128886-88-4

The profiling of kinases using established proteomics techniques is hampered by their non-covalent mode-of-action. One way to overcome this caveat is the use of probes featuring photo-labelling groups that can be activated by UV irradiation to generate a reactive species that will establish a covalent bond to the enzyme. In this study we have used the well-known kinase inhibitor H89 as a lead for the development of probes for the affinity-based profiling of clinically relevant kinases. A labelling protocol was established for recombinant kinases and more complex protein mixtures using gel-based techniques. We also show that the probes act in a competitive manner with other kinase inhibitors.

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The disclosure relates to a diazirine precursor mass tag compound represented by structural formula (I). Also disclosed is a method for detecting analytes in a sample, comprising derivatizing the analytes with the compound of formula (I), and detecting th

The first effective method for the introduction of a versatile substituent on 3-phenyl-3-trifluoromethyldiazirine has been developed. The simple preparation of a useful aldehyde intermediate allows easy access to various elaborated photoaffinity ligands, including a l-phenylalanine analog bearing a diazirine ring (TmdPhe). The asymmetric synthesis of TmdPhe was easily accomplished in gram quantities. Site-directed incorporation of this compound into the structure of a calmodulin-binding peptide using automated peptide synthesis afforded a photoreactive peptide that was successfully used for the specific labeling of calmodulin. Copyright

Synthesis of a 3-phenyl-3-trifluoromethyldiazirine derivative having a cleavable 1,2-diol moiety for photocross-linking technique is described. An ester of cinnamic acid derivative was prepared by the Wittig reaction of 2-methoxy-4-[3-trifluoromethyl-3H-diazirin-3-yl]benzaldehyde which can be readily prepared by the Friedel-Crafts reaction of corresponding phenyldiazirine. The osmium oxidation of the olefinic bond gave the desired diol derivative, ethyl 2,3-dihydroxy-3-[2-methoxy-4-(3-trifluoromethyl-3H-diazirin-3-yl)phenyl] propionate, in a high yield. The cleavability of the diol group was confirmed by the conventional periodate oxidation to give the starting aldehyde.

We introduce here a new photochemical probe suitable for labeling deep into the hydrophobic core of membranes: bis-phosphatidylethanolamine (trifluoromethyl)phenyldiazirine 19 (DIPETPD). This is a bipolar phospholipid provided with a covalently bonded chain designed to span the membrane and equipped with a centrally defined attachment point for the photolabeling group (trifluoromethyl)phenyldiazirine (TPD). This molecule was designed to enhance the geometrical resolution of photochemical labeling of membrane proteins by locating the photoreactive functionality in the center of the bilayer. The remarkable chemical stability of the photoreactive group TPD1 allowed the design of a straightforward and convergent synthetic strategy. The key steps developed for molecules of this new general kind are (a) the mild and efficient coupling of two moieties of N-tBOC-protected lysophosphatidylethanolamine methyl ester to the photoreactive symmetric dicarboxylic fatty acid mediated by dicyclohexylcarbodiimide and (dimethylamino)pyridine and (b) the smooth deprotection of the phosphate and amino functionalities with sodium iodide and trifluoroacetic acid, respectively, to yield the final product. DIPETPD has been successfully incorporated into small and large unilamellar vesicles of different lipid composition and prepared by a variety of procedures. The bilayer location of this reagent (transmembrane vs 'U'-shaped conformations) was assayed by reaction of the amino groups at the polar heads of the bipolar phospholipid with selected membrane-impermeable reagents. Photolysis of the probe incorporated into vesicles occurs readily upon irradiation with UV light (near 360 nm). These 'loaded' vesicles show adequate stability and appear uniform and unilamellar in electron micrographs. They undergo the fusion reaction with influenza virus as efficiently as reagent-free vesicles. Evidence is presented here that DIPETPD and a reductively methylated form efficiently label the peptide ion channel form of gramicidin A (and a chemical analogue) and the influenza virus hemagglutinin. DIPETPD may help to identify transmembrane regions of integral membrane proteins and map the lipid - protein interface in a region known to be deep in the membrane. A new radioactive version of this reagent ([3H]-DIPETPD)2 has been recently used to ascertain that the HA2 subunit of influenza virus hemagglutinin inserts into the target membrane prior to fusion.

The condensation of cysteine with 3-(4-formylphenyl) or 3-(4-cyanophenyl)-3-trifluoromethyldiazirine furnished thiazolidine and thiazoline derivatives in good yield. These heterocycles provide convenient access to photoaffinity probes an 35S ra