Enantioselective synthesis of (R)‐Cinacalcet via cobalt‐catalysed asymmetric Negishi cross‐coupling

A novel enantioselective synthesis of (R)‐cinacalcet with 99% enantiomeric excesses (ee) has been achieved. The main strategies of the approach include a gram‐scale cobalt‐catalysed asymmetric cross‐coupling of racemic ester with arylzinc reagent, Hoffman‐type rearrangement of acidamide, the amidation of chiral amine, and improving the ee of chiral amide from 87% to 99% via recrystallization.     

Resolution of 1- and 2-naphthylmethoxyacetic acids,NMR reagents for absolute configuration determination,by use of L-phenylalaninol

Racemic 1- and 2-naphthylmethoxyacetic acids (1NMA and 2NMA), the chiral anisotropic reagents used for absolute configuration determination of chiral secondary alcohols and primary amines, were conveniently resolved to enantiomers (>99% ee) by condensation with L-phenylalaninol (2-amino-3-phenylpropanol), chromatographic separation of the diastereomers, and hydrolysis. This method enables large-scale preparation of enantiomeric 1NMA and 2NMA.     

Collagen-induced arthritis

The collagen-induced arthritis (CIA) mouse model is the most commonly studied autoimmune model of rheumatoid arthritis. Autoimmune arthritis is induced in this model by immunization with an emulsion of complete Freund's adjuvant and type II collagen (CII). This protocol describes the steps necessary for acquisition, handling and preparation of CII, as well as selection of mouse strains, proper immunization technique and evaluation of the arthritis incidence and severity. Typically, the first signs of arthritis appear in this model 21-28 days after immunization, and identification of the arthritic limbs is not difficult. Using the protocol described, the investigator should be able to reproducibly induce a high incidence of CIA in various strains of genetically susceptible mice as well as learn how to critically evaluate the pathology of the disease. The total time for the preparation of reagents and the immunization of ten mice is about 1.5 h.     

Pyruvate aldolases in chiral carbon-carbon bond formation

A procedure for the preparation of optically pure alpha-keto-gamma-hydroxy carboxylic acids through stereospecific aldol addition catalyzed by pyruvate aldolases from the Entner-Doudoroff and the DeLey-Doudoroff glycolytic pathways is described. This highly versatile fragment serves as a precursor for a variety of commonly encountered functionalities, including beta-hydroxy aldehydes and carboxylic acids, alpha-amino-gamma-hydroxy carboxylic acids and alpha,gamma-dihydroxy carboxylic acids. The protocol described here uses recombinant His6-tagged KDPG aldolase for the synthesis of (S)-4-hydroxy-2-keto-4-(2'-pyridyl)butyrate. A protocol for evaluating enantiomeric excess through formation of the gamma-lactone of the dithioacetal followed by chiral-phase gas-liquid chromatography is also described. Enzyme expression and enzymatic synthesis can be accomplished in approximately 1 week. The enzymatic aldol addition proceeds in nearly quantitative yields with enantiomeric excesses greater than 99.7%.     

A convenient synthetic method of a 5,7-diarylcyclopenteno-[1,2-b]pyridine-6-carboxylate: a key intermediate for potent endothelin receptor antagonists

A convenient method for the synthesis of the title intermediate 4 was described. The key steps of this synthesis involved: (1) regioselective addition reaction of arylzinc reagent to quinolic anhydride in 42% isolated yield, (2) conversion of a ketoacid to an enone, which was achieved in 65% yield by intramolecular Knoevenagel reaction of beta-ketoester generated by condensation of an acid imidazolide with an ester enolate, followed by dehydration assisted with silica gel, and (3) stereoselective reduction of an allyl alcohol in 75% yield with zinc under acidic conditions. This synthesis enabled us to provide hundreds of grams of without chromatographic purification.     

Synthesis of enantiomerically enriched (R)-5-tert-butylazepan-2-one using a hydroxyalkyl azide mediated ring-expansion reaction

A procedure for the conversion of a symmetrical ketone to an enantiomerically pure lactam is described. The technique described here involves a ring-expansion reaction of a 4-substituted cyclohexanone accomplished with a chiral 1,3-azidopropanol derivative. The procedure entails first a one-step preparation of (R)-1-phenyl-3-azidopropanol from a commercially available halide precursor, which is then reacted with the ketone using BF(3) x OEt(2) as a Lewis acid promoter. The resulting lactam is subsequently converted into a chiral lactam of high enantiopurity via the two-stage removal of the chiral nitrogen substituent. The present protocol carries out the diastereoselective ring-expansion reaction with higher selectivity than competing processes and is generally useful for the preparation of 5-substituted caprolactams.     

