Deciphering the positional impact of chlorine in a new series of berberine analogues towards the superb-selective "turn-on" hydrophobic signaling of bovine serum albumin at physiological pH

Article abstract of DOI:10.1039/c9nj05642d

The optical signals of serum albumin (SA) provide precious information for realizing its native functions, in addition to developing related biomedical applications. Herein, we report a new class of easy synthesizable and water-soluble compounds (BZ₁-BZ₅) based on different chlorine positions on 9-O-benzyl-substituted berberine scaffolds for the selective detection of bovine serum albumin (BSA) in CP buffer solution (10 mM, pH 7.2) based on two competing factors: hydrophobic interactions and steric repulsion. The frail emission intensities of these probes were enhanced upon the addition of BSA; exceptionally, a remarkable increase in emission intensity (140-fold) and remarkable lifetime and quantum yield increases make BZ₄ an excellent fluorescence turn-on hydrophobic BSA sensor. Selectivity and co-existence studies involving other proteins, free tryptophan, etc. revealed that the microenvironment around the tryptophan moiety in BSA incites drastic spectral changes upon the introduction of BSA. Moreover, the most efficient lumino-probe, BZ₄, can detect bovine serum albumin at a nanomolar level (LOD = 3.3 nM) with a broadened range of linearity and slight altering of the secondary structure of the protein. Our experimental results and docking simulation studies show that the probe BZ₄ binds preferentially at "binding site II" of BSA. In addition, the binding and conformational alterations of BSA provoked by these analogues have been intensely investigated, and we fruitfully relate the binding results to the sensing outcome. The obtained results reveal how the different positioning of chlorine in benzyl-substituted berberine affects the hydrophobic sensing of BSA, making these probes a new category of BSA selective material with potential applications in proteome research.

Full text of DOI:10.1039/c9nj05642d

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Deciphering the positional impact of chlorine in a
new series of berberine analogues towards the
superb-selective ‘‘turn-on’’ hydrophobic signaling
of bovine serum albumin at physiological pH†
Cite this:DOI: 10.1039/c9nj05642d
Gopal Chandra Jana,a Sk Nayim,a Nandan Kumar Sahoo,a Somnath Das,a
a
Mt Nasima Aktara,a Anirudha Patra,a Md. Maidul Islamb and Maidul Hossain
*
The optical signals of serum albumin (SA) provide precious information for realizing its native functions,
in addition to developing related biomedical applications. Herein, we report a new class of easy
synthesizable and water-soluble compounds (BZ1–BZ5) based on different chlorine positions on
9-O-benzyl-substituted berberine scaffolds for the selective detection of bovine serum albumin (BSA)
in CP buffer solution (10 mM, pH 7.2) based on two competing factors: hydrophobic interactions and
steric repulsion. The frail emission intensities of these probes were enhanced upon the addition of BSA;
exceptionally, a remarkable increase in emission intensity (140-fold) and remarkable lifetime and quan-
tum yield increases make BZ4 an excellent fluorescence turn-on hydrophobic BSA sensor. Selectivity
and co-existence studies involving other proteins, free tryptophan, etc. revealed that the microenviron-
ment around the tryptophan moiety in BSA incites drastic spectral changes upon the introduction of
BSA. Moreover, the most efficient lumino-probe, BZ4, can detect bovine serum albumin at a nanomolar
level (LOD = 3.3 nM) with a broadened range of linearity and slight altering of the secondary structure
of the protein. Our experimental results and docking simulation studies show that the probe BZ4 binds
preferentially at ‘‘binding site II’’ of BSA. In addition, the binding and conformational alterations of BSA
provoked by these analogues have been intensely investigated, and we fruitfully relate the binding results
to the sensing outcome. The obtained results reveal how the different positioning of chlorine in benzyl-
substituted berberine affects the hydrophobic sensing of BSA, making these probes a new category of
BSA selective material with potential applications in proteome research.
Received 11th November 2019,
Accepted 27th December 2019
DOI: 10.1039/c9nj05642d
rsc.li/njc
Accordingly, the design and development of new promising
fluorescent sensors with high sensitivity for recognizing bio-
1. Introduction
In recent times, the notable development of a versatile bio- molecules is a central research area for protein biochemists due
sensor for both the unambiguous qualitative and quantitative to the ample application of these sensors in pathogen detection
labeling of proteins with high recognizing capabilities and good and medical diagnosis.3
binding linearity has attracted immense interest from chemical
Proteins are involved in a number of pivotal components
and biochemical researchers.1 Turn-on emission probes are valu- in living systems. Serum albumins, including bovine serum
able markers in proteomics as their emission intensities are albumin (BSA) and human serum albumin (HSA), are the most
amplified upon binding with proteins; these are mostly applicable plentiful carrier proteins in animal and human sera. They have
to the quantification of specific proteins and offer enough been implicated in a large number of physiological processes,
information for the in vivo imaging of proteins, for realizing like maintaining blood osmotic pressure; acting as major
the roles of proteins in cellular system and, in addition, for the transport proteins in blood plasma for many compounds, such
development of biomedical and biotechnological applications.2 as fatty acids; and being involved in initiating the transfer of
various ligands crossways through organ-circulatory interfaces,
such as in the kidneys, intestines, liver, and brain.4–7 Albumins
also act as covalent adducts, forming species with glutathione,
a Department of Chemistry and Chemical Technology, Vidyasagar University,
Midnapore, 721102, West Bengal, India. E-mail: hossainm@mail.vidyasagar.ac.in;
cysteine, and other different metals, like Cu(II), Ni(II), and so
Tel: +91-9432976277
b Department of Chemistry, Aliah University, Kolkata, West Bengal, India
on.8 Anomalous levels of serum albumins are strongly related
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9nj05642d to numerous diseases, such as diabetes mellitus,9 sepsis,10,11
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liver injuries,12 and kidney failure,13 and serum albumin levels and the emission intensity is amplified 140-fold upon the addi-
are presently viewed as diagnostic or analytical markers for tion of a saturated concentration of BSA to the probe BZ4 in CP
various diseases.14–16 The above facts have directed researchers buffer (10 mM, pH 7.2); a nanomolar detection value and strong
to deliberate the structural aspects of serum albumins and their eye-catching green fluorescence, observable via the naked eye,
interactions with drugs and various small biomolecules, and to were obtained under a UV lamp at 365 nm. Selectivity experiments
investigate the influence of albumins on the photophysical involving other proteins, like HSA, DNA, RNA, etc., show this to be
properties of the affected molecule. Structurally, serum albumins a superb selective BSA sensing probe. Another beauty of this work
have two binding sites, i.e., site I and site II, which are situated in is that it includes a detailed investigation of the interactions of
three homologous domains forming a heart-shaped protein.8,17 these synthesized analogues with BSA, and we fruitfully relate
The site I binding efficacy is chiefly based on hydrophobic these results to the sensing outcomes.
