Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process in a new derivative 2-Hydroxy-N-(2-phenylethyl)benzamide – Study on the effects of pH and medium polarity changes

RESEARCH ARTICLE

Spectroscopic and theoretical studies of

fluorescence effects induced by the ESIPT

process in a new derivative 2-Hydroxy-N-(2-

phenylethyl)benzamide – Study on the effects

of pH and medium polarity changes

2

3

4

Agnieszka Niemczynowicz ¹, Iwona Budziak , Sławomir Kulesza , Andrzej Gorecki ,

´

I D

Marcin Makowski⁵, Dariusz Karcz⁶, Karolina Starzak ⁶, Bożena Gładyszewska⁷,

a 1111111111

I D

Janusz Podleśny ⁸, Agnieszka I. Piotrowicz-Cieślak⁹, Arkadiusz Matwijczuk

7

I D

*

I D

1 Department of Analysis and Differential Equations, Faculty of Mathematics and Computer Science,

University of Warmia and Mazury, Olsztyn, Poland, 2 Department of Chemistry, University of Life Sciences in

Lublin, Lublin, Poland, 3 University of Warmia and Mazury in Olsztyn, Faculty of Mathematics and

Informatics, Chair of Relativistic Physics, Olsztyn, Poland, 4 Department of Physical Biochemistry, Faculty of

´

Biochemistry, Biophysics and Biotechnology of the Jagiellonian University, Krakow, Poland, 5 Department of

Theoretical Chemistry, Faculty of Chemistry, Jagiellonian University, Krakow, Poland, 6 Department of

´

Analytical Chemistry (C1), Faculty of Chemical Engineering and Technology, Cracow University of

Technology, Cracow, Poland, 7 Department of BioPhysics, University of Life Sciences in Lublin, Lublin,

Poland, 8 Institute of Soil Science and Plant Cultivation—State Research Institute, Puławy, Poland,

9 Department of Plant Physiology, Genetics and Biotechnology, Faculty of Biology and Biotechnology,

University of Warmia and Mazury in Olsztyn, Olsztyn, Poland

OPEN ACCESS

Citation: Niemczynowicz A, Budziak I, Kulesza S,

´

Gorecki A, Makowski M, Karcz D, et al. (2020)

Spectroscopic and theoretical studies of

fluorescence effects induced by the ESIPT process

in a new derivative 2-Hydroxy-N-(2-phenylethyl)

benzamide – Study on the effects of pH and

medium polarity changes. PLoS ONE 15(2):

e0229149. https://doi.org/10.1371/journal.

pone.0229149

* arkadiusz.matwijczuk@up.lublin.pl

Abstract

The paper presents the results of studies conducted with the use of stationary and time-

resolved fluorescence spectroscopy for the new derivative 2-Hydroxy-N-(2-phenylethyl)

benzamide (SAL-3) in aqueous solutions with various concentrations of hydrogen ions as

well as in solvent mixtures (i.e. media with changing polarity/polarizability). For the com-

pound selected for the study placed in aqueous solutions with varying concentrations of

hydrogen ions, the fluorescence emission spectra revealed a single emission band within

most of the pH range, however, at low pH (pH<3) a significant broadening (noticeable effect

of dual fluorescence) and shifting of the band was observed. Whereas, for water and polar

(protic) solvents, we observed a very interesting phenomenon of dual fluorescence never

before reported for this particular group of analogues (with the specific substituent system).

Based on the results of the experiments, it was observed that the presented effects may be

related both with conformational effects (related to the possible positioning of the–OH group

on the side of the carbonyl system, which facilitates the possibility of proton transfer) as well

as, most importantly, the effects of excited state intramolecular proton transfer (ESIPT–

Excited State Intramolecular Proton Transfer) related in this case with the necessary (new/

previously unobserved in published literature) presence of ionic and non-ionic forms of the

compound). Both the conducted quantum-mechanical [TD]DFT–Time-Dependent Density

Functional Theory) calculations and excited state dipole moment change calculations for

Editor: Danilo Roccatano, University of Lincoln,

UNITED KINGDOM

Received: October 2, 2019

Accepted: January 30, 2020

Published: February 25, 2020

open access article distributed under the terms of

the Creative Commons Attribution License, which

permits unrestricted use, distribution, and

reproduction in any medium, provided the original

author and source are credited.

Data Availability Statement: All relevant data are

within the manuscript and its Supporting

Information files.

Funding: This research was supported in part by

PL-Grid Infrastructure. The research was carried

out with the equipment purchased thanks to the

financial support of the European Regional

Development Fund in the framework of the Polish

PLOS ONE | https://doi.org/10.1371/journal.pone.0229149 February 25, 2020

1 / 28

Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

Innovation Economy Operational Program

(contract no. POIG.02.01.00-12-023/08). The

author Arkadiusz Matwijczuk acknowledges the

Cost project CA 15126.

the analyzed molecule in solvents with varying pH confirmed the association between the

observed fluorescence phenomena and the two aforementioned effects.

Competing interests: The authors have declared

that no competing interests exist.

1. Introduction

Salicylic acid (SA) and its derivatives (phenylpropanoids) are commonly found in plants. Met-

abolically, their changes are related to the plant’s defensive reaction to biotic and abiotic stress

[1]. The metabolism of phenylpropanoids shows high specificity and the respective paths of its

synthesis are activated under very specific conditions, in specific phases of cell differentiation

or under the influence of very specific environmental stimuli. Salicylic acid serves as the trans-

mitter in plant defensive reactions, it can be synthesized based via two separate and unrelated

synthesis paths taking advantage of different precursors: phenylalanine and the isochorismic

path present in chloroplasts [2].

Phenylpropanoids are commonly used against various pathogens. Some derivatives of sali-

cylic acid, such as 2-hydroxy-N-(arylalkyl)benzamides, show antimicrobial activity against

microscopic fungi and various bacterial strains, including mycobacteria [3], whereas several

other compounds are capable of inhibiting the proliferation and vitality of human cancer cells

[4]. The compound selected for the present study, 2-hydroxy-N-(2-phenylethyl)benzamide,

demonstrated 20% growth inhibition of Mycospharella pinodes species. Mycosphaerella blight

caused by Mycosphaerella pinodes (Berk. et Blox. Vestergr) is one of the main diseases encoun-

tered in pea cultivations [5]. It occurs commonly in the fields of Europe, North America, Aus-

tralia and New Zealand [6] and, on average, leads to a 10% decrease in yields, whereas in years

when the temperature and humidity conditions are particularly unfavorable, yield losses can

exceed 50% [5]. Limiting the incidence of the disease requires the use of adequate crop protec-

tion products. Hence, it is important to discover the photochemical/photophysical properties

of 2-hydroxy-N-(2-phenylethyl)benzamide in order to use it as a viable crop protection agent.

Due to its considerable potential related to the aforementioned properties, 2-hydroxy-N-

(2-phenylethyl)benzamide (SAL-3 –Fig 1A–1C) was selected for our study on the mechanisms

of molecular interactions. It was observed that the selected derivative of SA produces very

interesting effects of dual fluorescence whose mechanism in the selected new derivatives has

not been researched or associated with various other analogues already described in literature

to date [7–9]. Identifying the exact mechanism of dual fluorescence in the selected analogue

may prove significant to explaining the mechanisms of molecular interactions taking place in

both itself and the entire group of new SA derivatives [10–13].

