One-Step Synthesis of Functionalized Pyrazolo[3,4-b]pyridines via Ring Opening of the Pyrrolinium Ion.

Herein, we report a highly regioselective one-pot synthesis of pyrazolo[3,4-b]pyridines via the reaction of 3-arylidene-1-pyrrolines with aminopyrazoles. The reaction proceeds through the sequential nucleophilic addition/electrophilic substitution/C-N bond cleavage and provides easy access to pyrazolo[3,4-b]pyridine derivatives featuring a primary amino group. Moreover, the reaction can be terminated at the electrophilic substitution stage, thus providing convenient entry to the hardly accessible pyrazolopyrrolopyridine scaffold.

Silicas with Polyoxyethylene Branches for Modification of Membranes Based on Microporous Block Copolymers.

We have synthesized cubic and linear polysiloxanes containing polyoxyethylene branches (ASiP-Cu) using tetraethoxysilane, polyoxyethylene glycol, and copper chloride as precursors; the products are stable to self-condensation. The effect of copper chloride content on the chemical structure of ASiP-Cu has been established. A special study was aimed at defining the modifying effect of ASiP-Cu on the sorption characteristics of membranes based on microporous, optically transparent block copolymers (OBCs). These OBCs were produced using 2,4-toluene diisocyanate and block copolymers of ethylene and propylene oxides. The study demonstrated significantly increased sorption capacity of the modified polymers. On the basis of the modified microporous block copolymers and 1-(2-pyridylazo)-2-naphthol (PAN) analytical reagent, an analytical test system has been developed. Additionally, the modified OBCs have the benefit of high diffusion permeability for molecules of organic dyes and metal ions. It has been shown that the volume of voids and structural features of their internal cavities contribute to the complex formation reaction involving PAN and copper chloride.

Extent of N-Terminus Folding of Semenogelin 1 Cleavage Product Determines Tendency to Amyloid Formation.

It is known that four peptide fragments of predominant protein in human semen Semenogelin 1 (SEM1) (SEM1(86-107), SEM1(68-107), SEM1(49-107) and SEM1(45-107)) are involved in fertilization and amyloid formation processes. In this work, the structure and dynamic behavior of SEM1(45-107) and SEM1(49-107) peptides and their N-domains were described. According to ThT fluorescence spectroscopy data, it was shown that the amyloid formation of SEM1(45-107) starts immediately after purification, which is not observed for SEM1(49-107). Seeing that the peptide amino acid sequence of SEM1(45-107) differs from SEM1(49-107) only by the presence of four additional amino acid residues in the N domain, these domains of both peptides were obtained via solid-phase synthesis and the difference in their dynamics and structure was investigated. SEM1(45-67) and SEM1(49-67) showed no principal difference in dynamic behavior in water solution. Furthermore, we obtained mostly disordered structures of SEM1(45-67) and SEM1(49-67). However, SEM1(45-67) contains a helix (E58-K60) and helix-like (S49-Q51) fragments. These helical fragments may rearrange into ß-strands during amyloid formation process. Thus, the difference in full-length peptides' (SEM1(45-107) and SEM1(49-107)) amyloid-forming behavior may be explained by the presence of a structured helix at the SEM1(45-107) N-terminus, which contributes to an increased rate of amyloid formation.

Binding of Different Cyclosporin Variants to Micelles Evidenced by NMR and MD Simulations.

Peptides play a critical role in the life of organisms, performing completely different functions. The biological activity of some peptides, such as cyclosporins, can be determined by the degree of membrane permeability. Thus, it becomes important to study how the molecule interacts with lipid bilayers. Cyclosporins C, E, H and L were characterised molecular dynamics simulation; NMR spectroscopy studies were also carried out for cyclosporins C and E. The comparison of one- and two-dimensional spectra revealed certain similarities between spatial structures of the studied cyclosporin variants. Upon dissolving in water containing DPC micelles, which serve as model membranes, subtle changes in the NMR spectra appear, but in a different way for different cyclosporins. In order to understand whether observed changes are related to any structural modifications, simulation of the interaction of the peptide with the phospholipid micelle was performed. The onset of the interaction was observed, when the peptide is trapped to the surface of the micelle. Simulations of this kind are also of interest in the light of the well-known membrane permeability of cyclosporin, which is important for its biological action.

