Benzothiazole-Phenothiazine Conjugate Based Molecular Probe for the Differential Detection of Glycated Albumin

Article abstract of DOI:10.1002/ijch.202000098

Glycation of albumin proteins is considered the pathophysiological hallmark of several chronic fatal disorders, including diabetes mellitus (DM) and Alzheimer's disease (AD). The optical detection of glycated albumin is a simple and cost-effective tool with diagnostic implications for DM and AD. Herein, we developed a cyano derivative of the benzothiazole-phenothiazine conjugate (T_CNP) -based far-red fluorescence probe for the differential detection of glycated bovine serum albumin (BSA_G) over native BSA and oxidized BSA (BSA_O). The selective fluorescence enhancement of T_CNP in the presence of native BSA and BSA_O and significant quenching in the presence of BSA_G account for the distinct interaction between the probe and different structural variants of BSA. The high photostability and differential response towards native BSA and BSA_G infer that T_CNP is a potential molecular tool to assess the glycation induced structural changes of albumins.

Full text of DOI:10.1002/ijch.202000098

Communication
DOI: 10.1002/ijch.202000098
Benzothiazole-Phenothiazine Conjugate Based Molecular
Probe for the Differential Detection of Glycated Albumin
Ashish Kumar+,[a] Lakshmi Priya Datta+,[a] Sourav Samanta+,[a] Harshit Arora,[a] and Thimmaiah Govindaraju*[a]
Abstract: Glycation of albumin proteins is considered the oxidized BSA (BSAO). The selective fluorescence
pathophysiological hallmark of several chronic fatal disor- enhancement of TCNP in the presence of native BSA and
ders, including diabetes mellitus (DM) and Alzheimer’s BSAO and significant quenching in the presence of BSAG
disease (AD). The optical detection of glycated albumin is a account for the distinct interaction between the probe and
simple and cost-effective tool with diagnostic implications different structural variants of BSA. The high photostability
for DM and AD. Herein, we developed a cyano derivative of and differential response towards native BSA and BSAG infer
the benzothiazole-phenothiazine conjugate (TCNP) -based far- that TCNP is a potential molecular tool to assess the glycation
red fluorescence probe for the differential detection of induced structural changes of albumins.
glycated bovine serum albumin (BSAG) over native BSA and
Keywords: Albumin · Oxidation · Glycation · Far-red Fluorescence Probe · Differential Detection
DM is considered one of the most prevalent metabolic
disorders affecting more than 415 million individuals globally,
and the number of cases is estimated to cross over 642 million
by 2040.[1,2] The dreadful nature of DM is associated with
several neurological disorders like AD. DM triggers the
development of AD by promoting the blood-brain barrier
damage,[3,4] leading to the accumulation of misfolded amyloid
β (Aβ) peptides in the AD brain and related toxicities.[5–8] The
complications and evidence of DM link in neuropathological
diseases have led researchers to monitor blood glucose levels
critically. Quantitative determination of glycated proteins,
especially glycated hemoglobin A1c (HbA1c), is considered
one of the potential tools to detect hyperglycemia.[9] However,
the analysis has limitations regarding the use of HbA1c as a
short-term regular glycemic control.[10,11] The serum albumin
proteins (BSA: bovine serum albumin and HSA: human serum
albumin) account for ~50% of the total blood protein content
and exhibit several biomechanical properties, including on-
cotic pressure maintenance,[12,13] metal chelation,[14] interaction
with biomolecular entities,[15,16] transport of both endogenous
as well as exogenous cargo ligands, and immunomodula-
tion.[17] The remarkable ligand-binding features of albumin
proteins are attributed to undergoing glycation at multiple
sites, which is of significant interest to researchers and
clinicians as a credible indicator of serum blood glucose
level.[18,19] The nonenzymatic glycations of those albumin
proteins are a type of post-translational modifications where
the nucleophilic addition of reducing sugars to the free amine
or carbonyl groups of proteins forms reversible Schiff base
intermediate which rearranges to form the Amadori products
and further rearrangements like oxidation or cyclization trigger
formation of advanced glycated end products (AGEs).[20] The
serum albumin proteins of the native BSA consisting of 583
amino acids with 67% helix, 10% turn, and 23% extended
chain configuration with 17 disulfide bridges form a triple-
domain conformation.[21] The secondary structure of BSA is
vital to maintain the structure-function relationship and the
aberrant structural changes are implicated in various patho-
logical conditions (Figure 1A). In addition to glycation, BSA
undergoes substantial structural changes upon oxidation
(BSAO).[22] The formation of BSAO from native BSA triggers
the accumulation of reactive oxygen species (ROS) under
pathological conditions in the body and a critical indication of
oxidative stress, which is linked with disease conditions of
DM, age-related disorders, and neurological disorders.[23–26]
The nonenzymatic glycation of BSA is an inevitable repercus-
sion of increased blood glucose levels under the DM
conditions, potentially facilitating the formation of heteroge-
neous AGEs. The AGE-associated structural changes promote
the formation of neurofibrillary tangles (NFT) and amyloid
plaques, eventually leading to AD development.[24,27] In this
context, selective detection of different structural variants of
BSA is of foremost importance from the clinical perspective.
The conventional methods for detecting glycated proteins,
including glycated hemoglobin and albumin, are based on
various chromatography, electrochemistry, and immunochem-
istry techniques.[12,28–30] In the recent past, advanced immuno-
[a] A. Kumar,+ Dr. L. P. Datta,+ S. Samanta,+ H. Arora,
Prof. T. Govindaraju
Bioorganic Chemistry Laboratory, New Chemistry Unit and The
School of Advanced Materials (SAMat), Jawaharlal Nehru Centre
for Advanced Scientific Research (JNCASR), Jakkur P. O.,
Bengaluru-560064, Karnataka, India
E-mail: tgraju@jncasr.ac.in
[+] Equal first authorship (contributed equally to this work)
Supporting information for this article is available on the WWW
under https://doi.org/10.1002/ijch.202000098.
