Tethered Benzophenone Reagents for the Synthesis of Photoactivatable Ligands

Abstract

A new radiolabeled, bifunctional photoaffinity cross-linking reagent, N-succinimidyl p-benzoyl-[2,3-3H2]dihydrocinnamate, has been synthesized in high yield and with high specific activity. This reagent can be used to append the benzophenone photophore to amino groups of small molecules, such as O-aminoalkylinositol polyphosphates and polypeptides. The resulting tritiated photoaffinity labels can be purified and manipulated in ambient light and can be activated at 360 nm.

Specific Interaction of Golgi Coatomer Protein α-COP with Phosphatidylinositol 3,4,5-Trisphosphate

“…3 H]BZDC-NHS ester (38) and had the same nominal specific activity of 42.5 Ci/ mmol. This ensured that levels of radioactivity in each probe corresponded to equivalent concentrations, thereby allowing direct comparison of relative efficiencies of photoattachment.…”

“…InsP 6 was obtained from Sigma as the monopotassium salt. Anti-COP antisera were gifts from Dr. C. Harter 3 , PtdIns(4,5)P 2 , and PtdIns(3,4)P 2 was accomplished using the coupling reagent 2-cyanoethyl-N,N,NЈ,NЈ-tetra(isopropyl)phosphordiamidite (37,38,41). This introduced a reactive aminopropyl group at the P-1 phosphate to which the [ 3 H]BZDC moiety was then attached.…”

“…To address the InsP n and PtdInsP n binding specificities of individual COPI subunits, and to obtain evidence to support the roles of these high affinity interactions in vesicular trafficking, we employed a photoaffinity labeling approach with benzophenone-containing InsP n and PtdInsP n analogs (36). The benzophenone photophore allows handling in ambient light, activation at wavelengths Ͼ320 nm, and covalent labeling of active site residues in hydrophobic regions of proteins with high efficiency (37,38). Photoaffinity analogs of soluble inositol polyphosphates Ins(1,4,5)P 3 , Ins(1,3,4,5)P 4 , and InsP 6 (39,40) and the lipid-containing polyphosphoinositides PtdIns-(3,4,5)P 3 (41), PtdIns(4,5)P 2 (41), and PtdIns(3,4)P 2 (42) are used herein to determine the polyphosphoinositide selectivity for the COPI subunits in Golgi coatomer (39).…”

Selective Photoaffinity Labeling of the Inositol Polyphosphate Binding C2B Domains of Synaptotagmins

“…1) were used to probe the selectivity and specificity of the GST-Syt II-C2B interaction with IP n s. Each inositol polyphosphate photoaffinity label (25) was freshly prepared for this study from the corresponding 1-or 2-O-( -aminoalkyl) IP n derivative with the heterobifunctional reagent [ 3 H]BZDC-NHS ester (20). The P-1-linked 3-aminopropylphosphodiesters of Dmyo-IP 3 (18) and IP 4 (22), and the P-2-linked 6-aminohexylphosphodiester of meso-IP 6 (24) were prepared as described previously.…”

“…The peptide IHLMQNGKRLKKKKTTVKKKTLNPY-FNESFSF was synthesized by the Peptide Institute (Osaka, Japan) and was Ͼ98.1% homogeneous by HPLC, YMCPak ODS-AM, 10 -60% CH 3 CN in 0.1% trifluoroacetic acid. Ins(1,2,3,4,5,6)P 6 was prepared as described (24) and coupled with [ 3 H]BZDC-NHS in aqueous N,N-dimethylformamide (20). The following protocol was typical for several independent preparations of this photoactivatable ligand.…”