Synthesis of mimetic peptides containing glucosamine

Article abstract of DOI:10.1016/j.carres.2011.04.010

A convenient synthesis of 2-amino-3,4,6-tri-O-benzyl-2-deoxy-β-D- glucopyranoside was described from the readily available starting material 2-acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine). Herein, the coupling of different lipophilic amino acids w

Full text of DOI:10.1016/j.carres.2011.04.010

Carbohydrate Research 346 (2011) 1997–2003
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Synthesis of mimetic peptides containing glucosamine
⇑
Jianwei Zhang , Ming Zhao, Shiqi Peng
College of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A convenient synthesis of 2-amino-3,4,6-tri-O-benzyl-2-deoxy-b-
the readily available starting material 2-acetamido-2-deoxy-D-glucose (N-acetyl-D
D
-glucopyranoside was described from
-glucosamine). Herein,
the coupling of different lipophilic amino acids with 2-amino-3,4,6-tri-O-benzyl-2-deoxy-b- -glucose
Received 10 March 2011
Received in revised form 2 April 2011
Accepted 6 April 2011
D
was reported via an amide linkage as useful building blocks for the synthesis of glycopeptides. Of partic-
ular interest, bioactive peptide Arg-Gly-Asp (RGD) was incorporated into the building block containing
valine was also reported. The 15 examples of corresponding di-, tri- and tetra-peptides were obtained
Available online 13 April 2011
Keywords:
Amino sugar
Carbohydrate-mimetic peptide
N-Acetylglucosamine
as single aanomers.
Ó 2011 Elsevier Ltd. All rights reserved.
The carbohydrates of glycoproteins have been subjected to an
increasing interest during the last few years because they play
important roles in numerous biological processes including modi-
fication of proteins, the immune response,1–3 cell adhesion,4–7
inflammation, tumor metastasis,8 and substrate–receptor recogni-
tion.9,4,3,10 The effects of carbohydrates on biological activity and
stability of glycoproteins has been studied previously.11–13
Carbohydrates that coat the surfaces of bacteria are capable of
inducing an immune response that can recognize whole bacte-
ria,14–18 and some functional glycoproteins that are expressed on
tumor cell surfaces have been shown to induce specific antitumor
cell–antibody responses in mice and patients.19 There is a growing
interest in carbohydrate mimetic peptides as vaccines to target
cell-surface polysaccharides of infectious bacteria and tumor.20–23
Owing to the inherent complexity of carbohydrates, glycosyla-
tion can produce enormous structural diversity in proteins and in-
duce a variety of functional changes. Since glycosyl amino acids
and glycopeptides may be of value for medicinal chemistry, an-
other important issue is their stability toward enzymatic degrada-
tion under physiological conditions. The field of glycobiology has
grown explosively, and the interest in the development of methods
for the synthesis of glycoconjugates is rapidly expanding as their
biological and medical roles come into focus. Recent progress in
this area has been remarkable and a number of carbohydrate moi-
eties have been covalently attached to amino acids25 for incorpora-
tion into glycopeptide sequences. Thus, many efforts have been
devoted to establish easy and efficient methods for glycopeptide
synthesis.24–26
conjugates and this should facilitate a more rapid investigation of
this possibility. We were interested to develop compounds
reduced in carbohydrate character (monosaccharides). Amino
sugars are widely distributed in living organisms and occur as
constituents of glycoproteins, glycolipids, bacterial lipopolysaccha-
rides, and proteoglycans. 2-Amino-2-deoxy-D-glucose (glucosa-
mine) is the most common amino sugar and is generally found
as an N-acetylated and b-linked glycoside that is a natural compo-
nent of glycoproteins found in connective tissues and gastrointes-
tinal mucosal membranes. However, the ability to synthesize
glucosamine in the body declines with age and this, in turn,
incapacitates the generation of proteoglycan, which is known to
result in senile osteoarthritis (OA).27 Therefore, glucosamine (GlcN)
salts (sulfate and chloride) represent a new generation of drugs
that possess potentially chondroprotective or disease-modifying
properties and were originally suggested to promote the repair of
damaged cartilage. A publication by W. Bohne in 1969 showed that
GlcN can be used as a single pharmacologic agent to relieve the
symptoms of osteoarthritis. Glucosamine has recently received a
great deal of attention from the public as a potential treatment
for OA.28 Furthermore, the multiple antioxidant activity of glucosa-
mine was evident as it showed pronounced reducing power, and
superoxide/hydroxyl radical-scavenging ability. Because it is
non-toxic, it may be a desired food supplement as a potential
antioxidant. Experimental evidence was presented that glucosa-
mine possess a unique range of anti-inflammatory activities and
inhibits IL-1b- and TNF-a-induced NO production in normal
human articular chondrocytes. It is also a therapeutic agent for
The synthesis of monosaccharide conjugates is more straight-
forward than disaccharide or higher order oligosaccharide
inflammatory bowel diseases.29
Its amide bond is very stable in buffer solution at pH 7.4 and in
culture medium. Amides are, in general, important functional
groups widely found in natural products, pharmaceuticals, and
polymers. They may be prepared by coupling reactions between
⇑
Corresponding author. Tel.: +86 10 8391 1530; fax: +86 10 8391 1533.
