PROTEIN CONJUGATES OF DEFINED STRUCTURE - SYNTHESIS AND USE OF A NEW CARRIER MOLECULE

A new carrier molecule, NH2OCH2CO-(Gly)3-[Lys(H-Ser-)]5-Gly-OH, has be en synthesized to facilitate the preparation of protein conjugates of defined structure. Special features are as follows: (i)(aminooxy)-acet yl as a terminal group, which reacts specifically to form an oxime bon d under very mild conditions with an aldehyde group placed on a protei n in a prior step; (ii) a spacer group of three Gly residues; and (iii ) a set of five Lys residues, each of which is acylated with a Ser res idue. A second form of the carrier molecule, O-m-C6H4CH=NOCH2CO-(GlY)3 -[Lys(H-Ser)]5-Gly-OH,was also prepared. This form possesses a termina l aldehyde group which permits site-specific attachment by formation o f a hydrazone bond to the carboxyl termini of polypeptide chains which have been modified enzymatically with carbohydrazide in a prior step. Once the carrier is linked to protein in one of the above ways, i.e. through formation of either an oxime or hydrazone bond, the Ser residu es of the carrier (but not of the protein) may be oxidized by very mil d periodate treatment to generate aldehyde groups. Drugs possessing a hydrazide group (e.g. methotrexate gamma-hydrazide or desacetylvincale ukoblastine hydrazide) may then be conjugated via hydrazone formation to the aldehyde groups of the carrier. A cluster of five drug molecule s may thus be attached to a single site on a protein, giving a relativ ely homogeneous product in spite of the high drug conjugation ratio. S ynthesis of the carrier, formation of a pentadrug-protein conjugate, a nd wider implications of the chemistry are presented.