WO2008067997A1 - Préparation de 4-hydroxy-2,5-diméthyl-2,3-dihydrofuran-3-one - Google Patents

Préparation de 4-hydroxy-2,5-diméthyl-2,3-dihydrofuran-3-one Download PDF

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WO2008067997A1
WO2008067997A1 PCT/EP2007/010531 EP2007010531W WO2008067997A1 WO 2008067997 A1 WO2008067997 A1 WO 2008067997A1 EP 2007010531 W EP2007010531 W EP 2007010531W WO 2008067997 A1 WO2008067997 A1 WO 2008067997A1
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compound
group
formula
hydroxyalkyls
catalyst
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PCT/EP2007/010531
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Martin SCHÜRMANN
Daniel Mink
David John Hyett
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Isobionics B.V.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/60—Two oxygen atoms, e.g. succinic anhydride

Definitions

  • the invention relates to a method for preparing 4-hydroxy-2,5- dimethyl-2,3-dihydrofuran-3-one or an analogue thereof.
  • 4-hydroxy-2,5-dimethyl-2,3-dihydrofuran-3-one commonly known under the name Furaneol®
  • Furaneol® is a flavouring naturally found in e.g. strawberries and pineapples. It has a sweet, caramel-fruity, odour and flavour with fried meat aspects. It is mainly applied to fruity (strawberry), meat flavourings and ice cream formulations.
  • Several methods have been proposed to synthesise 4-hydroxy-2,5- dimethyl-2,3-dihydrofuran-3-one. According to WO 83/03846, a method to obtain Furaneol® from 6-deoxy-D-glucose is described in Matsui (Chem Abstr. 91 , 20309q 1979).
  • WO 83/03846 relates to a method for preparing 6-deoxy-D-fructose or 6-deoxy-L-sorbose, which is said to be suitable to be converted into Furaneol® in the presence of an organic nitrogen base and a carboxylic acid. Reaction conditions for such conversion are not shown.
  • WO 83/03846 focuses on a number of alternative methods to provide the 6-deoxy-D-fructose or 6-deoxy-L-sorbose.
  • fructose-1 ,6-bisphosphate is reacted at pH 7.0 with lactaldehyde in the presence of an enzymatic system composed of fructose 1 ,6-bisphosphate aldolase and those phosphate isomerase, followed by hydrolysis of the monophosphate salt thus obtained.
  • a drawback of this method is the need to have to hydrolyse the phosphate salt in order to obtain 6-deoxy-D-fructose or 6-deoxy-L-sorbose.
  • 1 ,3-dihydroxyacetone phosphate may be used instead of fructose-1 ,6-bisphosphate.
  • a hydrolysis step is required to obtain 6-deoxy-D-fructose or 6-deoxy-L-sorbose.
  • 1 ,3-dihydroxyacetone is treated with lactaldehyde in the presence of an anionic exchange resin.
  • a drawback of this method is the need for a non-natural chemical (the anionic exchange resin).
  • the method is not selective to the production of a specific hexose, as the product obtained is a mixture of 54 % of 6-deoxy-D-fructose, 36 % 6-deoxy-L-sorbose and 10 % unidentified components.
  • a method for preparing a deoxysugar in particular such a method which shows high selectivity to a specific deoxysugar, such as 1-deoxy-fructose or 6-deoxy-fructose.
  • the present invention relates to a method for preparing a deoxysugar, in particular a deoxyhexose, comprising reacting an ⁇ -hydroxyaldehyde or an ⁇ -ketoaldehyde (compound A) and an ⁇ -hydroxyketone (compound B) in the presence of an organo-catalyst (C) capable of catalysing an aldol reaction, thereby forming the deoxysugar.
  • the invention relates to a method for preparing a compound represented by the formula
  • R and R' each independently represent a hydrogen, a hydroxyl or a hydrocarbon moiety, which hydrocarbon moiety may comprise one or more heteroatoms, in particular O;
  • R" is selected from hydrogen and hydrocarbon moieties, which may comprise one or more heteroatoms, in particular from hydrogen and C1-C12 carboxylic acid residues, more in particular from hydrogen and C1-C6 carboxylic acid residues; the method comprising reacting an ⁇ -hydroxyaldehyde or an ⁇ -ketoaldehyde (compound A) and an ⁇ -hydroxyketone (compound B) in the presence of an organo- catalyst (C) capable of catalysing an aldol reaction and forming the compound represented by formula I.
