Synthesis, photophysical properties and systematic evaluations of new phenanthroimidazole fluorescent probe for bioimaging: Experimental and theoretical study

Article abstract of DOI:10.1016/j.jphotobiol.2016.11.008

In this study, the newly synthesized 4'-(1H-phenantro[9,10-d]-imidazol-2-yl)-bifenyl-4-carboaldehyde - PB2 was investigated as a fluorescent dye. For this reason, the spectroscopic properties in solvents of different polarity were studied. The experimenta

Full text of DOI:10.1016/j.jphotobiol.2016.11.008

Synthesis, photophysical properties and systematic evaluations of new
phenanthroimidazole fluorescent probe for bioimaging: Experimental and
theoretical study
Przemysław Krawczyk, Beata Jedrzejewska, Marek Pietrzak, Tomasz
Janek
PII:
DOI:
Reference:
S1011-1344(16)30602-9
doi:10.1016/j.jphotobiol.2016.11.008
JPB 10649
To appear in:
Received date:
Revised date:
Accepted date:
25 July 2016
22 September 2016
5 November 2016
Please cite this article as: Przemys ꢀl aw Krawczyk, Beata Jedrzejewska, Marek Pietrzak,
Tomasz Janek, Synthesis, photophysical properties and systematic evaluations of new
phenanthroimidazole fluorescent probe for bioimaging: Experimental and theoretical
study, (2016), doi:10.1016/j.jphotobiol.2016.11.008
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ACCEPTED MANUSCRIPT
Synthesis, photophysical properties and systematic evaluations of new
phenanthroimidazole fluorescent probe for bioimaging: experimental and
theoretical study
1
*
2
2,
3
Przemysław Krawczyk , Beata Jędrzejewska , Marek Pietrzak Tomasz Janek
1
Nicolaus Copernicus University, Collegium Medicum, Faculty of Pharmacy, Department of
Physical Chemistry, Kurpińskiego 5, 85-950 Bydgoszcz, Poland
2
UTP University of Science and Technology, Faculty of Chemical Technology and
Engineering, Seminaryjna 3, 85-326 Bydgoszcz, Poland
3
Wroclaw Medical University, Department of Inorganic Chemistry, Faculty of Pharmacy,
Borowska 211a, 50-556 Wroclaw, Poland
*Corresponding author: przemekk@cm.umk.pl; tel: +48 52 5853679
Abstract
In this study, the newly synthesized 4'-(1H-phenantro[9,10-d]-imidazol-2-yl)-bifenyl-4-
carboaldehyde - PB2 was investigated as a fluorescent dye. For this reason, the spectroscopic
properties in solvents of different polarity were studied. The experimental data were
supported by quantum-chemical calculations using density functional theory. Measurements
and theoretical calculations showed that PB2 compound is characterized by the non-
monotonic solvatochromism, strongly polar charge transfer excited state, large Stokes' shift,
high fluorescence quantum yield and high fluorescence lifetime. Simulations using AutoDock
presented in this study, showed that after conjugation with Concanavalin A in the active site
with LYS200, the PB2 possesses the highest probability of binding affinity. Circular
1

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dichroism (CD) measurement was performed to monitor the changes of the secondary
structure of Concanavalin A induced by the presence of PB2 fluorophore. These results
indicate that the addition of PB2 influences the secondary structure of Concanavalin A, but
does not affect the interactions with carbohydrate moieties. Finally, by using fluorescence
microscopy it was demonstrated that the PB2 is the photostable fluorescent probe and PB2
conjugate Concanavalin A exhibits a blue fluorescence. The results of this study have
implications in designing PB2-protein conjugate as a valuable alternative to commercial
probes designed for cellular labeling in biological and biomedical research.
Keywords
Fluorochrome; Solvatochromism; Conjugation and Bioimaging; Electronic excited state;
Density functional theory; AutoDock
2

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1
. Introduction
In recent years, one of the most developing branches of medical sciences is the
fluorescence technology. This method allows for sensitive and easy study of intermolecular
interactions and the transcriptional dynamics of the cell nucleus [1-2]. In particular,
fluorescent imaging and flow cytometry become the standard and highly sensitive methods
for detection of pathogens and their reliable location inside the human body [3-8]. In this
field, in vivo the fluorescent imaging in medicinal diagnostics is the visualization technique of
fluorescent probes pointing to the structural changes in tissues and biochemical processes.
This allows for a detailed analysis and monitoring of the location and dynamics of genes,
proteins, dyes expression and molecular interactions in cells, functionality of cells and
physiological parameters in a less time-consuming way [9-10]. The signal from the
fluorescent probe in site of interactions can be modulated and for this reason sensors relying
on activation and not just accumulation, can be utilized. Nevertheless, one of the major steps
in this science is to design and synthesize probes with the appropriate properties and thus this
technology is often limited. Primarily, the fluorescent dyes used herein should have
appropriate sensitivity and allow for the correct identification and quantitative determination
by non-destructive and non-invasive analysis of biochemical processes in vivo at the cellular
and molecular level [11-17]. Specific qualities are desirable for fluorescent nanostructures:
low toxicity and good solubility, high chemical and environmental stability, tolerance by
living cells and large penetration depth in living tissues, high photostability, large signal to
noise ratio, sharp absorption and emission under excitation, large Stokes' shift, bright
fluorescence, high quantum yield and resistance to photobleaching [18-21]. Furthermore, they
require an appropriate and specific reactive group allowing for an easy connection to
macromolecules. The most important ones are: -CHO, -NH , -SH, N-hydroxysuccinimide
2
ester and isothiocyjanate groups.
3