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Figure 1. (A) Schematic representation of BSA and its structural variants as pathophysiological markers. (B) The design strategy of D-π-A
architecture based far-red fluorescence probe (TCNP) and its application in the differential detection of different structural variants of BSA.
assay sensory platform,[31] enzymatic assay,[32] and
electroluminescence[33] based techniques have been reported
for the detection of glycated albumins. For bio-analyte
detection, the optical methods and assays are considered
advantageous due to their simple design, ease of handling, low
cost, and rapid quantification ability.[34] In practice, bio-analyte
responsive far-red fluorescent probes can outweigh visible
fluorescence probes for in vitro and in vivo applications, as
they cause minimal photodamage to the samples, exhibit
intense tissue penetration ability, and circumvent the problem
of autofluorescence from the surroundings.[35] In this aspect,
the tailored design of far-red (650 nm–900 nm) fluorescence
probes for the detection of various biological analytes and
markers have gained considerable attention from the scientific
community.[36–46] For in vivo applications, laser light source for
excitation and two-photon microscopy techniques will be
useful. While a range of fluorescence probes have been
reported for the detection of BSA or HSA,[36,47,48] far-red
fluorescence probe for the selective and differential detection
of structural variants of BSA has not been documented. For
instance, the extent of glycation and oxidation of BSA are
found to be elevated in DM patients, and these structural
alterations significantly impair the inherent binding affinity of
BSA to a range of external ligands, including drugs and
probes.[49,50] The conformational changes modify the three-
dimensional structure and make the ligand binding cavities
(site I and site II) narrow and shallow with leading to
compromised accessibility. Therefore, designing fluorescence
probes to differentially interact with the glycated albumin by
sensing the structural changes is challenging.
applicability of the probe (TCNP).[51–53] It has been established
that the cyano group substantially prevents the dye degradation
from singlet oxygen species[53] and a beneficial strategy to
improve the photostability. Incorporation of electron-with-
drawing cyano group in the design strategy influences the
energy difference profile between the highest occupied
molecular orbital (HOMO) and lowest unoccupied molecular
orbital (LUMO) of TCNP that triggers the far-red emission
along with improving the photostability. The probe was
synthesized by conjugating N-methyl phenothiazine (electron-
donor, D) with 2-(cyanobenzothiazol-2-yl) acetonitrile (elec-
tron-acceptor, A) moiety through a vinyl linkage (π-linker
unit) employing Knoevenagel-type condensation. The pheno-
thiazine was treated with sodium hydride (NaH) under ice-
cold condition followed by addition of methyl iodide (MeI)
°
and the reaction mixture was heated at 40 C to obtain 10-
methyl-10H-phenothiazine (1) (Scheme S1). The intermediate
1 was treated with phosphonium oxychloride (POCl3) in DMF
under heating conditions to obtain 10-methyl-10H-phenothia-
zine-3-carbaldehyde (2). Finally, the intermediate 2 was
condensed with 2-(cyanobenzothiazol-2-yl) acetonitrile by
Knoevenagel-type condensation in the presence of an organic
base to obtain the desired product TCNP as an orange-red solid.
All the intermediates and the final product were characterized
by NMR and HRMS analysis. The D-π-A molecular system
TCNP, exhibits a weak fluorescence signal in phosphate buffer
saline (PBS: 10 mM, pH=7.4) with λmax =672 nm upon
excitation at 480 nm (Figure S1). The observed spectral
behavior is particularly interesting from the perspective of an
ideal fluorescent molecular sensor (non-fluorescent in free-
state and strongly fluorescent in the analyte bound-state)
(Figure 1).[36,54,55] The weakly fluorescent TCNP show turn-on
emission in the presence of BSA. TCNP was found to
selectively detect BSA in mixed samples consisting of various
biological analytes. Subsequently, the detection of different
structural variants of BSA (BSAO and BSAG) was considered.
The differential sensing ability of TCNP towards BSA variants
was monitored through a series of spectroscopy measure-
ments.
In this work, we designed a far-red donor-π-acceptor
(D-π-A) system-based benzothiazole-phenothiazine probe
(TCNP) for the differential detection of structural variants of
BSA. The photostability of fluorescent probes is a primary
determinant for its practical utility. Incorporation of cyano
group in the bridge (vinyl linkage) of donor and acceptor
moieties was considered in our probe design.[51] This electron-
withdrawing cyano group suppresses the oxidative photo-
bleaching and significantly improve the photostability and
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The photophysical properties of fluorescent probe TCNP are
interesting due to the presence of donor and acceptor units
linked through the π-bridge, which is responsible for the
formation of an internal charge transfer (ICT) state in the polar
environment.[39] The probability of forming an ICT state and
response of TCNP towards different solvent systems was
assessed by recording the absorbance and fluorescence spectra
in solvents of varying polarity (Figure S2A and S2B). The
data showed a gradual shifting of absorption (λmax =464 nm in
toluene to 484 nm in PBS) and emission (λem =628 nm in
toluene to 672 nm in PBS) maxima towards longer wavelength
regions accompanied by the reduction in emission intensity
with increasing solvent polarity from toluene to PBS. The
observed bathochromic shift in polar solvents accounts for the
stabilization of the excited state and photoinduced ICT. The
observed data can be explained by the Lippert-Mataga theory
(effect of solvent polarity on fluorophore), which provides a
definitive approach to analyze the photophysical data of the
probe in solvents of varying polarity.[56] The mathematical
expression of the Lippert-Mataga equation is as follows:
showed minimal changes confirming the thermal stability of
TCNP (Figure S5). We incubated TCNP (10 μM) in solutions of
varying pH (1 to 13) for 10 min, and the fluorescent spectra
were recorded. The TCNP fluorescence did not change
significantly over a broad pH range, which confirmed pH
stability of the probe and its suitability under the physiological
conditions (Figure S6). Under extreme physical (temperature)
°
and chemical (pH) stimuli, viz., above 70 C and below pH 2,
marginal reduction in the basal fluorescence of TCNP was
observed. These are mostly outside the biologically relevant
conditions and not expected to affect the utility of the probe.
