Biomacromolecules 2004, 5, 1029-1037
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Accelerated Biodegradation of Poly(vinyl alcohol) by Glycosidations of the Hydroxyl Groups or Addition of Sugars Akinori Takasu,* Mizuho Takada, Hisashi Itou, Tadamichi Hirabayashi, and Takatoshi Kinoshita† Department of Environmental Technology and Urban Planning, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan Received December 14, 2003; Revised Manuscript Received February 13, 2004
Biodegradabilities of N-acetyl-D-glucosamine (GlcNAc)- (1) and chitobiose-substituted (2) poly(vinyl alcohol)s (PVA)s in a soil suspension (pH 6.5) were investigated at 25 °C for 40 days. Biochemical oxygen demand of 1 with a degree of substitution of 0.2-0.3 (DP ) 430-480) was higher than that of PVA under the degradation condition. Size exclusion chromatography, 1H NMR, and Fourier-transform infrared measurements of the recovered sample indicated that biodegradation of the PVA main chain was accelerated by partial glycosidation of hydroxyl groups in PVA. Similar acceleration was observed in a PVA/GlcNAc (50:50, w/w) mixture. Microbes which relate with degradation of the glycosidated polymers were grown in a culture medium including the soil suspension and the polymer as the carbon source. Polyacrylamide gel electrophoresis (SDS-PAGE) and IR measurements indicated that a cell-free extract derived from GlcNAcsubstituted PVA was different from that in the PVA/GlcNAc mixture. The results suggested that the PVA main chain in GlcNAc-substituted PVA was cleaved by a different microorganism or via a mechanism different from that in the mixture. Chitobiose-substituted PVA 2 showed more enhanced acceleration, indicating that the sugar length influenced the degradability. Introduction The increasing concern of the general public with the protection of our daily environment has largely contributed to the advent and growth of biodegradable polymers. However, the time required for their biodegradation is often unknown. Poly(vinyl alcohol) (PVA), which is highly crystalline even in the atactic form, has possible applications because it is water soluble and is an excellent barrier to scents, flavors, oils, and fat.1 Although PVA is the only carbon-carbon backbone polymer that is biodegradable under both aerobic2-6 and anaerobic7,8 conditions, the degradation rate under natural environmental conditions is too slow for it to be applied as an industrial degradable polymer.8,9 Exploring new chemical modification as well as building up miscible blends of PVA with other biodegradable polymers are the two most interesting approaches to improve the poor biodegradability of PVA.9 Although PVA/poly(3hydroxybutyrate),10 PVA/chitin derivatives,11 and PVA/ deoxyribonucleic acid12 have been reported as the examples for the latter, only a few study on the former subject have been published until now.13 On the other hand, greater attention has been directed to effective utilization of saccharide as alternative, renewable resources that can be steadily supplied and used for polymer synthesis. Recently, we succeeded in a new chemical modification of hydroxyl groups of PVA by a glycosidation reaction and * To whom correspondence should be addressed. Tel.: +81-52-7357159. Fax: +81-52-735-5342. E-mail address:
[email protected]. † Department of Materials Science and Engineering, Nagoya Institute of Technology.
