Delivery of 2-5A cargo into living cells using the Tat cell penetrating peptide: 2-5A-tat

Article abstract of DOI:10.1016/j.bmc.2006.07.058

2′,5′-Oligoadenylate tetramer (2-5A) has been chemically conjugated to short HIV-1 Tat peptides to provide 2-5A-tat chimeras. Two different convergent synthetic approaches have been employed to provide such 2-5A-tat bioconjugates. One involved generation

Full text of DOI:10.1016/j.bmc.2006.07.058

Bioorganic & Medicinal Chemistry 14 (2006) 7862–7874
Delivery of 2-5A cargo into living cells using the Tat cell
penetrating peptide: 2-5A-tat
Longhu Zhou,a Chandar S. Thakur,b Ross J. Molinaro,b Jayashree M. Paranjape,b
Rieuwert Hoppes,c Kuan-Teh Jeang,c Robert H. Silvermanb and Paul F. Torrencea,d,
*
aDepartment of Chemistry and Biochemistry, Northern Arizona University, Flagstaff, AZ 86011-5698, USA
bDepartment of Cancer Biology, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA
cMolecular Virology Section, Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, MD 20892, USA
dDepartment of Chemistry, Cleveland State University, Cleveland, OH 44115, USA
Received 8 June 2006; accepted 27 July 2006
Abstract—20,50-Oligoadenylate tetramer (2-5A) has been chemically conjugated to short HIV-1 Tat peptides to provide 2-5A-tat
chimeras. Two different convergent synthetic approaches have been employed to provide such 2-5A-tat bioconjugates. One involved
generation of a bioconjugate through reaction of a cysteine terminated Tat peptide with a a-chloroacetyl derivative of 2-5A. The
second synthetic strategy was based upon a cycloaddition reaction of an azide derivative of 2-5A with a Tat peptide bearing an
alkyne function. Either bioconjugate of 2-5A-tat was able to activate human RNase L. The union of 2-5A and Tat peptide provided
an RNase L-active chimeric nucleopeptide with the ability to be taken up by cells by virtue of the Tat peptide and to activate RNase
L in intact cells. This strategy provides a valuable vehicle for the entry of the charged 2-5A molecule into cells and may provide a
means for targeted destruction of HIV RNA in vivo.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
Interferon stimulated genes encode proteins which medi-
ate all of the biological effects of interferons.1,2 One set of
interferon stimulated genes comprise the 2-5A* synthe-
tase enzymes which produce a short oligonucleotide
called 2-5A (1, Fig. 1). This molecule plays a pivotal role
in the 2-5A system. Exposure of a cell to interferon induc-
es enhanced levels of 2-5A synthetase. Then dsRNA,
formed as an intermediate in viral replication, activates
the synthetase to generate 2-5A from ATP. 2-5A provides
an unambiguous signal to initiate RNA decay in mamma-
lian cells through activation of latent 2-5A-dependent
Abbreviations: 2-5A refers to a 20, 50-phosphodiester linker oligoade-
nylate of varying chain length of general formula (p50A20p)n50A; ds-
RNA, double-stranded ribonucleic acids; RNase L, ribonuclease L;
Tat, full-length peptide; tat, generic for truncated versions of Tat;
Figure 1. 2-5A tetramer, p50A20p50A20p50A20p50A.
2-5A-tat, generic for conjugates of 2-5A with tat peptides.
Keywords: Ribonuclease L; Interferon; Cycloaddition; Sulfhydryl
alkylation; RNA; 20,50-Oligoadenylate.
RNase L that degrades mRNA and rRNA, thereby inhib-
iting translation. RNase L is thus converted from a silent
to an active form by binding with 2-5A.
*
Corresponding author. Tel.: +928 523 0298; fax: +928 523 8111;
e-mail: Paul.Torrence@nau.edu
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.07.058

