Design of cell-permeable stapled peptides as HIV-1 integrase inhibitors

Article abstract of DOI:10.1021/jm4006516

HIV-1 integrase (IN) catalyzes the integration of viral DNA into the host genome, involving several interactions with the viral and cellular proteins. We have previously identified peptide IN inhibitors derived from the α-helical regions along the dimeric interface of HIV-1 IN. Herein, we show that appropriate hydrocarbon stapling of these peptides to stabilize their helical structure remarkably improves the cell permeability, thus allowing inhibition of the HIV-1 replication in cell culture. Furthermore, the stabilized peptides inhibit the interaction of IN with the cellular cofactor LEDGF/p75. Cellular uptake of the stapled peptide was confirmed in four different cell lines using a fluorescein-labeled analogue. Given their enhanced potency and cell permeability, these stapled peptides can serve as not only lead IN inhibitors but also prototypical biochemical probes or nanoneedles for the elucidation of HIV-1 IN dimerization and host cofactor interactions within their native cellular environment.

Full text of DOI:10.1021/jm4006516

Article
pubs.acs.org/jmc
Design of Cell-Permeable Stapled Peptides as HIV‑1 Integrase
Inhibitors
Ya-Qiu Long,*,† Shao-Xu Huang,†,∥ Zahrah Zawahir,‡,∥ Zhong-Liang Xu,† Huiyuan Li,†
Tino W. Sanchez,‡ Ying Zhi,† Stephanie De Houwer,§ Frauke Christ,§ Zeger Debyser,§
and Nouri Neamati*,‡
†CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi
Road, Shanghai 201203, China
‡Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, 1985 Zonal
Avenue, Los Angeles, California 90033, United States
§The Laboratory for Molecular Virology and Gene Therapy, KU Leuven and IRC KULAK, Kapucijnenvoer 33, B-3000 Leuven,
Flanders, Belgium
S
* Supporting Information
ABSTRACT: HIV-1 integrase (IN) catalyzes the integration of viral DNA into the host genome, involving several interactions
with the viral and cellular proteins. We have previously identified peptide IN inhibitors derived from the α-helical regions along
the dimeric interface of HIV-1 IN. Herein, we show that appropriate hydrocarbon stapling of these peptides to stabilize their
helical structure remarkably improves the cell permeability, thus allowing inhibition of the HIV-1 replication in cell culture.
Furthermore, the stabilized peptides inhibit the interaction of IN with the cellular cofactor LEDGF/p75. Cellular uptake of the
stapled peptide was confirmed in four different cell lines using a fluorescein-labeled analogue. Given their enhanced potency and
cell permeability, these stapled peptides can serve as not only lead IN inhibitors but also prototypical biochemical probes or
“nanoneedles” for the elucidation of HIV-1 IN dimerization and host cofactor interactions within their native cellular
environment.
preintegration complex (PIC)), as well as the integration itself,
are mediated by protein−protein interactions between relevant
viral proteins and host cell cofactors.8 IN is known to
participate in several of these functional interactions with
cellular proteins such as gemin2, cyclin-dependent kinase
inhibitor p21, transportin-SR2, and the extensively studied lens
epithelium-derived growth factor (LEDGF/p75).9−15 Selec-
tively targeting these vital interactions as well as the IN
dimerization/multimerization step as a therapeutic strategy
against HIV-1 using peptidomimetic, biologically stable
analogues of active peptides, or small-molecule inhibitors, is a
rapidly developing field of interest that benefits significantly
INTRODUCTION
■
HIV-1 integrase (IN) efficiently inserts viral DNA into the host
genome in two catalytic events. The first step, 3′-processing,
involves the cleavage of a dinucleotide adjacent to a conserved
CA on each terminus of the reverse-transcribed DNA, leaving
the recessed 3′-hydroxyl groups. In the subsequent strand
transfer step, these hydroxyl groups carry out a nucleophilic
attack on the host DNA.1−3 This results in a stable and
irreversible integration of the proviral DNA in the host genome.
Because IN has no mammalian counterpart, it is an attractive
therapeutic target. Currently, raltegravir and elvitegravir are the
two approved drugs selectively targeting IN and additional ones
are being tested in clinical studies.4−7 All catalytic events
preceding integration (from viral fusion to formation and
nuclear import of the large nucleoprotein complex termed the
Received: May 2, 2013
© XXXX American Chemical Society
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Figure 1. Optional positions on NL6 incorporating the α,α-disubstituted amino acids for side chain bridging. The red letters represent the essential
residues for the IN dimerization.
from novel research into biochemical probes or “nano-
needles”.12 Given the powerful ability of the ubiquitous α-
helical motif in mediating intracellular protein−protein
interactions across extensive biological pathways, such structur-
ally immutable chemical tools may have general utility in
further clarification and/or selective modulation of such
protein−protein interactions within their native cellular
environment. Unfortunately, most peptides are not suitable
probes for in vivo studies because of their poor cellular uptake.
However, when they are stapled into conformationally
restricted analogues, they can penetrate cellular membrane
like biological needles.
Previous studies from this group have demonstrated that
peptides can interfere with the IN catalysis, as well as with the
key LEDGF/p75−IN interaction necessary for successful
integration.16−18 A peptide derived from the reverse tran-
scriptase region of the HIV-1 HXB2 Pol sequence showed low
micromolar inhibition of IN activity.16 Inhibition of the
LEDGF/p75 interaction was demonstrated with both wild-
type and mutant IN proteins in vitro by a peptide derived from
the LEDGF/p75 protein sequence containing two IN contact
“hotspots”.18 Finally, we used a “sequence-walking” strategy to
pinpoint the areas of catalytic interest on wild-type IN protein.
Evaluation of the inhibition of IN catalysis by a series of
peptides spanning the IN protein sequence resulted in the
identification of two peptides, NL6 and NL9, with low
micromolar IC50 values for the inhibition of HIV-1 IN
catalysis.17 Each peptide was derived from the α1 and α3
helical domains of the IN protein, respectively. Alanine
scanning on these peptides further pinpointed the amino acid
residues that were later proven to be critical for IN dimerization
and at least one known HIV-1 IN protein−protein interaction,
that of LEDGF/p75 and IN.18,19 While these studies all provide
excellent examples of the potential of peptides in drug
development, the notorious issues of poor cell permeability,
secondary structure instability, and in vivo proteolysis severely
compromise peptides as successful therapeutic agents.
interaction and cell permeability. This all-hydrocarbon stapled
α-helix peptide strategy was first reported by the Verdine group
to enhance the helicity and metabolic stability of peptides,24
then was successfully used to generate a series of helical,
protease-resistant, cell permeable peptides derived from the
amphipathic helical BH3 region found in members of the family
of BCL-2 proteins.25−27 A stapled BH3 domain peptide from
the BID protein specifically activated the apoptotic pathway in
leukemia cells and human leukemia xenograft models.25
However, a more recent study concluded that hydrocarbon
stapled BimBH3 peptide neither increased binding affinity nor
enhanced cell permeability compared to the unstapled
peptide.27 This is in sharp contrast to the original studies by
Walensky et al.25 Recently, a stapled p53 peptide was shown to
directly modulate the transcriptional activity in hDM2-over-
expressing cancer cells by reactivation of the p53-mediated
tumor suppressor pathway.28,29 Both approaches apply to
cancer disease models. A proof-of-concept cell-penetrating
peptide targeting the HIV-1 capsid particle assembly was also
described that crossed the cell membrane and bound to the C-
terminal domain of the HIV-1 capsid protein.30 Cell
permeability enhancement strategies for peptides include the
conjugation of positively charged protein-transduction domains
such as the Tat-peptide conjugation, the antennapedia
homeodomain of Drosophila, and poly arginine segments.31,32
Cyclization techniques to enforce the α-helical nature of many
peptides have also been demonstrated. Furthermore, side chain
and backbone cyclization of peptides is another way to improve
the stability of linear peptides and examples of such peptides
were reported to inhibit the IN catalytic activities.33−35
However, polar cross-links often limit cell permeability and
leave the peptides open to degradation.36,37 The hydrocarbon
stapling technique elegantly combines enhanced cell pene-
tration of structurally stable α-helical peptides with an increased
resistance to proteolytic degradation. We thus synthesized a
series of NL6 derived peptides each containing hydrocarbon
“staples” of different lengths in an attempt to induce and
stabilize their helical conformation and consequently enhance
their cell permeability.
