3-nifro-3-deaza-2′-deoxyadenosine as a versatile photocleavable 2′-deoxyadenosine mimic

Article abstract of DOI:10.1021/ja047976m

A new photocleavable 2′-deoxyadenosine mimic, 3-nitro-3-deaza-2′-deoxyadenosine (NidA), was prepared and introduced into DNA fragments via its 6-O-trimethylphenyl precursor phophoramidite. Photocleavage of the resulting oligonucleotide is highly efficient

Full text of DOI:10.1021/ja047976m

Published on Web 07/20/2004
3-Nitro-3-deaza-2′-deoxyadenosine as a Versatile Photocleavable
2′-Deoxyadenosine Mimic
Caroline Crey-Desbiolles, Jean Lhomme, Pascal Dumy, and Mitsuharu Kotera*
LEDSS, UMR5616 CNRS, ICMG FR-2607 - BP53, UniVersite´ Joseph Fourier, 38041 Grenoble, France
Received April 8, 2004; E-mail: mitsu.kotera@ujf-grenoble.fr
Scheme 2
The photochemical manipulation of DNA is one of the most
demanded technologies in the current context for development of
new DNA-based nanotechnologies. Photochemical reactions are
performed in reagent-free medium and allow spatial and temporal
control. While many DNA-photocleaving agents are known,1 only
a few photocleavable “building blocks” of DNA have been
developed.2 Most are based on the photochemical properties of a
o-nitrobenzyl group attached to rings devoid of hybridization
properties. It is desirable to introduce new photochemical properties
into well-designed modified DNA with minimum structural modi-
fications and, in particular, preserving specific recognition capacity.
We report here a photocleavable 2′-deoxyadenosine mimic,
3-nitro-3-deaza-2′-deoxyadenosine (NidA, Scheme 1). The design
Scheme 1
of NidA is based on the properties of 7-nitroindole nucleoside 7Ni.
We reported 7Ni as a precursor for introduction of the deoxyribo-
nolactone lesion in oligonucleotides.3 Irradiation of oligonucleotides
containing the photosensitive nitroindole nucleoside 7Ni triggers a
radical process in which the excited nitro group induces intramo-
lecular H-1′ abstraction leading to deoxyribonolactone. Subsequent
mild basic or thermal treatment leads to total cleavage of the DNA
strand (Scheme 1). Following this strategy, we studied the nucleo-
side analogue NidA in which the nitroindole moiety in 7Ni was
replaced by the isosteric 3-nitro-3-deazaadenine nucleus. The new
nucleoside possesses the two recognition sites present in adenine,
i.e., the H-acceptor internal N-1 nitrogen and the H-donor primary
amino group, and is expected to possess both the cleavage properties
of 7Ni and the hydrogen bonding capacities of adenine. We report
here on the synthesis of nucleoside NidA, its incorporation in
oligonucleotides, and subsequent irradiation and piperidine treatment
leading to strand cleavage. We also show that NidA in DNA
possesses the hybridization properties of adenine.
The strategy for incorporation of NidA is outlined in Scheme 2.
3-Deazahypoxanthine, prepared in three steps from 3,4-diamino-
pyridine,4 was nitrated and chlorinated to afford 6-chloro-3-nitro-
3-deazapurine 1. Glycosylation of 1 with chloro sugar 2 using
Robins’ conditions5 gave the hardly separable mixture of 9-N- and
7-N-isomers 3a and 3b, respectively, in a 41/59 ratio. It appeared
that the most efficient strategy for preparing the modified oligo-
nucleotide involved the incorporation of NidA as its 6-O-trimeth-
ylphenyl precursor 5.6 The mixture of 3a and 3b was thus treated
with 2,4,6-trimethyl phenolate to give the mixture of the corre-
sponding ethers from which the 9-N-derivative 4 was easily isolated
by column chromatography. Basic hydrolysis of the toluoyl pro-
tections gave the key trimethylphenyl ether nucleoside 5. We found
that the aryl ether substituent was quantitatively replaced by -NH2
to afford nucleoside NidA when 5 was treated with NH3 in the
conditions of final deprotection classically used in oligonucleotide
