Synthesis of (3S,5R)-Carbapenam-3-carboxylic Acid and Its Role in Carbapenem Biosynthesis and the Stereoinversion Problem

Full text of DOI:10.1021/ja037665w

Published on Web 11/25/2003
Synthesis of (3S,5R)-Carbapenam-3-carboxylic Acid and Its Role in
Carbapenem Biosynthesis and the Stereoinversion Problem
Anthony Stapon, Rongfeng Li, and Craig A. Townsend*
Department of Chemistry, The Johns Hopkins UniVersity, 3400 North Charles Street, Baltimore, Maryland 21218
Received July 31, 2003; E-mail: ctownsend@jhu.edu
Scheme 2 a
(5R)-Carbapenem-3-carboxylic acid (4) is the simplest structur-
ally of over 60 naturally occurring carbapenem â-lactam antibiotics
isolated since the discovery of thienamycin,1 whose gene cluster
has been recently identified.2 Members of this family and their
derivatives are clinically important for their broad spectrum of
antibiotic activity and their relative resistance to most clinically
encountered â-lactamases.3
We determined that the assembly of all three â-lactams 2, 3,
and 4 produced by Erwinia carotoVora (now Pectobacterium
carotoVorum)2 and Serratia sp. ATCC 39006 is carried out through
the action of just three enzymes, CarA (carbapenam synthetase),
CarB, and CarC (Scheme 1).4-6 This process is accompanied by a
remarkable stereochemical inversion at C-5 of 2 to both 3 and 4,
which proceeds by loss of hydrogen label at that stereocenter
catalyzed by CarC (carbapenem synthase).6
a Reagents and conditions: (a) tert-butylbromo acetate, CH3CN, 40 h.
(b) triphenylphosphine, triethylamine, CH2Cl2, 20 h. (c) sodium cyanoboro-
hydride, acetic acid, CH3CN. (d) Pd(OH)2/C, H2, EtOH, 48 h. (e) (Boc)2dO,
t-BuOH/H2O. (f) RBr, DMF, CsCO3. (g) TFA (h) N-methyl-2-chloropyri-
dinium triflate, diisopropylethylamine, CH3CN, 4 h. (i) for 10b, Pd[PPh3]4,
p-toluenesulfinate sodium salt, MeOH/THF.
Scheme 1
carbapenam was unchanged after 15 h using our optimized
deprotection conditions6 (carboxyl group is exo). The absence of
reactivity by the epimeric (3S,5R)-carbapenam (PNB ester is endo)
is presumably due to the inability of the Pd catalyst to access the
ester C-O bond in this hindered bicyclic system.
Recognizing the utility of removing allyl esters in pH sensitive
substrates by Pd[PPh3]4 catalysts,16 we turned our attention to the
deprotection of (3S,5S)-carbapenam allyl ester 13 (Scheme 3) as a
test case prior to the synthesis of the (3S,5R)-carbapenam system
(Scheme 2). An added advantage in this instance is that the allyl
group acts as a two-atom spacer, potentially allowing better
interaction with the Pd catalyst during deprotection, compared to
the failed hydrogenolysis attempts.
While 3 has always been present in low amounts compared to 2
and 4 during isolation of the â-lactams from fermentation,4,7 no
direct evidence exists to establish its role as an intermediate, shunt
product, or product of the poorly understood self-resistance
mechanism.8 We report here the first functional analysis of (3S,5R)-
carbapenam carboxylic acid (3) as an intermediate in the assembly
of 4. In addition, we address the role of R-ketoglutarate in the
epimerization of 2 to 3 and subsequent desaturation to 4.
Recently6 we described the first successful synthesis of an
unprotected carbapenam-3-carboxylic acid skeletal system. The
instability/lability of the carbapenams is evident in previous
attempted syntheses9-11 and in the recent report of the carbapenem
synthase crystal structure12 in which 2 was produced enzymically
from 1. Having the sodium salt of 2 in hand, we attempted to
duplicate our deprotection strategy in a synthesis of 3.
Scheme 3 a
As shown in Scheme 2, starting from L-glutamate, thiolactam 5
was prepared according to a previously published procedure in four
steps.13 Eschenmoser coupling14 of 5 with tert-butylbromo acetate
provided the vinylogous amide 6. Reduction with sodium cyano-
borohydride/acetic acid yielded protected (2S,5R)-amino diacid 7,
which was separated from the (2S,5S)-amino diacid by silica gel
chromatography. Catalytic hydrogenation followed by treatment
with (Boc)2dO yielded 8. Derivatization with p-nitrobenzyl bro-
mide, deprotection with trifluoroacetic acid (TFA) gave 9a, followed
by â-lactam cyclization15 to yield 10a.
a Reagents and conditions: (a) NaOH, THF/H2O. (b) allyl bromide,
CsCO3, DMF. (c) TFA. (d) N-methyl-2-chloropyridinium triflate, diiso-
propylethylamine, CH3CN, 4 h. (e) Pd[PPh3]4, p-toluenesulfinate sodium
salt, MeOH/THF.
