Azido carbonyl compounds as DNA cleaving agents

Article abstract of DOI:10.1016/j.jphotobiol.2012.06.006

Irradiation of azido carbonyl compounds using UV light (≥310 nm) produced triplet alkyl nitrenes and aroyl radicals, which resulted in efficient cleavage of single strand DNA at pH 7.0. DNA cleaving ability of azido carbonyl compounds was found to be dependent on its concentration and substituents on its aromatic ring. Further, newly synthesized naphthalene based azido carbonyl compounds showed DNA cleavage ability at longer wavelength of UV light (≥350 nm) and also binding studies revealed that they bind to ct-DNA by weak intercalation mode.

Full text of DOI:10.1016/j.jphotobiol.2012.06.006

Journal of Photochemistry and Photobiology B: Biology 115 (2012) 25–34
Contents lists available at SciVerse ScienceDirect
Journal of Photochemistry and Photobiology B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Azido carbonyl compounds as DNA cleaving agents
Nilanjana Chowdhury, Sansa Dutta, S. Karthick, Anakuthil Anoop, Swagata Dasgupta,
⇑
N.D. Pradeep Singh
Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Irradiation of azido carbonyl compounds using UV light (P310 nm) produced triplet alkyl nitrenes and
aroyl radicals, which resulted in efficient cleavage of single strand DNA at pH 7.0. DNA cleaving ability
of azido carbonyl compounds was found to be dependent on its concentration and substituents on its aro-
matic ring. Further, newly synthesized naphthalene based azido carbonyl compounds showed DNA cleav-
age ability at longer wavelength of UV light (P350 nm) and also binding studies revealed that they bind
to ct-DNA by weak intercalation mode.
Received 17 January 2012
Received in revised form 3 May 2012
Accepted 12 June 2012
Available online 21 June 2012
Keywords:
Ó 2012 Elsevier B.V. All rights reserved.
Photoinduced DNA cleavage
Azido carbonyl compound
Triplet alkyl nitrene
Aroyl radical
DNA binding
1. Introduction
sensitizer resulted in efficient generation of triplet alkyl nitrenes
via intramolecular triplet energy transfer from ketone to azido
Aryl azide based photocrosslinking has been extensively used to
obtain information on the high order structure of protein–protein
and RNA–protein complexes [1–4]. Photoactivation of aryl azides
produces corresponding reactive singlet aryl nitrenes which isom-
erize to benzazirines and ketenimines [5–7]. At low temperature,
the singlet aryl nitrenes undergoes intersystem crossing to form
the corresponding triplet aryl nitrene [8,9]. Triplet aryl nitrenes
are known to undergo reactions like dimerization with a second
triplet nitrene or with an azide precursor to form azo dimer [8,9],
with molecular oxygen to produce nitroaryl compounds [10,11],
electron abstraction from the reducing agents [12] and hydrogen
atom abstraction from the solvent [13]. Recently, triplet 3-hydroxy
aryl nitrene have shown to induce RNA backbone cleavage based
on its hydrogen atom abstraction potential [14].
In comparison to aryl nitrenes, alkyl nitrene intermediates have
not been widely investigated. This is presumably due to the fact
that direct photolysis of alkyl azides in solution does not lead to
the formation of alkyl nitrene intermediates rather it undergoes a
concerted rearrangement from the singlet excited state of the azide
to form the imine derivatives with the loss of nitrogen [15,16]. Re-
cently, Gudmundsdottir’s [17] group for the first time showed that
photolysis of azido carbonyl compounds having in-build triplet
moiety. Further, same group also investigated the reactivity of trip-
let alkyl nitrene intermediates. In case of -azidoacetophenones,
the generated triplet alkyl nitrene were shown to be intercepted
by benzoyl radical (formed by -cleavage) to form photoproduct
I. Interestingly, triplet alkyl nitrene intermediates generated by
b-azidopropiophenones were found to undergo dimerization simi-
lar to triplet aryl nitrene, which later on cyclize to produce com-
a
a
pound II [18]. However in case of c-azidobutyrophenone, H-atom
abstraction by triplet ketone to generate ketyl radical is found to
be much more favorable compared to triplet nitrene formation
[19], the less favored triplet alkyl nitrene was shown to cyclize
to form compound III (Scheme 1).
Considering the ability of azido carbonyl compounds to effi-
ciently photogenerate triplet alkyl nitrene intermediates, together
with our recent research interest on exploring the ability of nitro-
gen centered radicals for DNA cleavage chemistry [20], made us to
explore the DNA cleaving ability of azido carbonyl compounds
using UV light.
Hence, we report for the first time
a-azidoacetophenones, b-
azidopropiophenones and -azidobutyrophenones as photoin-
c
duced DNA cleaving agents. In this paper, we discussed in detail
the synthesis and DNA cleaving ability of the above azido carbonyl
compounds. The effect of substituents on the aromatic ring of azido
carbonyl compounds on their DNA cleaving ability was assessed.
Further, we also showed naphthalene based azido carbonyl com-
pounds to act as DNA cleaving agents at longer wavelength of UV
light (P350 nm) and also studied their DNA binding behavior.
⇑
Corresponding author. Tel.: +91 3222 282324; fax: +91 3222 282252.
E-mail addresses: swagata@chem.iitkgp.ernet.in (S. Dasgupta), ndpradeep@
chem.iitkgp.ernet.in (N.D. Pradeep Singh).
1011-1344/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotobiol.2012.06.006

26
N. Chowdhury et al. / Journal of Photochemistry and Photobiology B: Biology 115 (2012) 25–34
O
N3
n
(n = 1,2 and 3)
intramolecular
energy transfer
hν
λ ≥ 310nm
O
N
O
O
H
N
(n = 2)
(n = 1)
NO2
n
I
O
bubbling with
oxygen
interception by
IV
benzoyl radical
(n = 3)
(n = 2)
dimerisation
intramolecular
cyclisation
Ph
O
N
N
Ph
Ph
N
III
II
Scheme 1. Photochemistry of azido carbonyl compounds.
