Maksim Royzen

Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels−Alder Reactivity

Described is a bioorthogonal reaction that proceeds with unusually fast reaction rates without need for catalysis: the cycloaddition of s-tetrazine and trans-cyclooctene derivatives. The reactions tolerate a broad range of functionality and proceed in high yield in organic solvents, water, cell media, or cell lysate. The rate of the ligation between trans-cyclooctene and 3,6-di-(2-pyridyl)-s-tetrazine is very rapid (k2 2000 M-1 s-1). This fast reactivity enables protein modification at low concentration.

Chemical characterization of the smallest S-nitrosothiol, HSNO; cellular cross-talk of H2S and S-nitrosothiols.

Dihydrogen sulfide recently emerged as a biological signaling molecule with important physiological roles and significant pharmacological potential. Chemically plausible explanations for its mechanisms of action have remained elusive, however. Here, we report that H2S reacts with S-nitrosothiols to form thionitrous acid (HSNO), the smallest S-nitrosothiol. These results demonstrate that, at the cellular level, HSNO can be metabolized to afford NO+, NO, and NO– species, all of which have distinct physiological consequences of their own. We further show that HSNO can freely diffuse through membranes, facilitating transnitrosation of proteins such as hemoglobin. The data presented in this study explain some of the physiological effects ascribed to H2S, but, more broadly, introduce a new signaling molecule, HSNO, and suggest that it may play a key role in cellular redox regulation.

A Photochemical Synthesis of Functionalized trans-Cyclooctenes Driven by Metal Complexation

Described is a scalable procedure for driving photochemical sytheses of trans-cyclooctene derivatives through metal complexation. During photoirradiation, reaction mixtures are continuously pumped through a column of a AgNO3-impregnated silica gel. The trans-cyclooctene derivative is selectively retained by the AgNO3-impregnated silica, but the cis-isomer elutes from the column back to the reaction flask, where it is photoisomerized and recirculated through the column. The method provides access to a range of usefully functionalized derivatives of trans-cyclooctene, including a derivative of 5-aza-trans-cyclooctene that underwent transannular cyclization upon treatment with bromine. The alkene stereochemistry is transferred with high fidelity to the hexahydropyrrolizine framework in the transannular cyclization.

Physical and structural properties of [Cu(BOT1)Cl]Cl, a fluorescent imaging probe for HNO

Nitroxyl, or HNO, is involved in a number of important physiological processes, such as vascular relaxation and neuroregulation. Effective imaging tools are required in order to gain a deeper understanding of the in vivo mechanisms of these processes and to identify the endogenous sources of HNO. Here, we further investigate the physical properties of our previously reported fluorescent nitroxyl sensor, [Cu(BOT1)Cl]Cl (J. Am. Chem. Soc.2010, 132, 5536; BOT1=BODIPY·triazole, a tetradentate ligand). A new high-yielding synthetic procedure for BOT1 is reported. The X-ray crystal structures of two Cu(II) complexes of BOT1 are described. These structural studies show that the BOT1 ligand can form Cu(II) coordination complexes of both square-pyramidal and trigonal-bipyramidal geometries. Cyclic voltammograms of [Cu(BOT1)Cl]Cl were acquired, revealing the presence of a quasi-reversible feature at 130 mV (vs the ferrocene/ferrocenium couple) in MeCN and at -40 mV (vs Ag/AgCl) in aqueous buffer, which is assigned to the Cu(II)/Cu(I) couple. The reactivity of [Cu(BOT1)Cl]Cl with Angelis salt, a stable source of HNO, was further investigated. A 1000-fold excess of Angelis salt elicits an immediate 10-fold emission turn-on response of the sensor, consistent with our previous report. A new observation, reported here, is that the intensity of this turn-on emission diminishes at longer incubation times. Fluorescent imaging of nitroxyl by [Cu(BOT1)Cl]Cl in HeLa cells was carried out. Upon treatment of the cells with Angelis salt, there was a modest 2-fold intracellular turn-on in emission intensity.

Synthesis and Characterization of Pt(IV) Fluorescein Conjugates to Investigate Pt(IV) Intracellular Transformations

Pt(IV) anticancer compounds typically operate as prodrugs that are reduced in the hypoxic environment of cancer cells, losing two axial ligands in the process to generate active Pt(II) species. Here we report the synthesis of two fluorescent Pt(IV) prodrugs of cisplatin in order to image and evaluate the Pt(IV) reduction process in simulated and real biological environments. Treatment of the complexes dissolved in PBS buffer with reducing agents typically encountered in cells, glutathione or ascorbate, afforded a 3- to 5-fold fluorescence turn-on owing to reduction and loss of their fluorescein-based axial ligands, which are quenched when bound to platinum. Both Pt(IV) conjugates displayed moderate cytotoxicity against human cancer cell lines, with IC50 values higher than that of cisplatin. Immunoblotting and DNA flow cytometry analyses of one of the complexes, Pt(IV)FL2, revealed that it damages DNA, causes cell cycle arrest in S or G2/M depending on exposure time, and ultimately triggers apoptotic cell death. Fluorescence microscopic studies prove that Pt(IV)FL2 enters cells intact and undergoes reduction intracellularly. The results are best interpreted in terms of a model in which the axial fluorescein ligands are expelled through lysosomes, with the platinum(II) moiety generated in the process binding to genomic DNA, which results in cell death.

Total synthesis of hyacinthacine A2: stereocontrolled 5-aza-cyclooctene photoisomerization and transannular hydroamination with planar-to-point chirality transfer

The total synthesis of hyacinthacine A2 is reported via a novel transannular hydroamination in which planar chirality of a 5-aza-trans-cyclooctene precursor is transferred to point chirality in the product. Key to the success of this strategy was the development of a method for establishing absolute planar chirality via stereocontrolled photoisomerization of a 5-aza-cis-cyclooctene. This was accomplished by constructing a 5-aza-cis-cyclooctene precursor with a trans-fused acetonide. The improved diastereoselectivity observed upon photoisomerization of this derivative is attributed to the conformational strain of the eight-membered ring in the minor diastereomer.

Supramolecular Metal Displacement allows On-Fluorescence Analysis of Manganese(II) in Living Cells

Due to the importance of Mn(2+) ions in biological processes, it is of growing interest to develop protocols for analysis of Mn(2+) uptake and distribution in cells. A supramolecular metal displacement assay can provide ratiometric fluorescence detection of Mn(2+), allowing for quantitative and longitudinal analysis of Mn(2+) uptake in living cells.