METHOD FOR PRODUCING MERCAPTANS BY DISULFIDE ENZYME HYDROGENOLYSIS

Abstract

Provided is an enzymatic process for preparing mercaptans from disulfides.

Claims

1-13: (canceled)

14. A process for the preparation of a mercaptan of formula RSH, comprising: (a) preparing a mixture, comprising: (1) a disulfide of formula RSSR, wherein R and R, independently, represent a linear, branched or cyclic hydrocarbon-based radical comprising from 1 to 20 carbon atoms, wherein the hydrocarbon-based radical is saturated or contains one or more unsaturations in the form of double or triple bond(s), or R and R form together, with the sulfur atoms bearing them, a cyclic group comprising from 4 to 22 atoms, (2) a catalytic amount of an amino acid bearing a thiol group or of a thiol-group-containing peptide, wherein the amino acid bearing a thiol group or the thiol-group-containing peptide may optionally be in the form of the corresponding disulfide, (3) a catalytic amount of an enzyme catalyzing the reduction of a disulfide bridge created between two equivalents of the amino acid bearing a thiol group or the thiol-group-containing peptide, (4) a catalytic amount of an enzyme catalyzing the dehydrogenation of the organic reducing compound in (b), (5) a catalytic amount of a cofactor common to the two enzymes catalyzing the reduction and the dehydrogenation, (b) adding an organic reducing compound in a stoichiometric amount relative to the disulfide of formula RSSR, (c) carrying out the enzymatic reaction, (d) recovering the mercaptan of formula RSH and the mercaptan of formula RSH, (e) optionally, separating and, optionally, purifying the mercaptan of formula RSH and/or the mercaptan of formula RSH.

15. The process of claim 14, comprising: (a) preparing of a mixture, comprising: a disulfide of formula RSSR, a catalytic amount of amino acid bearing a thiol group or of a thiol-group-containing peptide, a catalytic amount of a reductase enzyme corresponding to the amino acid bearing a thiol group or to the thiol-group-containing peptide, and a catalytic amount of NADPH, (b) adding an organic reducing compound in a stoichiometric amount relative to the disulfide with a catalytic amount of the corresponding dehydrogenase enzyme, (c) carrying out the enzymatic reaction, (d) recovering the mercaptan of formula RSH and the mercaptan of formula RSH, (e) separating and, optionally, purifying the mercaptan of formula RSH and the mercaptan of formula RSH.

16. The process of claim 14, wherein R and R, independently, represent a linear, branched or cyclic hydrocarbon-based radical comprising from 1 to 20 carbon atoms, wherein the hydrocarbon-based radical is saturated or contains one or more unsaturations in the form of double or triple bond(s), or R and R form together, and with the sulfur atoms bearing them, a cyclic group comprising from 5 to 10 atoms.

17. The process of claim 14, wherein R and R, independently, represent a linear or branched, saturated or unsaturated alkyl, cycloalkyl, aryl, alkylaryl or arylalkyl radical comprising from 1 to 20 carbon atoms and optionally functionalized by one or more functions chosen from alcohol, aldehyde, ketone, acid, amide, nitrile or ester functions or functions bearing sulfur, phosphorus, silicon or halogen.

18. The process of claim 14, wherein the disulfide of formula RSSR is dimethyl disulfide.

19. The process of claim 14, wherein the amino acid bearing a thiol group or the peptide bearing a thiol group is chosen from cysteine, homocysteine, glutathione and thioredoxin.

20. The process of claim 14, wherein the organic reducing compound is a hydrogen-donating organic reducing compound bearing a hydroxyl function and is chosen from alcohols, polyols, and sugars.

21. The process of claim 14, wherein the organic reducing compound is chosen from glucose, glucose 6-phosphate, and isopropanol.

22. The process of claim 14, wherein the pH of the reaction is between 6 and 8.

23. The process of claim 14, wherein the pH of the reaction is between 6.5 and 7.5.

24. The process of claim 14, wherein the organic reducing compound/disulfide molar ratio is between 0.01 and 100 over the whole of the reaction.

25. The process of claim 14, wherein the organic reducing compound/disulfide molar ratio is between 0.5 and 5 over the whole of the reaction.