The two most potent inhibitory IN-derived α-helical
peptides from the latter study are the focus of this report. By
examining the two-domain crystal structures of IN,20,21 we
found that the central core dimers are linked via interactions of
various types and the interfacial region of dimeric IN involves
the strong helix-to-helix contacts α1:α5′ and α5:α1′.22,23
Because our peptide NL6 just falls within the sequence of
the α1 helical domain, we propose that α-helix stabilization on
NL6 via a hydrocarbon “staple” might enhance the interfacial
This report identifies three α-helix stabilized IN-derived
peptides inhibiting the HIV-1 replication in the infected cells.
The corresponding unstapled peptides do not show therapeuti-
cally selective inhibition of the replication in cell-based assays,
although each pair of peptides has similar activity against IN in
our enzymatic assay. These peptides also inhibit the LEDGF/
p75−IN interaction in vitro. More significantly, the fluores-
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cently labeled stapled peptide is readily taken up by the cells.
To our knowledge this is the first report attempting to design
and synthesize all-hydrocarbon stapled peptides targeting the
integration events either by directly inhibiting IN or by
inhibiting its interaction with LEDGF/p75.
Scheme 1. Optimized Synthetic Route toward α-Methylated
α-Amino Acids Bearing an Olefinic Side Chain of Various
Lengths Suitably Protected for Solid Phase Peptide
a
Synthesis
RESULTS
■
The side chain bridging is an effective approach to induce and
stabilize the α-helix conformation of the linear peptide, and the
all-hydrocarbon bridge enables metabolic stability and cell
membrane permeability. Rather than stabilizing preferred
conformations, under certain circumstances, an all-hydrocarbon
staple can interact with the surface of the target protein and
contribute to the binding interaction.29 Incorporation of α,α-
disubstituted olefinic amino acids at i and i + 4 positions of the
12-amino acid NL6 sequence followed by a ruthenium-
catalyzed ring-closing metathesis (RCM) reaction generated
hydrocarbon stapled IN peptides.38 The α-methyl-α-alkenyla-
mino acid substitutions were made to flank three (substitution
positions i and i+4) amino acids within the NL6 peptide so that
reactive olefinic residues would reside on the same face of the
α-helix. Furthermore, the olefinic side chain varied from 3- to 6-
methylene to determine an optimal chain length for the
hydrocarbon staple.
a
Reagents and conditions: (i) PhCH(OMe)2, SOCl2, ZnCl2, THF, 0
°C, 4 h, 77%; (ii) allyl bromide for 2a, alkenyl iodide for 2b−d,
LiHMDS, THF/HMPA = 4:1, −78 °C, 4 h, 24−61%, dr 5:2; (iii)
LiOH·H2O, THF/H2O = 3:1, 0−25 °C, 12 h, 85−100%; (iv)
Ba(OH)2·8H2O (4.5 equiv for 3a−c, 2.5 equiv for 3d), H2O/DME =
5:3, under N2, refluxing, 12 h; (v) Fmoc-OSu, K2CO3, H2O/acetone =
1:1, 25 °C, 2 h, 59−94% overall yield for two steps.
We sought to optimize the NL6 peptide as a biological tool
and a prototype therapeutic by enforcing its α-helical structure
while preserving the key interacting residues that enable the
specific IN engagement. Therefore, the inserted position of the
α,α-disubstituted unnatural amino acid was chosen to avoid the
essential IN-binding residues based on our previous alanine
scanning results.17 As shown in Figure 1, three types of all-
hydrocarbon chain-tethered cyclic peptides were synthesized,
with N-terminal, middle, and C-terminal incorporation,
respectively, to determine which position is favorable for the
formation of the α-helix. Then the hydrocarbon cross-link
length was investigated to achieve the best helix-stabilizing and
potency-enhancing effects while the optimal inclusion positions
of the two α,α-disubstituted amino acids into NL6 peptide were
held constant.
Efficient Synthesis of Chiral α,α-Disubstituted Amino
Acids Bearing Olefinic Tethers of Various Lengths and a
Panel of Hydrocarbon-Stapled Peptides with Different
Inserted Position and Cross-Link Length. In order to
produce a series of hydrocarbon-stapled peptides for an
enhanced α-helicity and metabolic stability, we established an
efficient and facile methodology to synthesize optically pure α-
methyl-α-alkenylamino acids suitably protected for solid phase
peptide synthesis by optimizing the Seebach’s oxazolidinone
methods39−41 and screening the alkylation conditions. As
depicted in Scheme 1, starting from D-alanine, the condensation
of Nα-Cbz-D-alanine with benzaldehyde dimethyl acetal under
acidic conditions yielded predominantly cis-oxazolidinone
followed by the enantioretentive alkylation under basic
conditions.40 The resulting 2-phenyl-4-methyl-substituted
oxazolidinone underwent sequential hydrolysis and N-Fmoc
protection to afford the desired products (S)-4a−d. Since
previously modified conditions of benzaldehyde dimethyl acetal
and BF3·Et2O required 4 days under −40 °C to achieve good
yield and high diastereoselectivity,42 an optimized condition by
employing ZnCl2 as the Lewis acid in combination with thionyl
chloride greatly accelerated the predominant production of the
desired cis-(2R,4R)-2-phenyl-4-methyloxazolidinone 1 only
after 4 h of stirring at 0 °C.43
More effort was spent on the optimization of the alkylation
reaction to the oxazolidinone core by the inert alkenyl halide
that always gave poor yield when applying the reported
conditions.40,41 After the base, electrophile species, reagent
ratio, reaction temperature, and solvent were screened, the best
yield was obtained by using LiHMDS (2 equiv) as the base,
alkenyl iodide as the electrophile, and anhydrous THF/HMPA
(4:1, v/v) as the solvent at −78 °C. It is noteworthy that the
addition sequence of the reagents greatly impacted the
alkylation yield. Considering that the enolate intermediate
was not stable and the alkylation was slow, we added the base
separately to the prestirred mixture of the oxazolidinone and
the alkenyl halide in THF/HMPA. Gratifyingly, this resulted in
a stable and sufficient production of the alkylation products
2a−d bearing alkenyl side chain of various length at the α-
carbon, with a diastereomeric ratio of 5:2 regardless of the
alkenyl chain length. This is consistent with the previously
reported diastereoselectivity for a similar alkylation reaction.43
Then the sequential hydrolysis by LiOH, the removal of the
Nα-Cbz group with Ba(OH)2 in DME/H2O, and the Nα-Fmoc
reprotection readily furnished the chiral α-methylated α-amino
acids bearing an olefinic side chain with various chain lengths
and with orthogonal protection suitable for solid-phase peptide
synthesis based on Fmoc chemistry.
Subsequently, the α-methyl-α-alkenylamino acid containing
linear peptide was assembled on the solid phase and the RCM
reaction was performed on the resin with the assistance of
microwave irradiation by using CEM Liberty peptide
synthesizer (Scheme 2). However, for the introduction of the
bulky α-methyl-α-alkenylamino acid, manual and double
coupling was employed to secure the completion of the
assembly. Generally, the metathesis reaction proceeded
smoothly with the peptides containing the 4-carbon to 6-
carbon long alkenyl tethers to generate corresponding 6-carbon
to 10-carbon cross-linked peptides. However, the 3-carbon
olefinic tether was too short to metathesize (NLH10). Both cis
and trans double bond isomers were formed after the RCM
C
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Scheme 2. Fmoc-Based Solid-Phase Synthesis of Unstapled, Stapled, and Fluorescently Labeled Peptides, Exemplified by the
Synthesis of NLH5, NLH6, and NLX-1
reaction, which were directly used in the following studies
because they were not separable on HPLC.
