F O R M 2
THE PATENTS ACT, 1970 (39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
1. Title of the invention. –
ENZYME CATALYZED PREPARATION OF CARBAMIC ACID (R)-1-ARYL-2-TETRAZOLYL-ETHYL ESTER
2. Applicant(s)
(a) NAME : LUPIN LIMITED
(b) NATIONALITY : An Indian Company
(c) ADDRESS : Kalpataru Inspire, 3rd Floor,
Off Western Express Highway, Santacruz (East), Mumbai – 400 055, Maharashtra, India
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed :

FIELD OF INVENTION
The present invention provides process for preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester comprising preparation of (R)-1-aryl-2-tetrazolyl-ethyl alcohol by enzymatic reduction of the arylketone.
BACKGROUND OF THE INVENTION
The PCT application, WO 2006112685 (A1) discloses Carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester compounds (I) as neurotherapeutic agents. The specific compound [(1R)-1-(2-Chlorophenyl)-2-(tetrazol-2-yl) ethyl] carbamate (I’) is approved as Xcopri ® and it is indicated for the treatment of partial-onset seizures in adult patients. Another PCT application WO 2011046380 (A2) provides process for preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester compounds (I).

The present invention provides alternate, industrially feasible and cost-effective process for preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester compounds and compound [(1R)-1-(2-Chlorophenyl)-2-(tetrazol-2-yl) ethyl].
SUMMARY OF THE INVENTION
In one embodiment, the present invention relates to biocatalytic conversion of aryl ketone (II) to corresponding (R)-1-aryl-2-tetrazolyl-ethyl alcohol (III) and the polypeptides that mediate the conversion.


The biocatalysts used herein are ketoreductases derived from microorganisms such as Lactobacillus, particularly non-naturally occurring ketoreductases engineered for mediating the conversion with increased activity, high enantiomeric excess, high percent conversion in the presence of high substrate loading, and capable of regenerating the cofactor by its in situ activity as an alcohol dehydrogenase. Accordingly, in some embodiment, aryl ketone (II) are subjected to ketoreductase described herein, particularly a non-naturally occurring or engineered ketoreductase polypeptides, in the presence of cofactors NADPH or NADH under suitable reaction conditions to obtain corresponding (R)-1-aryl-2-tetrazolyl-ethyl alcohol (III).
In another embodiment, the present invention provides a process for preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester (I) wherein R is selected from hydrogen, halogen, alkoxy or halo alkyl comprising:

(i) subjecting aryl ketone (II) to ketoreductase, in the presence of NADPH or NADH cofactor under suitable reaction conditions to form (R)-1-aryl-2-tetrazolyl-ethyl alcohol (III) and


(ii) converting (R)-1 -aryl-2-tetrazolyl-ethyl alcohol (III) to carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester (I)
wherein, ketoreductase is non-naturally occurring ketoreductases engineered for mediating the conversion of aryl ketone (II) to (R)-1-aryl-2-tetrazolyl-ethyl alcohol (III).
DETAILED DESCRIPTION OF THE INVENTION
In the first embodiment, the present invention provides a process for preparation of (R)-1-aryl-2-tetrazolyl-ethyl alcohol (III) comprising subjecting aryl ketone (II) to ketoreductase in presence of cofactor under suitable reaction conditions, wherein the ketoreductase is capable of converting ketone (II) to corresponding alcohol (III) in enantiomeric excess and the ketoreductase has at least 80% homology with amino acid sequence SEQ ID NO: 1.
In the second embodiment, the present invention provides process for preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester (I) comprising:
(i) subjecting aryl ketone (II) to ketoreductase to form (R)-1-aryl-2-tetrazolyl-
ethyl alcohol (III) and (ii) converting (R)-1 -aryl-2-tetrazolyl-ethyl alcohol (III) to carbamic acid (R)-
1-aryl-2-tetrazolyl-ethyl ester (I)
wherein the ketoreductase is capable of converting ketone (II) to alcohol (III) in enantiomeric excess, in the presence of suitable cofactors and suitable reaction conditions and the ketoreductase has at least 80% homology with amino acid sequence SEQ ID NO: 1.

