Synthesis, photophysical, photochemical, DNA cleavage/binding and cytotoxic properties of pyrene oxime ester conjugates

Article abstract of DOI:10.1039/c2pp25033k

A new series of (E)-pyrene oxime ester conjugates of carboxylic acids including amino acids were synthesized by coupling with an environment sensitive fluorophore 1-acetylpyrene. (E)-Pyrene oxime esters exhibited strong fluorescence properties and interestingly their fluorescence properties were found to be highly sensitive to the surrounding environment. Direct irradiation of the (E)-pyrene oxime esters by UV light (≥350 nm) resulted in both the photo-Beckmann rearrangement product and products resulting from N-O bond homolysis. Photoproduct formation and their distribution were found to be solvent dependent. Further, we also showed (E)-pyrene oxime esters intercalated into DNA efficiently and photo-cleaved DNA. Finally we also showed these oxime esters can permeate cells efficiently and may cause cytotoxicity upon irradiation of light. The Royal Society of Chemistry and Owner Societies.

Full text of DOI:10.1039/c2pp25033k

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PAPER
Synthesis, photophysical, photochemical, DNA cleavage/binding and cytotoxic
properties of pyrene oxime ester conjugates†
Nilanjana Chowdhury,a Sansa Dutta,a Swagata Dasgupta,a N. D. Pradeep Singh,*a Mithu Baidyaa and
S. K. Ghoshb
Received 12th February 2012, Accepted 31st March 2012
DOI: 10.1039/c2pp25033k
A new series of (E)-pyrene oxime ester conjugates of carboxylic acids including amino acids were
synthesized by coupling with an environment sensitive fluorophore 1-acetylpyrene. (E)-Pyrene oxime
esters exhibited strong fluorescence properties and interestingly their fluorescence properties were found
to be highly sensitive to the surrounding environment. Direct irradiation of the (E)-pyrene oxime esters by
UV light (≥350 nm) resulted in both the photo-Beckmann rearrangement product and products resulting
from N–O bond homolysis. Photoproduct formation and their distribution were found to be solvent
dependent. Further, we also showed (E)-pyrene oxime esters intercalated into DNA efficiently and photo-
cleaved DNA. Finally we also showed these oxime esters can permeate cells efficiently and may cause
cytotoxicity upon irradiation of light.
pyrene conjugates such as pyrene dicarboxamide,16 pyrene dihy-
1
Introduction
drodioxin,17 viologen linked pyrene conjugates,18 pyrene
metallo porphyrins,19 1-hydroxy pyrene,20 pyrenyl boronic
acid21 were reported as DNA cleavage agents.
The development of synthetic organic molecules that can
efficiently interact or cleave DNA has been of considerable inter-
est since it enables us to design new drugs targeted for DNA as
well as to understand the electron and energy transfer properties
of DNA.1–3 Recently, synthetic molecules which can induce a
series of chemical reactions leading to DNA cleavage upon acti-
vation by light (DNA photocleavage agents) have been a subject
of growing interest. Several organic compounds including ene-
diynes,4 halogen containing compounds,5 riboflavins,6 naphtha-
limides7 and certain anthraquinone8 derivatives were
demonstrated as DNA photocleavage agents.
Pyrene, a well known fluorophore, because of its high fluor-
escence quantum efficiency as well as its environment sensitive
fluorescent properties has been used in a varied range of appli-
cations including high-affinity targeting of RNA and DNA using
modified oligonucleotides,9 detection of DNA and RNA hybrid-
ization by pyrene-labeled probe,10 in ligands for the measure-
ment of the proteolytic activity of enzymes11 and also as probes
for site-specific photocleavage of proteins.12 In recent times,
pyrene conjugates are targeted as DNA photocleavage agents
since pyrene derivatives act as intercalators13,14 and more impor-
tantly, they can be photochemically excited at a wavelength
longer than the absorption band of DNA.15 To date, several
The use of oxime conjugates of organic and inorganic mol-
ecules have gained great interest since they can be targeted as
novel therapeutic agents that can display acyl group transfer
capabilities and/or serve as specific inhibitors of a variety of
metalloenzymes. For example oximes of indole, isatin, quino-
line, indirubin, furan and derivatives of bis-indole alkaloid have
shown to possess anti tumour activity.22 In particular, furan
oxime was found to inhibit DNA, RNA and protein synthesis in
lipoid leukemia cells. Interestingly the oxime ester group is an
efficient pharmacophore and widely used in the field of
pesticides and drug design. Recently, Hwu et al.23 showed
irradiation of oxime esters of anthraquinone using UV light
(312 nm) undergoes homolysis of weak N–O bond to generate
nitrogen centered iminyl radicals, which resulted in
efficient DNA cleavage. The above interesting ability of oxime
esters to act as light induced DNA cleaving agents captured
our attention to develop oxime esters which can act as an
intercalator as well as induce DNA cleavage on excitation at
longer wavelengths.
Hence, we report for the first time pyrene oxime ester conju-
gates as intercalators which upon irradiation at longer wave-
lengths (≥350 nm) induces DNA cleavage. In this paper, we
discuss in detail the synthesis, characterization, photophysics
and photochemistry of the (E)-pyrene oxime ester conju-
gates. Further we also present the intercalation and DNA
cleaving behaviors of the (E)-pyrene oxime ester conjugates.