The most potent stapled peptide NLH6 was chosen to attach
a fluorescein tag to monitor its subcellular distribution. Thus,
the fluorescein-conjugated stapled peptide (NLX-1) was
synthesized on the resin after the RCM reaction (Scheme 2).
The coupling of 5-carboxyfluorescein with the side chain
protected peptide on resin was achieved by using DIC and
HOBt as the coupling agents with the microwave assistance.
Then treatment of the resin-bound fluorescein-conjugated
peptide with the cleavage cocktail afforded the fluorescently
labeled stapled peptide NLX-1. Similarly, the linear peptide
NL6 was conjugated with fluorescein to yield NLX-2 to serve as
a negative control.
Appropriate Hydrocarbon Stapling Increases the α-
Figure 2. CD spectra of the select peptides with incorporation of two
Helical Propensity of the IN Peptides. The circular
α,α-disubstituted amino acids (i, i + 4) in unstapled and stapled forms.
dichroism (CD) spectra of the linear and conformationally
constrained peptides were measured in the solvent system of
CH3CN/PBS (40% v/v), and the select CD spectra are shown
in Figure 2. The organic solvent acetonitrile was added to
improve the solubility of the peptide in the PBS buffer at pH
7.2. The presence of acetonitrile might interfere with the
formation of α-helix conformation of the peptide in the
solution. Studies on peptide fragments have revealed that
acetonitrile stabilizes and induces β-sheet conformation.44
However, considering the solubility and the small amount of
the acetonitrile employed, we believe that the CD spectra can
still reflect the real trend of the conformational change of the
stapled peptides. The α-helical content measured by CD is
based on a method reported previously (Table 1).45
As expected, for these 12-amino acid NLH series with i, i + 4
spanning pattern, the hydrocarbon stapled peptides generally
demonstrated an improved α-helical content (23−49% α-
helicity) compared to the parent NL6 linear peptide that
displayed 20% α-helical content in 40% CH3CN/H2O solution
and thus predominantly existed as a random coil (Figure 2 and
Table 1). The exception is the short hydrocarbon stapled
peptides (NLH11 and NLH12) that might be too rigid to
achieve the desired conformation of the whole molecule.
Interestingly, the fluorescein-tagged stapled peptide (NLX-1)
displayed the highest α-helical content, probably because of the
N-capping effect of the bulky hydrophobic fluorescent group.
On the other hand, the inserted position of the α,α-
disubstituted amino acid strongly affected the formation of α-
helix conformation, with the middle position being optimal for
the stabilization of α-helix geometry. For example, a consistent
increase in the α-helical content was observed on the middle
cross-linked peptides relative to the corresponding unstapled
modified peptides (NLH6 vs NLH5, NLH14 vs NLH13,
NLH16 vs NLH15). The chain length of the hydrocarbon
cross-link also affected the induction of α-helix conformation.
Among the 4-, 5-, and 6-methylene olefinic side chains, the
pentene side chain turned out to be the best length with respect
to inducing and stabilizing the α-helix conformation of the
HIV-1 IN 12-mer peptide NL6 (NLH6 vs NLH12, NLH14).
The hydrocarbon staple of 6 carbons is too short to permit the
D
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a
Table 1. Structures and the α-Helical Content of the Peptides with α,α-Disubstituted Amino Acid Incorporation
a
The CD spectra of NL-6 and NLH series were recorded in 10 mM PBS (pH 7.2)/40% MeCN (v/v) at room temperature. Peptides were dissolved
at 200 μM. PBS: phosphate buffered solution, 10 mM, pH 7.2.
formation of an unstrained helix and that of 10 carbons is
longer than necessary and therefore does not constrain the helix
as effectively as the 8-carbon tether. In short, for the i, i + 4
stapled peptide series, the incorporation of the α,α-
disubstituted amino acids at the middle positions of the
sequence and the cross-link of 8 carbons provide the optimal
fits for stapling the HIV-1 IN peptide to achieve enhanced
helicity.
α-Helical Peptide Pairs Inhibit IN Catalytic Activities.
The sequences of each stapled α-helix stabilized peptide and its
corresponding unstapled peptide are shown in Table 1, and
their activities are shown in Table 2. Each pair contains a
covalent hydrocarbon staple spanning the i, i + 4 positions
whose residues were not essential for the HIV-1 inhibitory
activity according to the alanine scanning results.17 The
position of residue i in each peptide pair varies, with i in the
first pair being the threonine residue at the N-terminus of the
E
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Table 2. Inhibition of HIV-1 IN Catalytic Activity, Its Interaction with LEDGF/p75, and Viral Replication by Conformationally
Constrained α-Helical Peptides and Their Corresponding Unrestrained Analogues
a
b
c
d
e
peptide
3′-processing IC50 (μM)
22
53 15
strand transfer IC50 (μM)
15
19 10
EC50 (μM)
CC50 (μM)
therapeutic index
LEDGF/p75−IN IC50 (μM)
f
NLH2
NLH3
NLH5
NLH6
NLH8
NLH9
NLH10
NLH11
NLH12
NLH13
NLH14
NLH15
NLH16
NLX-1
NLX-2
8
5
5
5
NA
26
4
25
28
50
6
>100
15
f
9
9
1
1
6
6
1
1
28
20
NA
3
14
g
g
f
g
>100
>100
>100
>111
68
NT
NT
NA
NT
g
g
f
g
>100
NT
NT
NA
NT
g
g
f
g
>333
NT
NT
NA
NT
f
74 11
90
5
>18
>40
18
40
NA
20
14
f
83
NA
g
g
f
g
61 12
48 10
43
5
4
3
7
NT
NT
NA
NT
g
g
f
g
44
NT
NT
NA
NT
15
22
5
2
3
11
11
18
1.6
8
>100
5
25
5
2
41 15
>125
70
1
6
1
11
13
18.7
>11
5
25
17.5 1.5
21
6.2 0.2
25
h
NL6
7
4
1
>100
>5
>25
a
b
c
IC50: 50% inhibition of the integrase enzymatic activity. EC50: 50% cytoprotective activity of the peptides in HIV-1 infected cells. CC50: 50% cell
d
e
death in same cells. Therapeutic index: ratio of CC50 to EC50 values. LEDGF/p75 IC50: 50% inhibition of the interaction between the two
proteins. NA: not applicable. NT: not tested. Catalytic activities are cited from ref 17.
f
g
h
sequence TAYFLLKLAGRW (the residues in bold represent
the irreplaceable hot spots). In the second peptide pair the
position of residue i moves to the middle leucine residue, and
the third peptide pair bears a C-terminal hydrocarbon staple
with i being lysine. This change in position of the hydrocarbon
linker has an effect on the inhibitory activity of the peptides,
which is highly correlated with the α-helical conformation and
the orientation of the essential residue side chain.
Stapling the NL6 (K7I) at the middle produced the active
peptide pair NLH15 and NLH16 with an increase in α-helicity,
thus generating moderate activity against the 3′-processing and
strand transfer reactions. Overall, the trends observed for the
IN inhibitory activity for these stapled and unstapled peptides
correlated reasonably well with their α-helical propensities
(Table 2). Since HIV IN exerts its function as a multimer (IN
dimers are sufficient for 3′-P, and tetrameric structures
participate in the ST step),46,47 presumably our active peptides
inhibit both 3′-P and ST reactions by blocking the multi-
merization of the HIV IN.
