Bovine serum albumin triggered waste-free aerobic oxidative coupling of thiols into disulphides on water: An extended synthesis of bioactive dithiobis(phenylene)bis(benzylideneimine) via sequential oxidative coupling-condensation reactions in one pot from aminothiophenol and benzaldehyde

Article abstract of DOI:10.1016/j.molcatb.2015.03.008

Bovine serum albumin (BSA) has been explored for aerobic oxidative coupling of thiols (aromatic, heterocyclic as well as aliphatic) "on water" towards formation of disulphides (SS) without using any metal/non-metal complexes, bases and additives, which renders the process environmentally benign and economically attractive with good recyclability (up to four cycles). The developed green protocol was further extended for synthesis of diallyldisulphide (DADS), an important constituent of natural occurring allicin. Among various synthesized disulphides, bis(2-aminophenyl)disulphide, obtained by oxidative coupling of 2-aminothiophenol in BSA, was further utilized for condensation with benzaldehyde in the same pot thus enabling easy access to bioactive dithiobis(phenylene)bis(benzyldeneimine). This is the first example of BSA catalysed sequential (oxidation/condensation) reaction where one SS and two CN bonds are formed solely "on water."

Full text of DOI:10.1016/j.molcatb.2015.03.008

Accepted Manuscript
Title: Bovine serum albumin triggered waste-free aerobic
oxidative coupling of thiols into disulfides on water: An
extended synthesis of bioactive
dithiobis(phenylene)bis(benzylideneimine) via sequential
oxidative coupling-condensation reactions in one pot from
aminothiophenol and benzaldehyde
Author: Saima Aditya G. Lavekar Rajesh Kumar Arun K.
Sinha
PII:
S1381-1177(15)00065-X
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2015.03.008
MOLCAB 3128
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
5-12-2014
12-3-2015
12-3-2015
Please cite this article as: Saima, A.G. Lavekar, R. Kumar, A.K. Sinha, Bovine serum
albumin triggered waste-free aerobic oxidative coupling of thiols into disulfides on
water: An extended synthesis of bioactive dithiobis(phenylene)bis(benzylideneimine)
via sequential oxidative coupling-condensation reactions in one pot from
aminothiophenol and benzaldehyde, Journal of Molecular Catalysis B: Enzymatic
(2015), http://dx.doi.org/10.1016/j.molcatb.2015.03.008
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Graphical Abstract:
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“Bovine serum albumin triggered waste-free aerobic oxidative coupling of thiols into disulfides
on water: An extended synthesis of bioactive dithiobis(phenylene)bis(benzylideneimine) via
sequential oxidative coupling-condensation reactions in one pot from aminothiophenol and
benzaldehyde”
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Saima, Aditya G. Lavekar, Rajesh Kumar and Arun K. Sinha *
Protein Catalysis
Oxidative Coupling Rection on water
Bovine Serum Albumin
SH
Ar
Ar
S
Ar
S
3
0 examples with good
S-S bond
Ar=Aromatic, Heteroaromatic
Aliphatic
to excellent yield
Merits:
Aerobic condition H O, R.T.
i. Use of Biocatalyst
ii. Waste free protocol
iii. Neutral reaction condition
,
2
Sequential Oxidative Coupling-Condensation Reaction
R'
NH2
N
S
SH
(i) BSA, H O, Air, 18 h
2
S
N
(
ii)
3
R'= -H, -OCH etc.
,
12 h
R
O
One-pot two-steps reaction on water
R'
One S-S and two C_N bonds
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A green and clean route for the aerobic oxidative coupling of thiols including aromatic,
heteroaromatic and aliphatic thiols employing BSA as a biocatalyst "on water" has been developed
towards formation of (S-S) disulfides under metal/non-metal- or base-free conditions. The
methodology also applicable for successful synthesis of diallyldisulfide (DADS), an important
precursor of bioactive allicin. Interestingly, bis(aminophenyl)disulfide generated by oxidation of
aminothiophenol could directly react with benzaldehyde to form bioactive bis(aminophenyl)
disulphidediimines in sequential (oxidation-condensation) manner with one S-S and two C-N bonds in
one pot.
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Page 1 of 36

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Highlights
 A mild and environmentally benign protocol was developed using Bovine Serum
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Albumin as a catalyst.
 Aerobic oxidation using water as green solvent.
 Broad substrates scope.
 Extended for the synthesis of bioactive bis(aminophenyl)disulphidediimines in one
pot from aminothiophenol and benzaldehydes in sequential manner.
 Good recyclability upto four cycles.
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Page 2 of 36

