Synthesis of oligonucleotides containing a new azobenzene fragment with efficient photoisomerizability

Article abstract of DOI:10.1016/S0968-0896(99)00244-8

The azobenzene derivatives possessing substituents of ROCH₂CH₂O- and-CH₂CH₂OR' or -CONHCH₂CH₂OR' at p,p'-positions, where R and R' are 4,4'-dimethoxytrityl and 2-cyanoethyl-N,N'-diisopropylphophoramidite, have been synthesized for linking two oligonucelotide segments. It has been found that the azobenzene linkers efficiently undergo trans-cis isomerization by exposing to UV light. The conversion efficiency showed slight dependence on structure or conformation of oligonucleotides attached to the azobenzene chromophore. The cis-form of the azobenzene in oligonucleotides was sufficiently stable at low temperature under dark. The present findings would open the way for light switch of nucleic acid structures. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00244-8

Bioorganic & Medicinal Chemistry 7 (1999) 2977±2983
Synthesis of Oligonucleotides Containing a New Azobenzene
Fragment with Ecient Photoisomerizability
Kazushige Yamana,* Katsushi Kan and Hidehiko Nakano
Department of Applied Chemistry, Himeji Institute of Technology, 2167 Shosha, Himeji, Hyogo 671-2201, Japan
Received 21 June 1999; accepted 11 August 1999
AbstractÐThe azobenzene derivatives possessing substituents of ROCH2CH2O- and-CH2CH2OR0 or -CONHCH2CH2OR0 at p,p0-
positions, where R and R0 are 4,40-dimethoxytrityl and 2-cyanoethyl-N,N0-diisopropylphophoramidite, have been synthesized for link-
ing two oligonucelotide segments. It has been found that the azobenzene linkers eciently undergo trans±cis isomerization by exposing
to UV light. The conversion eciency showed slight dependence on structure or conformation of oligonucleotides attached to the azo-
benzene chromophore. The cis-form of the azobenzene in oligonucleotides was suciently stable at low temperature under dark. The
present ®ndings would open the way for light switch of nucleic acid structures. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
attention has been paid for addition of photo-regulatory
function to these linker arms.
Azobenzene derivatives have been attracting current
interest as photo-responsive triggers for control of
macroscopic properties in biological systems. There
have been a number of papers concerning photo-reg-
ulation of enzyme activity,1,2 receptor binding,3 mem-
brane permeability,4±6 electron transfer,7,8 and peptide
conformation.9 However, little attempt has been made
to develop photo-responsive systems in relation to
nucleic acids. Komiyama et al. have recently reported
that oligonucleotides containing an azobenzene as a side
chain fragment could be used for photo-regulation of
nucleic acid binding.10 We have synthesized an azo-
benzene fragment for introduction into the main chain
of oligonucleotide strands.11,12 The oligonucleotides
connected by an azobenzene fragment appear attractive,
since two oligonucleotide strands attached to an azo-
benzene fragment would be in di€erent proximity
imposed by the photoisomerization. It is expected that
duplexes, triplexes, and ribozymes involving azo-
benzene-linked oligonucleotides would change their
conformation as a result of photo-illumination. Although
several non-nucleotide linkers have been shown to be
used as regulators for cooperative binding of oligo-
nucleotides to a structural RNA,13,14 hairpin duplex
and triplex formation,15±17 and ribozyme activity,18 little
Our investigation was initiated by design of the azo-
benzene-4,40-diamide derivatives that can be incorporated
between the terminal hydroxyl functions of two oligo-
nucleotide segments.11,12 However, it has been found that
azobenzene diamide underwent poor trans-cis isomeriza-
tion by exposure to UV light.12 In order to overcome this
problem, we reconsidered the structural element of the
azobenzene fragment. It is well known that azobenzene
derivatives with a combination of substituents such as
O-alkyl and alkyl, or O-alkyl and amide, at the p,p0-
positions can undergo trans±cis isomerization in a con-
version of over 90% under certain photo-illumination
conditions.19,20 We have, therefore, designed a new azo-
benzene fragment with appropriate substituents that is
useful for incorporation of an azobenzene chromophore
into oligonucleotide main chains. The present report
describes the details of the synthesis of oligoncucleotides
containing the new azobenzene fragment.
