NL1019622C2 - Preparing 2,4-dideoxyhexose/2,4,6-trideoxyhexose by reacting optionally substituted carbonyl compound with alpha-hydrogen atom and two carbon atoms and optionally substituted aldehyde at specific carbonyl concentration - Google Patents

Abstract

Preparing (M) 2,4-dideoxyhexose/2,4,6-trideoxyhexose (I), comprising reacting an optionally substituted carbonyl compound with at least 2 carbon atoms and at least one alpha-hydrogen atom and an optionally substituted aldehyde in the presence of aldolase and water, at a carbonyl concentration of at most 6 moles/l of reaction mixture, is new. Preparing (M) 2,4-dideoxyhexose/2,4,6-trideoxyhexose (I), comprising reacting an optionally substituted carbonyl compound with at least 2 carbon atoms and at least one alpha-hydrogen atom and an optionally substituted aldehyde in the presence of aldolase and water, at a carbonyl concentration of at most 6 moles/l of reaction mixture, is new. The final concentration of (I) is at least 2 mass% of the reaction mixture. An Independent claim is also included for use of 2,4-dideoxyhexose/2,4,6-trideoxyhexose (I), obtained by (I), in the preparation of a medicine.

Description

- 1 -

METHOD FOR THE PREPARATION OF 2,4-DIDEOXYHEXOSES AND 2.4.6-5 TRIDEOXYHEXOSES

The invention relates to a process for the preparation of a 2,4-dideoxyhexose or a 2,4,6-trideoxyhexose from a substituted or unsubstituted carbonyl compound with at least 2 carbon atoms and at least one α-hydrogen atom and an optionally or not substituted aldehyde in the presence of an aldolase and water.

Such a process is known from US-A-5,795,749, in which the synthesis of 2,4,6-trideoxyhexoses via a 4-substituted 3-hydroxybutanal intermediary is described. Acetaldehyde and a 2-substituted aldehyde are used as reactants and an aldolase as catalyst.

A known drawback of such enzyme catalyzed aldol condensations is that the production capacity is low. This is in particular due to the fact that such aldol condensations are generally carried out at low aldehyde concentrations, because it was generally assumed that the aldolase used would be highly inactivated at higher aldehyde concentrations. Another disadvantage of the low concentrations used is that at the end of the reaction the concentration of the 2,4,6-trideoxyhexose in the reaction mixture is low, which strongly influences the costs for, for example, work-up. Consequently, it was not expected that it would be possible to develop a commercially attractive process for the preparation of 2,4-dideoxyhexoses and 2,4,6-trideoxyhexoses based on such a technology.

The invention provides a commercially attractive process for the preparation of a 2,4-dideoxyhexose or a 2,4,6-trideoxyhexose using an enzyme catalyzed aldol condensation.

This is achieved according to the invention by carrying out the reaction between the optionally substituted carbonyl compound with at least 2 carbon atoms and at least one alpha hydrogen atom and the optionally substituted aldehyde at a carbonyl concentration of at most 6 mol / l reaction mixture such that the final concentration of the 1019622 -2- 2,4-dideoxyhexose or the 2,4,6-trideoxyhexose is at least 2 mass% of the reaction mixture.

In the known method for the synthesis of 2,4-dideoxyhexoses and 2,4,6-trideoxyhexoses, carbonyl concentrations of at most 0.4 mol per liter of reaction mixture are used. This results in final concentrations of the 2,4-dideoxyhexose or the 2,4,6-trideoxyhexose that are at most around 1 mass% of the reaction mixture. Surprisingly, the applicant has found that carbonyl concentrations up to 6 mol / l can be suitably used, while the degree of enzyme deactivation remains limited despite the high carbonyl concentration. As a result, higher final concentrations of the 2,4-dideoxyhexose or the 2,4,6-trideoxyhexose can be obtained, whereby the production capacity is increased and the costs for, among other things, processing of the product are reduced. As a result, it turned out to be possible to develop a commercially attractive process.

