Synthesis of side chain-protected amino acid phenylthiohydantoins and their use in quantitative solid-phase Edman degradation

Solid-phase Edman degradation of synthetic peptidyl-resins has been used advantageously to detect errors of deletion which might occur during Merrifield peptide synthesis. To facilitate complete quantitation of the resulting phenylthiohydantoin(PTH)-amino acids, the PTH derivatives of the following side chain-protected amino acid residues have been synthesized: Arg(Tos), Asp(OBzl), Cys(3,4-(CH3)2-Bzl), Glu(OBzl), Lys(2-ClZ), Ser(Bzl), Thr(Bzl), Tyr(2-BrZ), and Tyr(2,6-Cl2Bzl). For each derivative, a melting point, elemental analysis, and extinction coefficient were obtained. With these new compounds as HPLC standards, an unequivocal assignment and quantification of each side chain protected amino acid was possible. A quantitative analysis was performed for six model peptides with the general formula Ala-X-Leu-Y-Ala-Gly-NHCH2-resin (where X and Y represented different side chain-protected amino acyl residues). We have found solid-phase Edman degradation to be a useful aid for the characterization of peptides when they are used unpurified as synthetic antigens.     

Effect of alpha,alpha-dialkyl amino acids on the protease resistance of peptides

A tryptic [EC 3.4.21.4] digestion assay of 2-aminoisobutyric acid (Aib)-containing peptides was carried out to investigate the effect of alpha,alpha-dialkyl amino acid residues on the protease resistance. The introduction of Aib residues to the P1' positions exhibited a 19-fold higher protease resistance than the peptide with Aib residues introduced to the P2 position or the non-Aib peptide. The peptide having Aib residues introduced to the P1' and P2 positions resulted in complete resistance.     

The signal transfer regions of G alpha(s)

The crystal structure of soluble functional fragments of adenylyl cyclase complexed with G alpha(s) and forskolin, shows three regions of G alpha(s) in direct contact with adenylyl cyclase. The functions of these three regions are not known. We tested synthetic peptides encoding these regions of G alpha(s) on the activities of full-length adenylyl cyclases 2 and 6. A peptide encoding the Switch II region (amino acids 222-247) stimulated both adenylyl cyclases 2- to 3-fold. Forskolin synergized the stimulation. Addition of peptides in the presence of activated G alpha(s) partially inhibited G alpha(s) stimulation. Corresponding Switch II region peptides from G alpha(q) and G alpha(i) did not stimulate adenylyl cyclase. A peptide encoding the Switch I region (amino acids 199-216) also stimulated AC2 and AC6. The stimulatory effects of the two peptides at saturating concentrations were non-additive. A peptide encoding the third contact region (amino acids 268-286) located in the alpha 3-beta 5 region, inhibits basal, forskolin, and G alpha(s)-stimulated enzymatic activities. Since this region in G alpha(s) interacts with both the central cytoplasmic loop and C-terminal tail of adenylyl cyclases this peptide may be involved in blocking interactions between these two domains. These functional data in conjunction with the available structural information suggest that G alpha(s) activation of adenylyl cyclase is a complex event where the alpha 3-beta 5 loop of G alpha(s) may bring together the central cytoplasmic loop and C-terminal tail of adenylyl cyclase thus allowing the Switch I and Switch II regions to function as signal transfer regions to activate adenylyl cyclase.     

Synthesis and solubility properties of peptide fragments of human hemoglobin alpha-chain (123-136)

The solubility prediction method for protected peptides was successfully applied to relatively small peptide fragments of human hemoglobin alpha-chain (123-136) which contained various polar amino acid residues such as Asp(OBzl), Glu(OBzl), Lys(Z), Ser(Bzl), and Thr(Bzl). As reported previously for hydrophobic peptides and human proinsulin C-peptide fragments, solubility data indicated that the insolubility of protected peptides having a mean value of Pc value below 0.90 appeared to begin at the octa- or nonapeptide sequence level and that beta-sheet structure played an important role in the insolubility of peptides. When a peptide has a beta-sheet structure in the solid state, we can clearly determine the critical chain length for peptide insolubility, the solubility dependence on solvent properties, and the solubility independence of amino acid compositions of peptides.     

Synthesis by conventional methods of human growth hormone peptide fragments. I.

