Alkyl and aryl sulfonyl p-pyridine ethanone oximes are efficient DNA photo-cleavage agents

Article abstract of DOI:10.1016/j.jphotobiol.2016.02.017

Sulfonyloxyl radicals, readily generated upon UV irradiation of p-pyridine sulfonyl ethanone oxime derivatives, effectively cleave DNA, in a pH independent manner, and under either aerobic or anaerobic conditions. p-Pyridine sulfonyl ethanone oxime derivatives were synthesized from the reaction of p-pyridine ethanone oxime with the corresponding sulfonyl chlorides in good to excellent yields. All compounds, at a concentration of 100 μM, were irradiated at 312 nm for 15 min, after incubation with supercoiled circular pBluescript KS II DNA and resulted in extended single- and double- strand cleavages. The cleavage ability was found to be concentration dependent, with some derivatives exhibiting activity even at nanomolar levels. Besides that, p-pyridine sulfonyl ethanone oxime derivatives showed good affinity to DNA, as it was observed with UV interaction and viscosity experiments with CT DNA and competitive studies with ethidium bromide. The compounds interact to CT DNA probably by non-classical intercalation (i.e. groove-binding) and at a second step they may intercalate within the DNA base pairs. The fluorescence emission spectra of pre-treated EB-DNA exhibited a significant or moderate quenching. Comparing with the known aryl carbonyloxyl radicals the sulfonyloxyl ones are more powerful, with both aryl and alkyl sulfonyl substituted derivatives to exhibit DNA photo-cleaving ability, in significantly lower concentrations. These properties may serve in the discovery of new leads for "on demand" biotechnological and medical applications.

Full text of DOI:10.1016/j.jphotobiol.2016.02.017

Journal of Photochemistry & Photobiology, B: Biology 158 (2016) 30–38
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Alkyl and aryl sulfonyl p-pyridine ethanone oximes are efficient DNA
photo-cleavage agents
Nicolaos-Panagiotis Andreou a, Konstantinos Dafnopoulos a, Christos Tortopidis a, Alexandros E. Koumbis b,
Maria Koffa c, George Psomas d, Konstantina C. Fylaktakidou a,
⁎
a
Laboratory of Organic, Bioorganic and Natural Product Chemistry, Molecular Biology and Genetics Department, Democritus University of Thrace, University Campus, Dragana, 68100
Alexandroupolis, Greece
b
Laboratory of Organic Chemistry, Chemistry Department, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
Laboratory of Cellular Biology and Cell Cycle, Molecular Biology and Genetics Department, Democritus University of Thrace, University Campus, Dragana, 68100 Alexandroupolis, Greece
Laboratory of Inorganic Chemistry, Chemistry Department, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 August 2015
Received in revised form 17 February 2016
Accepted 18 February 2016
Available online 27 February 2016
Sulfonyloxyl radicals, readily generated upon UV irradiation of p-pyridine sulfonyl ethanone oxime derivatives,
effectively cleave DNA, in a pH independent manner, and under either aerobic or anaerobic conditions. p-
Pyridine sulfonyl ethanone oxime derivatives were synthesized from the reaction of p-pyridine ethanone
oxime with the corresponding sulfonyl chlorides in good to excellent yields. All compounds, at a concentration
of 100 μM, were irradiated at 312 nm for 15 min, after incubation with supercoiled circular pBluescript KS II
DNA and resulted in extended single- and double- strand cleavages. The cleavage ability was found to be concen-
tration dependent, with some derivatives exhibiting activity even at nanomolar levels. Besides that, p-pyridine
sulfonyl ethanone oxime derivatives showed good affinity to DNA, as it was observed with UV interaction and
viscosity experiments with CT DNA and competitive studies with ethidium bromide. The compounds interact
to CT DNA probably by non-classical intercalation (i.e. groove–binding) and at a second step they may intercalate
within the DNA base pairs. The fluorescence emission spectra of pre-treated EB–DNA exhibited a significant or
moderate quenching. Comparing with the known aryl carbonyloxyl radicals the sulfonyloxyl ones are more pow-
erful, with both aryl and alkyl sulfonyl substituted derivatives to exhibit DNA photo-cleaving ability, in signifi-
cantly lower concentrations. These properties may serve in the discovery of new leads for “on demand”
biotechnological and medical applications.
Keywords:
DNA photo-cleavage
DNA binding
Sulfonyl ketoximes
Sulfonyloxyl radicals
Photo-cleavage agents
© 2016 Elsevier B.V. All rights reserved.
1. Introduction
conjugates, since the alkyl ones suffer rapid decarboxylation and pro-
duce less drastic radicals [8].
Light as an “on–off trigger” has long been used as a tool to promote
and control reactions. It offers distinct advantages in several fields of
chemistry, including biomedicine, where it has been employed in “on
demand” therapeutic applications with profound benefits. Among
others, cancer treatment has been realized using photodynamic therapy
[1], combined in several cases with nanotechnological approaches [2].
Small synthetic organic molecules have been recognized as tools for
targeting a variety of DNA structural features, aiming to cause cancer
cell death [3]. Among DNA cleaving agents, those causing oxidative
cleavage upon UV light activation are called “photo-cleavers” [4]. O-
Acyl oximes represent one such class of compounds [5–7], which yield
photo-generated carbonyloxyl radicals (CRs) capable of causing oxida-
tive DNA damage. However, their use is so far mainly limited to aryl
Sulfonyl ketoximes (SKs) are useful organic precursors, which, upon
the Neber [9] or Beckmann [10] rearrangement reactions, provide ac-
cess to medicinally interesting scaffolds [11]. However, very limited
studies have been performed for the evaluation of the biological activity
of SKs themselves [12]. In the field of photosensitive polymeric systems,
SKs have found applications in microlithography, polymer, material and
computer sciences [13], and are considered photo-acid generators. The
production of the corresponding UV photo-generated sulfonyloxyl
radicals (SRs), which abstract a hydrogen atom to form sulfonic acid
derivatives, has been well documented [13].
