Highly solvatochromic fluorescent naphthalimides: Design, synthesis, photophysical properties and fluorescence switch-on sensing of ct-DNA

Article abstract of DOI:10.1016/j.bmcl.2012.11.003

We report the design, synthesis and photophysical properties of highly solvatochromic donor/acceptor substituted naphthalimide based fluorophores. The synthesized naphthalimides containing propargyl ends showed highly solvatochromic intramolecular charge

Full text of DOI:10.1016/j.bmcl.2012.11.003

Bioorganic & Medicinal Chemistry Letters 23 (2013) 96–101
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Highly solvatochromic fluorescent naphthalimides: Design, synthesis,
photophysical properties and fluorescence switch-on sensing of ct-DNA
⇑
Subhendu Sekhar Bag , Manoj Kumar Pradhan, Rajen Kundu, Subhashis Jana
Bioorganic Chemistry Laboratory, Department of Chemistry, Indian Institute of Technology Guwahati, Guwahati 781039, India
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the design, synthesis and photophysical properties of highly solvatochromic donor/acceptor
substituted naphthalimide based fluorophores. The synthesized naphthalimides containing propargyl
ends showed highly solvatochromic intramolecular charge transfer (ICT) feature as was revealed from
the UV–visible, fluorescence photophysical properties of these fluorophores and DFT/TDDFT calculation.
Fluorescence life times for the imide fluorophores were also measured in different solvents. The solid
state photophysical property of donor substituted naphthalimide 1 showed promising for future applica-
tion in material sciences. Furthermore, both the donor/acceptor substituted naphthalimide fluorophores
1–2 were exploited in sensing calf-thymus DNA via switch-on fluorescence response. The propargyl lin-
ker containing naphthalimides can further be exploited for the synthesis of labeled biomolecular building
blocks.
Received 11 September 2012
Revised 11 October 2012
Accepted 2 November 2012
Available online 12 November 2012
Keywords:
Propynyl naphthalimides
Fluorophores
Solvatochromic
Intra molecular charge transfer (ICT)
Probes of ct-DNA
Ó 2012 Elsevier Ltd. All rights reserved.
Switch-on sensing
Solvatochromic fluorescent molecules are widely known to
serve as extremely sensitive probes in biological systems for the
detection and probing of structures, dynamics, micropolarity
around a biomolecule and interbiomolecular interactions.1 There-
fore, the development of such fluorescent molecules is a very
important research target for understanding biological events
associated with interbiomolecular interactions. As for example,
highly solvatochromic fluorescent probes and fluorescently labeled
biomolecular building blocks such as solvofluorochromic nucleo-
sides/amino acids have been successfully utilized for the sensing
and detection of biomolecular microenvironment/biomolecules.2
However, many of such explored fluorophores emitted at a short
wavelength region, possessed low emission intensity and/or suf-
fered from a quenching incidence rendering them unsuitable for
practical use.3 Therefore, bright and long-wavelength emissive
fluorescent molecules with emission in the visible region have at-
tracted great interest in recent time as new fluorescent probes for
the detection and sensing of biomolecules without any interfer-
ence by the background signal generated from the autoabsorption
and autofluorescence of biomolecules.
the possibility of synthesizing 1,8-naphthalimide derivatives with
donor/acceptor substituents as new potential solvatochromic fluo-
rophores. Naphthalimide derivatives aroused the interest of chem-
ists, physicists and biologists for various reasons. The 4-amino and/
or alkyl amino-1,8-naphthalimide chromophore or their anologues
have been reported for their several interesting photophysical
properties and utilized for chemosensory applications.4i Because
of the ‘push–pull’ nature of its internal charge transfer (ICT) excited
state, they have been utilized in several biological applications.4
Naphthalenimides show a diversified reactivity towards biological
substrates including DNA and proteins. Furthermore, because of
their potential anti-tumor activity upon electronic excitation with
UV light 1,8-naphthalimides have attracted much attention in the
fields of biology and medicine. 4-Amino-1,8-naphthalimide also
have been utilized in supramolecular fields as well as DNA target-
ing molecule. Troger’s base compounds as well as metal complexes
of napthalimides have been developed and found to interact
strongly with DNA.4i–l However, there is no report of 1,8-naphthal-
imide in which 4-position is conjugated to donor/acceptor substi-
tuted phenylacetylenes to achieve much longer absorption
wavelength.
