Interaction of carborane-appended trimer with bovine serum albumin: A spectroscopic investigation

Article abstract of DOI:10.1016/j.ica.2019.03.035

The interaction of symmetrical triazine-cored carborane-appended trimer 5 with bovine serum albumin has been investigated using different spectroscopic methods. The intrinsic fluorescence of BSA was quenched by the carborane trimer 5 through the dynamic q

Full text of DOI:10.1016/j.ica.2019.03.035

Accepted Manuscript
Research paper
Interaction of carborane-appended trimer with bovine serum albumin: a spec-
troscopic investigation
Bibhuti Bhusan Jena, Lakkoji Satish, Chandra Sekhara Mahanta, Biswa Ranjan
Swain, Harekrushna Sahoo, Barada P. Dash, Rashmirekha Satapathy
PII:
S0020-1693(19)30165-3
https://doi.org/10.1016/j.ica.2019.03.035
ICA 18840
DOI:
Reference:
Inorganica Chimica Acta
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Revised Date:
Accepted Date:
4 February 2019
25 March 2019
25 March 2019
Please cite this article as: B. Bhusan Jena, L. Satish, C. Sekhara Mahanta, B. Ranjan Swain, H. Sahoo, B.P. Dash,
R. Satapathy, Interaction of carborane-appended trimer with bovine serum albumin: a spectroscopic investigation,
Inorganica Chimica Acta (2019), doi: https://doi.org/10.1016/j.ica.2019.03.035
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Interaction of carborane-appended trimer with
bovine serum albumin: a spectroscopic investigation
Bibhuti Bhusan Jena a, Lakkoji Satish b, Chandra Sekhara Mahanta a, Biswa Ranjan Swain a,
Harekrushna Sahoo b, Barada P. Dash c,*, Rashmirekha Satapathy a,*
a Department of Chemistry, Ravenshaw University, Cuttack, Odisha, 753003, India
b Department of Chemistry, NIT Rourkela, Odisha, 769008, India
c Department of Chemistry, Siksha “O” Anusandhan (Deemed to be University),
Bhubaneswar, Odisha 751030, India
*Corresponding authors. E-mail: barada.dash@gmail.com(BD);
rashmi.satapathy@gmail.com, rashmi@ravenshawuniversity.ac.in (RS)
Abstract
The interaction of symmetrical triazine-cored carborane-appended trimer 5 with bovine
serum albumin has been investigated using different spectroscopic methods. The intrinsic
fluorescence of BSA was quenched by the carborane trimer 5 through the dynamic quenching
mechanism and the estimated binding constant Kb value (3.3−4.1) × 105 M-1 is similar to
those obtained from the interaction of commercial drugs such as aspirin, ibuprofen and
ceftriaxone with BSA. The conformational changes of BSA were monitored by CD analysis
and temperature dependent CD measurements were done to study the thermal stability of
BSA in presence of carborane-appended trimer 5.
Keywords: O-Carborane, Bovine serum albumin, Fluorescence spectroscopy, Binding
constant
1

1. Introduction
Carboranes are a class of caged-boron compounds those contain both boron and carbon
atoms in the cluster. These are the widely explored polyhedral boron compounds in various
areas of chemistry [1]. Over the years, carborane clusters have attracted significant attention
for their use in the field of medicinal chemistry [2]. The advantage of carboranes and their
metal complexes is that they are chemically and biologically stable, resistant to catabolism
and hydrophobic in nature [3]. The anti-glioma activity of the carborane-anilinoquinazoline
conjugates has been found to be much higher than the parent cancer drug. Their stability in
the biological environment, ability to accumulate in glioma cells and cross the blood-brain
make them ideal candidates for the treatment of glioblastoma, the most malignant form of the
brain cancer [4]. In addition to their use in cancer therapy, they have been used as stable
hydrophobic pharmacophores, retinoid receptor ligands, steroid analogs, human blood
platelet function inhibitors, HIV protease inhibitors and imaging agents [2-5].
There are three widely used isomers of icosaheral carboranes. These are ortho-
carborane, meta-carborane and para-carborane [1c,2b]. The protons at the carbon atoms of
the cluster are acidic and it varies for isomers. The acidity of the protons increases in the
order ortho-carborane < meta-carborane < para-carborane [6]. Because of the acidic nature of
the protons attached to carbons in the clusters, organometallic bases, such as n-BuLi have
been routinely used in synthetic chemistry for modification of carbon atoms via lithiated
carboranyl species [3c]. The acidity of the cluster protons have been influenced by
substituents at either the boron or the carbon atoms [7-9]. The high hydrophobicity of many
boron clusters and their derivatives can be explained because of the presence of slightly
polarized hydrogen atoms on the boron atoms and their “hydride-like” character. This
prevents them to form hydrogen bonds, and makes the boron clusters hydrophobic. The
electronegativity of hydrogens enables boranes to form unconventional hydrogen bonds
2

