In vitro evolution of a ribozyme that contains 5-bromouridine.

The Tetrahymena group I ribozyme was modified by replacing all 99 component uridine residues with 5-bromouridine. This resulted in a 13-fold reduction in catalytic efficiency in the RNA-catalyzed phosphoester-transfer reaction compared to the behavior of the unmodified ribozyme. A population of 10(13) variant ribozymes was constructed, each containing 5-bromouridine in place of uridine. Five successive 'generations' of in vitro evolution were carried out, selecting for improved phosphoester transferase activity. The evolved molecules exhibited a 27-fold increase in catalytic efficiency compared to the wild-type bromouridine-containing ribozyme, even exceeding that of the wild-type ribozyme in the non-brominated form. Three specific mutations were found to be responsible for this altered behavior. These mutations enhanced activity in the context of 5-bromouridine, but were detrimental in the context of unmodified uridine. The evolved RNAs not only tolerated but came to exploit the presence of the nucleotide analogue in carrying out their catalytic function.     

Conversion of a ribozyme to a deoxyribozyme through in vitro evolution

An RNA ligase ribozyme was converted to a corresponding deoxyribozyme through in vitro evolution. The ribozyme was prepared as a DNA molecule of the same sequence, and had no detectable activity. A population of randomized variants of this DNA was constructed and evolved to perform RNA ligation at a rate similar to that of the starting ribozyme. When the deoxyribozyme was prepared as an RNA molecule of the same sequence, it had no detectable activity. Thus, the evolutionary transition from an RNA to a DNA enzyme represents a switch, rather than a broadening, of the chemical basis for catalytic function. This transfer of both information and function is relevant to the transition between two different genetic systems based on nucleic acid-like molecules, as postulated to have occurred during the early history of life on Earth.     

Consecutive approach to alkenes that combines radical addition of phosphorus hydrides with Horner-Wadsworth-Emmons-type reactions

[reaction: see text] Addition of diethyl thiophosphite to terminal alkenes, in the presence of a radical initiator, followed by deprotonation of the phosphonothioate and reaction with a ketone, offers a concise one-pot approach to substituted alkenes. This novel method, which can incorporate alkylation or acylation steps, can be applied to the stereoselective formation of sterically hindered tri- and tetrasubstituted alkenes.     

Evidence that protons can be the active catalysts in Lewis acid mediated hetero-Michael addition reactions

The mechanism of Lewis acid catalysed hetero‐Michael addition reactions of weakly basic nucleophiles to α,β‐unsaturated ketones was investigated. Protons, rather than metal ions, were identified as the active catalysts. Other mechanisms have been ruled out by analyses of side products and of stoichiometric enone–catalyst mixtures and by the use of radical inhibitors. No evidence for the involvement of π‐olefin–metal complexes or for carbonyl–metal‐ion interactions was obtained. The reactions did not proceed in the presence of the non‐coordinating base 2,6‐di‐tert‐butylpyridine. An excellent correlation of catalytic activities with cation hydrolysis constants was obtained. Different reactivities of mono‐ and dicarbonyl substrates have been rationalised. A ¹H NMR probe for the assessment of proton generation was established and Lewis acids have been classified according to their propensity to hydrolyse in organic solvents. Brønsted acid‐catalysed conjugate addition reactions of nitrogen, oxygen, sulfur and carbon nucleophiles are developed and implications for asymmetric Lewis acid catalysis are discussed.     

Identification of a gene cluster that directs putrebactin biosynthesis in Shewanella species: PubC catalyzes cyclodimerization of N-hydroxy-N-succinylputrescine

Putrebactin is a dihydroxamate iron chelator produced by the metabolically versatile marine bacterium Shewanella putrefaciens. It is a macrocyclic dimer of N-hydroxy-N-succinyl-putrescine (HSP) and is structurally related to desferrioxamine E, which is a macrocyclic trimer of N-hydroxy-N-succinyl-cadaverine (HSC). We recently showed that DesD, a member of the NIS synthetase superfamily, catalyzes the key step in desferrioxamine E biosynthesis: ATP-dependent trimerisation and macrocylization of HSC. Here we report identification of a conserved gene cluster in the sequenced genomes of several Shewanella species, including Shewanella putrefaciens, which is hypothesized to direct putrebactin biosynthesis from putrescine, succinyl-CoA and molecular oxygen. The pubC gene within this gene cluster encodes a protein with 65% similarity to DesD. We overexpressed pubC from Shewanella species MR-4 and MR-7 in E. coli. The resulting His6-PubC fusion proteins were purified by Ni-NTA affinity and gel filtration chromatography. The recombinant proteins were shown to catalyze ATP-dependent cyclodimerization of HSP to form putrebactin. The uncyclized dimer of HSP pre-putrebactin was shown to be an intermediate in the conversion of two molecules of HSP to putrebactin. The data indicate that pre-putrebactin is converted to putrebactin via PubC-catalyzed activation of the carboxyl group by adenylation, followed by PubC-catalyzed nucleophilic attack of the amino group on the carbonyl carbon of the acyl adenylate. This mechanism for macrocycle formation is very different from the mechanism involved in the biosynthesis of many other macrocyclic natural products, where already-activated acyl thioesters are converted by thioesterase domains of polyketide synthases and nonribosomal peptide synthetases to macrocycles via covalent enzyme bound intermediates. The results of this study demonstrate that two closely related enzymes, PubC and DesD, catalyze specific cyclodimerization and cyclotrimerization reactions, respectively, of structurally similar substrates, raising intriguing questions regarding the molecular mechanism of specificity.     