Results and Discussion
We have designed two azobenzene fragments whose
structures are shown in Scheme 1. Both azobenzene deri-
vatives possess O-alkyl and alkyl, or O-alkyl and amide
substituents at the p,p0-positions. The synthesis began with
diazo-coupling of 4-hydroxyethylaniline or 4-amino-N-
(hydroxyethyl)benzamide with phenol. The azobenzene
derivatives 1a and 1b thus obtained were allowed to react
with O-dimethoxytrityl bromoethanol in DMF in the
Key words: Azobenzene; phosphoramidite; oligonucleotide synthesis;
photoisomerization.
* Corresponding author. Tel.: +81-792-67-4895; fax: +81-792-67-
4895; e-mail: yamana@chem.eng.himeji-tech.ac.jp
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00244-8

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K. Yamana et al. / Bioorg. Med. Chem. 7 (1999) 2977±2983
Scheme 1.
presence of K2CO3, giving the azobenzene (2a and 2b)
possessing a hydroxyl group at one end and an O-dime-
thoxytrityl at the other. These compounds were converted
to the phosphoramidites 4a and 4b by using a standard
procedure. The azobenzene derivatives 3a and 3b as model
compounds were prepared by the reaction of 1a and 1b,
respectively, with ethyl iodide.
compound 3a due to photoisomerization. Before irra-
diation, the oligonucleotide 5 exhibits two major
absorption bands which appeared at 265 and 350 nm.
Based on the spectrum for compound 3a, the former
band is reasonably assigned to nucleic acid bases and
the latter to the azobenzene. Immediately after the illu-
mination, it was observed that the absorption at 350 nm
(due to the trans-form) decreased with increase in the
absorption at 440 nm (attributable to the cis-form).
After 2 min, no further spectral changes were observed.
The extent of the absorption changes were found to be
almost the same for both the oligomer and the model
compound, in which photoisomerization from trans to
cis of the azobenzene was ca. 80%. Similar ecient
photoisomerization (conversion eciency 80%) was
observed for oligomer 6 and its model 3b, indicating
that both the azo compounds synthesized here have
identical photoisomerizability by exposing to UV light.
The synthesis of oligonucleotides containing the azo-
benzene fragments 5±8 were carried out by a phosphor-
amidite chemistry on an automated DNA synthesizer. The
sequence of each oligomer is shown in Table 1. The stan-
dard protocol (25 equiv of amidite, 2 min) was used in the
coupling of normal deoxyribonucleoside amidites, while
an excess amount (60 equiv) of the reagent was employed
for 10 min in the coupling of azobenzene amidites 4. With
this protocol, the azobenzene amidites can be coupled to
the hydroxyl group of oligonucleotides on a CPG support.
After the usual deprotection, the azobenzene containing
oligonucleotides were puri®ed by denaturing poly-
acrylamide gel electrophoresis. The oligonucleotides thus
puri®ed were characterized by chromatographic behavior
and ion spray mass spectroscopy as indicated in Table 1.
The oligonucleotide integrity was veri®ed by the observa-
tion that the oligonucleotide exhibited single peak in
HPLC and expected molecular mass. Without UV illumi-
nation, the synthesized oligonucleotides consisted of only
the trans-form of the azobenzene because it is thermo-
dynamically more stable than the cis-form.21
The incorporated oligonucleotide segments whose
sequence is not likely to form a speci®c complex did not
a€ect the degree of photoisomerization of the azo-
benzene unit. In order to see whether photoisomeriza-
tion of the azobenzene fragment is a€ected by structure
or conformation of attached oligonucleotide segments,
two oligonucleotides 7 and 8 were examined. It is noted
that all binding experiments were carried out without
photo-illumination or in the trans form of the azo-
benzene chromophore. The UV melting curves for oli-
gomer 7 are shown in Figure 2(A). The oligonucleotide
solution of 7 at di€erent concentrations showed iden-
tical Tm (60ꢀC), which is dramatically higher than that
Figure 1 shows the UV±vis spectral changes for azo-
benzene containing oligonucleotide 5 and its model
Table 1. HPLC and ion spray mass data for oligonucleotides containing an azobenzene linker
Oligomer
RT (min)
Ion spray mass (negative) obs. (calcd.)