The synthesis of two protected peptides which correspond to positions 139-146 and 147-156 of the HGH primary structure is described. These peptides prepared by the stepwise procedure, are: Boc-Phe-Lys(Z)-Gln-Thr(Bzl)-Ser(Bzl)-Lys(Z)-Phe-OMe and Boc-Asp(OBzl)-Asn-Ser(Bzl)-His(Dnp)-Asn(OBzl)-Asp(OBzl)-Ala-Leu-OBzl. All protected intermediates were isolated and characterized for homogeneity.     

Optimized selective N-methylation of peptides on solid support.

Peptides containing N(alpha)-methylamino acids exhibit interesting therapeutic profiles and are increasingly recognized as potentially useful therapeutics. Unfortunately, their synthesis is hampered by the high price and nonavailability of many N(alpha)-methylamino acids. An efficient and practical three-step procedure for selective N-methylation of peptides on solid support is described. The procedure was based on the well known solid-phase N-methylation of N(alpha)-arylsulfonyl peptides, which was improved by using dimethylsulfate and the less expensive DBU as base. Every step of the procedure, amine activation by an o-nitrobenzenesulfonyl group, selective N-methylation and removal of the sulfonamide group, was optimized in respect of time and economy. The described optimized three-step procedure is performed in 35 min without solvent changes, instead of 3 h. Tripeptides (Fmoc-Phe-MeXaa-Leu-OH) containing N-methylated common amino acids were also prepared using the optimized procedure to demonstrate its compatibility with these amino acids. The described procedure allows an efficient synthesis of N(alpha)-methylamino acid containing peptides in a very short time using Fmoc solid-phase peptide synthesis.     

Amino acid sequences of the alpha and beta chains of adult hemoglobin of the brown lemur, Lemur fulvus fulvus.

Globin prepared from hemoglobin of the brown lemur (Lemur fulvus fulvus) was separated into alpha and beta chains by chromatography on a CM 52 column. The S-aminoethylated alpha and beta chains were each digested with trypsin and resulting peptides were isolated. The amino acid sequences of the tryptic peptides were established. The ordering of these peptides in the alpha and beta chains was deduced from the homology of their amino acid sequences with that of human adult hemoglobin. The primary structure of brown lemur hemoglobin thus obtained differs from that of human hemoglobin in 15 amino acids in the alpha chain and 26 in the beta chain.     

Identification of the site of interaction of the dihydropyridine channel blockers nitrendipine and azidopine with the calcium-channel alpha 1 subunit.

The dihydropyridine binding site of the rabbit skeletal muscle calcium channel alpha 1 subunit was identified using tritiated azidopine and nitrendipine as ligands. The purified receptor complex was incubated either with azidopine or nitrenidpine at an alpha 1 subunit to ligand ratio of 1:1. The samples were then irradiated by a 200 W UV lamp. The ligands were only incorporated into the alpha 1 subunit, which was isolated by size exclusion chromatography and digested either by trypsin (azidopine) or endoproteinase Asp-N (nitrendipine). Each digest contained two radioactive peptides, which were isolated and sequenced. The azidopine peptides were identical with amino acids 13-18 (minor peak) and 1428-1437 (major peak) of the primary sequence of the skeletal muscle alpha 1 subunit. The nitrendipine peptides were identical with amino acids 1390-1399 (major peak) and 1410-1420 (minor peak). The sequence from amino acids 1390 to 1437 is identical in the alpha 1 subunits of skeletal, cardiac and smooth muscle and follows directly repeat IVS6. These results indicate that dihydropyridines bind to an area that is located at the putative cytosolic domain of the calcium channel.     

Covalent structure of collagen: amino acid sequence of three cyanogen bromide-derived peptides from human alpha 1(V) collagen chain