the PVA derivative with a pendant sugar residue exhibiting different solubility and thermal properties from those of nonmodified PVA.14 Tokiwa et al. reported biodegradation of a sugar branched polymer consisting of glucose, fatty acid as a spacer arm, and PVA as the main chain, which was synthesized by radical polymerization of 6-O-vinyladipoylD-glucose.15 They reported that the polymer with a high molecular weight (DP > 100) scarcely degraded.15b Acceleration of the biodegradation of high-molecular-weight PVA is important from both environmental and industrial viewpoints. In this paper, we will report a new concept of acceleration of biodegradability of PVA by introducing sugar residues directly into the hydroxyl groups in PVA, that is, partial glycosidation or addition of sugar (mixing physically), although preliminary results were already reported as a communication.16 The accelerated biodegradation of the glycosidation was compared with that in the PVA/N-acetylD-glucosamine (GlcNAc) physical mixture. To clarify the degradation behavior, characterization of cell-free extracts from cultivation of the soil suspension using sugar-substituted PVAs and the PVA/GlcNAc physical mixture as screening substrates was carried out. Moreover, a biodegradation test of 2,4-pentadione and 4-hydroxy-4-methyl-2-pentanone as the degradation intermediates of the PVA main chain and cultivation of the soil suspension using them as the carbon sources were also performed. Experimental Section Materials. PVAs were purchased from Wako Pure Chemicals Co. (Osaka, Japan, DP ) 400-600) and Nacalai Tesque
10.1021/bm034527q CCC: $27.50 © 2004 American Chemical Society Published on Web 03/23/2004
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Scheme 1. Synthesis of GlcNAc and Chitobiose-Substituted PVAs
(Kyoto, Japan, DP ) 500). Their saponification values were determined as 97 and 89%, respectively, by 1H NMR measurement in dimethyl sulfoxide (DMSO)-d6. The former was composed of 27% syndiotactic, 49% heterotactic, and 24% isotactic triad sequences and the latter 28% syndiotactic, 49% heterotactic, and 24% isotactic triad.17 The number average molecular weight (Mn) and polydispersity index (Mw/ Mn) using size exclusion chromatography [SEC; poly(ethylene oxide) standards] estimation of the PVA with a 97% saponification value were 1.48 × 104 and 2.21, and those of 89% saponificated PVA were 1.20 × 104 and 2.23. Mercury(II) cyanide (purity, 95%) was obtained from Wako Pure Chemicals Co. (Osaka, Japan). Trifluoromethanesulfonic acid (TfOH, purity 98%) was purchased from Nacalai Tesque Co. (Kyoto, Japan). N,N-Dimethylacetamide (DMAc), 2-propanol, benzene, and chloroform (CHCl3) were purified by distillation. Measurements. Fourier transform infrared spectra were recorded in KBr using a JASCO FT/IR-430 spectrometer, and a 100% KBr disk was used as the blank. 1H NMR spectra were taken on a JEOL JNM-GX400 (400 MHz for 1H) at room temperature. Mn and Mw/Mn of the polymers were determined by SEC calibrated with poly(ethylene oxide) or globular protein standards using a system of JASCO model PU-980 with both JASCO 830-RI and UV-970 detectors and Shodex B-804+B-805 columns [eluent, 0.05 M K2HPO4(aq); temperature, 27 °C; concentration, 2-3 mg/mL]. Biodegradation Test. Biochemical oxygen demand (BOD) was determined by oxygen consumption with a BOD tester (model 200F, TAITEC Co., Koshigaya-shi, Japan), basically according to the ISO standard guidelines (ISO 14851) at 25 °C. The soil was obtained from the Nagoya University Farm. A soil suspension was prepared by stirring of the soil in water (150 g/L) at 25 °C for 30 min. Conditioned soil suspensions were prepared in a similar way expect for addition (200 mg/ L) of GlcNAc-substituted PVA 1b or a mixture of PVA and GlcNAc (47:53, w/w) and successive incubation at 25 °C for 12 h. The supernatants (15 mL) were added to the incubation media (150 mL). The incubation medium contained the following (mg/L): K2HPO4, 217; KH2PO4, 85;
