Synthesis of Carbapenems Containing Peptidoglycan Mimetics and Inhibition of the Cross-Linking Activity of a Transpeptidase of l,d Specificity

Article abstract of DOI:10.1002/chem.202004831

The carbapenem class of β-lactams has been optimized against Gram-negative bacteria producing extended-spectrum β-lactamases by introducing substituents at position C2. Carbapenems are currently investigated for the treatment of tuberculosis as these drugs are potent covalent inhibitors of l,d-transpeptidases involved in mycobacterial cell wall assembly. The optimization of carbapenems for inactivation of these unusual targets is sought herein by exploiting the nucleophilicity of the C8 hydroxyl group to introduce chemical diversity. As β-lactams are structure analogs of peptidoglycan precursors, the substituents were chosen to increase similarity between the drug and the substrate. Fourteen peptido-carbapenems were efficiently synthesized. They were more effective than the reference drug, meropenem, owing to the positive impact of a phenethylthio substituent introduced at position C2 but the peptidomimetics added at position C8 did not further improve the activity. Thus, position C8 can be modified to modulate the pharmacokinetic properties of highly efficient carbapenems.

Full text of DOI:10.1002/chem.202004831

Accepted Article
Accepted Article
Title: Synthesis of carbapenems containing peptidoglycan-mimetics
and inhibition of the cross-linking activity of a transpeptidase of
the L,D specificity
Authors: Saidbakhrom Saidjalolov, Zainab Edoo, Matthieu Fonvielle,
Louis Mayer, Laura Iannazzo, Michel Arthur, Mélanie Ethève-
Quelquejeu, and Emmanuelle Braud
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To be cited as: Chem. Eur. J. 10.1002/chem.202004831
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01/2020

10.1002/chem.202004831
Chemistry - A European Journal
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Synthesis of carbapenems containing peptidoglycan-mimetics
and inhibition of the cross-linking activity of a transpeptidase of
the L,D specificity
Saidbakhrom Saidjalolov,[a] Zainab Edoo,[b] Matthieu Fonvielle,[b] Louis Mayer,[b] Laura Iannazzo,[a]
Michel Arthur,*[b] Mélanie Etheve-Quelquejeu,*[a] and Emmanuelle Braud*[a]
[a]
[b]
S Saidjalolov, Dr L Iannazzo, Prof Dr M Etheve-Quelquejeu, Dr E Braud
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS
Université de Paris
45, rue des saints-pères, Paris, F-75006, France
E-mail: saidbakhrom.saidjalolov@etu.parisdescartes.fr, laura.iannazzo@parisdescartes.fr, melanie.etheve-quelquejeu@parisdescartes.fr,
emmanuelle.braud@parisdescartes.fr
Dr Z Edoo, Dr M Fonvielle, L Mayer, Dr M Arthur
INSERM UMRS 1138
Sorbonne Universités, UPMC Univ Paris 06, Sorbonne Paris Cité, Université de Paris
Centre de recherche des Cordeliers, Paris, F-75006, France
E-mail : zainab.edoo@crc.jussieu.fr, matthieu.fonvielle@crc.jussieu.fr, mayer.louis@outlook.fr, michel.arthur@crc.jussieu.fr
Supporting information for this article is given via a link at the end of the document.
Abstract: The carbapenem class of β-lactams has been optimized
against Gram-negative bacteria producing extended-spectrum β-
lactamases by introducing substituents at position C2. Carbapenems
are currently investigated for the treatment of tuberculosis since
these drugs are potent covalent inhibitors of L,D-transpeptidases
involved in mycobacterial cell wall assembly. We sought to optimize
carbapenems for inactivation of these unusual targets by exploiting
the nucleophilicity of the C8 hydroxyl group to introduce chemical
diversity. Since β-lactams are structure analogues of peptidoglycan
precursors, the substituents were chosen to increase similarity
between the drug and the substrate. Fourteen peptido-carbapenems
were efficiently synthesized. They were more effective than the
reference drug, meropenem, due to the positive impact of a
phenethylthio substituent introduced at position C2 but the
peptidomimetics added at position C8 did not further improve activity.
Thus, position C8 can be modified to modulate the pharmacokinetic
properties of highly efficient carbapenems.
lactam-resistant mutants of Enterococcus faecium[5] and
Escherichia coli[6] selected in vitro.
In the β-lactam family, members of the carbapenem class
(imipenem, meropenem, ertapenem, and doripenem) have been
developed and optimized for the treatment of infections due to
Gram-negative bacteria producing extended-spectrum β-
lactamases active on third-generation cephalosporins (Scheme
1C).[7] Optimization relied on introduction of various substituents
at the C2 position and of a methyl at position C1. The latter
modification increases the stability of the carbapenems with
respect to hydrolysis by a host enzyme, albeit at the expense of
a reduced reactivity of the drugs.[8] Carbapenems have been
shown to be active against Mycobacterium tuberculosis in
vitro[9,10] and in vivo[11] and are part of the recommended
treatment of pulmonary infections due to Mycobacterium
abscessus in cystic fibrosis patients.[12,13] The antibacterial
activity of carbapenems against M. tuberculosis and M.