2.3. General procedure for the synthesis of a-azidoacetophenones
2. Materials and methods
(3a–g)
2.1. General
Without further purification,
a-bromoacetophenone 2a (1.8 g,
6.02 mmol) was dissolved in acetone (15 mL) and solid NaN3
(0.5 g, 7.69 mmol) dissolved in water (3 mL) added to the reaction
mixture. The reaction mixture was stirred for 30 min. After the com-
pletion of the reaction, acetone was evaporated, diluted with EtOAc
and washed with water. The organic layer was separated and dried
with Na2SO4.The solvent was evaporated and purified by column
All reactions were conducted using oven-dried glassware under
an atmosphere of Argon (Ar). Commercial grade reagents were
used without further purification. Solvents were dried and distilled
following usual protocols. Flash chromatography was carried out
using Rankem Silica gel (230–400 mesh) purchased from Rankem,
India. TLC was performed on aluminum-backed plates coated with
Silica gel 60 with F254 indicator (Merck). The 1H NMR spectra were
measured with Bruker-200 (200 MHz), Bruker-400 (400 MHz) and
13C NMR spectra were measured with Bruker-200 (50 MHz),
Bruker-400 (100 MHz) using CDCl3. 1H NMR chemical shifts are ex-
pressed in parts per million (d) downfield to CDCl3 (d = 7.26), 13C
NMR chemical shifts are expressed in parts per million (d) relative
chromatography using pet ether-ethyl acetate (98:2) yielded
doacetophenone (3a). The above mentioned procedure was carried
out for the synthesis of substituted -azidoacetophenones (3b–g)
from their corresponding -bromoacetophenones.
a-azi-
a
a
2.4. General procedure for the synthesis of b-bromopropiophenones
(5a–f)
to the central CDCl3 resonance (d = 77.0). Coupling constants in 1
H
NMR are in Hz. Mass spectra were analyzed by Waters LCT mass
spectrometer. UV/vis absorption spectra were recorded on a
Shimadzu UV-2450 UV/vis spectrophotometer. IR data was deter-
mined on a Perkin–Elmer Spectrum One spectrometer (ATR tech-
nique). Melting points were measured in Reico (India) melting
point apparatus. pBR 322 DNA was used. Photolysis was carried
out using 125 W medium pressure mercury lamp supplied by SAIC
(India).
3-Bromopropionyl chloride (2.1 g, 12.25 mmol, 1.0 equiv), ben-
zene (5.4 mL, 61.25 mmol, 5.0 equiv) and dry dichloromethane
(15 mL) was taken in a round bottom flask under nitrogen atmo-
sphere. The solution was cooled to 0 °C and aluminum chloride
(1.8 g) was added in one portion. The resulting suspension was
stirred for 3 h at 25 °C. The reaction was quenched by pouring
the orange colored solution into an ice/water mixture. Dichloro-
methane was used to extract the desired product, and the organic
layer was washed with brine (10 mL), dried with Na2SO4, and con-
centrated in vacuo. The desired product (5a) was purified via col-
umn chromatography using pet ether-ethyl acetate (98:2).
Similar procedure was followed for the synthesis of other b-
bromopropiophenones (5b–f).
2.2. General procedure for the synthesis of a-bromoacetophenones
(2a–g)
Acetophenone 1a (1.0 g, 8.3 mmol) was dissolved in 20 mL
diethyl ether in a two neck round bottom flask and recrystallised
NBS (2.9 g, 16.8 mmol) was added portion wise. The reaction was
irradiated using Philips HPL-N (250 W, k = 200–600 nm) lamp fit-
ted with a water circulation arrangement at room temperature un-
der nitrogen atmosphere. After completion of the reaction, the
reaction mixture was filtered, diluted with ether and washed with
water. The organic layer was separated, dried with Na2SO4 and the
2.5. General procedure for the synthesis of b-azidopropiophenones
(6a–f)
The 3-bromopropiophenone 5a (2 g, 9.38 mmol) was dissolved
in DMF (20 mL) and solid NaN3 (0.73 g, 11.2 mmol) was added
and heated up to 50 °C and stirred for 2 h. After completion of
the reaction, the reaction mixture was diluted with EtOAc and
washed with water. The organic layer was separated and dried
solvent was removed under vacuum to yield
none (2a). Similar procedure was followed for the synthesis of
substituted -bromoacetophenones (2b–g).
a-bromoacetophe-
a

N. Chowdhury et al. / Journal of Photochemistry and Photobiology B: Biology 115 (2012) 25–34
27
with Na2SO4.The solvent was evaporated and purified by column
chromatography using pet ether-ethyl acetate (95:5) yielded b-azi-
dopropiophenone (6a). The above mentioned procedure was
carried out for the synthesis of substituted b-azidopropiophenones
(6b–f).
100
80
60
40
20
0
2.6. General procedure for the synthesis of c-chlorobutyrophenone
(7a–e)
4-Chlorobutyryl chloride (2.5 mL, 21.25 mmol, 1.0 equiv), ben-
zene (10 mL, 106.25 mmol, 5.0 equiv) and dry dichloromethane
(30 mL) was taken in a round bottom flask under nitrogen atmo-
sphere. The solution was cooled to 0 °C and aluminum chloride
(3.3 g) was added portion wise at ice cooled condition. The result-
ing suspension was stirred for 3 h at 25 °C. The reaction was
quenched by pouring the orange colored solution into an ice/water
mixture. Dichloromethane was used to extract the desired product,
and the organic layer was washed with brine (10 mL), dried with
Na2SO4, and concentrated in vacuo. The desired product (7a) was
purified via column chromatography using eluent pet ether-ethyl
acetate (98:2). Similar procedure was followed for the synthesis
0
50
100
150
200
250
Concentration
Fig. 1. Concentration study of 6d on the DNA cleaving ability in
phosphate buffer (pH 7.0) upon irradiation using P310 nm UV light for 45 min.
a sodium
of substituted c-chlorobutyrophenones (7b–e).