Pervaporation Polyurethane Membranes Based on Hyperbranched Organoboron Polyols.

On the basis of aminoethers of boric acid (AEBA), polyurethane vapor-permeable and pervaporative membranes were obtained. AEBAs, the structure of which is modified by bulk adducts (EM) of diphenylol propane diglycidyl ether and ethanolamine, were studied. It turned out that AEBA exists in the form of clusters, and the use of EM as a result of partial destruction of associative interactions leads to a significant decrease in the size of AEBA-EM particles and their viscosity compared to unmodified AEBA. The introduction of EM into the composition of AEBA leads to a threefold increase in the vapor permeability of polyurethanes obtained on their basis. The observed effect is explained by the fact that a decrease in the size of clusters leads to loosening of their dense packing. Areas of clustering due to associative interactions of hydroxyl groups, together with the hydrophilic nature of polyoxyethylene glycol, create channels through which water molecules can penetrate. The increase in vapor permeability is accompanied by a multiple increase in the permeability coefficients in the pervaporative dehydration of isopropanol.

Catalytic Etherification of ortho-Phosphoric Acid for the Synthesis of Polyurethane Ionomer Films.

The etherification reaction of ortho-phosphoric acid (OPA) with polyoxypropylene glycol in the presence of tertiary amines was studied. The reaction conditions promoting the catalytic activity of triethanolamine (TEOA) and triethylamine (TEA) in the low-temperature etherification of OPA were established. The catalytic activity of TEOA and TEA in the etherification reaction of phosphoric acid is explained by the hydrophobic-hydrophilic interactions of TEA with PPG, leading, as a result of collective interactions, to a specific orientation of polyoxypropylene chains around the tertiary amine. When using triethylamine, complete etherification of OPA occurs, accompanied by the formation of branched OPA ethers terminated by hydroxyl groups and even the formation of polyphosphate structures. When triethanolamine is used as a catalyst, incomplete etherification of OPA with polyoxypropylene glycol occurs and as a result, part of the phosphate anions remain unreacted in the composition of the resulting aminoethers of ortho-phosphoric acid (AEPA). In this case, the hydroxyl groups of triethanolamine are completely involved in the OPA etherification reaction, but the catalytic activity of the tertiary amine weakens due to a decrease in its availability in the branched structure of AEPA. The kinetics of the etherification reaction of OPA by polyoxypropylene glycol catalyzed by TEOA and TEA were studied. It was shown that triethanolamine occupies a central position in the AEPA structure. The physico-mechanical and thermomechanical properties of polyurethane ionomer films obtained on the basis of AEPA synthesized in a wide temperature range were studied.

Comparison of structural properties of cyclosporin A and its analogue alisporivir and their effects on mitochondrial bioenergetics and membrane behavior.

The paper considers the effect of the MPT pore inhibitor cyclosporin A (CsA) and its non-immunosuppressive analogue alisporivir (Ali) on the functioning of rat skeletal muscle mitochondria. We have shown that both agents at a standard in vitro concentration of 1 µM increase the calcium capacity of organelles and have no effect on the parameters of oxidative phosphorylation. However, an increase in their concentration to 5 µM leads to the suppression of oxygen consumption by mitochondria, which is more pronounced in the case of Ali. This effect is accompanied by a decrease in the membrane potential of organelles and, apparently, is based on the inhibition of electron transport along the mitochondrial respiratory chain due to limited mobility of coenzyme Q. We have noted that both agents do not affect the production of hydrogen peroxide by isolated mitochondria. NMR spectroscopy and molecular dynamics simulation did not reveal significant differences in the structure and backbone flexibility of CsA and Ali. Both agents decrease the overall fluidity of the membrane of DPPC liposomes, inducing an increase in laurdan generalized polarization parameter. A similar effect was also found in the case of mitochondrial membranes. We suggested that these effects of CsA and Ali, associated with their lipophilic nature and the ability to accumulate in the lipid phase of membranes, may cause a decrease in the efficiency of electron transport in the respiratory chain of mitochondria and suppression of the bioenergetics of these organelles.