abscessus involves inactivation of multiple targets including
PBPs and Ldts.[14-16] The drugs irreversibly inactivate these
enzymes by formation of a long-lasting covalent adduct resulting
from nucleophilic attack of the β-lactam carbonyl by the active-
site serine of PBPs or by the active-site cysteine of Ldts
(Scheme 1D).[17-19]
β-Lactams are structure analogues of the PG precursors and act
as suicide substrates of the transpeptidases. This conclusion
was initially based on the pioneering work of Tipper and
Strominger reported in 1965, who noticed that one of the
possible conformations of the terminal D-Ala4-D-Ala5 dipeptide of
stem pentapeptide is similar to that of penicillin (Figure 1A).[20]
By analogy, we present a carbapenem with a conformation that
simulates the D-Ala4-D-Ala5 (Figure 1A) or L-Lys3-D-Ala4 (Figure
1B) extremity of the pentapeptide or tetrapeptide stem peptides,
which are used as acyl donor by transpeptidases of the D,D and
L,D specificities, respectively.
Introduction
The biosynthesis of peptidoglycan (PG), the major constituent of
the bacterial cell wall, has been intensively studied to identify
targets for drug development. A number of inhibitors of the
constitutive enzymes have been described,[1,2] the most
successful as antibiotics being the β-lactams. These compounds
inhibit the enzymes that catalyze the final transpeptidation step
in which adjacent glycan chains made of alternating N-
acetylglucosamine (GlcNAc) and N-acetylmuramic acid
(MurNAc) are linked to each other by formation of an amide
bond between stem peptides carried by MurNAc residues
(Scheme 1A). D,D-transpeptidases, commonly referred to as
penicillin-binding proteins (PBPs), catalyze the formation of 4→3
cross-links connecting the fourth position of an acyl donor stem
peptide to the side chain amino group of a di-amino acid located
at the third position of an acyl acceptor stem peptide. Unusual
3→3 cross-links formed by unrelated L,D-transpeptidases[3]
(Ldts) (Scheme 1B) are predominant in mycobacteria[4] and in β-
Several groups have investigated the possibility of modifying the
structure of the substituents linked to the bicyclic core of β-
lactams to improve drug binding and antibacterial activity. This
was investigated not only with D,D-transpeptidases but also with
other members of the “D,D-peptidase” super family that share a
1
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Scheme 1. Transpeptidation reactions with the PG subunit of E. faecium, catalyzed by A) PBPs and B) Ldts. C) Examples of drugs belonging to the three main
classes of β-lactams. D) Formation of the covalent adduct (acyl-enzyme) resulting from nucleophilic attack of a carbapenem by the active-site serine of PBPs or
the active-site cysteine of Ldts.
common fold and catalytic mechanism but perform distinct
functions in PG metabolism (Figure 2). This diversity is relevant
to the current study since no general conclusion could be drawn
functions in vivo with branched PG precursors composed of a
linear stem peptide (L-Ala--D-Glx-L,L-DAP-D-Ala-D-Ala) and a
side chain composed of a single Gly residue linked to the ε
for the various representatives of the D,D-peptidases subclasses. amino group of L,L-DAP (Figure 1C).[23] Pratt et al. explored the
The premise of these investigations was that grafting groups
resembling to PG precursors onto D-Ala-D-Ala and β-lactams
should lead to parallel increases in the catalyzed acylation
reaction rates with PG precursor analogues and β-lactams. The
substituents of these peptidomimetics were designed to mimic
the side or main chain of the PG precursors (in green and in
blue in Figure 1C). Of note, the structure of the main chain is
conserved whereas that of the side chain is variable in different
bacterial species. Parallel increases in the acylation rates with
peptidomimetic substrates and -lactams were observed for the
R61 D,D-peptidase isolated form a bacterial strain belonging to
Streptomyces genus (L,L-DAP-Gly side chain). R61 is a class C
low-molecular weight D,D-peptidase of uncertain function with
respect to PG metabolism. In vitro, purified R61 catalyzes
formation of 4→3 cross-links (D,D-transpeptidase activity) and
hydrolysis of D-Ala5 (D,D-carboxypeptidase activity) by using as
substrates synthetic peptides containing the terminal acyl-D-Ala-
D-Ala moiety of PG precursors (Figure 1C).[22] The known
structure of the PG of Streptomyces spp. implies that R61
impact of grafting the L,L-DAP(Gly) dipeptide onto D-Ala-D-Ala
and representative β-lactams of the penam (penicillin) and
cephem classes (Figure 1C). Introduction of the peptidoglycan-
mimetic side chain specific of the Streptomyces spp. led to
dramatic parallel increases in the activity of the R61 D,D-
peptidase with PG precursor analogues and in the reactivity of
the enzyme with β-lactams.[21] The inactivation rate constant of
the R61 D,D-peptidase by the penicillin peptidomimetic
containing the L,L-DAP(Gly) motif (1.5 x 107 s-1.M-1) was three
orders of magnitude higher than that of penicillin (1.4 x 104 s-1.M-
1), approached the diffusion limit, and was the largest rate
constant yet reported for inactivation of a D,D-peptidase. The
fact that the L,L-DAP(Gly) substituent of the L,L-DAP(Gly)-
penicillin actually acted as a peptidomimetic was confirmed by
the crystal structures of R61 in complex with L,L-DAP(Gly)-
penicillin and L,L-DAP(Gly)-D-Ala-D-Ala.[24,25] These structures
showed that the L,L-DAP(Gly) moieties of these molecules
occupy nearly identical positions in the R61 catalytic cavity and
form the same interactions with enzyme residues.
