The pentafluoroethyltrihydridoborate anion: from shock sensitive salts to stable room temperature ionic liquids.

The reaction of pentafluoroethyllithium with BH3·thf yields [C2F5BH3]- that is stable against H2O, OH- and air due to the electron withdrawing C2F5 group. M[C2F5BH3] (M = K, Cs) are shock sensitive solids whereas salts with organic cations such as the room temperature ionic liquid [EMIm][C2F5BH3] are stable for years. [C2F5BH3]- is a mild reducing agent, which is also reflected by its electrochemical properties. Oxidation with I2 or Br2 in the presence of Lewis bases yields the first stable adducts C2F5BH2·L.

Analytical probabilistic modeling of RBE-weighted dose for ion therapy.

Particle therapy is especially prone to uncertainties. This issue is usually addressed with uncertainty quantification and minimization techniques based on scenario sampling. For proton therapy, however, it was recently shown that it is also possible to use closed-form computations based on analytical probabilistic modeling (APM) for this purpose. APM yields unique features compared to sampling-based approaches, motivating further research in this context. This paper demonstrates the application of APM for intensity-modulated carbon ion therapy to quantify the influence of setup and range uncertainties on the RBE-weighted dose. In particular, we derive analytical forms for the nonlinear computations of the expectation value and variance of the RBE-weighted dose by propagating linearly correlated Gaussian input uncertainties through a pencil beam dose calculation algorithm. Both exact and approximation formulas are presented for the expectation value and variance of the RBE-weighted dose and are subsequently studied in-depth for a one-dimensional carbon ion spread-out Bragg peak. With V and B being the number of voxels and pencil beams, respectively, the proposed approximations induce only a marginal loss of accuracy while lowering the computational complexity from order [Formula: see text] to [Formula: see text] for the expectation value and from [Formula: see text] to [Formula: see text] for the variance of the RBE-weighted dose. Moreover, we evaluated the approximated calculation of the expectation value and standard deviation of the RBE-weighted dose in combination with a probabilistic effect-based optimization on three patient cases considering carbon ions as radiation modality against sampled references. The resulting global γ-pass rates (2 mm,2%) are [Formula: see text]99.15% for the expectation value and [Formula: see text]94.95% for the standard deviation of the RBE-weighted dose, respectively. We applied the derived analytical model to carbon ion treatment planning, although the concept is in general applicable to other ion species considering a variable RBE.

Efficiency of analytical and sampling-based uncertainty propagation in intensity-modulated proton therapy.

The sensitivity of intensity-modulated proton therapy (IMPT) treatment plans to uncertainties can be quantified and mitigated with robust/min-max and stochastic/probabilistic treatment analysis and optimization techniques. Those methods usually rely on sparse random, importance, or worst-case sampling. Inevitably, this imposes a trade-off between computational speed and accuracy of the uncertainty propagation. Here, we investigate analytical probabilistic modeling (APM) as an alternative for uncertainty propagation and minimization in IMPT that does not rely on scenario sampling. APM propagates probability distributions over range and setup uncertainties via a Gaussian pencil-beam approximation into moments of the probability distributions over the resulting dose in closed form. It supports arbitrary correlation models and allows for efficient incorporation of fractionation effects regarding random and systematic errors. We evaluate the trade-off between run-time and accuracy of APM uncertainty computations on three patient datasets. Results are compared against reference computations facilitating importance and random sampling. Two approximation techniques to accelerate uncertainty propagation and minimization based on probabilistic treatment plan optimization are presented. Runtimes are measured on CPU and GPU platforms, dosimetric accuracy is quantified in comparison to a sampling-based benchmark (5000 random samples). APM accurately propagates range and setup uncertainties into dose uncertainties at competitive run-times (GPU [Formula: see text] min). The resulting standard deviation (expectation value) of dose show average global [Formula: see text] pass rates between 94.2% and 99.9% (98.4% and 100.0%). All investigated importance sampling strategies provided less accuracy at higher run-times considering only a single fraction. Considering fractionation, APM uncertainty propagation and treatment plan optimization was proven to be possible at constant time complexity, while run-times of sampling-based computations are linear in the number of fractions. Using sum sampling within APM, uncertainty propagation can only be accelerated at the cost of reduced accuracy in variance calculations. For probabilistic plan optimization, we were able to approximate the necessary pre-computations within seconds, yielding treatment plans of similar quality as gained from exact uncertainty propagation. APM is suited to enhance the trade-off between speed and accuracy in uncertainty propagation and probabilistic treatment plan optimization, especially in the context of fractionation. This brings fully-fledged APM computations within reach of clinical application.

