Synthesis of biologically active cyclic peptides

1. The extent of racemization and the coupling yield in peptide synthesis were studied under high dilution conditions. The azide method yielded the best results. 2. Five linear penta-peptide precursors related to gramicidin S were subjected to cyclization in order to study how the difference in the sequence influences the yield and the ratio of cyclic dimer to monomer. The azide with the sequence of -L -Pro-L -Val-L -Orn(Z)-L -Leu-D -Phe- afforded diZ-gramicidin S in a high yield of 63%. 3. Alternaria mali toxin III, a cyclotetradepsipeptide phytotoxin, was synthesized. The activated linear tetradepsipeptide containing a D -Dap(Z) (N³-Z-D -2,3-diaminopropionic acid) residue at the N-terminus afforded the cyclic precursor (53%). The Dap residue in the precursor was converted into a ΔAla residue by Hofmann degradation to give the desired product.     

Synthetic, biologically active amphiphilic peptides

Amphiphilic peptides typically consist of a peptide portion that may be 5-25 (or more) amino acids in length. The hydrophobic portion may be a single fatty acid residue, but can also be more elaborate. The main focus of this article lies on the family of synthetic anion binders (SATs) of the general structure (R(1))(2)N-COCH(2)OCH(2)CO-(Aaa)(n)-OR(3). The most-common R(1) group is the octadecyl (C(18)H(37)) group. The most studied peptide sequence in this family is (Gly)(3)-Pro-(Gly)(3), although different sequences (and longer and shorter peptides) have been prepared as well. The C-terminal ester residue providing the most effective anion release from liposomes is heptyl (C(7)H(15)), although many others have been examined. The compound (C(18)H(37))(2)N-COCH(2)OCH(2)CO-(Gly)(3)-Pro-(Gly)(3)-OBn (Bn=benzyl) was found to mediate Cl(-) transport in mouse epithelial cells.     

Prediction of location of active sites in biologically active peptides

In a previous paper we demonstrated that the short-range compact regions in atrial natriuretic factor (-hANF) predicted by the average distance map (ADM) correspond to its active sites [Kikuchi,J. Protein Chem.11, 579–581 (1992)]. In the present paper we apply the same method to other bioactive peptides and peptidic enzyme inhibitors. We again observe that active sites in each peptide are contained in short-range compact regions predicted by the ADM for the peptide. This demonstrates that the ADM method predicts the possible location of active sites in biologically active peptides in general. The possibility of practical application of the present method to rational drug design is also discussed.     

Lipotropin: precursor to two biologically active peptides.

Lipotropin appears to be the common precursor to β-MSH, a peptide with lipolytic activity, and C-Fragment, a peptide with potent opiate activity. The product formed is determined by the specificity of the activating enzymes.The amino acid sequence of β-MSH, the 18 residue melanocyte stimulating hormone, is contained within the central region of lipotropin (LPH), a 91 residue polypeptide. On this basis Li and his colleagues¹ suggested that LPH might be the prohormone of β-MSH. Bertagna, Lis and Gilardeau², on the other hand, were unable to demonstrate conversion of LPH to β-MSH $\text{in vitro}$ using pulse labelling techniques. If LPH is the precursor of β-MSH, formation of the hormone should be accompanied by release of the contiguous fragments of the prohormone and the fragments remain in the secretory particle of the gland. To obtain evidence on the biosynthetic origin of β-MSH, we have isolated peptides from pituitary in a search for the N- and C-fragments of the prohormone.     

Rodent submandibular gland peptide hormones and other biologically active peptides

The cervical sympathetic trunk-submandibular gland neuroendocrine axis plays an integral role in physiological adaptations and contributes to the maintenance of systemic homeostasis, particularly under the 'stress conditions' seen with tissue damage, inflammation, and aggressive behavior. The variety of polypeptides, whose release from acinar and ductal cells is under sympathetic nervous system control, offers coordinated and progressive levels of endocrine communication. Proteolytic enzymes (e.g. the kallikreins and furin maturases) are involved in the conversion of inactive precursors (e. g. Pro-EGF and SMR1) into biologically active molecules (e.g. EGF, SMR1-pentapeptide), which act on local or distant targets and thereby modulate the homeostatic process.     

