[Aerobic Denitrification and Microbial Community Shift in SBR Bioaugmented with Strains YH01 and YH02].

The enhanced aerobic denitrification capability of the mixed strains YH01+YH02 in utilizing potassium nitrate, and the dynamic changes in the microbial community component during the period of operation, were evaluated. The microbial community in different stages of the SBR was analyzed by using high-throughput sequencing technology after inoculation with YH01+YH02. The results showed that the NO3--N, TN, and COD removal efficiencies increased by 12.1%, 9.2%, and 9.4%, respectively. The relative abundances of the microbes in the microbial community increased at the genus level, and the diversity in the microbial community decreased after enhancement. Principal component analysis and UPGMA analysis revealed that the period of SBR operation was roughly divined into four phases. The relative abundances of Delftia and Acidovorax increased during the period of operation, and YH01+YH02 exhibited excellent compatibility with the SBR ecosystem and played an important part in aerobic denitrification.

[Analysis of the Microbial Community Structure in Continuous Flow Reactor Enhanced by Heterotrophic Nitrification and Aerobic Denitrification Bacterium Burkholderia sp. YX02].

To reveal the dynamic succession of microbial community structure along with time in bio-denitrification reactor, a continuous flow reactor containing immobilized heterotrophic nitrification-aerobic denitrification bacterium Burkholderia sp. YX02 was taken as a model. The microbial community structure in the bioreactor was analyzed by PCR-DCGE, and its correlations with environmental factors such as pH, NH4+ -N, NO2- -N, NO3- -N and COD were simultaneously investigated. The results showed that the microbial community was relatively rich during the early stage of 18 days. The similarity of community structure in different stages was not orderly declining with the operation. In addition, the structural similarity in adjacent stages firstly increased, then decreased, and eventually tended to be stable. Shannon-Wiener index firstly descended significantly, and then ascended with new microbial community emerging at the later stage. UPGMA clustering analysis roughly divided the process into three periods with certain relationship. Principal component analysis showed that during the operation of the bioreactor predominant bacterial community formed steadily and new microbial community dominated by Burkholderia sp. YX02 emerged at the later stage of the operation. Canonical correspondence analysis certificated that the structure of microbial community was most obviously affected by NO2- -N, followed by NO3- -N, NH4+ -N and COD, and pH had the least effect.

Peptide Bond Formation in Water Mediated by Carbon Disulfide.

Demonstrating plausible nonenzymatic polymerization mechanisms for prebiotic monomers represents a fundamental goal in prebiotic chemistry. While a great deal is now known about the potentially prebiotic synthesis of amino acids, our understanding of abiogenic polymerization processes to form polypeptides is less well developed. Here, we show that carbon disulfide (CS2), a component of volcanic emission and sulfide mineral weathering, and a widely used synthetic reagent and solvent, promotes peptide bond formation in modest yields (up to ∼20%) from α-amino acids under mild aqueous conditions. Exposure of a variety of α-amino acids to CS2 initially yields aminoacyl dithiocarbamates, which in turn generate reactive 2-thiono-5-oxazolidone intermediates, the thio analogues of N-carboxyanhydrides. Along with peptides, thiourea and thiohydantoin species are produced. Amino acid stereochemistry was preserved in the formation of peptides. Our findings reveal that CS2 could contribute to peptide bond formation, and possibly other condensation reactions, in abiogenic settings.

Chemically programmed antibodies targeting multiple alpha(v) integrins and their effects on tumor-related functions in vitro.

