Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
  • C12P13/12—Methionine; Cysteine; Cystine
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C319/00—Preparation of thiols, sulfides, hydropolysulfides or polysulfides
  • C07C319/02—Preparation of thiols, sulfides, hydropolysulfides or polysulfides of thiols
  • C07C319/08—Preparation of thiols, sulfides, hydropolysulfides or polysulfides of thiols by replacement of hydroxy groups or etherified or esterified hydroxy groups
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P11/00—Preparation of sulfur-containing organic compounds
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
  • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
  • C12Y205/01049—O-acetylhomoserine aminocarboxypropyltransferase (2.5.1.49)

Definitions

• the present invention relates to a process for the synthesis of functionalized mercaptans, as well as a composition allowing in particular the implementation of this process.
• Mercaptans are used in many industrial fields and many synthetic methods are known such as the sulfhydration of alcohols, the catalytic or photochemical addition of hydrogen sulfide to unsaturated organic compounds or substitution using hydrogen sulfurized with halides, epoxides or organic carbonates.
• substitution by hydrogen sulphide requires often high temperatures and pressures and leads to undesired by-products of the olefin, ether, sulphide and/or polysulphide type.
• the catalytic or photochemical addition of hydrogen sulfide to unsaturated compounds is generally done under slightly milder conditions but also leads to numerous by-products formed by isomerization of the raw material, by non-regioselective addition or by double addition. leading to the production of sulphides and/or polysulphides.
• the present invention makes it possible in whole or in part to overcome the drawbacks of the prior methods.
• An objective of the present invention is to provide a process for the synthesis of an improved functionalized mercaptan, in particular having a yield and/or a selectivity equivalent or superior to the known processes.
• An object of the present invention is to provide a process for the synthesis of a functionalized mercaptan with negligible, or even zero, co-production of by-products, in particular of sulphides and/or polysulphides.
• the present inventors have discovered that the functionalized mercaptans of formula (I) as defined below, in particular L-homocysteine, could be advantageously synthesized by reaction between compounds of formula (II) and a sulphhydrate salt and or a sulfide salt (hereinafter referred to as “salt”) as defined below or of HS, in the presence of a sulfhydrylase enzyme; said reaction taking place essentially in the absence of oxygen, or even in the absence of oxygen.
• the present inventors have thus discovered a process for the synthesis of functionalized mercaptans of formula (I) making it possible to limit or even avoid the co-production of sulphides and/or polysulphides, in particular disulphides.
• the method according to the invention makes it possible to produce L-homocysteine while limiting or even avoiding the co-production of L-homocysteine and/or L-homocysteine sulphide (also called 4,4'-sulfanediylbis( 2-aminobutanoic acid) / L- homolanthionine).
• L-homocysteine and/or L-homocysteine sulphide also called 4,4'-sulfanediylbis( 2-aminobutanoic acid) / L- homolanthionine.
• L-homocysteine has the following formula:
• L-homocysteine sulfide has the following formula:
• L-homocystine has the following formula:
• the functionalized mercaptan of formula (I) obtained according to the process of the invention can be enantiomerically pure.
• the process according to the invention is also easy to implement industrially. It can be carried out in solution under mild temperature and pressure conditions.
• the use of salts advantageously makes it possible to avoid the manipulation of hydrogen sulphide, which is a toxic gas, by the operators.
• the yield obtained may be greater than or equal to 85%, preferably greater than or equal to 90%, for example between 90% and 100%, limits included.
• the process according to the invention makes it possible in particular to obtain a yield of 100%, i.e. an increase of almost 20% compared to the other processes.
