Synthesis of peptidoglycan fragments and evaluation of their biological activity

Article abstract of DOI:10.1039/b511866b

The peptidoglycan (PG) bacterial cell wall glycoconjugate has been well known as a strong immunopotentiator. Partial structures of PG were chemically synthesized for elucidation of precise biological activities. Effective construction of distinct repeatin

Full text of DOI:10.1039/b511866b

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of peptidoglycan fragments and evaluation of their
biological activity
Seiichi Inamura,a Yukari Fujimoto,a Akiko Kawasaki,a Zenyu Shiokawa,a Eva Woelk,b Holger Heine,b
Buko Lindner,c Naohiro Inohara,d Shoichi Kusumotoa and Koichi Fukase*a
Received 23rd August 2005, Accepted 15th November 2005
First published as an Advance Article on the web 6th December 2005
DOI: 10.1039/b511866b
The peptidoglycan (PG) bacterial cell wall glycoconjugate has been well known as a strong
immunopotentiator. Partial structures of PG were chemically synthesized for elucidation of precise
biological activities. Effective construction of distinct repeating glycans of PG was accomplished by the
coupling of a key disaccharide glucosaminyl-b(1–4)-muramic acid unit. Stereoselective glycosylation of
disaccharide units was achieved by neighboring group participation of the N-Troc (Troc =
2,2,2-trichloroethoxycarbonyl) group and appropriate reactivity of N-Troc-glucosaminyl
trichloroacetimidate. By using an efficient synthetic strategy, mono-, di-, tetra- and octasaccharide
fragments of PG were synthesized in high yields. The biological activity of synthetic fragments of PG
was evaluated by induction of tumor necrosis factor-a (TNF-a) from human monocytes, and toll-like
receptor 2 (TLR2) and Nod2 dependencies by using transfected HEK293 cells, respectively. Here we
reveal that TLR2 was not stimulated by the series of synthetic PG partial structures, whereas Nod2
recognizes the partial structures containing the MDP moiety.
a bacterial cell wall. The polysaccharide chain is b(1–4) glycan
Introduction
composed of alternating N-acetylglucosamine (GlcNAc) and N-
The first line of defence against microorganisms is innate immunity
acetylmuramic acid (MurNAc) of which the carboxyl group is the
with molecular recognition of common bacterial components such
point of linkage to the peptide. Two research groups, including our-
as peptidoglycan (PG), lipopolysaccharide (LPS), lipoproteins
selves, have independently demonstrated that the minimum struc-
and bacterial DNA. These components induce many kinds of
mediator such as cytokines, prostaglandins, the platelet activating
ture required for the immunostimulation is N-acetylmuramyl-L-
alanyl-D-isoglutamine (muramyl dipeptide: MDP) 1.6,7,8
factor, and NO.1,2,3,4 The mediators stimulate immune competent
cells and, consequently, the above bacterial components show
immunopotentiating activity. Peptidoglycan (PG) has been well-
known as a potent immunopotentiator5 and numerous studies
have been performed to clarify the mechanism of stimulating the
immune system. However, the immunostimulating mechanism of
PG has not been well understood. The present work aims to clarify
this mechanism by using definite PG partial structures, which are
chemically synthesized.
Recent studies revealed that toll-like receptors (TLRs) play a
crucial role in innate immunity by recognizing microbial compo-
nents and activating immune cells against microbes. Currently,
eleven TLRs have been identified.9 The different TLRs are acti-
vated by different types of microbial components. Since microbial
components isolated from natural sources are usually contami-
nated with other microbial components, their biological activities,
especially their receptors, must be verified by using synthetic
specimens. TLR4 proved to be the receptor for lipopolysaccharide
(LPS).10 TLR9 is responsible for the recognition of the CpG
sequence of bacterial DNA.11 TLR2 is implicated in immunos-
timulation by PG, lipoprotein (BLP), and lipoarabinomannan,
though only BLP proved to be the ligand of TLR2 by using
synthetic lipopeptides, partial structures of BLP.12
Muramyl dipeptide (MDP) 1
PG consists of polysaccharide chains linked to a peptide net-
work to form a three-dimensional rigid structure constructing
aDepartment of Chemistry, Graduate School of Science, Osaka University,
1-1 Machikaneyama-cho, Toyonaka, Osaka, 560-0043, Japan. E-mail:
koichi@chem.sci.osaka-u.ac.jp; Fax: +81-6-6850-5391; Tel: +81-6-6850-
5419
bDepartment of Immunology and Cell Biology, Research Center Borstel,
23845, Borstel, Germany
cDepartment of Immunochemistry and Biochemical Microbiology, Research
Center Borstel, 23845, Borstel, Germany
dDepartment of Pathology, The University of Michigan Medical School, Ann
Arbor, Michigan, 48109, USA
Recently, Boneca et al. reported that highly purified PGs are not
sensed by TLR2.13 On the other hand, Dziarski’s group recently
re-evaluated the fact that peptidoglycan is a toll-like receptor 2
activator,14 and this is still controversial.
† Present address: Suntory Institute for Bioorganic Research, Shimamoto-
cho, Mishima-gun, Osaka 618–8503, Japan, and Department of Environ-
mental and Biotechnological Frontier Engineering, Fukui University of
Technology, Fukui, Fukui 910-8505, Japan.
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Membrane CD14 (mCD14) is a glycosylphosphatidylinositol-
anchored protein expressed on macrophage/monocyte and also
plays an important role in the recognition of microbial compo-
nents. It binds PG and other microbial components such as LPS,
and then transports them to their respective receptors.15 Recent
studies, however, indicate that MDP exhibited activity in a CD14-
and TLR2-independent manner.16,17,18
In addition, the activity of MDP was found to be not identical
with that of PG. Soluble peptidoglycan (sPG) obtained from
enzyme treatment of PG exhibited stronger immunostimulating
activity than MDP.15 The degree of cross-linking of the peptide
in sPG is low and, hence, sPG has a shorter peptide chain but
still has long glycan chains. On the other hand, a glycosidase
(=muramidase) digested mixture of sPG showed lower activity.19
These studies indicated that the glycan chain length might be
important for the activity. In the present study, therefore, we
synthesized various peptidoglycan partial structures with a longer
glycan chain, i.e., tetrasaccharides having di-, tri-, tetra-, and
pentapeptide units, and octasaccharide having dipeptide units,
and evaluated their biological activities and checked CD14
and TLR2 dependency. We also synthesized disaccharide ana-
logues with newer methods. Mobashery et al. also recently
reported the synthesis of PG partial structures composed of
tetrasaccharide.20
Scheme 1 Glycosylation of N-Troc donor with N-Troc acceptors.
Results and discussion
For the effective construction of distinct repeating glycans of
PG, we applied a sequential coupling reaction of a key dis-
accharide glucosaminyl-b(1–4)-muramic acid unit 4. Stereose-
lective glycosylation of the disaccharide units was achieved by
neighboring group participation of the N-Troc (Troc = 2,2,2-
trichloroethoxycarbonyl) group and appropriate reactivity of N-
Troc-glucosaminyl trichloroacetimidate. In a previous paper, we
have reported this sequential coupling method for the synthesis
of PG fragments, tetrasaccharide and octasaccharide having
dipeptide units, T-2P2 and O-2P4.21 The key intermediate for the
synthesis of these compounds was the disaccharide 4, which was
prepared by the glycosylation of the N-Troc-muramyl acceptor
3 with the N-Troc-glucosaminyl trichloroacetimidate donor 2 in
high yield (88%)21 as shown in Scheme 1. In this reaction, the
2-N-Troc group was introduced instead of an acetyl group with
expectation of high reactivity at the 4-hydroxy group in 2-N-Troc
acceptors. Glycosylation of the N-Troc-glucosaminyl donor 2 with
a N-Troc glucosaminyl acceptor 5 gave the disaccharide 6 in 91%
yield. On the other hand, the yield of the glycosylation of N-Troc
donor 2 with the correspoding N-acetyl-muramyl acceptor 7 did
not exceed 50%. Recently, Crich et al. systematically investigated
the reactivity of the 4-hydroxy group of the GlcNAc residue,
which is less reactive than that of N,N-diacetyl glucosamine,
N-acetyl-N-benzyl glucosamine, and 2-deoxy-2-azidoglucose
derivatives owing to possible intermolecular hydrogen bond
interaction.22
Scheme 2 Synthesis of disaccharide analogues: (a) Zn–Cu, AcOH then
Ac2O, Py., 69%; (b) Ir complex, H2, THF, 99%; (c) LiOH, dioxane : THF :
H2O = 2 : 4 : 1, rt, quant; (d) H–R(–OBn) (R: peptide, see Table 1), DIPC,
HOBt, Et3N, CH2Cl2, DMF, rt, 1 d, 86%; (e) TFA (20%), CH2Cl2, 0 ◦C,
44%; (f) Pd(OH)2, H2 (20 atm), AcOH, rt, 1 d. 68%.
H2O = 2 : 4 : 1, and an appropriate peptide (see Table 1) was
subsequently introduced to the liberated carboxylic acid using
N,Nꢀ-diisopropylcarbodiimide (DIPC), 1-hydroxybenzotriazole
(HOBt), and triethylamine in CH2Cl2. The cleavage of the vinyl
group and the benzylidene group with 20% TFA in CH2Cl2, and
a benzyl group at the C-terminus of peptide with hydrogenation
using a Pd(OH)2 catalyst gave compound 11.
