Complete Cleavage Research Articles

Symmetrical polymer systems prepared using a degradable bifunctional atom transfer radical polymerization initiator: Synthesis, characterization, and cleavage

ABSTRACTLinear (co)polymers and dimethacrylate‐end‐linked polymer networks of methyl methacrylate with 2‐(dimethylamino)ethyl methacrylate, cleavable in the middle of the polymer chain, either under thermolysis or alkaline hydrolysis conditions, were prepared via atom transfer radical polymerization (ATRP) using a specially designed bifunctional degradable initiator. This initiator was 2,6‐pyridinediethanol di(2‐bromo‐2‐methyl propanoate) (PyDEDBrMeP), bearing two 2‐(pyridin‐2‐yl)ethyl ester moieties, known for their thermal and hydrolytic (alkaline conditions) lability. As a control, a more stable bifunctional ATRP initiator, 2,6‐pyridinedimethanol di(2‐bromo‐2‐methyl propanoate) (PyDMDBrMeP), was also synthesized together with the corresponding linear polymers and polymer networks prepared from it. Thermal or hydrolytic treatment of the polymers prepared using PyDEDBrMeP led to a reduction in the molecular weights of the linear polymers by a factor of two, and to the conversion of the polymer networks to soluble branched (star) structures, consistent with the expected cleavage of the initiator residue located in the middle of the polymer chain. Thermal treatment of the polymers prepared using PyDMDBrMeP did not affect their molecular weight due to the thermal stability of the (pyridin‐2‐yl)methyl ester group, while treatment under alkaline hydrolysis conditions resulted in complete cleavage, similar to the PyDEDBrMeP‐prepared polymers. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017, 55, 2342–2355

The predictability of ocriplasmin treatment effects: is there consensus among retinal experts? Results from the EXPORT study.

To evaluate the agreement and predictability of ocriplasmin treatment effects among retinal experts (raters) by assessment of retinal imaging data of eyes treated for vitreomacular traction in nine different centers in Germany and Austria. Retrospective cohort study. Combined confocal near-infrared scanning laser ophthalmoscopy and spectral-domain optical coherence tomography images (Spectralis® device, Heidelberg Engineering GmbH, Germany) from 136 eyes of 135 subjects were reviewed by 14 raters using an internet-based grading database and a standardized questionnaire. In addition to the images taken within 2 days prior to treatment, age, gender, and lens status were disclosed to the raters. Treatment success was defined as a complete cleavage of the posterior vitreous cortex at day 28±5. Main outcome was the agreement and predictability among raters for assessment of treatment success. Raters generally accepted starting ocriplasmin treatment (chance for treatment success ≥ 1%) in 22.4 to 69.1% (median 53.2%) of eyes (moderate intra- and interrater agreements with kappa-values of 0.6 and 0.48). The likelihood for a high potential treatment success (equal or higher than 25%) was judged by the raters in 43.4% to 86.0% (median 62.6%) of eyes (moderate intra- and fair interrater agreements with kappa-values of 0.56 and 0.22). Allocating eyes for high potential treatment success overall increased the odds by 3.07, with odds ratios of single raters up to 4.06 to 6.16. These results underscore the importance of training health care providers in the evaluation of retinal imaging data and also to define characteristic morphological features better in the presence of vitreoretinal interface diseases. The better results of single raters in the predictability of treatment success by the allocation of eyes in the high-potential group indicates the high relevance of the meticulous analysis of retinal images.

Towards a glycosidase toolbox for glycan structure analysis: General Label‐Free Mass Spectrometry‐Based Assay to Identify Glycosidase Substrate Competence

Glycosidases are enzymes that catalyze the hydrolysis of glycosyl linkages and are widely used in oligosaccharide linkage analysis1. Common glycosidase assays, however, rely on using derivatized non‐natural substrates for analysis. Information as to the specificity of the leaving group and kinetic competency among natural substrates are lost during the process. Detection and quantitation of carbohydrate analytes without derivatization remains difficult to achieve. Herein we report two putative thermophilic glycosidase genes that were codon optimized and expressed, a mass‐differentiated substrate library2 to screen and identify the chemical function of the newly expressed glycosidases, and a novel mass spectrometric glycosidase assay developed to couple both the enzymatic hydrolysis of glycosidic linkages using newly characterized thermophilic glycosidases with the detection and quantitation of D‐galactose, D‐mannose and D‐glucose using a fixed ligand kinetic method3,4. Various natural substrates were screened using this new label‐free assay5 and kinetic competency information was first reported for two thermophilic enzymes. These newly characterized glycosidases were then combined with commercial glycosidases to form a glycosidase toolbox that enables a fast enzymatic screening for linkage analysis of glycan samples using a high throughput experiment platform. Overall, this assay is both fast and sensitive in that it only takes a few hours to detect nanogram quantities of monosaccharides and can quickly provide a complete cleavage profile for a glycosidase or oligosaccharide of interest.Support or Funding InformationWe appreciate the funding support of Joan and Marvin Carmack fund

