Peptides intermediates, synthesis

A van Vliet, RH Smulders, BH Rietman, GI Tesser. Protected peptide intermediates using a trityl linker on a solid support, in R Epton, ed. Innovations and Perspectives in Solid Phase Synthesis. Proceedings of the 2nd Symposium. Intercept, Andover,... 

It is interesting to note that serine peptidases can, under special conditions in vitro, catalyze the reverse reaction, namely the formation of a peptide bond (Fig. 3.4). The overall mechanism of peptide-bond synthesis by peptidases is represented by the reverse sequence f-a in Fig. 3.3. The nucleophilic amino group of an amino acid residue competes with H20 and reacts with the acyl-enzyme intermediate to form a new peptide bond (Steps d-c in Fig. 3.3). This mechanism is not relevant to the in vivo biosynthesis of proteins but has proved useful for preparative peptide synthesis in vitro [17]. An interesting application of the peptidase-catalyzed peptide synthesis is the enzymatic conversion of porcine insulin to human insulin [18][19]. 

Its use in solution synthesis is limited by the presence of sulfur-containing anoino acid residues as these poison metal catalysts, albeit methods have been proposed that partially overcome this serious drawback (see Section 2.1.1.1.1.1.3.1). This linoitation is also bypassed in cases where the target peptide molecule permits acidolysis with strong acids (HBr/AcOH or HF). As an additional limitation, the saponification of Al -Z-protected peptide esters under drastic conditions, such as those required for longer peptide intermediates, was found to induce decomposition of the Al -Z moiety with generation of N-ternninal hydantoins. ... 

The solid-phase technique has now been automated, and computer-controlled peptide synthesizers are available for automatically repeating the coupling and deprotection steps with different amino acids. Each step occurs in high yield, and mechanical losses are minimized because the peptide intermediates are never removed from the insoluble polymer until the final step. Among the many remarkable achievements recorded by Merri-field is the synthesis of bovine pancreatic ribonuclease, a protein containing 124 amino acid units. The entire synthesis required only 6 weeks and took place in 17% overall yield. 

Not only transpeptidation, but also a certain amount of peptide-peptide condensation, is possibly involved in the plastein reaction. With some of the proteases used for plastein formation, especially a-chymotrypsin (26, 52, 53, 54), the acyl-enzyme intermediate can be formed at pHs 5 from the reversal of the degradative reaction (Equation 1 E-OH + HOOC-CHR-NH E-O-OCCHR-NH- + H20). Once the acyl-enzyme intermediate is formed, the acyl group can be transferred to a nucleophile resulting in peptide bond synthesis. 

Aso et al. [95] studied a model system in order to obtain basic information on the mechanism of amino acid incorporation during an enzymatic modification reaction in the presence of papain. They found that the amino acid ester reacted as a nucleophile in the aminolysis of the acyl-enzyme intermediate to result in the formation of new peptides. Several proteases used in enzymatic peptide bond synthesis are known to form transitory acyl-enzyme intermediates during the hydrolysis of proteins. However, the acyl groups can be transferred to other nucleophiles (amino terminals of peptides or amino acids), synthesizing new peptide bonds [71]. With full knowledge of the above-mentioned facts, covalent amino acid enrichment of proteins can result in... 

While the heritability of flavor precursor accumulation in onions has not been determined, it is most likely a quantitatively inherited trait. As described earlier, the biosynthetic pathway is complex with many peptide intermediates. n addition, 11 proposed enzymes regulate compound synthesis in the flavor pathway U7). Further empirical evidence to support quantitative inheritance of the flavor... 

The methodology of solid phase peptide synthesis (SPPS) [65, 66] has been credited with the award of 1984 Nobel Prize in chemistry [67] to its inventor, Bruce R. Merrifield of the Rockefeller University. At the heart of the SPPS lies an insoluble polymer support or gel , which renders the synthetic peptide intermediates insoluble, and hence readily separable from excess reagents and by-products. In addition to peptide synthesis, beaded polymer gels are also being studied for a number of other synthetic and catalytic reactions [2]. Ideally, the polymer support should be chemically inert and not interfere with the chemistry under investigation. The provision of chemical inertiKss presents no difficulty, but the backbone structure of the polymer may profoundly influence the course of the reaction on the polymer support. This topic has attracted considerable interest, particularly in relation to the properties of polystyrene (nonpolar, hydrophobic), polydimethylacrylamide (polar, hydrophilic), and copoIy(styrene-dimethylaciylamide) (polar-nonpolar, amphiphilic) (see later). 

We have shown that upon applying our approach to the synthesis of soluble protected peptide intermediates, very hi yields per coupling step were obtained as compared to the classical solution methods. While it is commonly accepted that on using the standard solution synthetic mediod the average yield per coupling step is... 

Although ACMS I, II, aixl III contain all necessary sites for activation of 4 MHA and the hve amino acids, total cell-free synthesis of 4-MHA pentapeptide lactone with these three enzymes has not yet been accomplished. Data firom the one of our laboratories (UK) indicate that at least ACMS I and ACMS 11 are functionally intact, which allows the formation and accumulation of reaction intermediates on the surface of ACMS II when substrates are present. However, these intermediates were not found to be further transferred to ACMS III. Nevertheless, analysis of the peptide intermediates formed covalently bound to ACMS 11 in the additional presence of ACMS I enabled the cstab Ushment of the mechanisms of initiation, elongation, and epimerization in 4 MHA pen tapeptide lactone formation up to position 2 in the peptide ting sequence. 

The development of cell free systems has played an important role in attempts to decipher the mechanism of peptide antibiotic synthesis ". Most workers agree that this type of synthesis occurs in the complete absence of polynucleotides and that the amino acid sequence is determined by enzyme specificity and organisation in a multi-enzyme complex . The tyrocidines are produced in Bacillus brevis by an enzyme system which has been resolved into three complimentary fractions by Sephadex G-200 filtration a light and an intermediate component (molecular weight 100000 and 230000) which activate L-phenylalanine and L-proline respectively and a heavy fraction (460 000) which activates the remaining eight amino acids including L-phenylalanine. Each... 

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