Synthesis and Studies of Fluorescein Based Derivatives for their Optical Properties, Urease Inhibition and Molecular Docking

Article abstract of DOI:10.1007/s10895-018-2291-7

Herein, we design and synthesized new fluorescein based derivatives by insitu formation of fluorescein ester and further treated with corresponding hydrazide and amine to yield respective compounds i.e. FB1, FB2, FB3 and FB4. The spectral purity and chara

Full text of DOI:10.1007/s10895-018-2291-7

Journal of Fluorescence
https://doi.org/10.1007/s10895-018-2291-7
ORIGINAL ARTICLE
Synthesis and Studies of Fluorescein Based Derivatives for their Optical
Properties, Urease Inhibition and Molecular Docking
1
2
1
3
3
3
Prasad G. Mahajan & Nilam C. Dige & Balasaheb D. Vanjare & Hussain Raza & Mubashir Hassan & Sung-Yum Seo
&
4
1
Seong-Karp Hong & Ki Hwan Lee
Received: 13 July 2018 /Accepted: 28 August 2018
Springer Science+Business Media, LLC, part of Springer Nature 2018
#
Abstract
Herein, we design and synthesized new fluorescein based derivatives by insitu formation of fluorescein ester and further treated
with corresponding hydrazide and amine to yield respective compounds i.e. FB1, FB2, FB3 and FB4. The spectral purity and
characterization was done by using IR, NMR and Mass spectroscopies. The synthesized derivatives were examined for their
photophysical properties by using variety of organic solvents and results were discussed in details. The structural diversity of
synthesized compounds motivate us to evaluate these compounds for urease inhibition. The compound FB3 (IC = 0.0456 μM)
5
0
shows 100 fold more active against Jack bean urease than standard drug thiourea (IC50 = 4.7455 μM). Other synthesized
compounds showed potent activity. Free radical percentage scavenging assay further supported the capacity of compounds to
urease inhibition. While, molecular docking simulations helps to examine the molecular interactions of active compounds FB1,
FB2, FB3 and FB4 within the binding site of urease enzyme.
.
.
.
.
Keywords Synthesis Fluorescein derivatives Optical properties Urease inhibitors Molecular docking
Introduction
sites available in fluorescein stimulate the researchers to syn-
thesize its variety of derivatives followed by their application
[2, 3]. The number of derivatives originated from fluorescein
as base core are widely used as biomarkers [4, 5], probes for
reactive oxygen species (ROS) [6], to measure the pancreatic
esterase activity and for preparation of labeled oligonucleo-
tides (https://en.wikipedia.org/wiki/Fluorescein#Derivatives),
[7]. Now a days, because of the extent use and wide applica-
bility of fluorescein based organic compounds have received
much more attention in the field of medicinal science and
pharmacy [8, 9]. Therefore, design, synthesis and application
of fluorescein based organic compounds for studies on their
optical properties and utility in microbial evaluation is still
challenging task before the scientific community.
Fluorescein is one of the main and important member from the
dye family since last so many years. It is highly fluorescent,
hydrophilic and widely used as staining reagent in most of the
cell biology experiments [1]. Due to the diverse medicinal
applications, World Health Organization’s included fluoresce-
in is on the list of vital medicines in the category of ophthal-
mology. The excellent optical properties and active reactive
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s10895-018-2291-7) contains supplementary
material, which is available to authorized users.
*
Ki Hwan Lee
khlee@kongju.ac.kr
One of the natural enzyme belong to the amidohydrolases
family is urease [10, 11]. It is divalent nickel ion dependent
enzyme commonly originated in fungi, seeds of plants, algae,
soil and in human body parts such as guts and kidneys
1
2
3
4
Department of Chemistry, Kongju National University, Gongju,
Chungnam 32588, Republic of Korea
[12–14]. Urease are responsible for the increasing in the rate
Department of Chemistry, Shivaji University,
Kolhapur, Maharashtra 416004, India
of hydrolysis of ammonia (NH ) and carbon dioxide (CO )
3
2
and consequently increase in the pH of the medium and helps
to survive a harmful bacteria Helicobacter pylori [15, 16].
Such kinds of phenomenon are responsible for the number
of health disorders such as urine and gastrointestinal
Department of Biological Sciences, Kongju National University,
Gongju, Chungnam 32588, Republic of Korea
Division of Bio and Health Sciences, Mokwon University, Seo-gu,
Daejeon 35349, Republic of Korea

J Fluoresc
infections along with occurrence of kidney stone, pyelone-
phritis, hepatic encephalopathy, and ultimately hepatic uncon-
sciousness [17–19]. Soil infertility is also one of main things
that affected by the urease enzyme [20]. The range of organic
compounds from classes such as heterocycles [21], schiff ba-
ses [22], hydroxamic acids [23], heavy metal ions [24], qui-
nones [25] and thiols [26] were used so far as promising ther-
apies against hydrolysis of urease enzymes. Unfortunately,
most of the compounds fails to exhibits their action due to
hurdles such as low hydrolytic stability and toxic nature
evaporated under reduced vacuum pressure. Thus, orange-
yellow powder obtained was further washed with cold ethanol
to discharge the existence of starting material, if any. An ap-
preciable i.e. 78% yield of compound FB1 was recrystallized
using hot ethanol. The recrystallized product was further char-
acterized by spectral techniques viz., IR, NMR and mass
spectroscopy.