Protected amino-diacid6 11 was treated with base and allyl
bromide to yield 12 (Scheme 3). Treatment with TFA, followed
by â-lactam cyclization,15 gave 13. Standard allyl ester deprotection
conditions16 (i.e., Pd[PPh3]4, potassium 2-ethylhexanoate) yielded
2, but with large amounts of contaminating/side products that could
not be removed without causing further product decomposition.
Finally, utilizing the Pd[PPh3]4/p-toluenesulfinate sodium salt
Unfortunately, but in accord with literature reports,7,10 hydro-
genolysis of 10a to yield 3 was ineffectual as the protected (3S,5R)-
9
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J. AM. CHEM. SOC. 2003, 125, 15746-15747
10.1021/ja037665w CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
deprotection protocol,17 the labile 2 was obtained with minimal
amounts of decomposition. To complete the synthesis of the (3S,5R)
system, protected acid 8 (Scheme 2) was then treated with allyl
bromide, and TFA to give 9b, which was then cyclized15 to give
â-lactam 10b. Deprotection of the allyl ester17 10b, gave 3.
With the two diastereomeric carbapenams 2 and 3 in hand, carC
cloned from P. carotoVorum was overexpressed as its N-terminal
His6 tagged protein in Escherichia coli and purified under anaerobic
conditions by affinity chromatography on a Ni-NTA column using
standard procedures.
The ability of purified CarC to catalyze carbapenem synthesis
was monitored by paper disk assay on a plate of â-lactam-
supersensitive E. coli (SC12155) to detect production of hydro-
lytically unstable 4.18 As shown in Figure 1, incubation of 2 and 3
with CarC and added R-ketoglutarate and ascorbate gave clear zones
of inhibition (D and C, respectively). Controls with 3 itself (A), or
the assay mixture lacking R-ketoglutarate (B), failed to give zones
of inhibition in agreement with controls previously performed on
2.5,6 These observations show that 3 is indeed an intermediate in
the carbapenem biosynthetic pathway and that R-ketoglutarate is
required in the ultimate oxidative desaturation step to the carba-
penem nucleus 4.
2 according to our previous method,6 but replacing Superhydride
with Superdeuteride, to produce the carbapenam with a deuterium
at the C-5 bridgehead position. If CarC is capable of isomerizing
2 to 3 without the use of R-ketoglutarate, then we would observe
a mass change from [C5-2H]-carbapenam 2 (m/z 155 [M - H]) to
[C5-1H]-carbapenam 3 (m/z 154 [M - H]). No change in mass
was observed by ESI-MS analysis of CarC incubated with ascorbate
and [5-2H]-2 (m/z 155 [M - H]). However, upon addition of
R-ketoglutarate, production of 4 was observed (confirmed by
bioassay and ESI-MS m/z 152 [M - H]), accompanied by a small
but detectable level of [5-1H]-3 (m/z 154 [M - H]). Additionally,
[5-2H]-2 showed a slower rate of consumption in CarC assays as
compared to [5-1H]-2 as monitored by ESI-MS. This may indicate
that the isomerization process of 2 to 3 is the “slow step” in the
overall catalytic cycle rather than double bond formation in the
conversion of 3 to 4.
In sum, we take these findings to support the intermediacy of 3
in the overall conversion of 2 to 4 by CarC (Scheme 1). We propose
that the contrathermodynamic epimerization of 2 to 3 is coupled at
least to the binding of R-ketoglutarate and, while not strictly
demonstrated from the data, it is probably coupled to the reduction
of molecular oxygen and proceeds by way of radical abstraction at
C-5. The presumed Fe(IV)dO species formed in these processes19
is required to drive the subsequent desaturation process. How the
bridgehead hydrogen is replaced in the carbapenam 3 and the
stoichiometry and kinetics of reaction are the subjects of future
analyses.
Acknowledgment. This paper is dedicated with greatest respect
to mentors A. I. Scott and D. Arigoni on their 75th birthdays. We
are grateful to Dr. Barbara Gerratana for her help in carrying out
the LC/ESI-MS experiments and to the National Institutes of Health
(AI14937, RR13823) for financial support.
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