• the present invention relates to a process for the synthesis of at least one functionalized mercaptan of general formula (I) below:
• Ri and R 7 are a hydrogen atom or a hydrocarbon chain, saturated or unsaturated, linear, branched or cyclic, aromatic or not, of 1 to 20 carbon atoms and possibly comprising one or more heteroatoms;
• R3 being a hydrogen atom or a hydrocarbon chain, saturated or unsaturated, linear, branched or cyclic, aromatic or not, of 1 to 20 carbon atoms and which may comprise one or more heteroatoms,
• -NR4R5, R4 and R 5 identical or different, being a hydrogen atom or a hydrocarbon chain, saturated or unsaturated, linear, branched or cyclic, aromatic or not, of 1 to 20 carbon atoms and possibly include one or more heteroatoms; n is equal to 1 or 2; and * represents an asymmetric carbon; said method comprising the steps of: a) providing at least one compound of general formula (II) below:
• R10, R11, R I2 and R13 being independently of each other chosen from:
• R 7 and R 8 identical or different, being a proton, an alkali, an alkaline earth or an ammonium;
• R 9 is chosen from a proton, an alkali, an alkaline earth or an ammonium; b) supply of at least one sulphide salt and/or one sulphide or H 2 S salt; c) reaction between said at least one compound of formula (II) and said at least one sulfhydrate and/or sulfide salt or H 2 S in the presence of at least one enzyme chosen from sulfhydrylases, and preferably a sulfhydrylase associated with said compound of formula (II); said reaction being carried out essentially in the absence of oxygen, preferably in the absence of oxygen; d) obtaining at least one functionalized mercaptan of formula (I); e) optional separation of said at least one functionalized mercaptan of formula (I) obtained in step d); and f) optional additional functionalization and/or optional deprotection of the functionalized mercaptan of formula (I) obtained in step d) or e); and wherein steps a) and b) are
• Oxygen is understood in particular to mean dioxygen 0 2 .
• Step c) is thus carried out essentially in the absence of oxygen, or even in the absence of oxygen.
• the term “essentially in the absence of oxygen” means that there may remain a quantity of oxygen in the reaction mixture and/or in the gaseous phase (contained in the gas headspace of the reactor) such that the quantity of sulphides and/or polysulphides produced is less than or equal to 5% by weight relative to the total weight of the compound of formula (I) produced.
• the term “essentially in the absence of oxygen” preferably means that the reaction mixture contains less than 0.0015% oxygen (preferably strictly less than 0.0015%) by weight relative to the total weight of the reaction mixture and /or that the gaseous phase (contained in the gaseous headspace of the reactor) contains less than 21% oxygen (preferably strictly less than 21%) by volume relative to the total volume of said gaseous phase.
• step c) may alternatively be the following: c) reaction between said at least one compound of formula (II) and said at least one hydrosulphide and/or sulphide salt or H 2 S in the presence of at least one at least one enzyme chosen from sulfhydrylases, and preferably a sulfhydrylase associated with said compound of formula (II); said reaction taking place in a reactor in which the reaction mixture comprises between 0 and 0.0015% oxygen (preferably strictly less than 0.0015%) by weight relative to the total weight of the reaction mixture and/or the phase gas contained in the gas headspace of the reactor comprises between 0 and 21% oxygen (preferably strictly less than 21%) by volume relative to the total volume of the gas phase.
• the reaction mixture comprises between 0 and 0.0015% oxygen (preferably strictly less than 0.0015%) by weight relative to the total weight of the reaction mixture and/or the phase gas contained in the gas headspace of the reactor comprises between 0 and 21% oxygen (preferably strictly less than 21%) by volume relative to the total volume of the gas phase.
• the quantity of oxygen in the reaction mixture and/or in the gaseous phase is such that the quantity of sulphides and or polysulphides produced is less than or equal to 5% by weight relative to the total weight of the compound of formula (I) produced.
• step c) can be carried out in a closed reactor (i.e. without supplying oxygen from the air).
• the gaseous phase (contained in the gaseous headspace) does not include oxygen, in particular when HS is used.
• the gaseous phase (contained in the gas headspace) does not comprise oxygen and the reaction mixture comprises between 0 and 0.0015% oxygen (preferably strictly less than 0.0015%) by weight relative to the total weight of the reaction mixture.
• gas overhead means the space of the reactor located above the reaction mixture, preferably above the liquid reaction mixture. More specifically, the term “gas overhead” means the space located between the surface of the liquid reaction mixture and the top of the reactor (i.e. the upper part of the reactor comprising the gas phase when the lower part of the reactor comprises a liquid phase).
• the gas overhead comprises in particular a gas phase.
• the reactants are in particular introduced into the reactor in quantities such that a gaseous blanket is located above the reaction mixture contained in the reactor.
• reaction mixture may thus comprise: at least one compound of formula (II) as defined below, at least one sulphide and/or sulphide salt as defined below or H 2 S, at least one sulfhydrylase as defined below, optionally its cofactor as defined below, optionally a base as defined below, and optionally a solvent, preferably water.
• Said reaction mixture can be prepared by adding said compound of formula (II), said sulphhydrate and/or sulphide salt or H 2 S and said sulphydrylase in any order.
• the compound of formula (II) is in the form of a solution, more preferably in the form of an aqueous solution.