In a further embodiment herein, the ketoreductases used in the process are derived from Lactobacillus, particularly Lactobacillus kefir. In some embodiments, ketoreductases are naturally occurring ketoreductase of Lactobacillus kefir represented by SEQ ID NO: 1.
In another embodiments, the ketoreductases are non-natural, engineered ketoreductases that have residue differences as compared to the naturally occurring ketoreductase of Lactobacillus kefir represented by SEQ ID NO: 1. These differences occur at residue positions that can affect enzyme activity, stereoselectivity, thermostability, solvent stability, polypeptide expression, co-factor affinity, or various combinations thereof. In particular, the engineered polypeptides can have one or more residue difference as compared to SEQ ID NO:1 at the following residue positions: X17, X25, X29, X40, X43, X64, X71, X76, X80, X87, X94, X95, X96, X131, X144, X145, X147, X150, X152, X153, X157, X173, X190, X194, X195, X196, X199, X200, X226, X233, and X249. Guidance on amino acid residues that can be present at these positions as well as combinations of residue differences useful for generating enzymes with improved properties are described in detail in the descriptions herein.
In some embodiments, the engineered ketoreductase polypeptides capable of converting aryl ketone (II) or corresponding structural analog to (R)-1-aryl-2-tetrazolyl-ethyl alcohol (III) or corresponding structural analog in enantiomeric excess, comprises an amino acid sequence that has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a reference polypeptide selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18.
In some embodiments, the non-naturally occurring ketoreductase polypeptide for use in the processes disclosed herein and capable of converting ary ketone (II) to corresponding alcohol (III) in enantiomeric excess with activity that is equal to or with at least 2-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, or at least 35-fold or more activity of the polypeptide of SEQ

ID NO: 1 comprises an amino acid sequence that has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a reference amino acid sequence selected from any one of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19, with the proviso that the amino acid sequence comprises any one of the set of residue differences as compared to SEQ ID NO: 1 contained in any one of the polypeptide sequences of SEQ ID NO: 2 to SEQ ID NO: 19 listed in Table 1. In some embodiments, in addition to the set of amino acid residue differences of any one of the non-naturally occurring polypeptides of SEQ ID NO: 2 to SEQ ID NO: 19, the sequence of the non-naturally occurring polypeptide can further comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40 residue differences at other amino acid residue positions as compared to the SEQ ID NO: 1. In some embodiments, the residue differences can comprise conservative substitutions and non-conservative substitutions as compared to SEQ ID NO: 1.
Table 1:

Seq. ID No. Residue Differences
(Relative Sequence ID No. 1)
1 -
2 A80T, A94G, S96V, E145L, L153T, Y190P, V196L, I226V, Y249W
3 A80T, A94G, S96V, E145L, L153T, Y190P, V196L, I226V, Y249W
4 A80T, A94G, S96V, E145L, F147Q, L153T, Y190P, V196L, I226V, Y249W
5 A80T, A94G, S96V, E145L, L153T, Y190P, V196L, I226V
6 A80T, A94G, S96V, E145L, D150L, L153T, Y190P, V196M, L199M, I226V
7 A80T, S96V, I144V, E145L, L153T, Y190P, Y190P, V196M, L199M, I226V, Y249F

