Apeiron Synthesis SA

Wrocław, Poland

Apeiron Synthesis SA

Wrocław, Poland

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News Article | September 28, 2016
Site: www.nature.com

If not stated otherwise, all chemicals and reagents were obtained from Sigma Aldrich. Restriction enzymes were obtained from New England Biolabs. The AquaMet catalyst was purchased from Apeiron Synthesis S.A. All plasmids used in this study are collated in Supplementary Table 1. To construct the periplasmic expression vector for SAV, the gene for T7-tagged SAV was amplified by polymerase chain reaction (PCR) from pET-11b-SAV31 using primers 1 and 2 (Supplementary Table 2) to add the 21 amino acid OmpA signal peptide (MKKTAIAIAVALAGFATVAQA) to the N terminus of SAV. The PCR product was digested with restriction enzymes NdeI and BamHI, gel purified and ligated into the target vector pET-30b(+) (Merck Millipore) pre-treated with the same enzymes. The resulting expression vector, designated pET-30b-SAVperi, carries the gene for the OmpA::SAV fusion protein under the control of a P promoter. To construct a comparable cytoplasmic expression construct, the gene for T7-tagged SAV from pET11b-SAV was PCR-amplified without adding additional amino acids using primers 3 and 2 (Supplementary Table 2) and subsequently cloned into pET-30b(+) by restriction digest (NdeI and BamHI) and ligation, resulting in plasmid pET-30b-SAVcyto. All strains used in this study are summarized in Supplementary Table 3. A strain for periplasmic expression was constructed that combines the ease of library generation with the compatibility with the T7-expression system. Therefore, the gene of the T7 RNA polymerase was integrated into the chromosome of E. coli TOP10 (F− mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ−, Thermo Fisher Scientific) using the λDE3 Lysogenization Kit (Merck Millipore). The resulting lysogen was designated E. coli TOP10(DE3). E. coli TOP10(DE3) containing the respective expression plasmid for SAV (pET-30b-SAVperi or pET-30b-SAVcyto) was cultivated in a Luria–Bertani (LB) medium (50 ml)32 supplemented with kanamycin (50 mg l−1) in shake flasks (500 ml, 37 °C, 200 r.p.m.). An LB pre-culture was diluted 1:100 in fresh medium and incubation was performed until an optical density at 600 nm (OD ) of about 0.5 was reached. Subsequently, the cultivation temperature was lowered to 20 °C, and the expression of SAV was induced by addition of isopropyl-β-d-thiogalactopyranoside (IPTG, 50 μM) and cells were harvested by centrifugation (6,000 r.c.f., 2 min) after four hours of induction. The cell pellet was further processed either for fractionation to analyse the cellular protein content or for flow cytometry. To separate the periplasmic and the cytoplasmic fraction of cellular proteins, the PeriPreps Periplasting Kit (Epicentre Technologies) was used. For in-gel staining of SAV, biotin-4-fluorescein (10 μM) was added to the respective fractions before sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) analysis. Owing to the binding to the biotinylated fluorescent dye, SAV can be visualized under ultraviolet light in the gel (before staining with Coomassie blue). Owing to its high stability, SAV remains tetrameric and fully functional even under the otherwise denaturing conditions of SDS–PAGE. To stain SAV in whole cells of E. coli, the pellet of a shake-flask expression culture was resuspended in phosphate buffer saline (PBS32) to a final OD  = 1 before the addition of Atto-565-biotin (2 μM, Atto-Tec GmbH). Cells were incubated on ice (30 min) and subsequently subjected to three wash cycles by centrifugation (6,000 r.c.f., 2 min) and resuspension of the pellet in PBS. The resulting cell suspensions were analysed on a BD LSR Fortessa SORP (BD Biosciences) using a 561-nm laser for the excitation of Atto-565-biotin and a 610/20BP-600LP