Enantiomer Labeling Research Articles

Study on the Influence of Chirality in the Threading of Calix[6]arene Hosts with Dialkylammonium Axles

The influence of chirality in calixarene threading has been studied by exploiting the “superweak anion approach”. In particular, the formation of chiral pseudo[2]rotaxanes bearing a classical stereogenic center in their axle and/or wheel components has been considered. Two kind of pseudo[2]rotaxane stereoadducts, the “endo-chiral” and “exo-chiral” ones, having the stereogenic center of a cationic axle inside or outside, respectively, the calix-cavity of a chiral calixarene were preferentially formed with specifically designed chiral axles by a fine exploitation of the so-called “endo-alkyl rule” and a newly defined “endo-α-methyl-benzyl rule” (threading of a hexaalkoxycalix[6]arene with a directional (α-methyl-benzyl)benzylammonium axle occurs with an endo-α-methyl-benzyl preference). The obtained pseudorotaxanes were studied in solution by 1D and 2D NMR, and in the gas-phase by means of the enantiomer-labeled (EL) mass spectrometry method, by combining enantiopure hosts with pseudoracemates of one deuterated and one unlabeled chiral axle enantiomer. In both instances, there was not a clear enantiodiscrimination in the threading process with the studied host/guest systems. Possible rationales are given to explain the scarce reciprocal influence between the guest and host chiral centers.

Correlation between amino acid racemization and processing conditions for various wheat products, oil seed press cakes and lignin samples

The occurrence of d-amino acids in native and processed plant products is brought into context with the harshness of their treatment condition. It was found that already a small increase in processing harshness such as the milling efficiency of wheat straw or the increased pressure and duration of pumpkin seed oil extraction leads to traceable changes in the overall amino acid content as well as the racemization rate of free and protein bound amino acids. In the top position of our process harshness scale lies the strong alkali and heat-induced extraction of polyphenolic compound lignin from wood and other cellulose-rich plant fibers, e.g. wheat straw, during paper production. Since lignin accumulates in large quantities and its transformation into value-added “bio” products necessitates the recovery of native, functionally preserved lignin, milder pulping methods gain increasing interest. An unexpected drawback of such mild extraction conditions is however the high nitrogen content of such lignin products, which further increase with increasing mildness of the pulping procedure. The presence of amide bands in the FT-IR spectra of such mildly processed lignin has revealed that this nitrogen originated from co-extracted proteins and peptides. The total amino acid content as well as the free amino acid content and the degree of racemization was determined by gas chromatography–ion trap mass spectrometry (GC–Iontrap MS) employing enantiomer labeling for quantification. Cross-determination of the average amino acid content was calculated from nitrogen values obtained by elemental analysis and correlation factors were calculated from measured and calculated data. Since those amino acids with two chiral centers, such as isoleucine, threonine and hydroxyproline exhibit the highest possible diversity in terms of their relative amount to one another as well as their internal enantiomer distribution, this set of two-centered amino acids provided very distinct amino acid pattern, which proofed to be characteristic for the type of plant material as well as the harshness of the different processing conditions.

Structural characterization of a lipopeptide antibiotic A54145E(Asn3Asp9) produced by a genetically engineered strain of Streptomyces fradiae

A potent new lipopeptide antibiotic, A54145E(Asn(3)Asp(9)), was isolated from the fermentation broth of Streptomyces fradiae DA1489 engineered to delete genes encoding enzymes involved in hydroxylation of Asn(3) and methoxylation of Asp(9). The chemical structure predicted from the genetic changes in the biosynthetic pathway was determined by analyses of chemical transformations, D, L-amino acid quantitation by enantiomer labeling, tandem LC-MS/MS and 2D NMR techniques. These studies confirmed the primary amino acid sequence of A54145E(Asn(3)Asp(9)) predicted from the genetic engineering strategy, and also confirmed the structure and locations of three D-amino acids predicted from bioinformatic studies.

Structural Characterization of Daptomycin Analogues A21978C1-3(d-Asn11) Produced by a Recombinant Streptomyces roseosporus Strain

Three daptomycin-related lipopeptides, A21978C1-3(d-Asn11) (2-4), were purified from the fermentation broth of a recombinant Streptomyces roseosporus strain. Their chemical structures were determined by analyses of the biosynthetic pathway, chemical transformations, d,l-amino acid quantitation by enantiomer labeling, tandem LC-MS/MS, and 2D-NMR techniques. Compounds 2-4 exhibited potent antibacterial activity against Staphylococcus aureus with MIC values of 0.6, 0.3, and 0.15 microM, respectively, well correlated to the acyl tail chain length.

