METHOD FOR PRODUCING MERCAPTANS BY HYDROGEN-ASSISTED DISULFIDE ENZYME HYDROGENOLYSIS

Abstract

Provided is an enzymatic process for the preparation of a mercaptan of formula RSH from disulfides utilizing hydrogen.

Claims

1-12: (canceled)

13. 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 of the thiol-group-containing peptide, (4) a catalytic amount of an enzyme catalyzing the reduction of hydrogen, and (5) a catalytic amount of a cofactor common to the enzyme catalyzing the reduction of a disulfide bridge created between two equivalents of the amino acid bearing a thiol group or of the thiol-group-containing peptide and the enzyme catalyzing the reduction of hydrogen, (b) adding hydrogen with a catalytic amount of a hydrogen dehydrogenase enzyme, (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 of the mercaptan of formula RSH.

14. The process of claim 13, comprising: (a) preparing a mixture, comprising: (1) a disulfide of formula RSSR, (2) 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, (3) a catalytic amount of reductase enzyme corresponding to the amino acid bearing a thiol group or to the thiol-group-containing peptide, and (5) a catalytic amount of NADPH, (b) adding hydrogen with a catalytic amount of hydrogen 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/or of the mercaptan of formula RSH.

15. The process of claim 13, 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 5 to 10 atoms.

16. The process of claim 13, 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.

17. The process of claim 13, wherein the disulfide of formula RSSR is dimethyl disulfide.

18. The process of claim 13, wherein the amino acid bearing a thiol group or the peptide bearing a thiol group is chosen from cysteine, homocysteine, glutathione and thioredoxin.

19. The process of claim 13, wherein the hydrogen is introduced into the reaction medium via bubbling.

20. The process of claim 13, wherein the pH of the reaction is between 6 and 8.5.

21. The process of claim 13, wherein the hydrogen/disulfide molar ratio is between 0.01 and 100 over the whole of the reaction.

22. The process of claim 13, wherein the amino acid bearing a thiol group or thiol-group-containing peptide is glutathione.

23. The process of claim 13, wherein the cofactor is a flavinic cofactor or a nicotinic cofactor.

24. The process of claim 13, wherein the cofactor is NADPH.

25. The process of claim 13, wherein the disulfide of formula RSSR is dimethyl disulfide, the amino acid bearing a thiol group or thiol-group-containing peptide is glutathione, and the cofactor is NADPH.

26. 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 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 add bearing a thiol group or of the thiol-group-containing peptide, (4) optionally, a catalytic amount of an enzyme catalyzing the reduction of hydrogen, (5) a catalytic amount of a cofactor common to the enzyme catalyzing the reduction of a disulfide bridge created between two equivalents of the amino acid bearing a thiol group or of the thiol-group-containing peptide and the enzyme catalyzing the reduction of hydrogen, and (6) optionally, hydrogen.

27. The mixture of claim 26, wherein: the enzyme catalyzing the reduction of a disulfide bridge created between two equivalents of the amino acid bearing a thiol group or of the thiol-group-containing peptide is a reductase, and the cofactor is NADPH.

Description

EXAMPLE 1

[0091] 10 ml of glutathione enzymatic complex are introduced into a reactor containing 150 ml of buffered aqueous solution at pH 7.8. 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 hydrogen dehydrogenase enzyme. The reaction medium is brought to 35 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. At the same time, a 4 L.Math.h.sup.1 stream of hydrogen (measured under normal temperature and pressure conditions) is introduced into the reactor via bubbling. The reaction is carried out at atmospheric pressure. Gas chromatography analysis of the gases leaving the reactor shows virtually essentially the presence of hydrogen and methyl mercaptan (some traces of water). These outlet gases are trapped in 20% sodium hydroxide in water. The DMDS and the hydrogen (hydrogen/DMDS molar ratio over the whole of the reaction=10.7) are 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 of methyl mercaptan which has been driven out of the reactor by the excess hydrogen.

EXAMPLE 2

[0092] To the reaction medium of Example 1, 9.4 g (0.1 mol) of DMDS are reintroduced dropwise in 6 hours, but this time only a 1 l.Math.h.sup.1 hydrogen flow is introduced, also over 6 hours (hydrogen/DMDS molar ratio over the whole of the reaction=2.7). 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, and also shows that it is possible to work with hydrogen/DMDS molar ratios which are near to stoichiometry.

EXAMPLE 3

[0093] 10 ml of glutathione enzymatic complex are introduced into a reactor containing 70 ml of buffered aqueous solution at pH 6.8. The solution of enzymatic complex contains: 200 mg (0.65 mmol) of glutathione, 500 U of glutathione reductase, 100 mg (0.12 mmol) of NADPH and 50 U of hydrogen dehydrogenase. The latter is obtained from the culture of microorganisms (according to Biller et al., Fermentation Hyperthermophiler Mikroorganismen am Beispiel von Pyrococcus Furiosus, Shaker Verlag, Maastricht/Herzogenrath, 2002), using techniques well known to those skilled in the art.

[0094] The reaction medium is brought to 35 C. with mechanical stirring and nitrogen flushing. A first sample is taken at t=0. Subsequently, 20 g (0.22 mol) of dimethyl disulfide are added by means of a syringe.

[0095] At the same time, an amount of 4 l.Math.h.sup.1 of hydrogen (measured under normal temperature and pressure conditions) is introduced into the reaction medium via bubbling. The reaction is carried out at atmospheric pressure.

[0096] Gas chromatography analysis of the gases leaving the reactor shows virtually essentially the presence of hydrogen, nitrogen and and methyl mercaptan (some traces of water). These outlet gases are trapped in sodium hydroxide at 20% by weight in water. The DMDS and the hydrogen (hydrogen/DMDS molar ratio over the whole of the reaction=4.9) are 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.

[0097] The final analysis shows that the DMDS has been converted quantitatively into methyl mercaptan. In addition, a final gas chromatography analysis of the reaction medium confirms the absence of DMDS, and of methyl mercaptan which has been driven out of the reactor by the hydrogen.