Binding interactions and FRET between bovine serum albumin and various phenothiazine-/anthracene-based dyes: A structure-property relationship

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Binding interactions and FRET between bovine

serum albumin and various phenothiazine-/

anthracene-based dyes: a structure–property

relationship†

Cite this: RSC Adv., 2021, 11, 1679

*

Shouvik Bhuin,‡ Sayantan Halder,‡ Subit Kumar Saha

and Manab Chakravarty

The present study demonstrates binding interactions and Forster resonance energy transfer (FRET) between

¨

bovine serum albumin (BSA) and a series of structurally and electronically diverse phenothiazine (PTZ) and

anthracene (ANT) dyes. Upon selective excitation of tryptophan (Trp) residues of BSA, radiationless energy

transfer to a dye takes place, resulting in ﬂuorescence quenching of the former. Fluorescence quenching

mechanisms, FRET parameters, possible locations, and binding constants of dyes with the BSA have been

examined to deduce a structure–property relationship. The mechanism of quenching is apparently static

in nature. PTZ dyes with heteroatoms and a pentyl tail (C5-PTZ) attached to them were found to have

a stronger binding aﬃnity with BSA as compared to ANT dyes. Stronger binding aﬃnities of C5-PTZ dyes

with BSA result in greater energy transfer eﬃciencies (E_T). A dye with a strong electron-withdrawing

group present in it has shown better energy accepting capability. A FRET study with dicyanoaniline (DCA)

analogs of PTZ and ANT dyes (C5-PTZDCA and ANTDCA, respectively) revealed that E_Tdepends on

electronic and structural factors of molecules. An almost orthogonal geometry between ANT and DCA

moieties (ꢀ79^ꢁ) in ANTDCA induces the greater extent of electron transfer from ANT to DCA, showing

a higher E_Tfor this dye as compared to C5-PTZDCA in which the torsion angle is only ꢀ38^ꢁ. Further, the

observed facts have been validated by experimentally determined bandgaps (using cyclic voltammetry

experiments) for all the dyes. Thus, the hydrophobic character and the presence of interactive

substituents along with the electron-accepting abilities majorly control the FRET for such dyes with BSA.

Received 11th November 2020

Accepted 15th December 2020

DOI: 10.1039/d0ra09580j

rsc.li/rsc-advances

originated selectively from Trp residues of BSA. These Trp

residues act as donors in the energy transfer process with an

1. Introduction

acceptor, and this process can be radiative or non-radiative.

Serum albumin has been identied as the most abundant

protein in plasma. Many researchers remain focused on

studying the interactions of bioactive compounds with plasma

proteins, mainly serum albumin.¹Upon binding with the serum

albumin, the biological activities may be reduced or enhanced

and can provide a transportation path. Bovine Serum Albumin

(BSA) is a well-studied protein due to its structural homology

with human serum albumin and high abundance. BSA has

three structurally distinct domains, I, II, and III, with two sub-

domains (A and B) in each of the areas.²Out of two Trp residues

in BSA, Trp-134 is located on the surface of domain I, and Trp-

213 is situated in the hydrophobic pocket of domain II. Upon

excitation at ꢀ295 nm, the intrinsic uorescence can be

¨

Forster or uorescence resonance energy transfer (FRET) is

a radiationless process without emission and reabsorption of

photons. Energy exchange takes place between oscillating

dipoles of donor and acceptor of similar resonating frequen-

cies.³Since the discovery of the FRET, researchers have been

actively utilizing this potential non-radiative tool to unveil

numerous facts related to the eld of supramolecular materials

and biomedical research and development.^4,5There is a rapid

growth in the invention of donor–acceptor combinations for the

FRET applications. However, constructing an appropriate

donor–acceptor pair for the FRET is still a challenge because it

needs to meet primary inuencing criteria such as distance,

spectral overlap, and dipole orientation between donors and

acceptors.⁶There are numerous applications where donors and/

or acceptors in FRET are used as uorescence site markers.^7,8In

the present study, a series of phenothiazine and anthracenyl

molecules (Chart 1) are examined as acceptors for the energy

transfer from the donors i.e., Trp residues of BSA. To our

knowledge, both these structural motifs are not tested for FRET

Department of Chemistry, Birla Institute of Technology and Sciences-Pilani,

Hyderabad Campuses, Hyderabad-500078, Telangana, India. E-mail: manab@

hyderabad.bits-pilani.ac.in; sksaha@hyderabad.bits-pilani.ac.in

† Electronic supplementary information (ESI) available. CCDC 2041739 and

2041742. For ESI and crystallographic data in CIF or other electronic format see

DOI: 10.1039/d0ra09580j

‡ Shouvik Bhuin and Sayantan Haldar have Contributed equally.

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Chart 1 Phenothiazine and anthracenyl molecules that are studied herein for the FRET with BSA.

1

studies with BSA before. There are a few reports on complicated and H and ¹³C NMR data (see ESI; pages (3–6) and Fig. (S11–

phenothiazine-linked molecules for metal ion sensing through S16)†). PTZ, ANT, ANTA, and BSA were procured from Alfa-Aesar

FRET.^9,10As benzothiazole type systems are oen found to be and Sigma Aldrich. They were taken into use as received. BSA

suitable for FRET,^11,12the electron-rich phenothiazine core is of protein concentration was kept constant at 5 mM for all

our particular interest.

measurements. HEPES buﬀer was bought from SRL, India.

Additionally, the questions arise on the requirement of thia H₂SO₄and NaOH were procured from SDFCL, India, and SRL,

and aza part in a system for an eﬃcient FRET phenomenon. India, respectively. Quinine sulfate was obtained from Sigma

Therefore, we also intend to explore the FRET on anthracenyl- Aldrich. Spectral grade tetrabutylammonium perchlorate

based uorophores. Notably, anthracene and phenothiazine (TBAP) and MeCN were purchased from TCI, India.

are not analogous due to the fundamental structural diﬀer-

ences; however, we dealt with similar substituents for both the

2.2. Methods

skeletons. Apart from a few known dyes, the dicyanoaniline

A 10 mM solution of HEPES buﬀer was prepared with Milli-Q

(DCA) analogs [PTZDCA and ANTDCA, Chart 1] with acceptor–

water, and its pH was adjusted to 7.4. H₂SO₄and NaOH solu-

tion was used to adjust the pH of the buﬀer solution. BSA stock

solution was prepared with a freshly prepared buﬀer solution.

