Synthesis, conformation, and immunogenicity of monosaccharide-centered multivalent HIV-1 gp41 peptides containing the sequence of DP178

Article abstract of DOI:10.1016/j.bmc.2004.04.008

Several monosaccharide-centered multivalent HIV-1 gp41 peptides containing the sequence of DP178 were synthesized. Conformational studies showed that multivalent assembly enhanced the α-helical content of the peptide. Therefore, 2-, 3-, or 4-α-helix bundles of peptide DP178 could be obtained by assembling the peptide on a suitable bi-, tri-, or tetravalent template. Immunization studies indicated that while peptide DP178 alone was poorly immunogenic, the tetravalent peptide MVP-1 raised high titers of antibodies in mice that recognize not only peptide DP178 but also the native HIV-1 glycoprotein gp41, even in the absence of a carrier protein or adjuvant. The study suggests that carbohydrate-centered multivalent peptides provide not only a model for mimicking protein α-helix-bundle structure, but also an effective immunogen for raising high-titer antibodies against HIV-1 envelope glycoprotein gp41.

Full text of DOI:10.1016/j.bmc.2004.04.008

Bioorganic & Medicinal Chemistry 12 (2004) 3141–3148
Synthesis, conformation, and immunogenicity of
monosaccharide-centered multivalent HIV-1 gp41 peptides
containing the sequence of DP178
Jiahong Ni,a Robert Powell,a Ilia V. Baskakov,b Anthony DeVico,a
George K. Lewisa and Lai-Xi Wanga,*
aInstitute of Human Virology, University of Maryland Biotechnology Institute, University of Maryland, 725 Lombard Street,
Baltimore, MD 21201, USA
bMedical Biotechnology Center, University of Maryland Biotechnology Institute, University of Maryland, Baltimore, MD 21201, USA
Received 4 December 2003; revised 8 April 2004; accepted 8 April 2004
Available online 12 May 2004
Abstract—Several monosaccharide-centered multivalent HIV-1 gp41 peptides containing the sequence of DP178 were synthesized.
Conformational studies showed that multivalent assembly enhanced the a-helical content of the peptide. Therefore, 2-, 3-, or 4-a-
helix bundles of peptide DP178 could be obtained by assembling the peptide on a suitable bi-, tri-, or tetravalent template.
Immunization studies indicated that while peptide DP178 alone was poorly immunogenic, the tetravalent peptide MVP-1 raised high
titers of antibodies in mice that recognize not only peptide DP178 but also the native HIV-1 glycoprotein gp41, even in the absence
of a carrier protein or adjuvant. The study suggests that carbohydrate-centered multivalent peptides provide not only a model for
mimicking protein a-helix-bundle structure, but also an effective immunogen for raising high-titer antibodies against HIV-1
envelope glycoprotein gp41.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
of the ectodomain of gp41, which provides the driving
force for membrane fusion.4 Several pieces of evidence
suggest that the trimeric a-helical structure of the C-
terminal region of gp41 is an ideal target for HIV-1
vaccine design. First, the C-terminal peptide region of
gp41 is highly conserved among HIV-1 strains;4;5 sec-
ondly, the epitopes of several broadly neutralizing anti-
gp41 antibodies, including 2F5 and 4E10, were mapped
to the C-ectodomain region of gp41,6–10 suggesting that
the region is accessible to antibody neutralization; and
thirdly, synthetic peptides (C-peptides) derived from this
region, such as DP17811;12 and C34,5;13 are potent
inhibitors of HIV-1 infection. In particular, the sequence
of the potent HIV-1 inhibitor DP178, also called T20
that was recently approved as a new drug for the
treatment of AIDS, overlaps the epitope sequence NE-
QELLELDKWASLWN of neutralizing antibody
2F5.6;7 However, attempts to use peptide DP178 and its
conformation-constrained variants as immunogens
failed to raise neutralizing antibodies reactive to HIV-1
strains.14;15 The experimental data suggest that the
actual epitopes may be more complex in nature than a
single, linear peptide moiety.
The worldwide epidemic of HIV/AIDS urges develop-
ment of an effective HIV vaccine. It has been difficult to
design immunogens that are able to raise broadly neu-
tralizing antibodies against HIV-1 primary isolates.1–3
An essential step in HIV-1 entry involves virus-cell
membrane fusion mediated by HIV-1 envelope glyco-
protein gp41. On the envelope of HIV-1, gp41 forms a
trimeric complex associated with another envelope gly-
coprotein gp120. HIV-1 entry is initiated by the binding
of envelope glycoprotein gp120 to host cell receptor
CD4 and chemokine co-receptor CCR5 or CXCR4. The
binding triggers conformational changes in gp41, lead-
ing to the exposure of gp41, insertion of the N-terminal
fusion peptide into host membrane, and the subsequent
formation of the fusogenic, six a-helix-bundle structure
Keywords: Multivalent peptides; HIV; gp41; Conformation; Carbohy-
drate; Immunogenicity; Antibody; Vaccine.
* Corresponding author. Tel.: +1-410-706-4982; fax: +1-410-706-5068;
e-mail: wangx@umbi.umd.edu
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.04.008

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J. Ni et al. / Bioorg. Med. Chem. 12 (2004) 3141–3148
Considering that in membrane fusion and viral entry,
the functional unit of gp41 is an oligomeric (most likely
a trimeric), parallel stranded, coiled coil complex rather
than a monomeric gp41, and that the oligomeric gp41
complex undergoes conformational changes during viral
membrane fusion,4 we speculate that a conformation-
restrained, multivalent (oligomeric) structure consisting
of two or more gp41 C-peptide sequences may constitute
real epitopes of the broadly neutralizing antibodies
specific for the region. To capture or mimic the putative
multivalent structure, we attempted to assemble the
conserved gp41 C-peptides on a suitable scaffold to form
novel multivalent peptides in the hope that the multi-
valent assembly will form a-helix bundles resembling the
pre-fusogenic state of gp41. In this paper, we report the
synthesis, conformations, and immunization studies of
galactose-based, di-, tri-, and tetravalent HIV-1 gp41
peptides containing the sequence of DP178. We ob-
served that the template-assembled peptides form a-he-
lix bundles. Immunization studies showed that the
synthetic multivalent peptides raise high titers of anti-
bodies in mice that not only bind to the peptide im-
munogens used for the immunization, but also recognize
HIV-1 envelope glycoprotein gp41.