In continuation of our interest in the chemistry and biology of ox-
imes [7,14–17], we have decided to investigate the photo-activating be-
havior of SKs towards plasmid DNA. p-Pyridine ethanone oxime was
selected as the model carrier scaffold of the sulfonyl moiety in order
to facilitate comparison of the obtained results with the ones previously
reported for the p-pyridine oxime and amidoxime ester conjugates [7].
Additionally, after evaluating the derivative which exhibits the best
⁎
Corresponding author.
E-mail address: kfylakta@mbg.duth.gr (K.C. Fylaktakidou).
http://dx.doi.org/10.1016/j.jphotobiol.2016.02.017
1011-1344/© 2016 Elsevier B.V. All rights reserved.

N.-P. Andreou et al. / Journal of Photochemistry & Photobiology, B: Biology 158 (2016) 30–38
31
cleaving activity, the p-nitro-phenyl ester conjugate of p-pyridine
2.3. Data of Sulfonyl Ketoximes 3–14 and Carbonyl Ketoxime 15
2.3.1. (E)-1-(pyridin-4-yl)ethanone O-propylsulfonyl oxime (3)
White crystals, yield 90%, mp 135–136 °C (ethyl acetate/methanol);
IR (KBr): 3045, 2973, 1621, 1372 and 1172 (SO) cm−1; 1Η NMR
(500 μΗz, CDCl3) δ 1.10 (t, J = 7.5 Hz, 3H), 1.95 (sext, J = 7.6 Hz, 2H),
2.42 (s, 3H), 3.38 (t, J = 7.7 Hz, 2H), 7.57 (d, J = 6.2 Hz, 2H), 8.72
(d, J = 6.1 Hz, 2H); 13C NMR (125 μΗz, CDCl3) δ 12.9, 13.9, 17.1, 51.1,
120.8, 141.0, 150.5, 162.1; HRMS (ESI) Calc C10H14N2O3S [M + H]+
243.0798; found 243.0799.
ethanone oxime was also synthesized, allowing, thus, the direct reactiv-
ity comparison between sulfonyloxyl and carbonyloxyl radicals. A vari-
ety of both alkyl and aryl sulfonyl conjugates were prepared to further
perform structure activity relationship (SAR) studies. Nevertheless,
since SKs have the S0 → S1 and S0 → T1 transition states localized on
the oxime moiety [18], similar activity is expected to be observed with
other SKs as well, as long as they absorb at least partially at the wave-
length of irradiation, and have some kind of affinity to DNA [3,4].
2. Experimental
2.3.2. (E)-1-(pyridin-4-yl)ethanone O-decylsulfonyl oxime (4)
Beige crystals, yield 58%, mp 60 °C (ethyl acetate/methanol); IR
(KBr): 2957, 2923, 2855, 1596, 1364 and 1169 (SO) cm−1; 1Η NMR
(500 μΗz, CDCl3) δ 0.86 (t, J = 6.7 Hz, 3H), 1.22–1.35 (m, 12H), 1.45
(quin, J = 7.4 Hz, 2H), 1.90 (quin, J = 7.7 Hz, 2H), 2.41 (s, 3H), 3.38
(t, J = 7.8 Hz, 2H), 7.58 (d, J = 6.1 Hz, 2H), 8.71 (d, J = 5.9 Hz, 2H);
13C NMR (125 μΗz, CDCl3) δ 13.9, 14.0, 22.6, 23.2, 28.1, 28.9, 29.1,
29.4, 31.8, 49.5, 120.8, 141.2, 150.4, 162.1; HRMS (ESI) Calc
C17H28N2O3S [M + H]+ 341.1893; found 341.1894.
2.1. Materials, Physical Measurements and Instrumentation
All commercially available reagent-grade chemicals and solvents
were used without further purification. Dry solvents were prepared by
literature methods and stored over molecular sieves. Calf thymus (CT)
DNA, ethidium bromide (EB), NaCl and trisodium citrate were pur-
chased from Sigma-Aldrich Co. DNA stock solution was prepared by di-
lution of CT DNA to buffer (containing 15 mM trisodium citrate and
150 mM NaCl at pH 7.0) followed by 3 day stirring and kept at 4 °C for
no longer than two weeks. The stock solution of CT DNA gave a ratio
of UV absorbance at 260 and 280 nm (A260/A280) of 1.87, indicating
that the DNA was sufficiently free of protein contamination [19]. The
DNA concentration was determined by the UV absorbance at 260 nm
after 1:20 dilution using ε = 6600 M−1 cm−1 [20].
Melting points (mps) were measured on a Kofler hot-stage appara-
tus or a melting point meter M5000 Krüss, and are uncorrected. FT-IR
spectra were obtained in a Perkin–Elmer 1310 spectrometer using po-
tassium bromide pellets. NMR spectra were recorded on an Agilent
500/54 (500 MHz and 125 MHz for 1H and 13C respectively) spectrom-
eter using CDCl3, and/or DMSO-d6 as solvent. J values are reported in Hz.