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Bovine serum albumin triggered waste-free aerobic oxidative coupling of
thiols into disulfides on water: An extended synthesis of bioactive
dithiobis(phenylene)bis(benzylideneimine)
via
sequential
oxidative
coupling-condensation reactions in one pot from aminothiophenol and
benzaldehyde
[a],[b]
[a]
[c]
[a],[b]
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Saima,
Aditya G. Lavekar, Rajesh Kumar and Arun K. Sinha*
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[a] C.S.I.R.-Central Drug Research Institute (Council of Scientific and Industrial Research),
Lucknow- 226031 (U.P.), India. Fax: (+) 91-522-2771941.
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[b] Academy of Scientific and Innovative Research (AcSIR), Delhi
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[c] C.S.I.R.-Institute of Himalayan Bioresource Technology (Council of Scientific and
Industrial Research), Palampur-176061 (H.P.), India
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*E-mail: aksinha08@rediffmail.com
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Abstract: Bovine serum albumin (BSA) has been explored for aerobic oxidative coupling of
thiols (aromatic, heterocyclic as well as aliphatic) “on water” towards formation of disulfides
(S-S) without using any metal/non-metal complexes, bases and additives, which renders the
process environmentally benign and economically attractive with good recyclability (upto
four cycles). The developed green protocol was further extended for synthesis of
diallyldisulfide (DADS), an important constituent of natural occurring allicin. Among various
synthesized disulfides, bis(2-aminophenyl)disulfide, obtained by oxidative coupling of 2-
aminothiophenol in BSA, was further utilized for condensation with benzaldehyde in the
same pot thus enabling easy access to bioactive dithiobis(phenylene)bis(benzyldeneimine).
This is the first example of BSA catalyzed sequential (oxidation/condensation) reaction
where one S-S and two C-N bonds are formed solely "on water."
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Keywords: Bovine Serum Albumin, Disulfide, Oxidative Coupling-Condensation reactions,
Thiophenol, Waste-free Protocol, Water.
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1. Introduction
The organic synthesis with minimal environmental impact has become focus of chemists and
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the use of natural catalysts [1-6] (viz. proteins, enzymes, whole cell etc.) have steadily
ascended owing to inherited environmental and ecological benefits. In the same vein, the use
of water to facilitate eco-sustainable chemical reactions is also gaining momentum because
either its use as a solvent or its release as a by-product, will clearly have the least impact on
the environment [7-9]. Furthermore, there is representative "on water" [10-16] reactions
termed by Sharpless and co-workers [10] where conducting the reaction "on water" can be
substantially beneficial because of its high heat capacity, increase in reaction rate, selectivity
and an improved safety profile with ease of operation due to the insolubility of both reactants
and products in aqueous conditions. In this context, numerous organic transformations
including oxidation of aldehyde into carboxylic acid "on water" using molecular oxygen as
an environmentally benign and sustainable oxidant have surfaced [11]. Nevertheless, aerobic
oxidation reactions promoted by biocatalysts under neutral reaction conditions are rare and
lack generality "on water.”
Among various oxidation reactions, the selective oxidative coupling of thiols (-SH) to
corresponding disulfides (S-S bond) under mild reaction conditions is critical from biological
and chemical viewpoints [17-26]. The formation of disulfide bond is also a matter of interest
for the industrial production of some agrochemicals and pharmaceuticals [27-37] like
pyritinol [33], DADS (a major decomposition product of allicin found in Allium sativa) [36]
and dithiobis(phenylene)bis(benzyldeneimine), an antimicrobial agent against human
pathogens [34,35] (Fig.1).
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Conventionally, a large number of oxidative coupling protocols [38-55] including ball-
milling [38], Fe(BTC) [40], diaryltelluride [43], gold nanoparticles [46] and hypervalent
iodine(III) [47] have been documented for the synthesis of disulfides [52-55]. However, most
of these approaches suffer from inadequacies such as low selectivity due to over oxidation
[42] of final product into sulphoxides/sulphones, expensive and toxic nature of catalysts with
poor recovery and finally laborious work up procedures with generation of a lot of waste
materials. Recently, Wu and co-workers [51] have reported an effective and recyclable
photocatalytic method for selective conversion of thiols into disulphides with high turnover
number (TON) but visible-light irradiation of quantum dots is necessary. Against this milieu,
it is a surprise that only few biocatalysts [56-58] including horseradish peroxidase or
mushroom tyrosinase [56], baker’s yeast [57] and enzyme laccases [58] have been known for
aerobic oxidative coupling of thiols into disulfides but these reactions occurs in aqueous-
organic medium along with maintenance of pH conditions [56] and use of ABTS as radical
initiator [58]. So there is a strong need to develop such a biocatalytic system for oxidation of
thiols to disulfides which comprises all, i.e. inexpensive biocatalyst, aerobic oxygen and
water as a sole reaction medium besides recyclability of the biocatalyst (Fig. 2).
In this context, BSA [59-68] (also known as “Fraction V") an ubiquitous and
inexpensive, non-enzymatic transport protein derived from cows, occupies a unique position
due to its versatility to catalyze an array of chemical reactions [59-68, 69-71] including the
asymmetric thia-Michael [72] reaction. It is one of the most studied proteins that have a
strong affinity to bind organic molecules by reversible non-covalent complexation in its
hydrophobic pockets thus providing an environment for a number of organic transformations
including reduction [60], Knoevenagel condensation [61] and Gewald reaction [62]. Based
upon our ongoing interest on BSA catalyzed Aldol/Knoevenagel-Doebner condensation [69],
multicomponent Biginelli reaction [70] besides oxidation of tertiary amine to N-oxide at pH 9
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by Colonna et al [59], we ventured to explore BSA as a biocatalyst for waste-free aerobic
oxidation of thiols to disulfides "on water" which received considerable interest for various
chemical transformations [10-16] as water is a cheap, nontoxic solvent as well as it increases
the reactivity or selectivity of the reaction, often difficult to attain in organic solvents.
Among various disulfides (Fig. 1), bis(2-aminophenyl) disulfide [73-81] is
particularly known to participate towards formation of bis(2-aminophenyl)disulfidediimines,
a Schiff bases ligand [73] (Fig. 3) which possess sound antibacterial activity [74] and are also
used as basic scaffold in various chemical transformations [75-78]. Despite promising
biological profiles, synthesis of these Schiff bases requires two steps involving metal/base
[38,39,42,47] catalyzed oxidation of 2-aminothiophenol into corresponding bis(2-
aminophenyl)disulfide which upon isolation and purification followed by condensation with
benzaldehyde in anhydrous [73] ethanol affords the desired product bis(2-
aminophenyl)disulfidediimines (Fig. 3) having one S-S and two C-N bonds. Against this
drawback, creation of multiple bonds [82-84] in one pot without changing the reaction
condition and isolating the intermediate helps in minimizing waste, time and energy which
are most important aspects of green chemistry and generally accomplished by tandem [85-88]
(consecutive/sequential) reactions. It is worth mentioning that 2-aminothiophenol which is a
versatile synthon [73-81], on one side undergoes self-oxidative coupling into product bis(2-
aminophenyl)disulfide [47] while on the other side it undergoes condensation-cyclisation
with benzaldehyde leading to benzothiazole [89-91]. Hence it would be very challenging if
aerobic oxidative coupling-condensation between 2-aminothiophenol and benzaldehyde
undergoes in
a
tandem manner in one pot towards formation of bis(2-
aminophenyl)disulfidediimines (Schiff base) instead of benzothiazole on water (Fig. 3).
Further, formation of these Schiff bases [73] on water under neutral conditions would be an
interesting preposition as generally condensation between amine and carbonyl rather
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demands water removal [92-95] by azeotropic distillation [90] or use of lewis acid [94]
catalysts/dehydrating agents [95]. Hence the BSA possessing a rich diversity of surface
amino acids would seems effective towards formation of Schiff base as well as disulphides
ligands having miscellaneous medicinal and physicochemical profiles [33,35-36,61-62,73-74,
99-108].
In this context, we disclose for the first time, a neutral and waste free protocol for
direct aerobic oxidative coupling of thiols into disulfides "on water" utilizing BSA as a
biocatalyst which is successfully extended for the synthesis of bis(2-
aminophenyl)disulphidediimines via sequential (oxidative coupling-condensation) reaction in
one pot (Fig. 2 and 3) without using any metal oxidant/catalyst, base or organic solvents. The
recyclability of the biocatalyst is also noteworthy which emphatically demonstrates the
benign nature of the protocol.
2. Experimental Section
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2.1 Materials:-
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40
41
42
43
44
45
46
47
All the substrates/chemicals were obtained from commercial sources (Sigma Aldrich and
Alfa Aesar). The albumins (Bovine, Pig, Sheep, Chicken, Rabbit) and Lipases (PPL= Porcine
pancreas lipase, CAL-B= Candida antarctica lipase, CCL= Candida cylindracea; MJL=
Mucor javanicus, CRL= Candida rugosa lipase; TLL= Thermomyces lanugenosus lipase;
PCL= Pseudomonas cepacia lipase) used in this work have been purchased from Sigma
Aldrich. L-Arginine, Lysine and other amino acids were purchased from Sigma Aldrich and
Himedia. Distilled water was used in all reactions. The solvents used for isolation/purification
of compounds were obtained from commercial sources (Merck) and were used without
further purification.
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2.2 Analytical method:
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The GC-MS analysis was carried out on Shimadzu MS-QP-2010 system equipped with a
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BP-20 capillary column (SGE international). H (300 MHz and 400 MHz) and C (75.4 MHz
and 100 MHz) NMR spectra were recorded on Bruker Avance-300 and 400 spectrometer.
The following abbreviations were used to designate chemical shift multiplicities: s = singlet,
13
br = broad, d = doublet, t = triplet, q = quartet, m = multiplet. The C NMR spectras are
proton decoupled. Melting points were obtained manually by capillary methods and are
uncorrected. Column chromatography was done on silica gel (60-100 mesh size). Thin layer
chromatography (TLC) was performed on silica TLC plates and compounds were visualized
in iodine and under UV lamp.
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2.3 General Procedure
2.3.1 General procedure for the synthesis of disulfides (1b-27b):
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Thiophenol (0.25 mmol), BSA (50 mg) and deionised water (600 µL) were stirred at room
temperature for 12h or till completion of reaction (monitored by TLC). Then water (2 mL)
was added to reaction mixture and extracted with ethyl acetate (3mL X 2), organic part dried
over Na
2 4
SO , filtered and then concentrated under rotary evaporator and chromatographed
over silica gel by hexane/ethyl acetate (99:1). The obtained disulfides (1b-27b) were
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characterized and confirmed by comparing the H NMR and C NMR data with those
reported in the literature [38-51,109-117].
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2.3.2 General procedure for bis(2-aminophenyl)disulfidediimine Schiff bases (28b-30b):
2-Aminothiophenol (0.25 mmol), BSA (50 mg) and deionised water (600 µL) were stirred at
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room temperature for 18h to obtain the desired intermediate bis(2-aminophenyl)disulfide (4b)
followed by addition of benzaldehyde (1.1 equiv.) and BSA (50 mg) in the same pot. The
reaction mixture was further stirred at room temperature for 12h (monitored on TLC). After
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the completion of reaction, water (2 mL) was added to the reaction mixture and extracted
with ethyl acetate (3ml X 2), organic part dried over Na SO , filtered and concentrated under
rotary evaporator and recrystallized with methanol to obtained pure product. The obtained
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products (28b-30b) were characterized and confirmed by comparing the H NMR and
C
NMR data with those reported in the literature [75].
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2.4 ( H & C) NMR values of isolated compound (1b-30b):
2.4.1. Diphenyldisulfide (1b) [45]
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1
80
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White solid, mp: 58-61°C (lit [45] 61-62°C), H NMR (CDCl
3
, 400 MHz): δ 7.54-7.52 (4H,
13
3
m), 7.35-7.24 (6H, m); C NMR (CDCl , 100 MHz): δ 137.0, 129.0, 127.6, 127.2.
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2.4.2. Bis(2-naphthalenyl)disulfide (2b) [45]
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1
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84
85
3
White solid, mp: 133-136°C (lit [42] 136-137°C), H NMR (CDCl , 300 MHz): δ 8.03 (2H,
13
3
s), 7.83-7.75 (6H, m), 7.68 (2H, d, J = 8.63 Hz), 7.52 (4H, m); C NMR (CDCl , 75.4 MHz):
δ 134.7, 133.9, 132.9, 129.4, 128.2, 127.9, 127.2, 127.0, 126.7, 126.1.
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2.4.3. Bis(4-methoxyphenyl)disulfide(3b) [45]
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1
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White solid, mp: 33-36°C (lit [44] 34-35°C), H NMR (CDCl
3
, 300 MHz): δ 7.44 (4H, d, J =
, 75.4 MHz): δ 160.3,
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8.77 Hz), 6.88 (4H, d, J = 8.75 Hz), 3.83 (6H, s); C NMR (CDCl
3
133.1, 128.8, 115.0, 55.8.
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90
2.4.4. Bis(2-aminophenyl)disulfide (4b) [45]
1
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1
1
91
92
93
Yellow solid, mp: 90-94°C (lit [44] 93-94°C), H NMR (CDCl
3
, 300 MHz): δ 7.21-7.16 (4H,
13
m), 6.74 (2H, d, J = 8.48 Hz), 6.64 (2H, d, J = 8.48 Hz), 4.35 (4H, s); C NMR (CDCl
3
, 75.4
MHz): δ 149.1, 137.2, 132.0, 119.2, 118.7, 115.7.
1
94
2.4.5. Bis(4-hydroxyphenyl)disulfide (5b) [45]
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Yellow solid, mp: 152 –154 C (lit [44] 150–151 C), H NMR (DMSO-d
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, 400 MHz): δ 9.83
1
3
(2H, s), 7.29-7.27 (4H, m), 6.78-6.75 (4H, m); C NMR (DMSO-d
133.5, 125.6, 116.8.
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, 100 MHz): δ 158.8,
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98
2.4.6. Bis(3,4-dimethoxyphenyl)disulfide (6b) [109]
1
1
2
2
99
00
01
White solid, mp: 90-92°C (lit [109] 94°C), H NMR (CDCl
3
, 400 MHz): δ 7.09-7.04 (4H, m),
, 100 MHz): δ 149.3, 128.7,
13
6.82 (2H, d, J= 8Hz), 3.89 (6H, s), 3.85(6H, s); C NMR (CDCl
124.0, 114.2, 111.4, 56.0.
3
2
02
2.4. 7. Bis(2-methylphenyl)disulfide (7b) [45]
0
.
1
2
2
2
03
04
05
3
White solid, mp: 39-40 C (lit [48] 38-39°C), H NMR (CDCl , 300 MHz): δ 7.42 (4H, d, J =
13
8.14 Hz), 7.14 (4H, d, J = 8.02 Hz), 2.35 (6H, s); C NMR (CDCl3, 75.4 MHz): δ 137.8,
134.8, 130.2, 129.0, 21.5.
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06
2.4.8. Bis(2-methoxyphenyl)disulfide (8b) [41]
0
1
2
2
2
07
08
09
3
White solid, mp: 119–120 C (lit [41] 120°C), H NMR (CDCl , 400 MHz): δ 7.64-7.61 (2H,
13
3
m), 7.26-7.22 (2H, m), 6.99-6.95 (2H, m), 6.95–6.89 (2H, m) 3.93 (s, 6H); C NMR (CDCl ,
100 MHz,): δ 156.5, 127.7, 127.5, 124.5, 121.3, 110.4, 55.9.
2
10
2.4.9. Bis(4-methoxybenzyl)disulfide (9b) [110]
1
2
2
2
11
12
13
White solid, mp: 100-102°C (lit [110] 100°C) H NMR (CDCl
3
, 400 MHz): δ 7.20-7.18 (4H,
, 100 MHz): δ 159.0,
13
m), 6.90-6.86 (4H, m), 3.83 (3H, s) 3.63 (2H, s); C NMR (CDCl
130.5, 129.4, 113.9, 55.29, 42.81.
3
2
14
2.4.10. Bis(4-methylphenyl)disulfide(10b) [45]
1
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2
2
2
15
16
17
White solid, mp: 82-84°C (lit [44] 80-82°C), H NMR (CDCl
3
, 300 MHz): δ 7.43 (4H, d, J =
, 75.4 MHz): δ 137.9,
13
8.19 Hz), 7.15 (4H, d, J = 7.97 Hz), 2.35 (6H, s); C NMR (CDCl
134.3, 130.2, 129.0. 21.5.
3
2
18
2.4.11. Bis(3-chlorophenyl)disulfide (11b) [44]
1
2
2
2
19
20
21
White solid, mp: 70-72°C (lit [44] 70-72°C), H NMR (CDCl
3
, 300 MHz): δ 7.51 (2H, s),
13
7.39-7.33 (2H, m), 7.26-7.20 (4H, m); C NMR (CDCl
128.0, 127.4, 125.8.
3
, 75.4 MHz): δ 138.8, 135.6, 130.6,
2
22
2.4.12. Bis(2-nitrophenyl)disulfide, (12b) [111]
1
2
2
2
23
24
25
Yellow solid, mp: 192°C (lit [112] 192°C), H NMR (DMSO-d
6
, 400 MHz): δ 8.34-8.32 (2H,
13
6
m), 7.82-7.80 (2H, d, J=8 Hz), 7.7-7.66 (2H, m), 7.51-7.47(2H, m); C NMR (DMSO-d ,
100 MHz): δ 144.4, 133.8, 132.5, 126.3, 125.7, 125.1.
2
26
2.4.13. Bis(4-fluorophenyl)disulfide (13b) [45]
1
13
2
2
27
28
Yellowish oil, H NMR (CDCl
3
, 400 MHz): δ = 7.49-7.46 (4 H, m), 7.06-7.01 (4H, m); C
NMR (CDCl , 100 MHz) δ = 163.8, 161.4, 131.4, 131.3, 116.4, 116.2.
3
2
29
2.4.14. Bis(4-bromophenyl)disulfide(14b) [44]
1
2
2
30
31
White solid, mp: 92-96°C (lit [44] 94-95°C), H NMR (CDCl
3
, 300 MHz): δ 7.45-7.42 (4H,
13
3
m), 7.37-7.33 (4H, m); C NMR (CDCl , 75.4 MHz): δ 135.1, 132.6, 129.8, 121.9.
2
32
2.4.15. Bis(2-bromophenyl)disulfide (15b) [113]
1
2
2
2
33
34
35
White solid, mp: 95-97°C (lit [113] 97°C), H NMR (CDCl
3
, 400 MHz): δ 7.57-7.55 (4H, m),
13
7.31-7.27 (2H, m), 7.13-7.08 (2H, m); C NMR (CDCl
3
, 100 MHz): δ 136.2, 132, 128.2,
128.0, 127.0, 121.2.
1
1
Page 11 of 36

