Hydroxybenzotriazole

All the chemicals and reagents, including cetyltrimethylammonium bromide (CTAB, ≥98%, Sigma-Aldrich, St. Louis, MO, USA), sodium hydroxide (≥98%, Fluka, Hampton, NH, USA), tetraethyl orthosilicate (TEOS, ≥99%, Sigma-Aldrich), 3-aminopropyl)triethoxysilane (APTES, 99%, Sigma-Aldrich), 1-pyrenecarboxylic acid (97%, Sigma-Aldrich), hydroxy benzotriazole (≥97%, Sigma-Aldrich), 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide hydrochloride (≥99%, Sigma-Aldrich), triethylamine (>99%, Sigma-Aldrich), ethyl acetate (≥99.7%, Honeywell, Charlotte, NC, USA), were purchased and utilized without further purification.

All Fmoc-protected amino acids,
Wang resin, and hexafluorophosphate azabenzotriazole tetramethyl uronium
(HATU) were purchased from Matrix Innovation (Quebec, Canada). N,N′-dimethylformamide
(DMF), dichloromethane (DCM), N,N′-diisopropylethylamine (DIPEA),
piperidine, methanol, trifluoroacetic acid (TFA), diethyl ether, and
ethanol were purchased from Bio-Lab (Jerusalem, Israel). Triisopropylsilane
(TIPS), thioanisole, 1,2-ethanedithiol (EDT), acetic anhydride, hydroxybenzotriazole
(HOBT), N,N′-diisopropylcarbodiimide (DIC), 5(6)-carboxyfluorescein
[5(6)-FAM], and phenol were purchased from Sigma-Aldrich (St. Louis,
Missouri, USA). Paraffin oil (puriss meets the analytical specification
of Ph. Eur., BP, a viscous liquid), dimethylaminopropyl-N′-ethylcarbodiimide
hydrochloride (EDC), and MES hydrate were purchased from Sigma-Aldrich.
HPLC-grade water was purchased from Alfa Aesar and was used as received
without further purification. The Sonics Vibra-cell ultrasonic liquid
processor, Model-VCX 750 (Newtown, CT, USA) was used for ultrasonication.

All required organic solvents were purchased from Wilkem Scientific (Pawtucket, RI, USA). The Fmoc-protected building block of amino acids, including Fmoc-Trp(Boc)-OH, Fmoc-Arg(Pbf)-OH, Dde-K(Boc)-OH, Boc-βAla-OH, 3-(tritylthio)propionic acid, and H-Trp(Boc)-2-chlorotrityl resin, were purchased from Chem-Impex International Inc. (Wood Dale, IL, USA). Hydroxy-7-azabenzotriazole (HOAT), 1,3-diisopropylcarbodiimide (DIC), O-(benzotriazole-1-yl)-N,N,N’,N’-tetramethyluroniumhexafluorophosphate (HBTU), benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), and hydroxybenzotriazole (HOBt), glutaric anhydride (95%), triphosgene (99%), 4-dimethylaminopyridine (DMAP, 99%), N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), β-mercaptoethylamine hydrochloride (cystamine hydrochloride), and 2,2′-dithiodipyridine were purchased from Sigma-Aldrich Chemical Co. (Milwaukee, WI, USA). Camptothecin (CPT) was purchased from Santa Cruz Biotech, Inc. (Dallas, TX, USA) and Paclitaxel (PTX) was purchased from Euroasias Group Inc. (Woodbridge, NJ, USA).

All the EPs used in the current study, 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), hydroxybenzotriazole, (HOBT), isopropyl β-d-1-thiogalactopyranoside (IPTG), nutrient broth, glycerol, and CelLytic B Cell Lysis Reagent were purchased from Sigma-Aldrich (St. Louis, MO, USA). LCMS-grade solvents including water and acetonitrile, and hydrogen peroxide (30% w/v) were purchased from Millipore (Burlington, MA, USA). LCMS-grade formic acid was purchased from Fisher Chemical (Hampton, NH, USA), while kanamycin was purchased from Abcam (Cambridge, UK).

Graphite, potassium permanganate, sulphuric acid (36 M), hydrochloric acid (5%), sodium hydroxide, hydrogen peroxide (30%), hydroxy benzotriazole, dicyclohexyl carbodiimide, nicotinic acid hydrazide, potassium chloride, potassium ferrocyanide, citric acid, sodium citrate, caffeine powder, potassium dihydrogen phosphate, dipotassium hydrogen phosphate were purchased from Sigma Aldrich. All the reactions were carried out under a nitrogen atmosphere with the use of the standard Schlenk technique. All the solvents and reagents were of analytical grade and used without further purification. The O’cean one8 energy drink and imol plus were purchased from a local market and pharmaceuticals, respectively. The stock solutions of caffeine (100 × 10^–6 M) were prepared fresh each time of experiments. Aqueous solutions of different pH of buffers were prepared, diluted using millipore water.

Leucine aminopeptidase (EC 3.4.1.2) microsomal from porcine kidney (LAP), β-glucuronidase (EC 3.2.1.31) from helix pomatia type H-2 (GLU), N-(tert-Butoxycarbonyl)glycine, glycine, triglycine, σ-pinene, triphosgene, triethylamine, N-carboxyanhydride (NCA), 2-aminoethyl methacrylate hydrochloride (2-AMEA), allylamine, 4-nitrophenyl- β-D-glucuronide (PNPG), hydroxybenzotriazole (HOBt), N,N’-diisopropylcarboiimide (DIC), acrylic acid N-hydroxysuccinimide (Ac–NHS ester), deuterium oxide (D₂O), dimethylsulfoxide-d₆ (DMSO-d), chloroform-d (CDCl₃), 2,2-dihydroxyindane-1,3-dione (Ninhydrin) and tris(hydroxymethyl)aminomethane hydrochloride (Tris–HCl) was purchased from Sigma Aldrich (St. Louis, MO, USA). GGRSK was custom-synthesized from Lifetein (Somerset, NJ, USA) and Cosmogenetech (Seoul, South Korea). Urokinase (EC 3.4.4.21)-type plasminogen activator (uPA) was purchased from Fortunachem (Wuhan, China). Acrylic acid (AAc), sodium hydroxide (NaOH), ethyl acetate anhydrous (EA), dimethylformamide (DMF), tetrahydrofuran (THF), chloroform (CCl₄), methanol (MeOH), n-butanol (BuOH), acetic acid, acetonitrile, and trifluoroacetic acid were purchased from Daejung (South Korea). 2-hydroxy-2-methlypropiophenone (Darocur 1173) and dimethylsulfoxide anhydrous (DMSO) were purchased from TCI (Tokyo, Japan).