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The main aim of this study was the synthesis and spectroscopic characteristics of a
new compound belonging to the imidazole class, containing -CHO as a functional group: 4'-
(1H-phenantro[9,10-d]-imidazol-2-yl)-bifenyl-4-carboaldehyde - PB2. Imidazoles are very
popular in many areas of clinical medicine and are widely used as: antiepileptic agents,
antiparasitic, anthelmintic, anti-inflammatory, analgesics and platelet aggregation inhibitors
[
22-27], antineoplastic antibiotic, muscarinic receptor antagonist, antiulcerative, prohormone,
benzodiazepine antagonist, antihyperthyroid and hypnotic agents [28-31], as well as
ratiometric fluorescent probe for hydrogen sulfite [32], a ratiometric fluorescent probe for
cysteine and hemocysteine [33], antimicrobial drug [34], sensitive fluorescence ratiometric
probe for cyanide with its application for detection of cyanide in natural water samples and
biological samples [35], solar cells [36], multifunctional agents for treatment of Alzheimer's
disease [37]. Therefore, the primary objective of this work was to evaluate the usefulness of
the newly synthesized PB2 dye in potential applications as fluorescent probes in living cell
imaging technology and flow cytometry. The experimental data were supported by quantum-
chemical calculations using density functional theory (DFT) [38-44]. The choice of this
method is due to its adequate precision in reproducing experimental data, from structural to
spectroscopic parameters of many classes of compounds at relatively low computational costs
[
45-54]. The theoretical investigations were supplemented by molecular docking in exploring
the best binding site, to which the PB2 has the highest affinity.
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2
2
2
. Methodology section
.1. Experimental part
.1.1. Materials and synthesis
All reagents and solvents were obtained from Aldrich Chemical Co. The 2-(4-
bromophenyl)-phenanthro[9,10-d]imidazole was synthesized based on the procedures taken
from literature [55].
The 4'-(1H-phenanthro[9,10-d]-imidazol-2-yl)-biphenyl-4-carboaldehyde, PB2 was
obtained according to literature [56] using 2-(4-bromophenyl)-phenanthro[9,10-d]imidazole
(
2,7 mmol), 4-formylphenylboronic acid (3 mmol), P(Ph )Pd(OAc) (0,011 mmol), 1-
3 2
propanol (15-20 mL), Na CO3aq (6 mL, 2M), water (5 mL + 20 mL) and 2% NaHCO [Fig 1].
2
3
Fig.1.
o
C H N O; 398.45532 g/mol; mp 267.5 C; 36.9% yield; R = 0.36 for methanol-chloroform
28
18
2
f
0
.1:10 v/v.
1
H NMR (DMSO-d )  (ppm): 7.67 (m, 2H, Ar), 7.77 (m, 2H, Ar), 8.06 (m, 6H, Ar), 8.48-
6
8
8
8
.46 (d, J=8.0 Hz, 2H, Ar), 8.60-8.58 (d, J=8.0 Hz, 1H, Ar), 8.64-
.62 (d, J=8.0 Hz, 1H, Ar), 8.87-8.85 (d, 1H, J=8.0 Hz, Ar), 8.91-
.89 (d, J=8.0 Hz, 1H, Ar), 10.09 (s, 1H, CHO), 13.58 (s, 1H, NH)
1
3
C NMR (DMSO-d )  (ppm): 193.2 (CHO), 122.4, 122.5, 124.3, 124.6, 125.7, 126.0, 127.2,
6
1
1
27.6, 127.7, 127.8, 128.1, 130.7 (CH), 122.8, 127.4, 128.3, 128.4,
30.9, 135.7, 137.7, 139.6, 145.4, 148.9 (C)
-1
IR (KBr) (cm ): 3604, 3048, 1699, 1601, 1559, 1530, 1481, 1455, 1429, 1308, 1269, 1221,
1
169, 1044, 1003, 961, 820, 761, 746, 725, 691, 614, 499
Throughout the manuscript the following notation was adopted: 1.4-Dx - 1.4-dioxane;
Et O - diethyl ether; THF - tetrahydrofuran; MeCN - acetonitrile; DMF - N,N-
2
DiMethylFormamide; DMSO - DiMethylSulfoxide.
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2
.1.2. Measurements
1
13
The H (400 MHz) and C (100 MHz) NMR spectra were recorded on a Bruker
TM
Ascend 400 NMR spectrometers. Dimethylsulfoxide (DMSO-d ) was used as the solvent
6
and tetramethylsilane (TMS) as internal standard.
The IR spectra of the synthesized salts were recorded using a Bruker Vector 22 FT-IR
-1
spectrophotometer (Germany) in the range 400-4500 cm , by KBr pellet technique.
HPLC analysis was done by Waters HPLC systems equipped with Waters 2489 UV-Vis
detector (detection wavelength was 370 nm), Waters 1525 Binary HPLC Pump and a
Symmetry C18 column (3.5 m, 4.6  75 mm). Separation was conducted under isocratic
conditions with 0.8 ml/min flow rate, 25°C, 10 l injection volume and HPLC grade
acetonitrile as a mobile phase.
Melting points were determined on the Buchi M-565 Melting Point apparatus.
Absorption and emission spectra in solvents of different polarity were recorded at room
temperature using a Shimadzu UV-vis Multispec-1501 spectrophotometer and a Hitachi F-
4
500 spectrofluorimeter, respectively.
The fluorescence quantum yield of the tested compounds () was calculated using
equation (1):
IAref n2

 ref

(1)
2
I A n
ref
ref
where:
ref is the fluorescence quantum yield of the reference (Coumarin 1 in ethanol ref = 0.64
57]) sample in ethanol, A and Aref are the absorbencies of the compound under study and
[
reference sample at the excitation wavelength (404 nm), I and I are the integrated emission
ref
6

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intensities for the tested compounds and reference sample, n and n are the refractive indexes
ref
of the solvents used for the compounds and the reference, respectively.
The fluorescence lifetimes were measured using an Edinburgh Instruments single-
photon counting system (FLS920P Spectrometers). The excitation was provided by a
picosecond diode laser generating pulses of about 55 ps at 375 nm. The compounds were
studied at a concentration needed to provide absorbance of 0.2-0.3 in a 10 mm cell at 375 nm.
The fluorescence decays were fitted as sums of two exponentials. The average lifetime, av
was calculated as  = (   )/(  ), where  and  are the amplitudes and lifetimes.
av
i
i i
i
i
i
i
2
.1.3. General protein labeling procedures
Concanavalin A (carbohydrate-binding protein) was conjugated with a PB2-
fluorophore in phosphate-buffered saline (PBS, 10mM phosphate, 150mM NaCl, pH 7.4).
Briefly, two milligram of Concanavalin A was resuspended in 1 mL of buffer solution and 50
μL of 5 mg/mL PB2 in dimethyl sulfoxide (DMSO) was added during vigorous mixing. The
Schiff bases formed by amine-aldehyde coupling were reduced by adding 10 µL of 5M
sodium cyanoborohydride in 1 N NaOH per 1 mL reaction volume, and the reaction was
allowed to proceed overnight at 4 °C. After an overnight incubation at 4 °C in the dark,
unlabeled PB2 dyes were separated using a dialysis membrane centrifuge tube at 7000g for 10
min. The conjugated PB2-Concanavalin A solution retained in the dialysis tube was collected
and diluted in PBS to a protein concentration of 1 mg/mL. The degree of labeling was
determined by measuring the absorbance at 280 nm for Concanavalin A and at λABS of the
PB2 on a Varian Carry 50 Bio spectrophotometer (Varian, USA). The resultant product was
stored at 4 °C.
2
.1.4. Fluorescence imaging
Candida albicans ATCC 10231 and Yarrowia lipolytica A101 strains were grown in a
liquid yeast-peptone-glucose (YPG) medium consisting of (per liter) 10 g of yeast extract, 20
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g of peptone (both from Difco), and 20 g of glucose at 28 °C with constant shaking at 200
rpm. The overnight cultures of both yeast strains were centrifuged and washed twice with
PBS, pH 7.4. Then, the yeast cells were stained for 30 min at 28 °C with PB2-Concanavalin A
conjugate (final concentration 0.025 mg/mL) in the PBS. Images were obtained using a Zeiss
Axio Scope A1 microscope (Zeiss, Jena, Germany) with a 100× objective lens and Zeiss filter
set FS01 for fluorescence microscopy. Assays were carried out three times. Representative
images are presented in Fig. 5.
2
.1.5. Circular dichroism spectroscopy
Circular dichroism spectra of 0.1 mg/mL Concanavalin A solutions were recorded on
a Jasco model J-1500 spectropolarimeter (Jasco, Tokyo, Japan) at 25 °C under a constant flow
of nitrogen gas, in the absence and presence of a PB2 probe. The scanned wavelength domain
was 190–260 nm and the time constant, scan speed, bandwidth/resolution, 50 nm/min, 2 nm
and 200 millidegrees, respectively. The spectra represent the average of 9 scans. CD
3
-1
-1
intensities are expressed in Δɛ (dm ·mol ·cm ). The analysis of secondary structural contents
was performed using K2D3 web server. K2D3 web server is an online tool used to assess the
secondary structural elements in the form of α-helix and β-strand from the far-UV CD spectra
ranging from 190 to 240 nm [58].
2
.2. Computational details
The geometrical parameters of PB1 dye in its ground (S ) and excited (S ) state were
g
CT
optimized by using density functional theory (DFT) approach, with the PBE0 functional [59-
0] implemented in Gaussian 09 program package [61] with TIGHT threshold option.
6
Inclusion of this functional to designate the molecular structures is justified by the correct
description of the spectroscopic parameters in terms of experimental data for many classes of
D-π-A dyes [62-64]. For each optimized structure of the ground and excited state, the analysis
8