Na2HPO4, 447; NH4Cl, 5; CaCl2, 27; MgSO4‚7H2O, 23; and FeCl3‚6H2O, 0.25. The polymers were dissolved in the media, and the concentration was 100 mg/L. After the test, the suspension was filtered and the filtrate was evaporated and dialyzed against distilled water using a dialyzing tube (Seamless Cellulose Tubing, VT351, molecular weight cutoff 3500, Nacalai Tesque, Inc.) for 12 h. The solution was lyophilized to give polymeric material, which was purified by reprecipitation from water to methanol. The recovered sample was analyzed by SEC, IR, and 1H NMR measurements. Preparation of Chitobiose-Substituted PVA (2). A typical experimental procedure is as follows. The 89% saponificated PVA (0.20 g; 4.11 unit mmol; [-OH]0, 3.66 mmol) was dissolved in 5.8 mL of DMAc at 70 °C for 2 h. After cooling to room temperature, 2.25 g (3.66 mmol) of chitobiose oxazoline derivative20b (see Scheme 1) and TfOH (0.03 mL, 0.32 mmol) was added into the solution under nitrogen. After stirring at 80 °C for 6 h, the reaction mixture was poured into 500 mL of CHCl3/diethyl ether (1:2, v/v) and then purified by reprecipitation from CHCl3 to diethyl ether three times. After drying in vacuo, pentacetylated N,N′diacetylchitobiose-substituted PVA obtained was a powdery material (0.41 g, 98% yield). IR (KBr disk, cm-1): 3356 (νOsH), 2945 (νNsH), 1746 [νCdO(ester)], 1664 [νCdO(amide I)], 1550 [δNsH(amide II)], 1374 (δC-H), 1234, 1044 (νCs 1 OsC). H NMR (DMSO-d6, concentration, 2.0 wt %, 27 °C; ref., TMS, 400 MHz, δ): 1.10-1.16 (CH2CH-O-Sugar, and CH2CHOH), 1.76 [CH3CONH and CH2CHO(COCH3)], 1.90, 1.94, 1.99, 2.03 (CH3COO), 3.62 (H-4), 3.70-4.20 (H2, H-5, H-6a, H-2′, H-5′, H-6′a, CH2CH-O-sugar, and CH2CHOH), 4.20-4.40 (H-6b, H-6′b, CH2CHOH), 4.604.70 (H-1β, Η-1′β, CH2CHOH), 4.70-4.85 (CH2CHOCOCH3), 4.85-5.00 (H-4′), 5.00-5.20 (H-3, H-3′), 7.79 (NH′COCH3), 7.98 (NHCOCH3). 13C NMR (DMSO-d6, concentration, 10 wt %, 27 °C; ref., TMS, 100 MHz, δ): 20.4, 20.5, 20.6, 20.7, 20.9 (CH3COO), 22.7, 22.9 (CH3CONH), 43.6 (CH2CH-O-Sugar), 44.0-47.0 (CH2CHOH), 53.9, 55.1 (C-2, C-2′), 61.8, 63.0 (C-6, C-6′), 67.8, 68.4, 70.6, 71.1, 71.9, 72.5, 74.0, 76.3 (C-3, C-3′, C-4, C-4′, C-5, C-5′,
Accelerated Biodegradation of Poly(vinyl alcohol)
CH2CH in PVA backbone, CH2CH-O-Sugar), 94.6 (C-1′ β), 100.5 (C-1β), 169.2, 169.4, 169.6, 169.8, 170.2, 170.3 (CH3CO). Deacetylation of pentacetylated N,N′-diacetylchitobiosesubstituted PVA was carried out to give chitobiosesubstituted PVA (2). A typical experimental procedure is as follows. Pentaacetylated chitobiose-substituted PVA (0.20 g, 1.11 unit mmol, [sugar]/[-OH]0 ) 0.24) was dissolved in methanol (80 mL) at room temperature. A 2 N NaOH aqueous solution (1.9 mL, 3.8 mmol) was dropped into the solution. After 1 h, a resulting white powder was collected by filtration and washed with methanol. The precipitate was purified by reprecipitation with a water/methanol system and dried in vacuo to give a chitobiose-substituted PVA 2a (1.05 g, 76% yield, [sugar]/[-OH]0 ) 0.21). 