| The work presented in this thesis describes the development of novel methodology for the preparation of macromolecular conjugates using proteins and dendrimers as scaffolds. This methodology is based on the discovery that chemical modification of proteins can be achieved in vacuo, in the absence of solvents, viz., water. The results obtained represent the first attempts to use this methodology to achieve protein modifications that are otherwise difficult or impossible under aqueous conditions. While the inspiration for this research stemmed from practical objectives, much of the work undertaken developed into a proof of principle, not by design, but due to the fact that there is no precedence or established theoretical base for the in vacuo chemical modification of proteins.; Initially, the scope of the project involved the design and development of a novel detoxication construct for an artificial liver consisting of an enzyme coupled to lipophilic poly(propyleneimine) dendrimers as partitioning agents for the clearing of hydrophobic compounds from aqueous solution. While the lipophilic dendrimers proved to be feasible partitioning agents when tested with some drugs in aqueous solution, attempts to attach a model enzyme to the construct by established methods in solution met with limited success. The search for better, more efficient methods of tethering, immobilizing and cross-linking enzymes lead to the investigation of alternative conjugation methods.; p-Nitrophenylchloroformate is a common activating reagent used in organic synthesis and has been used to activate chemical modifiers for proteins. However, this reagent has not been used for direct chemical modification of proteins because of its insolubility in water. The results obtained show that this reagent, when used with the in vacuo procedure, can activate and cross-link proteins through chemical modification of protein carboxyl groups. In the course of these studies it was observed that a covalent dimer was present in ribonuclease A heated in the absence of reagent. This observation lead to an investigation which demonstrated that proton transfers between interacting carboxylate and ammonium groups in vacuo results in the formation of amide bonds.; Compared to solution methods, the in vacuo methods developed are experimentally simple, and may also be carried out without the use of reactive chemical reagents. Glycation of protein amino groups with reducing monosaccharides can readily be achieved by the formation of stable ketoamine derivatives using the in vacuo methodology. (Abstract shortened by UMI.)... |
