Evolution of class C β-lactamases: factors influencing their hydrolysis and recognition mechanisms

The most common bacterial resistance mechanism to β-lactam antibiotics is the production of β-lactamases. So far, β-lactamases have been classified into four different classes, three of them (A, C and D) have a serine in the active site as the nucleophilic group, which attacks to lactam antibiotic. Despite the large number of kinetic and theoretical studies and many native and complexed β-lactamases crystal structures, the mechanism by which they act is not well understood. The aim of this review is to show the different hypotheses which have been proposed to explain the hydrolysis mechanisms for class A and C lactamases and to cast light onto the interactions between the antibiotic and the Enterobacter cloacae P99 (a class C β-lactamase) in the Henry-Michaelis complex formed previous to the serine attack. Knowledge of these crucial points is essential for obtaining new β-lactam antibiotics not vulnerable to β-lactamases in order to minimize bacterial resistance.     

A highly efficient, asymmetric synthesis of blastidic acid: the β-amino acid component of the antibiotic, (+)-blasticidin S

A new approach for the asymmetric synthesis of blastidic acid, the β-amino acid component of the antibiotic, (+)-blasticidin S has been achieved in 11 steps and 30% overall yield. The key transformations involve a one-pot Swern/Wittig reaction sequence to prepare the carbon backbone followed by a stereoselective conjugate addition to introduce the β-amino functionality.     

饮用水中抗生素残留检测方法研究进展

抗生素作为一种杀灭或抑制细菌生长的药物,在食品、临床、兽药、农业等方面得到了广泛的应用。然而抗生素的大量应用导致其残留物进入地表水,进而污染自来水厂水源,而标准的水处理不能将其完全清除,从而对人体产生较大的危害,引起过敏、发热、耐药性甚至再生障碍性贫血等症状。因此,针对饮用水中抗生素残留检测的研究引起了人们广泛的关注。本文对近年来可应用于饮用水中抗生素残留分析方法(高效液相色谱、太赫兹时域光谱技术、免疫分析法、表面增强拉曼光谱法)的研究进展进行了综述,并对今后抗生素残留检测的发展方向作了展望。     

Stereoselective synthesis of methyl β-dl-novioside

Stereoselective synthesis of an antibiotic sugar, methyl β-dl-novioside from 2-(2-furyl)propan-2-ol is described. The key step involved transformation of 2,5-dimethoxy-2,5-dihydrofuran derivative into 2,3-unsaturated pyranos-4-ulose. Its glycosidation followed by reduction, methylation and hydroxylation afforded β-dl-novioside.It has been demonstrated that anomeric configuration of methyl noviosides and novobiocin are opposite to that reported in the literature.     

Theoretical calculations of β-lactam antibiotics. III. AM1, MNDO,and MINDO/3 calculations of hydrolysis of β-lactam compound (azetidin-2-one ring)

Semiempirical AM1, MINDO/3, and MNDO methods have been used in the study of the alkaline hydrolysis of β-lactam antibiotics through a base-catalyzed, acyl-cleavage, bimolecular mechanism. In this work, the hydroxyl ion has been chosen as nucleophilic agent and the azetidin-2-one ring like a model of β-lactam antibiotic. The MINDO/3 method does not predict correctly the energies of small rings. This, together with the fact that, like MNDO, it cannot detect the occurrence of hydrogen bonds, gives rise to uncertain estimates of energy barriers. The AM1 method can be considered the most suitable for studying the hydrolysis of β-lactam compounds.     

Study on the Conformation of Cyclodecapeptide Loloatin C with Obvious Antibiotic Activity

The conformation of cyclodecapeptide loloatin C with obvious antibiotic activity has been investigated in 2,2,2-trifluoroethanol/sodium acetate buffer solution and then characterized by Fr-IR, CD and NMR spectrum. The results of FT-IR show that there exists β-strand or β-tum secondary structure in the molecule. According to the CD spectrum, the helical turn is dominant but the β-turn structure also exists. Conformation of the whole molecule is probably a helical β-turn.The chemical shifts and coupling constants prove the existence of a β-structure in the regions of Val,Orn2 and Leu3. NOESY data and temperature gradients of amide protons suggest that the molecular conformation is a dumbbell-like structure with the waist located between ornithyl (position 2) and D-phenylalanyl (position 7) and β-turn on both ends.     