Some Peptide Pairs Inhibit LEDGF/p75−IN Interac-
tion. Interestingly, most of the peptide pairs active in inhibiting
IN catalytic activities inhibited the LEDGF/p75−IN interaction
as well, as shown in Table 2. Similar to the inhibition of IN
catalysis, these peptide pairs showed comparable IC50 values in
the low micromolar range. The stapled peptide NLH16 had an
IC50 of 5 μM. Its corresponding unstapled peptide NLH15
does not show significant inhibition below 100 μM. Unstapled
peptide NLH2 inhibits the LEDGF/p75−IN interaction with
an IC50 of 26 μM, while its corresponding stapled partner
NLH3 is inactive. The peptide pair NLH5 and NLH6 and
peptide pair NLH11 and NLH12, with inclusion of the two
α,α-disubstituted amino acids in the middle of the sequence,
are all moderately active (IC50 < 20 μM). Since the α1 helical
domain of HIV IN is also involved in the interaction with
LEDGF/p75 IBD (integrase binding domain),48 not surpris-
ingly, the α1 region-derived NL6 peptides inhibit the LEDGF/
p75−IN interaction probably by binding to the LEDGF/p75 in
a competitive fashion.
The most active peptide pair is NLH5 (unstapled) and
NLH6 (stapled) with IC50 values of 9 1 μM for 3′-processing
and 6 1 μM for strand transfer. The staple on this pair is
positioned linking the leucine residue (i) in the TAYFLL-
KLAGRW sequence with the alanine residue (i + 4), resulting
in a significant induction of α-helicity on the two peptides
NLH5 and NLH6. In contrast, the least active peptide pairs are
NLH8 (unstapled) and NLH9 (stapled), with IC50 > 100 μM
for both catalytic activities. They contain a hydrocarbon staple
linking the lysine (i) with the arginine residue (i + 4) and
possess the highest α-helical content. To understand the
observation that the C-terminal stapling induced high
propensity of α-helix conformation but caused a significant
loss of the inhibitory potency, we calculated the possible
conformations of the three peptides (NLH9, NLH6, and NL6)
by running MM2/minimize energy through Chemoffice
ChemBio3D Ultra 11.0. According to the calculated optimal
conformation with minimized energy of the three peptides (not
shown), the side chain of the essential residue Trp on peptide
NLH9 was distorted from that of the parent peptide NL6 and
of the active peptide NLH6, which might be the reason for the
loss in activity. This observation is consistent with a recent
report demonstrating that the addition of a hydrocarbon staple
to the BimBH3 peptide did not enhance its binding affinity or
biological activity presumably because of the loss of a network
of stabilizing intramolecular interactions on the peptide.27
Therefore, the orientation of the essential residue side chain is
another important issue besides the active conformation
stabilization for the protein−protein interactions.
Stapled α-Helical Peptides Inhibit HIV-1 Replication.
Six peptide pairs were each tested for the inhibition of HIV-1
replication in MT-4 cells. Cytotoxicity was also evaluated, and a
therapeutic index was calculated for each peptide as a ratio
CC50/EC50. Table 2 shows the EC50, CC50, and therapeutic
indices for each of the peptides evaluated in this assay. From
this group of pairs, the peptide showing the best overall
therapeutic index of 8 is NLH16 (stapled). NLH16 thus
inhibits the HIV-1 replication with the lowest corresponding
The single substitution (K7I) on the NL6 sequence was
previously found to enhance the HIV-1 inhibitory activity.17
F
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Figure 3. Uptake of the fluorescein-labeled peptides NLX1 (A, left) and NLX2 (B, right) in MT-4 cells. Images were generated using a Zeiss
LSM510 microscope with a Plan-Apochromat 63×/1.4 oil DIC objective. The three-dimensional stacks were performed with Z step size of 0.3 mm
using an argon laser for the measurement of FITC at 488 nm and a HeNe laser for the measurement of the cell membrane dye at 543 nm.
Figure 4. Strategy for design of cell permeable nanoneedles as probes toward elucidating regions of interest within a protein that are essential for
efficient catalysis or protein−protein interactions.
cytotoxicity in MT4 cells. It contains an optimal 8-carbon
hydrocarbon staple linking the leucine residue (i) in the middle
of the sequence with an alanine residue (i + 4). Its
corresponding unstapled peptide NLH15 does not show
therapeutically selective inhibition against the viral replication.
Similar antiviral activity difference between the “open” and
“closed” counterparts was observed on the peptide pairs NLH2
and NLH3, NLH5 and NLH6, in which the “closed” or stapled
peptides inhibit the HIV-1 replication with less cytotoxicity
than their corresponding open conformation derivatives.
Interestingly, NLH12 has the same sequence and contains
the hydrocarbon staple spanning the same positions as peptides
NLH11 (“open” linker), NLH5 (unstapled), and NLH6
(stapled, closed). However, the shorter cross-link itself appears
to cause a substantial loss in the inhibition of both IN catalysis
and HIV-1 replication, which was consistent with the
aforementioned observation that the 6-carbon staple of
NLH12 failed to stabilize the α helix conformation.
Peptides and peptide-like structures have the tendency to
bind to cell surfaces and in some cases block the entry step of
viral replication instead of acting on intracellular targets. We
therefore determined the antiviral activity of NLH16 against a
viral strain that is resistant to entry inhibitors.49 NLH16
remains active against the AMD310050 resistant strain (EC50
=
4.3 μM). This suggests that the peptide may indeed enter the
cell and show antiviral activity on an intracellular target.
Because the stapled peptides significantly inhibit purified
LEDGF/p75−IN, the antiviral effect is most likely due to the
dual inhibition of IN and its interaction with LEDGF/p75.
Peptides NL6 and NLX-2 do not cross the cell membrane (see
below), and their antiviral activities are most likely due to the
inhibition of viral entry. Future in-depth mechanistic studies
will further elucidate their mechanism of action.
Stapled Peptides Are Cell Permeable. Stapled peptides
are known to efficiently cross the plasma membrane. Because
IN is an intracellular target, it is important to demonstrate
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cellular uptake with any peptide targeting the integration
events. Therefore, we conjugated a fluorescein tag on the N-
terminus of the stapled peptide NLH6 to generate NLX-1 with
a 72% yield. We further demonstrated that this fluorescent
probe exhibits an enhanced α-helical content and significant IN
inhibitory activity. In fact, NLX-1 showed the highest (47%) α-
helical content among all the synthesized peptides. To
demonstrate the cellular uptake, we incubated two fluorescently
labeled peptides NLX-1 and NLX-2 with MT-4 cells for 1 h.
We observed a rapid accumulation of the stapled NLX-1
peptide but not the unstapled NLX-2 peptide in MT-4 cells
(Figure 3A versus Figure 3B). To further illustrate a general
trend for cellular uptake of the stapled peptide, we treated three
additional cell lines (CEM, HCT116, and Panc-1 cells) with
NLX-1 for 3 h. Confocal microscopy and nuclear staining with
Draq5 showed distinct cellular uptake of the stapled peptide
(Figure S1 in Supporting Information). The uptake of NLX-1
in all the cell lines was efficient and the peptide localized to the
cytoplasm with minimum distribution to the nucleus.
interaction is druggable. siRNA, shRNA, and antisense
technologies are not effective in validating the importance of
protein−protein interactions in a given disease, hampering
efforts to find new targets. On the other hand, designing small-
molecule inhibitors for protein−protein interaction is a
challenging endeavor with the technologies currently available.
This innovative concept of utilizing nanoneedles to both
validate protein−protein interaction targets and discover novel
therapeutics will prove to be highly efficient and cost-effective
and will lead to the identification of new targets leading to new
cures.