2.10. Comparison of DNA cleaving ability between a, b and c -azido
carbonyl compounds (3d, 6d and 8d)
2.7. General procedure for the synthesis of c-azidobutyrophenone
(8a–e)
The reaction mixtures (20
pBR322 DNA stock solution (form I, 62.5
6d and 8d) at 250 M, sodium phosphate buffer (10 mM, pH 7.0),
and 10% DMSO in a eppendrof were irradiated separately with
UV light (P310 nm, 125 W medium pressure mercury lamp) under
aerobic conditions at room temperature for 45 min. Bromophenol
blue was added to the reaction mixture and loaded on 1% agarose
gel, and then electrophoresis was carried out. The gel was visual-
ized by GEL LOGIC 200 Imaging System. The % of form II of com-
pound 3d, 6d and 8d was plotted.
l
L) containing supercoiled circular
The 4-chlorobutyrophenone 7a (2.5 g, 12.6 mmol) was dis-
solved in DMF (20 mL) and solid NaN3 (2.1 g, 25.2 mmol) was
added and heated up to 50 °C and stirred for 2 h. After completion
of the reaction, the reaction mixture was diluted with EtOAc and
washed with water. The organic layer was separated and dried
with Na2SO4.The solvent was evaporated and purified by column
chromatography using pet ether-ethyl acetate (95:5) yielded
lg/mL), compound (3d,
l
c-azidobutyrophenone (8a). The above mentioned procedure was
carried out for the synthesis of other
c-azidobutyrophenones
(8b–e).
2.11. Synthesis of
a, b and
c-azidonapthonones (10a–c)
2.8. Procedure for DNA cleaving ability of 6d at different
Concentrations
We have synthesized
a
, b and -azidonapthonones (10a–c)
c
similar way as described for the phenyl based azido carbonyl
compounds.
The reaction mixtures (20
pBR322 DNA stock solution (form I, 62.5
at different concentration (25–250 M) individually, sodium phos-
phate buffer (10 mM, pH 7.0), and 10% DMSO in a eppendrof were
irradiated with UV light (P310 nm, 125 W medium pressure mer-
cury lamp) under aerobic conditions at room temperature for
45 min. After addition of bromophenol blue to the reaction mix-
ture, gel electrophoresis was carried out and visualized by GEL LO-
GIC 200 Imaging System. Based on the above result we plotted % of
form II vs concentration of compound 6d (Fig. 1).
l
L) containing supercoiled circular
lg/mL), compounds (6d)
l
2.12. DNA cleavage study of naphthalene based azido carbonyl
compounds (10a, 10b and 10c)
The reaction mixtures (20
pBR322 DNA stock solution (form I, 62.5
(10a–c) (250 M), Sodium phosphate buffer (10 mM, pH 7.0), and
10% DMSO in eppendrof were irradiated with UV light
l
L) containing supercoiled circular
l
g/mL), compounds
l
a
(P310 nm, 125 W medium pressure mercury lamp) under aerobic
conditions at room temperature for 45 min. The sample buffer con-
taining bromophenol blue in glycerol was added to the reaction
mixture and loaded on 1% agarose gel. After addition of the tank
buffer (1X TAE), the electrophoresis apparatus was attached to a
power supply. The gel was visualized by GEL LOGIC 200 Imaging
System.
2.9. General procedure for photoinduced DNA cleavage by
a-azidoacetophenones (3a–g), b-azidopropiophenones (6a–f) and
c
-azidobutyrophenone (8a–e)
The reaction mixtures (20
pBR322 DNA stock solution (form I, 62.5
(3a–g, 6a–f and 8a–e) (250
l
L) containing supercoiled circular
g/mL), compounds
M), Sodium phosphate buffer
l
l
(10 mM, pH 7.0), and 10% DMSO in a eppendrof were irradiated
with UV light (P310 nm, 125 W medium pressure mercury lamp)
under aerobic conditions at room temperature for 45 min. The
sample buffer containing bromophenol blue in glycerol was added
to the reaction mixture and loaded on 1% agarose gel. After addi-
tion of the tank buffer (1X TAE), the electrophoresis apparatus
was attached to a power supply. The gel was visualized by GEL LO-
GIC 200 Imaging System.
2.13. Fluorimetric titration of azido naphthone compounds (10a, 10b
and 10c)
Fluorimetric titrations were performed in a 3 cm quartz cuvette
using an excitation wavelength of 340 nm having emission from
370 to 650 nm. The titration experiments were performed with
fixed concentrations of the compound (10a, 100
lM), with gradual
increasing the concentration of ct-DNA. The fluorescence of azido

28
N. Chowdhury et al. / Journal of Photochemistry and Photobiology B: Biology 115 (2012) 25–34
napthone compound quenched upon binding to DNA and increas-
ing concentration of DNA resulted in decrease in fluorescence
intensity of azido napthone compounds, which indicated binding
interaction of the compound with DNA. A quantitative estimation
of quenching experiments in term of the binding constant calcula-
tion is obtained from Stern Volmer equation
3. Results and discussion
3.1. Synthesis and UV/vis spectra of
b-azidopropiophenones (6a–f) and
a
-azidoacetophenones (3a–g),
c-azidobutyrophenones (8a–e)
We have synthesized three series of derivatives of azido car-
bonyl compounds as shown in Scheme 2. The series 1, series 2
and series 3 consists of
nones and -azidobutyrophenones respectively.