L. Zhou et al. / Bioorg. Med. Chem. 14 (2006) 7862–7874
7863
A major barrier to the fruitful application of 2-5A to
therapeutic situations has been the difficulty of delivery
to cells. Adequate uptake of the 2-5A oligonucleotide re-
quires treatments with hypotonic salts, calcium co-pre-
cipitates, or cationic lipids, none of which are suitable
for therapeutic applications.1 This paper reports a solu-
tion to this latter uptake dilemma and may provide an
approach for targeting of RNase L to HIV RNA in cul-
tured cells and potentially in vivo. This strategy is based
upon the molecular biology of HIV replication.
tion first was studied. AMP (2) was periodate oxidized
to the dialdehyde which was reacted with 3-azidopro-
panamine to yield the corresponding Schiff’s base. This
was reduced by sodium cyanoborohydride directly with-
out isolation. The product N-azidopropylmorpholine (3)
was obtained in 82% yield after HPLC purification, and
its structure was corroborated by proton NMR, phos-
phorus NMR, and high-resolution mass spectrometry.
To generate the corresponding modification to 2-5A,
similar reaction conditions were employed to provide
the 2-5A–propyl azide adduct (4) from 1 in 45% yield
after HPLC purification. Structure corroboration was
by MALDI-TOF mass spectrometry: (m/z, calcd for
C43H56N24O23P4: 1400.9; found 1401). The 1,3-dipolar
cycloaddition reaction of azido compounds with N-hexe-
nyl-modified tat peptide 5 was carried out smoothly in
the presence of CuSO4 (1 equiv) and sodium ascorbate
(2 equiv) at room temperature in water for 24 h. For
the reaction with azido-modified AMP (3), the tat adduct
(6) was obtained (after purification) in 60% yield. The
product’s structure (6) was confirmed by MALDI-TOF
mass spectrometry: (m/z, calcd for C83H152N45O20P:
2131; found 2131). The reaction between the azido-mod-
ified 2-5A tetramer 4 and tat peptide 5 was carried out
under similar conditions. The product 2-5A-tat (7) was
corroborated by MALDI-TOF mass spectrometry:
(m/z, calcd for C113H188N60O38P4: 3118; found 3118)
(see Fig. 2).
Interaction of the TAT protein with HIV RNA. For over
a decade, it has been known that the HIV Tat regulatory
protein stimulates transcription of the HIV RNA by
binding to the transactivation-responsive region (TAR)
located in the 50-terminus of the HIV RNA, just down-
stream of the transcriptional initiation site in the HIV
genome. Although the Tat protein is 101 amino acids
in length, the C-terminal domain, which contains a nine
amino acid arginine-rich region, binds to the TAR RNA
with the avidity of the full-length Tat.3 Additional stud-
ies have shown that nonapeptides (such as but not lim-
ited to Tat amino acids 49–57, RKKRRQRRR) can
bind to TAR RNA with affinity approximating the com-
plete Tat protein.4 The critical role played by the Tat
regulatory protein in HIV replication has made it an
attractive target for therapeutic interference in HIV/
AIDS.5–21 since interference with the Tat–TAR interac-
tion leads to inhibition of HIV replication.
The Tat protein and the truncated constituent arginine-
rich peptide GRKKRRQRRRPPQ, when fused to
bioactive peptides or proteins, are able to deliver these
molecules into intact cells. These so-called membrane
translocation signal (MTS) or cell-penetrating peptides
also effect internalization of peptide nucleic acids
(PNAs), plasmid DNA, antisense oligonucleotides,
paramagnetic cations, and superparamagnetic iron
oxide particles. Embedded in the MTS motif of Tat pro-
tein is the TAR recognition sequence, RKKRRQRRR.
The Tat activation domain, which promotes HIV repli-
cation and angiogenesis, occurs at Tat residues 21–40,
outside the cell-penetrating peptide sequence.
To obtain a 2-5A-tat conjugate by an independent alter-
nate route, the well-established nucleophilic attack of a
cysteine sulfhydryl on a chloroacetyl a-carbon was
employed (Fig. 3). Thus, tat peptide was elongated by
a cysteine group at the amino-terminus to provide com-
pound 14. To provide a 2-5A derivative with a reactive
chloroacetyl group, a model reaction of AMP with cys-
teine-functionalized Tat peptide was investigated. AMP
first was periodate-oxidized to the dialdehyde which was
then reacted with p-[(a-chloroacetyl)amino]benzylamine
(11). The latter was prepared in two steps from tert-Boc
protected p-amino-a-aminotoluene (8) and a-chloroace-
tyl chloride (9). The corresponding Schiff’s base interme-
diate from 11 and periodate-oxidized AMP was reduced
in situ with sodium cyanoborohydride, Na(CN)BH3.
After HPLC purification, this product (12) was obtained
in a yield of 85%. Its structure was corroborated by pro-
ton and carbon NMR and by electrospray mass spec-
trometry. These successful reaction conditions were
then applied to 2-5A tetramer (1) to provide the desired
2-5A-chloroacetyl conjugate (13) that, after HPLC puri-
fication, gave an experimental high resolution mass of
1499.443, in excellent agreement with the predicted mass
of 1499.279. Reaction of the chloroacetyl AMP (12) and
2-5A derivatives (13) with the cys-modified tat peptide
was accomplished under oxygen-free conditions to yield
both the AMP-tat conjugate (15) and the 2-5A-tat con-
jugate (16). The synthesis of a fluorescently labeled ver-
sion of conjugate 16 was achieved using essentially the
same technique by starting with a custom synthesized
peptide Cys-Gly-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-
Arg-Arg-fluorescein. The product’s structure (Fig. 4,
compound 17) was confirmed by MALDI-TOF.
Herein we describe the syntheses of two different 2-5A-
tat peptide conjugates that can be transported into
intact cells, activate RNase L, and effect the degradation
of cellular RNA.
2. Results
2.1. Synthesis of 2-5A-tat bioconjugates
Two different approaches were employed to join 2-5A
tetramer to Tat peptide. First, a chemoselective ligation
reaction was used; namely, 1,3-dipolar cycloaddition
employing cuprous ion catalyst. This methodology has
been widely applied.22–27 The Cu(I)-catalyzed reaction
of terminal alkynes with azides has many advantages
including very mild reaction conditions, high yields,
high chemoselectivity, and exclusive regioselectivity.
To apply it in the present circumstances, a model reac-

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L. Zhou et al. / Bioorg. Med. Chem. 14 (2006) 7862–7874
Figure 2. Synthesis of an AMP-tat and a 2-5A-tat conjugate through generation of a azide-functionalized AMP or 2-5A derivative followed by their
respective conjugations to an alkyne-functionalized tat peptide using Cu(I)-catalyzed 1,3-dipolar cycloaddition.
2.2. Activation of homogeneous human RNase L by
2-5A-tat peptide conjugates
the other end of the parent chain. RNA cleavage exper-
iments were carried out using several times of incuba-
tion with enzyme and activator (Table 1). Figure 5
displays the results of the experiment in which the incu-
bation time was 120 min. First, it is clear from Table 1
that the results at different times of incubation did not
vary significantly except for the somewhat higher values
obtained at 30 min. Comparison among the different
conjugates and 2-5A was based upon an average of
the four time determinations (column labeled ‘Relative’
in Table 1). Second, from Table 1 and Figure 4, it is
clear that the 2-5A-tat peptide chimeras 7 and 16 were
able to activate RNase L albeit with less potency than
parent 2-5A tetramer itself. Thus, the conjugate (16)
with the thioether linkage bond displayed an average
EC50 of 110 nM with the standard 2-5A tetramer (1)
showing an average EC50 of 2.5 nM. The conjugate (7)
with the triazole ring linkage moiety displayed an aver-
age EC50 of 300 nM. Thus, the thioether linkage-based
conjugate (16) was 44 times less effective as an RNase
L activator compared to parent 2-5A (1). The 2-5A-tat
peptide conjugate 7 was 120-fold less active than parent
2-5A (1) and threefold less active than conjugate 16.
When the simple AMP-Tat peptide adducts (com-
pounds 6 and 15) were evaluated in this RNA cleavage
assay, they were totally devoid of activity even at
300 nM, the highest concentration tested (data not
shown).
The ability of the 2-5A-tat peptide conjugates (7 and 16)
to activate RNase L was determined by a fluorescence
resonance energy transfer (FRET) method.28 This assay
employed recombinant human RNase L produced in in-
sect cells from a baculovirus vector and purified to
homogeneity by fast protein liquid chromatography
(FPLC).29 The cleavable substrate was composed of a
36 nucleotide synthetic oligoribonucleotide sequence de-
rived from respiratory syncytial virus with the fluoro-
phore, FAM, at the 50-terminus and black hole
quencher-1 (BHQ-1), at the 30-terminus (synthesized at
Integrated DNA Technologies). The RNA sequence
contains several cleavage sites for RNase L (UU or
UA). This RNA substrate does not provide a putative
target sequence for conjugates 7 or 16, but is used in this
context to evaluate the relative ability of these conju-
gates to activate human RNase L in comparison to
the unmodified activator, 2-5A tetramer itself. Relative
fluorescence as a monitor of RNase L enzymatic activity
is plotted versus concentration of either pure 2-5A tetra-
mer as a standard, or against the concentration of the
respective 2-5A-tat peptide conjugates 7 or 16 (Fig. 5).
Increasing fluorescence corresponds to RNA cleavage
as the quencher moiety is released as one fragment of
the RNA chain and cannot quench the fluorophore at

