Synthetic study of peptidoglycan partial structures. Synthesis of tetrasaccharide and octasaccharide fragments

Article abstract of DOI:10.1016/S0040-4039(01)01619-7

Partial structures of peptidoglycan, a potent immunostimulating glycoconjugate of bacteria, were synthesized for precise biological studies. A key disaccharide glucosaminyl-β(1-4)-muramic acid was prepared by stereoselective glycosylation of a N-Troc (Tro

Full text of DOI:10.1016/S0040-4039(01)01619-7

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 7613–7616
Synthetic study of peptidoglycan partial structures. Synthesis of
tetrasaccharide and octasaccharide fragments
Seiichi Inamura, Koichi Fukase* and Shoichi Kusumoto
Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka,
Osaka 560-0043, Japan
Received 21 August 2001; revised 31 August 2001; accepted 3 September 2001
Abstract—Partial structures of peptidoglycan, a potent immunostimulating glycoconjugate of bacteria, were synthesized for
precise biological studies. A key disaccharide glucosaminyl-b(1–4)-muramic acid was prepared by stereoselective glycosylation of
a N-Troc (Troc=2,2,2-trichloroethoxycarbonyl) muramic acid acceptor with a N-Troc-glucosaminyl trichloroacetimidate. The
disaccharide was converted to either a disaccharide acceptor or a donor. They were then coupled together by the same
glycosylation method to give a tetrasaccharide in a good yield. Octasaccharide was also obtained in a good yield in a similar
manner. N-Acetylation and coupling with the dipeptide moiety of L-alanyl-D-isoglutamine followed by deprotection afforded the
repeating peptidoglycan tetrasaccharide and octasaccahride peptide conjugates for the first time. © 2001 Elsevier Science Ltd. All
rights reserved.
Bacterial cell wall peptidoglycan (PGN) has been well-
known as a strong immunopotentiator.1 PGN induces
many kinds of mediators such as cytokines,
prostaglandins, platelet activating factor, and NO,
which stimulate the immune system and also cause
clinical manifestations of bacterial infections such as
fever, inflammation, and hypotension. Recent studies
have revealed the recognition mechanisms of PGN and
other bacterial immunopotentiator by immunocom-
petent cells. Membrane CD14 (mCD14), a glyco-
sylphosphatidylinositol-anchored protein expressed on
macrophage/monocyte, binds PGN and other microbial
products, e.g. lipopolysaccharide (LPS) of gram-nega-
tive bacteria.2,3 TLR4 (toll-like receptor 4) proved to
mediate cellular activation by LPS4,5 whereas TLR2
was shown to be the receptor for PGN,5–7 lipoproteins,8
and lipoteichoic acids.6
timulation is N-acetylmuramyl-L-alanyl-D-isoglutamine
(muramyl dipeptide: MDP) (1)9–11 (Fig. 1). We then
prepared a number of derivatives and structural
analogs of MDP to study their biological activities.
Since endopeptidase digests of PGN have been shown
to be more active than MDP and digests by glycosi-
dases,12 we synthesized two alternating disaccharide
dipeptides corresponding to the repeating units of PGN
but their biological activities were almost identical with
MDP.13 MDP was shown to inhibit the binding of
soluble PGN (sPGN) to human monocytes2 but a
recent study indicated MDP does not inhibit the bind-
ing potency of soluble CD14 to sPGN–agarose.3 These
results suggest that the binding of MDP to CD14 is
considerably weaker than that of PGN. In spite of their
biological significance, larger but defined partial struc-
tures of PGN were, however, not available even after
our above work. We therefore, undertook synthetic
approach to PGN fragments in order to clarify the role
of chain length in the binding with CD14 and TLR2. In
this paper, we describe a new synthetic route of tetra-
saccharide and octasaccharide fragments of PGN.
PGN consists of polysaccharide chains linked to a
peptide network to form a three-dimensional rigid
structure. The former is b(1“4)glycan composed of
alternating N-acetylglucosamine (GlcNAc) and N-
acetylmuramic acid (MurAc) whose carboxy group is
the point of linkage to the peptide. Two research
groups including ourselves independently demonstrated
that the minimum structure required for the immunos-
HO
O
HO
O
OH
AcHN
L-Ala D-isoGln
H3C CH CO
* Corresponding author.
