Stereochemistry of reactions of the inhibitor/substrates L- and D-beta-chloroalanine with beta-mercaptoethanol catalysed by L-aspartate aminotransferase and D-amino acid aminotransferase respectively

Two members of the alpha-family of PLP-dependent enzymes, L-aspartate aminotransferase and D-amino acid aminotransferase, have been shown to catalyse beta-substitution of L- and D-beta-chloroalanine respectively with beta-mercaptoethanol, reactions typical of the beta-family of PLP-dependent enzymes. The reaction catalysed by L-aspartate aminotransferase has been shown to occur with retention of stereochemistry, a typical outcome for reactions catalysed by beta-family enzymes. There are also indications that the reaction catalysed by D-amino acid aminotransferase may involve retention of stereochemistry. Both enzymes have been shown to catalyse exchange at C-3 when the appropriate enantiomer of beta-chloroalanine is the substrate.     

Complex Cascade Reaction Networks via Cross β Amyloid Nanotubes

Biocatalytic reaction networks integrate complex cascade transformations via spatial localization of multiple enzymes confined within the cellular milieu. Inspired by nature's ingenuity, we demonstrate that short peptide‐based cross‐β amyloid nanotubular hybrids can promote different kinds of cascade reactions, from simple two‐step, to multistep, to complex convergent cascades. The compartmentalizing ability of paracrystalline cross‐β phases was utilized to colocalize sarcosine oxidase (SOX) and hemin as an artificial peroxidase. Further, the catalytic potential of the amyloid nanotubes with ordered arrays of imidazoles were used as hydrolase mimic. The SOX‐hemin amyloid nanohybrids featuring a single extant enzyme could integrate different logic networks to access complex digital designs with the help of three concatenated AND gates and biologically relevant stimuli as inputs.     

Chiral induction effects in ruthenium(II) amino alcohol catalysed asymmetric transfer hydrogenation of ketones: an experimental and theoretical approach

The enantioselective outcome of transfer hydrogenation reactions that are catalysed by ruthenium(II) amino alcohol complexes was studied by means of a systematically varied series of ligands. It was found that both the substituent at the 1-position in the 2-amino-1-alcohol ligand and the substituent at the amine functionality influence the enantioselectivity of the reaction to a large extent: enantioselectivities (ee values) of up to 95% were obtained for the reduction of acetophenone. The catalytic cycle of ruthenium(II) amino alcohol catalysed transfer hydrogenation was examined at the density functional theory level. The formation of a hydrogen bond between the carbonyl functionality of the substrate and the amine proton of the ligand, as well as the formation of an intramolecular H...H bond and a planar H-Ru-N-H moiety are crucially important for the reaction mechanism. The enantioselective outcome of the reaction can be illustrated with the aid of molecular modelling by the visualisation of the steric interactions between the ketone and the ligand backbone in the ruthenium(II) catalysts.     

A G‐Quadruplex/Hemin Complex with Switchable Peroxidase Activity by DNA Hybridization

A hemin‐binding DNA G‐quadruplex (also known as a hemin aptamer or DNAzyme) has been previously reported to be able to enhance the peroxidase activity of hemin. In this work, we described a DNAzyme structure that had an effector‐recognizing part appearing as a single stranded DNA linkage flanked by two split G‐quadruplex halves. Hybridization of the single stranded part in the enzyme with a perfectly matched DNA strand (effector) formed a rigid DNA duplex between the two G‐quadruplex halves and thus efficiently suppressed the enzymatic activity of the G‐quadruplex/hemin complex, while the mismatched effector strand was not able to regulate the peroxidase activity effectively. With 2,2′‐azinobis(3‐ethylbenzthiazoline)‐6‐sulfonic acid (ABTS) as an oxidizable substrate, we were able to characterize the formation of the re‐engineered G‐quadruplex/hemin complex and verify its switchable peroxidase activity. Our results show that the split G‐quadruplex is an especially useful module to design low‐cost and label‐free sensors toward various biologically or environmentally interesting targets.     

