Synthesis, biophysical characterization, and anti-HIV-1 fusion activity of DNA helix-based inhibitors with a p-benzyloxyphenyl substituent at the 5′-nucleobase site

Article abstract of DOI:10.1016/j.bmcl.2018.04.012

DNA helix-based HIV-1 fusion inhibitors have been discovered as potent drug candidates. Introduction of hydrophobic groups to a nucleobase provides an opportunity to design inhibitors with novel structures and mechanisms of action. In this work, two novel nucleoside analogues (1 and 2) were synthesized and incorporated into four DNA duplex- and quadruplex-based inhibitors. All the molecules showed anti-HIV-1 fusion activity. The effect of the p-benzyloxyphenyl group and the attached linker on the helix formation and thermal stability were fully compared and discussed. Surface plasmon resonance analysis further indicated that inhibitors with the same DNA helix may still have variable reaction targets, mainly attributed to the different hydrophobic modifications.

Full text of DOI:10.1016/j.bmcl.2018.04.012

Bioorganic & Medicinal Chemistry Letters 28 (2018) 1842–1845
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, biophysical characterization, and anti-HIV-1 fusion activity of
DNA helix-based inhibitors with a p-benzyloxyphenyl substituent at the
50-nucleobase site
a,
Yongjia Tang a,c, Zeye Han a,c, Jiamei Guo b, Yangli Tian a, Keliang Liu a, , Liang Xu
⇑
⇑
a State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing 100850, China
b Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
DNA helix-based HIV-1 fusion inhibitors have been discovered as potent drug candidates. Introduction of
hydrophobic groups to a nucleobase provides an opportunity to design inhibitors with novel structures
and mechanisms of action. In this work, two novel nucleoside analogues (1 and 2) were synthesized
and incorporated into four DNA duplex- and quadruplex-based inhibitors. All the molecules showed
anti-HIV-1 fusion activity. The effect of the p-benzyloxyphenyl group and the attached linker on the helix
formation and thermal stability were fully compared and discussed. Surface plasmon resonance analysis
further indicated that inhibitors with the same DNA helix may still have variable reaction targets, mainly
attributed to the different hydrophobic modifications.
Received 2 January 2018
Revised 3 April 2018
Accepted 5 April 2018
Available online 6 April 2018
Keywords:
HIV-1
Fusion inhibitor
Duplex
Ó 2018 Elsevier Ltd. All rights reserved.
Quadruplex
SPR
Due to the quick drug-resistant mutations of HIV-1 strains,
developing inhibitors with novel structures or mechanisms of
action is of great interest in the field of anti-HIV-1 drug research.
Several DNA G-quadruplexes with various hydrophobic groups
modified at the 50- or 30-hydroxyl ends have been discovered as
potential HIV-1 fusion inhibitors. Both the G-quadruplex skeleton
and hydrophobic groups have proven important for the inhibitory
activity.1–9 Recently, our group demonstrated that DNA duplexes
and triplexes with the hydrophobic tert-butyldiphenylsilyl
(TBDPS) substituent had anti-HIV-1 fusion activity.10–12 As main-
taining the balance between the hydrophobic group and the
charged DNA helix is important for the activity, the TBDPS group
has been introduced at the nucleobase site via de novo synthesis
of nucleoside analogues. This modification strategy provides
another alternative for introducing hydrophobic groups into the
molecular structure, for duplex, triplex, and quadruplex inhibitors.
However, the structure design, especially for the duplex- and
triplex-based inhibitors, is still very limited. Only the TBDPS group
has been reported to be utilized by the nucleobase modification
strategy for these two types of DNA helix inhibitors, and the
described O-Si bond connecting the TBDPS group to the rest of
the nucleoside is easily hydrolyzed in the course of deprotection
of the oligonucleotide in aqueous ammonia at 55 °C.10,13,14
Here, two new nucleoside analogues (Fig. 1, 1 and 2) incorporat-
ing the hydrophobic moiety p-benzyloxyphenyl (PBP) at the nucle-
obase site were synthesized, aiming to facilitate the design of novel
anti-HIV-1 inhibitors with high efficiency or new mechanism of
action.
The reason for choosing PBP was that it had been introduced at
the 50-hydroxyl site in a classical ‘‘TGGGAG” quadruplex inhibitor
(G3, d(PBP-TGGGAG)4, Fig. S1), and this molecule showed higher
activity than any other quadruplex inhibitors ever reported.2,15
The details for the syntheses of 1, 2, and their corresponding phos-
phoramidites are shown in Scheme 1 and the supplementary data.
⇑
Corresponding authors.
E-mail addresses: keliangliu55@126.com (K. Liu), xuliang24998@yahoo.com
(L. Xu).
Fig. 1. Structures of nucleoside analogues 1 and 2.
c
The two authors contribute equally to this work.
https://doi.org/10.1016/j.bmcl.2018.04.012
0960-894X/Ó 2018 Elsevier Ltd. All rights reserved.

