The mechanism of Renilla-type luciferase reaction and its inhibition.

Martin Marek and Zbyněk Prokop Research Group

Significance

The widely used coelenterazine-powered Renilla luciferase was discovered over 40 years ago, but the oxidative mechanism by which it generates blue photons remains unclear. Here we decipher Renilla-type catalysis through crystallographic, spectroscopic and computational experiments. Structures of ancestral and extant luciferases complexed with the substrate-like analogue azacoelenterazine or a reaction product were obtained, providing molecular snapshots of coelenterazine-to-coelenteramide oxidation. Bound coelenterazine adopts a Y-shaped conformation, enabling the deprotonated imidazopyrazinone component to attack O₂ via a radical charge-transfer mechanism. A high emission intensity is secured by an aspartate from a conserved proton-relay system, which protonates the excited coelenteramide product. Another aspartate on the rim of the catalytic pocket fine-tunes the electronic state of coelenteramide and promotes the formation of the blue light-emitting phenolate anion. The results obtained also reveal structural features distinguishing flash-type from glow-type bioluminescence, providing insights that will guide the engineering of next-generation luciferase‒luciferin pairs for ultrasensitive optical bioassays.

Schenkmayerova, A., Toul, M., Pluskal, D., Baatallah, R., Gagnot, G., Pinto, G.P., Santana, V.T., Stuchla, M., Neugebauer, P., Chaiyen, P., Damborsky, J., Bednar, D., Janin, Y.L., Prokop, Z. & Marek, M.: Catalytic mechanism for Renilla-type luciferases, Nat. Catal. (2023). https://doi.org/10.1038/s41929-022-00895-z

A new technique for the digital mapping of biomarkers in tissues based on desorption and counting intact gold nanoparticle (Au NP) tags using infrared laser ablation single - particle inductively coupled plasma mass spectrometry (IR LA SP ICP MS).

Jan Preisler Research Group

Significance

In contrast to conventional UV laser ablation, Au NPs are not disintegrated during the desorption process due to their low absorption at 2940 nm. A mass spectrometer detects up to 83% of Au NPs. The technique is demonstrated on mapping a proliferation marker, nuclear protein Ki-67, in three-dimensional (3D) aggregates of colorectal carcinoma cells, and the results are compared with confocal fluorescence microscopy and UV LA ICP MS. Precise counting of 20 nm Au NPs with a single-particle detection limit in each pixel by the new approach generates sharp distribution maps of a specific biomarker in the tissue. Advantageously, the desorption of Au NPs from regions outside the tissue is strongly suppressed. The developed methodology promises multiplex mapping of low-abundant biomarkers in numerous biological and medical applications using multielemental mass spectrometers.

Stiborek, M., Jindřichová, L., Meliorisová, S., Bednařík, A., Prysiazhnyi, V., Kroupa, J., Houška, P., Adamová, B., Navrátilová, J., Kanický, V., and Preisler, J.:

Infrared Laser Desorption of Intact Nanoparticles for Digital Tissue Imaging

Anal. Chem. 2022 94, 51, 18114 – 18121, https://doi.org/10.1021/acs.analchem.2c05216

Labdane-type terpenes are a large family of bioactive natural products. Their often-complex structures present challenges to semisynthesis and de novo chemical synthesis in search of analogs with improved properties. To enable new modifications of the well-known tricyclic terpene forskolin, we have developed a distinct and potentially general synthetic scheme for the preparation of analogs of complex labdanes.

Jakub Švenda Research Group

Significance

We report a new synthetic strategy for the flexible preparation of forskolin-like molecules. The approach is different from the previously published works and employs a convergent assembly of the tricyclic labdane-type core from pre-functionalized cyclic building blocks. Stereoselective Michael addition enabled the fragment coupling with excellent control over three newly created contiguous stereocenters, all-carbon quaternary centers included. Silyl enol ether-promoted ring-opening metathesis paired with ring closure were the other key steps enabling concise assembly of the tricyclic core. Late-stage functionalization sequences transformed the tricyclic intermediates into a set of different forskolin-like molecules. The modular nature of the synthetic scheme described herein has the potential to become a general platform for the preparation of analogs of forskolin and other complex tricyclic labdanes.

Szczepanik, P. M., Mikhaylov, A. A., Hylse, O., Kučera, R., Daďová, P., Nečas, M., Kubala, L., Paruch, K., and Švenda, J.:

Convergent Assembly of the Tricyclic Labdane Core Enables Synthesis of Diverse Forskolin-like Molecules, Angew. Chem. Int. Ed. 2022, on-line version e202213183, https://doi.org/10.1002/anie.202213183