  • organo- catalyst C
  • R and R' may be independently selected from the group consisting of C1-C12 alkyls and C1-C12 hydroxyalkyls, more in particular from the group consisting of C1-C6 alkyls and C1-C6 hydroxyalkyls, even more in particular from the group consisting of C1-C3 alkyls and C1-C3 hydroxyalkyls.
  • carboxylic acid residues for R" are in particular selected from formate, acetate and propionate.
  • the compound represented by formula I is usually formed by cyclisation of a deoxysugar formed as an intermediate product from the reaction of compound A and compound B in the presence of the organo-catalyst. Hereby water is generally eliminated.
  • deoxysugar is used herein for any compound - at least conceptually - based on a carbohydrate, wherein at least one hydroxyl is replaced by hydrogen and wherein one or more hydroxyls are optionally replaced by one or more keto-groups.
  • the deoxysugar may be a deoxyhexose, i.e. a deoxysugar at least conceptually based on a hexose.
  • the deoxysugar may be represented by any of the formulas Ma and Mb: -A-
  • R 1 and R 2 are independently selected from the group consisting of hydrogen, hydroxyl and hydrocarbons which hydrocarbons may optionally contain one or more heteroatoms, in particular O.
  • R 1 and/or R 2 are independently selected from the group consisting of C1-C12 alkyls and C1-C12 hydroxyalkyls, in particular from the group consisting of C1-C6 alkyls and C1-C6 hydroxyalkyls, more in particular from the group consisting of C1-C3 alkyls and C1-C3 hydroxyalkyls.
  • R 1 and R 2 are both methyl, R 1 is methyl and R 2 is hydroxymethyl, or R 1 is hydroxymethyl and R 2 is methyl.
  • a compound selected from 4-hydroxy-2,5-dimethyl-2,3-dihydrofuran-3-one such as the commercially available Furaneol®
  • suitable starting compounds A and B can be selected.
  • compound A and/or compound B comprise a chiral carbon
  • the compound A and/or B may be used in a stereoisomerically (enantiomerically or diastereoisomerically) enriched form or as a racemic mixture of compound A respectively B.
  • stereoisomerically (enantiomerically) enriched is meant 'having an stereorisomeric excess (s.e.) (or enantiomeric excess (e.e.)) of either of the isomers (the (R)- or (S) -enantiomer in case of enantiomeric excess) of a compound 1 .
  • a preferred choice of compounds A and B also depends on factors such as availability of the compounds and/or the nature of the catalyst, namely whether it shows stereoselectivity.
  • stereoselectivity of a catalyst in particular an enzyme, such as an aldolase is meant herein that the catalyst (enzyme) preferentially uses one stereoisomer (enantiomer or diastereomer) of the substrate ( ⁇ -hydroxyaldehyde) to convert into the stereoisomerically enriched aldol reaction product.
  • the stereoselectivity of a catalyst in particular, an enzyme may be expressed in terms of E, the ratio of the specificity constants V max /K M of the two stereoisomers (enantiomers or diastereomers) as described in C-S. Chen, Y Fujimoto, G. Girdaukas, C. J. Sih., J. Am. Chem. Soc.
  • the aldolase has an E > 5, more preferably an E > 20, most preferably an E > 100.
  • the catalyst is stereoselective, preferably that stereoisomer of the substrate that is preferentially converted by the catalyst is used in the process of the invention.
  • the reaction mixture is preferably stereoisomerically enriched in the compound or compounds for which the catalyst shows a higher activity.
  • the stereoisomeric (enantiomeric) excess is preferably > 80%, more preferably > 85%, even more preferably > 90%, in particular >95%, more in particular > 97%, even more in particular > 98%, most in particular > 99%.
  • compound A may be an ⁇ -hydroxyaldehyde represented by the formula
  • R 1 has the above identified meaning; and/or compound B may be an ⁇ -hydroxyketone represented by the formula
  • R 2 has the above identified meaning.
