Carbohydrate-centered maleimide cluster as a new type of templates for multivalent peptide assembling: Synthesis of multivalent HIV-1 gp41 peptides

Article abstract of DOI:10.1016/S0968-0896(02)00339-5

This paper describes a facile synthesis of carbohydrate-centered maleimide clusters and their application as a new type of templates for multivalent peptide assembling. Simultaneous introduction of multiple maleimide functionalities onto a carbohydrate core was achieved through the reaction of carbohydrate-based polyamines with methoxycarbonylmaleimide or with the N-hydroxylsuccinimide ester of 6-maleimidohexanoic acid. The clustered maleimides placed on the carbohydrate core allow rapid and highly chemoselective ligation with multiple copies of cysteine-containing peptides under virtually neutral conditions at room temperature. This mild and highly efficient ligation method is extremely valuable for synthesizing large and complex multivalent peptides that may not be easily obtained by conventional ligation methods. The usefulness of the maleimide clusters as a new type of templates for multivalent peptide synthesis was exemplified by the synthesis of two tetravalent gp41 peptides incorporating the sequence of the potent HIV inhibitor, T20. The synthetic multivalent gp41 peptides are useful as novel immunogens to raise specific antibodies for HIV studies. They are also useful probes for studying HIV membrane fusion mechanisms.

Full text of DOI:10.1016/S0968-0896(02)00339-5

Bioorganic & Medicinal Chemistry 11 (2003) 159–166
Carbohydrate-Centered Maleimide Cluster as a New Type of
Templates for Multivalent Peptide Assembling:
Synthesis of Multivalent HIV-1 gp41 Peptides
Lai-Xi Wang,* Jiahong Ni and Suddham Singh
Institute of Human Virology, University of Maryland Biotechnology Institute, University of Maryland, 725 W. Lombard Street,
Baltimore, MD 21201, USA
Received 6 March 2002; accepted 16 April 2002
Abstract—This paper describes a facile synthesis of carbohydrate-centered maleimide clusters and their application as a new type of
templates for multivalent peptide assembling. Simultaneous introduction of multiple maleimide functionalities onto a carbohydrate
core was achieved through the reaction of carbohydrate-based polyamines with methoxycarbonylmaleimide or with the
N-hydroxylsuccinimide ester of 6-maleimidohexanoic acid. The clustered maleimides placed on the carbohydrate core allow rapid
and highly chemoselective ligation with multiple copies of cysteine-containing peptides under virtually neutral conditions at room
temperature. This mild and highly efficient ligation method is extremely valuable for synthesizing large and complex multivalent
peptides that may not be easily obtained by conventional ligation methods. The usefulness of the maleimide clusters as a new type
of templates for multivalent peptide synthesis was exemplified by the synthesis of two tetravalent gp41 peptides incorporating the
sequence of the potent HIV inhibitor, T20. The synthetic multivalent gp41 peptides are useful as novel immunogens to raise specific
antibodies for HIV studies. They are also useful probes for studying HIV membrane fusion mechanisms.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
multivalent peptide construction include cyclic peptide
and derivatives,13ꢀ15 porphyrin molecules,16,17 calix[4]-
arene core,10ꢀ12 and carbohydrates.18ꢀ21 Although multi-
valent peptide may be assembled by a stepwise solid-
phase peptide synthesis on an immobilized template, the
solid-phase synthesis itself and the purification of the
high molecular weight multivalent peptide product to
real homogeneity, after cleavage and de-protection,
present a clear challenge.1 The recent development of
techniques in chemoselective ligation of pre-assembled,
unprotected peptide segments has significantly enhanced
our ability to synthesize large, complex multivalent
peptides and proteins.3,7,8 Since polypeptides usually
contain a batch of functional groups (e.g., carboxyl,
amino, and hydroxyl groups, as well as aromatic side
chains), the success of the ligation approach relies on a
highly chemoselective reaction between a pair of
mutually reactive functionalities that are placed on the
unprotected peptide and the scaffold, respectively,
which should not react crossly with any other functional
groups in the polypeptides. Toward this end, two chemo-
selective reactions are most commonly used for multi-
valent peptide synthesis. One is the reaction of
Template assembled multivalent peptides have found
wide applications in recent years. For example, multiple
antigenic peptides (MAPs), in which 4–8 copies of an
antigenic peptide are attached to an oligo-lysine core,
were synthesized and used as synthetic vaccines against
HIV and other diseases.1ꢀ3 Compared to the conven-
tional vaccine preparations where an antigenic peptide
is attached to a carrier protein such as keyhole limpet
hemocyanin (KLH) and bovine serum albumin (BSA),
the MAPs constructs are chemically unambiguous and
immunologically focused, and often raise superior
immune responses.1,2,4ꢀ6 On the other hand, template
assembled multivalent peptides have been exploited to
design artificial proteins to study the folding and struc-
ture of proteins.7,8 Enhanced inhibitory activities of
peptide inhibitors were also attained through multi-
valent peptide assembly.9ꢀ12 In addition to oligo-lysine
core,3 other types of templates that were developed for
*Corresponding author. Tel.: +1-410-706-4982; fax: +1-410-706-
5068; e-mail: wangx@umbi.umd.edu
0968-0896/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00339-5

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L.-X. Wang et al. / Bioorg. Med. Chem. 11 (2003) 159–166
hydroxylamine or hydrazine nucleophiles with alde-
hydes/ketones, which results in the formation of oximes
and hydrazones, respectively.15,18,22,23 The reaction
should be performed under acidic conditions (pH 4–5),
and the oximes or hydrazones formed are chemically
stable under acidic to neutral conditions. The other
commonly used chemoselective reaction is the thioether
formation between thiol groups (usually from cysteine
residues) and bromoacetyl or chloroacetyl moieties
through nucleophilic substitution.24,25 Nevertheless, an
efficient nucleophilic substitution between a thiol-chloro-
acetyl (or bromoacetyl) reactive pair can efficiently take
place only under basic conditions (pH 8–9), and side
reactions between free amino groups in the polypeptides
and the haloacetyl groups may occur under the reaction
conditions.24
a cysteine residue at the C-terminus of the gp41 peptide
during solid phase synthesis, we found that a rapid and
highly chemoselective ligation between the cysteine-
containing gp41 peptide and a galactoside-based mal-
eimide cluster can be achieved under neutral conditions,
giving the target tetravalent peptides in high yield.
