Synthesis of Peptidoglycan Fragments from Enterococcus faecalis with Fmoc-Strategy for Glycan Elongation

Article abstract of DOI:10.1002/asia.201601357

Peptidoglycan (PGN) is an essential structural component of the bacterial cell wall conferring cell shape, which can be recognized by host-recognition proteins and receptors as well as bacterial surface proteins. In this work, the PGN partial structures from Enterococcus faecalis that contain a tetrasaccharide and an octasaccharide with a unique heptapeptide were synthesized via an Fmoc-strategy for elongation of the glycan chains. Namely, a 4′-O-Fmoc-protected disaccharide was utilized as the key intermediate in this efficient synthetic pathway for preparing various PGN fragments. Both the tetrasaccharide and octasaccharide with the unique heptapeptide were successfully synthesized for the first time.

Full text of DOI:10.1002/asia.201601357

CHEMISTRY
AN ASIAN JOURNAL
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Accepted Article
Title: Synthesis of peptidoglycan fragments from Enterococcus faecalis
with Fmoc-strategy for glycan elongation
Authors: Ning Wang, Hiroki Hasegawa, Cheng-yuan Huang, Koichi
Fukase, and Yukari Fujimoto
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To be cited as: Chem. Asian J. 10.1002/asia.201601357
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Synthesis of peptidoglycan fragments from Enterococcus
faecalis with Fmoc-strategy for glycan elongation
Ning Wang,[a] Hiroki Hasegawa,[a] Cheng-yuan Huang,[a] Koichi Fukase,*[a] and Yukari Fujimoto*[a],[b]
Abstract: Peptidoglycan (PGN) is an essential structural component
of the bacterial cell wall conferring cell shape, which can be
recognized by host recognition proteins and receptors as well as
bacterial surface proteins. In this work, the PGN partial structures
from Enterococcus faecalis that contain tetrasaccharide and
octasaccharide with a unique heptapeptide were synthesized with
Fmoc-strategy for elongation of the glycan chains. Namely, 4-O-
Fmoc-protected disaccharide was utilized as the key intermediate in
this efficient synthetic pathway for preparing various PGN fragments.
Both tetrasaccharide and octasaccharide with heptapeptide were
successfully synthesized for the first time.
E.
faecalis
PGN
fragments,
i.e.,
(GlcNAc-MurNAc)4
octasaccharide with the unique heptapeptide moiety were
synthesized for the first time by fragment condensation strategy
using Fmoc-protected glycans for elongation of the glycan
(Figure 1B), and the detailed scheme is shown in Scheme 1.
Bacterial cell wall peptidoglycan (PGN) is a huge polymer
composed of glycan chains and peptide chains that form three
dimensional mesh-like structures. Each glycan consists of
alternating N-acetylglucosamine (GlcNAc) and N-muramic acid
(MurNAc) linked with β(1→4) bond (Figure 1A). The glycans in
PGN are connected each other with peptide linkage at the
carboxylic acid of MurNAc. The glycan and the first three amino
acids connected to the glycan are quite common in bacteria.
LysM domain is a PGN recognition motif,[1] found in various
proteins from bacteriophages, bacteria, eukaryotes, plants, and
mammals.[2-9] In Enterococcus faecalis, there are three LysM-
containing proteins, named AtlA, AtlB and AtlC.[10,11] AtlA is an
N-acetylglucosaminidase, whereas AtlB and AtlC are N-
acetylmuramidase. Structure of a disaccharide fragment was
confirmed by AtlB digestion of E. faecalis PGN (Figure 1A)
followed by reduction with sodium borohydride.[12] Additionally, E.
faecalis PGN has a unique heptapeptide structure as the peptide
linkage; L-Ala-D-isoGln-L-Lys(L-Ala-L-Ala)-D-Ala-D-Ala, in which
the branched L-Ala-L-Ala dipeptide attaches to the amino group
of L-Lys in the typical L-Ala-D-isoGln-L-Lys-D-Ala-D-Ala
pentapeptide stem.[10]
In the present study, the PGN fragments from E. faecalis were
synthesized in order to investigate the precise recognition of
LysM domains of AtlB and AtlC with PGN. It was demonstrated
recently that longer glycan chains lead to stronger binding with
LysM domains. For example, LysM domain derived from Pteris
ryukyuensis chitinase-A bound to GlcNAc oligomers, (GlcNAc)n,
with 1:1 stoichiometry and (GlcNAc)5 showed higher affinity than
the shorter glycans.[13] The effect of the peptide chain length for
LysM recognition, however, has not been elucidated. Thus, the
[a] Dr. N. Wang, Mr. H. Hasegawa, Dr. C. Huang, Prof. Dr. Y. Fujimoto
Prof. Dr. K. Fukase,
Department of Chemistry
Graduate School of Science
Osaka University
1-1 Machikaneyama, Toyonaka, Osaka 560-0043 (Japan)
[b] Prof. Dr. Y. Fujimoto
Department of Chemistry
Faculty of Science and Technology
Keio University
3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522 (Japan)
E-mail: fujimotoy@chem.keio.ac.jp
Figure 1. A) Structure of peptidoglycan from Enterococcus faecalis, and the
structure of a disaccharide fragment obtained by AtlB digestion of E. faecalis
PGN followed by reduction with sodium borohydride. B) Synthetic targets 1
and 2 as the substrates of the AtlB.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/asia.