High resolution mass spectra (HRMS) were recorded on micrOTOF GC–
MS QP 5050 Shimadzu single-quadrupole mass spectrometer. Mass
spectra were determined on a Shimadzu LCMS-2010 EV system under
Electrospray Ionization (ESI) conditions. UV–vis spectra were recorded
on a Hitachi U–2001 dual beam spectrophotometer. Fluorescence emis-
sion spectra were recorded in solution on a Hitachi F–7000 fluorescence
spectrophotometer. Viscosity experiments were carried out using an
ALPHA L Fungilab rotational viscometer equipped with an 18 mL LCP
spindle. Samples containing plasmid DNA were irradiated in a
Macrovue 2011 transilluminator LKB BROMMA at 312 nm, T-15.M
90 W, 0.225 W/cm2, and 10 cm distance.
2.3.3. (E)-1-(pyridin-4-yl)ethanone O-hexadecylsulfonyl oxime (5)
White crystals, yield 53%, mp 66–68 °C (ethyl acetate/methanol); IR
(KBr): 2957, 2921, 2851, 1366 and 1168 (S = O) cm−1; 1Η NMR
(500 μΗz, CDCl3 + DMSO-d6) δ 0.79 (t, J = 6.7 Hz, 3H), 1.10–1.30
(m, 24H), 1.38 (quin, J = 7.3 Hz, 2H), 1.79 (quin, J = 7.6 Hz, 2H), 2.38
(s, 3H), 3.34 (t, J = 7.7 Hz, 2H), 7.71 (d, J = 5.5 Hz, 2H), 8.70 (d, J =
5.7 Hz, 2H); 13C NMR (125 μΗz, CDCl3 + DMSO-d6) δ 13.5, 13.7, 22.8,
27.7, 28.5, 28.8, 28.9, 29.0, 29.13, 29.17, 29.19, 29.20, 29.21, 29.22,
31.5, 49.0, 120.5, 140.7, 150.0, 161.8; HRMS (ESI) Calc C23H40N2O3S
[M + H]+ 425.2832; found 425.2833.
2.3.4. (E)-1-(pyridin-4-yl)ethanone O-phenylsulfonyl oxime (6)
Beige crystals, yield 93%, mp 156.3 °C (ethyl acetate/ethanol); IR
(KBr): 3048, 1592, 1377 and 1195 (SO) cm−1; 1Η NMR (500 μΗz,
CDCl3) δ 2.33 (s, 3H), 7.42 (bd, J = 4.6 Hz, 2H), 7.56 (t, J = 7.9 Hz,
2H), 7.66 (t, J = 8.1 Hz, 1H), 8.02 (d, J = 8.1 Hz, 2H), 8.63 (d, J =
4.6 Hz, 2H); 13C NMR (125 μΗz, CDCl3) δ 13.6, 120.7, 128.9, 129.0,
134.2, 135.2, 141.1, 150.3, 161.5; HRMS (ESI) Calc C13H12N2O3S
[M + H]+ 277.0641; found 277.0641.
2.3.5. (E)-1-(pyridin-4-yl)ethanone O-tosyl oxime (7) [22]
Brown crystals, yield 89%, mp 162.3 °C (ethyl acetate/ethanol); IR
(KBr): 1594, 1379 and 1195 (SO) cm−1; 1Η NMR (500 μΗz, CDCl3) δ
2.35 (s, 3H), 2.44 (s, 3H), 7.37 (d, J = 8.2 Hz, 2H), 7.54 (d, J = 6.2 Hz,
2H), 7.91 (d, J = 8.3 Hz, 2H), 8.67 (d, J = 5.9 Hz, 2H); 13C NMR
(125 μΗz, CDCl3) δ 13.6, 21.7, 121.2, 128.9, 129.7, 132.0, 142.6, 145.6,
149.0, 160.8; HRMS (ESI) Calc C14H14N2O3S [M + H]+ 291.0798;
found 291.0798.
All reactions were monitored on commercial available pre-coated
TLC plates (layer thickness 0.25 mm) of Kieselgel 60 F254. Silica gel
Merck 60 (40–60 mM) has been used for column chromatography.
Yields were calculated after recrystallization.
2.2. General Procedure for the Synthesis of Ethanone Sulfonyl Oximes
2.3.6. (E)-1-(pyridin-4-yl)ethanone O-[(4-nitrophenyl)sulfonyl] oxime (8)
Beige crystals, yield 90%, mp 117–119 °C (ethyl acetate/ethanol); IR
(KBr): 3105, 1531, 1381 and 1193 (SO) cm−1; 1Η NMR (500 μΗz,
CDCl3) δ 2.39 (s, 3H), 7.42 (d, J = 5.4 Hz, 2H), 8.24 (d, J = 8.5 Hz, 2H),
8.42 (d, J = 8.4 Hz, 2H), 8.68 (d, J = 5.4 Hz, 2H); 13C NMR (125 μΗz,
CDCl3) δ 14.0, 120.6, 124.2, 130.4, 140.6, 140.9, 150.6, 151.0, 162.9;
HRMS (ESI) Calc C13H11N3O5S [M + H]+ 322.0492; found 322.0493.
A solution of (E)-1-(pyridin-4-yl)ethanone oxime 1 [21] (274 mg,
2 mmol) in dry chloroform (15 mL) was cooled to 0 °C under an argon
atmosphere. Triethylamine (0.28 mL, 2 mmol) was added, followed by
the required sulfonyl chloride (2 mmol) and the mixture was stirred
for 1–3 h allowing the temperature to slowly rise to 25 °C. The reaction
was monitored by TLC and, upon completion, water (70 mL) was added
and the mixture was extracted with dichloromethane (2 × 70 mL). The
combined organic extracts were dried (Na2SO4) and concentrated
under vacuum (rotary evaporator) to give a residue which was purified
using column chromatography and a mixture of hexanes/ethyl acetate
as eluent.
2.3.7. (E)-1-(pyridin-4-yl)ethanone O-[(4-methoxyphenyl)sulfonyl] oxime
(9)
White crystals, yield 74%, mp 139–140 °C (ethyl acetate); IR (KBr):
1596, 1376 and 1174 (SO) cm−1; 1Η NMR (500 μΗz, CDCl3) δ 2.34 (s,
3H), 3.88 (s, 3H), 7.02 (d, J = 9.0 Hz, 2H), 7.52 (d, J = 6.2 Hz, 2H),
7.96 (d, J = 9.0 Hz, 2H), 8.68 (d, J = 5.8 Hz, 2H); 13C NMR (125 μΗz,
CDCl3) δ 13.6, 55.7, 114.3, 121.1, 126.3, 131.2, 142.4, 149.2, 160.7,
The same procedure was applied for the synthesis of the p-nitro-
benzoyl oxime 15, but, instead of sulfonyl chloride, PNP-benzoyl chlo-
ride was used. Reaction time: 1 h.