CONCLUSIONS
■
This study reports the first example of the all-hydrocarbon
stapled peptides derived from the α-helical region of HIV-1 IN
along the dimeric interface. We synthesized a series of stapled
peptides and their corresponding unconstrained partners to
compare and contrast their abilities to inhibit IN in enzymatic
and cell-based assays. The stapled peptides show antiviral
activity, while their corresponding unstapled peptides do not
show therapeutically selective inhibition of viral replication in
cell-based assays, although each pair of peptides has similar
activity against purified IN. These peptides also inhibit the
LEDGF/p75−IN interaction in vitro because of the fact that
the IN region from which the peptide was derived corresponds
to the site of the LEDGF/p75 binding. The active stapled
peptide inhibits a viral strain resistant to an entry inhibitor,
suggesting that its antiviral activity is due to intracellular
targeting. More significantly, the fluorescently labeled stapled
peptide was readily internalized by cells while the unstapled
peptide was not taken up, demonstrating that the stapling
enhances the cellular uptake. The antiviral activity of some
linear peptides is most likely due to the nonspecific interference
with viral entry but not due to the specific interaction with IN
inside the infected cell. To our knowledge this is the first report
on the design and synthesis of cell-permeable stapled peptides
targeting the integration events by directly inhibiting IN or its
interaction with the LEDGF/p75.
DISCUSSION
■
Our strategy in the synthesis of these stapled peptides was to
extrapolate to a cellular environment the idea established in our
previous study that peptides derived from the protein of
interest may be used to identify domains on the protein that are
essential for catalytic activity, protein−protein interactions,
and/or dimerization. Figure 4 summarizes this venture, from
the synthesis of the original peptides, through the optimization
for an enhanced activity, and the subsequent choice of peptides
for the helix stabilization. While this idea has proven to be
successful in our laboratory in vitro, we sought to extend it to
cell-based assays by synthesizing hydrocarbon-stapled α-helical
peptides. Such α-helix stabilized peptides mimicking the α-
helical BH3 domain of the BCL-2 protein have previously been
shown by Walensky et al. to have enhanced helicity compared
to their unstapled counterparts.25 These stapled peptides
exhibit in vivo stability, as evidenced by their ability to suppress
growth of human leukemia cells in mice by activating apoptotic
pathways. For recent reviews and select applications of stapled
peptides, see refs 51−55.
EXPERIMENTAL SECTION
Chemistry. H NMR spectra were recorded on a Varian Mercury
■
High throughput screening followed by structural optimiza-
tion was essential in identifying IN inhibitors suitable for
clinical use.56 The complex protein−protein interactions of IN
with cellular cofactors provide innovative therapeutic targets for
the development of small-molecule inhibitors of HIV-1, which
are regaded as an excellent alternative to the conventional
active site-directed HIV-1 inhibititors in the context of the rapid
emergence of the viral cross-resistance. However, these targets
are plagued by difficulties associated with the shallow,
hydrophobic nature of the protein surfaces involved in such
interactions.54,55 A previous study by Brass et al. using RNAi
and forward genetics techniques identified a plethora of HIV-
dependency factors that are required for the HIV-1 infection.57
We here introduce the concept of stapled optimized peptides as
cell-permeable nanoneedles that will enable the identification of
regions of interest in proteins that have thus far been identified
as interacting protein partners of IN, and other viral proteins.
The nanoneedles may themselves serve as a starting point for
the development of novel inhibitors and may be further
optimized for the inhibition of HIV-1 infection after
formulation into nanoparticles and immunoliposomes. More
importantly, nanoneedle technologies can be efficiently used to
test the hypothesis of whether a given protein−protein
1
300 or 400 MHz spectrometer. The data are reported in parts per
million relative to TMS and referenced to the solvent in which they
were run. EI mass spectra were obtained on a Finnigan MAT 95 mass
spectrometer, and ESI mass spectra were recorded on a Finnigan LCQ
Deca mass spectrometer. The solvent was removed by rotary
evaporation under reduced pressure, and flash column chromatog-
raphy was performed on silica gel H (10−40 μm). Anhydrous solvents
were obtained by redistillation over sodium wire. The purity of all
peptide products was >95% determined by analytical HPLC on a
Waters 1525EF with Vydac 218TP C18 reversed-phase column (4.6
mm × 250 mm) in two solvent systems (system 1, solvent A, 0.05%
TFA in water; solvent B, 0.05% TFA in 95% acetonitrile; system 2,
solvent A, 0.05% TFA in water; solvent B, 0.05% TFA in methanol).
(2R,4R)-2-Phenyl-3-(carbobenzyloxy)-4-methyloxazolidin-5-
one (1).42 To a solution of Cbz-D-alanine (5 g, 22.4 mmol) and
benzaldehyde dimethyl acetal (3.4 mL, 23 mmol) in dry THF (35 mL)
at 0 °C was added thionyl chloride (1.79 mL, 24.6 mmol). After the
mixture was stirred for 10 min, anhydrous ZnCl2 (3.36 g, 24.6 mmol)
was added, and the reaction mixture was stirred at this temperature for
4 h. The reaction mixture was quenched by dropwise addition of ice−
water and adjusted to pH 5 with saturated NaHCO3, then extracted
with ether. The ether layer was washed with a saturated solution of
NaHCO3 and brine, dried over Na2SO4, and concentrated. The crude
product was purified by crystallization from ethyl ether/n-hexane to
H
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give the title compound 1 as colorless needles (5.38 g, 77%). Mp 52−
53 °C. 1H NMR (CDCl3) δ: 1.59 (s, 3H), 4.47−4.52 (m, 1H), 5.14−
5.21 (m, 2H), 6.65 (bs, 1H), 7.34−7.41 (m, 10 H). MS-EI: 311 (M +).
(2R,4S)-2-Phenyl-3-(carbobenzyloxy)-4-methyl-4-(4-
pentenyl)oxazolidin-5-one (2c). To a stirring solution of (2R,4R)-
2-phenyl-3-(carbobenzyloxy)-4-methyloxazolidin-5-one 1 (2.446 g,
7.86 mmol) and 5-iodopent-1-en (2.004 g, 10.22 mmol) in dry
THF/HMPA = 4: 1 (20 mL) was added LiHMDS (1 M, 15.7 mL,
15.7 mmol) in dry THF slowly and portionwise under nitrogen at −78
°C. This light yellow solution was stirred at this temperature for 2 h.
Saturated NH4Cl aqueous solution was added to quench the reaction.
The THF was removed in vacuo. Then Et2O was added to extract the
solution. The organic layer was washed three times with saturated
NaHCO3 and NaCl solutions, respectively. The organic phase was
dried over Na2SO4 and evaporated. The residue was purified by flash
column chromatography with eluent of petroleum−ethyl acetate (5:1)
to give compound 2c (1.499 g, 50%) as a colorless oil. Further
purification by recrystallization with Et2O/hexane (3: 4) gave a
colorless lumpy solid. Mp 59−60 °C; [α20D] +64.4° (c 0.38, CHCl3).
1H NMR (400 MHz, DMSO-d6, 80 °C) δ: 1.16−1.31 (m, 2H), 1.67−
1.79 (m, 4H), 1.91−2.00 (m, 2H), 2.24−2.33 (m, 1H), 4.93−5.09 (m,
4H), 5.65−5.75 (m, 1H), 6.59 (s, 1H), 7.05−7.46 (m, 10H). 1H NMR
(300 MHz, CDCl3) two rotamers (5:2) δ: 1.16−1.39 (m, 4H), 1.72 (s,
0.85H), 1.82 (s, 2.14H), 2.05−2.24 (m, 1H), 2.48−2.70 (m, 1H),
4.90−5.09 (m, 3H), 5.54−5.84 (m, 1H), 6.44 (d, J = 24.7 Hz, 1H),
6.91 (d, J = 6.9 Hz, 1H), 7.15−7.50 (m, 10H). 13C NMR (100 MHz,
CDCl3) δ: 23.91, 33.27, 35.49, 37.23, 67.48, 67.97, 89.54, 115.54,
126.96, 128.06, 128.36, 128.57, 128.93, 130.10. EI-MS m/z: 379 (M+).