L. Zhou et al. / Bioorg. Med. Chem. 14 (2006) 7862–7874
7865
Figure 3. Synthesis of 2-5A-tat peptide via sulfhydryl-chloroacetyl ligation scheme.
2.3. Uptake of fluorescently labeled 2-5A-tat peptide
conjugate by intact cells (Fig. 6)
In Figure 6, panel A shows that fluorescent-tat is sur-
rounding the cells but is not yet taken up. In (B) fluores-
cent-tat has entered the cytoplasm and a slight
accumulation in the nucleus is observed (1 h). In panel
(C), after 2 h, fluorescent-tat appears to be most concen-
trated in the nucleolus and can also still be found in the
cytoplasm and nucleus. In panel (D), fluorescent 2-5A-
tat (compound 17) readily entered the cells and is ob-
served in the cytoplasm, nucleus, and nucleolus. Also,
it surrounds the cells in a similar manner as fluores-
cent-tat at the 10 min timepoint. In panel (E), After
one hour, the highest concentration of fluorescent 2-
5A-tat is in the nucleolus, the lowest concentration is
seen in the cytoplasm. The cells start to round up at this
stage. In panel (F), after two hours, the cells are mostly
rounded up, fluorescent 2-5A-tat concentration in the
cytoplasm has dropped and the compound is now clear-
ly most concentrated in the nucleolus. The rounding up
of cells seen for compound 17 may be related to the use
We first monitored by confocal microscopy to confirm
that both fluorescent-tat and fluorescent 2-5A-tat (Com-
pound 17) entered cells. Figure 6 provides visual confir-
mation that indeed both molecules are taken into cells
progressively over time. Both fluorescent-Tat and fluo-
rescent 2-5A-tat (compound 17) were applied to the cells
at a final concentration of 1 lM and images obtained at
the indicated times. Images of cellular uptake of com-
pounds were acquired with a Zeiss LSM410 laser scan-
ning confocal microscope equipped with a mixed-gas
laser (488-, 568-, and 647-nm excitation lines) in sequen-
tial scans and then analyzed with Microcosm Renais-
sance 410 software. Overlays of the acquired images
with corresponding differential interference contrast
(DIC) microscopy were generated using Adobe Photo-
shop CS software.

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L. Zhou et al. / Bioorg. Med. Chem. 14 (2006) 7862–7874
Figure 5. 2-5A-tat fusion molecules activate recombinant RNase L
in vitro. Tetrameric 2-5A (circles), and 2-5A-tat peptide compounds 7
and 16, (square and diamond symbols, respectively) were incubated at
20 ꢁC for 120 min with purified recombinant RNase L in the presence
of a cleavable FRET RNA probe (Section 5). The relative fluorescence
units (RFU) (abscissa), a measure of RNase L activity, is plotted as
function of compound concentration (ordinate). Error bars represent
standard deviations.
rRNA at unique sites in intact ribosomes.30 Therefore,
the endpoint for activation of RNase L in these experi-
ments was rRNA cleavage. All treatments were for 6 h
and were performed in duplicate. To provide an appro-
priate control, 2-5A tetramer (1) at a concentration of
5 lM was introduced into cells using the commercial
product FuGene 6 (Roche Diagnostics), a multi-compo-
nent lipid-based (non-liposomal) transfection reagent.
On the other hand, the 2-5A-tat peptide conjugate 16
was simply added to the cells in the absence of FuGene
or any other uptake enhancer. As is apparent from Figure
7, 2-5A transfection resulted in the characteristic cleav-
ages of 28S and 18S (lanes 5 and 6). No such rRNA
cleavage products were visible in the RNA isolated from
untreated or FuGene-treated cells. The same distinct
and characteristic rRNA cleavage products obtained
by 2-5A transfection also were apparent in the RNA iso-
lated from cells treated with 2-5A-tat peptide conjugate
16 at 250 and 500 nM concentrations (compare lanes 5
and 6 with lanes 9–12). Thus, conjugate 16, when
applied alone to these HeLa cells, was able to enter the
cells and then effect activation of RNase L leading to
rRNA degradation. The reduced level of rRNA cleavage
obtained with compound 16 as compared to 2-5A could
be due to the lower concentrations of compound 16 that
were used and to a reduced ability of this molecule to
activate RNase L (Fig. 5). The relative inactivity of com-
pound 16 for cleaving rRNA is actually an advantage in
that it could provide a therapeutic window to treat HIV
infections with minimal toxicity (Section 3).
Figure 4. Structure of fluorescent 2-5A-tat, compound 17.
of 1 lM concentration of this RNase L activator since
even 250 lM 2-5A-tat was able to effect rRNA degrada-
tion (vide infra Fig. 7).
2.4. Activation of RNase L in intact cells by 2-5A-tat
peptide conjugate
3. Discussion
Through the use of two very different conjugation chem-
istries, 2-5A tetramer has been ligated to Tat peptide.
One ligation chemistry involved an SN2 reaction of the
terminal cys of a modified Tat peptide with a 2-5A deriv-
ative modified with an a-chloroacetyl group. The second
To determine the ability of the 2-5A-tat conjugate, com-
pound 16, to both enter cells and activate intracellular
RNase L, a HeLa cell line that overexpressed RNase
L was employed (Section 5). RNase L is known to cleave

