Solvent Effects on Molecular Aggregation in 4-(5-Heptyl-1,3,4

Article pubs.acs.org/JPCB

Solvent Effects on Molecular Aggregation in 4‑(5-Heptyl-1,3,4thiadiazol-2-yl)benzene-1,3-diol and 4‑(5-Methyl-1,3,4-thiadiazol-2yl)benzene-1,3-diol Arkadiusz Matwijczuk,*,† Dariusz Kluczyk,⊥ Andrzej Górecki,§ Andrzej Niewiadomy,‡,∥ and Mariusz Gagoś*,⊥ †

Department of Biophysics, University of Life Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland Department of Chemistry, University of Life Sciences in Lublin, 20-950 Lublin, Poland § Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland ∥ Institute of Industrial Organic Chemistry, Annopol 6, 03-236 Warsaw, Poland ⊥ Department of Cell Biology, Institute of Biology, Maria Curie-Skłodowska University, 20-033 Lublin, Poland ‡

ABSTRACT: The article presents the results of spectroscopic studies of 4-(5-methyl-1,3,4-thiadiazol-2-yl)benzene-1,3-diol (C1) and 4-(5-heptyl-1,3,4-thiadiazol-2-yl)benzene-1,3-diol (C7) in organic solvent solutions. Depending on the concentration of the compound used, three bands were observed in the fluorescence emission spectra of the compounds in DMSO solutions. A single band was observed in methanol, propan-2-ol, or ethanol. The significantly shortened fluorescence lifetimes and the different shapes of circular dichroism (CD) spectra clearly indicate association of the fluorescence effects with the aggregation processes in the analyzed compounds. The differences in the course of the CD spectra also imply an effect of the substituent group structure on the molecule aggregation interactions. Therefore, it has been postulated that the occurrence of the different spectral forms induced by changes in the compound concentration may be related to the aggregation effects of C1 and C7 molecules, which are also induced by differences in the alkyl substituent structure.

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INTRODUCTION 1,3,4-Thiadiazoles with a substituted resorcyl fragment are extremely promising compounds in fighting diseases, in particular, neoplastic conditions. 1,3,4-Thiadiazoles were first described in 1882 by Fischer.1 Compounds comprised in this group have been widely applied as, for example, metal complexing agents,2,3 oxidation inhibitors, and various pigments. However, 1,3,4-thiadiazoles mainly exhibit anticancer,4 antifungal,5 antibacterial,6 anti-inflammatory,7 antioxidant,8 antihypertensive,9 and neuroprotective activity.10 Given their specific therapeutic properties, 4-(5-heptyl-1,3,4-thiadiazol-2-yl)benzene-1,3-diol (C7, Scheme 1A) and 4-(5-methyl-1,3,4-thiadiazol-2-yl)benzene-1,3-diol (C1, Scheme 1B) were chosen for the investigation of the molecular interactions presented in this study. The analyzed compounds exhibit interesting spectroscopic effects, which can be associated with the effects of keto/enol © 2016 American Chemical Society

Scheme 1. Chemical Structure of the C7 Molecule (A) and the C1 Molecule (B)

Received: June 22, 2016 Revised: July 25, 2016 Published: July 25, 2016 7958

DOI: 10.1021/acs.jpcb.6b06323 J. Phys. Chem. B 2016, 120, 7958−7969

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The Journal of Physical Chemistry B tautomerism induced by changes in medium polarizability,11,12 crystal polymorphism13 and solvatomorphism,14 and molecular interactions in model lipid systems.15 1,3,4-Thiadiazoles exhibit dual fluorescence effects16 or emit two separate fluorescence spectra, but to date, the mechanism of these phenomena has not been identified and fully elucidated. The association of the spectroscopic and conformational effects in the papers mentioned above may be important for elucidation of the pharmacological activity of the analyzed compounds. The aim of the investigations was to carry out a spectroscopic study of the molecular organization of 1,3,4-thiadiazole compounds at various concentrations in organic solvents and to elucidate the molecular mechanism involved in the effects observed in the spectra. On the basis of absorption spectroscopic and electron fluorescence methods (combined with the resonance light scattering (RLS) technique) as well as fluorescence lifetimes (time-correlated single-photon counting, TCSPC) and circular dichroism (CD) methods, the complexity of the physical processes involved in the appearance of three fluorescence bands in C7 and C1 was indicated. An attempt was made to elucidate the effect observed in both analogues with fluorescence methods and the exciton splitting theory combined with previous results of investigations of this group of compounds. Additionally, as evidenced by available results of crystallographic studies, it is possible that the resorcyl substituent of the compound is rotated, depending on the polarity of the solvent.14 The dual fluorescence effects or the effects of several fluorescence forms are associated with the appearance of two clearly separated emission bands upon electron excitation.17−21 These effects can be induced by solvent polarity and changes in pH,22 temperature, and concentration.19 The most common explanation of the dual fluorescence effect is the appearance of intramolecular charge-transfer (CT) states23 and, primarily, CT states with TICT (twisted intramolecular charge transfer).24 N,NDimethyl aminobenzonitryle (DMABN) is an example of a molecule with the TICT state. This model was introduced in the 1970s24 by K. Rotkiewicz, K. H. Grellmann, and Z. R. Grabowski. Despite some disagreement concerning its mechanism, the TICT approach has become one of the best-known models in experimental25 and theoretical26 studies in the field of photophysics and molecular spectroscopy. In this case, the ratio of the intensities of the emissions depended on the solvent polarity and temperature; in nonpolar solvents, the dual fluorescence disappeared.27 TICT states can be observed when a molecule in an excited state exhibits a very large dipole moment (∼20 D)27 achieved rapidly along an increase in the polarity of the medium. Furthermore, dual fluorescence can can be evoked by the existence of molecule tautomerism in excited states, that is, the socalled photoautomers. They have a characteristic emission spectrum differing from the spectrum of an ordinary excited molecule; these dual fluorescence effects are characteristic for compounds representing the groups of flavones and coumarins. Other causes of the appearance of multiple fluorescence bands include excimer emissions induced at an increasing concentration of the solution27 (which is, obviously, not contradictory to Kasha’s rule). Dual fluorescence can also be induced by protolytic reaction or proton attachment to the molecule, which corresponds to a change in the acid-alkaline properties.27 It is also worth mentioning at this point that an equally common explanation of the occurrence of multiple fluorescence bands is associated with the excited-state intramolecular proton transfer (ESIPT) process.28,29 A possibility of formation of

