Finely Selective Protections and Deprotections of Multifunctional Chitin and Chitosan (PDF)

Carbohydrate Polymers 81 (2010) 434–440 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www...

Carbohydrate Polymers 81 (2010) 434–440 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Finely selective protections and deprotections of multifunctional chitin and chitosan to synthesize key intermediates for regioselective chemical modifications Keisuke Kurita ∗ , Yuhya Yoshida, Tohru Umemura Department of Materials and Life Science, Faculty of Science and Technology, Seikei University, Musashino-shi, Tokyo 180-8633, Japan a r t i c l e i n f o a b s t r a c t Article history: Site-selective protection of chitin and chitosan has been studied in detail in order to distinguish the Received 16 September 2009 three kinds of functional groups, which would make possible finely controlled regiospecific structural Accepted 25 February 2010 modifications. To allow reliable chemical manipulations and to establish stable protection and facile Available online 7 March 2010 deprotection at high selectivity, benzyl was evaluated for the C-3 protection in combination with other protective groups including triphenylmethyl (trityl) for C-6 and acetyl or phthaloyl for C-2. Chitin was Keywords: first tritylated and then benzylated to give 3-O-benzyl-6-O-trityl-chitin, of which each of the trityl, benzyl, Chitin Chitosan and acetyl groups could be removed selectively with dichloroacetic acid, hydrogen-Pd/C, and aqueous Protection–deprotection sodium hydroxide, respectively, affording three kinds of derivatives having a reactive group at C-6, C- Chemical modification 3, or C-2. 2-N-Phthaloyl-chitosan was also tritylated at C-6 and benzylated at C-3; the resulting fully Benzylation protected product was detritylated, debenzylated, or dephthaloylated, similarly giving rise to three kinds Tritylation of precursors having a reactive group only at one position. The extents of all the substitution and removal Phthaloylation reactions proved quantitative under appropriate conditions to give structurally well-defined derivatives. They exhibited improved solubility in organic solvents, indicating high potential of these derivatives as novel convenient intermediates for designing diversified molecular architectures through regiospecific chemical modifications. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction N-phthaloyl-chitosan is the practical derivative currently used, since it is organosoluble and useful for the regioselective chem- Because of the presence of amino groups, polysaccharides chitin ical modifications (Nishimura et al., 1991). Several kinds of sugar and chitosan are expected to be particularly useful biopolymers in branches can be incorporated at the C-6 position to synthesize non- various fields (Domard, Guibal, & Vårum, 2007; Uragami, Kurita, & natural branched chitins and chitosans (Kurita, Akao, Kobayashi, Fukamizo, 2001; Uragami & Tokura, 2006). Although they are abun- Mori, & Nishiyama, 1997; Kurita, Shimada, Nishiyama, Shimojoh, dant and easily accessible, and therefore attracting much attention, & Nishimura, 1998; Kurita, Kojima, Nishiyama, & Shimojoh, 2000; their utilization has been quite limited. This is primarily due to Kurita, Akao, Yang, & Shimojoh, 2003). Many other substituents the difficulty in fabrication and structural transformation, which is have also been introduced for various purposes using 2-N- ascribed to the lack of solubility in suitable solvents. The various phthaloyl-chitosan (Holappa et al., 2004, 2005; Kurita, Hayakawa, distinctive biological and physicochemical functions of chitin and Nishiyama, & Harata, 2002; Nishimura et al., 1993, 1998; Nishiyama