LSA-50 paper: An alternative to P81 phosphocellulose paper for radiometric protein kinase assays.

Radiometric assays have widely been used for measuring protein kinase activity for decades. In addition, several non-radiometric kinase assay formats have been developed over the years, including luciferase-based and fluorescence-based assays. However, radiometric assays are still considered as the "gold standard" for protein kinase assays, because of their direct readout, high sensitivity, reproducibility, reliability, and very low background signals. These radiometric assays rely on P81 phosphocellulose paper to capture the phosphorylated substrate and wash out unreacted [γ-32P] ATP. However, recently the production of P81 was discontinued by the manufacturer, causing major concern within the protein kinase research community. The advantages of radiometric assays over other kinase assay methods call for an urgent alternative to the discontinued P81 paper. In this report, we demonstrate that the LSA-50 paper is a worthy alternative for radiometric protein kinase assays originally using P81 phosphocellulose paper.

Solvent-free N-Boc deprotection by ex situ generation of hydrogen chloride gas.

An efficient, scalable and sustainable method for the quantitative deprotection of the tert-butyl carbamate (N-Boc) protecting group is described, using down to near-stoichiometric amounts of hydrogen chloride gas in solvent-free conditions. We demonstrate the ex situ generation of hydrogen chloride gas from sodium chloride and sulfuric acid in a two-chamber reactor, introducing a straightforward method for controlled and stoichiometric release of HCl gas. The solvent-free conditions allow deprotection of a wide variety of N-Boc derivatives to obtain the hydrochloride salts in quantitative yields. The procedure obviates the need for any work-up or purification steps providing an uncomplicated green alternative to standard methods. Due to the solvent-free, anhydrous conditions, this method shows high tolerance towards acid sensitive functional groups and furnishes expanded functional group orthogonality.

Developments in the Discovery and Design of Protein Kinase D Inhibitors.

Protein kinase D (PKD) is a serine/threonine kinase family belonging to the Ca2+/calmodulin-dependent protein kinase group. Since its discovery two decades ago, many efforts have been put in elucidating PKD's structure, cellular role and functioning. The PKD family consists of three highly homologous isoforms: PKD1, PKD2 and PKD3. Accumulating cell-signaling research has evidenced that dysregulated PKD plays a crucial role in the pathogenesis of cardiac hypertrophy and several cancer types. These findings led to a broad interest in the design of small-molecule protein kinase D inhibitors. In this review, we present an extensive overview on the past and recent advances in the discovery and development of PKD inhibitors. The focus extends from broad-spectrum kinase inhibitors used in PKD signaling experiments to intentionally developed, bioactive PKD inhibitors. Finally, attention is paid to PKD inhibitors that have been identified as an off-target through large kinome screening panels.

Concomitant positive patch test reactions in FreeStyle-allergic patients sensitized to isobornyl acrylate.

BACKGROUND: Concomitant positive patch test reactions in patients sensitized to isobornyl acrylate (IBOA) have rarely been documented. OBJECTIVES: To report concomitant sensitizations in patients with allergic contact dermatitis (ACD) from the glucose sensor FreeStyle Libre and sensitized to IBOA. METHODS: In 2019, 26 patients with suspected ACD from FreeStyle Libre were patch tested to a baseline series and to a (meth) acrylate series containing IBOA and 2-phenoxyethyl acrylate (PEA) 0.1% pet. Diabetes devices and patch test preparations were analyzed with gas chromatography - mass spectrometry (GC-MS) for the presence of IBOA and PEA. RESULTS: Of the 26 patients, 18 (69%) were sensitized to IBOA, and eight (44%) and 11 (61%) of these were co-sensitized to sesquiterpene lactones and fragrances, respectively. Ten patients (56%) were co-sensitized to PEA, which, contrary to IBOA, could not be detected in any device. The PEA test material was shown to be contaminated with IBOA. CONCLUSIONS: Contact allergy to IBOA appears to be declining and IBOA-sensitized patients are most often co-sensitized to sesquiterpene lactones and fragrances. Vigilance is required when patch testing (acrylate) materials obtained from industry, as these might be contaminated and, hence, alter the results and their interpretation.

Design, synthesis and biological evaluation of pyrazolo[3,4-d]pyrimidine-based protein kinase D inhibitors.