2
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Figure 1. A) Structural similarity between β-lactams (penams and carbapenems) and the pentapeptide donor stem of PBPs. B) Structural similarity between
carbapenems and the tetrapeptide donor stem of Ldts. C) Structure of the peptidoglycan subunit used by the R61 D,D-peptidase in Streptomyces spp. and
peptidomimetic penams.[21] The third position of the stem pentapeptide is occupied by an L,L-diaminopimelyl (DAP) residue substituted by a Gly residue. D)
Structure of peptidomimetic carbapenems synthesized in this study. On the left: the substituents of the carbapenem core mimic the main chain of the PG subunit
of E. faecium (shown in blue). On the right: the substituents of the carbapenem core mimic the side chain of the PG subunit of E. faecium (shown in green). R1 = -
S(CH2)2Ph; R2 = H, L-Ala or L-Ala-D-Lac; R3 = H or CONH2; R4 = H, Me or NH2; R5 = H, Ac; n = 0-4. Inset: Structural analogy between carbapenem 28b and the L-
Lys-D-iAsn moiety of peptidoglycan precursors.
These observations suggest that the “perfect” penicillin with
Active-site Ser amidotransferases superfamily
defined by a conserved fold and conserved SxxK, SDN,
respect to inactivation of the R61 D,D-peptidase was obtained by
the peptidomimetic approach.[21] Similar results, although
and KTG motifs
quantitatively less impressive, were obtained with other
peptidomimetic specific of the various PG chemotypes but this
ß-Lactamases
only concerned class C D,D-peptidases[26-29] and the side chain
class A, C and D
of peptidoglycan precursors.[26,30,31]
The group of Mobashery explored the bi-substrate approach by
D,D-peptidases
synthesizing a complex molecule consisting of a cephalosporin
core substituted by mimics of the donor and acceptor stems of
the transpeptidation reaction. Crystallization of the R61 D,D-
peptidase acylated by this cephalosporin resulted in a model for
the binding of the donor and acceptor substrates of the
transpeptidation reaction.[32,33] The same approach adapted to
the structure of the E. coli peptidoglycan structure led to a
Involved in peptidoglycan metabolism
Class A and B High-Molecular
Class C Low-Molecular Weight
Weight (HMW)
(LMW)
D,D-peptidases
D,D-peptidases
peptidoglycan polymerization
(essential)
Hydrolytic enzymes (unessential)
cephalosporin that behaved as
a
time-dependent and
Class D,D-
irreversible inhibitor of E. coli PBP1b,
a
A
transpeptidase (first-order inactivation rate constant kinact of
0.0072 ± 0.0007 min-1 and inhibitor constant Ki of 2.5 ± 0.8
mM).[33]
The peptidoglycan mimetic approach has only been applied to
D,D-transpeptidases of the PBP family and to β-lactams of the
penam (penicillin) and cephalosporin classes. This prompted us
to design and develop the synthesis of carbapenem-based
peptidoglycan mimetics and to evaluate their efficacy in the
Class A
Class B
Endopeptidases
Hydrolysis of
D,D-
carboxypeptida
ses
D,D-
D,D-
transpeptidases
transpeptidases
4→3 cross-links
Hydrolysis of the
C-terminal D-Ala
of stem
Formation of 4→3
cross-links and
polymerization of
glycan chains
Formation of
4→3 cross-links
and
pentapeptides
morphogenesis
Figure 2. Classes of amidotransferases.
3
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inactivation of the prototypic L,D-transpeptidase from E.
faecium.[3,18,19] In the literature, the two main synthetic strategies
providing access to functionalized carbapenems are either
based on (i) the formation of the 4,5-fused nuclei from acyclic
precursors (i.e. the carbapenem core including the ß-lactam
ring)[34] or (ii) a direct addition of functionalities at the C2 and C8
positions on the preformed skeleton.[35] In this study, we took
advantage of the nucleophilicity of the hydroxyl group at position
C8 to connect various PG fragments via the formation of an ester
bond. The resulting carbapenem-based peptidoglycan mimetics
were divided into two categories containing portions of either the
main chain or the side chain of the E. faecium PG subunits
(Figure 1D, in blue and green, respectively). A functionalized
carbapenem with a phenethylthio group at the C2 position was
chosen as the model building block for modifications at C8 since
previous work indicated that the latter led to effective inactivation
of Ldts.[36]
reaction with aqueous ammonia provided compound 12 in 71%
yield. In the last step, deprotection of the carboxylic acid was
performed with LiOH affording the D-iso-glutamine derivative 13
in 87% yield.
