Penicillins as beta-lactamase-dependent prodrugs: enabling role of a vinyl ester exocyclic to the lactam ring

Incorporation of a vinyl ester exocyclic to the beta-lactam ring of a penicillin nucleus enables this to act as a beta-lactamase-dependent prodrug - rapid release of the (unactivated) alkoxy component of the vinyl ester is triggered by enzyme-catalysed hydrolysis of the beta-lactam ring, whilst buffer-catalysed hydrolysis of the structure at neutral pH is particularly slow.     

单分子层保护纳米 Au 粒子表面配位催化剂的制备及其应用

 贵金属纳米粒子由于其小尺寸效应而表现出特殊的催化性能. 综述了纳米 Au 粒子表面配位催化剂的制备方法及其在催化中的应用. 由于 Au 可与硫化物形成配位键, 所以硫化物可在 Au 表面形成有序单分子膜. 单分子膜保护的 Au 纳米粒子具有非常好的溶解性、分散性、稳定性, 以及由不同的表面功能团而导致的不同的催化性能. 该催化体系兼具均相催化剂和多相催化剂的特点, 这对开发新型催化剂具有重要的理论和实际意义.     

Immobilization of peroxidase glycoprotein on gold electrodes modified with mixed epoxy-boronic Acid monolayers

The development of bioelectronic enzyme applications requires the immobilization of active proteins onto solid or colloidal substrates such as gold. Coverage of the gold surface with alkanethiol self-assembled monolayers (SAMs) reduces nonspecific adsorption of proteins and also allows the incorporation onto the surface of ligands with affinity for complementary binding sites on native proteins. We present in this work a strategy for the covalent immobilization of glycosylated proteins previously adsorbed through weak, reversible interactions, on tailored SAMs. Boronic acids, which form cyclic esters with saccharides, are incorporated into SAMs to weakly adsorb the glycoprotein onto the electrode surface through their carbohydrate moiety. To prevent protein release from the electrode surface, we combine the affinity motif of boronates with the reactivity of epoxy groups to covalently link the protein to heterofunctional boronate-epoxy SAMs. The principle underlying our strategy is the increased immobilization rate achieved by the weak interaction-induced proximity effect between slow reacting oxyrane groups in the SAM and nucleophilic residues from adsorbed proteins, which allows the formation of very stable covalent bonds. This approach is exemplified by the use of phenylboronates-oxyrane mixed monolayers as a reactive support and redox-enzyme horseradish peroxidase as glycoprotein for the preparation of peroxidase electrodes. Quartz crystal microbalance, atomic force microscopy, and electrochemical measurements are used to characterize these enzymatic electrodes. These epoxy-boronate functional monolayers are versatile, stable interfaces, ready to incorporate glycoproteins by incubation under mild conditions.     

Gold‐Catalyzed Cyclization Processes: Pivotal Avenues for Organic Synthesis

Over the years, gold catalysis has materialized as an incredible synthetic approach among the scientific community. Due to the trivial reaction conditions and great functional compatibility, these progressions are synthetically expedient, because practitioners can implement them to build intricate architectures from readily amassed building blocks with high bond forming indices. The incendiary growth of gold catalysts in organic synthesis has been demonstrated as one of the most prevailing soft Lewis acids for electrophilic activation of carbon‐carbon multiple bonds towards a great assortment of nucleophiles. Nowadays, organic chemists consistently employ gold catalysts to carry out a diverse array of organic transformations to build unprecedented molecular architectures. Despite all these achievements and a plethora of reports, many vital challenges remain. In this account, we describe the reactivity of various gold catalysts towards cyclization processes developed over the years. These protocols give access to a wide scope of polyheterocyclic structures, containing different medium‐sized ring skeletons. This is interesting, as the quest for highly selective reactions to assemble diversely functionalized products has attracted much attention. We envisage that these newly developed chemo‐, regio‐, and diastereoselective protocols could provide an expedient route to architecturally cumbersome heterocycles of importance for the pharmaceutical industry.     