E-mail address: jwzhang2006@163.com (J. Zhang).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.04.010

1998
J. Zhang et al. / Carbohydrate Research 346 (2011) 1997–2003
carboxylic acids and amines.30–32 The synthetic design used for
preparation of glycosyl amides was based upon a number of rea-
sons: (1) glucosamine possess the biological activity; (2) N-acetyl-
glucosamine has several potential advantages over glucosamine as
a potential therapeutic anti-inflammatory agent. We report herein
the synthesis of peptidomimetics in which Boc-protected amino
acids were conjugated via an amide linkage to the amino sugar,
arginine–glycine–asparagine tripeptide sequence. Regarding
RGD-dependent integrins, vb3 and vb5 receptors have received
increasing attention as therapeutic targets, as the integrins vb3
and vb5 can be expressed by both tumor cells and tumor endothe-
a
a
a
a
lial cells.38–40 It is speculated that drugs that inhibit the adhesive
function of these integrins can inhibit tumor growth. Among selec-
tive receptor-targeting small peptides, integrinmediated RGD pep-
tides appear as attractive candidates. Moreover, it is well known
that RGD peptide can suppress platelet aggregation by blocking
the interaction between platelet and fibrinogen.41 In this study,
an interest has been increased in modifying the building blocks
by the addition of RGD and investigating the preparation for the
tetrapeptide from the building block containing valine and RGD
D
-glucosamine.33 The resulting peptide mimetics can be incorpo-
rated in a peptide structure to develop compounds. In this context,
we designed different lipophilic amino acids that were used as
building units for the preparation of a series of peptide mimetics:
these were
D-glucosamine derivatives N-linked via valine,
phenylalanine, and serine. Versatility is one of the major advanta-
ges of this stepwise procedure, which allows the synthesis of pep-
tidomimetics with variations in the peptide part. Asparagines may
be useful for the preparation of a diversity of structurally longer
peptides.34 As shown in Scheme 2 the building blocks 6a–c were
coupled with L-Boc-Asp (OBzl)-OH to furnish the asparagine conju-
gate dipeptides. After the protecting groups were removed in the
stepwise protocol, a wider range of amino acids were explored
peptide by adopting stepwise approach.
acetyl-
D-Glucosamine and N-
D
-glucosamine have been reported to possess interesting
biological activity,42–45 therefore, the creation of these compounds
would be useful for further biological evaluation, as well as for de-
tailed structure–activity studies that may lead to potential drugs or
provide leads for drug development.
The synthetic routes for the preparation of mimetic peptides
and introduced either into the side chain or into the
a
-amino group
containing
starting material are presented in Schemes 1 and 2. The free OH
groups of N-acetyl- -glucosamine (1) were benzylated with BaO,
Ba(OH)2ꢀ8H2O and BnBr to give benzyl 2-acetamido-1,3,4,6-tetra-
O-benzyl-2-deoxy-b- -glucopyranoside (2), which was easily
obtained as the single anomer by simple crystallization from
D-glycosamine from N-acetyl-D-glucosamine (1) as
of the asparagine to provide maximum diversity for peptide elon-
gation. Furthermore, some bioactive peptides containing aspara-
gines such as Arg-Gly-Asp (RGD) peptide were incorporated into
the building blocks. Peptides related to RGD are known to contrib-
ute to various biological functions. Integrin receptors constitute a
large family of proteins with structural characteristics of noncova-
D
D
methanol in 67% yield from 1.68 mmol (0.3 g) of N-acetyl-D-
lent heterodimeric glycoproteins formed of
a
and b subunits.35–37
gucosamine used. The 1H NMR spectrum of 2 showed the signal
for the anomeric proton at d 5.49 as a doublet with a coupling
One important recognition site for many integrins is the
OH
OBn
OBn
HCl
O
O
O
BnBr
HO
BnO
BnO
BnO
BnO
OH
OH
OBn
HO
NHAc
NH2
NHAc
3
1
2
Scheme 1.
OBn
OBn
O
OBn
HCl/EtOAc
O
O
4a-c
BnO
BnO
BnO
BnO
BnO
BnO
OH
NH2
AA
Boc AA NH
H
NH
OH
OH
4a
: Boc-L-Val-OH
3
5a-c
6a-c
4b
: Boc-L-Phe-OH
4c
: Boc-L-Ser(OBzl)-OH
Boc-L-Asp(OBzl)-OH
OBn
OBn
OBn
Boc-L-Gly-OH
HCl/EtOAc
O
O
O
BnO
BnO
BnO
BnO
BnO
BnO
H-(BzlO)Asp AA NH
Boc-Gly-(BzlO)Asp AA NH
Boc-(BzlO)Asp AA NH
OH
OH
OH
9a
7a-c
8a-c
HCl/EtOAc
OBn
OBn
O
Boc-L-Arg(NO2)-OH
O
BnO
BnO
BnO
BnO
Boc-(O2N)Arg-Gly-(BzlO)Asp AA NH
H-Gly-(BzlO)Asp AA NH
OH
OH
11a
10a
Scheme 2.

J. Zhang et al. / Carbohydrate Research 346 (2011) 1997–2003
1999
constant of 9.0 Hz, which was assigned as the b-anomer. It is
believed that b-benzylation proceeds via neighboring group partic-
1H NMR spectra of 5a–c showed signals for their anomeric protons
as doublets with coupling constants of 3.0–3.5 Hz. In all cases, a
ipation by an acyl group at N-2 of the
D
-gucosamine. Problems were
noteworthy point is that the aanomers are the sole products,
encountered with the removal of the acetyl group of intermediate 2
where using KOH in refluxing ethanol, NaOBu in butyl alcohol,
hydrazine hydrate, hydrazine in refluxing ethanol, and aqueous
methylamine, respectively, were ineffective. The acetyl group and
the anomeric benzyl groups of 2 were removed by subjecting the
2 to a mixture of 3 N hydrochloric acid and terahydrofuran to give
which can be attributed to the anomeric effect. Subsequently, re-
moval of the Boc group with hydrogen chloride in ethyl acetate
led to a set of amines 6a–c which were used as building blocks
for the facile preparation of longer peptides. We successfully pre-
pared di-, tri- and tetra-peptides. Removal of the Boc group and
subsequent coupling with the L-Boc-Asp (OBzl)-OH gave, sequen-
3 as a mixture of anomers46 (43% yield,
a:b = 1:0.5) (Scheme 1).
tially, the protected forms of the dipeptides 7a–c in 74%, 55% and
Furthermore, a wider range of amino acids and monosaccharide
derivatives 3 were explored to synthesize various glycosylated Boc
amino acid building blocks. Initially, the pure mixture of anomers 3
and different lipophilic amino acids valine, phenyalanine and ser-
ine were reacted by standard peptide coupling methods (DDC,
HOBt, and N-methylmolpholine) in solution phase. 1-Hydroxyben-
zotriazole (HOBt) was used to activate Boc-protected amino acids
and dicyclohexylcarbodiimide (DCC) was used as the coupling re-
agent in tetrahydrofuran (THF). The products obtained were puri-
fied by silica gel column chromatography to afford 5a–c as single
anomers in 95%, 92% and 65% yield, respectively.47 The assignment
of the configuration was performed by 1H NMR spectroscopy. The
53% yield, respectively. 1H NMR spectra indicated that 7a–c were
aanomers. Similarly, the iterative removal of the Boc group, to-
gether with the coupling reagents DCC/HOBt for peptide elonga-
tion, can be achieved. The coupling reaction of amine 8a with
Boc-protected glycine afforded only one
a anomer tripeptide 9a
in 71% yield. Finally, we synthesized the tetrapeptide 11a from
the building block containing valine and the biological activity of
a RGD peptide. Similarly, N-deprotection was performed and sub-
sequent coupling the N-terminus free tripeptide 10a and C-termi-
nus free L-Boc-Arg (NO2)-OH provided 11a in 33% yield as a pure a
anomer (Scheme 2). The Structures of mimetic peptides containing
glucosamine are shown in Table 1.