32
N.-P. Andreou et al. / Journal of Photochemistry & Photobiology, B: Biology 158 (2016) 30–38
164.4; HRMS (ESI) Calc C14H14N2O4S [M + H]+ 307.0747; found
307.0747.
2.4. DNA Cleavage Experiments
2.4.1. Cleavage of Supercoiled Circular pBluescript KS II DNA by Sulfonyl
Ketoximes
2.3.8. (E)-1-(pyridin-4-yl)ethanone O-[(4-(trifluoromethyl)phenyl]
sulfonyl) oxime (10)
The reaction mixtures (20 μL) containing supercoiled circular
pBluescript KS II DNA stock solution (Form I, 50 μM/base pair,
~500 ng), compounds, and Tris buffer (25 μM, pH 6.8) in Pyrex vials
were incubated for 30 min at 37 °C, centrifuged, and then irradiated
with UV light (312 nm, 90 W) under aerobic conditions at room temper-
ature for 15 min. After addition of the gel-loading buffer (6X Orange
DNA Loading Dye 10 mM Tris–HCl (pH 7.6), 0.15% orange G, 0.03% xy-
lene cyanol FF, 60% glycerol, and 60 mM EDTA, by Fermentas), the reac-
tion mixtures were loaded on a 1% agarose gel with ethidium bromide
staining. The electrophoresis tank was attached to a power supply at a
constant current (65 V for 1 h). The gel was visualized by 312 nm UV
transilluminator and photographed by an FB-PBC-34 camera vilber
lourmat. Quantitation of DNA-cleaving activities was performed by
integration of the optical density as a function of the band area using
the program “Image J” available at the site http://rsb.info.nih.gov/ij/
download.html.
Off white crystals, yield 79%, mp 115–116 °C (ethyl acetate/hex-
anes); IR (KBr): 3103, 3054, 1597, 1318, 1383 and 1196 (SO) cm−1
;
1Η NMR (500 μΗz, CDCl3) δ 2.39 (s, 3H), 7.53 (d, J = 3.8 Hz, 2H), 7.85
(d, J = 7.8 Hz, 2H), 8.17 (d, J = 7.8 Hz, 2H), 8.71 (d, J = 3.8 Hz, 2H);
13C NMR (125 μΗz, CDCl3) δ 13.9, 120.7, 122.6 (q, J = 272 Hz), 126.2
(q, J = 3.7 Hz), 129.5, 135.8 (q, J = 34 Hz), 138.8, 140.9, 150.4, 162.4;
HRMS (ESI) Calc C14H11F3N2O3S [M + H]+ 345.0515; found 345.0518.
2.3.9. (E)-1-(pyridin-4-yl)ethanone O-[(4-bromophenyl)sulfonyl] oxime
(11)
Off white crystals, yield 79%, mp 118–120 °C (ethyl acetate/hex-
anes); IR (KBr): 3086, 3015, 1569, 1176, 1379 and 1194 (SO) cm−1
;
1Η NMR (500 μΗz, CDCl3) δ 2.37 (s, 3H), 7.52 (d, J = 6.0 Hz, 2H), 7.72
(d, J = 8.5 Hz, 2H), 7.89 (d, J = 8.6 Hz, 2H), 8.69 (d, J = 5.3 Hz, 2H);
13C NMR (125 μΗz, CDCl3) δ 13.8, 121.1, 129.8, 130.4, 132.5, 134.1,
142.1, 149.3, 161.6; HRMS (ESI) Calc C13H11BrN2O3S [M + H]+
354.9747; found 354.9747, 356.9726.
2.4.2. Calculation of Single-strand Damage (ss)% and Double-strand
Damage (ds)%
ss% and ds% damage was calculated according to the following
Eqs. (1) and (2).
Form II
ðForm I þ Form II þ Form IIIÞ
2.3.10. (E)-1-(pyridin-4-yl)ethanone O-[(4-chlorophenyl)sulfonyl] oxime
(12)
ss% ¼
ds% ¼
ꢀ 100
ꢀ 100
ð1Þ
ð2Þ
Beige crystals, yield 72%, mp 108.2 °C (ethyl acetate); IR (KBr): 3092,
3013, 1592, 1175, 1382 and 1194 (SO) cm−1; 1Η NMR (500 μΗz, CDCl3)
δ 2.37 (s, 3H), 7.51 (d, J = 5.5 Hz, 2H), 7.55 (d, J = 8.5 Hz, 2H), 7.97 (d,
J = 8.6 Hz, 2H), 8.69 (d, J = 5.8 Hz, 2H); 13C NMR (125 μΗz, CDCl3) δ
13.8, 121.1, 129.5, 130.4, 133.5, 141.2, 142.1, 149.4, 161.6; HRMS (ESI)
Calc C13H11ClN2O3S [M + H]+ 311.0252; found 311.0258, 313.0227.
Form III
ðForm I þ Form II þ Form IIIÞ
As Form II we consider Form II of each series minus Form II of the ir-
radiated control DNA. As Form I we consider Form I of each series.
2.5. Interaction With CT DNA
2.3.11. (E)-1-(pyridin-4-yl)ethanone O-[(2,5-dichlorophenyl)sulfonyl]
oxime (13)
2.5.1. Study With UV Spectroscopy
Beige crystals, yield 41%, mp 106–108 °C (ethyl acetate/ethanol); IR
(KBr): 3083, 3060, 1593, 1389 and 1192 (SO) cm−1; 1Η NMR (500 μΗz,
DMSO-d6) δ 2.47 (s, 3H), 7.50 (d, J = 6.1 Hz, 2H), 7.82 (d, J = 8.6 Hz,
1H), 7.89 (dd, J = 8.6, 2.6 Hz, 1H), 8.12 (d, J = 2.6 Hz, 1H), 8.66 (d,
J = 6.1 Hz, 2H); 13C NMR (125 μΗz, DMSO-d6) δ 14.5, 121.4, 130.8,
132.1, 133.0, 134.4, 134.5, 136.5, 140.6, 150.8, 165.0; HRMS (ESI) Calc
C13H10Cl2N2O3S [M + H]+ 344.9862; found 344.9864, 346.9832,
348.9801.
