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. Author manuscript; available in PMC: 2020 May 1.
Published in final edited form as: J Photochem Photobiol A Chem. 2019 Mar 6;376:73–79. doi: 10.1016/j.jphotochem.2019.03.005

Photo-physical properties of substituted 2,3-distyryl indoles: Spectroscopic, computational and biological insights.

Ruwini D Rajapaksha 1, Danielle N Turner 3, Jade Vigil 1, Liliya V Frolova 1, Jeff Altig 1, Snezna Rogelj 2, Mahinda I Ranasinghe 1,*
PMCID: PMC6643299  NIHMSID: NIHMS1523582  PMID: 31333319

Abstract

The structural dependence of the photo-physical properties of substituted 2,3-distyryl (23DSI) indoles were studied using several spectroscopic techniques including steady-state UV-VIS spectroscopy, steady-state fluorescence spectroscopy, steady-state excitation spectroscopy, time correlated single photon counting (TCSPC) spectroscopy, and time-resolved fluorescence upconversion spectroscopy (TRFLS). Each of 23DSI derivatives investigated showed distinct fluorescence emission and UV-VIS spectra, indicating strong structural dependence of the emission and the excitation. The UV-VIS spectra of the 23DSI derivatives showed three main identical absorption bands with minor deviations in the absorbance caused by substituent groups on the distyryl rings. The time-resolved fluorescence up-conversion studies indicated that the fluorescence undergoes a mono-exponential decay whereas the calculated fluorescence lifetime showed relatively short fluorescence lifetimes of approximately 1 ns. All of the 23DSI derivatives showed two-photon absorption upon direct excitation of 1.6 W laser pulses at 800 nm. These studies suggest that the substituents, attached to distyryl core, are capable of boosting or hindering fluorescence intensities by distorting the π-conjugation of the 23DSI molecule. Our studies showed that 23DSI (p-F) has the highest fluorescence emission quantum yield. Theoretical calculations for the ground state of 23DSI derivatives confirmed differences in electron densities in 23DSI derivatives in the presence of different substituent attachments. The excellent fluorescence emission, high fluorescence quantum yield and two-photon absorption properties of these 23DSI molecules make them attractive candidates for potential applications in the fields of biological imaging, biomedicine, fluorescent probes, and photodynamic inactivation (PDI). B. subtilis samples, treated with micro molar solutions of 23DSI (p-OCH3) and 23DSI (p-CH3), showed very effective photodynamic inactivation (PDI) upon irradiation with white light.

Graphical Abstract

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1. Introduction

Heterocyclic compounds with an indole scaffold are of great interest due to their demonstrated potential applications in the fields of biology [1–3], medicine [4–8], photovoltaics [9–12], and as fluorescent dyes [13]. This class of indole-based compounds is well known for their antimicrobial activities [4, 5, 14]. Given these promising applications, our current research is focused on evaluation of a family of novel indole derivatives; since these derivatives are photo-activated, understanding of the photo-physics is crucial for advancement towards the applications listed above. We have studied and already reported our findings about the photo-physical properties of an antibacterial agent 2,3-distyrylindole (23DSI) [14]. In order to provide a comprehensive understanding of the photo-physics of these compounds, we have extended our studies to six different 23DSI derivatives. Differences in the photo-physical properties of 23DSI engendered by different substituent groups on the distyryl ring are the focus of this current report. The 23DSI derivatives are shown in Fig. 1. For facile comprehension between photophysical properties we include the previously reported characteristics of the lead compound 23DSI. Applied spectroscopic techniques, included in our current report, integrate information obtained from steady-state UV-VIS spectroscopy, steady-state fluorescence (FL) spectroscopy as well as high-end ultrafast time-resolved fluorescence upconversion spectroscopy (TRFLS) and time correlated single photon counting (TCSPC) spectroscopy. We believe these studies are crucial for understanding various derivatives’ optical and electronic properties, including photo-physical dynamics. Our experimental studies are further supported by our theoretical calculations. With these experimental and theoretical studies, it is our goal to provide complete insight into the photo-physical and photo-biological properties and thus the suitability for biological applications of this class of heterocycli derivatives.

