WO2008067997A1

WO2008067997A1 - Preparation of 4-hydroxy-2,5-dimethyl-2,3-dihydrofuran-3-one

Info

Publication number: WO2008067997A1
Application number: PCT/EP2007/010531
Authority: WO
Inventors: Martin SCHÜRMANN; Daniel Mink; David John Hyett
Original assignee: Isobionics B.V.
Priority date: 2006-12-05
Filing date: 2007-12-05
Publication date: 2008-06-12

Abstract

The present invention relates to a method for preparing a 4-hydroxy- 2,5-dimethyl-2,3-dihydrofuran-3-one or an analogue thereof, comprising reacting an α-hydroxyaldehyde or an α-ketoaldehyde (compound A) and an α-hydroxyketone (compound B) in the presence of an org a no-catalyst (C) capable of catalysing an aldol reaction, preferably selected from the group consisting of aldolase, proline, pyrrolidine and catalysts comprising a proline active site or a pyrrolidine active site, and forming 4-hydroxy-2,5-dimethyl-2,3-dihydrofuran-3-one or an analogue thereof.

Description

PREPARATION OF 4-HYDROXY-2,5-DIMETHYL-2,3-DIHYDROFURAN-3-ONE

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®, 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. In one approach 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.

Alternatively, 1 ,3-dihydroxyacetone phosphate may be used instead of fructose-1 ,6-bisphosphate. In this method too, a hydrolysis step is required to obtain 6-deoxy-D-fructose or 6-deoxy-L-sorbose.

In a different process, 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). Further, 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.

It is an object of the present invention to provide a novel method for preparing 4-hydroxy-2,5-dimethyl-2,3-dihydrofuran-3-one or an analogue thereof and/or a method to prepare a deoxysugar, such as a deoxyhexose, from which 4-hydroxy- 2,5-dimethyl-2,3-dihydrofuran-3-one or an analogue thereof can be prepared. It is a further object to provide such a method which is simple, in the sense that the method requires relatively few reaction steps.

It is a further object of the present invention to provide a novel method for preparing 4-hydroxy-2,5-dimethyl-2,3-dihydrofuran-3-one or an analogue thereof and/or a method to prepare a deoxysugar, from which 4-hydroxy-2,5-dimethyl- 2,3-dihydrofuran-3-one or an analogue thereof can be prepared, from starting materials which are readily available, in particular readily commercially available.

It is yet a further object to provide a novel method for preparing 4-hydroxy-2,5-dimethyl-2,3-dihydrofuran-3-one or an analogue thereof and/or a method to prepare a deoxysugar, from which 4-hydroxy-2,5-dimethyl-2,3- dihydrofuran-3-one or an analogue thereof can be prepared, which method can be carried out making use of reagents which are considered natural in the food processing industry.

Further, it is an object to provide 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.

One or more other objects which may be solved in accordance with the present invention will be apparent from the remainder of the description and/or claims. It has now been found possible to prepare a deoxysugar respectively

4-hydroxy-2,5-dimethyl-2,3-dihydrofuran-3-one or an analogue thereof by using specific starting material in the presence of a specific catalyst.

Accordingly, the present invention relates to a method for preparing a deoxysugar, in particular a deoxyhexose, comprising reacting an α-hydroxyaldehyde or an α-ketoaldehyde (compound A) and an α-hydroxyketone (compound B) in the presence of an organo-catalyst (C) capable of catalysing an aldol reaction, thereby forming the deoxysugar.

Further, the invention relates to a method for preparing a compound represented by the formula

[I] wherein

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 α-hydroxyaldehyde or an α-ketoaldehyde (compound A) and an α-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.

In particular 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.

Preferred examples of 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.

The term 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. For instance, the deoxysugar may be a deoxyhexose, i.e. a deoxysugar at least conceptually based on a hexose.

In particular, the deoxysugar may be represented by any of the formulas Ma and Mb: -A-

wherein R¹ and R² are independently selected from the group consisting of hydrogen, hydroxyl and hydrocarbons which hydrocarbons may optionally contain one or more heteroatoms, in particular O.

Preferably R¹ and/or R² 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. In a particularly preferred embodiment R¹ and R² are both methyl, R¹ is methyl and R² is hydroxymethyl, or R¹ is hydroxymethyl and R² is methyl.

A method of the invention may in particular be used to prepare a compound selected from 4-hydroxy-2,5-dimethyl-2,3-dihydrofuran-3-one (such as the commercially available Furaneol®), homofuronol (R=methyl, R' is ethyl, R" is hydrogen) or furonol acetate ((R=methyl, R' is methyl, R" is acetate).

