Rapid methods for the separation of natural mixtures of beauverolides, cholesterol acyltransferase inhibitors, isolated from the fungus Isaria fumosorosea.

Beauverolides (beauveriolides) are abundant, biologically active cyclodepsipeptides produced by many entomopathogenic fungi, including those that are used as biopesticides. Beauverolides act as cholesterol acyltransferase inhibitors in humans; thus, their mode of action has been the subject of pharmacological and clinical research. The cost-effective analytical methods are needed for fast, routine laboratory analysis of beauverolides. We isolated beauverolides from the fungal strain Isaria fumosorosea PFR 97-Apopka and opened the rings of the isolated beauverolides using a pyridine alkaline medium. We separated fractions of cyclic and linearized beauverolides by thin-layer chromatography, and found the chloroform-acetate (9:1, v/v) and chloroform-acetonitrile-acetate (8:1:1, v/v/v) mobile phases, respectively, to be the most efficient. We examined all the fractions by liquid chromatography-mass spectrometry using ion trap and Orbitrap high resolution mass spectrometry. For rapid screening of the contents of cyclic, and, particularly, linearized beauverolides, we developed a novel analytical method that consisted of using capillary electrophoresis coupled with contactless conductivity detection. Furthermore, we improved the separation of the peptides by applying capillary micellar electrokinetic chromatography with the N-cyclohexyl-2-aminoethanesulfonic acid:SDS:NaOH buffer, pH 9.8 as the background electrolyte. The described novel methods allow fast and cost-effective separation of chemically related groups of beauverolides.

Dihydrogen Bond in the Aminoborane Complex of a Nicergoline Intermediate.

An aminoborane side product from the nicergoline manufacture process was identified by single-crystal X-ray diffraction. As boranes of pharmaceutical molecules are quite rare, the binding potential of the BH3 group was investigated and compared with similar compounds using Cambridge Structural Database (CSD). Surprisingly, the packing was stabilized by a dihydrogen bond, which triggered a false alert for too-short contact of hydrogen atoms in IUCR checkCIF. As the dihydrogen bond concept is not widely known, such an alert might mislead crystallographers to force -CH3 optimal geometry to -BH3 groups. The B-H distances equal to or less than 1.0 Å (17% of the CSD structures) are substantially biased when analyzing the structures of aminoborane complexes in CSD. To conduct proper searching, B-H bond length normalization should be applied in the CSD search.

Structure and absolute configuration of natural fungal product beauveriolide I, isolated from Cordyceps javanica, determined by 3D electron diffraction.

Beauveriolides, including the main beauveriolide I {systematic name: (3R,6S,9S,13S)-9-benzyl-13-[(2S)-hexan-2-yl]-6-methyl-3-(2-methylpropyl)-1-oxa-4,7,10-triazacyclotridecane-2,5,8,11-tetrone, C27H41N3O5}, are a series of cyclodepsipeptides that have shown promising results in the treatment of Alzheimer's disease and in the prevention of foam cell formation in atherosclerosis. Their crystal structure studies have been difficult due to their tiny crystal size and fibre-like morphology, until now. Recent developments in 3D electron diffraction methodology have made it possible to accurately study the crystal structures of submicron crystals by overcoming the problems of beam sensitivity and dynamical scattering. In this study, the absolute structure of beauveriolide I was determined by 3D electron diffraction. The cyclodepsipeptide crystallizes in the space group I2 with lattice parameters a = 40.2744 (4), b = 5.0976 (5), c = 27.698 (4) Šand ß = 105.729 (6)°. After dynamical refinement, its absolute structure was determined by comparing the R factors and calculating the z-scores of the two possible enantiomorphs of beauveriolide I.

In-Silico Aided Screening and Characterization Results in Stability Enhanced Novel Roxadustat Co-Crystal.

Roxadustat (RXD) is an approved drug substances for the treatment of renal anemia. It has poor aqueous solubility and photochemical stability. This study employs a comprehensive approach to enhance the stability and physicochemical properties RXD through coformer selection and characterization. The investigation integrates delta pKa analysis, molecular complementary assessment, molecular electrostatic potential surface analysis, and machine learning techniques to predict potential co-crystal formation and binding interactions between drug molecules and coformers. The co-crystal screening which lead to in a novel RXD-nicotinamide co-crystal (RXD-NA). Experimental characterization underscores the physical and chemical stability of the co-crystals. To elucidate the supramolecular synthons and understand the intermolecular interactions in the RXD-NA co-crystal, Hirshfeld surfaces analysis, quantum theory of atoms in molecules (QTAIM) analysis and non-covalent interaction (NCI) analysis were performed. Computational analysis of photo-isomer formation aligns with experimental observations, further enhancing our understanding of RXD-coformer interactions. RXD-NA co-crystal was found photo-chemically stable as compared to free base API drug substance. This integrated methodology provides a systematic framework for informed co-crystal design, holding promise for optimizing RXD formulations based on molecular interactions and stability considerations. Consequently, this study contributes valuable insights to the field of rational drug design and formulation optimization.