Results and Discussion
Synthesis of carbapenem-based peptidoglycan mimetics
(peptido-carbapenems). The functionalized carbapenems were
synthesized according to the retrosynthesis outlined in Scheme
2
starting from the commercially available -methyl-vinyl-
phosphate 1. The phenethyl side chain was introduced at the C2
position by nucleophilic substitution of the phenyl-phosphate.[36]
The protected amino acids or peptides mimicking various
moieties of the stem peptide of PG precursors were incorporated
via esterification of the C8 hydroxyl group. In this strategy, the p-
nitrobenzyl (PNB) carbamate group was chosen as the main
protecting group for the synthesis of peptidoglycan mimetics,
enabling a single final deprotection step. Of note, the initial
syntheses of the former derivatives conducted with benzyl and
benzyloxycarbonyl protecting groups were unsuccessful since
these groups could not be efficiently removed during the final
deprotection step.
Scheme 3. A) Synthesis of N-CO2PNB carbamates protected compounds 3-
10. B) Synthesis of compound 13.
Peptidoglycan mimetic 16 was obtained by using as the starting
material 4-aminobutyric acid, which was first converted into the
corresponding methyl ester 14.[38] Coupling of 14 with alanine 8
assisted by K-oxyma® and EDC as coupling reagents afforded
15 in 86% yield. Deprotection of the methyl ester was performed
with LiOH leading to the corresponding acid 16 in 63% yield
(Scheme 4A). The synthesis of dipeptide derivative 20 started
with the formation of the amino benzyl ester 17 as previously
described.[39] Coupling with Boc-L-Ala-OH was achieved using
TBTU as the coupling agent. Compound 18 was treated with
TFA to remove the Boc group and reacted with (2R)-2-
methoxypropanoic acid affording compound 19 in 71% yield.
The final deprotection step was achieved by hydrogenolysis in
methanol providing acid 20 in 76% yield (calculated by NMR)
together with the corresponding methyl ester (Scheme 4B).
Scheme 2. Retrosynthesis of peptido-carbapenems from 1.
The general procedure for N-CO2PNB protection were found to
be highly efficient and versatile providing the protected amino
acids and analogues 3 to 10 in 63% to 99% yield (Scheme 3A).
D-iso-glutamine derivative 13 was prepared in a three-step
procedure (Scheme 3B). The -carboxylic acid of D-glutamic acid
was selectively protected using trimethylsilyl chloride and
methanol[37] followed by the introduction of the PNB group onto
the amine affording amino acid 11 in 69% yield. The carboxylic
acid was then activated with ethyl chloroformate and the
4
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Scheme 4. Synthesis of peptidomimetics 16 (A), 20 (B) and 26 (C).
To obtain peptide 26 (Scheme 4C), the -benzylated D-glutamic
acid 21 was protected with a Boc group affording 22 in 71%
yield. Subsequent amidation afforded 23 in 96% yield. The N-
Boc group of 23 was cleaved and coupling with Boc-L-Ala-OH
afforded 24 in 70% yield. Compound 24 was treated with TFA
and coupling with (2R)-2-methoxypropanoic acid afforded 25 in
72% yield. Final deprotection of the benzyl ester was achieved
by hydrogenolysis leading to peptide 26 in almost quantitative
yield.
cephalosporin nitrocefin, due to rupture of the β-lactam C-N
bond followed by protonation of the nitrogen in the case of
carbapenems.[42,43] Mass spectrometry analyses showed that all
carbapenems synthesized in this study formed the expected
acylenzymes without any secondary modification of the
compound following the acylation step, as is the case for other
classes of β-lactams (Table 2).[10,17] These results also indicated
that the ester link at the C8 position was not hydrolyzed in the
Ldtfm active site. This was further confirmed by incubating
peptido-carbapenem 28g in the presence or absence of Ldtfm for
0, 90, and 240 minutes. Loss of 114,2 Da expected for the
hydrolysis of the ester at C8 was observed in neither cases.
The efficacy of Ldtfm inactivation by peptidomimetic
carbapenems was evaluated by determining the k1 and k2 kinetic
constants for the formation of the amine anion and the
acylenzyme (Scheme 5), respectively.[8,17,41,44] These kinetic
constants were determined by stopped-flow fluorimetry as the
intrinsic fluorescence of the Ldtfm tryptophan residues varies for
the free enzyme, the amine anion, and the acylenzyme.[41]
Fluorescence kinetics were determined with a minimum of three
concentrations for each carbapenem. The rate constants k1, k-1
and k2 were determined by fitting differential equations to these
kinetics using the DynaFit software,[45] as described in section 5
of the Supplementary Information. Peptido-carbapenems 28a to
28n harbored a phenethylthio substituent at position C2. In
comparison to the reference carbapenem, meropenem, the
phenethyl side chain improved both the k1 and k2 kinetic
parameters (compare meropenem and compound 2’ in Table 3).
In comparison to 2’, introduction of peptidomimetics at the C8
The synthesis of peptido-carbapenems 28a-n was achieved in
two steps (Table 1). Esterification of carbapenem 2 was first
conducted with the PG analogues using
a
published
procedure[40] affording carbapenems 27a-n in 35 to 99% yield.
