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Excited-State Proton Transfer of Weak Photoacids Adsorbed on Biomaterials: 8‑Hydroxy-1,3,6-pyrenetrisulfonate on Chitin and Cellulose Ron Simkovitch and Dan Huppert* Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv University, Tel Aviv 69978, Israel S Supporting Information *

ABSTRACT: Time-resolved and steady-state florescence measurements were used to study the photoprotolytic process of an adsorbed photoacid on cellulose and chitin. For that purpose we used the 8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS) photoacid which transfers a proton to water with a time constant of 100 ps, but is incapable of doing so in methanol or ethanol. We found that both biopolymers accept a proton from the electronically excited acidic ROH form of HPTS. The excited-state proton-transfer (ESPT) rate of HPTS adsorbed on chitin is greater than that on cellulose by a factor of 5. The ESPT on chitin also occurs in the presence of methanol or ethanol, but at a slower rate. The transferred protons can recombine efficiently with the conjugate excited base, the RO− form of HPTS.

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INTRODUCTION Proton chemical reactions and proton conductivity play an essential role in biology. Hydroxy aryl compounds are a part of the family of photoacids. Photoacids are weak acids in their ground electronic state; however, their acidity increases by several orders of magnitude in their excited electronic state. The excited-state values of pKa* of photoacids range from −8 to about 3. Excitation of a photoacid leads to an excited-state proton transfer (ESPT) either to a base or to a solvent molecule. The ESPT process is extensively studied by timeresolved spectroscopical techniques.1−18 Weak photoacids can transfer a proton in the excited state to water but not to other regular protic solvents such as methanol, ethanol, or other alcohols. In the current study we use fluorescence techniques in order to study the ESPT rate of a common photoacid, 8-hydroxy1,3,6-pyrenetrisulfonate (HPTS),1,2 in contact with two of the most common biomaterials in nature, cellulose and chitin. Scheme 1a shows the chemical structure of cellulose. It composes the cell walls of green plants19−21 and is an abundant biopolymer in our ecosystem. Cellulose is a long linear polysaccharide polymer, (C6H10O5)n, consisting of several hundreds to more than 10 000 linked D-glucose units. Cellulose from wood pulp has chain lengths between 300 and 1700 units. Cellulose has both amorphous and crystalline regions. It can absorb a large amount of water molecules up to nearly 5:1 (H2O:cellulose ratio).22 Chitin is a long-chain polymer of N-acetyl-D-glucosamine (shown in Scheme 1b), a derivative of glucose, and can be found in many of nature’s creations. Chitin is the main component of the cell walls of fungi and also of the © 2015 American Chemical Society

Scheme 1. Molecular Structures of (a) Cellulose, (b) Chitin (Similar to Cellulose but with One Hydroxyl Group on Each Monomer Replaced by an Acetyl Amine Group), and (c) Chitosan, the Deacetylated Form of Chitin

exoskeletons of arthropods such as crabs, lobsters, and shrimps and of many insects.23 Chitosan, shown in Scheme 1c, is a polysaccharide polymer consisting of linked D-glucosamine (deacetylated unit) and Nacetyl-D-glucosamine (acetylated unit). It is prepared by a chemical reaction of crustacean shells with sodium hydroxide.23 It is used in agriculture as a plant-growth enhancer and also as a biopesticide.24 Received: February 10, 2015 Published: February 18, 2015 1973

DOI: 10.1021/acs.jpca.5b01398 J. Phys. Chem. A 2015, 119, 1973−1982

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The Journal of Physical Chemistry A In a recent study25 we have shown, by time-resolved fluorescence polarization spectroscopy, that photoacids are adsorbed on the scaffolds of chitosan and cellulose. This was deduced by using rotational diffusion26 measurements. We also used UV−vis steady-state and time-resolved methods to study the protic properties of the neutral pH cellulose and the basic pH chitosan.25 For this purpose, we adsorbed photoacids on the two biopolymers by spraying the acids in a methanol solution. Since the vapor pressure of methanol is high at room temperature, the methanol evaporates and other solvents can be sprayed on the biopolymer. Weak photoacids with a pKa* ≥ 0 transfer a proton to water within the excited lifetime of the protonated ROH form: about 3−10 ns. These weak photoacids can also transfer a proton to mild bases like acetate, F−, H2PO4−, etc. in aqueous solutions and in methanol and ethanol. We found that chitosan, which is composed mainly of D-glucosamine, can easily accept a proton from weak photoacids adsorbed on its scaffold. In the current study, we explore the possibility that the excited-state proton-transfer (ESPT) process can also take place from a photoacid to the N-acetyl-D-glucosamine of chitin. For this purpose, we used the 8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS) photoacid (shown in Scheme 2).