Tetrasaccharide 14 and octasaccharide 17 were then synthesized
from 4 (Scheme 3). Deprotection of the N-Troc group in 14
and 17, N-acetylation, saponification of ethyl esters, peptide
coupling, and then final deprotection afforded tetrasaccharide
with two dipeptide units 32 (T–2P2) and octasaccharide with four
dipeptide units 40 (O–2P4), respectively (Table 1).
We also synthesized a tetrasaccharide having tri-, tetra, and
pentapeptides 33 (T–3P2), 35 (T–4P2) and 37 (T–5P2). Conden-
sation of dicarboxylic acid 23 with appropriate peptide moieties
was effected by using 1-ethyl-3-(3-dimethylaminopropyl) carbodi-
imide hydrochloride (WSCI·HCl), HOBt, and triethylamine to
give protected tetrasaccharide containing two units of peptide
moieties 26 (78%), 27 (50%), and 28 (68%). All benzyl and
benzylidene groups of 26, 27, 28 were removed by catalytic
hydrogenation with Pd(OH)2 and H2 to give 33 (T–3P2), 35 (T–
4P2), 37 (T–5P2), respectively. The free amino group of the Lys
residue was then acetylated by treatment with Ac2O. Purification
with HP-20 column chromatography (elution with H2O) and
As shown in Scheme 2, disaccharide analogues were synthesized
via the key intermediate 4. After cleavage of the Troc group
with Zn–Cu in AcOH and subsequent acetylation with Ac2O,
isomerization of the allyl group to a vinyl group was performed
with H2-activated [Ir(cod)(MePh2P)2]PF6 to give compound 9.
The ethyl ester of 9 was cleaved with LiOH in dioxane : THF :
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Scheme 3 Synthesis of hexadecasaccharide: (a) Ir complex, H2, THF, then I2, H2O; (b) CCl3CN, Cs2CO3, CH2Cl2; (c) Me3N·BH3, BF3·Et2O, CH3CN;
(d) TMSOTf (0.1 eq), MS 4A, CH2Cl2, −15 ◦C.
Scheme 4 Synthesis of tetrasaccharide and octasaccharide peptides: (a) Zn–Cu, AcOH then Ac2O, Py; (b) LiOH, Dioxane, THF, H2O; (c) appropriate
peptide HCl salt, WSCI·HCl, HOBt, TEA, CH2Cl2; (d) Ir complex, H2, THF, then I2, H2O; (e) Pd(OH)2, AcOH, H2 (10 kg cm−2); (f) Ac2O,TEA, MeOH.
gel-permeation chromatography using Sephadex LH-20 (elution
with H2O/methanol) afforded a tetrasaccharide with two peptide
moieties 34 (T–3P2A), 36 (T–4P2A), 38 (T–5P2A). We also syn-
thesized glycan chains without peptide moieties. Tetrasaccharide
39 (T) and octasaccharide 41 (O) were synthesized by catalytic
hydrogenolysis of 23 and 24, respectively.
We further attempted the synthesis of hexadecasaccharide
having eight dipeptide units from octasaccharide 17. The allyl
glycoside in 17 was cleaved and the product was converted
to glycosyl trichloroacetimidate 18. Regioselective-ring opening
of the 4ꢀ,6ꢀ-O-benzylidene group in 17 with BH3·Me3N and
BF3·Et2O afforded the octasaccharide acceptor 19. Glycosylation
of 19 with 18 afforded the hexadecasaccharide 20. Unfortunately,
deprotection of sixteen Troc groups in 20 by Zn–Cu in AcOH
was not successful, giving a complex mixture of incompletely de-
protected products having a dichloroethoxycarbonyl group. Other
synthetic strategy and deprotection methods for the synthesis of
the hexadecasaccharide having eight dipeptide units are now under
investigation in our laboratory.
Biological activities of synthetic peptidoglycan fragments, i.e.,
tetrasaccharide with two dipeptide units 32 (T–2P2), octasaccha-
ride with four dipeptide units 40 (O–2P4), and the corresponding
tetra- and octasaccharide (T and O) devoid of peptide chains,
were evaluated by measuring cytokine induction from human
mononuclear cells (HMC). T–2P2 and O–2P4 showed potent
TNF-a inducing activity, whereas GlcNAc-MurNAc glycans T,
O without the peptides did not show this activity (Fig. 1).
Interestingly, T–2P2 showed stronger activity than octasaccharide
dipeptide O–2P4. Both compounds were more active than natural
peptidoglycan in this assay system. These results show that the
peptide parts are essential for the immunostimulating activity of
PG.
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Table 1 Synthesized peptidoglycan fragments
m
n
c
Yieldsa db,e
f
Compounds
Rꢀ
R
Abbreviations
0 (H)
0 (H)
0 (H)
1
1
1
1
1
1
1
1
1
1
1
1
3
3
—
—
—
—
25:86%
26:78%
—
27:50%
—
28:68%
—
—
29:31%
—
—
—
—
—
62%
50%
—
50%
—
50%
—
50%
48%
50%
—
—
—
—
—
—
98%
—
85%
—
1
H
H
H
H
L-Ala-D-isoGln
MDPc
M–3Pc
M–4Pc
D–3P
T–2P2
T–3P2
T–3P2A
T–4P2
T–4P2A
T–5P2
T–5P2A
T
30
31
11
32
33
34
35
36
37
38
39
40
41
L-Ala-D-isoGln L-Lys
L-Ala-D-isoGln-L-Lys-D-Ala
L-Ala-D-isoGln-L-Lys
L-Ala-D-isoGln
L-Ala-D-isoGln-L-Lys
L-Ala-D-isoGln-L-Lys(Ac)
L-Ala-D-isoGln-L-Lys D-Ala
L-Ala-D-isoGln-L-Lys(Ac) D-Ala
L-Ala-D-isoGln-L-Lys D-Ala-D-Ala
L-Ala-D-isoGln-L-Lys(Ac) D-Ala D-Ala
OH
1
1
1
1
1
1
1
1
1
1
1
H
a-Pr
a-Pr
a-Pr
a-Pr
a-Pr
a-Pr
a-Pr
a-Pr
a-Pr
85%
—
—
L-Ala-D-isoGln
OH
O–2P4
O
—
a Yields in Scheme 3. b Deprotection of allyl group was done only for tetrasaccharide dipeptide. c See reference 6.
Fig. 2 TNF-a inducing activity from HMC stimulated by different
synthetic PG fragments.
Fig. 1 TNF-a inducing activity in HMC. LPS: lipopolysaccharide, P3:
synthetic lipopeptide.
Biological activities of tetrasaccharides T–3P2A, T–4P2A, and
T–5P2A were then compared by measuring TNF-a inducing
activity from HMC. As shown in Fig. 2, tetrasaccharide including
tripeptide T–3P2A showed higher activity than the other tetrasac-
charides.
We checked the CD14 dependency by using anti-CD14 mono-
clonal antibody (mAb) MEM-18 and compound 406,15 as shown
in Fig. 3. Compound 406 is a synthetic lipid A partial structure
that shows antagonistic activity against LPS. It also inhibits the
binding of PG to CD14 and consequently suppresses the activity
of PG. As shown in Fig. 3, mAb MEM-18 and compound 406
partly inhibited the activation of synthetic PG partial structures
T–2P2 and O–2P4. The present results indicate that CD14 can
recognize the fundamental structure of PG.
Fig. 3 CD14 dependency of immunostimulation by synthetic peptidogly-
can partial structures.
the activity of PG was completely suppressed by anti-TLR2. These
synthetic PG fragments were also not active in TLR2 transfected
HEK293 cell (data not shown).
TLR2 dependency was then checked by using anti-TLR2
antibody (Fig. 4). The TNF-a inducing activity of all synthetic
PG partial structures was not inhibited by anti-TLR2, whereas
From the present study, tetra- and octasaccharide PG fragments
were excluded from the TLR2 ligand. So far, we have clarified that
partial structures having the typical peptidoglycan structure are
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might lead to the result that the sPG having longer glycan chains
have stronger activity than MDP. As in a case of human soluble
peptidoglycan recognition protein (hPGRP-S), it did not clearly
bind MDP, M–3P, M–4P and T–2P2 but T–3P2 and T–4P2, and
the binding constant of T–3P2 was approximately 70 times higher
than that of T–4P2.27
In conclusion, we have synthesized various PG fragments,
which include monosaccharide, disaccharide, tetrasaccharide and
octasaccharide with a series of peptide chains from di- to
pentapeptide. For the synthesis of a longer glycan, we developed
a sequencial glycosylation method and succeeded in extending
the glycan chain to hexadecasaccharide. With these PG partial
structures in hand, we observed the biological activity of the
compounds to estimate the recognition mechanism of PG in
the innate immune system. In the assay of immunostimulation
activity, TLR2 did not recognize the PG fragments, but Nod2
showed recognition ability for the PG fragments containing
the MDP moiety. We have also been using the PG fragments
for clarifying the recognized structure of other PG recognition
proteins such as hPGRP-S.27 It was also shown that hPGRP-L
recognizes M–3Pas aminimal structure tohydrolyze the fragments
at the linkage between muramic acid and L-Ala.28 PG, therefore,
proved to be a multifunctional immunopotentiator. Since PG and
its fragments derived from natural PG may be contaminated
with other immunostimulating components, the precise action
mechanism of PG will be solved by using structural definite
synthetic specimens.