Finally we also investigated the toxicity of the (E)-pyrene
oxime ester conjugates before and after irradiation against
cancer cells.
aDepartment of Chemistry, Indian Institute of Technology Kharagpur,
Kharagpur-721302, India. E-mail: ndpradeep@chem.iitkgp.ernet.in;
Fax: (+) 91-3222-282252; Tel: (+) 91-3222-282324
bDepartment of Biotechnology, Indian Institute of Technology
Kharagpur, Kharagpur-721302, India
†Electronic supplementary information (ESI) available. CCDC 856804
and 856952. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c2pp25033k
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ethanone O-benzoyl oxime 4a as a white solid. Other com-
pounds (4b–4e) of this series were prepared following the above
general procedure.
2
Materials and method
2.1 General
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 dis-
tilled following standard protocols. Flash chromatography was
carried out using Rankem Silica gel (230–400 mesh) purchased
from Rankem, India. TLC was performed on aluminium-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 were expressed in parts per
million (δ) downfield to CDCl3 (δ = 7.26), 13C NMR chemical
shifts are expressed in parts per million (δ) relative to the central
CDCl3 resonance (δ = 77.0). Coupling constants in 1H NMR are
in Hz. High-resolution mass spectra were recorded using a Qtof
Micro YA263 mass spectrometer. UV/vis absorption spectra
were recorded on a Shimadzu UV-2450 UV/vis spectropho-
tometer and fluorescence emission spectra were recorded on a
Hitachi F-7000 fluorescence spectrophotometer. FT-IR spectra
were recorded on a Perkin Elmer RXI spectrometer. Melting
points were measured in Toshniwal (India) melting point appar-
atus. pBR 322 DNA was used for DNA cleavage purpose. Pho-
tolysis was carried out using 125 W medium pressure mercury
lamp supplied by SAIC (India). RP-HPLC was performed using
Waters 2489 liquid chromatography on a C18 column (4.6 mm ×
250 mm) with a UV-vis detector.
2.4 General procedure for the synthesis of (E)-pyrene oxime
esters (4f–j)
Boc-protected amino acid 3f (350 mg, 1.4 mmol) was dissolved
in dry DCM (10 mL) and stirred under N2 atmosphere at 0 °C.
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(EDC-HCl) (320 mg, 1.6 mmol) and N-hydroxybenztriazole
(HOBt) (226 mg, 1.6 mmol) were added subsequently to the
reaction mixture at ice cold conditions and then the reaction
mixture was allowed to stir for 2 hours at rt. Again it was cooled
to 0 °C and a solution of 1-acetylpyrene oxime 2 (364 mg,
1.4 mmol) and N,N-diisopropylethylamine (1.2 mL, 7.0 mmol)
in dry DCM (10 mL) was added to the reaction mixture at ice
cold conditions. The reaction mixture was stirred for overnight,
diluted with DCM, washed with dilute HCl (5%) and then with
10% aqueous NaHCO3 and extracted with 50 mL DCM. The
organic portion was dried over anhydrous Na2SO4 and concen-
trated under vacuum. The crude mass was purified by flash
column chromatography using ethyl acetate–hexane (30 : 70)
solvent system to get 3,3-dimethyl-1-(2-((1-(pyren-1-yl)ethylide-
neaminooxy)carbonyl)pyrrolidin-1-yl)butan-1-one (E/Z 1.65 : 1)
4f as white solid. The other compounds 4g–j of this series were
prepared following the above general procedure.
2.5 Preparative photolysis of (E)-oxime ester (4a)
2.2 Cell culture
Photolysis of 4a (0.3 mM) in an oxygen-free MeOH/H2O
(90 : 10 v/v) using 125 W medium pressure Hg lamp as light
source (≥350 nm) and 0.1 M CuSO4 solution as UV cut-off
filter was carried out. The irradiation was monitored by TLC at
regular times interval. After completion of photolysis, the
solvent was removed under vacuum and the photoproducts were
isolated by column chromatography using EtOAc in hexane as
an eluent to give N-acetyl-N-(pyren-6-yl)benzamide (8a), 1-
acetyl pyrene (1) and benzoic acid (3a). The photoproducts were
analyzed by 1H NMR and compared with the authentic sample.
Normal fibroblast cells (3T3) and colon cancer cells (HT29)
were both cultured in Rosewell Park Memorial Institute-1640
(RPMI1640) media (Himedia, India) supplemented with 10%
foetal bovine serum (Hyclone, USA), penicillin (100 U mL−1
)
and streptomycin (100 μg mL−1), amphotericin B (250 μg
mL−1) (Himedia, India). Cells were cultured in a humidified
atmosphere with 5% CO2 in air at 37 °C in an incubator (New
Brunswick Scientific, USA).
