Conjugation of a nonspecific antiviral sapogenin with a specific HIV fusion Inhibitor: A promising strategy for discovering new antiviral therapeutics

Article abstract of DOI:10.1021/jm500763m

Triterpene saponins are a major group of active components in natural products with nonspecific antiviral activities, while T20 peptide (enfuvirtide), which contains a helix zone-binding domain (HBD), is a gp41-specific HIV-1 fusion inhibitor. In this paper, we report the design, synthesis, and structure-activity relationship (SAR) of a group of hybrid molecules in which bioactive triterpene sapogenins were covalently attached to the HBD-containing peptides via click chemistry. We found that either the triterpenes or peptide part alone showed weak activity against HIV-1 Env-mediated cell-cell fusion, while the hybrids generated a strong cooperative effect. Among them, P26-BApc exhibited anti-HIV-1 activity against both T20-sensitive and -resistant HIV-1 strains and improved pharmacokinetic properties. These results suggest that this scaffold design is a promising strategy for developing new HIV-1 fusion inhibitors and possibly novel antiviral therapeutics against other viruses with class I fusion proteins.

Full text of DOI:10.1021/jm500763m

Article
pubs.acs.org/jmc
Conjugation of a Nonspecific Antiviral Sapogenin with a Specific HIV
Fusion Inhibitor: A Promising Strategy for Discovering New Antiviral
Therapeutics
Chao Wang,† Lu Lu,‡ Heya Na,† Xiangpeng Li,† Qian Wang,‡ Xifeng Jiang,† Xiaoyu Xu,† Fei Yu,§
Tianhong Zhang,† Jinglai Li,† Zhenqing Zhang,† Baohua Zheng,† Guodong Liang,† Lifeng Cai,†
Shibo Jiang,*,‡,§ and Keliang Liu*,†
†Beijing Institute of Pharmacology & Toxicology, 27 Tai-Ping Road, Beijing 100850, China
‡Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, Shanghai Medical College and Institute of
Medical Microbiology, Fudan University, Shanghai 200032, China
§Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York 10065, United States
S
* Supporting Information
ABSTRACT: Triterpene saponins are a major group of active
components in natural products with nonspecific antiviral activities,
while T20 peptide (enfuvirtide), which contains a helix zone-binding
domain (HBD), is a gp41-specific HIV-1 fusion inhibitor. In this
paper, we report the design, synthesis, and structure−activity
relationship (SAR) of a group of hybrid molecules in which
bioactive triterpene sapogenins were covalently attached to the
HBD-containing peptides via click chemistry. We found that either
the triterpenes or peptide part alone showed weak activity against HIV-1 Env-mediated cell−cell fusion, while the hybrids
generated a strong cooperative effect. Among them, P26−BApc exhibited anti-HIV-1 activity against both T20-sensitive and
-resistant HIV-1 strains and improved pharmacokinetic properties. These results suggest that this scaffold design is a promising
strategy for developing new HIV-1 fusion inhibitors and possibly novel antiviral therapeutics against other viruses with class I
fusion proteins.
energy for virus−cell membrane fusion and entry into host
cells.12
INTRODUCTION
■
Antiviral agents can be designed by targeting different steps of
the enveloped virus replication cycle.1 Among the various life
cycle events, the entry of enveloped viruses into host cells by
fusion of the viral membrane with the cellular membrane is the
first step.2 This process has been considered as an attractive
target and has received wide attention.3,4 The human
immunodeficiency virus type 1 (HIV-1) features a common
mechanism to catalyze membrane fusion as other enveloped
viruses with class I fusion proteins.5 The envelope glycoproteins
(Env) of HIV-1, a class I membrane fusion protein, play
important roles in the early stage of HIV-1 entry.6,7 HIV-1 Env
is composed of a complex of noncovalently associated
glycoproteins surface subunit gp120 and transmembrane
subunit gp41.8 Its surface subunit gp120 engages in receptor
binding, and gp41 mediates fusion of the virus with the target
cell.9 With typical sequence motifs homologous to those
identified in class I enveloped viruses, gp41 contains an
extracellular domain, a transmembrane domain, and a
cytoplasmic tail.10,11 It is well-known that the formation of a
general six-helix bundle (6-HB) between three C-terminal
heptad repeats (CHRs) and an N-terminal heptad repeat
(NHR) trimer of the gp41 extracellular domain provides the
The synthetic peptides derived from CHR sequences can
bind to their counterpart, NHR, to prevent intramolecular
fusogenic 6-HB formation, thereby inhibiting membrane fusion
and viral infection.13,14 T20 (generic name, enfuvirtide; brand
name, Fuzeon) was approved by the U.S. Food and Drug
Administration in 2003 as the only HIV-1 fusion inhibitor for
the treatment of HIV-1 infections.15 Another peptide, C34,
which has a sequence that partially overlaps with that of T20,
exhibits promising potency in inhibiting HIV-1-mediated
membrane fusion.2 However, due to its poor solubility under
physiological conditions, C34 has only been used as a
laboratory tool to study the structure and function of HIV-1
gp41. Previous studies have shown that T20 contains a lipid-
binding domain (LBD), whereas C34 consists of a pocket-
binding domain (PBD), and both share a helix zone-binding
domain (HBD) (Figure 1A).16 The HBD exhibits only
marginal anti-HIV-1 activity. However, the addition of a PBD
to the N-terminus of the HBD or a LBD to the C-terminus of
the HBD makes the peptides much more potent than peptides
Received: May 16, 2014
© XXXX American Chemical Society
A
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Figure 1. Schematic representation of HIV-1 gp41 and the designed sapogenin−peptide conjugates. (A) HIV-1 gp41 contains a fusion peptide (FP),
an N-terminal heptad repeat (NHR), a C-terminal heptad repeat (CHR), a transmembrane domain (TM), and a cytoplasm domain (CP). The key
conformational change in gp41 can be blocked by CHR-derived fusion inhibitors T20 and C34. Both of them contain a partial helix zone-binding
domain (HBD), which is crucial for these CHR-based fusion inhibitors to interact with the helix zone in the NHR. (B) Chikusetsusaponin IVa
extracted from Alternanthera philoxeroides (Mart.) Griseb. (C) The designed sapogenin−peptide conjugates.
containing the HBD alone.17 Furthermore, some peptides
corresponded to the C-proximal region of α1-antitrypsin,
designated virus-inhibitory peptide (VIRIP),18,19 or derived
from the E2 envelope protein of GB virus C (GBV-C),20 can
interact with the HIV-1 gp41 fusion peptide and effectively
prevent the virus from entering into the host cell.
Saponins are a group of bioactive natural compounds mainly
produced by plants to counteract pathogens and herbivores; in
addition, they are a resource of lead compounds in drug
discovery due to their tremendous range of pharmacological
properties.21 Several pentacyclic triterpene saponins, such as
chikusetsusaponin IVa and glycyrrhizin, show wide antiviral
activities, and their administration to virus-infected patients
resulted in delayed progression of infection symptoms (Figure
1B).22,23 Although saponins have immense structural diversity,
they all share similar structural components: a hydrophobic
isoprenoid-derived sapogenin covalently linked to a hydrophilic
single saccharide or oligosaccharide moiety.24
Previous reports have indicated that the overall conformation
of saponins harmoniously constructed with both hydrophobic
and hydrophilic segments is essential for their biological
effects.25,26 Inspired by the strong cooperative effect of the
aglycone and sugar chain units generated in saponins, we
hypothesized that the triterpene sapogenin may serve as the
PBD or LBD and that its addition to the N- or C-terminus of a
peptide containing only the HBD may result in a significant
increase of the anti-HIV-1 activity of the peptides. Herein, we
designed novel HIV-1 fusion inhibitors that consisted of the
HBD-containing peptide P26 linked with a sapogenin derivative
at either the N- or C-terminus of P26 (Figure 1C). The
triterpene sapogenin−peptide conjugates were tested as
inhibitors of HIV-1 Env-mediated cell−cell fusion and
laboratory-adapted HIV-1IIIB (subtype B, X4) or HIV-1BaL
(subtype B, R5) replication as well as against both T20-
sensitive and resistant HIV-1 strains. One of these conjugates,
P26−BApc, which exhibited promising anti-HIV-1 activity, was
further subjected to in vitro and in vivo pharmacokinetic
studies. The results of this study will provide a promising
strategy for the rational design of novel antiviral peptides
against HIV-1 and other enveloped viruses with class I fusion
proteins.
