Incorporation of cyclic azobenzene into oligodeoxynucleotides for the photo-regulation of DNA hybridization

Article abstract of DOI:10.1016/j.bmcl.2015.10.043

Cyclic azobenzene carboxylic acid was synthesized using a shortened route. After reaction with d-threolinol, the resulting cyclic azobenzene-d-threolinol (cAB-Thr) building block was transformed into the corresponding DMTr-protected phosphoramidite, and incorporated into oligodeoxynucleotides at various positions and frequencies by solid phase synthesis. The melting temperatures of these modified oligonucleotides were determined by UV spectrometry. Photo-regulation of cAB-Thr-modified oligonucleotides with their complementary sequence was evaluated by Fluorescence Resonance Energy Transfer experiments using a fluorescein-Black Hole Quencher pair. Results suggest that while cis-cAB destabilizes DNA duplexes, trans-cAB can be accommodated in double stranded DNA.

Full text of DOI:10.1016/j.bmcl.2015.10.043

Bioorganic & Medicinal Chemistry Letters 25 (2015) 5594–5596
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Incorporation of cyclic azobenzene into oligodeoxynucleotides
for the photo-regulation of DNA hybridization
⇑
Fatma Eljabu, Joshi Dhruval, Hongbin Yan
Department of Chemistry, Brock University, 500 Glenridge Ave., St. Catharines, Ontario L2S 3A1, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclic azobenzene carboxylic acid was synthesized using a shortened route. After reaction with D-threoli-
Received 13 September 2015
Revised 13 October 2015
Accepted 15 October 2015
Available online 23 October 2015
nol, the resulting cyclic azobenzene–D-threolinol (cAB–Thr) building block was transformed into the
corresponding DMTr-protected phosphoramidite, and incorporated into oligodeoxynucleotides at various
positions and frequencies by solid phase synthesis. The melting temperatures of these modified
oligonucleotides were determined by UV spectrometry. Photo-regulation of cAB–Thr-modified
oligonucleotides with their complementary sequence was evaluated by Fluorescence Resonance Energy
Transfer experiments using a fluorescein–Black Hole Quencher pair. Results suggest that while cis-cAB
destabilizes DNA duplexes, trans-cAB can be accommodated in double stranded DNA.
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
Cyclic azobenzene
FRET
Hybridization
Photoisomerization
Spatiotemporal control
In the past decade or so, spatiotemporal regulation of nucleic
acid structures and functions has attracted increasing interest.
shown to give cyclic azoxybenzene 2 in moderate, but consistent,
yields (62%) on multiple gram scales. Further, with the shortened
synthesis, cyclic bromoazoxybenzene 3 was transformed directly
into cyclic bromoazobenzene 6 by the treatment with phosphorus
trichloride, which was subsequently converted to cyclic cyanoa-
zobenzene 7 with copper(I) cyanide, where in our original
synthetic route, a three step process was required to convert
1
–3
In this respect, light-responsive moieties have been incorporated
into nucleic acids to introduce either irreversible or reversible
transformation by light. Among the systems that have been
explored for this purpose, aromatic azobenzene has been shown
to be rather versatile in enabling light-regulated nucleic acid
hybridization, transcription and aptamer-binding.1,4–9 Of particu-
cyclic bromoazoxybenzene
3
to cyclic cyanoazobenzene 7
lar interest, cyclic azobenzene (cAB) has recently been shown to
(3 ? 4 ? 5 ? 7, Scheme 1).
also undergo light-driven isomerization.1
0,11
Prompted by the fact
As described in our previous Letter, cyclic azobenzene car-
11
that the absorption bands of cis- and trans-cAB in the visible region
are well resolved, and that isomerization can be effected by visible
light, instead of UV in the case of aromatic azobenzene, work in our
laboratory has been directed towards the investigation of the
potential of cAB in the regulation of nucleic acid functions by light.
We herein report our preliminary finding in the photoregulation of
DNA hybridization with the cAB-modified oligodeoxynucleotides.
boxylic acid 8 was subsequently reacted with D-threolinol to give
cAB–Thr building block 9. After the primary hydroxyl was pro-
tected with the dimethoxytrityl (DMTr) group, the product 10
was converted into the corresponding phosphoramidite 11. The
phosphoramidite was isolated as a typical mixture of two diastere-
3
1
omers, as indicated by two resonance signals in P NMR.
