Synthesis and characterization of the N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) alternate substrate analog N,N-dimethyl-l,l-SDAP.

Growing antibiotic resistance by pathogenic bacteria has led to a global crisis. The bacterial enzyme N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) provides a very attractive target for the discovery of a new class of antibiotics, as it resides exclusively in many pathogenic bacterial strains and is a key enzyme in the lysine biosynthetic pathway. This pathway is responsible for the production of lysine as well as meso-diaminopimelate (m-DAP), both of which are required for peptidoglycan cell-wall synthesis, and lysine for peptide synthesis. The enzyme DapE catalyzes the hydrolysis of N-succinyl-l,l-diaminopimelic acid (l,l-SDAP) to succinate and l,l-diaminopimelic acid (l,l-DAP), and due to its absence in humans, inhibition of DapE avoids mechanism-based side effects. We have executed the asymmetric synthesis of N,N-dimethyl-SDAP, an l,l-SDAP substrate analog and an analog of the synthetic substrate of our previously described DapE assay. Previous modeling studies advocated that N,N-dimethyl-SDAP might function as an inhibitor, however the compound behaves as a substrate, and we have demonstrated the use of N,N-dimethyl-SDAP as the substrate in a modified ninhydrin-based DapE assay. Thermal shift experiments of DapE in the presence of N,N-dimethyl-SDAP are consistent with a melt temperature (Tm) shifted by succinate, the product of enzymatic hydrolysis.

Carborane-Containing Hydroxamate MMP Ligands for the Treatment of Tumors Using Boron Neutron Capture Therapy (BNCT): Efficacy without Tumor Cell Entry.

New carborane-bearing hydroxamate matrix metalloproteinase (MMP) ligands have been synthesized for boron neutron capture therapy (BNCT) with nanomolar potency against MMP-2, -9 and -13. New analogs are based on MMP inhibitor CGS-23023A, and two previously reported MMP ligands 1 (B1) and 2 (B2) were studied in vitro for BNCT activity. The boronated MMP ligands 1 and 2 showed high in vitro tumoricidal effects in an in vitro BNCT assay, exhibiting IC50 values for 1 and 2 of 2.04 × 10-2 mg/mL and 2.67 × 10-2 mg/mL, respectively. The relative killing effect of 1 to L-boronophenylalanine (BPA) is 0.82/0.27 = 3.0, and that of 2 is 0.82/0.32 = 2.6, whereas the relative killing effect of 4 is comparable to boronophenylalanine (BPA). The survival fraction of 1 and 2 in a pre-incubation boron concentration at 0.143 ppm 10B and 0.101 ppm 10B, respectively, were similar, and these results suggest that 1 and 2 are actively accumulated through attachment to the Squamous cell carcinoma (SCC)VII cells. Compounds 1 and 2 very effectively killed glioma U87 delta EGFR cells after BNCT. This study is noteworthy in demonstrating BNCT efficacy through binding to MMP enzymes overexpressed at the surface of the tumor cell without tumor cell penetration.

Cyclobutanone Inhibitor of Cobalt-Functionalized Metallo-γ-Lactonase AiiA with Cyclobutanone Ring Opening in the Active Site.

An α-amido cyclobutanone possessing a C10 hydrocarbon tail was designed as a potential transition-state mimetic for the quorum-quenching metallo-γ-lactonase autoinducer inactivator A (AiiA) with the support of in-house modeling techniques and found to be a competitive inhibitor of dicobalt(II) AiiA with an inhibition constant of K i = 0.007 ± 0.002 mM. The catalytic mechanism of AiiA was further explored using our product-based transition-state modeling (PBTSM) computational approach, providing substrate-intermediate models arising during enzyme turnover and further insight into substrate-enzyme interactions governing native substrate catalysis. These interactions were targeted in the docking of cyclobutanone hydrates into the active site of AiiA. The X-ray crystal structure of dicobalt(II) AiiA cocrystallized with this cyclobutanone inhibitor unexpectedly revealed an N-(2-oxocyclobutyl)decanamide ring-opened acyclic product bound to the enzyme active site (PDB 7L5F). The C10 alkyl chain and its interaction with the hydrophobic phenylalanine clamp region of AiiA adjacent to the active site enabled atomic placement of the ligand atoms, including the C10 alkyl chain. A mechanistic hypothesis for the ring opening is proposed involving a radical-mediated process.

Carborane-Containing Matrix Metalloprotease (MMP) Ligands as Candidates for Boron Neutron-Capture Therapy (BNCT).

Based on the previously reported potent and selective sulfone hydroxamate inhibitors SC-76276, SC-78080 (SD-2590), and SC-77964, potent MMP inhibitors have been designed and synthesized to append a boron-rich carborane cluster by employing click chemistry to target tumor cells that are known to upregulate gelatinases. Docking against MMP-2 suggests binding involving the hydroxamate zinc-binding group, key H-bonds by the sulfone moiety with the peptide backbone residues Leu82 and Leu83, and a hydrophobic interaction with the deep P1' pocket. The more potent of the two triazole regioisomers exhibits an IC50 of 3.7 nM versus MMP-2 and IC50 of 46 nM versus MMP-9.