DESIGN
■
Because the interaction between NHR and CHR of HIV-1
gp41 plays a critical role in virus−cell membrane fusion, it is
conceivable that interfering with this interaction might prevent
fusogenic gp41 core formation and thus block HIV-1
infection.27 Indeed, both C34 and T20 peptides derived from
the CHR region exhibit anti-HIV-1 activities in the nanomolar
range.9 In our prior studies, we have shown that C34 and T20
contain a common 4−3 heptad repeat (HR) sequence, which
acts as a structure domain and is responsible for its interaction
with the helix zone in the gp41 NHR region.16,28 A synthetic
peptide CHR-3, which contains the entire 4−3 HR sequence
shared by C34 and T20 but lacks the PBD or LBD sequences,
exhibits only weak antiviral activity.17 These results suggest that
although the 4−3 HR sequence is essential for these C-peptides
to interact with the helix-zone domain in the viral gp41 NHR
region, the functional domains, e.g., the PBD in C34 and the
LBD in T20, are also critical for the interactions with their
corresponding targets. In this study, the HBD-derived peptide
B
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Figure 2. Chemical structures of triterpene derivatives.
a
Scheme 1. Synthesis of BA Derivatives
a
(i) TBDPSCl, imidazole, DMAP, DMF, 80 °C; (ii) 4-pentynoic acid, DIC, DMAP, DCM, rt; (iii) TBAF, THF, rt; (iv) propargyl bromide, K2CO3,
DMF, rt; (v) allyl bromide, K2CO3, DMF, rt; (vi) 4-pentynoic acid, DIC, DMAP, DCM, rt; (vii) acetic anhydride, pyridine, DMAP, DCM, rt; (viii)
propargyl bromide, K2CO3, DMF, rt.
P26 was used as the lead compound and fusion inhibitors with
novel scaffolds were synthesized by covalently attaching
bioactive triterpene sapogenins to the N- or C-terminus of
P26. We previously reported a set of conjugates in which the
small-molecule fusion inhibitors targeting the hydrophobic
cavity in the gp41 NHR-trimer can act as a substitute for the
pocket-binding domain (PBD) of the C34 peptide.29 We also
studied different linkers between a small molecule and a
peptide, in which a flexible linker, β-alanine (β-Ala), could
simply and nicely maintain the proper binding orientation and
conformation of the small molecules. Therefore, in this study,
the β-Ala spacer was inserted between the triterpene and the
target-specific peptide P26 to allow for flexibility.
Except for the hydrophobic aglycone precursor of triterpene
saponins, pentacyclic triterpenes exist in nature as free acids
and also possess a wide range of pharmacological profiles.21
Betulinic acid (BA) is a member of the triterpene family that is
present in a variety of plants.30 In an independent line of
investigation, Lee, Fujioka, and colleagues isolated BA from
Syzigium claviflorum and demonstrated its inhibitory potency
against HIV-1 replication in H9 lymphocytes.31 Subsequently,
Lee’s group designed a panel of BA derivatives with
C
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a
Scheme 2. Synthesis of OA and UA Derivatives
a
(i) TBDPSCl, imidazole, DMAP, DMF, 80 °C; (ii) 4-pentynoic acid, DIC, DMAP, DCM, rt; (iii) TBAF, THF, rt; (iv) propargyl bromide, K2CO3,
DMF, rt; (v) allyl bromide, K2CO3, DMF, rt; (vi) 4-pentynoic acid, DIC, DMAP, DCM, rt.
modifications at its C3 and C28 positions, respectively, and
identified lead compounds with enhanced anti-HIV-1 po-
tency.32,33 Structure−activity relationship (SAR) analysis34 has
shown that BA derivatives containing modifications to the
carboxylic acid group at C28, e.g., RPR103611 and IC9564
reported by Lee’s group, act at the entry step of infection. In
addition to the inhibitory effects on HIV-1 entry, BA derivatives
containing alterations to the hydroxyl group at the C3 position,
such as Bevirimat, have been demonstrated to inhibit viral
maturation specifically.35 Some other pentacyclic triterpenes
and their derivatives identified by Lee’s group, e.g., oleanolic
acid (OA) and ursolic acid (UA), which have a six-membered E
ring rather than the five-membered E ring found in BA, have
also exhibited antifusion or antimaturation activity.36,37 From
the previous SAR studies described above, the functional
groups at the C28 and C3 positions of these triterpene scaffolds
are critical for their bioactivities. Therefore, we designed and
synthesized four parallel series (series A−D) of triterpene
derivatives in which an alkyne group was introduced into the
parent structures of these sapogenins as the intermediate for
conjugation with the HBD-derived peptide P26 via a triazole
linkage by click chemistry to generate the sapogenin−peptide
conjugates (Figure 2).
resin with TFA. Unfortunately, this route was unsuccessful as a
result of the transformation of the olefinic bond of the
pentacyclic triterpenes in a TFA medium, which was consistent
with previous reports.38 Therefore, the triterpene derivatives
were covalently linked to the N- or C-terminus of the P26
peptide, respectively, via click chemistry.
The synthetic route to the triterpene derivatives is outlined
in Schemes 1 and 2. In general, to synthesize the triterpene
aglycone derivatives modified at the C3 position, the C28
carboxylic acid group was first protected by reacting it with tert-
butylchlorodiphenylsilane (TBDPSCl) to yield the silyl ester.
Next, the 4-pentynyl group was introduced at the C3 position
as a carboxylic ester. Finally, the TBDPS protecting group was
removed with tetrabutylammonium fluoride in tetrahydrofuran
(TBAF in THF) to afford the C3-modified series. On the other
hand, a propargyl group was directly introduced to the C28
carboxylic acid group to provide compounds in the C28-
modified series. As shown in Scheme 1, BApc and BApo were
also synthesized such that the C28 carboxylic acid group or the
C3 hydroxyl group on the scaffold of BA was permanently
protected by an allyl group or an acetyl group, respectively. As
shown in Scheme 2, UApc and OApc were also synthesized
such that the C28 carboxylic acid group on the scaffolds of UA
and OA was permanently protected by an allyl group,
respectively.
The azidoacetic acid was prepared in good yields by
substitution of sodium azide on the corresponding bromoacetic
acid in water. To synthesize sapogenin−peptide conjugates in
which a triterpene aglycone was attached to the N-terminus of
the P26 peptide, P26 peptide plus βAla as the linker was
initially synthesized on solid support using Rink Amide resin
following the standard Fmoc strategy synthesis procedure. This
βAla−P26 peptide was then functionalized on the N-terminus
CHEMISTRY
■
Because the unmodifed sapogenins have a carboxyl group
functionality, it would have been more straightforward to
couple the sapogenin derivatives to the amino group of the
peptides on the resin as the final step of solid-phase synthesis,
while the hydroxyl group of the sapogenins had been protected
as a tert-butyl ether during the reaction. All peptide protecting
groups, including the tert-butyl group on the nonpeptide
moiety, could be removed during peptide cleavage from the
D
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Figure 3. Strategy for the preparation of (A) BAc−P26 and (B) P26−BApc conjugates. Click chemistry provides an orthogonal and productive way
to link two large molecules.
Table 1. Inhibitory Activities of Sapogenin−Peptide Conjugates on HIV-1 Env-Mediated Cell−Cell Fusion
a
b
c
compd
sequence
EC50 (nM)
BAo−P26
UAo−P26
OAo−P26
BAc−P26
UAc−P26
OAc−P26
BApc−P26
BApo−P26
P26−BAo
P26−BAc
P26−BApc
P26−BApo
P26−UApc
P26−OApc
BA
BAo-a-NNYTSLIHSLIEESQNQQEKNEQELL
UAo-a-NNYTSLIHSLIEESQNQQEKNEQELL
OAo-a-NNYTSLIHSLIEESQNQQEKNEQELL
BAc-a-NNYTSLIHSLIEESQNQQEKNEQELL
UAc-a-NNYTSLIHSLIEESQNQQEKNEQELL
OAc-a-NNYTSLIHSLIEESQNQQEKNEQELL
BApc-a-NNYTSLIHSLIEESQNQQEKNEQELL
BApo-a-NNYTSLIHSLIEESQNQQEKNEQELL
NNYTSLIHSLIEESQNQQEKNEQELL-a-K(BAo)
NNYTSLIHSLIEESQNQQEKNEQELL-a-K(BAc)
NNYTSLIHSLIEESQNQQEKNEQELL-a-K(BApc)
NNYTSLIHSLIEESQNQQEKNEQELL-a-K(BApo)
NNYTSLIHSLIEESQNQQEKNEQELL-a-K(UApc)
NNYTSLIHSLIEESQNQQEKNEQELL-a-K(OApc)
betulinic acid
89.4 2.4
145 17
176 45
15.1 2.5
51.5 25
28.6 5.1
197 55
327 21
19.6 5.0
44.2 10
3.94 0.3
7.94 1.5
3.35 1.1
3.31 1.0
>100000
UA
ursolic acid
>100000
OA
oleanolic acid
>100000
P26
NNYTSLIHSLIEESQNQQEKNEQELL
Ptrz-a-NNYTSLIHSLIEESQNQQEKNEQELL
NNYTSLIHSLIEESQNQQEKNEQELL-a-K(Ptrz)
NNYTSLIHSLIEESQNQQEKNEQELL + BAo
YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF
3240 560
3580 156
2183 786
2390 612
10.1 1.4
Ptrz−P26
P26−Ptrz
P26 + BAo
T20
a
When a nonpeptide moiety is conjugated to the N-terminus of P26, the hybrid has carboxyamide at the C-terminus. When a nonpeptide moiety is
attached to the C-terminus of P26, the conjugate has an acetyl group at the N-terminus and carboxyamide at the C-terminus. P26 and T20 have an
b
c
acetyl group at the N-terminus and carboxyamide at the C-terminus. a, β-Alanine; Ptrz, 4-propyl-1H-1,2,3-triazol. Compounds were tested in
triplicate, and the data are presented as the mean standard deviation.
with an azido moiety by acylation with azidoacetic acid. Peptide
cleavage from the resin was carried out successfully, and the
crude azido-precursors were purified by reverse-phase HPLC.