The azido carbonyl compounds of series 1 were synthesized by
carrying out -bromination of substituted acetophenones (1a–g)
using N-Bromosuccinimide (NBS) under photolytic condition
[21]. The freshly prepared -bromoacetophenones (2a–g) were
then treated with sodium azide in acetone–water at room temper-
ature to form -azidoacetophenones (3a–g) [22]. While in the case
a-azidoacetophenones, b-azidopropiophe-
logðF0 ꢀ FÞ=F ¼ log Kb þ n log½DNAꢁ
c
where, Kb and n are the binding constant and the number of binding
sites, [DNA] = concentration of DNA, and F0, and F is the fluores-
cence intensities in absence and presence of DNA. From the linear
plot of log (F0 ꢀ F)/F vs log DNA, the binding constant (Kb) value
was calculated.
a
a
a
of series 2, b-bromopropiophenones (5a–f) were synthesized by
carrying out Friedel–Craft acylation of substituted arene (4a–f)
using 3-bromopropionyl chloride [23]. Later, treatment of 5a–f
with sodium azide in DMF provided the corresponding b-azidopro-
2.14. Ethidium bromide (EB) displacement assay of azido naphthone
compounds (10a, 10b and 10c)
piophenones (6a–f) [24]. The
c-azidobutyrophenones of (8a–e)
series 3 were synthesized by treatment of
c-chlorobutyrophenones
Fluorimetric titrations were performed in a 3 cm quartz cuvette
with NaN3 in DMF [24]. All the azido carbonyl compounds were
synthesized in moderate to good yields and further characterized
by 1H, 13C NMR, IR and mass spectral analysis.
The UV/vis absorption spectra (see Figs. 1–3s given in support-
ing information) of degassed 1.2 ꢃ 10ꢀ5 M solution of azido car-
bonyl compounds 3a–g, 6a–f and 8a–e in methanol were
recorded and the results were summarized in Table 1. The results
showed that all azido carbonyl compounds exhibited moderate
to strong absorption P310 nm, thus make them suitable for DNA
cleavage.
using an excitation wavelength of 480 nm. 3 mL of 10
lM ct-DNA
was saturated with 1 M of ethidium bromide (EB) solution in
l
10 mM phosphate buffer pH 7.0 containing 50 mM NaCl. The EB-
ct-DNA solution was then titrated by successive addition of com-
pounds 10a, 10b and 10c to reach a final concentration of
ꢂ32
lM for all the compounds. Emission spectra were recorded
from 500 nm to 750 nm. The fluorescence spectra of EB were mea-
sured using an excitation wavelength of 480 nm and the emission
range was set between 500 and 750 nm. The spectra were analyzed
according to the classical Stern–Volmer equation
F0=F ¼ 1 þ Ks ½Qꢁ
v
3.2. DNA cleaving ability of
a-azidoacetophenones (3a–g),
b-azidopropiophenones (6a–f) and
c
-azidobutyrophenones (8a–e)
where F0 and F are the fluorescence intensities at 608 nm in the ab-
sence and presence of the quencher, respectively, Ksv is the linear
Stern–Volmer quenching constant, [Q] is the concentration of the
quencher.
To find out the optimum concentration required for efficient
DNA cleavage by the azido carbonyl compounds, initially we car-
ried out irradiation of circular supercoiled pBR322 plasmid DNA
series 1: α-azidoacetophenones
O
O
O
NBS, Et2O
NaN3
N3
Br
Ar
Ar
Ar
1a-g
Me2CO-H2O
3a-g
hν
2a-g
Ar = Ph (3a; 85%), 2-OHC6H4 (3b; 90%), 2-BrC6H4 (3c; 92%), 3-NO2C6H4 (3d; 80%),
4-OMeC6H4 (3e; 90%), 3,4-OMeC6H3 (3f; 82%), 4-MeC6H4 (3g; 80%)
series 2:β-azidopropiophenones
O
O
O
Br
Cl
NaN3
ArH
4a-f
Ar
Br
Ar
N3
DMF, 50 °C
AlCl3 , DCM
5a-f
6a-f
Ar = Ph (6a; 75%), 4-BrC6H4 (6b; 82%), 4-OHC6H4 (6c; 80%), 3,4-OMeC6H3 (6d; 75%),
4-OMeC6H4 (6e; 80%), 4-MeC6H4 (6f;82%)
series 3
:
γ
-azidobutyrophenones
O
O
O
Cl
NaN3
N3
Cl
Ar
Cl
Ar
ArH
4a-e
DMF, 50 °C
AlCl3 , DCM
8a-e
7a-e
Ar= Ph (8a; 85%), 4-MeC6H4 (8b; 80%), 4-OMeC6H4 (8c; 75%), 3,4-OMeC6H3 (8d; 80%),
4-BrC6H4 (8e; 82%)
Scheme 2. Synthesis of a-azidoacetophenones (3a–g), b-azidopropiophenones (6a–f) and c- azidobutyrophenones (8a–e).

N. Chowdhury et al. / Journal of Photochemistry and Photobiology B: Biology 115 (2012) 25–34
29
Table 1
Single-strand cleavage of supercoiled circular pBR322 DNA (form I) to relaxed circular DNA (form II) by photolysis of compounds (3a–g, 6a–f 8a–e) under aerobic conditions at
room temperature using UV light P 310 nm for 45 min.
