US 9,469,861 B2Granted

Cell-free preparation of carbapenems

AssigneeGreenLight Biosciences, Inc.

InventorsWilliam Jeremy Blake, Daniel Klein-Marcuschamer

Life of this patent

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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.

Claims21

Dependent claims

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

or a salt or tautomer thereof, or a combination thereof;
the method comprising:
(1) providing one or more cell lysates comprising:
a. ProA and ProB, or γ-glutamyl kinase-GP-reductase multienzyme complex;
b. Glk;
c. Pgi;
d. PfkA;
e. Fba;
f. TpiA;
g. GapA;
h. Pgk;
i. GpmA;
j. Eno;
k. PykA;
l. PykF;
m. AceE;
n. AceF;
o. acetyl-CoA acetyltransferase;
p. acetyl-CoA carboxylase and malonyl-CoA reductase, or beta-alanine transaminase;
q. S-hydratase and 3-hydroxyacyl-CoA dehydrogenese, or beta-oxidation multienzyme;
r. crotonyl-CoA reductase;
s. threonine aldolase;
t. carboxymethyl-Pro synthase;
u. carbapenam synthetase or beta-lactam synthetase;
and
v. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and glycine, or salts thereof, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen, R2 is —CH2CH3, R3 is hydrogen, R4 is hydrogen, and R5 is —OH;
or
(2) providing one or more cell lysates comprising:
a. ProA and ProB, or γ-glutamyl kinase-GP-reductase multienzyme complex;
b. Glk;
c. Pgi;
d. PfkA;
e. Fba;
f. TpiA;
g. GapA;
h. Pgk;
i. GpmA;
j. Eno;
k. PykA;
l. PykF;
m. AceE;
n. AceF;
o. acetyl-CoA carboxylase and malonyl-CoA reductase, or beta-alanine transaminase;
p. threonine aldolase;
q. carboxymethyl-Pro synthase;
r. carbapenam synthetase or beta-lactam synthetase;
and
s. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and glycine, or salts thereof, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen, R2 is hydrogen, R3 is hydrogen, R4 is hydrogen, and R5 is —OH;
or
(3) providing one or more cell lysates comprising:
a. ProA and ProB, or γ-glutamyl kinase-GP-reductase multienzyme complex;
b. Glk;
c. Pgi;
d. PfkA;
e. Fba;
f. TpiA;
g. GapA;
h. Pgk;
i. GpmA;
j. Eno;
k. PykA;
l. PykF;
m. AceE;
n. AceF;
o. acetyl-CoA acetyltransferase;
p. S-hydratase and 3-hydroxyacyl-CoA dehydrogenese, or beta-oxidation multienzyme;
q. crotonyl-CoA reductase;
r. carboxymethyl-Pro synthase;
s. carbapenam synthetase or beta-lactam synthetase;
and
t. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and an optionally substituted glutamate of the formula (i):
or salts thereof, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen and R2 is —CH2CH3;
or
(4) providing one or more cell lysates comprising:
a. ProA and ProB, or γ-glutamyl kinase-GP-reductase multienzyme complex;
b. Glk;
c. Pgi;
d. PfkA;
e. Fba;
f. TpiA;
g. GapA;
h. Pgk;
i. GpmA;
j. Eno;
k. PykA;
l. PykF;
m. AceE;
n. AceF;
o. acetyl-CoA carboxylase;
p. carboxymethyl-Pro synthase;
q. carbapenam synthetase or beta-lactam synthetase;
and
r. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and an optionally substituted glutamate of the formula (i):
or salts thereof, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen and R2 is hydrogen;
or
(5) providing one or more cell lysates comprising:
a. Glk;
b. Pgi;
c. PfkA;
d. Fba;
e. TpiA;
f. GapA;
g. Pgk;
h. GpmA;
i. Eno;
j. PykA;
k. PykF;
l. AceE;
m. AceF;
n. acetyl-CoA acetyltransferase;
o. S-hydratase and 3-hydroxyacyl-CoA dehyrogenase, beta-oxidation multienzyme;
p. crotonyl-CoA reductase,
q. proline 3-hydroxylase;
r. proline oxidase;
s. carboxymethyl-Pro synthase;
t. carbapenam synthetase or beta-lactam synthetase;
and
u. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and a proline compound of formula (iv):
or salts thereof, wherein R5 is hydrogen, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen, R2 is —CH2CH3, and R5 is —OH;
or
(6) providing one or more cell lysates comprising:
a. Glk;
b. Pgi;
c. PfkA;
d. Fba;
e. TpiA;
f. GapA;
g. Pgk;
h. GpmA;
i. Eno;
j. PykA;
k. PykF;
l. AceE;
m. AceF;
n. acetyl-CoA carboxylase;
o. proline 3-hydroxylase;
p. proline oxidase;
q. carboxymethyl-Pro synthase;
r. carbapenam synthetase or beta-lactam synthetase;
and
s. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and a proline compound of formula (iv):
or salts thereof, wherein R5 is hydrogen, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen, R2 is hydrogen, and R5 is —OH;
or
(7) providing one or more cell lysates comprising:
a. Glk;
b. Pgi;
c. PfkA;
d. Fba;
e. TpiA;
f. GapA;
g. Pgk;
h. GpmA;
i. Eno;
j. PykA;
k. PykF;
l. AceE;
m. AceF;
n. acetyl-CoA acetyltransferase;
o. S-hydratase and 3-hydroxyacyl-CoA dehydrogenase, or beta-oxidation multienzyme;
p. crotonyl-CoA reductase,
q. proline oxidase;
r. carboxymethyl-Pro synthase;
s. carbapenam synthetase or beta-lactam synthetase;
and
t. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and a proline compound of formula (iv):
or salts thereof, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen and R2 is —CH2CH3;
or
(8) providing one or more cell lysates comprising:
a. Glk;
b. Pgi;
c. PfkA;
d. Fba;
e. TpiA;
f. GapA;
g. Pgk;
h. GpmA;
i. Eno;
j. PykA;
k. PykF;
l. AceE;
m. AceF;
n. acetyl-CoA carboxylase;
o. proline oxidase;
p. carboxymethyl-Pro synthase;
q. carbapenam synthetase or beta-lactam synthetase;
and
r. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and a proline compound of formula (iv):
or salts thereof, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen and R2 is hydrogen;
wherein:
the dashed line represents a double bond;
each instance of R3 and R4 is independently selected from the group consisting of hydrogen, 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;
R5 is hydrogen or —OR8;
each instance of R8 is independently selected from the group consisting of hydrogen, 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, optionally substituted heteroaryl, —C(═O)R8a, —C(═O)OR8a, —C(═O)SR8a, —C(═O)N(R8b)2, —C(═NR8b)R8a, —C(═NR8b)OR8a, —C(═NR8b)SR8a, —C(═NR8b)N(R8b)2, —C(═S)R8a, —C(═S)OR8a, —C(═S)SR8a, —C(═S)N(R8b)2, —C(═O)NR8bSO2R8a, —S(═O)R8a, —SO2R8a, —SO2N(R8a)2, —Si(R8a)3, —P(═O)(R8a)2, —P(═O)(OR8a)2, —P(═O)(R8a)(OR8a), —P(═O)(R8a)(N(R8b)2), —P(═O)(N(R8b)2)2, —P(═O)2R8a, —P(═O)2OR8a, —P(═O)2N(R8b)2, —B(R8a)2, —B(OR8a)2, and —BR8a(OR8a), wherein R8a 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, optionally substituted heteroaryl, if attached to an oxygen atom an oxygen protecting group, and if attached to a sulfur atom a sulfur protecting group, or two R8a groups or an R8a and R8b group are joined to form an optionally substituted heterocyclic ring;
and each instance of R8b is independently selected from the group consisting of hydrogen, 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, optionally substituted heteroaryl, and a nitrogen protecting group, or two R8b are joined to form an optionally substituted heterocyclic ring;
and
R7a is hydrogen;
wherein one or more enzymes selected from the group consisting of carboxymethyl-Pro synthase, carbapenam synthetase, beta-lactam synthetase, carbapenem synthase, and isozymes thereof, is sequestered in the periplasmic space of the cell prior to lysing;
and
wherein the one or more cell lysates are bacterial cell lysates.