Figure 1. MDP: muramyl dipeptide (1).
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01619-7

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S. Inamura et al. / Tetrahedron Letters 42 (2001) 7613–7616
The first key for the synthesis was stereoselective con-
struction of b(1–4) glycosidic linkage, in particular,
glycosylation at the hindered 4-hydroxy group of a
muramic acid (Mur) residue. In fact, glycosylation at
this position did not proceed at all when an oxazoline
derivative was used as a glycosyl donor in our previous
work.14 We employed the N-Troc-glucosaminyl
trichloroacetimidate which proved to be an effective
glycosyl donor for the formation of b-glucosaminyl
linkages in our synthesis of lipid A derivatives.15,16 The
second key was the regioselective ring opening of 4,6-
O-benzylidene of GlcN and Mur residues to form
6-O-benzyl derivatives by the combined used of
BH3·Me3N with BF3·Et2O.17 With these two key reac-
tions, we planned a simple strategy to construct the
repeating glycan chain as follows. A key disaccharide
b(1–4)GlcN-Mur was prepared as a common synthetic
intermediate, which was separately converted to either
a disaccharide acceptor or a donor. A tetrasaccharide
was obtained by the coupling of these disaccharide
components. An octasaccharide was synthesized from
the tetrasaccharide in a similar manner. Introduction of
TrocCl was then added to the reaction mixture to give
8. The undesired cyclic lactam was formed when
ammonium formate or dimedone was used as an addi-
tive. Regioselective reductive ring opening of the 4,6-O-
benzylidene of 8 was carried out by using BH3·Me3N
and BF3·Et2O in CH3CN to afford the glycosyl accep-
tor 10 with a free 4-hydroxy group in 67% yield.
N-Troc-glucosaminyl donor 15 was prepared from 3-O-
benzyl-4,6-O-benzylidene allyl glycoside 1320 via cleav-
age of the allyl glycoside and subsequent conversion to
the trichloroacetimidate. Glycosylation reaction of 10
with 15 (1.2 equiv. to 10) proceeded smoothly by using
TMSOTf (0.1 equiv.) as a catalyst at −15°C in the
,
presence of MS 4 A in CH2Cl2 under N2 to afford the
key disaccharide 16 in 98% yield. The corresponding
ethyl ester derivatives 7, 9, 11, 17 were also prepared in
similar manners (Scheme 1).
Tetrasaccharide 24 and octasaccharide 29 were then
synthesized from 16 as shown in Scheme 2. The allyl
glycoside in 16 was cleaved and the product with the
free 1-hydroxy group was converted to glycosyl
trichloroacetimidate 22. Regioselective ring opening of
the 4%,6%-O-benzylidene group in 16 with BH3·Me3N
and BF3·Et2O afforded the disaccharide acceptor 18 in
63% yield. Glycosylation of 18 with 22 (1.5 equiv. to
18) was carried out in a manner similar to the synthesis
of 16 by using TMSOTf as a catalyst. The tetra-
saccharide 24 was thus obtained in 55% yield. For the
synthesis of octasaccharide 29, tetrasaccharide ethyl
ester 25 was synthesized in a similar manner. Both
tetrasaccharide donor 28 and acceptor 26 were derived
from 25 in a manner similar to the synthesis of 22 and
18. Glycosyl coupling of 26 with 28 was then
attempted. In this case, 1.5 equiv. of donor 28 was used
against acceptor 26. The desired glycosylation was pro-
ceeded smoothly at −15°C to give octasaccharide 29 in
70% yield, which was a satisfactorily high yield for a
glycosylation reaction between large segments at a hin-
dered 4-hydroxy group.
the dipeptide moiety of
L-alanyl-D-isoglutamine fol-
lowed by deprotection afforded the alternating peptido-
glycan tetrasaccharide and octasaccharide with peptide
moieties.
