Rapid analysis of solvent effects on enamine formation by fluorescence: how might enzymes facilitate enamine chemistry with primary amines?

In order to determine factors that facilitate the use of primary amines in biological chemistry, solvent effects on enamine formation with glycine were studied. Solvent effects were rapidly analyzed by monitoring the increase in fluorescence resulting from the reaction between a fluorogenic maleimide and in situ-generated enamine of acetone. These studies suggest that in addition to a simple hydrophobic microenvironment, a microenvironment that also affords for hydrogen bond formation facilitates enaminebased reactions involving primary amines. 2003 Elsevier Ltd. All rights reserved. The enamine is one of the most important intermediates for carbon–carbon bond formation in both organic chemistry and the biological world. In organic synthesis, pyrrolidine derivatives are used to efficiently form enamines with carbonyl compounds in many reactions. In nature, on the other hand, enzymes such as aldolases and decarboxylases utilize the e-amino groups of lysine residues to form an enamine during their catalytic cycle. Antibodies and peptides have also been demonstrated to catalyze reactions via an enamine mechanism in aqueous solution. While biological catalysts can contain the pyrrolidine-based amino acid proline, the amine functionality is masked as an amide except when proline is the amino terminal residue of the polypeptide. How does nature use the e-amino group of lysine as effectively as organic chemists use pyrrolidinebased catalysts? The e-amino group in the free amino acid lysine has a pKa of 10.7 in aqueous solution and is protonated at neutral pH. Therefore the amino groups of lysine residues of proteins typically do not function as nucleophiles to form an enamine or imine in aqueous solution