Cholic acid as template for multivalent peptide assembly

Article abstract of DOI:10.1039/b307995c

Cholic acid, an amphiphilic steroid containing several selectively addressable functionalities, was exploited as a rigid template for multivalent peptide assembly. Thus, cholic acid-based templates suitable for chemoselective peptide ligation were synthesized, in which maleimide or bromoacetyl moieties were selectively introduced at the 3α, 7α, 12α-positions of cholic acid with varied length of linkers. Three peptides were chosen and tested for the chemoselective ligation. These include the HIV-1 peptide inhibitor DP178, the universal T-helper epitope derived from tetanus toxoid (830-844), and the minimum epitope sequence of the HIV-neutralizing antibody 2F5. It was found that the maleimide-functionalized templates are highly efficient for the ligation of all the peptides, while bromoacetyl templates led to low yield of ligation. Circular dichroism (CD) spectroscopic studies of the multivalent peptides (10a and 11a) containing three strands of peptide DP178 indicate that the template-assembled peptides form three α-helix bundles with significantly enhanced α-helix contents than the single peptide. The results suggest that cholic acid is a valuable template for constructing α-helix bundles that may be useful as mimics of conformational epitopes for vaccine development.

Full text of DOI:10.1039/b307995c

View Article Online / Journal Homepage / Table of Contents for this issue
Cholic acid as template for multivalent peptide assembly
Hengguang Li and Lai-Xi Wang*
Institute of Human Virology, University of Maryland Biotechnology Institute,
University of Maryland, 725 W. Lombard Street, Baltimore, MD 21201, USA
Received 16th July 2003, Accepted 1st September 2003
First published as an Advance Article on the web 17th September 2003
Cholic acid, an amphiphilic steroid containing several selectively addressable functionalities, was exploited as a
rigid template for multivalent peptide assembly. Thus, cholic acid-based templates suitable for chemoselective
peptide ligation were synthesized, in which maleimide or bromoacetyl moieties were selectively introduced at the
3α, 7α, 12α-positions of cholic acid with varied length of linkers. Three peptides were chosen and tested for the
chemoselective ligation. These include the HIV-1 peptide inhibitor DP178, the universal T-helper epitope derived
from tetanus toxoid (830–844), and the minimum epitope sequence of the HIV-neutralizing antibody 2F5. It was
found that the maleimide-functionalized templates are highly efficient for the ligation of all the peptides, while
bromoacetyl templates led to low yield of ligation. Circular dichroism (CD) spectroscopic studies of the multivalent
peptides (10a and 11a) containing three strands of peptide DP178 indicate that the template-assembled peptides form
three α-helix bundles with significantly enhanced α-helix contents than the single peptide. The results suggest that
cholic acid is a valuable template for constructing α-helix bundles that may be useful as mimics of conformational
epitopes for vaccine development.
Results and discussion
Introduction
Synthesis of functionalized templates
Template assembled multivalent peptides have been developed
predominately for two applications: as novel artificial proteins
to study the factors governing protein folding and stability,1,2
and as chemically defined immunogens for vaccine develop-
ment.3,4 Enhanced inhibitory activities of peptide inhibitors
were also attained through multivalent peptide assembly.5–9 So
far, several types of cyclic molecules have been exploited as
templates for multivalent peptide assembly. These include
cyclic peptide and derivatives,10–13 porphyrin molecules,14,15
calix[4]arene core,7,16 cavitand macrocycles,17,18 and mono-
saccharides.19–25 New types of templates are demanded in order
to control the topology of assembled peptides to achieve novel
properties of the assembled peptides.
We have previously prepared monosaccharide-centered
maleimide clusters and used them for the synthesis of multi-
valent HIV-1 gp41 peptides.25 The long term goal of our
template-assembled peptide project is to develop mimics of the
trimeric gp41 fusion intermediates to serve as vaccines and
inhibitors for blocking HIV-1 infection.26–28 We report in this
paper the use of cholic acid as a new template for multi-
valent peptide assembly. Cholic acid is a readily available,
rigid, and facial amphiphilic molecule. The hydrophilic
face contains 3 hydroxy groups at 3α, 7α, and 12α positions
with defined spatial orientations and the other face consisting
of the methyl groups and the backbone is hydrophobic.
Because of its unique molecular property, cholic acid has
been exploited for the design of artificial ion channel,29,30 for
drug targeting,31,32 and as scaffold for the assembly of
combinatorial libraries.33–35 We demonstrate here the modifi-
cation of cholic acid as a template for multivalent peptide
assembly. We found that cholic acid-based maleimide clusters
are highly efficient for chemoselective ligation of cysteine-
containing peptides. Moreover, assembly of three strands of the
potent HIV-1 inhibitor DP178, a 36-mer peptide,36,37 on the
cholic acid template led to the formation of a three-α-helix
bundle structure, which may serve as a mimic of trimeric
α-helical transition states of HIV-1 gp41 molecule during viral
membrane fusion.
For chemoselective ligation to cysteine-containing peptides, the
hydroxy groups of cholic acid (1) were selectively modified to
introduce thiol-reactive functionality such as maleimide and
bromoacetyl groups. The synthesis is summarized in Scheme 1.
The preparation of the 3α,7α,12α-tri-O-allyl intermediate 4 was
previously reported starting from methyl cholate.38 We used a
modified procedure for the synthesis. Instead of using methyl
cholate as starting material, cholic acid was directly reduced
with LiAlH4 in THF to give the tetra-hydroxycholane 2 in 98%
yield. Reaction of 2 with trityl chloride gave the trityl-protected
compound 3. Initial allylation of 3 with allyl bromide/NaH in
THF according to the literature38 was met with less success,
leading to a mixture of mono-, di-, and tri-allylated derivatives
even under reflux for 3 days with excess reagents. However,
replacement of the allyl bromide with allyl iodide under the
same conditions resulted in a quick and complete allylation of
the three hydroxy groups, from which the tri-O-allyl derivative 4
was isolated in almost quantitative yield. Compound 4 was then
subjected to photo-addition with cysteamine in the presence of
initiator AIBN in MeOH under UV irradiation (254 nm).39 The
reaction was monitored by TLC and NMR. It was found
that even after 6 days of reaction, only a small portion of the
tri-allyl derivative 4 was converted into a mixture of the corre-
sponding mono-, di-, and tri-amines with simultaneous de-O-
tritylation of the starting material. It is likely that the strong
UV absorbing trityl group might inhibit the radical reaction. To
avoid this, we removed the trityl group to provide 5 before
performing the photo-addition reaction. As a result, the photo-
addition of cysteamine to the allyl groups of 5 proceeded
rapidly and efficiently to give the tri-amine 6 in excellent yield
(Scheme 1).