Figure 1. The sequence and epitope mapping of the HIV-1 inhibitor
DP178.
membrane fusion, an essential step for viral entry. Sec-
ondly, the sequence overlaps the extended linear epitope
NEQELLELDKWASLWN (aa656-671 of gp160) of the
broadly neutralizing antibody 2F5. Thirdly, a search of
the database on T-helper epitopes in gp41 (http://hiv-
web.lanl.gov) reveals that the DP178 sequence contains
at least two T-helper epitopes, located at aa647-662 and
aa650-661 of HIV-1 envelope gp160 (Fig. 1). With both
B- and T-epitopes in the sequence, the resulting multi-
valent gp41 peptides containing DP178 are expected to
be immunogenic even in the absence of a carrier protein
or any other adjuvant.16
We previously reported the synthesis of carbohydrate-
centered maleimide clusters and their use as templates
for multivalent peptide assembly.17 The same approach
was used for preparing galactose-based bi-, tri-, and
tetravalent DP178 peptides. The tetravalent maleimide
cluster (2) was readily synthesized starting from methyl
2. Results and discussion
2.1. Synthesis of multivalent HIV-1 gp41 peptides
containing DP178
a–D-galactopyranoside (1) according to the reported
procedure.17 To prepare galactose-based bi- and triva-
lent maleimide clusters, a series of regio-selective mod-
We chose the sequence of the potent HIV-1 inhibitor
DP17811 for immunogen design for several reasons.
First, this sequence is conserved and is involved in viral
ifications
were
performed
on
methyl
a–
galactopyranoside (1). The synthesis was summarized in
Scheme 1. In the trivalent maleimide cluster (9), malei-
Scheme 1. Synthesis of maleimide clusters. Reactions and conditions: (a) tert-butyl(dimethyl)silyl chloride (1.2 mol equiv), pyridine, 0 ꢁC–rt; (b) 2,2-
dimethoxylpropane, p-TsOH (cat.), acetone, rt; (c) Mel, NaH, DMF, 0 ꢁC–rt; (d) tetrabutylammonium fluoride, THF, rt; (e) 90% acetic acid, 90 ꢁC;
(f) allyl bromide, NaH, DMF, 0–25 ꢁC; (g) cysteamine hydrochloride, MeOH, hm (254 nm), rt; (h) 6-maleimidohexanoic acid N-hydroxylsuccinimide
ester, aq NaHCO3, THF, 0–5 ꢁC.

J. Ni et al. / Bioorg. Med. Chem. 12 (2004) 3141–3148
3143
mide functionality was installed on the 3, 4, and 6-
positions of the galactose-core. In the bivalent malei-
mide cluster (14), the maleimide functionality was
selectively assembled on the 3 and 4-positions of the
galactose-core, with the hydroxyl groups at 2- and 6-
positions being protected by methyl groups (Scheme 1).
BVP-1, TVP-1, and MVP-1 were measured at 23 ꢁC in a
phosphate buffer (pH 7.2). Alpha-helical content was
calculated based on the mean residue ellipticity ½hꢀ
222
according to the proposed equation.24 The a-helical
contents for BVP-1, TVP-1, and MVP-1 were found to
be 30%, 32%, and 39%, respectively, in the phosphate
buffer. Under the same condition, the a-helical content
of DP178 was measured to be 18%, which is consistent
with the reported value.25 The tetravalent peptide MVP-
1 demonstrated the highest a-helical content among the
synthetic multivalent peptides. A comparison of the CD
spectra for DP178 and MVP-1 was shown in Figure 2. It
was reported that DP178 turns to aggregate in buffer
when the concentration is above 20 lM. We observed
that the CD spectra of DP178, BVP-1, TVP-1, and
MVP-1 were concentration independent in the range of
2–40 lM (data not shown). Thus, our data indicates that
the template-assembled bi-, tri-, and tetravalent peptides
all form a-helix bundles to some extent, with the tetra-
valent peptide MVP-1 possessing the highest content of
a-helical structure.
Chemoselective ligation between the respective male-
imide cluster and the cysteine-containing DP17817 gave
the bi-, tri-, and tetravalent peptides (Scheme 2). The
ligation was very efficient and normally completed
within 30 min in a mixed solvent consisting of MeCN
and phosphate buffer (pH 6.6) (1:1, v/v). The bi-, tri-,
and tetravalent peptides, BVP-1, TVP-1, and MVP-1
were obtained in 95%, 88%, and 91% isolated yields,
respectively, after HPLC purification. It should be
pointed out that peptide DP178-Cys has a tendency to
aggregate at 20 lM or higher concentration. For an
efficient ligation, we found that the addition of MeCN
was necessary to prevent the aggregation of the peptide
in a phosphate buffer. The resulting multivalent peptides
were readily purified by HPLC and characterized by
ESI-MS.
