BRCA1 mutation screening using restriction endonuclease fingerprinting-single-strand conformation polymorphism in an automated capillary electrophoresis system

Efficient mutation scanning techniques are needed for the rapid detection of novel disease-associated mutations and rare-sequence variants of putative importance. The large size of the breast cancer 1 gene (BRCA1) and the many mutations found throughout its entire coding sequence make screening for mutations in this gene particularly challenging. We have developed a method for screening exon 11 of the BRCA1 gene based on restriction enzyme digestion of fluorescence-labeled polymerase chain reaction (PCR) products followed by single-strand conformation polymorphism (SSCP) using an automated capillary electrophoresis system, denoted capillary restriction endonuclease fingerprinting (REF)-SSCP electrophoresis. Using this strategy on a control set of samples, we were able to detect 17 of 18 known sequence alterations. The method was then applied to screen 73 Norwegian females with family histories of breast and/or ovarian cancer. A total of 172 sequence alterations were detected, including substitutions, insertions, and deletions. One novel substitution of unknown function was identified. Sequencing of all samples negative in the capillary REF-SSCP system gave no additional mutations confirming the high sensitivity of the described methodology. Capillary REF-SSCP electrophoresis appeared as a technically convenient technique, requiring amplification of fewer PCR fragments than traditional SSCP. The novel strategy allows high-throughput mutation scanning without radioactive labeling and polyacrylamide gel electrophoresis (PAGE).     

4-metalated condensed pyrimidines: their preparation and reaction with aldehydes under Barbier-type conditions

[reaction: see text] The organometallic intermediate obtained from halogen-metal exchanges of 4-iodo-6-phenylthieno[2,3-d]pyrimidine under Barbier-type conditions was reacted with aldehydes to form the corresponding alcohols in moderate yields. The reaction involving an organolithium intermediate proceeded only at low temperature, whereas the reaction involving a magnesium ate intermediate also proceeded at room temperature. A crystal structure confirms that the expected constitutional alcohol isomer is formed, where no migration has taken place. The conditions were also suitable for 9-benzyl-6-iodopurine.     

Synthesis and reactions of 2-deoxy-β-D-ribofuranosyl derivatives of 3-aryl-4H-pyrrolo[2,3-d]pyrimidin-4-imines

Summary 3-Aryl-7-(2-deoxy--D-erythro-pentofuranosyl)-3,7-dihydro-4H-pyrrolo[2,3-d]-pyrimidin-4-imines (4) as well as 4-arylamino-7-(2-deoxy--D-erythro-pentofuranosyl)-2-methyl-5-phenyl-7H-pyrrolo[2,3-d]pyrimidines (7) have been synthesized by glycosylation of the sodium salt of the corresponding nucleobases with 2-deoxy-3,5-di-O-p-toluyl--D-erythro-pentofuranosyl chloride (2) followed by subsequent deprotection with sodium methoxide in methanol. The deprotected nucleoside4 undergoes aDimroth rearrangement on reflux for 24 h in water to furnish the 4-arylamino nucleoside7.

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Synthese und Reaktionen von 2-Deoxy--D-ribofuranosylderivaten von 3-Aryl-4H-pyrrolo[2,3-d]pyrimidin-4-iminen

Zusammenfassung 3-Aryl-7-(2-deoxy--D-erythro-pentafuranosyl)-3,7-dihydro-4H-pyrrolo[2,3-d]-pyrimidin-4-imine (4) und 4-Arylamino-7-(2-deoxy--D-erythro-pentofuranosyl)-2-methyl-5-phenyl-7H-pyrrolo[2,3,-d]pyrimidine (7) wurden durch Glycosylierung der Natriumsalze der entsprechenden Nucleosidbasen mit 2-Deoxy-3,5-di-O-p-toluyl--D-erythro-pentofuranosylchlorid (2) und anschließende Entfernung der Schutzgruppe mit Natriummethoxid in Methanol hergestellt. Das entschützte Nucleosid4 ergibt bei 24-stündigem Erhitzen in Wasser unter Rückfluß über eineDimroth-Umlagerung das 4-Aminonucleosid7.

     

Redox grafting of diazotated anthraquinone as a means of forming thick conducting organic films

Thick conductive layers containing anthraquinone moieties are covalently immobilized on gold using redox grafting of the diazonium salt of anthraquinone (i.e., 9,10-dioxo-9,10-dihydroanthracene-1-diazonium tetrafluoroborate). This grafting procedure is based on using consecutive voltammetric sweeping and through this exploiting fast electron transfer reactions that are mediated by the anthraquinone redox moieties in the film. The fast film growth, which is followed by infrared reflection absorption spectroscopy, atomic force microscopy, X-ray photoelectron spectroscopy, ellipsometry, and coverage calculation, results in a mushroom-like structure. In addition to varying the number of sweeps, layer thickness control can easily be exerted through appropriate choice of the switching potential and sweep rate. It is shown that the grafting of the diazonium salt is essentially a diffusion-controlled process but also that desorption of physisorbed material during the sweeping process is essentially for avoiding blocking of the film due to clogging of the electrolyte channels in the film. In general, sweep rates higher than 0.5 V s(-1) are required if thick, porous, and conducting films should be formed.     