interactions, while site II binding includes a combination of
hydrogen bonding, and electrostatic and hydrophobic inter-
actions.18,19 It has been reported previously that biomolecules
have good binding affinity to serum albumin mostly through
site II binding, and proficient photodynamic therapeutic (PDT)
applications have been shown.20,21
2. Materials and methods
2.1 Instrumentation
1H and 13C NMR spectra of compounds BZ2, BZ3, and BZ5 were
recorded using a 600 MHz (or 150 MHz for 13C) Bruker Lambda
spectrometer, and for compounds BZ1 and BZ4, a Bruker
AVANCE-III 500 MHz spectrometer was used at 25 1C, using
TMS as an internal reference in DMSO-d6 solvent. Mass data for
compounds BZ2, BZ3, and BZ5 were obtained using MALDI-ToF
apparatus (VOYGER-DE PRO, Applied Biosystems, USA), and
for compounds BZ1 and BZ4, a Bruker micro TOF QII high-
resolution mass spectrometer was used. UV-vis spectra were
acquired using a Shimadzu UV-1601 (Japan) spectrophoto-
meter, and a PerkinElmer LS-55 (USA) spectrofluorimeter was
used for fluorescence measurements with 2.5 nm excitation
and 5 nm emission slit. Fluorescence lifetime experiments were
performed using a DeltaFlext Modular fluorescence lifetime
system (Horiba scientific, UK).
Therefore, the development of proficient fluorescent probes
for selective-binding protein sensors is highly necessary and,
consequently, enormous efforts relating to the qualitative and
quantitative monitoring of albumins in biological fluids have
been reported over several decades. Various methods have been
developed for albumin detection, such as immune assays,
colorimetric techniques,22–24 cyclic voltammetry,25,26 surface-
enhanced Raman scattering,27 and chemiluminescence.28
Of the various methods available, fluorometric sensing has
received widespread consideration due to the potential for
rapid analysis, high sensitivity, and good selectivity, particu-
larly for real-time imaging in vivo. To date, a large number of
fluorescent probes for the quantification of bovine serum
albumins have been reported,29–32 however some of them suffer
from grim disadvantages such as complicated synthetic
procedures.33–35 Poor water solubility is also a factor for some
fluorescent probes like PRODAN, a neutral dye; on the other
hand, some probes have been designed as reactive probes
(reacting with the NH2 or SH groups in proteins) for serum
albumin labeling, which distorts the electronic effects of the
probes.36–38 Furthermore, some fluorescent sensors emit in a
somewhat lower wavelength region and it is not easy to reduce
the interference from the background.39 Additionally, a few
reported works have only focused on the detection of BSA but
not on the associated interactions with BSA, which seem to
have an effect on the quantification results. Even though it has
been reported that squaraine dyes interact with BSA leading
to fluorescent enrichment, no precise binding information
relating to site I or site II has been attained.40–42 To overcome
these deficiencies, we have successfully synthesized a series of
benzyl substituted berberine analogues (BZ1–BZ5) for interacting
with and quantifying BSA.
2.2 Reagents
Berberine chloride hydrate (BC), BSA (498%), other proteins,
nucleic acids, and enzymes were obtained from Sigma Aldrich
(St. Louis, MO, USA). The other chemicals, like cationic salts,
anionic salts, and all spectroscopic grade solvents, were
acquired from Merck Pvt. Ltd. The chemicals were used without
further purification, and solvents were distilled before use.
Double distilled water was used for the preparation of citrate-
phosphate (CP) buffer, containing 10 mM Na+ at pH 7.2, and
the berberine analogue, protein, enzyme, and nucleic acid
solutions were prepared in CP buffer.