Characterization of pseudacyclins A-E, a suite of cyclic peptides produced by Pseudallescheria boydii.

Pseudallescheria boydii sensu lato is an emerging fungal pathogen causing fatal infections in both immunocompromised and immunocompetent hosts. In this work, two P. boydii isolates (human and animal origin) have been identified as being producers of cyclic peptides. Five putative nonribosomal peptides with a unique structure, which have been named pseudacyclins, were characterized by nuclear magnetic resonance spectroscopy and mass spectrometry. The most abundant representative of the pseudacyclins was quantified also on fungal spores. The presence of these peptides on inhaled fungal spores creates the possibility for exploitation of pseudacyclins as early indicators of fungal infections caused by Pseudallescheria species.

Cyclosporins from Mycelium sterilae MS 2929.

The structures of two new cyclosporins were elucidated by NMR and MS methods as cyclo[-MeBmt(1)-Abu(2)-Sar(3)-MeLeu(4)-Val(5)-MeLeu(6)-Ala(7)-d-Ala(8)-MeLeu(9)-MeNva(10)-MeVal(11)-] and cyclo[-MeBmt(1)-Abu(2)-Sar(3)-MeLeu(4)-Abu(5)-MeLeu(6)-Ala(7)-d-Ala(8)-MeLeu(9)-MeLeu(10)-MeVal(11)-].

Methyl-ergometrine maleate from synchrotron powder diffraction data.

The title compound {systematic name: 9,10-didehydro-N-[1-(hydroxy-meth-yl)prop-yl]-d-lysergamide maleate}, C(20)H(26)N(3)O(2) (+)·C(4)H(3)O(4) (-), contains a large rigid ergolene group. This group consists of an indole plane connected to a six-membered carbon ring adopting an envelope conformation and N-methyl-tetra-hydro-pyridine where the methyl group is in an equatorial position. In the crystal, inter-molecular N-H⋯O, O-H⋯N and O-H⋯O hydrogen bonds form an extensive three-dimensional hydrogen-bonding network, which holds the cations and anions together.

Capecitabine from X-ray powder synchrotron data.

In the title compound [systematic name 5-de-oxy-5-fluoro-N-(pent-yloxycarbon-yl)cytidine], C(15)H(22)FN(3)O(6), the pentyl chain is disordered over two positions with refined occupancies of 0.53 (5) and 0.47 (5). The furan ring assumes an envelope conformation. In the crystal, inter-molecular N-H⋯O hydrogen bonds link the mol-ecules into chains propagating along the b axis. The crystal packing exhibits electrostatic inter-actions between the 5-fluoro-pyrimidin-2(1H)-one fragments of neighbouring mol-ecules as indicated by short O⋯C [2.875 (3) and 2.961 (3) Å] and F⋯C [2.886 (3) Å] contacts.

Metabolism of obeticholic acid in brown bullhead (Ameiurus nebulosus).

Analysis of brown bullhead (Ameiurus nebulosus) bile by ultra performance liquid chromatography high-resolution mass spectrometry (UPLC/HRMS) revealed a series of bile acids similar to those found in humans. Accordingly, we chose this fish as a model organism to examine the metabolism of obeticholic acid, a bile acid used to treat a number of human liver diseases and the one that has the potential to occur as an environmental contaminant. The taurine and glycine conjugates of obeticholic acid and keto-obeticholic acid were identified, as well as the D-cysteinolic acid conjugate of obeticholic acid, likely a metabolite specific to fish. In addition, metabolites of obeticholic acid (sulphate and glucuronide) and several hydroxy-obeticholic acid derivatives were found, representing typical pathways of primary and secondary steroid metabolism. Brown bullhead exposed to obeticholic acid at a dose of 100 mg/kg gave no overt signs of distress or toxicity.

Epoxidation is the preferred pathway of first-stage metabolism of abiraterone acetate in brown bullhead (Ameiurus nebulosus).

