Probing the Subunit-Subunit Interaction of the Tetramer of E. coli KDO8P Synthase by Electrospray Ionization Mass Spectrometry

Escherichia coli 3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation reaction between D-arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form KDO8P and inorganic phosphate (Pi). This enzyme exists as a tetramer in solution, which is important for catalysis. Two different states of the enzyme were obtained: i) PEP-bound and ii) PEP-unbound. The effect of the substrates and products on the overall structure of KDO8P synthase in both PEP-bound and unbound states was examined using electrospray ionization mass spectrometry. The analysis of our data showed that the complexes of the PEP-unbound enzyme with PEP (or Pi) favored the formation of monomers, while the complexes with A5P (or KDO8P) mainly favored dimers. The PEP-bound enzyme was found to exist in the monomer and dimer with a small amount of the tetramer, whereas the PEP-unbound form primarily exists in the monomer and dimer, and no tetramer was observed, suggesting that the bound PEP have a role in stabilization of the tetrameric structure. Taken together, the results imply that the addition of the substrates or products to the unbound enzyme may alter the subunit-subunit interactions and/or conformational change of the protein at the active site, and this study also demonstrates that the electrospray ionization mass spectrometric method may be a powerful tool in probing the subunit-subunit interactions and/or conformational change of multi-subunit protein upon binding to ligand.     

Design and Synthesis of 1-Benzyl-3-(a-hydroxy-(un)substituted benzylidene)pyrrolidine-2,4-diones as Potential Herbicides

The inhibitors of 4-hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) are an new kind of herbicides.[1]Generally, in structure, potent herbicides of this kind must possess: (1) a tricarbonyl methane structure and one of the three carbonyl groups must be a subustituted benzoyl group; (2) the compound must be able to enolise so that the enolate is capable of inhibiting HPPD enzyme by competitive combination with Fe2+, the reaction center of HPPD enzyme.[2]Recently, we noticed that the 3-acyltetramic acids form an expanding group of antibiotics and pigments from micro-organisms,[3] they display a range of biological activities[4] and all of this kind compounds possess all of the characters mentioned above. Tests of their antimicrobial activities indicated that the structure of the acyl substituent at the 3-position was important to many typical antibiotics.[4～6] These characters stimulated us to study this kind of compounds so as to discover new herbicides. In this report, we synthesized a series of compounds 3 and tested their herbicidal activities to investigate their structure-activity relationships.     

Characterization of 1,4-dihydroxy-2-naphthoyl-coenzyme A synthase (MenB) in phylloquinone biosynthesis of Synechocystis sp. PCC 6803

The gene product Sll1127 is a predicted 1,4-dihydroxy-2-naphthoyl-CoA synthase catalyzing an intramolecular Claisen condensation in the phylloquinone biosynthesis of the cyanobacterium Synechocystis sp.PCC 6803.This predicted catalytic function has been verified and the enzyme has been characterized for the first time with kcat = 0.013 s-1 and KM = 9μM.Its catalytic activity is found to strictly depend on externally added bicarbonate with an apparent KD = 0.60 mM.In addition,this enzyme is inhibited by its 1,4-dihydroxy-2-naphthoyl-CoA product through high-affinity binding,which causes a 18 nm shift of the inhibitor absorption at 392 to 410 nm and engenders a new absorption peak at 345 nm.All these properties of the cyanobacterial enzyme are closely similar to those of the Escherichia coli orthologue from the menaquinone biosynthetic pathway.These results provide additional supporting evidence for the essential role of bicarbonate as a catalytic base in the enzymatic reaction and the eubacterial origin of the enzymes in the cyanobacterial biosynthesis of phylloquinone.     

Determination of carcinoembryonic antigen using a novel amperometric enzyme-electrode based on layer-by-layer assembly of gold nanoparticles and thionine

Electrochemical sensing of carcinoembryonic antigen(CEA)on a gold electrode modified by the se- quential incorporation of the mediator,thionine(Thi),and gold nanoparticles(nano-Au),through co- valent linkage and electrostatic interactions onto a self-assembled monolayer configuration is de- scribed in this paper.The enzyme,horseradish peroxidase(HRP),was employed to block the possible remaining active sites of the nano-Au monolayer,avoid the non-specific adsorption,instead of bovine serum albumin(BSA),and amplify the response of the antigen-antibody reaction.Electrochemical ex- periments indicated highly efficient electron transfer by the imbedded Thi mediator and adsorbed nano-Au.The HRP kept its activity after immobilization,and the studied electrode showed sensitive response to CEA and high stability during a long period of storage.The working range for the system was 2.5 to 80.0 ng/mL with a detection limit of 0.90 ng/mL.The model membrane system in this work is a potential biosensor for mimicking the other immunosensor and enzyme sensor.     

