Probing the stereochemistry of E. coli 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (phenylalanine-sensitive)-catalyzed synthesis of KDO 8-P analogues

The five-carbon phosphorylated monosaccharide analogues, D-arabinose 5-phosphate, D-ribose 5-phosphate, and 2-deoxy-D-ribose 5-phosphate, were separately condensed with (Z)- and (E)-[3-(2)H]-phosphoenolpyruvate (PEP) in the presence of Escherichia coli 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH 7-P) synthase (phe) to give in the case of (Z)-[3-(2)H]-PEP (3S)-[3-(2)H]-3-deoxy-D-manno-octulosonate 8-phosphate, (3S)-[3-(2)H]-3-deoxy-D-altro-octulosonate 8-phosphate, and (3S)-[3-(2)H]-3,5-dideoxy-D-altro-octulosonate 8-phosphate, respectively, whereas incubation with (E)-[3-(2)H]-PEP gives the corresponding (3R)-monosaccharides. These results are in complete agreement with the observed facial selectivity of DAH 7-P synthase for its normal substrates D-erythrose 4-phosphate and PEP and provide direct evidence that DAH 7-P synthase (phe) catalyzes the si face addition of the C3 of PEP to the re face of C1 of the phosphorylated monosaccharides tested. Products formed by DAH 7-P synthase (phe)-catalyzed condensation of (Z)- and (E)-[3-F]-PEP with E 4-P were completely characterized by (1)H and (19)F NMR analysis for the first time. Results of our studies suggest that disappearence of the double bond between C2 and C3 of PEP and formation of a bond between C3 of PEP and C1 of the phosphorylated monosaccharide tested occur in concert during the DAH 7-P synthase-catalyzed condensation reaction.     

Conversion of aquifex aeolicus 3-deoxy-d-manno-octulosonate 8-phosphate synthase, a metalloenzyme, into a nonmetalloenzyme

The Aquifex aeolicus 3-deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS), a class II metalloenzyme, is converted into an active nonmetalloenzyme by a single amino acid mutation, namely, C11N. The result may provide insight into the evolutionary link between the two KDO8PS classes as well as the potential role of the metal and/or asparagine in the catalytic mechanism.     

Creation of a shikimate pathway variant

The competition between the Escherichia coli carbohydrate phosphotransferase system and 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase for phosphoenolpyruvate limits the concentration and yield of natural products microbially synthesized via the shikimate pathway. To circumvent this competition for phosphoenolpyruvate, a shikimate pathway variant has been created. 2-Keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases encoded by Escherichia coli dgoA and Klebsiella pneumoniae dgoA are subjected to directed evolution. The evolved KDPGal aldolase isozymes exhibit 4-8-fold higher specific activities relative to that for native KDPGal aldolase with respect to catalyzing the condensation of pyruvate and d-erythrose 4-phosphate to produce DAHP. To probe the ability of the created shikimate pathway variant to support microbial growth and metabolism, growth rates and synthesis of 3-dehydroshikimate are examined for E. coli constructs that lack phosphoenolpruvate-based DAHP synthase activity and rely on evolved KDPGal aldolase for biosynthesis of shikimate pathway intermediates and products.     

A snapshot of enzyme catalysis using electrospray ionization mass spectrometry

Insights into the early molecular events involving protein-ligand/substrate interactions such as protein signaling and enzyme catalysis can be obtained by examining these processes on a very short, millisecond time scale. We have used time-resolved electrospray mass spectrometry to delineate the catalytic mechanism of a key enzyme in bacterial lipopolysaccharide biosynthesis, 3-deoxy-d-manno-2-octulosonate-8-phosphate synthase (KDO8PS). Direct real-time monitoring of the catalytic reaction under single enzyme turnover conditions reveals a novel hemiketal phosphate intermediate bound to the enzyme in a noncovalent complex that establishes the reaction pathway. This study illustrates the successful application of mass spectrometry to reveal transient biochemical processes and opens a new time domain that can provide detailed structural information of short-lived protein-ligand complexes.     

