Synthesis and properties of triazole-linked RNA

Article abstract of DOI:10.1016/j.bmcl.2011.03.111

RNA oligonucleotides having triazole linkages between uridine and adenosine nucleosides have been prepared and studied using spectroscopic techniques. UV melting and CD showed that triazole strongly destabilized RNA duplex (7-14 °C per modification). NMR

Full text of DOI:10.1016/j.bmcl.2011.03.111

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3420–3422
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and properties of triazole-linked RNA
Daniel Mutisya a, Chelliah Selvam a, Scott D. Kennedy b, Eriks Rozners a,
⇑
a Department of Chemistry, Binghamton University, The State University of New York, Binghamton, NY 13902, USA
b Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
RNA oligonucleotides having triazole linkages between uridine and adenosine nucleosides have been
prepared and studied using spectroscopic techniques. UV melting and CD showed that triazole strongly
destabilized RNA duplex (7–14 °C per modification). NMR data suggested that, despite relative flexibility
around the modified linkage, all base pairs were formed.
Received 1 March 2011
Revised 25 March 2011
Accepted 29 March 2011
Available online 5 April 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
RNA
Modified nucleoside
Triazole internucleoside linkage
Huisgen dipolar cycloaddition
Click reaction
RNA interference may become a promising alternative to
conventional chemotherapy of genetic disorders, cancer, viral
infections and other diseases for which the target mRNA can be
identified.1 However, for short interfering RNAs (siRNAs) to be effi-
cient therapeutic agents they need to be chemically modified to
optimize nuclease resistance, crossing of cell membranes, biodis-
tribution and pharmacokinetics.2 We are interested in developing
RNA analogues that have the phosphate linkages replaced by
nonionic and hydrophobic modifications, which we believe may
improve the above properties of siRNAs. We have found that
amide3 (30-CH2–CO–NH-50) and formacetal4 (30-O–CH2–O-50) link-
ages have surprisingly little effect on the global A-type structure,
thermal stability and hydration of RNA and appear to be excellent
mimics of phosphodiester linkages. Meanwhile, preparation of
RNA bearing these modifications requires extensive synthetic
effort, which hinders rapid testing and development of modified
siRNAs. In a search for a nonionic modification that would be easy
to synthesize both at the modified nucleoside and siRNA level, we
became interested in triazole linkages that could be made via the
Huisgen [3 + 2] dipolar cycloaddition (the ‘click’ reaction).5,6
Von Matt et al.7 were the first to report synthesis and thermal
stability of triazole-linked DNA, where the triazole was derived
from 30-azidothymidine. They found that the triazole modification
of the DNA strand destabilized DNA–RNA heteroduplexes by about
1–2 °C per modification.7b Recently, Isobe et al.8 synthesized
triazole-linked dT10, using Huisgen cycloaddition of 30-azido and
50-ethynyl thymidine. The triazole in Isobe’s work was isomeric
RO
Base
RO
Base
RO
Base
O
N
O
O
O
OR
O
OR
N3
HO
OH
HO
Base
Base
N
Base
O
O
O
N
RO
OR
RO
OR
RO
OR
Figure 1. Retrosynthetic analysis of triazole-linked RNA.
to Von Matt’s structure and led to an exceptionally stable duplex
with complementary DNA.8a Similar triazole linkage was later used
by El-Sagheer and Brown to ligate synthetic DNA fragments and to
amplify the resulting construct using PCR.9 Dondoni and co-
workers10 also described preparation of trizole-linked DNA
trinucleosides, but did not report on biophysical properties of these
compounds.
Although triazole linkages prepared from 30-azidothymidine
have shown great promise in DNA, we expected that the presence
of 20-hydroxyl would complicate both preparation of 30-azidouri-
dine and synthesis of the corresponding triazole linked RNA. We
envisioned that the easiest approach to triazole linked RNA
would be a coupling of 30-O-propargyl and 50-azidoribonucleosides
(Fig. 1).
Zerrouki and co-workers11 incorporated the trizole linkage
shown in Figure 1 in DNA but did not report on biophysical prop-
erties of the modified oligonucleotides. El-Sagheer and Brown used
the ‘click’ reaction between 30-O-propargyl-5-methyldeoxycytidine
and 50-azidouridine to incorporate a single triazole linkage at
⇑
Corresponding author. Tel.: +1 607 777 2441; fax: +1 607 777 4478.
E-mail address: erozners@binghamton.edu (E. Rozners).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.111