Table 1
Structures of mimetic peptides containing 2-amino-2-deoxy-D-glucose (glucosamine)
No
Chemical structure
No
Chemical structure
OBn
OBn
O
O
2
3
BnO
BnO
BnO
BnO
OH
OBn
NH2
OBn
NHAc
OBn
O
O
BnO
BnO
Boc Val
BnO
BnO
Boc Phe
5a
5c
5b
6a
6c
NH
NH
OH
OH
OBn
OBn
O
O
BnO
BnO
BnO
BnO
Boc (BzlO)Ser
NH
H
Val
NH
OH
OH
OBn
OBn
O
O
BnO
BnO
BnO
6b
7a
7c
BnO
H
(BzlO)Ser
NH
OH
OBn
NH
Phe
H
OH
OBn
O
O
BnO
BnO
BnO
Boc-(BzlO)Asp
7b
8a
8c
BnO
Val NH
NH
Phe
Boc-(BzlO)Asp
OH
OBn
OH
OBn
O
O
BnO
BnO
BnO
BnO
Boc-(BzlO)-Asp-(BzlO)Ser
NH
Val
H-(BzlO)Asp
NH
OH
OH
OBn
OBn
O
O
BnO
BnO
BnO
BnO
H-(BzlO)-Asp-(BzlO)Ser
8b
9a
11a
NH
NH
H-(BzlO)Asp Phe
OH
OH
OBn
OBn
O
O
BnO
BnO
Boc-Gly-(BzlO)Asp
BnO
BnO
H-Gly-(BzlO)Asp
10a
NH
OH
NH
OH
OBn
O
BnO
BnO
Val
NH
Boc-(O2N)Arg-Gly-(BzlO)Asp
OH

2000
J. Zhang et al. / Carbohydrate Research 346 (2011) 1997–2003
Table 2
provided useful information for the further design of novel potent
agents.
Influenza virus neuraminidase inhibition for compounds 5a–c, 7a–c, 9a, and 11a
In summary, we have developed an effective procedure for the
Compound
Concentration (lg/mL)
Inhibition ratio (%)
preparation a series of peptide mimetics containing D-glucosamine
5a
5b
5c
7a
7b
7c
9a
11a
40
40
40
40
40
40
40
40
39.72
42.72
42.28
49.62
41.55
24.72
44.16
49.24
96.12
via valine, phenyalanine and serine derivatives as building blocks
which can be directly used for constructing parallel and combina-
torial glycopeptide libraries as well as the synthesis of various new
materials and peptide-based drug candidates. We synthesized the
tetrapeptide from the building block containing valine and bioac-
tive RGD peptide by adopting stepwise solution-phase approach.
We expect that the incorporation of a building block in a bioactive
peptide may induce intensifying effects. All tested compounds
exhibited a potent neuraminidase inhibitory effect. Therefore, it
was concluded that peptide mimetics as building blocks might be
essential for activity and were judged to be the most attractive
analogues for further optimization.
Zanamivir
0.0004
Both RGD and glucosamine have biological functions. We had
this in mind when trying to decipher the possible biological role
of the synthesized compounds 5a–c, 7a–c, 9a, and 11a, which were
screened for their antioxidant activity and antitumor activity in our
initial research. The results obtained exhibited undesired biological
activity. These derivatives do not have a biological function similar
to that of glucosamine and RGD in either antioxidant activity and
antitumor activity. Benzylation of N-acetylglucosamine led to the
disappearance of its antioxidant activity. It implied that the free
1. Experimental
1.1. General procedures
Unless otherwise stated, all reactions were carried out under a
nitrogen atmosphere (1 bar). The agents used in this work were
purchased from Sigma–Aldrich Chemical Co. (USA). Optical rota-
tions were determined with a Schmidt–Haensch Polartromic D
instrument (Germany). IR spectra were recorded with an Avatar
330 (Nicolet, USA) spectrometer.
hydroxyl group of N-acetyl-D-glucosamine was necessary for the
antioxidant activity. The biological activity of these derivatives
was further investigated. The anti-influenza virus neuraminidase
(NA) activities were evaluated and the biological data are summa-
rized in Table 2. A standard fluorimetric assay was used to measure
influenza virus NA activity .The substrate MUNANA was cleaved by
NA to yield a fluorescent product, which can be quantified. The
reaction mixture containing the test compounds and NA enzyme
from A/PR/8/34 (H1N1) in 32.5 mM MES buffer with 4 mM calcium
chloride (pH 6.5) was incubated for 1 h at 37 °C. After incubation,
the reaction was terminated by the addition of 34 mM NaOH.
The fluorescence was quantified at an excitation wavelength of
360 nm and emission wavelength of 450 nm. The inhibition ratio
(%) was the ratio of the decrease of the fluorescence intensity of
reaction mixture containing the tested compounds to the fluores-
cence intensity of reaction mixture containing virus but no inhibi-
tor. The assay results showed that all tested compounds possessed
1H and 13C NMR spectra were recorded at 300 MHz on a VXR-
300 instrument or at 500 MHz on a Bruker AM-500 instrument
in CDCl3 or in DMSO-d6 with Me4Si as the internal standard. Chem-
ical shifts are expressed in d-units (ppm). Chromatography was
performed on Qingdao silica gel H (Qingdao of China). The purities
of the intermediates and the products were confirmed by TLC (E.
Merck silica gel plates of type 60 F254, 0.25-mm layer thickness,
Germany) and HPLC (Waters, C18 column 4.6 ꢁ 150 mm, USA).