2.3 Synthesis of berberrubine (2)
Commercially available berberine chloride (1, 1000 mg) was
heated to 190 1C in a vacuum oven under a reduced pressure of
20–30 mmHg for 15 min to get berberrubine (2, 885 mg) in 90%
yield, according to a previously reported work.43
In this manuscript, the center of attention is focused on benzyl
substitution at the 9-O-position of berberine, an isoquinoline
quaternary plant alkaloid for the sensing of BSA. In Scheme 1
2.4 Synthesis of various benzyl substituted berberrubine
derivatives (3a, 3b, 3c, 3d, 3e)
we demonstrate the structures of the synthesized berberine Various examples of benzyl chloride (1.5 mM) were added to
analogues along with the different positions of chlorine (at the solutions of 2 (1 mM) in CH3CN in the presence of the base
2, 3, 4, and 2, 5 positions of the phenyl moiety) in order to control K2CO3 (1 mM); then the reaction mixtures were stirred for
the two effects of hydrophobic interactions and steric repulsion. 4–6 h at 80 1C. The crude products were obtained upon evapo-
Among the derivatives, BZ4 is exceedingly sensitive toward BSA rating the solvent under vacuum and washed with diethyl ether.
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Scheme 1 (a) The synthetic route to the berberine analogues (BZ1–BZ5). (b) A schematic presentation of the BSA sensing mechanism adopted by the
probe BZ4.
The products were purified via column chromatography on a protein and probe absorbance at the excitation and emission
silica gel column and eluted with varying concentrations of wavelengths, respectively.
a CHCl3–CH3OH solvent mixture to get the desired products
2.5.2 Fluorescence quenching. During fluorescence
(BZ1–BZ5). The berberine derivatives were well characterized using quenching experiments, 295 nm was chosen as the excitation
various methods, such as mass spectrometry, and 1H, and wavelength to discerningly excite the tryptophan moieties in
13C NMR spectroscopies (Fig. S27–S41, ESI†).
proteins.44 To nullify any contribution from the solvent to the
fluorescence intensity, the background emission intensity of
the blank buffer was deducted from every sample spectrum.
2.5.3 Absorbance measurements. UV-vis titration studies
(interaction studies) of the probes were achieved using 1 mL
of probe (5 mM) in 10 mM CP buffer at pH 7.2 in a 1 cm path
length quartz cell over the range of 200 to 600 nm with
increasing concentrations of BSA up to saturation.
2.5 The procedures for spectra measurements
2.5.1 Fluorescence measurements. Stock solutions of the
probes (BZ1–BZ5), BSA, and other proteins, enzymes, and amino
acids were prepared in CP buffer solution (10 mM, pH = 7.2).
The fluorometric titration experiments were carried out using
1 mL of probe stock solution (5 mM) in a 1 cm path length
quartz cell, and the various analytes stated above were gradually
added from a micropipette to obtain the spectrum. For titration
experiments, the excitation wavelength was fixed at 360 nm and
the emission spectrum was gathered from 400 to 650 nm; the
excitation and emission band widths were 5 nm and 5 nm,
respectively. We also consider the inner filter effect during the
gathering of fluorescence spectra via following equation
2.5.4 IR-experiments. FTIR spectral studies were done to
explore probe–BSA binding. Probe solution was gradually
added to a protein solution under constant stirring to ensure
the formation of a homogeneous solution and to achieve the
two desired probe to BSA concentration ratios of 0.5 : 1 and 1 : 1.
To ensure complex formation, spectra were collected after some
time at room temperature with spectra gathered over the spectral
range of 4000–600 cmꢁ1 at a nominal resolution of 2 cmꢁ1
.
AexiþAemi
2.5.5 Anisotropy measurements. Fluorescence polarization
anisotropy measurements of probes at the BSA saturation
2
Fcorr ¼ Fobs ꢀ e
(1)
where Fcorr and Fobs signify the corrected and observed concentration were made with the excitation wavelength and
intensities, respectively, and Aexi and Aemi are the sums of emission wavelength fixed at 360 and 531 nm, respectively.
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The spectra were recorded after some time to ensure homo- Sudlow site I (sub-domain IIA), and ibuprofen, which specifi-
geneous complex formation and averages of five consecutive cally binds to Sudlow site II (sub-domain IIIA).47 During
measurements are given.
performing this experiment, the site markers were increasingly
added to a mixture of BZ4 and BSA, while the other parameters,
like excitation wavelength, and excitation and emission
bandwidth, were identical to those used for the fluorescence
titration experiments.
2.6 Calculation of the detection limit
The detection limit of the probe BZ4 for BSA detection was
calculated based on fluorescence titration. To acquire the
detection limit, BZ4 (5 mM) was titrated with increasing con-
centrations of BSA (0–28 mM). The respective fluorescence
intensity of BZ4 was calculated 10 times without BSA, and the
standard deviation of the blank measurements was obtained.
To achieve the slope, the emission intensity at 517 nm was
plotted against the concentration of BSA. Then, the detection
limit was calculated using the equation: detection limit =
3.29s/k, where s and k signify the standard deviation of the
blank measurements and the slope of the I517 vs. concentration
of BSA plot, respectively.
2.10 Docking studies
For close observations of changes in the microenvironment of
BSA upon binding with BZ4, we performed molecular docking
studies. Accordingly, the three-dimensional (3D) structure of
BZ4 was drawn using MOE and energy optimized using the
AM1 semi-empirical method with
a RMS gradient of
0.001 kcal molꢁ1 48
.
The crystal structure of BSA (pdb id: 4f5s)
was collected from the Protein Data Bank. Before docking, the
binding sites were determined using site finder and 84 binding
sites were found in BSA. The compound was docked to the best
fitting domain of BSA, obtained via MOE, using the Alfa PMI
placement method.