Thereafter, sapogenin derivatives were attached to the azido-
precursors via click chemistry, which was achieved in the
presence of CuSO4·5H2O and sodium ascorbate in a mixture of
H2O and tert-butyl alcohol at room temperature (Figure 3A).
To synthesize conjugates in which a pentacyclic triterpene
was “capped” to the C-terminus of the P26 peptide, a lysine
(Lys) with allyloxycarbonyl (Alloc) group-modified side chains
was initially introduced to the C-terminus of the P26 peptide
through a βAla spacer. After routine synthesis, the Alloc
protecting group was selectively removed on-resin by
palladium-mediated deprotection. Next, an azido group was
introduced to the side chain of the Lys on-resin, and a click
reaction was applied after routine TFA-mediated cleavage and
purification (Figure 3B).
Click chemistry provides an orthogonal and productive way
for linking two large molecules. The reaction can be catalyzed
by cuprous ion from in situ reduction of copper(II) sulfate
pentahydrate through ascorbate.39 Although the formation of
byproducts resulting from Cu(II)-mediated oxidative side
reactions, e.g., Glaser-type alkyne coupling processes, is
suppressed as any dissolved dioxygen is reduced by ascorbate,
the ascorbate−Cu(II) system induced reactive oxygen species
have been shown to lead to histidine oxidation or produce
nondisulfide oxidative cross-linking.40,41 The tris-
(triazolylmethyl)amine ligands, e.g., N,N,N-Tris[(1-benzyl-1H-
1,2,3-triazol-4-yl)methyl]amine (TBTA), have been described
as a sacrificial reductant and a powerful stabilizing ligand for
E
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copper(I), intercepting hydroxyl radicals formation and
protecting Cu(I) from oxidation and disproportionation.40
Fortunately, neither histidine oxidation nor oxidative con-
nections was observed after 60 min in our experimental
condition (Supporting Information, Figure S1A). To analyze
the effect of TBTA on the copper-catalyzed azide−alkyne
cycloaddition (CuAAC), we further performed a cycloaddition
reaction between BAc and azido-precursor with addition of 5
equiv of TBTA relative to Cu. There were also no oxidized
byproducts observed. However, the reaction rate was reduced,
which was consistent with previous reports (Supporting
Information, Figure S1B).
2, a good agreement between fusion inhibition and inhibition of
HIV-1IIIB or HIV-1BaL infection was observed. P26−BApc,
Table 2. Anti-HIV-1 Activities and Cytotoxicities of the
a
Conjugates
EC50 (nM) for inhibiting
HIV-1IIIB
replication
HIV-1BaL
replication
b
compd
CC50 (μM)
SI
BAo−P26
UAo−P26
OAo−P26
BAc−P26
UAc−P26
OAc−P26
BApc−P26
BApo−P26
P26−BAo
P26−BAc
P26−BApc
P26−BApo
P26−UApc
P26−OApc
T20
475 87
565 130
369 2.0
133 53
387 252
94.0 15
242 11
501 122
154 9.0
61.6 16
4.28 0.7
41.4 1.0
2.18 0.8
3.11 0.1
10.9 0.4
456 72
288 32
498 87
98.0 26
113 55
150 21
519 98
99.0 12
135 15
83.1 8.3
6.90 0.1
20.8 5.5
2.51 0.5
0.271 0.01
2.88 0.1
>25
>25
>52
>44
>25
>68
>25
>187
>64
>25
RESULTS AND DISCUSSION
■
>25
>266
>103
>52
Inhibitory Activities of the Sapogenin−Peptide
Conjugates on HIV-1 Env-Mediated Cell−Cell Fusion.
First, we used an HIV-1 Env-mediated cell−cell fusion assay to
measure the inhibitory activities of these conjugates. The
activities of the compounds are shown in Table 1. In our assay,
BA, UA, and OA all exhibited no inhibitory activity in the cell−
cell fusion assay. P26 displayed moderate inhibitory activity,
with a 50% effective concentration (EC50) value of 3240 560
nM. Attaching BAo, UAo, or OAo to the N-terminus of P26
dramatically increased the activity, and this series of conjugates
had EC50 values of 89.4 2.4, 145 17, and 176 45 nM,
respectively, which were 18−40-fold greater than that of P26
alone. The inhibitory activity was further increased when the
compounds in series A were conjugated to P26, with EC50
>25
>25
>25
>50
>25
>406
3348
>603
3633
2643
>2293
14.3 1.0
>25
7.92 1.3
8.22 2.4
>25
a
Compounds were tested in triplicate, and the data are presented as
b
the mean standard deviation. SI (selectivity index) = CC50/EC50
for inhibiting HIV-1IIIB infection.
values of 15.1
2.5, 51.5
25, and 28.6
5.1 nM,
P26−UApc, and P26−OApc, which exhibited promising
inhibitory activities in the cell−cell fusion assay, also strongly
respectively, providing compounds with approximately 60-fold
more potency than that of P26 alone and reaching the potency
of T20. After the C28 carboxylic acid group and the C3
hydroxyl group were blocked with allyl and acetyl groups,
respectively, BApc−P26 exhibited 13-fold less potency than
BAc−P26, whereas BApo−P26 was only 3.6-fold less potent
than BAo−P26. These findings suggest that the C28 carboxylic
acid group has a more critical role than the C3 hydroxyl group
in inhibiting HIV-1 Env-mediated cell−cell fusion. In another
group of conjugates, the triterpene derivatives were linked to
the C-terminus of the P26 peptide. When the C28 or C3 group
was protected, the inhibitory potency was further increased.
However, the C28-protected hybrids (e.g., P26−BApc)
exhibited stronger inhibitory activities than the C3-protected
hybrids (e.g., P26−BApo). Compared with P26−BApc, P26−
UApc and P26−OApc showed similar inhibitory activities. It is
noteworthy that the triazole linkage makes limited contact with
the viral proteins at either the N- or C-terminus of P26. To
clarify whether P26 and sapogenin in a mixture state also
resulted in significant synergistic effect as in a covalently linked
state, the inhibitory activity of a mixture of P26 and BAo (1:1
ratio, unlinked) was tested as the control. Although P26−BAo
exhibited promising inhibitory activity, the synergy was lost
when simply mixing P26 with BAo in 1:1 ratio, which displayed
inhibited HIV-1IIIB infection with EC50 values of 4.28
0.7,
2.18
0.8, and 3.11
0.1 nM, respectively, reaching the
antiviral potency of T20. Furthermore, we also tested the
cytotoxicities of these compounds on MT-2 cells using an XTT
cytotoxicity assay (XTT: sodium 3′-[1-(phenylamino)-carbon-
yl]-3,4-tetrazolium-bis(4-methoxy-6-nitro)bezenesulfonic acid
hydrate). The low or absent cytotoxicity of conjugates on
MT-2 cells suggest that the sapogenin−peptide conjugates are
suitable for future study as drug candidates (Table 2).
The Function of BA Modification As Determined by a
Prime/Wash Assay in Cell−Cell Membrane Fusion.
Numerous biological effects of saponins have been ascribed
to their membrane perturbation.43 Previous reports have
indicated that saponins as well as sapogenins can incorporate
into lipid bilayers to alter the fluidity of the plasma membrane,
thus showing broad antiviral potency.44 To illustrate membrane
binding affinity, a prime/wash assay was performed. Conjugates
BApc−P26 and P26−BApc, as well as T20 and C34 as controls,
were preincubated with target cells (TZM-b1 cells) at 37 °C for
1 h, followed by washing to remove unbound peptide before
addition of the HL2/3 cells to initiate fusion (Figure 4). We
compared the EC50 values of peptide with or without washing
the cells to evaluate the binding ability of conjugates to target
cell membrane.45,46 We found that the antiviral activities of C34
and BApc−P26 were dramatically decreased (>769-fold and
similar potency as P26 alone (EC50 2390
612 nM).
Moreover, P26−BApc and P26−OApc showed solubility values
of 18.2 mg mL−1 and 12.4 mg mL−1, respectively, in pure water,
suggesting their potential as drug candidates (Supporting
Information, Table S1).
Inhibitory Activities of the Conjugated Peptides on
Viral Replication and Their Cytotoxicities. Next, we tested
the inhibitory activity of these compounds on the replication of
laboratory-adapted HIV-1 strains, IIIB (subtype B, X4) and BaL
(subtype B, R5), as described previously.42 As shown in Table
Figure 4. Strategy for the prime/wash assay of cell−cell membrane
fusion.
