A. Miyagawa et al.
Bull. Chem. Soc. Jpn. Vol. 79, No. 2 (2006)
355
stirred at 0 ꢂC for 30 min. The mixture was concentrated. Purifica-
tion by silica-gel column chromatography with 2:1 (v/v) chloro-
form–methanol was carried out on the residue to give 8 (481
mg, 55.8%): 1H NMR (D2O) ꢅ 6.26 (brd, 1H, –CH=C), 6.17 (brd,
1H, –C=CH), 5.74 (brd, 1H, –C=CH), 3.26 (brs, 2H, –CH2–N–
C–), 2.98 (brs, 2H, N–CH2–), 1.66 (brs, 2H, –CH2–C–N–), 1.55
(brs, 2H, –CH2–), 1.38 (brs, 4H, {CH2{ ꢄ 2).
to a carboxymethylated cellulose membrane was achieved by the
condensation reaction. Carboxymethylated cellulose membranes
(4 pieces) were soaked in a solution containing 1-[3-(dimethyl-
amino)propyl]-3-ethylcarbodiimide hydrochloride (11.4 mg) in
water (20 mL) and the mixture was stirred for 2 h at room temper-
ature. The glycoconjugate polymers (10 mg) were then dissolved
in water (10 mL) and added to the mixture. The mixture was stir-
red overnight at room temperature. The membranes were filtered,
washed thoroughly, and dried in air. Alternatively, the filtrate was
concentrated and dialyzed. After dialysis, the solution was lyophi-
lized and the amount of the polymers that had not been immobi-
lized to the membranes was determined. The dried membranes
were observed under an ultraviolet lamp (254 nm) to confirm
whether the polymers had been immobilized to the cellulose mem-
branes. The same procedure was performed for each polymer.
Protein Adsorption Assay. Protein quantification was per-
formed using a Bio-Rad Protein Assay based on the Bradford
method. The absorbance of protein solutions containing reagents
was measured at 590 nm. Comparison to a standard curve provid-
ed a relative estimate of protein concentration.
N-[6-(N-Acryloylaminohexyl)]-5-dimethylamino-1-naphtha-
lenesulfonamide (9):
Hexamethylenediamine (4.31 g, 37.1
mmol) was dissolved in 1,4-dioxane (100 mL) and the solution
was cooled to 0 ꢂC. After adding dansyl chloride (1.00 g, 3.71
mmol), the solution was stirred overnight at 0 ꢂC. The mixture
was concentrated and the residue was extracted with chloroform.
The chloroform solution was dried over anhydrous magnesium
sulfate, filtered, and evaporated in vacuo. The syrupy residue
was then dissolved in THF (60 mL) and sodium carbonate (789
mg, 7.44 mmol) was added to the mixture at 0 ꢂC. Acryloyl chlo-
ride (909 mL, 7.44 mmol) was added dropwise to the solution, and
the mixture was stirred at room temperature for 1 h. The mixture
was concentrated and extracted with chloroform. The chloroform
layer was washed successively with aqueous sodium hydrogencar-
bonate and brine, dried over anhydrous magnesium sulfate, fil-
tered, and evaporated in vacuo. Purification by silica-gel column
chromatography with 50:1 (v/v) of chloroform–methanol was car-
ried out on the residue to give 9 (1.12 g, 74.7%): 1H NMR
(CDCl3) ꢅ 8.54 (d, 1H, H-7), 8.30 (d, 1H, H-9), 8.23 (d, 1H, H-2),
7.54 (m, 2H, H-3,8), 7.19 (d, 1H, H-4), 6.27 (d, 1H, –CH=C),
6.10 (m, 1H, –C=CH), 5.78 (brs, 1H, –NH–CO–), 5.60 (d, 1H,
–C=CH), 3.23 (m, 2H, –CH2–N–S–), 2.88 (m, 8H, –CH2–N–C–,
{CH3 ꢄ 2), 1.37 (m, 4H, –CH2–C–N–, –CH2–C–S–), 1.18 (m, 4H,
{CH2{ ꢄ 2).
The cellulose membranes with immobilized glycoconjugate
polymers were set in the filter holder, and 200 mg mLꢃ1 ConA
or RCA120 PBS(ꢃ) solution (2 mL) was allowed to flow through
the holder. The holder was then washed with PBS(ꢃ) solution
(3 mL) and subsequently eluted with 0.2 M D-mannose, or D-lac-
tose, in PBS(ꢃ) solution (5 mL). The protein content of each of
the solutions was assayed using Bio-Rad Protein Assay reagents.