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of Hessians was conducted to verify that all the structures correspond to the minima on the
potential energy surface.
By applying the time-dependent density functional theory (TDDFT) [46-48] vertical
absorption and emission spectra were determined. The TDDFT treats the S and SCT states
g
with equilibrium solvation. For the best consideration of the solvent impact on the
fluorescence spectra, the ground state should be calculated with non-equilibrium solvation
[
65-66]. This was taken into account by including the state-specific (SS) corrected linear
response (cLR) approach [67] to the theoretical calculations. In the SS approach the solvent
dynamic polarizations are determined by the difference of the electron densities of the initial
and final states [68-70].
The polarity of the excited state was evaluated by numerical differentiation of the
excitation energies (E):
ꢋ
ꢋ
ꢋ
ꢋ
ꢅ
ꢈꢉꢊ ꢌꢍꢅ ꢈꢍꢊ ꢌ ꢅꢐꢈꢉꢊ ꢌꢍꢅꢐꢈꢍꢊ ꢌ
ꢆꢇ
ꢃ
ꢆꢇ
ꢀ
ꢄ
ꢏ
(2)
ꢁ
ꢂ
ꢋ
ꢋ
ꢍꢎꢊ
ꢍꢎꢊ
where i states for the Cartesian component of the dipole moment difference. In these
calculations there was used an electric field F of 0.001 a.u. strength.
Calculations of the spectroscopic parameters were conducted in connection with
standard-hybrid PBE0 functional, as well as long-range asymptotically corrected LC-ωPBE
[
71-73], CAM-B3LYP [74] and ωB97XD [75] functionals. The solvent effect on the linear
optical properties has been taken into account using the Integral Equation Formalism for the
Polarisable Continuum Model (IEF–PCM) [76-77].
The AutoDock 4.2 [78-80] simulations were applied to identify the site with the
highest affinity of PB2 dye relative to protein. The explorations of the binding site were
performed using a united-atom scoring function implemented in AutoDock Vina [81]. The
AutoDock Tools software package was used in order to prepare the PB2 molecule as a ligand
and Concanavalin A as a macromolecule. All the water molecules and Ca, Mg and Xe atoms
9

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were removed from the crystal structure of Concanavalin-A downloaded from the Protein
Data Bank in Europe (PDB ID: 2a7a) [82]. According to the literature [83], the conjugation of
molecules containing aldehyde as a functional group with a macromolecule occurs via
formation of a Shift base between the aldehyde and an amino group of a lysine residue. Then,
a reduction to a secondary or tertiary amine occurs, which gives a stable alkylamine bond. In
order to reflect this, the grid box was adjusted in such way that the space included the
individual -NH2 group of lysine's side chain, in subsequent simulations. The docking region
on the target protein was defined by establishing a grid box with the dimensions of X: 18; Y:
1
8; Z: 18 Å, with a grid spacing of 1 Å. In AutoDock 4.2 simulations, the Lamarkian genetic
algorithm was employed to identify appropriate binding energy and conformation of the PB1
dye. For each atom of the receptor molecule, Gasteiger charges were calculated. The docking
procedure was repeated ten times for each lysine.
The simulations of biological activity were performed using a combination of the
3
D/4D QSAR BiS/MC and CoCon algorithms developed by ChemoSophia company [84-86].
3
3
3
. Results and discussion
.1. Experimental
.1.1. Absorption and fluorescence spectra
Figure 1 shows the illustrative electronic absorption and the fluorescence spectra for the
tested compound in selected solvents. Table 1 collects the values of the absorption and
fluorescence maximum positions, molar absorption coefficients, Stokes shifts and
fluorescence quantum yields for the phenanthroimidazole derivative. Their electronic
absorption spectra displays a broad band with a maximum in the range from 370.5 to 377.5
-1
-1
-1
-1
nm and high molar absorption coefficients ranging from 35 900 M cm to 44 600 M cm .
The inspection of the data in Table 1 shows that the position of the -* absorption band
1
0

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depends slightly on solvent polarity. This indicates a less polar character of the compounds in
the ground state.
Table 1.
Fig. 2.
As indicated in Figure 2 and Table 1, the solvent polarity significantly affects the position of
fluorescence spectral maxima. The emission spectra are red-shifted by ca. 80 nm as the
solvent polarity increases. This, exemplified by the increase of emission maximum from 465
nm in diethyl ether to 545 nm in DMSO, observably indicates the change in charge
distribution within the molecules in the singlet excited state. Reasonable correlation has been
found between both the fluorescence peak maxima and the Stokes’ shift parameter and the
N
solvent polarity scale, ET (Fig.3).
The substantial red shift of the emission spectra and an increase of Stokes shift with
solvent polarity suggest an increase of the dipole moment in the excited state with respect to
that of the ground state.
Fig. 3.
Besides fluorescence band position also its intensity is affected by solvent polarity. As
shown in Fig. 4, the fluorescence quantum yield of PB2 decreases with increasing solvent
N
polarity E . Such behaviour is characteristic for compounds with a highly polar ICT excited
T
state [87]. The decrease in fluorescence intensity with increasing solvent polarity indicates
that the PB2 reveals a positive solvatokinetic behaviour. According to literature data [88-90],
in such a case a non-radiative decay is prominent in the compound due to efficient internal
1
*
conversion and/or intersystem crossing by extensive mixing between the close-lying (π-π )
1
*
and (n-π ) states. The observed linear correlation between the fluorescence quantum yields of
N
PB2 and ET scale demonstrates the opposite effect to the previously studied compound PB1
(4-(1H-phenanthro[9,10-d]-imidazol-2-yl)-benzaldehyde) [91]. This means that introduction
1
1