Directed evolution of 2-keto-3-deoxy-6-phosphogalactonate aldolase to replace 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase

Directed evolution of 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolase for microbial synthesis of shikimate pathway products provides an alternate strategy to circumvent the competition for phosphoenolpyruvate between 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase and the phosphoenolpyruvate:carbohydrate phosphotransferase system in Escherichia coli. E. coli KDPGal aldolase was evolved using a combination of error-prone polymerase chain reaction, DNA shuffling, and multiple-site-directed mutagenesis to afford KDPGal aldolase variant NR8.276-2, which exhibits a 60-fold improvement in the ratio kcat/KM relative to that of wild-type E. coli KDPGal aldolase in catalyzing the addition of pyruvate to d-erythrose 4-phosphate to form DAHP. On the basis of its nucleotide sequence, NR8.276-2 contains seven amino acid changes from the wild-type E. coli KDPGal aldolase. Amplified expression of NR8.276-2 in the DAHP synthase and shikimate dehydrogenase-deficient E. coli strain NR7 under fed-batch fermentor-controlled cultivation conditions resulted in synthesis of 13 g/L 3-dehydroshikimic acid in 6.5% molar yield from glucose. Increased coexpression of the irreversible downstream enzyme 3-dehydroquinate synthase increased production of 3-dehydroshikimic acid to 19 g/L in 9.7% molar yield from glucose. Coamplification with transketolase, which increases d-erythrose 4-phosphate availability, afforded 16 g/L 3-dehydroshikimic acid in 8.5% molar yield.     

19F NMR ligand perturbation studies on 6,7-bis(trifluoromethyl)-8-ribityllumazine-7-hydrates and the lumazine synthase complex of Bacillus subtilis. Site-directed mutagenesis changes the mechanism and the stereoselectivity of the catalyzed haloform-type reaction

The riboflavin synthase/lumazine synthase complex of Bacillus subtilis catalyzes the last two steps in riboflavin biosynthesis. The protein comprises a capsid of 60 beta subunits with lumazine synthase activity and a core of three alpha subunits with riboflavin synthase activity. The beta subunits catalyze the formation of 6,7-dimethyl-8-ribityllumazine (3) from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (1) and 3,4-dihydroxy-2-butanone 4-phosphate (2). Complexes of recombinant lumazine synthase (beta(60) capsids) with 6-trifluoromethyl-7-oxo-8-ribityllumazine (10) as well as 7S- or 7R-6,7-bistrifluoromethyl-8-ribityllumazine hydrate (11) were studied by (19)F NMR spectroscopy. Despite the large molecular weight of approximately 960 kDa of the protein, spectra with separated signals of free and bound ligand could be obtained. An unusually large shift difference of 8 ppm was observed between the 7-trifluoromethyl signals of free and bound ligand for epimer B of 11 and the enzyme. The signal is sensitive to the replacement of amino acid residues F22 and H88. Lumazine synthase catalyzes the elimination of the 7-trifluoromethyl group of R-diastereomer epimer A in a haloform-like reaction. The elimination reaction is also catalyzed by F22 mutants. The H88R mutant displays an opposite stereoselectivity for epimer B and a greatly enhanced reaction rate. From a model of the epimers in the active site of the protein, the main function of the side chain of F22 seems to be to keep the substrate ring in the correct position. H88 is in a position suited to act as proton acceptor in both the physiological as well as the haloform reaction. A different mechanism of the haloform-reaction is proposed in the case of the H88R mutant, initiated by hydrogen bonding of the 7-trifluorormethyl group and the guanidinium group of the arginine residue.     

Biosynthesis of 1-deoxy-1-imino-D-erythrose 4-phosphate: a defining metabolite in the aminoshikimate pathway.

