hCB2 ligand-interaction landscape: cysteine residues critical to biarylpyrazole antagonist binding motif and receptor modulation

The human cannabinoid 2 GPCR (hCB2) is a prime therapeutic target. To define potential cysteine-related binding motifs critical to hCB2-ligand interaction, a library of hCB2 cysteine-substitution mutants and a novel, high-affinity biarylpyrazole hCB2 antagonist/inverse agonist (AM1336) functionalized to serve as a covalent affinity probe to target cysteine residues within (or in the microenvironment of) its hCB2 binding pocket were generated. The data provide direct experimental demonstration that both hCB2 TMH7 cysteines [i.e., C7.38(284) and C7.42(288)] are critical to optimal hCB2-AM1336 binding interaction and AM1336 pharmacological activity in a cell-based functional assay (cAMP formation). Elongating the AM1336 aliphatic side chain generated another novel?hCB2 inverse agonist that binds covalently and selectively to C7.42(288) only. Identification of specific cysteine residues critical to hCB2 ligand interaction and function informs the structure-based design of hCB2-targeted medicines.     

Covalent inhibitors of human monoacylglycerol lipase: ligand-assisted characterization of the catalytic site by mass spectrometry and mutational analysis

The active site of recombinant hexa-histidine-tagged human monoacylglycerol lipase (hMGL) is characterized by mass spectrometry using the inhibitors 5-((biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701), and N-arachidonylmaleimide (NAM) as probes. Carbamylation of Ser(129) by AM6701 in the putative hMGL catalytic triad demonstrates this residue's essential role in catalysis. Partial NAM alkylation of hMGL cysteine residues 215 and/or 249 was sufficient to achieve approximately 80% enzyme inhibition. Although Cys(215) and/or Cys(249) mutations to alanine(s) did not affect hMGL hydrolytic activity as compared with nonmutated hMGL, the C215A displayed heightened NAM sensitivity, whereas the C249A evidenced reduced NAM sensitivity. These data conclusively demonstrate a sulfhydryl-based mechanism for NAM inhibition of hMGL in which Cys(249) is of paramount importance. Identification of amino acids critical to the catalytic activity and pharmacological modulation of hMGL informs the design of selective MGL inhibitors as potential drugs.     

A facile C-C bond cleavage in the epoxides and its use for the synthesis of oxygenated heterocycles by a ring expansion strategy

The bicyclic epoxy alcohols when treated with DAST gave a new class of rearranged organofluorine compounds, by a ring expansion via C-C bond cleavage of the oxirane ring. The outcome of this reaction with respect to ring size and stereochemical relation between the functionalities is presented here.     

Reaction monitoring in LPOS by 19F NMR. Study of soluble polymer supports with fluorine in spacer or linker components of supports

Various soluble polystyrene supports with fluorinated spacer or linker were prepared and studied by (19)F NMR for their use in LPOS reaction monitoring. Among three types of systems studied, the perfluoro Wang linker was found to be most efficient for this purpose. Substrates could be easily anchored to and cleaved from this new support-bound linker. The anchoring of the linker and the substrates on the polymer led to significant changes in the fluorine resonances. Therefore, the progress of these reactions could be both monitored and quantified. On the other hand, the chemical transformations on the anchored substrates led only to moderate changes in the fluorine resonances. Nevertheless, the reaction progress could also be monitored in this case. After cleavage of products, the polymer supports were recovered without loss in loading. Membrane separation technology was used to purify some polymer-bound products as well as to obtain the polymer-free cleaved product.     

Synchrotron far‐infrared spectroscopy of corroded steel surfaces using a variable angle of incidence

Far‐infrared spectroscopy, using a synchrotron source, has been used to study carbon steel corroded in CO₂‐saturated brine in the presence and absence of the corrosion inhibitor 2‐mercaptopyrimidine (MPY), which allowed the steel surface roughness to be modified. The effect of the angle of incidence (θ_i, 30–80°) on the band intensity and observed bands of the spectra from these surfaces has been determined. For the MPY‐treated steel (low surface roughness) the highest band intensity is observed at high θ_i (80°) and different bands were observed at different θ_i. In contrast, for the MPY‐free steel (high surface roughness) the highest band intensity is observed at low θ_i (30°) and spectral content changes were not observed. The results are explained in terms of the roughness of the MPY‐treated and MPY‐free steels, and their effect on the level of diffusely reflected light of the incident infrared beam.