F
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>85-fold, respectively) as a result of their insufficient interaction
with the cell membrane. Similar to T20, which interacts with
the target cell membrane via its LBD, P26−BApc only showed
an 11-fold decrease in potency, indicating a strong binding
affinity with the cell membrane (Table 3). These results suggest
that BApc located at the C-terminus, rather than the N-
terminus, of P26 may serve as a LBD.
concomitant increase in fluorescence according to their
potencies, could be detected in this assay. The positive control,
C34, strongly inhibited the 6HB formation, with IC50 values of
2.84 μM. As shown in Figure 5A, Ptrz−P26 alone exhibited no
competitive inhibition with env2.0, whereas triterpene−P26
hybrids exhibited inhibitory activities, with IC50 values ranging
from 8.31 to 15.5 μM (Supporting Information, Table S2).
P26−BAc exhibited much lower potency than BAc−P26,
although it could also block the 6HB formation, suggesting a
different mechanism of action. These results suggest that
triterpene located at the N-terminus of P26 may serve as a PBD
to bind to the pocket on the gp41 NHR region, resulting in the
increased inhibition of 6HB formation and HIV-1 Env-
mediated cell−cell fusion. The specific interactions between
BAc−P26 conjugates and HIV-1 gp41 NHR were further
confirmed by size-exclusion HPLC (SE-HPLC). Compared
with CHR−peptides alone, the complex formed between C34
or BAc−P26 and the N36 peptide eluted at the earlier retention
time. However, no new peak corresponding to the mixture of
P26−BAc peptide and N36 at the corresponding retention time
was observed (Figure 5B−D). These results indicate that BAc−
P26, similar to C34, is able to interact with N36 to form
complexes in PBS.
Sapogenin−Peptide Conjugates Inhibit HIV-1 Entry
by Time-of-Addition Assay. To determine whether P26−
BApc acquires another mechanism of action, a time-of-addition
assay was carried out to evaluate the inhibitory activity of P26−
BApc against HIV-1 replication when it was added to cells at
different intervals postinfection. As shown in Figure 6, P26−
BApc was highly effective in inhibiting HIV-1 replication when
it was added to the cells with virus together but exhibited
significantly decreased inhibitory activity when it was added 1 h
or longer after virus was added to the cells. However, the
nucleoside reverse transcriptase inhibitor zidovudine continued
to exert its full effect even when it was added 6 h postinfection.
These results suggest that P26−BApc exhibit anti-HIV-1
potency at an early stage of viral entry with a window of
approximately 1 h.
Table 3. Interaction of Conjugates with Lipid Bilayers
HIV-1 Env-mediated cell−cell fusion EC50 (nM)
a
compd
without washing
with washing
BApc−P26
P26−BApc
T20
117 24
13 5.9
14 5.5
1.3 0.7
>10000 (>85)
139 21 (11)
542 13 (39)
>1000 (>769)
C34
a
Compounds were tested in triplicate, and the data are presented as
the mean standard deviation; the values in parentheses indicate
relative changes (n-fold) in the EC50 compared with the EC50 without
washing.
Sapogenin−Peptide Conjugates Interact with HIV-1
gp41 NHR and Inhibit 6HB Formation of the gp41 NHR
and CHR. Another interesting feature of triterpene derivatives
is that the modified position (position C3 or C28 of the
triterpene scaffold) can orchestrate their mechanism of
action.22 Thus, a previously described fluorescence resonance
energy transfer (FRET) assay29 was used to test the inhibitory
activity of the triterpene−P26 conjugates on gp41 6HB
formation. Briefly, the ligand 2,2′-bipyridine-5′-carboxylate
(bpy) was linked to the N-terminus of the N-peptide
containing the hydrophobic pocket of gp41. The bpy−N-
peptide formed a trimeric coiled coil, namely env2.0, by
chelation with Fe(II), and acted as a fluorescence quencher of
the probe CP2-LY (MTWBEWDREIBNYTSLIC-LY), which
was modified with Lucifer yellow dye at its C-terminus and
specifically bound to env2.0. Competitive inhibition of env2.0−
probe interaction by sapogenin−peptide conjugates, with a
Figure 5. (A) Inhibitory activities of conjugates on 6HB formation and determination of specific interactions between (B) C34, (C) BAc−P26, (D)
P26−BAc and N36 using SE-HPLC analysis.
G
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and determined the in vitro and in vivo half-lives of P26−BApc
and T20, respectively. As shown in Figure 7, P26−BApc
exhibited a longer in vitro half-life than T20 in the liver (0.815
vs 0.516 h) and kidney homogenates (0.598 vs 0.281 h), as well
as with proteinase K (a broad-spectrum serine proteinase)
digestion (0.905 vs 0.324 h). Meanwhile, P26−BApc also
exhibited a longer in vivo half-life than T20 (3.13 vs 2.18 h)
(Table 5). T20 is the first peptidic HIV-1 fusion inhibitor
approved for the treatment of HIV-1 infections. It offers several
advantages, e.g., high activity, high specificity, and low toxicity,
which are similar to other peptide and protein drugs.48 Like
insulin, T20 cannot be administered orally as it is rapidly
inactivated by gastrointestinal enzymes. Furthermore, due to its
degradation by proteolytic enzymes in the blood, T20 also
suffer from short half-life in vivo. These weaknesses render T20
to be sc injected twice daily at high dosage (90 mg), resulting in
high cost to patients and serious local injection reactions.
Strikingly, our designed sapogenin−peptide conjugates showed
enhanced pharmacokinetic properties over T20, prolonging the
existence of hybrids in the circulatory system that may lead to a
lower frequency of injections or lower dose. Currently, oral
delivery of macromolecular drugs is still a challenge because of
the enzymatic degradation and poor penetration across the
intestinal membrane.49 In addition to pharmaceutical ap-
proaches, chemical modifications also have shown promising
results for enhancing oral bioavailability of peptides and
proteins. Lipidization of peptides (e.g., palmitoylation of insulin
and development of hexyl-insulin) has shown exciting results,
primarily due to enhanced enzymatic stability and improved
intestinal permeability.50 Similarly, our sapogenin−peptide
conjugates also exhibited prolonged in vitro and in vivo half-
lives due to the attachment of the strong hydrophobic
triterpenes to P26 peptide. We speculated that, despite meeting
with limited success to improve oral bioavailability, our strategy
to design the peptide conjugates opens a new avenue to
develop peptide fusion inhibitors with improved pharmaco-
logical properties.
Figure 6. Time-of-addition assay. P26−BApc (100 nM) was added at
different intervals postinfection by HIV-1IIIB in MT-2 cells. Zidovudine
(200 nM) was used as a control. The assay was done in triplicate, and
the experiment was repeated at least twice. The data are presented in
mean
SEM (standard error of mean) in one representative
experiment out of three.
Sapogenin−Peptide Conjugates Are Highly Potent
against T20-Sensitive and T20-Resistant HIV-1 Strains.
Although the first peptidic anti-HIV drug T20 has shown
promise in treating HIV/AIDS patients who have failed to
respond to the current reverse transcriptase inhibitors and
protease inhibitors, the successful clinical use of T20 has
stimulated efforts to develop potent peptides against T20-
resistant strains.47 Therefore, we tested our designed conjugates
against infection by T20-sensitive and resistant HIV-1 strains. A
panel of HIV-1NL4−3 mutants, including one T20-sensitive and
five T20-resistant strains, was used in our experiments. As
shown in Table 4, T20 was much less effective against T20-
Table 4. Activity of Conjugates against T20-Sensitive and
-Resistant Strains
a
NL4-3 mutant
D36G
T20 (EC50, nM)
P26−BApc (EC50, nM)
CONCLUSION
40.6 7.8
>2000 (>50)
>2000 (>50)
>2000 (>50)
>2000 (>50)
>2000 (>50)
29.2 1.3
■
In conclusion, we designed peptide conjugates by covalently
attaching a bioactive triterpene sapogenin to HBD-containing
peptide P26 derived from the middle portion of the HIV-1
gp41 CHR domain. In this newly designed scaffold, the
sapogenin unit may serve as PBD or LBD in the hybrid
molecule to interact with the HIV-1 gp41 pocket or the target
cell membrane, respectively. These conjugates showed a strong
cooperative effect between the sapogenin moiety and the P26
peptide, therefore, exhibited potent antiviral activity against
both HIV-1 X4 and R5 viruses as well as T20-resistant HIV-1
strains. This study thus provides an efficient strategy to design
new anti-HIV-1 compounds by combining natural products
with target-specific peptides. From a medicinal chemistry
perspective, triterpene saponins are a major group of active
components in natural products with universal antiviral
activities. Thus, exploiting their potential in anti-HIV-1
compound design will open new avenues for developing viral
fusion inhibitors against other viruses with class I membrane
fusion proteins.