Starting from the intermediate tri-amine 6, several templates
suitable for chemselective peptide ligation were prepared. Thus,
the three amino groups in 6 were directly converted into
maleimide groups through reaction with methoxycarbonyl-
maleimide to afford the trivalent maleimide cluster 7 in 78%
yield. On the other hand, reaction of 6 with 6-maleimido-
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 0 7 – 3 5 1 3
3507

View Article Online
Scheme 1 Reagents and conditions: (a) LiAIH4, THF, 0 ЊC to rt, 98%; (b) trityl chloride, Et3N, DBU, DMF, r.t., 93%; (c) allyl iodide, NaH, THF,
r.t. to 70 ЊC, 96%; (d) p-toluenesulfonic acid, DCM, MeOH, 100%; (e) 2-aminoethanethiol hydrochloride, ABIN, MeOH, UV (254 nm), r.t., 96%;
(f ) N-methoxycarbonylmaleimide, Et3N, DMF, r.t., 78%; (g) 6-maleimidohexanoic acid N-hydroxylsuccinimide ester, DCM, r.t., 76%; (h) bromo-
acetic anhydride, DCM, r.t., 89%.
hexanoic acid N-hydroxylsuccinimide ester gave another tri-
valent maleimide cluster, compound 8, with a longer spacer
between the maleimide group and the cholic acid core. In addi-
tion, a bromoacetyl group was introduced at each amino group
in 6 through N-bromoacetylation to give the bromoacetyl
derivative 9, which was prepared for the comparison of the
efficiency in ligation (Scheme 1).
peptides: P37C, T-helper, and P7C, respectively. In the case of
the T-helper sequence, a tetra-peptide spacer GSSS was intro-
duced at the N-terminus to increase the aqueous solubility of
the otherwise hydrophobic T-helper epitope. It was observed
that the ligation between the maleimide cluster 7 and the pep-
tide P37C or the T-helper peptide proceeded very rapidly in a
MeCN–phosphate buffer (1 : 1, v/v, pH 7.0) to give the trivalent
peptides 10a and 10b in 82 and 89% yields, respectively (Scheme
2). Similarly, peptide ligation using the maleimide cluster 8 with
a longer spacer afforded the trivalent peptides 11a and 11b in 83
and 91% yields, respectively (Scheme 2). Regardless of the
length and complexity of the peptides and maleimide clusters
used, all the ligation was complete within 3 hours under the
above conditions. The desired multivalent peptides were puri-
fied by reverse phase HPLC and characterized by electron spray
ionization mass spectrometry (ESI-MS). The typical ESI-MS
and HPLC profiles of the purified multivalent peptides 10a and
Chemoselective ligation of peptides to the cholic acid templates
To examine the chemoselective ligation, three antigenic pep-
tides were chosen and tested. These include the potent HIV
inhibitor DP178,36,37 a T-helper epitope from tetanus toxoid
(830–844),40 and a minimum epitope sequence ELDKWA for
HIV-neutralizing antibody 2F5.41 For ligation, a cysteine resi-
due was introduced at either the C- or N-terminus of the pep-
tides during solid phase synthesis to give the Cys-containing
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 0 7 – 3 5 1 3
3508

View Article Online
Scheme 2 Reagents and conditions: (a) phosphate buffer (pH 6.6)/MeCN (1 : 1, v/v), r.t.
Fig. 1 HPLC and ESI-MS profiles of trivalent peptides 10a and 11a.
11a are shown in Fig. 1. Together with the HPLC data, the
ESI-MS spectra showed the high purity and verified the correct
primary structure of the synthetic artificial proteins.
Next, we examined the ligation using the bromoacetyl deriv-
ative 9. Ligation between haloacetyl moiety and thiol function-
ality was used previously for constructing template-assembled
multivalent peptides.42,43 However, we found that neither the
longer peptide P37C nor the medium-sized peptide T-helper
gave the desired fully substituted product even when an excess
amount (6-fold) of the cysteine-containing peptide was used in
a mixed solvent (MeCN–borate buffer, pH 8.5). A mixture of
mono-substituted and trace di-substituted ligation products
were revealed by HPLC-MS analysis, together with the dimer-
ization of the cysteine-containing peptide under the alkaline
condition (data not shown). The reaction intermediates were
not purified for further characterization. When a short peptide
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 0 7 – 3 5 1 3
3509

View Article Online
Scheme 3 (a) Borate buffer (pH 8.5)/MeCN (1 : 1, v/v), r.t.