For the purposes of the invention, carbonyl concentration is understood to be the sum of the concentrations of the substituted or unsubstituted carbonyl compound with at least 2 carbon atoms and at least one α-hydrogen atom, the substituted or unsubstituted aldehyde and the intermediate formed in the reaction of the substituted or unsubstituted carbonyl compound with at least 2 carbon atoms and at least one α-hydrogen atom with the substituted or unsubstituted aldehyde.

The carbonyl concentration is essentially kept lower than 6 mol / l during the synthesis process. It will be clear to the person skilled in the art that slightly higher concentrations for a (very) short time will have little adverse effect, and are therefore still permissible within the scope of the invention. The carbonyl concentration is preferably between 0.1 and 5 moles per liter of reaction mixture, more preferably between 0.6 and 4 moles per liter of reaction mixture.

The final concentration of the 2,4-dideoxyhexose or the 2,4,6-30 trideoxyhexose is in practice usually between 5 and 50 mass%, for example between 8 and 40 mass%, in particular between 10 and 35 mass%. % calculated relative to the reaction mixture at the end of the reaction.

The order of addition of the substituted or unsubstituted carbonyl compound with at least 2 carbon atoms and at least one imoR 2 O 3 -3-α hydrogen atom, the substituted or unsubstituted aldehyde and the aldolase is not particularly critical. Preferably the aldolase is added to the reaction mixture before the addition of the substituted or unsubstituted carbonyl compound with at least 2 carbon atoms and at least one α-5 hydrogen atom. More preferably, the aldolase is mixed with a solution of at least a portion of the substituted or unsubstituted aldehyde before the substituted or unsubstituted carbonyl compound having at least 2 carbon atoms and at least one α-hydrogen atom is added, particularly when the al used then unsubstituted aldehyde reacts with itself to a lesser extent under the selected conditions than the used ai or unsubstituted carbonyl compound having at least 2 carbon atoms and at least one alpha hydrogen atom. Preferably more than 0.1 mol / l of reaction mixture of the substituted or unsubstituted aldehyde is submitted, more preferably more than 0.3 mol per liter of reaction mixture, in particular more than 0.6 mol per liter of reaction mixture. By this choice of the initial concentration of the substituted or unsubstituted aldehyde, the initial reaction rate of the enzymatic process is favorably influenced. Aldolase can be added or dosed in multiple portions during the reaction.

In one embodiment of the invention, both the unsubstituted or substituted carbonyl compound having at least 2 carbon atoms and at least one alpha hydrogen atom and the unsubstituted or substituted aldehyde and aldolase are each added to the reaction mixture in one go. The carbonyl compound with at least 2 carbon atoms and at least one alpha-hydrogen atom, the substituted or unsubstituted aldehyde and the aldolase can be added both simultaneously and in succession here. According to this embodiment, in general, the sum of the added amounts of substituted or unsubstituted carbonyl compound with at least 2 carbon atoms and at least one alpha hydrogen atom and substituted or unsubstituted aldehyde can be up to 6 moles per liter of reaction mixture and in practice usually becomes 30 final product concentrations reached between 7 and 15 mass% of the reaction mixture.

In another embodiment of the invention, the addition of the substituted or unsubstituted carbonyl compound with at least 2 carbon atoms and at least one α-hydrogen atom and / or the aldehyde optionally substituted in at least two portions, each subsequent addition taking place after at least a portion of the reactants from the previous addition have been converted. This ensures that the sum of the amounts of substituted or unsubstituted carbonyl compound with at least 2 carbon atoms and at least one α-hydrogen atom and unsubstituted or substituted aldehyde in the course of the reaction can be more than 6 moles per liter of reaction mixture, while the carbonyl concentration in the reaction mixture is also 6 moi / l at any time during the reaction to limit inactivation of the enzyme. The aldolase can, if desired, be added in one 10 times or in at least two portions.