2
36
2.4.16. Bis(4-chlorophenyl)disulfide (16b) [44]
1
2
2
2
37
38
39
White solid, mp: 62-65°C (lit [44] 65-66°C), H NMR (CDCl
3
, 300 MHz): δ 7.45 (4H, d, J =
13
8.65 Hz), 7.31 (4H, d, J = 8.65 Hz); C NMR (CDCl
129.7.
3
, 75.4 MHz); δ 135.6, 134.1, 129.8,
2
40
2.4.17. Bis(phenylmethyl)disulfide(17b) [45]
1
2
2
41
42
White solid, mp: 57-60°C (lit [44] 58-59 °C), H NMR (CDCl
3
, 300 MHz): δ 7.39-7.26 (10H,
13
3
m), 3.64 (4H, s); C NMR (CDCl , 75.4 MHz): δ 137.8, 129.8, 128.9, 127.8, 43.7.
2
43
2.4.18. Dicyclohexyldisulfide (18b) [44]
1
2
2
2
44
45
46
Colorless oil, H NMR (CDCl
3
, 300 MHz): δ 2.73-2.64 (2H, m), 2.08-2.04 (4H, m), 1.80-
13
1.79 (4H, m), 1.64-1.61 (2H, m), 1.35-1.26 (10H, m), C NMR (CDCl
41.3, 33.2, 26.5, 26.1.
3
, 75.4 MHz): δ 50.4,
2
47
2.4.19. L-Cystine( 19b) [49]
0
0
1
2
2
2
48
49
50
2
White solid, mp: 238-239 C (lit [43] 240-242 C) H NMR (D O+4%NaOD, 400 MHz): δ
13
2
3.59-3.57 (2H, m), 3.14-3.09 (2H, m), 2.94-2.88 (2H, m); C NMR (D O+4%NaOD, 100
MHz): δ 180.5, 54.8, 43.3.
2
51
2.4.20. Bis(2-pyridinyl)disulphide (20b) [45]
1
2
2
2
52
53
54
White solid, mp: 53-56°C (lit [45] 53-54°C), H NMR (CDCl
3
, 300 MHz): δ 8.50 (2H, m),
13
7.63 (4H, m), 7.16-7.13 (1H, m); C NMR (CDCl
120.0
3
, 75.4 MHz): δ 158.9, 149.5, 137.4, 121.1,
2
55
2.4.21. Bis(pyrimidin-2-yl)disulphide (21b) [45]
1
2
Page 12 of 36