Stock solutions of the diﬀerent dyes (phenothiazine and

donor–acceptor (A–D–A) architecture bring special attention to

this FRET study.^13,14Furthermore, the FRET analysis using non-

traditional donor–acceptor combinations is very much

appealing.⁶Most of these traditional compounds emit at UV-Vis

anthracene series of compounds) were prepared (ꢀ0.5 mM

region but with somewhat commendable Stokes shis

each) in pure methanol for recording the UV-vis absorption and

compared to the previous report.⁶Intriguingly, the FRET

uorescence spectra. All stock solutions were carefully degassed

with a pure N₂gas for 15 minutes before use. For the FRET study

between BSA and each individual dye, at a xed concentration

of BSA (5 mM), the concentration of dye was gradually increased

between phenothiazine or anthracene-based molecules and BSA

has enabled them to oﬀer as good acceptors. Such acceptors are

focused on their convincing applications in the FRET. Mecha-

nistic insights are specied to nd quenching of Trp-

in the range of 0 mM ꢂ15 mM. The total volume of each solution

uorescence by each of the dyes. An attempt has been made

was kept at 2 mL. A JASCO (model V-650) UV-vis spectropho-

tometer with a quartz cell of path length 1 cm with a Teon

stopper was used to record the absorption spectra. A FluoroLog-

TM (Horiba Scientic) spectrouorimeter was used for steady-

state uorescence measurements using a quartz cell of path

length 1 cm with a Teon stopper. For uorescence measure-

ments, all the samples were excited at 295 nm. Slit widths for

both excitation and emission were 2.5 nm, that along with the

to nd the possible relation between the structures of dyes and

various properties, such as binding strength/locations and

energy transfer eﬃciency. The governing eﬀect of substituted

hydrophobic and electron-withdrawing groups, the position of

substitution on the features mentioned above, has also been

elucidated.

scan rates, were maintained constant for all uorescence

measurements. All the uorescence spectra were corrected for

instrument sensitivity and also to avoid any inner lter eﬀect.

2. Experimental section

2.1. Materials

The known phenothiazine and anthracene compounds like C5- Electrochemical assessments were conducted using an Autolab

PTZ, C5-PTZA, and ANTCN were synthesized using reported PGSTAT128N potentiostat. Cyclic Voltammetry (CV) of the

procedures.^15,16C5-PTZCN, C5-PTZDCA, and ANTDCA are the samples were performed by switching the applied voltage in

compounds that were newly synthesized. Their structures were ꢂ3 V to +3 V; using a cleaned glassy carbon (GC) electrode as

veried by FT-IR, LCMS (Liquid Chromatography Mass a working electrode (WE). The Pt-wire and Ag/AgCl were used as

Spectrometry)/HRMS (High-Resolution Mass Spectrometry), a counter electrode (CE) and a reference electrode (RE),

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respectively, and a 0.1 M tetrabutylammonium perchlorate donor–acceptor systems, DCA was installed with the C5PTZ core

(TBAP) in acetonitrile was used as an electrolyte in a typical to aﬀord C5PTZDCA as an acceptor to elucidate the electronic

three-electrode system. A sample solution in acetonitrile was factor in the FRET process. For the comparative study with

mixed with electrolyte to study the electrochemical behavior of a similar tricyclic electron-rich system, anthracene is coupled

samples. The redox reaction that happened on the surface of with the DCA motif to aﬀord ANTDCA. Thus, we investigate the

GC, were recorded in NOVA 2.1.1. soware. HOMO/LUMO scope of changes in FRET with the same trend of functionalities

energy levels were determined from the onset of oxidation/ in these two diﬀerent scaﬀolds.

reduction potentials in the fullness of positive/negative scans.

A Jasco-J 1500 CD spectropolarimeter was utilized for recording

the far – UV circular dichroism (CD) spectra of appropriate

systems in the wavelength range of 190–260 nm. A cuvette

having path length as 0.1 cm was used. Each CD spectrum was

recorded at a scan speed of 50 nm min^ꢂ1with a spectral

bandwidth of 2.5 nm. The spectra obtained for diﬀerent BSA

systems were subtracted by buﬀer spectra for background

correction. All measurements were performed at 298.15 ꢃ 1 K

temperature.

3.1. Synthesis of the compounds

C5-PTZDCA compound was synthesized newly via a one-pot

reaction of aldehyde and malononitrile in the presence of trie-

thylamine (TEA) as depicted in Scheme 1. In the rst step, the

requirement of the base was not mandatory just because of the

highly active methylene group. The dicyano compound, C5-

PTZCN was not isolated separately herein. Instead, the addition

of excess malononitrile and TEA directly produced penta-

substituted benzene, C5-PTZDCA, in 63% yield. Thus, this

strategy is somewhat diﬀerent than the existing one,¹⁸hitherto

reported.

3. Results and discussions

The compound ANTDCA was synthesized in a manner

An electron-rich and inexpensive phenothiazine core is picked similar to C5-PTZDCA with a 25% yield, possibly due to the

up for the FRET-study with BSA, based on the earlier reports on various reaction scopes with anthracenyl core. A solvent-free

related thiazole as a well-established motif for FRET applica- twisted path¹⁹was also adopted to aﬀord ANTDCA, but the

tions.^9,17However, the presence of pyridyl nitrogen (electron- result was not pleasing.

withdrawing sp²-hydridized) in the thiazole type molecules

All the synthesized compounds are characterized by NMR,

displays the primary diﬀerence between phenothiazine and HRMS/LCMS, and FT-IR spectroscopy (see ESI† for details).

benzothiazole cores. To investigate beyond the conventional Mostly, the molecular structure for new compounds C5-

Scheme 1 Synthesis of C5-PTZDCA.

o

ꢀ

Fig. 1 The molecular structures of (a) C5-PTZDCA and (b) ANTDCA. The selected torsion angles ( ) and bond lengths (A) are also given. There are

two molecules in one asymmetric unit for both the systems. Hydrogens are omitted for the C5-PTZDCA for clarity. CCDC Number: ANTDCA

2041742; C5-PTZDCA: 2041739.

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and C5-PTZA, respectively. Still, the emission from C5-PTZA is

unexpected using l_ex¼ 295 nm. Albeit, a signicant extent of

overlap between the uorescence band of BSA and absorption

band of C5-PTZA (Fig. 2) along with the uorescence from both

the species indicate a denite possibility of FRET occurring

between them. Among various intrinsic uorophores as part of

the origin of BSA's uorescence, we have focused explicitly on to

excite only Trp residues to avoid the uorescence contribution

from the other amino acid residues of BSA such as phenylala-

nine (Phe) and tyrosine (Tyr). That is why the excitation wave-

length of 295 nm has been selectively preferred.²⁰Further,

between two Trp residues in BSA, Trp-134 resides in the

hydrophilic environment, whereas Trp-213 chooses a relatively

less polar part of BSA.²¹Intriguingly, such FRET behavior was

experienced with all the dyes used herein except molecular

anthracene (ANT).

Fig. 2 Normalized absorption and ﬂuorescence spectra of 5 mM BSA in

the presence of 5 mM C5-PTZA in an aqueous HEPES buﬀer solution.

l_exfor ﬂuorescence measurements is 295 nm.