With respect to the source of the nitrogen atom incorporated into the aminoshikimate pathway, d-erythrose 4-phosphate has been proposed to undergo a transamination reaction resulting in formation of 1-deoxy-1-imino-d-erythrose 4-phosphate. Condensation of this metabolite with phosphoenolpyruvate catalyzed by aminoDAHP synthase would then hypothetically form the 4-amino-3,4-dideoxy-d-arabino-heptulosonic acid 7-phosphate (aminoDAHP), which is the first committed intermediate of the aminoshikimate pathway. However, in vitro formation of aminoDAHP has not been observed. In this account, the possibility is examined that 3-amino-3-deoxy-d-fructose 6-phosphate is the source of the nitrogen atom of the aminoshikimate pathway. Transketolase-catalyzed ketol transfer from 3-amino-3-deoxy-d-fructose 6-phosphate to d-ribose 5-phosphate would hypothetically release 1-deoxy-1-imino-d-erythrose 4-phosphate. Along these lines, a chemoenzymatic synthesis of 3-amino-3-deoxy-d-fructose 6-phosphate was elaborated. Incubation of 3-amino-3-deoxy-d-fructose 6-phosphate in Amycolatopsis mediterranei crude cell lysate with d-ribose 5-phosphate and phosphoenolpyruvate resulted in the formation of aminoDAHP and 3-amino-5-hydroxybenzoic acid. 3-[15N]-Amino-3-deoxy-d-6,6-[2H2]-fructose 6-phosphate was also synthesized and similarly incubated in A. mediterranei crude cell lysate. Retention of both 15N and 2H2 labeling in product aminoDAHP indicates that 3-amino-3-deoxy-d-fructose 6-phosphate is serving as a sequestered form of 1-deoxy-1-imino-d-erythrose 4-phosphate.     

Biosynthesis of riboflavin. Single turnover kinetic analysis of 6,7-dimethyl-8-ribityllumazine synthase

6,7-dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyzes the condensation of 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate, affording the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine. Single turnover experiments monitored by multiwavelength photometry were performed with the recombinant lumazine synthase of Bacillus subtilis. Mixing of the enzyme with the pyrimidine type substrate is conducive to a hypsochromic shift as well as a decrease in absorbance of the heterocyclic substrate; the rate constant for that reaction is 0.02 s(-1) microM(-1). Rapid mixing of the complex between enzyme and pyrimidine type substrate with the second substrate, 3,4-dihydroxy-2-butanone 4-phosphate, is followed by the appearance of an early optical transient characterized by an absorption maxima at 330 nm of low intensity which was tentatively assigned as a Schiff base intermediate. The subsequent elimination of phosphate affords a transient with intense absorption maxima at 455 and 282 nm, suggesting an intermediate with an extended system of conjugated double bonds. The subsequent formation of the enzyme product, 6,7-dimethyl-8-ribityllumazine, is the rate-determining step.     

Synthesis of cyclophospho-glucoses and glucitols

The syntheses of cyclophosphodiesters of 5-C-(hydroxymethyl)-hexoses and hexitols and of 6-C-(hydroxymethyl)-hexoses are reported, along with that of 6-deoxy-gluco-heptose 7-phosphate. These compounds proved to be reasonable substrate mimics and show inhibitory activity against human d-myo-inositol 3-phosphate synthase.     

Investigation of the GeO2-1,6-diaminohexane-water-pyridine-HF phase diagram leading to the discovery of two novel layered germanates with extra-large rings