HRMS-ESI m/z: calcd for C23H25NO4 + Na 402.1681, found
402.1677.
THF/H2O (3:1, 20 mL) was added LiOH·H2O (83 mg, 1.97 mmol)
at 0 °C. The mixture was stirred overnight at 25 °C, and then
LiOH·H2O (20 mg, 0.48 mmol) was added again to adjust the pH to
9−10. After 3 h, 5% NaHCO3 was added to the reaction mixture. The
aqueous layer was washed with Et2O and then acidified to pH 2 using
2 N HC1. The mixture was extracted with Et2O, and the organic layer
was dried over Na2SO4. The solvent was removed in vacuo to yield
290 mg (quantitative) of 3c as a colorless oil which was used directly
1
for the next step without further purification. H NMR (300 MHz,
CDCl3) δ: 1.21−1.45 (m, 2H), 1.61 (s, 3H), 1.79−1.90 (m, 1H),
2.01−2.12 (m, 3H), 4.94−5.03 (m, 2H), 5.09 (s, 2H), 5.52 (s, 1H),
5.61−5.87 (m, 1H), 7.26−7.36 (m, 5H). 13C NMR (100 MHz,
CD3OD): δ 23.9, 24.3, 36.7, 38.0, 60.5, 67.9, 115.8, 129.2, 129.5,
130.0, 138.9, 140.0, 157.9, 178.0. [α]D20 +8.2 (c 1.0, CHCl3). ESI m/z:
calcd 291.2 for C16H21NO4, found 291.0.
Compounds 3a, 3b, and 3d were prepared according to procedures
similar to that for 3c.
(S)-2-(Benzyloxycarbonylamino)-2-methylpent-4-enoic Acid
(3a). Starting from 2a (762 mg, 2.17 mmol), LiOH·H2O (228 mg,
5.42 mmol), and 40 mL of THF/H2O (3/1, v/v), 3a (579 mg) was
1
obtained as a colorless oil in quantitative yield. H NMR (300 MHz,
CDCl3) δ: 1.61 (s, 3H), 2.62−2.69 (m, 1H), 2.75−2.77 (m, 1H),
5.06−5.16 (m, 4H), 5.48 (s, 1H), 5.63−5.77 (m, 1H), 7.29−7.39 (m,
5H).
(S)-2-(Benzyloxycarbonylamino)-2-methylbut-4-enoic Acid
(3b). Starting from 2b (366 mg, 1.9 mmol), LiOH·H2O (126 mg, 3
mmol), and 20 mL of THF/H2O (3/1, v/v), 3b (260 mg) was
obtained as a colorless oil in 94% yield.
(S)-2-(Benzyloxycarbonylamino)-2-methyloct-7-enoic Acid
(3d). Starting from 2d (896 mg, 2.28 mmol), LiOH·H2O (239 mg,
5.69 mmol), and 40 mL of THF/H2O (3/1, v/v), 3d (588 mg) was
obtained as a colorless oil in 85% yield. 1H NMR (300 MHz, DMSO-
d6): δ 1.08−1.27 (m, 4H), 1.31 (s, 3H), 1.64−1.76 (m, 2H), 1.98 (q, J
= 7.2 Hz, J = 6.6 Hz), 4.90−5.03 (m, 4H), 5.70−5.83 (m, 1H), 7.20−
7.44 (m, 5H).
(S)-N-(9-Fluorenylmethyl carbamate)-2-(2′-pentenyl)alanine
(4c). To a solution of 2-(benzyloxycarbonylamino)-2-methylhept-6-
enoic acid 3c (169 mg, 0.58 mmol) in 7.5 mL of DME and 4.5 mL of
water (DME/H2O = 5:3) was added Ba(OH)2·8H2O (733 mg, 2.32
mmol). After refluxing under nitrogen (65−90 °C) overnight, the
mixture was cooled to room temperature, and the barium salts were
precipitated. After addition of 8 mL of acetone and 5 mL of H2O and
K2CO3 to adjust the pH to 9−10, Fmoc-OSu (216 mg, 0.64 mmol)
was added to this solution at room temperature. The mixture was
stirred for 2 h and kept at pH 9−10 by adding K2CO3. The mixture
was then diluted with water and washed with Et2O. The aqueous layer
was acidified with 2 N HCl to pH 2 and extracted with Et2O. The
combined organic layers were dried (Na2SO4) and evaporated. The
residue was purified by flash column chromatography with elution of
CH2Cl2/CH3OH (5:1) to give compound 4c (130 mg, overall yield of
59% for two steps) as a white solid: [α20D] +10.6° (c 0.66, CHCl3). 1H
NMR (300 MHz, DMSO-d6) δ 1.33 (m, 5H), 1.99 (t, J = 6.9 Hz, 2H),
4.21−4.27 (m, 3H), 4.94−5.03 (m, 2H), 5.73−5.82 (m, 1H), 7.33 (td,
J = 7.2 Hz, 2H), 7.43 (t, J = 7.5 Hz, 2H), 7.72 (d, J = 7.8 Hz, 2H), 7.89
(d, J = 7.6 Hz, 2H). EI-MS m/z: 379 (M+).
Compounds 2a, 2b, and 2d were prepared according to procedures
similar to that for 2c.
(2R,4S)-Benzyl 4-Allyl-4-methyl-5-oxo-2-phenyloxazolidine-
3-carboxylate (2a). Starting from (2R,4R)-2-phenyl-3-(carbobenzy-
loxy)-4-methyloxazolidin-5-one (1) (3.113 g, 10 mmol), allyl bromide
(2.420 g, 20 mmol) in 25.0 mL of THF/HMPA (4/1, v/v), and
LiHMDS (1 M, 16.0 mL, 16 mmol), 2a (2.148 g) was obtained as
1
colorless oil in 61% yield. [α20D] +64.9° (c 1.14, CHCl3). H NMR
(400 MHz, DMSO-d6, 80 °C) δ: 1.72 (s, 3H), 2.48−2.50 (m, 1H),
3.06−3.09 (m, 1H), 5.01−5.20 (m, 4H), 5.61−5.72 (m, 1H), 6.46 (s,
1H), 7.12 (brs, 1H), 7.22 (s, 3H), 7.42−7.49 (m, 6H). 1H NMR (300
MHz, CDCl3) two rotamers (2.6:1) δ 1.71 (s, 0.82H), 1.80 (s, 2.15H),
2.53 (dd, J = 13.7, 6.2 Hz, 1H), 2.86−3.29 (m, 1H), 4.95 (s, 1H),
5.08−5.22 (m, 2H), 5.53−5.74 (m, 1H), 6.30 (d, J = 18.7 Hz, 1H),
6.87 (d, J = 6.2 Hz, 1H), 7.13−7.64 (m, 10H).
(2R,4S)-2-Phenyl-3-(carbobenzyloxy)-4-methyl-4-(but-3-
enyl)oxazolidin-5-one (2b). Starting from 1 (1.557 g, 5 mmol), 4-
iodobut-1-ene (1.365 g, 7.5 mmol) in 12.5 mL of THF/HMPA (4/1),
and LiHMDS (1 M, 7.5 mL, 7.5 mmol), 2b (0.431 g) was obtained as
yellow oil in 24% yield. [α20D] +71.5° (c 1.03, CHCl3). 1H NMR (400
MHz, DMSO-d6, 80 °C) δ: 1.67 (s, 3H), 1.81−1.96 (m, 3H), 2.36−
2.39 (m, 1H), 4.93−5.07 (m, 4H), 5.62−5.72 (m, 1H), 6.56 (s, 1H),
7.10 (brs, 1H), 7.26 (s, 3H), 7.40−7.43 (m, 6H). EI-MS m/z: 365
(M+).
