Benzimidazole quinoline derivatives - An effective green fluorescent dye for bacterial imaging

Article abstract of DOI:10.1139/V09-139

Abstract: A one-pot synthesis of benzimidazoles by condensing naphthyl or quinoline aldehyde with benzene- 1 ,2-diamine has been reported. IR, ¹H and ¹³C NMR, mass spectral, and CHN analyses were used to elucidate the structures of t

Full text of DOI:10.1139/V09-139

1692
Benzimidazole quinoline derivatives — An
effective green fluorescent dye for bacterial
imaging
Mahalingam Malathi, Palathurai Subramaniam Mohan, Raymond J. Butcher, and
Chidambaram Kulandaisamy Venil
Abstract: A one-pot synthesis of benzimidazoles by condensing naphthyl or quinoline aldehyde with benzene-1,2-diamine
1
has been reported. IR, H and 13C NMR, mass spectral, and CHN analyses were used to elucidate the structures of the
products. The molecular structural correlation in the optical properties of the quinoline and naphthalene benzimidazoles
was explored. The fluorescence quantum yield (ff) and time-resolved fluorescent lifetime of the quinoline benzimidazoles
derivatives were estimated. The influence of solvent polarity and pH on the optical property of quinoline derivatives was
illustrated. To explore the bioanalytical applicability, the thermal stability by TG–DTA analysis and the cytogenetic analy-
sis of 3-(1H-benzoimidazol-2-yl)-2-chloro-8-methyl-quinoline (1b) compound were carried out. The fluorescent staining
ability of 1b was analyzed and also compared with the normal Gram staining in the bacterium.
Key words: benzimidazole quinolines, optical property, fluorescence quantum yield, cytogenetic analysis, fluorescence
staining.
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Resume : On a realise une synthese monotope de benzimidazoles par condensation de naphtyl- et de quinoleinealdehydes
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avec de la benzene-1,2-diamine. On a fait appel aux analyses CHN et aux spectroscopies IR, RMN H et 13C et de masse
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pour elucider la structure des produits. On a explore la correlation de la structure moleculaire dans les proprietes optiques
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des benzimidazolylquinoleines et des benzimidazolylnaphtalenes. On a evalue le rendement quantique de fluorescence (ff)
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et le temps de vie fluorescent resolu en fonction du temps des derives benzimidazolylquinoleines. On a aussi illustre l’in-
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fluence de la polarite du solvant et du pH sur les proprietes optiques de ces derives de la quinoleine. Afin de pouvoir eva-
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luer les possibilites de l’utiliser dans le domaine des analyses biologiques, on a aussi effectue une analyse cytogenetique et
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on a evalue la stabilite thermique de la 3-(1H-benzimidazol-2-yl)-2-chloro-8-methylquinoleine (1b) par analyse thermogra-
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vimetrique (TG) et analyse thermique differentielle (ATD). On a analyse la capacite du compose 1b a agir comme colorant
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fluorescent et on a compare ses proprietes a celles de la coloration Gram normale dans les bacteries.
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Mots-cles : benzimidazolylquinoleines, propriete optique, rendement quantique de fluorescence, analyse cytogenetique, co-
loration de fluorescence.
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[Traduit par la Redaction]
shift away from the use of radioactive tracers in the bioana-
lytical study. Particularly, it eliminates the dangers of han-
dling carcinogenic dyes, radioactive materials, and the cost
of their proper disposal such as crystal violet, ethidium bro-
mide, and 32P in DNA staining analysis.
Accordingly, much research has been done to develop
one-photon fluorescent (OPF) probes because they are useful
tools for know how the functions in biological systems. One
of the major drawbacks of such probes is that the excitation
wavelengths are in the range of 350–560 nm, which may
cause damage to the substrates specifically in the living
Introduction
Rapid advances in optical technologies have recently led
to significant progress in medical diagnostics, drug discov-
ery, and (bio)chemical analysis, in particular for methods
that use fluorescence detection in microscopy, for microar-
rays or single-molecule tracking.1 These developments have
fueled the search for novel fluorophore (stains, labels, and
indicators)2 with high brightness that can be excited and
which could emit well within the visible or near-infrared
(NIR) region of the spectrum.3 This is a part of continual
Received 23 March 2009. Accepted 27 August 2009. Published on the NRC Research Press Web site at canjchem.nrc.ca on 6 November
2009.
Abbreviations: OPF: one-photon fluorescent. TPF: two-photon fluorescent. FTIR: fourier transform infrared. UV–vis: ultraviolet–visible.
NMR: nuclear magnetic resonance. XRD: X-ray diffraction. TG–DTA: thermogravimetric and differential thermal analysis. EtOAc: ethyl
acetate. EtOH: ethanol.
M. Malathi and P.S. Mohan.1 Department of Chemistry, Bharathiar University, Coimbatore 641 046, India.
R.J. Butcher. Department of Chemistry, Howard University, WA, USA.
C.K. Venil. Department of Environmental Science, Division of Microbiology, Bharathiar University, Coimbatore 641 046, India.
1Corresponding author (e-mail: ps_mohan_in@yahoo.com).
Can. J. Chem. 87: 1692–1703 (2009)
doi:10.1139/V09-139
Published by NRC Research Press

Malathi et al.
1693
cells.5 A fluorescent probe having higher excitation wave-
length6 overcomes these shortcomings in two ways: (i) using
lower energy photon can cause less damage to the cells or
tissues and (ii) lower energy photon has longer wavelength
that can penetrate deep inside the tissue. Many applications
of such materials have become reality, some of which in-
clude optical power limiting,7 two-photon (TP) upconversion
lasing,8 TP fluorescence excitation microscopy,9 three-di-
mensional optical-data storage,10 and photodynamic ther-
apy.11 An important addition to such applications would be
the development of TP probes for bioanalytical applications.