The interaction of compounds 3 and 8 with CT DNA was studied by
UV spectroscopy in order to investigate the possible binding modes to
CT DNA and to calculate the binding constants to CT DNA (Kb). The UV
spectra of the compounds (1 × 10−4 M) were recorded in the presence
of CT DNA at diverse [compound]/[DNA] mixing ratios (= r). Control
experiments with DMSO were performed and no changes in the spectra
of CT DNA were observed.
The binding constant, Kb, can be obtained by monitoring the changes
in the absorbance at the corresponding λmax with increasing concentra-
tions of CT DNA and it is given by the ratio of slope to the y intercept in
plots [DNA] / (εA −εf) versus [DNA], according to the Wolfe–Shimer
equation [23]:
2.3.12. (E)-4-[1-(pyridin-4-yl)ethylideneaminooxysulfonyl]benzonitrile
(14)
Beige crystals, yield 54%, mp 108.2 °C (ethyl acetate); IR (KBr): 3097,
3050, 2236, 1596, 1168, 1384 and 1195 (SO) cm−1; 1Η NMR (500 μΗz,
CDCl3 + DMSO-d6) δ 2.36 (s, 3H), 7.52 (d, J = 5.4 Hz, 2H), 8.05 (d, J =
8.3 Hz, 2H), 8.15 (d, J = 8.4 Hz, 2H), 8.63 (d, J = 5.5 Hz, 2H); 13C
NMR (125 μΗz, CDCl3 + DMSO-d6) δ 12.1, 115.4, 115.8, 119.3, 127.7,
131.7, 137.0, 139.0, 148.2, 161.6; HRMS (ESI) Calc C14H11N3O3S
[M + H]+ 302.0594; found 302.0599.
½DNAꢁ
½DNAꢁ
1
¼
þ
ð3Þ
ðεA−εf Þ εb−εf Kbðεb−εf Þ
where [DNA] is the concentration of DNA in base pairs, εA = Aobsd
/
[compound], εf = the extinction coefficient for the free compound and
εb = the extinction coefficient for the compound in the fully bound
form.
2.3.13. (E)-1-(pyridine-4-yl)ethan-1-one O-(4-nitrobenzoyl)oxime (15)
Off white crystals, yield 89%, mp 205.1 °C (ethyl acetate); IR (KBr):
3110, 3084, 1744, 1595 cm−1; 1Η NMR (500 μΗz, CDCl3) δ 2.55 (s,
3H), 7.70 (d, J = 5.8 Hz, 2H), 8.30 (d, J = 8.7 Hz, 2H), 8.37 (d, J =
8.7 Hz, 2H), 8.75 (d, J = 5.8 Hz, 2H); 13C NMR (125 μΗz, CDCl3) δ
14.3, 121.1, 123.9, 130.8, 134.1, 141.8, 150.5, 150.9, 161.6, 162.5;
HRMS (ESI) Calc C14H11N3O4 [M + H]+ 286.0822; found 286.0820.
2.5.2. Viscometry
Viscosity experiments were carried out using an ALPHA L Fungilab
rotational viscometer equipped with an 18 mL LCP spindle and the mea-
surements were performed at 100 rpm. The viscosity of DNA ([DNA] =
0.1 mM) in buffer solution (150 mM NaCl and 15 mM trisodium citrate
at pH 7.0) was measured in the presence of increasing amounts of the
compounds (up to the value of r = 0.35). All measurements were

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33
performed at room temperature. The obtained data are presented as
(η / η0)1/3 versus r, where η is the viscosity of DNA in the presence of
the compound, and η0 is the viscosity of DNA alone in buffer solution.
activity, we have synthesized its carbonyl analogue (15), in order to
compare the reactivity of the sulfonyl and the carbonyl radicals of sim-
ilar origin.
Compounds 3–14 were synthesized in good yields, as a single prod-
uct, upon the reaction of p-pyridine ethanone oxime 1 [21] with the cor-
responding alkyl or aryl sulfonyl chlorides 2a–l in dry CHCl3, in the
presence of Et3N and under an argon atmosphere (Scheme 2).
Compound 15 was similarly obtained, using the corresponding ben-
zoyl chloride, Scheme 3.
2.5.3. Competitive Studies With EB
The competitive studies of each compound with EB were investigat-
ed with fluorescence emission spectroscopy in order to examine wheth-
er the compound can displace EB from its DNA–EB complex. The DNA–
EB complex was prepared by adding 20 μM EB and 26 μM CT DNA in
buffer (150 mM NaCl and 15 mM trisodium citrate at pH 7.0). The pos-
sible intercalating effect of the compounds was studied by adding a cer-
tain amount of a solution of the compound step by step into a solution of
the DNA–EB complex. The influence of the addition of each compound
to the DNA–EB complex solution was monitored by recording the vari-
ation of fluorescence emission spectra with excitation wavelength at
540 nm.
All these compounds are new, with the exception of 7, which has
been previously reported [22].
3.2. DNA Cleavage Studies
DMF solutions of SKs 3–14 (100 μM) were mixed with a Tris buffer
solution (25 μM, pH = 6.8) containing the supercoiled circular
pBluescript KS II DNA (Form I) and this mixture was irradiated with
UV light (312 nm) at room temperature for 15 min, under aerobic con-
ditions. Plasmid DNA was analyzed by gel electrophoresis on 1% agarose
stained with ethidium bromide, Fig. 1. All experiments were performed
at least three times. None of the tested compounds showed any activity
towards DNA in the absence of UV irradiation.