  • Preferred compounds A in particular for preparing 4-hydroxy-2,5- dimethyl-2,3-dihydrofuran-3-one or an ester thereof, include pyruvaldehyde (natural, and commercially readily available), lactaldehyde (which may be prepared enzymatically from natural compounds such as lactic acid or propanediol) and glyceraldehyde.
  • Preferred compounds B in particular for preparing 4-hydroxy-2,5- dimethyl-2,3-dihydrofuran-3-one or an ester thereof, include monohydroxyacetone (can be prepared enzymatically with an alcoholdehydrogenase from a natural compound such as 1 ,2-propanediol, and commercially readily available) and 1 ,3- dihydroxyacetone (natural, and commercially readily available).
  • glyceraldehyde is reacted with monohydroxyacetone or lactaldehyde is reacted with 1 ,3-dihydroxyacetone.
  • concentrations for compounds A and B can be chosen in wide limits. For practical reasons, compounds A and B are normally used in about equimolar amounts.
  • the concentration of compounds A and/or B can be high, for instance about 2.5 M or more. It has been found that at least for some starting materials, notably pyruvaldehyde, the reaction rate by which compound A and B react is reduced if the concentration is increased to a high concentration. Therefore, the concentration is usually chosen to be about 1 M or less, preferably about 0.5 M or less, in particular about 0.25 M or less, more in particular about 0.15 M or less. A relatively low concentration may also be advantageous with respect to selectivity of the reaction. An optimum concentration also depends on the specific starting materials, the desired reaction rate and/or the desired conversion.
  • the concentration of each of compound A and/or compound B usually is at least about 5 mM, in particular at least about 10 mM.
  • concentration of each of compound A and/or compound B is at least about 25 mM, in particular at least about 50 mM.
  • the concentration of compounds A and/or B can be maintained at a desirable concentration by feeding compounds A and/or B to the reaction system during the reaction, in a controlled manner.
  • a catalyst is considered an organo-catalyst if it is substantially composed of one or more organic molecules, including biomolecules, such as polypeptides, including proteins and enzymes.
  • any organo-catalyst may be used that is capable of catalysing the addition of a ketone or aldehyde donor to an aldehyde acceptor, thereby forming an enamine.
  • Such catalysts are known to persons skilled in the art and can be found in literature, e.g. in Angew. Chem. Int. Ed, 2007, 46, 5572-5575; Ace. Chem. Res, 2004, 37, 546-557, and organic Letters, Vol. 9, No.17, p.3445-3448.
  • the organo-catalyst is a natural compound and/or generally regarded as safe to be present in a food or a drink.
  • Good results have in particular been achieved using an aldolase to catalyse the reaction, in particular in the preparation of 4-hydroxy-2,5-dimethyl-2,3- dihydrofuran-3-one.
  • any aldolase can be used.
  • the enzyme may be any aldolase that is regarded as a natural compound, according to food industry regulations.
  • an aldolase is meant a moiety comprising a polypeptide, in particular an enzyme, having aldolase activity, i.e. having the ability to catalyze the addition of a ketone or aldehyde donor to an aldehyde acceptor.
  • the moiety (enzyme) may be stereoselective.
  • a class I or class Il aldolase is used, for example a dihydroxyacetone dependent aldolase, for example fructose 1-6-bisphosphate aldolase (e.g. as used in WO83/03846), tagatose-1 ,6-bisphosphate aldolase, fuculose-1- phosphate aldolase, rhamnulose-1 -phosphate or 2-deoxy-D-ribose-5-phosphate aldolase.
  • a class I aldolase from the transaldolase family is chosen. More preferably the aldolase belongs to the type 3A subfamily (MipB/TalC subfamily). Even more preferably the aldolase chosen is a D-Fructose 6-phosphate aldolase (FSA 1 EC 4.1.2.).
