Photomodulation of PS-modified oligonucleotides containing azobenzene substituent at pre-selected positions in phosphate backbone

Article abstract of DOI:10.1016/j.bmc.2007.08.042

A new protocol has been developed for incorporation of a photoisomerizable azobenzene moiety into synthetic stereo-enriched [R_p] and [S_p] PS-oligonucleotides. The azobenzene pendant is attached at pre-selected positions in internucle

Full text of DOI:10.1016/j.bmc.2007.08.042

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 15 (2007) 7840–7849
Photomodulation of PS-modified oligonucleotides
containing azobenzene substituent at pre-selected
positions in phosphate backbone
Satyakam Patnaik,a P. Kumar,a B. S. Garg,b R. P. Gandhic and K. C. Guptaa,*
aNucleic Acids Research Laboratory, Institute of Genomics and Integrative Biology, Mall Road,
Delhi University Campus, Delhi 110 007, India
bDepartment of Chemistry, University of Delhi, Delhi 110 007, India
cDr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110 007, India
Received 27 April 2007; revised 21 August 2007; accepted 23 August 2007
Available online 28 August 2007
Abstract—A new protocol has been developed for incorporation of a photoisomerizable azobenzene moiety into synthetic stereo-
enriched [Rp] and [Sp] PS-oligonucleotides. The azobenzene pendant is attached at pre-selected positions in internucleotidic phosp-
horothioate oligonucleotides of both [Rp] and [Sp] diastereomers using a novel reagent, N-iodoacetyl-p-aminoazobenzene, 1. The
modified oligomers are purified on HPLC, characterized by LC–MS, and examined for their thermal and photoisomerization prop-
erties. The azobenzene moiety imparts greater stability to oligomer duplexes in (E) N@N configuration as compared to (Z) config-
uration. The placement of the azobenzene pendant close to 50-terminus (n ꢀ 1) and 30-terminus of the modified PS-oligos contributes
maximum stability to the duplex while a gradual decline in stability occurs with azobenzene moving toward middle of the duplex.
Circular Dichroism studies reveal that the chiral environment at the phosphorus center of the PS-oligos does not alter the global
conformation of the DNA duplex as such, suggesting conservation of conformation of the modified DNA strands.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
attracted considerable attention because of ease of its
preparation and efficient photoisomerization property
(E–Z transformation).2–5 Yamana et al. have success-
fully incorporated azobenzene moiety as a bridge be-
tween two oligo strands via the 50-end of one oligo
and the 30-end of the other.6 They further report incor-
poration of phosphoramidite reagents of various deriva-
tives of azobenzene having different substituents at
p-, p0-positions of the two phenyl rings for linking two
oligo strands7 and have examined photomodulation of
some duplex structures. In a series of papers, Komiyama
et al.8–18 have reported on photoregulation of enzyme
activity of T7 RNA polymerase by tethering an azoben-
zene to the promoter and regulating the activity of T7
DNA polymerase by photoresponsive oligonucleotide
as modulator,10,11 and have further studied triplex for-
mation and dissociation by tethering azobenzene to
the third strand of the triplex.12–14 Their approach
entails the use of phosphoramidite reagent based on
azobenzene-linked 2,2-bis (hydroxymethyl)-propionic
acid for subsequent incorporation into oligonucleotides
at various positions during synthesis. In the above
studies, the E-isomer has been demonstrated to be more
Recently, much attention has been focused on artificial
control of bio-chemical reactions.1 The most cited exam-
ple of this phenomenon is the regulation of gene expres-
sion, which can be achieved by addition of a short
oligonucleotide (12–15 mer) either at transcriptional
(antigene) or translational (antisense) stage. An alterna-
tive methodology is based on the use of isomerizable
structural moieties where regulation is controlled by
external means, i.e., light. Literature records sporadic
studies1–3 relating to control of enzyme activity, receptor
binding properties, membrane permeability, electron
transfer, peptide conformation, etc., where macroscopic
properties of biomolecules have been altered using UV–
vis radiation. Although, a number of photoisomerizable
groups have been developed, azobenzene molecule has
Keywords: PS-oligomers; Azobenzene; E–Z photoisomerization;
Hybridization; Circular Dichroism.
*
Corresponding author. Tel.: +91 11 27662491; fax: +91 11
27667471; e-mail: kcgupta@igib.res.in
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.08.042

S. Patnaik et al. / Bioorg. Med. Chem. 15 (2007) 7840–7849
7841
stable than the corresponding Z-isomer in both [Rp] and
[Sp] diastereomers. In another approach, Komiyama
et al.9 have tethered an azobenzene pendant at 20-posi-
tion of uridine followed by its incorporation into oligo-
nucleotides using phosphoramidite approach. However,
a differential stability behavior has been reported for the
synthetic photoresponsive oligonucleotide containing m-
aminoazobenzene substituent.18
aqueous NH4OH (30%, 55 ꢁC, 16 h). The purification
and characterization of each of the diasteromers of oli-
gonucleotides were done as described in Section 4. The
elution pattern shows that [Rp] isomer has less retention
time as compared to its [Sp] counterpart which elutes la-
ter having a higher retention time19,20 (Fig. 1a and b).
