2:
Theoretical details of Lippert-Mataga equation
Assuming the solute molecules spherical and centred in a closed spherical first solvation
shell, the solvent spectral shift is given by the following relation:
    mf ( , n)  const
a
f
(1)
where,
m
2( e   g ) 2
hca
(2)
3
 a and  are representatives of wave numbers of the absorption and emission maxima
f
respectively;  and  are the dipole moments in the ground state and excited state
g
e
correspondingly. Velocity of light in vacuum is represented by c and Planck’s constant by h.
Relation (3) gives the solvent polarity function for a spherical cavity with an Onsager radius
of a.
  1 n2  1
 2
2


1
2n  1
f ( , n) 
2   1
2 n 2  1
(1  3 .
)(1  3 . 2 )2
a 2  1
a 2n  1
(3)
Electric permittivity and refractive index are represented by  and n respectively whereas α
is the average polarizability (  e   g   ) of the solute. According to the theory proposed by
Lippert and Mataga, the polarizability of the solute is neglected (   0 ) and equation (3) is
simplified to the following relation.
3
f LM ( , n) 
  1 n2  1

2  1 2n2  1
(4)
To calculate dipole moment using either method,  a   f has been plotted against f ( , n)
and m is found from the slope (Eq. (1)). For a special case where the ground and excited-state
dipole moments (  g and  e ) are parallel, the relation (3) gives the dipole moment difference
 :
(5)
1
1
  ( e   g )  ( hca3 .m) 2
2
fLM ( , n) has been calculated for all the solvents used in this study and  a   f vs. f ( , n) has
been plotted for MTDCO in different protic and aprotic solvents.
5000
8000
[b]
-1
7500
/ cm
/ cm
-1
[a]
4500
7000
6500
4000
6000
3500
5500
0.20
0.22
0.24
0.26
0.28
0.30
0.32
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
Fig. 1. Plot of  a  f versus f LM ( , n) in a protic and b aprotic solvents for MTDCO.
The values of m have been obtained from the slopes of the plots (Fig. 1) in accordance with
Eq. 1. Then Eq. (5) has been used to calculate the changes in dipole moments of the
compounds in protic and aprotic solvents.
4
Supporting Information S3:
Kamlet-Taft Solvatochromic Comparison Method
Kamlet-Taft Solvatochromic Comparison Method (KTSCM) is usually used to measure
individual contributions of different modes of solute-solvent interactions. * is a measure of
the polarity/polarizability effects of the solvent; α scale is an index of solvent HBD
(hydrogen bond donor) acidity and β scale is an index of solvent HBA (hydrogen bond
acceptor) basicity. The coefficients s, a and b describe the sensitivity of the fluorophore to
each of the individual contributions mentioned. The benefit we obtain from this approach is
to sort out the quantitative role of properties such as hydrogen bonding. According to
KTSCM, the emission frequencies can be correlated using the following multiple linear
regression analysis approach.
   0  s *  a  b
(6)
 0 is the emission frequency (emission frequencies are expressed in cm−1) of the reference
solvent.
Supporting Information S4:
Catalan method
The shift in fluorescence spectrum of MTDCO is described as the function of the following
four properties of solvents: solvent acidity (SA), solvent basicity (SB), solvent polarizability
(SP) and solvent dipolarity (SdP). We can express the Catalan scale with the help of
following equation,
5
   0  bSA  cSB  dSP  eSdP
(7)
The coefficients b to e are the regression coefficients describing the sensitivity of property
fluorescence maxima to the different solute - solvent interaction mechanisms.
Supporting Information S5:
Absorbance
Absorption spectra of MTDCO in dioxane-water binary mixture
0.14 % water in
DOX-water mixture
0% water
0.12
10% water
0.10
40% water
60% water
0.08
100% water
0.06
0.04
0.02
0.00
275
300
325
350
375
Wavelength / nm
Fig. 2. Absorption spectra of MTDCO in Dioxane-water binary mixture. Concentration of the
compound is 1 × 10-6 M.
Supporting Information S6:
Table 2: Lifetime of MTDCO in dioxane-water binary mixture. Concentration of the compound is 1 ×
10-6 M.
% of water in
2
a
τ
χ
1
0.39
1.11
dioxane-Water
0
6
10
1
0.71
1.18
40
1
2.68
1.17
60
1
2.65
1.23
100
1
2.57
1.08
Supporting Information S7:
Table 3: Fluorescence decay parameters of MTDCO with increasing concentration of SDS.
Concentration of the compound is 1 × 10-6 M.
a
τ /ns
χ2
0M
1
2.57
0.99
0.0086 M
1
2.59
0.97
0.0180 M
1
2.70
0.99
0.0285 M
1
2.79
0.98
0.0456M