Possible Role of Carboxyl and Imidazole Groups in the Catalysis of Pummelo Limonoid Glucosyltransferase

 Limonoid bitterness is a serious problem in the citrus industry worldwide. Limonoid glucosyltransferase is an enzyme that catalyzes the conversion of bitter limonoid into non-bitter limonoid glucoside while retaining the health benefit of limonoids in the juice. The immobilization of this enzyme in a column can solve the juice bitterness problem. More information about the catalytic residues of the en-zyme is needed in this immobilization process. Glutamate/aspartate, histidine, lysine, tryptophan, serine, and cysteine residues were chemi-cally modified to investigate their roles in the catalytic function of limonoid glucosyltransferase. Inactivation of the enzyme following modi-fication of carboxyl and imidazole moieties was a consequence of a loss in substrate binding and catalysis in the glucosyltransfer reaction. The modification of a single histidine residue completely destroyed the ability of limonoid glucosyltransferase to transfer the D-glucopyranosyl unit. Tryptophan seemed to have some role in maintaining the active conformation of the catalytic site. Lysine also seemed to have some direct or indirect role in this catalysis but the modification of serine and cysteine did not have any effect on catalysis. There-fore, we conclude that the carboxyl and imidazole groups contain amino acids are responsible for the catalytic action of the enzyme.     

Wood cellulignin as an alternative matrix for enzyme immobilization

The objective of this work was to select an efficient methodology for preparing active samples of Candida rugosa lipase immobilized in wood cellulignin, to be applied in hydrolysis and ester reactions. For this purpose, lipase was immobilized in the matrix by physical adsorption (pure cellulignin) and covalent binding (activated cellulignin with glutaraldeyde or carbonyldiimidazole [CDI]) in the presence or absence of polyethylene glycol (PEG) (Molecular mass of 1500 Daltons) as stabilizing agent. The activating agent and the presence of PEG-1500 in the immobilization procedure showed a strong influence on enzyme retention in the support. The values for enzyme retention ranged from 20 to 68%, and the highest yield was obtained when the enzyme was immobilized in cellulignin activated with CDI in the presence of PEG-1500. This immobilized derivative presented high hydrolytic (193.27 μM/[mg·min]) and synthetic (522.92 μM/[g·min]) activities when compared with those obtained by other techniques. The superiority of this immobilized system was confirmed by additional analyses, such as infrared spectroscopy and elemental analysis, which demonstrated an appropriate enzyme fixation and the highest level of protein incorporation in the support. Further information on the immobilized derivative was obtained by assessing the recycle potential in both aqueous and nonaqueous media.     

Synthesis and Crystal Structure of 5-N-i-Propyl-2-(2′-nitrobenzenesulfonyl)-glutamine

IntroductionAcetohydroxy acid synthase(AHAS) is a perfecttarget for the design of environmentally benign herbi-cides because it is a key enzyme that is absent in ani-mals but is important for the biosynthesis of branched-chain amino acids in plants[1,2]. …     

Iron(III) Trifluoroacetate: Chemoselective and Recyclable Lewis Acid Catalyst for Diacetylation of Aldehydes,Thioacetalization and Transthioacetalization of Carbonyl Compounds and Aerobic Coupling of Thiols

Iron(III) trifluoroacetate [Fe(CF3CO2)3] was found to be a recyclable, highly efficient and chemoselective Lewis acid catalyst for protection of a variety of carbonyl compounds as thioacetals under nearly neutral conditions. With the use of this catalyst, 1,3-dithiolanes and 1,3-dithianes were obtained in high yields from various aldehydes. Under the same conditions ketones were similarly but more slowly thioketalized. This difference in reactivity between aldehydes and ketones was successfully utilized for the selective thioacetalization of aldehydes in the presence of ketones and also for the chemoselective conversion of β-diketone into the corresponding dithioacetal. Transthioacetalization of O,O-acetals and O,O-ketals into cyclic thioacetals was also achieved by using this catalyst. Additionally, iron(III) trifluoroacetate has been found to be efficient catalyst for the addition of acetic anhydride to both aromatic and aliphatic aldehydes to afford 1,1-diacetates (gem diacetates). Aerobic dimerization of thiols was achieved by this reagent mediated by sodium iodide and air atmosphere.     

A Novel, Simple and Efficient Synthesis of Ferrocenyl Enones and Alkynols

Propargylic alcohols are versatile precursors to many organic molecules including natural products and pharmaceutical compounds.[1] In our continuing interest in the development of practical methods for synthesis of ferrocene derivatives, we decided to investigate the efficiencies of ferrocenylacetylene addition to aromatic aldehydes under t-BuOKcatalyzed condition.[2] Interestingly, we found that the usually reliable coupling reaction between terminal alkyne of type 1 and aromatic aldehydes of type 2 does not furnish the expected propargylic alcohols, but that the isomeric aryl enones 3are found in high yield and very short reaction time (10～20 min). This provided a promising protocol for preparation of ferrocenyl chalcones in a practical, economical and mild sense compared with the traditional method. To the best of our knowledge, this unusual reaction was first observed for the addition of ferrocenylacetylene to aldehyde in a base-catalyzed manner (Eq. 1).     

