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
  • 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
  • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
• • 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)

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 sulfide halides, epoxides or organic carbonates.
• cysteine is currently produced biologically through a fermentation pathway (Maier T., 2003. Nature Biotechnology, 21: 422- 427). These biological pathways are softer and more suited to multifunctional molecules. However, these biological pathways often have low yields and/or are difficult to transpose and viable on an industrial scale. Moreover, here again, the production of the mercaptan of interest is accompanied by the corresponding sulphides and/or polysulphides such as the disulphides (cf. for example international application WO 2012/053777).
• An object of the present invention is to provide a process for the synthesis of an improved functionalized mercaptan, in particular having an improved yield, even a yield of at least 20%, preferably of at least 60%, of more preferably at least 80%, more preferably at least 90%.
• the present invention meets all or part of the above objectives.
• the functionalized mercaptans of formula (I) as defined below, in particular L-homocysteine are advantageously synthesized by reaction between compounds of formula (II) and H2S, in the presence of a sulfhydrylase enzyme, under a particular range of H2S partial pressure in the reactor where said reaction takes place.
• said H2S partial pressure is between 0.01 and 4 bars, for example between 0.01 and 3 bars, preferably between 0.1 and 3 bars, for example between 0.1 and 2.5 bars, and more preferably between 0.25 and 2 bars.
• the present inventors have thus discovered that the transformation of the compounds of formula (II) into functionalized mercaptans of formula (I) is highly dependent on the partial pressure of H2S in the reactor.
• the conversion and/or the yield is (are) between 80% and 100%, or even between 90% and 100%.
• the conversion and/or the yield is (are) 100%.
• the specific range of H 2 S partial pressure in the reactor according to the invention allows rapid reaction kinetics. For example, you can reach 100% efficiency in one hour.
• the reaction time can thus be between 0.15 h and 10 h, for example between 0.25 h and 4 h, preferably between 0.5 h and 1 h.
• the method according to the invention makes it possible to obtain better yields than a method using sulphide and/or sulphide salts as reagents.
• the use of hydrogen sulfide thus makes it possible to limit or even simplify the steps of purification and management of effluents which are necessary when such salts are used.
• the process according to the invention is therefore more respectful of the environment.
• the present invention relates to a process for the synthesis of at least one functionalized mercaptan of general formula (I) below: R 2 -XC*H(NRiR 7 )-(CH 2 )n-SH (I) in which,
• R1 and R7 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 which may comprise one or more heteroatoms;
• R2 is:
• G represents either (i) Re-C(O)-O-, or (ii) (R 7 O)(RsO)-P(O)-O-, or (iii) RgO-SO 2 -O-;
• R10, Ru, R12 and R13 being independently of each other chosen from:
• R 7 and Rs identical or different, being a proton, an alkali, an alkaline earth or an ammonium
• Rg being chosen from a proton, an alkali, an alkaline earth or an ammonium; b) supply of H 2 S; c) reaction between said at least one compound of formula (II) and H 2 S in the presence of at least one enzyme chosen from sulfhydrylases, preferably a sulfhydrylase associated with said compound of formula (II); said reaction taking place in a reactor with a partial pressure of H 2 S in the gas overhead of said reactor of between 0.01 and 4 bars, for example between 0.01 and 3 bars, preferably between 0.1 and 3 bars , for example between 0.1 and 2.5 bar, and more preferably between 0.25 and 2 bar, at the reaction temperature; 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
• heteroatom means in particular an atom chosen from O, N, S, P and halogens.
• inert gas means any gas having little or no reactivity in the context of the method according to the invention. Mention may be made, for example, of dinitrogen, argon or methane, preferably dinitrogen.
• reaction medium in particular a medium comprising at least one compound of formula (II), F S, and said at least one sulfhydrylase.
• Said reaction medium may thus comprise: at least one compound of formula (II) as defined below, 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.
• the reaction medium is liquid, for example in the form of an aqueous solution, in particular under the temperature and pressure conditions of step c).
• the H2S is in gaseous form, in particular under the temperature and pressure conditions of step c).
• part of the H2S is solubilized in the reaction medium so that the reaction of step c) takes place while the other part is found in gaseous form in the gaseous headspace of the reactor , at said partial pressure.
