Interactions of 4,4′-diaminoazobenzene derivatives with telomeric G-quadruplex DNA

Article abstract of DOI:10.1080/15257770.2018.1442578

The development of small molecules to stabilize the G-quadruplex structure has garnered significant attention for anticancer drug discovery. Herein, we report the synthesis of several 4,4′-diaminoazobenzene derivatives containing different substituent groups and their ability to bind and stabilize telomeric G-quadruplex DNA. Circular dichroism (CD) spectroscopy was performed to characterize the quadruplex topologies, measure stabilization effects, and evaluate their capabilities for conformational photoregulation. 4,4′-Diaminoazobenzene derivatives were found to moderately stabilize quadruplex structures but not affect conformational photoregulation. This work further develops the design and general understanding of the stabilization effects of small molecules with telomeric G-quadruplex DNA.

Full text of DOI:10.1080/15257770.2018.1442578

Nucleosides, Nucleotides and Nucleic Acids
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Interactions of 4,4′-diaminoazobenzene
derivatives with telomeric G-quadruplex DNA
Jeremy E. B. McCallum, Christopher W. Coyle, Ryan R. Elson & Blake A.
Titterington
To cite this article: Jeremy E. B. McCallum, Christopher W. Coyle, Ryan R. Elson & Blake A.
Titterington (2018): Interactions of 4,4′-diaminoazobenzene derivatives with telomeric G-quadruplex
DNA, Nucleosides, Nucleotides and Nucleic Acids, DOI: 10.1080/15257770.2018.1442578
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NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
, VOL. , NO. , –

https://doi.org/./..
ꢀ
Interactions of , -diaminoazobenzene derivatives with
telomeric G-quadruplex DNA
a
b
a
Jeremy E. B. McCallum , Christopher W. Coyle , Ryan R. Elson ,
c
and Blake A. Titterington
a
Department of Chemistry and Biochemistry, Loyola Marymount University, Los Angeles, CA, USA;
Molecular and Systems Pharmacology, Emory University, Atlanta, GA, USA; School of Medicine,
b
c
Creighton University, Omaha, NE, USA
ABSTRACT
ARTICLE HISTORY
Received  July 
Revised  February 
Accepted  February 
The development of small molecules to stabilize the
G-quadruplex structure has garnered significant attention
for anticancer drug discovery. Herein, we report the synthesis of
ꢀ
several 4,4 -diaminoazobenzene derivatives containing different
KEYWORDS
substituent groups and their ability to bind and stabilize telom-
eric G-quadruplex DNA. Circular dichroism (CD) spectroscopy
was performed to characterize the quadruplex topologies,
measure stabilization effects, and evaluate their capabilities
G-quadruplexes;
ꢀ

, -diaminoazobenzene;
photoisomerization; circular
dichroism
ꢀ
for conformational photoregulation. 4,4 -Diaminoazobenzene
derivatives were found to moderately stabilize quadruplex
structures but not affect conformational photoregulation. This
work further develops the design and general understanding
of the stabilization effects of small molecules with telomeric
G-quadruplex DNA.
Introduction
G-quadruplexes are secondary structures formed in guanine-rich sequences of
nucleic acids. G-tetrads, formed from the association of four guanine bases through
Hoogsteen base pairing, coordinate with cations and stack on top of each other to
form the quadruplex structure. These structures can assemble from one, two, or four
nucleic acid strands, forming a variety of topologies that can be classified as paral-
lel, antiparallel, and hybrid. The quadruplex structure can occur naturally in G-rich
sequences and have been identified in telomeres and gene promoter regions. Pro-
teins and ligands can bind G-quadruplexes, stabilizing the tertiary structure of DNA
and RNA, and inhibit or promote replication, transcription, and translation.[
As these structures are linked to such diverse functions, the development of small
molecules to stabilize the G-quadruplex structure for therapeutic purposes has gar-
nered significant attention. Small molecule stabilization of quadruplex formation in
1–4]
CONTACT Jeremy E. B. McCallum
Loyola Marymount University,  LMU Drive, Los Angeles, CA, , USA.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lncn.
 Taylor & Francis Group, LLC
jeremy.mccallum@lmu.edu
Department of Chemistry and Biochemistry,
©

