Fast, copper-free click chemistry: A convenient solid-phase approach to oligonucleotide conjugation

Article abstract of DOI:10.1039/b904185k

Solid-phase oligonucleotide conjugation by nitrile oxide-alkyne click cycloaddition chemistry has been successfully demonstrated; the reaction, compatible with all nucleobases, requires no metal catalyst and proceeds under physiological conditions.

Full text of DOI:10.1039/b904185k

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COMMUNICATION
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Fast, copper-free click chemistry: a convenient solid-phase approach
to oligonucleotide conjugationw
Ishwar Singh,a Joseph S. Vyleb and Frances Heaney*a
Received (in Cambridge, UK) 2nd March 2009, Accepted 9th April 2009
First published as an Advance Article on the web 22nd April 2009
DOI: 10.1039/b904185k
Solid-phase oligonucleotide conjugation by nitrile oxide–alkyne
click cycloaddition chemistry has been successfully demon-
strated; the reaction, compatible with all nucleobases, requires
no metal catalyst and proceeds under physiological conditions.
toward this goal have appeared in the literature. These too
have their limitations. First, a ruthenium-catalysed azide–
alkyne ‘‘fusion’’ reaction proceeds best in organic solvents with
yields and regioselectivity compromised in protic solvents.7
Second, Bertozzi et al.’s strain promoted azide–alkyne reaction
is not regiospecific. And third, the required strained cyclic
alkynes are available only following long synthetic sequences.8
Thus, a void exists in the field of chemical biology for a fast,
reliable, copper-free, solid-phase oligonucleotide conjugation
strategy which will proceed under physiological conditions.
We considered it unlikely that azides should be the only
family of Huisgen classified 1,3-dipoles9 with potential to act
as click cycloaddition partners and that the isoxazole generating
nitrile oxide–alkyne reaction has many attractions. Firstly, the
ease of dipole formation.10 Secondly, cycloaddition of nitrile
oxides to alkynes is calculated to be B6 kcal molꢀ1 lower than
that for similar azides, implying acceptable reaction rates at
room temperature.11 Thirdly, reactions of nitrile oxides with
monosubstituted alkynes are regioselective for formation of
3,5-disubstituted adducts.12 Finally, the isoxazole ring, present
in many bioactive compounds, has many possible modes of
action with biological molecules.13
The availability of reliable, robust and efficient chemistry for
the provision of oligonucleotide conjugates is central to much
of contemporary genomic research and to studies directed
toward nucleic acid therapies. Whilst both solution- and
solid-phase syntheses of such conjugates have been demon-
strated, the latter has many advantages, particularly in terms
of product purification. Generally, to encourage effective
coupling, the syntheses are conducted with large excesses of
the conjugating group and in the solid-phase approach excess
reagents, together with undesired side products, can simply be
removed by washing. To this end we wish to report an effective
strategy for chemical modification of DNA by copper-free,
click cycloaddition chemistry in the solid-phase.
Seminal insights into the application of the Huisgen–
Meldal–Sharpless azide–alkyne cycloaddition, considered the
prototype of click chemistry,1 for solution-phase oligonucleotide
bioconjugation have been provided by a number of labora-
tories.2 However, reports on solid-supported oligonucleotide
modification by Cu(I) promoted azide–alkyne click reactions
are limited2b,c and some proceed only with microwave
activation.2g Despite the successes of these reactions a number
of concerns persist. Apprehension exists over the handling of
potentially toxic and explosive organic azides3 and whilst in situ
dipole generation begins to address this problem,4 the require-
ment for a Cu(I) catalyst brings its own technical difficulties.5
To help deter the DNA degrading redox chemistry of Cu(I) the
reaction must be conducted under air-free conditions2f in the
presence of a tris-triazolylamine Cu(I) ligand.2d,6 Finally,
extensive washing is recommended to ensure quantitative
removal of the copper salt and the ligand during the reaction
work-up.2h Against this background a fast, copper-free, alter-
native to the azide–alkyne click reaction is highly desirable. To
date, no solid-phase alternative to the Cu(I) promoted reaction
has been reported and only two solution-phase approaches
The hypothesis that nitrile oxide–alkyne click chemistry
offers a reliable tool to chemically modify oligonucleotides
was initially tested in solution on the nucleoside 1. Chloramine-T
was selected as the dipole generating agent;10b its use in
antibody-catalysed 1,3-dipolar cycloaddition reactions14 and
in the preparation of 125I-labelled oligonucleotides15 suggests,
with judicious choice of reacting concentration, compati-
bility with biological systems. Treatment of 1 with benzonitrile
oxide, generated in situ from benzaldehyde oxime, and
chloramine-T afforded isoxazole-modified thymidine 2,
eqn (1). A singlet resonance at 6.59 ppm in the 1H NMR
spectrum, diagnostic for the 4-H proton, confirms the regio-
specificity of the reaction.
a Department of Chemistry, National University of Ireland, Maynooth,
Co. Kildare, Republic of Ireland. E-mail: mary.f.heaney@nuim.ie;
Fax: +353 1708 3815; Tel: +353 1708 3802
b School of Chemistry and Chemical Engineering, The Queen’s
University of Belfast, David Keir Building, Stranmillis Road, Belfast,
Northern Ireland, UK. E-mail: j.vyle@qub.ac.uk;
Fax: +44 (0)28 9097 652; Tel: +44 (0)28 9097 5485
