Phenylazonaphthalene as a superb photo-regulator for DNA-triplex formation

Article abstract of DOI:10.1016/S0040-4039(01)01347-8

Phenylazonaphthalene was tethered to the 5′-end of oligo(T) as photo-responsive moiety for the photo-regulation of DNA-triplex formation. The melting temperature of DNA-triplex changed as greatly as 40.5°C on trans?cis isomerization of phenylazonaphthalene. This change was much greater than that achieved by azobenzene.

Full text of DOI:10.1016/S0040-4039(01)01347-8

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 6723–6725
Phenylazonaphthalene as a superb photo-regulator for
DNA-triplex formation
Xingguo Liang, Hiroyuki Asanuma* and Makoto Komiyama*
Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku,
Tokyo 153-8904, Japan
Received 22 June 2001; revised 16 July 2001; accepted 19 July 2001
Abstract—Phenylazonaphthalene was tethered to the 5%-end of oligo(T) as photo-responsive moiety for the photo-regulation of
DNA-triplex formation. The melting temperature of DNA-triplex changed as greatly as 40.5°C on translcis isomerization of
phenylazonaphthalene. This change was much greater than that achieved by azobenzene. © 2001 Elsevier Science Ltd. All rights
reserved.
DNA-triplex formation has been regarded as one of the
most powerful techniques for the control of gene
expression.1 If this process is regulated by outer stimuli,
it undoubtedly widens the scope of application. Previ-
ously,2,3 we reported that triplex formation is photo-
regulated by cis–trans isomerization of the azobenzene
incorporated into the third strand. Planar trans-azoben-
zene stabilizes the triplex by stacking interaction,
whereas non-planar cis-azobenzene destabilizes it by
steric hindrance. It is indicated that more efficient
photo-regulation is plausible by using a photo-respon-
sive molecule which is larger than azobenzene. In this
paper, we show that a phenylazonaphthalene regulates
DNA-triplex formation far more efficiently than does
an azobenzene. The change of melting temperature (Tm)
on the cis–trans isomerization exceeds 40°C.
The phosphoramidite monomer 1 carrying a phenyl-
azonaphthalene moiety was synthesized according to
Scheme 1, and incorporated into oligonucleotides on an
automated synthesizer.4,5 The target for triple-helix for-
mation with XT13 is the T13/(dA)13 portion in the [a/t]
duplex, formed from two complementary 31-mer
oligonucleotides (see Scheme 2).
Before photo-irradiation, the phenylazonaphthalene in
XT13 mostly (>90%) takes the trans-form. The melting
curve (at 280 nm) of trans-XT13/a/t mixture is presented
N
a
c
N
b
NH2
N
N2+ Cl-
H2N
DMTO
NC
d
+
N
N2+Cl-
O2N
O2N
N
O
N
DMTO
O
O
N
HO
g
e
N
f
N
H
N
N
N
N
H
H
O
OH
P
OH
O
N
1
quant.
75%
40%
Scheme 1. Synthesis of the phosphoramidite monomer 1 involving phenylazonaphthalene residue. (a) HCl(aq); (b) H2SO4,
CH3COOH, NaNO2; (c) H3PO2(aq); (d) NaSH(aq), ethanol/H2O; (e) 2,2-bis(hydroxymethyl)propionic acid, dicyclohexylcarbodi-
imide, 1-hydroxybenzotriazole, DMF; (f) 4,4%-dimethoxytrityl (DMT) chloride, 4-dimethylaminopyridine, pyridine, CH2Cl2; (g)
2-cyanoethyl N,N,N%,N%-tetraisopropylphosphorodiamidite, 1H-tetrazole, CH3CN.
* Corresponding authors. Tel.: +81-3-5452-5200; fax: +81-3-5452-5209; e-mail: asanuma@mkomi.rcast.u-tokyo.ac.jp; komiyama@mkomi.rcast.
u-tokyo.ac.jp
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01347-8

