Novel photoresponsive cross-linking oligodeoxyribonucleotides having a caged α-chloroaldehyde

Article abstract of DOI:10.1016/j.bmc.2012.07.030

We have developed photoresponsive cross-linking oligodeoxyribonucleotides (ODNs) for sequence-selective interstrand covalent bond formation toward target nucleotides. A phosphoramidite derivative of α-chloroaldehyde whose carbonyl group was converted to a bis(2-nitrobenzyl)acetal group was prepared for the synthesis of photoresponsive α-chloroaldehyde (PCA)-conjugated ODN. The bis(2-nitrobenzyl)acetal group of a PCA-thymidine conjugate was completely removed by UV irradiation at 365 nm (400 mW/cm²) for 1 min. Photo-cross-linking studies revealed that PCA-ODN selectively reacted with the target nucleotides having an adenine or a cytosine moiety at the frontal position of the α-chloroaldehyde group.

Full text of DOI:10.1016/j.bmc.2012.07.030

Bioorganic & Medicinal Chemistry 20 (2012) 5071–5076
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Bioorganic & Medicinal Chemistry
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Novel photoresponsive cross-linking oligodeoxyribonucleotides having a caged
a-chloroaldehyde
⇑
Akio Kobori , Takemune Yamauchi, Yuko Nagae, Asako Yamayoshi, Akira Murakami
Department of Biomolecular Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We have developed photoresponsive cross-linking oligodeoxyribonucleotides (ODNs) for sequence-selec-
tive interstrand covalent bond formation toward target nucleotides. A phosphoramidite derivative of
Received 13 June 2012
Revised 13 July 2012
Accepted 13 July 2012
Available online 24 July 2012
a-chloroaldehyde whose carbonyl group was converted to a bis(2-nitrobenzyl)acetal group was prepared
for the synthesis of photoresponsive -chloroaldehyde (PCA)-conjugated ODN. The bis(2-nitroben-
a
zyl)acetal group of a PCA–thymidine conjugate was completely removed by UV irradiation at 365 nm
(400 mW/cm2) for 1 min. Photo-cross-linking studies revealed that PCA–ODN selectively reacted with
Keywords:
Cross-link
Antisense
the target nucleotides having an adenine or a cytosine moiety at the frontal position of the
hyde group.
a-chloroalde-
Ó 2012 Elsevier Ltd. All rights reserved.
a-Chloroaldehyde
1. Introduction
internal strand13,14 and used for photo-cross-linking with pyrimi-
dine moieties in complementary nucleotides. We have reported
that oligonucleotides having a Ps at the 20-O hydroxy group of
adenosine (20-Ps-eom15) recognize one base difference in the target
sequences under clinically relevant conditions. Using 20-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.16 Considering the potential benefits of photoresponsive
cross-linking reagent-nucleotide conjugates, it is important to
develop new photoresponsive groups that could give high yields
and exhibit specific reactive characteristics to target nucleobases
located in specific positions in the target sequence.
Oligonucleotide analogues forming covalent bonds with com-
plementary nucleotides in a sequence-specific manner under phys-
iological conditions are of potential clinical and biological
interest.1,2 In particular, photoresponsive oligonucleotide ana-
logues which cross-link with complementary nucleotides using
photo-irradiation as a trigger of the reaction have been developed
to investigate and control gene functions without damaging living
systems.3,4 These oligonucleotide analogues have photoresponsive
moieties introduced on a site which does not affect duplex or tri-
plex formation. The introduced photoresponsive moieties, which
are non-reactive or less reactive toward bio-molecules in their ori-
ginal form, generate highly reactive species after photo-irradiation.
