Posttranscriptional chemical functionalization of azide-modified oligoribonucleotides by bioorthogonal click and Staudinger reactions

Article abstract of DOI:10.1039/c1cc15659d

Direct incorporation of azide groups into RNA oligonucleotides by in vitro transcription reactions in the presence of a new azide-modified UTP analogue, and subsequent posttranscriptional chemical labeling of azide-modified oligoribonucleotide transcripts by click and Staudinger reactions are described. This postsynthetic labeling protocol is robust and modular, and offers an alternative access to RNA labeled with biophysical probes.

Full text of DOI:10.1039/c1cc15659d

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Chemical Communications
www.rsc.org/chemcomm
Volume 48 | Number 4 | 14 January 2012 | Pages 469–616
ISSN 1359-7345
COMMUNICATION
S. G. Srivatsan et al.
Posttranscriptional chemical functionalization of azide-modified
oligoribonucleotides by bioorthogonal click and Staudinger reactions
1359-7345(2012)48:4;1-8

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Cite this: Chem. Commun., 2012, 48, 498–500
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Posttranscriptional chemical functionalization of azide-modified
oligoribonucleotides by bioorthogonal click and Staudinger reactionsw
Harita Rao, Anupam A. Sawant, Arun A. Tanpure and Seergazhi G. Srivatsan*
Received 13th September 2011, Accepted 5th October 2011
DOI: 10.1039/c1cc15659d
Direct incorporation of azide groups into RNA oligonucleotides by
in vitro transcription reactions in the presence of a new azide-modified
UTP analogue, and subsequent posttranscriptional chemical labeling
of azide-modified oligoribonucleotide transcripts by click and
Staudinger reactions are described. This postsynthetic labeling
protocol is robust and modular, and offers an alternative access
to RNA labeled with biophysical probes.
elaborate chemical manipulations and protection–deprotection
steps. Such an endeavor would equip oligoribonucleotides with
azide groups, which could be further functionalized posttran-
scriptionally in a modular fashion by CuAAC, Staudinger
ligation and Staudinger reduction reactions. Here, we report
the synthesis of a new azide-modified ribonucleoside triphosphate
analogue, and its effective incorporation into oligoribonucleotides
by in vitro transcription reactions catalyzed by T7 RNA
polymerase. Reactions with various oligodeoxynucleotide templates
exemplify the utility of transcription reactions in functionalizing
oligoribonucleotides with more than one azide group. We further
illustrate the posttranscriptional chemical functionalization of
azide-modified oligoribonucleotides with biophysical probes by
click and Staudinger reactions.
Postsynthetic modification using bioorthogonal chemical
reactions has recently evolved as a valuable method to label
nucleic acids for diagnostic and therapeutic applications.1 In
this approach, a modified nucleoside containing a reactive
functionality is incorporated into an oligonucleotide either by
chemical or enzymatic methods, and further labeling is
achieved postsynthetically by a chemoselective reaction with
the reactive functionality. Coupling of aminoalkyl- or thioalkyl-
modified oligonucleotides with activated esters of the labels is one
of the standard procedures to postsynthetically functionalize
oligonucleotides.2 However, low chemoselectivity and propensity
of activated esters to hydrolysis reasonably affect the coupling
efficiency. Recently, bioorthogonal reactions such as copper(I)-
catalyzed alkyne–azide cycloaddition (CuAAC) and Staudinger
ligation between an azide and a phosphine have found wide utility
in labeling DNA postsynthetically.3 Despite such successes with
DNA, postsynthetic modification of RNA remains a challenge as
protocols developed for DNA modification do not necessarily
work for RNA due to its inherent instability.4
The azide-modified uridine analogue 6 and corresponding
triphosphate substrate 7 were synthesized as shown in Scheme 1.
5-Iodouridine was first perbenzoylated and then coupled with
propargyl alcohol in the presence of Pd(PPh3)4 catalyst and CuI to
afford the 5-(3-hydroxyprop-1-ynyl)-modified uridine 3. Catalytic
hydrogenation of
3 gave the 5-(3-hydroxypropyl)-modified
The azide group, which is the common reactive functionality in
both CuAAC and Staudinger reactions, is not easily incorporated
by conventional solid-phase oligonucleotide synthesis protocols as it
readily undergoes Staudinger-type reaction with phosphoramidite
substrates.5,6 Therefore, development of a method to incorporate
the azide group directly into oligoribonucleotides would greatly
enhance the accessibility of azide-modified RNA oligonucleotides
for postsynthetic labeling procedures. In this regard, incorporation
of an azide-modified ribonucleoside triphosphate by an in vitro
transcription reaction would be appropriate as it does not involve
Scheme 1 Reagents and conditions: (a) benzoyl chloride, DMAP,
pyridine, RT, 97%. (b) Propargyl alcohol, Pd(PPh3)4, CuI, Et3N,
DMF, RT, 74%. (c) Pd/C, H2, MeOH, RT, 83%. (d) (i) MsCl,
Et3N, CH2Cl2, 0 1C; (ii) NaN3, DMF, 40 1C, 87%. (e) Sodium
methoxide, MeOH, 60 1C, 52%. (f) (i) POCl3, (MeO)3PO, B4 1C;
(ii) tributylammonium pyrophosphate, Bu3N, B4 1C, 26%.7
Indian Institute of Science Education and Research,
Sai Trinity Building, Pashan, Pune 411021, India.