The aim of this study was to conduct spectroscopic and theoretical analyses of the molecu-

lar organization present in SAL-3 in aqueous solutions within the entire range of hydrogen

ions’ concentrations as well as in selected organic solvents and in mixtures thereof (with

water). In this context, the primary aim of the study was to identify the molecular mechanism

and propose the theoretical model for the effects of dual fluorescence observed in the fluores-

cence emission spectra of this derivative. By employing spectroscopic methods such as: elec-

tronic absorption and fluorescence spectroscopy (including the RLS—Resonance Light

Scattering) technique [14, 15], fluorescence lifetime measurements (TCSPC—Time-Correlated

Single Photon Counting), supported by M. Kasha’s exciton splitting theory [16], [TD]DFT cal-

culations, and determinations of the changes in excited state dipole moment changes, we were

able to evidence the complexity of the physical processes responsible for the emergence of the

dual fluorescence effect. The changes in dipole moments observed in the excited and ground

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

Fig 1. Panel A: Electronic absorption spectra of SAL-3 in aqueous solutions with various pH. The arrow marks the isosbestic point at the

wavelength of ~305 nm. The figure presents spectra for pH: 3; 6; 6.6; 7.1; 7.4; 8; 8.6, and 11. The experiments were conducted at 23 ^oC. Panel

B: Correlation between the absorbance ratio at the wavelength of 296 to 326 nm relative and the pH of the aqueous solution, based on the

spectra in Panel A.

https://doi.org/10.1371/journal.pone.0229149.g001

states, as well as their respective ratios, confirm the possible presence of an additional elec-

tronic state related to the ESIPT process, whereas the quantum-mechanical calculations con-

firmed the possibility of the balanced presence of various ionic and non-ionic forms of the

compound, which facilitates the possibility of dual fluorescence related to the ESIPT- phenom-

enon. It is noteworthy that previous literature reports for many analogues of this derivative

have not suggested this path in the explanation of the aforementioned effects.

The effects related to the dual nature of fluorescence [17, 18] may be induced by changes in

the solvent’s polarity, pH [17], temperature, or concentration of the analyzed compound [19,

20]. The most prevalent explanations offered for this type of effects include the emergence of

intramolecular CT (charge transfer) states [19], particularly the twisted intramolecular charge

transfer (TICT) [21, 22]. A characteristic feature of molecules showing this type of effects is the

observed high excited state dipole moment and relatively fast rate of stabilization with the

increase in the medium’s polarity. Another explanations can be made based on effects related

to the presence of excimer systems [23, 24], the processes of excited-state intramolecular pro-

ton transfer (ESIPT) [25–29] or breaking Kasha’s rule for polyatomic molecules (Brancato

et al. [30]). The most recent studies on the group of 1,3,4-thiadiazoles with the 2,4-dihydroxy-

phenyl substituent, where the effects of dual fluorescence are induced by processes related to

the phenomenon of molecular aggregation [31–33], which in turn lead to the emergence of CT

processes in certain analogues [17–19].

Apparently this is not the case in the compound investigated in this work and the fluores-

cence effects observed more likely originate from the excited-state intramolecular proton

transfer (ESIPT) [27, 34, 35]. It is also worth-emphasizing that the compound studied in this

work is characterized by conformational flexibility and can adopt two conformations, which

may either allow or suppress the proton transfer process. This structural feature may signifi-

cantly contribute to the fluorescence effects observed. The last important factor relates to the

possibility (depending on the polarity/pH of the medium) of the emergence of ionic and non-

ionic forms which most precisely correlate with the observed fluorescence effects, and which

have not yet been observed for any derivative described in worldwide literature.

Studies conducted with the use of fluorescence spectroscopy, both stationary and time-

resolved, on the SAL-3 compound, allowed us to observe the effect of dual fluorescence in

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

Table 1. Characteristics of the investigated compound (Fig 2).

Compound: Systematic name

(common name)

Molecular weight

[g^.mol^-1]

Melting point

[^oC]

Molar volume

[cm^3.mol^-1]

pK_a(temp. logP_oct

[^oC])

logD

(pH)

CAS registry

number

2-Hydroxy-N-(2-phenylethyl)

benzamide

137.14

140.2–141.2

106.5 3.0

8.37 (20)

1.06 1.06 (5.1)

65-45-2

https://doi.org/10.1371/journal.pone.0229149.t001

aqueous solutions complemented with various proportions of a protic solvent (as well as in

protic solvents themselves). In water solutions, with changing concentrations of hydrogen

ions, we registered only a single fluorescence emission band of varying intensity depending on

the changes in pH. Only at very low pH levels were we able to observe a shift of the main emis-

sion band and its broadening on the short wavelength side (a relatively clearly visible effect

indicating the possibility of a second emission band).

Uncovering the mechanism of the compound’s organization in media of varying pH may

significantly contribute to the determination of its potential pharmacological applications as

well as to future model studies aimed at identifying its specific molecular properties. Further-

more, the compound (and the entire group of analogues) may, due to its high quantum effi-

ciency, also find applications as a fluorescence probe sensitive to changes in the pH or polarity

of the medium.

2. Materials and methods

2.1. Materials

Molar volumes were calculated with ChemSketch software (2015). NMR spectra were recorded

with an NMR spectrometer Varian Unity Plus 500 MHz. Characteristics of the investigated

compound are presented in Table 1.

2-Hydroxy-N-(2-phenylethyl) benzamide. A mixture of 15.21 g (0.1 mol) of methyl salic-

ylate and 12.12 g (0.1 mol) of phenethylamine was left in a stoppered bottle for two weeks,

occasionally shaken, at room temperature. Next, the syrupy liquid was poured into diluted

hydrochloric acid. The precipitated mass was filtered and rinsed several times with cold water.

After air-drying, the yield was 19.53 g (80.2%). For further studies, the product was addition-

ally recrystallized two times from a mixture of chloroform-methanol (1:1) and dried in vac-

uum for 3 h at room temperature. The physicochemical characteristics were as follows: m.

p. 93.6–94.7 ^oC; ¹H-NMR (500 MHz, CDCl₃, δ): 11.97 (bs, 1H, -COOH), [7.33–7.42 (m, 3H),

7.23–7.31 (m, 3H), 7.21 (dd, 1H, J₁= 8.1 Hz, J₂= 1.5 Hz), 6.99 (d, 1H, J = 8.3 Hz), 6.81 (t, 1H,

J = 7.6 Hz) (phenyl)], 6.39 (bs, 1H, -NH-), 3.72 (q, 2H, J = 6.7 Hz), 2.95 (t, 2H, J = 6.9 Hz);

¹³C-NMR (125 MHz, CDCl₃, δ): 169.87 (-COOH), [161.46, 138.40, 134.14, 128.78, 128.72,

126,73, 125.12, 118.61, 118.55, 114.18 (phenyl)], [40.70, 35.47 (aliphatic)].

2.2. Methods

2.2.1 AFM measurements. Atomic Force Microscopy (AFM) relies on the mapping sur-

face geometry of solids determined at micro- and nano-levels by sampling the forces occurring

due to the interaction with a conical tip with an atomically-sharp apex. The probe is mounted

on an elastic cantilever, the bending of which can be detected by measuring laser beam

reflection.

Geometrical structure plays a major role in tribology, contact mechanics and dynamic pro-

cesses on the surface of films at various levels of complexity [36, 37]. In this aspect, AFM serves

as a tool for characterizing the spatial geometry of texturized films. In the current study, the

AFM method was employed to image the geometrical structure of SAL-3 crystal samples along

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

with the investigation of their nanomechanical properties (as additional properties of this

compound). AFM measurements were carried out using a Multimode 8 instrument with a

Nanoscope V controller (Bruker) and NSG11 scanning tips (NT-MDT) in air, under ambient

conditions. The estimated tip radius was 5 nm and the cantilever force constant was 5 N/m.

The scan area varied between 1 and 5 μm with 256 steps along each scan axis. The AFM data

were recorded in the Peak Force Quantitative Nanomechanical Mapping (PF-QNM) mode

that maps the surface topography together with selected mechanical properties such as:

reduced Young’s modulus, adhesion forces, and sample deformation. Here, the maximum

repulsive force between the tip and the surface was set to 2 nN.

2.2.2 Electronic absorption and fluorescence spectra. The electronic absorption spectra

of SAL3 were recorded on a double-beam UV-Vis spectrophotometer Cary 300 Bio (Varian)

equipped with a thermostatted tray holder with a 6×6 multi-cell Peltier block. The temperature

was controlled with a thermocouple probe (Cary Series II from Varian) placed directly in the

sample.

Fluorescence excitation, emission, and synchronous spectra were recorded with a Cary

Eclipse spectrofluorometer (Varian) at 22 ^oC. Fluorescence spectra were recorded at the reso-

lution of 0.5 nm and corrected for the lamp and photomultiplier spectral characteristics. Reso-

nance light scattering (RLS) measurements were performed as described by Pasternack and

Collings. The excitation and emission monochromators of the spectrofluorometer were

scanned synchronously (0.0 nm interval between the excitation and emission wavelengths);

the slits were set to obtain a spectral resolution of 1.5 nm. The spectral analysis was performed

with the use of Grams/AI 8.0 software (Thermo Electron Corporation).

