Synthesis of labeled meropenem for the analysis of M. tuberculosis transpeptidases

Article abstract of DOI:10.1016/j.tetlet.2009.10.124

A concise synthesis of ¹⁴C labeled meropenem prepared from ¹⁴C dimethylamine hydrochloride is described. Using a similar reaction sequence, the meropenem nucleus was also attached to biotin providing a probe for protein interaction studies.

Full text of DOI:10.1016/j.tetlet.2009.10.124

Tetrahedron Letters 51 (2010) 197–200
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of labeled meropenem for the analysis of M. tuberculosis
transpeptidases
*
David B. Kastrinsky, Clifton E. Barry III
The National Institutes of Health, National Institute of Allergy and Infectious Diseases, Tuberculosis Research Section, Bethesda, MD 20892, USA
a r t i c l e i n f o
a b s t r a c t
A concise synthesis of 14C labeled meropenem prepared from 14C dimethylamine hydrochloride is
described. Using a similar reaction sequence, the meropenem nucleus was also attached to biotin provid-
ing a probe for protein interaction studies.
Article history:
Received 5 September 2009
Revised 24 October 2009
Accepted 27 October 2009
Available online 30 October 2009
Published by Elsevier Ltd.
The mycobacterial cell wall is a complex, multi-layered hetero-
polymer composed of a peptidoglycan core that is covalently bound
to arabinogalactan and mycolic acids.1–3 Although the size and
hydrophobic nature of this complex contribute to the impermeabil-
ity of mycobacteria to many antibiotics,4,5 other features are likely to
contribute to the resistance to specific classes of antibiotics. The b-
lactams (penicillins, cephalosporins, and carbapenems) are the most
widely prescribed anti-infectives and derive their biologic activity
through the acylation and deactivation of the transpeptidases in-
volved in peptidoglycan crosslinking.6–8 Mycobacterium tuberculosis
OH
H
O
14C
NHHCl
OP(OPh)
2
N
O
HS
H314C
S
O
4
O
14 CH3
O
N
DIPEA
N
N
O
CH3CN
NO2
O
O
DIPEA
CH3CN
O
O
NO2
NO2
3
2
14CH3
14CH3
H314C
N
H314C
N
O
O
OH
O
OH
O
H
H
N
O
H2, Pd/C
EtOAc-
pH = 7 Phosphate
Buffer
NH
N
S
S
N
O
O
OH
O
O
NO2
1
5
NO2
Scheme 1. 14C labeled meropenem synthesis.
* Corresponding author. Tel.: +1 301 435 7509; fax: +1 301 402 0993.
E-mail address: cbarry@mail.nih.gov (C.E. Barry III).
0040-4039/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2009.10.124

198
D. B. Kastrinsky, C. E. Barry III / Tetrahedron Letters 51 (2010) 197–200
HOH2C
O
O
O
O
N-Glycolyl Muramic Acid
N-Acetyl Glucosamine
HOH2C
O
O
HN
OH
OH
O
O
NHCOCH 3
NH2
NH2
COOH
HO2C
O
COOH
NH
O
H
N
O
COOH
H
N
N
H
COOH
N
H
NH-i-Gln-Ala
Ala-i-Gln-NH
Ala-i-Gln-NH
O
O
4-3 D-Ala-DAP Crosslink
(4,3) D,D-transpeptidase
β-lactam Sensitive Pathway
Ala-i-Glu-DAP-D-Ala-D-Ala
(3,3) L,D-transpeptidase
β-lactam Insensitive Pathway?
NH-i-Gln-Ala
H2N
CO2H
NH2
O
NH
COOH
CO2H
D-Ala-D-Ala-DAP-i-Glu-Ala
H
N
NH-i-Gln-Ala
NH
CO2H
Ala-i-Gln-NH
O
CO2H
O
Adjacent Peptidoglycan strands
3-3 DAP-DAP Crosslink
Figure 1. Two types of peptidoglycan crosslink in M. tuberculosis.
OH
O
R1
H
N
H
N
N
R
H
N
S
S
O
O
S
O
NH
N
N
O
OR2
O
O
OH
OH
O
O
OH
Meropenem
Penicillins
Cephalosporins
NH2
COOH
H
N
Ala-i-Gln-NH
H
N
4,3
3,3
O
O
O
OH
Peptidoglycan D-Ala-D-Ala
Figure 2. Common b-lactam structures.
(Mtb) contains at least one chromosomal b-lactamase, Rv2068c, a
Class A, extended spectrum b-lactamase.9 Rv2068c can be inhibited
by clavulanic acid and thus combinations of antibiotic and b-lacta-
mase inhibitor or newer classes of b-lactamase resistant antibiotics
should prove effective for treating tuberculosis.10
extensively crosslinked and that b-lactams penetrate the cell wall
and inhibit transpeptidase targets in Mtb.11,12 A recent explanation
for this paradox suggested that the mycobacterial cell wall actually
contains two distinct types of crosslinks.13 The more intensively
studied 4–3 crosslinks (Fig. 1) are synthesized by a class of D,D
transpeptidases called penicillin-binding proteins that are inhib-
ited by the b-lactams. In several species of bacteria including M.
Historically the b-lactams have not been used in treating tuber-
culosis despite the fact that the peptidoglycan of mycobacteria is