L. Zhou et al. / Bioorg. Med. Chem. 14 (2006) 7862–7874
Table 1. RNase L activation at different times of incubation
7867
a
EC50 (nM)
Compound
At 30 min
At 60 min
At 90 min
At 120 min
Averageb
Relativec
2-5A (1)
3
200
350
2.7
80
2.2
80
2.1
90
2.5
110
300
1
44
16
7
300
300
280
120
a EC50, concentration (nM) of compound that was required to activate RNase L such that 50% of RNA substrate was degraded as evidenced by
change in fluorescence.
b Average EC50, average of all 4 determinations at different times.
c Relative EC50, ratio of EC50s of two activators where the potency of 2-5A tetramer is set arbitrarily to unity: EC50 (conjugate)/EC50 (2-5A). Thus,
relative EC50 is a measure of how much more conjugate is required to effect a 50% degradation of RNA as compared to 2-5A itself.
Fluorescence
A
DIC
Overlay
B
Fluorescence
DIC
Overlay
Figure 6. Proof that both 2-5A-tat and tat can be taken up into cells. Figure 6 monitors uptake over time from 10 mim to 2 h. Apparent is first the
attachment to the rim of the cell, and then entry of a more robust population of fluor-molecules into the cell and then finally into the nucleus. Panels
A, B, and C refer to control fluorescent-Tat peptide at 10 min, 1 h, and 2 h, respectively. The remaining panels (D, E, F) refer to the fluorescent 2-5A-
tat (compound 17) at the same time points. The top 3 images of each panel are a higher magnification (2.5·) of the lower 3 images.

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L. Zhou et al. / Bioorg. Med. Chem. 14 (2006) 7862–7874
Fluorescence
C
Overlay
DIC
D
Fluorescence
DIC
Overlay
Figure 6. (continued)
ligation strategy employed 1,3-dipolar cycloaddition of
a propargyl-cys-modified Tat peptide with a 2-5A deriv-
ative bearing a 20-terminal aliphatic azido moiety. Both
coupling reactions proceeded in good to excellent yields
(60–80%).
acids or nucleic acid analogues have been limited to liga-
tion through formation of disulfide bonds.
When targeting P-glycoprotein or aberrant splicing in
the Luciferase gene, Astriab-Fisher and co-workers
conjugated Tat and Antennapedia peptides with
oligodeoxyribonucleotide containing 50-terminal 2-
thiopyridyl groups.5,31 A similar approach was em-
ployed by Allinquant et al.32 in their generation of
Attennapedia peptide coupled to antisense oligode-
oxyribonucleotides against amyloid precursor protein
and also by Troy et al.33 for Attennapedia-coupled
antisense oligonucleotides against Cu/Zn superoxide
dismutase. Antopolsky et al.34 ligated oligonucleotide
phosphorothioates to the signal sequence of the
In contrast to the problems of precipitation of aggrega-
tion seen in the preparation of other CPP-oligonucleo-
tide conjugates, we observed no such issues with
2-5A-tat, perhaps due to the much shorter electronega-
tive oligonucleotide to which the electropositive Tat
peptide was ligated.
Thus far, with but one exception, the synthetic
approaches to CPP and Tat peptide-conjugated nucleic

L. Zhou et al. / Bioorg. Med. Chem. 14 (2006) 7862–7874
Fluorescence
Overlay
7869
E
DIC
F
Fluorescence
DIC
Overlay
Figure 6. (continued)
Kaposi fibroblast growth factor by disulfide exchange
with a mercaptoalkyl group tethered oligonucleotide
phosphorothioate and a peptide containing a pyridyl
disulfide functionality. Gait and co-workers35 used
the disulfide approach to prepare conjugates of
copolymeric locked nucleic acids with 20-O-methylat-
ed RNA with Tat peptide, transportan, penetratin,
and related peptides. Prater and Miller36 coupled
30-methylphosphonate-terminated 20-O-methyloligori-
bonucleotides to Tat peptide using a similar disulfide
coupling strategy.
reaction of tritylthio-terminated oligonucleotide with a
nitropyridylsulfenyl chloride.
The only exception to the use of disulfide chemistry in
the pursuit of oligonucleotide-CPP conjugates arises
from the efforts of Prater and Miller36 who prepared a
keto derivative of the Tat peptide by reacting a cys-
teine-terminated peptide with bromoacetone. This
allowed oxime formation with the oligonucleotide that
bore an introduced 50-reactive amino embodied as
4-(2-aminooxyethoxy-2-(ethylureido)quinoline.
Maurel et al.37 recently introduced a variation on the
disulfide chemistry theme by generating a nitropyridyl
disulfide functionalized oligonucleotide through the
The hypothesis has forwarded (e.g., Maurel et al.37) that
disulfide joined conjugates are advantageous because
they are stable in cellular media, but would be severed

