Synthesis of antisense oligonucleotides containing 2′-O-psoralenylmethoxyalkyl adenosine for photodynamic regulation of point mutations in RNA

Article abstract of DOI:10.1016/j.bmc.2008.12.001

2′-O-Psoralen-conjugated antisense oligonucleotide was able to recognize a point mutation of mRNA. It had outstanding ability to photo-cross-link only to oligoribonucleotides (ORN) having a point mutation. This type of antisense molecule is the only one o

Full text of DOI:10.1016/j.bmc.2008.12.001

Bioorganic & Medicinal Chemistry 17 (2009) 475–483
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of antisense oligonucleotides containing 20-O-psoralenylmethoxyalkyl
adenosine for photodynamic regulation of point mutations in RNA
*
Maiko Higuchi, Akio Kobori, Asako Yamayoshi, Akira Murakami
Department of Biomolecular Engineering, Kyoto Institute of Technology, Matsugasaki, Kyoto 606-8585, Japan
a r t i c l e i n f o
a b s t r a c t
20-O-Psoralen-conjugated antisense oligonucleotide was able to recognize a point mutation of mRNA. It
had outstanding ability to photo-cross-link only to oligoribonucleotides (ORN) having a point mutation.
This type of antisense molecule is the only one of its kind so far. To give high photo-cross-linking effi-
ciency and sequence selectivity to antisense molecules, we synthesized novel photo-reactive oligonucle-
otides (20-Ps-xom) containing psoralen at the 20-O-position adenosine via an ethoxymethylene (20-Ps-
eom), propoxymethylene (20-Ps-pom) and butoxymethylene (20-Ps-bom) linker, respectively. We evalu-
ated the photo-cross-linking efficiency and sequence selectivity in photo-cross-linking of 20-Ps-xom to
the complementary ORN and to an ORN having a mismatch base. Among them, 20-Ps-eom exhibited supe-
rior photo-cross-linking efficiency with high sequence selectivity.
Article history:
Received 10 October 2008
Revised 1 December 2008
Accepted 2 December 2008
Available online 6 December 2008
Keywords:
Photodynamic antisense therapy
Photo-cross-linking
Psoralen
Ó 2008 Elsevier Ltd. All rights reserved.
Point mutation
1. Introduction
Previously, a photo-reactive antisense oligonucleotide whose
50-O-position was modified with a 4,50,8-trimethylpsoralen (psora-
In recent years, it has been reported that point mutations in
genes are responsible for various cancers.1–4 The mutation affects
cellular proliferation and elicits tumorigenic properties. It has been
desired to inhibit the gene expression of disease-causing mutations
selectively without affecting the functions of normal genes. The
strict regulation of the expression of genes having point mutations
in their sequence could lead to the development of novel anti-can-
cer therapy. The point mutation of the ras gene is a typical example
which is reportedly responsible for various cancers.5–8 A consider-
able number of studies have been carried out using chemically
modified antisense oligonucleotides9–11 and siRNA.12–14 Some
groups have reported the successful suppression of mutant ras
mRNA using antisense oligonucleotides or siRNA, though these
methods inhibited the expression of not only mutant ras mRNA
but also wild-type ras mRNA.15–20 This undesired suppression
could lead to serious side effects in curing cancers. Thus, novel con-
ceptual attempts are needed.
We have attempted to regulate the expression of mRNA having
a point mutation using psoralen derivatives. They have an ability to
photo-cross-link covalently to pyrimidine bases, especially thy-
mine and uracil, upon UV irradiation (320–400 nm).21 When we
localize psoralen in the close vicinity of a mutationally-altered
pyrimidine base using psoralen-conjugated antisense oligonucleo-
tides, the sequence specific suppression of the functions of desig-
nated mRNA could be achieved.
len) via an aminoethylaminomethyl linker (50-Ps-oligo) was re-
ported.22,23 50-Ps-oligo complementary to the E6 region of human
papillomavirus type 18 (HPV-18) mRNA significantly elicited the
apoptotic death of HPV18-positive cervical carcinoma cells upon
UVA irradiation (365 nm) for 2 min in vitro and in vivo.24,25 With
these results, photodynamic antisense therapy (PDA-therapy) is
proceeding to the clinical stage. For future developments of PDA-
therapy, we have to minimize the side reactions of psoralen deriv-
atives with off-target mRNAs and the cytotoxicity of UVA used for
photo-cross-linking. The psoralen of 50-Ps-oligo in our previous
study could photo-cross-link to a pyrimidine base in the vicinity
of the psoralen even though the pyrimidine base is not the target
base. This might be due to the flexibility of the psoralen conjugated
at the 50-end. Instead of 50-end modification, we chose the 20-O-
position of adenosine as the modification site of psoralen and
synthesized a photo-reactive antisense oligonucleotide having a
20-O-psoralen-conjugated adenosine (20-Ps-oligo). Adenosine was
conjugated with a psoralen via a methylene linker at the 20-O-posi-
tion and the conjugated adenosine was incorporated into the des-
ignated position of the antisense oligonucleotides (20-Ps-met).26
The 20-Ps-met photo-cross-linked to the complementary oligoribo-
nucleotides (ORN) upon UVA irradiation (365 nm) with apprecia-
ble efficiency, but hardly at all to ORN having a mismatch base at
the opposite site of the 20-O-psoralen-conjugated adenosine. The
introduction of psoralen to the 20-O-position of adenosine greatly
enhanced the sequence specificity of the photo-cross-linking reac-
tion compared with 50-Ps-oligo. However the efficiency was largely
decreased. The photo-cross-linking efficiency of 20-Ps-met with
* Corresponding author. Tel./fax: +81 75 724 7814.
E-mail address: akiram@kit.ac.jp (A. Murakami).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.001

476
M. Higuchi et al. / Bioorg. Med. Chem. 17 (2009) 475–483
complementary ORN upon UVA irradiation was ca. 50% after
150 min irradiation, while the photo-cross-linking efficiency of
50-Ps-oligo was ca. 50% after 18 min irradiation. As UVA (320–
400 nm) is mutagenic and carcinogenic for cells,27 20-Ps-met is
not substantially applicable for clinical uses and therefore, photo-
irradiation time should be minimized by appropriate molecular de-
sign of antisense molecules.
(ver.8.0, Schrödinger).29 The initial position of psoralen was as fol-
lows:
site;
(*, designated intercalation
, target uracil;
, psoralen-conjugated adenosine). In
the case of 20-Ps-eom and 20-Ps-pom, it was demonstrated that
the psoralen was located in the close vicinity of the target uracil
(
). In the case of 20-Ps-bom, there is a possibility that the psora-
len is located in close range of both the target uracil ( ) and the
neighboring uracil ( ). To evaluate whether psoralen of 20-Ps-
xom can photo-cross-link to the target uracil ( ), the average dis-
tances between the 5,6-carbon of uracil and 3,4-carbon of 4,50,8-
trimethylpsoralen were estimated based on 3D-structures pre-
sented by MacroModel. The distances were 4.5 Å in 20-Ps-eom,
3.6 Å in 20-Ps-pom, 4.3 Å in 20-Ps-bom and 5.9 Å in 20-Ps-pnom.
The results suggest that psoralen of 20-Ps-eom, 20-Ps-pom, and 20-
Ps-bom can locate in the close range of the target uracil compared
with 20-Ps-met (5.6 Å); the psoralen of 20-Ps-eom and 20-Ps-pom
can cross-link only to the target uracil ( ) of the ORN-U; and
the psoralen of 20-Ps-bom can cross-link not only to the designated
uracil ( ) but also to the neighboring uracil ( ). On the other
hand, 20-Ps-pnom was supposed not to intercalate between base
pairs. According, we synthesized 20-Ps-eom, 20-Ps-pom, and 20-Ps-
bom, and examined their characteristics.