Ac-ELDKWAC-NH2 (P3C) was used to react with the bromo-
acetyl template 9 under the above ligation condition, the desired
trivalent peptide product 12 was formed together with several
mono- and di-substituted by-products, from which 12 was iso-
lated in 35% yield (Scheme 3). The results implicate a significant
difference in the reactivity between the bromoacetyl and male-
imide groups toward thiol functionality. The results again
demonstrate that maleimide clusters are particularly useful
for constructing very large and complex multivalent peptides
through the highly efficient thiol–maleimide ligation.25
Conformational studies
One goal of our project is to create three-α-helix bundle struc-
tures to mimic the trimeric fusion intermediates of the HIV-1
gp41 molecule. It is known that template-assembled construc-
tion may induce α-helix bundle formation when a suitable
template is applied.1,2,23 The 36-mer peptide DP178 was derived
from the C-terminal ectodomain of gp41, which is supposed
to expose as a trimeric α-helical intermediate during viral
membrane fusion.27,28 Nevertheless, free peptide DP178 exists
in solution in an almost random structure, with only 10–20%
α-helical content.44 Therefore, it will be interesting to investigate
whether the cholic acid-assembled peptides adopt an enhanced
α-helical structure, as exemplified by some other template-
assembled peptides.1,2,23 The circular dichroism (CD) spectra of
the cholic acid-based trivalent peptides 10a and 11a, together
with peptide DP178, were measured at 23 ЊC in a phosphate
buffer (pH 7.2). As can be seen in Fig. 2, both the trivalent
peptides 10a and 11a showed a significant degree of α-helicity,
while the DP178 showed a typical random structure in the buf-
fer. The content of α-helix was calculated to be 36, 41, and 18%
for 10a, 11a, and DP178, respectively, based on their mean resi-
due ellipticity [θ]222 and the proposed formula.45 The trivalent
peptide (11a) with a relatively long spacer between the peptide
Fig. 2 CD spectra of peptide DP178 and the trivalent peptides 10a
and 11a. The spectra were recorded at 5, 4, and 4 uM for peptides
DP178, 10a, and 11a, respectively, in a phosphate buffer (50 mM,
pH 7.2) at 23 ЊC.
strand and the cholic acid core showed slightly higher α-helical
content than the one with a relatively short spacer (10a). Since
an accurate calculation of the α-helical content relies on the
precise determination of peptide concentration, we carefully
quantified the peptide concentration through UV absorbance at
280 nm because of the presence of several tryptophan residues
in the peptide sequence. The α-helical content of DP178 in the
buffer is in good agreement with the reported value.44 It was
also found that the CD spectra of 10a, 11a, and DP178 were
concentration-independent in the range of 2–20 µM (data not
shown). The results clearly suggest that three-α-helix-bundle
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 0 7 – 3 5 1 3
3510

View Article Online
structure was formed when three strands of the HIV peptides
were installed on the cholic acid templates.
3ꢀ,7ꢀ,12ꢀ-Trihydroxy-24-trityloxy-5ꢁ-cholane (3). To a solu-
tion of compound 2 (6.23 g, 15.8 mmol) in DMF (100 mL)
were added trityl chloride (6.60 g, 23.7 mmol), Et3N (10 mL),
and DBU (0.90 g, 5.9 mmol) in sequence with stirring at room
temperature. After 24 h, water (400 mL) was added, and the
mixture was stirred vigorously for 2 h. The precipitate was
collected via filtration and washed with acetone. The crude
product was re-crystallized in acetone to give 3 (9.35 g, 93%)
as a white solid: m.p. 206–208 ЊC (lit.,38 m.p. 187 ЊC); 1H
NMR (300 MHz, CDCl3/TMS) δ 7.43 (d, 6H, J = 7.3 Hz,
phenyl H-2, H-6), 7.29–7.18 (m, 9H, phenyl H-3, H-4, H-5),
3.96 (bs, 1H, H–C(12)), 3.82 (bs, 1H, H–C(7)), 3.41 (m, 1H,
H–C(3)), 3.00 (m, 2H), 2.92 (s, 2H), 2.87 (s, 2H), 2.19 (m, 2H),
2.04 (s, 1H), 1.98–1.00 (series of multiplet, 16H), 0.96 (d, 3H,
J = 6.1 Hz, H3C–C(20)), 0.86 (s, 3H, H3C–C(10)), 0.64 (s, 3H,
H3C–C(13)).
Conclusion
Cholic acid was exploited as a new type of template for multi-
valent peptide assembly. The results suggest that cholic acid-
centered maleimide clusters are highly efficient for the construc-
tion of large and complex multivalent peptides. The cholic acid
template allowed α-helix-bundle formation when a suitable pep-
tide such as HIV-1 gp41 peptide DP178 was assembled. The
resulting three-α-helix bundles of DP178 may mimic the con-
formational epitopes of gp41 that are exposed during viral
membrane fusion, which should be useful for HIV-1 vaccine
development.
Experimental
3ꢀ,7ꢀ,12ꢀ-Triallyloxy-24-trityloxy-5ꢁ-cholane (4). Sodium
hydride (60% in mineral oil, 2.00 g, 29.2 mmol) was added in
portions to a solution of compound 3 (3.12 g, 4.8 mmol) in dry
THF (50 mL) under N2, and stirred for 2 h at room temper-
ature. Then allyl iodide (4.98 g, 30.0 mmol) was added dropwise
and the mixture was heated at 70 ЊC with stirring for 4 h. After
having cooled to room temperature, the reaction mixture was
partitioned between ethyl acetate (50 mL) and water (30 mL).
The organic layer was separated and washed with H2O (2 ×
20 mL) and brine, and dried over anhydrous Na2SO4, filtered,
and concentrated under vacuum. The residue was subject to
column chromatography on silica gel with hexane/ethyl acetate
(90 : 10, v/v) as the eluent to give 4 (3.49 g, 96%) as a pale
yellow syrup. 1H NMR (300 MHz, CDCl3/TMS) δ 7.44 (d, 6H,
J = 7.1 Hz, benzyl H-2, H-6), 7.29–7.20 (m, 9H, benzyl H-3,
General
All Fmoc-protected amino acids used for peptide synthesis
were purchased from Novabiochem. HATU, DIPEA and
Fmoc-PAL-PEG-PS were purchased form Applied Biosystems.
HPLC grade acetonitrile was purchased from Fisher Scientific.
DMF was purchased from B & J Biosynthesis. All other chem-
icals were purchased from Aldrich/Sigma and used as received.
1H and 13C NMR spectra were recorded on QE 300 or Inova
500 with Me4Si (δ 0) as the internal standard. The ESI-MS
spectra were measured on a Waters ZMD mass spectrometer.
