Β-elimination Reaction Research Articles

Dyeing properties of a reactive disperse dye carrying acetoxyethylsulphone group

The dyeing and color fastness characteristics of a reactive disperse dye on Nylon fabric, PET fabric and N/P mixture fabric have been examined. The most appropriate pH for the dyeing of Nylon fabric was 7 and suitable dyeing temperature was 100 °C. The build-up on Nylon fabric was good and various color fastnesses were good to excellent due to the formation of the covalent bond. The reactive disperse dye exhibited the similar dyeing properties on PET fabric to a conventional disperse dye. The rate of dyeing of the reactive disperse dye on Nylon fabric was faster than that on PET fabric when both fabrics were dyed simultaneously in a dye bath, but the difference in the color strength between the two fabrics decreased as the pH decreased. The reactive disperse dye was adequate to the one-step, one-bath dyeing of N/P mixture fabric with good color fastnesses. It was also found by HPLC analysis that the rate of β-elimination reaction of the reactive disperse dye at 100 °C increased 8 times as the pH increased by 1.

Crystallization and preliminary X-ray analysis of an exotype alginate lyase Atu3025 fromAgrobacterium tumefaciensstrain C58, a member of polysaccharide lyase family 15

Almost all alginate lyases depolymerize alginate in an endolytical fashion via a beta-elimination reaction. The alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, consisting of 776 amino-acid residues, is a novel exotype alginate lyase classified into polysaccharide lyase family 15. The enzyme was crystallized at 293 K by sitting-drop vapour diffusion with polyethylene glycol 4000 as a precipitant. Preliminary X-ray analysis showed that the Atu3025 crystal belonged to space group P2(1) and diffracted to 2.8 angstroms resolution, with unit-cell parameters a = 107.7, b = 108.3, c = 149.5 angstroms, beta = 91.5 degrees.

A biosystem for alginate metabolism in Agrobacterium tumefaciens strain C58: Molecular identification of Atu3025 as an exotype family PL-15 alginate lyase

The Gram-negative bacterium Sphingomonas sp. strain A1 (strain A1) has a peculiar biosystem to directly import and depolymerize a macromolecule, alginate, which is encoded by a cluster of genes on the genome. We identified five clustered ORFs homologous to some genes of the strain A1 cluster in the genome of Agrobacterium tumefaciens strain C58 (strain C58). These ORFs are Atu3021, Atu3022, Atu3023, and Atu3024, encoding a putative sugar ABC transporter system and Atu3025, which encodes a putative alginate lyase. We analyzed the involvement of this gene cluster in alginate metabolism. Strain C58 cells grew significantly on low-molecular-weight (LMW) alginate (average molecular weight, 1000), and we detected specific alginate-induced expression of Atu3024 and Atu3025. This strain does not grow on alginate (average molecular weight, 25 600), suggesting that the strain C58 gene cluster is involved in importing and degrading LMW alginate. One protein, Atu3025, purified from strain C58, was identified as an alginate lyase, and the enzyme overexpressed in Escherichia coli was further characterized. Atu3025 released monosaccharides specifically from alginate most efficiently at pH 7.3 and 30 °C through a β-elimination reaction, indicating that Atu3025 is an exotype alginate lyase potentially involved in the assimilation of LMW alginate in strain C58.

An efficient synthesis of ( E)-α,β-unsaturated ketones and esters with total stereoselectivity by using chromium dichloride

( E)-α,β-Unsaturated ketones 1 or esters 2 can be obtained with complete stereoselectivity by reaction of different 2-chloro-3-hydroxy ketones 3 or esters 4 and CrCl 2. A comparative study of the results of synthesis of ketones 1 with CrCl 2 or samarium is performed. A mechanism to explain both β-elimination reactions has been proposed.

Unsaturated Fatty Alcohol Derivatives as a Source of Substituted Allylzirconocene

From A to Z: Allylzirconocenes have been easily prepared in a one-pot procedure through a tandem allylic CH activation–β-elimination reaction from unsaturated fatty alcohol and ether starting materials (see scheme). The reaction proceeds rapidly under mild condition and is insensitive to the length of the carbon tether between the double bond and the alcohol moiety.

Manganese-Promoted β-Elimination Reactions: Totally Stereoselective Synthesis of (E)-α,β-Unsaturated Esters

Stereoselective β-elimination of 2-bromo-3-hydroxy­esters is achieved by using non-preactivated manganese and tri­methylsilyl chloride, to yield (E)-α,β-unsaturated esters with total diastereoselectivity.

