Laccase-catalyzed in situ generation and regeneration of N-phenyltriazolinedione for the aerobic oxidative homo-coupling of thiols to disulfides

Article abstract of DOI:10.1016/j.catcom.2018.06.007

The first report on aerobic in situ generation and regeneration of N-phenyltriazolinedione, a valuable oxidizing agent, from a catalytic amount of N-phenyl urazole in the presence of a laccase enzyme is presented. The application of a 4-phenyl urazole/Laccase/O₂ as a new cooperative catalytic oxidation system is reported for a transition-metal-free and halogen free oxidative homo-coupling reaction of structurally diverse thiols to their corresponding disulfides with good to excellent yields in a phosphate buffer solution under mild reaction conditions.

Full text of DOI:10.1016/j.catcom.2018.06.007

Accepted Manuscript
Laccase-catalyzed in situ generation and regeneration of N-
phenyltriazolinedione for the aerobic oxidative homo-coupling of
thiols to disulfides
Donya Khaledian, Amin Rostami, Seyed Amir Zarei
PII:
DOI:
Reference:
S1566-7367(18)30216-4
doi:10.1016/j.catcom.2018.06.007
CATCOM 5422
To appear in:
Catalysis Communications
Received date:
Revised date:
Accepted date:
3 March 2018
7 June 2018
8 June 2018
Please cite this article as: Donya Khaledian, Amin Rostami, Seyed Amir Zarei , Laccase-
catalyzed in situ generation and regeneration of N-phenyltriazolinedione for the aerobic
oxidative homo-coupling of thiols to disulfides. Catcom (2017), doi:10.1016/
j.catcom.2018.06.007
This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.

ACCEPTED MANUSCRIPT
Laccase-catalyzed in situ generation and regeneration of N-
phenyltriazolinedione for the aerobic oxidative homo-coupling of
thiols to disulfides
Donya Khalediana , Amin Rostami*a, Seyed Amir Zareib
aDepartment of Chemistry, Faculty of Science, University of Kurdistan, 66177-15175
Sanandaj, Iran. [Fax: +988716624004, Tel: +989183730910, E-mail: a.rostami@uok.com]
bDepartment of Chemistry, Faculty of Science, Sanandaj Branch, Islamic Azad University,
Sanandaj, Iran.

ACCEPTED MANUSCRIPT
Abstract
The first report on aerobic in situ generation and regeneration of N-phenyltriazolinedione, a
valuable oxidizing agent, from a catalytic amount of N-phenyl urazole in the presence of a
laccase enzyme is presented. The application of a 4-phenyl urazole/Laccase/O2 as a new
cooperative catalytic oxidation system is reported for a transition-metal-free and halogen free
oxidative homo-coupling reaction of structurally diverse thiols to their corresponding
disulfides with good to excellent yields in a phosphate buffer solution under mild reaction
conditions.
Keywords: Laccase enzyme, 4-Phenyl urazole, Cooperative catalytic system, N-
phenyltriazolinedione, Aerobic oxidative, Disulfides

ACCEPTED MANUSCRIPT
1. Introduction
Disulfides have a vital role in different fields such as: vulcanizing agents in industry,
efficacious agents in folding and stability of the proteins as well as their performance in
biochemistry, pharmaceutical activity and significant synthetic intermediates in organic
synthesis [1-2]. These applications can reflect the importance of thiols oxidative coupling to
their corresponding disulfides. Many oxidizing reagents have been reported in the literature to
accomplish this transformation [3-13]. However, these reagents show poor atom efficiency
and are often toxic. There is substantial interest towards the use of molecular oxygen or air as
the highly atom-economical, environmentally benign, and abundant oxidant in the oxidative
coupling of thiols in the presence of catalysts. Accordingly, various catalysts such as cobalt-
phthalocyanine [14], gold [15] and heterocyclic bismuth (III) compounds [16] have been used
for the oxidation of thiols using molecular oxygen as terminal oxidant. Although these
methods have been developed considerably, these procedures suffer from some problems
such as low catalytic efficiency, tedious catalyst preparation and the use of toxic organic
solvents. In addition, they may possibly leave toxic traces of heavy metals in the products.
Therefore, it is a purpose for chemists to gain more effective, non-metallic and
environmentally benign replacements.
4-Substituted 1,2,4-triazole-3,5-diones are notable for their ability to participate in a wide
range of concerted and stepwise reactions [17]. Also, they were introduced as one of the
efficient oxidative reagents due to its dehydrogenating feature [18-21]. Despite the versatility
of triazolediones, their low stability, high cost and relatively high toxicity and also challenges
that can arise in removing their reduced forms from the reaction products when used as
stoichiometric oxidant are disadvantages that could limit using of them in green organic
reactions. In order to avoid the abovementioned disadvantages, the best strategy is the in situ
generation of these valuable intermediates and organic oxidizing agents from the oxidation of
their corresponding urazoles that are stable, commercially available and cheap compounds.
Although various reagents have been reported for the oxidation of urazoles to the
corresponding triazolindiones (TADs) [22-25], these procedures suffer from one or more
disadvantages such as harsh conditions, the use of toxic and expensive stoichiometric
reagents and the production of large quantities of waste.
From the viewpoint of green and sustainable chemistry, O2 and air are highly atom-
economical, environmentally benign, and abundant oxidant that minimize the
generation of hazardous materials. Therefore, there is substantial interest towards the use

ACCEPTED MANUSCRIPT
of molecular oxygen or air as terminal oxidant. Direct aerobic oxidation of urazoles to the
corresponding triazolindiones is typically not feasible. In reality, a catalyst must be employed
for aerobic oxidation of urazoles with molecular oxygen. The employing of enzymes as safe
and biodegradable catalysts offer many benefits since biocatalytic reactions can be performed
under mild conditions [26-27]. In this context, the oxidative coupling of the various organic
compounds using aerial oxygen has been catalyzed by a laccase enzyme [28-29] (benzendiol;
O2 oxidoreductase, E.C. 1.10.3.2.). Higher redox potentials could be reached using laccase
and different cooperative agents to embrace the oxidation of diverse functional groups such
as alcohols, ethers and amines [30-32]. Recently, Beifuss and co-worker have reported
metal-free aerobic oxidative coupling of heterocyclic thiols using a laccase mediator system
(LMS) [33]. Although, this finding presented an interesting breakthrough in the field of
metal-free aerobic oxidation of thiols, this method requiers a large amount of laccase and the
use of mediators that are quite expensive and relatively toxic. Therefore, the discover of new
cooperative catalytic systems of laccase and cheap, commercially available in large
quantities, shelf-stable and active organic mediators for aerobic oxidation of thiols and other
organic compounds is in demand.
2. Experimental
The activity assays of commercial enzyme laccase from T. versicolor: The activity of
laccase
was
determined
spectrophotometrically
with
2,2’-azino-bis-(3-
-1
ethylbenzthiazoline-6-sulfonic acid) (ABTS, ε= 36 000 M-1.cm ) as substrate (5 mM,
100 µL) in acetate buffer (100 mM, pH=5.0). The absorbance change was observed at
420 nm for 5 min at room temperature [34]. One unit of activity is defined as the amount
of enzyme required to oxidize 1 µmol of ABTS per minute. The activity of laccase
enzyme batch applied in this investigation was evaluated at 0.87 U/mg.
General procedure for aerobic oxidation of sulfides to their corresponding disulfide in the
presence of laccase/4-phenyl urazole cooperative catalyst system
To a solution of 1 mmol of thiol in acetonitrile (2 mL), was added a solution of 8 mL of
phosphate buffer including laccase (40 U) and 4-phenyl urazole (10 mol%). The resulting
mixture was stirred under air at room temperature for the time specified. After completion of
the reaction (monitored by TLC), NaOH (10%, 10 mL) was added and the mixture was
stirred for 5 min. The products were extracted with diethyl ether (3 × 10 mL). The combined
organic phases were dried over anhydrous Na2SO4, filtered and evaporated under reduced
pressure. The crude products were purified by recrystallization or chromatography on silica