Type V collagen was prepared from human amnionic/chorionic membranes and separated into alpha 1(V) and alpha 2(V) polypeptide chains. The alpha 1(V) chain was digested with cyanogen bromide and nine peptides were obtained and purified. Three of the peptides, alpha 1(V)CB1, CB4, and CB7 having molecular weights of 5000, 8000, and 6000, respectively, were further analyzed by amino acid sequence analysis and thermolytic or tryptic digestions. CB1 contained 54 amino acids and identification of its complete sequence was aided by thermolysin digestion and isolation of two peptides, Th1 and Th2. CB4 contained 81 amino acids and sequence analysis of intact CB4 and five tryptic peptides provided us with its complete amino acid sequence. The peptide CB7 contained 67 amino acids and was cleaved into four tryptic peptides that were used for complete sequence analysis. The above results represent the first available covalent structure information on the alpha 1(V) collagen chain. These data enabled us to establish the location of these peptides within the helical structure of other collagen chains. CB4 was homologous to residues 66-145 in the collagen chain while CB1 represented residues 146-200 and CB7 was homologous with residues 201-269. This alignment was facilitated by identification of a helical collagen crossing site consisting of Hyl-Gly-His-Arg located at positions 87-90 in all collagen chains of this size thus far identified. Seventy-one percent homology (excluding Gly residues) was found between amino acids in this region of the alpha 1(XI) and of alpha 1(V) collagen chains while only 21 and 19% identity was calculated for the same region of alpha 2(V) and alpha 1(I) collagen chains, respectively.     

Amphipathic control of the 3(10)-/alpha-helix equilibrium in synthetic peptides.

A series of short, amphipathic peptides incorporating 80% C(alpha),C(alpha)-disubstituted glycines has been prepared to investigate amphipathicity as a helix-stabilizing effect. The peptides were designed to adopt 3(10)- or alpha-helices based on amphipathic design of the primary sequence. Characterization by circular dichroism spectroscopy in various media (1 : 1 acetonitrile/water; 9 : 1 acetonitrile/water; 9 : 1 acetonitrile/TFE; 25 mM SDS micelles in water) indicates that the peptides selectively adopt their designed conformation in micellar environments. We speculate that steric effects from ith and ith + 3 residues interactions may destabilize the 3(10)-helix in peptides containing amino acids with large side-chains, as with 1-aminocyclohexane-1-carboxylic acid (Ac(6)c). This problem may be overcome by alternating large and small amino acids in the ith and ith + 3 residues, which are staggered in the 3(10)-helix.     

Retinal cyclic-GMP phosphodiesterase gamma-subunit: identification of functional residues in the inhibitory region.

Previously, we have domain-mapped the 87 amino acid PDE gamma inhibitory subunit of the retinal phosphodiesterase (PDE) alpha beta gamma 2 complex using synthetic peptides. The PDE gamma subunit has a binding domain for transducin-alpha (T alpha) and for PDE alpha/beta within residues # 24-45 and an inhibitory region for PDE alpha/beta within residues # 80-87. In order to establish the role of individual amino acids in the function of the PDE gamma inhibitory subunit, peptides of PDE gamma # 63-87 and mutant peptides were synthesized and utilized in PDE inhibition assays. The following peptides exhibited a decreased ability to inhibit PDE alpha/beta: All were from PDE gamma # 63-87; PDE gamma Tyr 84----Gly, PDE gamma Phe 73----Gly and PDE gamma Gln 83----Gly.     

A novel lactococcal bacteriocin whose activity depends on the complementary action of two peptides.

A lactococcal bacteriocin, termed lactococcin G, was purified to homogeneity by a simple four-step purification procedure that includes ammonium sulfate precipitation, binding to a cation exchanger and octyl-Sepharose CL-4B, and reverse-phase chromatography. The final yield was about 20%, and nearly a 7,000-fold increase in the specific activity was obtained. The bacteriocin activity was associated with three peptides, termed alpha 1, alpha 2, and beta, which were separated by reverse-phase chromatography. Judging from their amino acid sequences, alpha 1 and alpha 2 were the same gene product. Differences in their configurations presumably resulted in alpha 2 having a slightly lower affinity for the reverse-phase column than alpha 1 and a reduced bacteriocin activity when combined with beta. Bacteriocin activity required the complementary action of both the alpha and the beta peptides. When neither alpha 1 nor beta was in excess, about 0.3 nM alpha 1 and 0.04 nM beta induced 50% growth inhibition, suggesting that they might interact in a 7:1 or 8:1 ratio. As judged by the amino acid sequence, alpha 1 has an isoelectric point of 10.9, an extinction coefficient of 1.3 x 10(4) M-1 cm-1, and a molecular weight of 4,346 (39 amino acid residues long). Similarly, beta has an isoelectric point of 10.4, an extinction coefficient of 2.4 x 10(4) M-1 cm-1, and a molecular weight of 4110 (35 amino acid residues long). Molecular weights of 4,376 and 4,109 for alpha 1 and beta, respectively, were obtained by mass spectrometry. The N-terminal halves of both the alpha and beta peptides may form amphiphilic alpha-helices, suggesting that the peptides are pore-forming toxins that create cell membrane channels through a "barrel-stave" mechanism. The C-terminal halves of both peptides consist largely of polar amino acids.     