In this report, we designed advanced 20-Ps-oligo to enhance the
photo-cross-linking efficiency and sequence selectivity by focusing
on the linker molecules. We synthesized novel photo-reactive oli-
gonucleotides (20-Ps-xom) containing psoralen at the 20-O-position
of adenosine via an ethoxymethylene (20-Ps-eom), propoxymethyl-
ene (20-Ps-pom) and butoxymethylene (20-Ps-bom) linker, and
examined their photo-cross-linking properties to ORNs. It was
found that the 20-Ps-eom and 20-Ps-pom exhibited an outstanding
ability to photo-cross-link only to complementary ORN. These re-
sults suggest that 20-Ps-eom and 20-Ps-pom were able to recognize
a point mutation of mRNA and selectively regulate the expression.
This type of antisense molecule is the only one of its kind so far.
2. Results and discussion
2.1. Molecular design
2.2. Synthesis of 20-O-psoralenylmethoxyalkyl adenosine
In a previous study, we reported that 20-Ps-met photo-cross-
linked to complementary ORN upon UVA irradiation but hardly
at all to ORN having a mismatch base.26 It was noteworthy that
20-Ps-met discriminated a single base mutation in RNA. However,
the long UVA irradiation time (150 min for 50% cross-linking yield)
must be avoided, because of the mutagenic and carcinogenic prop-
erties of UVA.27 We have also observed that the cell viability was
drastically decreased upon UVA irradiation.24 Accordingly, 20-Ps-
met is not practical for clinical purposes. To pursue PDA-therapy,
it is essential to enhance the photo-cross-linking efficiency of 20-
Ps-oligo without sacrificing its sequence specificity. We designed
other 20-Ps-oligos focusing on the length and flexibility of the link-
age that connects psoralen to the 20-O-position of adenosine. We
adopted alkoxymethylene as the linker molecule as shown in Fig-
ure 1. 20-Ps-oligos are denoted as 20-Ps-xom, where x represents
the first letter of the name of the linker molecules. As the target
of 20-Ps-xom, we chose a codon12 region of K-ras mRNA. A single
nucleotide mutation at codon 12 (12Gly?Val; GGU?GUU) is com-
mon among K-ras mRNA mutation. When K-ras underwent a point
mutation, the mutated K-ras transformed normal cells into cancer
cells. This type of event is frequently observed in various cancers
such as colorectal cancer and pancreas cancer.3 Selective suppres-
sion of mutated K-ras expression could lead to the development of
a novel methodology in cancer therapy. The sequences of 20-Ps-
xom are shown in Table 1. As the target, mutant-type K-ras ORN
(ORN-U) and wild-type K-ras ORN (ORN-G) were prepared. Prior
to the molecular design of 20-Ps-xom, the location of psoralen in
a duplex of 20-Ps-xom and ORN-U was evaluated by conforma-
tional calculations using the AMBER* force field28 in MacroModel
20-Ps-xom was synthesized by the introduction of 20-O-psora-
len-conjugated adenosine into the designated site of the oligode-
oxyribonucleotides. As the key adenosine derivative, 40-
chloroalkoxymethyl-4,50,8-trimethylpsoralens were synthesized
as shown in Scheme 1. 40-Chloromethyl-4,50,8-trimethylpsoralen
(1) and 40-hydroxyethoxymethyl-4,50,8-trimethylpsoralen (2a)
were synthesized according to the reported procedure.30,31 Com-
pound 1 was treated by a glycol as a linker molecule. Compound
1 was reacted with ethylene glycol, 1,3-propanediol, or 1,4-butane-
diol without solvent to give 2a, 2b or 2c, respectively. Chlorination
of 2a, 2b and 2c was then carried out in carbon tetrachloride with
triphenylphosphine to give 3a, 3b, and 3c. The overall yields of 3a,
3b, and 3c from 1 were 64%, 78%, and 62%, respectively.
20-O-Psoralen-conjugated adenosines were synthesized as
shown in Scheme 2. Unprotected adenosine was activated by so-
dium hydride in dry DMF followed by treatment with 3a, 3b or
3c at 80 °C for 32 h giving both 20-O-position and 30-O-position
modified adenosine. In this case, it was found that more than 90%
of the product was a 20-O-psoralen-modified product. 20-O-Psora-
len-conjugated adenosines (4a, 4b and 4c) were isolated by silica
gel chromatography in 18%, 16% and 28% yields, respectively. These
relatively low yields were attributed to the b-elimination reaction
of 40-chloroalkoxymethyl-4,50,8-trimethylpsoralens; further, the
suppression of the side reaction was unsuccessful. 4a, 4b and 4c
were treated with benzoyl chloride according to the general proce-
dures of Ti et al.32 to give 5a, 5b, and 5c. The 50-hydroxyl moieties of
5a, 5b and 5c were protected by dimethoxytrityl chloride in pyri-
dine followed by the treatment of 2-cyanoethyl-N,N,N0,N0-tetraiso-
propylphosphorodiamidite in the presence of 1H-tetrazole as a
catalyst in dry acetonitrile. Without purification, these reaction
mixtures were added to 50-detritylated d(ACCGCAT)-CPG, and the
mixtures were allowed to react at room temperature for 2 h. The
CPG support was further processed to give 20-Ps-xom with the des-
ignated sequence. The sequences of 20-Ps-xom complementary to
the codon12 region of K-ras mRNA are shown in Table 1.
NH2
N
N
n=2 : 2’-Ps-eom
O
N
N
O
n=3 : 2’-Ps-pom
n=4 : 2’-Ps-bom
n=5 : 2’-Ps-pnom
O
O
O
2.3. Interaction between 20-Ps-xom and ORNs
n
O
O
O
The behaviors of oligonucleotides to which psoralen had been
introduced were examined. The thermal stability of the duplexes
of 20-Ps-xom and ORNs were evaluated by UV melting profiles (Table
Figure 1. Structure of photo-reactive oligonucleotides containing 20-O-pso-
ralenylmethoxyalkyl adenosine.

M. Higuchi et al. / Bioorg. Med. Chem. 17 (2009) 475–483
477
20
10
20
a
b
10
0
0
-10
-20
-10
-20
220
270
320
370
220
270
320
370
wavelength (nm)
wavelength (nm)
Figure 2. CD spectra of the duplexes of 2’-Ps-xom and (a) ORN-U or (b) ORN-G. Key: Kr-ODN and ORN (thin line), 2’-Ps-eom and ORN (thick line), 2’-Ps-pom and ORN (gray
broken line) 2’-Ps-bom and ORN (black broken line), conditions : [2’-Ps-xom] = [Kr-ODN] = [ORN] = 2 mM in 0.1 M phosphate buffer containing 0.1 M NaCl (pH 7.0).
Measurements were carried out at 10 °C.
intensities around 465 nm were further quenched compared with
Table 1
20-Ps-xom alone. The degree of fluorescence quenching of 20-Ps-
Sequences of 20-Ps-oligos and ORNs
eom was most significant among the three molecules. Moreover,
Name
Sequence
ESI-MS (m/z)
the fluorescence spectra of 20-Ps-xom showed a slight blue shift.
It was reported that the fluorescence spectrum due to psoralen
was quenched and blue-shifted when psoralen was intercalated
between base pairs of the hybrid.33 These spectroscopic character-
izations suggest that 20-Ps-xom formed a stable A-form-like duplex
with ORNs and that the psoralen of 20-Ps-xom was intercalated be-
tween base pairs of the duplex.
Calcd
Found
a
a
a
20-Ps-eom
20-Ps-pom
20-Ps-bom
Kr-ODN
ORN-U
ORN-G
50 dTACGCCAAPsCAGCTCC 30
50 dTACGCCAAPsCAGCTCC 30
50 dTACGCCAAPsCAGCTCC 30
50 dTACGCCAACAGCTCC 30
50 rGGAGCUGUUGGCGUA 30
50 rGGAGCUGGUGGCGUA 30
([Mꢀ6H]ꢀ6) 795.642
([Mꢀ6H]ꢀ6) 797.977
([Mꢀ7H]ꢀ7) 685.839
([Mꢀ7H]ꢀ7) 638.963
([Mꢀ6H]ꢀ6) 813.107
([Mꢀ5H]ꢀ6) 968.125
795.623
798.069
685.903
638.907
813.131
968.155
a
Aps = 20-O-psoralen-conjugated adenosine.
