N n diisopropylcarbodiimide dic

All amino acid derivatives and Wang resin were purchased from Iris Biotech GmbH (Marktredwitz, Germany), whereas reagents for coupling and cleavage (N,N′-diisopropylcarbodiimide (DIC), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1-hydroxybenzotriazole hydrate (HOBt), 4-(dimethylamino)pyridine (DMAP), triisopropylsilane (TIS), trifluoroacetic acid (TFA)) and ninhydrin were delivered by Sigma-Aldrich Kft. (Budapest, Hungary). Aminooxyacetic acid (Aoa) and methoxyamine were TCI (Tokyo, Japan) products. The solvents (dichloromethane (DCM), N,N-dimethylformamide (DMF), acetonitrile (CH₃CN)) for synthesis and purification were obtained from Reanal (Budapest, Hungary) or VWR International Kft. (Debrecen, Hungary). Daunomycin (Dau) was donated from IVAX (Budapest, Hungary).

All reagents and solvents (anhydrous) were obtained from VWR International Kft. (Debrecen, Hungary). Fmoc-Rink Amide MBHA resin, N,N′-diisopropylcarbodiimide (DIC), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1-hydroxybenzotriazole hydrate (HOBt), and ninhydrin were purchased from Sigma-Aldrich Kft (Budapest, Hungary). Fmoc-Rink Amide 2CT resin and all amino acid derivatives used in this study were purchased either from Merck KGaA (Darmstadt, Germany) or Iris Biotech GmbH (Marktredwitz, Germany).

Rink Amide Protide resin (0.58 mmol/g), ethyl 2-cyano-2-(hydroxyimino) acetate (Oxyma Pure), and 9-fluorenylmethoxycarbonyl (Fmoc) amino acids were purchased from CEM Co (Matthews, NC, USA). Ciprofloxacin hydrochloride, ceftazidime, oxacillin, tobramycin, SYPRO red, Nile red, FITC-Con A, DAPI, crystal violet, triisopropylsilane, 2,2′-(ethylenedioxy)diethanethiol, trifluoroacetic acid, and N,N′-diisopropylcarbodiimide (DIC) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). HPLC-grade water, acetonitrile, and N,N-dimethylformamide (DMF) were bought from Daejung chemicals and metals Co., Ltd. (Siheung-si, Gyeonggi-do, Korea). Other chemicals used were of American Chemical Society reagent grade and used as received without any further purification.

MRBLEs coated with amine groups using 0.09% (v/v) pent-4-enylamine were stored in PBST and extensively washed with DCM, methanol, and DMF prior to biotin conjugation. For each conjugation reaction, we combined ~10,000 beads with 39 mg of biotin, 24 µL of N,N’-diisopropylcarbodiimide (DIC, Sigma-Aldrich) and 56 µL of DIPEA in 400 μL of DMF twice overnight, rotating at room temperature. After conjugation, we washed beads serially with 1 mL each of DMF, methanol, DCM, DMF, water and PBST. After washing, we passivated ~2000 beads with 5% BSA PBST for 1 h at room temperature on a rotator at 30 rpm; the other ~8000 beads were stored at 4 °C. We then exchanged the 5% BSA PBST solution to 2% BSA in PBST by washing and resuspension, added 1 µL of 1 mg/mL DyLight 650-tagged streptavidin (Abcam), and incubated for 30 min at 4 °C on a rotator at 30 rpm. Finally, we washed all beads three times with PBST and imaged them as described below.

All Fmoc amino acids, reagents, and solvents were used without purification. Fmoc amino acids and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo [4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) were purchase from AAPPTec, Bachem, Chem-Impex, ChemPep, and PurePep. The Rink amide ChemMatrix resin (catalog number: 7-600-1310) was purchased from Biotage. The 2-chlorotrityl chloride (2-CTC) resin (catalog number: 150301) were purchased from ChemPep. N,N-dimethylformamide (DMF), CH₂Cl₂, MeCN, MeOH, Et₂O, AcOH, Ac₂O, N,N-diisopropylethylamine (DIPEA), trifluoroacetic acid (TFA), NH₄HCl, NH₄HCO₃, urea, NaOH, 80% hydrazine hydrate, 2-mercaptoethanol were purchased from Fisher Scientific. Piperidine, triisopropylsilane (TIPS), hydroxybenzotriazole (HOBt), N,N′-diisopropylcarbodiimide (DIC), 2,2′-dithiodipyridine (DTDP), 2,2′-dithiobis (5-nitropyridine) (DTNP), I₂, Pd(PPh₃)₄, 1,3-dimethylbarbituric acid (DMBA), glucose, sodium ascorbate were purchased from Sigma Aldrich. 4-carboxy-3-fluorophenylboronic acid was purchased from Alfa Aesar. Agilent 6120 Quadrupole LC‒MS system was used to acquire the LC chromatograms and mass spectra of samples with Luna^® 5 μm C18 100 Å (50 × 2 mm) column (Phenomenex, CA, United States) at 0.4 ml/min with 5% of a H₂O/MeCN + 0.1% TFA solution for 1 min followed by a linear gradient from 5% to 95% of a H₂O/MeCN + 0.1% TFA solution over 5 min.

All chemicals were reagent grade and used without further purification. The protected chelator 2-(4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclo-dodecan-1-yl)acetic acid (DOTA-tris(tert-butyl) (^tBu) ester;) was supplied by CheMatech (Dijon, France). The L-amino acid precursors (9-fluorenylmethyloxycarbonyl (Fmoc))-Ala-OH, Fmoc-Gly-OH, Fmoc-Cys(triphenylmethyl) (Trt)-OH), Fmoc-Lys(tert-butyloxycarbonyl) (Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Phe-OH, Fmoc-Thr(^tBu)-OH, Fmoc-Ser(^tBu)-OH, the D-amino acid precursor Fmoc-DTrp(Boc)-OH, and H-L-Cys(S-acetamidomethyl) (Acm)-2-chlorotrityl resin (substitution 0.40 mmol/g) that was used in solid-phase peptide synthesis (SPPS) were purchased from CBL (Patras, Greece). The L-amino acid precursor Fmoc-Cys(Acm)-OH was purchased from Novabiochem (Darmstadt, Germany). For the coupling of the Fmoc-amino acids and the protected chelator DOTA-tris(^tBu)ester, the activating agents 1-hydroxybenzotriazol (HOBt) (CBL, Patras, Greece) and N,N′-diisopropylcarbodiimide (DIC) (Sigma Aldrich, Steinheim, Germany) were employed. The In-111 used for labeling was purchased from Mallinckrodt Medical B.V. (Petten, The Netherlands) in the form of [¹¹¹In]InCl₃ in a solution of 50 mM HCl at a 370 MBq/mL activity concentration on calibration date.