In yet another embodiment, during the reaction, the substituted or unsubstituted carbonyl compound having at least 2 carbon atoms and at least one α-hydrogen atom and / or the substituted or unsubstituted aldehyde is metered to the reaction mixture over time. Preferably in this embodiment at least a portion of the substituted or unsubstituted aldehyde is presented together with the aldolase. The aldolase can, if desired, be added in one go, added in multiple portions or dosed over time.

The latter two embodiments can lead to 20 final concentrations of the 2,4-dideoxyhexose or the 2,4,6-trideoxyhexose that are higher than 40% by mass. Moreover, it has been found that dosing the substituted or unsubstituted carbonyl compound with at least 2 carbon atoms and at least one alpha-hydrogen atom, the substituted or unsubstituted aldehyde and / or the aldolase to the reaction mixture can lead to considerable savings in enzyme costs.

Combinations of the embodiments described above are also possible.

The reaction temperature and the pH are not particularly critical and are both selected in function of the substrate. The reaction is preferably carried out in the liquid phase. The reaction can be carried out, for example, at a reaction temperature between -5 and 45 ° C, preferably between 0 and 10 ° C and a pH between 5.5 and 9, preferably between 6 and 8.

The reaction is preferably carried out at a more or less constant pH, using, for example, a buffer or automatic titration. Sodium and potassium bicarbonate, sodium and potassium phosphate, triethanolamine / HCl, bis-tris-propane / HCl and HEPES / KOH can be used as a buffer. A potassium or sodium bicarbonate buffer is preferably used, for example in a concentration between 5 and 400 mmol / l of reaction mixture.

The molar ratio of total added substituted or unsubstituted carbonyl compound with at least 2 carbon atoms and at least one α-hydrogen atom to total added substituted or unsubstituted aldehyde is not particularly critical and is preferably between 1.5: 1 and 4: 1, in particular between 1.8: 1 and 2.2: 1. Other ratios can also be applied but offer no benefits.

In the process according to the invention, an aldolase is used as the catalyst, which catalyzes the aldol condensation between two aldehydes or between an aldehyde and a ketone. Preferably 2-deoxyribose-5-phosphate aldolase (DERA, EC 4.1.2.4) or mutants thereof is used as aldolase, more preferably DERA from Escherichia coli or mutants thereof. The amount of DERA to be used is not particularly critical and is selected as a function of, for example, the reactants used, the reactant concentrations, the desired reaction rate, the desired reaction time and other economic factors. The amount of DERA to be used is, for example, between 50 and 5000 U / mmol of the substituted or unsubstituted aldehyde. Herein 1 U (unit) is a measure of enzymatic activity and corresponds to the conversion of 1 μιτιοΙ 2-deoxyribose-5-phosphate per minute at 37 ° C.

The substituted or unsubstituted carbonyl compound having at least 2 carbon atoms and at least one alpha hydrogen atom preferably has 2-6 carbon atoms, more preferably 2-4 carbon atoms. For example, aldehydes can be used, for example acetaldehyde and propanal, and ketones, for example acetone and fluoracetone. If an aldehyde is used as a substituted or unsubstituted carbonyl compound with at least 2 carbon atoms and at least one alpha hydrogen atom, this can of course also be in the form of its (hemi) acetal, for example as the acetal of an alcohol, in particular methanol, ethanol or glycol.

As substituted or unsubstituted aldehyde, there can be used, for example, aldehydes with 2-20 carbon atoms, for example acetaldehyde, propanal, butanal, an acetally protected dialdehyde or a substituted aldehyde, in particular a substituted acetaldehyde of the formula HC (0) CH 2 X wherein X represents a group that is the substituent desired in the end product or can be converted into it, for example a CN group, or a substituent that can be split off as a 'leaving group' in a follow-up reaction, for example a halogen, in particular Cl, Br or I; a tosylate group; a mesylate group; an acyloxy group, in particular an acetoxy group; a phenacetyloxy group; an alkyloxy group or an aryloxy group, with chloroacetaldehyde being particularly suitable. The acetaldehyde then substituted or unsubstituted can be used as such or in the form of its (hemi) acetal, for example as the acetal of an alcohol, for example methanol, ethanol or glycol.