Dependent upon the desired product, suitable starting compounds A and B can be selected.

In case 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.

In the framework of the invention with 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 compound¹.

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.

With 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 (α-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. 1982, 104, 7294-7299. Preferably, the aldolase has an E > 5, more preferably an E > 20, most preferably an E > 100. In case 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.

If the catalyst is not substantially stereoselective, the use of a racemic mixture may be preferred for practical reasons; in case the catalyst is substantially stereoselective, for a high yield, the reaction mixture is preferably stereoisomerically enriched in the compound or compounds for which the catalyst shows a higher activity. In such case 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%. In particular, compound A may be an α-hydroxyaldehyde represented by the formula

or an α-ketoaldehyde represented by the formula

wherein R¹ has the above identified meaning; and/or compound B may be an α-hydroxyketone represented by the formula

wherein R² 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).

In a preferred embodiment of the invention, glyceraldehyde is reacted with monohydroxyacetone or lactaldehyde is reacted with 1 ,3-dihydroxyacetone.

In particular, good results have been achieved in a method wherein pyruvaldehyde is reacted with monohydroxyacetone. Not only are these natural compounds that are readily available and relatively cheap, but it has also been found that the deoxysugar, in particular a deoxyhexose, and (if desired) 4-hydroxy-2,5- dimethyl-2,3-dihydrofuran-3-one or a derivative thereof, is formed in a good yield.

The 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.

In principle, 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.

For practical reasons, 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. Preferably the concentration of each of compound A and/or compound B is at least about 25 mM, in particular at least about 50 mM.

If desired, 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.

The reaction of compound A with compound B is carried out in the presence of an organo-catalyst. 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. In principle, 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.

Preferably, 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. In principle, any aldolase can be used. In particular, the enzyme may be any aldolase that is regarded as a natural compound, according to food industry regulations. With 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. In particular, the moiety (enzyme) may be stereoselective.

Preferably 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. Preferably, 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 (FSA₁ EC 4.1.2.).

Further, preferred 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 2 or of SEQ ID NO 4 and homologues thereof. 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 %.

For purpose of the present invention, 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].

Such aldolases can be employed without needing a cofactor. If desired, a reducing agent may be added, to protect the enzyme against oxidation.

Particular preferred is 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. 4) including wild-type enzymes or variants derived from natural mutations or artificial mutagenesis procedures (for example rational design or random mutagenesis) such as FsaA or FsaB variants Leu107Gln, Leu107Asn, and Ala129Ser as single amino acid residue exchanges or combinations of said exchanges), and 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. In particular for such an aldolase or a homologue thereof, 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.

Use can be made of mutants of naturally occurring (wild type) enzymes with aldolase activity in the process according to the invention. 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. Preferably, the aldolase has been isolated from the cells, i.e. a cell-free extract is used. For example, 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 Schϋrmann & Sprenger in J. Biol. Chem. 276 (14), 11055-11061 (2001). Preferably, 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. For practical reasons, 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).

In an embodiment, the organo-catalyst is selected from proline, catalysts comprising a proline-active site, pyrrolidine and catalysts having a pyrrolidine active site. In particular 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

wherein 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.

If several stereoisomers of the catalyst exist, a stereoisomerically enriched catalyst may be used or a racemic mixture. E.g. D-proline, L-proline or a mixture thereof can be used. Thus, 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. Advantages of using 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.

Preferred examples of such 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. Usually, the reaction mixture to form the deoxysugar is slightly acidic, neutral or slightly alkaline. In terms of the apparent pH (pH measured directly to the reaction mixture), the pH is usually chosen in the range of 5-12. Preferably the pH is at least 6. Preferably 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. Preferably, an acid and/or a base is used which is suitable for use in a food or drink. Examples of suitable acids and bases include phosphoric acids and phosphate salts (e.g. NaH₂PO₄, Na₂HPO4, Na₃PO₄), dihydrogencarbonate and carbonate salts (e.g. NaHCO₃ , Na₂CO₃), 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.

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 ⁰C is chosen. Preferably the temperature is up to 60 ⁰C, in particular up to 50 ⁰C in view of the catalyst stability, in particular in case an aldolase is used. A relatively low temperature, in particular of up to 40 ⁰C, more in particular up to 30 ⁰C is especially preferred, because of the low energy consumption, compared to a process wherein the reaction mixture is heated to a higher temperature.

In view of the reaction rate and/or selectivity, the temperature is usually at least about ambient temperature, in particular at least about 20 ⁰C, more in particular at least about 30 ⁰C.

