Oxygen-Sensing Chemiluminescent Iridium(III) 1,2-Dioxetanes: Unusual Coordination and Activity.

Next generation chemiluminescent iridium 1,2-dioxetane complexes have been developed which consist of the Schaap's 1,2-dioxetane scaffold directly attached to the metal center. This was achieved by synthetically modifying the scaffold precursor with a phenylpyridine moiety, which can act as a ligand. Reaction of this scaffold ligand with the iridium dimer [Ir(BTP)2(µ-Cl)]2 (BTP = 2-(benzo[b]thiophen-2-yl)pyridine) yielded isomers which depict ligation through either the cyclometalating carbon or, interestingly, the sulfur atom of one BTP ligand. Their corresponding 1,2-dioxetanes display chemiluminescent responses in buffered solutions, exhibiting a single, red-shifted peak at 600 nm. This triplet emission was effectively quenched by oxygen, yielding in vitro Stern-Volmer constants of 0.1 and 0.009 mbar-1 for the carbon-bound and sulfur compound, respectively. Lastly, the sulfur-bound dioxetane was further utilized for oxygen sensing in muscle tissue of living mice and xenograft models of tumor hypoxia, depicting the ability of the probe chemiluminescence to penetrate biological tissue (total flux ~ 106 p/s).

Synthesis of the Hypoxia-Inducible Factor-2α (HIF-2α) Inhibitor, 3-[(1S,2S,3R)-2,3-Difluoro-1-hydroxy-7-methylsulfonylindan-4-yl]oxy-5-fluorobenzonitrile (PT2977, Belzutifan); Efficient Replication of Established Approaches.

A concise linear synthesis of hypoxia inducible factor-2α (HIF-2α) inhibitor, belzutifan was achieved by reproducing key components of previous synthetic approaches to this molecule as described in several publications and patents. Belzutifan is an orally bioavailable small-molecule (HIF-2α) inhibitor for the treatment of von Hippel-Lindau (VHL) disease-associated renal cell carcinoma (RCC) that received FDA approval in 2021. Herein, we report a 13-step synthesis of PT2977 that proceeded in good overall yield with high diastereoselectivity. Separation of diastereomeric mixtures at two different stages of the synthesis proved advantageous in ease of separation. The X-ray structure of belzutifan was determined.

Evaluating the Effect of Dye-Dye Interactions of Xanthene-Based Fluorophores in the Fluorosequencing of Peptides.

A peptide sequencing scheme utilizing fluorescence microscopy and Edman degradation to determine the amino acid position in fluorophore-labeled peptides was recently reported, referred to as fluorosequencing. It was observed that multiple fluorophores covalently linked to a peptide scaffold resulted in a decrease in the anticipated fluorescence output and worsened the single-molecule fluorescence analysis. In this study, we report an improvement in the photophysical properties of fluorophore-labeled peptides by incorporating long and flexible (PEG)10 linkers at the peptide attachment points. Long linkers to the fluorophores were installed using copper-catalyzed azide-alkyne cycloaddition conditions. The photophysical properties of these peptides were analyzed in solution and immobilized on a microscope slide at the single-molecule level under peptide fluorosequencing conditions. Solution-phase fluorescence analysis showed improvements in both quantum yield and fluorescence lifetime with the long linkers. While on the solid support, photometry measurements showed significant increases in fluorescence brightness and 20 to 60% improvements in the ability to determine the amino acid position with fluorosequencing. This spatial distancing strategy demonstrates improvements in the peptide sequencing platform and provides a general approach for improving the photophysical properties in fluorophore-labeled macromolecules.

Synthesis of Carboxy ATTO 647N Using Redox Cycling for Xanthone Access.

A synthesis of the carbopyronine dye Carboxy ATTO 647N from simple materials is reported. This route proceeds in 11 forward steps from 3-bromoaniline with the key xanthone intermediate formed using a new oxidation methodology. The step utilizes an oxidation cycle with base, water, iodine, and more than doubles the yield of the standard permanganate oxidation methodology, accessing gram-scale quantities of this late-stage product. From this, Carboxy ATTO 647N was prepared in only four additional steps. This facile route to a complex fluorophore is expected to enable further studies in fluorescence imaging.

Improved Xanthone Synthesis, Stepwise Chemical Redox Cycling.

A base-catalyzed direct oxidation of rhodamine, carborhodamine, and siliconrhodamine pyronines to the corresponding xanthones is studied. This methodology utilizes addition of water to split pyronines into xanthone and reduced xanthene, the latter of which is returned to pyronine by oxidation with iodine. The transformation is general, working on the three most recalcitrant versions of N, N, N', N'-tetramethylpyronines in good to excellent yields.