Identification of specific carbonic anhydrase inhibitors via in situ click chemistry, phage-display and synthetic peptide libraries: comparison of the methods and structural study.

The development of highly active and selective enzyme inhibitors is one of the priorities of medicinal chemistry. Typically, various high-throughput screening methods are used to find lead compounds from a large pool of synthetic compounds, and these are further elaborated and structurally refined to achieve the desired properties. In an effort to streamline this complex and laborious process, new selection strategies based on different principles have recently emerged as an alternative. Herein, we compare three such selection strategies with the aim of identifying potent and selective inhibitors of human carbonic anhydrase II. All three approaches, in situ click chemistry, phage-display libraries and synthetic peptide libraries, led to the identification of more potent inhibitors when compared to the parent compounds. In addition, one of the inhibitor-peptide conjugates identified from the phage libraries showed greater than 100-fold selectivity for the enzyme isoform used for the compound selection. In an effort to rationalize the binding properties of the conjugates, we performed detailed crystallographic and NMR structural analysis, which revealed the structural basis of the compound affinity towards the enzyme and led to the identification of a novel exosite that could be utilized in the development of isoform specific inhibitors.

An Optimized Protocol for the Synthesis of Peptides Containing trans-Cyclooctene and Bicyclononyne Dienophiles as Useful Multifunctional Bioorthogonal Probes.

Despite the great advances in solid-phase peptide synthesis (SPPS), the incorporation of certain functional groups into peptide sequences is restricted by the compatibility of the building blocks with conditions used during SPPS. In particular, the introduction of highly reactive groups used in modern bioorthogonal reactions into peptides remains elusive. Here, we present an optimized synthetic protocol enabling installation of two strained dienophiles, trans-cyclooctene (TCO) and bicyclononyne (BCN), into different peptide sequences. The two groups enable fast and modular post-synthetic functionalization of peptides, as we demonstrate in preparation of peptide-peptide and peptide-drug conjugates. Due to the excellent biocompatibility, the click-functionalization of the peptides can be performed directly in live cells. We further show that the introduction of both clickable groups into peptides enables construction of smart, multifunctional probes that can streamline complex chemical biology experiments such as visualization and pull-down of metabolically labeled glycoconjugates. The presented strategy will find utility in construction of peptides for diverse applications, where high reactivity, efficiency and biocompatibility of the modification step is critical.

Designed To React: Terminal Copper Nitrenes and Their Application in Catalytic C-H Aminations.

Heteroscorpionate ligands of the bis(pyrazolyl)methane family have been applied in the stabilisation of terminal copper tosyl nitrenes. These species are highly active intermediates in the copper-catalysed direct C-H amination and nitrene transfer. Novel perfluoroalkyl-pyrazolyl- and pyridinyl-containing ligands were synthesized to coordinate to a reactive copper nitrene centre. Four distinct copper tosyl nitrenes were prepared at low temperatures by the reaction with SO2 tBuPhINTs and copper(I) acetonitrile complexes. Their stoichiometric reactivity has been elucidated regarding the imination of phosphines and the aziridination of styrenes. The formation and thermal decay of the copper nitrenes were investigated by UV/Vis spectroscopy of the highly coloured species. Additionally, the compounds were studied by cryo-UHR-ESI mass spectrometry and DFT calculations. In addition, a mild catalytic procedure has been developed where the copper nitrene precursors enable the C-H amination of cyclohexane and toluene and the aziridination of styrenes.

Design and Synthesis of Aza-Bicyclononene Dienophiles for Rapid Fluorogenic Ligations.

Fluorogenic bioorthogonal reactions enable visualization of biomolecules under native conditions with excellent signal-to-noise ratio. Here, we present the design and synthesis of conformationally-strained aziridine-fused trans-cyclooctene (aza-TCO) dienophiles, which lead to the formation of fluorescent products in tetrazine ligations without the need for attachment of an extra fluorophore moiety. The presented aza-TCOs adopt the highly strained "half-chair" conformation, which was predicted computationally and confirmed by NMR measurements and X-ray crystallography. Kinetic studies revealed that the aza-TCOs belong to the most reactive dienophiles known to date. The potential of the newly developed aza-TCO probes for bioimaging applications is demonstrated by protein labeling experiments, imaging of cellular glycoconjugates and peptidoglycan imaging of live bacteria.

Mechanism-Based Fluorogenic trans-Cyclooctene-Tetrazine Cycloaddition.

The development of fluorogenic reactions which lead to the formation of fluorescent products from two nonfluorescent starting materials is highly desirable, but challenging. Reported herein is a new concept of fluorescent product formation upon the inverse electron-demand Diels-Alder reaction of 1,2,4,5-tetrazines with particular trans-cyclooctene (TCO) isomers. In sharp contrast to known fluorogenic reagents the presented chemistry leads to the rapid formation of unprecedented fluorescent 1,4-dihydropyridazines so that the fluorophore is built directly upon the chemical reaction. Attachment of an extra fluorophore moiety is therefore not needed. The photochemical properties of the resulting dyes can be easily tuned by changing the substitution pattern of the starting 1,2,4,5-tetrazine. We support the claim with NMR measurements and rationalize the data by computational study. Cell-labeling experiments were performed to demonstrate the potential of the fluorogenic reaction for bioimaging.