Entry
Substrate
log
e
at 310 nm
Remaining amount of azide after
photolysis for 90 min (%)
Relative efficiency of product
Form I (%)
Form II (%)
formation K (10ꢀ3
)
1
2
3
4
5
6
7
8
3a
3b
3c
3de
3e
3f
3g
6a
6b
6c
6d
6e
6f
8a
8b
8c
8d
8e
6d
6d
6d
6d
2.81
3.18
2.80
2.88
3.18
3.87
3.03
2.84
2.92
3.20
3.88
3.12
2.56
2.82
3.28
3.22
4.06
3.03
3.88
3.88
3.88
3.88
61.9
57.4
65.8
92.5
47.0
44.5
50.1
59.2
63.9
57.8
44.2
45.5
48.8
64.4
55.5
60.0
62.5
89.2
–
5.3
6.2
4.5
–
38
35
66
90
10
7
18
37
82
35
3
62
65
34
9
8.5
9.0
7.7
5.7
4.9
6.2
9.0
8.8
8.1
4.9
6.5
5.5
5.3
3.9
–
89
92
82
63
18
65
97
94
92
14
56
35
27
12
26
28
97
97
9
10
11
12
13
14
15
16
17
18
19a
20b
21c
22d
6
8
86
45
65
73
88
74
72
2
–
–
–
–
–
–
2
a
b
c
DNA + compound 6d + ascorbic acid.
DNA + compound 6d + -cysteine.
DNA + compound 6d + (0.1 M) NaN3 solution.
DNA + compound 6d bubbling with argon gas.
L
d
e
Rate constant of compound 3d was not determined as the reaction is very slow.
(form I; 62.5
for a period of 45 min using different concentrations of 3,4-dime-
thoxy-b-azidopropiophenone (6d, 25–250 M) dissolved in a so-
dium phosphate buffer (pH 7.0, 10 mM) with 10% DMSO. The
results from gel electrophoresis on 1.0% agarose gel with ethidium
bromide staining indicated that 6d caused the single strand cleav-
age of supercoiled DNA (form I, SC) to give the relaxed circular DNA
(form II, NC). We noticed that DNA cleaving ability of 6d enhanced
with the increase of its concentration. For example the generated
l
g/mL) at room temperature under aerobic condition
of nicked (NC) DNA (Form II) was observed (Fig. 2, lane 9), whereas
the generated Form II DNA was found to be 97% (Fig. 2, lane 14 and
Table 1, entry 11) with 45 min irradiation of UV light, which clearly
suggest that UV light is key to initiate the DNA scission process.
To investigate the possibility of DNA cleavage by singlet oxygen,
l
we carried out the irradiation of DNA with 6d (250 lM) by adding
sodium azide (100 mM) (scavenger of singlet oxygen) as reported
in the literature [25,26] and the generated form II was found to
be 97 % (Fig. 3, lane 10 and Fig. 4) which is almost similar to the
ratio obtained for 6d under aerobic condition, which rules out
the involvement of singlet oxygen in DNA cleavage [27]. To con-
firm, we further increased the amount of NaN3 up to 500 mM
and we noticed the percentage of form II DNA remained almost
same.
form II was 5% at 50
lM whereas it was 42% and 69% at 150 and
200 M concentration respectively. Based on the above result we
l
plotted % of form II vs concentration of compound 6d (Fig. 1).
Based on above concentration study, the DNA cleaving ability of
all azido carbonyl compounds were investigated using 250 lM
concentration for a period of 45 min of irradiation and the results
were presented in Table 1. Results of control experiments were
also summarized in Table 1(entries 11, 19, 20, 21, 22).
Initially, to demonstrate the requirement of UV light to induce
DNA cleavage by the azido carbonyl compounds, we carried out
Further to understand the effect of molecular oxygen on the
DNA cleavage ability of azido carbonyl compounds, we carried
out the irradiation of DNA with compound 6d in argon saturated
solution (argon was bubbled to remove dissolved oxygen for a per-
iod of 30 min). Interestingly, the generated Form II DNA was found
to be 97% (Fig. 3, lane 11 and Fig. 4, Table 1, entry 22) in argon sat-
urated solution which is similar to the % of form II (97%) obtained
in case of aerobic condition (Table 1, entry 11). The above experi-
ment indicates that molecular oxygen did not affect the DNA cleav-
ing ability of azido carbonyl compounds, since triplet alkyl nitrenes
and aroyl radicals formed on photolysis of azido carbonyl
compounds are known to react with molecular oxygen [17,19].
the control experiment by treating compound 6d (250 lM) with
supercoiled (SC) DNA (Form I) in the dark for 45 min. No formation
Fig. 2. Single strand cleavage of supercoiled circular pBR322 DNA (form I) to
relaxed circular DNA (form II) was carried out by irradiation of
a-azido carbonyl
compounds (3a–g, 6a–f) (250 M) with P310 nm UV light in a sodium phosphate
l
buffer (pH 7.0) under aerobic condition at room temperature for 45 min. The
resultant products were subjected to electrophoresis on 1% agarose gel followed by
ethidium bromide staining under UV light. lane 1, DNA alone; lane 2, DNA + 3a; lane
3, DNA + 3b; lane 4, DNA + 3c; lane 5, DNA + 3d; lane 6, DNA + 3e; lane 7, DNA + 3f;
lane 8, DNA + 3g; lane 9, DNA + 6d in dark; lane 10, DNA irradiated; lane 11,
DNA + 6a; lane 12, DNA + 6b; lane 13, DNA + 6c; lane 14, DNA + 6d; lane 15,
DNA + 6e; lane 16, DNA + 6f.
Fig. 3. Lane 1, DNA alone; lane 2, DNA + 8a; lane 3, DNA + 8b; lane 4, DNA + 8c; lane
5, DNA + 8d; lane 6, DNA + 8e; lane 7, DNA irradiated; lane 8, DNA + ascorbic
acid + 6d; lane 9, DNA +
L-cysteine + 6d; lane 10, DNA + NaN3 + 6d; lane 11
DNA + compound 6d bubbling with Ar.

30
N. Chowdhury et al. / Journal of Photochemistry and Photobiology B: Biology 115 (2012) 25–34
100
90
80
70
60
50
40
30
20
10
0
pared to other azido carbonyl compounds. Among series 1 (a azide)
and series 2 (b azide), electron rich methoxy substituted azides 3e,
3f, 6d and 6e exhibited efficient DNA cleaving ability. On the other
hand, azido carbonyl compounds with electron poor bromo and ni-
tro substituted azides 3c, 6b and 3d showed lesser DNA cleaving
ability. For the series 3 compounds (c azide), we have found almost
% of SC
% of NC
similar observation, where methoxy substituted azide 8c and
methyl substituted azide 8b showed better DNA cleavage ability.
To understand the effect of substituents on the DNA cleaving
ability of azido carbonyl compounds, we studied the influence of
substituents on the reactivity of azido carbonyl compounds.
Benzene solutions containing azido carbonyl compounds
(3 ꢃ 10ꢀ3 M) were irradiated using UV light (P310 nm) for
90 min and the decomposition of the starting material was fol-
lowed by NMR spectroscopy at the interval of 30 min by using
1,2-dichloroethane as internal standard. From the decomposition
of the starting material from the NMR spectroscopy, we have plot-
ted ln C0/Ct vs time (t) and calculated the relative efficiency of
product formation (K) for the above compounds (details of relative
efficiency of product formation (K) given in the supporting infor-
mation, Figs. 4–6s). From the K values (Table 1) it is clear that for
1
2
3
4
5
6
Fig. 4. Single strand cleavage of supercoiled circular pBR322 DNA (form I) to
relaxed circular DNA (form II) was carried out by irradiation of -azido carbonyl
compound 6d (250 M) in presence of different additives with UV light (P310 nm)
in a sodium phosphate buffer (pH 7.0) under aerobic condition at room temperature
for 45 min. 1. DNA + 6d in dark; 2. DNA + 6d + ascorbic acid; 3. DNA + 6d +
a
l
L
-cysteine; 4. DNA + 6d+(0.1 M) NaN3; 5. DNA + 6d + bubbling with argon gas; 6.
a given series (a, b or c) of compounds, electron rich azides were
DNA + 6d + aerobic condition.
found to show better reactivity with respect to unsubstituted or
electron poor azides. The trends of reactivity order of azides were
found to be closely related to the DNA cleavage ability.
Higher reactive substrates were found to show high efficiency
in DNA scission process. The better reactivity of azido carbonyl
compounds having electron donating substituents can be probably
attributed due to two main reasons (i) higher molar absorption
Irradiation of supercoiled DNA with 6d in presence of ascorbic acid
and L-cysteine reduced the formation of form II DNA to 25% and
28% respectively, which indicates the generated radicals from 6d
were quenched and resulted in the poor DNA scission process
(Fig. 3, lane 8 and 9 and Fig. 4).
Finally, to investigate the role of aryl ketone in DNA cleavage
process, we carried out the DNA cleavage study of acetophenone,
coefficient (log e) of azido carbonyl compounds having electron
donating substituents at P310 nm compared to electron with-
drawing group (see Table 1), thus enabling them to absorb light
much more efficiently (ii) increase in the rate of intersystem cross-
ing (ISC) by electron donating substituents of azido carbonyl com-
pounds results in efficient generation of triplet alkyl nitrene
intermediate [28]. Further to support the above argument we cal-
p-methyl acetophenone with their corresponding
a-azido carbonyl
compounds 3a and 3g using 250 M concentration for a period of
l
45 min irradiation, individually as described earlier. The gel elec-
trophoresis results revealed that acetophenone and p-methyl ace-
tophenone showed poor DNA cleaving ability compared to their
corresponding azidocarbonyl compounds 3a and 3g (Fig. 5).
culated the excited-state energies of
a-azidoacetophenones (see
Table 2s supporting information) using TDDFT (B3LYP) with tri-
ple-zeta quality basis set (def2-TZVP). From the energy calculation,
it was found that, the substituent on the aromatic ring has great
influence on the singlet and triplet energy of azido carbonyl com-
pounds. In particular, singlet triplet energy gap is found to be smal-
3.3. Effect of substituents on the DNA cleaving ability of azido carbonyl
compounds
Analytical results from the gel electrophoresis (Table 1) showed
that substituents on the aromatic ring of azido carbonyl com-
pounds have influence on their DNA cleaving ability. We noticed
azido carbonyl compounds having electron donating substituent
on the aromatic ring exhibited better DNA cleaving ability com-
ler for
a-azidoacetophenones containing electron donating
substituents p-methoxy (3e), p-methyl (3g) and o-hydroxy (3b)
in compared to electron withdrawing substituent (m-nitro 3d).
Apart from the above two main reasons, the effect of substituents
on the electronic configuration of the excited ketone in azido car-
bonyl compounds also needs to be considered. Since, in case of
a
-azido acetophenone with electron donating substituent, the (
p,
pꢄ) triplet state is lower in energy than (n, pꢄ) triplet state and thus
80
70
60
50
40
30
20
10
0
facilitates the formation of triplet nitrene compared to
reaction [29].
a-cleavage
Interestingly, azido carbonyl compounds 3b and 6c having elec-
tron donating hydroxy substituent with good molar absorption
coefficient at P310 nm showed inferior DNA cleaving ability com-
pared to electron withdrawing substituent, since the hydroxy azi-
do carbonyl compounds can undergo phototautomerisation by
excited state intramolecular proton transfer reaction (ESIPT) simi-
lar to o-hydroxyacetophenone [30] rather than undergoing intra-
molecular triplet energy transfer to form triplet alkyl nitrenes.