Fig. 1:

Fig. 1:

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The structures of 23DSI derivatives.

As shown in the Fig. 1, 23DSI (H) does not have any substituent groups attached to the core 23DSI molecule. In 23DSI (p-OCH3) and 23DSI (p-CH3), strong and weak electron-donating group of methoxy (–OCH3) and methyl (-CH3) groups are attached to the para positions of 23DSI respectively. In the 23DSI (p-F) and 23DSI (p-Cl) respectively have fluorine (-F) and chlorine (-Cl) electron-withdrawing groups attached to the para position of 23DSI, respectively while in the 23DSI structures (o-Cl) and (m-Cl) the chloro substituent group is changed to the ortho and meta position, respectively. The motivation for these 23DSI (o-Cl) and (m-Cl) analogues was to compare photo-physical properties and biological activities with respect to the 23DSI (p-Cl) derivative. As we reported in our previous articles, the 23DSI (p-Cl) showed excellent photoactivated biological activities [5, 14]. Analogous structure-dependent biological activities of the novel 23DSI derivatives are under investigation and those results will be reported as a separate article.

2. Experimental

2.1. Spectroscopic studies

A 10.0 μM solution of each 23DSI derivative in dimethyl sulfoxide (DMSO) was used for steady-state UV-VIS spectroscopy and steady-state FL spectroscopy. The absorption spectra were recorded using the Cary 60 UV-VIS spectrophotometer while the fluorescence and the excitation measurements were measured using the Shimadzu RF-5301PC spectrofluorometer. The multiphoton absorption was measured by exciting the sample using Spectra Physics Mai Tai laser pulses and measured the emissions using an optical fiber which was coupled to an Ocean Optics HR2000+ spectrometer. For TCSPC spectroscopy, a 50 μM solution of each derivative in DMSO was used, whereas for the TRFLU spectroscopy, a 1 mM solution of each derivative in DMSO was used. The time-resolved fluorescence upconversion studies were performed utilizing the Newport time-resolved fluorescence spectrometer (TRFLS). The detailed description of TRFLS can be found in our previous research paper [15]. Briefly, TRFLS is a hybrid instrument capable of measuring both fluorescence upconversion and time-correlated single photon counting (TCSPC). The fundamental output beam from the oscillator (Spectra Physics Mai Tai HP) is split into two. One portion is used to generate a second or third harmonic excitation beam (Spectra Physics / GWU UHG), while the residual serves as the gate for upconversion. The gate beam is passed to a computer-controlled translation stage (New-port ILS250CC) where it makes six passes up and down the stage, resulting in a maximum delay time of 3.3 ns. The gate beam is focused by an anti-reflection coated lens (New-port AR.16) onto the upconversion crystal (BBO, type I). The excitation beam enters the TRFLS enclosure separately and is focused onto the sample. The fluorescence and the gate beams cross at a small angle in the upconversion crystal. The resulting upconverted beam is filtered and focused onto a computer-controlled monochromator. The upconverted light is detected and resolved as a particular wavelength by a PMT. Single photons are counted by a time-correlated single photon counting board (Becker & Hackle) and displayed in the TRFLS software or recorded in a file during data acquisition. Fluorescence lifetimes (τFL) of 23DSI derivatives were measured using TCSPC instrumentation of Horiba Fluorolog-3 (FLS-22) spectrofluorometer. Samples were excited using a 360 nm nano LED and emitted photons collected at 506 nm. The emitted photons were detected and the obtained fluorescence decay was deconvoluted with the instrumental response function that was determined using a light scattering solution. For this study we used LUDOX (40% SiO2 in water) as our scattering solution. The fluorescence lifetimes were calculated using decay analysis software (DAS6) fitting the signal to a single exponential decay function as shown in the Equation 1.

