R. B. Merrifield

Binding and action of cecropin and cecropin analogues: antibacterial peptides from insects.

The mechanism of action of cecropin was studied by using liposomes as a model system. The bilayer was efficiently destroyed if the liposome net charge was zero or negative. Cecropin analogues with an impaired N-terminal helix had reduced membrane disrupting abilities that correlate with their lower antibacterial activity. The reduced bactericidal activity of the analogues was rationalized in terms of reduced binding to bacteria. The stoichiometry of cecropin killing of bacteria suggests that amounts of cecropin sufficient to form a monolayer strongly modify the bacterial membrane. Although some bacteria were resistant to cecropin they did bind large amounts in a non-productive manner. In contrast, mammalian erythrocytes achieve resistance by avoiding the binding of cecropin.

Antibacterial and antimalarial properties of peptides that are cecropin-melittin hybrids

Solid phase synthesis was used to produce 5 hybrid peptides containing sequences from the antibacterial peptide, cecropin A, and from the bee venom toxin, melittin. Four of these chimeric peptides showed good antibacterial activity against representative Gram‐negative and Gram‐positive bacterial species. The best hybrid, cecropin A(1–13)‐melittin(1–13) was 100‐fold more active than cecropin A against Staphylococcus aureus. It was also a 10‐fold better antimalarial agent than cecropin B or magainin 2. Sheep red cells were lysed by melittin at low concentrations, but not by the hybrid molecules, even at 50 times higher concentrations.

Solid-Phase Peptide Synthesis

It is a privilege to be able to contribute to this volume in which Professor Zervas’ friends, students, and colleagues have joined together to honor him and his remarkable contributions to peptide chemistry. Although I did not know him until after his days in the Bergmann Laboratory at the Rockefeller Institute, I can claim the distinction of now working in those very same rooms that they occupied back in the mid-1930s. Like all peptide chemists, I am greatly indebted to Professor Zervas, having depended so heavily on the carbobenzoxy group and on the various modified urethan protecting groups which have been direct extensions of the revolutionary advance that Bergmann and Zervas made.

Biological activities of des-His1[Glu9]glucagon amide, a glucagon antagonist

Hyperglycemia in diabetes mellitus is generally associated with elevated levels of glucagon in the blood. A glucagon analog, des-His1[Glu9]glucagon amide, has been designed and synthesized and found to be an antagonist of glucagon in several systems. It has been a useful tool for investigating the mechanisms of glucagon action and for providing evidence that glucagon is a contributing factor in the pathogenesis of diabetes. The in vitro and in vivo activities of the antagonist are reported here. The analog bound 40% as well as glucagon to liver membranes, but did not stimulate the release of cyclic AMP even at 10(6) higher concentration. However, it did activate a second pathway, with the release of inositol phosphates. In addition, the analog enhanced the glucose-stimulated release of insulin from pancreatic islet cells. Of particular importance were the findings that the antagonist also showed only very low activity (less than 0.2%) in the in vivo glycogenolysis assay, and that at a ratio of 100:1 the analog almost completely blocked the hyperglycemic effects of added glucagon in normal rabbits. In addition, it reduced the hyperglycemia produced by endogenous glucagon in streptozotocin diabetic rats. Thus, we have an analog that possesses properties that are necessary for a glucagon antagonist to be potentially useful in the study and treatment of diabetes.

Orientation of Cecropin A Helices in Phospholipid Bilayers Determined by Solid-State NMR Spectroscopy

The orientation of the insect antibiotic peptide cecropin A (CecA) in the phospholipid bilayer membrane was determined using (15)N solid-state NMR spectroscopy. Two peptide samples, each specifically labeled with (15)N at Val(11) or Ala(27), were synthesized by solid phase techniques. The peptides were incorporated into phospholipid bilayers, prepared from a mixture of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol, and oriented on glass slides. The (15)N chemical shift solid-state NMR spectra from these uniaxially oriented samples display a single (15)N chemical shift frequency for each labeled residue. Both frequencies are near the upfield end of the (15)N chemical shift powder pattern, as expected for an alpha-helix with its long axis in the plane of the membrane and the NH bonds perpendicular to the direction of the magnetic field. These results support a mechanism of action in which CecA binds to and covers the membrane surface, thereby causing a general destabilization and leakiness of the lipid bilayer membrane. The data are discussed in relation to a proposed mechanism of membrane lysis and bacterial killing via an ion channel activity of CecA.

