11 azido 3 6 9 trioxaundecan 1 amine

The pili A109PrOF incorporating an NSAA, PrOF, containing an alkyne, were used to produce the AuNP-pili. 5 nm NHS-activated gold nanoparticles (Cytodiagnostics) were reacted with 11-Azido-3,6,9-trioxaundecan-1-amine (Sigma Aldrich) using the recommended protocol from Cytodiagnostics. In the last step, AuNP-linker-azide were obtained by buffer exchanging with PBS pH 7.4 (−) MgCl₂ (−) CaCl₂ using 100 kDa Amicon Ultra centrifugal filter units (three times, centrifuging at 14,000 x g for 15 min). Next, AuNP-linker-azide were coupled onto the pili A109PrOF through copper-catalyzed azide-alkyne cycloaddition reaction. Briefly, 20 µL of 50 mM THPTA and 10 µL of 20 mM CuSO₄ were premixed for 30 min at room temperature. Later, 10 µL of 150 µM pili were diluted in 30μL of PBS pH 7.4 (−) MgCl₂ (−) CaCl₂ and 2.5 µL of AuNP-linker-azide were added to the mixture. Then, 1 µL of the mixture of THPTA/CuSO4 was added to the entire 40μL pili/AuNP mixture, are combined. Finally, 2.5 µL of 100 mM aminoguanidine and 2.5 µL of 100 mM sodium ascorbate are added. The reaction mixture was shaken at 500 rpm tabletop shaker (Santa Cruz Biotechnology) for 1 h. To stop the reaction and obtain the AuNP-linker-pili, the samples were buffer exchanged to 150 mM ETA pH 10.5 using 100 kDa Amicon Ultra centrifugal filter units (three times, centrifuging at 14,000 × g for 15 min).

N-Carboxyanhydride of ε-trifluoroacetyl-L-lysine (NCA-Lys(TFA)) was purchased from Chuo Kasei Co., Ltd. (Osaka, Japan). MeAIB was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 11-Azido-3,6,9-trioxaundecan-1-amine, Boc-L-glutamic acid 1-benzyl ester (Boc-Glu-OBzl), Boc-L-glutamic acid 5-benzyl ester (Boc-Glu(OBzl)-OH), RPMI 1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), penicillin/streptomycin, BCH, and trypsin/EDTA were purchased from Sigma Aldrich Corporation (St. Louis, MO). Dimethylsulfoxide (DMSO), triethylamine (TEA), N-methyl-2-pyrolidone (NMP), BzlSer, Gln, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), and D-PBS(-) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). DMSO was purified by distillation under reduced pressure before use. Dibenzocyclooctyne-Cy5 (Cy5-DBCO) was purchased from Click Chemistry Tools (Scottsdale, AZ). Fetal bovine serum (FBS), and Hoechst 33342 were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA). Bovine serum albumin (BSA) was purchased from Nacalai Tesque, Inc. (Kyoto, Japan).