Flavin‐N5 Covalent Intermediate in a Nonredox Dehalogenation Reaction Catalyzed by an Atypical Flavoenzyme

The flavin‐dependent enzyme 2‐haloacrylate hydratase (2‐HAH) catalyzes the conversion of 2‐chloroacrylate, a major component in the manufacture of acrylic polymers, to pyruvate. The enzyme was expressed in Escherichia coli, purified, and characterized. 2‐HAH was shown to be monomeric in solution and contained a non‐covalent, yet tightly bound, flavin adenine dinucleotide (FAD). Although the catalyzed reaction was redox‐neutral, 2‐HAH was active only in the reduced state. A covalent flavin‐substrate intermediate, consistent with the flavin‐acrylate iminium ion, was trapped with cyanoborohydride and characterized by mass spectrometry. Small‐angle X‐ray scattering was consistent with 2‐HAH belonging to the succinate dehydrogenase/fumarate reductase family of flavoproteins. These studies establish 2‐HAH as a novel noncanonical flavoenzyme.     

Two‐Step Labeling of Endogenous Enzymatic Activities by Diels–Alder Ligation

A ligation strategy based on the Diels–Alder [4+2] cycloaddition for the two‐step activity‐based labeling of endogenously expressed enzymes in complex biological samples has been developed. A panel of four diene‐derivatized proteasome probes was synthesized, along with a dienophile‐functionalized BODIPY(TMR) tag. These probes were applied in a Diels–Alder labeling procedure that enabled us to label active proteasome β‐subunits selectively in cellular extracts and in living cells. We were also able to label the activity of cysteine proteases in cell extracts by utilizing a diene‐derivatized cathepsin probe. Importantly, the Diels–Alder strategy described here is fully orthogonal with respect to the Staudinger–Bertozzi ligation, as demonstrated by the independent labeling of different proteolytic activities by the two methods in a single experiment.     

New Bismaleimide-Silica Hybrid Materials: A Critical Assessment of Properties in Correlation with the Method of Synthesis

New hybrid materials have been prepared by sol–gel technique. They have been obtained from bismaleimide monomers either in reaction with N-(3-triethoxysilylpropyl)furan-2-carboxamide monomer, by a Diels–Alder reaction, or in reaction with (3-aminopropyl)triethoxysilane following a Michael addition reaction. The sol–gel process was conducted with or without adding different amounts of tetraethyl orthosilicate. The structures of the obtained compounds have been confirmed by proton nuclear magnetic resonance and Fourier transform infrared spectroscopy. A comparative study between Diels–Alder- and Michael addition-type products regarding their thermal and mechanical properties was also conducted for samples as obtained from synthesis. The thermoreversible character of the Diels–Alder hybrid materials has been demonstrated with the aids of differential scanning calorimetry and attenuated total reflectance Fourier transform infrared spectroscopy, the results from both methods being in good agreement with each other, and with literature data. The morphology of hybrid materials was studied by the atomic force microscopy, optical microscopy for three different stages: initial (24°C), at heating (150°C), and after cooling at 24°C, and scanning electron microscopy. All data confirmed the driving force for the dispersion of the Si-containing aggregates in the Michael addition series is the dynamic evolution of the sol–gel process, whereas the Diels–Alder series behavior is ruled by the thermoreversible character of the Diels–Alder cycloaddition.     

Biocatalytic Properties and Structural Analysis of Eugenol Oxidase from Rhodococcus jostii RHA1: A Versatile Oxidative Biocatalyst

Eugenol oxidase (EUGO) from Rhodococcus jostii RHA1 had previously been shown to convert only a limited set of phenolic compounds. In this study, we have explored the biocatalytic potential of this flavoprotein oxidase, resulting in a broadened substrate scope and a deeper insight into its structural properties. In addition to the oxidation of vanillyl alcohol and the hydroxylation of eugenol, EUGO can efficiently catalyze the dehydrogenation of various phenolic ketones and the selective oxidation of a racemic secondary alcohol—4‐(1‐hydroxyethyl)‐2‐methoxyphenol. EUGO was also found to perform the kinetic resolution of a racemic secondary alcohol. Crystal structures of the enzyme in complexes with isoeugenol, coniferyl alcohol, vanillin, and benzoate have been determined. The catalytic center is a remarkable solvent‐inaccessible cavity on the si side of the flavin cofactor. Structural comparison with vanillyl alcohol oxidase from Penicillium simplicissimum highlights a few localized changes that correlate with the selectivity of EUGO for phenolic substrates bearing relatively small p‐substituents while tolerating o‐methoxy substituents.     