Finally, the peptido-carbapenems 28a-n were obtained by
hydrogenolysis of 27a-n after HPLC purification. Using this
approach, a full mimetic of the main chain was obtained (28n).
In contrast, the best mimic of the side chain was 28b (inset in
Figure 2D) since incorporation of the D-iAsn extremity of
peptidoglycan precursor in the carbapenem could not be
obtained due to cyclisation of D-iAsn during the synthesis of the
L-Lys-D-iAsn dipeptide.
Efficacy of acylation of the Ldtfm L,D-transpeptidase by the
peptidomimetic
carbapenems.
Inactivation
of
L,D-
transpeptidases was previously shown to be
a
two-step
reaction[8] starting with nucleophilic attack of the β-lactam
carbonyl by the sulfur of the catalytic cysteine (Scheme 5). This
first step was proposed to lead to reversible formation of an
amine anion.[41] The second step is irreversible, except for the
5
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Table 1. Synthesis of peptido-carbapenems 28a-n.
STEP 1 - Coupling
STEP 2 - Deprotection
R1
Cpd
27a
R
Yield[a] (%)
Cpd
28a
Yield[d] (%)
3
89
21
Ac-Gly-OH[b]
27b
27c
93
96
28b
28c
26
6
4
Ac-ß-Ala-OH[b]
27d
27e
87
99
28d
28e
40
40
5
6
27f
97
28f
6
7
8
27g
27h
68
98
28g
28h
17
8
9
27i
90
28i
9
10
13
16
27j
27k
27l
84
82
86
28j
28k
28l
32
14
15
20
26
27m
27n
35[c]
87
28m
28n
31
21
[a] Isolated yield. [b] Commercially available. [c] NMR calculated yield. [d] Isolated yield after HPLC purification.
6
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meropenem indicating that the peptidomimetic did not improve
antibacterial activity.
Conclusion
We show that the C8 position of carbapenems can be efficiently
functionalized by esterification to introduce chemical diversity in
carbapenems. We added to this position mimics of the side
chain (L-Lys-D-iAsn) and main chain (D-Lac-L-Ala-D-iGln-L-Lys)
of peptidoglycan precursors from E. faecium. Biological
evaluation of the resulting series of 14 peptido-carbapenems
indicated that increasing similarity between the drug and the
stem peptide of PG precursors does not improve the efficacy of
inactivation of the L,D-transpeptidase produced by this species.
Similar negative results were previously reported for other
species-specific β-lactam peptidomimetics designed to inhibit
the transpeptidase activity of HMW class A and B PBPs (see
introduction section). As proposed by Josephine et al.,[27] this
could be accounted for by the mode of substrate recognition by
class A and B D,D-transpeptidases that may involve regions of
the enzyme other than the transpeptidase active site and the
glycan chains rather than the stem peptides. Such interactions
were identified by solid-state NMR analysis of an L,D-
transpeptidase from Bacillus subtilis.[48]
The carbapenems reported in this study were more effective
than the reference drug of this class, meropenem, due to the
positive impact of the phenethylthio substituent at position C2
combined to the mostly neutral impact of the substituents at
position C8. Thus, the latter position can be modified to modulate
the pharmacokinetic or other properties of carbapenems without
any deleterious effect on the efficacy of L,D-transpeptidase
inactivation or antibacterial activity. This strategy could be used
to optimize carbapenems for the treatment of lung infections due
to Mycobacterium tuberculosis and Mycobacterium abscessus
Scheme 5. Two-step acylation of L,D-transpeptidase Ldtfm by carbapenems.
The evidence for the formation of amine anion in the first step of the acylation
reaction is supported by previous studies.[8,43,44]
Table 2. Mass of adducts formed between Ldtfm and carbapenems.
Mass of adduct (Da)
Mass of
Cpd
carbapenem (Da)
Calculated
Observed
None
Not Applicable
16,639.3
16,640.2
Side chain mimetics
404.5
446.5
418.5
460.5
432.5
446.6
460.6
418.5
476.6
517.6
17 043.8
17 085.8
17 057.8
17 099.9
17 071.9
17 085.9
17 099.9
17 057.8
17 115.9
17 156.9
17 045.2
17 087.9
17 059.6
17 100.7
17 073.6
17 088.2
17 098.0
17 059.1
17 116.2
17 158.7
28a
28b
28c
28d
28e
28f
28g
28h
28i
28j
Main chain mimetics
475.6
503.6
589.7
17 114.9
17 142.9
17 259.0
17 116.3
17 143.6
17 259.5
28k
28l
since
L,D-transpeptidases
are
major
contributors
to
peptidoglycan polymerization in these bacteria.[4,10,11,49,50]
28m
632.7
17 272.1
17 273.4
28n
Experimental Section
General procedure for the synthesis of peptido-carbapenems 27a-n
(Step 1). Carbapenem 2 (1 equiv.) in presence of peptidoglycan mimetic
(4 equiv. up to 8 equiv.) was treated with DMAP (0.9 equiv.) and then
with small portions of EDC (8 equiv. up to 16 equiv.) at – 20 ˚C. The
reaction mixture was stirred for 1 – 24 hours while maintaining a
temperature between – 30 to – 15 ˚C. After the completion of the reaction,
EtOAc was added to dissolve the crude. The organic layer was washed
with a sat. solution of NaHCO3, brine, dried over MgSO4, filtered and
concentrated under reduced pressure. The crude mixture was purified by
silica gel chromatography (cyclohexane:EtOAc or DCM:MeOH as eluent
systems) to afford the desired peptido-carbapenem derivatives.