To understand the origin of uorescence quenching for Trp

residues of BSA, the radiative rate constant (k_r) of this emission

process is a vital parameter and calculated using Strickler–Berg

equation (eqn (1)) considering no solvent interactions and

PTZDCA and ANTDCA are determined unequivocally with the

help of single-crystal X-ray diﬀraction studies (Fig. 1). The X-ray

structures help to identify the exact position of cyano, amine,

and methyl groups within the dicyanoaniline (DCA) ring.

Moreover, the torsion angle between the DCA and anthracene or

phenothiazine could also be measured as depicted in Fig. 1.

change in the excited-state geometry:^22–24

ð

2

k_r¼ 3:13 ꢄ 10^ꢂ9y₀3dy x y₀f

(1)

where ꢁy₀represents the wavenumber when the absorption of the

BSA is maximum, 3 is the extinction coeﬃcient, and f denotes

the oscillator strength. The absorption spectra of BSA in the

absence and presence of each molecule (excluding ANT) as

quenchers were recorded to calculate k_rand f values in each case

and tabulated in Table 1.

Notably, the change in radiative rate constants of the BSA in

the presence or absence of a dye is not impressive. The changes

in the oscillator strength fall within the range of 0.01–4.61%,

which has an almost negligible contribution to the observed

quenching (29.0–66.9%, Table 1). Therefore, the uorescence

quenching of BSA by the molecules conrms the involvement of

the energy transfer process to a greater extent.

3.2. Systematic absorption and uorescence measurements

to study FRET

In this investigation, the FRET behavior between BSA and all the

organic dyes has been demonstrated by carrying out systematic

absorption and uorescence measurements. The detailed on

FRET are discussed in the ESI.† Before we deal in a sequence, let

us demonstrate the representative plot of FRET with one of the

above-listed compounds C5-PTZA (Fig. 2) to elucidate the

process. The normalized absorption and uorescence spectra of

5 mM BSA solution were recorded in the presence of an extrinsic

uorophore C5-PTZA (5 mM) in an aqueous HEPES buﬀer

solution. The peak maximum of the more extended wavelength

absorption band of BSA is located at 279 nm, while the

absorption peak maximum of C5-PTZA appeared at 373 nm.

Upon excitation of the sample (a mixture of BSA and C5-

PTZA) with a light of 295 nm, two uorescence bands at 347 nm

and 502 nm were found due to the emissions from both BSA

Next, the uorescence quenching of Trp residues in BSA was

studied at diﬀerent concentrations for each of the dyes, indi-

vidually except for ANT. This study has enabled us to explain the

mechanism of quenching, binding eﬃciency with the protein,

and location in the protein. Fluorescence quenching using

Table 1 The radiative rate constant (k_r), % change in oscillator strength (f), and % quenching of ﬂuorescence from Trp residues of BSA

%

Quencher (dye)

k_rꢄ 10^ꢂ10(without dye) (s^ꢂ1

)

k_rꢄ 10^ꢂ10(with dye) (s^ꢂ1

)

change in f

quenching

PTZ

C5-PTZ

1.45

1.49

1.51

1.46

1.49

1.42

1.41

0.01

3.81

4.61

0.11

3.87

2.93

2.26

3.30

31.7

34.9

66.9

49.6

29.0

34.9

30.7

35.8

C5-PTZA

C5-PTZCN

C5-PTZDCA

ANTA

ANTCN

ANTDCA

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quencher is represented by the Stern–Volmer (S–V) equation,³⁵

(eqn (2)):

F_o

¼ 1 þ K_SV½Qꢅ ¼ 1 þ k_qs_o½Qꢅ

(2)

F

where F_oand F are the uorescence intensities of Trp residues

of BSA in the absence and presence of a quencher, respectively;

K_SV, k_q, and s_orepresent S–V quenching constant, bimolecular

quenching rate constant, and the average lifetime of the uo-

rophore in absence of a quencher, respectively. The uores-

cence quenching of Trp residues is analyzed using the S–V plot,

as displayed in Fig. 4a. The observed downward curvature of the

S–V plot indicates the fractional accessibilities of two diﬀerent

Trp residues of BSA to PTZ.³⁵The downward curvature in

protein uorescence quenching generally occurs in the pres-

ence of a polar or charged quencher.³⁵Being polar/charged, the

quencher cannot penetrate the hydrophobic interior of the

protein. Here, the parent compound PTZ thus shows diﬀering

accessibility to the Trp residues. There is no signicant change

in the peak maxima of BSA with an increase in the concentra-

tion of PTZ. The S–V plot has been modied (Fig. 4b) based on

eqn S(1) in Note S1 in ESI† to nd the fraction of Trp residues

accessible to PTZ (f_ain eqn S(2)).†³⁵

Fig. 3 Fluorescence spectra of Trp residues of BSA in the presence of

diﬀerent concentrations of PTZ. [BSA] ¼ 5 mM. l_ex¼ 295 nm.

these molecules and analysis of quenching data in each case

has been attempted to decipher the observed facts.

3.2.1 FRET between BSA and phenothiazinyl compounds.

As intended, the series of molecules with phenothiazine (PTZ)

core (Chart 1; Fig. 1a) were initially studied. Upon incremental

addition of PTZ at a xed concentration of BSA (5 mM), there was

a diminution in the uorescence intensity of BSA, which can be

ascribed to the energy transfer from Trp residues to PTZ.

However, no increase in the uorescence intensity of PTZ

could be found, being a non-uorescent dye (Fig. 3). The above-

mentioned calculated values (Table 1) of the radiative rate

constants (k_r)/oscillator strength (f) and the analysis of data

have conrmed the energy transfer as a cause for the uores-

cence quenching. The FRET eﬃciency depends on donor–

acceptor proximity and spectral overlap, not necessarily that the

acceptor should be uorescent.²⁵There are recent reports on the

use of several non-uorescent acceptors/quenchers, e.g.,

Dabcyl,^26–28the black hole quenchers, BHQ,^28–30QSY-7, and

BHQ2,²⁸and QSY-9,³¹diﬀerent nucleotides,²⁸Guanosine,^32,33

and Au nanoparticles.^27,34The development of non-uorescent

dye derivatives-based FRET assays is a promising area that

can lead to broad applications in the receptor eld.²⁵A rela-

tionship between uorescence quenching and concentration of

The correlation coeﬃcient of the linear tting of the modi-

ed S–V plot (Fig. 4b) is found to be 0.9997. The calculated f_a

and K_avalues from the intercept and the slope of the modied

S–V plot are found to be 0.43 and 1.92 ꢄ 10⁵M^ꢂ1, respectively.