The proton of the ion-pair intermediate can undergo a second proton transfer process and form the RO−* form (the conjugated base). The ion pair may also recombine with the excited deprotonated state, RO−*, to re-form ROH* with a rate ka. The proton in the bulk recombines with a RO− by a diffusion-influenced geminate recombination process that can be described by the Debye−Smoluchowski equation (DSE).11,29 The fluorescence of the ROH* and the RO−* forms of the photoacid have different positions, and henceforth it is easy to observe the ROH and RO− species by timeresolved fluorescence. The diffusion assisted proton geminate recombination modifies the ROH* form population. As a consequence, the ROH time-resolved fluorescence exhibits a nonexponential long-time fluorescence tail.

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MATERIALS AND METHODS Chitin from crab shells (85% acetylated), 20 μm cellulose powder, and chitosan samples of medium molecular weight (190−310 kDa, 75−85% deacetylated) were purchased from Sigma-Aldrich. All solvents used in this study were of HPLC grade. To estimate the amount of water adsorbed on chitin under atmospheric conditions, we performed the following test: We dried 100 mg of chitin in an oven at 85 °C for 3 h. We then checked the water adsorbance under atmospheric conditions by measuring the weight of the sample for 36 min. We could not find a detectable change in the sample weight. We conclude that the water content is rather small in our chitin samples. For the time-correlated single-photon counting (TCSPC) measurements, we used for sample excitation a cavity-dumped titanium:sapphire femtosecond laser (Mira, Coherent). The laser output consists of 150 fs pulses in the spectral range of 760−840 nm. The second harmonic of the laser was used to excite the samples. The cavity dumper operated at a rate of 800 kHz. The TCSPC detection system was based on a Hamamatsu 3809U multichannel plate photomultiplier and an Edinburgh Instruments TCC 900 integrated TCSPC system. The instrument time response was approximately 40 ps (full width at half-maximum, fwhm). The excitation pulse energy was reduced by neutral-density filters to about 10 pJ. The steady-state fluorescence spectrum was measured by a Horiba Jobin Yvon FluoroMax-3 fluorescence spectrofluorimeter.

Scheme 2. Molecular Structure of 8-Hydroxy-1,3,6pyrenetrisulfonate (HPTS)

The major finding of this study is that an ESPT indeed takes place from the ROH form of adsorbed HPTS to the N-acetyl-Dglucosamine. We also note that the transferred proton on the chitin scaffold is mobile and can geminately recombine to reform the excited ROH* form of HPTS.

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RESULTS When HPTS is adsorbed on chitin already in the ground state, about half of the molecules are in the RO− state. This is seen from the excitation spectra shown in Figure 1a. HPTS is strongly adsorbed on the chitin’s scaffold. Chitin purchased from Sigma is composed of ∼85% N-acetyl-Dglucosamine and ∼15% or less D-glucosamine. The pKb of Dglucosamine is about 6, whereas that of N-acetyl-D-glucosamine is much greater, pKb ∼ 14. When HPTS with a pKa of 7.4 is added to chitin which is somewhat basic, the excitation spectrum shows the presence of both ROH and RO − absorption bands when the emission is measured at the RO− emission band at ∼525 nm. Shown in Figure 1a are five spectra: four of chitin samples and one for a sample of HPTS in methanol. Three of the five are wet samples in which HPTS is adsorbed on chitin and the solvent−chitin weight ratio is 0.8:1. The solvents are H2O, methanol, and ethanol. We also let the methanol evaporate, and

ESPT GEMINATE RECOMBINATION MODEL Scheme 3 shows a common photoprotolytic cycle of a photoacid.27,28 The excited-state photoacid, ROH*, transfers a proton to the solvent at a rate kPT to form an ion pair RO−*··· H+. Scheme 3. Photoprotolytic Cycle of Photoacidsa

a

DSE stands for Debye−Smoluchowski equation (see text). 1974

DOI: 10.1021/acs.jpca.5b01398 J. Phys. Chem. A 2015, 119, 1973−1982

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Figure 1. Steady-state spectra of HPTS on chitin. (a) Excitation spectra, emission collected at 530 nm. (b) Normalized emission spectra, excitation at 390 nm.

Figure 2. Time-resolved emission of the ROH form of HPTS in bulk solvents and on chitin with the same solvents, detected at 440 nm. (a) Normalized linear scale. (b) Normalized semilogarithmic scale.

intensity ratios, IFRO−/IFROH, of all samples shown in Figure 1 are greater than 2. We therefore conclude that in all chitin samples an efficient ESPT process occurs, even though in both methanol and ethanol solutions HPTS is incapable of transferring a proton to the solvent within the excited-state lifetime. For HPTS, the transition dipole moments and the fluorescence lifetimes of both ROH and RO− are about the same: τF ∼ 5.4 ns. A simple equation connects the fluorescence band intensity ratio and the ESPT rate constant,30 kPT:

only then do we measure both the steady-state spectrum and the time-resolved properties of a semidry sample under controlled atmospheric conditions of 23 °C and 50% humidity. As seen in Figure 1a, for the HPTS−methanol−chitin sample ex the intensity ratio Iex ROH/IRO− is about 2, whereas in ethanol, ex H2O, and semidry samples it is about the same, Iex ROH/IRO− ∼ 1, or even less. The sharp modulation of the excitation spectra in the spectral region of 440−480 nm arises from the xenon excitation lamp. Figure 1b shows the steady-state fluorescence spectra of the HPTS samples shown in Figure 1a. In methanol solutions, the spectrum consists of only the ROH band since ESPT is not taking place within the excited-state lifetime. The ESPT rate constant in methanol is estimated to be about 4 orders of magnitude smaller than in water. The ESPT rate in water27,28 is ∼1010 s−1 and the fluorescence lifetime of the ROH form is 5.4 ns (kF ≈ 1.8 × 108 s−1), and therefore ESPT from HPTS to methanol is not efficient. When HPTS is adsorbed on chitin in the presence of water, methanol, or ethanol, the steady-state fluorescence consists of two emission bands: a band of the ROH form with a maximum at ∼450 nm, and another emission band of the RO− form at 512 nm. The excitation wavelength is 390 nm, for which the RO− form absorptivity is about one-third of its maximum at 470 nm. Since chitin is slightly basic and the pKa of HPTS is about 7.4, then approximately half of the HPTS molecules are already in the RO− form in the ground state. The RO− form in the ground state contributes only a small fraction of the RO− emission band intensity, as seen in Figure 1b. The fluorescence

F F −1 −/ I kPT = IRO ROHτF

(1)

where τF is the fluorescence lifetime of the RO− form. Thus, we conclude that ESPT is taking place when HPTS is adsorbed on chitin. When water is present the ESPT rate is high, whereas for methanol and ethanol the rate is lower. Figure 2 shows, on linear and semilogarithmic scales, the time-resolved emission of the ROH form of HPTS of several samples. The signals were measured by the time-correlated singlephoton counting technique (TCSPC) with an instrument response, full width at half-maximum (fwhm), of ∼40 ps. Three of the six signals were measured for chitin samples in which HPTS was adsorbed in H2O, methanol, and ethanol solutions at a 1:1 solvent/chitin weight ratio. HPTS is a weak photoacid with pKa* ≈ 1.3. The ESPT rate constant, kPT, in water is 1010 s−1,27,28 and the time constant for proton transfer (PT) to bulk water is ∼100 ps. As seen in Figure 2b (semilog scale), the 1975

DOI: 10.1021/acs.jpca.5b01398 J. Phys. Chem. A 2015, 119, 1973−1982

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Figure 3. Time-resolved emission of the RO− form of HPTS in bulk H2O and on chitin with several solvents, detected at 525 nm. (a) Normalized linear scale. (b) Normalized semilogarithmic scale.

Figure 4. Time-resolved emission of the ROH form of HPTS in bulk H2O and D2O and on chitin with the same solvents, detected at 440 nm. (a) Normalized linear scale. (b) Normalized semilogarithmic scale.

397 nm, about one-third of the HPTS RO− signal arises from direct excitation of ground-state RO−. Figure S1 in the Supporting Information shows the time-resolved emission of the RO− signal, measured at 530 nm, of HPTS in basic and slightly acidic aqueous solutions (pH 10 and pH 6), with excitation at 397 nm. The RO− signal of HPTS at pH 6 is a result of the photoprotolytic process that begins with excitation of the ROH form and therefore shows a distinct signal rise with a rise time of 100 ps that is identical to the decay time of the ROH signal measured at 440 nm. Because of the large overlap of the ROH and RO− emission bands, the signal measured at 530 nm contains the contributions of both ROH and RO− that partially cancel part of the signal rise of the RO− component. The signal of the HPTS sample at pH 10 does not show a distinct rise except for the TCSPC instrument response of about 40 ps fwhm. Figure S1 in the Supporting Information also shows the result of a manipulation that combines the signals of pH 6 and pH 10 measured at 530 nm. This synthetic signal is the sum of half the normalized intensity of the pH 10 sample and the normalized signal of the pH 6 sample. It mimics the features of the signal obtained from HPTS adsorbed on chitin. The synthetic signal shows a significantly reduced amplitude of the rise component in a way similar to that of the RO− signal of HPTS adsorbed on chitin. The rise component of HPTS adsorbed on chitin in the presence of methanol (Figure 3) shows a rather long rise time of about 1 ns (compared to 100 ps in bulk water) and a subsequent exponential decay with a time constant of 5.4 ns. The signal rise fits nicely with the decay time of the HPTS

signal is nonexponential (not a straight line) as a consequence of an efficient reversible geminate recombination process. RO−* + H3O+ → ROH* + H 2O

(2)

−

The deprotonated HPTS RO * form is electrically charged by four electronic units (−4). The Coulomb potential between the diffusing geminate proton and RO− is large, and only at the great distance of 28 Å from RO− is the electrical-attraction potential equal to the thermal energy, kBT. The Debye radius, RD, is given in eq 3 and defines that distance the Coulomb interaction: RD =

Ze 2 εkBT

(3)

