Recognition of HIV-TAR RNA using neomycin-benzimidazole conjugates

Article abstract of DOI:10.1016/j.bmcl.2013.08.014

Synthesis of a novel class of compounds and their biophysical studies with TAR-RNA are presented. The synthesis of these compounds was achieved by conjugating neomycin, an aminoglycoside, with benzimidazoles modeled from a B-DNA minor groove binder, Hoechst 33258. The neomycin-benzimidazole conjugates have varying linkers that connect the benzimidazole and neomycin units. The linkers of varying length (5-23 atoms) in these conjugates contain one to three triazole units. The UV thermal denaturation experiments showed that the conjugates resulted in greater stabilization of the TAR-RNA than either neomycin or benzimidazole used in the synthesis of conjugates. These results were corroborated by the FID displacement and tat-TAR inhibition assays. The binding of ligands to the TAR-RNA is affected by the length and composition of the linker. Our results show that increasing the number of triazole groups and the linker length in these compounds have diminishing effect on the binding to TAR-RNA. Compounds that have shorter linker length and fewer triazole units in the linker displayed increased affinity towards the TAR RNA.

Full text of DOI:10.1016/j.bmcl.2013.08.014

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5689–5693
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Recognition of HIV-TAR RNA using neomycin–benzimidazole
conjugates
Nihar Ranjan a, Sunil Kumar a, , Derrick Watkins b, Deyun Wang c, Daniel H. Appella c, Dev P. Arya a,b,
⇑
a Laboratory of Medicinal Chemistry, Department of Chemistry, Clemson University, Clemson, SC 29634, United States
b NUBAD LLC, 900 B West Faris Road, Greenville, SC 29630, United States
c Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of a novel class of compounds and their biophysical studies with TAR-RNA are presented. The
synthesis of these compounds was achieved by conjugating neomycin, an aminoglycoside, with benzim-
idazoles modeled from a B-DNA minor groove binder, Hoechst 33258. The neomycin–benzimidazole con-
jugates have varying linkers that connect the benzimidazole and neomycin units. The linkers of varying
length (5–23 atoms) in these conjugates contain one to three triazole units. The UV thermal denaturation
experiments showed that the conjugates resulted in greater stabilization of the TAR-RNA than either neo-
mycin or benzimidazole used in the synthesis of conjugates. These results were corroborated by the FID
displacement and tat-TAR inhibition assays. The binding of ligands to the TAR-RNA is affected by the
length and composition of the linker. Our results show that increasing the number of triazole groups
and the linker length in these compounds have diminishing effect on the binding to TAR-RNA. Com-
pounds that have shorter linker length and fewer triazole units in the linker displayed increased affinity
towards the TAR RNA.
Received 25 May 2013
Revised 30 July 2013
Accepted 5 August 2013
Available online 14 August 2013
Keywords:
TAR
Neomycin
Aminoglycosides
tat-TAR inhibition
Benzimidazole
Ó 2013 Elsevier Ltd. All rights reserved.
Since the first reports of HIV-AIDS in the United States in the
early 1980s, research towards its cure have identified an RNA di-
rected strategy which targets the interactions of tat protein with
the Trans Activating Region (TAR) of the viral RNA.1 A 29-mer oli-
gonucleotide, which is a model of a full 59 mer TAR region of the
viral RNA, contains two of the most commonly found structural
features in the RNA-namely the hairpin loop and the short trinu-
cleotide bulge (Fig. 1a). The trinucleotide bulge region has a wide
major groove that is accessed by the tat protein for viral replication
and thus inhibition of this interaction has developed into a viable
approach to stop viral growth.2,3
Several DNA and RNA binders have been investigated to inhibit
tat-TAR interactions. These binders include polyamines (arginina-
mide),3 polyamides,4 peptides,5,6 peptidomimetics,7 intercalators,8
quinoline derivatives,9,10 quinolones,11 DNA minor groove bind-
ers,2 aminoglycosides12–14 and their derivatives.15–17 Neomycin is
an aminosugar (Fig. 1b) that has been known for decades for its
RNA binding.18,19 Neomycin has been shown to inhibit the tat-
TAR interaction by binding to the trinucleotide pyrimidine bulge
of the TAR-RNA14 and has the highest affinity among the selected
aminoglycosides studied.12 Neomycin has been observed to bind
with the TAR-RNA making contacts in the minor groove present
in the lower stem using its ring III and IV while rings I and II have
been found to interact with the trinucleotide bulge (Fig. 1a and b).
The bis-benzimidazole Hoechst 33258, a known B-DNA minor
groove binding molecule, has also been shown to interact with
the TAR-RNA at a site opposite to the bulge region where it recog-
nizes the helical region below the hairpin loop (Fig. 1a). RNAase A
footprinting analysis has suggested that Hoechst 33258 binds to
GCUCU bases of the TAR RNA in the upper stem.2
Aminoglycosides have emerged as versatile nucleic acid binders
over the past decade.20–26 Several aminoglycoside conjugates have
been synthesized27–30 and shown to have enhanced binding to
variety of DNA,31–34 RNA,35 and DNA:RNA hybrid36 structures.
We have recently reported recognition of TAR-RNA using a series
of dimeric neomycin conjugates.37,38 The neomycin dimers have
shown significant enhancement in the protection of MT-2 cells
from the cytopathic effects of HIV infection in comparison to
neomycin alone.37 These studies have opened new avenues for
multi-valent approaches for the recognition of TAR RNA by amino-
glycoside based small molecules.
Abbreviations: UV, ultra violet; FID, fluorescent intercalators displacement; CD,
circular dichroism; TAR, trans activating region; NMR, nuclear magnetic resonance;
MALDI-TOF, matrix assisted laser desorption and ionization-time of flight; NA, not
available; ND, not determined.
⇑
Corresponding author. Tel.: +1 (864) 656 1106; fax: +1 (864) 656 6613.
E-mail address: dparya@clemson.edu (D.P. Arya).
Present address: Department of Molecular Biophysics and Biochemistry, Yale
University, CT 06520, United States.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.08.014