So, 43% of Trp residues in BSA are only accessible to PTZ. The

estimated S–V quenching constant of an accessible fraction (K_a

¼ k_qs_o) is 1.92 ꢄ 10⁵M^ꢂ1. Considering the s_ofor BSA: ꢀ10^ꢂ8

s

(ref. 35–37) the bimolecular quenching rate constant, k_qappears

as 1.92 ꢄ 10¹³M^ꢂ1s^ꢂ1. However, the maximum value of k_qfor

a diﬀusion-controlled quenching process with a biopolymer is

ꢀ2.0 ꢄ 10¹⁰M^ꢂ1s^ꢂ1.³It implies that the mechanism of the

quenching of available fraction is static. By assuming the same

and independent binding sites for BSA in case of a static

quenching, the binding constant K⁰and the number of binding

sites (n) have been calculated based on eqn S(3) in Note S2.†³⁸

Fig. S1† displays the plot of log[(F_oꢂ F)/F] versus log(1/([PTZ]

– (F_oꢂ F)[BSA]/F_o)). The number of binding sites, n, and the

binding constant, K⁰calculated from the slope and intercept,

Fig. 4 (a) S–V plot for quenching of the Trp ﬂuorescence by PTZ in BSA, and (b) modiﬁed S–V plot for quenching the Trp ﬂuorescence by PTZ in

BSA. [BSA] ¼ 5 mM. l_ex¼ 295 nm.

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Fig. 5 (a) Fluorescence spectra of Trp residues of BSA with increasing concentration of C5 PTZ, and (b) S–V plot for quenching of the Trp

ﬂuorescence by C5-PTZ. [BSA] ¼ 5 mM. l_ex¼ 295 nm.

respectively of the linear tting [R²¼ 0.9997] of the data are higher binding constant of C5-PTZ than that for PTZ species

found to be 0.35 and 6.19 ꢄ 10³M^ꢂ1, respectively.

the binding of the former at the more hydrophobic site of the

Next, we dealt with the C5-PTZ, possibly more hydrophobic, BSA. Thus, the presence of a hydrocarbon tail enables the dye

as compared to PTZ due to the introduction of the pentyl chain. binding to a comparatively more hydrophobic site.³⁹

Notably, C5-PTZ is also non-uorescent like PTZ. The parame-

The next dye, C5-PTZA, with aldehyde functionality (Chart 1),

ters listed in Table 1 support the energy transfer as a reason for is found to be highly uorescent, unlike PTZ and C5-PTZ. Upon

uorescence quenching of Trp residues in the presence of C5- excitation at 295 nm, the uorescence from both Trp residues of

PTZ as a quencher. Fluorescence spectra of Trp residues of BSA BSA and C5-PTZA have been recorded at diﬀerent concentra-

at diﬀerent concentrations of C5-PTZ are shown by Fig. 5a, and tions of the latter, and the corresponding uorescence spectra

Fig. 5b displays the change in uorescence intensity with with an isoemissive point at 445 nm are shown by Fig. 6a.

increasing concentration of C5-PTZ based on eqn (2). The

Fig. 6a illustrates a gradual decrease in the uorescence

observed linear S–V plot (R²¼ 0.9995) indicates both the Trp intensity of the Trp residues (donor) of BSA with a concomitant

residues are equally accessible to the dye, C5-PTZ. Unlike PTZ, increase in the uorescence intensity of C5-PTZA (as an

C5-PTZ with a pentyl tail in the molecule can penetrate the acceptor) through the FRET process with increasing concen-

protein and bind to a comparatively interior site, and that tration of C5-PTZA. This trend is well displayed in Fig. 6b, which

facilitates equal accessibility of both the Trp residues.

shows the rise and fall in F/F_oof the acceptor and the donor,

The S–V constant, K_SV(¼ k_qs_o) calculated from the linear respectively. Detailed scrutiny revealed that the peak maximum

tting of the quenching data (Fig. 5b) is found to be 3.35 ꢄ 10⁴of Trp uorescence is blue-shied from 347 nm to 344 nm upon

M^ꢂ1. Taking s_o¼ 10^ꢂ8s, the k_qis calculated to be 3.35 ꢄ 10¹²the increasing concentration of C5-PTZA within the range of

M^ꢂ1s^ꢂ1. As the k_qvalue lies beyond the diﬀusion-controlled 0 mM to 15 mM. There could be two possible reasons for it. First,

limit, thus it is a static quenching. The binding constant (K⁰) the shi of peak maximum to a shorter wavelength with

and the number of binding sites, n calculated aer linear tting increasing C5-PTZA concentration could be due to the fact that

[Fig. S2,† R²¼ 0.9997] of the quenching data based on eqn S(3),† the Trp uorescence at a longer wavelength is getting quenched

are found to be 2.46 ꢄ 10⁴M^ꢂ1and 0.73, respectively. The more readily than that at a shorter wavelength.³⁵Trp-134 being

Fig. 6 (a) Fluorescence spectra of Trp residues of BSA and C5-PTZA at varying concentrations of C5-PTZA, and (b) decrement in ﬂuorescence

intensity of donor (Trp in BSA) with concomitant enhancement in ﬂuorescence intensity of acceptor (C5-PTZA) with increasing concentration of

C5-PTZA in the FRET process. [BSA] ¼ 5 mM. l_ex¼ 295 nm.

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Fig. 7 (a) S–V plot for quenching of the Trp ﬂuorescence by C5-PTZA in BSA, and (b) modiﬁed S–V plot for quenching of the Trp ﬂuorescence by

C5-PTZA in BSA. [BSA] ¼ 5 mM. l_ex¼ 295 nm.

present in a comparatively hydrophilic environment should (Fig. S4,† R²¼ 0.9999) of the data based on eqn S(3)† are found

have uorescence at a relatively longer wavelength as compared to be 1.30 ꢄ 10⁵M^ꢂ1and 1.06, respectively. The higher value of

to Trp-213. Thus as compared to Trp-213, Trp-134 is more binding constant for C5-PTZA as compared to C5-PTZ infers

accessible to C5-PTZA. Secondly, it could also be due to the that –CHO functionality in C5-PTZA induces added binding

changes in secondary structure of BSA in presence of a dye. To interactions with the protein.

verify it, the circular dichroism spectra for BSA in presence of

Subsequently, another dye, C5-PTZCN (Chart 1), has been

diﬀerent concentrations of C5-PTZA have been recorded and used with much higher electron-accepting capability due to the

given as Fig S3.† The % a-helix of the native BSA is found to be presence of di-cyano functionality linked through a p-bond.

changed from 62.7% to 55.4%.

