In the preceding paper [Mandal, D. K., Kishore, N., & Brewer, C. F. (1994) Biochem.] the trisaccharide 3,6-di-O-(a-D-mannopyranosyl)-D-mannose, which is present in all asparagine-linked carbohydrates, was shown by titrn. microcalorimetry to bind to the lectin Con A with nearly -6 kcal mol-1 greater enthalpy change (DH) than Me a-D-mannopyranoside (MeaMan). These results indicate that Con A possesses an extended binding site for the trisaccharide. In the present paper, the authors have investigated the binding of a series of synthetic analogs of the Me a-anomer of the trisaccharide using hemagglutination inhibition, solvent proton magnetic relaxation dispersion (NMRD), near UV CD, and titrn. microcalorimetry measurements. Four of the analogs tested possess an a-glucosyl or a-galactosyl residue substituted at either the a(1-6) or a(1-3) position. Anal. of the data indicates that the a(1-6) residue of the parent trimannoside binds to the so-called monosaccharide site and the a(1-3) residue to a weaker secondary site. Binding at the secondary site involves unfavorable interactions of the 2-equatorial hydroxyl of the a(1-3)Glc deriv. since this analog binds with 12-fold lower affinity and -3.4 kcal mol-1 lesser DH than the trimannoside, whereas the a(1-3)-2-deoxyGlc analog possesses essentially the same affinity and DH as the trimannoside. NMRD data show that the a(1-3) 2-, 3-, 4-, and 6-deoxy derivs. of the trimannoside induces essentially the similar conformational changes in the protein as that of the parent trimannoside. However, the calorimetry data show that only the 3-deoxy analog binds with .apprx.10-fold lower affinity and -3.4 kcal mol-1 lesser enthalpy change. This indicates that the 3-hydroxyl of the a(1-3)Man makes a specific hydrogen bond with the protein at a secondary binding site. The DH of -11 kcal mol-1 for the 3-deoxy analog is still, however, greater than that of MeaMan (-8.4 kcal mol-1), which indicates another site of contact between the trimannoside and Con A, most likely with the \"core\" Man residue. Thus, Con A has an extended binding site which includes a high-affinity site that recognizes the 3-, 4-, and 6-hydroxyl groups of the a(1-6)Man residue of the trimannoside (the monosaccharide site), a lower affinity site that binds the 3-hydroxyl of the a(1-3)Man residue, and a third site which appears to involve the \"core\" Man residue. [on SciFinder (R)]