Superbase‐Catalyzed [4+2] Cycloaddition of Acetylenes to 3,6‐Di(pyrrol‐2‐yl)‐1,2,4,5‐tetrazine: A Facile Synthesis of 3,6‐Di(pyrrol‐2‐yl)pyridazines

Acetylenes undergo the [4+2] cycloaddition to 3,6‐di(pyrrol‐2‐yl)‐1,2,4,5‐tetrazine in the potassium hydroxide/dimethyl sulfoxide or potassium tert‐butoxide/dimethyl sulfoxide systems (80 °C, 2.5–4 h) to afford (after extrusion of the nitrogen molecule from the intermediate) 3,6‐di(pyrrol‐2‐yl)pyridazines in up to 73% yield, while under non‐catalytic conditions this reaction does not take place. This unusual result substantially extends the scope of synthetic application and mechanistic diversity of the Diels–Alder reaction. The step‐wise mechanisms involving the formation of [OH/tetrazine]⁻ or [t‐BuO/tetrazine]⁻ anionic intermediate complexes or cycloaddition of tetrazine to the acetylide anion are considered.     

Structural Studies on the Reaction of Isopenicillin N Synthase with a Sterically Demanding Depsipeptide Substrate Analogue

Isopenicillin N synthase (IPNS) is a nonheme iron(II)‐dependent oxidase that catalyses the central step in penicillin biosynthesis, conversion of the tripeptide δ‐L ‐α‐aminoadipoyl‐L ‐cysteinyl‐D ‐valine (ACV) to isopenicillin N (IPN). This report describes mechanistic studies using the analogue δ‐(L ‐α‐aminoadipoyl)‐(3S‐methyl)‐L ‐cysteine D ‐α‐hydroxyisovaleryl ester (A^SmCOV), designed to intercept the catalytic cycle at an early stage. A^SmCOV incorporates two modifications from the natural substrate: the second and third residues are joined by an ester, so this analogue lacks the key amide of ACV and cannot form a β‐lactam; and the cysteinyl residue is substituted at its β‐carbon, bearing a (3S)‐methyl group. It was anticipated that this methyl group will impinge directly on the site in which the co‐substrate dioxygen binds. The novel depsipeptide A^SmCOV was prepared in 13 steps and crystallised with IPNS anaerobically. The 1.65 Å structure of the IPNS–Fe^II–A^SmCOV complex reveals that the additional β‐methyl group is not oriented directly into the oxygen binding site, but does increase steric demand in the active site and increases disorder in the position of the isovaleryl side chain. Crystals of IPNS–Fe^II–A^SmCOV were incubated with high‐pressure oxygen gas, driving substrate turnover to a single product, an ene‐thiol/C‐hydroxylated depsipeptide. A mechanism is proposed for the reaction of A^SmCOV with IPNS, linking this result to previous crystallographic studies with related depsipeptides and solution‐phase experiments with cysteine‐methylated tripeptides. This result demonstrates that a (3S)‐methyl group at the substrate cysteinyl β‐carbon is not in itself a block to IPNS activity as previously proposed, and sheds further light on the steric complexities of IPNS catalysis.     

Poly(ethylene glycol)‐Supported Chiral Imidazolidin‐4‐one: An Efficient Organic Catalyst for the Enantioselective Diels–Alder Cycloaddition

A tyrosine‐derived imidazolidin‐4‐one was immobilized on a modified poly(ethylene glycol) and converted in situ into a soluble polymer‐supported catalyst for the enantioselective Diels–Alder cycloaddition of acrolein to 1,3‐cyclohexadiene (up to 92% ee) and 2,3‐dimethyl‐1,3‐butadiene (73% ee). Catalyst recycling (up to four cycles) was accompanied by some loss of the chemical efficiency and marginal erosion of the enantioselectivity.     