3.4. Comparison of DNA cleaving ability of a, b and c azido carbonyl
compounds (3f, 6d, 8d)
p-Me acetophenone
acetophenone
3g
3a
To compare the DNA cleaving ability among the
a, b and c azido
Fig. 5. Comparision study between aryl ketones and
a-azidocarbonyl compounds.
carbonyl compounds, we irradiated dimethoxy substituted azides

N. Chowdhury et al. / Journal of Photochemistry and Photobiology B: Biology 115 (2012) 25–34
31
100
90
80
70
60
50
40
30
20
10
0
% of form II
Fig. 7. Single strand cleavage of supercoiled circular pBR322 DNA (form I) to
relaxed circular DNA (form II) was carried out by irradiation of -azido carbonyl
compounds 10a–c (250 M) with UV light (P310 nm) in a sodium phosphate
buffer (pH 7.0) under aerobic condition at room temperature for 45 min. The
resultant products were subjected to electrophoresis on 1% agarose gel followed by
ethidium bromide staining under UV light. lane 1, DNA alone; lane 2, DNA + 10a;
lane 3, DNA + 10b; lane 4, DNA + 10c.
a
l
3f
6d
8d
experiments were carried out using similar condition as described
for phenyl based azido carbonyl compounds keeping the irradia-
tion wavelength P350 nm. The results from gel electrophoresis
on 1.0% agarose gel with ethidium bromide staining indicated that
azido carbonyl compounds 10a, 10b and 10c caused the single
strand cleavage of DNA to give the relaxed circular DNA (Fig. 7
and Table 2).
Fig. 6. Comparison of
a
, b and
c
azido carbonyl compounds.
3f, 6d and 8d with DNA in presence of UV light for 45 min. We
noted that b azido carbonyl compound showed better DNA cleav-
ing ability compared to
a and c azido carbonyl compounds
(Fig. 6). The generated form II DNA was found to be 97% for dime-
thoxy substituted b-azide 6d (Table 1, entry 11) whereas for dime-
thoxy substituted
entry 6) and 27% (Table 1, entry 17) respectively.
a and c-azides 3f and 8d; it was 92% (Table 1,
3.7. DNA Binding Study of naphthalene based azido carbonyl
compounds (10a, 10b and 10c)
3.5. Synthesis and UV/vis spectra of a, b and c-azidonapthonones
3.7.1. Fluorimetric titration
(10a–c)
In order to understand the binding properties of azido carbonyl
compounds with ct-DNA, fluorometric titration was carried out by
addition of ct-DNA to a fixed concentration of compound 10a in
buffer solution. The fluorescence emission spectra are given in
Fig. 8 in absence and presence of ct-DNA. Compound 10a showed
an emission maximum at 475 nm in sodium phosphate buffer
(pH 7.0) solution on excitation at 340 nm (absorption maximum).
Characteristic changes were observed in fluorescence emission
spectra during the titration of compound 10a with ct-DNA. There
was a decrease in the fluorescence intensity of compound 10a,
compared to its free state, with the increase concentration of ct-
We have found that phenyl based azido carbonyl compounds
acts as efficient DNA cleaving agents. Hence, we were interested
to carry out DNA cleavage at longer wavelength of UV light
(P350 nm). For that purpose, we synthesized
derived azidocarbonyl compounds (Scheme 3).
a, b, c-naphthalene
The UV/vis absorption spectra of degassed 1.2 ꢃ 10ꢀ5 M solution
of azido carbonyl compounds 10a, 10b and 10c in methanol were
recorded and the results were given in supporting information
(Fig. 7s in the supporting information). The UV–vis spectra of these
compounds showed that they have moderate absorption P350 nm.
DNA (0–100 lM), which indicated the binding interaction of com-
pound 10a with ct-DNA. The binding constant (Kb) for the complex
formation was calculated from the plot of log (F0 ꢀ F)/F vs log
(DNA ꢃ 10ꢀ6), and was found to be 4.5 ꢃ 102 Mꢀ1. Similar way
the binding constants of compound 10b and 10c were calculated
and it was found to be 5.2 and 3.8 ꢃ 102 Mꢀ1 respectively (see
Table 3).
3.6. DNA cleavage ability of naphthalene based azido carbonyl
compounds (10a, 10b and 10c)
To investigate the DNA cleaving ability of naphthalene based
azido carbonyl compounds 10a, 10b and 10c, the DNA cleavage
O
O
N3
Br
i) AcCl, AlCl3, 0°C
NaN3
Me2CO-H2O
ii) NBS, Et2O, h
ν
9a
10a; 75%
O
Br
O
N3
O
Br
Cl
NaN3
AlCl3, DCM, 0°C
DMF, 50°C
8
9b
10b; 72%
O
O
N3
Cl
AlCl3, DCM, 0°C
NaN3
DMF, 50°C
Cl
Cl
9c
10c; 82%
O
Scheme 3. Synthesis of a, b and c-azidonapthonones (10a–c).

32
N. Chowdhury et al. / Journal of Photochemistry and Photobiology B: Biology 115 (2012) 25–34
Table 2
Single-strand cleavage of supercoiled circular pBR322 DNA (form I) to relaxed circular DNA (form II) by photolysis of compounds (10a–c) under aerobic conditions at room
temperature using UV light P 350 nm for 45 min.