Development of a receptor-based microplate assay for the detection of beta-lactam antibiotics in different food matrices

The penicillin-binding protein PBP 2x* from Streptococcus pneumoniae has been utilised to develop a novel microplate assay for the detection and determination of penicillins and cephalosporins with intact beta-lactam structure in milk, bovine and porcine muscle juice, honey and egg. In the assay, the receptor protein is immobilised to a microplate in the first step. To each sample a bifunctional reagent is added, with ampicillin and digoxigenin as functional groups (DIG-AMPI). The amount of bifunctional reagent, which is bound via its ampicillin part to the receptor protein, decreases with increasing beta-lactam concentration in the sample. The detection step uses anti-digoxigenin F(ab) fragments marked with horseradish peroxidase. The more bifunctional reagent is bound to the receptor protein, the more antibody fragments are bound via the digoxigenin part of the reagent. A maximum colour development with tetramethylbenzidine as chromogen for the peroxidase reaction is achieved, when no beta-lactam residues are present. A fractional factorial design was applied to detect chemometrically effects and interactions of the assay parameters. For optimisation of the significant parameters a Box-Behnken design was used. The assay has been developed for various food matrices as screening test with the option for a quantitative assay, when the identity of the residual beta-lactam is known (e.g. elimination studies). Cefoperazon, cefquinome, cefazolin, cloxacillin, ampicillin and benzylpenicillin could be detected at levels corresponding to 1/2 EU maximum residue limit (MRL) in milk, meat juice from muscle tissue of different species, egg and honey (where applicable) without needing lengthy and elaborate sample pre-treatment. Matrix calibration curves are presented, which show that quantitative analyses are possible.     

A theoretical study of the aminolysis reaction of lysine 199 of human serum albumin with benzylpenicillin: consequences for immunochemistry of penicillins

Herein, we present results of a computational study on benzylpenicillin attachment to Lys199 of human serum albumin via an aminolysis reaction. The internal geometry of the reactive part of the system was taken from previous work at the B3LYP/6-31+G* level on the water-assisted aminolysis reaction of a penicillin model compound (Díaz, N.; Suárez, D.; Sordo, T. L. J. Am. Chem. Soc. 2000, 122, 6710--6719). The protein environment around Lys199, the 6-acylamino side chain, and the 2-methyl groups of benzylpenicillin were relaxed by carrying out geometry optimizations with a hybrid QM/MM method (PM3/AMBER). Two different mechanistic routes were explored: a one-step water-assisted process and a carboxylate and water-assisted route in which the beta-lactam carboxylate and the ancillary water molecule mediate the proton transfer from the epsilon-amino group of Lys199 to the beta-lactam leaving N atom. The corresponding energy profiles in the protein combine the B3LYP/6-31+G* and PM3 energies of the reactive subsystem (benzylpenicillin + Lys199 side chain + the ancillary water molecule) and semiempirical PM3 energies of the entire system evaluated with a "divide and conquer" linear-scaling method. It is observed that penicillin haptenation to HSA can proceed through the water-assisted concerted mechanism which is calculated to have a high energy barrier of approximately 38 kcal/mol, in agreement with the experimentally observed slow reaction kinetics.     

Factors that determine the protein resistance of oligoether self-assembled monolayers --internal hydrophilicity,terminal hydrophilicity,and lateral packing density