chitosan suggest the prospect of synthesizing intelligent polymeric et al., 2000; Ouchi, Nishizawa, & Ohya, 1998; Yoksan, Akashi, materials having medicinal and pharmaceutical activities by appro- Hiwatari, & Chirachanchai, 2003; Yoksan, Matsusaki, Akashi, & priate chemical modifications (Kurita, 1997, 2001, 2006a, 2006b; Chirachanchai, 2004). Nishimura, Kohgo, Kurita, & Kuzuhara, 1991; Roberts, 1992). In the above procedures, the C-3 hydroxy was protected by For precise and well-controlled structural modifications of these acetylation, but the resulting ester linkage is somewhat vulner- polysaccharides to develop advanced materials with desirable able and may undergo hydrolysis and/or acetyl migration under bioactivities, it is necessary to clearly distinguish the three kinds certain reaction conditions, which limits its utility as a precursor. of functional groups in their repeating units. In this respect, 2- To further expand the scope of structural modifications of chitin and chitosan, stable protection and facile deprotection of the C-3 hydroxy group are undoubtedly key issues. In view of the reliabil- ∗ Corresponding author. ity for O-protection in chemical manipulations, benzyl would be a E-mail address: [email protected] (K. Kurita). promising candidate. It has, however, not been used for chitin and 0144-8617/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.carbpol.2010.02.042  K. Kurita et al. / Carbohydrate Polymers 81 (2010) 434–440 435 Table 1 Benzylation of 6-O-trityl-chitin (1)a. Base Base/pyranoseb BnCl/pyranoseb Repetition of reactionc ds for Bnd Yield (%) NaOH 2 6 2 0.85 65 NaOH 6 12 2 0.80 72 NaH 2 6 1 1.00 75 a 1, 0.30 g; DMSO, 4.5 mL; temp, rt; time, 24 h. b Mole ratio. c Number of repetition under the same reaction conditions. d Degree of substitution calculated from the C/N of elemental analysis. chitosan, and we have thus examined the possibility of benzyl as was added. After stirring the mixture in nitrogen at room tempera- a protecting group for C-3 in terms of both protection and depro- ture for 2 h, 0.51 g (4.04 mmol) of benzyl chloride (BnCl) was added tection in the presence of other protective groups to synthesize dropwise. The mixture was stirred at room temperature for 24 h, versatile intermediates for site-selective chemical modifications at and the resulting solution was poured into 30 mL of methanol to the three different fictional groups. precipitate the product. It was washed with water and methanol and dried to give 0.27 g (75%) of 3-O-benzyl-6-O-trityl-chitin as a 2. Experimental pale tan powdery material. IR (KBr):  3420 (NH), 3058 (arom), 1682 (amide I), 1150–1000 (pyranose), and 746 and 706 cm−1 (arom). 2.1. General procedures Anal. Calcd for C34 H33 NO5 ·0.5H2 O: C, 74.98; H, 6.29; N, 2.57. Found: C, 75.04; H, 6.39; N, 2.53. IR spectra were taken on a Shimadzu FTIR-8900 spectrometer. 1H NMR spectra were recorded with a JEOL JNM-LA400D FT-NMR 2.3.2. Detritylation of 3-O-benzyl-6-O-trityl-chitin in deuterated dimethyl sulfoxide (DMSO-d6 ) at 90 ◦ C. Elemental To 3 mL of a dichloroacetic acid/DMSO (1/1) mixed solvent was analysis was conducted with a Perkin-Elmer 2400 II instrument. added 30 mg (0.056 mmol) of 3-O-benzyl-6-O-trityl-chitin, and the Conductometric titration was carried out with a DKK·TOA CM- mixture was stirred at room temperature for 1 h. It was poured into 20J. HPLC was performed with a Waters 486 equipped with a 10 mL of acetonitrile/water (4/1), and the precipitate was washed Waters Controller 800, SIM Chromatocorder 21, and a Bondasphere with water and methanol. After drying, 13 mg (79%) of 3-O-benzyl- column (5 ␮m, C18, 100A) with a mobile phase of acetoni- chitin was obtained as a pale tan powdery