2.4. Photo-cross-linking properties of 20-Ps-xom
UVA irradiation of equimolar solutions of 20-Ps-xom and ORNs
was carried out at 35 °C on a transilluminator (365 nm, 1.6 mW/
cm2). To evaluate the photo-cross-linking efficiency and selectivity
in sequence recognition, these photo-reactions were monitored by
reversed-phase HPLC. Major photoproduct peaks (*) appeared in a
time-dependent manner (Fig. 4). As it was reported that psoralen–
pyrimidine adducts were regenerated to psoralen and pyrimidine
by irradiation at 240–290 nm,34 we used the photo-reaction to
identify the new peaks. Peaks with an asterisk were isolated and
then UV-irradiated at 254 nm for 30 min. The photo-regeneration
reactions of the photoproducts were analyzed by reversed-phase
HPLC (Fig. 5). It was observed that the peak with an asterisk in
the mixture of 20-Ps-eom and ORN-U regenerated both starting
ORN-U and 20-Ps-eom. Same reaction was observed in the mixture
of 20-Ps-pom and ORN-U. On the other hand, the photoproduct in
the mixture of 20-Ps-bom and ORN-U regenerated only starting
20-Ps-bom but not ORN-U, suggesting that the photoproduct in
the 20-Ps-bom mixture was an intramolecularly photo-cross-linked
products of 20-Ps-bom. The butoxymethylene linker in 20-Ps-bom
might be too long to photo-cross-link to the designated uracil of
ORN. Furthermore, the photoproduct in the mixture of 20-Ps-xom
and ORN-G regenerated only starting 20-Ps-xom. These results sug-
gest that 20-Ps-eom and 20-Ps-pom photo-cross-linked to ORN-U in
a sequence specific manner and that they were applicable for the
treatment of cancer cells having crucial point mutations in gene.
2). The meltingtemperatures (Tms) of the duplexesof 20-Ps-xom and
ORN-U suggested that the introduction of psoralen to adenosine via
alkoxymethylene linkers destabilized the duplex to some extent.
However, the Tms was high enough for 20-Ps-xom to function as an
antisenseoligonucleotide underphysiologicalconditions. Itwasalso
demonstrated that the duplex of 20-Ps-xom and ORN-G was also sta-
bly formed under physiological conditions.
UV spectra of the duplexes showed no appreciable spectrum
change, which might be due to the broad absorption band attrib-
uted to psoralen around 300–360 nm. The location of the psoralen
in the duplex was examined by circular dichroism (CD) spectros-
copy (Fig. 2). CD spectra of the duplexes of 20-Ps-xom and ORN-U
except for 20-Ps-bom and ORN-U showed substantially the same
spectra as the duplex of psoralen-unmodified ODN, suggesting that
the duplex was A-form-like. The duplex of 20-Ps-bom and ORN-U
also showed an A-form-like spectrum while the cotton effect was
significantly diminished. Induced circular dichroism (ICD) around
300–350 nm was not observed.
Fluorescence spectroscopy gave considerable information con-
cerning psoralen localization. Figure 3 shows the fluorescence
spectra of duplexes of 20-Ps-xom and ORNs. It was demonstrated
that the fluorescence intensity of the psoralen at 465 nm was sig-
nificantly quenched when psoralen was incorporated into ODN.
When 20-Ps-xom formed a duplex with ORN-U, the fluorescence
Cl
a
O
O
HO
Cl
b
n
n
O
O
O
O
O
O
O
O
O
1
n=2 : 2a
n=3 : 2b
n=4 : 2c
n=2 : 3a
n=3 : 3b
n=4 : 3c
Scheme 1. Reagents and conditions: (a) excess diol, 110 °C; (b) triphenylphosphine, carbon tetrachloride, pyridine, r.t.

Bz
N
478
M. Higuchi et al. / Bioorg. Med. Chem. 17 (2009) 475–483
NH
2
HN
N
N
N
NH2
N
N
N
N
N
N
N
HO
O
a
Cl
O
b
HO
O
HO
N
+
n
HO
O
O
O
HO
O
O
n
O
O
O
n
O
O
O
OH OH
O
O
O
n=2 : 3a
n=3 : 3b
n=4 : 3c
n=2 : 4a
n=3 : 4b
n=4 : 4c
n=2 : 5a
n=3 : 5b
n=4 : 5c
Bz
N
Bz
HN
HN
N
N
N
N
N
N
N
d
DMTrO
c
DMTrO
O
O
HO
O
O
P
O
n
O
O
n
(i-Pr) N
2
O
O
O
O
O
O
NC
O
n=2 : 6a
n=3 : 6b
n=4 : 6c
n=2 : 7a
n=3 : 7b
n=4 : 7c
Scheme 2. Reagents and conditions: (a) 40-chloroalkoxymethyl-4,50,8-trimethylpsoralen, NaH, DMF, 75 °C, (b) 1—TMSCl, pyridine, r.t; 2—BzCl, pyridine, r.t; 3—NH3, r.t, (c)
DMTrCl, pyridine, r.t; (d) 2-Cyanoethyl N,N,N0,N0-tetraisopropylphosphorodiamidite, 1H-tetrazole, acetonitrile, r.t.
or a propoxymethylene as a linker molecule of psoralen conjuga-
Table 2
tion dramatically enhanced the photo-cross-linking efficiency
and the sequence selectivity. The photo-cross-linking efficiency
of 20-Ps-eom was the highest among all types of psoralen-conju-
gated oligonucleotides ever reported,22–24 and the irradiation time
for photo-cross-linking could be allowed for clinical use.
Thermal stabilities of duplexes of 20-Ps-xom with ORNs
Tm (°C)
ORN-U
ORN-G
20-Ps-eom
20-Ps-pom
20-Ps-bom
Kr-ODN
63.1
62.7
64.0
67.3
59.1
58.8
60.2
58.9
3. Conclusion
In conclusion, we developed new types of photo-cross-linking
antisense oligonucleotides containing adenosine in which the 20-
O-position was modified with psoralen via alkoxymethylene link-
ers, and 20-Ps-eom and 20-Ps-pom showed the high photo-cross-
linking efficiency and the sequence selectivity. This is the first time
that we have come across such an antisense molecule, and it could
show the outstanding ability to control certain types of mutations.
Assuming that this photo-reaction proceeds in actual cancer cells
having point mutations in specified genes (e.g., G?U or A?U),
20-Ps-eom and 20-Ps-pom could regulate the proliferation of the
cancer cells. The attempt to apply these antisense molecules to
cancer cells having a point mutation in gene is now underway.
The photo-cross-linking efficiencies were evaluated based
on remaining intact ORN and the time courses are shown in
Figure 6. The photo-cross-linking efficiencies of 20-Ps-eom and
20-Ps-pom with ORN-U were ca. 75% and ca. 50% upon UVA irradi-
ation for 1 min at 35 °C, respectively. In the case of ORN-G, the effi-
ciencies of 20-Ps-eom and 20-Ps-pom were ca. 7% and ca. 9%,
respectively. In our previous report, the photo-cross-linking effi-
ciency of 20-Ps-met and ORN-U was ca. 3% upon UVA irradiation
for 1 min and ca. 35% for 120 min.26 Compared with the results
of 20-Ps-met, it was found that the adoption of an ethoxymethylene
100
80
60
40
20
0
100
80
60
40
20
0
100
a
b
c
80
60
40
20
0
340
440
540
640
340
440
540
640
350
450
550
650
wavelength (nm)
wavelength (nm)
wavelength (nm)
Figure 3. Fluorescence spectra of the duplexes of ORNs and (a) 20-Ps-eom, (b) 20-Ps-pom, (c) 20-Ps-bom. Key: the single-strand 20-Ps-xom (thin line), the duplex of 20-Ps-xom
and ORN-U (thick line), the duplex of 20-Ps-xom and ORN-G (gray line), 40-chloroalkoxymethyl-4,50,8-trimethylpsoralen (broken line). Conditions: [20-Ps-
xom] = [ORNs] = 2 lM in 0.1 M phosphate buffer containing 0.1 M NaCl (pH 7.0). Measurements were carried out at 10 °C. (kex = 330 nm).

