RESUMEN

Dipnictenes of the type RPn=PnR (Pn=P, As, Sb, Bi) can be viewed as dimers of the corresponding pnictinidenes R-Pn. Phosphanylidene- and arsanylidenephosphoranes (R-Pn(PMe3); Pn=P, As) have been shown to be versatile synthetic surrogates for the delivery of pnictinidene fragments. We now report that thermal treatment of 1 : 1 mixtures of R-P(PMe3) and R'-As(PMe3) gives access to arsaphosphenes of the type RP=AsR'. Three examples are presented and the properties and reactivity of Mes*P=AsDipTer (1) (Mes*=2,4,6-tBu3-C6H2; DipTer=2,6-(2,6-iPr2C6H3)2-C6H3) were studied in detail. Solid state 31P NMR spectroscopy revealed a large 31P NMR chemical shift anisotropy with a span of ca. 920 ppm for 1 while computational methods were employed to investigate this pronounced magnetic deshielding of the P atom in 1. In the presence of the carbene IMe4 (IMe4=:C(MeNCMe)2) 1 is shown to be split into the corresponding NHC adducts Mes*P(IMe4) and DipTerAs(IMe4), which is additionally shown for diarsenes.

RESUMEN

The synthesis of group 4 metal 1-metallacyclobuta-2,3-dienes as organometallic analogues of elusive 1,2-cyclobutadiene has so far been limited to SiMe3 substituted examples. We present the synthesis of two Ph substituted dilithiated ligand precursors for the preparation of four new 1-metallacyclobuta-2,3-dienes [rac-(ebthi)M] (M=Ti, Zr; ebthi=1,2-ethylene-1,10-bis(η5-tetrahydroindenyl)). The organolithium compounds [Li2(RC3Ph)] (1 b: R=Ph, 1 c: R=SiMe3) as well as the metallacycles of the general formula [rac-(ebthi)M(R1C3R2)] (2 b: M=Ti, R1=R2=Ph, 2 c: M=Ti, R1=Ph, R2=SiMe3; 3 b: M=Zr, R1=R2=Ph; 3 c: M=Zr, R1=Ph, R2=SiMe3) were fully characterised. Single crystal X-ray diffraction and quantum chemical bond analysis of the Ti and Zr complexes reveal ligand influence on the biradicaloid character of the titanocene complexes. X-band EPR spectroscopy of structurally similar Ti complexes [rac-(ebthi)Ti(Me3SiC3SiMe3)] (2 a), 2 b, and 2 c was carried out to evaluate the accessibility of an EPR active triplet state. Cyclic voltammetry shows that introduction of Ph groups renders the complexes easier to reduce. 13C CPMAS NMR analysis provides insights into the cause of the low field shift of the resonances of metal-bonded carbon atoms and provides evidence of the absence of the ß-C-Ti interaction.

RESUMEN

Reproducibility is at the heart of science. Nevertheless, with the advent of computer-based data processing and analysis, most spectroscopists have a hard time fully reproducing a figure from last year's publication starting from the raw data. Unfortunately, this renders their work eventually unscientific. To change this, we need to develop analysis tools that relieve their users from having to trace each processing and analysis step. Furthermore, these tools need to be modular, extendible, and easy to use in order to get used. To this end, we present here the open-source Python package cwepr based on the ASpecD framework for reproducible analysis of spectroscopic data. This package follows best practices of both, science and software development. Key features include an automatically generated gap-less record of each individual processing and analysis step from the raw data to the final published figure. Additionally, it provides a powerful user interface requiring no programming skills of the user. Due to its code quality, modularity, and extensive documentation, it can be easily extended and is actively developed by spectroscopists working in the field. We expect this approach to have a high impact in the field and to help fighting the looming reproducibility crisis in spectroscopy.

Asunto(s)

Programas Informáticos , Reproducibilidad de los Resultados

RESUMEN

Human 5-lipoxygenase (5-LO) is the key enzyme in the formation of leukotrienes (LTs), important mediators of inflammation. Cellular 5-LO activity is regulated in a complex manner, e.g. by calcium influx, the cellular redox status or 5-LO phosphorylation. Being a mobile enzyme, 5-LO migrates from the cytosol to the nuclear envelope where it is believed to interact with 5-lipoxygenase-activating protein (FLAP) and receives the substrate arachidonic acid (AA). 5-LO contains four cysteine residues located close to the AA entry site. In the present study, we show that in vitro glutathionylation of recombinant purified 5-LO wildtype (WT) as well as 5-LO 4C, a mutant where the four surface cysteines are replaced by serines (Cys159/300/416/418Ser), does not alter the product synthesis. However, in 5-LO/FLAP-transfected HeLa cells, treatment with the thiol-oxidizing agent diamide which promotes glutathionylation at surface Cys residues led to a decreased LT synthesis by 5-LO WT. In contrast to the WT enzyme, LT formation of the 4C mutant was stimulated by addition of diamide. Immunofluorescence studies in human monocytes and HEK293 cells, expressing 5-LO and FLAP, revealed that diamide prevented the translocation of 5-LO WT whereas it enhanced the translocation of the fourfold cysteine mutant. Therefore, we could demonstrate that the interface, involving the four cysteines 159, 300, 416 and 418, is important for the translocation to the nuclear membrane and the colocalization with FLAP.

Asunto(s)

Proteínas Activadoras de la 5-Lipooxigenasa/metabolismo , Araquidonato 5-Lipooxigenasa/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Leucocitos Mononucleares/metabolismo , Leucotrienos/metabolismo , Proteínas Activadoras de la 5-Lipooxigenasa/química , Proteínas Activadoras de la 5-Lipooxigenasa/genética , Sustitución de Aminoácidos , Araquidonato 5-Lipooxigenasa/química , Araquidonato 5-Lipooxigenasa/genética , Ácido Araquidónico/metabolismo , Sitios de Unión , Núcleo Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , Citosol/efectos de los fármacos , Citosol/ultraestructura , Diamida/farmacología , Regulación de la Expresión Génica , Glutatión/metabolismo , Células HEK293 , Células HeLa , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/ultraestructura , Mutación , Oxidación-Reducción , Cultivo Primario de Células , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal

RESUMEN

5-Lipoxygenase (5-LOX) is the key player of pro-inflammatory leukotriene biosynthesis. Its regulatory or so-called PLAT (polycystin-1, lipoxygenase, α-toxin) domain binds allosteric modulators like calcium, membranes, coactosin-like protein and Dicer, thereby influencing 5-LOX activity at the nuclear membrane by mediating translocation. The PLAT domain may also regulate cytosolic 5-LOX activity and possibly influence microRNA metabolism. Hence, it has also evolved as a promising target for anti-inflammatory therapy. Research focusing on this substructure of 5-LOX requires an assay system based on the isolated domain. However, we found that the isolated PLAT domain was highly prone to aggregation and therefore unsuitable for interaction studies. Substitution of the single, membrane-binding tryptophan 75 with glycine reduced aggregation and substantially increased its thermal stability. Calcium interaction of the single mutant was confirmed by differential scanning fluorimetry. Moreover, crosslinking experiments demonstrated the ability of the isolated PLAT domain to bind Dicer C-terminus whereas the interaction with coactosin-like protein required the interplay of the catalytic and the PLAT domain.