212 more elements

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

or a salt or tautomer thereof, or a combination thereof,
the method comprising:
(1) providing one or more cell lysates comprising:
a. ProA and ProB, or γ-glutamyl kinase-GP-reductase multienzyme complex;
b. Glk;
c. Pgi;
d. PfkA;
e. Fba;
f. TpiA;
g. GapA;
h. Pgk;
i. GpmA;
j. Eno;
k. PykA;
l. PykF;
m. AceE;
n. AceF;
o. acetyl-CoA acetyltransferase;
p. acetyl-CoA carboxylase and malonyl-CoA reductase, or beta-alanine transaminase;
q. S-hydratase and 3-hydroxyacyl-CoA dehydrogenase, or beta-oxidation multienzyme;
r. crotonyl-CoA reductase;
s. threonine aldolase;
t. carboxymethyl-Pro synthase;
u. carbapenam synthetase or beta-lactam synthetase;
and
v. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and glycine, or salts thereof, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen, R2 is —CH2CH3, R3 is hydrogen, R4 is hydrogen, and R5 is —OH;
or
(2) providing one or more cell lysates comprising:
a. ProA and ProB, or γ-glutamyl kinase-GP-reductase multienzyme complex;
b. Glk;
c. Pgi;
d. PfkA;
e. Fba;
f. TpiA;
g. GapA;
h. Pgk;
i. GpmA;
j. Eno;
k. PykA;
l. PykF;
m. AceE;
n. AceF;
o. acetyl-CoA carboxylase and malonyl-CoA reductase, or beta-alanine transaminase;
p. threonine aldolase;
q. carboxymethyl-Pro synthase;
r. carbapenam synthetase or beta-lactam synthetase;
and
s. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and glycine, or salts thereof, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen, R2 is hydrogen, R3 is hydrogen, R4 is hydrogen, and R5 is —OH;
or
(3) providing one or more cell lysates comprising:
a. ProA and ProB, or γ-glutamyl kinase-GP-reductase multienzyme complex;
b. Glk;
c. Pgi;
d. PfkA;
e. Fba;
f. TpiA;
g. GapA;
h. Pgk;
i. GpmA;
j. Eno;
k. PykA;
l. PykF;
m. AceE;
n. AceF;
o. acetyl-CoA acetyltransferase;
p. S-hydratase and 3-hydroxyacyl-CoA dehydrogenese, or beta-oxidation multienzyme;
q. crotonyl-CoA reductase,
r. carboxymethyl-Pro synthase;
s. carbapenam synthetase or beta-lactam synthetase;
and
t. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and an optionally substituted glutamate of the formula (i):
or salts thereof, wherein R5 is —OH, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen, R2 is —CH2CH3, and R5 is —OH;
or
(4) providing one or more cell lysates comprising:
a. ProA and ProB, or γ-glutamyl kinase-GP-reductase multienzyme complex;
b. Glk;
c. Pgi;
d. PfkA;
e. Fba;
f. TpiA;
g. GapA;
h. Pgk;
i. GpmA;
j. Eno;
k. PykA;
l. PykF;
m. AceE;
n. AceF;
o. acetyl-CoA carboxylase;
p. carboxymethyl-Pro synthase;
q. carbapenam synthetase or beta-lactam synthetase;
and
r. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and an optionally substituted glutamate of the formula (i):
or salts thereof, wherein R5 is —OH, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen, R2 is hydrogen, and R5 is —OH;
or
(5) providing one or more cell lysates comprising:
a. Glk;
b. Pgi;
c. PfkA;
d. Fba;
e. TpiA;
f. GapA;
g. Pgk;
h. GpmA;
i. Eno;
j. PykA;
k. PykF;
l. AceE;
m. AceF;
n. acetyl-CoA acetyltransferase;
o. S-hydratase and 3-hydroxyacyl-CoA dehydrogenese, or beta-oxidation multienzyme;
p. crotonyl-CoA reductase,
q. proline 3-hydroxylase;
r. proline oxidase;
s. carboxymethyl-Pro synthase;
t. carbapenam synthetase or beta-lactam synthetase;
and
u. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and a proline compound of formula (iv):