ACCEPTED MANUSCRIPT
gel (n-hexane/EtOAc, 5/1). All the products were known and characterized by comparison of
their spectral data (1H NMR) and physical properties (melting point) with those of authentic
samples.
3. Results and discussions
Very recently we have used a laccase enzyme as green catalyst to mediate aerobic oxidation
of urazoles to triazolindiones as valuable intermediate for the synthesis of 4-arylsulfonyl
urazoles [35]. Inspired by the successful results in this work and the ability of
triazolinediones to act as an effective stoichiometric oxidant for organic compounds [18-21],
herein, we want to present for the first time aerobic in situ generation and regeneration of 4-
phenyl triazoledione from a catalytic amount of 4-phenyl urazole in the presence of laccase as
biocatalyst for the oxidation of thiols in phosphate buffer/acetonitrile mixture at room
temperature (Scheme 1).
Laccase (Cat.), air (Oxidan.)
N
NH
N
HN
Cat.
O
O
O
O
N
N
Ph
Ph
R
S
S
2
R
S
R
H
phosphate buffer/acetonitrile, rt
Scheme 1. Aerobic oxidative coupling of thiol to disulfide catalyzed by a laccase/4-phenyl urazole cooperative
system
In order to optimize the reaction conditions, the aerobic oxidative coupling of 4-methyl
thiophenol was selected as a model reaction. Initially, oxidation of 4-methyl thiophenol was
conducted in the presence of laccase (40 U) as catalyst and air as an oxidant in MeCN/NaPBS
mixture (2:8) as solvent at room temperature for 3h. Under these conditions, low conversion
of 4-methyl thiophenol was observed (Table 1, entry 1). When the amount of laccase was
increased to 200 U, the reaction was completed after 24h (Table 1, entry 3). In order to
reduce the amount of laccase used and the reaction time, 4-phenyl urazole was used as
mediator. Upon further investigation (Table 1, entries 4-8), 40 U of laccase and 0.1 mmol of
4-phenyl urazole were selected as standard concentrations in all further reactions. The
oxidative coupling reaction of 4-methyl thiophenol in water, ethanol, water/ethanol (1/1; v/v),
THF and THF/water (1/1; v/v) media are proceeded with moderate yields ranging from 37%-
55% (entries 10-14).
Table 1. Optimization of the reaction conditions with respect to the effect of concentration of laccase and 4-
phenyl urazole and solvent on aerobic oxidative coupling of 4-methyl thiophenola

ACCEPTED MANUSCRIPT
SH
Laccase (Cat)/4-Phenyl urazole (Cat.), air
S
2
S
phosphate buffer/acetonitrile, r.t.
Entry
1
2
3
4
5
6
7
8
Solvent
Laccase (U)
4-Phenyl urazole (mmol)
-
-
Time (h)
3
3
24
5
1.5
3
3
3
3
GC Yield (%)
45
75
MeCN/NaPBS
MeCN/NaPBS
MeCN/NaPBS
MeCN/NaPBS
MeCN/NaPBS
MeCN/NaPBS
MeCN/NaPBS
MeCN/NaPBS
MeCN/NaPBS
Water
40
100
200
100
100
40
40
40
-
40
40
40
40
40
100
100
100
60
0.05
0.1
0.05
0.1
0.2
0.2
0.1
0.1
0.1
0.1
0.1
100b
100
45
9
10
11
12
13
14
3
3
3
3
37
45
50
55
Ethanol
Water/Ethanol (1:1)
THF
Water/THF (1:1)
3
55
aReaction conditions: 4-methyl thiophenol (1 mmol), phosphate buffer (0.1 M, 8 mL, pH = 5.0), acetonitrile (2 mL), air, rt,
bSelected reaction conditions.
Encouraged by the initial success, the generality and applicability of this method was
further examined for aerobic oxidative coupling of various thiols including aromatic (with
electron-withdrawing and electron-releasing groups), benzylic, heterocyclic and aliphatic
(cyclic and acyclic) thiols (Table 2).
Table 2. Aerobic oxidative coupling of thiols to disulfides catalyzed by laccase/4-phenyl urazole systema
Entry Thiol
Disulfide
Time Yield
(h)
M. p.
(°C)
M. p. (°C)
[Lit.]
(%)b
1
4
95
60-61
61-62 [3]
2
3
3
5
95
92
39-40
Oil
39-42 [4]
Oil [9]
4
6
95
69-71
70-72 [6]
5
6
7
90
85
69-70
Oil
69-70 [5]
Oil [6]
10

