Disulfide bond formation is the decisive event in the protein folding to determine the conformation and stability of protein. To achieve this disulfide bond formation in vitro, we took 2,5-dimercapto-1,3,4-thiadiazole (DMcT) as a model compound. We found that disulfide bond formation takes place between two sulfhydryl groups of DMcT molecules in methanol. UV-Vis, FT-IR and mass spectroscopic as well as cyclic voltammetry were used to monitor the course of reaction. We proposed a mechanism for the solvent induced disulfide bond formation on the basis of the results we obtained

Palanisamy Kalimuthu

Palraj Kalimuthu

S. Abraham John

English

Nature Precedings

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Solvent Induced Disulfide Bond Formation in 2,5-dimercapto-1,3,4-thiadiazole 
Palanisamy Kalimuthu, Palraj Kalimuthu and S. Abraham John*  
Department of Chemistry, Gandhigram Rural Institute-Deemed University 
Gandhigram-624 302, Dindigul, India. 
RECEIVED DATE (automatically inserted by publisher); e-mail: abrajohn@yahoo.co.in 
Disulfide bond formation is the decisive event in the protein 
folding to determine the conformation and stability of protein.1 
Many evidences have been shown for the role of disulfide bond 
formation in protein folding as a transient intermediate.2 It is 
worthwhile to mention here that there are recent reports on the co-
solvent  assisted disulfide bond formation in the production of 
biologically active α-Conotoxin ImI by methanol.3 The disulfide 
bonds must be formed between Cys2-Cys8 and Cys3-Cys12 of 
pro α-Conotoxin ImI to produce naturally occurring α-Conotoxin 
ImI.3 This α-Conotoxin is a selective antagonist of α7 nicotinic 
acetylcholine receptors.3 Thus it is very interesting to observe the 
disulfide formation in vitro. Can solvent alone do this excellent 
biological job without any added reagent? To answer this question 
we have done a bench work for the oxidation of sulfhydryl groups 
to disulfide by taking a hetero aromatic dithiol, 2,5-dimercapto-
1,3,4-thiadiazole (DMcT) in different solvents. Alhough DMSO 
is known for the oxidation of sulfhydryl oxidation to disulfide in 
the presence of added reagents,4 we found disulfide products with 
good yield in methanol in the absence of reagents when compared 
to DMSO and other solvents. Methanol offers good solubility of 
DMcT and easy isolation of disulfide products.  
We have observed a colour change for the methanolic solution 
of DMcT from pale yellow to intense yellow within three hours. 
On standing, the pale yellow colour of the methanolic solution of 
DMcT was further intensified to bright yellow fluorescent colour 
and then started to fade after 48h due to the formation of a pale 
yellow color solid in the solution. The pH of the fresh methanolic 
solution of DMcT is 2.1 and the pH of the solution increases as 
the time increases and attained a constant value of 6.8 after 48h. 
Figure 1A shows the UV-Vis spectra of DMcT in methanol 
recorded at various time intervals. The fresh methanolic solution 
of DMcT shows predominant peaks at 260 nm and 340 nm due to 
electronic transitions involving charge transfer hydrogen bonded 
(CTHB) type complex5 and   n→ pi* transition,6 respectively. The 
intensities of the peaks at 340 nm and 260 nm were decreased 
after 3 h and a new absorbance band was appeared at around 390 
nm responsible for the color intensification due to  n→ pi* 
transition (Fig 1A curve b). After 48h, the solution did not show 
any peak at 260 nm due to the transition involving CTHB type 
complex in addition to  enormous decrease in intensity of peak at 
340 nm indicating that DMcT molecules were converted into 
some products which were settled as a pale yellow solid in the 
solution. The disappearance of peak at 260 nm indicates that  the 
–S-H groups may be converted into -S-S- bond via deprotanation 
of sulfhydryl groups. 
To characterize the products formed in solution, we checked 
the 48h old methanolic solution with Thin Layer Chromatography 
(TLC) and found two spots corresponding to parent DMcT and a 
compound with higher molecular weight than DMcT. The pale 
yellow solid collected from the methanolic solution was found to 
be soluble in  DMSO and DMF.  EI Mass spectrum of pale yellow 
solid shows peaks at m/z 368 and 298.  
 