E-mail: srivatsan@iiserpune.ac.in
w Electronic supplementary information (ESI) available: Experimental
procedures, Fig. S1–S8, and NMR and mass spectra. See DOI:
10.1039/c1cc15659d
c
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uridine 4 in good yield. The hydroxypropyl group was activated
with mesyl chloride and converted into an azide by a reaction with
sodium azide. The azide-modified nucleoside 6 was then obtained
by deprotecting the benzoyl groups using sodium methoxide.
Finally, treating ribonucleoside 6 with POCl3, followed by a
reaction with bistributylammonium pyrophosphate under ice cold
conditions gave the azide-modified UTP 7.
The ability of T7 RNA polymerase to accept and incorporate
the modified UTP 7 into oligoribonucleotides was investigated by
performing in vitro transcription reactions in the presence of a series
of promoter–template deoxyoligonucleotide duplexes (Fig. 1). The
templates (S1–S5) contained one or two dA residues to guide the
incorporation of 7 into oligoribonucleotide transcripts at one or
two sites. Furthermore, at the 50-end of each template was placed a
dT residue to facilitate the incorporation of a unique adenosine
residue at the 30-end of each transcript. Radiolabeled oligori-
bonucleotide products obtained from successful transcription
reactions in the presence of GTP, CTP, UTP/7 and a-32P ATP
were resolved by analytical polyacrylamide gel electrophoresis
under denaturing conditions and phosphorimaged.8
Fig. 2 PAGE of oligoribonucleotide products obtained from in vitro
transcription reactions with templates S1–S5 in the presence of UTP
and modified UTP 7 under denaturing conditions. % incorporation of
7 is reported relative to a control reaction with UTP.7
digestion of 9 clearly revealed the incorporation of ribonucleoside 6
into the full-length oligoribonucleotide transcript (Fig. S3 and S4,
ESIw). In addition, mass measurement of HPLC fractions corres-
ponding to individual ribonucleosides also confirmed the integrity
of the natural as well as azide-modified ribonucleoside 6 present in
oligoribonucleotide 9 (Table S1, ESIw). Together, these results
unambiguously confirm the incorporation of azide-modified UTP
into oligoribonucleotides by transcription reactions.
A transcription reaction in the presence of template S1 and
UTP 7 afforded the full length oligoribonucleotide product 9
containing the modification at +7 position in an excellent
yield (Fig. 2, lane 2). A slight retardation in the migration of
modified transcript 9 compared to control transcript 8 revealed
the incorporation of the heavier ribonucleoside analogue 6 during
the transcription reaction (Fig. 2, compare lanes 1 and 2).
Importantly, the full length transcript was not detected in a
control reaction containing no UTP and 7, ruling out any
misincorporation during transcription reactions (Fig. 2, lane 3).
Interestingly, T7 RNA polymerase incorporated both natural
and modified UTP in a reaction containing these substrates in
an equimolar concentration (Fig. 2, lane 4, Fig. S1, ESIw). In
the presence of templates S2 and S3, the RNA polymerase
incorporated the triphosphate 7 near the promoter region
at +3 and +4 positions, respectively, with moderate efficiencies
(Fig. 2, lanes 6 and 8).9 Furthermore, reactions in the presence
of S4 and S5, which would result in the incorporation of 7 in
successive as well as in alternating sites, respectively, gave the
doubly-modified oligoribonucleotide products 12 and 13 in
good yields (Fig. 2, lanes 10 and 12).
Azide-modified oligoribonucleotide 9 was then subjected to
posttranscriptional chemical functionalization by CuAAC and
Staudinger reduction reactions (Scheme 2). Click reactions
were performed by incubating transcript 9 with alkyne building
blocks 1410 and 15 in the presence of a water-soluble Cu(I)
stabilizing ligand, tris-(3-hydroxypropyltriazolylmethyl)amine
(THPTA),4a,d CuSO4 and sodium ascorbate at 37 1C. Aliquots
of reaction mixtures (30, 60 and 180 min) were treated with calf
intestinal alkaline phosphatase (CIP) and resolved by analytical
PAGE under denaturing conditions. A reaction with 4-ethynyl-
1,8-naphthalimide derivative 14 resulted in the formation of the
fluorescent click product 16 in 30 min (Fig. 3, lanes 5–7).