filter combination for analysis of the emitted fluorescence signal (peak area). The histograms displayed comprise data of 100,000 ungated events for each sample. For expression of SAV (and mutants thereof) in the 96-deepwell format, LB medium (500 μl) supplemented with kanamycin (50 mg l−1) was inoculated from a single colony and grown until stationary phase (37 °C, 300 r.p.m., 50-mm shaking amplitude). This pre-culture (aliquot of 30 μl) was used to inoculate a main culture (1 ml) in a modified ZYM-5052 medium33 that lacked lactose as auto-induction agent, but contained kanamycin (50 mg l−1). Induction was performed by the addition of IPTG ([IPTG]  = 50 μM) and continued for four hours before harvesting. Subsequently, a fraction of the cultures (100 μl) was set aside for OD determination and the remaining suspension was subjected either to ICP-OES quantification or to metathesis with whole cells. The activity of the artificial metalloenzyme biot-Ru–SAV was quantified in the reaction buffer (100 mM sodium acetate, pH 4.0, 0.5 M MgCl , 2.5% DMSO) both in the presence and absence of 10 equiv. of glutathione (GSH or GSSG; 500 μM) relative to the amount of the cofactor biot-Ru (50 μM). The reaction mixture (total volume of 200 μl) was incubated shaking (16 h, 37 °C) and, after the reaction, 800 μl of methanol containing 2-phenylethanol (final concentration of 100 μM) as an internal standard was added. After centrifugation (5 min, 21,000 r.c.f., 4 °C), 500 μl of the supernatant were diluted with 500 μl of de-ionized water and the samples were subjected to UPLC analysis. UPLC analysis was performed using a Waters H-Class Bio using a BEH C18 1.7 μM column and a flow rate of 0.6 ml min−1 (eluent A, 0.1% formic acid in water; eluent B, 0.1% formic acid in acetonitrile; gradient at 0 min: 90% A, 10% B; at 0.5 min: 90% A, 10% B; at 2.5 min: 10% A, 90% B; at 3.5 min: 90% A, 10% B; at 4.5 min: 90% A, 10% B). The ultraviolet signal at 210 nm was used for quantification, and concentrations of the metathesis product umbelliferone 2 (retention time of 1.38 min) were determined on the basis of a standard curve with commercially available umbelliferone (Sigma Aldrich). To quantify the ruthenium content of cells, cultures from 96-deepwell plates were pelleted by centrifugation (3,220 r.c.f., 12 min, 4 °C) and resuspended in ice-cold Tris/HCl-buffer (1 ml, 50 mM, pH 7.4, 0.9% (w/v) NaCl) containing biot-Ru (10 μM). After incubation on ice (30 min), the cells were spun down (1,260 r.c.f., 8 min, 4 °C), the supernatant-containing excess cofactor was discarded, and the pellet was washed by resuspension in ice-cold Tris/HCl-buffer (1 ml), centrifugation and careful removal of the supernatant. Afterwards, the pellet was resuspended in de-ionized water (500 μl). Twelve replicates of this suspension were combined and concentrated nitric acid was added (550 μl, 65%) to fully digest the cellular material (48 h, 110 °C, pressure vials). The resulting clear solutions were diluted to a final volume of 10 ml with de-ionized water (containing 1 p.p.m. yttrium as internal standard) and subjected to ICP-OES quantification. The obtained p.p.b.-values (1 p.p.b. = 1 μg l−1) for ruthenium were transformed into a molar concentration and the average ruthenium atom count per cell was calculated from the OD of the cultures before decomposition assuming an OD -to-cell-number correlation for E. coli in complex medium of 7.8 × 108 ml−1 OD −1, as described in ref. 34. The cellular metathesis activity was quantified using a fluorescent assay. For this purpose, cell cultures from 96-deepwell plate cultivations (see above) were pelleted by centrifugation (3,220 r.c.f., 12 min) and resuspended in ice-cold Tris/HCl-buffer (500 μl, 50 mM, pH 7.4) containing biot-Ru (2.1 μM). This buffer was supplemented with NaCl (0.9% (w/v)) to adjust to a physiological NaCl