position had minor negative impacts on the k2 kinetic constant
for all compounds (28a to 28n). Thus, the peptidomimetics
reduced the efficacy of the chemical step of the reaction
involving the rupture of the β-lactam ring and protonation of its
nitrogen atom. The peptidomimetics had minor positive and
negative impacts on kinetic constant k1, which is the critical
constant determining the efficacy of Ldtfm inactivation.[41] These
results show that substitutions at the C8 position marginally
modulate the efficacy of inactivation of Ldtfm, as previously found
for high-molecular weight PBPs.
General procedure for the synthesis of peptido-carbapenems 28a-n
(Step 2). The corresponding carbapenem derivative (1 equiv.) was
Antibacterial activity. Peptido-carbapenems were tested by the
microdilution technique in 96-well plates against E. faecium
strain M5 in the presence of ampicillin (32 µg/ml), as previously
described.[46] Under such conditions, Ldtfm is the only functional
transpeptidase due to inhibition of PBPs by ampicillin.[47] The
minimal inhibitory concentrations (MICs) of peptido-
carbapenems 28a to 28n were equal to or higher than that of
treated with Pt/C 10% wt. (1 equiv. mass.) in
a mixture of
THF:triethylammonium bicarbonate buffer (1 M, pH = 8.5) (2:1) and the
reaction mixture was hydrogenated under 3.5 bars for 2 hours at room
temperature using the PARR apparatus. The crude mixture was filtered
through a celite pack to remove the catalyst. THF was removed under
reduced pressure, and H2O was added. The crude mixture was purified
by HPLC using a solvent system consisting of H2O and acetonitrile (linear
7
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Table 3. Efficacy of acylation of the Ldtfm L,D-transpeptidase by meropenem and peptido-carbapenems and antibacterial activity against E. faecium M512.
Cpd
R1
R2
k1 (µM-1min-1)[a]
k2 (min-1)[a]
MIC (µg ml-1)[b]
Meropenem
H
0.0819 ± 0.0004
1.59 ± 0.01
1
2‘[c]
H
0.292 ± 0.004
14.8 ± 0.18
2
Side chain Mimetics
28a
0.248 ± 0.004
0.347 ± 0.010
0.572 ± 0.007
0.343 ± 0.009
0.268 ± 0.003
0.109 ± 0.001
0.138 ± 0.002
0.379 ± 0.005
5.10 ± 0.05
4.68 ± 0.08
7.40 ± 0.06
3.92 ± 0.06
7.49 ± 0.07
3.16 ± 0.04
3.43 ± 0.03
5.20 ± 0.05
4
16
2
28b
28c
28d
28e
28f
16
4
8
28g
28h
8
1
28i
0.278 ± 0.005
0.168 ± 0.002
3.80 ± 0.04
2.70 ± 0.02
4
8
28j
Main chain Mimetics
28k
0.239 ± 0.010
0.192 ± 0.001
0.355 ± 0.006
0.121 ± 0.002
4.60 ± 0.11
4.79 ± 0.03
7.88 ± 0.11
9.07 ± 0.15
4
4
28l
28m
28n
8
> 16
[a] The methods used to determine kinetics parameters k1 and k2 are described in Section 4 of the Supplementary Information. [b] Median from three independent
experiments. [c] Synthesis of carbapenem 2’ was previously described.[36]
8
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gradient, 0 to 100% over 45 minutes) and the selected fractions were
collected and lyophilized to afford the final peptido-carbapenem
derivatives.
[21] H. R. Josephine, I. Kumar, R. F. Pratt, J. Am. Chem. Soc. 2004, 126,
8122-8123.
[22] M. Jamin, J. M. Wilkin, J. M. Frere, Biochemistry 1993, 32, 7278-7285.
[23] K. H. Schleifer, O. Kandler, Bacteriol. Rev. 1972, 36, 407-77.
[24] N. R. Silvaggi, H. R. Josephine, A. P. Kuzin, R. Nagarajan, R. F. Pratt,
J. A. Kelly, J. Mol. Biol. 2005, 345, 521-533.
Acknowledgements
[25] M. A. McDonough, J. W. Anderson, N. R. Silvaggi, R. F. Pratt, J. R.
Knox, J. A. Kelly, J. Mol. Biol. 2002, 322, 111-122.
The authors thank the „Ministère de l’Enseignement Supérieur,
de la Recherche et de l’Innovation“ for S. S. PhD grant.
[26] L. Dzhekieva, I. Kumar, R. F. Pratt, Biochemistry 2012, 51, 2804-2811.
[27] H. R. Josephine, P. Charlier, C. Davies, R. A. Nicholas, R. F. Pratt,
Biochemistry 2006, 45, 15873-15883.