material. IR (KBr):  trile/water (4/1). Chemicals were of reagent grade and used after 3406 (OH and NH), 3059 (arom), 1653 (amide I), 1523 (amide II), drying. Dimethyl sulfoxide (DMSO) and N,N-dimethylformamide 1150–1000 (pyranose), and 768, 748, and 702 cm−1 (arom). (DMF) were dried with calcium hydride and molecular sieves, Anal. Calcd for C15 H19 NO5 ·0.4H2 O: C, 59.95; H, 6.64; N, 4.66. respectively, and distilled. Pyridine was refluxed with potassium Found: C, 59.94; H, 6.47; N, 4.60. hydroxide and distilled. All the solvents were stored over molecular sieves. 2.3.3. Deacetylation of 3-O-benzyl-6-O-trityl-chitin A mixture of 20 mg (0.037 mmol) of 3-O-benzyl-6-O-trityl- 2.2. Structurally uniform chitin and chitosan chitin in 10 mL of 12 mol/L sodium hydroxide was stirred at 80 ◦ C for 8 h. After cooling to room temperature, the precipitate was Squid chitin with a degree of acetylation of 0.8–0.9 was N- washed by repeated decantations with water until neutral, filtered, acetylated with acetic anhydride in methanol, and a small amount and dried to give 15 mg (81%) of 3-O-benzyl-6-O-trityl-chitosan. IR of O-acetyl groups were selectively removed by treating with potas- (KBr):  3411 (NH2 ), 3059 (arom), 1599 (NH2 ), 1150–1000 (pyra- sium hydroxide/methanol to give white powdery chitin with a nose), and 746 and 700 cm−1 (arom). degree of N-acetylation of 1.00 as determined by conductometric Anal. Calcd for C32 H31 NO4 ·0.5H2 O: C, 76.47; H, 6.42; N, 2.79. titration (Kurita, Ishii, Tomita, Nishimura, & Shimoda, 1994). Found: C, 76.35; H, 6.41; N, 2.78. Pulverized shrimp chitin was deacetylated repeatedly with 40% aqueous sodium hydroxide to give fully deacetylated chitosan as 2.3.4. Debenzylation of 3-O-benzyl-6-O-trityl-chitin a white powder. Conductometric titration indicated the degree of To a solution of 3-O-benzyl-6-O-trityl-chitin (30 mg, deacetylation to be 1.00 (Nishimura, Matsuoka, & Kurita, 1990). 0.056 mmol) in 6 mL of DMSO/acetic acid (10:1) was added 30 mg of 5% Pd/C. The mixture was stirred under an atmosphere 2.3. 6-O-Trityl-chitin of hydrogen at 70 ◦ C for 24 h and filtered with Celite 545. The filtrate was concentrated under reduced pressure. The product Trimethylsilylated chitin with a degree of substitution (ds) 2.00 was precipitated in water, washed with water and methanol, and was treated with chlorotriphenylmethane to introduce triphenyl- dried to give 20 mg (80%) of 6-O-trityl-chitin. The spectral data methyl (trityl) group at C-6 according to the method reported were identical with those of the authentic sample. previously (Kurita, Sugita, Kodaira, Hirakawa, & Yang, 2005). The ds Anal. Calcd for C27 H27 NO5 ·0.4H2 O: C, 71.63; H, 6.19; N, 3.09. was 1.00 for the trityl as confirmed by spectroscopy and elemental Found: C, 71.70; H, 6.05; N, 3.08. analysis. IR (KBr):  3408 (OH and NH), 3057 (arom), 1670 (amide I), 1522 (amide II), 1150–1000 (pyranose), and 748 and 706 cm−1 2.4. 2-N-Phthaloyl-6-O-trityl-chitosan (arom). Anal. Calcd for C27 H27 NO5 ·1.4H2 O: C, 68.89; H, 6.38; N, 2.98. Chitosan was subjected to N-phthaloylation with phthalic anhy- Found: C, 68.72; H, 6.31; N, 2.92 dride in DMF/water (Kurita, Ikeda, Yoshida, Shimojoh, & Harata, 2002) followed by tritylation with chlorotriphenylmethane in pyri- 2.3.1. Benzylation of 6-O-trityl-chitin dine as reported (Nishimura et al., 1991). IR (KBr):  3472 (OH), 6-O-Trityl-chitin (0.30 g, 0.67 mmol) obtained above was dis- 3058 (arom), 1775 and 1716 (imide C O), 1150–1000 (pyranose), solved in 4.5 mL of DMSO, and 0.03 g (1.34 mmol) of sodium hydride and 743 and 700 cm−1 (arom). 