Pepsin as a catalyst for peptide synthesis: formation of peptide bonds not typical for pepsin substrate specificity.

Porcine pepsin in water solutions containing 15-28% of dimethylformamide at pH 5 and 20-37 degrees C catalysed the formation of peptide bonds between Z-Ala-Ala-Phe-OH and various amino acid or peptide derivatives. Substrate binding subsite S1' of pepsin demonstrated broad specificity in these reactions but revealed a certain preference for hydrophobic amino acid residues, including non-proteinous homophenylalanine, p-nitrophenylalanine, S-methylcysteine, as well as for those that contained, in addition to the hydrophobic elements, a group capable of donating a hydrogen bond, e.g. o-nitrotyrosine. This observation increases the range of peptides that might be prepared by pepsin-catalysed synthesis.     

肽合成中多对二硫键的形成策略及分析方法

二硫键的正确配对是富含二硫键多肽合成的关键。本文综述了含两对二硫键以上的多肽二硫键的形成策略,优化方法、以及二硫键配对方式的测定方法。     

How ribosomes make peptide bonds

Ribosomes are molecular machines that synthesize proteins in the cell. Recent biochemical analyses and high-resolution crystal structures of the bacterial ribosome have shown that the active site for the formation of peptide bonds--the peptidyl-transferase center--is composed solely of rRNA. Thus, the ribosome is the largest known RNA catalyst and the only natural ribozyme that has a synthetic activity. The ribosome employs entropic catalysis to accelerate peptide-bond formation by positioning substrates, reorganizing water in the active site and providing an electrostatic network that stabilizes reaction intermediates. Proton transfer during the reaction seems to be promoted by a concerted shuttle mechanism that involves ribose hydroxyl groups on the tRNA substrate.     

High-yield cleavage of tryptophanyl peptide bonds by o-iodosobenzoic acid.

A new procedure to cleave tryptophanyl peptide bonds in high yield is reported. The method involves treatment of the S-alkylated protein with o-iodosobenzoic acid. The procedure is highly selective for tryptophan and does not modify tyrosine or histidine, but may convert methionine to its sulfoxide derivative. The yields in the cleavage are 70--100%. Tryptophanyl bonds to alanine, glycine, serine, threonine, glutamine, arginine, and S-(pyridylethyl)cysteine are split in nearly quantitative yield, while those preceding isoleucine or valine are split in approximately 70% yield in the proteins examined in this work. The chemical mechanism for tryptophanyl bond cleavage has not been defined, but it is likely that oxidation of the indole ring occurs during the reaction with o-iodosobenzoic acid. Some problems with the quality of commercial preparations of the reagent are discussed.     

The partial charge of the nitrogen atom in peptide bonds.

A majority of the standard texts dealing with proteins portray the peptide link as a mixture of two resonance forms, in one of which the nitrogen atom has a positive charge. As a consequence, it is often believed that the nitrogen atom has a net positive charge. This is in apparent contradiction with the partial negative charge on the nitrogen that is used in force fields for molecular modeling. However, charges on resonance forms are best regarded as formal rather than actual charges and current evidence clearly favors a net negative charge for the nitrogen atom. In the course of the discussion, new ideas about the electronic structure of amides and the peptide bond are presented.     

Formation of peptide bonds by carboxypeptidase c from orange leaves.

Carboxypeptidase c partially purified from orange leaves was studied as a catalyst for enzymatic peptide synthesis. Various N-protected ester- and nucleophile compounds were evaluated in order to determine the substrate specificity. For further characterization of the synthetic reaction, optimum pH and the influence of the N-terminal protecting group were studied. Kinetic investigations revealed considerable differences in Km and Vmax for the nucleophile when the N-terminal protecting group of the substrate was varied.     

Enhancement of solubility by temporary dimethoxybenzyl-substitution of peptide bonds. Towards the synthesis of defined oligomers of alanine and of lysyl-glutamyl-glycine

The synthesis of the model compound Aloc-Ala-Ala-Dma-Ala-Ala-OMe has been described as an illustration of the fact that a large group reversibly alkylating the amido group of an oligomer can disturb the regularity of a peptide backbone, oppose its aggregation and thus enhance its solubility greatly, affording synthons for further oligomerization. Application of such a group not only affects the solubility, but alters also the properties of the intermediates. The concomitant change in reactivity may run to such an extent that N-alkylation of oligomers has to be abandoned (this was encountered in the attempted synthesis of Lys-Glu-Dmg). Consequently, the solubility of the growing protected peptide chain will become progressively less and in the mentioned example the oligomerization had to be terminated at the dodecapeptide level, indicating the severe need for reversible "structure-breaking" functions.     

The proteinase-catalysed synthesis of peptide hydrazides.

1. We report the trypsin-catalysed conversion, in high yield, of peptides to peptide hydrazides, t-butyloxycarbonylhydrazides and phenylhydrazides. The substitution is at the alpha-carboxy group. 2. We discuss the relative merits of carrying out the conversion either simultaneously with tryptic cleavage of the parent protein or after such cleavage. 3. We report analogous results with chymotrypsin, elastase and subtilisin. 4. We propose the use of such products in protein semi-synthesis and in the preparation of specific proteinase inhibitors.     

Influence of lithium cations on prolyl peptide bonds

The influence of lithium cations on the cis/trans isomerization of prolyl peptide bonds was investigated in a quantitative manner in trifluoroethanol (TFE) and acetonitrile, employing NMR techniques. The focus was on various environmental and structural aspects, such as lithium cation and water concentrations, the type of the partner amino acid in the prolyl peptide bond, and the peptide sequence length. Comparison of the thermodynamic parameters of the isomerization in LiCl/TFE and TFE shows a lithium cation concentration dependence of the cis/trans ratio, which saturates at cation concentrations >200 mM. A pronounced increase in the cis isomer content in the presence of lithium cations occurs with the exception of peptides with Gly‐Pro and Asp‐Pro moieties. The cation effect appears already at the dipeptide level. The salt concentration can considerably be reduced in solvents with a lower number of nucleophilic centers like acetonitrile. The lithium cation effect decreases with small amounts of water and disappears at a water concentration of about 5%. The isomerization kinetics under the influence of lithium cations suggests a weak cation interaction with the carbonyl oxygen of the peptide bond. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.