• sulphhydrate and/or sulphide salts are used in the form of a solution and more preferably in the form of an aqueous solution.
• HS sulphhydrate and/or sulphide salts
• it is generally in gaseous form. It can in particular be introduced into the reaction mixture by bubbling. The bubbling can be carried out by mixing the H 2 S with an inert gas, for example dinitrogen, argon or methane, preferably dinitrogen.
• the H 2 S can therefore be present in dissolved form in the reaction mixture.
• step c) In order to carry out step c) essentially in the absence of oxygen, or even in the absence of oxygen, conventional methods can be used.
• the oxygen is removed from the reaction mixture, for example by degassing.
• the oxygen is separately removed from each of the components or from the mixture of at least two of them which will form the reaction mixture.
• each of the solutions comprising the compound of formula (II), the sulfhydrate and/or sulfide salt when the latter is used, the sulfhydrylase and optionally the solvent is degassed.
• step c it is also possible to remove the oxygen from the top of the reactor in which step c takes place), preferably by degassing.
• the reactor can also be inerted with an inert gas such as nitrogen, argon or methane, preferably nitrogen.
• an inert gas such as nitrogen, argon or methane, preferably nitrogen.
• H 2 S which is gaseous
• degassing is of course not carried out for this reagent.
• H 2 S generally does not include oxygen.
• the absence of oxygen is obtained in the following way:
• the reactor is inert with an inert gas such as nitrogen, argon or methane, preferably nitrogen; and
• step c) the oxygen is present neither in dissolved form in a liquid (in particular in the reaction mixture), nor in gaseous form (in particular in the top of the reactor in which perform step c)).
• an inert gas e.g. argon, dinitrogen or methane.
• the sulphide salt and/or the sulphide salt or the H S is in excess, preferably in molar excess, relative to the compound of formula (II), preferably during step c) and more preferentially throughout the duration of step c).
• the hydrosulfide and/or sulfide salt or the H 2 S can therefore be in an over-stoichiometric quantity with respect to the quantity of the compound of formula (II), preferably during step c) and more preferably throughout the duration of step c).
• the [sulfhydrate salt and/or sulfide salt]/[compound of formula (II)] molar ratio or the H 2 S/compound of formula (II) molar ratio is between 1.5 and 10, preferably between 2 and 8, for example between 3.5 and 8, and even more preferably between 3.5 and 5, limits included, preferably during step c) and more preferably throughout the duration of step c). Said ratio can be kept constant throughout the duration of step c).
• Step c) can be carried out in solution, in particular in aqueous solution.
• the solution comprises between 50% and 99% by weight of water, preferably between 75% and 97% by weight of water relative to the total weight of the solution, limits included.
• the pH of the reaction mixture in step c) can be between 4 and 9, for example between 5 and 8, preferably between 6 and 7.5, and more particularly between 6.2 and 7.2, limits included, particularly when the reaction mixture is an aqueous solution.
• the pH can in particular be adjusted within the ranges mentioned above according to the optimum functioning of the sulfhydrylase chosen.
• the pH can be determined by conventionally known methods, for example with a pH-metric probe.
• step c) can be carried out according to the following two steps c1) and c2): c1) reaction between said at least one compound of formula (II) and said at least one sulphide salt and or sulphide or H 2 S in the presence of at least one enzyme chosen from sulfhydrylases, and preferably a sulfhydrylase associated with said compound of formula (II); said reaction being carried out essentially in the absence of oxygen, preferably in the absence of oxygen, and in solution; c2) adjustment of the pH of said solution by adding a base so as to obtain a pH of between 4 and 9, for example between 5 and 8, preferably between 6 and 7.5, and more particularly between 6.2 and 7.2, terminals included.
• any type of base can be used, preferably a base comprising a sulfur atom.
• base is meant in particular a compound or a mixture of compounds having a pH greater than 7, preferably between 8 and 14, limits included.
• the base can be chosen from salts of sulphides and/or salts of sulphides as defined below, sodium hydroxide, potassium hydroxide or aqueous ammonia.
• the base can be chosen from salts of sulphides and/or salts of sulphides as defined below.
• said base is the sulphide salt and/or the sulphide salt used in step c1).
• the preferred base is ammonium sulphhydrate (NH 4 SH).
• the base can be added at a concentration of between 0.1 and 10 M, preferably between 0.5 and 10 M, more preferably between 0.5 and 5 M, limits included.
• concentrated bases will be used so as to limit the dilution of the reaction mixture during the addition of the base.