Mass spectra (MS) were acquired on a Quattro Micro ZQ2000,
Waters, USA instrument. High-resolution mass spectra were re-
corded with a JEOL JMS-T100LC AccuTOF mass spectrometer.
1.2. Introducing 3 into
L-Val, L-Phe, and L-Ser
a weak NA inhibitory effect at the concentration of 40 lg/mL com-
pared with the effect of the positive compound zanamivir. The
tested compounds showed anti-influenza virus activity ranging
from 24.72% to 49.62%. It was observed that 5a–c as building
blocks showed 39.72%, 42.72% and 42.28% inhibition, respectively,
demonstrating that an amide bond linkage to a monosaccharide (D-
glucosamine) was necessary for achieving potency against influ-
enza virus. The compounds active against virus can be derived
1.2.1. Preparation of 5a
HOBt (90 mg, 0.667 mmol) and DCC (151 mg, 0.734 mmol) were
added to a solution of Boc-Val-OH 4a (145 mg, 0.668 mmol) in
anhyd THF (10 mL) at 0 °C. The reaction mixture was stirred at
0 °C for 0.5 h. The solution of 3 (300 mg, 0.668 mmol) in anhydrous
THF (5 mL) was added and adjusted to pH 9 with N-methyl-
morpholine. The reaction mixture obtained was keep at 0 °C for
2 h and at room temperature for 24 h. Precipitated DCU was re-
moved by filtration. The filtrate was evaporated under reduced
pressure and the residue was dissolved in EtOAc (50 mL). The solu-
tion was washed successively with satd aq NaHCO3, 5% KHSO4 and
satd aq NaCl and the organic phase was separated and dried over
Na2SO4. After filtration and evaporation of the solvent under re-
duced pressure, purification by chromatography (40:1 CH2Cl2–
from scaffolds containing D-glucosamine monosaccharide.
To understand the contribution of the asparagines in dipeptides
to the anti-influenza virus activities, 5a–c were used as reference
compounds, and the data indicated that when compared to 5a,
7a was more potent (49.62%), and when compared to 5b, 7b was
equally active (41.55%), and when compared to 5c, 7c exhibited
lower activity (24.72%). This comparison demonstrated that the
asparagines in the dipeptides had a significant contribution to
the activity. The dipeptides containing building blocks might allow
the asparagines to assume different conformations that render the
different activities observed for the analogues. The asparagine in
7a may be the active conformation. The subsequent peptide chain
elongation resulting tripeptide 9a and tetrapeptide 11a retained
the potency of analogue 7a. Structural changes do not affect bio-
logical activity. Compounds 9a and 11a showed 44.26%, and
49.24% inhibition, respectively, which is equivalent to that of 7a.
Further investigation is required on the biological activity of these
compounds in order to rationalize these observations. This study
MeOH) provided the product 5a (410 mg, 0.633 mmol, 95%). ½a D25
ꢂ
+53.0 (c 0.1, CHCl3); IR (KBr, neat): 3417, 3328, 1691, 1655 cmꢃ1
;
1H NMR (500 MHz, CDCl3): d 7.38–7.08 (15H, m, Ar-H), 6.17
(1H,d, J2, 9.0 Hz, N–H), 5.27 (1H,d, J1, 3.0 Hz, H-1), 5.09 (1H,
NH
2
d, JNH, 8.0 Hz, N–H), 4.86–4.52 (6H, m, PhCH2), 4.24 (1H, dt, J2, 3
a
10.0 Hz, J1, 3.0 Hz, H-2), 4.10 (1H, m, H-5), 3.88 (1H, t, J2, 3 = J3,
2
4 = 10.0 Hz, H-3), 3.81 (2H, m, H-6a, H-6b), 3.73 (1H, dd, J
a, b
12.5 Hz, JNH, 8.0 Hz, H-a), 3.66 (1H, t, J3, 4 = J4, 5 = 10.0 Hz, H-4),
a
2.03 (1H, m, CH), 1.47 (9H, s, CH3), 0.88 (3H, d, J 7.0 Hz, CH3),
0.85 (3H, d, J 7.0 Hz, CH3); 13C NMR (125 MHz, CDCl3): d 172.1,

J. Zhang et al. / Carbohydrate Research 346 (2011) 1997–2003
2001
156.1, 138.3, 138.0, 137.9, 128.7, 128.5, 128.4, 128.3, 128.2, 128.0,
127.9, 127.8, 127.7, 127.6, 91.7, 80.0, 79.5, 78.4, 74.8, 74.7, 73.5,
70.9, 68.8, 60.3, 53.4, 30.8, 28.3, 19.5, 17.6; ESIMS: m/z
671(M+Na), 649 (M+1); HRMS calcd for: (C27H48N6O12 + Na), m/z
(671.32279); found, m/z (671.32268).
was used immediately in the coupling reaction of the next step
without additional purification.