1
2
2
2
56
57
58
Yellow solid, mp: 144-145°C (lit [45] 143-145°C), H NMR (DMSO-d
6
+ CDCl
3
, 400 MHz):
13
δ 8.47-8.46 (4H, m), 7.08-7.06 (2H, m); C NMR (DMSO-d + CDCl , 100 MHz): δ 173.8,
6 3
163.0, 123.0
2
59
2.4.22. Bis(2-pyridinoxydyl)disulfide (22b) [114]
1
2
2
2
60
61
62
Yellow solid, mp: 202-204°C (lit [114] 205°C), H NMR (DMSO-d
6
, 300 MHz): δ 8.48 (2H,
13
s), 7.80.-7.3 (6H, m); C NMR (DMSO-d
6
, 75.4 MHz): δ 151.0, 139.5, 139.0, 127.9, 124.4,
122.7.
2
63
2.4.23. Bis(thiazol-2-yl)disulfide (23b) [115]
1
2
2
64
65
White solid, mp: 79°C (lit [115] 79-80°C), H NMR (CDCl
3
, 300 MHz):δ 7.80 (2H, d) 7.42
13
3
(2H, d); C NMR (CDCl , 75 MHz): δ 176.9, 144.3, 122.4.
2
66
2.4.24. Bis(benzo[d]thiazol-2-yl)disulfide (24b) [45]
0
0
1
2
2
2
67
68
69
White solid, mp: 176-177 C (lit [45] 176-178 C); H NMR (CDCl
3
, 400 MHz): δ 7.98-7.96
(2H, d, J= 8Hz), 7.81-7.79 (2H, d, J= 8Hz), 7.51-7.47 (2H, t, J= 8Hz), 7.40-7.36 (2H, t, J=
13
3
8Hz); C NMR (CDCl , 100 MHz): δ 167.8, 154.6, 136.2, 126.6, 125.3, 122.8, 121.3.
2
70
2.4.25. Bis(benzo[d]oxazol-2-yl)disulfide (25b) [45]
1
2
2
2
71
72
73
White solid, mp: 94-96°C (lit [45] 92-94°C), H NMR (DMSO-d
6
, 400 MHz): δ 7.77-7.75
13
(4H, m), 7.44-7.41 (4H, m); C NMR (DMSO-d
6
, 100 MHz): δ 159.8, 152.0, 141.2, 126.0,
125.3, 119.7, 111.0
2
74
2.4.26. 5,5'-disulfanediylbis(1,3,4-thiadiazol-2-amine) (26b) [116]
0
0
1
2
2
75
76
6
White solid, mp: 238-241 C (lit [116] 239 C), H NMR (DMSO-d , 300 MHz): δ 7.74 (2H,
13
6
s); C NMR (DMSO-d , 75.4 MHz): δ 173.0, 149.5.
1
3
Page 13 of 36