2.3 General procedure for the synthesis of (E)-pyrene oxime
esters (4a–e)
2.6 Photolysis of (E)-pyrene oxime esters (4a–i)
To a suspension of sodium hydride (66 mg, 2.4 mmol) in dry
THF (5 mL) 1-acetyl pyrene oxime 2 (300 mg, 1.0 mmol) was
added portion wise at ice cooled conditions and stirred for
30 min. Then the reaction mixture was allowed to stir at room
temperature for 30 min. The reaction mixture was recooled to
0 °C and benzoyl chloride (0.12 mL, 1.2 mmol) in 5 mL THF
was added slowly to the reaction mixture and it was allowed to
stir for 1 hour at room temperature. On completion of the reac-
tion, it was diluted with DCM and quenched with saturated
ammonium chloride solution and then it was extracted with
25 mL DCM (3 times). The organic portion was dried over anhy-
drous Na2SO4 and concentrated under vacuum. The crude mass
was purified by column chromatography using ethyl acetate/
hexane (15 : 85) solvent system to get (E)-1-(pyren-1-yl)-
Photolysis of oxime esters (4a–i, 0.3 mM) was carried out in
oxygen-free MeOH/H2O (90 : 10 v/v) using a 125 W medium
pressure Hg lamp as the light source (≥350 nm) and 0.1 M
CuSO4 solution as the UV cut-off filter. At regular time intervals,
20 μL of the aliquots were taken and analyzed by RP-HPLC
using A methanol mobile phase, at a flow rate of 1 mL min−1
(detection: UV 254 nm). Peak areas were determined by
RP-HPLC, which indicated a gradual decrease of the oxime ester
with time, and were the average of three runs. The reaction was
followed until the consumption of the oxime ester is less than
5% of the initial area. Based on HPLC data for each oxime ester,
we plotted normalized [A] (HPLC peak area) versus irradiation
time. We observed an exponential correlation for the disappear-
ance of the oxime ester which suggested a first order reaction.
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Further, the quantum yield for the photolysis of oxime esters
was calculated using eqn (1)
reference reading. Viscosities of ct-DNA (∼300 μM) and ct-
DNA complexed with the oxime ester at different molar ratios
were measured consecutively. The readings of sample mixtures
were noted after thermal equilibrium was achieved (∼15 min)
providing an additional incubation time of 15 min for each suc-
cessive addition. Each data point measured is the average of at
least four readings.
ðkpÞ
ðΦpÞ ¼
ð1Þ
I0F
where Φp is the photolysis quantum yield of pyrene oxime ester,
kp is the photolysis rate constant and I0 is the incident photon
flux and F is the fraction of light absorbed. Potassium ferrioxa-
late was used as an actinometer.
2.11 Docking studies of (E)-oxime esters 4c and 4h
The B-DNA crystal structure used for the docking studies was
obtained from the Protein Data Bank (PDB ID: 453D).25 The
DNA file was prepared for docking by removing water mol-
ecules and adding polar hydrogen atoms with Gasteiger charges.
The 3D structure of the ligands was generated in Sybyl 6.92
(Tripos Inc., St. Louis, USA) and its energy-minimized confor-
mation was obtained with the help of the MMFF94 force field
using MMFF94 charges. The rotatable bonds in the ligand were
assigned with AutoDockTools and docking was carried out with
AutoDock 4.0 Lamarckian Genetic Algorithm (GA).26 DNA was
enclosed in a grid having 0.375–0.431 Å spacing. Other miscel-
laneous parameters were assigned the default values given by
AutoDock. The output from AutoDock was rendered in
PyMol.27
2.7 Procedure for DNA cleaving ability of 4a at different
concentrations
The reaction mixtures (20 μL) containing supercoiled circular
pBR322 DNA stock solution (form I, 62.5 μg mL−1), com-
pounds 4a at different concentration (5, 10, 25, 50, 75, 100 μM)
individually, sodium phosphate buffer (10 mM, pH 7.0), and
10% DMSO were irradiated with UV light (≥350 nm, 125 W
medium pressure mercury lamp) under aerobic conditions at
room temperature for 30 min. After addition of bromophenol
blue to the reaction mixture, gel electrophoresis was carried out
and visualized by GEL LOGIC 200 Imaging System (Fig. 3).
2.8 Photoinduced DNA cleavage by (E)-pyrene oxime esters
(4a–j)
2.12 Cell cytotoxicity assay
The reaction mixtures (20 μL) containing supercoiled circular
pBR322 DNA stock solution (form I, 62.5 μg mL−1), oxime
esters 4a–j (50 μM), sodium phosphate buffer (10 mM, pH 7.0),
and 10% DMSO were irradiated with UV light at a fluence rate
of 2.6 W m−2 (≥350 nm, 125 W medium pressure mercury lamp
Cytotoxicity of 4g and 4h with and without irradiation on 3T3
(mouse embryonic fibroblast) and HT29 (human colon adenocar-
cinoma) cells was determined by conventional MTT assay.28
Cells in their exponential growth phase were trypsinised and
seeded in 96-well flat-bottom culture plates at a density of 3 ×
103 cells per well in 100 μL RPMI1640 complete medium. The
cells were allowed to adhere and grow for 24 hours at 37 °C in
an incubator and then the medium was replaced with 100 μL
fresh incomplete medium containing different concentrations of
4g and 4h (0–50 μM). The assay was performed in quadruplet
for each concentration. Cells were then incubated for 12 hours,
after which the culture medium was replaced with fresh medium.