2.2.3 Time-correlated single photon counting (TCSPC). Time-correlated single photon

counting (TCSPC) measurements were performed using a FluoroCube fluorimeter (Horiba,

France). The samples were excited with a pulsed Nano- LED diode at 294 nm (pulse duration

of 700 ps) operated with 1 MHz repetition. To avoid pulse pile-up, the power of the pulses was

adjusted to an appropriate level using a neutral gradient filter. Fluorescence emission was

recorded using a TBX-04 picosecond detector (IBH, JobinYvon, UK). The Data Station and

DAS6 software (JobinYvon (IBH, UK)) was used for data acquisition and signal analysis. All

fluorescence decays were measured in a 10 x 10 mm quartz tray using an emitter cut-off filter

408 nm. The excitation profiles required for the simplified analysis were measured without the

emitter filters on a light scattering tray. All measurements were performed at 20 ^oC in various

solvents. Each case of fluorescence decay was analyzed with the multi-exponential model

shown in the equation:

�

P

À t

I_t¼ _ia_iexp

=

ð1Þ

t

i

where α_iand τ_iare the pre-exponential factor and the decay time of component i, respectively.

The best-fitted parameters were obtained by minimizing the reduced χ²value as well as

through residual distribution of the experimental data. The fractional contribution (f_i) of each

decay time and the average lifetime of fluorescence decay (<τ>) were calculated with the fol-

lowing equations:

a_it_i

_ja_jt_j

P

f_i¼

ð2Þ

ð3Þ

P

hti ¼ _if_it_i

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

2.2.4 DFT calculations. A Gaussian 09 package [38], using B3LYP exchange-correlation

functional [39], aug-cc-pVDZ basis set [40] and very dense grids, was applied in the DFT cal-

culations. Dispersion effects were accounted for using Grimme’s D3 semi-empirical treatment

with the Becke-Johnson damping scheme [41]. The solvent effects were treated by Polarizable

Continuum Model (PCM) [42]. Charged species were modelled using Na⁺and Cl^-as counter-

ions. The excited state treatment was carried out using the standard random-phase approxi-

mation (RPA) approach to TD DFT formalism [43][A6]. The influence of the solvent on

energetics was determined according to the linear response approximation.

2.2.5 Estimation of the dipole moments. In the presented study, the ground and excited-

state dipole moment of the 2-hydroxy-N-(2-phenylethyl)benzamide (SAL-3) molecule was esti-

mated based on two methods based on the influence of the internal electric field (solvatochro-

mism). The influence of the solvents on the locations of the maxima of absorption and

fluorescence spectra is used to evaluate the excited-state dipole moments of the molecules.

Method 1. Based on the papers by Bakshiev and Kawski-Chamma-Viallet [44], the change

in the dipole moment can be calculated from the following two Eqs (4 and 5):

�

v_aÀ v_f¼ m₁F₁ðε; nÞ þ cste

ð4Þ

�

v_aþ v_f

¼ m₂F₂ðε; nÞ þ cste

ð5Þ

2

-1

�

Where v_aand v_fare the absorption and fluorescence maxima expressed in cm . The

expressions F₁(ε,n) (Baksviev’s polarity function) and F₂(ε,n) (Kawski-Chamma-Viallet func-

tion) for solvents are developed as Eqs (6 and 7).

�

2

2n þ 1 ε À 1 n À 1

F₁ðε; nÞ ¼

�

À

ð6Þ

ð7Þ

2

n þ 2

ε þ 2 n þ 2

�

2

4

2n þ 1

ε À 1 n À 1

ðn À 1Þ

2ðn þ 2Þ

F₁ðε; nÞ ¼

�

À

þ

2

2ðn þ 2Þ ε þ 2 n þ 2

Where n is the light refraction index and ε is the solvent’s dielectric constant. The dipole

moment change is calculated from the locations of absorbance and fluorescence maxima for

the given molecule in various solvents. Assuming that the symmetry of the analyzed molecule

remains unchanged following the electronic transition, and that the ground and excited-state

dipole moments are parallel, we obtain the following Eqs (8 and 9):

�

1=2

3

jm₁þ m₂j hca₀

m_g¼

�

ð8Þ

2

2m₁

�

1=2

3

jm₁À m₂j hca₀

m_e¼

�

ð9Þ

2

2m₁

Where μ_gand μ_eare the ground and exited-state dipole moments, respectively, h is Planck’s

constant, c is the velocity of light, and a₀is the radius of the molecule’s Onsager cavity. The

cavity radius for a dissolved substance – a₀- is calculated from Suppan’s equation [45] (10):

1=3

a₀¼ ð3M=4pdNÞ

ð10Þ

Where δ is the density of the dissolved substance, M is the molar mass, and N is the Avoga-

dro constant. The ration of the excited-state dipole moment to the ground-state dipole

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

moment can be expressed as (11):

m_ejm₁À m₂j

¼

ð11Þ

m_gjm₁þ m₂j

Where m₁and m₂are the respective slopes of straight lines obtained from the graphs repre-

�

senting the Stokes shift relative to the function of solvent polarity F₁(ε,n) and ðv_aþ v_fÞ=2 rela-

tive to the function of F₂(ε,n). The change in the molecule’s dipole moment is determined on

the basis of the difference between the excited-state dipole moment and the ground-state

dipole moment (Eq 12):

Dm ¼ m_eÀ m_g

ð12Þ

Method 2. The second method, first proposed by Reichardt [46], utilizes a scale describing

the microscopic polarity of solvents E^N_Twhich is an assessment based on the solvatochromic

effect present in the betaine pigment. The polarization dependence and presence of hydrogen

bonds in solvents are correlated with the polarity scale E^N_T. The theoretical basis for the correla-

tion between the spectral shift and the E_T^Nscale was provided by Ravi et al. [45]. The E^N_Tvalue is

defined by Eq (13), where water (E^N_T¼ 1) and tetramethylsilane (E_T^N¼ 0) are used as the refer-

ence solvents:

E_TðsolventÞ À E_TðTMSÞ E_TðsolventÞ À 30:7

E^N_T¼

¼

ð13Þ

E_TðwaterÞ À E_TðTMSÞ

32:4

Finally, the molecular dipole moment change Δμ can be described as (14):

vﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ

u

m � 81

u

Dm ¼ m À m ¼

ð14Þ

� �

t

Where m is the slope of the straight line obtained from the graph of the Stokes shift relative

e

g

3

6:2

a

11307:6 �

0

to the E^N_Tparameter.

3. Results and discussion

3.1 AFM study

The structure of the analogue selected for the study is presented in Fig 2A–2C.

As can be observed, the SAL-3 molecule is capable of forming an intramolecular hydrogen

bond between the–OH group and the adjacent carbonyl group. Fig 2B and 2C also evidence

the molecule’s capacity for proton transfer. Furthermore, the powder of the compound itself

was analyzed with the use of AFM measurements and the results were provided in S1–S5 Figs

enclosed in Supplementary Materials. The surface was found to be strongly layered with flat,

irregular terraces and sharp steps, each around 1 nm in hight, as best seen in S2 Fig. Apart

from that the above, structural inhomogeneities in the form of the irregular layers of foreign

material—2 nm thick and a few hundred nanometers in size (“A” marks)—can also be seen. In

addition, a large area of circular particles from 10 up to 100 nm in diameter can be spotted

(“B” marks). These particles are randomly distributed over the surface of the crystals with no

apparent alignment pattern, which excludes both molecular agglomeration and edge attraction

phenomena, and consequently surface diffusion processes, from further analysis.

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

Fig 2. Chemical structure of the SAL-3 molecule (A–non-ionized form, B–form with an H-bond between the–OH

and C = O group, C–form after proton transfer between the–OH and C = O group, in this case a carbonyl group is

formed and an–OH group emerges where the carbonyl group used to be).

https://doi.org/10.1371/journal.pone.0229149.g002

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

3.2 Spectroscopic studies

Fig 1, Panel A presents the results of absorption spectra measurements performed for SAL-3

with the use of electronic absorption spectroscopy in the full range of hydrogen ion concentra-

tions. The results indicate very clear changes in the shapes of the spectra, particularly in the

region important for physiological values. For better clarity of the presentation, the spectra

obtained for the analyzed compound at pH levels of: 3, 6, 6.6, 7.1, 8, 8.6, and 11 are presented.

