Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining Kd values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited Kd values of 27±3.9 nM and 4.2±0.48 nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The Ki values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the Ki values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for these MSH(4) constructs than was previously reported.

A flexible molecular scaffold bearing varying numbers of terminal alkyne groups was synthesized in five steps from solanesol. R(CO)-MSH(4)-NH 2 ligands, which have a relatively low affinity for binding at the human melanocortin 4 receptor (hMC4R), were prepared by solid phase synthesis and were N-terminally acylated using 6-azidohexanoic acid. Multiple copies of the azide N 3 (CH 2 ) 5 (CO)-MSH(4)-NH 2 were attached to the alkyne-bearing, solanesol-derived molecular scaffold via the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Control studies showed that the binding affinity of the triazole-containing ligand, CH 3 (CH 2 ) 3 (C 2 N 3 )(CH 2 ) 5 (CO)-MSH(4)-NH 2 , was not significantly diminished relative to the corresponding parental ligand, CH 3 (CO)-MSH(4)-NH 2 . In a competitive binding assay using a Eu-labeled probe based on the superpotent ligand NDP-α-MSH, the monovalent and multivalent constructs appear to bind to hMC4R as monovalent species. In a similar assay using a Eu-labeled probe based on MSH(4), modest increases in binding potency with increased MSH(4) content per scaffold were observed.

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Gadolinium triflate immobilized in room temperature ionic liquids (RTIL) 1-butyl-3methylimidazolium tetrafluoroborate ([bmim][BF 4 ]) and 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF 6 ]) was found to be a recyclable and green catalyst for acetylation of a variety of alcohols, phenols and amines. Acetylation reactions using acetic anhydride (Ac 2 O) as the reagent proceeded in excellent yields in the presence of catalytic amounts (0.2-0.5 mol%) of Gd(OTf) 3 immobilized in RTILs, at ambient temperature. In addition, the catalyst system Gd(OTf) 3 /[bmim][X] can be recovered and reused efficiently in these transformations.

A labeled variant of MSH(4), a tetrapeptide that binds to the human melanocortin 4 receptor (hMC4R) with low µM affinity, was prepared by solid phase synthesis methods, purified, and characterized. The labeled ligand, Eu-DTPA-PEGO-His-DPhe-Arg-Trp-NH 2 , exhibited a K d for hMC4R of 9.1±1.4 µM, approximately 10-fold lower affinity than the parental ligand. The labeled MSH(4) derivative was employed in a competitive binding assay to characterize the interactions of hMC4R with monovalent and divalent MSH(4) constructs derived from squalene. The results were compared with results from a similar assay that employed a more potent labeled ligand, Eu-DTPA-NDP-α-MSH. While results from the latter assay reflected only statistical effects, results from the former assay reflected a mixture of statistical, proximity, and/or cooperative binding effects.Early detection of many human cancers would be facilitated by the availability of imaging agents that selectively bind to cancer cells and report their existence and location by noninvasive techniques. 2-5 Development of such imaging agents involves covalently linking reporter moieties and multiple copies of ligands to produce "multivalent molecules" that will cooperatively bind to receptors expressed on the surface of cancer cells. [6][7][8][9][10][11][12] Multivalent molecules constructed from weakly binding ligands should display avidity for such cells when compared with the affinity of a single copy of the parental ligand. [6][7][8][9][10][11][12][13][14][15][16][17] The affinity of a molecule for binding to a receptor is often quantified by a competitive binding assay against a labeled ligand of known potency. For example, labeled forms of NDP-α-MSH, a superpotent ligand that binds to human melanocortin receptors,18 , 19 have been used for this purpose.20 ,21 In such assays one often assumes thermodynamic control, i.e., that the respective on-rates and off-rates of the competing ligands are similar. If this is not the case, details of how the assay is carried out (order and timing of reagent addition, © 2010 Elsevier Ltd. All rights reserved.Correspondence to: Eugene A. Mash. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. timing of measurements taken) can affect the outcome. Determination of on-rates and offrates for binding of multivalent molecules to living cells is difficult since labeled probes may be taken up by the cells and receptors may aggregate and cycle to and from the cell surface. In the absence of knowledge of ligand on-rates and off-rates, a close match between the affinity of the ligands used in constructio...