Tris(azidoethyl)amine hydrochloride; A versatile reagent for synthesis of functionalized dumbbell oligodeoxynucleotides

Article abstract of DOI:10.1021/ol400001w

Triazole-cross-linked oligodeoxynucleotides were synthesized using the Cu(I) catalyzed alkyne-azide cycloaddition with tris(azidoethyl)amine hydrochloride and oligodeoxynucleotides possessing N-3-(propargyl)thymidine at both the 3′- and 5′-termini. Further installation of a functional molecule to the dumbbell oligodeoxynucleotides was achieved by utilizing the remaining azide group.

Full text of DOI:10.1021/ol400001w

ORGANIC
LETTERS
2013
Vol. 15, No. 3
694–697
Tris(azidoethyl)amine Hydrochloride;
a Versatile Reagent for Synthesis
of Functionalized Dumbbell
Oligodeoxynucleotides
Satoshi Ichikawa,* Hideaki Ueno, Takuya Sunadome, Kousuke Sato, and
Akira Matsuda
Kita-12, Nishi-6, Kita-ku, Faculty of Pharmaceutical Sciences, Hokkaido University,
Sapporo 060-0812, Japan
ichikawa@pharm.hokudai.ac.jp
Received January 1, 2013
ABSTRACT
Triazole-cross-linked oligodeoxynucleotides were synthesized using the Cu(I) catalyzed alkyneꢀazide cycloaddition with tris(azidoethyl)amine
hydrochloride and oligodeoxynucleotides possessing N-3-(propargyl)thymidine at both the 30- and 50-termini. Further installation of a functional
molecule to the dumbbell oligodeoxynucleotides was achieved by utilizing the remaining azide group.
Our recent knowledge of novel aspects of DNA and
RNA research has been motivational for the further
development of nucleic acid based chemistry. Especially,
relatively short stranded nucleic acids have potential as
therapeutic agents including siRNAs, miRNAs, and apta-
mers, and much effort has been dedicated to functionalize
DNA and RNA with appropriate properties in order to
develop oligonucleotide therapeutics. In applying DNA or
RNA as therapeutic agents, there are several shortcomings
associated with the use of short stranded nucleic acids,
making their use in biological systems unfeasible as yet.
Generally, short double-stranded oligodeoxynucleic acids
(ODNs) used in these studies possess less thermal stability
under physiological conditions. Another drawback is the
presence of extra- and intracellular nucleases, which cleave
ODNs.1,2 Cross-linking double-stranded ODNs are one of
the solutions to achieve thermal stability and resistance to
nucleases.3ꢀ11 Dumbbell ODNs, which are circular nucleic
(2) Chu, B. C. F.; Orgel, L. E. Nucleic Acids Res. 1992, 20, 5857–5858.
(3) Cowart, M.; Benkovic, S. J. Biochemistry 1991, 30, 788–796.
(4) Milton, J.; Connolly, B. A.; Nikiforov, T. T.; Cosstick, R.
J. Chem. Soc., Chem. Commun. 1993, 779–780.
(5) Ferentz, A. E.; Keating, T. A.; Verdine, G. L. J. Am. Chem. Soc.
1993, 115, 9006–9014.
(6) Erlanson, D. A.; Chen, L.; Verdine, G. L. J. Am. Chem. Soc. 1993,
115, 12583–12584.
(7) Kool, E. T. Annu. Rev. Biophys. Biomol. Struct. 1996, 25, 1–28.
(8) Kool, E. T. Acc. Chem. Res. 1998, 31, 502–510.
(9) Murakami, A.; Yamamoto, Y.; Namba, M.; Iwase, R.; Yamaoka,
T. Bioorg. Chem. 2001, 29, 223–233.
(10) Glick, G. D. J. Org. Chem. 1991, 56, 6746–6747.
€
(11) Osborne, S. E.; Volker, J.; Stevens, S. Y.; Breslauer, K. J.; Glick,
(1) Uhlmann, E.; Peyman, A. Chem. Rev. 1990, 90, 543–583.