• the temperature during step c) can be between 10°C and 60°C, preferably between 20°C and 40°C, and more particularly between 25°C and 40°C, limits included.
• the pressure during step c) is usually atmospheric pressure.
• Step c) can be carried out in batch, semi-continuous or continuous. Any type of reactor may be suitable.
• the separation step e) can be carried out according to any technique known to those skilled in the art.
• the final product when the final product is a solid: by extraction and/or decantation with a solvent that is immiscible in the reaction medium followed by evaporation of said solvent; by precipitation (by partial evaporation of the solvents or by addition of a solvent in which the compound of interest is less soluble).
• This precipitation is generally followed by a filtration step according to any method known to those skilled in the art.
• the final product can then be dried; or by selective precipitation by adjusting the pH and depending on the respective solubilities of the different compounds.
• Homocysteine can in particular be recovered in solid form.
• the separation can be carried out by distillation or by distillation or evaporation preceded by a liquid/liquid extraction.
• Step f) of additional functionalization and/or possible deprotection can make it possible to obtain additional chemical functions and/or to deprotect certain chemical functions by conventional methods.
• XR 2 represents a function carboxylic
• the latter can be esterified, reduced to aldehyde, reduced to alcohol and then estherified, amidified, nitrile or others. All the functions can be obtained and/or deprotected by those skilled in the art depending on the end use intended for said functionalized mercaptan of formula (I).
• the functionalized mercaptan of formula (I) obtained at the end of step d) or e) can be subjected to one or more additional chemical reactions to obtain one or more mercaptan derivatives with different functionalities, said chemical reactions being reactions well known to those skilled in the art.
• heteroatom in particular means an atom chosen from O, N, S, P and halogens.
• unsaturated hydrocarbon chain means a hydrocarbon chain comprising at least one double or one triple bond between two carbon atoms.
• Ri and R 7 are a hydrogen atom or a hydrocarbon chain, saturated or unsaturated, linear, branched or cyclic, aromatic or not, of 1 to 20 carbon atoms and possibly comprising one or more heteroatoms;
• R 2 is:
• R 3 being a hydrogen atom or a hydrocarbon chain, saturated or unsaturated, linear, branched or cyclic, aromatic or not, of 1 to 20 carbon atoms and possibly comprising one or more heteroatoms,
• -NR4R5, R4 and R 5 identical or different, being a hydrogen atom or a hydrocarbon chain, saturated or unsaturated, linear, branched or cyclic, aromatic or not, of 1 to 20 carbon atoms and possibly include one or more heteroatoms; n is equal to 1 or 2; and * represents an asymmetric carbon.
• mercaptans are said to be functionalized because in addition to the —SH chemical function, they also comprise at least one —NR 1 R 7 function of the amine type.
• n is equal to 2.
• R 2 is -OR 3 with R 3 as defined above.
• R 3 can in particular be a hydrogen atom or a saturated hydrocarbon chain, linear or branched, of 1 to 10 carbon atoms, preferably of 1 to 5 carbon atoms.
• R 3 is H.
• Ri and R 7 which are identical or different, are a hydrogen atom or a hydrocarbon chain, saturated, linear or branched, of 1 to 10 carbon atoms, preferably of 1 to 5 carbon atoms.
• Ri and R ? are H.
• the functionalized mercaptans of formula (I) can be chosen from the group consisting of homocysteine, cysteine and their derivatives.
• the functionalized mercaptans of formula (I) are L-homocysteine and L-cysteine.
• a preferred functionalized mercaptan of formula (I) is homocysteine, and more particularly L-homocysteine of the following formula:
• n is equal to 2
• R 2 is -OR 3 with R 3 is H and Ri and R ? are H.
• the functionalized mercaptans of formula (I) are chiral compounds. They can be obtained in pure enantiomeric form by the process according to the invention. In the present description, when the enantiomeric form is not specified, the compound is understood whatever its enantiomeric form.
• the reaction mixture at the end of step c) does not comprise any sulphide or polysulphide and in particular no sulphide or polysulphide corresponding to the functionalized mercaptan of formula (I) obtained.
• the reaction mixture at the end of step c) comprises less than 10%, preferably less than 5% molar sulphides and polysulphides with respect to the total number of moles of compound of formula (II) converted into compound of formula (I).
• sulfide is meant in particular the sulfide corresponding to the compound of formula (I) which is itself of the following formula (III):
• polysulphide is understood in particular to mean the polysulphide corresponding to the compound of formula
• n is equal to 2 (which corresponds to a disulphide).