¹9F nuclear magnetic resonance screening of glucokinase activators.

Human hexokinase enzyme IV (EC 2.7.1.1) catalyzes the phosphorylation of glucose and regulates the level of glucose. This enzyme exhibits strong positive cooperativity due to an allosteric transition between an inactive form and a closed active form. This form can be stabilized by activators and, thus, can increase its turnover by a kinetic memory effect characterized by a slow decay to the inactive state. The structural details of this kinetic allostery are known. Several synthetic activators have been reported. We present a preliminary nuclear magnetic resonance (NMR) screening of a chemical library in search of molecules with some affinity for glucokinase (GK). The library, composed of eight molecules with known activity as well as molecules that display no interaction, has been tested using the FAXS (fluorine chemical shift anisotropy and exchange for screening) method, based on monitoring the R2 relaxation of the (19)F spin. To ensure a valid interaction measurement, the enzyme was placed in the presence of glucose and magnesium. The binding signal of one known fluorinated ligand was measured by determining the displacement of the known ligand. This simple measure of the (19)F signal intensity after an 80-ms spin echo correlates nicely with the EC50, opening a route for NMR screening of GK activators.

Applications of NMR screening techniques to the pharmaceutical target Checkpoint kinase 1.

Ligand screening techniques based on NMR spectroscopy are not as sensitive as other commonly used methods like fluorescence, radiolabeling and surface plasmon resonance. However, using modern NMR instrumentation, they can achieve reliable screening under near physiological condition using as little as 4.6 nmol of receptor and 100 nmol of ligand. Additionally, these NMR methods can also provide valuable and specific information on the ligand under investigation such as the dissociation constant KD, the binding epitope and most importantly some structural information on the actual conformation in the bound state. In this manuscript, we describe the use of NMR based screening techniques ("Saturation Transfer Difference" (STD) and "Water Ligand Observed via Gradient SpectroscopY" (WaterLOGSY)) to detect small therapeutic molecules that interact with the DNA damage checkpoint enzyme Checkpoint kinase 1 (Chk1). After the identification of the most potent ligand, we used specific NMR experiments to perform the epitope mapping of this ligand ("Group epitope mapping-STD" (GEM-STD), "Difference of Inversion REcovery rate with and without Target IrradiatiON" (DIRECTION)) and to characterize its bound conformation ("Transferred-Nuclear Overhauser Effect SpectroscopY" (tr-NOESY), "Transferred-Rotating frame Overhauser Effect SpectroscopY" (tr-ROESY)). Finally, we used molecular docking procedures to position the ligand within the active site of Chk1. On the experimental level, a comparison between NMR studies performed in a 90%H2O/10%D2O buffer and a 100% D2O buffer is also presented and discussed.

A novel class of oxylipins, sn1-O-(12-oxophytodienoyl)-sn2-O-(hexadecatrienoyl)-monogalactosyl Diglyceride, from Arabidopsis thaliana.

The cyclic derivative of 13(S)-hydroperoxolinolenic acid, 12-oxophytodienoic acid, serves as a signal transducer in higher plants, mediating mechanotransductory processes and plant defenses against a variety of pathogens, and also serves as a precursor for the biosynthesis of jasmonic acid, a mediator of plant herbivore defense. Biosynthesis of 12-oxophytodienoic acid from alpha-linolenic acid occurs in plastids, mainly in chloroplasts, and is thought to start with free linolenic acid liberated from membrane lipids by lipase action. In Arabidopsis thaliana, the glycerolipid fraction contains esterified 12-oxophytodienoic acid, which can be released enzymatically by sn1-specific, but not by sn2-specific, lipases. The 12-oxophytodienoyl glycerolipid fraction was isolated, purified, and characterized. Enzymatic, mass spectrometric, and NMR spectroscopic data allowed us to establish the structure of the novel oxylipin as sn1-O-(12-oxophytodienoyl)-sn2-O-(hexadecatrienoyl)-monogalactosyl diglyceride. The novel class of lipids is localized in plastids. Purified monogalactosyl diglyceride was not converted to the sn1-(12-oxophytodienoyl) derivative by the combined action of (soybean) lipoxygenase and (A. thaliana) allene oxide synthase, an enzyme ensemble that converts free alpha-linolenic acid to free 12-oxophytodienoic acid. When leaves were wounded, a significant and transient increase in the level of (12-oxophytodienoyl)-monogalactosyl diglyceride was observed. In A. thaliana, the major fraction of 12-oxophytodienoic acid occurs esterified at the sn1 position of the plastid-specific glycerolipid, monogalactosyl diglyceride.