concentration. To allow cellular uptake of biot-Ru, the suspensions were incubated on ice for 30 min, spun down (1,260 r.c.f., 8 min) and the supernatant with its excess of cofactor was discarded. The pellets were resuspended in the reaction buffer (160 μl, 100 mM sodium acetate, pH 4.0, 0.5 M MgCl ) and the reaction was initiated by addition of these cell suspensions (150 μl) to the substrate solution (50 μl reaction buffer containing 40 mM precursor 1 and 20% DMSO), leading to a final substrate concentration of 10 mM and 5% DMSO. As the metathesis product umbelliferone 2 is fluorescent, the reaction progress was monitored by fluorescence in a microtiter plate reader (excitation wavelength, 322 ± 4.5 nm; emission wavelength, 440 ± 10 nm; Infinite M1000 PRO, Tecan Group AG) at 37 °C and agitation (6-mm amplitude, orbital). Cell-specific metathesis activity is specified as the slope of the increasing fluorescence signal in the linear range of the reaction normalized by the OD of the respective culture. To generate diversity in the scaffold protein SAV, a focused, semi-rational strategy was pursued: the 20 amino acid residues closest to the ruthenium ion (see Supplementary Table 4) in a related artificial metalloenzyme structure8 were selected and individually randomized in SAVperi by site-saturation mutagenesis using NNK codons30. Degeneration was introduced by application of the Quikchange Site Directed Mutagenesis Protocol (Stratagene) using degenerate oligos (see Supplementary Table 4) and, after transformation, the libraries were checked for diversity by Sanger-sequencing of at least four individual clones before screening. To evaluate the performance of the SAVperi variants, the aforementioned fluorescent metathesis assay was carried out with at least 90 clones from the library in one 96-well plate (three replicates of strains producing ‘wild-type’ SAV (parent) and three replicates of strains carrying an empty vector control (pET-30b(+ ), lacking the gene for SAV ). Evaluating 90 members of an NNK library ensures a >94% likelihood of screening all 20 amino acid residues in the respective position29. To compensate for biological variance, promising clones were isolated and subjected to a replicate assay that was identical to the protocol described above, but using eight independent cultures per clone. After this first screening round, promising residues were ordered according to their potential impact on catalysis and mutations were combined using iterative saturation mutagenesis (ISM)30. To perform kinetic experiments on the artificial metathase, the initially used T7-tagged SAV as well as the quintuple mutant SAVmut that was isolated after ISM were cloned into a cytoplasmic expression vector (see Methods section ‘Cloning of SAV expression constructs’) and purified on an iminobiotin sepharose column as described elsewhere35. The biotin binding capacity was determined using a fluorescent quenching assay36. Kinetic measurements were performed in reaction buffer (200 μl total volume, 100 mM sodium acetate, pH 4.0, 0.5 M MgCl , 11.5% (v/v) DMSO) containing biot-Ru (50 μM) both in the presence and absence of purified SAV (100 μM binding sites of either T7-tagged SAV or SAVmut) as well as the substrate 1 (variable concentrations, 0–5 mM) for fluorescent RCM. The reaction was monitored in a microtiter plate reader as described in Methods section ‘Fluorescent metathesis assay with whole cells’. The maximum velocity of the reaction was determined from the fluorescent signal curve by linear regression. To retrieve kinetic parameters, the reaction velocities were plotted over the respective substrate concentrations using the software GraphPad Prism (GraphPad Software, version 6.05) and applying the integrated ‘Michaelis–Menten’ least-squares fit with no constraints for the maximum velocity V and substrate