The 2,4-dideoxyhexose or 2,4,6-trideoxyhexose is of formula 1

x Y

0)

H

for example a desired intermediate in the preparation of various drugs, in particular for the synthesis of HMG-CoA reductase inhibitors, more particularly for the synthesis of statins, for example lovastatin, cerivastatin, rosuvastatin, simvastatin, pravastatin and fluvastatin, in the particularly for ZD-4522 as described in Drugs of the Future, (1999), 24 (5), 511-513 by M. Watanabe et al., Bioorg. & Med. Chem. (1997), 5 (2), 437-444. The invention therefore provides a new, economically attractive route to 2,4-dideoxyhexoses or 2,4,6-trideoxyhexoses, which can be used for the synthesis of for example 2- (6-hydroxymethyl-1,3-dioxane-4) -yl) acetic acid, which is a desired intermediate for the preparation of medicines, in particular statins.

The 2,4-dideoxyhexose or 2,4,6-trideoxyhexose of formula I can be prepared by the process according to the invention from acetaldehyde and HC (O) CH 2 X, wherein X is defined as above, and can be 1019622 -7 - subsequently converted, for example, by known methods to the corresponding 4-hydroxy-tetrahydropyran-2-one of formula 2, V-5 wherein X is defined as above. Suitable reagents for this conversion are, for example, Br2 with calcium carbonate as base (Tetrahedron Lett. 30 (47), 1989 p.6503), pyridinium dichromate in dichloromethane (Carbohydrate Res. 300 1997 p. 301) and tetra-N-propylammonium tetra-oxoruthenate (J. Chem Soc, Chem Commun 1987 p. 1625).

Subsequently, the 4-hydroxy-tetrahydropyran-2-one obtained can, if desired, be converted into a 2- (1,3-dioxan-4-yl) acetic acid derivative of formula 3,

Rk / 2 / o (3 | 15 wherein X is as defined above and R 1, R 2 and R 3 each independently represent an alkyl group with 1-3 carbon atoms. This conversion can be effected, for example, in the presence of a suitable acetalizing agent and an acid catalyst .

Subsequently, the 2- (1,3-dioxan-4-yl) acetic acid derivative can be hydrolyzed in the presence of a base and water to a salt of formula 4,

«Due to W

wherein Υ represents an alkali metal, an alkaline earth metal, or a substituted or unsubstituted ammonium group, preferably Na, Ca or a tertraalkylammonium compound and wherein X, and R2 are as defined above. The hydrolysis can be followed by conversion to the corresponding 2- (1,3-dioxan-4-yl) acetic acid according to formula 4 with Y = H.

The salt of the 2- (1,3-dioxan-4-yl) acetic acid of formula 4 can be converted into a corresponding ester of the 2- (1,3-dioxan-4-10 yl) acetic acid of formula 3 with X , R 1 and R 2 are as defined above and wherein R 3 represents an alkyl group with 1-12 carbon atoms, an aryl group with 6-12 carbon atoms or an aralkyl group with 7-12 carbon atoms in a manner known per se. Preferably, the salt of the 2- (1,3-dioxan-4-yl) acetic acid is converted to the corresponding t-butyl ester of the 2- (1,3-dioxan-4-yl) acetic acid. The 2- (1,3-dioxan-4-yl) acetic acid derivative of formula 3, wherein R 3 represents an alkyl group with 1-12 carbon atoms, an aryl group with 6-12 carbon atoms or an aralkyl group with 7-12 carbon atoms can be converted to the acetic acid derivative of the corresponding 2- (6-hydroxymethyl-1,3-dioxan-4-yl) acetic acid of formula 5, wherein R 1, R 2 and R 3 are defined as above, for example as described in US-A-5594153

Rk / 2 A vUAr, m or in WO-A-200008011, via conversion to a compound of formula 3 with X = acyloxy, for example an acetoxy group, and R1, R2 and R3 as defined above, followed by replacement of the acyloxy group, in a generally known manner by a hydroxyl group.

