New chemical structures and liver-protective activity of the diterpenoids from Callicarpa rubella.

Callicarpa rubella is a characteristic folk herb in the genus Callicarpa, and has abundant ethnobotanical usage as indigenous medicine in Lingnan area of P. R. China. However, the phytochemical and pharmacological properties of C. rubella was rarely investigated. Now, three new diterpenoids, named rubellapene A-C (1-3), along with five known analogues (4-8), were isolated from C. rubella. Their structures were determined by spectroscopic methods, quantum chemical electronic circular dichroism calculations and single-crystal X-ray diffraction analysis. Notably, the norditerpenoids C18 of clerodane type (rubellapene B) was rarely found in the genus Callicarpa. The liver protective effects of all of the isolates (1-8) were evaluated by the changes of cell viability and transaminase content of AST and ALT in H2O2-induced BRL cells. Compound 1, 3-8 exhibited that potent liver protective effects at different levels.

Isolation, crystal structure, DFT calculation and molecular docking of uncinatine-A isolated from Delphinium uncinatum.

The main objective of our present research work was to explore molecular insight for potentially active new acetylcholinesterase inhibitor from the aerial parts of Delphinium uncinatum. New norditerpenoid alkaloids, uncinatine-A, was isolated from the basic alkaloidal fraction of D. uncinatum, based on bioactivity guided isolation. The structure of uncinatine-A was determined through latest spectroscopic techniques including single X-Ray diffraction technique. The structural data and electronic properties of uncinatine-A was also calculated by Density Functional Theory (DFT) using B3LYP/6-31þ G (p) basis set. The isolated natural product was evaluated for their acetyl cholinesterase inhibitory potential in dose dependent protocol (62.5-1000 µg/mL), followed by molecular docking studies. Significant competitive type inhibition activity (IC50 = 207.73 ± 0.3) was shown by isolated natural norditerpenoid against cholinesterase targets in comparison with standard drugs available in the market such as galanthamine. The molecular docking results showed that isolated natural product was well accommodated by AChE in the active site with docking scores -11.0326. This is the first report indicating uncinatine-A as a potent acetylcholinesterase inhibitor and can be used as a target drug in cerebral dementia and Alzheimer diseases.

The molybdenum storage protein forms and deposits distinct polynuclear tungsten oxygen aggregates.

Some N2-fixing bacteria store Mo to maintain the formation of the vital FeMo-cofactor dependent nitrogenase under Mo depleting conditions. The Mo storage protein (MoSto), developed for this purpose, has the unique capability to compactly deposit molybdate as polyoxometalate (POM) clusters in a (αß)3 hexameric cage; the same occurs with the physicochemically related tungstate. To explore the structural diversity of W-based POM clusters, MoSto loaded under different conditions with tungstate and two site-specifically modified MoSto variants were structurally characterized by X-ray crystallography or single-particle cryo-EM. The MoSto cage contains five major locations for POM clusters occupied among others by heptanuclear, Keggin ion and even Dawson-like species also found in bulk solvent under defined conditions. We found both lacunary derivatives of these archetypical POM clusters with missing WOx units at positions exposed to bulk solvent and expanded derivatives with additional WOx units next to protecting polypeptide segments or other POM clusters. The cryo-EM map, unexpectedly, reveals a POM cluster in the cage center anchored to the wall by a WOx linker. Interestingly, distinct POM cluster structures can originate from identical, highly occupied core fragments of three to seven WOx units that partly correspond to those found in MoSto loaded with molybdate. These core fragments are firmly bound to the complementary protein template in contrast to the more variable, less occupied residual parts of the visible POM clusters. Due to their higher stability, W-based POM clusters are, on average, larger and more diverse than their Mo-based counterparts.

Structural and functional relationship of Cassia obtusifolia trypsin inhibitor to understand its digestive resistance against Pieris rapae.

