AcidâBase Equilibriums of Lumichrome and its 1-Methyl, 3-Methyl

Article pubs.acs.org/JPCA

Acid−Base Equilibriums of Lumichrome and its 1-Methyl, 3-Methyl, and 1,3-Dimethyl Derivatives Dorota Prukała,*,† Ewa Sikorska,‡ Jacek Koput,† Igor Khmelinskii,§ Jerzy Karolczak,∥,⊥ Mateusz Gierszewski,† and Marek Sikorski*,† †

Faculty of Chemistry, A. Mickiewicz University, Grunwaldzka 6, 60-780 Poznań, Poland Faculty of Commodity Science, Poznań University of Economics, al. Niepodleglości 10, 60-967 Poznań, Poland § Universidade do Algarve, FCT, DQF and CIQA, Campus de Gambelas, 8005-139 Faro, Portugal ∥ Faculty of Physics and ⊥Centre of Ultrafast Laser Spectroscopy, A. Mickiewicz University, Umultowska 85, 61-614 Poznań, Poland ‡

S Supporting Information *

ABSTRACT: Lumichrome photophysical properties at different pH were characterized by UV−vis spectroscopy and steady-state and timeresolved fluorescence techniques, in four forms of protonation/ deprotonation: neutral form, two monoanions, and dianion. The excited-state lifetimes of these forms of lumichrome were measured and discussed. The results were compared to those obtained for similar forms of alloxazine and/or isoalloxazine, and also to those of 1-methyland 3-methyllumichrome and 1,3-dimethyllumichrome. The absorption, emission, and synchronous spectra of lumichrome, 1-methyl- and 3-methyllumichrome, and 1,3-dimethyllumichrome at different pH were measured and used in discussion of fluorescence of neutral and deprotonated forms of lumichrome. The analysis of steady-state and time-resolved spectra and the DFT calculations both predict that the N(1) monoanion and the N(1,3) dianion of lumichrome have predominantly isoalloxazinic structures. Additionally, we confirmed that neutral lumichrome exists in its alloxazinic form only, in both the ground and the excited state. We also confirmed the existence and the alloxazinic structure of a second N(3) monoanion. The estimated values of pKa = 8.2 are for the equilibrium between neutral lumichrome and alloxazinic and isoalloxazinic monoanions, with proton dissociation from N(1)−H and N(3)−H groups proceeding at the almost the same pH, while the second value pKa = 11.4 refers to the formation of the isoalloxazinic dianion in the ground state.

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fluoride or acetate anions, which seem to promote this process.11 Tautomerization of lumichrome in the ground state in aqueous solutions occurred upon addition of cucurbit[7]uril.12 The acid−base behavior of lumichrome in aqueous solutions has been studied by Lasser and Feitelson,13 who presented the absorption and emission spectra of lumichrome as a function of pH. However, studies of lumichrome at different pH are difficult due to both participation of protonated, neutral, and deprotonated species and transformations between alloxazineand isoalloxazine-like species. Therefore, we need to establish what forms exist in solution, which are predominant, and when one transforms into another. The structures and abbreviations of the lumichromes discussed here are presented in Figure 1. Despite the fact that acid−base properties of lumichrome have already been studied, presently we are focusing on a more systematic study of the effect of pH on spectral and fluorescent

INTRODUCTION Lumichrome, the main product of the photodecomposition and biodegradation of riboflavin,1−3 has been extensively studied because its alloxazine scaffold undergoes photoinduced tautomerization, changing its lumichrome-like emission into isoalloxazine-like emission. Hydrogen bonding with pyridine or acetic acid was found to promote this rearrangement via double proton transfer in the excited state.4−6 However, there are some controversies on lumichrome tautomerization in water or in its presence. Recently Penzkofer and Tyagi concluded that at pH >7 a partial ground-state tautomerization of lumichrome to 7,8-dimethylisoalloxazine occurs by N1 to N10 intramolecular proton transfer;7 also recently Mitra et al.8 have proposed water assisted excited-state proton-transfer reaction of lumichrome. These results are in apparent disagreement with our earlier paper, where no proton transfer in the ground state had been detected. Neither could we detect any excited-state proton transfer caused by water molecules.9 Our results have been recently confirmed by Douhal et al.10 Tautomerization in the ground state has been achieved for lumichrome dissolved in acetonitrile, mediated by © 2012 American Chemical Society