5:
6:
7:
8:
50-dTTTTTTTT-L1-TTTTTTTTa
50-dTTTTTTTT-L2-TTTTTTTTa
32.5
Ð
31.1
29.9
5153.1 (5154.4)
5197.3 (5197.5)
5225.2 (5226.6)
7093.6 (7093.7)
50-dTTTTTTTT-L1-AAAAAAAAa
50-dCCCTAGGAA-L1-AATCGGATCTCGGa
a
L1 and L2 indicate the azobenzene linkers derived from the amidites 4a and 4b, respectively. Reversed HPLC (cosmosil 5C18-AR-300, 4.6Â150
mm) was carried out by (i) 5% CH3CN (5 min), (ii) a linear gradient of CH3CN (5±25% in 30 min), and then (iii) a linear gradient of CH3CN (25±
70% in 45 min) each at a ¯ow rate of 1.0 mL/min in 50 mM triethylammmonium acetate (pH 7).

K. Yamana et al. / Bioorg. Med. Chem. 7 (1999) 2977±2983
2979
Figure 1. Changes in the UV±vis spectra for azobenzene containing oligonucleotide 5 (A) in a phosphate bu€er and its model compound 3a in
CH3CN (B) due to photoisomerization. The solution of the azobenzene derivative in a Pyrex test tube was irradiated in a cooled water bath (5ꢀC) by
a high-pressure Hg lamp using band-path ®lter to transmit light at around 350 nm. UV±vis spectra were obtained at the same temperature at the
indicated time. After 120 s, no further spectral changes were observed for both derivatives.
5
5
Figure 2. (A) UV melting curves for azobenzene containing oligonucleotide 7 at di€erent strand concentrations (*:5Â10 M, *:1Â10 M) in
0.1 M NaCl and 0.01 M sodium phosphate, adjusted to pH 7. The melting experiments were carried out without photo-illumination. (B) Changes in
the UV±vis spectra for azobenzene containing oligonucleotide 7 in the phosphate bu€er due to photoisomerization. The photoirradiation was done
in the same way as described in Figure 1. After 60 s, no further spectral changes were observed.
(8ꢀC) for the duplex of oligothymidylate (T8) and oli-
godeoxyadenylate (dA8). The observed melting tem-
perature is almost identical to that for the hairpin
duplex capped by the azobenezene diamide derivative.12
These observations clearly indicate that the oligomer 7
with 18-mer target are shown in Figure 3(A). Each
melting pro®le exhibited a typical sigmoidal curve as
seen for a DNA duplex. The duplex of the 24-mer and
the 18-mer target, which is known to form a bulge
structure,23 showed a relatively lower Tm than that
for the full-match 18-mer duplex. Similarly, the azo-
benzene containing duplex exhibited the lowered Tm.
These observations suggested that the azobenzene
containing duplex forms a bulge structure. The azo
chromophore, which may be looped out from the
duplex, was isomerized by exposing of UV light in ca.
50% conversion from trans to cis form as shown in
Figure 3(B). The single-stranded azobenzene-oligo-
nucleotide (8) was found to be photoisomerized in a
similar conversion (80%) for oligomer 5 under the same
conditions.
containing self-complementary sequence formed
a
remarkably stable hairpin duplex. By analogy of the
stilbene-linked hairpin duplex,22 the azobenzene chro-
mophore is suggested to be stacked with the base-pair of
the duplex. As shown in Figure 2(B), the UV±vis spec-
tral changes of 7 upon UV illumination were observed
similarly for oligonucleotide 5. In this oligomer, how-
ever, the azobenzene fragment was isomerized in slightly
less eciency to a€ord cis-isomer (ca. 60%) under the
photo-illumination conditions.
