meta-Aminoazobenzene as a thermo-insensitive photo-regulator of DNA- duplex formation

Article abstract of DOI:10.1016/S0040-4039(99)02233-9

meta-Aminoazobenzene has been introduced in the side chain of oligonucleotides as a photo-responsive molecule. Compared with the para- aminoazobenzene which was previously used, the thermal cis→trans isomerization was much slower: the half-lives of the ci

Full text of DOI:10.1016/S0040-4039(99)02233-9

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 1055–1058
meta-Aminoazobenzene as a thermo-insensitive photo-regulator
of DNA-duplex formation
Hiroyuki Asanuma, Xingguo Liang and Makoto Komiyama ∗
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Bunkyo-ku,
Tokyo, 113-8656 Japan
Received 1 November 1999; accepted 25 November 1999
Abstract
meta-Aminoazobenzene has been introduced in the side chain of oligonucleotides as a photo-responsive mole-
cule. Compared with the para-aminoazobenzene which was previously used, the thermal cis→trans isomerization
was much slower: the half-lives of the cis-isomers of m- and p-aminoazobenzene were 13.2 h and 20 min at 50°C,
respectively. By using the present oligonucleotides, duplex formation and dissociation was efficiently regulated on
photo-irradiation without being disturbed by the thermal isomerization. © 2000 Elsevier Science Ltd. All rights
reserved.
Keywords: azo; azo compounds; isomerization; phosphoramidites; photochemistry.
Up to now, a variety of photo-responsive molecules have been synthesized for the artificial regulation
of chemical and biological systems.1 Among many photo-responsive molecules, azobenzene has been
widely used due to (i) its large structural change induced by photo-irradiation and (ii) its easiness of
chemical modification.1 In almost all the cases, the azobenzene residues were introduced through para-
substituted functional groups (such as -NH2, -COOH, and -OH). This is mainly because these para-
substituted azobenzenes are commercially available or easy to synthesize. We have also introduced p-
aminoazobenzene in the side chain of oligonucleotides through the para-amino groups and successfully
regulated the DNA duplex formation and dissociation by photo-irradiation.2 However, one that is incurred
with the use of azobenzene as a photo-regulator is the thermally induced cis→trans isomerization. The
isomerization is rapid especially when functional groups are covalently attached to the para-position of
azobenzene. In order to achieve strict photo-regulation with azobenzene, the thermal isomerization must
be minimized.
According to the literature,3 rapid thermal isomerization of para-substituted cis-azobenzene is attri-
buted to the electronic effect of the substituent that weakens -N_N- bond. Thus, thermal stability of cis-
azobenzene should be much improved when substituents are attached to the meta-position of azobenzene.
However, little has been reported on a modified azobenzene with a meta-substituent, especially on the
∗
Corresponding author.
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(99)02233-9
tetl 16161

