Cell-free preparation of carbapenems

• US 9,469,861 B2
• Filed: 09/07/2012
• Issued: 10/18/2016
• Est. Priority Date: 09/09/2011
• Status: Active Grant

• Current Assignee: GreenLight Biosciences, Inc.
• Original Assignee: GreenLight Biosciences, Inc.
• Inventors: Blake, William Jeremy, Klein-Marcuschamer, Daniel
• Primary Examiner(s): Tsay, Marsha
• Application Number:
  US13/606,911
• Publication Number:
  US 20130065878A1
• Time in Patent Office: 1,502 Days
• US Class Current: 1/1
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12N 9/0026 acting on CH-NH groups of donors (1.5)
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12N 9/1217 Phosphotransferases with a carboxyl group as acceptor (2.7.2)
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12P 17/184 containing a beta-lactam ring, e.g. thienamycin
• CPC Class Codes: C12Y 102/01041 Glutamate-5-semialdehyde dehydrogenase (1.2.1.41)
• CPC Class Codes: C12Y 105/99008 Proline dehydrogenase (1.5.99.8)
• CPC Class Codes: C12Y 203/01 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12Y 207/02011 Glutamate 5-kinase (2.7.2.11)
• CPC Class Codes: C12Y 603/03004 (Carboxyethyl)arginine beta-lactam-synthase (6.3.3.4)
• CPC Class Codes: C12Y 603/04016 Carbamoyl-phosphate synthase (ammonia) (6.3.4.16)
• CPC Class Codes: C12Y 603/05005 Carbamoyl-phosphate synthase (glutamine-hydrolysing) (6.3.5.5)
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

Provided herein are cell-free systems for generating carbapenems, e.g., a compound of the Formula (I):

or salts thereof; wherein , R¹, R², R³, R⁴, R⁵, and R⁶ are defined herein. Also provided are pharmaceutical compositions comprising a compound generated by the inventive cell-free system, and use of these compounds and compositions for the treatment of bacterial infections.

Claims

1. A method of generating a compound of Formula (I-a):

2. The method of claim 1, wherein R³ is hydrogen or —

CH₃.

3. The method of claim 1, wherein R⁴ is hydrogen.

4. The method of claim 1, wherein one or more enzymes selected from the group consisting of ProA, ProB, γ

-glutamyl kinase-GP-reductase multienzyme complex, Glk, Pgi, PfkA, Fba, TpiA, GapA, Pgk, GpmA, Eno, PykA, PykF, AceE, Ace, acetyl-CoA acetyltransferase, beta-oxidation multienzyme, S-hydratase, 3-hydroxyacyl-CoA dehydrogenase, crotonyl CoA reductase, acetyl-CoA carboxylase, malonyl-CoA reductase, threonine aldolase, beta-alanine transaminase, proline 3-hydroxylase, proline oxidase, and isozymes thereof, were present in the cell cytoplasm prior to lysing.

5. The method of claim 1, wherein the cell lysate is a lysate of an E. coli organism engineered to overexpress the group of enzymes.

6. The method of claim 1, wherein R⁵ is hydrogen or —

OH, and the proline compound of formula (iv);

7. The method of claim 1, wherein the proline compound of formula (iv):

8. The method of claim 1, wherein R⁵ is —

OH, and the glucose and glycine or salts thereof are enzymatically converted to an optionally substituted glutamate semialdehyde of formula (ii);

9. The method of claim 1, wherein R⁵ is hydrogen or —

OH, and the optionally substituted glutamate of formula (i) or salt thereof is enzymatically converted to an optionally substituted glutamate semialdehyde compound of formula (ii);

10. The method of claim 1, wherein the optionally substituted glutamate of formula (i):

12. The method of claim 1, wherein R⁵ is hydrogen or —

OH, and the combination of glucose or salt thereof and the proline compound of formula (iv) or salt thereof is enzymatically converted to an optionally substituted CoA compound of formula (iii), or salt thereof, and a compound of formula (v), or salt thereof;

11. A method of generating a compound of Formula (I-a):

13. The method of claim 11, wherein the compound of Formula (I-a):

14. The method of claim 13, wherein the compound of Formula (I-e) is contacted with a compound of the formula HS—

R⁸,

wherein R⁸ is selected from the group consisting of optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heteroalkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;

to provide a thiol-containing compound of Formula (I-c);

15. The method of claim 11, wherein R² is —

CH₂CH₃.

16. The method of claim 15, further comprising contacting the compound with an oxygenase enzyme to provide a hydroxylated compound wherein R² is —

CH(OH)CH₃.

17. The method of claim 11, wherein R¹ and R² are both hydrogen.

18. The method of claim 17, further comprising contacting the compound with a methyltransferase enzyme to provide a compound wherein R¹ is hydrogen and R² is —

CH₃ or —

CH₂CH₃.

19. The method of claim 18, further comprising contacting the compound wherein R² is —

CH₂CH₃ with an oxygenase enzyme to provide a hydroxylated compound wherein R² is —

CH(OH)CH₃.

20. The method of claim 14, wherein R⁸ is an optionally substituted alkyl, optionally substituted heteroalkyl, or optionally substituted heterocyclyl.

21. The method of claim 20, wherein R⁸ is selected from the group consisting of: