Application of cyanuric chloride-based six new chiral derivatizing reagents having amino acids and amino acid amides as chiral auxiliaries for enantioresolution of proteinogenic amino acids by reversed-phase high-performance liquid chromatography

Six dichloro-s-triazine (DCT) reagents having l-Leu, d-Phg, l-Val, l-Met, l-Ala and l-Met-NH₂ as chiral auxiliaries in cyanuric chloride were introduced for enantioseparation of 13 proteinogenic amino acids. Four other DCTs and six monochloro-s-triazine (MCT) reagents having amino acid amides as chiral auxiliaries were also synthesized. These 16 chiral derivatizing reagents (CDRs) were used for synthesis of diastereomers of all the 13 analytes using microwave irradiation, which were resolved by reversed-phase high-performance liquid chromatography (RP-HPLC) using C18 column and gradient eluting mixture of aqueous TFA and acetonitrile with UV detection at 230 nm. It required only 60–90 s for derivatization using microwave irradiation. Better resolution and lower retention times were observed for the diastereomers prepared with CDRs having amino acids as chiral auxiliaries as compared to counterparts prepared with reagents having amino acid amides as chiral auxiliaries. As the best resolution of all the 13 analytes was observed for their diastereomers prepared using the DCT reagent having l-Leu as chiral auxiliary, this CDR was further employed for derivatization of Lys, Tyr, His and Arg followed by RP-HPLC analysis of resulting diastereomers. The results are discussed in light of acid and amide groups of chiral auxiliaries constituting CDRs, electronegativities of the atoms of achiral moieties constituting CDRs and hydrophobicities of side chains of amino acids constituting CDRs and analytes.     

Application of (S)-N-(4-Nitrophenoxycarbonyl) phenylalanine methoxyethyl ester as a chiral derivatizing reagent for reversed-phase high-performance liquid chromatographic separation of diastereomers of amino alcohols, non-protein amino acids, and PenA

Indirect enantioresolution of 15 primary and secondary amino group containing compounds (amino alcohols, non-protein amino acids, PenA) was done using the reagent (S)-N-(4-Nitrophenoxycarbonyl) phenylalanine methoxyethyl ester [(S)-NIFE] by reversed-phase high-performance liquid chromatography. The diastereomeric derivatives were analyzed under reversed-phase conditions using linear gradient. The detection was at 205 nm and sharp peaks were obtained. The reagent used is comparatively economic than the other derivatizing reagents. Method validation was also done.     

Synthesis and application of chiral triazine condensing reagents prepared from esters of amino acids

Treatment of cyanuric chloride with chiral amines or esters of chiral amino acids gave chiral 2,4-dichloro-6-alkylamino-1,3,5-triazines (2-5) in 49-69% yield, which were found useful as coupling reagents. Enantioselective activation and enantioselective aminolysis in the presence of 2-5 was observed.     

high-performance liquid chromatography of amino acids, peptides and proteins : LXIII. Reversed-phase high-performance liquid chromatographic characterisation of several polypeptide and protein hormones

The chromatographic behaviour on alkylsilicas of a variety of hormonal proteins is described. Optimization of resolution and recovery of these protein hormones, which included porcine relaxins, human chorionic gonadotropin, human placental lactogen, pituitary derived growth hormone and adenohypophyseal glycoprotein hormones, was achieved by manipulation of both mobile and stationary phase parameters. With standard stainless-steel analytical columns (10–30 cm × 0.4 cm) packed with meso- or macro-porous n-alkylsilica supports these proteins can be readily fractionated at the semi-preparative level with separation times generally under 90 min using elution systems directly compatible with subsequent methods of primary structure determination or biological functional analysis. The effects of changes in several experimental parameters on peak symmetry, retention and recovery are described.     

High-performance liquid chromatographic enantioseparation of unusual secondary amino acids on a D-penicillamine-based chiral ligand exchange column

The application of a chiral ligand-exchange column (CLEC) for the direct high-performance liquid chromatographic enantioseparation of unusual secondary amino acids using D-penicillamine-Cu(II) complex as chiral selector is reported. The amino acids investigated were pyrrolidine-2-carboxylic acid, piperidine-2-carboxylic acid, piperazine-2-carboxylic acid, morpholine-3-carboxylic acid, and thiomorpholine-3-carboxylic acid analogs. Chromatographic results are given as the retention, separation, and resolution factors. The chromatographic conditions were varied to achieve optimal separation. The elution sequence of the enantiomers was determined and in most cases the S isomer eluted before R.     

