Asymmetric synthesis of pipecolic acid derivatives using the aza-Diels-Alder reaction

Imines of the type R---N=CHCO₂Et can be coerced into undergoing a (4+2) cycloaddition with substituted dienes if the reaction is carried out in DMF in the presence of both water and acid: these reactions show extremely high regio- and diastereoselectivity. Use of the 1-phenylethyl group as a chiral auxiliary leads to moderate asymmetric induction (typical d.e. ca. 70%); moreover, the diastereoisomers are surprisingly easy to separate, giving a short general route to optically pure substituted pipecolic acid derivatives.     

Determination of methylmalonic acid and glutaric acid in urine by aqueous-phase derivatization followed by headspace solid-phase microextraction and gas chromatography-mass spectrometry

In this work, a novel technique of aqueous-phase derivatization followed by headspace solid-phase microextraction and gas chromatography-mass spectrometry was developed for the determination of organic acids in urine. The analytical procedure involves derivatization of organic acids to their ethyl esters with diethyl sulfate, headspace sampling, and GC/MS analysis. The proposed method was applied to the determination of methylmalonic acid and glutaric acid in urine. The experimental parameters and method validation were studied. Optimal conditions were obtained: PDMS fiber, extraction temperature 55 degrees C, extraction time 30 min, and 60 microL of diethyl sulfate as derivatization reagent with 2 mg of the ion pairing agent tetrabutylammonium hydrogensulfate. The method was linear over three orders of magnitude, and detection limits were 21 nM for methylmalonic acid and 34 nM for glutaric acid, respectively. Consequently, in-situ derivatization/HS-SPME/GC/MS is an alternative and powerful method for determination of organic acids as biomarkers in biological fluids.     

An efficient access to both enantiomers of pipecolic acid

An efficient and convenient synthesis of both enantiomers of pipecolic acid has been developed using the intramolecular cyclization of 2-amino-6-bromohexanoic acid under mild conditions.     

Quantification by selected ion monitoring of pipecolic acid, proline, gamma-aminobutyric acid and glycine in rat brain

A procedure for the simultaneous analysis of brain pipecolic acid, proline, gamma-aminobutyric acid and glycine--amino acids with potent inhibitory actions on the central nervous system--was developed. The identification and quantification of the amino acids were performed with a gas chromatographic--mass spectrometric--computer system using deuterium-labelled amino acids as the internal standards. After separation of the amino acids by high-performance liquid chromatography, the methyl ester heptafluorobutyryl derivatives were prepared. The lower limit of quantification for this method is at the picomole level. The usefulness of this chromatographic procedure has been demonstrated by measurement of trace amounts of pipecolic acid in rat brain.     

Assay of ambroxol in biological fluids by capillary gas-liquid chromatography

A sensitive and rapid method for the determination of ambroxol in biological fluids is described. It comprises a single extraction step, derivatization and selective determination with capillary gas-liquid chromatography (in split-mode) and electron-capture detection. The limit of quantification in plasma is ca. 3 ng/ml. The method is applied to the pharmacokinetics of ambroxol in humans.     

固相萃取-衍生化-气相色谱法测定血液中的3种苯氧羧酸类除草剂

建立了一种血液中2,4-二氯苯氧乙酸(2,4-D)、2-(2,4-二氯苯氧)-丙酸(2,4-DP)和4-氯-2-甲基-苯氧乙酸(MCPA)3种苯氧羧酸类除草剂的分析方法.血样用0.1 mol/L盐酸稀释后用GDX401大孔树脂吸附、用乙醚洗脱,萃取物用二氯丙醇在硫酸催化下进行酯化衍生,衍生物经气相色谱-电子捕获检测.2,4-D、2,4-DP和MCPA的检测限分别为20,8和40 ng/mL.定量分析用2,4-二氯苯乙酸作内标,线性关系和回收率结果均令人满意.     

