WO2000023608A1

WO2000023608A1 - Preparation of amino alcohols

Info

Publication number: WO2000023608A1
Application number: PCT/NL1999/000655
Authority: WO
Inventors: Martin Karutz; Marcel Gerardus Wubbolts; Holger Paschold
Original assignee: DSM Biotech GmbH
Current assignee: DSM Verwaltungs GmbH
Priority date: 1998-10-20
Filing date: 1999-10-20
Publication date: 2000-04-27
Anticipated expiration: 2001-04-20
Also published as: AU6373199A; NL1010354C2

Abstract

Process for the preparation of an amino alcohol, in which a corresponding α-hydroxy keto compound is brought into contact with a dehydrogenase with the aid of a cofactor in the presence of a compound that contains an -NH group. The hydroxy keto compound is preferably converted in the presence of a micro-organism or parts of it belonging in particular to one of the following genera are used: Candida, Zygoascus, Pseudomonas, Burkholderia, Comomonas, Vibrio, Enterobacter, Yersinia, Klebsiella, and Citrobacter, and especially: Pseudomonas fluorescens, Pseudomonas luteola, Pseudomonas putida, Pseudomonas species, Burkholderia cepacia, Comomonas testosteroni, Vibrio fluvialis, Vibrio species, Enterobacter cloacae, Enterobacter agglomerans, Yersinia species, Klebsiella species, Citrobacter fruendii, Zygoascus hellenicus, Candida species, with special preference for Candida sp. MUCL 41424 and more particularly deriving from Candida sp. MUCL 41424. The hydroxy keto compound is preferably prepared from the corresponding olefinically unsaturated compound, and the cofactor is regenerated.

Description

PREPARATION OF AMINO ALCOHOLS

Description

The present invention relates to a process - for the preparation of an amino alcohol, in which the corresponding α-hydroxy keto compound is brought into contact with a dehydrogenase with the aid of a cofactor in the presence of a compound that contains an -NH group .

The literature does not give any examples of enzymatic reactions in which an α-hydroxy keto compound is converted into an amino alcohol with the aid of an (aminating) dehydrogenase. The Applicant has now found that this conversion can be achieved with a high yield and with a high selectivity.

It has also been found that, if a prochiral compound is used as the α-hydroxy keto compound, the amino alcohol can be obtained in the optically active form. It was thus found that 2-hydroxy- 1-indanone gives (IS, 2R) -cis-aminoindanol selectively out of the four possible diastereomers of l-amino-2-indanol . Amino alcohols are important building blocks in the preparation of e.g. pharmaceuticals. For example, cis-l-amino-2-indanol is used in the preparation of HIV protease inhibitors used as anti- AIDS drugs, as described for example by Thompson et al . in J. Med. , 35, 1992.

The present invention also relates to the preparation of α-hydroxy keto compounds from the corresponding amino alcohols with the aid of a dehydrogenase in the presence of a cofactor.

The enzymatic reaction is often carried out in practice at a temperature of 10-70°C and preferably 20-40°C. The pH at which the enzymatic reaction is conducted is similarly not critical and preferably lies in the range between 4 and 9.

The micro-organism from which the dehydrogenase used in the preparation of amino alcohols is derived can be for example a bacterium, a yeast or a fungus. The micro-organisms in question preferably belong to one of the following genera: Candida, Zygoascus, Pseudomonas, Burkholderia, Comomonas, Vibrio, Enterobacter, Yersinia, Klebsiella, and Citrobacter, and especially: Pseudomonas fluorescens, Pseudomonas luteoia, Pseudomonas putida, Pseudomonas species, Burkholderia cepacia, Comomonas testosteroni , Vibrio fluvialis, Vibrio species, Enterobacter cloacae, Enterobacter agglomerans, Yersinia species, Klebsiella species, Citrobacuer fruendii, Zygoascus hellenicus, Candida species, with special preference for Candida sp. MUCL 41424; it has been found that, in the presence of such a micro-organism, the α-hydroxy keto compound can readily be prepared in si tu from the corresponding diol and even from the corresponding olefinically unsaturated compound. It has thus been found also possible to prepare amino alcohols from the corresponding olefinically unsaturated compounds in a one-pot process. The invention therefore also relates to such a one-pot process. The enzyme preparation used in the present invention is not restricted by purity and the like, so both a crude enzyme solution and a purified enzyme can be used, and the preparation can also consist of (permeabilized and/or immobilized) cells that possess the required activity or of a homogenate of cells with such an activity. The enzyme can also be used in immobilized form or in chemically modified form. If the enzyme preparation used also contains some undesirable, contrary enzymatic activity, it is recommended that this undesirable activity should be removed or suppressed in order to ensure a maximum selectivity. The invention is not limited in any way by the form in which the enzyme is used for the present invention. It is of course also within the scope of the invention to use a related enzyme that is derived e.g. from a mutant or a genetically modified micro-organism.