The multiple roles of protein kinase D (PKD) in various cancer hallmarks have been repeatedly reported. Therefore, the search for novel PKD inhibitors and their evaluation as antitumor agents has gained considerable attention. In this work, novel pyrazolo[3,4-d]pyrimidine based pan-PKD inhibitors with structural variety at position 1 were synthesized and evaluated for biological activity. Starting from 3-IN-PP1, a known PKD inhibitor with IC50 values in the range of 94-108 nM, compound 17m was identified with an improved biochemical inhibitory activity against PKD (IC50 = 17-35 nM). Subsequent cellular assays demonstrated that 3-IN-PP1 and 17m inhibited PKD-dependent cortactin phosphorylation. Furthermore, 3-IN-PP1 displayed potent anti-proliferative activity against PANC-1 cells. Finally, a screening against different cancer cell lines demonstrated that 3-IN-PP1 is a potent and versatile antitumoral agent.

Measurements of Atmospheric Proteinaceous Aerosol in the Arctic Using a Selective UHPLC/ESI-MS/MS Strategy.

In this article, an analytical methodology to investigate the proteinaceous content in atmospheric size-resolved aerosols collected at the Zeppelin observatory (79 °N, 12 °E) at Ny Ålesund, Svalbard, from September to December 2015, is proposed. Quantitative determination was performed after acidic hydrolysis using ultrahigh-performance liquid chromatography in reversed-phase mode coupled to electrospray ionization tandem mass spectrometry. Chromatographic separation, as well as specificity in the identification, was achieved by derivatization of the amino acids with N-butyl nicotinic acid N-hydroxysuccinimide ester prior to the analysis. The chromatographic run was performed within 11 min and instrumental levels of detection (LODs) were between 0.2 and 8.1 pg injected on the column, except for arginine which exhibited an LOD of 37 pg. Corresponding method LODs were between 0.01 and 1.9 fmol/m3, based on the average air sampling volume of 57 m3. The sum of free amino acids and hydrolyzed polyamino acids was shown to vary within 6-2914 and 0.02-1417 pmol/m3 for particles in sizes < 2 and 2-10 µm in equivalent aerodynamic diameter, respectively. Leucine, alanine, and valine were the most abundant among the amino acids in both aerosol size fractions. In an attempt to elucidate source areas of the collected aerosols, 5- to 10-day 3D backward trajectories reaching the sampling station were calculated. Overall, the method described here provides a first time estimate of the proteinaceous content, that is, the sum of free and polyamino acids, in size-resolved aerosols collected in the Arctic. Graphical Abstract ᅟ.

Synthesis of N-Acyl Sulfamates from Fluorosulfates and Amides.

A novel synthetic strategy toward  N-acyl sulfamates was developed. Interestingly, fluorosulfates, a new emerging class of electrophiles, were used to construct the sulfamate core. This precludes handling of chlorosulfonyl isocyanate and sulfamoyl chloride. In combination with amides, a wide and diverse set of N-acyl sulfamates was synthesized, including functionalized bioactive compounds. Furthermore, initial results showed that this method is also amenable to access N-thioacyl sulfamates.

Ex Situ Generation of Sulfuryl Fluoride for the Synthesis of Aryl Fluorosulfates.

A convenient transformation of phenols into the corresponding aryl fluorosulfates is presented: the first protocol to completely circumvent direct handling of gaseous sulfuryl fluoride (SO2F2). The proposed method employs 1,1'-sulfonyldiimidazole as a precursor to generate near-stoichiometric amounts of SO2F2 gas using a two-chamber reactor. With NMR studies, it was shown that this ex situ gas evolution is extremely rapid, and a variety of phenols and hydroxylated heteroarenes were fluorosulfated in good to excellent yields.

Carbonylation as a novel method for the assembly of pyrazine based oligoamide alpha-helix mimetics.

The design and synthesis of oligoamide α-helix peptidomimetics is reported. The oligoamide type systems are prepared in a modular fashion by coupling the monomers using palladium-catalyzed carbonylation chemistry. This enabled us to use substrates with a low nucleophilicity, leading to previously unreported pyrazine based oligoamide α-helix mimetics. The proof of principle is given by synthesizing a small set of compounds. Various end-capping groups were introduced and also a mixed multimer was successfully prepared.