M. Higuchi et al. / Bioorg. Med. Chem. 17 (2009) 475–483
479
Figure 4. Time course of photo-cross-linking reactions of equimolar mixture of 20-Ps-xom and ORNs analyzed by reversed-phase HPLC. (a) 20-Ps-eom and ORN-U, (b) 20-Ps-
eom and ORN-G, (c) 20-Ps-pom and ORN-U, (d) 20-Ps-pom and ORN-G, (e) 20-Ps-bom and ORN-U, (f) 20-Ps-bom and ORN-G. Key: absorbance at 260 nm (black line), absorbance
at 330 nm (gray line), Conditions: [20-Ps-xom] = [ORNs] = 2
lM in 0.1 M phosphate buffer containing 0.1 M NaCl (pH 7.0), HPLC conditions: the acetonitrile gradient in TEAA
(pH 7.0) was as follows; 0–10 min, a linear gradient from 7.5% to 15%; 10–20 min, a linear gradient from 15% to 50%; 20–30 min maintained at 50%, column oven; 40 °C.
3.69 (t, J = 5.86, 2H), 3.75–3.78 (t, J = 5.71, 2H), 4.64 (s, 2H), 6.21
(d, J = 1.05, 1H), 7.58 (s, 1H); 13C NMR (CDCl3) d 8.4, 12.3, 19.3,
32.2, 61.5, 63.3, 68.8, 109.1, 111.4, 111.8, 112.7, 116.1, 125.0,
149.1, 153.4, 154.6, 154.8, 161.5. HRMS m/z: calcd for C18H20NaO5
([M+Na]+) 339.121, found 339.119.
4. Experimental
4.1. General
All reagents and solvents were purchased from commercial
sources and used without further purification. Silica gel column
chromatography was carried out on Wako gel C-200. NMR spectra
were measured with Bruker Biospin DRX500 spectrometer at
500 MHz for 1H and 125 MHz for 13C with a deuterated solvent
and TMS as an internal standard. ESI-MS spectra were recorded
on Bruker DALTONICSÒ microTOF. Oligodeoxyribonucleotides and
oligoribonucleotides were synthesized via the conventional
procedure using a Beckmen Oligo1000 DNA synthesizer. Abbrevia-
tion of some reagents: Bz, benzoyl; DMTr, 4,40-dimethoxytri-
phenylmethyl.
4.2.2. 40-(400Hydroxybutoxymethyl)-4,50,8-trimethylpsoralen
(2c)
Compound 1 (767 mg, 2.77 mmol) was suspended in 1,4-butane-
diol (25 ml, 282 mmol), and heated up to 110 °C to dissolve 1.
The mixture was stirred at 110 °C for 10 min and after cooling to
room temperature, water (40 ml) was added. The solution was ex-
tracted four times with CH2Cl2 (60 ml). The organic phase was
dried over Na2SO4, filtrated, and concentrated todryness. The crude
residue was dissolved in acetonitrile (50 ml). The solution was kept
at 0 °C overnight, and a white precipitate was collected. The crystal
was isolated by filtration to give 2c (652 mg, 71%). 1H NMR (CDCl3)
d 1.65–1.74 (m, 4H), 2.49 (s, 6H), 2.54 (s, 3H), 3.52–3.55 (t, J = 5.93,
2H), 3.62–3.64 (t, J = 6.03, 2H), 4.63 (s, 2H), 6.21 (d, J = 1.10, 1H),
7.59 (s, 1H); 13C NMR (CDCl3) d 8.4, 12.3, 19.3, 26.6, 30.0, 62.5,
63.0, 70.0, 109.0, 111.6, 111.9, 112.7, 116.1, 125.0, 149.1, 153.4,
154.6, 154.8, 161.5. HRMS m/z: calcd for C19H22NaO5 ([M+Na]+)
353.137, found 353.113.
4.2. Synthesis
4.2.1. 40-(300-Hydroxypropoxymethyl)-4,50,8-trimethylpsoralen
(2b)
Compound 1 (1.59 g, 5.76 mmol) was suspended in 1,3-pro-
panediol (40 ml, 553 mmol), and heated up to 110 °C to dissolve
1. The mixture was stirred at 110 °C for 10 min and after cooling
to room temperature, water (60 ml) was added. The solution was
extracted four times with CH2Cl2 (80 ml). The organic phase was
dried over Na2SO4, filtrated, and concentrated to dryness. The
crude residue was dissolved in acetonitrile (70 ml). The solution
was kept at 0 °C overnight, and a white precipitate was collected.
The crystal was isolated by filtration to give 2b (1.52 g, 83%). 1H
NMR (CDCl3) d 1.85–1.89 (m, 2H), 2.50 (s, 6H), 2.54 (s, 3H), 3.67–
4.2.3. 40-(200-Chloroethoxymethyl)-4,50,8-trimethylpsoralen (3a)
Compound 2a (1.49 mg, 4.95 mmol) was dissolved in dry pyri-
dine (50 ml). To this solution, triphenylphosphine (3.25 g,
12.4 mmol) and carbon tetrachloride (l.60 ml, 12.4 mmol) were
added and stirred at room temperature for 7 h followed by evapo-
ration to dryness. The residue was resolved in CH2Cl2 (120 ml) and

480
M. Higuchi et al. / Bioorg. Med. Chem. 17 (2009) 475–483
a
b
*
*
0
5
10
15
20
25
30
35
0
5
10
15
20
25
30
35
retention time (min)
retention time (min)
c
d
*
*
0
5
10
15
20
25
30
35
0
5
10
15
20
25
30
35
retention time (min)
retention time (min)
e
f
*
*
0
5
10
15
20
25
30
35
0
5
10
15
20
25
30
35
retention time (min)
retention time (min)
Figure 5. Photo-regeneration reactions were analyzed by reversed-phase HPLC. Isolated products obtained from the reaction mixture of (a) 20-Ps-eom and ORN-U, (b) 20-Ps-
eom and ORN-G, (c) 20-Ps-pom and ORN-U, (d) 20-Ps-pom and ORN-G, (e) 20-Ps-bom and ORN-U, (f) 20-Ps-bom and ORN-G, were irradiated at 254 nm for 30 min. Key:
absorbance at 260 nm (black line), absorbance at 330 nm (gray line). HPLC conditions: the acetonitrile gradient in TEAA (pH 7.0) was as follows; 0–10 min, a linear gradient
from 7.5% to 15%; 10–20 min, a linear gradient from 15% to 50%; 20–30 min maintained at 50%, column oven; 40 °C. HPLC conditions: the acetonitrile gradient in TEAA (pH
7.0) was as follows; 0–10 min, a linear gradient from 7.5% to 15%; 10–20 min, a linear gradient from 15% to 50%; 20–35 min maintained at 50%, column oven; 40 °C.
100
75
50
25
0
100
75
50
25
0
100
75
50
M atch-RNA
25
Msim atch-RNA
a
b
c
0
2
4
0
2
4
0
2
4
UV irradiation time (min)
UV irradiation time (min)
UV irradiation time (min)
Figure 6. Time course of photo-cross-linking efficiencies of (a) 20-Ps-eom, (b) 20-Ps-pom, (c) 20-Ps-bom, and ORN-U (thin line) or ORN-G (broken line).
washed with aqueous 5% NaHCO3 (120 ml). The organic layer was
dried over Na2SO4, filtrated, and concentrated to dryness. The res-
idue was purified by flash chromatography to give 3a (1.37 g, 87%)
as white powder. 1H NMR (CDCl3) d 2.49 (d, J = 1.35, 6H), 2.56 (s,
3H), 3.67–3.69 (m, 2H), 3.76–3.78 (m, 2H), 4.71 (s, 2H), 6.23 (d,
J = 1.10, 1H), 7.68 (s, 1H); 13C NMR (CDCl3) d 8.4, 12.3, 19.4, 43.2,
63.6, 69.9, 109.0, 111.6, 111.9, 112.8, 116.2, 124.9, 149.2, 153.4,
154.6, 154.7, 161.5. HRMS m/z: calcd for C17H17NaO4 ([M+Na]+)
343.071, found 343.074.
was purified by flash chromatography to give 3b (896 mg, 94%)
as white powder. 1H NMR (CDCl3) d 2.02–2.07 (m, 2H), 2.49 (s,
6H), 2.55 (s, 3H), 3.64–3.67 (m, 4H), 4.62 (s, 2H), 6.22 (d, J = 1.00,
1H), 7.57 (s, 1H); 13C NMR (CDCl3) d 8.4, 12.3, 19.4, 32.5, 41.8,
63.3, 66.2, 109.1, 111.5, 111.9, 112.8, 116.2, 125.1, 149.2, 153.3,
154.7, 154.8, 161.5. HRMS m/z: calcd for C18H19NaO4 ([M+Na]+)
357.087, found 357.092.
