2 1h benzotriazol 1 yl 1 1 3 3 tetramethyluronium hexafluorophosphate hbtu

N-Fluorenylmethyloxycarbonyl (Fmoc) protected amino acids, amino acid attached 2-chlorotrityl resins, 1-[bis(dimethylamino) methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluoroph osphate (HATU), and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were purchased from Chem-Impex International, Inc. (Wood Dale, IL) and AAPPTEC LLC (Louisville, KY). Potassium hexacyanoferrate (II) trihydrate, potassium nitrate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysulfosuccinimide (Sulfo-NHS), 6-mercapto-1-hexanol, and 6-mercapto-1-hexanoic acid were obtained from Sigma-Aldrich (St. Louis, MO). Dithiothreitol (DTT), sodium phosphate dibasic heptahydrate (Na₂HPO₄•7H₂O), sodium phosphate monobasic (NaH₂PO₄) and 2-(4-morpholino)ethanesulfonic acid (MES) were purchased from Fisher Scientific (Hampton, NH). Purified recombinant human cathepsin B (~60% 37 kDa inactive form and ~40% 25 kDa active form) was acquired from R&D Systems Inc. (Minneapolis, MN). Cathepsin B solutions were activated by incubation in 25 mM MES buffer (pH = 5.0) containing 5 mM DTT for 15 min before proteolysis experiments.

4-Diethylaminosalicylaldehyde, diethylmalonate diisopropylethylamine (DIPEA) and trifluoroacid (TFA) were purchased from Oakwood Chemical. Benzyl alcohol was bought from Mallinckrodt. Succinic anhydride, 4-dimethylaminopyridine (DMAP), 1,3-diisopropylcarbodiimide (DIC), 1- hydroxybenzotriazole (HOBt), triethylsiliane (TES), 2-(1H-benzotriazol-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate (HBTU) and amino acids were purchased from Chem Impex Int’l Inc. Rink amide resin was purchased from ChemPep. Benzoic acid and all solvents were purchased from Fisher Scientific. The synthesis of starting materials and peptides, including 7-(diethylamino)-3-coumarin carboxylic acid (DAC) [49 (link)] and benzyl succinic acid [50 (link)] are outlined (Fig. S1) and described in the Supplementary Materials.