• gas overhead means the space of the reactor situated above the reaction medium, preferably above the liquid reaction medium. More particularly, the term “gas overhead” means the space situated between the surface of the liquid reaction medium and the top of the reactor (ie 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 comprising H 2 S at said partial pressure.
• the reaction medium and the H 2 S are notably introduced into the reactor in quantities such that a gaseous blanket is located above the reaction medium contained in the reactor.
• step c) can be described as follows: c) reaction between said at least one compound of formula (II) and H 2 S in the presence of at least one enzyme chosen from sulfhydrylases , preferably a sulfhydrylase associated with said compound of formula (II); said reaction being carried out in a reactor with a partial pressure of H 2 S above the reaction medium of between 0.01 and 4 bars, for example between 0.01 and 3 bars, of preferably between 0.1 and 3 bars, for example between 0.1 and 2.5 bars, and more preferably between 0.25 and 2 bars, at the reaction temperature.
• at least one enzyme chosen from sulfhydrylases preferably a sulfhydrylase associated with said compound of formula (II)
• said reaction being carried out in a reactor with a partial pressure of H 2 S above the reaction medium of between 0.01 and 4 bars, for example between 0.01 and 3 bars, of preferably between 0.1 and 3 bars, for example between 0.1 and 2.5 bars, and more preferably between 0.25 and 2 bars, at
• said H2S partial pressure corresponds to the total pressure of the gaseous phase present in the gaseous headspace (i.e. only the H2S is present in the gaseous headspace of the reactor).
• said H2S partial pressure can be kept constant throughout step c). This can be obtained by continuous introduction of F S into the reactor or by occasional regular or irregular additions of F S to the reactor, during step c). Indeed, the H2S being consumed during the reaction, it is thus possible to compensate for the decrease in the partial pressure of H2S.
• said H2S partial pressure can be reached before or during step c), then the introduction of F S into the reactor is stopped.
• the H2S partial pressure therefore decreases during step c), preferably until the reaction stops.
• the H 2 S partial pressure can be controlled throughout step c), in particular by any known technique, for example using a manometer.
• the H 2 S can be added so that a state of equilibrium between the liquid phase (reaction medium) and the gaseous phase (comprising the H 2 S at said partial pressure) is reached in the reactor.
• the total pressure of the gaseous phase in the gaseous headspace corresponds approximately to atmospheric pressure (approximately 1.01325 bar). It is also possible to choose to work at underpressure or at overpressure with respect to atmospheric pressure depending on the desired operating conditions.
• a vacuum is created in the reactor and then the H2S is introduced at the partial pressure according to the invention.
• a vacuum is drawn down to - 1 bar and then an F S pressure of 0.25 bar is applied.
• the following steps are carried out: the top of the reactor is swept with the aid of an inert gas, such as N2; then a partial vacuum is carried out; then the H2S is introduced at partial pressure according to the invention.
• an inert gas such as N2
• a vacuum is created at -0.25 bar then 0.25 bar of F S is added.
• a mixture of inert gas, such as N2, and H2S at the partial pressure according to the invention.
• a mixture of 0.25 bar of F S and 0.75 bar of N2 can be introduced.
• an inert gas into the reactor (scavenging of the gaseous headspace), such as N2, then to add the F S at the partial pressure according to the invention.
• an inert gas for example, one can apply a pressure of 1 bar of N2 then add 0.25 bar of F S.
• 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.
• the reactor used for step c) can be of any type. It is preferably chosen from piston type reactors or continuous reactors, preferably agitated and/or with recirculation of the gaseous phase and/or with recirculation of the liquid phase. Preferably, said reactor allows recirculation (or recycling) of the gaseous phase present in the gaseous overhead.
• Steps a) and b) can be simultaneous or performed in any order.
• Said reaction medium can be prepared by adding said compound of formula (II), said sulfhydrylase and optionally its cofactor in any order. It is preferable to introduce the F S afterwards. This makes it possible in particular to more easily manage the H2S pressure introduced into the reactor.
• the compound of formula (II) and/or the sulfhydrylase is (are) in the form of a solution, more preferably in the form of an aqueous solution.
• the H 2 S can be introduced into the reactor by any known method and in particular by bubbling into the reaction medium, preferably by bubbling into the reaction medium from the bottom of the reactor.