As a result, the design and synthesis of fluorescent probes
with higher excitation wavelength have attained the central
position of the current research.12
Scheme 1. Quinoline and naphthalene benzimidazoles.
A detailed search of structural correlation in the fluorescent
probes reveals that the benzimidazole derivatives could act as
an effective fluorescent probe.13 In addition to the biological
activity,14 many of benzimidazole derivatives are studied as
luminescent singling sensors for anions.15 Recently, Zang et
al.16 have reported a Y-shaped TP-active material based on
the imidazole core. Consecutively, Iyer et al.17 have reported
that the benzimidazole ring in a heteroaromatic system could
exhibit efficient fluorescence-light-emitting property. All the
reports reveal that the benzimidazole and its derivatives have
different fluorescence efficiencies, depending on the molecu-
lar structure. Consequently, the molecular-structure-based de-
signing is one of the convenient tools for the innovation of the
new efficient fluorescent probes.
In this connection, quinoline system gains importance due
to its recent revolution in optical chemistry.18 Basically, qui-
noline derivatives have been synthesized and widely utilized
as drugs for antimalarial, anticancer, and also as DNA inter-
calators.19 In contrary, there has been a paucity of impera-
tive papers account that the quinoline derivatives could be
used as selective fluorescence chemosensors for biologically
important metal ions, as fluorescent dopent in OLEDs and
electrochemiluminescence,20 and so forth. This kindles inter-
est to study the optical property of the benzimidazole-substi-
tuted quinoline derivatives. Consecutively, the synthesis of
benzimidazole naphthalene derivatives also intended to
study the role of quinoline system in the optical property of
the synthesized quinoline benzimidazole derivatives.4
cording to the reported procedure.21 The naphthalene
carbaldehyde starting material was purchased from Sigma-
Aldrich chemicals and used without further purification.
FTIR spectra (4000–400 cm–1) of compounds were recorded
as KBr pellets with a Nicolet Avatar Model FTIR spectro-
1
photometer. H and 13C NMR spectra were recorded on a
Bruker Avance 400 (400 MHz) spectrometer in CDCl3 as a
solvent and tetramethylsilane (TMS) as an internal standard.
Mass spectra were recorded on a Trace DSQ mass spectro-
graph. Microanalyses (C, H, and N) were performed on a
Vario EL-III Elementar elemental analyzer. UV–vis absorp-
tion spectra (200–800 nm) were recorded using a Shimadzu
UV/2501PC spectrophotometer. The fluorescence spectra
were recorded using JASCO FP-6600 spectrofluorometer
(Xenon lamp). The fluorescence microscopy analysis was
done by using Nikon fluorescence microscopes.
Synthesis of benzimidazole compounds
Scheme 2 outlines the general procedure for the synthesis
of quinoline and naphthalene benzimidazole derivatives. The
aldehyde compound (3 or 4) in chloroform was stirred with
two drops of glacial acetic acid. To this, benzene-1,2-dia-
mine (5) was added, and the stirring was continued for about
15 min. The formation of product was confirmed by TLC.
Excess CHCl3 was evaporated, and the resulting crude was
washed with water. The dried crude was adsorbed for col-
umn chromatography using petroleum ether and EtOAc mix-
ture as eluent. The benzimidazole product was isolated in
petroleum ether/EtOAc (9:1) medium. The obtained product
On par with our objective, we propose a conventional
synthetic methodology for the synthesis of quinoline and
naphthalene benzimidazole derivatives (BIQ, BIN) in high
yield (see Scheme 1 for structures). The optical properties
of the synthesized compounds were analyzed by using ab-
sorption and emission spectral studies. Based on the fluores-
cence property, the applicability of the compounds in the
bioanalytical studies is focused.
1
was characterized by IR, H and 13C NMR, mass, and ele-
mental analyses.
Synthesis of 3-(1H-benzoimidazol-2-yl)-2-chloro-quinoline
(1a)
The compound was prepared as described above by the
reaction of 2-chloro-quinoline-3-carbaldehyde (10 mmol,
1.73 g) and benzene-1,2-diamine (5 mmol, 0.541 g). Off-
white solid (yield: 1.25 g, 89.33%); mp 202 8C. IR (KBr):
Experimental section
1
Materials and physical measurements
3424, 1607, 1405, 1034, 741. H NMR (400 MHz, CDCl3)
All reagents and chemicals were purchased from Ran-
chem chemicals. All the solvents were HPLC-grade. Thin-
layer chromatography (TLC) was performed using glass
plates coated with silica gel-G containing 13% calcium sul-
phate as binder. Melting points were determined using a
Raga melting point apparatus and are uncorrected. The start-
ing quinoline carbaldehyde compounds were synthesized ac-
d: 7.27 to 7.38 (m, 2H, C6–quinoline–Q–H, C3’–benzo–BE–
H), 7.60–7.64 (m, 2H, C2’ and C5’ of BE–H), 7.79–7.83 (m,
2H, C7–Q–H and C4’–BE–H), 7.92–7.94 (d, 1H, J = 8 Hz,
C5–Q–H), 8.03–8.05 (d, 1H, J = 8 Hz, C8–Q–H), 9.26 (s,
1H, C4–Q–H), 10.59 (bs, NH–imidazole–IMD–H). 13C
NMR d: 120.22, 122.66, 123.84, 126.31, 127.10, 127.45,
127.81, 130.59, 131.30, 131.80, 136.21, 140.83, 141.11,
Published by NRC Research Press

1694
Can. J. Chem. Vol. 87, 2009
Scheme 2. Preparation of quinoline and naphthalene benzimidazole derivatives.