Orange-yellow powder, Observed melting point: 198 °C,
IR Fig. S1: 3022 (-NH), 2961 (OH), 1713, 1667 (C=O), 1436,
1395, 1321, 1312, 1232, 1202, 1192, 967, 903, 808,
−1 1
[
12]. Therefore, urease inhibition is of crucial importance
682 cm ; H NMR (DMSO-d 400 MHz) Fig. S2 δ: 10.81
6,
and has attracted advanced courtesy due to their extensive
applications in medicinal industry. The present research work
aims to design and synthesis of fluorescein based derivatives
for studies on their optical properties and biomedical applica-
tion. All synthesized fluorescein based new derivatives have
significant potential inhibition capacity against jack bean ure-
ase enzyme. The activity results were supported by the free
radical scavenging assay and molecular docking simulations.
(s, 1H, -NH), 10.42 (s, 2H, -OH), 7.43 (m, 6H, ArH), 7.12–
7.17 (m, 8H, ArH), 4.45 (s, 1H, -CH), 4.06 (s, 2H, -CH ) ppm;
2
Mass (ESI MS) Fig. S3: 549 [M + 1], 514, 442, 402, 385, 325,
309 m/z.
Synthetic Procedure for FB2
This compound was synthesized using a fluorescein
(
(
0.515 g, 1.55 mmol) which was dissolved in methanol
20 mL) in 50 mL round bottom flask. 1 mL of conc.
Experimental
H SO was added to the flask. The whole mixture was
2 4
refluxed for 6 h and further 2-(thiophen-3-
yl)acetohydrazide (0.242 g, 1.55 mmol) was added in
it. The resultant reaction mixture was refluxed another
3 h. The completion of reaction was confirmed using
TLC technique. The reaction mixture was allowed to
cool at room temperature and solvent was evaporated
under reduced vacuum pressure. Thus, orange-yellow
powder obtained was further washed with cold ethanol
to discharge the existence of starting material, if any.
An appreciable i.e. 69% yield of compound FB2 was
recrystallized using hot ethanol. The recrystallized prod-
uct was further characterized by spectral techniques viz.,
IR, NMR and mass spectroscopy.
Reagents
Fluorescein, 2-benzimidazoyl-thioacetic acid hydrazide,
2
-(thiophen-3-yl)acetohydrazide, thiourea and thiophene-3-
carbohydrazide used in the synthesis part were purchased
from Alfa Aesar. Conc. H SO was procured from Sigma-
2
4
Aldrich, Korea. The di-methyl sulphoxide (DMSO), di-
methyl formamide (DMF), Tetrahydrofuran (THF), methanol,
ethanol, ethyl acetate, chloroform, acetonitrile (ACN), dichlo-
romethane (DCM) and acetone were procured from Samchun
Chemicals, Korea and used after further distillation. Double
distilled water was used to carry out all experiments.
Orange-yellow powder, Observed melting point: 177 °C,
IR Fig. S4: 3022 (-NH), 2961 (OH), 1713, 1638 (C=O), 1585,
1448, 1395, 1202, 1192, 1118, 967, 913, 903, 819, 808, 754,
Method for the Synthesis of Fluorescein Based
Compounds
−1 1
6
82 cm ; H NMR (DMSO-d 400 MHz) Fig. S5 δ: 10.07(s,
6,
Scheme 1 shows the synthetic route for FB1, FB2, FB3 and
FB4 derived from fluorescein.
1H, -NH), 8.25 (d, 2H, ArH), 7.82–7.88 (m, 2H, ArH), 7.51
(d, 2H, ArH), 7.30 (s, 2H, -OH), 6.99–7.02 (m, 2H, ArH),
6
.86 (s, 1H, ArH), 6.77–6.80 (m, 2H, ArH), 6.54 (s, 2H,
Synthetic Procedure for FB1
ArH), 3.38 (s, 2H, -CH ) ppm; Mass (ESI MS) Fig. S6: 470
2
[M+], 280, 124, 97 m/z.
This compound was synthesized using a fluorescein (0.515 g,
1
5
.55 mmol) which was dissolved in methanol (20 mL) in
0 mL round bottom flask. 1 mL of conc. H SO was added
Synthetic Procedure for FB3
2
4
to the flask. The whole mixture was refluxed for 6 h followed
by addition of 2-benzimidazoyl-thio-acetic acid hydrazide
This compound was synthesized using a fluorescein
(0.515 g, 1.55 mmol) which was dissolved in methanol
(20 mL) in 50 mL round bottom flask. 1 mL of conc.
H SO was added to the flask. The whole mixture was
refluxed for 6 h with further addition of thiourea
(0.118 g, 1.55 mmol) in water to it. The resultant
(
0.363 g, 1.55 mmol). The resultant reaction mixture was
refluxed further for 3 h. The completion of reaction was con-
firmed using TLC technique. The reaction mixture was
allowed to cool at room temperature and solvent was
2
4

J Fluoresc
Scheme 1 Schematic
representation of synthesis route
for FB1, FB2, FB3 and FB4
Code
FB1
R-NH
2
Product
Yield (%)
78
FB2
FB3
6
9
82
FB4
7
2
reaction mixture was refluxed for 3 h. The completion
of reaction was confirmed using TLC technique. The
reaction mixture was allowed to cool at room tempera-
ture and solvent was evaporated under reduced vacuum
pressure. Thus, orange-yellow powder obtained was fur-
ther washed with cold ethanol to discharge the existence
of starting material, if any. An appreciable i.e. 82%
yield of compound FB3 was recrystallized using hot
ethanol. The recrystallized product was further charac-
terized by spectral techniques viz., IR, NMR and mass
spectroscopy.
Synthetic Procedure for FB4
This compound was synthesized using a fluorescein (0.515 g,
1.55 mmol) which was dissolved in methanol (20 mL) in
50 mL round bottom flask. 1 mL of conc. H SO was added
2
4
to the flask and whole mixture was refluxed for 6 h followed
by addition of thiophene-3-carbohydrazide (0.220 g,
1.55 mmol) in it. The resultant reaction mixture was refluxed
for 3 h. The completion of reaction was confirmed using TLC
technique. The reaction mixture was allowed to cool at room
temperature and solvent was evaporated under reduced vacu-
um pressure. Thus, orange-yellow powder obtained was fur-
ther washed with cold ethanol to discharge the existence of
starting material, if any. An appreciable i.e. 72% yield of com-
pound FB4 was recrystallized using hot ethanol. The recrys-
tallized product was further characterized by spectral tech-
niques viz., IR, NMR and mass spectroscopy.
