Practical synthesis of a neuropeptide Y antagonist via stereoselective addition to a ketene

[Reaction: see text]. The synthesis of neuropeptide Y antagonist 1, currently under clinical investigation for the treatment of obesity, is described. The convergent synthesis from trans-spirolactone carboxylic acid intermediate 2a and aminopyrazole 3 is predicated on a stereoselective route to the former. The coupling reaction of ethyl 4-oxocyclohexanecarboxylate (10a) with lithiated isonicotinamide 11 was investigated in detail, but even optimized conditions only provided a 45:55 ratio of trans:cis isomers (12a:12b). While selective crystallization schemes were developed to isolate the thermodynamically less stable trans isomer 2a, improved stereocontrol was subsequentially achieved by the application of ketene chemistry. The ketene formation and quench was investigated under a variety of conditions aimed at maximizing the trans:cis ratio. Reacting a mixture of carboxylic acids 2a and 2b with POCl3 in THF, followed by concomitant addition of tert-butyl alcohol in the presence of TMEDA at 35 degrees C provided a 4:1 ratio of trans:cis tert-butyl esters (18a:18b) via in situ ketene formation. Ester hydrolysis, followed by selective crystallization of undesired 2b as the HCl salt, led to isolation of 2a in 47% overall yield. Aminopyrazole intermediate 3 was synthesized via the condensation reaction of 2-fluorophenylhydrazine hydrochloride (4a) with acrylonitrile derivative 5 in 65-70% yield. Coupling of advanced intermediates 2a and 3b via activation with thionyl chloride gave a 92% yield of 1.     

CAST/CNMR: highly accurate C NMR chemical shift prediction system considering stereochemistry

Accurate, practical prediction of ¹³C NMR chemical shifts has been achieved with a new system, CAST/CNMR, taking account of stereochemistry. The CAST/CNMR system has solved the critical problem of the accurate distinction of differences and similarities in stereochemical structures around a specific carbon, which has not yet been achieved by any other database-oriented system for prediction of ¹³C NMR chemical shifts. CAST/CNMR uses a three-dimensional structural database together with a ¹³C NMR spectral database. Absolute/relative configurational and conformational structural information are described by the CAST (CAnonical-representation of STereochemistry) coding method. This paper provides an overview of the CAST/CNMR system, and describes its application to two natural products as examples.     

Preparative high-yield electroelution of proteins after separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and its application to analysis of amino acid sequences and to raise antibodies.

A method for the preparative high-yield electroelution of proteins from sodium dodecyl sulphate (SDS) polyacrylamide gel strips was established. The method consisted of SDS-polyacrylamide gel electrophoresis, detection of proteins with sodium acetate and electrophoretic elution at 200 V for 3 h by utilizing a horizontal flat-bed gel electrophoresis apparatus. Standard proteins with molecular masses of 14-66 kilodalton (cytochrome c, aldolase, ovalbumin and bovine serum albumin) were recovered with an average yield of 73.6 +/- 2.3%. A membrane-bound protein, rat skeletal muscle Ca(2+)-ATPase (100 kilodalton) was also well recovered (over 60%). This method was applicable to the purification of proteins required for N-terminal amino acid sequencing and to raise antibodies.     

ALL-trans β-CAROTENE-5,6-EPOXIDE IN THYLAKOID MEMBRANES

All- trans β-carotene-5,6-epoxide has been found in the thylakoid membranes of spinach and of the cyanobacterium Synechococcus vulcanus Copeland. The epoxide was extracted from the thylakoid membranes with acetone, and was isolated by high-performance liquid chromatography (HPLC). The structure of the epoxide was identified by means of mass, Raman, and electronic absorption spectroscopy. Changes in the amount of the epoxide, as a result of epoxidation and (apparent) de-epoxidation reactions in the membranes, were traced by analysis of extracts on HPLC. In isolated thylakoid membranes, only the epoxidation reaction took place. The reaction was caused by irradiation or by the addition of ferricyanide, suggesting that electron transport reactions in the membranes are involved in the epoxidation. In intact spinach leaves, however, both epoxidation and de-epoxidation took place; the extent of epoxidation correlated with the intensity of light incident on the leaves. The epoxidation and de-epoxidation of all- trans β-carotene are contrasted with those of xanthophylls (in the violaxanthin cycle).     

Preparation and characterization of spherical polymer packings from polybutadiene for size-exclusion chromatography

Porous polymer spherical particles for column packings in nonaqueous size-exclusion chromatography (SEC) were prepared from 1,2-syndiotactic polybutadiene by suspension and evaporation method. The polymer microbeads obtained were crosslinked by radical reaction between 2-vinyl groups in polybutadiene with ultraviolet radiation, to render them insoluble. These microbeads have wider chromatographic separation width than polystyrene column packings. In addition, the polybutadiene microbeads did not show the excessive retention observed with commercial polystyrene columns for polycyclic aromatic compounds. Therefore, a close correlation between the elution volume and M, for polycyclic aromatic compounds was observed with polybutadiene microbeads columns.     