2.7 Fluorescence quantum yield calculations
The fluorescence quantum yields of the synthesized analogues
(BZ1–BZ5) in the absence and presence of BSA were obtained
using an optically comparable solution of quinine sulfate
(Fr = 0.546 in 1 N H2SO4) as a standard (excitation wavelength
of 365 nm). The quantum yield (Fs) is estimated using the given
equation45
3. Result and discussion
3.1 UV-vis absorption study
Primarily, the absorption spectra of BZ1–BZ5 in the presence of
bovine serum albumin were examined via UV-vis spectroscopy.
The compound BZ4 showed two absorption maxima at 423 nm
and 345 nm in the wavelength region of 300–600 nm in CP
buffer (10 mM, pH = 7.2). Upon the addition of BSA, the
absorbance at 345 nm gradually decreased approaching satura-
tion, with three clearly visible isosbestic points at 357, 364, and
373 nm (Fig. 1a). This result indicates the existence of interac-
tions between the p electronic cloud of the probe and the
tryptophan moiety in BSA,49 and the notable hypochromic shift
indicates that the probe is closer to BSA.50 This may be
attributed to the formation of a complex between the probe
and BSA via electrostatic and hydrophobic interactions. Simi-
larly, we performed the UV-vis titration of other probes with a
gradual increase in the BSA concentration (Fig. S1, ESI†).
Accordingly, the binding constant (KBH) values for all probe
(BZ1–BZ5)–BSA composites were evaluated from Benesi–Hildebrand
plots (Fig. 1b), and data analysis was used to calculate a binding
constant of 7.3 ꢀ 104 for the complex formed between BZ4 and BSA
(Table S1, ESI†).
ꢀ
ꢁꢀ
ꢁ
ArFs
AsFr
Zs2
Zr2
Fs ¼ Fr
(2)
where Ar and As are, respectively, the absorbance of the reference
and sample solutions, Fr and Fs signify the integrated emission
intensities (areas) at the same excitation wavelength, and Z
represents the refractive index of the solvent.
2.8 Lifetime measurements
Lifetime decay data were assessed via the time-correlated single
photon counting (TCSPC) method using CP buffer at pH 7.2 at an
excitation wavelength of 372 nm from a laser LED source. The
decay profile, I(t), was calculated using the following equation46
ꢀ
ꢁ
a
X
ꢁt
IðtÞ ¼
ai exp
(3)
ti
i¼1
where ai is the early emission intensity of the ith component having
lifetime t. The chi-square (w2) value specifies the best fitting
parameter. The obtained data were scrutinized using EZ-time
software. The mean lifetime (tm) of the sample under various
experimental conditions was determined via the following equation
3.2 Fluorescence titration experiments
P
Moreover, a strong indication of compound–BSA interaction
comes from fluorescence spectral studies. All the compounds
(BZ1–BZ5) were weakly fluorescent with insignificant fluores-
cence quantum yields upon excitation at their corresponding
excitation wavelengths. Remarkable emission enhancement
with a notable hypsochromic shift was observed upon the
aiti
i
P
tm
¼
(4)
ai
i
2.9 Site marker experiments
To scrutinize the binding site of the analogue towards BSA, site gradual addition of BSA up to saturation for all compound
marker displacement experiments were done using two site- (BZ1–BZ5) solutions (Fig. S2, ESI†), and the comparatively high-
specific markers, namely, warfarin, which specifically binds to est emission intensity increase (B140 fold) was noticed in the
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Fig. 1 (a) UV-vis spectra of BZ4 (5 mM) with an increasing concentration of BSA, ranging from 0–68 mM, in CP buffer (pH 7.2, 10 mM). (b) Benesi–
Hildebrand plots of all synthesized analogues (BZ1–BZ5) upon the addition of varying concentrations of BSA.
case of probe BZ4. This drastic change in the fluorescence decreases for the 3-chlorobenzyl, 2,5-dichlorobenzyl and 2-chloro-
signal could offer the potential for ocular BSA sensing, as benzyl substituents in respective order.
depicted in Scheme 1. Plots of the emission intensities of the
3.3 Selectivity and sensitivity
probes against BSA concentration (Fig. S3, ESI†) demonstrated
that the probe BZ4 showed a good linear relationship compared
to the other compounds (Table S2, ESI†) and the fluorescence
quantum yield (F) after binding with BSA was highest for
compound BZ4 (Table 1). Therefore, all the above factors led
us to speculate that the derivative BZ4 (3d) had the highest
sensitivity towards BSA (Fig. 2a) and in this manuscript, we
used the analogue BZ4 for the quantification of BSA during
in vitro analysis. The linearity range for BSA sensing using
probe BZ4 extended up to 2.5 mM (Fig. 2b) and the detection
limit according to IUPAC conventions (3.29s/K) was found to be
3.3 nM, making this a superior BSA detection probe. The
differences in the BSA sensing capabilities of these compounds
(BZ4 4 BZ3 4 BZ5 4 BZ2 4 BZ1) are rationalized based on the
different positions of the chlorine group in the benzyl substi-
tuent. We argue that competition between two factors, namely
hydrophobic interactions and steric repulsion, is responsible
for the sensing abilities of the probes. When Cl is situated at
the 4-position, minimum steric repulsion exists; however,
maximum hydrophobicity provokes compound BZ4 to efficiently
interact with BSA. Steric repulsion increases and hydrophobicity
The naked eye visualization under a UV lamp of all these probe
solutions (5 mM) upon the addition of BSA and HSA (28 mM)
revealed that BZ4 was a superb selective probe for BSA (Fig. 3a).