1.4. Introducing 3 into L-Val-Asp, L-Phe-Asp, and L-Ser-Asp
1.4.1. Preparation of dipeptide derivative 7a
Compound 7a was prepared by a method similar to the prepa-
ration of 5a using 6a and Boc- -Asp (OBzl)-OH (123 mg,
1.2.2. Preparation of 5b
Compound 5b was prepared by a method similar to the prepa-
ration of 5a using 3 (140 mg, 0.312 mmol), and Boc-Phe-OH 4b
(83 mg, 0.312 mmol), HOBT (42 mg, 0.312 mmol), and DCC
(71 mg, 0.343 mmol). The residue was purified by silica gel column
chromatography (40:1 CH2Cl2–MeOH) to provide the product 5b
L
0.380 mmol), HOBT (51 mg, 0.380 mmol), and DCC (86 mg,
0.418 mmol). The residue was purified by silica gel column chro-
matography (40:1 CH2Cl2–MeOH) to provide the product 7a
(240 mg, 0.281 mmol, 74%). ½a D25
ꢂ
+21.4 (c 0.1, CHCl3); IR (KBr,
;
(200 mg, 0.287 mmol, 92%). ½a D25
ꢂ
+25.6 (c 0.1, CHCl3); IR (KBr,
neat): 3435, 3311, 1699, 1645 cmꢃ1
1H NMR (300 MHz, CDCl3):
neat): 3427, 3336, 1693, 1654,698 cmꢃ1
;
1H NMR (500 MHz,
d 7.36–7.11 (20H, m, Ar-H), 7.04 (1H, d, JNH, 8.7 Hz, N–H), 6.58
a
CDCl3): d 7.36–7.14 (20H, m, Ar-H), 6.32 (1H, d, J2, NH = 9.0 Hz, N–
H), 5.20 (1H, d, J1, 2 3.5 Hz, H-1), 4.96 (1H, d, JNH, 8.0 Hz, N–H),
4.81–4.47 (6H, m, PhCH2), 4.28 (1H, q, J1 7.0 Hz, H-a), 4.20 (1H,
(1H, d, J2,
9.0 Hz, N–H), 5.70 (1H, d, JNH, 8.1 Hz, N–H), 5.16
NH
a
a
(1H, d, J1, 3.3 Hz, H-1), 5.10 (2H, s, PhCH2), 4.85–4.48 (7H, m,
2
PhCH2, H-a), 4.24 (2H, m, H-a, H-2), 4.08 (1H, m, H-5), 3.91 (1H,
td, J2, 3 10.0 Hz, J1, 2 3.5 Hz, H-2), 4.08 (1H, m, H-5), 3.81 (1H, t, J2,
3 = J3, 4 = 10.0 Hz, H-3), 3.76 (2H, m, H-6a, H-6b), 3.67 (1H, t, J3,
4 = J4, 5 = 10.0 Hz, H-4), 3.08 (1H, dd, J1 14.0 Hz, J2 6.0 Hz, CH2),
2.99(1H, dd, J1 14.0 Hz, J2 7.0 Hz, CH2), 1.37 (9H,s, CH3); 13C NMR
(125 MHz, CDCl3): d 171.7, 155.5, 138.3, 138.0, 137.9, 136.6,
136.3, 129.3, 128.6, 128.4, 128.1, 127.9, 127.7, 126.9, 91.7, 80.4,
79.6, 78.2, 74.8, 74.7, 73.5, 70.9, 68.8, 53.6, 49.4, 33.8, 28.2; ESIMS:
m/z 719 (M+Na), 697 (M+1); HRMS: calcd for (C30H52N2O16 + Na),
m/z (719.32155); found, m/z (719.32131).
t, J1 9.0 Hz, H-3), 3.80–3.71 (2H, m, H6a, H6b), 3.67 (1H, t, J1
9.0 Hz, H-4), 2.98 (1H, dd, J1 16.8 Hz, J2 4.5 Hz, CH2), 2.82 (1H, dd,
J1 16.8 Hz, J2 6.3 Hz, CH2), 2.14 (H, m, CH), 1.44 (9H, s, CH3), 0.92
(3H, d, J 6.6 Hz, CH3), 0.84 (3H, d, J 6.6 Hz,CH3); 13C NMR
(75 MHz, CDCl3): d 171.6, 171.1, 170.9, 155.6, 138.5, 138.2, 138.0,
135.3, 135.2, 128.6, 128.5, 128.4, 128.3, 128.2, 127.9, 127.8,
127.7, 127.6, 127.5, 127.4, 91.6, 80.9, 79.5, 78.5, 74.8, 74.7, 73.4,
70.7, 68.7, 67.0, 58.7, 53.6, 51.1, 35.7, 30.1, 28.3, 19.5, 17.6; ESIMS:
m/z 876 (M+Na), 854 (M+1); HRMS: calcd for (C43H59N5O13 + Na),
m/z (876.40070); found, m/z (876.40063).
1.2.3. Preparation of 5c
Compound 5c was prepared by a method similar to the prepa-
ration of 5a using 3 (400 mg, 0.891 mmol), Boc-Ser (OBzl)-OH 4c
(263 mg, 0.891 mmol), HOBT (120 mg, 0.891 mmol), and DCC
(202 mg, 0.980 mmol). The residue was purified by silica gel col-
umn chromatography (40:1 CH2Cl2–MeOH) to provide the product
1.4.2. Preparation of dipeptide derivative 7b
Compound 7b was prepared by a method similar to the prepa-
ration of 5a using 6b and Boc-L-Asp (OBzl)-OH (79 mg,
0.244 mmol), HOBT (33 mg, 0.244 mmol), and DCC (55 mg,
0.268 mmol). The residue was purified by silica gel column chro-
matography (40:1 CH2Cl2–MeOH) to provide the product 7b
5c (420 mg, 0.579 mmol, 65%). ½a D25
ꢂ
+36.9 (c 0.1, CHCl3); IR (KBr,
1H NMR (500 MHz, CDCl3): d
9.5 Hz, N–H), 5.45
neat): 3328, 1695, 1655 cmꢃ1
;
(120 mg, 0.133 mmol, 55%). ½a D25
ꢂ
+13.7 (c 0.1, CHCl3); IR (KBr,
7.36–7.14 (20H, m, Ar-H), 6.79 (1H, d, J2,
neat): 3401, 3315, 3064, 1735, 1647, 1697, 1519 cmꢃ1
;
1H NMR
NH
(1H, d, JNH, 9.0 Hz, N–H), 5.21 (1H, d, J1, 3.0 Hz, H-1), 4.87–
a
2
(300 MHz, CDCl3): d 7.37–7.03 (25H, m, Ar-H), 6.85 (1H, d, JNH,
a
a
4.42 (6H, m, PhCH2), 4.29 (2H, dt, J2, 10.0 Hz, J1, 3.0 Hz, H-2),
3
2
8.1 Hz, N–H), 6.47 (1H, d, J2,
8.7 Hz, N–H), 5.47 (1H, d, JNH,
NH
4.23 (1H, m, H-a), 4.11 (1H, m, H-5), 3.856–3.622 (5H, m, H-3,
8.7 Hz, N–H), 5.17 (1H, d, J1, 3.3 Hz, H-1), 5.10 (2H, t, J 4.0 Hz,
2
H-4, H6a, H6b, CH2), 3.51 (1H, dd, J1 9.5 Hz, J2 6.5 Hz, CH2), 1.44
(9H, s, CH3); 13C NMR (125 MHz, CDCl3): d 172.9, 170.3, 156.9,
155.5, 138.4, 138.1, 138.0, 137.9, 137.4, 128.5, 128.4, 128.3,
127.9, 127.8, 127.7, 127.6, 127.5, 92.1, 80.3, 78.2, 74.9, 74.8, 73.5,
73.4, 73.2, 70.9, 69.7, 69.0, 53.6, 49.2, 28.3; ESIMS: m/z 749
(M+Na), 727 (M+1); HRMS: calcd for (C31H54N2O17 + Na), m/z
(749.33202); found, m/z (749.33120).