Y. Tang et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 1842–1845
1843
Scheme 1. The syntheses of 1, 2, and their corresponding phosphoramidites. (I) 3-Chloropropyne, DMF, rt; (II) CuI, Pd(PPh3)4, Et3N, DMF, rt; (III) DMT-Cl, pyridine, rt; (IV)
[(iPr)2N]2P(OCH2CH2CN), (iPr)2EtN, CH2Cl2, rt; (V) Pd/C, H2, CH3OH, 30 °C, 5 kgf/cm2; (VI) DMT-Cl, pyridine, rt; (VII) [(iPr)2N]2P(OCH2CH2CN), (iPr)2EtN, CH2Cl2, rt.
It should be pointed out that, we had intended to get compound 2
from 1 directly by hydrogenation, however, compound 1 was
found hard to dissolve in methyl alcohol and a primary byproduct
(9, Scheme 1) was generated in the hydrogenation course. As an
alternative method, compound 1 was reacted with DMT-Cl first
and then taken hydrogenation to give compound 2, the synthetic
route was found in good purity and yield.
Next, four oligonucleotides (d1, d2, g1, and g2, Table 1) contain-
ing 1 or 2, separately, and two natural oligonucleotides (d0 and g0)
were synthesized on an ABI 392 DNA/RNA synthesizer (Applied
Biosystems, Foster City, CA), according to the standard protocol.
Deprotection of the sequences was conducted with aqueous
ammonia at 55 °C for 15 h. The oligonucleotides were purified by
reverse-phase HPLC (A: 0.1 M acetic acid/triethylamine containing
5% acetonitrile; B: acetonitrile; B in A from 10% to 80% in 20 min, at
1 mL/min), and were characterized by MALDI-TOF MS (Table 1).
Three duplex structures (D0, D1 and D2) and three tetramolec-
ular G-quadruplex structures (G0, G1 and G2) were obtained by
annealing the corresponding oligonucleotide sequences (d0, d1,
d2, g0, g1, or g2, separately) in phosphate-buffered saline buffer
containing 20 mM MgCl2 (for D0, D1 and D2) or 100 mM KCl (for
G0, G1 and G2). The buffers were heated to 90 °C, held for 5 min,
and cooled to 10 °C at a rate of 1 °C/min. The samples were main-
tained at 10 °C overnight before tests.
Table 2
The melting temperatures and in vitro anti-cell fusion activities of D0 to G2.
No.
Sequence
Melting Temperature (°C)c
IC50
>20
6.68 0.69
2.19 0.56
>20
(lM)
D0a
D1
D2
G0b
G1
d(TGGTCGACCT)2
d(1GGTCGACC1)2
d(2GGTCGACC2)2
d(TGGGAG)4
d(1GGGAG)4
d(2GGGAG)4
54.4 1.6
51.8 1.7
55.6 0.8
54.7 0.5
59.5 1.2
70.6 2.6
1.97 0.23
0.78 0.04
G2
a
b
c
D represents duplex.
G represents G-quadruplex.
Three parallel experiments were performed to derive the means standard
deviations (SD).
path length, 4.0-s response time, and 50-nm/min scanning speed.
Oligonucleotides were at a concentration of 8
g2) or 4 M (d0, d1 and d2). The wavelength was at 280 nm for
lM (g0, g1, and
l
D0, D1 and D2, and 260 nm for G0, G1, and G2. It was found that
the melting curves of G-quadruplex were not superimposable with
the annealing curve, which might mainly attribute to the slow
kinetics of G-quadruplex folding.15,18 As a result, the Tm of quadru-
plex and duplex were both evaluated by the melting curves, and
the tests were carried out after annealing and maintaining at 10
°C overnight. As shown in Table 2, all helixes showed Tm values
greater than 37 °C, indicating stable helix structures at room tem-
perature. D1 and D2 showed comparable Tm values to natural D0
without significant difference (p > 0.05), however, increased ther-
mal stability was observed for G1 and G2 when compared to
unmodified G0 (p < 0.01). This result indicated the positive effect
of hydrophobic modification on thermal stability of quadruplex,
while not on that of duplex, and the difference might mainly attri-
bute to the discrepant helix assembly pattern. Former researches
reported that hydrophobic modification at the 50-hydroxyl end of
TGGGAG quadruplex would greatly increase the thermal stabil-
ity;1,2,4,7,8 relatively, our results indicated that modification at the
50-end of TGGGAG from nucleobase site also showed considerable
effect. Moreover, G2 was with a much higher promotion extent
than that of G1 (p < 0.01). The more hydrophobicity and flexibility
of the propyl linker (in 2) than the propenyl linker (in 1), as well as
The melting temperatures (Tm) of D0, D1, D2, G0, G1, and G2
were measured on CD spectropolarimeter (MOS-450; Bio-Logic
Inc., France) with temperature rising from 15 °C to 90 °C, at step
of 1 °C/min, using 4.0-nm bandwidth, 0.1-nm resolution, 0.1-cm
Table 1
MALDI-TOF characterization of the oligonucleotides.
No.
Sequence (50-30)
MS (calc)
MS (found)
d0
d1
d2
g0
g1
g2
TGGTCGACCT
1GGTCGACC1
2GGTCGACC2
TGGGAG
1GGGAG
2GGGAG
3019.0
3463.6
3471.6
1872.3
2095.0
2099.0
3019.2
3463.0
3472.5
1872.0
2094.2
2098.6