Analytical TLC was performed on glass plates coated with
silica gel 60 F254 (E. Merck) by visualizing the spots with UV
light (254 nm) irradiation, 10% ethanolic sulfuric acid,
ninhydrin spraying and/or iodine coloration. Flash column
chromatography was performed with silica gel 60 (EM Science,
particle size 0.040–0.063 mm, 230–400 mesh). Photo-addition
reaction was carried out in a quartz flask under N2. Analytical
HPLC was carried out with a Waters 626 HPLC instrument on
a Waters Nova-Pak C18 column (3.9 × 150 mm) at 40 ЊC. 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
minϪ1. Peptides were detected by UV absorbance at 214 and/or
280 nm. Preparative HPLC was performed with a Waters 600
HPLC instrument with a Waters C18 column (Symmetry 300,
19 × 300 mm). The column was eluted with a suitable gradient
of water–MeCN containing 0.1% TFA. Peptides purified from
HPLC were lyophilized and kept under nitrogen in a freezer
(Ϫ20 ЊC). For the determination of concentrations of peptide
DP178 and its multivalent derivatives, the UV absorbance was
run on Beckman DU 640 spectrophotometer at 280 nm, and
the concentration was calculated according to the equation
proposed in the literature.46 CD spectra were measured on
JASCO-810 (CD-ORD) spectropolarimeter using quartz
cuvette of 1 mm path length at 23 ЊC.
H-4, H-5), 5.88 (m, 3H, HC᎐CH ), 5.23 (m, 3H, HHC᎐CHC),
᎐
᎐
2
5.11 (m, 3H, HHC᎐CHC), 4.13 (m, 4H, OH CHC᎐CH , (C-7,
᎐
᎐
2
2
C-12)), 3.99 (bs, 2H, OH CHC᎐CH , C-3), 3.88–3.50 (series of
᎐
2
2
multiplet, 6H), 3.35 (t, 2H, J = 8.3 Hz, H2–C(24)), 3.32 (bs, 1H,
H–C(12)), 3.16 (m, 1H, H–C(7)), 3.03 (m, 1H, H–C(3)), 3.07
(m, 1H, H–C(3)), 2.30–1.02 (series of multiplet, 18H), 0.92 (d,
3H, J = 6.0 Hz, H3C–C(20)), 0.89 (s, 3H, H3C–C(10)), 0.66 (s,
3H, H3C–C(13)).
3ꢀ,7ꢀ,12ꢀ-Triallyloxy-24-hydroxy-5ꢁ-cholane (5). p-Toluene-
sulfonic acid (45.0 mg, 0.23 mmol) was added to a solution of
compound 4 (1.20 g, 1.6 mmol) in a mixed solvent of dichloro-
methane (40 mL) and EtOH (10 mL). The mixture was stirred
at room temperature overnight, then diluted with dichloro-
methane (20 mL). The mixture was washed with 0.5 NaHCO3
(15 mL), H2O (2 × 15 mL) and brine. The organic layer was
dried over anhydrous Na2SO4 and concentrated under vacuum.
The residue was subject to flash column chromatography on
silica gel with hexane/ethyl acetate (70 : 30, v/v) as the eluent to
give 5 (0.83 g, 100%) as a colorless syrup. 1H NMR (500 MHz,
CDCl /TMS) δ 5.91 (m, 3H, HC᎐CH ), 5.25 (m, 3H, HHC᎐
᎐
᎐
3
2
CHC), 5.11 (m, 3H, HHC᎐CHC), 4.07 (d, 2H, J = 12.5 Hz,
᎐
3ꢀ,7ꢀ,12ꢀ,24-Tetrahydroxycholane (2). To a suspension of
LiAlH4 (3.85 g, 100 mmol) in dry THF (100 mL) at 0 ЊC was
added dropwise a solution of cholic acid 1 (15.03 g, 35 mmol) in
dry THF (200 mL) with vigorous stirring under nitrogen
atmosphere. The reaction mixture was then stirred at room
temperature overnight. Celite (3.00 g) was added to the sluggish
mixture. The precipitate was removed via filtration and washed
with hot methanol. The filtrate was concentrated, and the
product was crystallized with acetone to compound 2 as a white
solid (13.20 g, 98%): m.p. 238–239 ЊC (lit.,38 m.p. 236–238 ЊC);
1H NMR (500 MHz, CDCl3/TMS) δ 3.96 (bs, 1H, HO–C(12)),
3.82 (bs, 1H, HO–C(7)), 3.79 (d, 2H, J = 2.5 Hz, H2–C(24)),
3.25 (b, 1H, HO–C(24)), 3.21 (m, 1H, H–C(12)), 3.15 (m, 1H,
H–C(7)), 3.07 (m, 1H, H–C(3)), 2.15–1.10 (series of multiplet,
25H), 0.92 (d, 3H, J = 6.5 Hz, H3C–C(20)), 0.89 (s, 3H, H3C–
C(10)), 0.65 (s, 3H, H3C–C(13)); ESI-MS: 395.58 (M ϩ H)ϩ.