Convergent synthesis of complex diketopiperazines derived from pipecolic acid scaffolds and parallel screening against GPCR targets

A convergent approach to highly functionalized diketopiperazines (DKPs) using enantioenriched pipecolic acids is described. Scandium triflate-catalyzed [4 + 2] aza-annulation was employed to produce stereochemically well-defined building blocks. A resin "catch and release" strategy was devised to convert annulation products to pipecolic acid monomers. Complex diketopiperazines were efficiently assembled utilizing one-pot cyclodimerization of pipecolic acids. Massively parallel screening of the complex DKPs against a panel of molecular targets identified novel ligands for a number of G-protein-coupled receptors (GPCRs).     

Determination of busulfan in human plasma by gas chromatography with electron-capture detection

A simple and highly sensitive gas chromatographic method has been developed for the determination of busulfan in human plasma. After extraction of plasma specimens (clinical or spiked) with ethyl acetate, busulfan and the internal standard [1,8-bis(methanesulfonyloxy)octane] were derivatized with 2,3,5,6-tetrafluorothiophenol to yield compounds monitored by a 63Ni electron-capture detector. Sample recoveries from extraction and derivatization were greater than 78 and 91%, respectively. The limit of quantitation was 0.01 microgram/ml (0.04 microM) in 1 ml of plasma with a linear relationship over the 0.01-1.0 micrograms/ml (0.04-4 microM) concentration range. The method has been applied to analyze the plasma versus time profile of busulfan in human subjects following administration of an oral dose of 4 mg/kg per day as a marrow ablative chemotherapy for bone marrow transplantation.     

A short enantioselective synthesis of pipecolic acid

A simple enantioselective route to pipecolic acid is described. The key step involves the Sharpless asymmetric epoxidation of an N-protected aminoheptenol which spontaneously cyclises to a piperidine derivative on deprotection.     

Diastereoselective functionalizations of enecarbamates derived from pipecolic acid towards 5-guanidinopipecolates as arginine mimetics

Various substituents could be diastereoselectively introduced into the 5-position of pipecolic acid via electrophilic or free-radical-initiated addition to the carbon-carbon double bond of endocyclic enecarbamates derived from pipecolic acid. This study allowed the diastereoselective synthesis of both cis- and trans-5-guanidino pipecolates, which were designed as constrained arginine mimetics and whose potential inhibition of nitric oxide synthase (NOS) was evaluated with three NOS isoforms.     

Determination of mefloquine by electron-capture gas chromatography after phosgene derivatization in biological samples and in capillary blood collected on filter paper

Mefloquine is determined in 100-microliter samples of whole blood, plasma and capillary blood collected on filter paper by gas chromatography with electron-capture detection after derivatization with phosgene. Sample preparation for whole blood and plasma involves a protein precipitation step that uses a combination of zinc and acetonitrile, followed by simultaneous extraction with methylene chloride and derivatization with phosgene at pH 9.50. Filter paper spots are immersed for 12-24 h in 0.1 M hydrochloric acid, followed by simultaneous extraction with methyl tert.-butyl ether and derivatization. After evaporation of the organic phase and reconstitution with ethyl acetate, 1 microliter of the extract is injected into a megabore capillary column. Because of the high sensitivity of the method, mefloquine concentrations down to 25 nmol/l (9.5 micrograms/l) are determined in 100-microliters samples with a relative standard deviation of 12% at the 25 nmol/l level. Excellent precision was obtained over the range of concentrations tested, 0.10-3 mumol/l (45-1100 micrograms/l), in both plasma and whole blood and from filter-paper-collected capillary blood. The day-to-day relative standard deviation in plasma at the therapeutic level (1-3 mumol/l) was 4.5% (n = 8).     

Highly sensitive and simple method for measurement of pipecolic acid using reverse-phase ion-pair high performance liquid chromatography with tris(2,2'-bipyridine)ruthenium(III) chemiluminescence detection

A new, highly sensitive chemiluminescence method for measurement of pipecolic acid in various substances such as human serum, cow's milk, beer, and apple juice has been developed. The method is based on reverse-phase ion-pair high performance liquid chromatographic separation and subsequent tris(2,2'-bipyridine)ruthenium(III) chemiluminescence detection. It was confirmed that imino acids show strong chemiluminescence upon mixing with tris(2,2'-bipyridine)ruthenium(III). A calibration graph, based on a standard pipecolic acid solution, was linear over the range 5.0x10(-9)M to 2.0x10(-5)M and the detection limit was 24fmol (signal-to-noise ratio=3). This highly sensitive and selective determination method can be applied to selected samples without purification or pre-concentration procedures. Compared to the previous HPLC methods, the proposed method is easier, more sensitive, and time-saving.     