Subsequently, a large-scale click reaction was performed, and
the band corresponding to the click product was isolated and
confirmed by mass analysis (Fig. S5, ESIw). Furthermore, the
fluorescent oligoribonucleotide 16 displayed an emission profile
similar to that of the click product (20) of a reaction between
ribonucleoside 6 and alkyne 14 (Fig. 4).7 However, a reaction
Oligoribonucleotide transcript 9, isolated from large-scale
transcription reactions with template S1, was further characterized
to confirm the presence of ribonucleoside 6 in the transcription
product (Fig. S2, ESIw). MALDI-TOF MS analysis of 9 and
HPLC analysis of ribonucleoside products arising from enzymatic
Fig. 1 Incorporation of azide-modified UTP 7 into oligoribonucleotides
by transcription reactions. Synthetic DNA template sequences (S1–S5)
and corresponding transcripts are also shown.7
Scheme 2 (a) CuAAC reactions with alkyne substrates 14 and 15.
(b) Staudinger reduction reaction.7
c
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cellular RNAs posttranscriptionally by click reaction using
ribonucleoside 6.11
We thank Prof. S. Galande for providing the facility to
carry out radiolabeling experiments, and Prof. K. N. Ganesh
for his valuable comments. S.G.S. is grateful to the Depart-
ment of Science and Technology, India, for financial support.
A.A.S. and A.A.T. are thankful to CSIR, India, for graduate
fellowship.
Fig. 3 PAGE of oligoribonucleotide products obtained from click
reactions between transcript 9 and alkyne substrates 14 and 15.
Lane 1, CIP treated 9. Lanes 2–4, reaction with 15. Lanes 5–7, reaction
with 14. Bands corresponding to the biotinylated- (17) and fluorescent-
(16) click products are indicated with arrows.7
Notes and references
1 (a) P. M. E. Gramlich, C. T. Wirges, A. Manetto and T. Carell,
Angew. Chem., Int. Ed., 2008, 47, 8350; (b) S. H. Weisbrod and
A. Marx, Chem. Commun., 2008, 5675; (c) M. D. Best, Biochemistry,
2009, 48, 6571; (d) E. Lallana, R. Riguera and E. Fernandez-Megia,
Angew. Chem., Int. Ed., 2011, 50, 8794.
2 (a) S. Jin, C. V. Miduturu, D. C. McKinney and S. K. Silverman,
J. Org. Chem., 2005, 70, 4284; (b) G. M. Blackburn, M. J. Gait,
D. Loakes and D. M. Williams, Nucleic Acids in Chemistry
and Biology, RSC, UK, 3rd edn., 2006.
with biotin-alkyne substrate 15 gave a small amount of the
expected click product 17 and also an unknown slower migrating
oligoribonucleotide as the major product (Fig. 3, lanes 2–4 and
Fig. S6, ESIw). Rewardingly, when the overall incubation time was
reduced by eliminating the dephosphorylation step, the biotinylated
click product was obtained as the major product, which was
confirmed by MALDI-TOF MS analysis (Fig. S7, ESIw).
Next, postsynthetic conversion of the azide to amine
functionality by Staudinger reduction reaction was investigated
(Scheme 2). Reduction of azide-modified oligoribonucleotide 9
was performed in the presence of triphenylphosphine-3,30,
30 0-trisulfonic acid (TPPTS), at 55 1C for 15 h. The reduced
product 18, after treatment with CIP, was purified by PAGE
under denaturing conditions and confirmed by mass analysis
(Fig. S8, ESIw). Importantly, under these conditions there
was no discernible degradation of the oligoribonucleotide
product. Together, these results clearly demonstrate that the
azide-modified oligoribonucleotides synthesized by transcription
reactions are highly suitable for posttranscriptional chemical
labeling protocols.
3 Selected examples: (a) S. H. Weisbrod and A. Marx, Chem.
Commun., 2007, 1828; (b) V. R. Sirivolu, P. Chittepu and
F. Seela, ChemBioChem, 2008, 9, 2305; (c) P. M. E. Gramlich,
S. Warncke, J. Gierlich and T. Carell, Angew. Chem., Int. Ed.,
2008, 47, 3442; (d) R. M. Franzini and E. T. Kool, J. Am. Chem.