2.2. Covalent attachment of azobenzene pendant to PS-
oligonucleotides
In this communication, we report a new convenient pro-
tocol for tethering an azobenzene moiety to synthetic
PS-oligos at pre-selected positions in the backbone. This
method is based on the use of a novel reagent, N-iodo-
acetyl-p-aminoazobenzene, 1, which is attached to puri-
fied diastereomers [Rp] and [Sp] of phosphorothioate
oligonucleotides. The paper also records a detailed study
of stability characteristics of duplexes of modified
PS-oligonucleotides and unmodified complementary
oligonucleotides as a function of the position of the
azobenzene (which moves from 30-end toward the
50-end of the duplex), both under experimental condi-
tions (D) as well as under photoirradiations (hm).
The covalent attachment of the azobenzene pendant to
the PS-oligonucleotides was achieved by treating the
PS-oligonucleotides with an excess of 1 (Scheme 3).
The purification and characterization were done vide
experimental to yield the oligonucleotide conjugates in
almost quantitative yield. Figure 1a and b shows HPLC
profiles of [Rp] and [Sp] diastereomers of PS-4 phosp-
horothioate oligonucleotide while, Figure 1c and d de-
picts a co-injection of [Rp] and [Sp] diastereomers of
PS-4 oligomer and azobenzene linked [Rp] diastereomer
of PS-4 oligonucleotide, respectively.
Similarly, other conjugates were prepared, purified, and
analyzed.
2. Results and discussion
2.3. Photomodulation of single stranded oligonucleotides
containing azobenzene moiety
In designing a facile methodology for incorporation of
azobenzene moiety into the oligonucleotide chains, we
preferred incorporation of PS-linkage in the oligomer
sequence at pre-selected positions based on the knowl-
edge that iodo-alkyl reagents are specific toward PS-
linkages. This synthetic protocol eliminates the steps
associated with the phosphoramidite monomer (used
by the Komiyama’s group) and reduces the formation
of intermediates in the synthetic sequence. The synthesis
of the reagent, N-iodoacetyl-p-aminoazobenzene, 1, was
accomplished through coupling of p-aminoazobenzene
with iodoacetic acid in the presence of dicyclohexylcar-
bodiimide in 80% yield (Scheme 1). This was character-
The azobenzene-tethered modified oligonucleotides ex-
hibit two distinct absorption bands at ꢁ343 and
ꢁ434 nm along with the characteristic band at around
260 nm, which has not been shown for the sake of clar-
ity. The former band is attributed to p–p* transition in
azobenzene and, under normal conditions, all phosp-
horothioate oligomers with azobenzene residue predom-
inantly comprise of the thermodynamically stable
E-isomer. Figure 2 (top) shows UV and (bottom) HPLC
profiles of purified [Sp] diastereomer of PS-1. It was
observed that, on irradiating (365 nm) PS-1 [Sp] diaste-
reomer over a period of time (curve a, top), the intensity
of the band at ꢁ343 nm (E-isomer, 91.6%) decreased
with concurrent increase in intensity of band at
ꢁ434 nm, Z-isomer (curve b, top). The photostationary
state was obtained within 30 min of irradiation (365 nm)
(17.7% of Z-isomer), which reflects no further increment
in the band intensity at ꢁ434 nm. When the above sam-
ple was further exposed to visible light (k > 400 nm), the
peak corresponding to Z-isomer of the PS-1 [Sp]
diastereomer displayed a decrease in intensity (with
corresponding increase in the peak at 343 nm due to
the E-isomer, i.e., 89%) [curve c]. HPLC profiles
(bottom) also corroborate the above photoreversibility
effect. Profile (a), Figure 2 (bottom) shows the elution
pattern of PS-1 ([Sp] diastereomer) prior to irradiation
1
ized through IR, H NMR, ES-MS mass spectrometry,
and elemental analysis.
2.1. Modified oligonucleotides: synthesis and purification
PS-oligonucleotides (Table 1) were synthesized on nor-
mal and modified supports (A, Scheme 2) on a Pharma-
cia Gene Assembler Plus using the phosphoramidite
approach. The phosphorothioate linkages (PS) were
introduced in the machine itself at desired sites using
tetraethylthiuram disulfide (TETD) in place of a con-
ventional oxidation reagent. The cleavage of modified
PS-oligonucleotides from the solid support and removal
of protecting groups from the backbone and nucleo-
bases were achieved in a single step by treatment with
N
N
O
DCC, THF
0 ˚C
N
N
+
I
NH
OH
H2N
I
O
1
Scheme 1. Preparation of azobenzene pendant 1.

7842
S. Patnaik et al. / Bioorg. Med. Chem. 15 (2007) 7840–7849
Table 1. Oligonucleotide sequencesa
Oligomers
Diastereomeric mixed sequences
Observed mass (M + Na+)
Internucleotide linkage
PO-1
PO-2
PS-1
d(TTTTTTTT)
d(AAAAAAAA)
2394
2474
2410
2410
2410
2410
2410
2410
2410
2473
2490
2490
2490
2490
2490
2490
2490
2553
Phosphate
Phosphate
d(TTTTTTTpsT) [Rp/Sp]
d(TTTTTTpsTT) [Rp/Sp]
d(TTTTTpsTTT) [Rp/Sp]
d(TTTTpsTTTT) [Rp/Sp]
d(TTTpsTTTTT) [Rp/Sp]
d(TTpsTTTTTT) [Rp/Sp]
d(TpsTTTTTTT) [Rp/Sp]
d(TTTTTTTTps)
Phosphorothioate
Phosphorothioate
Phosphorothioate
Phosphorothioate
Phosphorothioate
Phosphorothioate
Phosphorothioate
Thiophosphate
PS-2
PS-3
PS-4
PS-5
PS-6
PS-7
PS-8b
PS-9
d(AAAAAAApsA) [Rp/Sp]
d(AAAAAApsAA) [Rp/Sp]
d(AAAAApsAAA) [Rp/Sp]
d(AAAApsAAAA) [Rp/Sp]
d(AAApsAAAAA) [Rp/Sp]
d(AApsAAAAAA) [Rp/Sp]
d(ApsAAAAAAA) [Rp/Sp]
Phosphorothioate
Phosphorothioate
Phosphorothioate
Phosphorothioate
Phosphorothioate
Phosphorothioate
Phosphorothioate
Thiophosphate
PS-10
PS-11
PS-12
PS-13
PS-14
PS-15
PS-16b
d(AAAAAAAAps
)
a ps denotes position of the phosphorothioate/thiophosphate linkage in T-modified and A-modified strands.
b These oligonucleotides do not have diastereomers.
O
O
O
O
O
O
O
O
S
S
OH
O
DMTrO
OH
DMTrO
O
DMAP, TEA
DMAP,
BTCM,
TPP
NH2
O
O
O
S
NH
DMTrO
O
O
(A)
Scheme 2. Preparation of polymer support (A) for synthesis of thiophosphorylated oligonucleotides.
while profile (b) depicts the pattern when PS-1 [Sp] dia-
stereomer is irradiated (365 nm). Photoreversible E–Z
isomerization is depicted in profile (c) when the above
sample was again exposed to visible radiation
(k > 400 nm). The above observations also lend credence
to the fact that azobenzene moiety is tethered to the PS-
modified oligonucleotides.
ward-oriented [Sp] PS-moiety in the B-helix of the dia-
stereomeric analogues of DNA.21,22
The Tm of native A8/T8 was found to be 22.3 ꢁC. The Tm
of the PS-modified duplex, T7XT/A8 ([Rp] isomer), was
observed at 35.2 ꢁC (+12.9ꢁ and +9.8 ꢁC with respect
to Tm of A8/T8 and T7psT/A8, respectively). When the
same duplex was irradiated at 365 nm, the Tm changed
to 28.2 ꢁC (ꢀ7.0 ꢁC with respect to E-isomer and
+5.9 ꢁC with respect to A8/T8 duplex). This substantial
difference in the Tm value suggests that, in E-configura-
tion, azobenzene, as a dangling aromatic residue, sup-
posedly intercalates between the base pairs of the two
complementary strands of the duplex, thereby enhanc-
ing stability of the duplex, whereas the duplex destabi-
lizes with N@N attaining Z-configuration. This
enhanced stability imparted by the azobenzene pendant,
in E-configuration, may be related to preponderance of
planar conformation of the E-isomer23 that facilitates
better base stacking effect as compared to the Z-isomer.
The observation further suggests that the azobenzene
moiety, in its E-configuration, effectively intercalates
2.4. Photomodulation of duplexes containing azobenzene
moiety
The Tm values for duplexes of a series of PS-modified
homothymidines [TppsTq] and homoadenosines [AxpsAy]
with their corresponding complementary strands are
given in Table 2, while Table 3 incorporates Tm data
on azobenzene-tethered phosphorothioate modified
homothymidines [TpXTq] and homoadenosines [AxXAy].
Of particular significance is the difference in Tm values
for [Rp] and [Sp] isomers (Table 2). Such differential
stability characteristics of [Rp] and [Sp] isomers have
been attributed to the difference in conformational
adjustments caused by inward-oriented [Rp] vs out-

S. Patnaik et al. / Bioorg. Med. Chem. 15 (2007) 7840–7849
7843
Figure 1. HPLC profiles of PS-4 phosphorothioate oligomer (a) [Rp] diastereomer, Rt = 8.148; (b) [Sp] diastereomer, Rt 8.451; (c) a co-injection of (a)
and (b); (d) Azobenzene linked [Rp] diastereomer, Rt = 12.275. Conditions: Column, RP-18 Hypersil Gold; Gradient, 0–20% B in 15 min, A = 0.1 M
HCOONH4 buffer, pH 7.1, B = MeCN; flow rate, 1.0 ml/min.
BASE
O
BASE
BASE
O
O
O
O
O
O
O
P
O
O
P
N
N
X
O
N
N
Phosphate buffer
+
1
X
O
S
O
P S
O
N
N
H
O
S
H
pH8
BASE
BASE
BASE
hν, 365nm
hν, 430nm
O
O
O
O
O
O
O
O
O
P
O
O
P
O
O
O
P
O
O
O
Z
- isomer
E
- isomer
Scheme 3. Incorporation of azobenzene pendant 1 into synthetic oligonucleotides and photomodulation of azobenzene linked oligonucleotides under
UV–vis light.
between the neighboring base pairs of the oligonucleo-
tide duplex, while, in Z-configuration, it creates spatial
distortion in the nearest base pairs, resulting in a
destabilization of the duplex as compared to the E-con-
figuration.24–26 In case of PS-modified duplex, A7XA/T8
([Rp] isomer), the same trend was observed although to a
lesser extent where the E-isomer has a Tm of 28.3 ꢁC
(+6.0ꢁ and +5.1 ꢁC with respect to A8/T8 and A7psA/
T8, respectively) as against its Z-isomer which has a
Tm of 23.3 ꢁC (ꢀ5.0 ꢁC with respect to E-isomer and
+1.0 ꢁC with respect to A8/T8). When the azobenzene
moiety moves toward the middle of the modified strand,
the stability of duplexes registers further decrease as
evident in T5XT3/A8 ([Rp] isomer) where the Tm value
of the E-isomer was observed to be 28.3 ꢁC (+6.0 ꢁC
with respect to A8/T8), while in case of Z-isomer, the
Tm value of 23.1 ꢁC (ꢀ5.2 ꢁC with respect to E-isomer
and +0.8 ꢁC with respect to A8/T8) was recorded due
to a decrease in duplex stability.
Interestingly, with azobenzene placed in the middle of
the duplex, in T4XT4/A8 ([Rp] isomer), the Tm value of
the E-isomer was found to be 26.8 ꢁC (+4.5ꢁ and
+1.6 ꢁC with respect to A8/T8 and T4psT4/A8, respec-
tively), whereas a small decrease in stability results in
case of Z-isomer, with Tm value of 22.8 ꢁC (ꢀ4.0 ꢁC
with respect to E-isomer and +0.5 ꢁC with respect to
A8/T8). Likewise, when the azobenzene pendant is

7844
S. Patnaik et al. / Bioorg. Med. Chem. 15 (2007) 7840–7849
1
λ= 365 nm
1
0.8
0.6
0.4
0.2
0
0.8
c
a
0.6
0.4
b
0.2
λ>400 nm
0
300
350
400
450
500
300
350
400
450
500
Wavelength (nm)
Wavelength (nm)
mAU
18.537
mAU
140
18.359
mAU
a
b
c
70
60
50
40
30
20
10
0
18.58
150
125
100
75
120
100
80
15.250
60
40
50
15.14
25
20
0
0
0
5
10
15
20
25
30
35 m/r
0
5
10
15
20 25
30
35 m/r
0
5
10
15
20
25
30
35 m/r
Time (min)
Time (min)
Time (min)
Figure 2. UV absorption patterns (top) and HPLC profiles (bottom) of PS-1 phosphorothioate oligo-([Sp] diastereomer), (a) before, and (b) after
irradiation at k = 365 nm. (c) Depicts spectral pattern when (b) is exposed to visible light (k > 400 nm). Irradiation was carried out for 1 h at 303 K.
Conditions: Column, RP-18 Hypersil Gold; Gradient, 0–15% B in 25 min, A = 0.1 M HCOONH4 buffer, pH 7.1, B = MeCN; flow rate, 1.0 ml/min.
Table 2. Melting temperature (Tm) of duplexes between stereo-
enriched oligos {[Rp] and [Sp]}, and their corresponding complemen-
tary sequencesa
with its passage into Z-isomer, that is, Tm of 22.9 ꢁC
(ꢀ4.9 ꢁC with respect to E-isomer and +0.6 ꢁC with re-
spect to A8/T8). Figure 3a shows a typical thermal melt-
ing profile of the modified duplex A3XA5/T8 ([Sp]
isomer) before and after photoisomerization. In the
above examples, the E–Z transformation of the azoben-
zene pendant (with attendant change in spectral charac-
teristics) appeared reversible indicating that the
azobenzene moiety is acting as a photomodulator of
the oligomer duplexes. The E–Z isomerization is further
supported by the observation that when the Z-isomer of
A3XA5/T8 ([Sp] isomer) was illuminated under visible
light (k > 400 nm) its melting profile almost superim-
posed with that of its E-counterpart (Fig. 3b).
Duplex
Tm (ꢁC)
[Rp]
isomer
DTm
[Sp]
isomer
DTm
(DTm w.r.t.
native)
(DTm w.r.t.
native)
A7psA/T8
A4psA4/T8
T7psT/A8
T4psT4/A8
23.2
23.3
25.4
25.2
(+0.9)
(+1.0)
(+3.1)
(+2.9)
23.8
23.5
25.7
25.8
(+1.5)
(+1.2)
(+3.4)
(+3.5)
[AxpsAy] = [TppsTq] = [A8] = [T8] = 2 lM, 10 mM phosphate buffer
containing 1.0 M NaCl, pH 7.1. ps denotes position of the phosp-
horothioate linkage in T-modified and A-modified strands.
a Values in Table 2 are average values of two observations per each
oligo sequence. The reproducibility of Tm values was within
[Tm 0.5 ꢁC].
In the modified duplex, T8X/A8, both in E- as well as Z-
configurations, no significant change in stability oc-
curred [+0.8 ꢁC (E) to ꢀ0.9 ꢁC (Z)] with respect to A8/
T8, perhaps because of weak intra-strand stacking effect
through the homopyrimidine strand.27 However, in case
of modified duplex, A8X/T8, the dangling azobenzene
moiety does seem to impart a degree of stability
[+1.8 ꢁC (E) to ꢀ0.1 ꢁC (Z)] to it.
moved from the middle of the duplex towards the 50-
end, stability of the duplex shows an increase in the
Tm value. This trend is observed in both the homopurine
and homopyrimidine modified duplexes. Thus, in
T2XT6/A8 ([Rp] isomer), the Tm value of the E-isomer
was found to be 31.0 ꢁC (+8.7 ꢁC with respect to A8/
T8), while that of its Z-isomer, the observed Tm value
was 25.2 ꢁC (ꢀ5.8 ꢁC with respect to E-isomer and
+2.9 ꢁC with respect to A8/T8). Subsequently, in the case
of AXA7/T8 ([Rp] isomer) duplex, the E-isomer had a Tm
of 27.8 ꢁC (+5.5 ꢁC with respect to A8/T8) while, the sta-
bility of the above duplex, decreases (upon irradiation),
From the data presented in Table 3, we conclude that in
both the [Rp] and [Sp] isomeric series, the trans isomer
enhances the stabilization of the duplexes with respect
to the corresponding cis isomer; the azobenzene pen-
dant, placed closed to 30- and 50-termini, confers maxi-
mum stability to the above duplexes, which registers a
fall as the azobenzene pendant is moved towards the
middle of the duplex. However, in all the above observa-

S. Patnaik et al. / Bioorg. Med. Chem. 15 (2007) 7840–7849
7845
Table 3. Melting temperature (Tm) of duplexes between stereo-enriched oligos {[Rp] and [Sp]}, and their corresponding complementary sequencesa
Duplex A8/T8 (native)
Tm (ꢁC) = 22.3
[Rp] isomer
[Sp] isomer
E (DTm w.r.t. native) Z (DTm w.r.t. native) DTm (E–Z) E (DTm w.r.t. native) Z (DTm w.r.t. native) DTm (E–Z)
A7XA/T8
A6XA2/T8
A5XA3/T8
A4XA4/T8
A3XA5/T8
A2XA6/T8
AXA7/T8
28.3 (+6.0)
24.1 (+1.8)
20.6 (ꢀ1.7)
18.4 (ꢀ3.9)
21.0 (ꢀ1.3)
24.5 (+2.2)
27.8 (+5.5)
24.1 (+1.8)
35.2 (+12.9)
31.6 (+9.3)
28.3 (+6.0)
26.8 (+4.5)
27.9 (+5.6)
31.0 (+8.7)
33.4 (+11.1)
23.1 (+0.8)
23.3 (+1.0)
18.6 (ꢀ3.7)
16.8 (ꢀ5.5)
15.7 (ꢀ6.6)
17.1 (ꢀ5.2)
19.2 (ꢀ3.1)
22.9 (+0.6)
22.2 (ꢀ0.1)
28.2 (+5.9)
25.9 (+3.6)
23.1 (+0.8)
22.8 (+0.5)
23.0 (+0.7)
25.2 (+2.9)
27.2 (+4.9)
21.4 (ꢀ0.9)
5.0
5.5
3.8
2.7
3.9
5.3
4.9
1.9
7.0
5.7
5.2
4.0
4.9
5.8
6.2
1.7
26.9 (+4.6)
23.6 (+1.3)
17.7 (ꢀ4.6)
16.3 (ꢀ6.0)
17.9 (ꢀ4.4)
23.2 (+0.9)
27.1 (+4.8)
—
23.8 (+1.5)
19.8 (ꢀ2.5)
15.4 (ꢀ6.9)
14.6 (ꢀ7.7)
16.0 (ꢀ6.3)
19.0 (ꢀ3.3)
23.4 (+1.1)
—
3.1
3.8
2.3
1.7
1.9
4.2
3.7
—
b
A8X/T8
T7XT/A8
T6XT2/A8
T5XT3/A8
T4XT4/A8
T3XT5/A8
T2XT6/A8
TXT7/A8
29.5 (+7.2)
26.2 (+3.9)
24.9 (+2.6)
23.1 (+0.8)
24.2 (+1.9)
26.0 (+3.7)
27.9 (+5.6)
—
24.5 (+2.2)
22.4 (+0.1)
21.6 (ꢀ0.7)
19.6 (ꢀ2.7)
21.8 (ꢀ0.5)
23.1 (+0.8)
23.0 (+0.7)
—
5.0
3.8
3.3
3.5
2.4
2.9
4.9
—
b
T8X/A8
[AxXAy] = [TpXTq] = [A8] = [T8] = 2 lM, 10 mM phosphate buffer containing 1.0 M NaCl, pH 7.1. X = N-acetyl-p-aminoazobenzene linked to
phosphorothioate backbone. [Rp] and [Sp] represent diastereomers of PS-oligos.