OH CHC᎐CH , (C-12)), 4.00 (d, 2H, J = 5.5 Hz, OH CHC᎐
᎐ ᎐
2
2
2
CH2, (C-7)), 3.77 (dt, 1H, J = 7.5, 5.0 Hz, HH–C(24)), 3.71
(ddd, 1H, J = 6.0, 4.5, 1.5 Hz, HH–C(24)), 3.61 (b, 2H,
OH CHC᎐CH , (C-3)), 3.54 (bs, 1H, H–C(12)), 3.32 (d, 1H,
᎐
2
J =2 2.5 Hz, H–C(7)), 3.13 (m, 1H, H–C(3)), 2.26 (dd, 1H,
J = 13.0, 12.0 Hz, H–C(11)), 2.18 (ddd, 1H, J = 8.0, 8.0, 4.0 Hz,
H–C(8)), 2.01 (dd, 1H, J = 10.0, 9.5 Hz, H–C(11)), 1.87–0.96
(series of multiplet, 24H), 0.92 (d, 3H, J = 6.5 Hz, H3C–C(20)),
0.89 (s, 3H, H3C–C(10)), 0.65 (s, 3H, H3C–C(13)); 13C NMR
(500 MHz, CDCl3/TMS) 136.26, 116.45, 115.69, 95.00, 80.98,
79.34, 75.07, 68.58, 69.47, 68.93, 63.93, 63.90, 46.58, 46.55,
42.81, 42.25, 42.23, 40.03, 35.76, 35.62, 35.60, 35.24, 35.23,
32.02, 29.69, 29.11, 28.22, 27.86, 27.69, 23.44, 23.22, 18.03,
12.79; ESI-MS calcd. for C33H54O4 (M): 514.40; Found: 515.57
(M ϩ H)ϩ, 457.52 (M Ϫ 58 ϩ H)ϩ, 399.47 (M Ϫ 2 × 58 ϩ H)ϩ,
341.47 (M Ϫ 3 × 58 ϩ H)ϩ.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 0 7 – 3 5 1 3
3511

View Article Online
3ꢀ,7ꢀ,12ꢀ-Tri-(6-amino-3-thiahexyoxyl)-24-hydroxy-5ꢁ-
cholane (6). To a mixture of compound 5 (300 mg, 0.58 mmol)
and 2-aminoethanethiol hydrochloride (397 mg, 3.50 mmol) in
methanol (20 mL) in a Quartz flask was added AIBN (9.4 mg,
0.057 mmol). The solution was degassed by bubbling N2, and
irradiated by UV (254 nm) with stirring under N2 for 20 h when
1H NMR revealed that no residue allyl group existed. Dichloro-
methane (30 mL) was added and the mixture was washed with 0.5
M NaHCO3 (2 × 10 mL), H2O (2 × 5 mL) and brine. The organic
layer was dried over anhydrous NaSO4 and concentrated under
vacuum. The residue was subject to flash column chromato-
graphy on silica gel with ethyl acetate/methanol (0 : 100 to 100 : 0,
HO–C(24)), 3.21 (m, 1H, H–C(12)), 3.15 (m, 1H, H–C(7)), 3.07
(m, 1H, H–C(3)), 2.66–2.59 (m, 12H, OCH2CH2CH2S, SCH2-
CH2N), 2.18 (m, 6H, NCOCH2CH2), 2.15–1.06 (series of
multiplet, 48H), 0.92 (d, 3H, J = 6.5 Hz, H3C–C(20)), 0.89 (s,
3H, H3C–C(10)), 0.65 (s, 3H, H3C–C(13)); 13C NMR (500
MHz, CDCl3/TMS) 182.11, 178.58, 175.72, 174.17, 174.22,
165.45, 165.50, 164.76, 164.58, 163.88, 163.59, 138.62, 138.61,
137.85, 136.67, 136.20, 135.96, 86.51, 75.43, 72.47, 67.51, 67.29,
66.91, 63.38, 49.95, 48.27, 45.13, 45.91, 46.11, 43.35, 41.56,
40.88, 41.09, 41.82, 39.45, 37.76, 36.58, 35.33, 34.91, 34.96,
34.54, 34.57, 34.58, 34.30, 34.37, 34.89, 33.52, 33.62, 32.28,
31.17, 30.18, 29.18, 29.23, 29.56, 28.85, 28.63, 28.83, 28.12,
27.91, 27.03, 26.41, 26.68, 26.76, 25.83, 25.67, 25.31, 24.67,
17.89, 11.76; ESI-MS calcd. for C69H108N6O13S3 (M): 1325.70;
found: 1348.09 (M ϩ Na)ϩ, 1326.17 (M ϩ H)ϩ, 663.82
1
v/v) as the eluent to give 6 (415 mg, 96%) as a syrup. H NMR
(500 MHz, CDCl3/TMS) δ 3.74 (m, 1H, H–C(24)), 3.68 (m, 1H,
H–C(24)), 3.61 (m, 2H, OCH2CCH2CH2S), 3.56 (bs, 2H, OCH2-
CCH2CH2S), 3.53 (t, 2H, J = 7.0 Hz, OCH2CCH2CH2S), 3.35 (m,
7H, CH2CH2NH2, HO–C(24)), 3.22 (m, 1H, H–C(12)), 3.16 (m,
7H, NCH2CH2S, H–C(7)), 3.06 (m, 1H, H–C(3)), 2.86 (m, 6H,
OCH2CH2CH2S), 2.74 (m, 3H, OCH2CHHCH2S), 2.68 (t, 3H, J
= 7.0 Hz, OCH2CHHCH2S), 2.20–1.05 (series of multiplet, 30H),
0.98 (d, 3H, J = 6.0 Hz, H3C–C(20)), 0.94 (s, 3H, H3C–C(10)),
0.72 (s, 3H, H3C–C(13)); 13C NMR (500 MHz, CDCl3/TMS)
80.82, 79.95, 76.15, 66.19, 65.82, 62.47, 47.36, 46.46, 46.29, 42.86,
42.10, 40.64, 39.87, 38.91, 38.86, 35.72, 35.45, 35.12, 34.82, 32.11,
30.14, 29.93, 29.83, 28.93, 28.79, 28.61, 28.54, 28.47, 28.33, 28.00,
27.65, 27.59, 23.32, 22.72, 22.16, 17.52, 17.13, 14.47, 11.75;
ESI-MS calcd. for C39H75N3O4S3 (M): 745.48. Found: 746.58
(M ϩ 2H)2ϩ
.