Z is the RO− electronic charge (4 for the RO− form of HPTS), ε is the dielectric constant of the medium (εH2O = 78) and e, kB, and T are the electronic charge, the Boltzmann factor, and the absolute temperature. In bulk water RD is 28 Å. At shorter distances r < RD, the Coulomb potential is greater than the thermal energy, and therefore the probability of geminate recombination is enhanced when the proton−RO− distance r is shorter than RD. Figure 3 shows the time-resolved fluorescence of the RO− form of HPTS measured at 525 nm, close to the band peak position at 512 nm for several samples. Three chitin samples and one sample of HPTS in bulk H2O were examined. About 50% of the HPTS adsorbed on chitin is already in the ground state in the RO− form. When excited at 1976

DOI: 10.1021/acs.jpca.5b01398 J. Phys. Chem. A 2015, 119, 1973−1982

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The Journal of Physical Chemistry A ROH signal measured at 440 nm (see Figure 2). The semidry sample and the ethanol sample show a smaller amplitude of the rise component since the ground-state RO− population is much larger than in methanol (see Figure 1a). The rise times of the HPTS RO− signals of all the chitin samples in the presence of methanol and ethanol are comparable. HPTS in the water− chitin sample shows a much faster ESPT rate and thus the rise time is much shorter. Figures 1−3 clearly show that ESPT occurs in the HPTS/ chitin sample at a reduced rate compared to HPTS in bulk water. In previous studies,7−9,31−33 it was shown that ESPT occurs not only to solvent molecules but also to weak bases in solution. We suggest that the N-acetylamine group of the Nacetyl-D-glucosamine is a weak base but strong enough to accept the proton from the ROH* of HPTS. We devote a section in the Supporting Information to presenting results of an ESPT experiment in which a proton is transferred from HPTS to a mild base (acetate ion) in methanol solution. Figure S2 in the Supporting Information shows both steady-state and time-resolved-emission results of HPTS in several methanol acetate solutions. The results clearly show the direct proton transfer from HPTS to the acetate ion. In conclusion, photoacids including HPTS transfer a proton to mild bases in solution. When adsorbed on chitin, HPTS transfers the proton to the N-acetylamine of chitin. Kinetic Isotope Effect. Figure 4 shows, on linear and semilogarithmic scales, the time-resolved emission of the ROH form of HPTS measured at 440 nm in H2O and D2O solutions as well as of HPTS adsorbed on chitin in the presence of H2O and D2O at a 1:1 weight ratio. The ESPT rate of HPTS in an aqueous solution has a kinetic isotope effect of about 3. The proton-transfer (PT) time constants in H2O and D2O are 100 and 300 ps, respectively. As seen in Figure 4, the kinetic isotope effect (KIE) is somewhat smaller in the chitin samples. The smaller KIE may arise from a small amount of H2O adsorbed on chitin. It may also arise from a smaller KIE of the proton-transfer rate to chitin and not to water itself. The kinetic isotope effect of the ESPT process from HPTS to acetate ions in aqueous solution34 is about 2. The existence of a kinetic isotope effect rules out the possibility that the shorter fluorescence lifetime of HPTS ROH adsorbed on chitin arises from other nonradiative processes not related to the ESPT process. KIE measurements are important in the identification of ESPT in complex systems, in which adsorption may enhance or prevent nonradiative processes. As an example, the fluorescence intensities of molecular-rotor molecules like thioflavin-T (ThT) and auramine-O are enhanced by 3 orders of magnitude when these molecules are adsorbed on cellulose and the fluorescence decay rate decreases by the same amount.35 In these compounds, the nonradiative rate is a result of intramolecular rotation and is not related to an ESPT process. HPTS Adsorbed on Cellulose. Figure 5 shows the steadystate emission spectrum of HPTS adsorbed on cellulose in the presence of methanol (1:1 ratio). As seen in Figure 5, the spectrum consists of both the ROH and RO− bands but the ESPT process is much less efficient than that of HPTS adsorbed on chitin, since the intensity ratio of ROH to RO− is about 3:1. The weak intensity of the RO− spectral band may result from the presence of atmospheric water adsorbed on cellulose or because of the proton-accepting power of the glucose itself. For comparison, Figure 5 also shows the HPTS spectrum in bulk methanol and in a water−methanol

Figure 5. Steady-state measurements of HPTS on cellulose−methanol, bulk methanol and water−methanol mixture of xwater = 0.32, normalized emission spectra, excitation at 390 nm.