Fig. 4 Involvement of TLR2 in TNF-a inducing activity of synthetic
Peptidoglycan partial structures in HMC.
not recognized by TLR2. We, however, think it is still possible that
natural PGs associated with a lipid moiety would be the TLR2
ligands. Mycobacterium PG (BCG-PG) is covalently bound to
mycolic acid via arabinogalactan. Gram-positive PG is physically
associated with lipoteichoic acids. In fact, the biological activity
of various 6-O-acylated MDP proved to be different from that
of MDP.23,24 Recently, 6-O-acyl-MDP derivatives were found to
activate TLR2 and TLR4 on human dendritic cells as is the case
with BCG-PG, although MDP expressed no ability to activate
TLRs.25 Synthetic studies of peptidoglycan partial structures are
still in progress for identification of the structure that is sensed
through TLR2.
Experimental
Recently, the intracellular protein Nod2 was identified as a
cellular receptor for MDP, and the above partial structures
containing tetra- and octasaccharide T–2P2 and O–2P4 also
showed Nod2 dependent immunostimulatory activity, whereas
only glycan chains T and O did not.26 In these PG fragments,
MDP showed more potent activity than T–2P2 and O–2P4. We
also observed the effect of a structural difference, that is the
peptide length connecting the monosaccharide, with the same
assay system previously reported,26 as shown in Fig. 5. MDP also
showed the most potent activity in the fragments, monosaccharide
with dipeptide (MDP), tripeptide (M–3P), and tetrapeptide (M–
4P). These results suggest that once the PG fragments enter a
cell, Nod2 recognizes PG structures containing the MDP moiety.
However, outside cells, TLR2 or other recognition proteins would
recognize rather longer or lipophilic PG fragments, and this fact
NMR spectra were measured with Varian INOVA-600, Varian
UNITY-600, JEOL JNM-LA500, and JEOL JNM-GSX 400
spectrometers. The chemical shifts in CDCl3 are given in d
values from tetramethylsilane as an internal standard. For the
measurement in D2O and CD3OD, the HDO signal (4.66 ppm
at 35 ◦C) and CD3OH (4.76 ppm) were used as a reference,
respectively. Mass spectra were obtained on a PerSeptive Biosys-
tem MarinerTM Biospectrometry Workstation or a PerSeptive
Biosystem Voyager Elite XL (MALDI-TOF-MS, Matrix: a-
Cyano-4-hydroxycinnamic acid). Specific rotations were measured
on a Perkin-Elmer 241 polarimeter. HPLC analysis was carried
out with CLASS-VP system by the SHIMADZU CORPORA-
TION. Elemental analyses were performed with a Yanaco CHN
corder MT-3, MT-5 and MT-6. Silica-gel column chromatography
was carried out using a Kieselgel 60 (Merck, 0.040–0.063 mm) at
medium pressure (2–4 kg cm−2). Dry CH2Cl2, THF, DMF, toluene,
and benzene were purchased from Kanto Chemicals, Tokyo. Dis-
tilled water used for HPLC and lyophilization was purchased from
Otsuka (Tokyo, Japan) or prepared by a combination of Tolay Pure
LV-308 (Tolay) and GSL-200 (Advantec, Tokyo, Japan). Molecu-
lar Sieves (MS) 4A was activated by heating at 250 ◦C in vacuo for
3 h. Tetrakis (triphenylphosphine) Palladium (0) was prepared just
before use. All other commercially obtained materials were used
as received. [Ir(cod)(MePh2P)2]PF6 activated with H2 was used
for isomerization of the 1-O-allyl group to the 1-O-(1-propenyl)
group as [Ir(cod)(H)(MePh2P)2]PF6. The activation was carried
out as follows. A solution of [Ir(cod)(MePh2P)2]PF6 in THF was
degassed and then the iridium complex was activated with H2 for
30 min. H2 in the reaction vessel was then replaced with N2 and
Fig. 5 Stimulation of Nod2 by PG fragments. HEK293T cells were
transfected with Nod2 (Nod2) or vector control (−), and the indicated
amount of each compound was added to the cells and the ability of each
compound to activate NF-jB was determined.
236 | Org. Biomol. Chem., 2006, 4, 232–242
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=
the solution was used for the isomerization. The lactic acid moiety
is designated Lac in the NMR measurement.
CH2–), 5.87–5.82(m, 1H, –CH2–CH CH2), 5.58(s, 1H, C6H5–
=
CH–), 5.31–5.13 (m, 3H, H1, –CH2–CH CH2), 4.89–4.82(m,
The high resolution mass spectra of the final compounds were
obtained on an Electro Spray Ionization Fourier Transform Ion
Cyclotron Resonance (ESI FT-ICR) mass spectrometer, APEX II
(Bruker Daltonics, Billerica, USA). The Instrument is equipped
with a 7 T actively shielded magnet and an Apollo ion source.
For the positive ion spectra samples (∼10 ng ll−1) the lyophilized
substances were dissolved in a 30 : 10 (v/v) mixture of water and
acetonitrile adjusted with acetic acid to pH 3.0. The samples were
sprayed at a flow rate of 2 lL min−1. The capillary entrance voltage
was set to 3.8 kV, and drying gas temperature to 150 ◦C. For
the interpretation of the plural samples, the positive or negative
ion mass spectra obtained were charge deconvoluted and mass
numbers refer to neutral molecules.
2H, C6H5–CH2–), 4.66–4.60 (m, 2H, CH3–CH2–O–CO–), 4.45–
4.34(m, 3H, C6H5–CH2–, H6ꢀ), 4.26–4.12(m, 2H, H1ꢀ, Lac-aH),
=
4.08–4.06(m, 2H, –CH2–CH CH2), 3.96–3.92 (m, 2H, –CH2–
ꢀ
ꢀ
ꢀ
=
CH CH2, H3), 3.78–3.44 (m, 9H, H2, H4, H6, H3 , H4 , H6 ),
3.32–3.29 (m, 1H, H5ꢀ), 2.01 (s, 3H, –NH–CO–CH3), 1.74 (s, 3H, –
NH–CO–CH3), 1.74–1.26 (m, 6H, Lac-CH3, CH3–CH2–O–CO–).
To a solution of the 2,2ꢀ-N,N-diacetyl-disaccharide (100 mg,
0.12 mmol) in dry THF (2.5 mL) was added H2-activated
[Ir(cod)(MePh2P)2]PF6 (3.1 mg, 3.6 × 10−3 mmol) in dry THF
(1 mL). After being stirred under an argon atmosphere at room
temperature for 7 h, the reaction mixture was quenched with sat-
urated aqueous NaHCO3 (20 mL) and the mixture was extracted
with AcOEt (30 mL × 2). The organic layer was washed with
sat. NaHCO3 aq (30 mL) and brine (30 mL), dried over Na2SO4,
and concentrated in vacuo. The residue was purified by silica-gel
flash chromatography (5 g, CHCl3 : acetone = 7 : 1) to give 9 as
a white solid (100 mg, 99%). ESI-TOF-MS (positive) m/z 832.4
[M + Na]+;1H-NMR 500 MHz, CDCl3 d = 7.53–7.14(m, 15 H,
Allyl 6-O-benzyl-4-O-(3-O-benzyl-4,6-O-benzylidene-2-deoxy-2-
(2,2,2-trichloroethoxycarbonylamino)-b-D-glucopyranosyl)-3-O-
((R)-1-(ethoxycarbonyl)ethyl)-2-deoxy-2-(2,2,2-trichloroethoxy-
carbonylamino)-a-D-glucopyranoside 4
=
C6H5–CH2–), 6.10 (d, J = 12.2 Hz, 1H, –CH CH–CH3), 5.59(s,
To a mixture of the imidate 2 (26.0 g, 38 mmol), the acceptor 3
(17.0 g, 30 mmol), and MS4A in dry CH2Cl2 (300 mL) at −15 ◦C
was added TMSOTf (340 lL, 3.0 mmol). After being stirred at
the same temperature for 10 min, the reaction was quenched with
chilled saturated aqueous NaHCO3 (30 mL), and the mixture was
extracted with CHCl3 (250 mL). The organic layer was washed
with NaHCO3 (60 mL) and brine (60 mL), dried over Na2SO4,
and concentrated in vacuo. The residue was purified by silica-gel
flash chromatography (600 g, toluene : EtOAc = 10 : 1) to give 4
as a pale yellow solid (29.0 g, 88%).
1H, C6H5–CH–), 5.51 (d, J = 1.0 Hz, 1H, H1), 5.08–5.05(m, 1H,
=
–CH CH–CH3), 4.89–4.82(dd, J = 12.6 Hz, 2H, C6H5–CH2–),
4.66–4.62 (m, 2H, CH3–CH2–O–CO–), 4.44–4.32(m, 3H, C6H5–
CH2-, H6ꢀ), 4.24–4.15(m,2H, H1ꢀ, Lac-aH), 3.96 (t, J = 9.l Hz,
1H, H3), 3.78–3.43 (m,9H, H2, H4, H5,H6, H2ꢀ, H3ꢀ, H4ꢀ, H6ꢀ),
3.30–3.29 (m,1H, H5ꢀ), 2.00 (s, 3H, –NH–CO–CH3), 1.52 (d, J =
=
6.9 Hz, 3H, –CH CH–CH3), 1.73 (s, 3H, –NH–CO–CH3), 1.37–
1.26 (m, 6H, Lac-CH3, CH3–CH2–O–CO–).