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N. Ranjan et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5689–5693
Neomycin
(a)
(b)
NH2
Hairpin Region
O
G G
OH
OH
I
U
G
A
H2N
C
NH2
O
II
OH
O
C
G
NH2
O
5'''
G C
A U
U G C
HO
III
C
NH2
OH
O
U
Upper Stem
A U
Bulge region
G C
A U
C G
IV
H2N
O
OH
OH
G C Lower Stem
G C
5' 3'
NH2
TAR RNA
(c)
O
OH
OH
H2N
N
NH
2O
O
O
NH2
Spacer
O
N
H
OH
N
N
NH2
OH
O
Neomycin-benzimidazole conjugate
O
H2N
OH
OH
Figure 1. (a) Illustration of 29 mer short oligonucleotide mimic of the TAR-RNA. (b,c) Chemical structures of neomycin and neomycin–benzimidazole conjugates.
In our continuing effort to develop novel small molecules for
TAR RNA recognition, we herein report a series of neomycin–benz-
imidazole conjugates modeled from the bisbenzimidazole dye
Hoechst 33258. Hoechst 33258 has been shown to interact with
the TAR RNA through intercalative binding.39 However, the planar
surface of Hoechst 33258 is wider than that of RNA base pairs.
Therefore, a smaller benzimidazole was hypothesized to be a more
complimentary surface that favors facile entry between the helical
bases in addition to reducing the molecular weight and polarity of
the conjugated ligand.
The monobenzimidazole derivatives modeled from Hoechst
33258 (referred to as benzimidazoles henceforth), like its parent
structure, penetrates easily in the cell and can be fluorescently de-
tected (unpublished results). The benzimidazoles (DPA 101 and
DPA 102, Scheme 1) lack one benzimidazole unit in comparison
to Hoechst 33258 which is a bisbenzimidazole dye. These smaller
benzimidazoles are much easier to synthesize and purify than
the larger and more polar bisbenzimidazoles. The synthesis of neo-
mycin–benzimidazole conjugates was achieved using a divergent
strategy. The neomycin units (azide40,41/alkyne units) and the
benzimidazole units (containing the complementary alkyne/azide)
were prepared separately (Supplementary data Scheme S1).
Scheme shows the synthesis of alkyne/azide terminated
1
benzimidazoles. The synthesis of clickable benzimidazoles rested
on selective incorporation of the terminal alkyne or azide units.
DPA 101 was synthesized in three steps from commercially
available 5-chloro-2-nitroaniline (1) as shown in Scheme 1. This
transformation was achieved by substitution of the chloro
substituent in 1 with N-methyl piperazine in the presence of
K2CO3. The synthesized compound 5-(4-methylpiperazin-1-yl)-2-
nitroaniline was then hydrogenated to its corresponding diamine42
2 followed by condensation with 4-(prop-2-ynyloxy) benzalde-
hyde (3)43 in the presence of Na2S2O5 to afford the desired ligand
DPA 101. Similarly, DPA 102 was synthesized by condensation of
the above mentioned diamine (2) with 4-(2-azidoethoxy) benzal-
dehyde (4)44 in the presence of the oxidant sodium metabisulfite.45
DPA 101 and DPA 102 were then reacted with various bisazides
(5a–d) or bisalkynes (6a–e) to afford the azide or alkyne termi-
nated benzimidazole derivatives (DPA 103–DPA 112) containing
various linker lengths. The ligands DPA 103–DPA 112 were then
reacted with Boc protected neomycin azide (7) or Boc protected
neomycin alkyne (8) (see Supplementary data S2a and b for the
N
N
N3
n
(v)
O
O
N
N
N
N
N
H
N
NH2
H
NH2
N
N
N
n = 0 (
), n = 4 (
)
DPA 105
DPA 103
NO2
(i,ii)
DPA 101
NH2
(iii)
n = 5 (DPA 106), n = 9 (DPA 107)
N
X
Cl
N
N
N
(iv)
N
(vi)
O
N
N
O
N3
N
H
N
(1)
( )
2
N
H
N
N
N
X =
DPA 108 X = -CH2OCH2-, DPA 109
,
DPA 102
, n = 3 (
n = 6 (
), n = 4 (
),
DPA 111
DPA 110
X =
)
DPA 112
n
Scheme 1. Reagents and conditions: (i) DMF, N-methylpiperazine, 100 °C, 5 h, 60%; (ii) Pd–C (10%), ethanol, H2, 6 h, qaunt.; (iii) 4-(Prop-2-ynyloxy) benzaldehyde (3),
Na2S2O5, H2O, reflux, 12h, 65%; (iv) 4-(2-azidoethoxy) benzaldehyde (4), Na2S2O5, H2O, reflux, 12h, 82%; (v) sodium ascorbate, copper(II) sulfate, 50 fold excess bisazides [N3–
CH2–(CH2)n–N3; n = 0(5a), n = 4(5b), n = 5(5c), n = 9(5d)], ethanol, room temperature, overnight,65–80%; (vi) sodium ascorbate, copper(II) sulfate, 50-fold excess bisalkynes
[1,4 diethynyl benzene (6a), propargyl ether (6b), HC–(CH2)n–CH; n = 3(6c), n = 4(6d), n = 6(6e)] ethanol, temperature, overnight, 75–90%.