Design, Spectral Characteristics, Photostability, and Possibilities for Practical Application of BODIPY FL-Labeled Thioterpenoid.

This paper presents the design and a comparative analysis of the structural and solvation factors on the spectral and biological properties of the BODIPY biomarker with a thioterpene fragment. Covalent binding of the thioterpene moiety to the butanoic acid residue of meso-substituted BODIPY was carried out to find out the membranotropic effect of conjugate to erythrocytes, and to assess the possibilities of its practical application in bioimaging. The molecular structure of the conjugate was confirmed via X-ray, UV/vis-, NMR-, and MS-spectra. It was found that dye demonstrates high photostability and high fluorescence quantum yield (to ~100%) at 514-519 nm. In addition, the marker was shown to effectively penetrate the erythrocytes membrane in the absence of erythrotoxicity. The conjugation of BODIPY with thioterpenoid is an excellent way to increase affinity dyes to biostructures, including blood components.

Unraveling the Mechanism of Platelet Aggregation Suppression by Monoterpenoids.

Platelet aggregation causes various diseases and therefore challenges the development of novel antiaggregatory drugs. In this study, we report the possible mechanism of platelet aggregation suppression by newly synthesized myrtenol-derived monoterpenoids carrying different heteroatoms (sulphur, oxygen, or nitrogen). Despite all tested compounds suppressed the platelet aggregation in vitro, the most significant effect was observed for the S-containing compounds. The molecular docking confirmed the putative interaction of all tested compounds with the platelet's P2Y12 receptor suggesting that the anti-aggregation properties of monoterpenoids are implemented by blocking the P2Y12 function. The calculated binding force depended on heteroatom in monoterpenoids and significantly decreased with the exchanging of the sulphur atom with oxygen or nitrogen. On the other hand, in NMR studies on dodecyl phosphocholine (DPC) as a membrane model, only S-containing compound was found to be bound with DPC micelles surface. Meanwhile, no stable complexes between DPC micelles with either O- or N-containing compounds were observed. The binding of S-containing compound with cellular membrane reinforces the mechanical properties of the latter, thereby preventing its destabilization and subsequent clot formation on the phospholipid surface. Taken together, our data demonstrate that S-containing myrtenol-derived monoterpenoid suppresses the platelet aggregation in vitro via both membrane stabilization and blocking the P2Y12 receptor and, thus, appears as a promising agent for hemostasis control.

Conjugate of meso-carboxysubstituted-BODIPY with thioterpenoid as an effective fluorescent probe: Synthesis, structure, spectral characteristics, and molecular docking.

This paper is devoted to the design of a fluorescent probe based on meso-carboxysubstituted-BODIPY with a thioterpene fragment. The functional replacement of the methoxy group in the BODIPY molecule on a thioterpene fragment was carried out in order to find out the antiplatelet and anticoagulant action mechanisms of thioterpenoids and to assess the membrane and receptor factors contributions. The molecular structure of the conjugate was confirmed via UV/vis-, NMR- and MS-spectra. It is found that the probe is a high fluorescence quantum yield (to âˆ¼ 100%) in the blue-green region at 509-516 nm. Molecular docking of all studied molecules showed that the BODIPY with terpenoid conjugation is an excellent way to increase their affinity to platelet receptor P2Y12.

Interaction of the pitavastatin with model membranes.

Pitavastatin is a statin drug that, by competitively inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A reductase, can lower serum cholesterol levels of low-density lipoprotein (LDL) accompanied by side effects due to pleiotropic effects leading to statin intolerance. These effects can be explained by the lipophilicity of statins, which creates membrane affinity and causes statin localization in cellular membranes. In the current report, the interaction of pitavastatin with POPC model membranes and its influence on the membrane structure were investigated using H, H and P solid-state NMR spectroscopy. Our experiments show the average localization of pitavastatin at the lipid/water interface of the membrane, which is biased towards the hydrocarbon core in comparison to other statin molecules. The membrane binding of pitavastatin also introduced an isotropic component into the 31P NMR powder spectra, suggesting that some of the lamellar POPC molecules are converted into highly curved structures.