26. The process of claim 14, wherein the amino acid bearing a thiol group or a thiol-group-containing peptide is glutathione.

27. The process of claim 14, wherein the cofactor is a flavinic cofactor or a nicotinic cofactor.

28. The process of claim 14, wherein the disulfide of formula RSSR is dimethyl disulfide (DMDS), the amino acid bearing a thiol group or the peptide bearing a thiol group is chosen from cysteine, homocysteine, glutathione and thioredoxin, the organic reducing compound is a hydrogen-donating organic reducing compound bearing a hydroxyl function and is chosen from alcohols, polyols, and sugars, and the cofactor is a flavinic cofactor or a nicotinic cofactor.

29. The process of claim 14, wherein the disulfide of formula RSSR is dimethyl disulfide (DMDS), the amino acid bearing a thiol group or the peptide bearing a thiol group is glutathione, the organic reducing compound is glucose, and the cofactor is NADPH.

30. A mixture, comprising: a disulfide of formula R S S R, wherein R and R, independently, represent a linear, branched or cyclic hydrocarbon-based radical comprising from 1 to 20 carbon atoms, wherein the hydrocarbon-based radical is saturated or contains one or more unsaturations in the form of double or triple bond(s), or R and R form together, and with the sulfur atoms bearing them, a cyclic group comprising from 4 to 22 atoms, a catalytic amount of amino acid bearing a thiol group or of a thiol-group-containing peptide, wherein the amino acid bearing a thiol group or the thiol-group-containing peptide may optionally be in the form of the corresponding disulfide, a catalytic amount of an enzyme catalyzing the reduction of a disulfide bridge created between two equivalents of the amino acid bearing a thiol group or the thiol-group-containing peptide, optionally, a catalytic amount of an enzyme catalyzing the dehydrogenation of an organic reducing compound, and a catalytic amount of a cofactor common to the two enzymes catalyzing the reduction and the dehydrogenation.

31. The mixture of claim 30, comprising: a disulfide of formula RSSR, a catalytic amount of the amino acid bearing a thiol group or the thiol-group-containing peptide, a catalytic amount of a reductase enzyme corresponding to the amino acid bearing a thiol group or the thiol-group-containing peptide, and a catalytic amount of NADPH.

Description

EXAMPLE 1

[0098] 10 ml of glutathione enzymatic complex (Aldrich) and 19.2 g (0.1 mol) of glucose are introduced into a reactor containing 150 ml of 0.1 mol/l phosphate buffer at pH 7.30. The solution of enzymatic complex contains: 185 mg (0.6 mmol) of glutathione, 200 U of glutathione reductase, 50 mg (0.06 mmol) of NADPH and 200 U of glucose dehydrogenase. The reaction medium is brought to 25? C. with mechanical stirring. A first sample is taken at t=0. Subsequently, the dimethyl disulfide (9.4 g, 0.1 mol) is placed in a burette and added dropwise to the reactor; the reaction begins. A stream of nitrogen is placed in the reactor. Gas chromatography analysis of the gases leaving the reactor shows virtually essentially the presence of nitrogen and methyl mercaptan (some traces of water). These outlet gases are trapped in 20% sodium hydroxide in water. The DMDS is introduced in 6 hours and the reaction is monitored by potentiometric argentometric titration of the methyl mercaptan sodium salt in the trap at the outlet of the reactor. In addition, a final gas chromatography analysis of the reaction medium confirms the absence of DMDS, and by UPLC/mass spectrometry traces of glucose and the virtually exclusive presence of gluconolactone are found.

EXAMPLE 2

[0099] To the reaction medium of Example 1, 19.2 g (0.1 mol) of glucose are reintroduced in one go, and 9.4 g (0.1 mol) of DMDS are reintroduced dropwise in 6 hours. The reaction is monitored in the same way as in Example 1, after having changed the 20% sodium hydroxide solution at the outlet of the reactor. The analyses at the end of the reaction confirm the complete disappearance of the DMDS, totally converted into methyl mercaptan found in sodium salt form in the sodium hydroxide solution. Only the gluconolactone is analysed and found in the reaction medium at the end of the reaction. This example shows the robustness of the catalytic system through its reproducibility.