804
Published on the web July 28, 2012
Synthesis and Crosslinking Activity of 4-N-(4,5¤,8-Trimethylpsoralen-4¤-ylmethyl)-
2¤-deoxycytidine-containing Oligodeoxyribonucleotides
Akio Kobori,* Tomita Kohji, Yuko Nagae, Asako Yamayoshi, and Akira Murakami
Department of Biomolecular Engineering, Graduate School of Science and Technology,
Kyoto Institute of Technology, Goshokaido-cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585
(Received June 14, 2012; CL-120644; E-mail: akobori@kit.ac.jp)
Photocrosslinking oligonucleotides have been developed to
investigate and control gene functions without damaging living
systems. Here, 4-N-(4,5¤,8-trimethylpsoralen-4¤-ylmethyl)-2¤-de-
oxycytidine-containing oligodeoxyribonucleotide (ODN(P))
was newly synthesized. Photocrosslinking studies revealed that
ODN(P) sequence-selectively crosslinked to the target with the
uridine in the U-C mismatch base-pair.
Ras proteins1 are guanine nucleotide binding proteins that
transduce growth signals from the cell surface to the interior of
the cell. In the past, three mutant RAS genes: K-RAS, H-RAS,
and N-RAS, have been identified in humans.2 The K-RAS gene
has a G to U transversion mutation in codon 12 and is found
in adenocarcinomas of the pancreas, colon, and lung.3 K-ras
protein is an oncogenic protein showing a persistent GTPase
activity and activates several effectors, such as PI3 kinase,
Raf-1, and RalGEFs.4 The signaling pathways including these
proteins are related to cell survival and proliferation.
Figure 1. (a) 4-N-(4,5¤,8-Trimethylpsolaren-4¤-ylmethyl)-2¤-
deoxycytidine. (b) 4,5¤,8-Trimethylpsoralen-containing oligo-
deoxynucleotides: ODN(P), and target oligonucleotides:
ORN(U) and ORN(G) used in this study. (c) Schematic repre-
sentation of the bulge structures of the duplex formed by
ODN(P) with ORN(U) or ORN(A).
Previously, we5 and others6 have reported sequence-selec-
tive crosslinking studies using oligonucleotides containing
4,5¤,8-trimethylpsoralen derivatives. Among them, oligonucleo-
tides containing a 4,5¤,8-trimethylpsoralen derivative at the 2¤-O
hydroxy group of adenosine (2¤-Ps-eom5) recognize one base
difference in the target sequences under clinically relevant
conditions. By using 2¤-Ps-eom, we successfully achieved the
inhibition of K-ras-immortalized cell proliferation (K12V) but
not of Vco cells that contain the wild-type K-ras gene.5
Considering potential benefits of 4,5¤,8-trimethylpsoralen-nu-
cleotide conjugates, it is important to develop oligonucleotides
having 4,5¤,8-trimethylpsoralen at a suitable position for the
crosslinking reaction.
4¤-ylmethyl group and an amino group at the 4-position of the
pyrimidine ring was observed in H-H COSY spectra, indicating
that a psoralen-4¤-ylmethyl group was introduced at the 4-
position. Following protection of the 5¤-hydroxy group and
phosphitylation of the 3¤-hydroxy group, a phosphoramidite unit
4 was prepared and used for the synthesis of ODN(P)11 using the
standard phosphoramidite DNA synthesis procedures.
In 1996, Pedersen et al.7 reported the fluorescent properties
of 4-N-pyrenylmethyl-2¤-deoxycytidine introduced in the one
nucleotide bulge structure. These results suggested that a pyrene
moiety was efficiently intercalated in the duplexes. In this study
we newly synthesized 4-N-(4,5¤,8-trimethylpsoralen-4¤-ylmeth-
yl)-2¤-deoxycytidine (P) and examined the crosslinking proper-
ties of ODN containing P in a single nucleotide bulge structure
with oligoRNA.8 (Figure 1)
The synthetic route of a 4-N-(4,5¤,8-trimethylpsoralen-4¤-
ylmethyl)-2¤-deoxycytidine phosphoramidite unit is shown as
Scheme 1. Activation of C4 of 3¤,5¤-bisTBDMS-2¤-deoxyuridine
as the tetrazolyl derivative 1 was achieved by following a
previous method.9 Conversion of 1 to 4-N-(4,5¤,8-trimethylpsor-
alen-4¤-ylmethyl)-2¤-deoxycytidine derivative was accomplished
in good yield using (4,5¤,8-trimethylpsolaren-4¤-yl)methyl-
amine.10 After removal of the TBDMS group, the chemical
structure of P was assigned from 1D and 2D NMR measure-
ments (Figure S112). A cross-peak between CH2 of the psoralen-
Photocrosslinking properties of the duplex containing a P in
a single nucleotide bulge structure were studied (Figures 2a-2c).
To estimate the reaction conditions of crosslinked duplex
formation, the duplex stabilities of ODN(P) hybridized with
ORN(U) or ORN(G), which are 15nt oligoRNA having
sequences of the K-RAS mutation, were examined. Although
the duplex stability of ODN(P)/ORN(U) (Tm = 47 °C) was
lower than that of ODN(P)/ORN(G) (Tm = 57 °C), both of
the duplexes were stable under the crosslinking conditions.
The photoinduced crosslinking activities of ODN(P) were
examined in the presence of ORN(U) (Figure 2a) and ORN(G)
(Figure 2b). An equimolar mixture of 5¤-32P-labeled ODN(P)
and ORN(U) or ORN(G) was incubated in 100 mM phosphate
buffer (0.1 M NaCl, pH 7.0) at 37 °C. After the incubation,
the reaction mixtures were UV-irradiated for 0-120 min on
a
transilluminator (FUNAKOSHI FTI-LW, 365 nm, 1.6
mJ cm¹2 s¹1), and then analyzed by denaturing PAGE. As
shown in Figure 2a, a new single band in the high-molecular-
Chem. Lett. 2012, 41, 804-805
© 2012 The Chemical Society of Japan
Kobori, Akio
