WO2007068498A1

WO2007068498A1 - Process for the preparation of delta lactones

Info

Publication number: WO2007068498A1
Application number: PCT/EP2006/012141
Authority: WO
Inventors: Daniel Mink; Michael Wolberg; Martin SCHÜRMANN; Iris Hilker
Original assignee: Isobionics B.V.
Priority date: 2005-12-15
Filing date: 2006-12-15
Publication date: 2007-06-21
Also published as: EP1969131A1

Abstract

The invention relates to a process for the preparation of δ-lactones by reacting acetaldehyde and a substituted acetaldehyde in the presence of an aldolase, preferably a DERA from Escherichia coli, from Bacillus subtilis or from a Geobacillus species, δ-lactones, such as saturated δ-lactones, α, β-unsaturated δ-lactones and 3-hydroxy- δ-lactones can suitably be used as (intermediates in the production of) flavors and/or fragrances and/or pharmaceuticals.

Description

PROCESS FOR THE PREPARATION OF DELTA LACTONES.

The invention relates to a process for the preparation of enantiomerically enriched δ-lactones. The invention also relates to use of said δ- lactones in food or cosmetics or as pharma intermediates. δ-lactones, such as saturated δ-lactones, α, β-unsaturated δ-lactones and 3-hydroxy- δ-lactones can suitably be used as (intermediates in the production of) flavors and/or fragrances and/or pharmaceuticals.

For example, the α, β-unsaturated (-)-(R)-massoialactone is useful as a flavoring agent with a butter and milk-like flavor (JP 08002891 B4, JP 07080867 B4). It is used as a flavor enhancer (JP 3523127 B2) and as an antibacterial agent (KR 2000066367 A). The saturated (+)-(R)-δ-decalactone is for example used as flavor in dairy products, beverages, dentifrice, chewing gum, food and as part of fragrance compositions for colognes, perfumed articles, detergents, fabric softeners and hair preparations (US 6271194 B1 ). It is used as a fungal growth inhibitor (US 6060507 A). The α, β-unsaturated (-)-(R)-dodec-2-enolactone, for example, is a flavor-enhancer of sweet fermentation flavor for traditional Japanese food (JP 3523127 B2). Saturated (+)- (R)-δ-dodecalactone, for example, is a widely used flavor and is part of fragrance compositions (US 6271194 B1 ). The α, β-unsaturated δ-pentadec-2-enolactone, for example, is a testosterone-5a-reductase inhibitor and used as such in hair growth stimulants (JP 2525179 B2).

Several processes for the preparation or isolation of δ-lactones are known in the art.

For example, some δ-lactones can be produced via chemical synthesis, for example, the compound ((-)-(R)-massoialactone) of formula (I)

can be prepared via the asymmetric allylboration of appropriate aldehydes to form acrylic esters of homoallylic alcohols, which after refluxing in dichloromethane in the presence of Grubbs' catalyst provided the natural enantiomer of amongst others (R)-(-)-massoia lactone (P. Ramachandran et al., Tetrahedron Letters 41 (2000), 583- 586)).

A general synthesis route for the preparation of racemic compounds of formula (II)

wherein R² stands for CH₃, CH₃(CH₂)₂, CH₃(CH₂)₄, CH₃(CH₂)₆ was described by A. Nobuhara, Agr. Biol. Chem., (1968) vol. 32, no. 8, p1016-1020. In this route, the corresponding carbonyl compound was reacted in several steps (e.g. Reformatsky reaction, Grignard reaction, reduction etc.) to form the desired racemic compound of formula (3).

There are also examples of processes, wherein δ-lactones were isolated from natural sources. For example, a compound ( (-)-(R)- δ-Oct-2-enolactone) of formula (III)

was isolated from extracts from the seedling roots and stems of Cryptocarya ashersoniana (M. A. G. Ricardo et al., ARKIVOC (2004) (vi), 127-136). Other examples of δ-lactones isolated from natural sources are 2-decen-5-olide and 2-dodecen-5-olide, which can be isolated from the bark of Cryptocaria massoia. Subsequently these compounds were hydrogenated by Saccharomyces cerevisiae to form 5-decanolide and 5-dodecanolide (N. ter Burg et al., (1994) Developments in Food Science, vol. 35, Trends in flavour research, p. 481-486).

Many of these processes have one or more of the following disadvantages: many steps are required, yields are low, expensive and/or hazardous reagents (for example resolving agents or (chiral) precursors, catalysts or metalorganic intermediates) are necessary, natural sources are scarce, it is difficult to obtain enantiomerically enriched products, hence making these processes economically unattractive.

Therefore, it is the object of the invention to provide an improved process for the preparation of enantiomerically enriched δ-lactones.

This object is achieved by a process comprising the step of a) reacting acetaldehyde and a substituted acetaldehyde of formula HC(O)CH₂R, wherein R stands for an alkyl of at least 5 C-atoms, a cycloalkyl, an alkenyl, an alkynyl or for an aryl, wherein R may be substituted, in the presence of an aldolase to form the corresponding enantiomerically enriched compound of formula (1 )

It has surprisingly been found that by using the process of the invention, δ-lactones can be prepared with a high enantiomeric excess (e.e.) without the need for expensive resolution agents or chiral precursors.

Furthermore, the yield obtained is usually high. Moreover, the process can be performed in water, making this process very attractive from an environmental point of view. Also, the δ-lactones may be prepared from readily available and cost effective starting materials.

R preferably stands for an alkyl of at most 20 C-atoms, more preferably of at most 12 C-atoms; a cycloalkyl of at least 3 and at most 7 C-atoms, preferably at least 5 C-atoms, more preferably of at most 6 C-atoms; an alkenyl of at most 20 C-atoms, preferably at least 5 C-atoms, more preferably of at most 12 C- atoms; an alkynyl of at most 20 C-atoms, preferably at least 5 C-atoms, more preferably of at most 12 C-atoms; or for phenyl. The alkyl, alkenyl or alkynyl may be branched, but is preferably linear. Examples of linear alkyls in the definition of R include n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl. Examples of branched alkyls in the definition of R include 1 -methyl-butyl, 2-methyl-butyl, 3-methyl-butyl, 1-methyl-pentyl, 2- methyl-pentyl, 3-methyl-pentyl, 4-methyl-pentyl, 1 -ethyl-butyl, 2-ethyl-butyl, 1-methyl- hexyl, 2-methyl-hexyl, 3-methyl-hexyl, 4-methyl-hexyl, 5-methyl-hexyl, 1-ethyl-pentyl, 2- ethyl-pentyl, 3-ethyl-pentyl. Examples of linear alkenyls in the definition of R include pent-1-enyl, pent-2-enyl, pent-3-enyl, pent-4-enyl etc. Examples of branched alkenyls in the definition of R include 1-methyl-but-1-enyl, 2-methyl-but-1-enyl, 3-methyl-but-1- enyl, 1-methyl-but-2-enyl, 2-methyl-but-2-enyl, 3-methyl-but-2-enyl, 1-methyl-but-3- enyl, 2-methyl-but-3-enyl, 3-methyl-but-3-enyl, 1-methyl-pent-1-enyl, 2-methyl-pent-1- enyl, 3-methyl-pent-1-enyl, 4-methyl-pent-1-enyl, 1-methyl-pent-2-enyl, 2-methyl-pent- 2-enyl, 3-methyl-pent-2-enyl, 4-methyl-pent-2-enyl, 1-methyl-pent-3-enyl, 2-methyl- pent-3-enyl, 3-methyl-pent-3-enyl, 4-methyl-pent-3-enyl, 1-methyl-pent-4-enyl, 2- methyl-pent-4-enyl, 3-methyl-pent-4-enyl, 4-methyl-pent-4-enyl, 1-ethyl-buM-enyl, 2- ethyl-but-1-enyl, 1-ethyl-but-2-enyl, 2-ethyl-but-2-enyl, 1-ethyl-but-3-enyl, 2-ethyl-but-3- enyl. Examples of linear alkynyls in the definition of R include pent-1-ynyl, pent-2-ynyl, pent3-ynyl, pent-4-ynyl etc. Preferably R stands for a linear alkyl, a linear alkenyl or for phenyl. For purpose of this invention, in the calculation of the amount of C-atoms in a group represented by R, C-atoms of possible substituents are not included.