Although digestive resistance of Kunitz protease inhibitors has been reported extensively, the molecular mechanism is not well established. In the present study, the first X-ray structure of Cassia obtusifolia trypsin inhibitor (COTI), a member of Kunitz protease inhibitors, was solved at a resolution of 1.9 Å. The structure adopted a classic ß-trefoil fold with the inhibitory loop protruding from the hydrophobic core. The role of Phe139, a unique residue in Kunitz protease inhibitors, and Arg63 in the COTI structure was verified by F139A and R63E mutants. COTI was a specific inhibitor of bovine trypsin and the result was also verified by COTI-trypsin complex formation. Meanwhile, COTI showed equivalent inhibitory activity with that of soybean trypsin inhibitor against bovine trypsin and midgut trypsin from Pieris rapae. The F139 and R63E mutants further indicated that inhibitory specificity and efficiency of COTI were closely related to the global framework, the conformation and the amino acid composition of reactive loop. Finally, a midgut trypsin from P. rapae (PrSP40), which might be involve in the food digestion, was proposed to be a potential target of COTI and might be a promising target for future crop-protection strategy. The results supported the digestive resistance of COTI.

4-Hydroxy-pyran-2-one and 3-hydroxy-N-methyl-2-oxindole derivatives of Salinispora arenicola from Brazilian marine sediments.

Three new 3-hydroxy-N-methyl-2-oxindole (1 and 2) and 4-hydroxy-pyran-2-one (3) derivatives, along with the known 3-hydroxy-N-methyl-2-oxindole (4) and 6-methoxy-N-methylisatin (5) were isolated from a marine Salinispora arenicola strain from sediments of the St. Peter and St. Paul Archipelago, Brazil. The structures of the new compounds were elucidated by a combination of spectroscopic (1D and 2D NMR and HR-ESIMS) data, including single-crystal X-ray diffraction analysis for 2 and 3. Compounds 1 to 5 were assayed for their antimicrobial properties, but only 4 and 5 were active against Enterococcus faecalis with MIC value of 15.6 µg/mL.

Tetra-glucopyranosyl Diterpene ent-Kaur-16-en-19-oic Acid and ent-13(S)-Hydroxyatisenoic Acid Derivatives from a Commercial Extract of Stevia rebaudiana (Bertoni) Bertoni.

Stevia rebaudiana and its diterpene glycosides are one of the main focuses of food companies interested in developing novel zero calorie sugar substitutes since the recognition of steviol glycosides as Generally Recognized as Safe (GRAS) by the United States Food and Drug Administration. Rebaudioside A, one of the major steviol glycosides of the leaves is more than 200 times sweeter than sucrose. However, its lingering aftertaste makes it less attractive as a table-top sweetener, despite its human health benefits. Herein, we report the purification of two novel tetra-glucopyranosyl diterpene glycosides 1 and 3 (rebaudioside A isomers) from a commercial Stevia rebaudiana leaf extract compounds, their saponification products compounds 2 and 4, together with three known compounds isolated in gram quantities. Compound 1 was determined to be 13-[(2-O-ß-d-glucopyranosyl-6-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl) oxy]ent-kaur-16-en-19-oic acid-ß-d-glucopyranosy ester (rebaudioside Z), whereas compound 3 was found to be 13-[(2-O-ß-d-glucopyranosyl-3-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-oic acid -ß-d-glucopyranosy ester. Two new tetracyclic derivatives with no sugar at position C-19 were prepared from rebaudiosides 1 and 3 under mild alkaline hydrolysis to afford compounds 2 13-[(2-O-ß-d-glucopyranosyl-6-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl) oxy]ent-kaur-16-en-19-oic acid (rebaudioside Z1) and 4 13-[(2-O-ß-d-glucopyranosyl-3-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-oic acid. Three known compounds were purified in gram quantities and identified as rebaudiosides A (5), H (6) and J (7). Chemical structures were unambiguously elucidated using different approaches, namely HRESIMS, HRESI-MS/MS, and 1D-and 2D-NMR spectroscopic data. Additionally, a high-quality crystal of iso-stevioside was grown in methanol and its structure confirmed by X-ray diffraction.