Protected disaccharide tripeptide 10: (R=L-Ala-D-
ESI-TOF-MS (positive) m/z 1119.2 [M + Na]+; 1H NMR
isoGln-L-Lys(Z)–OBn)
(400 MHz, CDCl3) d = 7.45–7.29 (15H, m, (C6H5)–CH2–),
=
=
5.85–5.78 (1H, m, –CH2–CH CH2), 5.57 (1H, s, Ph–CH ),
To a solution of 9 (110 mg, 0.13 mmol) in dioxane : THF : H2O
(2 : 4 : 1, 4.0 mL) was added LiOH (19 mg, 0.79 mmol) and
stirred at room temperature for 1 h. The solution was neutralized
with Dowex H+ (Dowex 50W × 8 200–400 mesh H form,
DowChemicals) and then applied to an HP-20 column (2 cm ×
20 cm). Organic and inorganic salts were removed by elution with
H2O (160 mL), then eluted with MeOH and concentrated in vacuo
to give a disaccharide with a free lactic acid moiety as a white
solid (105 mg, quant.). ESI-TOF-MS (positive) m/z 805.4 [M +
H]+;1H-NMR (500 MHz, CDCl3) d = 7.48–7.23(m, 15 H, C6H5–
=
5.28–5.13 (3H, m, H-1, –CH2–CH CH2), 4.89–4.59 (10H, m,
CCl3–CH2–OCO–, CH3–CH2–OCO–, Ph–CH2–), 4.43–4.39 (1H,
m, H-6ꢀ), 4.26–4.04 (5H, m, Lac-aH, Ph–CH2–, H-1ꢀ, –CH2–
=
=
CH CH2), 3.98–3,93 (2H, m, –CH2–CH CH2, H-3), 3.77–3.56
(6H, m, H-2, H-4, H-4ꢀ, H-6, H-6ꢀ), 3.43–3.41 (2H, m, H-2ꢀ, H-
5), 3.25–3.21 (2H, m, H-3, H-5ꢀ), 1.34–1.25 (6H, m, Lac-CH3,
CH3–CH2–OCO). Found: C, 51.42; H, 4.90; N, 2.60. Calcd for
C47H54Cl6N2O15: C, 51.33; H, 4,95; N, 2.55%.
=
CH2–), 6.13 (d, J = 10.5 Hz, 1H, –CH CH–CH3), 5.64(s, 1H,
1-Propenyl 2-acetylamino-6-O-benzyl-4-O (2-acetylamino-3-O-
benzyl-4,6-O-benzylidene-2-deoxy-b-D-glucopyranosyl-3-O-((R)-
1-(ethoxycarbonyl)ethyl)-2-deoxy-a-D-glucopyranoside 9
C6H5–CH–), 5.43 (d, J = 3.1 Hz, 1H, H1), 5.06–5.02(m, 1H,
=
–CH CH–CH3), 4.87–4.79(m, 2H, C6H5–CH2–), 4.64–4.56 (m,
3H, C6H5–CH2-, H6ꢀ, Lac-aH), 4.31–4.28(m, 1H, H1ꢀ), 4.07–3.49
(m, 11H, H2, H3, H4, H5,H6, H2ꢀ, H3ꢀ, H4ꢀ, H6ꢀ), 3.30–3.29 (m,
1H, H5ꢀ), 1.98 (s, 3H, –NH–CO–CH3), 1.87 (s, 3H, –NH-CO-
To a solution of 4 (2.1 g, 1.9 mmol) in AcOH (2.5 mL) was added
Zn–Cu (prepared from 5.6 g of Zn) and the mixture was stirred
at room temperature for 4 h. The insoluble materials were filtered
off and the filtrate was concentrated in vacuo. The residual solvent
was removed by coevaporation with toluene (10 mL) The residue
was dissolved in pyridine (40 mL) and acetic anhydride (42 mL)
and the solution was stirred at room temperature. After 1 day,
the solution was removed by concentration with toluene (10 mL).
The residue was purified by silica-gel flash chromatography (150 g,
CHCl3 : acetone = 7 : 1) to give 2,2ꢀ-N,N-diacetyl-disaccharide as
a white solid (1.1 g, 69%). ESI-TOF-MS (positive) m/z 832.4 [M +
Na]+;1H-NMR 500 MHz, CDCl3 d = 7.53–7.15(m, 15 H, C6H5–
=
CH3), 1.50 (d, J = 1.5 Hz, 3H, –CH CH-CH3), 1.38 (d, J =
6.9 Hz, 3H, Lac-CH3).
To a solution of the above disaccharide (105 mg, 0.13 mmol)
in dry DMF : CH2Cl2 (1 : 1, 4.0 mL) was added HOBt
(24 mg, 0.18 mmol) and DIPC (2.8 × 10−2 mL, 0.18 mmol) and
stirred under an argon atmosphere at room temperature for 1 h.
To a solution of HCl·H-L-Ala-D-isoGln-L-Lys(Z)–OBn (71 mg,
0.12 mmol) in dry DMF : CH2Cl2 (1 : 1, 4.0 mL) was added
Et3N (1.6 × 10−2 mL, 0.12 mmol) and the above mixture, and the
reaction mixture was stirred under an argon atmosphere at room
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temperature overnight. The reaction mixture was concentrated
in vacuo and the residue was dissolved in CHCl3 (50 mL). The
CHCl3 solution was washed with 10% aqueous citric acid (50 mL),
saturated aqueous NaHCO3 (80 mL) and brine (80 mL), dried over
Na2SO4, and concentrated in vacuo. The residue was purified by
silica-gel flash chromatography (50 g, CHCl3 : MeOH = 10 : 1) to
give 10 as a white solid (138 mg, 87%). ESI-TOF-MS (positive) m/z
1378.7 [M + Na]+;1H-NMR (500 MHz, CDCl3) d = 7.48–7.23(m,
and the reaction mixture was stirred for additional 10 min. To
the reaction mixture was rapidly added aqueous Na2S2O3 (5%,
100 ml). The mixture was then extracted with EtOAc (50 mL). The
organic layer was washed with aqueous Na2S2O3 (5%, 50 mL ×
2), aqueous sat. NaHCO3 (100 mL × 2), brine (50 mL), dried over
Na2SO4 and then concentrated in vacuo. The residue was purified
by silica-gel flash chromatography (180 g, toluene : EtOAc = 4 :
1) to give 1-liberated-disaccharide as a pale yellow solid (2.72 g,
93%).
=
25 H, C6H5–CH2–), 6.13 (d, J = 12.0 Hz, 1H, –CH CH–CH3),
5.61(s, 1H, C6H5–CH–), 5.36 (d, J = 3.5 Hz, 1H, H1), 5.22–
[a]2D3 = + 8.4 (c 1.00, CHCl3); ESI-MS (positive) m/z = 1079.0
5.09(m, 6H, –CH CH–CH3, C6H5–CH2–, C6H5–CH2–OCO–),
[M + Na]+; 1H NMR (400 MHz, CDCl3) d = 7.54–7.27 (m, 15H,
=
=
4.89–4.87(d, J = 11.5 Hz, 1H, C6H5–CH2–), 4.73–4.61 (m, 3H,
C6H5–CH2–), 4.54–4.23 (m, 5H, H6ꢀ, Lac-aH, Lys-aH, Gln-aH,
Ala-aH), 4.23–3.93(m, 2H, H1ꢀ, H3), 3.84–3.56(m,8H, H2, H4,
H5,H6, H2ꢀ, H4ꢀ, H6ꢀ), 3.40–3.31 (m, 2H, H3ꢀ, H5ꢀ), 3.18–3.07
(m,2H, Lys-eCH2), 2.42–2.28 (m, 2H, Gln-cCH2), 2.25–2.20 (m,
2H, Gln-bCH2), 1.97 (s, 3H, –NH–CO–CH3), 1.90 (s, 3H, –NH–
CO–CH3), 1.80–1.57 (m, 4H, Lys-bCH2, Lys-d CH2), 1.55–1.35
(C6H5)–CH2–), 5.60 (br.s, 1H, H-1), 5.57 (s, 1H, Ph–CH ), 4.89–
4.60 (m, 8H, CCl3–CH2–OCO–, CH3–CH2–OCO–, Ph–CH2–),
4.43–4.39 (m, 1H, H-6ꢀꢀꢀ), 4.31–3.91 (m, 4H, Ph–CH2–, H-1ꢀ, Lac-
aH), 3.95–3.91 (m, 1H, H-3), 3.82–3.63 (m, 6H, H-2, H-4, H-6,
H-4ꢀ, H-6ꢀ), 3.43–3.41 (m, 2H, H-2ꢀ, H-5), 3.22–3.21 (m, 2H, H-
3ꢀ, H-5ꢀ), 1.35–1.27 (m, 6H, Lac-Me, CH3–CH2–OCO). Found:
C, 51.68; H, 5.33; N, 2.35. Calcd for C44H50Cl6N2O15: C, 51.24; H,
5.12; N, 2.54%.
=
(m, 4H, Lys-cCH2, Ala-CH3), 1.28–1.12 (m, 6H, –CH CH–CH3,
Lac-CH3).
To a solution of 1-liberated-disaccharide (2.72 g, 2.56 mmol)
in dry CH2Cl2 (6 mL) at rt were added Cs2CO3 (417 mg,
1.28 mmol) and CCl3CN (3.7 mL, 25.6 mmol). After being stirred
for 1 h, insoluble materials were filtered off through celite and
concentrated. The residue was lyophilized from benzene to give
12 as a pale yellow solid (3.04 g), which was used for subsequent
glycosylation without purification.
