A new carrier molecule, NH2OCH2CO-(Gly)3-[Lys(H-Ser-)]5-Gly-OH, has been synthesized to facilitate the preparation of protein conjugates of defined structure. Special features are as follows: (i) (aminooxy)-acetyl as a terminal group, which reacts specifically to form an oxime bond under very mild conditions with an aldehyde group placed on a protein 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 residue. A second form of the carrier molecule, HCO-m-C6H4CH = NOCH2CO-(Gly)3-[Lys(H-Ser)]5-Gly-OH, was also prepared. This form possesses a terminal aldehyde group which permits site-specific attachment by formation of 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 residues of the carrier (but not of the protein) may be oxidized by very mild periodate treatment to generate aldehyde groups. Drugs possessing a hydrazide group (e.g. methotrexate gamma-hydrazide or desacetylvincaleukoblastine hydrazide) may then be conjugated via hydrazone formation to the aldehyde groups of the carrier. A cluster of five drug molecules may thus be attached to a single site on a protein, giving a relatively homogeneous product in spite of the high drug conjugation ratio. Synthesis of the carrier, formation of a pentadrug-protein conjugate, and wider implications of the chemistry are presented.
A two-step approach to the production of well-defined protein conjugates is described. In the first step, a linker group, carbohydrazide, having unique reactivity (a hydrazide group) is attached specifically to the carboxyl terminus by using enzyme-catalyzed reverse proteolysis. Since the hydrazide group exists nowhere else on the protein, specificity is assured in a subsequent chemical reaction (formation of a hydrazone bond) of the modified protein with a molecule (chelator, drug, or polypeptide) carrying an aldehyde or keto group. The product is sufficiently stable at neutral pH, no reduction of the hydrazone bond being necessary for the hydrazones described. Protein modification is thus restricted to the carboxyl terminus and a homogeneous product results. With insulin as a model, conditions are described for producing such well-defined conjugates in good yields. The use of other linker groups besides carbohydrazide, and applications of these techniques to antibody fragments, are discussed.
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