3ꢀ,7ꢀ,12ꢀ-Tri-[6-(2-bromoacetylamido-3-thiahexyoxyl)]-24-
hydroxy-5ꢁ-cholane (9). To a solution of free amine 6 (12 mg, 16
µmol) in dichloromethane (10 mL) was added bromoacetic
anhydride (17 mg, 64 µmol). The mixture was stirred at room
temperature for 3 h when TLC showed that no more free amine
remained in the reaction mixture. The solvent was removed
under vacuum. The residue was subject to column chromato-
graphy on silica gel with ethyl acetate/hexane (60 : 40, v/v) as the
eluent to give 9 (16 mg, 89%) as a pale white oil. 1H NMR (500
MHz, CDCl3/TMS) δ 4.04 (m, 1H, H–C(24)), 4.00 (m, 1H, H–
C(24)), 3.93 (m, 6H, OCH2CCH2CH2S), 3.89 (m, 6H,
CH2CH2NH), 3.61 (m, 6H, OCH2CH2CH2S), 3.32 (m, 1H, H–
C(12)), 3.25 (b, 1H, HO–C(24)), 3.14 (m, 7H, NCH2CH2S, H–
C(7)), 3.08 (m, 1H, H–C(3)), 2.71 (m, 3H, OCH2CHHCH2S),
2.63 (m, 3H, OCH2CHHCH2S), 2.20–1.05 (series of multiplet,
28H), 1.82 (bs, 6H, OCCH2Br), 0.92 (d, 3H, J = 6.0 Hz, H3C–
C(20)), 0.89 (s, 3H, H3C–C(10)), 0.66 (s, 3H, H3C–C(13)); 13C
NMR (500 MHz, CDCl3/TMS) 168.53, 168.51, 168.51, 80.82,
79.95, 76.15, 66.19, 65.82, 65.81, 62.47, 46.52, 46.29, 42.86,
42.10, 40.64, 39.87, 38.91, 38.86, 35.72, 35.45, 35.12, 34.82,
32.11, 30.14, 29.93, 29.83, 28.93, 28.79, 28.61, 28.54, 28.47,
28.33, 28.00, 27.65, 27.59, 27.34, 25.60, 23.32, 22.72, 22.16,
17.52, 17.13, 14.47, 13.55, 11.75; ESI-MS calcd. for C45H78Br3-
N3O7S3 (M): 1109.25. Found: 1110.52 (M ϩ H)ϩ, 555.58
(M ϩ H)ϩ, 373.98 (M ϩ 2H)2ϩ
.
3ꢀ,7ꢀ,12ꢀ-Tri-(6-maleimido-3-thiahexyoxyl)-24-hydroxy-5ꢁ-
cholane (7). To a solution of free amine 6 (10 mg, 13 µmol)
in DMF (10 mL) were added N-methoxycarbonylmaleimide
(20 mg, 0.13 mmol) and Et3N (1.0 mL) dropwise with stirring.
After 6 h, the reaction mixture was diluted with ethyl acetate
(20 mL) and washed with H2O (2 × 20 mL) and brine. The
organic layer was separated, dried over anhydrous Na2SO4, fil-
tered, and concentrated. The residue was subject to column
chromatography on silica gel with ethyl acetate/hexane (5 : 95,
v/v) as the eluent to provide 7 (10 mg, 78%) as a pale-yellow oil.
1H NMR (500 MHz, CDCl /TMS) δ 6.73 (s, 6H, OCHC᎐CH),
᎐
3
3.79 (d, 2H, J = 2.5 Hz, H2–C(24)), 3.74 (m, 6H, CH2CH2-
NCO), 3.68–3.57 (m, 6H, OCH2CCH2CH2S), 3.57–3.42 (m,
6H, SCH2CH2N), 3.22 (b, 1H, HO–C(24)), 3.20 (m, 1H,
H–C(12)), 3.17 (m, 1H, H–C(7)), 3.05 (m, 1H, H–C(3)), 2.72
(m, 6H, OCH2CH2CH2S,), 2.69–2.60 (m, 6H, OCH2CH2CH2S),
2.15–0.98 (series of multiplet, 24H), 0.92 (d, 3H, J = 6.5 Hz,
H3C–C(20)), 0.88 (s, 3H, H3C–C(10)), 0.65 (s, 3H, H3C–C(13));
13C NMR (500 MHz, CDCl3/TMS) 187.05, 176.13, 170.73,
170.72, 170.75, 166.02, 160.98, 157.37, 156.08, 155.85, 134.12,
134.27, 134.30, 134.36, 135.06, 136.6, 104.75, 102.52, 99.57,
94.82, 94.80, 93.96, 93.91, 91.26, 90.05, 85.27, 60.01, 57.73,
50.85, 49.91, 48.23, 37.06, 37.02, 37.10, 36.13, 36.01, 36.00,
34.78, 34.66, 34.63, 34.29, 29.14; 29.10, 29.00, 25.67, 25.31,
24.67, 19.22, 18.55, 18.08, 11.86. ESI-MS calcd. for C51H75N3-
O10S3 (M): 985.45. Found: 986.84 (M ϩ H)ϩ, 771.73 (M Ϫ 215
ϩ H)ϩ, 556.59. (M Ϫ 2 × 215ϩ H)ϩ.
(M ϩ 2H)2ϩ
.
Peptides
All peptides were synthesized using the standard Fmoc-based
solid phase peptide synthesis on a Fmoc-PAL-PEG-PS resin.25
The P37C, Ac-YTSLIHSLIEESQNQQEKNEQELLELDKW-
ASLWNWFC-NH2, was reported previously.25 The character-
istic data for the T-helper epitope and the minimum epitope
sequence of the HIV-neutralizing antibody 2F5 are shown
below.
T-helper
CGSSSQYIKANSKFIGITEL-NH2, tR 8.6 min (under the
analytical HPLC conditions described in the general methods);
ESI-MS: Calcd. M = 2145.48; Found: 1073.90 (M ϩ 2H)2ϩ
,
716.42 (M ϩ 3H)3ϩ
.
3ꢀ,7ꢀ,12ꢀ-Tri-[6-(6-maleimidohexanamido-3-thiahexyoxyl)]-
24-hydroxy-5ꢁ-cholane (8). To a solution of free amine 6 (7.2
mg, 9.6 µmol) in dichloromethane (10 mL) was added 6-male-
imidohexanoic acid N-hydroxylsuccinimide ester (30 mg, 96
µmol). The mixture was stirred at room temperature for 3 h and
the solvent was evaporated under vacuum. The residue was sub-
ject to column chromatography on silica gel with ethyl acetate/
methanol (95 : 5, v/v) as the eluent to afford 8 (9.6 mg, 76%) as a
P-7C
Ac-ELDKWAC-NH2, tR 7.9 min (under the analytical HPLC
conditions described in the general methods). ESI-MS: Calcd.