4.2.5. 40-(400-Chlorobutoxymethyl)-4,50,8-trimethylpsoralen (3c)
Compound 2c (586 mg, 1.77 mmol) was dissolved in dry pyri-
dine (10 ml). To this mixture, triphenylphosphine (1.40 g,
4.2.4. 40-(300-Chloropropoxymethyl)-4,50,8-trimethylpsoralen
(3b)
5.31 mmol) and carbon tetrachloride (510 ll, 5.32 mmol) were
Compound 2b (900 mg, 2.84 mmol) was dissolved in dry pyri-
dine (20 ml). To this solution, triphenylphosphine (1.28 g,
4.87 mmol) and carbon tetrachloride (530 ll, 5.49 mmol) were
added and stirred at room temperature for 6 h followed by evapo-
ration to dryness. The residue was resolved in CH2Cl2 (70 ml) and
washed with aqueous 5% NaHCO3 (70 ml). The organic layer was
dried over, filtrated, and concentrated to dryness. The residue
added and stirred at room temperature for 9 h followed by evapo-
ration to dryness. The residue was dissolved in CH2Cl2 (70 ml)
washed with aqueous 5% NaHCO3 (70 ml). The organic phase was
dried over Na2SO4, filtrated, and concentrated to dryness. The res-
idue was purified by flash chromatography to give 3c (546 mg,
88%) as white powder. 1H NMR (CDCl3) d 1.75–1.79 (m, 2H),
1.85–1.91 (m, 2H), 2.50 (m, 6H), 2.56 (s, 3H), 3.51–3.55 (t,

M. Higuchi et al. / Bioorg. Med. Chem. 17 (2009) 475–483
481
J = 5.93, 4H), 4.62 (s, 2H), 6.23 (d, J = 0.82, 1H), 7.58 (s, 1H); 13C
NMR (CDCl3) d 8.4, 12.3, 19.3, 27.0, 29.6, 44.8, 63.1, 69.1, 109.1,
111.5, 112.0, 112.8, 116.1, 125.1, 149.2, 153.3, 154.7, 161.5. HRMS
m/z: calcd for C19H21NaO4 ([M+Na]+) 371.103, found 371.120.
3.36–3.64 (m, 6H), 3.95 (d, J = 3.1, 1H), 4.26 (s, 1H), 4.42–4.44 (t,
J = 5.6, 1H), 4.50 (s, 2H), 4.60 (s, 1H), 5.39 (s, 1H), 5.74 (s, 2H),
5.94 (d, J = 6.2, 1H), 6.30 (s, 1H), 7.65 (s, 1H), 8.09 (s, 1H), 8.35 (s,
1H); 13C NMR (CDCl3) d 8.7, 12.5, 19.1, 26.1, 26.4, 26.8, 61.9,
62.2, 69.4, 69.5, 70.0, 81.3, 86.5, 86.8, 108.2, 112.6, 112.7, 112.8,
116.2, 119.7, 125.3, 140.1, 148.9,149.4, 152.9, 154.1, 154.3, 155.1,
156.6, 160.5. HRMS m/z: calcd for C29H33NaO8 ([M+Na]+)
602.223, found 602.207.
4.2.6. 20-O-[[(4,50,8-trimethyl) psoralen-40-ylmethoxy] ethyl]
adenosine (4a)
Adenosine (7.23 g, 26.8 mmol) was stirred with 60% NaH
(1.23 g, 32.2 mmol) in dry DMF (50 ml) at room temperature for
1 h, then 3a (980 mg, 2.8 mmol) in dry DMF (15 ml) was added.
The mixture was stirred at 75 °C for 27 h. Phosphate buffer
(1.0 ml, 100 mM sodium phosphate, pH 7.0) was added to the reac-
tion mixture, the solution was evaporated to near dryness, and the
residue was dissolved in CH2Cl2. The resulting solution was
washed with aqueous 5% NaHCO3 (150 ml ꢁ 3). The organic layer
was dried over Na2SO4, filtrated, and concentrated to dryness.
The residue was purified by silica gel column chromatography. Elu-
tion with CH2Cl2–MeOH (25:1, v/v) gave fractions of 4a (270 mg,
18% yield). 1H NMR (CDCl3) d 2.50 (s, 3H), 2.53 (s, 3H), 2.58 (s,
3H), 3.49 (m, 1H), 3.55–3.59 (m, 2H), 3.72 (m, 2H), 3.86 (s, 1H),
3.93 (d, J = 12.8, 1H), 4.32 (s, 1H), 4.51 (d, J = 4.35, 1H), 4.67 (t,
J = 3.35, 2H), 4.79 (m, 1H), 5.76 (s, 2H), 5.80 (d, J = 7.6, 1H), 6.26
(s, 1H), 6.70 (d, J = 11.9, 1H), 7.60 (s, 1H), 7.77 (s, 1H), 8.30 (s,
1H); 13C NMR (CDCl3) d 8.5, 12.3, 19.3, 60.4, 63.2, 63.4, 68.4,
69.6, 70.8, 81.6, 88.1, 89.6, 109.3, 111.0, 111.4, 113.0, 116.3,
124.7, 141.0, 149.3, 152.4, 153.3, 154.6, 155.5, 156.0, 161.4. HRMS
m/z: calcd for C27H29NaO8 ([M+Na]+) 574.191, found 574.198.
4.2.9. 6-N-Bz-20-O-[[(4,50,8-Trimethyl) psoralen-40-ylmethoxy]
ethyl] adenosine (5a)
Compound 4a (260 mg, 0.5 mmol) was dried by repeated coeva-
poration with pyridine and was dissolved in dry pyridine (5 ml). To
the solution was added trimethylchlorosilane (300
After the mixture was stirred for 15 min, benzoyl chloride
(275 l, 2.4 mmol) was added and maintained at room tempera-
ll, 2.4 mmol).
l
ture for 2 h. The mixture was then cooled in ice bath and water
(1 ml) was added. After 5 min, 28% aqueous ammonia (2.5 ml)
was added and stirred at room temperature for 0.5 h. The reaction
mixture was then evaporated to near dryness and the residue was
dissolved in CHCl3 (60 ml). The solution was washed three times
with water (60 ml). The organic layer was dried over Na2SO4, fil-
tered, and concentrated to dryness. The residue was purified by
flash chromatography to give 5a (284 mg, 93%) as yellow powder.
1H NMR (DMSO-d6) d 2.48 (m, 9H), 3.55 (s, 3H), 3.65 (m, 2H), 3.67
(m, 1H), 3.97 (d, J = 3.7, 1H), 4.57 (d, J = 7.05, 3H), 5.15 (s, 2H), 6.14
(d, J = 5.4, 1H), 6.30 (s, 1H), 7.54 (m, 3H), 8.68 (s, 1H), 8.69 (s, 1H),
11.17 (s, 1H); 13C NMR (DMSO-d6) d 8.7, 12.5, 19.1, 61.4, 61.7, 66.1,
66.9, 67.4, 69.1, 73.9, 81.6, 86.2, 86.3, 93.4, 107.6, 108.4, 112.6,
112.9, 116.2, 125.2, 128.9, 129.0, 132.9, 133.7, 143.2, 148.9,
150.9, 154.3, 154.3, 155.3, 160.5. HRMS m/z: calcd for C34H33NaO9
([M+Na]+) 678.218, found 678.209.