  • aldolases are dihydroxyacetone-phosphate independent aldolases, in particular FsaA or FsaB, more in particular FsaA or FsaB from Escherichia coli K12 (EC 4.1.2). Specifically preferred are aldolases having the sequence of SEQ ID NO:
  • SEQ ID NO 1 A nucleic acid sequence encoding the aldolases of SEQ ID No 2 and SEQ ID No 4 is given in SEQ ID NO 1 respectively 3.
  • Homologues are in particular aldolases having a sequence identity of at least 60%, preferably at least 65%, more preferably at least 70 %, more preferably at least 75%, more preferably at least 80%, in particular at least 85 %, more in particular at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 %.
  • sequence identity is determined in sequence alignment studies using ClustalW, version 1.82 http://www.ebi.ac.uk/clustalw multiple sequence alignment at default settings (matrix: Gonnet 250; GAP OPEN: 10; END GAPS: 10; GAP EXTENSION: 0.05; GAP DISTANCES: 8).
  • Amino acid residues of wild-type or mutated protein sequences corresponding to positions of the amino acid residues in the wild-type amino sequence of the E. coli K12 FsaA [SEQ ID No.2] or E. coli K12 FsaB [SEQ ID No.4] can be identified by performing ClustalW version 1.82 multiple sequence alignments (http://www.ebi.ac.uk/clustalw) at default settings (matrix: Gonnet 250; GAP OPEN: 10; END GAPS: 10; GAP EXTENSION: 0.05; GAP DISTANCES: 8).
  • Amino acid residues which are placed in the same column as an amino acid residue as given in [SEQ ID No.2] respectively [SEQ ID No.4] in such alignments are defined to be positions corresponding to this respective amino acid residue of the E. coli K12 wild-type FsaA [SEQ ID No.2] respectively FsaB [SEQ ID No.4].
  • aldolases can be employed without needing a cofactor.
  • a reducing agent may be added, to protect the enzyme against oxidation.
  • an aldolase selected from FsaA (accession number P78055) from Escherichia coli K12 (SEQ ID No. 2), FsaB (accession number P32669) from Escherichia coli K12 (SEQ ID No.
  • wild-type enzymes or variants derived from natural mutations or artificial mutagenesis procedures such as FsaA or FsaB variants Leu107Gln, Leu107Asn, and Ala129Ser as single amino acid residue exchanges or combinations of said exchanges
  • FsaA or FsaB variants Leu107Gln, Leu107Asn, and Ala129Ser as single amino acid residue exchanges or combinations of said exchanges
  • variants of FSAs from other organisms in which the corresponding positions to FsaA or FsaB from Escherichia coli K12 in their respective FSA sequences have been exchanged.
  • compound A is preferably enantiomerically enriched with the R- enantiomer, as such enzyme tends to have a higher reaction rate with respect to the R- enantiomer.
  • Mutants of wild-type enzymes can for example be made by modifying the DNA encoding the wild type enzymes using mutagenesis techniques known to the person skilled in the art (random mutagenesis, site-directed mutagenesis, directed evolution, gene shuffling, etc.) so that the DNA encodes an enzyme that differs by at least one amino acid from the wild type enzyme and by effecting the expression of the thus modified DNA in a suitable (host) cell.
  • Mutants of the aldolase may for example have improved properties with respect to (stereo)selectivity and/or activity and/or stability and/or solvent resistance and/or pH profile and/or temperature profile.
  • the aldolase may be used in any form.
  • the aldolase has been isolated from the cells, i.e. a cell-free extract is used.
  • the aldolase may be used - for example in the form of a dispersion, a solution or in immobilized form - as crude enzyme, as a commercially available enzyme, as an enzyme further purified from a commercially available preparation, as an enzyme obtained from its source by a combination of known purification methods, in whole (optionally permeabilized and/or immobilized) cells that naturally or through genetic modification possess the required aldolase activity, or in a lysate of cells with such activity.
  • the aldolase is usually used in a amount corresponding to an activity of at least 0.1 U per mmole acceptor substrate (compound A), wherein the activity is determined by using a spectro-photometrical assay as described by Sch ⁇ rmann & Sprenger in J. Biol. Chem. 276 (14), 11055-11061 (2001).
  • the aldolase is used in an amount corresponding to at least 1 U per mmol acceptor substrate, in particular at least 10 U per mmol acceptor substrate, more in particular of at least 25 U per mmol acceptor substrate.