y=Aexp(−xτ)+y0 (1)

A is the amplitude associated with decay, τ is the time constant and yo is the background offset. The time-resolved fluorescence upconversion experiments were carried out as follows: 1.0 mL of 1 mM 23DSI solution was placed into a rotating cell (50 rpm). The gate beam was set to 800 nm and a 400 nm excitation beam was generated using second harmonic generation. All samples were excited at an excitation power of 25 mW. The upconverted signal was generated by mixing the fluorescence and the fundamental frequencies in a nonlinear crystal. For all samples, upconversion spectra were generated for parallel polarizations of excitation by doing at least five scans for the first 550 ps with time intervals of 2.0 ps. The scans for 506 nm were added, averaged, and normalized. Deconvoluting the decay with the instrumental response function (IRF) and fitting the signal to the first order decay equation, shown in the Equation 1 yielded decay fit.

2.2. Computational Studies

A theoretical investigation was performed to study the ground state properties of 23DSI derivatives utilizing the Gaussian 16 computational package [16]. The Gauss View 06 molecular building package was used to build the molecule [17]. The ground state geometry optimization using Gaussian 16 was performed using the hybrid functional Becke, 3-parameter, Lee-Yang-Parr (B3LYP) of the density functional method with a 6-311+ G (2d, p) diffused basis set [18–21]. The calculations were performed in solvent phase using DMSO as a solvent. For solvent phase calculations in Gaussian 16, the conductor-like polarizable continuum model (CPCM) with a self-consistent reaction field (SCRF) method was employed [22, 23]. The optimized structures for 23DSI molecules are shown in Fig. S1 and the generated frontier molecular orbitals for HOMO and LUMO for ground state optimized geometry are shown in Fig. S2 in the Supplementary Information. The energies of HOMO and LUMO molecular orbitals were used to analyze the possible electronic transitions and hence the possible excitation wavelengths [24, 25]. The analyzed MO energies and assigned electronic transitions are shown in Table S2 and Fig. S3 in the Supplementary Information.

2.3. Bacterial Culture, Treatment, and Photodynamic Inactivation (PDI)

Bacillus subtilis was inoculated from a glycerol stock onto a Mueller-Hinton agar plate (MHA) and grown overnight at 37°C. A broth suspension of B. subtilis was grown in Mueller-Hinton broth (MHB) at 37°C on a rotary shaking incubator (100 rpm) overnight at 37°C. The optical density of the overnight cell culture was taken at OD600 and the cell concentration adjusted to ~1×106 colony forming units per milliliter (CFU/mL); one milliliter of cell suspension was added to a borosilicate glass culture tube and treated with 0.1 μM or 1.0 μM 23DSI (p-OCH3), 23DSI (p-CH3), or 23DSI (p-Cl) and vortexed to mix. Following the treatment, the samples were incubated at 37°C on a rotary shaking incubator (100 rpm) for 45 minutes. Immediately after the incubation, the cell samples were inoculated as a lawn onto MHA plates and a mask with a center cut-out was placed 1 cm above the surface of the agar plate and irradiated with white light from a Lumacare LC-122 unit (85 J/cm2 of 400 – 700 nm light) for 2 minutes at 3 cm height above the sample lawn. The MHA plates were incubated at 37°C overnight. Qualitative observations were noted [26–28].

