Excited-State Intramolecular Proton Transfer (ESIPT) of Fluorescent

Shenzhen Key Laboratory of Special Functional Materials, College of Materials Science and. Engineering, Shenzhen University, Shenzhen 518060, China. â...
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Excited-State Intramolecular Proton Transfer (ESIPT) of Fluorescent Flavonoid Dyes: A Close Look by Low Temperature Fluorescence Xiaoman Bi,†,§ Bin Liu,‡,§ Lucas McDonald,† and Yi Pang*,† †

Department of Chemistry & Maurice Morton Institute of Polymer Science, The University of Akron, Akron, Ohio 44325, United States ‡ Shenzhen Key Laboratory of Special Functional Materials, College of Materials Science and Engineering, Shenzhen University, Shenzhen 518060, China ABSTRACT: Flavonoids have emerged to be an important molecular frame for chemical sensors, due to their capability to give dual emission and their compatibility to biological cells. Two flavonoid compounds with Me2N- and Et2N-substituents were examined by acquiring their fluorescence at different temperatures, in order to evaluate the impact of intramolecular charge transfer (ICT) on ESIPT. By freezing the sample solution in liquid N2, the study detected the “locally excited” state at λem ≈ 460 nm, in which both ICT and ESIPT processes were not present. As temperature was warmed up (to about −90 °C), ICT process became gradually allowed and emission was shifted to λem ≈ 510 nm, which was attributed to the normal form N* of flavonoid. Emission from tautomeric form T* (λem ≈ 575 nm) could only be observed at a higher temperature, when ESIPT became allowed. With the aid of a model compound (without R2N-substituent) and computational study, the study led to improved understanding on the photophysical properties of flavonoid materials in general.

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similar flavonoids are also known to be useful for DNA recognition.12,13 Presence of a hydroxyl group at the 3-position of flavonoid 1 allows the molecule to undergo excited-state intramolecular proton transfer (ESIPT), resulting in desirable large Stokes’ shift. Another notable feature in the flavonoid 1 is the potential intramolecular charge transfer (ICT) which occurs between the electron-donor (e.g., amino group on ring B) and the acceptor (e.g., the carbonyl group on ring C). Both features are important properties for flavonoid sensors. In the event of ICT, separated charges are located on the different rings, i.e., on the B-ring and C-ring (see Scheme 2). This is in contrast to ESIPT, where the separated charges are only on the C-ring. Although the two events are quite different, little is known about their relative importance, as it is difficult to separate them in an experiment. It should be noted that the ICT is not only coexistent with ESIPT, but also across the path of ESIPT in flavonoid 1. A rational sensor design is depending on fundamental understanding about the ICT and ESIPT interaction that ultimately controls the sensor performance. In a system with ESIPT, the ESIPT emission is typically enhanced without notable spectral shift, when the sample is frozen in liquid nitrogen.14,15 Meanwhile, in a π-conjugated molecule, the content of ICT process would be decreased or inhibited when the sample is frozen, which could lead to a large

INTRODUCTION As an important natural pigment, flavonoids constitute a major portion of natural products present in fruits and vegetables,1,2 and are responsible for the colors (e.g., red and orange) in fruits and vegetables.3 There are significant interests in utilizing the flavonoids for chemical sensors, as flavonoids provide a chromophore frame with low toxicity and attractive properties of selective binding to proteins.4,5 Most sensor designs have been focusing on the substitution at C-2 and C-6 positions on the chromen-4-one segment of flavonoid 1 (Scheme 1). For Scheme 1. Structure of Flavonoid Derivatives

example, the fluorescence probe 1c exhibited selectivity for phospholipid membranes, whose surfaces bear negative charges.6−8 The derivatives with an amino group at the C-6 position can be used for sensing polarity change9 and proteins.10 Our recent study also shows that the substituent could have a large impact on the fluorescence response to albumin proteins.11 For example, 1d with an acetamide group on ring-B gives a significant less fluorescence turn-on than 1a. In addition to protein detection mentioned above, structurally © 2017 American Chemical Society