Octadecanoid and hexadecanoid signalling in plant defence.

Plants respond to situations requiring the initiation of inducible defence reactions with a complex array of signalling events that ultimately result in the activation of sets of defence genes. Among the chemical signals involved in the induction of defence reactions are cyclic oxylipins derived from C18- or C16-unsaturated fatty acids, the octadecanoids and the hexadecanoids. Key to understanding octadecanoid biology are the C18-metabolite 12-oxophytodienoic acid (OPDA) and the C12-compound jasmonic acid which is biosynthetically derived from 12-oxophytodienoic acid. Different octadecanoids likely have different biological functions. The bouquet of signalling compounds, rather than any single compound, is probably decisive for the biological response that results. This means that the processes regulating the pool sizes of different octadecanoids and their distribution within the plant are key to understanding octadecanoid biology. Recent results, including the cloning of several enzymes of the octadecanoid biosynthetic pathway, have provided first insights into these processes and into how the octadecanoid system is linked to other defence-related signalling pathways of the plant cell.

12-Oxophytodienoate-10,11-reductase: occurrence of two isoenzymes of different specificity against stereoisomers of 12-oxophytodienoic acid

The reduction of 12-oxophytodienoic acid (OPDA) to 3-oxo-2(2'[Z]-pentenyl)-cyclopentane-1-octanoic acid is catalyzed by 12-oxophytodienoate-10,11-reductase (OPR). Analysis of the isomer preference of OPR has indicated that the activity is composed of two isoenzymes exhibiting different stereoselectivities. The two isoforms of OPR have been separated, using protein extracts of Rock Harlequin (Corydalis sempervirens) as the starting material. OPRI, the enzyme reported earlier from the same species and corresponding to the cloned OPR from Arabidopsis, utilized 9R,13R-OPDA >> 9S, 13R-OPDA but not the 13S-configured isomers, whereas the new activity, OPRII, effectively reduced all four OPDA isomers, including the natural 9S,13S-OPDA (cis-[+]-OPDA). OPRII activity is characterized in detail. The enzyme's enzymatic, biochemical, and immunological properties prove that it is a close relative of OPRI. The roles of OPRI and OPRII in octadecanoid biology are discussed.

A zinc chelator inhibiting gelatinases exerts potent in vitro anti-invasive effects.

Matrix metalloproteinases are zinc metalloenzymes involved in remodelling of the extracellular matrix. We compared the anti-invasive properties of a zinc ejector matrix metalloproteinase inhibitor with those of reference compounds (hydroxamic acid-based BB-94 and Ro-31-9790) which form inactive ternary complexes with the enzymes and the catalytic zinc. We show that the compound undecadenedioic acid bis-[[2-(3 H-imidazol-4-yl)-ethyl]-amide] (S 30372) is active against gelatinases, chelates zinc and exhibits enzymatic features compatible with the potential to extract zinc from gelatinases. We then used five invasive cell lines in the Matrigel invasion chamber assay (NIH-3T3 fibroblasts, Lewis lung carcinoma cells, EJ138 and J82 bladder carcinoma and HT1080 fibrosarcoma cells). With the exception of J82 cells which were unaffected by the three inhibitors, all remaining cells were substantially more sensitive to S 30372 in terms of maximal inhibition of invasion attained. This suggests that matrix metalloproteinase inhibitors with zinc chelating/ejecting properties may be more efficient in preventing tumor progression.

Quantitation of the octadecanoid 12-oxo-phytodienoic acid, a signalling compound in plant mechanotransduction.