a Values in Table 3 are average values of two observations per each oligo sequence. The reproducibility of Tm values was within [Tm 0.5 ꢁC]. The
values in parentheses indicate DTm of each of the entries with respect to Tm of the native.
b These entries do not have diastereomers.
1.2
0.8
0.4
0
1.2
0.8
0.4
0
a
b
cis
cis
trans
trans
10
20
30
40
50
10
20
30
40
50
°
°
Temperature
C
Temperature C
Figure 3. Thermal melting profile of (a) duplex A3XA5/T8 ([Sp] isomer) before and after photoisomerization, (b) duplex A3XA5/T8 ([Sp] isomer) (—)
when exposed to UV irradiation (365 nm) and (----) when exposed to visible light (>400 nm).
tions the [Rp] isomers record greater stabilization as
compared to the corresponding [Sp] isomers.
the conjugated duplexes in the present investigation,
the CD spectra were recorded at 10 ꢁC ensuring
complete association of the PS-modified oligo strand
with its corresponding complementary strand. The
normalized CD spectra of the PS-modified duplexes,
after buffer subtraction, are shown in Figure 4. The
CD spectrum of the unmodified duplex was similar
to that of the native DNA having positive and nega-
tive CD bands of moderate magnitudes at wavelengths
above 220 nm and a crossover point at ꢁ242 nm. In
addition, the PS-modified oligonucleotides exhibited
weak CD (positive as well as negative) bands in the
300–400 nm region. The apparent difference of CD
signatures, in this region, is perhaps due to increased
flexibility in the phosphorothioate modified oligonu-
cleotide strand.
2.5. Circular Dichroism studies
It is well established that binding of an achiral ligand
with DNA induces optical activity which is registered
in induced Circular Dichroism (ICD) in the long-
wavelength range of absorption of complex.27 The
binding of such a ligand or adduct along the helix axis
of B-DNA induces ICD of two different categories: (i)
groove-localized chromophores show strong positive
ICD from transition moments oriented along the
groove, and (ii) intercalated chromophores may
display weaker positive or negative ICD.28,29 To deter-
mine spatial orientation of the azobenzene moiety in

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S. Patnaik et al. / Bioorg. Med. Chem. 15 (2007) 7840–7849
Figure 4. CD spectra of (a) native duplex (A8/T8), (b) [Rp]-A5XA3/T8, (c) [Rp]-T5XT3/A8; (—-) before UV irradiation, and (----) after irradiation.
The spectra of PS-modified A5XA3/T8 ([Rp] isomer) and
T5XT3/A8 ([Rp] isomer) duplexes in Figure 4b and c,
respectively, show more or less similar CD pattern in
the 250–280 nm region. This suggests that the global
conformation of the PS-modified duplex is relatively
insensitive to the stereochemistry of the PS-oligo
backbone.
are observed, albeit, the spectra exhibit overall similar-
ity: a positive signal at 280 nm and a negative band cen-
tered at 255 nm. These spectral features perhaps reflect
intercalation involving azobenzene and chirally stacked
DNA base pairs.27
3. Conclusion
However, the CD spectra of the modified PS-oligos dif-
fered significantly from that of the native duplex
(Fig. 4). They are marked by disappearance (in the spec-
trum of the latter) of a positive band at around 250 nm
and a negative band around 277 nm, with appearance
(in the spectrum of the former), respectively, of a nega-
tive band at 250 nm and a positive band at 280 nm,
reflective of the influence on the modification on either
of the strands.18b
An efficient and simple strategy to tether an azobenzene
moiety to oligonucleotides has been designed. The pro-
tocol provides flexibility concerning incorporation of
azobenzene moiety at pre-selected sites in PS-oligomer
sequences. Using the azobenzene-linked PS-oligonucleo-
tides, modulation of the duplex was realized under UV–
vis light. Owing to special importance of PS-oligos in
antigene and antisense technology, the present investiga-
tions provide a potential methodology for affecting
photomodulations in-vitro and in-vivo studies.
The CD spectra of A8X/T8 and T8X/A8 duplexes, in Fig-
ure 5a and b, respectively, reveal no significant change in
the pattern (albeit, the spectra are marked by change in
intensity), upon UV irradiation suggesting that incorpo-
ration of azobenzene, at the 30-end of modified strands
of the above duplexes, exerts little influence on the glo-
bal conformation of the duplexes.