Dithiophosphate-Induced Redox Conversions of Reduced and Oxidized Glutathione.

Phosphorus species are potent modulators of physicochemical and bioactive properties of peptide compounds. O,O-diorganyl dithiophoshoric acids (DTP) form bioactive salts with nitrogen-containing biomolecules; however, their potential as a peptide modifier is poorly known. We synthesized amphiphilic ammonium salts of O,O-dimenthyl DTP with glutathione, a vital tripeptide with antioxidant, protective and regulatory functions. DTP moiety imparted radical scavenging activity to oxidized glutathione (GSSG), modulated the activity of reduced glutathione (GSH) and profoundly improved adsorption and electrooxidation of both glutathione salts on graphene oxide modified electrode. According to NMR spectroscopy and GC-MS, the dithiophosphates persisted against immediate dissociation in an aqueous solution accompanied by hydrolysis of DTP moiety into phosphoric acid, menthol and hydrogen sulfide as well as in situ thiol-disulfide conversions in peptide moieties due to the oxidation of GSH and reduction of GSSG. The thiol content available in dissolved GSH dithiophosphate was more stable during air oxidation compared with free GSH. GSH and the dithiophosphates, unlike DTP, caused a thiol-dependent reduction of MTS tetrazolium salt. The results for the first time suggest O,O-dimenthyl DTP as a redox modifier for glutathione, which releases hydrogen sulfide and induces biorelevant redox conversions of thiol/disulfide groups.

NMR resonance assignment and backbone dynamics of a C-terminal domain homolog of orange carotenoid protein.

Photoprotection in cyanobacteria is mediated by the Orange Carotenoid Protein (OCP), a two-domain photoswitch which has multiple natural homologs of its N- and C-terminal domains. Recently, it was demonstrated that C-terminal domain homologs (CTDHs) of OCP are standalone carotenoproteins participating in multidirectional carotenoid transfer between membranes and proteins. Non-covalent embedment of a ketocarotenoid causes dimerization of the small 16-kDa water-soluble CTDH protein; however, dynamic interactions of CTDH with membranes and other proteins apparently require the monomeric state. Although crystallography recently provided static snapshots of the Anabaena CTDH (AnaCTDH) spatial structure in the apo-form, which predicted mobility of some putative functional segments, no crystallographic information on the holo-form of CTDH is presently available. In order to use NMR techniques to cope with the dynamics of the AnaCTDH protein, it was necessary to obtain 1H, 13C and 15N resonance assignments. AnaCTDH samples enriched with 13C and 15N isotopes were prepared using recombinant protein expression, and NMR resonance assignment was achieved for more than 90% of the residues. The obtained results revealed that the structure of AnaCTDH in solution and in the crystal are largely equivalent. Together with 15N NMR relaxation experiments, our data shed light on the AnaCTDH dynamics and provide the platform for the subsequent analysis of the holo-CTDH structure in solution, for the better understanding of light-triggered protein-protein interactions and the development of antioxidant nanocarriers for biomedical applications in the future.

Design, Spectral Characteristics, and Possibilities for Practical Application of BODIPY FL-Labeled Monoterpenoid.

This article describes the design and biological properties of a BODIPY FL-labeled monoterpenoid BF2-meso-(4-((1″R)-6″,6″-dimethylbicyclo[3.1.1]hept-2″-ene-2″)yl-methoxycarbonylpropyl)-3,3',5,5'-tetramethyl-2,2'-dipyrromethene conjugate (BODIPYmyrt). The fluorophore was characterized using X-ray, NMR, MS, and UV/vis spectroscopy. The conjugate exhibits a high quantum yield (to ∼100%) in the region 515-518 nm. BODIPYmyrt effectively penetrates the membranes of the bacterial and fungal cells and therefore can be used to examine the features of a broad spectrum of Gram-positive and Gram-negative bacteria and pathogenic fungi as well. Moreover, BODIPYmyrt exhibits a moderate tropism to the subcellular structures in mammalian cells (e.g., mitochondria), thereby providing an attractive scaffold for fluorophores to examine these particular organelles.