(2R,4S)-2-Phenyl-3-(carbobenzyloxy)-4-methyl-4-(hex-5-
enyl)oxazolidin-5-one (2d). Starting from 1 (2.179 g, 7 mmol), 6-
iodohex-1-ene (1.900 g, 9.1 mmol) in 17.5 mL of THF/HMPA (4/1),
and LiHMDS (0.5 M, 22.4 mL, 11.2 mmol), 2d (1.122 g) was
obtained as yellow oil in 41% yield. [α20D] +66.0° (c 0.97, CHCl3). 1H
NMR (400 MHz, DMSO-d6, 80 °C) δ: 1.09−1.33 (m, 4H), 1.67 (s,
3H), 1.70−1.78 (m, 1H), 1.95 (q, 2H), 2.27 (br, 1H), 4.92−5.09 (m,
4H), 5.70−5.80 (m, 1H), 6.56 (s, 1H), 7.12 (brs, 1H), 7.28 (s, 3H),
Compounds 4a, 4b, and 4d were prepared according to procedures
similar to that for 4c.
(S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-2-methyl-
pent-4-enoic Acid (4a). Starting from 3a (238 mg, 0.9 mmol),
Ba(OH)2·8H2O (998 mg, 3.2 mmol), 21 mL of DME/H2O (5/3, v/
v), and Fmoc-OSu (364 mg, 1.08 mmol), 4a (231 mg) was obtained as
a white powder in 73% yield. 1H NMR (300 MHz, (CD3)2SO) δ: 1.27
(s, 3H), 2.35−2.42 (m, 1H), 2.49−2.60 (m, 1H), 4.19−4.27 (m, 3H),
4.99−5.06 (m, 2H), 5.62−5.71 (m, 1H), 7.28−7.47 (m, 4H), 7.69 (d,
2H, J = 7.5 Hz), 7.86 (d, 2H, J = 7.5 Hz).
1
7.42 (s, 6H). H NMR (300 MHz, CDCl3) two rotamers (2.5:1) δ:
0.98−1.38 (m, 4H), 1.68 (s, 0.85H), 1.77 (s, 2.14H), 1.78−1.95 (m,
2H), 2.01−2.12 (m, 1H), 2.38−2.65 (m, 1H), 4.97 (m, 3H), 5.53−
5.77 (m, 1H), 6.40 (d, J = 18.0 Hz, 1H), 6.87 (d, J = 6.3 Hz, 1H),
7.13−7.50 (m, 10H). EI-MS m/z: 393 (M+).
2-(Benzyloxycarbonylamino)-2-methylhept-6-enoic Acid
(3c). To a stirred solution of (2R,4S)- 2-phenyl-3-(carbobenzyloxy)-
4-methyl-4-(4-pentenyl)oxazolidin-5-one 2c (374 mg, 0.99 mmol) in
(S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-2-methyl-
but-5-enoic Acid (4b). Starting from 3b (260 mg, 0.94 mmol),
Ba(OH)2·8H2O (1.035 g, 3.28 mmol), 24 mL of DME/H2O (5/3, v/
v), and Fmoc-OSu (364 mg, 1.08 mmol), 4b (324 mg) was obtained
1
as a white powder in 94% yield. [α20D] +7.0° (c 1.02, CHCl3). H
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NMR (300 MHz, DMSO-d6) δ: 1.33 (s, 3H), 1.72−1.78 (m, 1H),
1.84−1.96 (m, 2H), 4.17−4.27 (m, 3H), 4.92−5.02 (m, 2H), 5.73−
5.82 (m, 1H), 7.29−7.34 (m, 2H), 7.39−7.50 (m, 2H), 7.71 (d, 2H, J
= 7.8 Hz), 7.88 (d, 2H, J = 7.2 Hz). EI-MS m/z: 365 (M+).
acids, and the N-Fmoc group in the N-terminus of the resin-bound
peptide was removed with 20% piperidine/DMF (12 min). Then the
side chain protected resin-bound peptide was mixed with the Grubbs
catalyst (second generation) (0.2 equiv). The reaction mixture was
stirred at room temperature for 5 h for peptides NLH3, NLH6, and
NLH9 or under microwave irradiation (23 W, 75 °C) for 3 min for
peptides NLH14 and NLH16 in the solvent 1,2-dichloroethane. The
peptide was then cleaved from the resin, and meanwhile the side
chains were deprotected using the cleavage system: TFA/EDT/H2O
(9.5:0.25:0.25) or TFA/TES/H2O (9.5:0.25:0.25) at room temper-
ature for 2 h. The crude peptides were purified to homogeneity by RP-
HPLC.
NLH3. tR = 16.7 min (gradient: 10−20% B over 2 min, 20−60% B
over 20 min). ESI-MS m/z (C75H112N18O13, calcd 1472.9): 1474.1
([M + H]+), 737.9 ([M + 2H]2+/2), 1472.1 ([M − H]−). Yield: 58%.
Purity: 100% (determined by HPLC).
NLH6. tR = 21.3 min (gradient: 10−20% B over 2 min, 20−60% B
over 20 min). ESI-MS m/z (C76H114N18O14, calcd 1502.9): 1504.1
([M + H]+), 752.9 ([M + 2H]2+/2), 1502.0 ([M − H]−). Yield: 57%.
Purity: 100% (determined by HPLC).
NLH9. tR = 26.8 min (gradient: 10−20% B over 2 min, 20−60% B
over 20 min). ESI-MS m/z (C73H106N14O14, calcd 1402.8): 1426.1
([M + Na]+), 1402.2 ([M − H]−). Yield: 43%. Purity: 100%
(determined by HPLC).
NLH12. tR = 15.19 min (gradient: 10−20% B over 2 min, 20−53% B
over 18 min). ESI-MS m/z (C74H110N18O14, calcd 1474.9): 1475.9
([M + H]+), 1473.9 ([M − H]−). Yield: 25%. Purity: 100%
(determined by HPLC).
NLH14. tR = 26.6 min (gradient: 10−22% B over 2 min, 22−60% B
over 23 min, 60−90% B over 3 min). ESI-MS m/z (C74H110N18O14,
calcd 1530.9): 1531.8 ([M + H]+), 1553.8 ([M + Na]+), 777.8 ([M +
Na + H]2+/2), 766.7 ([M + 2H]2+/2). Yield: 43%. Purity: 100%
(determined by HPLC).
NLH16. tR = 16.11 min (gradient: 10−30% B over 2 min, 30−53% B
over 23 min). ESI-MS m/z (C76H113N17O14, calcd 1487.9): 1489.0
([M + H]+), 1486.9 ([M − H]−). Yield: 40%. Purity: 100%
(determined by HPLC).
(S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-2-methyl-
oct-7-enoic Acid (4d). Starting from 3d (274 mg, 0.90 mmol),
Ba(OH)2·8H2O (708 mg, 2.24 mmol), 15.1 mL of DME/H2O (5/3,
v/v), and Fmoc-OSu (363 mg, 1.08 mmol), 4d (268 mg) was obtained
1
as a white powder in 76% yield. H NMR (300 MHz, DMSO-d6) δ:
1.11−1.29 (m, 7H), 1.61−1.77 (m, 2H), 1.97 (q, 2H, J = 6.6 Hz),
4.14−4.24 (m, 3H), 4.87−4.99 (m, 2H), 5.67−5.81 (m, 1H), 7.27−
7.41 (m, 4H), 7.68 (t, 2H, J = 7.5 Hz), 7.86 (d, 2H, J = 7.8 Hz).
