Evaluation of new base-labile 2-(4-nitrophenylsulfonyl) ethoxycarbonyl (Nsc)-amino acids for solid-phase peptide synthesis.

The 2-(4-nitrophenylsulfonyl)ethoxycarbonyl (Nsc) group is a new base-labile protecting group for solid-phase peptide synthesis, completely interchangeable with the fluorenylmethoxycarbonyl (Fmoc) protecting group, but with certain advantages. In this paper, we report a methodology with Nalpha-Nsc-protected amino acids for the synthesis of some melanotropins important to our research, namely, gamma-melanocyte-stimulating hormone (gamma-MSH), its [Nle3]-analogue, and a cyclic alpha-MSH/beta-MSH hybrid. We developed an efficient protocol for the synthesis of the cyclic MSH analogue that yielded this peptide in >98% purity. The gamma-MSH synthesis, which gave problems with both the Boc and Fmoc strategies, yielded the desired peptide by Nsc-chemistry but was accompanied by side products. Finally, the Nle3-gamma-MSH analogue was synthesized more efficiently using the Fmoc strategy, suggesting that Nsc-chemistry might not be the best methodology for certain sequences.

Conformational and topographical considerations in designing agonist peptidomimetics from peptide leads.

The design of peptidomimetic ligands with agonist biological activities in vitro and in vivo has been challenging. Lofty goals have been set for this research including high potency, high receptor type selectivity, high stability in vitro and in vivo, and high efficacy in vitro and in vivo for agonists. A systematic stepwise strategy has been developed to accomplish these goals. These include determining the primary amino acid side chain residues required for molecular recognition and, in the case of agonist activity, those required for information transduction. In addition to determining the preferred backbone conformation which can serve as a template for the bioactive conformation (an alpha-helix, beta-turn, beta-sheet, etc.), a strategy has been developed to examine and determine the preferred side chain conformations in chi space (chi1, chi2, etc.). These include specific covalent and non-covalent constraints which can place the constrained side chains at highly preferred gauche (-), or gauche (+), or trans conformations. Examples are provided that illustrate this methodology and provide insight into the topographical requirements for ligand receptor interactions. Often, at this juncture one can obtain a quite precise 3D pharmacophore for the ligand, as well as high stability to agonist biodegradation and good bioavailability including the ability to cross membrane barriers. If a non-peptide ligand is desired, efforts are in progress to develop templates, and aspects of conformational design that permit assembling of all components necessary for molecular recognition and transduction. Here the proper choice of template that can place the key side chain residue in 3D space is still difficult, and thus only partial success has been achieved in terms of potent and selective ligands. A few of these approaches are presented and discussed in some detail.

D-Amino acid scan of gamma-melanocyte-stimulating hormone: importance of Trp(8) on human MC3 receptor selectivity.

In our search for potent and receptor-selective agonists and antagonists, we report here the results of D-amino acid substitution at each position of the short peptide gamma-melanocyte-stimulating hormone (gamma-MSH). The native gamma-MSH shows weak binding at all three receptors (i.e., the human MC3, MC4, and MC5) and a selectivity of 1-2 orders of magnitude at the MC3R over the MC4R and MC5R. Sequential replacement of each residue in the gamma-MSH sequence with the corresponding D-isomer results in analogues which mostly have weaker binding affinity than the native peptide, except for two analogues. For the DTrp(8) analogue, there is an increase in binding affinity by about 1 order of magnitude (IC(50) = 6 nM) at the MC3R compared with that of the natural molecule and an increase in selectivity for the MC3R by 2 orders of magnitude compared with the activity at the MC4R and MC5R. The DPhe(6) analogue is about 10-fold more potent (IC(50) = 8.8 nM) at the MC3R compared with the native peptide but lacks subtype selectivity. Measurement of the intracellular cAMP accumulation in human MC3R, MC4R, and MC5R revealed that the native peptide shows potent activity at the MC3R (EC(50) = 5.9 nM) and is about 50-100-fold selective at this receptor compared with the MC4R and MC5R. The DArg(10) (EC(50) = 35 nM) and DPhe(11) (EC(50) = 11 nM) analogues are selective for the MC3R by 1 and 2 orders of magnitude compared with the MC4R and MC5R, respectively. The DTrp(8) compound (EC(50) = 0.33 nM) shows about 300- and 250-fold increase in selectivity at the MC3R compared with the MC4R and MC5R, respectively. Finally, the DTyr(1) peptide is selective for the MC3R (EC(50) = 12 nM) by 40-200-fold compared with the MC4R and MC5R. In general, the trend is that D-amino acid substitutions of the aromatic residues 1, 6, 8, and 11 and the basic residue Arg(10), but not Arg(7), result in an increase in MC3R selectivity over the MC4R and MC5R and only agonist activity is observed. Thus, the key residues of gamma-MSH identified in this study include the aromatic residues 1, 6, 8, and 11 and the basic residue Arg(10) (but not Arg(7)), as important for MC3 selectivity over the MC4 and MC5 subtypes. Further, the study reveals the extreme importance of DTrp at position 8 in imparting potency and selectivity since this is the most selective analogue for the human MC3R reported thus far.