intramolecular hydrogen bonding is a condition for this type of effect to arise.30 In the case of the analyzed compounds, the fluorescence effects observed may clearly be influenced by the aggregation factor combined with the specific molecule conformation, which changes depending on the medium;16 aggregation can induce specific interactions of the analyzed molecules, leading to spectral effects. An equally important factor is the structure and properties of the molecules selected for the investigations. The investigations of the C1 and C7 compounds conducted with stationary and excitation fluorescence spectroscopy methods revealed the existence of three distinct fluorescence bands induced by different concentrations of the compounds and the solvent type. Elucidation of the mechanism of organization of the 1,3,4-thiadiazole compounds in different-polarity organic solvents may be important for determination of their pharmacological applications and for further model research aimed at identification of molecular traits that have an impact on pharmacological properties. Furthermore, it can be assumed that, due to their high quantum efficiency, the analyzed compounds can also be used as fluorescent probes with sensitivity to changes in pH or ambient temperature.

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MATERIAL AND METHODS Materials. The 4-(5-methyl-1,3,4-thiadiazol-2-yl)benzene1,3-diol (C1) and 4-(5-heptyl-1,3,4-thiadiazol-2-yl)benzene-1,3diol (C7) (see Scheme 1A,B) were synthesized in the Department of Chemistry of the University of Life Sciences in Lublin; details of the procedure are described elsewhere.31 The purification procedures of the 1,3,4-thiadiazoles compound are described in detail in refs 13 and 14. Methods. Electronic Absorption and Fluorescence Spectra. Electronic absorption spectra of C1 and C7 were recorded on a double-beam UV−vis spectrophotometer Cary 300 Bio (Varian) equipped with a thermostated cuvette holder with a 6 × 6 multicell 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 °C. Fluorescence spectra were recorded at 0.5 nm resolution and corrected for the lamp and photomultiplier spectral characteristics. RLS measurements were performed as in Pasternack and Collings.32,33 The excitation and emission monochromators of the spectrofluorimeter were scanned synchronously (0.0 nm interval between excitation and emission wavelengths), and the slits were set to obtain spectral resolution of 1.5 nm. The spectral analysis was performed with the use of Grams/AI 8.0 software (Thermo Electron Corporation). Time-Correlated Single-Photon Counting (TCSPC). TCSPC measurements were performed on a FluoroCube fluorimeter (Horiba, France). The samples were excited with a pulsed NanoLED diode at 372 nm (pulse duration of 150 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 picosecond detector TBX-04 (IBH, JobinYvon, UK). The DataStation and the DAS6 software (JobinYvon (IBH, U.K.)) were used for data acquisition and signal analysis. All fluorescence decays were measured in a 10 × 10 mm quartz cuvette using an emitter cutoff filter with transmittance for wavelengths longer than 408 nm. The excitation profiles required for the deconvolution analysis were measured without the emitter filters on a light scattering cuvette. 7959


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The Journal of Physical Chemistry B

Figure 1. (A,C) Normalized electron absorption spectra in the selected organic solvents: methanol (dashed blue line), ACN (red line), ethanol (solid blue line), propanol (dashed black line), butanol (solid black line), and DMF (dashed gray line) for C7 (A) and C1 (C). (B,D) Fluorescence emission bands normalized in the maximum for C1 (D) and C7 (B) in the same organic solvents as those described above (see Table 1 for the fluorescence lifetime data). The emission spectra for all analyzed samples were obtained at an excitation wavelength in the maximum of the absorption band. All absorption and fluorescence emission spectra were obtained at a temperature of 23 °C.