The fluorescence intensity, as well as the emission maximum,
is almost the same upon the addition of various analytes,
such as different cationic and anionic salts (28 mM), to the
probe (BZ4) solution (Fig. S4–S7, ESI†). Interestingly, upon
the addition of other proteins or enzymes, like hemoglobin,
ribonuclease A, insulin, tryptophan, trypsin, etc., the emission
signal was not significantly altered; however, in the presence of
DNA and RNA, the fluorescence intensity was somewhat
increased (Fig. 3b and c). We have already discussed that
hydrophobic interactions are the foremost factor responsible
for BSA sensing and, accordingly, selectivity studies reflect that
other proteins and enzymes do not have appropriate hydro-
phobic regions to bind with the probe.51 The hydrophobicity of
these proteins depends on various factors, like the amino acid
sequences, protein structures, and side chains, and BSA is
assumed to have higher hydrophobicity than the other proteins/
enzymes used. However, berberine, a DNA/RNA interacting plant
alkaloid, and, similarly, the synthesized berberine analogues
also interact with DNA/RNA; therefore, the emission intensity
increased a little but not significantly compared to the addition
of BSA. Competitive experiments, i.e., studying the emission
intensity of BZ4 upon the addition of BSA in the presence of
other proteins or enzymes, indicate that the presence of these
proteins/enzymes was insignificant to hamper the detection of
BSA (Fig. 3b).
Table 1 The photophysical properties of the berberine analoguesa in the
absence and presence of BSA at 25 1C
Fb
tm (ns) knr ꢀ 108 (sꢁ1) rd
Ka ꢀ 104 (Mꢁ1
)
c
Entry
BZ1
BZ2
BZ3
BZ4
BZ5
4.2 ꢀ 10ꢁ3 0.98
6.4 ꢀ 10ꢁ3 0.95
5.2 ꢀ 10ꢁ3 0.93
4.0 ꢀ 10ꢁ3 0.68
5.3 ꢀ 10ꢁ3 1.81
10.16
10.45
10.69
14.64
5.4
1.54
1.83
1.38
0.90
1.40
0.1126
0.1129
0.1128
0.113
—
—
—
—
—
0.1127
0.1149 2.2 ꢀ 104
0.1163 3.1 ꢀ 104
0.1195 5.2 ꢀ 104
0.12337 5.6 ꢀ 104
0.1188 4.3 ꢀ 104
3.4 Binding stoichiometry (Job’s plot)
BZ1 + BSA 0.032
BZ2 + BSA 0.042
BZ3 + BSA 0.104
BZ4 + BSA 0.17
BZ5 + BSA 0.08
6.28
5.22
6.46
9.20
6.56
To determine the binding stoichiometry between the probe BZ4
and BSA, we obtained a Job’s plot, where the concentration of
the both probe and BSA remain fixed, but the mole fraction of
both is altered. Here, we plot the fluorescence intensity of the
solution, keeping a fixed concentration (5 mM) of probe and BSA
a
b
5 mM analogue. Quantum yield with respect to quinine sulfate.
Average lifetime. Anisotropy.
c
d
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Fig. 2 (a) The emission spectra of BZ4 (5 mM) upon the addition of increasing concentrations of BSA (0–28 mM) in CP buffer (pH 7.2 and 10 mM).
(b) The fluorescence intensity at 517 nm of probe BZ4 (5 mM), which was linearly related to the concentration of BSA (0–2.5 mM) in CP buffer (pH 7.2, 10 mM);
lex = 360 nm.
Fig. 3 (a) The fluorescence intensities of the synthesized analogues (5 mM) upon the addition of 28 mM HSA/BSA; corresponding optical images
under UV light at 365 nm are depicted in the inset. (b) The fluorescence responses of probe BZ4 (5 mM) to other biological interferents, (DNA, RNA,
ribonuclease A, insulin, cytochrome C, b-lactoglobulin, lysozyme, hemoglobin, proteinase K, trypsin, tryptophan, transferrin, amylase; 28 mM) in CP buffer
(pH 7.2, 10 mM), lex = 360 nm. (c) Optical photos of BZ4 probe solution upon the addition of various proteins/enzymes/amino acids under a UV lamp
at 365 nm.
while varying the mole fraction; the fit lines intersected nicely at a decrease in emission intensity upon the introduction of ibuprofen
binding stoichiometry of 1: 1 (Fig. S8, ESI†). These results further to the BZ4–BSA complex indicates that probe BZ4 was replaced
clarified that the analogue molecule bound to one binding site by ibuprofen from the protein microenvironment. Overall, 65%
of BSA.
replacement and 25% replacement of BZ4 were realized upon
the addition of ibuprofen and warfarin to the BZ4–BSA complex,
respectively (Fig. 4a and Fig. S9, S10, ESI†). The results indicate that
3.5 Site-selective binding
Site marker experiments were carried out to gain insight into the probe BZ4 has stronger binding affinity towards site II than site I.
probe–BSA interactions. For this experiment, warfarin and ibuprofen The greater binding affinity of BZ4 for site II was predominantly due
were chosen as site I and site II markers, respectively. The abrupt to a combination of electrostatic and hydrophobic interactions.44
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3.6 Fluorescence anisotropy
decay constant (knr) values of BZ4, with the help of lifetime and
fluorescence quantum yield data, were established as 14.64 ꢀ 108
and 0.90 ꢀ 108 sꢁ1 in the absence and presence of BSA,
respectively, which suggests that migration from an aqueous to
a protein environment decreased the knr values. Thus, the
decrease in the non-radiative rate in the presence of BSA tunes
the lifetime enhancement of BZ4 in the protein environment,
and the results reveal that probe BZ4 strongly binds to BSA
via hydrophobic interactions. We also performed the above-
mentioned experiments on the other compounds (Fig. S12, ESI†),
and it was observed that the lifetime enhancement is highest in
the case of BZ4 in the presence of BSA (Table 2), which strongly
corroborates our previous experiments.