The Stern–Volmer constant (KSV) is used to evaluate the quenching
efficiency for each compound according to the Stern–Volmer equation
[24]:
Io
I
¼ 1 þ KSV½Qꢁ
ð4Þ
In the presence of compounds 3–14 the supercoiled plasmid DNA
(Form I) sustained single-stranded (ss) nicks of the double helix, gener-
ating the relaxed circular DNA (Form II) found in all experiments,
whereas the linear DNA (Form III) was formed as well, generated by
double-stranded (ds) nicks, Fig. 1.
where Io and I are the emission intensities in the absence and the pres-
ence of the quencher, respectively, [Q] is the concentration of the
quencher (compounds 3 and 8); KSV is obtained from the Stern–
Volmer plots by the slope of the diagram Io/I versus [Q]. Subsequently,
the quenching constant kq may be calculated by the equation:
The results presented in Fig. 1 show that most of the aryl SKs are in
general considerably reactive. The most important observation is that
alkyl SRs are also effective, and, comparing to the aryl ones, they are
similarly efficient at the given concentration, contrary to what was ob-
served for the carbonyl analogues [7,8]. As for the aromatic ones, a
SAR study revealed that lack of substitution on the aromatic ring (6) re-
sults in reduced activity. The activity also decreases when the CH3 group
is replaced with the electron withdrawing CF3 group (7 and 10, respec-
tively). Additionally, regarding the p-halogenated compounds, the
bromo-derivative 11 is not as efficient comparing to the chloro-one 12
[4], whereas the dichloro-derivative 13 was the less reactive. Calcula-
tion of the energies of the N–O bonds of these SKs indicates that their
variation lies within ~2 Kcal/mol (45.4–47.7 Kcal/mol).
The observations that a) the energy provided by UV irradiation is
more than sufficient for the cleavage of all N–O bonds; b) the length
of the aliphatic chain is almost irrelevant to the activity; c) the substitu-
ents of the aromatic ring with electron donating effect (7 and 9) and
electron withdrawing effect (8) have approximately the same activity
(Form II + Form III); and d) all aromatic substituents with electron
withdrawing effects have not the same activity (8 and 10–14) shows
that the variations of activity might also be dependable on affinity
DNA interactions of the sulfonyl conjugates. This is in accordance to pre-
viously reported conclusions regarding the dependence of DNA cleav-
age mechanism on the structure of the photo-cleavage agent,
primarily because of the geometrical requirements for H-abstraction re-
actions [4a].
KSV ¼ kqτo
ð5Þ
where τo = the average fluorescence lifetime of the EB–DNA system.
3. Results and Discussion
3.1. Chemistry
Photolysis causes homolytic cleavage of the N–O bond of SKs and
production of iminyl and sulfonyloxyl radicals (Scheme 1). As it has
been demonstrated the iminyl radical may abstract a hydrogen atom
to give an imine, which is further hydrolyzed to give the corresponding
ketone. The same ketone is obtained when the iminyl radical reacts with
molecular oxygen. Finally, a third option is its dimerization to a
bisalkylidene hydrazine derivative. On the other hand, the SR abstracts
a hydrogen atom and gives the corresponding acid [13].
The mixture of oxime sulfonyl esters photo-cleaved material is ex-
pected to contain iminyl and sulfonyloxyl radicals, which, potentially,
are both capable of attacking DNA, under various mechanisms. Since
there is no precedent of SRs acting as DNA photo-cleaving agents, we
wish to provide data which correspond to the reactivity of SRs them-
selves. For this reason we have synthesized derivatives 3–14 in order
to establish a structure–activity relationship study within the SRs series.
Additionally, based on the fact that derivative 8 exhibited the best
Scheme 1. The fate of radicals generated by the photo-cleavage of the N–O bond of oxime sulfonyl esters.

34
N.-P. Andreou et al. / Journal of Photochemistry & Photobiology, B: Biology 158 (2016) 30–38
15 was 86( 5.8)%, including a ~3% of double-strand cleavage, at a con-
centration of 100 μM [Fig. 2(A), Lane 9]. Although both 8 and 15
completely consume supercoiled plasmid DNA at the given concentra-
tion, sulfonyl ester 8 causes many more nicks, which eventually leads
to the formation of linear DNA and a high ds/ss ratio.
Furthermore, we have also examined the lowest concentration
which is sufficient for the 50% cleavage of the supercoiled plasmid
DNA caused by each one of compounds 3–8. The results at concentra-
tions of 10 and 1 μM are given in Fig. 2(A) and (B), respectively. Τhe di-
agram with the average cleavage for all experiments is depicted in
Fig. 2(C). All three aromatic sulfonic conjugates exhibit good cleavage
ability (~70–80%) at a concentration of 10 μM, whereas the aliphatic
ones were found less reactive at both concentrations.
Scheme 2. Synthesis of p-pyridine-ethanone oxime sulfonyl esters 3–14. Reagents and con-
ditions: RSO2Cl (1.1 eq.), dry Et3N (1.1 eq.), dry CHCl3, 0 °C to r.t., 1–3 h, 41–93%.
In brief, the cleavage caused by SKs is concentration dependent.
Comparing the activity of carbonyl analogue 15 at a concentration of
100 μM with the sulfonyl analogue 8 at a concentration of 10 μM we
conclude that the latter has about the same activity in approximately
10-fold lower concentration [86( 5.8)% and 77.5( 1.1)%, respective-
ly], Fig. 2(A) lanes 8 and 9. Compound 8 is effective for 50% DNA
photo-cleavage in a concentration of approximately 1 μM. Most impor-
tantly, according to these results all compounds are expected to have
some activity even in the nanomolar range.
Scheme 3. Synthesis of the p-pyridine-ethanone PNP oxime 15. Reagents and conditions:
PNPCOCl (1.1 eq.), dry Et3N (1.1 eq.), dry CHCl3, 0 °C to r.t., 1 h, 89% (PNP = p-NO2–C6H4–).
