A novel method for purification of biologically active C-terminal fragment of human recombinant anti-mullerian hormone

Anti-mullerian hormone (AMH) is one of the least studied members of transforming growth factor beta superfamily showing pro-apoptotic activity against cells positive for hormone type II receptor overexpressed by malignant cells in many cancer cases. Here, we propose an improved method for isolation of recombinant C-terminal AMH fragment (C-rAMH) to obtain homogeneous preparations of this protein with high biological activity. In contrast to our previously developed C-rAMH purification technology based on reversed-phase HPLC, the key stage of the new approach is hydrophobic interaction chromatography using Toyopearl Butyl-650S resin performed under more benign conditions. This modification of the previously developed method allowed highly purified C-rAMH to be obtained that is characterized by twice the specificity estimated as the ability to bind to the recombinant analog of AMH type II receptor and by significantly higher biological activity, that is, the ability to induce the death of target cells. Thus, we made the purification technology even more cost-effective and suitable for the production of drug forms based on C-rAMH.     

Application of electrochemical detection in high-performance liquid chromatography to the assay of biologically active compounds

Electrochemical detection in liquid chromatography was introduced in 1973 for the assay of catecholamines, the electroactive hormones and neurotransmitters, and continues to grow in popularity for the assay of trace amounts of biologically active compounds in complex samples in biology and medicine.     

Fluorination in superacids: a novel access to biologically active compounds

In HF-SbF₅, reaction of various alkaloids with NBS, NCS, H₂O₂ yields fluoroderivatives: anti-addition is observed at the C6C7 double bond with tabersonine, but a more complex reaction is operative with vindoline, leading to 7-substituted (Br, Cl, OH)-20-fluoro derivatives. A completely different reaction pathway is observed with bis indole alkaloids (vinblastine, anhydrovinblastine, vinorelbine) in HF-SbF₅. In the presence of NBS (or better of CCl₄ or CHCl₃), 20′,20′-difluoroderivatives are obtained. Vinflunine (20′,20′-difluoro-3′,4′-dihydrovinorelbine) has been selected for its promising antitumor activity.     

Isolation and sequencing of a new biologically active peptide from human lung carcinoma

A biologically active peptide designated hLCP has been isolated and purified to homogeneity from human lung carcinoma by means of acidic extraction and successive chromatography on Sephadex G-50, Toyopearl HW-40 F and reverse-phase high performance liquid chromatography columns. Analysis showed that peptide consists of thirteen amino acids. Primary structure of hLCP has been deduced by double-coupling Edman degradation combined with enzyme digestion as H-Ser-Pro-Pro-Asp-Gly-Lys-Lys-Glx-Ser-Ala-Asp-Val-Lys-OH. hLCP possessed significant excitatory activity on an electrical stimulation induced contraction. No hLCP could be detected in normal lung tissue. The possibility of using hLCP as a biochemical marker in the clinic for the early detection of lung carcinoma is being investigated.     

A rapid liquid chromatography-tandem mass spectrometry method for quantification of a biologically active molecule camboginol in the extract of Garcinia cambogia

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of camboginol in the extract of fruit rinds of Garcinia cambogia has been developed. Separation was achieved isocratically on an RP C(18) column using a solvent system consisting of a mixture of acetonitrile-water (9:1) and methanol-acetic acid (99.5:0.5) in the ratio of 30:70 as mobile phase at a flow rate of 0.4 mL/min. A multiple reaction monitoring (MRM) method was developed for quantification of camboginol in the fruit rinds extract of G. cambogia using MRM transitions of m/z 601.4 --> m/z 176.7 and m/z 601.4 --> m/z 448.9, respectively. The calibration curve based on peak area against concentration was linear up to 50 ng/mL with a detection limit of 0.5 ng/mL. The method showed satisfactory reproducibility with a coefficient of variation of less than 6%. The method was successfully applied for quantification of camboginol in different Garcinia extracts.     

Interaction of metallic nanoparticles with a biologically active molecule, dopamine

We present the results of first-principles molecular orbital calculations describing the interaction of metallic nanoparticles, represented by Mn(13), Ag(13), and Al(13) atomic clusters, with a biologically active molecule, dopamine. The interaction strength, determined in terms of the nanoparticle-molecule complex binding energy, is found to be higher for Mn than either Ag or Al and can be explained in terms of the degree of the hybridization of the (metal) atomic orbitals with the molecular orbitals in the complex. Furthermore, smaller interaction strength of these metallic nanoparticles with water compared to that with dopamine predicts the preference of forming a complex of dopamine with the metallic nanoparticles in the aqueous solution. The calculated results may therefore suggest that the presence of these metallic nanoparticles could induce different levels of dopamine depletion in solution.     