1844
Y. Tang et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 1842–1845
Fig. 2. CD spectrum results of duplex (D0, D1, and D2, left) and quadruplex (G0, G1, and G2, right).
the resulting more spatial freedom of the connecting hydrophobic
group brought by propyl linker (correspondingly, the geometry of
the propenyl linker extruded the PBP outward the helix more lin-
early), were supposed to be responsible for the better promotion
effect.
was very slow, the distinct results might mainly contribute to their
14 days of incubation before anti-HIV assays. Besides, the paper
also supposed different target and action mechanism of
d
(TGGGAG)4 from traditional 50-end conjugated ODNs.
Unlike the quadruplex inhibitors first discovered about 30 years
ago; limited information is known about the mechanisms of action
of the newly established DNA duplex inhibitors. Therefore, surface
plasmon resonance (SPR) analysis was carried out in this work, for
the first time, to explore the interaction between the duplex inhi-
bitors and the HIV-1 surface glycoproteins gp120 and gp41
directly. The test was performed on a Biacore T100 instrument
(GE Healthcare, Uppsala, Sweden) at 25 °C in HBS-P buffer (10
mM HEPES, 150 mM NaCl, 10 mM CaCl2, and 0.05% surfactant
P20; pH 7.4). Recombinant HIV-1(IIIB) gp120 (Immuno Diagnostics
Inc., Woburn, MA) or HIV-1(IIIB) gp41 (ProSpec-Tany TechnoGene
Ltd., Israel) was covalently immobilised on a CM5 sensor chip using
standard amine coupling chemistry, separately. Samples were
The CD spectrum of each helix (2 lM) was obtained at 25 °C on
same spectropolarimeter (MOS-450). The results are shown in
Fig. 2. D1 and D2 showed positive peaks at approximately 280
nm and negative absorption peaks at approximately 240 nm,
which were in accordance with typical CD spectra of unmodified
duplex D0. G1 and G2 showed positive peaks at approximately
260 nm and negative peaks at approximately 235 nm, which were
also similar to unmodified parallel quadruplex G0. This result indi-
cated that the introduction of hydrophobic PBP either by propyl or
propenyl linker at nucleobase site was with little influence to
duplex and quadruplex structural motif, in other words, the regular
helix structures were not greatly disturbed.
In addition, the slow mobility of helixes than single strand con-
trol T6 and T8 in native polyacrylamide gel electrophoresis (N-
PAGE) further confirmed the formation of the duplex or quadru-
plex structure (Fig. S2).
injected for 1 min at a flow rate of 30
sociation phase of 2 min. The CM5 sensor chip surface was regen-
erated with injection of 2.5 M NaCl at a flow rate of 30
1 min.
lL/min and followed by a dis-
lL/min for
As reported previously, an HIV-1 envelope protein mediated
cell-cell (HL2/3 and TZM-bl cells) fusion assay was used to deter-
mine the anti-fusion activities. HL2/3 were effector cells expressed
HIV-1 Gag, Env, Tat, Rev, and Nef proteins at the cell surface; HL2/3
were obtained through the AIDS Reference and Reagent Program,
NIH, from Barbara K. Felber and George N. Pavlakis. TZM-bl were
target cells expressing CD4 and coreceptors; TZM-bl cells were
obtained through the AIDS Reference and Reagent Program, NIH,
from John C. Kappes, Xiaoyun Wu, and Tranzyme, Inc. The cell-cell
fusion was quantitated by a luciferase assay 8 h after cells mixing
and inhibitor adding.16,17 The results are shown in Table 2. D1, D2,
G1, and G2 all showed anti-fusion activity. D2 demonstrated
slightly greater activity than D1, and G2 also had greater activity
than G1 (p < 0.05), indicating that nucleoside analogue 2 might