DMF, and butanol. With the increasing solvent polarity, a hypsochromic (panels A and C and Figure 2A,B) spectral shift (from 317 to 323 nm for C7 (panel A) and from 316 to 323 nm for C1, panel C)) was observed. In the case of both compounds, a broad band in the wavelength range from 300 to 350 nm with a maximum at ∼320 nm, which is characteristic for the π → π* electron transition, can be observed. In both compounds, there are clear bands with considerably lower absorption and a maximum at ∼360 nm. The intensity of these bands (substantially lower than that of the bands with a maximum at 320 nm) and the corresponding bathochromic shift imply potential presence of aggregated forms of the compounds (dimers, N-aggregates; see Figure 1A,C) in the analyzed samples. Panels B and D in Figure 1 present the fluorescence emission spectra of the analyzed compounds corresponding to the respective absorption spectra shown in panels A and C. The excitation wavelength for all samples corresponds to the maximum of the absorption spectra (Figure 1A,C). The fluorescence emission spectra reveal bathochromic shift effects. All of the presented fluorescence emission spectra exhibit a single fluorescence band with a maximum from 361 nm (C7 in DMF) to 374 nm (C7 in Et−OH) and from 362 nm (C1 in DMF) to 371 nm (C1 in Et−OH) (Figure 1B,D). It can be noted that the change in the medium polarity exerts a greater impact on the stability of the molecule excitation state in the case of C7 than that in C1 (Figure 1B,D and Table 1). To clarify the mechanism of the bathochromic shift of the analyzed compounds (Figure 1), in Figure 2, we show the

All measurements were performed in DMSO at room temperature. Fluorescence decay was analyzed with a multiexponential model according to the equation It =

⎛ −t ⎞ ⎟ ⎝ τi ⎠

∑ αi exp⎜ i

(1)

where αi and τi are the pre-exponential factor and the decay time of component i, respectively. Best-fit parameters were obtained by minimization of the reduced χ2 value as well as residual distribution of experimental data. The average lifetime of fluorescence decay was calculated according to the following equation ⟨τ ⟩ =

∑i αiτi2 ∑i αiτi

(2)

CD Measurements. CD measurements were carried out on a J710 spectrometer (JASCO, Japan) using a 1 mm path length quartz cell. The spectra were collected in the range of 240−600 nm with a 1 nm data pitch, 20 nm/min scanning speed, 4 s response time, and 2 nm bandwidth and averaged over three acquisitions. DMSO compounds were used as blanks, and their spectra were subtracted from the raw data.

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RESULTS AND DISCUSSION Figure 1A,C presents electron absorption spectra for C7 and C1 obtained in methanol, acetonitrile (ACN), ethanol, propan-2-ol, 7960


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2 presents a change in the Stokes shift as a function of the polarizability of the analyzed medium (known as the Lippert− Mataga equation). Evidently, the presented dependencies are not linear22 for either of the analyzed compounds. This may indicate changes in the value of the dipole moment in the excited state or a change in its direction relative to the ground state.34 It should be emphasized that in the analysis of solvatochromic effects the Lippert−Mataga function is more accurate in the description of the interaction of molecules with the solvent and the intermolecular interactions in the ground and excited states than other dependencies. The presence of the solvent modifies absorption and fluorescence spectra of the analyzed compounds, usually leading to a shift of their maxima toward longer rather than shorter wavelengths; however, the presence of intermolecular hydrogen bonds (often resulting in compound aggregation) may cause deviation from this model.34 In the present study, special attention should be paid to the effects shown in Figures 3 and 4 (Panels D,E,F), which present fluorescence emission spectra at different concentrations of the compounds in DMSO analyzed in order to determine the interactions in the solutions for both compounds. Figure 3Ad−Af presents the appearance of three fluorescence bands for C7 depending on the changes in the concentration of the analyzed compound. In turn, Figure 3Aa−Ac shows electron absorption spectra corresponding to the concentrations in the fluorescence emission spectra. The electron absorption spectra exhibit a characteristic band with a maximum at 318 nm (Figure 1) and bands with maxima at 365 and 435 nm appearing at an increasing concentration of the compound, which should be associated with molecular aggregation. The first fluorescence band with a maximum at 371 nm can be observed in the fluorescence emission spectra at an excitation wavelength of 318 nm (panels Aa and Ad). With the increase in the concentration in the range of c1− c3, the fluorescence intensity gradually increases and then begins to decline at c4, which is characteristic for the effect of concentration-dependent fluorescence quenching. At concentration c5 and an excitation wavelength of 318 nm, only a residual fluorescence emission spectrum with a maximum at 371 nm can be observed. The second fluorescence with a maximum at 408 nm starts to be visible at an excitation wavelength of 365 nm (Figure 3Ab,Ae). With the increase in the compound concentration, the first fluorescence band is completely quenched and the emission band with a maximum at 408 nm becomes more intense. A further increase in the concentration of the compound results in a dual fluorescence effect, that is, fluorescence with a maximum at 408 nm and a new band shifted toward longer wavelengths with a maximum at 510 nm (Figure 3Ae,Af). At higher concentrations of the solution and the high c7−8 concentrations (Table 2), the second fluorescence with a maximum at 408 nm is completely quenched and only an increase in the fluorescence with a maximum at 510 nm can be observed (panel Af). Its intensity rises particularly rapidly at sample excitation at a wavelength of 435 nm (see panels Ac and Af; the band intensity was divided by 3 for better presentation of the results). Panels Ba−Bc show changes in the intensity of the fluorescence emission of the respective bands, depending on the concentration of the analyzed solution. Evidently, the change in the concentration is accompanied by a change in the intensity of the consecutive fluorescence bands of the analyzed compound, and dual fluorescence with a maximum at 408 nm shifted toward 430 nm with long-wave emission at 510 nm can be observed at certain c5−6 concentration values. The appearance of the bands at 365 and 435 nm in the absorption spectrum of the analyzed compound together with the increase in