The key disaccharide 16 was synthesized as shown in
Scheme 1. A new N-Troc-muramic acid acceptor 10
was prepared via a new effective route. N-Alloc-GlcN
allyl glycoside (alloc=allyloxycarbonyl)
5
was
employed as a precursor for the Mur residue, since the
alloc group is stable under the basic conditions for
introduction of the lactic acid moiety. Treatment of 5
with NaH followed by addition of trifluoromethanesul-
fonyl-L-(S)-2-propionic acid benzyl ester afforded Mur
derivative 6 in a good yield.18 The alloc group of 6 was
then replaced with the Troc group, since isomerization
of the 1-O-allyl group to 1-O-2-propenyl group did not
proceed at all by an Ir complex19 in the presence of the
N-alloc function. Deprotection of the N-alloc group
was carried out by treatment with Pd (PPh3)4 (0.3
equiv.) in the presence of acetic acid as an additive.
Condensation of the dipeptide with the glycan part 24
and 29 was then investigated (Scheme 3). Using Zn–Cu
BnO
HO
O
O
Ph
Ph
O
HO
HO
HO
O
Ph
O
O
O
a, b, c
e
f
O
d
O
O
O
O
O
O
HO
AllocHN
5, 57% (for three steps)
OH
NH2•HCl
AllocHN OAllyl
CH3 CH CO2R
TrocHN OAllyl
TrocHN OAllyl
OAllyl
CH3 CH CO2R
CH3 CH CO2R
4
10: R = Bn, 58%
11: R = Et, 87%
6: R = Bn, 86%
7: R = Et, 98%
8: R = Bn, 91%
9: R = Et, 58%
Ph
Ph
O
Ph
O
O
O
BnO
TrocHN
14: R =H, 99%
15: R = C(=NH)CCl3, quant.
O
g
h
O
O
O
O
BnO
HO
OR
j
TrocHN OAllyl
TrocHN OAllyl
Ph
12
13, 87%
O
BnO
O
O
i
O
O
O
16: R = Bn, 98%
17: R = Et, 88%
BnO
TrocHN
TrocHN OAllyl
H3C CH CO2R
Scheme 1. Synthesis of a key disaccharide: (a) allocCl, NaHCO3, H2O; (b) AllylOH, DOWEX 50 W X8 200–400 mesh, 80°C; (c)
PhCH(OMe)2, p-TsOH; (d) NaH then trifluoromethanesulfonyl- -(S)-2-propionic acid benzyl ester; (e) Pd(PPh3)4, AcOH,
CH2Cl2, then TrocCl; (f) Me3N·BH3, BF3·Et2O, CH3CN; (g) BnBr, AgO, CH2Cl2; (h) Ir complex, H2, THF, then I2, H2O; (i)
L
,
CCl3CN, Cs2CO3, CH2Cl2; (j) TMSOTf (0.1 equiv.), MS 4 A, CH2Cl2, −15°C.

