High-performance thin-layer chromatography chemical fingerprinting: a modern technique for comparative assessment of a multivariate chromatogram analysis of Padina boergesenii

JPC – Journal of Planar Chromatography – Modern TLC - Gujarat’s coastline holds immense storage of valuable marine macroalgae. Padina boergesenii is normally familiar as leafy...     

Determination of the natural abundance δ15N of nortropane alkaloids by gas chromatography–isotope ratio mass spectrometry of their ethylcarbamate esters

An important route for the detoxification of tropane alkaloids involves N-demethylation to the nor-compounds followed by further degradation. In order to study the mechanisms of the pertinent reactions, a suitable means to determine the isotope ratios of the substrates and products is required. However, the polarity and functionality of the nortropane compounds makes their analysis as free bases difficult. A method is described which allows both the quantification of nortropane alkaloids and the determination of their natural abundance δ¹⁵N values. The protocol exploits the derivatisation of the alkaloids by reaction with ethyl chloroformate in aqueous medium and the quantitative extraction of the ensuing ethylcarbamate esters. The improved chromatographic properties of these derivatives gives ample separation of the isomeric nortropine and norpseudotropine for measurement of their δ¹⁵N (‰) values by isotope ratio mass spectrometry interfaced to gas chromatography. Adequate separation could not be achieved with the underivatised compounds. Repeatability and precision are sufficient to allow differences in the δ¹⁵N values (∆δ¹⁵N) > 0.8‰ to be measured, with a standard deviation routinely ∼0.3‰. The methodology has been tested by determining the changes in the δ¹⁵N values of nortropine and norpseudotropine during degradation by cell suspension cultures of a Pseudomonas strain expressing a specific capacity for tropine catabolism. The precision and reproducibility are shown sufficient to allow the evolution of the δ¹⁵N values to be followed during the fermentation.     

Reaction of C-silylated alpha-diazophosphines as nucleophiles toward carbonyl compounds: a mechanistic study and application to the synthesis of alkynes and alpha-hydroxyphosphonamides

Diversely substituted alpha-hydroxyphosphonamides and alkynes have been efficiently synthesized through the reaction of C-silylated alpha-diazophosphines with different types of aldehydes (2 equiv) in a neutral medium under very mild conditions. The reaction with some chiral aldehydes is highly diastereoselective leading to phosphonamides as single diastereomers. The novel reaction is influenced by electronic and steric effects being precluded for aromatic aldehydes containing electron-releasing substituents on the phenyl ring and for bulky aliphatic aldehydes. The mechanistic studies of these processes, which are highly exothermic, provide evidence for a nucleophilic attack of the diazophosphine to the aldehyde leading to a betaine that rapidly rearranges to a diazomethylenephosphorane, which has been detected or captured in some instances. The diazomethylenephosphorane reacts with a second molecule of aldehyde according to a Wittig-type condensation, and the rate-determining step of the whole process is believed to be the decomposition of the resultant oxaphosphetane to afford the hydroxyphosphonamide and a diazocumulene. Finally, this intermediate loses molecular nitrogen giving a transient carbene that rapidly evolves toward the alkyne.     

Phase evolution,structural characteristics and mechanism of vesicle formation from a synthetic amphiphile: Controlled morphology by tuning solution phase parameters

We report herein, the aggregation behavior of 3, 4-di(dodecyloxy)benzoic acid-4-hydroxy phenyl ester (DDBE), a synthetic amphiphile and a true non-ionic surfactant system as per the geometrical considerations. The true surfactant nature of the system stems from its hydrophilic-lipophilic-balance (HLB?=?4.7), comparable to that of Span-60, also a true non-ionic surfactant. This compound undergoes spontaneous vesicle formation in THF:water binary solvent mixtures which further underwent fission at lower DDBE concentrations and fusion at higher concentrations, leading to giant vesicles of the order of 3000?nm. These vesicles are sensitive to the polarity of their environment. The predominant mode of interaction as observed from the molecular dynamics simulations were found to be π-π stacking with the phenyl rings of the molecule. Further, the system, upon complete extraction into water, formed spherical aggregates of size 50?nm based on the good solvent-poor solvent combination as the necessary condition for the vesicle formation.GRAPHICAL ABSTRACT     

Theoretical and experimental investigation of the basicity of phosphino(silyl)carbenes

[reaction: see text] The reactivity of phosphino(trimethylsilyl)carbenes 1 with several organic acids has been examined in order to evaluate the pKa values of the conjugate acids. Carbenes 1 react efficiently with C-organic acids such as 1,3-dimesitylimidazolium chloride, phenylacetylene, acetonitrile, and acetyltrimethylsilane, which have pKa's in DMSO in the range 18-31. However, the reaction of the conjugate acids 1H+ with the anion perturbs the determination of the genuine basicity of 1. Theoretical calculations have been performed in order to quantify the basicity of phosphino(trimethylsilyl)carbenes 1 and to compare them with that of N-heterocyclic carbenes 2. The pKa of 1H+ in DMSO has been computed to be in the 23.0-23.4 range, so that 1 is not strong enough as a base to spontaneously deprotonate organic acids such as phenylacetylene, acetonitrile, or acetyltrimethylsilane. However, its conjugate acid 1H+ is a strong electrophile and easily reacts with the nucleophilic conjugate bases of these acids leading to the formation of the corresponding phosphorus ylides.     