PhCH2), 4.81–4.48 (7H, m, PhCH2, H-a), 4.41 (1H, m, H-a), 4.19
(1H, dt, J1 9.6 Hz, J2 = 3.3 Hz, H-2), 4.03 (1H, m, H-5), 3.85 (1H, t, J
9.6 Hz, H-3), 3.74–3.61 (3H, m, H6a, H6b, H-4), 3.12 (1H, dd, J1
14.1 Hz, J2 6.0 Hz, CH2), 3.03 (1H, dd, J1 14.1 Hz, J2 6.9 Hz, CH2),
2.91 (1H, dd, J1 17.1 Hz, J2 4.8 Hz, CH2), 2.75 (1H, dd, J1 17.1 Hz,
J2 6.3 Hz, CH2), 1.41 (9H, s, CH3); 13C NMR (75 MHz, CDCl3): d
171.5, 170.8, 170.7, 155.5, 138.6, 138.2, 138.0, 136.5, 135.2,
129.3, 128.7, 128.6, 128.5, 128.4, 128.3, 127.9, 127.8, 127.6,
127.5, 126.9, 91.6, 81.1, 79.7, 78.3, 74.8, 74.7, 73.4, 70.7, 68.7,
67.1, 54.1, 53.9, 51.0, 36.7, 35.8, 28.2; ESIMS: m/z 924 (M+Na),
902 (M+1); HRMS: calcd for (C37H59N9O17 + Na), m/z
(924.39266); found, m/z (924.39006).
1.3. Deprotection of 5a–c
1.3.1. Preparation of 6a
A solution of 5a (246 mg, 0.380 mmol) in HCl in EtOAc (8 mL,
4 mol/L) was stirred at 0 °C for 3 h. The solvent was evaporated
and the residue was dissolved in EtOAc (20 mL) and again evapo-
rated to dryness. The resulting solid was used immediately in the
coupling reaction of the next step without additional purification.
1.4.3. Preparation of dipeptide derivative 7c
Compound 7c was prepared by a method similar to the prepa-
ration of 5a using 6c and Boc-L-Asp (OBzl)-OH (120 mg,
0.372 mmol), HOBT (50 mg, 0.372 mmol), and DCC (84 mg,
0.409 mmol). The residue was purified by silica gel column chro-
matography (40:1 CH2Cl2–MeOH) to provide the product 7b
1.3.2. Preparation of 6b
Compound 6b was prepared by a method similar to the prepa-
ration of 6a using 5b (170 mg, 0.244 mmol). The resulting solid
was used immediately in the coupling reaction of the next step
without additional purification.
(184 mg, 0.197 mmol, 53%). ½a D25
ꢂ
+24.1 (c 0.1, CHCl3); IR (KBr,
;
neat): 3334, 1733, 1699, 1643 cmꢃ1
1H NMR (500 MHz, CDCl3):
d 7.38–7.13 (25H, m, Ar-H), 6.89 (1H, d, JNH, 9.5 Hz, N–H), 5.56
a
(1H, d, JNH, 7.5 Hz, N–H), 5.17 (1H, d, J2,
10.5 Hz, N–H), 5.16
a
NH
1.3.3. Preparation of 6c
Compound 6c was prepared by a method similar to the prepa-
ration of 6a using 5c (270 mg, 0.372 mmol). The resulting solid
(1H, t, J1, 2 3.0 Hz, H-1), 5.12 (2H, dd, J1 22.0 Hz, J2 12.0 Hz, PhCH2),
4.85–4.34 (8H, m, PhCH2, H- 1, H- 2), 4.28 (1H, dt, J1 9.5 Hz, J2
3.0 Hz, H-2), 4.08(1H, m, H-5), 3.90 (1H, t, 9.5 Hz, H-3),
a
a
J

2002
J. Zhang et al. / Carbohydrate Research 346 (2011) 1997–2003
3.81–3.58 (4H, m, H-4, H-6a, H-6b, CH2), 3.53 (1H, dd, J1 9.5 Hz, J2
6.0 Hz, CH2), 3.01 (1H, dd, J 17.0 Hz, J2 4.5 Hz, CH2), 2.82 (1H, dd, J1
17.0 Hz, J2 5.0 Hz, CH2), 1.45 (9H, s, CH3); 13C NMR (125 MHz,
CDCl3): d 171.5, 171.3, 170.8, 169.6, 155.5, 138.6, 138.2, 137.9,
137.5, 137.4, 135.3, 135.1, 129.8, 128.6, 128.4, 128.3, 128.2,
127.9, 127.8, 127.7, 127.5, 92.0, 80.9, 80.2, 78.3, 74.9, 74.8, 73.4,
73.3, 70.8, 69.1, 68.9, 67.0, 53.8, 53.1, 51.1, 36.1, 28.3; ESIMS:
m/z 954 (M+Na), 932 (M+1); HRMS: calcd for (C43H61N7O16 + Na),
m/z (954.40725); found, m/z (954.40714).
d 172.7, 171.3, 170.5, 170.3, 169.4, 159.8, 155.9, 139.2, 138.8,
136.5, 128.8, 128.7, 128.6, 128.4, 128.3, 128.1, 127.9, 127.7, 91.3,
79.4, 79.2, 78.7, 74.5, 74.1, 72.8, 70.2, 69.6, 66.6, 66.2, 57.9, 54.4,
53.9, 49.8, 42.4, 36.3, 31.4, 29.6, 28.7, 25.3, 19.8, 17.7; ESIMS:
m/z 1134 (M+Na), 1112 (M); HRMS: calcd for (C55H77N5O19 + Na),
m/z (1134.51104); found, m/z (1134.51073).
Acknowledgments
This work was supported by National Natural Scientific Founda-
tion of China (20642004) and Educational Council Foundation of
Beijing (KM200710025012).