436 K. Kurita et al. / Carbohydrate Polymers 81 (2010) 434–440 Scheme 1. Anal. Calcd for C33 H27 NO6 ·1.1H2 O: C, 68.89; H, 6.38; N, 2.98. washed with water by decantation until neutral, filtered, and Found: C, 68.72; H, 6.31; N, 2.92. dried to give 21 mg (88%) of 3-O-benzyl-6-O-trityl-chitosan as a white powder. IR (KBr):  3450 (NH2 ), 3057 (arom), 1595 (NH2 ), 2.4.1. Benzylation of 2-N-phthaloyl-6-O-trityl-chitosan 1150–1000 (pyranose), and 748 and 704 cm−1 (arom). 1 H NMR A mixture of 50 mg (2.1 mmol) of sodium hydride and 4.5 mL of (DMSO-d6 ): ı 3.5–5.2 (pyranose) and 7.2–7.9 ppm (phenyl and DMSO was heated at 70 ◦ C in nitrogen for 1 h to give an almost phthaloyl). homogeneous solution and cooled to room temperature. A por- Anal. Calcd for C33 H31 NO4 ·0.8H2 O: C, 76.22; H, 6.32; N, 2.69. tion (1.2 equiv.) of the solution was added to 0.30 g (0.56 mmol) Found: C, 76.05; H, 6.24; N, 2.73. of 2-N-phthaloyl-6-O-trityl-chitosan dissolved in 4.5 mL of DMSO. The mixture was stirred at room temperature for 2 h, and 0.43 g 2.4.4. Debenzylation of (3.40 mmol) of benzyl chloride was added dropwise. After stirring 3-O-benzyl-2-N-phthaloyl-6-O-trityl-chitosan the mixture at room temperature for 24 h, the resulting solution 3-O-Benzyl-2-N-phthaloyl-6-O-trityl-chitosan (80 mg, was poured into 30 mL of methanol to precipitate the product, 0.128 mmol) was dissolved in 4 mL of DMSO/acetic acid (10/1), which was washed with water and methanol and dried to give and 80 mg of 5% Pd/C was added. The mixture was heated in 0.27 g (77%) of 3-O-benzyl-2-N-phthaloyl-6-O-trityl-chitosan as a a hydrogen atmosphere with stirring at 80 ◦ C for 24 h. It was pale tan powdery material. IR (KBr):  3059 (arom), 1778 and 1719 filtered with Celite 545, and the filtrate was concentrated under (imide C O), 1150–1000 (pyranose), and 746 and 704 cm−1 (arom). reduced pressure. The viscous solution was poured into 30 mL of 1 H NMR (DMSO-d ): ı 3.5–5.2 (pyranose), 6.8–7.3 (phenyl), and ethanol, and the precipitate was collected by centrifugation. It was 6 7.5–7.9 ppm (phthaloyl). washed with water and methanol, and dried to give 65 mg (95%) of Anal. Calcd for C40 H33 NO6 ·0.2H2 O: C, 76.59; H, 5.37; N, 2.23. 2-N-phthaloyl-6-O-trityl-chitosan as a white powder. IR (KBr):  Found: C, 76.78; H, 5.21; N, 2.25. 3425 (OH), 3059 (arom), 1776 and 1717 (imide C O), 1150–1000 (pyranose), and 746 and 704 cm−1 (arom). 1 H NMR (DMSO-d6 ): ı 2.4.2. Detritylation of 3.5–5.2 (pyranose), 6.92 (phenyl), and 7.5–7.7 ppm (phthaloyl). 3-O-benzyl-2-N-phthaloyl-6-O-trityl-chitosan Anal. Calcd for C33 H27 NO6 ·0.5H2 O: C, 73.05; H, 5.20; N, 2.58. To 2 mL of dichloroacetic acid/DMSO (2/3) was added 20 mg Found: C, 73.02; H, 5.07; N, 2.57. (0.032 mmol) of 3-O-benzyl-2-N-phthaloyl-6-O-trityl-chitosan, and the solution was stirred at room temperature for 1 h. The 3. Results and discussion product was precipitated in acetonitrile/water (4:1), washed with water and methanol, and dried to give 3-O-benzyl-2-N-phthaloyl- In order to discuss the influence of reaction conditions on the chitosan. The yield of a white powdery material was 9 mg (74%). structures of products and to synthesize well-defined derivatives, IR (KBr):  3481 (OH and NH), 3059 (arom), 1778 and 1719 (imide structurally uniform chitin and chitosan should be used as starting C O), 1150–1000 (pyranose), and 746 and 712 cm−1 (arom). 1 H materials. Because of the presence of some free amino groups in iso- NMR (DMSO-d6 ): ı 3.5–5.2 (pyranose) and 7.2–7.9 ppm (phenyl lated chitin, acetylation was conducted to give fully N-acetylated and phthaloyl). chitin. Fully N-deacetylated chitosan was prepared by repeated Anal. Calcd for C21 H19 NO6 ·0.3H2 O: C, 65.21; H, 5.11; N, 3.62. alkaline treatments, from which N-phthaloyl-chitosan was derived. Found: C, 65.28; H, 4.99; N, 3.56. 