Biomacromolecules 2004, 5, 453-457
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Laser Photolysis of Carboxymethylated Chitin Derivatives in Aqueous Solution. Part 1. Formation of Hydrated Electron and a Long-Lived Radical Maolin Zhai,*,† Hisaaki Kudoh,† Guozhong Wu,†,‡ Radoslaw A. Wach,† Yusa Muroya,† Yosuke Katsumura,† Naotsugu Nagasawa,§ Long Zhao,§ and Fumio Yoshii§ Nuclear Engineering Research Laboratory, School of Engineering, University of Tokyo, Shirakata-Shirane 2-22, Tokai-mura, Naka-gun, Ibaraki-ken, 319-1188 Japan, and Takasaki Radiation Chemistry Research Establishment, Japan Atomic Energy Research Institute, Watanuki-machi, Takasaki-shi, Gunma-ken, 370-1292 Japan Received August 1, 2003; Revised Manuscript Received November 19, 2003
Laser photolysis experiments on carboxymethylated chitin derivatives, such as carboxymethyl chitin (CM-chitin) and carboxymethyl chitosan (CM-chitosan), in aqueous solution by a 248 nm excimer laser were carried out for the first time. The transient absorption spectra of photolyzed CM-chitin or CM-chitosan solutions revealed a strong band with the maximum at 720 nm, which was assigned to the hydrated electron (eaq- ). In the presence of argon, the eaq- decays by reacting with CM-chitin or CM-chitosan, and the rate constants are (6.1 ( 0.1) × 107 M-1 s-1 and (3.7 ( 0.1) × 107 M-1 s-1, respectively. Long-lived radicals with relatively weak absorption intensity were detected in the near-UV region. The absorption band was not notably characteristic and showed only an increasing absorption toward shorter wavelengths. It is similar to the signal of •CM-chitin or •CM-chitosan macroradicals formed by the reaction of CM-chitin or CM-chitosan with an OH• radical. It was assigned to •CM-chitin- or •CM-chitosan- macroradicals formed by eaq- + CM-chitin or CM-chitosan reaction. CM-chitin aqueous solutions were further examined by pulse radiolysis in order to confirm the site of the long-lived radical. Introduction Chitin is the most abundant natural amino polysaccharide, whose structure is similar to cellulose, containing an acetamido group instead of the C-2 hydroxyl group. Chitosan is the deacetylated product of chitin, showing enhanced solubility in dilute acids (degree of deacetylation is larger than 40%). Chitin and chitosan have been investigated widely and have application in the health care industry, the food and beverage industry, agriculture, pharmacy, biotechnology, etc.1-4 For many applications cited above, the modification of chitin or chitosan is desired. For example, chitin and chitosan have poor solubility, so water-soluble chitin or chitosan derivatives, such as CM-chitin and CM-chitosan, being soluble in both acidic and basic physiological media, might be good candidates for the applications in pharmacology and biotechnology.5-7 Radiation technology is an important method for the modification of chitin derivatives. It includes radiationinduced degradation, grafting, and cross-linking.6-10 Chitosan is a radiation-degraded polysaccharide, and after radiation treatment, chitosan has good antimicrobial activity. It has been applied as a plant growth promoter, as a protectant of * To whom correspondence should be addressed. Present address: College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871 People’s Republic of China. Fax: (+86) 10-62759191. E-mail:
[email protected]. † University of Tokyo. ‡ Present address: Applied Radiation Center, Shanghai Institute of Applied Physics, P.O. Box 800-204, Shanghai, 201800 China. § Japan Atomic Energy Research Institute.
plants from environmental stress, for food production, and for fruit protection.11 CM-chitin and CM-chitosan undergo degradation during irradiation in the solid state and diluted solution (below 10%). However, when the concentration is more than 10%, the so-called paste-like state, cross-linking is observed. Hydrogels formed by radiation cross-linking have antibacterial activity, which is an important feature for biomedical materials.6 Radiation-induced changes in chitin and its derivatives are attributed to free-radical reactions. The pulse radiolysis technique has been used for investigating chain scission reactions and mechanisms of other radicalinduced reactions in chitosan.12,13 Because chitosan can dissolve only in an acidic medium, some reactions cannot be followed unless one introduces a substitution group into chitosan molecules, which makes it soluble in solutions of a wide pH range. In our work, carboxymethylated chitin derivatives are chosen to investigate the free radical reactions of chitin derivatives induced by laser pulse photolysis. Laser photolysis is in several cases a convenient technique similar to pulse radiolysis for the study of free radical reaction, e.g., free radical reactions in protein and their subunits, the amino acids.14 Carboxymethylated chitin derivatives have a structure similar to amino acids. Thus, is there also a similar photochemistry behavior in carboxymethylated chitin derivatives? In this paper, the photochemistry of carboxymethylated chitin derivatives induced by a KrF excimer laser will be studied. Such an investigation is also very helpful for the understanding of the behavior of chitin derivatives upon UV irradiation, for their potential modifica-
10.1021/bm034272g CCC: $27.50 © 2004 American Chemical Society Published on Web 01/09/2004
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Figure 1. Structures of CM-chitin or CM-chitosan. Table 1. Characteristic Parameters of CM-Chitin and CM-Chitosan Used in This Studya
sample
DS
DDA
intrinsic viscosity (dL/g)
CM-chitin CM-chitosan
0.83 0.91
31.4% 84.0%
2.32 2.43
a
Mw 2.9 × 104 3.1 × 104
DS, degree of substitution; DDA, degree of deacetylation.