Twenty juvenile individuals of brown bullhead (Ameiurus nebulosus), average weight 77 g, were fed by abiraterone acetate prodrug dissolved in olive oil via gastric probe. Dose applied was 3 mg/10 g fish weight. After feeding, they were let out into aquarium and kept there for 3 days. Aquarium water containing excreted metabolites was extracted, and sample was purified and finally analyzed by means of HPLC/MS. Expected both primary (products of hydroxylation) and secondary (products of glucuronidation and sulfatation) metabolites of abiraterone acetate were identified. The NMR measurement of one of the prevailing metabolites presumed to be one of possible hydroxy-abiraterones discovered that it is not hydroxy-abiraterone but abiraterone 16,17-epoxide. Closer analysis of MS2 and MS3 spectra revealed that one of presumed hydroxy-abiraterone acetates and also some secondary metabolites are probably 16,17-epoxides.

Calcium-induced generation of reactive oxygen species in brain mitochondria is mediated by permeability transition.

Mitochondrial uptake of calcium in excitotoxicity is associated with subsequent increase in reactive oxygen species (ROS) generation and delayed cellular calcium deregulation in ischemic and neurodegenerative insults. The mechanisms linking mitochondrial calcium uptake and ROS production remain unknown but activation of the mitochondrial permeability transition (mPT) may be one such mechanism. In the present study, calcium increased ROS generation in isolated rodent brain and human liver mitochondria undergoing mPT despite an associated loss of membrane potential, NADH and respiration. Unspecific permeabilization of the inner mitochondrial membrane by alamethicin likewise increased ROS independently of calcium, and the ROS increase was further potentiated if NAD(H) was added to the system. Importantly, calcium per se did not induce a ROS increase unless mPT was triggered. Twenty-one cyclosporin A analogs were evaluated for inhibition of calcium-induced ROS and their efficacy clearly paralleled their potency of inhibiting mPT-mediated mitochondrial swelling. We conclude that while intact respiring mitochondria possess powerful antioxidant capability, mPT induces a dysregulated oxidative state with loss of GSH- and NADPH-dependent ROS detoxification. We propose that mPT is a significant cause of pathological ROS generation in excitotoxic cell death.

Absolute Configuration Determination of a Taxol Precursor Based on Raman Optical Activity Spectra.

Enantiomeric purity of drugs is essential for their biological activity. In the present study, we investigate the performance of Raman optical activity (ROA) spectroscopy in distinguishing four possible stereoisomers of the synthetic precursor used for the production of taxol from baccatin III. Taxol is one of the best-selling medicaments used in the treatment of ovarian, lung, and breast cancers and Kaposi's sarcoma. In a low yield, it may be isolated from the bark of the Pacific yew tree (Taxus brevifolia); however, its industrial production is largely dependent on the precursor. It is shown that for reliable comparison of the experimental and computed Raman and ROA intensities a large number of conformers had to be averaged, to properly account for molecular flexibility in solution. In addition, if combined with the density functional theory computations, ROA spectra provide convenient and economic means of absolute configuration determination.

Nonribosomal cyclic peptides: specific markers of fungal infections.

Some cyclic peptides and depsipeptides are synthesized in microorganisms by large multienzymes called nonribosomal peptide synthetases. The structures of peptide products originating in this way are complex and diverse and are microorganism-specific. This work proposes the use of fungal cyclic peptides and depsipeptides as extremely specific markers of fungal infections. Since a reliable molecular tool for diagnosing fungal infections at an early stage is still missing, we present mass spectrometry as a new, modern, broadband (with respect to fungal strain) and specific tool for clinical mycologists. More than 40 different fungal species can be rapidly characterized according to specific families of cyclic peptides, and in some cases, a particular fungal strain can be identified on the basis of its cyclopeptide profile. This paper is also aimed at initiating discussion on the biological role of these secondary metabolites, especially of those synthesized by medically important strains. Proven cytotoxic, anti-inflammatory or immunosuppressive activities of some cyclic peptides indicate that these molecules may contribute to the synergistic array of fungal virulence factors and support microbial invasion during fungal infection. In addition to an overview on recent mass spectrometric protocols for cyclic peptide sequencing, the structures of new peptides from Paecilomyces and Pseudallescheria are presented.

Erratum: Capecitabine from X-ray powder synchrotron data. Corrigendum.

[This corrects the article DOI: 10.1107/S1600536809017905.].

Role of amino acid N-methylation in cyclosporins on ring opening and fragmentation mechanisms during collisionally induced dissociation in an ion trap.

The product ion mass spectra (collisionally induced dissociation mass spectra) of 12 different cyclosporins modified at every N-methylated amino acid residue with respect to cyclosporin A were compared and the effect of N-demethylation on ring opening mechanisms was evaluated. The four preferential protonation sites were identified in [MeBmt(1)]-cyclosporins. Three sites represented the N-methylated nitrogens of Sar(3), MeLeu(6) and MeLeu(9), while the remaining one represented the lactone group formed by the intramolecular N,O-acyl shift. Selective N-demethylation resulted either in the deletion of the entire fragment ion series or its substantial attenuation. The structures of three new natural cyclosporins in the study were supported by NMR data.

