As described in Chapter 6, the most versatile fluorescence-quenched pair with respect to both ease of synthesis and efficiency of energy transfer is the Abz/Tyr (3-NO₂) pair (2-aminobenzoyl/3-nitrotyrosme). The synthesis of the surtably protected building blocks that can be used in a flexrble way in multrple-column peptide synthesis (MCPS) mvolves the preparation of Fmoc-Tyr(NO₂-OH (fluorene-9-ylmethyloxycarbonyl-Tyr(NO₂)-OH) and Fmoc-Lys(Boc-Abz)-OPfp (Fmoc-Lys(tert-butyloxycarbonyl-Abz)-O-pentafluorophenyl ester) as described (in Subheadings 2.2.1.–2.2.4.). The Fmoc-Tyr(NO₂-OH can be activated in situ since protection of the acidtc phenol is drffrcult and any acylation of the nitrotyrosme side-chain during syntheses 1s completely reverted in the subsequent treatment with prperrdme. The MCPS is particularly useful for portion mixing since all that IS needed is to add a mixing chamber above the open columns of the synthesizer. A detailed procedure for the synthesis of a fluorescence-quenched substrate library is described below and demonstrated for the serme protease subtiltsm Carlsberg substrate with the structure H-Y(NO₂)X¹X²PX³X⁴X⁵K(Abz)-(PEGA) where X can be any of the 20 naturally encoded amino acids. Prolme has been inserted to direct the cleavage since prolme is only accepted in S₂; however, it is not a requrrement to dnect the cleavage and subsequent substrate libraries have been randomized in all positions