4.2.7. 20-O-[[(4,50,8-Trimethyl) psoralen-40-ylmethoxy] propyl]
adenosine (4b)
Adenosine (8.86 g, 33.1 mmol) was stirred with 60% NaH
(1.60 g, 39.8 mmol) in dry DMF (50 ml) at room temperature for
1 h, then 3b (740 mg, 2.20 mmol) and potassium iodide (512 mg
3.10 mmol) in dry DMF (20 ml) were added. The mixture was stir-
red at 75 °C for 27 h. Phosphate buffer (1.0 ml, 100 mM sodium
phosphate, pH 7.0) was added to the reaction mixture, the solution
was evaporated to near dryness, and the residue was dissolved in
CH2Cl2. The resulting solution was washed with aqueous 5% NaH-
CO3 (100 ml ꢁ 3). The organic layer was dried over Na2SO4, fil-
trated, and concentrated to dryness. The residue was purified by
silica gel column chromatography. Elution with CH2Cl2–MeOH
(25:1, v/v) gave fractions of 4b (203 mg, 16% yield). 1H NMR
(CDCl3) d 2.02 (m, 2H), 2.47 (s, 3H), 2.48 (s, 3H), 2.49 (s, 3H),
3.36–3.52 (m, 6H), 4.01 (m, 1H), 4.13 (s, 1H), 4.26 (s, 1H), 4.46 (s,
2H), 4.79 (m, 1H), 5.42 (s, 1H), 5.70 (s, 2H), 5.93 (m, 1H), 6.30 (d,
J = 11.9, 1H), 7.67 (s, 1H), 8.08 (s, 1H), 8.34 (s, 1H); 13C NMR (CDCl3)
d 8.7, 12.4, 19.2, 30.2, 61.8, 62.3, 63.8, 66.5, 67.3, 67.8, 72.9, 79.7,
86.7, 87.9, 108.3, 112.6, 112.8, 116.2, 129.1, 132.0, 140.0, 140.3,
148.9, 152.9, 153.2, 154.1, 155.3, 156.6, 160.5. HRMS m/z: calcd
for C28H31NaO8 ([M+Na]+) 588.207, found 588.211.
4.2.10. 6-N-Bz-20-O-[[(4,50,8-Trimethyl) psoralen-40-ylmethoxy]
propyl] adenosine (5b)
Compound 4b (200 mg, 0.35 mmol) was dried by repeated
coevaporation with pyridine and was dissolved in dry pyridine
(4 ml). To the solution was added trimethylchlorosilane (230
1.82 mmol). After the mixture was stirred for 15 min, benzoyl chlo-
ride (205 l, 1.80 mmol) was added and maintained at room tem-
ll,
l
perature for 2 h. The mixture was then cooled in ice bath and water
(1 ml) was added. After 5 min, 28% aqueous ammonia (2 ml) was
added and stirred at room temperature for 0.5 h. The reaction mix-
ture was then evaporated to near dryness and the residue was dis-
solved in CHCl3 (60 ml). The solution was washed three times with
water (25 ml). The organic layer was dried over Na2SO4, filtered,
and concentrated to dryness. The residue was purified by flash
chromatography to give 5b (122 mg, 52%) as yellow powder. 1H
NMR (DMSO-d6) d 1.97–2.00 (m, 2H), 2.42 (d, 6H), 2.48 (s, 3H),
3.39–3.40 (m, 2H), 3.55–3.68 (m, 4H), 3.96 (d, J = 3.9, 1H), 4.30
(m, 1H), 4.44 (m, 1H), 4.48 (s, 2H), 5.13–5.18 (m, 2H), 6.07 (d,
J = 5.5, 1H), 6.28 (s, 1H), 7.50–7.54 (m, 2H), 7.62 (m, 1H), 7.67 (s,
1H), 7.99–8.02 (m, 2H), 8.70 (m, 2H),11.2 (s, 1H); 13C NMR
(DMSO-d6) d 8.7, 12.5, 19.1, 29.9, 61.4, 62.3, 66.5, 66.8, 67.4, 69.1,
73.9, 81.6, 86.2, 86.5, 93.5, 107.6, 108.3, 112.6, 112.8, 116.2,
125.2, 128.9, 129.0, 132.9, 133.7, 143.2, 148.9, 150.9, 154.2,
154.3, 155.3, 160.5. HRMS m/z: calcd for C35H35NaO9 ([M+Na]+)
692.233, found 692.222.
4.2.8. 20-O-[[(4,50,8-Trimethyl) psoralen-40-ylmethoxy] butyl]
adenosine (4c)
Adenosine (5.70 g, 21.6 mmol) was stirred with 60% NaH
(1.02 g, 25.7 mmol) in dry DMF (30 ml) at room temperature for
1 h, then 3c (500 mg, 1.43 mmol) and potassium iodide (332 mg
2.00 mmol) in dry DMF (20 ml) were added. The mixture was stir-
red at 75 °C for 31 h. Phosphate buffer (1.0 ml, 100 mM sodium
phosphate, pH 7.0) was added to the reaction mixture, the solution
was evaporated to near dryness, and the residue was dissolved in
CH2Cl2. The resulting solution was washed with aqueous 5% NaH-
CO3 (100 ml ꢁ 3). The organic layer was dried over Na2SO4, fil-
trated, and concentrated to dryness. The residue was purified by
silica gel column chromatography. Elution with CH2Cl2–MeOH
(25:1, v/v) gave fractions of 4c (232 mg, 28% yield). 1H NMR
(CDCl3) d 2.02–2.10 (s, 2H), 2.44 (s, 3H), 2.47 (s, 3H), 2.49 (s, 3H),
4.2.11. 6-N-Bz-20-O-[[(4,50,8-Trimethyl) psoralen-40-ylmethoxy]
butyl] adenosine (5c)
Compound4b (208 mg, 0.36 mmol) was driedby repeated coeva-
poration with pyridine and was dissolved in 4 ml of dry pyridine. To
the solution was added trimethylchlorosilane (228
After the mixture was stirred for 15 min, benzoyl chloride (210
1.8 mmol) was added and maintained at room temperature for 2 h.
ll, 1.8 mmol).
l
l,

482
M. Higuchi et al. / Bioorg. Med. Chem. 17 (2009) 475–483
The mixture was then cooled in ice bath and water (0.8 ml) was
added. After 5 min, 28% aqueous ammonia (1.5 ml) was added and
stirred at room temperature for 0.5 h. The reaction mixture was then
evaporated to near dryness and the residue was dissolved in CHCl3
(50 ml). The solution was washed three times with water (30 ml).
The organic layer was dried over Na2SO4, filtered, and concentrated
to dryness. The residue was purified by flash chromatographyto give
5b (248 mg, 94%) as yellow powder. 1H NMR (DMSO-d6) d 2.20 (m,
4H), 2.42–2.48 (m, 9H), 3.39–3.40 (m, 2H), 3.46–3.78 (m, 6H), 3.97
(d, J = 3.4, 1H), 4.30 (s, 1H), 4.48 (m, 1H), 4.50 (s, 2H), 5.16 (m, 1H),
5.23 (d, J = 5.5, 1H), 6.09 (d, J = 5.8, 1H), 6.29 (s, 1H), 7.48–7.53 (m,
3H), 7.69 (s, 1H), 7.98–8.02 (m, 2H), 8.69 (s, 1H),8.72 (s, 1H); 13C
NMR (DMSO-d6) d 8.7, 12.5, 19.0, 26.1, 26.3, 26.8, 61.9, 62.2, 69.4,
69.7, 70.0, 81.3, 86.5, 86.8, 108.2, 112.6, 112.7, 112.8, 116.2, 119.7,
125.3,128.8, 130.0, 132.7, 133.7, 140.1, 148.7,149.5, 152.9, 154.1,
154.3, 155.0, 156.6, 160.5. HRMS m/z: calcd for C36H37NaO9
([M+Na]+) 706.249, found 706.252.