Received: February 27, 2017 Published: April 6, 2017 4981

DOI: 10.1021/acs.jpcb.7b01885 J. Phys. Chem. B 2017, 121, 4981−4986

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The Journal of Physical Chemistry B Scheme 2. Flavonoids 2-3 in the Normal and Tautomer Forms, which Are Capable of Undergoing ESIPT and ICT, and Schematic Illustration of the Related Photophysical Process

Figure 1. Fluorescence of flavonoids 2 and 3 in CH2Cl2 (10 μM) at room temperature, while being excited at 410 nm. Inset (top) shows the UV−vis absorption.

spectral blue shift (e.g., > 100 nm in a terpyridine derivative).16 Observation of ESIPT and ICT emission at different temperature thus could provide valuable information about the photophysical process in the flavonoid emission. A recent study by Demchenko et al.17 examined the temperaturedependent ESIPT in flavonoid 1a and 1c, revealing that relative fluorescence from N* (on the basis of FN*/FT* ratio) is basically not changed with temperature (in the range of 0−60 °C) in DMF solution.17 While the previous study shed some light on the ESIPT, no report is found to probe the impact of ICT in the flavonoid derivatives. Our recent study shows that flavonoid 2 exhibited unusual selectivity toward endoplsmic reticulum (ER), when it is used to stain eukaryotic cells.18 In this contribution, the fluorescence of 2−3 were examined in a wide range of temperatures (from 20 °C to −196 °C) in CH2Cl2. The study detected two emission peaks at 495−516 nm and 570−575 nm, which were attributed to emission from N* and T* tautomers, respectively. In addition, a third peak revealed at 457−463 nm when the temperature was below −123 °C at the expense of N* and T* emission, exhibiting the strong ICT effect. The result provided the experimental evidence that ICT played an important role in the emission of N* tautomer, thus further advancing our understanding on the photophysical process of flavonoids.

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RESULTS AND DISCUSSION At room temperature, the flavonoid 2 and 3 in CH2Cl2 exhibited a weak emission at ∼495 nm and a stronger emission peak at ∼570 nm (Figure 1), which were assigned to the normal (N*) and tautomer (T*) emission, respectively. While 2 and 3 gave dominant T* emission, the relative content of N* and T* emission from them was notably different, indicating the impact of substituent on ESIPT pathway. In order to freeze the molecular conformation, the flavonoid solution in a quartz tube (with 3 mm inside diameter) was quickly cooled by immersing into liquid nitrogen in a quartz Dewar. The spectra were then acquired as the temperature was gradually raised (within a few hours). Interestingly, only emission band at 457 nm was observed from 2 at the extremely low temperature (−186 °C to −100 °C) (Figure 2a). However, the N* emission band at 509 nm was predominant, when the temperature was warmed to −90 °C. It appeared that the molecules were still in a relatively rigidity environment that prevented the H atom migration (a necessary condition for

Figure 2. (a) Fluorescence spectra of flavonoid 2 in CH2Cl2 at different temperatures (excitation wavelength at 420 nm). (b) Fluorescence intensity ratio at 509 and 571 nm at different temperatures.

tautomer formation). The tautomer emission (T*) became visible when the temperature was warmed to −80 °C and the solvent was completely melted (mp of CH2Cl2 is −96 °C). The T* emission gradually became stronger, as the temperature was rising to room temperature. Detection of only emission at 509 nm at about −90 °C provided the direct evidence of forming the excited normal form N* upon photon absorption. Observation of the T* emission at a slightly higher temp 4982