ACCEPTED MANUSCRIPT
7
13
85
123-124
125-128 [7]
8
9
9
87
88
Oil
Oil
Oil [8]
Oil [6]
10
10
12
85
Oil
Oil [10]
11
12
14
15
75
75
176-178
84-86
177-180 [4]
84-88 [4]
aReaction conditions: Laccase (40 U), thiol (1 mmol), 4-phenylurazole (0.1 mmol), phosphatebuffer (0.1 M, 8 mL, pH = 5.0),
acetonitrile (2 mL), air, rt. bIsolated yield.
As shown in Table 2, the corresponding disulfides were obtained in good to high yields, it
seems that the resulting yields for oxidative coupling of aromatic thiols are slightly higher in
comparison with aliphatic thiols. It was shown that aromatic thiols oxidized more quickly
than aliphatic thiols. It is also observed that aromatic thiols with electron-donating groups
(Entry 2) are more reactive than thiols with electron-withdrawing groups (Entries 3, 4).
Interestingly, 2-mercaptobenzothiazole and 2-mercaptobenzimidazole which are relatively
unreactive thiols were also converted to the corresponding disulfides in good yields under the
same reaction conditions (Table 2, entries 11-12).
Although the exact mechanism of this reaction is not clear, based on previously reported
mechanisms about application of laccase in oxidation of catechol in tandem reactions [29-33,
35] and oxidative coupling of thiols using 4-phenyl-1,2,4-triazole-3,5-dione as stoichiometric
oxidant [20], the proposed mechanism and catalytic cycle for the aerobic oxidative coupling
reaction of thiol to corresponding disulfide in the presence of the laccase-urazole cooperative
catalyst system has been depicted in Scheme 2. It is supposed that the reaction begins with
the oxidation conversion of 4-phenyl urazole to 4-phenyl-1,2,4-triazole-3,5-dione by laccase
that is accompanied by the reduction of oxygen to water. Then, the oxidative coupling
reaction of thiol leads to the formation of disulfide that is motivated by the reduction
conversion of 4-phenyl-1,2,4-triazole-3,5-dione to 4-phenyl urazole.

ACCEPTED MANUSCRIPT
O
NH
H2O
Laccase (ox)
S
R
Ph
N
R
S
NH
O
O
O
1/2O2
Laccase (red)
S
NH
N
N
N
N
R
H
Ph
Ph
N
R
S
O
O
S
H
R
Scheme 2. Proposed mechanism and catalytic cycle for the aerobic oxidation
4. Conclusion
In summary, we have found the simple, green and efficient method for aerobic oxidative
coupling of structurally diverse thiols to their corresponding disulfides in the presence of
laccase/4-phenyl urazole as a novel cooperative catalytic system in phosphate buffer (pH =
5.0)/ acetonitrile solution at room temperature. This present method is more eco-friendly
and practical than other currently available methods due to being free from any transition
metal and halide and the quantitative conversion under mild reaction conditions. Further
studies on the scope and synthetic application of this new cooperative catalyst system are in
progress in our laboratory.
Acknowledgments
We are grateful to Kurdistan University Council for the financial support of this research.
Appendix A. Supplementary data
Supplementary data to this article can be found online at.

ACCEPTED MANUSCRIPT
References
[1] M. Taha, N. H. Ismail, S. Imran, A. Wadood, F. Rahim, M. Riaz, Bioorg. Med. Chem. 23
(2015) 7211-7218.
[2] T. Tankam, K. Poochampa, T. Vilaivan, M. Sukwattanasinitt, S. Wacharasindhu,
Tetrahedron 72 (2016) 788-793.
[3] M. Hirano, S. Yakabe, M. Fukami, T. Morimoto, Synth. Commun. 27 (1997) 2783-2787.
[4] A. Ghorbani-Choghamarani, S. Sardari, J. Sulfur Chem. 32 (2011) 63-69.
[5] B. Karami, M. Montazerozohori, M. H. Habibi, Molecules 10 (2005) 1358-1363.
[6] A. Talla, Photoredox aerobic oxidation of thiols to disulfides in a photomicroreactor,
M.Sc. Thesis, Eindhoven University of Technology, 2014.
[7] D. L. Holbrook, Handbook of Petroleum refining Processes, R. A. Meyers Ed., McGraw
Hill, 1996.
[8] P. Zhong, M. P. Guo, Synth. Commun. 31 (2001) 1825-1828.
[9] A. Ghorbani-Choghamarani, M. Nikoorazm, H. Goudarziafshar, M. Abbasi, Bull. Korean
Chem. Soc. 32 (2011) 693-696.
[10] Z. Lasemi, R. Hosseinzadeh, M. Tajbakhsh, M. Mohadjerani, Bulg. Chem. Commun. 45
(2013) 379-384.
[11] S. Sheriff Shah, S. Karthik, N. D. Pradeep Singh, RSC Adv. 5 (2015) 45416-45419.
[12] L. Shiri, A. Gorbani-Choghamarani, M. Kazemi, Aust. J. Chem. 70 (2017) 9-25.
[13] A. Rostami, S. Moradi, Z. Shokri, C. R Chim. 21 (2018) 80-87.
[14] Y. Dou, X. Huang, H. Wang, L. Yang, H. Li, B. Yuan, G. Yang, Green Chem. 19 (2017)
2491-2495.
[15] A. Coma, T. Rodenas, M. J. Sabater, Chem. Sci. 3 (2012) 398-404.
[16] A. M. Toma, C. I. Rat, O. D. Pavel, C. Hardacre, T. Ruffer, H. Lang, M. Mehring, A.
Silvestru, V. I. Parvulescu, Catal. Sci. Technol. 7 (2017) 5353-5353.
[17] K. D. Bruycker, S. Billiet, H. A. Houck, S. Chattopadhyay, J. M. Winne, F. E. D. Prez,
Chem. Rev. 116 (2016) 3919-3974.
[18] M. A. Zolfigol, A. Ghorbani Choghamarani, M. Shahamirian, M. Safaiee, I.
Mohammadpoor-Baltork, S. Mallakpour, M. Abdollahi-Alibeik, Tetrahedron Lett. 46
(2005) 5581-5584.
[19] M. A. Zolfigol, D. Azarifar, S. Mallakpour, I. Mohammadpoor-Baltork, A. Forghanih,
B. Maleki, M. Abdollahi-Alibeik, Tetrahedron Lett. 47 (2006) 833-836.
[20] A. Christoforou, G. Nicolaou, Y. Elemes, Tetrahedron Lett. 47 (2006) 9211-9213.