tion by UV irradiation.15 In the next part of this work, OH• and SO4• radicals are used to study the influence of the substituted group on the reactivity of carboxymethylated chitin derivatives.
Figure 2. Transient absorption spectra observed on laser photolysis of 10 mM CM-chitin solution saturated with argon at pH 8.9 (100 mJ/ pulse).
Experimental Section Both of CM-chitin and CM-chitosan used in this study were obtained from Koyou Chemical Industrial Co., Ltd., Japan. Their structures are shown in Figure 1, and the characteristic parameters are summarized in Table 1. DS and DDA of the samples were determined from Fourier transform infrared (FTIR) absorption spectra.16,17 The FTIR spectra of samples were recorded with FTIR4000 (Shimadzu Co., Ltd., Kyoto, Japan) by the KBr tablet method at a resolution of 4 cm-1 and an accumulation of 10 scans. DS was determined from the absorption ratio of the stretching vibration of CdO at 1735 cm-1 to the amide I band at 1665 cm-1 using with a calibration curve.16 In the case of DDA, the adsorption ratio of amide II band at 1550 cm-1 to the CH stretching vibration at 2878 cm-1 was used with a calibration curve.17 The intrinsic viscosity [η] of the sample was measured with an Ubbelohde-type viscometer in a 0.1 M NaCl aqueous solution at 25 °C. The molecular weight (Mw) of the sample was estimated on the basis of the Mark-Houwink-Sakurada equation, [η] ) kMR, where k ) 7.92 × 10-5 and R ) 1.00.18 Other chemicals were analytical reagents, and CM-chitin or CM-chitosan solutions were made with Millipore water. The pH was adjusted by addition of HClO4 or NaOH. HClO4 was used because the perchlorate ion is inactive with the OH• radical and thus does not interfere with the results. Prior to irradiation, solutions were saturated with high purity argon or N2O gas to remove oxygen or scavenge the eaq-, respectively. Steady-state absorption was determined with a UV-3300 spectrophotometer. The equipment for laser photolysis was described in detail in a previous paper.19 A 248 nm (KrF) excimer laser was used with the maximum energy of 330 mJ per 20 ns pulse. The analyzing light was perpendicular with the laser beam, and the optical length was 15 mm. The solution was supplied to the optical cell through a flowing system at about 4 mL/min.
Figure 3. Transient absorption spectra observed at the end of laser pulse for 10 mM CM-chitin solution. (100 mJ/pulse).
Pulse radiolysis experiments were performed using an electron beam (10 ns) of 35 MeV delivered from a linear accelerator at the University of Tokyo.20 The absorbed dose was determined using an N2O-saturated solution of 10 mM KSCN as the dosimeter (G ) 5.2 × 104 m2 J-1 at 472 nm).21 The preparation and analysis of the samples is the same as that in laser photolysis, except that the optical path length was 18 mm. Results and Discussion Transient Absorption Spectra of CM-Chitin and the Assignation of eaq-. The ground state absorption spectrum of CM-chitin in water drops suddenly from 210 to 230, nm and molar absorption coefficient at 248 nm is 100 M-1 cm-1. The laser induced transient absorption spectra of CM-chitin solution at pH 8.9 are shown in Figure 2. Two absorption bands are indicated: a strong band in the visible range and a weak one in the near-UV region. The absorption band in the visible range has the λmax of 720 nm. To assign this band, the solution was irradiated in different conditions, and the results are shown in Figures 3 and 4. This band disappeared when the solution was bubbled with N2O, which is known as an effective eaq- scavenger [reaction 1, k ) 9.1 × 109
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Photolysis of CM-Chitin Derivatives. Part 1
Figure 4. Representative decay profiles of the absorbance at 720 nm after laser photolysis of 10 mM CM-chitin solution at pH 8.9 (100 mJ/pulse).