2.3. Immunogenicity of the synthetic multivalent DP178
peptides
2.2. Conformational studies
Since the tetravalent peptide MVP-1 forms an a-helix
bundle with the highest a-helix content among the syn-
thetic multivalent peptides, we predict that MVP-1 may
partially mimic the pre-fusogenic state of the oligomeric
gp41 during membrane fusion. Therefore, MVP-1 and
DP178 were chosen to immunize mice in order to eval-
uate and compare their immunogenicity. Mice were
primed with MVP-1 or DP178 (10 lg each), and boosted
at days 14 (1ꢁ boost), 28 (2ꢁ boost), and 90 (3ꢁ boost)
with the same amount of immunogen without any
adjuvant. Serum was collected 10 days after each boost
and the antibody responses were evaluated with enzyme-
linked immunosorbent assays (ELISAs). As shown in
Figure 3, the peptide DP178 alone was found to be
poorly immunogenic. In contrast, the tetravalent format
of DP178, MVP-1 was highly immunogenic even in
the absence of any adjuvant. After the third boost,
the anti-DP178 antibody titers reached 1:2 · 106 for the
sera from mice immunized with MVP-1, while the
It has been demonstrated that template-assembled pep-
tides may form a-helix bundles when a suitable template
is applied.18–20 We and others have recently reported
that the 36-mer peptide DP178 formed three a-helix
bundles when assembled on a cholic acid scaffold or on
an oligolysine template.21;22 On the other hand, Brask
et al.23 recently reported that assembly of a model
peptide on several monosaccharide templates (Galp,
Glcp, and Altp) resulted in four a-helix-bundle forma-
tion. Therefore, it is interesting to investigate how the
DP178 peptides behave in the bi-, tri-, and tetravalent
formats on the galactopyranoside scaffold. The circular
dichroism (CD) spectra of the multivalent peptides
Figure 2. CD spectra of MVP-1 and DP178. The spectra were
recorded with 10 lM of DP178 and 2.5 lM of MVP-1 in a phosphate
buffer (50 mM, pH 7.2) at 23 ꢁC.
Scheme 2. Synthesis of the multivalent peptides.

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J. Ni et al. / Bioorg. Med. Chem. 12 (2004) 3141–3148
Figure 3. Antibody responses. Mice were primed with 10 lg of MVP-1 or DP178, and boosted at days 14 (1ꢁ boost), 28 (2ꢁ boost), and 90 (3ꢁ boost),
respectively, with the same amount of immunogen without any adjuvant. Serum was collected 10 days after each boost and the antibody responses
were evaluated with enzyme-linked immunosorbent assays (ELISAs). A, antibody response against immobilized DP178; B, antibody response against
immobilized HIV-1 gp41.
anti-DP178 titer for the sera from mice immunized with
DP178 was 1:9 · 103 after the third boost (Fig. 3A).
Moreover, it was found that the antibodies raised from
mice immunized with MVP-1 also recognized native
HIV-1 envelope glycoprotein gp41, and the anti-gp41
antibody titer reached 1:4 · 105 after third boost (Fig.
3B). These results suggest that multivalent peptides
containing both B- and T- epitopes are highly effective
immunogens even in the absence of a carrier protein or
any adjuvant. This raises the possibility that multivalent
gp41 peptide immunogen may raise conformation-spe-
cific antibody targeting the prefusogenic state of gp41.
The next step is to characterize these antibodies and to
evaluate whether the antibodies can neutralize various
HIV-1 isolates.
measured on a Waters ZMD mass spectrometer. Ana-
lytical TLC was performed on glass plates coated with
silica gel 60 F254 (E. Merck). Carbohydrates were de-
tected 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). Photo-addition reactions were
carried out in a quartz flask under N2. Automatic solid-
phase peptide synthesis was performed as described
previously.17 The final peptides were purified by reverse-
phase HPLC. For the biotinylation of DP178, after re-
moval of Fmoc at the last step in the solid-phase syn-
thesis, the peptide on the resin was treated with biotin
and HBTU in DMF. After coupling, the peptide was
deprotected and cleaved from the resin, and finally
purified by HPLC. Analytical HPLC was carried out
with a Waters 626 HPLC instrument using a Waters
Nova-Pak C18 column (3.9 · 150 mm) at 40 ꢁC. The
column was eluted with a linear gradient of acetonitrile
(10–90%) containing 0.1% TFA in 20 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 600 HPLC instrument of a Waters C18
column (Symmetry 300, 19 · 300 mm). The column was
eluted with a suitable gradient of water–MeCN con-
taining 0.1% TFA. Peptides purified from HPLC were
lyophilized and kept under nitrogen in a freezer
()20 ꢁC). For the determination of concentrations of
peptide DP178 and its multivalent derivatives, the UV
absorbance was run on Beckman DU 640 spectropho-
tometer at 280 nm, and the concentration was calculated
according to the equation proposed in the literature.26
CD spectra were measured on JASCO-810 (CD-ORD)
spectropolarimeter using quartz cuvette of 1 mm path
length at 23 ꢁC.
3. Conclusion
Several monosaccharide-centered multivalent HIV-1
gp41 peptides containing the sequence of DP178 were
synthesized. It was found that multivalent assembly
enhanced the a-helical content of the peptide, and led to
the formation of 2-, 3-, or 4-a-helix bundles when a bi-,
tri-, or tetravalent template was used. Immunization
studies indicated that while peptide DP178 in the ab-
sence of a carrier protein or adjuvant was poorly
immunogenic, the tetravalent peptide MVP-1 was able
to raise high titers of antibodies in mice that recognize
not only peptide DP178 but also the native HIV-1 gly-
coprotein gp41. The study suggests that the carbohy-
drate-centered multivalent peptides provide not only a
model for mimicking protein a-helix-bundle structure,
but also an effective immunogen that raises high-titer
anti-gp41 antibodies, which may be exploited for HIV-1
vaccine development.