A novel mimetic peroxidase catalyst by using magnetite-containing silica nanoparticles as carriers

A method for coating magnetite and mimetic enzyme (hemin) with amorphous silica to form a novel mimetic peroxidase (magnetite–hemin/SiO₂) has been developed by combining reverse microemulsion and the modified Stöber method. The magnetic silica nanoparticle supported hemin has a long-term stability toward temperature and good reusability. They can be easily separated from the reaction solution by using an external magnetic field and reused directly for next round of reaction. The peroxidase activity of the magnetite–hemin/SiO₂ was studied based on its catalytic effect on the reaction of p-hydroxyphenylacetic acid and H₂O₂. The results indicated that the catalytic activity of the new mimetic enzyme catalyst is higher than that of the free hemin. The possibility of its application was proven by the determination of H₂O₂, with the detection limits of 7.3 nmol L^?1 H₂O₂.     

Single‐Step Microwave‐Mediated Synthesis of Oxazoles and Thiazoles from 3‐Oxetanone: A Synthetic and Computational Study

The direct microwave‐mediated condensation between 3‐oxetanone and primary amides and thioamides has delivered moderate to good yields of (hydroxymethyl)oxazoles and (hydroxymethyl)thiazoles. The reactions use a sustainable solvent and only require short reaction times. These are highly competitive methods for the construction of two classes of valuable heteroarenes, which bear a useful locus for further elaboration. Electronic structure calculations have shown that the order of events involves chalcogen atom attack at sp³ carbon and alkyl–oxygen cleavage. The critical role of acid catalysis was shown clearly, and the importance of acid strength was demonstrated. The calculated barriers were also fully consistent with the observed order of thioamide and amide reactivity. Spontaneous ring opening involves a modest degree of C? O cleavage, moderating the extent of strain relief. On the acid‐catalysed pathway, C? O cleavage is less extensive still, but proton transfer to the nucleofuge is well advanced with the carboxylic acid catalysts, and essentially complete with methanesulfonic acid.     

The selective addition of water to C=C bonds; enzymes are the best chemists

Water is the liquid of life. Nature has therefore evolved countless enzymes that catalyse the addition of water to C=C bonds, isolated or conjugated. These reactions are regio- and enantioselective, they are part of primary metabolism as well as the secondary metabolism. The enzymes that catalyse these reactions (hydratases or hydro-lyases) are applied industrially in selected cases. However, they are not generally used in the laboratory although they outperform all currently available catalytic chemical methodologies. This feature article highlights the potential that hydratases have for chemistry compared to the acid catalysed addition of water.     

Protein–Inorganic Array Construction: Design and Synthesis of the Building Blocks

Herein we describe the design and synthesis of the first series of di‐functional ligands for the directed construction of inorganic‐protein frameworks. The synthesized ligands are composed of a metal‐ion binding moiety (terpyridine‐based) conjugated to an epoxysuccinyl peptide, known to covalently bind active cysteine proteases through the active‐site cysteine. We explore and optimize two different conjugation chemistries between the di‐functionalized metal‐ion ligand and the epoxysuccinyl‐containing peptide moiety: peptide‐bond formation (with limited success) and Cu^I‐catalysed click chemistry (with good results). Further, the complexation of the synthesized ligands with Fe^II and Ni^II ions is investigated: the di‐functional ligands are confirmed to behave similarly to the parent terpyridine. As designed, the peptidic moiety does not interfere with the complexation reaction, in spite of the presence of two triazole rings that result from the click reaction. ES‐MS together with NMR and UV/Vis studies establish the structure, the stoichiometry of the complexation reactions, as well as the conditions under which chemically sensitive peptide‐containing polypyridine ligands can undergo the self‐assembly process. These results establish the versatility of our approach and open the way to the synthesis of di‐functional ligands containing more elaborated polypyridine ligands as well as affinity labels for different enzyme families. As such, this paper is the first step towards the construction of robust supramolecular species that cover a size‐regime and organization level previously unexplored.     