The fluorescence anisotropy indicates the rigidity of a fluoro-
phore microenvironment and provides additional evidence in
favor of interactions between the probes and BSA. An enhance-
ment in the fluorescence anisotropy reflects greater rigidity in
the surrounding fluorophore environment. Plots of the fluores-
cence anisotropy (r) of the compounds against the concentration
of added BSA show that upon increasing the BSA concentration,
the anisotropy increases for all analogues (Fig. 4b), and the
respective r values are depicted in Table 1. The greater increase
in the anisotropy in the case of probe BZ4 compared to the other
examples suggests that the restricted of rotation is maximum
upon the introduction of BSA and, furthermore, that probe BZ4 is
more strongly bound with BSA compared to the other probes.
3.9 Fluorescence lifetime decay of BSA in the presence of
the analogues
3.7 Effects of pH on the probe–BSA complex
Fluorescence lifetime measurements also enhance our knowledge
of the interactions between probes and proteins;44 accordingly, we
perform time-correlated lifetime experiments involving BSA in the
absence and presence of the analogues. The best-fit decay profile
of native BSA exhibited a triexponential function, where the
corresponding lifetimes are 2.03, 4.79, and 7.66 ns, and the average
lifetime is 5.86 ns, in good concurrence with the literature value.41
Upon the successive addition of the analogues (BZ1–BZ5) to BSA
solution, the mean lifetime values of BSA showed few changes, as
shown in Fig. S13 and in Table S4 (ESI†). The results signify the
formation of complexes between BSA and the analogues, followed
by the alteration of the microenvironment around BSA. The
insignificant changes in the lifetime values indicate that the
fluorescence quenching process is static in nature, which is mainly
due to ground state complex formation between the protein and
berberine analogues.
Changes in the fluorescence intensity during probe BZ4–BSA
complex formation at various pH levels could also occur if our
probe is applied in biological systems. The fluorescence inten-
sity of the BZ4–BSA complex in the physiological pH range,
acidic pH range, and basic pH range were investigated and the
probe showed maximum emission intensity enhancement in
the presence of a BSA microenvironment at physiological pH
(Fig. S11, ESI†). We chose a physiological pH value of 7.2 for the
experiments in this article.
3.8 The fluorescence lifetimes of analogues in the presence
of BSA
Fluorescence lifetime measurements enhance our knowledge
about the microenvironment in the region of a fluorophore and
can be used to explore excited state interaction phenomena.3
Accordingly, we performed time-correlated single photon
counting (TCSPC) experiments on all analogues in the absence
3.10 Proposed sensing mechanism
and presence of BSA. The TCSPC (lifetime) data of BZ4 (Fig. 5b) Taking together all of the above experimental findings, such
demonstrated that in the absence of BSA, tri-exponential decay as absorption titration, fluorescence titration, fluorescence
was exhibited with a mean lifetime (tm) of 0.83 ns; in the quenching and lifetime measurements, it is indicated that
presence of BSA, the tri-exponential decay significantly increased the positional difference of the Cl atom affects the interactions
with a mean lifetime (tm) of 6.12 ns (Table 2). The non-radiative between the synthesized probe molecules (BZ1–BZ5) and BSA;
Fig. 4 (a) Site marker experiments: plots of F0/F for BZ4 + BSA complexes upon the addition of two site markers, namely warfarin and ibuprofen. (b) The
variation of the fluorescence anisotropy (r) of the probes (5 mM) upon the addition of various concentration of BSA at an emission wavelength of 517 nm
(pink: BZ4; green: BZ3; blue: BZ5; orange: BZ2; purple: BZ1).
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Fig. 5 (a) TCSPC spectra of BZ4 (5 mM) in the absence and presence of BSA (28 mM) in CP buffer (pH 7.0, 10 mM Na+). (b) TCSPC spectra of BSA in the
presence of different concentrations of probe BZ4 in CP buffer (pH 7.0, 10 mM Na+).
Table 2 Lifetime data from the berberine analogues in the absence and we performed a control experiment involving the addition of
presence of BSA
tryptophan to BZ4 solution and no noteworthy spectral changes
were observed (Fig. S14, ESI†); this points out the fact that
the microenvironment around the tryptophan moiety in BSA
provokes the drastic spectral changes upon the introduction of
BSA to probe solution.
Entry
BZ1
t1 (ns) a1
t2 (ns) a2
0.10
t3 (ns) a3
tm (ns) w2
0.27 0.82 2.05
6.99 0.08 0.98
1.06
1.03
BZ1 + BSA 0.60
0.25 3.71
0.32 11.5 0.43 6.28
BZ2
0.25
0.81 1.73
0.38 3.74
0.11
0.29 11.7
7.02 0.08 0.95
0.33 5.22
1.07
1.08
BZ2 + BSA 0.74
3.11 Three-dimensional fluorescence spectral studies
3D fluorescence spectroscopy was carried out by concurrently
changing the excitation and emission wavelengths, reflecting
changes in the microenvironment of the protein upon inter-
action with the compounds (BZ1–BZ5).52 The 3D spectra of BSA
in the absence and presence of the compounds are presented in
Fig. S15–S20 (ESI†) and the results are listed in Table S5 (ESI†).