Keywords: Carbapenem • Esterification • Peptidoglycan •
Peptidomimetics • Transpeptidase
[28] P. H. Bentley, A. V. Stachulski, J. Chem. Soc. Perk. Trans 1 1983, 6,
1187-1192.
[29] S. Hanessian, C. A. Couture, N. Georgopapadakou, Bioorg. Med.
Chem. Lett. 1993, 3, 2323-2328.
[1]
[2]
L. L. Silver, Curr. Opin. Microbiol. 2003, 6, 431-438.
[30] V. V. Nemmara, L. Dzhekieva, K. S. Sarkar, S. A. Adediran, C. Duez,
R. A. Nicholas, R. F. Pratt, Biochemistry 2011, 50, 10091-10101.
[31] E. Sauvage, A. J. Powell, J. Heilemann, H. R. Josephine, P. Charlier,
C. Davies, R. F. Pratt, J. Mol. Biol. 2008, 381, 383-393.
S. Jovetic, Y. Zhu, G. L. Marcone, F. Marinelli, J. Tramper, Trends
Biotechnol. 2010, 28, 596-604.
[3]
J. L. Mainardi, M. Fourgeaud, J. E. Hugonnet, L. Dubost, J. P. Brouard,
J. Ouazzani, L. B. Rice, L. Gutmann, M. Arthur, J. Biol. Chem. 2005,
280, 38146-38152.
[32] W. Lee, M. A. McDonough, L. P. Kotra, Z. H. Li, N. R. Silvaggi, Y.
Takeda, J. A. Kelly, S. Mobashery, Proc. Natl. Acad. Sci. U.S.A. 2001,
98, 1427-1431.
[4]
[5]
M. Lavollay, M. Fourgeaud, J. L. Herrmann, L. Dubost, A. Marie, L.
Gutmann, M. Arthur, J. L. Mainardi, J. Bacteriol. 2011, 193, 778-782.
E. Sacco, J. E. Hugonnet, N. Josseaume, J. Cremniter, L. Dubost, A.
Marie, D. Patin, D. Blanot, L. B. Rice, J. L. Mainardi, M. Arthur, Mol.
Microbiol. 2010, 75, 874-885.
[33] M. Lee, D. Hesek, M. Suvorov, W. Lee, S. Vakulenko, S. Mobashery, J.
Am. Chem. Soc. 2003, 125, 16322-16326.
[34] A. H. Berks, Tetrahedron 1996, 52, 331-375.
[35] M. Sunagawa, A. Sasaki, J. Syn. Org. Chem. Jpn 1996, 54, 761-771.
[36] L. Iannazzo, D. Soroka, S. Triboulet, M. Fonvielle, F. Compain, V.
Dubee, J. L. Mainardi, J. E. Hugonnet, E. Braud, M. Arthur, M. Etheve-
Quelquejeu, J. Med. Chem. 2016, 59, 3427-3438.
[6]
J. E. Hugonnet, D. Mengin-Lecreulx, A. Monton, T. den Blaauwen, E.
Carbonnelle, C. Veckerle, Y. V. Brun, M. van Nieuwenhze, C. Bouchier,
K. Tu, L. B. Rice, M. Arthur, Elife 2016, 5.
[7]
[8]
K. M. Papp-Wallace, A. Endimiani, M. A. Taracila, R. A. Bonomo,
Antimicrob. Agents Chemother. 2011, 55, 4943-4960.
[37] S. S. More, R. Vince, J. Med. Chem. 2009, 52, 4650-4656.
[38] C. P. Gordon, N. Dalton, N. Vandegraaff, J. Deadman, D. I. Rhodes, J.
A. Coates, S. G. Pyne, R. Griffith, J. B. Bremner, P. A. Keller,
Tetrahedron 2018, 74, 1253-1268.
N. Bhattacharjee, S. Triboulet, V. Dubee, M. Fonvielle, Z. Edoo, J. E.
Hugonnet, M. Etheve-Quelquejeu, J. P. Simorre, M. J. Field, M. Arthur,
C. M. Bougault, Antimicrob. Agents Chemother. 2019, 63, e02039-18.
J. E. Hugonnet, L. W. Tremblay, H. I. Boshoff, C. E. 3rd Barry, J. S.
Blanchard, Science 2009, 323, 1215-1218.
[39] T. J. Kop, J. Dordevic, M. S. Bjelakovic, D. R. Milic, Tetrahedron 2017,
73, 7073-7078.
[9]
[40] S. B. Singh, D. Rindgen, P. Bradley, T. Suzuki, N. Wang, H. Wu, B.
Zhang, L. Wang, C. Ji, H. Yu, R. M. Soll, D. B. Olsen, P. T. Meinke, D.
A. Nicoll-Griffith, J. Med. Chem. 2014, 57, 8421-8444.
[10] N. Dhar, V. Dubee, L. Ballell, G. Cuinet, J. E. Hugonnet, F. Signorino-
Gelo, D. Barros, M. Arthur, J. D. McKinney, Antimicrob. Agents
Chemother. 2015, 59, 1308-1319.