A DNA bulge structure would be formed by the mixing of
the oligonucleotide 8 with the sequence of 50-dCCGA-
GATCCGACC TAGGG (18-mer target), since there are
excess residues of-AA-L1-AA- on one side of the duplex.23
The UV melting pro®les for duplexes of oligomer 8, 50-
We next examined the thermal stability of cis-form of
the azo chromophore in the oligonucleotides pro-
duced by the UV illumination. Figure 4(A) showed
the time-course of the absorbance changes at 350 nm
for the photoisomerized oligomer 5 at the di€erent
temperatures. After a prolonged time, the absorbance at
dCCCTAGGAAAAAATCGGATCTCGG
(24-mer),
and 50-dCCCTAGGTCGGATCTCGG (18-mer) formed

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K. Yamana et al. / Bioorg. Med. Chem. 7 (1999) 2977±2983
Figure 3. (A) UV melting pro®les for duplexes of (*) oligomer 8, (*) 50-dCCCTAGGAAAAAATCGGATCTCGG (24-mer), and (&) 50-
M
dCCCTAGGTCGGATCTCGG (18-mer) formed with 18-mer target (50-dCCGAGATCCGACCTAGGG) at a strand concentration of 5Â10
5
in 1.0 M NaCl and 0.01 M sodium phosphate, adjusted to pH 7. The melting experiments were carried out without photo-illumination. (B) Changes
in the UV±vis spectra for azobenzene containing duplex 8 in the phosphate bu€er due to photoisomerization. The photoirradiation was done in the
same way as described in Figure 1. After 120 sec, no further spectral changes were observed.
Figure 4. (A) Time-dependent changes in absorption at 350 nm for photoisomerized oligomer 5 at di€erent temperatures: (*) 80ꢀC, (&) 70ꢀC, (^)
60ꢀC, (x) 50ꢀC, (*) 40ꢀC. (B) Arrhenius plot for the cis-trans isomerization of oligomer 5.
350 nm was returned to the original obtained before
illumination. Based on these absorbance changes, the
apparent half-life for the cis azo chromophore at each
temperature can be estimated to yield the ®rst order
kinetic constant for the cis±trans isomerization. The
analysis of these data from the Arrhenius plot (Fig.
4(B)) provided the activation energy to be 20 kcal/mol.
Similar kinetic parameters were obtained for the cis-
trans isomerization in the duplexes of oligomers 7 and 8.
These results indicated that the cis form of the azo
chromophore incorporated into oligonucleotides or
duplexes is suciently stable at low temperature. We,
therefore, anticipate that photo-induced structural or
conformational changes that occurred in azobenzene-
oligonucleotide conjugates would be locked for a con-
siderable period under the dark conditions.
It has been found that the azobenzene linker eciently
undergoes trans±cis isomerization by exposing to UV
light. The conversion eciency showed slight dependence
on structure or conformation of oligonucleotides attached
to the azobenzene chromophore. The cis form of the azo-
benzene in oligonucleotides was stable at low temperature
in the dark. The present ®ndings would open the way for
light switch of nucleic acid structures.
Experimental
General methods. 31P NMR spectra were obtained on a
JEOL GX-400 spectrometer using 85% H3PO4 as an
external standard.1H NMR spectra were measured on a
JEOL EX-270 spectrometer, in which chemical shifts
(d ppm) were determined on the basis of a residual peak
of solvent (2.49 for DMSO-d6 or 7.26 for CDCl3).
High-performance liquid chromatography (HPLC)
was performed on a Waters 600E model apparatus
with a Hitachi LC 4200 UV±VIS detector at 345 nm
Conclusion
We have accomplished the synthesis of a novel photo-
isomerizable linker for linking oligonucleotide segments.

K. Yamana et al. / Bioorg. Med. Chem. 7 (1999) 2977±2983
2981
using a reversed phase Cosmosil 5C18-AR-300 column
(4.6Â150 mm). Column chromatography and thin-layer
chromatography (TLC) were carried out on Merck
silica gel 60 and Merck 60 PF254, respectively. Ultra-
violet (UV) spectra were recorded with a Hitachi U-
3000 spectrophotometer equipped with a thermoelec-
trically controlled cell holder (Hitachi SPR-10).
EtOAc:hexane, v/v). The appropriate fractions were
collected and then the solvent was removed to give a
orange solid material (0.7 g, 63%). TLC (3:7, EtOA-
c:hexane, v/v) Rf 0.64; 1H NMR (DMSO-d6): d=2.81 (t,
2H), 3.30 (t, 2H), 3.66 (m, 2H), 3.72 (s, 6H), 4.28 (t,
2H), 4.67 (t, 1H), 6.86±6.93 (m, 4H), 7.05±7.42 (m,
13H), 7.76±7.88 (m, 4H); FABMS (positive) m/z 589
[M+1].
