This report describes the first direct comparison between the reactive immunization and transition state analogue hapten manifolds for catalytic antibody production. In an initial communication (Janda et al J. Am. Chem. Soc. 1997, 119, 10251) we described the use of a phosphonate diester hapten 5, in a reactive immunization approach, that elicited a panel of proficient biocatalysts for the hydrolysis of S-(+)-naproxen p-methylsulfonylphenyl ester (3b) [k cat (3b)/k uncat (3b) ) 0.05-6.60 × 10 5 ]. However, only moderate enantioselectivity was obtained when the panel of antibody catalysts was studied in a kinetic resolution of rac-3a, the best result leading to S-(+)-4a in 90% ee for 35% conversion of rac-3a. This report details a transition state analogue hapten approach to elicit antibody catalysts for this same process by employment of phosphonate monoester 6. This strategy has yielded a library of catalysts with excellent turnover numbers [k cat (3b)/k uncat (3b) ) 0.14-19.0 × 10 5 ] and enantioselectivities. Three of these catalysts, 6G6, 12C8, and 12D9, perform a useful kinetic resolution of rac-3a, generating S-(+)-naproxen 4a in >98% ee with up to 50% conversion. Comparing the two hapten strategies reveals that the antibodies, although elicited for the same reaction with the same substrate, exhibit quite different catalytic behavior. The transition state analogue approach has furnished better catalysts, in terms of turnover numbers and enantiomeric discrimination, but which possess varying degrees of product inhibition by phenol 9. Thermodynamic evaluation reveals that their catalytic power is derived almost entirely as a function of differential stabilization of the transition state over the ground state: K m (3b)/K i (8). By contrast, the reactive immunization approach has elicited more proficient biocatalysts that couple an efficient "catalytic" mechanism and improved substrate recognition with no product inhibition.

Seroprevalence in the DMHDS declined from age 11 to 21 predominantly in females. The decline involved a greater rate of seroreversion and lower rate of seroconversion in females than males.

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Pharmacokinetic and metabolite identification studies were conducted to understand the clearance pathways of EPZ011652 [(2-aminoethyl)(methyl)({3-[4-(propan-2-yloxy)phenyl]-1H-pyrazol-4-yl} methyl)amine], a potent protein arginine N-methyltransferase inhibitor. Metabolic clearance was the major pathway of EPZ011652 elimination in rats with structural elucidation of metabolites via liquid chromatography -mass spectrometry (LC-MS n ) accurate mass measurement revealing the formation of a novel aliphatic N-acetylated metabolite (M1) located on the terminal nitrogen of the ethylenediamine side chain. EPZ015564, a synthetic standard of the N-acetyl product, was prepared and was also generated by human and rat, but not dog hepatocytes. In rat hepatocytes, on incubation with EPZ011652, the concentration of EPZ015564 initially increased before decreasing with incubation time, suggesting that the metabolite is itself a substrate for other metabolizing enzymes, in agreement with the identification of metabolites M2, M3, and M4 in rat bile, all N-acetylated metabolites, undergoing sequential phase I (demethylation, oxidation) or phase II (sulfation) reactions. Reaction phenotyping with recombinant human N-acetyltransferase (NAT) isoforms revealed that both NAT1 and NAT2 are capable of acetylating EPZ011652, although with different catalytic efficiencies. Kinetic profiles of EPZ015564 formation followed classic Michaelis-Menten behavior with apparent K m values of >1000 mM for NAT1 and 165 6 14.1 mM for NAT2. The in vitro intrinsic clearance for EPZ011652 by NAT2 (110 mL/min/mg) was 500-fold greater than by NAT1. In summary, we report the unusual N-acetylation of an aliphatic amine and discuss the implications for drug discovery and clinical development.

The stereochemistries at positions 2 and 3 of the title compound, C10H10BrCIO2, have been confirmed by Xray crystal structural analysis. The halogen atoms show an antiperiplanar arrangement.