As can be observed in Fig 1, Panel A, dissociation of the–OH group from the hydroxybenzene

ring in the ortho position (Fig 2) causes a hypsochromic spectral shift by 996 cm^-1(9 nm) at

pH 1 and bathochromic shift by 2112 cm^-1(21 nm) in the compound’s spectra at pH 12, rela-

tive to the spectra observed in polar (protic) solvents. Furthermore, in the case of the analogue

selected for the study, the process of ionization can be accompanied by slight (but noticeable)

processes of aggregation [16]. At approx. pH 7 (in Fig 1, Panel A, clearly for spectra at pH 7.1

or pH 7.4) there is a clear widening of the SAL-3 absorption bands (dashed blue and solid red

lines), indicating the possible presence of spectral forms other than monomeric [16]. In the

case of the compound’s spectra at low pH levels (e.g. pH 3, below pH 6), the spectrum absor-

bance is decreasing, moreover the band on the longwave side of the spectrum disappears

(which in the analyzed case suggests, based on the [TD]DFT calculations, more likely the

effects of various ionic forms of the compound as opposed to aggregation effects), which can

indicate the significant prevalence of monomeric SAL-3 forms emerging in the given ionic

form in the medium in question (see Panel A in Fig 1). Observed broadening of the bands at

pH 7 compared to the respective spectra in the solvents shows the impact of heterogeneity of

the system. The effects indicate (as confirmed in the [TD]DFT calculations) the occurrence of

different ionic forms of the compound depending on the pH of the medium. Furthermore, we

can also observe a clearly defined isosbestic point at the wavelength of 305 nm, which may

indicate that the transition between the respective ionic (and non-ionic) forms of the com-

pound is very fluid (which is further corroborated by the results of fluorescence spectra mea-

surements). At pH levels such as 7.1 or 7.4 (as well as others not presented but within the same

pH range) we can observe the aforementioned strong widening of the band on the longwave

side with the maximum at approx. 326 nm, which suggests the prevalence of a specific ionic

form (with the–O^-group) whose content remains at a probably continuously (slightly) increas-

ing level with the increasing pH. This also indicates the prevalence of ionized forms of the

SAL-3 molecule, the spectra where the widening disappears correlate with the prevalence of

non-ionized forms present mostly as monomers (as will be further discussed in the text and

which has a strong impact on fluorescence effects, Fig 3). It is also noteworthy that at low pH

levels, protonation of the carbonyl group is also possible with the associated additional ionic

form. Although not observed in the absorption spectra, the possible evidence of its presence

was noted in the fluorescence spectra.

Panel B in Fig 1 presents the relationship between the absorbance maximum at 296 nm

(prevalence of the monomeric form–non-ionized/or ionized in the carbonyl group) to 326 nm

(prevalence of the associated form–and ionized in pH higher than physiological, with the–O^-

group) for SAL-3 depending on the pH value of the analyzed aqueous solution. We can clearly

observe that the level of monomerization (with the prevalence of the form containing an ion-

ized–O^-group) for the selected analogue is the highest in high pH (to approx. pH 8), generally

higher than physiological. Next, with the decreasing pH, within the range of 8 to 4.5, we

observe the presence of both ionized and non-ionized forms, with the decreasing participation

of the former. Meanwhile, at pH below 4, we once again observe the prevalence of ionized

forms, and at very low pH levels (1, 2, or 3), there is the possibility of protonation also of the

carbonyl group, emergence of an additional ionic form, which is not, however, seen in the

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

Fig 3. Fluorescence emission spectra for SAL-3 in aqueous solutions with various pH. The figure presents the results for pH: 1; 6.5; 7.1; 8;

8.9; and 11. The spectra were obtained at 23˚C in the spectral range from 305 to 600 nm.

https://doi.org/10.1371/journal.pone.0229149.g003

absorption or fluorescence emission spectra. It should be emphasized that the most significant

changes of the discussed ratio in the analyzed analogue are observed primarily within the phys-

iological pH range, as can be clearly seen in the absorption spectra presented in Fig 1, Panel A.

The subsequent stage of the study entailed an analysis of the SAL-3 analogue with the use of

fluorescence spectroscopy. One should point out the very interesting fluorescence effects illus-

trated in Fig 3.

It presents the fluorescence emission spectra for SAL-3, corresponding to the absorption

spectra in Fig 1 (Panel A), relative to changes in the pH of the aqueous solution (the results are

presented, respectively, for pH 1, 3, 6, 7.1, 8, and 8.9 –similarly to the absorption spectra in Fig

1, Panel A). The excitation wavelength for all the analyzed samples corresponded to the respec-

tive absorption spectra in Fig 1 (Panel A). As can be observed in the case of aqueous solutions

with pH levels lower than physiological condition, the study revealed a clear shift of the emis-

sion band maximum to 430 nm and the emergence of a clear band widening on the shortwave

side, with the maximum at approx. 386 nm (Fig 3). In the case of SAL-3, the effect is clearly

present within the pH range from 1 to ~6. Above pH 6, we observe only a single fluorescence

band with the maximum shifted to approx. 411 nm (also with significantly higher intensity).

After longwave excitation (corresponding to the ionized forms with the–O^-group) with the

maximum at approx. 326 nm for the samples in pH lower than 6, we observe only a single fluo-

rescence emission band with the maximum at approx. 411 nm. The effect clearly indicates the

possible coexistence of ionized and non-ionized forms of the molecule in the solution.

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

3.3 RLS study

In order to determine the impact of the coexistence of various ionic forms of the analyzed

compound on the observed fluorescence effect, we performed decomposition of a selected

absorption spectrum (e.g. for pH 7.4 in S6 Fig in SM) and analysis (only qualitative) of its

respective constituents, we also analyzed the RLS spectra for SAL-3 within the entire range of

hydrogen ion concentrations (Fig 4A).

As can be observed in S6 Fig presenting the decomposition of the absorption spectrum of

SAL-3 in pH 7.4, the bands were resolved into their respective constituents, with the ones with

the maxima at approx. 296 nm (at this pH) and 326 nm (solid red and green lines) identified

as the most significant contributors. It is clearly apparent that the higher the contribution of

the constituent corresponding to the longwave part of the spectrum, the more quickly we can

observe quenching of the effect of emission spectra widening on in the shortwave side in the

fluorescence emission spectra, accompanied by a shift of the emission band to approx. 411

nm. The above effects confirm the earlier assumption that in order for the effect of dual fluo-

rescence to be observed in the case of SAL-3, the additional condition of a relatively low con-

centration of the analyzed compound must be met, while aggregation effects are more likely to

quench rather than induce it. The key question remains, however, whether the molecule is ion-

ized or non-ionized, as this determines the emergence of the specific effects related to the wid-

ening of the fluorescence emission bands. In the example decomposition of the absorption

bands, we can also observe the necessity of the presence (in order to obtain a good decomposi-

tion) of additional bands–such as the one with the maximum at approx. 285 nm. However, the

emission from this band does not cause any clear changes in the emission spectra.

This fact, i.e. the coexistence of ionic and non-ionic forms, was further corroborated by the

performed RLS [14] spectra measurements conducted in aqueous solutions at various concen-

trations of hydrogen ions, as already discussed above (Fig 4A). However, their presence was

usually associated with the possibility of chromophoric aggregation of compounds present in

the solution [14]. But as can be observed in Fig 4A, the RLS bands for the respective pH ranges

vary in intensity and shape. They appear for various pH ranges, which suggests that the aggre-

gation (chromophoric) slightly evidenced in the electronic absorption spectra has no signifi-

cant impact on the observed effect of dual fluorescence, and its influence is mainly related to

quenching the same. At the same time, the observed oscillatory structure of the RLS bands

may evidence the potential presence of different forms: ionic and non-ionic, depending on the

pH, whose relative prevalence may fluctuate [14] in various pH ranges, and therefore change

the character of their mutual interaction. It is noteworthy that for pH levels of ~6 and lower,

there is an additional broadening (very intensive) of the RLS bands, which indicates the possi-

ble presence of additional ionized forms (described above) at low pH levels, which could

explain the additional effects observed in the fluorescence emission spectra at low pH.

Fig 5 presents fluorescence excitation spectra corresponding to the emission spectra shown

in Fig 3.