The cAB–Thr building block was then incorporated into a
20-mer oligodeoxynucleotide by the phosphoramidite chemistry-
based solid phase synthesis. Note that up to four cAB–Thr units
were incorporated at varying positions of the 20-mer sequence.
In order to study hybridization by Fluorescence Resonance Energy
Transfer (FRET), fluorescein (FAM) and Black Hole Quencher-1
We previously reported the synthesis of cAB–D-threolinol
conjugate 9 (cAB–Thr), starting from the commercially available
0
2
,2 -dinitrodibenzyl 1. The synthesis involved seven steps of trans-
formation. One of the reactions, that is, formation of cyclic azoxy-
benzene 2, was later found to occasionally give inconsistent
results, as a volatile organic buffer (step i, Scheme 1) was used in
the reaction. By using sodium carbonate (step ii, Scheme 1), instead
of triethylammonium acetate, this transformation has now been
0
(BHQ-1) were incorporated to the 5 -end of the FAM sequence,
0
and 3 -ends of nine complementary sequences (AB
0 4
–AB )
with varying number of cAB–Thr, respectively (Table 1). All
sequences were purified by polyacrylamide gel electrophoresis
(
PAGE), and their molecular weights were confirmed by mass
⇑
Corresponding author. Tel.: +1 905 688 5550x3545.
E-mail address: tyan@brocku.ca (H. Yan).
spectrometry.
http://dx.doi.org/10.1016/j.bmcl.2015.10.043
960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
0

F. Eljabu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5594–5596
5595
Scheme 1. Reagents and conditions: (i) Pb, pH 9.5, NEt
3
–CH
3
COOH, MeOH; (ii) Pb, Na
2
CO
3
, MeOH; (iii) Br
2
, CH
3
COOH; (iv) CuCN, DMF; (v) PCl
3
, C
6
H
6
; (vi) Al, NH
OP(Cl)N(i-Pr)
2
NH
2
; (vii)
TiCl
3
, aq. HBr, H
2
O
2 2
; (viii) KOH, EtOH, H O; (ix)
D-threolinol, DCC, N-hydroxysuccinimide, DMF; (x) DMTr–Cl, pyridine; (xi) Hünig’s base, NCCH
2
CH
2
2
ꢀTHF.
Table 1
Oligonucleotide sequences incorporating cAB–Thr
0
0
Isolated yielda in OD260
b
M.W. calculatedc
M.W. foundd
Oligo ID
FAM
Sequence (5 –3 )
Scale of synthesis (
lmol)
T
m
(°C)
FAM-GGTATATCTCCTTCTTAAAG
1.0
1.0
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
27.7
56.8
—
6620.2
6703.6
7104.9
7104.9
7104.9
7506.3
7506.3
7506.3
7907.7
8309.0
6619.8
6703.8
7105.5
7103.5
7104
7505
7506
7505.5
7907
8308.5
AB
AB
0
CTTTAAGAAGGAGATATACC-BHQ-1
CTTTAAGAAGXGAGATATACC-BHQ-1
CTXTTAAGAAGGAGATATACC-BHQ-1
CTTTAAGAAGGAGATATAXCC-BHQ-1
CTTTAXAGAAGGAGATXATACC-BHQ-1
CTXTTAAGAAGXGAGATATACC-BHQ-1
CTTTAAGAAGXGAGATATAXCC-BHQ-1
CTTTAXAGAAGXGAGATXATACC-BHQ-1
CTTTXAAGAXAGGAXGATAXTACC-BHQ-1
48
43
44
42
36
37
36
28
21
f
1
9.7
5.5
e
AB1-1
AB1-2
f
17.6
e
AB
2
2.7
1.9
e
AB2-1
AB2-2
f
10.9
f
AB
AB
3
4
4.0
f
6.1
a
b
c
d
e
f
Isolated by PAGE.
of duplexes with FAM-sequence. Cyclic azobenzene exist in the cis-form.
M.W. of neutral species.
Mass deconvoluted for neutral species.
Deprotected by incubation with concentrated ammonium hydroxide at 55 °C for 15 h.
