Biflex 3

Benzimidazole derivatives (BnzAc-OH, NBnzAc-OH, PBnzAc-OH and ClBnzAc-OH) were obtained by reacting t-butyl bromoacetate (1 eq) with an appropriate substrate (1.5 eq): benzimidazole, 2-nonylbenzimidazole, 2-(2-pyridyl) benzimidazole and 2-(2-chlorophenyl) benzimidazole in the presence of K₂CO₃ (1.2 eq) and catalytic amounts of tetrabutylammonium iodide (TBAI). Reactions were carried out in acetonitrile (ACN) for 20 h at 60 °C. After the syntheses had been completed, t-butyl groups were removed using 50% trifluoroacetic acid (TFA) in dichloromethane (DCM) for 2 h at room temperature. Crude products were purified by column chromatography on silica gel using methanol:ethyl acetate as the eluent. Fractions of the highest purity (>98%) were analyzed by an analytical reversed-phase high-performance liquid chromatography (RP-HPLC) system (Prominence, Shimadzu, Duisburg, Germany) using a Kinetex XB-C18 column (Phenomenex, 4.6 × 150 mm, 5 µm particle size) with several gradients of ACN with 0.08% TFA at a flow rate of 1 mL/min. Identities of compounds were confirmed by MALDI-TOF mass spectrometry (Bruker BIFLEX III, Bruker Daltonics, Billerica, MA, USA) using the α-CCA (α-cyano-4-hydroxycinnamic acid) matrix. Thus, the obtained benzimidazole derivatives (Figure 1) were attached on-resin to the ε-amino group of Lys⁷ residue within the TP10 structure.

Solvents were dried using traditional methods, and freshly distilled prior to use. Reactions were monitored by thin-layer chromatography (TLC) on silica gel GF254-precoated plates and RP-HPLC (Agilent Technologies, Santa Clara, CA, USA). Compounds were detected under UV light and/or visualized by phosphomolybdic acid in ethanol solution. Solvents were evaporated under reduced pressure and below 50 °C. Mass spectra were obtained on Bruker APEX (Bruker Daltonics, Billerica, MA, USA). High-resolution MS (HRMS) were performed with Bruker BIFLEX III (Bruker Daltonics, Billerica, MA, USA) and Bruker APEX II (Bruker Daltonics, Billerica, MA, USA). ¹H-NMR and ¹³C-NMR spectra were recorded on a BrukerAV400 (Bruker Biospin, Ettlingen, Germany) spectrometer or Bruker Avance III-HD 600 NMR Spectrometer (Bruker Biospin, Ettlingen, Germany) using TMS as internal standard. Chemical shifts are reported in parts per million and coupling constants quoted in Hz. Alkyne (a, b, c, d, e, f) CuSO₄·5H₂O, Sodium ascorbate and copper sheet from Alfa Aesar (Tianjin, China). Peptide 8 was purchased from GL Biochem (Shanghai, China) Ltd. Starting materials and copies of NMR spectrum of compound 6, 7a to 7i and 9 were put in the Supplementary Materials of this paper.