Synthesis of enantiopure 2-(aminoalkyl)phenol derivatives and their application as catalysts in stereoselective reactions

Enantiopure 2-(aminoalkyl)phenol derivatives are an interesting class of compounds widely used in homogeneous ligand accelerated catalysis. A series of practical and convenient methods available for their preparation are revised, together with the methodologies for the determination of their configuration. The uses of these compounds in metal catalysed asymmetric reactions in the addition of dialkyl zinc reagents to aldehydes and in the reduction of ketones with borane are described. Moreover 2-(aminoalkyl)phenol derivatives have found use also as chiral shift reagents for carboxylic acids.     

Three-layer sandwich gel electrophoresis: a method of salt removal and protein concentration in proteome analysis

Sample preparation plays a critical role in successful proteomic applications. Features of electrospray mass spectrometry impose limits on the types of buffers, detergents and other reagents that can be used in sample preparation. Unfortunately, many of these mass spectrometry incompatible reagents significantly enhance protein recoveries from complex matrices. This problem prompted our search for a better cleanup protocol. Our data suggest that the Three-layer Sandwich Gel Electrophoresis (TSGE) protocol can solve this problem and provide near quantitative recovery of extremely low concentration proteins from harsh solutions, a feature not available from other cleanup protocols. The hallmark of the TSGE protocol is the combination of the properties of agarose gels (that serve as the matrix to immobilize the proteins of interest) with low- and high-percentage polyacrylamide gels (that serve as the concentration and sealing layers, respectively). By electrophoretically driving the proteins of interest from the agarose matrix into the concentration layer, the TSGE protocol simultaneously concentrates the sample in the concentration layer and provides an environment amenable to downstream buffer exchange and proteolytic digestion. In combination with 2D-LC-MS/MS, the TSGE protocol was evaluated in the analysis of a whole cell extract from the protozoan parasite Toxoplasma gondii. Comparison of our experimental proteomic results with in silico predictions from gene data indicated that TSGE did not bias the protein identification.     

Immunoassay for thyroxine (T4) in serum using capillary electrophoresis and micromachined devices

As part of our ongoing effort to develop electrophoretic assay technology for clinical diagnostics, we describe a competitive immunoassay for the determination of serum thyroxine (T4) based on electrophoresis and laser induced fluorescence (LIF). Measurements of total T4 are useful for the clinical evaluation of thyroid function. A fluorescein thyroxine conjugate was utilized in conjunction with a polyclonal antibody preparation as assay reagents. Capillary electrophoresis (CE) conditions tolerant of the direct injection of serum without extraction or other sample preparation steps were developed and used for quantitation of total T4 in serum. We have been exploring the use of micromachined devices with arrays of channels for high assay throughput. Our assay protocol was carried in a microchip format. The results illustrate that gains in speed can be additionally achieved, with the electrophoretic separation of free from bound labelled T4 being performed in about 15 s for serum samples.     

Catalytic enantioselective addition of indoles to arylnitroalkenes: an effective route to enantiomerically enriched tryptamine precursors

A new valuable catalytic protocol for the preparation of synthetically useful beta-indolyl nitro compounds bearing benzhydryl stereocenters is presented. The combined use of catalytic amounts of a commercially available chiral [SalenAlCl] complex and pyridine allowed, for the Friedel-Crafts alkylation reaction of indoles with aromatic nitro-olefins to be carried out in good yields and enantioselectivity (up to 63% ee).     

A high-throughput fluorimetric assay for angiotensin I-converting enzyme

Angiotensin I-converting enzyme (ACE) assays are commonly used for measuring enzymatic activity in clinical and biological samples. The fluorimetric procedure described is sensitive, rapid and involves unsophisticated procedures and inexpensive reagents. It uses the substrate hippuryl-L-histidyl-L-leucine, and the fluorescent adduct of the enzyme-catalyzed product L-histidyl-L-leucine is quantified fluorimetrically. This assay has been adapted for a 96-well plate format that produces comparable data to previously described assays and has the advantage of greater efficiency with respect to both time and reagents. The protocol can be used for routine purposes or for more detailed kinetic analyses. The apparent Km and kcat values for purified testis ACE determined from a double reciprocal plot were 3.0 mM and 195.7 s(-1), respectively. The protocol can be completed within 4 h.     