M = 905.86; Found, 905.53 (M ϩ H)ϩ, 453.42 (M ϩ 2H) 2ϩ
.
Ligation of cysteine-containing peptide to the cholic acid-based
maleimide clusters
1
colorless oil. H NMR (500 MHz, CDCl3/TMS) δ 6.72 (s, 6H,
HC᎐CH), 6.61 (t, 1H, J = 6.5 Hz, HN), 6.48 (t, 1H, J = 6.5 Hz,
The peptides (1.5 mol. equivalent per maleimide functionality)
were dissolved in degassed phosphate buffer (pH 6.6) contain-
ing 50% acetonitrile to a final concentration of ca. 2 µmol
mLϪ1. The mixture was gently shaken under N2 at room
᎐
HN), 6.19 (t, 1H, J = 6.5 Hz, HN), 3.59 (td, 2H, J = 13.0, 6.5
Hz, H2–C(24)), 3.52 (t, 6H, J = 7.0 Hz, OCH2CCH2CH2S), 3.47
(bs, 6H, CH2CH2NCO) 3.43 (m, 6H, NCH2CH2S), 3.25 (b, 1H,
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 0 7 – 3 5 1 3
3512

View Article Online
3 J. P. Tam and J. C. Spetzler, Methods Enzymol., 1997, 289, 612–637.
temperature. The processing of ligation was monitored with
analytic HPLC. The reaction mixture was purified with
RP-HPLC. The peptides were lyophilized and their identity was
characterized by ESI-MS.
4 J. P. Tam, J. Immunol. Methods, 1996, 196, 17–32.
5 Y. Wei, G. L. McLendon, A. D. Hamilton, M. A. Case, C. B.
Purring, Q. Lin, H. S. Park, C. S. Lee and T. Yu, Chem. Commun.,
2001, 1580–1581.
6 H. S. Park, Q. Lin and A. D. Hamilton, Proc. Natl. Acad. Sci.
U. S. A., 2002, 99, 5105–5109.
Trivalent peptide (10a)
7 M. A. Blaskovich, Q. Lin, F. L. Delarue, J. Sun, H. S. Park,
D. Coppola, A. D. Hamilton and S. M. Sebti, Nat. Biotechnol., 2000,
18, 1065–1070.
8 J. P. Tam, Y. A. Lu and J. L. Yang, Eur. J. Biochem., 2002, 269, 923–
932.
9 J. P. Tam and Q. Yu, Org. Lett., 2002, 4, 4167–4170.
10 G. Tuchscherer, C. Servis, G. Corradin, U. Blum, J. Rivier and
M. Mutter, Protein Sci., 1992, 1, 1377–1386.
11 G. Tuchscherer, Tetrahedron Lett., 1993, 34, 8419–8422.
12 A. Nefzi, X. Sun and M. Mutter, Tetrahedron Lett., 1995, 36, 229–
230.
13 H. K. Rau, N. DeJonge and W. Haehnel, Proc. Natl. Acad. Sci.
U. S. A., 1998, 95, 11526–11531.
14 K. S. Akerfeldt, R. M. Kim, D. Camac, J. T. Groves, J. D. Lear and
W. F. DeGrado, J. Am. Chem. Soc., 1992, 114, 9656–9657.
15 T. Sasaki and E. T. Kaiser, J. Am. Chem. Soc., 1989, 111, 380–381.
16 H. S. Park, Q. Lin and A. D. Hamilton, J. Am. Chem. Soc., 1999,
121, 8–13.
17 A. S. Causton and J. C. Sherman, Bioorg. Med. Chem., 1999, 7,
23–27.
18 A. S. Causton and J. C. Sherman, J. Pept. Sci., 2002, 8, 275–282.
19 J. Brask and K. J. Jensen, J. Pept. Sci., 2000, 6, 290–299.
20 J. Brask and K. J. Jensen, Bioorg. Med. Chem. Lett., 2001, 11, 697–
700.
82% yield; tR 15.1 min (under the analytical HPLC conditions
described in the general methods); ESI-MS: Calcd. M =
14770.88; Found, 1847.83 (M ϩ 8H)8ϩ, 1642.74 (M ϩ 9H)9ϩ
,
1478.59 (M ϩ 10H)10ϩ, 1344.26 (M ϩ 11H)11ϩ, 1232.32
(M ϩ 12H)12ϩ, 1137.72 (M ϩ 13H)13ϩ, 1056.34 (M ϩ 14H)14ϩ
.
Trivalent peptide (10b)
20% yield; tR 12.0 min (under the analytical HPLC conditions
described in the general methods); ESI-MS: Calcd. M =
7421.90; Found, 1485.67 (M ϩ 5H)5ϩ, 1238.18 (M ϩ 6H)6ϩ
,
1061.51 (M ϩ 7H)7ϩ, 928.94 (M ϩ 8H)8ϩ, 825.98 (M ϩ 9H)9ϩ
.
Trivalent peptide (11a)
83% yield; Retention time (tR) 14.4 min (under the analytical
HPLC conditions described in the general methods); ESI-MS:
Calcd. M = 15110.13; Found, 1889.74 (M ϩ 8H)8ϩ, 1680.20
(Mϩ 9H)9ϩ, 1512.32 (M ϩ 10H)10ϩ, 1374.92 (M ϩ 11H)11ϩ
,
1260.44 (M ϩ12H)12ϩ
(Mϩ14H)14ϩ
, , 1080.46
1163.64 (M ϩ13H)13ϩ
.
21 K. J. Jensen and G. Barany, J. Pept. Res., 2000, 56, 3–11.
22 R. P. McGeary, I. Jablonkai and I. Toth, Tetrahedron, 2001, 57,
8733–8742.
23 K. J. Jensen and J. Brask, Cell. Mol. Life Sci., 2002, 59, 859–869.
24 J. Brask, J. M. Dideriksen, J. Nielsen and K. J. Jensen, Org. Biomol.
Chem., 2003, 1, 2247–2252.
25 L. X. Wang, J. Ni and S. Singh, Bioorg. Med. Chem., 2003, 11, 159–
166.
