Methods for hydroxylyation of chemical compounds

1. A process for preparing a product compound of formula (Ia),

the process comprising:

i. contacting a substrate compound of formula (IIa)

with an engineered polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2,

ii. in the presence of α-ketoglutaric acid at a concentration equimolar or higher than that of the substrate compound concentration, under suitable reaction conditions to prepare a product compound of formula (Ia);

wherein for the compound of formula (Ia) and the compound of formula (IIa),

Q is (C 1 -C 5 )alkylene;

L is (C 1 -C 4 )alkylene;

R 6 is halo;

and

wherein the sum of ring carbon atoms of said Q +L is an integer from 2 to 5;

with the provisos that

(i) when the sum of ring carbon atoms of said Q +L is 2, then L is a methylene; and

(ii) when the sum of ring carbon atoms of said Q +L is 3, then L is either a bond or ethylene; and

wherein the engineered polypeptide comprises a mutation of X166T/Q/L.

2. The process of claim 1 , in which the compound of formula (Ia) comprises the compound of formula (Ib),

the process comprising:

i. contacting substrate compound of formula (IIb)

with an engineered polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2,

ii. in the presence of α-ketoglutaric acid at a concentration equimolar or higher than that of the substrate compound concentration, under suitable reaction conditions to prepare a product compound of formula (Ib);

wherein for the compound of formula (Ib) and the compound of formula (IIb),

R 6 is halo;

k is an integer from 1 to 5;

r is an integer from 0 to 4; and

wherein k +r is 3, 4 or 5;

with the proviso that when k +r is 3, then k is 1 or 3; and

wherein the engineered polypeptide further comprises one or more residue differences at residue positions selected from the group consisting of: X2, X3, X4, X5, X9, X13, X25, X26, X29, X30, X36, X42, X52, X57, X58, X59, X66, X86, X92, X95, X103, X112, X113, X115, X116, X121, X131, X150, X151, X225, X230, X270, and X271.

3. The process of claim 1 , in which the compound of formula (Ia) comprises the compound of formula (Ic),

the process comprising:

i. contacting substrate compound of formula (IIc)

with an engineered polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2,

ii. in the presence of α-ketoglutaric acid at a concentration equimolar or higher than that of the substrate compound concentration, under suitable reaction conditions to prepare a product compound of formula (Ic);

wherein for the compound of formula (Ic) and the compound of formula (IIc),

R 6 is halo; and

wherein the engineered polypeptide further comprises one or more residue differences at residue positions selected from the group consisting of: X2, X3, X4, X5, X9, X13, X25, X26, X29, X30, X36, X42, X52, X57, X58, X59, X66, X86, X92, X95, X103, X112, X113, X115, X116, X121, X131, X150, X151, X225, X230, X270, and X271.

4. The process of claim 1 , in which the compound of formula (Ia) comprises the compound of formula (Ie),

the process comprising:

i. contacting substrate compound of formula (IIe)

with an engineered polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2,

ii. in the presence of α-ketoglutaric acid at a concentration equimolar or higher than that of the substrate compound concentration, under suitable reaction conditions to prepare a product compound of formula (Ie);

wherein for the compound of formula (Ie) and the compound of formula (IIe),

R 6 is halo; and

wherein the engineered polypeptide further comprises one or more residue differences at residue positions selected from the group consisting of: X2, X3, X4, X5, X9, X13, X25, X26, X29, X30, X36, X42, X52, X57, X58, X59, X66, X86, X92, X95, X103, X112, X113, X115, X116, X121, X131, X150, X151, X225, X230, X270, and X271.

5. The process of claim 1 , in which the suitable reaction conditions comprise Fe +2 , optionally in which the Fe +2 is in the form of (NH 4 ) 2 (FeSO 4 ) 2 .

6. The process of claim 1 , in which the suitable reaction conditions comprise a reductant that reduces Fe +3 to Fe +2 , optionally in which the reductant comprises ascorbic acid present in at least about 0.1 fold, 0.2 fold, 0.3 fold, 0.5 fold, 0.75 fold, 1 fold, 1.5 fold, or at least 2 fold the molar concentration of substrate compound.

7. The process of claim 1 , in which the suitable reaction conditions comprise O 2 , optionally in which the O 2 is provided by forced aeration.

8. The process of claim 1 , in which the suitable reaction conditions comprise (a) substrate loading at about 10 g/L to 100 g/L; (b) about 1 g/L to about 50 g/L of the engineered polypeptide; (c) α-ketoglutarate at a concentration of about 1 to 2 molar equivalents of the substrate compound; (d) ascorbic acid at a concentration of about 0.25 to 0.75 molar equivalents of the substrate compound; (e) about 0.5 mM to about 12 mM of FeSO 4 ; (f) a pH of about 6 to 8; (g) a temperature of about 20° to 40° C.; and (h) a reaction time of 6 to 120 h.

9. The process of claim 1 , wherein said engineered polypeptide further comprises at least one mutation at a residue selected from the group consisting of 2K, 2T, 3S, 4Q, 4L, 4E, 4S, 5I, 5L, 5M, 91, 13T, 25R, 26T, 29A, 30V, 30P, 36T, 42E, 52P, 57T, 57A, 58A, 59G, 66Q, 86S, 92V, 95M, 103L, 103Q, 112T, 112V, 113E, 115E, 115H, 115D, 115G, 115S, 115A, 116L, 121F, 131Y, 131F, 150S, 1515, 225L, 225Y, 225W, 230V, 270E, 271K, and 271, wherein said residue positions are numbered relative to SEQ ID NO:2.

10. The process of claim 1 , wherein said engineered polypeptide further comprises a mutation or combination of mutations, as compared to the sequence of SEQ ID NO: 2, selected from the group consisting of:

(a) 103L;

(b) 52P and 255Y;

(c) 4E/L/S and 115A;

(d) 25R and 58A;

(e) 29A;

(f) 115H/D/G and 121F;

(g) 3S and 103L;

(h) 103L and 131Y/F;

(i) 26T and 103L;

(j) 25R, 66Q, 92V and 115E;

(k) 25R, 66Q, 92V, 103L, and 115E; and

(l) 3S, 25R, 66Q, 92V, 103L, and 115E.

11. The process of claim 1 , wherein said engineered polypeptide further comprises one or more differences in at least one residue position, as compared to the sequence of SEQ ID NO: 2 at residue positions selected from the group consisting of: 17, 24, 26, 62, 88, 98, 114, 140, 151, 186, 188, and 205.

12. The process of claim 1 , wherein said engineered polypeptide further comprises at least one difference in at least one residue position, as compared to the sequence of SEQ ID NO: 2, wherein said residue position is selected from the group consisting of: 17V, 24R, 24S, 26R, 26W, 62Q, 88R, 98F, 98T, 114N, 140L, 151A, 151H, 186G, 188G, and 205V.