Results and Discussion
Agalactoside-based, tetravalent maleimide cluster ( 7)
was synthesized through a series of efficient chemical
transformations (Scheme 1). First, compound (1) was
allylated with allyl bromide to give the tetra-O-allyl
galactoside (2) (94%). The tetra-allyl derivative was
then subject to regioselective hydroboration with 9-bora-
bicyclo-[3.3.1]nonane (9-BBN), and subsequent alkaline
oxidation with H2O2 to give the tetra-O-(3-hydroxy-
propyl) galactoside (3) in 84% yield. To synthesize the
tetra-amino derivative (6) that is required for introducing
maleimide groups, the tetraol (3) was reacted with tri-
phenylphosphine-iodine in DMF to give the tetraiodide
(4) (70%), which was then converted into the azido-
compound (5) by treatment with NaN3 in DMF. Catalytic
hydrogenation of 5 afforded the tetra-amino derivative
(6) in a quantitative yield. Finally, Simultaneous intro-
duction of 4 maleimide groups was achieved by treating
the amine (6) with methoxycarbonylmaleimide in aqueous
MeCN containing NaHCO3 to give the tetravalent
maleimide cluster (7) in 76% yield (Scheme 1).
As part of our ongoing project on the development of
multivalent peptides as HIV vaccines and viral mem-
brane fusion inhibitors, we are interested in assembling
novel multivalent gp41 peptides on a carbohydrate
scaffold. The HIV-1 gp41 is an envelope glycoprotein
that mediates the fusion of viral and cellular mem-
branes, a critical step for HIV entry and infection. It
was reported that synthetic gp41 C-peptides (peptides
corresponding to the C-terminal ectodomain of gp41)
such as T20 and C34, potently inhibit membrane fusion
by both laboratory-adapted strains and primary isolates
of HIV-1.26ꢀ28 The critical roles of certain peptide
domains of gp41 in mediating membrane fusion provide
ideal targets for developing therapeutic and preventative
agents against HIV/AIDS.29 On the other hand, we
chose carbohydrates as the scaffold for displaying the
gp41 peptides because the rigid ring structure and the
distinct configurations of multiple functionalities in
carbohydrates make them unique platforms for topo-
logical accommodations of peptide chains. In fact,
monosaccharides and oligosaccharides have been
extensively exploited as scaffolds for the construction of
dendritic glycoligands30,31 and, more recently, for the
synthesis of carbopeptides and carboproteins.18ꢀ20
Although the reactive pairs of aminooxy-aldehyde and
thiol-haloacetyl have been widely used for chemoselec-
tive ligation in multivalent peptide synthesis, we found
that some of the gp41 peptides, such as the potent HIV
fusion inhibitor T20 (a 36-mer peptide from the C-term-
inal ectodomain of HIV-1 gp41), are either insoluble
under acidic conditions, thus preventing the application of
the popular oxime chemistry, or are not stable under basic
conditions and causing side reactions with haloacetyl
derivatives, making the nucleophilic substitution not fea-
sible for the ligation. Taking advantage of the well-estab-
lished, highly efficient Michael-type addition of a thiol
group to a maleimide moiety,32,33 we reasoned that the
reaction between a carbohydrate-centered maleimide
cluster and a thiol-containing peptide should allow a
highly chemoselective ligation under virtually neutral
conditions, thus providing a new and efficient approach
for multivalent peptide assembling. In this paper, we
report a facile synthesis of carbohydrate-centered mal-
eimide clusters. In addition, we report here the synthesis
of two tetravalent gp41 peptides, each of which contains 4
strands of the 36-mer gp41 peptide, T20. By introducing
The synthesis of maleimide clusters with variable length
of spacers between the carbohydrate core and the mal-
eimide was easily achievable by extending the spacers
during the synthesis. The length of spacers between the
carbohydrate core and the peptide chains is an important
factor to determine the orientation and intra-molecular
interaction of the peptide chains, which will eventually
affect the properties of the resulting multivalent pep-
tides.3,7,34 For the purpose, two tetravalent maleimide
clusters (compounds 9 and 10) that have longer spacers
between the maleimide and the carbohydrate core were
synthesized (Scheme 2). Briefly, four amino functional-
ities were introduced into the tetra-O-allyl derivative (2)
by photoaddition with cysteamine in MeOH.35 Instead
of using a large excess of cysteamine hydrochloride as
previously reported,35 we used only 3 molar equivalent
per OH of cysteamine hydrochloride and monitored the
1
reaction by measuring H NMR. When the reaction is
proceeding, the signals at d 5.10–6.05 (for the allyl
groups) decrease and the new signals at d 2.68–2.90 (for
SCH2) increase. After disappearance of the allyl signals,
the resulting product (8) was readily isolated in 80%
yield by Sephadex G-15 gel filtration chromatography.