D. B. Kastrinsky, C. E. Barry III / Tetrahedron Letters 51 (2010) 197–200
199
tuberculosis, a different type of 3–3 crosslink (Fig. 1) has been ob-
served. These crosslinks are formed by L,D transpeptidases, a
new class of cysteine transpeptidase previously thought to be
insensitive to b-lactams but now known to be inhibited by the
carbapenem class of b-lactam antibiotics. Conceivably, the 3–3
crosslinks provide an alternative architectural modification and
some evidence suggests that these linkages play a role in stationary
phase rigidification providing benefits for the long term survival of
nonreplicating bacilli.14–20
The carbapenems, a class of four FDA-approved antibiotics (imi-
penem, meropenem, ertapenem, and doripenem)are b-lactams with
a structure derived from the natural product thienamycin.21 This
class received much attention due to its broad spectrum potency
notably toward gram negative and anaerobic bacteria, its stability
to clinically significant b-lactamases, and its rapid, bactericidal
activity.22 The penicillins and cephalosporins contain fused bicyclic
six-membered dihydrothiazoline ring of the cephalosporins reveal
common precursors in valine and cysteine. While the differences
in core structure and N-acyl substitution account for differences in
reactivity, b-lactamase stability, and spectrum of activity, the puta-
tive targets of these compounds are still D,D transpeptidases. The
structural similarity of the carbapenem core, the hydroxyethyl and
sulfide-linked proline sidechains excepted, suggested that the car-
bapenems also inhibited the D,D transpeptidases. This fact was cor-
roborated in several studies,23–25 however recent evidence suggests
the L,D transpeptidases are another possible target.
The recent discovery that the peptidoglycan of stationary phase
cultures of M. tuberculosis contains up to 80% 3–3 crosslinks con-
trasted strikingly with the traditional view of mycobacterial pepti-
doglycan and strongly supported the notion that the L,D
transpeptidases contribute to the resistance to the b-lactam antibi-
otics.26 More intriguingly, a putative L,D transpeptidase, Mtb
Rv0116c, (LdtMtb), was inhibited by meropenem, and from studies
conducted in our laboratory, meropenem demonstrated efficacy
against extensively drug resistant (XDR)-TB when co-administered
with the b-lactamase inhibitor clavulanic acid.27 This combination
structures, and are suicide substrates reminiscent of D-Ala-D-Ala, the
substrate for the 4–3 transpeptidation reaction, as originally
suggested by Tipper and Strominger (Fig. 2).6 The saturated five-
membered thiazolidine ring of the penicillins and the saturated
O
HN
1. DPPA,Et3N,
t-BuOH, 74%
H
N
-
H
CF3CO2
+H3N
O
O
NH
H
H
HO
NH
S
2. TFA, 87%
S
H
6
Biotin
O
O
O
S
HN
HN
H
H
NH
NH
H
HS
F3CCO - +H3N
H
N
H
2
N
S
S
6
N
O
O
O
DIPEA
DMF, 80%
O
O
NO2
O2N
2
7
OH
O
H
O
OH
H
O
OP(OPh)
2
HN
N
O
H
S
NH
H
4
N
H
N
O
O
S
O
O
N
NO2
O
O
DIPEA
O
O2N
8
DMF, 71%
O2N
O
S
HN
H
NH
H
H2, 10% Pd/C
OH
O
H
N
HN
pH = 7 KH2PO4 buffer
ethyl acetate
75%
S
O
NH
OH
O
9
Scheme 2. Synthesis of a biotinylated carbapenem.