w Electronic supplementary information (ESI) available: Experimental
procedures, NMR spectra, fluorescence spectra, melting temperature
determinations, MALDI-TOF MS data and HPLC data. See DOI:
10.1039/b904185k
ð1Þ
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Scheme 1 Solid-phase synthesis of isoxazole-ligated oligonucleotides.
(a) DNA = thymidine, (b) DNA = T10, (c) DNA = 50-TCGCACA-
CACGC-30. (i) 4, BMT, CH3CN, rt, 30 min then I2 (0.1 M) THF–
pyridine–H2O; (ii) chloramine-T, aq NaHCO3 (4%), EtOH, rt
(a) 30 min (b) 30 min (c) 10 min.
Fig. 2 Reversed-phase HPLC analysis of crude reaction products
(UV absorbance at 260 nm vs. time). (a) Linear oligonucleotide (7b),
(b) isoxazole-oligonucleotide (9b), (c) isoxazole-oligonucleotide (10a),
(d) isoxazole-oligonucleotide (10b), (e) linear oligonucleotide (7c), (f)
isoxazole-oligonucleotide (9c).
The inherent advantages of the solid-phase approach to oligo-
nucleotide conjugation, viz less intensive purification, lead
to demonstration of the resin-supported nitrile oxide click
reaction. Commercially available 500 A CPG-succinyl nucleoside
support was selected. The phosphoramidite alkyne 4 was
synthesised, eqn (2), and attached directly to the 50-position
of CPG-loaded thymidine 5a, Scheme 1. Reversed-phase
HPLC analysis of a sample of 7a, obtained following
the deprotection–cleavage protocol, indicated quantitative
conversion to the thymidine-alkyne 6a, Fig. 1. The reactivity
of the support-bound alkyne 6a in nitrile oxide click cyclo-
addition chemistry was tested by exposing it to benzaldehyde
oxime and chloramine-T in ethanolic NaHCO3, Scheme 1.
Following deprotection and cleavage, near-quantitative
conversion to the isoxazole-nucleotide conjugate 9a was evidenced
by HPLC analysis. MALDI-TOF-MS of 7a and 9a confirmed
the structural integrity of both products.
A fluorogenic version of the reaction confirms its efficacy
for biolabelling applications. Since polycyclic aromatic
compounds are known base surrogates for DNA16 and since
anthracenes intercalate DNA strands and their fluorescent
properties are tunable,17 in situ generated naphthalene 1-nitrile
oxide and anthracene 9-nitrile oxide were selected as click
partners for CPG-T10-alkyne 6b. Employing the chloramine-T
methodology, quantitative conversion to the clicked products
was judged from HPLC analysis on 10a,b, obtained after
deprotection–cleavage, Fig. 2c,d. MALDI-TOF-MS data
confirmed the structures of 10a,b. The fluorescence spectrum
of the naphthalene bearing single stranded oligonucleotide 10a
showed a strong fluorescence with an emission maximum at
376 nm and an excitation at 310 nm whilst its anthracene
analogue 10b showed an emission maximum at 429 nm and an
excitation at 350 nm.
To have real value for oligonucleotide bioconjugation the
solid-phase click reaction must be compatible with nucleobases
more susceptible to mutagenic modification, i.e. cytosine,
guanine and adenine. Thus, the resin-bound dodecamer 6c
was prepared. HPLC analysis, Fig. 2e, and MALDI-TOF-MS
characterisation of 7c confirmed successful attachment of the
alkyne. However, the hetero-oligonucleotide was sensitive
to the conditions used to effect the click reaction; optimal
conditions involved reaction of 6c (0.2 mM) in ethanolic
aqueous NaHCO3 (1 : 2) with chloramine-T (114 mg) and
oxime (31 mg) for 10 minutes at room temperature. HPLC
analysis showed good conversion to the isoxazole-modified
DNA, 9c, Fig. 2f and the MALDI-TOF-MS unambiguously
confirmed the expected mass of the isoxazole-ligated product
9c. The thermal consequences of isoxazole introduction were
evaluated by UV melting experiments. The unmodified
oligonucleotide 11, 50-TCGCACACACGC-30, and the click
functionalized derivative 9c were hybridised with the comple-
mentary strand 12, 50-GCGTGTGTGCGA-30. The duplex 9cꢂ
12 shows a Tm value of 69.7 ꢃ 0.7 1C whilst the reference
duplex 11ꢂ12 shows a Tm value of 67.0 ꢃ 0.9. Thus, it is clear
that the isoxazole moiety enhances DNA duplex stability.
In conclusion, a solid-phase nitrile oxide–alkyne click
reaction has been used to form isoxazole conjugated oligo-
nucleotides. The procedure, which has potential for application
in bioconjugation, polymer and materials science and drug
discovery, is selective, convenient and fast. It occurs under
ð2Þ
A parallel reaction sequence with CPG-supported decathy-
midylate, 5b, confirmed compatibility with longer chain oligo-
nucleotides and the isoxazole-ligated decamer 8b formed from
the resin-bound DNA-alkyne 6b. Quantitative yields for the
coupling and the click reactions were evident from HPLC
analysis, Fig. 2a,b. MALDI-TOF-MS data confirmed the
structure of the alkyne 7b and the isoxazole-ligated-T10, 9b.
A
control experiment verifies the chemoselectivity of
the reaction, and CPG-supported decathymidylate 5b was
returned unchanged following experimentation under the
conditions of the nitrile oxide–alkyne click reaction, thus
confirming that the nitrile oxide reacts only with the alkyne.
Fig. 1 Structures of modified oligonucleotides following cleavage
from the support and deprotection. (a) DNA
(b) DNA = T10, (c) DNA = 50-TCGCACACACGC-30.
= thymidine,
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atmospheric conditions, in aqueous solvents, within minutes
not hours, and is high yielding and highly regioselective.
Importantly, it does not require a Cu(I) catalyst nor an
oxygen-free environment. Thus, the solid-phase nitrile
oxide–alkyne reaction offers a valuable click alternative to
azide–alkyne chemistry for applications in oligonucleotide
bioconjugation.2
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