Peptide-lipid interaction monitored by spin labeled biologically active melanocortin peptides

The present work comparatively analyzes the interaction of alpha-MSH and its more potent and long-acting analog [Nle4, D-Phe7]alpha-MSH (NDP-MSH) with lipid bilayers. The peptides were spin labeled with Toac (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at the N-terminal, as those derivatives had been previously shown to keep their full biological activity. Due to the special rigidity of the Toac covalent binding to the peptide molecule, this spin label is highly sensitive to the peptide backbone conformation and dynamics. The peptides were investigated both by the electron spin resonance (ESR) of Toac0 and the time resolved fluorescence of Trp9 present in the peptides. The Toac0 ESR of the membrane-bound peptides indicates that the two peptides are inserted into the bilayer, close to the bilayer surface, in rather similar environments. A residue titration around pKa 7.5, possibly that of His6, can be clearly monitored by peptide-lipid partition. Trp9 time resolved fluorescence indicates that the peptides, and their Toac-labeled derivatives, present rather similar conformations when membrane bound, though Trp9 in NDP-MSH, and in its Toac-labeled derivative, goes somewhat further down into the bilayer. Yet, Toac0 ESR signal shows that the Toac-labeled N-terminal of NDP-MSH is in a shallower position in the bilayer, as compared to the hormone.     

Release of biologically active peptides from Escherichia coli at subzero temperatures

Moss, C. Wayne (North Carolina State University, Raleigh), and M. L. Speck. Release of biologically active peptides from Escherichia coli at subzero temperatures. J. Bacteriol. 91:1105-1111. 1966.-Freezing and storage of Escherichia coli at -20 C in phosphate buffer resulted in loss of cell viability and a pronounced leakage of cellular material which had maximal absorption at 260 mmu. Greater loss in cell viability occurred when cells were frozen in distilled water, but only small amounts of 260 mmu absorbing material were detected. Unfrozen cells stored at 2 and 22 C in each menstruum showed little loss in viability, but cells in phosphate buffer released significant amounts of material during storage. Leakage material from cells in phosphate buffer contained greater amounts of ribonucleic acid and amino acids than did material from cells in distilled water. Leakage material from frozen cells contained protein in the form of peptides of relatively small molecular weight; this was not observed for unfrozen cells. These compounds protected a dilute cell suspension from the lethal effects of freezing, and also possessed biological activity for the recovery of cells which had been "injured" by freezing. Direct cell counts indicated that the material released was not a result of cell lysis.     

Conformational and topographical considerations in the design of biologically active peptides

An outline of the basic considerations that are under development for the rational design of biologically active peptides and peptidomimetics is given. The necessary interplay of biophysical, chemical, and biological considerations is emphasized. The importance of properly designed biological assays to provide chemical information analogous to that from biophysical studies is discussed. The development of asymmetric synthesis in conjunction with conformational considerations for the preparation of specialized amino acids and amino acid mimetics is a critical aspect of the approach. The overall approach is illustrated with three examples from our laboratory: (1) the redesign of somatostatin to a highly potent and selective μ-opioid receptor antagonist using conformational and topographical considerations in design and for obtaining insights into the pharmacophor; (2) the use of topographical considerations for obtaining oxytocin antagonists; and (3) the application of designer amino acids prepared by asymmetric synthesis to obtain insight into the topographical requirements at δ-opioid receptors. © 1993 John Wiley & Sons, Inc.     