• reaction mixture at the end of step c) does not comprise L-homocysteine sulphide or L-homocysteine when the compound of formula (I) is L-homocysteine.
• a functionalized mercaptan of formula (I ) as defined below and a compound of formula (V) GH with G as defined above i.e., a compound of the type: (i') R 6 -C(0)-OH, (ii') (R 7 0)(R 8 0)-P(O)-OH, or (iii') R 9 0- S0 2 -OH; with R 6 , R 7 , Rs and R 9 as defined below.
• R 6 , R 7 , Rs and R 9 as defined below.
• (II) is O-acetyl-L-homoserine, we get L-homocysteine and acetic acid.
• the compounds of formula (V) may be responsible for the acidification of the reaction mixture during step c). Also, it is possible to maintain the pH of the reaction mixture between 4 and 9, for example between 5 and 8, preferably between 6 and 7.5, and more particularly between 6.2 and 7.2, in particular during the step c) as mentioned above and in particular by adding a base as defined above.
• the present invention can be carried out in the presence of a sulphhydrate and/or sulphide salt or in the presence of H 2 S (hydrogen sulphide).
• Said salt is generally supplied in the form of a solution, preferably aqueous.
• Said at least one sulfhydrate and/or sulfide salt may be chosen from the group consisting of: ammonium sulfhydrate, alkali metal sulfhydrates, alkaline-earth metal sulfides, alkali metal sulfides and alkaline-earth metal sulfides.
• alkali metals lithium, sodium, potassium, rubidium and cesium, preferably sodium and potassium.
• alkaline-earth metals beryllium, magnesium, calcium, strontium and barium, preferably calcium.
• said at least one sulphide salt and/or sulphide salt can be chosen from the group consisting of: ammonium sulphide NH 4 SH, sodium sulphide NaSH, potassium sulphide KSH, calcium sulphide Ca(SH) 2 , sodium sulfide Na 2 S, ammonium sulfide (NH 4 ) 2 S, potassium sulfide K 2 S and calcium sulfide CaS.
• the preferred sulphide is ammonium sulphide NH 4 SH.
• the ammonium released during the reaction can for example be reused as a source of nitrogen for the growth of microorganisms, in particular of microorganisms expressing or overexpressing sulfhydrylase.
• the microorganisms can be chosen from the group consisting of: cells of bacteria such as Escherichia coli, Bacillus sp., or Pseudomonas, cells of yeasts such as Saccharomyces cerevisiae or Pichia pastoris, cells of fungi such as Aspergillus niger, Penicillium funiculosum or Trichoderma reesei, insect cells such as Sf9 cells, or even mammalian (in particular human) cells such as HEK 293, PER-C6 or CHO cell lines.
• bacteria such as Escherichia coli, Bacillus sp., or Pseudomonas
• yeasts such as Saccharomyces cerevisiae or Pichia pastoris
• fungi such as Aspergillus niger, Penicillium funiculosum or Trichoderma reesei
• insect cells such as Sf9 cells
• mammalian (in particular human) cells such as
• bacterial cells More particularly, bacterial cells and even more preferably d ⁇ cells will be used. coli.
• R 7 and R 8 identical or different, being a proton, an alkali, an alkaline earth or an ammonium, preferably a proton or an alkali, and more particularly H + or Na + ;
• R 9 is chosen from a proton, an alkali, an alkaline earth or an ammonium, preferably a proton or an alkali, and more particularly an H + or Na + proton;
• G represents either R 6 -C(0)-0- or R 9 0-S0 2 -0-; preferably G is R 6 -C(0)-0-
• R 2 and R 13 are H.
• aromatic group is preferably meant the phenyl group.
• the compound of general formula (II) is in particular a derivative of serine (when n is equal to 1) or homoserine (when n is equal to 2), in particular L-serine or L-homoserine .
• It can for example be chosen from the group consisting of: O-phospho-L-homoserine, O-succinyl-L-homoserine, GO-acetyl-L-homoserine, GO-acetoacetyl-L-homoserine, O-propio-L-homoserine, GO-coumaroyl-L-homoserine, GO-malonyl-L-homoserine, GO-hydroxymethylglutaryl-L-homoserine, O-pimelyl-L-homoserine O-sulfato-L-homoserine , GO-phospho-L serine, GO-succinyl-L serine, GO ace
• O-phospho-L-homoserine O-succinyl-L-homoserine, O-acetyl-L-homoserine, I ⁇ - acetoacetyl-L-homoserine , O-propio-L-homoserine, O-coumaroyl-L-homoserine, I ⁇ -malonyl-L-homoserine, O-hydroxymethylglutaryl-L-homoserine, O-pimelyl-L-homoserine and l 'O-sulfato-L-homoserine.