Formation and Reactivity of a C-P-N-Sc Four-Membered Ring: H2 , O2 , CO, Phenylsilane, and Pinacolborane Activation.

Reaction of scandium dimethyl complex LScMe2 (L=[2-{N(DIPP)C(Ph)N}C6 H4 CH=N(DIPP)]- , DIPP=2,6-(iPr)2 C6 H3 ) (2) with bulky phosphine ArPH2 (Ar=2,6-{3,5-(Me)2 C6 H3 }2 C6 H3 ) gives an unprecedented scandium 2,3-azaphosphametallacyclobutane complex L'Sc (L'=[2-{N(DIPP)C(Ph)N}C6 H4 C(H)P(2,6-{3,5-(CH3 )2 C6 H3 }2 C6 H3 )N(DIPP)]3- ) (3) that contains a C-P-N-Sc four-membered ring. Complex 3 was characterized by X-ray crystallography, revealing a short distance between the Sc and P atoms (2.786(1) Å), but without a direct Sc-P bonding interaction. The formation of 3 involves a complete cleavage of the imino bond of the tridentate ligand L and C-P and N-P bond coupling. Complex 3 not only activates H2 under mild conditions to give a H-H bond cleavage product, but also reacts with O2 , CO, phenylsilane, and pinacolborane to produce novel products through cleavage and formation of various bonds.

Fetal hemoglobin is much less prone to DNA cleavage compared to the adult protein

Hemoglobin (Hb) is well protected inside the red blood cells (RBCs). Upon hemolysis and when free in circulation, Hb can be involved in a range of radical generating reactions and may thereby attack several different biomolecules. In this study, we have examined the potential damaging effects of cell-free Hb on plasmid DNA (pDNA). Hb induced cleavage of supercoiled pDNA (sc pDNA) which was proportional to the concentration of Hb applied. Almost 70% of sc pDNA was converted to open circular or linear DNA using 10µM of Hb in 12h. Hb can be present in several different forms. The oxy (HbO2) and met forms are most reactive, while the carboxy-protein shows only low hydrolytic activity. Hemoglobin A (HbA) could easily induce complete pDNA cleavage while fetal hemoglobin (HbF) was three-fold less reactive. By inserting, a redox active cysteine residue on the surface of the alpha chain of HbF by site-directed mutagenesis, the DNA cleavage reaction was enhanced by 82%. Reactive oxygen species were not directly involved in the reaction since addition of superoxide dismutase and catalase did not prevent pDNA cleavage. The reactivity of Hb with pDNA can rather be associated with the formation of protein based radicals.

Physiopathology and Management of Gluten-Induced Celiac Disease.

Proline- and glutamine-rich gluten proteins are one of the major constituents of cereal dietary proteins, which are largely resistant to complete cleavage by the human gastrointestinal (GI) digestive enzymes. Partial digestion of gluten generates approximately 35 amino acids (aa) immunomodulatory peptides which activate T-cell-mediated immune system, followed by immunological inflammation of mucosa leading to the onset of celiac disease (CD). CD is an autoimmune disease associated with HLA-DQ2/DQ8 polymorphism and dysbiosis of gut microbiota. CD is either diagnosed using duodenal mucosal biopsis or serological testing for transglutaminase 2 (TG2) specific antibodies (IgA and IgG). Current therapy for CD management is gluten-free diet, while other therapies like glutenase, probiotics, immunomodulation, jamming of HLA-DQ2, inhibition of TG2, and gluten tolerance aided by gluten tolerizing vaccines are being developed.