Chemically treated cells were then irradiated with UV-ray for
30 min and further incubated for about 48 hours at 37 °C.
Medium was then aspirated and 100 μL of 1 mg mL−1 MTT
reagent in PBS was added to each well and incubated for
4 hours; during this period active mitochondria of viable cells
reduce MTT to purple formazan. Unreduced MTT were dis-
carded and the formazan precipitate was dissolved in DMSO
(100 μL). Optical density (OD) was measured spectrophotome-
trically using a microplate reader at 570 nm. The cytotoxic effect
of each treatment was expressed as percentage of cell viability
relative to the untreated control cells.
having incident intensity (I0) = 5.767 × 1017 quanta S−1 24
under
)
aerobic conditions at room temperature for 30 min resulted in an
incident energy fluence of 4.6 × 103 J m−2. The sample buffer
containing bromophenol blue in glycerol was added to the reac-
tion mixture and loaded on 1% agarose gel. After addition of the
tank buffer (1× TAE), the electrophoresis apparatus was attached
to a power supply. The gel was visualized by GEL LOGIC 200
Imaging System.
2.9 Ethidium bromide (EB) displacement assay of (E)-oxime
esters 4c and 4h
Fluorimetric titrations were performed in a 3 cm quartz cuvette
using an excitation wavelength of 545 nm. 3 mL of 20 μM ct-
DNA was saturated with 2 μM of ethidium bromide (EB) sol-
ution in 10 mM phosphate buffer pH 7.0 containing 50 mM
NaCl. The EB-ct-DNA solution was then titrated by successive
addition of the oxime ester to reach a final concentration of
∼32 μM for both the esters. Emission spectra were recorded
from 550 nm to 750 nm.
2.13 Fluorescence microscopy
2.10 Viscosity measurements of (E)-oxime esters 4c and 4h
Cells were seeded in a 24 well plate and were allowed to grow
for 24 hours. Cells were then treated with different concen-
trations of 4g and 4h for 1 hour. Media was then aspirated and
was thoroughly washed with phosphate buffer saline (PBS) (1×)
Viscosity measurements were carried out at a constant tempera-
ture (28 °C) using a viscometer connected to a thermostat. The
viscosity of the phosphate buffer (10 mM) was used as the
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Scheme 1 Synthesis of (E)-pyrene oxime esters (4a–j).
Table 1 Synthetic yields, UV/vis and fluorescence data of (E)-pyrene oxime esters (4a–j) in absolute ethanol
UV/vis
Fluorescence
f
Entry
Oxime ester
Synthetic yielda (%)
λmaxb (nm)
log εc
λmaxd (nm)
Stokes’ shifte (nm)
φF
1
2
3
4
5
6
7
8
9
10
4a
4b
4c
4d
4e
4f
4g
4h
4i
90
76
91
92
70
80
75
79
82
76
341
340
341
340
341
340
341
340
341
340
4.45
4.61
4.65
4.60
4.60
4.58
4.65
4.53
4.55
4.45
419
418
420
416
418
415
419
417
415
416
78
78
79
76
77
75
78
77
74
76
0.050
0.048
0.055
0.042
0.038
0.035
0.042
0.046
0.034
0.035
4j
a Based on isolated yield. b Maximum absorption wavelength. c Molar absorption coefficient (in mol−1 L cm−1) at the maximum absorption
wavelength. d Maximum emission wavelength. e Difference between maximum absorption wavelength and maximum emission wavelength.
f Fluorescence quantum yield (error limit within 5%).
and was analyzed with a fluorescent microscope (Olympus
1X51, Japan).
N,N-diisopropylethylamine (DIPEA) yielded the (E)-pyrene
oxime esters (4f–j). All pyrene oxime esters were found to have
the (E)-configuration (determined from NMR spectroscopy, X-
ray diffraction study). HRMS, LCMS and elemental analysis
supported the structure and purity of oxime esters. Detailed pro-
cedures for the synthesis were given in the Materials and method
section and analytical data of the (E)-oxime esters (4a–j) is pro-
vided in the ESI.†
3
Results and discussion
3.1 Synthesis of (E)-pyrene oxime esters (4a–j)
A series of (E)-pyrene oxime esters of carboxylic acids including
amino acids were synthesized as outlined in Scheme 1. (E)-1-
Acetylpyrene oxime (2) was prepared from commercially avail-
able 1-acetylpyrene (1) by reaction with hydroxylamine hydro-
chloride in the presence of NaOH. Treatment of freshly prepared
2 with various acid chlorides (3a–e) in the presence of sodium
hydride in dry THF at 0 °C afforded the corresponding (E)-
pyrene oxime esters (4a–e) in good to excellent yields (Table 1).