1.5. Deprotection of tripeptide derivatives
1.5.1. Preparation of 8a
Compound 8a was prepared by a method similar to the prepa-
ration of 6a using 7a (160 mg, 0.188 mmol). The resulting solid was
used immediately in the coupling reaction of the next step without
additional purification.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2011.04.010.
1.6. Introducing 3 into L-Val-Asp-Gly
References
1. Yao, N.; Fung, G.; Malekan, H.; Ye, L.; Kurth, M. J.; Lam, K. S. Carbohydr. Res.
2010, 345, 2277–2281.
2. Inaba, Y.; Kawakami, T.; Aimoto, S.; Ikegami, T.; Takeuchi, T.; Nakazawa, T.;
Yano, S.; Mikata, Y. Carbohydr. Res. 2009, 344, 613–626.
3. Dwek, R. A. Chem. Rev. 1996, 96, 683–720.
Compound 9a was prepared by a method similar to the prepa-
ration of 5a using 8a and Boc- -Gly-OH (33 mg, 0.188 mmol), HOBT
L
(25 mg, 0.188 mmol), and DCC (43 mg, 0.207 mmol). The residue
was purified by silica gel column chromatography (40:1 CH2Cl2–
MeOH) to provide the product 9a (121 mg, 0.133 mmol, 71%).
4. Varki, A. Glycobiology 1993, 3, 97–130.
5. Wong, C. Chem. Res. 1999, 32, 376–385.
½
a 2D5
ꢂ
+10.4 (c 0.1, CHCl3); IR (KBr, neat): 3436, 3282, 1718,
6. Herzner, H.; Reipen, T.; Schultz, M.; Kunz, H. Chem. Rev. 2000, 100, 4495–4537.
7. Lis, H.; Sharon, N. Eur. J. Biochem. 1993, 218, 1–27.
8. Collins, B. E.; Paulson, J. C. Curr. Opin. Chem. Biol. 2004, 8, 617–625.
9. Berger, E. G.; Boddecke, E.; Kamerhg, J. P.; Kobata, A.; Paulson, J. C.;
Vliegenthart, J. F. G. Experientia 1982, 38, 1129–1258.
1639 cmꢃ1 1H NMR (300 MHz, CDCl3): d 7.82 (1H, d, JNH,
;
a
7.8 Hz, N–H), 7.76 (1H, d, JNH, 7.8 Hz, N–H), 7.44–7.14 (20H, m,
a
Ar-H), 6.64 (1H, br s, N–H), 5.45 (1H, br s, N–H), 5.18 (1H, d, J1, 2
3.3 Hz, H-1), 5.10 (2H, m, PhCH2), 4.89–4.49 (7H, m, PhCH2,
10. Blithe, D. L. Trends Glycosci. Glycotech. 1993, 5, 81–98.
11. Taken, M.; Takasaki, S.; Shimada, M. J. Biol. Chem. 1990, 265, 12127–12130.
12. Takeuchi, M.; Inoue, N.; Stlckiand, T. W.; Kubota, M.; Wada, M.; Sbimizo, R.;
Hopi, S.; Kozutsumi, H.; Taksbashi, S.; Kobata, A. Proc. Natl. Acad. Sci. U.S.A.
1989, 86, 7819–7822.
13. Nisbi, T.; Bob, S. Glycotechnology 1992, 4, 336–344.
14. Jennings, H. J. Curr. Top. Microbiol. Immunol. 1990, 150, 97–127.
15. Jennings, H. J. Adv. Exp. Med. Biol. 1988, 228, 495–550.
16. Lee, C. J. Mol. Immunol. 1987, 24, 1005–1019.
H-a
1), 4.24 (2H, m, H-2, H-
a2), 4.10 (1H, m, H-5), 3.99–3.60 (6H,
m, H-3, H-4, H-6a, H-6b,
a-CH2), 2.99 (1H, m), 2.98 (1H, dd, J1
16.8 Hz, J2 7.2 Hz, CH2), 2.82 (1H, dd, J1 16.8 Hz, J2 6.0 Hz, CH2),
2.18 (1H, m, CH), 1.45 (9H, s, CH3), 0.83 (3H, d, J 6.6 Hz,CH3),
0.89 (3H, d, J 6.6 Hz,CH3); 13C NMR (75 MHz, CDCl3): d 171.4,
171.2, 170.5, 156.4, 138.5, 138.2, 138.1, 135.2, 128.6, 128.3,
127.8, 127.7, 127.6, 127.5, 126.8, 111.3, 91.5, 80.9, 79.6, 78.5,
74.8, 73.4, 70.8, 68.9, 67.1, 60.0, 53.9, 50.8, 50.1, 35.4, 29.5, 28.3,
19.5, 17.6; ESIMS: m/z 933 (M+Na); HRMS: calcd for
(C38H66N6O19 + Na), m/z (933.42804); found, m/z (933.42609).
17. Lindberg, A. A. Vaccine 1999, 17, 28–36.
18. Weintraub, A. Carbohydr. Res. 2003, 338, 2539–2547.
19. Wang, L. X.; Ni, J.; Singh, S.; Li, H. Chem. Biol. 2004, 11, 127–134.
20. Johnson, M. A.; Pinto, B. M. Top. Curr. Chem. 2008, 273, 116–126.
21. Johnson, M. A.; Pinto, B. M. Aust. J. Chem. 2002, 55, 13–25.
22. Borrelli, S.; Hossany, R. B.; Pinto, B. M. Clin. Vaccine Immunol. 2008, 15, 1106–
1117.
1.7. Deprotection of the tripeptide derivative
23. Yao, Nianhuan; Fung, Gabriel; Malekan, Hamed; Ye, Long; Kurth, Mark J.; Lam,
Kit S. Carbohydr. Res. 2010, 345, 2277–2281.
24. Nakahara, Y. Trends Glycosci. Glycotechnol. 2003, 15, 257–273.
25. Routenberg, L. K.; Seeberger, P. H. Angew. Chem. 2004, 116, 612–615.
26. Plante, O. J.; Palmacci, E. R.; Seeberger, P. H. Adv. Carbohydr. Chem. Biochem.
2003, 58, 35–54.
27. McDevitt, C. A.; Muir, H. J. Bone Joint Surg. B 1976, 58, 94–101.
28. Kelly, G. Altern. Med. Rev. 1998, 3, 27–29.
Compound 10a was prepared by a method similar to the prep-
aration of 6a using 9a (100 mg, 0.1094 mmol). The resulting solid
was used immediately in the coupling reaction in the next step
without purification.
