Biocatalysts and methods for the synthesis of (1R,2R)-2-(3,4-dimethoxyphenethoxy)cyclohexanamine
The disclosure provides transaminase polypeptides capable of converting the substrate, 2-(3,4-dimethoxyphenethoxy)cyclohexanone to the trans diastereomer product (1R,2R)-2-(3,4-dimethoxyphenethoxy)cyclohexanamine in at least a 2:1 diastereomeric ratio relative to the cis diastereomer (1R,2S)-2-(3,4-dimethoxyphenethoxy)cyclohexanamine. The disclosure also provides polynucleotides, vectors, host cells, and methods of making and using the transaminase polypeptides in processes for preparing (1R,2R)-2-(3,4-dimethoxyphenethoxy)cyclohexanamine and its analogs, which can product compounds can be further used to prepare the aminocyclohexylether compound, (3R)-1-[(1R,2R)-2-[2-(3,4-dimethoxyphenyl)ethoxy]cyclohexyl]pyrrolidin-3-ol, which is an ion channel blocker.
1. An engineered transaminase comprising an amino acid sequence having at least 90% sequence identity to the reference polypeptide of SEQ ID NO: 6 and an amino acid residue difference as compared to SEQ ID NO: 6 at the positions X69V, X124I, and X136W.
2. The polypeptide of claim 1 in which the amino acid differences as compared to SEQ ID NO: 6 comprise X69V, X124I, and X136W.
3. The polypeptide of claim 2 in which the polypeptide amino acid sequence further comprises one or more amino acid residue differences as compared to SEQ ID NO: 6 selected from the following: X2K; X2Q; X2S; X4I; X4L; X5H; X5I; X5L; X5N; X5S; X5T; X5V; X7A; X8T; X9N; X9Q; X9S; X10V; X11K; X14R; X28P; X37R; X38G; X41F; X42A; X44Q; X44V; X52K; X54K; X54N; X54P; X54R; X55L; X56G; X56L; X56S; X58L; X94L; X99L; X108V; X126A; X126T; X135Q; X141L; X142R; X142T; X150A; X150F; X150N; X155A; X156A; X156F; X156G; X156S; X156T; X157L; X164A; X165N; X171A; X182T; X199F; X199R; X199Y; X209C; X209D; X209E; X210S; X213P; X215F; X215Y; X217S; X218M; X22I; X223I; X223L; X223M; X223N; X245S; X257F; X265T; X267V; X296S; and X328I.
4. The polypeptide of claim 1 in which the polypeptide is capable of converting a racemic mixture of compound (1) to compound (2a) in at least a 2:1 diastereomeric ratio relative to compound (2c) under suitable reaction conditions:
5. The polypeptide of claim 1 in which the polypeptide amino acid sequence comprises SEQ ID NO:206.
6. A polynucleotide encoding the polypeptide of claim 1 .
7. An expression vector comprising the polynucleotide of claim 6 .
8. A host cell comprising the polynucleotide of claim 6 .
9. A method for preparing a polypeptide comprising culturing a host cell of claim 8 and isolating the polypeptide from the cell.
10. A method for preparing (1R,2R)-2-(3,4-dimethoxyphenethoxy)cyclohexanamine (compound (2a)),
comprising contacting (R)-2-(3,4-dimethoxyphenethoxy)cyclohexanone (compound (1a))
with a transaminase polypeptide of claim 1 in the presence of an amino donor under suitable reactions conditions.
11. The method of claim 10 , wherein the suitable reaction conditions comprise at least about 10 g/L compound (1a), at least about 1 g/L polypeptide, at least about 1 M isopropylamine, at least about 1 g/L pyridoxal 5′-phosphate, 0.2 M borate, at least about 20% (v/v) DMSO, pH 10.5, and 45° C.
12. The method of claim 10 , in which the method further comprises one or more steps selected from: extraction of compound (2a); isolation of compound (2a); forming a salt of compound (2a); purification of compound (2a); and crystallization of compound (2a).