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of the second phenyl ring to the structure of the PB1 affects the fluorescence properties to a
great extent.
Fig. 4.
For the tested compound two-exponential fluorescence decays were observed (Table
2
), with first components lifetime close to 2 ns and the second of about 0.3-0.6 ns. Moreover,
the shortest average fluorescence lifetimes are observed in diethyl ether. The correlation
N
between the fluorescence average lifetimes and ET solvent polarity parameter for PB2 clearly
demonstrates the sensitivity of its fluorescence behaviour to the solvents. The fluorescence
lifetimes increase with an increase of solvent polarity. What is more, for PB2 the fluorescence
quantum yield is indirectly proportional to its lifetime i.e. as the  decreases with increase of
Fl
solvent polarity, the  increases. This is probably due to the strong interaction between the
Fl
strongly polarized molecule and solvents in the excited state.
Table 2.
Based on the average lifetimes and quantum yields of fluorescence data, radiative (k ) and
r
nonradiative (k ) rate constants have been calculated according to Eqs. 3 and 4. The values
nr
are collected in Table 3.
Fl
kr 
(3)
(4)
av

1 Fl

k 
nr
av
Analysis of the data collected in Table 3 indicates that the k /k ratio changes with
r
nr
solvent polarity. For PB2 compound the ratio increases with increasing polarity of the
medium pointing to the increasing role of non-radiative processes, due to the enhancement of
intersystem crossing, internal conversion and vibrational deactivation [92-94]
Table 3.
1
2

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The obtained spectral results suggest that PB2 may be available for use as a
fluorescence labeling reagent for the circular dichroism measurements and fluorescence
microscopy of glycoproteins in biological samples.
3
.1.2. Protein labeling with PB2 fluorophore
The binding of the probes to protein has gained importance in a lot of different fields
in biomedical research. This method is based on the fluorescence detection of
macromolecules (such as proteins or antibodies) labeled with probes [95-96]. The PB2 probes
with reactive aldehyde groups formed Schiff bases with the primary amines of Concanavalin
A. The reaction efficiency and bond stability between aldehydes and primary amines was
increased by the addition of sodium cyanoborohydride.
The internalization of the synthesized probe was examined at cellular level of the
Candida albicans and Yarrowia lipolytica cells using fluorescence microscopy to achieve the
optimal concentration and time exposition. The spectral study shows that the PB2-
Concanavalin A conjugates accumulated selectively in yeast cells exhibits a blue fluorescence
with excitation filter BP 365/12 nm and LP 397 nm emission filter (Fig. 5). On the basis of
our results, it is concluded that the PB2-Concanavalin A conjugate is characterized by strong
and stable fluorescence, what is a promising sign for serious application for this compound as
a fluorophore for biomolecules. Our data were compared to those obtained performing a
similar systematic study on the interaction of fluorescein isothiocyanate (FITC), one of the
most used fluorescent probes for labeling of proteins and antibodies [97-98]. Our results
recommend PB2 as a possible valuable alternative to FITC for use as fluorescent label for
proteins. Our further studies will include the labeling of antibodies, by taking advantage of the
reactive group on PB2 (−CHO) able to bind covalently to amines, lysine ε-amines and N-
terminal α-amines groups of the antibody, and studying the antigen–antibody interaction.
Fig. 5.
1
3

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3
.1.3. Effects of PB2 on the Concanavalin A secondary structure
The circular dichroism spectrum of Concanavalin A is that of a typical lectin, with a
negative band near 220 nm revealing a content of β-sheet structure [99]. Fitting the spectrum
of native Concanavalin A at 25 °C yielded a 40.04% β-sheet and only 2.97% α-helical
structure. The PB2-Concanavalin A conjugate caused a secondary structural change of the
lectin to 26.55% β-sheet/14.22% α-helical. These spectral changes suggest the occurrence of
bulk conformational modifications due to fluorophore binding to the primary amines of
Concanavalin A (Fig. 6).
Fig. 6.
3
.2. Theoretical aspect
For the investigated dye, the lowest-lying singlet excited state is the CT state (bright state).
Structures and geometrical parameters of the PB2 dye after optimization were shown in Fig. 7
and Table S1. Firstly, the CT state is more planar than the ground state. The S is twisted on
g
C8-C10 bond and during excitation this twisting is not observed. For SCT dihedral angles 7-8-
1
0-11 and 9-8-10-15 become smaller by more than 2.2° while 7-8-10-15 and 9-8-10-11 angles
become larger by more than 2.2°. After the excitation, the twisting on the C13-C16, which is
present in S and S , undergoes a slight straightening. In this case, for the ground state
g
CT
dihedral angles 12-13-16-17 and 14-13-16-21 are longer by 22° and 12-13-16-21 and 14-13-
6-17 are shorter by more than 22°. Although in the CT state bonds linking aromatic parts
C8-C10, C13-C16, C19-C22) are reduced by more than 0.025Å, the structure of the excited
1
(
state seems to be longer (C1/2-C22). This is due to the fact that aromatic rings are slightly
flattened and bonds such as N7-C8 and N9-C8 are lengthened by over 0.017. Not without
significance is the effect of solvent on the structure of the tested dye. Primarily, the structure
of ground state is shortened while for the SCT it is lengthened in the function of the solvent
polarity. Mainly, this is the result of reduction of the dihedral angles for S and increasing
g
1
4

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them for S . Moreover, for both structures there can be discerned a linear increase or
CT
decrease of the bond lengths and angles relative to the polarity of the environment. The
exception is water where this relation is often disrupted. This observation is in correlation
with the solubility of the PB1 molecule, in relation to the free energy of solvation (ΔGsolvation
)
[
100] (see Table 4). In water the ΔGsolvation adopts the smallest value, decreased by more than
4
5% relative to the MeCN for which it has the greatest value. This growth in conjunction with
structural changes significantly affects the solubility in water. The PB2 dyes is very weakly
-7
soluble in water and the calculated solubility is: 5.4·10 mol/l (in 25°C, 298.15 K, 760
mmHg).
Fig. 7.
Table 4.
According to Fig. 8, the charge transfer excitation is dominated by HOMO-LUMO
transition. The HOMO is distributed over the entire molecule while the LUMO is mainly
located on the benzaldehyde fragment and partially on the imidazole part. The -CHO group is
a moderately deactivating substituent but after excitation the electron-cloud mainly
accumulates on benzaldehyde. For this reason, the lowest-lying excited state can be assigned
as a π-π* transition mixed with ICT process. In this system, the C8-Ar-C22 plays the role of
the system with π-conjugated double bonds and may be favourable for nonlinear optical
properties. Moreover, in all solvents the studied dye is characterized by high value of the EGAP
(over 3 eV, see Table S2) which indicates a high fluorescent efficiency and good stability
[
93]. The value of chemical hardness (ƞ), predicted from HOMO-LUMO, is relatively low
and therefore the PB2 should be treated as a soft molecule. For this reason, its reactivity is
very high and it undergoes uni-molecular reaction (such as with proteins) relatively quickly
and easily.
Fig. 8.
1
5