3.1. Benzylation of 6-O-trityl-chitin 2.4.3. Dephthaloylation of 3-O-benzyl-2-N-phthaloyl-6-O-trityl-chitosan 6-O-Trityl-chitin (1) prepared by the tritylation of 3,6- 3-O-Benzyl-2-N-phthaloyl-6-O-trityl-chitosan (30 mg, O-bistrimethylsilyl-chitin was benzylated in the presence of 0.048 mmol) was added to 10 mL of hydrazine monohydrate, pulverized sodium hydroxide in DMSO under various conditions. and the mixture was heated at 90 ◦ C for 24 h. The product was The substitution was, however, not quantitative, even after repeat-  K. Kurita et al. / Carbohydrate Polymers 81 (2010) 434–440 437 Fig. 2. HPLC profiles of the reaction mixtures of 2 with dichloroacetic acid (DCA)/DMSO (A, 10/0; B, 7/3; C, 5/5; D, 3/7). Fig. 1. IR spectra of chitin derivatives (KBr): A, chitin; B, 6-O-trityl-chitin (1); C, 3-O-benzyl-6-O-trityl-chitin (2); D, 3-O-benzyl-chitin (3); E, 3-O-benzyl-6-O-trityl- Table 2 chitosan (4). Detritylation of 3-O-benzyl-6-O-trityl-chitin (2)a. Dichloroacetic acid/DMSO (v/v) dsb Yield (%) ing the reaction two times with excess base and benzyl chloride Tr Bn (Table 1), probably because of the high hygroscopicity of the base in the powder form. With sodium hydride, the reaction proceeded 10/0 0.00 0.27 88 7/3 0.00 0.75 92 smoothly, and the ds reached 1.0 (Scheme 1, Table 1). In the IR 6/4 0.00 0.92 87 spectrum of the product, 3-O-benzyl-6-O-trityl-chitin (2), bands 5/5 0.00 1.00 91 due to the hydroxy disappeared, and strong aromatic bands were 4/6 0.07 1.00 92 observed (Fig. 1). 3/7 – – –c a 2, 30 mg; dichloroacetic acid/DMSO, 3 mL; temp, rt; time 1 h. b 3.2. Detritylation of 3-O-benzyl-6-O-trityl-chitin (2) Degree of substitution calculated from the C/N of elemental analysis. c Aromatic bands in the IR spectrum were strong, and the peak due to trityl alcohol in the HPLC profile of the reaction mixture was small (Fig. 2). O-Trityl is usually removed with acid, and dichloroacetic acid was suited for the deprotection of 3-O-acetyl-2-N-phthaloyl- 6-O-trityl-chitosan to afford a precursor for C-6 modifications (Nishimura et al., 1991). The acid is, however, rather strong and The reaction was thus performed in 12 mol/L sodium hydroxide to may possibly interfere with the benzyl of 2. As expected, the HPLC give a fully deacetylated product, 3-O-benzyl-6-O-trityl-chitosan analysis of the supernatant of the reaction mixture indicated the (4) (Table 3, Scheme 1). As evident in Fig. 1, amide I and II bands presence of both trityl and benzyl alcohols as shown in Fig. 2. disappeared completely in the IR spectrum, and aromatic bands On dilution of dichloroacetic acid with DMSO, the peak due to remained strong. benzyl alcohol became small, and a 1/1 mixture did not cause debenzylation. Elemental analysis also revealed debenzylation in addition to detritylation at high acid concentrations, and the 1/1 Table 3 mixture proved appropriate for selective full detritylation to give Deacetylation of 3-O-benzyl-6-O-trityl-chitin (2)a. 3-O-benzyl-chitin (3) (Table 2, Scheme 1). The IR spectrum in Fig. 1 showed hydroxy bands, and the bands due to aromatic became NaOHaq (mol/L) Temperature (◦ C) Time (h) ds for Acb Yield (%) weak. 6 60 8 – –c 6 70 5 – –c 6 70 8 0.17 80 3.3. Deacetylation of 3-O-benzyl-6-O-trityl-chitin (2) 6 80 8 0.19 91 12 80 5 0.02 81 To transform 2 into the derivative having free amino groups, it 12 80 8 0.00 86 was treated with aqueous alkali to remove the acetyl. The hydroly- a 2, 20 mg; NaOHaq, 10 mL. sis of the amide group was, however, rather sluggish, and strong b Degree of substitution calculated from the C/N of elemental analysis. c amide bands remained in the IR spectra under mild conditions. Strong amide bands were observed in the IR spectra. 