Development Of Novel Protein Modification Methods For Protein-Polymer Coupling Studies

Proteins with non-natural functions play a critical role in many frontier researches.Proteins which carrying isotopes or imaging tags are widely used in the field of biomedicine.Latterly much attention has also been focused on the enzyme immobilization and polymerization in biomass conversion.Therefore,the development of simple and efficient modification reactions to obtain more novel modified peptides and proteins has become a cross-disciplinary research.In the last few years,significant advances have been made in protein modification through the use of hydrazone ligation strategy and diaminodiacid strategy.For hydrazone ligation strategy,it has been suffering from the slow kinetics under physiological conditions and catalyst toxicity.Although promising results have been derived by the use of fast-reacting substrates,this catalyst-free concept has not been applied to the application of protein modification.For the diaminodiacid strategy,design and synthesize cystine derivative diaminodiacid is the key.However,the types of these derivatives are limited and these compounds can only be used under drastic conditions.These disadvantageous factors are detrimental to the further application of diaminodiacid strategy in protein modification.In order to overcome these problems and improve the usability and practicability of these two strategies in protein modification,we carried out the two main researches in this doctoral dissertation.The first part explores the catalyst-free imine formation and develops the catalyst-free bioorthogonal hydrazone ligation via ortho-halo-substituted benzaldehyde.This new strategy permits catalyst-free hydrazone ligation of proteins for the first time.Two important proteins,ubiquitin and autophagy-related protein LC3,are labeled with fluorophores.However,we only obtained fluorophore-modified proteins using this initial version of the catalyst-free hydrazone ligation and didn't achieve complex molecule modified proteins.Obviously,the conjugation between complex molecules(polymers,drugs,etc)and proteins are more practical.Therefore,we designed a new ortho-halo-benzaldehyde reagent to solve the problem of poor modifiers scope.It is expected to be robust and useful functionalization while maintaining the reactivity.The fluorophore/PEG/Drug modified proteins were successfully obtained.This study provides a solid foundation for the preparation of polymer-modified cellulose.The second part focuses on generation of new types of cystine derivatives which called diaminodiacids and can be used in mild reaction conditions.Different linkages and protected groups were introduced to four diaminodiacids.These diaminodiacids have versatile linkages and mild deprotection conditions compared to the previously reported diaminodiacids.Moreover,these new compounds are compatible with solid phase peptide synthesis.In addition,we successfully synthesized oxytocin derivatives using the new diaminodiacid,indicating that it is suitable for peptide/protein modification.This study provides some new user-friendly diaminodiacids and extends the scope of diaminodiacid strategy.