La(III), Ce(III), Gd(III), and Eu(III) Complexation with Tris(hydroxymethyl)aminomethane in Aqueous Solution.

Lanthanides such as cerium(III), europium(III), and gadolinium(III) are widely used for designing fluorescent probes or magnetic resonance imaging contrasting agents for biological systems. The synthesis and study of lanthanide complexes in buffer solutions imitating biological fluids are often complicated because of a lack of data on the lanthanide interactions with buffer solution components. Therefore, Ln(III) [where Ln(III) = La(III), Ce(III), Gd(III), Eu(III)] complexation with a widely used buffer agent, tris(hydroxymethyl)aminomethane (Tris), in aqueous solution is studied using potentiometry, spectrofluorimetry, and 139La NMR spectroscopy. The stoichiometric composition of complexes is determined using mass spectrometry. The thermodynamic stability constants of Ln(III)-Tris complexes are calculated from potentiometric and spectral data; the difficulties in the study of these systems, reliability, and accuracy of the obtained constants are discussed. The possible structures of free Tris and its complexes with lanthanides(III) are optimized on the density functional theory/PBE0 level; the peculiarities of metal-ligand bonds were studied by Quantum Theory Atoms in Molecules analysis.

Backbone resonance assignment and dynamics of 110 kDa hexameric inorganic pyrophosphatase from Mycobacterium tuberculosis.

Family I soluble inorganic pyrophosphatases (PPases; EC 3.6.1.1) are enzymes essential for all organisms. They hydrolyze inorganic pyrophosphate, thus providing the driving force for numerous biosynthetic reactions. Soluble PPases retain enzymatic activity only in multimeric forms. PPases from various organisms are extensively studied by X-ray crystallography but until now there was no information on their structure and dynamics in solution. Hexameric 110 kDa (6 × 18.3 kDa) PPase from Mycobacterium tuberculosis (Mt-PPase) is a promising target for the rational design of potential anti-tuberculosis agents. In order to use NMR techniques in functional studies of Mt-PPase and rational design of the inhibitors for this enzyme, it is necessary to have information on the backbone 1H, 13C and 15N resonance assignments. Samples of Mt-PPase enriched with 99% of 13C and 15N isotopes, and 95% of 2H were obtained using recombinant protein expression in an isotopically-labeled medium and effective heat-shock protocol for the deuterium-to-hydrogen exchange of the amide groups. Backbone resonance assignment was achieved for more than 95% of the residues. It was found that the secondary structure of Mt-PPase in solution corresponds well to the crystal structure of this protein. Protein backbone dynamics were studied using 15N NMR relaxation experiments. Determined resonance assignments and dynamic properties provide the basis for the subsequent structure-based design of novel inhibitors of Mt-PPase-potential anti-tuberculosis drugs.

Williams-Beuren syndrome-related methyltransferase WBSCR27: cofactor binding and cleavage.

Williams-Beuren syndrome, characterized by numerous physiological and mental problems, is caused by the heterozygous deletion of chromosome region 7q11.23, which results in the disappearance of 26 protein-coding genes. Protein WBSCR27 is a product of one of these genes whose biological function has not yet been established and for which structural information has been absent until now. Using NMR, we investigated the structural and functional properties of murine WBSCR27. For protein in the apo form and in a complex with S-(5'-adenosyl)-l-homocysteine (SAH), a complete NMR resonance assignment has been obtained and the secondary structure has been determined. This information allows us to attribute WBSCR27 to Class I methyltransferases. The interaction of WBSCR27 with the cofactor S-(5'-adenosyl)-l-methionine (SAM) and its metabolic products - SAH, 5'-deoxy-5'-methylthioadenosine (MTA) and 5'-deoxyadenosine (5'dAdo) - was studied by NMR and isothermal titration calorimetry. SAH binds WBSCR27 much tighter than SAM, leaving open the question of cofactor turnover in the methylation reaction. One possible answer to this question is the presence of weak but detectable nucleosidase activity for WBSCR27. We found that the enzyme catalyses the cleavage of the adenine moiety from SAH, MTA and 5'dAdo, similar to the action of bacterial SAH/MTA nucleosidases. We also found that the binding of SAM or SAH causes a significant change in the structure of WBSCR27 and in the conformational mobility of the protein fragments, which can be attributed to the substrate recognition site. This indicates that the binding of the cofactor modulates the folding of the substrate-recognizing region of the enzyme.