The octadecanoid 12-oxo-phytodienoic acid (OPDA) is an intermediate in biosynthesis of jasmonic acid in plants. A technique for the quantitation of this compound is described which has a limit of detection of 20 pg cis-OPDA corresponding to 4 ng g-1 tissue for the overall procedure and which uses high isotopic abundance [2H5]cis-(+/-)-OPDA, synthesized enzymatically by recombinant allene oxide synthase, as internal standard. The levels of cis-OPDA have been determined in a wide variety of monocotyledonous and dicotyledonous angiosperms and were found to vary considerably among different species. In mechanically stimulated tendrils of Bryonia dioica, the level of cis-OPDA increases several-fold, correlating with the initiation and progression of the free coiling response. In Phaseolus vulgaris internodes undergoing a thigmomorphogenic response, the levels of cis-OPDA were also found to increase several-fold well before the development of thigmomorphogenic symptoms. The thigmomorphogenic reaction could also be triggered by application of the octadecanoid structural analog, coronatine. Coronatine did not induce OPDA accumulation in treated tissues and is thus active per se. In both species, Bryonia dioica and Phaseolus vulgaris, the (+)-enantiomer of cis-OPDA is found and accumulates after mechanical stimulation. Our results establish 12-oxo-phytodienoic acid as a signalling compound in higher plant mechanotransduction.

Analysis of 12-oxo-phytodienoic acid enantiomers in biological samples by capillary gas chromatography-mass spectrometry using cyclodextrin stationary phases.

The octadecanoid plant growth regulator 12-oxo-phytodienoic acid (12-oxo-PDA), which is also an intermediate in the biosynthesis of jasmonic acid, is obtained from 13-hydroperoxylinolenic acid via an unstable allene oxide generated by the enzyme allene oxide synthase. Recombinant, bacterially expressed and purified allene oxide synthase from Arabidopsis thaliana yields racemic 12-oxo-PDA as a mixture of 94:6 cis:trans diastereomers. In the presence of allene oxide cyclase from Solanum tuberosum, a product of high enantiomeric purity was obtained, which was shown to be (+)-cis-12-oxo-PDA (98:2 cis:trans diastereomers). Based on this coupled reaction, a preparative procedure was developed that yields pure (+)-cis-12-oxo-PDA in milligram quantities. Furthermore, an analytical technique employing capillary gas chromatography-mass spectrometry and beta- or gamma-cyclodextrin stationary phases was developed that enables the direct analysis of nanogram amounts of enantiomers of 12-oxo-PDA, as their methyl esters, in plant tissues. In the species analyzed, endogenous cis-12-oxo-PDA is the (+)-enantiomer.

Design and synthesis of new linear and cyclic bradykinin antagonists.

We report here on the synthesis and pharmacological properties of a new series of small linear and cyclic peptides derived from the five C-terminal amino acid residues of second-generation bradykinin receptor antagonists. Variations of the two first residues of the pentapeptide (Thi-Ser-D-Tic-Oic-Arg) were shown to modulate the biological activities of the analogs on bradykinin-induced smooth muscle contractions in rabbit jugular vein (RJV), a tissue preparation specific of the B2 bradykinin receptor. Several analogs showed pA2 values around 7 on this tissue preparation, and one cyclic compound, c[-Gly-Thi-D-Tic-Oic-Arg-], 24, in which Thi-Ser was replaced by Gly-Thi, displayed a pA2 of 7.4 on RJV. On the basis of these results, three cyclic molecules and their linear counterparts (compounds 22-24 and 4-6, respectively) were tested on human umbilical vein, a tissue specific of the human B2 receptor. The pKB values obtained for these compounds on these tissue preparations were equivalent to those obtained for the decapeptide NPC 567 (4.8 < pA2 < 5.1). NMR and molecular modeling studies performed on compound 24 clearly demonstrated a type II' beta-turn structure. This analog may serve as a new lead for the design of nonpeptide ligands of the bradykinin B2 receptor subtype.

Solution conformation by NMR and molecular modeling of three sulfide-free somatostatin octapeptide analogs compared to angiopeptin.

The conformation in dimethylsulfoxide of the somatostatin derivative angiopeptin and of three disulfide-free analogs was estimated by two-dimensional nuclear magnetic resonance spectroscopy at room temperature. The resulting 3D molecular graphics were compared and shown to reflect the observed differences in the inhibition of restenosis after rat aorta balloon injury by these octapeptide inhibitors. Angiopeptin and its active analog 2 displayed a relatively rigid conformation of the cyclic hexapeptide backbone due to the presence of two well-defined hydrogen bonds, further stabilized by a third hydrogen bond outside the ring. No such constraints were detected for the two biologically inactive analogs, which, compared to 2, had a two-atom longer or shorter hexapeptide ring. The well-defined structure of compound 2 may serve as an improved pharmacophore for this new class of drugs.

