Methods for hydroxylyation of chemical compounds

Patent metadata

Field	Value
Publication number	US-10370688-B2
Application number	US-201615357668-A
Country	US
Kind code	B2
Filing date	Nov 21, 2016
Priority date	May 8, 2012
Publication date	Aug 6, 2019
Grant date	Aug 6, 2019

Abstract

The present disclosure provides engineered proline hydroxylase polypeptides for the production of hydroxylated compounds, polynucleotides encoding the engineered proline hydroxylases, host cells capable of expressing the engineered proline hydroxylases, and methods of using the engineered proline hydroxylases to prepare compounds useful in the production of active pharmaceutical agents.

First claim

What is claimed is:

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 leas

Assignees

• Codexis Inc

Inventors

• Chen Haibin
• Bong Yong Koy
• Cabirol Fabien L
• Gohel Prafulchandra Anupam
• Li Tao
• Moore Jeffrey C
• Quintanar-Audelo Martina
• Hong Yang
• Collier Steven J
• Smith Derek

Classifications

• C12N9/0071 Primary

  acting on paired donors with incorporation of molecular oxygen (1.14) · CPC title

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title

• C12Y114/11002

  Procollagen-proline dioxygenase (1.14.11.2), i.e. proline-hydroxylase · CPC title

• C12P17/12 Primary

  containing a six-membered hetero ring · CPC title

• C12P13/24

  Proline; Hydroxyproline; Histidine · CPC title