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Excited-State Proton Transfer of Weak Photoacids Adsorbed on Biomaterials: Proton Transfer to Glucosamine of Chitosan Ron Simkovitch, and Dan Huppert J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/jp511349j • Publication Date (Web): 04 Jan 2015 Downloaded from http://pubs.acs.org on January 12, 2015

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The Journal of Physical Chemistry

Excited-State Proton Transfer of Weak Photoacids Adsorbed on Biomaterials: Proton Transfer to Glucosamine of Chitosan Ron Simkovitch and Dan Huppert* Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv University, Tel Aviv 69978, Israel *Corresponding author: Dan Huppert E-mail: [email protected] Phone: 972-3-6407012 Fax: 972-3-6407491 Abstract UV-vis steady-state and time-resolved techniques were employed to study the excited-state proton-transfer process from two weak photoacids positioned next to the surface of chitosan and cellulose. Both chitosan and cellulose are linear polysaccharides; chitosan is composed mainly of D-glucosamine units. In order to overcome the problem of the high basicity of the glucosamine, we chose 2-naphthol (pKa*≈2.7) and 2-naphtholo-6-sulfonate (pKa*≈1.7) as the proton emitters because of their ground state pKa (≈9). Next to 1:1 cellulose: water weight ratio, the ESPT rate of these photoacids is comparable to that of bulk water. We found that the ESPT rate of 2-naphthol (2NP) and 2-naphtholo-6-sulfonate (2N6S) next to chitosan in water (1:1) weight ratio samples is higher than in bulk water by a factor of about 5 and 2 respectively. We also found an efficient ESPT process that takes place from these photoacids in methanol environment next to the chitosan scaffold, whereas ESPT is not observed in methanolic bulk solutions of these photoacids. We therefore conclude that ESPT occurs from these photoacids to the D-glucosamine units that make up chitosan.

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Introduction Proton reactions and proton mobility are ubiquitous in life science. Photoacids are a class of molecules that are weak acids in their ground electronic state with pKa values ranging from 5 to 9. In their excited electronic state, their acidity increases, usually by more than seven pKa units, and thus their values of pKa* range from -8 to about 3. Photo-excitation of a photoacid by a brief pulse of light leads to an excited-state proton transfer (ESPT) to the solvent. This field has been widely researched by timeresolved optical techniques1,2,3,4,5,6,7,8,9,10,11,12,13,14,15. It was found that photoacids with pKa*>0 can transfer a proton efficiently within its excited-state lifetime only to water but not to linear alcohols like methanol or longer chain monols or diols. In the current study we explore by steady-state and time-resolved fluorescence techniques, the ESPT rate of two weak photoacids next to two biomaterials. The two biomaterials are cellulose and chitosan. Cellulose, shown in Scheme 1b, can be found in the cell walls of green plants16,17 and hence it is the most abundant organic polymer on Earth18.

a)

b)

Scheme 1: a) Molecular structure of chitosan, where R is randomly either a hydrogen atom,

or a

group. b) Molecular structure of cellulose

Cellulose is a polysaccharide (C6H10O5)n consisting of a long linear chain with several hundred to over ten thousand linked D-glucose units that condense through β(1→4)glycosidic bonds. Cellulose is a straight-chain polymer with no coiling or branching. Many properties of cellulose depend on its chain length or degree of polymerization. Cellulose from wood pulp has typical chain lengths of between 300 and 1700 units. It 2 Environment ACS Paragon Plus

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has both crystalline and amorphous regions, and upon hydration, can absorb water molecules at a ratio of nearly 5:1 (H2O: cellulose)19. Chitosan is a linear polysaccharide composed of randomly distributed β-(1-4)linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit), shown in Scheme 1a. It is synthesized by treating crustacean shells with sodium hydroxide. The molecular weight of commercially produced chitosan is between 3800 and about 106 daltons. In agriculture, chitosan is used primarily as a natural seedtreatment and plant-growth enhancer, and as an ecologically friendly biopesticide that boosts the innate ability of plants to defend themselves against fungal infections20. We chose two weak photoacids for the current study - 2-naphthol (2NP) and 2-naphthol-6-sulfonate (2N6S), shown in Scheme 2.

b)

a)

Scheme 2: a) Molecular structure of 2-naphthol (2NP). b) Molecular structure of 2-naphthol6-sulfonate (2N6S)

The ground-state pKa values of 2NP and 2N6S are 9.4 and 9.0, respectively; their excited-state pKa* values are 2.7 and 1.7. We chose these two photoacids because of two important properties they possess. The first one is their high pKa values, (pKa≥9), that enable keeping them in the ground state in their protonated form - ROH - next to the chitosan surface. The amine group in the D-glucosamine unit of chitosan is a mild base and so it will deprotonate acidic molecules nearby. We found that these two photoacids next to chitosan are indeed predominantly ~5:1 in their ROH form and therefore are capable of undergoing the photoprotolytic cycle described below. Upon excitation (see the full photoprotolytic cycle in Scheme 3), the excitedstate acid, ROH*, can undergo an intermolecular proton transfer (kPT) from the acid group to nearby solvent molecules to form, as a first step, an RO-*···H+ ion pair. We



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refer to this process as an intermolecular excited-state proton transfer (ESPT) to the solvent. When the photoacid molecule also has a base group, like a heterocyclic nitrogen atom or an amine group, excited-state intramolecular proton transfer (ESIPT) may occur. k PT

-

ROH*

DSE

[RO *···H ]

ka hν

+

-

RO * + H

+

kq ' k PT

ROH

ka'

-

+

DSE

[RO ···H ]

-

RO + H

+

Scheme 3: Photoprotolytic cycle of photoacids. DSE stands for Debye-Smoluchowski Equation (see text).

