Oligopeptide active esters

There are several ways to prepare peptides, polypeptides, and artificial proteins carrying these aromatic amino acids as their constituents. Homopolypeptides can be prepared by the polymerization of amino acid N-carboxyanhydrides (NCAs) derived from the corresponding amino acids ( ). Sequential polypeptides of the form (ABC...W) are prepared by the polymerization of the corresponding oligopeptide active esters ABC...W-X. Small peptides of any sequence can be synthesized by a step-by-step procedure (5). Finally, cultivation of some bacteria in the presence of artificial amino acids will possibly incorporate them into the proteins produced by the bacteria. In the following, attention will be focused on homopolypeptides and sequential copolypeptides carrying one type of chromophore on a chain. 

Fig. 6.15. Carboxylic acid activation with DCC. [1,3] means the intramolecular substitution of the oxygen atom 01 by the N atom "3" via a cyclic four-membered tetrahedral intermediate. From the point of view of the heteroatoms, this SN reaction corresponds to a migration of the acyl group R-C=0 from the oxygen to the nitrogen. (Examples for amino acid activations in the form of the pentafluorophenyl ester C or the benzotriazolyl ester D are given in Figure 6.32 (oligopeptide synthesis) and Figure 6.31 (dipeptide synthesis), respectively.

The L-substtate was hydrolyzed 1.5—2 times faster than the D-substrate with either of the oligopeptides (ii or 12), and the flexible oligopeptide 12 was 2 to 3 times more efficient than imidazole. The observed stereoselectivity is noteworthy, e )ecia]ly since tiieie was no stereoselectivity in the solvofysis of some a mmetric phenyl esters 13,14,15 by an optically active vin lymer containing inudazole group 16 134). 

Thermitase, a thermostable extracellular serine protease from the thermophrhc microorganism Thermoactinomyces vulgaris, with an esterase/protease activity ratio >1000 1 shows a broad amino acid side-chain tolerance and cleaves methyl, ethyl, benzyl, ethox-ybenzyl, and ferf-butyl esters from a variety of Nps-, Boc-, Bpoc-, and Z-protected di- and oligopeptides in high yields at pH 8 and 33-55 °C (Scheme 15 ).[28,29,60-62] jjj addition, it is specific for the a-carboxy groups of Asp and Glu. 

The role of silylated reagents in the formation of oligopeptides has been explored . Here, the bis(trimethylsilyl) ester of the [l-(trimethylsilylamino)alkyl]phosphonic acid is coupled with an activated A -cbz-amino acid and the silyl groups are subsequently removed under aqueous conditions the process can then be repeated. Oligopeptides have also been obtained as the result of enzyme catalysis when the condensations between amino carboxylic esters and (a) A -protected (aminoalkyl)phosphonic esters or (b) A -protected [(aminoalkyl)methyl]phosphinic esters is brought about in the presence of (a) alkaline phosphatase (Ej) and phosphodiesterase (E2) and (b) alkaline phosphatase and total bee venom (E3) (the latter aiding in the removal of both carboxylate ester and A -acetyl groups) ... 

Basic to protease catalyzed oligopeptide synthesis is equilibrium- or thermodynamic control to direct reversal of proteolysis . Difficulties encountered include low reaction rates, high stoichiometric amounts of enzyme, and the need to apply direct approaches to shift the reactions towards formation of desired products. Reaction conditions that lead to product precipitation or extraction increase efficiency of the reverse reaction. Kinetically controlled syntheses has proved useful for serine and cysteine proteases that form activated acyl enzyme intermediates during catalysis. This approach generally involves use of activated acyl moieties, such as esters, as donor components which significantly accelerate the reaction rate. This study makes use of principles from both kinetic and thermodynamically controlled reactions in that, reactants are activated by formation of esters and products precipitate fi om reactions. 

Scheme 8.11 Schematics of the polymerization in aqueous solutions of mixtures of enantiopure leucine-thioethyl ester with racemic valine, that was activated in situ with solid diimidazole, to yield a mixture of (D)-valine oligopeptides with a single L-leucine thioester residue at the C terminus and copeptides of (L)-valine and (L)-leucine.

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