In silico and in vitro metabolism studies support identification of designer drugs in human urine by liquid chromatography/quadrupole-time-of-flight mass spectrometry

Human phase I metabolism of four designer drugs, 2-desoxypipradrol (2-DPMP), 3,4-dimethylmethcathinone (3,4-DMMC), α-pyrrolidinovalerophenone (α-PVP), and methiopropamine (MPA), was studied using in silico and in vitro metabolite prediction. The metabolites were identified in drug abusers’ urine samples using liquid chromatography/quadrupole-time-of-flight mass spectrometry (LC/Q-TOF/MS). The aim of the study was to evaluate the ability of the in silico and in vitro methods to generate the main urinary metabolites found in vivo. Meteor 14.0.0 software (Lhasa Limited) was used for in silico metabolite prediction, and in vitro metabolites were produced in human liver microsomes (HLMs). 2-DPMP was metabolized by hydroxylation, dehydrogenation, and oxidation, resulting in six phase I metabolites. Six metabolites were identified for 3,4-DMMC formed via N-demethylation, reduction, hydroxylation, and oxidation reactions. α-PVP was found to undergo reduction, hydroxylation, dehydrogenation, and oxidation reactions, as well as degradation of the pyrrolidine ring, and seven phase I metabolites were identified. For MPA, the nor-MPA metabolite was detected. Meteor software predicted the main human urinary phase I metabolites of 3,4-DMMC, α-PVP, and MPA and two of the four main metabolites of 2-DPMP. It assisted in the identification of the previously unreported metabolic reactions for α-PVP. Eight of the 12 most abundant in vivo phase I metabolites were detected in the in vitro HLM experiments. In vitro tests serve as material for exploitation of in silico data when an authentic urine sample is not available. In silico and in vitro designer drug metabolism studies with LC/Q-TOF/MS produced sufficient metabolic information to support identification of the parent compound in vivo.

Detecting proteins complex formation using steady-state diffusion in a nanochannel

In this work, we present theoretical and experimental studies of nanofluidic channels as a potential biosensor for measuring rapid protein complex formation. Using the specific properties offered by nanofluidics, such as the decrease of effective diffusion of biomolecules in confined spaces, we are able to monitor the binding affinity of two proteins. We propose a theoretical model describing the concentration profile of proteins in a nanoslit and show that a complex composed by two bound biomolecules induces a wider diffusion profile than a single protein when driven through a nanochannel. To validate this model experimentally, we measured the increase of the fluorescent diffusion profile when specific biotinylated dextran was added to fluorescent streptavidin. We report here a direct and relatively simple technique to measure the affinity between proteins. Figure We present theoretical and experimental studies of nanofluidic channels as potential biosensors for rapidly measuring protein complex formation. Our system is based on steady-state diffusion effects which are observed inside a nanoslit.     

Solubility-Driven Isolation of a Metastable Nonagold Cluster with Body-Centered Cubic Structure

The conventional synthetic methodology for atomically precise gold nanoclusters by using reduction in solution offers only the thermodynamically most stable nanoclusters. Herein, a solubility-driven isolation strategy is reported to access a metastable gold cluster. The cluster, with the composition of [Au₉(PPh₃)₈]⁺ ( 1 ), displays an unusual, nearly perfect body-centered cubic (bcc) structure. As revealed by ESI-MS and UV/Vis measurements, the cluster is metastable in solution and converts to the well-known [Au₁₁(PPh₃)₈Cl₂]⁺ ( 2 ) within just 90 min. DFT calculations revealed that although both 1 and 2 are eight-electron superatoms, there is a driving force to convert 1 to 2 as shown by the increased cohesion and larger HOMO–LUMO energy gap of 2 . The isolation and crystallization of the metastable gold cluster were achieved in a biphasic reaction system in which reduction of gold precursors and crystallization of 1 took place concurrently. This synthetic protocol represents a successful strategy for investigations of other metastable species in metal nanocluster chemistry.     

Divergent CH Insertion–Cyclization Cascades of N‐Allyl Ynamides

Gold carbene reactivity patterns were accessed by ynamide insertion into a C(sp³)? H bond. A substantial increase in molecular complexity occurred through the cascade polycyclization of N‐allyl ynamides to form fused nitrogen‐heterocycle scaffolds. Exquisite selectivity was observed despite several competing pathways in an efficient gold‐catalyzed synthesis of densely functionalized C(sp³)‐rich polycycles and a copper‐catalyzed synthesis of fused pyridine derivatives. The respective gold–keteniminium and ketenimine activation pathways have been explored through a structure–reactivity study, and isotopic labeling identified turnover‐limiting C? H bond‐cleavage in both processes.     