mixture of xH2O = 0.32. In methanol solution, the spectrum consists of the ROH emission band alone. This observation clearly shows that the ESPT process from the ROH of HPTS does not take place within the excited-state lifetime (5.4 ns) in neat methanol, whereas in neat water the rate constant is 1010 s−1. In the water−methanol mixture of xH2O ≈ 0.32 the ESPT rate is similar to that of HPTS adsorbed on cellulose. Cellulose powder is somewhat hygroscopic, and atmospheric water vapor may be adsorbed on the semidry samples. Only for water−methanol mixtures of mole fraction xH2O ≥ 0.3 is the steady-state emission spectrum of HPTS equal to that of HPTS adsorbed on cellulose in the presence of methanol (1:1 ratio) shown in Figure 5. Figure S3 in the Supporting Information shows the steadystate emission spectra of HPTS in several water−methanol mixtures in which the water content is in the range of molar ratios of 0−0.5. As seen in Figure S3 (Supporting Information), at a low mole ratio of water (xH2O < 0.1), the emission spectrum consists of the ROH band alone (IFROH/IFRO− >25:1). Only at higher water content does the spectrum show two emission bands, that of the ROH and that of the RO−, a consequence of an efficient photoprotolytic process. We found that the emission spectrum of HPTS at a water mole ratio of xH2O ≈ 0.32 is similar to that of HPTS adsorbed on cellulose in the presence of methanol (1:1 weight ratio). Figure S4 in the Supporting Information shows the ROH and RO− timeresolved emission of HPTS in cellulose−methanol and in water−methanol solutions in the mole fraction range of 0.28− 0.42. Both the ROH signal decay curve and the RO− signal rise of the methanol−water solutions are similar to those of the cellulose−methanol sample. We measured the amount of atmospheric water vapor adsorbed on 100 mg of dried cellulose (at 85 °C for several hours). We found that only 3 mg of water is adsorbed on the cellulose sample at 25 °C and 47% humidity. We therefore conclude that cellulose promotes the ESPT process of adsorbed photoacid molecules. Figure 6a shows the time-resolved emission of the HPTS ROH band measured at 440 nm adsorbed on cellulose in the presence of methanol at a 1:1 weight ratio. For comparison, we also added the fluorescence decay curve of HPTS in methanol solution. As seen in Figure 6a, the fluorescence decay rate of the HPTS ROH band when adsorbed on cellulose in the presence of methanol is greater than that obtained for methanol solution. The shorter decay time of the fluorescence signal shows that an ESPT process 1977

DOI: 10.1021/acs.jpca.5b01398 J. Phys. Chem. A 2015, 119, 1973−1982

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Figure 6. Time-resolved emission of (a) ROH form of HPTS in bulk MeOH, chitin with methanol and cellulose with methanol samples, detected at 440 nm, shown on a normalized semilogarithmic scale, and (b) RO− form of HPTS in bulk MeOH, chitin with methanol and cellulose with methanol, detected at 525 nm shown on a normalized semilogarithmic scale.

adsorbed on chitin. The amplitude of the second decay component is 0.1 for bulk H2O and 0.32 for HPTS on chitin. The second and the third time components are assigned to the repopulation of the ROH* form by the reversible geminate recombination process. The larger amplitude of the second time component in the chitin sample reflects the greater probability of the proton geminate recombination. The time constants of τ2 are 0.6 and 0.8 ns for the bulk and chitin samples. The decay time of the third time component is rather long but shorter than the fluorescence decay time in the absence of PT, which is 5.4 ns in bulk water. The amplitude of the third time component is rather small in water, ∼0.01, and is much larger when HPTS is adsorbed on chitin, ∼0.05. The third time exponent arises from the nonexponential nature of the diffusion-assisted reversible proton geminate recombination process.27,28 The formalism of bimolecular second-order kinetics cannot account for the geminate recombination process. Below, we analyze the time-resolved data using the diffusionassisted reversible geminate recombination model that was briefly described before. The first step in the model is the intermolecular excited-state proton transfer with a unimolecular rate constant, kPT. The proton geminate recombination repopulates the excited-state ROH* which may subsequently undergo a second protolytic cycle. Since the recombination reaction is influenced by the proton diffusion, the decay of the ROH fluorescence tail signal is not exponential. The long-time tail of the fluorescence, when compensated by multiplying the signal by exp[t/τF] to account for the finite excited-state lifetime, decays according to a power law, t−α. The asymptotic long-time expression for the amplitude and the decay law is given by11,27,28

occurs when HPTS is adsorbed on cellulose in the presence of methanol. When HPTS is adsorbed on chitin, the ESPT rate is much larger than that on cellulose samples (see Figure 6a). These findings are in accord with the steady-state spectrum that shows the existence of an RO− band. Figure 6b shows the time-resolved emission of the RO− band HPTS, measured at 525 nm. HPTS is adsorbed on cellulose in the presence of methanol and on a semidry sample. The TCSPC HPTS signals of both samples measured at 525 nm show a distinct rise component that is a consequence of a slow ESPT process that takes place when HPTS is adsorbed on cellulose. For comparison, we also added the signal of HPTS RO− in water−methanol mixtures of xH2O = 0.32. On the basis of the steady-state fluorescence spectra, the time-resolved emission studies and the control experiments, we conclude that an ESPT process occurs from HPTS to glucose when HPTS is adsorbed on cellulose.