The spectra demonstrate four distinct peaks: peak ‘‘a’’ signifies
first-order Rayleigh scattering (lem = lex), and peak ‘‘b’’ is
the second-order Rayleigh scattering peak (lem = 2lex). Peak 1
(lex = 280 nm, lem = 345 nm) is due to the characteristic spectra
of both Tyr and Trp as BSA was excited at 280 nm, and peak 2
(lex = 230 nm, lem = 345 nm) is due to the fluorescence spectrum
of the polypeptide backbone in BSA. In the case of peak 1, there was
a considerable Stokes shift with a decrease in the fluorescence
intensity; in the case of peak 2, there was also a decrease in the
fluorescence intensity, and significant Stokes shifts were observed in
the order BZ4 4 BZ3 4 BZ5 4 BZ2 4 BZ1. This type of Stokes shift
indicates that the polarity of both components (Trp and Tyr)
decreased and BSA was suppressed into a hydrophobic pocket
(Table S5, ESI†). The decreases in the emission intensities of peaks
1 and 2 indicate the occurrence of interactions between the probes
and BSA, leading to changes in the microenvironment around Trp
along with the structural alteration of BSA.
BZ3
0.30
0.82 2.1
0.28 3.79
0.10
0.26 11.5
6.72 0.07 0.93
0.46 6.46
1.09
1.05
BZ3 + BSA 0.67
BZ4
0.24
0.83 1.36
0.20 5.97
0.10
0.21 13.1
5.01 0.07 0.68
0.59 9.20
1.03
1.05
BZ4 + BSA 1.13
BZ5
0.35
0.74 2.26
0.27 3.71
0.14
0.28 11.9
7.32 0.17 1.81
0.45 6.56
1.01
1.09
BZ5 + BSA 0.64
consequently, the sensing abilities of these probes are affected.
Furthermore, a control experiment involving the addition of
BSA to berberine solution resulted in no significant alterations
to the fluorescence spectrum, while the addition of BSA to a
benzyl group-containing berberine analogue resulted in lesser
changes to the spectra. The above-mentioned control experi-
ments further indicate the effects of the position of the Cl atom
in the benzyl moiety. The above-mentioned experiments
reflect that the order of binding affinity of the probes was
BZ4 4 BZ3 4 BZ5 4 BZ2 4 BZ1, and the fluorescence titration
studies revealed that sensing ability for BSA of these probes was
in the order BZ4 4 BZ3 4 BZ5 4 BZ2 4 BZ1. Thus, we assume that
competition between two factors, i.e., hydrophobic interactions
and steric repulsion, is accountable for the sensing capabilities
of the probes. When Cl is situated at the 4-position, minimum
steric repulsion and maximum hydrophobicity tunes the
compound, BZ4, to efficiently interact with BSA; however, steric
3.12 Fluorescence quenching studies
repulsion increases and hydrophobicity gradually decreases The quenching of the fluorescence of a protein upon binding
for the 3-chlorobenzyl, 2,5-dichlorobenzyl and 2-chlorobenzyl with a ligand offers significant information about changes in
substituents. To gain insight into the interactions between the the microenvironment of the protein in close proximity with
compounds and BSA, it is necessary to inspect the interactions the ligand molecule. The fluorescence properties of BSA are
between the free amino acid tryptophan and BZ4. Consequently, predominantly due to the presence of two tryptophan moieties,
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namely Trp-134 and Trp-212, in the I and II domains, where F0, F, and [Q] denote the terms stated above; the obtained
respectively. The emission spectrum of BSA exhibits an binding constant and n values are listed in Table S6 (ESI†). The
enhanced fluorescence signal at 345 nm upon excitation at binding constant value of the BZ4–BSA complex was found to be
295 nm in buffer solution, and it should be noted that we higher than those of the other probe–BSA complexes, indicating
selectively chose an excitation wavelength of 295 nm rather greater quenching efficiency, and the n values for all the probe–BSA
than 280 nm to minimize the fluorescence contribution from complexes were almost one, indicating that the probes are situated
the tyrosine moiety present in BSA. The fluorescence spectrum at one binding site.
of BSA was remarkably changed upon the addition of different
concentrations of probes. Fig. 6a shows that the steady state
fluorescence intensity of BSA decreased gradually upon the
successive introduction of BZ4 up to saturation, with a large
red shift of 27 nm. Also, the fluorescence intensity of BSA
decreased up to saturation accompanied by a red shift upon
the addition of the other probes (Fig. S20 and S21, ESI†). This
type of spectral change, a red shift with a decrease in the emission
intensity, indicates a strong interaction between the probe and BSA,
and the microenvironment of tryptophan may change from hydro-
phobic or less hydrophilic to a hydrophilic environment. The
maximum decrease in the fluorescence intensity of BSA and the
larger red shift upon the addition of BZ4 indicate its strong
interaction with BSA compared to the other probes. Consequently,
we calculate the quenching constant by applying the Stern–Volmer
equation in the form
3.13 Binding study via FT-IR
Further conformation regarding berberine analogue–BSA com-
plexation comes through FT-IR spectral analysis. The changes
in the intensities at 1656 cmꢁ1 (amide I band), a mainly CQO
stretching mode, and at 1543 cmꢁ1 (amide II band), C–N
stretching coupled with N–H bending modes of native BSA,
were monitored upon the addition of the analogues.52 Also, IR
derivative plot and curve fitting methods were applied to
investigate changes in the secondary structure of the protein
upon the addition of the analogues. The intensities of
the stretching frequencies at 1653 and 1546 cmꢁ1 decreased
gradually with an increase in the concentration of the analogue
BZ4 (Fig. 7a). The decrease in the intensities of the amide I and
II bands signifies a decrease in the a-helix content of BSA upon
the addition of analogue BZ4. The IR-derivative plot is shown in
Fig. 7b, and the corresponding results are given in Table S8
(ESI†), which also indicate a reduction in a-helix content upon
BSA–BZ4 complexation. Similarly, IR spectral analyses of BSA
in the presence of the other analogues are given in the ESI†
(Fig. S23–S26).