• 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 F S is introduced pure (without being mixed with another gas).
• the H2S can also be introduced through the top of the reactor and for example then equilibrate with the reaction medium, the reaction medium preferably being under stirring.
• the F S is in excess, preferably in molar excess, relative to the compound of formula (II), preferably during step c) and more preferably throughout the duration of step vs).
• the H2S can therefore be in an over-stoichiometric quantity relative to the quantity of the compound of formula (II), preferably during step c) and more preferably throughout the duration of step c).
• the H2S/compound of formula (II) molar ratio is between 1.1 and 20, preferably between 1.1 and 10, preferably between 2 and 8, for example between 3.5 and 8, and even more preferably between 3.5 and 5, 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.
• the pH of the reaction medium 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, in particular when the reaction medium 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.
• the pH can in particular be adjusted by adding a base, preferably throughout the reaction of step c). 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.
• the base can be chosen from sulphide salts and/or sulphide salts, sodium hydroxide, potassium hydroxide or ammonia.
• the preferred base is ammonium sulphhydrate (NH 4 SH).
• the sulphide and/or sulphide salt can be chosen from the group consisting of: ammonium sulphide, alkali metal sulphides, alkaline earth metal sulphides, alkali metal sulphides and alkaline earth metal sulphides.
• alkali metals lithium, sodium, potassium, rubidium and cesium, preferably sodium and potassium.
• alkaline earth metals means beryllium, magnesium, calcium, strontium and barium, preferably calcium.
• the sulphide salt and/or sulphide salt can be chosen from the group consisting of: ammonium sulphide NH4SH, sodium sulphide NaSH, potassium sulphide KSH, calcium sulphide Ca(SH)2, sodium sulfide Na2S, ammonium sulfide (NFL ⁇ S, potassium sulfide K2S and calcium sulfide CaS.
• the preferred sulfide is ammonium sulfide NH4SH.
• 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. concentrated bases so as to limit the dilution of the reaction medium during the addition of the base.
• Step c) can be carried out in batch, semi-continuously or continuously.
• Step c) carried out essentially in the absence of oxygen:
• Oxygen is understood to mean in particular dioxygen O2.
• step c) is carried out essentially in the absence of oxygen, or even in the absence of oxygen.
• step c) is carried out essentially in the absence of oxygen (or even in the absence of oxygen O2), this makes it possible, if necessary, to limit (or even avoid) the co-production of sulphides and/ or polysulphides, in particular disulphides, unwanted by-products (cf. application FR2007577).
• the term “essentially in the absence of oxygen” means that there may remain a quantity of oxygen in the reaction medium 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” means that the reaction medium contains less than 0.0015% oxygen (preferably strictly less than 0.0015%) by weight relative to the weight total of the reaction medium and/or that the gaseous phase (contained in the gaseous headspace) contains less than 21% oxygen (preferably strictly less than 21%) by volume relative to the total volume of said gaseous phase.
• the reaction medium may contain between 0 and 0.0015% oxygen (preferably strictly less than 0.0015%) by weight relative to the total weight of the reaction medium and/or the gaseous phase (contained in the gas overhead) may contain 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 medium and/or in the gaseous phase (contained in the gaseous headspace) is such that the quantity of sulphides and/or polysulphides produced is less than or equal to 5% by weight relative to the weight total 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.
• 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 total weight of the reaction mixture.
• the O2/H2S mixture can present a risk of explosion, which obviously implies a risk for the safety of the operators.
• step c) when step c) is also carried out essentially in the absence of oxygen (or even in the absence of oxygen), this makes it possible, if necessary, to produce L-homocysteine while limiting (or even avoiding) the co-production of L-homocystine and/or L-homocysteine sulfide (also called 4,4'-sulfanediylbis(2-aminobutanoic acid) / L-homolanthiine), unwanted by-products.
• L-homocystine and/or L-homocysteine sulfide also called 4,4'-sulfanediylbis(2-aminobutanoic acid) / L-homolanthiine
• L-homocysteine sulfide has the following formula:
• L-homocystine has the following formula:
• step c) In order to perform 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 medium, 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 medium.
• each of the solutions comprising the compound of formula (II), the sulfhydrylase and optionally the solvent is degassed.
