Noninvasive continuous monitoring of solid-phase peptide synthesis by acid-base indicator.

Merrifield's solid-phase peptide synthesis'r' requires almost quantitative acylation of amino groups upon the peptidyl resin. Several methods of monitoring the course and completion of this reaction have been described (for a review see e.g,"), the ninhydrin test being the one most commonly used'. Recently, we have employed an indicative amount of 3,4-dihydro-3-hydroxy-4-oxo-I,2,3-benzotriazine to monitor the acylation of amino groups in our configuration of continuous-flow solid-phase peptide synthesis'v. This triazine has been recommended by Atherton et a\.6 for the preparation of self-indicating activated esters. However, the colour change from yellow to white is not sufficiently conspicuous if one works on standard polystyrene resin that tends to be yellowish itself. Therefore, we attempted to find an alternative indicator that would be deprotonated by free amino groups on peptidyl resin and whose ionized form would be deeply coloured. Among the several indicators tested, bromophenol blue (3',3",5',5"-tetrabromophenolsulfophtaleiu) displayed the best properties: the colour change from yellow (}'ll1a, 429 nm) to dark blue (}'ll1a, 600 nm) is remarkable, the sensitivity is high (molar absorption coefficient "600 91 800). The resin lacking free amino groups exposed to the solution of bromophenol blue becomes