Mechanistic Aspects of Phosphate Diester Cleavage Assisted by Imidazole. A Template Reaction for Obtaining Aryl Phosphoimidazoles

Phosphoimidazole-containing compounds are versatile players in biological and chemical processes. We explore catalytic and mechanistic criteria for the efficient formation of cyclic aryl phosphoimidazoles in aqueous solution, viewed as a template reaction for the in situ synthesis of related compounds. To provide a detailed analysis for this reaction a series of <i>o</i>-(2′-imidazolyl)­naphthyl (4-nitrophenyl) phosphate isomers were examined to provide a basis for analysis of both mechanism and the influence of structural factors affecting the nucleophilic attack of the imidazolyl group on the phosphorus center of the substrate. Formation of the cyclic aryl phosphoimidazoles was probed by NMR and ESI-MS techniques. Kinetic experiments show that cyclization is faster under alkaline conditions, with an effective molarity up to 2900 M for the imidazolyl group, ruling out competition from external nucleophiles. Heavy atom isotope effect and computational studies show that the reaction occurs through a S_N2­(P)-type mechanism involving a pentacoordinated phosphorus TS, with apical positions occupied by the incoming imidazolyl nucleophile and the <i>p</i>-nitrophenolate leaving group. The P–O bond to the leaving group is about 50–60% broken in the transition state

Structural and Kinetic Properties of the Aldehyde Dehydrogenase NahF, a Broad Substrate Specificity Enzyme for Aldehyde Oxidation

The salicylaldehyde dehydrogenase (NahF) catalyzes the oxidation of salicylaldehyde to salicylate using NAD⁺ as a cofactor, the last reaction of the upper degradation pathway of naphthalene in <i>Pseudomonas putida</i> G7. The naphthalene is an abundant and toxic compound in oil and has been used as a model for bioremediation studies. The steady-state kinetic parameters for oxidation of aliphatic or aromatic aldehydes catalyzed by 6xHis-NahF are presented. The 6xHis-NahF catalyzes the oxidation of aromatic aldehydes with large <i>k</i>_cat/<i>K</i>_m values close to 10⁶ M^–1 s^–1. The active site of NahF is highly hydrophobic, and the enzyme shows higher specificity for less polar substrates than for polar substrates, e.g., acetaldehyde. The enzyme shows α/β folding with three well-defined domains: the oligomerization domain, which is responsible for the interlacement between the two monomers; the Rossmann-like fold domain, essential for nucleotide binding; and the catalytic domain. A salicylaldehyde molecule was observed in a deep pocket in the crystal structure of NahF where the catalytic C284 and E250 are present. Moreover, the residues G150, R157, W96, F99, F274, F279, and Y446 were thought to be important for catalysis and specificity for aromatic aldehydes. Understanding the molecular features responsible for NahF activity allows for comparisons with other aldehyde dehydrogenases and, together with structural information, provides the information needed for future mutational studies aimed to enhance its stability and specificity and further its use in biotechnological processes