146.68, 147.03, 147.38. EI-MS: m/z = [M+] 279, [M + 2]+
281. Anal. calcd. for C16H10N3Cl: C, 68.70; H, 3.60; N,
15.02. Found: C, 68.55; H, 3.48; N, 14.78.
Synthesis of 3-(1H-benzoimidazol-2-yl)-2-chloro-6-methyl-
quinoline (1d)
The compound was prepared as described above by the
reaction of 2-chloro-6-methyl-quinoline-3-carbaldehyde
(9 mmol, 1.85 g) and benzene-1,2-diamine (4.5 mmol,
0.4866 g). Off-white solid (yield: 1.305 g, 88.80%); mp
Synthesis of 3-(1H-benzoimidazol-2-yl)-2-chloro-8-methyl-
quinoline (1b)
1
220 8C. IR (KBr): 3443, 1605, 1334, 1024, 762. H NMR
The compound was prepared as described above by the
reaction of 2-chloro-8-methyl-quinoline-3-carbaldehyde
(9 mmol, 1.85 g) and benzene-1,2-diamine (4.5 mmol,
0.4866 g). Off-white solid (yield: 1.4 g, 89.82%); mp
(400 MHz, CDCl3) d: 2.64 (s, 3H, C6–Q–CH3–H), 7.34–
7.38 (m, 2H, C3’ and C4’–BH–H), 7.48–7.52 (m, 2H, C5
and C7–Q–H), 7.76–7.81 (d, 3H, C8–Q–H, C2’ and C5’–
BH–H, J = 8 Hz), 9.25 (s, 1H, C4–Q–H), 10.42 (bs, NH–
IMD–H). 13C NMR d: 22.03, 120.25, 122.67, 123.02,
126.32, 127.11, 127.45, 127.81, 130.59, 131.32, 131.81,
136.21, 140.83, 141.11, 146.69, 147.03, 147.40. EI-MS: m/
z = [M+] 293, [M + 2]+ 295. Anal. calcd. for C17H12N3Cl:
C, 69.51; H, 4.12; N, 14.31. Found: C, 69.32; H, 3.89; N,
14.14.
1
215 8C. IR (KBr): 3432, 1604, 1330, 1024, 760. H NMR
(400 MHz, CDCl3) d: 2.78 (s, 3H, C8–Q–CH3–H), 7.34–
7.38 (m, 2H, C3’ and C4’ BE–H), 7.48–7.52 (t, 1H, C6–Q–
H, J = 8 Hz), 7.62–7.64 (d, 1H, C7–Q–H, J = 8 Hz), 7.74–
7.76 (d, 3H, C5–Q–H, C2’ and C5’ BE–H, J = 8 Hz), 9.22
(s, 1H, C4–Q–H), 10.40 (bs, NH–IMD–H). 13C NMR d:
22.05, 120.31, 122.26, 123.42, 126.27, 126.99, 127.52,
127.82, 130.59, 131.34, 131.81, 136.22, 140.84, 141.11,
146.69, 147.12, 147.38. EI-MS: m/z = [M+] 293, [M + 2]+
295. Anal. calcd. for C17H12N3Cl: C, 69.51; H, 4.12; N,
14.31. Found: C, 69.43; H, 3.98; N, 14.18.
Synthesis of 2-(3-chloro-naphthalen-2-yl)-1H-
benzoimidazole (2a)
The compound was prepared as described above by the
reaction of 3-chloro-naphthalene-2-carbaldehyde (9 mmol,
1.72 g) and benzene-1,2-diamine (4.5 mmol, 0.4866 g). Yel-
lowish orange solid (yield: 1.125 g, 89.70%); mp 210 8C. IR
Synthesis of 3-(1H-benzoimidazol-2-yl)-2-chloro-7-methyl-
quinoline (1c)
1
(KBr): 3470, 1614, 1321, 744. H NMR (400 MHz, CDCl3)
The compound was prepared as described above by the
reaction of 2-chloro-7-methyl-quinoline-3-carbaldehyde
(9 mmol, 1.85 g) and benzene-1,2-diamine (4.5 mmol,
0.4866 g). Off-white solid (yield: 1.305 g, 88.80%); mp
d: 7.25–7.34 (m, 4H, C6 and C7–naphthalene–NA, C3’ and
C4’–BH–H), 7.65–7.67 (d, 2H, C5 and C8–NA, J = 8 Hz),
7.69–7.75 (m, 3H, C4–NA, C2’ and C5’–BE–H), 7.84 (s,
1H, C1–NA–H), 9.5 (bs, NH–IMD–H). 13C NMR d: 115.4,
115.5, 122.9, 123.0, 126.1, 126.9, 127.0, 127.1, 127.8,
128.0, 130.1, 132.2, 134.3, 134.7, 137.9, 138.0, 141.5. EI-
MS: m/z = [M+] 278, [M + 2]+ 280. Anal. calcd. for
C17H11N2Cl: C, 73.26; H, 3.98; N, 10.05. Found: C, 73.15;
H, 3.79; N, 9.85.
1
220 8C. IR (KBr): 3435, 1606, 1370, 1044, 752. H NMR
(400 MHz, CDCl3) d: 2.57 (s, 3H, C7–Q–CH3–H), 7.33 to
7.38 (m, 3H, C6–Q–H, C3’ and C4’–BE–H), 7.48–7.45 (dd,
1H, J1 = 8 Hz, J2 = 4 Hz, C5–Q–H), 7.83–7.86 (m, 3H, C8–
Q–H, C2’ and C5’–BE–H), 9.25 (s, 1H, C4–Q–H), 10.42 (bs,
NH–IMD–H). 13C NMR d: 22.03, 120.25, 121.92, 123.39,
126.32, 124.82, 127.15, 127.78, 130.13, 131.32, 131.81,
136.21, 140.82, 142.75, 145.80, 147.39, 147.66. EI-MS: m/
z = [M+] 293, [M + 2]+ 295. Anal. calcd. for C17H12N3Cl: C,
69.51; H, 4.12; N, 14.31. Found: C, 69.42; H, 3.97; N, 14.12.