  • the aldolase is usually added in an amount corresponding to an activity of up to 1000 U per mmol acceptor substrate, in particular up to 500 U, more in particular up to 250 U, even more in particular up to 100 U per mmol acceptor substrate.
  • 1 U is defined herein as the amount of enzyme (aldolase) that splits 1 mM of D-fructose-6-phosphate in dihydroxyacetone and glyceraldehyde-3-phosphate in 1 minute at 37°C in 5OmM glycyl/glycine buffer containing 1 mM dithiothreitol (pH 8.0).
  • aldolase enzyme that splits 1 mM of D-fructose-6-phosphate in dihydroxyacetone and glyceraldehyde-3-phosphate in 1 minute at 37°C in 5OmM glycyl/glycine buffer containing 1 mM dithiothreitol (pH 8.0).
  • the organo-catalyst is selected from proline, catalysts comprising a proline-active site, pyrrolidine and catalysts having a pyrrolidine active site.
  • catalysts having a pyrrolidine active site or a proline active site are catalysts comprising a secondary amine in a 5-membered ring as an active site, such as represented by the following formula
  • Y can be a hydrogen (then the compound is pyrrolidine), a carboxylic acid group (proline), an ester or a support material to which the five-membered ring is bonded.
  • a stereoisomerically enriched catalyst may be used or a racemic mixture.
  • D-proline, L-proline or a mixture thereof can be used.
  • a different stereoselectivity may be achieved for a (stereoisomeric) reagent or product.
  • the amount of organo-catalyst can be chosen within a wide range. Usually the concentration is at least 0.01 mol %, with respect to the total amount of compound A and B. In particular the concentration may be at least 0.1 mol %, at least 1 mol %, at least 5 mol %, or at least 10 mol % in particular for non-enzymatic organo- catalyst. The concentration is usually up to 25 mol %, in particular up to 20 mol %, in particular for non-enzymatic organo-catalysts.
  • proline preferably L-proline as L-proline is indigenous to the human body, and thus does not need to be removed in case the product made is to be used as a food additive, include satisfactory yield and selectivity, and the fact that proline is a naturally occurring compound. Moreover, proline, in particular L-proline, is readily available and relatively cheap.
  • the catalytic reaction of compound A and B can be carried out in any suitable solvent. In particular when using an aldolase, the reaction is preferably carried out in water or an aqueous solvent, wherein aqueous means that the water content is at least 50 wt. %, preferably at least 90 wt. %, even more preferably at least 99 wt.%. In particular for non-enzymatic organic catalysts reaction of compound A and B may preferably be carried out in the presence of one or more organic solvents or a mixture of one or more organic solvents and water.
  • Suitable solvents are in particular those wherein compound A and B dissolve.
  • Particular suitable are polar solvents, in particular polar solvents that are fully water-miscible, i.e. that can be mixed with water and form a single phase with water, at least under the reaction conditions.
  • solvents include tetrahydrofuran, dimethylsulfoxide, water-miscible alcohols, such as methanol, ethanol and propanol, and mixtures thereof.
  • the pH is suitably chosen, inter alia dependent upon factors such as the organo-catalyst activity and stability.
  • the reaction mixture to form the deoxysugar is slightly acidic, neutral or slightly alkaline.
  • the pH is usually chosen in the range of 5-12.
  • the pH is at least 6.
  • the pH is up to 9. Good results have in particular been achieved with a pH from about 7 to about 8.
  • the pH may be adjusted as desired with one or more acids and/or bases.
  • the acids and bases may be selected from inorganic and organic acids and bases.
  • an acid and/or a base is used which is suitable for use in a food or drink.
  • acids and bases examples include phosphoric acids and phosphate salts (e.g. NaH 2 PO 4 , Na 2 HPO4, Na 3 PO 4 ), dihydrogencarbonate and carbonate salts (e.g. NaHCO 3 , Na 2 CO 3 ), sulphuric acid, hydrochloric acid, sodium hydroxide, potassium hydroxide, citric acid and salts thereof (such as sodium salts) and acetic acid and salts thereof (such as sodium acetate). Acids and/or bases may be selected such that a pH-buffer is formed. The skilled person will know how to select suitable acids and/or bases to form a suitable buffer, depending on the Ka value(s) of the acids and/or bases.