3. Results and discussion

The steady-state absorption and emission spectra for all 10.0 μM solutions of 23DSI derivatives dissolved in DMSO are shown in Fig. 2 and Fig. 3 respectively. All absorption spectra show three main distinct absorption bands at around 380 nm, 335 nm, and 260 nm. Interestingly, the absorption band at 260 nm is not very pronounced in the 23DSI (p-OCH3) and 23DSI (p-CH3) derivatives. The absence of the 260 nm absorption band is due to the electron-donating nature of 23DSI (p-OCH3) and 23DSI (p-CH3) substituents that facilitates the relatively larger electron delocalization length and thus narrowing the HOMO-LUMO band gap. Hence increase the energy loss as excited state vibration [29]. The lack of the 260 nm absorption peak of 23DSI (p-OCH3) and 23DSI (p-CH3) support the idea that the optical properties of 23DSI exquisitely depend on the positional and structural characteristics of the appended substituents.

Fig. 2:

Fig. 2:

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UV-VIS spectra of 10.0 μM solutions of 23DSI in DMSO. The black line shows 23DSI (H), red line shows 23DSI (p-OCH3), magenta line shows 23DSI (p-CH3), blue line shows 23DSI (p-F), yellow line shows 23DSI (o-Cl), orange line shows 23DSI (m-Cl), and green line shows 23DSI (p-Cl) analogs.

Fig. 3:

Fig. 3:

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Emission spectra of 10 μM solutions of 23DSI derivatives in DMSO upon excitation at the three different absorption wavelengths . Black line shows 23DSI (H), red line shows 23DSI (p-OCH3), magenta line shows 23DSI (p-CH3), blue line shows 23DSI (p-F), yellow line shows 23DSI (o-Cl), orange line shows 23DSI (m-Cl), and green line shows 23DSI (p-Cl) analogs.

Further, we observed a profound change in the absorbance cross-section with a change in the substituent groups attached to 23DSI derivatives. The electron density studies of ground state molecular orbitals of each substituent demonstrate that each substituent is facilitating and hindering the electron delocalization as a function of its molecular nature and position on the distyryl rings. This dissimilarity of electron densities in ground state HOMO and LUMO molecular orbitals are shown in Fig. S2 in the Supplementary Information.

The p-Cl derivatives showed the highest absorptions while the p-F derivative showed only a minimal increase in same absorbance compared to 23DSI (H) which has no substituent group attached to dystyryl rings. This can be explain by the similar steric effect observed in 23DSI (p-F) and 23DSI (H). The 23DSI (p-CH3) and the 23DSI (p-OCH3) showed the lowest absorbance. The experimental absorption data can be explained by using the calculated oscillator strengths for each absorption band of these molecules (Table 1) [30].

Table 1.

The Calculated Oscillator Strengths of Each Absorption Band.

23DSI Derivative Oscillator Strength
HOMO to LUMO (380 nm) HOMO−1 to LUMO (335 nm) HOMO−1 to LUMO+1 (260 nm)
23DSI (H) 0.3520 0.472 0.1299
23DSI (p-OCH3) 0.0547 0.1676 0.0457
23DSI (p-CH3) 0.1914 0.2465 0.007
23DSI (p-F) 0.3589 0.4846 0.0765
23DSI (o-Cl) 0.4596 0.5111 0.3196
23DSI (m-Cl) 0.7596 1.4764 0.5217
23DSI (p-Cl) 0.5271 0.7249 0.3846
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The p-F derivative and 23DSI (H) have identical oscillator strengths giving similar absorbance spectra. The very low oscillator strengths for absorbance at 260nm for the 23DSI (p-CH3) and the 23DSI (p-OCH3) resulted in very low absorbance at 260nm. Although, the 23DSI (p-OCH3) has stronger electron donating ability compared to the 23DSI (p-CH3) derivative, the ground state geometry of 23DSI (p-OCH3) is non-planar leading to relatively poor delocalization of electrons (Fig. S1 in Supplementary Information). It is well know fact that the increase in the electron delocalization length results in a red shift in the absorption bands. Since a red shift is not observed, this supports the notion of non-planar ground state geometry of these moelcules. Among the chloro-subsituted 23DSI derivatives, the m-Cl substituted 23DSI (m-Cl) showed the highest absorbance whereas the o-Cl substituted 23DSI (o-Cl) showed the lowest absorbance. This observation also clearly demonstrates the positional dependence of oscillator strength and thus the absorbance. The excitation at these three distinct absorption bands resulted in the same emission for all 23DSI derivatives albeit with different emission intensities. These emissions are shown in Fig. 3. According to the fluorescence emission spectra, the para-substituted, electron-withdrawing 23DSI derivatives shows higher emission intensities than the para-substituted electron-donating 23DSI derivatives. The chloro-substituted 23DSI derivatives showed a slight red shift of the emission bands. The 23DSI (p-OCH3) did not show the regular emission band at excitation of 260 nm; this is due to the lack of the absorption band at 260 nm in our UV-VIS spectra. However, 23DSI (p-CH3) showed stronger emission at this excitation wavelength compared to the 23DSI (p-OCH3). Among the three chloro-derivatives, the 23DSI (p-Cl) showed the highest emission, whereas the 23DSI (o-Cl) showed the least emission. This is due to the differences in conjugation length of the molecules. The 23DSI (o-Cl) has the lowest conjugation length while 23DSI (p-Cl) has the highest conjugation length. These observations additionally support the idea that the position of the substituent group in the distyryl rings will influence the optical properties of these molecules. The change of the emission intensity, as well as the slight shift of the emission peaks of the para substituted derivatives, mainly due to the effects imposed by the substituent groups.