Figure 2. (A) Position of the absorption maximum for C1 and C7 depending on the average electric dipole polarizability (in 10−24 cm3). (B) Relationships between the position of the Stokes shift for C1 and C7 and the average electric dipole polarizability (in 10−24 cm3). The numbers in both panels denote the following solvents: 1, H2O; 2, Mt−OH; 3, ACN; 4, Et−OH; 5, acetone; 6, DMSO; 7, propan-2-ol; 8, butanol; and 9, chloroform (see Table 3). In both panels, straight lines were fitted to the experimental points for the observed electron transition. All parameters are presented in Table 3. White dots, values for C1; black dots, values for C7.

Table 1. Fluorescence Lifetimes of C1 and C7 in the Selected Solvents solvents

C1 τ ± Δ τ [ns]

C7 τ ± Δ τ [ns]

methanol propanolol butanol ACN DMF DMSO

2.507 ± 0.025 2.157 ± 0.024 2.508 ± 0.019 2.436 ± 0.098 2.815 ± 0.021 2.036 ± 0.046

1.873 ± 0.037 2.186 ± 0.021 2.599 ± 0.021 2.354 ± 0.171 2.878 ± 0.020 3.493 ± 0.054

relationships between the position of the absorption maximum for the π → π* electron transition (panel A) and Stokes shift (panel B), depending on changes in the polarizability of the solvent used. As is evident for C1 and C7 (panel A), the linear slope coefficients are negative for the selected polar solvents, which may be associated with an increase in the dipole moment of the molecule in an excited state and indicate that its direction is retained during the electron transition. A considerably lower fit of the relationships is obtained by description of the correlations between the absorption maximum and the Kirkwood polarity function (related to the dielectric constant of the medium) or the Lorenz−Lorentz function (associated with changes in the refractive index of the medium) (not shown). Panel B in Figure 7961


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Figure 3. (Aa−Ac) Electron absorption spectra for C7 in DMSO at different concentration values (Table 2). (Ad−Af) Fluorescence emission spectra for C7 in DMSO at the selected concentration values (Table 2). The excitation wavelengths are 318 nm in (Ad), 318 and 365 nm in (Ae), and 365 and 435 nm for the spectra in (Af). (Ba−Bc) Fluorescence emission intensity at 370 (Ba), ∼430 (Bb), and 510 nm (Bc) depending on the concentration of the analyzed compound in DMSO. In (Af), the intensity of the spectrum with a maximum at 510 nm was divided by 3 for better visualization of the results. All absorption and fluorescence emission spectra were obtained at a temperature of 23 °C.

fluorescence emission spectra depending on the changes in the compound concentration. The relationships between the changes in fluorescence intensity and the compound concentration are presented in panels Ba−Bc. Similar effects depending on changes in the concentration can be observed, but in this case, the impact of the alkyl substituent on the fluorescence effects is evident. The long-wave spectral bands in the electron absorption spectra corresponding to the aggregation process are not so clearly visible but noticeable. Additionally, all three fluorescence forms with maxima at 370, 425, and 550 nm can be observed in the fluorescence emission spectra, but they are presented more selectively. In the case of C1, the intermediate states, in which it would be possible to observe a similar dual fluorescence effect as in the case of C7, cannot be captured (Figure 3Ae,Af). In conclusion, the impact of the two factors described above on the

the concentration implies a probability of the presence of other than monomeric spectral forms of the compound.35 The RLS technique most likely reveals chromophore aggregation effects in C7 and C1.36 On the basis of exciton splitting theory and spectral shifts, it was possible to calculate the distance between the adjacent chromophores of the C1 and C7 molecules.37 The distance between the adjacent chromophores is 3.91 Å in the case of the C7 dimers (in the DMSO solution) and 4.34 Å for C1.38 These results are in agreement with crystallographic data demonstrating slightly smaller distances, which is reasonable, given the substantially denser packing of the molecules in the crystalline structure. Figure 4 presents the results of an analogical experiment as that for the C1 compound shown in Figure 3. Panels Aa−Ac show electron absorption spectra, and panels Ad−Af present 7962


