The Peptide Bond Formation

After deprotection and liberation of the amino groups, the polymer-bound peptide is prepared to become acylated by another N-protected building block, which usually is an amino acid. The dissolved carboxylic component is activated before the admixture with the polymer or in the presence of it, followed by intense mixing of the phases. 

Most of the activation methods known from conventional peptide synthesis in solution were explored on polymer concerning their desired reactivity in relation to side reactions, which are particularly unfavorable if the by-products are also bound to the support. Though urethane-masked amino acids generally are shielded from racemization under controlled conditions, this problem limits the use of peptidic building blocks C-terminally activated, since they tend to form the redoubtable oxazolinone intermediate from which the abstraction of a -proton is facilitated (Fig. 40). Recent results indicated a chance to overcome this problem and will be mentioned in the proper section of this chapter. 

To meet the fundamental demand for completion of all reactions on polymer, the activated carboxylic component usually is added in great excess, which naturally enhances the possibility of side reactions on the solute and on polymer as well. In the following, the main methods employed in the Merrifield synthesis, including those very opposing aspects like complete transformations, however free from side reactions, have to be judged and taken into consideration. 

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To direct the synthesis so that only Phe-Gly is formed, the amino group of phenylalanine and the car boxyl group of glycine must be protected so that they cannot react under the conditions of peptide bond formation. We can represent the peptide bond formation step by the following equation, where X and Y are amine- and carboxyl-protecting groups, respectively ... 

Pretazettine (395) has been the subject of numerous biological studies, and it has been shown to exhibit a number of interesting activities (96,97,101,178-187). For example, 395 was found to inhibit HeLa cell growth as well as protein synthesis in eukaryotic cells by interfering with the peptide bond formation step (97,101). Furthermore, pretazettine inhibited the purified RNA-dependent DNA polymerase (reverse transcriptase) from avian myeloblastosis virus, a typical C-type virus (178), in an unusual fashion since it physically combined with the polymerase enzyme itself rather than interacted with the nucleic acid template. Pretazettine also exhibited antiviral activity against the Rauscher leukemia virus in mouse embryo cell cultures by suppressing viral replication (179). 

The peptides 3 were prepared by condensation of phtaloyl,N-carbobenzoxy,N-t-butoxycarbonyl or N-fomyl-amino acids with 1-aminoalkanephosphonic acids as well as with their dialkyl or diphenyl esters /Scheme/. Special attention was payed on theefectiveness of the peptide bond formation and on the methods for selective and total removal of the blocking groups. 

The so-called ribozymes (Box 22) were discovered in 1982 by T. Cech and S. Altman. The naturally occurring species catalyze predominantly one reaction type -hydrolysis or transesterification of phosphodiester bonds in RNA. A very important natural ribozyme is the ribosome. On the basis of X-ray crystallographic investigations it was recently shown that the active site for the peptide bond-formation reaction is composed exclusively of RNA. 

Trp, Leu, Met), and it was selected for making the Tyr-Gly, Phe-Leu, and Phe-Met peptide bonds. Papain was selected for Gly-Gly and Gly-Phe peptide bonds. Bromelain is very similar to papain as far as its specificity. All these proteases are serine or cysteine type, and the peptide bond formation can be done under kinetically controlled conditions. Thermolysin is an asparyl protease, and it was selected mainly for Phe-Leu and Gly-Phe bonds. MeCN, EtOAc, and methyl caproate were used as solvents with a controlled amount of buffer or at fixed water amount. 

As shown in Fig. 12.5-8 the equilibrium of a peptidase-catalyzed reaction is normally shifted to the thermodynamically more stable cleavage products. In contrast to proteolysis, the peptide bond formation is a two-substrate reaction and requires not only a specificity-dependent insertion of the carboxyl component into the S-subsites of the active site, but also an optimal binding of the amine component in the S region. To shift the equilibrium in favor of fragment product formation various... 

Inhibitor of protein synthesis, blocking the peptide bond formation (272,273)... 

The oxidation-reduction method, developed initially by Mukaiyama et al. [133] and related to the previously described organophosphorus methods, has permitted a variety of important solid-phase applications. The mechanism of the activation is complex and involves the oxidation of the triaryl/ alkyl-phosphine to the oxide as well as reduction of the disulfide to the mercapto derivative. However, different active species, such as 81 (Fig. 11), the 2-pyridyl thioester, or even the symmetrical anhydride, have been postulated to form. For the intermediate 81, the peptide bond formation may proceed through a (cyclic transition state. The method has been used for conventional stepwise synthesis [134], acylation of the first protected amino acid to a hydroxymethyl resin, and to achieve segment condensation on a solid support in the opposite direction (N C) [135,136]. Lastly, it has been used for efficient grafting of a polyethylene glycol (molecular weight 2000) derivative to an aminomethyl resin to prepare PEG-PS resins [137]. 

The ribosomal protein biosynthetic machinery encompasses all three types of modnlarity. The ribosome catalyst can be separated from the element determining the snbstrate specificity, i.e. the mRNA template. The two acylated tRNA snbstrates for the peptide-bond formation bind to different ribosomal sites, the A and P sites, and the mnltistep pathway catalyzed by the modnlar system may be reprogrammed by codon choice. 

The first formal studies of peptide bond formation can be traced back to 1881, when Curtius obtained Bz-Gly-Gly through the treatment of glycine silver salt with benzoyl chloride [3]. A notable later milestone was Fisher s synthesis of an octadecapeptide LGGGLGGGLGGGGGGGGG in 1907, where the a-halogen was utilized as the precursor of a-amine as a means of protection during the peptide bond formation [4]. 

Figure I Schematic views of reactions involved in peptide biosynthesis. (1) Adenylate formation involving nucleophilic attack of the carboxyl group at the a-phosphate of the MgATP --complex with release of MgPP. - (2) aminoacylation of the pantetheine cofactor by formation of the thio-late anion, attack of the mixed anhydride, and release of AMP (3) tentative view of the peptide bond formation by nucleophilic attack of the aminoacyl-nitrogen at the preceding thioester-car-boxyl, involving deprotonaiion-protonation (4) epimerization of an aminoacyl-thioester, a reaction differing from those catalyzed by the well-characterized amino acid racemases. (Altered from Ref. 13. )...

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