The cellular oxidative stress is a common phenomenon under
disease conditions, and it is essential to investigate the fate of
T
CNP under these conditions. We recorded fluorescence spectra
*
À
of TÀCNP in the presence of different ROS (H2O2, HO , NO2 ,
NO3 and ClOÀ ). For the experiment, TCNP (10 μM) was
incubated with ROS (50 μM) in PBS (10 mM, pH=7.4) for
10 min, and the TCNP emission was monitored upon excitation
at 480 nm. Interestingly, the TCNP emission remained mostly
unaffected by ROS at their high concentrations, which
affirmed the suitability of the probe for practical applications
under cellular conditions (Figure S7).
�
�
e À 1
n2 À 1
2
=
À
=
ðmE À mGÞ
2e þ 1
2 n2 þ 1
Next, the utility of TCNP as a molecular probe to sense the
albumins was evaluated. The structural homology between
BSA and HSA makes them identical in terms of ligand binding
ability. The selectivity of the probe TCNP towards BSA and
HSA was investigated in comparison with other biological
analytes such as trypsin, glutathione, and skimmed milk. The
fluorescence spectra were recorded by exciting TCNP (10 μM)
at 480 nm in PBS buffer and maintaining the stoichiometric
ratio of probe:analyte at 1:1 (Figure 2A). TCNP showed a
hypsochromic shift (~55 nm) in its fluorescence maxima
(λmax =672 nm) upon interaction with BSA or HSA. A
significant enhancement in the fluorescence emission intensity
(~7- and 6-fold increment for BSA and HSA, respectively)
was also observed. While, negligible changes in the
fluorescence emission intensity of TCNP in the presence of
trypsin and glutathione were observed, which revealed the
selectivity of the probe towards albumins. Notably, the slight
fluorescence response of the probe observed for skimmed milk
is attributed to the presence of albumin proteins. The crystal
structure analysis of BSA and HSA protein revealed three
different drug-binding domains (domains I, II, and III), and the
drug molecules generally bind to the sudlow 1 and sudlow 2
sites present in domain II and III.[57,58]
2
vA À vB ¼
=
a3
hc
where, h is the Planck’s constant, c is the speed of light, a is
the radius of the cavity in which the fluorophore resides, n is
the refractive index of the solvent, and ɛ is the dielectric
constant of the solvent, νA, and νF are the wavenumbers (cmÀ 1)
of absorption and fluorescence, and μE and μG correspond to
the dipole moments of the probe in the respective ground and
excited states. The change in the spectral shifts is verified by
analyzing the reorientation of solvents upon interaction with
the probe molecule, which is termed as orientation polar-
izability [Δf(e,n)] and expressed by the mathematical formula,
�
�
e À 1
n2 À 1
=
½Dfðe; nÞ� ¼
=
À
2e þ 1
2 n2 þ 1
The non-linear Lippert plot validates specific interaction
between TCNP and solvents of different polarity, and the
change in energy of the fluorophore is dependent on the
dielectric constant of the medium (Figure S3). The photo-
stability of the probe was investigated by irradiating TCNP
(10 μM, PBS:DMSO, 1:1, v/v) with a 100 W high power light
source. The negligible changes in the absorbance intensity
upon irradiation for 1 h confirmed the excellent photostability
of TCNP (Figure S4). The observed photostability of the probe
TCNP reinforced the design strategy of incorporating cyano-
group in the probe. Further, we investigated the stability of
The sudlow site I is known for the entrapment of hydro-
phobic drugs within its large hydrophobic cavity, and the
hydrophobic nature of TCNP accounts for its potential binding
in sudlow site I.[36] The fluorescence titration experiment was
performed to gain insight into the binding interactions between
BSA and TCNP. Increasing concentrations of BSA (0–900 μM)
TCNP in different external physical and chemical conditions
such as temperature, pH, and in the presence of various
reactive oxygen species (ROS). To assess the thermal stability,
we recorded the fluorescent spectra of TCNP (10 μM) in PBS
were titrated against a fixed concentration of the probe TCNP
(10 μM), and the changes in fluorescence emission intensity at
619 nm was monitored upon excitation at 480 nm (Figure 2B).
With BSA titration, the λmax value of the probe shifted towards
a lower wavelength accompanied by the gradual increase in
°
over the temperature range of 20–80 C upon exciting at
480 nm. The emission spectra at different temperatures
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Figure 2. (A) Selective detection of albumins (HSA and BSA) by TCNP (λex =480 nm λem =619 nm) over other biological analytes (trypsin, GSH,
skimmed milk). (B) The change in emission spectra of TCNP (10 μM) upon addition of increasing concentration of BSA (0–900 μM). (C) The
change in intrinsic fluorescence of BSA (10 μM) (λex =278 nm, λem =340 nm) with increasing concentration of TCNP (0–700 μM) and (D) the
corresponding Stern-Volmer plot of intrinsic fluorescence change of BSA with increasing concentration of TCNP to calculate Ksv (quenching
constant). NFI: Normalized fluorescence intensity.
the emission intensity. The enhancement in emission intensity
and hypsochromic shift in the emission maxima validates the
confinement of the probe in one of the hydrophobic pockets of
I0=I ¼ 1 þ Kqtq½Q� ¼ 1 þ KSV ½Q�
BSA. The entrapment of TCNP within the hydrophobic cavity
of BSA is anticipated to influence the conformation of the
protein and potentially alter the protein microenvironment.