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DATA ANALYSIS The decay curves of the time-resolved emission signal of the ROH form of HPTS in bulk H2O and HPTS adsorbed on chitin, shown in Figures 2 and 4, are nonexponential. They can be approximated by a three-exponential fitting function: 3

IF(t ) =

∑ ai exp(t /τi)

(4)

i=1

Table 1 provides the three exponential fitting parameters. Table 1. Fitting Parameters by a Three-Exponential Function of HPTS ROH TCSPC Signals chitin bulk

H2O D2O H2O D2O

a1

τ1 (ns)

a2

τ2 (ns)

a3

τ3 (ns)

0.63 0.57 0.89 0.85

0.12 0.18 0.11 0.3

0.32 0.36 0.10 0.14

0.8 1.2 0.6 1.05

0.05 0.07 0.01 0.01

2.7 3.7 2.5 3.7

IfROH(t ) exp[t /τF] ∼

πa 2ka exp[−V (a)] 2kPT(πD)d /2

t −d /2 (5)

a is the reaction-sphere radius, kPT and ka are the intrinsic ESPT and geminate recombination rates occurring on the reaction sphere, −V(a) (=RD/a) is the Coulomb potential at the reaction sphere radius, D is approximately the proton diffusion coefficient, d is the dimension of the diffusion space, and RD is the Debye radius and is given by eq 3. Equation 5 shows that the long-time ROH fluorescence decay fits a power law of t−d/2. Equation 5 also indicates that the amplitude depends on several parameters, but the decay law depends only on the dimension of the diffusion space.

In both bulk and chitin samples, the component with the shortest time constant has the largest amplitude. In bulk water, the amplitude is 0.89 ± 0.04, while in chitin it is much smaller, 0.63 ± 0.03. We attribute the short time component to the ESPT rate constant, kPT. In bulk water, the proton is transferred to a nearby water molecule which is hydrogen-bonded to the HPTS hydroxyl group. The value of kPT in bulk water is ∼9 × 109 s−1, and in previous studies,11,27,28 the quoted values are about 1.0 × 1010 s−1. We find a similar decay time for HPTS 1978

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The Journal of Physical Chemistry A Table 2. Fitting Parameters of the Geminate Recombination Modela kPT [109 s−1] bulk H2O bulk D2O chitin H2O D2O MeOH EtOH a

ka [Å·ps−1]

D [cm2 s−1]

a0 [Å]

−4

dim.

τf−1 [109 s−1]

added exponentialb

9 2.8

6 2.7

10 0.66 × 10−4

6 6

3 3

0.18 0.18

− −

3 2 1.05 1.05

6 4 2 2

0.66 0.35 0.28 0.28

10−4 10−4 10−5 10−5

6 6 6 6

2.7 2.75 2.85 2.85

0.22 0.22 0.25 0.24

+0.2 exp[−t/0.12] +0.17 exp[−t/0.2] +0.1 exp[−t/0.26] +0.15 exp[−t/0.22]

× × × ×

RD = 28 Å. bTime in nanoseconds.

We used the program of Krissinel’ and Agmon36 to fit the time-resolved emission of HPTS on chitin. The program solves the spherically symmetric diffusion problems (SSDP) under the proper initial and boundary conditions that are appropriate for the reversible proton geminate recombination. There are several fitting parameters involved in the sphericalsymmetric geminate recombination model. There are two intrinsic rate constants: the ESPT kPT and the geminate recombination ka (see Scheme 3). The reaction-sphere radius a represents a sphere that mimics the size of the ROH form of a photoacid surrounded by one or more layers of water molecules. The proton in the solvent diffuses with a diffusion coefficient which is approximately that of the proton DH+. In bulk water at room temperature, it is DH+ = 9.1 × 10−5 cm2/s. The RO− form of the HPTS molecule is 4 times negatively charged, and thus the Coulomb attraction potential between the RO− and the geminate proton is large. In water, with a large dielectric coefficient of 78, it is about 28 Å. The Coulomb potential increases the probability of geminate recombination, and this is seen as a large amplitude of the long-time fluorescence tail of the ROH* time-resolved signal. Table 2 shows the fitting parameters of the ROH fluorescence signal of HPTS in bulk H2O and D2O as well as that of HPTS adsorbed on chitin in the presence of H2O, D2O, methanol, and ethanol. The geminate recombination model provides an excellent fit of HPTS ROH decay signals in both bulk H2O and D2O. We could not achieve a good fit of the early times of the ROH signal decay of the chitin samples. Table 2 provides the fitting parameters of the ROH of HPTS time-resolved signals in bulk H2O and D2O as well as of HPTS on chitin in the presence of H2O, methanol, and ethanol. In order to obtain a reasonable fit for the HPTS-on-chitin signal, we added to the SSDP fit an exponential short decay time of small amplitude −0.2 ± 0.05. The time constant of this component is similar to that of the ESPT in bulk water, τPT = 0.12 ns. We assign this time component to a proton transfer from HPTS to a water pool next to the chitin scaffold. In cellulose−water samples it was found that water is in two states:37 bound water which is next to the glucose, and free water that forms water pools. We are unaware of any similar study that describes the state of water next to chitin. We therefore adopt the cellulose−water sample picture. The major part (a ≈ 0.8) of the ROH fluorescence decay fits nicely with the geminate recombination model. An alternative explanation of the small-amplitude, short-decay-time component is that it arises from the proton transfer from HPTS to the small fraction of chitin’s D-glucosamine groups. The relative concentration of these groups is estimated to be less than 15% in chitin.38 The amplitude of the rapid component is about the same as that of the D-glucosamine in chitin. The ESPT rate constant, kPT, is only 3 × 109 s−1, about one-third of that in bulk water. The intrinsic recombination rate is