Fig. 8a shows changes in uorescence spectra of both donor and

The S–V plot, in this case, exhibits an upward curvature, acceptor parts with increasing concentration of C5-PTZCN with

concave towards the y-axis at higher concentrations of the an isoemissive point at ꢀ447 nm. Like the previous dye, C5-

quencher (C5-PTZA) (Fig. 7a). This trend can be well described PTZA, a gradual diminution in the uorescence intensity of the

by using concurrent static and dynamic quenching mecha- Trp residues of BSA with a simultaneous rise in the uorescence

nisms.³⁵The modied S–V equation for a quenching process intensity of C5-PTZCN are presented in Fig. 8b.

through both static and dynamic quenching can be written as

The blue shi in the uorescence peak maximum of BSA is

eqn S(4) in Note S3.† which can be further modied through due to the same reason, as discussed in the case of C5-PTZA.

eqn S(5) and S(6).†

The S–V plot for this quenching of uorescence presents

Fig. 7b presents the modied S–V plot (R²¼ 0.9999) based on a downward curvature (Fig. 9a). The modied S–V plot based on

eqn S(6).† Then, by solving the quadratic equation, we get the eqn S(1)† is shown in Fig. 9b (R²¼ 0.9999).

values of the S–V dynamic quenching constant (K_D) as 5.96 ꢄ

The fractional accessibility of the dye to the Trp residues of

10⁴M^ꢂ1and the S–V static quenching constant (K_S) as 4.02 ꢄ BSA indicated by the downward curvature in the S–V plot leads

10⁴M^ꢂ1. Assuming that eqn S(3)† is applicable in this case as us to calculate f_aand K_afrom the intercept and the slope of the

static quenching is in place, the binding constant (K⁰) and the plot in Fig. 9b based on eqn S(1)†. The f_aand K_avalues are

number of binding sites (n) calculated from the linear tting estimated to be 0.87 and 2.06 ꢄ 10⁵M^ꢂ1, respectively. So, 87%

Fig. 8 (a) Fluorescence spectra of Trp residues of BSA and C5-PTZCN with increasing concentration of the dye, and (b) decrement in ﬂuo-

rescence intensity of donor (Trp residues in BSA) with concomitant enhancement in ﬂuorescence intensity of acceptor (C5-PTZCN) with

increasing concentration of C5-PTZCN in the FRET process. [BSA] ¼ 5 mM. l_ex¼ 295 nm.

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Fig. 9 (a) S–V plot for quenching of Trp ﬂuorescence by C5-PTZCN in BSA, and (b) modiﬁed S–V plot for quenching of the Trp ﬂuorescence by

C5-PTZCN in BSA. [BSA] ¼ 5 mM. l_ex¼ 295 nm.

of Trp residues of BSA are accessible to the C5-PTZCN. The based on eqn S(1)† was scrutinized (R²¼ 0.9965) to nd the

bimolecular quenching constant, k_q¼ 2.06 ꢄ 10⁵M^ꢂ1/10^ꢂ8s ¼ fractional accessibility (f_a), as well as the S–V quenching

2.06 ꢄ 10¹³M^ꢂ1s^ꢂ1, which is beyond the diﬀusion-controlled constant (K_a) those, are found out to be 0.85 and 3.59 ꢄ 10⁴

limit. Thus, the mechanism of quenching appears to be static. M^ꢂ1. So, 85% of Trp residues of BSA are accessible to the C5-

The binding constant (K⁰) and the number of binding sites (n) PTZDCA. The quenching rate constant, k_qis found to be 3.59 ꢄ

calculated from the linear tting (Fig. S5,† R²¼ 0.9999) of the 10¹²M^ꢂ1s^ꢂ1, reecting the static quenching mechanism. The

quenching data based on eqn S(3)† are found to be 1.21 ꢄ 10⁵respective values of binding constant (K⁰) and a number of

M^ꢂ1and 0.63, respectively. Of note, the binding of C5-PTZCN is binding sites (n) calculated from the linear tting of the data in

almost the same as that of C5-PTZA.

Fig. S6† (R²¼ 0.9999) based on eqn S(5)† are calculated to be

Besides, the new dye studied in this series is C5-PTZDCA 2.81 ꢄ 10⁵M^ꢂ1and 0.97.

(Chart 1), dicyanoaniline (DCA) moiety attached to the C5-PTZ

3.2.2 FRET between BSA and anthracenyl compounds.

core. Fig. 10a shows the uorescence spectra of donor–acceptor Similar studies, as discussed above, have also been carried out

pair with increasing concentration of the acceptor, C5-PTZDCA, with an anthracenyl series of molecules given in Chart 1. At the

with an isoemissive point at ꢀ403 nm. The dye, C5-PTZDCA, very beginning, we took a representative example of FRET using

with a strong acceptor–donor–acceptor unit along with a methyl the ANTA molecule. Electron-rich molecular anthracene, ANT,

group, is found to be highly uorescent. The gradual decrease does not act as an acceptor in the FRET process with BSA as

in the uorescence intensity of the Trp residues of BSA with a donor. However, each of its derivatives in the series, such as

a simultaneous rise in the uorescence intensity of C5-PTZDCA ANTA, ANTCN, and ANTDCA with signicant electron-

is represented by Fig. 10b. The blue-shi in the emission peak withdrawing substituents, has shown the FRET phenomenon

maximum of BSA indicating fractional accessibility of Trp as an acceptor. In each case, the energy transfer is responsible

residues and/or changes in secondary structure of BSA is noted for the uorescence quenching of Trp residues of BSA, as sup-

in this case as well.

ported by data given in Table 1.

The S–V plot for this system also displayed a slight down-

Fluorescence quenching of Trp residues by ANTA (a weak

ward curvature (Fig. 11a). The modied S–V plot (Fig. 11b) uorescent molecule) through the FRET process has been

Fig. 10 (a) Fluorescence spectra of Trp residues of BSA and C5-PTZDCA with increasing concentration of the dye, and (b) dropping in ﬂuo-

rescence intensity of donor (Trp in BSA) with concomitant enhancement in ﬂuorescence intensity of acceptor (C5-PTZDCA) with increasing

concentration of C5-PTZDCA in the FRET process. [BSA] ¼ 5 mM. l_ex¼ 295 nm.

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Fig. 11 (a) S–V plot for quenching of the Trp ﬂuorescence by C5-PTZDCA in BSA, and (b) modiﬁed S–V plot for quenching of the Trp ﬂuo-

rescence by C5-PTZDCA in BSA. [BSA] ¼ 5 mM. l_ex¼ 295 nm.