Combinatorial peptide libraries: robotic synthesis and analysis by nuclear magnetic resonance, mass spectrometry, tandem mass spectrometry, and high-performance capillary electrophoresis techniques.

Combinatorial peptide libraries are a new source of compounds from which a large number of pharmacological leads will emerge in the next few years. A large body of literature shows that this approach is of considerable interest in many areas of biological sciences for the search of enzyme substrates and inhibitors, of receptor agonists or antagonists, or of antigen sequences. Nevertheless, the analytical investigation of such complex mixtures as libraries which contain up to millions of individual molecules is still poorly documented. In this work, we present analytical solutions for their characterization. NMR and tandem mass spectrometry (MS/MS) can provide an in deep description of any type of combinatorial libraries, while MS and high-performance capillary electrophoresis can bring a rapid and overall information at the routine level, sufficient to ensure a first assessment of their composition. MS in the fast atom bombardment mode was used to describe the libraries O1X2X3X4X5 or O1X2X3X4 (Oi and Xi are fixed and random residue in position i, respectively). Advantage was taken of the high proton affinity of arginine and of its induction of charge remote fragmentation to interpret the MS spectra of whole libraries and neutral losses (MS/MS) in the model sublibraries ArgGlyX3X4 and NipValX3X4 (Nip,4-nitrophenylalanine). Two-dimensional NMR allowed the incorporation of the individual residues during synthesis to be tested in 24 sublibraries O1X2X3X4. While from the pharmacological point of view, impressive discoveries made with combinatorial peptide libraries have already been reported, our results show that they should be complemented by appropriate analytical tools, crucial for the proper characterization and exploitation of these libraries.

In vivo and in vitro glucuronidation of the flavonoid diosmetin in rats.

Flavonoids form an important family of compounds widely present in plants and therefore in food, sometimes as substitutes for synthetic antioxidants. Although the excretion routes of flavonoids in animals has been explored, little is known about the details of their conjugation in the xenobiotic metabolism pathway. In this study, we investigate the metabolism of diosmetin as a model compound in the rat, particularly its level in blood after treatment (100 mg/kg, po) and the presence of its glucuronide(s) in both blood and urine. We demonstrate that after po treatment of the rat, a rapid glucuronidation takes place and that diosmetin circulates as glucuronides, whereas no free diosmetin is present in blood and urine. The glucuronides formed are present in the blood plasma at a high level (approximately 10 micrograms/ml), for at least 6 hr after the treatment and the conjugates are excreted in urine. We have detected four different glucuronides in blood and characterized the two major ones after purification by a combination of MS, NMR, and UV spectroscopy: diosmetin-7,3'-diglucuronide and diosmetin-3'-glucuronide. A brief characterization of the in vitro glucuronidation of some flavonoid compounds using liver microsomal preparations suggests that this important class of natural compounds might be conjugated by a specific isoform of UDP-glucuronosyltransferase. Thus, this work brings evidence that diosmetin is rapidly glucuronoconjugated in the rat and provides an explanation for the low po bioavailability of the unchanged compound that can be generalized to this important class of natural compounds.

Isolation, sequence, and conformation of seven trichorzianines B from Trichoderma harzianum.

From the antagonistic fungus Trichoderma harzianum, a group of acidic new peptides, trichorzianines B (TB), was isolated in addition to neutral trichorzianines A (TA) previously studied. TA and TB exhibit various biological activities related to their membrane properties and a different behaviour of the two groups was noticed. As observed for other peptaibols, TB consist in a microheterogeneous mixture which was resolved into pure peptides by reversed-phase C18 HPLC. The sequence of the seven main isolated TB, namely TB IIa, TB IIIc, TB IVb, TB Vb, TB VIa, TB VIb, TB VII, was determined by the combined use of positive ion FAB mass spectrometry and 2D 1H n.m.r. spectroscopy, including COSY and NOESY experiments. TB differ from the corresponding TA only by the replacement of Gln 18 in the TA sequence by a glutamic acid. The 1H n.m.r. data suggested that the TB are mainly organized in an alpha helix.