Sensitive determination of pipecolic acid in serum by high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride as a fluorescent labelling reagent

A simple and highly sensitive high-performance liquid chromatography (HPLC) for the determination of pipecolic acid (PA) in serum was developed. Pipecolic acid and nipecotic acid (internal standard (IS)) were derivatised with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride (DMS-Cl) to produce fluorescent sulfonamides. The labelling reaction was carried out at 70 °C for 15 min at pH 9.0. The fluorescent derivatives were separated on a reversed-phase column (45 °C) with a stepwise elution using methanol/acetonitrile/10 mmol l⁻¹ acetic acid (42:5:53) and methanol at a flow rate of 1.0 ml/min and detected at excitation and emission wavelengths of 316 and 403 nm, respectively. The labelling yield was 100.8%. The detection limit of pipecolic acid was 4 fmol at signal-to-noise ratio of 3. The within-day and day-to-day relative standard deviations were 3.3-8.1 and 1.4-6.4%, respectively. The concentration of pipecolic acid in normal human serum was 1.09±0.37 μmol l⁻¹.     

Biosynthesis of pipecolic acid by RapL, a lysine cyclodeaminase encoded in the rapamycin gene cluster

Rapamycin, FK506, and FK520 are immunosuppressant macrolactone natural products comprised of predominantly polyketide-based core structures. A single nonproteinogenic pipecolic acid residue is installed into the scaffold by a nonribosomal peptide synthetase that also performs the subsequent macrocyclization step at the carbonyl group of this amino acid. It has been assumed that pipecolic acid is generated from lysine by the cyclodeaminases RapL/FkbL. Herein we report the heterologous overexpression and purification of RapL and validate its ability to convert L-lysine to L-pipecolic acid by a cyclodeamination reaction that involves redox catalysis. RapL also accepts L-ornithine as a substrate, albeit with a significantly reduced catalytic efficiency. Turnover is presumed to encompass a reversible oxidation at the alpha-amine, internal cyclization, and subsequent re-reduction of the cyclic delta1-piperideine-2-carboxylate intermediate. As isolated, RapL has about 0.17 equiv of tightly bound NAD+, suggesting that the enzyme is incompletely loaded when overproduced in E. coli. In the presence of exogenous NAD+, the initial rate is elevated 8-fold with a Km of 2.3 microM for the cofactor, consistent with some release and rebinding of NAD+ during catalytic cycles. Through the use of isotopically labeled substrates, we have confirmed mechanistic details of the cyclodeaminase reaction, including loss of the alpha-amine and retention of the hydrogen atom at the alpha-carbon. In addition to the characterization of a critical enzyme in the biosynthesis of a medically important class of natural products, this work represents the first in vitro characterization of a lysine cyclodeaminase, a member of a unique group of enzymes which utilize the nicotinamide cofactor in a catalytic manner.     

Determination of the new monoamine oxidase inhibitor brofaromine and its major metabolite in biological material by gas chromatography with electron-capture detection

A sensitive gas chromatographic assay for the simultaneous determination of brofaromine [4-(7-bromo-5-methoxy-2-benzofuranyl)piperidine hydrochloride], a new monoamine oxidase-A inhibitor, and its major metabolite was developed and validated. After addition of 4-(5-bromo-2-benzofuranyl)piperidine as internal standard, the compounds were isolated from biological fluids by liquid-liquid extraction at basic pH. After derivatization with heptafluorobutyric anhydride the compounds were chromatographed using a packed column (OV-17) and an electron-capture detector. The limit of quantitation was ca. 0.03 nmol per sample (10 ng) for both compounds. analysis of spiked samples demonstrated the good accuracy and precision of the method, which is suitable for use in pharmacokinetic and bioavailability studies. The method was applied to samples from an experiment in a healthy volunteer treated with a single oral dose of 75 mg of brofaromine hydrochloride. Plasma profiles before and after enzymic hydrolysis showed that about one-third of the total brofaromine in plasma and practically all of the major metabolite (O-desmethylbrofaromine) were present in the conjugated form.     

