“Tag and Modify” Protein Conjugation with Dynamic Covalent Chemistry

Abstract

The development of small protein tags that exhibit bioorthogonality, bond stability, and reversibility, as well as biocompatibility, holds great promise for applications in cellular environments enabling controlled drug delivery or for the construction of dynamic protein complexes in biological environments. Herein, we report the first application of dynamic covalent chemistry both for purification and for reversible assembly of protein conjugates using interactions of boronic acid with diols and salicylhydroxamates. Incorporation of the boronic acid (BA) tag was performed in a site-selective fashion by applying disulfide rebridging strategy. As an example, a model protein enzyme (lysozyme) was modified with the BA tag and purified using carbohydrate-based column chromatography. Subsequent dynamic covalent "click-like" bioconjugation with a salicylhydroxamate modified fluorescent dye (BODIPY FL) was accomplished while retaining its original enzymatic activity.

Boronic acids as building blocks for the construction of therapeutically useful bioconjugates

“…27). 155 BA-SHA linkage has also found valid applications with smaller protein fragments, functioning as a linker unit for the attachment of a nucleic acid payload to tumour-targeting peptides.…”

2nd PSL Chemical Biology Symposium (2019): At the Crossroads of Chemistry and Biology

“…By reversible complex formation,with diols or hydroxamic acids, these functions work as at ag. [25] Engineered amino acids containing such motifs provide interesting opportunities for dynamic sequence-specific recognition of peptides. Using these amino acids, ac ore-shell zymogen associating ab oronic acid-modified enzyme with ac omplementary dendron was synthesized; [26] theses tructures are able to control enzymatic activity through pH modulation.…”

“…Using this method, proteins have been coupled with small molecules containing one boronic acid. By reversible complex formation, with diols or hydroxamic acids, these functions work as a tag . Engineered amino acids containing such motifs provide interesting opportunities for dynamic sequence‐specific recognition of peptides.…”

Protein‐inorganic calcium‐phosphate supraparticles as a robust platform for enzyme co‐immobilization

“…Affinity binding pairs or charged domains can lead to more predictable binding events with the immobilization support, but such sequences are not available in all enzymes. Covalent immobilization of enzyme on the material can be achieved by utilizing reactive natural or nonnatural amino acids in the protein (Al‐Lolage, Meneghello, Ma, Ludwig, & Bartlett, 2017; Meldal & Schoffelen, 2016; Wang, 2017; Xiang et al, 2014; Zegota et al, 2018). Most covalent interactions are irreversible, which minimizes undesired leaching of enzyme, but renders the immobilization support useless if the enzyme loses activity (Sadana, 1988; Sheldon & Woodley, 2018).…”

“…Using fusion proteins, an affinity binding domain may be directly added to the terminus of an enzyme, spatially decoupling catalytic activity and binding. The affinity binding partner may be incorporated on the immobilization material via random adsorption, covalent chemistry, or by composing the material of these domains (Banta et al, 2010; Bolivar et al, 2016; Park & Champion, 2016; Shen et al, 2005; Zegota et al, 2018). With this design strategy, it is possible to create a reversible and modular immobilization scheme that can be applied to a variety of supports and enzymes.…”