Fig. 12 (a) Fluorescence spectra of Trp residues of BSA and ANTA with increasing concentration of ANTA, and (b) decrement in ﬂuorescence

intensity of donor (Trp residues in BSA) with concomitant enhancement in ﬂuorescence intensity of acceptor (ANTA) with increasing

concentration of ANTA in the FRET process. [BSA] ¼ 5 mM. l_ex¼ 295 nm.

shown in Fig. 12a. On the gradual treatment of ANTA, the applicable for concurrent static and dynamic quenching

uorescence intensity for Trp residues of BSA is decreased with mechanism, the quenching data were plotted. Still, we failed to

an increase in uorescence intensity of ANTA, showing an iso- t the data to the said equation. It led us to follow the sphere-of-

emissive point at ꢀ462 nm (Fig. 12a).

action model as an alternate mechanism of the static part of

An upward curvature noted in the S–V plot (Fig. 13a) indi- quenching. The modied S–V equation for the sphere-of-action

cates either a combined dynamic and static mechanism of model is written as eqn S(7) in Note S4.†³⁵Further modication

quenching or static quenching with the sphere-of-action (Per- done has been shown through eqn S(8) to S(10) and has been

rin) model. Based on a modied S–V equation (eqn (S6)†) discussed in Note S4.†⁴⁰

Fig. 13 (a) S–V plot for quenching of Trp ﬂuorescence of BSA by ANTA, (b) plot of (1ꢂ (F/F_o))/[ANTA] versus F/F_o,and (c) modiﬁed S–V plot for

quenching of the Trp ﬂuorescence of BSA by ANTA. [BSA] ¼ 5 mM. l_ex¼ 295 nm.

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Fig. 14 (a) Fluorescence spectra of Trp residues of BSA and ANTCN with increasing concentration of ANTCN, and (b) S–V plot for quenching of

Trp ﬂuorescence of BSA by ANTCN. [BSA] ¼ 5 mM, l_ex¼ 295 nm.

Fig. 15 (a) Fluorescence spectra of Trp residues in BSA with increasing concentration of ANTDCA., and (b) lowering in ﬂuorescence intensity of

donor (Trp in BSA) with concomitant enhancement in ﬂuorescence intensity of acceptor (ANTDCA) with increasing concentration of ANTDCA in

the FRET process. [BSA] ¼ 5 mM. l_ex¼ 295 nm.

Based on eqn S(10),† the values of K_Dand V have been quenching data have been plotted based on the actual S–V

calculated from the slope and intercept, respectively by linear equation for the sphere-of-action model (eqn S(7)†) and shown

tting (R²¼ 0.9988) of the data in the plot of (1 ꢂ (F/F_o))/[ANTA] by Fig. 13c. The S–V dynamic quenching constant was calcu-

versus F/F_oshown by Fig. 13b. The values of K_Dand V are found lated from the slope of the linear tting (R²¼ 0.9996) of the data

to be 1.79 ꢄ 10⁴M^ꢂ1and 1.27 ꢄ 10^ꢂ17liters, respectively. The in the plot of F_o/F(e^VN^[ANTA]/1000) versus [ANTA] based on eqn

AV

radius of the sphere of action is calculated to be 144 nm. Aer S(7).† The K_Dvalue is found to be 1.94 ꢄ 10⁴M^ꢂ1, which is

calculating the approximate value of V with an assumption of diﬀerent from that calculated from the plot in Fig. 13b because

the low value of the term, VN_AV[ANTA]/1000 in eqn S(8),† the of the assumption involved in the derivation of corresponding

Fig. 16 (a) S–V plot for quenching of the Trp ﬂuorescence in BSA by ANTDCA, and (b) modiﬁed S–V based on eqn S(3).† [BSA] ¼ 5 mM, l_ex

¼

295 nm.

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eqn S(10).† Assuming that the fraction of molecules presents

within the sphere of action at the time of excitation is non-

uorescent and undergoing static uorescence quenching,

eqn S(3)† has been used to calculate the binding constant (K⁰)

and the number of binding sites (n) upon linear tting of the

data in Fig. S7† (R²¼ 0.9999). The values of K⁰and n are found to

be 3.95 ꢄ 10⁴M^ꢂ1and 1.31.

Next, ANTCN with conjugated dicyano substituents is also

non-uorescent. The uorescence from Trp residues of BSA in

the presence of diﬀerent concentrations of ANTCN is shown in

Fig. 14a. The radiative rate constant (k_r) and oscillator strength

(f) of the donor in the absence and presence of ANTCN and %

quenching data given in Table 1 support the energy transfers as

a cause for the quenching of Trp uorescence. It has been dis-

cussed above that energy transfer is possible even if the acceptor

is non-uorescent. A linear S–V plot (R²¼ 0.9995) for the Trp

uorescence quenching is obtained and displayed in Fig. 14b.

The value of S–V quenching constant, K_SVcalculated from the

slope of the plot is found to be 3.13 ꢄ 10⁴M^ꢂ1, with a corre-

sponding uorescence quenching rate constant (k_q) value as

3.13 ꢄ 10¹²M^ꢂ1s^ꢂ1. A k_qvalue beyond the diﬀusion-controlled

limit denotes the static quenching mechanism. The binding

constant (K⁰) of ANTCN with BSA and the number of binding

sites (n) have been calculated from the linear tting (R²

¼

0.9999) of the data in Fig. S8 based on eqn S(3)† and the values

are found to be 3.15 ꢄ 10⁴M^ꢂ1and 1.10, respectively. Thus, the

binding constant of ANTCN is very close to that of ANTA, as

noticed in the case of C5-PTZ analogs.

With our interest in studying a new electron-rich donor–

acceptor–donor (D–A–D) molecular system, ANTDCA molecule

is considered here for the FRET with BSA. There is a consider-

able amount of FRET with an isoemissive point at ꢀ409 nm

(Fig. 15a). The blue-shi in the emission spectrum of Trp resi-

dues of BSA is also observed as discussed above. A trend of

decrease in the uorescence intensity of Trp residues of BSA

and an increase of the uorescence intensity of the dye,

ANTDCA, are represented by Fig. 15b.

Based on the quenching of the BSA–Trp uorescence by

ANTDCA, the S–V plot is identied with slight downward

curvature (Fig. 16a). Therefore, quenching data have plotted

based on modied S–V eqn S(1)† and are shown by Fig. 16b (R²

¼ 0.9997). The values of fractional accessibility (f_a) and the S–V

quenching constant (K_a) are calculated to be 0.25 and 5.85 ꢄ 10⁵

M^ꢂ1, respectively. So, 25% of Trp residues in BSA are only

accessible to the quencher, ANTDCA. The quenching rate

constant (k_q) is found to be 5.85 ꢄ 10¹³M^ꢂ1s^ꢂ1, indicating

a static quenching mechanism. The binding constant (K⁰) and

a number of binding sites (n) are found to be 2.38 ꢄ 10⁴M^ꢂ1

and 0.63, respectively [calculated from the linear tting

(Fig. S9,† R²¼ 0.9998) of the data based on eqn S(3)].†

¨

3.2.3 FRET parameters. The Forster's distance (R_o), the

eﬃciency of energy transfer (E_T) and the distance (r) between an

acceptor quencher dye and donor Trp residues of BSA have been

calculated to create a quantitative analysis of FRET between Trp

residues of BSA and diﬀerent dyes.³⁶Details of calculation of

these parameter along with the calculation of the overlap inte-

gral, J(l) have been given in ESI as Note S5.† It is pertinent to

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note that the presence of the two donor moieties needs the

assumption of an average distance between donors and accep-

tors.³Also, even though the donor is covalently attached to the

protein, but the acceptor dyes are not. However, the high

binding constant of each dye with the BSA reduces the possi-

bility of any high uncertainty allowing us to estimate the average

distance between a donor and an acceptor. It is noteworthy that

all FRET calculations have been done with a ratio of concen-

tration of a dye to BSA of 1 : 1 taking 5 mM concentration of

each. With this concentration of each of the donors and the

acceptors, the intensity ratios, [Int_acceptor/Int_donor] are found to

vary from 0.05 to 0.72. Lowest intensity ratio is found in case of

ANTA. However, its relative quantum yield is calculated to be

0.08 which is not very low so that it could contribute a signi-

cant error in calculations. The intensity ratios mentioned above

are increased with increasing concentration of a dye further

reducing the chances of errors in the quenching data.