Received: January 16, 2012 Revised: June 24, 2012 Published: June 25, 2012 7474

dx.doi.org/10.1021/jp300522h | J. Phys. Chem. A 2012, 116, 7474−7490

The Journal of Physical Chemistry A

Article

is maintained between the two. This technique is frequently used in analysis of food products and other heterogeneous mixtures possessing strong autofluorescence. Our attention was attracted by the recent reports on acid− base properties of lumazine and especially by the controversy on the values of pKa = 10.5 reported by Huppert et al.14 vs the pKa = 8.0 value reported in an elegant and valuable comment made by Oliveros and Lorente and others.15 In fact, the value pKa = 8.0 is in agreement with values reported for lumazine decades ago and found to be 7.91, 7.76, and 7.95, see ref 15 and the references cited therein. Also recently Penzkofer and Tyagi have reported pKa = 12.5 for the neutral−anionic equilibrium of lumichrome. However, earlier for the equilibrium between neutral and anionic forms of lumichrome (N(1)−H and N(3)− H deprotonate with similar probabilities) pKa = 8.2816 had been reported by Koziołowa, and pKb = 12.916 for the equilibrium between monoanionic and bianionic forms (both N1 and N3 deprotonated). It is clear that the values reported by Penzkofer and Tyagi (12.5) are in contradiction to those previously reported by Koziołowa (8.28),16 as are also different the values previously reported for lumazine. In view of the controversies on possible water assisted tautomerization of lumichrome and on values of pK for the equilibrium between neutral and anionic forms of lumichrome, we believe that this report will provide a substantial contribution to the discussion. In this paper we took full advantage of the synchronous fluorescence spectra to study different species of lumichrome at

Figure 1. Structures of lumichrome, 1-methyllumichrome, 3methyllumichrome, and 1,3-dimethyllumichrome.

properties of lumichrome, using both steady-state and timeresolved techniques. Among other approaches, we are taking full advantage of synchronous fluorescence spectra to study different species (protonated, neutral, and deprotonated) of the same molecule, existing at different pH. In this method excitation and emission monochromators are scanned simultaneously, synchronized so that a constant wavelength difference

Figure 2. The absorption spectra of lumichrome (A), 3-methyllumichrome (B), 1-methyllumichrome (C), and 1,3-dimethyllumichrome (D) at different pH. Constant concentrations were maintained throughout the 2 to 12 pH range. 7475



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Figure 3. Fluorescence spectra of lumichrome derivatives at different pH: (A) lumichrome, (B) 3-methyllumichrome, (C) 1-methyllumichrome, and (D) 1,3-dimethyllumichrome. All spectra registered in the same conditions; λexc = 390 nm.

different pH, combining it with traditional measurements of absorption, fluorescence, and fluorescence excitation spectra, together with fluorescence lifetime measurements. The study is supported by a systematic study of lumichrome derivatives, namely, 1-methyllumichrome, 3-methyllumichrome, and 1,3dimethyllumichrome. This synthetic approach made possible selective observation of species present at different pH.

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The values of pH were measured with a Hanna Instruments pH-meter. Only freshly prepared solutions were used in order to minimize any photolytic or hydrolysis reactions. UV−vis absorption spectra were recorded on a Varian Cary 5E spectrophotometer and on a Jobin Yvon-Spex Fluorolog 3 spectrofluorometer, using the option to measure absorption on the latter. Steady-state fluorescence excitation, emission, and synchronous spectra were recorded on a Jobin Yvon-Spex Fluorolog 3-22 spectrofluorometer. The synchronous spectra were recorded with offsets of 10, 20, and 30 nm, all showing similar results, therefore spectra with 20 nm offset were chosen for presentation as having the best quality. The synchronous fluorescence spectra are plotted as a function of the emission wavelength. We always checked whether the absorption spectra in the beginning and in the end of the experiments were the same, because of the eventual hydrolysis of alloxazines (including lumichrome) and isoalloxazines to the 6,7-dimethyl-3-(N′methylureido)quinozaline-2-carboxylic acid20−22 or to other compounds.20 However, we have not detected any indications of hydrolysis in our experiments. All fluorescence lifetime measurements were performed at the Centre for Ultrafast Laser Spectroscopy in Poznan, with the respective fluorescence lifetime spectrophotometer setup, using the single-photon timing technique. Magic-angle detection was employed to avoid artifacts associated with the rotational difussion in the sample. Detailed description of the experiments is available in the literature.23 Briefly, a Spectra-Physics pico/ femtosecond laser system was used as the source of exciting pulses. That included a Tsunami Ti:sapphire laser, pumped with a BeamLok 2060 argon ion laser, which generated 1−2 ps pulses at a repetition rate of about 82 MHz and average power

MATERIALS AND METHODS

Lumichrome (7,8-dimethylalloxazine) (Aldrich) was recrystallized from methanol before usage. Samples of 1-methyllumichrome, 1,3-dimethyllumichrome,17,18 and 3-methyllumichrome19 were available from previous work. Water was triply distilled. Lumichrome, 1-methyllumichrome, 3-methyllumichrome, and 1,3-dimethyllumichrome were saturated in 4 M HCl and then filtrated for the pH-dependent measurements. 10 mL aliquots of this saturated solution were titrated with 2 or 3 M NaOH to the appropriate pH values as shown in Figure 2 and Figure 3. The concentrations of lumichrome, 1-methyllumichrome, 3methyllumichrome, and 1,3-dimethyllumichrome presented in Figures 2 and 3 were 0.9 × 10−5 M, 3 × 10−5 M, 0.8 × 10−5 M, and 0.5 × 10−5 M respectively. Note that 1,3-dimethyllumichrome is poorly soluble in water, therefore its absorption and emission spectra are of lower quality (Figures 2D and 3D). The concentrations of the appropriate solutes for the lifetime measurements were slighty lower; we maintained the absorbance in the 0.05−0.1 range in the maxima of the two lowest-energy bands. 7476