We found that using less cysteamine hydrochloride did
not affect the efficiency of the reaction but greatly
facilitated the purification of the product by gel filtration.
Treatment of 8 with methoxycarbonylmaleimide gave
the tetravalent maleimide cluster (9) in 71% yield after
chromatographic purification. On the other hand, coupling
amine (8) with the N-hydroxylsuccinimide ester of
6-maleimidohexanoic acid afforded the tetravalent mal-
eimide cluster (10) in 43% yield (Scheme 2).

L.-X. Wang et al. / Bioorg. Med. Chem. 11 (2003) 159–166
161
Scheme 1. (a) Allyl bromide, NaH/DMF, 0 ꢁC to rt, 94%; (b) 9-BBN, THF, reflux, then 3 M aq NaOH, 30% H2O2, 0 ꢁC to rt, 84%; (c) PPh3/I2,
DMF, 80 ꢁC, 70%; (d) NaN3, DMF, rt, 78%; (e) Pd/C, H2, MeOH, rt, 100%; (f) methoxycarbonylmaleimide, 1 M aq NaHCO3, MeCN, rt, 76%.
Scheme 2. (a) Cysteamine hydrochloride, MeOH, hv, rt, 80%; (b) methoxycarbonylmaleimide, 1 M aq NaHCO3, MeCN, rt, 71%; (c) 6-mal-
eimidohexanoic acid N-hydroxylsuccinimidyl ester, 1 M aq NaHCO3, THF, 0 ꢁC to rt, 43%.
The established synthetic schemes are equally useful for the
synthesis of maleimide clusters on different carbohydrate
cores, which will allow the presentations of peptide chains
in distinct orientations as well as in different valencies. As
an example, a b-glucopyranoside-based, pentavalent mal-
eimide cluster was readily synthesized (Scheme 3). Briefly,
the penta-O-allyl b-glucoside (11), which was prepared
according to the reported procedure,36 was converted
into the amino-compound (12) in 80% yield by photo-
addition with cysteamine. Compound (12) was then
reacted with the N-hydroxylsuccinimide ester of 6-mal-
eimidohexanoic acid, giving the penta-valent maleimide
cluster (13) in 39% yield (Scheme 3).
The usefulness of the synthetic maleimide clusters was
exemplified by the synthesis of the tetravalent gp41
peptides 15 and 16, each of which contains four strands
of sequence of the peptide inhibitor T20 (Scheme 4).
T20 is a 36-mer peptide from the C-terminal ectodo-
main of HIV-1 gp41.26,27 The peptide exhibits potent
and broadly inhibitory activities against different strains
of HIV through blocking viral membrane fusion, and is
currently in clinical trials for the treatment of AIDS.37
In order to ligate the peptide to the maleimide cluster, a
cysteine residue was introduced at the C-terminus of the
peptide during solid-phase peptide synthesis. The
cysteine-containing peptide (14) was thus synthesized

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L.-X. Wang et al. / Bioorg. Med. Chem. 11 (2003) 159–166
Scheme 3. (a) Cysteamine hydrochloride, MeOH, hv, rt, 80%; (b) 6-maleimidohexanoic acid N-hydroxylsuccinimide ester, 1 M aq NaHCO3, THF,
0 ꢁC to rt, 39%.
using the Fmoc chemistry and purified by reverse-phase
HPLC. While peptide (14) is insoluble in aqueous buf-
fers below pH 6.5, it is readily soluble under neutral to
slightly alkaline conditions (pH 7.0–7.5). To our sur-
prise, the initial attempt to ligate the peptide (14) with
the maleimide cluster (7) in a phosphate buffer (50 mM,
pH 7.2) failed to give the desired tetravalent peptide.
However, the reaction resulted in a polymer-like solid
that, after lyophilization, can hardly dissolve again in
aqueous buffers or aqueous organic solvents, or in pure
organic solvents such as DMF and acetonitrile. Reverse
phase HPLC analysis of the reaction mixture reveals a
very broad peak following the peak of peptide (14).
Mass spectrometric analysis of the solid failed to give
any useful information. We assume that the solid mate-
rial may result from aggregation of the peptide, which
has a concentration of 1 mM in the reaction buffer. The
clustered maleimide may serve as a cross-link reagent
for the non-covalently aggregating peptides, promoting
further and eventually irreversible aggregations of the
peptides. It was previously revealed that, in aqueous
buffer (pH 7.0), T20 is monomeric at the concentration
below 10 mM, but exhibits complicated monomer/tetra-
mer equilibrium and other aggregation patterns when
the concentration is above 20 mM.38 We also observed
that, under native gel filtration conditions (PBS, pH 7.2)
and at 0.5 mM, both T20 and the synthetic peptide (14)
appear as a series of broad peaks that have molecular
weights corresponding to the oligomeric and polymeric
forms of the parent peptides (data not shown), indicat-
ing that indeed the peptides form aggregates at rela-
tively high concentrations. We eventually found that the
ligation of peptide (14) and the maleimide cluster (7)
can proceed very smoothly in a 1:1 acetonitrile-phos-
phate buffer (pH 7.0) to give the desired tetravalent
peptide (15) (Scheme 4). In the presence of high con-
centration of acetonitrile, the peptide could exist in
monomeric form. The ligation is rapid and highly effi-
cient. HPLC monitoring indicates that the reaction is
actually finished within 30 min. The tetravalent peptide
Scheme 4. (a) 1:1 MeCN–phosphate buffer (pH 7.0), room temp,
Yields: 82% for 15; 84% for 16.
