Abstract
3-arylpropiolonitriles (APN) are promising alternatives to maleimide for chemo-selective thiol conjugation, because the reaction product has a remarkably hydrolytic stability compared with that of thiol-maleimide reactions in vitro. However, whether cysteine modification with APN enhances stability in vivo compared to thiol-maleimide reactions remains unclear, probably due to the too short in vivo serum half-life of a protein to observe significant cleavage of thiol-maleimide/-APN reaction products. The conjugation of human serum albumin (HSA) to a therapeutic protein reportedly prolongs the in vivo serum half-life. To evaluate the in vivo stability of the thiol-APN reaction product, we prepared HSA-conjugated Arthrobacter globiformis urate oxidase (AgUox), a therapeutic protein for gout treatment. Site-specific HSA conjugation to AgUox was achieved by combining site-specific incorporation of tetrazine containing an amino acid (frTet) into AgUox and a crosslinker containing trans-cyclooctene and either thiol-maleimide (AgUox-MAL-HSA) or -APN chemistry (AgUox-APN-HSA). Substantial cleavage of the thioester of AgUox-MAL-HSA was observed in vitro, whereas no cleavage of the thiol-APN product of AgUox-APN-HSA was observed. Furthermore, the in vivo serum half-life of AgUox-APN-HSA in the late phase was significantly longer than that of AgUox-MAL-HSA. Overall, these results demonstrate that the thiol-APN chemistry enhanced the in vivo stability of the HSA-conjugated therapeutic protein.
Highlights
IntroductionPublisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations
To evaluate the in vivo stability of the thiol-APN reaction product, we investigated the in vivo stability of a human serum albumin (HSA)-therapeutic protein conjugate
We previously reported that site-specific albumin conjugation to the permissive site of a protein results in in vivo serum half-life extension without loss of activity [30,31]
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. The thiol group (-SH) has been widely used for the chemical modification of biomolecules [1,2,3,4,5]. Because cysteine residues of proteins are relatively rarer than other reactive amino acids, such as lysines, they are often used for site-specific modifications [6,7,8,9,10]. Maleimides are widely used as the reaction partners of cysteines of proteins [11,12,13,14]. The thiol-maleimide reaction generates a thioether bond at a pH range of
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