Entry
Substrate
log
e
at 350 nm
Remaining amount of azide after
photolysis for 90 min (%)
Form I (%)
Form II (%)
1
2
3
10a
10b
10c
3.13
3.14
3.12
70.1
75.1
92.0
45
20
85
55
80
15
140
120
100
80
-1.4
-1.6
-1.8
-2.0
-2.2
-2.4
-2.6
Kb = 4.6 X 102
60
40
20
0
400
450
500
550
600
650
-5.0
-4.8
-4.6
-4.4
-4.2
-4.0
-6
Wavelength (nm)
log (DNA X 10 )
Fig. 8. Fluorescence emission spectra of compound 10b (100
lM) in 10 mM phosphate buffer, pH 7.0 containing 50 mM NaCl with increasing concentration of ct-DNA. The
DNA concentrations were from top to bottom (0–100
lM). The excitation wavelength, kex, was 340 nm.
3.7.2. Ethidium bromide displacement study
Table 3
To ascertain the mode of binding of azido naphthones to the calf
thymus DNA (ct-DNA) competitive EB displacement assay was per-
formed using 10a, 10b and 10c. The fluorescence intensity of ethi-
dium bromide (EB), a well-known classical intercalator is enhanced
upon complexation with the DNA [32,33]. As the concentration of
azido napthones increased, we observed fluorescence quenching of
EB DNA complex, indicating that azido naphthones partially dis-
places the ethidium bromide via weak intercalative mode (Figs. 9
and 10).
The quenching of EB bound to ct-DNA by the above compounds
was in good agreement with the linear Stern–Volmer equation [34],
which provides further evidence that the azido naphthone com-
pounds bind to DNA. The ksv value of compound 10a was
3.0 ꢃ 103 Mꢀ1 and for 10b it was found to be 4.6 ꢃ 103 Mꢀ1. How-
ever the quenching is more prominent in the case of 10b compared
to 10a. The quenching pattern of compound 10c (Kb = 4.3 ꢃ
Calculated binding constant (Kb) and Stern–Volmer constant (Ksv) for compound 10a,
10b and 10c.
Compound
Kb (Mꢀ1
)
Ksv (Mꢀ1
)
10a
10b
10c
4.5 ꢃ 102
5.2 ꢃ 102
3.8 ꢃ 102
3.0 ꢃ 103
4.6 ꢃ 103
4.3 ꢃ 103
The binding constants of compound 10a, 10b and 10c were
compared with the well known classical intercalating agent meth-
ylene blue, acridine orange and ethidium bromide reported in lit-
erature [31] and it was found that the Kb value of azido
naphthones was two orders magnitude smaller than the reported
Kb values of classical intercalators, which indicated the weak inter-
calation binding of naphthalene based azidocarbonyl compounds
with DNA.
250
200
150
100
50
1.12
3
Ksv = 3.0 X 10 M-1
1.10
1.08
1.06
1.04
1.02
1.00
0
500
0
5
10
15
20
25
30
35
550
600
650
700
750
6
[Concentration] X 10
Wavelength (nm)
Fig. 9. Fluorescence emission spectra of EB (dotted), EB bound to DNA (top solid line) and quenching of EB bound DNA by 10a in 10 mM phosphate buffer, pH 7.0 containing
50 mM NaCl. [EB] = 2 M; [DNA] = 20 M; [10a] = 3–32 M, kex = 480 nm, kem = 608 nm.
l
l
l

N. Chowdhury et al. / Journal of Photochemistry and Photobiology B: Biology 115 (2012) 25–34
33
1.2
250
200
150
100
50
Ksv = 4.6 X 103 M-1
1.1
1.0
0
500
550
600
650
700
750
0
5
10
15
20
25
30
35
6
Wavelength (nm)
[concentration] X 10
Fig. 10. Fluorescence emission spectra of EB (dotted), EB bound to DNA (top solid line) and quenching of EB bound DNA by 10b in 10 mM phosphate buffer, pH 7.0 containing
50 mM NaCl. [EB] = 2 M; [DNA] = 20 M; [10b] = 3–32 M, kex = 480 nm, kem = 608 nm.
l
l
l
103 Mꢀ1) was found similar to 10b.The experimental results indi-
cate that the azido carbonyl compounds can bind to ct-DNA by weak
intercalation mode.
[6] W.V.E. Doering, R.A. Odum, Ring enlargement in the photolysis of phenyl azide,
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4. Conclusions
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computational study of singlet phenylnitrene, J. Am. Chem. Soc. 121 (1999)
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We have demonstrated for the first time, azido carbonyl com-
pounds as efficient photo-induced DNA cleavage agents. We also
showed that DNA cleaving ability of azido carbonyl compounds
were dependent both on their concentration and substituents on
the aromatic ring. Among
a, b and c azido carbonyl compounds,
b azido carbonyl compound showed better DNA cleaving ability.
Further, we have also showed naphthalene based azido carbonyl
compounds weakly intercalated with ct-DNA and efficiently
cleaved DNA at long-wavelength of UV light. Among all the azido
carbonyl compounds, 3,4-dimethoxy-b-azidopropiophenone 6d
showed best DNA cleaving ability at 250
irradiation of 45 min.
lM concentration and
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York, 1984.
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Acknowledgements
[17] S.M. Mandel, J.A.K. Bauer, A.D. Gudmundsdottir, Photolysis of
a-
We thank DST (SERC Fast Track Scheme) for financial support,
DST-FIST for 400 MHz NMR. Nilanjana is thankful to UGC for re-
search fellowship. We thank Prof. S. Hajra for helping with LC-MS.
azidoacetophenones:trapping of triplet alkyl nitrenes in solution, Org. Lett. 3
(2001) 523–526.
[18] N.D.P. Singh, S.M. Mandel, J. Sankaranarayanan, S. Muthukrishnan, M. Chang,
R.M. Robinson, P.M. Lahti, B.S. Ault, A.D. Gudmundsdottir, Selective formation
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Appendix A. Supplementary material
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Gudmundsdottir,
Intramolecular
H-atom
abstraction
in
c-azido-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jphotobiol.2012.
06.006.
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