M-1 s-1].22 Decay of the band is faster in an aerated system than that in argon due to the reaction of hydrated electron with oxygen [reaction 2, k ) 1.9 × 1010 M-1 s-1].22 Therefore, this band was assigned to the eaq-, indicating the photoionization of CM-chitin [reaction 3]. From Figure 3, it is also noted that the apparent yield of eaq- observed immediately after the laser pulse decreased with increasing the pH of CM-chitin solution (i.e., the decrease of H+ and -NH3+ concentrations) due to the fast reaction of eaq- with free H+ or the -NH3+ group of CM-chitin [reaction 4, k ) 2.3 × 1010 M-1 s-1]22 eaq- + N2O f •OH + -OH + N2
(1)
eaq- + O2 f O2-•
(2)
hV
CM-chitin 98 +CM-chitin + eaq-
(3)
eaq- + H+ f •H
(4)
CM-chitin + eaq- f •CM-chitin-
(5)
Macroradicals of CM-Chitin. From Figure 2 (or Figure 5a), a weak signal of the long-lived radical at 7.0 µs was observed in the near-UV region. It is not particularly characteristic; it shows only an increasing absorption toward shorter wavelengths. It may be the band of •CM-chitinmacroradicals formed by eaq- + CM-chitin reaction 5 or CM-chitin+ macroradicals formed by photoionization of CM-chitin as the counterpart of eaq- (3). To assign the longlived radical, a pulse radiolysis experiment of the CM-chitin aqueous solution was carried out. In pulse radiolysis, the direct effect of irradiation on CM-chitin is negligible (low polymer concentration in solution) and the effect mainly occurs via reactive products of water radiolysis (6), i.e., the OH• radical, H atom, and eaqH2O f OH•, H•, eaq-, H+, H2O2, H2
(6)
For studying the eaq- + CM-chitin reactions, Ar-saturated solutions of CM-chitin were irradiated by electron pulse in the presence of 0.2 M tert-butyl alcohol, which was used as an OH radical scavenger (7)
Figure 5. a. Transient absorption spectra (7 µs) observed on laser photolysis of 10 mM CM-chitin solution at pH 8.9 (100 mJ/pulse). b. Transient absorption spectra (10 µs) of CM-chitin radicals recorded after electron pulsing of 20 mM CM-chitin aqueous solution at pH 8.0 (115 Gy, pulse duration 10 ns).
OH• + tert-butyl alcohol f H2O + t-butyl alcohol• (7) Formed tert-butyl alcohol radicals are unreactive toward polymers. The absorption band of CM-chitin- macroradicals formed by eaq- + CM-chitin reaction is shown in Figure 5b. The band is similar to that raised from laser photolyzed CM-chitin aqueous solution (Figure 5a). It suggests that longlived radicals observed in laser photolysis were formed by indirect reaction, i.e., the reaction of CM-chitin and eaq-. Although photoionization of CM-chitin should lead to the formation of CM-chitin+ macroradicals as the counterpart of ejected electron, because there are no absorption bands in the transient spectra that could be assigned to CM-chitin+, the fate of this species is unknown. In N2O-saturated solutions, eaq- was completely converted to OH• radical by reaction 1. Therefore, in N2O saturated CM-chitin solutions, the macroradicals could be formed by the reaction of CM-chitin with the OH• radical OH• + CM-chitin f •CM-chitin + H2O
(8)
The bands of CM-chitin in N2O-saturated solution in laser photolysis and pulse radiolysis are shown in Figure 5, parts a and b, respectively. It is easy to find that these bands are
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Figure 6. Rate constants of the pseudo-first-order reaction of eaqreacted with H+, oxygen, or CM-chitin (100 mJ/pulse).
