NMRium: Teaching nuclear magnetic resonance spectra interpretation in an online platform.

NMRium is the first web-based software that allows displaying, processing, interpretation, and teaching of 1D and 2D NMR data in a user-friendly interface. It can import the most common data formats (e.g., JCAMP-DX, Bruker, Varian, and Jeol). While the scope for the use of NMRium encompasses a variety of applications such as being a component in data repositories or electronic lab notebooks (ELN), performing structure elucidation or preparing raw spectral data for publication, it also excels in enhancing teaching of NMR interpretation. In this paper, we present some current possibilities of this new tool. Several series of exercises are already provided on https://www.nmrium.org/teaching.

Biotransformation of anabolic androgenic steroids in human skin cells.

In comparison to well-known drug-metabolizing organs such as the liver, the metabolic capacity of human skin is still not well elucidated despite the widespread use of topical drug application. To gain a comprehensive insight into anabolic steroid metabolism in the skin, six structurally related anabolic androgenic steroids, testosterone, metandienone, methyltestosterone, clostebol, dehydrochloromethyltestosterone, and methylclostebol, were applied to human keratinocytes and fibroblasts derived from the juvenile foreskin. Phase I metabolites obtained from incubation media were analyzed by gas chromatography-mass spectrometry. The 5α-reductase activity was predominant in the metabolic pathways as supported by the detection of 5α-reduced metabolites after incubation of testosterone, methyltestosterone, clostebol, and methylclostebol. Additionally, the stereochemistry structures of fully reduced metabolites (4α,5α-isomers) of clostebol and methylclostebol were newly confirmed in this study by the help of inhouse synthesized reference materials. The results provide insights into the steroid metabolism in human skin cells with respect to the characteristics of the chemical structures.

Twenty years of nmrshiftdb2: A case study of an open database for analytical chemistry.

In October 2003, 20 years ago, the open-source and open-content database NMRshiftDB was announced. Since then, the database, renamed as nmrshiftdb2 later, has been continuously available and is one of the longer-running projects in the field of open data in chemistry. After 20 years, we evaluate the success of the project and present lessons learnt for similar projects.

Formation of Breslow Intermediates from N-Heterocyclic Carbenes and Aldehydes Involves Autocatalysis by the Breslow Intermediate, and a Hemiacetal.

Under aprotic conditions, the stoichiometric reaction of N-heterocyclic carbenes (NHCs) such as imidazolidin-2-ylidenes with aldehydes affords Breslow Intermediates (BIs), involving a formal 1,2-C-to-O proton shift. We herein report kinetic studies (NMR), complemented by DFT calculations, on the mechanism of this kinetically disfavored H-translocation. Variable time normalization analysis (VTNA) revealed that the kinetic orders of the reactants vary for different NHC-to-aldehyde ratios, indicating different and ratio-dependent mechanistic regimes. We propose that for high NHC-to-aldehyde ratios, the H-shift takes place in the primary, zwitterionic NHC-aldehyde adduct. With excess aldehyde, the zwitterion is in equilibrium with a hemiacetal, in which the H-shift occurs. In both regimes, the critical H-shift is auto-catalyzed by the BI. Kinetic isotope effects observed for R-CDO are in line with our proposal. Furthermore, we detected an H-bonded complex of the BI with excess NHC (NMR).

Prediction of chemical shift in NMR: A review.

Calculation of solution-state NMR parameters, including chemical shift values and scalar coupling constants, is often a crucial step for unambiguous structure assignment. Data-driven (sometimes called empirical) methods leverage databases of known parameter values to estimate parameters for unknown or novel molecules. This is in contrast to popular ab initio techniques that use detailed quantum computational chemistry calculations to arrive at parameter estimates. Data-driven methods have the potential to be considerably faster than ab inito techniques and have been the subject of renewed interest over the past decade with the rise of high-quality databases of NMR parameters and novel machine learning methods. Here, we review these methods, their strengths and pitfalls, and the databases they are built on.