For a high activity a temperature in the range of 37-50 ⁰C 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. In case the compound of formula I is not formed with a sufficient yield, and/or at a sufficient rate in the reaction mixture wherein the organo-catalysed reaction of compound A with compound B takes place, the formation of the compound with formula I may be enhanced by a manner known in the art.

In a preferred embodiment, 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. Suitable acids and bases that may be used to adjust the pH include acids and bases, as described above. In particular 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 ⁰C) of 5 or less. Preferably 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. When using an alkaline solution, the pH is usually at least 8, in particular at least 9, more in particular at least about 11.

Usually 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 ⁰C, in particular at least ambient temperature, e.g. about 20 ⁰C. Usually the temperature is up to 70 ⁰C, in particular up to 50 ⁰C more in particular up to 30 ^CC.

The compound of formula I may be isolated from the reaction mixture in a manner known in the art. In case the compound of formula I that is to be prepared is a compound wherein R" is not hydrogen, 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. In particular, 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. In a preferred embodiment an acetate ester is formed, especially in case R¹ and R² each represent a methyl group. The resultant compound ("Furonol acetate") is in particular suitable as a flavouring ingredient. The invention will now be illustrated by the following examples.

EXAMPLE 1 - Enzymatic synthesis of 4-hvdroxy-2.5-dimethyl-3(2H)-furanone PCR-Amplification of fsa genes from Escherichia coli K12 For the Gateway cloning (Invitrogen, Great-Britain) of the fsaA (bO825) [SEQ ID No. 1] and fsaB (b3946) [SEQ ID No. 2] genes of E. coli K12, encoding the D-fructose 6-phosphate aldolases FsaA (P78055) [SEQ ID No. 3] and FsaB (P32669) [SEQ ID No. 4], gene specific primers containing attB sites were developed [SEQ ID No. 5-8] (attB sequences underlined): fsaA_Eco_for [SEQ ID No. 5]:

5' ACAAGTTTGT ACAAAAAAGC AGGCTAGGAG GAATTAACCA TGGAACTGTA TCTGGATACT TCAGACG 3' fsaA_Eco_rev [SEQ ID No. 6]:

5' ACCCAGCTTT CTTGTACAAA GTGGTTTAAA TCGACGTTCT GCCAAACGCT CC 3' fsaB_Eco_for [SEQ ID No. 7]: 5¹ ACAAGTTTGT ACAAAAAAGC AGGCTAGGAG GAATTAACCA TGGAACTGTA TCTGGACACC GCTAACG 3' fsaB_Eco_rev [SEQ ID No. 8]:

5' ACCCAGCTTT CTTGTACAAA GTGGTTTAGA GATGAGTAGT GCCAAATGCG GC 3¹ These primers were used in four independent PCR reactions for each gene, respectively, with genomic DNA of E. coli K12 as template. Proofreading AccuPrime Pfx DNA Polymerase (Invitrogen, Great-Britain) was used according to the supplier's procedure with an annealing temperature gradient of 52 to 64°C. For all annealing temperatures only specific amplification products of the expected size of about 700 base pairs (bp) were obtained. The fsaA and fsaB amplification products were pooled and purified (QiaQuick PCR purification kit, Qiagen, Hilden, Germany), respectively.

Cloning of fsa genes into expression plasmid pBAD-DEST. The purified PCR products were used in the Gateway BP cloning reactions to insert the target genes into the intermediate cloning vector pDONR201 (Invitrogen) generating the respective entry vectors pENTR-fsaA and pENTR-fsaB. After transformation of competent E. coli DH5α cells (Invitrogen), the resulting transformand clones (> 500 for both fsaA and fsaB) were pooled and the total plasmid DNA isolated (Plasmid DNA Spin Mini Kit, Qiagen). 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.

Overexpression and activity determination of FsaA and FsaB The E coli TOP10 pBAD-fsaA and E. coli TOP10 pBAD-fsaB clones were cultivated in 2.5 ml LB medium (containing 100 μg/ml carbenicillin) at 28°C on a gyratory shaker over night. These precultures were used to inoculate 100 ml expression cultures (LB medium with 100 μg/ml carbenicillin) to a start optical density of OD₆₂o_nm = 0.05. The cultures were incubated at 37°C and horizontal shaking at 180 revolutions per minute (rpm). 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 OD₆₂O ~ 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).

