Automated synthesis of peptides.
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Automation↗
Biomedical subjects
R B Merrifield
Publications and source records attributed to R B Merrifield.
Automated peptide synthesis.
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Amino Acid Sequence↗
Hydrophobic effects on antibacterial and channel-forming properties of cecropin A-melittin hybrids.
The design of cecropin-melittin hybrid analogues is of interest due to the similarities in the structure of the antimicrobial peptides cecropin and melittin but differences in their lytic properties. We suspected that a hydrophobic residue in position 2 of milittin (Ile8 in the hybrid) plays an important role in the activity of the 15-residue hybrid, KWKLFKKIGAVLKVL-NH2, [CA(1-7)M(2-9)NH2] and have now examined its role in the analogue toward five test bacteria. Deletion of Ile8 reduced activity, and it was not restored by lengthening to 15 residues by addition of another threonine at the C-terminus. Replacement of Ile8 by a hydrophobic leucine maintained good activity and Ala8 was equally active for four organisms, although less active against Staphylococcus aureus. Replacement by the hydrophilic Ser8 strongly reduced potency against all five organisms. Deletion of Leu15 decreased activity, but addition of Thr16 maintained good activity. The presence of hydrophobic residues appears to have a significant effect on the process of antibacterial activity. These peptide analogues showed voltage-dependent conductance changes and are capable of forming ion-pores in planar lipid bilayers. The antibacterial action of the peptides is thought to be first an ionic interaction with the anionic phosphate groups of the membrane followed by interaction with the hydrocarbon core of the membrane and subsequent reorientation into amphipathic alpha-helical peptides that form pores (ion-channels), which span the membrane. The analogue also showed an increase in alpha-helicity with an increase in hexafluoro 2-propanol concentration.
Amino Acid Sequence↗
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.
Adenylyl Cyclases↗
D-enantiomers of 15-residue cecropin A-melittin hybrids.
The all-D enantiomers of six 15-residue hybrids of cecropin A and melittin were synthesized. They contained the seven N-terminal residues of cecropin A, followed by eight residues from the N-terminal region of melittin. They were pure and of the correct composition and structure. The peptides were compared with their all-L enantiomers. The L and D isomer pairs were each exact mirror images by circular dichroism at several concentrations of hexafluoroisopropanol, and at 12 or 20% were highly helical. The L analogs were rapidly hydrolyzed by trypsin but the D analogs were very resistant, making them suitable candidates for orally active drugs. These 15-mers did not form ion channels in normal lipid bilayers made in decane, but those bilayers made in squalene were thinner and the peptides did form ion-conducting channels. The D/L pairs of peptides were very active antibiotics against five representative Gram-negative and Gram-positive bacteria. In each case the D and L isomers were essentially equally active within experimental error. This is interpreted to mean that the peptides do not act by tight interactions with chiral receptors, enzymes or lipids. The action of these peptides against these organisms is best explained by self-aggregation and the formation of ion-conducting pores across bacterial membranes.
Anti-Bacterial Agents↗
Mechanisms of aspartimide formation: the effects of protecting groups, acid, base, temperature and time.
Factors affecting aspartimide formation, such as protecting groups, acidity, basicity, and temperature, were studied using the model tetrapeptide, Glu-Asp-Gly-Thr. The aspartyl carboxyl side chain in this tetrapeptide was either free or protected as a benzyl or cyclohexyl ester. Our results showed that the cyclohexyl ester led to far less aspartimide formation during acidic or tertiary amine treatment than the corresponding benzyl ester. The rate constants of aspartimide formation in HF-anisole (9:1, v/v) for the tetrapeptide protected as the benzyl ester were found to be 6.2 x 10(-6) and 73.6 x 10(-6) s-1 at -15 degrees and 0 degrees C respectively. These values were about three times faster than the corresponding free- or cyclohexyl ester-protected tetrapeptide. Little difference was seen when the studies were carried out at room temperature. The cyclohexyl protected tetrapeptide gave only 0.3% aspartimide in diisopropylethylamine treatment in 24 h, a 170-fold reduction of imide formation when compared with the benzyl protected tetrapeptide. Thus, using the cyclohexyl ester for aspartyl protection, our studies showed aspartimide formation could be significantly reduced to less than 2% under standard peptide synthesis conditions. Furthermore, with these model peptides, the mechanism of acid catalyzed aspartimide was studied in a range of HF concentrations. In dilute HF cleavage conditions (HF:dimethylsulfide 1:3, v/v), the mechanism was found to be of the AAC2 type, with the rate of aspartimide formation increasing very slowly with increasing acid concentration. In concentrated HF solutions (HF greater than 70% by volume), the rate of aspartimide formation increased rapidly with the increase in acid concentration. However, from model studies, the mechanism of aspartimide formation in concentrated HF was AAC2 rather than AAC1.
Acids↗