[41] S. Triboulet, M. Arthur, J. L. Mainardi, C. Veckerle, V. Dubee, A.
Nguekam-Moumi, L. Gutmann, L. B. Rice, J. E. Hugonnet, J. Biol.
Chem. 2011, 286, 22777-22784.
[11] A. H. Diacon, L. van der Merwe, M. Barnard, F. von Groote-
Bidlingmaier, C. Lange, A. L. Garcia-Basteiro, E. Sevene, L. Ballell, D.
Barros-Aguirre, N. Engl. J. Med. 2016, 375, 393-394.
[42] Z. Edoo, M. Arthur, J. E. Hugonnet, Sci. Rep. 2017, 7, 9136.
[43] S. Triboulet, Z. Edoo, F. Compain, C. Ourghanlian, A. Dupuis, V.
Dubee, L. Sutterlin, H. Atze, M. Etheve-Quelquejeu, J. E. Hugonnet, M.
Arthur, ACS Infect. Dis. 2019, 5, 1169-1176.
[12] E. Le Run, M. Arthur, J. L. Mainardi, Antimicrob. Agents Chemother.
2019, 63, e01915-18.
[13] R. A. Floto, K. N. Olivier, L. Saiman, C. L. Daley, J. L. Herrmann, J. A.
Nick, P. G. Noone, D. Bilton, P. Corris, R. L. Gibson, S. E. Hempstead,
K. Koetz, K. A. Sabadosa, I. Sermet-Gaudelus, A. R. Smyth, J. van
Ingen, R. J. Wallace, K. L. Winthrop, B. C. Marshall, C. S. Haworth,
Thorax 2016, 71, 88-90.
[44] N. Bhattacharjee, M. J. Field, J. P. Simorre, M. Arthur, C. M. Bougault,
J. Phys. Chem. B 2016, 120, 4767-4781.
[45] P. Kuzmic, Methods Enzymol. 2009, 467, 247-280.
[46] K. Peters, M. Pazos, Z. Edoo, J. E. Hugonnet, A. M. Martorana, A.
Polissi, M. S. VanNieuwenhze, M. Arthur, W. Vollmer, Proc. Natl. Acad.
Sci. U. S. A. 2018, 115, 10786-10791.
[14] M. Cordillot, V. Dubee, S. Triboulet, L. Dubost, A. Marie, J. E.
Hugonnet, M. Arthur, J. L. Mainardi, Antimicrob. Agents Chemother.
2013, 57, 5940-5945.
[47] J. L. Mainardi, V. Morel, M. Fourgeaud, J. Cremniter, D. Blanot, R.
Legrand, C. Frehel, M. Arthur, J. Van Heijenoort, L. Gutmann, J. Biol.
Chem. 2002, 277, 35801-35807.
[15] P. Kumar, K. Arora, J. R. Lloyd, I. Y. Lee, V. Nair, E. Fischer, H. I.
Boshoff, C. E. 3rd Barry, Mol. Microbiol. 2012, 86, 367-381.
[16] R. Gupta, M. Lavollay, J. L. Mainardi, M. Arthur, W. R. Bishai, G.
Lamichhane, Nat. Med. 2010, 16, 466-469.
[48] P. Schanda, S. Triboulet, C. Laguri, C. M. Bougault, I. Ayala, M. Callon,
M. Arthur, J. P. Simorre, J. Am. Chem. Soc. 2014, 136, 17852-17860.
[49] M. Lavollay, M. Arthur, M. Fourgeaud, L. Dubost, A. Marie, N. Veziris,
D. Blanot, L. Gutmann, J. L. Mainardi, J. Bacteriol. 2008, 190, 4360-
4366.
[17] S. Triboulet, V. Dubee, L. Lecoq, C. Bougault, J. L. Mainardi, L. B. Rice,
M. Etheve-Quelquejeu, L. Gutmann, A. Marie, L. Dubost, J. E.
Hugonnet, J. P. Simorre, M. Arthur, PLoS One 2013, 8, e67831.
[18] L. Lecoq, V. Dubee, S. Triboulet, C. Bougault, J. E. Hugonnet, M.
Arthur, J. P. Simorre, ACS Chem. Biol. 2013, 8, 1140-1146.
[19] J. L. Mainardi, J. E. Hugonnet, F. Rusconi, M. Fourgeaud, L. Dubost, A.
N. Moumi, V. Delfosse, C. Mayer, L. Gutmann, L. B. Rice, M. Arthur, J.
Biol. Chem. 2007, 282, 30414-30422.
[50] E. Le Run, M. Arthur, J. L. Mainardi, Antimicrob. Agents Chemother.
2018, 62, e00623-18.
[20] D. J. Tipper, J. L. Strominger, Proc. Natl. Acad. Sci. U. S. A. 1965, 54,
1133-1141.
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Entry for the Table of Contents
A synthetic route to carbapenems modified at position C8 was developed to assess the impact of mimetics of the main and side
chains of peptidoglycan precursors on cross-linking activity of L,D-transpeptidase from E. faecium. Substitution at position C8 can be
therefore used to modulate the properties of carbapenem without deleterious effect on the efficacy of L,D-transpeptidase inhibition.
10
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