Prediction of liquid chromatographic retention for differentiation of structural isomers

A liquid chromatography (LC) retention time prediction software, ACD/ChromGenius, was employed to calculate retention times for structural isomers, which cannot be differentiated by accurate mass measurement techniques alone. For 486 drug compounds included in an in-house database for urine drug screening by liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOFMS), a retention time knowledge base was created with the software. ACD/ChromGenius calculated retention times for compounds based on the drawn molecular structure and given chromatographic parameters. The ability of the software for compound identification was evaluated by calculating the retention order of the 118 isomers, in 50 isomer groups of 2–5 compounds each, included in the database. ACD/ChromGenius predicted the correct elution order for 68% (34) of isomer groups. Of the 16 groups for which the isomer elution order was incorrectly calculated, two were diastereomer pairs and thus difficult to distinguish using the software. Correlation between the calculated and experimental retention times in the knowledge base tested was moderate, r² = 0.8533. The mean and median absolute errors were 1.12 min, and 0.84 min, respectively, and the standard deviation was 1.04 min. The information generated by ACD/ChromGenius, together with other in silico methods employing accurate mass data, makes the identification of substances more reliable. This study demonstrates an approach for tentatively identifying compounds in a large target database without a need for primary reference standards.     

New methodological criteria in rapid multichannel microspectrofluorometry

Summary The application of microspectrofluorometric techniques to the study of various metabolic pathways in the dynamic context of intracellular interactions has gained further versatility through the use of better correlated microtopographic and spectral observations, rapid automatic data processing and a more accurate definition of specific conditions at fluorescence excitation and detection to minimize the action of exciting wavelengths on the stability of fluorochrome emission. The kinetic parameters of metabolic transients due to microinjection of substrate, are evaluated from 50–200 intracellular sites simultaneously at a temporal resolution of ~ 64 msec. Thus, regional asynchronicities and kinetic discrepancies of the metabolic response may be recognized in correlation with intracellular structure. Sequential changes in the topographic fluorescence pattern or in the fluorescence spectra may be used to define various intracellular pathways associated with the catabolism of natural metabolites or the intracellular distribution and conversion of exogenous molecules. The combination with automated data processing makes possible the uninterupted studies at the level of various intracellular compartments simultaneously on a topographic mode or the identification via spectral analysis of rapid intracellular conversion undergone by natural and exogenous fluorochromes.

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Zusammenfassung Die Anwendung spektralfluorometrischer Mikromethoden für das Studium verschiedener Stoffwechselvorgänge im dynamischen Zusammenhang intrazellulärer Reaktionen hat durch besser abgestimmte mikrotopographische und spektrale Beobachtungsmöglichkeiten, rasche automatische Datenermittlung, genauere Angabe spezifischer Bedingungen zur Fluoreszenzanregung und Herabsetzung der Einwirkung bestimmter Wellenlängen auf die Stabilität der Fluorochromemission zu weiteren Fortschritten geführt. Die kinetischen Parameter metabolischer Übergänge infolge einer Mikroinjektion von Substrat wurden gleichzeitig an 50–200 intrazellulären Orten mit einer zeitlichen Auflösung von etwa 64 msec ausgewertet. So können regionale Ungleichzeitigkeiten und kinetische Diskrepanzen der metabolischen Beantwortung in Korrelation zur intrazellulären Struktur erkannt werden. Aufeinander folgende Veränderungen der topographischen Fluoreszenzmuster oder der Fluoreszenzspektren lassen sich zur Definition verschiedener intrazellulärer Vorgänge beim Abbau natürlicher Metaboliten oder der intrazellulären Verteilung und Umsetzung exogener Moleküle verwenden. Die Kombination mit automatischer Datenverarbeitung ermöglicht ununterbrochene Untersuchungen in der Größenordnung verschiedener intrazellulärer Räume gleichzeitig auf topographischem Wege oder den spektralanalytischen Nachweis rascher intrazellulärer Umsetzungen natürlicher oder exogener Fluorochrome.