The systematic exploration of the phase diagram of the GeO(2)-1,6-diaminohexane-water-pyridine-HF system has allowed the identification of specific roles of the HF, H(2)O contents, and HF/H(2)O ratio in the formation of Ge(7)X(19) (Ge(7)), Ge(9)X(25-26) (Ge(9)), and Ge(10)X(28) (Ge(10)) clusters (X = O, OH, F). This work has led to the discovery of two novel structures with extra-large 18-membered rings accommodating 1,6-diaminohexane (DAH): SU-63, |1.5H(2)DAH|[Ge(7)O(14)X(3)]·2H(2)O, a layered germanate constructed from Ge(7) clusters with the Kagome? topology, and SU-64, |11H(2)DAH|[Ge(9)O(18)X(4)][Ge(7)O(14)X(3)](6)·16H(2)O, a germanate built of two-dimensional slabs containing both Ge(7) and Ge(9) clusters (X = OH or F). We also put SU-64 in context with previously reported cluster germanate compounds with related topologies by means of a simple crystal deconstruction study.     

Biosynthesis of terpenoids: efficient multistep biotransformation procedures affording isotope-labeled 2C-methyl-D-erythritol 4-phosphate using recombinant 2C-methyl-D-erythritol 4-phosphate synthase.

This paper describes the recombinant expression of the ispC gene of Escherichia coli specifying 2C-methyl-D-erythritol 4-phosphate synthase in a modified form that can be purified efficiently by metal-chelating chromatography. The enzyme was used for the preparation of isotope-labeled 2C-methyl-D-erythritol 4-phosphate employing isotope-labeled glucose and pyruvate as starting materials. The simple one-pot methods described afford numerous isotopomers of 2C-methyl-D-erythritol 4-phosphate carrying (3)H, (13)C, or (14)C from commercially available precursors. The overall yield based on the respective isotope-labeled starting material is approximately 50%.     

Synthesis and characterization of six new quaternary actinide thiophosphate compounds: Cs(8)U(5)(P(3)S(10))(2)(PS(4))(6)(,) K(10)Th(3)(P(2)S(7))(4)(PS(4))(2), and A(5)An(PS(4))(3), (A = K, Rb, Cs; An = U, Th)

Six new actinide metal thiophosphates have been synthesized by the reactive flux method and characterized by single-crystal X-ray diffraction: Cs(8)U(5)(P(3)S(10))(2)(PS(4))(6) (I), K(10)Th(3)(P(2)S(7))(4)(PS(4))(2) (II), K(5)U(PS(4))(3) (III), K(5)Th(PS(4))(3) (IV), Rb(5)Th(PS(4))(3) (V), and Cs(5)Th(PS(4))(3) (VI). Compound I crystallizes in the monoclinic space group P2(1)/c with a = 33.2897(1) A, b = 14.9295(1) A, c = 17.3528(2) A, beta = 115.478(1) degrees, Z = 8. Compound II crystallizes in the monoclinic space group C2/c with a = 32.8085(6) A, b = 9.0482(2) A, c = 27.2972(3) A, beta = 125.720(1) degrees, Z = 8. Compound III crystallizes in the monoclinic space group P2(1)/c with a = 14.6132(1) A, b = 17.0884(2) A, c = 9.7082(2) A, beta = 108.63(1) degrees, Z = 4. Compound IV crystallizes in the monoclinic space group P2(1)/n with a = 9.7436(1) A, b = 11.3894(2) A, c = 20.0163(3) A, beta = 90.041(1) degrees, Z = 4, as a pseudo-merohedrally twinned cell. Compound V crystallizes in the monoclinic space group P2(1)/c with a = 13.197(4) A, b = 9.997(4) A, c = 18.189(7) A, beta = 100.77(1) degrees, Z = 4. Compound VI crystallizes in the monoclinic space group P2(1)/c with a = 13.5624(1) A, b = 10.3007(1) A, c = 18.6738(1) A, beta = 100.670(1) degrees, Z = 4. Optical band-gap measurements by diffuse reflectance show that compounds I and III contain tetravalent uranium as part of an extended electronic system. Thorium-containing compounds are large-gap materials. Raman spectroscopy on single crystals displays the vibrational characteristics expected for [PS(4)](3)(-), [P(2)S(7)](4-), and the new [P(3)S(10)](5)(-) building blocks. This new thiophosphate building block has not been observed except in the structure of the uranium-containing compound Cs(8)U(5)(P(3)S(10))(2)(PS(4))(6).     