Examples of substituents, if R stands for an alkyl, alkenyl or alkynyl include: alkyls, for example methyl, ethyl; alkoxy groups, for example methoxy, ethoxy; oxo, halogens, for example Cl, F, Br, I; aryl, for example phenyl; cycloalkyl, for example cyclohexyl; Examples of substituents, if R stands for an aryl, preferably phenyl; or if R stands for a cycloalkyl, preferably cyclohexyl; include: alkyl, preferably an alkyl of 1-3 C-atoms, for example methyl or ethyl; alkoxy, for example methoxy, ethoxy; halogens, for example Cl, F, Br, I; hydroxy; NO₂.

In the framework of the invention with 'aldolase' (an enzyme with aldolase activity) is meant an enzyme having the ability to catalyse an aldol condensation between two aldehydes or between an aldehyde and a ketone.

Preferably the aldolase used is 2-deoxy-D-ribose 5-phosphate aldolase (DERA, EC 4.1.2.4) or mutants hereof, more preferably DERA from Escherichia coli or mutants hereof. The amino acid sequence of DERA from Escherichia coli K12 is given in SEQ ID No. 1 (SEQ ID No. 1 : wild-type DERA from Escherichia coli K12). The gene sequence from Escherichia coli K12 encoding this amino acid sequence is given in SEQ ID No. 2. (SEQ ID No. 2: wild-type deoC gene from Escherichia coli K12). Preferably, the aldolase used in the process of the present invention is a DERA from Escherichia coli, Bacillus subtilis or from a Geobacillus species, or a mutant thereof. An example of an aldolase from a Geobacillus species or a mutant thereof is disclosed in Greenberg, W. A. et al, 2004, PNAS, 5788-5793.

Wild-type enzymes are enzymes as can be isolated from natural sources or environmental samples; naturally occurring mutants of such enzymes, i.e. mutants as also can be isolated from natural sources or environmental samples, within the scope of this patent application are also considered to be wild-type enzymes. The term mutants, for this patent application, therefore solely will intend to indicate that they have been or are being obtained from wild-type enzymes by purposive mutations of the DNA (nucleic acid) encoding said wild-type enzymes (whether by random mutagenesis, for instance with the aid of PCR or by means of UV irradiation, or by site-directed mutation, e.g. by PCR methods, saturation mutagenesis etc. as are well-known to the skilled man, optionally with recombination of such mutations). Mutants of the aldolase may have improved properties, for example with respect to selectivity for the substrate and/or activity and/or stability and/or solvent resistance and/or pH profile and/or temperature profile.

If a mutant aldolase is used, preferably the mutant has at least one amino acid substitution at one or more of the positions K13, T19, Y49, N80, D84, A93, E127, A128, K146, K160, 1166, A174, M185, K196, F200, or S239 in SEQ ID No.1 , or at positions corresponding thereto, preferably at position F200 or at a position corresponding thereto, and/or a deletion of at least one amino acid at one of the positions S258 or Y259 in SEQ ID No.1 , optionally in combination with C-terminal extension, preferably by one of the fragments TTKTQLSCTKW (SEQ ID No.11 , C- terminal extension 2) and KTQLSCTKW [SEQ ID No.13, C-terminal extension 3] and/or in combination with N-terminal extension. An example of a nucleic acid sequence encoding SEQ ID No.1 1 is given in SEQ ID No. 12 (SEQ ID No. 12, coding sequence for C-terminal extension 2). An example of a nucleic acid sequence encoding SEQ ID No. 13 is given in SEQ ID No. 14 (SEQ ID No. 14, coding sequence for C-terminal extension 3).

Amino acid residues of other aldolase sequences corresponding to positions of the amino acid residues in the wild-type amino sequence of the E. coli K12 DERA [SEQ ID No.1] 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 of the E. coli K12 wild-type DERA sequence as given in [SEQ ID No.1] in such alignments are defined to be positions corresponding to this respective amino acid residue of the E. coli K12 wild-type DERA [SEQ ID No.1].

As used herein, the amino acids in the sequences and at the various positions therein, are indicated by their one letter code (respectively by their three letter code) as follows:

Most preferably, the mutant aldolase used has at least one of the amino acid substitutions in, or corresponding to the substitutions in SEQ ID No.1 selected from the group consisting of: a. K13 and/or K196 replaced by a positively charged amino acid, preferably by R or H; b. T19 and/or M185 replaced by another amino acid, preferably by another amino acid selected from the groups consisting of hydrophilic amino acids, in particular consisting of S, T, C, Q, and N, and/or hydrophobic amino acids, in particular consisting of V, L and I; c. Y49 replaced by an aromatic amino acid selected from the group consisting of F and W; d. N80 and/or 1166 and/or S239 replaced by another amino acid selected from the group of hydrophilic amino acids consisting of T, S, C, Q and N; e. D84 and/or A93 and/or E127 replaced by another, preferably smaller, amino acid selected from the group of small amino acids consisting of, in order of decreasing size, E, T, N, P, D, C, S, A, and G; f. A128 and/or K146 and/or K160 and/or A174 and/or F200 replaced by another amino acid selected from the group of hydrophobic amino acids consisting of I, L, M, V, F, and Y; and/or has a deletion of at least one amino acid at the positions S258 and Y259 in SEQ ID No.1 or at positions corresponding thereto, optionally in combination with C-terminal extension, preferably by one of the fragments TTKTQLSCTKW [SEQ ID No.11] and KTQLSCTKW [SEQ ID No.13] and/or in combination with N-terminal extension. If a mutant aldolase is used, the mutant aldolase used may be truncated by deletion of at least one amino acid residue from SEQ ID No. 1 , e.g. by deletion of S258 and/or Y259 or of positions corresponding thereto and then extended, preferably by one of the fragments TTKTQLSCTKW [SEQ ID No.11] and KTQLSCTKW [SEQ ID No.13].

If a mutant aldolase is used, even more preferably, the mutant aldolase has one or more of the mutations in, or corresponding to the mutations in SEQ ID No.1 selected from the group of K13R, T19S, Y49F, N80S, D84G, A93G, E127G, A128V, K146V, K160M, I166T, A174V, M185T, M185V, K196R, F200I, F200M, F200V, S239C, ΔS258, ΔY259, C-terminal extension by TTKTQLSCTKW [SEQ ID No.11], and C-terminal extension by KTQLSCTKW [SEQ ID No.13]. If a mutant aldolase is used, preferably, the mutant aldolase has at least the following two mutations in, or corresponding to the two mutations in SEQ ID No. 1 selected from the group of F200I and ΔY259; F200M and ΔY259; F200V and ΔY259; F200I and C-terminal extension by KTQLSCTKW [SEQ ID No.13]; F200M and C-terminal extension by KTQLSCTKW [SEQ ID No.13]; and F200V and C-terminal extension by KTQLSCTKW [SEQ ID No.13];

The amino acid sequence of Escherichia coli K12 DERA, wherein the F200I position is mutated is given in SEQ ID No. 3 (SEQ ID No. 3: F200I mutant of Escherichia coli K12 DERA). The aldolase used in the process of the present invention is preferably the aldolase of SEQ ID No. 3. An example of a gene encoding the amino acid sequence of SEQ ID No. 3 is given in SEQ ID No. 4 (SEQ ID No. 4: mutated cfeoC gene encoding F200I mutant of Escherichia coli K12 DERA).

The amino acid sequence of Escherichia coli K12 DERA, wherein the F200I position is mutated and the Y259 position is deleted is given in SEQ ID No. 5 (SEQ ID No. 5: F200I/ΔY259 mutant of Escherichia coli K12 DERA). An example of a gene encoding the amino acid sequence of SEQ ID No. 5 is given in SEQ ID No. 6 (SEQ ID No. 6: mutated deoC gene encoding F200I/ΔY259 mutant of Escherichia coli K12 DERA).