• the compound of general formula (II) can be chosen from the group consisting of: O-phospho-L-homoserine, O-succinyl-L-homoserine, O-acetyl-L-homoserine, O -sulfato-L-homoserine and O-propio-L-homoserine.
• the compound of general formula (II) can be chosen from the group consisting of: O-phospho-L-homoserine, O-succinyl-L-homoserine, O-acetyl-L-homoserine.
• OAHS O-acetyl-L-homoserine
• They can be obtained by a fermentation process from a hydrocarbon source and nitrogen, for example as described in application WO 2008/013432.
• renewable raw material can be chosen from glucose, sucrose, starch, molasses, glycerol, bioethanol, preferably glucose.
• L-serine derivatives can also be produced from the acetylation of L-serine, as L-serine can itself be obtained by fermentation of a renewable raw material.
• the renewable raw material can be chosen from glucose, sucrose, starch, molasses, glycerol, bioethanol, preferably glucose.
• L-homoserine derivatives can also be produced from the acetylation of L-homoserine, L-homoserine, which itself can be obtained by fermentation of a renewable raw material.
• the renewable raw material can be chosen from glucose, sucrose, starch, molasses, glycerol, bioethanol, preferably glucose.
• the reaction between said at least one compound of formula (II) and said at least one sulphide and/or sulphide salt as defined above or H S is carried out in the presence of at least one enzyme chosen from sulfhydrylases, preferably a sulfhydrylase associated with said compound of formula (II).
• sulfhydrylases preferably a sulfhydrylase associated with said compound of formula (II).
• the sulfhydrylase associated with a compound of formula (II) is easily identifiable because it shares the same name, for example O-acetyl-L-homoserine sulfhydrylase (OAHS Sulfhydrylase) is associated with O-acetyl-L-homoserine.
• the sulfhydrylase makes it possible in particular to catalyze the reaction between said compound of formula (II) and said salt or H 2 S.
• catalyst generally means a substance accelerating a reaction and which is found unchanged at the end of this reaction.
• the sulfhydrylase, and optionally its cofactor can be used in a catalytic amount.
• catalytic amount is meant in particular an amount sufficient to catalyze a reaction. More particularly, a reactant used in a catalytic quantity is used in a smaller quantity, for example between about 0.01% and 20% by weight, limits included, relative to the quantity by weight of a reactant used in a stoichiometric proportion.
• Said sulfhydrylase enzyme preferably belongs to the class of transferases, in particular designated by the nomenclature EC 2.X.X.XX (or denoted EC 2).
• the EC nomenclature for “Enzyme Commission numbers” is widely used and can be found at https://enzyme.expasy.org/.
• said enzyme is chosen from sulfhydrylases of class EC 2.5.X.XX (or denoted EC 2.5.), ie transferases which transfer an alkyl or aryl group other than a methyl group.
• the sulfhydrylases are in particular of class EC 2.5.1.XX (with XX which varies according to the substrate of the enzyme).
• O-acetylhomoserine sulfhydrylase is of the EC 2.5.1.49 type.
• - GO-phosphoserine sulfhydrylase is EC 2.5.1.65 type.
• O-succinylhomoserine sulfhydrylase is of type EC 2.5.1.49.
• O-acetyl-L-homoserine sulfhydrylase is of type EC 2.5.1.49.
• - GO-phospho-L-serine sulfhydrylase is of the EC 2.5.1.65 type.
• O-succinyl-L-homoserine sulfhydrylase is of type EC 2.5.1.49.
• the sulfhydrylase used can be chosen from O-phospho-L-homoserine sulfhydrylase, GO-succinyl-L-homoserine sulfhydrylase, O-acetyl-L-homoserine sulfhydrylase, GO-acetoacetyl-L-homoserine sulfhydrylase, GO-propio-L-homoserine sulfhydrylase, GO-coumaroyl-L-homoserine sulfhydrylase, GO -malonyl-L-homoserine sulfhydrylase, GO-hydroxymethylglutaryl-L-homoserine sulfhydrylase, O-pimelyl-L-homoserine sulfhydrylase, GO-s
• the sulfhydrylase used can be chosen from O-phospho-L-homoserine sulfhydrylase, O-succinyl-L-homoserine sulfhydrylase, O-acetyl-L-homoserine sulfhydrylase, O-acetoacetyl-L -homoserine sulfhydrylase, O-propio-L-homoserine sulfhydrylase, GO-coumaroyl-L-homoserine sulfhydrylase, O-malonyl-L-homoserine sulfhydrylase, GO-hydroxymethylglutaryl-L-homoserine sulfhydrylase, O-pimelyl- L-homoserine sulfhydrylase, GO-sulfato-L-homoserine sulfhydrylase.