2
77
2.4.27. 1,2-diallyldisulphide (27b) [117]
1
2
2
78
79
Yellow oil, H NMR (CDCl
3
, 400MHz): δ 5.92-5.75 (2H, m) 5.24-5.10 (4H, m), 3.16-3.14
13
(4H, m); C NMR (CDCl
3
, 100 MHz): δ 134.39, 118.5, 42.4.
2
2
80
81
Spectral data of bis(2-aminophenyl)disulfidediimine Schiff bases (28b-30b):
2.4.28. 2,2´-disulfanediylbis(N-benzylideneaniline) (28b) [73]
0
1
2
2
2
2
82
83
84
85
Yellow solid, mp: 140 C (lit [73] 140°C), H NMR (CDCl
3
, 400 MHz): δ 8.54 (s, 2H), 8.03-
8.00 (m, 4H), 7.71-7.69 (m, 2H), 7.53-7.49 (m, 6H), 7.28-7.16 (m, 4H), 7.09-7.07 (m, 2H);
13
C NMR (100 MHz; CDCl
3
): δ 160.0, 148.9, 136.0, 132.1, 131.7, 129.1, 128.8, 126.9, 126.0
, 117.2.
2
86
2.4.29. 2,2´-disulfanediylbis(N-(4-methoxybenzylidene)aniline) (29b)[73]
0
1
1
2
2
2
87
88
89
Yellow solid, mp: 140 C (lit [73] 139 -140°C), H NMR (CDCl
3
, 400 MHz): δ 8.44 ( H, s),
13
7.97-7.95 (4H, m), 7.66 (1H, m),7.28–7.02 (12H, m), 3.91 (6H, s); C NMR (CDCl
3
, 100
MHz) : δ 162.5, 159.2, 149.2, 131.9, 130.9, 129.2, 126.8, 126.4, 125.8, 117.1, 114.2, 55.3.
2
90
2.4.30. 2,2'-disulfanediylbis(N-(pyridin-2-ylmethylene)aniline) (30b)[74]
0
1
2
2
2
2
91
92
93
94
Yellow solid, mp: 141 C (lit [74] 139-140°C), H NMR (DMSO-d
6
, 400 MHz,) δ 8.72 (s,
13
2H), 8.30 – 8.21 (m, 2H), 7.62 – 7.50 (m, 5H), 7.44 – 7.23 (m, 9H); C NMR (DMSO-d
6
,
100 MHz) : 160.4, 154.5, 149.7, 147.8, 136.8, 132.7, 127.9, 127.1, 126.0, 125.5, 122.2,
117.3.
2
95
3.0 Results and Discussion
2
2
96
97
Inspired by the outcomes of recent reports [69-72] on the decisive role of BSA catalyzed (i)
enone formation in ionic liquid [69] and (ii) thia-Michael addition to chalcone in water [72],
1
4
Page 14 of 36