approximately that of bulk water, but the proton diffusion coefficient is smaller and its value is about two-thirds of that of bulk water. The relative amplitude of the fluorescence longtime tail of HPTS on chitin increases by a factor of about 3 (compared to that of HPTS in bulk water); the relative ratio ka/ kPT increases by the same factor, since the ESPT rate on chitin decreases by a factor of 3. This is easily seen in eq 5, which quantifies the amplitude and the decay law of the long-time fluorescence tail. The fitting parameters of the signals of the adsorbed HPTS ROH on chitin in the presence of methanol or ethanol are also given in Table 2. The ESPT rate constant, kPT, and the recombination rate, ka, are about one-third of their values in the presence of H2O. The diffusion coefficient is about half that of the water sample. We also found that a smallamplitude short-decay-time component is needed in order to obtain a good fit of the fluorescence decay curves of the ROH form of HPTS on chitin in the presence of methanol and ethanol.

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MAIN FINDINGS The main findings of this paper are the following: 1. 8-Hydroxy-1,3,6-pyrenetrisulfonate (HPTS) adsorbed on chitin shows photoprotolytic activity. 2. Excited-state proton transfer (ESPT) from HPTS, presumably to chitin acetyl glutamate, occurs at a rate slower than the rate to bulk water. 3. The ESPT process also occurs in semidry chitin samples where only a small amount of water is adsorbed from the surrounding air, as well as in samples covered with methanol or ethanol. 4. In aqueous chitin samples, the ESPT rate is greater by a factor of 3 than in methanol and ethanol. 5. A rather small kinetic isotope effect (KIE) of ∼1.7 is observed when H2O is replaced by D2O, whereas the KIE of the ESPT of HPTS in water is ∼3.1. 6. When HPTS is adsorbed on cellulose in a methanol sample of 1:1 weight ratio, ESPT occurs at a rate 10 times slower compared to ESPT from HPTS to bulk water. In contrast, HPTS does not transfer a proton to methanol in bulk methanol solutions.

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DISCUSSION In recent studies25,39 we reported on the photoprotolytic activity of photoacids adsorbed on two biopolymers: cellulose and chitosan (see Scheme 1). The latter is the deacetylated form of chitin also shown in Scheme 1. Cellulose, composed of glucose monomers, showed mild protic activity when a small amount of water was added to dry HPTS adsorbed on the cellulose. HPTS was added by spraying it in a methanol solution on the 20 μm size cellulose powder. The water was added after the methanol evaporated. 1979

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Chitosan is composed of D-glucosamine units and is a basicpH material. To conduct the photoprotolytic experiments on a photoacid adsorbed on chitosan, we used 2-naphthol with pKa ∼ 9.4. About two-thirds of the 2-naphthol molecules were in the ROH form, and thus a photoprotolytic experiment could be conducted even on the basic-pH chitosan. The results show a direct proton transfer not only to the water molecules next to chitosan but also to the amine group of the D-glucosamine. In the current study, we measure the photoprotolytic activity of HPTS adsorbed on chitin. Chitin is composed of N-acetyl-Dglucosamine, which is the main component of the cell walls of fungi and also in the exoskeletons of arthropods such as crabs, lobsters, and shrimps and of many insects. The steady-state and time-resolved optical spectroscopy results show unequivocally that photoprotolytic processes indeed occur from the adsorbed HPTS molecules to the N-acetyl-D-glucosamine. The amide chemical bond is usually considered as not involved in protic activity. In protein protic chemistry and hydrogen-bond formation, the peptide bond does not respond to small pH changes and does not form strong hydrogen bonds with other amino acids of the protein. The results of the current study show more activity of the amide bond than anticipated. The Nacetyl-D-glucosamine units accept a proton from a photoacid within the excited-state lifetime of the ROH form of HPTS (τF ≈ 5.4 ns). This result is not at all trivial. As mentioned before, the amide bond in proteins is considered to be inert in terms of proton chemistry. Only the acidic or the basic amino acids are important in the proton chemistry of a protein. Chitin is not a protein but certainly has an amide bond that is active in accepting a proton from the excited ROH* form of HPTS. The proton docks on the amide bond and is mobile. The mechanism of proton mobility is hopping to a nearby acetylglucosamine monomer. The diffusion coefficient in chitin− H2O, 1:1 by weight, is about two-thirds of that in bulk water. In the presence of methanol or ethanol, the proton diffusion constant near the chitin scaffold is reduced by a factor of 2. In a previous article39 we studied the ESPT process of HPTS adsorbed on cellulose in the presence of a small amount of water. We found that ESPT occurs in these samples. In wet samples of less than1:1 by weight of water:cellulose, the ESPT rate is rather low, and thus, within the excited-state lifetime, the quantum efficiency of the ESPT is also low. When the water content is higher, the ESPT rate is almost that of bulk water. We explain these results by the existence of pools of water in cellulose of high water content, in which the triple negatively charged HPTS molecules desorb from the cellulose surface to these pools. In the current study, we extended the ESPT measurements of adsorbed HPTS on cellulose to cellulose covered with methanol and ethanol. We found that ESPT from HPTS also occurs in the presence of methanol and ethanol. In bulk methanol HPTS solutions, the ROH form is incapable of transferring a proton to the methanol molecules. Figure 5 shows the ROH and RO− steady-state fluorescence bands, and the time-resolved signals of the ROH and RO− forms are shown in Figure 6. Figures 5 and 6 show that an ESPT process indeed occurs when methanol is added to HPTS adsorbed on cellulose. We therefore conclude that cellulose promotes ESPT processes from adsorbed photoacids. The proton acceptor is most likely the glucose itself and not the methanol. Methanol may assist in the process as a mediator. The groups of the glucose most likely to accept the proton are the ether-oxygen groups.