Synthesis of 2-(3-chloro-5-methyl-naphthalen-2-yl)-1H-
benzoimidazole (2b)
The compound was prepared as described above by the
Published by NRC Research Press

Malathi et al.
1695
reaction of 3-chloro-5-methyl-naphthalene-2-carbaldehyde
(9 mmol, 1.84 g) and benzene-1,2-diamine (4.5 mmol,
0.4866 g). Yellowish orange solid (yield: 1.132 g, 85.65%);
mp 225 8C. IR (KBr): 3456, 1607, 1332, 756. 1H NMR
(400 MHz, CDCl3) d: 2.61 (s, 3H, C6–Q–CH3–H), 7.20–
7.29 (m, 4H, C6 and C7–NA–H, C3’ and C4’–BE–H), 7.59–
7.63 (d, 2H, C5 and C8–NA–H, J = 8 Hz), 7.65–7.72 (m,
3H, C4–NA–H, C2’ and C5’–BE–H), 7.82 (s, 1H, C1–NA–
H), 9.2 (bs, 1H, NH–IMD–H). 13C NMR d: 20.63, 114.2,
114.5, 121.9, 122.0, 125.1, 125.9, 127.2, 127.5, 127.9,
128.0, 130.2, 132.2, 134.2, 134.5, 137.5, 138.2, 140.5. EI-
MS: m/z = [M+] 292, [M + 2]+ 294. Anal. calcd. for
C17H11N2Cl: C, 73.85; H, 4.48; N, 9.57. Found: C, 73.65;
H, 4.34; N, 9.47.
viding cells were arrested in the metaphase by adding
0.05 mL of colchicine solution (w = 0.01%). The contents
in the vial were centrifuged at 1200 rpm for 5 min at the
end of the colchicine treatment. The supernatant was dis-
carded, and 5 mL of pre-warmed hypotonic solution
(0.075 mol L–1 KCl) was added to the cell button. The cells
were incubated for 20 min, sedimented after centrifugation
at 1200 rpm for 10 min, and then fixed in a freshly prepared
fixative (methanol/glacial acetic acid, 3:1, v/v). Two or three
changes of the fixative were applied. Slides were prepared
by placing a drop of the cell suspension on a clean chilled
slide and dried immediately at 40 8C for a few seconds.
The slides were routinely stained in a 2% buffered solution
of Giemsa.23 Three-hundred-and-fifty well-banded meta-
phases were analyzed for each treatment under an oil im-
mersion lens.
Optical measurements
For the absorption analysis, stock solution was prepared
by dissolving 10 mg of BIQ derivatives in 100 mL ethanol.
The solution was diluted to 10–5 mol/L concentration for the
absorption and 10–7 mol/L for the emission analysis. But in
the case of solvent polarity study of 1b, the compounds in
the specified solvent medium were only used for both ab-
sorption and emission analysis. The fluorescence intensity
vs. pH titration was carried out using 10% aqueous alcoholic
solution of the BIQ-1b compound. 0.1 mol/L HCL and
0.1 mol/L NaOH were used to adjust the pH of the medium.
Fluorescence decays were recorded using TCSPC method
using the following setup. A diode-pumped millena CW la-
ser (Spectra Physics) 532 nm was used to pump the Ti:sap-
phire rod in Tsunami picosecond mode locked laser system
(Spectra Physics). The 750 (80 MHz) was taken from the
Ti:sapphire laser and passed through pulse picker (Spectra
Physics, 3980 2S) to generate 4 MHz pulses. The seond har-
monic output (375 nm) was generated by a flexible har-
monic generator (Spectra Physics, GWU 23PS). The
vertically polarized 375 nm laser was used to excite the
sample. The fluorescence emission at magic angle (54.78)
was dispersed in a monochromator (f/3 aperture), counted
by a MCP PMT (Hamamatsu R 3809), and processed
through CFD, TAC, and MCA. The instrument response
function for this system is ~52 ps. The fluorescence decay
was analyzed by using the software provided by IBH (DAS-
6) and PTI global analysis software.22
Staining analysis in the bacterial microbe
Potent strains of bacteria were isolated from the gut of
sulfur butterfly (Kricogonialyside). The isolation and cultur-
ing experimental procedures of the bacteria were carried out
using the reported methods.24 The confirmed Serratia mar-
cescens SB08 was used in the staining process. Initially, the
bacterium was heat-fixed in the clean glass slide. Two or
three drops of 10% ethanolic solution of 1b (10–9 mol/L)
was added to the heat-fixed bacterial smear and allowed to
bind for 15–60 s. After that, the slide was rinsed with dis-
tilled water and air-dried. Then, the slide was examined
under the fluorescence microscopy.
Results and discussion
Synthesis of benzimidazole derivatives
Recent exploration of pyrene- and benzimidazole-based
compounds as long-wavelength fluorophores25 raise our in-
terest to synthesize quinoline and naphthalene derivatives
containing benzimidazole unit and study their structural cor-
relation in the optical property. To achieve our objective, the
work is started by synthesizing benzimidazole compounds
from the derivatives of quinoline and naphthalene aldehydes
with 1,2-diaminobenzene (Scheme 2). Such quinoline benzi-
midazole derivative was first prepared by Bhanumathi et
al.26 in two-stage approach. Their synthetic method was
low-yielding, time-consuming, and did not seem amenable
for the synthesis of benzimidazole compounds as demanded
by the need of their application. Additionally, Kidwai et
al.27 reported a bisquinoline formation in the condensation
reaction of quinoline aldehyde with benzene-1,2-diamine.