contribute more to the activity than 1. Considering the thermal sta-
bility of D2 being higher than D1 (p < 0.05), and G2 being higher
than G1 (p < 0.01), although it was not clear whether the thermal
stability was related to the anti-fusion activity, the discrepancy
both in activity and thermal stability might be explained by the
different spatial freedoms of the hydrophobic group in 1 and 2.
As mentioned above, a former most potent quadruplex inhibi-
tor, d(PBP-TGGGAG)4 (named as G3, synthesized according to
Fig. S1 and Ref. 2), was tested for anti-fusion activity as a control.
In this work, D2 and G2 showed equivalent or slightly greater
The results of the duplex-based inhibitors (D1 and D2) interact-
ing with gp120 and gp41 are shown in Fig. S3 and Table 3. D2 and
D1 both showed an interaction with gp41; however, D2 showed a
much higher absorption and KD value than D1. Besides, D2 showed
a KD value when interacting with gp120, while D1 did not. As D1
and D2 have the same sequence composition and the same
hydrophobic group, PBP, the conformational difference between
D1 and D2 at the PBP connection site might be the key factor to
explain these results. To the best of our knowledge, these findings
are the first to indicate that inhibitors possessing the same duplex
structural motif may bind to their reaction targets differently,
based on the structure and spatial conformation of the hydropho-
bic substituent. In addition, D0, which has the same sequence com-
position as D1 and D2 but does not contain a hydrophobic group,
showed no interaction with gp120 or gp41. This result further indi-
cated the importance of the hydrophobic group for the interaction
between the inhibitor and receptor.
Similar results were observed for G1 and G2. For gp41, G2
demonstrated a higher absorption and KD value than those of G1.
For gp120, G2 also showed an interaction, while G1 did not. It
should be mentioned that the G2 and D2 containing the nucleoside
Table 3
a
The KD (mol/L) values of different molecules.
activity than G3 (1.84 0.12 lM) under the same test condition,
indicating their potential use as novel lead compounds.
No.
gp120
gp41
D1
D2
G1
G2
Very low or not detected
5.25 ꢀ 10ꢁ6
2.80 ꢀ 10ꢁ5
5.37 ꢀ 10ꢁ6
1.27 ꢀ 10ꢁ6
7.67 ꢀ 10ꢁ7
In addition, the natural control, D0 and G0, showed no obvious
activities in our test condition, indicating the importance of
hydrophobic groups. However, very recently, Romanucci reported
the surprising anti-HIV activity of the natural G-quadruplex d
(TGGGAG)4.19 As the kinetic for natural G-quadruplex folding
Very low or not detected
6.61 ꢀ 10ꢁ6
a
KD represents the dissociation constant.

Y. Tang et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 1842–1845
1845
Fig. 3. Inhibition of formation of the HIV-1 gp41 6HB. For lanes 1–7, N36, C34, D1, D2, G1, and G2 at a concentration of 50 mM were used.
analogues 2, had higher activities than G1 and D1 containing the
nucleoside analogues 1. These results further indicated that the
anti-fusion activity could be attributed to different interactions
between the helix inhibitor and the receptors.
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Acknowledgements
Financial support from the Beijing Nova Program
(Z171100001117095) and the National Natural Science Foundation
of China (81573345) is gratefully appreciated.
A. Supplementary data
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sequence: Surprising anti-HIV-1 activity. Eur J Med Chem. 2018;145:425–430.
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2018.04.012.