The effects of intracerebroventricular administration of biologically active peptides in conscious sheep

The present study records the physiological effects of 24-hour intracerebroventricular infusion of a variety of biologically active peptides in conscious sheep. A number of peptides including AVP and TRH produced increases in mean arterial pressure, heart rate and body temperature. There was an overall positive correlation between peptide-induced changes in body temperature and changes in either mean arterial pressure or heart rate. TRH and β-endorphin had marked effects on behaviour and several peptides reduced food and water intake. Several peptides increased urinary sodium excretion, however, few peptides changed plasma electrolyte concentrations. TRH produced small effects on plasma ACTH and plasma glucose concentrations. The peptides in this study produced physiological changes which were probably mediated by their actions on the central nervous system.     

High-performance immobilized metal ion affinity chromatography of peptides: analytical separation of biologically active synthetic peptides

The separation of more than 30 biologically active synthetic peptides and their analogs on a high-performance immobilized metal ion affinity chromatography column is described. The metal chelating gel (TSK gel chelate-5PW) contains iminodiacetic acid (IDA) covalently coupled to a hydrophilic, resin-based matrix with a bead diameter of 10 micron. The retention of the peptides on Cu(II), Ni(II), and Zn(II) ions immobilized on the chelating gel showed that some of them can be separated by isocratic elution while the majority of them are retained and are separated into distinct fractions by elution with a linear imidazole gradient or with a continuously decreasing pH gradient. Of the three immobilized metal ions investigated here, the IDA-Cu(II) chelate column gave the best resolution irrespective of the type of gradient used. This is amply illustrated by the resolution of angiotensins I and II and their seven synthetic analogs. The results obtained here serve as guidelines for the future exploitation of this separation method for the efficient fractionation of a wide variety of peptides on an analytical or preparative scale.     

Selection of biologically active peptides by phage display of random peptide libraries

Random peptide libraries displayed on phage are used as a source of peptides for epitope mapping, for the identification of critical amino acids responsible for protein—protein interactions and as leads for the discovery of new therapeutics. Efficient and simple procedures have been devised to select peptides binding to purified proteins, to monoclonal and polyclonal antibodies and to cell surfaces in vivo and in vitro.     

Separation of biologically active peptides by capillary electrophoresis and high-performance liquid chromatography

HPLC and CE have been applied to the separation of some newly synthesized substances, including nonapeptides from the intrachinary region of insulin, insulin-like growth factors I and II (IGF I and II) and some penta- and hexapeptides. All the peptides are satisfactorily separated using a reversed-phase HPLC system with a C₁₈ stationary phase and mobile phases of 20–40% acetonitrile (v/v) and 0.2% trifluoroacetic acid in water (v/v). The best CE separation of IGF I and II has been achieved in a 30 mM phosphate buffer (pH 4–5), whereas 150 mM phosphoric acid (pH 1.8) is optimal for the insulin nonapeptides. The latter electrolyte is also suitable for the CE separation of the hexapeptides, as is a micellar system containing 20 mM borate-50mM sodium dodecyl sulfate (pH 9.0). Complete CE resolution of the d- and l-forms is possible in a 50 mM phosphate buffer (pH 2.5) containing 10 mM β-cyclodextrin. UV spectrophotometric detection was used throughout, at wavelengths from 190 to 215 nm. The CE procedures are, in general, preferable to HPLC separations, as they exhibit better separation efficiencies, are faster and consume smaller amounts of analytes and reagents.     

Conformational restrictions of biologically active peptides via amino acid side chain groups

Determining the relationships between conformation and biological activity in peptide hormones and neurotransmitters is an important goal of contemporary biology. A major difficulty in these studies is the conformational flexibility of most peptides and the high dependence of the conformations on environment. The question arises whether conformations determined in solution are relevant to those important to the peptide at the membrane receptor(s). One recent approach to overcome these difficulties has been the use of conformational constraints by covalent bonding of side chain groups of residues in the peptide. In this manner linear peptides are rendered cyclic, and cyclic peptides are further conformationally constrained either by ring contractions or by other conformational constraints. Biologically active peptides specifically designed by this approach have been found to possess several useful properties including: 1) greater conformational integrity; 2) increased agonist or antagonist potency; 3) prolonged biological activity; 4) increased enzymatic stability; and 5) increased specificity for a particular receptor. Careful applications of this approach have provided important new designs features for peptide structure-function studies, and new insights into peptide conformation-activity relationships for oxytocin, somatostatin, enkephalin, bradykinin, vasopressin, and other biologically active peptides.     

