Coordination‐Assisted Bioorthogonal Chemistry: Orthogonal Tetrazine Ligation with Vinylboronic Acid and a Strained Alkene

Abstract

Bioorthogonal chemistry can be used for the selective modification of biomolecules without interfering with any other functionality that might be present. Recent developments in the field include orthogonal bioorthogonal reactions to modify multiple biomolecules simultaneously. During our research, we observed that the reaction rates for the bioorthogonal inverse-electron-demand Diels-Alder (iEDDA) reactions between nonstrained vinylboronic acids (VBAs) and dipyridyl-s-tetrazines were exceptionally higher than those between VBAs and tetrazines bearing a methyl or phenyl substituent. As VBAs are mild Lewis acids, we hypothesised that coordination of the pyridyl nitrogen atom to the boronic acid promoted tetrazine ligation. Herein, we explore the molecular basis and scope of VBA-tetrazine ligation in more detail and benefit from its unique reactivity in the simultaneous orthogonal tetrazine labelling of two proteins modified with VBA and norbornene, a widely used strained alkene. We further show that the two orthogonal iEDDA reactions can be performed in living cells by labelling the proteasome by using a nonselective probe equipped with a VBA and a subunit-selective VBA bearing a norbornene moiety.

Two‐Step Bioorthogonal Activity‐Based Protein Profiling of Individual Human Proteasome Catalytic Sites

“…This led to the development of potential two‐step ABPs 8 – 14 (Figure ) as strained, electron‐rich alkenes for bioorthogonal labeling using iEDDA as the ligation strategy. Compound 12 was included in the research of bioorthogonal iEDDA reactions between non‐strained vinylboronic acids and pyridyl‐substituted tetrazines …”

“…In case the inhibitors are covalent and irreversible, they can be modified to contain reporter entities for activity‐based proteasome profiling purposes. Recently, we disclosed a set of inhibitors selective for one of each of the six catalytic subunits of human constitutive proteasomes and immunoproteasomes (Figure ),– whereas activity‐based proteasome probes have also been reported by other research groups –. With this set of subunit‐selective inhibitors 1 – 7 (Figure A), each human proteasome activity can be inhibited at will.…”

Isomeric triazines exhibit unique profiles of bioorthogonal reactivity

“…4,17 Such mutually orthogonal reagents are in demand for chemical tagging of multiple biomolecules. [5][6][7][8][9][10][11][12][13][14] We recently reported that a 6-substituted 1,2,4-triazine constitutes a new privileged scaffold (Fig. 1A).…”

“…In fact, there are only a handful of bioorthogonal reaction pairs that can be used simultaneously. [3][4][5][6][7][8][9][10][11][12][13][14][15][16] We are developing "privileged" scaffolds that not only meet the requirements for bioorthogonality, but are also compatible with existing reagents to enable tandem application. 4,17 Such mutually orthogonal reagents are in demand for chemical tagging of multiple biomolecules.…”

“…18 This result is in sharp contrast to related tetrazine motifs, which react vigorously with a variety of alkenes and alkynes. 12,13,[20][21][22][23][24][25][26][27][28] To expand upon the unique features of triazines, we aimed to synthesize and examine the reactivities of alternatively substituted rings. 1,2,4-Triazines react with dienophiles to form new bonds across C3 and C6.…”