The Stereoselective Oxidation of para-Substituted Benzenes by a Cytochrome P450 Biocatalyst

The serine 244 to aspartate (S244D) variant of the cytochrome P450 enzyme CYP199A4 was used to expand its substrate range beyond benzoic acids. Substrates, in which the carboxylate group of the benzoic acid moiety is replaced were oxidised with high activity by the S244D mutant (product formation rates >60 nmol.(nmol-CYP)⁻¹.min⁻¹) and with total turnover numbers of up to 20,000. Ethyl α-hydroxylation was more rapid than methyl oxidation, styrene epoxidation and S-oxidation. The S244D mutant catalysed the ethyl hydroxylation, epoxidation and sulfoxidation reactions with an excess of one stereoisomer (in some instances up to >98 %). The crystal structure of 4-methoxybenzoic acid-bound CYP199A4 S244D showed that the active site architecture and the substrate orientation were similar to that of the WT enzyme. Overall, this work demonstrates that CYP199A4 can catalyse the stereoselective hydroxylation, epoxidation or sulfoxidation of substituted benzene substrates under mild conditions resulting in more sustainable transformations using this heme monooxygenase enzyme.     

Synthesis,characterization and biomimetic oxygenations of manganese(II), iron(III) and copper(II) pyridyl hydrazone complexes

The preparation and characterization of MnII, FeIII and CuII complexes of three tridentate pyridyl hydrazones are reported. The ligands were prepared via Schiff base condensation of 6-chloro-2-hydrazopyridine with alpha-formyl-(L1), alpha-acetyl-(L2), or alpha-benzoyl-(L3) pyridine. The structural characterization of the compounds prepared was based on elemental analyses, electrical conductance and magnetic moment measurements, 1H-n.m.r., i.r., u.v.-vis. and e.s.r spectroscopic methods. The overall structure and reactivity of the metal chelates critically depend on the ligand substituents within the carbonyl moiety. Octahedral and tetrahedral monomeric species were proposed for MnII complexes, and an octahedral environment for the FeIII complexes. Regarding the copper(II) complexes, a monomeric square-planar and a dimeric structure with a chloride bridge in square-pyramidal geometry were suggested. In the presence of molecular oxygen, MnII and CuII complexes catalyse the oxidative transformation of catechol (benzene-1,2-diol) to the corresponding o-benzoquinone. Iron(III) complexes catalyse the aerobic oxidation of catechol to the intradiol cleavage product. The catalytic activity has been correlated with the Lewis acidity of the metal centres created according to the nature of the ligand substituents. The probable mechanistic implications of the catalysed oxidation reactions are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Staining of fluid-catalytic-cracking catalysts: localising Brønsted acidity within a single catalyst particle

A time‐resolved in situ micro‐spectroscopic approach has been used to investigate the Brønsted acidic properties of fluid‐catalytic‐cracking (FCC) catalysts at the single particle level by applying the acid‐catalysed styrene oligomerisation probe reaction. The reactivity of individual FCC components (zeolite, clay, alumina and silica) was monitored by UV/Vis micro‐spectroscopy and showed that only clay and zeolites (Y and ZSM‐5) contain Brønsted acid sites that are strong enough to catalyse the conversion of 4‐fluorostyrene into carbocationic species. By applying the same approach to complete FCC catalyst particles, it has been found that the fingerprint of the zeolitic UV/Vis spectra is clearly recognisable. This almost exclusive zeolitic activity is confirmed by the fact that hardly any reactivity is observed for FCC particles that contain no zeolite. Confocal fluorescence microscopy images of FCC catalyst particles reveal inhomogeneously distributed micron‐sized zeolite domains with a highly fluorescent signal upon reaction. By examining laboratory deactivated FCC catalyst particles in a statistical approach, a clear trend of decreasing fluorescence intensity, and thus Brønsted acidity, of the zeolite domains is observed with increasing severity of the deactivation method. By comparing the average fluorescence intensities obtained with two styrenes that differ in reactivity, it has been found that the Brønsted acid site strength within FCC catalyst particles containing ZSM‐5 is more uniform than within those containing zeolite Y, as confirmed with temperature‐programmed desorption of ammonia.     

Heteroatom‐embedded Medium‐Sized Cycloalkynes: Concise Synthesis,Structural Analysis,and Reactions

A variety of medium‐sized cycloalkynes were efficiently synthesized by the double Nicholas reaction of cobalt complex and bis(hetero)substituted acyclic compound. The alkyne moiety within the ring has a unique bent structure and high reactivity toward cycloaddition reactions. Furthermore, preparation of multifunctionalized alkynes was achieved by embedding the cycloalkyne within a peptide chain.     