Spectroscopic, electrochemical and DNA binding studies of some monomeric copper(II) complexes containing N2S(thiolate)Cu core and N4S(disulfide)Cu core

We report the synthesis, characterization and spectroscopic results of the Cu(II) complexes [Cu(pabt)(OH2)](ClO4) (1), [Cu(pabt)(Imz)](ClO4) (2), [Cu(pabt)(N-MeImz)](ClO4) (3), [Cu(pabt)Cl] (4), [Cu(pma)Cl] (5), [Cu(pdta)Cl]Cl (6) and [Cu(reduced-pdta)Cl]Cl (7), (Hpabt=N-(2-mercaptophenyl)-2′-pyridylmethylenimine, Hpma=N-(2-pyridylmethyl)-2-mercaptoaniline, pdta=2,2′-di(pyridyl-2-methyleneimine)diphenyl disulfide, reduced-pdta=2,2′-di(pyridyl-2-methylamine)diphenyl disulfide, Imz=imidazole, N-MeImz=N-methylimidazole). Electronic spectra of all these compounds display strong LMCT bands in the visible region mainly associated with S→Cu(II), and consistent with TDDFT results. A four-line EPR pattern originating from the interaction of the unpaired electron with the central 63/65Cu nucleus (I=3/2, natural abundances: 63Cu, 69.17%; 65Cu, 30.83%) with the isotropic coupling constant (Aiso) values of 80±1.5G at RT for all these complexes suggests monomeric nature in solution. The redox behavior of these compounds show either nearly reversible or quasi-reversible Cu(II)/Cu(I) couple with redox potentials within the range −0.08 to −0.20V versus Ag/AgCl. Some of these compounds show strong intercalative DNA binding and its complete cleavage. 1–3 exhibit remarkable cytotoxicity against C6 glioma cell line and human cervical cancer HeLa cell line. IC50 values of 2 and 3 for the cervical cancer HeLa cell line reveal that they exhibit higher cytotoxicity than many reported Cu(II) compounds.

Ethanolysis of Kraft lignin to platform chemicals on a MoC1-x/Cu-MgAlOz catalyst

The complete conversion of Kraft lignin is examined over a MoC1-x/Cu-MgAlOz catalyst in pure ethanol to give value-added chemicals with small molecular weight, including C6 alcohols, C8C10 esters, benzyl alcohols and arenes, without the formation of char or tar. MoC1-x/Cu-MgAlOz exhibited much higher activity than the previously reported MoC1-x/AC, CuMgAlOy and Cu-MgAlOz catalysts and achieved the highest yield of aromatic compounds, 575mg/g lignin, at 330°C. The complete cleavage of aryl-O bonds in phenols is observed at temperatures over 300°C. Furthermore, the MoC1-x/Cu-MgAlOz catalyst is reusable with a 22.4% loss in the yield of aromatic compounds after 5 cyclic runs.

Light-Triggered Release of Biomolecules from Diamond Nanowire Electrodes.

The controlled release of biomolecules from a substrate surface is a challenging task. Photocleavable linkers appear as attractive candidates for light-triggered delivery. We show here the possibility of creating photoactivable diamond nanowire interfaces, from which molecules can be photochemically released upon irradiation at 365 nm for several minutes. The approach is based on the covalent modification of boron-doped diamond nanowires (BDD NWs) with o-nitrobenzyl containing ligands, to which different biomolecules can be attached via amide bond formation. The photodecomposition reaction and the subsequent release of small proteins such as lysozyme or enzymes such as horseradish peroxidase (HRP) are investigated using electrochemical impedance spectroscopy. Using a colorimetric assay, we demonstrate that, while complete cleavage of HRP was achieved upon irradiation for 10 min at 1 W cm(-2), this exposure time resulted in a partial loss of enzymatic activity.

Highly Tactic Cyclic Polynorbornene: Stereoselective Ring Expansion Metathesis Polymerization of Norbornene Catalyzed by a New Tethered Tungsten-Alkylidene Catalyst.

The tungsten alkylidyne [(t)BuOCO]W≡C((t)Bu) (THF)2 (1) reacts with CO2, leading to complete cleavage of one C═O bond, followed by migratory insertion to generate the tungsten-oxo alkylidene 2. Complex 2 is the first catalyst to polymerize norbornene via ring expansion metathesis polymerization to yield highly cis-syndiotactic cyclic polynorbornene.

Engineering Periodic shRNA for Enhanced Silencing Efficacy.