1019622 -9-

The 4-hydroxy-tetrahydropyran-2-one of formula 2 / γγ * Va where X is defined as above can also be converted to the corresponding dihydroxyhexanoic acid of formula 6 with the aid of an acid or a base.

OH OH O

(6) The dihydroxyhexanoic acid of formula 6 can then be converted to a 2- (1,3-dioxan-4-yl) acetic acid derivative of formula 3, wherein X, R 1 and R 2 are as defined above and R 3 is an alkyl group of 1- 12 carbon atoms, an aryl group with 6-12 carbon atoms or an aralkyl group with 7-12 carbon atoms, for example in the presence of a suitable acetalizing agent and an acid catalyst.

The invention is further elucidated with reference to the examples, without however being limited thereby.

Examples

Preparation and measurement methods 1. Preparation of an amount of 6-chloro-2,4,6-trideoxhexose as a reference material for GC analysis

To 0.62 L of 100 mM NaHCO 3 solution was added at 10 ° C 76 mL (0.60 mol) of a 50% chloroacetaldehyde solution and 84 mL (1.47 mol) of acetaldehyde, after which the pH was measured by e.g. 4N NaOH was adjusted to pH 7.0. The enzymatic reaction was started by the addition of 220 g% n 1 qr22 -10 enzyme solution (2050 U / g). After approximately 18 hours of stirring, a maximum conversion to 6-chloro-2,4,6-trideoxyhexose was measured by GC. The reaction was stopped by adding 1.7 L of acetone. To this mixture, 4 g of dicalite was added and after stirring for about 30 minutes, the mixture was filtered. The filtrate was evaporated in vacuo at 40 ° C for 5 hours. The oil thus formed was purified by means of column chromatography on a silica gel column (400 ml) and eluted with an eluent consisting of a mixture of petroleum ether (b.p. 40-70 ° C) and ethyl acetate in a volume ratio of 1: 2. After evaporating the fraction with an Rf value of 0.33, a total of 26 g of the 6-chloro-2,4,6-trideoxyhexose was isolated. Here, the term Rf value of a fraction is known to the person skilled in the art and is defined as the distance that a fraction travels in the column divided by the distance that the running agent travels in the column or the time it takes a fraction to reach a certain distance. in the column divided by the time it takes for the eluant to cover the same distance in the column.

The purity of the 6-chloro-2,4,6-trideoxyhexose was determined by means of 1 H NMR according to a method described by P.P. Lankhorst in Pharmacopeial Forum, 22, (3), 2414-2422.

2. GC analysis

The concentration of the 6-chloro-2,4,6-trideoxyhexose during the enzymatic reaction was determined by means of gas chromatographic analysis. Use was made of a Hewlett Packard GG with flame ionization detector, type HP5890 series II, equipped with a Chrompack CP-Sil 5 CB column (25 m * 25 0.25 mm, df 1.2 μπι). Concentrations were determined on the basis of a calibration curve of the 6-chloro-2,4,6-trideoxyhexose with known chemical purity (1) in acetone.

