mPEG-NHS carbonates: Effect of alkyl spacers on the reactivity: Kinetic and mechanistic insights

Abstract

Nowadays, the chemical conjugation to mPEG, also known as PEGylation, is a well-recognized technology used to improvethe pharmaceutical properties of the therapeutic proteins. Over the last 20 years, more than 10 PEGylated macromolecules reached themarket with tremendous success, whereas various other bioconjugates are under advanced clinical trials. mPEG-N-hydroxysuccinimidylcarbonate is an important reagent of widespread application for the PEGylation of biomacromolecules. One of the most important challengesin this technology is the development of more selective PEGylation reagents aimed to provide more consistent polymer-proteinconjugates. One approach followed to improve the selectivity of PEGylation reagents is the design of less reactive derivatives, for example,by incorporation of alkyl spacers between the polymer chain and the terminal reactive group. In this work, we prepared a family ofmPEG?N-hydroxysuccinimidyl carbonates bearing spacers of up to 6 carbon atoms. The kinetics of hydrolysis of the carbonates wasstudied under different experimental conditions, as a straight measure of the influence of the length of the spacer on the reactivity.By DFT calculations, we propose a detailed mechanism for the hydrolysis reaction. The influence of the length of alkyl spacer on thereactivity of the carbonates and related esters is studied and discussed in detail. Finally, to further evaluate the reactivity, selectedN-hydroxysuccinimidyl carbonates were studied in the conjugation reaction of bovine lactoferrin.