G. D. J. Am. Chem. Soc. 1996, 118, 11993–12003.
r
10.1021/ol400001w
Published on Web 01/22/2013
2013 American Chemical Society

acids consisting of double-stranded stem region and nu-
cleotide loops at both of their termini, possess increased
exonuclease resistance because they have no terminal
nucleotide residues.12ꢀ15 Chemical modification includ-
ing cross-linking of ODNs by click chemistry is a grow-
ing field.16 Recently we have reported that triazole-
cross-linked ODNs were synthesized using the Cu(I) cata-
lyzed alkyneꢀazide cycloaddition (CuAAC)17,18 with ODNs
possessing N-3-(azidoethyl)thymidine and N-3-(propargyl)-
thymidine at the 30- and 50-termini (Figure 1a).19 The
newly synthesized ODNs possess thermal stability and
showed excellent resistance against snake venom phos-
phodiesterase (30-exonuclease), whose properties are
necessary for decoy molecules to achieve biological re-
sponses leading to alteration of gene expression. However,
the N-3-(iodoethyl)thymidine 30-phosphoramidite was ne-
cessarily used as a precursor of the N-3-(azidoethyl)-
thymidine residue because azido groups are known to be
readily reduced by a trivalent phosphorus atom during the
automated DNA synthesis.20 Once introduced in solid
phase DNA synthesis via the general phosphoramidite
method, the iodo group is converted to the corresponding
azide group via the on-column application of NaN3. In
order to overcome this shortcoming, we report herein the
development of tris(azidoethyl)amine hydrochloride (1)
as a novel cross-linking reagent of ODNs by the CuAAC
(Figure 1b).
Our improved strategy to access the dumbbell ODNs
is to use the multivalent azide linker as an external cross-
linking source and N-3-(propargyl)thymidine as a partner
unit, which is incorporated into the ODN at both termini.
This allowed us to avoid using any precursor amidite units
thatneededto be convertedtothe corresponding azide unit
during the automated ODN synthesis. Furthermore, in-
stallation of a functional molecule to the dumbbell ODNs
is now possible by utilizing the remaining azide group.
Scheme 1. Preparation of Tris(azidoethyl)amine Hydrochloride
Tris(azidoethyl)amine hydrochloride (1) was prepared
as shown in Scheme 1. Tris(ethanol)amine hydrochloride
(2) was treated with SOCl2 in CHCl3 at reflux for 3 h,
and the resulting tris(2-chloroethyl)amine (3) was reacted
with NaN3 in DMF to give tris(azidoethyl)amine. After
the aqueous workup, the free amine21 was converted to
its hydrochloride salt, which was crystallized from AcOEt.
The salt has good solubility in water and is suitable for
cross-linking biomolecules in aqueous media.
With the cross-linking reagent 1 in hand, the formation
of the dumbbell ODN was examined. As in the case of our
previous system, most of the strategies to cross-link ODNs
are donein anintramolecular fashion. Different from these
strategies, the first step of the reaction sequence in this
study is an intermolecular CuAAC of 1 with either of the
propargylgroups atthe termini of double-stranded ODNs.
The following CuAAC is in the intramolecular mode.
The intermolecular CuAAC could be challenging be-
cause simultaneous CuAAC at both propargyl groups at
one terminal could proceed, resulting in termination of the
cross-linking or oligomerization. In order to see the ability
of 1 to cross-link ODN, a single stranded hairpin ODN
(hpODN), where two N-3-(propargyl)thymidines were
attached at the 50- and 30-ends, was first investigated for
cross-linking at the single terminus (Scheme 2). Thus, after
the annealing of hpODN, the CuAAC was carried out by
(16) Recent review: El-Sagheer, A. H.; Brown, T. Chem. Soc. Rev.
2010, 39, 1388–1405. See also the following works: Pujari, S. S.; Xiong,
H.; Seela, F. J. Org. Chem. 2010, 75, 8693–8696. Kocalka, P.;
El-Sagheer, A. H.; Brown, T. ChemBioChem 2008, 9, 1280–1285.
El-Sagheer, A. H.; Brown, T. Int. J. Pept. Res. Ther. 2008, 14, 367–372.
(17) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002,
67, 3057–3064.
Figure 1. Formation of triazol cross-linked dumbbell ODNs by
CuAAC.