Integrins αvß3 and αvß6 are highly expressed on tumor cells and/or by the tumor vasculature of many human cancers, and represent promising targets for anticancer therapy. Novel chemically programmed antibodies (cpAbs) targeting these integrins were prepared using the catalytic aldolase Antibody (Ab) programming strategy. The effects of the cpAbs on cellular functions related to tumor progression were examined in vitro using tumor cell lines and their cognate integrin ligands, fibronectin and osteopontin. The inhibitory functions of the conjugates and their specificity were examined based on interference with cell-cell and cell-ligand interactions related to tumor progression. Cell binding analyses of the anti-integrin cpAbs revealed high affinity for tumor cells that overexpressed αvß3 and αvß6 integrins, and weak interactions with αvß1 and αvß8 integrins, in vitro. Functional analyses demonstrated that the cpAbs strongly inhibited cell-cell interactions through osteopontin binding, and they had little or no immediate effects on cell viability and proliferation. On the basis of these characteristics, the cpAbs are likely to have a broad range of activities in vivo, as they can target and antagonize one or multiple αv integrins expressed on tumors and tumor vasculatures. Presumably, these conjugates may inhibit the establishment of metastastatic tumors in distant organs through interfering with cell adhesion more effectively than antibodies or compounds targeting one integrin only. These anti-integrin cpAbs may also provide useful reagents to study combined effect of multiple αv integrins on cellular functions in vitro, on pathologies, including tumor angiogenesis, fibrosis, and epithelial cancers, in vivo.

Multiple catalytic aldolase antibodies suitable for chemical programming.

Chemical programming of nine murine antibodies with catalytic aldolase activity was examined using compounds, equipped with diketone or pro-vinyl ketone linkers that inhibit integrin adhesion receptor functions. The results showed that most Abs were programmed using the diketone compounds in a manner similar to previously reported catalytic antibody 38C2. On the other hand, only those antibodies, which catalyzed the retro aldol reaction of the pro-vinyl ketone linkers efficiently, were programmed. Conjugated to integrin targeting compounds, at least three new antibodies, including 84G3, 85H6, and 90G8, exhibited high specific binding to human tumor cells expressing integrin alpha(v)beta(3.).

Alteration of the bis-tetrahydrofuran core stereochemistries in asimicin can affect the cytotoxicity.

A systematic analysis using 10 synthetic asimicin stereoisomers revealed that the stereochemistry of the bis-tetrahydrofuran core, including the tetrahydrofuran rings and the adjacent hydroxy functions, had significant effect on its cytotoxicity. Our findings set to rest the highly controversial perception that the stereochemistry of the tetrahydrofuran core has little effect on the activity, which is not true for its cytotoxic effect, and also reinforces the previous conclusion that asimicin is a highly potent anticancer compound.

Functional and mechanistic analyses of biomimetic aminoacyl transfer reactions in de novo designed coiled coil peptides via rational active site engineering.

Ribosomes and nonribosomal peptide synthetases (NRPSs) carry out instructed peptide synthesis through a series of directed intermodular aminoacyl transfer reactions. We recently reported the design of coiled-coil assemblies that could functionally mimic the elementary aminoacyl loading and intermodular aminoacyl transfer steps of NRPSs. These peptides were designed initially to accelerate aminoacyl transfer mainly through catalysis by approximation by closely juxtaposing four active site moieties, two each from adjacent noncovalently associated helical modules. In our designs peptide self-assembly positions a cysteine residue that is used to covalently capture substrates from solution via transthiolesterification (substrate loading step to generate the aminoacyl donor site) adjacent to an aminoacyl acceptor site provided by a covalently tethered amino acid or modeled by the epsilon-amine of an active site lysine. However, through systematic functional analyses of 48 rationally designed peptide sequences, we have now determined that the substrate loading and intermodular aminoacyl transfer steps can be significantly influenced (up to approximately 103-fold) by engineering changes in the active site microenvironment through amino acid substitutions and variations in the inter-residue distances and geometry. Mechanistic studies based on 15N NMR and kinetic analysis further indicate that certain active site constellations furnish an unexpectedly large pK(a) depression (1.5 pH units) of the aminoacyl-acceptor moiety, helping to explain the observed high rates of aminoacyl transfer in those constructs. Taken together, our studies demonstrate the feasibility of engineering efficient de novo peptide sequences possessing active sites and functions reminiscent of those in natural enzymes.