Further, we studied the likelihood of two photon absorption (2PA) characteristics for all these derivatives since 23DSI (p-Cl) showed multiphoton absorption [14]. For these studies we excited 1 mM solution of each of the 23DSI derivatives at 800 nm using pulsed laser with the power of 1.6 W and detected the emitted fluorescence using an Ocean Optics HR2000+ spectrophotometer. These 2PA spectra are shown in Fig. 4.

Fig. 4:

Fig. 4:

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Emission spectra of 10 μM solutions of 23DSI derivatives in DMSO, obtained by exciting the sample at 800 nm laser pulses. Black line shows 23DSI (H), red line shows 23DSI (p-OCH3), magenta line shows 23DSI (p-CH3), blue line shows 23DSI (p-F), yellow line shows 23DSI (-o-Cl), orange line shows 23DSI (m-Cl), and green line shows 23DSI (p-Cl) analogs.

The strong 2PA characteristics of these 23 DSI molecules suggest potential applications in the fields of fluorescence microscopy [31], 3-D optical storage [32], photodynamic therapy (PDT) [26, 33, 34] and photodynamic inactivation (PDI) [27, 35–37]. Our biological studies (discussed below) show that 23DSI analogue can be used as photosensitizers for PDI. After studying their photophysical properties we are particularly interested in 23DSI (p-OCH3) and 23DSI (p-CH3) analogs and their PDI properties as presented in this article in later section.

The ultrafast time-resolved fluorescence upconversion studies showed that all these derivatives experience a single exponential decay with several hundreds of picoseconds time constants. The observed fluorescence decay graphs are shown in Fig. 5 and the calculated decay parameters are listed in the Table 2.

Fig. 5:

Fig. 5:

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The time-resolved fluorescence upconversion spectroscopic decay at 506 nm fluorescence emission wavelength. 1 mM solution of each sample was used and all samples were excited at 400 nm with 25 mW excitation power.

Table 2.

Calculated Fluorescence Decay Parameters Using Time-resolved Fluorescence Upconversion Spectroscopy and Fitted into A Single Exponential Decay Function.