referred as dihydrogen bonds. However, such bonds are weaker than classical hydrogen
bonds and a net repulsive effect toward surrounding water molecules prevails making the
boron clusters hydrophobic in nature. The hydrophobicity of carboranes also varies with the
dipole moment which is dependent on the position of the carbon atoms in the carborane cage.
Consequently, the hydrophobicity of carborane isomers increases in the order ortho-
carborane < meta-carborane < para-carborane [10]. It has also been established that C-
substituted icosahedral carboranes are most hydrophobic and the presence of the acidic free
CH groups in unsubstituted carboranes lower the hydrophobicity. Nonetheless, attachment of
substituents to one of the boron atoms lowers the hydrophobicity [11]. Both acidity and
hydrophobicity of outer hydrogen atoms play a crucial role on intermolecular interactions of
unsubstituted clusters. As mentioned, carboranes have two sets of hydrogen atoms: acidic CH
groups and hydridic BH groups. The acidic CH group typically forms hydrogen bonds with
elements O, N, S, F and π system [12-14]. The hydridic character of the BH units forms
weaker dihydrogen bonds with electronegative elements. Such bonds have been observed in
crystal structures of smaller carborane-containing molecules [15].
Serum albumin is the most abundant protein in the mammalian blood. It is often
considered as a transport protein possessing the binding ability for a broad variety of
compounds (endogenous and exogenous). The binding of drug molecules with albumin
proteins, generally influences the pharmacodynamics and pharmacokinetics of these drugs.
Hence, a proper investigation of the binding affinity of drug molecules to albumin could be
helpful in designing novel biologically active molecules. Bovine serum albumin (BSA)
consists of 583 amino acid residues with around 60% helix and 17 disulfide bridges.
Moreover, it is composed of three domains (I, II, and III) and each domain further divided
into two subdomains A and B (Figure 1) [16]. It is structurally similar to human serum
albumin (HSA).
3

Figure 1. Structure of BSA (PDB-3v03).
Therefore, a thorough investigation of the binding affinity of icosahedral carborane
clusters to BSA can provide useful information for designing carborane-based biologically
active molecules. However, there are only few reports on interaction studies of polyhedral
boron clusters with serum albumin. The interaction between BSA and polyhedral boron
clusters such as cobalt bis(dicarbollide), iron bis(dicarbollide), para-carboranes,
dodecaborate anion and their derivatives was studied using measurements of fluorescence
quenching of BSA in increasing concentrations of the boron clusters and their derivatives.
The observed strength of boron cluster interactions with BSA decreases in order:
metallabis(dicarbollides) [M(C2B9H11)2]− > para-carborane (C2B10H12) >> dodecaborate
anion [B12H12]2− [17]. Hydrophobicity of metallabis(dicarbollides) depends on the cation
while para-carboranes are always hydrophobic [18]. The binding constant of ortho-carborane
appended chlorins with HSA was found to be four times higher than the binding constant
observed for chlorins without ortho-carborane clusters [19]. The study indicated enhanced
interaction between HSA and chlorin derivatives due to the presence ortho-carborane. Ortho-
carborane shows the lowest hydrophobicity among the three icosahedral carboranes.
Therefore, low serum albumin interaction is expected from ortho-carborane derivatives.
Among the icosahedral carboranes, it is easy to prepare ortho-carborane derivatives and thus
it is cost effective to use it for medicinal applications. The aim of our present study is to
4

understand the interaction and mechanism involved in the interaction between ortho-
carborane and BSA. For this purpose we chose to study the interaction between a boron-rich
triazine-cored trimer containing three ortho-carborane clusters in the periphery with BSA. A
symmetrical carborane-appended trimer 5 (Scheme 1) has been synthesized and different
spectrophotometric methods have been employed to understand molecular level interactions
and the effect of ortho-carborane-appended trimer 5 on the structure and stability of BSA.
2. Results and discussion
OH
CF3SO3H
CHCl3
NC
OH
1
N
N
N
Br
K2CO3
Acetone
2
HO
OH
Br
NC
O
K2CO3
Acetone
3
CF3SO3H
CHCl3
O
N
N
N
O
4
O
B10H14
CH3CN
H
C
C
O
H
C
N
N
H
N
C
C
O
O
C
5
Scheme 1. Synthesis of triazine-cored carborane-appended trimer 5.
5