Oxidative addition reactions in cluster complexes of osmium: Intracluster reactions of benzyne and a novel 'phenyl' complex

The structures of HOs₃(CO)₇(PPh₂)(PPh₃)(C₆H₄), HOs₃(CO)₈(PPh₃)(PPh₂C₆H₄) and HOs₃(CO)₇(PPh₂)(PPh₂C₆H₄C₆H₃) are described, the latter illustrating an intracluster reaction of the coordinated benzyne ligand.     

Alkynoates as a source of reactive alkylinides for aldehyde addition reactions

[see reaction]. The reaction of activated alkynes with carbonyl compounds in the presence of a catalytic amount of a nucleophile leads to enol-protected functionalized propargyl alcohols and 1,3-dioxolane compounds by way of a mild carbon-carbon bond formation reaction.     

Absolute rate constants of alkene addition reactions of a fluorinated radical in water

Absolute rate constants of *R(f)SO(3)(-) radical addition to a series of water-soluble alkenes containing ionic, carboxylate substituents were measured by laser flash photolysis experiments in water. The observed rate constants were all considerably larger than those of structurally similar analogues in a nonpolar organic solvent, with rate factors of 3-9-fold being observed. It is concluded that such rate enhancements derive at least in part from stabilization of the polar transition state for addition of the electrophilic fluorinated radical to alkenes by the polar solvent water.     

醛不对称加成反应的进展

综述了近年来醛不对称加成反应的进展,包括加氘还原、与有机金属化合物加成、与有机硼化合物加成、与氰化物加成、与磷酸酯加成和aldol加成等反应。     

The kinetics of successive addition of oxirane to alkanethiols in the presence of basic catalysts

Rate constants and activation parameters of direct reaction of oxirane with certain alkyl-2-hydroxyethyl sulfides and/or successive oxyethylene adducts in the presence of basic catalyst were studied at temperatures 323–353 K. The reactions were found to be of the first order with respect to oxirane, sulfide, and catalyst concentrations. The kinetics are consistent with a termolecular mechanism.     

Experimental evidence that halogen bonding catalyzes the heterogeneous chlorination of alkenes in submicron liquid droplets

A key challenge in predicting the multiphase chemistry of aerosols and droplets is connecting reaction probabilities, observed in an experiment, with the kinetics of individual elementary steps that control the chemistry that occurs across a gas/liquid interface. Here we report evidence that oxygenated molecules accelerate the heterogeneous reaction rate of chlorine gas with an alkene (squalene, Sqe) in submicron droplets. The effective reaction probability for Sqe is sensitive to both the aerosol composition and gas phase environment. In binary aerosol mixtures with 2-decyl-1-tetradecanol, linoleic acid and oleic acid, Sqe reacts 12–23× more rapidly than in a pure aerosol. In contrast, the reactivity of Sqe is diminished by 3× when mixed with an alkane. Additionally, small oxygenated molecules in the gas phase (water, ethanol, acetone, and acetic acid) accelerate (up to 10×) the heterogeneous chlorination rate of Sqe. The overall reaction mechanism is not altered by the presence of these aerosol and gas phase additives, suggesting instead that they act as catalysts. Since the largest rate acceleration occurs in the presence of oxygenated molecules, we conclude that halogen bonding enhances reactivity by slowing the desorption kinetics of Cl₂ at the interface, in a way that is analogous to decreasing temperature. These results highlight the importance of relatively weak interactions in controlling the speed of multiphase reactions important for atmospheric and indoor environments.

The heterogeneous chlorination rate of an alkene is unexpectedly accelerated in the presence of spectator molecules containing oxygenated functional groups, which suggests weak halogen bonds can catalyze reactions at liquid surfaces.     

Continuous hydrogenation reactions in a tube reactor packed with Pd/C

Continuous flow of the substrate solution and hydrogen gas through a tube reactor packed with Pd/C catalyst brings about a highly reactive and efficient hydrogenation system, which converts 4-cyanobenzaldehyde to the benzyl alcohol derivatives at 25 degrees C, and at 90 degrees C, the cyano group becomes reduced to give the corresponding amine and toluene derivatives within 2 min.     