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Figure 4. As in Figure 2, (Aa−Ac) Electron absorption spectra for C1 in DMSO at different concentration values (see Table 2). (Ad−Af) Fluorescence emission spectra for C1 in DMSO at the selected concentration values (see Table 2). The excitation wavelengths are 317 nm in (Ad), 317 and 370 nm in (Ae), and 370 and 410 nm for the spectra in (Af). (Ba−Bc) Fluorescence emission intensity at 370 (Ba), ∼425 (Bb), and 550 nm (Bc) depending on the changes in the concentration of the analyzed compound in DMSO. All absorption and fluorescence emission spectra were obtained at a temperature of 23 °C.

of the alkyl substituent in the analyzed 1,3,4-thiadiazoles. The multitude of the spectral structures also suggests that in the case of both C1 and C7 compounds dimerization takes place first and is followed by the N-aggregation effect upon the concentration increase (discussed further in the text). We can postulate that the fluorescence emission spectrum with a maximum at 371 nm may be typical of the monomeric structures of the analyzed compounds, whereas the bands with maximum emissions at 410 and 510 nm (510 nm for C7 and 550 nm for C1) result from two types of aggregation. The first stage involves dimerization, in which fluorescence with a maximum at 408 nm (410 nm for C1) and dual fluorescence with maxima at 408 and 510 nm for C7 as well as 410 and 550 nm for C1 are observed. In the final interaction stage, long-wave fluorescence is only observed in the spectra of both compounds at N-aggregation.

Table 2. Fluorescence Lifetimes of C1 and C7 at the Different Concentrations of the Compound DMSO −6

c1 = 8.88 × 10 c2 = 2.14 × 10−5 c3 = 7.86 × 10−5 c4 = 1.90 × 10−4 c5 = 7.43 × 10−4 c6 = 1.8 × 10−3 c7 = 1.30 × 10−2 c8 = 3.26 × 10−2 c9 = 2.56 × 10−1

C1 τ ± Δ τ [ns]

C7 τ ± Δ τ [ns]

2.233 ± 0.036 0.956 ± 0.014 0.291 ± 0.005 0.292 ± 0.009 0.284 ± 0.009 0.272 ± 0.006 0.272 ± 0.006 0.166 ± 0.017 0.323 ± 0.011

6.854 ± 0.063 2.199 ± 0.019 1.441 ± 0.011 1.374 ± 0.009 1.011 ± 0.006 1.001 ± 0.006 0.963 ± 0.006 0.937 ± 0.005 1.187 ± 0.006

observed spectral effects should be underlined, primarily the molecular aggregation phenomenon and changes in the structure 7963


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Figure 5. Effect of the concentration on C7 (A) and C1 (B) fluorescence decay. Dotted lines show decays of fluorescence emission in DMSO at a given concentration of the fluorophore, while solid lines are triple exponential fits. (C,D) Plots of the residuals determined for the presented fits for C7 and C1, respectively. The excitation pulse profile set up at 372 is shown by the dotted black line.

Figure 6. Effect of the concentration on the fluorescence lifetime in DMSO for C1 (A−C) and C7 (D−F). The dependence of the mean fluorescence lifetimes of triple exponential fits at a given concentration of the fluorophore is shown in (A,D), while fluorescence lifetimes of components and their fractional contributions are presented in (B,C) for C1 and (E,F) for C7, respectively.

TCSPC Measurements. Figure 5A,B presents the results of measurements of fluorescence lifetimes for the different concentrations of the C1 and C7 samples in DMSO. Light with

a wavelength of 372 nm was applied for induction of excitation. The selected excitation wavelength is suitable for the long-wave edge of absorption of the monomeric form and resonant 7964


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Figure 7. Fluorescence excitation spectra (Ex) for C1 (A) and for C7 (B) obtained in DMSO for the selected values of the compound concentrations. Excitation emission was recorded at a wavelength of 370 nm (concentration c2) in both panels and at 410 nm (c7, dashed red line), 540 nm (c9, solid blue line), and 540 nm (c10, dashed blue line) in (A) and at 426 nm (c7, dashed red line), 525 nm (c9, dashed blue line), and 525 nm (c10, solid blue line) in (B). All fluorescence emission spectra were obtained at a temperature of 23 °C.

