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Abstract: Various fluorescent nucleoside agonists of the A3 adenosine receptor (AR) were compared as high affinity probes using radioligands and flow cytometry (FCM). They contained a fluorophore linked through the C2 or N 6 position and rigid A3AR-enhancing (N)-methanocarba modification. A hydrophobic C2-(1-pyrenyl) derivative MRS5704 bound nonselectively. C2-Tethered cyanine5-dye labeled MRS5218 bound selectively to hA3AR expressed in whole CHO cells and membranes. By FCM, binding was A3AR-mediated (blocked by A3AR antagonist, at least half through internalization), with t 1/2 for association 38min in mA3AR-HEK293 cells; 26.4min in sucrose-treated hA3AR-CHO cells (K d 31nM). Membrane binding indicated moderate mA3AR affinity, but not selectivity. Specific accumulation of fluorescence (50nM MRS5218) occurred in cells expressing mA3AR, but not other mouse ARs. Evidence was provided suggesting that MRS5218 detects endogenous expression of the A3AR in the human promyelocytic leukemic HL-60 cell line. Therefore, MRS5218 promises to be a useful tool for characterizing the A3AR. [Copyright &y& Elsevier]

Abstract: The physiological role of the A3 adenosine receptor (AR) was explored in cardiac ischaemia, inflammatory diseases and cancer. We report a new fluorophore-conjugated human (h) A3AR antagonist for application to cell-based assays in ligand discovery and for receptor imaging. Fluorescent pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-ylamine (pyrazolo-triazolo-pyrimidine, PTP) and triazolo[1,5-c]quinazolin-5-yl)amine (triazolo-quinazoline, TQ) AR antagonists were compared. A chain-extended and click-conjugated Alexa Fluor-488 TQ derivative (MRS5449) displayed a radioligand binding K i value of 6.4±2.5nM in hA3AR-expressing CHO cell membranes. MRS5449 antagonized hA3AR agonist-induced inhibition of cyclic AMP accumulation in a concentration-dependent manner (K B =4.8nM). Using flow cytometry (FCM), MRS5449 saturated hA3ARs with very high specific-to-nonspecific binding ratio with an equilibrium binding constant 5.15nM, comparable to the K d value of 6.65nM calculated from kinetic experiments. K i values of known AR antagonists in inhibition of MRS5449 binding in whole cell FCM were consistent with radioligand binding in membranes, but agonist binding was 5-20 fold weaker than obtained with agonist radioligand [125I]I-AB-MECA. Further binding analysis of MRS5549 suggested multiple agonist binding states of the A3AR. Molecular docking predicted binding modes of these fluorescent antagonists. Thus, MRS5449 is a useful tool for hA3AR characterization. [Copyright &y& Elsevier]

Photoresponsive materials offer excellent spatiotemporal control over biological processes and the emerging phototherapeutic methods are expected to have significant effects on targeted cancer therapies. Recent examples show that combination of photoactivatable approaches with bioorthogonal chemistry enhances the precision of targeted phototherapies and profound implications are foreseen particularly in the treatment of disperse/diffuse tumors. The extra level of on-target selectivity and improved spatial/temporal control considerably intensified related bioorthogonally assisted phototherapy research. The anticipated growth of further developments in the field justifies the timeliness of a brief summary of the state of the art., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Tetrazine-bearing fluorescent labels enable site-specific tagging of proteins that are genetically manipulated with dienophile modified noncanonical amino acids. The inverse electron demand Diels-Alder reaction between the tetrazine and the dienophile fulfills the criteria of bioorthogonality allowing fluorescent labeling schemes of live cells. Here, we describe the detailed synthetic and labeling protocols of a near infrared emitting siliconrhodamine-tetrazine probe suitable for super-resolution imaging of residue-specifically engineered proteins in mammalian cells.

The synthesis of a set of tetrazine-bearing fluorogenic dyes suitable for intracellular labeling of proteins in live cells is presented. The red excitability and emission properties ensure minimal autofluorescence, while through-bond energy-transfer-based fluorogenicity reduces nonspecific background fluorescence of unreacted dyes. The tetrazine motif efficiently quenches fluorescence of the phenoxazine core, which can be selectively turned on chemically upon bioorthogonal inverse-electron-demand Diels-Alder reaction with proteins modified genetically with strained trans-cyclooctenes., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

4-Alkyloxyimino derivatives of pyrimidine nucleotides display high potency as agonists of certain G protein-coupled P2Y receptors (P2YRs). In an effort to functionalize a P2Y 6 R agonist for fluorescent labeling, we probed two positions ( N 4 and γ-phosphate of cytidine derivatives) with various functional groups, including alkynes for click chemistry. Functionalization of extended imino substituents at the 4 position of the pyrimidine nucleobase of CDP preserved P2Y 6 R potency generally better than γ-phosphoester formation in CTP derivatives. Fluorescent Alexa Fluor 488 conjugate 16 activated the human P2Y 6 R expressed in 1321N1 human astrocytoma cells with an EC 50 of 9 nM, and exhibited high selectivity for this receptor over other uridine nucleotide-activated P2Y receptors. Flow cytometry detected specific labeling with 16 to P2Y 6 R-expressing but not to wild-type 1321N1 cells. Additionally, confocal microscopy indicated both internalized 16 ( t 1/2 of 18 min) and surface-bound fluorescence. Known P2Y 6 R ligands inhibited labeling. Theoretical docking of 16 to a homology model of the P2Y 6 R predicted electrostatic interactions between the fluorophore and extracellular portion of TM3. Thus, we have identified the N 4 -benzyloxy group as a structurally permissive site for synthesis of functionalized congeners leading to high affinity molecular probes for studying the P2Y 6 R.

Effective syntheses of endo- and exocyclic alpha,beta-unsaturated ketones as CC dipolarophiles were carried out in the 13alpha-estrone series. The 1,3-dipolar cycloadditions of 15,16alpha,beta-unsaturated ketones of 13alpha-estrone 3-methyl and 3-benzyl ether with nitrilimines stereoselectively furnished two regioisomers of new condensed pyrazolines in a ratio of 2:1. The main product was the isomer obtained by the attack of the N-terminus of the 1,3-dipole on the carbon atom beta to the carbonyl group of the dipolarophile. The nitrilimine cycloadditions to the 16-methylene-17-ketones of 13alpha-estrone 3-methyl and 3-benzyl ether stereo- and regioselectively furnished spiropyrazolines. The attack of the N-terminus of the dipole occurred on the alpha-carbon of the alpha,beta-unsaturated ketones. The reactions were performed under both homogeneous and heterogeneous conditions. Silver acetate as a base proved more effective than its triethylamine counterpart. Changes in regio- and stereoselectivities were not observed on variation of the conditions of the cycloaddition reactions. The structures of the new products were determined by NMR (one- and two-dimensional) and MALDI TOF MS techniques, with C(70) fullerenes as matrix in the latter case.