1H NMR (D2O, concentration, 1.0 wt %, 27 °C; ref., sodium 2,2-dimethyl2-silapentane-5-sulfonate, 400 MHz, δ): 1.28-1.87 (CH2CH-O-chitobiose and CH2CHOH), 2.06 (NHCOCH3), 3.35-4.29 (H-2, H-3, H-4, H-5, H-6, H-2′, H-3′, H-4′, H-5′, H-6′, CH2CHOH, CH2CH-O-chitobiose), 4.58 (H-1β, Η′1β). IR (KBr disk): 3399 (νOsH), 2943 (νNsH), 1653 [νCd O(amide I)], 1559 [δNsH(amide II)], 1377 (νNsH), 1071 cm-1(νCsOsC). Preparation of Isopropyl 2-Acetamido-2-deoxy-β-Dglucopyranoside (3). In a 100-mL three-necked flask, 2-acetamido-3,4,6-tri-O-acetyl-R-D-glucopyranosyl chloride,19 2.0 g (5.5 mmol), and Hg(CN)2, 2.0 g (7.9 mmol), were dissolved in dry benzene (20 mL) under a nitrogen atmosphere. Into the solution was poured 0.5 mL (6.5 mmol) of 2-propanol, and the mixture was placed in an oil bath at 80 °C for 20 h. The reaction mixture was filtered and the filtrate was evaporated. The powder obtained was dissolved in CHCl3 and washed with 30% KI aqueous solution. The organic layer was condensed and purified by recrystallization in ethanol to give isopropyl 2-acetamido-3,4,6-tri-O-acetyl2-deoxy-β-D-glucopyranoside (0.48 g, 22% yield). The acetylated GlcNAc derivative, 300 mg (0.78 mmol), was dissolved in methanol (3.0 mL). and then 12 mg (0.22 mmol) of NaOMe was added into the solution at 27 °C. After the deacetylation proceeded for 3 h, the solution was made neutral with Dowex 50WX-8-400, and the suspension was filtered. The filtrate was evaporated to give isopropyl 2-acetamido-2-deoxy-β-D-glucopyranoside (3, 158 mg, 80% yield). IR (KBr disk, cm-1): 3285 (νOsH), 2969 (νCsH), 1655 (νCdO), 1553 (δNsH), 1431 (δCsH), 1130, 1081, 1029 (νCs 1 O). H NMR (400 MHz, D2O, δ): 1.13 (d, 3H, J ) 6.0 Hz, CHCH3), 1.20 (d, 3H, J ) 6.0 Hz, CHCH3), 2.04 (s, 3H, NHCOCH3), 3.44 (br, 2H, H-4 and H-5), 3.55 (t, 1H, J ) 8.8 Hz, H-3), 3.63 (t, 1H, J ) 8.0 Hz, H-2), 3.74 (br d, 1H, J ) 9.6 Hz, H-6a), 3.92 (br d, 1H, J ) 12.4 Hz, H-6b), 4.02 (m, 1H, CHCH3), 4.54 (d, 1H, J ) 8.4 Hz, H-1b). 13C NMR (D2O, concentration, 1.0 wt %, 27 °C; ref., sodium 2,2dimethyl-2-silapentane-5-sulfonate, 400 MHz, δ): δ ) 23.8 (CH3CONH), 24.8, 24.9 (CHCH3), 58.5 (C-2), 63.2 (C-6), 72.6 (C-4), 76.2 (C-3), 78.4 (C-5), 76.5 (CHCH3), 102.2 (C-1 β), 100.5 (C-1 β), 177.1 (CH3CO). Preparation of Isopropyl O-(2-Acetamido-2-deoxy-βD-glucopyranosyl)-(1f4)-2-acetoamido-2-deoxy-β-D-glucopyranoside (4). In a 100-mL three-necked flask, chitobiose
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oxazoline derivative,20b 0.83 g (1.35 mmol), and CSA, 0.18 g (0.08 mmol), were dissolved in dry CH2Cl2 (12 mL) under a nitrogen atmosphere. Into the solution was poured 0.5 mL (6.5 mmol) of 2-propanol, and the mixture was placed in an oil bath at 90 °C for 5 h. The reaction mixture was filtered, and the filtrate was evaporated. The powder obtained was dissolved in CHCl3 and washed with a 30% KI aqueous solution. The organic layer was condensed to give isopropyl O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1f4)-2-acetoamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranoside (0.21 g, 23% yield). The acetylated chitobiose derivative, 63 mg (0.09 mmol), was dissolved in methanol (3.0 mL), and then 11 mg (0.03 mmol) of NaOMe was added into the solution at 27 °C. After the deacetylation proceeded for 9 h, the solution was made neutral with Dowex 50WX8-400, and the suspension was filtered. The filtrate was evaporated to give isopropyl O-(2-acetamido-2-deoxy-β-Dglucopyranosyl)-(1f4)-2-acetoamido-2-deoxy-β-D-glucopyranoside (4, 27 mg, 62% yield). IR (KBr disk, cm-1): 3404 (νOsH), 2939 (νCsH), 1649 (νCdO), 1560 (δNsH), 1410 (δCs 1 H), 1130, 1081, 1029 (νCsO). H NMR (400 MHz, D2O, δ: 1.12 (d, 3H, J ) 6.2 Hz, CHCH3), 1.22 (d, 3H, J ) 6.2 Hz, CHCH3), 2.04, 2.08 (s, 3H × 2, NHCOCH3), 3.38-4.06 (m, 11H, H-2, 4, 5, and 6, H-2′, 4′, 5′, and 6′, CHCH3), 4.55 (br, 2H, H-1, H-1′β). Microorganism and Growth Conditions. The isolated GlcNAc-substituted PVA assimilating microbe was obtained from a soil suspension by an enrichment culture technique. The condition of the basal medium (pH 7.5) for the culture was as follows: 