Ketoreductase polypeptides for the preparation of phenylephrine

1. An engineered polypeptide capable of converting 1-(3-hydroxyphenyl)-2-(methylamino)ethanone to (R)-phenylephrine, wherein the amino acid sequence of the polypeptide has at least 95% identity to SEQ ID NO: 4 and comprises at least one residue difference selected from C190G, A202F, M206C, and Y249F.

2. The engineered polypeptide of claim 1 , wherein the polypeptide is capable of stereospecifically converting 1-(3-hydroxyphenyl)-2-(methylamino)ethanone to (R)-phenylephrine in an enantiomeric excess of at least 99%.

3. An engineered polynucleotide encoding the engineered polypeptide of claim 1 .

4. A method for producing a compound of Formula I from a compound of Formula II:

wherein R 2 is a group selected from: —H, —Cl, —Br, —I, —F, —CH 3 , —OH, —OCH 3 , —SH, —SCH 3 , —NH 2 , —NHCH 3 , or a long chain alkyl;

wherein R 3 is a group selected from: —H, —Cl, —Br, —I, —F, —CH 3 , —OH, —OCH 3 , —SH, —SCH 3 , —S(O)CH 3 , —NH 2 , —NHCH 3 , —N(CH 3 ) 2 , —OR, —SR, —NR 2 , —SO 2 NR 2 (wherein R═—H, —CH 3 , or alkyl), ethyl, propyl, isopropyl, cyclopropyl, or a long chain alkyl;

wherein R 4 is a group selected from: —H, —Cl, —Br, —I, —F, —CH 3 , —OH, —OCH 3 , —SH, —SCH 3 , —S(O)CH 3 , —SO 2 CH 3 , —NH 2 , —NHCH 3 , —N(CH 3 ) 2 , SO 2 NR 2 (wherein R═—H, —CH 3 );

wherein R 5 is a group selected from: —H, —Cl, —Br, —I, —F, —CH 3 , —OH, —OCH 3 , —SH, —SCH 3 , —S(O)CH 3 , —SO 2 CH 3 , —NH 2 , —NHCH 3 , —N(CH 3 ) 2 , —OR, —SR, —NR 2 , —SO 2 NR 2 (wherein R═—H, —CH 3 , or alkyl), ethyl, propyl, isopropyl, or cyclopropyl;

wherein R 6 is a group selected from: —H, —Cl, —Br, —I, —F, —CH 3 , —OH, —SH, or —NH 2 ;

wherein R 2 and R 3 , R 3 and R 4 , or R 4 and R 5 can optionally be connected as part of a 5 or 6 membered ring;

wherein R α is a group selected from: —H, —CH 3 , ethyl, propyl, isopropyl, cyclopropyl, or a long chain alkyl; wherein R β is a group selected from: —H, —CH 3 , ethyl, propyl, isopropyl, or cyclopropyl;

wherein R α and R β can form a ring, or wherein the R α -R β unit is a carbonyl or imino functional group;

wherein R N1 and R N2 can be independently a group selected from: —H, —CH 3 , —OH, —OCH 3 , —OR, —C(O)R (wherein R═—H, —CH 3 , or alkyl), ethyl, propyl, isopropyl, cyclopropyl, long chain alkyl, carbonyl, or carboxy;

the method comprising contacting a mixture comprising the compound of Formula II with a polypeptide of claim 1 , under reaction conditions suitable to convert the compound of Formula II to the compound of Formula I.

5. The method of claim 4 , comprising:

contacting a polypeptide of claim 1 with a mixture comprising a 1-(3-hydroxyphenyl)-2-(methylamino)ethanone substrate and a buffer under reaction conditions suitable to convert 1-(3-hydroxyphenyl)-2-(methylamino)ethanone to (R)-phenylephrine.

6. The method of claim 4 , wherein the reaction conditions comprise a temperature of about 25° C. to about 35° C.

7. The method of claim 4 , wherein the reaction conditions comprise a temperature of about 30° C.

8. The method of claim 4 , wherein the reaction conditions comprise a pH of about 6.5 to about 7.0.

9. The method of claim 4 , wherein the reaction conditions comprise an initial pH of about 7.0 and then adjusting the initial pH to about 6.75 after about 2 hours.

10. The method of claim 4 , wherein the mixture comprises 50% isopropyl alcohol.

11. The method of claim 4 , wherein the buffer is selected from 0.1 M triethanolamine and 0.05 M potassium phosphate.

12. The method of claim 4 , wherein the mixture comprises about 0.05 g/L NADP.

13. The method of claim 4 , wherein the reaction conditions comprise an inert atmosphere.

14. The method of claim 4 , wherein the reaction conditions comprise a pH of about 6.75-7.0, a temperature of about 30° C., about 50% isopropyl alcohol, about 0.05 g/L NADP, about 100 g/L 1-(3-hydroxyphenyl)-2-(methylamino)ethanone, and about 0.9-1.1 g/L of the polypeptide, wherein the reaction is carried out under N 2 for about 19-24 hours.