N-Isobutyryl-l-cysteine

OPA in combination with chiral thiols is one method used to determine amino acid enantiomers. A highly fluorescent diastereomeric isoindole is formed and can be separated on a reverse-phase column. Some of these chiral thiols include N-acetyl-L-cysteine (NAC), N-tert-butyloxy-carbonyl- L-cysteine (Boc-L-Cys), N-isobutyryl- L-cysteine (IBLC), and N-isobutyryl- D -cysteine (IBDC). Replacing OPA-IBLC with OPA-IBDC causes a reversal in the elution order of the derivatives of D- and L-amino acids on an ODS column (Hamase et al., 2002). Nimura and colleagues (2003) developed a novel, optically active thiol compound, N-(tert-butylthiocarbamoyl)- L-cysteine ethyl ester (BTCC). This reagent was applied to the measurement of D-Asp with a detection limit of approximately 1 pmol, even in the presence of large quantities of L-ASP. 

Fig. 18 VA top) and VCD bottom) spectra of N-isobutyryl-cysteine protected AuNPs in NaOH/D20. Solutions were made from 7 mg of sample in 50 pL of solution. The dashed (solid) lines correspond to the spectra of the particles covered by the L-enantiomer (D-enantiomer). The VA spectrum of the particles covered with N-isobutyryl-L-cysteine was offset for clarity. Adapted with permission from [155], Copyright (2006) American Chemical Society...

The derivatives are used for amino acid analysis via HPLC separation. Instead of mercapto-ethanol, a chiral thiol, e.g., N-isobutyryl-L-cysteine, is used for the detection of D-amino acids. The detection hmit lies at 1 pmol. The very fast racemizing aspartic acid is an especially suitable marker. One disadvantage of the method is that proline and hydroxyproline are not detected. This method is apphed, e.g., in the analysis of fruit juices, in which high concentrations of D-amino acids indicate bacterial contamination or the use of highly concentrated juices. Conversely, too low concentrations of D-amino acids in fermented foods (cheese, soy and fish sauces, wine vinegar) indicate unfermented imitations. Fluorescamine reacts with primary amines and amino acids - at room temperature under alkaline conditions - to form fluorescent pyrrolidones (Aex = 390 nm, Aem - 474 nm). The detection limit lies at 50-100 pmol ... 

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