Kinetic and regiospecific interrogation of covalent intermediates in the nonribosomal peptide synthesis of yersiniabactin

For interrogation of enzyme-bound intermediates in nonribosomal peptide synthetases (NRPSs), mass spectrometry is used to read out the kinetics and substrate specificity of this medicinally important class of enzymes. The protein HMWP2 (230 kDa) catalyzes 11 chemical reactions, four of which could be resolved by fast quench approaches combined with mass spectrometry. The rate of complex intermediate accumulation at the PCP1 active site was observed to occur with a rate of 19 s(-1), with the rate of cysteine acylation faster than that of intermediate translocation. Use of alternative substrates for salicylic acid (at the ArCP carrier domain) and l-cysteine (at the PCP1 carrier domain) revealed a high penalty for omission of the salicyl alcohol. For some substrates, large discrepancies were found between prior adenylation assays and the current MS-based readouts. Indirect evidence for condensation via a thiolate attack (vs an amino group) was also accumulated. This is the first report to correlate the percent occupancy of multiple active sites in parallel with kinetic and structural resolution of intermediates and provides new evidence of interdomain and intermodule communication within thiotemplate assembly lines.     

SITE-DIRECTED MUTAGENESIS OF THE LH2 LIGHT-HARVESTING COMPLEX OF Rhodobacter sphaeroides: CHANGING βLys23 TO Gin RESULTS IN A SHIFT IN THE 850 nm ABSORPTION PEAK

The present study describes the construction of a Rhodobacter sphaeroides light-harvesting (LH2) mutant in which the charged residue βSLys23 is changed by site-directed mutagenesis to a Gin residue, and the characterization of the resulting mutant complex by a range of spectroscopic techniques. In the 77 K absorption spectrum of the mutant, the peak equivalent to the 850 nm peak in the wild-type membrane is blue-shifted by approximately 18 nm to 837 nm; except for this blue-shift, the 77 K. fluorescence excitation and emission spectra and the circular dichroism spectrum of the mutant are very similar to the equivalent spectra from the wild-type membranes, suggesting that the mutation βLys23 → Gin probably does not cause any major changes in the conformation or aggregation state of these membranes. Possible causes of the 18 nm blue-shift in the absorption spectrum are discussed.     

First organophosphorus radical-mediated cyclisations to afford medium-sized rings: eight-membered lactones and seven- and eight-membered lactams

The phosphorus based radical precursors N-ethylpiperidine hypophosphite (EPHP) and diethylphosphine oxide (DEPO) are efficient reagents for carrying out the formation of seven- and eight-membered rings. Esters and amides were successfully converted into the corresponding eight-membered lactones and seven- and eight-membered lactams in good to excellent yields.     

Synthesis and biological evaluation of new inhibitors of UDP-Galf transferase--a key enzyme in M. tuberculosis cell wall biosynthesis

Two iminosugars have been designed and synthesized as potential inhibitors of UDP-Galf transferase, an enzyme involved in Mycobacterium tuberculosis cell wall biosynthesis. The design is based on a proposed model of the transition state for the transferase reaction. One of the two racemic compounds is the first reported inhibitor of the target enzyme from M. smegmatis.     

Determination of lactate or oxalate using injected lactate oxidase and peroxidase by capillary electrophoresis with UV detection

Two reactions, catalyzed by lactate oxidase (LO) and peroxidase, are initiated by a single injection of the enzymes and the substrate 2,2'-azino-bis(3-ethylene-thiazoline-6-sulfonic acid) (ABTS) into the capillary previously filled with the sample (lactate or lactate-oxalate mixture) and the run buffer containing NADH. The oxidized ABTS product upon reaction with NADH is converted to NAD(+) which is separated and detected in less than 2 min at 266 nm with a sample throughput of 7 min (including wash steps between samples). Simplex trade mark software is used to optimize the enzyme concentrations and reaction temperature. Consumption of the more expensive LO enzyme is only 1.4 x 10(-3) U per assay assuming 27 nL per injection. Linearity is established within the range from 0.0025 to 1 mM with R(2) of 0.9982. Recoveries of lactate from five spiked serum samples averaged 101%. Application of this method for the determination of oxalate as an inhibitor of LO is demonstrated.     