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Scheme 1. Synthesis of octasaccharide with heptapeptide.
We previously synthesized the octasaccharide dipeptide
containing four GlcNAc-MurNAc units.[14,15] One of the key
reactions was the regioselective ring opening reduction of the
benzylidene group in long glycans (such as 14 to 15 in Scheme
2). However, in the case of longer glycan, the yields and
reproducibility of the regioselective ring opening reaction for the
oligosaccharide intermediates were not sometimes satisfactory.
glycosyl donors.[17-20] In our previous synthetic study for the
tetrasaccharide containing two MurNAc-GlcNAc repeating units,
we found that the typical trichloacetimidate donors were more
readily decomposed in glycosylation with low reactive hydroxy
groups than the corresponding N-phenyltrifluoroacetimidates.[21]
The improved stability of N-phenyltrifluoroacetimidates also
showed advantage to the glycosylation between large
tetrasaccharide fragments in present study. In addition, the basic
condition for the preparation of the trichloacetimidate was not
tolerated by the glycosylation donor, leading to the removal of
Fmoc group. We thus used the N-phenyltrifluoroacetimidates as
the suitable glycosyl donors in this study.
The efficient construction of heptapeptide 4 was performed
using sequential coupling method as shown in Scheme 3. Since
the solubility of the protected linear pentapeptide in organic
solvents was very low, we designed the synthetic route where
the side-chain L-Ala-L-Ala was introduced to the tripeptide 16
which was prepared in lab (see SI). Since the direct coupling of
Boc-L-Ala-L-Ala-OH and 16 did not proceed, the two branched
L-Ala moieties were attached to 16 step by step. Condensation
of Boc-L-Ala-OH with the tripeptide 16 was carried out by using
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
Scheme 2. Preparation of glycosyl acceptor via benzylidene protected
intermediate.
To improve the approach to the synthesis of the
octasaccharide, we employed a disaccharide with 4’-O-Fmoc
protection (3a) as the key intermediate, since the O-Fmoc group
(water-soluble
carbodiimide;
WSCD·HCl),
1-
hydroxybenzotriazole (HOBt) and TEA to give tetrapeptide 17.
The second L-Ala in the L-Lys branch was introduced by the
coupling with Z-L-Ala-OH to form pentapeptide 18. Fmoc group
of 18 was then removed by treatment with piperidine in THF and
the resulted pentapeptide amine was coupled with the dipeptide
can be cleaved by
a mild base, such as piperidine or
trimethylamine, to give the 4-OH glycosyl acceptor in better
yields. The Fmoc-method is also suitable for solid-phase
synthesis of PGN glycans.[16] The other improvement of the
synthesis was the use of the N-phenyltrifluoroacetimidates as
(Boc-L-Ala-D-isoGln-OH)
using
O-(7-azabenzotriazol-1-yl)-
N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) to
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give the protected heptapeptide 19 in 77% yield. Although the
solubility in organic solvents was low, purification of 19 with
silica-gel column chromatography was achieved by using CHCl3-
MeOH as an eluent. Cleavage of Boc group of 19 and treatment
with HCl in Et2O afforded hydrochloride salt of heptapeptide 4,
which was used for further condensation with glycan fragments
(Scheme 1). The fully deprotected heptapeptide 20 was also
obtained by catalytic hydrogenation of 4 with Pd(OH)2 and H2 in
acetic acid.
with LiOH was carried out to give 11. Introduction of
heptapeptide 4 to the liberated four carboxyl groups was also
the key reaction for the synthesis. We avoided the usage of
carbodiimides as condensation reagents since they sometimes
afford N-acylurea by-products. Hence, condensation of 11 with 4
by
using
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-
and
tetramethyluronium hexafluorophosphate (HATU)
triethylamine (TEA) in DMF successfully afforded the glycan
peptide conjugate 12. Deprotection of all of benzyl-type
protecting groups by catalytic hydrogenolysis (H2, 2 MPa) in
acetic acid (AcOH) using palladium hydroxide on carbon
successfully gave fully deprotected octasaccharide with
heptapeptide 2. The liberated octasaccharde 13 was also
prepared from 11 with the catalytic hydrogenolysis in 90% yield.