Effects on electrophoretic mobility and antibacterial spectrum of removal of two residues from synthetic sarcotoxin IA and addition of the same residues to cecropin B

Cecropin B and cecropin IA (sarcotoxin IA) are 35‐ and 39‐residue antibacterial peptides from a silk moth and a meat fly, respectively. Using solid phase synthesis we have made these peptides as well as two 37‐residue analogs, one containing a deletion of leucine and lysine (residues 2a and 2b) as compared to cecropin IA, the other containing an insertion of leusine and lysine at the corresponding place in cecropin B. This addition and removal of a lysine residue did not cause the expected change in electrophoretic mobility. When tested for antibacterial spectra, the insertion analog was found to be as active as the parent compound while the deletion analog had lost most of its antibacterial capacity. In addition it was shown that the C‐terminal amide contributes to the broad spectrum properties of the cecropins.

A chromatographic method for the quantitative analysis of the deprotection of dithiasuccinoyl (Dts) amino acids

Abstract The dithiasuccinoyl (Dts)-protecting group for amino acids is revoved by thiols (2 equivalents) through the intermediacy of an open-chain carbamoyl disulfide. Starting materials, intermediates, and products can be separated from one another on the standard amino acid analyzer 0.9 × 54-cm column of sulfonated polystyrene resin with 0.2 n sodium citrate buffers. Compounds are detected with the standard ninhydrin-hydrindantin reagent because the hydrindantin acts as a reducing agent and the released amino acid reacts in situ with the ninhydrin to give a purple color. Elution times, integration constants, and the ratios of absorbances at 570 and 440 nm are tabulated. Quantitative conversion of dithiasuccinoyl amino acids to the parent amino acids can be achieved with 0.1 n sodium hydroxide, 0.01 m alcoholic sodium borohydride, 0.1 m triphenylphosphine or 2 m m tri-n-butylphosphine in dioxane-H2O (9:1) or water, and with a variety of thiols under various conditions. The chromatographic methodology is applicable to the determination of rate constants of the pseudo-first-order reductive deprotection of dithiasuccinoyl amino acids.

Specificity of Peptides: New aspects of the specificity of peptides with vitamin and hormone action are described.

The data now at hand show that specificity among biologically active peptides is probably not as exquisite as might have been deduced from experiences with some of the water-soluble vitamins. It is plain that a given biological effect can be evoked by peptides which differ considerably. It is likewise plain that the effect cannot be evoked by a number of other peptides. There is apparently some specificity, but more is involved than that incorporated in the classical view.

Positively Charged Residues at Positions 12, 17, and 18 of Glucagon Ensure Maximum Biological Potency

Glucagon is a peptide hormone that plays a central role in the maintenance of normal circulating glucose levels. Structure-activity studies have previously demonstrated the importance of histidine at position 1 and the absolute requirement for aspartic acid at position 9 for transduction of the hormonal signal. Site-directed mutagenesis of the receptor protein identified Asp64 on the extracellular N-terminal tail to be crucial for the recognition function of the receptor. In addition, antibodies generated against aspartic acid-rich epitopes from the extracellular region competed effectively with glucagon for receptor sites, which suggested that negative charges may line the putative glucagon binding pocket in the receptor. These observations led to the idea that positively charged residues on the hormone may act as counterions to these sites. Based on these initial findings, we synthesized glucagon analogs in which basic residues at positions 12, 17, and 18 were replaced with neutral or acidic residues to examine the effect of altering the positive charge on those sites on binding and adenylyl cyclase activity. The results indicate that unlike N-terminal histidine, Lys12, Arg17, and Arg18 of glucagon have very large effects on receptor binding and transduction of the hormonal signal, although they are not absolutely critical. They contribute strongly to the stabilization of the binding interaction with the glucagon receptor that leads to maximum biological potency.

BIOPOLYMER SYNTHESIS ON SOLID SUPPORTS

The concept of solid phase synthesis began as a new approach to the preparation of peptides, but has been extended to the synthesis of polynucleotides, polysaccharides, polyamides and a variety of other classes of compounds. The solid support simplifies and accelerates the synthetic process, aids the separation of reagents and by-products, and reduces problems of insolubility of intermediates. The synthesis may involve a stepwise addition of monomer units or the condensation of preformed oligomer fragments. Large numbers of peptides and several small proteins have been synthesized by the solid phase method. To facilitate the synthesis We have developed a new, more acid-stable attachment of the peptide to the polystyrene support, in which the chain is anchored through a hydroxymethylphenylacetamidomethyl group. Alternatively, the peptide chain can be anchored through a more labile alkoxybenzyl ester linkage, if the very acid-sensitive Bpoc group is used for Nα-protection. The ideal design for a multi-step synthesis will depend on a combination of protecting groups that are removable by completely independent reaction mechanisms. Such a system is called orthogonal. A new amino protecting group, the dithiasuccinoyl (Dts) group, has been designed for that purpose. It is removed by reduction, but is stable to acids and photolysis and can be used in conjunction with these two orthogonal modes of deprotection. The synthesis of biopolymers on solid supports was illustrated by studies of the role of arginine in the neurotoxin, apamin, and of the substrate specificity of a histone deacetylase and by a recent synthesis of crystalline glucagon.