4. Experimental
4.1.1. Ethyl 6-O-t-butyldimethylsilyl-a-D-galactopyrano-
side (3). Compound 1 (210 mg, 1.0 mmol) was dissolved
in pyridine (5 mL), tert-butyldimethylsilyl chloride
(182 mg, 1.2 mmol) was added at 0–5 ꢁC. After stirring
the resulting mixture at 0–5 ꢁC for 30 min, and then at
room temperature for 2 h, MeOH (1 mL) was added and
4.1. General methods
1H NMR spectra were recorded on QE 300 with Me4Si
ðd0Þ as the internal standard. The ESI-MS spectra were

J. Ni et al. / Bioorg. Med. Chem. 12 (2004) 3141–3148
3145
the solvent was removed under reduced pressure. Col-
umn chromatography (CH2Cl2/MeOH 95:5) of the res-
idue gave compound 3 (270 mg, 83%). Rf 0.43 (CH2Cl2/
MeOH 90:10); 1H NMR (300 MHz, CDCl3/TMS): d
4.92 (d, 1H, J ¼ 3:4 Hz, H-1), 4.09 (br s, 1H, H-4), 3.94–
3.74 (m, 6H, H-2, H-3, H-5, H-6, H-6þ, and
OCHHCH3), 3.62–3.49 (m, 1H, OCHHCH3), 3.20 (br s,
1H, OH-4), 3.04 (d, 1H, J ¼ 4:9 Hz, OH-3), 2.31 (dd,
1H, J ¼ 9:8 Hz, OH-2), 1.24 (dd, 3H, R ¼ 7:1, 6.8 Hz,
OCH2CH3), 0.90 (s, 9H, (CH33CSi)), 0.09 (s, 6H,
CH3SiCH3); ES-MS: 667.47 (2M+Na)þ, 345.24
(M+Na)þ, 323.25 (M+H)þ.
4.1.4. Ethyl 2-O-methyl-a-
D
-galactopyranoside (6).
Compound 5 (221 mg, 0.59 mmol) was dissolved in
THF (5 mL) and tetrabutylammonium fluoride in THF
(1 M, 0.71 mL) was added. The mixture was stirred at
room temperature for 1 h, and the solvent was then
evaporated. Column chromatography (CH2Cl2/MeOH
95:5) of the residue gave ethyl 3,4-O-isopropylidene-2-
O-methyl-a- -galactopyranoside (146 mg, 95%): Rf 0.70
D
(CH2Cl2/MeOH 92:8). 1H NMR (300 MHz, CDCl3/
TMS): d 5.00 (d, 1H, J ¼ 2:9 Hz, H-1), 4.38–4.22 (m,
2H, H-3, H-4), 4.10–3.70 (m, 4H, H-5, H-6, H-6þ, and
OCHHCH3), 3.66–3.49 (m, 4H, OCHHCH3, 2-OCH3),
3.37 (dd, 1H, J ¼ 7:6, 2.9 Hz, H-2), 2.29 (dd, 1H,
J ¼ 10:7, 3.8 Hz, OH-6), 1.54 (s, 3H, CCH3), 1.36 (s,
3H, CH3C), 1.25 (dd, 3H, J ¼ 7:1, 6.9 Hz, OCH2CH3);
ES-MS: 285.12 (M+Na)þ. Treatment of the product
(92 mg, 0.35 mmol) with 90% acetic acid (3 mL) at 90 ꢁC
for 2 h gave compound 6 (77 mg, 99%) after column
chromatography. Rf 0.54 (CH2Cl2/MeOH 80:20); 1H
NMR (300 MHz, D2O): d 5.21 (d, 1H, J ¼ 3:4 Hz, H-1),
4.04–3.74 (m, 4H, H-3, H-4, H-5, OCHHCH3), 3.74 (d,
2H, J ¼ 6:1 Hz, H-6, and H-6þ), 3.68–3.48 (m, 2H, H-2,
and OCHHCH3), 3.47 (s, 3H, 2-OCH3), 1.24 (dd, 3H,
J ¼ 7:1, 6.9 Hz, OCH2CH3); ES-MS: 245.16 (M+Na)þ.
4.1.2. Ethyl 6-O-t-butyldimethylsilyl-3,4-O-isopropylid-
ene-a-D-galactopyranoside (4). Compound 3 (431 mg,
1.34 mmol) was dissolved in acetone (10 mL). 2,2-di-
methoxypropane (278.5 mg, 2.68 mmol) and pTsOH
(cat.) were added. The resulting mixture was stirred at
room temperature for 3 h. The reaction mixture was
then neutralized with aqueous NaHCO3 and concen-
trated. The residue was taken up in EtOAc (50 mL) and
washed with aqueous NaHCO3, brine, and water. The
organic layer was dried (Na2SO4) and concentrated.
Column chromatography (CH2Cl2/MeOH 95:5) of the
residue gave compound 4 (460 mg, 95%). Rf 0.67
(CH2Cl2/MeOH 93:7). 1H NMR (300 MHz, CDCl3/
TMS): d 4.85 (d, 1H, J ¼ 3:2 Hz, H-1), 4.28–4.16 (m,
2H, H-3, H-4), 4.03 (m, 1H, H-5), 3.92–3.75 (m, 4H, H-
2, H-6, H-6þ, and OCHHCH3), 3.62–3.49 (m, 1H,
OCHHCH3), 2.28 (dd, 1H, J ¼ 7:1 Hz, OH-2), 1.50 (s,
3H, CCH3), 1.34 (s, 3H, CH3C), 1.24 (dd, 3H, J ¼ 7:1,
6.6 Hz, OCH2CH3), 0.90 (s, 9H, (CH33CSi)), 0.08 (s, 6H,
CH3SiCH3); ES-MS: 385.26 (M+Na)þ, 363.26 (M+H)þ.
4.1.5. Ethyl 3,4,6-tri-O-allyl-2-O-methyl-a-D-galactopy-
ranoside (7). A solution of compound 6 (77 mg,
0.35 mmol) in anhydrous DMF (5 mL) was added
dropwise to a stirred suspension of sodium hydride (60%
dispersion in mineral oil, 416 mg, 10.4 mmol) in anhy-
drous DMF (15 mL). The reaction mixture was stirred
at 0 ꢁC for 30 min and allyl bromide (1 mL) was then
added dropwise. The resulting mixture was stirred at
0 ꢁC to room temperature for 5 h. The reaction was
quenched by slow addition of MeOH (5 mL). The mix-
ture was concentrated to dryness under reduced pres-
sure. The residue was partitioned between EtOAc and
diluted HCl. The organic layer was separated and
washed with brine and water, dried over Na2SO4, and
concentrated. Column chromatography (hexane/EtOAc
75:25) of the residue afforded compound 7 (100 mg, 87
%). Rf 0.42 (hexane/EtOAc 70:30); 1H NMR (300 MHz,
CDCl3/TMS): 6.02–5.86 (m, 3H, OCH2CH@CH2),
5.36–5.10 (m, 6H, OCH2CH@CH2), 5.00 (d, 1H,
J ¼ 3:2 Hz, H-1), 4.39 (dd, 1H, J ¼ 12:3, 5.4 Hz,
OCHHCH ¼ CH2), 4.26–3.98 (m, 2H, OCH2CH@CH2),
3.91 (m, 1H, H-5), 3.83 (br s, 1H, H-4), 3.80–3.68 (m,
3H, H-3, H-6, and OCHHCH3), 3.64–3.49 (m, 6H, H-2,
H-6þ, OCHHCH3, and 2-OCH3), 1.24 (br t, 3H,
J ¼ 6:9 Hz, OCH2CH3); ES-MS: 365.28 (M+Na)þ.