Reversed-phase high-performance liquid chromatographic analysis of hydroxyproline and proline from collagen by derivatization with dabsyl chloride

A high-performance liquid chromatographic method for the analysis of hydroxyproline and proline has been developed. The method is based on the derivatization of the secondary amino group with dabsyl-chloride after blocking of the primary amino group with o-phthalaldehyde. Dabsyl-hydroxyproline and dabsyl-proline were separated from other amino acids by high-performance liquid chromatography in the gradient elution mode, and eluted at 10.27 and 16.02 min, respectively. The correlations between the peak areas of dabsyl-hydroxyproline and dabsyl-proline were linear in the range from 20–200 pmol, with equations y = 1.10x − 0.80 (r = 0.999) and y = 1.12x − 0.52 (r = 0.999), respectively. The method was applied to the analysis of rat tail collagen, and the contents of hydroxyproline and proline were 1.55 ± 0.04 and 2.03 ± 0.04 nmol/μg, respectively.     

Resolution of chiral drugs by liquid chromatography based upon diastereomer formation with chiral derivatization reagents

Chiral derivatization reagents for resolution of biologically important compounds, such as chiral drugs by high-performance liquid chromatography (HPLC), based upon pre-column derivatization and diastereomer formation, are reviewed. The derivatization reagents for various functional groups, i.e., amine, carboxyl, carbonyl, hydroxyl and thiol, are evaluated in terms of reactivity, stability, wavelength, handling, versatility, sensitivity, and selectivity. The applicability of the reagents to the analyses of drugs and bioactive compounds are included in the text.     

A convenient,rapid method for the resolution of enantiomeric amino acids using chiral phases on stainless-steel capillary gas chromatographic columns

An improved method is described for the resolution of enantiomeric isopropyl esters of N-trifluoroacetyl-α-amino acids of nonbasic amino acids using N-docosanoyl-l-valyl-t-butylamide and N-octadecanoyl-l-valyl-l-valine cyclohexyl ester as mixed chiral phases on 150-ft stainless-steel capillary columns. Enantiomers of Ala, Val, Ile, Leu, Ser, Thr, Asp, Met, Glu, and Phe are resolved in 105 min. This method avoids the fractionation problems and high costs encountered with the diastereometric method and difficulties and costs encountered in loading and maintaining glass capillary columns. It is particularly useful for studies involving a large number of resolutions as in a study of the kinetics of racemization of amino acids.     

Reverse-phase liquid chromatographic analysis of amino acids after reaction with o-phthalaldehyde

Precolumn formation of o-phthalaldehyde (OPA) derivatives of amino acids, followed by reverse-phase separation and fluorescent detection, provides rapid, sensitive amino acid analysis. Eighteen OPA-amino acid derivatives are resolved on a Micropack MCH 5 column and can be measured at picomole levels. Ease of derivative preparation and separation makes liquid chromatographic analysis of OPA-amino acids a convenient and improved technique for measuring or confirming the presence of low levels of amino acids in aqueous solutions. Use of the method was demonstrated by measuring low concentrations of amino acids released from zooplankters stressed by contaminants.     

Simple and rapid quantitative high-performance liquid chromatographic analysis of plasma amino acids

A simple and rapid high performance liquid chromatographic method for the determination of plasma amino acids was developed. The method uses minimal sample volume and automated online precolumn derivitization of amino acids with o-phthalaldehyde and fluorescent detection. Amino acids are separated by a simplified gradient without column heating. The assay is linear from 5 to 1000 micromol/L for all amino acids. Recovery of amino acids was between 91 and 108%, intra-assay coefficient of variation (CV) was 1-7%, and inter-assay CV was 2-12%. The simple sample preparation and minimal sample volume make the method useful for the quantitation of amino acids in both patient and experimental animal samples.     