(15) was purified in 82% yield by reverse-phase HPLC,
and the purity and identity of the product was con-
firmed by analytic HPLC and ES–MS, respectively. We
also prepared several short gp41 peptides that contain
the broadly neutralizing antibody 2F5’s epitope
ELDKWA39 and have a cysteine residue at either the

L.-X. Wang et al. / Bioorg. Med. Chem. 11 (2003) 159–166
163
C- or N-termini, and used them for the ligation. We
found that these peptides, which do not have solubility
or aggregation problems, can be ligated to the clustered
maleimides very rapidly in the range of pH 6.5–7.4 in an
aqueous buffer, giving the desired multivalent peptides
in virtually quantitative yields (data not shown).
40 ꢁC. The column was eluted with a linear gradient of
acetonitrile (10–90%) containing 0.1% TFAin 25 min
at a flow rate of 1 mL/min. Peptides were detected by
UV absorbance at 214 and/or 280 nm. Preparative
HPLC was performed with a Waters 600HPLC instru-
ment on a Waters C18 column (Symmetry300, 19ꢂ300
mm) and/or on a Delta-Pak C18 column [Delta-Pak
RCM 2ꢂ(2.5ꢂ10 cm)]. The column was eluted with a
linear gradient of acetonitrile (10–60%) containing
0.1% TFAat a flow rate of 10 mL/min. The peptides
were detected at 214/280 nm. Purified peptides were
lyophilized and kept under nitrogen in a freezer
(ꢀ20 ꢁC).
Similarly, ligation of the peptide (14) to a different
template, the tetravalent maleimide cluster (10), under
the same reaction conditions as described for the synth-
esis of 15 gave the tetravalent gp41 peptide (16) in 84%
yield after preparative HPLC (Scheme 4).
Ethyl 2,3,4,6-tetra-O-allyl-ꢀ-D-galactopyranoside (2). A
solution of ethyl a-d-galactopyranoside 1 (416.4 mg, 2.0
mmol) in anhydrous DMF (5 mL) was added dropwise
to a stirred suspension of sodium hydride (60% disper-
sion in mineral oil, 960.0 mg, 24.0 mmol) in anhydrous
DMF (15 mL). After 30 min, the reaction mixture was
cooled to 0 ꢁC, and allyl bromide (2.90 g, 24.0 mmol)
was added dropwise. The resulting mixture was stirred
for 1 h at 0 ꢁC, and 3 h at room temperature. After the
mixture was cooled in an ice-bath, excess sodium
hydride was quenched by slow addition of methanol (5
mL). The volatiles were evaporated to dryness, and then
the residue was mixed with ethyl acetate (150 mL) and
washed with brine (3ꢂ30 mL). The organic phase was
dried over Na2SO4, filtered, and concentrated. The oily
residue was purified by flash chromatography (hexane/
EtOAc 85:15) to afford compound 2 (690.0 mg, 94%) as
Conclusion
Afacile synthesis of carbohydrate-centered maleimide
clusters was described. The clustered maleimides placed
on the carbohydrate core allow rapid and highly chemo-
selective ligation with multiple copies of cysteine-contain-
ing peptides under virtually neutral conditions. The
mildness and high efficiency of the ligation reaction
make the method extremely valuable for synthesizing
large and complex multivalent peptides that may not be
easily obtained by conventional ligation methods. Par-
ticularly, the use of carbohydrates as the core molecule
allows the topological display of multiple peptide chains
with defined spatial orientations, depending on the nat-
ure of the sugar molecules and the length of the spacers.
The usefulness of the approach was exemplified by the
synthesis of two tetravalent gp41 peptides incorporating
the sequence of the potent HIV inhibitor, T20. The
immunogenicity of the synthetic multivalent gp41 pep-
tides are currently being evaluated and the results will
be reported elsewhere. The synthetic multivalent gp41
peptides are also useful probes for studying the HIV
membrane fusion mechanisms.
1
a colorless oil; Rf 0.65 (hexane/EtOAc 7:3); H NMR
(300 MHz, CDCl3/TMS)
d
6.05–5.84 (m, 4H,
OCH2CH¼CH2), 5.36–5.10 (m, 8H, OCH2CH¼CH2),
4.94 (d, 1H, J=3.7 Hz, H-1), 4.39 (dd, 1H, J=12.6, 5.6
Hz,
OCHHCH¼CH2),
4.30–3.98
(m,
7H,
OCH2CH=CH2), 3.93 (dd, 1H, J=7.1, 6.4 Hz, H-5),
3.84 (s, 1H, br, H-4), 3.83 (dd, 1H, J=9.8, 3.7 Hz, H-2),
3.80–3.60 (m, 2H, H-3, and OCHHCH3), 3.64–3.55 (m,
2H, H-6 and OCHHCH3), 3.52 (dd, 1H, J=9.0, 6.1 Hz,
H-60), 1.24 (t, 3H, J=7.1 Hz, OCH2CH3); ES–MS:
391.36 (M+Na)+, 323.32 (MꢀOEt)+, 207.14
Experimental
General methods
(M+2Na)2+
.