or salts thereof, wherein R5 is hydrogen, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen, R2 is —CH2CH3, and R5 is —OH;
or
(6) providing one or more cell lysates comprising:
a. Glk;
b. Pgi;
c. PfkA;
d. Fba;
e. TpiA;
f. GapA;
g. Pgk;
h. GpmA;
i. Eno;
j. PykA;
k. PykF;
l. AceE;
m. AceF;
n. acetyl-CoA carboxylase;
o. proline 3-hydroxylase;
p. proline oxidase;
q. carboxymethyl-Pro synthase;
r. carbapenam synthetase or beta-lactam synthetase;
and
s. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and a proline compound of formula (iv):
or salts thereof, wherein R5 is hydrogen, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen, R2 is hydrogen, and R5 is —OH;
or
(7) providing one or more cell lysates comprising:
a. Glk;
b. Pgi;
c. PfkA;
d. Fba;
e. TpiA;
f. GapA;
g. Pgk;
h. GpmA;
i. Eno;
j. PykA;
k. PykF;
l. AceE;
m. AceF;
n. acetyl-CoA acetyltransferase;
o. S-hydratase and 3-hydroxyacyl-CoA dehydrogenese, or beta-oxidation multienzyme;
p. crotonyl-CoA reductase,
q. proline oxidase;
r. carboxymethyl-Pro synthase;
s. carbapenam synthetase or beta-lactam synthetase;
and
t. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and a proline compound of formula (iv):
or salts thereof, wherein R5 is —OH, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen, R2 is —CH2CH3, and R5 is —OH;
or
(8) providing one or more cell lysates comprising:
a. Glk;
b. Pgi;
c. PfkA;
d. Fba;
e. TpiA;
f. GapA;
g. Pgk;
h. GpmA;
i. Eno;
j. PykA;
k. PykF;
l. AceE;
m. AceF;
n. acetyl-CoA carboxylase;
o. proline oxidase;
p. carboxymethyl-Pro synthase;
q. carbapenam synthetase or beta-lactam synthetase;
and
r. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and a proline compound of formula (iv):
or salts thereof, wherein R5 is —OH, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen, R2 is hydrogen, and R5 is —OH;
wherein:
the dashed line represents a double bond;
each instance of R3 and R4 is independently selected from the group consisting of hydrogen, 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;
R7a is hydrogen;
and
wherein the one or more cell lysates are bacterial cell lysates;
wherein the combination of glucose and glycine, or salts thereof, or the combination of glucose and an optionally substituted glutamate of the formula (i), or salts thereof, is enzymatically converted to an optionally substituted CoA compound of formula (iii), or salt thereof, and an optionally substituted glutamate semialdehyde of formula (ii), or salt thereof:
wherein —SCoA is Coenzyme A monoradical;
and
wherein the optionally substituted glutamate semialdehyde of formula (ii) or salt thereof and the optionally substituted CoA compound of formula (iii) or salt thereof are enzymatically converted to a pyrrolidinyl compound of Formula (II-a):
or salt thereof, upon contact with carboxymethylPro synthase or an isozyme thereof;
further wherein the pyrrolidinyl compound (II-a) or salt thereof is enzymatically converted to a β-lactam compound of Formula (III-a):
or salt thereof, upon contact with carbapenam synthetase or beta-lactam synthetase, or an isozyme thereof;
and
further wherein the β-lactam compound of Formula (III-a) or salt thereof generates a compound of Formula (I-a):
or salt thereof, wherein represents a double bond, upon contact with carbapenem synthase or isozyme thereof.