D. Mutisya et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3420–3422
3421
the active site of a hammerhead ribozyme.12 The modified ribo-
zyme was fully functional suggesting that the triazole linkage in
Figure 1 may be well tolerated in RNA despite being two atoms
longer than the phosphate. Our own recent results, suggesting that
RNA may tolerate hydrophobic nonionic modifications better than
DNA,3,4 inspired us to test the properties of triazole-linked RNA.
We found that the triazole linkage strongly destabilized RNA dou-
ble helix by about 7–14 °C per modification depending on the se-
quence and location of the modifications. Although satisfactory
melting curves could be recorded for all model systems, NMR
and CD spectra suggested that the double helical structure is rela-
tively flexible.
MMTO
Ura
O
MMTO
Ura
MMTO
Ura
O
N
O
N
O
OAc
O
OAc
O
OAc
(a)
(b)
AdeBz
4
N
N
AdeBz
O
O
N
N
N3
AdeBz
O
HO
OTOM
O
O
OTOM
NC
P
N
6
HO
OTOM
5
TOM =
7
O
Si
Scheme 2. Synthesis of triazole-linked r(UTRA) phosphoramidite. Steps: (a) CuSO4,
sodium ascorbate, THF/ethanol/water, 60 °C, 12 h, 79%; (b) iPr2NEt, ClP(OCH2-
CH2CN)N(iPr)2, CH2Cl2, rt, 6 h, 47%.
The key consideration in our design was minimizing the syn-
thetic effort. While preparation of 50-azidoribonucleosides was
straightforward,13 synthesis of 30-O-propargyl ribonucleosides in-
volved alkylation of the 20,30-cis-diol system, which we expected
to proceed with low selectivity and require separation of isomeric
products.14 However, we envisioned that the quick access of the
alkyne coupling partner (only three to four steps from ribonucleo-
sides) would offset the difficulties and low yields of the alkylation
step.
Table 1
Thermal stability of triazole modified RNA
Entry
Sequence
tm TRa
tm RNAb
Dtm
RNA1
RNA2
RNA3
RNA4
GCGUAUTRACGC
GCGUTRAUACGC
GUGUTRACAC
39.5
38.7
16.0
29.5
53.7
53.7
40.8
58.3
–7.1
–7.5
–12.4
–14.4
Treatment of the known 50-O-methoxytrityluridine 1 with dibu-
tyltin oxide followed by propargyl bromide gave the expected mix-
ture of 30- and 20-O-propargyluridine (2 and 3, in an approximate
ratio of 1:1.3, respectively) from which the target compound was
isolated in 28% yield (Scheme 1). To complete the synthesis of
the alkyne coupling partner 4, 20-OH was protected with acetyl
group.
GCGUTRACGC
a
tm (°C) of triazole-modified RNA, TR denotes position of the triazole linkage.
tm (°C) of unmodified RNA control.
b
10
5
A
We recently developed synthesis of selectively protected 50-azi-
do-20-O-TOM adenosine 5,3a which in the present study served well
as the coupling partner for 4 in the Huisgen cycloaddition reaction
to prepare the triazole linked r(UTRA) dimer 6 (Scheme 2). The
structure of the triazole linkage was confirmed using 2D NOESY
NMR spectroscopy. In particular, the NOEs of adenosine H50, tria-
zole aromatic H and 30-OCH2 of uridine confirmed the assigned
structure of 6 (for details see Supplementary data, Figs. S1 and
S2). Standard phosphoramidite synthesis gave dimer 7 suitable
for solid phase synthesis (Expedite 8909) of RNA sequences bear-
ing the triazole modification.
To evaluate the effect of triazole linkage on thermal stability of
RNA we synthesized several self-complementary sequences
(Table 1) that had performed well in our previous thermodynamic
and structural studies.3a,4a UV thermal melting (Fig. S7) was done
as previously reported.3a,4a Results in Table 1 show that triazole
linkage strongly destabilized RNA helices. The effect was more dra-
0
-5
GCGUAUACGC
GCGUAUtrACGC
GCGUtrAUACGC
-10
-15
200
220
240
260
wavelength (nm)
280
300
320
10
B
matic for RNA3 and RNA4 (D m
triazole linkages were placed opposite to each other in the double
helix than for RNA1 and RNA2 (D m ca. 7 °C per modification)
t >10 °C per modification) where the
5
0
t
where the triazoles were shifted relative to each other.
To gain insights into how the triazole linkage affected the struc-
ture of modified RNA we compared the circular dichroism (CD)
spectra of RNA1–RNA4 with the spectra of the corresponding
unmodified RNAs (Fig. 2). RNA2 gave the expected CD spectrum
(Fig. 2A) similar to the unmodified RNA. Despite the similar tm
and melting curves (Fig. S7) RNA1 gave almost no CD signal
-5
-10
-15
-20
GCGUACGC
GCGUtrACGC
200
220
240
260
wavelength (nm)
280
300
320
MMTO
Ura
MMTO
Ura
MMTO
1.3
Ura
O
O
O
(a)
+
:
Figure 2. Comparison of CD spectra of triazole-linked RNAs (broken lines) and the
unmodified RNA controls (solid lines). Recorded at A 20 °C, B 0 °C.
HO
OH
O
OR
HO
O
1
1
2 R = H
4 R = Ac
3
(b)
( Fig. 2A). A similar result was obtained with RNA3 (Fig. S8).
RNA4 gave CD spectrum similar to but less intense than the
unmodified control (Fig. 2B). Overall, the CD data suggested that
Scheme 1. Propargylation of 50-O-methoxytrityluridine. Steps: (a) Bu4NBr, Bu2SnO,
DMF 100 °C, 2 h, then propargyl bromide, 45 °C, 24 h, 28%; (b) acetic anhydride,
pyridine, rt, 5 h, 98%.