New chiral polyfunctional cyclobutane derivatives from (−)-verbenone: possible surfactant behaviour

New enantiopure cyclobutane derivatives have been synthesized from a chiral precursor derived from (?)-verbenone. The cyclobutane moiety acts as a chiral platform to afford a γ-amino acid function in a branched side-chain containing an additional stereogenic centre as well as additional C₆ or C₁₆-alkyl chains linked to the ring by means of an amine or an amide function. One of these compounds, obtained as a 1:2 mixture with its TFA salt has been investigated, suggesting behaviour as a good surfactant and its critical micellar concentration has been determined.     

Strategy for choosing extraction procedures for NMR-based metabolomic analysis of mammalian cells

Metabolomic analysis of mammalian cells can be applied across multiple fields including medicine and toxicology. It requires the acquisition of reproducible, robust, reliable, and homogeneous biological data sets. Particular attention must be paid to the efficiency and reliability of the extraction procedure. Even though a number of recent studies have dealt with optimizing a particular protocol for specific matrices and analytical techniques, there is no universal method to allow the detection of the entire cellular metabolome. Here, we present a strategy for choosing extraction procedures from adherent mammalian cells for the global NMR analysis of the metabolome. After the quenching of cells, intracellular metabolites are extracted from the cells using one of the following solvent systems of varying polarities: perchloric acid, acetonitrile/water, methanol, methanol/water, and methanol/chloroform/water. The hydrophilic metabolite profiles are analysed using ¹H nuclear magnetic resonance (NMR) spectroscopy. We propose an original geometric representation of metabolites reflecting the efficiency of extraction methods. In the case of NMR-based analysis of mammalian cells, this methodology demonstrates that a higher portion of intracellular metabolites are extracted by using methanol or methanol/chloroform/water. The preferred method is evaluated in terms of biological variability for studying metabolic changes caused by the phenotype of four different human breast cancer cell lines, showing that the selected extraction procedure is a promising tool for metabolomic and metabonomic studies of mammalian cells. The strategy proposed in this paper to compare extraction procedures is applicable to NMR-based metabolomic studies of various systems.     

Searching for new cell-penetrating agents: hybrid cyclobutane-proline γ,γ-peptides

Two generations of hybrid γ,γ-peptides containing cyclobutane amino acids and cis-γ-amino-L-proline joined in alternation have been synthesized and their capacity to cross the eukaryotic cell membrane has been evaluated. The first generation consists of di-, tetra- and hexapeptides, and their properties have been analyzed as well as the influence of peptide length and chirality of the cyclobutane residues. Results have shown that the absolute configuration of the cyclobutane amino acid does not have a relevant influence. The second generation consists of hybrid γ,γ-hexapeptides with a common backbone and distinct side chains introduced with different linkage types through the α-amino group (N(α)) of the proline monomers. These peptides have been shown to be non-toxic towards HeLa cells and to internalize them effectively, the best results being obtained for the peptides with a spacer of five carbons between the N(α) atom and the guanidinium group. The introduction of cyclobutane residues inside the sequence affords a good balance between charge and hydrophobicity, reducing the number of positive charges. This results in lower toxicity and similar cell-uptake properties when compared to previously described peptide agents.     

Acebutolol and alprenolol metabolism predictions: comparative study of electrochemical and cytochrome P450-catalyzed reactions using liquid chromatography coupled to high-resolution mass spectrometry

A comparative study of the electrochemical conversion and the biotransformation performed by the cytochrome P450 (CYP450) obtained by rat liver microsomes has been achieved to elucidate the oxidation mechanism of both acebutolol and alprenolol. For this purpose, a wide range of reactions such as N-dealkylation, O-dealkoxylation, aromatic hydroxylation, benzyl hydroxylation, alkyl hydroxylation, and aromatic hydroxylation have been examined in this study, and their mechanisms have been compared. Most of the results of the electrochemical oxidation have been found to be in accordance with those obtained by incubating acebutolol and alprenolol in the presence of CYP450, i.e., N-dealkylation, benzyl hydroxylation, and O-dealkoxylation reactions catalyzed by liver microsomes were found to be predicted by the electrochemical oxidation. The difficulty for the electrochemical process to mimic both aromatic and alkyl hydroxylation reactions has also been discussed, and the hypothesis for the absence of aromatic hydroxylated and alkyl hydroxylated products, respectively, for alprenolol and acebutolol, under the anodic oxidation has been supported by theoretical calculation. The present study highlights the potential and limitation of coupling of electrochemistry–liquid chromatography–high-resolution mass spectrometry for the study of phase I and phase II reactions of acebutolol and alprenolol.