Development of enzyme-linked immunosorbent assays for the detection of deacetoxycephalosporin C and isopenicillin N synthase activity

Although there are a number of existing assays for monitoring the activity of both isopenicillin N synthase (IPNS) and deacetoxycephalosporin C synthase (DAOCS), none have demonstrated the qualities required for screening a mutant library. Hence, enzyme-linked immunosorbent assays (ELISAs) for IPNS and DAOCS were developed based on the detection of the catalytic turnover products isopenicillin N and cephalexin/phenylacetyl-7-aminodeacetoxycephalosporanic acid (G-7-ADCA), respectively. These assays are relatively fast compared to existing assays, such as the hole-plate bioassay, and are amenable with high-throughput screening. Both the IPNS and DAOCS-ELISAs were optimised for use with crude protein extracts rather than purified protein, thereby eliminating any additional time required for purification. The ELISA developed for the detection of cephalexin had an IC₅₀ value of 154 ± 9 ng mL⁻¹ and LOD of 7.2 ± 2.2 ng mL⁻¹ under conditions required for the assay. Good recoveries and correlation was observed for spiked samples when the concentration of crude protein was kept below 1 mg mL⁻¹. The DAOCS-ELISA was found to have increased sensitivity compared to the hole-plate bioassay (10.3 μg mL⁻¹). The IPNS-ELISA did not significantly increase the sensitivity (approximately 5 μg mL⁻¹) compared to that of the hole-plate bioassay (16 μg mL⁻¹) for isopenicillin N. The minimum amount of crude protein extract required for producing detectable amounts of product for both assays was below 0.5% of the maximum amount of protein that the assay could contain without any effect on the ELISA. This suggests that when screening a mutant library, mutants producing low amounts of the product could still be detected using these assays.     

Diastereoselective approach to novel octahydroisoquinolones and an extension to its one-pot synthesis

The present Letter describes the use of β-lactams for the synthesis of functionalized β-amino esters and their transformation to trisubstituted octahydroisoquinolone derivatives in good yields with an extension to their one-pot synthesis.     

Biosensor analysis of penicillin G in milk based on the inhibition of carboxypeptidase activity

The β-lactam antibiotics, including penicillins, are the most important antimicrobial substances used for mastitis treatment. Consequently, this is also the most frequently occurring type of antibiotic residues in milk. Today, in addition to the traditional microbial inhibitor tests, rapid and sensitive receptor and immunoassays are used in residue control. Due to the limitations in throughput capacity of these tests, recent applications of automated biosensor technology in food analysis are of great interest.A surface plasmon resonance (SPR)-based biosensor (Biacore) was used to design an inhibition assay to detect β-lactam antibiotics in milk. A microbial receptor protein with carboxypeptidase activity was used as detection molecule. One advantage of using this receptor protein over antibodies that are more commonly used is that only the active, intact β-lactam structure is recognized, whereas most antibodies detect both active and inactive forms. In the presence of β-lactam antibiotics the formation of a stable complex between receptor protein and antibiotic inhibits the enzymatic activity of the protein. The decrease in enzymatic activity was measured using an antibody against the degraded substrate and penicillin G in milk samples was quantitatively determined. The limit of detection of the assay for penicillin G was determined to 2.6 μg kg⁻¹ for antibiotic-free producer milk, which is below the European maximum residue limit (MRL) of 4 μg kg⁻¹. The coefficient of variation at 4 μg kg⁻¹ penicillin G, ranged between 7.3 and 16% on three different days.     

