Angewandte
Chemie
DOI: 10.1002/anie.201410270
Natural Product Synthesis
Total Synthesis and Biological Evaluation of the Antibiotic Lysocin E
and Its Enantiomeric, Epimeric, and N-Demethylated Analogues**
Motoki Murai, Takuya Kaji, Takefumi Kuranaga, Hiroshi Hamamoto, Kazuhisa Sekimizu, and
Masayuki Inoue*
Abstract: Lysocin E, a macrocyclic peptide, exhibits potent
antibacterial activity against methicillin-resistant Staphylococ-
cus aureus (MRSA) through a novel mechanism. The first total
synthesis of lysocin E was achieved by applying a full solid-
phase strategy. The developed approach also provides rapid
access to the enantiomeric, epimeric, and N-demethylated
analogues of lysocin E. Significantly, the antibacterial activity
of the unnatural enantiomer was comparable to that of the
natural isomer, suggesting the absence of chiral recognition in
its mode of action.
A solid-phase synthetic route to 1a would make it possible
to prepare a wider variety of lysocin E analogues than with
a solution-phase route, thereby enabling a comprehensive
structure–activity relationship (SAR) study of 1a in search of
[6–8]
more potent analogues.
Herein, the first total synthesis of
lysocin 1a by a solid-phase approach is disclosed. The
developed route enabled the preparation of not only the
enantiomer of 1a (ent-1a), but also of epimeric (1b) and
N-demethylated analogues (1c). A preliminary comparative
study of the antibacterial properties of 1a, ent-1a, 1b, and 1c
sheds light on the structural factors that are relevant to the
lysocin E molecular mode of action.
I
nfectious diseases caused by antibiotic-resistant bacteria
[
1,2]
pose a threat to public health around the world.
Nosoco-
The core structure of 1a is a 37-membered macrocycle
comprising twelve amino acid residues with an N-methylated
amide and an ester linkage (Scheme 1). There are four types
of d-configured amino acids (d-Gln, d-Trp, d-Arg, d-N-Me-
Phe) present within the sequence, and (R)-3-hydroxy-5-
mial infections caused by methicillin-resistant Staphylococcus
aureus (MRSA) in hospitals have become an especially
serious clinical problem. Recently, Sekimizu and co-workers
isolated a novel peptidic natural product, lysocin E (1a,
Scheme 1), from a culture supernatant of a Lysobacter species
and showed that 1a was a potent growth inhibitor of MRSA
[9]
methylhexanamide is appended to the macrocyclic core.
To enable a full solid-phase synthesis, this complex structure
was retrosynthetically disconnected into the eleven building
blocks 3–5, 6a, 7, 8, 9a, and 10–13, the protective groups of
which were designed to possess three dimensions of orthog-
with
mgmL . Furthermore, 1a was found to possess a potent
therapeutic effect in mice infected with S. aureus. The ED
a minimum inhibitory concentration (MIC) of
ꢀ1 [3]
4
5
0
ꢀ
1
[10]
[11]
value of 1a (0.5 mgkg ) was even smaller than that of
vancomycin (5.8 mgkg ), which is widely used to treat
MRSA infections.
onal reactivity. Acid-labile groups (Wang resin of 3, Pbf
ꢀ
1
of 4, tBu of 8 and 10, TBS of 9a, Boc of 12, and Trt of 13) were
chosen for protection of the side chains, whereas the base-
[12]
Menaquinone, a co-enzyme in the bacterial respiratory
labile Fmoc group was used for temporary N protection.
a
[4]
chain, has been assumed to be the main target molecule of
The allyl group, cleavable under neutral conditions, was
[3]
1
a (Scheme 1). Peptide 1a selectively forms an equimolar
selected for the carboxylic acid moiety of 3. Therefore, our
[
13]
complex with menaquinone present in the cytoplasmic
membrane of S. aureus, effecting cell death through mem-
brane disruption. Although the involvement of other mole-
cules in the mechanism has not been ruled out, the mode of
action of 1a is apparently distinct from those of previously
known antibiotics. As an antibiotic that functions by a novel
mechanism is less likely to show cross-resistance with existing
antibiotics, 1a is viewed as a useful lead compound for the
strategy involved the following operations:
1) stepwise
solid-phase assembly of the linear sequence using Fmoc
chemistry from side-chain-anchored 3, 2) orthogonal depro-
tection of the allyl group of 2a to selectively liberate the
C carboxylic acid, 3) intramolecular amidation between the
a
free C carboxylic acid and the N amine, taking advantage of
a
a
[
14]
the pseudo-high dilution of the resin-bound molecule, and
4) simultaneous acid-promoted global deprotection and
cleavage to release the targeted peptide 1a. This strategy
would allow for the unified and efficient preparation of the
lysocin E derivatives ent-1a, 1b, and 1c by simply replacing
the building blocks of 1a for their enantiomeric counterparts,
[
5]
development of a suitable drug candidate.
[
*] Dr. M. Murai, T. Kaji, Dr. T. Kuranaga, Dr. H. Hamamoto,
Prof. Dr. K. Sekimizu, Prof. Dr. M. Inoue
Graduate School of Pharmaceutical Sciences
The University of Tokyo
9
b, and 6c, respectively.
As a prelude to the solid-phase synthesis of 1a, the
branched fragment 9a was prepared (Scheme 2). LDA-
promoted addition of ethyl acetate (14) to isovaleryl chloride
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)
E-mail: inoue@mol.f.u-tokyo.ac.jp
[
**] This research was financially supported by the Funding Program for
Next Generation World-Leading Researchers (JSPS) to M.I., a Grant-
in-Aid for Young Scientists (B) of the JSPS to T.Ku., and JST A-STEP
to T.Ku. and K.S.
(
15) afforded b-ketoester 16. Asymmetric Noyori reduction
[
15]
of the ketone using [RuCl {(R)-BINAP}] converted 16 into
2
enantiomerically pure (R)-ethyl-3-hydroxy-5-methylhexa-
[
16]
noate (17). Saponification of 17 provided carboxylic acid
18, which was condensed with l-Thr(Bn)-OBn (19) to give
Angew. Chem. Int. Ed. 2014, 53, 1 – 6
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
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