A diazirine-based nucleoside analogue for efficient DNA interstrand photocross-linking

Article abstract of DOI:10.1021/ja805445j

A diazirine-based nucleoside analogue (DBN) efficiently forms DNA interstand cross-linking under near-UV irradiation. This new base analogue may find broad applications in biotechnology and phototherapy. Copyright

Full text of DOI:10.1021/ja805445j

Published on Web 10/09/2008
A Diazirine-Based Nucleoside Analogue for Efficient DNA Interstrand
Photocross-Linking
Zhihai Qiu, Lianghua Lu, Xing Jian, and Chuan He*
Department of Chemistry, The UniVersity of Chicago, 929 East 57th Street, Chicago, Illinois 60637
Received July 14, 2008; E-mail: chuanhe@uchicago.edu
A common strategy for cancer therapy is to induce DNA damage
and subsequent apoptosis of cancer cells. Some of the most effective
anticancer agents in clinical use, such as cyclophosphamide,
mitomycin C, and cisplatin, exert their effects by creating DNA
interstrand cross-links.1-4 We report here the design and synthesis
of a diazirine-based nucleoside analogue (DBN, Figure 1) that can
be incorporated into DNA by solid phase oligonucleotide synthesis.
The DBN-containing dsDNA efficiently forms an interstranded
cross-link upon photoirradiation.
Figure 1. A diazirine-based nucleoside analogue (DBN) that would form
a DNA interstrand cross-link under photoirradiation.
Scheme 1. Synthesis of Diazirine-Based Nucleoside Analoguea
Diazirine derivatives have been widely used to investigate the
structure and function of nucleic acids, proteins, and various
macromolecular complexes.5-18 These compounds possess several
noticeable advantages over other photoaffinity groups: first, they
can be rapidly photolyzed at wavelengths (350-360 nm) beyond
the absorbance region of most biomacromolecules; second, they
form highly reactive carbenes upon photolysis, which readily cross-
link to various functional groups including the inert aliphatic C-H
bonds; third, the cross-linking reaction with carbenes produces
carbon-based bonds that are stable under typical experimental
conditions; finally, the diazirine derivatives are sterically less
hindered than other photoactive groups and possess excellent
chemical stability to be handled under moderate laboratory
illumination.
Various approaches have been developed to tether the aryl(tri-
fluoromethyl)diazirine group to the base (pyrimidine7-12 or
15,16
purine13,14) or the ribose part
of nucleoside analogues. All
these approaches, however, lead to extrahelical attachment of the
diazirine group to the DNA double helices. We envisioned that
direct installation of an aryl(trifluoromethyl)diazirine group to the
ribose may provide an intrahelical attachment of this group to the
DNA duplex (Figure 1). Upon UV irradiation the diazirine group
forms a carbene intermediate that could cross-link to the opposite
strand. In addition, the lack of steric hindrance of DBN may allow
it to be recognized by DNA modification or repair proteins to
facilitate protein-nucleic acid photocross-linking.
We developed a synthetic scheme that allows the production of
the corresponding phosphoramidite at a reasonable efficiency
(Scheme 1). The starting materials furanoid glycal 119,20 and 3-(4-
iodophenyl)-3-(trifluoromethyl)-3H-diazirine 210,21,22 were prepared
from commercially available thymidine and 1,4-diiodobenzene,
respectively. The critical C-glycoside bond formation was achieved
by ꢀ-anomeric selectivity using Heck chemistry23,24 under Jeffery’s
conditions.25 The free nucleoside analogue 5 was then obtained
from 3 by deprotection of the tert-butyldimethylsilyl group and
reduction with sodium triacetoxyborohydride.24 Two subsequent
steps following standard procedures were carried out to prepare
the 5′-O-dimethoxytrityl, 3′-O-phosphoramidite derivative 7 that
can be used for the solid phase oligonucleotide synthesis.
a
Reagents and conditions: (a) Pd(OAc)2, nBu4N+Cl-, Cy2NCH3, DMF,
60 °C (46%); (b) nBu4N+F-, THF/HOAc, rt (92%); (c) Na(OAc)3BH,
CH3CN/HOAc, -10 °C (56%); (d) DMTrCl, DMAP, pyridine, 0 °C (71%);
(e) NC(CH2)2OP(Cl)N(iPr)2, DIPEA, CH2Cl2, 0 °C (84%).
(Figure S1), which correspond to the mass of the trimer, the mass
of the trimer with the loss of N2, and the mass of the trimer reacted
with the matrix, respectively. When the deprotected product was
irradiated (near-UV, hV > 300 nm) in water for 10 min, its MALDI-
TOF mass spectrum gave a new peak corresponding to the product
of the carbene reacted with water, together with the disappearance
of the previous three peaks (Figure S2). This observation clearly
indicated the presence of the diazirine group in the trimer.
A 15-mer oligonucleotide sequence B1 (5′-ATG AAC CBG
GAA AAC-3′) was synthesized and purified following the same
protocol. It was annealed with complementary strands with A, T,
G, or C opposite B, respectively. The melting temperatures (Tm)
of the dsDNAs (in 10 mM Tris-HCl, pH 7.4, 100 mM NaCl) were
measured by differential scanning calorimetry (Table 1). With the
exception of the B:A pair (entry 1, Table 1), in which case the
melting behavior was not detected, the other three pairs (entries 2,
3, and 4, Table 1) lowered the Tm values by 18-21% (10-12 °C)
A trimer T-B(DBN)-T was synthesized and deprotected with
concentrated ammonia at room temperature overnight. The MALDI-
TOF mass spectrum of the deprotected product gave three peaks
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10.1021/ja805445j CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
photolyzed rapidly and the lifetime of its photolysis product carbene
is on a nanosecond time scale.
In summary, we have developed a nucleoside analogue that
efficiently forms interstrand cross-linking in dsDNA upon near-
UV irradiation. This nucleoside analogue may find broad applica-
tions in biotechnology such as probing nucleic acid-nucleic acid
and protein-nucleic acid interactions and in phototherapy. Further
studies of the details of the photocross-linking of this and analogous
nucleosides and their applications are currently ongoing in our
laboratory.
Figure 2. Interstrand photocross-link for the DBN paired with A, T, G,
and C, respectively (30 µM). B represents the 15-mer ssDNA containing
DBN; A, T, G, and C represent the complementary ssDNA strands shown
in Table 1.
Acknowledgment. We are grateful to Dr. P. R. Chen, Mr. U. K.
Shigdel, and Dr. J. Zhang for assistance and discussions. This
research is supported by the W. M. Keck Foundation, the Arnold
and Mabel Beckman Foundation, the Research Corporation, and a
Camille Dreyfus Teacher Scholar Award.
Table 1. Thermodynamic Properties of dsDNAs
∆H
∆S
Tm
(kcal·mol-1) (cal·K-1 ·mol-1) (°C)
entry dsDNA
sequences
Supporting Information Available: A detailed experimental sec-
tion, Figures S1-S9. This material is available free of charge via the
Internet at http://pubs.acs.org.
1
2
3
4
5
6
(B:A) 5′-ATG AAC CBG GAA AAC-3′
3′-TAC TTG GAC CTT TTG-5′
(B:T) 5′-ATG AAC CBG GAA AAC-3′
n.a.
63.0
47.5
66.4
-
n.a.
200
147
198
-
n.a.
45
48
46
55
58
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3′-TAC TTG GTC CTT TTG-5′
(B:G) 5′-ATG AAC CBG GAA AAC-3′
3′-TAC TTG GGC CTT TTG-5′
(B:C) 5′-ATG AAC CBG GAA AAC-3′
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