Chiral synthesis via organoboranes. 41. The utility of B-chlorodiisopinocampheylborane for a general synthesis of enantiomerically pure drugs

A general approach to the synthesis of enantiomerically pure α-phenyl amino alcohols via the asymmetric reduction of α-phenyl haloalkyl ketones or α-phenyl aminoalkyl ketones with B-chlorodiisopinocampheylborane is described. Using this approach, an improved synthesis of a potential antipsychotic, α-(4-fluorophenyl)-4-(2-pyrimidinyl)-1-piperazinebutanol in ⩾98% ee, and the broncholdilator 1-(2-methoxy-2-phenethyl)-4-(3-hydroxy-3-phenylpropyl)piperazine (eprozinol) in ⩾99% ee is achieved. © 1995 Wiley-Liss, Inc.     

A fast, simple, and reliable high-yielding method for DNA extraction from different plant species

Genetic studies and pathogen detection in plants using molecular methods require the isolation of DNA from a large number of samples in a short time span. A rapid and versatile protocol for extracting high-quality DNA from different plant species is described. This method yields from 1 to 2 mg of DNA per gram of tissue. The absorbance ratios (A₂₆₀/A₂₈₀) obtained ranged from 1.6 to 2.0. A minimal presence of contaminating metabolites (as polymerase chain reaction [PCR] inhibitors) in samples and a considerable savings in reagents are characteristics of this protocol, as well as the low cost of the analysis per sample. The quality of the DNA was suitable for PCR amplification.     

Combining regio- and enantioselectivity of lipases for the preparation of (R)-4-chloro-2-butanol

Preparation of 98% ee (R)-4-chloro-2-butanol was carried out by the enzymatic hydrolysis of chlorohydrin esters, using fungal resting cells and commercial enzymes. Hydrolyzes were carried out using lipases from Candida antarctica (Novozym 435), C. rugosa, Rhizomucor miehei (Lipozyme IM), Burkolia cepacia, and resting cells of Rhizopus oryzae and Aspergillus flavus. The influence of the enzyme, the solvent, the temperature, and the alkyl chain length on the selectivity of hydrolyzes of isomeric mixtures of chlorohydrin esters is described. Regioselectivity was higher than 95% for some of the tested lipases. Novozym 435 allowed preparation of the (R)-4-chloro-2-butanol after 15 min of reaction at 30-40 degrees C.     

An ELISA method to measure inhibition of the COX enzymes

The cyclooxygenase (COX) reaction can be monitored by measurement of oxygen consumption, peroxidase co-substrate oxidation or prostaglandin (PG) detection. This protocol describes a procedure measuring cyclooxygenase activity by quantifying PGE2 produced by enzymatic conversion of arachidonic acid, in the presence or absence of potential inhibitors. This high-throughput method has the advantage that it directly measures cyclooxygenase activity and requires little enzyme. The first part of the assay consists of incubating arachidonic acid, cyclooxygenase and the test samples to generate prostaglandins. The second part uses an ELISA method to quantify the amount of PGE2 produced by the enzymatic reaction. The isolation of COX-1 and COX-2 enzymes is also described. This protocol can be completed in approximately 23 h, including 16-h and 4-h incubation phases. This does not include enzyme preparation (3 h for COX-1 and 24 h for COX-2) or preparation of ELISA plates (23 h, including incubation).     