Disaccharide tripeptide 11
A solution of 10 (2.8 mg, 2.1 × 10−3 mmol) in TFA : CH2Cl2 (1 :
4, 0.5 mL) was stirred at 0 ◦C for 1.5 h. The reaction mixture
was quenched with saturated aqueous NaHCO3 (20 mL) and the
mixture was extracted with AcOEt (50 mL). The organic layer was
washed with saturated aqueous NaHCO3 (20 mL) and dried over
Na2SO4, and concentrated in vacuo. The residue was purified by
silica-gel flash chromatography (5 g, CHCl3 : MeOH = 8 : 1) to give
1-liberated disaccharide as a white solid (1.1 mg, 44%). ESI-TOF-
MS (positive) m/z 1250.7 [M + Na]+;1H-NMR (500 MHz, CDCl3)
d = 7.28–7.17(m, 25 H, C6H5–CH2–), 5.17(d, J = 3.5 Hz, 1H, H1),
5.07–4.81(m, 4H, C6H5–CH2–, C6H5–CH2–OCO–), 4.56–4.42 (m,
8H, C6H5–CH2–, H6ꢀ), 4.30–4.18 (m, 5H, Lac-aH, Lys-aH, Gln-
aH, Ala-aH, H1ꢀ), 3.97–3.95(m, 1H, H3), 3.80–3.32(m,12H, H2,
H4, H5, H6, H2ꢀ, H3ꢀ, H4ꢀ, H5ꢀ, H6ꢀ,), 3.12–2.96 (t, J = 6.8 Hz,
2H, Lys-eCH2), 2.26–2.22 (m, 2H, Gln-cCH2), 2.10–2.06 (m, 2H,
Gln-bCH2), 1.85 (s, 3H, –NH–CO–CH3), 1.78 (s, 3H, –NH–CO–
CH3), 1.73–1.50 (m, 4H, Lys-bCH2, Lys-dCH2), 1.37–1.33 (m, 8H,
Lys-cCH2, Ala-CH3, Lac-CH3).
To a solution of the 1-librarated disaccharide (1.1 mg, 9.0 ×
10−4 mmol) in acetic acid (2.0 mL) was added palladium hydroxide
(2.0 mg, 1.4 × 10−2 mmol) in acetic acid (1.0 mL), and the mixture
was stirred under H2 (20 atm) at room temperature overnight.
The Pd catalyst was removed by filtration and the filtrate was
concentrated in vacuo to give 11 as a white solid (0.5 mg, 68%). ESI-
TOF-MS (positive) m/z 824.4 [M + Na]+. HRMS-ESI FT-ICR
(positive): (M) calcd for C33H57N7O17, 823.381; found, 823.393.
4ꢀ-O-Deprotected-disaccharide 13
To a solution of 4 (1.5 g, 1.36 mmol) and trimethyl◦amine-borane
(150 mg, 2.05 mmol) in dry CH3CN (13 mL) at 0 C was added
boron trifluoride diethyl etherate (960 mg, 6.80 mmol) dropwise
and the mixture was stirred at rt for 30 min. The reaction was then
quenched with ice and saturated aqueous NaHCO3 (100 mL) and
the mixture was extracted with EtOAc (100 mL × 2). The organic
layer was washed with aqueous 10% citric acid (15 mL × 4),
saturated aqueous NaHCO3 (150 mL), and brine (100 mL), dried
over Na2SO4, and concentrated in vacuo. The residue was purified
by silica-gel flash chromatography (180 g, toluene : AcOEt = 4 :
1) to give 13 as a colorless solid (1.13 g, 73%).
[a]2D3 = + 25.6 (c 1.00, CHCl3); ESI-TOF-MS (positive) m/z =
1
1121.6 [M + Na]+; H NMR (400 MHz, CDCl3) d = 7.43–7.27
=
(15H, m, (C6H5)–CH2–), 5.85–5.79 (1H, m, –CH2–CH CH2),
=
5.26–5.13 (3H, m, –CH2–CH CH2, H-1), 4.86–4.50 (9H, m,
CCl3–CH2–OCO–, CH3–CH2–OCO–, Ph–CH2–), 4.33–4.04 (5H,
m, Ph–CH2–, Lac-aH, H-1 , –CH2–CH CH2), 3.97–3.57 (10H,
m, –CH2–CH CH2, H-3, H-4, H-6, H-3 , H-4 , H-6 ), 3.46–3.36
(2H, m, H-2ꢀ, H-5), 3.27–3.23 (1H, m, H-5ꢀ), 1.29–1,20 (6H, m,
Lac-CH3, CH3–CH2–OCO). Found: C, 51.68; H, 5.33; N, 2.35.
Calcd for C47H56Cl6N2O15: C, 51.24; H, 5.12; N, 2.54%.
ꢀ
=
ꢀ
ꢀ
ꢀ
=
Disaccharide 1-O-trichloroacetimidate 12
Fully protected tetrasaccharide 14
To a degassed solution of 4 (3.0 g, 2.7 mmol) in dry THF (6 mL)
was added [Ir(cod)(MePh2P)2]PF6 (23 mg, 0.027 mmol) activated
with H2. After being stirred under a nitrogen atmosphere at room
temperature for 1 h, the solution of [Ir(cod)(MePh2P)2]PF6 (23 mg,
0.027 mmol) activated with H2 in dry THF (3 mL) was added. After
being stirred under a nitrogen atmosphere at room temperature
for 1 h, iodine (690 mg, 2.7 mmol) and water (10 mL) were added
To a mixture of the imidate 12 (2.7 g, 38 mmol), the acceptor 13
(1.65 g, 30 mmol), and MS4A in dry CH2Cl2 (75 mL) at −15 ◦C
was added TMSOTf (18 lL, 0.15 mmol). After being stirred at
the same temperature for 10 min, the reaction was quenched with
ice cold saturated aqueous NaHCO3 (100 mL), and the mixture
was extracted with CHCl3 (100 mL). The organic layer was washed
238 | Org. Biomol. Chem., 2006, 4, 232–242
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with NaHCO3 (60 mL × 2) and brine (60 mL), dried over Na2SO4,
and concentrated in vacuo. The residue was purified by silica-gel
flash chromatography (300 g, toluene : EtOAc = 6 : 1) to give 14
as a pale yellow solid (2.33 g, 79%).
The residue was purified by silica-gel flash chromatography (200 g,
toluene : AcOEt = 4 : 1) to give 16 as a colorless solid (1.4 g, 74%).
23
[a]D = − 24.3 (c 1.00, CHCl3); ESI-TOF-MS (positive) m/z
1
2161.4 [M + Na]+; H NMR (400 MHz, CDCl3) d = 7.43–7.28
23
=
[a]D = − 1.7 (c 1.00, CHCl3); ESI-TOF-MS (positive) m/z =
(30H, m, (C6H5)–CH2–), 5.86–5.80 (1H, m, –CH2–CH CH2),
1
2159.0 [M + Na]+; H NMR (400 MHz, CDCl3) d = 7.51–7.27
5.28–5.11 (1H, m, –CH2–CH CH2), 5.57 (1H, d, J = 12,2 Hz, Ph–
=
=
(30H, m, (C6H5)–CH2–), 5.86–5.76 (1H, m, –CH2–CH CH2),
CH2–), 4.88–4.50 (19H, m, CCl3–CH2–OCO–, CH3–CH2–OCO–,
Ph–CH2–), 4.45–3.86 (14H, m, Ph–CH2–, CCl3–CH2–OCO–, H-
1, H-1 , H-1 , H-1 , Lac-aH, –CH2–CH CH2), 3.83–2.84 (24H,
m, H-2, H-3, H-4, H-5, H-6, for each four pyranose), 1.31–1,24
(12H, m, Lac-Me, CH3–CH2–OCO).
=
=
5.56 (1H, s, Ph–CH ), 5.25–4.98 (4H, m, –CH2–CH CH2, Ph–
CH2–, H-1), 4.88–4.33 (17H, m, CCl3–CH2–OCO–, CH3–CH2–
OCO–, Ph–CH2–), 4.32–3.90 (12H, m, Ph–CH2–, Lac-aH, H-1ꢀꢀ,
ꢀ
ꢀꢀ
ꢀꢀꢀ
=
ꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
=
H-1 , H-1 , H-6 , –CH2–CH CH2), 3.87–3.01 (24H, m, Ph–
CH2–, H-2, H-3, H-4, H-5, H-6, H-2ꢀ, H-3ꢀ, H-4ꢀ, H-5ꢀ, H-6ꢀ,
H-2ꢀꢀ, H-3ꢀꢀ, H-4ꢀꢀ, H-5ꢀꢀ, H-6ꢀꢀ, H-2ꢀꢀꢀ, H-3ꢀꢀꢀ, H-4ꢀꢀꢀ, H-5ꢀꢀꢀ, H-6ꢀꢀꢀ),
1.32–1,27 (12H, m, Lac-CH3, CH3–CH2–OCO).