affinity K . Crystals of SAV and variant SAVmut were obtained at 20 °C within two days by the sitting-drop vapour diffusion technique mixing 1 μl crystallization buffer (1.5 M ammonium sulfate, 0.1 M sodium acetate, pH 4.0) and 4 μl protein solution (26 mg ml−1 lyophilized protein in water). The droplet was equilibrated against a reservoir solution of 100 μl crystallization buffer. Subsequently, single crystals were soaked for two days at 20 °C in a soaking buffer, which was prepared by mixing 1 μl of a 10 mM stock solution of complex biot-Ru (in 50% aqueous DMSO) and 9 μl crystallization buffer. After the soaking, crystals were transferred for 30 s into a cryo-protectant solution consisting of 25% (v/v) glycerol in crystallization buffer. Next, crystals were shock-frozen in liquid nitrogen. Additional soaking of the above metathase crystals with substrate surrogate 1 did not lead to fluorescence. We therefore conclude that the multiple catalytic steps (for example, ligand displacement and cross-metathesis) required to ultimately liberate umbelliferone 2 cannot take place within a crystal. X-ray diffraction data were collected at the Swiss Light Source beam line X06DA at a wavelength of 1 Å and processed with the software XDS37 and AIMLESS (CCP4 Suite)38. The structure was solved by molecular replacement using the program PHASER (CCP4 Suite)38 and the structure 2QCB from the PDB as an input model with ligand and water molecules removed. For structure refinement REFMAC5 (CCP4 Suite)39 and PHENIX.REFINE40 were used. Ligand manipulation was carried out with the program REEL using the small-molecule crystal structure ABEJUM from the Cambridge Structural Database as an input model40. For water picking and electron density and structure visualization, the software COOT41 was used. Figures were drawn with PyMOL (the PyMOL Molecular Graphics System, version 1.5.0.5, Schrödinger, LLC). Crystallographic details, processing and refinement statistics are given in Supplementary Table 5. There is one SAVmut monomer in the asymmetric unit from which a tetramer can be generated by application of two orthogonal crystallographic two-fold symmetry axes. The 12 N-terminal residues of the T7-tag and 25 residues at the C terminus are not resolved, probably owing to disorder. Residual electron density in the biotin-binding site and the biotin vestibule as well as two strong anomalous dispersion density peaks in the biotin vestibule (Extended Data Fig. 3) suggested modelling of complex biot-Ru in two conformations I and II (56% and 44% occupancy, respectively). This projects the ruthenium atom in either one of the two densities, in close proximity to a crystallographic two-fold symmetry axis (Fig. 3b, Extended Data Figs 3, 4c, d). Only partial or no electron density was present for the mesityl linker and the terminal mesityl group, probably owing to high flexibility. In conformer I, the lengthy dimesitylimidazolidine (DMI)-Ru head group reaches into the neighbouring cis-related SAVmut monomer. The I conformer is stabilized mostly by hydrophobic interactions between the distal face of the DMI ligand and amino acid side chains within two neighbouring cis-related 7,8-loops (L1101, S1121, T114Q1, K121R1, L1241, S1122, W1202, K121R2, L1242; superscripts refer to monomers 1 or 2 of the tetrameric SAVmut; Extended Data Fig. 4c). Besides the DMI ligand, two chloride ions could be modelled binding to the ruthenium, but no density was found for the alkylidene, presumably owing to high flexibility and/or low occupancy. The orientation of the chlorides was very similar to that in a small-molecule crystal structure of the Grubbs–Hoyveda second-generation catalyst (ABEJUM from the Cambridge Structural Database), placing them nearly in trans position to each other. The ruthenium is largely solvent-exposed, which could facilitate substrate binding