Diverse carbene transferase enzyme catalysts derived from a P450 enzyme

Patent metadata

Field	Value
Publication number	US-11525123-B2
Application number	US-202117200394-A
Country	US
Kind code	B2
Filing date	Mar 12, 2021
Priority date	Mar 12, 2020
Publication date	Dec 13, 2022
Grant date	Dec 13, 2022

Abstract

The present disclosure provides cytochrome P450 variants useful for carrying out in vivo and in vitro carbene insertion reactions. Methods for preparing carbene insertion products including cyclopropenes, cyclopropanes, bicyclobutanes, substituted lactones, cyclized compounds, and substituted amines are also described.

First claim

What is claimed is:

1. A polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 and optionally 1-30 mutations at positions 2, 47, 70, 72, 74, 78, 80, 82, 87, 88, 92, 118, 142, 162, 190, 226, 240, 252, 263, 264, 267, 279, 327, 328, 332, 366, 401, 436, 437, and 474 of SEQ ID NO:1.

2. The polypeptide of claim 1 , wherein the polypeptide comprises 1-7 mutations at positions 70, 72, 74, 263, 332, 436, and 437.

3. The polypeptide of claim 1 , wherein the polypeptide comprises 1-6 mutations at positions 87, 264, 267, 327, 332, and 437.

4. The polypeptide of claim 1 , wherein the polypeptide contains 1-7 mutations at positions 47, 72, 118, 264, 327, 437, and 474.

5. The polypeptide of claim 1 , wherein the polypeptide comprises:
1-5 mutations at positions 78, 88, 401, 436, and 437; or
1-8 mutations at positions 70, 78, 162, 190, 328, 401, 436, and 437.

6. The polypeptide of claim 1 , wherein the polypeptide comprises 1-19 mutations at positions 2, 72, 74, 80, 87, 92, 142, 162, 226, 240, 252, 263, 279, 327, 328, 332, 366, 436, and 437.

7. The polypeptide of claim 1 , wherein the polypeptide comprises 1-6 mutations at positions 327, 437, 332, 87, 264, and 327.

8. A method for forming a carbene insertion product, the method comprising:
forming a reaction mixture comprising an enzyme catalyst and one or two enzyme substrates, and
incubating the mixture to form the carbene insertion product,
wherein at least one of the substrates comprises a carbene precursor moiety,
wherein the enzyme catalyst comprises the amino acid sequence set forth in SEQ ID NO:1 and optionally 1-30 mutations at positions 2, 47, 70, 72, 74, 78, 80, 82, 87, 88, 92, 118, 142, 162, 190, 226, 240, 252, 263, 264, 267, 279, 327, 328, 332, 366, 401, 436, 437, and 474 of SEQ ID NO:1.

9. The method of claim 8 , wherein:
the reaction mixture comprises a first enzyme substrate according to Formula I

and a second enzyme substrate according to Formula II

the carbene insertion product is a cyclopropene according to Formula III

and
the enzyme catalyst optionally comprises 1-11 mutations at positions 70, 72, 74, 87, 263, 264, 267, 327, 332, 436, and 437;
and wherein:
R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently selected from the group consisting of H, optionally substituted C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl, optionally substituted C 6-10 aryl, optionally substituted 6- to 10-membered heteroaryl, optionally substituted 6- to 10-membered heterocyclyl, cyano, halo, nitro, N(R 8 ) 2 , B(R 9 ) 2 , Si(R 9 ) 3 , C(O)OR 7 , C(O)SR 7 , C(O)N(R 7 ) 2 , C(O)R 7 , C(O)ON(R 7 ) 2 , C(O)NR 7 OR 8 , C(O)C(O)OR 7 , and P(O)(OR 7 ) 2 ; and
each R 7 , R 8 , and R 9 is independently selected from the group consisting of H, C 1-6 alkyl and optionally substituted C 6-10 aryl.

10. The method of claim 9 , wherein the enzyme catalyst comprises 1-7 mutations at positions 70, 72, 74, 263, 332, 436, and 437.

11. The method of claim 9 , wherein the enzyme catalyst comprises 1-6 mutations at positions 87, 264, 267, 327, 332, and 437.