438 K. Kurita et al. / Carbohydrate Polymers 81 (2010) 434–440 Table 4 Debenzylation of 3-O-benzyl-6-O-trityl-chitin (2)a. Temperature (◦ C) dsb Yield (%) Bn Tr 50 0.16 1.00 70 60 0.08 1.00 63 70 0.00 1.00 80 a 2, 30 mg; Pd/C (5%), 0.030 g; solvent (DMSO/AcOH (10/1)), 6 mL; H2 , 1 atm; temp, rt; time 24 h. b Degree of substitution calculated from the C/N of elemental analysis. 3.4. Debenzylation of 3-O-benzyl-6-O-trityl-chitin (2) Removal of the benzyl was then examined to demonstrate facile regeneration of the C-3 hydroxy after desired modification reac- tions based on 2. Catalytic hydrogenation of 2 was carried out at various temperatures, and as listed in Table 4, complete deben- zylation was possible at 70 ◦ C without any interference with the other substituents (Scheme 1). The spectroscopy data were identi- cal with those of the authentic sample, and the elemental analysis also supported the structure of 1. 3.5. Benzylation of 2-N-phthaloyl-6-O-trityl-chitosan (6) 2-N-Phthaloyl-chitosan (5) and the derived 2-N-phthaloyl-6-O- trityl-chitosan (6) are key precursors for controlled modifications, and the C-3 hydroxy of 6 can be protected by acetylation (Nishimura et al., 1991). In order to explore for an alkali-resistive and yet read- Fig. 3. IR spectra of chitosan derivatives (KBr): A, 2-N-phthaloyl-chitosan (5); B, 2-N- phthaloyl-6-O-trityl-chitosan (6); C, 3-O-benzyl-2-N-phthaloyl-6-O-trityl-chitosan ily removable protective group for C-3, benzyl was chosen as a (7); D, 3-O-benzyl-2-N-phthaloyl-chitosan (8). candidate. Benzylation of 6 was first attempted with powdered sodium hydroxide, but the reaction proceeded only to low extents, Table 6 as evidenced by strong hydroxy bands in the IR spectra, as in the Detritylation of 3-O-benzyl-2-N-phthaloyl-6-O-trityl-chitosan (7)a. benzylation of 1. Sodium hydride appeared to be superior as a base, Acid/DMSO (v/v) Time (h) dsb Yield (%) but even after two repeated reactions, the ds was 0.72 (Table 5). Benzylation was then conducted according to the methyl- Tr Bn sulfinyl carbanion method (Corey & Chaykovsky, 1962; Greenwald, Dichloroacetic acid (10/0) 1 0.00 0.46 56 Chaykovsky, & Corey, 1963; Sjöberg, 1966). As included in Table 5, Dichloroacetic acid (7/3) 1 0.00 0.70 70 full substitution was achieved to give 3-O-benzyl-2-N-phthaloyl-6- Dichloroacetic acid (6/4) 1 0.00 0.86 76 O-trityl-chitosan (7). Complete disappearance of the hydroxy bands Dichloroacetic acid (5/5) 1 0.00 0.97 74 Dichloroacetic acid (4/6) 1 0.00 1.00 83 in the IR spectrum (Fig. 3), as well as the elemental analysis, con- Dichloroacetic acid (3/7) 1 0.10 1.00 69 cluded the full substitution (Scheme 2). Trifluoroacetic acid (5/5) 1 0.00 0.92 67 Trifluoroacetic acid (4/6) 1 0.03 1.00 80 3.6. Detritylation of Acetic acid (4/6) 24 – – –c 3-O-benzyl-2-N-phthaloyl-6-O-trityl-chitosan (7) a 7, 20 mg; acid/DMSO, 2 mL; temp, rt. b Degree of substitution calculated from the C/N of elemental analysis. c No appreciable change was observed in the IR spectrum. To synthesize a precursor having a reactive group at C-6, 3-O- benzyl-2-N-phthaloyl-chitosan (8), detritylation of 7 was studied with dichloroacetic, trifluoroacetic, and acetic acids. As in the while retaining the benzyl (Scheme 2). Trifluoroacetic acid was sim- detritylation of 2, dichloroacetic acid removed the benzyl in addi- ilarly effective. Selective detritylation in these solvents was also tion to the trityl as summarized in Table 6. However, a mixture confirmed by HPLC as in the case of 2 mentioned above. Bands of about equal volumes of the acid and DMSO removed the trityl ascribable to the hydroxy were observed in the IR spectrum of the Table 5 Benzylation of 2-N-phthaloyl-6-O-trityl-chitosan (6)a. Methodb Base Base/pyranonec BnCl/pyranosec Repetition of reactiond dse Yield (%) A NaOH 6 12 1 – –f A NaH 2 6 1 – –f A NaH 6 12 1 – –f A NaH 1.2 6 2 0.72 65 B NaH/DMSO 1.2 6 1 1.00 76 a 6, 0.30 g; DMSO, 4.5 mL; temp, rt; time, 24 h. b Base (method A) or NaH/DMSO (methylsulfinyl carbanion; method B) was added to a solution of the chitosan derivative in DMSO. c Mole ratio. d Number of repetition under the same reaction conditions. e Degree of substitution calculated from the C/N of elemental analysis. f Strong hydroxy bands were observed in the IR spectra.  