(36G)V38A
135 29 (4.6)
34.4 3.7 (1.2)
192 10 (6.5)
322 52 (11)
308 5.5 (10)
(36G)V38A/N42D
(36G)N42T/N43K
(36G)V38E/N42S
(36G)V38A/N42T
a
Compounds were tested in triplicate, and the data are presented as
the mean standard deviation; the values in parentheses indicate
relative changes (n-fold) in the EC50 compared with the EC50 in the
presence of the D36G substitution; HIV-1NL4−3(D36G) is a T20-sensitive
strain and the others are T20-resistant strains.
resistant strains, even at a concentration as high as 2000 nM.
However, P26−BApc was highly effective against both T20-
sensitive and resistant strains, with EC50 values ranging from
29.2 to 322 nM, indicating that these conjugates have the
potential to treat patients for whom T20 therapy is ineffective.
Pharmacokinetic Studies. One of the major limitations of
using synthetic peptide T20 for the treatment of HIV/AIDS
patients is its high sensitivity to proteolytic enzymes in the
blood, leading to its poor pharmacokinetics in vivo.2 We
speculated that our designed sapogenin−peptide conjugates
would be more resistant than T20 to proteolytic enzymes.
Therefore, we investigated the pharmacokinetic profile of P26−
BApc in rats after intravenous administration of a single dose
EXPERIMENTAL SECTION
Chemistry. Melting points were measured with a RY-1 melting
point apparatus without correction. 1H (400 MHz) and 13C (100
MHz) NMR spectra were measured on a JNM-ECA-400 spectrometer
■
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Figure 7. Metabolic stability of P26−BApc and T20 (8 μg/mL) in (A) liver homogenate, (B) kidney homogenate, and (C) a proteinase K digestion
assay. (D) Pharmacokinetic profile of P26−BApc and T20 in plasma following the administration of a single intravenous dose (4 mg/kg) to rats.
Table 5. Pharmacokinetic Parameters of P26−BApc and T20 in Rats Following a Single Dose iv Administration Derived from
Model-Independent Analysis
compd
AUC (0−t) (μg/(ml·h))
MRT (0−t) (h)
t1/2 (h)
Vd (L/kg)
Cmax (mg/L)
P26−BApc
T20
101 24
3.18 0.19
2.10 0.68
3.13 0.45
2.18 0.86
0.17 0.05
0.44 0.14
40.4 18
25.5 2.7
26.9 2.6
using TMS as the internal standard. The solvent used was CDCl3
unless otherwise indicated. Mass spectra (MS) were measured on an
API-150 mass spectrometer with an electrospray ionization source
from ABI Inc. TLC was performed on silica gel GF254 plates. Silica gel
(200−300 mesh) from Qingdao Haiyang Chemical Company was
used for column chromatography. All chemicals were obtained from
Beijing Chemical Works or Sigma-Aldrich, Inc., and solvents were
distilled and dried before use according to literature procedures. For
tested compounds, purity was determined by analytical RP-HPLC
using an Agilent 1200 series and a Phenomenex Jupiter C4 column
(150 mm × 4.6 mm, 5 μm) using two different solvent systems
(methods A and B in the Supporting Information) and a flow rate of 1
mL/min with detection wavelength at 210 nm. All tested compounds
were purified, which resulted in ≥95% purity for assay testing.
Synthesis of 2 and 10a−b. Under enclosed conditions flushed
with N2, TBDPSCl (1.2 equiv, 0.23 mL, 0.79 mmol) was added to a
stirred solution of BA, OA, or UA (1.0 equiv, 0.30 g, 0.66 mmol),
imidazole (2.5 equiv, 0.11 g, 1.65 mmol), and DMAP (0.1 equiv, 8.0
mg, 0.06 mmol) in N,N-dimethylformamide (DMF) (2 mL). The
mixture was continuously stirred, heated to reflux overnight, and
monitored by TLC. After the reaction was finished, DCM (25 mL)
was added. The organic phase was washed with 1 M HCl, and the
aqueous phase was extracted with DCM three times (20 mL × 3). The
organic phase was pooled, washed with brine, and dried over
anhydrous Na2SO4. After removal of the solvent under reduced
pressure, the residue was purified by silica gel column chromatography
using petroleum ether/ethyl acetate (30:1) as the eluent to afford 2
and 10a−b as a white solid.
tert-Butyldiphenylsilyl 3β-Hydroxylup-20(29)-en-28-oate (2).
Yield 0.35 g (78%); mp 139−141 °C. MS (m/z): calcd for
1
C46H66O3Si, 694. LC-MS (m/z (rel intens)): 695 (M + H, 14). H
NMR (CDCl3): δ 7.69 (m, 4H), 7.36 (m, 6H), 4.66 (s, 1H), 4.55 (s,
1H), 3.17 (dd, J = 11.2, 4.76 Hz, 1H), 2.40 (m, 1H), 2.23 (m, 1H),
2.04 (m, 1H), 1.80 (m, 1H), 1.13−1.65 (m, 32H), 0.95 (m, 9H), 0.75
(m, 8H). 13C NMR (CDCl3): δ 175.21, 150.66, 135.40, 135.25,
132.10, 129.95, 127.66, 109.50, 78.96, 77.32, 77.00, 76.69, 57.73,
55.33, 50.60, 48.93, 46.27, 42.51, 40.69, 38.83, 37.76, 37.15, 37.02,
34.37, 32.38, 30.40, 29.88, 27.96, 27.39, 27.02, 25.54, 20.82, 19.36,
19.30, 18.28, 16.20, 15.85, 15.34, 14.61.
tert-Butyldiphenylsilyl 3β-Hydroxyolean-12-en-28-oate (10a).
Yield 0.33 g (73%); mp 114−116 °C. MS (m/z): calcd for
1
C46H66O3Si, 694. LC-MS (m/z (rel intens)): 695 (M + H, 18). H
NMR (CDCl3): δ 7.67 (m, 4H), 7.34 (m, 6H), 5.25 (t, J = 3.36 Hz,
1H), 3.18 (dd, J = 10.6, 5.04 Hz, 1H), 2.87 (dd, J = 12.8, 3.92 Hz, 1H),
2.00 (m, 1H), 1.80 (m, 3H), 1.11−1.64 (m, 36H), 0.90−0.97 (m,
15H). 13C NMR (CDCl3): δ 176.59, 143.57, 135.77, 135.73, 132.05,
132.05, 131.93, 129.88, 127.51, 127.47, 122.37, 79.00, 77.31, 77.00,
76.68, 55.14, 47.89, 47.53, 46.27, 41.86, 41.61, 39.18, 38.71, 38.44,
36.94, 33.07, 32.38, 30.72, 28.08, 27.16, 27.08, 25.66, 23.51, 23.34,
19.33, 18.26, 16.97, 15.58, 15.32.
tert-Butyldiphenylsilyl 3β-Hydroxyurs-12-en-28-oate (10b). Yield
0.31 g (68%); mp 134−136 °C. MS (m/z): calcd for C46H66O3Si, 694.
1
LC-MS (m/z (rel intens)): 695 (M + H, 21). H NMR (CDCl3): δ
I
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7.67 (m, 4H), 7.36 (m, 6H), 5.21 (t, 1H), 3.20 (m, 1H), 2.23 (d, 1H),
0.81−1.92 (m, 56H). 13C NMR (CDCl3): δ 176.24, 137.79, 135.85,
135.80, 131.99, 129.86, 127.48, 127.45, 125.62, 79.01, 77.31, 77.00,
76.69, 55.17, 53.27, 49.35, 47.53, 42.26, 39.41, 39.36, 38.95, 38.70,
38.65, 36.88, 36.66, 33.21, 30.83, 28.13, 27.89, 27.21, 27.11, 24.72,
23.17, 21.21, 19.33, 18.25, 17.25, 17.05, 15.63, 15.50.
32.09, 30.49, 29.63, 27.92, 25.36, 23.65, 20.80, 19.30, 18.10, 16.48,
16.13, 15.97, 14.63, 14.51.
3β-(Pent-4′-ynoyloxy)olean-12-en-28-oic Acid (OAc). Yield 0.17 g
(95%); mp 199−202 °C. MS (m/z): calcd for C35H52O4, 536. LC-MS
1
(m/z (rel intens)): 536 (M+, 100). H NMR (CDCl3): δ 5.27 (t, J =
3.36 Hz, 1H), 4.53 (t, J = 8.12 Hz, 1H), 2.80 (dd, J = 13.7, 4.2 Hz,
1H), 2.54 (m, 4H), 0.58−1.97 (m, 46H). 13C NMR (CDCl3): δ
183.64, 171.49, 159.63, 145.66, 143.57, 122.51, 82.61, 81.30, 69.01,
56.83, 55.25, 47.49, 46.50, 46.20, 41.52, 40.21, 39.23, 37.92, 37.71,
36.94, 33.82, 33.04, 32.47, 30.64, 28.03, 27.98, 25.89, 23.54, 23.32,
22.89, 18.08, 17.12, 16.68, 15.33, 14.52.