The detailed information on the structural changes can be
obtained by examining the intrinsic fluorescence at 340 nm,
which corresponds to the characteristic signal of tryptophan
units of BSA. BSA (10 μM) was titrated with the increasing
concentration of TCNP (0–700 μM), and the change in the
fluorescence was measured at 340 nm (λex =278 nm). The
increasing concentrations of TCNP showed a gradual decrease
in the fluorescence emission intensity at 340 nm, indicating
possible changes in the microenvironment of aromatic residues
of BSA upon binding with TCNP (Figure 2C). In most of the
cases, upon interaction with external chemical probes, proteins
get denatured, which is validated by the distinct hyperchromic
shift of emission maxima at the tryptophan region.[59] How-
ever, the absence of any characteristic shift in the intrinsic
fluorescence band (λex =340 nm) confirmed that the interac-
tion of TCNP with BSA does not cause the unfolding or
denaturation of the protein. The Stern-Volmer plot investigated
the fluorescence quenching data, and the extent of quenching
was quantified by the following equation 2.
Where, I0 and I are the steady-state fluorescence intensities
in the absence and presence of quencher, respectively, KSV is
the Stern-Volmer quenching constant, τq is the average lifetime
of the protein without the quencher, and [Q] is the concen-
tration of the quencher (TCNP). The KSV value was determined
by plotting I0/I at 340 nm against the probe concentration
(Figure 2D) and was found to be 5.26×105 MÀ 1 as measured
by the linear regression assessment, which ensures effective
interaction between BSA and TCNP. BSAO accounts for several
pathological conditions in the body,[60] and investigating the
ability of the probe to interact with BSAO is of great interest.
To assess the interaction, the native BSA was subjected to
oxidation by incubating BSA (10 μM) with hydrogen peroxide
(H2O2, 100 μM), ferrous sulfate (FeSO4, 10 μM) and EDTA
(10 μM) in PBS at room temperature in the dark. The
oxidation was monitored by the spectral change and cyclic
voltammetric analysis. As observed from the fluorescence
spectroscopy data, the emission band of BSA corresponds to
the tryptophan fluorescence (λem =340 nm, λex =278 nm)
significantly reduced indicating the changes in the micro-
environment of the tryptophan region upon oxidation (Fig-
ure 3A). Cyclic voltammetry analysis confirmed the oxidation
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discussed (vide supra), TCNP exhibits fluorescence response
with BSA, and we were intended to understand the probe
response to BSAO in comparison to native BSA. TCNP (10 μM)
was treated with BSA or BSAO (10 μM) in PBS at room
temperature for 15 min, and emission spectra were recorded
upon excitation at 480 nm. The data showed that the emission
intensity of TCNP in the presence of BSAO is significantly
higher compared to native BSA (Figure 3C), suggesting that
TCNP differentially interacts with BSA and BSAO. The
observed photophysical changes revealed that TCNP exhibits
moderate differential sensing ability to detect BSAO over
native BSA.
As discussed earlier, BSAG is a biomarker for DM and
provides information on the average glucose level in the
blood.[61] As an endogenous glycemic control, the differential
detection of BSAG by TCNP can be an important diagnostic
tool for the early detection of DM. The glycation of albumin
proceeds via nonenzymatic attachment of glucose residues to
the free amino groups of lysine and arginine moieties followed
by the formation of Schiff base intermediate, which further
undergoes Amadori-rearrangement to form more stable keto-
amine or fructosyl-lysine products known as Amadori product
(Figure 4A). The glycation of BSA (50 μM) was carried out in
°
the presence of glucose (5 mM) in PBS at 37 C under
continuous incubation for 28 days. Concentration-dependent
photophysical studies monitored the extent of glycation of the
incubated BSA. The change in emission intensity of trypto-
phan residue of BSA at 340 nm was recorded upon excitation
at 278 nm (Figure 4B). The data showed a decrease in
emission intensity at 340 nm in the case of BSAG, which
indicates the changes in the tryptophan microenvironment
upon glycation. Further, the change in emission intensity in
the range of 400–550 nm was measured upon excitation at
370 nm. The characteristic fluorescence signals in the region
400–550 nm are the predictor of the formation of glycated
products (Figure S8). The glycation of BSA and the formation
of AGEs are accompanied by major structural changes in the
protein with globular to β sheet-like conversion.[62]
In this context, the thioflavin-T (ThT) assay is a reliable
method to probe the structural changes in BSAG due to the
high affinity of ThT dye towards β sheet-like structures.[28]
The assay was performed by treating different concentrations
of native BSA or BSAG (2, 5, and 10 μM) with ThT (10 μM),
and the change in emission intensity at 480 nm was recorded
upon excitation at 440 nm (Figure S9). There are no significant
changes in the ThT fluorescence intensities in the presence of
native BSA. While, BSAG showed a concentration-dependent
enhancement in ThT fluorescence intensities, and fluorescence
enhancement was significant at 10 μM concentration of BSAG,
which revealed alterations in the secondary structure of BSA
due to glycation. The structural changes of BSA due to
glycation were further confirmed by CD spectroscopy meas-
urement (Figure 4C). The CD spectra of BSA (10 μM) and
BSAG (10 μM) were recorded in PBS. The CD spectrum of
native BSA showed characteristic peaks at 205 nm and
225 nm corresponding to the α-helix structure. The CD
Figure 3. (A) The characteristic changes in intrinsic fluorescence of
BSA upon oxidation (λex =278 nm λmax =340 nm). (B) Cyclic voltam-
metry analysis to assess the changes in current density of BSA
(inset) upon oxidation (BSAO). (C) Emission spectra of TCNP with
native BSA and BSAO in PBS (λex =480 nm λem =619 nm). NFI:
Normalized fluorescence intensity.