Crystallographic studies on the reaction of isopenicillin N synthase with an unsaturated substrate analogue

Isopenicillin N synthase (IPNS) catalyses conversion of the linear tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N (IPN), the central step in biosynthesis of the beta-lactam antibiotics. The unsaturated substrate analogue delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-vinylglycine (ACvG) has previously been incubated with IPNS and single product was isolated, a 2-alpha-hydroxymethyl isopenicillin N (HMPen), formed via a monooxygenase mode of reactivity. ACvG has now been crystallised with IPNS and the structure of the anaerobic IPNS:Fe(II):ACvG complex determined to 1.15 A resolution. Furthermore, by exposing the anaerobically grown crystals to high-pressure oxygen gas, a structure corresponding to the bicyclic product HMPen has been obtained at 1.60 A resolution. In light of these and other IPNS structures, and recent developments with related dioxygenases, the [2 + 2] cycloaddition mechanism for HMPen formation from ACvG has been revised, and a stepwise radical mechanism is proposed. This revised mechanism remains consistent with the observed stereospecificity of the transformation, but fits better with apparent constraints on the coordination geometry around the active site iron atom.     

The Vilsmeier reagent as an efficient acid activator for the synthesis of β-lactams

The Vilsmeier reagent (chloromethylenedimethylammonium chloride) has been used as an efficient and cheap acid activator for the one-step Staudinger reaction of substituted acetic acids and imines under mild conditions. This reaction is clean and the by-products are DMF and triethylamine hydrochloride which were removed by simple aqueous work-up.     

Evaluating substrate specificity of glutathione peroxidase mimic by molecular dynamics simulations and kinetics

The substrate specificities of glutathione peroxidase (GPX) mimic, 6,6′-ditellurobis(6-deoxy-β-cyclodextrin) (6-TeCD), for three hydroperoxides (ROOH), H₂O₂, tert-butyl hydroperoxide (t-BuOOH) and cumene hydroperoxide (CuOOH), are investigated through molecular dynamics (MD) simulations. The most stable conformations and the total interaction energies of complex of 6-TeCD with ROOH are used to evaluate the substrate specificity of 6-TeCD. The steady-state kinetics of 6-TeCD is studied and the Michaelis-Menten constant (K _m) and second-order rate constant k _max/K _ROOH show that 6-TeCD displays different affinity and specificity to ROOH. These results of experiments are well consistent with ones obtained by MD simulations, indicating that MD simulations could be applied to evaluation substrate specificity of small-molecule enzyme mimics.     

4-CF3-ezetimibe analogs: design,synthesis, and biological evaluation of cholesterol absorption inhibitions

On the purpose of looking for better cholesterol absorption inhibitors, several trifluoromethyl substituted ezetimibe analogs 1a–d were designed and synthesized. The key steps in the synthesis of these optically pure trans-4-CF₃-β-lactams include chiral auxiliary induced asymmetric hydrogenation and substrate controlled stereoselective alkylation. The inhibitory activities of these target compounds were evaluated on the cholesterol absorption in Caco-2 cells. The result showed that the inhibitory activity of compound 1a was comparable to ezetimibe.     

2-Acylimino-3-alkyl-3H-thiazoline derivatives: one-pot, three-component condensation synthesis of novel β-turn mimics

One-pot, three-component condensation of aroylthiourea, primary amine and α-halocarbonyl derivatives for the synthesis of 2-acylimino-3-alkyl-3H-thiazoline derivatives is described. This method is useful for simultaneously incorporating diverse functional groups at four positions in the 3H-thiazoline skeleton to obtain β-turn tripeptide mimics.     

A gram-scale laboratory synthesis of Annonaceous acetogenin mimic AA005

AA005(1),a potent anticancer mimicking molecule of natural Annonaceous acetogenin,features a central C₂-symmetric triglycol moiety and three stereogenic centers.To support ongoing animal studies,a scalable 10-step synthesis of AA005(1) has been studied,optimized and accomplished in this work,starting from commercially available economic materials ethylene glycol and(R)-epichlorohydrin.A regio-and stereo-controlled BF₃·Et₂O-promoted epoxide opening of(R)-epichlorohydrin with ethylene glycol was investigated,optimized and successfully applied to the scalable preparation of the crucial fragment 6 a with C₂-symmetry.Further successive epoxide openings elaborated both two side chains and the whole skeleton with proper nucleophiles and electrophiles.Comparison and optimization of the reaction sequences finally led to completion of a new multi-gram synthesis ofAA005(1) with satisfactory enantiomeric and diastereomeric purity.     