Orange-yellow powder, Observed melting point: 139 °C,
IR Fig. S7: 3265 (-OH), 3022 (-NH), 1661, 1601 (C=O),
−
1
1
395, 1192, 1118, 1038, 967, 913, 903, 808, 728, 682 cm ;
1
H NMR (DMSO-d 400 MHz) Fig. S8 δ: 9.81 (s, 2H, -NH ),
6,
2
7
2
.73–7.76 (m, 2H, ArH), 7.54 (m, 2H, ArH), 7.20–7.22 (m,
H, -OH), 7.10–7.13 (m, 4H, ArH), 6.52–6.54 (s, 2H, ArH)
ppm; Mass (ESI MS) Fig. S9: 391 [M + 1], 256, 192, 160,
28, 116,96, 76 m/z.
Orange-yellow powder, Observed melting point: 222 °C,
IR Fig. S10: 3322 (-OH), 3022 (-NH), 2930, 2851, 1713,
1

J Fluoresc
1
661 (C=O), 1579, 1395, 1311, 1270, 1202, 1192, 1142,
FB1, FB2, FB3 and FB4.
−1 1
1118, 1056, 967, 903, 820, 754, 703, 682 cm ; H NMR
FP: Aref : n2
Φ ¼ Φ
(
(
DMSO-d 400 MHz) Fig. S11 δ: 10.23 (s, 1H, -NH), 8.23
d, 1H, ArH), 7.78–7.89 (m, 3H, ArH), 7.64 (s, 1H, ArH),
6
,
ð1Þ
f
f ðref Þ
Fref : AP: n2
ref
7
6
.49 (d, 1H, ArH), 7.21 (d, 2H, -OH), 7.10 (m, 1H, ArH),
.88 (d, 1H, ArH), 6.63–6.69 (m, 1H, ArH), 6.54 (s, 1H,
where, Φ and Φ
are the fluorescence quantum yield of
f
f (ref)
corresponding compound and reference quinine sulphate, re-
spectively. The area under the emission spectrum of the re-
spective synthesized compound and quinine sulphate are de-
noted as F and F , respectively. Absorbances shown as A
ArH), 6.51 (d, 1H, ArH), 6.46 (d, 2H, ArH) ppm; Mass (ESI
MS) Fig. S12: 456 [M+], 252, 234, 142, 128, 111 m/z.
P
ref
ref
Characterization Techniques
and A for reference quinine sulphate and individual com-
P
pound, respectively. The refractive indexes of solvents used
to dissolve the compounds FB1, FB2, FB3 and FB4 and
quinine sulphate given as n and n , respectively.
ref
The formation of designed fluorescein derivatives were con-
firmed by using spectral analysis such as FT-IR, NMR and
mass spectroscopies. FT-IR spectra were noted on a Frontier
1
IR (Perkin Elmer) spectrometer using KBr pellets. H NMR
Urease Inhibition Activity
spectra were recorded on a Bruker Avance 400 MHz spec-
trometer with TMS as an internal standard. Mass spectrum
were recorded on Shimadzu QP 2010, Japan. The melting
point was observed on Digimelt (SRS, USA) melting point
apparatus.
The already reported method was used to measure the quan-
tity of ammonia produced with indophenols used to evaluate
the Jack bean urease activity [29, 30]. In a 96-well plate,
20 μL of enzyme (Jack bean urease, 5 U/mL) and 20 μL of
respective compounds (FB1, FB2, FB3, FB4) was dissolved
in 50 μL potassium phosphate buffer (100 mM urea, 0.01 M
UV-Vis. Absorption and Fluorescence Spectroscopy
K HPO , 1 mM EDTA and 0.01 M LiCl, pH 8.2), followed
by incubation for 30 min at 37 °C. After the time, each
solution was filled with the 50 μL each of phenol reagents
UV–visible absorption spectra of compounds FB1, FB2, FB3
and FB4 were recorded on a Shimadzu Spectrophotometer
using variety of organic solvents at concentration of 1 μM
for each. The absorption spectra were scanned for 350 to
2
4
(1%, w/v phenol and 0.0.5%, w/v sodium nitroprusside) and
5
0 μL of alkali reagent (0.5%, w/v NaOH and 0.1% sodium
7
1
00 nm wavelength range using 1 cm quartz cuvette and a
nm slit width. The fluorescence emission spectra of com-
hypochlorite NaOCl) and let the solution stands for 10 min.
The microplate reader (OPTI Max, Tunable) was used to
measure the absorbance at 625 nm. The experiment and
the measurement of absorbance was accomplish in triplicate.
The urease inhibition activities were calculated according to
the following formula:
pounds were examined by using FS-2 fluorescence spectrom-
eter (Scinco, Korea) in individual solvent. The fluorescence
measurement was done at respective excitation wavelength
and slit width of 1 nm for emission and excitation bandwidths.
Fluorescence Lifetime
ODcontrol−ODcompound
Urease inhibition activity ð%Þ ¼
 100 ð2Þ
The synthesized compounds FB1, FB2, FB3 and FB4 were
examined to acquire their fluorescence lifetime. For this study,
decay curves were obtained using a Time Correlated Single
Photon Counting (TCSPC) spectrophotometer (HORIBA-
iHR320) employing a nanosecond diode laser with time cor-
relation of 0.0548 ns per channel and operated at its respective
excitation wavelength. The monitoring decay curves thus re-
ceived were analyzed using DAS6, data station software
equipped with the instrument.