• the sulfhydrylase can be chosen from O-phospho-L-homoserine sulfhydrylase, GO-succinyl-L-homoserine sulfhydrylase, O-acetyl-L-homoserine sulfhydrylase, O-sulfato-L-homoserine sulfhydrylase and O-propio-L-homoserine sulfhydrylase.
• the sulfhydrylase can be selected from GO-phospho-L-homoserine sulfhydrylase, GO-succinyl-L-homoserine sulfhydrylase and O-acetyl-L-homoserine sulfhydrylase.
• the enzyme is O-acetyl-L-homoserine sulfhydrylase (OAHS Sulfhydrylase).
• Said sulfhydrylase and in particular O-acetyl-L-homoserine sulfhydrylase, can come from or be derived from the following bacterial strains: Pseudomonas sp., Chromobacterium sp., Leptospira sp. or Hyphomonas sp..
• Sulfhydrylases can function, as well known to those skilled in the art, in the presence of a cofactor such as pyridoxal-5'-phosphate (also called PLP) or one of its analogues, preferably pyridoxal-5 '-phosphate.
• a cofactor such as pyridoxal-5'-phosphate (also called PLP) or one of its analogues, preferably pyridoxal-5 '-phosphate.
• a sulfhydrylase cofactor can be added to the reaction mixture.
• a sulfhydrylase cofactor for example pyridoxal-5'-phosphate
• step c) can be carried out in aqueous solution
• the enzyme and optionally its cofactor can be dissolved beforehand in water before being added to said solution.
• cells for example bacterial or other, can produce or even overproduce said cofactor at the same time as they express or overexpress the sulfhydrylase enzyme so as to avoid a step of supplementing said cofactor.
• the sulfhydrylase, and optionally its cofactor are:
• the isolation and/or purification of said enzyme produced can be carried out by any means known to those skilled in the art. It may be, for example, a technique chosen from electrophoresis, molecular sieving, ultracentrifugation, differential precipitation, for example with ammonium sulphate, ultrafiltration, membrane or gel filtration, exchange of ions, a separation by hydrophobic interactions, or an affinity chromatography, for example of the IMAC type.
• the enzyme of interest may or may not be overexpressed in said cells, hereinafter called host cells.
• the host cell can be any suitable host for the production of the enzyme of interest from the expression of the corresponding coding gene. This gene can then be found either in the host genome or carried by an expression vector.
• the term "host cell” within the meaning of the present invention is a prokaryotic or eukaryotic cell.
• Host cells commonly used for the expression of recombinant or non-recombinant proteins include in particular bacterial cells such as Escherichia coli or Bacillus sp., or Pseudomonas, yeast cells such as Saccharomyces cerevisiae or Pichia pastoris, fungal cells such as Aspergillus niger, Penicillium funiculosum or Trichoderma reesei, insect cells such as Sf9 cells, or even mammalian (in particular human) cells such as HEK 293, PER-C6 or CHO cell lines.
• the enzyme of interest and optionally the cofactor are expressed in the bacterium Escherichia coli.
• the enzyme of interest is expressed within a strain of Escherichia coli such as for example Escherichia coli BL21 (DE3).
• the cell lysate can be obtained using various known techniques such as sonication, pressure (French press), via the use of chemical agents (eg xylene, triton) etc...
• the lysate obtained corresponds to a crude extract crushed cells.
• the amount of biomass expressing the sulfhydrylase enzyme relative to the mass of the compound of formula (II) is between 0.1% and 10% by weight, of preferably between 1% and 5% by weight, and/or the amount of cofactor relative to the compound of formula (II) is between 0.1% and 10% by weight, preferably between 0.5% and 5% by weight, terminals included.