However, for the coupling of amino acids with 1-acetylpyrene
oxime (2), we followed a slightly different protocol. Boc-pro-
tected amino acids (3f–j) were activated by treatment with
EDC–HCl and HOBt. Later, addition of 1-acetyl pyrene oxime
(2) to the activated Boc-protected amino acids in the presence of
3.2 Photophysical properties of (E)-pyrene oxime esters (4a–j)
The UV/vis absorption and emission spectra of the degassed 4 ×
10−6 M solution of (E)-oxime esters (4a–j) in absolute ethanol
were recorded. The absorption and emission maxima, molar
absorptivities (ε) and fluorescence quantum yield (ΦF) of the
above oxime esters were summarized in Table 1. Fluorescence
quantum yields were calculated using anthracene as standard
(ΦF = 0.27 in ethanol).29
Fig. 1 showed the normalized absorption and the emission
spectra of (E)-oxime ester 4a in ethanol. The absorption
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Fig. 1 Normalized UV/vis absorption spectrum (blue line) and emis-
sion spectrum (red line) of 4a in ethanol (4.0 × 10−6 M).
Fig. 2 Corrected fluorescence spectra of 4a in ACN and in increasing
percentage of water in ACN (4.0 × 10−6 M).
spectrum of 4a showed an intense band centered at 341 nm
(log ε = 4.45 mol−1 L cm−1) while in the emission spectrum the
emission maxima was red shifted to about 419 nm. We observed
similar absorption and emission maxima for all other oxime
esters (Table 1) which clearly suggest that pyrene moiety only
dictated the position of the absorption and emission maxima
ruling out the influence of its counterpart carboxylic acids. The
Stokes’ shift was calculated from the difference in the absorption
and the emission maxima and the magnitude of the Stokes’ shift
of all the oxime esters varies between 75–79 nm. Further,
oxime esters also showed moderate fluorescence quantum yield
(0.035 < Φ < 0.055).
cut-off filter. The course of the photolysis was monitored by
HPLC study. We found that after 10 min of irradiation, (E)-
oxime ester (4a) provided N-acetyl-N-(pyren-6-yl)benzamide
(8a, 60%), acetyl pyrene (1, 30%) and benzoic acid (3a, 2.5%)
as three major photoproducts isolated after column purification
(Scheme 2).
The formation of acetyl pyrene and benzoic acid was attribu-
ted to photoinduced N–O bond cleavage chemistry,32whereas N-
acetyl-N-(pyren-6-yl)benzamide was from the photo-Beckmann
rearrangement. The above photoproducts, 1 and 3a, were ident-
ified by isolating and comparing with that of corresponding auth-
entic samples. The photo-Beckmann rearrangement product 8a
was established by NMR spectroscopy and single-crystal X-ray
diffraction.
3.3 Fluorescence spectra of (E)-oxime ester 4a in binary
solvent.
3.4.2 Solvent effect on the photolysis of pyrene oxime ester
conjugate (4a). To study the effect of solvent on the photopro-
duct formation and distribution of (E)-pyrene oxime esters, we
carried out the photolysis of 4a (0.3 mM) in benzene, dichloro-
methane (DCM), acetonitrile (ACN), methanol, and MeOH/H2O
for 10 min and the results are displayed in Table 2. We observed
oxime ester 4a produced exclusively photo-Beckmann rearrange-
ment product (8a) in benzene. While in acetonitrile, we noticed
formation of both photo-Beckmann rearrangement product (8a)
and photo fragmentation products. Interestingly both in methanol
and aqueous methanol (10%) we noticed the increase in the
yield of photofragmentation products.
To investigate environment-sensitive behavior of fluorescent
pyrene oxime esters, we recorded the emission spectra of 4a in
acetonitrile–water solvent mixtures. Similar to pyrene,30,31 we
also noted enhancement in the intensity of the forbidden vibronic
O–O band as we increased the percentage of water in the solvent
system (Fig. 2). The above fact was due to the more perturbation
of the first singlet excited state of pyrene oxime ester because of
the greater interaction of polar solvent with pyrene, which sub-
sequently diminished the forbidden nature and resulted in
enhancement of O–O band intensity. The photophysical studies
showed that (E)-pyrene oxime esters exhibited good fluor-
escence, large Stokes’ shift, moderate fluorescence quantum
yield and moreover their fluorescence properties were sensitive
to its surrounding environment.
3.4.3 Mechanism for the photolysis of (E)-pyrene oxime
esters (4a). Direct irradiation of (E)-oxime ester (4a) resulted in
N–O bond scission from the triplet excited state32 to generate a
pyrene iminyl (5) and benzoyloxy cage radical pair (Scheme 3).
The above generated radical pair in aqueous polar protic solvent,
escapes from the cage and subsequently abstracts hydrogen from
the solvent to produce photofragmentation products (1-acetyl
pyrene (1) and benzoic acid (3a)). On the other hand, formation
of the photo-Beckmann rearrangement product can be under-
stood in terms of fission at the oxygen–carboxyl bond at the
3.4 Photochemical study of (E)-pyrene oxime esters (4a–j)
3.4.1 Photolysis of (E)-oxime ester (4a). To understand the
photochemistry of (E)-pyrene oxime esters, we carried out the
photolysis of 4a (0.3 mM) in an oxygen-free MeOH/H2O
(90 : 10 v/v) solution using a 125 W medium pressure Hg lamp
as light source (≥350 nm) and 0.1 M CuSO4 solution as a UV
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Scheme 2 Photolysis of (E)-pyrene oxime ester (4a).