K. Kurita et al. / Carbohydrate Polymers 81 (2010) 434–440 439 Scheme 2. Table 7 was supported by comparing the spectral data with those of the Dephthaloylation of 3-O-benzyl-2-N-phthaloyl-6-O-trityl-chitosan (7)a. authentic sample and by elemental analysis. Temperature (◦ C) ds for Phthb Yield (%) 80 – –c 3.9. Solubility of the derivatives 90 0.04 83 100 0.00 95 Qualitative solubilities of the products were examined in excess a 7, 30 mg; hydrazine monohydrate, 10 mL; time, 24 h. organic solvents at room temperature. Though chitin and chitosan b Degree of substitution calculated from the C/N of elemental analysis. were insoluble in common solvents, protected derivatives were c Weak imide bands were observed in the IR spectrum. soluble in common aprotic polar organic solvents such as DMSO, DMF, and pyridine, except 4 that was only partially soluble in these product (Fig. 3). Acetic acid was, however, too weak an acid for solvents. detritylation. 4. Conclusions 3.7. Dephthaloylation of 3-O-benzyl-2-N-phthaloyl-6-O-trityl-chitosan (7) Properly protected derivatives of chitin and chitosan are indis- pensable to attain finely controlled chemical manipulations for Reactive free amino groups were expected to regenerate by designing complicated and yet well-defined molecular environ- dephthaloylation of 7 to give a chitosan derivative having protec- ments. The results described here revealed the efficient synthesis tive groups at both C-3 and C-6. Derivative 7 was thus heated in of site-selectively and quantitatively protected derivatives of chitin hydrazine at 90 ◦ C to give an almost dephthaloylated product. The and chitosan having only one reactive group in the repeating units. full hydrazinolysis was possible at 100 ◦ C (Table 7, Scheme 2) result- The perfect discrimination of the different functional groups and ing in the formation of 4, identical with the compound prepared improved solubility are significant in the chemical aspects of these from 2. In the IR spectrum, strong bands at 1778 and 1719 cm−1 almost unutilized biomass resources, and the derivatives will be of characteristic of imide disappeared completely. practical utility as convenient and versatile key intermediates for the regioselective chemical modifications under mild conditions. 3.8. Debenzylation of 3-O-benzyl-2-N-phthaloyl-6-O-trityl-chitosan (7) Acknowledgment Catalytic hydrogenation of 7 also proceeded thoroughly at 80 ◦ C This work was partially supported by “High-Tech Research Cen- with Pd/C to remove the benzyl as in the case of 2, resulting in the ter” Project for Private Universities: matching fund subsidy from formation of 6 (Table 8, Scheme 2). The structure of the product MEXT, 2004–2008. References Table 8 Debenzylation of 3-O-benzyl-2-N-phthaloyl-6-O-trityl-chitosan (7)a. Corey, E. J., & Chaykovsky, M. (1962). Journal of the American Chemical Society, 84, Temperature (◦ C) ds for Bnb Yield (%) 866. Domard, A., Guibal, E., & Vårum, K. M. (Eds.). (2007). Advances in Chitin Science, Vol. 60 0.11 97 9. 10th ICCC-EUCHIS’06, Montpellier. 70 0.04 96 Greenwald, R., Chaykovsky, M., & Corey, E. J. (1963). Journal of Organic Chemistry, 28, 80 0.00 95 1128. 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