217 more elements

Dependencies are inferred from the claim text.

Facts

Application: 13/606,911Filed 7 Sep 2012 · us provisional application
Priority: 9 Sep 2011
Published: US 2013/0065878 A114 Mar 2013
Granted: 18 Oct 2016
Term ends: Not estimatedThe source records don’t support an estimate.

Classification22 codes

C12P 17/184
Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system: containing a beta-lactam ring, e.g. thienamycin
A61P 31/04
Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics: antibacterial agents
C12N 9/0008
Oxidoreductases: acting on the aldehyde or oxo group of donors
C12N 9/0026
Acting on nitrogen containing compounds as donors: acting on CH-NH groups of donors

Family

Application61/533,039Us provisional application · filed 9 Sep 2011
US 2013/0065878 A1Pre-grant publication · published 14 Mar 2013
US 9,469,861 B2This patent

From the US filing history. Foreign family members aren’t listed.

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Abstract

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

or a salt or tautomer thereof, or a combination thereof;
the method comprising:
(1) providing one or more cell lysates comprising:
a. ProA and ProB, or γ-glutamyl kinase-GP-reductase multienzyme complex;
b. Glk;
c. Pgi;
d. PfkA;
e. Fba;
f. TpiA;
g. GapA;
h. Pgk;
i. GpmA;
j. Eno;
k. PykA;
l. PykF;
m. AceE;
n. AceF;
o. acetyl-CoA acetyltransferase;
p. acetyl-CoA carboxylase and malonyl-CoA reductase, or beta-alanine transaminase;
q. S-hydratase and 3-hydroxyacyl-CoA dehydrogenese, or beta-oxidation multienzyme;
r. crotonyl-CoA reductase;
s. threonine aldolase;
t. carboxymethyl-Pro synthase;
u. carbapenam synthetase or beta-lactam synthetase;
and
v. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and glycine, or salts thereof, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen, R2 is —CH2CH3, R3 is hydrogen, R4 is hydrogen, and R5 is —OH;
or
(2) providing one or more cell lysates comprising:
a. ProA and ProB, or γ-glutamyl kinase-GP-reductase multienzyme complex;
b. Glk;
c. Pgi;
d. PfkA;
e. Fba;
f. TpiA;
g. GapA;
h. Pgk;
i. GpmA;
j. Eno;
k. PykA;
l. PykF;
m. AceE;
n. AceF;
o. acetyl-CoA carboxylase and malonyl-CoA reductase, or beta-alanine transaminase;
p. threonine aldolase;
q. carboxymethyl-Pro synthase;
r. carbapenam synthetase or beta-lactam synthetase;
and
s. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and glycine, or salts thereof, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen, R2 is hydrogen, R3 is hydrogen, R4 is hydrogen, and R5 is —OH;
or
(3) providing one or more cell lysates comprising:
a. ProA and ProB, or γ-glutamyl kinase-GP-reductase multienzyme complex;
b. Glk;
c. Pgi;
d. PfkA;
e. Fba;
f. TpiA;
g. GapA;
h. Pgk;
i. GpmA;
j. Eno;
k. PykA;
l. PykF;
m. AceE;
n. AceF;
o. acetyl-CoA acetyltransferase;
p. S-hydratase and 3-hydroxyacyl-CoA dehydrogenese, or beta-oxidation multienzyme;
q. crotonyl-CoA reductase;
r. carboxymethyl-Pro synthase;
s. carbapenam synthetase or beta-lactam synthetase;
and
t. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and an optionally substituted glutamate of the formula (i):
or salts thereof, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen and R2 is —CH2CH3;
or
(4) providing one or more cell lysates comprising:
a. ProA and ProB, or γ-glutamyl kinase-GP-reductase multienzyme complex;
b. Glk;
c. Pgi;
d. PfkA;
e. Fba;
f. TpiA;
g. GapA;
h. Pgk;
i. GpmA;
j. Eno;
k. PykA;
l. PykF;
m. AceE;
n. AceF;
o. acetyl-CoA carboxylase;
p. carboxymethyl-Pro synthase;
q. carbapenam synthetase or beta-lactam synthetase;
and
r. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and an optionally substituted glutamate of the formula (i):
or salts thereof, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen and R2 is hydrogen;
or
(5) providing one or more cell lysates comprising:
a. Glk;
b. Pgi;
c. PfkA;
d. Fba;
e. TpiA;
f. GapA;
g. Pgk;
h. GpmA;
i. Eno;
j. PykA;
k. PykF;
l. AceE;
m. AceF;
n. acetyl-CoA acetyltransferase;
o. S-hydratase and 3-hydroxyacyl-CoA dehyrogenase, beta-oxidation multienzyme;
p. crotonyl-CoA reductase,
q. proline 3-hydroxylase;
r. proline oxidase;
s. carboxymethyl-Pro synthase;
t. carbapenam synthetase or beta-lactam synthetase;
and
u. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and a proline compound of formula (iv):
