Past, present, and future developments in enantioselective analysis using capillary electromigration techniques.

Enantioseparation of chiral products has become increasingly important in a large diversity of academic and industrial applications. The separation of chiral compounds is inherently challenging and thus requires a suitable analytical technique that can achieve high resolution and sensitivity. In this context, CE has shown remarkable results so far. Chiral CE offers an orthogonal enantioselectivity and is typically considered less costly than chromatographic techniques, since only minute amounts of chiral selectors are needed. Several CE approaches have been developed for chiral analysis, including chiral EKC and chiral CEC. Enantioseparations by EKC benefit from the wide variety of possible pseudostationary phases that can be employed. Chiral CEC, on the other hand, combines chromatographic separation principles with the bulk fluid movement of CE, benefitting from reduced band broadening as compared to pressure-driven systems. Although UV detection is conventionally used for these approaches, MS can also be considered. CE-MS represents a promising alternative due to the increased sensitivity and selectivity, enabling the chiral analysis of complex samples. The potential contamination of the MS ion source in EKC-MS can be overcome using partial-filling and counter-migration techniques. However, chiral analysis using monolithic and open-tubular CEC-MS awaits additional method validation and a dedicated commercial interface. Further efforts in chiral CE are expected toward the improvement of existing techniques, the development of novel pseudostationary phases, and establishing the use of chiral ionic liquids, molecular imprinted polymers, and metal-organic frameworks. These developments will certainly foster the adoption of CE(-MS) as a well-established technique in routine chiral analysis.

Trans-cyclosulfamidate mannose-configured cyclitol allows isoform-dependent inhibition of GH47 α-d-mannosidases through a bump-hole strategy.

Class I inverting exo-acting α-1,2-mannosidases (CAZY family GH47) display an unusual catalytic itinerary featuring ring-flipped mannosides, 3S1 → 3H4‡ → 1C4. Conformationally locked 1C4 compounds, such as kifunensine, display nanomolar inhibition but large multigene GH47 mannosidase families render specific "isoform-dependent" inhibition impossible. Here we develop a bump-and-hole strategy in which a new mannose-configured 1,6-trans-cyclic sulfamidate inhibits α-d-mannosidases by virtue of its 1C4 conformation. This compound does not inhibit the wild-type GH47 model enzyme by virtue of a steric clash, a "bump", in the active site. An L310S (a conserved residue amongst human GH47 enzymes) mutant of the model Caulobacter GH47 awoke 574 nM inhibition of the previously dormant inhibitor, confirmed by structural analysis of a 0.97 Å structure. Considering that L310 is a conserved residue amongst human GH47 enzymes, this work provides a unique framework for future biotechnological studies on N-glycan maturation and ER associated degradation by isoform-specific GH47 α-d-mannosidase inhibition through a bump-and-hole approach.

Structure-Activity Relationship Studies of α-Ketoamides as Inhibitors of the Phospholipase A and Acyltransferase Enzyme Family.

The phospholipase A and acyltransferase (PLAAT) family of cysteine hydrolases consists of five members, which are involved in the Ca2+-independent production of N-acylphosphatidylethanolamines (NAPEs). NAPEs are lipid precursors for bioactive N-acylethanolamines (NAEs) that are involved in various physiological processes such as food intake, pain, inflammation, stress, and anxiety. Recently, we identified α-ketoamides as the first pan-active PLAAT inhibitor scaffold that reduced arachidonic acid levels in PLAAT3-overexpressing U2OS cells and in HepG2 cells. Here, we report the structure-activity relationships of the α-ketoamide series using activity-based protein profiling. This led to the identification of LEI-301, a nanomolar potent inhibitor for the PLAAT family members. LEI-301 reduced the NAE levels, including anandamide, in cells overexpressing PLAAT2 or PLAAT5. Collectively, LEI-301 may help to dissect the physiological role of the PLAATs.