質量分析法による糖類とカチオン種との錯形成の研究

The chiral discrimination abilities of various permethylated oligosaccharides were evaluated toward ammonium ions of esters of amino acids using the fast atom bombardment (FAB) mass spectrometry (MS)/enantiomer labeled (EL) guest method. The complexation properties of the selected permethylated oligosaccharides were examined by other spectroscopic methods such as 1H-nuclear magnetic resonance, and the structures of their complexes were discussed. The artificial chiral hosts were synthesized on the basis of the obtained information, and then one of them was applied to evaluation of optical purity of chiral ammonium salts using FAB mass spectrometry.

ESIおよびFABマススペクトロメトリーを用いるアミン類およびジペプチド類のエナンチオマー過剰度の決定

The enantiomeric excess (e.e.) determination of organic amines and dipeptides has been examined using ESI and FAB mass spectrometry. Here, an equimolar amount of labeled and unlabeled enantiomeric host pair compounds is mixed with a given e.e.-unknown guest compound. This is called the enantiomer labeled (EL)-host method. The enantiomeric host pairs employed were (1) chiral podands having galactose end-groups and (2) chiral crown ethers. Three sets of the Ie (intensity excess)-e.e. plot showed excellent linear relationships, indicating that the e.e.-value of e.e.-unknown amine compounds can be determined by the simple ESIMS or FABMS coupled with the EL-host method, within the mean error of 1.5-4%e.e.

Matrix Effects on the Chiral Recognition Determined by the Relative Peak Intensity of Diastereomeric Host-Guest Complex Ions Using the FAB Mass Spectrometry/Enantiomer Labeled Guest Method.

The FAB mass spectrometry (MS)/enantiomer labeled (EL) guest method is one of the useful technique to easily evaluate chiral discrimination ability of a chiral host (H) toward chiral guests (GR+, GS-dn+). In this paper, the matrix dependency of the relative peak intensity (IRIS) of the diastereomeric host-guest complex ions [(H+GR)+ and (H+GS-dn)+] was investigated (host, MeFruNys; guest, Phe-O-iPr+). The IRIS values of spectra measured using NBA, NPOE, and DTDE matrices agreed almost with the ratio of concentrations of the complex ions calculated from the association constants (KR and KS) in chloroform at 298 K. While, the IRIS values of spectra using glycerol matrix reached almost unity. It was assumed that the IRIS values depend upon the matrices used since the difference of solvation ability of the matrices affects the actual concentration of the guest concerning in the complexation. And then, it was recommended that NBA is the best matrix for chiral recognition mass spectrometry studies.

定量的キラル認識マススペクトロメトリーの進歩

The developments of our quantitative chiral recognition studies in the host-guest complexations between chiral crown ethers and chiral amino acid esters by FAB, ESI, and MALDI mass spectrometry have been described. Both the enantiomer labeled (EL)-guest method and the EL-host method, which had been previously proposed by us, were successfully advanced. The relative peak intensity (IRIS) values of the diastereomeric host-guest complex ions appeared in mass spectra were especially noted, and their use to chiral recognition was illustrated. Generally, the former method was used to chiral recognition ability-determination of a given host, and the latter one was used to enantiomeric excess (e.e.)-determination of a given guest. Through the manuscript, emphasis has been put on the finding that one can detect correctly the concentration ratio of the two diastereomeric host-guest complex ions formed in a matrix solution by FAB mass spectrometry and then, that FABMS is a powerful tool for detecting chiral host-guest interactions in the host-guest complexation systems. Recent ,e.e.-determination methods by ESI mass spectrometry etc. were also summarized.

Development of an improved automated gas-chromatographic chiral analysis system: application to non-natural amino acids and natural protein hydrolysates.