or salts thereof, wherein R5 is hydrogen, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen, R2 is —CH2CH3, and R5 is —OH;
or
(6) providing one or more cell lysates comprising:
a. Glk;
b. Pgi;
c. PfkA;
d. Fba;
e. TpiA;
f. GapA;
g. Pgk;
h. GpmA;
i. Eno;
j. PykA;
k. PykF;
l. AceE;
m. AceF;
n. acetyl-CoA carboxylase;
o. proline 3-hydroxylase;
p. proline oxidase;
q. carboxymethyl-Pro synthase;
r. carbapenam synthetase or beta-lactam synthetase;
and
s. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and a proline compound of formula (iv):
or salts thereof, wherein R5 is hydrogen, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen, R2 is hydrogen, and R5 is —OH;
or
(7) providing one or more cell lysates comprising:
a. Glk;
b. Pgi;
c. PfkA;
d. Fba;
e. TpiA;
f. GapA;
g. Pgk;
h. GpmA;
i. Eno;
j. PykA;
k. PykF;
l. AceE;
m. AceF;
n. acetyl-CoA acetyltransferase;
o. S-hydratase and 3-hydroxyacyl-CoA dehydrogenase, or beta-oxidation multienzyme;
p. crotonyl-CoA reductase,
q. proline oxidase;
r. carboxymethyl-Pro synthase;
s. carbapenam synthetase or beta-lactam synthetase;
and
t. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and a proline compound of formula (iv):
or salts thereof, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen and R2 is —CH2CH3;
or
(8) providing one or more cell lysates comprising:
a. Glk;
b. Pgi;
c. PfkA;
d. Fba;
e. TpiA;
f. GapA;
g. Pgk;
h. GpmA;
i. Eno;
j. PykA;
k. PykF;
l. AceE;
m. AceF;
n. acetyl-CoA carboxylase;
o. proline oxidase;
p. carboxymethyl-Pro synthase;
q. carbapenam synthetase or beta-lactam synthetase;
and
r. carbapenem synthase;
or isozymes thereof;
wherein the one or more cell lysates are contacted with glucose and a proline compound of formula (iv):
or salts thereof, to provide a compound of Formula (I-a) or a salt or tautomer thereof or a combination thereof, wherein R1 is hydrogen and R2 is hydrogen;
wherein:
the dashed line represents a double bond;
each instance of R3 and R4 is independently selected from the group consisting of hydrogen, 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;
R5 is hydrogen or —OR8;
each instance of R8 is independently selected from the group consisting of hydrogen, 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, optionally substituted heteroaryl, —C(═O)R8a, —C(═O)OR8a, —C(═O)SR8a, —C(═O)N(R8b)2, —C(═NR8b)R8a, —C(═NR8b)OR8a, —C(═NR8b)SR8a, —C(═NR8b)N(R8b)2, —C(═S)R8a, —C(═S)OR8a, —C(═S)SR8a, —C(═S)N(R8b)2, —C(═O)NR8bSO2R8a, —S(═O)R8a, —SO2R8a, —SO2N(R8a)2, —Si(R8a)3, —P(═O)(R8a)2, —P(═O)(OR8a)2, —P(═O)(R8a)(OR8a), —P(═O)(R8a)(N(R8b)2), —P(═O)(N(R8b)2)2, —P(═O)2R8a, —P(═O)2OR8a, —P(═O)2N(R8b)2, —B(R8a)2, —B(OR8a)2, and —BR8a(OR8a), wherein R8a 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, optionally substituted heteroaryl, if attached to an oxygen atom an oxygen protecting group, and if attached to a sulfur atom a sulfur protecting group, or two R8a groups or an R8a and R8b group are joined to form an optionally substituted heterocyclic ring;
and each instance of R8b is independently selected from the group consisting of hydrogen, 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, optionally substituted heteroaryl, and a nitrogen protecting group, or two R8b are joined to form an optionally substituted heterocyclic ring;
and
R7a is hydrogen;
wherein one or more enzymes selected from the group consisting of carboxymethyl-Pro synthase, carbapenam synthetase, beta-lactam synthetase, carbapenem synthase, and isozymes thereof, is sequestered in the periplasmic space of the cell prior to lysing;
and
wherein the one or more cell lysates are bacterial cell lysates.

212 more elements

Facts

Application

13/606,911Filed 7 Sep 2012 · us provisional application

Priority

9 Sep 2011

Published

US 2013/0065878 A114 Mar 2013

Granted

18 Oct 2016

Term ends

Not estimatedThe source records don’t support an estimate.

Classification22 codes

C12P 17/184
Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system: containing a beta-lactam ring, e.g. thienamycin
A61P 31/04
Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics: antibacterial agents
C12N 9/0008
Oxidoreductases: acting on the aldehyde or oxo group of donors
C12N 9/0026
Acting on nitrogen containing compounds as donors: acting on CH-NH groups of donors