(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 27 January 2011 (27.01.2011) WO 2011/009849 A2 (51) International Patent Classification: (74) Common Representative: BASF SE; 67056 Lud C12P 7/62 (2006.01) wigshafen (DE). (21) International Application Number: (81) Designated States (unless otherwise indicated, for every PCT/EP2010/060458 kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (22) Date: International Filing CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, 20 July 2010 (20.07.2010) DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (25) Filing Language: English HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, (26) Publication Language: English ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, (30) Priority Data: NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, 09166015.9 2 1 July 2009 (21 .07.2009) EP SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, 61/227,797 23 July 2009 (23.07.2009) US TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (71) Applicants (for all designated States except US): BASF (84) Designated States (unless otherwise indicated, for every SE [DE/DE]; 67056 Ludwigshafen (DE). MAX- kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN E.V. [DE/DE]; Hofgarten- ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, str. 8, 80539 Mϋnchen (DE). TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, (72) Inventors; and LV, MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, (75) Inventors/Applicants (for US only): STURMER, Rain- SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, er [DE/DE]; Hauptstrasse 153, 67127 Rόdersheim- GW, ML, MR, NE, SN, TD, TG). Gronau (DE). SCHNEIDER, Nina [DE/DE]; Carl-Blos- Str. 3, 77654 Offenburg (DE). BOY, Matthias [DE/DE]; Published: Ernst-Ludwig-Str. 39 a, 64625 Bensheim (DE). — without international search report and to be republished ACHATZ, Brigitte [DE/DE]; Windeckstr. 26, 681 63 upon receipt of that report (Rule 48.2(g)) Mannheim (DE). RABUS, RaIf [DE/DE]; Hinrich- — with sequence listing part of description (Rule 5.2(a)) Schmidt-Str. 52, 26 160 Bad Zwischenahn (DE). HEI- DER, Johann [DE/DE]; Spiegelslustweg 24b, 35039 Marburg (DE). (54) Title: METHOD FOR PREPARING OPTICALLY ACTIVE HYDROXY ACID ESTERS (57) Abstract: The invention relates to a method for preparing optically active 2-hydroxy acid ester derivatives of the formula Rl- C(OH)-C(O)-O-R2 (I),comprising the bringing into contact of 2-oxo acid ester derivatives of the formula R1-C(O)-C(O)-O-R2 (II) with an enzyme (E) selected from the class of dehydrogenases, in the presence of reduction equivalents, where the compound of the formula (II) is enzymatically reduced to the compound of the formula (I), and the reduction equivalents consumed in the course of the reaction are regenerated again by converting a reducing agent (RA) to the corresponding oxidation product (OP) with the help of the enzyme (E). The invention further comprises a dehydrogenase which reduces 2-oxo acid esters in the presence of reduction equivalents to the corresponding S- 2-hydroxy acid esters, and also a nucleic acid encoding the dehydrogenase. Method for preparing optically active hydroxy acid esters The present invention relates to a method for obtaining optically active hydroxy compounds through enantioselective reduction of organic keto compounds and methods for obtaining these hydroxy compounds in the two-phase system using enzymes with dehydrogenase activity. Background of the invention Optically active hydroxy compounds, such as, for example, ethyl 2-hydroxy-4- phenylbutyrate, of very high enantiomer purity are important precursors in the synthesis of drugs, in particular inhibitors of the so-called angiotensin converting enzyme (ACE), which are used for treating patients with hypertension. Many of these inhibitors, such as enalapril, ramipril, cilazapril, quinapril and lysinapril, have a common general structural feature which is responsible for improved application properties. The feature common to such inhibitors is the S-enantiomer form of the 2-amino-4-phenylbutyrate with the structural formula (Ia). The S-2-hydroxy-4-phenylbutyric acid of the formula Ib (R enantiomer) is used for preparing isomeric compounds. Obtaining chiral compounds through stereospecific microbiological reduction is known (for an overview cf. Simon et al., Angew. Chemie 97, 541 , 1985). The biocatalysts used are often intact microorganisms, for example fungi (e.g. Mucor, Geotrichum, Saccharomyces, Candida) or bacteria (e.g. Proteus, Pseudomonas). It is also possible to use microbial extracts. Electron donors are, for example, carbohydrates (e.g. glucose), formate, ethanol, hydrogen or the cathode of an electrochemical cell. Reduction of the