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There is almost no effect of pH on the absorption spectra of 1,3-dimethyllumichrome in the pH range from 1 to 11. The emission spectra of lumichrome and its derivatives at different pH are presented in Figure 3. These spectra were obtained by excitation at 390 nm, exhibiting one band at λ = 473 nm, very similar for all compounds, of relatively small intensity in the acidic and neutral conditions in the 1 to 6 pH range. Two bands with their maxima at about 460 and 530 nm appeared for lumichrome in the 7 to 12 pH range (Figure 3A). There is once again only one emission band with the maximum at 536 nm at still higher pH values. Emission spectra of 3-methyllumichrome at different pH are shown in Figure 3B. In contrast to lumichrome, here we can see only one band at about 473 nm at pH 5 and 6. The band at about 460 nm predominates at pH ≥8. Figure 3C presents the emission of 1-methyllumichrome at different pH. There is only one emission band in all values of pH examined. However, in contrast to lumichrome and 3methyllumichrome, the changes are only observed in shorter wavelengths (from 475 to 462 nm). The intensity is almost constant in the pH range from 1 up to 5, growing at higher pH. Figure 3D presents the emission of 1,3-dimethyllumichrome at different pH. There is only one emission band, virtually independent of pH. Absorption and fluorescence spectra of lumichrome at different pH together with the corresponding excitation and synchronous spectra recorded at selected pH of 5, 7, and 13 are presented in Figures 4, 5, and 6. Further on, the A frames in the same Figures 4−6 show the normalized fluorescence excitation spectra at the same pH values, compared to the respective absorption spectra, with the emission wavelengths listed in the respective figures. The B frames in the same figures show the relative emission spectra excited at different wavelengths and the synchronous spectra, all at the same pH. Note that all of the spectra were recorded using the same excitation and emission slits, for uniform spectral resolution and sensitivity. At pH 5 lumichrome exists mainly as the neutral form (Figure 4A).18 The absorption spectra of lumichrome in the pH 1−7 range show a long-wavelength maximum near 350 nm and a shoulder near 388 nm, and are independent of pH in that region. The two experimentally observed absorption bands arise from two independent π−π* transitions.28,29 The fluorescence excitation spectra overlap well the absorption spectra when the emission is observed at 450 and 540 nm, see Figure 4A. The fluorescence excitation spectrum reveals another band at about 300 nm when the emission is observed at 400 nm. Lumichrome solution excited at 350 nm gives an emission spectrum with a maximum at 473 nm and a shoulder at 399 nm while the excitation at 390 nm gives only one maximum in the emission spectra. Excitation of lumichrome solution at 450 nm gives a small emissive component with a maximum at about 540 nm. The synchronous spectra recorded in acidic conditions reveal two emitting species with their spectral maxima at ca. 370 and 436 nm, indicated in Figure 4B. At pH 7 the absorption spectra of lumichrome still maintain their shape, while the fluorescence excitation spectra show that there are three emitting species absorbing in this region (Figure 5A). An interesting situation is revealed by the emission spectra at pH 7 (Figure 5B).

of over 1 W, tunable in the 720−1000 nm range. The repetition rate of the excitation pulses was variable from 4 MHz to singleshot by using a model 3980-2S pulse selector. Second and third harmonics of the picosecond pulse obtained on a GWU-23PS harmonic generator could be used for excitation, giving great flexibility in the choice of the excitation wavelength. Elements of an Edinburgh Instruments FL900 system were used in the optical and control components of the system. The pulse timing and data processing systems employed a biased TAC model TC 864 (Tenelec) and the R3809U-05 MCP-PMT emission detector with thermoelectric cooling and appropriate preamplifiers (Hamamatsu). All experiments were carried out at room temperature. TD-DFT Calculations. The electronic structure of lumichrome and its 1- and 3- anions and 1,3-dianion has been studied by means of the density-functional theory (DFT).24 In this work, DFT calculations were performed by using the PBE0 hybrid method25 in conjunction with a modest 6-31G* splitvalence polarized basis set.26 Excitation energies and transition intensities were calculated using the time-dependent DFT approach for the optimized ground-state geometries. Oscillator strengths were calculated in the dipole length representation. Calculations were performed by using the Gaussian 09 package of ab initio programs.27