3. Preparation of the DERA solution

Standard techniques were applied to the enzyme DERA

to overexpress in recombinant Escherichia coli cells. Such techniques are described in Sambrook et al., "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor, NY, 1989. In the present case, the cteoC gene from Escherichia coli W3110 was cloned into the commercial λ η 1 afi 9 2 -11 - available expression vector pBADftfyc-HisB (Invitrogen, Groningen, NL). The gene (including the stop codon) was multiplied by a polymerase chain reaction and introduced into the Nco and Eco RI sites of the vector in such a way that the ATG start codon of deoC was equal to the ATG codon on the Λ / col location of the vector (translation fusion). Because the deoC stop codon was co-cloned, a His-tag fusion protein, which is also encoded on the vector, was prevented. Escherichia coli DH10B cells (Life Technologies, Breda, NL) with the resulting plasmid pBAD_DERA2.2 were grown in a 10 L aerated and stirred tank reactor 10 on a complex Luria Bertani medium with 100 pg / mL carbenicillin and induced by addition of arabinosetot 0, 01%. To increase cell density, after the cells had consumed the LB medium, two additional aqueous solutions were added containing pure glycerol (feed 1) and 13% yeast extract and 1.3% trypton (Difco, Michigan, US) (feed 2) contain. In this way, approximately 350 g of wet cells were obtained from a 10 L culture, which were recovered by centrifugation, resuspended in a potassium phosphate buffer of pH 7.2 and centrifuged again. The supernatant was removed and the pellet was stored at -20 ° C.

Various methods have been described in the literature for the purification of DERA from recombinant E. coli cells, for example in Chen et al, J. Am. Chem.

Soc. Vol 114, 1992, 741-748 and Wong et al., J. Am. Chem. Soc. Vol 117, 1995, 3333-3339). However, purification is not a condition for efficient use of DERA in aldol reactions, see, for example, Wong et al., J. Am. Chem. Soc. Vol 117.1995, 3333-3339. For the partial purification of DERA, 25 standard techniques were used. Frozen pellets were suspended in a 100 mM potassium phosphate buffer at pH 7.2 (1 part by weight of wet cells and 2 parts by weight of buffer), after which the cell walls were destroyed by continuous ultrasonification. The clear crude extract was obtained by centrifugation and used directly or stored at -20 ° C. The solution can be further concentrated if desired by ultrafiltration, for example with the aid of Millipore ultrafiltration membranes with a cut-off value of 10,000 Dalton (Millipore, Bedford, USA). Typical specific activities obtained by this method are between 1400 and 5300 units per g of solution.

a in 4 m © O O

-12-

4. Determination of the activity of DERA

Recombinant E. coli cells synthesizing DERA were sonicated in a 50 mM potassium phosphate buffer and centrifuged (18,500 rpm, 30 min) to remove the cell material. The resulting free cell extract was diluted and used in a coupled spectrophotometric test. Hereby 2-deoxyribose-5-phosphate was converted to D-glyceraldehyde-3-phosphate and acetaldehyde, after which the D-glyceraldehyde-3-phosphate was converted to dihydroxyacetone phosphate in the presence of triosephosphate isomerase (TIM, EC 10 5.3.1.1). Dihydroxyacetone phosphate was then converted to glycerol-3-phosphate in the presence of glycerol phosphate dehydrogenase (GDH, EC 11.1.8). In the final step, nicotine aminedenine dinucleotide in reduced form (NADH) was consumed, which was followed spectrophotometrically by determining the decrease in absorbance at 340 nm. Typically a total volume of 3 ml was used, containing 2878.5 μΜ of a 50 mM triethanolamine buffer with pH 7.2, 30 μΙ of a 12 mM solution of NADH in water, 11.5 μΙ of an enzyme suspension containing 30 units of GDH and 500 units of TIM in 3.2 M aqueous ammonium phosphate, and 30 μΙ of a 50 mM solution of 2-deoxyribose-5-phosphate in water. The reaction was started by adding 50 μ of free cell extract. The temperature was 37 ° C. The activity, expressed in U (nits), in which 1 unit equals the enzymatic activity corresponding to the conversion of 1 μηηοΙ 2-deoxyribose-5-phosphate per minute at 37 ° C, was calculated from the linear decrease in absorption at 340 nm.