Nanoporous block copolymer micelle/micelle multilayer films with dual optical properties

We introduce a novel and versatile approach for preparing self-assembled nanoporous multilayered films with tunable optical properties. Protonated polystyrene-block-poly(4-vinylpyridine) (PS-b-P4VP) and anionic polystyrene-block-poly(acrylic acid) (PS-b-PAA) block copolymer micelles (BCM) were used as building blocks for the layer-by-layer assembly of BCM multilayer films. BCM film growth is governed by electrostatic and hydrogen-bonding interactions between the opposite BCMs. Both film porosity and film thickness are dependent upon the charge density of the micelles, with the porosity of the film controlled by the solution pH and the molecular weight (M(w)) of the constituents. PS(7K)-b-P4VP(28K)/PS(2K)-b-PAA(8K) films prepared at pH 4 (for PS(7K)-b-P4VP(28K)) and pH 6 (for PS(2K)-b-PAA(8K)) are highly nanoporous and antireflective. In contrast, PS(7K)-b-P4VP(28K)/PS(2K)-b-PAA(8K) films assembled at pH 4/4 show a relatively dense surface morphology due to the decreased charge density of PS(2K)-b-PAA(8K). Films formed from BCMs with increased PS block and decreased hydrophilic block (P4VP or PAA) size (e.g., PS(36K)-b-P4VP(12K)/PS(16K)-b-PAA(4K) at pH 4/4) were also nanoporous. This is attributed to a decrease in interdigitation between the adjacent corona shells of the low M(w) BCMs, thus creating more void space between the micelles. Multilayer films with antireflective and photochromic properties were obtained by incorporating a water-insoluble photochromic dye (spiropyran) into the hydrophobic PS core of the BCMs assembled in the films. The optical properties of these films can be modulated by UV irradiation to selectively and reversibly control the transmission of light. Light transmission of higher than 99% was observed with accompanying photochromism in the (PS(7K)-b-P4VP(28K)/PS(2K)-b-PAA(8K)) multilayer films assembled at pH 4/6. Our approach highlights the potential to incorporate a range of materials, ranging from conventional hydrophilic materials with specific interactions to hydrophobic compounds, into the assembled BCMs to yield multifunctional nanoporous films.     

Fluorogenic stereochemical probes for transaldolases

Transaldolase catalyzes the transfer of dihydroxyacetone from, for example, fructose 6-phosphate to erythrose 4-phosphate. As a potential probe for assaying fluorescent transaldolase, 6-O-coumarinyl-fructose (1) was prepared in six steps from D-fructose. The corresponding 6-O-coumarinyl-5-deoxy derivative 2 was prepared stereoselectively from acrolein and tert-butyl acetate by a chemoenzymatic route involving Amano PS lipase for the kinetic resolution of tert-butyl 3-hydroxypent-4-enoate (7) and E. coli transketolase for assembly of the final product. The corresponding stereoisomer related to D-tagatose was obtained by a chemical synthesis starting from D-ribose. Indeed, transaldolases catalyze the retro-aldolization of substrate 1 to give dihydroxyacetone and 3-O-coumarinyl-glyceraldehyde. The latter primary product undergoes a beta-elimination in the presence of bovine serum albumin (BSA) to give the strongly fluorescent product umbelliferone. A similar reaction is obtained with the 5-deoxy analogue 2, but there is almost no reaction with its stereoisomer 3. The stereoselectivity of transaldolases can be readily measured by the relative rates of fluorescence development in the presence of the latter pair of diastereomeric substrates.     