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RESULTS AND DISCUSSION Spectral Characteristics of Lumichrome and Its Derivatives at Different pH. The UV−vis absorption spectra for lumichrome, 1-methyllumichrome, 3-methyllumichrome, and 1,3-dimethyllumichrome in aqueous solutions at different pH are shown in Figure 2. Absorption spectra recorded at different pH for every lumichrome derivative show different but characteristic behavior. The most pronounced changes of absorption spectra as a function of pH are observed for lumichrome, while almost no changes are observed for 1,3dimethyllumichrome. Absorption spectra for both 1-methyllumichrome and 3-methyllumichrome change as a function of pH. All lumichrome derivatives, including lumichrome itself, have almost identical absorption spectra in the pH range of up to 7, and show one band with λmax around 352 nm and a shoulder at around 388 nm. Note the low solubility of 1,3-dimethyllumichrome in this range of pH. Spectra with two separate bands with their maxima at about 350 and 395 nm for lumichrome are present in the more basic pH range (8−10). The absorption spectra of lumichrome change most markedly in the pH 12−14 range, where a growing absorption band with its maximum at about 425 nm is seen. The locations of both absorption maxima and their intensities change in the pH range of 1−12, with a red shift of the longer-wavelength band at higher pH. Note that we see no well-defined isosbestic points in the absorption spectra of Figure 2A. The first absorption maximum for 3-methyllumichrome (Figure 2B) shifts hypsochromically to λmax around 342 nm, while the second absorption maximum shifts bathochromically to λmax around 421 nm, while pH grows to 13. Figure 2B shows a single isosbestic point at λ = 397 nm (see the discussion below). The shape and location of the 1-methyllumichrome maxima also change with pH (Figure 2C). The first absorption band shifts hypsochromically to λ = 348 nm and the second to λ = 391 nm, while pH grows from 5 to 12.5. There is also an isosbestic point at about λ = 368 nm. 7477



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Figure 4. (A) Absorption and normalized fluorescence excitation spectra of lumichrome at pH 5; arrows indicate wavelengths used for excitation in the fluorescence lifetime measurements. (B) Relative emission and synchronous spectra of lumichrome at pH 5; arrows indicate wavelengths used for detection in the fluorescence lifetime measurements.

Figure 5. (A) Absorption and normalized fluorescence excitation spectra of lumichrome at pH 7; arrows indicate wavelengths used for excitation in the fluorescence lifetime measurements. (B) Normalized emission and synchronous fluorescence spectra of lumichrome at pH 7; arrows indicate wavelengths used for detection in the fluorescence lifetime measurements.

We see that excitation at 320 nm gives dual emission with the first maximum at 398 nm, and a second intensive but very broad band, with its maximum difficult to pinpoint precisely. Excitation at 390 nm once more gives the broad emission band, while the excitation at 450 nm gives a third band with its maximum at 528 nm. When we subtract the emission spectra obtained when exciting at 450 nm from those obtained when exciting at 390 nm, a new band shows up with the maximum at 462 nm: therefore, the broad band in the range of 400−650 nm is a superposition of two individual emission bands. On the other hand, the synchronous fluorescence spectrum clearly confirms the existence of three emissive forms of lumichrome at this pH, with each of the synchronous peaks appearing at the onset of the respective emission band. Similar absorption and emission spectra of lumichrome were recorded for pH 8 and pH 9 (Figure 2s in the Supporting Information) although this time the emission with maximum at ca. 400 nm was absent. Naturally, the fluorescence excitation spectra reveal only two predominant emitting species instead of three. The fluorescence excitation bands observed at 450 nm vary only slightly in the 5−8 pH range, maintaining their shapes (Figures 4 and 5 and Figures 1s and 2s in the Supporting Information). However the spectrum at pH 9 (Figure 2s in the Supporting Information) seems to be different; therefore we believe this may indicate the existence of a different form. Similar absorption and emission spectra of lumichrome were recorded in the pH range 10−11, while at pH >12 only one emission band was present. Examples of spectra at pH 12 and

13, showing the pronounced differences, are given in Figure 3sA,B in the Supporting Information (at pH 12) and Figure 6A,B (at pH 13). The absorption spectra of lumichrome with two maxima at 341 and 413 nm and the fluorescence excitation spectra at pH 12 still reveal two forms, while the absorption spectrum at pH 13 is different. The two maxima in the latter appear at 338 and 429 nm, with the excitation spectra confirming the existence of a single emitting species. The emission and the synchronous fluorescence spectra also confirm that only one emitting species exists at pH 13. We also conclude that the changes in the absorption spectrum at pH 13 as compared to pH 12 are more noticeable than those in the emission spectrum. The most interesting result is that the synchronous spectra work quite well in the detection of number of different forms present in the ground state at different pH values. The same result is confirmed by traditional absorption, emission, and excitation spectra. However, the main advantages of using synchronous spectroscopy as compared to traditional absorption, emission, and excitation spectra are the ease of use and the more complete results in less time. Using the lumichrome derivatives with the methyl group in positions N(1), N(3), and N(1,3) allowed us to better understand the spectral behavior of lumichrome at different pH, by selectively disabling one or both of the deprotonation steps. The absorption spectra of 1-methyllumichrome and of 3methyllumichrome at pH 5 (Figure 7A,C) are very similar to 7478