Efficient synthesis of a new pipecolic acid analogue with a bicyclic structure

This report describes the synthesis of 2-azabicyclo[2.2.2]octane-1-carboxylic acid, a constrained pipecolic acid analogue. The route gives a very good total yield starting from cheap and readily available compounds and uses very easy reactions.     

Synthesis of new triazole substituted pyroaminoadipic and pipecolic acid derivatives

Racemic 5-(4,5-substituted-1H-1,2,3-triazol-1yl)-pyroaminoadipic and pipecolic acid derivatives were synthesized from meso dimethyl-α,α′-dibromoadipate 1 in good yields using mild reaction conditions. The key step of this reaction sequence was the 1,3-dipolar cycloaddition of an acetylenic compound on α-azido-α′-bromoadipate 2. A reactive α-(substituted-1H-1,2,3-triazol-1-yl)-α′- bromoadipate derivative 3a-d was generated and reacted with sodium azide followed by Pd/C-catalyzed hydrogenation to provide lactams 5a-d. The chemoselective reduction of the amide carbonyl group of 5a-d with BH₃ followed by acid hydrolysis provided 5-(4,5-substituted-1H-1,2,3-triazol-1-yl) pipecolic acids in racemic form.     

Gas chromatographic quantitation of methoxyphenamine and three of its metabolites in plasma

Sensitive gas chromatographic procedures for the determination of methoxyphenamine and three of its metabolites in plasma have been developed. The metabolites were measured using an electron-capture detector. This simple procedure is based on the precipitation of protein from a 1-ml plasma sample with 10% trichloroacetic acid, followed by aqueous derivatization with pentafluorobenzoyl chloride at pH 9.2 and a single-step cyclohexane extraction. The lower limit of detection for the N-desmethyl, O-desmethyl and aromatic 5-hydroxy metabolites of methoxyphenamine were 1.6, 3.1 and 2.2 ng ml-1, respectively, with coefficients of variation less than 10%. The poor electron-capture response of fluorinated derivatives of methoxyphenamine necessitated the use of nitrogen-phosphorus detection. Extractive derivatization with pentafluorobenzoyl chloride, without the need for protein precipitation, enabled quantitation of methoxyphenamine down to 3.8 ng ml-1 from a 2-ml aliquot of plasma. In a pilot study involving healthy volunteers who received a single oral dose of methoxyphenamine hydrochloride plasma concentration could be followed in all three subjects for at least 24, 32, 12 and 4 h for methoxyphenamine and the O-desmethyl, 5-hydroxy and N-desmethyl metabolites, respectively.     

Synthesis of chiral nonracemic 4-trans-substituted pipecolic acid derivatives

The syntheses and resolutions of enantiomerically enriched 4-phenyl, 4-tert-butyl, and 4-isopropyl pipecolic acids are described. Optically active diastereomers were prepared by diastereomeric salt formation with the chiral base, l-tyrosine hydrazide, to provide Cbz or Boc protected 4-cis-d-pipecolic acid derivatives in >98% ee. Subsequent esterification followed by sodium methoxide catalyzed epimerization provided the isomeric 4-trans-l-pipecolic esters. In addition, an efficient synthesis of 4-phenyl-cis-pipecolic acid is described.     

Simultaneous extraction and derivatization of 2-chlorovinylarsonous acid from soils using supercritical and pressurized fluids

Supercritical carbon dioxide and pressurized fluids are compared for the extraction with in situ derivatization of 2-chlorovinylarsonous acid (CVAA) from a series of seven spiked soils. Samples are allowed to age (up to 42 days) and periodically extracted. Sample ageing leads to a recovery decrease due to the development of strong interactions between CVAA and matrix active sites, as time elapses. A similar behavior is observed when usual ultrasonic extraction is performed. Supercritical fluid extraction (SFE) with in situ derivatization leads to the highest recovery. Moreover, SFE allows a solvent consumption reduction. A limit of detection of 0.2 microg/g is reached with the SFE method.