2.1. Synthesis
The Ccage-appended C3-symmetric trimer containing three ortho-carborane clusters was
synthesized by employing two synthetic methods (Scheme 1). In the first approach compound
4 was prepared from commercially available 4-hydroxy benzonitrile 1 via treatment with
propargyl bromide and subsequent triflic acid-mediated trimerization reaction with yield of
73%. In the alternate approach, first trimer 2 was prepared from the 4-hydroxy benzonitrile
via triflic acid-mediated trimerization reaction in chloroform and subsequent treatment with
propargyl bromide in presence of K2CO3 in acetone produced the desired compound 4 in 92%
yield. Finally, the three-alkyne moieties in compound 4 underwent reaction with decaborane
in presence of acetonitrile to produce symmetrical triazine-cored carborane-appended trimer
5 in 31% overall yield [20].
2.2. Characterization
The newly synthesized compounds 4 and 5 were characterized by NMR, IR and mass
spectral analysis. Compound 5 showed characteristic B−H stretching frequency at 2587 cm-1
13
in IR spectra. In C {1H} NMR the cage carbon showed peak at 78.1 ppm and 76.0 ppm
1
respectively. The C−H proton of cage appeared at 4.04 ppm in H NMR spectra. Finally, the
MALDI-TOF mass spectral analysis showed peaks at m/z 472 and 827 respectively,
confirming the formation compounds 4 and 5.
2.3. Fluorescence spectra
Intrinsic fluorescence can provide valuable information about the change in structure
and the micro-environment in the vicinity of the fluorophores during the interaction of the
ligand molecules with the protein. BSA has three different intrinsic fluorophore residues:
tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe). Out of the three groups, Trp has
highest quantum yield whereas Phe has minimum. BSA has two Trp residues; Trp134 and
Trp212. The former is on the surface or in a more hydrophilic environment of domain IB and
6

the later is in the hydrophobic pocket of domain IIA [16]. We fixed the wavelength 295 nm to
excite BSA and with this we avoided the interference from Tyr and Phe residues, so that the
emission spectra can provide valuable information about the change in the micro-
environment of Trp residues after interaction with carborane trimer 5. The fluorescence
spectra have been analyzed using two important fluorescence properties i.e., maximum
intensity (Imax) and maximum emission wavelength (max) as these two properties are highly
global and local environmental sensitive. Figure 2a shows the fluorescence spectra of BSA in
presence of different concentration of carborane trimer 5.
Figure 2. Fluorescence spectra (a) and absorption spectra (b) of BSA in presence of
carborane trimer 5 with concentration (0, 2, 4, 6, 8, 10 µM). Far UV circular dichroism (c)
and melting temperature at wavelength 222 nm (d) of BSA in presence of carborane trimer 5
with concentration (0, 2, 10 µM).
7

It is clear from the Figure 2a that emission intensity decreases gradually with the
increase in concentration of carborane trimer 5 to the protein solution. The emission maxima
of native BSA in the buffer solution were found to be at 344 nm. With the addition of
carborane trimer 5, there was a slight red shift in emission maxima (347 nm at 10 µM). Red
shifting of emission maxima indicates that the polarity around the Trp residues increased
after the interaction of carborane trimer 5 with BSA, which might be possible due to the
conformational changes in BSA that leads to the exposure of Trp residues to the more polar
environment. Furthermore, the decrease in fluorescence intensity with increase in the
concentration of carborane trimer 5 could be due to the fluorescence quenching by a variety
of molecular interactions such as ground-state complex formation, excited-state reactions,
energy transfer, molecular rearrangement, and collision quenching.
Therefore, the analysis of fluorescence quenching would reveal the mechanism of protein-
carborane trimer 5 binding and can provide an idea about nature of the binding phenomenon.
The static quenching occurs when there is a ground-state complex formation between the
fluorophore and quencher whereas dynamic quenching occurs by the collision between
fluorophore and quencher at excited-states. These two quenching mechanisms can be
differentiated from their dependency on temperature, viscosity, and excited-state lifetime.
Here, we employed the temperature-dependent measurements, where in general, the
bimolecular quenching constants increase with the rise in the temperature during the dynamic
quenching, whereas, the reverse effect is observed in static quenching. Stern-Volmer equation
(Equation 2) was used to study the mechanism of quenching [21].
8