and product release. In Fig. 3b, the alkylidene was modelled binding to the ruthenium (magenta stick model) to highlight its orientation in the biotin vestibule. Conformer II is different from I by a rotation (about 60°) of the DMI-Ru moiety around an axis parallel to the cylinder axis of the SAVmut β-barrel (Fig. 3b, Extended Data Fig. 4d). This rotation places the hydrophobic distal side of the DMI-ligand in proximity to amino acid side chains within loop-5,61 (A86, H87), loop-7,81 (S112, T114Q) and loop-4,53 (D67, S69, A65) of a neighbouring trans-related SAV monomer. Atom N49K-Nε is located in close proximity to ruthenium (2.4 Å). No electron density was found to model chloride ions and the alkylidene ligand bound to the ruthenium. Because the cofactor is bound in close proximity to a two-fold crystal symmetry axis, formation of a cis-symmetry-related neighbouring cofactor by application of the crystal symmetry operation results in extensive steric clashes between the two cofactors in the orientation I. In contrast, coexistence of cofactor pairs I–I or II–II orientation is sterically accessible (Extended Data Fig. 3a, c, e). Normalized B factors of residues within cofactor-flanking loop-7,8 are increased by up to about 0.5 when compared to related SAV structures that crystalized in the same space group and in a very similar unit cell (Extended Data Fig. 5b). This suggests increased loop-7,8 flexibility (Fig. 3b, Extended Data Fig. 4c, d). This flexibility is likely to be caused by three factors: (i) mutation T114Q cleaves a hydrogen-bond between threonine-OγH and T115-carbonyl oxygen (green dashed line in Fig. 3b and Extended Data Fig. 4a, b) and leads to a new hydrogen-bond between T114Q-glutamine-Nε and S112-OγH (red dashed line in Fig. 3b and Extended Data Fig. 4c, d); (ii) mutation A119G in the loop leads to increased entropy; and (iii) mutation V47A reduces steric hindrance between V47A in loop-3,42 and W120 in loop-7,81 (Fig. 3b and Extended Data Fig. 4c, d). The overall structure of complex biot-Ru–SAV is virtually identical to that of complex biot-Ru–SAVmut (root-mean-square deviation, r.m.s.d. = 0.25 Å). As in the mutant, two strong residual electron density peaks (F  − F cofactor omit map: 12σ (conformer I) and 11σ (conformer II)) and two anomalous dispersion density peaks (9σ (conformer I) and 5σ (conformer II)) were located within the biotin vestibule of a SAV monomer at the interface between two symmetry-related SAV monomers (Extended Data Fig. 3b, d, f). The same two cofactor conformations I and II found in mutant SAVmut were modelled in the biotin binding vestibule of SAV with an occupancy of 50% for each conformer I and II (Extended Data Fig. 4a, b). The side chain of residue L110 adopts two conformations with 50% occupancy each (Extended Data Fig. 4a, b). The close proximity of the terminal methyl group in L110 conformation A to the aromatic mesityl ring (distance between L110-CδH and mesityl of 4.4 Å) of cofactor conformation I suggests a stabilizing σ–π interactions (Extended Data Fig. 4a, red star). The L110 side chain conformation B can coexist only with cofactor conformation II (Extended Data Fig. 4b). This hypothesis is supported by the fact that the same L110 side chain conformation A is found in complex [(Cp*)Ir(Biot-p-L)Cl]-SAV-S112A (PDB, 3PK2), which has an aromatic ring located in the same position as the mesityl linker in structure biot-Ru–SAV, suggesting a similar σ–π interaction. In contrast, the side chain of L110 in apo-SAV (PDB, 2BC3) adopts conformation B. In complex biot-Ru–SAV, a water molecule is bound in proximity to L110 with 50% occupancy (Extended Data Fig. 4b). Steric clashes with L110 side chain in conformation A and the NHC ligand of biot-Ru conformer I suggest that the water is present only with L110 conformation B and biot-Ru conformer II. Additionally, the