ODcontrol
Where, ODcontrol and ODcompound denotes the optical densi-
ties in the absence and presence of each compound, respec-
tively. Thiourea was used as the standard inhibitor for this
experiment.
Free Radical Scavenging Assay
Radical scavenging activity was determined by modifying
already reported method [31, 32] with the aid of 2, 2-
diphenyl-1 picrylhydrazyl (DPPH) assay. The assay solution
composed by the addition of 100 μL of DPPH (150 μM) and
20 μL of increasing concentration of test compounds with
constant final volume was adjusted to 200 μL in each well
using MeOH followed by 30 min incubation at room temper-
ature. The reference inhibitor used for the assay was ascorbic
Fluorescence Quantum Yield
The fluorescence quantum yield (Φ ) for compounds FB1,
FB2, FB3 and FB4 were determined at same concentration
f
(
1 μM) using quinine sulphate as reference (Φ = 0.58) [27].
f
The equation given below [28] was further used to estimate
the fluorescence quantum yield for synthesized compound

J Fluoresc
acid (vitamin C). The microplate reader (OPTI Max, Tunable)
at 520 nm used to carried out the assay analysis in three inde-
pendent experiments. The reaction rates have been compared
and the percent inhibition resulting from the presence of tested
inhibitors was calculated.
and hydrogen atoms were added to the protein. The structure
was then minimized to reach the converged root mean square
deviation (RMSD) of 0.30 Å with the OPLS_2005 force field.
The active site of the enzyme is defined from the co-
crystallized ligands from Protein Data Bank and literature data
[37]. Furthermore, docking experiment was performed against
all synthesized compounds (here termed as ‘ligands’) and tar-
get protein by using Glide docking protocol [37]. The predict-
ed binding energies (docking scores) and conformational po-
sitions of ligands within active region of protein were also
performed using Glide experiment. Throughout the docking
simulations, both partial flexibility and full flexibility around
the active site residues are performed by Glide/SP/XP and
induced fit docking (IFD) approaches [38].
Computational Methodology
Selection of Jack Bean Urease Structure from PDB
The Protein Data Bank (PDB) (www.rcsb.org) with PDBID
4
H9M provides Jack bean urease structure. The UCSF
Chimera 1.10.1 tool was employed to minimize the selected
crystal structure of urease [33]. Furthermore, Molprobity
server [34] and Protparam [35] was accessed for the stereo-
chemical properties of urease structure and
Ramachandran plot and calculations on values. The on-
line server VADAR 1.8 predicts the protein architecture
and statistical percentage values of receptor proteins helices,
beta-sheets, coils and turn [36].
Results and Discussion
Spectroscopic Studies: Optical Properties
To investigate the photophysical properties of synthesized
fluorescein derivatives, absorption spectroscopy, fluorescence
spectroscopy and fluorescence lifetime was employed. The
excellent solubility of all compounds in most of the common-
ly used organic solvents led us to study their photophysical
properties in each solvent at room temperature. Fig. 1a, b, c, d
Grid Generation and Molecular Docking
Prior to molecular docking, the optimized carbonic anhydrase
structure was prepared using the BProtein Preparation Wizard^
workflow in Schrödinger Suite. Bond orders were assigned
Fig. 1 Absorption spectra of FB1 (a), FB2 (b), FB3 (c) and FB4 (d) in variety of solvents [1 μM]

J Fluoresc
Fig. 2 Fluorescence spectra of FB1 (a), FB2 (b), FB3 (c) and FB4 (d) in variety of solvents [1 μM]
shows absorption spectra of compound FB1, FB2, FB3 and
FB4 in corresponding organic solvents, respectively. From the
spectral observation, it is seen that all compounds have max-
imum absorption wavelength in visible range. The polarity of
solvents plays a crucial role to decide their photophysical
properties. An increase in the solvent polarity simultaneously
shows a bathochromic shift in absorption spectrum for each
compound. The red shift of wavelength maxima is assigned to
the interaction of polar nature of solvent with the fluorescein-
based molecules and responsible for decrease the excited state
energy level that results in bathochromic shift in absorption
maxima [39–41]. The less pronounced absorption bands for
compounds FB2, FB3 and FB4 in region of 470–550 nm in
Fig. 1b, c, d is because of π → n* transitions, while it is sig-
nificant in case of FB1 at 511–528 nm. The other bands in
respective solvents are in favor of π → π* transitions. The
calculated molar excitation coefficient (ε) and absorption
spectrum of all compounds shows FB1, FB3 and FB4 has
Table 1 Photophysical properties of FB1, FB2, FB3 and FB4 in respective solvent
Solvent
FB1
FB2
FB3
FB4
6
6
6
6
λ
abs
λ
(nm)
FL
ε × 10
λ
(nm)
abs
λ
(nm)
FL
ε × 10
λ
(nm)
abs
λ
(nm)
FL
ε × 10
λ
(nm)
abs
λ
(nm)
FL
ε × 10
−
1
−1
−1
−1
−1
−1
−1
−1
(nm)
(M cm )
(M cm )
(M cm )
(M cm )
Water
528
521
520
511
525
512
513
522
515
520
555
541
538
522
550
524
528
543
529
534
2.76
1.56
1.20
0.14
2.59
0.49
1.02
1.71
0.62
0.78
458
450
450
448
454
448
449
452
449
450
555
523
522
502
550
510
515
523
518
520
0.43
0.27
0.23
0.01
0.4
454
453
450
440
454
446
448
453
450
450
546
528
526
516
528
517
519
528
520
526
3.6
457
452
451
446
455
446
447
455
448
448
552
521
520
518
525
518
519
522
520
520
1.9