8 A80T, A94G, S96V, E145L, F147Q, L153T, Y190P, V196M, L199M, I226V,
9 A80T, A94G, V95M, S96V, I144V, E145L, F147Q, L153T, Y190P, V196M, L199M, I226V, Y249F
10 A80T, A94G, V95M, S96V, I144V, E145L, L153T, Y190P, V196L, L199M, I226V
11 L17M, A80T, A94G, S96V, I144V, E145L, F147Q, D150L, L153T, Y190P, P194, V196L, L199M, I226V
12 V43R, A64V, T71P, A80T, V87L, A94G, S96V, E145L, F147Q, L153T, D173L, Y190P, V196M, L199M, I226V
13 V43R, A64V, A80T, A94G, S96V, E145L, F147Q, L153T, D173L, Y190P, V196M, L199M, I226V, D233G
14 H40R, V43R, A64V, T71P, A80T, V87L, A94G, S96V, E145L, F147Q, L153T, D173L, Y190P, V196M, L199M, I226V
15 E29T, H40R, V43R, A64V, T71P, T76A, A80T, V87L, A94G, V95Y, S96R, E145L, F147Q, T152L, L153T, N157C, D173L, Y190P, V196M, L199M, E200P, I226V
16 E29T, H40R, V43R, A64V, T71P, A80T, V87L, A94G, V95Y, S96R, N131C, E145L, F147Q, T152A, L153T, D173L, Y190P, V196M, L199M, I226V
17 E29T, H40R, A64V, T71P, A80T, V87L, A94G, V95Y, S96R, N131C, E145L, F147Q, T152A, L153T, D173L, Y190P, V196M, L199M, E200P, I226V
18 L17H, D25T, E29T, H40R, A64V, T71G, A80T, V87L, A94G, V95Y, S96R, N131C, E145L, F147Q, T152A, L153T, N157S, D173L, Y190P, V196M, L199M, E200P, I226V
19 L17H, E29T, H40R, A64V, T71P, A80T, V87L, A94G, V95Y, S96R, N131C, E145L, F147Q, T152A, L153T, N157S, D173L, Y190P, V196M, L199M, E200P, I226V

The ketoreductases referred herein are procured from Codexis Inc., and are described in greater details including polynucleotides encoding these ketoreductases, expression vectors and host cells and methods to express, isolate and purification of these ketoreductases in the PCT application WO2012/142302 by Codexis, which is incorporated by reference herein.
Suitable reaction conditions employing the ketoreductases, cofactors are provided in greater details below.
The ketoreductase is added to the reaction mixture in the form of the purified enzymes, whole cells transformed with gene(s) encoding the enzymes, and/or cell extracts and/or lysates of such cells. The reaction may be carried out in single phase system or using biphasic system such as aqueous-organic solvent system.
The Aryl ketone (II) is treated with the enzyme in presence of suitable buffer, cofactor and an alcohol and optionally mixture of alcohol and a solvent.
The cofactor used in present invention is preferably nicotineamide adenine dinucleotide phosphate (NADP) or nicotineamide adenine dinucleotide (NAD), which are utilized in the reduced state, i.e. NADPH or NADH, respectively.
The Buffer can be selected from Phosphate buffer, triethanol amine and the like. The alcohol can be selected from methanol, ethanol, propanol, butanol and solvent can be selected from organic solvent such as dimethyl sulfoxide.
The Phosphate buffer preparation was typically prepared as (0.2M, pH 7.00, 1mM NADP, 2 mM MgSO4 7H2O): 1.36 g KH2PO4 was dissolved in 50 ml demineralized water and 1.74 g K2HPO4 was dissolved in 50 ml demineralized water, both the solutions were mixed to adjust the pH 7.00. The preparation of Triethanol amine-MgSO4 buffer is typically prepared by mixture of 19.07 g of triethanol amine and 0.320 g MgSO4 7H2O in 1280 ml demineralized water.
In the present invention, the enzyme and co-factor loading with respect to the substrate is very low, enzyme is in the range of 5 mg per gram of substrate and cofactor is in the range of 1 mg per gram of substrate.