Purification and Characterization of an Enantioselective Amidase from Pseudomonas chlororaphis B23

An amidase produced by Pseudomonas chlororaphis B23 was purified and characterized. The purification procedure used included ammonium sulfate precipitation and hydrophobic, anion-exchange, gel filtration, and ceramic hydroxyapatite chromatography steps. This amidase has a native molecular mass of about 105 kDa and is a homodimer whose subunits have a molecular mass of 54 kDa. The enzyme exhibited maximal activity at 50(deg)C and at pH values ranging from 7.0 to 8.6. We found no evidence that metal ions were required, and the enzyme was inhibited by several thiol reagents. This amidase exhibited activity against a broad range of aliphatic and aromatic amides and exhibited enantioselectivity for several aromatic amides, including 2-phenylpropionamide (enantiomeric excess [ee] = 100%), phenylalaninamide (ee = 55%), and 2-(4-chlorophenyl)-3-methylbutyramide (ee = 96%), but not 2-(6-methoxy-2-naphthyl)propionamide (the amide form of naproxen) (ee = 0%). The characteristics of the P. chlororaphis B23 amidase are the same as the characteristics of enantioselective amidases described by Mayaux et al. (J. F. Mayaux, E. Cerbelaud, F. Soubrier, D. Faucher, and D. Petre, J. Bacteriol. 172:6764-6773, 1990; J. F. Mayaux, E. Cerbelaud, F. Soubrier, P. Yeh, F. Blanche, and D. Petre, J. Bacteriol. 173:6694-6704, 1991) and Kobayashi et al. (M. Kobayashi, H. Komeda, T. Nagasawa, M. Nishiyama, S. Horinouchi, T. Beppu, H. Yamada, and S. Shimizu, Eur. J. Biochem. 217:327-336, 1993).     

由有机硼烷合成桃小食心虫性信息素 Z-7-二十烯-11-酮和 Z-7-十九烯-11-酮

研究了用有机硼烷的转化反应立体选择合成 Z-4-十一烯-1-醛(7)以及将其应用于桃小食心虫性信息素 Z-7-11-二十烯-11-酮(1)和 Z-7-十九烯-11-酮(2)的简便合成方法。用前文报告的经由有机硼烷一锅反应制备法获得的 Z-4-十一烯-1-醇(6),经氯铬酸吡啶(PCC)氧化生成 Z-4-十一烯-1-醛(7),后者与 Grignard 试剂作用,得中间物仲醇 Z-7-链烯-11-醇(8),然后使之氧化,即得预期的 Z-7-链烯-11-酮(1)和(2)。从起始原料1-已烯出发,总产率分别为36.9％和35.0％.     

Polymer supported synthesis of aminooxyalkylated oligonucleotides,and some applications in the fabrication of microarrays

A new protocol has been described for solid phase preparation of 3′- and 5′-aminooxylalkylated oligonucleotides using commercially available reagents. This involves attachment of linker 4 either with an LCAA-CPG support via succinoylation followed by synthesis (3′-aminooxyalkylated oligomers) or formation of its phosphoramidite 6 followed by coupling with desired oligomer (for generating 5′-aminooxyalkylated oligomers). Both the routes produced modified oligonucleotides in sufficiently high yields and purity (on HPLC) via conventional oligonucleotide synthesis on an automated synthesizer and deprotection step using aqueous ammonia (16 h, 60 °C). Aminooxyalkylated oligonucleotides were used to construct microarrays on glass surface (biochips). The performance of the biochips was evaluated by immobilizing modified oligonucleotides on epoxylated glass microslides under different sets of conditions with respect to pH, temperature and time. Further, the constructed microarrays were successfully used for detection of nucleotide mismatches and bacterial typhoid.     

Rapid high resolution western blotting: from gel to image in a single day.

A streamlined protocol is described that allows high sensitivity antigen detection by Western blotting in a single day. The choice of membrane blotting matrix, as well as blocking reagents, has been optimized in order to allow rapid development of the blot with chemiluminescent reagents. The entire process, from gel to blot to a permanent, hard copy image on x-ray film, can be accomplished within six hours.     

Head-to-tail macrocyclization of cysteine-free peptides using an o-aminoanilide linker

A head-to-tail macrocyclization protocol for the preparation of cysteine-free cyclic peptides was investigated. The o-aminoanilide linker constructed in the peptide sequence by a standard Fmoc-based peptide synthesis procedure was subjected to nitrite-mediated activation under acidic conditions toward N-acyl benzotriazole as the active ester species. The subsequent cyclization smoothly proceeded by neutralization in the presence of additives such as 1-hydroxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt) to afford the expected cyclic pentapeptide, a CXCR4 antagonist. The cyclization efficiencies were dependent on the precursor open-chain sequence. The application of this step-wise activation-cyclization protocol to microflow reaction systems is also described.