Synthesis of α-2′-deoxynucleosides

α-Thymidine (4) was synthesized from thymidine (1) in 3 steps in 36% overall yield without using chro-matography and with the possibility of increasing the yield to 85% by reusing the remaining α,β-mixture. 1-(2-Deoxy-3,5-di-O-p-toluoyl-α-D-erythro-pentofuranosyl)thymine (3) was further converted to 1-(2-deoxy-α-D-erythro-pentofuranosyl)-5-methylcytosine (5) .     

Selective Inversion of the Proximal or Distal Hydroxyl Groups in syn,syn-3-[N-(Alkoxycarbonyl)amino] 1,2-Diols via Cyclic Sulfates

The formation of cyclic sulfates (4) from syn,syn-3-[N-(benzyloxycarbonyl)amino] 1,2-diols provides a common intermediate to access other diastereomers via two inversion procedures. Thermolysis of the cyclic sulfates in acetonitrile normally leads to inversion of the distal hydroxyl group to form a 1,3-oxazin-2-one (6). Catalytic hydrogenation of the cyclic sulfates under basic conditions (NEt(3)) results in inversion at the proximal hydroxyl group to form a 1,3-oxazolidin-2-one (5).     

HPLC Method for Determination of Rosiglitazone in Plasma

A fast and sensitive high performance liquid chromatography method for quantitative determination of rosiglitazone in human plasma has been developed. The extraction from plasma was performed using solid-phase extraction (SPE) on C4 silica (100 mg) disposable extraction cartridges (DEC). The separation of rosiglitazone and two metabolites was achieved on a Phenomenex® Synergi 4 µm MAX-RP (150 × 4.6 mm) column, protected by a guard column. The mobile phase was 0.01 M ammonium acetate, pH 7.0 - acetonitrile (65:35, v/v). (3S)-3-OH-quinidine was used as internal standard. The analytes were detected using fluorescence detection. The method was validated. The limit of quantitation was 1 ng mL⁻¹ and the detection limit was 0.25 ng mL⁻¹ for rosiglitazone in human plasma. The recovery was 90% for rosiglitazone. Linearity was observed over a range of 1-1000 ng mL⁻¹ (r²=0.9959). The intra- and inter-day precision (C.V.) did not exceed 8.7 %. Applicability of the method was demonstrated by a clinical pharmacokinetic study. A healthy volunteer received in two separate phases 4 mg and 8 mg rosiglitazone maleate as a single oral dose. Plasma concentrations were measured for 24 h in both phases.     

Identification and Directed Development of Non‐Organic Catalysts with Apparent Pan‐Enzymatic Mimicry into Nanozymes for Efficient Prodrug Conversion

Nanozymes, nanoparticles that mimic the natural activity of enzymes, are intriguing academically and are important in the context of the Origin of Life. However, current nanozymes offer mimicry of a narrow range of mammalian enzymes, near‐exclusively performing redox reactions. We present an unexpected discovery of non‐proteinaceous enzymes based on metals, metal oxides, 1D/2D‐materials, and non‐metallic nanomaterials. The specific novelty of these findings lies in the identification of nanozymes with apparent mimicry of diverse mammalian enzymes, including unique pan‐glycosidases. Further novelty lies in the identification of the substrate scope for the lead candidates, specifically in the context of bioconversion of glucuronides, that is, human metabolites and privileged prodrugs in the field of enzyme‐prodrug therapies. Lastly, nanozymes are employed for conversion of glucuronide prodrugs into marketed anti‐inflammatory and antibacterial agents, as well as “nanozyme prodrug therapy” to mediate antibacterial measures.     

Unexpected Isolation of 4‐Isothiocyanatomethylene‐4H‐pyridine‐1‐carboxylic Acid Ethyl Ester as Potential Template in Organic Synthesis

The synthesis of 4‐isothiocyanatomethyl‐pyridine 4 in 36% yield by Hasegawa and Kotani (Japanese patent 49088878, 1974) has spurred us to investigate this preparation in detail. In addition to this compound, 4‐isothiocyanatomethylene‐4H‐pyridine‐1‐carboxylic acid ethyl ester 3 can be isolated. The synthesis of both compounds 3 and 4 were optimized to 75% and 50% yield respectively. Reaction of compound 3 with methylamine gave thiourea derivatives 5, the same product obtained on reacting 4‐isothiocyanatomethyl‐pyridine 4 with methylamine. We succeed in adjusting the reaction conditions to obtain high yield either from compound 3 or isothiocyanate derivatives 4.