N. Ranjan et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5689–5693
5691
Table 1
reaction scheme) under click chemistry conditions leading to the
formation of Boc protected neomycin benzimidazole conjugates
(DPA 113–123). Deprotection of the Boc groups was achieved with
4 M HCl in dioxane (Scheme 2, S2a and b). The purity of new com-
pounds was checked with TLC and 1HNMR; the identity of the syn-
thesized compounds was further corroborated by (1H/13C) NMR
and mass (MALDI-TOF) spectroscopic analysis. A reaction scheme
for the synthesis of DPA 123 and the structures of all ligands syn-
thesized are displayed in Scheme 2.
These neomycin–benzimidazole conjugates (DPA 113–123)
were then evaluated for their affinity towards TAR RNA. The rela-
tive affinities of these ligands were evaluated by two methods. In
the FRET analysis,46 a tat peptide containing fluorescein and rhoda-
mine as FRET pair was complexed with TAR RNA. The tat-TAR com-
plex was incubated with ligand of interest to displace bound tat
protein which is reflected by the quenching of fluorescein emis-
sion. The tat-TAR inhibitory affinity can be expressed as IC50 which
reflects a ligand’s ability to displace 50% bound tat peptide from
TAR. In another assay based on fluorescent intercalator displace-
ment assay (FID),47 a TAR bound intercalator (EtBr) was succes-
A table showing the DC50, IC50 and thermal stabilization afforded by ligands used in
the study
Ligand
Linker
No. of triazoles DC50 (nM) IC50 (nM)
DTm (°C)
lengtha
Neomycin NA
NA
NA
1
1474 79
ND
2.0
1.0
5.6
DPA 101
DPA 123
DPA 119
DPA 120
DPA 121
DPA 118
DPA 122
DPA 113
DPA 115
DPA 116
DPA 117
NA
5
ND
33
272
8
11
12
13
13
15
17
20
21
23
2
2
2
2
2
3
3
3
78
83
140
180
81
184
150
200
180
1285 155 3.0
337 43
507
3.4
4.1
3.0
5.4
2.5
2.1
3.8
9
379 20
949 92
411 51
851 80
1368 50
3
1380 150 4.0
a
The linker length was assigned by counting the number of main chain atoms
between the 5000-carbon on ribose ring (ring III, see Fig. 1) of neomycin and oxygen
atom on the phenolic end of the benzimidazole.
NHR
O
HO
NHR
NHR
HO
N
NHR
O
O
NHR
O
O
N3
N
OH
N
+
H
N
DPA 101
OH
7
O
R = Boc
(i, ii)
O
NHR
NH2
OH
OH
O
HO
HO
NH2
NH2
O
N
O
NH2
N
O
N
OH
O
H
N
OH
NH2
N
O
N
N
O
DPA 123
NH2
OH
OH
N
N
NH2
N
N
N
O
Benzimidazole
HO
HO
O
4
N
N
NH2
Neomycin
N
N
NH2
O
N
O
n = 0, DPA 113; n =4, DPA 115
n
N
NH2
O
DPA 116
DPA 117
; n=9,
n = 5,
OH
N
N
N
O
OH
Benzimidazole
NH2
OH
O
Neomycin
Neomycin
N
O
NH2
DPA 118
OH
N
N
N
N
N
N
Neomycin
N
N
Benzimidazole
Benzimidazole
O
O
H
N
N
DPA 119
N
N
N
Neomycin
N
N
N
O
Benzimidazole
m
DPA 120
DPA 121;
DPA 122
m = 4,
m = 0,
; m =1,
Scheme 2. Reagents and conditions: (i) Sodium ascorbate, copper(II) sulfate, ethanol, room temperature, overnight; (ii) dichloromethane, 4 M HCl in 1,4 dioxane, 49%
cumulative yield for two steps.