Soc., 2009, 131, 16021; (e) Z. Pianowski, K. Gorska, L. Oswald,
C. A. Merten and N. Winssinger, J. Am. Chem. Soc., 2009,
131, 6492; (f) K. Furukawa, H. Abe, K. Hibino, Y. Sako,
S. Tsuneda and Y. Ito, Bioconjugate Chem., 2009, 20, 1026;
(g) Y. Xu, Y. Suzuki and M. Komiyama, Angew. Chem., Int.
Ed., 2009, 48, 3281; (h) C. Beyer and H.-A. Wagenknecht, Chem.
Commun., 2010, 46, 2230; (i) A. K. Sharma and J. M. Heemstra,
J. Am. Chem. Soc., 2011, 133, 12426.
4 Most of the methods use alkyne-modified RNA oligonucleotides.
(a) A. H. El-Sagheer and T. Brown, Proc. Natl. Acad. Sci. U. S. A.,
2010, 107, 15329; (b) P. van Delft, N. J. Meeuwenoord,
S. Hoogendoorn, J. Dinkelaar, H. S. Overkleeft, G. A. van der
Marel and D. V. Filippov, Org. Lett., 2010, 12, 5486;
(c) K. N. Jayaprakash, C. G. Peng, D. Butler, J. P. Varghese,
M. A. Maier, K. G. Rajeev and M. Manoharan, Org. Lett., 2010,
12, 5410; (d) H. Peacock, O. Maydanovych and P. A. Beal, Org.
Lett., 2010, 12, 1044; (e) E. Paredes and S. R. Das, ChemBioChem,
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S. Sasaki, Chem. Commun., 2011, 47, 5004.
In summary, the versatile azide group has been effectively
incorporated into RNA oligonucleotides by transcription
reactions in the presence of a new azide-modified ribonucleotide
analogue. This modification has allowed the postsynthetic chemical
functionalization of oligoribonucleotides by chemoselective
reactions such as CuAAC and Staudinger reactions. Our
results demonstrate that this facile and modular labeling
protocol will provide an alternative access to functionalized
oligoribonucleotides for applications in a variety of areas
ranging from diagnostics to therapeutics and material sciences.
We are currently investigating the possibility of labeling
5 T. Wada, A. Mochizuki, S. Higashiya, H. Tsuruoka, S. Kawahara,
M. Ishikawa and M. Sekine, Tetrahedron Lett., 2001, 42, 9215.
6 Few examples are available in which the azide group has been
introduced postsynthetically or during solid-phase chemical synthesis.
These methods are either time-consuming or involve elaborate
chemical manipulations. (a) G. P. Miller and E. T. Kool, J. Org.
Chem., 2004, 69, 2404; (b) A. Kiviniemi, P. Virta and H. Lonnberg,
¨
Bioconjugate Chem., 2008, 19, 1726; (c) G. Pourceau, A. Meyer,
J.-J. Vasseur and F. Morvan, J. Org. Chem., 2009, 74, 6837;
(d) M. Aigner, M. Hartl, K. Fauster, J. Steger, K. Bister and
R. Micura, ChemBioChem, 2011, 12, 47.
7 See ESIw for experimental details.
8 Failed reactions resulting in truncated transcripts would not carry
the radiolabel, and hence, would remain undetected.
9 Significant reduction in transcription efficiency is observed with
templates that result in a modification near the promoter region.
(a) S. G. Srivatsan and Y. Tor, J. Am. Chem. Soc., 2007, 129, 2044;
(b) M. G. Pawar and S. G. Srivatsan, Org. Lett., 2011, 13, 1114.
10 1,8-Naphthalimide derivatives are very useful fluorescent probes.
(a) M. Sawa, T.-L. Hsu, T. Itoh, M. Sugiyama, S. R. Hanson,
P. K. Vogt and C.-H. Wong, Proc. Natl. Acad. Sci. U. S. A., 2006,
103, 12371; (b) Y. Tian, F. Su, W. Weber, V. Nandakumar,
B. R. Shumway, Y. Jin, X. Zhou, M. R. Holl, R. H. Johnson
and D. R. Meldrum, Biomaterials, 2010, 31, 7411.
11 The ability of T7 RNA polymerase to incorporate both UTP and
7 suggests that cellular RNAs can be possibly functionalized with
azide groups in vivo by the ribonucleoside salvage pathway.
C. Y. Jao and A. Salic, Proc. Natl. Acad. Sci. U. S. A., 2008,
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Fig. 4 Fluorescence spectra (2 mM) of click products 16 and 20.7
500 Chem. Commun., 2012, 48, 498–500
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This journal is The Royal Society of Chemistry 2012