Fully-protected octasachharide 17
To a mixture of the imidate 15 (3.8 g, 1.7 mmol), the acceptor 16
(2.4 g, 1.1 mmol), and MS4A in dry CH2Cl2 (112 mL) at −15 ◦C
was added TMSOTf (13 lL, 0.11 mmol). After being stirred at
the same temperature for 10 min, the reaction was quenched with
chilled saturated aqueous NaHCO3 (100 mL), and CHCl3 (50 mL)
was added. The organic layer was washed with NaHCO3 (60 mL ×
2) and brine (60 mL), dried over Na2SO4, and concentrated in
vacuo. The residue was purified by silica-gel flash chromatography
(300 g, toluene : EtOAc = 5 : 1) to give 17 as a pale yellow solid
Tetrasaccharide 1-O-trichloroacetimidate 15
To a degassed solution of 14 (5.4 g, 2.5 mmol) in dry THF
(50 mL) was added H2 activated (30 min) [Ir(cod)(MePh2P)2]PF6
(100 mg, 0.12 mmol) in dry THF (3 mL). After being stirred
under a nitrogen atmosphere at room temperature for 1 h, iodine
(1.26 g, 5.0 mmol) and water (10 mL) were added and the reaction
mixture was stirred for additional 10 min. To the reaction mixture
was added rapidly aqueous Na2S2O3 (5%, 150 ml). The mixture
was then extracted with EtOAc (100 mL). The organic layer was
washed with aqueous Na2S2O3 (5%, 100 mL × 2), aqueous sat.
NaHCO3 (100 mL × 2) and brine (50 mL) then dried over Na2SO4
and concentrated in vacuo. The residue was purified by silica-gel
flash chromatography (300 g, toluene : EtOAc = 4 : 1) to give
1-liberated tetrasaccharide as a pale yellow solid (4.3 g, 81%).
(3.2 g, 70%).
23
[a]
= − 20.2 (c 1.00, CHCl3); MALDI-TOF-MS (positive)
D
m/z = 4253.42 [M + Na]+; Found: C, 50.68; H, 4.96; N, 2.70.
Calcd for C179H204Cl24N8O57·1H2O: C, 50.60; H, 4.81; N, 2.63%.
Octasaccharide 1-O-trichloroacetoimidate 18
To a degassed solution of 17 (300 mg, 0.071 mmol) in dry THF
(2 mL) was added [Ir(cod)(MePh2P)2]PF6 (6 mg, 0.007 mmol)
activated with H2 in THF (3 mL). After being stirred under
nitrogen atmosphere at room temperature for 1 h, iodine (35 mg,
0.142 mmol) and water (0.5 mL) were added and the reaction
mixture was stirred for additional 10 min. To the reaction mixture
was added rapidly aqueous Na2S2O3 (5%, 10 ml). The mixture
was then extracted with EtOAc (20 mL). The organic layer was
washed with aqueous Na2S2O3 (5%, 10 mL × 2), aqueous sat.
NaHCO3 (50 mL × 2), and brine (20 mL, dried over Na2SO4,
and then concentrated in vacuo. The residue was purified by silica-
gel flash chromatography (20 g, toluene : EtOAc = 5 : 1) to give
1-liberated-octasaccharide as a pale yellow solid (260 mg, 88%).
MALDI-TOF-MS (positive) m/z = 4213.8 [M + Na]+.
23
[a]D = − 11.0 (c 1.00, CHCl3); ESI-TOF-MS (positive) m/z =
1
2119.7 [M + Na]+; H NMR (400 MHz, CDCl3) d = 7.62–7.27
(30H, m, (C6H5)–CH2–), 5.62 (1H, br.s, H-1), 5.56 (1H, s, Ph–
=
CH ), 5.11 (1H, d, J = 12,2 Hz, Ph–CH2–), 4.87–4.46 (19H,
m, CCl3–CH2–OCO–, CH3–CH2–OCO–, Ph–CH2–, H-1ꢀꢀ, H-6ꢀꢀꢀ),
4.31–3.12 (7H, m, Ph–CH2–, H-1ꢀ, H-1ꢀꢀꢀ, Lac-aH), 3.96–3.44
(19H, m, Ph–CH2–, H-2, H-3, H-4, H-5, H-6, H-2ꢀ, H-4ꢀ, H-
6ꢀ, H-3ꢀꢀ, H-4ꢀꢀ, H-5ꢀꢀ, H-6ꢀꢀ, H-2ꢀꢀꢀ, H-4ꢀꢀꢀ, H-6ꢀꢀꢀ), 3.36–3.18 (5H,
m, H-3ꢀ, H-5ꢀ, H-2ꢀꢀ, H-3ꢀꢀꢀ, H-5ꢀꢀꢀ). 1.36–1,25 (12H, m, Lac-CH3,
CH3–CH2–OCO).
To a solution of 1-liberated tetrasaccharide (4.3 g, 2.0 mmol) in
dry CH2Cl2 (20 mL) at rt were added Cs2CO3 (325 mg, 1.0 mmol)
and CCl3CN (2.0 mL, 20.0 mmol). After being stirred for 1 h,
insoluble materials were filtered off through celite and the filtrate
was concentrated. The residue was lyophilized from benzene to
give 15 as a pale yellow solid (3.8 g), which was used for subsequent
glycosylation without purification.
To
a solution of 1-liberated-octasaccharide (260 mg,
0.062 mmol) in dry CH2Cl2 (1.2 mL) at rt were added Cs2CO3
(4 mg, 0.62 mmol) and CCl3CN (62 lL, 0.62 mmol). After being
stirred for 1 h, insoluble materials were filtered off through celite
and the filtrate was concentrated. The residue was lyophilized from
benzene to give 18 as a pale yellow solid (240 mg).
4ꢀꢀꢀ-O-Deprotected-tetrasaccharide 16
To a solution of 14 (1.9 g, 0.89 mmol) and trimethylamine-borane
(128 mg, 1.77 mmol) in dry CH3CN (36 mL) at rt was added
boron trifluoride diethyl etherate (461 lL, 3.56 mmol) diluted
with CH3CN (1.4 mL) dropwise and the mixture was stirred at rt
for 1 h. The reaction was then quenched with saturated aqueous
NaHCO3 (50 mL) and the mixture was extracted with EtOAc
(100 mL). The organic layer was washed with aqueous 10% citric
acid (150 mL × 4), saturated aqueous NaHCO3 (150 mL), and
brine (100 mL), dried over Na2SO4, and concentrated in vacuo.
4-O-Deprotected octasaccharide 19
To a solution of 17 (100 mg, 0.023 mmol) and trimethylamine-
borane (5.4 mg, 0.026 mmol) in dry CH3CN (4.8 mL) at 0 ◦C
was added boron trifluoride diethyl etherate (9.6 lL, 0.071 mmol)
in dry CH3CN (28 lL) dropwise and the mixture was stirred
at rt for 1 h. The reaction was then quenched with saturated
aqueous NaHCO3 (5 mL) and the mixture was extracted with
EtOAc (10 mL). The organic layer was washed with aqueous 10%
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citric acid (15 mL × 4), saturated aqueous NaHCO3 (15 mL),
and brine (10 mL), dried over Na2SO4, and concentrated in vacuo.
The residue was purified by silica-gel flash chromatography (10 g,
toluene : AcOEt = 4 : 1) to give 19 as a colorless solid (85 mg,
85%).
were removed by elution with H2O (300 mL), then eluted with
MeOH to give 23 as a white solid (170 mg, quant.).
ESI-TOF-MS (positive) m/z = 1575.1 [M + Na]+.
Octasaccharide with liberated carboxylic acid 24
MALDI-TOF-MS (positive) m/z = 4255.00 [M + Na]+.
To a solution of 22 (28 mg, 9.0 lmol) in dioxane : THF : H2O (2 :
4 : 1, 4 mL) was added LiOH (3 mg, 0.073 mmol) and stirred at
rt for 1 h. The solution was neutralized with Dowex H+(Dowex
50W-X8 200–400 mesh H form, DowChemicals) and then applied
to HP-20 column (2 cm × 40 cm). Organic and inorganic salts were
removed by elution with H2O (300 mL), then eluted with MeOH
to give 24 as a white solid (28 mg, quant.).
Hexadecasaccharide 20
To a mixture of the imidate 18 (85 mg, 0.02 mmol), the acceptor 19
(240 mg, 0.05 mmol), and MS4A in dry CH2Cl2 (3 mL) at −15 ◦C
was added TMSOTf (0.4 mg, 2.0 lmol). After being stirred at the
same temperature for 10 min, the reaction was quenched with ice
cold saturated aqueous NaHCO3 (5 mL), and the mixture was
added with CHCl3 (5 mL). The organic layer was washed with
NaHCO3 (5 mL × 2) and brine (5 mL), dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica-gel flash
chromatography (80 g, toluene : EtOAc = 4 : 1) to give 20 as a
pale yellow solid (87 mg, 56%).
MALDI-TOF-MS (positive) m/z = 3115.26 [M + Na]+.
Protected tetrasaccharide dipeptide 25
To a solution of 23 (122 mg, 0.078 mmol, HCl·H-L-Ala-D-
Glu(OBn)–NH2 (83 mg, 0.24 mmol), and HOBt (33.5 mg,
0.25 mmol) in CH2Cl2(14 mL) were added WSCI·HCl (37 mg,
0.25 mmol) and triethylamine (48 lL, 0.47 mmol) at 0 ◦C and the
mixture was stirred at rt overnight. The mixture was diluted with
AcOEt and insoluble materials were filtered off. The filtrate was
concentrated and the residue was dissolved in CHCl3. The CHCl3
solution was washed with citric acid (1 M, 20 mL), H2O(20 mL),
saturated aqueous NaHCO3 (20 mL), and brine (20 mL). The
organic layer was dried over Na2SO4 and concentrated in vacuo.