S. Inamura et al. / Tetrahedron Letters 42 (2001) 7613–7616
7615
Ph
BnO
HO
BnO
Ph
O
BnO
O
BnO
O
O
BnO
O
O
O
O
O
O
a
NH
b
O
O
c
O
O
16: R = Bn
17: R = Et
O
O
BnO
BnO
OCCCl3
OH
TrocHN
TrocHN
OAllyl
H3C CH CO2R
TrocHN
TrocHN
TrocHN
TrocHN
H3C CH CO2R
H3C CH CO2R
20: R = Bn, 95%
21: R = Et, 91%
18: R = Bn, 63%
19: R = Et, 52%
22: R = Bn, 92%
23: R = Et, 85%
d
BnO
O
BnO
BnO
BnO
Ph
O
O
O
O
O
O
O
O
O
O
BnO
TrocHN
TrocHN
OAllyl
TrocHN
TrocHN
H3C CH CO2R
H3C CH CO2R
24: R = Bn, 55%
25: R = Et, 79%
a
b
25
BnO
O
BnO
O
BnO
BnO
O
O
O
BnO
HO
BnO
BnO
BnO
BnO
Ph
O
O
O
O
O
O
O
O
O
O
BnO
O
O
O
O
BnO
O
OR
TrocHN
TrocHN
TrocHN
TrocHN
TrocHN
TrocHN
TrocHN
TrocHNOAllyl
H3C CH CO2Et
H3C CH CO2Et
H3C CH CO2Et
H3C CH CO2Et
d
27: R =H, 81%
c
26, 60%
28: R = C(=NH)CCl3, 83%
BnO
BnO
BnO
BnO
BnO
BnO
BnO
O
O
Ph
O
O
BnO
O
O
O
O
O
O
O
O
O
O
BnO
O
BnO
O
BnO
O
O
O
O
O
TrocHN
H3C
TrocHN
CH CO2Et
TrocHN
H3C
TrocHN
CH CO2Et
TrocHN
TrocHN
TrocHN
TrocHN OAllyl
H3C
CH CO2Et
H3C
CH CO2Et
29, 70%
Scheme 2. Synthesis of tetra-, octasaccharide: (a) Me3N·BH3, BF3·Et2O, CH3CN; (b) Ir complex, H2, THF, then I2, H2O; (c)
,
CCl3CN, Cs2CO3, CH2Cl2; (d) TMSOTf (0.1 equiv.), MS 4 A, CH2Cl2, −15°C.
Ph
O
Ph
O
BnO
BnO
O
BnO
BnO
BnO
O
BnO
BnO
O
O
O
O
O
O
O
O
O
O
a
c
O
O
O
O
BnO
TrocHN
O
O
BnO
O
O
BnO
n
AcHN
AcHN
AcHN
AcHNOAllyl
TrocHN
H3C CH CO2R
TrocHN
TrocHN
H3C CH CO2R
OAllyl
n
H3C CH CO2R
H3C CH CO2R
24: n = 1, R = Bn
29: n = 3, R = Et
30: n = 1, R = Bn, 86%
32: n = 1, R = H, 73%
31: n = 3, R = Et, 82%
33: n = 3, R = H, 97%
b
b
O
Ph
BnO
O
O
BnO
O
BnO
HO
HO
HO
HO
HO
O
HO
O
O
O
O
O
O
O
e
O
O
O
O
O
O
BnO
BnO
OR
O
O
HO
OR
AcHN
AcHN
AcHN
AcHN
n
AcHN
AcHN
AcHN
n
AcHN
L-Ala D-GluNH2
H3C CH CO
L-Ala
H3C CH CO
D-GluNH2
OBn
H3C CH
L-Ala D-isoGln
CO
H3C CH CO L-Ala D-isoGln
OBn
34: n = 1, R = α-Allyl, 86%
35: n = 1, R = H, 70%
2: n = 1, R = H, 70%
3: n = 3, R = α-Propyl, 48%
d
36: n = 3, R = α-Allyl, 31%
Scheme 3. Synthesis of tetra-, octasaccharide dipeptide: (a) Zn–Cu, AcOH, then Ac2O, Py; (b) for 32: 1 M NaOMe, THF, H2O;
for 33: LiOH, dioxane, THF, H2O; (c) HCl·H- -Ala- -Glu(OBn)-NH2, WSCI·HCl, HOBt, TEA, CH2Cl2; (d) Ir complex, H2,
L
D
THF, then I2, H2O; (e) Pd(OH)2, AcOH, H2 (10 atm).