Fmoc-protected amino acids were purchased from
Novabiochem. HATU, DIPEA and Fmoc-PAL-PEG-
PS were purchased from Applied Biosystems. HPLC
grade acetonitrile was purchased from Fisher Scientific.
DMF was purchased from B & J Biosynthesis. All other
chemicals were purchased from Aldrich/Sigma and used
Ethyl 2,3,4,6-tetra-O-(3-hydroxypropyl)-ꢀ-D-galactopyr-
anoside (3). To a stirred solution of 2 (230.0 mg, 0.62
mmol) in dry THF (10 mL) was added dropwise a
solution of 9-BBN in THF (0.5 M, 10.0 mL, 5.0 mmol)
at room temperature. The reaction mixture was heated
under reflux for 2 h, and excess of 9-BBN was then
destroyed by dropwise addition of water at 0 ꢁC. The
hydroboration mixture was oxidized by treatment with
3 M aq NaOH (11.0 mL) and 30% H2O2 solution (11.0
mL) at 0 ꢁC, followed by stirring overnight at room
temperature. The mixture was saturated with K2CO3,
and the phases were separated. The aqueous phase was
extracted with THF (2ꢂ30 mL) and the combined
organic phases were dried over Na2SO4, filtered, and
concentrated. The oily residue was purified by column
chromatography (ethyl acetate/Methanol 8:2) to yield
tetraol 3 (231.0 mg, 84%) as a colorless oil; Rf 0.49
(EtOAc/MeOH 7:3); 1H NMR (D2O) d 5.10 (d, 1H,
J=3.4 Hz, H-1), 4.02 (dd, 1H, J=5.8, 4.8 Hz,
1
as received. H NMR spectra were recorded on QE 300
with Me4Si (d 0) as the internal standard. The ES–MS
spectra were measured on a Waters ZMD mass spec-
trometer. Analytical TLC was performed on glass plates
coated with silica gel 60 F254 (E. Merck). Carbohydrates
were detected by charring with 10% ethanolic sulfuric
acid. Amines were detected by ninhydrin spraying.
Flash chromatography was performed on silica gel 60
(200–400 mesh, EM Science). Gel filtration was carried
out on Sephadex G-15 (Pharmacia) using de-ionized
water as the eluent. Photo-addition reactions were car-
ried out in a quartz flask under N2. Analytical HPLC
was carried out with a Waters 626HPLC instrument on
a Waters Nova-Pak C18 column (3.9ꢂ150 mm) at

164
L.-X. Wang et al. / Bioorg. Med. Chem. 11 (2003) 159–166
OCHHCH2CH2OH), 3.94 (s, 1H, br, H-4), 3.86–3.50
(m, 22H, H-2, H-3, H-5, H-6, H-60, OCH2CH2CH2OH,
OCH2CH2CH2OH and OCH2CH3), 1.86–1.75 (m, 8H,
OCH2CH2CH2OH), 1.19 (t, 3H, J=7.1 Hz,
OCH2CH3); ES–MS: 463.45 (M+Na)+, 441.47
stirred at room temperature for 4 h. After adding
CH2Cl2 (50 mL), the organic layer was separated and
washed with brine (3ꢂ10 mL). The organic layer was
then dried (Na2SO4), filtered, and concentrated. The
oily residue was purified by flash chromatography
(CH2Cl2/MeOH 95:5) to give maleimide 7 (23.2 mg,
76%); Rf 0.42 (CH2Cl2/MeOH 95:5); 1H NMR (CDCl3/
TMS) d 6.70 (s, 2H, CH=CH), 6.69 (s, 4H, CH=CH),
6.68 (s, 2H, CH=CH), 4.98 (d, 1H, J=3.4 Hz, H-1), 3.95–
3.82 (m, 2H, H-4, and OCHHCH2CH2N), 3.80ꢃ3.35 (m,
22H, H-2, H-3, H-5, H-6, H-60, OCH2CH2CH2N,
OCH2CH2CH2N, and OCH2CH3), 1.96–1.78 (m, 8H,
OCH2CH2CH2N), 1.25 (t, 3H, J=7.1 Hz, OCH2CH3);
(M+H)+, 395.43 (MꢀOEt)+, 243.26 (M+2Na)2+
.
Ethyl 2,3,4,6-tetra-O-(3-azidopropyl)-ꢀ-D-galactopyra-
noside (5). To a stirred solution of Ph3P (1.00 g, 3.81
mmol) in dry DMF (3 mL) was added iodine (0.97 g,
3.81 mmol). After 10 min, a solution of tetraol 3 (210.0
mg, 0.48 mmol) in DMF (2 mL) was added dropwise.
The resulting mixture was stirred for 2 h at room tem-
perature and another 2 h at 80 ꢁC. Heating was then
discontinued and the mixture was concentrated under
reduced pressure to remove DMF. The residue was
purified by flash chromatography (hexane/EtOAc 8:2)
to give tetraiodide 4 (293.0 mg, 70%). The iodide 4 thus
obtained was used immediately for the next step. Iodide
4: Rf 0.65 (hexane/EtOAc 7:3); 1H NMR (CDCl3/TMS)
d 4.97 (d, 1H, J=3.4 Hz, H-1), 3.96–3.83 (m, 2H, H-4,
and OCHHCH2CH2I), 3.80–3.47 (m, 14H, H-2, H-3, H-
5, H-6, H-60, OCH2CH2CH2I, and OCH2CH3), 3.36–
3.24 (m, 8H, OCH2CH2CH2I), 2.20–1.98 (m, 8H,
OCH2CH2CH2I), 1.23 (t, 3H, J=7.1 Hz, OCH2CH3).