200
D. B. Kastrinsky, C. E. Barry III / Tetrahedron Letters 51 (2010) 197–200
was also active against nonreplicating bacilli, suggesting an essen-
tial remodeling or recycling function of these enzymes in metabol-
ically static bacteria. With the immediate goal of identifying the
protein targets of the carbapenems in whole cells of M. tuberculosis,
we undertook the synthesis of two labeled forms of meropenem for
use as probes.
meropenems offer tools to identify and characterize the targets
of the carbapenems in other organisms.
Acknowledgments
We gratefully acknowledge Dr. Kriti Arora and Dr. Pradeep Ku-
mar for help with scintillation counts and biological studies. We
would like to thank Dr. Bong-Jin Kim of the Korean Research Insti-
tute for Chemical Technology (KRICT) for helpful suggestions and
Dr. Noel Whittaker of the National Institute of Diabetes, Digestive,
and Kidney Diseases (NIDDK) for high resolution mass spectral
analysis. Funding was provided by the Intramural Research Pro-
gram of the National Institutes of Health, National Institute of Al-
lergy and Infectious Diseases.
The synthesis of 14C labeled meropenem (1, Scheme 1) utilizes
bicyclic intermediate 2 (prepared in 2 steps from trans 4-hydro-
xy-L
-proline)28,29 which is synthesized in kilogram quantities for
the production of MerremÒ. From 2, the synthesis was optimized
at milligram scale for introduction of the radiolabel. Unlabeled
intermediates were prepared as TLC standards and spectral data
are contained in the supporting information. A 250 lCi sample of
14C-labeled Me2NH–HCl (4.0 mg) was obtained from American
Radiolabeled Chemicals specially prepared in acetonitrile. This re-
Supplementary data
agent was treated with DIPEA (18
52 mol) for 2 h at 0 °C. The solution was subsequently treated
with unlabeled Me2NH–HCl (4.4 mg, 54 mol), DIPEA (18 L,
107 mol) and stirred for an additional 2 h at 0 °C. While there
are reports of in situ opening of 2 and coupling to 4, we purified
the thiol intermediate 3 by flash chromatography (SiO2, 5%
MeOH–CH2Cl2). Labeled 3 was coupled to the carbapenem enol
lL, 107 lmol) and 2 (16 mg,
l
Supplementary data (1H NMR spectra and experimental infor-
mation) associated with this Letter can be found, in the online ver-
sion, at doi:10.1016/j.tetlet.2009.10.124.
l
l
l
References and notes
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(20 L, 112 mol) in acetonitrile (0.5 mL, 3 h, 25 °C) to provide 5
l
l
(19 mg, 56% two steps). Hydrogenolysis of 5 (10 mg, H2, 10% Pd/
C, 1 atm, 2 h), in a biphasic (1:1, 2 mL) solvent system of ethyl ace-
tate/aqueous potassium phosphate buffer (0.050 M, pH 7) followed
by the removal of the catalyst by filtration through Celite, and
washing of the aqueous layer with ethyl acetate (2 Â 1 mL) pro-
vided an aqueous solution of labeled meropenem 1 which was
pure by nonradiographic TLC (Rf = 0.25) 7:3 MeOH–CH2Cl2), radio-
graphic TLC is contained in the supporting info. The aqueous phase
containing the pure, labeled antibiotic was adjusted with addi-
tional phosphate buffer to a volume of 1.0 mL (29.3
radioactive yield) and was stored in 100
at À30 °C for future use.
lCi/mL, 22%
lL aliquots and frozen
We also considered the use of an alternative, nonradioactive la-
bel for utilization in a protein pull-down strategy (Scheme 2). Since
the radiolabeled meropenem synthesis relied on the introduction
of a labeled amine, it would be advantageous to introduce an alter-
native label in a similar fashion with the only constraints that the
label be compatible with the carbapenem nucleus and not impart
any significant steric demand. Biotin does not contain an amine
handle, but previous reports documented its introduction by Cur-
tius rearrangement of the carboxylic acid.30 Treatment of biotin
(1.0 g, 4.2 mmol) in t-BuOH (15 mL, distilled from CaH2) with DPPA
(1.0 mL, 4.6 mmol) and Et3N (0.64 mL, 4.6 mmol) at 95 °C for 14 h
provides N-Boc norbiotinamine (1.0 g, 74%). Deprotection of this
compound (1.6 g, 5.1 mmol) in neat TFA (3.0 mL) at 25 °C provides
the salt 7 isolated as a white solid (1.2 g, 87%). Insoluble in aceto-
nitrile, the reaction of
1.6 mmol) in DMF (8.0 mL) with DIPEA (310
25 °C for 2 h provides the thiol 8 (0.450 g, 80%). Coupling of 8
(0.181 g, 0.347 mmol) to 4 (0.206 g, 0.347 mmol) with DIPEA
(63 lL, 0.364 mmol) provided 9 (0.215 g, 71%). Hydrogenolysis
(H2, 10% Pd/C, 45 psi, 2 h) of 9 (0.233 g, 0.268 mmol) in the bipha-
sic solvent system detailed above (5 mL ethyl acetate: 5 mL phos-
phate buffer) removed the p-nitrobenzyl ester and carbamate. In
this case, the aqueous layer was collected, washed with ethyl ace-
tate (2 Â 5 mL), and concentrated in vacuo. The crude residue was
dissolved in methanol, filtered to remove the precipitated salts,
and concentrated again to provide the biotin-labeled meropenem
9 (111 mg, 75%) as a yellow solid, pure by 1H NMR (supporting
info). Studies to indentify the targets of meropenem in M. tubercu-
losis are underway. It is worth noting that 14C and biotin-labeled
7
(0.56 g, 1.7 mmol) with
2 (0.50 g,
lL, 1.8 mmol) at
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