4.1.3. Ethyl 6-O-t-butyldimethylsilyl-3,4-O-isopropylid-
ene-2-O-methyl-a-D-galactopyranoside (5). A solution
of compound 4 (284 mg, 0.78 mmol) in anhydrous DMF
(5 mL) was added dropwise to a stirred suspension of
sodium hydride (60% dispersion in mineral oil, 312 mg,
7.80 mmol) in anhydrous DMF (15 mL). After stirring
the resulting suspension for 30 min, the reaction mixture
was cooled to 0 ꢁC, and then iodomethane (0.5 mL) was
added. The resulting mixture was stirred at 0 ꢁC for 1 h
and at room temperature for 3 h. After the mixture was
cooled in an ice bath, excess sodium hydride was con-
sumed by slow addition of MeOH (5 mL). The mixture
was concentrated to dryness under reduced pressure.
The residue was then partitioned between EtOAc and
water. The organic layer was washed with diluted HCl,
brine, and water, dried over Na2SO4, and concentrated.
The product was purified by column chromatography
(hexane/EtOAc 85:15) to provide compound 5 (277 mg,
95%); Rf 0.45 (hexane/EtOAc 80:20). 1H NMR
(300 MHz, CDCl3/TMS): d 4.94 (d, 1H, J ¼ 2:9 Hz, H-
1), 4.29–4.20 (m, 2H, H-3, H-4), 4.02 (m, 1H, H-5),
3.94–3.72 (m, 3H, H-6, H-6þ, and OCHHCH3), 3.60–
3.50 (m, 4H, OCHHCH3, 2-OCH3), 3.35 (dd, 1H,
J ¼ 7:6, 3.4 Hz, H-2), 1.53 (s, 3H, CCH3), 1.34 (s, 3H,
CH3C), 1.26 (dd, 3H, J ¼ 7:1, 6.6 Hz, OCH2CH3), 0.90
(s, 9H, (CHR33CSi)), 0.08 (s, 6H, CH3SiCH3); ES-MS:
399.37 (M+Na)þ.
4.1.6.
methyl-a-
Ethyl 3,4,6-tri-O-(6-amido-3-thiahexyl)-2-O-
D-galactopyranoside trihydrochloride (8). To a
solution of compound 7 (100 mg, 0.29 mmol) and AIBN
(10 mg) in MeOH (15 mL) in a quartz flask was added
cysteamine hydrochloride (198 mg, 1.74 mmol). After
being degassed by bubbling N2 into solution for 30 min,
the solution was stirred and irradiated (UV, 254 nm)
under N2 atmosphere for 24 h. The solvent was evapo-
rated and the residue was subject to gel filtration on a
column of Sephadex G-15 using water as the eluent.

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J. Ni et al. / Bioorg. Med. Chem. 12 (2004) 3141–3148
Fractions containing the product were pooled and
lyophilized to give compound 8 (190 mg, 96%). 1H
NMR (D2O): d 5.19 (d, 1H, J ¼ 3:2 Hz, H-1), 4.10–4.04
(m, 1H, SCH2CH2CHHO), 4.03–3.99 (m, 1H, H-4),
3.96 ꢁ 3.52 (m, 12H, H-2, H-3, H-5, H-6, H-6þ, SCH2
CH2CHHO, SCH2CH2CH2O, and OCH2CH3), 3.47 (s,
3H, 2-OCH3), 3.32–3.24 (m, 6H, ClHNH2CH2CH2S),
2.96–2.86 (m, 6H, SCH2CH2NH2HCl), 2.76–2.68 (m,
6H, OCH2CH2CH2S), 1.98–1.88 (m, 6H, OCH2CH2-
CH2S), 1.25 (br t, 3H, J ¼ 6:7 Hz, OCH2CH3); ES-MS:
574.41 (M+H)3HCl)þ.
to 0 ꢁC, and iodomethane (0.7 mL) was added. The
resulting mixture was stirred for at 0 ꢁC to room tem-
perature for 5 h. After the mixture was cooled with an
ice bath, the reaction was quenched by slow addition of
MeOH (5 mL). The mixture was concentrated to dryness
under reduced pressure. The residue was partitioned
between EtOAc and water. The organic layer was
washed with diluted HCl, brine, and water, dried over
Na2SO4, and concentrated. The residue was subject to
column chromatography (hexane/EtOAc 70:30) to give
compound 11 (243 mg, 88%). Rf 0.34 (hexane/EtOAc
1
70:30). H NMR (300 MHz, CDCl3/TMS): d 4.97 (d,
1H, J ¼ 3:0 Hz, H-1), 4.26 (dd, 1H, J ¼ 7:9, 5.4 Hz, H-
3), 4.22–4.14 (m, 2H, H-4, H-5), 3.85–3.70 (m, 1H,
OCHHCH3), 3.66 (d, 2H, J ¼ 4:8 Hz, H-6 and H-6þ),
3.64–3.50 (m, 4H, OCHHCH3, 2-OCH3), 3.43 (s, 3H, 6-
OCH3), 3.36 (dd, 1H, J ¼ 7:9, 3.0 Hz, H-2), 1.54 (s, 3H,
CCH3), 1.34 (s, 3H, CH3C), 1.25 (br t, 3H, J ¼ 7:0 Hz,
OCH2CH3); ES-MS: 299.17 (M+Na)þ.