The micro-organisms can be cultured on various mixtures of compounds that make the growth of the cells possible in the composition used under the conditions described above. According to the procedure adopted, it is possible to use as a carbon source made from carbohydrates (e.g. glucose or sucrose), organic acids (e.g. acetic, lactic or succinic acid) or salts thereof. It is also possible to employ substrates that are converted along the metabolic route and contain the required enzymes. The nitrogen source can be provided by organic nitrogen compounds (e.g. yeast extract, casamino acids and peptone) , inorganic nitrogen compounds (e.g. ammonium sulphate or ammonium phosphate) or amino alcohols that form the product of the reaction involved. If necessary, vitamins and trace amounts of organic or inorganic salts can also be added to the medium.

The micro-organisms are often cultured in practice at a temperature of between 20 and 50°C and preferably at 25-30°C. The cultures are often incubated at a pH of 4-9, the preferred pH range being 4.5-5.5 for yeasts and fungi, and 6.5-7.5 for bacteria. The cultures are e.g. aerated by passing through them a gas containing molecular oxygen, or else by introducing into them another gaseous or liquid compound that can be used by the organisms as a source of oxygen. Another alternative is to stir or shake the culture mechanically. The micro-organisms can be cultured in a reaction mixture that contains a single aqueous liquid phase or in a mixture of two liquid phases, one of which is aqueous and the other is an organic solvent such as an alcohol (particularly decanol) , an alkane (e.g. decane) , or a synthetic or natural oil (e.g. silicone oil, rapeseed oil or sunflower- seed oil etc.) .

The micro-organisms can be cultivated in a batch process, a fed-batch process or a continuous culture. In the case of a batch process, all the substrates are introduced into the reaction mixture at the beginning of the reaction. In zhe fed-batch process, on the other hand, the substrates are partly introduced during the process, and f the reaction mixture comprises two phases, the substrates can also be added as a solution in the organic solvent or in the oil. In the case of a continuous process, the intended substrates and the other components of the medium can be added separately in order to decouple the product formation from the residence time.

The concentration of both the substrate and the product can be chosen so that both are present in dissolved form in the reaction mixture. The concentrations can also be chosen to be so high that part of the substrate and/or of the product is present in solid form.

Single-stage processes (one-pot synthesis) are characterized by a substrate conversion that is simultaneous with cell growth. In the case of a two- stage process, the micro-organisms are first cultured, and the resulting biomass or parts of it is brought into contract with the substrate, optionally after the cell residues have been removed. This approach also makes cross-feeding possible, that is to say specific components such as buffer salts are added to further improve the conversion of the substrate in the second stage of the process, as is known to the person skilled in the art. α-Hydroxy keto compounds can also exist in the tautomeric form, and therefore this form is also included in the term "α-hydroxy keto compounds". E.g. the following α-hydroxy keto compounds can be used in the process according to the present invention: α-hydroxy ketones and α-hydroxy aldehydes, especially primary and secondary aromatic hydroxy ketones, for instance: 2 -hydroxy- 1-phenylethan- 1 -one , 2 -hydroxy- 1 - phenylpropan-1-one, 2 -hydroxy- 1-phenylbutan-l-one , 2- hydroxy-2 , 3 , 4-trihydronaphthalen-l-one , 3-hydroxy-4- phenylbutan-2-one, 2 -hydroxy-1-phenylpentan-3 -one, 2- hydroxy-l-phenylhexan-3 -one, and 2 -hydroxy-1-indanone primary and secondary aliphatic hydroxy ketones, for instance: 1-hydroxyacetone, 3-hydroxybutan-2-one, 1- hydroxybutan-2-one, 2-hydroxypentan-3-one, 1- hydroxypentan-2-one, 2-hydroxyhexan-3 -one, 3- hydroxypent-4-en-2 -one, and 4-hydroxyhex-5-en-3-one, saturated and unsaturated aliphatic cyclic hydroxy ketones, for instance: 2 -hydroxycyclopentan-1-one, 2- hydroxycyclohexan-1-one, 5-hydroxycyclopent-2-en-l-one, 2 -hydroxycyclopent-3 -en- 1 -one, 6-hydroxycylohexa-2 , 4- dien-1-one, 2-hydroxycyclohex-3-en-l-one , and 6- hydroxycyclohex-3 -en-l-one, primary and secondary aromatic hydroxy aldehydes, for instance: 2 -hydroxy-2 - phenylethanal, 2 -cyclopenta-2 , 4-dienyl-2- hydroxyethanal , 2 -hydroxy-3 -phenylpropanal , and 2 - hydroxy-4 -phenylbutanal , primary and secondary aliphatic hydroxy aldehydes, for instance: 2- hydroxypro anal , 2-hydroxybutanal , and 2- hydroxypen anal, and saturated and unsaturated cyclic aliphatic hydroxy aldehydes, for instance: 2- cyclohexyl-2-hydroxyethanal and 2-cyclopentyl-2- hydroxyethanal .