Following the ESPT process, the proton in the ion pair can either diffuse to the bulk solvent, RO-* + H+, which can be described by the Debye-Smoluchowski Equation (DSE)7,21, or it may return by a random walk and recombine with the excited deprotonated state, RO-*, to form ROH* by what is known as the geminate recombination process. Since the ROH* and the RO-* states have different positions of the emission-band peak (for 2NP, ~360 and 417nm, respectively), it is relatively easy to follow each of the species by time-resolved fluorescence in order to follow the ESPT process. The second important property of these weak photoacids is the high sensitivity of the ESPT rate to the properties of the proton acceptor and the physical-chemistry parameters of the solvent. As already mentioned, the weak photoacids (pKa*≥0) are incapable of transferring a proton to methanol and other alcohols within the lifetime of the excited state, whereas stronger photoacids with pKa*420nm) arises mostly from the cellulose background emission. When water is added to the cellulose 2NP sample, the spectrum consists of two emission bands, the ROH and RO- bands, as expected from an efficient ESPT rate of 2NP adsorbed or next to the cellulose scaffold. Previous studies have shown19 that water of a weight of up to about 200% of the weight of cellulose is in two states. A small fraction is in the form of bound water next to the cellulose scaffold and the rest is in water pools next to the cellulose. The bound water molecules do not participate in the ESPT process, whereas the water molecules in the water pools that are enclosed within the cellulose scaffold do participate in the photoprotolytic process and accept the photoacid proton. Figure 4c shows the emission spectrum of 2NP in 30mg of chitosan in the presence of 30µL of methanol. The spectrum consists of both the RO- and the ROH F F bands, with an amplitude ratio of I RO − / I ROH ≈ 2.5 . This high ratio is mainly due to the

large basicity of chitosan24 pKb≈6.3. As seen in Scheme 1a, chitosan is a linear polysaccharide composed of 75-85% randomly distributed D-glucosamine and 2515% N-acetyl-D- glucosamine. However ESPT does occur in 2NP in chitosan in the presence of methanol, which is unexpected. We mentioned above that ESPT from 2NP to methanol is not observed in a neat methanol solution and the ESPT rate is estimated to be only of the order of 104s-1. As we will show, the time-resolved fluorescence measurements indeed



confirm that an ESPT process is taking place next to chitosan, even in methanol solution. Figure 4d shows the steady-state emission spectrum of 2NP on chitosan in the presence of 30µL of water. The amplitude ratio between the ROH and RO- bands for F F this sample is I RO − / I ROH ≈ 4.2 and ESPT occurs at a greater rate than in pure water

solution. Figure 5 shows the steady-state excitation and emission spectra of 2naphtholo-6 sulfonate (2N6S) in several media. a)

b)

Normalized Signal

1.0

Bulk

0.8

H2O Ex 310nm

0.6

MeOH Ex 310nm MeOH Em 400nm

0.4

H2O Em 440nm

H2O H2O

Cellulose Ex 310nm H2O

0.8

Ex 310nm MeOH

0.6

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0.0 300

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600

c)

350 1.0

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Ex 310nm

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600

Chitosan H2O 0.8

0.6

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0.4

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500

d)

Chitosan MeOH Normalized Signal

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Ex 310nm

0.0 350

400

450

500

Wavelength [nm]

550

600

350

400

450

500

550

600

Wavelength [nm]

Figure 5: a. Emission and excitation spectra of 2N6S in methanol and water solutions. b. Steady-state emission spectra of 2N6S on cellulose and in the presence of methanol or water. c. Emission spectrum of 2N6S on chitosan/methanol sample d. Emission spectrum of 2N6S on chitosan/water sample

Figure 5a shows the emission and excitation spectra of 2N6S in methanol and water solutions. The emission spectrum of 2N6S in methanol solution consists of the ROH band only, with a maximum at ~360nm; in water the spectrum consists of both the ROH and the RO- bands. These results are explained by the fact that the ESPT process takes place in water but not in methanol, and so in water the emission