25

EXAMPLE 1

Preparation of 6-chloro-2,4,6-trideoxhexose in a batch experiment

To 37 ml of a 100 mM buffer solution of NaHCO 3 was added 4.3 ml (0.03 mol) of a 45% solution of chloroacetaldehyde and 3.4 ml (0.06 mol) of acetaldehyde at a temperature of 4 ° C. added. The pH of this mixture was 7.3. The enzymatic reaction was started by adding 6.3 mL (5270 U / g) enzyme solution. The reaction was carried out at a constant pH of 7.5. The mixture was analyzed at regular intervals by means of GC. The reaction was stopped by adding 100 mL of acetone after a 1019622 -13 concentration of the 6-chloro-2,4,6-trideoxyhexose of about 80 g / L (8 mass%) was reached.

EXAMPLE II

Preparation of 6-chloro-2,4,6-trideoxhexose in a repetitive batch experiment

The experiment from example I was repeated. However, after a 6-chloro-2,4,6-trideoxyhexose concentration of about 80 g / L was reached, a further 4.3 mL (0.03 mol) of a 45% solution of chloroacetaldehyde, 3.4 mL ( 0.06 mol of acetaldehyde and 6.3 ml (5270 U / g) of enzyme solution added. This was repeated three more times, each time the conversion had reached a constant value and did not increase further. In this way a total of 0.15 mol of a 45% solution of chloroacetaldehyde and 0.30 mol of acetaldehyde was added. The reaction was carried out at a constant pH of 7.5. Samples from the reaction mixture were analyzed using.

GC. The final concentration of 6-chloro-2,4,6-trideoxyhexose was approximately 240 g / L (24 mass%).

EXAMPLE III

Preparation of 6-chloro-2,4,6-trideoxhexose by means of dosing of the 20 reactants.

To 6.2 L of demineralized water was added 84 g (1.0 mol) of NaHCO 3, after which the solution was cooled to approximately 2 ° C. Subsequently, 72.3 mL (0.4 mol) of a 45% solution of chloroacetaldehyde was added thereto, after which the solution was brought to pH 7.3 with the aid of 32% HCl. An enzyme solution (6.8x10® U) was added to this mixture, after which the volume was made up to 7.5 L. For a total of 1.3 hours a total of 1.3 L (9.5 mol) of a 45% chloroacetaldehyde solution was added to this mixture and 0.9 L (9.9 mol) of a 50% acetaldehyde solution dosed. After this, 0.86 L (9.5 mol) of a 50% acetaldehyde solution was added for 5 hours. The reaction mixture was stirred overnight at 2 ° C and a constant pH of 7.5. The total weight of the reaction mixture was 10.9 kg. The content of 6-chloro-2,4,6-trideoxyhexose was determined by means of gas chromatography and was 126 g / L (12.6 mass%).

1019622 - 14- »

EXAMPLE IV

Preparation of 6-chloro-2,4,6-trideoxhexose by means of dosing of the reaction components.

To 0.44 L of demineralized water was added 11.8 g (0.14 mol) of NaHCO 3, after which the solution was cooled to approximately 2 ° C. Subsequently, 10 ml (0.07 mol) of a 45% solution of chloroacetaldehyde was added thereto, after which the solution was brought to pH 7.3 with the aid of 32% HCl. An enzyme solution (1.03 x 10 6 U) was added to this mixture. 0.93 L (1.35 mol) of a 45% chloroacetaldehyde solution and 0.127 L (1.40 mol) of a 50% acetaldehyde solution were added to this mixture for 2 hours. After this, 0.134 L (1.46 mol) of a 50% acetaldehyde solution was added for 5 hours. The reaction mixture was stirred overnight at 2 ° C at a constant pH of 7.5. The content of 6-chloro-2,4,6-trideoxyhexose was determined by means of gas chromatogralia and was 180 g / L (18.0 mass%).