In this case, the excitation wavelength corresponded to the first or second (in the context of

bands widening on the shortwave side of the emission spectra) maximum observed in the fluo-

rescence emission spectra. The fluorescence excitation spectra, due to their selectiveness,

reveal various spectral forms originating from, in our case, e.g.: iodized or non-ionized forms.

This fact is confirmed by the aforementioned deconvolution analysis performed on the

absorption spectra, as well as (partially) the RLS spectra. In this case we are dealing with bands

originating from the ionized or non-ionized form of the SAL-3 molecule. As clearly indicated

by Fig 5, at pH levels higher than ~6, one can observe a significantly higher intensity of the

band on the longwave side–due to the prevalence of ionized forms observed at high pH, as

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

Fig 4. Panel A: Resonance light scattering (RLS) spectra for selected values of aqueous solution pH, analogically to the previous

experiments. Panel B: RLS spectra for selected mixtures of H₂O and propan-2-ol.

https://doi.org/10.1371/journal.pone.0229149.g004

well as the possible presence of an additional ionic form at low pH levels (as already discussed

above). Meanwhile, at pH lower than mentioned above, a band maximum emerges which cor-

responds to the maximum of the main absorption band with the maximum at approx. 300 nm.

The band is characteristic of the non-ionized form.

Fig 5. Fluorescence excitation spectra for selected pH of the aqueous solution. Excitation emission was set at the respective maximum of

the longwave fluorescence emission spectra. The spectra were obtained at 23˚C in the spectral range from 270 to 390 nm.

https://doi.org/10.1371/journal.pone.0229149.g005

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

3.4 Spectroscopic effects in mixed solvents

Next, in order to determine the impact of changing polarity/polarizability of the medium on

the observed fluorescence effects (presented in Fig 3), Panel A in Fig 6 presents the results of

the electronic absorption spectra measurements conducted on SAL-3 in various ratios of water

to propan-2-ol.

The spectra were normalized at the absorption band maximum. As we can see, the band

maximum, corresponding to the π!π^�electronic transition [47] (approx. 300 nm), clearly

shifts towards the shorter wavelengths, from 301 to 296 nm, depending on the addition of the

polar (protic) solvent. Furthermore, on the longwave side of the bands, at approx. 320 nm,

there is a visible slight broadening of the absorption band (affecting its half width) evidencing

the coexistence of various forms of the compound (ionic, non-ionic or aggregation effects,

although in this case most likely the fact of the presence of aforementioned forms–as will also

be discussed further in the text). In turn, Panel B in Fig 6 presents the shift of the absorption

maximum (from Panel A) relative to the ratio of the solvents used (water/propan-2-ol). As can

be observed, the relationship remains at roughly the same level up to 6:4 (Pr-OH: H₂O). Above

that value, there is a fast shift of the band towards the longwave side and the effect of dual fluo-

rescence is no longer observed in the fluorescence emission spectra (further in the text).

However, the most noteworthy effects are those presented in Fig 7 (Panel A).

Panels A and B illustrate the fluorescence emission spectra for the studied analogue in vari-

ous ratios of water to propan-2-ol. Panel A shows the fluorescence emission spectra after exci-

tation at the main maximum of the absorption spectra (as presented in Fig 6, Panel A), i.e. at

approx. 300 nm. In this case, we can very clearly observe a very interesting effect of dual fluo-

rescence with the maxima at approx. 331 nm and 429 nm in samples where the solutions con-

tained more propan-2-ol (i.e. more polar and protic media). By comparing the spectra in Fig

7, Panel B (with Fig 6), we can clearly observe that the effect of dual fluorescence in SAL-3

emission spectra occurs up to the H₂O to Pr-OH ratio of approx. 6:4. The addition of the polar

protic solvent into the analyzed system enforced the emergence of dual emission related, in

this particular case, to the balance between ionic and non-ionic forms of the analyzed ana-

logue. With decreasing content of propan-2-ol, the band on the shortwave side of the emission

spectra quickly disappears, we also observe decreasing intensity of the band with the maximum

at approx. 430 nm which also shifts towards shorter wavelengths to approx. 412 nm.

The correlations presented in the graphs used for the calculation of ground and excited-

state dipole moments as well as their respective ratios clearly evidence the differentiation

between polar protic solvents (in whose presence the effects of dual fluorescence are observed)

and non-polar or polar aprotic solvents, in which only a single emission band in the fluores-

cence spectrum is observed (further in the text). The results obtained in terms of changes in

the dipole moments of SAL-3 molecules, as well as the quantum-mechanical [TD]DFT calcula-

tions, indicate the fact that the dual emission effects observed for this molecule are caused by

the ESIPT effect combined, in this case, with the coexistence of the ionic and non-ionic forms

of the analyzed analogue. Panel B in Fig 7 presents the analogical spectra in Panel A, but in this

case for at approx. 320 nm (band widening in the absorption spectra corresponding to the ion-

ized form of the given molecule). As can be seen, in this case we no longer observe the effect of

dual fluorescence, which excludes the contribution of aggregation effects to the phenomenon–

as already discussed above. Or, more precisely, aggregation effects can be said to quench the

observed effect of dual fluorescence. These results are consistent with those presented in Figs 1

and 3.

Fig 8 illustrates the fluorescence excitation spectra corresponding to the emission spectra

presented in Fig 7.

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

Fig 6. Panel A: Electronic absorption spectra for selected ratios of water to propan-2-ol, normalized at the wavelength maximum. Panel B:

Position of the maximum of the electronic absorption band for the spectra presented in Panel A.

https://doi.org/10.1371/journal.pone.0229149.g006

In this case, the excitation wavelength corresponded to the first (Panel A) or second (Panel

B) maximum in the fluorescence emission spectrum (for dual emission). Fluorescence excita-

tion spectra, due to their selectiveness, display various spectral forms originating from differ-

ent forms, in this case from ionized or non-ionized forms. As can be observed in Fig 8,

depending on the ratio of the solvents used (water:propan-2-ol) and clearly depending on the

excitation, we observe the presence of bands which can be associated with either the appropri-

ate type of ionic or non-ionic forms. At the excitation wavelength corresponding to the short-

wave fluorescence emission maximum from Fig 7, we observe basically only a change in the

intensity of the bands relative to the changing ratio of the solvents. However, in the case of

longwave excitation (most likely corresponding to the increasing prevalence of the ionic

form), as we add water, we observe that the band is shifted towards longer wavelengths, up to

approx. 312 nm, as well as a significant increase in band intensity. The effects correspond to

the changes in the absorption/emission spectra and confirm the contribution of various ionic

forms of the compound to the fluorescence emission.

Fig 7. Panel A: Fluorescence emission spectra corresponding to the spectra in Fig 6A. The excitation wavelength corresponded to the

maximum of the absorption band. Panel B: Fluorescence emission spectra analogous to those in Panel A but with the excitation wavelength

corresponding to the slopes of the absorption band–as described in the paper.

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

Fig 8. Fluorescence excitation spectra corresponding exactly to the emission spectra in Fig 7. Panel A: Excitation spectrum with emission

detected at 331 nm; Panel B: Excitation spectrum with emission detected at 429 nm.

https://doi.org/10.1371/journal.pone.0229149.g008

Similarly to the aqueous solutions, also in this case the spectra of resonance light scattering

(RLS) were measured. Fig 4B presents RLS spectra corresponding to the results in Figs 6–8 for

the analyzed SAL-3 solutions in various solvents. As shown, with increasing content of water

we observed a significant increase in the intensity of the RLS spectra, which may also confirm

the presence of aggregation effects that may coincide with various forms of the compound, e.g.

ionic and non-ionic. As can be seen in Fig 4B, the RLS spectra for the respective ranges of the

ratio vary in terms of intensity and shape. One can also note the oscillative structure of the

observed RLS spectra indicating the potential presence of different ionic and non-ionic struc-

tures depending on the changes in the quantitative proportions of the solvents. It is also note-

worthy that for the solvent ratio of 5:5 and with increasing amount of water, there is a band

between 300–350 nm which very quickly gains in intensity. It can be associated (as confirmed

by the above results) with the growing content of ionic forms of SAL-3 in the analyzed system.

The remaining RLS spectra (in the longwave direction) change in intensity but not in shape,

which further confirms that the analyzed system may contain both mentioned forms of the

compound.

