Investigation of the Formaldehyde-Catalyzed NNitrosation of Dialkyl Amines: An Automated Experimental and Kinetic Modelling Study Using Dibutylamine.

The potential for drug substances and drug products to contain low levels of N-nitrosamines is of continued interest to the pharmaceutical industry and regulatory authorities. Acid-promoted nitrosation mechanisms in solution have been investigated widely in the literature and are supported by kinetic modelling studies. Carbonyl compounds, particularly formaldehyde, which may be present as impurities in excipients and drug product packaging components or introduced during drug substance manufacturing processes are also known to catalyze nitrosation, but their impact on the risk of N-nitrosamine formation has not been systematically investigated to date. In this study, we experimentally investigated the multivariate impact of formaldehyde, nitrite and pH on N-nitrosation in aqueous solution using dibutylamine as a model amine. We augmented a published kinetic model by adding formaldehyde-catalyzed nitrosation reactions. We validated the new kinetic model vs. the experimental data and then used the model to systematically investigate the impact of formaldehyde levels on N-nitrosamine formation. Simulations of aqueous solution systems show that at low formaldehyde levels the formaldehyde-catalyzed mechanisms are insignificant in comparison to other routes. However, formaldehyde-catalyzed mechanisms can become more significant at neutral and high pH under higher formaldehyde levels. Model-based sensitivity analysis demonstrated that under high nitrite levels and low formaldehyde levels (where the rate of formaldehyde-catalyzed nitrosation is low compared to the acid-promoted pathways) the model can be used with kinetic parameters for model amines in the literature without performing additional experiments to fit amine-specific parameters. For other combinations of reaction parameters containing formaldehyde, the formaldehyde-catalyzed kinetics are non-negligible, and thus it is advised that, under such conditions, additional experiments should be conducted to reliably use the model.

On-column trace-level formation of N-nitrosamine in a liquid chromatography-mass spectrometry analytical system.

A model reaction between di-n-butylamine and sodium nitrite was studied to investigate trace-level N-nitrosamine formation. Liquid chromatography-mass spectrometry (LC-MS) analysis of kinetic time points from an in-progress reaction showed a systematic offset in nitrosamine concentration between quenched and unquenched samples. By combining samples of amine and nitrite in the needle of the autosampler it was demonstrated that N-nitrosamine was formed in the LC-MS system. Further experimentation indicated that nitrosation was occurring on-column.

Post-translational site-selective protein backbone α-deuteration.

Isotopic replacement has long-proven applications in small molecules. However, applications in proteins are largely limited to biosynthetic strategies or exchangeable (for example, N-H/D) labile sites only. The development of postbiosynthetic, C-1H → C-2H/D replacement in proteins could enable probing of mechanisms, among other uses. Here we describe a chemical method for selective protein α-carbon deuteration (proceeding from Cys to dehydroalanine (Dha) to deutero-Cys) allowing overall 1H→2H/D exchange at a nonexchangeable backbone site. It is used here to probe mechanisms of reactions used in protein bioconjugation. This analysis suggests, together with quantum mechanical calculations, stepwise deprotonations via on-protein carbanions and unexpected sulfonium ylides in the conversion of Cys to Dha, consistent with a 'carba-Swern' mechanism. The ready application on existing, intact protein constructs (without specialized culture or genetic methods) suggests this C-D labeling strategy as a possible tool in protein mechanism, structure, biotechnology and medicine.

Simvastatin activates single skeletal RyR1 channels but exerts more complex regulation of the cardiac RyR2 isoform.

BACKGROUND AND PURPOSE: Statins are amongst the most widely prescribed drugs for those at risk of cardiovascular disease, lowering cholesterol levels by inhibiting 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase. Although effective at preventing cardiovascular disease, statin use is associated with muscle weakness, myopathies and, occasionally, fatal rhabdomyolysis. As simvastatin, a commonly prescribed statin, promotes Ca2+ release from sarcoplasmic reticulum (SR) vesicles, we investigated if simvastatin directly activates skeletal (RyR1) and cardiac (RyR2) ryanodine receptors. EXPERIMENTAL APPROACH: RyR1 and RyR2 single-channel behaviour was investigated after incorporation of sheep cardiac or mouse skeletal SR into planar phospholipid bilayers under voltage-clamp conditions. LC-MS was used to monitor the kinetics of interconversion of simvastatin between hydroxy-acid and lactone forms during these experiments. Cardiac and skeletal myocytes were permeabilised to examine simvastatin modulation of SR Ca2+ release. KEY RESULTS: Hydroxy acid simvastatin (active at HMG-CoA reductase) significantly and reversibly increased RyR1 open probability (Po) and shifted the distribution of Ca2+ spark frequency towards higher values in skeletal fibres. In contrast, simvastatin reduced RyR2 Po and shifted the distribution of spark frequency towards lower values in ventricular cardiomyocytes. The lactone pro-drug form of simvastatin (inactive at HMG-CoA reductase) also activated RyR1, suggesting that the HMG-CoA inhibitor pharmacophore was not responsible for RyR1 activation. CONCLUSION AND IMPLICATIONS: Simvastatin interacts with RyR1 to increase SR Ca2+ release and thus may contribute to its reported adverse effects on skeletal muscle. The ability of low concentrations of simvastatin to reduce RyR2 Po may also protect against Ca2+ -dependent arrhythmias and sudden cardiac death.