F0
¼ 1 þ Kqt0½Qꢂ ¼ 1 þ KSV½Qꢂ
(5)
F
where F0 and F represent the fluorescence intensities of BSA in the
absence and presence of the quencher, respectively, [Q] indicates
the concentration of quencher, and KSV is the Stern–Volmer
quenching constant. Stern–Volmer plots of F0/F against the probe
concentration for all probes are depicted in Fig. S21 and S22 (ESI†)
and each plot is linear in nature; the KSV values for each probe are
shown in Table S6 (ESI†). We can also calculate the probe–BSA
binding constant (KA) values and number of binding sites (n) from
quenching experiments (Fig. 6b), assuming that BSA has the same
independent binding sites, using the following equation
3.14 Molecular docking studies
The molecular docking of BZ4 with BSA was performed to gain
insight into changes in the protein microenvironment and
provide further confirmation about site-specific binding during
BZ4–BSA binding. As a previous experiment (the site marker
study) concluded that the probe (BZ4) bound to sub-domain
IIIA (the ibuprofen binding site), we positioned BZ4 at the sites
of this domain. The lowest MOE energy outcome (Table S8,
ESI†) specified that the ligand bound to sub-domain IIIA better
F0 ꢁ F
log
¼ log KA þ log½Qꢂ
(6)
F
Fig. 6 (a) Fluorescence spectra of 5 mM BSA in the presence of increasing concentrations of probe BZ1 (0 to 40 mM) at pH 7.2 in 10 mM CP buffer.
(b) Modified Stern–Volmer plots of log[(F0 ꢁ F)/F] vs. log[BSA] for all BSA–probe complexes at pH 7.2.
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Fig. 7 (a) FT-IR spectra in the region of 1800 to 800 cmꢁ1 from hydrated films (pH 7.2, 10 mM Na+ CP buffer, 25 1C) of free BSA and its complexes
(BSA + BZ4) at different concentrations. (b) Second derivative resolution enhancement and the curve-fitted amide I region (1700–1600 cmꢁ1). (c) A 3D
binding picture of BZ4 when docked with BSA. (d) A 2D representation of the conformation of BZ4 when docked with BSA. (e) The binding distance
between BZ4 and tryptophan (Trp) 212 of BSA.
than the other sites. This result indicates that the ligand interacts upon the addition of BSA, while changes in the emission signal
with BSA at the hydrophobic cavity of sub-domain IIIA of BSA upon the addition of various other proteins/enzymes are insignif-
(Fig. 7c). The analogue was surrounded by hydrophobic and icant under a similar set of investigation conditions; this heralds a
negatively charged residues, such as Arg194, Tyr 149, Ser 191, Glu potential new, hydrophobic, turn-on fluorescence sensor (BZ4) for
152, and Glu 191. From Fig. 7d, it is clear that a hydrogen bond the selective and nanomolar quantification of BSA. Dye displace-
between Ser 191 and an oxygen atom of the analogue stabilizes the ment experiments and molecular docking simulations revealed
complex. Besides, an arene-cation interaction between Arg 194 and that probe BZ4 has strong binding affinity towards site II of BSA.
the analogue aromatic ring also played a vital role in complex Experimental tools like absorption titration, fluorescence titration,
formation. The positive charge of the nitrogen present in the ligand fluorescence quenching, fluorescence anisotropy and lifetime mea-
was also an important factor. From the arrangement of the surements showed that the microenvironment produced by bovine
analogue, it was observed that most of the analogue penetrates serum albumin and the structural restriction of the probe, invol-
into the binding pocket of BSA, suggesting that the binding was ving steric, H-bonding and electronic factors, is the decisive factor
mainly hydrophobic in nature. The distance between the Trp 212 determining the fluorescence signaling of BSA, and we usefully
residue and the ligand was about 5.66 Å (Fig. 7e), and the free showed how two factors, namely steric effects and the hydropho-
energy was ꢁ9.34 kcal molꢁ1, which further affirms the obtained bicity of these analogues, affect the detection of BSA. From the
experimental results.
above results, we suppose that this work definitely provides an idea
for designing plant alkaloid-based probes for SA in the future, and
we strongly deem that these BSA selective probes will have great
impact in future biomedical research.
4. Conclusions
In summary, we have synthesized a new series of berberine
analogues via the different positioning of
a chlorobenzyl
Conflicts of interest
molecule at the 9-O position of the mother berberine, and the
protein binding and structure-optical sensing properties of these
analogues towards BSA detection were investigated in depth. The
investigation revealed that the analogues interact with BSA non-
covalently and cause an increase in the emission intensity. The
There are no conflicts to declare.
Acknowledgements
superiority of this study lies in the huge increase in the emission Sk. Nayim and Mt. Nasima Aktara acknowledge the UGC for
intensity (140-fold) as well as the quantum yield (43 fold) of BZ4 MANF fellowships and Somnath Das also acknowledges the
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