Article

SUMMARY AND CONCLUSIONS

In the current research we have investigated the photoprotolytic processes of an adsorbed photoacid on two biopolymers: chitin and cellulose. Chitin is composed of Nacetyl-D-glucosamine and is the main component of the cell walls of fungi and also in the exoskeletons of arthropods such as crabs, lobsters, and shrimps and of many insects. We used steady-state and time-resolved fluorescence techniques to follow the photoprotolytic processes of the photoacid in this study. 8-Hydroxy-1,3,6-pyrenetrisulfonate (HPTS), a common photoacid with a pKa ∼ 7.4, was chosen for this purpose. HPTS is a mild photoacid with strong absorption and emission properties. The fluorescence of HPTS has two bands: one of the ROH form with a maximum at ∼450 nm, and the other band of the RO− form with a maximum at ∼512 nm. The major finding of the current study is that HPTS adsorbed on chitin transfers a proton to the acetyl-glucosamine monomer in the presence of a small amount of water and also when chitin is covered with methanol or ethanol. The ROH* signal decay is composed of two contributions: a small-amplitude (∼0.2) fast decay time component and a major amplitude (∼0.8) with a slower decay that can be fitted by the reversible proton geminate recombination model.27,28 The short-time decay component may arise from proton transfer to solvent pools next to the chitin scaffold or more likely to Nglucosamine (base) that exists also in chitin at a much lower concentration38 (85%). The ESPT rate from HPTS to Nacetyl-D-glucosamine in the presence of water is slower by a factor of 3 than the rate of HPTS to bulk water. In chitin HPTS samples covered with a small amount of methanol or ethanol, the ESPT rate is lower by a factor of 3 than the samples covered with water. The proton mobility next to the chitin scaffold is about two-thirds of that of a proton in bulk water. In a previous study,39 we measured, by the same techniques, the photoprotolytic processes of HPTS adsorbed on cellulose− water samples. We found that the time-resolved fluorescence decay of ROH is composed of two time components; the first one has a rapid decay and the decay rate of the second component is much slower. Their relative amplitudes depended on the water content of the sample. When the water/cellulose weight ratio is 2:1, the amplitude of the rapid component is much greater than that of the slow component. In the current study, we explore further the ESPT process in cellulose. We measured the time-resolved signal of the ROH form of HPTS adsorbed on cellulose with either methanol or ethanol. In bulk solutions of these solvents, HPTS is incapable of undergoing a photoprotolytic cycle, but when HPTS is adsorbed on cellulose, an ESPT process does occur, but at a much slower rate. A general conclusion can be drawn from the current study of the two abundant biopolymers, cellulose and chitin, both of which are able to promote proton activity. These biopolymers can accept a proton from an acidic compound at a rate that is lower than the transfer rate of the proton from the photoacid to bulk water. The protic reaction described in this article occurs in the excited state, but the same should happen for regular acids. Protic reactions of regular acids with chitin or cellulose may also occur, but at a much slower rate than to bulk water. The protons accepted by these biopolymers are mobile and can participate in further protic reactions of molecules next to or adsorbed on these biopolymers. 1980

DOI: 10.1021/acs.jpca.5b01398 J. Phys. Chem. A 2015, 119, 1973−1982

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Since plants, fungi, and arthropods are exposed to sunlight, the ESPT processes we report in the current study may occur also in these biomaterials in nature. There are several reports of solar energy harvesting in the epicuticle of the Oriental hornet.40−43 This photovoltaic activity may arise from ESPT processes similar to the one we report here.

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ASSOCIATED CONTENT

S Supporting Information *

ESPT of HPTS in aqueous sodium acetate bulk solutions, ESPT of HPTS to a mild base in methanolic solutions, and ESPT of HPTS in water−methanol solutions. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: 972-3-6407012. Fax: 972-3-6407491. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from the Israel Science Foundation. REFERENCES

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DOI: 10.1021/acs.jpca.5b01398 J. Phys. Chem. A 2015, 119, 1973−1982