Synthesis of 3 and 11a−b. DIC (5.0 equiv, 0.23 g, 1.8 mmol)
dissolved in dry DCM (4 mL) was added dropwise over 2 min to a
stirred solution of 2 or 10a−b (1.0 equiv, 0.25 g, 0.36 mmol), DMAP
(1.0 equiv, 0.04 g, 0.36 mmol), and pentinoic acid (3.0 equiv, 0.11 g,
1.08 mmol) in dry DCM (6 mL). The mixture was stirred at room
temperature and monitored by TLC. After the reaction was complete,
25 mL of DCM was added, and the organic phase was washed with
10% citric acid, saturated sodium bicarbonate solution, and brine and
dried over Na2SO4. After the mixture was filtered and the filtrate was
evaporated under reduced pressure, the residue was purified by silica
gel column chromatography using petroleum ether/ethyl ether (30:1)
as the eluent to yield 3 and 11a−b as a white solid.
3β-(Pent-4′-ynoyloxy)urs-12-en-28-oic Acid (UAc). Yield 0.16 g
(92%); mp 242−245 °C. MS (m/z): calcd for C35H52O4, 536. LC-MS
1
(m/z (rel intens)): 536 (M+, 100). H NMR (CDCl3): δ 5.24 (t, J =
3.36 Hz, 1H), 4.53 (t, J = 8.16 Hz, 1H), 2.54 (m, 4H), 2.21 (d, J = 7.84
Hz, 1H), 0.77−1.97 (m, 45H). 13C NMR (CDCl3): δ 176.84, 143.36,
122.57, 78.98, 74.36, 55.16, 51.61, 47.57, 46.73, 45.80, 43.85, 41.66,
41.24, 39.33, 38.72, 38.40, 36.99, 33.78, 33.06, 32.70, 32.14, 30.66,
28.06, 27.62, 27.15, 25.81, 23.58, 23.37, 22.97, 18.30, 17.06, 15.55,
15.31.
Synthesis of BAo, OAo, and UAo. To a solution of BA (1.0
equiv, 0.5 g, 1.1 mmol) in 10 mL of DMF were added potassium
carbonate (3.0 equiv, 0.46 g, 3.3 mmol) and propargyl bromide (2.0
equiv, 171 μL, 2.2 mmol). The mixture was stirred at room
temperature for 4 h. The reaction was monitored by TLC. After the
reaction was finished, the remaining potassium carbonate and
generated potassium bromide were removed by filtration, and the
solvent was concentrated under reduced pressure. The crude product
was purified by silica gel column chromatography using ethyl acetate/
petroleum ether (10:1) as the eluent to afford BAo as a white solid.
The above method was used to synthesize compounds OAo and UAo
by using OA and UA instead of BA.
tert-Butyldiphenylsilyl 3β-(Pent-4′-ynoyloxy)lup-20(29)-en-28-
oate (3). Yield 0.21 g (78%); mp 172−174 °C. MS (m/z): calcd for
1
C51H70O4Si, 774. LC-MS (m/z (rel intens)): 797 (M + Na, 7). H
NMR (CDCl3): δ 7.68 (m, 4H), 7.36 (m, 6H), 4.66 (s, 1H), 4.55 (s,
1H), 4.49 (m, 2H), 2.52 (m, 4H), 2.23 (m, 1H), 2.12 (m, 1H), 1.96
(m, 1H), 1.79 (m, 1H), 1.11−1.65 (m, 37H), 0.73−0.94 (m, 19H).
13C NMR (CDCl3): δ 175.21, 171.50, 150.62, 135.39, 135.24, 132.08,
129.93, 127.65, 109.54, 82.64, 81.34, 77.31, 77.00, 76.68, 68.99, 57.71,
55.39, 50.48, 48.90, 46.29, 42.50, 40.69, 38.33, 37.80, 37.72, 37.04,
34.28, 33.81, 32.35, 30.38, 29.85, 27.93, 27.01, 25.48, 23.66, 19.33,
19.29, 18.13, 16.50, 16.23, 15.84, 14.57, 14.51.
tert-Butyldiphenylsilyl 3β-(Pent-4′-ynoyloxy)olean-12-en-28-oate
(11a). Yield 0.22 g (80%); mp 103−105 °C. MS (m/z): calcd for
1
C51H70O4Si, 774. LC-MS (m/z (rel intens)): 775 (M + H, 25). H
Prop-2′-ynyl 3β-Hydroxylup-20(29)-en-28-oate (BAo). Yield 0.42
NMR (CDCl3): δ 7.66 (m, 4H), 7.34 (m, 6H), 5.25 (t, J = 3.08 Hz,
1H), 4.51 (t, J = 9.24 Hz, 1H), 2.87 (d, J = 17.6 Hz, 1H), 2.53 (m,
4H), 0.85−1.97 (m, 67H). 13C NMR (CDCl3): δ176.59, 171.49,
143.59, 135.77, 135.73, 129.88, 127.51, 127.47, 122.25, 82.63, 81.32,
77.31, 77.00, 76.69, 69.00, 55.22, 47.88, 47.44, 46.24, 41.86, 41.60,
39.19, 38.08, 37.69, 36.81, 33.81, 33.06, 30.72, 28.04, 27.08, 25.62,
23.51, 23.34, 19.33, 16.95, 16.73, 15.37, 14.52.
g (78%); mp 185−187 °C. MS (m/z): calcd for C33H50O3, 494. LC-
1
MS (m/z (rel intens)): 495 (M + H, 28). H NMR (CDCl3): δ 4.65
(m, 4H), 3.17 (dd, J = 5.04, 11.52 Hz, 1H), 3.00 (m, 1H), 2.42 (t, J =
2.52 Hz, 1H), 2.19 (m, 2H), 1.90 (m, 2H), 0.73−1.67 (m, 40H). 13C
NMR (CDCl3): δ 175.23, 150.43, 109.64, 78.93, 74.32, 56.54, 55.28,
51.31, 50.50, 49.41, 46.80, 42.33, 40.73, 38.82, 38.65, 38.20, 37.13,
36.76, 34.26, 31.89, 30.44, 29.59, 27.94, 27.34, 25.47, 20.81, 19.33,
18.23, 16.11, 15.96, 15.33, 14.68.
Prop-2′-ynyl 3β-Hydroxyolean-12-en-28-oate (OAo). Yield 0.44 g
(82%); mp 158−160 °C. MS (m/z): calcd for C33H50O3, 494. LC-MS
(m/z (rel intens)): 495 (M + H, 25). 1H NMR (CDCl3): δ 5.30 (t, J =
3.64 Hz, 1H), 4.60 (qd, 2H), 3.22 (m, 1H), 2.85 (m, 1H), 2.41 (t, J =
2.24 Hz, 1H), 1.85−1.98 (m, 3H), 0.74−1.64 (m, 42H). 13C NMR
(CDCl3): δ 176.85, 143.36, 122.57, 78.98, 78.09, 74.37, 55.14, 51.61,
47.55, 46.73, 45.78, 41.65, 41.22, 39.33, 38.72, 38.39, 36.98, 33.77,
33.05, 32.69, 32.14, 30.64, 28.06, 27.62, 27.15, 25.80, 23.57, 23.38,
22.96, 18.29, 17.04, 15.55, 15.32.
Prop-2′-ynyl 3β-Hydroxyurs-12-en-28-oate (UAo). Yield 0.39 g
(80%); mp 145−147 °C. MS (m/z): calcd for C33H50O3, 494. LC-MS
(m/z (rel intens)): 495 (M + H, 18). 1H NMR (CDCl3): δ 5.27 (t, J =
3.64 Hz, 1H), 4.60 (qd, 2H), 3.20 (dd, J = 5.04, 10.64 Hz, 1H), 2.42
(t, J = 2.52 Hz, 1H), 2.25 (d, J = 10.9 Hz, 1H), 2.03 (m, 1H), 1.89 (m,
2H), 1.31−1.69 (m, 18H), 0.76−1.08 (m, 25H). 13C NMR (CDCl3):
δ 176.66, 137.70, 125.82, 78.99, 78.06, 74.39, 55.14, 52.76, 51.57,
48.13, 47.49, 42.00, 39.54, 39.02, 38.73, 38.58, 36.91, 36.38, 32.99,
30.58, 28.09, 27.97, 27.18, 24.14, 23.45, 23.25, 21.15, 18.26, 17.15,
16.97, 15.61, 15.45.