ES–MS: 779.28 (M+Na)+, 401.29 (M+2Na)2+
.
Ethyl 2,3,4,6-tetra-O-(6-amino-3-thia-hexyl)-ꢀ-D-galac-
topyranoside tetrahydrochloride (8). To a solution of the
tetra-O-allyl derivative 2 (404.6 mg, 1.10 mmol) and
AIBN (30.0 mg) in methanol (15 mL) in a Quartz flask
was added cysteamine hydrochloride (1.50 g, 13.20 mmol).
A fter being degassed by bubbling N into solution for 30
2
min, the resulting mixture was stirred and irradiated (UV,
254 nm) under N2. The reaction was monitored by mea-
1
suring the H NMR of a small portion of the reaction
mixture, which was dried and deuterium-exchanged with
D2O before recording the NMR. During the progress of
the reaction, the signals at d 5.10–6.05 (for the allyl
groups) decreased and the new signals at d 2.68–2.90
(for SCH2) increased. After 24 h, NMR indicated the
disappearance of the allyl signals. MeOH was then evapo-
rated and the residue was purified by gel filtration on a
Sephadex G-15 column using water as the eluent. Fractions
containing the product were pooled and lyophilized to give
amine 8 (723.4 mg, 80%) as a colorless glass-like solid;
1H NMR (D2O) d 5.08 (d, 1H, J=3.4 Hz, H-1), 4.06–
3.98 (m, 1H, OCHHCH2CH2S), 3.96–3.92 (m, 1H, H-4),
3.90ꢃ3.48 (m, 14H, H-2, H-3, H-5, H-6, H-60,
OCH2CH2CH2S, and OCH2CH3), 3.26ꢃ3.14 (m, 8H,
SCH2CH2NH2HCl), 2.90–2.82 (m, 8H, SCH2CH2NH2-
HCl), 2.72–2.68 (m, 8H, OCH2CH2CH2S), 1.96–1.78 (m,
8H, OCH2CH2CH2S), 1.20 (t, 3H, J=7.1 Hz, OCH2CH3);
Amixture of iodide 4 (260.0 mg, 0.30 mmol) and NaN3
(1.24 g, 19.07 mmol) in dry DMF (10 mL) was stirred
overnight at room temperature. The mixture was eva-
porated at reduced pressure to dryness, and the residue
was partitioned in CH2Cl2 (100 mL) and water. The
organic layer was washed with brine (3ꢂ20 mL), dried
(Na2SO4), filtered, and concentrated. The oily residue
was purified by flash chromatography (hexane/EtOAc
8:2) to provide tetraazide 5 (124.0 mg, 78%); Rf 0.41
1
(hexane/EtOAc 7:3); H NMR (CDCl3/TMS) d 4.95 (d,
1H, J=3.4 Hz, H-1), 3.96–3.85 (m, 2H, H-4, and
OCHHCH2CH2N3), 3.80–3.47 (m, 14H, H-2, H-3, H-5,
H-6, H-60, OCH2CH2CH2N3, and OCH2CH3), 3.47–
3.35 (m, 8H, OCH2CH2CH2N3), 1.95–1.76 (m, 8H,
OCH2CH2CH2N3), 1.25 (t, 3H, J=7.1 Hz, OCH2CH3);
ES–MS: 563.39 (M+Na)+, 467.42 (MꢀOEtꢀN2)+.
ES–MS: 677.1 (M+H-4HCl)+, 339.1 (M+2H-4HCl)2+
.
Ethyl 2,3,4,6-tetra-O-(3-aminopropyl)-ꢀ-D-galactopyra-
noside (6). Azide 5 (61.0 mg, 0.11 mmol) was hydro-
genated with Pd/C (10%, 10 mg) in methanol (5 mL)
overnight at room temperature. The mixture was fil-
tered through a bed of Celite, and the filtrate was then
Ethyl 2,3,4,6-tetra-O-(6-maleimido-3-thia-hexyl)-ꢀ-D-ga-
lactopyranoside (9). Asolution of amine 8 (23.9 mg,
29.0 mmol) dissolved in 1 M aqueous solution of
NaHCO3 (1 mL) was treated with methoxycarbonyl-
maleimide (27.03 mg, 17.4 mmol) at 0 ꢁC. After 5 min,
the mixture was diluted with water (1 mL) and acetoni-
trile (2 mL), and then stirred at room temperature for 1
h. The mixture was diluted with CH2Cl2 (50 mL) and
washed with brine (3ꢂ10 mL). The organic phase was
dried over Na2SO4, filtered and concentrated. The oily
residue was purified by flash chromatography (CH2Cl2/
MeOH 96:4) to give maleimide 9 (20.3 mg, 71%); Rf
1
concentrated to give tetraamine 6 (51.5 mg, 100%); H
NMR (D2O) d 5.10 (d, 1H, J=3.4 Hz, H-1), 4.06–3.98
(m, 1H, OCHHCH2CH2NH2), 3.96–3.92 (m, 1H, H-4),
3.90ꢃ3.48 (m, 14H, H-2, H-3, H-5, H-6, H-60,
OCH2CH2CH2NH2, and OCH2CH3), 2.89–2.70 (m, 8H,
OCH2CH2CH2NH2), 1.90–1.65 (m, 8H, OCH2CH2-
CH2NH2), 1.18 (t, 3H, J=7.1 Hz, OCH2CH3); ESꢀMS:
437.48 (M+H)+, 219.16 (M+2H)2+
.