1056
thermal durability of its cis-form. In the present communication, m-aminoazobenzene is introduced in the
side chain of the oligonucleotides. This modification provides the incorporated cis-azobenzene residue
with pronounced thermal stability. By using this oligonucleotide, the formation and dissociation of the
duplex with its complementary counterpart can be sufficiently photo-regulated by the cis–trans isomeri-
zation of azobenzene. The meta-substitution effect on the thermo-insensitivity is clearly demonstrated.
The azobenzene residue was incorporated through the centered carboxyl group of trimethylene
chain to minimize the perturbation of the backbone structure from the natural deoxyribose.2 The
phosphoramidite monomer 2 carrying m-aminoazobenzene was synthesized according to Scheme 1.
First, m-nitroaniline was coupled with nitrosobenzene in acetic acid. The obtained m-nitroazobenzene
was converted to m-aminoazobenzene through the reduction with sodium hydrosulfide in ethanol/water.
Then, 2,2-bis(hydroxymethyl)propionic acid was coupled with the azobenzene in the presence of dicyclo-
hexylcarbodiimide and 1-hydroxybenzotriazole. The obtained diol was converted to the phosphoramidite
monomer according to the previous method.2,4 All the intermediates and the product were purified by
either recrystallization or silica gel column chromatography, and characterized by NMR spectroscopy.5
The modified oligonucleotide, prepared from 2 and the conventional monomer, was 50-AAAXmAAAA-
30 (abbreviated as A3XmA4; Xm denotes the residue coming from the monomer 2) which was purified by
the reversed-phase HPLC, and characterized by MALDI-TOFMS. Two diastereomers derived from the
chirality of the phosphoramidite monomer 2, and the trans–cis isomers with respect to the incorporated
azobenzene residue, were completely resolved by the HPLC.6
Scheme 1. Synthesis of phosphoramidite monomer 2. (a) nitrosobenzene, acetic acid; (b) NaSH, ethanol/H2O;
(c) 2,2-bis(hydroxymethyl)propionic acid, dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, pyridine, DMF;
(d) 4,40-dimethoxytrityl (DMT) chloride, 4-dimethylaminopyridine, pyridine, CH2Cl2; (e) 2-cyanoethyl
N,N,N0,N0-tetraisopropylphosphorodiamidite, 1H-tetrazole, CH3CN
The UV–vis spectrum of trans-A3XmA4 (fraction (c); retention time 17.1 min) is shown as the dotted
line in Fig. 1(a).7 On UV-light irradiation (300 nm<λ<400 nm),8 it was promptly isomerized to cis-
A3XmA4 (the broken line in Fig. 1(a)).9 This process is reversible so that cis-A3XmA4 was again iso-
merized to the trans-form on visible-light irradiation (400 nm<λ). The thermal cis→trans isomerization
hardly took place. The UV–vis spectrum of cis-A3XmA4 after being kept at 50°C for 1 h virtually super-
imposed on that before the treatment (the solid line in Fig. 1(a)). In case of p-aminoazobenzene-tethered
oligonucleotides (50-AAAXpAAAA-30; A3XpA4),10 photo-isomerization (trans→cis and cis→trans) oc-
curred also smoothly (Fig. 1(b)). However, thermal cis→trans isomerization promptly occurred when the
cis-isomer was situated at 50°C for 1 h. The UV–vis spectrum after the warming was almost the same
as that of the trans-A3XpA4 (the solid line in Fig. 1(b)). These results clearly demonstrate the thermal
durability of the meta-substituted cis-azobenzene: half-life of cis→trans isomerization was 13.2 h (790

1057
min) for the meta-substituted aminoazobenzene, while it was only 20 min for the para-substituted one.
The rate constants and half-lives of cis→trans isomerization at various temperatures are listed in Table
1.11 Under physiological conditions (37°C, pH 7.0), the half-life of the cis-A3XmA4 was as long as 64 h.
Thus, thermo-stability was greatly improved by the meta-substitution.12
Fig. 1. UV–vis spectra of A3XmA4 (a) and A3XpA4; (b) in the trans-form (dotted line) and the cis-form (broken line). The solid
lines show the spectra of cis-A3XmA4 and cis-A3XpA4 after being kept at 50°C for 1 h11
Table 1
The rate constants and half-lives of thermal cis→trans isomerization11
The melting temperature of the duplex between the present modified oligonucleotide and its count-
erpart (T8: 50-TTTTTTTT-30) was reversibly altered by the cis–trans isomerization. The Tms of trans-
A3XmA4/T8 and cis-A3XmA4/T8 were 24.3°C and 16.7°C, respectively.13 These values were compar-
able to those of the p-aminoazobenzene-tethered oligonucleotides: Tms of trans-A3XpA4/T8 and cis-
A3XpA4/T8 were 24.8°C and 15.9°C, respectively. Thus, formation and dissociation of the DNA duplex
could be sufficiently regulated even when the amino group was attached to the meta-position.
In conclusion, a thermo-insensitive and photo-responsive oligonucleotide was successfully synthesiz-
ed from m-aminoazobenzene. By using this oligonucleotide, regulation of the duplex formation and
dissociation was achieved only by photo-irradiation.