3422
D. Mutisya et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3420–3422
that pair may be slightly distorted or dynamic allowing some
water access. The relative flexibility of the central AU pair is consis-
tent with reduced intensity of the CD signal for RNA4 (Fig. 2B).
In summary, we found that the triazole 30-O–CH2–C@CH–N-50
linkage strongly destabilized RNA double helix (D m –7 to –14 °C
t
per modification) and is not a good mimic of the phosphate linkage
in RNA. Our results on the triazole linkage that has four bonds (30-
O–CH2–C@CH–N-50) instead of the two bonds long phosphate (30-
O–P(O)2–O-50) linkage are in contrast to results reported by Isobe
et al.8 who found that the two bonds long triazole (30-N–CH@CH-
50) linkage strongly stabilized DNA. The most likely explanation
for these differences is that the significantly longer linkage adopts
a conformation that is less than optimal for RNA double helix and
causes the flexibility of the UA base pairs as suggested by the NMR
and CD spectra of RNA4. Depending on the sequence the duplex
may become disordered, as suggested by the lack of CD signal for
RNA1 and RNA3. The striking observation that modified duplexes
with similar melting curves gave different CD spectra underlines
the need for careful and comprehensive biophysical studies before
conclusions can be made about the properties of a nucleic acid
modification.
Acknowledgments
We thank National Institutes of Health (R01 GM071461) for
financial support of this research and Dr. Pradeep S. Pallan
(Vanderbilt University) for help with MALDI-TOF MS.
Figure 3. NMR spectra and 2D water–NOESY spectrum of RNA4 duplex (0.1 mM).
Upper left: 1D spectra of the imino proton region of the duplex (upper right with
numbering) showing that U4 imino is only observed at low temperature. Bottom:
2D water–NOESY acquired at 1 °C with mixing time of 150 ms. Red circles indicate
A5 H2 NOEs to U4 imino, C6 H10, and cross-strand A5 H10. Cytosine intramolecular
amino–amino cross-peaks typical in Watson–Crick GC pairs are labeled, including
C8 in the terminal base pair for which no G imino is observed. Strong cross-peaks
from G3 and G7 iminos to C6 and C2 aminos, respectively, indicate GC
Watson–Crick pairs.
Supplementary data
Supplementary data (experimental procedures, copies of HPLC
traces, UV melting curves, CD and NMR spectra) associated with
this article can be found, in the online version, at doi:10.1016/
j.bmcl.2011.03.111.
References and notes
triazole modification made RNA duplexes more flexible and that
RNA1 and RNA3 might be significantly disordered even at temper-
atures well below the tm obtained by UV melting (20 and 0 °C,
respectively).
To gain insight into base pair formation in the triazole modified
RNA we studied RNA4 using NMR techniques.15 Spectra in 95%
H2O/5% D2O exhibit three clear imino proton signals (Fig. 3, top),
one of which is clear only at low temperature where exchange of
the imino proton with water is sufficiently slowed.
Strong NOEs to cytosine amino protons allow assignment of the
two temperature-stable imino protons to G3 and G7 in GC base
pairs. An NOE to the H2 proton of the lone adenine, despite the
high rate of exchange with water, identifies the third imino peak
as belonging to uracil in an AU base pair. Further evidence of this
AU pair is contained in typical NOEs from adenine H2 to the
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similar to that observed for C2 and C6 amino protons, is typical
of Watson–Crick GC pairs. Thus, the NMR evidence indicates
formation of a duplex that includes the central AU pair, although
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