Application of a Thermal β-Elimination Reaction toN-Alkoxy-3,3-dinitro­isoxazolidines: Synthesis of 3-Nitroisoxazolines

A facile general method has been developed for the synthesis of 5-substituted 3-nitroisoxazolines by the application of a thermal β-elimination reaction to N-alkoxy-3,3-dinitroisoxazolidines in chlorobenzene.

First report of a lyase for cepacian, the polysaccharide produced by Burkholderia cepacia complex bacteria

Bacteria belonging to the Burkholderia cepacia complex (Bcc) are interesting for their involvement in pulmonary infections in patients affected by cystic fibrosis (CF) or chronic granulomatous disease. Many Bcc strains isolated from CF patients produce high amounts of exopolysaccharides (EPS). Although different strains sometimes biosynthesise different EPS, the majority of Bcc bacteria produce only one type of polysaccharide, which is called cepacian. The polymer has a unique heptasaccharidic repeating unit, containing three side chains, and up to three O-acetyl substituents.. We here report for the first time the isolation and characterisation of a lyase active towards cepacian produced by a Bacillus sp., which was isolated in our laboratory. The enzyme molecular mass, evaluated by size-exclusion chromatography, is 32,700 ± 1500 Da. The enzyme catalyses a β-elimination reaction of the disaccharide side chain β- d-Gal p-(1 → 2)-α- d-Rha p-(1→ from the C-4 of the glucuronic acid residue present in the polymer backbone. Although active on both native and de-acetylated cepacian, the enzyme showed higher activity on the latter polymer.

Evidence of a Borderline Region between E1cb and E2 Elimination Reaction Mechanisms: A Combined Experimental and Theoretical Study of Systems Activated by the Pyridine Ring

We report a combined experimental and theoretical study to characterize the mechanism of base-induced beta-elimination reactions in systems activated by the pyridyl ring, with halogen leaving groups. The systems investigated represent borderline cases, where it is uncertain whether the reaction proceeds via a carbanion intermediate (E1cb, A(xh)D(H) + D(N)) or via the concerted loss of a proton and the halide (E2, A(N)D(E)D(N)) upon base attack. Experimentally, the Taft correlation for H/D exchange, in OD(-)/D(2)O with noneliminating substrates (1-methyl-2-(2-Xethyl)pyridinium iodide), is used to predict the expected values of the rate constants for the elimination reactions with N-methylated substrates and F, Cl, Br as the leaving group. The comparison indicates an E1cb irreversible mechanism with F, but the deviation observed with Cl and Br does not allow a conclusive assignment. The theoretical calculations show that for the N-methylated substrate with a fluoride leaving group the elimination proceeds via formation of a moderately stable carbanion. No stable anionic intermediate is instead found when the leaving group is Cl or Br, as well as for any of the nonmethylated species, indicating a concerted elimination. The methylated substrate with Cl shows however only a moderate increase in reactivity compared to the fluorinated substrate, despite the change in mechanism. Very interestingly, our analysis of the computed two-dimensional potential energy surface for the reaction with a F leaving group indeed evidences the lack of a net distinction between the E1cb and E2 reaction paths, which appear to merge smoothly into each other in these borderline cases.

Structural insight into the DNA polymerase β deoxyribose phosphate lyase mechanism

A large number of biochemical and genetic studies have demonstrated the involvement of DNA polymerase β (Pol β) in mammalian base excision repair (BER). Pol β participates in BER sub-pathways by contributing gap filling DNA synthesis and lyase removal of the 5′-deoxyribose phosphate (dRP) group from the cleaved abasic site. To better understand the mechanism of the dRP lyase reaction at an atomic level, we determined a crystal structure of Pol β complexed with 5′-phosphorylated abasic sugar analogs in nicked DNA. This DNA ligand represents a potential BER intermediate. The crystal structure reveals that the dRP group is bound in a non-catalytic binding site. The catalytic nucleophile in the dRP lyase reaction, Lys72, and all other potential secondary nucleophiles, are too far away to participate in nucleophilic attack on the C1′ of the sugar. An approximate model of the dRP group in the expected catalytic binding site suggests that a rotation of 120° about the dRP 3′-phosphate is required to position the ɛ-amino Lys72 close to the dRP C1′. This model also suggests that several other side chains are in position to facilitate the β-elimination reaction. From results of mutational analysis of key residues in the dRP lyase active site, it appears that the substrate dRP can be stabilized in the observed non-catalytic binding conformation, hindering dRP lyase activity.