Synthesis and application of methyleneoxy pseudodipeptide building blocks in biologically active peptides

Summary. Pseudodipeptides H-Phe[CH₂O]Phe-OH, H-Tyr[CH₂O] Asp-OH and H-Pro[CH₂O]-D-Thr-OH were synthesized using the intramolecular Williamson reaction via substituted morpholin-3-one ring with the nitrogen atom protected with bulky Boc group. This protection and the substituent at C₅ position induced the stereospecific alkylation at the C₂ position introducing the side chain of the C-terminal amino acid mimetic. In the first pseudodipeptide a quenching of the enolate with benzaldehyde was followed by dehydration and corresponding double bond was hydrogenated with high stereospecific purity. In the other pseudodipeptides, this alkylation was carried out directly by tert-butyl 2-bromoacetate or acetaldehyde. However, in the latter reaction an R configuration of C₃ substituent in conjugated lactame ring was determined using a NOE NMR. Consequently, after opening this ring by acidic hydrolysis, the C-terminal part of corresponding pseudodipeptide possessed the side-chain of D-Thr mimetic, contrary to former one. Synthesized pseudodipeptides were introduced into HIV protease inhibitors and into peptides with oostatic activity.     

Cloning of prepronociceptin has led to the discovery of other biologically active peptides

Among the opioid receptors family, the cloning of the mu, kappa and delta receptors was followed by that of another member, named ORL1 (Opiate Receptor Like 1). In spite of obvious homologies with the mu, kappa and delta receptors, ORL1 does not display a relevant affinity for the endogenous ligands of these former receptors (beta endorphin, enkephalins, dynorphin A...). This observation has prompted to search for an endogenous ligand of ORL1. A heptadecapeptide which fulfils this function, with a nanomolar affinity, has been found. It was named either nociceptin or orphanin FQ. It demonstrates, according either to the dose or to the route of administration, hyperalgesic, allodynic, antiopioidergic or even analgesic effects. It displays also many behavioural effects, modifying especially locomotion, exploratory behaviour, motivation, anxiety, memory, food intake. Nociceptin results from the cleavage of a large precursor protein, prepronociceptin (PPNOC). In this latter, nociceptin is flanked on its C-terminal region by another peptide which may be regarded either as a heptadecapeptide (NocII), or a bidecapeptide (NocIII) according to the inclusion or not of a fragment constituted by 3 arginine residues. Investigating the functions modulated by NocII, we observed that it stimulates locomotor activity of mice and shortens the forepaws licking latency in the hot plate test (55 degrees C); these effects are not shared by NocIII. The simultaneous administration of NocII and nociceptin resulted in animals put on the hot plate to the appearance of their respective effects, not modified by the presence of the other. A 41 amino acid peptide flanks nociceptin on its N-terminal region in PPNOC. It may be cleaved to generate a heptadecapeptide, named nocistatin on account of its antagonist effect on the hyperalgesia/allodynia induced by nociceptin. Thus, the discovery of ORL1 has led to that of nociceptin, that of its precursor PPNOC, and thereby to that of NocII/NocIII and nocistatin. The functions modulated by these peptides are being investigated whereas their receptors are yet unknown. These multiple targets allow to expect new strategies to modulate their functions.     

Role of proteolysis in posttranslational modifications of biologically active peptides and polypeptides]

Data from literature about the precursors of biologically active peptides and polypeptides in cells are summarized. Their processing by limited proteolysis and the enzymes which take part in this process are presented.