Figure 2. Structure of tetrasaccharide fragments 1 and 21.
The tetrasaccharide fragments 1 and 21 (Figure 2) were also
synthesized for the analysis of the binding with LysM domain of
the AtlB (The details of the synthesis are in the Supporting
Information, Scheme SI-2). The synthesized PGN fragments
was used for the elucidation of the interaction between PGN and
LysM domains from E. faecalis.[22] The results of the binding with
a single LysM module was as flollows; the tetrasaccharide
(GlcNAc-MurNAc)2 21 (KD values 650 µM) showed higher
affinitiy than the (GlcNAc-MurNAc-dipeptide(L-Ala-D-isoGln))2 or
(GlcNAc-MurNAc-heptapeptide)2 1 (KD values 1.8±0.5 mM or >1
mM, respectively), while the binding affinity increased with the
length of the glycan chains (KD = 80 µM for (GlcNAc-MurNAc)4
2). [22]
In conclusion, we have developed an Fmoc-strategy for the
synthesis of bacterial PGN with utilizing 4-O-Fmoc protected N-
phenyltrifluoroacetimidates as the glycosyl donors. The PGN
fragments containing heptapeptide stem was synthesized for the
first time. The octasaccharide with heptapeptide was the largest
PGN fragment so far synthesized. The synthetic PGN fragments
have been used for the elucidation of the interaction between
PGN and LysM domains from E. faecalis[22] and further analysis
will be reported in elsewhere.
Scheme 3. Synthesis of heptapeptide.
Synthesis of the tetrasaccharide and octasaccharide
fragments with the heptapeptide was then carried out. In our
strategy, 3a and 6 served as the precursors for glycosylation
donors (3b and 7) as well as acceptors. Specifically, cleavage of
Fmoc group in disaccharide 3a and tetrasaccharide 6 was
achieved by using 20% piperidine in THF to afford the
corresponding acceptors 5 and
8 in 80% or 84% yield,
respectively. Removal of allyl glycoside in 3a followed by N-
phenyltrifluoroacetimidation gave 3b in 93% yield for 3 steps
(Scheme 1). Glycosylation of 3b and 5 was then carried out by
using TMSOTf as a catalyst at -20 ˚C to give the tetrasaccharide
6 in good yield. Trace amount of the byproducts, such as the
OTMS replacement of OH group in 5 induced by TMSOTf, could
be observed in the reaction system. The high stability of glycosyl
donor 3b during the storage was another advantage of this
synthetic pathway (stable for 3 months under -20°C). The
tetrasaccharide donor 7 and acceptor 8 were then synthesized
from 6. Glycosylation of 7 with 8 also proceeded successfully to
give octasaccharide 9 in 78% yield. The yield was slightly higher
than the glycosylation by using the corresponding benzylidene
Experimental Section
The synthetic procedures and characterization of the compounds are
described in the Supporting Information.
Acknowledgements
protected
tetrasaccharide
trichloacetimidate
(70%).[15]
Considering the stability during the storage and reproducibility of
the glycosylation, the present results showed the versatility of N-
phenyltrifluoroacetimidates for fragment condensation of longer
glycans.
After cleavage of the Troc group with Zn–Cu in AcOH and
subsequent acetylation with Ac2O, the cleavage of ethyl ester
The authors wish to thank Dr. Stéphane Mesnage for his
valuable suggestions on the structures of Enterococcus faecalis
peptidoglycan. This work was supported in part by Grants-in-Aid
for Scientific Research (Nos. JP26102732, JP26282211, and
JP16H01162) from the Japan Society for the Promotion of
Science (JSPS). We also received support from a funding
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Entry for the Table of Contents
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Ning Wang, Hiroki Hasegawa, Cheng-
yuan Huang, Koichi Fukase,* Yukari
Fujimoto*
Page No. – Page No.
Synthesis of peptidoglycan
fragments from Enterococcus
faecalis with Fmoc-strategy for
glycan elongation
The peptidoglycan fragments, octasaccahride heptapeptide, from Enterococcus
faecalis were synthesized with using the Fmoc-strategy for long glycan elongation.
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