(18) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596–2599.
(19) Nakane, M.; Ichikawa, S.; Matsuda, A. J. Org. Chem. 2008, 73,
1842–1851.
(12) Hosoya, T.; Takeuchi, H.; Kanesaki, Y.; Yamakawa, H.; Miyano-
Kurosaki, N.; Takai, K.; Yamamoto, N.; Takaku, H. FEBS Lett. 1999,461,
136–140.
(13) Erie, D.; Sinha, N.; Olson, W.; Jones, R.; Breslauer, K. Bio-
chemistry 1987, 26, 7150–7159.
(14) Chu, B. C. F.; Orgel, L. E. Nucleic Acids Res. 1992, 20, 5857–
(20) Wada, T.; Mochizuki, A.; Higashiya, S.; Tsuruoka, H.; Kawa-
hara, S.; Ishikawa, M.; Sekine, M. Tetrahedron Lett. 2001, 42, 9215–
9219.
5858.
(21) Free amine is a known compound. See: Wltuckl, E. F.; Wilson,
E. R.; Flanagan, J. E.; Frankel, M. B. J. Chem. Eng. Data 1983, 28, 285–
286.
(15) Abe, N.; Abe, H.; Ito, Y. J. Am. Chem. Soc. 2007, 129, 15108–
15109.
Org. Lett., Vol. 15, No. 3, 2013
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Scheme 2. Formation of Dumbbell ODN by CuAACa
a P = N-3-(propargyl)thymidine residue; T = N-3-(substituted)
thymidine residue. Reaction was conducted under the conditions with
1 (20 equiv), CuSO4 (10 equiv), TG-TBTA(10 equiv), Na ascorbate
(20 mM), NaCl (50 mM) in MOPS buffer (pH 7.0, 2 mM) at room
temperature for 6 h.
adding a solid-supported copper(I) catalyst immobilized
with tris(benzyltriazolyl)amine onto a Tentagel resin22
(TG-TBTA, 10 equiv) to a solution of hpODN (0.1 μM)
and 1 (10equiv) in2 mMMOPSbuffer (pH7.0) containing
20 mM sodium ascorbate and 50 mM NaCl at room
temperature. The progress of the reaction was monitored
by HPLC (Figure 2a,c). A new peak was observed at a
retention time of 9.5 min (10 min for hpODN), and the
reaction was complete within 6 h. The structure of the
newly formed ODN (tzODN) was determined by analyz-
ing MALDI-TOF MS and a composition of nucleosides
as follows. Thus, the resulting tzODN was purified by
reversed-phase HPLC (33% isolated yield),23 and enzy-
matic digestion of the ODN showed a composition
of nucleosides. As a result, no peak corresponding to
N-3-(propargyl)thymidine (P) was detected by HPLC
analysis, and the bistriazole-bridged thymidine 4 was
formed instead (Figure 2b,d).24 The resulting nucleoside
composition was in good accordance with the desired one,
and mono- or tris-triazole derivative was not observed in
the reaction mixture. Of note is a rather preferred intra-
molecular CuAAC with ring closure for hpODN. The use
Figure 2. Reversed-phase HPLC chromatograms (UV absor-
bance vs time) in the CuAAC (a, c, and e) and their nucleoside
composition analysis (b, d, and f). (a) hpODN in the absence of
Cu[I] catalysis; (b) enzymatic digestion of hpODN; (c) after the
CuAAC of hpODN; (d) enzymatic digestion of tzODN; (e) after
the CuAAC of tzODN with 5; (f) enzymatic digestion of dansyl-
tzODN.
