Controlled primary functionalization of agarose affinity supports by carboxymethylation and subsequent addition of...

Experimental details are given for the O-carboxymethylation of cross-linked agarose beads with chloroacetate in aqueous sodium hydroxide, formation of a tandem 2-amino-ethylamide derivative, and coupling of beta-alanine residues to the aminoethyl groups using a new reagent, N-phthalimidyl N'-ethylsulfonylethoxycarbonyl-beta-alaninate. An ideal support for attaching affinity ligands results. Extent of derivatization was controlled by the time and temperature of the first reaction. Functional group densities were estimated by using hydrogen ion binding measurements for carboxyls and a new reagent, N-succinimidyl 3-[2-(4-nitrophenylamino)ethyldithio]propionate, for assaying reactive amino groups on solid supports. Synthesis and use of the new reagents are described.