Arylazido5 and ziazirine6 derivatives that generate highly reactive
electron-deficient species after UV irradiation have been intro-
duced to nucleotide analogues and used for site-specific cross-
linking to complementary nucleotides in a sequence specific
manner. Thionucleobase7 derivatives, which mainly give 2+2
photo-adducts of the excited C–S bond onto the 5,6 double bond
of pyrimidines under UV irradiation, have been incorporated into
functionalized nucleotides, such as ribozymes8 and protein-recog-
nized RNA,9 to elucidate active-structures and protein-RNA inter-
actions. 5-Cyanovinyldeoxyuridine10 incorporated to the 50-end
of oligoODN reacted to an adenine residue in a complementary
ODN to yield end-capping DNA duplexes. 4,50,8-Trimethylpsoralen
(Ps) derivatives have been introduced at the 50-end,11,12 and the
a
-Chloroacetaldehyde (CAA), which is generated by the metab-
olism of vinylchloride,17,18 reacts with deoxycytidine (dC), deoxya-
denosine (dA) under slightly acidic and neutral conditions, yielding
1,N6-ethenoadenine (
e
A), and 3,N4-ethenocytosine (
e
C), respec-
tively.19–23 Under neutral conditions, CAA subtly reacts with guan-
ine, yielding N2,3-ethenoguanine (
G), with decomposition of CAA.
e
Quantitative analysis of the formation of etheno adducts in DNA
duplexes by the CAA reaction reveals that the relationship can be
represented as follows:
reported25 a sequence-selective cross-linking reaction that formed
a triple helix by using an -haloacetamide-attached ODN and the
eC > eA > e
G.24 Grant and Dervan
a
target DNA duplex. Detailed product analysis of the target strand
using alkaline treatment for reactive species-selective cleavage
revealed that an
selectively reacted with guanine-N7 in the target DNA duplex.
Summerton and Barlet also reported that an -haloketone-conju-
gated ODN26 forms a guanine-N7 adduct in the DNA duplexes.
Although cross-linking studies on -haloacetamides and -haloke-
tones having chemical structures similar to -chloroaldehyde have
a
-haloacetamide attached to the 50-end of ODN
a
a
a
⇑
Corresponding author. Tel./fax: +81 75 724 7849.
E-mail address: akobori@kit.ac.jp (A. Kobori).
a
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.030

5072
A. Kobori et al. / Bioorg. Med. Chem. 20 (2012) 5071–5076
previously been reported, we believe that
a
-haloaldehyde has not
previously been incorporated into nucleotides and has not been
used for the sequence-selective modification of target
nucleotides.27
In this study, we newly synthesized photo-cross-linking ODNs
having a photoresponsive
50-end of the ODN. The PCA group was comprised of an
bis(2-nitrobenzyl)acetal group, which was converted to an
a
-chloroaldehyde (PCA) group at the
-chloro
-chlo-
a
a
roaldehyde group after 1 min of UV irradiation. Photo-cross-link-
ing studies revealed that the oligonucleotide conjugates
underwent sequence-selective cross-linking to target nucleotides
in a time-dependent manner under physiological conditions.
2. Results and discussion
2.1. Synthesis of PCA-conjugated ODNs
Figure 1. Oligonucleotides used in this study.
The synthetic route of a-chloroaldehyde derivatives is shown as
Scheme 1. Photo-labile protective groups known as caging group
have been widely introduced to append the photo-responsivity
to the bio-active reagents for controlling living systems in a spatial
and temporal fashion. Therefore, the bis(2-nitrobenzyl)acetal
group, which was first reported in 196328 as a photo-removal pro-
tective group of carbonyl groups, was chosen for the protection of
2.2. Deprotection conditions of the bis(2-nitrobenzyl)acetal
group
Deprotection conditions of the bis(2-nitrobenzyl)acetal group
attached to the 50-end of thymidine (PCA-T) were examined by re-
versed-phase HPLC. After UV irradiation at 365 nm (400 mW/cm2)
for 30 s, the peak of PCA-T (Fig. 2a) almost disappeared, concomi-
tant with an increase in the new product that peaked at approxi-
mately 18 min. The high-resolution mass of the purified product
the a-chloroaldehyde group, which is usually unstable and extre-
mely reactive with nucleophiles.29,30 N-Chlorosuccinimide was
used for the chlorination of 6-oxohexanoyl benzoate,31 which
was prepared using the reported procedures. Because 2 was inher-
ently unstable and purification of 2 from a mixture of the starting
material and the bis-chlorinated derivative did not succeed, crude
products were used for introduction of the bis(2-nitrobenzyl)acetal
group. After the protection of the carbonyl group using 2-nitroben-
zyl alcohol and naphthalene sulfonic acid, the benzoyl group was
deprotected by methanolic ammonia. Under such basic conditions,
precisely corresponded to deprotected PCA-T,
a-chloroaldehyde-
conjugated thymidine (CA-T); calcd 453.08, found 453.08. Kinetic
study of the deprotection reaction revealed that deprotection of
the bis(2-nitrobenzyl)acetal group was completed within 1 min
at a kinetic rate t1/2 = 1.9 s (Fig. 2b). Therefore, 1 min UV irradiation
at 365 nm (400 mW/cm2) was used for the cross-linking studies
described below.