DMSO
DMF
2.72
2.38
0.82
3.5
0.86
0.56
0.14
1.59
0.185
0.24
1.21
0.29
0.4
THF
Methanol
EA
0.026
0.03
3.01
0.06
0.1
0.64
2.06
3.12
1.20
1.59
Chloroform
ACN
DCM
Acetone

J Fluoresc
Table 2 Estimated Stoke’s shift, quantum yield (Φ
f
) and fluorescence lifetime (τ) values for FB1, FB2, FB3 and FB4 in respective solvent
Solvent
FB1
FB2
FB3
FB4
Stoke’s
Φ
f
τ
Stoke’s
Φ
f
τ
Stoke’s
Φ
f
τ
Stoke’s
Φ
f
τ
Shift
(ns)
Shift
(cm )
(ns)
Shift
(cm )
(ns)
Shift
(cm )
(ns)
−
1
−1
−1
−1
(
cm )
Water
921.375
709.569
643.408
412.383
865.800
447.280
553.783
740.881
513.884
504.177
0.067
0.118
0.133
0.663
0.09
3.06
3.01
2.78
2.65
2.66
2.54
2.51
2.49
2.38
2.29
3816.043
3101.763
3065.134
2401.109
3844.613
2713.458
2854.239
3003.434
2966.695
2991.452
0.044
0.297
0.323
0.793
0.059
0.667
0.466
0.203
0.4
2.72
2.71
2.70
2.69
2.68
2.64
2.60
2.58
2.54
2.49
3711.413
3135.661
3210.815
3347.427
3087.037
3079.164
3053.605
3135.661
2991.452
3210.815
0.052
0.072
0.087
0.437
0.053
0.302
0.147
0.054
0.125
0.115
3.84
3.79
3.78
3.69
3.67
3.54
3.53
3.51
3.47
3.46
3765.896
2930.035
2942.179
3116.505
2930.402
3116.505
3103.541
2820.933
3090.659
3090.659
0.056
0.135
0.167
0.847
0.119
0.756
0.376
0.129
0.26
3.21
3.17
3.14
3.13
3.12
3.10
3.09
3.04
2.91
2.84
DMSO
DMF
THF
Methanol
EA
0.349
0.345
0.109
0.272
0.143
Chloroform
ACN
DCM
Acetone
0.331
0.169
distinct photophysical properties than FB2 in almost each or-
ganic solvent studied.
value to the lower one with increase in the solvent polarity.
Fluorescence lifetime values are also dependent in accordance
with the polarity, dielectric constant and dipole moments of
the solvents used for the photophysical studies. Even-though
the changes observed for the fluorescence lifetime values for
each compound in respective solvent is small, it plays a vital
role in excited state phenomenon of compounds and changes
in their spectral properties. In an overall compared to FB1,
FB2 and FB4 compound, the FB3 shows a significant absorp-
tion, fluorescence emission, quantum yield and lifetime prop-
erties in all organic solvents under studied.
Fluorescence emission spectra of compounds FB1, FB2,
FB3 and FB4 is given as Fig. 2a, b, c, d in respective solvents.
The emission spectrum of all compounds noted significant
single broad peak in spectral region 480–600 nm is because
of transition of excited state to ground state i.e. n* → π and
π* → π transitions. The increase in polar nature of the solvent
shifts the emission spectrum towards red side and vice-versa.
Fluorescence properties guide to estimate the Stoke’s shift
values and quantum yield for each compound. These proper-
ties plays significant role in photophysical properties of organ-
ic compound in any solvent. Table 1 and Table 2 illustrates the
overall estimation of photophysical properties for each com-
pound in respective solvent. From the results, it concluded that
Stoke’s shift founds more in FB2, FB3 and FB4 than FB1.
The change in Stoke’s shift value with change in solvent for
each compound suggest that there were probability of struc-
tural changes in excited state through one of competing pro-
cess with the fluorescence. Another property of compounds
that emphasis to be act as good photophysical aspirant is fluo-
Urease Inhibition Activity
To examine the potent applicability in the form of urease in-
hibitor, all newly synthesized fluorescein derivatives were
accessed for Jack bean urease. Table 3 shows comparative
study of all compounds FB1, FB2, FB3 and FB4 with thio-
urea for urease activity along with their IC50 values. Amongst
them, fluorescein derivative FB3 shows excellent activity
with IC50 = 0.0456 μM. A free -C=O and –OH functional
group on the structure of synthesized compounds led to inter-
acts with Arg639 and Ala636, respectively. However, other
fluorescein derivatives shows potent urease activity in order to
rescence quantum yield (Φ ). The calculated quantum yield
f
values in each solvent suggest when the solvent polarity in-
creased the quantum yield value decreases is because of the
charge transfer phenomena [42]. The transportation of elec-
tron from the structural moiety possessing –NH group towards
the xanthene moiety of compound led to occurs intermolecu-
lar charge transfer (ICT) [43, 44]. This simultaneously in-
creases the dipole moment via relaxation process to solvent
molecules in the excited state [45]. The changes in dipole
moments and difference in dielectric constants of solvents
responsible to shift the spectral properties of compounds along
with change in fluorescence lifetime that decreases the radia-
tive pathways from excited state. This decrease in the radiative
pathways from the excited state disturbs the quantum yield
Table 3 Urease
inhibition activity of
Compound
Urease activity
FB1, FB2, FB3 and FB4
IC50 ± SEM (μM)
FB1
0.1752 ± 0.006155
0.0963 ± 0.004815
0.0456 ± 0.000860
0.0887 ± 0.007625
4.7455 ± 0.054590
FB2
FB3
FB4
Thiourea
*SEM = Standard Error of Mean

J Fluoresc
The compounds FB1, FB3 and FB4 show excellent activity
as compared to standard drug Ascorbic Acid (vitamin C) how-
ever, the FB2 compound did not show significant radical
scavenging ability even at the higher concentration (100 μg/
mL). Figure 3 shows that derivative FB3 was with higher
percentage scavenging activity and almost same as standard
drug used in this assay i.e. vitamin C (ascorbic acid).