As a part of our ongoing program for the design and synthesis of
highly solvatochromic fluorophores with a linker unit for possible
future use in labeling of biomolecular building blocks and taking
into consideration of the importance of naphthalimide derivatives4
in biological applications, we were thus very much interested in
Therefore, in this particular we report the syntheses and photo-
physical properties of donor/acceptor containing 1,8-naphthali-
mide derivatives 1–2 in which chromophores have free acetylene
arms which can be exploited further for future applications such
as for the synthesis of labeled biomolecular building blocks. We ex-
plored the sensitivity in fluorescence response upon changing the
solvent polarity of these fluorophores. We also wanted to examine
⇑
Corresponding author. Fax: +91 361 258 2324.
E-mail address: ssbag75@iitg.ernet.in (S.S. Bag).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.003

S. S. Bag et al. / Bioorg. Med. Chem. Lett. 23 (2013) 96–101
97
the ability of the fluorophores 1–2 in possible sensing of biomole-
cule like ct-DNA.
compound 1 as red crystalline solid and 2 as yellow powdered so-
lid in very good yields. The structures of the final products were
characterized by NMR, mass spectrometry, and by X-ray single
crystal analysis of compound 1.
The crystal structure of fluorophore 1 showed a very interesting
arrangement (Fig. 1). Thus, the donor substituted planar naphthal-
imide with the pendent propargyl arm crystallized in a layered
sheet-like arrangement. The planar sheets are layered on top of
Therefore, we have chosen 4-bromo-1,8-naphthalic anhydride
as our starting material. The reason behind our choice was to
achieve charge transfer fluorescence response upon incorporating
a donor unit by a substitution at 4-bromo functionality. Thus, there
might be a possibility of getting emission at longer wavelength re-
gion because of the conjugation between the donor substituent
and the acceptor naphthalimide core leading to a long range charge
transfer (CT) process and hence we expectedly would observe an
excellent solvofluorochromicity.4,5 On the other hand we also
wanted to know the effect of an acceptor substituent and compare
the photophysical behavior with that of the donor substituted one.
Therefore, the fluorophores might show high solvatofluorochrom-
icity which could be utilized for probing of biomolecular
microenvironment.
each other, with slip-stacked ArCH–
internal alkyne and aromatic C–H of naphthalimide core. The lay-
ers are held by C–C– (3.37 Å) bonding between donor/acceptor
aromatics and -stacking between the donor unit in one layer
p-bonding (3.29 Å) between
p
p
and the acceptor naphthalimide core of the other layer (Fig. 1a).
Head-to-tail arrangements of donor N,N-dimethylanilino unit of
one molecule in one layer and acceptor naphthalimide core of a
separate molecule in second layer makes the system stable by
With this background and aim we have first synthesized the
fluorophores 1–2. The routes adopted for the synthesis of target
compounds 1–2 is shown in Scheme 1. Thus, a Pd(0)-mediated
‘click-reagent version’ of Sonogashira coupling4b,6 of anhydride 3
with 4-ethynyl-N,N-dimethylaniline and 4-ethynylbenzonitrile,
respectively, in dry DMF at 80 °C for 4 h, yielded 4-substituted-
1,8-naphthalic anhydrides, 6–7. After recrystalization from hot tol-
uene pure naphthalic anhydride derivatives 6–7 in 65% and 70%
yields, respectively, were afforded. Next, conjugated anhydrides
6–7 were allowed to react with propargyl amine in dry
ethanol under reflux for 50 h to afford 4-(4-N,N-dimethylamino-
phenylethynyl)-N-(2-propynyl)-1,8-naphthalimide (1, 4-DMAPENI)
and 4-(4-cyanophenylethynyl)-N-(2-propynyl)-1,8-naphthalimide
(2, 4-CPENI), respectively, with good yields (Scheme 1). Washing
the products in water and ethanol and recrystalization gave pure
forming a strongly
layer like structure.7 The layers are so arranged that the donor-N,N-
dimethylphenyl moiety interacts via -stacking and charge trans-
fer interactions with the acceptor naphthalimide ring in the mole-
cule in the layer above and below with an average interlayer
distance being 3.31 Å. The pendant terminal acetylenic-H involved
in H-bonding with –C@O of naphthalimide group (2.37 Å) of a mol-
ecule in a different layer giving rise to a bent-stair-like arranged
third stacked layer (Fig. 1a and b).