The ketoreductase reaction provides up to 100% conversion of the substrate with high enantioselectivity. The reaction provides “enantiomerically pure” (R)-1-aryl-2-tetrazolyl-ethyl alcohol (III).
The term “ketoreductase” means enzyme catalyst reduction reaction of the ketone functional group of the compound.
The term “enantiomerically pure” (R)-1-aryl-2-tetrazolyl-ethyl alcohol (II) refers to (R)-1-aryl-2-tetrazolyl-ethyl alcohol (III) with a chiral purity of greater than 95%, 96%, 97%, 98%, 99%, preferably greater than 99%, more preferably greater than 99.5 to 99.9%, by HPLC.
In some embodiments, the non-naturally occurring ketoreductases showed improved enzymatic properties for the conversion of aryl ketone (II) to (R)-1-aryl-2-tetrazolyl-ethyl alcohol (III), relative to the naturally occurring ketoreductase polypeptide of SEQ ID NO: 1, including, among others, increased conversion rates, high stereoselectivity, increased solvent stability, and increased thermal stability.
In some embodiments, the non-naturally occurring, engineered ketoreductase polypeptides disclosed herein are capable of carrying out the conversion with high enantiomeric excess (e.g., at least about 99% e.e.), increased activity, high percent conversion (e.g., at least about 90%) in 2 to 24 hours. In majority of the experiments complete conversions were achieved in 2 to 3 hours, short reaction cycles would result in high production in reduced time span making the process commercially beneficial.
The (R)-1-aryl-2-tetrazolyl-ethyl alcohol (III) of the present invention can be further converted to carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester (I) with methods known in WO 2006112685 (A1) and WO 2011046380 (A2).
The present invention provides “enantiomerically pure” (R)-1-aryl-2-tetrazolyl-ethyl alcohol (II), this compound is highly pure and can be utilized in the next step without purification. The (R)-1-aryl-2-tetrazolyl-ethyl alcohol (II) is converted to compound (I) with high purity. Compound (I) is obtained with chiral purity of

greater than 98%, 99%, preferably greater than 99%, more preferably greater than 99 %, by HPLC.
The present invention provides simple and economical preparation of highly pure Carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester compounds. The present invention is further illustrated by the following representative examples and does not limit the scope of the invention. All the enzymes mentioned herein are procured from Codexis.
Example 1: preparation of (R)-1-aryl-2-tetrazolyl-ethyl alcohol (III).
To a slurry of enzyme (5 mg) and buffer solution (0.9 ml) (0.2 M potassium phosphate buffer, pH 7.00, 1 mM NADP, 2 mM MgSO4) at 30oC was added 0.1 ml substrate [aryl ketone (II) (5 mg) in isopropanol (0.1 ml) and dimethyl sulfoxide (0.01 ml)]. The reaction mixture was maintained at 30oC for 24 hours and samples were analyzed by chiral HPLC.
Enzymes used in Example 1 and the corresponding results are listed in the table below:

S.No. Name of the enzyme (commercially available Codexis enzyme) Chiral purity (%)

(R)-1-aryl-2-tetrazolyl-ethyl alcohol (II). (S)-1-aryl-2-tetrazolyl-ethyl alcohol (II).
1 KRED-P1-B02 99.41 0.59
2 KRED-P1-B05 97.68 2.32
3 KRED-P1-B10 98.21 1.79
4 KRED-P1-C01 89.92 10.08
5 KRED-P2-C02 73.62 26.38
6 KRED-P2-D03 46.47 53.53
7 KRED-P2-D11 98.41 1.59
8 KRED-P2-D12 82.36 17.64