Controlled administration of dehydrochloromethyltestosterone in humans: Urinary excretion and long-term detection of metabolites for anti-doping purpose.

Dehydrochloromethyltestosterone (DHCMT) is an anabolic-androgenic steroid that was developed by Jenapharm in the 1960s and was marketed as Oral Turinabol®. It is prohibited in sports at all times; nevertheless, there are several findings by anti-doping laboratories every year. New long-term metabolites have been proposed in 2011/12, which resulted in adverse analytical findings in retests of the Olympic games of 2008 and 2012. However, no controlled administration trial monitoring these long-term metabolites was reported until now. In this study, DHCMT (5 mg, p.o.) was administered to five healthy male volunteers and their urine samples were collected for a total of 60 days. The unconjugated and the glucuronidated fraction were analyzed separately by gas chromatography coupled to tandem mass spectrometry. The formation of the described long-term metabolites was verified, and their excretion monitored in detail. Due to interindividual differences there were several varieties in the excretion profiles among the volunteers. The metabolite M3, which has a fully reduced A-ring and modified D-ring structure, was identified by comparison with reference material as 4α-chloro-17ß-hydroxymethyl-17α-methyl-18-nor-5α-androstan-13-en-3α-ol. It was found to be suitable as long-term marker for the intake of DHCMT in four of the volunteers. In one of the volunteers, it was detectable for 45 days after single oral dose administration. However, in two of the volunteers M5 (already published as long-term metabolite in the 1990s) showed longer detection windows. In one volunteer M3 was undetectable but another metabolite, M2, was found as the longest detectable metabolite. The last sample clearly identified as positive was collected between 9.9 and 44.9 days. Furthermore, the metabolite epiM4 (partially reduced A-ring and a modified D-ring structure which is epimerized in position 17 compared to M3) was identified in the urine of all volunteers with the help of chemically synthesized reference as 4-chloro-17α-hydroxymethyl-17ß-methyl-18-nor-androsta-4,13-dien-3ß-ol. It may serve as additional confirmatory metabolite. It is highly recommended to screen for all known metabolites in both fractions, glucuronidated and unconjugated, to improve identification of cheating athletes. This study also offers some deeper insights into the metabolism of DHCMT and of 17α-methyl steroids in general.

New Insights into the Metabolism of Methyltestosterone and Metandienone: Detection of Novel A-Ring Reduced Metabolites.

Metandienone and methyltestosterone are orally active anabolic-androgenic steroids with a 17α-methyl structure that are prohibited in sports but are frequently detected in anti-doping analysis. Following the previously reported detection of long-term metabolites with a 17ξ-hydroxymethyl-17ξ-methyl-18-nor-5ξ-androst-13-en-3ξ-ol structure in the chlorinated metandienone analog dehydrochloromethyltestosterone ("oral turinabol"), in this study we investigated the formation of similar metabolites of metandienone and 17α-methyltestosterone with a rearranged D-ring and a fully reduced A-ring. Using a semi-targeted approach including the synthesis of reference compounds, two diastereomeric substances, viz. 17α-hydroxymethyl-17ß-methyl-18-nor-5ß-androst-13-en-3α-ol and its 5α-analog, were identified following an administration of methyltestosterone. In post-administration urines of metandienone, only the 5ß-metabolite was detected. Additionally, 3α,5ß-tetrahydro-epi-methyltestosterone was identified in the urines of both administrations besides the classical metabolites included in the screening procedures. Besides their applicability for anti-doping analysis, the results provide new insights into the metabolism of 17α-methyl steroids with respect to the order of reductions in the A-ring, the participation of different enzymes, and alterations to the D-ring.

NMReDATA: Tools and applications.

The nuclear magnetic resonance extracted data (NMReDATA) format has been proposed as a way to store, exchange, and disseminate nuclear magnetic resonance (NMR) data and physical and chemical metadata of chemical compounds. In this paper, we report on analytical workflows that take advantage of the uniform and standardized NMReDATA format. We also give access to a repository of sample data, which can serve for validating software packages that encode or decode files in NMReDATA format.