Sample τ / ps A
23DSI (H) 278.03 ± 15.102 0.434 ± 0.009
23DSI (p-OCH3) 320.05 ± 41.033 0.363 ± 0.019
23DSI (p-CH3) 507.91 ± 67.411 0.649 ± 0.049
23DSI (p-F) 384.02 ± 29.887 0.366 ± 0.013
23DSI (o-Cl) 434.34 ± 34.351 0.466 ± 0.019
23DSI (m-Cl) 425.32 ± 31.191 0.479 ± 0.018
23DSI (p-Cl) 571.77 ± 78.233 0.634 ± 0.053
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According to the calculated decay constants, the 23DSI (H) analog has the fastest fluorescence decay constant whereas the 23DSI (p-Cl) has the highest fluorescence decay constant. The p-Cl substituted 23DSI (p-Cl) shows an extended π-conjugation compounded with the excellent electron-withdrawing ability of -Cl group. We belive these structural properties could be the reason for the observed longest time constant of this derivative. The fluoro-substituted 23DSI (p-F) shows a moderate fluorescence decay constant compared to chloro-substituted 23DSI derivatives. The originally-expected longer fluorescence decay for the 23DSI (p-F) was not observed. This is likely due to an inductive effect within the 23DSI (p-F) due to the substituted fluorine atoms. However, 23DSI (p-Cl) shows a higher uncertanity in the time constant; this could be due to the scattering of the sample.

Suprisingly, the 23DSI (p-CH3) showed a longer decay constant, comparable to 23DSI (p-Cl). The fluorescence lifetime (τFL) of these derivatives were calculated and are listed in the Table 3. 10 μM Solution of 23DSI derivatives in DMSO were used to study fluorescence lifetimes. Average of three trials were used to calculate τFL. The electron-donating groups attached to 23DSI (p-OCH3) and 23DSI (p-CH3) show the longest fluorescence lifetimes while 23DSI (p-F) and 23DSI (p-Cl) show the next longest lifetimes.

Table 3:

Calculated Fluorescence Lifetimes (τFL) of 23DSI Derivatives

Sample τFL / ns % Error
23DSI (H) 0.815 0.24
23DSI (p-OCH3) 1.33 3.16
23DSI (p-CH3) 1.05 2.95
23DSI (p-F) 0.964 0.93
23DSI (o-Cl) 0.365 3.29
23DSI (m-Cl) 0.615 0.81
23DSI (p-Cl) 0.949 1.79
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The τFL is correlated with the electron donating/withdrawing nature of the substituent groups. As shown in Table 2, 23DSI (p-OCH3) has the longest fluoresence lifetime whereas 23DSI (p-Cl) has the lowest fluorescence lifetime among the para substituted derivatives. The τFL data nicely correlate with the substituent’s attachment position on the distyryl ring. As shown in Table 3, the τFL increased with o-Cl, m-Cl, and p-Cl respectively. We belive this is because of the increase in the conjugation length of these 23DSI derivatives. The fluorescence lifetime data clearly demonstrate the structural dependency of photophysical properties of these derivatives. Further, fluorescence quantum yield (ϕFL) of these derivatives were calculated using coumarine 153 in ethanol as a reference. The 10 μM solutions were used and the samples were excited at the 380 nm excitation wavelength. The reference quantum yield 0.509 was used at 380 nm excitation; 1.3617 refractive index was used for ethanol and 1.4772 refractive index was used for the solvent DMSO. The calculated ϕFL are listed in the Table 4.

Table 4:

Fluorescence Quantum Yields (ϕF) of 10 μM Solution of 23DSI Derivatives.

Sample ϕF
23DSI (H) 0.38
23DSI (p-OCH3) 0.37
23DSI (p-CH3) 0.58
23DSI (p-F) 0.46
23DSI (o-Cl) 0.07
23DSI (m-Cl) 0.13
23DSI (p-Cl) 0.25
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Our calculated ϕF shows the 23DSI (p-CH3) has the highest ϕF. This observation again suggests the potential use of this compound as a photosensitizer for PDI applications. Our ground state (GS) geometry optimization followed by frequency calculations show that the 23DSI (p-Cl) possess the minimum energy among all these derivatives. The calculated GS energies are listed in the Table S1 in the Supplementary Information. According to GS energy studies, the 23DSI (H) has the highest energy and the chioro-substituted derivatives have the lowest optimized GS energies. These results show that the chioro-substituent stabilizes 23DSI (H) compared to the other substituents. The plotted energy digaram is shown in Fig. 6.