0.45 (CH2Cl2/MeOH 96:4); H NMR (CDCl3/TMS) d
1
6.72 (s, 8H, CH¼CH), 4.94 (d, 1H, J=3.4 Hz, H-1),
3.95–3.82 (m, 2H, H-4, and OCHHCH2CH2N),
3.80ꢃ3.45 (m, 22H, H-2, H-3, H-5, H-6, H-60,
OCH2CH2CH2S, SCH2CH2N, and OCH2CH3), 2.90–
2.60 (m, 16H, OCH2CH2CH2SCH2CH2N), 1.96–1.82 (m,
8H, OCH2CH2CH2S), 1.23 (t, 3H, J=7.1 Hz,
OCH2CH3); ES–MS: 1019.49 (M+Na)+, 951(M-OEt)+.
Ethyl 2,3,4,6-tetra-O-(3-maleimidopropyl)-ꢀ-D-galacto-
pyranoside (7). Asolution of amine 6 (17.8 mg, 0.04
mmol) in 1 M aqueous solution of NaHCO3 (1 mL) was
treated with methoxycarbonylmaleimide (37.94 mg, 0.24
mmol) at 0 C. After 5 min, the mixture was diluted
with water (1 mL) and acetonitrile (2 mL), and then
ꢁ

L.-X. Wang et al. / Bioorg. Med. Chem. 11 (2003) 159–166
165
Ethyl
2,3,4,6-tetra-O-[(6-(6-maleimidohexanamido)-3-
maleimide 13 (76.7 mg, 39%); Rf 0.35 (CH2Cl2/MeOH
95:5); 1H NMR (CDCl3/TMS)
6.69 (s, 10H,
thia-hexyl)]-ꢀ-D-galactopyranoside (10). Asolution of
amine 8 (138.6 mg, 0.17 mmol) in 1 M aqueous solution
of NaHCO3 (1.5 mL) was added dropwise to a stirred
solution of 6-maleimidohexanoic acid N-hydroxyl-
succinimide ester (272.5 mg, 0.88 mmol) in THF (3 mL),
which was cooled with a bath of ice-water. The resulting
mixture was stirred for 1 h at 0–5 ꢁC. The mixture was
then diluted with CHCl3 (70 mL) and washed with brine
(3ꢂ10 mL). The organic phase was dried over Na2SO4,
filtered, and concentrated. The oily residue was purified
by flash chromatography (CH2Cl2/MeOH 95:5) to give
maleimide 10 (103.6 mg, 43%); Rf 0.62 (CH2Cl2/MeOH
d
CH¼CH), 6.28–6.15 (m, 5H, NH), 4.18 (d, 1H, J=7.8
Hz, H-1), 3.95–3.50 (m, 13H, H-5, H-6, H-60,
OCH2CH2CH2S), 3.50 (t, 10H, J=7.2 Hz,
NCH2(CH2)4), 3.50–3.38 (m, 10H, SCH2CH2N), 3.24–
3.16 (m, 2H, H-3, H-4), 3.04–2.96 (m, 1H, H-2), 2.70–
2.58 (m, 20H, OCH2CH2CH2SCH2CH2N), 2.18 (t, 10H,
J=7.3 Hz, N(CH2)4CH2 ), 1.92–1.78 (m, 10H,
OCH2CH2CH2S), 1.74–1.54 (m, 20H, NCH2CH2CH2-
CH2CH2), 1.38–1.24 (m, 10H, N(CH2)2CH2(CH2)2);
ES–MS: 1733.16 (M+H)+, 866.84 (M+2H)2+
.
1
9:1); H NMR (CDCl3/TMS) d 6.68 (s, 8H, CH¼CH),
Peptide (14). The peptide 14 was synthesized on a Pioneer
Peptide Synthesizer (Applied Biosystems, Foster City,
CA, USA) using Fmoc-chemistry on Fmoc-PAL-PEG-PS
resin. 4-fold excess of Na-Fmoc-protected amino acids were
used for each coupling and HATU/DIPEA were used as
the coupling reagents. The N-terminus of the peptide was
capped with an acetyl group. Cleavage of the peptide from
the resin with simultaneous deprotection was performed
with TFA/thioanisole/EDT/anisole (90:5:3:2) (cocktail R),
and the crude peptide was precipitated with cold ether.
Purification of the peptide was carried out by preparative
HPLC as described in the general methods. The purified 14
appeared as a single peak on analytic HPLC. Under the
analytic conditions (General Methods), the peptide was
6.26–6.20 (m, 2H, NH), 6.12 (t, 1H, J=6.8 Hz, NH),
6.04 (t, 1H, J=6.8 Hz, NH), 4.92 (d, 1H, J=3.2 Hz, H-
1), 3.95–3.86 (m, 2H, H-4, and OCHHCH2CH2N),
3.80ꢃ3.40 (m, 30H, H-2, H-3, H-5, H-6, H-60,
OCH2CH2CH2S, SCH2CH2N, NCH2(CH2)4 and
OCH2CH3), 2.75–2.60 (m, 16H, OCH2CH2CH2-
SCH2CH2N), 2.18 (t, 8H, J=7.3 Hz, N(CH2)4CH2),
1.96–1.82 (m, 8H, OCH2CH2CH2S), 1.74–1.54 (m, 16H,
NCH2CH2CH2CH2CH2),
N(CH2)2CH2(CH2)2), 1.22 (t, 3H, J=7.1 Hz, OCH2CH3);
ES–MS: 1450.01 (M+H)+, 725.56 (M+2H)2+
1.37–1.24
(m,
8H,
.