Liquid chromatographic resolution of N-acyl-alpha-amino acids as their anilide derivatives on a chiral stationary phase based on (S)-leucine

A chiral stationary phase (CSP 1) derived from N-(3,5-dinitrobenzoyl)leucine N-phenyl N-alkylamide was used for the liquid chromatographic resolution of anilide derivatives of N-acyl-alpha-amino acids and the chromatographic resolution results were compared with those from four other commercial CSPs. The chromatographic resolution results showed that CSP 1 was most effective among five CSPs used in this study. The chiral recognition mechanism exerted by CSP 1 for the resolution of anilide derivatives of N-acyl-alpha-amino acids is proposed to involve a face-to-face pi-pi interaction and two hydrogen bonding interactions between the CSP and the analytes from the chromatographic resolution behaviors of slightly modified anilide derivatives of N-acyl-alpha-amino acids. The chiral recognition mechanism proposed is quite similar to that advanced previously for the resolution of N-(3,5-methoxybenzoyl)-alpha-amino acids on CSP 1, even though the interaction sites of the two types of analytes were totally different from each other. The apparent similarity of the two chiral recognition mechanisms was assumed to stem from the identical interaction modes of the two types of analytes with the CSP. In addition, the dependence of the enantioselectivity of anilide derivatives of N-acyl-alpha-amino acids on the length of the alkyl tail of the N-acyl group of analytes was rationalized to stem from the intercalation of the N-acyl group of the (R)-enantiomer of analytes between the tethers of the CSP.     

The reactions of phenylglyoxal and related reagents with amino acids.

1. The reaction of phenylglyoxal (PGO), glyoxal (GO), and methylglyoxal (MGO) with amino acids were investigated at mild pH values at 25 degrees. These aldehydes reacted most rapidly with arginine and the rate of reaction increased with increasing pH values. Histidine, cystine, glycine, tryptophan, asparagine, glutamine, and lysine reacted with these aldehydes at significant but various rates, depending on the pH and the kind of the reagent used. The reactions with these amino acids seemed to involve both the alpha-amino groups and the side chain groups, and no significant reaction appeared to occur with the side chain alone except with those of arginine, lysine, and cysteine. These reagents were similarly reactive with the guanidinium group of arginine, but PGO appeared to be much less reactive with the epsilone-amino group of lysine than MGO and GO. The other ordinary amino acids were very much less reactive or did not react at all with these reagents, with the exception of cysteine. 2. Di-PGO-L-arginine was prepared from Nalpha-benzyloxycarbonyl-L-arginine, and di-PGO-methylguanidine from methylguanidine, and the stoichiometry of the reaction of two PGO molecules with one guanidino group was confirmed. A glyoxal derivative of L-arginine (GO-arginine) was prepared by reaction of glyoxal with arginine. GO-arginine was fairly unstable, especially at higher pH values. A similar derivative (MGO-arginine) was also found to be formed by reaction of MGO with L-arginine, and was similarly unstable. These derivatives, however, did not regenerate arginine upon acid hydrolysis.     

High-performance liquid chromatographic analysis of amino acids in physiological fluids: on-line precolumn derivatization with o-phthaldialdehyde

A cation-sensing electrode can be used to monitor enzymatic reactions if their substrates and products differ in the ability to bind the cation. In the first method, the electrode potential vs time curve is recorded. The second, metal-stat method consists of measuring the rate at which substrate must be added to the reaction medium in order to keep the electrode potential at a constant level. These approaches have been used to assay inorganic pyrophosphatase, carboxypeptidase A, and hexokinase with the Mg2+ and Cu2+ ions as the indicators.     

High-performance liquid chromatographic enantioseparation of Betti base analogs on a newly developed isopropyl carbamate-cyclofructan6-based chiral stationary phase