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L. Zhou et al. / Bioorg. Med. Chem. 14 (2006) 7862–7874
to be related to a proximity effect due to creation of a
new specific high-affinity binding site (the antisense do-
main of the 2-5A-antisense chimera) on RNase L.41 In
this case, it is clear from Figure 1 that 2-5A-tat possesses
similar RNase L activation competence to previously
described molecules of this genre. As expected, AMP-
S-tat and AMP-N-tat conjugates did not show the abil-
ity to activate RNase L (data not shown). This latter re-
sult is congruent with early reports that three intact
adenine rings are needed for full RNase L binding
ability.1
Figure 7. Compound 16 is able to enter cells unassisted and to activate
intracellular RNase L. HeLa cells overexpressing RNase L were
untreated, mock transfected with Fugene, transfected with trimer 2-5A
using Fugene, or treated with compound 6 without uptake enhancer
(as indicated in the figure). RNA was isolated and separated on an
RNA Chip (Section 5). Positions of intact 28S and 18S rRNA (left)
and specific rRNA cleavage products (arrow to the right of figure) are
indicated. Lanes 1 and 2, untreated cells; lanes 3 and 4, mock treated
cells; lanes 5 and 6, 2-5A (5 lM) plus Fugene; lanes 7 and 8, 125 nM
2-5A-tat; lanes 9 and 10, 250 nM 2-5A-tat; lanes 11 and 12, 500 nM
2-5A-tat.
4. Conclusions
2-5A-tat conjugates provide a solution to one of chief
roadblocks to the use of 2-5A as a therapeutic entity;
namely, ineffective uptake of 2-5A itself. Historically,
heroic and cell-unfriendly methods have been used to
introduce 2-5A oligonucleotides into cells.1 This has lim-
ited the experimental investigation of the biological ef-
fects of 2-5A as such methods are cumbersome, toxic
to cells, and often of problematic reproducibility. The
existence of a membrane-transportable version of 2-5A
should permit a wider investigation of the biochemistry
of 2-5A and RNase L. In addition, this conjugation ap-
proach might be extended to 2-5A-antisense itself to ad-
vance the potential of 2-5A-antisense as both a drug
discovery target validation tool and as a potential
therapeutic.
to component oligonucleotide and peptide in the reduc-
ing environment of the cells. From the perspective of the
eventual hoped-for in vivo therapeutic application of
nucleic acid- or nucleic acid analogue-CPP constructs,
disulfide linkages will likely be counterproductive.
Kwok et al.38 studied the biodistribution, metabolic sta-
bility, and transient gene expression of sulfhydryl cross-
linked peptide DNA formulations. They found that
despite significant reductive stability of such disulfide-
linked nucleic acid-peptides as measured in vitro, the
intracellular reducing environment of the liver released
DNA prematurely resulting in rapid metabolism. Thus,
alternative more stable ligation chemistries, as devel-
oped herein, may be valuable additions to the bioconju-
gation schemes available for nucleic acid-CPPs, when
eventual therapeutic endpoints are involved.
Another potential extension of this work applies to HIV
therapeutics. While antiretroviral drugs effective against
the HIV reverse transcriptase and protease enzymes rep-
resent milestones in the therapy of HIV infection, and
have converted HIV infection into a chronic disease that
requires lifelong therapy, highly active antiretroviral
therapy (HAART),42 once regarded by some as a poten-
tial cure for HIV infection, is limited by emerging prob-
lems43–45 of HIV infection.46 Such considerations make
it imperative that new antiretroviral drugs be developed
to provide less toxic treatments and to allow options for
salvage therapy.47,48 Ideally these agents should possess
mechanisms of action that depart from the established
reverse transcriptase and protease inhibitors. Indeed,
promising options are being pursued including integrase
inhibitors,49,50 oligonucleotides,51–53 fusion inhibi-
tors,54–58 and agents that interfere with HIV gene regu-
lation.59–64
2-5A-tat conjugates were reduced significantly in RNase
L activation ability as compared to the unligated 2-5A
tetramer. These 30- to 100-fold reductions in RNase L
activation ability, compared to unmodified 2-5A itself,
are similar reductions in RNase L activation to that seen
for chimeric 2-5A-antisense constructs of similar chain
length. This comparison is valid when evaluation was
carried out against RNA substrates that were non-com-
plementary to the antisense domain of 2-5A-antisense
analogues. Thus for instance, in the case of the 2-5A-an-
tisense chimera with an all DNA phosphodiester DNA
antisense domain that did not complement the substrate
RNA, RNase L activation ability was reduced by about
30-fold compared to parent 2-5A.29 When the antisense
domain was a PNA39 or morpholino analogue40 that did
not complement substrate RNA, then RNase L activa-
tion was reduced 60- to 100-fold. Greater activity has
been observed when the antisense domain of the chimera
targets specifically the RNA substrate. Thus, a 2-5A-an-
tisense molecule with an all phosphodiester DNA target-
ing a specific sequence of PKR RNA was only three
times less active than parent 2-5A.41 This increase in
activity against targeted substrate has been hypothesized
This research makes possible employment of a peptide
as the targeting agent for 2-5A. Specifically, the consti-
tutive, latent, and potent 2-5A-dependent RNase L
could be targeted to the HIV RNA by the accom-
plished conjugating 20,50-oligoadenylates (2-5A) with
Tat peptides to provide 2-5A-tat. As with the previous-
ly demonstrated 2-5A-antisense strategy, this may re-
sult in the selective ablation of HIV RNA and
inhibition of HIV replication. The Tat-derived pep-
tide(s) domain of the 2-5A-tat conjugate would bind
to TAR of HIV RNA, and the appended 2-5A domain
would attract and activate the 2-5A-dependent RNase
L to this region of HIV RNA, resulting in its selective