Deprotected by incubation with aqueous dimethylamine–concentrated ammonium hydroxide (1:1, v/v) at 60 °C for 20 min. X: cAB–Thr.
T
m
As noted in the isolated yields of the sequences in Table 1, the
spectroscopic results of the AB
pling yield for the synthesis of AB
the trityl assay, however, the mass spectroscopy indicated the
presence of the cleavage product (as HOR in Scheme 2) that is
approximately six times higher in intensity than that of the desired
AB product (Fig. S1 in the Supplementary material).
The overall yields can be improved by using milder deprotec-
1
sequence, where the overall cou-
yields for all cAB–Thr modified sequences are quite low, specially
when the deprotection was carried out by incubation with concen-
trated ammonium hydroxide at 55 °C for 15 h (Footnote e, Table 1).
These low yields are presumably attributed to cleavages of the
amide bond under the basic deprotection conditions, leading to
eventual cleavages of internucleotide phosphodiester linkages
1
was 90%, as determined by
2
1
(Scheme 2). This hypothesis corroborates with the mass
tion conditions, such as incubation with AMA, that is, aqueous
dimethylamine–concentrated ammonium hydroxide (1:1, v/v), at
6
0 °C for 20 min. The PAGE image shown in Figure S2 in the
Supplementary material clearly shows that the latter deprotection
conditions gave much better yields, which is also consistent with
the data shown in Table 1. In future studies, however, ultramild
1
2,13
14
phosphoramidites
together with the Q-linker will be used
towards the synthesis of cAB–Thr modified oligonucleotides, as
this combination allows for fast cleavage of oligonucleotide
products from the solid support and removal of N-acyl protecting
Scheme 2. Proposed chain cleavage under basic conditions.

5
596
F. Eljabu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5594–5596
FRET measurement of AB with controls
3
70000
60000
50000
40000
30000
20000
10000
0
AB0-ss
FAM-ss
AB0-FAM
cisAB3-FAM
tranAB3-FAM
500
510
520
530
540
550
560
570
580
590
600
Wavelength (nm)
3
Figure 2. FRET measurement of fluorescent quenching by AB .
Figure 1. Back-isomerization of trans- to cis-cAB in DMSO (1.5 mg/1.5 ml) at 37 °C.
Table 2
Percentages of fluorescent quenching as a measure of degree of hybridization for the
DNA duplexes incorporating cAB–Thr
group on nucleobases under very mild conditions, such as incubation
with aqueous ammonium hydroxide at room temperature for 2 h.
Entry
Duplex
% quenching
cis-)
% quenching
(trans-)
% difference
(trans–cis)
Melting temperatures (T
and complementary sequences (AB
UV spectroscopy. In all cases, cAB was in the cis-form. It can be
seen from the T s (Table 1) that incorporation of cAB–Thr leads
m
) of duplexes formed between FAM
(
0
–AB ) were determined by
4
1
2
3
4
5
6
7
8
9
FAM-AB
FAM-AB
FAM-AB1-1
FAM-AB1-2
0
—
—
—
5
1
90
90
83
63
65
58
37
22
95
106
104
90
94
87
73
58
m
16
21
27
29
29
36
36
to destabilization of DNA duplexes. It appears that incorporation
of each cAB–Thr unit into the sequence leads to a decrease of 5–
FAM-AB
2
FAM-AB2-1
FAM-AB2-2
8
°C in T
of the position at which cAB–Thr is incorporated.
of duplexes formed from the trans-cAB were not
m
, and that the destabilization is relatively independent
FAM-AB
FAM-AB
3
m
T s
4
determined as trans-cAB undergoes back-isomerization to cis-cAB
at elevated temperatures (Fig. 1). As is shown in Figure 3S in the
Supplementary material, back-isomerization from trans- to cis-
cAB is temperature-dependent. Thus, it takes approximately
transcription, in a turn-on fashion. Work in this direction is
currently underway in this laboratory.
1
00 h, 24 h, and 100 min for trans-cAB to completely isomerize
back to the cis-form at 25, 37, and 55 °C, respectively. This time-
scale of ‘relaxation’ will be an important consideration for future
applications of cAB in regulating DNA functions.