the
ramidite derivative was prepared using the standard procedures
and was used for the synthesis of the -chloroaldehyde-conjugated
a-chloro bis(2-nitrobenzyl)acetal group was stable. A phospho-
2.3. Cross-linking studies of PCA-conjugated ODNs
a
ODNs, PCA-T, PCA–ODN1, and PCA–ODN2 (Fig. 1). PCA–ODN1
and PCA–ODN2 contain the antisense sequences of bcl2 mRNA32
at positions 401–421 and 402–422, respectively.
The specificity and efficiency of the cross-linking reaction of
-chloroaldehyde-conjugated ODNs were examined by gel
a
NO2
O
O
4 OBz
O
(a)
(b)
H
OBz
NO2
H
OBz
(d)
Cl
4
4
2
Cl
3
1
2
OCE
NO2
P
4 O
NiPr2
O
(c)
(e)
4 OH
O
Cl
Cl
2
2
4
5
R: d(GTT CTC CCA GCG TGC G)
(PCA-ODN1)
NO2
O
P
:
d(TTC TCC CAG CGT GCG C)
4 O
O
R
O
(PCA-ODN2)
Cl
O-
:
d(T)
2
(PCA-T)
6
Scheme 1. (a) N-chlorosuccinimide,
L
-proline, 10-camphorsulfonic acid, CH2Cl2, ꢀ5 °C, 20 h, 99%; (b) 2-nitrobenzyl alcohol(HONB), 2,2-dimethoxypropane, 2-naphthalene-
sulfonic acid, 95 °C, 24 h, 57%; (c) 1% NaOH/MeOH, rt, 12 h, 89%; (d) O-cyanoethyl-N,N,N0,N0-tetraisopropylphosphorodiamidite, 1H-tetrazole, rt, acetonitrile, 1 h, 59%; (e)
standard phosphoramidite method, d(GTT CTC CCA GCG TGC G)-CPG and d(TTC TCC CAG CGT GCG C)-CPG for PCA–ODN1 and PCA–ODN2, respectively.

A. Kobori et al. / Bioorg. Med. Chem. 20 (2012) 5071–5076
5073
derivatives. They also reported that the cyclic intermediate (8)
showed higher stability in comparison with that of (7) and that
isolation of the intermediate (7) was difficult. In consideration of
these results, a cross-linking product containing an intermediate
(8) and that containing 3,N4-ethenocytosine might be distinctly
observed during the 3,N4-ethenocytosine-forming reaction in
Figure 3b and c. Although an N-7 adduct, 1,N2-etheno adduct,
and N2,3-etheno adduct of chloroacetaldehyde with guanine were
reported by Bartsch and co-workers,36 cross-linking products in
the reaction between PCA–ODN1 and G-ODN1 were scarcely
observed under the reaction conditions used (Fig. 3e). To quantify
the cross-linking reaction between PCA–ODN1 and N-ODN1, the
cross-linking efficiency was estimated and is summarized in
Figure 5a. The cross-linking yield of PCA–ODN1 to A-ODN1 was
increased with time and reached 60% after 48 h of incubation at
37 °C at a kinetic rate of t1/2 = 22 h. To determine the cross-linking
efficiency of PCA–ODN1 and C-ODN1, the combined intensity of
both generated bands was used. The results showed that C-ODN1
had modest reactivity with PCA–ODN1. In the case of N = T or G
(Fig. 3d and e), the cross-linking efficiency was less than 5% at
Figure 2. Deprotection of PCA-T under UV irradiation. (a) Reversed-phase HPLC
spectra of PCA-T (upper: without UV irradiation. lower: after 30 s UV irradiation at
365 nm). (b) Variation of relative mole fractions of PCA-T and CA-T over time were
determined by analyzing the reversed-phase HPLC profiles of the reaction mixtures.