like those of •CM-chitin- macroradicals formed by eaq- + CM-chitin reaction. In the pulse radiolysis experiment of the chitosan solution, the similar band of •chitosan macroradicals formed by the reaction of chitosan with OH• radical also has been observed.13 The macroradical species last for a long time. Because the duration of pulsed light from the xenon lamp was 50 µs, the entire decay process was not followed. Kinetic Analyses of the eaq- Decay. The decay kinetics of the eaq- formed under laser pulse of the CM-chitin aqueous solution were examined from 500 to 900 nm (Figure 6). Because the absorption of long-lived radical is much weaker than that of eaq-, its influence on the decay of eaq- can be neglected. The rate constants of the pseudo-first-order decay of eaq-, constant from 500 to 900 nm, in argon (pH ) 8.9), argon (pH ) 4.7), and air (pH ) 8.9) are (5.9 ( 0.2) × 105, (1.3 ( 0.1) × 106, and (4.8 ( 0.1) × 106 s-1, respectively. The decay rate of eaq- is faster in the aerated system or acidic medium due to its reaction with oxygen or H+ (-NH3+). If we assume the concentration of oxygen in the solution is 0.25 mM, the rate constant of second-order decay of eaq- in air (pH ) 8.9) evaluated in this experiment is (1.9 ( 0.1) × 1010 M-1 s-1, which is the same as the quoted value.22 In the presence of argon, eaq- disappears in the reaction with CM-chitin. The rate constant of the eaq- + CM-chitin reaction, estimated from the slope of the pseudo first-order decay constant versus polymer concentration (Figure 7), is (6.1 ( 0.1) × 107 M-1 s-1. The rate constant of the eaq- + CM-chitin reaction was also determined by pulse radiolysis (Figure 7), k ) (5.6 ( 0.3) × 107 M-1 s-1, which is nearly the same as that from laser photolysis. In general, the reactivity of eaq- toward the carbohydrates is low (typically k < 5 × 106 M-1 s-1).23 Because CM-chitin contained a carbonyl group (affinity for electron), the rate constant is about 10-fold higher. Concentration of eaq- Against Laser Energy. Figure 8 showed that the apparent yield of eaq- increased linearly with the beam energy from 55 to 230 mJ pulse-1. Although the eaq- formation in pure water via the multiphoton process was also observed under the same conditions, its yield was negligible as compared to the eaq- formed by photoionization of CM-chitin aqueous solution. The quantum yield of eaq-
Zhai et al.
Figure 7. Plot of the first-order decays of eaq- against CM-chitin concentration after laser photolysis of CM-chitin solution saturated with argon at pH 8.9 (100 mJ/pulse) and after electron pulse of CMchitin aqueous solution saturated with argon containing 0.2 M tertbutyl alcohol at pH 8.0 (112 Gy per pulse).
Figure 8. Plot of the absorbance (720 nm) measured for eaq- against laser pulse energy for a 10 mM CM-chitin solution saturated with argon at pH 8.9.
was not determined because this value varied with many factors such as pH, Mw, DS, DDA, and CM-chitin origin. Influence of Substituent Group and Mechanisms of Photoionization of CM-Chitin. Chitin cannot dissolve in water, and its deacetylated product, chitosan, only dissolves in acidic solution (pH < 4). In this experiment, we used another carboxymethylated chitin derivative, CM-chitosan, with higher degree of deacetylation (Table 1). CM-chitosan is a perfect counterpart for studying on mechanisms of photoionization of CM-chitin. The photolyzed CM-chitosan aqueous solution also has a very strong band of eaq- formed by the photoionization of CM-chitosan (9), but its yield is lower than that of CM-chitin (Figure 9). Because the content of -NHCOCH3 groups in CM-chitosan is also lower than that in CM-chitin, it is suggested that upon laser irradiation electrons may be ejected from -NHCOCH3 groups of the CM-chitin chain and then captured by water to form eaq-. So the yield of eaq- in CM-chitosan is lower than that in CM-chitin. Due to a weak signal on the long-lived radical, it is impossible to compare CM-chitin macroradicals with CM-chitosan macroradicals here. The rate constant of the eaq- + CM-chitosan reaction (10) was measured in the
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Photolysis of CM-Chitin Derivatives. Part 1
Conclusions Photolysis of aqueous solutions of CM-chitin derivatives by 248 nm excimer laser leads to the formation of eaq- and macroradicals. The apparent yield of eaq- increased linearly with the beam energy, and in the absence of oxygen, eaqdecayed by reacting with CM-chitin or CM-chitosan, and the rate constants of decay are (6.1 ( 0.1) × 107 and (3.7 ( 0.1) × 107 M-1 s-1, respectively. The difference is attributed to the influence of the carbonyl group (affinity for electron) in CM-chitin or CM-chitosan. A weak band of CM-chitin derivatives macroradicals shows an increasing absorption toward shorter wavelengths. References and Notes Figure 9. Transient absorbance spectra observed on laser photolysis of 10 mM CM-chitosan solution saturated with argon at pH 8.9 (100 mJ/pulse).
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15)
eaq-
Figure 10. Plot of the first-order decay of the against CM-chitin or CM-chitosan concentration after laser photolysis saturated with argon at pH 8.9 (100 mJ/pulse).
presence of argon, and the result was (3.7 ( 0.1) × 107 M-1 s-1. It is lower than that of the eaq- + CM-chitin reaction (Figure 10). The difference can be attributed to the influence of the carbonyl group (affinity for electron) in CM-chitin or CM-chitosan hV +
-
CM-chitosan 98 CM-chitosan + eaq
(9)
CM-chitosan + eaq- f •CM-chitosan-
(10)
(16) (17) (18) (19) (20) (21) (22) (23)
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BM034272G
