Evidence for nonstatistical dynamics in the Wolff rearrangement of a carbene

Two 13C-labeled isomers of the formal Diels−Alder adduct of acetylmethyloxirene to tetramethyl 1,2,4,5-benzenetetracarboxylate have been synthesized. Flash vacuum thermolysis of these adducts leads to various isotopic isomers of acetylmethylketene, the ratios of which have been determined by NMR. The surprising finding that the principal product comes from methylpyruvoyl carbene rather than its more stable isomer diacetylcarbene is explained by MPWB1K density functional calculations, which show that the reactant probably undergoes a unimolecular rearrangement to a norcaradiene derivative prior to its fragmentation. Coupled-cluster calculations on the methylpyruvoyl carbene show that it is capable of undergoing three unimolecular isomerizations. The fastest is 1,2-acetyl migration to give acetylmethylketene directly. The next is rearrangement via acetylmethyloxirene to diacetylcarbene and thence by Wolff rearrangement to acetylmethylketene. The least-favorable reaction is degenerate rearrangement via 1,3-dimethyl-2-oxabicyclo[1.1.0]butan-4-one (the epoxide of dimethylcyclopropenone). The combined experimental and computational results indicate that Wolff rearrangement of the diacetylcarbene occurs with a 2.5:1 ratio of the methyl groups despite the fact that they are related by a twofold axis of symmetry in the carbene. Preliminary molecular dynamics simulations are consistent with this conclusion. Taken together, the results suggest that the Wolff rearrangement is subject to the same kind of nonstatistical dynamical effects detected for other kinds of thermally generated reactive intermediates

A R T I C L E S

The predominant biosynthetic route to vitamin B6 is catalyzed by a single enzyme. The synthase subunit of this enzyme, Pdx1, operates in concert with the glutaminase subunit, Pdx2, to catalyze the complex condensation of ribose 5-phosphate, glutamine and glyceraldehyde 3-phosphate to form pyridoxal 5¢-phosphate, the active form of vitamin B6. In previous studies it became clear that many if not all of the reaction intermediates were covalently bound to the synthase subunit, thus making them difficult to isolate and characterize. Here we show that it is possible to follow a single turnover reaction by heteronuclear NMR using 13 C-and 15 N-labeled substrates as well as 15 N-labeled synthase. By denaturing the enzyme at points along the reaction coordinate, we solved the structures of three covalently bound intermediates. This analysis revealed a new 1,5 migration of the lysine amine linking the intermediate to the enzyme during the conversion of ribose 5-phosphate to pyridoxal 5¢-phosphate. Vitamin B6 is a composite term for six different vitamers recognized as pyridoxine (1), pyridoxal (2), pyridoxamine (3) and their corresponding 5¢-phosphorylated derivatives (4, 5 and 6, respectively). The vitamer pyridoxal 5¢-phosphate (PLP; 5) is known for its catalytic versatility 1 . In most cases PLP acts as an enzyme-bound cofactor that participates in diverse biochemical reactions and pathways, including amino acid biosynthesis, carbohydrate metabolism and the modification of many amine-containing compounds. It has been estimated that at least 140 different PLP-dependent enzymes exist, and approximately 1.5% of the genes in a typical prokaryote encode PLP-using enzymes 2 . A number of these enzymes are already targets for therapeutic agents, and many more are thought to be good candidates 3 . In addition to these well-documented roles in enzyme catalysis, PLP has recently been implicated in singlet oxygen resistance As vitamin B6 is required for various processes in all organisms, it is either biosynthesized de novo, as is the case for most microorganisms and plants, or acquired externally, as is necessary for animals 6 . Two independent de novo pathways for the biosynthesis of PLP are currently known. The best understood pathway, found in Escherichia coli (1-deoxyxylulose 5-phosphate (7)-dependent), is rarely used compared with the route present in most other species (ribose 5-phosphate-dependent) 7 . Pdx1-Pdx2, the biosynthetic enzyme found in Bacillus subtilis, catalyzes the condensation reaction shown in Scheme 1 using either D-ribose 5-phosphate (R5P; 8) or D-ribulose 5-phosphate (Ru5P; 9), glutamine (10) and D-glyceraldehyde 3-phosphate (G3P; 11) Previous high-resolution mass spectrometric analysis of Pdx1 revealed that the first substrate used, R5P or Ru5P, becomes covalently attached to the enzyme through an active site lysine concomitant with the loss of water (Pdx1 + 212 Da) The first hint that the Pdx1-Z 1 intermediate was not bound via an imine was provided by the observation of its relatively high stability. It was shown that ESI-FTMS spectra of Pdx1-Z 1 could be collected even without NaBH 4 reduction 23 . This was surprising because the preparation of the sample for analysis included a reversed-phase desalting step that was performed under mildly acidic conditions that should result in imine hydrolysis 23 . Furthermore, the addition of only NH 4 Cl (or glutamine if Pdx2 was present) to Pdx1-Z 1 resulted in the accumulation of the chromophoric species, I 320 , which appeared to be bound to the enzyme via C5 (ref. 22). Based on the fact that only one molecule of water is lost during the reaction of Pdx1 with R5P to form Pdx1-Z 1 , it was difficult to imagine that the substrate was initially covalently bound to C5. The data suggested the possibility of a C-N bond shift in going from Pdx1-Z 1 to I 320 , but there was no direct structural data supporting this claim. In addition to these two intermediates, a thir