Synthesis of BApc, OApc, and UApc. To a solution of BA, OA,
or UA (1.0 equiv, 1.0 g, 2.0 mmol) in 10 mL of DMF were added
potassium carbonate (2.0 equiv, 0.61 g, 4.0 mmol) and allyl bromide
(1.6 equiv, 284 μL, 3.3 mmol). The mixture was stirred at room
temperature for 4 h. The reaction was monitored by TLC. After the
reaction was finished, the remaining potassium carbonate and
generated potassium bromide were removed by filtration, and the
solvent was concentrated under reduced pressure. The product was
directly used for the next step without further purification. In the next
step, DIC (6.0 equiv, 0.23 g, 1.8 mmol) dissolved in dry DCM (4 mL)
tert-Butyldiphenylsilyl 3β-(Pent-4′-ynoyloxy)urs-12-en-28-oate
(11b). Yield 0.18 g (68%); mp 98−99 °C. MS (m/z): calcd for
1
C51H70O4Si, 774. LC-MS (m/z (rel intens)): 775 (M + H, 57). H
NMR (CDCl3): δ 7.66 (m, 4H), 7.34 (m, 6H), 5.20 (t, J = 3.64 Hz,
1H), 2.53 (m, 4H), 2.25 (d, J = 10.9 Hz, 1H), 1.96 (m, 2H), 1.76 (m,
5H), 0.83−1.56 (m, 47H). 13C NMR (CDCl3): δ 176.25, 171.48,
137.83, 135.85, 135.80, 132.00, 129.86, 127.48, 127.45, 125.48, 82.63,
81.33, 77.31, 77.00, 76.68, 69.00, 55.24, 53.25, 49.34, 47.44, 42.25,
39.41, 39.35, 38.94, 38.28, 37.68, 36.76, 33.81, 30.82, 28.08, 27.87,
27.11, 24.71, 23.54, 23.20, 23.14, 21.21, 19.33, 17.23, 17.08, 15.79,
15.54, 14.52.
Synthesis of BAc, OAc, and UAc. Under enclosed conditions
flushed with N2, tetrabutylammonium fluoride (TBAF) (1.5 equiv,
0.14 g, 0.52 mmol) was added to a solution of 3 (1.0 equiv, 0.27 g,
0.34 mmol) in 5 mL of THF. The mixture was stirred for 3 h at room
temperature with monitoring by TLC. After the reaction was
complete, 15 mL of DCM was added. The organic phase was washed
with 1 M HCl and brine and dried over Na2SO4. After the mixture was
filtered and the filtrate was evaporated under reduced pressure, the
residue was purified by silica gel column chromatography using
petroleum ether/ethyl acetate (8:1) as the eluent to yield BAc as a
white solid. The same procedure described above was used to obtain
OAc and UAc from 11a−b.
3β-(Pent-4′-ynoyloxy)lup-20(29)-en-28-oic Acid (BAc). Yield 0.16
g (90%); mp 253−255 °C. MS (m/z): calcd for C35H52O4, 535. LC-
1
MS (m/z (rel intens)): 536 (M+, 100). H NMR (CDCl3): δ 4.73 (s,
1H), 4.61 (s, 1H), 4.50 (m, 1H), 2.53 (m, 4H), 2.25 (m, 2H), 1.96 (m,
2H), 0.83−1.69 (m, 41H). 13C NMR (CDCl3): δ 181.75, 181.62,
171.55, 150.38, 109.73, 82.63, 81.33, 69.01, 56.31, 55.34, 50.30, 49.17,
46.90, 42.36, 40.62, 38.33, 38.24, 37.80, 37.05, 36.99, 34.15, 33.79,
J
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was added dropwise over 2 min to a stirred solution of 6 or 14a−b
(1.0 equiv, 0.15 g, 0.3 mmol), DMAP (1.0 equiv, 0.04 g, 0.3 mmol),
and pentinoic acid (3.0 equiv, 0.09 g, 0.9 mmol) in dry DCM (6 mL).
The mixture was stirred at room temperature and monitored by TLC.
After the reaction was complete, 25 mL of DCM was added, and the
organic phase was washed with 10% citric acid, saturated sodium
bicarbonate solution, and brine and dried over Na2SO4. After the
mixture was filtered and the filtrate was evaporated under reduced
pressure, the residue was purified by silica gel column chromatography
using ethyl acetate/petroleum ether (20:1) as the eluent to yield
BApc, OApc, and UApc as a white solid.
used. Coupling of the amino acids was achieved using O-benzotriazol-
1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU,
GL Biochem, Shanghai, China) and diisopropylethylamine (DIEA,
Acrose) as an activator and an active base, respectively, in N,N-
dimethylformamide (DMF) solution. The Fmoc protection group was
removed using 20% piperidine/DMF. Between every coupling or
Fmoc removal, the resin was washed five times with DMF and three
times with dichloromethane (DCM). The carboxyl termini were
amidated upon cleavage from the resin, and the amino termini were
capped with acetic acid anhydride. The peptides were cleaved from the
Rink Amide resin and deprotected with Reagent K, which contained
82.5% trifluoroacetic acid (TFA), 5% thioanisole, 5% m-cresol, 5%
water, and 2.5% ethanedithiol. The crude peptide products were
precipitated with cold diethyl ether, lyophilized, and purified by
preparative reverse-phase high-performance liquid chromatography
(HPLC) using a Waters preparative HPLC system (PrepLC 4000):
gradient elution of 30−50% solvent B in solvent A (0.1% TFA in H2O,
solvent A; 0.1% TFA in 70% CH3CN/H2O, solvent B) over 60 min at
16 mL/min on a Waters X-bridge C8, 10 μm, 19.5 mm × 250 mm
column. Analytical RP-HPLC was performed on a RP-C8 column
(Zorbax Eclipse XDB-C8, 5 μm, 4.6 mm × 150 mm) with gradient
elution of 5−100% solvent B in solvent A over 25 min at a flow rate of
1 mL/min. Compounds were detected by UV absorption at 210 nm
with a Shimadzu SPD-10A detector. All peptides were purified to
>95% purity. The molecular weight of the peptides was confirmed by
matrix-assisted laser desorption/ionization-time-of-flight mass spec-
trometry (MALDI-TOF-MS; Autoflex III, Bruker Daltonics).
Conjugates Synthesis (Click Chemistry). In brief, purified
azido-peptide precursor (1.0 equiv, 0.005 mmol) was dissolved in 1
mL of H2O, to which triterpene derivatives (1.2 equiv, 0.006 mmol)
dissolved in 1 mL of tert-butyl alcohol was added. Then CuSO4·5H2O
(1.0 equiv, 0.005 mmol) and sodium ascorbate (5.0 equiv, 0.025
mmol) were added to the mixture, which was left stirring at room
temperature for 4 h. The reaction was monitored by analytical RP-
HPLC with a Phenomenex Jupiter C4 column (150 mm × 4.6 mm, 5
μm) using 0.1% TFA in water (A) and 0.1% TFA in 90% CH3CN/
H2O (B) as the eluents, and the linear gradients as follows: 50−80%
(B) in 5 min to 100% (B) in 3 min, washing step at 100% (B) for 20
min, flow 1 mL/min. After 4 h incubation, the reaction was complete
and the resulting conjugated peptide was purified to >95% purity as
described above (65−80 yields) and confirmed by MALDI-TOF-MS.
Cell−Cell Fusion Assay. Cell−cell fusion assays were performed
as previously described.51 HL2/3 cells, which stably express HIV Gag,
Env, Tat, Rev, and Nef proteins, and TZM-bl cells, which stably
express large amounts of CD4 and CCR5, were obtained from the
NIH AIDS Reference and Reagent Program (contributed by Drs.
Barbara Felber and George Pavlakis or Drs. John C. Kappes and
Xiaoyun Wu, respectively). TZM-bl cells (2.5 × 104/well) and HL2/3
cells (7.5 × 104/well) were coincubated in 96-well plates (Corning
Costar) at 37 °C in 5% CO2 in the presence of different
concentrations of inhibitors. After incubation for 6−8 h, the medium
was aspirated, the cells were washed and lysed, and luciferase activity
was measured using the Luciferase Assay System (Promega, Madison,
WI) on a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale,
CA).
Allyl 3β-(Pent-4′-ynoyloxy)lup-20(29)-en-28-oate (BApc). Yield
0.78 g (68%); mp 120−122 °C. MS (m/z): calcd for C38H56O4, 576.
1
LC-MS (m/z (rel intens)): 577 (M + H, 10). H NMR (CDCl3): δ
5.92 (m, 1H), 5.24 (q, 2H), 4.73 (s, 1H), 4.57 (m, 4H), 3.00 (m, 1H),
2.53 (m, 4H), 2.25 (m, 2H), 1.96 (m, 3H), 0.83−1.68 (m, 38H). 13C
NMR (CDCl3): δ 175.52, 171.50, 150.53, 132.54, 118.08, 109.61,
82.63, 81.35, 69.00, 64.56, 56.53, 55.40, 50.42, 49.41, 46.93, 42.17,
40.71, 38.21, 38.17, 37.21, 37.07, 36.98, 34.20, 33.80, 32.09, 30.32,
29.60, 27.92, 24.21, 23.11, 20.87, 19.32, 18.13, 16.51, 16.14, 15.93,
14.65, 14.51.
Allyl 3β-(Pent-4′-ynoyloxy)olean-12-en-28-oate (OApc). Yield
0.66 g (58%); mp 140−142 °C. MS (m/z): calcd for C38H56O4,
576. LC-MS (m/z (rel intens)): 577 (M + H, 7). 1H NMR (CDCl3): δ
5.89 (m, 1H), 5.29 (m, 3H), 4.52 (m, 3H), 2.86 (dd, J = 13.5, 3.84 Hz,
1H), 2.53 (m, 4H), 0.72−1.67 (m, 44H). 13C NMR (CDCl3): δ
177.33, 171.47, 143.69, 132.53, 122.28, 117.67, 82.60, 81.31, 69.01,
64.77, 55.26, 47.92, 46.98, 45.81, 41.65, 41.30, 39.02, 38.06, 37.78,
36.88, 33.81, 33.07, 32.61, 32.52, 30.09, 28.03, 27.62, 25.83, 23.60,
23.50, 23.37, 23.00, 18.18, 16.92, 16.82, 15.33, 14.53.