The cell-free extracts (CFEs) were obtained by subsequent sonification in a MSE Soniprep 150 sonificator (small probe, 5 min in an ice/acetone bath), centrifugation for one hour at 4°C and 39,00Ox g, and recovery of the supernatant. The protein content of the CFEs was determined using a modified protein- dye binding method as described by Bradford in Anal. Biochem. 72, 248-254 (1976). Of each sample 50 μl in an appropriate dilution was incubated with 950 μl reagent (100 mg Brilliant Blue G250 dissolved in 46 ml ethanol and 100 ml 85% ortho- phosphoric acid, filled up to 1 ,000 ml with milli-Q water) for at least five minutes at room temperature. The absorption of each sample at a wavelength of 595 nm was measured in a Perkin-Elmer Lambda20 UV/VIS spectrometer. Using a calibration line determined with solutions containing known concentrations of bovine serum albumin (BSA, ranging from 0.025 mg/ml to 0.25 mg/ml) the protein concentration in the samples was calculated. The fructose 6-phosphate aldolase activity in the cell-free extracts was determined using a spectro-photometrical assay as described by Schϋrmann & 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 KH₂PO₄, 12.54 g/l K₂HPO₄, pH 7.0 containing 100 μg/ml carbenicillin) in an ISF-200 laboratory fermentor (Infors). For the inoculation of the fermentor an over night starter culture (20 h, 28°C, 180 rpm) in 0.5 I TB was used, which itself had been inoculated with colonies of E. coli TOP 10 freshly transformed with pBAD-fsaB .

The expression of the fsaB gene was induced by addition of 0.1% (w/v) L-arabinose at a cell density of OD₆₂₀ = 1.02 (after 1.5 h). After about 6 hours of cultivation (OD₆₂₀ = 15.6) the cells were harvested by centrifugation (12 minutes at 12,227x g at 5°C). The wet cells (310 g) were washed with 0.1 M K-phosphate buffer (pH 7.0) and resuspended in 930 ml of the same buffer. Cells were disrupted in a nanojet homogeniser (Haskel) at 1300 bar (3 runs of 30 min) and subsequently centrifuged (35,00Ox g for 45 min at 4°C) to obtain the cell-free extract (supernatant). Part of the CFE was frozen and stored at -20⁰C until further use. The other part was kept at 4°C for the direct use.

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. co// TOP 10 pBAD-fsaB containing 33.5 U of FSA activity (measured as described above with D-fructose 6-phosphate as substrate) at room temperature in a total volume of 5 ml with a 50 mM Na-phosphate buffer pH 7.5 containing 1 mM dithiothreitol (DTT). As control reactions identical reactions with FsaB but containing only one of the substrates hydroxyacetone or pyruvaldehyde as well as reactions containing both substrates, in which the cell-free extract containing FsaB was replaced by an equal amount of 50 mM Na-phosphate buffer pH 7.5 plus 1 mM DTT or by a cell-free extract of E. co// TOP 10 pBAD/Myc-HisC (plasmid without insert from Invitrogen), were set up. The reactions were incubated in a screw cap glass bottles with stirring using magnetic stirring bars. After 3 days samples of the reactions were taken, diluted in 2OmM K₂HPO₄ (pH 3.0) and analysed by HPLC on an lnertsil ODS-3- column (Varian) for the formation of 4-hydroxy-2,5-dimethyl-3(2H)-furanone using a DAD detector / UV detector at 280 nm with commercial 4-hydroxy-2,5-dimethyl-3(2H)-furanone (Fluka) as reference material.

The formation of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (0.74 g/l) could only be detected in the reaction containing FsaB and both substrates hydroxyacetone and pyruvaldehyde. The smell of 4-hydroxy-2,5-dimethyl-3(2H)- furanone was also clearly noticeable from the reaction mixture.

EXAMPLE 2 - Synthesis 4-hvdroxy-2.5-dimethyl-3(2H)-furanone with proline

Hydroxyacetone and pyruvaldehyde (each 100 g/l) were incubated with 10 mol% (with respect to both substrates) of L-proline in 5 ml of total volume with tetrahydro-furan (THF) or dimethyl-sulphoxide (DMSO) as solvents. As control reactions identical reactions with L-proline but containing only one of the substrates hydroxyacetone or pyruvaldehyde as well as reactions with both substrates but without L-proline were set up. The reactions were incubated in a screw cap glass bottles with stirring using magnetic stirring bars. After 3 days samples of the reactions were taken, diluted in diluted in 2OmM K₂HPO₄ (pH 3.0) and analysed by HPLC on an lnertsil ODS- 3- column (Varian) for the formation of 4-hydroxy-2,5-dimethyl-3(2H)-furanone using a DAD detector/ UV detector at 280 nm with commercial 4-hydroxy-2,5-dimethyl-3(2H)- furanone (Fluka) as reference material.

The formation of significant amounts of 4-hydroxy-2,5-dimethyl-3(2H)- furanone could only be detected in the reactions containing L-proline and both substrates hydroxyacetone and pyruvaldehyde. In THF 0.58 g/l and in DMSO 0.27 g/l of 4-hydroxy-2,5-dimethyl-3(2H)-furanone were detected.