2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
we planned to carry out the BSA catalyzed one-pot multicomponent reaction among
benzaldehyde, acetone and thiophenol in water via enone formation followed by thia-Michael
addition in the same pot (see supporting information). Unpredictably, we got some competing
side product (58% on GC-MS basis) which upon isolation and characterization by NMR, GC-
MS and HPLC comparison with standard [44], was confirmed to be diphenyldisulfide (see
S.I.). This result encouraged us to stir a mixture of thiophenol (0.25 mmol) (1a) and bovine
serum albumin [69,70] (50 mg, 0.00075 mmol, molecular weight = 66000 [59]) "on water"
[10] under aerobic [11] conditions for 12h, thereby providing diphenyldisulfide (1b) in 85%
yield (Entry 1, Table 1, GC-MS basis). Delighted by this serendipitous BSA protein catalysis
of 1a, we screened different commercially available serum proteins viz. rabbit, sheep, pig,
chicken, egg albumin to improve the yield of 1b, however, in all cases 1b was obtained in
poor yield (Entry 2-6, Table 1).
During the screening of different serum proteins (Entries 1-6, Table 1), it was realized that
conversion of thiol (1a) into disulfide (1b) with protein BSA (Entry 1, Table 1) was found
optimum which may be due to the rich varieties of surface amino acids [61,62] present in it.
Recently, Laccase enzyme [58] has also been utilized for oxidative coupling of thiols into
disulfides, therefore, we further planned to employ some well exploited and commercially
available enzymes [71] (Entries 7-12, Table 1) like CAL-B (Candida antarctica lipase), CRL
(Candida rugosa lipase), MJL (Mucor javanicus lipase), PCL (Pseudomonas cepacia lipase),
and CCL(Candida cylindracea lipase) for oxidation of thiol (1a) into disulfide (1b) on water
but none of these lipases showed any significant product (1b) formation (Entries 7-12, Table
1) in comparison to protein BSA (Entry 1, Table 1).
Next to improve the yield of 1b beyond 85% (Entry 1, Table 1), variations in time,
effect of temperature, solvents and amount of BSA were studied (Entries 2-15,Table 2).
During variations in time (Entries 1-3, Table 2), it was found that 12 h ( Entry 1) was
1
5
Page 15 of 36

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3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
sufficient for the oxidative coupling of 1a while increase (24 h) or decrease (6 h) in reaction
time did not show any improvement in yield of 1b (Entries 2-3, Table 2). Further, variations
in reaction temperature (Entries 4-5,Table 2) and amount of BSA (Entries 6-7) did not show
any influence on the improvement of yield of 1b. Thereafter, the change in reaction medium
from water to organic solvents (Entries 8-12,Table 2) were taken into account but no
significant improvement in the yield of 1b was observed. Further to validate our perception
regarding the crucial role of BSA, dissolved oxygen in water or aerobic oxygen for oxidation
of 1a into 1b, we performed a set of three reactions i) aerobic oxidation of 1a without BSA in
water (Entry 13, Table 2), ii) oxidation of 1a with BSA and water under N
ward off oxygen) (Entry 14, Table 2) and iii) oxidation of 1a with BSA in degassed water (to
ward off oxygen) under N atmosphere (Entry 15) wherein yield of 1b was obtained in 0%,
2
atmosphere (to
2
37% and traces amount respectively (on the basis of GC-MS analysis). These findings clearly
highlighted the crucial role of BSA and aerial oxygen for facile oxidative coupling of 1a into
1b. Among various proposed reaction pathway for oxidative coupling of thiols, a thiyl radical
[58] pathway is generally known for S-S bond formation. To ascertain whether the aerobic
oxidation of 1a follows a free radical pathway or not, an experiment with 10 mol% BHT (a
free radical quencher) (Entry 16, Table 2) as well as oxidation of 1a in dark (Entry 17, Table
2) were conducted and in both cases 1b was obtained in good yield, hence, ruling out the
possibility of free radical pathway.
After optimal reaction conditions in hand for oxidative coupling of 1a, we examined
the scope of different substituted thiols to form the disulfides (1b-18b, Table 3) including
synthesis of a physiologically important cystine (19b, Table 3). As shown in Table 3 (1b-
19b), a wide range of thiophenols with electron-donating (2b-10b, Table 3) and electron-
withdrawing (11b-16b) substituents have provided good to excellent yields (upto 97%) of
disulfides [96-98] wherein some of disulfides possess cytotoxic [97], leishmanial [98] and
1
6
Page 16 of 36