Allyl 3β-(Pent-4′-ynoyloxy)urs-12-en-28-oate (UApc). Yield 0.80 g
(70%); mp 150−151 °C. MS (m/z): calcd for C38H56O4, 576. LC-MS
1
(m/z (rel intens)): 577 (M + H, 21). H NMR (CDCl3): δ 5.87 (m,
1H), 5.21 (m, 3H), 4.50 (m, 3H), 2.53 (m, 4H), 2.24 (d, J = 11.2 Hz,
1H), 1.90 (m, 4H), 0.72−1.67 (m, 40H). 13C NMR (CDCl3): δ
177.10, 171.43, 138.06, 132.51, 125.46, 117.71, 82.58, 81.28, 68.99,
64.77, 55.24, 52.83, 48.06, 47.40, 41.98, 39.52, 39.01, 38.99, 37.89,
37.67, 35.88, 35.82, 33.79, 33.00, 30.61, 28.03, 27.93, 24.16, 23.48,
23.23, 21.15, 18.13, 17.00, 16.73, 15.45, 14.48.
Synthesis of BApo. A mixture of BA (1.0 equiv, 4.0 g, 8.76
mmol), pyridine (25 mL), and acetic anhydride (2.0 equiv, 3.5 mL)
was stirred at room temperature overnight until it became
homogeneous. The mixture was then poured into ice-cold water and
stirred for 20 min. The intermediate 8 was filtered off and directly used
for the next step without further purification. In the next step, to a
solution of 8 (1.0 equiv, 1.0 g, 2.0 mmol) in 10 mL of DMF were
added potassium carbonate (3.0 equiv, 0.91 g, 6.0 mmol) and
propargyl bromide (2.0 equiv, 342 μL, 4.0 mmol). The mixture was
stirred at room temperature for 4 h. The reaction was monitored by
TLC. After the reaction was finished, the remaining potassium
carbonate and generated potassium bromide were removed by
filtration, and the solvent was concentrated under reduced pressure.
The crude product was purified by silica gel column chromatography
using ethyl acetate/petroleum ether (10:1) as the eluent to afford 0.8 g
of BApo as a white solid in 78% yield.
Prop-2′-ynyl 3β-Acetoxylup-20(29)-en-28-oate (BApo). Melting
HIV-1 Infection Assay. For measuring the inhibitory activity of
the peptides on infection of HIV-1IIIB, HIV-1BaL, and T20-resistant
HIV-1 strains, 1 × 104 MT-2 cells were infected with 100 TCID50 of a
virus in the presence or absence of the peptides at graded
concentrations. On the fourth day postinfection, the culture
supernatants were collected for detection of p24 antigen using an
enzyme-linked immunosorbent assay (ELISA). The percent inhibition
by the peptides and 50% effective concentration (EC50) values were
calculated using Calcusyn software.52,53
point 180−182 °C. MS (m/z): calcd for C35H52O4, 536. LC-MS (m/z
1
(rel intens)): 537 (M + H, 17). H NMR (CDCl3): δ 4.64 (q, 4H),
4.45 (m, 1H), 3.00 (m, 1H), 2.43 (m, 1H), 2.24 (m, 2H), 2.04 (s, 3H),
1.90 (m, 2H), 0.82−1.68 (m, 40H) . 13C NMR (CDCl3): δ 175.16,
171.00, 150.41, 109.69, 80.92, 78.12, 74.28, 56.57, 55.40, 51.30, 50.45,
49.45, 46.84, 42.36, 40.17, 38.38, 38.22, 37.77, 37.08, 36.77, 34.21,
31.91, 30.46, 29.31, 27.91, 25.47, 23.67, 21.31, 20.87, 19.33, 18.14,
16.46, 16.18, 15.99, 14.73.
Peptide Synthesis. Peptides were synthesized using a Liberty
automated microwave peptide synthesizer (CEM Co., Matthews, NC)
with a standard solid-phase N-(9-fluorenyl)methoxycarbonyl (Fmoc)
chemistry protocol. All protected amino acids used were purchased
from GL Biochem Ltd. (Shanghai, China). Rink Amide resin (0.38−
0.45 mmol/g, Nankai Hecheng S&T Co. Ltd., Tianjin, China) was
Cytotoxicity Assay. The in vitro cytotoxicity of conjugated
peptides to virus target cells (MT-2) was measured by the XTT assay.
Briefly, 100 μL of conjugates at graded concentrations were added to
equal volumes of cells (5 × 105/mL) in wells of 96-well plates. After
incubation at 37 °C for 4 days, 50 μL of XTT solution (1 mg/mL)
containing 0.02 μM phenazine methosulfate (PMS) were added. After
K
dx.doi.org/10.1021/jm500763m | J. Med. Chem. XXXX, XXX, XXX−XXX

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Article
4 h, the absorbance at 450 nm (A450) was measured with an ELISA
reader. The 50% cytotoxicity concentrations (CC50) were calculated
using the CalcuSyn software.
81072581 and 81373456) and a National Science and
Technology Major Project of China grant (2012ZX09301003).
The Solubility of Peptide−Sapogenin Conjugates. We
estimated the solubility of the peptide−sapogenin conjugates as
previously described.12 Approximately 1 mg of each peptide was
weighed, and 10 μL of dd-H2O or phosphate-buffered saline (PBS)
were added to dissolve the samples. The solutions were centrifuged,
and the supernatants were recovered. The concentration of each
tyrosine-containing hybrid was calculated using a NanoDrop-2000c
UV spectrophotometer (Thermo Scientific, USA) by measuring the
UV absorbance of the supernatants at 280 nm.
A Prime/Wash Assay. To illustrate the key mechanism of action
of the peptide−sapogenin conjugates, a prime/wash assay of cell−cell
membrane fusion was performed. First, BAc−P26, P26−BApc, T20,
and C34 were incubated with TZM-b1 cells at 37 °C for 1 h, followed
by washing. Then, HL2/3 cells were added to initiate infection. After
incubation for 48 h, the EC50 values of the peptide with or without
washing were calculated to evaluate the ability of the residual peptides
to inhibit cell−cell fusion.
Fluorescence Resonance Energy Transfer (FRET) Assay.
Briefly, the ligand 2,2′-bipyridine-5′-carboxylate (bpy) was linked to
the N-terminus of the N-peptide containing the hydrophobic pocket of
gp41 (a 31-mer NHR peptide: Bpy-GQAVEAQQHLLQLTVW-
GIKQLQARILAVEKK-NH2). The bpy−N-peptide formed a trimeric
coiled coil, namely env2.0, by chelation with Fe(II), and acted as a
fluorescence quencher of the probe CP2-LY (MTWBEWDREIB-
NYTSLIC-LY), which was modified with Lucifer yellow dye at its C-
terminus and specifically bound to env2.0. Competitive inhibition of
env2.0-probe interaction by sapogenin−peptide conjugates, with a
concomitant increase in fluorescence according to their potencies,
could be detected in this assay. Typically, 7 μM binding sites (three
per receptor trimer) and 1 μM CP2-LY were used in the assay.
Binding Assays by Size-Exclusion Chromatography. N36 was
mixed with conjugated peptides (final concentration = 0.20 mM) at a
molar ratio of 1:1 in 50 mM sodium phosphate/150 mM NaCl (pH
7.2) and incubated at 37 °C for 30 min. The mixture or peptide (20
μL) was applied to the Phenomenex BioSep-SEC-s2000, 300 mm ×
7.80 mm HPLC column equilibrated with H2O and eluted at 0.8 mL/
min, and fractions were monitored at 210 nm.
ABBREVIATIONS USED
■
CHR, C-terminal heptad repeat; NHR, N-terminal heptad
repeat; 6HB, six-helix bundle; DCC, N,N′-dicyclohexylcarbo-
diimide; DMAP, 4-dimethylaminopyridine
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ASSOCIATED CONTENT
* Supporting Information
■
S
Analytical HPLC methods and characterization data of key
compounds, HPLC analysis of BAc−P26 conjugate synthesis,
the solubility of peptide−sapogenin conjugates, inhibitory
activity of peptide conjugates on 6HB formation, and
pharmacokinetic experimental procedures. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*For K.L.: phone, 86-10-6816-9363; fax, 86-10-6821-1656; E-
mail, keliangliu55@126.com.
■
*For S.J.: phone, 86-21-54237673; fax, 86-21-54237465; E-
mail, shibojiang@fudan.edu.cn.
Notes
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contains multifunctional domains. Relation to mechanisims of action
of anti-HIV peptides. J. Biol. Chem. 2007, 282 (13), 9612−9620.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This research was supported, in part, by grants from the
National Science Foundation of China (81373266, 81273434,
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