Materials and solvent. 2-Cyanoethyl-N,N,N0,N0-tetra-
isopropyl phosphorodiamidite was obtained from
Aldrich. Protected deoxynucleoside 30-(2-cyanoethyl)-
N,N0-diisopropylphosphoramidites and nucleoside-loa-
ded controlled pore glass (CPG) supports were pur-
chased from Cruachem. Oligonucleotides were prepared
by a phosphoramidite chemistry on a Pharmacia LKB
Gene Assembler Plus DNA/RNA synthesizer. For
synthesis of the modi®ed oligonucleotides, the X-bottle
was used to supply the modi®ed amidite solution.
CH2Cl2, triethylamine (TEA), and diisopropylethyl-
amine (DIEA) were re¯uxed over CaH2 for 5 h, dis-
tilled, and stored over CaH2. DMF was distilled under
the reduced pressure over ninhydrin and stored over
molecular sieves. All other reagents and solvents were
used as received.
Synthesis of 4-hydroxyethyl-40-ethoxyazobenzene (3a). 1a
(0.47 g, 2 mmol) was allowed to react with ethyl iodide
(0.5 g, 3 mmol) in the presence of K2CO3 (0.5 g, 3.6
mmol) in DMF (30 mL) at 40ꢀC for 5 h. The reaction
mixture was evaporated to nearly dryness. The residue
was dissolved in EtOAc which was washed with water.
After the organic layer was dried with Na2SO4, the sol-
vent was removed in vacuo. The residual material was
applied on a silica gel column which was eluted by
EtOAc:hexane (4:1, v/v). The appropriate fractions were
collected and then the solvent was removed in vacuo to
give a orange crystalline (0.48 g, 86%). TLC (4:1,
EtOAc:hexane, v/v) Rf 0.61; 1H NMR (DMSO-d6):
d=1.36 (t, 3H), 2.80 (t, 2H), 3.65 (m, 2H), 4.13 (q, 2H),
4.68 (t, 1H), 7.10 (d, 2H), 7.40 (d, 2H), 7.75 (d, 2H),
7.85 (d, 2H); EIMS (positive) m/z 270.
Synthesis of 4-aminophenetyl alcohol. To a solution of
4-nitrophenetyl alcohol (2.0 g, 12 mmol) in methanol
(60 mL), hydrogen gas was passed through over Pd-C
(0.2 g) for 4 h. After monitoring the completion of the
reaction by TLC, the reaction mixture was ®ltered. The
®ltrate was evaporated to give a white powder (1.4 g,
87%). TLC (4:1, EtOAc:hexane, v/v) Rf 0.31; H NMR
(DMSO-d6): d=2.73 (t, 2H), 3.78 (t, 2H), 6.64 (d, 2H),
7.02 (d, 2H).
Synthesis of 4-(20-O-dimethoxytrityl ethyl-oxy)azoben-
zene-40-ethyl (2-cyanoethyl)-N,N0-diisopropylphosporami-
dite (4a). To the solution of 2a (0.28 g, 0.5 mmol) and
1H-tetrazole (0.039 g, 1.1 equiv) and diisopropylamine
(0.078 mL) in 2.5 mL of dry CH2Cl2 was added 2-cya-
noethyl-N,N,N0,N0-tetraisopropyl phosphorodiamidite
(0.35 mL, 2.0 equiv). The solution was continued to stir
at room temperature for 2 h. MeOH (2 mL) was added to
terminate the reaction and then the solution was diluted
with 3.0 mL of EtOAc containing a small amount of
DIEA. The solution was washed in 10% NaHCO3 and
the organic phase was dried by Na2SO4 and con-
centrated in vacuo. The residual solution was applied to
silica gel column chromatography (30:60:10, EtOA-
c:hexane:TEA, v/v). The appropriate fractions were
collected and evaporated in vacuo to give the phos-
phoramidite (0.38 g, 92%). TLC (30:60:10, EtOAc:
hexane:TEA, v/v) Rf 0.70; 31P NMR (CH3CN): 148.0
ppm; FABMS (positive) m/z 789 [M+1].