The amino acid sequence of Escherichia coli K12 DERA, wherein the F200I position is mutated and wherein the C-terminus is extended by the amino acid sequence of SEQ ID No. 13 is given in SEQ ID No. 7 (SEQ ID No. 7: F200I and C- terminal extension 3 mutant of Escherichia coli K12 DERA). An example of a gene encoding the amino acid sequence of SEQ ID No. 7 is given in SEQ ID No. 8 (SEQ ID No. 8: mutated deoC gene encoding F200I and C-terminal extension 3 mutant of Escherichia coli K12 DERA). The amino acid sequence of Escherichia coli K12 DERA, wherein the

F200I position is mutated, the S258 and Y259 positions are deleted and wherein the C- terminus is extended by the amino acid sequence of SEQ ID No. 11 is given in SEQ ID No. 9 (SEQ ID No. 9: F200I, ΔS258, ΔY259 and C-terminal extension 2 mutant of Escherichia coli K12 DERA). An example of a gene encoding the amino acid sequence of SEQ ID No. 9 is given in SEQ ID No. 10 (SEQ ID No 10: mutated deoC gene encoding F200I, ΔS258, ΔY259 and C-terminal extension 2 mutant of Escherichia coli K12 DERA).

The aldolase (wild type or mutant) may be used in any form. For example, the aldolase may be used - for example in the form of a dispersion, emulsion, 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 aldolase obtained from its source by a combination of known purification methods, in whole (optionally pemneabilized and/or immobilized) cells that naturally or through genetic modification possess aldolase activity, or in a lysate of cells with such activity etc. etc.

The process of the invention may for example be performed using the reaction conditions as described in US 5,795,749, for instance in column 4, lines 1-18 or for instance using fed-batch reaction conditions as described in W. A. Greenberg et a/., PNAS, vol. 101 , pp 5788-5793, (2004). For 2-substituted aldehydes having a low solubility in water, it may be advantageous to feed those 2-substituted aldehydes to the reaction mixture. Also, it may be possible to feed acetaldehyde to the reaction mixture. Preferably both acetaldehyde and the 2-substituted aldehyde are fed to the reaction mixture.

Preferably, the process of the invention is performed under the reaction conditions as described in WO03/006656: the carbonyl concentration, that is the sum of the concentration of acetaldehyde, aldehyde of formula HC(O)CH₂R¹ and the intermediate product formed in the reaction between the aldehyde and the aldehyde of formula HC(O)CH₂R¹, is preferably held at a value below 6 moles/l during the synthesis process. It will be clear to one skilled in the art that slightly higher concentrations for a (very) short time will have little effect. More preferably, the carbonyl concentration is chosen between 0.01 and 5 moles per liter of reaction mixture, most preferably between 0.05 and 4 moles per liter of reaction mixture.

The reaction temperature and the pH are not critical and both are chosen as a function of the substrate. Preferably the reaction is carried out in the liquid phase. The reaction can be carried out for example at a reaction temperature between -5 and +50⁰C, preferably between 0 and 40⁰C, more preferably between 5 and 37°C; and at a pH between 5.5 and 9, preferably between 6 and 8.

The reaction is preferably carried out at more or less constant pH, use for example being made of a buffer or of automatic titration. As a buffer for example sodium and potassium bicarbonate, sodium and potassium phosphate, triethanolamine/HCI, bis-tris-propane/HCI and HEPES/KOH can be applied. Preferably a potassium or sodium bicarbonate buffer is applied, for example in a concentration between 10 and 400 mmoles/l of reaction mixture.

The molar ratio between the total quantity of aldehyde and the total quantity of 2-substituted aldehyde is not very critical and preferably lies between 1.5:1 and 4: 1 , in particular between 1.8:1 and 2.2: 1.

The amount of aldolase used in the process of the invention is in principle not critical. It is routine experimentation to determine the optimal concentration of enzyme for an enzymatic reaction and so the person skilled in the art can easily determine the amount of aldolase to be used.

It is also possible to add additives such as for example surfactants or cosolvents to the reaction mixture. For example surfactants may be used to enhance the reaction rate. Examples of surfactants include anionic surfactants, for example linear alkyl sulphates, in particular sodium dodecylsulphate (SDS) or sodium hexadecylsulphate (SHS); cationic surfactants, for instance cetyl trimethylammoniumbromide (CTAB); and nonionic surfactants, for instance ethoxylated alcohols, in particular tetra(ethyleneglycol) tetradecylether. Also, cosolvents may be added to help solubilize the substrate. Examples of cosolvents include dimethylsulphoxide, dimethylformamide, methanol, ethanol, isopropanol, tert- butylmethyl ether, i-butyl-methylketone.

In a preferred embodiment of the invention, step a) of the process of the present invention is performed in an emulsion. This may especially be advantageous in case the substrates which should be converted have a low solubility in water. For purpose of the present invention, an emulsion is defined as a ternary mixture of water, a surfactant and an oil phase, which may be an aliphatic alkane.

Examples of aliphatic alkanes which may be used as oil phase in an emulaion include: cyclohexane, isooctane, tetradecane, hexadecane, octadecane, squalane. The oil-in- water (O/W) emulsion is formed by intense mixing which leads to an increased internal surface and thus facilitates mass transfer between the phases. Especially interesting emulsions are microemulsions that are thermodynamically stable and have a domain size in the nanometer range (see for instance Clapes et al., Chem. Eur. J. 2005, 11 , 1392-1401 and Schwuger et al., Chem. Rev. 1995, 95, 849-864.).

The enantiomerically enriched compound of formula (1) may be oxidized to form the corresponding enantiomerically enriched compound of formula (5)

wherein R is as defined above.

Therefore, the invention also relates to a process comprising the steps of a) reacting acetaldehyde and a substituted acetaldehyde of formula HC(O)CH₂R, wherein R stands for an alkyl of at least 5 C-atoms, a cycloalkyl, an alkenyl, an alkynyl or for an aryl, wherein R may be substituted, in the presence of an aldolase to form the corresponding enantiomerically enriched compound of formula (1)^"

and b) oxidizing the enantiomerically enriched compound of formula (1 ) by a suitable oxidizing agent to form the corresponding enantiomerically enriched compound of formula (5) wherein R stands for an alkyl of at least 5 C-atoms, a cycloalkyl, an alkenyl, an alkynyl or for an aryl as described above, wherein R may be substituted as described above.

As a suitable oxidizing agent to be used in the oxidization of the enantiomerically enriched compound of formula (1), in principle all oxidizing agents known to the skilled person to be applicable in the oxidation of an alcohol to a ketone can be applied. For example, the enantiomerically enriched compound of formula (1) may be oxidized by peroxides or by other oxidizing agents. Examples of peroxides include hydrogen peroxide and tert-butyl hydroperoxide. Examples of other oxidizing agents include peracids (e.g. meta-chlorobenzoic peracid or peracetic acid), hypochlorites (e.g. bleach or t-butyl hypochlorite), perborates, N-oxides, permanganates, chromates, for instance K₂Cr₂O₇; halogens, for instance Br₂ and Cl₂; chlorates, for instance KCIO₃ ; bromates, for instance KBrO₃; perchlorates, periodates; peroxymonosulphates, for instance K₂HSO₅ (e.g. oxone®); peroxodisulphates, NiO₄ and air/oxygen. Preferably, the oxidizing agent used in the process of the invention is Br₂. If desired, the oxidation may be carried out in the presence of an appropriate catalyst, such as for example salts or oxides of the metals V, Ce, Mn, Ni, Fe, Cu, Os, Mo, W, Re, or Ru; or organic catalysts, for example isobutyraldehyde in the case of air/oxygen or for example tetramethylpiperidine N-oxide (TEMPO) in the case of bleach. The oxidation by peroxides or other oxidizing agents is generally performed in a solvent, such as for instance dichloromethane, chloroform, 1 ,2- dichloroethane, methanol, ethanol, 2-propanol, acetonitrile, acetic acid, toluene, water, N-methyl-2-pyrrolidone (NMP), dimethylsulphoxide (DMSO), dimethylformamide (DMF), tetrahydrofurane (THF), or tert-butyl methyl ether (TBME). It is also possible to use biphasic solvent systems consisting of an aqueous phase and an organic phase in the presence of a phase-transfer catalyst, such as for instance quarternary ammonium salt or quarternary phosphonium salt (for instance tetraalkylammonium halide, e.g. tetrabutylammonium bromide) or crown ether (for example 18-crown-6). Another class of suitable solvents comprises ionic liquids such as, for example, 1 ,3-dialkyl imidazolium salts or N-alkyl pyridinium salts of acids such as for example hexafluorophosphoric acid or tetrafluoroboric acid or trifluoromethane sulphonic acid, or with (CF₃SO₂)₂N^' as anionic counterpart.

