The study describes chemo-enzymatic synthesis of organic disulphide compounds. The reaction was initiated by hydrolysis of thiol acetates using hydrolytic enzyme lipase (PPL) immobilized on to magnetic nanoparticles and subsequent formation of organic disulphide compounds. Lipase was immobilized on to the magnetic nanoparticles by co-precipitation method via epichlorohydrin chitosan cross-linking, under mild and eco-friendly conditions. The immobilized lipase enzyme exhibited broad range of substrate specificity in synthesizing disulphide compounds, which involves both intra and inter-molecular disulphide bond formation under anaerobic conditions. The disulphide compounds synthesized also show a promising antimicrobial activity.
The disulphide linkages being an integral part of the macromolecules, mainly present in functional proteins like enzymes, peptides, hormones and antibodies. These disulphide bonds provide configurational specificity and also help in stabilizing the protein molecules to carry out their specific biological functions [
All the chemicals used in the study were AR grade and were purchased from Merck co and Sigma-Aldrich Chemicals USA. Lipases obtained from porcine pancreas (PPL) 3000 U/mg of protein (Biuret) EC 232-619-9 from Sigma―Aldrich USA. All the microbial strains used were procured from ATCC microbial culture collection center―USA.
The disulphide products were quantified by HPLC (Gilson system) using silica column (inside diam. 4.6 × 250 mm, film thickness 4 µm); with a mobile phase of Hexane: Ethyl acetate (70:30); flow rate 0.8 ml/min at a UV wave length of 254 nm.
The magnetic nanoparticles (Magnetite (Fe3O4)-chitosan (CS)-poly[N-benzyl-2-(methacryloxy)-N,N-dimethy- lethanaminium bromide] (PQ) nanoparticles (Fe3O4-CS-PQ) were synthesized as reported in the literature [
The lipase activity for free and immobilized enzyme was measured by the hydrolysis of olive oil esters as described [
The enzymatic hydrolysis of thiophenyl compounds i.e., the substituted aromatic, heterocyclic and aliphatic thio esters (1a-1h) (1m mol) were taken in 10 ml of solvent cyclohexane, to the above reaction medium the dispersed lipase enzyme immobilized on to the magnetic nanoparticles was added and the reaction was incubated under nitrogen atmosphere on shaking incubator at 35˚C. The rate of product formation was monitored by TLC/HPLC. On completion of reaction (~55% conversion), the lipase (PPL) immobilized on magnetic nanoparticles present in the reaction mixture was separated with the help of an external magnetic bar, and the immobilized enzyme thus obtained was reused. The crude disulphide product obtained was purified by flash chromatography and the structures of the products were confirmed by NMR, Mass spectral studies.
The antimicrobial and antifungal activities of the disulphide compounds were determined using the dilution technique. Minimum inhibitory concentrations (MICs) were determined [
The hydrolytic enzyme lipase immobilized on magnetic nano particles, has shown broad substrate specificity in hydrolyzing both aromatic & aliphatic thiol acetates and simultaneously forms high yield of the respective disulphide compounds (
In conclusion, we have demonstrated the lipase assisted oxidative coupling of thiol acetate into disulphide
S. No | Substrate | Product | Yield % |
---|---|---|---|
1 | C6H5-SCOCH3 | C6H5-S-S-C6H5 (2a) | 42 |
2 | C6H5-S-CO-t-Bu | 2a | 36 |
3 | C6H5-S-CO-C2H3 | 2a | 48 |
4 | C6H5-S-CO-C2H3 | 2a | 40 |
5 | C6H5-S-CO-C15H31 | 2a | 52 |
6 | C6H5-S-CO-C6H5 | 2a | 36 |
7 | C6H5-S-CO-C6H4-p-Cl | 2a | 47 |
8 | p-C6H5-S-CO-C6H4-CO-S-C6H5 | 2a | 50 |
9 | p-Cl-C6H4-S-CO-CH3 | p-Cl-C6H4-S-S-C6H4-Cl-p (2b) | 55 |
10 | 45 | ||
11 | 38 | ||
12 | n-C3H7-S-CO-CH3 | n-C3H7-S-SC3H7-n (2e) | 40 |
13 | n-C12H25-S-CO-CH3 | (n-C12H25-S)2 (2f) | 43 |
14 | n-C12H25-S-CO-t-Bu | 2f | 39 |
15 | n-C12H25-S-CO-C3H5 | 2f | 30 |
16 | 38 | ||
17 | 33 |
aAll the compounds were characterized by the 1HNMR, MS, IR Spectral studies. bYeilds refer to pure after column chromatography.