After getting all the compounds in hand in very pure form we
turned our attention to study their photophysical properties. Thus,
the UVꢀvis absorption spectra of the naphthalimide fluorophores
1–2 were studied in various solvents of different polarity. The lon-
gest wavelength absorption of compound 1 was characterized by a
broad band with the maxima appearing between 450 and 467 nm
p-stacked and charge transfer stabilized stacked
p
O
N
O
O
O
O
O
(i)
O
(ii)
R
R
Br
O
R
NH2
6, R = -NMe2 (65%)
7, R = -CN (70%)
3
1, R = -NMe2 (75%)
2, R = -CN (80%)
8
4, R = -NMe2
5, R = -CN
Reagents and conditions: (i) Pd(PPh3)4/Et3N/DMF/CuSO4/Na-ascorbate/80 °C, 4 h; (ii) EtOH/Reflux, 50 h.
Scheme 1. Synthesis of donor/acceptor labeled naphthalimide fluorophores 1–2.
Figure 1. Crystal structure (a–b) showing inter planar distance and the two planes formed by two pendent acetylenic arms. (c) Crystal network of the molecule 1 (the CCDC
number is CCDC 882241).

98
S. S. Bag et al. / Bioorg. Med. Chem. Lett. 23 (2013) 96–101
(a)
(b)
140 nm shift
(c)
1.2
1600000
1200000
800000
400000
0
Hex
Hex
CyHex
Toluene
Dioxane
Ether
CyHex
Toluene
Dioxane
Ether
EtOAc
CHCl3
THF
0.9
0.6
0.3
0
CHCl3
445
545
645
745
445
545
645
745
845
Wavelength (nm)
Wavelength (nm)
Figure 2. (a) Emission and (b) normalized emission spectra of 1 (4-DMAPENI) in different solvents (10
after irradiation of 1 in UV light (k = 254 nm) under a UV-transilluminator.
lM, rt; kex = kmax = 445–465 nm). (c) Colors:colours in different solvents
(a)
(b)
(c)
1.E+04
1.E+02
1.E+00
1.2
0.8
0.4
0
1.E+04
Absorption
Emission
CHCl3_Em=610
Solid_Em=710
IRF
CH3CN
MeOH
10% wt
30% wt
50% wt
TFE
1.E+02
1.E+00
IRF
0
10
20
30
40
50
0
10
20
30
300
500
700
900
Wavelength (nm)
Time/ns
Time/ns
Figure 3. (a) Solid state UV–visible/fluorescence spectra and (b) time resolved fluorescence traces in solution and solid states of fluorophore 1. (c) Time resolved fluorescence
traces of fluorophore 2 in various solvents.
depending on the polarity of the solvents (see Supplementary
data). This band was unambiguously assigned as intramolecular
charge transfer (ICT) band that was generated out of charge trans-
fer from the donor 4-N,N-dimethylaminophenyl group to the
acceptor naphthalimide ring system via the internal alkyne. The
origin of ICT was also evident from the fairly broad, intense and
the large solvatochromicity of the band.5b,8 Thus, an increase in
polarity of the media led to a red shift of the absorption maxima
of about 14–24 nm when changing the solvent from hexane to
chloroform to DMSO suggesting that the ground state of the mole-
cule was significantly polar. However, for the fluorophore 2, a blue
shift of about 17 nm of the absorption band on changing the sol-
vent from dioxane to trifluoroethanol (between 389 and 372 nm)
was observed which might be because of the presence of electron
acceptor cynophenyl unit in conjugation with the naphthalimide
ring system (see Supplementary data). This observation indicated
that the ground state was not so polar and the fluorophore 2 was
lipophilic in nature. Therefore, the donor N,N-dimethylaminophe-
nyl unit was responsible for the enhancement of the charge sepa-
ration in the molecule 1 which ultimately led to a large red shift of
the absorption maxima.
