Carmali, S; (2015) New Bis-Alkylation Reagents for Protein Conjugation. Doctoral thesis , UCL (University College London).

Abstract

Bis-alkylation for disulfide-bridging PEGylation has emerged as a valid strategy for protein conjugation. Proteins can be efficiently modified to add a three-carbon methylene bridge between the two sulfurs in a disulfide bond. The so-called C3 bis-sulfone reagent is a linear poly (ethylene glycol) (PEG) that has been functionalised at one terminus with a latently reactive bis-alkylation moiety capable of undergoing sequential Michael reactions. Latency is achieved by utilising leaving groups that must undergo elimination to unmask an ,-unsaturated double bond needed for Michael addition. Structural modifications of these reagents are thought to alter the solvent availability or electrophilic character of the Michael acceptor to modulate conjugation reactivity with a protein. It was therefore hypothesised that by modifying the structure of C3 bis-sulfone reagent, it would be possible to obtain reagents with different reactivity. This variable reactivity can then be exploited with bifunctional reagents to allow the preparation of protein-protein conjugates in an efficient manner. Several synthetic targets and strategies were examined to prepare different types of di-PEG and multifunctional reagents for protein conjugates. A small family of di-PEG bis-alkylating reagents with different molecular weights was prepared and the conjugation efficiency was compared to linear C3 PEG reagents of the same overall molecular weight. Molecular dynamic studies were used to understand how the PEG chain affected the linker reactivity. Results showed that PEG-linker interactions were found to be less pronounced for reagents that contained two 10 kDa PEG chains (di-PEG2×10) when compared to a linear C3 reagent with a single PEG of 20 kDa (PEG20) with the same overall molecular weight. While the presence of a second PEG chain was found to influence conjugation efficiency, the modification of the bis-alkylating Michael acceptor in C3 reagent was also explored as a means to vary reactivity. Acetylenic ketones were examined as bis-Michael acceptors in the preparation of two C1 reagents with distinct structural features (aliphatic and aromatic). An aliphatic C1 reagent was prepared without leaving groups but was found to have less reactivity when compared to an aromatic C3 reagent. Semi-empirical studies suggested that this lower reactivity could be attributed to less electron-withdrawing aliphatic structure and to stereoelectronic effects. Aliphatic C1, while less reactive was found to undergo a double Michael addition and consequently allowed re-bridging of a reduced disulfide. In contrast, aromatic C1 required leaving groups to modulate the higher reactivity observed but was not found to re-bridge a reduced disulfide. The C3 bis-sulfone reagent is known to undergo elimination much more slowly at slightly acidic pH values. This is important because conjugation will not proceed until elimination has occurred. The need for elimination was used as a basis for the synthesis of hetero-bifunctional reagents that could be used for hetero-functional protein-protein conjugates, such as bispecific Fab-PEG-Fab conjugates. For potential scalability, effort was focused on preparing mono-sulfone-PEG-bis-sulfone hetero-bifunctional reagents (MpB reagents) that could be utilised in a one-pot reaction sequence to give hetero-functional protein conjugates. Reactions using the MpB reagent showed the potential to allow the sequential conjugation of two Fab molecules by altering the pH conditions of the reaction mixture in a single reaction vessel. This variable reactivity can provide a synthetic platform for controlled sequential conjugation that can allow the efficient preparation of protein-protein conjugates.

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