Fragmentation studies of pentacoordinated bisaminoacylspirophosphoranes by negative electrospray ionization mass spectrometry

Fragmentation pathways of a series of pentacoordinated bisaminoacylspirophosphoranes were elucidated by electrospray ionization multistage mass spectrometry (ESI-MS(n)) in negative mode. The deprotonated ions of pentacoordinated bisaminoacylspirophosphoranes tend to eliminate a corresponding amino acid to form base peak. The hydrogen/deuterium exchange experiment, the high-resolution mass spectrometry, (13)C stable isotope labeling experiment and theoretical calculations were used to rationalize the proposed fragmentation pathways and to verify the differences between the fragmentation pathways. The results indicate that the negative molecular ions of pentacoordinated bisaminoacylspirophosphoranes dissociate through its open-chain tricoordinated tautomers. The relative Gibbs free energies (ΔG) of the product ions and proposed fragmentation pathways were estimated using the B3LYP/6-31 + + G(d, p) model. The results have some potential applications in the identification structures of similar spirophosphorane compounds by ESI-MS(n).     

Synthesis and application of isocyanates radiolabeled with carbon-11

Carbon-11 labeled isocyanates are efficiently prepared by dehydration of [(11) C]carbamate salts, which in turn are easily formed from cyclotron-produced [(11) C]CO(2) and amines in the presence of a CO(2) fixation agent. The [(11) C]isocyanates are useful radiosynthons for the synthesis of a variety of [carbonyl-(11) C]-labeled asymmetrical ureas and carbamate esters. The method is well suited to incorporate any isotope of carbon, and is especially useful for positron emission tomography (PET) radiotracers for in vivo imaging. This is demonstrated by using the method to make [carbonyl-(11) C]-6-hydroxy-[1,1'-biphenyl]-3-yl cyclohexylcarbamate which is a novel radiotracer for PET imaging of fatty acid amide hydrolase.     

Biosynthesis of Streptolidine Involved Two Unexpected Intermediates Produced by a Dihydroxylase and a Cyclase through Unusual Mechanisms

Streptothricin‐F (STT‐F), one of the early‐discovered antibiotics, consists of three components, a β‐lysine homopolymer, an aminosugar D ‐gulosamine, and an unusual bicyclic streptolidine. The biosynthesis of streptolidine is a long‐lasting but unresolved puzzle. Herein, a combination of genetic/biochemical/structural approaches was used to unravel this problem. The STT gene cluster was first sequenced from a Streptomyces variant BCRC 12163, wherein two gene products OrfP and OrfR were characterized in vitro to be a dihydroxylase and a cyclase, respectively. Thirteen high‐resolution crystal structures for both enzymes in different reaction intermediate states were snapshotted to help elucidate their catalytic mechanisms. OrfP catalyzes an Fe^II‐dependent double hydroxylation reaction converting L ‐Arg into (3R,4R)‐(OH)₂‐L ‐Arg via (3S)‐OH‐L ‐Arg, while OrfR catalyzes an unusual PLP‐dependent elimination/addition reaction cyclizing (3R,4R)‐(OH)₂‐L ‐Arg to the six‐membered (4R)‐OH‐capreomycidine. The biosynthetic mystery finally comes to light as the latter product was incorporation into STT‐F by a feeding experiment.     

Global quantification of phosphoproteins combining metabolic labeling and gel‐based proteomics in B. pumilus

Differential proteomics targeting the protein abundance is commonly used to follow changes in biological systems. Differences in localization and degree of post‐translational modifications of proteins including phosphorylations are of tremendous interest due to the anticipated role in molecular regulatory processes. Because of their particular low abundance in prokaryotes, identification and quantification of protein phosphorylation is traditionally performed by either comparison of spot intensities on two‐dimensional gels after differential phosphoprotein staining or gel‐free by stable isotope labeling, sequential phosphopeptide enrichment and following LC‐MS analysis. In the current work, we combined in a proof‐of‐principle experiment these techniques using ¹⁴N/¹⁵N metabolic labeling with succeeding protein separation on 2D gels. The visualization of phosphorylations on protein level by differential staining was followed by protein identification and determination of phosphorylation sites and quantification by LC‐MS/MS. This approach should avoid disadvantages of traditional workflows, in particular the limited capability of peptide‐based gel‐free methods to quantify isoforms of proteins. Comparing control and stress conditions allowed for relative quantification in protein phosphorylation in Bacillus pumilus exposed to hydrogen peroxide. Altogether, we quantified with this method 19 putatively phosphorylated proteins.     

In Situ Labeling and Distance Measurements of Membrane Proteins in E. coli Using Finland and OX063 Trityl Labels

In situ investigation of membrane proteins is a challenging task. Previously we demonstrated that nitroxide labels combined with pulsed ESR spectroscopy is a promising tool for this purpose. However, the nitroxide labels suffer from poor stability, high background labeling, and low sensitivity. Here we show that Finland (FTAM) and OX063 based labels enable labeling of the cobalamin transporter BtuB and BamA, the central component of the β-barrel assembly machinery (BAM) complex, in E coli. Compared to the methanethiosulfonate spin label (MTSL), trityl labels eliminated the background signals and enabled specific in situ labeling of the proteins with high efficiency. The OX063 labels show a long phase memory time (T_M) of ≈5 μs. All the trityls enabled distance measurements between BtuB and an orthogonally labeled substrate with high selectivity and sensitivity down to a few μm concentration. Our data corroborate the BtuB and BamA conformations in the cellular environment of E. coli.     