Molecular evolution of bacterial β-lactam resistance

Background: Two groups of penicillin-destroying enzymes, the class A and class C β-lactamases, may have evolved from bacterial transpeptidases that transfer x-d-Ala-d-Ala peptides to the growing peptidoglycan during cell wall synthesis. Both the transpeptidases and the β-lactamases are acylated by β-lactam antibiotics such as penicillin, which mimic the peptide, but breakdown and removal of the antibiotic is much faster in the β-lactamases, which lack the ability to process d-Ala-d-Ala peptides. Stereochemical factors driving this evolution in specificity are examined.Results: We have compared the crystal structures of two classes of β-lactamases and a β-lactam-sensitive d-alanyl-d-alanine-carboxypeptidase/transpeptidase (DD-peptidase). The class C β-lactamase is more similar to the DD-peptidase than to another β-lactamase of class A.Conclusions: The two classes of β-lactamases appear to have developed from an ancestral protein along separate evolutionary paths. Structural differentiation of the β-lactamases from the DD-peptidases appears to follow differences in substrate shapes. The structure of the class A β-lactamase has been further optimized to exclude d-alanyl peptides and process penicillin substrates with near catalytic perfection. Keywords: drug resistance, enzymology, penicillin antibiotics, protein ancestry Received: 7 October 1996     

The structure of hitachimycin,a novel macrocyclic lactam involving β-phenylalanine

The structure of hitachimycin ($\text{1}$) has been determined by NMR spectrometry and chemical reactions. The antibiotic is a novel 19-membered macrocyclic lactam possessing a β-phenylalanine moiety.     

Enantioselective Synthesis of β-Lactam Carried C(3) Functionality via Ester Enolate-Imine Condensation

Enantioselective synthesis of optically active β-lactam via ester enolate-imine condensation using homochiral ester 16 and imine 17 provided β-lactam 18a in 77% cc. Lactam 18a, carried C(3) functionality on β-lactam nuclei, provided a potential precursor toward a synthesis of dipeptide antibiotic.     

Desulfation and rearrangement of tigemonam to an isoxazolidin-5-one and the synthesis of the rearrangement product

The β-lactam antibiotic Tigemonam 2 undergoes desulfation to the N-hydroxyazetidinone 4 , which rearranges to the isoxazolidin-5-one 6 . The structure of the rearrangement product 6 was confirmed by synthesis.     

An Easy Preparation of Chemically Pure,High-Activity Tritium-Labelled N-Propionylampicillin for the Analysis of Penicillin-Binding Proteins

Abstract

The laboratory preparation of chemically pure, ³H-labelled β-propionylampicillin suitable for studies of the mode of action of β-lactam antibiotics has been investigated. The chemical purity of the labelled antibiotic is a crucial factor for the quantitative determination of penicillin-binding proteins. Ampicillin (D-α-aminobenzylpenicillin) was N-[³H]propionylated by N-succinimidyl [³H]propionate, the labelled reaction products were analyzed by thin-layer chromatography followed by fluorography. The synthesis and the purification of the resulting N-[³H]propionylampicillin have been optimized, the best conditions for the acylation reaction were at pH 8.25 for 2 h, giving both a high yield of the labelled antibiotic and minimal amounts of unwanted by-products. The reaction mixture was fractionated by a two-step procedure, the purified radioactive antibiotic did not contain any residual reactant, especially free ampicillin, or degradation products such as D-α.-[³H]propionamidobenzylpenicilloic acid. This improved procedure allows an easy, fast and rather inexpensive preparation of a good quality radiolabeled antibiotic for studies of penicillin-interacting proteins.     

Thienamycin. A solution of the stereochemical problem

A new nitrone-based synthesis of β-lactams is described which makes provision for the 1-hydroxyethyl moiety in the potent antibiotic thienamycin. Moreover, the relative stereochemical features of the natural product are defined in a step involving the cycloaddition of a nitrone with methyl crotonate.     

An aziridine route to chiral β-lactams a novel entry to (+)-thienamycin

Enantiomerically pure 2,3- epoxy alcohols are transformed readily and stereospecifically to the corresponding aziridines, regioselective ring-opening of which allows subsequent conversion to chiral β-lactams suitable for elaboration to the title antibiotic.     

A new spirocyclic proline-based lactam as efficient type II′ β-turn inducing peptidomimetic

A new proline-based spirotricyclic lactam is reported as an efficient type II′ β-turn inducing peptidomimetic. After investigations of the reverse turn properties by computational techniques, the scaffold has been synthesized by a straightforward sequence relying on a key RCM reaction for the construction of the spirocyclic lactams ring. For its conformational properties, the scaffold can be considered a privileged structure to be employed as a mimic of the β-turn motif of the potent antibiotic Gramicidin S.     

Unconvential construction of a substituted benzene ring

A substituted biphenyl suitable for elaboration to the antibiotic resistomycin ($\text{1}$) has been synthesised by a novel double Michael process involving dimethyl β-oxoglutarate and the diacetylenic diester ($\text{4}$).     