Example 2: preparation of (R)-1-aryl-2-tetrazolyl-ethyl alcohol (III).
A slurry of aryl ketone (II) (40 g) triethanol amine-MgSO4 buffer (550 ml) and isopropanol (240 ml) was heated to 45-50°C. A mixture of enzyme (0.4 g) and NADP (0.4 g) in Triethanol amine-MgSO4 buffer (10 ml) was added to the slurry and was stirred for about 4 hours. The solution was distilled and extracted with ethyl acetate . The organic layer was concentrated to obtain the residue (40 g) with HPLC (chiral) purity of 99.8%.
Example 3: Preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester compounds (I).
A mixture of (R)-1-aryl-2-tetrazolyl-ethyl alcohol (III) (100 g), 1,1'-Carbonyldiimidazole (108 g) and dichloromethane (550 ml) was stirred at 25-30°C for about 6 hours. Aqueous ammonia (50 ml) was added and the mixture was further stirred for another 6 hours. The reaction mixture was washed with water, the organic layer was separated and concentrated. Diisopropyl ether (400 ml) was added to the residue and the mixture was stirred at 5-10°C for about 4 hours. The solid was filtered and recrystallized from isopropanol to obtain compound (I) with HPLC (chiral) purity of 99.0%.
Sequence Listing: Amino acid sequences
>SEQ ID NO.:1
MTDRLKGKVAIVTGGTLGIGLAIADKFVEEGAKVVITGRHADVGEKAAKSIGGTDVIRFVQHDASD EAGWTKLFDTTEEAFGPVTTVVNNAGIAVSKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKNK GLGASIINMSSIEGFVGDPTLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDD LEGAEEMMSQRTKTPMGHIGEPNDIAWICVYLASDESKFATGAEFVVDGGYTAQ
>SEQ ID NO.:2
MTDRLKGKVAIVTGGTLGIGLAIADKFVEEGAKVVITGRHADVGEKAAKSIGGTDVIRFVQHDASD EAGWTKLFDTTEETFGPVTTVVNNAGIGVVKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKNK GLGASIINMSSILGFVGDPTTGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGPIKTPLLDD LEGAEEMMSQRTKTPMGHIGEPNDIAWVCVYLASDESKFATGAEFVVDGGWTAQ

>SEQ ID NO.:3
MTDRLKGKVAIVTGGTLGIGLAIADKFVEEGAKVVITGRHADVGEKAAKSIGGTDVIRFVQHDASD EAGWTKLFDTTEETFGPVTTVVNNAGIGVVKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKNK GLGASIINMSSILGFVGDPTTGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGPIKTPLLDD LEGAEEMMSQRTKTPMGHIGEPNDIAWVCVYLASDESKFATGAEFVVDGGWTAQ
>SEQ ID NO.:4
MTDRLKGKVAIVTGGTLGIGLAIADKFVEEGAKVVITGRHADVGEKAAKSIGGTDVIRFVQHDASD EAGWTKLFDTTEETFGPVTTVVNNAGIGVVKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKNK GLGASIINMSSILGQVGDPTTGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGPIKTPLLDD LEGAEEMMSQRTKTPMGHIGEPNDIAWVCVYLASDESKFATGAEFVVDGGWTAQ
>SEQ ID NO.:5
MTDRLKGKVAIVTGGTLGIGLAIADKFVEEGAKVVITGRHADVGEKAAKSIGGTDVIRFVQHDASD EAGWTKLFDTTEETFGPVTTVVNNAGIGVVKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKNK GLGASIINMSSILGFVGDPTTGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGPIKTPLLDD LEGAEEMMSQRTKTPMGHIGEPNDIAWVCVYLASDESKFATGAEFVVDGGYTAQ
>SEQ ID NO.:6
MTDRLKGKVAIVTGGTLGIGLAIADKFVEEGAKVVITGRHADVGEKAAKSIGGTDVIRFVQHDASD EAGWTKLFDTTEETFGPVTTVVNNAGIGVVKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKNK GLGASIINMSSILGFVGLPTTGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGPIKTPLMDD MEGAEEMMSQRTKTPMGHIGEPNDIAWVCVYLASDESKFATGAEFVVDGGYTAQ
>SEQ ID NO.:7
MTDRLKGKVAIVTGGTLGIGLAIADKFVEEGAKVVITGRHADVGEKAAKSIGGTDVIRFVQHDASD EAGWTKLFDTTEETFGPVTTVVNNAGIAVVKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKNK GLGASIINMSSVLGFVGDPTTGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGPIKTPLMDD MEGAEEMMSQRTKTPMGHIGEPNDIAWVCVYLASDESKFATGAEFVVDGGFTAQ
>SEQ ID NO.:8
MTDRLKGKVAIVTGGTLGIGLAIADKFVEEGAKVVITGRHADVGEKAAKSIGGTDVIRFVQHDASD EAGWTKLFDTTEETFGPVTTVVNNAGIGVVKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKNK GLGASIINMSSILGQVGDPTTGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGPIKTPLMDD MEGAEEMMSQRTKTPMGHIGEPNDIAWVCVYLASDESKFATGAEFVVDGGYTAQ
>SEQ ID NO.:9
MTDRLKGKVAIVTGGTLGIGLAIADKFVEEGAKVVITGRHADVGEKAAKSIGGTDVIRFVQHDASD EAGWTKLFDTTEETFGPVTTVVNNAGIGMVKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKNK GLGASIINMSSVLGQVGDPTTGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGPIKTPLMDD LEGAEEMMSQRTKTPMGHIGEPNDIAWVCVYLASDESKFATGAEFVVDGGFTAQ
>SEQ ID NO.:10
MTDRLKGKVAIVTGGTLGIGLAIADKFVEEGAKVVITGRHADVGEKAAKSIGGTDVIRFVQHDASD EAGWTKLFDTTEETFGPVTTVVNNAGIGMVKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKNK

GLGASIINMSSVLGFVGDPTTGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGPIKTPLLDD MEGAEEMMSQRTKTPMGHIGEPNDIAWVCVYLASDESKFATGAEFVVDGGYTAQ
>SEQ ID NO.:11
MTDRLKGKVAIVTGGTMGIGLAIADKFVEEGAKVVITGRHADVGEKAAKSIGGTDVIRFVQHDASD EAGWTKLFDTTEETFGPVTTVVNNAGIGVVKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKNK GLGASIINMSSVLGMVGLPTTGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGPIKTRLMDD MEGAEEMMSQRTKTPMGHIGEPNDIAWVCVYLASDESKFATGAEFVVDGGYTAQ
>SEQ ID NO.:12
MTDRLKGKVAIVTGGTLGIGLAIADKFVEEGAKVVITGRHADRGEKAAKSIGGTDVIRFVQHDVSD EAGWPKLFDTTEETFGPVTTLVNNAGIGVVKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKNK GLGASIINMSSILGQVGDPTTGAYNASKGAVRIMSKSAALLCALKDYDVRVNTVHPGPIKTPLMDD MEGAEEMMSQRTKTPMGHIGEPNDIAWVCVYLASDESKFATGAEFVVDGGYTAQ
>SEQ ID NO.:13
MTDRLKGKVAIVTGGTLGIGLAIADKFVEEGAKVVITGRHADRGEKAAKSIGGTDVIRFVQHDVSD EAGWTKLFDTTEETFGPVTTVVNNAGIGVVKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKNK GLGASIINMSSILGQVGDPTTGAYNASKGAVRIMSKSAALLCALKDYDVRVNTVHPGPIKTPLMDD MEGAEEMMSQRTKTPMGHIGEPNDIAWVCVYLASGESKFATGAEFVVDGGYTAQ
>SEQ ID NO.:14
MTDRLKGKVAIVTGGTLGIGLAIADKFVEEGAKVVITGRRADRGEKAAKSIGGTDVIRFVQHDVSD EAGWPKLFDTTEETFGPVTTLVNNAGIGVVKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKNK GLGASIINMSSILGQVGDPTTGAYNASKGAVRIMSKSAALLCALKDYDVRVNTVHPGPIKTPLMDD MEGAEEMMSQRTKTPMGHIGEPNDIAWVCVYLASDESKFATGAEFVVDGGYTAQ
>SEQ ID NO.:15
MTDRLKGKVAIVTGGTLGIGLAIADKFVTEGAKVVITGRRADRGEKAAKSIGGTDVIRFVQHDVSD EAGWPKLFDATEETFGPVTTLVNNAGIGYRKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKNK GLGASIINMSSILGQVGDPLTGAYCASKGAVRIMSKSAALLCALKDYDVRVNTVHPGPIKTPLMDD MPGAEEMMSQRTKTPMGHIGEPNDIAWVCVYLASDESKFATGAEFVVDGGYTAQ
>SEQ ID NO.:16
MTDRLKGKVAIVTGGTLGIGLAIADKFVTEGAKVVITGRRADRGEKAAKSIGGTDVIRFVQHDVSD EAGWPKLFDTTEETFGPVTTLVNNAGIGYRKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKCK GLGASIINMSSILGQVGDPATGAYNASKGAVRIMSKSAALLCALKDYDVRVNTVHPGPIKTPLMDD MEGAEEMMSQRTKTPMGHIGEPNDIAWVCVYLASDESKFATGAEFVVDGGYTAQ
>SEQ ID NO.:17
MTDRLKGKVAIVTGGTLGIGLAIADKFVTEGAKVVITGRRADVGEKAAKSIGGTDVIRFVQHDVSD EAGWPKLFDTTEETFGPVTTLVNNAGIGYRKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKCK GLGASIINMSSILGQVGDPATGAYNASKGAVRIMSKSAALLCALKDYDVRVNTVHPGPIKTPLMDD MPGAEEMMSQRTKTPMGHIGEPNDIAWVCVYLASDESKFATGAEFVVDGGYTAQ

