Atomic-Resolution 1.3 Å Crystal Structure, Inhibition by Sulfate, and Molecular Dynamics of the Bacterial Enzyme DapE.

We report the atomic-resolution (1.3 Å) X-ray crystal structure of an open conformation of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE, EC 3.5.1.18) from Neisseria meningitidis. This structure [Protein Data Bank (PDB) entry 5UEJ] contains two bound sulfate ions in the active site that mimic the binding of the terminal carboxylates of the N-succinyl-l,l-diaminopimelic acid (l,l-SDAP) substrate. We demonstrated inhibition of DapE by sulfate (IC50 = 13.8 ± 2.8 mM). Comparison with other DapE structures in the PDB demonstrates the flexibility of the interdomain connections of this protein. This high-resolution structure was then utilized as the starting point for targeted molecular dynamics experiments revealing the conformational change from the open form to the closed form that occurs when DapE binds l,l-SDAP and cleaves the amide bond. These simulations demonstrated closure from the open to the closed conformation, the change in RMS throughout the closure, and the independence in the movement of the two DapE subunits. This conformational change occurred in two phases with the catalytic domains moving toward the dimerization domains first, followed by a rotation of catalytic domains relative to the dimerization domains. Although there were no targeting forces, the substrate moved closer to the active site and bound more tightly during the closure event.

O-Cyclopropyl hydroxylamines: gram-scale synthesis and utility as precursors for N-heterocycles.

O-Cyclopropyl hydroxylamines, now accessible via a novel and scalable synthetic route, have been demonstrated to be bench-stable and practical precursors for the synthesis of N-heterocycles via a di-heteroatom [3,3]-sigmatropic rearrangement. In order to study the reactivity of these compounds in depth, a robust synthesis of both ring-substituted and ring-unsubstituted O-cyclopropyl hydroxylamines has been developed. Metal-free conditions for the facile N-arylation of these precursors were also identified. It was found that the N-arylated O-cyclopropyl hydroxamates can efficiently undergo a one-pot [3,3]-sigmatropic rearrangement/cyclization/rearomatization cascade under base-mediated conditions to furnish a structurally diverse set of substituted tetrahydroquinolines.

Titanium-Mediated Synthesis of Spirocyclic NH-Azetidines from Oxime Ethers.

The TiIV -mediated synthesis of spirocyclic NH-azetidines from oxime ethers using either an alkyl Grignard reagent or terminal olefin ligand exchange coupling partner is described. Through a proposed Kulinkovich-type mechanism, a titanacyclopropane intermediate forms and serves as a 1,2-aliphatic dianion equivalent, inserting into the 1,2-dielectrophilc oxime ether to ultimately give rise to the desired N-heterocyclic four-membered ring. This transformation proceeds in moderate yield to furnish previously unreported and structurally diverse NH-azetidines in a single step.

Antimicrobial activity of a natural compound and analogs against multi-drug-resistant Gram-positive pathogens.

The increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) has sparked global concern due to the dwindling availability of effective antibiotics. To increase our treatment options, researchers have investigated naturally occurring antimicrobial compounds and have identified MC21-A (C58), which has potent antimicrobial activity against MRSA. Recently, we have devised total synthesis schemes for C58 and its chloro-analog, C59. Here, we report that both compounds eradicate 90% of the 39 MRSA isolates tested [MIC90 and minimum bactericidal concentration (MBC90)] at lower or comparable concentrations compared to several standard-of-care (SoC) antimicrobials including daptomycin, vancomycin, and linezolid. Furthermore, a stable, water-soluble sodium salt of C59, C59Na, demonstrates antimicrobial activity comparable to C59. C59, unlike vancomycin, kills stationary-phase MRSA in a dose-dependent manner and completely eradicates MRSA biofilms. In contrast to vancomycin, exposing MRSA to sub-MIC concentrations of C59 does not result in the emergence of spontaneous resistance. Similarly, in a multi-step study, C59 demonstrates a low propensity of resistance acquisition when compared to SoC antimicrobials, such as linezolid and clindamycin. Our findings suggest C58, C59, and C59Na are non-toxic to mammalian cells at concentrations that exert antimicrobial activity; the lethal dose at median cell viability (LD50) is at least fivefold higher than the MBC90 in the two mammalian cell lines tested. A morphological examination of the effects of C59 on a MRSA isolate suggests the inhibition of the cell division process as a mechanism of action. Our results demonstrate the potential of this naturally occurring compound and its analogs as non-toxic next-generation antimicrobials to combat MRSA infections. IMPORTANCE: The rapid emergence of methicillin-resistant Staphylococcus aureus (MRSA) isolates has precipitated a critical need for novel antibiotics. We have developed a one-pot synthesis method for naturally occurring compounds such as MC21-A (C58) and its chloro-analog, C59. Our findings demonstrate that these compounds kill MRSA isolates at lower or comparable concentrations to standard-of-care (SoC) antimicrobials. C59 eradicates MRSA cells in biofilms, which are notoriously difficult to treat with SoC antibiotics. Additionally, the lack of resistance development observed with C59 treatment is a significant advantage when compared to currently available antibiotics. Furthermore, these compounds are non-toxic to mammalian cell lines at effective concentrations. Our findings indicate the potential of these compounds to treat MRSA infections and underscore the importance of exploring natural products for novel antibiotics. Further investigation will be essential to fully realize the therapeutic potential of these next-generation antimicrobials to address the critical issue of antimicrobial resistance.

The application of modern reactions in large-scale synthesis.

In the past decade, the field of organic synthesis has witnessed tremendous advancements in the areas of photoredox catalysis, electrochemistry, C-H activation, reductive coupling and flow chemistry. While these methods and technologies offer many strategic advantages in streamlining syntheses, their application on the process scale is complicated by several factors. In this Review, we discuss the challenges that arise when these reaction classes and/or flow chemistry technology are taken from a research laboratory operating at the milligram scale to a reactor capable of producing kilograms of product. We discuss how these challenges have been overcome through chemical and engineering solutions. Specifically, this Review will highlight key examples that have led to the production of multi-hundred-gram to kilogram quantities of active pharmaceutical ingredients or their intermediates and will provide insight on the scaling-up process to those developing new technologies and reactions.

Practical access to axially chiral sulfonamides and biaryl amino phenols via organocatalytic atroposelective N-alkylation.

The importance of axial chirality in enantioselective synthesis has been widely recognized for decades. The practical access to certain structures such as biaryl amino phenols known as NOBINs in enantiopure form, however, still remains a challenge. In drug delivery, the incorporation of axially chiral molecules in systematic screening has also received a great deal of interest in recent years, which calls for innovation and practical synthesis of structurally different axially chiral entities. Herein we present an operationally simple catalytic N-alkylation of sulfonamides using commercially available chiral amine catalysts to deliver two important classes of axially chiral compounds: structurally diverse NOBIN analogs as well as axially chiral N-aryl sulfonamides in excellent enantiopurity. Structurally related chiral sulfonamide has shown great potential in drug molecules but enantioselective synthesis of them has never been accomplished before. The practical catalytic procedures of our methods also bode well for their wide application in enantioselective synthesis.

Transition metal-free direct dehydrogenative arylation of activated C(sp3)-H bonds: synthetic ambit and DFT reactivity predictions.

A transition metal-free dehydrogenative method for the direct mono-arylation of a wide range of activated C(sp3)-H bonds has been developed. This operationally simple and environmentally friendly aerobic arylation uses tert-BuOK as the base and nitroarenes as electrophiles to prepare up to gram quantities of structurally diverse sets (>60 examples) of α-arylated esters, amides, nitriles, sulfones and triaryl methanes. DFT calculations provided a predictive model, which states that substrates containing a C(sp3)-H bond with a sufficiently low pK a value should readily undergo arylation. The DFT prediction was confirmed through experimental testing of nearly a dozen substrates containing activated C(sp3)-H bonds. This arylation method was also used in a one-pot protocol to synthesize over twenty compounds containing all-carbon quaternary centers.