4.1.7. Ethyl 3,4,6-tri-O-(6-(6-maleimidohexanamido)-3-
-galactopyranoside (9).
thiahexyl)-2-O-methyl-a-
D
A
solution of compound 8 (34 mg, 49.8 lmol) in 1 M
aqueous solution of NaHCO3 (1.0 mL) was added
dropwise to a stirred solution of N-succimidyl 6-ma-
leimidohexanoic acid ester (90 mg, 293 lmol) in THF
(3 mL) at 0 ꢁC. The resulting mixture was stirred at 0–
5 ꢁC for 1 h, then diluted with CHCl3 (50 mL). The
solution was washed with brine and water. The organic
phase was dried over Na2SO4 and concentrated. The
residue was subject to column chromatography
(CH2Cl2/MeOH 95:5) to give compound 9 (26.01 mg,
46%). Rf 0.58 (CH2Cl2/MeOH 90:10); 1H NMR
(CDCl3/TMS): d 6.69 (s, 6H, CH ¼ CH), 6.20–6.04 (m,
3H, NHC(O)), 4.97 (d, 1H, J ¼ 3:1 Hz, H-1), 3.95–3.38
(m, 29H, H-2, H-3, H-4, H-5, H-6, H-6þ, NCH2CH2-
CH2CH2CH2C(O), (O)CNHCH2CH2S, SCH2CH2-
CH2O, 2-OCH3 and OCH2CH3), 2.72–2.58 (m, 12H,
OCH2CH2CH2SCH2CH2NH), 2.18 (t, 6H, J ¼ 7:3 Hz,
NCH2CH2CH2CH2CH2C(O)), 1.94–1.80 (m, 6H, OCH2-
CH2tlsbCH2S), 1.74–1.54 (m, 12H, NCH2CH2-
CH2CH2CH2C(O)), 1.39–1.20 (m, 9H, NCH2CH2-
CH2CH2CH2C(O), OCH2CH3); ESI-MS: 1153.63
(M+H)þ.
4.1.10. Ethyl 2,6-di-O-methyl-a-D-galactopyranoside
(12). A solution of compound 11 (193 mg, 0.70 mmol)
in 90% acetic acid was heated at 90 ꢁC for 2 h. The
solvent was evaporated and the residue was subject to
column chromatography (CH2Cl2/MeOH 90:10) to give
compound 12 (164 mg, 99%). Rf 0.41 (CH2Cl2/MeOH
1
90:10); H NMR (300 MHz, CDCl3/TMS): d 5.10 (d,
1H, J ¼ 3:2 Hz, H-1), 4.10–4.05 (m, 1H, H-4), 3.98–3.90
(m, 2H, H-3, H-5), 3.85–3.72 (m, 1H, OCHHCH3), 3.68
(d, 2H, J ¼ 4:9 Hz, H-6 and H-6þ), 3.67–3.50 (m, 2H,
H-2, and OCHHCH3), 3.49 (s, 3H, 2-OCH3), 3.42 (s,
3H, 6-OCH3), 1.26 (br t, 3H, J ¼ 7:0 Hz, OCH2CH3);
ES-MS: 259.28 (M+Na)þ.
4.1.11. Ethyl 3,4-di-O-allyl-2,6-di-O-methyl-a-D-galacto-
pyranoside (13). A solution of compound 12 (142 mg,
0.60 mmol) in anhydrous DMF (8 mL) was added slowly
to a stirred suspension of sodium hydride (60% disper-
sion in mineral oil, 480 mg, 12 mmol) in anhydrous
DMF (12 mL). The reaction mixture was stirred at 0 ꢁC
for 30 min and allyl bromide (1 mL) was then added
dropwise. The resulting mixture was stirred at 0 ꢁC to
room temperature for 6 h. The reaction was quenched
by slow addition of MeOH (5 mL). The mixture was
concentrated to dryness under reduced pressure. The
residue was partitioned between EtOAc and diluted
HCl. The organic layer was separated and washed with
brine and water, dried over Na2SO4, and concentrated.
Column chromatography (hexane/EtOAc 70:30) of the
residue afforded compound 13 (161 mg, 85 %). Rf 0.38
(hexane/EtOAc 70:30); 1H NMR (300 MHz, CDCl3/
TMS): 6.02–5.86 (m, 2H, OCH2CH@CH2), 5.36–5.14
(m, 4H, OCH2 CH@CH2), 5.00 (d, 1H, J ¼ 3:5 Hz, H-
1), 4.39 (dd, 1H, J ¼ 12:2, 5.2 Hz, OCHHCH@CH2),
4.28–4.06 (m, 3H, OCH2CH@CH2), 3.89 (dd, 1H,
J ¼ 6:4, 5.9 Hz, H-5), 3.80 (s, 1H, br, H-4), 3.80–3.68
(m, 3H, H-3, H-6, and OCHHCH3), 3.64–3.49 (m, 6H,
H-2, H-6þ, OCHHCH3, and 2-OCH3), 3.38 (s, 3H, 6-
OCH3), 1.25 (br t, 3H, J ¼ 7:0 Hz, OCH2CH3); ES-MS:
339.25 (M+Na)þ.
4.1.8. Ethyl 3,4-O-isopropylidene-a-D-galactopyranoside
(10). To a solution of compound 4 (250 mg, 0.69 mmol)
in THF (10 mL) was added tetrabutylammonium fluo-
ride in THF (1 M, 1 mL). The solution was stirred at
room temperature for 2 h and the solvent was evapo-
rated. Column chromatography (CH2Cl2/MeOH 90:10)
of the residue gave compound 10 (168 mg, 98%). Rf 0.53
(CH2Cl2/MeOH 90:10); 1H NMR (300 MHz, CDCl3/
TMS): d 4.91 (d, 1H, J ¼ 3:0 Hz, H-1), 4.24–4.20 (m,
2H, H-2, H-3), 4.12–4.44 (m, 1H, H-5), 3.99–3.74
(m, 4H, H-3, H-6, H-6þ, and OCHHCH3), 3.66–3.49
(m, 1H, OCHHCH3), 2.55–2.30 (br s, 2H, OH-2, and
OH-6), 1.51 (s, 3H, CCH3), 1.36 (s, 3H, CH3C), 1.25 (br
t, 3H, J ¼ 7:0 Hz, OCH2CH3); ES-MS: 271.13
(M+Na)þ.