Mechanistic studies performed with compound 8 (Fig. 3, left part)
showed that SKs can react under anaerobic conditions (Lane 4) and
that the mode of action under aerobic conditions most probably in-
volves hydroxyl radicals and singlet oxygen (reaction with DMSO—Lane
5 and NaN3—Lane 6, respectively). Nevertheless, the cleavage of plasmid
DNA is not enhanced when D2O was used as solvent (Lane 7). These re-
sults indicate that not a single mechanistic pathway is implicated in the
cleavage. Additional experiments to clarify the behavior of the system
are in due course. Finally, a variation of pH (range 5–8) does not affect
the photo-cleavage activity (Fig. 2, right part, Lanes 8–11, respectively).
The ability of SKs to act under anaerobic conditions and acidic pH
may be very useful in the treatment of solid tumors were hypoxic con-
ditions as well as low acidity dominate [28].
A high ratio of ds to ss cleavages is a very important characteristic of
DNA cleaving ability [25], since ds cleavages are more difficult to be
repaired and may trigger self-programmed cell death. The UV generated
SRs cause ss and ds cleavages, with the one derived from p-pyridine p-
nitrophenyl sulfonyl ethanone oxime 8 yielding the best ratio of
double- to single-strand damages [Fig. 1(A), Lane 8 and Fig. 1(B)]. More-
over, compared to the p-pyridine p-nitrobenzoyl oxime and amidoxime
derivatives, the sulfonyl ones were found to exhibit a superior effect on
cleavage: approximately 10-fold lower concentrations were sufficient
to lead to the same activity, along with similar ds/ss ratio cleavage
(the effective concentration was 100 μM and 1000 μM, respectively)
[7]. This is in accordance with previous literature kinetic results where
SRs are reported to be more reactive compared to CRs [26]. The role of
the p-nitro-phenyl conjugate was also found to be important in both
types of radicals [4,7,27]. DNA photo cleavage of carbonyl compound
3.3. DNA Binding Studies
The interaction of SKs with CT DNA, which proves the affinity of SKs
with DNA, was examined by UV spectroscopic titrations and DNA
Fig. 1. (A) Comparative dose measurement results from DNA cleavage by the SKs derivatives 3–14 at a concentration of 100 μM. Gel electrophoresis data: Lane 1: DNA without UV irra-
diation; Lane 2: DNA with UV irradiation; Lanes 3–14: DNA + 3–14, respectively, and with UV irradiation. (B) (ss + ds)% DNA photo-cleavage caused by SKs 3–14 (100 μM). Blue column
ss%, red column ds%. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

N.-P. Andreou et al. / Journal of Photochemistry & Photobiology, B: Biology 158 (2016) 30–38
35
Fig. 2. (A) Comparative dose measurement results from DNA cleavage by the SKs derivatives 3–8 at concentration of 10 μM. Gel electrophoresis data: Lane 1: DNA without UV irradiation;
Lane 2: DNA with UV irradiation; Lanes 3–14: DNA + 3–8, respectively, and with UV irradiation; Lane 9: DNA + 15 (100 μM). (B) Comparative dose measurement results from DNA cleav-
age by the SKs derivatives 3–8 at concentration of 1 μM. Gel electrophoresis data: Lane 1: DNA without UV irradiation; Lane 2: DNA with UV irradiation; Lanes 3–14: DNA + 3–8, respec-
tively, and with UV irradiation. (C) % DNA photo-cleavage caused by SKs 3–8 (1 μM and 10 μM, dark gray and light gray, respectively). (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
0.12, the viscosity did not exhibit any appreciable change in the pres-
ence of the compounds; for higher concentrations, however, the viscos-
ity presented a significant increase.
Considering the overall changes of the DNA viscosity in the presence
of compounds 3 and 8, we may suggest that, initially, the compounds in-
teract to CT DNA probably by non-classical intercalation (i.e. groove–
binding) and at a second step they may intercalate within the DNA
base pairs [31]. This behavior may elucidate the findings from the UV
spectroscopic studies.
Fig. 3. Gel electrophoresis data. Left part: Mechanistic studies involved at the DNA
cleavage by derivative 8 (100 μM). Lane 1: DNA without UV irradiation; Lanes 2–13:
DNA with UV irradiation; Lane 3: DNA + 8; Lane 4: DNA + 8 + argon; Lane 5:
DNA + 8 + DMSO (20%); Lane 6: DNA 8 + NaN3; Lane 7: DNA + 8 + D2O. Right part:
Effect of pH. Lanes 8–13: DNA + 8 at pH 5, 6, 7, 8, 9, and 10, respectively.
The ability of the compounds to displace EB from the EB–DNA com-
plex may shed light in their intercalating ability. The competition be-
tween compounds 3 and 8 with EB for the intercalation sites of DNA
viscosity measurements and was carried out representatively for the
alkyl derivative 3 and the aryl one 8, which exhibited the best activity.
The slight hypochromism (2% for 3 and 3% for 8) observed in the UV
spectra of the compounds in the presence of increasing amounts of CT
DNA (Fig. 4) is indicative of the existing interaction.
was monitored by fluorescence emission spectroscopy with λexcitation
=
540 nm. The fluorescence emission spectra of pre-treated EB–DNA
([EB] = 20 μM, [DNA] = 26 μM) as recorded in the presence of increas-
ing amounts of compounds 3 or 8 [Fig. 6(A) and (B)] exhibit a significant
or moderate quenching [60.2% for 3 and 48% for 8, Fig. 6(C)], respective-
ly. This quenching is due to the ability of the compounds to displace EB
from the DNA–EB complex and indirectly reveals that their interaction
with CT DNA via an intercalative mode may exist [31].
The binding constant of the compounds with CT DNA (Kb) was calcu-
lated by the Wolfe–Shimer equation (Eq. (3)) [23] and plots {[DNA] /
(εA – εf)} versus [DNA] (Insets in Fig. 4). The Kb value of 3 [Kb
=
=
4.82( 0.25) × 105 M−1] was much higher than that of 8 [Kb
2.44( 0.10) × 105 M−1], suggesting that 3 exhibits higher affinity
than 8 for CT DNA. These Kb values are of the same order to that of classic
DNA-intercalator ethidium bromide (EB) (Kb = 1.23 × 105 M−1) as pre-
viously reported by our group [29].