Protein resistance of oligoether self-assembled monolayers (SAMs) on gold and silver surfaces has been investigated systematically to elucidate structural factors that determine whether a SAM will be able to resist protein adsorption. Oligo(ethylene glycol) (OEG)-, oligo(propylene glycol)-, and oligo(trimethylene glycol)-terminated alkanethiols with different chain lengths and alkyl termination were synthesized as monolayer constituents. The packing density and chemical composition of the SAMs were examined by XPS spectroscopy; the terminal hydrophilicity was characterized by contact angle measurements. IRRAS spectroscopy gave information about the chain conformation of specific monolayers; the amount of adsorbed protein as compared to alkanethiol monolayers was determined by ellipsometry. We found several factors that in combination or by themselves suppress the protein resistance of oligoether monolayers. Monolayers with a hydrophobic interior, such as those containing oligo(propylene glycol), show no protein resistance. The lateral compression of oligo(ethylene glycol) monolayers on silver generates more highly ordered monolayers and may cause decreased protein resistance, but does not necessarily lead to an all-trans chain conformation of the OEG moieties. Water contact angles higher than 70 degrees on gold or 65 degrees on silver reduce full protein resistance. We conclude that both internal and terminal hydrophilicity favor the protein resistance of an oligoether monolayer. It is suggested that the penetration of water molecules in the interior of the SAM is a necessary prerequisite for protein resistance. We discuss and summarize the various factors which are critical for the functionality of "inert" organic films.     

Self-assembled monolayers on electrode surfaces: a probe for redox kinetics

The formation and characterization of self-assembled monolayers of organosulfur compounds like alkanethiols and dialkyl (di)sulfides on metal surfaces such as gold are areas of current research interest. The presence of an aromatic ring in a thiol molecule can enhance the binding between Au and the thiol, resulting in the formation of compact and impervious self-assembled monolayers. The formation of a monolayer of 2-mercaptobenzothiazole (MBT), containing an aromatic group with a fused thiazole ring but no long alkyl chain, is achieved on a gold electrode surface. Voltammetric investigations of ferro/ferricyanide and ferrous/ferric redox systems carried out on this Au|MBT electrode are reported. Further, the possibility of using such an Au|MBT electrode to distinguish between inner and outer sphere electron transfer reactions is indicated. Received: 2 January 1998 / Accepted: 14 May 1998     

Molecular modeling and chemical reactivity of sanfetrinem and derivatives

The indiscriminate use of beta-lactams has considerably diminished their efficiency as a result of bacteria developing effective defense mechanisms against them. Recent pharmaceutical research has led to the synthesis of tricyclic beta-lactam antibiotics known as "tricyclic carbapenems" or "trinems". In this work, we studied the chemical reactivity, an essential property for antibiotic action, of trinems and found it to be similar to that of cephalosporins. Also, we elucidated the interaction pattern for sanfetrinem and 4beta-methoxy trinem and compared it to that for classical beta-lactams. The behavior of both trinems was found to be similar to that of penicillin G toward Staphylococcus aureus PC1, and that of cephalothin and imipenem toward Enterobacter cloacae P99.     

Orientation modulation of a synthetic polypeptide in self-assembled monolayers: a TOF-SIMS study

Structure and orientation of molecules are key properties of functionalized surfaces. Using time-of-flight secondary ion mass spectrometry (TOF-SIMS), here we investigate how to modulate these parameters upon the immobilization process varying the conditions of self-assembly. The molecule of interest, a template-assembled synthetic protein (TASP), consists of a central peptide ring with orthogonally arranged residues. Thioalkane chains allow the directed self-assembly of the molecule on a gold surface; four serine residues on the opposite side of the ring can be used as anchoring sites for various functional sensing molecules. The TASP conformation and its orientation in self-assembled monolayers (SAMs) play a central role for the accessibility of these serine residues. To study the influence of the self-assembly conditions, two series of samples were prepared. Pure TASP monolayers of different surface densities are compared to mixed TASP/alkanethiol monolayers prepared by sequential adsorption varying sequence and particular incubation times as well as by coadsorption modifying incubation times and TASP/alkanethiol mass ratios. Switching the TASP orientation from a state where the molecules are lying flat on the surface to an upright orientation turned out to be possible by inserting the TASP into a preformed alkanethiol monolayer of an appropriate surface density. This study demonstrates that TOF-SIMS is an excellent tool not only to investigate the surface composition, but also the molecular structure of functionalized surfaces.     