substrate takes place through a reductase, e.g. through a substrate-specific dehydrogenase. In general, the reduction equivalents required by the reductase are supplied by a coenzyme, e.g. by pyridine nucleotides such as NADH (nicotinamide adenine dinucleotide) and NADPH (nicotinamide adenine dinucleotide phosphate) or by flavin nucleotides such as FMNH (flavin mononucleotide) and FADH (flavin adenine dinucleotide). The reduced nucleotides for their part are usually formed in a series of enzyme-catalyzed steps with the formation of competing electron acceptors or by electron transfer via natural or synthetic mediators (e.g. ferredoxin, viologens). Also known are final reductases which can absorb electrons directly from the mediators. For example, Lacerda et al., Tetrahedron: Asymmetry 17, 2006, pages 1186-1 188, describes a method for the microbial reduction of 2-oxo-4-phenylbutyrates using various bacterial strains. The European patent specification EP 0 347 374 describes a method of preparing R-2- hydroxy-4-phenylbutyric acid in which the substrate is reduced with the enzyme D-lactate dehydrogenase from Staphylococcus epidermidis in the presence of an electron donor and an enzyme-substrate system for regenerating the electron donor. Furthermore, WO2005/049816 discloses a NADPH-dependent dehydrogenase from Metschnikowia zobellii which, in the presence of water and NADPH, catalyzes the stereoselective reduction of carbonyl compounds to the corresponding chiral hydroxy compounds. EP 0 645 453 discloses an enantioselective alcohol dehydrogenase which is suitable for the reduction of organic keto compounds to the corresponding hydroxy compounds, this reduction leading enantioselectively to the corresponding R compounds. Short description of the Invention: The object of the invention was to find a route for the enantioselective reduction of 2-oxo acid esters, in particular ethyl 2-oxo-4-phenylbutyrate, wherein the reaction method should lead as quantitatively as possible to the product by a cost-effective route. This object was achieved by the surprising finding that enzymes with dehydrogenase activity, in particular those which can be prepared from microorganisms of the genus Azoarcus, are capable of the enantioselective catalysis of the above reaction with simultaneous cofactor regeneration. The invention firstly provides a method for preparing optically active 2-hydroxy acid ester derivatives of the formula (I), R1-C(OH)-C(O)-O-R2 or a salt thereof, wherein R 1 and R2, independently of one another, are a) -(C1-C20)-alkyl, in which alkyl is straight-chain or branched, b) -(C2-C20)-alkenyl, in which alkenyl is straight-chain or branched and comprises one, two, three or four double bonds depending on chain length, c) -(C2-C20)-alkynyl, in which alkynyl is straight-chain or branched and optionally comprises one, two, three or four triple bonds, d) -(C6-C14)-aryl, e) -(C1-C8)-alkyl-(C6-C14)-aryl, f) -(C5-C14)-heterocycle which is unsubstituted or mono- to trisubstituted by halogen, hydroxyl, amino or nitro, or g) -(C3-C7)-cycloalkyl, wherein the residues specified above under a . to g . are unsubstituted or are mono- to trisubstituted, independently of one another, by 1. -OH, 2 . halogen, such as fluorine, chlorine, bromine or iodine, 3 . -NO2, 4 . -C(O)-O-(CI -C20)-alkyl, in which alkyl is straight or branched and is unsubstituted or mono- to trisubstituted by halogen, hydroxyl, amino or nitro, or 5 . -(C5-C14)-heterocycle which is unsubstituted or mono- to trisubstituted by halogen, hydroxyl, amino or nitro the method comprising the bringing into contact of 2-oxo acid ester derivatives of the formula (II) R1-C(O)-C(O)-O-R2 with an enzyme (E) selected from the class of the dehydrogenases, in the presence of reduction equivalents, wherein the compound of the formula (II) is enzymatically reduced to the compound of the formula (I), and the reduction equivalents consumed in the course of the reaction are regenerated again by converting a reducing agent (RA) to the corresponding oxidation product (OP) with the help of the enzyme (E). Depending on the dehydrogenase used, the -OH group of the formula (I) is in the S configuration (Ia) or in the R configuration (Ib) relative to the carbon atom to which it is bonded. Enzymes (E) suitable according to the invention are in particular the enzymes of the families of the aldo-keto reductases of the aldo-keto reductase superfamily (K.M.Bohren, B.Bullock, B.Wermuth and K.H.Gabbay J.Biol. Chem. 1989, 264, 9547-9551) and of the short-chain alcohol dehydrogenases/reductases (SDR). The latter enzyme group is described in detail, for example, in H.Jδrnvall, B.Persson, M.Krook, S.Atrian, R.Gonzalez-Duarte, J.Jeffery and D.Ghosh, Biochemistry, 1995, 34, pp.