1058
Acknowledgements
This work was partially supported by a Grant-in-Aid for Scientific Research from the Ministry
of Education, Science, and Culture, Japan (Molecular Synchronization for Design of New Materials
System). Support by the Grant from ‘Research for the Future’ Program of the Japan Society for the
Promotion of Science (JSPS-RFTF97I00301) is also acknowledged.
References
1. (a) Shinkai, S.; Minami, T.; Kusano, Y.; Manabe, O. J. Am. Chem. Soc. 1983, 105, 1851. (b) Würthner, F.; Rebek Jr., J. J.
Chem. Soc. Perkin Trans. 2 1995, 1727. (c) Shimomura, M.; Kunitake, T. J. Am. Chem. Soc. 1987, 109, 5175. (d) Willner,
I.; Rubin, S.; Zor, T. J. Am. Chem. Soc. 1991, 113, 4013. (e) Hohsaka, T.; Kawashima, K.; Sisido, M. J. Am. Chem. Soc.
1994, 116, 413. (f) Hamachi, I.; Hiraoka, T.; Yamada, Y.; Shinkai, S. Chem. Lett. 1998, 537. (g) Yamana, K.; Yoshikawa,
A.; Nakano, H. Tetrahedron Lett. 1996, 37, 637. (h) Yamana, K.; Yoshikawa, A.; Noda, R.; Nakano, H. Nucleosides and
Nucleotides 1998, 17, 233. (i) Lee, S.-Y.; Lee, H.; Cheong, C.-M.; Kim, J.-M.; Ahn, K.-D. Polym. Bull. 1998, 40, 1.
2. (a) Asanuma, H.; Ito, T.; Komiyama, M. Tetrahedron Lett. 1998, 39, 9015. (b) Asanuma, H.; Ito, T.; Yoshida, T.; Liang, X.;
Komiyama, M. Angew. Chem., Int. Ed. Engl. 1999, 38, 2393.
3. Hartley, G. S. J. Chem. Soc. 1938, 633.
4. Endo, M.; Azuma, Y.; Saga, Y.; Kuzuya, A.; Kawai, G.; Komiyama, M. J. Org. Chem. 1997, 62, 846.
5. Compound 1: 1H NMR [CDCl3 (TMS), 270 MHz] δ 9.43(s, 1H, -NHCO-), 7.94–6.83 (m, 22H, aromatic protons of DMT
and azobenzene), 3.76 (s, 6H, -OCH3), 3.68 and 3.64 (d, 2H, Jgem=11.2 Hz -CH2OH), 3.42 and 3.40 (d, 2H, Jgem=6.6 Hz,
DMT–OCH2-), 1.34 (s, 3H, -CH3).
6. The HPLC conditions: a Merck LiChrospher 100 RP-18(e) column, 260 nm, 0.5 cm3 min−1, a linear gradient 5–25% (25
min) acetonitrile/water containing 50 mM ammonium formate. Under these conditions, four isomers were eluted at (a) 15.9
min, (b) 16.2 min, (c) 17.1 min, (d) 17.7min, respectively. The fractions (a) and (c) are cis- and trans-isomers of A3XmA4
with the same configuration, and (b) and (d) are cis- and trans-isomers of another configuration, respectively. MALDI
TOFMS analysis: (c) fraction: obsd (negative mode) 2508, calcd 2504, (d) fraction; obsd 2508, calcd 2504.
7. Before UV irradiation, 75% of A3XmA4 existed as the trans-form.
8. The light from a 150 W Xenon lamp was irradiated for 20 min through an appropriate filter. Infrared light was cut off by
using a water filter.
9. 85% of cis-form was generated by this treatment as determined by HPLC.
10. This oligonucleotide was synthesized according to Ref. 2. Four isomers of A3XpA4 were completely resolved by the HPLC,
and the relationship between these isomers were identical with that for A3XmA4. In this paper, the fraction (c) of A3XpA4
was used.
11. The specimen was as follows: [A3XmA4]([A3XpA4])=50 µmol dm−3, [NaCl]=1 mol dm−3, pH 7.0 (10 mmol dm−3
phosphate buffer). The rate constants of the thermal cis→trans isomerization were determined by the UV–vis spectroscopy
from the change of absorbance at 325 nm (for A3XmA4) or 350 nm (for A3XpA4).
12. The present results indicate that thermal stability will be also improved by meta-substitution with other functional groups
such as -OH.
13. The absorbance at 260 nm was monitored on a JASCO model V-530 spectrophotometer, equipped with a programmed
temperature-controller. The rate of temperature change was 1.0°C/min. The concentrations of the modified oligonucleotides
and 50-TTTTTTTT-30 were 50 µmol dm−3, and the ionic strength was kept constant at 1 mol dm−3 by using NaCl at pH 7.0
(10 mmol dm−3 phosphate buffer). The Tm values were determined from the maximum in the first derivative of the melting
curve.