4. Experimental
4.1. N-Iodoacetyl-p-aminoazobenzene 1
To a pre-cooled solution of iodoacetic acid (576 mg,
3.1 mmol), dissolved in anhydrous THF (15 ml), was
added p-aminoazobenzene (610 mg 3.1 mmol) followed
by dropwise addition of a solution of DCC (640 mg,
3.1 mmol), dissolved in THF (10 ml), with constant stir-
ring over a period of 15 min. The mixture was allowed
to stir at 0 ꢁC for 1 h followed by another 3 h at rt.
Prior to photomodulation, the CD spectrum of E-iso-
mer of T7XT/A8 ([Sp]) duplex (Fig. 5c) displays a maxi-
mum at 280 nm and a minimum at 255 nm along with
weakly induced bands around 340–360 nm (p–p* transi-
tion of the E-isomer). When the duplex is exposed to UV
light, weak differences in the intensity of the CD bands
Figure 5. CD spectra of (a) duplex (A8X/T8), (b) T8X/A8, (c) [Sp]-T7XT/A8; (—-) before UV irradiation, and (----) after UV irradiation.

S. Patnaik et al. / Bioorg. Med. Chem. 15 (2007) 7840–7849
7847
The reaction was monitored on TLC and the precipi-
tated dicyclohexyl urea was removed by filtration. The
filtrate was evaporated to dryness under vacuum and
purified by silica gel chromatography. The compound
was eluted with EDC and fractions containing the de-
sired compound pooled together and concentrated to
obtain 1 in 80% yield as a yellowish orange flaky mate-
was replaced by a sulfurization step using (TETD)
(0.5 M in MeCN)33 in the machine. For introduction
of thiophosphate at 30-end, the oligomer was assembled
on the modified polymer support A (Scheme 2). The
cleavage of modified oligonucleotides from the solid
support and simultaneous removal of protecting groups
from internucleotide phosphates and exocyclic amino
groups of nucleobases were achieved by treatment with
aqueous NH4OH (ꢁ30%) at 55 ꢁC for 16 h. After depro-
tection, the ammoniacal solution was concentrated in
speed vac, the residue dissolved in water (200 ll) and ap-
plied onto a desalting column (RP C-18 Silica Gel col-
umn). The oligomer was eluted with 30% MeCN in
water, concentrated in a speed vac, and purified by an-
ion exchange chromatography (FPLC). The configura-
tional assignment of diastereomers, [Rp] and [Sp], and
their separation from the stereo-mixture sequences were
achieved by RP-HPLC.19,20 The mass profiles of modi-
fied oligonucleotides were recorded on a Hewlett-Pack-
1
rial (903 mg). H NMR (CDCl3) d: 7.83–7.93 (m, 4H,
Ar-H), 7.46–7.53 (q, 3H, Ar-H), 7.67–7.69 (d, 2H, Ar-
H), 3.89 (s, 2H, –CH2–), 7.96 (s, 1H, –NHCO–). ESI-
MS calculated for C14H12N3OI (M): 365.0. Found:
365.18. Mp: 172 ꢁC. IR bands (thin film),
m cmꢀ1 = (1100–1400), (1423–1440), 1498, 1538, 1605,
1636, (2923–3284). Elemental Anal. Calcd (%) for
C14H12N3OI: C, 46.05; H, 3.31; N, 11.51. Found: C,
45.93; H, 3.41; N, 11.39.
4.2. Preparation of polymer support for 30-thiophosphated
oligonucleotides
ard
1100
MSD
Electron
Spray
Mass
4.2.1. Mono 2-(4,40-dimethoxytrityloxyethanesulfonyl)-
ethyl succinate.
Spectrophotometer in the linear negative mode, which
have been tabulated in Table 1.
A
mixture of 2-(4,40-dimethoxy-
trityloxyethylsulfonyl)-ethanol (456 mg, 1.0 mmol),30
succinic anhydride (200 mg, 2.0 mmol), dry TEA
(165 ll, 1.2 mmol), and DMAP (61 mg, 0.5 mmol) was
dissolved in dry EDC (4 ml) and stirred for 30 min at rt.
The reaction was monitored on TLC and quenched by
addition of MeOH (0.5 ml). The reaction mixture was fur-
ther diluted with EDC (20 ml) and washed successively
with cold 5% aqueous citric acid (2· 15 ml) and saturated
solution of NaCl (2· 15 ml). The organic phase was col-
lected, dried over anhydrous Na2SO4, and concentrated
to obtain the title compound (540 mg; 97% yield).