In the use of combinatorial chemistry as a novel strategy for drug discovery, the chirality assessment of building blocks used for library construction is particularly important in the evaluation of biological actions of generated libraries. The procedure for chiral analysis of lead compounds in screening may be of a high priority, particularly in the case of the protection of intellectual rights. Previously, an automated amino acid analysis system using enantiomer labeling was developed. The system incorporates a reactor, which allows automated esterification and acylation of amino acids, and is connected to an on-line gas chromatographic system. A capillary column coated with a chiral phase is employed for the separation of the enantiomers. This particular system is improved with a newly constructed "high-throughput auto-derivatizer" in combination with a new GC-system. The resulting data can be processed by newly constructed software. The analyses of amino acid derivatives or hydrolysates of proteins and peptides are carried out routinely within ca. 45 min, including derivatization. Using this system several non-natural amino acids were tested with respect to the stereoisomeric configuration. In addition, acid hydrolysates of food proteins and tissues obtained by autopsy were analyzed as an application to proteome research.

Quantitative determination of isoflurane enantiomers in blood samples during and after surgery via headspace gas chromatography–mass spectrometry

The quantitative analysis of the chiral volatile anesthetic isoflurane ( 1) for biomedical applications by means of enantioselective gas chromatography (mass sensitive detector, selected ion monitoring) was studied. Two methods for the quantification of the enantiomers in blood samples drawn during and after narcosis were compared. Either the isomeric enflurane ( 2) was selected as an internal standard or a single enantiomer of 1 was used for the standard addition method, an approach referred to as ‘enantiomer labeling’. Concentrations up to 0.3 μmol/l of the single enantiomers could be differentiated two days after anesthesia. The presented data imply that the body clearance for (+)-( S)- 1 and (−)-( R)- 1 proceeds to a measurable degree of enantioselectivity.

ＦＡＢＭＳによるキラル分子認識化合物の探求 キラルクラウンエーテル類のキラル識別能

The detection method of chiral discrimination ability of some macrocycles toward amino acid ester hydrochlorides using FAB mass spectrometry has been explained in this paper. The FABMS enantiomer labeled (EL) guest method, which was recently proposed by Sawada et al., was a useful and facile technique to detect chiral discrimination ability of new chiral hosts. A host (H) was complexed with 1 : 1 amino acid guest mixture of an (R)-enantiomer (GR+) and a deuterium labeled (S)-enantiomer (GS-dn+). The enantioselectivity of the host for the guest was quantitatively estimated from the relative peak intensity (I (H-GR)+/I (H-GS-dn)+=IRIS) values of the two host-guest complex ion peaks at a difference of m/z=n.

Enantiomer separation by gas chromatography on chiral stationary phases

The analytical separation of enantiomers by capillary gas chromatography on chiral stationary phases (CSPs) is reviewed. Inherent principles and methodological advances are described. Three principal CSPs tailored for hydrogen bonding, coordination and inclusion are currently employed. Cyclodextrin derivatives have proved to be the most versatile and universal CSPs in gas chromatography. Anchoring the CSPs to a polysiloxane backbone leads to Chirasil-type stationary phases with improved temperature stability, efficiency and lifetimes. Immobilization of Chirasil-type stationary phases allows enantiomer separations also in capillary supercritical fluid chromatography and capillary electrochromatography. Thermodynamic parameters of enantioselectivity in gas chromatography, kinetic studies of enantiomerization and methods of enantiomer labelling are discussed.

Determination of d, l-Amino Acids in Natural Water by Gas Chromatography with Enantiomer Labeling

Amino acids in natural water have been determined quantitatively in form of L-isomers. At the same time their D/L ratios have been determined by gas chromatography with enantiomer labeling. The water was sampled at Biwako, Kinokawa and Takihata, For an analysis of free amino acids, a batch technique was used to preconcentrate the amino acids by ion-exchange resin of Dowex 50W-X8. For total dissolved amino acids, the water sample was first heated in 6 M HC1 to hydrolyze the bound amino acids, and then purified by a column packed with Dowex 50W-X8. The major amino acids in natural water samples were found to be Thr, Ser, Asp and Orn in free form, and Gly, Ser, Asp and Glu in total dissolved form. In addition, some amino acids in free form showed high D/L ratio that might be due to the pollution.

Plasma l-arginine levels in a rabbit model of hypercholesterolaemia

A novel method for the quantitation of l-arginine in plasma samples has been developed, based on the technique of enantiomer labelling coupled to the capillary gas chromatographic separation of amino acids. Using this technique plasma l-arginine levels have been measured in rabbits fed a 1% cholesterol-containing diet and compared with rabbits fed a standard diet. Blood cholesterol levels were significantly elevated in the rabbits fed cholesterol but no significant difference was found in plasma l-arginine levels between the cholesterol-fed and control animals. No d-arginine was found in any of the plasma samples. It is concluded that the impairment in endothelium-dependent relaxation previously described in this animal model of hypercholesterolaemia is not due to deficiency of plasma l-arginine.