Fig. 6:

Fig. 6:

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The calculated GS energy for optimized structures of 23DSI derivatives. The energies are shown in the Hartree units.

The superior stability of 23DSI (o-Cl), 23DSI (m-Cl), and 23DSI (p-Cl) is due to the excellent π-overlapping of the molecular orbitals in these derivatives. The generated ground state molecular orbital energies were further analyzed inorder to understand the possible electronic transitions observed in the experimental UV-VIS Spectra. The energies of the highest-occupied molecular orbitals (HOMO), the energies of the lowest-unoccupied molecular orbitals (LUMO) and the difference in HOMO and LUMO energies were plotted to visualize the HOMO and LUMO energies as a function of these distinct substituents. The plotted graphs are shown in Fig. 7.

Fig. 7:

Fig. 7:

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The energies of HOMO, LUMO and the difference of energy of LUMO and HOMO. The HOMO energies are shown as blue squares, the energies of LUMO are shown as red circles, and the difference of energies of HOMO and LUMO are shown as green triangles.

As shown in Fig. 7 the HOMO and LUMO energies vary slightly among the distinct 23DSI derivatives. However, the differences between the HOMO and LUMO energies are almost the same for all 23DSI derivatives. The calculated wavelength for this difference of energy is about 380 nm, the wavelength responsible for HOMO to LUMO transition on all 23DSI derivatives and in complete agreement with our experimentally observed UV-VIS absorption band at 380 nm. To identify the other possible transitions, we studied the energies of HOMO-1, HOMO-2, LUMO+1, and LUMO+2. These results are summerized in the Table S2 and Fig. S3 in the Supplementary Information. This allowed us to assign the possible transitions for the three main excitations bands observed in our experimentally derived UV-VIS spectra. As mentioned above, the 380 nm band is due to the HOMO to LUMO transition, the 335 nm band is due to the combined transitions of HOMO-1 to LUMO and HOMO to LUMO+1 transitions, and the 260 nm band is due to the combined transitions of HOMO-1 to LUMO+1, HOMO-2 to LUMO and HOMO to LUMO+2 transitions. The electron-rich nature of these derivatives and excellent π-conjugations may promote these strong absorption bands as well as the strong fluorescence emission relaxation of the singlet excited state (S1) to the ground state (S0). Further, HOMO and LUMO showed different electron densities when the distinct substituent groups were attached to 23DSI rings. This is supported by the idea that the fundamental structure-dependent optical properties are exhibited in these 23DSI derivatives. As shown in the Fig. 3, all transitions relax to the S1 state by internal conversion and subsequently relax to S0 by fluorescence emission. The emission intensities differ with the attached substituent and the position of the substituent in the distyryl rings of 23DSI derivatives; this is because of alteration in the π-conjugation and/or the magnitude of the π-conjugation. As shown by our fluorescence upconversion studies, this is quite a rapid process with several hundreds of picosends time constants and even shorter fluorescence lifetimes.

Our studies have shown that 23DSI compounds are activated by direct excitation. As such, 23DSI (p-CH3) and 23DSI (p-OCH3) analogs could serve as additional photosensitizers for PDI applications. To confirm this hypothesis, biological studies are needed to verify the photosensitizing activity. In order to investigate the structure-dependent activity of the most promising 23DSI derivatives, a qualitative PDI experiment was performed on a culture of Bacillus subtilis treated with either 23DSI (p-Cl), a previously studied, highly photoactivatable compound, with 23DSI (p-OCH3), or 23DSI (p-CH3). A center cut-out mask was placed over each agar plate containing B. subtilis (1×106 CFU/mL) treated with varying concentrations of 23DSI (p-OCH3), 23DSI (p-CH3), or 23DSI (p-Cl) for 45 min before being irradiated with white light for 2 minutes. Following a 24 hour incubation at 37°C, a photograph of the plates was captured. The photograph shown in Fig. 8 demonstrates PDI of the B. subtilis samples that were treated with either 0.1 μM and 1μM of 23DSI (p-OCH3), 23DSI (p-CH3), and 23DSI (p-Cl), whereby bacterial growth is present only where bacterial cells were covered by the mask to prevent irradiation, 23DSI excitation and thus bacterial killing.