Their reaction conditions and medium are similar to the
Bhanumathi et al. synthetic methodology. Recently, Chen
reported a one-pot synthetic methodology for the synthesis
of 2-benzimidazolyl quinoline by the direct condensation of
diamine with quinoline acid.28 In this sequence, the explora-
tion of reaction conditions and temperature for the synthesis
of benzimidazole derivatives from the diamine and alde-
hydes could be significant. It may give a valuable outlook
for the synthesis of complex benzimidazole derivatives.
Cytogenetic analysis
Cytogenetic analysis was performed for compound 1b in
human peripheral blood lymphocyte culture (PBLC). They
were obtained from peripheral blood set-up following the
method of Hungerford.23 Two experiments were carried out
using four smoking and non-smoking healthy male donors,
aged 23, 24, and 28 years. The testing compound was dis-
solved in 1% methanol. Its four different concentrations of
0.02, 0.2, 2, and 20 mg mL–1 were added to the culture me-
dium (0.1 mL solution of compound 1b per 8 mL of the me-
dium) after 48 h culture initiation. Duplicate cultures for
each dose were maintained for the study of chromosomal
aberrations. Then, cultures were incubated at 37 8C for a pe-
riod of 24 (total 72 h) h. Methanol (1%) was applied to con-
trol I and II, respectively.
Initially, the reaction was carried out with the 1:1 molar
ratio of reactants in chloroform with catalytic amount of gla-
cial acetic acid. This reaction conditions resulted in the for-
mation of a mixture of products. However, 2:1 molar ratio
The cultures were shaken periodically up to three times a
day. During the two hours before the culture harvest, the di-
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Can. J. Chem. Vol. 87, 2009
1
Table 1. Yields, NH H NMR, nNH IR, and absorption lmax values of quinoline, naphthalene benzoimidazoles 1a–1d and 2a, 2b.
a
Serial No.
Compound
Yield (%)
89.33
89.82
88.80
88.80
dH (ppm)
10.59
10.40
10.42
10.42
9.5
IR n(NH) (cm–1)
3424
3432
3435
3443
3470
3456
lmax in nmb (3 ꢁ 105 M–1 cm–1)
346 (1.89)
354 (1.94)
351 (1.92)
352 (1.93)
317 (2.55)
332 (2.62)
1
2
3
4
5
6
1a
1b
1c
1d
2a
2b
89.70
85.65
9.2
aNH proton of the benzimidazole ring in the BIQ and BIN (400 MHz, CDCl3).
bThe 30 mmol/L ethanolic solution of BIQ and BIN compounds were used in the UV–vis absorption analysis.
of the reactants, i.e., aldehyde and diamine, respectively,
produced a single product in the same reaction medium after
15 min. The purification of the products from the crude mix-
ture was done by column chromatography using petroleum
ether:ethyl acetate (9:1) mixture as eluent. The structure of
the products was confirmed by IR, NMR, mass, and elemen-
tal analyses. The single-crystal structure of one of the quino-
line benzimidazole derivatives (1b) was solved by X-ray
crystallographic technique and was reported.29 The charac-
terization analysis supports the formation of benzimidazole
derivatives using the 2:1 molar ratio of reactants. The same
reaction was carried out smoothly in a beaker without
changing the environmental condition, such as the high tem-
perature, inert atmosphere, and expensive catalysts. It is be-
lieved that the presence of excess aldehydes might help to
stop the backward reaction.
Fig. 1. The UV–vis absorption spectra of the BIQ and BIN com-
pounds in 30 mmol/L concentration: (i) absorption spectra of BIQ-
1b with lmax = 336 and 354; (ii) absorption spectra of BIN-2b with
lmax = 332 and 350 nm.
In the characterization analysis, a broad spectral correla-
tion could be observed specifically in the benzimidazole N–
H functional group of BIQ and BIN derivatives (Table 1).
The IR spectrum of the BIQ and BIN derivatives shows nNH
band in the region of 3420 –3445 and 3455–3470 cm–1, re-
spectively. The lower nNH wavelength for BIQ compounds
illustrates the existence of strong hydrogen bonding (Imida–
1
N–HꢀꢀꢀCl–Q) in their molecular structure. In addition, the H
excitation), exhibits two fluorescence peaks around lmax 432
and 851 nm (Fig. 2a). But the intensity of the first fluores-
cence peak is 10 times higher compared with the following
peak (10:1). The same solution, on excitation in the range of
lmax 700 –710 nm (double excitation), exhibits a fluores-
cence peak and a hump (Fig. 2b) around lmax 433 and
849 nm, respectively. In this double excitation, the intensity
ratio of the fluorescence peak and hump is also found to be
10:1. However, the fluorescence peak intensity in the single
excitation (350 –355 nm) is five times higher than the dou-
bly excited (700 –710 nm) fluorescence peak. This shows
the sensitivity of the double excitation process. All the spec-
trofluorimetric experiments of 1b were repeated in different
solvents, i.e., in CH2Cl2, CHCl3, DMF, DMSO, and EtOH.
The combined spectral data expose the double-excitation
property of 1b in the above mentioned solvent. Additionally,
it illustrates that the intensity of the fluorescent lmax is
higher in polar solvents than in non-polar solvents.
NMR spectra of BIQ and BIN derivatives show a pro-
nounced deshielding effects in the d value of BIQ–N–H
functional group (d 10.40 –10.59 and 8.52–8.55, respec-
tively), which further confirms the existence of strong hy-
drogen bonding (Imida–N–HꢀꢀꢀCl–Q) in the BIQ derivatives.
It is concluded that the nitrogen lone-pair electrons in the
quinoline ring could facilitate the existence of strong hydro-
gen bonding in the BIQ derivatives. This specific molecular
structural conformation will play a vital role in the optical
behavior of BIQ compounds compared with BIN com-
pounds.