Compound | E. coli | P. aeruginosa | K. pneumoniae | B. subtilis | S. aureus | C. albicans | C. tropicals |
---|---|---|---|---|---|---|---|
2a | - | - | - | - | - | >2.5 | >2.5 |
2b | 15.0 | 20.0 | 10.0 | 10.5 | 30.5 | >2.5 | >2.5 |
2c | 20.5 | 25.0 | 35.0 | 20.0 | 50.0 | >2.5 | >2.5 |
2d | 10.0 | 50.0 | 50.0 | 25.0 | 70.0 | >2.5 | >2.5 |
2e | 35.0 | 35.0 | 30.0 | 15.0 | 20.0 | >2.5 | >2.5 |
2f | 12.5 | 20.5 | 25.0 | 60.0 | 50.0 | >5.0 | >5.0 |
Amphot-ericin B | - | - | - | - | - | 3.00 | 3.00 |
Netilmicin | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | - | - |
aResults obtained were the mean of three independent experiments, with standard deviation of 10%.
compounds using immobilized lipase on to magnetic nanoparticles. Thus the methodology developed and carried out in mild and environment friendly condition. Further, we believe that the application of this methodology can be used in the synthesis of biologically important compounds having disulphide linkage and this methodology is an attractive alternative to the existing method.
Authors are thankful to CSIR-IICT XII FYP Project CSC-0106 “BIAGOS” for funding the research project.
Phenyl disulphide (2a): White solid; mp: 57˚C - 59˚C [mp: 58˚C - 60˚C]. 1H NMR (300 MHz, CDCl3): δ = 7.40 - 7.35 (m, 4 H), 7.21 - 7.10 (m, 6 H). 13C NMR (300 MHz, CDCl3): δ = 137.02, 129.05, 127.45, 127.11. MS: m/z = 219 (M + H).
p-Chlorophenyl disulphide (2b): White solid; mp: 72˚C - 74˚C [mp: 72˚C - 73˚C]. 1H NMR (300 MHz, CDCl3): δ = 7.33 (d, J = 8.8 Hz, 4 H), 7.18 (d, J = 8.8 Hz, 4 H). 13C NMR (300 MHz, CDCl3): δ = 135.12, 133.66, 129.33, 129.32. MS: m/z = 286 (M+, 64)
2-Naphthlenyl disulphide (2c): White solid; mp: 136˚C - 137˚C [mp: 135˚C - 138˚C]. 1H NMR (300 MHz, CDCl3): δ 7.98 (d, J = 0.9 Hz, 2 H), 7.80 - 7.22 (m, 4 H), 7.75 - 7.61 (m, 2 H), 7.47 - 7.42 (m, 4 H). 13C NMR (300 MHz, CDCl3): δ = 134.00, 133.52, 132.50, 128.89, 127.80, 127.51, 126.70, 126.66, 126.20, 125.74. MS: m/z = 318 (M+)
1, 2-dithiane (2d): White solid. m.p: 30˚C - 32˚C. 1H NMR (300 MHz, CDCl3): 1.6 - 1.7 (m, 4 H), 2.5 - 2.8 (4 H, m). 13C NMR (300 MHz, CDCl3): δ 26.70, 37.45. MS: m/z = 120 (M+,61)
n-Propyl disulphide (2e): Colourless oil. 1H NMR (300 MHz, CDCl3): δ = 2.61 (t, J = 7.1 Hz, 4 H), 1.65 (sp, J = 7.5 Hz, 4 H), 0.99 (t, J = 7.3 Hz, 6 H). 13C NMR (300 MHz, CDCl3): δ 40.90, 22.34, 12.90. MS: m/z = 150 (M+, 90)
Dodecyl disulphide (2f): Colourless oil. 1H NMR (300 MHz, CDCl3): δ = 2.48 (t, J = 6.9 Hz, 4 H), 1.64 - 1.54 (m, 4 H), 1.35 - 1.27 (m, 36 H), 0.89 (t, J = 6.3 Hz, 6 H). 13C NMR (300 MHz, CDCl3): δ 34.50, 31.90, 29.60, 29.60, 29.50, 29.50, 29.30, 29.10, 28.42, 24.60, 22.61, 14.00. MS: m/z = 402 (M+).