Synthesis and photoinduced cross-linking reactions of 4,5',8- trimethylpsoralen-incorporated oligodeoxyribonucleotide

Article abstract of DOI:10.1246/cl.2009.272

Photo-induced cross-linking reaction at a certain position in nucleic acids, occurring in a sequence specific manner, would be the key to inhibitor the growth of carcinoma cells, which has a point mutation in the ras gene. We newly synthesized 4,5',8-trim

Full text of DOI:10.1246/cl.2009.272

272
Chemistry Letters Vol.38, No.3 (2009)
Synthesis and Photoinduced Cross-linking Reactions of
4,50,8-Trimethylpsoralen-incorporated Oligodeoxyribonucleotide
Akio Kobori,ꢀ Kazutaka Takaya, Maiko Higuchi, Asako Yamayoshi, and Akira Murakami
Kyoto Institute of Technology, Graduate School of Science and Technology, Department of Biomolecular Engineering,
Goshokaido-cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585
(Received December 9, 2008; CL-081144; E-mail: akobori@kit.ac.jp)
Photo-induced cross-linking reaction at a certain position in
The synthetic route of psoralen nucleoside (Ps) is shown in
Scheme 1. ꢀ-Selective glycosyl bond formation was the key re-
action in the synthesis of psolaren nucleoside. We used palladi-
um-catalyzed coupling of vinyl halide with TBDMS-protected
glycal according to Coleman’s procedure.7 Glycal was prepared
from 30,50-O-bis(tert-butyldimethylsilyl)thymidine by fragmen-
tation in the presence of hexamethyldisilazane followed by se-
lective desilylation of the primary ether by treatment of 1 equiv-
alent of n-Bu4NF.8 Our initial experiment on palladium-cata-
lyzed coupling using vinyl bromide9 with glycal was unsuccess-
ful because of the low reactivity of 40-bromo-4,50,8-trimeth-
ylpsoralen.10 To address this failure, the bromo group introduced
to trimethylpsoralen was changed to a more reactive iodo group.
Then, the coupling partner of 2 was obtained by regioselective
iodination at 40-position of 4,50,8-trimethylpsoralen by N-iodo-
nucleic acids, occurring in a sequence specific manner, would be
the key to inhibitor the growth of carcinoma cells, which has a
point mutation in the ras gene. We newly synthesized 4,50,8-tri-
methylpsoralen nucleoside and examined its photoinduced
cross-linking activities in DNA duplexes after UVA irradiation.
Psoralens are a family of furocoumarine derivatives and are
widely used for PUVA (psolarel and UVA) treatments for skin
disease.1 Psoralen reacts with TA-rich sequences in DNA duplex
and forms cross-linking products under photoirradiation at
365 nm.2 Though 8-methoxypsoralen, a naturally occurring
psoralen derivative that is cost effective to obtain, is generally
used for PUVA, 4,50,8-trimethylpsoralen has much higher reac-
tivity toward DNA.3
1
succinimide. The position of iodination was confirmed by H–
1
For decade, we4 and other researchers5 have studied 4,50,8-
trimethylpsoralen-derivatized oligonucleotides as an antisense
molecule for sequence-selective gene regulation. Oligonucleoti-
des having a 4,50,8-trimethylpsoralen at the 50-end hybridized to
the complementary RNA and formed a covalent bond after UV
irradiation. As a result, psoralen-derivatized oligonucleotides
can drastically inhibit carcinoma cell growth (IC50 ¼ 16 nM),
whereas scramble oligonucleotides having the psoralen inhibit
growth slightly.4 20-O-Psoralen derivatized oligoribonucleoti-
des4 that are also used to inhibit cell growth with a point muta-
tion in the ras gene,6 which is responsible for various cancers.
Cross-linking activity highly depends on the distance and direc-
tion of double bonds in the psoralen and pyrimidine bases. Al-
though the hydrophobicity of psoralen ring might support the
intercalation between nucleobases in duplex DNA or RNA, the
use of linkers tethered to oligonucleotides caused uncertainty
in the location of interaction. Because the position of psoralen
attachment is significant for the formation of photoinduced cova-
lent bonds with high sequence selectivity, we newly designed C-
nucleoside bearing 4,50,8-trimethylpsoralen as nucleobase surro-
gates (Figure 1). Herein, we report the synthesis and properties
of psoralen nucleoside, as well as its photoinduced cross-linking
activities in DNA duplexes.
1H NOESY and H–1H COSY. Reaction of 2 with glycal in the
presence of Pd(OAc)2, n-Bu3N, NaOAc, and LiCl afforded Heck
product 3.11 The enol TBDMS protecting group was voluntarily
removed in the glycosylation. The stereochemistry of the C100
glycosidic center of 3 was confirmed by the observation of an
NOE of the C400–H when C100–H was irradiated. Ketone 3 was
Scheme 1. (a) 1.5 equiv of N-iodosuccinimide, trifluoroacetic
acid–CHCl3 (1:1, v/v), 3 h, rt, 71%, (b) 2.2 equiv of glycal,
0.5 equiv of Pd(OAc)2, 0.7 equiv of n-Bu3N, 1.6 equiv of
NaOAc, 1.0 equiv of LiCl, N,N-dimethylformamide, 18.5 h,
30 ꢁC, 91%, (c) 1.3 equiv of NaBH(OAc)3, CH3CN–AcOH
(1:1, v/v), 2 h, 0 ꢁC, 75%, (d) 2.0 equiv of DMTrCl, pyridine,
5 h, rt, 55%, (e) 2.4 equiv of i-Pr2NPClOCE, 5.0 equiv of N,N-
diisopropylethylamine, CH3CN, 2 h, rt, quant.
Figure 1. Psoralen nucleoside (Ps) and oligodeoxyribonucleoti-
des used in this study.
Copyright Ó 2009 The Chemical Society of Japan