DOI: 10.1021/acs.jpcb.7b01885 J. Phys. Chem. B 2017, 121, 4981−4986

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The Journal of Physical Chemistry B (−80 °C) indicated that the initially generated “excited normal form N*” could be easily transformed to the tautomer form T* via ESIPT process. The results also showed that the proton transfer was a quite effective process during the tautomerization N* → T*. Plotting the fluorescence ratio of 509 to 571 nm (FN*/FT*) revealed that the relative content of N* emission became significantly higher at low temperature (below −80 °C) (Figure 2b). Consistent higher ratio of FN*/FT* in the temperature range (−90 °C to −80 °C) pointed to that ESIPT became less effective even in solution at the low temperature. Although the N* emission λem was slightly blueshifted (to 495 nm) with increasing temperature, the fluorescence ratio of 509 to 571 nm should still represent FN*/FT* in good approximation as the emission peak was broad. Fluorescence of 2 at different temperature (Figure 2a) revealed three distinctively different species, including nearly exclusive N*-emission at 509 nm (at −90 °C), predominant T*-emission at 571 nm (at 25 °C), and a clear transition from N* to T* states. The intriguing question is the nature of the third emission at 457 nm, which occurred when the temperature was extremely low (below −100 °C). The emission spectra of 2 at 457 nm was notably blue-shifted from the N* emission (at 509 nm). In order to characterize the species that gave emission at 457 nm, we decided to acquire fluorescence excitation spectra that should reveal the absorption profile of the emitting chromophore.19,20 The corrected excitation spectrum of 2 at −198 °C was very similar to that at room temperature (Figure 3), which also matched the

N* emission involved significant ICT interaction (as shown in II, see Scheme 2). On the basis of the above evidence, it was assumed that the molecular geometry of excited 2 was frozen at liquid N2 temperature, giving predominant LE emission at 457 nm (species I). Once the temperature was above the melting point of solvent, the restriction on molecular motion was removed, which enabled the subsequent ICT process. This led to N* emission, which included strong ICT interaction (species II). The ESIPT process appeared to have a higher barrier than ICT, as the former could only occur at a higher temperature (above −80 °C). The spectroscopic response was further examined by freezing 3 in CH2Cl2, in order to verify what was observed from 2 (Figure 2). Emission of 3 revealed the similar pattern in responding to temperature change (Figure 4a). When the

Figure 3. Fluorescence excitation spectra (solid line, detection wavelength at 572 nm), and fluorescence emission spectra (broken line) of 2 in CH2Cl2 at 25 °C and liquid N2 temp.

Figure 4. (a) Fluorescence spectra of compound 3 in DCM at various temperatures (from −196 to 20 °C). Excitation at 420 nm. (b) Fluorescence intensity ratio at 516 and 575 nm at different temperatures.

absorption spectrum of 2 at room temperature (λmax = 407 nm, see inset of Figure 1). The diminished Stokes shift at −198 °C (liquid N2) suggests that the excited species at −198 °C could be associated with the locally excited (LE) state (structure I in Scheme 2), as the molecule was frozen in the solvent matrix (Franck−Condon principle).21 The complete shift of emission from 457 to 509 nm occurred within a narrow range of temperature change (from −100 °C to −90 °C), as a consequence of melting the rigid solvent matrix, which permits the bond alternation/rotation in the subsequent ICT process. In the flavonoid molecule, an electron donor (i.e., − NR2) was interacting with an electron acceptor, as shown in II (Scheme 2). Such intramolecular charge transfer (ICT) was responsible for the observed red-shifted emission (from 457 to 509 nm as shown in Figure 2a). In other words, the observed

solution of 3 was frozen in liquid N2 (at −196 °C), only one emission band (at 463 nm) was observable, corresponding to the LE state. No spectral shift was observed when the sample was warmed to −145 °C, indicating that the ICT was not allowed at this point. When temperature was further warmed to −123 °C, the emission was gradually red-shifted to about 520 nm, as ICT process gradually became allowed. The emission at ∼520 nm was from the excited normal form N*, which includes ICT character as described by species II. The emission from the tautomer T* was detected only after the solution was warmed to above −123 °C, giving both emission bands from N* (at 516 4983

DOI: 10.1021/acs.jpcb.7b01885 J. Phys. Chem. B 2017, 121, 4981−4986

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Figure 5. (a) Fluorescence spectra of flavonoid 4 in CH2Cl2 at different temperatures (excitation wavelength at 350 nm). Inset is the UV−vis absorption (at 20 °C, λmax = 308, 343, 357 nm). (b) Fluorescence intensity ratio at 441 and 516 nm at different temperatures. Inset is the proposed photophysical process for ESIPT.