Table 2 Photolysis of pyrene oxime ester conjugate (4a) in various
solvents
different concentrations of 4a (5–100 μM) dissolved in a sodium
phosphate buffer (pH 7.0, 10 mM) with 10% DMSO. The
results from the gel electrophoresis on 1.0% agarose gel with
ethidium bromide staining indicated that 4a caused the single
strand cleavage of DNA (form I) to give the relaxed circular
DNA (form II). We noticed that the DNA cleaving ability of 4a
enhanced with the increase of its concentration (see Table 1s in
the ESI†). For example, at a 10 μM concentration, the % of form
II was 27 (lane 2, Fig. 3), whereas for concentrations of 25 μM
and 50 μM, it was 65 and 82 respectively. The % of form II was
found to be 98 when the concentration of 4a was 100 μM (lane
7, Fig. 3).
Remaining Acetylpyrene PhCO2H Rearranged
% of 4a
Solvent
(1)a
(3a)a
product (8a)a
Benzene
DCM
ACN
MeOH
MeOH–H2O
9.20
47.8
5.5
1.1
—
—
2.2
23.5
31.5
31.7
—
—
2.0
2.5
3.5
82.6
44.9
68.5
63.5
62.8
a Photoproducts yield were calculated from 1H-NMR of crude reaction
mixture using 1,2-dichloroethane as internal standard.
3.5.2 Photoinduced DNA cleavage of (E)-pyrene oxime
esters (4a–j). Based on the above concentration study, the DNA
cleaving ability of all oxime esters (4a–j) were investigated
using a 50 μM concentration and the results are presented in
Fig. 4. Based on Fig. 4 the % of form I, form II and the ratio of
both forms of the DNA molecule for all the oxime esters (4a–j)
were calculated and the results are tabulated in Table 4.
triplet excite state33 to form a pyrene iminyloxy (6) and benzoyl
radical pair which is recombined in-cage to form the oxaziridin
intermediate (7).34 The newly formed oxaziridines intermediate
then reorganize to form N-acetyl-N-(pyren-6-yl)benzamide (8a).
3.4.4 Photolysis of (E)-oxime esters (4a, 4c, 4g and
4h). Based on the above studies, we carried out the photolysis of
(E)-oxime esters (4a, 4c, 4g and 4h) in 10% MeOH/H2O as
described earlier and the results are tabulated in Table 3. The
quantum yield (Table 2s in ESI†) was calculated using potass-
ium ferrioxalate as an actinometer.29
Table 3 indicated that the carboxylic acids have an influence
on both the photoproduct distribution and quantum yield of the
oxime esters. In the case of the amino acids derived from oxime
esters (4g and 4h), we observed only photo-fragmentation pro-
ducts and moreover the quantum efficiency was found to be
tenfold times lesser as compared to oxime esters of simple aro-
matic carboxylic acids (4a and 4c).
Table 4 indicates that the potency of the oxime esters to cleave
DNA varied to a great extent, among the aromatic acids, derived
oxime esters (4a–e), 4-cyanobenzoic acid (4b) and 3,4-
dimethoxy benzoic acid (4c) derived oxime ester showed better
DNA cleaving activity as compared to their counter parts. In the
case of amino acids, phenylalanine (4g) and phenyl glycine (4h)
derived oxime esters resulted in better DNA cleaving ability.
Although the above oxime esters generate the common pyrene
iminyl radical (5), the difference in DNA-cleaving ability must
depend on the stability of the carboxyl radicals (3a–j) generated
by the oxime esters. These results led us to believe that the car-
boxyl radicals acted as the important DNA-cleaving species
along with iminyl radical (5). On the other hand, the role of
small amounts of the decarboxylated aryloxy species on DNA
scission cannot be excluded. To confirm that the radicals are
involved in DNA cleavage, we irradiated DNA with 4c in the
presence of radical quencher ascorbic acid and found that
ascorbic acid inhibited the cleavage of DNA (Table 4, entry 8,
Fig. 4, lane 8). To demonstrate the requirement of UV light for
pyrene oxime esters to induce DNA cleavage, we carried out the
control experiment by treating oxime ester (4c, 50 μM) with
3.5 DNA cleaving ability of (E)-pyrene oxime esters (4a–j)
3.5.1 Photoinduced DNA cleavage of 4a at different concen-
tration. To assess the DNA cleaving ability of (E)-oxime esters,
initially we carried out irradiation of circular supercoiled
pBR322 plasmid DNA (form I; 62.5 μg mL−1) at room tempera-
ture under aerobic conditions for a period of 30 min using
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Scheme 3 Possible photocleavage pathway of (E)-oxime esters (4a).
Table 3 Photolytic data of (E)-pyrene oxime esters (4a, 4c, 4g and 4h)
in 10% MeOH/H2O solvent system
Photo productsa (%)
Pyrene
oxime
ester
Time for 50%
decomposition
(min)
Acetyl
pyrene
(1)
Rearranged
product (8)
Released
acid
Fig. 3 Single strand cleavage of supercoiled circular pBR322 DNA
(form I) to relaxed circular DNA (form II) on irradiation of 4a at differ-
ent concentrations (5–100 μM) using UV light (≥350 nm) for 30 min.