EXAMPLE 3 - Synthesis 4-hvdroxy-2,5-dimethyl-3(2H)-furanone with pyrrolidine

Hydroxyacetone and pyruvaldehyde (each 100 g/l) were incubated with 10 mol% (with respect to both substrates) of L pyrrolidine in 5 ml of total volume with tetrahydro-furan (THF) as solvent. As control reactions identical reactions with pyrrolidine but containing only one of the substrates hydroxyacetone or pyruvaldehyde as well as a reaction with both substrates but without pyrrolidine were set up. The reactions were incubated in a screw cap glass bottles with stirring using magnetic stirring bars. After 3 days samples of the reactions were taken, diluted in diluted in

2OmM K₂HPO₄ (pH 3.0) and analysed by HPLC on an lnertsil ODS-3- column (Varian) for the formation of 4-hydroxy-2,5-dimethyl-3(2H)-furanone using a DAD detector / UV detector at 280 nm with commercial 4-hydroxy-2,5-dimethyl-3(2H)-furanone (Fluka) as reference material. The formation of significant amounts of 4-hydroxy-2,5-dimethyl-3(2H)- furanone (0.46% (w/v)) could only be detected in the reactions containing pyrrolidine and both substrates hydroxyacetone and pyruvaldehyde.

Claims

Method for preparing a compound represented by the formula

wherein

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 a hydrocarbon moiety selected from C1-C12 alkyl and C1-C12 hydroxyalkyls, more in particular selected from C1-C6 alkyl and

C1-C6 hydroxyalkyls; and

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 α-hydroxyaldehyde or an α-ketoaldehyde (compound A) and an α-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.

Method according to claim 1 , wherein the catalyst is selected from the group consisting of aldolases, proline, pyrrolidine and catalysts comprising a proline active site or a pyrrolidine active site.

Method according to claim 2, wherein the aldolase is selected from the group consisting of aldolases from EC-class 4.1.2, preferably from the group consisting of FsaA from Escherichia coli K12 (SEQ ID No. 2) or FsaB from

Escherichia coli K12 (SEQ ID No. 4) and homologues thereof having a sequence identity of at least 60% with SEQ ID No. 2 or with SEQ ID No. 4.

Method according to any of the preceding claims wherein compound A is a α-hydroxyaldehyde represented by the formula

or wherein compound A is a α-ketoaldehyde is represented by the formula

wherein R¹ is selected from the group consisting of C1-C12 alkyls and C1-C12 hydroxyalkyls, preferably from the group consisting of C1-C6 alkyls and C1-C6 hydroxyalkyls, more preferably from the group consisting of C1-C3 alkyls and C1-C3 hydroxyalkyls, in particular methyl. Method according to any of the preceding claims, wherein the α-hydroxyketone is represented by the formula

wherein R² is selected from the group consisting of C1-C12 alkyls and C1-C12 hydroxyalkyls, preferably from the group consisting of C1-C6 alkyls and C1-C6 hydroxyalkyls, more preferably from the group consisting of C1-C3 alkyls and C1-C3 hydroxyalkyls, in particular methyl. Method according to any of the preceding claims, wherein the α-hydroxyaldehyde or the α-ketoaldehyde (compound A) and the α-hydroxyketone (compound B) is reacted with the organo-catalyst (C) capable of catalysing an aldol reaction, thereby forming a deoxysugar; and forming the compound according to formula I from the deoxysugar by cyclisation and elimination, and wherein the cyclisation and elimination reaction is preferably carried out in water or an aqueous solution, in particular an acidic aqueous solution or an alkaline aqueous solution.

7. Method according to any of the preceding claims, wherein a compound of according to formula I - wherein R" is hydrogen - is prepared and thereafter the compound is esterified with a carboxylic acid, in particular a carboxylic acid selected from formic acid, acetic acid and propionic acid.

8. Method according to any of the preceding claims, wherein the compound represented by formula I is selected from 4-hydroxy-2,5-dimethyl-2,3- dihydrofuran-3-one, homofuronol and furonol acetate. 9. Method for preparing a deoxysugar, in particular a deoxyhexose, comprising reacting an α-hydroxyaldehyde or an α-ketoaldehyde (compound A) and an α-hydroxyketone (compound B) in the presence of a organo-catalyst (C) capable of catalysing an aldol reaction, thereby forming the deoxysugar. 10. Method according to claim 9, wherein compound A is as defined in claim 4, compound B is as defined in claim 5 and/or the organo-catalyst is as defined in claim 2 or claim 3.