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3
3
3
3
3
3
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3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
antibacterial activities [74]. Delightedly, amino or hydroxy substituted thiophenol (4a and 5a,
Table 3) also successfully underwent oxidative coupling to form corresponding disulfide (4b
and 5b, Table 3) in good yield (upto 85%) whereas previous protocols [44-46] documented
the formation of the same disulphides (4b and 5b) in poor yield due to additional interaction
of -NH
Interestingly, BSA possesses rich variety of amino acids which will interact efficiently with
-SH of thiophenol in water whereas strong hydrogen bonding ability of –OH and –NH in
2
or -OH proton verses -SH proton of thiophenol with catalysts/reagents [44].
2
water may attenuate their interaction with biocatalyst (BSA) thereby permitting smooth
oxidative coupling of thiophenols (4a and 5a) into disulfides (4b and 5b, Table 3).
Apart from aromatic disulphides (1b-17b, Table 3), many heteroaromatic disulfides (20b-26b,
Table 4) are privileged scaffolds of some medicinally active compounds [27-37], thus high
therapeutic profile and their recent biocatalyzed synthesis by U. Beifuss et al [58] propelled
us to target synthesis of these heteroaromatic disulfides (20b-26b, Table 4) in our neutral
catalytic system (BSA/water) devoid of any additives or use of organic solvent. We
successfully obtained six membered heterocyclic disulfides with excellent yield (upto 90%)
having pyridine and pyrimidine ring (20b-26b, Table 4) which are documented for production
of a dendritic cell-based vaccine for HIV [35] as well as a drug for neuropsychological
disorders [33]. Similarly, five membered heterocyclic disulfides (23b-26b, Table 4) were also
obtained in moderate yield (upto 65%).
In order to further extend the application of developed protocol for oxidation of aliphatic
thiols, the synthesis of bioactive [36, 99-101] diallyl-disulfide (DADS) (27b), a well-known
secondary metabolite of garlic (Allium sativa) was targeted. Thus, BSA catalyzed oxidation
of allylthiol (Scheme1) on water provided diallyldisulfide (DADS) in 78% yield (isolated)
under environmentally benign aerobic condition.
1
7
Page 17 of 36

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3
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3
3
3
3
3
3
3
3
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3
3
3
3
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
Mechanistically, we hypothesized that acidic and basic amino acid functionalities in the side
chain of BSA [64-67] might be responsible for oxidative coupling of thiophenols into
disulfides in aqueous condition. In fact, structurally the BSA, a non-redox carrier protein,
contains side chains of acidic (Asp, Glu) [64] and basic (Arg, Lys, His)[64] amino acids
which could act as an ambiphile [102-104] in water. In this view, disulfides formation might
be driven through transition state {A} (Scheme 2) wherein sulfur of thiophenol interacts with
acidic hydrogen of surface amino acid side chains of BSA analogues to carbonyl oxygen
activation by oxyanion hole [105-108] in enzyme catalysis [59-68, 105-107] or amino acid
catalysis [97] thereby imparting the partial positive character to sulfur of thiophenol. On the
other side the basic amino side chains [59-71] of BSA as reported previously [70,102] by
Zhao et al. [62] or heterocyclic amino acids like histidine [62,63,105] present in BSA could
somewhat behave like base thereby their interaction with -SH proton (of another molecule of
thiophenol) impart nucleophilic character to sulfur. Overall this ambiphilic [49,102-104]
(electrophilic and nucleophilic) activation triggers interaction of nucleophilic (δ-) and
electrophilic (δ+) sulfur, which was further assisted by aerobic oxygen and water through
hydrogen bonding (HB) leading to diphenyldisulfide with removal of water as the only by
product.
Furthermore to investigate an insight in the crucial role of amino acids (acidic or basic) side
chain present in BSA, we carried out oxidative coupling of thiophenol (1a) with
commercially available acidic (Glutamic acid, Aspartic acid) and basic (Arginine and Lysine)
amino acids in water (see S.I.). To our delight, arginine provided maximum yield (52%)
while glutamic acid provided 37% yield of 1b (see S.I.) which overall supports the synergetic
role of both acidic and basic amino acid functionalities present in BSA which might be
responsible for high yield of disulfide (85%, 1b, Table 3) under above catalytic system (i.e.
1
8
Page 18 of 36

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3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
BSA-water-air). Further, reduction in yield of disulfide (1b) with acylated and denatured [70]
BSA also confirms our mechanistic postulate (see S.I.).
Having established substrates scope and plausible mechanism, we proceeded to assess
the recyclability of the biocatalytic system for oxidation of 4-methoxy thiophenol (3a) into
bis(4-methoxyphenyl) disulfide (3b, Table 3) where the recovered catalyst retained activity
upto four cycles, thereafter yield of 3b decreased below 90% (Fig. 4) due to coagulation of
BSA. The E-factor (see S.I.) of our biocatalytic system were also found to be low for 3b
which further confirms environmental compatibility of this biocatalytic protocol according to
green chemistry metrics.
To probe the practical applicability of our catalytic system (Table 3) for disulfides synthesis,
preparative scale oxidative coupling reaction of 4-methoxythiophenol (3a, 1g, 7.1 mmol)
using BSA (200 mg) in water (2 mL), at room temperature (16 h) formed corresponding
disulphide, bis(4-methoxyphenyl)disulfide (3b, Table 3) in excellent yield upto 92% (see S.I.)
precluding any metal/non-metal complexes, bases and additives as shown in Table 5 which
clearly demonstrates its applicability in comparison to reported protocols [38-47, 56-58,118-
133].
After the successful oxidation of thiols (aromatic, heterocyclic, aliphatic) into corresponding
disulfides (1b-27b, Table 3, 4 and scheme 1) and establishing the mechanistic study (Scheme
2), we envisioned that bis(2-amino diphenyl)disulfide, an amino functionalised disulfide (4b,
Table 3), can further be reacted with benzaldehyde to form bis(aminophenyl)disulfidediimine
which possesses good antimicrobial activity against human pathogens [73] and also
documented as an important ligand in synthetic chemistry [74] besides acting as a basic
scaffold for various organic synthesis [75-79]. Thus, in order to examine the application of
our developed biocatalytic green protocol for tandem [85-88] oxidative coupling as well as
condensation reactions between aminothiophenol and benzaldehyde, we particularly added
1
9
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4
21
22
23
24
25
26
27
28
29
30
31
32
33
34
benzaldehyde in parts (to avoid benzothiazole formation) [89-91] to a well stirred mixture of
2-aminothiophenol and BSA in water but unfortunately, the desired bis(2-
aminophenyl)disulfidediimine was formed only in trace amount with many side-products
(Scheme 3). So in order to elude the multiple product formation due to competitive reactions
(Scheme 4), we next followed tandem sequential [88] reaction wherein initial formation of
diiminedisulfide (4b) was achieved in 18 h. After that benzaldehyde (1.1eq) was added in the
same pot and further stirred for 12h at r.t. which successfully furnished desired product (28b)
in 77% yield (Scheme 3) via oxidative coupling and condensation reaction in one pot.
Similarly, disulfidediimines (29b-30b) were also obtained with good yields via tandem-
sequential oxidative coupling-condensation in the same pot under aerobic aqueous conditions
(Scheme 4) while reported methods [73-74] involve multi steps and demand anhydrous
conditions. Thus among the richness of various organic transformations catalyzed by BSA
[134], our method may be employed as a mild approach for disulfides synthesis along with
extended approach towards Schiff bases
4
35
4.0 Conclusions
4
4
4
4
4
4
4
4
4
36
37
38
39
40
41
42
43
44
In summary, this is the first report on bovine serum albumin (BSA) catalyzed oxidative
coupling of a series of thiols (aromatic, aliphatic and heterocyclic) into disulfides (S-S bonds)
“on water” under neutral conditions whose significant features are: (i) metal and base-free
protocol, (ii) air as a green oxidant, (iii) water as a green solvent, (iv) mild reaction
conditions and easy isolation of the products, (v) waste free protocol and (vi) good
recyclability with high atom economy and low E factor. More importantly, the versatility of
protocol finds application for synthesis of 4,5-dithia-1,7-octadiene (diallyldisulphide
commonly known as DADS), a biologically important secondary metabolite of garlic (Allium
sativa). Furthermore, the utility of the methodology for an efficient construction of
2
0
Page 20 of 36

