Boron trifluoride-etherate (BF3), one of the Lewis acids, was found to be an efficient acid in the Edman sequencing method, affording the retention of the N-terminal amino acid configuration at the cyclization/cleavage reaction from peptides. Examples for the sequencing of D-Leu-Gly and L-Pro-L-Leu are described, utilizing 7-N,N-dimethylaminosulphonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS) as a fluorogenic Edman reagent. The peptide labelled with DBD-NCS was cyclized/cleaved with 1% BF3 in dichloroethane containing 0.02% ethanethiol at 50 degrees C for 5 min. The resultant DBD-thiazolinone (TZ)-amino acid was separated on a phenylcarbamoylated cyclodextrin column with fluorometric detection at 524 nm with excitation at 387 nm. DBD-TZ-amino acids thus obtained showed remarkable retention of their configuration of alpha-carbon atoms of amino acids (DBD-TZ-L-Leu; 3%, -D-Pro; 6%). Even after 30 min of the cyclization/cleavage reaction at 50 degrees C, DBD-TZ-amino acids retained their configuration to the same degree. On the other hand, DBD-TZ-amino acid obtained by cyclization/cleavage reaction with TFA at 50 degrees C for 5 min was racemized to a great extent (DBD-TZ-L-Leu; 31%, -D-Pro; 48%). BF3 should be the most recommendable acid at the cyclization/cleavage reaction for amino acid sequence and configuration determination of peptides containing D-amino acid residues.

Amino Acid Sequence↗

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4-(N-hydrazinoformylmethyl-N-methyl)-amino-7-N, N-dimethylaminosulphonyl-2,1,3-benzoxadiazole (DBD-CO-Hz) as a precolumn fluorescence derivatization reagent for carboxylic acids in high-performance liquid chromatography.

A new fluorescent reagent for carboxylic acids, 4-(N-hydrazinoformylmethyl-N-methyl)amino-7-N,N-dimethylaminosu lphonyl-2, 1,3-benzoxadiazole (DBD-CO-Hz) was synthesized and its applicability as a precolumn derivatization reagent in high-performance liquid chromatography (HPLC) was examined. DBD-CO-Hz reacted with saturated fatty acids in the presence of a condensing agent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and pyridine at room temperature for 30 min to give fluorescent products. The reaction solution was subjected to a reversed-phase HPLC and detected fluorometrically at a wavelength of 550 nm with an excitation of 440 nm. The detection limits of the derivatives were at low fmol range on column.

Carboxylic Acids↗

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Sensitive detection of near-infrared fluorescent dyes using high-performance liquid chromatography with peroxyoxalate chemiluminescence detection system.

The highly sensitive detection of four near-infrared (near-IR) fluorescent dyes, methylene blue, pyridine 1, oxazine 1 and 3,3'-diethylthiadicarbocyanine iodide (DTDCI) by high-performance liquid chromatography (HPLC) with post-column peroxyoxalate chemiluminescence (PO-CL) detection utilizing bis(4-nitro-2-(3, 6,9-trioxadecyloxycarbonyl)phenyl) oxalate (TDPO) and hydrogen peroxide was examined. The detection limits for methylene blue, pyridine 1, oxazine 1 and DTDCI were 120, 27, 31, 0.19 fmol on-column at a signal-to-noise ratio of 2, respectively. The sensitivity for DTDCI was 250 times that obtained by HPLC with the conventional fluorescent detection.

Chromatography, High Pressure Liquid↗

PubMedDate not supplied

Analytical chemistry and biochemistry of D-amino acids.

The methodologies for the analysis of D-amino acids in biological materials have been reviewed, including the use of enzymes, gas and liquid chromatography with chiral stationary phases and diastereomer derivatization with chiral reagents followed by GC or HPLC separation. The distribution of D-amino acids in the body, their origin, metabolism and possible roles in human diseases are discussed.

Amino Acids↗

PubMedDate not supplied

Optimization of the reaction conditions for the peroxyoxalate chemiluminescence detection system of fluorescent compounds in HPLC.

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Chromatography, High Pressure Liquid↗