Characterization of O-glycosylation sites in MUC2 glycopeptides by nanoelectrospray QTOF mass spectrometry

Sequencing of eight O-glycosylated peptides by nanoESI-QTOF-MS/MS was carried out to provide a sensitive general characterization method for determination of glycosylation site(s) and of the type of the attached carbohydrate moiety in a single experiment. The glycopeptide structures were chosen to demonstrate the feasibility of this sensitive and accurate approach, where isobaric peptide structures either (i) with the same number of attachment sites in different position in the peptide backbone, and (ii) with the same number of sugar moieties distributed on different attachment sites in the peptide backbone, can be clearly distinguished. Beside the B-type carbohydrate sequence ions of high abundance, it is possible to register diagnostic b- and y-type glycosylated peptide ions of lower abundance due to high dynamic range of the QTOF analyser. The applicability of this approach for detailed analysis of highly clustered O-glycan structures as found in biological mucin samples is discussed.     

DNA-enhanced peroxidase activity of a DNA aptamer-hemin complex

Background: In vitro selection (SELEX) previously identified short single-stranded DNAs that specifically bound N-methylmesoporphyrin IX (NMM), a stable transition-state analogue for porphyrin-metallation reactions. Interestingly, iron (III)-protoporphyrin (hemin) was a good competitive inhibitor for the DNA-catalyzed metallation reaction, and appeared to bind strongly to the NMM-binding DNA aptamers. We investigated the peroxidase activity of the aptamer-hemin complexes to see if the DNA component of the complex, like the apoenzymes in protein peroxidases, could enhance the low intrinsic peroxidatic activity of hemin.Results: Two porphyrin-bind ing DNA aptamers bound hemin with submicromolar affinity. The aptamer-hemin complexes had significantly higher peroxidase activity than hemin alone, under physiological conditions. The V_obs of the PS2.M-hemin complex was 250 times greater than that of hemin alone, and significantly superior to a previously reported hemin—catalytic-antibody complex. Preliminary spectroscopic evidence suggests the coordination of the hemin iron in the complex changes, such that the complex more closely resembles horseradish peroxidase and other heme proteins rather than hemin.Conclusions: A new class of catalytic activity for nucleic acids is reported. The aptamer-hemin complexes described are novel DNA enzymes and their study will help elucidate the structural and functional requirements of peroxidase enzymes in general and the ways that a nucleic acid ‘apoenzyme’ might work to enhance the intrinsic peroxidatic ability of hemin. These aptamer-hemin complexes could be regarded as prototypes for redox-catalyzing ribozymes in a primordial ‘RNA world’.     

Heteroatom‐embedded Medium‐Sized Cycloalkynes: Concise Synthesis,Structural Analysis,and Reactions

A variety of medium‐sized cycloalkynes were efficiently synthesized by the double Nicholas reaction of cobalt complex and bis(hetero)substituted acyclic compound. The alkyne moiety within the ring has a unique bent structure and high reactivity toward cycloaddition reactions. Furthermore, preparation of multifunctionalized alkynes was achieved by embedding the cycloalkyne within a peptide chain.     

氯化血红素作为过氧化物模拟酶与HRP的比较研究

用荧光光谱和紫外－可见吸收光谱研究了氯化血红素（hemin)催化过氧化氢与对羧基苯丙酸反应的动力学和反应产物,表明hemin同时具有模拟过氧化物酶、过氧化氢酶的活性,在反应过程中hemin自身被氧化分解。hemin催化反应的产物除与天然的辣根过氧化物酶（HRP）催化的产物相同（λex/λem=310nm/406nm）外,还有激发峰在258nm和300nm,发射峰在360nm的荧光物质。通过对产物光谱特性的比较,研究了hemin和HRP的特异性差异。该研究结果对进一步筛选和研究高特异性过氧化物模拟酶具有重要的指导意义。     

Catalytic oxidative degradation of s-triazine and phenoxyalkanoic acid based herbicides with metalloporphyrins and hydrogen peroxide: Identification of two distinct reaction schemes

