Recent Advances in Bioorthogonal Ligation and Bioconjugation

Abstract

The desire to create biomolecules modified with functionalities that go beyond nature’s toolbox has resulted in the development of biocompatible and selective methodologies and reagents, each with different scope and limitations. In this overview, we highlight recent advances in the field of bioconjugation from 2016 to 2023. First, (metal-mediated) protein functionalization by exploiting the specific reactivity of amino acids will be discussed, followed by novel bioorthogonal reagents for bioconjugation of modified biomolecules.

Ultrafast Au(III)-Mediated Arylation of Cysteine

“…46,48,53−55 Importantly, the mechanistic landscape of these organometallic arylation reagents is distinct from their organic counterparts, which often rely on S N Ar, S N 2, or Michael additions, which can be kinetically slow, reversible, or both. 6,20,26,56 Previously developed Pd(II) and Au(III) organometallic OA reagents can selectively transfer aryl groups onto Cys residues in peptides and proteins under mild, aqueous conditions, reach reaction completion within minutes, and display high chemoselectivity for Cys compared with other nucleophilic residues, even when used in excess. 31,32,34,[40][41][42]44,57 These reagents can be synthesized in one step, are air-stable, and show little to no decrease in reactivity after months of storage on the bench.…”

“…By decoupling the slow OA from the significantly faster subsequent elementary steps, many of the challenges associated with the compatibility of biological systems and transition metal-mediated catalytic processes (e.g., the use of organic solvents, elevated temperatures, rigorously anaerobic and/or moisture-free conditions, high catalyst loading, and careful selection of ligands) are simplified or eliminated. − Mechanistically, the overall Cys arylation process facilitated by organometallic OA complexes is hypothesized to rely on the combination of two elementary steps: bioconjugation via Cys thiol coordination to the metal center and subsequent reductive elimination to produce highly stable S–C aryl bonds. Together, these two mechanistic steps are generally assumed to be fast and effectively irreversible under the reaction conditions required for Cys modification. ,,− Importantly, the mechanistic landscape of these organometallic arylation reagents is distinct from their organic counterparts, which often rely on S N Ar, S N 2, or Michael additions, which can be kinetically slow, reversible, or both. ,,, …”

Study of uricase-polynorbornene conjugates derived from grafting-from ring-opening metathesis polymerization

“…‘PEGylation’ or covalently modifying proteins with hydrophilic polyethylene glycol (PEG), a technique pioneered by Abuchowski et al in 1977 has been the golden standard in addressing these challenges. 4–6 PEGylation enhances protein solubility and stability while mitigating immunogenicity. 7–9 As of October 2023, there are 28 FDA approved PEGylated protein therapeutics used for the treatment of diseases such as cancer, diabetes and hepatitis and many more in various stages of development.…”

“…Advancements in new chemical and biochemical technologies including controlled polymerizations, conjugation chemistries 6,31–33 and genetic engineering (XTEN, 34 PASylation®, 35,36 HESylation 37,38 ) have facilitated developments of new safer and effective approaches beyond PEGylation. 23,39 Among these, controlled polymerization techniques such as atom transfer radical polymerization (ATRP), reversible-addition fragmentation chain transfer (RAFT), ring-opening metathesis polymerization (ROMP) and nitroxide mediated polymerization (NMPs) have enabled the synthesis of polymers with low dispersity, allowing precise control over chemical functionality, properties, architecture and molecular weights.…”

Facile Access to Branched Multispecific Proteins

“…Advances in bioconjugation have facilitated the generation of highly homogeneous protein–protein, protein–peptide, and protein–small-molecule chimeras. − Importantly, the site-specific generation of such chimeras has enabled the production of fusion proteins that would be inaccessible through biosynthesis alone. For example, bespoke protein–protein conjugates, joined N-to-N, C-to-C, or via nonterminal residues, can be produced with relative ease .…”