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Synlett 2003(9): 1327-1330
DOI: 10.1055/s-2003-40329
LETTER
© Georg Thieme Verlag Stuttgart ˙ New York

Synthetic Analogs of the Lewisb-Tetrasaccharide and their Binding to Griffonia Simplicifolia Lectin-Part I

Vivekanand Kamath, Joanna Sadowska, Shahul Nilar, David R. Bundle, Ole Hindsgaul*
Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada
e-Mail: ole.hindsgaul@ualberta.ca;
Further Information

Publication History

Received 12 March 2003
Publication Date:
30 June 2003 (online)

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Abstract

The lewis b tetrasaccharide binds specifically to the Griffonia simplicifolia lectin GS-IV. In an effort to obtain tighter binding derivatives, we have synthesized a series of analogs of this tetrasaccharide and studied their binding to the lectin. This paper (part I in a series of two) describes the synthesis of and binding studies with analogs where the galactose unit (b) has been altered at the 6-position. Even though this position does not make direct contact with the protein in the crystal structure, the binding was very sensitive to these substitutions.

Key words

lewisb-tetrasaccharide - lectins - galactose unit

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100 g of peeled and finely ground seeds were stirred with 200 mL of MeOH (5 × 15min) and CH2Cl2 (3 × 15 min). The filtrates were discarded and the powder was dried overnight at r.t. All subsequent procedures were carried out at 4 °C. The dry meal (54 g) was extracted with 400 mL P1 buffer for 2 h with gentle stirring. The extract was centrifuged for 1 h at 12k rpm and the sediment was re-extracted with P1 buffer (400 mL) overnight. The aqueous extracts were combined and solid ammonium sulfate was added to 30% saturation. The mixture was gently stirred for 2 h. The precipitated proteins were spun (12k rpm, 20 min) and then discarded. More ammonium sulfate was added to 75% saturation and left stirring overnight. The precipitate was collected by centrifugation, suspended in 25 mL P1 buffer and dialyzed against P1 buffer. The extract was loaded on the Synsorb-Leb affinity column (30 g) and the column was washed with P1 buffer until the OD280 reading was less than 0.02. The lectin was eluted with 0.05 M carbonate/bicarbonate buffer pH 10 with 0.015M NaCl (yield 26mg). The lectin was dialyzed against P2 buffer, and then concentrated using a Centriprep10 (Amicon). The concentrated lectin was loaded on Synsorb A affinity column (30 g) and recovered flow through was then run through Synsorb B affinity column (30 g). In both cases P2 buffer was used as eluent. The purity of lectin obtained was confirmed on SDS-PAGE with 2% 2ME where two protein bands were observed of apparent molecular weights of approximately 28 and 26 KD. (P1 buffer: 0.08 M Na2HPO4, 0.02 M KH2PO4, 0.15 M NaCl, 0.015 M NaN3, 0.5 mM Na2S2O4, 0.14 mM CaCl2. P2 buffer: 0.072 M Na2HPO4, 0.028 M NaH2PO4, 0.15 M NaCl, 3 mM NaN3, 0.1 mM CaCl2, and 0.1 mM MnCl2). pH of both buffers was adjusted to 7.2.