Lane 1, DNA alone; lane 2, DNA + 4c (5 μM); lane 3, DNA + 4c
(10 μM); lane 4, DNA + 4c (25 μM); lane 5, DNA + 4c (50 μM); lane
6, DNA + 4c (75 μM); lane 7, DNA + 4c (100 μM).
4a
4c
4g
4h
6
4
37
35
27.5
28.0
nd
18.2
18.8
41.8
42.5
1.2
1.6
4.8
5.2
nd
a Photoproducts yield were calculated after column purification, nd = not
detected by HPLC.
3.6 DNA-binding studies of (E)-pyrene oxime esters (4a–j)
The DNA-binding properties of the oxime esters were investi-
gated by fluorescence, viscosity and docking studies using
oxime esters 4c and 4h.
DNA in the dark for 1 hour. We found the % of form II (Table 4,
entry 2, Fig. 4, lane 2) in the dark was 8 whereas in presence of
UV light % of form II was 92 (Table 4, entry 5, Fig. 4, lane 5)
after 30 min, which suggest that UV light is the key to initiate
the DNA scission process. Further, to investigate the possibility
of DNA cleavage by singlet oxygen, we carried out the
irradiation of DNA with 4c by adding sodium azide (scavenger
of singlet oxygen) and the % of form II was found to be 90
(Table 4, entry 14, Fig. 4, lane 14), which is similar to the % of
form II for 4c under the aerobic conditions, which ruled out the
involvement of singlet oxygen in DNA cleavage.
3.6.1 Ethidium bromide (EB) displacement assay of (E)-
oxime esters 4c and 4h. To ascertain the mode of binding of
pyrene oxime esters to the calf thymus DNA (ct-DNA), a com-
petitive EB displacement assay has been performed using 4c and
4h. The fluorescence intensity of EB, a well-known classical
intercalator is enhanced upon complexation with the DNA.35
Upon intercalation into the DNA helix by the pyrene oxime
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ester, the fluorescence intensity of the EB–DNA complex was
quenched with increasing concentration of the oxime ester.
Fig. 5 showed considerable decreases in the fluorescence inten-
sity of the EB DNA complex upon addition of oxime esters 4c
and 4h, which was characteristic of the intercalative mode of
binding. However, the quenching was more prominent in the
case of oxime ester 4h compared to 4c at the same
concentration.
3.6.2 Viscosity measurements of (E)-oxime esters 4c and 4h.
Viscosity measurements provide a simple, theoretically sound
means of distinguishing the DNA binding mode. A classical
intercalation generally leads to lengthening of the DNA helix as
base pairs are separated to accommodate the intercalator, which
is reflected in an increase of DNAviscosity.36–39 The relative vis-
cosity of DNA and the DNA pyrene oxime ester was plotted
against the ratio of pyrene oxime ester and DNA concentrations
as shown in Fig. 6. As expected, the oxime esters dramatically
increase the length of DNA, resulting in an increased viscosity.
Among the oxime esters, 4h intercalated the most into ct-DNA
as compared to 4c.
Table 4 Single-strand cleavage of supercoiled circular pBR322 DNA
(form I) to relaxed circular DNA (form II) on irradiation of pyrene
oxime esters (4a–j)
Entry
Oxime ester
% of form I
% of form II
Form II/form I
1a
2b
3
4
5
—
4c
4a
4b
4c
4d
4e
4c
4f
4g
4h
4i
4j
4c
100
92
18
10
7
18
14
81
39
8
—
8
—
0.08
4.50
9.00
13.3
4.55
6.14
0.23
1.56
11.5
10.1
1.27
1.86
9.00
81
90
93
82
86
19
61
92
91
56
65
90
6
3.6.3 Docking studies of (E)-oxime esters 4c and 4h. Mol-
ecular docking studies were performed to substantiate the inter-
action and preferred binding mode of the pyrene oxime ester
with DNA. The docking analysis reveals that oxime ester 4c and
4h may intercalate into the helical DNA. The pyrene ring of
both the oxime esters intercalates between the base pairs of the
DNA helix, while the side chains interact with the phosphate
backbone as reflected in the Fig. 7. Thus the docking study cor-
roborates the experimental findings, which suggest that the com-
pounds are behaving as intercalators of DNA.
7
8c
9
10
11
12
13
14d
9
44
35
10
a DNA alone. b DNA + compound 4c in the dark. c DNA + 4c +
ascorbic acid. d DNA + 4c + NaN3 solution (0.1 M).
Fig. 4 Single strand cleavage of supercoiled circular pBR322 DNA (form I) to relaxed circular DNA (form II) on irradiation of pyrene oxime esters
(4a–j) at a concentration of 50 μM, using UV light (≥350 nm) for 30 min. Lane 1, DNA alone; lane 2, DNA + 4c in the dark; lane 3, DNA + 4a; lane
4, DNA + 4b; lane 5, DNA + 4c; lane 6, DNA + 4d; lane 7, DNA + 4e; lane 8, DNA + 4c + ascorbic acid; lane 9, DNA + 4f; lane 10, DNA + 4g;
lane 11, DNA + 4h; lane 12, DNA + 4i; lane 13, DNA + 4j; lane 14, DNA + 4c + NaN3.