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Fig. 9.
Fig. 9 presents the molecular electrostatic potential (MEP). The MEP analysis is very
useful in designing new fluorescent probes because localizing the active electrophilic and
nucleophilic region specifies the sites where the molecule can participate in the formation of
H-bonds as well as determines the intermolecular interaction. The PB2 dye has two strong
regions for electrophilic attack (red sites) localized on C20 and at the C14-C15 bond. Thus,
the extensions of the molecule by the introduction of an additional benzene ring relative to the
4
-(1H-phenanthro[9,10-d]-imidazol-2-yl)-benzaldehyde (PB1) [91] causes a constant negative
site at C12 (benzaldehyde part) and the appearance of an additional region in the middle ring.
In addition, the external imidazole rings adopt a more negative value than in the case of PB1.
Maximum positive (blue) sites for nucleophilic reactivity are localized on the hydrogen atom
at the N7-H. However, in contrast to PB1 where only one strong positive region was
observed, in the case of PB2 dye strongly positive electrostatic sites also appear at the C13-
C16 bond linking the benzene rings and in the central imidazole ring. Thus, increasing the
structure of the molecule is associated with increasing the potential sites from where the dye
can experience intermolecular as well as can undergo non-covalent interactions.
Table 5.
Theoretically determined one-photon absorption spectra (ꢑ ꢒꢕ ꢓꢖ ꢔꢒ, OPA) are listed in
Tables 5 and S3. Among the vertical excitation energies (ΔE), the values closest to
experimental ones are obtained using PBE0 functional. Its application shifts the maximum of
ꢕꢖ
the ꢑ
towards longer wavelengths, however this bathochromic shift is not large and on
ꢒꢓꢔꢒ
average is 27 nm. In turn, the long-corrected functionals lead to significantly overestimated
ΔE and the average error is 36 nm, 62.6 nm and 46 nm for CAM-B3LYP, LC-ωPBE and
ωB97XD, respectively. As mentioned earlier, in the absorption process the ground state
should be considered in non-equilibrium solvation and therefore the cLR-TDDFT calculations
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have been performed (see Table S4). Firstly, the cLR approach gives the OPA maxima shifted
towards longer wavelengths and the values obtained for the long-corrected functionals are
approaching the experimental data. In the case of PBE0, including the state-specific solvation
correction causes that the error for PBE0 becomes larger and the overestimation is on average
4
7.6 nm. This time, the functional giving the most reliable results turned out to be ωB97XD,
which induces a bathochromic shift by only 14 nm. Such correct predictions for this
functional were not observed in the case of PB1 dye. What is more important, both models in
an analogous way describe the solvatochromic behaviour. Presented data shows, that PB2
dye is characterized by non-monotonic solvatochromism. The transition from gas phase to the
weakly polar solvent is associated with the red shift and for theoretical data it occurs up to
THF. In a more polar MeCN the opposite is visible but in DMF there is a positive
solvatochromism. In DMSO there are some inconsistencies, because in terms of
solvatochromic behaviour the results consistent with the measured values are obtained with
TDDFT, while cLR-TDDFT provides a hypsochromic shift. In water, for both approaches
there is observed a re-negative solvatochromism. The PB1 dye was characterized by the same
solvatochromic behaviour. Taking into account the measured data, placing an additional
benzene ring in the structure of the PB2 dye results in a shift of the maximum absorption
bands in the direction of longer wavelengths by only 1 nm in weakly polar and 4 nm in polar
solvents. For TDDFT calculations these values are 1nm and 3nm but for cLR-TDDFT they
increase to 12nm and 6 nm, respectively.
According to HOMO-LUMO plots, photoexcitation to the charge-transfer state leads
to substantial changes in the electron density of the solute and these differences in the solute-
solvent electrostatic interaction energy between S and SCT induce a strong solvatochromic
g
shift. For this reason, a linear dependence of the solvatochromic shift on the dipole moment
difference (Δꢀꢁꢏꢗꢘ) between ground (µ ) and CT states (µ ) should be excepted. For
g
CT
1
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compounds with positive solvatochromism, µ > µ and the excited state is characterized by a
CT
g
high polarity which results in a significant decrease of the ΔE. In contrast, the blue shift
occurs when the ground state is better stabilized by polar solvents than the CT state and µ >
g
µCT what intensifies the ΔE of the solute. Moreover, the hypsochromic shift occurs also when
H-bond interactions are present which additionally enhances the intensity of the CT
absorption band [102]. For the tested PB2 dye, a µCT > µ dependency is observed in all
g
solvents (see Table S5). Dipole moments of the ground and CT states increase in the function
of solvent polarity, except for DMF where µCT is reduced by about 0.1D. The PB2 exhibits
strongly polar charge-transfer state (ꢙꢀꢁꢏꢗꢘꢚ˃ than 13 D and 8 D for PBE0 and CAM-B3LYP
respectively) but this uptrend cannot be concluded explicitly. For PBE0, the ꢙꢀꢁꢏꢗꢘ enhances
with the solvent polarity and only in DMF a disorder of this behaviour is observed. For other
functionals, the increase of ꢙꢀꢁꢏꢗꢘ follows up to Et O while from THF it is reduced and only
2
in water there is observed an enhancement of the CT state polarity. For this reason, the pure
electrostatic contributions to the solvent-solute interactions do not occur and the short-range
specific interactions such as H-bond or self-aggregation should be expected. The comparison
of the vertical OPA determined theoretically and experimentally (see Fig.10) also reveals
some inconsistencies. In the region of interest, an additional peak in the measured absorption
spectra is visible in the main band, near 350nm. For TDDFT this peak is more separated and
located at 310 nm. However, in all cases this peak as well as the main band have the same
intensity and occur independently of the solvent polarity. Therefore, the reasons for its
*
formation should be sought in a n-π transition or the presence of the aldehyde group
(
additionally calculated maxima of the ꢑ ꢒꢕ ꢓꢖ ꢔꢒ are: benzaldehyde - 195.17 nm; acetaldehyde -
06.73 and 245.50 nm; formaldehyde - 223.70 nm, see Fig. S1). In this connection it should
2
be assumed that PB2 dye, as well as PB1, tend to self-aggregate in medium and polar
environments which induces the reversal solvatochromism [103-104]. This is in accordance
1
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with the MEP plots where there are regions from which the studied compound can undergo
not only H-bond interactions with protein but also with other molecules. As it can be derived
from previously presented research [64] this situation does not have a significant effect on the
in-vivo studies.
Fig. 10.
The calculated emission spectra (ꢑ ꢛꢒ ꢓꢔ ꢔꢒ) were shown in Tables 5 and S6. As in the case
of the OPA spectra, closest to the experimental data are ꢑꢛꢔ obtained using PBE0. This
ꢒꢓꢔꢒ
functional overestimates the de-excitation energy in weakly polar 1,4-Dx and Et O and
2
underestimates it in polar MeCN, DMF and DMSO. In each case Δꢑ ꢛꢜ ꢔꢝ ꢞꢏꢟꢠꢜꢡ> 12 nm. The
asymptotically corrected functionals shifted ꢑꢛꢔ towards shorter wavelengths by more than
ꢒꢓꢔꢒ
3
0 nm and among them the values closer to measured ones are given by CAM-B3LYP. In
THF the used functionals provide the fluorescence maximum with an error at the lowest level,
while for MeCN and DMSO discrepancies are the highest in relation to the experimental data.
After state-specific correction, PBE0 shifts the bands maxima in the direction of longer
wavelengths and as a result increases the difference to the measured values. For other
functionals, the SS correction does not work correctly. After using cLR-TDDFT, the
differences in ꢑꢛꢔ between computed and measured values are significantly increased and
ꢒꢓꢔꢒ
the hypschromic effect is enhanced. On the other hand, predictability of solvatochromic
behvior is similar in both approaches. Transition from the vacuum to weakly polar 1,4-Dx is
associated with a bathochromic shift. From Et2O the non-monotonic behavior in function of
the solvent polarity is observed. The reversal solvatochromism visible in Et O and in polar
2
solvents confirms the conclusions for the OPA about solute-solvent interactions. Moreover,
similarly to the ꢑ ꢒꢕ ꢓꢖ ꢔꢢꢒ , the electric permittivity of the environment does not affect the intensity
of the fluorescence spectra and the additional peak disappears. These observations do not
allow to clearly exclude the possibility of H-bonds formation. However, they confirmed that
1
9