(6-Amino-3-thia-hexyl) 2,3,4,6-tetra-O-(6-amino-3-thia-
hexyl)-ꢁ-D-glucopyranoside pentahydrochloride (12). To
a solution of penta-O-allyl derivative 1136 (628.5 mg,
1.65 mmol) and AIBN (50.0 mg) in methanol (15 mL) in
a Quartz flask was added cysteamine hydrochloride
(2.80 g, 24.67 mmol). After being degassed by bubbling
N2 into the solution for 30 min, the resulting mixture
was stirred and irradiated (UV, 254 nm) under N2. The
eluted at 15.84 min. ES–MS of 14: 1532.84 (M+3H)3+
1149.80 (M+4H)4+, 920.12 (M+5H)5+
,
.
Tetravalent peptide (15). To a solution of peptide 14
(20.0 mg, 4.35 mmol) in phosphate buffer (50 mM, pH
7.0, 3.0 mL) and acetonitrile (3.0 mL) was added drop-
wise a solution of maleimide 7 (0.52 mg, 0.69 mmol) in
DMF (104 mL). After shaken at room temperature for 2
h, the reaction mixture was lyophilized. The residue was
purified by RP-HPLC as described in general methods,
giving the tetravalent peptide 15 (10.7 mg, 82%) as a
white powder. Analytical HPLC showed that the purified
15 showed up at 16.47 min as a single peak. ES–MS of
1
reaction was monitored by H NMR, After 24 h when
NMR showed the disappearance of the allyl signals, the
solvent removed by evaporation and the residue was
subjected to gel filtration on a column of Sephadex G-
15 using water as the eluent. Fractions containing the
product were pooled and lyophilized to give amine 12
1
(1.25 g, 80%) as a colorless glass-like solid; H NMR
15: 1914.75 (M+10H)10+
1596.01 (M+12H)12+, 1473.36 (M+13H)13+, 1368.29
(M+14H)14+, 1276.76 (M+15H)15+
, ,
1741.00 (M+11H)11+
(D2O) d 4.43 (d, 1H, J=7.8 Hz, H-1), 4.04–3.40 (m,
13H, H-5, H-6, H-60, OCH2CH2CH2S), 3.40 (dd, 1H,
J=8.5, 9.1 Hz, H-4), 3.36 (dd, 1H, J=9.1, 9.3 Hz, H-3),
3.26–3.16 (m, 10H, SCH2CH2NH2HCl), 3.11 (dd, 1H,
J=7.8, 9.3 Hz, H-2), 2.90–2.82 (m, 10H, SCH2-
CH2NH2HCl), 2.72–2.64 (m, 10H, OCH2CH2CH2S),
1.96–1.84 (m, 10H, OCH2CH2CH2S); ES–MS: 766.1
.
Tetravalent peptide (16). To a solution of peptide 14
(20.0 mg, 4.35 mmol) in phosphate buffer (50 mM, pH 7.0,
3.0 mL) and acetonitrile (3.0 mL) was added a solution of
maleimide 10 (1.05 mg, 0.72 mmol) in DMF (210 mL). After
2 h at room temperature, the reaction mixture was lyophi-
lized and the residue was purified by preparative HPLC as
described in General Methods to afford the tetravalent
peptide 16 (12 mg, 84%). Analytical HPLC showed a sin-
gle peak for 16 with a retention time of 16.85 min. ES–MS
(M+Hꢀ5HCl)+, 383.7 (M+2Hꢀ5HCl)2+
.
[6-(6-Maleimidohexanamido)-3-thia-hexyl] 2,3,4,6-tetra-
O-[6-(6-maleimidohexanamido)-3-thia-hexyl)-ꢁ-D-gluco-
pyranoside (13). Asolution of amine 12 (109.8 mg, 0.12
mmol) in 1 M aqueous solution of NaHCO3 (1.2 mL)
was added dropwise to a stirred solution of 6-mal-
eimidohexanoic acid N-hydroxylsuccinimide ester
(214.2 mg, 0.70 mmol) in THF (3 mL) that was cooled
with a bath of ice-water. The resulting mixture was
stirred for 1 h at 0–5 ꢁC, and then diluted with CHCl3
(70 mL). The organic layer was separated and washed
with brine (3ꢂ10 mL), dried (Na2SO4), filtered, and
concentrated. The oily residue was subjected to column
chromatography (CH2Cl2/MeOH 95:5) to give penta-
of 16: 1653.72 (M+12H)12+, 1526.60 (M+13H)13+
,
1417.63 (M+14H)14+, 1323.20 (M+15H)15+
.
Acknowledgements
The work was financially supported by the Institute of
Human Virology, University of Maryland Biotechnology
Institute. We thank Prof. George Lewis for stimulating
discussions.

166
L.-X. Wang et al. / Bioorg. Med. Chem. 11 (2003) 159–166
20. Jensen, K. J.; Barany, G. J. Pept. Res. 2000, 56, 3.
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