2
J. E. B. MCCALLUM ET AL.
telomeric DNA was one of the first targets as the G-quadruplex structure has been
shown to decrease telomerase activity. Telomerase, which is responsible for main-
taining the length of telomeres, is expressed in approximately 85% of cancer cells,
[
5–7]
providing inhibition as an attractive candidate for anticancer therapies.
Several
quadruplex stabilizers such as telomestatin, RHPS4, and BRACO-19 have shown
[
8–10]
promising results.
Our group is interested in developing small-molecule quadruplex stabilizers and
targeted azobenzene derivatives for their photoregulation potential. Azobenzene
derivatives can photoisomerize to the cis isomer under ultraviolet light and back to
ꢀ
the trans isomer under visible light. Several 4,4 -dihydroxyazobenzene derivatives
have been synthesized and shown to stabilize or destabilize the quadruplex struc-
ture dependent upon formation of the trans or cis isomer.[
11–14]
Derivatization of
azoaniline moieties have not, as of yet, been investigated. We here report derivati-
ꢀ
zation of 4,4 -diaminoazobenzene and the effect of several amino side chains on the
stabilization of the DNA quadruplex structure.
Results and discussion
Synthesis
ꢀ
4
,4 -Diaminoazobenzene (1) was synthesize by the sodium sulfide reduction of dis-
[
15]
perse orange 3 as previously described.
A one-pot preparation of diaminoa-
ꢀ
zobenzene derivatives 2–7 (DAAB 2–7) was achieved upon treatment of 4,4 -
diaminoazobenzene with the corresponding chloroacyl chloride (2-chloroacetyl
chloride or 3-chloropropionyl chloride) followed by the addition of diethy-
lamine, pyrrolidine, piperidine, or morpholine in moderate to good overall yields
(Scheme 1).
H
N
NH
2
O
n
NR
2
Cl
1.
Cl
3
, Et N, THF
N
n
N
O
N
O
N
2. R
2
NH
2
R N
H
2
N
N
H
1
n
2-7 (n = 1-2)
H
H
N
N
N
N
N
O
N
N
O
O
O
N
O
O
N
N
N
H
N
H
2
3
5
H
N
H
N
N
N
N
O
N
N
O
O
N
O
N
N
H
N
N
H
6
H
N
H
N
N
N
N
O
N
N
O
O
N
O
N
N
H
N
N
H
4
7
ꢀ
Scheme . Synthesis of , -diaminoazobenzene derivatives 2–7.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
CD spectroscopy
Telomeric DNA has been shown to be polymorphic, capable of forming several
topologies with specific circular dichroism (CD) spectroscopic signatures. Parallel,
anti-parallel, and hybrid quadruplex structures can be determined by specific posi-
[
16–18]
tive and negative peaks within 180–300 nm range where DNA absorbs light.
Two DNA strands, TEL6 (d(TTAGGG)) and TEL24 (d(TTAGGG) ), were found
4
to form stable G-quadruplex structures in Tris-HCl-KCl buffer. The CD spectrum
of TEL6 showed a negative peak at 240 nm, a positive peak at 260 nm, and a weak
negative peak at 284 nm characteristic of a parallel, tetramolecular G-quadruplex
structure (Fig. 1a). The spectrum of TEL24 exhibited a positive peak at 288 nm with
a shoulder at 270 nm, indicative of a hybrid-type, monomolecular G-quadruplex
(Fig. 1b). The CD spectrum of TEL6 and TEL24 in the presence of compound 7
a)
2
2
1
1
5
0
5
0
5
0
5
220
240
260
280
300
320
-
-10
-15
-20
Wavelength (nm)
b)
2
2
1
1
5
0
5
0
5
0
5
220
270
320
370
420
470
-
-10
Wavelength (nm)
Figure . CD spectra of  µM telomeric quadruplex DNA in Tris-HCl-KCl buffer. The solid blue spec-
trum is quadruplex DNA without ligand while the red dashed spectrum contains compound 7 in a :
ratio of ligand:[quadDNA]. a) CD spectra of TEL6 in the presence and absence of compound 7. b) CD
spectra of TEL24 in the presence and absence of compound 7.