1
2.85
1.00
0.0620M
1
2.91
0.98
0.0655 M
1
2.93
0.99
SDS
(concentration)
Table 4: Fluorescence decay parameters of MTDCO with increasing concentration of TX-100.
Concentration of the compound is 1 × 10-6 M.
Triton-X
(a1) /(a2)
(Concentration)
τ1(SD)
χ2
τav
/ns
0M
(1) / (0)
2.574
1.00
2.574
0.001 M
(1) / (0)
2.627
0.98
2.627
7
0.002M
(1) / (0)
2.637
1.01
2.637
0.01M
(1) / (0)
2.708
0.96
2.708
Table 5: Fluorescence decay parameters of KTHC-57with increasing concentration of CTAB.
Concentration of the compound is 1 × 10-6 M.
Concentration
(CTAB)
0M
(a)
τ (SD) /ns
χ2
τav
(1) / (0)
2.574
0.98
2.574
0.0005M
(1) / (0)
2.612
0.94
2.612
0.0007M
(1) / (0)
2.622
1.01
2.622
Table 6: Fluorescence decay parameters of MTDCO with increasing concentration aqueous β-CD.
Concentration of the compound is 1 × 10-6 M.
Concentration
(β-CD)
0M
(a1)/(a2)
τ1 /ns
(1) / (0)
2.574
0.0025M
(0.65)/(0.35)
0.0100M
0.0200M
τ2 /ns
χ2
τav
---------
0.99
2.574
2.372
3.045
0.98
2.607
(0.18)/(0.82)
1.819
3.211
1.00
2.958
(0.10)/(0.90)
1.449
3.326
1.22
3.1381
Supporting Information S8:
Determination of binding constant values using the methods described by Almgren et al.
Here we have the relation, (I  I0 ) /  Ix  I0   1  ( K[ M ])1 where I0, Ix and I∞ are the
fluorescence intensities of MTDCO considered in the absence of surfactant, at an
8
intermediate surfactant concentration and at a condition of complete micellization. K is the
binding constant and [M], the micellar concentration. The micellar concentration [M] is
determined by, [M] 
 S  CMC / N
(8)
S represents the surfactant concentration. The values of N (aggregation number of a micellar
system) for SDS, CTAB and TX-100 are 62, 60 and 143 respectively . Binding constant (K)
values have been determined from the slope of the plots of (I  I0 ) /  I x  I0  against [ M ]
1
1.8
1.6
[b]
[a]
1.6
1.5
1.5
1.4
(I-I0) / (Ic-I0)
(I-I0) / (Ic-I0)
1.7
1.4
1.3
1.2
1.1
1.3
1.2
1.1
1.0
1.0
0.9
2000
3000
4000
-1
5000
[M] L mol
6000
0
7000
20000
40000
60000
80000 100000 120000
-1
-1
[M] L mol
-1
3.5
[c]
3.0
(I-I0) / (Ic-I0)
2.5
2.0
1.5
1.0
0
200000 400000 600000 800000 1000000 1200000
-1
[M] L mol
-1
Fig. 3. Plot of (Iα-Io)/(Ic-I0) against [M]-1 in (a) SDS, (b) CTAB and (c) TX-100.
Supporting Information S9:
Modified version of Benesi–Hildebrand equation to assess the binding constant for inclusion
complex formation between MTDCO and β-CD
9
We have used the following equation:
(I  I0 ) /  Ix  I0   1  ( K[  CD])1
(9)
I0, Ix and I∞ are the emission intensities of MTDCO in the absence of β-CD, at an
intermediate concentration of β-CD and at a concentration of complete interaction
respectively. We have obtained a linear regression [Fig. 4] in the plot of (I  I0 ) /  I x  I0  vs
[  CD]1 .
2.0
(I-I0) / (Ic-I0)
1.8
1.6
1.4
1.2
1.0
0
50
100
150
200
250
-1
[M] L mol
300
350
400
450
-1
Fig.4. Modified Benesi– Hildebrand plot of (I∞- I0)/(Ix -I0) vs. [β-CD]-1 for inclusion complex
formation between MTDCO and β-CD. Concentration of the compound is 1×10-6 M. The linear
regression indicates a 1:1 stoichiometry.
Supporting Information S10:
Effect of denaturant:
Denaturation is the breaking up and possible annihilation of the secondary and/or tertiary
structures of proteins. Conversely, the primary structure (amino acid sequence) remains
10
unaffected. This is because of the feeble process of denaturation that fails to break the peptide
bonds. The denaturation results in modification of the normal alpha helix and beta sheets of a
protein. Additionally, the protein uncoils to form a random shape. Desolvation of the guest
molecule occurs because of this destabilization and further leads to its expulsion into the bulk
aqueous phase. As is evident, a substance that is able to disrupt, denature and destroy the
three dimensional macromolecular structure of proteins, DNA, RNA, etc. is obtained. Such a
substance is called a chaotropic agent or chaotrope. Chaotropic agents interrupt the
stabilizing effect leading to the aforementioned denaturation. Chaotropic agents include urea,
guanidine hydrochloride, lithium perchlorate, etc.
470
470
[a]
HSA
BSA
465
460
[b]
em
max
460
455
455


em
max
465
BSA
HSA
450
450
445
445
440
0
1
2
3
4
5
6
7
[Urea] M
0
2
4
6
[GuHCl] (M)
Fig.5. Variation of emission wavelength of protein bound MTDCO against the (a) concentration of
urea and (b) concentration of guanidine hydrochloride.
11