Solution structure of the N-terminal domain of the Staphylococcus aureus hibernation promoting factor.

Staphylococcus aureus hibernation promoting factor (SaHPF) is a 22,2 kDa protein which plays a crucial role in 100S Staphylococcus aureus ribosome formation during stress. SaHPF consists of N-terminal domain (NTD) that prevents proteins synthesis by binding to the 30S subunit at the P- and A-sites, connected through a flexible linker with a C-terminal domain (CTD) that keeps ribosomes in 100S form via homodimerization. Recently obtained 100S ribosome structure of S. aureus by cryo-EM shown that SaHPF-NTD bound to the ribosome active sites, however due to the absence of SaHPF-NTD structure it was modeled by homology with the E. coli hibernation factors HPF and YfiA. In present paper we have determined the solution structure of SaHPF-NTD by high-resolution NMR spectroscopy which allows us to increase structural knowledge about HPF structure from S. aureus.

Effect of triphenylphosphonium moiety on spatial structure and biointeractions of stereochemical variants of YRFK motif.

Chemical modification of therapeutic peptides is an important approach to improving their physicochemical and pharmacokinetic properties. The triphenylphosphonium (TPP) cation has proved to be a powerful modifier; however, its effects on peptide structure and activity remain uncharacterized. In this study, cytoprotective tetrapeptides based on the YRFK opioid motif with L- or D-Arg residues were linked to (triphenylphosphonio)carboxylic acids with ethylene and pentylene spacers (TPP-3 and TPP-6 groups, respectively). The three-dimensional structure of the oligopeptides was analyzed by NMR spectroscopy, computational methods and circular dichroism (CD). A more compact and bent structure with segregated aromatic groups was revealed for the D-arginine-containing tetrapeptide and its TPP-6 derivative. The TPP moiety caused structure-organizing effect on the tetrapeptides, resulting in transition from random coil to ß-sheet structures, and decreased the peptide backbone flexibility up to ten times. The TPP-3-modified oligopeptide with the lowest RMSD value (ca. 0.05 Å) was characterized by intrapeptide hydrophobic interactions between the TPP and side groups of Tyr and Phe residues accompanied by strong CD induction. The TPP-6-modified oligopeptides showed enhanced ability to form intermolecular associates and disturb liposomal membranes. The relationship between the spatial structure of the oligopeptides and some of their biologically relevant interactions were additionally revealed and are discussed.

Backbone and side chain NMR assignments for the ribosome binding factor A (RbfA) from Staphylococcus aureus.

Ribosome binding factor A (RbfA) is a 14.9 kDa adaptive protein of cold shock, which is important for bacterial growth at low temperatures. RbfA can bind to the free 30S ribosomal subunit and interacts with the 5'-terminal helix (helix I) of 16S rRNA. RbfA is important for the efficient processing of 16S rRNA and for the maturation (assembly) of 30S ribosomal subunits. Here we report backbone and side chains 1H, 13C and 15N chemical shift assignments of RbfA from Staphylococcus aureus. Analysis of the backbone chemical shifts by TALOS+ suggests that RbfA contains four α-helixes and three ß-strands with α1-ß1-ß2-α2-α3-ß3-α4 topology. Secondary structure of RbfA have KH-domain fold topology with ßααß subunit which is characterized by a helix-kink-helix motif in which the GxxG sequence is replaced by a conserved AxG sequence, where an Ala residue at position 70 forming an interhelical kink. The solution of the structure of this protein factor and its complex with the ribosome by NMR spectroscopy, X-ray diffraction analysis and cryo-electron microscopy will allow further development of highly selective substances for slowing or completely stopping the translation of the pathogenic bacterium S. aureus, which will interfere with the synthesis and isolation of its pathogenicity factors.