ACCEPTED MANUSCRIPT
[21] R. C. Cookson, I. D. R. Stevens, C. T. Watts, Chem. Commun. (1966) 744-744.
[22] M. A. Zolfigol, M. Torabi S. E. Mallakpour, Tetrahedron 57 (2001) 8381-8384.
[23] P. Zhong, M. P. Guo, Synth. Commun. 31 (2001) 1825-1828.
[24] M. A. Zolfigol, G. Chehardoli S. E. Mallakpour, Synth. Commun. 33 (2003) 833-841.
[25] M. A. Zolfigol, E. Madrakian E. Ghaemi, S. Mallakpour, Synlett (2002) 1633-1636.
[26] K. Faber, Biotransformations in Organic Chemistry, Springer, Berlin, 6th edn., 2011.
[27] S. K. Dey, A. Mukherjee, Coord. Chem. Rev. 310 (2016) 80-115.
[28] M. Mogharabi, M. A. Faramarzi, Adv. Synth. Catal. 356 (2014) 897-927.
[29] D. Habibi, A. Rahimi, A. Rostami, S. Moradi, Tetrahedron Lett. 58 (2017) 289-293.
[30] H. T. Abdel-Mohsen, J. Conrad, U. Beifuss, Green Chem. 16 (2014) 90-95.
[31] M. D. Cannatelli, A. J. Ragauskas, Tetrahedron Lett. 57 (2016) 3749-3753.
[32] H. T. Abdel-Mohsen, K. Sudheendran, J. Conrad, U. Beifuss, Green Chem. 15 (2013)
1490-1495.
[32] Q. Cao. L.M. Dornan, L. Rogan, N. L. Hughes, M. J. Muldoon, Chem. Commun. 50
(2014) 4524-4543.
[33] H. T. Abdel-Mohsen, K. Sudheendran, J. Conrad, U. Beifuss, Green Chem. 15 (2013)
1490-1495.
[34] C. Bohlin, K. Lundquist, L. Jönsson, J. Bioorg. Chem. 37 (2009) 143-148.
[35] A. Rahimi, D. Habibi, A. Rostami, M. A. Zolfigol, S. Mallakpour, Tetrahedron Lett. 59
(2018) 383-387.

ACCEPTED MANUSCRIPT
Graphical Abstract
Laccase-catalyzed in situ generation and regeneration of N-phenyltriazolinedione for the
aerobic oxidative homo-coupling of thiols to disulfides
Donya Khaledian, Amin Rostami, Seyed Amir Zarei
Laccase (Cat.), air (Oxidan.)
N
NH
HN
N
Cat.
O
O
O
O
N
N
Ph
Ph
R
S
S
H2O
+
R
S
2
R
H
phosphate buffer/acetonitrile, rt
R=Ar (Het), benzylic, linear, cyclic

ACCEPTED MANUSCRIPT
Highlights
 Aerobic in situ generation and regeneration of N-phenyltriazolinedione catalyzed by
laccase
 Discovering a new cooperative catalytic system consisting of laccase and 4-phenyl
urazole
 The first report application of a laccase/4-phenyl urazole for the aerobic oxidation of
thiols
 This catalyst system allows for greener methodology that affords high reaction yields.