RNA interference (RNAi) provides a versatile therapeutic approach via silencing of specific genes, particularly undruggable targets in cancer and other diseases. However, challenges in the delivery of small interfering RNA (siRNA) have hampered clinical translation. Polymeric or periodic short hairpin RNAs (p-shRNAs)-synthesized by enzymatic amplification of circular DNA-are a recent development that can potentially address these delivery barriers by showing improved stability and complexation to enable nanoparticle packaging. Here, we modify these biomacromolecules via structural and sequence engineering coupled with selective enzymatic digestion to generate an open-ended p-shRNA (op-shRNA) that is cleaved over ten times more efficiently to yield siRNA. The op-shRNA induces considerably greater gene silencing than p-shRNA in multiple cancer cell lines up to 9 days. Furthermore, its high valency and flexibility dramatically improve complexation with a low molecular weight polycation compared to monomeric siRNA. Thus, op-shRNA provides an RNAi platform that can potentially be packaged and efficiently delivered to disease sites with higher therapeutic efficacy.

Synthesis, characterization, and biological evaluation of new biotinylated (99m) Tc/Re-tricarbonyl complexes.

The synthesis and biological evaluation of three new biotinylated fac-[(99m)Tc/Re(CO)3](+) complexes with the tridentate ligands L1, L2, and L3 are reported. L1-L3 contain the chelators 2-((5-aminopentyl)(pyridin-2-ylmethyl)amino)acetic acid, 2-(2-aminoethylthio)-3-(1H-imidazol-4-yl)propanoic acid, and 2-amino-3-(1-carboxy-2-(1H-imidazol-4-yl)ethylthio)propanoic acid, respectively, which are conjugated to biotin's carboxylate via their amine group. The fac-[Re(CO)3(L1-L3)] complexes were synthesized and characterized by NMR and IR, where the (N,N,O) coordination for ReL1 and the (N,S,O) coordination for ReL2 and ReL3 were confirmed. The tracer complexes fac-[(99m)Tc(CO)3(L1-L3)] were synthesized in high yield and were found highly stable in 10(-3) M L-histidine and L-cysteine over 24 h. Furthermore, they exhibited high binding affinity (>90%) for avidin. Rat plasma studies showed complete cleavage of biotin from (99m)TcL1 after 1 h and a low percentage of intact (99m)TcL2 and (99m)TcL3 with no biotin cleavage metabolites present, over 24 h. Similarly, rat urine analysis showed the presence of intact (99m)TcL2 and (99m)TcL3, while (99m)TcL1 was cleaved. Biodistribution studies of (99m)TcL2 and (99m)TcL3 revealed fast blood and tissue clearance.

Thermal catalytic oxidation of octachloronaphthalene over anatase TiO2 nanomaterial and its hypothesized mechanism.

As an environmentally-green technology, thermal catalytic oxidation of octachloronaphthalene (CN-75) over anatase TiO2 nanomaterials was investigated at 300 °C. A wide range of oxidation intermediates, which were investigated using various techniques, could be of three types: naphthalene-ring, single-benzene-ring, and completely ring-opened products. Reactive oxygen species on anatase TiO2 surface, such as O2−• and O2−, contributed to oxidative degradation. Based on these findings, a novel oxidation degradation mechanism was proposed. The reaction at (101) surface of anatase TiO2 was used as a model. The naphthalene-ring oxidative products with chloronaphthols and hydroxyl-pentachloronaphthalene-dione, could be formed via attacking the carbon of naphthalene ring at one or more positions by nucleophilic O2−. Lateral cleavage of the naphthalene ring at different C1-C10 and C4-C9, C1-C2 and C4-C9, C1-C2 or and C3-C4 bond positions by electrophilic O2−• could occur. This will lead to the formation of tetrachlorophenol, tetrachloro-benzoic acid, tetrachloro-phthalaldehyde, and tetrachloro-acrolein-benzoic acid, partially with further transformation into tetrachlorobenzene-dihydrodiol and tetrachloro-salicylic acid. Unexpectedly, the symmetric half section of CN-75 could be completely remained with generating the intricate oxidative intermediates characteristically containing tetrachlorobenzene structure. Complete cleavage of naphthalene ring could produce the ring-opened products, such as formic and acetic acids.

Evolutionary well-conserved region in the signal peptide of parathyroid hormone-related protein is critical for its dual localization through the regulation of ER translocation.