Separation and quantitation of milk whey proteins of close isoelectric points by on-line capillary isoelectric focusing--electrospray ionization mass spectrometry in glycerol-water media

On-line coupling between CIEF and ESI/MS based on the use of bare fused-silica capillaries and glycerol-water media, recently developed in our laboratory, has been investigated for the separation of milk whey proteins that present close pI values. First, a new rinsing procedure, compatible with MS detection, has been developed to desorb these rather hydrophobic proteins (α-casein (α-CN), bovine serum albumin (BSA), lactoferrin (LF)) from the inner capillary wall and to avoid capillary blockages. Common hydrochloric acid washing solution was replaced by a multi-step sequence based on the use of TFA, ammonia and ethanol. To achieve the separation of major whey proteins (β-lactoglobulin A (β-LG A), β-lactoglobulin B (β-LG B), α-lactalbumin (α-LA) and BSA, which possess close pI values (4.5-5.35), CIEF parameters i.e. carrier ampholyte nature, capillary partial filling length with ampholyte/protein mixture and focusing time, have been optimized with respect to total analysis time, sensitivity and precision on pI determination. After optimization of sheath liquid composition (80:20 (v/v) methanol-water+1% HCOOH), quantitation of β-LG A, β-LG B, α-LA and BSA was performed. The limits of detection obtained from extracted ion current (EIC) and single ion monitoring (SIM) modes were in the 57-136 nM and 11-68 nM range, respectively. Finally, first results obtained from biological samples demonstrated the suitability of CIEF-MS as a potential alternative methodology to 2D-PAGE to diagnose milk protein allergies.     

pH诱导烟草多酚氧化酶二级结构变化的光谱学研究

In order to obtain that more information on the secondary and active site structure of polyphenol oxidase from nicotinan tobacco,FT-IR,synchronous fluorescence and Vis-UV have bee employed to study the secondary structure changes of polyphenol oxidase induced by pH. The results show that,compared with native PPO（pH=7）,PPO at pH=2 contains less both a-helix and anti-parallel β-sheet,more random coil,and almost the same amount of β-turn,and that PPO at pH = 11 contains less a-helix,anti-parallel β-sheet,β-turn,and more random coil. The microenvironments of both Trp and Tyr residues in PPO undergo some changes with pH changing. With the decrease in pH values at the range of pH=7.0-3.38,the coordination between Cu2+ in the active site and imidazoles of histidine are strenghtened. Different LMCT bands of the PPO active site have been observed by changing pH,which result from the structure of Cu（Ⅱ）-imidazole and Cu（Ⅱ）-imidazolate.     

Rhodium(III)-catalyzed C-H alkylation of heterocycles with allylic alcohols in water: A reusable catalytic system for the synthesis of β-aryl ketones

A highly efficient, green and sustainable protocol for rhodium(III)-catalyzed C-H alkylation of heterocycles with allylic alcohols has been achieved, which affords a series of β-aryl ketones in high yields. The reaction proceeds smoothly in water under air, and works well with heterocycles such as indoles, indolines, pyrroles and carbazoles. Notably, the expensive rhodium catalyst in water could be easily separated from the organic products, and reused for at least five times without loss of its catalytic activity and selectivity, which is a promising, green and sustainable pathway for the synthesis of β-aryl ketones. To the best of our knowledge, this is the first example for the reuse of expensive rhodium catalyst in water.     

Fluorescent Mesoporous Nanoparticles for β-Lactamase Screening Assays

We present a sensitive and rapid screening method for the determination of β-lactamase activity of antibiotic-resistant bacteria, by designing a pH-sensitive fluorescent dye-doped mesoporous silica nanoparticle encapsulated with penicillin G as a substrate. When penicillin G was hydrolysed by β-lactamase and converted into penicilloic acid, the acidic environment resulted in fluorescence quenching of the dye. The dye-doped mesoporous nanoparticles not only enhanced the β-lactamase-catalyzed reaction rate but also stablized the substrate, penicillin G, which degrades into penicilloic acid in a water solution without β-lactamase. Twentyfive clinical bacterial samples were tested and the antibiotic resistant and susceptible strains were identified. The proposed method may detect the presence of β -lactamases of clinically relevant samples in less than 1 hour. Moreover, the detection limit of β-lactamase activity was as low as 7.8×10⁻⁴ U/mL, which was determined within two hours.     

Construction of highly enantiopure β,β-diaryl substituted glycine containing two contiguous stereocenters via asymmetric 1,6-conjugate addition

Asymmetric construction of β,β-diaryl substituted glycine bearing two contiguous chiral centers remains a significant challenge. Herein, the application of the para-quinone methides (p-QMs) and Ni(II)- complex of glycine via 1,6-conjugate addition to achieve asymmetric synthesis of β,β-diaryl substituted glycine has been explored successfully. This protocol features operational simplicity, high diastereoselectivity, and recyclable chiral ligand, which provides a versatile approach to the synthesis of highly enantiopure β,β-diaryl substituted glycine.