Figure 8. RLS spectra for C1 (A) and for C7 (B) obtained at different concentrations of the analyzed compounds in a DMSO solution. The concentrations correspond to the values presented in Figures 2, 3, and 7 (see also Table 3). All spectra were obtained at a temperature of 23 °C.

components of fluorescence lifetimes. The component with the longest fluorescence lifetime is reduced from ∼13 to ∼2 ns for C1 and from ∼19 to ∼3 ns in the case of C7. In turn, the shorterlifetime component is reduced from ∼2 to ∼0.5 ns for C1 and from ∼3 to ∼1 ns for C7. A highly significant impact on the shortening of the fluorescence lifetime is also exerted by the increase in the contribution of the shortest fractional component, which takes place already for the second concentration (increased proportion from ∼25 to ∼60% for C7 and from ∼60 to ∼80% for C1). If the occurrence of the individual components is identified with the monomeric fraction and different oligomeric forms of the fluorophore, the shorter component is certainly characteristic for the oligomeric form, which is consistent with literature data. The change in the lifetimes of the different fractions may be related to the different sizes of resulting oligomers or the presence of a greater number of forms with characteristic constant lifetimes, which could not be properly separated due to the limitation of the method (in this case, the fractions could not be directly identified with the individual forms of the fluorophore). Nevertheless, it should be emphasized that the clear reduction in both recorded fluorescence lifetimes evidently suggests involvement of aggregation processes in the fluorescence effects.

excitation of the aggregated form. The fluorescence decay was monitored using the TCSPC method at a temperature of 22 °C for light with a wavelength greater than 408 nm, that is, fluorescence emitted by all oligomeric forms was observed (see Figures 3 and 4). The results were analyzed with deconvolution of the fluorescence decay using the apparatus profile and eq 1, each time for i = 1, 2, 3, and 4. A three-exponential fluorescence decay model was most optimal for both compounds at all of the concentrations analyzed. The analysis, based on a lower number of components, did not allow accurate representation of the fluorescence intensity decay, and addition of the fourth component did not improve the quality of the fit, which was verified by the value of the fit parameter and residual distribution analysis (Figure 5C,D). Lifetime measurement is an excellent method for assessment of the state of fluorophores at different concentrations because the measured parameters are not directly dependent on it. Therefore, the changes accompanying the concentration gradation indicate the occurrence of different forms of fluorophores, whose proportion changes as a concentration function. The reduction of the average fluorescence lifetime observed for both compounds is mostly visible at the initial increase in the concentration (Figure 6A,B). This is caused by the significant shortening of the two longer 7965


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The Journal of Physical Chemistry B Figure 7 shows fluorescence excitation spectra (Ex) for C1 (panel A) and for C7 (panel B) obtained in DMSO solutions of the compounds. The excitation emission was recorded at a wavelength of 370 nm (concentration c2) in both panels, at 410 nm (c7, dashed red line), 540 nm (c9, solid blue line), and 540 nm (c10, dashed blue line) in panel A and at 426 nm (c7, dashed red line), 525 nm (c9, dashed blue line), and 525 nm (c10, solid blue line) in panel B, that is, at a maximum of the fluorescence emission spectra from Figures 3 and 4. At excitation of the C7 compound (panel B) with a wavelength of 370 nm, an initial increase in the band intensity with a maximum at 319 nm and its slight bathochromic shift to 323 nm are observed (not shown). Next, the increase in the compound concentration is accompanied by a decline in the band intensity and very distinct splitting of the band at 319 nm to 345 and 261 nm. Along the increasing compound concentration, virtually complete quenching of these bands and appearance of long-wave bands with a maximum at 385 nm (excitation at 525 nm) and 450 nm (excitation at 525 nm) are observed. It can be noted that a long-wave band with a maximum at 365 nm (shifting toward 385 nm) appears at an excitation wavelength of 425 nm (dashed red line at concentration c7). Similar effects are observed in the case of the excitation spectra recorded for C1 (see Figure 7A). On the basis of exciton splitting theory, the characteristic long-wave bands should clearly be assigned to the different aggregated forms of the analyzed compounds, which is also evident in the case of the RLS spectra presented in Figure 8 (discussed below). As can be seen, the bands