References
´
R. Axen, J. Porath, Nature 1966, 23, 367.
W. C. Lee, C. C. Hsiao, R. C. Ruaan, J. Chem. Technol.
1
2
Copolymerization. Solution of the glycosyl monomer 3 or 7,
amine monomer 8, fluorescent monomer 9, or acrylamide in
deionized water or DMSO was degassed using a diaphragm pump,
and TEMED (0.1 equiv for glycosyl monomer) and APS (0.04
equiv for glycosyl monomer) were added to the solution. The
reaction mixture was continuously stirred overnight at room tem-
perature. The resulting product was purified by reprecipitation
with a mixed solution of methanol and water and lyophilized to
give a water-soluble copolymer as a powder. This procedure
was performed for each of the monomer ratios in Table 1.
Modification of Membrane. Carboxymethylation of the cel-
lulose membrane was achieved by the addition of bromoacetic
acid. Cellulose membranes (20 pieces) were soaked in a mixture
of a 1, 3, and 6 M NaOH solution (30 mL) and saturated NaCl
solution (30 mL), and then stirred for 1 h at room temperature.
To the reaction mixture was added bromoacetic acid (0.10, 0.50,
1.0, or 2.0 g) and the mixture was then stirred overnight at room
temperature. Next, the membranes were washed thoroughly and
dried in air.
Biotechnol. 1995, 64, 66.
3
878.
4
I. Chibata, T. Tosa, T. Sato, Appl. Microbiol. 1974, 27,
G. Broun, D. Thomas, G. Gellf, D. Domurado, A. M.
Berjonneau, C. Guillon, Biotechnol. Bioeng. 1973, 15, 359.
N. Weliky, F. S. Brown, E. C. Dale, Arch. Biochem. Bio-
phys. 1969, 131, 1.
5
6
7
W. Guo, E. Ruchenstein, J. Membr. Sci. 2003, 211, 101.
W. Brummer, N. Hennrich, M. Klochow, H. Lang, H. D.
¨
Orth, Eur. J. Biochem. 1972, 25, 129.
T. Sato, T. Mori, T. Tosa, I. Chibata, Arch. Biochem. Bio-
phys. 1971, 147, 778.
8
9 J. Lui, J. Wang, L. G. Bachas, D. Bhattacharrya, Biotech-
nol. Prog. 2001, 17, 866.
10 E. A. Peterson, H. A. Sober, J. Am. Chem. Soc. 1956, 78,
751.
11 D. Stollner, F. W. Scheller, A. Warsinke, Anal. Biochem.
¨
2002, 304, 157.
Determination of the Carboxyl Content in the Cellulose
Membrane. The carboxyl content in the membrane was deter-
mined by titration. Initially, 4 pieces of the modified cellulose
membranes were soaked for 30 min in a 1 M HCl solution. The
membranes were washed thoroughly and cut in small pieces and
placed in a 25 mL flask, and then water was added. Several drops
of 0.04% phenolphthalein indicator were added to the flask. The
solution containing the pieces of membrane was then titrated with
a 2.0 mM NaOH solution. The concentration of the NaOH solution
used was calibrated by titration with a commercial 0.1 M acetic
acid solution.
12 S. M. C. Ritchie, L. G. Bachas, T. Olin, S. K. Sikdar, D.
Bhattacharyya, Langmuir 1999, 15, 6346.
13 W. Guo, E. Ruchenstein, J. Membr. Sci. 2002, 197, 53.
14 E. Klein, J. Membr. Sci. 2000, 179, 1.
15 W. B. Turnbull, J. F. Stoddart, Rev. Mol. Biotechnol. 2002,
90, 231.
16 R. Roy, Curr. Opin. Struct. Biol. 1996, 6, 692.
17 Y. C. Lee, R. T. Lee, Acc. Chem. Res. 1995, 28, 321.
18 S. Elgavish, B. Shaanan, Trends Biochem. Sci. 1997, 22,
462.
´
19 R. Auzely, M. Cristea, M. Rinaudo, Biomacromolecules
2002, 3, 998.
Immobilization. Immobilization of glycoconjugate polymers