Participation of lipid peroxidation in rat pertussis vaccine pleurisy. III. Thiobarbituric acid (TBA) reactant and lysosomal enzyme

Activity of lysosomal enzymes, such as N-acetyl-beta-D-glucosaminidase (NAG), was assayed in exudate on a rat model of Bordetella pertussis vaccine pleurisy. Thiobarbituric acid (TBA)-reactive substances (TBA.R) and superoxide dismutase (SOD) activity were then monitored in the exudate on the acute phase response in this inflammatory model. Retention of the exudate in the pleural space increased rapidly after the challenge, and the exudate volume at 24 h reached about three times the volume at 6 h. The activity of SOD at 6 h was shown to be higher than that at 24 h after the challenge, thus showing negative correlations with TBA-R levels and exudate volume. The levels of TBA.R rapidly increased and reached maximum values at 24 h. It was concluded that the above three parameters correlated to the acute phase response in this inflammatory model.     

Spin-probe ESR study on the dynamics of liquid molecules in the MCM-41 nanochannel: temperature dependence on 2-propanol and water

A spin-probe ESR study has been made on the dynamics of 2-propanol and water molecules in the nanochannel of MCM-41 at various temperatures. In the former system, 2-propanol is separated into two phases: one with molecules immobilized in the ESR time scale and the other with mobile ones, even at temperatures more than 40 degrees higher than the bulk melting point. In the case of water, on the other hand, only the "immobilized" water was detected at a temperature as high as 313 K. At higher temperature, spin-probe molecule undergoes anisotropic rotational diffusion to reduce resistance from the solvent molecules in the nanochannel. These results are explained in relation to the intermolecular network intensified in the nanochannel. Static as well as dynamic structures of these solutions have been discussed.     

Identification of conjugation positions of urinary glucuronidated vitamin D3 metabolites by LC/ESI–MS/MS after conversion to MS/MS‐fragmentable derivatives

A liquid chromatography/electrospray ionization–tandem mass spectrometry‐based method was developed for the identification of the conjugation positions of the monoglucuronides of 25‐hydroxyvitamin D₃ [25(OH)D₃] and 24,25‐dihydroxyvitamin D₃ [24,25(OH)₂D₃] in human urine. The method employed derivatization with 4‐(4‐dimethylaminophenyl)‐1,2,4‐triazoline‐3,5‐dione to convert the glucuronides into fragmentable derivatives, which provided useful product ions for identifying the conjugation positions during the MS/MS. The derivatization also enhanced the assay sensitivity and specificity for urine sample analysis. The positional isomeric monoglucuronides, 25(OH)D₃‐3‐ and ‐25‐glucuronides, or 24,25(OH)₂D₃‐3‐, ‐24‐ and ‐25‐glucuronides, were completely separated from each other under the optimized LC conditions. Using this method, the conjugation positions were successfully determined to be the C3 and C24 positions for the glucuronidated 25(OH)D₃ and 24,25(OH)₂D₃, respectively. The 3‐glucuronide was not present for 24,25(OH)₂D₃, unlike 25(OH)D₃, thus we found that selective glucuronidation occurs at the C24‐hydroxy group for 24,25(OH)₂D₃.     

Synthesis of end‐functionalized polyethers by phosphazene base‐catalyzed ring‐opening polymerization of 1,2‐butylene oxide and glycidyl ether

For the living ring‐opening polymerization (ROP) of epoxy monomers, the catalytic activity of organic superbases, tert‐butylimino‐tris(dimethylamino)phosphorane, 1‐tert‐butyl‐2,2,4,4,4‐pentakis(dimethylamino)‐2Λ⁵,4Λ⁵‐catenadi(phosphazene), 2,8,9‐triisobutyl‐2,5,8,9‐tetraaza‐1‐phosphabicyclo[3.3.3]undecane, and 1‐tert‐butyl‐4,4,4‐tris(dimethylamino)‐2,2‐bis[tris(dimethylamino)phosphoranylidenamino]‐2Λ⁵,4Λ⁵‐catenadi(phosphazene) (t‐Bu‐P₄), was confirmed. Among these superbases, only t‐Bu‐P₄ showed catalytic activity for the ROP of 1,2‐butylene oxide (BO) to afford poly(1,2‐butylene oxide) (PBO) with predicted molecular weight and narrow molecular weight distribution. The results of the kinetic, post‐polymerization experiments, and MALDI‐TOF MS measurement revealed that the t‐Bu‐P₄‐catalyzed ROP of BO proceeded in a living manner in which the alcohol acted as the initiator. This alcohol/t‐Bu‐P₄ system was applicable to the glycidol derivatives, such as benzyl glycidyl ether (BnGE) and t‐butyl glycidyl ether, to afford well‐defined protected polyglycidols. The α‐functionalized polyethers could be obtained using different functionalized initiators, such as 4‐vinylbenzyl alcohol, 5‐hexen‐1‐ol, and 6‐azide‐1‐hexanol. In addition, the well‐defined cyclic‐PBO and PBnGE were successfully synthesized using the combination of t‐Bu‐P₄‐catalyzed ROP and click cyclization. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Cyclic sample pooling using two‐dimensional liquid chromatography system enhances coverage in shotgun proteomics

We report a cyclic sample pooling technique devised in two‐dimensional liquid chromatography–electrospray ionization mass spectrometry (LC‐ESI‐MS) shotgun proteomics that renders deeper proteome coverage; we combined low pH reversed‐phase (RP) LC in trifluoroacetic acid in the first dimension, followed by cyclic sample pooling of the eluate and low‐pH RP‐LC in formic acid in the second dimension. The new protocol has a significantly higher resolving power suitable for LC‐ESI‐MS/MS shotgun proteomics. Copyright © 2013 John Wiley & Sons, Ltd.