• the reaction mixture may also include:
• solvents chosen from water, buffers such as phosphate buffers, Tris-HCl, Tris-base, ammonium bicarbonate, ammonium acetate, HEPES (4-(2-hydroxyethyl)-1 acid piperazine ethane sulfonic), CHES (N-cyclohexyl-2-aminoethane sulfonic acid), or salts such as sodium chloride, potassium chloride, or mixtures thereof;
• buffers such as phosphate buffers, Tris-HCl, Tris-base, ammonium bicarbonate, ammonium acetate, HEPES (4-(2-hydroxyethyl)-1 acid piperazine ethane sulfonic), CHES (N-cyclohexyl-2-aminoethane sulfonic acid), or salts such as sodium chloride, potassium chloride, or mixtures thereof;
• additives such as surfactants, in particular to promote the solubility of one or more reagent(s) or substrate(s).
• step c) The various components which can be used for the reaction of step c) above are easily accessible commercially or can be prepared according to techniques well known to those skilled in the art. These different elements can be in solid, liquid or gaseous form and can very advantageously be dissolved or dissolved in water or any other solvent to be used in the process of the invention.
• the enzymes used can also be grafted onto a support (case of supported enzymes).
• said compound of formula (II) is O-acetyl-L-homoserine
• the enzyme used is GO-acetyl-L-homoserine sulfhydrylase
• the functionalized mercaptan of formula (I) obtained is L-homocysteine.
• said compound of formula (II) is O-acetyl-L-homoserine
• the salt is ammonium sulfhydrate
• the enzyme used is O-acetyl-L-homoserine sulfhydrylase
• the functionalized mercaptan of formula (I) obtained is L-homocysteine.
• the present invention also relates to a composition, preferably an aqueous solution, comprising: a compound of formula (II) as defined above; a sulfhydrylase, preferably a sulfhydrylase associated with the compound of formula (II), as defined above; and a hydrosulfide salt and/or a sulphide salt as defined above or excess HS, preferably excess NH 4 SH.
• said composition comprises: O-acetyl-L-homoserine; O-acetyl-L-homoserine sulfhydrylase; and excess NH 4 SH OR H 2 S.
• composition corresponds in particular to the reaction mixture as defined above.
• the conditions, characteristics and optional additional components are the same as those defined for the reaction mixture as defined above.
• the composition according to the invention does not comprise dissolved oxygen.
• the sulphide and/or sulphide salt or the H 2 S is in excess, preferably in molar excess, relative to the compound of formula (II).
• the sulphide and/or sulphide salt or the H 2 S can therefore be in an over-stoichiometric quantity with respect to the quantity of the compound of formula (II).
• the molar ratio [sulfhydrate salt and/or sulfide salt]/[compound of formula (II)] or H 2 S/compound of formula (II) is between 1.5 and 10, preferentially between 2 and 8, for example between 3.5 and 8, and even more preferably between 3.5 and 5, limits included.
• the composition may also comprise a sulfhydrylase cofactor as defined above.
• composition according to the invention allows the implementation of the method according to the invention.
• O-acetyl-L-homoserine was synthesized from L-homoserine and acetic anhydride according to the protocol described in the work of Sadamu Nagai, “Synthesis of O-acetyl-L-homoserine”, internationale Press, ( 1971), vol.17, p. 423-424.
• the analyzes by potentiometry, HPLC and NMR show a progressive disappearance of the reagents (OAHS and NaSH) and the progressive appearance of several products over time.
• the compounds thus formed are mainly:
• the molar selectivities with respect to the transformed OAHS obtained are as follows:
• homocysteine sulphide L-homolantionine / 4,4'-sulfanediylbis(2-aminobutanoic) acid
• homocystine disulphide / L-4,4'-Dithiobis(2-aminobutanoic) acid
• O-acetyl-L-homoserine was synthesized from L-homoserine and acetic anhydride according to the protocol described in the work of Sadamu Nagai, “Synthesis of O-acetyl-L-homoserine”, internationale Press, ( 1971), vol.17, p. 423-424.
• homocysteine sulphide (L-homolantionine) is not formed, because it is not detectable in the final reaction medium, even in trace amounts.
• O-acetyl-L-homoserine was synthesized from L-homoserine and acetic anhydride according to the protocol described in the work of Sadamu Nagai, “Synthesis of O-acetyl-L-homoserine”, internationale Press, ( 1971), vol.17, p. 423-424.
• the OAHS, NaSH and OAHS sulfhydrylase solutions and the water are previously degassed separately by nitrogen sparging at the reaction temperature (before mixing) to eliminate the presence of dissolved oxygen.
• the reactor is also inerted under dinitrogen.

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