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We present normalized fluorescence excitation, relative emission, and synchronous spectra of 1-methyllumichrome at pH 12 in Figure 9A,B. The shape of the absorption spectrum has changed in comparison to spectra at lower pH. Now two separate bands with their maxima at 348 and 391 nm are observed, with the excitation spectrum overlapping the absorption and confirming that only one emitting species absorbs in this range. The emission with the maximum at 462 nm and the synchronous fluorescence spectrum also confirm that only one emitting species exists at pH 12. The acid−base reactions of lumichrome in aqueous solutions are summed up in Figure 10, while those of 1-methyllumichrome and 3-methyllumichrome are shown in Figure 11. According to Lasser and Feitelson,13 the fluorescence of protonated lumichrome could not be observed at room temperature. However, the excitation spectra measured by us at pH up to pH 7 indicate an emissive species with its absorption maximum at 315 nm. The emission of this species appears with the maximum at 399 nm. Unfortunately, due to its low fluorescence quantum yields and possibly short lifetimes, we were unable to record its fluorescence kinetics. Penzkofer and Tyagi7 also observed absorption and emission in this region. However, they attributed these absorption and emission bands to unidentified impurities. Lasser and Feitelson13 suggested that dissociation at position N(1) leads to the B3 type of structure, instead of the B2 form, with the B3 absorption spectrum red-shifted, similar to what should occur upon transition to an isoalloxazinic system. Recently, Penzkofer and Tyagi7 have concluded than in the range from pH 7 to about pH 12 lumichrome was converted into 7,8-dimethylisoalloxazine by intramolecular ground-state proton transfer (tautomerization) from N1 to N10. They estimated that in the pH range from 10 to 12 approximately 40% of 7,8-dimethylisoalloxazine exists in solution. However, the existence of the “isoalloxazinic” form of lumichrome A3 at this range of pH had not been confirmed by other groups. According to these authors, the pH dependent absorption and fluorescence behavior of lumichrome indicate that neutral lumichrome is the predominant species in the range from pH −0.53 to pH 8, while forms A and A3 exist from pH 8 to pH 12.5 ≈ pKa. They have also postulated conversion of A to B1 and of A3 to N3 anion with 7,8-dimethylisoalloxazine structure around pH = pKa, and tautomerization of B1 to N3 anion with 7,8-dimethylisoalloxazine structure above pH 13, with complete conversion at pH ≥14.7 Contrary to that, Lasser and Feitelson13 conclude that a dianion with the B5 structure is formed at pH >12. They believe that the spectra at pH 13 and 14 most closely correspond to that of the dianion, with the two absorption maxima at about 340 and 428 nm. According to our own results,6 we believe that lumichrome exists as a neutral form (A) at pH range ≤7, where its absorption and emission spectra remain unchanged in water (Figure 2A and Figure 3A). At pH from 8 to 10 both absorption and emission spectra change, showing two separate bands, as has also been found previously.30 We believe that in this pH range the forms B1 and B3 are predominant for lumichrome. We can identify the absorption and emission bands for the isoalloxazinic species quite precisely (we believe that it is the form B3). The fluorescence excitation band observed at 540 nm equally confirms the existence of the isoalloxazinic form in this pH

Figure 6. (A) Absorption and normalized fluorescence excitation spectra of lumichrome at pH 13; arrows indicate wavelengths used for excitation in the fluorescence lifetime measurements. (B) Normalized emission and synchronous fluorescence spectra of lumichrome at pH 13; arrows indicate wavelengths used for detection in the fluorescence lifetime measurements.

each other and to the absorption spectra of neutral lumichrome itself, having two maxima in the same wavelength range. The fluorescence excitation spectra observed at 450 and 540 nm are very similar to the absorption spectra for both 3methyllumichrome and 1-methyllumichrome. It has to be noted that a second component may be present in the fluorescence excitation spectra, with its maximum at about 300 nm when observed at 400 nm. However, the respective emissions are very weak and cannot be isolated in Figure 7C,D. Indeed, in these figures we can only see one emission band with its maximum at 473 nm, attributed to a single species. Similarly, the synchronous spectra reveal only one emitting species predominant for both compounds at pH 5. Absorption and fluorescence excitation spectra of 1methyllumichrome and 3-methyllumichrome at pH 8 are presented in Figure 8A,B. We see that the absorption spectra of 1-methyllumichrome are not exactly the same as the fluorescence excitation spectra, with the fluorescence maximum shifted hypsochromically by about 10 nm as compared to the same spectra at pH 5. Additionally, only a single band is present in the synchronous spectrum, hypsochromically shifted as compared to the previous spectrum. Figure 8C,D presents the absorption and the fluorescence excitation spectra at pH 8 for 3-methyllumichrome. The absorption spectrum is different from that at pH 5; note the growing band with its maximum at about 430 nm. The synchronous spectrum shows two separate bands with the maxima at 437 and 483 nm, overlapping the onsets of the two emission bands. 7479



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Figure 7. (A) Absorption and normalized fluorescence excitation spectra of 1-methyllumichrome at pH 5. (B) Relative emission and synchronous spectra of 1-methyllumichrome at pH 5. (C) Absorption and normalized fluorescence excitation spectra of 3-methyllumichrome at pH 5. (D) Relative emission and synchronous spectra of 3-methyllumichrome at pH 5.