of BSA. The native BSA showed a characteristic oxidation
peak at À 0.63 V and the less intense reduction peak at
À 0.70 V for electrochemical oxidation and reduction cycles,
respectively (Figure 3B inset). However, BSAO showed a
broad maximum in the range of À 0.65 to À 0.61 V for the
oxidation, while no peak was observed for the reduction
(Figure 3B). In general, the disulfide bridges of BSA are
susceptible to oxidation, and the reduction leading to a
possible rearrangement of the disulfide bonds of BSA. In the
case of BSAO, the absence of any reduction peaks indicates the
complete oxidation of BSA. Next, we monitored the inter-
action of TCNP with BSAO by spectrophotometry method. As
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Figure 4. (A) Schematic representation of glycation of BSA under high blood glucose levels and the formation of AGEs. (B) Intrinsic
fluorescence change of BSA upon glycation (λmax =340 nm, λex =278 nm). (C) Circular dichroism (CD) spectra of native BSA and BSAG
confirming the changes in helical content upon glycation. (D) The change in fluorescence intensity of TCNP (10 μM) with different
concentration of BSA and BSAG (10 and 100 μM) (λex =480 nm, λem =656 nm). (E) Intrinsic fluorescence change of BSAG (10 μM) monitored
at 530 nm upon the increasing concentration of the probe (0 to 100 μM) (λex =480 nm, λem =656 nm). (F) Intrinsic fluorescence change of
BSAG (10 μM) (λex =278 nm, λem =340 nm) recorded with increasing concentration of TCNP (0–100 μM). Inset is the enlarged view of the box,
which shows changes in intrinsic fluorescence intensity of BSAG in the presence of varying concentrations of the probe. NFI: Normalized
fluorescence intensity.
spectrum of glycated BSA showed a decrease in the peak
intensities at 205 nm and 225 nm indicating a reduction in the
helical content. This result is in good agreement with ThT
fluorescence assay data and confirmed major structural
changes to native BSA upon glycation. Next, we performed
the matrix-assisted laser desorption and ionization (MALDI)
analysis of BSA and BSAG using 2,5-dihydroxybenzoic acid/
sinapinic acid (20:80) as the matrix (Figure S10). The MALDI
spectrum showed an increased molecular mass peak for
glycated BSA compared to native BSA. The increased
molecular weight suggests BSA modification upon addition of
more than four glucose residues, which subsequently undergo
rearrangements to form the glycated derivatives. Next, we
investigated the utility of TCNP to differentially probe BSAG
against native BSA. TCNP (10 μM) was treated with native
BSA (10 and 100 μM) or BSAG (10 and 100 μM) and
incubated for 15 min at room temperature. The emission
spectra of TCNP was recorded upon excitation at 480 nm
(Figure 4D). The characteristic enhancement in the emission
spectra of TCNP at 619 nm along with hypsochromic shift was
unchanged for native BSA. However, significant quenching of
fluorescence emission with a bathochromic spectral shifting (~
55 nm) was observed in the case of BSAG in comparison to
native BSA. Notably, the emission spectra of BSAG showed a
characteristic band at 530 nm (Figure 4E), which is a signature
band corresponding to the glycation of arginine units in BSA
that unambiguously confirmed the formation of AGEs and
Arg-194, Arg-196, Arg-198, Arg-217, Arg-409, Lys-204 and
Lys-413 present in sudlow I and sudlow II sites are typically
responsible for ligand interaction.[46] The glycation predictably
makes significant structural alterations and conformational
changes in the sudlow sites due to covalent attachment of
glucose units to the amine groups of lysine and arginine
residues followed by the cyclic rearrangement to form AGEs.
The concentration-dependent study was performed, where the
concentration of probe was varied from 10 to 100 μM, and
BSAG concentration was fixed at 10 μM (Figure 4E). The
careful observation of the fluorescence data revealed that the
characteristic signature band of AGEs at 530 nm was
decreased upon interacting with the probe. A subsequent
increase in the probe concentration results in the disappearance
of the AGE band and reappearance of the intense fluorescence
signal at 655 nm, which is gradually enhanced with probe
concentration. The glycation-induced modifications of amino
acid residues in the drug-binding sites alter the surface area,
width, and volume of the binding pockets, which prevents the
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1
probe accessibility and alters the binding interactions. To gain
further insights on the probe-analyte interaction, the changes
in intrinsic tryptophan fluorescence (λem =340 nm, λex =
278 nm) was monitored by titrating increasing concentrations
of the probe (10 to 100 μM) to a fixed concentration of BSAG
(10 μM). As discussed earlier, the glycation induced structural
changes (helix to β-sheet transition) exposes the drug-specific
sudlow sites and alters the tryptophan microenvironment of
the protein, which reflected in the quenching of tryptophan
fluorescence intensity of BSAG as compared to native BSA
(vide supra). The observed increased trend in tryptophan
fluorescence quenching as a function of probe concentration
(10–100 μM) suggests that the probe interaction further
contributes to the structural alteration of BSAG (Figure 4F).
The glycation and oxidation induce significant structural
alteration to native BSA, which is believed to significantly
change the conformation of drug-binding sites (sudlow I and
sudlow II). TCNP binds to the hydrophobic drug-binding pocket
of native BSA, as revealed by the changes in intrinsic
fluorescence signal of protein and turn-on emission of the
probe. The conformational changes of BSA upon glycation
and oxidation significantly affect the intrinsic protein and
probe fluorescence responses, which result in the observed
differential detection patterns.
We have developed a fluorescence probe TCNP with a D-π-
A molecular system for the differential detection of structural
variants of BSA. The detailed photophysical studies performed
with TCNP established the selective and differential detection
of BSA/HSA, BSAO, and BSAG. The photostable TCNP exhibit
highly specific turn-on fluorescence emission and distinct
photophysical properties in the presence of BSA/HSA and
BSAO, with a maximum enhancement for the latter. The
glycation of native BSA resulted in significant conformational
changes and showed a prominent fluorescence quenching
response in the presence of the probe as compared to other
BSA analytes (BSA/HSA or BSAO). The structural modifica-
tion of BSA upon oxidation (BSAO) and glycation (BSAG)
have distinctively influenced the binding interaction of TCNP,
which resulted in differential fluorescence response. This study
underlines the importance of differential detection of structural
variants of BSA as the alternative method to blood glucose
level measurements for the diagnosis of various pathological
conditions. Further, modifications and improvements to the
methods and protocols reported here are anticipated to provide
novel diagnostic tools for DM and associated disease con-
ditions.
eluent to yield a white solid 1 in good yield (60%). H NMR
(400 MHz, DMSO d6): δ 7.22 (t, J=7.7 Hz, 2H), 7.16 (d, J=
7.8 Hz, 2H), 6.96 (dd, J=7.7 Hz, 4.1 Hz, 4H), 3.31 (s, 3H).
13C NMR (100 MHz, DMSO d6): δ 145.3, 127.7, 126.7, 122.4,
122.0, 114.5, 35.0. HRMS (ESI) m/z: calcd for C13H11NS [M
+H]+, 214.0684; Found, 214.0671.