Synthesis of beta-hydroxy-beta-(fluoronitrophenyl)alanines: vital components in the assembly of biologically active cyclic peptides

[formula: see text] Numerous biologically active cyclic peptides, such as the antibiotic vancomycin, contain amino acid residues connected through side-chain biaryl or aryl-alkyl ether linkages. Nucleophilic aromatic substitution reactions have recently been shown to provide a general method for the formation of such ether linkages, and consequently the synthesis of functionalized fluoronitro-substituted aromatic amino acids is of great interest. Herein, a method for the stereospecific synthesis of 3-fluoro-4-nitro- and 4-fluoro-3-nitro-threo-beta-hydroxyphenylalanine is described.     

Loss of resistance to decay of scots pine heartwood in a biologically active environment

The heartwood ofPinus sylvestris (Scots pine or Baltic redwood) is known to contain natural chemicals (extractives) that confer a measure of resistance to decay by cellulolytic and lignolytic wood-destroying fungi. It is also known that the heartwood nearest the pith (juvenile heartwood) is frequently less resistant than the mature heartwood and can become rapidly decayed under appropriately damp conditions. Window joinery in the UK is manufactured substantially fromP. sylvestris and the extent of natural resistance to decay of the heartwood, and of any changes that may occur with time in service, is therefore of great practical and economic relevance. Investigations have been carried out at PRL in which small blocks (20 × 5 × 5 mm) have been excised from the relevant zones of new, joinery-grade timber and from timber previously in service as window joinery for periods of up to 10 yr. After sterilization, the blocks were exposed to attack byConiophora puteana (FPRL No HE) for a period of 4 wk and the weight loss of the blocks was assessed. Results with new timber confirmed that the mature heartwood is significantly more durable than the juvenile heartwood, which in turn is significantly more durable than the sapwood (0.1% probability level in each case). With timber from service, different results were obtained. From regions protected by paint and remote from the effects of water penetration via the joint, the mature heartwood was still more durable than juvenile heartwood, but the difference was less significant (5% probability level). However in the region of the joints, which are subject to wetting and the microbial colonization that accompanies it, the mature heartwood had become significantly less durable than the mature heartwood from the protected region. Furthermore, it was no longer more durable than the juvenile wood associated with it. The precise mechanism of the change in durability of the mature wood is still not known. Leaching of water-soluble extractives is thought unlikely since the main extractives present inP. sylvestris (pinosylvins) are not markedly water-soluble and in any case, although water enters the timber components in the liquid phase, subsequent loss occurs in the vapor phase and not by mass flow. It seems more likely that the effect is a result of the activity of the mixed microflora of bacteria, yeasts, molds, and blue-stain fungi that have already been shown to be the early colonizers of pine joinery in service. Possibly the microbial activity serves to detoxify the inhibitory extractives or to render the heartwood more susceptible to cellulolytic and lignolytic systems by some nutritional or microstructural means.     

Experimental and in silico characterization of a biologically active inosose

Inositols have been recently reported to show a biological activity as inhibitors of both glycosidase and amyloid-β protein. After having harvested good crystals suitable for single crystal X-ray diffraction, we performed a comparison with the data inferred by means of a molecular dynamics simulation, based on the use of an appropriate Force Field coupled to the most performing charging scheme. This approach allowed a detailed analysis extended to ultra-fine details, such as atomic displacement parameters. It confirmed the good validity of a robust approach already tested by us in previous studies. A NMR analysis of the molecule in solution was also carried out, to compare the structural findings suggested by the X-ray analysis with the ones in solution and avoid confining them to the solid-state. In this framework, we investigated the above-mentioned inhibiting activity of a class of inososes, by means of a molecular docking investigation, which proved the suggested validity of the studied compound as inhibitor of the α-glucosidase.     

