Enzymatic fragment condensation

The synthetic scheme is shown in Fig. 15 (138), which was based on 4 + 1 enzymatic fragment condensation. A -Cbz-Tyr-Gly-Gly-Phe-Leu-NH2 and A -Boc-Tyr-Gly-Gly-Phe-Met-NH2 were prepared in good yield. 

Fragment condensation of peptides corresponds to a reverse protease reaction -peptide synthesis instead of cleavage - and this is well known in the hterature as well. In fact, proteases have been used extensively for peptide coupling (Jakubke etal, 1985 1996 Jakubke, 1987 1995 Luisi etal, 1977b). This work has shown that even small proteins can be synthesized by block-wise enzymatic couphng (see also Kullmann, 1987, and, for some more recent developments, Celovsky and Bordusa, 2000). 

Celovsky, V. and Bordusa, F. (2000). Protease-catalyzed fragment condensation via substrate mimetic strategy a useful combination of solid-phase peptide synthesis with enzymatic methods. /. Pept. Res., 55, 325-9. 

In order to synthesize longer polypeptides and small proteins, the condensation of suitable starting fragments is an essential prerequisite. Since chemical fragment condensations suffer from the danger of enantiomerization, enzymatic ligations are pronoising alternatives (see... 

Machauer, R. Waldmann, H. Synthesis of lipi-dated eNOS peptides by combining enzymatic, noble metal- and acid-mediated protecting group techniques with solid phase peptide synthesis and fragment condensation in solution. Chemistry... 

DNA adducts with PAA, at concentrations of the order of 1 per 107 bases, can be determined after hydrolysis of the DNA to adducts such as 33 and 34 (Section n.B.2), by HPLC with tandem ESI-MS-MS detection. The LOD are in the order of 50 fmol on the column, for both analytes. Quantitative analysis of DNA adducts with PAA requires investigation of the MS of each individual adduct to be obtained after hydrolysis of DNA. For example, adducts 33 and 34, formed on enzymatic condensation of benzidine (7b) and 1-aminofluorene (10a) with adenine residues of DNA, can be determined by tandem ESI-MS-MS analysis, following the fragmentations depicted in 105 and 106, which gives optimum intensities when operating under suitable conditions. Additional support for the assignments can be obtained from adducts derived from deuteriated 7b and 10a28. 

Terpenoids do not necessarily contain exact multiples of five carbons and allowance has to be made for the loss or addition of one or more fragments and possible molecular rearrangements during biosynthesis. In reality the terpenoids are biosynthesized from acetate units derived from the primary metabolism of fatty acids, carbohydrates and some amino acids (see Fig. 2.10). Acetate has been shown to be the sole primary precursor of the terpenoid cholesterol. The major route for terpenoid biosynthesis, the mevalonate pathway, is summarized in Fig. 2.16. Acetyl-CoA is involved in the generation of the C6 mevalonate unit, a process that involves reduction by NADPH. Subsequent decarboxylation during phosphorylation (i.e. addition of phosphate) in the presence of ATP yields the fundamental isoprenoid unit, isopentenyl pyrophosphate (IPP), from which the terpenoids are synthesized by enzymatic condensation reactions. Recently, an alternative pathway has been discovered for the formation of IPP in various eubacteria and plants, which involves the condensation of glyceraldehyde 3-phosphate and pyruvate to form the intermediate 1-deoxy-D-xylulose 5-phosphate (Fig. 2.16 e.g. Eisenreich et al. 1998). We consider some of the more common examples of the main classes of terpenoids below. 

Enzymatic condensation of cholecystokinin CCK-8 (4-6) and CCK-8 (7-8) peptide fragments in organic media. Biotechnol. Bioeng., 56 (4), 456-463. 

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