• the reactor can also be inerted with an inert gas such as dinitrogen, argon or methane, preferably dinitrogen.
• an inert gas such as dinitrogen, argon or methane, preferably dinitrogen.
• the substantial absence, or even the total absence, of oxygen is obtained in the following way: the reactor is inert with an inert gas such as dinitrogen, argon or methane, preferably dinitrogen; and each of the solutions comprising the compound of formula (II), the sulfhydrylase and optionally the solvent is degassed.
• an inert gas such as dinitrogen, argon or methane, preferably dinitrogen
• Industrial degassing methods are well known and the following can be cited, for example: pressure reduction (vacuum degassing), thermal regulation (increasing the temperature for an aqueous solvent and lowering the temperature for an organic solvent ), membrane degassing, degassing by alternating freeze-pump-thaw cycles, degassing by bubbling an inert gas (for example argon, dinitrogen or methane).
• pressure reduction vacuum degassing
• thermal regulation increasing the temperature for an aqueous solvent and lowering the temperature for an organic solvent
• membrane degassing degassing by alternating freeze-pump-thaw cycles
• degassing by bubbling an inert gas for example argon, dinitrogen or methane
• step c) the oxygen is present neither in dissolved form in a liquid (in particular in the reaction medium), nor in gaseous form (in particular in said gas phase) .
• the separation step e) can be carried out using 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.
• the 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.
• X-R2 represents a carboxylic function
• the latter can be esterified, reduced to aldehyde, reduced to alcohol then esterified, amidated, 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 several mercaptan derivatives with different functionalities, said chemical reactions being well-known reactions.
• 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;
• R2 is:
• 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,
• mercaptans are said to be functionalized because in addition to the —SH chemical function, they also comprise at least one —NRI R 7 function of the amine type.
• n is equal to 2.
• R 2 is -OR3 with R3 as defined above.
• R3 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.
• R3 is H.
• Ri and R 7 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 7 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 most particularly L-homocysteine of the following formula:
• n is equal to 2
• R2 is -OR3 with R3 being H and Ri and R7 being H.
• the functionalized mercaptan of formula (I) obtained according to the process of the invention can be enantiomerically pure.
• the functionalized mercaptans of formula (I) are chiral compounds. In the present description, when the enantiomeric form is not specified, the compound is understood whatever its enantiomeric form.
• the reaction medium 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 medium 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).
• sulphide in particular means the sulphide corresponding to the compound of formula (I) which is itself of the following formula (III):
• polysulphide means in particular the polysulphide corresponding to the compound of formula (I) which is itself of the following formula (IV):
• n is equal to 2 (which corresponds to a disulphide).
• the reaction medium 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 above and a compound of formula (V) GH with G as defined below i.e.
• reaction medium it is possible to maintain the pH of the reaction medium 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.
• R10, Ru, R12 and R13 being independently of each other chosen from:
• R 7 and Rs 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 + ;
• Rg 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 Re-C(O)-O- or R9O-SO2-O-; preferably G is R 6 -C(O)-O-,
• Rw and Rn are H.
• Ri2 and R 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 of homoserine (when n is equal to 2), in particular of L-serine or of L-homoserine. It can for example be chosen from the group consisting of: O-phospho-L-homoserine, O-succinyl-L-homoserine, O-acetyl-L-homoserine, O-acetoacetyl-L- homoserine, O-propio-L-homoserine, O-coumaroyl-L-homoserine, O-malonyl-L-homoserine, O-hydroxymethylglutaryl-L-homoserine, O-pimelyl-L- homoserine O-sulfato-L-homoserine, O-phospho-L serine, O-succinyl-L serine, O acetyl-L serine,
• O-phospho-L-homoserine O-succinyl-L-homoserine, O-acetyl-L-homoserine, O- acetoacetyl-L-homoserine, O-propio-L-homoserine, O-coumaroyl-L-homoserine, O-malonyl-L-homoserine, O-hydroxymethylglutaryl-L-homoserine, O- pimelyl-L-homoserine and 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
• 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, L-serine itself being able to 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 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.
• the reaction between said at least one compound of formula (II) and H2S 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 H2S (enzymatic reaction).
• catalyst generally means a substance which accelerates 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 catalytic quantity is used in a smaller quantity, for example between about 0.01% and 20% by weight, relative to the quantity by weight of a reactant used in 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 varying according to the substrate of the enzyme).