We agree with Lasser and Feitelson13 that in the pH 12−14 range the predominant form is B5. Its absorption spectrum is completely different from those at lower pH, having the maxima at about 340 and 425 nm. The fluorescence excitation spectra observed at 540 nm confirm that a single emitting form is predominant in this pH range. The emission spectra of B5 are also different from those at lower pH values, showing a single distinct band. The band is bathochromically shifted as compared to 530 nm of the isoalloxazine monoanion. The same conclusions are also obtained from the synchronous spectra. Consider Figure 12, which presents raw synchronous spectra, allowing better perception of the appearance and disappearance of the various fluorescent components. The spectra can be described expressing the synchronous fluorescence intensity Is as a function of the analyte concentration c:31

range. However, we are unable to precisely determine the absorption and emission maxima of the alloxazine monoanion (B1) based on common absorption and emission spectra because the recorded emission (λ ex = 390 nm) has contributions from more than one form, depending on pH. Still, the absorption and the emission spectra of these monoanionic and neutral forms (B1 and A respectively) of lumichrome should be very similar, as inferred by comparison to the respective spectra of 1-methyllumichrome and 1,3dimethyllumichrome (see below). At pH closer to neutral there is still some neutral lumichrome present, distorting the spectrum of the alloxazinic monoanion. The alloxazinic emission band can also be seen as a shoulder with the maximum that shifts from 476 to about 462 nm in function of pH; we attribute this shift to a growing emission from the alloxazinic monoanion, as also based on detailed investigation presented in Figures 4 to 6 and Figures 1s to 3s in the Supporting Information. Note that the fluorescence excitation spectra in this pH range with fluorescence observed at 450 nm (see Figure 2s in the Supporting Information) do not overlap the absorption spectra. Therefore, we believe that this indicates appearance of a new absorbing species, which we identify as the B1 form. Thus, the apparent absorption is a superposition of the spectra of B1 and B3 forms in this pH range.

Is = Kcb Ex(λex )Em(λex + Δλ)

(1)

Here, Is is the synchronous fluorescence intensity; c the concentration of the analyte; Ex the intensity of the excitation spectrum at λex; Em the intensity of the emission spectrum at λex + Δλ; b the thickness of the sample; and K the constant describing the instrumental factors, including geometry and other parameters. The synchronous fluorescence spectrum is 7480



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As we see, the bands with their maxima at 436−435 nm have low intensity, being attributed mostly to neutral lumichrome in its alloxazinic form. This synchronous band overlaps the emission spectra of neutral lumichrome as we can see in Figures 4 to 6 (and Figures 1s to 3s in the Supporting Information). Note that the total concentration of lumichrome is kept the same in the entire 2 to 12 pH range, thus the growing synchronous bands at higher pH come from new emitting species. At higher pH the maximum of the alloxazinic component is also shifting to shorter wavelengths (431 nm), indicating the appearance of the B1 monoanion. Another growing emission band at 485−487 nm is attributed to the isoalloxazinic monoanion in its B3 form. At pH 12 we still have the alloxazinic monoanion, with lower intensity than before, with a predominant contribution of the isoalloxazinic monoanion; however, at this pH the dianionic species should already be present as well. At pH 13 there is only one band with the maximum at 488 nm that we attribute to the isoalloxazinic dianion. We believe that the emission bands of the dianion are very similar to those of the isoalloxazinic monoanion. However, we can still note the disappearance of the emission of the alloxazinic monoanion at pH 13, converting entirely into the dianion, as evidenced by both spectral and lifetime changes. The absorption and emission spectra of 1-methyllumichrome are presented in Figures 2, 3, 7, 8, and 9 showing “alloxazinic character”. Only two forms, 1A and 1B1, may exist at pH 5 to 12.5 for this compound, both of alloxazinic structure. At higher pH the neutral form of 1-methyllumichrome should be absent

therefore a function of both the emission and the excitation spectrum of the compound or compounds of interest. As a result, it can be also plotted as a function of wavelength of excitation (λex) or wavelengths of emission (λex + Δλ) used in recording it. Therefore, we should state whether the excitation or the emission wavelength is used on the plot, to avoid misunderstanding. Indeed, Horiba Jobin-Yvon have decided to present synchronous fluorescence spectra as a function of excitation on their first spectrofluorimeters of the Fluorolog 2 series, later switching to using the emission wavelength instead, on their later machines. We are presenting our synchronous fluorescence spectra as a function of the emission wavelength. The selected value of Δλ has a significant impact on the nature and shape of the spectrum recorded. Normally, a single band is observed in the synchronous scan spectrum for a single emitting species present, regardless of the structure that its conventional emission spectrum may have. For example, the fluorescence spectrum of tetracene has a characteristic structure in the broad range from 460 up to 600 nm, with maxima at 473, 507, and 546 nm. However, the synchronous fluorescence scan of tetracene has only one narrow band at 473 nm.32 There is no general rule for choosing Δλ; however, the bandwidth increases with Δλ. A simple rule is to use small values, 10 or 20 nm (the slit widths should be not exceed half of Δλ) for compounds with small Stokes shift, and larger values for compounds with larger Stokes shift. Thus, the best way to start studies of an unknown system, containing a mixture of compounds with different fluorescence characteristics, is to record total synchronous fluorescence spectra, for an entire set of Δλ values. 7481