Acyl Donor Intermediates in N-Heterocyclic Carbene Catalysis: Acyl Azolium or Azolium Enolate?

Azolium enolates and acyl azolium cations have been proposed as intermediates in numerous N-heterocyclic carbene (NHC) catalyzed transformations. Acetyl azolium enolates were generated from the reaction of 2-propenyl acetate with both saturated (SIPr) and aromatic (IPr) NHCs, isolated, and characterized (NMR, XRD). Protonation with triflic acid gave the corresponding acetyl azolium triflates which were isolated and characterized (NMR, XRD). Acyl azolium cations have been proposed as immediate precursors of the ester product, for example, in the redox esterification of α,ß-enals. Studies with d3 -acetyl azolium triflate suggest that ester formation originates instead from an azolium enolate intermediate. Furthermore, the acetyl azolium enolate selectively reacted with alcohol nucleophiles in the presence of amines. While the acetyl azolium cation did not react with alcohols, an ester-selective reaction was induced by addition of base, by intermediate formation of the acetyl azolium enolate.

Pursuing Experimental Reproducibility: An Efficient Protocol for the Preparation of Cerebrospinal Fluid Samples for NMR-based Metabolomics and Analysis of Sample Degradation.

NMR-based metabolomics investigations of human biofluids offer great potential to uncover new biomarkers. In contrast to protocols for sample collection and biobanking, procedures for sample preparation prior to NMR measurements are still heterogeneous, thus compromising the comparability of the resulting data. Herein, we present results of an investigation of the handling of cerebrospinal fluid (CSF) samples for NMR metabolomics research. Origins of commonly observed problems when conducting NMR experiments on this type of sample are addressed, and suitable experimental conditions in terms of sample preparation and pH control are discussed. Sample stability was assessed by monitoring the degradation of CSF samples by NMR, hereby identifying metabolite candidates, which are potentially affected by sample storage. A protocol was devised yielding consistent spectroscopic data as well as achieving overall sample stability for robust analysis. We present easy to adopt standard operating procedures with the aim to establish a shared sample handling strategy that facilitates and promotes inter-laboratory comparison, and the analysis of sample degradation provides new insights into sample stability.

Technical Synthesis of 1,5,9-Cyclododecatriene Revisited: Surprising Byproducts from a Venerable Industrial Process.

The synthesis of 1,5,9-cyclododecatriene by selective trimerization of butadiene catalyzed by TiCl4 and ethylaluminum sesquichloride has been commercially used since 1965. Although thoroughly investigated, not all details of the mechanism are completely understood. The recent development of a new process to produce cyclododecanone involving oxidation of 1,5,9-cyclododecatriene with N2O has led to the serendipitous discovery of an array of hitherto unknown byproducts, formed in the trimerization of butadiene: eleven tricyclic C12H20 and one tetracyclic C12H18 hydrocarbons, three of which had never been described before. The identification of these byproducts became possible by using a combination of chemical enrichment, high-resolution distillation, 13C-2D-INADEQUATE NMR, and comparison with ab initio calculated spectra, thus demonstrating the power of these combined techniques. The identification of these byproducts contributes to a better understanding of the mechanism of this centrally important reaction.

Breslow Intermediates from Aromatic N-Heterocyclic Carbenes (Benzimidazolin-2-ylidenes, Thiazolin-2-ylidenes).

We report the first generation and characterization of elusive Breslow intermediates derived from aromatic N-heterocyclic carbenes (NHCs), namely benzimidazolin-2-ylidenes (NMR, X-ray analysis) and thiazolin-2-ylidenes (NMR). In the former case, the diamino enols were generated by reaction of the free N,N-bis(2,6-diisopropylphenyl)- and N,N-bis(mesityl)-substituted benzimidazolin-2-ylidenes with aldehydes while the dimer of 3,4,5-trimethylthiazolin-2-ylidene served as the starting material in the latter case. The unambiguous NMR identification of the first thiazolin-2-ylidene-based Breslow intermediate rests on double 13 C labeling of both the NHC and the aldehyde component. The acyl anion reactivity was confirmed by benzoin formation with excess aldehyde.