Next, we evaluated the fluorescence photophysical properties of
the fluorophores. Thus, for all the cases it was observed that the ef-
fect of the solvent polarity on the emission maxima was more pro-
nounced than that on the absorption maxima (Figs. 2–4). The
fluorescence spectra of all the compounds consisted of structure-
less broad band except in highly low polar solvent like hexane,
cyclohexane where some structured bands appeared. However,
an increase in the polarity of the solvent led to large Stokes shift
of emission maxima for compound 1 and comparatively less for
compound 2. Thus, from the fluorescence spectra of imide fluoro-
phore 1 (Fig. 2a and b), it was clear that as the solvent polarity in-
creases a strong red shift of about 140 nm (kmax,hexane 474 nm, and
kmax,chloroform 611 nm) was observed with decrease in both the fluo-
rescence intensity and the quantam yield (Table 1). Therefore, the
fluorophore 1 is highly solvatochromic. The red shift and signal
broadening of the spectra indicated that the conjugation is ex-
tended well between the acceptor imide carbonyl group and the
donor N,N-dimethylanilino unit through internal alkyne. Same
observation was reflected in the fluorescence images of the com-
pound in different solvents at a wavelength of 254 nm under a
transilluminator (Fig. 2c). Thus, in hexane/cyclohexane it showed
a strong greenish blue color while the color changed from greenish
blue to yellow in toluene, pink in dioxane and ultimately to red in
chloroform. Surprisingly, no emission was observed when polarity
of the solvent increased to methanol through ethylacetate.
Red colored crystalline solid nature, highly ICT character and
non-emissive nature of the fluorophore 1 in protic polar solvent
drew our interest to evaluate its’ solid state photophysical property
for possible future material applications. Thus, the solid state UV–
visible spectra showed two absorption bands at 444 and 560 nm
(Fig. 3a). The solid state emission spectra revealed a strong emis-
sion at 721 nm with a Stokes shift of 161 nm when excited at its
long wavelength absorption band (Fig. 3b). The time resolved fluo-
rescence in solid state showed a longer life time (s1 = 5.59 nm)
compared to its solution state (Fig. 3c). More interestingly, the
gas phase/solution phase energy gap between HOMO and LUMO
(2.6–2.7 eV) remained very closer to that of a band gap of an organ-
ic semiconductor.9 Thus, our observations on solid state photo-
physical property clearly showed that the fluorophore 1 might

S. S. Bag et al. / Bioorg. Med. Chem. Lett. 23 (2013) 96–101
99
(a)
(b)
(c)
1.2
1.2
0.8
0.4
0
3000000
2000000
1000000
0
CyHex
Toluene
CH3CN
5% wt
25 nm shift
CyHex
50 nm shift
Toluene
Dioxane
EtOAc
CHCl3
EtOH
CH3CN
MeOH
TFE
Dioxane
EtOAc
CHCl3
EtOH
CH3CN
MeOH
TFE
10% wt
15% wt
20% wt
25% wt
40% wt
50% wt
60% wt
0.8
0.4
0
375
475
575
375
475
Wavelength (nm)
575
380
480
580
Wavelength (nm)
Wavelength (nm)
Figure 4. (a) Emission and (b) normalized emission spectra of 2 (4-CPENI) in different solvents. (c) Normalized emission spectra in CH3CN titrated with water (10
kex = 370 nm).