The overlap between the corrected uorescence spectrum of

Trp residues of BSA as donor and absorption spectrum of C5-

PTZA as acceptor has been shown as a representative one in

Fig. 2. Similar overlapping has also been noted in cases of all

other dyes for which overlap integral values have been calculated.

The calculated FRET parameters are given in a tabular form

in Table 2. The R_ovalues measured are in the range of 24.2–29.4

ꢀ

A, which corroborates well with the range (20–90 A) for FRET to

occur.³Thus, the data reect that dyes are binding close to the

Trp residues of BSA. In addition, the band gaps for all dyes have

been calculated based on the data obtained from CV experi-

ments to compare the E_Tvalues of the dyes in phenothiazine

and anthracenyl series (see Fig. S10† for details). To further

support the band gaps data, the absorption peak maxima

(l^m_ab^a_s^x) of all dyes have been determined. The band gaps and

l^m_ab^a_s^xvalues along with l^m_^axvalues are also tabulated in Table 2.

In order to have an overall discussion on the quenching

mechanism, energy transfer processes with diﬀerent dyes as an

acceptor, and binding aﬃnities of dyes with the BSA, the

quenching data are compiled in Table 3. Few FRET parameters

and band gap data tabulated in Table 2 have been used. It can

be seen that the quenching mechanism is apparently static. In

phenothiazine series, combined static and dynamic quenching

has been noted only in the case of C5-PTZA. Surprisingly,

a combined quenching mechanism has been shown only by

ANTA among all dyes in anthracenyl series following a sphere of

action model of the static part of quenching in this case. Thus,

–CHO functionality has some connection with the combined

quenching mechanism, possibly because of the interaction

between the –CHO group and some active functional group of

amino acids of BSA, such as the –NH functionality of Trp resi-

dues. The binding aﬃnities of dyes in phenothiazine series are

stronger than the corresponding dyes in anthracenyl series

could be due to the presence of pentyl tail as well as hetero

atoms inducing a greater extent of hydrophobic as well as

hydrogen bonding and coulombic attractive interactions in the

former than that in the latter. A higher binding constant value

of C5-PTZ as compared to PTZ is due to the presence of a pentyl

tail that enhances the hydrophobicity in the former. Among all

dyes, including phenothiazine and anthracenyl series, the

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energy transfer eﬃciency, E_Tis found to be maximum in the also noticed the expected trend [C5-PTZ < PTZ < C5-PTZDCA <

case of C5-PTZCN (E_T¼ 0.45), proving itself as the most eﬃcient C5-PTZA < C5-PTZCN] in the increase of l^m_ab^a_s^xof pure dyes. The

ꢀ

FRET acceptor with a distance of 28.8 A from the donor, which increase in E_Twith decreasing the band gap except for C5-

is minimum among all other donor–acceptor distances. The PTZDCA can be described with the molecular structural factor,

next dye which shows E_Tclosest to C5-PTZCN is C5-PTZA, with dominating over the electronic factor.

an E_Tof 0.35. The binding constants for C5-PTZCN and C5-PTZA

As far as anthracenyl series are concerned, their binding

with BSA are found to be very much comparable with the value aﬃnities with BSA are lower than that for dyes in phenothiazine

of 1.21 ꢄ 10⁵and 1.30 ꢄ 10⁵M^ꢂ1, respectively. Diﬀerent series as described above. Due to the absence of pentyl tail in

substituents present in these two dyes must be responsible for anthracenyl series of dyes, they probably bind to the compara-

the diﬀerence in E_T. The presence of more potent electron- tively less hydrophobic/interior sites of the protein. That is why

withdrawing functionalities (two –CN groups) for C5-PTZCN the average distances between the donor and each of dyes,

ꢀ

facilitates the compound to be a more eﬃcient acceptor in ANTA (35.2 A) and ANTCN (36.4 A) are longer than the corre-

ꢀ

comparison to C5-PTZA that carries one electron-withdrawing sponding above-mentioned analogs, C5-PTZA (32.6 A) and C5-

ꢀ

–CHO group. The presence of an electron-withdrawing cyano- PTZCN (28.8 A), with eventually smaller E_Tvalues (Table 2).

ester group in a molecule makes it an eﬃcient acceptor, While the respective E_Tfor C5-PTZA and C5-PTZCN are 0.35 and

which has been reported by Vijayakumar et al.⁴¹In phenothia- 0.45, the E_Tfor ANTA and ANTCN are only 0.10 and 0.11,

zine series, the lowest E_Tis observed for C5-PTZDCA (E_T¼ 0.12) respectively. Now, if we compare between the other two new

though it has got the highest binding constant with BSA (K⁰¼ dyes, C5-PTZDCA and ANTDCA, the fact that the binding

2.81 ꢄ 10⁵M^ꢂ1). A high value of the binding constant of this dye constant of ANTDCA is almost one-tenth of that of C5-PTZDCA,

and high accessibility (85%) of Trp residues to this dye imply the former one binds at the comparatively less hydrophobic

that the dye binds to the interior of the protein, perhaps with region, and as a result, its fractional accessibility to the Trp

a drive of additional hydrophobic interaction due to the pres- residues is lesser than that of the latter one. However, inter-

ence of a benzene ring and hydrogen bonding and coulombic estingly the E_Tof ANTDCA is higher than that of C5-PTZDCA,

interactions with the BSA as a result of the presence of diﬀerent and it can be deciphered by the diﬀerence in electronic and

functional groups such as –NH₂and –CN. However, the lowest structural factors between these two systems. Anthracene is

E_Tfor this dye can be attributed to the attachment of electron- a at polyaromatic hydrocarbon without any heteroatoms,

donating groups such as amine and methyl that can signi- whereas phenothiazine is a middle ring folded electron-rich

cantly enrich the electron density to the molecule and hamper core with two specic electron donor atoms such as nitrogen

the accepting ability of cyanobenzene.⁴²Also, the reason due to and sulfur. In our recent research, phenothiazine appears to be

the presence of acceptor moiety, cyano benzene at a distant more electron-rich compared to anthracene.⁴³From the

position as compared to other dyes cannot be ruled out. This is molecular structure (Fig. 1), the torsion angle between ANT ring

further supported by the longest r (¼35.6 A, Table 2) in this case. and DCA ring is measured to be ꢀ79^ꢁ, whereas the torsion angle

