Cobalt-catalyzed annulation of styrenes with α-bromoacetic acids.

We report a method for addition of α-bromophenylacetic acids to vinyl C[double bond, length as m-dash]C bonds in styrenes to afford γ-lactones. Reactions employed a simple cobalt catalyst Co(NO3)2·6H2O in the presence of dipivaloylmethane (dpm) ligand. Many functionalities including halogen, ester, and nitro groups were compatible with reaction conditions. If α-bromoesters were used, vinylacetates were the major products.

Synthesis of primary N-arylthioglyoxamides from anilines, elemental sulfur and primary C-H bonds in acetophenones.

A simple method for coupling of anilines, acetophenones, and elemental sulfur to afford N-arylthioglyoxamides has been developed. Reactions proceeded in the presence of Na2SO3 and DMSO, thus eliminating the need for transition metals and external oxidants. Functionalities such as halogen, ester, methylthio, and heterocycle groups were compatible with the conditions. Electron-poor acetophenones sometimes gave isosteric glyoxamides.

Functionalization of activated methylene C-H bonds with nitroarenes and sulfur for the synthesis of thioamides.

We report a method to obtain arylthioamides by the functionalization of sp3 C-H bonds in phenylacetic acids and benzyl alcohols. Reactions proceeded without the use of any solvents and were compatible with many functionalities and heterocycles. These conditions allow for a rapid synthesis of thioamides from simple, commercial substrates.

A new route to substituted furocoumarins via copper-catalyzed cyclization between 4-hydroxycoumarins and ketoximes.

A new route to substituted furocoumarins via copper-catalyzed cyclization between 4-hydroxycoumarins and ketoximes was developed. CuBr2 exhibited higher activity than other copper salts, affording the desired furocoumarins in high yields. The transformation proceeded readily in the absence of stoichiometric external oxidants. The significance of this synthetic strategy would be (1) the easily available starting materials; (2) low cost catalyst CuBr2; and (3) being without stoichiometric external oxidants. This protocol is complementary to previous approaches in the synthesis of substituted furocoumarins.

An efficient access to ß-ketosulfones via ß-sulfonylvinylamines: metal-organic framework catalysis for the direct C-S coupling of sodium sulfinates with oxime acetates.

A copper-based framework Cu2(OBA)2(BPY) was synthesized and used as a recyclable heterogeneous catalyst for the synthesis of ß-sulfonylvinylamines from sodium sulfinates and oxime acetates via direct C-S coupling reaction. The transformation was remarkably affected by the solvent, and chlorobenzene emerged as the best option. This Cu-MOF displayed higher activity than numerous conventional homogeneous and MOF-based catalysts. The catalyst was reutilized many times in the synthesis of ß-sulfonylvinylamines without considerably deteriorating in catalytic efficiency. These ß-sulfonylvinylamines were readily converted to the corresponding ß-ketosulfones via a hydrolysis step with aqueous HCl solution. To the best of our knowledge, this direct C-S coupling reaction to achieve ß-sulfonylvinylamines was not previously conducted with a heterogeneous catalyst.

Emergency room surgical workload in an inner city UK teaching hospital.

BACKGROUND: Emergency admissions may account for over 50% of surgical admissions. The impact on service provision and implications for training are difficult to quantify. We performed a cohort study to analyse these workload patterns. METHODS: Data on emergency room (ER) surgical admissions over six months was collected including patient demographics, referral sources, diagnosis, operation and length of stay and analysed according to sub-speciality and age-groups. RESULTS: There were 1392 (median age 41 (IQR 28-60) years, M:F = 1.7:1) emergency surgical admissions over six months; 45% were under 40 years of age and 48% patients self-referred to the ER. The commonest diagnoses were abscesses (11%), non-specific abdominal pain (9.7%) and neuro-trauma (9.6%). The median length of stay was 4 (IQR 2-8) days; with older (>80 years) patient staying significantly longer than those <40 years of age (median 8 vs 2 two days, P < 0.0001, Kruskal-Wallis test). Vascular patients remained in hospital longer than trauma or general surgery patients (median 14 vs 3 days, P < 0.0001, Kruskal-Wallis test). A high proportion (43.5%) of the patients required operative intervention and service implications of various diagnoses and operative interventions are highlighted. CONCLUSION: With the introduction of shortened training period in Europe and World over, trainees may benefit from increased exposure to trauma and surgical emergencies. Resource planning should be based on more comprehensive, prospective data such as these.