 
Figure 1. A) UV-Vis spectra of methanolic solution of 1mM DMcT         
a) fresh solution b) 3h  c) 48h d) 72h and e) 96h old solutions.  B)  FT-IR 
spectra of  a) DMcT  b) 3h old  methanolic solution of  DMcT and c) pale 
yellow color solid from methanolic solution of DMcT.  
The m/z 298 confirms the presence of dimer DMcT and the 
molecular ion peak at m/z 368 shows the presence of trimer minus 
CS2. The formation of –S-S− bond was further confirmed by 
FTIR spectrum of the pale yellow solid. When compared to FT-IR 
spectrum of DMcT, spectra of 3h old solution of DMcT in 
methanol      (Figure 1B) and the pale yellow  solid    ( Fig 1B 
curve c ) clearly showing a new weak absorption band at 471 cm-1 
due to –S-S− bond formation7 and the absorption band at 2475 
cm-1 of  –S-Hstr8 is undetectable for the pale yellow solid since 
most of the -S-H groups were consumed for the formation of 
disulfide. All other stretching frequencies of DMcT were retained 
for the pale yellow solid. From the above results we concluded 
that the formed pale yellow solid is the mixture of disulfide dimer 
and disulfide trimer of DMcT.  
The next task is to reveal the nature of existence of DMcT in 
methanol and then the mechanism of formation of disulfide dimer 
and disulfide trimer in methanol. We have carried out the 
electrochemical studies of methanolic solution of DMcT at 
different time intervals. Figure 2 shows the cyclic voltammograms 
(CVs) recorded for the methanolic solution of DMcT at different 
time intervals. CV of fresh methanolic solution of DMcT shows 
three oxidation peaks at 0.29V, 0.69V and 0.86V corresponding 
to the oxidations of DMcT2-, DMcT- and DMcT, respectively 
(Figure 2a).9 Close examination of the CV indicates that the 
predominant existence of  DMcT2- and DMcT-, make us to 
conclude that the DMcT molecules were deprotanated to DMcT2- 
and DMcT- immediately after dissolved it in methanol. 
The oxidation peaks corresponding to DMcT2- and DMcT- 
decreases when the CVs were recorded after 3h and 72h      
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 –1 0 1 2
–100
0
/ µ
Α
/ V vs.Ag/AgCl( KCl sat )
a
b
E
I
–1 0 1 2
–10
0
10 a b
c
I/
 