NMReDATA, a standard to report the NMR assignment and parameters of organic compounds.

Even though NMR has found countless applications in the field of small molecule characterization, there is no standard file format available for the NMR data relevant to structure characterization of small molecules. A new format is therefore introduced to associate the NMR parameters extracted from 1D and 2D spectra of organic compounds to the proposed chemical structure. These NMR parameters, which we shall call NMReDATA (for nuclear magnetic resonance extracted data), include chemical shift values, signal integrals, intensities, multiplicities, scalar coupling constants, lists of 2D correlations, relaxation times, and diffusion rates. The file format is an extension of the existing Structure Data Format, which is compatible with the commonly used MOL format. The association of an NMReDATA file with the raw and spectral data from which it originates constitutes an NMR record. This format is easily readable by humans and computers and provides a simple and efficient way for disseminating results of structural chemistry investigations, allowing automatic verification of published results, and for assisting the constitution of highly needed open-source structural databases.

Kirmse-Doyle- and Stevens-Type Rearrangements of Glutarate-Derived Oxonium Ylides.

A novel chemoenzymatic synthetic cascade enables the preparation of densely decorated tetrahydrofuran building blocks. Here, the lipase-catalyzed desymmetrization of 3-alkoxyglutarates renders highly enantioenriched carboxylic acid intermediates, whose subsequent activation and oxonium ylide rearrangement by means of rhodium or copper complexes furnishes functionalized O-heterocycles with excellent diastereoselectivity. The two-step protocol offers a streamlined and flexible synthesis of tetrahydrofuranones bearing different benzylic, allylic or allenylic side chains with full control over multiple stereogenic centers.

Exploring the Role of Solvent on Carbohydrate-Aryl Interactions by Diffusion NMR-Based Studies.

Carbohydrate-protein interactions play an important role in many molecular recognition processes. An exquisite combination of multiple factors favors the interaction of the receptor with one specific type of sugar, whereas others are excluded. Stacking CH-aromatic interactions within the binding site provide a relevant contribution to the stabilization of the resulting sugar-protein complex. Being experimentally difficult to detect and analyze, the key CH-π interaction features have been very often dissected using a variety of techniques and simple model systems. In the present work, diffusion NMR spectroscopy has been employed to separate the components of sugar mixtures in different solvents on the basis of their differential ability to interact through CH-π interactions with one particular aromatic cosolute in solution. The experimental data show that the properties of the solvent did also influence the diffusion behavior of the sugars present in the mixture, inhibiting or improving their separation. Overall, the results showed that, for the considered monosaccharide derivatives, their diffusion coefficient values and, consequently, their apparent molecular sizes and/or shapes depend on the balance between solute/cosolute as well as solute/solvent interactions. Thus, in certain media and in the presence of the aromatic cosolute, the studied saccharides that are more suited to display CH-π interactions exhibited a lower diffusion coefficient than the noncomplexing sugars in the mixture. However, when dissolved in another medium, the interaction with the solvent strongly competes with that of the aromatic cosolute.

1,4-Bis-Dipp/Mes-1,2,4-Triazolylidenes: Carbene Catalysts That Efficiently Overcome Steric Hindrance in the Redox Esterification of α- and ß-Substituted α,ß-Enals.

As reported by Scheidt and Bode in 2005, sterically nonencumbered α,ß-enals are readily converted to saturated esters in the presence of alcohols and N-heterocyclic carbene catalysts, e.g., benzimidazolylidenes or triazolylidenes. However, substituents at the α- or ß-position of the α,ß-enal substrate are typically not tolerated, thus severely limiting the substrate spectrum. On the basis of our earlier mechanistic studies, a set of N-Mes- or N-Dipp-substituted 1,2,4-triazolium salts were synthesized and evaluated as (pre)catalysts in the redox esterification of various α- or ß-substituted enals. In particular the 1,4-bis-Mes/Dipp-1,2,4-triazolylidenes overcome the above limitations and efficiently catalyze the redox esterification of a whole series of α/ß-substituted enals hitherto not amenable to NHC-catalyzed transformations. The synthetic value of 1,4-bis-Mes/Dipp-1,2,4-triazolylidenes is further demonstrated by the one-step bicyclization of 10-oxocitral to (racemic) nepetalactone in diastereomerically pure form.