Direct evidence of methylglucose lipopolysaccharides/palmitoyl-CoA noncovalent complexes by capillary zone electrophoresis-electrospray/mass spectrometry.

Mycobacterial methylglucose lipopolysaccharides (MGLPs) play an important regulatory role in the biosynthesis of long-chain fatty acids by forming complexes with neosynthesized acyl-CoA fatty acid derivatives. The MGLPs from Mycobacterium smegmatis were purified by high-performance anion-exchange chromatography and characterized by LSIMS and CE/ESI-MS. We investigated their interaction with palmitoyl-CoA using capillary zone electrophoresis with both direct and indirect UV detection. In the latter mode, the signal of the UV-transparent MGLPs decreased upon addition of increasing amounts of palmitoyl-CoA; while using direct UV detection, the addition of palmitoyl-CoA to the MGLPs revealed characteristic profiles. The major peak was assigned to the noncovalent MGLP/palmitoyl-CoA complex on the basis of its electrophoretic mobility. The abundance of the complex was found to increase until the MGLP/palmitoyl-CoA molar ratio reached a 1/1 stoichiometry. The existence of and the stoichiometry of this complex were assessed by CE/ESI mass spectrum analysis, showing pseudomolecular ions of the MGLP/palmitoyl-CoA complex. These results confirm that CE/ESI-MS is a powerful tool to characterize noncovalent molecular association.

Purification and characterization of a novel protease from culture filtrates of a Streptomyces sp.

A fibrinolytic protease has been isolated from Streptomyces sp. culture filtrate by successive chromatography on Mono S and Sephadex G50. The purified protease had a molecular mass of 33 kDa and had an isoelectric point of 6.7. It showed a sharp pH optimum at 7.8 with maximal protease activity between 35 degrees C and 50 degrees C. Its amino acid composition and amino-terminal sequence (17 residues) were determined. The protein exhibited marked hydrolytic activity toward the substrates N-Succ-(Ala)2-Pro-Phe-pNA (K(m) = 0.77 mM, Vmax = 24.2 mumol mg-1 min-1) and N-Succ-(Ala)2-Pro-Leu-pNA (K(m) = 0.92 mM, Vmax = 7.7 mumol mg-1 min-1). It was totally inhibited by alpha 1-antitrypsin, D-Phe-Pro-Arg-chloromethylketone and sodium dodecyl sulfate but was insensitive to EDTA, dithiothreitol, phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, pepstatin or elastatinal. In this respect, this protease differed in its physico-chemical and biochemical properties from other extracellular proteases previously found in bacteria and fungi. The results suggest that it has properties of chymotrypsin-like serine-type proteases.

Characterization of the deamidated forms of recombinant hirudin.

Recombinant hirudin variant rHV2-Lys 47 (MW = 6906.5) was intentionally deamidated by incubation in pH 9 phosphate buffer at 37 degrees C. Anion-exchange HPLC analysis showed that 11 forms could be generated. These were isolated and purified by combined anion-exchange and reversed-phase HPLC. Acid-catalyzed carboxyl methylation was used to introduce a mass shift of +15 amu per deamidated residue present in the molecule before analysis by liquid secondary ion mass spectrometry (LSIMS). Methylation enhanced, in particular, the abundance of the sequence ions in the LSIMS spectra. This permitted the determination of both the number (three) and the localization of the deamidated residues: Asn 52, Asn 53, and a residue located in the N-terminal 1-39 domain. Complementary sequencing techniques proved that the latter residue was Asn 33. Altogether four mono-, three di-, and four tri-deamidated forms were identified. The heterogeneity of the forms having identical deamidation positions but being chromatographically separable is thought to arise from the generation of alpha- and beta-aspartyl iso forms during the nonenzymatic deamidation process.

Liquid secondary ion mass spectrometry applied to structural confirmation of enzymically prepared C-terminal-truncated derivatives of recombinant hirudin.