4.1.9. Ethyl 3,4-O-isopropylidene-2,6-di-O-methyl-a-D-
galactopyranoside (11). A solution of compound 10
(248 mg, 1.0 mmol) in anhydrous DMF (5 mL) was
added slowly to a stirred suspension of sodium hydride
(60% dispersion in mineral oil, 328 mg, 8.2 mmol) in
anhydrous DMF (15 mL). After stirring the resulting
suspension for 30 min, the reaction mixture was cooled

J. Ni et al. / Bioorg. Med. Chem. 12 (2004) 3141–3148
3147
4.1.12. Ethyl 3,4-di-O-(6-maleimido-3-thiahexyl)-2,6-di-
O-methyl-a- -galactopyranoside dihydrochloride (14). To
solution was lyophilized and the product was purified by
reverse-phase HPLC.
D
a solution of compound 13 (110 mg, 0.35 mmol) and
AIBN (15 mg) in MeOH (15 mL) in a quartz flask was
added cysteamine hydrochloride (227 mg, 2.0 mmol).
After being degassed by bubbling N2 into the solution
for 30 min, the solution was stirred and irradiated (UV,
254 nm) under a N2 atmosphere for 24 h. The solvent
was evaporated and the residue was subject to gel fil-
tration on a column of Sephadex G-15 using water as
the eluent. Fractions containing the product were
pooled and lyophilized to give compound 14 as its
Tetravalent peptide MVP-1: yield, 91%; ESI-MS:
1653.72 (M+12H)12þ, 1526.60 (M+13H)13þ, 1417.63
(M+14H)14þ, 1323.20 (M+15H)15þ
.
Trivalent peptide TVP-1: yield 95%; ESI-MS: 2135.70
(M+7H)7þ, 1868.98 (M+8H)8þ, 1661.43 (M+9H)9þ
1495.23 (M+10H)10þ, 1359.47 (M+11H)11þ, 1246.31
(M+12H)12þ, 1150.49 (M+13H)13þ
,
.
1
hydrochloride (171 mg, 90%). H NMR (D2O): d 5.19
Bivalent peptide BVP-1: yield 88%; ESI-MS: 2011.08
(M+5H)5þ, 1675.98 (M+6H)6þ, 1436.80 (M+7H)7þ
(d, 1H, J ¼ 3:2 Hz, H-1), 4.10–4.04 (m, 1H,
SCH2CH2CHHO), 4.03–3.98 (m, 1H, H-4), 3.96–3.52
(m, 10H, H-2, H-3, H-5, H-6, H-6þ, SCH2CH2CHHO,
SCH2CH2CH2O, and OCH2CH3), 3.47 (s, 3H, 2-
OCH3), 3.42 (s, 3H, 6-OCH3), 3.32–3.24 (m, 4H, ClH-
NH2CH2CH2S), 2.96–2.86 (m, 4H, SCH2CH2NH2HCl),
2.76–2.68 (m, 4H, OCH2CH2CH2S), 1.98–1.88 (m, 4H,
OCH2CH2CH2S), 1.24 (br t, 3H, J ¼ 6:6 Hz,
OCH2CH3); ES-MS: 471.20 (M+H)2HCl)þ.
,
1006.04
1257.36 (M+8H)8þ
,
1117.81 (M+9H)9þ
,
(M+10H)10þ, 914.76 (M+11H)11þ
.
4.3. Circular dichroism (CD)
Multivalent gp41 peptides (MVP-1, TVP-1, and BVP-1)
and peptide DP178 were dissolved in a phosphate buffer
(50 mM, pH 7.2) at concentrations ranging from 4–
40 lM. The actual concentration of the peptides was
measured by UV absorption and calculated according to
the proposed formulation based on the absorbance of
Trp residues at 280 nm.26 The CD spectra were mea-
sured at 23 ꢁC and an average of three scans was taken.
Alpha-helical content was calculated based on the mean
4.1.13. Ethyl 3,4-di-O-(6-(6-maleimidohexanamido)-3-
thiahexyl)-2,6-di-O-methyl-a-D-galactopyranoside (15).
A solution of compound 14 hydrochloride (60 mg,
0.11 mmol, 49 lmol) in 1 M aqueous solution of
NaHCO3 (1.0 mL) was added dropwise to a stirred
solution of N-succimidyl 6-maleimidohexanoic acid es-
ter (101 mg, 0.33 mmol) in THF (3 mL) at 0 ꢁC. The
resulting mixture was stirred at 0–5 ꢁC for 1 h, then di-
luted with CHCl3 (40 mL). The solution was washed
with brine and water. The organic layer was dried over
Na2SO4 and concentrated. The residue was subject to
column chromatography (CH2Cl2/MeOH 90:10) to give
compound 15 (39.5 mg, 42%).Yield: 42%; Rf 0.49
residue ellipticity ½hꢀ and the proposed equation.24
222
4.4. Immunization of mice
Groups of five female BALB/c mice (8–10 weeks old
from Jackson Laboratory) were immunized intraperi-
toneally with 10 lg each of the synthetic peptide
immunogen. The mice were boosted with the same
quantity (10 lg) of individual immunogen at days 14
(first boost), 28 (second boost), and 90 (third boost).
The immunogens were formulated in a phosphate-buf-
fered saline (PBS) (10 lg/200 lL for each injection)
without any adjuvant. Blood was drawn from the retro-
orbital plexus 10 days after each boost. Sera from the
same group were pooled and used for detecting epitope-
specific antibodies by enzyme-linked immunosorbent
assay (ELISA).