Stereoselective synthesis of 3-alkylidene/alkylazetidin-2-ones from azetidin-2,3-diones

Azetidin-2,3-diones have been used as synthons for the synthesis of C-3 alkylidene/alkylazetidin-2-ones. Some of the 3-alkylazetidin-2-ones are well known for their cholesterol absorption inhibitor activity. A regio and stereoselective Grignard reaction on a keto group followed by dehydration using PPh₃/CCl₄ reagent is a key step in this synthesis. Hydrogenation of the 3-alkylideneazetidin-2-ones provided stereoselectively cis-3-alkylazetidin-2-ones in very good yields.     

A stereodivergent route to epimeric 2-piperidinylglycines: application to the synthesis of carbocyclic β-lactam derivatives

A route to two epimeric hitherto unknown 2-piperidinylglycine derivatives, as precursors of carbocyclic β-lactam derivatives, has been developed, which features diastereoselective addition of allyl metal reagents to an N-allylimine derivative of Garner’s aldehyde and ring-closing metathesis as key steps.     

Enantioselective total synthesis of (2S,3R,4R)-d-xylo-phytosphingosine from substituted azetidin-2-one

Enantiomerically pure (2S,3R,4R)-d-xylo phytosphingosine is synthesized in 36% overall yield in seven steps from known β-lactam (8) derived from d-mannitol triacetonide.     

Facile diastereoselective synthesis of functionally enriched hydantoins via base-promoted intramolecular amidolysis of C-3 functionalized azetidin-2-ones

Synthesis of functionally enriched hydantoins has been developed and validated via base-assisted intramolecular amidolysis of C-3 functionalized β-lactams.     

Design and synthesis of novel triazole derivatives containing γ-lactam as potential antifungal agents

A series of novel triazole derivatives containing γ-lactam were designed and synthesized, and their structures were confirmed by ~1H NMR,~(13)CNMR and HRMS. The in vitro antifungal activities of the target compounds were evaluated. The results showed that all of the compounds exhibited stronger activity against the six clinically important fungi tested than fluconazole. 3D and 3E showed comparative activity against the fungi tested except for Candida glabrata and Aspergillus fumigatus as voriconazole. In addition,the docking model for 2A and CYP51 was investigated.     

Design, synthesis and functional analysis of dansylated polytheonamide mimic: an artificial Peptide ion channel

We report herein the design, total synthesis, and functional analysis of a novel artificial ion channel molecule, designated as dansylated polytheonamide mimic (3). The channel 3 was designed based on an exceptionally potent cytotoxin, polytheonamide B (1). Our strategy for the development of synthetic ion channels, which could be easily derivatized for various functions, involved two key features. First, the structure of 1 was simplified by replacing many of nonproteinogenic amino acid residues which required multistep synthesis by commercially available amino acids while retaining those residues necessary for folding. It significantly reduced the number of synthetic steps and facilitated a practical chemical construction of 3. Second, the introduction of propargyl glycine at residue 44 enabled facile installation of dansyl group as a reporter of the membrane localization of 3. Application of a newly designed protective group strategy provided efficient construction of the 37 amino acid sequence of residues 12-48 through one automatic solid-phase peptide synthesis. After peptide cleavage from the resin, 3 was synthesized via dansyl group introduction and one fragment-coupling reaction with residues 1-11, followed by the global deprotection. The simplified mimic 3 exhibited potent cytotoxicity toward p388 mouse leukemia cells (IC(50) = 12 nM), effectively induced ion transport across the lipid bilayers of liposomes, and displayed H(+) and Na(+) ion channel activities. Because of its simplified yet functional scaffold structure with a potential for diversification, our rationally designed ion channel molecule should be useful as a novel platform for developing various cytotoxic channel molecules with additional desired functions.     