However, other FB1 and FB4 shows moderate scavenging
activity as compared to FB3 and vitamin C.
Structural and Physiochemical Evaluation of Jack
Bean Urease
Jack bean urease is a one of the class of hydrolase protein
consists of different numbers of amino acids. The overall pro-
tein structure is mentioned in Fig. 4. The protein architecture
has 27% helices, 31% β sheets and 41% coils and that was
investigated by VADAR analysis. Moreover, 97.5% of resi-
dues were present in favored regions illustrated by
Ramachandran plots (Fig. S13) signifies the precision of phi
Fig. 3 Free radical % scavenging activity of FB1, FB2, FB3 and FB4.
Values were represented as mean ± SEM. All compounds concentrations
were 100 μg/mL)
(
(φ), and psi (ψ) angles among the coordinates of jack bean
urease. The aggregation of average isotopic masses of resi-
dues was used to calculate physiochemical properties
(Table 4) of jack bean urease viz. molecular weight (MW).
Whereas, pK values of amino acids of target protein were
useful to compute the theoretical pI [46]. The predicated pI
value (6.05) is in aggrement with the computational predicted
pI range values (4.31 to 11.78) of proteins [47] that showed
the accuracy of targeted protein. The consistency also ob-
served in case of predicted value of aliphatic and instability
indexes and relative volume engaged by aliphatic side chains
of targeted protein. The sum of hydropathic values of all ami-
no acids present in the protein was indicated by GRAVYvalue
Fig. 4 Protein structure
standard thiourea as FB4 > FB2 > FB1. From the results we
can conclude that derivative FB3 was 100 times more active
than standard control, thiourea (IC = 4.7455 μM). The ex-
[48]. If it is more negative value then it’s behavior is more
hydrophilic rather than hydrophobic [46].
5
0
cellent activity against urease enzyme for FB3 was further
supported by free radical scavenging assay.
Molecular Docking Analysis
Binding Energy Evaluation of Synthesized Compounds
Free Radical Scavenging
Each synthesized fluorescein derivative was docked
against urease enzyme protein to predict the best conforma-
tional position. The glide docking energy values (Kcal/mol)
The synthesized FB1, FB2, FB3 and FB4 fluorescein deriva-
tives were evaluated for DPPH free radical scavenging ability.
Table 4 Physiochemical
properties of Urease by
Sr. No.
Parameters
Values
ProtParam
1
2
3
4
5
6
Molecular weight (MW)
Theoretical pI
Extinction coefficient* (assuming all Cys residues are reduced)
Aliphatic index
Instability index
Gran average of hydropathicity (GRAVY)
90,747.7 Da
6.05
53,290
90.48
31.75
−0.152
Extinction Coefficient units M− cm−1 at 280 nm
1
*

J Fluoresc
Ala636, respectively. The observed binding interactions be-
tween residues and functional groups are in comparable with
the literature data that fortify our docking results [49, 50]. The
comparative binding energy and SAR analysis are in favor to
conclude that compound FB3 would be the potent ure-
ase inhibitor against jack bean protein enzyme. The oth-
er docking complexes are given in supplementary data
(
Fig. S14-S16).
Conclusions
Fig. 5 Graphical interpretation of docking energy values
In present study, new fluorescein-based derivatives were syn-
thesized from fluorescein, hydrazides and amine. All com-
pounds were assessed for their spectral characteristics using
NMR, IR and Mass spectroscopies. All synthesized com-
pounds exhibits excellent optical properties in most of the
commonly used organic solvents. The significant Stoke’s shift
values, quantum yield and fluorescence lifetime values are in
support of superior optical properties for synthesized com-
pounds. It was found that the compound FB3 shows effective
urease inhibition with IC50 value 0.0456 μM compared to
other compound and standard drug. Molecular docking stud-
ies and free radical scavenging assay are also found in accor-
dance with the experimental results and thus strengthen the
experimental data of present studies. The advantage of syn-
thesized compounds in terms of optical properties is that hy-
drophilicity. This study can open new window in direction to
use of these compounds in cell biology or sensing applica-
tions. In an overall, from the urease inhibition, free radical
scavenging and molecular docking studies, it can be conclud-
ed that all compounds shows excellent but different properties
compared to each other and standard positive control drug.
However, compound FB3 can serve as structural model in
scheming novel drugs counter to urease enzyme.
and bonding interaction (hydrogen/hydrophobic) pattern
inspected and scrutinized to generate docked complexes.
The best conformational position of each compound within
the active region of target protein was recognized on the basis
of binding energy value. Figure 5 displays the comparative
study of docking energy values for synthesized derivatives
FB1, FB2, FB3 and FB4. The docking results shows that
derivative FB3 show best energy value (−5.11 Kcal/mol)
compared to energy values of other derivatives FB1, FB2
and FB4 as −5.01, −4.21 and − 4.23 Kcal/mol, respectively.
Ligand-Binding Analysis of Urease Docked Complexes
All synthesized fluorescein derivatives were restrained in the
active binding region of receptor molecule exhibits different
conformational postures, which was supported by molecular
docking studies. Figure 6 illustrates the docked complex struc-
ture of compound FB3 (ligand) with urease protein enzyme.