Peptide Synthesis and Characterization. The linear peptides
were synthesized manually or instrumentally (CEM Liberty peptide
synthesizer) using Fmoc-based SPS protocols on PAL amide resin
(0.53 mmol/g) or Rink amide−MBHA resin. The N-Fmoc group of
the resin was removed with 20% piperidine in DMF (30 min at room
temperature), then coupled with Nα-protected amino acid (3 equiv) in
the presence of DIPCDI (3 equiv) and HOBt or HOAt (3 equiv) at
room temperature for 2 h. The cycle of deprotection and coupling was
repeated until the whole sequence was assembled. The side chain
protections of the Fmoc-AA are as follows: Arg(Pmc), Lys(Boc),
Thr(t-Bu), Tyr(t-Bu), Trp(Boc). DIPCDI/HOBt (or HOAt)
activation of Nα-protected amino acids was employed for the coupling,
and 20% piperidine/DMF was used for Fmoc deprotection. TFA/
EDT(or TES)/H2O (9.5:0.25:0.25) was used for the resin cleavage
and side chain deblocking. For α,α-disubstituted amino acids, double
coupling using HOAt was conducted to achieve a complete reaction.
The crude peptides were purified to homogeneity by reverse-phase
high-performance liquid chromatography (RP-HPLC). HPLC con-
ditions were as follows: Vydac C18 column (10 mm × 250 mm);
solvent A, 0.05% TFA in water; solvent B, 0.05% TFA in 95%
acetonitrile in water with gradient indicated below; flow rate, 2.5 mL/
min; UV detector, 225 nm. ESI-MS was performed on a Finnigan
LCQDeca mass spectrometer. The purity of products was
characterized by analytical HPLC and ESI-MS and was >98% as
determined by HPLC analyses with Vydac 218TP C18 column (4.6
mm × 250 mm) in two solvent systems (system 1, solvent A, 0.05%
TFA in water; solvent B, 0.05% TFA in 95% acetonitrile; system 2,
solvent A, 0.05% TFA in water; solvent B 0.05% TFA in methanol).
NLH2. tR = 18.9 min (gradient: 10−20% B over 2 min, 20−60% B
over 20 min). ESI-MS m/z (C77H116N18O13, calcd 1500.9): 1502.4
([M + H]+), 752.1 ([M + 2H]2+/2). Yield: 65%. Purity: 100%
(determined by HPLC).
Fluorescein-Labeled Peptides. After the Nα-Fmoc deprotection,
the side chain protected resin-bound peptide (NLH-6 or NL-6) was
coupled with 5-carboxyfluorescein by using DIC and HOBt as the
coupling reagents under microwave irradiation (23 W, 75 °C, 10 min)
and double coupling. Then treatment of the resin-bound peptide with
the cleavage cocktail afforded the fluorescently labeled stapled or linear
peptide (NLX-1 or NLX-2).
NLX1. tR = 19.8 min (gradient: 10−20% B over 2 min, 20−60% B
over 20 min). ESI-MS m/z (C97H124N18O20, calcd 1862.1): 1862.9
([M + H]+), Yield: 72%. Purity: 100% (determined by HPLC).
NLX2. tR = 16.8 min (gradient: 10−20% B over 2 min, 20−60% B
over 20 min). ESI-MS m/z (C92H118N18O20, calcd 1796.1): 1796.9
([M + H]+). Yield: 37%. Purity: 99% (determined by HPLC).
NLH5. tR = 22.5 min (gradient: 10−20% B over 2 min, 20−60% B
over 20 min). ESI-MS m/z (C78H118N18O14, calcd 1530.9): 1532.2
([M + H]+), 1554.2 ([M + Na]+), 766.8 ([M + 2H]2+/2), 1530.1 ([M
− H]−). Yield: 61%. Purity: 100% (determined by HPLC).
NLH8. tR = 26.2 min (gradient: 10−20% B over 2 min, 20−60% B
over 20 min). ESI-MS m/z (C75H110N14O14 calcd 1430.8): 1431.8 ([M
+ H]+), 1454.1 ([M + Na]+), 1430.1 ([M − H]−). Yield: 34%. Purity:
100% (determined by HPLC).
NLH10. tR = 23.4 min (gradient: 0−20% B over 2 min, 20−55% B
over 26 min). ESI-MS m/z (C74H110N18O14, calcd 1474.8): 1476.2
([M + H]+), 1474.1 ([M − H]−). Purity: 100% (determined by
HPLC).
NLH11. tR = 16.9 min (gradient: 10−20% B over 2 min, 20−53% B
over 18 min). ESI-MS m/z (C76H114N18O14, calcd 1503.9): 753.1 ([M
+ 2H]2+/2). Yield: 75%. Purity: 100% (determined by HPLC).
NLH13. tR = 28.2 min (gradient: 10−22% B over 2 min, 22−60% B
over 23 min, 60−90% B over 3 min). ESI-MS m/z (C80H122N18O14,
calcd 1558.9): 1560.2 ([M + H]+), 1582.2 ([M + Na]+), 781.1 ([M +
2H]2+/2). Yield: 42%. Purity: 100% (determined by HPLC).
NLH15. tR = 28.2 min (gradient: 10−20% B over 2 min, 20−60% B
over 23 min). ESI-MS m/z (C78H117N17O14, calcd 1515.9): 1517.1
([M + H]+), 1538.9 ([M + Na]+), 1514.9 ([M − H]−). Yield: 96%.
Purity: 100% (determined by HPLC).
ASSOCIATED CONTENT
■
S
* Supporting Information
Details of circular dichroism (CD) measurements, in vitro assay
for HIV-1 IN catalytic activity inhibition, in vitro assay for
inhibition of LEDGF/p75-HIV-1 IN interaction, and confocal
1
microscopy protocol and H NMR spectra of the alkylation
products 2. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*For Y.-Q.L.: phone, 86-21-50806876; fax, +86-21-50806876;
e-mail, yqlong@mail.shcnc.ac.cn. For N.N.: phone, 323-442-
2341; fax, 323-442-1390; e-mail, neamati@usc.edu.
Stapled Peptides. By use of the fluorenylmethoxycarbonyl
(Fmoc) protection strategy, the linear peptide was synthesized
manually on the PAL amide resin coupled with the respective amino
Author Contributions
∥S.-X.H. and Z.Z. contributed equally to this manuscript.
J
dx.doi.org/10.1021/jm4006516 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The work in Long’s laboratory was supported by funds from
the National Natural Science Foundation of China (Grants
81021062, 81072527, and 81123004), and Science and
Technology Commission of Shanghai Municipality (Grant
08JC1422200). The work in the Neamati and Debyser
laboratories were supported by NIH/NIAID (Grant
R21AI081610). We thank N. J. Van Der Veken, B. Van
Remoortel, and M. Michiels and Multiphoton Unit at KU
Leuven for excellent technical assistance.
ABBREVIATIONS USED
■
Cbz, carboxybenzoyl; CD, circular dichroism; CC50, cytotoxic
concentration 50%; DIPCDI, diisopropylcarbodiimide; DME,
dimethyl ether; EC50, effective concentration 50%; EDT, 1,2-
ethanedithiol; Fmoc, fluorenylmethoxycarbonyl; HIV-1 IN,
human immunodeficiency virus type 1 integrase; IBD, integrase
binding domain; LEDGF, lens epithelium-derived growth
factor; LDA, lithium diisopropylamide; LDEA, lithium
diethylamide; LiHMDS, lithium bis(trimethylsilyl)amide;
KHMDS, lithium hexamethyldisilazide; PBS, phosphate buf-
fered saline; PIC, preintegration complex; RCM, ring-closing
metathesis; THF/HMPA, tetrahydrofuran/hexamethylphos-
phoramide; THF/DME, tetrahydrofuran/ethylene glycol di-
methyl ether; TES, triethylsilane; TFA, trifluoroacetic acid;
TFE, trifluoroethanol; TI, therapeutic index; TMS, tetrame-
thylsilane
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