Supramolecular nano drug delivery systems mediated via host-guest chemistry of cucurbit[n]uril (n = 6 and 7)

As a novel family of macrocyclic molecules,cucurbit[n]urils(CB[n]s) have emerged as promising building blocks of supramolecular nano drug delivery systems(SNDDS) in recent years.Direct encapsulation of amphiphilic guests by CB[6] and CB[7] can modulate their amphiphilicity,resulting in formation of supramolecular amphiphiles that self-assemble into supramolecular nanoparticles for drug delivery.Additionally,CB[n]'s host-guest chemistry on the surface of mesoporous nanoparticles makes CB[n] an ideal blocking agent to control drug release from delivery vehicles.These SNDDS possess intrinsic stimuli responsiveness towards external guest or host,which can further incorporate re s ponsiveness to a variety of other stimuli including pH,thermal,redox,photo and enzyme,to realize multiple stimuli-responsive drug release.Moreover,the recent breakthrough in direct functionalization of CB[n]s has provided a feasible method for preparing superior CB[6] and CB[7] derivatives that can be employed to build multifunctional SNDDS with unoccupied macrocycles located on surface,which could be decorated with various functional "tags" through host-guest chemistry.In this review,we summarized the recent progress of CB[6] and CB[7] based SNDDS through formation of supramolecular amphiphiles,supramolecular nanovalves as well as supramolecularly tailorable surface,which we hope to further promote the development of CB[n]s family as building blocks for advanced SNDDS.     

BrΦnsted acid-promoted ‘on-water’ C(sp^3)-H functionalization fot the synthesis of isoindolinone/[1,2,4]triazolo[1,5-a]pyrimidine derivatives targeting the SKP2-CKS1 interaction

The isoindolinone and biaryl scaffolds are prevalent in natural products and drug molecules,which have showed broad and interesting biological activities.The efficient construction of such hybridized molecules and biological evaluation are of great interest to medicinal chemistry community.In this communication,we report an efficient BrΦnsted acid-promoted C(sp^3)-H functionalization approach that enables the rapid construction of biologically important isoindolinone/[1,2,4]triazolo[1,5-a]pyrimidine hybrids from 5-methyl-7-(2,4,6-trimethoxyphenyl)-[1,2,4]triazolo[1,5-a]pyrimidine,2-formylbenzoic acid and various anilines.The title compounds were generated in high to excellent yields(up to 96%)regardless of the electronic nature and steric effects of the substituents.In this reaction,an isoindolinone scaffold,one C-C single bond,and two C-N bonds were formed simultaneously with high atom economy.In this work,we have envisioned that the methyl group linked to the electron-deficient Nheterocycles could be used as a new synthetic handle for late-state diversification and may have broad applications in the field of organic and medicinal chemistry.Besides,the title compounds have exhibited promising activity against the SKP2-CKS1 interaction.     

A New Route for the Synthesis of Thymidylate Synthase Inhibitor Raltitrexed

Raltitrexed (8), a new quinazoline-based inhibitor of thymidylate synthase (TS), has been registered widely for the first-line treatment of advanced colorectal cancer. [1,2] As reported in the literature, [3,4] it can be prepared from 2-thiophenecarboxylic acid via 7 steps in 3% overall yield, but n-BuLi and the low temperature at - 78 ℃ was needed for the introduction of 5-carboxyl group into thiophene ring through lithiation of 2-(N-Boc-N-methylamino) thiophene followed by the addition of CO2. Here we wish to report a new route for the synthesis of Raltitrexed which was obtained from 2,5-thiophenedicarboxylic acid via 6 steps in 18.2% overall yield (Scheme 1). The mild conditions utilized in the synthetic route avoid the use of n-BuLi, NaH and the experimental conditions of low temperature at - 78 ℃ and strictly free of water, and are suitable for the large-scale preparation.     

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.     

A convenient one-pot synthesis of new chromeno[3,4-c]chromene and chromeno[3,4-c]pyridine derivatives in the presence of high surface area of magnesium oxide

In this investigation a new strategy involves the one-pot,three-component reaction of malononitrile,salycilaldehyde and phenol derivatives in the presence of high surface area of MgO is extended to the formation of chromeno[3,4-c]chromene derivatives in good to excellent yields in a short reaction time.Also,the three component reactions of an aldehyde such as salycilaldehyde and ketones with malononitril for the formation of chromeno[3,4-c]pyridines are investigated.     

