The initial assessment of newly designed fluorescent nonapeptide substrates. (A) The structures of reference nonapeptide substrate ne-T9 and five new designed nonapetide substrates, ne-H9, on-H9, ne-P9, ne-K9, and ne-D9. (Qu: Quencher, Fl: Fluorophore) (B) Fluorescence enhancement velocity due to the fluorescence increment of variant fluorescent nonapeptide substrates reacting with SIRT1, full-length SIRT2, SIRT3, and SIRT6 for 30 min. Enzymatic reactions were performed with 40 μmol/L of fluorescent nonapeptide substrate (ne-T9, ne-H9, on-H9, ne-P9, ne-K9, or ne-D9), 1 mmol/L of NAD+, and 0.5 μmol/L enzyme (SIRT1, full-length SIRT2, SIRT3, or SIRT6) in assay buffer at 37 °C. Data are presented as mean±SD, n=3.