side chain of L124 adopts two conformations, each with 50% occupancy. Only conformation L124A does not undergo steric clashes with a methyl group of the bridging mesityl moiety of cofactor conformer I (Extended Data Fig. 4a, b). Together, the conformational side chain flexibility of residues L110 and L124 reflects the presence of the two cofactor conformations I and II. In contrast to artificial metalloenzyme biot-Ru–SAVmut, the normalized B factors of residues within the cofactor-flanking loop-7,8 in complex biot-Ru–SAV do not show increased values when compared to those in related crystal structures (Extended Data Fig. 5b). Indeed, a hydrogen-bond is formed between T114-OγH and T115-carbonyl oxygen in complex biot-Ru–SAV that could rigidify the loop (Extended Data Fig. 4a, b). The conversion for the product 4 was quantified by 1H-NMR. For this purpose, a deuterated reaction buffer was prepared from acetic acid-d , dry MgCl and D O with the same concentrations as for the reaction buffer used for substrate 1 (100 mM acetate, 0.5 M MgCl ). The pH was adjusted to 3.6 by addition of 1 M NaOD in D O (with respect to pD = pH + 0.4). For the reaction, 300 μl of a substrate 3 stock solution (100 mM in deuterated reaction buffer) was mixed with 291 μl of either a solution of SAV, SAVmut, or SAVmut2 (200 μM binding sites in deuterated reaction buffer) or plain deuterated reaction buffer (for samples without SAV or any of the SAV variants). Afterwards, 9 μl of a biot-Ru (or HGII/AQM) stock solution (3.34 mM in DMSO-d ) was added to obtain a final concentration of 50 μM and the reaction was performed for 16 h at 37 °C and 200 r.p.m. The mixture was analysed by 1H-NMR and the yield of the reaction product 4 was quantified by comparing integrals (I) of the product 4 peaks at 3.41 p.p.m. and 2.05 p.p.m. and the substrate 3 peaks at 3.33 p.p.m. and 1.91 p.p.m. using: yield = I /(I  + I ). To quantify the conversion of substrate 5, a 97 μl aliquot of either SAV solution (200 μM SAV binding sites in reaction buffer) or plain reaction buffer (for samples without SAV variants) was mixed with 100 μl of a stock solution of substrate 5 (20 mM in reaction buffer). Subsequently, 3 μl of the respective catalyst/cofactor stock solution (3.34 mM in DMSO) was added to obtain a final concentration of 50 μM. The reaction was performed for 16 h at 37 °C and 200 r.p.m. Then, an aqueous solution of benzyltriethylammonium chloride (100 μl, 10 mM) was added as an internal standard and 700 μl of methanol was added. The mixture was cleared by centrifugation and 250 μl of the supernatant was mixed with 750 μl of water for the final quantification of product 6 by UPLC-MS. For the kinetic experiment, the reaction mixture was scaled up to a total volume of 1 ml and 50 μl aliquots of this mixture were collected at different time points and immediately injected into 950 μl of a quenching solution (0.5 mM potassium cyanoacetate, 0.25 mM benzyltriethyl-ammonium chloride (internal standard) in 50% aqueous methanol). After removal of precipitated protein by centrifugation, the supernatant was analysed by UPLC-MS. To quantify the cellular metathesis activity for substrate 5, a protocol analogous to that applied for the umbelliferone precursor 1 was applied. The substrate 5 (final concentration 10 mM) was added to whole cells and the samples were incubated at 37 °C and 300 r.p.m. for 16 h. To quantify the conversion for the non-fluorescent product 6, an extraction was performed: 800 μl of methanol was added to each sample and an extraction was carried out (one hour with vigorous shaking, 800 r.p.m. at room temperature). The samples were cleared by centrifugation, the supernatant was diluted with water (factor four) and analysed by UPLC-MS using a calibration curve recorded for product 6. No statistical methods were used to predetermine sample size.