SHIMADZU-CAT MODEL DLAA-1, A NOVEL AMINO ACID ANALYZER FOR D/L AND QUANTITATIVE ANALYSIS

Chirality analysis of amino acids is of particular interest in bioindustries. Especially in many pharmaceutical industries it is very important to evaluate synthetic bioactive peptides and to analyze proteins in processing in food industries. Enantiomer labelling methodology is a new, generation of quantitative amino acid analysis which allows determination of the exact composition of amino acids. The Shimadzu-CAT Model DLAA-1 was constructed as an amino acid analyzer for both D/L and quantitative analysis.

Gas chromatographic determination of amino acids in river water by enantiomer labeling

In this study we examined a determination of total dissolved amino acids in river water by the enantiomer labeling method. The method involves the use of a given amino acid antipode as an internal standard, and is based on the fact that natural amino acids occur exclusively in the L-form

Capillary Gas Chromatographic Analysis of Amino Acids by Enantiomer Labeling

The optical isomers of amino acids can be easily separated by gas chromatography using capillary columns coated with the chiral polysiloxane peptide, Chirasil-Val. Quantitative trace amino acid analysis in complex mixtures such as biological fluids, sea water, or protein hydrolysates can be achieved by enantiomer labeling: The D-amino acid enantiomers, which do not occur naturally, are added to the sample prior to analysis as internal standards. Because the D-enantiomers show the same physical and chemical properties as the natural L-enantiomers, they are ideal standard references. In routine analysis, the derivatization is achieved with a new automated derivatization robot. The D-standard serves as overall internal standard for the whole analytical procedure from sample enrichment to derivatization, chromatography, and response of the detector.

Automated pre-column derivatization in gas chromatography amino acid analysis via enantiomer labelling: An application of autoderivat 100

An apparatus for the automatic derivatization of samples for chromatography is described. When coupled with an autosampler, gaschromatograph and calculating integrator, the system may be used as a fully automatic amino acid analyser, utilizing the advantages of enantiomer labelling.

Amino acid determination by capillary gas chromatography on chirasil-val : Enantiomer labelling nitrogen-selective detection

Amino acid analysis by enantiomer labelling and capillary gas chromatography on Chirasil-Val is superior to conventional gas chromatography and ion-exchange chromatography with respect to sensitivity, accuracy and speed. Employment of an alkali flame-ionization detector allows the selective detection of amino acids and suppression of background peaks; in addition, the delectability of amino acids is enhanced. Most nitrogen-selective detectors require meticulous adjustment of the operating conditions, but in combination with enantiomer labelling this is less critical. Maximum sensitivity and selectivity of the alkali-bead flame-ionization detector is achieved with a minimal flow of hydrogen. When using it as the carrier gas in capillary gas chromatography, flow control instead of the common pressure regulation is recommended to avoid a continuous fall of the baseline during temperature programming. Accurate flow control is achieved with a micro-aperture. The benefit of nitrogen-selective detection is especially apparent for histidine and arginine.

The determination of amino-acid enantiomers in human saliva with Chirasil-Val

The use of polysiloxanes as stationary phase in glass-capillary gas-chromatography carrying chiral groups enables the separation and quantitation of most amino-acid enantiomers and a variety of other compounds in a much shorter time than previously. This phase exhibits low volatility and high thermal stability and may be used in routine analysis with open tubular columns at temperatures of at least 175 °C. Most protein amino acids are separated in a temperature programme between 90 and 175 °C, thus obviating the need for multiple injections. Resolution factors are lower than those of other diamide phases containing the l-valine-t. butyl-amide group, but are sufficient for resolution of almost all protein amino-acid enantiomers. Incomplete recovery from the sample, incomplete derivatization, hydrolysis and thermal decomposition of the derivative and shifting response factors can be compensated for by adding the unnatural enantiomer. The accuracy of amino-acid analysis by enantiomer labelling is equal or superior to that of known methods and permits complete analysis of peptides with respect to both amino-acid composition and the optical purity of each amino acid. The application of this method to human saliva provided information concerning enzymic and bacterial process in the mouth.