Multifrequency electron paramagnetic resonance characterization of PpoA, a CYP450 fusion protein that catalyzes fatty acid dioxygenation

PpoA is a fungal dioxygenase that produces hydroxylated fatty acids involved in the regulation of the life cycle and secondary metabolism of Aspergillus nidulans . It was recently proposed that this novel enzyme employs two different heme domains to catalyze two separate reactions: within a heme peroxidase domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic acid [(8R)-HPODE]; in the second reaction step (8R)-HPODE is isomerized within a P450 heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. In the present study, pulsed EPR methods were applied to find spectroscopic evidence for the reaction mechanism, thought to involve paramagnetic intermediates. We observe EPR resonances of two distinct heme centers with g-values typical for Fe(III) S = (5)/(2) high-spin (HS) and Fe(III) S = (1)/(2) low-spin (LS) hemes. (14)N ENDOR spectroscopy on the S = (5)/(2) signal reveals resonances consistent with an axial histidine ligation. Reaction of PpoA with the substrate leads to the formation of an amino acid radical on the early millisecond time scale concomitant to a substantial reduction of the S = (5)/(2) heme signal. High-frequency EPR (95- and 180-GHz) unambiguously identifies the new radical as a tyrosyl, based on g-values and hyperfine couplings from spectral simulations. The radical displays enhanced T(1)-spin-lattice relaxation due to the proximity of the heme centers. Further, EPR distance measurements revealed that the radical is distributed among the monomeric subunits of the tetrameric enzyme at a distance of approximately 5 nm. The identification of three active paramagnetic centers involved in the reaction of PpoA supports the previously proposed reaction mechanism based on radical chemistry.     

碳碳不饱和键氟化加成反应的研究进展

选择性地向有机物中引入氟原子越来越受到重视，近年来通过碳碳不饱和键的 氟化加成来引入氟原子发展迅速。综述了碳碳不饱和键氟化加成反应的研究进展。     

Continuous addition of reagent technique: a new approach to differential reaction-rate methods

The potential of the continuous addition of reagent technique in differential reaction-rate methods was evaluated. The approach was tested on the simultaneous determination of copper and iron through their fast complex-formation reaction with pyridoxal thiosemicarbazone. Mixtures of these metal ions at the sub-μg ml^?1 level with copper/iron ratios from 5:1 to 1:2 can be analysed with a precision (relative standard deviation) of 0.25 and 0.45%, respectively. The method was successfully applied to the determination of both metals in serum samples with a purpose-designed reaction mini-chamber.     

Thiazolium-catalyzed additions of acylsilanes: a general strategy for acyl anion addition reactions

A strategy utilizing N-heterocyclic carbenes (NHCs) derived from thiazolium salts has been developed for the generation of carbonyl anions from acylsilanes. Synthetically useful 1,4-diketones and N-phosphinoyl-alpha-aminoketones have been prepared in good to excellent yields via NHC-catalyzed additions of acylsilanes to the corresponding alpha,beta-unsaturated systems and N-phosphinoylimines. These organocatalytic reactions are air- and water-tolerant methods to execute robust carbonyl anion addition reactions. Additionally, polysubstituted aromatic furans and pyrroles have been efficiently synthesized in a one-pot process using this carbonyl anion methodology. The addition of alcohols to the reaction renders the process catalytic in thiazolium salt. In an effort to synthesize a potential intermediate along the proposed reaction pathway, silylated thiazolium carbinols have been identified to provide good yields of carbonyl anion addition products when subjected to the standard reaction conditions in the presence of suitable electrophiles.     

Detection of a proton-transfer process by kinetic solvent isotope effects in NH(4)(+)-mediated reactions catalyzed by a hammerhead ribozyme

Hammerhead ribozymes have been considered to be metalloenzymes. However, this proposal was recently questioned by the finding that the reaction proceeds in the presence of high concentrations of monovalent ions such as NH(4)(+) ions and in the absence of any divalent metal ions. Our present analysis based on solvent isotope effects indicates that (1) a proton transfer(s) occurs only in the NH(4)(+)-mediated reaction but not in metal-ion-mediated reactions such as Mg(2+)- and Li(+)-mediated reactions, (2) the catalyst that stabilizes the 5' leaving group in the NH(4)(+)-mediated reaction is different from that in the metal-ion-mediated HH ribozyme reactions, (3) an NH(4)(+) ion seems to act as a general acid catalyst, and (4) a nucleobase alone should not be the catalyst.     

Forty years of in vitro evolution

It has been 40 years since Spiegelman and co-workers demonstrated how RNA molecules can be evolved in the test tube. This result established Darwinian evolution as a chemical process and paved the way for the many directed evolution experiments that followed. Chemists can benefit from reflecting on Spiegelman's studies and the subsequent advances, which have taken the field to the brink of the generation of life itself in the laboratory. This Review summarizes the concepts and methods for the directed evolution of RNA molecules in vitro.