The α-hydroxy keto compounds that can be used in the process according to the present inventions are in some cases commercially available and can be prepared by a conventional method, e.g. chemically as described by Baskaran et al . (1986) in J.O.C. 54, 5182 and by N.S. Srinivasan and D.G. Lee (1979) in Synthesis (7), 520-1, or enzymatically as described by S. Ui et al . (1996) in J. Ferment. Bioeng., 81 (5), 386-389.

The α-hydroxy keto compounds can also be prepared in si tu from the corresponding diol, as described above. The diols are in some cases commercially available or can be prepared conventionally as described by J.M. Brand et al . (1992) in Appl . Environ. Microbiol . , 58 (10), 3407-9 and by T. Hudlicky et al . (1996) in J. Am. Chem. Soc . , 118 (44), 10752-65.

In addition, the diols can also be prepared in si tu , as described above, from the corresponding olefinically unsaturated compound which as a rule is available commercially per se . Any compound acting as a donor of a primary or secondary amino group can be used as the amino donor, for example ammonia, inorganic ammonium compounds (e.g. ammonium salts, and especially ammonium sulphate or ammonium phosphate) or, preferably in the case of whole cells, an (organic) compound containing a primary or secondary amino group, for instance glutamine .

The cofactor can be a hydrogen-transferring cofactor, for example NADH, NADPH or FADH₂. The cofactor is preferably regenerated, e.g. by the conventional methods, especially enzymatically, for example with the aid of formate dehydrogenase or alcohol dehydrogenase, or else by an electrochemical method. The invention is further explained below with reference to the following examples but is not limited by them.

Example 1 - Screening experiment

Natural samples of sewage, sewage sludge and soil were used as the starting material in the selection of a biocatalyst capable of converting 1- keto-2-hydroxyindane (chosen as an example of an α- hydroxy keto compound) into l-amino-2-hydroxyindane, representing an example of an amino alcohol. These samples were incubated in a complex medium and in a mineral medium. The complex medium consisted of: 10 g/1 yeast extract, 10 g/1 peptone and 5.0 g/1 sodium chloride. The mineral medium contained the following constituents: 2.6 g/1 trisodium citrate dihydrate, 1 g/1 magnesium sulphate heptahydrate , 0.5 g/1 sodium chloride, 0.08 g/1 ferrous sulphate heptahydrate, 0.1 g/1 calcium chloride dihydrate, and 5.5 g/1 phosphoric acid. A stock solution (1 ml/1) was added containing the following further minerals: 2.0 g/1 aluminium sulphate.18 H₂0, 0.7 g/1 cobalt (II) sulphate hexahydrate, 2.5 g/1 copper (II) sulphate pentahydrate, 0.5 g/1 boric acid, 20.0 g/1 manganese (II ) chloride tetrahydrate, 3.0 g/1 Na₂Mo0₄.2H₂0, 2.0 g/1 NiS0₄.3H₂0, and 15.0 g/1 ZnS0₄.7H₂0.