Facile synthesis of benzamides to mimic an α-helix

A new α-helix mimetic was designed by using a benzamide as a rigid scaffold. It presents three functional groups corresponding to side chains of amino acids found at the i, i + 4, and i + 7 positions of an ideal α-helix, which are displayed on the same helical face. Its efficient synthesis was achieved by employing simple alkylation and amidation reactions which can be easily adapted for solid-phase synthesis. As a result, two tris-benzamides were produced to mimic two helical regions found in a peptide hormone, glucagon.     

Computational modeling of the kinetics and mechanism of tellurium-based glutathione peroxidase mimic

A new generation of glutathione peroxidase enzyme mimic based on organotellurium was introduced. The catalytic cycles of these mimics, tellura and tellenol, were clarified by density functional theory and solvent-assisted proton exchange procedure as an indirect proton exchange chain. From the kinetic viewpoint, the oxidation of tellura (ΔG^≠ = 23.55 kcal mol⁻¹) was considered as the rate-determining step using a single-step process. Various behaviors of tellenol were examined in the reduction of tellurenylsulfide based on methanethiol nucleophilicity. On the basis of the turnover frequency calculations, during the catalytic cycles of tellura and tellenol, the rate of the catalytic cycle of tellura is faster than that of tellenol. A decrease in the electron density and an increase in the Laplacian from the reactant to the transition states are evidence of the bond rupture, whereas an opposite change is evidence of the bond formation. Finally, different analyses of the electron location function and localized orbital locator within the quantum theory of atoms in molecules were applied and discussed. The covalent nature of the intramolecular interactions suggests that the Te⋯N interaction is stronger than that of Te⋯H. Finally, based on different analyses, tellura can be considered the more reactive GPx mimic than tellenol.     

One‐step synthesis of polysiloxane graft polyethylene mimic: Non‐expensive compatibilizer for polyethylene/polysiloxane blend

Polysiloxane/polyolefin copolymers have drawn much attention recently and emerged as a new group of functional polyolefin since they possess distinctive properties and find great potential applications in many areas (eg, compatibilizer, processing aid and surface modifier). However, traditional routes to synthesize polysiloxane/polyolefin copolymers generally require multi‐step labor‐consuming procedures. Herein, we report a novel one‐step synthesis of polydimethylsiloxane graft polyethylene (PDMS‐g‐PE) mimics. It was found that PDMS‐g‐PE mimics, namely vinylmethylsiloxane‐dimethylsiloxane‐(C30‐45 alkyl)methylsiloxane copolymers (short for VD‐AMS), could be formed via a one‐step synthetic procedure based on the siloxane equilibrium process between silanol‐terminated vinylmethylsiloxane‐methylsiloxane copolymer and (C30‐45 alkyl)methylsilicone. The chemical structures of VD‐AMS were characterized unambiguously using Fourier transform infrared spectroscopy, nuclear magnetic resonance, gel permeation chromatography, differential scanning calorimetry. The correlation between reaction conditions and the structural parameters of VD‐AMS was established. Based on our experimental results, a plausible mechanism for the synthesis of VD‐AMS was proposed. Scanning electron microscopy micrographs showed that VD‐AMS could function as an efficient compatibilizer for immiscible PE/silicone blend. Given that the precursors of VD‐AMS are commercially available with low prices and that VD‐AMS can be easily synthesized under mild conditions, we believe VD‐AMS can represent as a competitive potential compatibilizer due to its relatively low cost.     

N,N,N-Trimethylchitosan chloride as a gene vector: synthesis and application

N,N,N-Trimethylchitosan chloride with different degrees of quaternization has been synthesized and characterized by (1)H NMR spectroscopy. The particle size ranges from 150 to 600 nm, which is dependent on the N/P ratio and is less influenced by the degree of quaternization. The majority of the particles have a spherical morphology. The zeta potential of the particles increases with the N/P ratio and the quaternization degree of TMC. Short-term contact experiments show good biocompatibility of TMC, but long-term contact experiments reveal its high toxicity. This study suggests that TMC is a promising gene carrier, but further modification is still required to improve its cytocompatibility.