Enantio- and diastereoselective syntheses of cyclic Calpha-tetrasubstituted alpha-amino acids and their use to induce stable conformations in short peptides

C(alpha)-tetrasubstituted alpha-amino acids are widely used to design and prepare peptides and peptide mimics with constrained conformations. Subcategories of these compounds are cyclic C(alpha)-tetrasubstituted alpha-amino acids, in which both alpha-substituents are covalently connected. This survey presents recent advances in the synthesis and application of cyclic C(alpha)-tetrasubstituted alpha-amino acids in a systematic order beginning with cyclopropane amino acids, continuing with four, five, six membered rings, and ring structures larger than six-membered. We discuss synthetic routes to the cyclic C(alpha)-tetrasubstituted alpha-amino acids and their use as conformation determining elements in peptides.     

Stimulation of yeast mating hormone activity by synthetic oligopeptides.

The biological activities of two synthetic oligopeptides (His-Trp-Leu-Gln-Leu and Trp-Leu-Gln-Leu), which represent part of the primary structure of the mating hormone alpha factor from Saccharomyces cerevisiae, were studied. The peptides did not exhibit hormonal activity by themselves. However, both intensified the mating-type-specific inhibitory effect of native alpha factor on the division of haploid cells of mating type a. Random peptides or mixtures of the corresponding amino acids did not stimulate alpha factor activity. Likewise, a synthetic peptide representing another part of the alpha factor sequence was ineffective. In addition, the activity of a factor, the mating hormone produced by a cells, was not influenced by the synthetic peptides, indicating that the compounds specifically affect the interaction between alpha factor and its target cells. The analysis of the utilization of the tetrapeptide as a source of amino acids for auxotrophic a strains suggested an extracellular site of action for the observed enhancement of alpha factor activity.     

Role of acidic amino acids in peptide substrates of the beta-adrenergic receptor kinase and rhodopsin kinase.

The beta-adrenergic receptor kinase (beta-ARK) phosphorylates G protein coupled receptors in an agonist-dependent manner. Since the exact sites of receptor phosphorylation by beta-ARK are poorly defined, the identification of substrate amino acids that are critical to phosphorylation by the kinase are also unknown. In this study, a peptide whose sequence is present in a portion of the third intracellular loop region of the human platelet alpha 2-adrenergic receptor is shown to serve as a substrate for beta-ARK. Removal of the negatively charged amino acids surrounding a cluster of serines in this alpha 2-peptide resulted in a complete loss of phosphorylation by the kinase. A family of peptides was synthesized to further study the role of acidic amino acids in peptide substrates of beta-ARK. By kinetic analyses of the phosphorylation reactions, beta-ARK exhibited a marked preference for negatively charged amino acids localized to the NH2-terminal side of a serine or threonine residue. While there were no significant differences between glutamic and aspartic acid residues, serine-containing peptides were 4-fold better substrates than threonine. Comparing a variety of kinases, only rhodopsin kinase and casein kinase II exhibited significant phosphorylation of the acidic peptides. Unlike beta-ARK, RK preferred acid residues localized to the carboxyl-terminal side of the serine. A feature common to beta-ARK and RK was a much greater Km for peptide substrates as compared to that for intact receptor substrates.     

Cyclic peptides. XXVI. Synthesis of AM-toxin II analogs by cyclization through ester bond formation

In order to explore the route for the preparation of cyclodepsipeptide by cyclization through an ester bond formation, two analogs of AM-toxin II, cyclotetradepsipeptide, were synthesized. As a preliminary experiment, synthesis of [L-Phe3, L-Ser(Bzl)4]-AM-toxin II, containing L-Phe and L-Ser(Bzl) in place of L-App (2-amino-5-phenyl-pentanoic acid) and delta Ala (alpha, beta-dehydroalanine), respectively, was attempted. Cyclization of H-L-Hmb-L-Phe-L-Ser(Bzl)-L-Ala-OH in CH2Cl2 at 10 mM concentration using water-soluble carbodiimide (EDC) and 4-dimethylaminopyridine (DMAP) successfully gave a cyclic monomer in 16% yield. Cyclization of H-L-Hmb-L-App-L-Ser(Bzl)-L-Ala-OH under the same conditions also afforded a cyclic monomer, [L-Ser(Bzl)4]AM-toxin II, in 19% yield. Analytical parameters of these cyclic monomers obtained were identical to those of the authentic samples obtained by cyclization through a peptide bond formation.     