The residue was purified by silica-gel flash chromatography (20 g,
CHCl3 : MeOH = 20 : 1) to give 25 as a white solid (143 mg, 86%).
ESI-TOF-MS (positive) m/z = 2153.52 [M + Na]+;1H NMR
(500 MHz, CDCl3) d = 7.52–7.15 (40H, m), 5.58 (1H, m), 5.57
(1H, s), 5.56–5.07 (6H, m), 4.86 (1H, d, J = 12.3 Hz), 4.83 (1H,
d, J = 3.7 Hz), 4.74 (1H. dd, J = 12.1 Hz), 4.66–4.57 (4H, m),
4.35–4.24 (8H, m), 4.09–3.92 (6H, m), 3.83–3.59 (12H, m), 3.53–
3.43 (10H, m), 3.39–3.35 (1H, m), 3.34–3.20 (1H, m), 2.56–2.41
(4H, m), 2.17–2.03 (7H, m), 1.93 (3H, s), 1.88 (3H, s), 1.73 (3H,
s), 1.57–1.53 (3H, m), 1.43 (1H, d, J = 6.9 Hz), 1.37–1.33 (3H,
m), 1.26 (1H, m). Found: C, 62.03; H, 6.63; N, 6.38. Calcd for
C113H138N10O31·3H20: C, 62.08; H, 6.64; N, 6.41%.
MALDI-TOF-MS (positive) m/z 8427.3 [M + Na]+
2-N-Acetyl-tetrasaccharide 21
To a solution of 14 (1.05 g, 0.46 mmol) in AcOH (10 mL) was added
Zn–Cu (prepared from 1 g of Zn), and the mixture was stirred at
rt for 1 h. The insoluble materials were filtered off and the filtrate
was concentrated in vacuo. The residual solvent was removed by
coevaporation with toluene (5 mL × 3). The crude product was
dissolved in pyridine (7 mL) and acetic anhydrate (7 mL) and the
solution was stirred at rt. After 30 min, the solution was removed
by concentration in vacuo. The residual solvent was removed by
coevaporation with toluene (5 mL × 3). The residue was purified
by silica-gel flash chromatography (80 g, CH3Cl : acetone = 3 : 1)
to give 21 as a white solid (750 mg, quant.).
23
[a]D = − 7.4 (c 1.00, CHCl3); ESI-TOF-MS (positive) m/z =
1609.2 [M + H]+, 1631.6 [M + Na]+.
2-N-Acetyl-octasacchride 22
Proteced tetrasaccharide tripeptide 26. ESI-TOF-MS (posi-
tive) m/z = 1330.49 [M + 2H]2+, tetrapeptide 27 ESI-TOF-MS
(positive) m/z = 1400.53 [M + 2H]2+, pentapeptide 28 ESI-
TOF-MS (positive) m/z = 1471.47 [M + 2H2+], octasaccharide
dipeptide 29 ESI-TOF-MS (positive) m/z = 4228.27 [M + Na]+
were synthesized in a manner similar to 25.
To a solution of 17 (1.0 g, 0.23 mmol) in AcOH (15 mL) was
added Zn–Cu (prepared from 2 g of Zn), and the mixture was
stirred at rt for 1 h. The insoluble materials were filtered off and
the filtrate was concentrated in vacuo. The residual solvent was
removed by coevaporation with toluene (5 mL × 3). The crude
product was dissolved in pyridine (15 mL) and acetic anhydrate
(16 mL) and the solution was stirred at rt. After 30 min, the
solution was concentrated in vacuo. The residual solvent was
removed by coevaporation with toluene (5 mL x3). The residue
was purified by silica-gel flash chromatography (80 g, CH3Cl :
acetone = 3 : 1) to give 22 as a white solid (600 mg, 83%).
Tetrasaccharide 39. To a solution of 23 (50 mg, 0.032 mmol)
in acetic acid (3 mL) was added palladium hydroxide on carbon
(100 mg) in acetic acid(1 mL) and the mixture was stirred under
H2 (10 atm) for one day. The Pd catalyst was removed by filtration
and the filtrate was concentrated. The residue was purified with
LH-20 column by elution with MeOH to give 39 as a white solid
(7.3 mg, 22%). MALDI-TOF-MS (negative) m/z = 1015.9 [M –
H]−.
ESI-TOF-MS (positive) m/z = 3183.71 [M + Na]+.
Tetrasaccharide with librareted carboxylic acid 23
To a solution of 21 (180 mg, 0.11 mmol) in dioxane : THF : H2O
(2 : 4 : 1, 1.2 mL) was added LiOH (28 mg, 0.66 mmol) and stirred
at rt for 1 h. The solution was neutralized with Dowex H+(Dowex
50 W × 8 200–400 mesh H form, DowChemicals) and then applied
to an HP-20 column (2 cm × 40 cm). Organic and inorganic salts
Tetrasaccharide dipeptide 32. To a degassed solution of
25 (300 mg, 0.071 mmol) in dry THF (2 mL) was
[Ir(cod)(MePh2P)2]PF6 (6 mg, 0.007 mmol) activated with H2
in dry THF (3 mL). The solution was stirred under a nitrogen
atmosphere at rt for 1 h, iodine (35 mg, 0.142 mmol) and water
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1
(0.5 mL) were added and the reaction mixture was stirred for
additional 10 min. The reaction was quenched by the addition of
aqueous Na2S2O3 (5%, 10 ml). The mixture was then extracted
with EtOAc (20 mL). The organic layer was washed with aqueous
Na2S2O3 (5%, 10 mL × 2), aqueous sat. NaHCO3 (50 mL × 2), and
brine (20 mL), dried over Na2SO4, and then concentrated in vacuo.
The residue was purified by silica-gel flash chromatography (20 g,
toluene : EtOAc = 5 : 1) to give 1-liberated-tetrasaccharide as a
pale yellow solid (260 mg, 88%). ESI-TOF-MS (positive) m/z =
2113.6 [M + Na]+.
found, 1954.939; H NMR (500 MHz, D2O): d = 4.86–4.80 (m,
1H, H-1), 4.46–4.40 (m, 3H), 4.36–4.0 (m, 12H, Lac-a-CH, Ala-a-
CH, iGln-a-CH, Lys-a-CH), 3.86–3.30 (m, 26H), 2.95–2.91 (t, J =
7.5, 4H, Lys-e-CH2), 2.40–2.31 (t, J = 7.0, 4H, iGln-c-CH2), 2.09–
1.82 (m, 22H, iGln-b-CH2 × 2, NHC(O)CH3 × 4, Lys-b-CH2 ×
2, Lys-d -CH × 2), 1.78–1.4 (m, 8H, Lys-d -CH × 2, Lys-c-CH2 ×
2, Propyl CH3–CH2), 1.40–1.22 (m, 12H, Ala-b-CH3 × 6, Lac-b-
CH3 × 2), 0.85–0.80 (m, 3H, Propyl CH3).
N-Acetylated tetrasaccharide tripeptide 34. To a solution of
33 (5 mg, 0.025 mmol) in methanol (0.5 mL) were added TEA
(10 lL) and Ac2O (5 lL). After being stirred for 2 h, the
mixture was concentrated and the residue was lyophilized from
acetonitrile/H2O to give 34 (5.13 mg, 98%) as white powder.
ESI-TOF-MS (negative) m/z = 876.4 [M – 2H]2−; HRMS-ESI
FT-ICR (negative): (M) calcd for C73H122N14O35, 1754.820; found,
To a solution of the above 1-liberated-tetrasaccharide (86 mg,
0.04 mmol) in acetic acid (3 mL) was added palladium hydroxide
on carbon (100 mg) in acetic acid(1 mL) and the mixture was
stirred under H2 (20 atm) for one day. The Pd catalyst was removed
by filtration and the filtrate was concentrated and lyophilized from
H2O to give 32 (T–2P2) as a white solid (39 mg, 70%). ESI-TOF-
MS (negative) m/z = 685.3 [M – 2H]2−; HRMS-ESI FT-ICR
(negative): (M) calcd for C54H88N10O31, 1372.561; found, 1372.555;
1H NMR (600 MHz, D2O): d = 5.16–5.15 (d, J = 3.0 Hz, 1H,
H-1), 4.46–4.42 (m, 3H), 4.36–4.32 (m, 2H), 4.30–4.27 (m, 2H,
iGln-a-CH), 4.26–4.19 (m, 2H), 3.86–3.30 (m, 24H), 2.31 (m, 4H,
iGln-c-CH2), 2.12–2.03 (m, 4H, iGln-b-CH2), 1.96–1.95 (s, 12H,
NHC(O)CH3 × 4), 1.37–1.35 (m, 6H, Ala-b-CH3), 1.31–1.28 (m,
6H, Lac-b-CH3).
1
1754.848; H NMR (500 MHz, D2O): d = 4.86–4.80 (m, 1H, H-
1), 4.46–4.40 (m, 3H), 4.36–4.05 (m, 8H, Lac-a-CH, Ala-a-CH,
iGln-a-CH, Lys-a-CH), 3.86–3.30 (m, 26H), 3.10–3.02 (m, 4H,
Lys-e-CH2), 2.37–2.31 (t, J = 9.5, 4H, iGln-c-CH2), 2.09–2.0 (m,
4H, iGln-b-CH2), 2.02–1.81 (m, 22H, NHC(O)CH3 × 4, Lys-e-
NHC(O)CH3 × 2, Lys-b-CH × 2, Lys-d-CH × 2), 1.78–1.4 (m,
8H, Lys-d -CH × 2, Lys-c-CH2 × 2, Propyl CH3–CH2), 1.40–1.35
(m, 6H, Ala-b-CH3), 1.31–1.24 (m, 6H, Lac-b-CH3), 0.85–0.80 (t,
J = 9.3, 3H, Propyl CH3).