Black line: relative mole fraction of PCA-T. Gray line: relative mole fraction of CA-T.
electrophoresis and autoradiography. As target sequences of PCA–
ODN1, we used 21-mer ODNs (N-ODN1), which consist of
sequences complementary to PCA–ODN1 and a 5-nucleotide dan-
gling end containing one of the four nucleotides (N = A, G, C, and
T) at the frontal position of the PCA. Reaction mixtures of PCA–
ODN1 and 32P-labeled N-ODN1 in 10 mM sodium phosphate buffer
(pH 7.0) containing 100 mM NaCl were irradiated for 1 min at
365 nm (400 mW/cm2) and incubated at 37 °C for 192 h. Analyses
of these reactions by gel electrophoresis followed by autoradiogra-
phy are shown in Figure 3a–e. In the case of A-ODN1(N = A), a new
single band in the high-molecular-weight region was observed in
the first 6 h of incubation (Fig. 3a, lane 2), whereas the control
reaction mixture (Fig. 3a, lane 1), which was not irradiated, showed
no new bands even after 7 days. The intensity of the new band was
increased in a time-dependent manner with a concomitant de-
crease in that of A-ODN1. PCA–ODN1 also reacted with C-ODN1
(Fig. 3b) and gave two new bands, Band 1 and Band 2, in the
high-molecular-weight region under the same reaction conditions.
The intensity variation of obtained the new bands was further ana-
lyzed by changing the reaction temperature in accordance with
previously reported methods33,34 (Fig. 3c). The intensity of the
higher-molecular-weight band (Band 1) was decreased and that
of the lower-molecular-weight band (Band 2) was increased after
incubation initially at 37 °C for 24 h, and later at 80 °C for 4 h. Wie-
wiorowski and co-workers35 reported that the reaction of cytosine
and adenine derivatives with CAA consisted of two steps (Fig. 4):
[1]. Alkylation of the endocyclic nitrogen and cyclization by addi-
tion of an exoamine group to the carbonyl group, resulting in the
formation of cyclic intermediates (7), and (8); and [2] double bond
formation by dehydration, resulting in the formation of etheno
48 h in both cases. These results indicate that
a-chloroaldehyde-
conjugated ODNs preferentially formed the cross-linking products
with the target nucleotides having A or C at the frontal position of
the PCA after UV irradiation and the quantitative relationship is
represented as follows: A > C >> G = T. In addition, the cross-linking
reactivity of PCA–ODN1 to the complementary RNA (A-ORN) was
analyzed (Fig. 3f). PCA–ODN1 reacted with A-ORN just as did with
A-ODN1, and the cross-linking efficiencies at 48 h and 168 h were
68% and 76%, respectively. These results suggest that the cross-
linking reactions of PCA–ODN1 were applicable to not only the
complementary DNA strand but also the complementary RNA
strand.