L. Zhou et al. / Bioorg. Med. Chem. 14 (2006) 7862–7874
7871
cleavage by a catalytic mechanism. This possibility of
selective inhibition of HIV replication is presently un-
der active investigation.
5.2. Modification of AMP with azido functional group;
synthesis of compound 3
Compound 3 was synthesized from 50-AMP (2) and ex-
cess 3-azidopropanamine. Thus, sodium periodate solu-
tion (0.6 mL of 0.1 M in water, 60.0 lmol) was added in
one portion to a solution of AMP (17.35 mg in 0.5 mL
water, 50.0 lmol), and the resulting mixture was stirred
in the dark at 0 ꢁC for 30 min. To destroy the excess
periodate, a Na2S2O3 solution (1.0 mL of 0.1 M,
100.0 lmol) was added, and the resultant solution was
maintained at 0 ꢁC for an additional 10 min in the dark.
3-Azidopropanamine (25.0 mg, 250.0 lmol) was added,
and the pH of the reaction mixture was adjusted to 8.6
with 5% acetic acid. The resulting mixture was stirred
at 0 ꢁC for 2 h and then a sodium cyanoborohydride
solution (1 mL of 0.5 M in water, 0.5 mmol) was added.
This reaction mixture was kept at 0 ꢁC for 1 h during
which time the pH was maintained at 6.6 by periodic
addition of 0.5% HOAc. The reaction mixture was stir-
red at 4ꢁ overnight after which HPLC analysis showed
the reaction was complete. The product 3 was purified
by HPLC on semi-preparative Adsorbosphere C-18 col-
umn (10 · 250 mm) (82% yield) and its structure corrob-
orated by 1H NMR, 13P NMR, and high resolution
mass spectroscopy (HRMS). 1H NMR (CD3OD) d
(ppm) 1.85 (m, 2 H, –CH2–); 2.37 (t, J = 10.8 Hz, 1H,
H-30); 2.71 (t, J = 7.2 Hz, 2H, –CH2N), 2.81 (m, 1H,
H-20); 3.15–3.31 (m, 2H, H-20, H-30); 3.40 (t,
J = 7.2 Hz, 2H, –CH2N3); 3.99 (m, 2H, H-50, H-50);
4.20 (m, 1H, H-40); 6.00 (dd, J = 10.0, 2.4 Hz, 1H, H-
10); 8.19 (s, 1H); 8.25 (s, 1H). 31P NMR (CD3OD) d
(ppm) 1.94. HRESI MS calcd for C13H20N9O5P:
414.1403 (M+1)+; found: 414.1391.
5. Experimental
Except where otherwise noted, all reversed-phase HPLC
analysis and purifications were conducted on a Varian
Pro Star 210 instrument using Adsorbosphere ODS
analytical
(4.6 · 250 mm)
and
semi-preparative
(10 · 250 mm) C-18 and C-8 columns (Alltech Compa-
ny) for analysis and purification with the following buf-
fers: (buffer A) 50 mM NH4OAc (pH 7.00); (buffer B)
50% MeOH/H2O. Linear gradient elution was employed
with flow rate of 1 mL/min for analytic and 5 mL/min
for preparative application. 1H NMR (400.14 MHz),
31P NMR (169.99 MHz), and 13C NMR (100.62 MHz)
spectra were recorded on Varian (Palo Alto, CA)
VNMR 400 spectrometer. High resolution ESI mass
spectrometry (HRESI) was recorded on an IonSpec
Fourier Transform Mass Spectrometer at University of
Arizona (Tucson, AZ). Matrix-assisted laser desorp-
tion/ionization time-of-flight mass spectrometry (MAL-
DI-TOF/MS) was carried out on Kratos MALDI III
instrument at HT laboratories (San Diego, CA). The
chemical shifts are reported in parts per million (ppm)
relative to internal tetramethylsilane in CDCl3 or
DMSO-d6 (1H NMR) or relative to H3PO4 (31P
NMR). The coupling constants (J) are reported in Hertz
(Hz). UV spectra were determined on a Hewlett Packard
8452 spectrophotometer. The reagents 50-AMP,
NaBH3CN, CuSO4, NaIO4, sodium ascorbate, and
6-heptynoic acid were purchased from Aldrich–Sigma
(St Louis, MO). All amino acids and resin were pur-
chased from Advanced Chemtech.
5.3. Coupling between 2-5A tetramer and 3-azidoprop-
anylamine: synthesis of compound 4
5.1. Peptide synthesis
A solution of NaIO4 (0.1 M 4.0 lL, 0.4 lmol) was added
to a cooled solution of p50A20p50A20p50A20p50A (80 lL,
8.8 OD, 0.2 lmol). The oxidation was carried out at
0 ꢁC in the dark for 90 min. Then a 2-fold excess of
Na2S2O3 over NaIO4 was added to the solution to
remove the excess oxidant. The reaction mixture was
stirred for another 10 min. After that, 3-azidopropanyl-
amine (16.0 lL of 0.05 M, 0.8 lmol) was added, and the
pH was adjusted to 8.6 with 0.5% HOAc. After 3.5 h,
4.0 lL of 0.5 M NaBH3CN was added, and the pH
was adjusted to about 6.6. After overnight incubation,
the product was purified by HPLC to afford 4 in 45%
yield. The product 4 was analyzed by MALDI-TOFF
mass spectrometry: calcd for C43H56N24O23P4: 1400.9;
found: 1401).
Peptide 5 containing the translocation sequence of the
tat peptide and alkyne functional group was synthesized
using 0.25 mmol scale on a ABI 431 peptide synthesizer
using standard FOMC chemistry with HOBt/HBTU/
DIEA as activating reagents and using 20% piperidine
for FMOC removal. The peptide sequence is 6-hepty-
noic-Gly-Gly-Arg-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-
Arg-COOH (the italicized amino acids correspond to
residues 48–57 RKKRRQRRR of the tat protein).
The resin used was FMOC-Arg-Wang resin with
0.61 mmol/g loading. All arginines were double coupled
and the alkyne moiety was coupled using 6-heptynoic
acid. The peptide was cleaved with TFA/TIPS/water
(93:5:2) for 5 h and purified by reversed-phase HPLC
with an YMC-Pack ODS column (150 · 20 mm,
Waters). MALDI-MS (M+1)+: calcd for 1719.04;
found: 1719.1.
5.4. Conjugation products from azide-alkyne cycloaddi-
tion: synthesis of compounds 6 and 7
Similar procedures were employed for the synthesis and
purification of peptide 14 that contained two spacer
glycine residues and then the terminal cysteine moiety.
The product’s structure was corroborated by ESI-MS.
MS (M+1)+: calcd for C60H118N33O14S: 1557; found:
1557.
Compound 3 (0.8 mg, 2 lmol) and tat peptide 5 (1.7 mg,
1 lmol) were dissolved in water (30 lL). Sodium ascor-
bate (1 lmol, 1 lL of freshly prepared 1 M solution in
water) was added, followed by copper(II) sulfate solu-
tion (1 lmol, 10 lL of 0.1 M in water). The mixture
was stirred vigorously for 24 h. The reaction mixture