4
J. E. B. MCCALLUM ET AL.
Table . Changes in melting curves of quadruplex DNA. ꢀ Tm = Tm (DNA+ligand) – Tm (DNA). Ther-
mal melting of TEL6 was monitored at  nm and TEL24 was monitored at . The ratio represents
[ligand]:[quadDNA].
ꢀ
Tm (°C)
TEL 
TEL 
Compound
:
:
:
:
2
3
4
5
6
7
.
.
.
.
.
.
.
.
—
—
.
.
.
.
.
.
.
.
.
.
—
—
.
.
(
consistent for compounds 2–6, data not shown) exhibited similar peaks, indicat-
ing the diaminoazobenzene derivatives formed the same topologies. Compound 7
(
(
DAAB 7) exhibited strong induced CD bands at 374 nm (positive) and 454 nm
negative) which confirm the interaction of 7 with the quadruplex DNA. Induced
CD bands have been shown to aid in deciphering specific binding modes as end-
stacking, intercalation, or groove-binding.[
19–27]
The bisignate shape in the visible
range, containing one positive and one negative band, is suggestive of an exciton
effect and indicative of the formation of dimer or higher order DAAB 7-complexes
associated with the quadruplex DNA.
Thermal stability studies
After confirming quadruplex formation, the quadruplex stabilization effects for each
derivative were measured. Melting curve profiles were conducted for both TEL6 and
TEL24 in the absence and presence of compounds 2–7 at two different concentra-
tions. The changes in melting temperature (ꢀ T ) of the quadruplex DNA were
m
calculated and reported in Table 1.
ꢀ
4
,4 -Diaminoazobenzene derivatives displayed minor to moderate quadruplex
stabilization effects with greater ꢀ T temperatures for the hybrid G-quadruplex
m
structure of TEL24. Not surprisingly, changing amino side chains affected quadru-
plex stabilization. Compounds 3 and 7, both with pyrrolidinyl side chains, showed
the largest stabilization effects for both quadruplex topologies. Compound to DNA
ratios greater than 2:1 were required to see stabilization effects greater than 4°C,
confirming the importance that dimer or higher-order DAAB derivative-complexes
bound to DNA contribute to the observed stabilization effects.
Quadruplex DNA mobility assay
A native gel electrophoresis experiment (Figure 2) was performed to investigate
the possibility of higher order (dimer, trimer) DNA quadruplex assemblies upon
complexation with DAAB derivatives. TEL24 migrated faster than the marker dyes
(bromophenol blue, xylene cyanol) which respectively migrate to the same point as

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
Figure . NativepolyacrylamidegelelectrophoresisofofcompoundandTEL24DNA. Lane, TEL24
with dye markers (bromophenol blue, xylene cyanol); lane , TEL24; lane , TEL24 with  (:); lane

, TEL24 with  (:).
double-stranded DNA of 12 bp and 45 bp in a 20% polyacrylamide nondenatur-
[
28–29]
ing gel as previously demonstrated.
In the presence of compound 7, TEL24
predominantly migrated the same as the TEL24 DNA alone. These results sup-
port that higher order DAAB-templated DNA assemblies (dimer, trimer, etc.) are
not observed and that DAAB derivative-complexation occurs on single quadruplex
monomer units.
Photoregulation
As the best stabilizer, compound 7 was chosen for evaluation of photoregulation
of the quadruplex structure. When irradiated with light at 350 nm, compound 7
(
in the presence of TEL24) photoisomerized from the trans to the cis isomer as
indicated by changes in the UV/VIS spectrum and in agreement with previous
2
1
1
1
1
.8
.6
.4
.2
1
0
0
0
0
.8
.6
.4
.2
0
2
00
250
300
350
400
450
500
550
600
Wavelenth (nm)
Figure . UV/Vis spectrum of compound 7 in TEL24 DNA in Tris-HCl-KCl buffer. The solid blue line
represents compound 7 before irradiation while the dashed red line represents compound 7 after

min irradiation.

6
J. E. B. MCCALLUM ET AL.
work (Figure 3).[11–12,14] While the UV/VIS spectra demonstrated successful pho-
toisomerization, no changes were observed in the CD spectrum below 300 nm as
expected, indicating a lack of regulation (dissociation or conformation change) of
the hybrid quadruplex conformation. As TEL24 formed hybrid-type quadruplex
structures in the presence and absence of compound 7, a conformational change is
not expected, assuming the cis isomer does not substantially bind DNA to induce
a conformational change. Indeed Wang et al. and Xing et al. demonstrated rela-
tively similar CD spectra for quadruplex DNA in the absence and presence of cis-
ꢀ
ꢀ
4
,4 -dihydroxyazobenzene derivatives while their trans-4,4 -dihydroxyazobenzene
[
11–12]
derivatives induced quadruplex DNA conformational changes.
In our case,
the only change in CD spectra occurred for the induced CD bands which were
diminished immediately after photoisomerization but returned to the same values
within 5 minutes, indicating an initial reduction in higher order DAAB 7 to DNA
complexation. Presumably, the cis isomer of 7 would decrease quadruplex DNA sta-
bilization as compared to the trans isomer, resulting in a decreased ꢀT , but iso-
m
merization back to the trans form occurs faster than the time in which the melting
curve measurements can be completed.
Conclusions
Six 4,4-diaminoazobenzene derivatives were synthesized and found to bind to
and stabilize quadruplex DNA. In the presence of TEL6, (d(TTAGGG)), paral-
lel quadruplexes were formed while TEL24, (d(TTAGGG) ), formed hybrid-type
4
quadruplex structures. DNA stabilization effects were weak to moderate with the
greatest effect demonstrated by compound 7 containing a pyrrolidinyl side chain,
stabilizing the hybrid-type quadruplex most effectively. Induced CD bands indicated
stabilization appeared to occur through a dimer or higher order complex associated
with quadruplex DNA for TEL24. Photoisomerization from the trans to cis isomer
had no apparent effect on the quadruplex conformation. This work further devel-
ops the design and general understanding of the stabilization effects of azobenzene
derivatives with telomeric G-quadruplex DNA.
Experimental section
General information
All reagents were purchased from Aldrich Chemical Company and used without
further purification. DNA was purchased from Integrated DNA Technologies with
standard desalting purification and used without further purification. NMR spec-
tra were recorded on a Bruker Avance-400 Spectrometer. Circular dichroism spec-
tra were collected on a JASCO J-815 Circular Dichroism spectrophotometer using
0
.1-cm path length quartz cuvettes. Electrospray ionization mass spectra were per-
formed on an Agilent 6530 Q-TOF ESI mass spectrometer or a PerkinElmer Flexar
SQ 300 ESI mass spectrometer. UV/Vis spectra were collected on an Agilent Cary