     

Analysis of metabolic transients in intact cells by rapid microfluorimetry

Summary The analysis of NAD⁺ (or NADP⁺) reduction-reoxidation transients in single living cells by rapid microfluorimetry provides a mean to screen the activity of various intracellular enzymes, the interplay of regulating cofactors as well as the influence of structural compartmentalization or membrane barriers. Examples of possible applications refer to the analysis of transient parameters, the pattern of enzymatic pathways in relation to cell growth, effects of cofactors or metabolic preloading, etc. Through the incorporation of a nitrogen chamber the method has been extended to cells (e. g. L cells, human astrocytoma) which require anaerobiosis for glycolytic reduction of NAD⁺. When glucose-6-phosphate is replaced by glucose-1-phosphate the lag which precedes NAD⁺ reduction is prolonged from 100–200 msec up to 500–1000 msec. This can be shortened in presence of glucose-1,6-phosphate (a coenzyme for phosphoglucomutase). Differences in the flux pattern of the forward reaction at the phosphoglucomutase are found in a pleiomorphic population of L cells: e. g. glucose-1-phosphate more easily channeled towards the Embden-Meyerhof sequence in the larger non-dividing individuals. Preincubation with glycerol or xylitol leads to a prolongation of all parameters in the fluorescence transients, while cyclic AMP and ethionine lead to the opposite. The pattern of fluorescence transients makes possible a differentiation between reversible and irreversible inhibitors of LDH. Thus, by rapid microfluorimetry it is possible to resolve the early and later phases of fluorescence transients into components corresponding to characteristic steps in the sequence of intracellular events or control states.

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Zusammenfassung Die Analyse von NAD⁺- (oder NADP⁺-)Reduktions-Reoxidationsübergängen in einzelnen lebenden Zellen durch schnelle Mikrofluorimetrie bietet eine Möglichkeit, die Aktivität verschiedener intrazellulärer Enzyme zu testen, das Zwischenspiel regulatorischer Faktoren zu studieren und den Einfluß struktureller Abteilungsbildung oder von Membranbarrieren zu untersuchen. Anwendungsbeispiele werden gebracht für die Analyse von Übergangsparametern, für die Untersuchung des Musters enzymatischer Stoffwechselschritte während des Zellwachstums und für die Untersuchung der Wirkung von Cofaktoren oder von Stoffwechselvorbelastungen. Durch Einbeziehung einer Stickstoffkammer konnte die Methode auf Zellen angewendet werden, die für die glykolytische Reduktion von NAD⁺ anaerobe Bedingungen verlangen (z. B. L-Zellen, menschliches Astrocytom). Wird Glucose-6-phosphat durch Glucose-1-phosphat ersetzt, so wird die NAD⁺-Reduktion von 100 bis 200 msek bis auf 500 bis 1000 msek verzögert. Diese Zeit kann bei Anwesenheit von Glucose-1,6-phosphat (einem Coenzym für Phosphoglucomutase) verkürzt werden. Differenzen im Fließmuster der vorgenannten Reaktion der Phosphoglucomutase werden in pleiomorphen Populationen von L-Zellen gefunden. So ging z. B. Glucose-1-phosphat leichter in die Embden-Meyerhof-Reaktionsfolge ein in den größeren, sich nicht teilenden Individuen.Vorinkubation mit Glycerin oder Xylit führt zu einer Verlängerung aller Parameter der Fluoreszenzübergänge, während cyklisches AMP und Äthionin das Gegenteil bewirken. Das Muster der Fluoreszenzübergänge ermöglicht eine Differenzierung zwischen reversiblen und irreversiblen Inhibitoren von LDH. So können durch schnelle Mikrofluorimetrie die frühen und späteren Phasen der Fluoreszenzübergänge in Teilschritte aufgelöst werden, die für den Ablauf intrazellulärer Vorgänge oder für Kontrollzustände charakteristisch sind.

     

Direction specific interactions of 1,4-dicarboxylic acid with calcite surfaces

Interactions of succinic acid (SUC) with the {104} cleavage faces of calcite show a strong preference in crystallographic directions. In situ atomic force microscopy revealed that the morphology of etch pits on the crystal surfaces experienced a transition from the common rhombus to a hexagon upon the introduction of SUC. The pit shape further evolved from 6-sided to 7-sided and eventually to 5-sided with increasing concentrations of SUC. Analysis indicates that the morphology changes may result from SUC preferentially binding to the [42] and [010] edges of the (104) plane to selectively slow down their step speed.