Two Rapid Catalyst-Free Click Reactions for In Vivo Protein Labeling of Genetically Encoded Strained Alkene/Alkyne Functionalities

Abstract

Detailed kinetic analyses of inverse electron-demand Diels–Alder cycloaddition and nitrilimine-alkene/alkyne 1,3-diploar cycloaddition reactions were conducted and the reactions were applied for rapid protein bioconjugation. When reacted with a tetrazine or a diaryl nitrilimine, strained alkene/alkyne entities including norbornene, trans-cyclooctene, and cyclooctyne displayed rapid kinetics. To apply these “click” reactions for site-specific protein labeling, five tyrosine derivatives that contain a norbornene, trans-cyclooctene, or cyclooctyne entity were genetically encoded into proteins in Escherichia coli using an engineered pyrrolysyl-tRNA synthetase-tRNACUAPyl pair. Proteins bearing these noncanonical amino acids were successively labeled with a fluorescein tetrazine dye and a diaryl nitrilimine both in vitro and in living cells.

Fluorescent Probes for Tracking the Transfer of Iron–Sulfur Cluster and Other Metal Cofactors in Biosynthetic Reaction Pathways

“…An even more dramatic binding signal may be generated by placing the fluorophore near the metal binding site using artificial amino acid technology. 38 The sensitivity of labeled Fe–S acceptor proteins to cluster binding, but insensitivity to Fe 2+ /sulfide/GSH/NADPH (and low sensitivity to cysteine), along with the ability to monitor a labeled protein in the presence of unlabeled Fe–S proteins, will make these probes transformative new tools for investigating in vitro Fe–S cluster assembly reactions. Furthermore, the ability to detect other metal ions suggests that this labeling approach may also have applications in the in vitro studies of additional metal transfer reactions.…”

Phospha‐Michael Addition as a New Click Reaction for Protein Functionalization

“…[17] Strained alkenes such as norbornene, trans-cyclooctene and cyclopropene react rapidly with tetrazine or nitrilimine. [18–21] Non-strained olefins also react with tetrazine and nitrilimine readily. [12, 16] …”

Potassium Acyltrifluoroborate (KAT) Ligations are Orthogonal to Thiol‐Michael and SPAAC Reactions: Covalent Dual Immobilization of Proteins onto Synthetic PEG Hydrogels

“…We also developed and synthesized reagents for introducing the necessary reaction partners – KAT, hydroxylamine and cyclooctyne – into bovine serum albumin (BSA) as a model protein ( Scheme ) . Cyclooctyne derivatives have been previously incorporated into proteins using synthetic bioconjugation techniques such as lysine or cysteine modification as well as genetic encoding methods, where an unnatural amino acid bearing a cyclooctyne residue is inserted into a protein by cellular machinery …”