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spectrum consists of both bands. The band intensity ratio in water solution F F 25,26 ( I RO , it was − / I ROH ≈ 8 ) indicates that the ESPT rate is high. In previous studies

found that the ESPT rate coefficient of 2N6S, kPT≈109s-1, is about an order of magnitude greater than that of 2-naphthol. Figure 5b shows the steady-state emission spectra of 2N6S when added to 20µm-size cellulose powder in the presence of methanol or water. In the case of methanol, the RO- band intensity is small and ESPT may not take place. By contrast, 2N6S in water (20 µL H2O in 20 mg cellulose), shows ESPT at a rate similar to that for pure water solution. In the 2N6S chitosan/methanol sample, the steady-state emission spectrum consists of both ROH and RO- bands of nearly the same intensity. If the values of pKa of both chitosan and 2N6S are similar (~9), it is expected that the populations of the ROH and RO- forms of 2N6S in the ground state will be similar. The steady-state emission spectrum of 2N6S in water shows a rather weak ROH band and a tenfold stronger RO- band. This clearly indicates that the ESPT rate is high. Time-resolved measurements

Figure 6 shows the time-resolved emission of both the ROH and RO- bands of 2-naphthol (2NP) in several media.

a)

b)

Normalized Signal

1

1

430 nm

430 nm

0.1

0.1

360 nm

Bulk

0.01

Cellulose

0.01

Bulk H2O 360nm Bulk H2O 430nm

360 nm

20µl H2O

360 nm

20µl H2O

Bulk MeOH 360nm

1E-3 0 1

5

10

15

20

25

1E-3 30

c)

1

0

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d)

430 nm Normalized Signal

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430 nm 0.1

0.1

360 nm

0.01

0.01

360

nm

Chitosan H2O

Chitosan MeOH 1E-3

1E-3 0

5

10

15

Time [ns]

20

25

30

0

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15

Time [ns]


20

25

30


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Figure 6: Time-resolved emission of both the ROH and RO- bands of 2-naphthol (2NP) in a. bulk H2O and methanol, b. time-resolved emission signals of the ROH and RO- forms of 2NP next to a cellulose surface in water samples of 1:1 weight ratio, c. TCSPC signals of 2NP in 1:1 weight ratio of chitosan with methanol, d. TCSPC signals of 2NP in 1:1 weight ratio of chitosan with water.

The time-resolved emission is measured by the time-correlating single-photoncounting technique with a full width at half maximum instrument-response time of about 40ps. Figure 6a shows, for 2NP in water and in methanol solutions, both the ROH band emission measured at 360nm and the RO- band measured at 430nm. ESPT does not take place in methanol since kPT is lower than the fluorescence decay rate by about four orders of magnitude. In water, kPT~ kF and thus ESPT does take place, although with reduced efficiency, and about half of the excited molecules decay radiatively to the ground state prior to proton transfer. The emission signal measured at 430nm contains the signals of both the ROH and RO- forms of the 2NP photoacid because of the spectral overlap between the two emission bands and the reduced efficiency of the ESPT process. At about 4ns, about 70% of the measured signal is that of the RO-. The rise component measured at 430nm fits nicely with the decay rate of the ROH signal measured at 360nm. Figure 6b shows the time-resolved emission signals of both the ROH and ROforms of 2NP next to a 1:1 ratio by weight of a cellulose/water surface of 20µm powder of cellulose and water. The decay rate of the ROH signal and the rise and decay rates of the RO- signal are similar to those of 2NP in water solution. We explain this result by the fact that the 2NP molecules are next to pools of water and therefore the rate of ESPT to water in wet cellulose is as efficient as in pure water solution. Figure 6c shows the TCSPC signals of 2NP in 1:1 (by weight) chitosan/methanol samples. Surprisingly, the decay rate of the ROH emission is much greater than expected from the ROH fluorescence decay rate of 2NP in methanol solution. The signal measured at 430nm shows a rise component followed by a long exponential decay. This kind of time-resolved emission signal is expected for photoacids that can transfer a proton within the lifetime of their first excited state. We therefore conclude that an ESPT process indeed occurs in the chitosan/methanol samples.


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Figure 6d shows the time-resolved fluorescence of 2NP on a chitosan/water sample (1:1 ratio by weight). The ESPT process occurs, as expected, at a rate greater than in a methanol/chitosan sample of the same weight ratio, and at a much greater rate than in a bulk water sample. Figure S2 in the SI section shows the time-resolved fluorescence of the RO- form of 2NP measured at 450nm in H2O, on samples of cellulose and chitosan in either methanol or water environments. As seen in the figure, the rise of the signal is fastest on chitosan/H2O samples. The rise-time of the RO- signal is assigned to the ESPT process and therefore fits the decay time of the ROH signals shown in Figure 6. Figure 7 shows the time-resolved fluorescence of 2N6S measured at the ROH and RO- bands in several media.

Normalized Signal

1

a) 450 nm

0.01

1

Bulk

360 nm MeOH

0.1

360 nm H2O

0.01

0.1

Bulk H2O

b) Cellulose

MeOH +20µl H2O +40µl H2O

Bulk H2O

+60µl H2O

Bulk MeOH

1E-3 0

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1

450 nm

450 nm 0.1

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Chitosan (H2O)

360 nm 0.01

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Chitosan (MeOH) 1E-3

1E-3 0

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Figure 7: a. TCSPC signals of 2N6S in methanol and H2O solutions. b. TCSPC signals of the ROH form of 2N6S on a cellulose sample. c. Time-resolved fluorescence of the ROH and RO- forms of 2N6S on chitosan in the presence of methanol. d. As panel c, only in the presence of water.