of the 1-O-allyl group of 34 by treatment with an Ir
, all the benzyl and benzylidene groups
couple in AcOH effected deprotection of the four N-
Troc groups in 24. Free amino groups were then acetyl-
ated with Ac2O and pyridine. Saponification of the two
benzyl esters in the resulting tetrasaccharide 30 gave 32,
which was isolated in 73% yield by adsorption chro-
matography using a Diaion HP-20 column followed by
purification by silica-gel column chromatography. Con-
complex and I2
were removed by catalytic hydrogenation with Pd(OH)2
and H2. Purification with a HP 20 column (elution with
H2O) followed by gel-permeation chromatography
using Sephadex LH-20 (elution with H2O) afforded
tetrasaccharide dipeptide 2 in 70% yield. ESI-MS (nega-
tive mode, m/z=685.3 [M−2H]2−, 1371.8 [M−H]−) and
NMR provided evidences for the structure 2. The cor-
rect anomeric configurations of the protected precursor
34 were confirmed by the chemical shift values of the
1H NMR signals of each anomeric protons (H-1 at
densation of 32 with H-L-Ala-D-Glu(OBn)-NH2 was
effected by using 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide (WSCI), 1-hydroxybenzotriazole (HOBt),
and triethylamine to give protected tetrasaccharide
dipeptide conjugate 34 in 86% yield. After deprotection

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S. Inamura et al. / Tetrahedron Letters 42 (2001) 7613–7616
l=4.83 and H-1%, 1%%, 1%%% at 4.45–4.30), which corre-
spond to a and b anomers, respectively.
Golenbock, D. T. J. Clinic. Invest. 2000, 105, 497–504 and
references cited therein.
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H.; Ogawa, T.; Takeda, K.; Akira, S. Immunity 1999, 11,
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Protected octasaccharide dipeptide 36 was prepared in
a similar manner. Since the anomeric configuration at
the terminal 1-position in long glycan chain was not
expected to influence the biological activity, catalytic
hydrogenation of 36 was carried out without cleavage
of the 1-O-allyl group to give octasaccharide dipeptide
conjugate 3 in the form of a-propyl glycoside in 48%
yield (MALDI-MS, positive mode, m/z=2792.13[M+
Na]+).
9. Kotani, S.; Watanabe, Y.; Kinoshita, F.; Shimono, T.;
Morisaki, I.; Shiba, T.; Kusumoto, S.; Tarumi, Y.; Ike-
naka, K. Biken J. 1975, 18, 105–111.
As described tetra- and octasaccharide peptide conju-
gates were synthesized corresponding to the dimer and
tetramer of the basic structural unit of peptidoglycan,
i.e. GlcNAc-MurNAc disaccharide linked to the dipep-
10. Kusumoto, S.; Tarumi, Y.; Ikenaka, K.; Shiba, T. Bull.
Chem. Soc. Jpn. 1976, 49, 533–539.
11. Ellouz, F.; Adam, A.; Ciorbaru, R.; Lederer, E. Biochem.
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tide of
L
-alanyl-
D
-isoglutamine. The present study was
the first success for the synthesis of peptidoglycan frag-
ments composed of more than three saccharide
residues. In view of the sufficiently high yield of each
synthetic transformation, the present work provided
synthetic routes to more complex various partial struc-
tures of peptidoglycan.
12. (a) Ciorbaru, R.; Petit, J. F.; Lederer, E.; Zissman, E.;
Bona, C.; Chedid, L. Infect. Immun. 1976, 13, 1084–1090;
(b) Takada, H.; Tsujimoto, M.; Kotani, S.; Kusumoto, S.;
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Kawasaki, A.; Takada, H.; Kotani, S.; Inai, S.; Nagaki, K.;
Matsumoto, M.; Yokogawa, K.; Kawata, S.; Kusumoto,
S.; Shiba, T. Microbiol. Immunol. 1987, 31, 551–569; (d)
Yoshida, H.; Kinoshita, K.; Ashida, M. J. Biol. Chem.
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A biological study of peptidoglycan fragments 2 and 3
will be reported in due course.
13. Kusumoto, S.; Yamamoto, K.; Imoto, M.; Inage, M.;
Tsujimoto, M.; Kotani, S.; Shiba, T. Bull. Chem. Soc. Jpn.
1986, 59, 1411–1417.
Acknowledgements
14. Kusumoto, S.; Imoto, M.; Ogiku, T.; Shiba, T. Bull. Chem.
The present work was financially supported in part by
‘Research for the Future’ Program No. 97L00502 from
the Japan Society for the Promotion of Science.
Soc. Jpn. 1986, 59, 1419–1423.
15. Liu, W.-C.; Oikawa, M.; Fukase, K.; Suda, Y.; Kusumoto,
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