Complete 1H NMR assignment of cholesteryl benzoate.

The 750 MHz 1H NMR spectrum of cholesteryl benzoate (1b) could be assigned completely, which means all chemical shifts and all coupling constants, including some long-range values, were established. This task was possible by extracting many approximate coupling constant values in the overlapped spectrum region from an HSQC experiment, and using these values in the 1H iterative full spin analysis integrated in the PERCH NMR software. The task was facilitated using our published data for 3ß-acetoxypregna-5,16-dien-20-one (3), the assignment data of the sesquiterpene benzoquinone dihydroperezone (2), also performed in the present study, which contains the same carbon atoms chain than cholesterol (1a), and an HSQC study of (25R)-27-deuteriocholesterol (1c) we prepared some 40 years ago. The HSQC values of 1c in combination with the coupling constants of 1b also allowed to completely assigning the spectrum of 1c. The complete assignment of 1b and 1c further provided the opportunity to estimate the hydrogen shifts induced upon benzoylation of cholesterol. Comparison of the experimental vicinal coupling constants of 1b with the values calculated using the Altona software provides an excellent correlation. In addition, a single crystal X-ray diffraction study of 1b provided the molecular conformation in the solid state, which revealed the side chain adopts an extended conformation.

Structure of a Kunitz-type potato cathepsin D inhibitor.

Potato cathepsin D inhibitor (PDI) is a glycoprotein of 188 amino acids which can inhibit both the aspartic protease cathepsin D and the serine protease trypsin. Here we report the first X-ray structure of PDI at a resolution of 2.1 Å showing that PDI adopts a ß-trefoil fold, which is typical of the Kunitz-family protease inhibitors, with the inhibitory loops protruding from the core. Possible reactive-site loops including one involving a unique disulphide and another involving a protruding 310 helix are identified and docking studies indicate the mode of action of this unusual bi-functional inhibitor.

Synthesis, characterization, microbiological evaluation, genotoxicity and synergism tests of new nano silver complexes with sulfamoxole: X-ray diffraction of [Ag2(SMX)2]·DMSO.

The synthesis and microbiological evaluation of two new Ag(I) complexes with sulfamoxole (SMX), [Ag2(SMX)2]·H2O and [Ag4(SCN)3(SMX)]·H2O are described. Both were characterized by elemental analysis, thermogravimetry, powder and single crystal X-ray diffraction, NMR, Raman and experimental and theoretical IR spectroscopies. Their antibacterial and antifungal properties were evaluated by agar and broth dilution assays, respectively. In addition, synergism tests for Pseudomonas aeruginosa were performed, and genotoxicity studies were carried out employing the Allium cepa test. Both complexes displayed good activity against Escherichia coli, Staphylococcus aureus, P. aeruginosa, and 10 fungi strains, with lower minimum inhibitory concentrations (MICs) than that of free SMX in all cases. The nanometrical crystallite particle size determined from XRPD, DLS and TEM might explain the good microbiological activity in spite of the low solubility of both complexes. The fractional inhibitory concentration (FIC) calculated from the P. aeruginosa test data indicated that the activity of the complexes is not due to synergism of the free components in the concentration ratios studied. Moreover, none of the complexes displayed cytotoxic effects on onions in the concentration range tested, and chromosome aberrations were not observed.

Fragment-based discovery of focal adhesion kinase inhibitors.

Chemically diverse fragment hits of focal adhesion kinase (FAK) were discovered by surface plasmon resonance (SPR) screening of our in-house fragment library. Site specific binding of the primary hits was confirmed in a competition setup using a high-affinity ATP-site inhibitor of FAK. Protein crystallography revealed the binding mode of 41 out of 48 selected fragment hits within the ATP-site. Structural comparison of the fragment binding modes with a DFG-out inhibitor of FAK initiated first synthetic follow-up optimization leading to improved binding affinity.