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in less polar solvents the PB2 dye tends mainly to self-aggregate, while in strongly polar ones
the specific solute-solvent interactions may occur. Similar conclusions came from the analysis
of the PB1 dye.
One of the coefficients specifying the usefulness of a dye as a fluorescent probe in
bioimaging is the Stokes' shift. As mentioned in experimental part, the PB2 is characterized
ꢤꢥ
by a large value of Δꢣ ꢚwhich increases as a function of solvent polarity but is slightly
decreased in water. For ab inito calculations, only PBE0 effectively reflects the behavior of
Stokes' shift in relation to the solvent. For asymptotically corrected functionals a decline in
ꢤꢥ
the Δꢣ ꢚvalue occurs from MeCN while in water these values grow. In the case of PBE0, the
Stokes' shift increases with increasing dielectric constant up to water, where it is reduced.
However, the values nearest to the experimental ones are obtained with CAM-B3LYP and
ꢤꢥ
-1
Δꢣ ꢚ is underestimated, except for water, and the average relative error is 147.6 cm . For
-1
PBE0 the average overestimation is 1881.6 cm . The use of state-specific approach leads to
underestimation of the Δꢣꢤꢥ which enhances the difference to the measured data. Despite the
fact that both functionals correctly reproduce the behaviour of the Stokes' shift relative to the
ꢤꢥ
-1
-1
solvent, the
are 6338.2 cm and 2098.9 cm for CAM-B3LYP and PBE0
ꢜꢝꢞꢏꢒꢦꢧꢏꢘꢨꢨꢛꢘ
respectively. Analogous dependence was observed for the PB1 dye. Therefore it should be
noted that PBE0 is almost a universal tool in the description of spectroscopic properties of
fluorescent probes.
As mentioned in the methodology section, the conjugation of PB2 with Conacavalin A
occurs via formation of a Shift base between the aldehyde of the dye and amino groups of a
lysine residue and next by reduction (with participation of NaBH which acts as the reductive
4
agent) to a secondary or tertiary amine to give a stable alkylamine bond. Thus, it is possible to
connect the dye to a macromolecule in several places. In order to identify the most probable
site for dye-protein conjugation, the AutoDock procedure was employed. As shown in Table
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S7, the lowest binding free energy (ΔG in kcal/mol) characterizes the sites with LYS200 and
b
the calculated inhibition constant (K ) is 0.24 mM. In this active site, the PB2 dye posses the
i
highest probability of potential binding affinity with the active site of the Concanavalin A [105-
1
06]. As it was shown in Fig. 11, in this location the fluorochrome probe is efficiently
inserted into the aromatic cage formed by LYS200 and GLY45 interacts with aldehyde group;
SER201 directed toward C16 and SER164 interacting with imidazole part. This binding is not
stabilized by the π-π stacking interaction and also the presence of H-bonds is not observed.
The slightly higher value of ΔG is observed for the binding active site created by the VAL79,
b
ILE29, LYS30 and ASN237, where LYS30 is directed towards C11 in central ring. However,
the ΔΔG between these locations is only 0.2 kcal/mol and the K value is doubled and
b
i
increases to 0.45 mM. As before, a π-π stacking interaction is not observed, as well as no H-
bonds. For the third binding active site (LYS114) with a relatively high affinity, the ΔGb
increases to -5.4 kcal/mol and K is increased to 0.70 mM. The PB2 is sandwiched between
i
the aromatic cage of LYS14, THR123, THR112, HIS127, GLU192 and MET129. The
fluorochrome is positioned by CHO group towards THR123 and LYS114 interacts with C16
of benzaldehyde. Moreover, in this location a H-bond forms between N7-H and the oxygen
atom of the THR112 (3.125 Å) which is consistent with the MEP analysis. The formation of a
H-bond may have an effect on the growth of the inhibition constant. For the active site with
LYS101 with lower affinity and where H-bond is not observed, the K is reduced by 0.29 mM.
i
In all active binding sites the PB2 dye is twisted with respect to the reference geometry (Sg).
This twisting occurs on the C8-C10 and C13-C16 bonds. The biggest twisting is observed in
the location with LYS200 where the torsion angle C8-C10-C16-O23 changes form -34.2° for
the S structure to 69.4° after docking. Additionally, the O23 in the aldehyde group is oriented
g
in a line with N7-H hydrogen. For other mentioned binding modes this angle is decreased and
the oxygen atom remains in the orientation analogous to Sg.
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Fig. 11.
Requirements for fluorescent probes are not only the specific characteristics of their
spectroscopic properties, but also specific physicochemical features. One of them is the
bioavailability [107]. The possibility and rate of penetration through biological membranes
and consequently, probability of achieving an appropriate concentration in the region of
action is referred to LogP. This value determines the absorption and the permeability across
cell membranes. For the PB2 dye the calculated LogP = 4,85 ±0.36 and according to the
Lipinski "5" rule, is characterized by relatively good bioavailability [108]. The studied
compound is characterized by high metabolism at CYP-450 (the calculated probabilities are
0
.6668 and 0.9938 for the CYP450-2D6 and CYP450-3A4, respectively). So, after
completing the task as a fluorescent marker, it will be quickly removed from the body without
interacting with other drugs [109]. Another important parameter indicating bioconcentration is
LogBCF. It refers to the process of uptake and build up of chemicals in living organisms. The
calculated LogBCF = --4.89558 and suggests that PB2 molecule will not be bioaccumulative
in living organisms. Moreover, the calculated probability of toxicity is 0.46 ±0.1 what
indicates a moderate toxicity (the range 0.2-08). Therefore, insertion of an additional benzene
ring relative to the PB2 dye does not amend significant difference to LogP (ΔLogPPB2-PB1
=
0
.04) and LogBCF (ΔLogBCFPB2-PB1 = 1.01). However, it reduces the probability of toxicity
by 0.23. On the other hand, the PB2 can be used in many other areas of medical sciences. This
dye is characterized by many biological activities and exhibits such properties as: Anti-Tumor
Cycline-dependent kinase 4 inhibitory activity; Anti-Tumor DNA anti-metabolitic activity;
HIV1-proteaze inhibitory activity and others (see Fig.S2).
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Conclusions
Synthesis, spectral, photophysical and some of biological activities in different
solvents of new phenanthroimidazole derivatives: 4'-(1H-phenantro[9,10-d]-imidazol-2-yl)-
bifenyl-4-carboaldehyde (PB2) were presented. The experimental measurements showed a
significant effect on the position of absorption and emission spectra. The emission spectra are
red-shifted by ca. 80 nm resulting in a large Stokes' shift. The high molar absorption
coefficient, indicating intramolecular charge transfer (ICT) characteristics of the transition
and decreasing the fluorescence quantum yield in function of solvent polarity, suggests that
PB2 is characterized by a highly polar ICT excited state. For this dye, the fluorescence
quantum yield is indirectly proportional to the lifetime which may be due to some interaction
between the strongly polarized molecule and solvents in the excited state. The theoretical
calculated vertical excitation energies, as well as the state-specific corrected linear response
approach clearly indicate that PB2 compound exhibits the non-monotonic solvatochromism.
By contrast, the correlations of the polarity of the CT state in function of solvent polarity
showed that this dye exhibits strongly polar charge transfer excited state and a red shift should
occur. Therefore, the observed non-monotonic behaviour is associated with a tendency of
self-aggregation in some environments which also may affect the solubility. The AutoDock
simulations pointed out that the active site near LYS200 is the most probable location where
the conjugation of PB2 with Conconavalin A will occur. This binding active site is
characterized by lowest ΔG values and the inhibition constant is only 0.24 mM. Interactions
b
with macromolecules are purely van der Waals interactions and no π-π stacking nor H-bonds
are observed. Moreover, theoretical calculations showed that CT state is more planar than the
ground state and after docking the structure of PB2 dye undergoes greater twisting, which
occurs on the C8-C10 and C13-C16 bonds. The usefulness of this compound in biomedical
imaging was confirmed by fluorescence microscopy imaging. The in-vitro study showed that
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the PB2 conjugate Concanavalin A exhibited a blue fluorescence with high stability and
intensity. Therefore this compound has potential applicability as a fluorescent probe for
protein detection, for example for labelling of lectins, for detection of specific carbohydrate
moieties and antibodies for immunofluorescence assays. On the other hand, the tested dye is
characterized by moderate toxicity, good bioavailability and low bioaccumulative in living
organisms. All of these properties make the PB2 dye a promising potential compound for
fluorescence bioimaging.
Acknowledgements
This research was supported by the Computational Grant No. 249, PCSS (Poznan, Poland); in
part by PL-Grid Infrastructure; BS 9/2014. Yarrowia lipolytica A101 was a kind gift of Dr.
Aleksandra Mirończuk (Department of Biotechnology and Food Microbiology, Wrocław
University of Environmental and Life Sciences, Wroclaw, Poland).
Supporting Information Available: Analytical data, molecular structure and spectroscopic
properties for investigated compound.
Submission declaration
Submitting the article " Synthesis, photophysical properties and systematic evaluations of new
phenanthroimidazole fluorescent probe for bioimaging: experimental and theoretical study" to
The Journal of Photochemistry and Photobiology B: Biology has not been published
previously, is not under consideration for publication elsewhere, publication is approved by
all authors and it will not be published elsewhere including electronically in the same form,
in English or in any other language, without the written consent of the copyright-holder.
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Conflict of interest
The authors declare that there are no conflicts of interest.
Declaration of interest
Submitting the article "Synthesis, photophysical properties and systematic evaluations of new
phenanthroimidazole fluorescent probe for bioimaging: experimental and theoretical study" to
The Journal of Photochemistry and Photobiology B: Biology does not have any potential
conflicts of financial and non-financial interests.
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References:
1] P. Mitra, M. Banerjee, S. Biswas, S. Basu, Protein interactions of Merocyanine 540:
[
spectroscopic and crystallographic studies with lysozyme as a model protein, J. Photochem.
Photobiol. B 121 (2013) 46-56, DOI: 10.1016/j.jphotobiol.2013.02.010.
[
2] N. Romanov, C. Anastasi, X. Liu, Cyanine dyes for labeling molecular ligands with
improved fluorescence intensity and photostability, 2013: WO2013041117 A1.
3] M. Fernández-Suárez, A.Y. Ting, Fluorescent probes for super-resolution imaging in
living cells, Nat. Rev. Mol. Cell. Biol. 9 (2008) 929-943, DOI: 10.1038/nrm2531.
4] C. Basford, N. Forraz, C. McGuckin, Optimized multiparametric immunophenotyping of
umbilical cord blood cells by flow cytometry, Nat. Protoc. 5(7) (2010) 1337-1346. DOI:
0.1038/nprot.2010.88
5] A. Tanimura, Development and application of fluorescent protein-based indicators for live
cell imaging, J. Oral. Biosci. 57 (2015) 54 – 60, DOI: 10.1016/j.job.2015.02.006.
6] X. Zhang, S. Li, Z. Liu, S. Wang, J. Xiao Self-assembled multicolor nanoparticles based
on functionalized twistacene dendrimer for cell fluorescent imaging, NPG Asia Mater. 7
[
[
1
[
[
(2015) 230-10, DOI: 10.1038/am.2015.126.
[
7] J. Liu, Y.Q. Sun, H. Zhang, H. Shi, Y. Shi, W. Guo, Sulfone-Rhodamines: A new class of
near-infrared fluorescent dyes for bioimaging." ACS Appl. Mater. Interfaces. 8(35) (2016)
2953–22962, DOI: 10.1021/acsami.6b08338.
8] Y.-J. Gong et al, A unique approach toward near-infrared fluorescent probes for
bioimaging with remarkably enhanced contrast, Chemical Science 7 (2016) 2275-2285, DOI:
0.1039/C5SC04014K.
9] S. Lindström, Flow cytometry and microscopy as means of studying single cells: a short
introductional overview, Methods. Mol. Biol. 853 (2012) 13-15, DOI: 10.1007/978-1-61779-
67-1_2.
2
[
1
[
5
2
6