Scission of the Lactyl Ether Bond of N-Acetylmuramic Acid by Escherichia coli “Etherase”

The ubiquitous bacterial cell wall sugar N-acetylmuramic acid (MurNAc) carries a unique D-lactyl ether substituent at the C3 position. Recently, we proposed an etherase capable of cleaving this lactyl ether to be part of the novel bacterial MurNAc dissimilation pathway (Dahl, U., Jaeger, T., Nguyen, B. T., Sattler, J. M., Mayer, C. (2004) J. Bacteriol. 186, 2385-2392). Here, we report the identification of the first known MurNAc etherase. The encoding gene murQ is located at 55 min on the Escherichia coli chromosome adjacent to murP, the MurNAc-specific phosphotransferase system. A murQ deletion mutant could not grow on MurNAc as the sole source of carbon and energy but could be complemented by expressing murQ from a plasmid. The mutant had no obvious phenotype when grown on different carbon sources but accumulated MurNAc 6-phosphate at millimolar concentrations from externally supplied MurNAc. Purified MurQ-His6 fusion protein and extracts of cells expressing murQ both catalyze the cleavage of MurNAc 6-phosphate, with GlcNAc 6-phosphate and D-lactate being the primary products. The 18O label from enriched water is incorporated into the sugar molecule, showing that the C3-O bond is cleaved and reformed by the enzyme. Moreover, an intermediate was detected and identified as an unsaturated sugar molecule. Based on this observation, we suggested a lyase-type mechanism (beta-elimination/hydration) for the cleavage of the lactyl ether bond of MurNAc 6-phosphate. Close homologs of murQ were found on the chromosome of several bacteria, and amino acid sequence similarity with the N-terminal domain of human glucokinase-regulatory protein (GckR or GKRP) was recognized.

A Structural Basis for Depolymerization of Alginate by Polysaccharide Lyase Family-7

Alginate lyases depolymerize alginate, a heteropolysaccharide consisting of alpha-L-guluronate and beta-D-mannuronate, through a beta-elimination reaction. Their structure/function relationships are expected to provide information valuable to future industrial alginate processing and drug design for Pseudomonas aeruginosa alginate biofilm-dependent infection, but much remains unknown. Here, we present the crystal structure at 1.0 A resolution and the results of mutational analysis of Sphingomonas sp. A1 alginate lyase A1-II', which is grouped into the polysaccharide lyase (PL) family-7. The overall structure of A1-II' uses a beta-sandwich fold, and it has a large active cleft covered by two short flexible loops. Comparison with other family PL-7 structures indicated that loop opening is necessary for substrate binding when the catalytic reaction is initiated. In contrast to the disorder in many side-chains on the protein surface, the three adjacent beta-strands at the center of the active cleft are well ordered. This results from hydrogen bond networks and stacking-like associations identical with those in other family PL-7 structures. Disruption of these interactions by site-directed mutagenesis (R146A, E148A, R150A, Q189A, and K280A) makes the protein insoluble or greatly decreases its activity. The A1-II' structure includes two sulfate ions in the active cleft. Ammonium sulfate was a potent inhibitor with a Ki of 2.5 mM, indicating that our structure represents a model of the inhibitory state. Results of mutational analysis and continuous hydrogen bond networks suggest that Arg146, Gln189, His191, and Tyr284 form an active center. Tyr284OH appears particularly crucial to the catalytic reaction, which is supported by sulfate ion binding and the proximity to the C5 and O4 atoms of subsite +1 in the model obtained by energy minimization calculations using tri-mannuronate. The structural basis shown by this study is similar in many respects to that of the family PL-5 enzymes.