Figure 7a shows the TCSPC signals of 2N6S in methanol and H2O solutions. In bulk methanol, ESPT does not take place and only the ROH emission band exists. The fluorescence decay is exponential with a decay time of ~6ns, which is determined by the fluorescence rate and some oxygen fluorescence quenching that takes place in



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atmospheric ambient conditions. In water solution, the ESPT rate is efficient and the fluorescence decay measured at the ROH-band maximum at 360nm is about 1.6ns. Because of the overlap with the RO- band (whose maximum is at 430nm), the signal at long times shows also a ~1% of the long RO- emission. Figure 7b shows the TCSPC signals of the ROH form of 2N6S on cellulose. When a 20mg sample of cellulose powder is sprayed with 20µL of 2N6S in methanol, the ROH fluorescence lifetime is long and ESPT does not take place. After more than 20 minutes, most of the methanol evaporates and 20µL of water is added to the cellulose. The ROH signal of 2N6S in the cellulose/water sample consists of a fast 1ns decay component followed by a weak (amplitude 0.2) and long (~6ns) decay component. When more water is added to the sample, the decay time of the short-time component is almost unaffected but its amplitude increases from 0.8 in 20µL of water to ~0.9 for 40µL and ~0.92 for the 60µL water sample. The RO- TCSPC signal measured at 450nm is shown in Figure S3 in the SI. The RO- signals of 2N6S, shown in Figure S3 in the SI, are similar to those of 2NP shown in Figure S2. Figure 7c shows the ROH and RO- time-resolved fluorescence signals of 2N6S of a 30mg chitosan sample sprayed with 30µL of 10-3M 2N6S in methanol solution. The ROH signal shows that a rather slow ESPT process occurs at a rate of 1.3×108 s-1 in the sprayed chitosan solution. The RO- signal measured at 450nm consists of three contributions. The major contributor is the RO- form produced by the ESPT process, with a smaller contribution resulting from direct excitation from the ground state of the RO-. The third contribution is that of the ROH band that is due to band overlaps and also contributes to the 450nm emission. Figure 7d shows the ROH and RO- time-resolved fluorescence signals of 2N6S of a chitosan sample in the presence of 30µL of H2O. The ROH signal consists of a short decay-time component of ~800ps with large amplitude of ~0.92, followed by a weak, ~0.08, non-exponential long fluorescence tail. In bulk water, the ESPT rate is 5×108s-1, whereas the ESPT rate of 2N6S on chitosan is about twice that. This major result of our study is consistent with the increase in the ESPT rate of 2-naphthol on chitosan (see Figures 6a and 6d). The ESPT rate of 2NP on a chitosan/water sample is about five times that in a pure water sample. 16 Environment ACS Paragon Plus

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Data analysis We derived the ESPT rate coefficient, kPT, from a simple approximation that neglects the contribution of reversible proton-geminate recombination27,28 (see scheme 3), RO- *+H +  → ROH* , to the long-time fluorescence of the ROH form.

kPT = k − kF

(6)

where k is the fluorescence-decay rate coefficient and kF is the fluorescence-decay rate coefficient in the absence of proton transfer. The value of kF is obtained from the fluorescence decay of the two photoacids in bulk methanol or in ethanol. k is calculated from the average lifetime of the ROH fluorescence, τ av = ∫ I F (t ) dt , where

IF is the normalized time-resolved emission signal of the ROH form. The experimental time-resolved emission signals of the ROH form of both 2NP and 2N6S in bulk water and in methanol are nearly exponential. The diffusion-assisted geminate-recombination (GR) rate that follows the initial ESPT process for 2NP in water is rather small, since the Coulomb attraction between RO- (a single negative charge) and H+ in kBT units is about one at 7Å, the traditional contact radius of the reaction. Also, in 2NP the values of kPT and the fluorescence rate coefficient, kF, are comparable and thus the GR process is less effective since most of the excited RO-* molecules decay radiatively prior to the GR, which is usually a relatively slow process. Table 2 provides the average emission lifetimes of ROH* of the two photoacids in bulk water, methanol and on cellulose and chitosan samples. Table 2: τav [ns] of the ROH form of 2-naphthol (2NP) and 2-naphthol-6-sulfonate (2N6S) in several media

τ av2 NP [ns ]

τ av2 N 6 S [ns ]

Bulk MeOH

6.2

5.8

Bulk H2O

5.4

1.6

Cellulose MeOH

6.9

5.9

Cellulose H2O

6.6

1.7

Chitosan MeOH

3.4

3.4



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Chitosan H2O

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1.9

1.1

Table 3 provides the ESPT rate coefficient, kPT, of the samples for which τav are given in Table 2. Table 3: ESPT rates a,b 2 NP −1 k PT [s ]