lM, rt;
Table 1
Photophysical data of substituted naphthalimide 1 and 2
Ufa
s1 (ns)
Fluorophore 1 (4-DMAPENI)
v2
kr (109 sꢀ1
)
knr (109 sꢀ1
)
max
abs
max
em
Solvents
k
k
Hexane
Cyclohexane
Toluene
Dioxane
Ether
CHCl3
EtOAc
THF
DMSO
453
459
454
448
446
467
449
456
470
453
459
474
480
548
572
577
611
—
—
—
—
—
0.79
0.66
0.53
0.24
0.23
0.14
—
—
—
—
—
2.80
2.84
3.74
3.75
—
2.58
—
—
1.05
1.02
1.03
1.17
—
1.04
—
—
0.28
0.23
0.14
0.06
—
0.05
—
—
0.07
0.12
0.13
0.20
—
0.33
—
—
—
—
—
—
—
—
—
—
—
—
—
—
CH3CN
MeOH
Ufa
s1 (ns)
v2
kr (109 sꢀ1
)
knr (109 sꢀ1
)
max
abs
max
k
k
em
Fluorophore 2 (4-CPENI)
Dioxane
Toluene
EtOAc
CHCl3
389
392
387
393
387
387
387
387
387
372
403
408
406
411
423
431
436
441
434
442
0.57
1.45
1.62
1.52
1.38
1.66
1.74
1.83
1.91
1.79
2.05
1.11
1.03
1.17
1.16
1.13
1.08
1.07
0.99
1.04
1.10
0.39
0.29
0.36
0.35
0.32
0.30
0.22
0.16
0.30
0.27
0.30
0.32
0.29
0.37
0.28
0.27
0.32
0.36
0.25
0.22
0.48
0.56
0.49
0.54
0.53
0.41
0.32
0.54
0.55
CH3CN
10% H2O in CH3CN
30% H2O in CH3CN
50% H2O in CH3CN
MeOH
TFE
a
The fluorescence quantum yields (Uf) were determined using perylene10a,10b as a reference with the known Uf = 0.94 in cyclohexane for fluorophore 1 and in quinine
sulphate10c as a reference with the known Uf = 0.54 in 0.1 molar solution in sulfuric acid for 2.
find applications in material sciences such as in organic semicon-
ductors/electronic devices.
acceptor played an important role in the large dipole change dur-
ing excitation and made fluorophore 1 highly solvatochromic.
To get an insight into the strong solvent polarity sensitive emis-
sion behaviors of compounds 1–2, the fluorescence spectral depen-
After investigating the photophysical properties of fluorophore
1 both in solution and in solid state we next evaluated the fluores-
cence photophysical property of fluorophore 2. Thus, for the case of
acceptor containing naphthalimide fluorophore 2, upon changing
the solvent polarity, both the intensity and the quantam yield of
emission were found to be increased with a moderate Stokes shift
of about 50 nm when compared between the solvents cyclohexane
and TFE (Fig. 4a–c).
dency on solvent polarity parametres (
Df) was studied on the basis
of Lipert-Mataga model.11 Thus, a good linear correlation of the
abs
fl
~
~
absorption and fluorescence maxima (mmax, and mmax respectively,
in cmꢀ1) of fluorophores 1–2 with the solvent polarity functions
~
D
f indicated that for the fluorophore 1, the mabs values apparently
correlated linearly with
Df. This result suggested that the ground
Overall, it was observed that the fluorescence quantum yields
markedly decreased with increase in solvent polarity for the case
of fluorophore 1. Protic solvents, like methanol, are known to in-
duce fluorescence quenching through non-radiative pathways via
hydrogen bonding. Thus, the fluorescence in methanol was died
away due to protic solvent–solute interactions. These properties
indicated that compound 1 has strong charge transfer (CT) charac-
ter. The conjugation between the N,N-dimethyl aminophenyl moi-
ety as an electron donor and the naphthalimide core as an electron
state of 1 was moderately polar in nature. However, a less correla-
tion was observed in case of the fluorophore 2, suggesting a nonpo-
lar ground state (see Supplementary data). The reasonably high
~
slopes of mfl versus Df plots for all the fluorophores 1–2 further sug-
gested that the fluorescent states of these fluorophores were highly
polar in nature, most possibly of intramolecular charge transfer
(ICT) character.12,13 Thus, the change of emission wavelength of
the fluorophores with solvent polarity indicated their potential
~
ICT features. A good linear correlation with large slopes of the Dm

100
S. S. Bag et al. / Bioorg. Med. Chem. Lett. 23 (2013) 96–101
versus
D
f plots suggested that the fluorescence states were highly
located at 445 and 395 nm, respectively, showed a negligible ef-
fect on the change in wavelength position and intensity as ct-
DNA was added gradually (see Supplementary data).16 Next,
fluorescence titration experiment was carried out. Thus, upon
polar in nature for all the fluorophores (See Supplementary data).
Substantially high values of (le g) obtained from the plot
ꢀ
l
(22.6 D for 1 and 13.9 for 2) indicated that the fluorescence state
of 1 is of higher ICT character than 2 (see Supplementary data).