nm) and T* form (at 575 nm) (from −123 °C to room temperature). Relative fluorescence ratio (FN*/FT*) was basically constant in the temperature range from 20 °C to −70 °C (Figure 4b), indicating that ESIPT rate was not affected while the emission intensity was increased for both N* and T*. The ESIPT appeared to be slowed only when the temperature was lower than −80 °C. Fluorescence of 3 started to exhibit spectral shift when temperature was higher than −140 °C and predominant N* emission (at ∼516 nm) existed over a wide temperature range (−134 °C to −110 °C, Figure 4a). This was in sharp contrast to the fluorescence of 2, where the spectral shift occurred when temperature was higher than −90 °C (Figure 2a), and dominant N* emission only existed in a narrow temperature window (−90 °C to −80 °C, Figure 2a). It appeared that the transition from species I to II (Scheme 2) was affected by the size of alkyl group in the donor substituent −NR2. A possible explanation was that a larger alkyl group (i.e., ethyl) could lead to a large solvent cavity near the donor −NR2 group. Although the entire ethyl was more difficult to move than methyl group, the alignment of the −NR2 group (necessary for forming species II) could be facilitated through partial C−C bond rotation within the solvent cavity. This might allow the formation of II at a lower temperature, which led to a more pronounced dependence on temperature from 3 (in comparison with 2). Fluorescence of Model Compound 4. Spectral study from both 2 and 3 suggests that ICT should be fully allowed in the normal tautomer when ESIPT is operational. In order to further verify the contribution of ICT, we decided to examine the fluorescence of 4, in which the impact of ICT is absent from the amino group. At room temperature, 4 exhibited predominant emission at ∼516 nm (Figure 5a), which was attributed to emission from T* (via ESIPT). At −96 °C (just below the mp of solvent), a broad new emission band occurred at ∼441 nm, which was blue-shifted from T* emission and was assigned to N* emission. The emission ratio of 441 to 516 nm was used to represent the relative ratio of FN*/FT*, and plotted against the temperature (Figure 5b). Although N* emission (at 441 nm) increased at a faster pace as temperature decreased to about −117 °C, the T* emission also increased slightly, which was accompanied by a spectral red-shift from ∼516 to 528 nm. As the temperature was below −125 °C, T* emission decreased drastically but did not completely disappear (even at −198 °C),

in contrast to what was observed from 2 and 3. These results suggested that ESIPT in 4 could be a more competitive process (in comparison with 2−3). The higher T* emission for 4 led to its lower FN*/FT* ratio (≈0.3, see Figure 5b), in comparison with FN*/FT* ≈0.5 for 2 (Figure 4b) at 20 °C. When the temperature was below −117 °C, the emission became structured, as typically observed from rigid aromatic molecules.22,23 The emission at 385 nm could be associated with the (0,0) vibronic band of a rigid chromophore, whose relative intensity could become stronger as the temperature decreased to −196 °C.22 The development of vibronic band at 385 nm showed that temperature decreases (from −117 °C to −196 °C) did not cause notable spectral shift. The observed vibronic bands (at 385 and 402 nm) could be attributed to the “locally excited” state that was associated with the rigid “chromen-4-one” segment. As temperature was warmed up, the excited 4 could undergo the “intracyclic charge transfer” to form 6 (see inset in Figure 5b), as ICT could occur before the proton transfer is allowed. In other words, the N* emission of a flavonoid actually involved significant charge transfer as shown in 6. Molecular Modeling. As shown in the structure 4, ESIPT involves electronic interaction between the oxygen atom and carbonyl group in the C-ring of “chromen-4-one” segment. In order to gain further understanding on the importance of this interaction and substituent effect, molecular HOMO and LUMO orbitals were generated from the normal form of 2 and 4 by using Gaussian 09 at B3LYP/6-31+G level (Figure 6). From the HOMO to LUMO orbitals of 4, there was a notable decrease in the orbital lope on the “chromen oxygen” (indicated by cyan arrow), which was accompanied by an increase on the “carbonyl oxygen” (Figure 6). The result indicated that some extent of charge transfer was occurring within the “chromen-4-one” unit of 4, which contributed to its higher tendency to undergo ESIPT. On the contrary, the orbital lope on the “chromen oxygen” was very small in the HOMO orbital of 2. Thus, predominant ICT was occurring between the carbonyl of “chromen-4-one” unit and 4-aminophenyl group, which led to higher electron density on the carbonyl oxygen and facilitated the ESIPT process. The observation was consistent with the assumption that the normal excited state N* of 2−3 involved significant charge transfer. 4984