The structuring and properties of hydrogels prepared with a biologically active macromonomer

The copolymerization of a macromonomeric inhibitor of proteolytic enzymes with acrylamide and N,N′-methylenebis(acrylamide) in an aqueous solution in the presence of a redox catalyst has been studied. It has been shown that the addition of mercaptoacetic acid as a chain-transfer agent to the polymerization system results in a change in the structure of the forming hydrogel, i.e., a decrease in the number of large pores. Simultaneously, a substantial increase in the degree of the macromonomer incorporation into the hydrogel is attained and favorable conditions for the development of the biological activity of the macromonomer are created.     

Effect of sulfur-containing amino acids and peptides on the free-radical fragmentation of biologically active phospho derivatives of glycerol

The effects of sulfur-containing amino acids and peptides (cysteine, N-acetylcysteine (NAC), methionine, methionine sulfoxide (MetO), taurine (Tau), glutathione (GSH, GSSH), cysteinylglycine, and glutamylcysteine) on the free-radical degradation of glycero-1-phosphate and dimiristoyl phosphatidylglycerol with phosphoether bond rupture induced by γ-radiation or the Fe²⁺(EDTA)(Cu²⁺)–H₂O₂–(ascorbate) systems have been studied. In the case of a radiation-initiated process, thiols exert the greatest radioprotective effect in a concentration-dependent manner, and the action of MetO and Tau is neutral. In the case of Cu²⁺- mediated fragmentation, all of the test compounds with exception of Tau and NAC inhibit the process. The action of the test compounds on the fragmentation mediated by the Fe²⁺/EDTA–H₂O₂ (Fe²⁺–H₂O₂–ascorbate) systems can be either anti(pro)oxidant or neutral. In this case, all of the thiols other than glutamylcysteine exert an activating effect.     

Simultaneous determination of three active components in rat plasma by UPLC–MS/MS: Application to pharmacokinetic study after oral administration of Herba Sarcandrae extract

A rapid, specific and sensitive ultra‐performance liquid chromatography tandem mass spectrometry (UPLC‐MS/MS) method was developed and validated for determination of isofraxidin, rosmarinic acid and kaempferol‐3‐O ‐glucuronide in rat plasma using warfarin as an internal standard (IS). Separation was conducted on a Thermo Hypersil GOLD C₁₈ column with linear gradient elution using methanol and water. Mass spectrometric detection was conducted using selected reaction monitoring (SRM) via an electrospray ionization (ESI) source. All analytes exhibited good linearity within their concentration ranges (r > 0.9990). The lower limits of quantitations of isofraxidin, rosmarinic acid, and kaempferol‐3‐O‐ glucuronide were 1.31, 0.67 and 0.92 ng/mL, respectively. Intra‐ and inter‐day precisions of these investigated components exhibited an RSD within 11.7%, and the accuracy ranged from −12.5 to 15.0% at all QC levels. The developed method was successfully applied to a pharmacokinetic study of isofraxidin, rosmarinic acid, and kaempferol‐3‐O‐ glucuronide in rats after oral administration of Herba Sarcandrae Extract.     

Pharmacokinetics of anthraquinones in rat plasma after oral administration of a rhubarb extract

A sensitive and specific LC‐MS/MS method was developed for simultaneous determination of aloe‐emodin, rhein, emodin, chrysophanol and physcion and their conjugates in rat plasma. The lower limit of quantitation of each anthraquinone was 0.020–0.040 µm . Intra‐day and inter‐day accuracies were 90.1–114.3% and the precisions were <14.6%. The matrix effects were 104.0–113.2%. The method was successfully applied to a pharmacokinetic study in rats receiving a rhubarb extract orally. The area under the concentration–time curve (AUC_0–t) and peak concentration (C_max) of free aloe‐emodin and emodin in rat plasma were much lower than those of rhein. The amounts of chrysophanol and physcion were too low to be continuously detected. After treating the plasma samples with β‐glucuronidases, each anthraquinone was detectable throughout the experimental period (36 h) and showed much higher plasma concentrations and AUC_0–t. The free/total ratios of aloe‐emodin, rhein and emodin were 6.5, 49.0 and 1.7% for C_max and 3.7, 32.5 and 1.1% for AUC_0–t, respectively. The dose‐normalized AUC_0–t and C_max of the total of each anthraquinone were in the same descending order: rhein > emodin > chrysophanol > physcion > aloe‐emodin. These findings reveal phase II conjugates as the dominant in vivo existing forms of rhubarb antharquinones and warrant a further study to evaluate their contribution to the herbal activity. Copyright © 2013 John Wiley & Sons, Ltd.     