The pH was adjusted to 5 by adding KOH, and the medium was treated m an autoclave for 15 min at 121°C. A solution of 0.01 g/1 thiamme hydrochloride and 0.001 g/1 biotin was added to the sterile medium after passing the latter through a microfilter. 2 g/1 of a racemic mixture of (IS, 2R) -cis-l-amino-2-indanol and (1R, 2S) -cis-l-amino-2 -indanol were added as the sole nitrogen source before sterilizing the medium. 4 g/1 glucose were introduced in the form of a sterile solution as the carbon source. A mineral medium without sodium citrate was also used, which - unlike the medium described above - was titrated to pH 7 with potassium hydroxide. The cuituring was done at 28 and 37°C. Organisms that could grow on the mineral medium by utilizing l-amino-2 -indanol were streaked out and kept on an agar plate prepared with a mineral medium and agar (15 g/1) .

An example of an organism capable of growing on l-amino-2 -indanol was the isolate Candida sp. MUCL 41424, which had been isolated from a Canadian soil sample contaminated with bitumen.

Example 2 The isolate Candida s . MUCL 41424 was cultured on the mineral medium at pH 5 with 3 g/1 1- amino-2 -indanol as the nitrogen source. 10 ml of the medium were introduced in a 100-ml shaker flask, which was placed on a shaker and incubated at 25 °C. The extent of cell growth was determined by measuring the optical density of the medium with a spectrophotometer at 620 nm. High-performance liquid chromatography (HPLC) was used to determine the concentration of the l-amino-2 -indanol (c) under the following chromatographic conditions: column: Nucleosil 120-5 C₁₈,

25 x 4 mm, eluent A: methanol, eluent B: acetonitrile, temperature: 40°C, pre-column derivatizing with o-phthalaldehyde in B(OH)₃, fluorescence detection: extinction at 230 nm, emission at 455 nm.

The culture reached an optical density of 10 after incubation for 110 h. This indicated that the cells could utilize l-amino-2 -indanol as a nitrogen source and convert it into l-keto-2-hydroxyindane (see Fig. 1, wherein c is given as a function of time (in hours) ) .

Example 3

The isolate Candida sp . MUCL 41424 was cultured on a complex medium containing 11.7 g/1 Difco Yeast Carbon Base (YCB) and 6.7 g/1 Difco Yeast Nitrogen Base (YNB) . The cells were harvested at an optical density of 18 (measured at 620 nm) by centrifuging the medium at 6000 rpm for 5 min. The cells were diluted with potassium phosphate buffer (pH 7.2) to an optical density of 220. 0.6 ml of diluted cells were then mixed with 1.2 g of 0.5 -mm glass beads and shaken for 12 min in a Retsch type laboratory glass-bead mill. The beads were then removed by sedimentation in a bench centrifuge.

The presence of an aminating dehydrogenase converting the α-hydroxy keto compounds into amino alcohols was demonstrated with the aid of a spectrophotometer containing a cuvette with 1000 μl of potassium phosphate buffer (pH 7.2) 10 μl of NADH (2.5 mM), 50 μl of YNB (67 g/1) and 50 μl of cell extract. The reaction was started by adding 100 μl of l-keto-2-hydroxyindane (3 g/1) and was carried out at 25°C. The conversion was determined by monitoring the decrease in the NADH concentration (c, measured as the extinction at 340 nm, as a function of time t in minutes (see fig. 2)) . As Fig. 2 shows, the rate of reaction during the first 10 min was 2.5 times as high in the presence of the α-hydroxy keto compound (♦) as in the control reaction (■) . The introduction of the α- hydroxy keto compound into the mixture used in the control reaction (at a point in time indicated by an arrow in Fig. 2) resulted in a marked increase in the rate of reaction.

Example 4 The following substances were used as the α-hydroxy keto compound instead of l-keto-2- hydroxyindane in an experiment comparable with Example 3: hydroxyacetone (■) , 2 -hydroxy- 1-indanone (•) and 3- hydroxy-2-butanone (♦) (and control reaction (o) ) . Fig. 3 shows the decrease in the NADH concentration, determined with a spectro photometer at 340 nm as a function of time (t) in minutes. These results indicate that all the components were converted by the enzyme, causing a decrease in the NADH concentration. Concommittent formation of amino groups by the enzyme has been observed by HPLC analysis of the assay incubation mixture (precolumn derivatization carried out as in example 2) .