4 th% Λ Λ 15 OO

Claims (19)

Process for the preparation of a 2,4-dideoxyhexose or a 2,4,6-trideoxyhexose from a substituted or unsubstituted carbonyl compound having at least 2 carbon atoms and at least one alpha hydrogen atom and a substituted or unsubstituted aldehyde in presence of an aldolase and water, characterized in that the reaction between the substituted or unsubstituted carbonyl compound with at least 2 carbon atoms and at least one alpha hydrogen atom and the unsubstituted or substituted aldehyde is carried out at a carbonyl concentration of at most 6 mol / l reaction mixture and the final concentration of the 2,4-dideoxyhexose or the 2,4,6-trideoxyhexose is at least 2 mass% of the reaction mixture. 2. Process according to claim 1, characterized in that the final concentration of the 2,4-dideoxyhexose or the 2,4,6-trideoxyhexose is between 5 and 50 mass% of the reaction mixture. Process according to claim 2, characterized in that the final concentration of the 2,4-dideoxyhexose or the 2,4,6-trideoxyhexose is between 10 and 35 mass% of the reaction mixture. Process according to any one of claims 1-3, characterized in that the carbonyl concentration in the reaction mixture during the synthesis process is between 0.6 and 4 mol / l of reaction mixture. 5. A method according to any one of claims 1-4, characterized in that at least 0.1 mol / l of the substituted or unsubstituted aldehyde is submitted. Process according to one of claims 1 to 5, characterized in that the carbonyl compound, whether or not substituted, with at least 2 carbon atoms and at least one alpha hydrogen atom and / or the aldehyde, whether or not substituted, is added in at least two portions 30 to the reaction mixture. Process according to any one of claims 1-5, characterized in that the carbonyl compound, whether or not substituted, with at least 2 carbon atoms and at least one α-hydrogen atom and / or the 4 η λ ήϋΟΟ ψ -16- substituted or not aldehyde are dosed over time to the reaction mixture. 8. A method according to any one of claims 1-7, characterized in that the molar ratio of total added substituted or unsubstituted carbonyl compound with at least 2 carbon atoms and at least one α-hydrogen atom to total added substituted or unsubstituted aldehyde between 1, 5: 1 and 4: 1. The method according to any of claims 1 to 8, characterized in that a 2-deoxyribose-5-phosphate alkase is used as the aldolase. The method according to claim 9, characterized in that the 2-deoxyribose-5-phosphate aldolase is from Escherichia coli. 11. A method according to any one of claims 1-10, characterized in that as substituted or unsubstituted aldehyde a substituted acetaldehyde of the formula HC (O) CH 2 X is used, wherein X represents CN, a halogen, a tosylate group, a mesylate group , an acyloxy group, a phenacetyloxy group, an alkyloxy group or an aryloxy group. Method according to claim 11, characterized in that X is a leaving group. A method according to any one of claims 1 to 12, characterized in that acetaldehyde is used as the carbonyl compound substituted or unsubstituted. The method according to claims 12 and 13, wherein the 2,4-dideoxyhexose or 2,4,6-trideoxyhexose obtained is subsequently converted to the corresponding 4-hydroxy-tetrahydropyran-2-one. The method of claim 14, wherein the 4-hydroxy-tetrahydropyran-2-one is subsequently converted to a 2- (1,3-dioxan-4-yl) acetic acid derivative. The method of claim 15, wherein 2- (1,3-dioxan-4-yl-acetic acid derivative) is subsequently converted to the corresponding 2- (6-hydroxymethyl-1,3-dioxan-4-yl) acetic acid derivative. A method according to claim 15 or claim 16, wherein the 2- (1,3-dioxan-4-yl) acetic acid derivative is the t-butyl ester of the 2- (1,3-dioxan-4-yl-acetic acid). Use of a 2,4-dideoxyhexose or a 2,4,6-trideoxyhexose obtained by a method according to any one of claims 1-13 in the preparation of a medicament. 19. Use according to claim 17, characterized in that the drug is an HMG-CoA reductase inhibitor, preferably a statin. <niafi22