Synthesis of 10-methyl-10H-phenothiazine-3-carbalde-
hyde (2). To a stirred solution of 1 (500 mg, 2.34 mmol) in
DMF (2 mL), POCl3 (1.5 mL, 1.81 g, 11.83 mmol) was added
°
dropwise under nitrogen atmosphere at 0 C. The reaction
mixture was stirred under the same conditions for 40 min
before it was brought to room temperature and stirred for
°
20 min followed by 3 h at 60 C and monitored by TLC. After
completion of reaction, the reaction mixture was poured into
ice-cold water to obtain a yellow-brown precipitate of 2 in
1
good yield (62%). H NMR (400 MHz, DMSO d6): δ 9.80 (s,
1H), 7.75 (d, J=8.4 Hz, 1H), 7.61 (s, 1H), 7.25 (t, J=7.7 Hz,
1H), 7.18 (d, J=7.6 Hz, 1H), 7.10 (d, J=8.4 Hz, 1H), 7.02 (t,
J=7.4 Hz, 2H), 3.39 (s, 3H). 13C NMR (100 MHz, DMSO d6):
δ 190.6, 150.4, 143.7, 130.9, 130.4, 128.0, 127.2, 126.9, 123.5,
122.4, 121.1, 115.4, 114.5, 35.6. HRMS (ESI) m/z: calcd for
C14H11NOS [M]+, 241.0561; found, 241.0967.
Synthesis of 2-(benzothiazole-2-yl)-3-(10-methyl-10H-
phenothiazin-3-yl) acrylonitrile (TCNP). To a stirred solution
of 2 (500 mg, 2.07 mmol) in ethanol (30 mL), piperidine
(283 μL, 244 mg, 2.86 mmol) was added dropwise. The
reaction mixture was stirred at room temperature for 20 min,
to which ethanol solution of 2-(benzo-thiazol-2-yl) acetonitrile
(690 mg, 3.96 mmol) was added. The reaction mixture was
stirred at room temperature for 24 h and monitored by TLC.
After completion of reaction, the reaction mixture was kept at
°
À 20 C overnight for precipitation. The obtained dark-orange
precipitate was washed with hexane and purified by column
chromatography on silica gel using ethyl acetate: hexane as
eluent. The product obtained as an orange solid in excellent
1
yield (80%). H NMR (400 MHz, DMSO d6): δ 8.24 (s, 1H),
8.15 (d, J=7.9 Hz, 1H), 8.02 (dd, J=16.9 Hz, 8.4 Hz, 2H),
7.90 (s, 1H), 7.56 (t, J=7.6 Hz, 1H), 7.49 (t, J=7.6 Hz, 1H),
7.26 (t, J=7.7 Hz, 1H), 7.20 (d, J=7.3 Hz, 1H), 7.13 (d, J=
8.7 Hz, 1H), 7.03 (t, J=7.5 Hz, 2H), 3.40 (s, 3H). 13C NMR
(100 MHz, DMSO d6): δ 163.5, 152.9, 148.6, 146.4, 143.5,
128.1, 126.9, 123.5, 122.8, 122.3, 115.4, 114.8, 101.8, 35.5.
HRMS (ESI) m/z: calcd. for C23H15N3S2 [M+H]+, 398.0779;
found, 398.0837.
BSA Oxidation and Characterization. For the oxidation
of BSA, BSA (10 μM) was pretreated with FeSO4 (10 μM)
and EDTA (10 μM) in PBS (10 mM, pH=7.4) for 5 min and
then H2O2 (100 μM) was added and the sample was incubated
for another 10 min at room temperature under dark
condition.[63,64] The oxidation of BSA was monitored by cyclic
voltammetry (CV) and fluorescence measurements. The
samples were purged with argon for 30 min to degas the
electrolyte. The cyclic voltammetry was performed in PBS
(10 mL, 10 mM, pH=7.4) using Ag/AgCl (1 M KCl) as a
reference electrode, glassy carbon as a working electrode and
Pt coil as the counter electrode. The CV spectra were recorded
Synthesis of 10-methyl-10H-phenothiazine (1). To a
stirred solution of 10H-phenothiazine (500 mg, 2.50 mmol) in
DMF in a high-pressure sealed tube, NaH (60 mg, 2.50 mmol)
°
was added at 0 C. The reaction mixture was stirred for 30 min
°
at 0 C followed by 15 min at room temperature. To the same
solution, MeI (2 mL, 4.55 g, 32.11 mmol) was added dropwise
°
and stirred at 40 C for 24 h and the reaction was monitored by
thin layer chromatography (TLC). After completion of the
reaction, the crude reaction mixture was purified by column
chromatography on silica gel using ethyl acetate: hexane as
©
Isr. J. Chem. 2021, 61, 222–230
2021 Wiley-VCH GmbH
www.ijc.wiley-vch.de
228

Communication
[15] E. L. Gelamo, C. H. T. P. Silva, H. Imasato, M. Tabak, Biochim.
Biophys. Acta 2002, 1594, 84–99.
[16] T.-T. Zhu, Y. Zhang, X.-A. Luo, S.-Z. Wang, M.-Q. Jia, Z.-X.
Chen, J. Agric. Food Chem. 2018, 66, 1242–1250.
