Visualization of Endogenous ERK1/2 in Cells with a Bioorthogonal Covalent Probe

The RAS–RAF–MEK–ERK pathway has been intensively studied in oncology, with RAS known to be mutated in ∼30% of all human cancers. The recent emergence of ERK1/2 inhibitors and their ongoing clinical investigation demands a better understanding of ERK1/2 behavior following small-molecule inhibition. Although fluorescent fusion proteins and fluorescent antibodies are well-established methods of visualizing proteins, we show that ERK1/2 can be visualized via a less-invasive approach based on a two-step process using inverse electron demand Diels–Alder cycloaddition. Our previously reported trans-cyclooctene-tagged covalent ERK1/2 inhibitor was used in a series of imaging experiments following a click reaction with a tetrazine-tagged fluorescent dye. Although limitations were encountered with this approach, endogenous ERK1/2 was successfully imaged in cells, and “on-target” staining was confirmed by over-expressing DUSP5, a nuclear ERK1/2 phosphatase that anchors ERK1/2 in the nucleus

Terminal aziridines by alpha-deprotonation/electrophile trapping of N-protected aziridine.

N-tert-Butylsulfonyl and N-tert-butylsulfinyl aziridine undergo alpha-lithiation/electrophile trapping providing a new entry to terminal aziridines. With N-tert-butylsulfinyl aziridine complete asymmetric induction is observed alpha to nitrogen

Convenient preparations of racemic and enantiopure methyl 6-oxopipecolate

Short, convenient syntheses of racemic and enantiopure methyl 6-oxopipecolate are described, starting from either pipecolic acid or (S)-lysine respectively. The sequence for the latter compound relies upon improved methodology for the oxidation of C-6 of lysine

Diastereoselectivity in the alkylations of bicyclic piperidinones

The synthesis of substituted [4.3.0] bicyclic lactams derived from 6- oxo-2-hydroxymethylpiperidine is described. The enolate derived flora these systems can be alkylated with a range of reactive electrophiles; the diastereoselectivity which can be achieved depends on the substitution pattern of the oxazolidine ring system and the nature of the alkylating reagent, and can vary from 1:1 to as much as 10:1

Inhibition of the histone demethylase JMJD2E by 3-substituted pyridine 2,4-dicarboxylates.

Based on structural analysis of the human 2-oxoglutarate (2OG) dependent JMJD2 histone N(ε)-methyl lysyl demethylase family, 3-substituted pyridine 2,4-dicarboxylic acids were identified as potential inhibitors with possible selectivity over other human 2OG oxygenases. Microwave-assisted palladium-catalysed cross coupling methodology was developed to install a diverse set of substituents on the sterically demanding C-3 position of a pyridine 2,4-dicarboxylate scaffold. The subsequently prepared di-acids were tested for in vitro inhibition of the histone demethylase JMJD2E and another human 2OG oxygenase, prolyl-hydroxylase domain isoform 2 (PHD2, EGLN1). A subset of substitution patterns yielded inhibitors with selectivity for JMJD2E over PHD2, demonstrating that structure-based inhibitor design can enable selective inhibition of histone demethylases over related human 2OG oxygenases

A structural view of the action of Escherichia coli (lacZ) beta-galactosidase.

The structures of a series of complexes designed to mimic intermediates along the reaction coordinate for beta-galactosidase are presented. These complexes clarify and enhance previous proposals regarding the catalytic mechanism. The nucleophile, Glu537, is seen to covalently bind to the galactosyl moiety. Of the two potential acids, Mg(2+) and Glu461, the latter is in better position to directly assist in leaving group departure, suggesting that the metal ion acts in a secondary role. A sodium ion plays a part in substrate binding by directly ligating the galactosyl 6-hydroxyl. The proposed reaction coordinate involves the movement of the galactosyl moiety deep into the active site pocket. For those ligands that do bind deeply there is an associated conformational change in which residues within loop 794-804 move up to 10 A closer to the site of binding. In some cases this can be inhibited by the binding of additional ligands. The resulting restricted access to the intermediate helps to explain why allolactose, the natural inducer for the lac operon, is the preferred product of transglycosylation

Enantiopure bicyclic piperidinones: stereoselectivity in lactam enolate alkylations.

The synthesis and alkylation of [4.3.0]-bicyclic lactams, derived from 6-oxopipecolic acid, have been investigated. Alkylation can proceed with predominantly exo-diastereoselectivity, but the efficiency of this process depends on the substitution at the hemiaminal ether system. These products can be readily deprotected to give substituted hydroxymethyl lactams in good yield