The reactivity of the adjacent nucleoside to the frontal base of
the
a-chloroaldehyde was examined using PCA–ODN2 and
N-ODN2, which contain one of the four nucleotides (N = A, G, C,
and T) at the frontal position of the PCA and deoxyadenosine next
to the frontal position of PCA. The cross-linking reactions of PCA–
ODN2 with A-ODN2 and C-ODN2 proceeded in the same manner as
those of PCA–ODN1 with A-ODN1 and C-ODN1. The cross-linking
efficiencies of T-ODN2 and G-ODN2 at 48 h were approximately
20% (Fig. 5b), whereas those of T-ODN1 and G-ODN1 were less
than 5% (Fig. 5a). Although further detailed conformational studies
will be needed, these results might suggest that the a-chloroalde-
hyde group can react with a neighboring 20-deoxyadenosine at the
frontal base. Finally, the fluorescence spectra of the reaction mix-
tures between PCA–ODN1 and N-ODN1 (N = A or T) after 48 h
incubation at 37 °C were measured using an excitation wavelength
Figure 3. Photo-induced cross-linking study of PCA-ODN1 with target nucleotides. (a–f) N-ODN1 and A-ORN were labeled at the 50-end with 32P and hybridized with PCA–
ODN1 in 100 mM of sodium phosphate buffer (pH 7.0) containing 150 mM of NaCl. The reaction mixture was irradiated at 365 nm for 1 min and then incubated for up to
7 days at 37 °C. Autoradiograms of 20% polyacrylamide/7 M urea gel were obtained to analyze the cross-linking reaction of PCA–ODN1 in the presence of 100 nM of N-ODN1:
(a) N = A, (b) N = C, (d) N = T, and (e) N = G. Lane 1, without UV irradiation; Lane 2, 6 h; Lane 3, 12 h; Lane 4, 24 h; Lane 5, 48 h; Lane 6, 96 h; Lane 7, 168 h. (c) Photo-induced
cross-linking study of the duplex between PCA–ODN1 and C-ODN1. The reaction mixtures were irradiated at 365 nm for 1 min, then incubated initially at 37 °C for 24 h, and
later at 80 °C for several hours. Lane 1, incubation at 37 °C for 24 h; Lane 2, incubation at 37 °C for 24 h, and at 80 °C for 1 h ; lane 3, incubation at 37 °C for 24 h, and at 80 °C
for 2 h ; lane 4, incubation at 37 °C for 24 h, and at 80 °C for 4 h. (f) Autoradiogram of the cross-linking reaction mixture in the presence of A-ORN.

5074
A. Kobori et al. / Bioorg. Med. Chem. 20 (2012) 5071–5076
Figure 4. The crosslinking reaction of adenine and cytosine with a chloroaldehyde derivative.
Figure 5. (a) Time course of the cross-linking efficiency of PCA–ODN1. The cross-linking efficiencies were obtained as follows: (band density of duplex)/(band density of
duplex + band density of N-ODN1). Black solid line: N = A; black broken line: N = C; gray solid line: N = G; gray broken line: N = T. (b) Time course of the cross-linking
efficiency of PCA–ODN2. (c) Fluorescence spectra of PCA–ODN1 in the presence of N-ODN1 (N = A, black line; N = T, gray line) after 48 h of incubation at 37 °C. The excitation
wavelength was 282 nm.
at 282 nm (Fig. 5c). A typical fluorescent signal derived from C10-
substituted 1,N6-ethenoadenine34 with a peak (maximum) at a
wavelength of approximately 420 nm was observed only in the
case of N = A, whereas no fluorescence was observed in the case
of N = T. These results strongly suggested that 1,N6-ethenoadenine
was formed in the reaction between PCA–ODN1 and A-ODN1.
plates precoated with Merck silica gel 60 F254. Spots were visual-
ized with UV light or staining with molybdic acid (a solution of
10 g (NH4)6Mo7O24 and 4 g Ce(NH4)4(SO4)4 in 360 mL of 10% aque-
ous H2SO4 in 330 mL ethanol). Wakogel C-200 was used for silica
gel flash chromatography. 1H and 13C NMR spectra were measured
on a BRUKER ADVANCE II 300 (1H spectra at 300 MHz; 13C spectra
at 75 MHz; 31P spectra at 121 MHz) spectrometer. FAB mass spec-
tra were recorded on a JEOL JMS-700 MStation. Gel permiation
chromatography (JAI) was performed using an LC 9201 recycling
preparative HPLC system with JAIGEL-GS310 GPC column. DNA
and RNA oligonucleotides were purchased from GeneDesign, Inc.