Fig. 5 Fluorescence quenching spectra of EB–ct-DNA with oxime ester (A) 4c and (B) 4h; Red line indicates the fluorescence emission spectra of
EB in absence of ct-DNA and top black line indicates the fluorescence emission spectra of EB bound to ct-DNA; [EB] = 2 μM, [ct-DNA] = 20 μM,
[4c] = 0–32 μM and [4h] = 0–32 μM; arrow indicates the quenching of fluorescence with increasing amount of compounds 4c and 4h.
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3.7 Cell permeability and localization studies of (E)-pyrene
oxime esters 4g and 4h
3T3 mouse embryonic fibroblast cells and HT29 human colon
adenocarcinoma cells. Cells were treated with various concen-
trations of the oxime esters (0–50 μM) and their cytotoxicity was
analysed with or without the radiation. Cells exposed to
irradiation along with the oxime esters have significantly lower
cell counts 48 hours after irradiation as compared to unexposed
cells treated with the pyrene oxime esters. The cell count for the
3T3 and HT29 irradiated cells were found to be as low as ∼24%
and ∼17% at a concentration as low as 10 μM of 4g for which
unexposed cells survived ∼87% and ∼79% respectively after
48 hours (Fig. 9a). Similarly at the same concentration of 4h
treatment, cell survival values were ∼29% and ∼32% for ir-
radiated cells and ∼60% and ∼83% for cells that were only
treated with 4h (Fig. 9b). At higher concentrations cell survival
reduced to more drastic levels as compared to untreated cells. In
fact, for 3T3 cells, their IC50 value drastically dropped from
29.83 μM to 5.07 μM for 4g and 15.14 μM to 6.30 μM after
irradiation. Similar treatment of HT29 cells led to a reduction of
the IC50 values, from 23.45 μM to 4.40 μM for 4g and 18.36 μM
to 7.30 μM for 4h, indicating an enhancement of its toxicity many
fold when oxime esters were irradiated with UV light (Table 5).
The energy fluence rate of irradiation was 2.6 W m−2 and the
30 min irradiation resulted in an incident energy influence of
4.6 × 103 J m−2 for cell cytotoxicity study.
To study the cell permeability of pyrene oxime esters 4g and 4h,
3T3 mouse embryonic fibroblast cells were treated with various
concentrations (1, 2 and 5 μM) of both compounds 4g and 4h
for about an hour. Fluorescent microscopy (Fig. 8) revealed that
compounds 4g and 4h could enter the cell at a concentration as
low as 1 μM and the trend remained similar for other concen-
trations as well.
3.7.1 Cell cytotoxicity studies of (E)-pyrene oxime esters 4g
and 4h after irradiation. The above studies have highlighted the
potential of the oxime esters as genotoxic agents and so to study
their cytotoxic effects we carried out the cytotoxicity studies in
4
Conclusions
The newly synthesized (E)-pyrene oxime esters displayed charac-
teristic properties, such as (i) exhibited good fluorescence and
more interestingly their fluorescence properties were found to be
sensitive to the environment (ii) underwent both photo-Beckman
Fig. 6 The increase in relative viscosity of ct-DNA upon gradual
addition of the pyrene oxime ester at 28 °C; [DNA] = 300 μM; [pyrene
oxime ester conjugate]/[DNA] ratio was varied from 0–0.35.
Fig. 7 Docked conformations of 4c and 4h with ct-DNA. Panels correspond to A: pyrene oxime ester 4c; B: pyrene oxime ester conjugate 4h.
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Fig. 8 Intracellular localisation of the 4g and 4h oxime esters in 3T3 mouse embryonic fibroblast cells incubated at 5 μM of the conjugate for 1 hour
at 37 °C. Images were obtained using an inverted fluorescence microscope. Row A has bright field images and row B has corresponding fluorescent
images.
Fig. 9 (a) Cytotoxicity of 4g in 3T3 fibroblast cells and HT29 colon cancer cells. Data shows the percentage of cell survival counted 48 hours after
the treatment with 4g at 0 °C without and with UV irradiation (30 min). (b) Cytotoxicity of 4h in 3T3 fibroblast cells and HT29 colon cancer cells.
Data shows the percentage of cell survival counted 48 hours after the treatment with 4h at 0 °C without and with UV irradiation (30 min).
rearrangement and N–O bond cleavage on irradiation (iii) exhib-
ited a greater potency as DNA cleavage agents, particularly at
longer wavelengths of UV light (≥350 nm) (iv) acted as DNA
intercalators and (v) finally, permeated the cell effectively and
were found to be more cytotoxic upon photoexcitation. These
conjugates did not show any significant specificity to cancer
cells in terms of cytotoxicity and hence further modifications
Table 5 IC50 values (in μM) of 3T3 and HT29 cells treated with
different combination of conjugates and UV light
Cells
4g
4g + irradiation
4h
4h + irradiation
3T3
HT29
29.83
23.45
5.07
4.40
15.14
18.36
6.29
7.30
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