Absorption and emission spectral studies
The optical analyses of BIQ and BIN compounds were re-
corded in ethanol. Figure 1 shows the absorption spectra of
compounds 1b and 2b. In general, UV–vis absorption spec-
tra of BIQ and BIN ethanolic solutions exhibit peaks with
lmax in the range of 340 to 355 nm (Table 1). lmax values
of the respective compounds was used to excite the corre-
sponding compound in the spectrofluorimetric analysis. The
resulting fluorescence spectra of BIQ and BIN compounds
prove the existence of fluorescence property only in BIQ
compounds.
All the observed results propose that the BIQ derivatives
should have intramolecular charge transfer (ICT) interaction
in the solution form. ICT occurs between the quinoline and
imidazole ring systems during electronic excitation. It re-
stricts the internal rotation of imidazole unit within the mol-
The absorption lmax of 1b is 354 nm in ethanol. This sol-
ution, on excitation in the range of lmax 350–355 nm (single
ecule, which creates a self-organized supramolecular
architecture. This molecular conformational arrangement of
Published by NRC Research Press

Malathi et al.
1697
Fig. 2. Fluorescence spectra of ethanolic solution of BIQ-1b in 0.3 mmol/L concentration: (a) Solid line: emission spectra of 1b in the
excitation wavelength lexc 354 nm and the resulting emission lemi = 434 and 852 nm; dashed line: emission spectra of 1b in the excitation
wavelength lexc = 708 nm and the resulting emission lemi = 447 nm. (b) Expansion form of fluorescence spectra of 2a from 750 to
1000 nm.
excited molecules can facilitate radiative decay during the
electronic relaxation process, while in the case of naphtha-
lene bearing imidazole moiety, the absence of ICT can sus-
tain the internal rotation, thereby the electronic relaxation
frequently takes place as non-radiative decay process.30
Spectrofluorimetric analysis of other derivatives of BIQ (1a,
1c, and 1d) also exhibits the higher wavelength excitation
and fluorescence emission property in solution.
Fig. 3. Fluorescence quantum-yield measurement of 1b (mmol/L
concentration range). (i) Fluorescent vs. absorption intensities of 2-
aminopyridine in 0.1 mol/L H2SO4 solution, lexc = 335 nm and
lemi = 370 nm. (ii) Fluorescent vs. absorption intensities of BIQ-1b
in ethanol, lexc = 354 nm and lemi = 434 nm.
Fluorescence quantum-yield measurement
Fluorescence quantum yield (ff) of the BIQ derivatives
(x) in ethanol was determined using 2-amino pyridine as a
standard (std), since both compounds have excitation wave-
length in the range of 340–450 nm. 0.1 mol/L H2SO4 solu-
tion was used in the preparation of 2-amino pyridine
solution in different concentrations. For quantum-yield
measurement, the absorption and fluorescence spectra of 2-
amino pyridine solution were recorded in the following con-
centrations: 0 (blank), 0.2, 0.4, 0.6, 0.8, and 1 mmol/L. The
spectral experimental procedures were repeated in the etha-
nolic solution of BIQ derivatives in the same concentrations.
The integrated fluorescence intensity vs. absorbance was
plotted (Fig. 3), and the slope was calculated. The quantum
yields of the benzimidazolyl quinoline compounds were cal-
culated by applying the observed experimental data in the
following equation:
idine in 0.1 mol/L H2SO4 solution is 0.6. The refractive in-
dex of the 0.1 mol/L H2SO4 and ethanol is 1.36 and 1.003,
respectively. By doing the calculation, the fluorescence
quantum yields (ff) of the BIQ derivatives are determined,
and the values are tabulated (Table 2).
ꢀ
ꢁꢀ
ꢁ
Gradstd
Gradx
hx2
h2std
½1ꢂ
fx ¼ fstd
Here, fx and fstd denote the fluorescence quantum yields
Fluorescence lifetime measurement
of unknown (BIQ derivatives) and standard (2-amino pyri-
dine) compounds, respectively. The ‘‘Grad’’ and h denote
the gradient (slop) of the graph and refractive index of the
respective solvent, respectively. The fstd of the 2-amino pyr-
Time-dependent measurements of receptors have been
performed using time-correlated single-photon counting
(TCSPC) technique (Fig. 4). The fluorescecent lifetime of
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1698
Can. J. Chem. Vol. 87, 2009
Fig. 4. Fluorescence decay and residual curves profiles of BIQ-1b
in ethanol, lexc = 354 nm and lemi = 432 nm. The solid lines re-
present the best fit to the data.
each BIQ derivative was measured in ethanol using the cor-
responding solution absorption lmax wavelength as the exci-
tation wavelength. The fluorescence lifetimes (tf) of the
compounds are found to be in the range of 2–3 ns (Table 2).
Fluorescence efficiency
To optimize the fluorescence efficiency of BIQ deriva-
tives, the influence of solvent polarity and pH of the me-
dium on the optical property of 1b was studied. In this
fluorescence-optimizing spectrofluorimetric analysis, the sin-
gle excitation of 1b (428–435 nm) is mainly accounted.
Solvent-polarity study
The absorption and emission of 1b were studied in differ-
ent solvents with increasing polarity (toluene, CH2Cl2,
CHCl3, DMSO, DMF, EtOH, and 10% EtOH). Absorption
spectra of the quinoline benzimidazole derivatives showed a
gradual red shift with the increase in solvent polarity. This
shift is much pronounced in the last three solvents in the fol-
lowing order: DMF < EtOH < H2O (10% ethanol) (Fig. 5).
Absorption lmax values of 1b in different solvents were used
to record fluorescence spectra of the corresponding solu-
tions. The recorded fluorescence spectrum shows red shift
with the increase in solvent polarity. This illustrates the util-
ity of this compound as a polar fluorescent probe (Fig. 6).31
The experiment was repeated with double-excitation wave-
length lmax. The resulting spectra in the combined form
were shown in Fig. 7. Absorbance and fluorescence wave-
length lmax of all other BIQ derivatives (1a, 1c, and 1d) in
DMF, EtOH, and water (10% ethanol) were measured and
tabulated (Table 2).
