Mass spectrometric characterization of the selective androgen receptor modulator (SARM) YK-11 for doping control purposes.

RATIONALE: Selective androgen receptor modulators (SARMs) represent an emerging class of therapeutics targeting inter alia conditions referred to as cachexia and sarcopenia. Due to their anabolic properties, the use of SARMs is prohibited in sports as regulated by the World Anti-Doping Agency (WADA), and doping control laboratories test for these anabolic agents in blood and urine. In order to accomplish and maintain comprehensive test methods, the characterization of new drug candidates is critical for efficient sports drug testing. Hence, in the present study the mass spectrometric properties of the SARM YK-11 were investigated. METHODS: YK-11 was synthesized according to literature data and three different stable-isotope-labeled analogs were prepared to support the mass spectrometric studies. Using high-resolution/high-accuracy mass spectrometry following electrospray ionization as well as electron ionization, the dissociation pathways of YK-11 were investigated, and characteristic features of its (product ion) mass spectra were elucidated. These studies were flanked by density functional theory (DFT) computation providing information on proton affinities of selected functional groups of the analyte. RESULTS AND CONCLUSIONS: The steroidal SARM YK-11 was found to readily protonate under ESI conditions followed by substantial in-source dissociation processes eliminating methanol, acetic acid methyl ester, and/or ketene. DFT computation yielded energetically favored structures of the protonated species resulting from the aforementioned elimination processes particularly following protonation of the steroidal D-ring substituent. Underlying dissociation pathways were suggested, supported by stable-isotope labeling of the analyte, and diagnostic product ions for the steroidal nucleus and the D-ring substituent were identified. Further, trimethylsilylated YK-11 and its deuterated analogs were subjected to electron ionization high-resolution/high-accuracy mass spectrometry, complementing the dataset characterizing this new SARM. The obtained fragment ions resulted primarily from A/B- and C/D-ring structures of the steroidal nucleus, thus supporting future studies e.g. concerning metabolic pathways of the substance. Copyright © 2017 John Wiley & Sons, Ltd.

The MoSGrid Science Gateway - A Complete Solution for Molecular Simulations.

The MoSGrid portal offers an approach to carry out high-quality molecular simulations on distributed compute infrastructures to scientists with all kinds of background and experience levels. A user-friendly Web interface guarantees the ease-of-use of modern chemical simulation applications well established in the field. The usage of well-defined workflows annotated with metadata largely improves the reproducibility of simulations in the sense of good lab practice. The MoSGrid science gateway supports applications in the domains quantum chemistry (QC), molecular dynamics (MD), and docking. This paper presents the open-source MoSGrid architecture as well as lessons learned from its design.

On the sigma,pi-energy separation of the aromatic stabilization energy of cyclobutadiene.

The separation of the aromatic stabilization energy (ASE) of cyclobutadiene (CBD) into a sigma- and a pi-component is reinvestigated. Eight different reactions are considered for this purpose. As expected, the total destabilization energies that result from these reactions depend only on the reference compound and not on the reaction itself. The heats of formation that can be obtained from the calculated reaction energies are in excellent agreement with the recently determined experimental value of 102.3 +/- 3.8 kcal mol(-1) (A. Fattahi, L. Liz, Z. Thian and S. R. Kass, Angew. Chem., Int. Ed., 2006, 45, 4984-4988). Evaluation of the angular strain in CBD from a newly considered reaction confirms earlier estimates and yields a strain energy of 34 +/- 3 kcal mol(-1). If referred to s-cis-butadiene this leads to an ASE of -37 +/- 4 kcal mol(-1) in close agreement with estimates provided by A. Fattahi, L. Liz, Z. Thian and S. R. Kass, Angew. Chem., Int. Ed., 2006, 45, 4984-4988; and by K. B. Wiberg, Chem. Rev., 2001, 101, 1317-1332. With s-trans-butadiene as reference we obtain -42 +/- 4 kcal mol(-1). This value is 8 to 10 kcal mol(-1) less destabilizing than recent estimates of A. A. Deniz, K. S. Peters and G. J. Snyder, Science, 1999, 286, 1119-1112; and Kovacevic, D. Baric, Z. B. Maksic, T. Müller, J. Phys. Chem. A, 2004, 108, 9126-9133. Attempts to separate ASE(CBD) and E(strain)(CBD) into a sigma- and a pi-component do not lead to useful results. In contrast to ASE and E(strain) themselves, the sigma- and pi-components depend strongly on the applied reaction. A detailed analysis reveals that it is not possible to associate these components with only one of the molecules that participate in the reaction. The components depend on all of these molecules and therefore on the underlying reaction. Generally, components that result from a formal sigma,pi-energy separation of aromatic stabilization energies or strain energies cannot be considered as the sigma- and pi-components of these energies.