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temperature change, the study provides a deep understanding about the photophysical process of flavonoids. The N* emission includes the full extent of ICT permitted by molecular structures. In addition, ESIPT could be turned off while ICT is partially allowed as a consequence of lower temperature. By identifying the LE, N*, and T* states, the study thus provides the experimental evidence to illustrate the impact of ICT on the ESIPT process. Comparison between 2 and 4 further revealed that the N* emission was significantly lower in the latter, in the absence of an electronic donor group. The study suggests that an electronic donor group could actively perturb the ESIPT process, through donor−acceptor interaction, and could significantly affect the N*/T* emission ratio.

Figure 6. Molecular HOMO and LUMO orbitals of 2 and 4 in normal form. The orbitals were generated by using DFT at B3LYP/631+G(d,p) setting, with geometry optimization followed by energy minimization. The orbital lope on the “chromen oxygen” and “carbonyl oxygen” are indicated by cyan and pink arrows, respectively.

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ORCID

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

Bin Liu: 0000-0003-2211-5557

EXPERIMENTAL SECTION Reagents and Instrumentation. All solvents were purchased from commercial sources and used without further purification. 3-Hydroxy-2-phenyl-4H-chromen-4-one (4) was synthesized by using a literature procedure.24 1H NMR and 13C NMR spectra were obtained using a Bruker AVANCE II. UV− vis spectra were acquired on a Hewlett-Packard 8453 diodearray spectrometer. Fluorescence spectra were measured by using FluoroMax-4 spectrometer. Synthesis of 3. Compound 3 was synthesized similarly as 218 by mixing N-(3-acetyl-4-hydroxyphenyl)butyramide (40 mmol) and 4-(diethylamino)benzaldehyde (40 mmol) in 150 mL of ethanol, followed by slow addition of 40 mL of aqueous NaOH (15 g, 375 mmol) solution. The reaction mixture was stirred at 70 °C for 1 h, and then cooled to room temperature for 24 h. H2O2 solution (20 mL of 30%) was slowly added into the reaction solution, which was cooled in an ice−water bath. After stirring at room temperature for 12 h, the mixture was poured into ice water and then placed into the refrigerator overnight. The precipitate was collected via filtration, and washed with ethanol. The product was purified by recrystallization from hexane/ethanol (v/v = 3/1). Yield= 16%. 1H NMR (d6-DMSO, 500 MHz): δ = 10.15 (s, 1H, -OH), 9.02 (s, 1H, -NH), 8.39 (d, 1H, J = 2.5 Hz), 8.08 (d, 2H, J = 9.0 Hz), 7.88 (m, 1H), 7.65 (d, 1H, J = 9.0 Hz), 6.80 (d, 2H, J = 9.0 Hz), 3.44 (m, 4H), 2.33 (t, 2H), 1.66 (m, 2H), 1.15 (t, 6H), 0.93 (t, 3H). 13C NMR (d6-DMSO, 125 MHz): d = 172.07, 171.76, 150.68, 148.89, 147.32, 137.25, 136.16, 132.29, 129.68, 125.31, 121.95, 118.92, 117.52, 113.26, 111.25, 44.17, 38.77, 18.98, 14.08, 12.91. MS, calcd for [C23H26N2O4+H]+ m/z: 395.2; found: 395.2.

Author Contributions §

X.B. and B.L. contributed equally.

Notes

The authors declare no competing financial interest.

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CONCLUSION Fluorescence spectra of flavonoids 2 and 3 were acquired at different temperatures by freezing their solutions in liquid nitrogen. When the flavonoid was frozen to −198 °C, the molecule in a rigid solvent matrix permits to detect a locally excited (LE) state without the ICT, giving blue-shifted fluorescence (emission peak at ∼457 nm). When the temperature was gradually increased to about −90 °C, predominant emission was observed from the normal form (N*) of flavonoid (λem ≈ 509−516 nm), and the tautomer emission (from T*) was detected at λem ≈ 571−575 nm. Through monitoring the response of LE, N*, and T* states to 4985

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DOI: 10.1021/acs.jpcb.7b01885 J. Phys. Chem. B 2017, 121, 4981−4986