Fluoro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate: a reagent for formation of interchain carboxylic anhydrides on self-assembled monolayers

In this paper, we report the reactivity of fluoro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate (TFFH), a reagent for transformation of carboxylic acids into acid fluorides in solution, toward self-assembled monolayers (SAMs) of 16-mercaptohexadecanoic acid on gold. Contrary to the solution-based reactions, we found that only interchain carboxylic anhydrides (ICAs), not acid fluorides (AFs), were obtained at surfaces by the facile interchain reaction under most reaction conditions studied. AFs were found to be formed only when tetrabutylammonium fluoride, a reagent inducing fast decomposition of ICAs, was added to the reaction mixture. The reactivity of TFFH toward carboxylic acid-terminated SAMs was different from that of cyanuric fluoride, which has been reported previously (Langmuir 2005, 21, 11765-11772). This study provides more insight into the role of the proximity effect in SAM-based reactions as well as another approach to the formation of ICAs from carboxylic acid-terminated SAMs.     

Activation for catalysis of penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus by bacterial cell wall

Methicillin-resistant Staphylococcus aureus (MRSA) has acquired a unique penicillin-binding protein (PBP), PBP 2a, which has rendered the organism resistant to the action of all available beta-lactam antibiotics. The X-ray structure of PBP 2a shows the active site in a closed conformation, consistent with resistance to inhibition by beta-lactam antibiotics. However, it is known that PBP 2a avidly cross-links the S. aureus cell wall, which is its physiological function. It is shown herein that synthetic fragments of the bacterial cell wall bind in a saturable manner to PBP 2a and cause a conformational change in the protein that makes the active site more accessible to binding to a beta-lactam antibiotic. These observations and measurements point to a novel strategy by nature to keep the active site of PBP 2a sheltered from the inhibitory activity of the antibiotics, yet it becomes available to the polymeric cell wall by a requisite conformational change for the critical cell wall cross-linking reaction.     

Simple Synthetic Routes to N-Heterocyclic Carbene Gold(I)–Aryl Complexes: Expanded Scope and Reactivity

The discovery of sustainable and scalable synthetic protocols leading to gold–aryl compounds bearing N-heterocyclic carbene (NHC) ligands sparked an investigation of their reactivity and potential utility as organometallic synthons. The use of a mild base and green solvents provide access to these compounds, starting from widely available boronic acids and various [Au(NHC)Cl] complexes, with reactions taking place under air, at room temperature and leading to high yields with unprecedented ease. One compound, (N,N′-bis[2,6-(di-isopropyl)phenyl]imidazol-2-ylidene)(4-methoxyphenyl)gold, ([Au(IPr)(4-MeOC₆H₄)]), was synthesized on a multigram scale and used to gauge the reactivity of this class of compounds towards C−H/N−H bonds and with various acids, revealing simple pathways to gold–based species that possess attractive properties as materials, reagents and/or catalysts.     

Loosely packed self-assembled monolayer of N-hexadecyl-3,6-di(p-mercaptophenylacetylene)carbazole on gold and its application in biomimetic membrane research

Dithiols of N-hexadecyl-3,6-di(p-mercaptophenylacetylene)carbazole (HDMC) have been synthesized and employed to form self-assembled monolayers (SAMs) on gold. One characteristic of the HDMC molecule is its peculiar molecular structure consisting of a large and rigid headgroup and a small and flexible alkyl-chain tail. HDMC adsorbates can attach to gold substrates by a strong Au-S bond with weak van der Waals interactions between the alkyl-chain tails, leading to a loosely packed hydrophobic SAM. In this way we can couple hybrid bilayer membranes (HBMs) to gold surfaces with more likeness to a cell bilayer than the conventional HBMs based on densely packed long-chain alkanethiol SAMs. The insulating properties and stability of the HDMC monolayer as well as the HDMC/lipid bilayer on gold have been investigated by electrochemical techniques including cyclic voltammetry and impedance spectroscopy. To test whether the quality of the bilayer is sufficiently high for biomimetic research, we incorporated the pore-forming protein alpha-hemolysin) and the horseradish peroxidase into the bilayers, respectively. Experimental results demonstrated that this type of loosely packed hydrophobic SAM has great potential in biomimetic bilayer research and biosensor application.     