Tetrasaccharide tripeptide 33. To a solution of 27 (95 mg,
0.036 mmol) in AcOH (3 mL) was added palladium hydroxide
(100 mg) in AcOH and stirred under H2 (20 atm) for 1 day. The
reaction was monitored by TLC analysis and the hydrogenolysis
was continued until deprotection was completed. The Pd catalyst
was filtered off by celite and the filtrate was concentrated.
The residue was lyophilized from acetonitrile-H2O to give 33
(39 mg, 50%) as a white powder. ESI-TOF-MS (negative) m/z =
834.5 [M – 2H]2−; HRMS-ESI FT-ICR (negative): (M) calcd for
N-Acetylated tetrasaccharide tetrapeptide 36 and N-acetylated
tetrasaccharide pentapeptide 38 were synthesized from compound
35 (5 mg, 0.025 mmol) in a manner similar to the preparation of 33.
36. ESI-TOF-MS (negative) m/z = 947.5 [M–2H]2−; HRMS-
ESI FT-ICR (negative): (M) calcd for C79H132N16O37, 1812.873;
1
found, 1812.896; H NMR (500 MHz, D2O): d = 4.86–4.80 (m,
1H, H-1) 4.46–4.40 (m, 3H), 4.40–4.08 (m, 10H, Lac-a-CH, Ala-
a-CH, D-iGln-a-CH, Lys-a-CH), 3.90–3.30 (m, 26H), 3.10–3.02
(m, 4H, Lys-e-CH2), 2.39–2.18 (m, 4H, iGln-c-CH2), 2.09–1.40
(m, 34H, iGln-b-CH2, NHC(O)CH3 × 4, Lys-e-NHC(O)CH3 ×
2, Lys-b-CH2 × 2, Lys-d -CH2 × 2, Lys-d -CH × 2, Lys-c-CH2 ×
2, Propyl CH3–CH2), 1.40–1.22 (m, 12H, Ala-b-CH3 × 4, Lac-b-
CH3 × 2), 0.85–0.80 (t, J = 9.3, 3H, Propyl CH3).
1
C69H118N14O33, 1670.798; found, 1670.817; H NMR (500 MHz,
D2O): d = 4.86–4.80 (m, 1H, H-1) 4.46–4.40 (m, 3H), 4.36–4.05
(m, 8H, Lac-a-CH, Ala-a-CH, iGln-a-CH, Lys-a-CH), 3.86–3.30
(m, 26H), 3.00–2.90 (t, J = 11.4, 4H, Lys-e-CH2), 2.37–2.31 (t,
J = 9.5, 4H, iGln-c-CH2), 2.09–2.0 (m, 4H, iGln-b-CH2), 2.02–
1.81 (m, 18H, NHC(O)CH3 × 4, Lys-b-CH × 2, Lys-d -CH ×
2), 1.78–1.69 (m, 2H, Lys-d -CH × 2), 1.58–1.70 (m, 4H, Lys-c-
CH2 × 2), 1.61–1.4 (m, 2H, Propyl CH3–CH2), 1.40–1.35 (m, 6H,
Ala-b-CH3), 1.31–1.28 (m, 6H, Lac-b-CH3), 0.85–0.80 (t, J = 9.3,
3H, Propyl CH3).
38. ESI-TOF-MS (negative) m/z = 1018.5 [M – 2H]2−; HRMS-
ESI FT-ICR (negative): (M) calcd for C85H142N18O39, 2038.968;
1
found, 2038.962; H NMR (500 MHz, D2O): d = 4.86–4.80 (m,
1H, H-1), 4.46–4.40 (m, 3H), 4.40–4.0 (m, 14H, Lac-a-CH, Ala-
a-CH, D-iGln-a-CH, Lys-a-CH), 3.90–3.30 (m, 26H), 3.10–3.02
(m, 4H, Lys-e-CH2), 2.39–2.30 (m, 4H, iGln-c-CH2), 2.09–1.40
(m, 34H, iGln-b-CH2, NHC(O)CH3 × 4, Lys-e-NHC(O)CH3 ×
2, Lys-b-CH2 × 2, Lys-d -CH2 × 2, Lys-d -CH × 2, Lys-c-CH2 ×
2, Propyl CH3–CH2), 1.40–1.20 (m, 18H, Ala-b-CH3 × 6, Lac-b-
CH3 × 2), 0.85–0.79 (t, J = 7.5, 3H, Propyl CH3).
Tetrasaccharide tetrapeptide 35 and tetrasaccharide pentapep-
tide 37 was synthesized from 27 in a manner similar to the synthesis
of 33
35. ESI-TOF-MS (negative) m/z = 905.1 [M – 2H]2−; HRMS-
ESI FT-ICR (negative): (M) calcd for C75H128N16O35, 1812.873;
1
found, 1812.896; H NMR (500 MHz, D2O): d = 4.86–4.80 (m,
Octasaccharide dipeptide 40. To a solution of 29 (140 mg,
0.033 mmol) in acetic acid (3 mL) was added palladium hydroxide
on carbon (140 mg) in acetic acid(1 mL) and the mixture was
stirred under H2 (20 atm) for one day. The Pd catalyst was removed
by filtration and the filtrate was concentrated. The residue was
lyophilized from H2O to give 40 as a white solid (74 mg, 70%).
MALDI-TOF-MS (negative) m/z = 2767.63 [M – H]−; HRMS-
ESI FT-ICR (negative): (M) calcd for C111H180N20O61, 2769.159;
1H, H-1), 4.46–4.40 (m, 3H), 4.36–3.95 (m, 10H, Lac-a-CH, Ala-
a-CH, D-iGln-a-CH, Lys-a-CH), 3.86–3.30 (m, 26H), 3.00–2.90
(t, J = 7.5, 4H, Lys-e-CH2), 2.40–2.31 (t, 4H, iGln-c-CH2), 2.09–
1.82 (m, 22H, iGln-b-CH2 × 2, NHC(O)CH3 × 4, Lys-b-CH2 ×
2, Lys-d -CH × 2), 1.78–1.4 (m, 8H, Lys-d -CH × 2, Lys-c-CH2 ×
2, Propyl CH3–CH2), 1.40–1.22 (m, 12H, Ala-b-CH3 × 4, Lac-b-
CH3 × 2), 0.85–0.80 (m, 3H, Propyl CH3).
37. ESI-TOF-MS (negative) m/z = 976.64 [M – 2H]2−; HRMS-
found, 2769.157; H NMR (600 MHz, D2O) d = 4.94–4.88 (m,
1
ESI FT-ICR (negative): (M) calcd for C81H138N18O37, 1954.947;
1H), 4.54–4.46 (m, 4H), 4.43–4.24 (m, 16H), 3.94–3.34 (m, 53H),
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2.40–2.30 (m, 8H, c-Gln-CH2 × 4), 2.19–2.09 (m, 4H, iGln-
b−CHH × 4), 2.06–1.90 (m, 28H, NHC(O)CH3 × 8, iGln-
b−CHH × 4), 1.44–1.34 (m, 24H, Ala-b-CH3 × 4, Lac-b-CH3 ×
4).
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Octasaccharide 41. Compound 41 was synthesized from com-
pound 24 in a manner similar to the preparation of 40.
HRMS-ESI FT-ICR (negative): (M) calcd for C79H128N8O49,
1972.777; found, 1972.773; 1H NMR (600 MHz, D2O) d = 5.23–
5.21 (m, 1H), 4.54–4.46 (m, 4H), 4.43–3.92 (m, 56H), 1.99–2.10 (m,
24H, NHC(O)CH3 × 8), 1.21–1.40 (m, 12H, Lac-b-CH3, CH3–
CH2–OCO × 4); 13C NMR (150 MHz, D2O) d = 182.1, 181.4,
174.8, 174.5, 174.4, 174.2, 105.5, 102.2, 100.1, 79.9, 79.1, 78.8,
77.4, 76.1, 75.8, 75.3, 78.2, 74.9, 73.4, 72.0, 71.3, 70.3, 69.7, 61.2,
60.5, 59.6, 56.1, 55.4, 54.6, 54.3, 22.3, 22.0, 21.6, 18.4.
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Acknowledgements
We thank Mr. S. Adachi and Mr. M. Doi for NMR measurements,
and Ms. K. Hayashi and Ms. T. Ikeuchi for elemental analysis, at
Osaka University. The present work was financially supported
in part by ‘Research work in Future’ program No. 97L00502 and
Grants-in Aid for Scientific research (No.14380288, No.17510178)
from the Japan Society for the Promotion of Science, and by the
Deutsche Forschungsgemeinschaft (GRK288 to HH, EW).
27 J. H. Cho, J. P. Fraser, K. Fukase, S. Kusumoto, Y. Fujimoto, G. L.
Stahl and R. A. Ezekowitz, Blood, 2005, 106, 2551–2558.
28 Z. M. Wang, X. Li, R. R. Cocklin, M. Wang, K. Fukase, S. Inamura,
S. Kusumoto, D. Gupta and R. Dziarski, J. Biol. Chem., 2003, 278,
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