Covalent attachment active-esters method

Figure 2. General scheme for covalent attachment of amino acids to proteins by the active-esters method. Reaction conditions are described in Ref. 22 TFA = trifluoroacetic acid.

In the dendritic [Co(salen)] complexes prepared by Breinbauer and Jacobsen the dendrimer again serves as - covalent - support material for the catalytic entities attached to the periphery [62]. These dendritic Jacobsen catalysts were obtained by reaction of the corresponding PAMAM dendrimers with active ester derivates of chiral ]Co(II)-(salen)] units according to standard peptide coupling methods. In hydrolytic kinetic resolution of vinylcyclohexane oxide the dendrimer 14 (Fig. 6.40) showed a dramatically increased reactivity compared to the commercially available monomeric Jacobsen catalyst [63-67]. Whereas the latter merely gave a conversion of less than 1% with an indeterminable ee, 14 afforded a conversion of 50% with an ee of 98 2. 

The library was designed not to contain amino acids that could be self-acylated, thus Ser, Thr, Lys, and Cys were not part of the library. His was incorporated as a known weak acylation catalyst and was expected to be selected. After reacting the library with the dye-marked Pfp ester, and thorough washing, several beads remained red, indicating covalent attachment of the dye to the resin beads. Analysis of the peptides on these red beads showed that each active bead carried at least one His. There is, therefore, no cross catalysis between different beads. The method can be used to search for catalysts of any bimolecular reaction in which one reaction partner can be attached to a solid support and the other labeled with a dye, a fluorophore, or radioactivity. 

Limiting essential amino acids covalently attached to proteins by using activated amino acid derivatives can improve the nutritional quality and change the functional properties of proteins. The best chemical methods for incorporating amino acids into water-soluble proteins involve using car-bodiimides, N-hydroxysuccinimide esters of acylated amino acids, or N-carboxy-a-amino acid anhydrides. The last two methods can give up to 75% incorporation of the amount of amino acid derivative used. With the anhydride method, as many as 50 residues of methionine have been linked to the 12 lysine residues of casein. The newly formed peptide and isopeptide bonds are hydrolyzed readily by intestinal aminopeptidase, making the added amino acids and the lysine from the protein available nutritionally. 

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