synthesis. Accordingly, 5 was tritylated and phosphitylated to give
phosphoramidite 6 that was used in solid-phase DNA synthesis.
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C O M M U N I C A T I O N S
Figure 1. PAGE analysis of irradiation (λ > 320 nm, pH 8, time and
treatment indicated) of 5′-32P-labeled 11-mer containing NidA 7 (0.5 µM)
(lanes 1-4, single stranded, and lanes 9-12, double stranded) and of 5′-
32P-labeled 11-mer containing 7Ni 8 (0.5 µM) (lanes 5-8, single stranded,
and lanes 13-16, double stranded). Bands a and b are assigned to secondary
products resulting from piperidine and DTT (present in labeling buffer)
addition on lactone in 9 as observed previously.8
Figure 3. Melting curves of duplex 10 [5 µM]. pH 7 in PO4 buffer (10
mM), [EDTA] ) 1 mM, [NaCl] ) 100 mM. R and Tm values were
determined according to ref 9.
These results show that the nucleoside analogue NidA constitutes
a remarkable photocleavable 2′-deoxyadenosine mimic. It can be
efficiently prepared and introduced into any preselected site in
oligonucleotides. Photocleavage is quantitative and rapid, both in
single and double strands. Its hybridization properties are very
similar to those of the natural adenosine nucleoside. Enzymatic
incorporation in DNA is currently under way.
Acknowledgment. We thank Dr. C. Philouze for crystal-
lographic analysis of NidA, Dr. J.-F. Constant and Dr. N. Berthet
for their assistance with PAGE analysis as well as helpful comments
and discussions regarding these analyses, and Dr. William Moneta
and M. Vincent Steinmez for MS.
Figure 2. Perspective view of the crystallographic structure of NidA
showing the distance between the oxygen of the nitro group and the
anomeric hydrogen.
The 11-mer 5′-d(CGCAC-NidA-CACGC)-3′ 7 was thus prepared
using standard conditions.7 ES-MS analysis of the oligonucleotide
confirmed its structure, which additionally proved the introduction
of the NH2 amino group in the NidA moiety during the last step of
the synthesis (see Supporting Information).
Oligonucleotide 7 was irradiated at λ > 320 nm in dilute aqueous
solution at room temperature. After 60 min, the NidA-containing
oligonucleotide was transformed into the deoxyribonolactone-
containing oligonucleotide 5′-d(CGCAC-dL-CACGC)-3′ 9 that was
characterized by ES-MS (see Supporting Information). Mild pi-
peridine treatment of 9 led to total cleavage of the modified strand
(Figure 1). Quite interestingly, PAGE analysis shows that the photo-
induced cleavage process is also efficient in the double-stranded
state (T facing NidA in the complementary strand). It also reveals
that the process is at least as efficient as that observed for the
previously reported 7Ni-containing oligonucleotide 5′-d(CGCAC-
7Ni-CACGC)-3′ 8.
The efficiency of the photoreaction may be attributed to the
highly favorable conformation of the NidA nucleotide that presents,
at least in the solid state, an anti conformation in which the H-1′
to be abstracted is located at a 2.1 Å distance from the oxygen of
the nitro group (Figure 2).
Hybridization properties of NidA were examined by measuring
the melting temperatures (Tm) of duplex 5′-d(GCGTG-X-GTGCG)-
3′/5′-d(CGCAC-Y-CACGC)-3′10 (Figure 3) X ) T, C; Y ) A,
NidA. Very similar Tm values were determined respectively for
the matched duplexes T:A and T:NidA (Tm ) 59 and 60 °C,
respectively) and for the mismatched duplexes 9 C:A and C:NidA
(Tm ) 46 and 46 °C, respectively). CD spectra of the A- and NidA-
containing oligomers were also similar (see Supporting Informa-
tion).
Supporting Information Available: Detailed synthetic procedures
for 6 and 7, ES-MS and HPLC analyses of 7 and 8, and CD spectrum
of duplex 10 (PDF), as well as X-ray crystallographic data in CIF format
for NidA. This material is available free of charge via the Internet at
http://pubs.acs.org.
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