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.

Abstract-An efficient synthesis of tetrazole analogues of amino acids starting from N a -Fmoc amino acid in a three-step protocol is reported. The free amino tetrazoles were obtained in good yields and with excellent purity after removal of the Fmoc group. The synthesis of analogues of aspartic and glutamic acids in which the 5-tetrazolyl moiety is inserted at the b/c carboxyl group starting from Fmoc-Asn and Fmoc-Gln and the incorporation of these tetrazoles into peptides are also described.

The N(alpha)-Fmoc-peptide isocyanates 3a-q, 4a-c, and 5a-c were prepared by the Curtius rearrangement of N(alpha)-Fmoc-peptide acid azides in toluene under thermal, microwave, and ultrasonic conditions. All the N(alpha)-Fmoc-oligo-peptide isocyanates made were isolated as stable crystalline solids with 71 to 94% yield and were fully characterized by 1H NMR, 13C NMR, and mass spectroscopy. Their utility for the synthesis of oligo-alpha-peptidyl ureas 7a-f and 8a-c by the divergent coupling approach was demonstrated. The coupling of N(alpha)-Fmoc-dipeptide isocyanates with amino acid ester or with N,O-bis(trimethylsilyl)amino acids resulted in N(alpha)-Fmoc-tripeptidyl urea ester and acids containing one each of peptide bond and urea bond. The divergent approach is extended to the synthesis of tetrapeptidyl ureas by the 2 + 2 strategy using bis-TMS-peptide acid as an amino component. To incorporate urea bonds in adjacent positions, N(alpha)-Fmoc-peptidyl urea isocyanates 9a-d were prepared and employed in the synthesis of three tetrapeptidyl ureas 10a-b and 11 containing one peptide bond and two urea bonds in series from the N-terminal end. The protocol was then employed for the synthesis of five urea analogues 13-15, 18, and 21 of [Leu5]enkephalin containing urea bonds at the 2, 3, 4 positions as well as at the 2, 4 and 2, 3, 4 positions. The analogue 2l was made by the convergent synthesis by the N --> C terminal chain extension. Finally, two urea analogues 22 and 23 of repeat units of bioelasto polymers, namely Val-Pro-Gly-Val-Gly-OH and Pro-Gly-Val-Gly-Val-OH, were synthesized incorporating the urea bond by the concomitant isocyanate generation and urea bond formation under thermal conditions.

An epimerization free and efficient total synthesis of immunosuppressant cyclosporin O (CsO) by step-by-step assembly of amino acids in solution phase is reported. The couplings were performed by employing Fmoc-amino acid chlorides and were mediated by zinc dust under neutral conditions. The yield and purity of the coupling of sterically hindered N-methylamino acids to N-methylamino acids at positions 8, 9, 10, and 11 were enhanced by repeating the coupling thrice at these particular junctures. All the 10 intermediate peptides pertaining to CsO and the final CsO were isolated and completely characterized through IR, 1H NMR, mass spectrometry, and HPLC techniques.

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...