Interstrand photocrosslinking of DNA via p-carbamoylvinyl phenol nucleoside

Article abstract of DOI:10.1016/j.bmcl.2005.01.029

We report a novel interstrand photocrosslinking of oligodeoxynucleotides (ODNs). In this system, a modified ODN containing p-carbamoylvinyl phenol nucleoside reacts by photoirradiation at 366 nm with adenine residue of a complementary template ODN to yield a crosslinked ODN in 97% yield.

Full text of DOI:10.1016/j.bmcl.2005.01.029

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1299–1301
Interstrand photocrosslinking of DNA via p-carbamoylvinyl
phenol nucleoside
Yoshinaga Yoshimura,b,* Yoshiaki Itoa and Kenzo Fujimotoa,b,
*
aThe School of Materials Science, Japan Advanced Institute of Science and Technology, Ishikawa 923-1292, Japan
bPRESTO, Japan Science and Technology Agency, Ishikawa 923-1292, Japan
Received 26 November 2004; revised 11 January 2005; accepted 13 January 2005
Abstract—We report a novel interstrand photocrosslinking of oligodeoxynucleotides (ODNs). In this system, a modified ODN con-
taining p-carbamoylvinyl phenol nucleoside reacts by photoirradiation at 366 nm with adenine residue of a complementary template
ODN to yield a crosslinked ODN in 97% yield.
ꢀ 2005 Elsevier Ltd. All rights reserved.
The linkage of DNA binding agents to ODNs has be-
come a popular strategy to enhance the efficiency of
antisense and triplex inhibition of cellular processes.1
Psoralens have been used for many years as interstrand
photocrosslinker of nucleic acids.2 Psoralens form a
reversible intercalative complex with DNA and can un-
dergo subsequent photoactivation with near-UV light
and then crosslink through a [2 + 2] cycloaddition with
thymine residues. However, interstrand photocrosslink
occurs between thymine residues at the duplex site 50-
TA-30 preferentially.3 To overcome such a sequence
dependence, the study of photocrosslinker has been
developed. The photochemical [2 + 2] cycloaddition of
cinnamic acid and its methyl esters with alkenes is one
of the most extensively investigated and synthetically
useful of photochemical reactions.4 These photoreac-
tions of cinnamic acid are expected to use as photocross-
linker for the study of interstrand photocrosslink of
nucleic acids. Template directed reversible DNA photo-
ligation via 5-vinyldeoxyuridine has already reported as
a tool for DNA engineering and nanotechnology.5
The synthesis of the phosphoramidite of methyl ester of
p-CVP is outlined in Scheme 1. Compound 3 was synthe-
sized from 4-iodophenol and Hofferꢀs a-chlorosugar.
Deprotection of 3 with sodium methoxide afforded 4.
Compound 4 was coupled with methylacrylate to afford
5.7 Compound 5 was dimethoxytritylated, and con-
verted into the nucleoside phosphoramidite 7. The
assignments of b-stereochemistry at C10 for 5 was based
on COSY and NOESY spectra, which showed the
cross-peak between H10 and H40. The modified ODN
Here we report the development of a novel interstrand
photocrosslink via p-carbamoylvinyl phenol nucleoside
(
p-CVP) in duplex DNA.6 We also demonstrate that the
modified ODN containing p-CVP was photocrosslinked
with adjacent adenine residue by UV irradiation.
Scheme 1. Reagents and conditions: (a) NaH, chlorosugar, THF,
room temperature, 19 h, 62%; (b) NaOCH3, THF, room temperature,
15 h, 49%; (c) methyl acrylate, Pd(OAc)2, PPh3, Et3N, dioxane, 115 ꢁC,
4 h, 41%; (d) 4,40-dimethoxytrityl chloride, DMAP, pyridine, room
temperature, 16 h, 47%; (e) (iPr2N)2PO(CH2)2CN, 1H-tetrazole, ace-
tonitrile, room temperature, 1 h, quant.
Keywords: Photocrosslink; Nucleoside; Cycloaddition.
*
Corresponding authors. Tel.: +81 761511673; fax: +81 761511665;
e-mail: yosinaga@jaist.ac.jp
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.01.029