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N. Ranjan et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5689–5693
triazole units (DPA 113–DPA 117) as displayed by their DC50 and
IC50 values. DPA 123, with a short linker and a single triazole,
emerges as the best ligand in binding to the TAR with DC50 and
IC50 values of 33 and 257 nM, respectively. To study the selectivity
of DPA 123 for TAR over other HIV RNA targets, we investigated its
ability to affect the Rev-RRE (rev response element) complexation.
We observed that DPA 123 displays much better ability to affect
tat-TAR binding, when compared to its ability to displace Rev from
RRE, another RNA target of HIV virus. The IC50 value for inhibition
of RRE RNA-HIV-1 Rev peptide complex was (768 309) nM (see
Supplementary data), significantly higher than IC50 value for inhi-
bition of tat-TAR complexation.
The nucleic acid binding of these ligands was also evaluated
using thermal denaturation experiments. The thermal denatur-
ation temperatures are listed in Table 1. In the presence of benz-
imidazole DPA 101 alone, the thermal stabilization afforded was
1.0 °C while the same with neomycin was 2.0 °C. All conjugates
afforded higher thermal stabilization of TAR RNA than any of the
building blocks used to build the conjugates, with DPA 123 provid-
ing the highest thermal stabilization (5.6 °C). The highest thermal
stabilization afforded by DPA 123 also corroborated the results ob-
tained from FID experiments and the tat-TAR inhibition assay. In
some cases, however, we did not observe correlation between
Figure 2. A representative plot showing the decrease in the fluorescence of
fluorescein labeled tat-TAR complex upon ligand (DPA 123) binding of tat-TAR RNA
complex. A 1:1 mixture of tat peptide (100 nM) and TAR RNA (100 nm) were mixed
in Tris buffer containing 50 mM Tris, 20 mM KCl at pH 7.4. The resulting complex
was titrated with DPA 123 until no change in fluorescence was observed. The
resulting binding isotherm was fitted with a dose response curve using Origin 5.0
(see Supplementary data for fitting details).
the thermal binding data and the DC50/IC50 values. As these DC50
/
IC50 values are obtained by evaluating two entirely different as-
says, the lack of correlation could be attributed to the differences
in the binding modes of the two probes with TAR RNA. EtBr inter-
calates nonspecifically whereas the fluorescein tagged tat protein
binds more specifically in a 1:1 fashion. The thermal denaturation
profiles of the RNA, on the other hand, reflect a more complex tem-
perature dependence of ligand:RNA equilibrium constants and are
not expected to always correlate with isothermal room tempera-
ture Kd or IC50 measurements.
sively displaced by the addition of ligand of interest. The resulting
binding isotherm can then be analyzed to obtain DC50 values (DC50
corresponds to the concentration of the ligand required to displace
50% of the bound intercalator). The results of these assays are sum-
marized in Table 1 and a representative plot is shown in Figure 2.
The data shown in Table 1 reveals that affinity of these ligands to-
wards TAR is dependent on both linker length and their composi-
tion. In general, ligands with shorter linker and fewer triazoles
(e.g. DPA 123, DPA 120) displayed a stronger affinity to TAR RNA
in comparison to conjugates with longer linker length and more
We also performed circular dichroism studies with DPA 123 to
investigate the ligand induced changes in the conformation of the
TAR RNA. As shown in Figure 3, the titration of DPA 123 led to
Figure 3. CD titration profile of TAR RNA with DPA 123. The ligand was added serially to the RNA solution in small increments as displayed on the graph. After each
successive addition, the nucleic acid ligand-complex was stirred and equilibrated for five minutes before the spectrum was recorded. Each spectrum shown in the figure is an
average of three scans. The experiments were performed in cacodylate buffer containing 10 mM sodium cacodylate, 0.5 mM EDTA, 100 mM KCl at pH 6.8 (T = 20 °C). The
boxed regions have been expanded for clarity.

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Supplementary data
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