ACCEPTED MANUSCRIPT
[
10] M. Díaz, M. Herrero, L.A. Garcia, C. Quirós, Application of flow cytometry to industrial
microbial bioprocesses, Biochem. Eng. J. 48(3) (2010) 385-407, DOI:
0.1016/j.bej.2009.07.013.
11] R. Weissleder, V. Pittet, Imaging in the era of molecular oncology, Nature 4522 (2008)
80-589, DOI: 10.1038/nature06917.
12] L.E. Jennings, N.J. Long, ‘Two is better than one’-probes for dual-modality molecular
imaging, Chem. Commun. 24 (2009) 3511-3524, DOI: 10.1039/B821903F.
13] J.-M. Liu, J.-T. Chen, X.-P. Yan, Near infrared fluorescent trypsin stabilized gold
1
[
5
[
[
nanoclusters as surface plasmon enhanced energy transfer biosensor and in vivo cancer
imaging bioprobe, Anal. Chem. 85 (2013) 3238-3245, DOI: 10.1021/ac303603f.
[
14] H. Guo, N.M. Idris, Y. Zhang, LRET-based biodetection of DNA release in live cells
using surface-modified upconverting fluorescent nanoparticles, Langmuir 27 (2011) 2854-
860, DOI: 10.1021/la102872v.
15] Z. Li, Y. Zhang, S. Jiang, Multicolor core/shell-structured upconversion fluorescent
nanoparticles, Adv. Mater. 20 (2008) 4765-4769, DOI: 10.1002/adma.200801056.
16] F. Rijke, H. Zijlmans, S. Li, T. Vail, A.K. Raap, R.S. Niedbala, H.J. Tanke, Up-
converting phosphor reporters for nucleic acid microarrays, Nat. Biotechnol. 19 (2001) 273-
76. DOI: 10.1038/85734.
17] J. Kim, H.S. Kim, N. Lee, T. Kim, H. Kim, T. Yu, I.C. Song, W.K. Moon, T. Hyeon,
2
[
[
2
[
Multifunctional uniform nanoparticles composed of a magnetite nanocrystal core and a
mesoporous silica shell for magnetic resonance and fluorescence imaging and for drug
delivery, Angew. Chem. Int. Ed. Engl. 47(44) (2008) 8438-8441, DOI:
1
0.1002/anie.200802469.
2
7