of an excess amount of tris(azidoethyl)amine (up to 1000
equiv) still gave the tzODN as a main product. It would be
because the increased number of the reactive sites with two
azide groups, which can be cross-coupled with the alkyne
in the opposite strand, and these reactive groups are in
close proximity in space at the terminus of the strand for
the second CuAAC. Next, cross-linking by 1 was investi-
gated with a double-stranded ODN (dsODN), which has
the cross-linking site at both termini. When the same
conditions to prepare tzODN were applied to dsODN,
the desired tz2ODN was afforded supported by a composi-
tion analysis of nucleosides (Figures S2, S5). This would
imply that the CuAAC sequence was successfully achieved
and the triazole cross-linking indeed occurred at both
termini providing a dumbbell ODN. UV melting experi-
ments were then conducted to characterize the thermally
induced denaturation of ODNs. Comparing the Tm values
for cross-linked tzODN (79.4 °C) or tz2ODN (87.9 °C)
with that of the control hpODN (47.6 °C) or dsODN
(44.1 °C) revealed a dramatic increase (>30 °C) in thermal
(22) Chan, T. R.; Fokin, V. V. QSAR Comb. Sci. 2007, 26, 1274–1279.
(23) The isolated yields of dumbbell ODNs after CuAAC were
moderate (33ꢀ41% yield after HPLC purification) although the HPLC
analysis of the reaction mixture showed relatively clean conversion (see
Figure 2c). This is true for any CuAAC reactions in this study although
the reason is unclear.
(24) Authentic triazole derivatives were also prepared as shown in
Scheme S1.
696
Org. Lett., Vol. 15, No. 3, 2013

suggest that the terminal nucleobase modifications do not
significantly distort the helical geometry of the duplex.
Having established the cross-linking of hpODN by
the CuAAC, the next CuAAC was examined on tzODN.
As representative examples, a fluorophore and a peptide
were chosen as the functional molecules. The CuAAC of
tzODN and N-propargyldansylamide26 (5, 100 equiv) was
carried out (Scheme 3). As a result, complete consumption of
tzODN occurred, and a new peak possessing absorption at
340 nm was observed in the HPLC analysis (Scheme S2b).
MALDI-TOF MS and a composition analysis of the newly
formed peak unambiguously determined the formation of
the desired dansyl-tzODN (41% isolated yield, Figure 2e,f),23
a functionalized dumbbell ODN. Reaction of tzODN with
peptide 6 also gave peptidyl-tzODN (44% isolated yield,
Figure S3).
Scheme 3. Functionalization of Dumbbell ODN by CuAACa
In conclusion, tris(azidoethyl)amine hydrochloride (1)
was developed as a novel cross-linking and functionalizing
reagent of ODNs by the CuAAC.
Triazole-cross-linked functionalized dumbbell ODNs
were successfully synthesized by two sequential CuAACs
with ODNs possessing N-3-(propargyl)thymidine at the 30-
and 50-termini. This strategy is effective for the preparation of
cross-linked ODNs because the precursor ODNs are stable
and easy to prepare. This study should now enable us to
functionalize DNA and RNA with appropriate properties in
order to develop oligonucleotide therapeutics.
a Reaction was conductedunder the conditions with alkyne derivative
(100 equiv), CuSO4 (10 equiv), TG-TBTA(10 equiv), Na ascorbate
(20 mM), NaCl (50 mM) in MOPS buffer (pH 7.0, 2 mM) for 6 h.
Acknowledgment. This work was supported by a JSPS
Grant-in-Aid for Scientific Research A (AM, Grant No.
23249008) and Grant-in-Aid for Challenging Exploratory
Research (SI, Grant No. 22659020). We thank Ms. S. Oka
and Ms. A. Tokumitsu (Center for Instrumental Analysis,
Hokkaido University) for measurement of the mass spectra.
stability with the triazole cross-link. The control ODNs
(hpODN and dsODN) displayed a characteristic B-DNA
spectrum,25 and tzODN and tz2ODN possessing the tria-
zole cross-linked thymidines at the end(s) of the helix
displayed similar CD spectra (Figure S7). These results
Supporting Information Available. Experimental pro-
cedures, NMR spectra for new compounds, HPLC chro-
matograms, CD spectra. This material is available free of
charge via the Internet at http://pubs.acs.org.
(25) Ivanov, V. I.; Minchenkova, L. E.; Schyolkina, A. K.; Poletayev,
A. I. Biopolymers 1973, 12, 89–110.
(26) Bolletta, F.; Fabbri, D.; Lombardo, M.; Prodi, L.; Trombini, C.;
Zaccheroni, N. Organometalics 1996, 15, 2415–2417.
The authors declare no competing financial interest.
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