Indirect electron-donating effects governing the concerted bimolecular elimination processes

The concerted bimolecular β-elimination (E2) reaction of substituted alkanes (X–C α H 2–C β H 2–…) is studied using the semilocalized quantum-chemical approach suggested previously (V. Gineityte, J. Mol. Struct. (Theochem), 588 (2002) 99; V. Gineityte, Int. J. Quant. Chem., 94 (2003) 302) and based on the power series for the one-electron density matrix. Electron density redistributions among separate bonds of both the reactant and the external base ( B ⋅⋅ ) are analyzed and interpreted in this approach in terms of direct (through-space) and indirect (through-bond) interactions of bond orbitals (BOs). The main aim of the study lies in revealing the principal ways of the indirect influence of the base B ⋅⋅ upon characteristics of the heteroatom containing (X–C α ) bond (The direct interactions between respective orbitals are expected to be negligible in the E2 processes). The above-specified indirect influence is shown to consist of two components: First, of the additional indirect electron-donating effect of the initially occupied (bonding) orbital of the H–C β bond upon the X–C α bond, wherein the orbital of the base participates as a mediator, and, second, of the indirect electron-donating effect of the base itself upon the same bond by means of orbitals of the H–C β bond. The known predominance of the trans β elimination over the relevant cis process is accordingly related to higher efficiencies of the above-enumerated indirect electron-donating effects for the case of trans-arranged X–C α and H–C β bonds. The results obtained along with those of an analogous investigation of the S N 2 process (V.Gineityte, J.Mol.Struct. (Theochem), 541 (2001) 1) yield a unified description of both reactions, wherein the direct and indirect attacks of the reagent upon the X–C α bond underly the S N 2 and E2 reactions, respectively. Consequently, the result of competition between these processes is expected to depend upon relative efficiencies of direct and indirect influences of the given reagent upon characteristics of the nucleofuge-containing (X–C α ) bond.

β‐Elimination Reactions by Using Samarium Diiodide

AbstractFor Abstract see ChemInform Abstract in Full Text.

New Short Route to Unsaturated Fluoroketonucleosides: Case of 5-Fluoro-1-(6-O-Acetyl-3,4-Di-Deoxy-3-Fluoro-β-d-Glycero-Hex-3-Eno-Pyranos-2-Ulosyl) Uracil

The proposed short synthesis involves two key steps: Oxidation of the isopropylidene derivative of the 3-fluoronucleoside possessing a free hydroxyl group in 2-position and acetylation of deprotected 3-fluoro-2-ketonucleoside which, after a β-elimination reaction, gives the desired unsaturated ketonucleoside 5.

Molecular identification of Sphingomonas sp. A1 Alginate lyase (A1-IV′) as a member of novel polysaccharide lyase family 15 and implications in alginate lyase evolution

Sphingomonas sp. A1 (strain A1) produces three endotypes (A1-I [65 kDa], A1-II [25 kDa], and A1-III [40 kDa]) and an exotype (A1-IV [86 kDa]) alginate lyases in cytoplasm. These four enzymes cooperatively depolymerize alginate into constituent monosaccharides. In addition to the genes for these lyases, novel genes encoding hypothetical proteins homologous with A1-IV were found in the genomes of many bacteria including strain A1. One such protein, A1-IV′ (90 kDa) of strain A1, was overexpressed in Escherichia coli cells, purified, and characterized. A1-IV′ catalyzed the cleavage of glycosidic bonds in alginate through a β-elimination reaction and released unsaturated di- and trisaccharides as main products, thus indicating that the enzyme is an endotype alginate lyase. A1-IV′, which differed from A1-IV in some enzymatic properties, was not expressed in strain A1, suggesting that A1-IV′ has no significant role in alginate metabolism. A1-IV′ and other A1-IV homologs facilitate the creation of novel polysaccharide lyase family 15 based on their primary structures, implying the evolution route of alginate lyases in family PL-15.

Methylglyoxal, a Metabolite Derived from Glycolysis, Functions as a Signal Initiator of the High Osmolarity Glycerol-Mitogen-activated Protein Kinase Cascade and Calcineurin/Crz1-mediated Pathway in Saccharomyces cerevisiae

Methylglyoxal (MG) is a typical 2-oxoaldehyde derived from glycolysis, although it inhibits the growth of cells in all types of organism. Hence, it has been questioned why such a toxic metabolite is synthesized via the ubiquitous energy-generating pathway. We have previously reported that expression of GLO1, coding for the major enzyme detoxifying MG, was induced by osmotic stress in a high osmolarity glycerol (HOG)-mitogen-activated protein (MAP) kinase-dependent manner in Saccharomyces cerevisiae. Here we show that MG activates the HOG-MAP kinase cascade. Two osmosensors, Sln1 and Sho1, have been identified to function upstream of the HOG-MAP kinase cascade, and we reveal that MG initiates the signal transduction to this MAP kinase cascade through the Sln1 branch. We also demonstrate that MG activates the Msn2 transcription factor. Moreover, MG activated the uptake of Ca(2+) in yeast cells, thereby stimulating the calcineurin/Crz1-mediated Ca(2+) signaling pathway. We propose that MG functions as a signal initiator in yeast.