Preferably, in the oxidation of an enantiomerically enriched compound of formula (2) a reaction temperature of at least -20⁰C is applied. More preferably, a temperature of at least O⁰C is applied, even more preferably a temperature between

18-25°C is applied. Preferably, the oxidation of an enantiomerically enriched compound of formula (2) is performed at a temperature lower than 150⁰C, more preferably lower than 10O⁰C, even more preferably lower than 6O⁰C, most preferably lower than 40⁰C. In principle the optimal amount of oxidizing agent used for the oxidation of the enantiomerically enriched compound of formula (1 ) can be easily obtained by the person skilled in the art through routine experimentation. Preferably, the molar amount of oxidizing agent used is at least 0.5 with respect to the amount of compound of formula (1), more preferably at least 1 with respect to the amount of compound of formula (1). Preferably, the molar amount of oxidant used is lower than 20 with respect to the amount of compound of formula (1), more preferably lower than 10 with respect to the amount of compound of formula (1), most preferably lower than 5 with respect to the amount of compound of formula (1).

In another aspect, the invention relates to a process comprising the steps of: a) reacting acetaldehyde and a substituted acetaldehyde of formula HC(O)CH₂R¹, wherein R¹ stands for H, an alkyl, a cycloalkyl, an alkenyl, an alkynyl or for an aryl, wherein R¹ may be substituted, in the presence of an aldolase to form an enantiomerically enriched compound of formula (6)

and b) further oxidizing the enantiomerically enriched compound of formula (6) by a suitable oxidizing agent to form the corresponding enantiomerically enriched compound of formula (7)

and c) dehydrating the enantiomerically enriched compound of formula (7) to form the corresponding enantiomerically enriched compound of formula (3)

R¹ preferably stands for an alkyl of 1 - 20 C-atoms, more preferably at least 5, and also more preferably of at most 12 C-atoms; a cycloalkyl of at least 3 and at most 7 C-atoms, more preferably of at most 6 C-atoms; an alkenyl of at most 20 C-atoms, at least 2 C-atoms, more preferably of at most 12 C-atoms; an alkynyl of at most 20 C-atoms, more preferably of at most 12 C-atoms; or for phenyl. Preferably, if used in the process as described above, R¹ stands for an alkyl, alkenyl or alkynyl chain of at least 2, 3, 4 or of at least 5 C-atoms. The alkyl, alkenyl or alkynyl may be branched, but is preferably linear. Examples of linear alkyls include ethyl, n-propyl, n- butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl. Examples of branched alkyls include i-propyl and i-butyl Examples of linear alkenyls include but-1-enyl, but-2-enyl, but-3- enyl, pent-1-enyl, pent-2-enyl, pent-3-enyl, pent-4-enyl etc. Examples of branched alkenyls include i-propenyl, i-but-1-enyl, i-but-2-enyl. Examples of linear alkynyls include but-1-ynyl, but-2-ynyl, but-3-ynyl, pent-1-ynyl, pent-2-ynyl, pent3-ynyl, pent-4- ynyl etc. Preferably R¹ stands for a linear alkyl, a linear alkenyl or for phenyl. For purpose of this invention, in the calculation of the amount of C-atoms in a group represented by R¹, C-atoms of possible substituents are not included.

Examples of substituents, if R¹ stands for an alkyl, alkenyl or alkynyl include: alkyls, for example methyl, ethyl; alkoxy groups, for example methoxy, ethoxy; oxo, halogens, for example Cl, F, Br, I; aryl, for example phenyl; cycloalkyl, for example cyclohexyl; Examples of substituents, if R¹ stands for an aryl, preferably phenyl; or if R¹ stands for a cycloalkyl, preferably cyclohexyl; include: alkyl, preferably an alkyl of 1-3 C-atoms, for example methyl or ethyl; alkoxy, for example methoxy, ethoxy; halogens, for example Cl, F, Br, I; hydroxy; NO₂.

In principle all methods for the dehydration of alcohols known to the person skilled in the art may be used for the dehydration of the enantiomerically enriched compound of formula (7). For example, the dehydration may be performed by heating the enantiomerically enriched compound of formula (7). Preferably, the compound of formula (7) is dehydrated by heating in the presence of a dehydration catalyst, for instance an Lewis or Brønsted acid or a base. Preferably, the compound of formula (7) is dehydrated by heating in the presence of an acid, more preferably in the presence of a strong acid, that is an acid with a pKa of 4 or smaller. Examples of Brønsted acids which may be used for dehydration include hydrochloric acid, sulphuric acid, methanesulphonic acid, oxalic acid, formic acid, acetic acid, trifluoro acetic acid, p-toluenesulphonic acid and boric acid. Examples of Lewis acids and other dehydration catalysts which may be used for dehydration include hydrogen sulphates, for instance KHSO₄, anhydrous CuSO₄, ZnCI₂, iodine, P₂O₅, BF₃-etherate, sodium acetate/acetic acid, phthalic anhydride, acetic anhydride, trifluoroacetic acid anhydride, thionyl chloride/pyridine, phosphorylchloride (POCI₃), mesyl chloride, tosyl chloride, carbodiimides or acidic ion exchange resins.

The dehydration of an enantiomerically enriched compound of formula (7) may be further promoted by removal of the unsaturated reaction product, for example by distillation. Alternatively, the dehydration of an enantiomerically enriched compound of formula (7) may be further promoted by removal of the water formed during dehydration, for example by azeotropic distillation.

The dehydration may be performed without solvent, in the neat dehydration catalyst/reagent or in an aqueous solution of the dehydration catalyst. Preferably, the dehydration is performed in a solvent, more preferably in an organic solvent for instance in DMSO₁ DMF, alcohol, for instance methanol, ethanol, isopropanol; TBME, THF, chloroform, dichloromethane, 1 ,2-dichloroethane, acetonitrile or acetone. More preferably, in order to facilitate removal formed during dehydration, the dehydration is performed in a solvent capable of forming azeotropes with water, such as for instance toluene, benzene, xylene or ethyl acetate. The reaction temperature of the dehydration of an enantiomerically enriched compound of formula (1) is preferably at least O⁰C. More preferably, a temperature of at least 40°C is applied, even more preferably a temperature between 60 and 1 15°C is applied. Preferably, the dehydration of an enantiomerically enriched compound of formula (1 ) is perfomed at a temperature lower than 200⁰C, more preferably lower than 150⁰C.

The optimal amount of dehydration catalyst used in the dehydration of an enantiomerically enriched compound of formula (7) can be easily obtained by the person skilled in the art through routine experimentation. The concentration of the dehydration catalyst in the dehydration reaction mixture is preferably at least 0.05% (w/w). More preferably, it is at least 0.1% (w/w), even more preferably at least 0.5% (w/w) and most preferably at least 1% (w/w).

Preferably, the concentration of dehydration catalyst is lower than 95% (w/w), more preferably lower than 50%, even more preferably lower than 20% (w/w), most preferably lower than 10% (w/w). The optimal amount of dehydration catalyst used may depend on the combination of the choice of dehydration catalyst, the solvent and the substrate. For example, if para-toluenesulphonic acid is used, the concentration of dehydration catalyst concentration is typically between 1 and 5% (w/w).

If a dehydration catalyst is capable of binding 1 molar equivalent of water, it is called a stoichoimetric dehydration catalyst. Examples of stoichoimetric dehydration catalysts include for instance thionylchloride, acetic anhydride or mesyl chloride. If a stoichoimetric dehydration catalyst is used for the dehydration, it is preferably used in a molar amount that is at least equivalent to the amount of compound of formula (7). More preferably, the molar ratio of stoichoimetric dehydration catalyst and the compound of formula (7) is chosen between 10:1 and 1 :1 , even more preferably between 5:1 and 1 :1 , most preferably between 2:1 and 1 :1.

If a dehydration catalyst is capable of binding more than 1 molar equivalent of water, it may be advantageous to use a molar amount that is less than the equivalent amount of compound of formula (7), for example in a molar ratio of dehydration catalyst and the compound of formula (7) of between 0.5: 1 and 1 : 1. An example of a dehydration catalyst capable of binding more than 1 molar equivalent of water is phosphoryl chloride.