(3 ml). To the solution was added 4,40-dimethoxytrityl chloride
(230 mg, 0.69 mmol), and the mixture was stirred at room temper-
ature for 6 h, then water (20 ml) was added. The solution was ex-
tracted three times with CH2Cl2 (20 ml). The organic layer was
dried over Na2SO4, filtered, and concentrated to dryness. The resi-
due was purified by flash chromatography to give 6c (105 mg, 31%)
as white foam. 1H NMR (CDCl3) d 2.15 (m, 4H), 2.47–2.48 (t, 9H),
3.16 (s, 1H), 3.48–3.52 (m, 5H), 3.75–3.76 (m, 8H), 4.22 (d,
J = 3.0, 2H), 4.52 (m, 2H), 4.59–4.62 (m, 2H), 6.14–6.15 (d, J = 3.5,
2H), 6.79–6.82 (m, 4H), 7.25–7.33 (m, 4H), 7.41–7.43 (m, 4H),
7.48–7.51 (m, 2H), 8.05 (d, J = 6.8, 1H), 8.16 (s, 1H), 8.69 (s, 1H),
9.12 (s, 1H); 13C NMR (CDCl3) d 8.4, 12.3, 19.3, 26.1, 26.3, 26.8,
30.9, 55.2, 62.9, 63.0, 69.7, 69.8, 70.9, 81.7, 84.1, 86.6, 87.0, 109.2,
11.5, 111.9, 112.8, 113.2, 116.1, 127.0, 127.4, 127.9, 128.0, 128.1,
128.5,128.8, 130.0, 130.1, 131.9, 132.8, 133.4, 135.5, 141.4, 144.4,
149.1, 153.3, 154.6, 154.9, 158.6, 161.5; HRMS m/z: calcd for
C57H55NaO11 ([M+Na]+) 1008.380, found 1008.375.
4.2.12. 6-N-Bz-50-O-DMTr-20-O-[[(4,50,8-Trimethyl) psoralen-40-
ylmethoxy] ethyl] adenosine (6a)
4.2.15. Synthesis of 6-N-Bz-50-O-DMTr-20-O-{[(4,50,8-trimethyl)
psoralen-40-ylmethoxy] alkyl} adenosine 30-(2-cyanoethyl-N,N0-
diisopropylphosphoramidite) (7a,7b,7c) and introduction to
oligonucleotide
Compound 5a (82 mg, 0.12 mmol) was dried by repeated coeva-
poration with pyridine and was dissolved in dry pyridine (1.5 ml).
To the solution was added 4,40-dimethoxytrityl chloride (104 mg,
0.30 mmol), and the mixture was stirred at room temperature for
8 h, then water (20 ml) was added. The solution was extracted
three times with CH2Cl2 (20 ml). The organic layer was dried over
Na2SO4, filtered, and concentrated to dryness. The residue was
purified by flash chromatography to give 6a (63 mg, 55%) as white
foam. 1H NMR (DMSO-d6) d 2.48 (m, 9 H), 3.58–3.68 (m, 2H), 3.69
(s, 6H), 3.81 (m, 1H), 4.01 (m, 1H), 4.42 (d, J = 7.05, 1H), 4.59 (s, 1H),
4.67 (t, J = 7.05, 1H), 5.15 (d, 1H), 6.18 (d, J = 5.4, 1H), 6.27 (s, 1H),
6.78–6.81(m, 4H), 7.17–7.23 (m, 6H), 7.30–7.32 (m, 3H), 7.53–7.55
(m, 2H), 7.62 (m, 1H), 7.70 (s, 1H), 8.03 (d, J = 3.7, 2H) 8.52 (s, 1H),
8.60 (s, 1H), 11.18 (s, 1H); 13C NMR (CDCl3) 8.4, 12.3, 19.3, 55.2,
62.9, 63.0, 69.8, 70.9, 81.7, 84.2, 86.6, 87.1, 109.2, 11.5, 111.9,
112.6, 113.2, 116.1, 127.0, 127.4, 127.9, 128.0, 128.2, 128.5,
128.8, 130.0, 130.2, 131.9, 132.8, 134.4, 135.5, 141.4, 144.4,
149.1, 153.3, 154.6, 154.9, 158.6, 161.5. HRMS m/z: calcd for
C55H51NaO11 ([M+Na]+) 980.348, found 980.366.
The fully protected 50-O-DMTr-ACCGCAT (1.0
l
mol) was syn-
thesized on CPG using a DNA synthesizer. Each compound, 7a,
7b, and 7c (10 mol) in dry acetonitrile (72 l) was allowed to re-
act with 2-cyanoethyl N,N,N0,N0-tetraisopropylphosphorodiamidite
l
l
(4
ll, 11
lmol) in the presence of 0.5 M 1H-tetrazole (24 ll,
11
lmol), and these mixtures were stirred at room temperature
for 1 h. Without purification, the reaction mixtures were added
to 50-detritylated ACCGCAT-CPG in the presence of 1 equiv. of
1H-tetrazole in dry acetonitrile, and the mixtures were allowed
to react at room temperature for 2 h. The DMTr cations released
from the CPG were quantified by UV spectroscopy and the coupling
yield was ca. 50%. After capping, the CPG support was then set to
the DNA synthesizer and the oligonucleotide synthesis was contin-
ued. The CPG support was treated with concentrated 28% aqueous
ammonia at 55 °C for 7 h, and the solution was concentrated to
dryness. Purification of 20-Ps-xom was performed with a reverse-
phase HPLC with an acetonitrile gradient in TEAA (pH 7.0). Column,
CAPCELL PAK C18, 5
l
m, 4.6 U ꢁ 150 mm, (Shiseido, Co. Ltd., To-
4.2.13. 6-N-Bz-50-O-DMTr-20-O-[[(4,50,8-Trimethyl) psoralen-40-
ylmethoxy] propyl] adenosine (6b)
kyo, Japan); mobile phase, (A) 0.1 M triethylammonium acetate
(TEAA, pH 7.0) and (B) 50% CH3CN in 0.1 M TEAA at the flow rate
of 0.8 ml/min (acetonitrile gradient: 7.5–15% for 15 min, 15–50%
for 10 min, 50% for 10 min). The fractions containing the oligonu-
cleotides with DMTr groups were concentrated, and then the DMTr
groups were removed by treatment with 80% acetic acid for
30 min.
Compound 5b (56 mg, 0.08 mmol) was dried by repeated coeva-
poration with pyridine and was dissolved in dry pyridine (1 ml). To
the solution was added 4,40-dimethoxytrityl chloride (45 mg,
0.13 mmol), and the mixture was stirred at room temperature for
5 h, then water (20 ml) was added. The solution was extracted three
times with CH2Cl2 (20 ml). The organic layer was dried over Na2SO4,
filtered, and concentrated to dryness. The residue was purified by
flash chromatography to give 6b (49 mg, 58%) as white foam. 1H
NMR (CDCl3) d 1.90 (m, 2H), 2.40 (s, 3H), 2.49 (s, 3H), 2.51 (s, 3H),
3.24 (s, 1H), 3.38 (m, 1H), 3.47 (s, 1H), 3.55–3.60 (m, 2H), 3.78 (s,
8H), 3.92 (m, 1H), 4.15 (s, 1H), 4.41 (s, 1H), 4.44 (s, 1H), 4.59–4.63
(m, 2H), 5.96 (s, 1H), 6.15 (s, 1H), 6.80–6.82 (m, 4H), 7.21–7.31(m,
6H), 7.41 (s, 1H), 7.43 (s, 1H), 7.52–7.57 (m, 5H), 8.03 (d, J = 6.8,
1H), 8.17 (s, 1H), 8.69 (s, 1H), 9.12 (s, 1H); 13C NMR (CDCl3) d 8.5,
12.3, 19.3, 29.6, 55.2, 62.8, 62.9, 66.5, 68.3, 69.6, 81.7, 84.1, 86.6,
87.0, 109.2, 111.4, 11.5, 112.8, 113.2, 116.1, 124.9, 127.0, 127.8,
127.9, 128.1, 128.9, 130.0, 130.1, 132.8, 135.5, 135.6, 141.4, 144.4,
152.6, 153.1, 155.2, 158.6, 161.4, 164.6. HRMS m/z: calcd for
C56H53NaO11 ([M+Na]+) 994.364, found 994.380.
4.3. Measurements of UV-melting profiles of duplexes of 20-Ps-
oligo and ORN
UV melting profiles of the duplex 20-Ps-xom and ORN were ob-
tained by a UV spectrophotometer equipped with a programmed
thermal controller at an increase rate of 1.0 °C/min. The sample
solutions were prepared in 100 mM phosphate buffer (pH 7.0) con-
taining 0.1 M NaCl, and the concentration of the oligonucleotides
was fixed at 2.0 lM.