[17] G. J. Quinlan, G. S. Martin, T. W. Evans, Hepatology 2005, 41,
1211–1219.
[18] R. Dolhofer, H. Wieland, FEBS Lett. 1979, 103, 282–286.
[19] R. Dolhofer, O. H. Wieland, Diabetes 1980, 29, 417–422.
[20] I. Sadowska-Bartosz, I. Stefaniuk, S. Galiniak, G. Bartosz, Redox
Biology 2015, 6, 93–99.
[21] S. K. Swain, D. Sarkar, Appl. Surf. Sci. 2013, 286, 99–103.
[22] S. Chesne, P. Rondeau, S. Armenta, E. Bourdon, Biochimie 2006,
88, 1467–1477.
[23] D. A. Butterfield, F. Di Domenico, A. M. Swomley, E. Head, M.
Perluigi, Biochem. J. 2014, 463, 177–189.
[24] N. T. Moldogazieva, I. M. Mokhosoev, T. I. Mel’nikova, Y. B.
Porozov, A. A. Terentiev, Oxid. Med. Cell. Longev. 2019, 2019,
3085756.
[25] L. M. Sayre, M. A. Smith, G. Perry, Curr. Med. Chem. 2001, 8,
721–738.
[26] L. M. Sayre, G. Perry, M. A. Smith, Chem. Res. Toxicol. 2008,
21, 172–188.
at a scan rate of 0.1 V/s in the range of À 1 V to À 0.24 V. The
change in the fluorescence intensity was measured at λem
340 nm upon excitation at 278 nm.
=
Glycation of BSA. BSA (50 μM) was incubated with
°
glucose (5 mM) in PBS (10 mM, pH=7.4) at 37 C for
28 days under constant agitation.[65] The glycation of BSA was
monitored by the change in the fluorescence spectra at λem
=
340 nm upon excitation at 278 nm and λem =485 nm upon
excitation at 370 nm, respectively, which are the characteristic
region to monitor glycation of BSA. After glycation of BSA,
the sample was extensively dialyzed in PBS (10 mM, pH=
7.4) using the PVDF membrane (14 kDa) for 48 h to remove
excess glucose molecules. The concentration of glycated BSA
was measured by Bradford’s assay. The stock solutions were
°
stored at À 20 C.
Acknowledgment
We thank JNCASR, SwarnaJayanthi Fellowship grant (DST/
SJF/CSA-02/2015-2016), the Department of Science and
Technology (DST), India, Sheikh Saqr Laboratory (SSL),
ICMS-JNCASR financial support, and CSIR and UGC for
student fellowships.
[27] G. Münch, J. Thome, P. Foley, R. Schinzel, P. Riederer, Brain
Res. Rev. 1997, 23, 134–143.
[28] A. Schmitt, J. Schmitt, G. Münch, J. Gasic-Milencovic, Anal.
Biochem. 2005, 338, 201–215.
[29] E. Schleicher, O. H. Wieland, J. Clin. Chem. Clin. Biochem.
1989, 27, 577–587.
[30] M. Rendell, K. Rasbold, J. Nierenberg, R. Krohn, G. Hermanson,
D. Klenk, P. K. Smith, Clin. Biochem. 1986, 19, 216–220.
[31] H. Ki, J. Oh, G.-R. Han, M.-G. Kim, Lab Chip 2020, 20, 844–
851.
[32] T. Kouzuma, T. Usami, M. Yamakoshi, M. Takahashi, S.
Imamura, Clin. Chim. Acta 2002, 324, 61–71.
[33] Y. Inoue, M. Inoue, M. Saito, H. Yoshikawa, E. Tamiya, Anal.
Chem. 2017, 89, 5909–5915.
[34] L. Unger-Angel, B. Rout, T. Ilani, M. Eisenstein, L. Motiei, D.
Margulies, Chem. Sci. 2015, 6, 5419–5425.
[35] L. Yuan, W. Lin, K. Zheng, L. He, W. Huang, Chem. Soc. Rev.
2013, 42, 622–661.
[36] K. Rajasekhar, C. J. Achar, T. Govindaraju, Org. Biomol. Chem.
2017, 15, 1584–1588.
[37] Y. V. Suseela, N. Narayanaswamy, S. Pratihar, T. Govindaraju,
Chem. Soc. Rev. 2018, 47, 1098–1131.
[38] K. Rajasekhar, N. Narayanaswamy, N. A. Murugan, K. Viccaro,
H. G. Lee, K. Shah, T. Govindaraju, Biosens. Bioelectron. 2017,
98, 54–61.
References
[1] C. Bommer, E. Heesemann, V. Sagalova, J. Manne-Goehler, R.
Atun, T. Bärnighausen, S. Vollmer, Lancet Diabetes Endocrinol.
2017, 5, 423–430.
[2] C. C. Patterson, S. Karuranga, P. Salpea, P. Saeedi, G. Dahlquist,
G. Soltesz, G. D. Ogle, Diabetes Res. Clin. Pract. 2019, 157,
107842.
[3] S. Prasad, R. K. Sajja, P. Naik, L. Cucullo, J. Pharmacovigilance
2014, 2, 125.
[4] M. Bogush, N. A. Heldt, Y. Persidsky, J. Neuroimmune Pharma-
col. 2017, 12, 593–601.
[5] C. Sims-Robinson, B. Kim, A. Rosko, E. L. Feldman, Nat. Rev.
Neurol. 2010, 6, 551–559.
[6] A. M. Vincent, B. C. Callaghan, A. L. Smith, E. L. Feldman, Nat.
Rev. Neurol. 2011, 7, 573–583.
[39] K. Rajasekhar, N. Narayanaswamy, N. A. Murugan, G. Kuang,
H. Ågren, T. Govindaraju, Sci. Rep. 2016, 6, 23668.
[40] N. Narayanaswamy, S. Narra, R. R. Nair, D. K. Saini, P.