Where, F0 and F are the fluorescence intensities of BSA in the absence and presence of
quencher respectively. KSV is the Stern-Volmer quenching constant, τ0 is the fluorescence
lifetime of protein without any quencher, kq is the bimolecular quenching rate constant, and
[Q] is the quencher concentration. The calculated KSV and kq values for BSA-carborane
trimer 5 complex at different temperatures are presented in Table 1 and Stern−Volmer plots
are shown in Figure 3a. From Table 1, it is observed that calculated KSV values are increasing
with increase in temperature. This suggests that major quenching mechanism is due to
dynamic quenching rather than static quenching. But, the values of bimolecular quenching
rate constants are observed to be higher than 2.0×1010 M−1 s−1. Therefore, the static
component of fluorescence quenching cannot be ignored. Absorption spectra of BSA in the
presence carborane trimer 5 could differentiate the static and dynamic quenching mechanism.
In general, the absorption spectra changes during static quenching due to the formation of a
ground-state complex between the fluorophore and quencher. In case of dynamic quenching,
only excited-states of the fluorophore are involved in the collision with quenchers, thus it is
expected that there is no change in the absorption spectra.
2.4. UV-Vis spectra analysis
UV-Vis spectra measurements were used to distinguish the quenching mechanism and
to analyze the conformational changes induced by the carborane trimer 5 after interaction
with BSA. The absorption spectra of BSA in the absence and presence of carborane trimer 5
are shown in Figure 2b. There are two peaks in the wavelength region of 200–230 nm and
260–290 nm. The peak in the lower wavelength region corresponds to the π–π* transition of
the backbone framework of the protein and the one in the higher wavelength region refers to
the n–π* transitions of aromatic amino acids such as Trp, Tyr, and Phe [22]. In the higher
wavelength region, the maximum appears at 278 nm. Any changes in the intensity and
9

shifting of this maximum indicate the alteration of the micro-environment of aromatic
chromophores. It was observed that there was no shifting in the peak but a marginal change
in the absorbance noticed in presence of carborane trimer 5 (Figure 2b). This indicates that
the micro-environment of aromatic chromophores are not changed, however, a ground-state
complex is formed between BSA and carborane trimer 5. It also suggests that the
fluorescence quenching is due to the static quenching.
Table 1. Stern-Volmer constant (KSV) and quenching constant (kq) for the interaction of
carborane trimer 5 with BSA at 298, 303 and 308 K.
T (K)
298
KSV (M-1)
3.35 × 104
3.98 × 104
4.61 × 104
kq(M-1 s-1)
3.36 × 1012
3.98 × 1012
4.61 × 1012
r2
0.9952
0.9943
0.9929
303
308
2.5. Determination of binding parameters
Although the fluorescence quenching is mostly dynamic in nature, the absorbance studies
indicate that static quenching is also involved in the quenching mechanism. Therefore, we
now use the following equation (Equation 3) considering the binding of carborane trimer 5 to
a set of equivalent sites on protein molecule to obtain the binding constant (K) and a number
of binding sites (n) [22, 23].
Where, n is the number of binding sites and Kb is the binding constant. The values of n and
Kb were obtained from the slope and intercept of the plot of log(F0 F)/F vs log[Q]
respectively. The calculated results for the carborane trimer 5 binding at different
temperatures (298, 303 & 308 K) are given in Table 2. The value of n was found to be closer
10

to unity indicating there was one independent class of binding sites on BSA. The estimated
Kb value was found to be in the order of 105 M-1, which is similar to the values of the binding
constant for small organic drugs like aspirin, ibuprofen and ceftriaxone that have a strong
binding force with BSA [24]. The value of the binding constant also matches with the binding
constant obtained from the interaction of virstatin drug with HSA [25].
Figure 3. (a) Stern-Volmer plots for the binding of BSA with carborane trimer 5 at various
temperatures (25 oC – Black, 30 oC – Red, 35 oC – Blue), (b) Double-logarithmic plots for the
binding of BSA with carborane trimer 5 at various temperatures (25 oC – Black, 30 oC – Red,
35 oC – Blue) and (c) Van’t Hoff plots for the binding of BSA with carborane trimer 5.
11