Characterisation of a Recombinant NADP‐Dependent Glycerol Dehydrogenase from Gluconobacter oxydans and its Application in the Production of L‐Glyceraldehyde

The acetic acid bacterium Gluconobacter oxydans has a high potential for oxidoreductases with a variety of different catalytic abilities. One putative oxidoreductase gene codes for an enzyme with a high similarity to the NADP⁺‐dependent glycerol dehydrogenase (GlyDH) from Hypocrea jecorina. Due to this homology, the GlyDH (Gox1615) has been cloned, over‐expressed in Escherichia coli, purified and characterised. Gox1615 shows an apparent native molecular mass of 39 kDa, which corresponds well to the mass of 37.213 kDa calculated from the primary structure. From HPLC measurements, a monomeric structure can be deduced. Kinetic parameters and the dependence of the activity on temperature and pH were determined. The enzyme shows a broad substrate spectrum in the reduction of different aliphatic, branched and aromatic aldehydes. Additionally, the enzyme has been shown to oxidize a variety of different alcohols. The highest activities were observed for the conversion of D ‐glyceraldehyde in the reductive and L ‐arabitol in the oxidative direction. Since high enantioselectivities were observed for the reduction of glyceraldehyde, the kinetic resolution of glyceraldehyde was investigated and found to yield enantiopure L ‐glyceraldehyde on preparative scale.     

Catalytic Scope of the Thiamine‐Dependent Multifunctional Enzyme Cyclohexane‐1,2‐dione Hydrolase

The thiamine diphosphate (ThDP)‐dependent enzyme cyclohexane‐1,2‐dione hydrolase (CDH) was expressed in Escherichia coli and purified by affinity chromatography (Ni‐NTA). Recombinant CDH showed the same C?C bond‐cleavage and C?C bond‐formation activities as the native enzyme. Furthermore, we have shown that CDH catalyzes the asymmetric cross‐benzoin reaction of aromatic aldehydes and (decarboxylated) pyruvate (up to quantitative conversion, 92–99 % ee). CDH accepts also hydroxybenzaldehydes and nitrobenzaldehydes; these previously have not (or only in rare cases) been known as substrates of other ThDP‐dependent enzymes. On a semipreparative scale, sterically demanding 4‐(tert‐butyl)benzaldehyde and 2‐naphthaldehyde were transformed into the corresponding 2‐hydroxy ketone products in high yields. Additionally, certain benzaldehydes with electron withdrawing substituents were identified as potential inhibitors of the ligase activity of CDH.     

A New Class of Enantioselective Catalytic 2‐Pyrone Diels–Alder Cycloadditions

A highly enantioselective catalytic Diels–Alder (DA) cycloaddition of 2H‐pyran‐2,5‐diones (synthon of 5‐hydroxy‐2‐pyrones) has been developed with a Cinchona‐derived thiourea as the catalyst. The conditions were optimized by using 0.2 equiv. of the catalyst and 0.1 equiv. of formic acid in 2‐propanol at room temperature, which afforded the DA products in yields of up to 90% (exo/endo=5.5:1, 98% ee) with trans‐β‐nitrostyrene derivatives as the dienophiles. The structure/activity relationships of the bifunctional catalyst and the effects of the steric, electronic and hydrogen‐bonding properties of the dienophiles have been studied.     

Synthesis and polymerization of anthracene‐based itaconimides

Two new anthracene‐based itaconimides, ie N‐(1‐ and 2‐anthryl) itaconimides, were synthesized by the reaction of aromatic amines with itaconic anhydride, followed by itaconamic acid imidization. The same reaction failed in the case of anthracene‐ring substitution at the position 9′. Radical and thermal polymerization of N‐(1‐ and 2‐anthryl) itaconimides led to polymers with anthracene pendant groups. No self‐polymerization by Diels–Alder cycloaddition of the itaconic function (dienophile) and anthracene nucleus (diene) was observed, as is the case for N‐(anthryl) maleimides and citraconimides. Copyright © 2004 Society of Chemical Industry     

Detailed Structure–Function Correlations of Bacillus subtilis Acetolactate Synthase