Diastereoselectivity-switchable and highly enantioselective 1,3-dipolar cycloaddition of nitrones to alkylidene malonates.

Trisoxazoline 1/Co(ClO(4))(2).6H(2)O catalyzed the 1,3-cycloaddition between nitrones 3 with alkylidene malonates 2 at 0 degrees C to give the isoxazolidines with both high enantioselectivity and high exo selectivity. However, when the temperature was lowered from 0 to -40 degrees C, the same cycloaddition afforded endo isomers as the major products with good to high enantioselectivity. A mechanism is provided.

Controllable enantioselective Friedel-Crafts reaction between indoles and alkylidene malonates catalyzed by pseudo-C3-symmetric trisoxazoline copperII complexes.

Pseudo-C(3)-symmetric trisoxazoline copper(II) complexes prove to be excellent catalysts in the Friedel-Crafts alkylation of indoles with alkylidene malonates. The absolute stereochemistry of this reaction is shown to be dependent on the solvent. Reactions in isobutyl alcohol afford the Friedel-Crafts alkylation adducts in excellent yields and with up to +98% ee. In 1,1,2,2-tetrachloroethane (TTCE), however, the opposite enantiomers of the products are obtained in good yields with up to -89% ee. Water tolerance of chiral catalyst trisoxazoline 2a/Cu(OTf)(2) is examined, and it is found that the addition of up to 200 equiv of water relative to catalyst in isobutyl alcohol has almost no effect on enantioselectivity but slows down the reaction. The reaction scope is studied as well. The roles of alcohol as the solvent to accelerate the reaction are discussed. The stereochemical models of asymmetric induction for reactions both in isobutyl alcohol and in TTCE are also developed.

Chiral tris(oxazoline)/Cu(II) catalyzed coupling of terminal alkynes and nitrones.

Novel chiral iPr-tris(oxazoline)/Cu(ClO4)2 x 6H2O catalyzed coupling of terminal acetylenes and nitrones to afford cis-disubstituted beta-lactams is described; the choice of base proves essential to both the diastereoselectivity and the enantioselectivity.

Unexpected catalyst for Wittig-type and dehalogenation reactions.

A novel catalyst 2 for Wittig-type and dehalogenation reactions was developed. In the presence of triphenyl phosphite, a wide variety of aldehydes could react with alpha-bromoacetates to afford alpha,beta-unsaturated esters or ketones in high yields with excellent E-stereoselectivity when 1-2 mol % of compound 2 was used. Compound 2 was also an effective catalyst for reductive dehalogenation of alpha-bromocarbonyl compounds. The mechanisms for the above reactions were also proposed.

Wittig-type olefination catalyzed by PEG-telluride.

Soluble poly(ethylene glycol) (PEG)-supported telluride 2 was designed and synthesized for catalytic Wittig-type reactions. It was found that the catalytic loading could be reduced from 20 to 2 mol % by the introduction of PEG (even to 0.5 mol % when some telluride salts were used as the catalyst). Under the catalytic reaction conditions, a wide variety of aldehydes with different structures could react with bromoacetate to afford beta-substituted or alpha,beta-disubstituted unsaturated esters in high yields with excellent E-stereoselectivity. The modified process, by using sodium bisulfite instead of triphenyl phosphite, represented a very simple product isolation procedure. The roles of PEG for promoting the ylide formation and stabilizing the catalytic species were disclosed. The mechanism was also studied.

A novel chiral sulfonium yilde: highly enantioselective synthesis of vinylcyclopropanes.

A newly designed chiral sulfonium allylide, generated in situ from the corresponding sulfonium salt in the presence of KOBu(t), reacted with alpha,beta-unsaturated esters, ketones, amides, and nitriles to afford trans-2-silylvinyl-trans-3-substituted cyclopropyl esters, ketones, amides, and nitriles with outstanding diastereoselectivity and excellent enantioselectivity in good to high yields. A mechanistic rationale is proposed.