Oxidative degradation of the herbicides atrazine (1), atraton (2), ametryn (3) and mecoprop (4), was carried out with hydrogen peroxide and metalloporphyrins as catalysts. Two different reaction conditions were studied, the first involving Mn(TDCPP)Cl in an aprotic solvent with buffer (S-I), and the second using Fe(TPFPP)Cl in a protic solvent (S-II). Reaction products were characterized, and based on these it is shown that there are two distinct reaction schemes.In the case of the S-I conditions, it is suggested that the s-triazines were oxidized through hydroxylation of the alkyl side chains followed by dealkylation, while S-II was ineffective for these reactions. In contrast, mecoprop, was oxidized with high efficiency by S-II, leading to decarboxylation and further oxidation, while in the presence of S-I, low substrate conversion was observed, and reaction resulted mainly from oxidation at the benzyl position. Sulfoxidation of ametryn was observed with both systems.The different reactivity shown by the two systems supports the involvement of different reactive species, which we assign to the oxo and hydroperoxy complexes. These routes show similarities with metabolic pathways, with the reactivity pattern of S-I analogous to the reported metabolism of these pollutants with cytochrome P450 enzymes, while S-II catalyses mecoprop decarboxylation via a similar pathway to that seen with peroxidase catalysed reactions.     

Architectures, mechanisms and molecular evolution of natural product methyltransferases

Covering: up to January 2012The addition of a methyl moiety to a small chemical is a common transformation in the biosynthesis of natural products across all three domains of life. These methylation reactions are most often catalysed by S-adenosyl-l-methionine (SAM)-dependent methyltransferases (MTs). MTs are categorized based on the electron-rich, methyl accepting atom, usually O, N, C, or S. SAM-dependent natural product MTs (NPMTs) are responsible for the modification of a wide array of structurally distinct substrates, including signalling and host defense compounds, pigments, prosthetic groups, cofactors, cell membrane and cell wall components, and xenobiotics. Most notably, methylation modulates the bioavailability, bioactivity, and reactivity of acceptor molecules, and thus exerts a central role on the functional output of many metabolic pathways. Our current understanding of the structural enzymology of NPMTs groups these phylogenetically diverse enzymes into two MT-superfamily fold classes (class I and class III). Structural biology has also shed light on the catalytic mechanisms and molecular bases for substrate specificity for over fifty NPMTs. These biophysical-based approaches have contributed to our understanding of NPMT evolution, demonstrating how a widespread protein fold evolved to accommodate chemically diverse methyl acceptors and to catalyse disparate mechanisms suited to the physiochemical properties of the target substrates. This evolutionary diversity suggests that NPMTs may serve as starting points for generating new biocatalysts.     

N-Terminal peptide aldehydes as electrophiles in combinatorial solid phase synthesis of novel peptide isosteres

N-Terminal peptide aldehydes were synthesized on a solid support and utilized as electrophiles in nucleophilic reactions in order to furnish novel and diverse peptide isosteres. The aldehyde moiety of the peptide was synthesized by coupling a protected aldehyde building block to the peptide and deprotecting it quantitatively in less than 3 min. It was found that protection of the two succeeding amide nitrogens was necessary in order to avoid reaction between the aldehyde and backbone amides. The N-terminal peptide aldehydes were successfully reacted in the following way: (a) reductive amination with a large variety of amines, leading to N-alkyl-gamma-aminobutyric peptide isosteres positioned centrally in the peptide; (b) reductive amination with amino esters, leading to N-terminal 2,5-diketopiperazine peptides; (c) Horner-Wadsworth-Emmons olefination, leading to unsaturated peptide isosteres positioned centrally in the peptide; and (d) Pictet-Spengler condensations, leading to tetrahydro-beta-carbolines either positioned centrally in a peptide or fused with a diketopiperazine ring in the N-terminus of the peptide.     

Enzymatic Promiscuity： Escherichia coli BioH Esterase-catalysed Aldol Reaction and Knoevenagel Reaction

Esterase BioH,which is obligatory for biotin synthesis in Escherichia coli,was found to exhibit a promiscuous ability to catalyse Aldol and Knoevenagel reactions with moderate to good yields.The reaction conditions including organic solvent,molar ratio of ketone to aldehyde,enzyme amount,and reaction time were investigated to evaluate the effect of different reaction conditions on yield.Target compounds were afforded in the best yield of 91.2％ for Aldol reaction and 54.7％ for Knoevenagel reaction.In addition,because the enzyme could be prepared with a low cost,this protocol could provide an economic route to conduct Aldol and Knoevenagel reactions,which expand the field of enzymatic promiscuity.