Biocatalysts and methods for the synthesis of substituted lactams

1. An engineered transaminase polypeptide comprising an amino acid sequence having at least 80% sequence identity to reference sequence SEQ ID NO:4 and at least an amino acid residue difference as compared to SEQ ID NO:4 at residue position X192, and the substitution X136L.

2. The engineered polypeptide of claim 1 , in which X192 is selected from A, G, H, K, N, Q, R, and S.

3. The engineered polypeptide of claim 2 , in which the amino acid sequence further comprises one or more residue differences as compared to SEQ ID NO:4 selected from: X2M; X4F, X4L, X4Y; X5C, X5F, X5I, X5H, X5K, X5L, X5M, X5N, X5P, X5S, X5T, X5V, X5Y; X6F, X6R, X6S, X6T; X8H; X9L, X9I, X9Q, X9F; X1OL; X12H; X13A, X13F; X17L; X181; X21L; X22A, X22H; X25A; X27L, X27H, X27P; X28N; X29N, X29T; X30F, X30G; X31H, X31R; X34V; X37S; X42G; X43S; X46R; X47R; X48A, X48G, X48R, X48S, X48W; X49I, X49P; X50S; X52H; X54A, X54L, X54M, X54P, X54R; X55M; X56D, X56E, X56W; X61G; X61W; X62A, X62L, X62N, X62S; X64C; X66A, X66S; X68I; X69A, X69G, X69T; X72A; X80Q; X81K; X84T; X85L, X85R; X88W; X89L; X97P, X97T; X99L; X101G, X101L, X101R; X102H, X102R; X102T, X102W; X103G, X103N, X103V; X106L, X106S, X106T, X106V; X107V; X108T; X115R; X117V; X120F; X122F, X122M, X122W, X122Y; X124F, X124K, X124L, X124M, X124N, X124R, X124S, X124W; X126A, X126C, X126G, X126I, X126T; X127T; X128A; X131F; X132H; X134G, X134L, X134S, X134V, X134Y, X134W; X139E; X140A, X140K; X140M, X140T, X140V; X141A; X142M; X143V; X144D, X144I; X146R; X150L, X150S; X152A, X152C, X152F, X152G, X152I, X152L, X152R, X152S, X152W; X155C, X155I, X155L, X155T; X156A, X156F, X156S, X156T; X157L; X159G; X160L; X161M, X161N; X165L, X165N, X165V; X168E, X168R; X176C; X179K; X185A; X190M; X191A, X191G, X191S; X196L, X196V; X199L, X199V; X203M; X208K; X209F, X209M, X209Q, X209V; X210G, X210S; X215F, X215H, X215L; X217L; X223A, X223S, X223T, X223V; X227I; X231T; X233D; X234L; X241R; X256R; X260Q; X263V; X265Y, X265W; X266N; X267V; X269L, X269T; X270R, X270T; X273D, X273H, X273M, X273R, X273V; X274F, X274L, X274R, X274T; X282I, X282L, X282T, X282V; X284A, X284P, X284S, X284T, X284V; X295N, X295S; X296D, X296L, X296R, X296S, X296W; X297D; X297G; X300G, X300L; X305Q, X305T; X308A; X309L, X309T, X309W; X311Q, X311T; X312C, X312M; X316L, X316R; X319R, X319T, X319Y; X320G, X320Q; X323C, X323E, X323N, X323R; X324Q; X325M, X325P, X325Q, X325V; X327L, X327R; and X329P.

4. The engineered polypeptide of claim 1 , wherein the amino acid sequence further comprises a combination of residue differences selected from:

(a) X192A/X192S and X215H;

(b) X124W, X126A, and X192A/X192N/X192S;

(c) X124W, X126A, X152L/X152W/X152I, and X192A;

(d) X54L, X124W, X126A, X192A, X215H, and X223S;

(e) X54L, X64C, X124W, X126A, X192A, and X215H;

(f) X124W, X126A, X140K, X150S, X155I, X192A, X196L, X215H, and X227I;

(g) X54L, X124W, X126A, X155I, X156T, X157L, X192A, X215H, and X223S;

(h) X54P, X64C, X124W, X126A, X155I, X156T, X157L, X192A, and X215H;

(i) X124W, X126A, X143V, X150S, X156T, X159G, X192A, X215H, X223S, and X227I;

(j) X54P, X56E, X64C, X124W, X126A, X139E, X140K, X143V, X150S, X156T, X192A, and X215H; and

(k) X124W, X126A, X140K, X143V, X155I, X156T, X157L, X192A, X199V, X215H, X223S, X227I, and X231T.

