Supporting Information

Transcription

1 Supporting Information Metal Coordinative-crosslinked Polysaccharide Nanogels with Redox Sensitivity Yoshihiro Sasaki, 1,2 Tai irakura, 1 Shin-ichi Sawada, 1 and Kazunari Akiyoshi* 1,3 1 Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Kanda-Surugadai, Chiyoda-ku, Tokyo PREST, Japan Science and Technology Agency, oncho, Kawaguchi, Saitama Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto (Received November 22, 2010; CL ; akiyoshi@bio.polym.kyoto-u.ac.jp) Copyright The Chemical Society of Japan

2 Synthesis of imidazole-bearing polysaccharide General rganic solvents were purified, dried by a standard procedure and kept over a drying agent before use. Unless otherwise mentioned, reagents were commercially available as a guaranteed grade and used without further purification. The synthetic scheme is shown in Scheme S1. CP, which was substituted with 1.1 cholesteryl groups per 100 glucose units of the parent pullulan (M w 100,000), was obtained as previously described (K. Akiyoshi, S. Deguchi, N. Moriguchi, S. Yamaguchi, J. Sunamoto, Macromolecules 1993, 26, 3062). C 2 R C 2 R m X = CN(C 2 ) 6 NC CP R = or X: Pullulan R = 4-Nitropheynylchloroformate DMAP DMS/Pyridine C 2 R C 2 R m Y = C N 2 activated CP R =, X, or Y: activated Pullulan R = or Y istamine DMAP DMS/Pyridine 0.01 N Na C 2 R C 2 R m Z = CN(C 2 ) 2 N ImCP R =, X, or Z: ImP R = or Z N Scheme S1. Synthesis of imidazole-bearing polysaccharides. 4-Nitrophenylchloroformate activation A typical example is as follows: For activation of CP, 4-nitrophenylchloroformate

3 (2.41 mg, 12 mmol) was added to a solution of CP (2.0 g, 12 mmol equivalent of anhydroglucoside units) in 80 ml of dry DMS/pyridine mixture (1/1 by vol.) at 0 C. To this solution was added 4-dimethylaminopyridine (DMAP, 60 mg, 0.50 mmol) as a catalyst for acylation. The reaction mixture was stirred for 4.0 h at 0 C and subsequently added to an excess of absolute ethanol. The white precipitate was collected and washed with an excess of ethanol and finally dried overnight under vacuum at 25 C. Activated CP (5 10 mg) was dissolved in 10 ml of 0.1 N Na. The concentration of p-nitrophenyl groups substituted to CP was spectrophotometrically determined on a itachi U-3300 spectrophotometer (Tokyo, Japan). The 4-nitrophenolate moiety had an absorption maximum at 400 nm and a molar absorption coefficient ε 400 = L mol 1 cm 1. The degree of substitution was 25 mol%. IR (KBr): 1765 (ν; C=), 1526 (ν as ; N 2 ), 1350 (ν s ; N 2 ) and 862 cm 1 (ν; C=N) on a Shimadzu FTIR-8100S infrared spectrophotometer (Kyoto, Japan). The activation of pullulan was also performed by the same procedures, and the degree of substitution of 4-nitrophenolate moiety was determined to be 22 mol%. Synthesis of imidazole-bearing polysaccharides The obtained pullulan or CP derivative was reacted with histamine to introduce imidazolyl groups. A typical example is as follows: For imidazole-substituted CP, an activated CP derivative (1.23 g, 5.88 mmol equivalent of anhydroglucoside units; degree of activation 25 mol%) was dissolved in DMS/pyridine. To this solution, a two-fold excess of histamine (330 mg, 3.0 mm) and DMAP (30 mg, 0.25 mm) were added. The reaction mixture was stirred for 3 days at room temperature. To the reaction

