Photo‐induced and Rapid Labeling of Tetrazine‐Bearing Proteins via Cyclopropenone‐Caged Bicyclononynes

Inverse electron‐demand Diels–Alder cycloadditions (iEDDAC) between tetrazines and strained alkenes/alkynes have emerged as essential tools for studying and manipulating biomolecules. A light‐triggered version of iEDDAC (photo‐iEDDAC) is presented that confers spatio‐temporal control to bioorthogonal labeling in vitro and in cellulo. A cyclopropenone‐caged dibenzoannulated bicyclo[6.1.0]nonyne probe (photo‐DMBO) was designed that is unreactive towards tetrazines before light‐activation, but engages in iEDDAC after irradiation at 365 nm. Aminoacyl tRNA synthetase/tRNA pairs were discovered for efficient site‐specific incorporation of tetrazine‐containing amino acids into proteins in living cells. In situ light activation of photo‐DMBO conjugates allows labeling of tetrazine‐modified proteins in living E. coli. This allows proteins in living cells to be modified in a spatio‐temporally controlled manner and may be extended to photo‐induced and site‐specific protein labeling in animals.     

Laser‐Initiated Radical Trifluoromethylation of Peptides and Proteins: Application to Mass‐Spectrometry‐Based Protein Footprinting

Described is a novel, laser‐initiated radical trifluoromethylation for protein footprinting and its broad residue coverage. ^.CF₃ reacts with 18 of the 20 common amino acids, including Gly, Ala, Ser, Thr, Asp, and Glu, which are relatively silent with regard to ^.OH. This new approach to footprinting is a bridge between trifluoromethylation in materials and medicinal chemistry and structural biology and biotechnology. Its application to a membrane protein and to myoglobin show that the approach is sensitive to protein conformational change and solvent accessibility.     

Vancomycin‐Dependent Response in Live Drug‐Resistant Bacteria by Metabolic Labeling

The surge in drug‐resistant bacterial infections threatens to overburden healthcare systems worldwide. Bacterial cell walls are essential to bacteria, thus making them unique targets for the development of antibiotics. We describe a cellular reporter to directly monitor the phenotypic switch in drug‐resistant bacteria with temporal resolution. Vancomycin‐resistant enterococci (VRE) escape the bactericidal action of vancomycin by chemically modifying their cell‐wall precursors. A synthetic cell‐wall analogue was developed to hijack the biosynthetic rewiring of drug‐resistant cells in response to antibiotics. Our study provides the first in vivo VanX reporter agent that responds to cell‐wall alteration in drug‐resistant bacteria. Cellular reporters that reveal mechanisms related to antibiotic resistance can potentially have a significant impact on the fundamental understanding of cellular adaption to antibiotics.