Facilitating quality control for spectra assignments of small organic molecules: nmrshiftdb2--a free in-house NMR database with integrated LIMS for academic service laboratories.

nmrshiftdb2 supports with its laboratory information management system the integration of an electronic lab administration and management into academic NMR facilities. Also, it offers the setup of a local database, while full access to nmrshiftdb2's World Wide Web database is granted. This freely available system allows on the one hand the submission of orders for measurement, transfers recorded data automatically or manually, and enables download of spectra via web interface, as well as the integrated access to prediction, search, and assignment tools of the NMR database for lab users. On the other hand, for the staff and lab administration, flow of all orders can be supervised; administrative tools also include user and hardware management, a statistic functionality for accounting purposes, and a 'QuickCheck' function for assignment control, to facilitate quality control of assignments submitted to the (local) database. Laboratory information management system and database are based on a web interface as front end and are therefore independent of the operating system in use.

Studies on the collision-induced dissociation of adipoR agonists after electrospray ionization and their implementation in sports drug testing.

AdipoR agonists are small, orally active molecules capable of mimicking the protein adiponectin, which represents an adipokine with antidiabetic and antiatherogenic effects. Two adiponectin receptors were reported in the literature referred to as adipoR1 and adipoR2. Activation of these receptors stimulates mitochondrial biogenesis and results in an improved oxidative metabolism (via adipoR1) and increased insulin sensitivity (via adipoR2). Hence, adipoR agonists are potentially performance enhancing substances and targets of proactive and preventive anti-doping measures. In this study, two adipoR agonists termed AdipoRon and 112254 as well as two isotopically labeled internal standards (ISTDs) were synthesized in three-step reactions. The products were fully characterized by nuclear magnetic resonance spectroscopy (NMR), mass spectrometry (MS) and density functional theory (DFT) computation. Collision-induced dissociation pathways following electrospray ionization were suggested based on the determined elemental compositions of product ions, comparison to product ions derived from labeled analogs (ISTDs), H/D-exchange experiments and the results of DFT calculations. The most abundant product ions were found at m/z 174, tentatively assigned to protonated 1-benzyl-1,2,3,4-tetrahydropyridine for AdipoRon, and m/z 207, suggested as protonated 1-(4-methoxybenzyl)piperazine, for 112254. Notably, the loss of the heterocyclic ring (i.e. piperazine and piperidine, respectively) in a supposedly intramolecular elimination reaction was observed in both cases. A qualitative determination of both AdipoR agonists in human plasma was established and fully validated for doping control purposes. Validation items such as recovery (86-89%), specificity, linearity, lower limit of detection (1 ng/ml), intraday (3-18%) and interday (5-16%) precision as well as ion suppression or enhancement were determined. Based on these findings adipoR agonists can be implemented in sports drug testing procedures.

Carbene catalyzed umpolung of α,ß-enals: a reactivity study of diamino dienols vs. azolium enolates, and the characterization of advanced reaction intermediates.

Since their discovery by Bode and Glorius in 2004, N-heterocyclic carbene catalyzed conjugate umpolung reactions of α,ß-enals have been postulated to involve the formation of diamino dienols ("homoenolates") and/or azolium enolates ("enolates"), typically followed by addition to electrophiles, e.g. Michael-acceptors. In this article, we provide evidence, for the first time, for the postulated individual and specific reactivity patterns of diamino dienols (γ-C-C-bond formation) vs. azolium enolates (ß-C-C-bond formation). Our study is based on the pre-formation of well defined diamino dienols and azolium enolates, and the in situ NMR monitoring of their reactivities towards enone electrophiles. Additionally, reaction intermediates were isolated and characterized, inter alia by X-ray crystallography.