7872
L. Zhou et al. / Bioorg. Med. Chem. 14 (2006) 7862–7874
was purified by a DEAE Sephadex column and the elut-
ed fraction was lyophilized to give the product 6 (9.2
OD, 60%). The product was analyzed by MALDI-
TOFF mass spectrometry: calcd for C83H152N45O20P:
2131; found: 2131).
of 0.5% HOAc. After the reaction mixture was stirred at
4 ꢁC overnight, HPLC analysis showed the reaction was
complete. The product 12 was purified by HPLC on
semi-preparative Adsorbosphere C-18 column (10·
250 mm) (85% yield) and its structure corroborated by
1
1H NMR, 13P NMR, and mass spectroscopy (MS). H
The reaction between the azido-functionalized 2-5A tet-
ramer 4 and tat peptide 5 was carried out under similar
reaction conditions with a similar purification protocol
The product’s structure was corroborated by MALDI-
TOFF mass spectrometry: calcd for C113H188N60O38P4:
3118; found: 3118.
NMR (D2O) d (ppm) 2.28 (t, J = 12.0 Hz, 1H, H-30);
2.67 (t, J = 10.4 Hz, 1H, H-20); 2.90–2.99 (m, 2H, H-
20, H-30); 3.52–3.70 (m, 2H, H-50, H-50); 3.78 (s, 2H,
CH2); 4.06 (m, 1H, H-40); 4.11 (s, 2H, CH2); 5.72 (d,
J = 9.6, Hz, 1H, H-10); 7.20–7.28 (m, 4H, ArH); 8.03
(s, 1H); 8.07 (s, 1H). 13C NMR (D2O) d (ppm) 43.5,
53.7, 55.1, 61.0, 64.4, 75.2, 79.0, 118.4, 119.2, 129.4,
132.6, 137.5, 138.9, 148.8, 152.7, 155.9, 164.4; 31P
NMR (D2O) d (ppm) 2.01. ESI MS calcd for (M+1)+
C19H23ClN7O6P: 512; found: 512.
5.5. Synthesis of p-(N-chloroacetamido)benzylamine
(compound 11)
Chloroacetyl chloride (9, 0.64 mL, 8.0 mmol) in 5 mL
dry THF was added dropwise into a mixture of sodium
bicarbonate (1.0 g, 10.0 mmol), 4-[(N-Boc)aminometh-
yl]aniline (8, 473 mg, 97%, 2 mmol), and dry THF
(15 mL) at 0 ꢁC (ice-bath) under N2 atmosphere over a
period of 10 min. The resultant mixture was stirred for
another 2 h as the temperature was allowed to rise from
0 ꢁC to room temperature. The reaction mixture was
then poured into a sodium bicarbonate solution, and
extracted with ethyl acetate (3· 50 mL). The combined
organic solvent was dried over sodium sulfate and then
evaporated to produce a white powder 10 (quantitative
5.7. Synthesis of compound 13
NaIO4 solution (0.1 M, 4.0 lL, 0.4 lmol) was added to a
cooled solution of 2-5A tetramer (1) (80 lL, 8.8 OD,
0.2 lmol), and the oxidation was allowed to proceed
at 0 ꢁC in the dark for 90 min. Then a twofold excess
of Na2S2O3 over NaIO4 was added to the solution to re-
move the excess oxidant. The reaction mixture was stir-
red for another 10 min. After that, compound 11
(20.0 lL of 0.1 M, 2.0 lmol) was added, and the pH
was adjusted to about 8.4 with 0.1 M NaOH. After
the mixture had been stirred at 0 ꢁC for 2.5 h, 4.0 lL
of 0.5 M NaBH3CN was added, and the pH was adjust-
ed to 6.4 with 0.5% HOAc. After overnight incubation,
the product was purified by HPLC to afford com-
pound13. The product was analyzed by HRMS mass
spectrometry: calcd for C49H60ClN22O24P4: 1499.279;
found: 1499.4439).
1
yield). H NMR (CDCl3) d (ppm) 1.45 (s, 9H, CH3);
4.18 (s, 2H, CH2); 4.28 (s, 2H, CH2); 4.84 (s, 1H,
NH); 7.25–7.28 (dd, 4H, ArH); 8.23 (s, 1H, NH). 13C
NMR (CDCl3) d (ppm) 28.3, 42.8, 44.1, 79.5, 120.3,
128.2, 135.8, 135.9, 155.8, 163.7.
Next a hydrogen chloride solution in dioxane (2.5 mL,
4 M) was added into a suspension of compound 10
and dry ethyl acetate (2.5 mL) at 0 ꢁC. The mixture
was allowed to stir for 2 h at ice temperature. After
removal of the organic solvent under reduced pressure,
a white solid product 11 was obtained (200 mg, 85%
5.8. Synthesis of Tat peptide conjugation products,
compounds 15 and 16
Peptide 14 and compound 12 (3 mg, 5.8 lmol) were dis-
solved in acetonitrile (50 lL). Then buffer (200 lL of
0.1 M NaH2PO4) was added into the mixture under a
nitrogen atmosphere. After the mixture was stirred at
room temperature for 4 h under N2, the product was
separated using a DEAE Sephadex A-25 column with
acetonitrile/water (50:50) solution as eluent. The collect-
ed fractions were lyophilized to give the product 15 (6.4
OD, 73% yield). The product was analyzed by ESI mass
spectrometry: calcd for C79H139N40O20PS: 2032; found:
2032).
1
yield). H NMR (D2O d (ppm) 4.06 (s, 2H, CH2); 4.17
(s, 2H, CH2); 7.34–7.42 (m, 4H, ArH). 13C NMR
(D2O) d (ppm) 42.8, 43.0, 122.6, 129.9, 130.2, 137.1,
168.5.
5.6. Synthesis of compound 12 from AMP and p-[N-(a-
chloroacetamido)]benzylamine (11)
Sodium periodate solution (0.6 mL 0.1 M in water,
60.0 lmol) was added in one portion to a solution of
AMP (17.35 mg in 0.5 mL water, 50.0 lmol), and the
resulting mixture was stirred in the dark at 0 ꢁC for
30 min. To destroy the excess periodate, a Na2S2O3
solution (1.0 mL of 0.1 M, 100 lmol) was added, and
the resultant solution was maintained at 0 ꢁC for an
additional 10 min in the dark. An aqueous solution of
compound 11 (1 mL, 23.5 mg, 100.0 lmol) was added,
and the pH of the reaction mixture was adjusted to 8.6
with 2 N NaOH. The resulting mixture was stirred at
0 ꢁC for 2.5 h and then a sodium cyanoborohydride
solution (0.5 mL of 0.5 M in water, 0.25 mmol) was add-
ed. This mixture was kept at 0 ꢁC for 1 h during which
time the pH was maintained at 6.3 by periodic addition
The reaction between the chloroacetyl-functionalized 2-
5A tetramer 13 and tat peptide 14 was carried out under
similar reaction conditions. The product’s structure was
corroborated by MALDI-TOF mass spectrometry:
calcd for C109H175N55O38P4S: 3019; found: 3017).
5.9. Synthesis of 2-5A-S-Tat-fluorescein (compound 17)
Acetonitrile (50 lL) was added to a mixture of the chlo-
roacetamido derivative (13, 0.27 lmol) of 2-5A and the
cysteine-terminated tat peptide labeled with fluorescein
(0.09 lmol). Then buffer (200 lL, 0.1 M NaH2PO4)