A practical synthesis of (+/-)-alpha-isosparteine from a tetraoxobispidine core

[reaction: see text] The title alkaloid was synthesized in racemic form from 3,7-diallyl-2,4,6,8-tetraoxo-3,7-diazabicyclo[3.3.1]nonane (7) by a regioselective diallylation reaction followed by double ring-closing olefin metathesis and exhaustive reduction. Tetraoxobispidine 7 was itself prepared in three simple operations from dimethyl malonate. The entire sequence to alpha-isosparteine was conducted on a multigram scale and proceeded without recourse to chromatography.     

Recovery of intact proteins from silver stained gels

Silver stained proteins of a wide molecular weight (MW) range (20-116 kDa) were successfully recovered by both electroblot and electroelution. The recovery was demonstrated for nanogram loads of proteins separated by SDS-PAGE and visualized by silver staining methods compatible and incompatible with mass spectrometry (MS). It was shown that the alcohol/acid and glutaraldehyde fixation steps present in a number of staining procedures did not prevent recovery of intact proteins from gels. It was found that the recovery of intact proteins from silver stained gels was substantially increased upon pre-equilibration in a buffer containing the reducing agent, dithiothreitol (DTT). The effect of destaining on the recovery of silver stained proteins was also investigated. Comparable recovery of intact proteins within a wide MW range from silver stained gels with and without destaining step was demonstrated. Recovery of model proteins from gels visualized using silver staining method compatible with MS showed 52 to 76% yield of that from the unstained gel, depending upon method of the transfer. Comparison of the recovery of intact proteins from gels visualized using other staining procedures was also made. The above findings have implications as to the supposed irreversible nature of protein "fixation" inside polyacrylamide matrix, and confirm lack of binding of proteins in the gel to metal silver deposited on its surface. This method has the potential to be suitable for direct characterization of proteins by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) without additional purification steps.     

The solution photochemistry of αβ-unsaturated sulphones

1-Phenyl-2-(benzenesulphonyl)-ethylene and 1-phenyl-2-(benzenesulphonyl)-prop-1-ene have been shown to undergo $\text{Z}$,$\text{E}$-photoisomerisation, whereas 2-benzenesulphonylindene readily forms [_π2 + _π2] photoadducts with 2,3-dimethylbut-2-ene, cyclopentene, and cyclohexene.     

Aspects of the inorganic chemistry of rubber vulcanization. Part 61 Compounds of benzothiazole-2-selenone; a selenium-containing analogue of 2-mercaptobenzothiazole

Summary Benzothiazole-2-selenone, BTSeH, was prepared by treating 2-chlorobenzothiazole with Na₂Se, and the 6-ethoxy analogue (EtOBTSeH) was obtained similarly. From BTSeH, the species [R₄N][BTSe] (R=Me, Et Bu-n), Zn-(BTSe)₂(pyr)₂, (BTSe^–)₂, Zn(BTSe-)₂I₂ and Me₂C=CH-CH(SSeTB)Me were obtained. Addition of [n-Bu₄N] [BTSe] to [Zn(BTSe)₂] _n afforded [n-Bu₄N] [Zn(BTSe)₃(H₂O)]. The Se n.m.r. spectra of these species are briefly reported.     

Rearrangement of epoxynitriles: a convenient homologation of acyclic and cyclic ketones to carboxylic acids

A convenient two-step homologation of both aliphatic and aromatic ketones to the corresponding carboxylic acid has been developed. First ketones were converted to epoxynitriles with the Darzens reaction. Second, a Lewis acid mediated rearrangement of these epoxynitriles with lithium bromide was achieved to give homologated secondary alkanoic acids (as well as aryl-alkanoic) in good yields. The mechanism and the scope of the rearrangement reaction were investigated. This strategy constitutes a two-step homologation of ketones to secondary carboxylic acids.