Application of ligand-exchange capillary electrophoresis to the chiral separation of alpha-hydroxy acids and beta-blockers

The application of the principle of ligand-exchange capillary electrophoresis to two substance classes is described. As chiral selector N-(2-hydroxyoctyl)-L-4-hydroxyproline-copper(II) complex was used. This principle was applied to the chiral separation of alpha-hydroxy acids and drugs containing amino alcohol structure such as beta-blockers. The enantioselectivity was found to be strongly dependent on pH corresponding to the optimal conditions for complex formation for each structure class.     

Kinetic method for the simultaneous chiral analysis of different amino acids in mixtures

The kinetic method has been extended to enantiomeric excess (ee) determinations on amino acids present in mixtures. Singly charged trimeric clusters [Cu(II)(ref*)(2)(A(m)) - H](+) are readily generated by electrospraying solutions containing Cu(II), a chiral reference ligand (ref*), and the amino acids (analytes A(m), m = 1-3). A trimeric cluster ion for each amino acid is individually mass-selected and then collisionally activated to cause dissociation by competitive loss of either the reference ligand or the analyte. For each analyte in the mixture, as shown from separate experiments, the logarithm of the ratio of the fragment abundances for the complex containing one enantiomer of this analyte expressed relative to that for the fragments of the corresponding complex containing the other enantiomer is linearly related to the enantiomeric composition of the amino acid. Formation and dissociation of each trimeric complex ion are shown to occur independently of the presence of other analytes. Chiral selectivity appears to be an intrinsic property and the chiral selectivity R(chiral(m)) measured from the mixture of analytes is equal to R(chiral) measured for the pure analyte. The sensitive nature of the methodology and the linear relationship between the logarithm of the fragment ion abundance ratio and the optical purity, characteristic of the kinetic method, allow the determination of chiral impurities of less than 2% ee in individual compounds present in mixtures by simply recording the ratios of fragment ion abundances in a tandem mass spectrum.     

A new chiral lanthanide NMR probe for the determination of the enantiomeric purity of alpha-hydroxy acids and the absolute configuration of alpha-amino acids in water

A water-soluble, enantiopure lanthanide complex, SSS-[Ln x L3], has been assessed as an effective chiral derivatizing agent for the determination of the enantiomeric purity of alpha-hydroxy acids in aqueous solution. The complex displays superior chemical shift non-equivalence (DeltaDeltadelta approximately 2-11 ppm) for the diastereomeric resonances of interest compared to lanthanide shift reagents reported in the literature (DeltaDeltadelta <0.1 ppm, typically). 1H NMR studies have also revealed that SSS-[Ln x L3] can be used to determine the absolute configuration of alpha-amino acids at physiological pH, in water. The ability of SSS-[Ln x L3] to signal anion binding and, in particular, to distinguish between diastereomers through optical techniques such as lanthanide luminescence and circular dichroism has also been assessed.     

Synthesis of all nineteen appropriately protected chiral alpha-hydroxy acid equivalents of the alpha-amino acids for Boc solid-phase depsi-peptide synthesis

[reaction: see text] The preparation of depsi-peptides, amide-to-ester-substituted peptides used to probe the role of hydrogen bonding in protein folding energetics, is accomplished by replacing specific l-alpha-amino acid residues by their alpha-hydroxy acid counterparts in a solid-phase synthesis employing a t-Boc strategy. Herein we describe the efficient stereoselective synthesis of all 19 appropriately protected alpha-hydroxy acid equivalents of the l-alpha-amino acids, employing commercially available materials, expanding the number of available alpha-hydroxy acids from 9 to 19.     

Ligand anatomy: probing remote substituent effects in asymmetric catalysis through NMR and kinetic analysis

A series of structurally related beta-amino alcohols only differing in the steric bulk of a remote alkoxy group exhibit striking differences in kinetic behavior when used as ligands in the asymmetric diethylzinc addition to benzaldehyde (R = Trityl, much more active). A combination of NMR titration studies and kinetic analysis allows the quantitative decomposition of the remote substituent effect into a lower dimerization constant of the active species and a much faster ethyl addition step.     

