Glutathione peptide synthesis

The preparation of glutathion by methods of peptide synthesis is expansive and gives 20-30% yield of GHS. For the first time synthetic glutathion was prepared by M. Bergmann et al. 

This chapter provides a manual for a laboratory-hased short course to introduce the common techniques of solid-phase peptide synthesis (SPPS). The course provides students the opportunity to design and manually synthesize analogs of glutathione using relatively simple equipment available in any unsophisticated laboratory. The manual provides compact protocols for both the different steps of SPPS and the final cleavage of peptides from resin supports. We also introduce a simple method for the synthesis of combinatorial libraries of glutathione analogs that is suitable for those relatively unfamiliar with the field of peptide chemistry. 

Key Words Solid-phase peptide synthesis r-Boc chemistry Fmoc chemistry glutathione. 

The study of de novo peptide bond formation by isolated enzyme systems is restricted at the present time to a few low-molecular-weight compounds. Of the various compounds that have been used as models for the investigation of enzymatic peptide synthesis, glutathione (GSH) is of particular interest, since the synthesis of this tripeptide has remained the only system which lends itself to the study of de novo formation of a peptide bond from free amino acids. Whether the mechanism of this synthesis is representative of peptide bond formation in general is at present unknown. 

The possible role of glutathione in peptide synthesis in isolated enz3rmes has also been shown negligible by the studies of Waelsch and of Hanes. The most important function of glutathione thus remains that of a regulator of cellular metabolism. This regulation is maintained by the equilibrium value of the reversible system... 

Acylation of amines, synthesis of linear and cyclic peptides, including glutathione and bradykinin Peptide synthesis... 

The enzymes which catalyze the incorporation of glycine into glutathione are extractable. In pigeon liver, it is only these soluble enzymes which take part in the synthesis, whereas the insoluble particulate matter shows no enzymatic activity (325). The number of glutathione peptide bonds formed was calculated from experiments in which glutathione... 

ATP is involved in the synthesis of hippuric acid and its analogs. In the case of hippuric acid, and very probably of its analogs, the role of ATP is to form the aromatic acyl-Co A, which then condenses with glycine to form the quasi-peptide. But in the synthesis of glutamine from glutamic acid and ammonia Co A is not required, ATP is. And in the synthesis of glutathione, the one case so far elucidated of an enzymatic peptide synthesis from amino acids, again Co A appears not to be required, while ATP is. 

In contrast to the preceding observations and conclusions, there are reports from one laboratory that the mechanism of peptide formation resembles that of protein synthesis. Cellfree preparations of B. hrevis were observed to synthesize gramicidin and tyrocidine provided that both a 140,000 X g supernatant solution and a ribosomal preparation are present (in addition to Mg+, ATP, phosphoenol-pyruvic acid, pyruvic kinase, glutathione, and amino acid mixture). The supernatant solution presumably supplies s-RNA and amino acid activating enzymes. In this system, peptide synthesis is suppressed by exposure of either the supernatant or ribosomal portions to RNase (Uemura et al., 1963)- Furthermore, neither chloramphenicol nor puromycin are capable of differentiating protein from peptide synthesis in this system the formation of each of the products is inhibited 98% by either 10 (xg/ml of the former or 100 fi.g/ml of the latter antibiotic (Okuda et al., 1964b). 

Aminoacid peptides (c.f. Z-/ -Ala-/ -Ala-OC2H5, 75% yield) have also been prepared by this pyrazolide method. 31 A glutathione synthesis was accomplished by the pyr-azolide method in the following way [32]... 

They are required for synthesis of peptides or proteins that have a specific role in trauma e.g. antibodies, acute-phase proteins, cytokines, glutathione. 

Deprotection of peptides. In a synthesis of bovine pancreatic ribonucleasc, the 33 protective groups required were removed by three treatments with l M TEMSA thioanisole at 0° for 60 minutes. The groups were Cbz, r-butyl, p-methoxybenzyl, and p-methoxybenzenesulfonyl. The final step involved air oxidation of the cysteine residues to disulfides mediated by glutathione.2... 

Peptide Fluoromethyl Ketones Fluoroalkyl derivatives of the peptide chloromethyl ketones have been prepared in an attempt to improve specificity by reducing nonspecific alkylation at cysteine residues (Rasnick, D., Synthesis of peptide fluoromethyl ketones and the inhibition of human cathepsin B, Anal. Biochem. 149, 461 65, 1985). Nonspecific reaction with sulfydryl groups such as those in glutathione was reduced there was still reaction with active site cysteine although at a slower rate than with the chloroalkyl derivative (16,200 M s vs. 45,300 1 2 21.9 min. vs. 

It is important to further develop the concept of structure-activity relationships to precisely define the structural requirements of glutathione action. Thus, this section introduces the design, synthesis, and screening of a peptide combinatorial library to obtain multiple glutathione analogs. Combinatorial libraries will be composed of mixtures of peptides (consisting of natural or noncoded amino acids) on solid support. After cleavage from the resin, the mixtures of the peptides will be screened directly in different specific assays. 

You will synthesize a small peptide library based on glutathione, introducing three different substitutions by mixtures of four natural or noncoded amino acids. This synthesis creates a mixture of four peptides at the first step of the synthesis, 16 at the second step, and 64 at the third step (Fig. 2). 

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