2.6. Thermodynamic parameters and mode of interaction
Various binding forces engaged in the binding of drugs with biomolecules include
electrostatic forces, hydrogen bonding, hydrophobic interactions, and Van derWaals
interactions. The distinction between these forces can be made based on the signs and
magnitude of thermodynamic parameters such as enthalpy change (ΔH0), entropy change
(ΔS0), and free energy change (ΔG0). The thermodynamic parameters can be calculated by
the Equations (4) and (5).
Where, K is the equilibrium binding constant (L mol-1), T is the temperature (K), and R is the
gas constant (8.314 J mol-1 K-1). According to Ross’s theory, if ΔH0 > 0 and ΔS0 > 0, the
major binding force is hydrophobic in nature; whereas if ΔH0 < 0 and ΔS0 < 0 then hydrogen
bonding or van derWaals forces plays a major role; otherwise, if ΔH0 < 0 and ΔS0 > 0, the
electrostatic forces play a major role [26,27]. The calculated thermodynamic parameters for
the interaction carborane trimer 5 with BSA are given in Table 2. The negative value of ∆G
indicates the interaction is sponatneous process. Moreover, the positive values of ΔH and ΔS
suggest that the binding between carborane trimer 5 and BSA is mainly driven by
hydrophobic forces. Similar results were observed in previous studies where boron clusters
interact with serum albumin protein through hydrophobic bonding [17].
12

Table 2. Binding parameters (binding constant – Kb and binding sites – n) and
thermodynamic parameters (Gibbs free energy - ∆G0, enthalpy - ∆H0and entropy - ∆S0) for
the interaction of carborane trimer 5 with BSA at 298, 303 and 308 K.
T (K)
298
logKb
5.52
5.55
5.61
n
r2
Kb (M-1)
- ∆G0 (kJ mol-1) ∆H0(kJ mol-1) ∆S0(J mol-1 K-1)
1.19
1.18
1.18
0.9961 3.3 × 105
0.9977 3.6 × 105
0.9982 4.1 × 105
31.5
32.2
33.1
15.3
156.9
303
308
2.7. Circular dichroism spectral analysis
CD spectroscopy has been employed to study the changes in the secondary structure of
BSA after the interaction with carborane trimer 5. The secondary structure of BSA exhibits
two characteristic negative bands in the far-UV region (190-250 nm). The negative bands at
208 and 222 nm appear due to the π–π* and n–π* transitions of peptide backbone in α-helices
[28, 29]. Alterations in the band intensity and position would indicate the conformational
changes of BSA after interaction with carborane trimer 5. Figure 2c shows the secondary
structure of BSA in the absence and presence of carborane trimer 5. It was observed that the
structure of BSA remains intact after interaction with carborane trimer 5. However, to
investigate the slight changes induced by the carborane trimer 5, we calculate the percentage
of α-helix in BSA using Equation-6 [30].
Where,
[Cp is the molar concentration of the protein, n is the number of amino acid residues (583 for
BSA) and l the path length (0.1 cm)]. The observed α-helix values are found to be 64.7, 64.0,
13

63.6% in case of BSA, BSA+2µM carborane trimer 5, BSA+10µM respectively. This
suggests that there was a slight decrease in the α-helicity of BSA after interaction with
carborane trimer 5. The insignificant changes in the secondary structure of BSA reveal that
the carborane trimer 5 binds to the external surface of proteins through weak intermolecular
interactions.
2.8. Thermal stability of BSA
Thermal CD analysis was employed to investigate the relative denaturation of BSA in
presence of carborane trimer 5 by measuring the changes in the CD spectra (at 222 nm) with
increasing temperature (Figure 2d). The obtained results were fitted to a two-state
equilibrium sigmoidal curve between the folded state (N) and the unfolded state (U), from
which the melting temperature (Tm) was calculated using the Boltzmann function [20]. The
melting temperature of BSA in the presence of carborane trimer 5 (0, 2, 10 µM) was found to
be 70.1±0.2, 70.9±0.4, 70.5±0.5 oC respectively.
3. Experimental section
3.1. Materials
Decaborane and BSA were purchased from Sigma-Aldrich, and were used without any
further purification. All samples are prepared using PBS buffer (0.01 M). Milli-Q water (18
M cm, Millipore, Bedford, MA) was used to prepare PBS buffer. All the chemicals required
for the synthesis of carborane trimer 5 and buffer preparation are of analytical grade
purchased from the local suppliers.
14

