In vivo and in vitro olefin cyclopropanation catalyzed by heme enzymes

Patent metadata

Field	Value
Publication number	US-9493799-B2
Application number	US-201514625449-A
Country	US
Kind code	B2
Filing date	Feb 18, 2015
Priority date	Oct 9, 2012
Publication date	Nov 15, 2016
Grant date	Nov 15, 2016

Abstract

The present invention provides methods for catalyzing the conversion of an olefin to any compound containing one or more cyclopropane functional groups using heme enzymes. In certain aspects, the present invention provides a method for producing a cyclopropanation product comprising providing an olefinic substrate, a diazo reagent, and a heme enzyme; and admixing the components in a reaction for a time sufficient to produce a cyclopropanation product. In other aspects, the present invention provides heme enzymes including variants and fragments thereof that are capable of carrying out in vivo and in vitro olefin cyclopropanation reactions. Expression vectors and host cells expressing the heme enzymes are also provided by the present invention.

First claim

What is claimed is:

1. A reaction mixture for producing a cyclopropanation product, the reaction mixture comprising an olefinic substrate, a carbene precursor, and a heme enzyme, wherein the cyclopropanation product is a compound according to Formula I:

wherein:
R 1 is independently selected from the group consisting of H, optionally substituted C 1-18 alkyl, optionally substituted C 6-10 aryl, optionally substituted 5- to 10-membered heteroaryl, C(O)OR 1a , and C(O)R 8 ;
R 2 is independently selected from the group consisting of H, optionally substituted C 1-18 alkyl, optionally substituted C 6-10 aryl, optionally substituted 5- to 10-membered heteroaryl, C(O)OR 2a , and C(O)R 8 ; and
R 3 , R 4 , R 5 , and R 6 are independently selected from the group consisting of H, C 1-18 alkyl, optionally substituted C 6-10 aryl, optionally substituted C 1 -C 6 alkoxy, NR 7 C(O)R 8 , C(O)R 8 , C(O)OR 8 , and N(R 9 ) 2 ;

wherein:
R 1a and R 2a are independently selected from the group consisting of H and optionally substituted C 1-18 alkyl;
each R 7 and R 8 is independently selected from the group consisting of H, optionally substituted C 1-12 alkyl, and optionally substituted C 6-10 aryl; and
two R 9 moieties, together with the nitrogen atom to which they are attached, form an optionally substituted 5- to 10-membered heterocyclyl.

2. The reaction mixture of claim 1 , wherein each R 1a and R 2a is independently an optionally substituted C 1-3 alkyl.

3. The reaction mixture of claim 1 , wherein each R 8 is independently an optionally substituted C 1-3 alkyl.

4. The reaction mixture of claim 1 , wherein R 8 is a phenyl group.

5. The reaction mixture of claim 1 , wherein the carbene precursor is a diazo reagent selected from the group consisting of diazomethane, an α-diazoester, an α-diazoamide, an α-diazonitrile, an α-diazoketone, and an α-diazoaldehyde.

6. The reaction mixture of claim 5 , wherein the diazo reagent has a formula selected from the group consisting of:

wherein:
R 1a is selected from the group consisting of H and optionally substituted C 1 -C 6 alkyl; and
each R 8 is independently selected from the group consisting of H, optionally substituted C 1-12 alkyl, and optionally substituted C 6-10 aryl.

7. The reaction mixture of claim 5 , wherein the diazo reagent is ethyl diazoacetate.

8. The reaction mixture of claim 1 , wherein the olefinic substrate and the carbene precursor are part of the same substrate.

9. The reaction mixture of claim 1 , wherein the cyclopropanation product is produced in vitro.

10. The reaction mixture of claim 1 , wherein the reaction mixture further comprises a reducing agent.

11. The reaction mixture of claim 1 , wherein the heme enzyme is localized within a whole cell and the cyclopropanation product is produced in vivo.

12. The reaction mixture of claim 1 , wherein the cyclopropanation product is produced under anaerobic conditions.

13. The reaction mixture of claim 1 , wherein the heme enzyme is a variant thereof comprising a mutation at the axial position of the heme coordination site.

14. The reaction mixture of claim 1 , wherein the heme enzyme is a cyctochrome P450 enzyme or a variant thereof.

15. The reaction mixture of claim 14 , wherein the cyctochrome P450 enzyme is a P450 BM3 enzyme or a variant thereof.

16. The reaction mixture of claim 15 , wherein the P450 BM3 enzyme comprises the amino acid sequence set forth in SEQ ID NO: 1.

17. The reaction mixture of claim 1 , wherein the olefinic substrate has the following formula:

wherein:
R 10 is selected from the group consisting of H, optionally substituted C 1 -C 6 alkyl, optionally substituted C 1 -C 6 alkoxy, C(O)N(R 7 ) 2 , C(O)OR 8 , N(R 9 ) 2 , halo hydroxy, and cyano;
R 11 and R 12 are independently selected from the group consisting of H, optionally substituted C 1-6 alkyl, and halo;
R 13 is selected from the group consisting of C 1-6 alkyl, optionally substituted C 1-6 alkoxy, halo, and haloalkyl; and
the subscript n is an integer from 0 to 2.

18. The reaction mixture of claim 17 , wherein:
R 10 , R 11 and R 12 are H;
R 13 is halo; and
the subscript n is 2.

19. The reaction mixture of claim 18 , wherein each R 13 group is a fluoro.

20. The reaction mixture of claim 5 , wherein the diazo reagent is diazoacetone.

Assignees

• California Inst Of Techn

Inventors

• Coelho Pedro S
• Brustad Eric M
• Arnold Frances H
• Wang Zhan
• Lewis Jared C

Classifications

• C12P7/62 Primary

  Carboxylic acid esters · CPC title

• C12N9/0004

  Oxidoreductases (1.) · CPC title

• C12P13/02 Primary

  Amides, e.g. chloramphenicol {or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes (peptides C12P21/00 or C07K)} · CPC title

• Y02P20/52

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

• C12Y106/02004

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