A new method for the synthesis of chiral beta-branched alpha-amino acids

A new method for the synthesis of chiral beta-branched alpha-amino acids based on a copper-mediated directed allylic substitution reaction with Grignard reagents is reported. This is the first case in which a delta-stereogenic center is controlling the diastereoselectivity of an o-DPPB-directed allylic substitution. Depending on the alkene geometry of the starting material either diastereomer, anti or syn, is accessible with good levels of acyclic stereocontrol.     

Characterization of the cereulide NRPS alpha-hydroxy acid specifying modules: activation of alpha-keto acids and chiral reduction on the assembly line

Several nonribosomal peptide natural products are composites of alpha-hydroxy acid and alpha-amino acid monomers. Cereulide, the emetic toxin from the human pathogen Bacillus cereus, and valinomycin, from Streptomyces spp., are closely related macrocyclic K+ ionophores. The macrocyclic core of each natural product contains alternating peptide (six) and ester (six) bonds, and their cyclododecadepsipeptide structures consist of a tetradepsipeptide unit repeated three times. Here we overexpress the cereulide NRPS alpha-hydroxy acid specifying modules from CesA and CesB and demonstrate that each contains an alpha-keto acid activating adenylation domain and a chiral alpha-ketoacyl-S-carrier protein reductase (alpha-KR). The logic used by the cereulide NRPS is likely at work in the valinomycin NRPS and may be the general strategy used in bacterial NRPSs to form alpha-hydroxy acid containing natural products.     

A new method for production of chiral 2-aryl-2-fluoropropanoic acids using an effective kinetic resolution of racemic 2-aryl-2-fluoropropanoic acids

We report a new method for the preparation of chiral 2-aryl-2-fluoropropanoic acids, including 2-fluoroibuprofen, a fluorinated analogue of non-steroidal anti-inflammatory drugs (NSAIDs), by the kinetic resolution of racemic 2-aryl-2-fluoropropanoic acids using enantioselective esterification. By applying pivalic anhydride (Piv2O) as a coupling agent, bis(α-naphthyl)methanol [(α-Np)2CHOH] as an achiral alcohol, and (+)-benzotetramisole (BTM) as a chiral acyl-transfer catalyst, a series of racemic 2-aryl-2-fluoropropanoic acids were kinetically separated to afford the optically active carboxylic acids and the corresponding esters with good to high enantiomeric excesses. This technology can provide a convenient approach to furnish the chiral α-fluorinated drugs containing quaternary carbons at the α-positions in the 2-aryl-2-fluoropropanoic acid structure.     

Ligand exchange chromatography for analysis and preparative separation of tritium-labelled amino acids

Racemic tritium-labelled amino acids were separated into optical isomers by chromatography on a chiral polyacrylamide sorbent filled with copper ions. The polyacrylamide sorbent is synthesized by Mannich's reaction through the action of formaldehyde and L-phenylalanine upon polyacrylamide Biogel P-4 in an alkali phosphate buffer. Tritiumlabelled amino acids are eluted by a weak alkali solution of ammonium carbonate. Data are presented on the ligand exchange chromatography of amino acids depending on the degree to which the sorbent is filled with copper ions and on the eluent concentration. Conditions are suggested for the quantitative separation of amino acid racemates. Amino acids are isolated from the eluent on short columns filled with sulfonated cation exchanger in the H⁺ form. HPLC on modified silica gel sorbents is also used for the analysis of tritium-labelled optically active amino acids. Amino acids are eluted by a weakly acidic water-methanol solution containing ammonium acetate. UV and scintillation flow type detectors are used.     

