Transamidation peptides

NH3 or RNH2 or R2NH Amide formation from carboxylic acid derivatives (mild) or from carboxylic acids (A technical synthesis of nylon-6,6) transamidation [capro-lactame —> nylon-6 (perlon)] Peptide synthesis (Section 6.4.3)... 

Proteins destined to be GPI-linked are synthesized in the endoplasmic reticulum and attached to preassembled GPI anchors within 1 min after the protein s synthesis [97,98]. The newly synthesized C-terminal sequence is rapidly replaced by the GPI anchor in what appears to be a transamidase reaction [99]. ATP and GTP stimulate processing of the C-terminal peptide in an in vitro microsome assay system [100], suggesting that a nucleotide-dependent step occurs prior to transamidation. However, it is not clear what role this nucleotide-dependent step would have in attaching a GPI anchor. 

Transpeptidation, transamidation, a reaction involving the transfer of one or more amino acids from one peptide chain to another. This term was first coined by Fruton, in 1950, by analogy with transglycosidation for the papain-catalyzed displacement reaction between Bz-Gly-NH2 and aniline forming Bz-Gly-NHPh. Of special importance in relation to protease-catalyzed transpeptidation reactions in a preparative scale is the one-step tryptic conversion of porcine insulin into human insulin, despite the controversial interpretation of the mechanism involved. A bacterial transpeptidase, serim-type u-Ala-u-Ala carhoxypeptidase (EC... 

In 2012, Ohshima and co-workers reported microwave-assisted transamidative deacylation of unactivated amides with a combination of an ammonium salt and ethylene diamine at 50-90°C [105]. In 2014, the same group realized hydrazinolysis of unactivated amide bonds [106]. The reaction proceeded at moderate temperature (50-70°C) to provide M-acyl hydrazines and amines in good yield. They applied the reaction conditions to cleave peptides (Fig. 16). Selective cleavage of the Gly-Phe bonds in two peptides was realized. No epimerization at the a-position of the Phe residue was observed. The site-selectivity was likely caused by both steric and electronic factors. 

Factor X, fibrin-stabilizing factor the last clotting factor to act in the blood coagulation cascade. It is an a2-plasma globulin of M, 350,000 and contains 2a- and 2P-chains of M, 100,000 and 77,000, respectively. It is activated by thrombin in the presence of Cs to factor Xllla, which catalyses the formation of y-glutamyl-E-lysine peptide bonds in a calcium-dependent transamidation reaction. These bonds serve to cross-link the fibrin chains into a 3-dimensional network, the clot. 

In summarizing, it must be realized that most of all acidic conditions to remove a synthetic peptide from its gel phase support include the possibility for undesired attacks on either protected or free peptide side functions as well as on the backbone, causing fissions and conversions also during work-up manipulations of already detached raw products. This is the case because most of the usually employed protecting principles — urethanes, esters, and ethers as well as some functional sites of a peptide such as alcoholic, thioUc, and amide side chain groups — can be involved in proton catalyzed eliminations, transesterification, transamidations, and cyclol formations, though some of these side reactions usually are rather feared under basic conditions. 

A similar problem associated with synthetic peptides of chain length > 5 amino acid residues is the identification of side reactions. Those involve substitutions on imidazole-, phenol- and indole moieties of histidine, tyrosine, and tryptophane as well as conversions, transamidation, and cyclizations of aspartic and glutamic side chains. As long as structural variations are stable under the reaction conditions of an Edman degradation, they can be detected from the proper phenylthiohydantoines in combination with H-NMR- and mass spectrometry. Quantitative amino acid analyses of impure peptides after acidic total hydrolysis do not indicate those structural deviations between main product and contaminations. 

Figure 5.12 Generation and screening of P-endorphin peptides generated by protease-catalyzed transamidation.

All the known transpeptidation and transamidation reactions are of the carboxyl-transfer type and so far no example has been reported of the alternative type which might be envisaged, namely transfer of the amino moiety of a donor peptide to new linkage ivith the carboxyl group of an acceptor. 

Peptide Synthesis in an Exchange Reaction during Hydrolysis (Transamidation and Transpbptidation)... 

All peptide bonds are cleaved by transamidation to give all component amino acids as their corresponding hydrazides. Only the carboxy end retains its free carboxy function. 

Johnston,. Mycek, and Fruton (95) extended these observations and found that transamidation with papain proceeded more rapidly at pH 8 than at pH 5. Furthermore, hydroxylamine was found to be an effective replacement agent, yielding hydroxamic acids. The specificity of papain toward the substrate in these reactions was similar to that for the hydrol rtic reaction. In a later study (97) it was demonstrated that amino acid amides or peptides could serve as replacing agents, e.g., papain catalyzed the following synthesis ... 

On this basis the extent to which an enzyme-catalyzed transamidation will occur will depend on the relative concentration of water and the replacement reagent, and the relative affinity of the replacement agent for the ES-complex. Durell and Fruton (51) have studied papain-catalyzed hydroxamic acid formation from o-benzoyl-L-argininamide. Their calculations show that hydroxylamine is about 420 times more efficient in its reaction with the enzyme-substrate complex than is water. Preliminary observations suggest that when amino acid amides or peptides are the attacking molecule the efficiency is even greater. Moreover, papain is a much more effective catalyst for transamidation than trypsin. 

The addition of the GPI anchor to the C-terminal domain of glycoproteins occurs post-translationally and involves a C-terminal GPI-directing signal sequence (10-30 residues at the C-terminus that are largely hydrophobic), which directs the specific attachment of a GPI anchor [46], The removal of the C-terminal peptide and addition of the GPI anchor is catalyzed in the ER by an unidentified enzyme termed a transamidase that catalyzes a transamidation reaction [47-49]. 

A number of hydrolytic enzymes, for example the plant proteolytic enzymes papain and ficin, are also able to permit transfer reactions, in these cases transamidations. The evidenee in such cases is that there occurs co-valent bonding of an intermediate to the enz)rme and that this enzyme-intermediate compound retains much of the bond energy of the peptide bond. The intermediate compound in these cases is an enzyme bound thioester involving the sulphydryl group of a cysteine residue of the enzyme. 

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