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
6
0 spectrophotometer. Photoirradiation was performed using a UVP, 5 watt UV
lamp at 365 nm and maximum isomerization was achieved after 5 minutes. Auto-
mated column chromatography was achieved through flash chromatography using
a CombiFlash system.
®
Circular dichroism
A 40 µM solution [10 µM quadruplex] of TEL6 (d(TTAGG) and 10 µM of TEL24
(
1
d(TTAGGG) were prepared in 10 mM HCl-Trizma base buffer solution with
00 mM KCl at a pH of 7.5. Stock solutions of compounds 2–7 were prepared in
4
®
DMSO with final concentrations of 20 µM, 40 µM, and 80 µM for the correspond-
ing studies. Full spectra scans (200 nm – 600 nm) were recorded at 25°C and melting
curve profiles were performed at ramp rate of 1°C/min with a D.I.T. of 16 sec. and
a band width of 2 nm.
Gel electrophoresis
Gel electrophoresis experiments were performed on a native gel containing 20%
polyacrylamide (acrylamide/bis-acrylamide = 37.5:1) in TBE buffer. 40 mM KCl
was added to the buffer and gel to maintain G-quadruplex structure formation. The
gel was run at 4°C and viewed by UV shadowing.
Synthesis
ꢀ

8
, -diaminoazobenzene ()
.
.66 g (0.036 mol) of Na S 9H O was solubilized in 300 mL of H O and added drop-
2
2
2
wise to a solution of 3.63 g of disperse orange 3 (0.015 mol) in 300 mL of MeOH over
hour. The resulting mixture was refluxed at 85°C for 3 hours. The product was pre-
1
cipitated in 600 mL of cold H O, vacuum filtered, and purified by automated flash
2
chromatography (hexanes/ethyl acetate) to yield the final product in 87% yield.
1
H NMR (400 MHz; CDCl ): δ 3.94 (br. s, 4H), 6.73 (d, J = 8.2 Hz, 4H), 7.73 (d,
3
13
J = 8.2 Hz, 4H) ppm. C NMR (100 MHz; CDCl ): δ 114.9, 124.5, 146.0, 148.7 ppm.
3
+
ESI-MS: Calcd for C H N [M+H] 213.11, found: 213.06.
12
13
4
General synthesis of compounds –
ꢀ
0
.425 g (0.002 mol) of 4,4 -diaminoazobenzene was dissolved in 100 mL of anhy-
drous THF and cooled with an ice bath. 1.2 mL (4 eq) of triethylamine and 3 eq. of
the corresponding acid chloride was added sequentially. The reaction was warmed
to room temperature and allowed to react for 2 hours. The reaction mixture was
then cooled in an ice bath and 6 equivalents of the corresponding amine (pyrroli-
dine, diethylamine, piperidine, or morpholine) was added and stirred at room tem-
perature overnight. After concentration, the crude product was dissolved in DCM
and washed 3 times with an equal volume of saturated sodium bicarbonate solution.