ACCEPTED MANUSCRIPT
[
18] D. Janczewski, Y. Zhang, G.K. Das, D.K. Yi, P. Padmanabhan, K.K. Bhakoo, T.T. Tan,
S.T. Selvan, Bimodal magnetic-fluorescent probes forbioimaging, Microsc. Res. Tech. 74
(2011) 563-576. DOI: 10.1002/jemt.20912.
[
19] M. Tsuji, S. Ueda, T. Hirayama, K. Okuda, Y. Sakaguchi, A. Isono, H. Nagasawa,
FRET-based imaging of transbilayer movement of pepducin in living cells by novel
intracellular bioreductively activatable fluorescent probes, Org. Biomol. Chem. 11 (2013)
3
030-3037, DOI: 10.1039/C3OB27445D.
20] F. Auzel, Upconversion and anti-stokes processes with f and d ions in solids, Chem. Rev.
04 (2004) 139-174, DOI: 10.1021/cr020357g.
21] A. Germond, H.Fujita, T. Ichimura, T.M. Watanabe, Design and development of
[
1
[
genetically encoded fluorescent sensors to monitor intracellular chemical and physical
parameters, Biophys. Rev. 8 (2) (2016) 121-138, DOI: 10.1007/s12551-016-0195-9.
[
22] U. Uçucu, N.G. Karaburun, I. Işikdağ, Synthesis and analgesic activity of some 1-benzyl-
2
1
-substituted-4,5-diphenyl-1H-imidazole derivatives, Farmaco. 56 (2001) 285-290, DOI:
0.1016/S0014-827X(01)01076-X.
[
23] A. Yeşilada, S. Koyunoğlu, N. Saygili, E. Kupeli, E. Yeşilada, E. Bedir, I. Khanc,
Synthesis, anti-inflammatory and analgesic activity screening of some new 4-(3H)-
quinazolinone derivatives, Arch. Pharm. Pharm. Med. Chem. 337 (2004) 96-104, DOI:
1
0.1002/ardp.200200752.
24] L. Quattara, M. Debaert, R. Cavier, Synthesis and antiparasitic activity of new nitro-5-
imidazole derivatives, Farmaco. (sci) 42(6) (1987) 449-456, PMID:2904887
25] S. Dutta, Synthesis and anthelmintic activity of some novel 2-substituted-4,5-diphenyl
imidazoles, Acta. Pharm. 60(2) (2010) 229-235. DOI: 10.2478/v10007-010-0011-1.
[
[
2
8

ACCEPTED MANUSCRIPT
[
26] A.K. Sengupta, T. Bhattacharya, Synthesis and antimicrobial activity of some
substituted 2-phenyl-3-arylquinazol-4-ones, J. Indian. Chem. Soc. 60 (1983) 373-376, DOI:
0.1002/chin.198349246.
27] L. Navidpour, H. Shadnia, H. Shafaroodi, M. Amini, R. Dehpour, A. Shafiee, Design,
synthesis and biological evaluation of substituted 2-alkylthio-1,5-diarylimidazoles as selective
COX-2 inhibitors, Bioorg. Med. Chem. 15 (2007) 1976-1982, DOI:
0.1016/j.bmc.2006.12.041.
28] G. Fluoret, Synthesis of pyroglutamylhistidylprolineamide by classical and solid phase
methods, J. Med. Chem. 13 (1970) 843-845, DOI: 10.1021/jm00299a011.
29] R.N. Brogden, R.C. Heel, T.M. Speight, G.S. Avery, Metronidazole in anaerobic
infections: A review of its activity, pharmacokinetics and therapeutic use, Drugs 16(5) (1978)
87-417, PMID:363399.
30] R.W. Brimblecombe, W.A.M. Duncan, G.J. Durant, J.C. Emmett, C.R. Ganellin, M.E.
1
[
1
[
[
3
[
Parsons, J.W. Black, The pharmacology of cimetidine, a new histamine H -receptor
2
antagonist, Br. J. Pharmacol. 53 (1975) 435–436, DOI: 10.1111/j.1476-5381.2010.00854.x.
[
31] W. Hunkeler, H. Möhler, L. Pieri, P. Polc, E.P. Bonetti, R. Cumin, R. Schaffner, W.
Haefely, Selective antagonists of benzodiazepines, Nature 290 (1981) 514-516, DOI:
0.1038/290514a0.
32] X. Cheng, H. Jia, J. Feng, J. Qin, Z. Li, "Reactive” probe for hydrogen sulfite: Good
ratiometric response and bioimaging application, Sensors and Actuators B 184 (2013) 274-
80, DOI: 10.1016/j.snb.2013.04.070.
33] W. Lin, L. Long, L. Yuan, Z. Cao, B. Chen, W. Tan, A Ratiometric fluorescent probe for
cysteine and homocysteine displaying a large emission shift, Org. Lett. 10(24) (2008) 5577-
580. DOI: 10.1021/ol802436j.
1
[
2
[
5
2
9