Conformational properties of the proline region of porcine neuropeptide Y by CD and 1H-nmr spectroscopy.

We synthesized porcine neuropeptide Y (pNPY) N-terminal fragments by solid-phase synthesis techniques and analyzed them for solution conformational properties by CD and 1H-nmr spectroscopy. The analogues pNPY1-9 and pNPY1-14 displayed CD spectra indicative of random structures and showed no evidence for induced alpha-helical structures in trifluoroethanol (TFE) up to 50%. However, the CD spectra of pNPY1-9 suggested a conformational shift in tetrahydrofuran. Although in aqueous solution the CD spectra of pNPY1-21 indicated random structures with induction of only a small percentage of alpha-helix in aqueous TFE, pNPY1-25 displayed 13% alpha-helical structure in aqueous solution that increased to 40 and 41% by the addition of TFE and methanol, respectively. The nmr spectra of pNPY1-9 and the proline region of pNPY1-25 indicated extended structures with all-trans conformers at Pro5 and Pro8 for pNPY1-9 and at Pro5, Pro8, and Pro13 for pNPY1-25; in each case the Tyr1-Pro2 amide bond was in both cis and trans conformations. However, observed nuclear Overhauser effect correlations and HN exchange experiments indicated an alpha-helical segment in pNPY1-25 initiated by Pro13 and extending from residues 14 to 25. Thus, the N-terminal polyproline region of NPY has no propensity to fold into a regular secondary structure, although Pro13 is a helix initiator, a result consistent with the proposed role of this amino acid in the NPY structural model.

Neuropeptide Y N-terminal deletion fragments: correlation between solution structure and receptor binding activity at Y1 receptors in rat brain cortex.

N alpha-Acetyl (Ac), N-terminal deletion fragments of porcine neuropeptide Y (NPY) have been synthesized and characterized for solution conformation properties by circular dichroism and for receptor binding activity at benextramine-sensitive Y1 binding sites in rat brain cortex. Sequential deletion of Tyr1, Pro2, and Ser3 had no effect on the structural (alpha-helical content of 32.5, 30.6, and 30.7%, respectively, at 1 x 10(-5) M) or aggregation (monomer to dimer transition for N alpha-Ac-NPY3-36 and N alpha-Ac-NPY4-36) properties of NPY. In contrast, deletion of Tyr1 decreased receptor binding activity in rat brain cortex by 4-fold (IC50 = 13.0 nM versus 3.75 nM for NPY), but further deletion of Pro2-Ser3 had no additional detrimental effect on receptor binding activity relative to the desTyr1 analog. Thus, Pro2 and Ser3 do not contribute either to the stability of the NPY tertiary structure nor directly to the receptor-ligand interactions. Additional removal of N-terminal amino acids Lys4-Pro5 decreased the helical content and abolished aggregation to a dimeric form of the resultant analog, results suggesting that the residues around Pro5 are important for formation of NPY's compact, pancreatic polypeptide (PP)-fold structure. This loss in structure also correlated with a further 2-3 fold drop in receptor binding activity. These structure-activity correlations provide evidence for the importance of the PP-fold structure in the activity of NPY at Y1 receptors in rat brain cortex.