3.5 TCSPC study

Time-resolved spectroscopic measurements were performed for SAL-3 in the same pH range

as in the experiments described above. The excitation wavelength used was 294 nm as it is the

most effectively absorbed by the most intensive absorption component (the most intensive

peak with the maximum at approx. 300 nm–presented in Fig 1 or 6A). It will also be absorbed

by the other two components–however, significantly less efficiently. Fluorescence emission

was measured for wavelengths higher than 408 nm, i.e. only one fluorescence component was

monitored–the one characterized by the greater wavelength.

The analysis of fluorescence intensity decay was conducted using deconvolution for a 1, 2,

and 3 component model. For all the analyzed pH values, the two-component model proved to

be optimum. The analysis based on a singular decay was insufficient, whereas the addition of

the third component did not significantly improve the quality of fit (Fig 9).

In all the analyzed cases, the long-life component had a characteristic time of approx. 3 ns,

which decreased slightly with growing pH. For the second, short-life component, considerably

greater pH-dependent variability was observed. At low pH, it was approx. 0.1 ns, whereas at

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

Fig 9. Panels A-B: TCSPC analysis for SAL3 in water at various pH. The top panel presents the decay of fluorescence intensity. Black color

denotes the apparatus profile. Points correspond to measurement results and the continuous line to the double component analysis. The

bottom panel presents the distribution of residuals. The excitation wavelength was 294 nm. Panels C-D: TCSPC analysis for SAL-3 in

water in various solution compositions. The top panel presents the decay of fluorescence intensity for a selected propanol content. Black

color denotes the apparatus profile. Points correspond to measurement results and the continuous line to the double component analysis.

The bottom panel presents the distribution of residuals. The excitation wavelength was 294 nm.

https://doi.org/10.1371/journal.pone.0229149.g009

high pH it increased to approx. 0.9 ns. A clear change in the lifetime of this component was

observed for pH in the range of 8–10 (Fig 10, Panel B).

The contribution of the long-life component was greater than that of the short-life compo-

nent, approx. 80%, and increased to approx. 90% with growing pH. Only for the lowest pH lev-

els was an inversely proportional relationship between the components observed, as in this

case the contribution of the long-life component was only approx. 30%. A significant change

of this parameter was also observed at around pH 7 (Fig 9, Panel C). The described values have

an obvious impact on the mean fluorescence lifetime which increased from approx. 1.5 ns to

approx. 2.7 ns with increasing pH. In this case, the most significant change was observed for

pH 2 and pH 7 (Panel A in Fig 10, Table 2).

An identical measurement system was used to determine the impact of the solvent’s (polar-

ity/polarizability) on the spectroscopic parameters of SAL-3 (Fig 9D–9F). In particular, the

excitation wavelength and range of emission wavelengths considered were the same as in the

time-resolved experiments discussed above. The solvent was a mixture of water and propan-

2-ol constituting between 10% and 90% of the solvent’s volume. Similarly, to the previous

cases, the analysis was conducted based on deconvolution in a 1, 2, and 3 component model of

fluorescence intensity decay. In each of the cases considered, the two-component model

proved the most adequate, which evidences the presence of two classes of fluorophores. The

contribution of the first, characterized by a shorter fluorescence lifetime, was 27% for the low–

10% content of propanol, and increased to 81% at the 80% concentration. A further increase of

the concentration resulted in a decrease of the component’s contribution–to 68% (Fig 9, Panel

F). The fluorescence lifetime of this component increased monotonically with the increasing

concentration of propan-2-ol in the entire range of the concentrations considered, form 0.18

ns to 1.5 ns. The lifetime of the second constituent, characterized by a linger fluorescence

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

Fig 10. Panels A-C: Effect of pH on the fluorescence lifetime for SAL3 in water. Panel A presents fluorescence lifetimes of the

mean values observed at various pH levels, while panel B shows fluorescence lifetimes of the first (τ1) and the second (τ2)

component, observed for various pH. The dependences of the fraction of both components in pH are presented in Panel C. The

data analysis was carried out for the two-component model (Eq (1) for i = 2) for data presented in Fig 9A and 9B. Panels: D-F:

Effect of the content of propan-2-ol on SAL-3 fluorescence lifetime. Panel A presents fluorescence lifetimes of the mean values

observed in various propanol mixtures, while panel B shows fluorescence lifetimes of the first (τ1) and the second (τ2) component.

The dependences of the fraction of both components in solution composition are presented in Panel C. The data analysis was

carried out for the two component model (Eq (1) for i = 2) for data presented in Fig 9C and 9D.

https://doi.org/10.1371/journal.pone.0229149.g010

Table 2. Fluorescence lifetimes of SAL-3 in different aqueous solvents at different pH with F408 cut-off filter and

diode 294 nm.

SAL-3 in different pH

F408

pH

1.8

3

α

τ

Δτ

1.523 0.023

2.955 0.033

2.432 0.060

2.529 0.075

2.427 0.045

2.591 0.036

2.703 0.014

2.696 0.016

2.699 0.014

2.681 0.016

4.6

6

7.1

8

8.9

10

11

12.1

https://doi.org/10.1371/journal.pone.0229149.t002

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

lifetime, was 3.6 ns in 10% propanol, and increased to 5 ns in the concentration range of 40–

60%, after which it decreased again to 2.8 ns as the propanol concentration continued to

increase (Fig 10, Panel D). The mean fluorescence lifetime, determined on the basis of said

results from Eq 3, showed a monotonic decrease with the growing concentration of propanol,

from 2.8 to 1.9 ns within the concentration range of 20–90%. Decreasing the concentration of

this component to 10% resulted in a slight decrease in the mean fluorescence lifetime to 2.7 ns

(Fig 10, Panel C, Table 3).

The above results confirm the possible e existence/coexistence of two forms of the analyzed

compound, ionic and non-ionic, depending on the changes in the pH of the system or the

polarity of the medium used.

3.6 Computational results

The conducted quantum-mechanical calculations corroborated the conclusions reached on

the basis of the experimental data. In the case of SAL-3, we are dealing with a pH-dependent

balance/equilibrium of various forms of the compound. At very low pH, the SAL3H⁺form is

predominant (Fig 11A), whereas at high pH, the SAL3^-form is prevalent (Fig 11C).

At the remaining pH values, the undissociated form SAL-3 is predominant. The locations

and intensities obtained from the [TD]DFT calculations were highly consistent with the

changes observed in the absorption and fluorescence spectra. The observed shift of the absorp-

tion maximum towards longer wavelengths with increasing pH, the shift of fluorescence emis-

sion bands towards longer wavelengths accompanied by a decrease in intensity between

medium and very low pH levels, as well as the shift towards shorter wavelengths between

medium and high pH values–were all confirmed in the results of [TD]DFT calculations (Fig

11 and Table 4).

3.7 Estimation of ground state and excited state dipole moment

In order to determine how the dipole moments of the analyzed molecule vary between its

ground state and excited state, in the present study, the ground and excited-state dipole

moment of the SAL-3 molecule was estimated for two different positions of the fluorescence

maxima (Em 1 –maximum at approx. 330 nm, Em 2 –maximum at approx. 430 nm). In fluo-

rescence measurements for polar solvents, the analyzed molecule produced two bands of vary-

ing intensity, whereas in nonpolar solvents, we observed only a single band, as already

Table 3. Fluorescence lifetimes of SAL-3 in different aqueous solvents at different pH with F408 cut-off filter and

diode 294 nm.

SAL-3 in H₂O : Pr-OH

F408

pH

9 : 1

8 : 2

7 : 3

6 : 4

5 : 5

4 : 6

3 : 7

2 : 8

1 : 9

α

τ

Δτ

2.693 0.023

2.837 0.016

2.741 0.014

2.620 0.014

2.444 0.014

2.264 0.013

2.054 0.012

1.923 0.013

1.893 0.023

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

Fig 11. Ground state structures of various protonation forms of the SAL-3 molecule as optimized using B3LYP/aug-cc-

pVDZ and PCM treatment with water as a solvent. Hydrogen bonds depicted by red dashed lines. Panel A: SAL-3H⁺form,

Panel B: SAL-3 form and Panel C: SAL-3^-form.

https://doi.org/10.1371/journal.pone.0229149.g011

discussed above in this text. The positions of the SAL-3 molecule’s absorbance and fluores-

cence maxima in various solvents are presented in Table 5.