The changes in the compound concentrations are accompanied by a considerable change in the shape of the RLS bands of both compounds. In the concentration range in which only the first fluorescence emission band with a maximum at ∼370 nm is observed, the RLS bands of the compounds change only inconsiderably. With the increasing compound concentrations as well as the disappearance of the first band of fluorescence and simultaneous appearance of the second band, a decline in a part of the RLS signal with a maximum at ∼300 nm (for both compounds) is noted. Drastic changes in the RLS spectra take place at a concentration with the longest-wave fluorescence band only. Evidently, the RLS signal decays completely from a major part of the recorded range in both cases, and very distinct bands appear in the region above 450 nm in the case of both compounds. In conclusion, it should be noted that the RLS bands assigned to the chromophore aggregation of molecules C1 and C7 are very characteristic and the observed fluorescence effects are clearly associated with the chromophore aggregation of molecules C1 and C7 because distinct RLS bands are visible in both C1 and C7 (in DMSO). The oscillatory structure of the RLS bands indicates a multitude of structures of the aggregated compounds differing in sizes33 (this may confirm our earlier assumption of the existence of several aggregated forms of the investigated molecules). Energy Level Schemes. On the basis of the presented fluorescence studies, several spectral forms accompanying the change in the concentration of the analyzed compounds in DMSO were noted and are shown in Figure 7. Given the calculations performed according to exciton splitting theory and the results presented in Figures 3, 4, 7, and 8, we attempted a determination of energy level schemes for C1 and C7, which is shown in Figure 9A (for C7) and B (for C1). Assuming the presence of the different aggregated forms of the compound, emergence of several types of aggregated forms of both compounds with the increase in their concentration can be postulated. In the case of the aggregate I type, the width of the band gap for C7 between electron states at 296 nm (33784 cm−1) and 408 nm (24510 cm−1) is 4β1 = 9274 cm−1 (panel A), under the assumption of the presence of aggregated structures (Naggregates, in accordance with the exciton splitting theory). Hence, it can be assumed that this is the first aggregated form, aggregate I. In this case, there is a slight decrease in the electron level ΔΣ1 = 2196 cm−1 in relation to the electron energy level characteristic for the monomeric form with a maximum at 319 nm (31347 cm−1), which can be clearly seen both in the electron absorption spectrum and in the fluorescence excitation spectrum (Ex(Em370)). Emission of the monomeric form is observed with a maximum at 371 nm (26954 cm−1). Very distinct bands associated with strong aggregation (characteristic for long-wave aggregates) with maxima at ∼345 nm (28986 cm−1) and 450 nm (22222 cm−1) can be observed in the fluorescence excitation spectra as well. With the increase in the compound concentration, subsequent exciton band splitting can be seen between 345 and 430 nm (23256 cm−1), that is, 4β2 = 5730 cm−1. In this case, the reduction of the energy level relative to the level characteristic for the monomeric form is ΔΣ2 = 5227 cm−1, which is approximately 2-fold greater than that in the first reduction characteristic for aggregates I. On the basis of the analysis of the fluorescence spectra, the presence of type III aggregated forms can be postulated with exciton splitting between 450 and 510 nm (from the fluorescence emission spectrum) (19608 cm−1) and 4β3 = 2614 cm−1. In this case, a ΔΣ3 = 10433 cm−1 reduction of the energy level is observed. In accordance with the theoretical assumptions, the RLS bands should be located near the

Table 3. Values of the Molar Extinction Coefficient and the Transition Dipole Moment for C1 and C7 C1

C7

solvents

ε [M−1 cm−1]

μ [Deby]

ε [M−1 cm−1]

μ [Deby]

DMSO methanol ethanol 2-propanol butanol ACN DMF chloroform

14576.91 12531.75 13756.65 14257.45 15173.54 11082.22 13776.74 12332.79

3.85 3.77 3.89 4.93 4.14 3.40 3.81 3.63

16638.97 14136.16 14698.89 14982.26 15601.43 12834.31 14007.11 13930.36

4.13 3.93 4.02 4.12 4.22 3.67 3.91 3.80

are present for both C1 (panel A) and C7 (panel B), but they exhibit different shapes, which may indicate differences in the structure of the aggregated forms. According to exciton splitting theory, these shifts are associated with two aggregation types, that is, the hypsochromic shift is related to emergence of “card pack” aggregates and the bathochromic shift is associated with “head-totail” aggregation.39,40 Therefore, it can be concluded that the fluorescence effects observed are associated with the aggregation effects of the C1 and C7 molecules in the selected solvents. Below, we present the results of RLS (Δλ = 0) analyses, which largely confirm these assumptions. Resonance Light Scattering (RLS) Study. In order to investigate the effect of aggregation on fluorescence, RLS spectra were obtained for C7 and C1 in DMSO, depending on the changes in the medium concentration. Panel A in Figure 8 presents RLS spectra for C1 generated in the DMSO solution at the different compound concentrations. In turn, panel B shows analogous RLS spectra for C7 also obtained in the DMSO solution at the selected compound concentrations. The presence of the RLS bands should be associated with chromophore aggregation of the components contained in the solution.32,33 7966


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Figure 9. Diagram of the energy levels of monomeric and aggregated forms for C1 (B) and C7 (A). Symbols A, F, and RLS denote absorption (upward vertical solid lines), emission (downward dashed lines), and resonance light scattering (vertical lines with curvature), respectively. Parameter β describes the dipole−dipole interaction energy of aggregated C1 and C7 molecules. ΔΣ denotes the distance between the S1 level and the center of the band of aggregated forms. Wavy lines denote nonradial internal conversion.

Figure 10. Effect of the C1 and C7 concentrations on the CD spectra of the analyzed compound. The measurements were performed in DMSO for the different concentrations indicated in the graph. (A) Results for C1; (B) results for C7.

