Bioorthogonal labelling of living bacteria using unnatural amino acids containing nitrones and a nitrone derivative of vancomycin

Abstract

Unnatural D-amino acids bearing endocyclic nitrones were developed for live-cell labelling of the bacterial peptidoglycan layer. Metabolic incorporation of D-Lys and D-Ala derivatives bearing different endocyclic nitrones was observed in E. coli, L. innocua, and L. lactis. The incorporated nitrones of these bacteria then rapidly underwent strain-promoted alkyne-nitrone cycloaddition (SPANC) reactions affording chemically modified bacteria.

Cycloadditions of Trans‐Cyclooctenes and Nitrones as Tools for Bioorthogonal Labelling

“…The highly reactive tetrazine and TCO scaffolds can be used by pairing them with smaller, alternative reactive tags. We have previously shown that nitrones are small enough to be tagged onto unnatural amino acids (UAAs) and glycans, which are then readily taken up into the peptidoglycan wall and cell membrane, respectively . As the UAAs bearing nitrones proved to be stable and demonstrated robust incorporation, our work expands the bioorthogonal labelling toolbox to incorporate fluorophores bearing a s‐TCO moiety.…”

“…Nitrones have been shown to be excellent 1,3‐dipoles as they react with a number of bioorthogonal partners, and demonstrate rates faster than the parallel reactions with azides . Moreover, cyclic nitrones have the advantage of providing additional sites for manipulating strain and electronics, and are stable under both acidic and basic aqueous conditions . Similar to cyclopropenes and azides, nitrones are small and can overcome issues of diene size, hydrophobicity and stability; factors limiting the use of tetrazines.…”

“…We have previously shown that nitrones are small enough to be tagged onto unnatural amino acids (UAAs) and glycans, which are then readily taken up into the peptidoglycan wall and cell membrane, respectively. [28,34] As the UAAs bearing nitrones proved to be stable and demonstrated robust incorporation, our work expands the bioorthogonal labelling toolbox to incorporate fluorophores bearing a s-TCO moiety. The bioorthogonal potential of the TCO-nitrone cycloaddition was investigated by using unnatural amino acids bearing cyclic nitrones and an Alexa-488 fluorophore appended to s-TCO.…”

“…[13] Moreover, cyclic nitrones have the advantage of providing additional sites for manipulating strain and electronics, and are stable under both acidic and basic aqueous conditions. [28,29] Similar to cyclopropenes and azides, nitrones are small and can overcome issues of diene size, hydrophobicity and stability; factors limiting the use of tetrazines. Probing metabolic processes can be achieved by incorporating unnatural functionality using traceless reagents bearing bioorthogonal reactive groups that can be further tagged for analysis.…”

Conferring Phosphorogenic Properties on Iridium(III)‐Based Bioorthogonal Probes through Modification with a Nitrone Unit

“…[7] Nitrone-modified biomolecules have been applied to sitespecific modification of proteins [8] and labeling of mammalian cell surfaces [9] and bacterial cell walls. [10] Second, C = N isomerization is known to provide af acile non-radiative deactivation pathway for fluorescent [11] and phosphorescent [12] compounds.T hus,w hen the isomerization of the C= Ng roup is inhibited, these compounds are expected to resume their emission behavior. To our knowledge,n itrone derivatives have never been explored for the construction of bioorthogonal probes.…”