1
Synthesis of 4-hydroxyethyl-4-hydroxyazobenzene (1a).
To an ice-cooled aqueous solution (90 mL) of 4-amino-
phenetyl alcohol (1.4 g, 10 mmol) and NaNO2 (0.7 g, 10
mmol) in 0.3 N HCl, a solution (20 mL) of phenol (1.0
g, 10 mmol) in 10% NaOH was added with stirring.
After the dark-red precipitate was removed by ®ltration
the ®ltrate was neutralized with 2 N HCl. The orange
colored precipitate was ®ltered and washed well with
water. The solid was dissolved in a small amount of
EtOAc and then applied on a silica gel column. The
elution was carried out by using a mixed solvent of
EtOAC:hexane (4:1, v/v). The appropriate fractions
were collected and the solvent was removed in vacuo
a€ording a bright orange solid (0.9 g, 37%). TLC (4:1,
EtOAc:hexane, v/v) Rf 0.75; 1H NMR (DMSO-d6):
d=2.80 (t, 2H), 3.66 (m, 2H), 4.70 (br, 1H), 6.94 (d,
2H), 7.39 (d, 2H), 7.75 (dd, 4H), 10.25 (s, 1H); EIMS
(positive) m/z 242.
Synthesis of 4-amino-N-hydroxyethylbenzamide. 2-Ami-
noethanol (4.5 mL, 75 mmol) was allowed to react with
4-nitrobenzoyl chloride (9.3 g, 50 mmol) in CH2Cl2 (75
mL) containing TEA (15 mL) at 0ꢀC for 30 min. The
precipitate was ®ltered and then dried in vacuo over
P2O5. The solid material obtained was treated with H2
over Pd-C in a similar manner described for 4-amino-
phenetyl alcohol, a€ording a white solid (7.3 g, 82%).
TLC (9:1, CH2Cl2:MeOH, v/v) Rf 0.18; 1H NMR
(CDCl3): d=3.60 (t, 2H), 3.81 (t, 2H), 6.68 (d, 2H), 7.61
(d, 2H).
Synthesis of 4-hydroxyethyl-40-(20-O-dimethoxytrityl eth-
yl-oxy)azobenzene (2a). 1a (0.47 g, 2 mmol) was
allowed to react with O-dimethoxytrityl protected bro-
moethanol (1.3 g, 3 mmol) in the presence of K2CO3
(0.5 g, 3.6 mmol) in DMF (30 mL) at 40ꢀC for 5 h.
After the solvent was evaporated, the residue was sepa-
rated into EtOAc and water. The organic layer was
dried with Na2SO4 and then evaporated to nearly dry-
ness. The residue was applied on a silica gel column (3:7,
Synthesis of 4-hydroxy-40-(N-hydroxyethyl)azobenza-
mide (1b). The synthesis and puri®cation of 1b was car-
ried out by essentially the same procedures described for
1a to give a bright orange crystalline (46%). TLC (9:1,
CH2Cl2:MeOH, v/v) Rf 0.32; 1H NMR (CDCl3):

2982
K. Yamana et al. / Bioorg. Med. Chem. 7 (1999) 2977±2983
d=3.35 (t, 2H), 3.51 (t, 2H), 4.75 (t, 1H), 6.99 (d, 2H),
7.82 (m, 4H), 8.01 (d, 2H), 8.58 (t, 1H); FABMS (posi-
tive) m/z 286 [M+1].
spectra were measured with an increase in temperature
from 0 to 80ꢀC at a rate of 0.5ꢀC/min.
Analysis of photoisomerization of azobenzene. The solu-
tions of azobenzene containing oligonucleotides in a
phosphate bu€er and model compounds 3a and b in
CH3CN were irradiated in a cooled water bath (5ꢀC) by
a high-pressure Hg lamp using band-path ®lter to
transmit light at around 350 nm. The aliquot of the
solution was analyzed by UV±vis spectra at 5ꢀC after an
appropriate time interval.