and FASMAC Co., Ltd, respectively. DNA oligonucleotides contain-
ing PCA were prepared and purified as described below. N-ODN1,
N-ODN2, and A-ORN were purified by preparative 20% denaturing
polyacrylamide gel electrophoresis. The 50 radiolabeling was done
3. Conclusions
We have prepared synthetic ODNs containing photoresponsive
a
-chloroaldehyde at the 50-end. Photo-irradiation is the key to
the cross-linking reaction between the caged ODN and the target
nucleotides. From the quantitative analysis of the cross-linking
products, a-chloroaldehyde-conjugated ODNs can be seen to react
with the target nucleotides having an adenine and a cytosine at the
frontal position of the PCA. Preliminary product studies using fluo-
rescence analysis indicated that 1,N6-ethenoadenine derivatives
were generated in the reaction. Although the efficiency and kinetic
rate of cross-linking reactions need to be improved, the caged
with [c-32P] ATP (PerkinElmer) and T4 polynucleotide kinase,
which was obtained from TaKaRa Bio Inc. A Milli-Q PF system
was used to purify the distilled deionized water that was used to
make all the buffers. Autoradiography was performed on a BAS-
1800II (Fuji Film. Data analysis was performed with Image Gauge
(version 3.4) and rate constants and dissociation constants were
determined with Kaleidagraph (version 3.6.1) software.
a-chloroaldehyde analogues are still applied to the antisense
therapy and nucleotide manipulation in vivo, and biological assays
using this principle are now in progress.
4.2. 5-Chloro-6-oxohexanoyl benzoate(2)
4. Experimental section
(+)-10-Camphorsulfonic acid (2.4 mg, 0.01 mmol) and L-proline
4.1. General methods and materials
(20.2 mg, 0.04 mmol) in CH2Cl2 (2 mL) were added to the solution
of 6-oxohexanoyl benzoate (1) (42.2 mg, 0.2 mmol) in CH2Cl2
(1 mL) at ꢀ10 °C under nitrogen atmosphere and stirred for
Reagents and solvents were purchased from Wako Pure Chem-
ical Industries, TCI, or Sigma. Reactions were monitored with TLC

A. Kobori et al. / Bioorg. Med. Chem. 20 (2012) 5071–5076
5075
5 min, and then N-chlorosuccinimide in CH2Cl2 (2 mL) was added.
After stirring for 20 h at ꢀ10 °C, triethylamine (200 L, 1.4 mmol)
128.5, 128.4, 128.3, 124.8, 124.7, 117.7, 104.9, 67.0, 66.3, 63.4,
63.2, 61.7, 58.4, 58.2, 43.1, 42.9, 32.1, 30.7, 30.6, 29.7, 24.7, 24.6,
24.5, 22.7, 20.4, 20.3, 19.9; 31P NMR (300 MHz CDCl3) d 147.35.;
l
was added and the reaction mixture was washed with saturated
sodium thiosulfate three times, dried over sodium sulfate, and
evaporated under reduced pressure. 5-Chloro-6-oxohexanoyl ben-
zoate (2) (50.5 mg, 99%) was obtained without further purification.
1H NMR (300 MHz CDCl3) d 9.51 (1H, d, J = 2.1 Hz), 8.03 (2H, d,
J = 7.8 Hz), 7.56 (1H, t, J = 6.3 Hz), 7.44 (2H, t, J = 5.9 Hz), 4.34
(2H, t, J = 6.3 Hz), 4.20 (1H, m), 2.12–2.03 (1H, m), 1.98–1.58 (7H,
m); 13C NMR (300 MHz, CDCl3) d 195.1, 166.6, 132.9, 130.2,
129.5, 128.4, 64.4, 63.7, 31.6, 28.2, 22.3. (ESI-TOF Mass) Found
[M+Na]+ = 277.043, Calcd [M+Na]+ = 277.061.
(ESI-TOF
Mass)
Found
[M+Na]+ = 661.217,
Calcd
[M+Na]+ = 661.217.
4.6. Synthesis and characterization of PCA-T, PCA–ODN1, and
PCA–ODN2
PCA phosphoramidite (5) was introduced to 0.1 lmole of thy-
midine, 50-d(GTT CTC CCA GCG TGC G)-30 and 50-d(TTC TCC CAG
CGT GCG C)-30 attached on CPG, which were purchased from Gene-
Design, Inc., using standard b-cyanoethyl phosphoramidite chem-
istry. The coupling times for the PCA phosphoramidites (5) was
600 s. All oligonucleotides were deprotected in concentrated
ammonium hydroxide (55 °C, 16 h) and purified by reversed-phase
HPLC on a SHIMADZU 10A system with a SHISEIDO CAPCELL PAK
C-18 reversed-phase column (HPLC conditions: 0.8 ml/min; sol-
vent A = 0.1 M TEAA; solvent B = 50% CH3CN/0.1 M TEAA linear gra-
dient from 0% to 60% over 30 min, monitored at 260 nm). All
oligonucleotides were characterized by ESI-TOF mass spectrome-
try. PCA-T, Calcd [MꢀH]1ꢀ = 741.158, Found = 741.181.; PCA–
ODN1, Calcd [Mꢀ6H]6ꢀ = 890.143, Found =890.105.; PCA–ODN2,
Calcd [Mꢀ6H]6ꢀ = 887.311, Found = 887.261.