Figure 8 reveals a clear increase in the RLS signal intensity at ∼350 and 450 nm (in both panels of Figure 8) and a strong maximum at ∼517 nm. This indicates that the band represents the bottom of the exciton band, probably of aggregate III, a “super aggregate”. As already mentioned in the discussion of the fluorescence emission

absorption bands of the aggregated form33 associated with transition to the lowest exciton level. Therefore, in the case of aggregate form III, the band with a maximum at 510 nm can be assigned to the “bottom” of the exciton band (highly distinct in the RLS spectra, Figure 8). The analysis of the RLS bands from 7967


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fluorescence spectroscopy, fluorescence lifetimes, and CD spectroscopy show that this phenomenon indicates strong dependence of this effect on molecular aggregation. The measurements of the fluorescence spectra of the compounds in DMSO at different concentrations demonstrate the presence of the effect at the specified concentration values. The analyses carried out with the use of the RLS and CD techniques confirm the relationship between the observed effects and chromophore aggregation of the selected C1 and C7 molecules. Most probably, the three fluorescence emission bands observed for C1 and C7 are associated with the molecular aggregation phenomenon as well as their structure, in particular, the difference in the alkyl chain structure, which evidently changes the intermolecular interactions in the analyzed systems. The calculations based on exciton splitting theory and the measurements of relevant synchronous spectra and fluorescence excitation spectra contributed to recognition of the pattern of the electron levels in the analyzed molecules. The synchronous spectra exhibit clear bands that show an exciton spectrum bottom, which is characteristic for the highly aggregated form of the investigated compounds. Furthermore, the relevant fluorescence emission spectra obtained at the various proportions of the organic solvents used in the study indicate an impact of the molecular structure of C1 and C7 on the aggregation pattern.

spectra, in this case, the dimerization process may have taken place in the first phase (or, more likely, aggregates have already been formed), and stronger aggregation may proceed in the subsequent phase of the concentration increase. Figure 9 presents the electron level schemes for the different potential aggregated forms. It should be emphasized here that the relatively close position of the bottom of the exciton band of the aggregate to the monomeric form may be explained by the similar intensities of the fluorescence emission spectra of the investigated compounds. In the case of the energy level scheme for C1 presented in Figure 9B, there is a similar situation as that for C7, that is, absorption to the singlet state of the monomeric form at 318 nm (31447 cm−1) and fluorescence emission at 370 nm (27027 cm−1) are observed first. Next, three aggregate forms are noted, energy level splitting by 4β1 = 9280 cm−1 occurs between 297 nm (33670 cm−1) and 410 nm (24390 cm−1), and a ΔΣ1 = 2417 cm−1 reduction of the energy level relative to the monomeric form is observed. In the case of aggregate II, there is energy level splitting by 4β2 = 5711 cm−1 between 342 nm (29240 cm−1) and 425 nm (23529 cm−1) and a ΔΣ2 = 5062 cm−1 reduction of the energy level relative to the monomer. For the next aggregate form III, energy level splitting by 4β3 = 3323 cm−1 between 465 nm (21505 cm−1) and 550 nm (18182 cm−1) is observed as well as a ΔΣ3 = 11603 cm−1 reduction of the energy level. It is worth noting that, in the case of the longest-wave aggregates, a bottom of the exciton band can be observed in the RLS spectra, where a strong increase in the intensity of the spectrum is noted at ∼500 nm. Moreover, each time, the reduction of the energy levels is approximately 2-fold greater than that in the preceding form. Given the β value, it was possible to calculate the distance between adjacent molecules in the dimer. In order to investigate the aggregation of the analyzed molecules in greater detail, the spectra of CD were measured and presented in Figure 10A,B. As can be seen, at the low concentrations of the compound, the CD signals derived from the two investigated samples are very weak. This indicates that they do not exhibit clear chiral centers in the unaggregated state (or at a low aggregation level). However, the increase in the compound concentration leads to a marked change in the CD spectrum. Negative Cotton effects appear at concentration c3 for C1 and at concentration c4 for C7 at wavelengths of 293 and 333 nm, respectively. A further increase in the concentration of C1 does not result in changes of the spectrum in this range. Therefore, it can be concluded that only one of the oligomeric forms exhibits this spectral trait and its proportion does not change with the increasing concentration of the compound (which may have reached its threshold concentration value). However, at the higher concentrations of the compound, there is an additional asymmetric center and a negative Cotton effect with lower intensity at a wavelength of 391 nm. In the case of C7, the Cotton effect is more strongly dependent on the concentration as it is shifted toward longer wavelengths at 333 nm and concentration c4, and in the case of concentration c5, the effect is observed at a wavelength of 378 nm. It should be emphasized that the effect of the alkyl substituent on the spectral effects in the CD spectrum is more pronounced in the case of C7 than C1.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Fax: +(48 81) 4456684. Phone: +(48 81) 445 69 37 (A.M.). *E-mail: [email protected]. Phone: +(48 81) 537 59 04 (M.G.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was mainly financed by a grant from the University of Life Science in Lublin (TKF/MN/5 to A.M.). REFERENCES

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CONCLUSIONS The present study conducted with the use of, for example, electron fluorescence spectroscopy has revealed the presence of three fluorescence emission bands in the spectra of the analyzed compounds, two of which occur as dual fluorescence at the specified concentration range. The investigations based on 7968


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Solvent Effects on Molecular Aggregation in 4-(5-Heptyl-1,3,4

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