Interpolation method for treatment of mutual kinetic effects

An interpolation method for resolution of binary mixtures subject to mutual kinetic effects is reported. For a mixture of two components A and B reacting under pseudo-first-order conditions with a reagent to yield the same product, the determination of the concentration of A is based on obtaining several calibration graphs from plots of final absorbance of component B in the presence of different known amounts of A. The slope of these plots is dependent on the concentration of component A. The concentration of B is calculated from the plots of the difference in the initial rates of the concentration/time curves for the sample and several reference solutions vs. the concentration of B. The method is accurate for the resolution of binary mixtures subject to synergic effects. Fast, direct complex-formation reactions between cobalt and nickel with pyridoxal thiosemicarbazone are used to evaluate the performance of the proposed method with use of a home-made stopped-flow mixing module. The determination of cobalt/nickel mixtures is feasible in the weight ratio range 2.5:1.0 to 1.0:5.0, in the μg ml^?1 region with an accuracy and precision less than 5 and 2%, respectively.     

Cerate oxidimetry of alpha-hydroxy and alpha-keto carboxylic acids in acetic acid

Methods for the preparation and use of a solution of ammonium hexanitratocerate(IV) in acetic acid as a titrant for the oxidation of alpha-hydroxy and alpha-keto carboxylic acids in acetic acid are presented. The applicability of this reagent with respect to the stoichiometry, products and reaction times obtained is discussed.     

Simple enzymatic detection method for urinary sulfated 7 alpha-hydroxy bile acids in normal subjects and in patients with acute hepatitis

Urinary sulfated primary bile acids, 7 alpha-hydroxy bile acids, are detected by an enzymatic method using 7 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.-, 7 alpha-HSD) after chromatographic fractionation on Sephadex G-25. Urinary sulfated or glucuronated bile acids are hydrolyzed by beta-glucuronidase/sulfatase (EC 3.2.1.31/EC 3.1.6.1) from Helix pomatia and then released 7 alpha-hydroxy bile acids are detected with 7 alpha-HSD in the presence of beta-ND+, diaphorase (EC 1.6.99.2, from Clostridium kluyveri) and 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride. The absorbance of formazan formed during the enzymic reaction is measured at 500 nm. Excretion values of 7 alpha-hydroxy bile acids in normal subjects and in patients with acute hepatitis were compared. This enzymatic detection method for the excretion pattern of urinary 7 alpha-hydroxy bile acids may be useful for clinical diagnosis.     

Correlation analysis of substituent effects on the acidity of benzoic acids by the AM1 method

Theoretical analysis of the electronic effect of aromatic substituents was done with the use of the AM1 computational procedure. The gas-phase acidity of substituted benzoic acids was linear with the difference in the heat of formation between corresponding benzoic acids and benzoate anions, the energy of the highest occupied molecular orbital, and the net charge on the acidic oxygen atoms of the corresponding benzoate anions. The Hammett σ constant was linearly correlated with the net charge on the atoms of the acid moiety of substituted benzoic acids. The AM1 computational procedure satisfactorily reproduced the electronic properties of a wide variety of substituents.     

A simple method for the determination of enantiomeric excess and identity of chiral carboxylic acids

The association between an achiral copper(II) host (1) and chiral carboxylate guests was studied using exciton-coupled circular dichroism (ECCD). Enantiomeric complexes were created upon binding of the enantiomers of the carboxylate guests to the host, and the sign of the resultant CD signal allowed for determination of the configuration of the studied guest. The difference in magnitudes and shapes of the CD signals, in conjunction with linear discriminant analysis (LDA), allowed for the identity of the guest to be determined successfully. A model was created for the host-guest complexes which successfully predicts the sign of the observed CD signal. Further, Taft parameters were used in the model, leading to rationalization of the observed magnitudes of the CD signals. Finally, the enantiomeric excess (ee) of unknown samples of three chiral carboxylic acid guests was determined with an average absolute error of ±3.0%.     

A novel spectrophotometric method for the simultaneous kinetic analysis of ternary mixtures by mean centering of ratio kinetic profiles

In this paper, a novel and very simple method was developed for the simultaneous spectrophotometric determination of ternary mixtures, without prior separation steps. The method is based on the mean centering of ratio kinetic profiles. The mathematical explanation of the procedure is illustrated. In order to investigate the applicability of the proposed method, it was applied to the simultaneous spectrophotometric determination of hydrazine, phenylhydrazine and acetylhydrazine based on their condensation reactions with p-(dimethylamino)benzaldehyde (DAB) and p-nitrobenzaldehyde (NB) in micellar sodium dodecyl sulfate (SDS) media as a typical ternary mixture. The analytical characteristics of the method such as accuracy, precision, relative standard deviation (R.S.D.) and relative standard error (R.S.E.) were calculated.     