Chemistry Letters Vol.38, No.3 (2009)
273
Figure 2. Photoinduced cross-linking study of Ps in DNA duplexes. Autoradiograms of 20% polyacrylamide/7 M urea gel were ob-
tained to analyze the cross-linking reaction of Ps-ODN in the presence of 200 nM of H-ras(N); (a) N ¼ T, (b) N ¼ C, (c) N ¼ A, and (d)
N ¼ G. Ps-ODN was labeled at the 50-end with 32P and hybridized with complementary DNA in 100 mM of sodium phosphate buffer
(pH 7.0) containing 150 mM of NaCl. The reaction mixture were irradiated at 365 nm for 0–60 min. Lane 1, 0 min; lane 2, 1 min;
lane 3, 5 min; lane 4, 10 min; lane 5, 30 min; lane 6, 60 min. (e) Autoradiogram of the cross-linking reaction mixture in the absence
of the target DNA. (f) Time course of remaining H-ras(N) in the reaction. The ratios were calculated using the band density determined
by densitomeric analysis. Black solid line: N ¼ T, black broken line: N ¼ C, gray solid line: N ¼ A, gray broken line: N ¼ G.
References and Notes
stereoselectively reduced to the ribo-glycoside Ps using mild
reducing agent, sodium triacetoxyboronhydride.12 Conventional
tritylation afforded 50-O-DMTr psoralen nucleoside in 27%
overall yield from 4,50,8-trimethylpsoralen. Phosphoramidite
unit 5 was employed in the solid-phase synthesis of an oligo-
deoxyribonucleotide (Ps-ODN), which has antisence sequence
of the mutation site in the H-ras gene, without purification by
silica gel column chromatography.
1
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The photoinduced cross-linking activities of psoralen
nucleoside in DNA duplexes were examined in the presence of
H-ras(N) (Figure 2). An equimolar mixture of 50-32P labeled
PS-ODN and H-ras(N) was incubated in 100 mM of phosphate
buffer (0.15 M NaCl, pH 7.0) at 37 ꢁC. After the incubation,
the reaction mixtures were UV-irradiated for 0–60 min on a
transilluminator (FUNAKOSHI FTI-LW, 365 nm, 1.6 mJ/
cm2ꢂs), and then analyzed by denaturing PAGE. As the time
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molecular region, except Ps-ODN in the absence of H-ras(N)
(Figure 2e). Figure 2a suggested that cross-linked products
formed from one-minute UV irradiation in the presence of
H-ras(T), and the cross-linking reaction was completed within
30 min (half-life of remaining H-ras(T): 3 min). In the case of
H-ras(C), the cross-linked product was also observed with a
much slower reaction rate (half-life of remaining H-ras(C):
30 min), whereas cross-linked products were hardly observed
in the presence of H-ras(A) or H-ras(G). These results suggested
that psoralen nucleoside in DNA duplex react with pyrimidine
nucleoside, and that the cross-linking efficiency was extremely
high in the presence of thymidine in front of psoralen nucleoside.
In conclusion, we have revealed the high reactivity and se-
lectivity of psoralen nucleoside toward pyrimidine bases in du-
plex DNA. Further studies targeted RNA strands are under way.
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10 Examples of unsuccessful reaction conditions for coupling of glycal
with 40-bromo-4,50,8-trimethylpsoralen are include the following:
Pd(OAc)2 (0.25 equiv), NaHCO3 (2 equiv), and Bu4NCl (7 equiv),
DMF, 48 h; 1,3-(diphenylphosphino)propane (0.6 equiv), Pd(OAc)2
(0.25 equiv), NaHCO3 (2 equiv), DMF, 24 h.
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Published on the web (Advance View) February 21, 2009; doi:10.1246/cl.2009.272