Reductive Degradation Behavior of Bioreducible Poly(disulfide amine) for Enhancing SiRNA Efficiency

Bioreducible cationic polymer poly(CBA‐DAH) containing repeated disulfide linkages on the polymer backbone was synthesized through Michael‐type polyadditions of CBA to DAH monomers. Poly(CBA‐DAH) could spontaneously form nanoscale polyelectrolyte complexes through electrostatic interactions with siRNA in an aqueous phase. These nanoparticles were rapidly degraded under the reductive cytoplasmic environment with subsequently releasing the siRNA cargo into the cytoplasm where RNAi takes place, as a result of the breakdown of disulfide bonds in the polymers. The reductive degradation behavior of the poly(CBA‐DAH)/siRNA polyplexes is more likely to increase RNAi activity with enhancing the cytoplasmic localization of siRNA molecules. Poly(CBA‐DAH) may have great potential as a gene carrier especially for therapeutic applications of siRNAs owing to the reductive degradation characteristics.

     

Inhibition mode of a bisubstrate inhibitor of KDO8P synthase: a frequency-selective REDOR solid-state and solution NMR characterization

In this report the mode of inhibition of mechanism-based inhibitor (2, K(i) = 0.4 microM) of 3-deoxy-d-manno-2-octulosonate-8-phosphate synthase (KDO8PS), which was designed to mimic the combined key features of its natural substrates arabinose-5-phosphate (A5P) and phoshoenolpyruvate (PEP) into a single molecule, was investigated. Our earlier solid-state NMR observations identified the inhibitor to bind in a way that partly mimics A5P, while the phosphonate moiety of its PEP-mimicking part exhibits no interactions with enzyme residues. This result was apparently in disagreement with the competitive inhibition of 2 against PEP and with the later solved crystal structure of KDO8PS-2 binary complex identifying the interactions of its PEP-mimicking part with the enzyme residues that were not detected by solid-state NMR. To solve this discrepancy, further solid-state REDOR NMR and (31)P solution NMR experiments were applied to a variety of enzyme complexes with the substrates and inhibitor. In particular, a novel frequency-selective REDOR experiment was developed and applied. Integration of the solution and solid-state NMR data clearly demonstrates that under conditions of stoichiometric enzyme-ligand ratio at thermodynamic equilibrium (a) PEP binding is unperturbed by the presence of 2 and (b) both PEP and 2 can bind simultaneously to the synthase, i.e., form a ternary complex with PEP occupying its own subsite and 2 occupying A5P's subsite. The latter observation suggests that under the conditions used in our NMR measurements, the inhibition pattern of 2 against PEP should have a mixed type character. Furthermore, the NMR data directly demonstrate the distinction between the relative binding strength of the two moieties of 2: enzyme interactions with PEP-mimicking moiety are much weaker than those with the A5P moiety. This observation is in agreement with KDO8PS-2 crystal structure showing only remote contacts of the phosphonate due to large structural changes of binding site residues. It is concluded that these phosphonate-enzyme interactions evidenced by both (31)P solution NMR and X-ray are too weak to be preserved under the lyophilization of KDO8PS-2 binary complex and therefore are not evidenced by the solid-state REDOR spectra.     

CRYSTAL STRUCTURE OF {Ni[(C_4H_9O)_2PS_2](C_(12)H_8N_2)_2} -[(C_4H_9O)_2PS_2]

<正> {Ni C(C4H9O)2PS2] (C12H8N2)2} [C(C4H9O)2PS2], Mr= 795. 60, Monocinic,P2,/n,a=16. 806(2), b=12. 720(2), c=21. 248(2) A. β=98. 454(7)°, V = 4493A3,Z=4,Dc= 1. 18g·cm-3,Mo Ka radiation,A=0. 71069A ,F(000) = 1664, R=0. 102 for 4154 reflections with I≥3σ(I).The crystal structure consists of complex cation {NiC(C4H9O)2PS2](C12H8N2)2}+ and complex anion C(C4H9O)2PS2]-. In the cation, nickel (Ⅱ) atom is coordinated by four nitrogen atoms and two sulfur atoms to form a distorted octahedron.     