Identification of alternative products and optimization of 2-nitro-5-thiocyanatobenzoic acid cyanylation and cleavage at cysteine residues

The reagent 2-nitro-5-thiocyanatobenzoic acid (NTCB) is commonly used to cyanylate and cleave proteins at cysteine residues, but this two-step reaction requires lengthy incubations and produces highly incomplete cleavages. In previous reports, incomplete cleavage was attributed to a competing β-elimination reaction that converts cyanylated cysteine to dehydroalanine. In this study, previously unidentified side reactions of the NTCB cleavage were discovered and β-elimination was not the major reaction competing with peptide bond cleavage. A major side reaction was identified as carbamylation of lysine residues. Carbamylation could be minimized by desalting the cyanylation reaction before cleavage or by reducing the reactant concentrations, but both methods suffered from further reductions in cleavage efficiency. Based on model peptide studies, poor cleavage was primarily caused by a mass neutral rearrangement of the cyanylated cysteine which produced a cleavage-resistant, nonreducible product. The formation of this product could be minimized by using stronger nucleophiles for the cleavage reaction. We discovered that base-catalyzed nucleophilic cleavage could be achieved with many amino-containing compounds. Most notably, glycine is capable of promoting efficient cleavage. In addition, efficient NTCB cleavage can be performed in a simple one-step method without a prior cyanylation step, rather than the previously described two-step reaction.

Purification and characterization of a novel glycoprotein from wheat germ water-soluble extracts

A novel glycoprotein, referred to as WGGP, was isolated from wheat germ water-soluble extracts, and purified by ion-exchange and gel filtration column chromatography. The homogeneity of WGGP was demonstrated by gel filtration on Sepharose CL-6B. The glycoprotein contains 56.4% of protein, rich in glutamic acid, asparagic acid, alanine, glycine, valine and leucine. Mannose accounted for 94% of the neutral sugar moiety, and traces of arabinose, xylose, glucose and galactose were found in WGGP. The molecular weight was estimated to be about 40,000 on SDS–PAGE. The existence of O-glycosidic linkage in WGGP was demonstrated with a β-elimination reaction.

Crystal Structure of Unsaturated Glucuronyl Hydrolase, Responsible for the Degradation of Glycosaminoglycan, from Bacillus sp. GL1 at 1.8 Å Resolution

Unsaturated glucuronyl hydrolase (UGL) is a novel glycosaminoglycan hydrolase that releases unsaturated d-glucuronic acid from oligosaccharides produced by polysaccharide lyases. The x-ray crystallographic structure of UGL from Bacillus sp. GL1 was first determined by multiple isomorphous replacement (mir) and refined at 1.8 A resolution with a final R-factor of 16.8% for 25 to 1.8 A resolution data. The refined UGL structure consists of 377 amino acid residues and 478 water molecules, four glycine molecules, two dithiothreitol (DTT) molecules, and one 2-methyl-2,4-pentanediol (MPD) molecule. UGL includes an alpha(6)/alpha(6)-barrel, whose structure is found in the six-hairpin enzyme superfamily of an alpha/alpha-toroidal fold. One side of the UGL alpha(6)/alpha(6)-barrel structure consists of long loops containing three short beta-sheets and contributes to the formation of a deep pocket. One glycine molecule and two DTT molecules surrounded by highly conserved amino acid residues in UGLs were found in the pocket, suggesting that catalytic and substrate-binding sites are located in this pocket. The overall UGL structure, with the exception of some loops, very much resembled that of the Bacillus subtilis hypothetical protein Yter, whose function is unknown and which exhibits little amino acid sequence identity with UGL. In the active pocket, residues possibly involved in substrate recognition and catalysis by UGL are conserved in UGLs and Yter. The most likely candidate catalytic residues for glycosyl hydrolysis are Asp(88) and Asp(149). This was supported by site-directed mutagenesis studies in Asp(88) and Asp(149).