Synthesis and Evaluation of Novel Ring-Strained Noncanonical Amino Acids for Residue-Specific Bioorthogonal Reactions in Living Cells.

Bioorthogonal reactions are ideally suited to selectively modify proteins in complex environments, even in vivo. Kinetics and product stability of these reactions are crucial parameters to evaluate their usefulness for specific applications. Strain promoted inverse electron demand Diels-Alder cycloadditions (SPIEDAC) between tetrazines and strained alkenes or alkynes are particularly popular, as they allow ultrafast labeling inside cells. In combination with genetic code expansion (GCE)-a method that allows to incorporate noncanonical amino acids (ncAAs) site-specifically into proteins in vivo. These reactions enable residue-specific fluorophore attachment to proteins in living mammalian cells. Several SPIEDAC capable ncAAs have been presented and studied under diverse conditions, revealing different instabilities ranging from educt decomposition to product loss due to ß-elimination. To identify which compounds yield the best labeling inside living mammalian cells has frequently been difficult. In this study we present a) the synthesis of four new SPIEDAC reactive ncAAs that cannot undergo ß-elimination and b) a fluorescence flow cytometry based FRET-assay to measure reaction kinetics inside living cells. Our results, which at first sight can be seen conflicting with some other studies, capture GCE-specific experimental conditions, such as long-term exposure of the ring-strained ncAA to living cells, that are not taken into account in other assays.

Debugging Eukaryotic Genetic Code Expansion for Site-Specific Click-PAINT Super-Resolution Microscopy.

Super-resolution microscopy (SRM) greatly benefits from the ability to install small photostable fluorescent labels into proteins. Genetic code expansion (GCE) technology addresses this demand, allowing the introduction of small labeling sites, in the form of uniquely reactive noncanonical amino acids (ncAAs), at any residue in a target protein. However, low incorporation efficiency of ncAAs and high background fluorescence limit its current SRM applications. Redirecting the subcellular localization of the pyrrolysine-based GCE system for click chemistry, combined with DNA-PAINT microscopy, enables the visualization of even low-abundance proteins inside mammalian cells. This approach links a versatile, biocompatible, and potentially unbleachable labeling method with residue-specific precision. Moreover, our reengineered GCE system eliminates untargeted background fluorescence and substantially boosts the expression yield, which is of general interest for enhanced protein engineering in eukaryotes using GCE.

Rapid synthesis and antimicrobial activity of novel 4-oxazolidinone heterocycles.

The synoxazolidinone family of marine natural products bear an unusual 4-oxazolidinone heterocyclic core and promising antimicrobial activity against several strains of pathogenic bacteria. As part of our research program directed at the synthesis and chemical biology of this family of natural products we have developed a one-step method for the generation of variously substituted 4-oxazolidinone scaffolds from readily available materials. These studies revealed the importance of an electron deficient aromatic ring for antimicrobial activity and serve as the basis for future SAR studies around the 4-oxazolidinone core.

A rapid synthesis of 4-oxazolidinones: total synthesis of synoxazolidinones A and B.

A five-step total synthesis of the marine natural product synoxazolidinone A was achieved through a diastereoselective imine acylation/cyclization cascade. Synoxazolidinone B and a series of analogues were also prepared to explore the potential of these 4-oxazolidinone natural products as antimicrobial agents. These studies confirmed the importance of the chlorine substituent for antimicrobial activity and revealed simplified dichloro derivatives that are equally potent against several bacterial strains.

Stereoselective Synthesis of Quaternary Pyrrolidine-2,3-diones and ß-Amino Acids.

A facile, diastereoselective synthesis of highly substituted pyrrolidine-2,3-diones is reported, along with the one-step conversion of these heterocycles to novel ß-amino acids and further functionalized derivatives. This method involves an unusually mild, one-pot, three-component cyclization/allylation followed by a Claisen rearrangement to provide unusual pyrrolidinone products that are densely functionalized and contain an all-carbon quaternary stereocenter. The reported reaction sequence is operationally simple, exquisitely diastereoselective, and provides gram-scale access to valuable heterocyclic scaffolds and ß-amino acids not readily accessible via existing approaches.

5-Benzylidene-4-oxazolidinones potently inhibit biofilm formation in Methicillin-resistant Staphylococcus aureus.

Investigation into the biological function of 5-benzylidene-4-oxazolidinones revealed dose-dependent inhibition of biofilm formation in Methicillin-resistant S. aureus (MRSA). This structurally unusual class of small molecules inhibit up to 89% of biofilm formation with IC50 values as low as 0.78 µM, and disperse pre-formed biofilms with IC50 values as low as 4.7 µM. Together, these results suggest that 4-oxazolidinones represent new chemotypes to enable the study of bacterial biofilms with small molecule chemical probes.