Kondaiah, T. Govindaraju, Chem. Sci. 2016, 7, 2832–2841.
[41] D. Maity, A. Raj, P. K. Samanta, D. Karthigeyan, T. K. Kundu,
S. K. Pati, T. Govindaraju, RSC Adv. 2014, 4, 11147–11151.
[42] N. Narayanaswamy, M. Kumar, S. Das, R. Sharma, P. K.
Samanta, S. K. Pati, S. K. Dhar, T. K. Kundu, T. Govindaraju,
Sci. Rep. 2014, 4, 6476.
[43] D. Maity, T. Govindaraju, Org. Biomol. Chem. 2013, 11, 2098–
2104.
[44] N. Narayanaswamy, S. Das, P. K. Samanta, K. Banu, G. P.
Sharma, N. Mondal, S. K. Dhar, S. K. Pati, T. Govindaraju,
Nucleic Acids Res. 2015, 43, 8651–8663.
[7] K. Rajasekhar, M. Chakrabarti, T. Govindaraju, Chem. Commun.
2015, 51, 13434–13450.
[8] H. Arora, M. Ramesh, K. Rajasekhar, T. Govindaraju, Bull.
Chem. Soc. Jpn. 2020, 93, 507–546.
[9] D. Li, Z. Zhang, H. Chang, Sensors Actuators B: Chem. 2019,
291, 433–440.
[10] T. Kohzuma, M. Koga, Mol. Diagn. Ther. 2010, 14, 49–51.
[11] K. L. Costa, Z. A. Taboza, G. B. Angelino, V. R. Silveira, R.
Montenegro Jr, A. N. Haas, R. O. Rego, J. Periodontol. 2017, 88,
17–25.
[12] F. K. Yeboah, V. A. Yaylayan, Food / Nahrung 2001, 45, 164–
171.
[13] T. W. Evans, Aliment. Pharmacol. Ther. 2002, 16, 6–11.
[14] L. G. Naso, L. Lezama, M. Valcarcel, C. Salado, P. Villacé, D.
Kortazar, E. G. Ferrer, P. A. M. Williams, J. Inorg. Biochem.
2016, 157, 80–93.
[45] D. Maity, A. Raj, D. Karthigeyan, T. K. Kundu, T. Govindaraju,
Supramol. Chem. 2015, 27, 589–594.
©
Isr. J. Chem. 2021, 61, 222–230
2021 Wiley-VCH GmbH
www.ijc.wiley-vch.de
229

Communication
[46] D. Maity, A. K. Manna, D. Karthigeyan, T. K. Kundu, S. K. Pati,
T. Govindaraju, Chem. Eur. J. 2011, 17, 11152–11161.
[47] S. Lee, D.-B. Sung, S. Kang, S. Parameswaran, J.-H. Choi, J. S.
Lee, M. S. Han, Sensors (Basel) 2019, 19, 5298.
[48] C. Liu, W. Yang, Q. Gao, J. Du, H. Luo, Y. Liu, C. Yang, J.
Lumin. 2018, 197, 193–199.
[57] G. Sudlow, D. J. Birkett, D. N. Wade, Mol. Pharmacol. 1975, 11,
824–832.
[58] G. Sudlow, D. J. Birkett, D. N. Wade, Mol. Pharmacol. 1976, 12,
1052–1061.
[59] R. Joshi, M. Jadhao, H. Kumar, S. K. Ghosh, Bioorg. Chem.
2017, 75, 332–346.
[49] J. Baraka-Vidot, A. Guerin-Dubourg, E. Bourdon, P. Rondeau,
Biochimie 2012, 94, 1960–1967.
[50] S. Awasthi, N. A. Murugan, N. T. Saraswathi, Mol. Pharm. 2015,
12, 3312–3322.
[51] N. I. Shank, K. J. Zanotti, F. Lanni, P. B. Berget, B. A. Armitage,
J. Am. Chem. Soc. 2009, 131, 12960–12969.
[52] T. Chen, Z. Chen, R. Liu, S. Zheng, Org. Biomol. Chem. 2019,
17, 6398–6403.
[60] R. Misra, S. Kar, Chem. Phys. 2012, 397, 65–73.
[61] Z. Turk, S. Ljubic, N. Turk, B. Benko, Clin. Chim. Acta 2001,
303, 105–115.
[62] B. Bouma, L. M. Kroon-Batenburg, Y.-P. Wu, B. Brünjes, G.
Posthuma, O. Kranenburg, P. G. de Groot, E. E. Voest, M. F.
Gebbink, J. Biol. Chem. 2003, 278, 41810–41819.
[63] S. Guedes, R. Vitoriro, R. Domingues, F. Amado, P. Domingues,
Rapid Commun. Mass Spectrom. 2009, 23, 2307–2315.
[64] C. P. Baron, H. E. F. Retsgaard, L. H. Skibsted, M. L. Andersen,
Free Radical Res. 2006, 40, 409–417.
[53] A. Toutchkine, D. V. Nguyen, K. M. Hahn, Org. Lett. 2007, 9,
2775–2777.
[54] S. Samanta, T. Govindaraju, ACS Chem. Neurosci. 2019, 10,
4847–4853.
[65] A. Schmitt, J. Schmittb, G. Münchc, J. Gasic-Milencovica, Anal.
Biochem. 2005, 338 201–215.
[55] N. Narayanaswamy, S. Das, P. K. Samanta, K. Banu, G. P.
Sharma, N. Mondal, S. K. Dhar, S. K. Pati, T. Govindaraju,
Nucleic Acids Res. 2015, 43, 8651–8663.
[56] B. Bagchi, D. W. Oxtoby, G. R. Fleming, Chem. Phys. 1984, 86,
257–267.
Manuscript received: November 11, 2020
Revised manuscript received: January 11, 2021
Version of record online: January 26, 2021
©
Isr. J. Chem. 2021, 61, 222–230
2021 Wiley-VCH GmbH
www.ijc.wiley-vch.de
230