L. Zhou et al. / Bioorg. Med. Chem. 14 (2006) 7862–7874
7873
was added to the above mixture under a nitrogen atmo-
sphere. The mixture was stirred at room temperature for
24 h under N2, and the product was separated using a
DEAE–Sephadex A-25 column with CH3CN/H2O
(50:50) as eluent. The collected fraction was lyophilized
to give the product 2-5A-S-Tat-fluorescein (1.2 OD,
30% yield). The conjugate product was confirmed
by MALDI-TOFF mass spectrometry: calcd for
C132H188N56O45P4S: 3435; found: 3435).
prior to treatment. The pipette tip was dipped in serum
prior to pipetting compound 16 to avoid attachment to
plastic tip. Compound 16 was diluted with complete
medium plus 2% FBS prior to adding to cells. After
6 h incubation with compound 16 at 37 ꢁC, media were
removed and cells were washed twice with PBS. Isolated
RNA (1 lg) was separated on RNA chips (Agilent Tech-
nologies) and analyzed with an Agilent Bioanalyzer
2100 (Agilent Technologies).
5.10. RNase L assay
5.12. Uptake of Tat
RNase L activity was determined by a fluorescence res-
onance energy transfer (FRET) method (35, Thakur
2005). The assay uses recombinant human RNase L pro-
duced in insect cells from a baculovirus vector and puri-
fied by FPLC columns (36). The cleavable substrate
consists of a 36 nucleotide synthetic oligoribonucleotide
sequence derived from respiratory syncytial virus with
the fluorophore, FAM, at the 50-terminus and black
hole quencher-1 (BHQ-1), at the 30-terminus (synthe-
sized at Integrated DNA Technologies). The RNA se-
quence contains several cleavage sites for RNase L
(UU or UA). The test compounds or 2-5A were diluted
to final concentrations of 1, 3, 10, 30, 100, 300, 1000,
and 3000 nM in DEPC-treated water. Five microliters
of each diluted sample was added in triplicate to 96-well
black polystyrene microtiter plates (Corning) on ice.
Forty-five microliters of the reaction mixture was added
to each sample and the plates were gently agitated for
10 s to mix, then were centrifuged briefly at 500 rpm.
The assays contained 100 nM RNA probe, 25 nM
RNase L, 25 mM Tris–HCl (pH 7.4), 100 mM KCl,
10 mM MgCl2, 50 lM ATP, and 7 mM 2-mercap-
toethanol with and without 2-5A or other compounds.
RNase L was the last component added to the reaction
mixtures. The plates were incubated at 20 ꢁC protected
from light. Fluorescence was measured at 5, 30, 60, 90,
and 120 min with a Wallac 1420 fluorimeter (Perkin El-
mer) (absorption 485 nm/emission 535 nm).
HeLa cells were cultured in 200 ll DMEM (Cambrex),
supplemented with 10% fetal bovine serum (FBS) (Hy-
Clone), glutamine, penicillin/streptomycin (Gemini Bio
Products), and 0.01% protamine (Sigma P4005). Fluo-
rescently labeled 2-5A-tat peptide conjugate (2-5A-tat)
and fluorescent-tat-SH were added to cells to a final
concentration of 1 lM. Before adding the peptides to
the cells, pipette-tips were dipped in serum; tat-peptides
will otherwise stick to the plastic tips. After 10 min,
one hour, and two hours, the medium was replaced
with PBS (Cambrex). Images of cellular uptake of
our compounds were acquired with a Zeiss LSM410
laser scanning confocal microscope equipped with a
mixed-gas laser (488-, 568-, and 647-nm excitation
lines) in sequential scans then analyzed with Micro-
cosm Renaissance 410 software. Overlays of the ac-
quired images were generated using Adobe Photoshop
CS software.
Acknowledgments
PFT thanks the National Institute of Allergy and Infec-
tious Diseases for support from Grant 5 R03 AI054184-
02, Robert Smith for excellent technical assistance, and
Edgar Civitello for advice in peptide synthesis.
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