Protein conjugates of defined structure: Synthesis and use of a new carrier molecule

Abstract

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.

Reversible Covalent Chemistry in Drug Delivery

“…In subsequent work, a more elaborate linker strategy was developed with the goal of increasing the DAVLB loading per conjugate [98]. A model protein was enzymatically functionalised with a hydrazine group, which was then coupled to aldehyde functionalised peptide 5 (Fig.…”

A Fluorescent Interleukin‐8 Receptor Probe Produced by Targetted Labelling at the Amino Terminus

“…Tertbutyloxycarbonyl-aminooxyacetyl N-hydroxysuccinimide ester was prepared from aminooxyacetate (Fluka) (Villaseca et al, 1993) and 300 mg added to 100 mg (W-trifluoroacetyl-lysine) (NovaBiochem) dissolved in 1 ml dimethylsulfoxide. The apparent pH was adjusted to 8.0 with N-methyl morpholine.…”

Aminooxyacetic Acid

“…This method has been used to successfully attach clusters of potential drugs to antitumor monoclonal antibodies for use in vivo. 16 Oxime linkages have been applied to polypeptide macromolecules linked by successive generations of amino acids. One example of this has been the construction of a choleratoxin B subunit with a molecular mass of 20550 Da (eq 18).…”