Endohedral Mixed Aggregates: Sodium Alkoxide Cages with Organic or Inorganic Central Anions and Variable Hull

Alkali metal alkoxides are widely used in chemistry due to their Brønsted basic and nucleophilic properties. Potassium alkoxides assist alkyllithium in the metalation of hydrocarbons in Lochmann-Schlosser-bases. Both compounds form mixed aggregates, which enhance the thermal stability, solubility, and the basic reactivity of these mixtures. A very unusual spherical mixed alkoxy aggregate was discovered by Grützmacher et al., where a central dihydrogen phosphide anion is surrounded by a highly dynamic shell of thirteen sodium atoms and a hull of twelve tert-butoxide groups. This structural motif can be reproduced by a reaction of trimethylsilyl compounds of methane, halogens, or pseudo-halogens with excess sodium tert-butoxide. A nucleophilic substitution releases the corresponding anion, which is then encapsulated by the sodium alkoxide units. The compounds are soluble in hydrocarbon solvents, enabling studies of solutions by high-resolution NMR spectroscopy and IR/Raman studies of the crystalline materials.     

Addressing the metabolic activation potential of new leads in drug discovery: a case study using ion trap mass spectrometry and tritium labeling techniques

Metabolic activation of drug candidates to electrophilic reactive metabolites that can covalently modify cellular macromolecules may result in acute and/or idiosyncratic immune system-mediated toxicities in humans. This presents a significant potential liability for the future development of these compounds as safe therapeutic agents. We present here an example of an approach where sites of metabolic activation within a new drug candidate series were rapidly identified using online liquid chromatography/multi-stage mass spectrometry on an ion trap mass spectrometer. This was accomplished by trapping the reactive intermediates formed upon incubation of compounds with rat and human liver microsomes as their corresponding glutathione conjugates and mass spectral characterization of these thiol adducts. Based on the structures of the GSH adducts identified, potential sites and mechanisms of bioactivation within the chemical structure were proposed. These metabolism studies were interfaced with iterative structural modifications of the chemical series in order to block these bioactivation sites within the molecule. This strategy led to a significant reduction in the propensity of the compounds to undergo metabolic activation as evidenced by reductions in the irreversible binding of radioactivity to liver microsomal material upon incubation of tritium-labeled compounds with this in vitro system. With the efficiency and throughput achievable with such an approach, it appears feasible to identify and address the metabolic activation potential of new drug leads during routine metabolite identification studies in an early drug discovery setting.     

A highly efficient synthesis of [1-C, O]- and [1-C, H2]-glycerol for the elucidation of biosynthetic pathways

Labeled glycerol is a widely used biochemical probe to investigate biosynthetic pathways. A highly efficient synthesis of [1-¹³C, ¹⁸O]- and [1-¹³C, ²H₂]-glycerol is described in which the ¹³C label is introduced using cyanide. The ¹⁸O label was introduced by a Pinner synthesis and reduction of the ester 5 allowed incorporation of the ²H labels.     

Pd‐Catalyzed Reaction of Allyl Carbonate with Polyols: The Role of CO2 in Transesterification versus Etherification of Glycerol

An intermolecular Pd/PPh₃‐catalyzed transesterification of diallyl carbonate with glycerol to generate glycerol carbonate has been developed. Analysis of the reaction kinetics in THF indicates a first‐order dependence on Pd and diallyl carbonate, that the Pd bears two phosphines during the turnover limiting event, and that increasing the glycerol concentration inhibits reaction, possibly via change in the polarity of the medium. ¹³C isotopic labeling studies demonstrate that the Pd‐catalyzed transesterification requires at least one allyl carbonate moiety and that there is rapid equilibrium of the allyl carbonate with CO₂ in solution, even when present only at low concentrations. A mechanism that is consistent with these results involves oxidative addition of the allyl carbonate to Pd followed by reversible decarboxylation, with the intermediate η¹‐ and η³‐allyl Pd alkoxides mediating direct and indirect transesterification reactions with the glycerol. Using this model, successful simulations of the kinetics of reactions conducted under atmospheres of N₂ or CO₂ could be achieved, including switching in selectivity between etherification and transesterification in the early stages of reaction. Reactions with the higher polyols threitol and erythritol are also efficient, generating the terminal (1,2) monocarbonates with high selectivity.     

Minimization of side reactions during Lys Tag derivatization of C-terminal lysine peptides

Several issues need to be considered concerning chemical labeling strategies in proteomics. Some of these are labeling specificity, possible side reactions, completeness of reaction, recovery rate, conserving integrity of sample, hydrolysis of peptide bonds at high pH, and signal suppression in mass spectrometry (MS). We tested the effects of different reaction conditions for 2-methoxy-4,5-dihydro-1H-imidazole (Lys Tag) derivatization of the ?-amine group of lysine (K) residues. By using nanoflow LC–electrospray ionization-MS (LC–ESI-MS) and MS/MS in combination with MSight 2-D image analysis, we found that standard Lys Tag derivatization processes and conditions induce side reactions such as (i) Lys Tag labeling of the N-terminus, (ii) methylation of internal aspartic acid (D), glutamic acid (E) and C- and N-peptide termini and (iii) deamidation of asparagine (N) and glutamine (Q). We found temperature and pH to be the main variables to control side reactions. Lowering the reaction temperature from 55 °C to room temperature reduced deamidation from 22.8 ± 1.4% (SEM) to 7.7 ± 5.5% (SEM) and almost totally blocked methylation (7.0 ± 1.2% (SEM) to 0.4 ± 0.4% (SEM) of the internal acidic amino acids (D and E) at high pH. We conclude that lowering the reaction temperature minimizes undesired side reactions during Lys Tag derivatization in solution.