Extraribosomal cyclic tetradepsipeptides beauverolides: profiling and modeling the fragmentation pathways.

Profiling of cyclic tetradepsipeptides beauverolides was tested as a chemotaxonomic tool for fungal strain identification/discrimination. Two new tetradepsipeptides, beauverolides Q and R, were characterized by tandem mass spectrometry. Specific elimination of 113 atomic mass units from both protonated and sodiated molecules of beauverolides is ubiquitous for all 12 most dominant congeners evaluated in this profiling study. Reconstruction of the total ion chromatogram, according to this neutral fragment release, was used for data filtering and selectivity enhancement. Selective ring opening and fragment ion formation of beauverolide I are discussed in detail utilizing high-level theoretical modeling of the fragmentation pathways.

Profiling of cyclic hexadepsipeptides roseotoxins synthesized in vitro and in vivo: a combined tandem mass spectrometry and quantum chemical study.

High-performance liquid chromatography and tandem mass spectrometry (HPLC/MS/MS) was used for the detection of cyclic hexadepsipeptides roseotoxins produced by Trichothecium roseum. Roseotoxins were found in both submerged standard cultivation on CzapekDox medium and in vivo cultivation extract obtained from an apple. Roseotoxin chromatographic profiles from these two experiments were compared. Product-ion collision-induced dissociation (CID) spectra obtained on an ion trap (electrospray ionisation, ESI) were used for the identification of natural roseotoxins A, B, C and of minor destruxins A and B. The dissociation behavior of roseotoxins is discussed in terms of a fragmentation scheme proposed for describing the dissociation pathways of cyclic peptides. This scheme involves opening of the cyclopeptide ring via formation of oxazolone derivatives and fragmentation of the resulting linear species, which have a free N-terminus and an oxazolone ring at the C-terminus. Some aspects of this fragmentation scheme are underlined by modeling the dissociation channels of roseotoxin A using quantum chemical calculations. The structures of roseotoxin A and destruxin B were verified by nuclear magnetic resonance (NMR) spectroscopy. Structures of three new minor natural roseotoxins [Val(4)]RosA, [MeLxx(4)]RosA and [MeLxx(4)]RosB were deduced by ion cyclotron resonance Fourier transform mass spectrometry (ICR-FT-MS) and ion trap tandem mass spectrometry by examining the pre-separated roseotoxin fraction.

Thiosugars--derivatization agents for chiral resolution of homoleucines.

o-Phthaldialdehyde was used in combination with a series of six thiosugars for the pre-column chiral derivatization of selected primary amino acids. The diastereomers were resolved on a conventional reversed-phase column and with fluorescence detection. A surprisingly effective resolution of 1-isoindolyl-(1-thioglycosides) derived from 1-thio-beta-L-fucose was observed.

Direct evidence of plant-pathogenic activity of fungal metabolites of Trichothecium roseum on apple.

Apples were exposed to various concentrations of roseotoxins - metabolites of Trichothecium roseum and kinetic fluorescence imaging was used to detect the area influenced by the phytotoxin. Contrast was quantified within these images between the areas exposed to roseotoxins and the untreated areas. It was proved that roseotoxin B is able to penetrate apple peel and produce chlorotic lesions. Activity of roseotoxin B is similar as the activity of destruxins, host specific phytotoxins of Alternaria brassicae parasitic on canola.

Pharmacological profile of 4-(2',4'-difluorobiphenyl-4-yl)-2-methylbutyric acid (deoxoflobufen).

4-(2',4'- Difluorobiphenyl-4-yl)-2-methylbutyric acid (deoxoflobufen, VUFB 19053, CAS 847475-35-8) has been developed as a new omega-biphenyl-alkanoic acid and studied in comparison with the racemic form of 4-(2',4'-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acid (flobufen, CAS 112344-52-2). The compounds were tested in a series of models including acute inflammation induced by carrageenan, adjuvant arthritis, in vitro inhibition of the leuktotriene B4 (LTB4) production, reaction of the graft versus the host (GVHR), production of specific antibodies against ovalbumin, peritoneal exudate formation induced by thioglycollate and phagocytosis of thioglycollate-stimulated mouse peritoneal macrophages. Deoxoflobufen exhibited strong anti-inflammatory, antiarthritic and immunomodulatory effects in most of the performed tests. Anti-inflammatory and antiarthritic effects are fully comparable with those of flobufen, however, the compound is less toxic and has apparently stronger immunomodulating effects.