Figures 5, 6, and 7 point out that 1b in the excited state is
more polar than in the ground state. For the increase of the
polarity of the solvent, the energy level which corresponds
to a more polar molecular conformation will be more inten-
Published by NRC Research Press

Malathi et al.
1699
Fig. 6. Emission spectra of 1b (single excitation) in (i) toluene,
Fig. 5. UV–vis absorption spectra of BIQ-1b in (i) toluene, lmax
=
lexc = 312 nm and lemi = 370 nm; (ii) DMF, lexc = 342 and lemi
425 nm; (iii) EtOH, lexc = 354 nm and lemi = 434 nm; (iv) 10%
aqueous ethanol, lexc = 375 nm and lemi = 442 nm.
=
312 nm; (ii) DMF, lmax = 326 and 342 nm; (iii) EtOH, lmax = 330
and 354 nm; (iv) 10% aqueous ethanol, lmax = 354 and 375 nm.
sively lowered.32 Hence, a red shift of the wavelength is ob-
served in the absorption and emission spectra of 1b with the
increase in solvent polarity.
In addition to the red shift of wavelength, Figs. 6 and 7
illustrate marked difference in the lmax of the solution dur-
ing double excitation compared with the single excitation.
This further confirms the sensitivity of the double-excitation
processes. This type of shifting also suggests that the excited
geometrical conformation of 1b may be suitable for the
model of twisted intramolecular charge transfer (TICT).32
Fig. 7. Emission spectra of 1b (double excitation) in (i) toluene,
lexc = 624 nm and lemi = 385 nm; (ii) DMF, lexc = 684 nm and
lemi = 421 nm; (iii) EtOH, lexc = 708 nm and lemi = 447 nm;
(iv) 10% aqueous ethanol, lexc = 750 nm and lemi = 456 nm.
Fluorescence pH titration
Quantitative pH titration was performed to find the fluo-
rescent efficiencies of BIQ compounds in the wide range of
pH. In this analysis, the intensity of first fluorescence peak
during the single excitation of 1b (lexc = 354 nm and
lemi = 432 nm) was accounted. The fluorescence spectrum
of the 3 mmol/L aqueous ethanol solution of 1b was re-
corded in the pH range of 2 to 14, and a plot was drawn be-
tween the fluorescence intensity and pH. Figure 8 shows the
fluorescent intensity vs. pH titration plot of 1b. From the
graph, it is observed that the fluorescent intensity is higher
in the lower pH range and continuously decreases in higher
pH range, owing to the deprotonation of the N atoms in the
basic medium. However, it should be noted that the differ-
ence in the fluorescent intensity is very little when the pH
is greater than 6.5. The fluorescent stability of 1b in this
pH range might be helpful to avoid the interference of pos-
sible pH change induced by biological stimulation.
factor which determines the suitability of the compound in
cell-line analysis. With this necessity, the cytogenetic analy-
sis was performed by using one of the BIQ compounds 1b
to scale up the utility of the derivatives in living-cells analy-
sis. The preparation of the metaphase plates is detailed in
the Experimental section.
Bioanalytical study of 1b
All the prepared slides in well metaphase plates were
screened for chromosomal aberrations (Table 3). The aberra-
tion includes endoreduplication, fragments, chromatid
breaks, and chromatid gaps. The clastogenic properties of
the compound were studied by investigating the effects of
the compounds on human chromosomes through in vitro
Cytogenetic analysis
The observed results in the optical measurements support
the suitability of BIQ derivatives as efficient polar fluores-
cent probes for bioanalytical study. But in the case of in
vivo analysis, the cytotoxicity of the compound is the main
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Can. J. Chem. Vol. 87, 2009
significant applications in the industrial biotech processes,
such as biocatalysis,33 bioremediation34, and so forth, in
such a way that leads to the implementation of green chem-
istry in various industrial processes. Additionally, the
enzyme-producing bacteria have promising medicinal activ-
ity.35 In this regard, many enzyme-producing bacteria are
analyzed in anticancer studies.36
Fig. 8. Fluorescence pH titration of BIQ-1b (3 mmol/L of 10%
aqueous ethanolic solution) with lexc = 354 nm and lemi = 432 nm.
The pH of the solution was adjusted by using 0.1 mol/L HCl and
0.1 mol/L NaOH to get acidic and basic pH, respectively.
In this margin, S. marcescens a Gram-negative bacterium
produces L-asparaginase enzyme that has anti-proliferative
and anti-tumor activities.37 Accordingly, its identification,
proliferation, and its structural analysis become significant
in the bacterial research. Recently, Venil et al.24 discussed
the factors affecting the production of enzyme L-asparagi-
nase from the bacterium S. marcescens SB and optimized
an effective medium for its production. In this progress, the
fluorescence imaging of the bacteria should have significant
value. The surface morphological study will reveal many
fundamental features of this organism and produce novel
tools to study previously inaccessible problems. Addition-
ally, this staining analysis can give a fundamental idea about
the affinity of the BIQ compounds in the cell wall and also
decides its applicability for further bioanalytical studies,
such as optical imaging of living systems, detection of intra-
cellular free metal ions, and so forth. With this intension
preliminarily, we concentrate the staining analysis in the
Gram-negative bacterium S. marcescens SB08.
analysis in the lymphocyte cultures. The data of the com-
pound are pooled and shown in Table 3. It was noted that
the increase in the chromosomal aberrations is dose-
dependent. Chromosomal aberrations like chromatid gaps
and chromatid breaks were also observed. Polyploid cells
were absent in all examined slides. From Table 3, it is in-
ferred that the compound did not show any aberration up to
10–4 mol/L concentration, which is essential for cell-lines in
vitro studies.