2 N 6 S −1 k PT [s ]

Bulk H2O

8.7×107

4.8×108

Cellulose H2O

5.3×107

4.5×108

Chitosan MeOH

1.4×108

1.3×108

Chitosan H2O

4.2×108

8.0×108

kPT deduced by eq. 6, a τF of 2NP, b τF of 2N6S

Main findings 1. The ESPT process from the weak 2-naphthol and 2-naphthol-6-sulfonate photoacids to water next to a cellulose scaffold occurs at about the same time as in bulk water. 2. The ESPT rate of these photoacids to bulk methanol is slower by four orders of magnitude than in water, their fluorescence rate is greater by more than three orders of magnitude and therefore could not be observed. 3. ESPT occurs efficiently from both 2-naphthol (2NP) and 2-naphthol-6sulfonate (2N6S) situated next to a chitosan surface in the presence of small amounts of water or methanol. 4. Direct ESPT from the two photoacids, 2NP and 2N6S, to the amine group of the D-glucosamine of chitosan also occur with high efficiencies. 5. The ESPT rates from both 2NP and 2N6S to water next to the chitosan surface are greater by factors of 5 and 1.7 from their values in bulk water.

Discussion In the current study, we find that 2NP sprayed on chitosan, in the presence of methanol or water, effectively transfers a proton within the lifetime of the excited


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state. It is expected that ESPT to water may occur if the water content as a proton acceptor will be high enough to form a hydronium ion, H3O+(H2O)n, where n>4. However, it was not expected that the ESPT process would take place from these weak photoacids to methanol next to the chitosan scaffold. We explain this unexpected result by suggesting that the proton-transfer process occurs to the amino groups of D-glucosamine (75-85% coverage) or to a lesser extent to the N-acetyl-Dglucosamine (25-15%) of chitosan. From the results of the current study, we know that when 2NP is sprayed on cellulose that is composed of only D-glucose units and in the presence of methanol, ESPT does not take place. Weak photoacids with pK a* >0 transfer a proton within the lifetime of the excited state to bulk water, but not to linear alcohols like methanol to decanol. The ESPT rate of weak photoacids to water varies logarithmically with their K a* values. The ESPT rate of 2-naphthol (2NP) ( pK a* 2.7) is about 108s-1, while that of 2naphthol-6-sulfonate (2N6S) ( pK a* ~1.7) is ~109s-1. Thus one pKa unit changes the ESPT rate by a factor of ten. The ESPT rate of weak photoacids to methanol is lower by about four orders of magnitude and even more to ethanol and the longer linear alcohols. Thus for 2NP we estimate an ESPT rate of about 104s-1 and ~105s-1 for 2N6S. Therefore these low rates do not affect the steady-state emission spectrum of these photoacids in alcohols, which consist only of the ROH band. It also does not affect the time-resolved emission decay rate of the ROH band which is determined by the fluorescence rate and in a methanol solution, also by oxygen quenching. To summarize, the fluorescence spectra of 2NP and 2N6S in alcohols, when excited as ground-state ROH, consist of only a single ROH band. In water, the spectra consist of two emission bands, that of ROH and that of RO-. ESPT to a mild base in methanolic solutions In previous studies4,5,9,29,30,31, it was shown that ESPT occurs not only to solvent molecules but also to weak bases in solution. At low concentrations of base (CB< 0.5M), the reaction rate with the base in solution depends on the time it takes to bring the proton donor and acceptor to close proximity. Since molecular translational motion in the liquid phase is governed by a random walk, the proton-transfer reaction rate is limited by the mutual diffusion motion, and the rate coefficient at long times is



determined by the second-order diffusion-controlled rate coefficient that is given approximately by

k D ≈ 4π N ' Da

(7)

Where NA is Avogadro's number and N'=NA/1000. D is the mutual diffusion coefficient (D=DA+DB). DA and DB are the diffusion coefficients of ROH and the base and a is the contact radius. For small molecules in a non-viscous medium like water, the kD values are of the order of 0.5-2×109M-1s-1. Since the excited-state lifetime of many photoacids is about 5ns, the weak-base concentrations that affect the fluorescence decay time are greater than 0.1M. Figure 8a shows the steady-state emission spectrum of 2NP in neat methanol and in methanol solutions of a weak base, sodium acetate. b)

a) 1M NaAc 0.5M NaAc 0.25M NaAc 0.14M NaAc MeOH 0.14M NaAc excitation

0.8 0.6 0.4

1

Normalized Signal

1.0

Normalized Signal

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0.1 1M NaAc 0.5M NaAc 0.25M NaAc 0.14M NaAc MeOH

0.2 0.0

0.01

300

350

400

450

500

550

600

0

Wavelength [nm]

1

2

3

4

5

6

7

8

9

Time [ns]

Figure 8: 2NP in methanol and in methanol solutions of sodium acetate. a. Steady-state emission spectrum. b. Time-resolved emission of both the ROH and the RO- emission bands.

