In vivo and in vitro olefin cyclopropanation catalyzed by heme enzymes

Patent metadata

Field	Value
Publication number	US-10208322-B2
Application number	US-201615278561-A
Country	US
Kind code	B2
Filing date	Sep 28, 2016
Priority date	Oct 9, 2012
Publication date	Feb 19, 2019
Grant date	Feb 19, 2019

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 diazo reagent, and a heme enzyme variant; wherein the heme enzyme variant can cyclopropanate an olefinic substrate.

2. The reaction mixture of claim 1 , 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 6- to 10-membered heteroaryl, halo, cyano, C(O)OR 1a , C(O)N(R 7 ) 2 , C(O)R 8 , C(O)C(O)OR 8 , and Si(R 8 ) 3 ;
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 6- to 10-membered heteroaryl, halo, cyano, C(O)OR 2a , C(O)N(R 7 ) 2 , C(O)R 8 , C(O)C(O)OR 8 , and Si(R 8 ) 3 ;
wherein
R 1a and R 2a are independently selected from the group consisting of H, optionally substituted C 1-18 alkyl and -L-R c , wherein
each L is selected from the group consisting of a bond, —C(R L ) 2 -, and —NR L —C(R L ) 2 -,
each R L is independently selected from the group consisting of H, C 1-6 alkyl, halo, —CN, and -SO 2 , and
each R C is selected from the group consisting of optionally substituted C 6-10 aryl, optionally substituted 6- to 10-membered heteroraryl, and optionally substituted 6- to 10-membered heterocyclyl; and

R 3 , R 4 , R 5 , and R 6 are independently selected from the group consisting of H, C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl, optionally substituted C 6-10 aryl, optionally substituted C 1 -C 6 alkoxy, halo, hydroxy, cyano, C(O)N(R 7 ) 2 , NR 7 C(O)R 8 , C(O)R 8 , C(O)OR 8 , and N(R 9 ) 2 ,
wherein
each R 7 and R 8 is independently selected from the group consisting of H, optionally substituted C 1-12 alkyl, optionally substituted C 2-12 alkenyl, and optionally substituted C 6-10 aryl; and
each R 9 is independently selected from the group consisting of H, optionally substituted C 6-10 aryl, and optionally substituted 6- to 10-membered heteroaryl, or two R 9 moieties, together with the nitrogen atom to which they are attached, can form 6- to 18-membered heterocyclyl;

or R 3 forms an optionally substituted 3- to 18-membered ring with R 4 ;
or R 5 forms an optionally substituted 3- to 18-membered ring with R 6 ;
or R 3 or R 4 forms a double bond with R 5 or R 6 ;
or R 3 or R 4 forms an optionally substituted 5- to 6-membered ring with R 5 or R 6 .

3. The reaction mixture of claim 1 , wherein the olefinic substrate is selected from the group consisting of an alkene, a cycloalkene, and an arylalkene.

4. The reaction mixture of claim 3 , wherein the olefinic substrate has the formula:

wherein R 3 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 5 and R 6 are independently selected from the group consisting of H, optionally substituted C 1-6 alkyl, and halo;
R 10 is selected from the group consisting of optionally substituted C 1 -C 6 alkyl, optionally substituted C 1 -C 6 alkoxy, halo, and haloalkyl; and
the subscript r is an integer from 0 to 2.

5. The reaction mixture of claim 1 , wherein the diazo reagent has a structure according to the formula:

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 6- to 10-membered heteroaryl, halo, cyano, C(O)OR 1a , C(O)N(R 7 ) 2 , C(O)R 8 , C(O)C(O)OR 8 , and Si(R 8 ) 3 ; and
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 6- to 10-membered heteroaryl, halo, cyano, C(O)OR 2 a, C(O)N(R 7 ) 2 , C(O)R 8 , C(O)C(O)OR 8 , and Si(R 8 ) 3 ;
wherein
R 1a and R 2a are independently selected from the group consisting of H, optionally substituted C 1-18 alkyl and -L-R C , wherein
each L is selected from the group consisting of a bond, —C(R L ) 2 -, and —NR L —C(R L ) 2 -,
each R L is independently selected from the group consisting of H, C 1-6 alkyl, halo, —CN, and -SO 2 , and
each R C is selected from the group consisting of optionally substituted C 6-10 aryl, optionally substituted 6- to 10-membered heteroraryl, and optionally substituted 6- to 10-membered heterocyclyl; and
each R 7 and R 8 is independently selected from the group consisting of H, optionally substituted C 1-12 alkyl, optionally substituted C 2-12 alkenyl, and optionally substituted C 6-10 aryl.

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

7. The reaction mixture of claim 1 , wherein the heme enzyme is a globin or a variant thereof.

Assignees

• California Inst Of Techn

Inventors

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

Classifications

• Y02P20/52

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

• C12N9/0042

  NADPH-cytochrome P450 reductase (1.6.2.4) · 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

• C12N9/0004

  Oxidoreductases (1.) · CPC title

• C12P7/62 Primary

  Carboxylic acid esters · CPC title