A method for continuous and stable perfusion of tissue and single cell preparations with accurate concentrations of volatile anaesthetics.

BACKGROUND: It is difficult to design a system to reliably deliver volatile anaesthetics such as halothane or isoflurane to in vitro preparations such as tissues or cells cultures: the very volatility of the drugs means that they can rapidly dissipate from even carefully-prepared solutions. Furthermore, many experiments require the control of other gases (such as oxygen or carbon dioxide) which requires constant perfusion. NEW METHOD: We describe a constant perfusion system that is air-tight (i.e., allows the accurate administration of hypoxic or hypercapnic gas mixtures), in which volatile anaesthetic is delivered via calibrated vaporisers by constant bubbling into the perfusing solution (and continuously monitored for stability by infrared spectroscopy in the headspace above the solution). RESULTS: We have confirmed the accuracy (i.e., linear relationship of dissolved concentrations with vapour dial settings) and stability (i.e., over time) of the anaesthetic concentrations in solutions in samples taken from the bottles into which anaesthetic is bubbled, and from samples taken from the tissue perfusion bath, using gas chromatrography-mass spectrometry (GC-MS). CONCLUSIONS: It is possible to deliver volatile anaesthetics in accurate concentrations to cell/tissue preparations whilst adjusting ambient air composition rapidly, stable over sustained time periods.

A cell-permeable ester derivative of the JmjC histone demethylase inhibitor IOX1.

The 2-oxoglutarate (2OG)-dependent Jumonji C domain (JmjC) family is the largest family of histone lysine demethylases. There is interest in developing small-molecule probes that modulate JmjC activity to investigate their biological roles. 5-Carboxy-8-hydroxyquinoline (IOX1) is the most potent broad-spectrum inhibitor of 2OG oxygenases, including the JmjC demethylases, reported to date; however, it suffers from low cell permeability. Here, we describe structure-activity relationship studies leading to the discovery of an n-octyl ester form of IOX1 with improved cellular potency (EC50 value of 100 to 4 µM). These findings are supported by in vitro inhibition and selectivity studies, docking studies, activity versus toxicity analysis in cell cultures, and intracellular uptake measurements. The n-octyl ester was found to have improved cell permeability; it was found to inhibit some JmjC demethylases in its intact ester form and to be more selective than IOX1. The n-octyl ester of IOX1 should find utility as a starting point for the development of JmjC inhibitors and as a use as a cell-permeable tool compound for studies investigating the roles of 2OG oxygenases in epigenetic regulation.

Adduction of solvent molecules by ions isolated within an ion trap mass spectrometer under atmospheric pressure ionisation conditions.

Benzylpyridine and papaverine, an alkyl quinoline, both produce product ions containing an azepinium ring during atmospheric pressure chemical ionisation or electrospray multistage mass spectrometry. By controlling the trapping conditions, an isolated azepinium ion was held within the trap for an extended period of time without excitation. A subsequent analytical scan revealed a mass spectrum containing ions at two mass-to-charge (m/z) ratios, the first at the m/z of the isolated product ion and the second at an m/z ratio corresponding to the adduction of a molecule of solvent. Isolation and resonance excitation of the adduct ion remove the solvent molecule, resulting in recovery of the azepinium ion at the same signal intensity as the adduct ion. Isolating and trapping the ion for a further period allowed the solvent adduct ion to be re-formed. Modulation of the solvent flowing into the source while the ion was trapped allowed variation in the solvent molecule adducted to the trapped ion. The proportion of the ion current due to the adduct ion depends on the nature of the isolated ion, the proton affinity of the solvent and the length of time for which the ion was trapped. Adduct ion formation, deliberately maximised in this study, can occur to a significant extent under standard ion trap operating conditions, reducing the ion current of product ions of interest and, ultimately, the response in tandem mass spectrometric assays.

Atmospheric pressure ionisation mass spectrometric fragmentation pathways of noscapine and papaverine revealed by multistage mass spectrometry and in-source deuterium labelling.

Two opium alkaloids, noscapine and papaverine, show good response as [M+H]+ ions in positive ion electrospray mass spectrometry and atmospheric pressure chemical ionisation mass spectrometry. The two compounds exhibit markedly different fragmentation pathways and behaviour under multistage mass spectrometry (MSn), with papaverine displaying a wealth of ions in MS2 and noscapine providing a single dominant ion at each stage of MSn prior to MS4. Elucidation of the fragmentation pathways using the MSn capability of the ion trap was aided by spraying the analytes in 2H2O to incorporate an isotopic label. Simplex optimisation allowed optimum trapping and fragmentation parameters to be determined, leading to a six-fold improvement in response for one transition and a seven-fold improvement for one transition sequence.