4.4. Incorporation of azobenzene pendant into oligo-
nucleotides
A stock solution of 2.73 mM concentration was pre-
pared by dissolving 1 (1 mg) in DMF (1.0 ml). For
coupling the azo derivative, 1, with each of the modi-
fied phosphorothioate oligonucleotides, a pre-calcu-
lated amount of the oligomer was taken and 50-fold
excess of the azo derivative was added to it. In a typ-
ical reaction, PS-4 (0.21 OD A254) was dissolved in
phosphate buffer (50 ll, pH 8.0). To this was added a
solution of 1 (50 ll). The reaction mixture was then left
on a shaker overnight. The solvent was removed in a
speed vac, the residue dissolved in water (100 ll) and
applied onto a Sephadex G-25 desalting column. The
oligomer conjugate was eluted with 50.0 mM triethy-
lammonium formate buffer, pH 7.1, and concentrated
in a speed vac. Subsequently, the oligomer was sub-
jected to purification by RP-HPLC. In a standard
experiment, the elution pattern of [Rp] and [Sp] diaste-
reomers of PS-4 phosphorothioate oligomer is shown
in Figure 1a and b, where-as Fig. 1c and d depicts a
co-injection of [Rp] and [Sp] diastereomers of PS-4 oli-
gomer and azobenzene linked [Rp] diastereomer PS-4
oligomer, respectively. HPLC Conditions: Column,
RP-18 Hypersil Gold; Gradient, 0–20% B in 15 min,
A = 0.1 M HCOONH4 buffer, pH 7.1, B = MeCN; flow
rate, 1.0 ml/min.
4.2.2. Attachment of the above succinate to 3-aminopropy-
lated CPG. Mono 2-(4,40-dimethoxytrityloxyethane-
sulfonyl)-ethyl succinate (28 mg, 50 lmol) was dissolved
in MeCN (1.0 ml) and to this were added bromotrichlo-
romethane (5 ll, 50 lmol) and DMAP (12.2 mg,
100 lmol). To the above, triphenylphosphine (6.55 mg,
25 lmol), dissolved in MeCN (0.5 ml), was added and
the mixture was swirled for 30 s followed by addition of
3-aminopropylated CPG (250 mg).31 The suspension
was stirred for 30 min at rt. The support was recovered
on a sintered glass funnel, washed with MeCN (5·
10 ml) and diethyl ether (2· 5 ml), respectively. This was
dried under vacuum and subjected to capping (residual
amino groups) with a solution consisting of Ac2O/TEA/
N-methylimidazole/DCM 1:1:0.4:6, v/v/v/v), for 30 min
at rt. The functionalized polymer support was then
washed, dried, and stored at 4 ꢁC until used for oligonu-
cleotide synthesis.
Other PS-oligos conjugates were prepared and purified
similarly.
4.3. Oligonucleotide synthesis and purification
Modified and unmodified oligonucleotide sequences
(Table 1) were synthesized at 0.2 lmol scale on a Phar-
macia Gene Assembler Plus using the standard phos-
phoramidite approach following the manufacturer’s
protocol.32 The phosphorothioate (PS)-linkage was
incorporated at pre-selected positions in the oligonucle-
otides during synthesis cycle in a manner analogous to
that in the normal cycle except that the oxidation step
4.5. Photomodulation of azobenzene-linked PS-oligo-
nucleotides
Photomodulation of azobenzene conjugated modified
PS-oligos was monitored using UV spectroscopy and
RP-HPLC. In a typical experiment, the diastereomers,
[Rp] and [Sp], of modified PS-oligo (PS-1) were separated
by RP-HPLC. The attachment of azobenzene residue to

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S. Patnaik et al. / Bioorg. Med. Chem. 15 (2007) 7840–7849
[Sp] diastereomer thus obtained, and its subsequent puri-
fication were achieved as described above. The azoben-
zene-tethered oligomer (0.5 OD A254nm) was dissolved
in distilled water and kmax of E- and Z-isomers at 343
and 434 nm, respectively, were determined to calculate
the molar extinction coefficients. Prior to irradiation,
the molar extinction coefficient (e) value for the E-iso-
mer was found to be 2.4 · 104 cmꢀ1 Mꢀ1 (at 343 nm),
which accounted for ꢁ91.6% of E-isomer and 8.4% of
Z-isomer (at 434 nm). Subsequently, the stoppered opti-
cal cell was placed inside a photoreactor and irradiated
for 30 min. Upon irradiation with UV light (365 nm),
the value of E-isomer decreased to 1.14 · 104 cmꢀ1 Mꢀ1
(82.3%) with concurrent increase in the Z-isomer
(17.7%) indicating E–Z isomerization. The photosta-
tionary state, in this reaction, was achieved within
30 min (prolonged exposure of the sample did not dis-
play any further change in the spectral pattern). On
exposure of the above sample (photostationary state)
to visible light (k > 400 nm), the value of e for E-isomer
increased to 1.82 · 104 cmꢀ1 Mꢀ1 (corresponding to
89% E-isomer), showing reversible E–Z isomerization.
values were taken over a range of 220–400 nm, at
ꢁ10 ꢁC below the melting temperatures of all the du-
plexes. At least three runs were performed for record-
ing the CD spectrum of each of the PS-modified
duplexes. The final spectrum in each case was ob-
tained after baseline subtraction and normalization
of the respected CD curves.
Acknowledgment
Authors gratefully acknowledge financial support from
the Department of Biotechnology, New Delhi, India.
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