The enantiomerically enriched compound of formula (3) prepared in the process of the invention may be hydrogenated by a suitable hydrogenating agent to form the corresponding enantiomerically enriched compound of formula (4)

wherein R¹ is as defined above. Therefore, the invention also relates to a process comprising the steps of: a) reacting acetaldehyde and a substituted acetaldehyde of formula HC(O)CH₂R¹, wherein R¹ stands for H, an alkyl, a cycloalkyl, an alkenyl, an alkynyl or for an aryl, wherein R¹ may be substituted, in the presence of an aldolase to form an enantiomerically enriched compound of formula (6)

and b) further oxidizing the enantiomerically enriched compound of formula (6) by an oxidizing agent to form the corresponding enantiomerically enriched compound of formula (7)

(3) and d) hydrogenating the enantiomerically enriched compound of formula (3) by reacting the enantiomerically enriched compound of formula (3) with a suitable hydrogenating agent to form the corresponding enantiomerically enriched compound of formula (4)

Methods for the hydrogenation of an enantiomerically enriched compound of formula (3) are known to the person skilled in the art. As a suitable hydrogenating agent to be used in the hydrogenation of the enantiomerically enriched compound of formula (3), in principle all hydrogenating agents known to the skilled person to be applicable in the hydrogenation of a double bond may be applied. For example, the enantiomerically enriched compound of formula (3) may be hydrogenated with hydrogen, with hydrides or with combinations of alkalimetals and ammonia or amines. Examples of hydrides include sodium hydride; aluminum hydrides, for instance LiAIH₄, LiHAI(OMe)₃, AIH, /-Bu₂AIH, HAI(OR)₂ (R= CH₂CH₂OCH₃, t-butyl, i-propyl), HAI(N(/-Pr)₂)₂; tin hydrides, for instance SnH, tributyl stannane, triphenyl stannane, diphenyl stannane; borohydrides, for instance NaBH₄ or NaCNBH₃, LiHB(sec-Bu)₃, KHBR₃ (R=/-butyl, phenyl); silanes, for instance R₃SiH (R=methyl, ethyl), phenylsilane, diphenylsilane, triphenylsilane or polymethylhydrosiloxane. Examples of combinations of alkali metals, for example Li, Na; and ammonia or amines include ethyl amine or hexamethylphosphoramide; or zinc with hydrochloric acid or nickel(ll)chloride; or magnesium in methanol; or sodium dithionite with sodium hydrogencarbonate. Preferably, the hydrogenation of the enantiomerically enriched compound of formula (3) (step d) ) is performed in the presence of a hydrogenation catalyst. For example, the hydrogenation may be performed in the presence of a chemical hydrogenation catalyst. Alternatively the hydrogenation may be performed in the presence of a biocatalyst. Examples of chemical hydrogenation catalysts include metals, for example the transitions metals Ni, Pd, Pt, Co, Rh, Ir, Ru, Mo, Cu; or salts or complexes thereof. A widely used catalyst for the hydrogenation of enantiomerically enriched compounds of formula (3) is Pd/C (see for instance JP 10158257 A2 which describes the hydrogenation of enantiomerically enriched δ-decenolactone and δ- dodecenolactone using Pd/C). In case of hydrogen as the hydrogenating agent, the hydrogenation of the enantiomerically enriched compound of formula (3) may be carried out in a hydrogen containing atmosphere. This atmosphere may for example be applied at pressures lower than 35 MPa, preferably lower than 1 MPa, more preferably between 0.1 and 0.6 MPa. The temperature in the hydrogenation may for example be in the range of -20⁰C to 150⁰C, preferably between 0⁰C and 100⁰C, more preferably from 20°C to 50°C.

The hydrogenation of an enantiomerically enriched compound of formula (3) may be carried out in bulk or in a solvent, for example in an alcohol, for instance methanol, ethanol or isopropanol; ethyl acetate, THF, dioxane, diethyl ether, MTBE, water or acetic acid.

Examples of biocatalyst which can be used in the hydrogenation of the enantiomerically enriched compound of formula (3) are known to the skilled person. See for a specific example for instance ter Burg, N and van der Schaft, P. H., Trends in Flavour Research, 1994, p481-486, who describe the hydrogenation of 2-decen-5-olide by Baker's yeast. In general, as a biocatalyst, for example the cells of Saccharomyces cerevisiae or other species, for example Polyporus durus, lschnoderna benzoinum, Bjerkandera adusta, Poria xantha, Pleurotus ostreatus, Saccharomyces delbrueckii, Pichia ohmeri, Pichia anomala, Pichia stipitis, Debaryomyces hansenii, Zymomonas mobilis, Zygosaccharomyces rouxii, Schawnniomyces occidentalis, Sarcina lutea or Geotrichum candidum; may be used. The hydrogenation may be performed under conditions known to the person skilled in the art to be applicable to whole cell biocatalytic reactions, for example in a buffer at a pH of between 2.5 and 7, preferably at a pH of between 3 and 6.5. The temperature during the process may for example be in the range of 20-37⁰C₁ preferably in the range of 27-37°C. A cosubstrate may be added to provide the reduction equivalents such as NADH and NADPH in the whole cells used as biocatalyst. As a cosubstrate for instance a sugar, preferably glucose, may be used. The cells may for example be used in free or immobilised form at concentrations of 2-30 g/L dry cell weight, preferably 5-20 g/L dry cell weight. If desired, the cells may be aerated. In order to avoid substrate and/or product toxicity, the enantiomerically enriched compound of formula (3) may be added to the cells following a feeding protocol, and/or additives, such as for example adsorbent resins, cyclodextrins or organic solvents, may be employed.

In the framework of the invention with enantiomerically enriched is meant 'having an enantiomeric excess (e.e.) of either the (R)- or (S) -enantiomer of a compound'. Preferably, the enantiomeric excess is > 80%, more preferably > 85%, even more preferably > 90%, in particular >95%, more in particular > 97%, even more in particular > 98%, most in particular > 99%.

The stereochemistry of the excess of enantiomer (the (R)-enantiomer of the compound of formula (6) or the (S)-enantiomer of the compound of formula (6)) formed depends on the choice of aldolase. The person skilled in the art knows which aldolases preferably catalyze the formation of the (R)-enantiomer of the compound of formula (6) and which aldolases preferably catalyze the formation of the (S)-enantiomer of the compound of formula (6).

For example, if an (f?)-selective aldolase is used, an example of which is DERA from Escherichia coli K12, the formed enantiomerically enriched compound of formula (6) will be an excess of a compound of formula (6A)

wherein R¹ is as described herein. The corresponding enantiomerically enriched compound of formula (7) will then be an excess of a compound of formula (7A)

wherein R¹ is as described herein. The corresponding enantiomerically enriched compound of formula (3) will then be an excess of a compound of formula (3A)

wherein R¹ is as described herein. The corresponding enantiomerically enriched compound of formula (4) will then be an excess of a compound of formula (4A)

wherein R¹ is as described herein.

For example, if an (S)-selective aldolase is used, the formed enantiomerically enriched compound of formula (6) will be an excess of a compound of formula (6B)

wherein R¹ is as described herein. The corresponding enantiomerically enriched compound of formula (7) will, in case of the use of an (S)-selective aldolase, be an excess of a compound of formula (7B)

wherein R¹ is as described herein. The corresponding enantiomerically enriched compound of formula (3) will, in case of the use of an (S)-selective aldolase, be an excess of a compound of formula (3B)

wherein R¹ is as described herein. The corresponding enantiomerically enriched compound of formula (4) will, in case of the use of an (S)-selective aldolase, be an excess of a compound of formula (4B)

wherein R¹ is as described herein.

In yet another aspect, the invention also relates to the use of an enantiomerically enriched δ-lactone obtained in a process according to the invention in food, cosmetics or as a pharma intermediate.

The invention will now be elucidated by way of the following examples, without however being limited thereto.