Sigma-pi energy separation in homodesmotic reactions.

A well-established quantity for specifying the aromaticity or antiaromaticity of cyclic conjugated molecules is the so-called aromatic stabilization energy (ASE), which can be derived-either experimentally or theoretically-from appropriate homodesmotic reactions. To gain further insight into the origin of aromaticity, several schemes have been devised to partition ASE into nuclear and electronic as well as sigma and pi contributions, some of which have resulted in contradictory statements about the driving force of aromatic stabilization. Currently, these contradictions have not been resolved and have resulted in a confusing distinction between two different types of aromaticity: extrinsic and intrinsic aromaticity. By investigating different homodesmotic reactions we show that, in contrast to ASE itself, the individual contributions that enter the ASE can strongly depend on the type of reaction. Caution is therefore advised if conclusions or physical interpretations are derived from the individual components. The contradictions result from the fact that some reactions suffer from an imbalance in the number of interaction terms at the two sides of the reaction equation. The concept of isointeractional reactions is introduced and results in the elimination of the imbalance. For these reactions, the contradictions disappear and the distinction between intrinsic and extrinsic aromaticity becomes unnecessary. As far as the sigma-pi partitioning is concerned, several schemes proposed in the literature are compared. Contradictory results are obtained depending on the partitioning scheme and reaction used. In this context, it is demonstrated that for the partitioning of the electron-electron interaction, the scheme introduced by Jug and Köster is the one that is most theoretically grounded.

Structural dynamics of the DnaK-peptide complex.

The molecular chaperone DnaK recognizes and binds substrate proteins via a stretch of seven amino acid residues that is usually only exposed in unfolded proteins. The binding kinetics are regulated by the nucleotide state of DnaK, which alternates between DnaK.ATP (fast exchange) and DnaK.ADP (slow exchange). These two forms cycle with a rate mainly determined by the ATPase activity of DnaK and nucleotide exchange. The different substrate binding properties of DnaK are mainly attributed to changes of the position and mobility of a helical region in the C-terminal peptide-binding domain, the so-called LID. It closes the peptide-binding pocket and thus makes peptide binding less dynamic in the ADP-bound state, but does not (strongly) interact with peptides directly. Here, we address the question if nucleotide-dependent structural changes may be observed in the peptide-binding region that could also be connected to peptide binding kinetics and more importantly could induce structural changes in peptide stretches using the energy available from ATP hydrolysis. Model peptides containing two cysteine residues at varying positions were derived from the structurally well-documented peptide NRLLLTG and labelled with electron spin sensitive probes. Measurements of distances and mobilities of these spin labels by electron paramagnetic resonance spectroscopy (EPR) of free peptides or peptides bound to the ATP and ADP-state of DnaK, respectively, showed no significant changes of mobility nor distance of the two labels. This indicates that no structural changes that could be sensed by the probes at the position of central leucine residues located in the center of the binding region occur due to different nucleotide states. We conclude from these studies that the ATPase activity of DnaK is not connected to structural changes of the peptide-binding pocket but rather only has an effect on the LID domain or other further remote residues.