29. Rong, X.; Song, L.; Zhan y, G.; Hua, H. Y.; Cui, p. L.; Xia, Ji.; Jin, H. F.; Peng, C. L.
Bioorg. Med. Chem. 2006, 14, 1706–1709.
1.8. Introducing 3 into L-Val-Asp-Gly-Arg
30. Pozzo, A. D.; Ni, M. H.; Esposito, E.; Dallavalle, S.; Musso, L.; Bargiotti, A.;
Pisano, C.; Vesci, L.; Bucci, F.; Castorina, M.; Foderà, R.; Giannini, G.; Aulicino,
C.; Penco, S. Bioorg. Med. Chem. 2010, 18, 64–72.
31. Seo, J. W.; Michaelian, N.; Owens, S. C.; Dashner, S. T.; Wong, A. J.; Barron, A. E.;
Carrasco, M. R. Org. Lett. 2009, 11, 5210–5213.
32. Cho, C. C.; Liu, J. N.; Chien, C. H.; Shie, J. J.; Chen, Y. C.; Fang, J. M. J. Org. Chem.
2009, 74, 1549–1556.
33. Scaffidi, A.; Stubbs, K. A.; Vocadlo, D. V.; Stick, R. V. Carbohydr. Res. 2008, 343,
2744–2753.
34. Swarts, B. M.; Chang, Y. C.; Hu, H. G.; Guo, Z. W. Carbohydr. Res. 2008, 343,
2894–2902.
35. Arnaout, M. A.; Goodman, S. L.; Xiong, J. P. Curr. Opin. Cell Biol. 2002, 14, 641–
651.
36. Plow, E. F.; Haas, T. A.; Zhang, L.; Loftus, J.; Smith, J. W. J. Biol. Chem. 2000, 275,
21785–21788.
37. Humphries, M. J. Biochem. Soc. Trans. 2000, 28, 311–339.
38. Trabocchi, A.; Menchi, G.; Cini, N.; Bianchini, F.; Raspanti, S.; Bottoncetti, A.;
Pupi, A.; Calorini, L.; Guarna, A. J. Med. Chem. 2010, 53, 7119–7128.
39. Varner, J. A.; Cheresh, D. A. Curr. Opin. Cell Biol. 1996, 8, 724–730.
40. Yin, R. T.; Zheng, H.; Xi, T.; Xu, H. M. Bioconjugate Chem. 2010, 21, 1142–1147.
41. Chen, H. S.; Mo, W.; Zhang, Y. L.; Su, H. B.; Ma, J. W.; Yao, R. M.; Zhang, S. H.; Ge,
J. B.; Song, H. Y. Eur. J. Pharm. 2007, 566, 137–144.
Compound 11a was prepared by a method similar to the prep-
aration of 5a using 10a, Boc- -Arg (NO2)-OH (35 mg, 0.1094 mmol),
L
HOBT (15 mg, 0.1094 mmol), and DCC (25 mg, 0.120 mmol). The
residue was purified by silica gel column chromatography (40:1
CH2Cl2–MeOH) to provide the product 11a (40 mg, 0.0361 mmol,
33%). ½a 2D5
ꢂ
+39.4 (c 0.1, CHCl3); 1H NMR (300 MHz, DMSO): d 8.44
(1H, br s, N–H), 8.27 (1H, d, JNH, 7.8 Hz, N–H), 8.16 (1H, d, JNH,
a
a
7.8 Hz, N–H), 8.06–7.81(2H, br s, N–H), 7.62 (1H, d, J2,
9.6 Hz,
NH
N–H), 7.33–7.14 (20H, m, Ar-H), 6.93 (1H, d, JNH, 7.2 Hz, N–H),
a
6.76 (1H, d, J 4.2 Hz, N–H), 5.06 (2H, m, PhCH2), 4.92 (1H, t, J1,
2
3.6 Hz, H-1), 4.76–4.39 (7H, m, PhCH2, H-
9.0 Hz, J2 5.5 Hz, H- 2), 4.19–3.40 (9H, m, H-2, H-3, H-4, H-5, H-
6a, H-6b, H- 3, H- 4), 3.12 (2H, m, CH2), 2.82 (1H, dd, J1 16.5 Hz,
a1), 4.31 (1H, dd, J1
a
a
a
J2 6.0 Hz, CH2), 2.62 (1H, dd, J1 16.5 Hz, J2 8.1 Hz, CH2), 1.94 (1H,
m, CH), 1.50 (4H, m, –CH2–CH2–), 1.37 (9H, s, CH3), 0.75 (3H, d, J
6.6 Hz, CH3), 0.89 (3H, d, J 6.6 Hz, CH3); 13C NMR (75 MHz, CDCl3):

J. Zhang et al. / Carbohydrate Research 346 (2011) 1997–2003
2003
}
}
42. Somsák, L.; Kovács, L.; Tóth, M.; Osz, E.; Szilágyi, L.; Gyorgydeák, Z.; Dinya, Z.;
45. Temelkoff, D. P.; Smith, C. R.; Kibler, D. A.; McKee, S.; Duncan, S. J.; Zeller, M.;
Hunsen, M.; Norris, P. Carbohydr. Res. 2006, 341, 1645–1656.
46. Yi, C. Z.; Ying, X. L.; Hua, S. G. Chin. Chem. Lett. 2005, 16, 179–182.
47. Sirion, U.; Purintawarrakun, S.; Sahakitpichan, P.; Saeeng, R. Carbohydr. Res.
2010, 345, 2401–2407.
Docsa, T.; Tóth, B.; Gergely, P. J. Med. Chem. 2001, 44, 2843–2848.
43. Murphy, P. V.; Pitt, N.; O’Brien, A.; Enright, P. M.; Dunne, A.; Wilson, S. J.;
Duane, R. M.; O’Boyle, K. M. Bioorg. Med. Chem. Lett. 2002, 12, 3287–3290.
44. Pitt, N.; Duane, R. M.; O’Brien, A.; Bradley, H.; Wilson, S. J.; O’Boyle, K. M.;
Murphy, P. V. Carbohydr. Res. 2004, 339, 1873–1887.