Addition ofo-aminobenzoic acid during Fmoc solid phase synthesis of a fluorogenic substrate containing 3-nitrotyrosine

Following the Fmoc-tBu synthesis of an intramolecularly quenched substrate with the sequence NH2-Abz-Gly-Ala-Ala-Pro-Phe-Tyr(3-NO2)-Asp-OH, in addition to the target peptide a side product accounted for more than 50% of the crude product as measured by the integrated peak area by RP-HPLC analysis.1H NMR and ESI-MS analysis confirmed the presence of two Abz residues in this side product. The enzymatic and chemical methods revealed the position of this modification to be the hydroxyl function of Tyr(3-NO2). The side reaction was entirely prevented upon treatment of the resin bound peptide with piperidine prior to final TFA cleavage.