General procedures

Cultivation & Expression

Chemically competent E. coli TOP10 cells (Invitrogen) were freshly transformed according to the suppliers procedure with pBAD//Wyc-HisC plasmids (Invitrogen), in which the wild-type deoC gene from E. coli K12 (SEQ ID No. 2) and a mutated deoC gene from E. coli K12 (SEQ ID No. 4), containing a A598T nucleotide transversion resulting in the exchange of Phe at position 200 of the wild-type DERA amino acid sequence of E. coli K12 (SEQ ID No. 1 ) for lie at position 200 (SEQ ID No. 3), had been cloned into the Λ/col and EcoRI sites, respectively. Two 50 ml precultures in Luria Bertani medium (LB; 10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCI containing 100 μg/ml carbenicillin) were inoculated with single colonies from the respective transformation agar plates, and incubated overnight on a gyratory shaker at 180 revolutions per minute (rpm) and 28^CC.

The next day sterile Erlenmeyer flasks containing 1 I LB medium each with 100 μg/ml carbenicillin were inoculated with the 50 ml pre-cultures to a start cell density of OD₆₂₀ = 0.05 and incubated with shaking (180 rpm) at 28°C. At cell densities of OD_62O = 0.6 the expression of wild-type DERA of E. coli K12 and the thereof derived mutant DERA₁ containing the amino acid exchange F200I, was induced by addition of 0.1 % (w/v) L-arabinose. The cultures were further incubated under the same conditions over night until a final cell density of OD₆₂₀ = 4 was reached (total cultivation time of 21 h).

Preparation of cell-free extract (small scale)

After 21 hours of incubation both cultures were harvested by centrifugation (5 minutes at 5,00Ox g, 4°C) and the cell pellets were resuspended in volumes of a 50 mM triethanolamine buffer (pH 7.2) corresponding to three times their wet cell weight. The cell-free extracts (cfe) were obtained by sonification of the cell suspensions in a MSE Soniprep 150 sonificator for 2 times 5 minutes (10 seconds pulse followed by 10 seconds pause, large probe) and centrifugation for one hour at 4°C and 39,00Ox g. The supernatants were transferred into new flasks (cell-free extracts). The specific activities of the cell-free extracts of both wild-type and F200I mutant DERA determined by the DERA Activity Assay as described below were in the same range. The cell-free extracts (cfes) were kept at 4°C until further use.

Fermentation

For enzymatic syntheses on preparative scale, fermentations for the production of wild-type and F200I mutant DERA were carried out on ten liter scale in an ISF-200 laboratory fermentor (Infors). For the inoculation of the fermentor an over night (24 h) starter culture in 0.5 I Terrific Broth (TB; 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) was used, which itself had been inoculated with 0.1 ml of the respective glycerol stock culture.

The expression of the wild-type and mutant genes, coding for wild- type and F200I mutant DERA, respectively, were induced by addition of 0.1% (w/v) L- arabinose at cell densities of OD₆₂₀ = 1. After 18 hours of cultivation (OD₆₂O generally around 20) the cells were harvested by centrifugation (12 minutes at 12,227x g at 5°C). The wet cells (generally more than 300 g) were washed with 0.1 M K-phosphate buffer (pH 7.0) and resuspended in volumes of a 50 mM triethanolamine buffer (pH 7.2) corresponding to three times their wet cell weight. Cells were disrupted in a nanojet homogeniser (Haskel) at 1600 bar (double run) and subsequently centrifuged (34,524x g for 60 min at 4°C) to obtain the cell-free extracts (supernatant).

The DERA activities measured by the DERA Activity Assay as described below were 1.6 kU/ml cfe for both wild-type and F200I mutant DERA. The specific DERA activity given in U/mg total protein in the cfes was determined to be 53 and 55 for wild-type and F200I mutant DERA, respectively.

The cfes were frozen and stored at -20°C until further use.

DERA Activity Assay

For the estimation of DERA activity in the cell-free extracts containing wild-type and F200I mutant DERA, the initial activity in the physiological reaction of DERA, the aldol cleavage of 2-deoxy-D-ribose 5-phosphate to acetaldehyde and D- glyceraldehyde 3-phosphate, was determined at room temperature (RT = 25°C). In this assay, the formed D-glyceraldehyde 3-phosphate is subsequently converted by the auxiliary enzyme triosephosphate isomerase to dihydroxyacetone phosphate, which is reduced to L-glycerol 3-phosphate by L-glycerol 3-phosphate dehydrogenase (both auxiliary enzymes from Roche Diagnostics) with an equimolar amount of NADH as cosubstrate. The splitting of one molecule of 2-deoxy-D-ribose 5-phosphate therefore corresponds to the formation of one molecule of NAD from NADH, which in turn can be quantified by measuring the absorbance of the samples at 340 nm wavelength with a molar absorption coefficient for NADH of ε = 6.22 μmol ^* cm^'2.

1 Unit of DERA activity is defined as the enzyme amount necessary to convert 1 μmol 2-deoxy-D-ribose 5-phosphate in 1 minute at room temperature (25° C) in 50 mM triethanolamine buffer pH 7.2.

The activity assays were carried out with 10 μl cfe of a suitable dilution in 50 mM triethanolamine pH 7.2 (usually more than 1 , 000-fold for wilde-type and mutant DERA), 0.3 mM NADH and 5 mM 2-deoxy-D-ribose 5-phosphate as substrate concentration in 50 mM triethanolamine pH 7.2 and an excess of both auxiliary enzymes (more than 1.5 U/ml L-glycerol 3-phosphate dehydrogenase and more than 4.5 U/ml triosephosphate isomerase) in a total reaction volume of 1 ml. The assay was started by adding 10 μl of 500 mM 2-deoxy-D-ribose 5-phosphate stock solution to the reaction mixture in a 1 ml cuvette and by subsequent mixing. The consumption of NADH was followed using a PerkinElmer Lambda 20 spectrophotometer at 340 nm.

The specific activities of the cell-free extracts of both wild-type and F200I mutant DERA determined by this DERA activity assay were in the same range.

Example I

Synthesis of 6-propyl-5, 6-dihvdro-pyran-2-one

Step 1. Aldolase reaction to 6-propyl-tetrahvdro-pyran-2,4-diol (6PThpD)

50 mmol butanal (3.61 g) and 100 mmol acetaldehyde (4.40 g) were dissolved in 427 mL of a weak sodium hydrogencarbonate buffer (10 mM, pH 7.2). If necessary, the pH was set to 7.2 by titration with diluted aqueous sodium hydroxide. Cell free extract of DERA enzyme of SEQ ID No. 1 (1600 U/mL, 63 mL) was added to the reaction mixture to obtain an aldolase concentration of 200 kU/L. The reaction was stirred at 22-25°C for 4 hours, then once more 50 mmol butanal (3.61 g) and 100 mmol acetaldehyde (4.4 g) were added slowly over 5 min while keeping the pH at 7.2. The reaction was stirred for another 4 hours until complete conversion. It was confirmed by GC-MS (gas chromatography-mass spectrometry) analysis that besides 6PThpD, only traces (<5%) of the monoaldol product 3-hydroxy-hexanal and of the acetaldehyde self- condensation product 6-methyl-tetrahydropyran-2,4-diol were formed as side products.

Following the same protocol, also 6-pentyl-tetrahydropyran-2,4-diol was prepared from hexanal and acetaldehyde (conversion 70%).

Step 2. Oxidation of ΘPThpD to 4-hvdroxy-6-propyl-tetrahvdro-pyran-2-one

ΘPThpD was subsequently oxidized with bromine in an aqueous solution in presence of BaCO₃. The aqueous suspension was prepared by addition of two equivalents of the carbonate (39.5 g) to the aqueous solution containing the ΘPThpD (ca. 100 mM). After cooling it to to 0⁰C, two equivalents of bromine (200 mmol, 10.2 ml) were fed to the suspension over 2 hours. Then the mixture was allowed to warm to 25°C for 4 hours after which GC showed full conversion of the starting material. The reaction was quenched by addition of sodium thiosulphate. The carbonate salt was filtered off and the aqueous phase was extracted three times with ethyl acetate. After drying over sodium sulphate and evaporation of the solvent, a product mixture containing 4-hydroxy-6-propyl-tetrahydro-pyran-2-one and traces of oxidized side products was obtained. The 4-hydroxy-6-propyl-5,6-dihydro-pyran-2-one was separated from the oxidized side products by column chromatography over silica with heptane/ethyl acetate (3:7) for NMR analysis. ¹H-NMR (300 MHz, CDCI₃): δ= 4.69 (m, 1 H, H-6), 4.35 (m, 1 H, H-4),

2.69 (J=17.7, 4.8 Hz, dd, 1 H, H-3), 2.59 (17.7, 3.6, -1.5 Hz, ddd, 1 H, H-3), 1.91-2.00 (m, 1 H, H-5), 1.71-1.75 (m, 1 H, H-5, overlapping), 1.35-1.70 (m, 4H, CH₂, overlapping), 0.94 (t, 3H, CH₃) ppm.