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4
4
4
4
4
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45
46
47
48
49
50
51
52
53
biologically active bis(aminophenyl)disulfidediimine is also demonstrated where BSA
catalyzed one S-S and two C-N bonds are formed for the first time solely "on water" in one
pot via sequential oxidative coupling of 2-aminothiophenol into intermediate bis(2-
aminophenyl)disulfide followed by condensation with benzaldehyde without isolation of
intermediate, thus the developed protocol is of great value from the green chemistry
perspective and organic synthesis due to inexpensive nature of BSA, air as a green oxidant
and water as a solvent. Work is underway toward the further development of relevant
reactions.
Acknowledgements
4
4
4
4
4
54
55
56
57
58
Authors are gratefully acknowledge the Director (s) of C.S.I.R.-C.D.R.I., Lucknow and
C.S.I.R.-I.H.B.T., Palampur for their kind cooperation and encouragement. Some part of
work carried out at I.H.B.T. (3537). The authors are also grateful to SAIF division of CDRI
and Mr. S Kumar/Mrs. V. Pathania of I.H.B.T. for providing spectral data. CDRI
Communication No. 36/2015.
4
4
59
60
†Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/b000000x
4
61
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4
4
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64
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[2] T. Hudlicky, J.W. Reed, Chem. Soc. Rev. 38 (2009) 3117-3132.
[3] D. J. Pollard, J. M. Woodley, Trends Biotechnol. 25 (2007) 66-73.
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[4] A. Schmid, J. S. Dordick, B. Hauer, A. Kiener, M. Wubbolts, B. Witholt, Nature 409
(2001) 258-268.
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68
[5] A. Russo, C. De Fusco, A. Lattanzi, ChemCatChem. 4 (2012) 901–916.
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Figure Captions
Figure 1: Biological active scaffolds containing S-S bond
6
6
66
67
Figure 2: BSA catalysed first oxidative coupling of thiols into disulfides (S-S bond)
under neutral condition
6
6
68
69
Figure 3: Synthesis of Schiff bases with one S-S and two C-N bonds via oxidative
coupling-condensation reactions in one pot
6
70
Figure 4: Recyclability of BSA in developed protocol
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72
Table 1: Screening of different serum proteins and enzymes for oxidation of thiophenol
[a]
(1a) into disulfide (1b)
6
6
6
6
73
74
75
76
2
[a] Experimental conditions: 0.25 mmol 1a, 600 µL H O for 12 hr; [b] yield on the basis
of GC-MS; (BSA=Bovine serum albumin; CAL-B= Candida antarctica lipase-B; CRL=
Candida rugosa lipase; MJL=Mucor javanicus lipase; TLL= Thermomyces lanugenosus
lipase; PCL= Pseudomonas cepacia lipase; CCL=Candida cylindracea lipase.)
[a]
6
77
Table 2: Optimization of BSA catalyzed disulfide synthesis
[
a]
6
6
6
78
79
80
Experimental conditions: 0.25 mmol 1a, 600 µL solvent; BSA= Bovine serum albumin
[b]
[c]
[d]
[e]
2
50 mg; Yield on the basis of GC-MS; without BSA; under N atmosphere; in
[f]
the presence of BHT ( Butylatedhydroxytoluene); reaction in dark
6
6
81
82
Table 3: Substrate scope for oxidative coupling of aromatic thiols into disulfides in
[a]
BSA-water-oxygen catalytic system
[
a]
6
6
83
84
Reaction conditions: Thiol 0.25 mmol, BSA 50 mg, water 600 µL, rt, reaction time
[b]
[c]
(12-48 h); Isolated yield; yield on GC-MS basis
6
6
85
86
Table 4: Substrate scope for oxidative coupling of heterocyclic thiols into disulfides in
[a]
BSA-water-oxygen catalytic system
6
6
87
88
[a] Experimental conditions: Thiol 0.25 mmol, BSA= 50 mg, Water= 600 µL; [b]
Isolated yield
6
6
89
90
Table 5: Substrate scope for oxidative coupling of heterocyclic thiols into disulfides in
[a]
BSA-water-oxygen catalytic system
6
6
91
92
Scheme 1: Synthesis of diallyldisulfide (DADS)
2
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Page 29 of 36