1300
Y. Yoshimura et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1299–1301
containing p-CVP was prepared according to the
standard phosphoramidite chemistry on a DNA synthe-
sizer using phosphoramidite 7. After HPLC purification,
50-d(p-CVPGCGTG)-30 (ODN 1) was characterized by
the nucleoside composition and MALDI-TOF MS
[calcd 1858.3528 for (MꢀH)ꢀ, found 1858.3695]. The
modified ODN 50-d(TGTGCCp-CVPGCGTG)-30 (ODN
2) was characterized by the nucleoside composition
and MALDI-TOF MS [calcd 3706.49 for (M+H)+,
found 3706.34] (Fig. 1).
uct of ODN 1 and ODN 3 [calcd 5485.74 for (M+H)+,
found 5485.98]. The isolated ODN 4 was digested with
AP, svPDE, and P1 nuclease at 37 ꢁC for 4 h. Enzymatic
digestion of isolated ODN 4 showed the formation of
dC, dG, dI, and dT in a ratio of 6:6:3:1 together with
dI–p-CVP photoadduct that was a deaminated product
of dA–p-CVP adduct during enzymatic digestion
process.10 The dI–p-CVP adduct was confirmed by MAL-
DI-TOF MS [calcd 532.2043 for (M+H)+, found
532.1096]. We examined molecular modeling studies
of the duplex 1 Æ 3. As shown in Figure 3, the vinyl group
of p-CVP is stacked on N7–C8 double bond of adjacent
adenine residue of ODN 3. It is known that photoreac-
tion of adenine-thymine dinucleotide analog proceeds
via [2 + 2] cycloaddition between the C5–C6 double
bond of thymine and the N7–C8 double bond of ade-
nine.11 As judged from the molecular modeling, it is
strongly suggested that the photocrosslink reaction pro-
ceed via [2 + 2] cycloaddition between the double bond
of p-CVP and the N7–C8 double bond of adenine giving
rise to the formation of aza-cyclobutane structure.
When 50-d(p-CVPGCGTG)-30 (ODN 1) was irradiated at
366 nm for 30 min in the presence of template ODN 3
(Scheme 2), ODN 4 was produced in 96% yield as deter-
mined by HPLC analysis (Fig. 2).8 The reaction mixture
(total volume 60 lL) containing ODN 1 (30 lM, strand
concn) in the presence of template ODN 3 (20 lM,
strand concn) in 50 mM sodium cacodylate buffer
(pH 7.0) and 100 mM sodium chloride was irradiated
with transilluminator (366 nm) at 0 ꢁC for 30 min.9
MALDI-TOF MS indicated that isolated ODN 4 ob-
tained from HPLC purification was a crosslinked prod-
To demonstrate the feasibility of this photocrosslink, we
examined two photoreactions of interstrand crosslink of
the modified ODN containing p-CVP. When ODN 1 was
irradiated at 366 nm for 30 min in the presence of tem-
plate ODN 3 together with ODN 5, ODN 4 was pro-
duced in 96% yield as determined by HPLC analysis
(Scheme 3).8 The reaction mixture (total volume 60
lL) containing ODN 1 (30 lM, strand concn) and
ODN 5 (20 lM, strand concn) in the presence of tem-
plate ODN 3 (20 lM, strand concn) in 50 mM sodium
cacodylate buffer (pH 7.0) and 100 mM sodium chloride
was irradiated with transilluminator (366 nm) at 0 ꢁC
for 30 min. When ODN 2 was irradiated at 366 nm for
30 min in the presence of template ODN 3, ODN 6
was produced in 97% yield as determined by HPLC
analysis (Scheme 4).8 The reaction mixture (total volume
60 lL) containing ODN 2 (34 lM, strand concn) in the
Figure 1. Structure of p-carbamoylvinyl phenol nucleoside (p-CVP).
Scheme 2. Interstrand photocrosslink of ODN 1 in the presence of
template ODN 3.
Figure 3. Molecular modeling of stacked geometry in B-form DNA.
The model was optimized by AMBER* force field in water by using
MacroModel version 8.1.
Figure 2. HPLC analysis of 366 nm irradiated ODN 1 and template
ODN 3; (a) before photoirradiation, (b) irradiation at 366 nm for
30 min, 96% yield. The progress of photoreaction was monitored by
HPLC on a 5-ODS-H column (4.6 · 150 mm, elution with a solvent
mixture of 50 mM ammonium formate, pH 7.0, linear gradient over
30 min from 3% to 20% acetonitrile at a flow rate 1.0 mL/min).
Scheme 3. Interstrand photocrosslink using ODN 1.