Isobutanol is deemed to be a next‐generation biofuel and a renewable platform chemical. 1 Non‐natural biosynthetic pathways for isobutanol production have been implemented in cell‐based and in vitro systems with Bacillus subtilis acetolactate synthase (AlsS) as key biocatalyst. 2 – 6 AlsS catalyzes the condensation of two pyruvate molecules to acetolactate with thiamine diphosphate and Mg²⁺ as cofactors. AlsS also catalyzes the conversion of 2‐ketoisovalerate into isobutyraldehyde, the immediate precursor of isobutanol. Our phylogenetic analysis suggests that the ALS enzyme family forms a distinct subgroup of ThDP‐dependent enzymes. To unravel catalytically relevant structure‐function relationships, we solved the AlsS crystal structure at 2.3 Å in the presence of ThDP, Mg²⁺ and in a transition state with a 2‐lactyl moiety bound to ThDP. We supplemented our structural data by point mutations in the active site to identify catalytically important residues.     

Dendrimer‐encapsulated Pt nanoparticles/polyaniline nanofibers for glucose detection

A novel amperometric glucose biosensor based on self‐assembling glucose oxidase (GOx) and dendrimer‐encapsulated Pt nanoparticles (Pt‐DENs) on nanofibrous polyaniline (PANI) was described. PANI nanofibers were synthesized via an interfacial polymerization method. A sulfonated polyelectrolytes‐poly(sodium 4‐styrenesulfonate) (PSS) was used to form the negative PANI/sulfonated polyelectrolyte complex, which had good disperse in aqueous solution. GOx was immobilized on the PANI/PSS surface by alternatively assembling a cationic Pt‐DENs layer and an anionic GOx layer. The unique sandwich‐like layer structure (Pt‐DENs/GOx/Pt‐DENs/PANI/PSS) formed by self‐assembling provides a favorable microenvironment to keep the bioactivity of GOx and to prevent enzyme molecule leakage. The fabricated Pt‐DENs/GOx/Pt‐DENs/PANI/PSS electrode exhibited excellent response performance to glucose with a detection limit of 0.5 μM, wide linear range from 10 μM to 4.5 mM, short response time within 5 s, improved sensitivity of 39.63 μA/(mM cm²), and good stability (85% remains after 20 days). © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Block and Random Living,Ring‐Opening Metathesis Copolymerization of Functionally Differentiated Carbazole‐Containing Norbornene Monomers

The synthesis of novel diblock polymers containing both a potential charge transport and a non‐linear optic block has been accomplished. The synthesis exploits the living, ring‐opening metathesis block copolymerization of two norbornene type monomers, one of which contains an unsubstituted N‐carbazolyl ring while the other has a bromo substituent at the 3‐position of the carbazole ring. Conversion of the bromo functionality to a 2,2‐dicyanovinyl group introduces the non‐linear optic property. The first monomer was prepared by the previously reported efficient cation radical Diels–Alder cycloaddition of N‐trans‐1‐propenylcarbazole to 1,3‐cyclopentadiene, while the second was obtained by N‐bromosuccinimide bromination of the first monomer. For purposes of comparison, the corresponding random copolymer was also synthesized.     

Comparative study of poly(vinyl alcohol)‐based support materials for the immobilization of glucose oxidase

BACKGROUND: The performances of four types of glucose oxidase (GOD) immobilization materials based on poly(vinyl alcohol) (PVA) were compared. The matrices of interest were chemically‐linked PVA, freeze‐thawed PVA cryogel, tetramethoxysilane (TMOS) sol‐gel‐PVA hybrid material, and alumina sol‐gel‐PVA hybrid material. RESULTS: Overall, the membranes showed good sensitivity except for the chemically cross‐linked PVA. However, the main differences with the enzyme immobilization methods were enzyme leakage and values of K_m^app. CONCLUSION: Freeze‐thawed PVA‐GOD membranes and TMOS‐PVA, which showed satisfactory sensitivity and adequate value of K_m^app, were quite promising as support materials for immobilizing GOD. Copyright © 2007 Society of Chemical Industry     

Free‐radical copolymerization of [(4‐isopropyl phenyl) oxycarbonyl] methyl methacrylate with acrylonitrile and methyl methacrylate