The measurements were carried out at several concentrations ranging from 0.14 – 1M. As seen in the figure, the spectrum of 2NP in methanol consists of the ROH band only, while in the presence of sodium acetate (NaAc) the spectrum shows the contribution of two emission bands, that of ROH and that of RO-. The greater the F F NaAc concentration, the larger the band-amplitude ratio, I RO − / I ROH . The pH of these

acetate solutions is kept below the pKa value of 2NP (≈9.45)1 by adding a small amount of acetic acid to obtain a buffer of pH ~6.7. The spectra shown in Figure 8a are the result of an efficient ESPT process from 2NP ROH to the Ac- ion. kD ROH*+Ac-  → RO- *+HAc


10

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The pseudo-first-order reaction-rate coefficient, k D ⋅ C Ac- , increases with acetate F F concentration ( C Ac- ) and therefore the amplitude ratio I RO . − / I ROH increases with C Ac-

Figure 8b shows the time-resolved emission of both the ROH and the ROemission bands of 2NP in methanol solutions of NaAc. The NaAc concentrations are the same as for the samples for which the steady-state emission spectra are shown in Figure 8a. As seen in Figure 8b, the fluorescence-decay rate of the ROH form increases with the NaAc concentration. The ESPT results of 2NP to Ac- ions in methanol solution, shown in Figure 8, strongly support this study's main conclusion: the weak base, namely the amine groups on the chitosan scaffold, play a major role in the unexpectedly efficient ESPT of the weak photoacids when sprayed on chitosan. When water is added to 2NP or 2N6S on chitosan, the overall ESPT reaction rate is greater than in methanol. This is to be expected, since two ESPT channels exist in chitosan water samples. The first channel is the direct ESPT from a photoacid to a nearby water molecule; the second is the ESPT to the amino groups of chitosan. In previous studies5,9, it was found that at high base concentration (CB>1M) the ESPT mechanism of mild photoacids to a base involves one or more water molecules that bridge between the proton donor and acceptor such as [ROH (H2O)n B]. In the extreme case of a direct-contact ROH·B- complex, the ESPT time coefficient in aqueous solution is rather small, a few hundreds of femtoseconds. When one water molecule bridges between ROH and B- (in our case, Ac- ion), the PT time- coefficient is of the order of 6ps. In longer water bridges, of a few water molecules, the protontransfer time coefficient increases with the amount of water-bridge molecules. There exists a distribution of n, the number of water molecules that make up the bridge and the average number of water molecules n depends on the base concentration. The proton transfer rate of 2NP on chitosan is 5 times shorter than in bulk water. This higher rate indicates a proton transfer to the amine of chitosan. Proton transfer does not take place in bulk methanol, but does occur when 2NP is adsorbed on chitosan and is in a methanol environment. In our current study of chitosan and 2NP or 2N6S photoacids, it is difficult to provide a structural mechanism for how the ESPT process proceeds, but it is clear that a proton is transferred to the amine groups of the glucosamine. 21 Environment ACS Paragon Plus


Table 4 provides the values of the ESPT process to a mild base of 2NP in sodium acetate/methanol solutions. Table 4: τav and kPT of the ROH form of 2-naphthol in NaAc –methanol solutions a

a

1M NaAc

0.5M NaAc

0.25M NaAc

0.14M NaAc

τav [ns]

1.2

1.6

2.3

3.1

kPT (s-1)

7.1×108

4.7×108

2.7×108

1.6×108

kF=1.6×108 s-1 ; k=1/τav

kPT=k-kF;

ESPT in semi-dry samples From the results presented so far, we conclude that ESPT takes place from photoacids to the amine groups of chitosan. Figure 9a shows the steady-state emission spectrum of 2NP sprayed on chitosan by adding 30µL of methanol solution to 30mg of chitosan, at several times after the chitosan sample was exposed to the methanolic solution of the photoacids. b)

a)

1

0.8

Norm. Fluorescence intensity

Normalized Signal

1.0 Semidry 1 hour Wet 0.6 0.4 30min 0.2

Wet 1 hour 2 hours

0.1

0.01

0.0 350

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Wavelength (nm)

550

0

02/10/14

c) 1.0

Norm. Fluorescence intensity

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Wet 1 hour 2 hours 3 hours

0.8 0.6 0.4 0.2 0.0 0

10

20

30

Time (ns)


10

Time (ns)

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Figure 9: a. Steady-state emission spectrum of 2NP on chitosan in the presence of small amounts of methanol at several times. b. Time-resolved emission of 2NP ROH on chitosan in the presence of methanol, at three time intervals after the addition of the photoacid: wet, 60 and 120 minutes. c. TCSPC signal of the RO- form measured at 430nm, on the same sample as in 6b, wet, 1 hour, 2 hours and three hours.

The sample is measured during and while the methanol evaporates. We measure the emission spectrum of 2NP immediately after it is sprayed on chitosan and after 30 minutes, an hour and two hours. The sample is kept under a controlled atmosphere - 47% humidity and 23°C. We find that the steady-state spectrum is almost time-independent after an hour. Figure 9b shows the time-resolved emission of 2NP ROH in methanol solution sprayed on a sample of chitosan, measured at three time intervals: 30 minutes after the addition of the photoacid, 60 minutes and 120 minutes. The time-resolved emission signal measured at 360nm is almost unaffected as time progresses. The signal decay is nonexponential with an average time of ∞

τ av = ∫ I f (t )dt ≈1 ns. 0

Figure 9c shows the TCSPC signal of the RO- form of the same sample, measured at 430nm. The time required to accumulate the TCSPC measurement of the ROH signal to about 3000 counts was about 6 minutes. The RO- measurement was acquired immediately after the ROH measurement and took only about 4 minutes. The ROsignal of 2NP on semidry chitosan samples shows both fast and slow rise components. The major signal rise component has an amplitude of ~0.7 and shows a fast rise time that is comparable with the instrument response time of about 50ps. The second, smaller component with an amplitude of ~0.3, exhibits a much longer rise time of about 1ns. We attribute this longer time component to the ESPT process from the 2NP ROH form to the glucosamine group. We attribute the large amplitude and short risetime component to: 1) a large overlap of the ROH and RO- bands at 430nm, the



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measured wavelength of the TCSPC signal, 2) the large ground-state population of the RO- form in semi-dry samples. Thus, RO- is directly excited from the ground state.