Metal–Free Ring–Opening Metathesis Polymerization with Hydrazonium Initiators

A new strategy for the ring–opening metathesis polymerization (ROMP) of cycloalkenes using hydrazonium initiators is described. The initiators, which are formed by the condensation of 2,3-diazabicyclo[2.2.2]octane and an aldehyde, polymerize cyclopropene monomers by a sequence of [3+2] cycloaddition and cycloreversion reactions. This process generates short chain polyolefins (Mn ≤ 9.4 kg/mol) with relatively low dispersities (Đ ≤ 1.4). The optimized conditions showed efficiency comparable to that achieved with Grubbs’ catalyst. A positive correlation between monomer to initiator ratio and degree of polymerization was revealed through NMR spectroscopy

Hydrazine-Catalyzed Ring-Opening Metathesis Polymerization of Cyclobutenes

Materials formed by the ring-opening metathesis polymerization (ROMP) of cyclic olefins are highly valued for industrial and academic applications but are difficult to prepare free of metal contaminants. Here we describe a highly efficient metal-free ROMP of cyclobutenes using hydrazine catalysis. Reactions can be initiated via in situ condensation of a [2.2.2]-bicyclic hydrazine catalyst with an aliphatic or aromatic aldehyde initiator. The polymerizations show living characteristics, achieving excellent control over molecular weight, low dispersity values, and high chain-end fidelity. Additionally, the hydrazine can be used in substoichiometric amounts relative to the aldehyde chain-end while maintaining good control over molecular weight and low dispersity values, indicating that a highly efficient chain transfer mechanism is occurring

Anodically Coupled Electrolysis For The Heterodifunctionalization Of Alkenes

The emergence of new catalytic strategies that cleverly adopt concepts and techniques frequently used in areas such as photochemistry and electrochemistry has yielded a myriad of new organic reactions that would be challenging to achieve using orthodox methods. Herein, we discuss the strategic use of anodically coupled electrolysis, an electrochemical process that combines two parallel oxidative events, as a complementary approach to existing methods for redox organic transformations. Specifically, we demonstrate anodically coupled electrolysis in the regio- and chemoselective chlorotrifluoromethylation of alkenes

Evidence for Nonstatistical Dynamics in the Wolff Rearrangement of a Carbene

Two ¹³C-labeled isomers of the formal Diels−Alder adduct of acetylmethyloxirene to tetramethyl 1,2,4,5-benzenetetracarboxylate have been synthesized. Flash vacuum thermolysis of these adducts leads to various isotopic isomers of acetylmethylketene, the ratios of which have been determined by NMR. The surprising finding that the principal product comes from methylpyruvoyl carbene rather than its more stable isomer diacetylcarbene is explained by MPWB1K density functional calculations, which show that the reactant probably undergoes a unimolecular rearrangement to a norcaradiene derivative prior to its fragmentation. Coupled-cluster calculations on the methylpyruvoyl carbene show that it is capable of undergoing three unimolecular isomerizations. The fastest is 1,2-acetyl migration to give acetylmethylketene directly. The next is rearrangement via acetylmethyloxirene to diacetylcarbene and thence by Wolff rearrangement to acetylmethylketene. The least-favorable reaction is degenerate rearrangement via 1,3-dimethyl-2-oxabicyclo[1.1.0]butan-4-one (the epoxide of dimethylcyclopropenone). The combined experimental and computational results indicate that Wolff rearrangement of the diacetylcarbene occurs with a 2.5:1 ratio of the methyl groups despite the fact that they are related by a twofold axis of symmetry in the carbene. Preliminary molecular dynamics simulations are consistent with this conclusion. Taken together, the results suggest that the Wolff rearrangement is subject to the same kind of nonstatistical dynamical effects detected for other kinds of thermally generated reactive intermediates

A diffusible signal factor of the intestine dictates Salmonella invasion through its direct control of the virulence activator HilD.

Successful intestinal infection by Salmonella requires optimized invasion of the gut epithelium, a function that is energetically costly. Salmonella have therefore evolved to intricately regulate the expression of their virulence determinants by utilizing specific environmental cues. Here we show that a powerful repressor of Salmonella invasion, a cis-2 unsaturated long chain fatty acid, is present in the murine large intestine. Originally identified in Xylella fastidiosa as a diffusible signal factor for quorum sensing, this fatty acid directly interacts with HilD, the master transcriptional regulator of Salmonella, and prevents hilA activation, thus inhibiting Salmonella invasion. We further identify the fatty acid binding region of HilD and show it to be selective and biased in favour of signal factors with a cis-2 unsaturation over other intestinal fatty acids. Single mutation of specific HilD amino acids to alanine prevented fatty acid binding, thereby alleviating their repressive effect on invasion. Together, these results highlight an exceedingly sensitive mechanism used by Salmonella to colonize its host by detecting and exploiting specific molecules present within the complex intestinal environment