The present investigation has been achieved in accordance with the Diels–Alder reaction (1,4 cycloaddition) to produce a new halogenated bicyclic adduct. ortho‐Bromoallylbenzoate is a new dienophile that was prepared in a pure form, and its structure was confirmed. The Diels–Alder syntheses of hexachlorocyclopentadiene and the new dienophile were studied to determine the optimum condensation reaction conditions under a temperature range of 90–160°C, reaction times of 1–8 h, and molar diene/dienophile ratios from 1:1 to 5:1 as a consequence. The optimum conditions reached were a temperature of 140°C, an initial diene/dienophile molar ratio of 3:1, and a duration time of 6 h. The maximum stoichometric yield under these optimum conditions (82.5%) was obtained. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 2331–2338, 2003     

Studies on the Biosynthesis of the Stephacidin and Notoamide Natural Products: A Stereochemical and Genetic Conundrum

The stephacidin and notoamide natural products belong to a group of prenylated indole alkaloids containing a bicyclo[2.2.2]diazaoctane core. Biosynthetically, this bicyclic core is believed to be the product of an intermolecular Diels–Alder (IMDA) cycloaddition of an achiral azadiene. Since all of the natural products in this family have been isolated in enantiomerically pure form to date, it is believed that an elusive Diels–Alderase enzyme mediates the IMDA reaction. Adding further intrigue to this biosynthetic puzzle is the fact that several related Aspergillus fungi produce a number of metabolites with the opposite absolute configuration, implying that these fungi have evolved enantiomerically distinct Diels–Alderases. We have undertaken a program to identify every step in the biogenesis of the stephacidins and notoamides, and by combining the techniques of chemical synthesis and biochemical analysis we have been able to identify the two prenyltransferases involved in the early stages of the stephacidin and notoamide biosyntheses. This has allowed us to propose a modified biosynthesis for stephacidin A, and has brought us closer to our goal of finding evidence for, or against, the presence of a Diels–Alderase in this biosynthetic pathway.     

An Efficient Synthesis of Ring‐Opening Metathesis Monomers and Polymers Containing Carbazole and Other Electron‐Rich Moieties

An efficient synthesis of ring‐opening metathesis monomers and polymers containing ionizable moieties such as N‐carbazolyl, 2‐dibenzofuranyl, 2‐dibenzothiophenyl, and 4‐anisyl functionalities has been developed, using cation radical Diels‐Alder cycloaddition chemistry to generate the appropriate norbornene‐type monomers.     

Exploring the Potential of Norbornene‐Modified Mannosamine Derivatives for Metabolic Glycoengineering

Metabolic glycoengineering (MGE) allows the introduction of unnaturally modified carbohydrates into cellular glycans and their visualization through bioorthogonal ligation. Alkenes, for example, have been used as reporters that can react through inverse‐electron‐demand Diels–Alder cycloaddition with tetrazines. Earlier, norbornenes were shown to be suitable dienophiles; however, they had not previously been applied for MGE. We synthesized two norbornene‐modified mannosamine derivatives that differ in the stereochemistry at the norbornene (exo/endo linkage). Kinetic investigations revealed that the exo derivative reacts more than twice as rapidly as the endo derivative. Through derivatization with 1,2‐diamino‐4,5‐methylenedioxybenzene (DMB) we confirmed that both derivatives are accepted by cells and incorporated after conversion to a sialic acid. In further MGE experiments the incorporated sugars were ligated to a fluorophore and visualized through confocal fluorescence microscopy and flow cytometry.     

Inhibition Studies of Mycobacterium tuberculosis Salicylate Synthase (MbtI)

Mycobacterium tuberculosis salicylate synthase (MbtI), a member of the chorismate‐utilizing enzyme family, catalyses the first committed step in the biosynthesis of the siderophore mycobactin T. This complex secondary metabolite is essential for both virulence and survival of M. tuberculosis, the etiological agent of tuberculosis (TB). It is therefore anticipated that inhibitors of this enzyme may serve as TB therapies with a novel mode of action. Herein we describe the first inhibition study of M. tuberculosis MbtI using a library of functionalized benzoate‐based inhibitors designed to mimic the substrate (chorismate) and intermediate (isochorismate) of the MbtI‐catalyzed reaction. The most potent inhibitors prepared were those designed to mimic the enzyme intermediate, isochorismate. These compounds, based on a 2,3‐dihydroxybenzoate scaffold, proved to be low‐micromolar inhibitors of MbtI. The most potent inhibitors in this series possessed hydrophobic enol ether side chains at C3 in place of the enol‐pyruvyl side chain found in chorismate and isochorismate.