5. The engineered polypeptide of claim 1 , in which the amino acid sequence further comprises residue differences selected from:

(a) X192A;

(b) X124L and X192A;

(c) X124W, X126A, X192A, and X284A;

(d) X122W, X124F, X126A, X192A and X284V;

(e) X124W, X126A, X143V, X150S, X156T, X159G, X192A, X215H, X223S, and V227I; and

(f) X54P, X64C, X124W, X126A, X139E, X143V, X155I, X157L, X159G, X192A, X199V, X215H and X227I.

6. The engineered polypeptide of claim 1 , in which the amino acid sequence comprises SEQ ID NO:824.

7. The engineered polypeptide of claim 1 , capable of converting compound 2 to compound 1,

under suitable reaction conditions.

8. The engineered polypeptide of claim 1 , capable of converting compound 2 to compound 1d and compound 1a

with at least 1.5 fold the activity of SEQ ID NO:4 under suitable reaction conditions.

9. The engineered polypeptide of claim 8 , in which the amino acid sequence further comprises at least one or more residue differences as compared to SEQ ID NO:4 selected from: X54A; X54L; X56D; X56E; X61G; X61W; X62A; X62L; X62N; X62S; X64C; X68I; X122F, X122W; X122Y; X124K; X124M; X124S; X124W; X126C; X126I; X139E; X140K; X143V; X150L; X155I; X155L; X159G; X160L; X176C; X192A; X192G; X192H; X192K; X192N; X192Q; X192R; X192S; X199L; X199V; X209F; X209M; X209Q; X209V; X223S; X223T; X223V; X227I; X282L, X282V; X284A; X284P; X284S; X284T; and X284V.

10. The engineered polypeptide of claim 1 , capable of converting compound 2 to compound 1d and compound 1a

in a diastereomeric ratio greater than 8:1 over compound 1b and compound 1c.

11. The engineered polypeptide of claim 10 , in which the amino acid sequence further comprises one or more residue differences as compared to SEQ ID NO:4 selected from: X62N, X62A, X62S; X62L; X124W, X124F; X124K; X124L; X124M; X124N; X124R; X124S; X136Y; X152A; X152C; X152F; X152G; X152I; X152L; X152R; X152S; X152W; X192A; X192G; X192H; X192K; X192N; X192Q; X192R; X192S; X215H; X215L; X284V; X284A; X284P; X284S and X284T.

12. A polynucleotide encoding the engineered polypeptide of claim 1 .

13. A polynucleotide comprising SEQ ID NO:823.

14. An expression vector comprising the polynucleotide of claim 12 .

15. The expression vector of claim 14 , comprising a control sequence.

16. A host cell comprising the polynucleotide of claim 12 .

17. A method of preparing a transaminase polypeptide, comprising culturing the host cell of claim 16 , under conditions suitable for expression of the polypeptide.

18. The method of claim 17 , further comprising isolating the polypeptide.

19. A process for preparing a compound of formula I,

wherein

L is a leaving group;

A is an optionally substituted cycloalkyl, heterocycloalkyl, aryl, heteroaryl, fused aryl, or fused heteroaryl;

R 1 is H or an optionally substituted (C 1 -C 6 ) alkyl;

Q is O or S;

M is —CR a R b —, wherein each M is independent of the other and R a and R b are selected from H, halo, —OR 3 , —SR c , —CN, —NO 2 , —NR d R e , alkyl, alkenyl, alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, cycloalkylalkyl, cycloalkylalkenyl, cycloalkylalkynyl, heterocycloalkylalkyl, heterocycloalkylalkenyl, and heterocycloalkylalkynyl, wherein R c is selected from H, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, and R d and R e are selected from H, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, and a protecting group; and

n is an integer from 0 to 4,

comprising contacting a substrate compound of formula II,

wherein L, A, R 1 , Q, M and n are defined above, with a transaminase polypeptide of claim 1 , in the presence of an amino donor under suitable reaction conditions.

20. The process of claim 19 , wherein L is selected from: Cl, Br, —OR f , —OC(O)R f , —SR f , and —OPO 3 , wherein each R f is, independently of the others, selected from (C 1 -C 6 )alkyl or aryl.