Furthermore, the observed quenching is in good agreement (R =
0.99) with the linear Stern–Volmer equation (Eq. (4)) [24]. The values
of the Stern–Volmer constant (KSV) as calculated from the Stern–
Volmer plots (Insets in Fig. 6) are 6.07( 0.18) × 104 M−1 for 3
However, it is generally believed that it is not quite safe to propose
the exact mode of binding to CT DNA merely from UV spectroscopic ti-
tration studies [30]. Thus, the viscosity of a CT DNA solution (0.1 mM)
was monitored upon addition of increasing amounts of the compounds
3 and 8 (up to the value of r = 0.35) (Fig. 5). Initially and up to r value of
and 3.72( 0.10)
×
104 M−1 for 8; they may be considered
rather high and verify the tight binding of compounds 3 and 8 to DNA
[29,32]. Taking τo = 23 ns as the fluorescence lifetime of EB–DNA sys-
tem [33], the quenching constants of the compounds as calculated
Fig. 4. UV spectra of a DMSO solution (1 × 10−4 M) of compounds (A) 3 and (B) 8 in the presence of increasing amounts of CT DNA. The arrows show the changes upon increasing amounts
½DNAꢁ
of CT DNA. Insets: Plot of ðε
Þ versus [DNA].
A −εf

36
N.-P. Andreou et al. / Journal of Photochemistry & Photobiology, B: Biology 158 (2016) 30–38
show better activity. The reasons may be that, at least in comparable de-
rivatives, SKs show lower N–O bond energy (~5 Kcal/mol), as well as
better intercalation as it was measured in binding affinity studies.
4. Conclusions
We have shown that SRs are effective DNA photo-cleaving agents.
The herein provided experimental results evident a superiority of SRs
compared to the known CRs as DNA photo-cleavers. SK 8 had similar
ratio of ds to ss cleavage, in an approximately 10-fold lower concentra-
tion than a comparable oxime ester.
Additionally, another factor differentiating SRs from CRs, is that alkyl
SRs are very potent DNA photo-cleavers. This property was not so far
observed for the carbonyl derivatives. SAR and DNA binding affinity
studies within the 4-pyridine SK series revealed that the aliphatic SK 3
exhibited better affinity to DNA than the aromatic SK 8, but approxi-
mately the same total photo-cleavage ability, although with a lower
ratio of ds to ss cleavage.
Sulfonyl ketoximes are organic compounds easily accessible in large
scale from ketone derived oximes, and are stable in the absence of light
for prolonged periods of time. The DNA cleavage is concentration de-
pendent and occurs simply by irradiation without the need of external
additives. 4-Pyridine ethanone oxime has been used as a model com-
pound and it proved the efficacy of the SRs as novel DNA photo-
cleavage agents, in concentrations close to the nanomolar scale.
Fig. 5. Relative viscosity (η / ηo)1/3 of CT DNA (0.1 mM) in buffer solution (150 mM NaCl
and 15 mM trisodium citrate at pH 7.0) in the presence of compounds 3 and 8 at
increasing amounts (r = [compound] / [DNA]).
with Eq. (5) are kq(3) = 2.64( 0.08) × 1012 M−1 s−1 and kq(8)
=
1.62( 0.04) × 1012 M−1 s−1; the values of the kq constants are signif-
icantly higher than 1010 M−1 s−1 suggesting, thus, the quenching via a
static mechanism [34].
A current study from our group, concerning the photo-cleavage of
sulfonyl amidoximes [35], show results which are in general agreement
with the observations of the present. However, SKs presented herein
Fig. 6. Emission spectra (λexcit = 540 nm) for EB–DNA ([EB] = 20 μM, [DNA] = 26 μM) in buffer solution in the absence and presence of increasing amounts of (A) compound 3 and
(B) compound 8 (up to the value of r = 0.06). The arrows show the changes of intensity upon increasing amounts of the compounds. Insets: Stern–Volmer quenching plot of EB
bound to CT DNA for (A)
3 (equation of linear fitting: y = 6.07( 0.18) ∗ ∗ x + 0.93 (r = = 0.985)) and (B) 8 (equation of linear fitting: y =
104 0.993, R2
3.72( 0.10) ∗ 104 ∗ x + 0.95 (r = 0.994, R2 = 0.987)). (C) Plot of EB relative fluorescence emission intensity at λ = 592 nm (I/Io, %) versus r (r = [compound] / [DNA]) (150 mM
NaCl and 15 mM trisodium citrate at pH = 7.0) in the presence of compound 3 and 8 (up to 39.8% of the initial EB–DNA fluorescence intensity for 3 and 52% for 8).

N.-P. Andreou et al. / Journal of Photochemistry & Photobiology, B: Biology 158 (2016) 30–38
37
(c) P. Blom, A.X. Xiang, D. Kao, E.A. Theodorakis, Design, synthesis, and evaluation of
N-aroyloxy-2-thiopyridones as DNA Photocleaving reagents, Bioorg. Med.
Chem. 7 (1999) 727–736.
The possibility of introducing a variety of properly designed SKs
which, depending on their individual DNA interactions, could yield
highly reactive SRs, may be fruitful in the fields of pharmacy, molecular
biology and medicine. Thus, enriching the pool of possible applications
with more powerful weapons, capable of acting at various pHs and
under aerobic or anaerobic conditions, may provide novel biotechnolog-
ical and therapeutic approaches for anticancer and antimicrobial
therapies.
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We thank Assoc. Prof. A. K. Zarkadis (Univ. of Ioannina, Chemistry
Department) and Dr. P. Varras and Dr. P. Gritzapis for the calculation
of the N–O bond energies of the compounds. The authors declare no
competing financial interest.
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