The possibility for trans-cAB–Thr to be accommodated in DNA
duplexes was subsequently assessed by Fluorescence Resonance
Energy Transfer (FRET) experiments. Thus, FAM and nine comple-
Acknowledgment
This work was funded by the Natural Science and Engineering
Research Council of Canada.
mentary sequences (AB
0
–AB
4
) were individually hybridized, and
Supplementary data
then subjected to isomerization by exposure to light at 395 nm
for 2 h, and the fluorescence of each duplex sample was deter-
mined immediately. Figure 2 shows the FRET quenching results
Supplementary data (procedures for compound syntheses and
characterization reported in this work) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/j.
bmcl.2015.10.043.
3
of AB . The FRET quenching of the FAM/BHQ-1 FRET pair was used
as an indication for the degree of hybridization, which is measured
by Eqs. 1 for cis-cAB–Thr and 2 for trans-cAB–Thr (F.I.: fluorescent
intensity), and the values of percentages of fluorescent quenching
for each of the duplexes are summarized in Table 2.
References and notes
1.
Mayer, G.; Heckel, A. Angew. Chem., Int. Ed. 2006, 45, 4900.
F:I:ðFAMÞ ꢁ F:I:ðFAM ꢁ cisAB Þ
x
2. Tang, X. J.; Dmochowski, I. J. Mol. BioSyst. 2007, 3, 100.
%
quenchingðcisÞ ¼
ꢂ 100%
ð1Þ
3
4
.
.
Young, D. D.; Deiters, A. Org. Biomol. Chem. 2007, 5, 999.
Asanuma, H.; Liang, X.; Nishioka, H.; Matsunaga, D.; Liu, M.; Komiyama, M. Nat.
Protoc. 2007, 2, 203.
F:I:ðFAMÞ ꢁ F:I:ðFAM ꢁ AB
0
Þ
F:I:ðFAMÞꢁF:I:ðFAMꢁtransAB
x
Þ
5. Dohno, C.; Uno, S. N.; Sakai, S.; Oku, M.; Nakatani, K. Bioorg. Med. Chem. 2009,
7, 2536.
Liang, X.; Takenaka, N.; Nishioka, H.; Asanuma, H. Chem. Asian J. 2008, 3, 553.
7. Liu, M.; Asanuma, H.; Komiyama, M. J. Am. Chem. Soc. 2006, 128, 1009.
Nishioka, H.; Liang, X.; Kashida, H.; Asanuma, H. Chem. Commun. 2007, 4354.
9. Beharry, A. A.; Woolley, G. A. Chem. Soc. Rev. 2011, 40, 4422.
%
quenchingðtransÞ ¼
ꢂ100%
1
F:I:ðFAMÞꢁF:I:ðFAMꢁAB Þ
0
6
.
ð2Þ
8
.
As Table 2 shows, compared with cis-cAB–Thr, the trans-isomer is
better accommodated in DNA duplexes. While the differences in
fluorescent quenching between duplexes containing cis- and
trans-cAB–Thr might appear to be moderate, it should be borne in
mind that the cis ? trans isomerization in cyclic azobenzene will
likely find applications as a ‘turn-on’, instead of a ‘turn-off’, switch.
Thus, these results do suggest that cyclic azobenzene could be used
for the spatiotemporal regulation of DNA functions, such as
10. Siewertsen, R.; Neumann, H.; Buchheim-Stehn, B.; Herges, R.; Näther, C.; Renth,
F.; Temps, F. J. Am. Chem. Soc. 2009, 131, 15594.
11. Joshi, D. K.; Mitchell, M. J.; Bruce, D.; Lough, A. J.; Yan, H. Tetrahedron 2012, 68,
8670.
1
1
2. Schulhof, J. C.; Molko, D.; Teoule, R. Tetrahedron Lett. 1987, 28, 51.
3. Sinha, N. D.; Davis, P.; Usman, N.; Perez, J.; Hodge, R.; Kremsky, J.; Casale, R.
Biochimie 1993, 75, 13.
14. Pon, R. T.; Yu, S. Tetrahedron Lett. 1997, 38, 3327; Ling, R.; Yoshida, M.; Mariano,
P. S. J. Org. Chem. 1996, 61, 4439.