Phosphacarborane chemistry: the 7,8,9,11-, 7,9,8,10- and 7,8,9,10-isomers of nido-P2C2B7H9--diphosphadicarbaborane analogues of 7,8,9,10-C4B7H11

The reaction between the carborane arachno-4,6-C2B7H13 (1) and PCl3 in dichloromethane in the presence of a "proton sponge" (PS = 1,8-dimethylaminonaphthalene) resulted in the isolation of the eleven-vertex nido-diphosphadicarbaboranes 7,8,9,11-P2C2B7H, (2) and 3-Cl-7,8,11-P2C2B7H, (3-Cl-2) in yields of 54 and 7%, respectively. Replacement of the PS by NEt3 in the same reaction gave diphosphadicarbaboranes 2 and 3-CI-2 together with the isomeric species nido-7,9,8,10-P2C2B7H, (3) in yields of 28, 15 and 3%, respectively. The reaction between the isomeric carborane arachno-4,5-C2B7H13 (4) and PCl3 in dichloromethane in the presence of PS gave the asymmetrical isomer, nido-7,8,9,10-P2C2B7H, (5). along with the chloro derivatives 4-Cl-7,8,9,10-P2C2B7H8 (4-Cl-5) and 11-Cl-7,8,9,10-PC2B7,H8 (11-Cl-5) (yields of 21, 1 and 13%, respectively). The structures of the chlorinated derivatives 3-Cl2 and 11 -Cl-5 were determined by X-ray diffraction analysis. In addition, the structures of all compounds isolated were geometry-optimised and confirmed by comparison of experimental 11B chemical shifts with those calculated by the GIAO-SCF/II//RMP2(fc)/6-31G* method. The calculations also include the structure and 11B NMR shifts of the isomer nido-7,10,8,9-P2C2B7H9 (6) which has not yet been isolated.     

High-performance liquid chromatography assay for 1-deoxy-D-xylulose 5-phosphate synthase activity using fluorescence detection

A high-performance liquid chromatography assay for activity of 1-deoxy-D-xylulose 5-phosphate synthase, an early enzyme in the recently discovered 2-C-methyl-D-erythritol-4-phosphate pathway, was developed. In this assay, the enzymatic product 1-deoxy-D-xylulose was first derivatized with a fluorescent reagent 2-anthranilic acid, followed by separation using HPLC on a Nova-Pak phenyl column with a mobile phase containing CH3CN-water-1-butylamine-tetrahydrofuran-H3PO4 (2:97:0.125:0.5:0.25, v/v). The eluate was monitored by fluorescence detection at an excitation wavelength of 320 nm and an emission wavelength of 425 nm for quantitation of the fluorescent derivative. A linear response was obtained between 5 and 200 ng of 1-deoxy-D-xylulose. This assay was successfully applied to measure the 1-deoxy-D-xylulose 5-phosphate synthase activity in a recombinant E. coli overexpressing dxs gene. It demonstrated that this assay is simple, sensitive and selective compared to the methods used at present.     

Facile and Controlled Fabrication of Functional Gold Nanoparticle‐coated Polystyrene Composite Particle

Gold nanoparticles‐coated polystyrene (AuNPs‐coated PS) composite particles with raspberry‐like morphology are successfully prepared with the aid of a unique thermodynamically driving effect. It is of considerable interest that the AuNPs generate and self‐assemble with raw, ordinary PS microspheres that preexist in the oxidation–reduction systems. The synthesized AuNPs‐coated PS composite particles have been extensively characterized using scanning electron microscope, transmission electron microscope, and UV–Vis‐NIR spectroscopy. The results indicate that the morphology of the resultant composite particles is governed by simply changing the amount and type of reductants and the concentration of PS microspheres. The AuNPs‐coated PS composite particles also exhibit the good surface‐enhanced Raman scattering and catalytic performances.