Granted Patent B2

US 12,247,233 · App. 17/751,200 · Granted Mar 11, 2025

Inventors: Gregory Hughes (Scotch Plains, NJ); Paul N. Devine (Tinton Falls, NJ); Fred J. Fleitz (Germantown, WI); Brendan T. Grau (Warrington, PA); John Limanto (Metuchen, NJ); Christopher Savile (Santa Clara, CA); Emily Mundorff (Garden City, NY)

Assignee: Codexis, Inc.

C12P17/10C12N9/1096C12P13/001C12P13/04C12P17/12C12P17/182C12Y206/01Y02P20/52

Abstract

Claims (96)

1. A process for preparing an amine product of structural formula (I):

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

R 1 is optionally substituted aryl or heteroaryl;

R 2 is an optionally substituted C 1 -C 6 alkyl, —R 3 C(O)R 4 , or —R 3 OC(O)R 5

R 3 is an optionally substituted C 1 -C 4 alkyl, and R 4 is H, an optionally substituted C 1 -C 4 alkyl, NR 6 R 7 , or OR 8 , where R 5 , R 6 , R 7 , and R 8 are independently H or C 1 -C 4 alkyl;

the process comprising contacting a ketone substrate of structural formula (II):

with a transaminase polypeptide in the presence of an amino donor under reaction conditions suitable for converting the ketone substrate to the amine product, wherein the transaminase polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 102 and is capable of converting the ketone substrate to the amine product of structural formula (I) at a rate that is increased as compared to SEQ ID NO:2.

2. The process of claim 1 , wherein:

i) R 1 is an optionally substituted phenyl;

ii) R 1 is an optionally substituted pyridinyl;

iii) R 1 is a substituted aryl or heteroaryl;

iv) the substitution on the C 1 -C 6 alkyl and R 3 are selected from halogen, NR 5 R 6 , or OR 8 , where R 5 and R 6 are defined above and R 8 is H or C 1 -C 4 alkyl; or

v) R 2 is methyl or halo substituted methyl, wherein the halo substituted methyl optionally is CF 2 H or CF 3 .

3. The process of claim 1 , wherein:

i) the amine product of structural formula (I) is:

wherein R 9 is H, Cl, Br, F, CH 3 , CF 3 , CN, SO 2 , —OCH 3 , —C(O)CH 3 , or NO 2 , and the ketone substrate of structural formula (II) is:

wherein R 9 optionally is in the para position on the phenyl ring;

ii) the amine product of structural formula (I) is (S)-1-(4-bromophenyl)-2,2,2-trifluoroethanamine:

and the ketone substrate of structural formula (II) is 1-(4-bromophenyl)-2,2,2-trifluoroethanone:

iii) the amine product of structural formula (I) is (S)-2,2,2-trifluoro-1-p-tolylethanamine:

and the ketone substrate of structural formula (II) is 2,2,2-trifluoro-1-p-tolylethanone:

iv) the amine product of structural formula (I) is (S)-2,2,2-trifluoro-1-(4-(trifluoromethyl)phenyl)ethanamine:

and the ketone substrate of structural formula (II) is 2,2,2-trifluoro-1-(4-(trifluoromethyl)phenyl)ethanone:

v) the amine product of structural formula (I) is:

and the ketone substrate of structural formula (II) is:

wherein R 7 is optionally substituted C 1 -C 4 alkyl, and R 10 is R 9 defined above;

vi) the amine product of structural formula (I) is (R)-ethyl-3-amino-3-(pyridin-2-yl)propanoate:

and the ketone substrate of structural formula (II) is ethyl 3-oxo-3-(pyrindin-2-yl)propanoate:

vii) the amine product of structural formula (I) is:

and the ketone substrate of structural formula (II) is:

wherein R 11 is halogen, OH, —C(O)R 4 , —OC(O)R, or NR 6 R 7 , wherein R 4 , R 5 , R 6 , R 7 , R 7 and R 10 are defined above; or

viii) wherein the amine product of structural formula (I) is (S)-4-chloro-1-(2-fluorophenyl)butan-1-amine:

and the ketone substrate of structural formula (II) is 4-chloro-1-(2-fluorophenyl)butan-1-one:

4. The process of claim 1 , wherein:

i) the reaction conditions comprise a temperature of 20° C. to 65° C.;

ii) the reaction conditions comprise a temperature of 40° C. to 65° C.;

iii) the amine product of structural formula (I) is produced in at least 90% enantiomeric excess;

iv) the amine product of structural formula (I) is produced in at least 99% enantiomeric excess;

v) the amino donor is selected from isopropylamine, alanine, 3-aminobutyric acid, or methylbenzylamine;

vi) the reaction conditions comprise a pH of 7.0 to a pH of 11.0, wherein the pH optionally is maintained by adding isopropylamine;

vii) the reaction conditions comprise a solvent of dimethylsulfoxide (DMSO), wherein the DMSO is between 10% to 40% (v/v); and/or

viii) wherein the amino donor is present at 5 to 25 g/L.

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

6. The process of claim 5 , wherein the amino donor is an amino acid and the carbonyl by-product is a keto acid.

7. The process of claim 6 , wherein the carbonyl by-product has a vapor pressure higher than water, and removal of the carbonyl byproduct is by sparging with a non-reactive gas or by applying a vacuum, wherein:

i) the non-reactive gas optionally is nitrogen gas; or

ii) the amino donor optionally is isopropylamine and the carbonyl by-product is acetone.

8. The process of claim 1 , wherein the transaminase comprises an amino acid sequence having a residue difference as compared to SEQ ID NO:2 at one or more residue positions selected from: X4; X5; X8; X18; X25; X26; X27; X28; X30; X41; X42; X48; X49; X50; X54; X55; X60; X61; X62; X65; X81; X94; X96; X102; X117; X120; 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, X292; X297; X306; X321; and X329, wherein:

i) the residue difference optionally occurs at one or more residue positions selected from: X62, X69, X122, X136, X137, X195, X199, X208, X209, X223, X225, X282, and X284;

ii) wherein the type of amino acid residue at the position of the residue difference 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;

X160 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;

X223 is a constrained 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;

iii) the amino acid residue at the position of the residue difference optionally 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; X223 is P; 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; and optionally the residue difference 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; X223 is P; X269 is P; X273 is Y; X282 is S; X284 is G; X297 is S; X321 is P and X329 is H; or

iii) the transaminase polypeptide optionally has the amino acid sequence of SEQ ID NO: 58, 72, 74, 80, 86, 96, 98, 100, or 102.

Assignments (1)

Continuity (8)

References Cited (136)