Summary and conclusions Chitosan is a polysaccharide composed of mainly D-glucosamine units (75-85%) and much less N-acetyl-D-glucosamine. It is prepared from the natural product chitin, which has mainly N-acetyl-D-glucosamine units. Photoacids are weak acids in the ground state, with pKa values ranging from 5-9.4. 2-naphthol and 2-naphthol sulfonate derivatives have pKa values of ~9 and are therefore suitable for the exploration of the photoprotolytic process next to the chitosan scaffold which is a weak base and may react already in the ground-state with the ROH form of photoacids with pKa≤7. In weak photoacids, ESPT occurs only to water and not, for example, to linear alcohols like methanol - decanol. Photoacids also efficiently transfer a proton within their excited-state lifetime to a mild base dissolved in water or linear alcohols like methanol and ethanol. In the current study, we used 2-naphthol and 2-naphthol-6sulfonate to study the photoprotolytic process next to the chitosan scaffold. We found that ESPT occurs in chitosan/water samples of 1:1 weight ratio at a rate three times greater than in bulk water. We also found, that in a chitosan/methanol sample of 1:1 weight ratio, ESPT occurs efficiently, whereas in bulk methanol the ESPT rate is much too low and therefore cannot be observed, since the fluorescence rate blocks the observation-time window and limits the observation of ESPT processes to high ESPT rates where kPT>0.1kF, (kPT and kF are the ESPT and fluorescence rates). We also find that in semi-dry chitosan samples in which most of the methanol has evaporated, ESPT is still effective. We therefore conclude that an ESPT process also occurs in semi-dry samples from the photoacid to the glucosamine units of the chitosan scaffold. This finding raises the possibility that protons in living cells can easily move from one place to another, not only by diffusion of a water-hydronium complex, as in bulk water, but also between water and an adjunct-base scaffold.

Acknowledgement This work was supported by grants from the James-Franck German-Israeli Program in Laser-Matter Interaction and by the Israel Science Foundation.


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Supporting Information Available A. Rotational diffusion of HPTS B. Time-resolved emission of 2-naphthol and 2-naphthol-6-sulfonate This information is available free-of-charge via the internet at http://pubs.acs.org

References

1. Ireland, J. F.;Wyatt, P.A. Acid-Base Properties of Electronically Excited States of Organic Molecules. Adv. Phys. Org. Chem. 1976, 12, 131–221. 2. Gutman, M.; Nachliel, E. The Dynamic Aspects of Proton Transfer Processes .Biochem. Biophys. Acta 1990, 1015, 391–414. 3.Tolbert, L. M.; Solntsev, K. M Excited-State Proton Transfer: From Constrained Systems to “Super” Photoacids to Superfast Proton Transfer. Acc. Chem. Res. 2002, 35, 19–27 4. Rini, M.; Magnes, B. Z.; Pines, E.; Nibbering, E.T. Real-Time Observation of Bimodal Proton Transfer in Acid-Base Pairs in Water. J. Science 2003, 301, 349–352. 5.Mohammed, O. F.; Pines, D.; Dreyer, J.; Pines, E.; Nibbering, E. T. Sequential Proton Transfer Through Water Bridges in Acid-Base Reactions. J. Science 2005, 310, 83–86. 6. Tran-Thi, T. H.; Gustavsson, T.; Prayer, C.; Pommeret, S.; Hynes, J. T. Primary Ultrafast Events Preceding the Photoinduced Proton Transfer from Pyranine to Water. Chem. Phys. Lett. 2000, 329, 421–430. 7. Agmon, N. Elementary Steps in Excited-State Proton Transfer. J. Phys. Chem. A

2005, 109, 13–35. 8. Spry, D. B.; Fayer, M. D. Charge Redistribution and Photoacidity: Neutral Versus Cationic Photoacids. J. Chem. Phys. 2008, 128, 084508-1-084508-9. 9. Siwick, B. J.; Cox, M. J.; Bakker, H. J. Long-Range Proton Transfer in Aqueous Acid−Base Reactions. J. Phys. Chem. B 2008, 112, 378–389. 10. Mohammed, O. F.; Pines, D.; Nibbering, E. T. J.; Pines, E. Base-Induced Solvent Switches in Acid–Base Reactions. Agnew. Chem. Int. Ed. 2007, 46, 1458–1461.