26 L. X. Wang, Curr. Pharm. Des., 2003, 9, 1771–1787.
27 S. Jiang, Q. Zhao and A. K. Debnath, Curr. Pharm. Des., 2002, 8,
563–580.
28 D. C. Chan and P. S. Kim, Cell, 1998, 93, 681–684.
29 R. Balasubramanian and U. Maitra, J. Org. Chem., 2001, 66, 3035–
3040.
30 C. Goto, M. Yamamura, A. Satake and Y. Kobuke, J. Am. Chem.
Soc., 2001, 123, 12152–12159.
31 P. W. Swaan, K. M. Hillgren, F. C. Szoka Jr. and S. Oie, Bioconjugate
Chem., 1997, 8, 520–525.
32 M. Kagedahl, P. W. Swaan, C. T. Redemann, M. Tang, C. S. Craik,
F. C. Szoka Jr. and S. Oie, Pharm. Res., 1997, 14, 176–180.
33 H. De Muynck, A. Madder, N. Farcy, P. J. De Clercq, M. N. Perez-
Payan, L. M. Ohberg and A. P. Davis, Angew. Chem. Int. Ed., 2000,
39, 145–148.
34 J. F. Barry, A. P. Davis and M. N. Perez-Payan, Tetrahedron Lett.,
1999, 40, 2849–2852.
35 X. T. Zhou, A. Rehman, C. Li and P. B. Savage, Org. Lett., 2000, 2,
3015–3018.
36 C. T. Wild, D. C. Shugars, T. K. Greenwell, C. B. McDanal and
T. J. Matthews, Proc. Natl. Acad. Sci. U. S. A., 1994, 91, 9770–9774.
37 J. M. Kilby, S. Hopkins, T. M. Venetta, B. DiMassimo, G. A. Cloud,
J. Y. Lee, L. Alldredge, E. Hunter, D. Lambert, D. Bolognesi,
T. Matthews, M. R. Johnson, M. A. Nowak, G. M. Shaw and
M. S. Saag, Nat. Med., 1998, 4, 1302–1307.
Trivalent peptide (11b)
91% yield; tR 12.4 min (under the analytical HPLC conditions
described in the general methods); ESI-MS: Calcd. M =
7761.16; Found, 1553.33 (M ϩ 5H)5ϩ, 1294.71 (M ϩ 6H)6ϩ
,
1109.88 (M ϩ 7H)7ϩ, 971.31 (M ϩ 8H)8ϩ, 863.56 (M ϩ 9H)9ϩ
.
Trivalent peptide (2)
The peptide P-7C (9.7 mg, 11 µmol) was dissolved in degassed
sodium borate buffer (pH 8.5) containing 50% acetonitrile
(3 mL). The bromoacetyl derivative 9 (2.6 mg, 2.4 µmol) in
acetonitrile was added with gentle shaking. The processing of
ligation was monitored with analytic HPLC. The desired tri-
valent peptide 12 was purified with RP-HPLC and lyophilized
to afford a white solid (3 mg, 35%). tR 14.5 min (under the
analytical HPLC conditions described in the general methods);
ESI-MS: Calcd. M = 3582.65; Found, 1792.26 (M ϩ 2H)2ϩ
,
1195.14 (M ϩ 3H)3ϩ, 896.74 (M ϩ 4H)4ϩ, 717.68 (M ϩ5H)5ϩ
.
Circular dichroism (CD)
The samples of trivalent peptide 10a, trivalent peptide 11a, pep-
tides DP178, and the control sample 3α,7α,12α-tri-(6-amino-3-
thiahexyoxyl)-24-hydroxy-5β-cholane 6 were prepared in a
concentration range from 8 to 18 µM in 50 mM phosphate
buffer (pH 7.2). The accurate concentration of the peptides was
measured by UV-absorption and calculated based on the pro-
posed formulation.46 The measurement was run at 23 ЊC. Blank
was subtracted from the CD spectra, which were the average of
three-time scans.
38 C. Li, A. S. Peters, E. L. Meredith, G. W. Allman and P. B. Savage,
J. Am. Chem. Soc., 1998, 120, 2961–2962.
39 J. Ni, S. Singh and L. X. Wang, Carbohydr. Res., 2002, 337, 217–220.
40 P. Panina-Bordignon, A. Tan, A. Termijtelen, S. Demotz,
G. Corradin and A. Lanzavecchia, Eur. J. Immunol., 1989, 19, 2237–
2242.
41 T. Muster, F. Steindl, M. Purtscher, A. Trkola, A. Klima,
G. Himmler, F. Ruker and H. Katinger, J. Virol., 1993, 67, 6642–
6647.
42 H. K. Rau and W. Haehnel, J. Am. Chem. Soc., 1998, 120, 468–476.
43 Y. A. Lu, P. Clavijo, M. Galantino, Z. Y. Shen, W. Liu and J. P. Tam,
Mol. Immunol., 1991, 28, 623–630.
Acknowledgements
We thank Ms Haijing Song for technical assistance; Dr Jiahong
Ni for providing peptide T20; and Dr Ilia V. Baskakov for
assistance in measuring the CD spectra. The work was
supported by the Institute of Human Virology, University of
Maryland Biotechnology Institute, and National Institutes of
Health (grant AI51235 to LXW).
44 M. K. Lawless, S. Barney, K. I. Guthrie, T. B. Bucy, S. R. Petteway
Jr. and G. Merutka, Biochemistry, 1996, 35, 13697–13708.
45 P. C. Lyu, J. C. Sherman, A. Chen and N. R. Kallenbach, Proc. Natl.
Acad. Sci. U. S. A., 1991, 88, 5317–5320.
46 C. N. Pace, F. Vajdos, L. Fee, G. Grimsley and T. Gray, Protein Sci.,
1995, 4, 2411–2423.
References
1 G. Tuchscherer and M. Mutter, J. Pept. Sci., 1995, 1, 3–10.
2 G. Tuchscherer, D. Grell, M. Mathieu and M. Mutter, J. Pept. Res.,
1999, 54, 185–194.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 0 7 – 3 5 1 3
3513