Practical spectrophotometric assay for the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase, a potential antibiotic target.

A new enzymatic assay for the bacterial enzyme succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18) is described. This assay employs N6-methyl-N2-succinyl-L,L-diaminopimelic acid (N6-methyl-L,L-SDAP) as the substrate with ninhydrin used to detect cleavage of the amide bond of the modified substrate, wherein N6-methylation enables selective detection of the primary amine enzymatic product. Molecular modeling supported preparation of the mono-N6-methylated-L,L-SDAP as an alternate substrate for the assay, given binding in the active site of DapE predicted to be comparable to the endogenous substrate. The alternate substrate for the assay, N6-methyl-L,L-SDAP, was synthesized from the tert-butyl ester of Boc-L-glutamic acid employing a Horner-Wadsworth-Emmons olefination followed by an enantioselective reduction employing Rh(I)(COD)(S,S)-Et-DuPHOS as the chiral catalyst. Validation of the new ninhydrin assay was demonstrated with known inhibitors of DapE from Haemophilus influenza (HiDapE) including captopril (IC50 = 3.4 [± 0.2] µM, 3-mercaptobenzoic acid (IC50 = 21.8 [±2.2] µM, phenylboronic acid (IC50 = 316 [± 23.6] µM, and 2-thiopheneboronic acid (IC50 = 111 [± 16] µM. Based on these data, this assay is simple and robust, and should be amenable to high-throughput screening, which is an important step forward as it opens the door to medicinal chemistry efforts toward the discovery of DapE inhibitors that can function as a new class of antibiotics.

Rearrangement of cyclotriveratrylene (CTV) diketone: 9,10-diarylanthracenes with OLED applications.

Electroluminescent 9,10-diaryl anthracenes have been shown to be promising host and hole-transporting materials in organic electroluminescence due to their high thermal stability, electrochemical reversibility, and wide band gap useful for organic light-emitting diodes (OLEDs), especially blue OLEDs. Oxidation of cyclotriveratrylene (CTV) to the corresponding diketone and subsequent bromination resulted in an unexpected rearrangement to a highly functionalized 9-aryl-10-bromoanthracene derivative, which was employed in Suzuki couplings to synthesize a series of 9,10-diaryl compounds that are structural analogues of anthracene derivatives used in the preparation of OLEDs but are more highly functionalized, including electron-donating methoxy groups in addition to substitution by a carboxylic acid moiety. The UV/fluorescence solution spectra show strong emissions at 446, 438, and 479 nm, respectively, for the anthracene 10-phenyl, 10-naphthyl, and 10-pyrenyl adducts containing a benzoic acid functional group, whereas the analogues bearing the hydroxymethylene moiety from reduction of the benzoic acid to the corresponding alcohols gave much shorter emission wavelengths of 408, 417, and 476 nm, respectively, and had somewhat higher quantum yields, suggesting they are better candidates for OLED applications.

A thermodynamic and kinetic characterization of the solvent dependence of the saddle-crown equilibrium of cyclotriveratrylene oxime.

The equilibration of the saddle conformer of cyclotriveratrylene (CTV) oxime to the corresponding crown conformer was followed by (1)H NMR in five separate solvents, and kinetic and thermodynamic parameters were determined from the NMR data. The oxime saddle conformers of 3 are favored in CDCl(3) (K(eq) = [saddle]/[crown] = 1.4), whereas the CTV oxime crown conformer 3a is favored in three more polar solvents studied (DMSO-d(6), acetonitrile-d(3), acetone-d(6)). Surprisingly, the CTV oxime crown conformer is also slightly favored in the nonpolar solvent 1,4-dioxane-d(8). These behaviors are discussed in terms of hydrogen bonding, entropy, and possible host-guest considerations. An X-ray crystal structure was obtained for CTV monoketone, and structures of the different conformers of CTV, CTV ketone, and CTV oxime were calculated with semiempirical AM1 methods for direct comparison of their ground-state energies.

An aza-cyclophane stacked in racemic columnar assemblies: whole-molecule disorder in a two-dimensional solid solution.

The oxime derivative of [1.1.1]cyclophane cyclotriveratrylene (CTV) was ring expanded utilizing a Beckmann rearrangement to provide a ten-membered N-acetyl macrocyclic amide that crystallizes as a chloroform monosolvate in columnar assemblies manifesting an unusual disorder within the crystal. Columns made up of this structure consist of infinite columnar assemblies of alternating D and L enantiomers and therefore necessarily are made up of a racemate, yet the chiralities of individual molecules in adjacent columns are independent of one another, leading to the overall formation of a two-dimensional solid solution. The random arrangement of the columns within the structure leads to the emergence of a crystallographic mirror plane not reflected by the molecular symmetry, to a change of symmetry from Pna2(1) to Pnma and to whole-molecule disorder of the bowl-shaped molecules within the columns.