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11. Mondal, S.K.; Sahu, K.; Sen, P.; Roy, D.; Ghosh, S.; Bhattacharyya, K. Excited State Proton Transfer of Pyranine in a γ-cyclodextrin Cavity. Chem. Phys. Lett. 2005, 412, 228–234. 12 Prasun, M. K.; Samanta, A. Evidence of Ground-State Proton-Transfer Reaction of 3-Hydroxyflavone in Neutral Alcoholic Solvents. J. Phys. Chem. A 2003, 107, 6334– 6339. 13. Bhattacharya, B.; Samanta, A. Excited-State Proton-Transfer Dynamics of 7Hydroxyquinoline in Room Temperature Ionic Liquids. J. Phys. Chem. B 2008, 112, 10101–10106. 14. Pérez Lustres, J. L.; Kovalenko, S. A.; Mosquera, M.; Senyushkina, T.; Flasche, W.; Ernsting, N. P. Ultrafast Solvation of N-Methyl-6-Quinolone Probes Local IR Spectrum. Angew. Chem., Int. Ed. 2005, 44, 5635-5639. 15. Pérez -Lustres, J.; Rodriguez-Prieto, F.; Mosquera, M.; Senyushkina, T.; Ernsting, N.; Kovalenko, S. Ultrafast Proton Transfer to Solvent: Molecularity and Intermediates from Solvation-and Diffusion-Controlled Regimes. J. Am. Chem. Soc.

2007, 129, 5408-5418. 16. Crawford, R. L. In Lignin biodegradation and transformation; Wiley New York:

1981 17. Updegraff, D. M. Semimicro Determination of Cellulose In Biological Materials. Anal. Biochem. 1969, 32, 420-424. 18. Klemm, D.; Heublein, B.; Fink, H.; Bohn, A. Cellulose: Fascinating Biopolymer and Sustainable Raw Material. Angew. Chem., Int. Ed. 2005, 44, 3358-3393. 19. Agrawal, A. M.; Manek, R. V.; Kolling, W. M.; Neau, S. H. Water Distribution Studies within Microcrystalline Cellulose and Chitosan using Differential Scanning Calorimetry and Dynamic Vapor Sorption Analysis. J. Pharm. Sci. 2004, 93, 17661779. 20. Linden, J. C.; Stoner, R. J.; Knutson, K. W.; Gardner-Hughes, C. A. Organic Disease Control Elicitors. Agro Food Industry Hi-Tech 2000, 11, 32-34. 21. Debye, P. Reaction Rates in Ionic Solutions. Trans. Electrochem. Soc. 1942, 82, 265-272.


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22. Huppert, D.; Kolodney, E. Picosecond Proton Transfer Studies in Water-Alcohols Solutions. Chem. Phys. 1981, 63, 401-410. 23. Ofran, M.; Feitelson, J. Time Dependence of Dissociation in the Excited State of β-Naphthol. Chem. Phys. Lett. 1973, 19, 427-431. 24. Pillai, C.; Paul, W.; Sharma, C. P. Chitin and Chitosan Polymers: Chemistry, Solubility and Fiber Formation. Prog. Polym. Sci. 2009, 34, 641-678. 25. Genosar, L.; Leiderman, P.; Koifman, N.; Huppert, D. Effect of Pressure on the Proton-Transfer Rate from a Photoacid to a Solvent. 2. DCN2 in Propanol. J. Phys. Chem. A 2004, 108, 309-319. 26. Genosar, L.; Leiderman, P.; Koifman, N.; Huppert, D. Effect of Pressure on Proton Transfer Rate from a Photoacid to a Solvent. 3. 2-Naphthol and 2-Naphthol Monosulfonate Derivatives in Water. J. Phys. Chem. A 2004, 108, 1779-1789. 27. Pines, E.; Huppert, D.; Agmon, N. Geminate Recombination in Excited‐State Proton‐Transfer Reactions: Numerical Solution of the Debye–Smoluchowski Equation with Backreaction and Comparison with Experimental Results. J. Chem. Phys. 1988, 88, 5620-5630. 28. Agmon, N.; Pines, E.; Huppert, D. Geminate Recombination in Proton‐Transfer Reactions. II. Comparison of Diffusional and Kinetic Schemes. J. Chem. Phys. 1988, 88, 5631-5638. 29. Genosar, L.; Cohen, B.; Huppert, D. Ultrafast Direct Photoacid-Base Reaction. J. Phys. Chem. A 2000, 104, 6689-6698. 30. Cohen, B.; Huppert, D.; Agmon, N. Non-Exponential Smoluchowski Dynamics in Fast Acid-Base Reaction. J. Am. Chem. Soc. 2000, 122, 9838-9839. 31. Cohen, B.; Huppert, D.; Agmon, N. Diffusion-Limited Acid-Base Nonexponential Dynamics. J. Phys. Chem. A 2001, 105, 7165-7173.



TOC Graphic Chitosan MeOH Bulk MeOH cellulose MeOH

1

Norm. Signal

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

OH O HO

HO O

N H2

N H2 O O OH

Chitosan

0

1

2

3

4

n

5

6

7

8

9 10

Time [ns]


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Excited-State Proton Transfer of Weak Photoacids ... - ACS Publications

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