Parathyroid hormone-related protein (PTHrP) has two different targeting signals: an N-terminal signal peptide for the endoplasmic reticulum (ER) targeting and an internal nuclear localization signal. The protein not only functions as a secretory protein, but is also found in the nucleus and/or nucleolus under certain conditions. PTHrP signal peptide is less hydrophobic than most signal peptides mainly due to its evolutionarily well-conserved region (QQWS). The substitution of four tandem leucine residues for this conserved region resulted in a significant inhibition of the signal peptide cleavage. At the same time, proportion of nuclear and/or nucleolar localization decreased, probably due to tethering of the protein to the ER membrane by the uncleaved mutant signal peptide. Almost complete cleavage of the signal peptide accompanied by a lack of nuclear/nucleolar localization was achieved by combining the hydrophobic h-region and an optimized sequence of the cleavage site. In addition, mutational modifications of the distribution of charged residues in and around the signal peptide affect its cleavage and/or nuclear/nucleolar localization of the protein. These results indicate that the well-conserved region in the signal peptide plays an essential role in the dual localization of PTHrP through ER targeting and/or the membrane translocation.

Elucidation of the Molecular Mechanism Driving Duplication of the HIV-1 PTAP Late Domain.

HIV-1 uses cellular machinery to bud from infected cells. This cellular machinery is comprised of several multiprotein complexes known as endosomal sorting complexes required for transport (ESCRTs). A conserved late domain motif, Pro-Thr-Ala-Pro (PTAP), located in the p6 region of Gag (p6(Gag)), plays a central role in ESCRT recruitment to the site of virus budding. Previous studies have demonstrated that PTAP duplications are selected in HIV-1-infected patients during antiretroviral therapy; however, the consequences of these duplications for HIV-1 biology and drug resistance are unclear. To address these questions, we constructed viruses carrying a patient-derived PTAP duplication with and without drug resistance mutations in the viral protease. We evaluated the effect of the PTAP duplication on viral release efficiency, viral infectivity, replication capacity, drug susceptibility, and Gag processing. In the presence of protease inhibitors, we observed that the PTAP duplication in p6(Gag) significantly increased the infectivity and replication capacity of the virus compared to those of viruses bearing only resistance mutations in protease. Our biochemical analysis showed that the PTAP duplication, in combination with mutations in protease, enhances processing between the nucleocapsid and p6 domains of Gag, resulting in more complete Gag cleavage in the presence of protease inhibitors. These results demonstrate that duplication of the PTAP motif in p6(Gag) confers a selective advantage in viral replication by increasing Gag processing efficiency in the context of protease inhibitor treatment, thereby enhancing the drug resistance of the virus. These findings highlight the interconnected role of PTAP duplications and protease mutations in the development of resistance to antiretroviral therapy. Resistance to current drug therapy limits treatment options in many HIV-1-infected patients. Duplications in a Pro-Thr-Ala-Pro (PTAP) motif in the p6 domain of Gag are frequently observed in viruses derived from patients on protease inhibitor (PI) therapy. However, the reason that these duplications arise and their consequences for virus replication remain to be established. In this study, we examined the effect of PTAP duplication on PI resistance in the context of wild-type protease or protease bearing PI resistance mutations. We observe that PTAP duplication markedly enhances resistance to a panel of PIs. Biochemical analysis reveals that the PTAP duplication reverses a Gag processing defect imposed by the PI resistance mutations in the context of PI treatment. The results provide a long-sought explanation for why PTAP duplications arise in PI-treated patients.

Positive B cell flow cytometry crossmatch/full HLA matched renal transplant pair

Background An ABO incompatible non sensitized male renal transplant patient was worked-up with his full HLA matched brother. The patient was desensitized with rituximab (500 mg 2 weeks prior to transplant) and four sessions of plasmapheresis and small dose intravenous immunoglobulin (100 mg/kg after each plasma session). The crossmatch included both current serum (the day before transplant) and previous serum (before the start of desensitization). Method A standard three color flow cytometry crossmatch was performed against the potential donor (1 mg/ml pronase treated cells) and the serum was tested by Luminex PRA screen (One Lambda Inc.). An evaluation of the pronase effectiveness was assessed using anti- CD20 monoclonal antibodies. Results The patient PRA screen was negative for HLA class I and II and the crossmatch results were negative for T cells and positive B cell IgG (133 MCS) for the current serum and negative T and B cell IgG for the previous serum. Staining the B cells used in the crossmatch with anti-CD20 demonstrated the complete absence of the CD20 receptors. The auto B cell IgG flow crossmatch was not assessed due to absence of patients’ B cells as a result of rituximab effect. Conclusion This case demonstrates that the desensitization using rituximab, plasmapheresis, and intravenous immunoglobulin for depleting antibodies in ABO incompatible renal transplant patients prior to transplantation can cause B cell nonspecific binding despite treating the cells with pronase. This nonspecific binding results in positive B cell IgG flow crossmatch. Using different anti-CD20 monoclonal antibodies for the evaluation efficacy of pronase activity is suggested to confirm complete cleavage of the CD20 receptors.