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entirely. The absorption spectra of 1A show one band at ca. 355 nm with a shoulder at ca. 390 nm at pH 5 to 7. At higher pH values 1B1 form becomes predominant, its absorption bands changing slightly and absorption maxima shifting to ca. 350 and 400 nm. The emission spectra with maxima at about 473 nm at pH 5−7 and at 463 nm at higher pH are identified as emissions from the respective 1A and 1B1 forms. We present raw synchronous spectra of 1-methyllumichrome in Figure 12. Note that only one band exists in the entire range of pH. However, neutral and anionic forms of this compound should also appear at certain pH values. Therefore, we believe that the absorption and emission bands are very similar for neutral and monoanionic species of 1-methyllumichrome. We identify the low-intensity components at pH 5 to 7 with their maxima at 442−439 nm as the neutral 1-methyllumichrome in its alloxazinic form (form 1A). This alloxazinic component is growing with pH, simultaneously shifting hypsochromically (437 nm). The neutral form of 1-methyllumichrome should be absent at higher pH; therefore we attribute this band to the alloxazinic monoanion at these pH values. This also confirms that the growing emission band with its maximum at 431 nm in the synchronous spectra of lumichrome (Figure 12, top panel) is attributable to the B1 monoanion (Figure 10). 3-Methyllumichrome at pH from 5 to 12 can exist only as two forms, neutral and the N(1) monoanion, as it has a single proton dissociation site. Its monoanion has two possible structures, 3B2 and 3B3 (Figure 11). The spectra presented in

Figures 2, 3, 7, 8, and 9 show that this compound has typically alloxazinic absorption and emission at pH 5 and 6. Therefore, we attribute the 3A structure to its neutral form (Figure 11). The typical isoalloxazinic absorption and emission appear at higher pH values, bathochromically shifted in comparison to the neutral molecule; therefore, we conclude that the monoanion of this compound has the 3B3 structure. Figure 12 shows raw synchronous spectra of 3-methyllumichrome at different pH. These spectra have two bands at pH 5 to 9, and only one band at pH >9. We attribute the lowintensity component with its maximum at 437 nm to the neutral 3-methyllumichrome in its alloxazinic form. Note that this maximum does not shift with pH, therefore, the monoanions with alloxazinic structure are absent, as opposed to lumichrome and 1-methyllumichrome. A new band with growing intensity and stationary maximum appears at pH ≥8, attributed to the 3B3 isoalloxazinic monoanion. Note that the two maxima in these spectra almost coincide with the respective bands in the synchronous spectra of lumichrome (Figure 12), attributable to the respective neutral and isoalloxazinic forms. The important conclusion based on these spectra is that the N(1) monoanion exists in its isoalloxazinic form only. Equilibrium Constants of Lumichrome and Its Derivatives. The pKa values of lumichrome and its derivatives in the ground state were determined using the DATAN software package for the total titration experiment; the spectrophotometric titration curve of lumichrome is present in Figure 13, together with the reconstruction of the absorption 7482



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Figure 10. The steps of protonation and deprotonation of lumichrome, with different species proposed by different authors (see the text for details).

also the spectra of 1-methyllumichrome and 3-methyllumichrome, reconstructed using the DATAN software package. The pKa values of these compounds for the equilibriums between their neutral and monoanionic forms (1B1 for 1methyllumichrome and 3B3 for 3-methyllumichrome) are almost the same, pKa = 8.6 and 8.5 respectively. Excitation of the neutral lumichrome molecule into its first singlet state S1 changes the electronic density distribution, with the net charge values in the excited state decreasing in the order N(5) > N(10) > N(3) > N(1).34 These changes in the electronic density distribution upon excitation produce an increase in basicity at the N(10) and in acidity at N(1), as obtained from the quantum chemical calculations.34,35 According to previous publications, the pKa* value for deprotonation of lumichrome at N(1) in the S1 state is lower than that in the ground state by 2 to 6 units,33 whereas the deprotonation pKa at N(3) is only slightly lower than that in the ground state.34 According to our measurements, the pKa* values in the excited state are quite similar to those in the ground state. Indeed, the experimental results for lumichrome obtained from the titration curves in the excited state (Figure 14) produced pKa* = 7.7 for the equilibrium between neutral and the two deprotonated species (B1 and B3); similarly to what happened in the ground state, the dissociation occurs both at N1 and N3. This value for lumichrome is only slightly lower than that estimated for the ground state. The calculated pKa* = 11.2 is the equilibrium constant for the formation of dianion B5 in the excited state, according to

spectra of the species present in aqueous solution of lumichrome at different pH (right panel). The spectra of individual species presented in Figure 13 were obtained using multivariate analysis using the same software package. Note that the spectra of four individual species presented for lumichrome in Figure 13 correspond quite well to the experimental data at selected pH points (Figure 2). We believe that the pKa values for formation of B1 and B3 monoanions from neutral lumichrome are very close to each other, which is also supported by pKa values determined by titration experiments performed for 1-methyllumichrome and 3-methyllumichrome. Our opinion is also supported by others, who proposed proton abstraction from the N(3)−H group as the first dissociation step. They believe it leads to the alloxazinic structure B1, with the dissociation constant very close to that of the N(1)−H group.33 We believe that the pKa values for the equilibriums between neutral lumichrome and the respective B1 and B3 species are approximately equal, therefore the red curve in Figure 13 should be a superposition of the spectra of these two monoanions. The estimated value of pKa = 8.2 is the equilibrium constant between neutral lumichrome and monoanions B1 and B3. It means that the dissociation of N(1)−H and N(3)−H groups proceeds at almost the same pH. The estimated pKa = 11.4 is the equilibrium constant for the formation of dianion B5 in the ground state. The spectrophotometric titration curves in the ground state of 1-methyllumichrome and 3-methyllumichrome are also shown in Figure 13. The right panels of Figure 13 present 7483



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Figure 11. The steps of protonation and deprotonation of 1-methyllumichrome and 3-methyllumichrome.