The excited state dipole moment determined from Stokes shift
was found to be more than the ground state for fluorophore 1. This
might be associated with partial transfer of electron from the do-
nor N,N-dimethylamino group to the acceptor naphthalimide core.
To understand the fluorescence behavior more precisely, we
measured fluorescence life time in different solvents and found
that the decay followed a single exponential fitting for all the fluo-
rophores. The effect of polarity was very similar to what was ob-
served for the fluorescence quantum yield (Table 1). While in
case of compound 1 increase in the polarity of the solvent led to
a shortening of the lifetime, in case of compound 2, life time in-
creased as the polarity of the solvent was increased. The depen-
dence of the quantum yield of fluorescence of 1 on the nature of
the solvent was mainly dictated by changes in the rate of radiation-
less deactivation as was revealed from the change in the rate con-
stants of radiationless deactivation on going from hexane to
chlorofom (Table 1). However, the opposite was obviously the re-
sult in case of fluorophore 2 correlating its dependency of quantum
yield with the change in solvent polarity. Time dependent DFT cal-
culation14 also revealed that the emissive state of 1 was character-
ized with more significant electron redistribution, that is, ICT
feature and rationalized the explanation of ICT origin and the sol-
vent polarity dependency of the fluorophores’ emission (see Sup-
plementary data).
Being inspired by the interesting solvatochromic emission
property, we next turned our attention to explore the possible
sensing of microenvironment of ct-DNA using fluorophore 1 and
2. Naphthalimides are well known as probes of nucleic acids and
many naphthalimide chromophores have been utilized in fluores-
cence based elucidation of structure, dynamics and conformation
of proteins.4c,3d,4d–h Therefore, we envisaged that the naphthali-
mide moiety of our probes might significantly interact with the
aromatic bases in DNA via intercalative stacking/H-bonding and/
or electrostatic interaction. This idea along with the intense emis-
sion of the probes in low polar media compared to high polar med-
ia and/or buffer drew our attention to exploit fluorophores 1–2 in
the possible sensing of microenvironment of calf-thymus DNA (ct-
DNA) which is easily available biomolecules with widespread
applications.15
gradual
addition
of
ct-DNA,
the
emission
intensity
(kem = 525 nm) of the fluorophore 2 was found to be significantly
enhanced with slightly red shifted pattern. The emission became
saturated at 75 lM concentration of ct-DNA compared to 30 lM
probe concentration (Fig. 5a). This observation clearly indicated a
well-defined binding of the probe 2 with ct-DNA. On the con-
trary, fluorophore 1 which is of greater ICT character compared
to fluorophore 2, showed a sudden enhancement of fluorescence
intensity with very little blue shifted pattern at probe: ct-DNA
concentration ratio of 1:1. Beyond this ratio no regular trend
in decreased emission upon addition of increasing amount of
ct-DNA was observed when excited at 460 nm (Fig. 5c). The
emission response of fluorophore 2 upon binding with ct-DNA
represented the possible binding of the probes along the groove
side thereby facing more polar microenvironment leading to an
enhancement of fluorescence intensity of probe 2 and an anom-
alous behavior in emission for the case of fluorophore 1. The
quenching incidence of probe 1 could easily be explained if we
consider the non-radiative deactivation via solvent solute H-
bonding interaction when exposed to the more polar microenvi-
ronment along the groove side. These observations are in accord
with the intrinsic emission behaviors of the probes as was rev-
eled from the solvent polarity dependent emissions of each fluo-
rophores.15 The association constant of probe 2 with ct-DNA was
also determined by a Benesi-Hildebrand plot (inset, Fig. 5a)
which was found to be 3.98 ꢁ 104 Mꢀ1 with an experimental free
energy of binding, G = ꢀ6.3 kcal/mol.
Thermal denaturation experiment indicated no destabilization
of ct-DNA upon binding with the probe 2 suggesting that the probe
possibly bind to a groove of ct-DNA (see Supplementary data).16 To
investigate the binding mode of the probes with ct-DNA an inter-
calation or a groove binding, a competitive binding experiment
was performed. Thus, upon addition of increasing amount of probe
1 and/or 2, no significant change in fluorescence of ethidium bro-
mide of EB–ct-DNA complex was observed indicating that both
the probes were not interacting with ct-DNA as intercalators but
may be groove binders (see Supplementary data).17 Though, naph-
thalimides are known as intercalators,4c,3d,4d–h because of ap-
pended propergyl units the probes were unable to intercalate
sterically between the DNA bases rather they preferred to bind
Therefore, we studied the interaction behavior of our probes
with ct-DNA by spectroscopic means in aqueous phosphate buf-
fer (pH 7.0). Thus, the absorption maxima of fluorophore 1 and 2
along side the groove position of ct-DNA via possible p-stacking/
H-bonding interactions which was also supported from the fluo-
rescence experiment.