The direct separation of the enantiomers of 1-(α-aminoarylmethyl)-2-naphthol, 1-(α-aminoalkyl)-2-naphthol, 2-(α-aminoarylmethyl)-1-naphthol analogs, and 2-(1-amino-2-methylpropyl)-1-naphthol) was performed on a newly developed chiral stationary phase containing isopropyl carbamate-cyclofructan6 as chiral selector, with n-heptane/alcohol/trifluoroacetic acid as mobile phase. The effects of the mobile-phase composition, the nature and concentration of the alcoholic and acidic modifiers, and the structures of the analytes on the retention and resolution were investigated. In some cases, separations were carried out at constant mobile-phase compositions in the temperature range 5-40°C. Thermodynamic parameters and T(iso) values were calculated from plots of ln k' or ln α versus 1/T. -Δ(ΔH°) ranged from 2.8 to 3.2 kJ mol(-1) , -Δ(ΔS°) from 7.7 to 10.1 J mol(-1) K(-1) , and -Δ(ΔG°) from 0.2 to 0.5 kJ mol(-1) . It was found that the enantioseparations were enthalpy driven. The sequence of elution of the stereoisomers determined in some cases was (R) < (S).     

A novel chiral thiol reagent for automated precolumn derivatization and high-performance liquid chromatographic enantioseparation of amino acids and its application to the aspartate racemase assay

A novel optically active thiol compound, N-(tert-butylthiocarbamoyl)-L-cysteine ethyl ester (BTCC), is synthesized as a chiral derivatization reagent. This compound and o-phthalaldehyde react with amino acid enantiomers to produce fluorescent diastereomers that are readily separable on a reverse-phase column by HPLC. Enantioseparation of acidic amino acids in particular is markedly improved using BTCC. In this study, the HPLC method for enantioseparation with the novel compound is applied to the aspartate (Asp) racemase assay. Derivatized D-Asp is eluted before the L-Asp derivative. Consequently, a small amount of D-Asp produced by the activity of racemase on a large quantity of L-Asp substrate may be quantified accurately, even at very low activity. Since the derivatization reaction proceeds rapidly at room temperature, a fully automated system is established for derivatization and sample injection. The automated method is practical and successfully applied to the archaeal Asp racemase assay. We presume that the procedure is additionally applicable to the enantioseparation of other amino acids, amino alcohols, and catecholamines.     

Liquid chromatographic enantiomer resolution of N-hydrazide derivatives of 2-aryloxypropionic acids on a crown ether derived chiral stationary phase

The liquid chromatographic separation of the enantiomers of several N-hydrazide derivatives of 2-aryloxypropionic acids was performed on a crown ether type chiral stationary phase derived from (18-crown-6)-2,3,11,12-tetracarboxylic acid. The behavior of chromatographic parameters by the change of mobile phases and additives for the resolution of these analytes was investigated. The enantiomers of all analytes were base-line resolved with a mobile phase of 100% methanol containing 20 mM H2SO4. These results are the first reported for enantiomer resolution of chiral acids of 2-aryloxypropionic acids as their N-hydrazide derivatives.     

High-performance liquid chromatographic analysis of physiological amino acids in human brain tumors by pre-column derivatization with phenylisothiocyanate

A reversed-phase high-performance liquid chromatographic technique for the determination of free amino acids in five biopsies of human brain tumors (two meningiomas, one glioblastoma and two oligodendrogliomas) is described. The frozen tissues were homogenized, deproteinized with perchloric acid and neutralized with potassium hydroxide. Aliquots of the supernatant containing the physiological amino acids are used for pre-column derivatization with phenylisothiocyanate. The derivatized PTC-amino acids (phenylthiocarbamyl derivatives) are stable for a five day period if stored as a powder at −20°C in an inert atmosphere and they can be analyzed on a reversed-phase column (PicoTag) using a gradient of two eluents with absorption detection at a wavelength of 254 nm. Good resolution of several amino acids (>30) is achieved within ca. 60 min. For most amino acids this method is suitable for an accurate measurement over a wide range of physiological concentrations (50–400 pmol) starting from a very small amount of sample.     

Enantiomeric separation of five amino acids by high-performance liquid chromatography on a chiral crown ether column

A chiral liquid chromatographic method was validated to analyze the D-and L-enantiomers of five amino acids contained in a commercial solution: aspartic acid, leucine, lysine, phenylalanine, and valine. These 10 compounds were separated on a chiral crown ether column with a mobile phase composed of water adjusted to pH 1.5 with perchloric acid, with ultraviolet detection at 220 nm. The method was applied to the commercial amino acid solution before and after sterilization by 5 kGy irradiation; no stereoconversion was observed following sterilization. Chirality 9:150–152, 1997. © 1997 Wiley-Liss, Inc.