The thrombin-specific inhibitor, hirudin variant rHV2-Lys 47 (rHirudin), is a 65-amino acid polypeptide produced by recombinant DNA technology in yeast. Previous studies have shown that the acidic C-terminal segment of hirudin is susceptible to enzymic degradation. To address the question of C-terminal-truncated forms of the protein in terms of by-products or metabolites, well-defined reference compounds are needed. We prepared nine derivatives by carboxypeptidase Y digestion of rHirudin followed by a two-step chromatographic purification. Liquid secondary ion mass spectrometric measurements performed on peptides collected after reversed-phase high-performance liquid chromatography showed three pure forms (1-64, 1-63 and 1-56) and three mixtures of two forms each (1-62 + 1-61, 1-58 + 1-57 and 1-55 + 1-54), which were readily distinguished from one another by their mass spectra. Further purification of these co-eluted samples was achieved by ion-exchange chromatography and their structures were confirmed by liquid secondary ion mass spectrometry. Preliminary studies conducted on intact rHirudin indicated that this is an excellent analytical tool for mass measurements of hirudin-related proteins. Indeed, it allowed rapid (within 10-15 min), precise (0.50 a.m.u. relative to expected value), reproducible (mean MH+ = 6907.64 +/- 0.42 a.m.u.), sensitive (up to 500 ng, i.e. 72 pmol) and specific measurement of the quasi-molecular ion (MH+) of the protein, and was thus readily applicable to the analysis of several derivatives.

Site specific radioiodination of recombinant hirudin.

Recombinant hirudin variant rHV2-Lys47 was radioiodinated using the chloramine-T method. Depending on the reaction pH, the two tyrosine residues, Tyr3 and Tyr63, responded differently to iodination but without change in total iodination yield. Of the incorporated -125 iodine 80% was located on Tyr3 at pH 7.4, but 65% was found on Tyr63 at pH 4. These distinct iodination patterns suggest the existence of a pH-dependent multimerization and/or important conformational changes in the tertiary structure with pH. Each radiotracer was purified to high specific activity by simple low-pressure chromatography including gel filtration and reverse-phase separation, both on short cartridges. The method was validated by reverse-phase and anion-exchange HPLC with on-line radioactivity detection. The iodination sites were characterized following carboxypeptidase Y cleavage coupled with radio-HPLC.

Metabolism of ticlopidine in rats: identification and quantitative determination of some its metabolites in plasma, urine and bile.

The fate of Ticlopidine, a new inhibitor of platelet aggregation, has been studied in rats following single oral doses. The compound was quickly absorbed as evaluated by the time of the peak plasma concentration. The metabolism of Ticlopidine involved N-oxidation, cleavage of the N-C bond, oxidation of aliphatic carbon followed by glycine conjugation. Methods for quantitation of Ticlopidine and of its metabolites, Ticlopidine-N-oxide, Tetrahydrothienopyridine, 2-cloro-hippuric acid, and the metabolite named Ticlopidine-M, are reported. Urinary excretion took place essentially in the first 24 hours and biliary excretion was most pronounced in the hour following dosing. Small amounts of Ticlopidine were excreted unchanged. The major urinary metabolites were 2-chlorohippuric acid (16% of the dose) and tetrahydrothienopyridine (8%), while Ticlopidine-M predominated in the bile (2% in 0.5h). The peak plasma concentration of Ticlopidine occurred at 0.5 hour. The plasma concentration/time curve displayed a biphasic profile and the terminal half-life of Ticlopidine was tentatively estimated to be 6-10 h in the rat. Ticlopidine-N-oxide was also quantified in plasma: the levels were inferior to those of Ticlopidine throughout the experimental period.

Quantitative determination of p-chlorophenoxyisobutyric acid in blood plasma by gas-liquid chromatography.

A gas-liquid chromatographic method for the determination of p-chlorophenoxyisobutyric (CPIB) acid in blood plasma is described. The substance is extracted from acidified plasma into benzene, the extract is evaporated to dryness and the residue is methylated with an alcohol-free solution of diazomethane and submitted to chromatography on a glass column packed with 3% OV-17. Data on the recovery, reproducibility and sensitivity of the method are given. CPIB acid plasma levels in rats after acute and prolonged treatments with the aluminium salt of CPIB acid (Atherolip) are also presented.