Patent
Apeiron Synthesis S.A. and University Waszawski | Date: 2013-08-14

The object of the invention is a novel metal complex of the formula (1), the parameters of which are as defined in the description of the invention, as well as use of the metal complex of the formula (1) in the olefin metathesis reactions.


PubMed | Apeiron Synthesis SA, Wroclaw University of Technology, Nicolaus Copernicus University and Warsaw University of Technology
Type: Journal Article | Journal: Angewandte Chemie (International ed. in English) | Year: 2016

The state-of-the-art in olefin metathesis is application of N-heterocyclic carbene (NHC)-containing ruthenium alkylidenes for the formation of internal C=C bonds and of cyclic alkyl amino carbene (CAAC)-containing ruthenium benzylidenes in the production of terminal olefins. A straightforward synthesis of bis(CAAC)Ru indenylidene complexes, which are highly effective in the formation of both terminal and internal C=C bonds at loadings as low as 1ppm, is now reported.


Pastva J.,J. Heyrovsky Institute of Physical Chemistry | Skowerski K.,Apeiron Synthesis S.A. | Czarnocki S.J.,Apeiron Synthesis S.A. | Zilkova N.,J. Heyrovsky Institute of Physical Chemistry | And 3 more authors.
ACS Catalysis | Year: 2014

Ruthenium olefin metathesis catalysts bearing a polar quaternary ammonium group in N-heterocyclic ligand were immobilized on silica and siliceous mesoporous molecular sieves with different pore sizes (SBA-15 and MCM-41). The activity of the heterogeneous catalysts was found to increase with an increase in pore size of the support used, with the best results observed for SBA-15-supported catalyst. The influence of reaction conditions (temperature, solvent, catalyst, and substrate concentration) on the efficiency of new heterogeneous catalysts was established. A significant influence of the counterion present in the ruthenium complex on the activity of immobilized catalysts was also found: those derived from chloride containing ion exhibited the highest activity. High activity in ring-closing metathesis of substrates as citronellene, 1,7-octadiene, and diallyl compounds as well as in cross-metathesis of unsaturated aliphatic compounds with methyl acrylate was observed under optimized conditions. In some cases, heterogenization led to catalysts with efficiency higher than those observed for corresponding homogeneous complexes. © 2014 American Chemical Society.


Skowerski K.,Apeiron Synthesis SA | Pastva J.,Apeiron Synthesis SA | Czarnocki S.J.,Apeiron Synthesis SA | Janoscova J.,J. Heyrovsky Institute of Physical Chemistry | Janoscova J.,University of Pardubice
Organic Process Research and Development | Year: 2015

The ammonium tagged Hoveyda-type catalyst bearing sterically enlarged N-heterocyclic carbene ligand was synthesized and supported on SBA-15. The obtained heterogeneous olefin metathesis catalyst forms unprecedentedly stable Ru-methylidenes and provides products of ring-closing and cross metathesis with turnover numbers (TONs) up to 35 000 and turnover frequencies (TOFs) up to 1590 min-1. The catalyst proved to be truly recyclable and effective in a continuous flow mode. © 2015 American Chemical Society.


Kozlowska A.,University of Warsaw | Dranka M.,Warsaw University of Technology | Zachara J.,Warsaw University of Technology | Pump E.,University of Graz | And 3 more authors.
Chemistry - A European Journal | Year: 2014

Cyclic Ru-phenolates were synthesized, and these compounds were used as olefin metathesis catalysts. Investigation of their catalytic activity pointed out that, after activation with chemical agents, these catalysts promote ring-closing metathesis (RCM), enyne and cross-metathesis (CM) reactions, including butenolysis, with good results. Importantly, these latent catalysts are soluble in neat dicyclopentadiene (DCPD) and show good applicability in ring-opening metathesis polymeriyation (ROMP) of this monomer. © 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.


This disclosure relates to new metal complexes, such as compounds of Formula 1, and their application in olefin or alkyne metathesis and to methods of carrying out olefin metathesis reactions.


This disclosure relates to new metal complexes, such as compounds of Formula 1, and their application in olefin or alkyne metathesis and to methods of carrying out olefin metathesis reactions.


Patent
Apeiron Synthesis S.A. | Date: 2014-06-24

The invention concerns use of metal scavengers of the formula (1), wherein the variables are as defined in the description of the invention, for removal of ruthenium residues, compounds, or complexes thereof, from the post-reaction mixtures, from the products of reactions catalysed with ruthenium complexes, as well as from organic compounds contaminated with ruthenium.


The invention is related to the metal complexes of the general formula (1). The invention is related also to the use of metal complexes of the formula 1 as (pre)catalysts for the olefin metathesis reactions, as well as to the process for carrying out the olefin metathesis reaction.

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