Transaminase reactions

1. A process for preparing the compound of formula (III):

having the indicated stereochemical configuration at the stereogenic center marked with an * in an enantiomeric excess over the opposite enantiomer,

wherein, R 10 is Cl, Br, F, CH 3 , CF 3 , CN, SO 2 , —OCH 3 , or NO 2 ,

the process comprising:

(a) contacting a ketone substrate of formula:

wherein R 11 is halogen, OH, C(O)R 4 , OC(O)R 5 , or NR 6 R 7 , wherein R 4 , R 5 , R 6 , and R 7 is H or C 1 -C 4 alkyl, and R 10 is as defined above,

with a transaminase polypeptide in the presence of an amino donor, wherein said amino donor comprises isopropylamine, alanine, 3-aminobutyric acid, or methylbenzylamine, under reaction conditions suitable for converting the ketone substrate to an amine product of formula:

wherein the amino acid sequence of the transaminase polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2, wherein the amino acid residue of the transaminase polypeptide which corresponds to the residue at position 223 of SEQ ID NO: 2 is proline, and wherein the transaminase polypeptide is capable of converting the ketone substrate to the amine product at a rate that is greater than that of a transaminase polypeptide comprising the amino acid sequence of SEQ ID NO:2; and

(b) cyclizing the amine product under suitable conditions to form the compound of formula (III).

2. The process of claim 1 , wherein the ketone substrate is 4-chloro-1-(2-fluorophenyl)butan-1-one:

and the amine product is (S)-4-chloro-1-(2-fluorophenyl)butan-1-amine:

thereby forming (R)-2-(2-fluorophenyl)pyrrolidine:

in enantiomeric excess.

3. The process of claim 1 , wherein a reaction condition comprises a temperature of 20° C. to 65° C.

4. The process of claim 3 , wherein a reaction condition comprises a temperature of 40° C. to 65° C.

5. The process of claim 1 , wherein the amine product is produced in at least 90% enantiomeric excess.

6. The process of claim 1 , wherein the amine product is produced in at least 99% enantiomeric excess.

7. The process of claim 1 , further comprising the step of removing a carbonyl by-product of the reaction.

8. The process of claim 7 , wherein the amino donor is alanine or 3-aminobutyric acid and the carbonyl by-product is a keto acid.

9. The process of claim 7 , wherein the carbonyl by-product has a vapor pressure higher than water, and removal of the carbonyl byproduct is by sparging with a nonreactive gas or by applying a vacuum.

10. The process of claim 9 , wherein the non-reactive gas is nitrogen gas.

11. The process of claim 9 , wherein the amino group donor is isopropylamine and wherein the carbonyl by-product is acetone.

12. The process of claim 1 , wherein a reaction condition is a pH of 7.0 to a pH of 11.0.

13. The process of claim 12 , wherein the pH is maintained by adding isopropylamine.

14. The process of claim 1 , wherein a reaction condition comprises dimethylsulfoxide (DMSO) as a solvent.

15. The process of claim 14 , wherein the concentration of DMSO is between 10% to 40% (v/v).

16. The process of claim 1 , wherein the ketone substrate is present at 5 to 25 g/L.

17. The process of claim 1 , wherein one or more residue positions corresponding to the residue positions of SEQ ID NO: 2 selected from: X4; X5; X8; X18; X25; X26; X27; X28; X30; X41; X42; X48; X49; X50; X54; X55; X60; X61; X62; X65; X69; X81; X94; X96; X102; X117; X120; XI22; X124; X126; X136; X137; X138; X146; X148; X150; X152; X155; X156; X160; X163; X164; X169; X174; X178; X195; X199; X204; X208; X209; X211; X215; X217; X225; X230; X252; X269; X273; X282; X284; X292; X297; X306; X321; and X329 is/are substituted in the transaminase polypeptide.

18. The process of claim 17 , wherein the one or more residue positions corresponding to the residue positions of SEQ ID NO:2 selected from: X62, X69, X122, X136, X137, X195, X199, X208, X209, X225, X282, and X284 is/are substituted in the transaminase polypeptide.

19. The process of claim 17 , wherein the type of amino acid substitution is selected from:

X4 is an aromatic residue,

X8 is a constrained residue;

X26 is an aromatic or constrained residue;

X48 is a polar, acidic, aliphatic or non-polar residue;

X60 is an aromatic residue;

X61 is an aromatic residue;

X62 is an aromatic or polar residue;

X65 is an aliphatic residue;

X69 is a cysteine (C) or non-polar, polar, or aliphatic residue;

X81 is a non-polar residue;

X94 is an aliphatic residue;

X96 is an aliphatic residue;

X102 is an aliphatic or basic residue;

X122 is a constrained, non-polar or aliphatic residue;

X124 is a polar or constrained residue;

X136 is an aromatic residue;

X137 is a polar or aliphatic residue;

X150 is aromatic, constrained or polar residue;

X152 is cysteine (C), non-polar, aliphatic, or polar residue;

XI60 is an aliphatic residue;

X163 is an aliphatic or constrained residue;

X169 is an aliphatic residue;

X174 is an aliphatic residue;

X178 is a polar residue;

X195 is an aromatic or polar residue;

X199 is an aliphatic or aromatic residue;

X208 is cysteine (C) or constrained, non-polar, aromatic, polar, or basic residue;

X209 is an aliphatic residue;

X211 is an aliphatic residue;

X215 is a cysteine (C);

X217 is a polar residue;

X225 is an aromatic residue;

X230 is an aliphatic residue;

X252 is an aromatic or aliphatic residue;

X269 is a constrained residue;

X273 is an aromatic residue;

X282 is a polar residue;

X284 is a non-polar residue;

X292 is a polar residue;

X297 is a polar residue;

X306 is an aliphatic residue;

X321 is a constrained residue, and

X329 is a constrained or aromatic residue.

20. The process of claim 17 , wherein the amino acid substitution is selected from: X4 is Y; X8 is P; X26 is H; X48 is Q, D, V, G, or A; X60 is F; X61 is Y; X62 is T, Y or F; X65 is A; X69 is G, C, T, A, or S; X81 is G; X94 is I or L; X96 is L; X102 is L or K; X122 is M, I, L, V, or H; X124 T, H or N; X136 is Y or F; X137 is T or I; X150 is F, H, or S; X152 is I, L, S or C; X160 is L; X163 is H or V; X169 is L; X174 is A; X178 is S; X195 is F or Q; X199 is W or I; X208 is H, C, G, K, N, Y, D or S; X209 is L; X211 is I; X215 is C; X217 is N; X225 is Y; X230 is V; X252 is F; X269 is P; X273 is Y; X282 is S; X284 is G; X292 is T; X297 is S; X306 is L; X321 is P; and X329 is H.

21. The process of claim 20 , wherein the amino acid substitution is selected from: X8 is P; X60 is F; X61 is Y; X62 is T, Y or F; X65 is A; X69 is G, C, T, A, or S; X81 is G; X94 is I or L; X96 is L; X122 is M, I, L, V, or H; X124 T, H or N; X136 is Y or F; X169 is L; X178 is S; X199 is W or I; X209 is L; X215 is C; X217 is N; X269 is P; X273 is Y; X282 is S; X284 is G; X297 is S; X321 is P and X329 is H.

22. The process of claim 17 , wherein the transaminase polypeptide comprises the amino acid sequence of SEQ ID NO: 74.