Schiff base-forming and related reagents

Pyridoxal and pyridoxal 5-phosphate are the only naturally occurring compounds which have been used as fluorescent labels. These compounds form Schiff bases with primary amino groups. The reduction of the C=N bond with sodium borohydride (NaBH4) leads to the formation of fluorescent pyridoxyl derivatives [260,261], 

The following procedure has been suggested. Amino acids and primary amines are dissolved in 0.5 M phosphate buffer, pH 9.3. This solution is mixed with the same volume of a freshly prepared 0.4 M pyridoxal solution in the same buffer (pyridoxal/amino acids = 5 1). 

After heating for 30 min at 80 °C, 100 mg of NaBH4 dissolved in 1 ml of 0.1 M NaOH is added. The excess of NaBH4 is decomposed by acidification to pH 1-2 with hydrochloric acid. 

The yield of a-amino acid derivatives is 90%. Lysine and ornithine form dipyridoxyl derivatives. Recovery of the proline and hydroxyproline derivatives is about 25%. 

For derivative formation with amines, pyridoxal 5-phosphate is preferred to pyridoxal in order to allow ion echange chromatographic separation. The reaction mixtures are submitted immediately to chromatographic separation. 

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