Fluorescence Resonance Energy Transfer Substrate (FRETS) Library for Determining Protease Specificity

Each substrate (SFA-3701-v, SFV-3719-v) in the FRETS-25Xaa series contains a highly fluorescent 2-(N-methylamino)benzoyl (Nma) group linked to the side chain of the amino-terminal D-2,3-diamino proprionic acid (D-A2pr) residue, which is efficiently quenched by a 2,4- dinitrophenyl (Dnp) group linked to the ε-amino function of Lys. Xaa represents a fixed position of each of the 19 natural amino acids excluding Cys (noted in product name, code SFA-3701-v - SFV-3719-v). A mixture of 5 amino acid residues (P, Y, K, I, and D) is at the Yaa position along with a mixture of 5 amino acid residues (F, A, V, E, and R) at the Zaa position for each fixed Xaa. This provides a peptide mixture of 25 combinations of each Xaa series resulting in a combinatorial library totaling 475 peptide substrates. Both Nma and Dnp groups are linked to the side chain of the individual residues, allowing for the determination of the cleavage site by a specific enzyme, through mass spectrometric analysis and Edman degradation as well.