4.3. 5-Chloro-6,6-bis(2-nitrobenzyl)hexanoyl benzoate (3)
2-Nitrobenzyl alcohol (HONB) (118.3 mg, 0.7 mmol), 2,2-dime-
thoxypropane (28 lL, 0.31 mmol), and 2-naphthalenesulfonic acid
(8.0 mg, cat) were added to the solution of 2 (70.5 mg, 0.28 mmol)
in anhydrous toluene (3 mL) under a nitrogen atmosphere and stir-
red for 48 h at 80 °C. After addition of triethylamine (300 lL,
2.1 mmol), CHCl3 (3 mL) was added and the organic solution was
washed with saturated sodium bicarbonate three times, dried over
sodium sulfate, and evaporated under reduced pressure. The resi-
due was purified by silica column chromatography (hexane/chlo-
roform 4:1) to yield 3. (92.0 mg, 57% from 1) 1H NMR (300 MHz
CDCl3) d 8.03 (4H, m), 7.85 (1H, d, J = 7.5 Hz), 7.80 (1H, d,
J = 7.8 Hz), 7.64 (2H, m), 7.53 (1H, m), 7.42 (4H, m), 5.10 (4H, s),
4.93 (1H, d, J = 5.1 Hz), 4.35 (2H, t, J = 5.7 Hz), 4.13 (1H, m), 2.04–
1.64 (6H, m); 13C NMR (300 MHz, CDCl3) d 166.6, 147.1, 133.9,
132.9, 130.3, 129.5, 129.1, 128.9, 128.3, 124.8, 104.9, 67.1, 66.3,
64.6, 61.5, 32.0, 28.2, 22.8, 21.5; (ESI-TOF Mass) Found
[M+Na]+ = 565.134, Calcd [M+Na] + = 565.135.
4.7. Molar absorption coefficient of PCA-T
0.2 OD of PCA-T was fully digested with calf intestine alkaline
phosphatase (25 units, 1
lL) and snake venom phophodiesterase
(1 unit, 30 L) in alkaline phosphatase buffer at 37 °C for 40 h.
l
The digested solution was analyzed by reversed-phase HPLC. The
molar absorption coefficient of PCA-T was determined as 16,700
by comparing peak areas with a standard solution containing 0.1
OD of 20-deoxyadenosine.
4.4. 5-Chloro-6,6-bis(2-nitrobenzyl)hexan-1-ol (4)
Compound
3
(92 mg, 0.16 mmol) and sodium hydroxide
4.8. Deprotection conditions of PCA-T
(30 mg, 0.75 mmol) in MeOH (3 mL) were stirred at room temper-
ature for 12 h. CHCl3 (10 mL) was added and the organic solution
was washed with brine (3 ꢁ 50 mL), dried over sodium sulfate,
and evaporated under reduced pressure. The residue was purified
by silica column chromatography (hexane/chloroform 4:1) to yield
0.50 OD of PCA-T was dissolved in 50 lL of 10 mM phosphate
buffer containing 100 mM NaCl. The solution was irradiated at
365 nm on an LED Spot Light (HLV2, 365 nm, 400 mW/cm2, CCS
Inc.) for up to 1 min and analyzed by reversed-phase HPLC. The
concentration of PCA-T was determined by comparing peak areas
with a standard solution containing 0.30 OD of T.