0.4% (NH4)2SO4, 0.1% KH2PO4, 0.2% K2HPO4, and 0.05% MgSO4‚7H2O. Test tubes containing 5 mL of the basal medium and 10 mg of sugar-substituted PVAs or PVA powder were inoculated with the soil suspension (50 µL) used for the BOD test. Measuring the absorbance at 660 nm monitored the cell growth. The strain was maintained on agar slants using a 0.5% sugar-substituted PVAs or PVA concentration of the culture medium, and this concentration was used throughout this work. The composition of the culture medium was as follows: 0.1% (NH4)2SO4, 0.02% KH2PO4, 0.1% K2HPO4, 0.02% MgSO4‚7H2O, 0.01% NaCl, 0.01% CaCl2, 0.001% FeSO4‚7H2O, 0.05% peptone, 0.005% yeast extract, 1 µg/mL (final concentration) PQQ, and 1.5% agar. The final pH was adjusted to 7.5. Purification of GlcNAc-Substituted PVA 1 Degrading Enzyme (Preparation of Cell-Free Extracts). The basal medium for cell cultivation was the same as that for the slants without agar using a 0.2% KH2PO4, 1.0% K2HPO4, and 1.0% polymer concentration. An antifoaming agent (0.03%) was added before shaking. Cultivation was carried out with 200 mL of the culture medium in a 500-mL shake flask at 30 °C with reciprocal shaking for 5 days. Cells were harvested by centrifugation at 13 000g (at 4 °C for 35 min) to obtain wet cells from a 200-mL culture broth. The cells were resuspended in 20 mL of distilled water containing 1 mM ethylenediaminetetraacetic acid‚2Na, 1 mM phenylmethylsulfonyl fluoride, and 0.5% Tween 80 and divided into some 30-mL tubes. The cell suspension was then disrupted with an ultrasonic oscillator (19 kHz, 125 W, 50% pulse) for 10 min at 0 °C. The cell debris was removed by centrifugation
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Figure 1. Structures of GlcNAc-substituted PVA 1 and chitobiose-substituted PVA 2 used in this study. Scheme 2. Preparation of Model Compounds 3 and 4
at 20 000g (at 4 °C for 20 min) to obtain the supernatant (20 mL). The supernatant was dialyzed against a 10 mM phosphate buffer (pH 7.2) at 4 °C using a dialyzing tube (Seamless Cellulose Tubing, UC36-32-100, Viskase Sales Corp.) to obtain the cell-free extracts. For further purification of the extract, supernatant was dialyzed by using Tris-HCl buffer (pH 8.5). Results and Discussion Preparation of GlcNAc-Substituted PVA 1 and Chitobiose-Substituted PVA 2 with Various Degrees of Substitution. The structures of 1 and 2 used in this study are shown in Figure 1. According to our procedure for GlcNAc-substituted PVA,14 chitobiose-substituted PVAs 2 were also synthesized by a reaction of hydroxyl groups in PVA with sugar oxazoline derivatives, readily prepared by the trimethylsilyl trifluoromethanesulfonate method20 starting from chitobiose, and subsequent deacetylations by the Zemplen method (Scheme 1).21 The ring opening involves a nucleophilic attack of the hydroxyl group in PVA at C5 of the oxazoline ring with inversion of the configuration at the carbon based on SN2 reaction. The structures were determined by IR, 1H, and 13C NMR spectra. The NMR assignments of sugar-substituted PVAs 1 and 2 are achieved by comparison with model compounds 3 and 4, respectively. The model compounds 3 and 4 were prepared by the glycosidation reaction of 2-propanol with 2-acetamido-3,4,6-tri-O-acetyl-R-D-glucopyranosyl chloride19 and chitobiose oxazoline derivative,20 respectively. The β-selective glycosidation was also confirmed by 1H NMR measurement. The anomeric proton was observed at 4.58 ppm in the 1H NMR of 2a, indicating β-selective glycosidation.14 The sugar content ([sugar]/
Figure 2. Biodegradability of isopropyl glucopyranoside 3 as the model compound of GlcNAc-substituted PVA 1 in the soil suspension detected by BOD.