Electrospray Ionization Mass Spectral Characteristics of Polyamide Containing 1,4,7,10-Tetraazacyclododecane

The study of the fragmentations of the protonated molecular species[M H]~ and selected fragment ions al-lowed proposals for the main fragmentation pathways of the title compound.In order to better understand the frag-mentation pathways,the MS~3 and MS~4 spectra of the title compound were obtained.The main fragmentation path-ways occur by the cleavage of the C-CO bonds between N-methylpyrrole and carbonyl groups,CO-NH amidebonds,NH-C bonds between the NH groups and the N-methylpyrrole rings,and N-C bonds in1,4,7,10-tetraazacyclododecane.Electrospray ionization was proven to be a good method for the structural charac-terization and identification of this kind of compound.     

Quantitative High-Performance Liquid Chromatographic Determination of Neurotoxin β-ODAP and α- isomer in Grass Pea(Lathyrus sativus)

Grass pea (Lathyrus sativus) is leguminous crop cultivated in India,Bangladesh and Ethiopia, where it is a major protein source for people in the lowest income groups[1] .However, excessive ingestion of this pulse can lead to irreversible paralysis of the legs-a disease known as neurolathyrism or lathyrism.the causative agent was reported to be the non-protein amino acids,β-N-L-oxalyl-α,β-diaminopropionic acid (β-ODAP, sometimes, referred to as BOAA)[2]. The α-isomer of ODAP, α-N-oxaly 1-α,β-diaminopropionic acid, has been shown to be nontoxic to chicks and mice[3]. Lathyrus sativus shows good adaptation to the low rainfall conditions of northwestern China. It grows also in poor soil and is resistant to salt and flood. Our group was exploring to breed low or zero toxin varieties of Lathyrus sativus as grain crops for human consumption and as protein-rich feed for animals. So it is necessary to develop a method to detemine the toxin suitablefor differenth pu rpose.     

One-pot aqueous-phase synthesis of quinoxalines through oxidative cyclization of deoxybenzoins with 1,2-phenylenediamines catalyzed by a zwtterionic Cu(Ⅱ)/calix[4]arene complex

A green protocol for the synthesis of quinoxalines has been developed from catalytic oxidative cyclization of deoxybenzoins with 1,2-phenylenediamines in water.The optimal conditions are involved in the use of a water-soluble mononuclear copper(Ⅱ) complex of a zwitterionic calix[4]arene[Cu(Ⅱ)LCH_2O)]I_2(1,H_4L=[5,ll,17,23-tetrakis(trimethylammonium)-25,26,27,28-tetrahydroxycalix[4]arene]) as a catalyst in alkali solution after refluxing for 15 h in O_2.The target quinoxaline and its derivatives were obtained in good yields(up to 88%).The procedure described in this paper is simple,practical and environmentally benign.     

Mo／MCM－49催化剂上甲烷的脱氢芳构化反应

In recent years, the catalytic dehydroaromatization of methane to benzene has been a challenging and intriguing research subject in heterogeneous catalysis and natural gas chemical industry. Since the first report on non-oxidative conversion of methane to benzene on Mo/HZSM-5[1], many attractive studies regarding the development of new catalyst, the catalyst characterization[2～4], the reaction mechanism and the bifunctional role of Mo carbide and HZSM-5 have been reported. However, Mo/HZSM-5 catalyst was thought to have the best catalytic performance for CH4 dehydroaromatization to benzene. Recently, another kind of zeolite, MCM-22, was found to be a promising catalyst support for formation of benzene from methane[5]. Xu et al[6] studied the catalytic performance of MoO3/MCM-49 for this reaction without accounting for coke formation. In this paper, we present MCM-49 as a support, and the catalyst Mo/MCM-49 shows high ratio of benzene to aromatics in selective dehydroaromatization of methane.     

Two-parameter stochastic resonance in a model of electrochemical oxidation of formic acid on Pt

Stochastic resonance (SR) is shown in a two-parameter system, a model of electrochemical oxidation of formic acid on Pt. The driving current and the saturation coverage for carbon monoxide are two control parameters in this model. Modulation of an excitable focal stable state close to a Hopf bifurcation by a weak periodic signal in one parameter and noise in the other parameter is found to give rise to SR. The results indicate that the noise can enlarge a weak periodic signal and lead the system to be ordered. The scenario and novel aspects of SR in this system are discussed.     

Photochemical multicomponent transformation of acceptor-only diazoalkanes by merging their cycloaddition and carbene reactivities

Herein, we report an efficient photochemical method for the synthesis of poly-substituted pyrazoles through a multicomponent reaction of acceptor-only diazoalkanes, alkynes, and solvents(cyclic ethers or nitriles). The key to this success was driven by the photolysis of acceptor-only diazoalkanes to form free carbene species and the fast in situ [3 + 2]-cycloaddition formation of nucleophilic N–H pyrazole derivatives. This work also serves as an entry to allow future reaction design on the combi...