Bacterial staining analysis
The fluorescent microscopic view shows that 1b is an ex-
cellent green-fluorescence light-emitting material (Fig. 10a).
The details of the slide preparation are explained in the Ex-
perimental section. In addition to the fluorescence staining,
normal Gram staining of the bacterium were also done. The
normal Gram-stained bacterial smear in the slide was
viewed in the microscope (Fig. 10b), whereas the BIQ-
stained one was viewed in the fluorescence microscope.
The clean rod-shape image (Fig. 10c) of the bacterium con-
firms the binding of 1b to the surface of the bacterium.
Thermal analysis
BIQ compounds are remarkably stable towards air. It can
be kept under air for a long time without any noticeable de-
composition. Figure 9 shows the TG–DTA curves of 1b.
Compound 1b exhibits two steps of decomposition in TG.
The endothermic peak at 200 8C corresponds to the melting
point of the compound. The first exothermic peak at 275 8C
corresponds to the loss of chlorine. Then, a complete exo-
thermic decomposition of the molecule occurs between 500
and 800 8C. During the decomposition, the masses of the
compound, intermediate, and final product are those which
best fit with observed mass loss in TG curve. The TG results
are in good agreement with the DTA data. From this study,
it is inferred that the compound is highly stable up to
250 8C.
Conclusion
A specific molar ratio is implemented for the synthesis of
BIQ and BIN compounds at room temperature. This short-
reaction-time method offers high yield with reference to the
reported procedures. All the synthesized compounds are
characterized by various spectral and analytical techniques
1
(IR, H, 13C NMR, mass, and CHN analyses).
The preliminary optical analysis confirms the fluores-
cence property of the BIQ derivatives and also reveals that
such property is silent in the BIN derivatives. This optical
behavior of BIQ derivatives can be attributed to the quino-
line moiety facilitating a strong charge-transfer interaction
with the imidazole group during the electronic relaxation.
Due to that, its derivatives alone show intense absorption
and fluorescence bands with ff approximately close to 1. In
addition to the fluorescence property, the spectrofluorimetric
analysis of BIQ derivatives exhibits a higher excitation
wavelength (lexc = 708 nm) and emission property (l =
434 and 850 nm) in different solvents, since it is also an es-
sential parameter for the recognition of TP fluorescence
probes. This low energy excitation and emission photophys-
Staining study
All the photophysical, cytotoxicity, and thermal analyses
of BIQ derivatives support its applicability for bioanalytical
studies. This creates an intention to carry out the staining
analysis using BIQ compounds as staining dye. The appreci-
able fluorescence efficiency (high ff, water solubility, and
thermal stability analysis) of 1b makes it attractive for the
staining analysis. For the initial staining analysis, we prefer
small microorganism selectively bacteria because they are
one of the primary microbes used in the drug discovery.
Apart from the pathogenic activity of bacteria, they have
Published by NRC Research Press

Malathi et al.
1701
Table 3. Cytogenetic analysis of the BIQ compound 1b.
1b (24 h)a
0.02 mg mL–1
Control
0.2 mg mL–1
2 mg mL–1
20 mg mL–1
400
Mean number of aberrations
0 h
400
24 h
400
Average No. of metaphases (n = 8)
Endoreduplication
Fragments
Chromatid breaks
Chromatid gaps
400
400
400
—
—
—
—
0.57 0.20
0.32 0.35
0.89 0.318 0.94 0.20
0.02 0.17 0.05 0.10
0.59 0.23
0.34 0.30
0.60 0.25
0.37 0.22
0.97 0.19
0.07 0.10
0.62 0.27
0.45 0.28
1.02 0.15
0.09 0.14
1.69 0.24
1.49 0.52
1.34 0.47
0.69 0.29
aChromosomal aberrations after 24 h incubation of 48 h initiated human peripheral blood lymphocyte culture with compound 1b.
Fig. 9. TG–DTA of BIQ-1b: (i) TG of BIQ-1b; (ii) DTA of BIQ-1b.
Fig. 10. Staining analysis of BIQ-1b: (a) fluorescent green-light-emitting view of BIQ-1b; (b) Gram-stained microscopic view of the S.
marcescens SB; (c) BIQ-1b-stained fluorescence microscopic view of the S. marcescens SB.
ical property of BIQ compounds is significantly economic in
the preliminary bioanalytical study of the living systems.
In general, the combined optical spectral data of the BIQ
and BIN derivatives conclude that the methyl derivatives
have better photophysical properties in comparison with the
unsubstituted derivatives. In addition to the optical property,
a vast difference also exists in the IR and NMR spectral data
of the substituted and unsubstituted benzimidazole deriva-
tives. This is mainly due to the extension of conjugation
(hyperconjugation)38 facilitated by the methyl protons.
Based on the optical properties, one of the BIQ deriva-
The fluorescence quantum yield (ff) and time-resolved
fluorescent lifetime (Tf) of the BIQ derivatives were meas-
ured to evaluate its fluorescence efficiency. The optical
analysis with increasing solvent polarity exposed the polar
fluorescent probe nature of the BIQ derivatives. The fluores-
cence pH-titration measurement confirms the applicability of
the BIQ derivatives in a wide range of pH.
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Can. J. Chem. Vol. 87, 2009
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Supplementary data for this article are available on the
journal Web site (canjchem.nrc.ca) or may be purchased
from the Depository of Unpublished Data, Document Deliv-
ery, CISTI, National Research Council Canada, Ottawa, ON
K1A 0R6, Canada. DUD 5304. For more information on ob-
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Acknowledgement
One of the authors (MM) thanks the Council of Science
and Industrial Research (CSIR), New Delhi, for the award
of Senior Research Fellowship (SRF). Authors are thankful
to the analytical instrumentation support from the NMR re-
search center, IISc., Bangalore, India, and National Centre
for Ultrafast Processes, University of Madras, Taramani
Campus, India.
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