4 product was added excess ethanol/diethylether mixture (1/1 by vol.) was added to the reaction product, and the mixture was stored at 4 C overnight. The white precipitates were collected and dissolved in 0.05 N Na. The resulting solution was placed in a Spectra/Por 6 dialysis bag (MW cut-off = 3500, Spectrum, ouston, TX, USA) and dialyzed against distilled water and lyophilized. The product was identified based on its IR and 1 NMR spectra: IR (KBr): 1700 cm -1 (ν; urethane C=), 1550 cm -1 (δ; N), 1 NMR (DMS d 6 /D 2 (10:1 v/v)), TMS): δ (m, 4, C 2 C 2 Im), 4.68 (s, 33 per 100 glucose units, pullulan C 1 (1-6)), 5.05 (d, 66 per 100 glucose units, pullulan C 1 (1-4)), 6.88 (s, 1, Im-5), 7.55 (s, 1, Im-2). The degree of substitution of imidazole coupled to the polysaccharides per 100 glucose units was quantitatively determined using the integrated areas of the glucopyranosyl rings and the imidazolyl group to be 21. The imidazole-bearing pullulan was also synthesized by the same procedures, and the degree of substitution of the imidazolyl groups was determined to be 23. Preparation of the nanogel solution The nanogel solution was prepared as previously reported (K. Akiyoshi, S. Deguchi, N. Moriguchi, S. Yamaguchi, J. Sunamoto, Macromolecules 1993, 26, 3062). Briefly, ImCP was suspended in Milli-Q water or buffer by stirring for 6 h at 25 C to form a turbid suspension. The suspension was then sonicated with a probe-type sonicator (UR-200P, TMY; tip diameter 2 mm) at 40 W for 10 min on ice, and filtered through a membrane filter (Sterile acrodisk 25, Gelman Science, pore size 0.22 μm) to remove dust or metallic particles shedded from the titanium transducer tip. A clear aqueous

5 nanogel suspension was obtained. Appropriate amounts of CuCl or CoCl was directly added to the nanogel suspension. For oxidation of Co(II) to obtain Co(III), 0.3% (w/w) 2 2 was added to the solution containing CoCl 2 and incubated for 30 min at 25 C. Size-Exclusion Chromatography (SEC)-Multiangle Laser Light Scattering (MALS). SEC was carried out on a chromatography system (Tosoh Co., Ltd., Tokyo, Japan) composed of a CCPD dual pump, a C-8010 column oven and a RI-8010 refractive index detector on a Q sepharose (Amersham Pharmacia Biotech., Uppsala, Sweden) connected to a MALS detector (DAWN DSP, Wyatt Technology, Santa Barbara, CA). An aqueous solution of 100 mm EPES buffer (p 7.5) containing 100 mm NaCl was used as the mobile phase. The molecular weight and the z-average root-mean-square radius of gyration were determined using ASTRA software on the basis of Zimm s equation. The increase in the refractive index (dn/dc) was determined by ptilab DSP (Wyatt Technology, Santa Barbara, CA). The value of dn/dc for ImP and ImCP was The sample solutions were passed through a 0.45 μm filter (Ekicrodisk 3, Gelman Science Japan, Ltd., Tokyo, Japan) before being applied to the column.

6 Fig. S1. IR spectrum for activated pullulan (KBr method) Fig. S2. IR spectrum for activated CP (KBr method)

7 Fig. S3. IR spectrum for ImP (KBr method) Fig. S4. IR spectrum for ImCP (KBr method)

8 Fig. S5. 1 NMR spectrum for ImP. Fig. S6. 1 NMR spectrum for ImCP.

9 Abs640 / a.u. [Imidazole unit] / [Cu(II)] Fig. S7. Changes in the absorbance at 640 nm (Abs 640 ), reflecting the complexation of ligands and metal ions, upon addition of ImP (open circle), ImCP (closed circle) and 4-methylimidazole (closed square) at 25 ºC. [Cu(II)] = 1.0 mm.

10 Fig. S8. Schematic view of the effect of cobalt ions and cyclodextrin on the association behaviour of ImCP.