The ground and excited dipole moment for SAL-3 was estimated using two methods with

the use of two solvent polarity functions F₁(ε, n) and F₂(ε, n) (Eqs 6 and 7) as well as the

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

Table 4. TD-DFT predictions for various protonation forms of the SAL-3 molecule in a solvent (water) treated as

per the PCM approach. Energetics given in a nm scale, dimensionless oscillatory strengths given in parentheses. Low-

lying absorbing states shown as well as the lowest excited state for emission.

Form

SAL-3H⁺

Vertical absorption

303 (0.15); 297 (0.05)

293 (0.18)

Vertical emission

443 (0.08)

SAL-3

SAL-3^-

423 (0.27)

318 (0.17)

376 (0.24)

https://doi.org/10.1371/journal.pone.0229149.t004

microscopic solvent function E^N_T(Eq 13) for various solvents (n-heptane, n-hexane, chloro-

form, butan-1-ol, propan-2-ol, ethanol, DMF, DMSO, methanol, acetonitrile, H₂O). The val-

ues of the light refraction index, the dielectric constant ε, and the functions of F₁(ε, n), F₂(ε, n)

and E^N_Tare presented in Table 6.

�

The graph presenting the Stokes shifts for the F₁(ε, n) function and ðv_aþ v_fÞ=2 for the

F₂(ε, n) function are presented in Fig 12 (Panels A and B), whereas the correlation between the

Stokes shift and the E^N_Tparameter is presented in Fig 13.

In method 1, based on the publications by Bakshiev and Kawski-Chamma-Viallet, in the

case of the first fluorescence maximum (Em 1) the estimated value of Δμ for polar solvents was

4.493 and 3.314 depending on the linear fit applied (Table 7).

Whereas in the case of the second fluorescence maximum (Em 2), the change in the dipole

moment was Δμ 2.220. Table 6 also presents the values of slopes (m₁and m₂) and coefficients

of determination r². In the case of the graph in Panel B, good fit of the model was observed in

both cases (r²in the range of 0.87–0.90), whereas in the case of the Panel A graph, a somewhat

worse fit was observed for the first fluorescence maximum (Em1) (r²= 0.56).

In the second method based on the Dimroth-Reichardt methodology, the changes of the

dipole moment values for the two analyzed shifts were similar: for Em 1 Δμ was 1.344, for Em

2 Δμ was 1.384 (Table 8).

The value of r²was within the range of 0.89 to 0.98, which evidences good linear fit. As

could be expected, the value of the excited-state dipole moment was higher than the ground-

state dipole moment, which indicates that the SAL-3 molecule is more polar in its singlet

excited state as compared to its ground state. The above calculations of the changes in the

SAL-3 molecule dipole moments suggest that solvatochromic shifts lead to an important effect

of crisscrossing states with different distributions of electronic density relative to the polarity

of the medium used. In polar solvents, electronic states with high dipole moments (often the

states associated with e.g. excited-state proton transfer) are stabilized relative to the low dipole

moment states. If the energies of such states are similar, a change in the polarity/polarizability

of the medium may trigger a reversal of the states’ order. Photochemical properties are deter-

mined by the character of the lowest excited state with the given multiplicity (Kasha’s rule),

consequently they can be affected (as observed in our study) by very significant changes of sol-

vent polarity. And in the case of our study, we observed effects associated with the phenome-

non of dual fluorescence in SAL-3 molecules with the changing medium polarity.

It can be therefore preliminarily posited that the presence of the interesting effect of dual

fluorescence observed for the SAL-3 compound, based on the results presented below in Figs

1–13 relative to both stationary spectroscopy and fluorescence lifetime measurements as well

as, most importantly, the conformational potential of the SAL-3 molecule, is connected to the

effect of excited-state intramolecular proton transfer (ESIPT) in this particular particle. Let us

observe that for SAL-3, the effects of dual fluorescence occur in a medium (protic) where with

adequate rotation of the molecule there is a possibility of forming a hydrogen bond. In envi-

ronments characterized by the prevalence of ionized forms of SAL-3, i.e. e.g. at pH higher than

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

Table 5. Positions of the absorption spectra and fluorescence maxima for SAL-3 in different solvents.

Solvent

H₂O

Maximum absorbance [nm] I Maximum fluorescence [nm] II Maximum fluorescence [nm] I Stokes shift [cm^-1] II Stokes shift [cm^-1]

330

301

302

303

302

304

302

308

309

306

370

330

332

335

333

332

430

432

433

432

436

431

436

434

3276.003

2919.561

2992.101

2882.818

3152.554

3082.553

2864.707

2992.101

7047.216

9966.777

9964.435

9908.612

9855.152

9964.435

9958.957

9910.727

9531.753

9426.68

Propan-2-ol

Ethanol

DMSO

DMF

Butan-1-ol

Acetonitrile

Methanol

n-Heptane

n-Hexane

Chloroform

9638.264

https://doi.org/10.1371/journal.pone.0229149.t005

6.5 or media composed of mixed solvents with increasing water content, the effect tends to dis-

appear very quickly. Both the techniques of resonance light scattering (RLS) and band decom-

position with the analysis of the contribution of the respective components also excluded, with

high probability, the contribution of chromophoric aggregation to the induction of the dis-

cussed effect. In fact, the latter seems to be a quenching factor in this case. As mentioned

above, in the emission spectra registered after excitation at the wavelength corresponding to

the band which may partially originate from aggregated forms, we observed only a single fluo-

rescence emission band.

The ionized form, prevalent at pH higher than 6.5 (with excitation at the wavelength of the

absorption band maximum), corresponds to longwave fluorescence emission. The non-ion-

ized form, present at pH lower than 6.5 (mainly at very low pH levels), corresponds to a single

shortwave fluorescence emission band emerging in protic media. In such media, SAL-3 mole-

cules can occur primarily with the ionized–OH group. In protic media (pH under 6.5) as well

as in low concentrations, where the prevalent forms of the analyzed system are monomeric

(ionic or non-ionic), the aforementioned hydroxyl group is located on the right-hand side of

the carbonyl group and an intramolecular hydrogen bond is formed. Along the same, in the

excited state, a proton is transferred to the–OH group and a carbonyl group is formed in its

place, whereas an–OH group is formed in the original position of the carbonyl group (see Fig

2B and 2C and Fig 11).

Table 6. Solvent parameters F₁(ε,n), F₂(ε,n), dielectric constant ε, index of refraction n and E^N_Tfor different solvents.

E_T^N

Solvent

H₂O

ε

n

F₁(ε,n)

0.914

0.781

0.817

0.842

0.836

0.753

0.862

0.856

-0.001

0.002

0.372

F₂(ε,n)

0.532

0.476

0.49

80.10

20.18

25.30

47.24

36.70

17.80

36.64

33.00

1.92

1.3333

1.3772

1.3594

1.4773

1.4305

1.3993

1.3416

1.3265

1.3876

1.3723

1.4429

1.000

0.552

0.654

0.444

0.386

0.586

0.460

0.765

0.012

0.006

0.259

Propan-2-ol

Ethanol

DMSO

0.529

0.515

0.467

0.508

0.502

0.087

0.085

0.286

DMF

Butan-1-ol

Acetonitrile

Methanol

n-Heptane

n-Hexane

Chloroform

1.89

4.81

https://doi.org/10.1371/journal.pone.0229149.t006

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

Fig 12. Stokes shift to F₁(ε,n) using Bakhshiev’s equation (panel A), ðv�_aþ v�_fÞ=2 to F₂(ε,n) using Kawski-Chamma-

Viallet’s equation (panel B) for SAL-3 dissolved in different solvents (1—n-heptane, 2—n-hexane, 3 –chloroform, 4—

butan-1-ol, 5—propan-2-ol, 6—ethanol, 7—DMF, 8—DMSO, 9—methanol, 10 –acetonitrile, 11—H₂O). Black square–

Em 1 (maximum of fluorescence ~ 330 nm), blue circle–Em 2 (maximum of fluorescence ~ 430 nm).

https://doi.org/10.1371/journal.pone.0229149.g012

4. Conclusions

The results presented in this paper obtained primarily with the use of stationary spectroscopy

(measurements of absorption and fluorescence spectra), time-resolved spectroscopy (TCSPC

measurements of fluorescence lifetimes), as well as resonance light scattering (RLS) measure-

ments, indicate that the fluorescence emission spectra of the SAL-3 compound belonging to

the group of salicylic derivatives are characterized by a very interesting effect of dual fluores-

cence. The discussed effects are present at pH ranges lower than physiological and at low con-

centrations of the analyzed analogue. At pH higher than physiological, only a single

fluorescence emission maximum is observed. At low pH, we observe widening of the main

fluorescence emission band on the shortwave side and a significant shift of the main emission

band towards longer wavelengths. In the case of mixed solvents, we begin to observe a clear

effect of dual fluorescence with increasing content of the protic solvent. In the case of dual

emission, the respective bands are present at the wavelengths of approx. 330 nm and 430 nm.