µΑ
E / V vs. Ag/AgCl (KCl sat )
(curves 2b and 2c). The presence of disulfide species in 
methanolic solution of DMcT was confirmed by running the CVs 
of 48h old methanolic solution of 1mM DMcT from 0V to -0.8V 
where electrochemical oxidation of DMcT2-/DMcT- is not 
possible (Figure  2 inset (b)) and fresh methanolic solution of 
DMcT in the presence of excess triethylamine (TEA) (Figure  2 
inset (a)). It has been reported that DMcT or DMcT- or DMcT2- 
oxidized to form dimer disulfide or polymer disulfide, but the 
reduction of both occurs at the same potential.10 As we can see 
from Figure  2 (inset) that the reduction potential of 48h old 
methanolic solution (Figure 2 inset(b)) matches with the reduction 
potential of dimer/polymer disulfide peak of DMcT/TEA    
(Figure 2 inset (a)), suggesting  the presence of disulfide 
containing species in the 48h old methanolic solution of DMcT.   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 2.  Cyclic voltammograms of 1mM DMcT in methanol at various 
time intervals. a) Fresh solution b) 3h old solution c) 72h old solution.             
Inset: Cyclic voltammograms of  a) 1mM DMcT in methanol with excess 
TEA b) 48h old solution of 1mM DMcT in methanol. 
From the above results we conclude that the oxidation of 
sulfhydryl groups  to disulfide bond was induced by methanol. It 
is suggested that initially hydrogen bonding between the oxygen 
atom of methanol and hydrogen atom of sulfhydryl group of 
DMcT was formed. In the second step, proton transfer from the 
sulfhydryl group of DMcT to methanol leads to the formation of 
DMcT- and DMcT2-. The protonated methanol molecules back 
donating the proton to harvest two electrons from DMcT2- and  
DMcT- and hence disulfide bond formation takes place (Scheme 
1). The released two electrons from deprotanated molecules of 
DMcT during the course of disulfide dimer or trimer formation 
utilized for the molecular hydrogen formation. Since the reaction 
is extremely slow we could not observe hydrogen evolution. The 
formed trimer separates out along with some dimer as a pale 
yellow solid due to very low solubility in methanol. 
     The formation of disulfide bond in methanol can be 
explained by the proton donor and acceptor properties of the 
solvents.11 The donor number12 of solvent plays a main role in the 
present work. The high donor number of methanol (19.0)13 
implying good basicity and proton acceptor characters.11 DMcT 
behaves as strong acid in solvents having good basicity  and 
proton acceptor characters.10 In those solvents the  dissociation   
of thiol groups of DMcT is facilitated. Methanol facilitate the auto 
oxidation of sulfhydryl groups to disulfide by deprotanation. The 
formation of disulfide bond was not observed  in chloroform and 
acetonitrile because of the poor proton donor and acceptor 
properties of these solvents.9 
 
 
Scheme 1 
 
 
 
 
 
 
Note: The hydrogen bonds between methanol and DMcT are shown in 
bold. 
 
Since DMcT contains two acidic sulfhydryl groups and 
intuitive to form disulfide bond, it is possible to incorporate  the 
DMcT in between two cyesteine units of a protein via disulfide 
bond formation, there by we can alter the conformation of the 
protein. Further the slow rate of  formation of disulfide bond has 
to be accelerated by changing from pure methanol solvent to 
mixture of methanol. These works are currently underway in our 
laboratory. 
Acknowledgment. Palanisamy Kalimuthu thanks UGC, 
NewDelhi for the award of Junoir Research Fellowship. SAJ 
thanks the Department of Science and Technology, New Delhi for 
financial support under the Fast Track Scheme for Young 
Scientists (SR/FTP/CS-09/2002).  
Supporting Information Available: The detailed experimental 
procedure, pH values of solutions, colour changes of DMcT solution 
and mass spectrum of the products available as pdf file. 
References 
 
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Disulfide dimer
Disulfide trimer
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Solvent Induced Disulfide Bond Formation in 2,5-dimercapto-1,3,4-thiadiazole 
Palanisamy Kalimuthu, Palraj Kalimuthu and S. Abraham John 
 
 
 
 
 
 
            
 
Disulfide bond formation is the decisive event in the protein folding to determine the conformation and stability of protein. To 
achieve this disulfide bond formation in vitro, we took 2,5-dimercapto-1,3,4-thiadiazole (DMcT) as a model compound. We 
found that disulfide bond formation takes place between two sulfhydryl groups of DMcT molecules in methanol. UV-Vis, FT-IR 
and mass spectroscopic as well as cyclic voltammetry were used to monitor the course of reaction. We proposed a 
mechanism for the solvent induced disulfide bond formation on the basis of the results we obtained.  
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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S SS
HH
CH3OH
NN
S SS
H
NN
S SS
H
NN
S SS
H
NN
S SS
NN
S SS
H
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Solvent Induced Disulfide Bond Formation in 2,5-dimercapto-1,3,4-thiadiazole

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