3-methyllumichrome. Finally, only the emission attributed to the neutral molecule has been observed for 1,3-dimethyllumichrome in the entire pH range from 3 to 11. Time Resolved Fluorescence Spectra of Lumichrome at Different pH. The measured fluorescence lifetimes of lumichrome at different pH are given in Table 1 and in Tables 1as and 1bs in the Supporting Information. Additionally, Table 2 lists the measured fluorescence lifetimes of 1-methyllumichrome, 3-methyllumichrome, and 1,3-dimethyllumichrome at selected pH points, and selected emission and excitation wavelengths. The decay times were measured at λem = 480 nm (and 540 nm; see Supporting Information) by excitation at 390 nm (and 425 nm; see Supporting Information). The fluorescence lifetimes of each of the forms of lumichrome and their derivatives were attributed to the respective neutral and ionic forms on the basis of the above analysis of absorption, emission, fluorescence, and synchronous spectra in different pH and of understanding which of the forms were excited and observed in lifetime measurements. The sum of contributions of these forms may be different from unity, as we attribute some of the extracted lifetimes to the background. The arrows in Figure 4, Figure 5, and Figure 6 (and Figures 1s to 3s in the Supporting Information) indicate the wavelengths used for fluorescence excitation and fluorescence detection in the lifetime measurements. Note that the selected excitation wavelengths, 390 and 425 nm, provide for light absorption by alloxazinic and/or isoalloxazinic forms of lumichrome and/or its derivatives, respectively, independently of their ionization

results also obtained using the DATAN software package. This value is slightly lower than that in the ground state. The pKa* values for deprotonations reactions of 1-methyllumichrome and 3-methyllumichrome are 8.4 and 8.3 respectively, while the second dissociation at around pH of 11 is absent. Once more, the pKa* 8.4 and 8.3 values are also almost the same as calculated for the ground state. The spectrophotometric titration curves for the excited state of 1-methyllumichrome and 3-methyllumichrome are also presented in Figure 14. Note that four emissive species are present in the reconstructed emission spectra of Figure 14, with each either emitting in a separate spectral region or having very different emission intensity. Note also that the red curve in Figure 14 is a sum of emissions originating in two different deprotonated forms of lumichrome, with B1 and B3 structures, present in the pH range from about 8 to about 11. This conclusion is supported by the results obtained for methyl-substituted lumichrome derivatives. The reconstructed spectra of 1-methyllumichrome and 3-methyllumichrome show only two emitting species throughout the entire pH range investigated. Emission from neutral form of both 1-methyl- and 3-methyllumichrome derivatives is dominant at pH 8. Similarly, only the emission from the 3B3 anion is observed for 3-methyllumichrome at pH >8. The dianion cannot be formed for either of these two derivatives, therefore the dianion emission, similar to that observed in lumichrome, never appears for either 1-methyllumichrome or 7484



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fluorescence observed in solutions at different pH values. All of the results presented in Table 1 and Tables 1as, 1bs, and 1cs in the Supporting Information uniformly and univocally indicate that equilibrium of different species exists in the ground state, and that there is no reaction or relaxation in the excited state. Figure 15 is the graphical presentation of data included in Table 1 and shows a plot of the measured lifetimes of different forms of lumichrome as a function of pH with lumichrome excited at 390 nm and emission observed at 480 nm. Figure 4s and Tables 1as and 1bs in the Supporting Information show similar information, for excitation at 390 nm and emission at 540 nm and for excitation at 425 nm and emissions at 480 and 540 nm. The fluorescent decays were fitted by double or triple exponential functions. In each case the goodness of fit was evaluated using reduced chi-square, χ2, Durbin−Watson, and ordinary-runs tests, as well as by inspection of the distribution of the weighted residuals and of the autocorrelation functions. The goodness of fit analysis suggests that two or three emitting species are present at each pH. According to the literature and our own results the absorption and emission spectra of lumichrome at pH 1−7 are mostly attributable to the neutral molecule.13 On the other hand, the fluorescence of the neutral molecule is quenched by protons at lower pH values: the rate constant of this reaction is (2.5 ± 0.5) × 1010 M−1 s−1.13 Accordingly, Grodowski et al.35 reported a fluorescence lifetime of 1.4 ns for lumichrome in aqueous solutions at pH 2.2, noting also a minor component with 3 ns lifetime. However Penzkofer and Tyagi7 explain the decrease of fluorescence quantum yield and fluorescence lifetime of lumichrome at pH

AcidâBase Equilibriums of Lumichrome and its 1-Methyl, 3-Methyl

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