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

Fig 13. Stokes shift variation with the normalized value of solvent polarity E^N_Tfor SAL-3. Black square–I Stokes

Shift, red circle–II stokes shift in different solvents (1—n-hexane, 2—n-heptane, 3 –chloroform, 4—DMF, 5—DMSO,

6—acetonitrile, 7 –propan-2-ol, 8 –butan-1-ol, 9—ethanol, 10 –methanol, 11—H₂O).

https://doi.org/10.1371/journal.pone.0229149.g013

The RLS results of synchronic spectra measurements and interpretation of the bands identified

in the course of decomposition of selected absorption spectra, also clearly indicate decay of the

effect in systems with increasing concentrations of the compound. The measurements of fluo-

rescence lifetimes indicate that for the pH ranges where said fluorescence effects are observed,

as well as for adequate proportions of solvents that allow the effect of dual fluorescence, the

observed fluorescence lifetimes are only slightly different from the ranges registered in systems

where the effects are not present. However, in (mixed solvent) media where dual fluorescence

does occur, we observe an additional lifetime component corresponding to the shortwave

form of the emission.

Therefore, after analyzing the presented results, we posited that the observed fluorescence

phenomena are related to the effects of excited-state electron transfer (ESIPT) taking place in

the molecule. The mechanism of dual fluorescence in SAL-3 is primarily due to the coexistence

of its ionized and non-ionized forms, which become predominant at pH levels higher/lower

than 6.5. The ionized form corresponds to a single longwave band of fluorescence emission.

Table 7. Onsager cavity radius a₀for SAL3. Slopes S₁and S₂determined by Stokes shift and ðv�_aþ v�_fÞ=2 in the function of F₁(ε,n) and F₂(ε,n), ground state μ_g, excited

state μ_e, change in dipole moment Δμ and coefficient of determination r²for SAL-3.

a₀[Å]

4,336

m₁

m₂

μ_e/μ_g

1.863

1.392

1.441

μ_e[D]^a

9.700

μ_g[D]^b

5,207

8.450

5.033

Δμ [D]^c

4.493

r²

Em 1

Em 2

2492.268

1355.502

608.444

-8268.586

-3367.164

0.72 : 0.90

0.56 : 0.90

0.96 : 0.87

11.763

7.253

3.314

2.220

1 D = 3.33564 10⁻³⁰Cm = 10⁻¹⁸statC cm

^aValue calculated from the plot of Stokes shift vs. F₁(ε,n)

b

�

Value calculated from the plot of ðv_aþ v_fÞ=2 shift vs. F₂(ε,n)

^cThe dipole change calculated from the experimental values of μ_gand μ_e

https://doi.org/10.1371/journal.pone.0229149.t007

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

Table 8. Slope m, change in dipole moment Δμ and coefficient of determination r²for SAL-3.

M

Δμ [D]

1.344

r²

Em 1

Em 2

737.859

780.620

0.98

0.89

1.383

https://doi.org/10.1371/journal.pone.0229149.t008

The non-ionized form, having assumed the correct molecular conformation with the hydroxyl

group from the hydroxybenzene ring on the side of the carbonyl group, forms a hydrogen

bond which facilitates the processes of excited-state proton transfer.

To recapitulate, it should once again be strongly emphasized that the effects of excited-state

proton transfer taking place in SAL-3 molecules are of key significance in the context of the

observed fluorescence phenomena. As such, they will be further researched in future studies

aimed at developing a more exact theoretical model of the phenomena observed in this group

of new SA analogues. The ability to provide a concise description of the mechanisms applicable

to those particular compounds may contribute to the explanation and better utilization of the

mechanisms of their bioactivity. Additionally, doe to their particular photophysical properties,

the compounds from this group may also potentially be used as effective fluorescence probes

or molecular switches, which continue to be in high demand in the context of molecular

biology.

Supporting information

S1 Fig. Topographical and nanomechanical AFM mapping of SAL-3 sample (5 μm scan

length).

(PDF)

S2 Fig. 3D surface topography of the sample from S1 Fig.

(PDF)

S3 Fig. Topographical and nanomechanical AFM mapping of a SAL-3 sample (2 μm scan

length).

(PDF)

S4 Fig. Topographical and nanomechanical AFM mapping of a SAL-3 sample (2 μm scan

length).

(PDF)

S5 Fig. Topographical and nanomechanical AFM mapping of a SAL-3 sample (1 μm scan

length).

(PDF)

S6 Fig. Example of electronic absorption spectra decomposition for SAL-3 at pH 7.4. Solid

black line–experimental spectrum; solid grey line–spectrum fitted with the decomposition

components; blue, red, and green lines–respective components of the given band after decom-

position.

(PDF)

S7 Fig. Graphic symbols. Top–variety of the possible forms of the investigated SAL-3 deriva-

tive. Bottom–Fluorescence emission spectra of SAL-3 recorded in H₂O : 2-propanol mixture

with dual emission marked in green.

(PNG)

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process

Author Contributions

Conceptualization: Arkadiusz Matwijczuk.

´

Data curation: Agnieszka Niemczynowicz, Iwona Budziak, Sławomir Kulesza, Andrzej Gor-

ecki, Marcin Makowski, Dariusz Karcz, Janusz Podleśny, Agnieszka I. Piotrowicz-Cieślak,

Arkadiusz Matwijczuk.

´

Formal analysis: Agnieszka Niemczynowicz, Iwona Budziak, Sławomir Kulesza, Andrzej Gor-

ecki, Marcin Makowski, Karolina Starzak, Bożena Gładyszewska, Janusz Podleśny,

Agnieszka I. Piotrowicz-Cieślak, Arkadiusz Matwijczuk.

Funding acquisition: Bożena Gładyszewska, Janusz Podleśny, Arkadiusz Matwijczuk.

´

Investigation: Agnieszka Niemczynowicz, Iwona Budziak, Sławomir Kulesza, Andrzej Gor-

ecki, Dariusz Karcz, Arkadiusz Matwijczuk.

´

Methodology: Agnieszka Niemczynowicz, Iwona Budziak, Sławomir Kulesza, Andrzej Gor-

ecki, Marcin Makowski, Dariusz Karcz, Karolina Starzak, Bożena Gładyszewska, Janusz

Podleśny, Agnieszka I. Piotrowicz-Cieślak.

Project administration: Dariusz Karcz, Karolina Starzak, Bożena Gładyszewska, Janusz

Podleśny, Arkadiusz Matwijczuk.

Resources: Agnieszka Niemczynowicz, Agnieszka I. Piotrowicz-Cieślak.

´

Software: Iwona Budziak, Sławomir Kulesza, Andrzej Gorecki, Marcin Makowski, Karolina

Starzak, Bożena Gładyszewska.

Supervision: Marcin Makowski, Arkadiusz Matwijczuk.

´

Validation: Agnieszka Niemczynowicz, Sławomir Kulesza, Andrzej Gorecki, Marcin

Makowski, Dariusz Karcz, Karolina Starzak, Bożena Gładyszewska, Janusz Podleśny,

Agnieszka I. Piotrowicz-Cieślak, Arkadiusz Matwijczuk.

Visualization: Arkadiusz Matwijczuk.

Writing – original draft: Agnieszka Niemczynowicz, Iwona Budziak, Sławomir Kulesza, Mar-

cin Makowski, Agnieszka I. Piotrowicz-Cieślak, Arkadiusz Matwijczuk.

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Spectroscopic and theoretical studies of fluorescence effects induced by the ESIPT process in a new derivative 2-Hydroxy-N-(2-phenylethyl)benzamide – Study on the effects of pH and medium polarity changes

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