3.2. Sample preparation
The stock carborane trimer 5 solutions were prepared by dissolving the required
amount of 5 in PBS buffer containing 1% ethanol. BSA-carborane trimer 5 solutions for the
spectroscopic studies were prepared by adding a fixed amount of stock carborane trimer 5
solution to the protein solution to obtain final desired concentration of carborane trimer 5
(The details of carborane trimer 5 and BSA concentrations were given in the respective
section). After addition of carborane trimer 5, the solutions were sonicated followed by
incubation at room temperature for 0.5 hours to attain equilibrium before measurements.
3.3. General synthetic information
All reactions were generally performed under argon in oven-dried flasks using Schlenk
lines. Solvents and reagents were added by syringes. Solvents were dried using standard
procedures. Reagents were used as purchased without further purification unless indicated
otherwise. Products were purified by column chromatography on silica gel (60-120 mesh),
1
and yields refer to analytically pure samples. While the H NMR spectra were recorded on a
13
Fourier-Transform multinuclear NMR spectrometer at 400 MHz, the C {1H} NMR spectra
were recorded at 100.6 MHz. The chemical shifts are reported relative to tetramethylsilane
(TMS) (1H: δ = 0.00 ppm) and CDCl3 (13C: δ = 77.0 ppm). The integrals are in accordance
11
with the assignments, the coupling constants (J) are all given in Hz. The B {1H} NMR
spectra were recorded at 128.4 MHz and the chemical shifts are relative to BF3.Et2O
standard. The Infrared (IR) spectra of all compounds were recorded on an FT-IR
spectrophotometer. Melting points of the solids were measured using a standard apparatus
and the data were uncorrected. Mass spectral analyses were carried out using MALDI-TOF
mass spectrometers.
15

3.4. Syntheses
3.4.1. Compound 4
Method A: Compound 2 (500 mg, 1.399 mmol) was solubilised in 25 mL of dry
acetone. Then propargyl bromide (0.65 mL, 6.296 mmol) and K2CO3 (1158 mg, 8.395 mmol)
were added to this solution. The reaction mixture was refluxed at 60−70 oC for 15 h and then
the mixture was filtered through a cotton plug. The solvent was evaporated by using rotary
evaporator and the crude product was purified by silica gel column chromatography with
10−20% EtOAC in hexane as eluent to obtain 605 mg of pure compound 4 as colorless solid.
o
Yield: 92%. mp: 176 C. Method B: Compound 3 (100 mg, 0.632 mmol) was dissolved in 2
mL of dry chloroform. To this solution, CF3SO3H (0.35 mL, 3.164 mmol) was added to it at 0
o
oC. The mixture was stirred at 0 C for 10 min, and then at room temperature for 24 h. The
reaction mixture was quenched with dropwise addition of water containing small amount of
NH4OH. The reaction mixture was filtered over a cotton plug. The residue was dissolve in
acetone and then recrystallised from acetone to obtain 172 mg of pure compound 3 as a
o
colorless solid. Yield: 73%. mp: 176 C. 1H NMR (CDCl3, 400 MHz): δ 8.63 (d, 6H, J = 8.0
Hz), 7.05 (d, 6H, J = 8.0 Hz), 4.73 (s, 6H), 2.50 (t, 3H, J = 4.0 Hz). 13C {1H} NMR (CDCl3,
100.6 MHz): δ 170.7, 161.0, 130.7, 129.9, 114.7, 78.1, 76.0, 55.9. IR (KBr): 3290, 2918,
2114, 1708, 1606, 1521, 1448, 1224, 1022, 924, 814 cm-1. MALDI-TOF-MS (m/z): calc. for
C30H22N3O3: 471.2, found 472.3 [M+H]+.
3.4.2. Compound 5
Decaborane (374 mg, 0.302 mmol) was refluxed in 4 mL of dry acetonitrile for 3 h.
The reaction mixture was allowed to cool to room temperature. Then compound 4 (400 mg,
0.849 mmol) in dry acetonitrilie 3 mL was added to the reaction mixture with stirring. Again
o
it was refluxed at 100 C for 12 h. The mixture was purified by silica gel chromatography
with 14% EtOAC in hexane as eluent to obtained 217 mg of pure compound 5 as colourless
o
1
solid. Yield: 31%. mp: > 230 C. H NMR (CDCl3, 400 MHz): δ 8.57−8.64 (m, 6H),
16