12. The method of claim 8 , wherein:
the reaction mixture comprises a first enzyme substrate according to Formula IV

and
a second enzyme substrate according to Formula II

and
the carbene insertion product is a cyclopropane according to Formula V

and wherein:
R 3 , R 4 , R 10 , R 11 , R 12 and R 13 are independently selected from the group consisting of H, optionally substituted C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl, optionally substituted C 6-10 aryl, optionally substituted 6- to 10-membered heteroaryl, optionally substituted 6- to 10-membered heterocyclyl, cyano, halo, nitro, N(R 8 ) 2 , B(R 9 ) 2 , Si(R 9 ) 3 , C(O)OR 7 , C(O)SR 7 , C(O)N(R 7 ) 2 , C(O)R 7 , C(O)ON(R 7 ) 2 , C(O)NR 7 OR 8 , C(O)C(O)OR 7 , and P(O)(OR 7 ) 2 ; and
each R 7 , R 8 , and R 9 is independently selected from the group consisting of H, C 1-6 alkyl and optionally substituted C 6-10 aryl.

13. The method of claim 8 , wherein:
the reaction mixture comprises a first enzyme substrate according to Formula VI

and
a second enzyme substrate according to Formula II

the carbene insertion product is a bicyclobutane according to Formula V

and
the enzyme catalyst optionally comprises 1-7 mutations at positions 47, 72, 118, 264, 327, 437, 474;
and wherein:
R 3 , R 4 , R 14 , R 15 , R 16 and R 17 are independently selected from the group consisting of H, optionally substituted C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl, optionally substituted C 6-10 aryl, optionally substituted 6- to 10-membered heteroaryl, optionally substituted 6- to 10-membered heterocyclyl, cyano, halo, nitro, N(R 8 ) 2 , B(R 9 ) 2 , Si(R 9 ) 3 , C(O)OR 7 , C(O)SR 7 , C(O)N(R 7 ) 2 , C(O)R 7 , C(O)ON(R 7 ) 2 , C(O)NR 7 OR 8 , C(O)C(O)OR 7 , and P(O)(OR 7 ) 2 ; and
each R 7 , R 8 , and R 9 is independently selected from the group consisting of H, C 1-6 alkyl and optionally substituted C 6-10 aryl.

14. The method of claim 13 , wherein the enzyme catalyst contains 1-7 mutations at position 47, 72, 118, 264, 327, 437, and 474.

15. The method of claim 8 , wherein:
the reaction mixture comprises a first enzyme substrate according to Formula VIII

and
a second enzyme substrate according to Formula IX

the carbene insertion product is a substituted lactone according to Formula X

and
the enzyme catalyst optionally comprises 1-8 mutations at positions 327, 437, 332, 87, 264, 327, 267, 328;
and wherein:
R 18 , R 19 , R 20 , and R 21 are independently selected from the group consisting of H, optionally substituted C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl, optionally substituted C 6-10 aryl, optionally substituted 6- to 10-membered heteroaryl, optionally substituted 6- to 10-membered heterocyclyl, cyano, halo, nitro, N(R 8 ) 2 , B(R 9 ) 2 , Si(R 9 ) 3 , C(O)OR 7 , C(O)SR 7 , C(O)N(R 7 ) 2 , C(O)R 7 , C(O)ON(R 7 ) 2 , C(O)NR 7 OR 8 , C(O)C(O)OR 7 and P(O)(OR 7 ) 2 ;
each R 7 , R 8 , and R 9 is independently selected from the group consisting of H, C 1-6 alkyl, and optionally substituted C 6-10 aryl; and
subscript n is an integer ranging from 0 to 10.

16. The method of claim 15 , wherein the enzyme comprises 1-8 mutations at positions 327, 437, 332, 87, 264, 327, 267, and

Assignees

• California Inst Of Techn

Inventors

• Chen Kai
• Dunham Noah P
• Wackelin Daniel J
• Zhou Andrew Z
• Liu Zhen

Classifications

• C12N9/0042 Primary

  NADPH-cytochrome P450 reductase (1.6.2.4) · CPC title

• C12P17/04

  containing a five-membered hetero ring, e.g. griseofulvin {, vitamin C} · CPC title

• C12P17/16

  containing two or more hetero rings {(thiamine open chain analogs C12P17/167, i.e. not condensed among themselves or through a common carbocyclic ring system)} · CPC title

• C12Y106/02004

  NADPH-hemoprotein reductase (1.6.2.4), i.e. NADP-cytochrome P450-reductase · CPC title

• C12P7/62

  Carboxylic acid esters · CPC title