[-OH]0) was determined by the intensity ratio of the peaks at 1.28-1.87 (-CH2-) and 2.06 (-NHCOCH3) ppm. Biodegradability of Model Compound 3. Isopropyl 2-acetamido-2-deoxy-β-D-glucopyranoside (3) was prepared as a model compound for the GlcNAc-substituted PVA 1. As shown in Scheme 2, 3 was synthesized by the KoenigsKnorr reaction22 (see Experimental Section). In the biodegradation test using the soil suspension, model compound 3 [BOD/theoretical oxygen demand (TOD) ) 41%] as well as GlcNAc (BOD/TOD ) 46%) was easily degraded in 20 days (Figure 2), although about 10 days retardation of the degradation was observed. We expected that the induction time is due to the derivatization at the anomeric carbon (hemiacetal to acetal). The degradation rate was also influenced by the derivatization to some extent. In the BOD curves, the maximum degradation rate of 3 estimated from the slope was little lower than that of GlcNAc. The results indicated that microorganisms in the soil suspension could catabolize low-molecular-weight model compound 3 as well as GlcNAc within 20 days.
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Accelerated Biodegradation of Poly(vinyl alcohol)
Table 1. Chracterization of PVA and Sugar-Substituted PVAs (1 and 2) before and after the Biodegradation Test in the Soil Suspensiona before degradation
after degradation
sample
[sugar]/ [-OH]0b
M nc × 10-4
Mw/ M nc
[sugar]/ [-OH]0b
Mnc × 10-4
Mw/ M nc
BOD/TOD (%)
PVA 1a 1b 1c 2a 2b
0 0.31 0.24 0.21 0.21 0.08
1.48 4.60 4.53 4.18 3.96d 4.27
2.21 2.53 2.76 2.66 1.44d 3.12
0 0.16 0.12 0.08 0.08 0.04
1.21 1.78 2.09 2.04 1.26 1.48
2.56 2.45 2.57 2.30 2.87 2.76
2 18 17 16 16 10
a Polymer concentration, 100 mg/L; pH 6.5, 25 °C for 40 days. b Degree of substitution, determined by 1H NMR in D2O. c Determined by SEC in a 0.05 M K2HPO4 aqueous solution using poly(ethylene oxide) standards. d Soluble part.
Figure 3. SEC traces of (A) PVA, (B) PVA after the biodegradation test for 40 days in the soil suspension, (C) GlcNAc-substituted PVA 1a, and (D) GlcNAc-substituted PVA 1a after the biodegradation test for 40 days in the soil suspension detected by differential RI. Flow rate, 1.0 mL/min; temperature, 27 °C.
Biodegradation of Sugar-Substituted PVAs (1 and 2) and the PVA/GlcNAc Physical Mixture in the Soil Suspension. Biodegradations of PVA and sugar-substituted PVAs (1 and 2) in the soil suspension (pH 6.5, 25 °C) were monitored by BOD, and the degradation percentages were calculated from the BOD values and TOD value, in which the polymers were dissolved in the media. In the BOD measurement, GlcNAc-substituted PVA 1a with a degree of substitution ([sugar]/[-OH]0) of 0.31 [Mn ) 4.60 × 104, DP ) 427, Mw/Mn ) 2.53] was degraded by 18% of BOD/TOD in 40 days, while PVA was hardly degraded (2%). In 1a, the PVA main chain content is 28 wt %. SEC measurement with a differential refractive index (RI) detector was carried out to confirm the cleavage of the PVA main chain. The SEC chromatogram of PVA after 40 days of the degradation test did not show an important change, compared with PVA before the test (Figure 3A,B). On the other hand, there was an important change for the RI-detected chromatogram of the GlcNAc-substituted PVA 1a (Figure 3C,D). After the degradation test, the SEC curve shifted to a low molecular weight [Mn ) 1.78 × 104, Mw/Mn ) 2.45] and became bimodal accompanied with a clear peak at a molecular weight of 0.3 × 104 to 0.4 × 104 (arrow mark in Figure 3D). Although the shift of the SEC curve is mainly due to the deglycosidation, formation of the intermediate molecular weight polymer between that of the starting polymer and that of low-molecular-weight degradation compounds was confirmed interestingly. The clear peak ascribed to intermediate molecular compounds (Mn ) 0.3 × 104 to 0.4 × 104) indicated that endo-type PVA main chain scission surely occurred because the recovered sample had the [sugar]/[-OH]0 value of 0.16 even after the biodegradation test. We speculated that degradation of the main chain proceeded via random scission. As recovered samples were
purified by dialysis using cellulose tubing (molecular weight cutoff 3500), the low-molecular-weight fraction (