6.91−6.94 (m, 6H), 4.46 (s, 6H), 4.04 (s, 3H). 13C {1H} NMR (CDCl3, 100.6 MHz): δ 171.3,
11
157.5, 130.3, 123.4, 114.8, 71.4, 69.5, 57.9. B {1H} NMR: −2.67, −4.47, −9.04, −11.69,
−13.13. IR (KBr): 3069, 2918, 2587 (B-H), 1606, 1514, 1448, 1236, 814 cm−1. MALDI-
TOF-MS (m/z): calc. for C30H52B30N3O3: 827.08; found 827.4 [M+H]+.
3.5. Fluorescence spectral measurements
The fluorescence measurements were done using Horiba Jvon Spectrometer (Fluoromax-
4P). The excitation wavelength was fixed at 295 nm to avoid the interference from Tyr
(tyrosine) and Phe (Phenylalanine) and the emission was collected as a function of
wavelength in the range of 305−450 nm. Excitation and emission slit widths were set at 5 nm.
The concentration of BSA was 2 µM and the carborane trimer 5 concentrations were varied
from 0 to 10 µM with an interval of 2 µM. The fluorescence intensities are corrected to
eliminate the inner filter effect using the following relationship [31].
(1)
Where, Icor and Iobs are the corrected and observed fluorescence intensity respectively, and
Aex and Aem are the absorption values of carborane trimer 5 at the excitation and emission
wavelengths, respectively.
3.6. Absorption measurements
The absorption spectra are performed on a double beam Cary 100 UV−vis
spectrophotometer (Agilent Technologies, USA) and 1.0 cm quartz cells are used for the
measurements. The concentration of BSA was 2 µM and the carborane trimer 5
concentrations were varied from 0 to 10 µM with an interval of 2 µM.
3.7. Circular dichroism (CD) spectroscopy
Secondary structure of BSA was studied with JASCO-1500 circular dichroism (CD)
spectrophotometer. The scan speed (200 nm/min) was fixed with a response time of 1s and 1
nm band width. Each spectrum was collected by averaging two spectra. The concentration of
17

BSA was fixed at 2 µM and carborane trimer 5 was maintained at 2 and 10 µM. Each sample
spectrum was background corrected by subtracting the blank (without protein) from the
protein spectra.
4. Conclusions
Triazine-cored symmetrical trimer 5 containing three ortho-carborane clusters in the
periphery has been synthesized. The binding affinity between carborane trimer 5 and BSA,
and the effect of carborane trimer 5 on protein conformation has been investigated using
different spectroscopic methods (including fluorescence, UV-Vis, and CD). Fluorescence
analysis along with absorption measurement showed that dynamic quenching as major
quenching process over static quenching. The estimated binding constant value of carborane
trimer 5 with BSA is similar to the binding constant values observed for small drugs like
aspirin, ibuprofen and ceftriaxone with BSA. The negative value of free energy change
elucidates that the interaction process is spontaneous. Thermodynamic binding parameters
reveal that the binding of carborane trimer 5 with BSA is driven mainly by hydrophobic
forces and thus the hydrophobicity of ortho-carborane clusters seems to play an important in
it. These all findings can be useful in the development of new bioactive compounds
containing ortho-carborane clusters.
Acknowledgements
Authors thank the DST-SERB, UGC and CSIR, govt. of India for financial support.
Appendix. Supplementary data
Supplementary data to this article (NMR and mass spectra of the compounds) can be found
online at https:// doi.org/xxxx/xxxx.
18

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 The interaction of a carborane-appended trimer with bovine serum albumin has been
reported.
 Spectroscopic methods such as fluorescence spectroscopy and circular dichroism have
been used for the evaluation.
 The observed binding constant value is found to be similar to those obtained for
commercial drugs like aspirin, ibuprofen and ceftriaxone.
23