Y. Yoshimura et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1299–1301
1301
References and notes
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1. (a) Helene, C. Curr. Opin. Biotechnol. 1993, 4, 29–36; (b)
Luce, R. A.; Hopkins, P. B. Methods Enzymol. 2001, 340,
396–412.
2. (a) Takasugi, M.; Guendouz, A.; Chassignol, M.; Decout,
Scheme 4. Interstrand photocrosslink using ODN 2.
´ `
J. L.; Lhomme, J.; Thuong, N. T.; Helene, C. Proc. Natl.
Acad. Sci. U.S.A. 1991, 88, 5602–5606; (b) Thuong, N. T.;
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E. J.; Glazer, P. M. Nucleic Acids Res. 1994, 22, 2845–
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3. (a) Zhen, W.-P.; Buchardt, O.; Nielsen, H.; Nielsen, P. E.
Biochemistry 1986, 25, 6598–6603; (b) Weidner, M. F.;
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Am. Chem. Soc. 2000, 122, 5646–5647; (b) Fujimoto, K.;
Matsuda, S.; Ogawa, N.; Hayashi, M.; Saito, I. Tetrahe-
dron Lett. 2000, 41, 6451–6454; (c) Fujimoto, K.; Mat-
suda, S.; Hayashi, M.; Saito, I. Tetrahedron Lett. 2000, 41,
7897–7900; (d) Fujimoto, K.; Ogawa, N.; Hayashi, M.;
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Figure 4. Melting curves of the 2 Æ 3 duplex (a) and ODN 6 (b). Tm
values of the 2 Æ 3 duplex (5.0 lM) and ODN 6 (5.0 lM) were measured
in 50 mM sodium cacodylate and 100 mM sodium chloride, pH 7.0.
6. For recent reports for the synthesis of crosslinked duplex
DNA, see: (a) Harwood, E. A.; Sigurdsson, S. T.; Edfeldt,
N. B.; Reid, B. R.; Hopkins, P. B. J. Am. Chem. Soc. 1999,
121, 5081–5082; (b) Nakatani, K.; Yoshida, T.; Saito, I.
J. Am. Chem. Soc. 2002, 124, 2118–2119.
7. Spectroscopic data for selected compounds are as follows.
Compound 5: 1H NMR (CDCl3, 300 MHz) d 2.32 (dt, 1H,
J = 14.2 Hz, 5.7 Hz), 2.55 (ddd, 1H, J = 14.2 Hz, 6.9 Hz,
1.7 Hz), 3.61–3.75 (m, 2H), 3.77 (s, 3H), 4.07–4.11 (m,
1H), 4.62–4.68 (m, 1H), 5.90 (dd, 1H, J = 5.7 Hz, 1.7 Hz),
6.30 (d, 1H, J = 16.2 Hz), 7.05 (d, 2H, J = 8.6 Hz), 7.44 (d,
2H, J = 8.6 Hz), 7.62 (d, 1H, J = 16.2 Hz), HRMS
(MALDI): calcd for C15H18O6Na [(M+Na)+] 317.0996,
found 317.0938, UV (H2O/CH3OH = 1:1) kmax (e) 306 nm
(14,300 Mꢀ1 cmꢀ1), e366 = 155.
presence of template ODN 3 (20 lM, strand concn) in
50 mM sodium cacodylate buffer (pH 7.0) and 100 mM
sodium chloride was irradiated with transilluminator
(366 nm) at 0 ꢁC for 30 min. MALDI-TOF MS indi-
cated that isolated ODN 6 obtained from HPLC purifi-
cation was a crosslinked product of ODN 2 and ODN 3
[calcd 7330.92 for (M+H)+, found 7331.57].
To examine the influence of photocrosslink reaction on
the thermal stability, the melting temperature (Tm) of
the duplex 2 Æ 3 or ODN 6 was determined by UV-mon-
itored thermal denaturation. As shown in Figure 4, the
duplex 2 Æ 3 showed a melting temperature of 50.5 ꢁC,
whereas ODN 6 melted at over 80 ꢁC. Example of this
behavior has been seen for crosslinked ODNs by the
introduction of thiol groups.12 Thus, photocrosslinking
increased Tm of ODN, a dramatic stabilization of the
duplex form.
8. The yield was calculated on the basis of ODN 3.
9. Isolation of each interstrand crosslinked photoproducts
that were produced in two photoreactions of ODN 1 in the
presence of template ODN 50-d(CACGCTGGCACA)-30
or 50-d(CACGCCGGCACA) -30 was not successful due to
furnish lower conversions of photo-induced crosslinking
reactions.
In conclusion, we demonstrate here that a modified
ODN containing p-CVP can be crosslinked by irradiating
at 366 nm with adjacent adenine residue in a [2 + 2]
manner. This feature of the photoreactivity may provide
the intriguing methodology that is applicable to anti-
sense and antigene.
10. dI–p-CV
P
adduct: UV (H2O) kmax (e) 256 nm
(7540 Mꢀ1 cmꢀ1).
11. Paszyc, S.; Skalski, B.; Wenska, G. Tetrahedron Lett.
1976, 6, 449–450.
12. Ferentz, A. E.; Keating, T. A.; Verdine, G. L. J. Am.
Chem. Soc. 1993, 115, 9006–9014.