  • phosphoric acids and phosphate salts e.g. NaH 2 PO 4 , Na 2 HPO4, Na 3 PO 4
  • dihydrogencarbonate and carbonate salts e.g. NaHCO 3 , Na 2 CO 3
  • sulphuric acid hydrochloric acid, sodium hydrox
  • the temperature is suitably chosen, inter alia dependent upon factors such as the organo-catalyst activity and stability. Usually a temperature in the range of 0-75 0 C is chosen. Preferably the temperature is up to 60 0 C, in particular up to 50 0 C in view of the catalyst stability, in particular in case an aldolase is used. A relatively low temperature, in particular of up to 40 0 C, more in particular up to 30 0 C is especially preferred, because of the low energy consumption, compared to a process wherein the reaction mixture is heated to a higher temperature.
  • the temperature is usually at least about ambient temperature, in particular at least about 20 0 C, more in particular at least about 30 0 C.
  • a temperature in the range of 37-50 0 C has been found particularly suitable, especially if the aldolase is FsaB (from E. coli K12).
  • the product formed from compounds A and B can be used to prepare a compound according to formula I, which may be used as a flavouring agent.
  • a compound according to formula I which may be used as a flavouring agent.
  • the formation of the compound with formula I may be enhanced by a manner known in the art.
  • the formation of the compound with formula I may be accomplished by subjecting the intermediate reaction product of compounds A and B to a cyclisation and elimination reaction in a water-containing solution, in particular an aqueous solution as described above, which may be acidic, neutral or alkaline.
  • a water-containing solution in particular an aqueous solution as described above, which may be acidic, neutral or alkaline.
  • Suitable acids and bases that may be used to adjust the pH include acids and bases, as described above.
  • good results with respect to the reaction rate have been achieved in an aqueous acidic solution having a pH (measured to the fluid at 25 0 C) of 5 or less.
  • the pH is at least 1 , in particular at least 2, in view of fully or at least substantially avoiding degradation of the compound of formula I.
  • Particular preferred is a pH of at least about 3.
  • the pH is usually at least 8, in particular at least 9, more in particular at least about 11.
  • the pH is 13 or less, in particular 12 or less, in view of avoiding degradation of the compound of formula I.
  • the temperature may be chosen within wide limits, usually the temperature is at least 10 0 C, in particular at least ambient temperature, e.g. about 20 0 C. Usually the temperature is up to 70 0 C, in particular up to 50 0 C more in particular up to 30 C C.
  • the compound of formula I may be isolated from the reaction mixture in a manner known in the art.
  • the R" group is preferably attached to the compound after the elimination and cyclisation. Suitable manners to achieve this can be based on methodology known in the art, such as a known esterification reaction in case an ester is to be formed.
  • the compound may be esterified with an organic acid having 1-12 carbon atoms, preferably having 1-6 carbon atoms, in particular formate, acetate or priopionate.
  • an acetate ester is formed, especially in case R 1 and R 2 each represent a methyl group.
  • the resultant compound (“Furonol acetate”) is in particular suitable as a flavouring ingredient.
  • EXAMPLE 1 Enzymatic synthesis of 4-hvdroxy-2.5-dimethyl-3(2H)-furanone PCR-Amplification of fsa genes from Escherichia coli K12
  • the pooled plasmids pENTR-fsaA and pENTR-fsaB were then applied in the Gateway LR cloning reactions with pBAD//Wyc-His-DEST (obtained as described in EP1513946) to obtain the expression vectors pBAD-fsaA and pBAD-fsaB, respectively.
  • the transformation of E. coli TOP10 with the LR reactions yielded more than hundred individual colonies, respectively. From both transformation agar plates 4 individual clones were selected and tested for fructose 6-phosphate aldolase activity.