In detail, structural activity relationship (SAR) study showed
that two active hydrogen bonds were seen in ligand (com-
pound FB3) docking complex. The carbonyl oxygen and ben-
zyl hydroxyl groups forms good interactions with Arg639 and
Fig. 6 Docking complex of FB3

J Fluoresc
Acknowledgments This work was supported by a grant from Mid-Career
Researcher Program (NRF- 2016R1A2B4016552) through the National
Research Foundation of Korea (NRF) funded by the Ministry of Science,
ICT & Future Planning (MSIP).
20. Saeed A, Rehman S, Channar PA, Larik FA, Abbas Q, Hassan M,
Raza H, Flörke U, Seo SY (2017) Long chain 1-acyl-3-
arylthioureas as jack bean urease inhibitors, synthesis, kinetic
mechanism and molecular docking studies. J Taiwan Inst Chem
Eng 77:54–63
2
1. Ibrar A, Khan I, Abbas N (2013) Structurally diversified heterocy-
cles and related privileged scaffolds as potential urease inhibitors: a
brief overview. Arch Pharm (Weinheim, Ger) 346:423–−446
2. Aslam M, Mahmood S, Shahid M, Saeed A, Iqbal J (2011)
Synthesis, biological assay in vitro and molecular docking studies
of new Schiff base derivatives as potential urease inhibitors. Eur J
Med Chem 46:5473–5479
References
2
1
.
Mondal SB, Gao S, Zhu N, Liang R, Achilefu S (2014) Chapter five
real-time fluorescence image-guided oncologic surgery. Adv
-
Cancer Res 124:171–211
2
3
4
.
.
.
Moore GE (1947) Fluorescein as an agent in the differentiation of
normal and malignant tissues. Science 106:130–131
Efron N (2012) Chapter 16 - corneal staining, Contact Lens
Complications (Third Edition) 155–166
23. Dixon NE, Gazzola C, Watters JJ, Blakeley RL, Zerner B (1975)
Inhibition of jack bean urease (EC 3.5.1.5) by acetohydroxamic
acid and by phosphoramidate. Equivalent weight for urease. J Am
Chem Soc 97:4130–4131
24. Shaw WHR, Raval DN (1961) The inhibition of urease by metal
ions at pH 8.9. J Am Chem Soc 83:3184–3187
25. Zaborska W, Kot M, Superata K (2002) Inhibition of jack bean
urease by 1,4-benzoquinone and 2,5-dimethyl-1,4-benzoquinone.
Evaluation of the inhibition mechanism. J Enzyme Inhib Med
Chem 17:247–253
Shinoda J, Yano H, Yoshimura S, Okumura A, Kaku Y, Iwama T
(
2003) Fluorescence-guided resection of glioblastoma multiforme
by using high-dose fluorescein sodium, technical note. J Neurosurg
9:597–603
9
5
.
.
Moore GE, Peyton WT, French LA, Walker WW (1948) The clin-
ical use of fluorescein in neurosurgery: the localization of brain
tumors. J Neurosurg 5:392–398
Grimm JB, Heckman LM, Lavis LD (2013) Chapter one - the
chemistry of small-molecule fluorogenic probes. Prog Mol Biol
Transl Sci 113:1–34
Brush CK (1991) Fluorescein labelled phosphoramidites, US
Patent 5,583,236
Folli S, Wagnieres G, Pelegrin A, Calmes JM, Braichotte D,
Buchegger F (1992) Immunophotodiagnosis of colon carcinomas
in patients injected with fluoresceinated chimeric antibodies against
carcinoembryonic antigen. Proc Natl Acad Sci U S A 89:7973–
2
2
2
6. Ambrose JF, Kistiakowsky GB, Kridl A (1950) Inhibition of urease
by sulfur compounds. J Am Chem Soc 72:317–321
7. Fletcher AN (1696) Qunine sulphate as fluorescence quantum yield
standard. Photochem Photobiol 9:439–444
8. Rojkiewicz R, Kus P, Kozub P, Kempa M (2013) The synthesis of
new potential photosensitizers [1]. Part 2. Tetrakis-
6
7
8
.
.
(hydroxyphenyl)porphyrins with long alkyl chain in the molecule.
Dyes Pigments 99:627–635
2
3
9. Weatherburn MW (1967) Phenol-hypochlorite reaction for determi-
nation of ammonia. Anal Chem 39:971–974
0. Raza H, Qamar A, Mubashir H, Seong-Hui E, Zaman A, Kim D,
Phull AR, Kim SJ, Kang SK, Seo SY (2017) Isolation, characteri-
zation, and in silico, in vitro and in vivo antiulcer studies of isoim-
peratorin crystallized from Ostericum koreanum. Pharm Biol 55:
7
977
9
.