¹³C-NMR (300 MHz, CDCI₃): δ=171.7 (C-2), 76.3 (C-6), 62.9 (C-4), 39.0 (C-3), 38.1 (C-5), 36.4 (C-7), 18.7 (C-8), 14.5 (C-9) ppm.

Step 3. Dehydration of 4-hvdroxy-6-propyl-tetrahvdro-pyran-2-one to 6-propyl-5.6- dihvdro-pyran-2-one

The mixture of 4-hydroxy-6-propyl-tetrahydro-pyran-2-one and traces of oxidized side products obtained in step 2 was dissolved in toluene and heated to reflux in presence of 3 mol% of p-toluenesulphonic acid for one hour. The formed water was distilled off azeotropically. The organic phase was washed with saturated aqueous sodium hydrogencarbonate and brine and dried over sodium sulphate. After evaporation of the toluene, a product mixture was obtained out of which the product 6- propyl-5,6-dihydro-pyran-2-one was purified by column chromatography over silica with heptane/ethyl acetate (1 :1). The overall yield of the three reaction steps with respect to butanal was 34%.

1 H-NMR (300 MHz, CDCI₃): δ= 6.85 (m, 1 H, H-4), 5.95 (J=9.6, 2.4, -1.5 Hz, ddd, 1 H, H-3), 4.38 (m, 1 H, H-6), 2.28 (m, 2H, H-5), 1.66-1.82 (m, 1 H, CH₂), 1.32-1.62 (m, 3H, CH₂), 0.89 (J=7.2 Hz, t, 3H, CH₃) ppm.

13C-NMR (300 MHz, CDCI₃): δ= 164.9 (C-2), 145.8 (C-4), 121.7 (C- 3), 78.1 (C-6), 37.24 (C-7), 29.7 (C-5), 18.4 (C-8), 14.1 (C-9) ppm.

Example Il

Comparison of DERA WT (wild type) of SEQ ID No. 1 with DERA mutant F200I (of SEQ ID No. 3) in the synthesis of 6-propyl-tetrahvdrooyran-2.4-diol (6PThpD). Wildtype DERA of SEQ ID No. 1 and the mutant DERA (F200I exchange) were tested under the same reaction conditions on butanal as an acceptor aldehyde. Two different aldolase concentrations, 60 kU/L and 200 kU/L of DERA activity respectively, were tested. The experiments were carried out in closed vials with magnetic stirring on a 5 ml scale. The appropriate volume (x μl_) of a NaHCO₃ buffer (10 mM, pH7.2), butanal (y1 = 44.5 μl_) and acetaldehyde (y2 = 55.9 μl_) were added into each vial. Finally, DERA was added as a cell free extract containing overexpressed WT DERA or mutant DERA F200I to start the reactions. Both cell free extract contained a DERA activity of 1600 U/ml, determined by the DERA Activity Assay as described above. For 60 kU/L of DERA activity in the reaction, z = 187.5 μL of WT DERA or, respectively, the F200I mutant DERA was added to the vial. In case of the reactions with 200 kU/L DERA activity, the volume of cell free extract to add was z = 625 μL.

According to these volumes, the amount of buffer was calculated as x = (5000-y1-y2-z) μL. The reactions were stirred at 25°C. After 4h, the concentration of 6-propyl- tetrahydropyran-2,4-diol (6PThpD) was determined by GC. The initial reaction rate for 200 kU/L DERA activity was obtained by linear regression from four samples in which the 6PThpD concentration (mM) was determined. These four samples were taken at 15, 30, 45 and 60 min after the start of the reaction and analyzed by GC. The results are shown in Table 1 below. Table 1. Comparison of activity of DERA WT of SEQ ID No. 1 with DERA mutant F200I of SEQ ID No. 3 in the synthesis of 6-propyl-tetrahydropyran-2,4-diol (6PThpD).

The above results show that the DERA mutant F200I performs better than wild-type DERA with butanal as an acceptor aldehyde. The productivities at 200 kU/L are comparable, but at 60 kU/L the F200I mutant DERA is much more efficient in ΘPThpD production. Thus the aldolase concentration can be reduced significantly as compared to the wild-type in the process of the present invention. Therefore use of the F200I mutant DERA is commercially even more interesting.

Example III

DERA reaction in microemυlsion for different acceptor aldehydes.

Wild type DERA was tested in a microemulsion system according to Clapes et al, Chem. Eur. J. 2005, 11 , 1392-1401. In a closed 5 ml_ vial with a reaction volume of 2.5 mL was the volumetric ratio of buffer/surfactant/hexadecane = 90/4/6. The octanal concentration was 100 mM and the acetaldehyde concentration 200 mM in all experiments. A 5 M acetaldehyde stock solution in water was freshly prepared and its pH was titrated to 7.2. Hexadecane (150 mg), the surfactant tetra(ethylene glycol) tetradecyl ether C14E4 (100 mg) and 0.25 mmol octanal (y mg) were weighed into the vial and this oil phase was mixed intensively on a vortex mixer (1 min at 2500 rpm). Acetaldehyde stock solution (100 μl_) was mixed with x μl_ of triethanolamine buffer pH (7.2, 50 mM) (x = 2500-150-100-100-313-y) μl_ separately. This aqueous solution was then added dropwise to the oil phase while further mixing on the vortex mixer so that an emulsion formed. Then a cell free extract of DERA WT (313 μl_, DERA activity 1600 U/mL) was added and the reaction was mixed again for 1 min. The closed vial was then placed on a horizontal shaker (480 rpm) for reaction. After 6 hours, a sample was taken from each vial and analyzed by GC. Conversion was estimated based on the peak areas of acceptor aldehyde and 6-heptyl-tetrahydro-pyran-2,4-diol product. Results of other acceptor aldehydes tested according to this protocol are shown in Table 2 below. Table 2. Aldolase reaction in emulsion for different acceptor aldehydes.

No. Product Acceptor aldehyde Conversion

Butanal >90%

(R)-β-propyl- tetrahydro-pyran-2,4- diol

Pentanal 70-90%

(R)-6-butyl-tetrahydro- pyran-2,4-diol

Hexanal 45-55%

(R)-θ-pentyl-tetrahydro- pyran-2,4-diol

Phenylacetaldehyde 50-60%

(R)-θ-phenyl- tetrahydro-pyran-2,4- diol Example IV

Synthesis of 6-ethyl-3, 4, 5, 6-tetrahydro-pyran-2-one

Step 1. Aldolase reaction to 6-ethyl-tetrahvdro-pyran-2,4-diol (ΘEThpD)

A mixture of 340 ml bicarbonate buffer (final concentration 50 mol/l), 14 ml acetaldehyde (0.25 mol) and 18 ml propanal (0.25 mol) was stirred until all aldehydes were dissolved. At 28°C, 47.5 ml cell-free extract containing DERA variant F200I (1600 U/ml measured with 2-deoxy-D-ribose 5-phosphate as substrate as described in the general part) was added. During 3.5 hours 14 ml acetaldehyde (0.25 mol) was dosed to the solution. The conversion of propanal was approximately 80%. The conversion to ΘEThpD was approximately 60-70%. The ratio ΘEThpD to θ-methyl- tetrahydro-pyran-2,4-diol was above 5:1 judged by thin layer chromatography (TLC₁ Silicagel Θ0 F_2S4; developed in CH₂CI₂/acetonitril = 1/1 and stained with KMnO₄ solution. R_f θ-methyl-tetrahydro-pyran-2,4-diol: 0.34; R_f 6EThpD: 0.45).

The enzyme was removed from the reaction mixture by precipitation with the same volume of ice-cold acetone followed by filtration. The enzyme solution was dosed during 0.5 h to 400 ml acetone (-20°C to 0°C). Stirring was continued for 1 hour at 0⁰C and the resulting enzyme slurry was filtered over a pre-coated dicalite filter. The acetone in the clear filtered solution was removed via evaporation under vacuum at 40⁰C.