(c)
(a)
(b)
20000
15000
10000
5000
0
900000
3.0
Eqv. of ct-DNA
R = 0.98304
0.0
K = 3.98 x 104 M-1
0.0
2.5
0.25
0.5
0.25
2.0
0.50
0.75
1.0
0.75
1.5
600000
1.0
1.5
1.5
2.0
2.5
1.0
1
2
3
4
5
2.0
[ct-DNA]-1 104 M-1
2.5
300000
0
400
500
600
700
475
525
575
625
675
Wavelength (nm)
Wavelength (nm)
Figure 5. (a) Fluorescence titration of probe 2 (kex = 362 nm). Inset: Benesi-Hildebrand plot for determination of association constant in respective media. (b) Amber⁄ energy
minimized geometry of probe 2 with DNA, showing the minor groove binding mode of the probe. The DNA sequence was 50-d(⁄CP⁄GP⁄CP⁄GP⁄AP⁄AP⁄TP⁄TP⁄CP⁄GP⁄CP⁄G)-30,
(PDB Id: 1DNH). (c) Fluorescence titration of probe 1 (kex = 460 nm) with various concentration of ct-DNA ([probe] = 30 lM, phosphate buffer, pH 7.0, rt).

S. S. Bag et al. / Bioorg. Med. Chem. Lett. 23 (2013) 96–101
101
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To support minor groove binding event, we have carried out
MacroModel calculation by Maestro, version 9.0 with AMBER⁄
force field in ⁄water.1⁄8 For the opti⁄mizati⁄on we ⁄chose the DNA se-
quence [50-d( CP⁄GP CP⁄GP⁄AP⁄AP TP⁄TP CP⁄GP CP⁄G)-30, (PDB Id:
1DNH)] where Hoechst 33258 dye enter into the minor groove.
Thus, Amber⁄ optimized geometry of our probe 2 with the model
DNA sequence (Fig. 5b) showed and support our experimental
observation of groove binding event especially in the minor groove.
Therefore, it was clear from the above findings that the probe 2
was more efficient compared to probe 1 in sensing microenviron-
ment of ct-DNA via the generation of enhanced fluorescence signal.
The low fluorescence intensity of the probes in phosphate buffer in
absence of biomolecules is not due to the insolubility of the probe
but may be attributed to the radiationless channel assisted by
intermolecular hydrogen bonding present in aqueous solution.5a,b
However, as the probes bound to the groove side they experienced
restricted radiationless channel inside the groove of ct-DNA ulti-
mately leading to a fluorescence switch-on signal with high inten-
sity and quantum yield.
In conclusion, we developed new donor/acceptor conjugated
fluorescent naphthalimide based fluorophores. The fluorescence
of naphthalimide containing terminal alkynes were of highly ICT
character and very sensitive to solvent polarity. We showed that
the died down fluorescence of probe 1 in buffer could be recovered
in presence of ct-DNA. We also investigated that both the probes
are capable of sensing of microenvironment of ct-DNA via a gener-
ation of enhanced fluorescence signal. The solid state fluorescence
property of the probe 1 might find application in materials sci-
ences. The exploitation of the terminal acetylenes to the synthesis
of fluorescently labeled biomolecular building blocks such as la-
beled nucleosides and amino acids and their applications thereof
is our current research target.
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Acknowledgments
We thank Department of Science and Technology [DST: SR/SI/
OC-69/2008], Govt. of India, for a financial support. M.K.P. and
S.J. thank UGC and CSIR respectively, Government of India and
R.K. thanks IIT Guwahati for their fellowships.
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Supplementary data
Supplementary data (experimental procedure, characterization,
photophysical spectra, DFT calculation, macromodel structures and
crystal structure) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmcl.2012.11.003.
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