ꢀ

It indicates that the phenothiazine ring with pentyl tail is between the C5-PTZ ring and DCA ring is only ꢀ38^ꢁ. Thus,

aligned towards the protein's hydrophobic interior, and the a greater extent of charge transfer in ANTDCA might be favor-

substituted benzene ring is protruded towards the outer able for it to be a better acceptor than C5-PTZDCA resulting in

hydrophilic site. Moreover, the molecular structure depicts that a higher E_Tin FRET.⁴⁴Moreover, as the DCA is attached to the

the dicyanoaniline part is tilted with ꢀ38^ꢁtorsion angle with C5 carbon of ANTDCA rather than the C2 carbon of C5-PTZDCA,

folded (ꢀ41^ꢁfolded middle ring) phenothiazine core (Fig. 1). On the former one remains closer to the donor in BSA (r ¼ 30.9) as

the other hand, C5-PTZCN without any such DCA ring present compared to the latter (r ¼ 35.6). The increasing order of E_Tof

in the structure shows maximum E_T,and almost the same dyes in anthracenyl series is found to be as follows: ANTA <

accessibility (87%) as that is found for C5-PTZDCA that oﬀers ANTCN < ANTDCA (Table 2), which is correlated agreeably with

ꢀ

the shortest average distance (28.8 A) from the Trp residues of the decreasing order of band gaps (Table 2) i.e. ANTA > ANTCN >

max

abs

BSA. The lack of electron-withdrawing substituents for PTZ and ANTDCA while the increasing order of l

of pure dyes is

C5-PTZ compounds can attribute to lower E_Tas compared to C5- established as (Table 2): ANTA < ANTCN < ANTDCA. In contrast

PTZA and C5-PTZCN. Quenching data also dictate that the to C5-PTZDCA, ANTDCA shows the anticipated energy transfer

binding constants for PTZ and C5-PTZ are comparatively lower eﬃciency from the order of band gap values in anthracenyl

than the other three dyes in this series. So, the functional series. It depicts that the electronic factor for ANTDCA plays an

groups present in the other three dyes feasibly undergo some important role.

kind of binding interactions with the protein. To check whether

From the results of fractional accessibilities to the four dyes,

the E_Thas any relation with the band gap of a dye, the band gap PTZ, C5-PTZCN, C5-PTZDCA, and ANTDCA, it can be seen that

values of all dyes have been calculated (Table 2) from CV the % accessibilities are high and almost same for C5-PTZCN

measurements (Fig. S10† displays CV diagrams for pure dyes). and C5-PTZDCA, 87% and 85%, respectively. Thus these two

The increasing E_Torder for the dyes of phenothiazine series dyes bind to a site from where both the Trp residues are quite

(Table 2) is found with the following trend: C5-PTZDCA < C5- accessible. The binding sites must be in the hydrophobic

PTZ < PTZ < C5-PTZA < C5-PTZCN. However, the decreasing region; otherwise, Trp-213 would not be accessible. For the

order of band gap is as follows (Table 2): C5-PTZ > PTZ > C5- other two dyes, PTZ and ANTDCA fractional accessibilities are

PTZDCA > C5-PTZA > C5-PTZCN. With decreasing band gap, we only 43% and 25%, respectively. Therefore, they bind to

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hydrophilic sites. Thus, hydrophobic substitution on dyes campus, for helping in the CV analysis. CAL lab facilities

makes them more accessible to the Trp residues present in available at BITS-Pilani Hyderabad is acknowledged.

a hydrophobic pocket. Earlier, Karukstis et al.⁴⁴also noted the

higher fraction of accessibility of Chlorophyll uorescence by

hydrophobic substitution of naphthoquinones. Linear S–V plot

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the FRET parameters have been estimated. However, as

a strategy, the variable mechanisms of quenching in presence of

diﬀerent dyes as quenchers as noted aer a detailed analysis of

the quenching data have been described.

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8 L. Wu, C. Huang, B. P. Emery, A. C. Sedgwick, S. D. Bull,

X. P. He, H. Tian, J. Yoon, J. L. Sessler and T. D. James,

A few easily accessible, structurally and electronically diverse

anthracene and phenothiazine dyes are explored for binding-

studies with BSA. FRET between Trp residues of BSA and the

dyes, mechanism of uorescence quenching, and their possible

locations are examined to deduce a structure–property rela-

tionship. The binding eﬃciency of phenothiazine dyes with

a pentyl tail is stronger than that of anthracenyl dyes due to the

presence of a hydrocarbon tail and also hetero atoms in the

former inducing hydrophobic as well as hydrogen bonding and

coulombic attractive interactions. Energy transfer between Trp

residues of BSA as a donor and each dye as an acceptor is found

to be responsible for uorescence quenching. Energy transfer

has been noted even in cases of non-uorescent acceptors, PTZ,

C5-PTZ, and ANTCN. The mechanism of quenching is static.

Concurrent static and dynamic mechanisms have been distin-

guished only in cases of C5-PTZA and ANTA with a sphere of

action model of the static part of quenching for the latter dye.

Dyes with a hydrophobic entity (pentyl tail herein) present in

their structures bind with the hydrophobic sites of protein,

showing better energy acceptors. Although the binding constant

is more signicant for C5-PTZDCA than ANTDCA, the favored

energy transfer for ANTDCA could be due to its relatively higher

electron-transfer capabilities within the molecules and lower

acceptor–donor distances. The band gap values of all the dyes

are also very much supportive of the observed behavior. Thus,

the hydrophobic character and the presence of interactive

substituents along with the electron-accepting abilities majorly

control the FRET for such acceptor molecules with BSA.

¨

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Conﬂicts of interest

The authors declare no conict of interest.

Acknowledgements

˘

14 E. Yalçin, Y. C. Kutlu, V. Korkmaz, E. ¸Sahin and Z. Seferoglu,

SB and MC thank CSIR 02(-0289)/17/EMR-II for the nancial

assistance. The authors thank Susmita Roy, PhD Research

Scholar, Department of Chemistry, BITS Pilani, Hyderabad

2,6-Dicyanoaniline based donor–acceptor compounds: the

facile synthesis of uorescent 3,5-diaryl/hetaryl-2,6-

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RSC Adv., 2021, 11, 1679–1693 | 1693

Article Doi

Binding interactions and FRET between bovine serum albumin and various phenothiazine-/anthracene-based dyes: A structure-property relationship

DOI: 10.1039/d0ra09580j

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Article abstract of DOI:10.1039/d0ra09580j

Full text of DOI:10.1039/d0ra09580j

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