21. The process of claim 19 , in which L is —OR f , wherein R f is selected from an optionally substituted methyl, ethyl, n-propyl, isopropyl, -butyl, iso-butyl, sec-butyl, and tert-butyl.

22. The process of claim 19 , wherein A is selected from an optionally substituted phenyl, pyridinyl, indolyl, and napthyl.

23. The process of claim 19 , wherein R 1 is an optionally substituted (C 1 -C 6 )alkyl.

24. The process of claim 19 , wherein R a and R b are selected from H and —NR d R e .

25. The process of claim 19 , in which A has one or more substitutions selected from —OR g , —COOH, —SO 2 , —SR h , —NR i R j , —NO 2 , —CN, halo, alkyl, and aryl, wherein R g , R h , R i and Rj are selected from H and (C 1 -C 6 )alkyl.

26. The process of claim 19 , in which the protecting group is selected from tert-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), trichloroethyl chloroformate (Troc), p-methoxybenzyl carbonyl (Moz), 3,4-dimethoxybenzyl (DMPM), p-methoxybenzyl (Pmb), tosyl (Ts) and carbobenzyloxy (Cbz).

27. The process of claim 19 , in which n is 2 or 3.

28. The process of claim 19 , wherein the substrate compound of formula II comprises the compound of formula 2p

wherein PG is a protecting group, to produce the compound of formula 1p

29. A process for preparing compound 1d1 and compound 1a1

wherein PG is a protecting group, in diastereomeric ratio greater than 8:1 over compound 1b1 and compound 1c1, comprising contacting a substrate compound 2p,

with a transaminase polypeptide of claim 1 , in the presence of an amino donor under suitable reaction conditions.

30. The process of claim 19 , in which the substrate compound is at a loading of 0.5 to 200 g/L.

31. The process of claim 30 , in which the substrate compound is at a loading selected from 0.5, 1, 5, 10, 20, 30, 40, 50, 100, 150, and 200 g/L.

32. The process of claim 19 , in which the suitable reaction conditions comprise a DMSO concentration of 1% to 80% (v/v).

33. The process of claim 32 , in which the amino donor is isopropylamine.

34. The process of claim 33 , in which the isopropylamine is at a concentration of 0.1 to 3.0 M.

35. The process of claim 19 , in which the suitable reaction conditions comprise (a) substrate loading of about 10 to 100 g/L substrate compound; (b) transaminase polypeptide concentration of about 1 to 40 g/L; (c) IPM concentration of about 0.1 to 10 M; (d) PLP cofactor concentration of about 0.1 to 1 mM; (e) pH of about 8.5 to 11; and (f) temperature of about 30 to 60° C.

36. The process of claim 19 , in which the suitable reaction condition comprises: about 50 g/L substrate compound, about 20 g/L or less of transaminase polypeptide, about 50% (v/v) dimethylsulfoxide (DMSO), about 1 M isopropylamine (IPM), about 1 mM pyridoxal phosphate (PLP), about 0.2 M borate, about pH 10.5 and about 55° C.

37. A process for preparing the compound of formula III

wherein,

A is an optionally substituted cycloalkyl, heterocycloalkyl, aryl, heteroaryl, fused aryl, or fused heteroaryl;

R 1 is H or an optionally substituted(C 1 -C 6 ) alkyl;

Q is O or S;

M is —CR a R b —, wherein each M is independent of the other and R a and R b are selected from H, halo, —OR c , —SR c , —CN, —NO 2 , —NR d R e , alkyl, alkenyl, alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, cycloalkylalkyl, cycloalkylalkenyl, cycloalkylalkynyl, heterocycloalkylalkyl, heterocycloalkylalkenyl, and heterocycloalkylalkynyl, wherein R c is selected from H, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, and R d and R e are selected from H, alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and a protecting group; and

n is an integer from 0 to 4,

comprising (a) carrying out the process of claim 19 , to prepare the compound of formula I, and (b) cyclizing the compound of formula I.

38. The process of claim 37 , in which the compound of formula III comprises compound 3p,

wherein PG is a protecting group, the process comprising (a) carrying out the process of claim 28 for preparing compound 1p and (b) cyclizing the compound 1p.

39. The process of claim 37 , in which the compound of formula III comprises compound 3d1 and compound 3a1

wherein compound 3d1 and compound 3a1 is produced in diastereomeric ratio greater than 8:1 over compound 3b1 and compound 3c1,

the process comprising (a) carrying out the process of claim 29 , and (b) cyclizing the products of step (a).