• O-acetylhomoserine sulfhydrylase is of type EC 2.5.1.49.
• O-phosphoserine sulfhydrylase is type EC 2.5.1.65.
• O-succinylhomoserine sulfhydrylase is EC type 2.5.1.49.
• O-acetyl-L-homoserine sulfhydrylase is of type EC 2.5.1.49.
• O-phospho-L-serine sulfhydrylase is of type EC 2.5.1.65.
• the O-succinyl-L-homoserine sulfhydrylase is of the EC 2.5.1.49 type.
• 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, O-coumaroyl-L-homoserine sulfhydrylase, O-malonyl-L-homoserine sulfhydrylase, O-hydroxymethylglutaryl-L-homoserine sulf
• 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, O-coumaroyl-L-homoserine sulfhydrylase, O-malonyl-L-homoserine sulfhydrylase, O-hydroxymethylglutaryl-L-homoserine sulfhydrylase , O-pimelyl-L-homoserine sulfhydrylase, O-sulfato-L-homoserine sulfhydrylase.
• the sulfhydrylase can be chosen from O-phospho-L-homoserine sulfhydrylase, O-succinyl-L-homoserine sulfhydrylase, O-acetyl-L-homoserine sulfhydrylase, O-sulfato -L-homoserine sulfhydrylase and O-propio-L-homoserine sulfhydrylase.
• the sulfhydrylase can be chosen from O-phospho-L-homoserine sulfhydrylase, O-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 medium.
• a sulfhydrylase cofactor for example pyridoxal-5'-phosphate
• a sulfhydrylase cofactor for example pyridoxal-5'-phosphate
• 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 either in isolated and/or purified form, for example in aqueous solution;
• 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. either included in a crude extract, i.e. in an extract of crushed cells (lysate);
• 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. is included in whole cells. For this, the same techniques as above can be used without performing the cell lysis step.
• 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, 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.
• the reaction medium may also comprise: optionally one or more solvents chosen from water, buffers such as phosphate buffers, Tris-HCl, Tris-base, ammonium bicarbonate, ammonium acetate, HEPES (acid 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic), CHES (N-cyclohexyl-2-aminoethanesulfonic acid), or salts such as sodium chloride, potassium chloride, or their media; optionally additives such as surfactants, in particular to promote the solubility of one or more reagent(s) or substrate(s).
• buffers such as phosphate buffers, Tris-HCl, Tris-base, ammonium bicarbonate, ammonium acetate, HEPES (acid 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic), CHES (N-cyclohexyl-2-aminoethanesulfonic acid), or salts such
• 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 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 solubilized FkS, preferably in excess.
• said composition comprises: O-acetyl-L-homoserine; O-acetyl-L-homoserine sulfhydrylase; and the solubilized F S, preferably in excess.
• composition corresponds in particular to the reaction medium as defined above.
• the composition according to the invention does not comprise dissolved oxygen.
• the F S is in excess, preferably in molar excess, relative to the compound of formula (II).
• the F S can therefore be in an over-stoichiometric quantity with respect to the quantity of the compound of formula (II).
• the F S / compound of formula (II) molar ratio is between 1.1 and 20, preferably between 1.1 and 10, preferably between 2 and 8, for example between 3.5 and 8, and even more preferably between 3.5 and 5.
• 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.
• Figure 1 represents the yield (%) of the enzymatic synthesis reaction of L-homocysteine after 1 hour of reaction as a function of the partial pressure of H 2 S (bars).
• 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.
• reaction medium is then degassed by bubbling with nitrogen for about ten minutes.
• the yield of the reaction is measured after 1 hour of reaction via an approach for quantification of the mercaptan formed by argentimetric potentiometry titration (results also confirmed by NMR and HPLC analyses).
• 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 reactor is placed under vacuum in order to eliminate all the gases present in the top of the reactor and thus finely control the pressure of added hydrogen sulphide. Then an H 2 S pressure of 0.25 bar is applied. The start of the reaction is confirmed by an acidification gradual release of the reaction medium (due to the gradual release of the acetic acid co-product) and the pH of the solution is maintained at about 6.5 via the gradual addition of ammonia (4M).

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