The potential use of these derivatives 23DSI (p-Cl) as multiphoton absorbing photosensitizers for generating reactive oxygen species (ROS) for PDI applications is under further investigation and the findings will be published in a separate manuscript. Furthermore, we intend to develop these derivatives as fluorescent probes for biological imaging applications.

4. Conclusion

In this article, we present the spectroscopic analysis of seven novel 23DSI derivatives. These experimental findings are further supported by our theoretical calculations. Each derivative shows a slight variation in absorption and emission as a function of the specific substituent attached to the distyryl ring. Further, we observed position-dependent optical properties with the same substituent group. We believe these structure and position-dependent optical properties arise due to the alteration in the π-conjugation lengths and magnitude, compounded by the pronounced π-molecular orbital overlapping of these 23DSI derivatives. In our studies, we observed two photon absorption for all these derivatives; this property offers applications in PDI, photodynamic theraphy (PDT), biological imaging, and fluoprescence microscopy. Our theoretical calculations show dissimilar electron densities in the examination of the HOMO and LUMO properties, this further supports the notion the structure-dependent optical characteristics. The fluorescence decay studies with ultrafast time-resolved fluorescence upconversion spectroscopy showed analogue-specific fluorescence decay within the range of several hundreds picoseconds. The TCSPC spectroscopic studies resulted in different fluorescence lifetimes for distinct substituent within 23DSI derivatives, nonetheless all fluorescence lifetimes are in the magnitude of 1 ns. From our theoretical calculations we are able to assign the possible electron transitions to the experimentally-observed absorption bands.

Supplementary Material

1

Fig. 8.

Fig. 8.

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PDI of B. subtilis cells treated with 23DSI (p-OCH3), 23DSI (p-CH3), and 23DSI (p-Cl), and exposed to white light. A) B. subtilis cells pre-treated with 23DSI (p-OCH3) (0.1μM (top half of plate) and 1μM (bottom half of plate)). B) B. subtilis cells pre-treated with 23DSI (p-CH3) (1μM (top half of plate) and 0.1μM (bottom half of plate)). C) B. subtilis cells pre-treated with 1% v/v DMSO (vehicle control). D) B. subtilis cells pre-treated with 23DSI (p-Cl)(0.1μM (top half of plate) and 1μM (bottom half of plate)). All cell samples were spread over an agar plate and irradiated with white light through a center cut-out mask for 2 minutes and incubated at 37°C for the bacterial lawn to develop.

Research Highlights.

  • Steady state and time-resolved fluorescence spectroscopic investigations of a class of organic molecules

  • Theoretical calculations using Gaussian to support the photo-physics of these molecules

  • Biological assay investigation to support the photodynamic inactivation by these organic molecules upon light activation.

Acknowledgments

The authors would like to thank NSF for funding support through NM-EPSCOR solar energy nexus (#IIA-1301346) and National Institute of General Medical Sciences (Grant P20GM103451). The NMT start up grant also contributed to establishing the “ultrafast laser spectroscopy lab”. S.R. acknowledges Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103451 and S.R. and L.V.F. acknowledge their NMT Presidential Research Support. Authors also would like to thank Prof. Michael D. Heagy for use of the Fluorolog instrument for the fluorescence lifetime measurements.

Footnotes

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Conflicts of interest

There are no conflicts to declare.

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