4. (60.0 mg, 88%) 1H NMR (300 MHz CDCl3)
d 8.06 (2H, d,
J = 6.6 Hz), 7.86 (1H, d, J = 7.8 Hz), 7.80 (1H, d, J = 7.8 Hz), 7.65
(2H, m), 7.45 (2H, t, J = 7.8 Hz), 5.10 (4H, s), 4.92 (1H, d,
J = 5.4 Hz), 4.12 (1H, m), 3.68 (2H, t, J = 6.0 Hz), 1.89–1.50 (6H,
m); 13C NMR (300 MHz, CDCl3) d 147.1, 133.8, 129.0, 128.4,
124.8, 104.8, 67.0, 66.3, 62.6, 61.6, 32.1, 22.5.; (ESI-TOF Mass)
Found [M+Na]+ = 461.084, Calcd [M+Na] + = 461.109.
4.9. Photo-cross-linking reactions of PCA–ODNs
1.0 pmol of the N-ODNs and A-ORN were radiolabeled on the
50-end using [
c-32P]-ATP with T4 polynucleotide kinase (TaKaRa
Bio Inc.) at 37 °C. Excess [ -32P]-ATP was removed using a C-18
c
4.5. O-(5-Chloro-6,6-bis(2-nitrobenzyl)hexan-1-yl)-O0-cyano-
ethyl-N,N-diisopropylphosphoramidite (5)
reversed-phase column according to the manufacturer’s protocol.
For crosslinking studies, additional nonradioactive N-ODNs or
A-ORN was added to the labeled strand to yield a concentration
of 2% labeled nucleic acids. An equimolar solution of N-ODNs
(N = A, C, G, T) or A-ORN and PCA–ODNs (100 nM each) was dena-
tured at 95 °C for 5 min and slowly cooled at 37 °C in 10 mM phos-
phate buffer (pH 7.0) containing 100 mM NaCl. The reaction
mixtures were irradiated by UVA on an LED Spot Light (HLV2,
365 nm, 400 mW/cm2, CCS Inc.) for 1 min, then incubated at
37 °C up to 7 days. The reaction mixtures were analyzed by dena-
tured PAGE (20% polyacrylamide, 7 M Urea, 25% formamide/TBE,
500 V, 45 °C, 90 min). The separation of high-molecular-weight nu-
cleic acid fragments arising from the product and the 21-nucleo-
tide fragment originating from the N-ODNs or A-ORN was
visualized using autoradiography by exposure to an Image Plate
(Fuji Film) for 1 h. The resulting autoradiograms were quantitated
using Image Gauge (version 3.4) software to generate plots of prod-
To a solution of 4 (60 mg, 0.14 mmol) in anhydrous CH3CN was
added O-cyanoethyl-N,N,N0,N0-tetraisopropylphosphorodiamidite
(27 lL, 0.15 nmol) and 1H-tetrazole (6.1 mg, 0.15 mmol). After
stirring for 1 h at room temperature, the reaction mixture was
quenched with 5% sodium bicarbonate solution and extracted with
diisopropyl ether. The organic layer was washed with 0.1 N NaOH
aq, dried over sodium sulfate, and concentrated in vacuo. The crude
product (4) (70.9 mg, 79%) was purified by gel permiation chroma-
tography (GPC). 1H NMR (300 MHz CDCl3) d 8.06 (2H, d, J = 8.1 Hz),
7.87 (1H, d, J = 7.8 Hz), 7.81 (1H, d, J = 7.5 Hz), 7.66 (2H, t,
J = 7.5 Hz), 7.45 (2H, t, J = 7.8 Hz), 5.10 (4H, s), 4.91 (1H, d,
J = 5.4 Hz), 4.11 (1H, m), 3.83 (2H, m), 3.61 (2H, m), 2.63 (2H, t,
J = 6.3 Hz), 1.89–1.50 (6H, m), 1.19–1.12 (12H, m); 13C NMR
(300 MHz, CDCl3) d 147.1, 147.0, 133.9, 133.9, 133.8, 128.9,

5076
A. Kobori et al. / Bioorg. Med. Chem. 20 (2012) 5071–5076
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This work was supported by Grant-in-Aid for Young scientists
(B) (22750154) from the Japan Society for the Promotion of Science
(JSPS) and by a grant from A-STEP (AS231Z03776G) of the Japan
Science and Technology Agency (JST).
Supplementary data
Supplementary data associated with this article can be found, in
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