Synthesis of 4-(20-O-dimethoxytrityl ethyl-oxy)-40-(N-hy-
droxyethyl)azobenzamide (2b). The synthesis and pur-
i®cation of 2b was carried out by essentially the same
procedures described for 2a to give an orange crystalline
(67%). TLC (9:1, CH2Cl2:MeOH, v/v) Rf 0.62; 1H
NMR (CDCl3): d=3.27±3.55 (m, total 6H), 3.72 (s,
6H), 4.30 (m, 2H), 4.74 (t, 1H), 6.86±6.93 (m, total 5H),
7.19±7.42 (m, total 12H), 7.89±8.04 (m, 4H), 8.58 (t,
1H); FABMS (positive) m/z 632 [M+1].
The calculations of the cis% in the oligonucleotides
after the irradiation were carried out by using the
absorbance changes at 350 nm based on the assumption
that the absorbance of the cis-isomer at the employed
wavelength is negligible. The model compound 3a was
Synthesis of 4-ethoxy-40-(N-hydroxyethyl)azobenzamide
(3b). The synthesis and puri®cation of 3b was carried
out by essentially the same procedures described for 3a
to give an orange crystalline (70%). TLC (9:1,
CH2Cl2:MeOH, v/v) Rf 0.45; 1H NMR (CDCl3):
d=1.37 (t, 3H), 3.28±3.54 (m, total 4H), 4.15 (m, 2H),
4.73 (t, 1H), 7.13 (d, 2H), 7.87±8.04 (m, total 6H), 8.57
(t, 1H); EIMS (positive) m/z 313.
1
analyzed by H NMR in CDCl3 before and after the
UV illumination. Before irradiation, the compound
exhibited four doublets (7.02, 7.36, 7.84 and 7.91 ppm)
at aromatic region, indicating only the trans of the azo
chromophore. Four new doublets (6.77, 6.93, 7.04 and
7.18 ppm) attributable to the cis form of the azobenzene
appeared after exposing of UV light. The integration of
these peaks provided the content of cis-form (60%). The
UV±vis spectral measurements were done for the same
solution, giving the cis content to be 50% based on the
absorbance changes at 350 nm. It is therefore noted that
the calculated cis% based on the absorbance changes
may be slightly lower than expected.
Synthesis of 4-(20-O-dimethoxytrityl ethyl-oxy)-40-{N-
ethyl (2-cyanoethyl)-N,N0-diisopropylphosporamidite}azo-
benzamide (4b). The synthesis and puri®cation of 4b
was carried out by essentially the same procedures
described for 4a to give the phosphoramidite (97%).
TLC (60:30:10, EtOAc:hexane:TEA, v/v) Rf 0.81; 31P
NMR (CH3CN): 148.1 ppm; FABMS (positive) m/z 832
[M+1].
The thermal back reaction (in dark) from cis to trans
was monitored by UV±vis spectra at di€erent tempera-
tures. After a prolonged time, the absorbance at 350 nm
was returned to the original obtained before illumina-
tion. Based on these absorbance changes, the apparent
half-life for the cis azo chromophore at each tempera-
ture can be estimated, yielding the ®rst order kinetic
constants. The analysis of these data from the Arrhenius
plot (Fig. 4(B)) provided the activation energy for the
cis±trans isomerization.
Synthesis of oligonucleotides containing an azobenzene
fragment. The synthesis of azobenzene modi®ed oligo-
nucleotides was accomplished by a phosphoramidite
chemistry beginning with 50-DMT-nucleoside (1.3 mmol)
bound to a CPG support. For the coupling of normal
deoxyribonucleoside phosphoramidites, the standard
protocol (120 mL of 0.1 M amidite and 120 mL of 0.1 M
tetrazole in acetonitrile, 1.5 min) was used. For the
coupling of azobenzene phosphoramidites, 120 mL of
the 0.12 M amidite and 120 mL of 0.1 M tetrazole in
acetonitorile (10 min) were used. With these conditions,
the amidites 4a and b can be coupled in eciencies of
40±65% with the hydroxyl functions of oligonucleotide
chains attached to CPG. The CPG bound oligonucleo-
tides were treated with concentrated ammonium hy-
droxide at 55ꢀC for 12 h. Puri®cation of the modi®ed
oligonucleotides was performed with 20% denaturing
polyacrylamide gel electrophoresis as described.12 The
integrity of oligonucleotide structure was veri®ed with
spray mass analysis.