Example 5 Example 3 was repeated on a larger scale to make it possible to determine the formation of 1-amino- 2 -indanol directly. For this purpose, the reaction was carried out at 25°C in a mixture containing 10 ml of potassium phosphate buffer (pH 7.2), 100 μl of NADH

(2.5 mM) , 1 ml of Yeast Nitrogen Base (67 g/1), and 500 μl of cell extract.

The reaction was initiated by the addition of 1 ml of 3 g/1 of l-keto-2-hydroxyindane, and it was stopped after 3 h of reaction time by the addition of

500 μl of 4N potassium hydroxide. 5 ml of the resulting mixture were combined with 5 ml of dichloromethane to extract the product from the aqueous phase. The organic phase was separated from the aqueous phase and evaporated to dryness at 60°C. The dry residue thus obtained was dissolved in 500 μl of methanol for analysis by HPLC. The results showed that a significant amount of l-amino-2 -indanol had been formed.

Example 6

The isolate Candida sp . MUCL 41424 was incubated in a 1 -litre shaker flask containing 100 ml of the Yeast Nitrogen Base medium and 0.1 ml of indene, with mechanical stirring. After this first stage, the following two substances were added to the culture at set times every day for 14 days: 1C ml of Yeast Nitrogen Base (67 g/1) and 1-2 ml cf a l:5-mixture (by volume) of indene and silicone oil.

The presence of 1-aminc-2 -indanol was detected by HPLC analysis as described in Example 2. The enantiomeric purity of the resulting 1- amino-2 -indanol was then determined by a different HPLC method, carried out under the following conditions: column: Nucleosil 120-5-C₁₈, 25 x 4 mm; eluent A: 50 mM phosphate buffer at pH 7; eluent B: a 50:50 methanol buffer mixture; temperature: 40°C; pre-column derivatizing with o-phthalaldehyde and N-acetylcysteine (L-NAC; 30 mM) , fluorescence detection: extinction at 230 nm, emission at 455 nm. The results shown in Fig. 4 indicate that the isolate Candida sp . MUCL 41424 can convert indene into (IS , 2R) -cis-l-amino-2-indanol with a high stereospecificity and a high enantioselectivity. The upper part represents the reference material racemic cis-l-amino-2-indanol and racemic trans-1- amino-2- indanol ; the lower part represents the sample.

Claims

1. Process for the preparation of an amino alcohol, characterized in that the corresponding α-hydroxy keto compound is brought into contact with a dehydrogenase with the aid of a cofactor in the presence of a compound that contains an -NH group.

2. Process according to claim 1, in which a dehydrogenase derived from a micro-organism belonging to one of the following genera Candida, Zygoascus, Pseudomonas, Burkholderia, Comomonas, Vibrio, Enterobacter, Yersinia, Klebsiella, and Citrobacter is used.

3. Process according to claim 2 wherein the dehydrogenase is derived from Pseudomonas fluorescens, Pseudomonas luteola, Pseudomonas putida, Pseudomonas species, Burkholderia cepacia, Comomonas testosteroni , Vibrio fluvialis, Vibrio species, Enterobacter cloacae, Enterobacter agglomerans, Yersinia species, Klebsiella species, Citrobacter fruendii, Zygoascus hellenicus or Candida species.

4. Process according to claim 3, in which a dehydrogenase derived from Candida sp . MUCL 41424 is used.

5. Process according to any one of claims 1-4, in which the hydroxy keto compound is converted in the presence of a micro-organism or parts of it.

6. Process according to claim 5, in which the α- hydroxy keto compound is prepared in si tu from the corresponding diol .

7. Process according to claim 6, in which the corresponding diol is prepared in si tu from the corresponding olefinically unsaturated compound.

8. Process according to any one of claims 1-7, in which the cofactor is regenerated.

9. Process according to any one of claims 1-8, in which the reaction is carried out in a two-phase system.

10. Process for the preparation of an α-hydroxy keto compound, characterized in that the corresponding amino alcohol is brought into contact with a dehydrogenase in the presence of a cofactor.