1
(CH2Cl2/MeOH 90:10); H NMR (CDCl3/TMS): d 6.69
(s, 4H, CH@CH), 6.05 (br s, 2H, NHC(O)), 4.98 (d, 1H,
J ¼ 3:1 Hz, H-1), 3.95–3.41 (m, 23H, H-2, H-3, H-4, H-
5, H-6, H-6þ, NCH2CH2CH2CH2CH2C(O), (O)CNH-
CH2CH2S, SCH2CH2CH2O, 2-OCH3, and OCH2CH3),
3.38 (s, 3H, 6-OCH3), 2.72–2.60 (m, 8H, OCH2CH2-
CH2SCH2CH2NH),
2.18
(t,
4H,
J ¼ 7:3 Hz,
NCH2CH2CH2CH2CH2C(O)), 1.94–1.80 (m, 4H,
OCH2CH2CH2S), 1.74–1.54 (m, 8H, NCH2CH2CH2-
CH2CH2C(O)), 1.39–1.19 (m, 7H, NCH2CH2-
CH2CH2CH2C(O), OCH2CH3); ESI-MS: 857.46
(M+H)þ.
4.5. Enzyme-linked immunosorbent assay (ELISA)
To detect DP178-specific antibody, microtiter ELISA
plates were coated with 8 lg/mL Neutravidin (strepta-
vidin) in PBS and incubated at 4 ꢁC overnight. After
washings with PBS/0.5% Tween-20, nonspecific binding
was blocked with 5% (w/v) BSA in PBS at room tem-
perature for 1 h. Plates were then washed and 2 lg/mL of
biotinylated DP178 in 1% BSA was added. Plates were
incubated at 37 ꢁC for 1 h. The plates were again washed
and mouse serum was added in a 1:2 or 1:3 serial dilu-
tion in 1% BSA/PBS. The plates were incubated at 37 ꢁC
for 2 h and washed. To the plates was added a solution of
1:2000 diluted horseradish peroxidase (HRP)-conjugated
4.2. Synthesis of multivalent gp41 peptides––General
procedures
Peptide DP178-Cys was dissolved in a mixed solvent of
phosphate buffer (50 mM, pH 6.6) and acetonitrile (1:1,
v/v) to make a 5 mg/mL solution. A solution (2 mg/mL)
of the synthetic maleimide cluster (2 or 9 or 15) in DMF
was added to the peptide solution. Generally, 1.5-molar
equivalent of the peptide was used to react with 1-molar
equivalent maleimide functionality. The resulting mix-
ture was gently shaken at room temperature for 2 h. The

3148
J. Ni et al. / Bioorg. Med. Chem. 12 (2004) 3141–3148
8. Zwick, M. B.; Labrijn, A. F.; Wang, M.; Spenlehauer, C.;
Saphire, E. O.; Binley, J. M.; Moore, J. P.; Stiegler, G.;
Katinger, H.; Burton, D. R.; Parren, P. W. J. Virol. 2001,
75, 10892.
9. Stiegler, G.; Kunert, R.; Purtscher, M.; Wolbank, S.;
Voglauer, R.; Steindl, F.; Katinger, H. AIDS Res. Hum.
Retroviruses 2001, 17, 1757.
10. Xu, W.; Smith-Franklin, B. A.; Li, P. L.; Wood, C.; He, J.;
Du, Q.; Bhat, G. J.; Kankasa, C.; Katinger, H.; Cavacini,
L. A.; Posner, M. R.; Burton, D. R.; Chou, T. C.;
Ruprecht, R. M. J. Hum. Virol. 2001, 4, 55.
11. Wild, C. T.; Shugars, D. C.; Greenwell, T. K.; McDanal,
C. B.; Matthews, T. J. Proc. Natl. Acad. Sci. U.S.A. 1994,
91, 9770.
12. Kilby, J. M.; Hopkins, S.; Venetta, T. M.; DiMassimo, B.;
Cloud, G. A.; Lee, J. Y.; Alldredge, L.; Hunter, E.;
Lambert, D.; Bolognesi, D.; Matthews, T.; Johnson, M.
R.; Nowak, M. A.; Shaw, G. M.; Saag, M. S. Nat. Med.
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goat anti-mouse IgG. After incubation for 1 h at
37 ꢁC, the plates were washed again and a solution of
3,3þ,5,5þ-tetramethyl benzidine (TMB) was added.
Color was allowed to develop for 5 min, and the reaction
was then quenched by adding a solution of 0.5 M H2SO4
to each well. The optical density was then measured at
450 nm. To detect gp41-specific antibody in the serum,
the plates were coated with 1 lg/mL gp41 in PBS at 4 ꢁC
overnight. After washing, blocking, and washing, the
plates were incubated with a series dilution of the mouse
serum. The antibody titers were then measured as de-
scribed above. Antibody titers were calculated as
reciprocals of the highest dilutions, which gave optical
density values at least twofold above the negative con-
trol.
13. Chan, D. C.; Chutkowski, C. T.; Kim, P. S. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 15613.
14. McGaughey, G. B.; Citron, M.; Danzeisen, R. C.;
Freidinger, R. M.; Garsky, V. M.; Hurni, W. M.; Joyce,
J. G.; Liang, X.; Miller, M.; Shiver, J.; Bogusky, M. J.
Biochemistry 2003, 42, 3214.
15. Joyce, J. G.; Hurni, W. M.; Bogusky, M. J.; Garsky, V.
M.; Liang, X.; Citron, M. P.; Danzeisen, R. C.; Miller, M.
D.; Shiver, J. W.; Keller, P. M. J. Biol. Chem. 2002, 277,
45811.
Acknowledgements
We thank Dr. Hengguang Li for assistance in measuring
the CD spectra. The work was supported in part by the
Institute of Human Virology, University of Maryland
Biotechnology Institute, and National Institutes of
Health (NIH grant AI51235 to LXW).
16. Tam, J. P. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 5409.
17. Wang, L. X.; Ni, J.; Singh, S. Bioorg. Med. Chem. 2003,
11, 129.
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