Chiral analysis by mass spectrometry using the kinetic method in flow systems

Chiral analysis is an important task of analytical chemistry. Besides separation techniques, mass spectrometry can be applied in this field. One mass spectrometric approach is based on Cooks' kinetic method. The method was successfully applied in a static system in which the concentration of the analyte as well as the chiral selector solution was constant during the experiment. The application of the kinetic method in dynamic systems (changing concentration of analyte) is presented. Such systems allow the speeding up of the analytical process (flow injection analysis (FIA)) or the use of the kinetic method for chiral detection after liquid chromatographic separation.The influence of the concentration of the components of the chiral selector solution as well as its flow rate on the recognition of enantiomers was evaluated. A new procedure for correction for the differences between ratio of enantiomers in the liquid phase and their observed ratio in the gas phase is also described. A significant improvement in accuracy using this procedure was achieved. Applicability of the method was demonstrated in the analysis of amino acids using FIA as well as HPLC/MS. After an achiral separation of leucine and isoleucine, chiral mass spectrometric detection was successfully used for enantiomeric recognition.     

Quantitative chiral analysis of phthaloylglutamic acid and related analogs by a single ratio kinetic method using electrospray ionization and matrix-assisted laser desorption techniques

A rapid method for quantitative chiral analysis of phthaloylglutamic acid and its dimethyl ester by Cook's kinetic method is demonstrated using electrospray ionization (ESI) and matrix-assisted laser desorption techniques. Transition-metal-bound complex ions containing the chiral phthaloylglutamic acid and its dimethyl ester are generated by ESI mass spectrometry and subjected to collision-induced dissociation. The ratio of the two competitive dissociation rates is related to the enantiomeric composition of the drug mixture. A seven-point calibration curve, derived from the kinetic method, allowed rapid quantitation of the enantiomeric excess of drug mixtures. In this paper, matrix-assisted laser desorption/ionization (MALDI) coupled with the linear ion trap (LIT) technique is evaluated for its applicability as a complementary technique to ESI for chiral discrimination and quantitation.     

Relative calcium-binding strengths of amino acids determined using the kinetic method

We have measured the relative calcium-binding energies of amino acids using tandem mass spectrometry of Ca(2+)-bound trimeric amino acids. Although calcium-bound dimeric amino acid complexes coordinated too strongly to allow observation of the two competing dissociation products (calcium-bound monomeric ions) required for analysis of their metal binding affinities using the conventional kinetic method, the Ca(2+)-bound trimeric cluster ions dissociated readily to form dimeric cluster ions through simple ligand losses. The calcium-binding energies were obtained by comparing the ratio of the [Ca(2+)(A(1))(2) - H(+)](+) and [Ca(2+) (A(1))(A(2)) - H(+)](+) ions that dissociated from the [Ca(2+) (A(1))(2)(A(2)) - H(+)](+) ion and the ratio of the [Ca(2+)(A(2))(2) - H(+)](+) and [Ca(2+)(A(1)) (A(2)) - H(+)](+) ions that dissociated from the [Ca(2+)(A(1))(A(2))(2) - H(+)](+) ion, where A(1) and A(2) represent two amino acids. The energies deduced from this analysis represent the relative average binding energies of complexes having the form [Ca(2+)(A(1))(2) - H(+)](+). The relative Ca(2+)-binding strengths of the alpha-amino acid complexes follow the order Cys < Ser < Thr < Ile < Leu < Val < Gly < Ala < Pro < Phe < Met < Tyr < Asn < His < Gln < Trp < Lys < Arg. To our knowledge, this report provides the first example of using kinetic methods to determine the relative binding strengths of divalent metal-amino acid complexes.