Synthetic Analogs of the Lewis^b-Tetrasaccharide and their Binding to Griffonia Simplicifolia Lectin-Part I

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.

lewis^b-tetrasaccharide - lectins - galactose unit

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29

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-Le^b 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 Na₂S₂O_4, 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.

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