4.4. CD spectra of duplexes of 20-Ps-oligo and ORN
Circular dichroism (CD) spectra were obtained on a CD spectro-
photometer (J-720, JASCO, Tokyo, Japan) equipped with a thermal
controller (RET-100, Neslab, Portsmouth, NH, USA). An equimolar
4.2.14. 6-N-Bz-50-O-DMTr-20-O-[[(4,50,8-Trimethyl) psoralen-40-
ylmethoxy] butyl] adenosine (6c)
solution of 20-Ps-xom (or Kr-ODN) and ORNs (2.0
lM each) was
Compound 5c (235 mg, 0.34 mmol) was dried by repeated
coevaporation with pyridine and was dissolved in dry pyridine
prepared in a buffer containing 100 mM sodium phosphate (pH
7.0) and 0.1 M NaCl.

M. Higuchi et al. / Bioorg. Med. Chem. 17 (2009) 475–483
483
4.5. Fluorescence spectra of duplexes of 20-Ps-oligo and ORN
References and notes
1. Hollstein, M.; Sidransky, D.; Vogelstein, B.; Harris, C. C. Science 1991, 253, 49.
2. Greenblatt, M. S.; Bennett, W. P.; Hollstein, M.; Harris, C. C. Cancer Res. 1994, 54,
4855.
3. Wolf, D. M.; Jordan, V. C. Breast Cancer Res. Treat. 1994, 31, 129.
4. England, G. M.; Bilimoria, M. M.; Chen, Z.; Assikis, V. J.; Muenzner, H. D.; Jordan,
V. C. Int. J. Oncol. 1998, 12, 981.
Fluorescence spectra were obtained by a Fluorescence spectro-
photometer (RF-5300PC, Shimadzu Co. Kyoto, Japan) equipped
with a thermal controller at 15 °C. An equimolar solution of 20-
Ps-oligo and ORN (2.0 lM each) in 100 mM phosphate buffer (pH
7.0) containing 0.1 M NaCl was heated at 85 °C for 5 min and
slowly cooled to the designated temperature prior to measure-
ments. No attempt was made to eliminate dissolved oxygen in
the buffer. The excitation bandwidth was 5 nm. The emission
bandwidth was 5 nm.
5. Barbacid, M. Annu. Rev. Biochem. 1987, 56, 779.
6. Johannes, L. B.; Fearon, E. R.; Hamilton, S. R. Nature 1987, 327, 293.
7. Kahn, S.; Yamamoto, F.; Almoguera, C.; Winter, E.; Forrester, K.; Jordano, J.;
Perucho, M. Anticancer Res. 1987, 7, 639.
8. Johannes, L. B. Cancer Res. 1989, 49, 4682.
9. Kurreck, J. Eur. J. Biochem. 2003, 270, 1628.
10. Manoharan, M. Biochem. Biophys. Acta 1999, 1489, 117.
11. Vester, B.; Wengel, J. Biochemistry 2004, 43, 13233.
12. Elbashir, S. M.; Martinez, J.; Patkaniowska, A.; Lendeckel, W.; Tuschl, T. EMBO J.
2001, 23, 6877.
4.6. Photo-cross-linking reaction of psoralen-conjugated
oligonucleotide (Ps-oligo) and ORN
13. Amarzguioui, M.; Holen, T.; Babaie, E.; Prydz, H. Nucleic Acids Res. 2003, 31, 589.
14. Braasch, D. A.; Jensen, S.; Liu, Y.; Kaur, K.; Arar, K.; White, M. A.; Corey, D. R.
Biochemistry 2003, 42, 7967.
An equimolar solution of 20-Ps-xom and ORNs (2.0
lM each)
was denatured at 85 °C for 5 min and slowly cooled to 37 °C in a
buffer containing 100 mM sodium phosphate (pH 7.0) and 0.1 M
NaCl. The reaction mixture was irradiated by UVA on a transillumi-
nator (FTI-LW, 365 nm, 1.6 mW/cm2, Funakoshi Co., Ltd., Tokyo, Ja-
pan), and analyzed by reversed-phase HPLC with an acetonitrile
15. Chang, E. C.; Miller, P. S.; Cushman, C.; Devadas, K.; Pirollo, K. F.; Ts0o, P. O. P.;
Yu, Z. P. Biochemistry 1991, 30, 8283.
16. Mukhopadhyay, T.; Tainsky, M.; Cavender, A. C.; Roth, J. A. Cancer Res. 1991, 51,
1744.
17. Zhang, Y.; Mukhopadhyay, T.; Donehower, L. A.; George, R. N.; Roth, J. A. Hum.
Gene Ther. 1993, 4, 451.
18. Cowsert, L. M. Anti-Cancer Drug Dev. 1997, 12, 359.
19. Kita, K.; Saito, S.; Morikawa, C. Y.; Watanabe, A. Int. J. Cancer 1999, 80, 554.
20. Andreyev, H. J. N.; Ross, P. J.; Cunningham, D.; Clarke, P. A. Gut 2001, 48, 230.
21. Cimino, G. D.; Gamper, H. B.; Isaacs, S.; Hearst, J. Annu. Rev. Biochem. 1985, 54,
1151.
22. Lee, B. L.; Murakami, A.; Blake, K. R.; Lin, S. B.; Miller, P. S. Biochemistry 1988, 27,
3197.
23. Kean, J. M.; Murakami, A.; Blake, K. R.; Cushman, C. D.; Miller, P. S. Biochemistry
1988, 27, 9113.
24. Murakami, A.; Yamayoshi, A.; Iwase, R.; Nishida, J.; Yamaoka, T.; Wake, N. Eur. J.
Pharm. Sci. 2001, 13, 25.
25. Yamayoshi, A.; Kato, K.; Suga, S.; Ichinoe, A.; Arima, T.; Matsuda, T.; Kato, H.;
Murakami, A.; Wake, N. Oligonucleotide 2007, 17, 66.
26. Higuchi, M.; Yamayoshi, A.; Yamaguchi, T.; Iwase, R.; Yamaoka, T.; Murakami,
A. Nucleosides, Nucleotides Nucleic Acids 2007, 26, 277.
27. Stary, A.; Sarasin, A. Methods Enzymol. 2000, 319, 153165.
28. McDonald, D. Q.; Still, W. C. Tetrahedron Lett. 1992, 33, 7743.
29. Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.; Lipton, M.; Canfield,
C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput. Chem. 1990, 11, 440.
30. Isaacs, S. T.; Shen, C. J.; Hearst, J. E.; Rapoport, H. Biochemistry 1977, 16, 1058.
31. Pieles, U.; Englisch, U. Nucleic Acids Res. 1989, 17, 285.
32. Ti, G. S.; Gaffney, B. L.; Jones, R. A. J. Am. Chem. Soc. 1982, 104, 1316.
33. Salet, C.; De Sa E Melo, T. M.; Bensasson, R.; Land, E. J. Biochem. Biophys. Acta
1980, 607, 379.
gradient in TEAA (pH 7.0). Column, CAPCELL PAK C18, 5 lm, 4.6
U ꢁ 150 mm, (Shiseido, Co. Ltd., Tokyo, Japan); column oven,
40 °C; mobile phase, (A) 0.1 M triethylammonium acetate (TEAA,
pH 7.0) and (B) 50% CH3CN in 0.1 M TEAA at a flow rate of
0.8 ml/min (acetonitrile gradient: 7.5–15% for 15 min, 15–50% for
10 min, 50% for 10 min). The photo-cross-linking yields were eval-
uated from the amount of the intact ORNs.
Photo-regeneration reaction was carried out as follows: isolated
samples by HPLC were dried up and then irradiated by a short
wavelength illuminator (MINERALIGHTÒ LAMP, UVGL-58,
254 nm, Funakoshi Co., Ltd., Tokyo, Japan). The photo-reversal
samples were dissolved in 0.1 M TEAA and analyzed by reversed-
phase HPLC.
Acknowledgments
This research was partly supported by a Grant-in-Aid for Scien-
tific Research (C) of The Ministry of Education, Science, Sports and
Culture of Japan (19550164, A.M., A.K., A.Y.; 20750136, A.Y).
34. Rabin, D.; Crothers, D. M. Nucleic Acids Res. 1979, 7, 689.