Application of photoelectron spectroscopy to biologically active molecules and their constituent parts IV. Methylnitroimidazoles

Photoelectron (PE) spectra of imidazole ( 1), 1 -methylimidazole ( 2 ), 2-methylimidazole ( 3 ), 4(5)-nitroimidazole ( 4 ), 2-methyl-4(5)nitroimidazole ( 5 ), 1,2-dimethyl-5-nitroimidazole ( 6 ), 1-ethyl-2-methyl-5-nitroimidazole ( 7 ), 1-bromoethyl-2-methyl-5-nitroimidazole ( 8 ) and 1-hydroxyethyl-2-methyl-5-nitroimidazole ( 9 ) have been recorded using Hel excitation. The electronic structure of the potent antitrichomonal agent 9 is discussed in comparison with compounds 1–8 allowing for the study of the influence of substituents on the imidazole ring.     

Is it biologically relevant to measure the structures of small peptides in the gas-phase?

Recent developments in sample introduction of biologically relevant molecules have heralded a new era for gas-phase methods of structural determination. One of the biggest challenges is to relate gas-phase structures, often measured in the absence of water and counter ions, with in vivo biologically active structures. An advantage of gas-phase based techniques is that a given peptide can be analysed in a variety of different forms, for example, as a function of charge state, or with additional water molecules. Molecular modelling can provide insight into experimental findings and help elucidate the differences between structural forms. Combining experiment and theory provides a thorough interrogation of candidate conformations. Here two important naturally occurring peptide systems have been examined in detail and results are assessed in terms of their biological significance.The first of these is gonadotropin-releasing hormone (GnRH), a decapeptide which is the central regulator of the reproductive system in vertebrates. We have examined several naturally occurring variants of this peptide using Ion Mobility Mass Spectrometry and Electron Capture Dissociation (ECD) in conjunction with Fourier Transform Ion Cyclotron Mass Spectrometry (FT-ICR-MS). Candidate conformations are modelled using the AMBER force field. Single amino acid changes, for example Gly6 → Ala6, or Ala6 → D-Ala6, have observable effects on the gas phase structure of GnRH. It has been shown that evolutionary primary sequence variations are key to the biological activity of GnRH, and it is thought that this is due to different binding affinities at target receptors. This work provides strong evidence that this activity is structurally based. The second system examined is the relationship between the quaternary structure and activity of two novel β-defensins. FT-ICR mass spectrometry has been employed to characterize di-sulphide bridging and dissociation based experiments utilised to investigate their structural core. Defr1, with five cysteines, exists as a covalently bound disulphide linked dimer; Defr1 Y5C with six cysteines also is observed as a dimer, but non-covalently bound, suggesting that this defensin has a tendency to aggregate. The activity of Defr1 is 10 times higher than that of Defr1 Y5C when tested against the pathogen Pseudomonas aeruginosa. The results from these studies could inform future design of novel GnRH type ligands and anti-microbial agents, and illustrate the power of gas-phase based techniques for solving peptide structures.     

Recent advances in the synthesis of biologically active spirooxindoles

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Cocyclotrimerization of 6-alkynylpurines with alpha,omega-diynes as a novel approach to biologically active 6-arylpurines

Transition metal complex catalyzed cocyclotrimerization of 6-alkynylpurines 1 with various diynes enables the preparation of a plethora of substituted 6-arylpurines 3 in good yields. The most general catalyst for the reaction is a user-friendly system based on a nickel-phosphine complex and reductant (NiBr(2)(dppe)/Zn) in MeCN. The reaction conditions are compatible with various protective groups on the purine moiety (Bn, THP). As far as other potential catalysts were concerned, only CoBr(PPh(3))(3) showed reasonable activity in cocyclotrimerization of alkynylpurines with dipropargyl ether. A comparison of catalytic with stoichiometric approaches and the ligand effect in the catalyst is also given. Cytostatic activity screening of title 6-arylpurines was performed and several moderately active compounds were found.     

Mechanocomposites of biologically active acids on the basis of a natural biopolymer

Mechanical activation of the natural biopolymer arabinogalactan with organic acids (succinic, aminoacetic) was studied by IR spectroscopy and X-ray phase analysis. Evidence was obtained for the formation of mechanocomposites with a chemical bond between the natural biopolymer and organic acids. The nature of this bond depends on the nature of the acid. The composites obtained by mechanical activation of such mixtures are stable disperse systems with a uniformly distributed active ingrediaent.