8
J. E. B. MCCALLUM ET AL.
The final product was purified using automated column flash chromatography (hex-
anes/ethyl acetate) to afford the final derivatives in good yields. For compounds 6–7,
trimethylamine was omitted and 10 eq. of the corresponding amines were used.
N,N’-(diazene-,-diylbis(,-phenylene))bis(-(diethylamino)acetamide) ()
1
H NMR (400 MHz; CDCl ): δ 1.11 (t, J = 6.4 Hz, 12H), 2.68 (q, J = 6.4 Hz, 8H), 3.18
3
13
(
s, 4H), 7.73 (d, J = 7.8 Hz, 4H), 7.91 (d, J = 7.8 Hz, 4H), 9.53 (br. s, 2H) ppm. C
NMR (100 MHz; CDCl ): δ 12.6, 49.1, 58.4, 119.5, 124.1, 140.2, 149.2, 170.5 ppm.
3
+
ESI-MS: Calcd for C H N O [M+H] 439.28, found: 439.03. 62% yield.
24
35
6
2
N,N’-(diazene-,-diylbis(,-phenylene))bis(-(pyrrolidin--yl)acetamide) ()
1
H NMR (400 MHz; CDCl ): δ 1.88 (br. s, 8H), 2.72 (br. s, 8H), 3.31 (s, 4H), 7.74
3
13
(
(
d, J = 7.6 Hz, 4H), 7.91 (d, J = 7.6 Hz, 4H), 9.31 (br. s, 2H) ppm. C NMR
100 MHz; CDCl ): δ 24.3, 54.8, 60.0, 119.7, 124.0, 140.2, 149.2, 169.5 ppm. ESI-
3
+
MS: Calcd for C H N O [M+H] 435.25, found: 435.13. 65% yield.
2
4
31
6
2
N,N’-(diazene-,-diylbis(,-phenylene))bis(-(piperidin--yl)acetamide) ()
1
H NMR (400 MHz; CDCl ): δ 1.52 (br. s, 4H), 1.68 (br. s, 8H), 2.58 (br. s, 8H),
3
3
.12 (s, 4H), 7.73 (d, J = 7.6 Hz, 4H), 7.92 (d, J = 7.6 Hz, 4H), 9.51 (br. s, 2H) ppm.
13
C NMR (100 MHz; CDCl ): δ 23.7, 51.7, 55.1, 63.0, 119.6, 124.1, 140.2, 149.2,
3
+
169.2 ppm. ESI-MS: Calcd for C H N O [M+H] 463.28, found: 463.31. 60%
24
33
8
2
yield.
N,N’-(diazene-,-diylbis(,-phenylene))bis(-morpholinoacetamide) ()
1
H NMR (400 MHz; CDCl ): δ 2.65 (br. s, 8H), 3.18 (s, 4H), 3.80 (br. s, 8H), 7.73
3
13
(
(
d, J = 8.0 Hz, 4H), 7.92 (d, J = 8.0 Hz, 4H), 9.26 (br. s, 2H) ppm. C NMR
100 MHz; CDCl ): δ 54.0, 62.7, 67.2, 119.7, 124.1, 139.9, 149.3, 168.2 ppm. ESI-
3
+
MS: Calcd for C H N O [M+H] 467.24, found: 467.20. 57% yield.
2
4
31
6
4
N,N’-(diazene-,-diylbis(,-phenylene))bis(-(diethylamino)propanamide) ()
1
H NMR (400 MHz; CDCl ): δ 1.14 (t, J = 6.8 Hz, 12H), 2.53 (t, J = 6.7 Hz, 4H),
3
2
(
4
[
.69 (q, J = 6.8 Hz, 8H), 2.79 (t, J = 6.7 Hz, 4H), 7.77 (d, J = 7.8 Hz, 4H), 7.88
1
3
d, J = 7.8 Hz, 4H), 11.62 (br. s, 2H) ppm. C NMR (100 MHz; CDCl ): δ 11.6, 33.3,
3
6.1, 49.0, 119.6, 124.0, 141.3, 148.8, 171.3 ppm. ESI-MS: Calcd for C H N O
24 35 6 2
+
M+H] 467.3, found: 467.2. 42% yield
N,N’-(diazene-,-diylbis(,-phenylene))bis(-(pyrrolidin--yl)propanamide) ()
1
H NMR (400 MHz; CDCl ): δ 1.88 (br. s, 8H), 2.53 (t, J = 6.7 Hz, 4H), 2.72 (br. s,
3
8
(
1
4
H), 2.79 (t, J = 6.7 Hz, 4H), 7.74 (d, J = 7.6 Hz, 4H), 7.91 (d, J = 7.6 Hz, 4H), 11.62
1
3
br. s, 2H) ppm. C NMR (100 MHz; CDCl ): δ 24.3, 33.0, 54.8, 56.0, 119.7, 124.0,
3
+
40.2, 149.2, 171.5 ppm. ESI-MS: Calcd for C H N O [M+H] 463.3, found:
24
31
6
2
63.1. 38% yield.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
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[
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