  • the expression of the fsa genes was induced by addition of 0.02% (w/v) L-arabinose in the middle of the logarithmic growth phase (cell densities of OD 62 O ⁇ 0.6). After overnight incubation under identical conditions the cells were harvested by centrifugation (15 min at 500Ox g, 4°C) and resuspended in 4 ml 50 mM glycyl-glycine buffer pH 8.0 containing 1 mM dithiothreitol (DTT).
  • DTT dithiothreitol
  • CFEs cell-free extracts
  • MSE Soniprep 150 sonificator small probe, 5 min in an ice/acetone bath
  • centrifugation for one hour at 4°C and 39,00Ox g
  • recovery of the supernatant was determined using a modified protein- dye binding method as described by Bradford in Anal. Biochem. 72, 248-254 (1976).
  • the fructose 6-phosphate aldolase activity in the cell-free extracts was determined using a spectro-photometrical assay as described by Sch ⁇ rmann & Sprenger in J. Biol. Chem. 276 (14), 11055-11061 (2001).
  • the specific activities of 0.8 and 0.6 U/mg protein as determined for the CFEs containing FsaA or FsaB were comparable with the reported literature values.
  • DNA sequencing proved that the fsaA and fsaB inserts of pBAD-fsaA and pBAD-fsaB were identical to the genomic sequences.
  • a 10 I scale fermentation of wild-type-FsaB was performed at 37°C in TB-medium (terrific broth; 12 g/l tryptone, 24 g/l yeast extract, 4 g/l glycerol, 2.31 g/l KH 2 PO 4 , 12.54 g/l K 2 HPO 4 , pH 7.0 containing 100 ⁇ g/ml carbenicillin) in an ISF-200 laboratory fermentor (Infors).
  • ISF-200 laboratory fermentor ISF-200 laboratory fermentor
  • the fermentation yielded a cell-free extract with a specific FSA activity of 1.5 U/mg cell-free extract protein with a protein concentration of 22 mg/ml and volumetric activity of 33.5 U/ml.
  • Aldolase reactions with hydroxyacetone and pyruvaldehyde 0.1 mol/l of each hydroxyacetone and pyruvaldehyde were incubated with cell-free extract of E.

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Abstract

La présente invention concerne un procédé pour préparer une 4-hydroxy-2,5-diméthyl-2,3-dihydrofuran-3-one ou un analogue de celle-ci, comprenant la réaction d'un α-hydroxyaldéhyde ou d'un α-cétoaldéhyde (composé A) et d'une α-hydroxycétone (composé B) en présence d'un catalyseur organique (C) capable de catalyser une réaction d'aldolisation, de préférence choisi dans le groupe constitué par l'aldolase, la proline, la pyrrolidine et des catalyseurs comprenant un site actif de proline ou un site actif de pyrrolidine, et la formation d'une 4-hydroxy-2,5-diméthyl-2,3-dihydrofuran-3-one ou d'un analogue de celui-ci.
PCT/EP2007/010531 2006-12-05 2007-12-05 Préparation de 4-hydroxy-2,5-diméthyl-2,3-dihydrofuran-3-one WO2008067997A1 (fr)

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CN112047911A (zh) * 2020-09-18 2020-12-08 厦门欧米克生物科技有限公司 一种呋喃酮的催化合成方法

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2343448A1 (es) * 2009-01-29 2010-07-30 Consejo Superior De Investigaciones Cientificas (Csic) (50%) Procedimiento quimo-enzimatico para la sintesis de 1-deoxi-d-xilulosa.
WO2010086482A1 (fr) * 2009-01-29 2010-08-05 Consejo Superior De Investigaciones Científicas (Csic) Procédé chimio-enzymatique pour la synthèse de 1-déoxy-d-xylulose
CN110563676A (zh) * 2019-08-15 2019-12-13 安徽金禾实业股份有限公司 2,5-二甲基-4-羟基-3(2h)-呋喃酮的制备方法
CN110698618A (zh) * 2019-10-15 2020-01-17 陕西师范大学 水溶性共聚物固载l-脯氨酸催化剂及其制备方法和应用
CN112047911A (zh) * 2020-09-18 2020-12-08 厦门欧米克生物科技有限公司 一种呋喃酮的催化合成方法

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