Acknowledgements
We are very grateful to Professors Isao Saito, Hiroshi
Sugiyama, and Kazuhiko Nakatani for mass spectra.
We thank Dr. Takashi Sugimura for use of 31P NMR
spectrometer.
References
1. Hosaka, T.; Kawashima, K.; Sisido, M. J. Am. Chem. Soc.
1994, 116, 413.
2. Hamachi, I.; Hiraoka, T.; Yamada, Y.; Shinkai, S. Chem.
Lett. 1998, 6, 537.
3. Wasermann, N. H.; Erlanger, B. F. NATO Adv. Sci. Inst.
Ser., Ser. A 1983, 68, 269.
4. Kano, K.; Tanaka, Y.; Ogawa, T.; Shimomura, M.; Kuni-
take, T. Photochem. Photobiol. 1981, 34, 323.
5. Yamaguchi, H.; Nakanishi, H. Biochim. Biophys. Acta 1993,
1148, 179.
Preparation of oligonucleotide solution for UV melting
measurements. Oligonucleotide solutions were prepared
using a bu€er containing 0.1 or 1.0 M NaCl and 0.01 M
sodium phosphate, adjusted to pH 7.0. Oligonucleotide
concentrations were determined by absorbance at 260
nm and the calculated single-strand extinction coe-
cients based on a nearest neighbor model.12 Before
melting experiments, the solutions were heated to 80ꢀC,
kept there for 5 min, and then gradually cooled down to
room temperature. All duplex melting curves by UV
6. Tanaka, M.; Sato, T.; Tonezawa, Y. Langmuir 1995, 11,
2834.

K. Yamana et al. / Bioorg. Med. Chem. 7 (1999) 2977±2983
2983
7. Archut, A.; Azzellini, G. C.; Balzani, V.; Cola, L. D.;
Volgtle, F. J. Am. Chem. Soc. 1998, 120, 12187.
8. Tsuchiya, S. J. Am. Chem. Soc. 1999, 121, 48.
9. Ulysee, L.; Cubillos, J.; Chmielewski, J. J. Am. Chem. Soc.
1995, 117, 8466.
10. Asanuma, H.; Ito, T.; Komiyama, M. Tetrahedron Lett.
1998, 39, 9015.
11. Yamana, K.; Yoshikawa, A.; Nakano, H. Tetrahedron
Lett. 1996, 37, 637.
16. Letsinger, R. L.; Wu, T. J. Am. Chem. Soc. 1995, 117,
7323.
17. Rumney, S.; Kool, E. T. J. Am. Chem. Soc. 1995, 117,
5635.
18. Komatsu, Y.; Koizumi, M.; Nakamura, H.; Ohtsuka, E. J.
Am. Chem. Soc. 1994, 116, 3692.
19. Matsumoto, M.; Miyazaki, D.; Tanaka, M.; Azumi, R.;
Manda, E.; Kondo, Y.; Yoshino, N.; Tachibana, H. J. Am.
Chem. Soc. 1998, 120, 1497.
12. Yamana, K.; Yoshikawa, A.; Noda, R.; Nakano, H.
Nucleosides and Nucleotides 1998, 17, 233.
13. Richardson, P. L.; Schepartz, A. J. Am. Chem. Soc. 1991,
113, 5109.
20. Fujimaki, M.; Matsuzawa, Y.; Hayashi, Y.; Ichimura, K.
Chem. Lett. 1998, 165
21. Adamson, A. A.; Vogler, A.; Kunkely, H.; Wachter, R. J.
Am. Chem. Soc. 1978, 100, 1298.
14. Cload, S. T.; Schepartz, A. J. Am. Chem. Soc. 1991, 113,
6324.
15. Giovannangeli, C.; Thoung, N. T.; Helene, C. Proc. Natl.
Acad. Sci. USA 1993, 90, 10031.
22. Lewis, F. D.; Wu, T.; Zhang, Y.; Letsinger, R. E.; Green-
®eld, M. R.; Wasielewski, M. R. Science 1997, 277, 673.
23. Gohlke, C.; Murchie, A. I. H.; Lilley, D. M. J.; Clegg, R.
M. Proc. Natl. Acad. Sci. USA 1994, 91, 11660.