Step 2. Oxidation of 6EThpD to 4-hvdroxy-6-ethyl-tetrahvdro-pyran-2-one

During 1 hour 40.2 g Br₂ (0.25 mol) was dosed at 25°C to the remaining water solution of step 1. During the dosing the pH was kept between pH = 5.0 to 5.5 by dosing solid Na₂CO₃. After complete Br₂ addition, stirring was continued while adding Na₂CO₃ until the pH remained stable. In total 26.5 g Na₂CO₃ (0.25 mol) was added. The conversion yield of 4-hydroxy-6-propyl-tetrahydro-pyran-2-one was approximately 80% based on thin layer chromatography analysis (TLC, Silicagel Θ0 F₂₅₄; developed in CH₂CI₂/acetonitril = 1/1 and stained with KMnO₄ solution. R_f 4- hydroxy-β-methyl-tetrahydro-pyran-2-one: 0.5Θ; R_f 4-hydroxy-θ-ethyl-tetrahydro-pyran- 2-one: 0.Θ4). The reaction mixture was filtered to remove the inorganic salts. The salts were washed with 50 ml ethyl acetate (EtOAc). The filtrate was extracted continuously with 250 ml EtOAc for 17 hours until no 4-hydroxy-6-ethyl-tetrahydro- pyran-2-one was left in the water phase. The organic phase was evaporated under vacuum at 40^cC. The ratio 4-hydroxy-6-ethyl-tetrahydro-pyran-2-one to 4-hydroxy-6- methyl-tetrahydro-pyran-2-one was above 5:1 judged by thin layer chromatography (Silicagel 60 F₂₅₄; developed in CH₂CI₂/acetonitril = 1/1 and stained with KMnO₄ solution. R_f values as above). H¹-NMR:

4-hydroxy-6-methyl-tetrahydro-pyran-2-one (300MHz, CDCI₃):δ 4.84 (1 H,m), 4.34 (1 H,m), 2.8 (1 H₁S)₁ 2.68 (2H,m), 2.0-1.6 (2H,m), 1.39 (3H,d).

4-hydroxy-6-ethyl-tetrahydro-pyran-2-one (300MHz, CDCI₃):δ 4.60 (1H,m), 4.30 (1 H,m), 3.6 (1 H,s), 2.59 (2H,m), 2.0-1.6 (4H,m), 0.95 (3H,t).

Step 3. Dehydration of 4-hvdroxy-6-ethyl-tetrahvdro-pyran-2-one to 6-ethyl-5,6-dihydro- pyran-2-one

The resulting 24.4 g product of step 2 was dissolved in 250 ml toluene. 360 mg p-toluenesulphonic acid (pTsOH.H₂O) was added. The reaction mixture was refluxed for 3 hours (1 10^cC) and the resulting reaction water was removed azeotropically. The conversion of 4-hydroxy-6-ethyl-tetrahydro-pyran-2-one to 6-ethyl-5,6-dihydro-pyran-2- one was above 95% judged by TLC (Silicagel 60 F₂₅₄; developed in EtOAc/heptane = 1/1 and stained with KMnO₄ solution. R_f 6-methyl-5,6-dihydro-pyran-2-one: 0.38; R_f 6- ethyl-5,6-dihydro-pyran-2-one: 0.51).

The toluene solution was washed once with 50 ml saturated NaHCO₃ solution and twice with 50ml water. To the remaining toluene phase was added 2.5 g charcoal (Norit SX P145). The toluene solution was filtered over a precoated dicalite filter, evaporated and distilled at top temperature = 78°C (0.5 mbar). Isolated yield of 6-ethyl- 5,6-dihydro-pyran-2-one was 80%. H¹-NMR: 6-methyl-5,6-dihydro-pyran-2-one (300MHz,CDCI₃):δ 6.85 (1 H,m), 5.96 (1 H,m),

4.52 (1 H,s), 2.3-1.6 (2H,m), 1.42 (3H,d).

6-ethyl-5,6-dihydro-pyran-2-one (300MHz,CDCI₃):δ 6.85 (1 H,m), 5.96 (1 H,m), 4.30 (1 H,m), 2.3-1.6 (4H,m), 0.98 (3H,t). C¹³-NMR: 6-ethyl-5,6-dihydro-pyran-2-one (300MHz, CDCI₃): δ 165.0, 145.5, 121.7, 79.6, 29.3, 28.2, 9.6

Step 4. Hydrogenation of 6-ethyl-5,6-dihvdro-pyran-2-one to 6-ethyl-3,4,5,6-tetrahydro- pyran-2-one

The distilled 6-ethyl-5,6-dihydro-pyran-2-one (23.4 g) was dissolved in 120 ml toluene. 1.95 g 5% Pd/C (Degussa E196, 50% H₂O) was added. The mixture was hydrogenated during 5 hours at 30⁰C and 10 bar H₂. The conversion of 6-ethyl- 5,6-dihydro-pyran-2-one to 6-ethyl-3,4,5,6-tetrahydro-pyran-2-one was higher than 95% as judged by TLC (Silicagel 60 F₂S₄; developed in EtOAc/heptane = 1/1 and stained with KMnO₄ solution. R_f 6-methyl-3,4,5,6-tetrahydro-pyran-2-one: 0.35; R_f 6- ethyl-3,4,5,6-tetrahydro-pyran-2-one: 0.46). After filtration and evaporation of the toluene, 6-ethyl-3,4,5,6-tetrahydro-pyran-2-one was distilled as a colourless liquid. The yield of 6-ethyl-3,4,5,6-tetrahydro-pyran-2-one was 80%. The ratio of 6-ethyl-3,4,5,6- tetrahydro-pyran-2-one to 6-methyl-3,4,5,6-tetrahydro-pyran-2-one was approximately 9:1. H¹-NMR:

6-methyl-3,4,5,6-tetrahydro-pyran-2-one (300MHz,CDCI₃):δ 4.35 (1 H,m), 2.3- 2.6 (2H,m), 1.4-1.9 (6H,m), 1.31 (3H,d). 6-ethyl-3,4,5,6-tetrahydro-pyran-2-one (300MHz,CDCI₃):δ 4.15 (1 H,m), 2.3-2.6

(2H,m), 1.4-1.9 (6H,m), 0.95 (3H,t).

Claims

Process, comprising the step of a) reacting acetaldehyde and a substituted acetaldehyde of formula HC(O)CH₂R, wherein R stands for an alkyl of at least 5 C-atoms, a cycloalkyl, an alkenyl, an alkynyl or for an aryl, wherein R may be substituted, in the presence of an aldolase to form the corresponding enantiomerically enriched compound of formula (1)

Process according to claim 1, further comprising the step of b) oxidizing the enantiomerically enriched compound of formula (1) by a suitable oxidizing agent to form the corresponding enantiomerically enriched compound of formula (5)

wherein R has the meaning described above

Process comprising the steps of: a) reacting acetaldehyde and a substituted acetaldehyde of formula HC(O)CH₂R¹, wherein R¹ stands for H, an alkyl, a cycloalkyl, an alkenyl, an alkynyl or for an aryl, wherein R¹ may be substituted, in the presence of an aldolase to form an enantiomerically enriched compound of formula (6)

4. Process according to claim 3, further comprising the step of d) hydrogenating the enantiomerically enriched compound of formula (3) by reacting the enantiomerically enriched compound of formula (3) with a suitable hydrogenating agent to form the corresponding enantiomerically enriched compound of formula (4)

5. Process according to any one of claims 1-4, wherein R or R¹ stands for a linear alkyl, a linear alkenyl or for phenyl.

6. Process according to any one of claims 1-5, wherein the aldolase is a DERA from Escherichia coli, from Bacillus subtilis or from a Geobacillus species or a mutant thereof.

7. Process according to any one of claims 1-6, wherein the aldolase is the aldolase of SEQ ID No. 3 or a mutant thereof.

8. Process according to any one of claims 1-7 wherein step a) is performed in an emulsion.

9. Process according to any one of claims 4-8, wherein step d) is performed in the presence of a hydrogenation catalyst..

10. Use of an enantiomerically enriched δ-lactone obtained in a process according to any one of claims 1-9 in food or cosmetics or as a pharma intermediate.