>SEQ ID NO.:18
MTDRLKGKVAIVTGGTHGIGLAIATKFVTEGAKVVITGRRADVGEKAAKSIGGTDVIRFVQHDVSD EAGWGKLFDTTEETFGPVTTLVNNAGIGYRKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKCK GLGASIINMSSILGQVGDPATGAYSASKGAVRIMSKSAALLCALKDYDVRVNTVHPGPIKTPLMDD MPGAEEMMSQRTKTPMGHIGEPNDIAWVCVYLASDESKFATGAEFVVDGGYTAQ
>SEQ ID NO.:19
MTDRLKGKVAIVTGGTHGIGLAIADKFVTEGAKVVITGRRADVGEKAAKSIGGTDVIRFVQHDVSD EAGWPKLFDTTEETFGPVTTLVNNAGIGYRKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKCK GLGASIINMSSILGQVGDPATGAYSASKGAVRIMSKSAALLCALKDYDVRVNTVHPGPIKTPLMDD MPGAEEMMSQRTKTPMGHIGEPNDIAWVCVYLASDESKFATGAEFVVDGGYTAQ

WE CLAIM :
1. A process for preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl
ester (I) wherein R is selected from hydrogen, halogen, alkoxy or halo alkyl comprising:

(iii) subj ecting aryl ketone (II) to ketoreductase, in the presence of NADPH or NADH cofactor to form (R)-1-aryl-2-tetrazolyl-ethyl alcohol (III), and

(iv) converting (R)-1-aryl-2-tetrazolyl-ethyl alcohol (III) to carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester (I) wherein, ketoreductase is non-naturally occurring ketoreductases.
2. A process for preparation of (R)-1-aryl-2-tetrazolyl-ethyl alcohol (III) comprising subjecting aryl ketone (II) to ketoreductase in presence of cofactor, wherein the ketoreductase is capable of converting ketone (II) to corresponding alcohol (III) in enantiomeric excess and the ketoreductase has at least 80% homology with amino acid sequence SEQ ID NO: 1.
3. The process as claimed in claim 1 wherein, ketoreductase has at least 80% homology with amino acid sequence SEQ ID NO: 1.

4. The process as claimed in claim 1 wherein, aryl ketone (II) is treated with the ketoreductase in presence of buffer and an alcohol or mixture of alcohol and solvent.
5. The process as claimed in claim 4 wherein, the buffer is a phosphate or triethanol amine buffer.
6. The process as claimed in claim 4 wherein, the alcohol is methanol, ethanol, propanol, or butanol.
7. The process as claimed in claim 4 wherein, the solvent is dimethyl sulfoxide.
8. The process as claimed in claim 1 wherein, (R)-1-aryl-2-tetrazolyl-ethyl alcohol (III) has chiral purity of 99.5 to 99.9%, by HPLC.
9. The process as claimed in claim 1 wherein, ketoreductase has amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19.