Proteolytic Degradation of Bovine Submaxillary Mucin (BSM) and Its Impact on Adsorption and Lubrication at a Hydrophobic Surface.

The effects of proteolytic digestion on bovine submaxillary mucin (BSM) were investigated in terms of changes in size, secondary structure, surface adsorption, and lubricating properties. Two proteases with distinctly different cleavage specificities, namely trypsin and pepsin, were employed. SDS-PAGE analysis with staining for proteins and carbohydrate moieties showed that only the unglycosylated terminal regions of BSM were degraded by the proteases. Size exclusion chromatography (SEC) and dynamic light scattering (DLS) analyses indicated that tryptic digestion mainly led to the reduction in size, whereas pepsin digestion rather caused an increase in the size of BSM. Less complete cleavage in terminal peptide regions by pepsin and subsequent aggregation were possibly responsible for the increased size. Far-UV circular dichroism (CD) spectra of the protease-treated BSM showed a slight change in the secondary structure owing to the removal of terminal domains, but the overall random coil conformation adopted by the central glycosylated domain remained dominant and essentially unchanged. Surface adsorption properties as characterized by optical waveguide lightmode spectroscopy (OWLS) showed that tryptic digestion of BSM resulted in a decrease in the adsorbed mass, but pepsin digestion led to a slight increase in the adsorbed mass onto a hydrophobic surface compared to intact BSM. This is in agreement with the partial preservation of peptide segments in the terminal regions after pepsin digestion as confirmed by SEC and DLS studies. Despite a contrast in the adsorbed amount of the protease-treated BSMs onto the surface, both proteases substantially deteriorated the lubricating capabilities of BSM at a hydrophobic interface. The present study supports the notion that the terminal domains of BSM are critical to the adsorption and lubricating properties of BSM at hydrophobic interfaces.

Acid hydrolysis of xylan

The public interest in usage of lignocellulosic biomass as a renewable resource has grown strongly during the last decade. Hemicelluloses are the second most abundant constituents of wood biomass, but have yet received less attention, compared to cellulose. In this work hydrolysis of xylan, as an example of hemicelluloses, was studied at moderate temperatures between 70 and 90°C and pH values ranging from 0.5 to 3 with different liquid (HCl, H2SO4, triflouroacetic acid, oxalic acid) and solid (Smopex-101) catalysts. Experimental results showed complete cleavage of xylan with xylose yields of up to 95%.

Methods, setup and safe handling for anhydrous hydrogen fluoride cleavage in Boc solid-phase peptide synthesis.

Solid-phase peptide synthesis (SPPS) using tert-butyloxycarbonyl (Boc)/benzyl (Bzl) chemistry is an indispensable technique in many laboratories around the globe, and it provides peptides to the pharmaceutical industry and to thousands of scientists working in basic research. The Boc/Bzl strategy has several advantages, including reliability in the synthesis of long and difficult polypeptides, alternative orthogonality regarding protecting groups and ease of producing C-terminal thioesters for native chemical ligation applications. In this process, anhydrous hydrogen fluoride (HF) is used to remove the side chain protecting groups of the assembled peptide and to release the peptide from the resin, a process typically described as 'HF cleavage'. This protocol describes the general methodology, apparatus setup and safe handling of HF, with the aim of providing comprehensive information on the safe use of this valuable, well-studied and validated cleavage technique. We explain the cleavage mechanism, the physicochemical properties and risks of HF, first aid measures and the correct use of the apparatus. In addition, we provide advice on scavenger selection, as well as a troubleshooting section and video material illustrating key steps of the procedure. The protocol comprises precleavage sample preparation (30 min-2.5 h), complete HF cleavage procedure (2 h) and reaction workup (30 min).

Reductive Cleavage of Carbon Monoxide by a Disilenide.

The complete reductive cleavage of the triple bond in carbon monoxide was achieved using a lithium disilenide at room temperature. The C-C-coupled product can be regarded as a silanone dimer with pending alkyne and silirene moieties and incorporates two equivalents of CO per disilenide unit. A formation mechanism via ketenyl intermediates is proposed on the basis of DFT calculations and elucidated experimentally by employing Group 6 metal carbonyls as both stabilizing entity and source of CO in the reaction with disilenide. The isolation of cyclic silylene complexes with weakly donating ketenyl donor groups further supports the mechanistic scenario.