Van Dam GM, Themelis G, Crane LM, Harlaar NJ, Pleijhuis RG,
Kelder W (2011) Intraoperative tumor-specific fluorescence imag-
ing in ovarian cancer by folate receptor-alpha targeting: first in-
human results. Nat Med 17:1315–1319
2
18–226
3
3
1. Reddy CVK, Sreeramulu D, Raghunath M (2010) Antioxidant ac-
tivity of fresh and dry fruits commonly consumed in India. Food
Res Int 43:285–288
2. Qamar A, Raza H, Hassan M, Phull AR, Kim SJ, Seo SY (2017)
Acetazolamide inhibits the level of tyrosinase and melanin: an en-
zyme kinetic, in vitro, in vivo and in silico studies. Chem Biodivers
1
0. Holm L, Sander C (1997) An evolutionary treasure: unification of a
broad set of amidohydrolases related to urease. Proteins Struct
Funct Genet, 28:72–82
1
1. Krajewska B, Ureases I (2009) Functional, catalytic and kinetic
properties: a review. J Mol Catal B Enzym 59:9–21
1
2. Macegoniuk K, Grela E, Palus J, Rudzinska-Szostak E,
Grabowiecka A, Biernat M, Berlicki Ł (2016) 1,2-
Benzisoselenazol-3(2H)-one derivatives as a new class of bacterial
urease inhibitors. J Med Chem 59:8125–8133
3. Mobley HL, Island MD, Hausinger RP (1995) Molecular biology
of microbial ureases. Microbiol Rev 59:451–480
1
4. https://doi.org/10.1002/cbdv.201700117
3. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM
2004) UCSF chimera–a visualization system for exploratory re-
3
3
(
search and analysis. J Comput Chem 25:1605–1612
1
1
4. Chen VB, Arendall WB 3rd, Headd JJ, Keedy DA, Immormino
RM (2010) MolProbity: all-atom structure validation for macromo-
lecular crystallography. Acta Crystallogr D Biol Crystallogr 66:12–
4. Mobley HL, Hausinger RP (1989) Microbial ureases: significance,
regulation, and molecular characterization. Microbiol Rev 53:85–
2
1
1
08
3
3
3
5. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR,
Appel RD, Bairoch A (2005) (In) John M. Walker (ed): The prote-
omics protocols handbook, Humana Press. 571–607
6. Willard L, Ranjan A, Zhang H, Monzavi H, Boyko RF (2003)
VADAR: a web server for quantitative evaluation of protein struc-
ture quality. Nucleic Acids Res 31:3316–3319
7. Friesner RA, Murphy RB, Repasky MP, Frye LL, Greenwood JR,
Halgren TA, Sanschagrin PC, Mainz DT (2006) Extra precision
glide: docking and scoring incorporating a model of hydrophobic
enclosure for protein-ligand complexes. J Med Chem 49:6177–
6196
1
5. Follmer C (2010) Ureases as a target for the treatment of gastric and
urinary infections. J Clin Pathol 63:424–430
6. Fatima A, Pereira C, Dau C, Olímpio G, Oliveira BGF, Franco LL,
Silva PHC (2018) Schiff bases and their metal complexes as urease
inhibitors – a brief review. J Adv Res. https://doi.org/10.1016/j.jare.
1
2
018.03.007
1
1
7. Amtul Z, Siddiqui RA, Choudhary MI (2002) Chemistry and mech-
anism of urease inhibition. Curr Med Chem 9:1323–1348
8. Zambelli B, Musiani F, Benini S, Ciurli S (2011) Chemistry of Ni2+
in urease: sensing, trafficking, and catalysis. Acc Chem Res 44:
5
20–530
38. Farid R, Day T, Friesner RA, Pearlstein RA (2006) New insights
about HERG blockade obtained from protein modeling, potential
energy mapping, and docking studies. Bioorg Med Chem 14:3160–
3173
1
9. Marzadori C, Miletti S, Ciurli GS (1998) Immobilization of jack
bean urease on hydroxyapatite: urease immobilization in alkaline
soils. Soil Biol Biochem 30:1485–1490

J Fluoresc
3
4
4
9. Seth D, Setua P, Chakraborty A, Sarkar N (2007) Solvent relaxation
46. Bjellqvist B, Hughes GJ, Pasquali C, Paquet N, Ravier F, Sanchez
JC, Frutiger S, Hochstrasser D (1993) The focusing positions of
polypeptides in immobilized pH gradients can be predicted from
their amino acid sequences. Electrophoresis 14:1023–1031
47. Xiong X, Huang S, Zhang H, Li J, Shen J, Xiong J, Lin Y, Jiang L,
Wang X, Liang S (2009) Enrichment and proteomic analysis of
plasma membrane from rat dorsal root ganglions. Proteome Sci 5:
41
6
of a room-temperature ionic liquid [bmim] [PF ] confined in a
ternary microemulsion. J Chem Sci 119:105–111
0. Park KH, Park JW, Hamilton AD (2007) Solvent and pH effects on
fluorescence of 7-(Dimethylamino)-2-fluorenesulfonate. J Fluoresc
1
7:361–369
1. Haidekker MA, Brady TP (2005) Effect of solvent polarity and
solvent viscosity on the fluorescence properties of molecular rotors
and related probes. Bioorg Chem 33:415–425
4
8. Kyte J, Doolittle RF (1982) A simple method for displaying the
hydropathic character of a protein. J Mol Biol 157:105–132
4
4
2. Valeur B (2001) Molecular fluorescence: principles and applica-
tions. Wiley-VcH Verlag GmbH,
3. Muińo PL, Callis PR (2009) Solvent effects on the fluorescence
quenching of tryptophan by amides via Electron transfer.
Experimental and computational studies. J Phys Chem B 113:
4
9. Abdul FT, Saeed A, Channar PA, Ashraf Z, Abbas Q, Hassan M,
Larik FA (2018) Synthesis, enzyme inhibitory kinetics, and com-
putational studies of novel 1-(2-(4-isobutylphenyl) propanoyl)-3-
arylthioureas as Jack bean urease inhibitors. Chem Biol Drug Des
2
572–2577
9
1:434–447
4
4. Huang Y, Cheng T, Li F, Luo C, Huang CH (2002) Photophysical
studies on the mono- and dichromophoric hemicyanine dyes II.
Solvent effects and dynamic fluorescence spectra study in chloro-
form and in LB films. J Phys Chem 106:10031–10040
5. Petit J, Denis-Gay M, Ratinaud MH (1993) Assessment of fluoro-
chromes for cellular structure and function studies by flow cytom-
etry. Biol Cell 78:1–13
5
0. Channar PA, Saeed A, Albericio F, Larik FA, Abbas Q, Hassan M,
Raza H, Seo SY (2017) Sulfonamide-linked ciprofloxacin, sulfadi-
azine and amantadine derivatives as a novel class of inhibitors of
Jack bean urease; synthesis, kinetic mechanism and molecular
docking. Molecules 22:1352. https://doi.org/10.3390/
molecules22081352
4