Preparation of beta- and gamma-lactams from carbamoyl radicals derived from oxime oxalate amides

A general synthetic route to oxime oxalate amides was developed and applied to the preparation of molecules incorporating N-benzyl-N-alkenyl amides linked with acetone oxime or benzaldoxime units. In addition, 2-substituted-thiazolidine-4-carboxylic acid methyl ester amides of oxalyl benzaldoxime were also prepared. It was shown by EPR spectroscopy that the oxalyl benzaldoxime amides dissociated to produce benziminyl and carbamoyl (aminoacyl) radicals when photolysed with 4-methoxyacetophenone as a photosensitizer. Carbamoyl radicals derived from N-alk-3-enyl oxime oxalate amides underwent ring closure to afford pyrrolidin-2-ones. The analogous N-alk-2-enyl precursors afforded azetidin-2-ones. Reactions of the cyclohexenyl and cinnamyl oxime oxalate amides afforded a bicyclic beta-lactam and a 3-benzyl-substituted beta-lactam respectively. Interestingly, both products were isolated as hydroxylated compounds. A thiazolidine-derived oxime oxalate amide containing an isobutenyl side chain also dissociated with production of the corresponding thiazolidinyl-carbamoyl radical, as shown by EPR spectroscopy. GC-MS evidence indicated that this radical cyclised to afford some of the corresponding penicillin derivative     

Co-opting the cell wall in fighting methicillin-resistant Staphylococcus aureus: potent inhibition of PBP 2a by two anti-MRSA beta-lactam antibiotics

Methicillin-resistant Staphylococcus aureus (MRSA) is a global bacterial scourge that has become resistant to many classes of antibiotics, and treatment options for MRSA infections are limited. The cause of MRSA resistance to all commercially available beta-lactam antibiotics is the acquisition of the gene mecA, which encodes penicillin-binding protein 2a (PBP 2a). PBP 2a is a transpeptidase, which in contrast to the other transpeptidases of S. aureus does not experience inhibition by beta-lactam antibiotics. The lack of inhibition is due to a closed conformation for the active site for PBP 2a, which opens up only in the course of the catalytic function of the protein. Here we show that two new anti-MRSA antibiotics now undergoing clinical trials, ceftaroline and ME1036, are able to inhibit PBP 2a effectively, a process that is enhanced in the presence of a cell wall structural surrogate. It is likely that in the course of bacterial growth the occupancy of the allosteric site for the cell wall is co-opted by these antibiotics, and under these conditions the second-order rate constant for the encounter of the antibiotic and PBP 2a approaches the clinically useful value of 10(4)-10(5) M-1 s-1. These compounds are potent inhibitors of PBP 2a as well as PBPs from other species, and have potential as therapeutic agents for treatment of serious infections by MRSA and other resistant bacterial pathogens.     

L-半胱氨酸和胱胺共自组装膜活体检测抗坏血酸

自组装单分子膜（SAM）由于其独特的物理化学性质近年来受到了极大的关注. SAM通过金硫键在电极表面形成高度有序的单分子膜,该稳定的分子膜不仅可以调节表面的亲疏水性质,而且可以促进电极表面氧化还原活性分子的反应速率. 本论文提出了一种简单有效的方法,在金微电极上构建半胱氨酸和胱胺共自组装单分子膜用于活体内抗坏血酸的检测. 研究发现,当混合单分子层中半胱氨酸和胱胺的摩尔比为1:1时,可以在低电位下（约为0.10 V）显著增强抗坏血酸氧化的电子转移动力学,同时该膜能在一定程度上抵抗蛋白质在电极表面的非特异性吸附. 将共自组装单分子膜应用到活体检测中,作者检测到鼠纹状体中抗坏血酸的基准值为257±30mmol·L^-1（n = 3）. 本论文为活体电化学检测提供了一种简单、有效的方法.     

