Ethyl- (7), benzyl- (8), tert-butyl- (9), and fluorenylmethyl-4-chlorobenzoyloxycarbamates (10) have been prepared as storable and easy-to-prepare nitrogen sources for use in the intermolecular Sharpless aminohydroxylation reaction and its asymmetric variant. These reagents were found to be effective under base-free reaction conditions. The scope and limitations of these methods have been explored using a variety of alkenes, among which, trans-cinnamates, in particular, proved to be good substrates.

Mycobacterium tuberculosis and many other members of the Actinomycetes family produce mycothiol, i.e., 1-D-myo-inosityl-2-(N-acetyl-L-cysteinyl)amido-2-deoxy-α-D-glucopyranoside (MSH or AcCys-GlcN-Ins), to act against oxidative and antibiotic stress. The biosynthesis of MSH is essential for cell growth, and has been proposed to proceed via a biosynthetic pathway involving four key enzymes, MshA-D. The MSH biosynthetic enzymes present potential targets for inhibitor design. With this as a long-term goal, we have carried out a kinetic and mechanistic characterization, using steady state and pre-steady state approaches, of the recombinant Mycobacterium smegmatis MshC. MshC catalyzes the ATP-dependent condensation of GlcN-Ins and cysteine to form CysGlcN-Ins. Initial velocity and inhibition studies show that the steady state kinetic mechanism of MshC is a Bi Uni Uni Bi Ping Pong mechanism, with ATP binding followed by cysteine binding, release of PP i , binding of GlcN-Ins, followed by the release of Cys-GlcN-Ins and AMP. The steady state kinetic parameters were determined to be: k cat equal to 3.15 s −1 , and K m values of 1.8, 0.1, and 0.16 mM for ATP, cysteine, and GlcN-Ins, respectively. A stable bisubstrate analog, 5′-O-[N-(Lcysteinyl)sulfamonyl]adenosine, exhibits competitive inhibition versus ATP and non-competitive inhibition versus cysteine, with an inhibition constant of ~306 nM versus ATP. Single-turnover reactions of the first and second half reactions were determined using rapid quench techniques, giving rates of ~9.4 s −1 and ~5.2 s −1 , respectively, consistent with the cysteinyl adenylate being a kinetically competent intermediate in the reaction by MshC. KeywordsMycobacterium; mycothiol; cysteine ligase Mycothiol (MSH, acetyl-Cys-GlcN-Ins), a conjugate of N-acetylcysteine (AcCys) and , is the predominant low molecular weight thiol that protects actinomycetes against oxidative stress and cellular electrophilic toxins (1-4). Among actinomycetes, mycobacteria generate the highest intracellular levels of MSH (5). Studies have shown that Mycobacterium smegmatis mutants lacking MSH become more sensitive towards oxidizing agents, electrophiles, and antibiotics (1-3), indicating the critical role of MSH in the survival and pathogenicity of mycobacteria (1). The detoxification mechanism of electrophiles by MSH has been proposed (Scheme 1, path a) to involve the formation of a MSH S-conjugate of the electrophile (1). The conjugate *AUTHOR EMAIL ADDRESS: blanchar@aecom.yu.edu. CORRESPONDING AUTHOR FOOTNOTE. Phone: (718) Fax: (718) It has been proposed that MSH is synthesized via a series of enzymatic reactions (6-9), as illustrated in Scheme 1, path b. In brief, the process is initiated by an N-acetyl-glucosamine transferase (MshA) to generate 3-phospho-GlcNAc-Ins, which is subsequently dephosphorylated to form GlcNAc-Ins by an unknown phosphatase (9). GlcNAc-Ins is subsequently deacetylated by MshB. The resulting GlcN-Ins is ligated with cysteine in a reaction catalyzed by a cysteine ligase, MshC. ...

Decoupling the roles of the farnesoid X nuclear receptor and Takeda G-protein-coupled bile acid receptor 5 is essential for the development of novel bile acid therapeutics targeting metabolic and neurodegenerative diseases. Herein, we describe the synthesis of 12β-methyl-18-nor-bile acids which may serve as probes in the search for new bile acid analogues with clinical applicability. A Nametkin-type rearrangement was applied to protected cholic acid derivatives, giving rise to tetra-substituted Δ13,14- and Δ13,17-unsaturated 12β-methyl-18-nor-bile acid intermediates (24a and 25a). Subsequent catalytic hydrogenation and deprotection yielded 12β-methyl-18-nor-chenodeoxycholic acid (27a) and its 17-epi-epimer (28a) as the two major reaction products. Optimization of the synthetic sequence enabled a chromatography-free route to prepare these bile acids at a multi-gram scale. In addition, the first cis-C-D ring-junctured bile acid and a new 14(13 → 12)-abeo-bile acid are described. Furthermore, deuteration experiments were performed to provide mechanistic insights into the formation of the formal anti-hydrogenation product 12β-methyl-18-nor-chenodeoxycholic acid (27a).

The involvement of protein kinase CK1δ in the pathogenesis of severe disorders such as Alzheimer’s disease, amyotrophic lateral sclerosis, familial advanced sleep phase syndrome, and cancer has dramatically increased interest in the development of effective small molecule inhibitors for both therapeutic application and basic research. Unfortunately, the design of CK1 isoform-specific compounds has proved to be highly complicated due to the existence of six evolutionarily conserved human CK1 members that possess similar, different, or even opposite physiological and pathophysiological implications. Consequently, only few potent and selective CK1δ inhibitors have been reported so far and structurally divergent approaches are urgently needed in order to establish SAR that might enable complete discrimination of CK1 isoforms and related p38α MAPK. In this study we report on design and characterization of optimized 4,5-diarylimidazoles as highly effective ATP-competitive inhibitors of CK1δ with compounds 11b (IC50 CK1δ = 4 nM, IC50 CK1ε = 25 nM), 12a (IC50 CK1δ = 19 nM, IC50 CK1ε = 227 nM), and 16b (IC50 CK1δ = 8 nM, IC50 CK1ε = 81 nM) being among the most potent CK1δ-targeting agents published to date. Inhibitor compound 11b, displaying potential as a pharmacological tool, has further been profiled over a panel of 321 protein kinases exhibiting high selectivity. Cellular efficacy has been evaluated in human pancreatic cancer cell lines Colo357 (EC50 = 3.5 µM) and Panc89 (EC50 = 1.5 µM). SAR is substantiated by X-ray crystallographic analysis of 16b in CK1δ and 11b in p38α.

An early step of the inflammatory response, the rolling of leukocytes on activated endothelial cells, is mediated by selectin/carbohydrate interactions. The tetrasaccharide sialy Lewisx is a ligand for E-, P-, and L-selectin and therefore serves as a lead structure for the development of analogues. A combination of synthesis and structure-based design allowed rapid optimization. The current lead 2a was evaluated in our E-selectin cell flow chamber assay where it proved to inhibit rolling and adhesion with an IC50 of 28+/-7 microM. The assays used are predictive for the in vivo efficacy of test compounds as shown for 2a in a proteose peptone induced peritonitis model of acute inflammation in mice.