6724
X. Liang et al. / Tetrahedron Letters 42 (2001) 6723–6725
HO
O
HO
HO
O
O
N
N
N
N
H
N
N
N
H
N
H
N
O
O
O
O
P OH
O
O
O
P OH
O
P OH
O
Ym
Yp
X
YmT13 :
5′-YmT-TTT-TTT-TTT-TTT-3′
YpT
YpT13 :
XT13 :
T13 :
5′-
-TTT-TTT-TTT-TTT-3′
5′-XT-TTT-TTT-TTT-TTT-3′
5′-T-TTT-TTT-TTT-TTT-3′
a:
5′-cgtcggttt-A-AAA-AAA-AAA-AAA-tttcgtggc-3′
t
: 3′-gcagccaaa-T-TTT-TTT-TTT-TTT-aaagcaccg-5′
Scheme 2. Sequences of the oligonucleotides used in this study.
by the solid line in Fig. 1.6 The Tm for the triplex
formation is 45.5°C. Upon irradiating the solution with
UV light (375–400 nm),7 the phenylazonaphthalene is
rapidly isomerized to the cis-form (>70%), as confirmed
by UV–visible spectroscopy and HPLC analysis (Supple-
mentary Material available). Concurrently, the melting
curve drastically shifts towards the lower temperature
side, with respect to that of the trans-form (compare the
broken line with the solid one). The Tm value is 5.0°C.
Thus, the change of Tm (DTm), induced by the cis–trans
isomerization of the phenylazonaphthalene, is as large as
40.5°C. When the cis-isomer is further isomerized back
to the trans-form by visible light irradiation (>420 nm),7
the melting curve is almost superimposed with that
before the UV irradiation. The present photo-regulation
is reversible.
Table 1. Tm values of triplexes formed from the modified
oligonucleotides and the [a/t] duplexa,b
Modified
oligonucleotide
Tm (°C)
cis-Form
DTm (°C)
trans-Form
XT13
45.5
29.5
30.2
5.0
13.5
10.7
40.5
16.0
19.5
YpT13
YmT13
a pH 7.0 (10 mM HEPES buffer), 0.2 M MgCl2, [a]=2.0 mM, [t]=2.4
mM, and [the third strand]=2.0 mM.
b Tm of the native triplex T13/a/t is 22.5°C.
greater conjugate system, trans-phenylazonaphthalene
shows stronger stacking interaction with adjacent DNA
base-pairs than does trans-azobenzene. Thus, the Tm of
trans-XT13/a/t is much higher (by 15–16°C) than those
of trans-YpT13/a/t and trans-YmT13/a/t. This triplex is
more stable than native triplex T13/a/t (Tm=22.5°C) and
is efficiently formed under physiological conditions.
These arguments are supported by CD and UV–visible
spectroscopy,8 as well as by computer molecular model-
ing. Furthermore, cis-phenylazonaphthalene induces
more significant steric hindrance against the target
duplex, so that cis-YpT13/a/t triplex is even less stable (by
5–7°C) than either cis-YpT13/a/t or cis-YmT13/a/t. These
two factors render the phenylazonaphthalene highly
potent for photo-regulation of DNA-triplex formation.
For the purpose of comparison, the results for the
azobenzene-tethered oligonucleotides YpT13 and YmT13
(see Scheme 2 for the structures) are listed in Table 1. The
DTm values (16.0°C for YpT13 and 19.5°C for YmT13) are
notably smaller than the corresponding value for XT13.
Superiority of phenylazonaphthalene as a photo-regula-
tor of triplex formation is conclusive. Because of the
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) and the ‘Research
for the Future’ Program of the Japan Society for the
Promotion of Science (JSPS-RFTF97I00301). Support
by Iwatani Naoji Foundation (for X.G.L.) is also
acknowledged.
Figure 1. Melting curves for trans-XT13/a/t triplex (solid line)
and cis-XT13/a/t triplex (dotted line): pH 7.0 (HEPES), 0.2 M
MgCl2, [a]=2.0 mM, [t]=2.4 mM, [XT13]=2.0 mM. The arrows
in the Figure indicate the Tms.

X. Liang et al. / Tetrahedron Letters 42 (2001) 6723–6725
6725
6. The absorbance at 280 nm was monitored on a JASCO
model V-530 spectrophotometer, equipped with a pro-
grammed temperature controller. The rate of temperature
change was 1.0°C min−1. The Tm value was determined as
the maximum of the first derivative of melting curve. The
t:a ratio was kept at 1.2 in order to ensure the absence of
free a, which would form a duplex with the third strand
DNA and perturb the measurement.
7. The photo-isomerization was accomplished by irradiating
light from a 150 W xenon lamp for 10 min through an
appropriate filter: a P10-390-R band pass filter from Koyo
Corporation for UV irradiation (375–400 nm), and a Y-43
color glass filter from Asahi Technoglass Corporation for
visible-light irradiation (>420 nm). The photochemical
properties of phenylazonaphthalene are similar to those of
azobenzene.
8. When the trans-XT13/a/t triplex is formed, circular
dichromism (CD) is negatively induced at around 390 and
480 nm. This indicates that the trans-phenylazonaph-
thalene intercalates between the base pairs and stabilizes
the triple helix by hydrophobic stacking, as described in
Ref. 2. This argument is further certified by the
bathochromic shift (16 nm) of the phenylazonaphthalene.
References
1. (a) Wang, G.; Seidman, M. M.; Glazer, P. M. Science
1996, 271, 802; (b) Barre, F. X.; Giovannangeli, C.;
He´le`ne, C.; Harel-Bellan, A. Nucleic Acids Res. 1999, 27,
743; (c) Cogoi, S.; Papozzi, V.; Quadrifoglio, F.; Xodo, L.
Biochemistry 2001, 40, 1135.
2. Asanuma, H.; Liang, X. G.; Yamazawa, A.; Yoshida, T.;
Komiyama, M. Angew. Chem., Int. Ed. Engl. 2000, 39,
1316.
3. Photo-regulation of DNA duplex formation by using
azobenzene has been also reported: Asanuma, H.; Ito, T.;
Yoshida, T.; Liang X. G.; Komiyama, M. Angew. Chem.,
Int. Ed. Engl. 1999, 38, 2393.
4. MALDI-TOFMS of XT13: calcd [M−H+]: 4314.7; obsd.:
4315.0. All the products and reaction intermediates in
Scheme 1 were sufficiently characterized by usual methods
(see Supplementary Material).
5. YpT13 and YmT13 were synthesized as described previously:
Asanuma, H.; Ito, T.; Komiyama, M. Tetrahedron Lett.
1998, 39, 9015; Asanuma, H.; Liang X. G.; Komiyama, M.
Tetrahedron Lett. 2000, 41, 1055.