Exploring the chemistry of penicillin as a beta-lactamase-dependent prodrug

The penam nucleus can be modified to behave as a beta-lactamase-dependent 'prodrug' by incorporation of a vinyl ester side chain at the 6-position. Enzyme-catalysed hydrolysis of the beta-lactam ring uncovers the thiazolidine-ring nitrogen as a nucleophile that drives a rapid intramolecular displacement on the side chain. Attachment of 7-hydroxy-4-methylcoumarin as the releasable group of this side chain generated a penicillin structure that can function as a fluorescence-based reporter substance/diagnostic for the presence of low levels of beta-lactamase enzyme in solution. Mechanistic details of the reaction pattern are documented and the scope and limitations of exploiting the structural modification are discussed.     

Self-assembled monolayers as a base for immunofunctionalisation: unequal performance for protein and bacteria detection

Biosensor development strongly depends on the optimisation of surface functionalisation strategies. When gold surfaces are considered, immunofunctionalisation by modification of self-assembled monolayers (SAMs) is one of the preferred approaches. In this respect, SAM-based antibody (Ab) incorporation has shown better performance than Ab physisorption for the detection of proteins and small targets. Reports on bacteria detection are less frequent. In this work, we assess the performance of various SAM-based gold immunofunctionalisation strategies, currently applied to protein detection, in the field of bacteria determination. We present the results for Ab chemical conjugation on mercaptopropanoic acid and mercaptoundecanoic acid SAMs, as well as on a dextranized cysteamine SAM. All the modified surfaces studied were shown to be appropriate for the direct detection of an enzyme-labelled protein, but none succeeded in detecting a bacterial target in a sandwich assay format. Conversely, gold functionalised by Ab physisorption allowed E. coli detection when a sandwich enzyme-linked assay was carried out. The implications of bacteria size and wall complexity are discussed. These results indicate that immunofunctionalisation strategies appropriate for protein detection are not necessarily transferable to work with more complex targets such as bacteria. In this respect, Ab physisorption appears to be a suitable alternative to SAM-based gold functionalisation for bacteria detection.     

Mixed monolayers to promote g-protein adsorption: alpha2A-adrenergic receptor-derived peptides coadsorbed with formyl-terminated oligopeptides

Pure and mixed monolayers of a synthetic peptide, GPR-i3n, derived from the third intracellular loop of the alpha2 adrenergic receptor and a shorter inactive oligopeptide, N-formyl-(Gly)3-(Cys) (called 3GC), were prepared on gold surfaces. The mixing ratio of the GPR-i3n and 3GC was used to control G-protein binding capability. The GPR-i3n peptide is specially designed for bovine G-protein selectivity and has been proven to have high affinity to G-proteins [Vahlberg, C.; Petoral, R. M., Jr.; Lindell, C.; Broo, K.; Uvdal, K. Langmuir 2006, 22 (17), 7260-7264]. Pure 3GC monolayers show very low protein adsorption capability. In this study, 3GC is chosen as a coadsorbent, with the aim to induce molecular conformational changes during monolayer formation to enhance G-protein adsorption. A full characterization of the mixed monolayers was done. The monolayer thickness and the mass-related surface coverage for both GPR-i3n and 3GC were investigated using radio labeling. The GPR-i3n was labeled by 125I-targeting tyrosine, and the activity was measured by using radioimmunoassay (RIA). The formation and chemical composition of GPR-i3n and 3GC monolayers were investigated using X-ray photoelectron spectroscopy, and it is shown that both GPR-i3n and 3GC bind chemically to the gold surface. The interaction between the mixed monolayers and G-proteins was investigated by means of real-time surface plasmon resonance. There is a higher protein binding capacity to the monolayer when the GPR-i3n peptide is intermixed with the 3GC coadsorbent, despite the fact that the 3GC itself has a very low G-protein binding capability. This supports a molecular reorientation at the surface, while 3GC is intermixed with GPR-i3n.