Goodpasture syndrome. Localization of the epitope for the autoantibodies to the carboxyl-terminal region of the alpha 3(IV) chain of basement membrane collagen.

The autoantibodies of patients with Goodpasture syndrome are primarily targeted to the noncollagenous (NC1) domain of the alpha 3(IV) chain of basement membrane collagen (Saus, J., Wieslander, J., Langeveld, J. P. M., Quinones, S., and Hudson, B. G. (1988) J. Biol. Chem. 263, 13374-13380). In the present study, the location of the Goodpasture epitope in human alpha 3NC1 was determined, and its structure was partially characterized. This was achieved by identification of regions of alpha 3NC1 which are candidates for the epitope and which are structurally unique among the five known homologous NC1 domains (alpha 1-alpha 5); amino acids that are critical for Goodpasture antibody binding, by selective chemical modifications; and regions that are critical for Goodpasture antibody binding, by synthesis of 12 alpha 3NC1 peptides and measurement of their antibody binding capacity. The carboxyl-terminal region, residues 198-233, was identified as the most likely region for the epitope. By experiment, lysine and cysteine were identified as critical amino acids for antibody binding. Three synthetic peptides were found to inhibit Goodpasture antibody binding to alpha 3NC1 markedly: a 36-mer (residues 198-233), a 12-mer (residues 222-233), and a 5-mer (residues 229-233). Together, these results strongly indicate that the Goodpasture epitope is localized to the carboxyl-terminal region of alpha 3NC1, encompassing residues 198-233 as the primary antibody interaction site and that its structure is discontinuous. These findings provide a conceptual framework for future studies to elucidate a more complete epitope structure by sequential replacement of residues encompassing the epitope using cDNA expression products and peptides synthesized chemically.     

Extensive homology between the carboxyl-terminal peptides of mouse alpha 1(IV) and alpha 2(IV) collagen

We have determined the complete primary structure for the carboxyl-terminal peptides of mouse alpha 1(IV) and alpha 2(IV) collagen; which have 229 and 227 amino acids, respectively. The amino acid sequences are 63% identical and conservatively substituted in 28 positions. A striking feature of these peptides is that the first half of each sequence is homologous with the second half, 37% in alpha 1(IV) and 36% in alpha 2(IV). These results suggest that the carboxyl-terminal peptides of type IV collagen are closely related in their structure and evolution. Presumably, they were first derived by internal duplication of a common ancestral DNA sequence which later, by gene duplication, gave rise to the two different but homologous carboxyl-terminal peptides of type IV collagen.     

Structure of a yeast pheromone gene (MF alpha): a putative alpha-factor precursor contains four tandem copies of mature alpha-factor

We have cloned and sequenced a gene (MF alpha) coding for alpha-factor, a tridecapeptide mating factor secreted by yeast alpha cells. A plasmid carrying the MF alpha gene was identified by screening for production of alpha-factor by mat alpha 2 mutants, which fail to secrete alpha-factor because of simultaneous synthesis and degradation of the factor. The cloned segment codes for four mature alpha-factor within a putative precursor of 165 amino acids. The putative precursor begins as a signal sequence for secretion. The next segment, of approximately 60 amino acids, contains three potential glycosylation sites. The carboxy-terminal half of the precursor contains four tandem copies of mature alpha-factor, each preceded by spacer peptides of six or eight amino acids (variations of Lys-Arg-Glu-Ala-Asp-Ala-Glu-Ala), which are hypothesized to contain proteolytic processing signals.     

Synthesis of chicken galanin and its N- and C-terminal segments, and preparation of their antisera

We describe the synthesis of the first aviangalanin (GAL), chicken GAL, and its N-terminal and C-terminal segments by solid-phasesynthesis, using Boc/Bzl amino acidprotection groups and MBHA resin. The three peptides were prepared with purities of over97%, as determined by RP-HPLC, HPCE, FAB-MS or ESI-MSand amino acid analysis.Antibodies against these synthetic peptides wereraised in rabbits and used forimmunohistochemical localization of GAL-immunoreactiveneurons in chicken brain.