Biocatalysts and methods for the synthesis of armodafinil
View Patent ↗The present invention relates to non-naturally occurring polypeptides useful for preparing armodafinil, polynucleotides encoding the polypeptides, and methods of using the polypeptides. The non-naturally occurring polypeptides of the present invention are effective in carrying out biocatalytic conversion of the (i) 2-(benzhydrylsulfinyl)acetamide to (−)-2-[(R)-(diphenyl-methyl)sulfinyl]acetamide (armodafinil), or (ii) benzhydryl-thioacetic acid to (R)-2-(benzhydrylsulfinyl)acetic acid, which is a pivotal intermediate in the synthesis of armodafinil, in enantiomeric excess.
1. A non-naturally-occurring, recombinant polypeptide having cyclohexanone monooxygenase (CHMO) activity wherein the amino acid sequence of the polypeptide has at least 95% sequence identity to SEQ ID NO: 136.
2. The polypeptide of claim 1 , wherein the polypeptide is capable of converting the acid substrate (1b) to compound (2b) (R-enantiomer) or its opposite enantiomer compound (S-enantiomer) with at least 2-fold improved activity relative to the wild-type polypeptide of SEQ ID NO: 2
3. The polypeptide of claim 2 , wherein the polypeptide is capable of converting the acid substrate of compound (1b) to the R-enantiomer compound (2b) in at least 50% enantiomeric excess.
4. The polypeptide of claim 1 , wherein the polypeptide is capable of converting compound (1a) to compound (2a)
in at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% enatiomeric excess under suitable reaction conditions.
5. The polypeptide of claim 2 , wherein the polypeptide is capable of converting compound (1a) to compound (2a) with an activity increased at least 2-fold, at least 4-fold, at least 10-fold, at least 25-fold, at least 40-fold, or at least 60-fold relative to the activity of the polypeptide of SEQ ID NO: 2 under suitable reaction conditions.
6. The polypeptide of claim 2 , which is capable of converting compound (1b) to compound (2b)
in enantiomeric excess under suitable reaction conditions.
7. The polypeptide of claim 6 , wherein the polypeptide is capable of converting compound (1b) to compound (2b) in at least 75%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% enantiomeric excess under suitable reaction conditions.
8. The polypeptide of claim 2 , wherein the polypeptide is capable of converting compound (1b) to compound (2b) with an activity increased at least 2-fold, at least 4-fold, at least 10-fold, at least 25-fold, at least 40-fold, or at least 60-fold relative to the activity of the polypeptide of SEQ ID NO: 38 under suitable reaction conditions.
9. The polypeptide of claim 2 , wherein the polypeptide is capable of at least 90% or greater conversion of compound (1b) to compound (2b) in 24 h with a substrate loading of about 50 g/L.
10. The polypeptide of claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 90, 96, 98, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140 or 142.
11. A method for preparing compound (2a) from compound (1a) in enantiomeric excess,
comprising contacting compound (1a) with the polypeptide of claim 1 in the presence of cofactor NADPH or NADH under suitable reaction conditions.
12. The method of claim 11 , further comprising converting NADP + to NADPH with a cofactor recycling system.
13. The method of claim 12 , wherein the cofactor recycling system is selected from a ketoreductase and a secondary alcohol, or glucose dehydrogenase and glucose.
14. The method of claim 13 , wherein the secondary alcohol is isopropyl alcohol.
15. The method of claim 11 , wherein the suitable reaction conditions comprise a co-solvent selected from the group consisting of MeOH, EtOH, isopropanol (IPA), acetone, toluene, MeCN, methyl tert-butyl ether (MTBE), N-methyl-2-pyrrolidone (NMP), dimethylacetamide (DMAc), dimethylformamide (DMF), propylene glycol, polyethylene glycol (PEG), tetramethylurea, N-ethylpyrollidinone, tetraglyme, 1,3-Dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU), DMIU, hexamethylphosphoramide (HMPA) and dimethylsulfoxide (DMSO).
16. The method of claim 11 , wherein the suitable reaction conditions comprise a co-solvent of about 2% to about 7.5% (v/v) NMP.
17. The method of claim 11 , wherein the suitable reaction conditions comprise a co-solvent of about 5-15% (v/v) PEG200, or about 10% (v/v) PEG200.
18. A method for preparing compound (2b) from compound (1b) in enantiomeric excess,
comprising contacting compound (1b) with the polypeptide of claim 1 in the presence of cofactor NADPH or NADH under suitable reaction conditions.
19. The method of claim 18 , wherein the suitable reaction conditions comprise loading compound (1b) at a concentration of at least 20 g/L, at least 40 g/L, at least 50 g/L, at least 75 g/L, or at least 100 g/L.
20. The method of claim 18 , further comprising converting NADP + to NADPH with a cofactor recycling system.
21. The method of claim 20 , wherein the cofactor recycling system is selected from a ketoreductase and an alcohol, or glucose dehydrogenase and glucose.
22. The method of claim 21 , wherein the alcohol is isopropyl alcohol.
23. The method of claim 18 , wherein the suitable reaction conditions comprise a co-solvent selected from the group consisting of MeOH, EtOH, isopropanol (IPA), acetone, toluene, MeCN, methyl tert-butyl ether (MTBE), N-methyl-2-pyrrolidone (NMP), dimethylacetamide (DMAc), dimethylformamide (DMF), propylene glycol, polyethylene glycol (PEG), tetramethylurea, N-ethylpyrollidinone, tetraglyme, 1,3-Dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU), DMIU, hexamethylphosphoramide (HMPA) and dimethylsulfoxide (DMSO).
24. The method of claim 18 , wherein the suitable reaction conditions comprise a co-solvent of about 2% to about 7.5% (v/v) NMP.
25. The method of claim 18 , wherein the suitable reaction conditions comprise a co-solvent of about 5-15% (v/v) PEG200, or about 10% (v/v) PEG200.
26. A method for preparing compound (2a),
comprising the steps of:
(a) preparing compound (2b) from compound (1b) by contacting compound (1b) with the polypeptide of claim 1 ,
(b) preparing compound (2a) from compound (2b) by esterification and amidation.
27. A method for preparing compound of formula (II) from compound of formula (I) in enantiomeric excess,
wherein R 1 is —OH, —OCH 3 , —OCH 2 CH 3 , —NH 2 , —NHCH 3 , —N(CH 3 ) 2 , or —NHOH, wherein Ar 1 and Ar 2 are each independently a monocyclic aryl, a bicyclic aryl, or a heteroaryl group having 5-6 ring atoms, wherein Ar 1 and Ar 2 are each optionally, independently substituted 1 to 3 times with groups R 2 and/or R 3 , wherein R 2 and R 3 are independently —H, —F, —Cl, —Br, —CF 3 , —CH 3 , —CH 2 CH 3 , —NH 2 , —NHCH 3 , or —N(CH 3 ) 2 , and wherein Ar 1 and Ar 2 are optionally connected either
(i) via a group X, wherein X is O, NR, S, CH 2 , CH 2 CH 2 , or CH═CH; or
(ii) directly via a bond and X is absent,
the method comprising contacting compound of formula (I) with the polypeptide of claim 1 in the presence of cofactor NADPH or NADH under suitable reaction conditions.