Isolera one instrument

The phenol (200 mg) was dissolved in CH₂Cl₂ (5 mL), and 4-dimethylaminopyridine (0.1 equiv.) was added. 2-Azidoacetic acid (1.2 equiv.) was dissolved in CH₂Cl₂ (5 mL) with N,N’-diisopropylcarbodiimide (1.2 equiv.) and stirred for 15 min, before it was added carefully to the phenol solution. The reaction mixture was stirred under N₂. After 2 h, additional 2-azidoacetic acid (1.2 equiv.) and N,N’-diisopropylcarbodiimide (0.6 equiv.) were added to the reaction mixture, and stirring was continued for 2 h. Then, the reaction mixture was concentrated by rotary evaporation, and the crude product was purified using an Isolera One instrument from Biotage™ equipped with a 25 g KP-Sil column. The product was eluted with CH₂Cl₂, and fractions containing the pure product were combined and concentrated by rotary evaporation. The product was dried in vacuo.

¹H-NMR spectra were recorded at 400 MHz with a JNM-ECS400 NMR spectrometer (JEOL, Tokyo, Japan). For all NMR measurements, chemical shifts were referenced to the solvent values (δ = 2.49 or 7.26 ppm for DMSO-d₆ or CDCl₃, respectively). Silica gel column chromatography was performed using a Biotage Isolera One instrument (Biotage AB, Uppsala, Sweden) equipped with a SNAP Ultra Column cartridge. Gel permeation chromatography (GPC) measurements of DOPE-pAA-Cys5 was carried out using a high performance liquid chromatography (HPLC) system (CBM-20A/LC-20AD/SIL-10AXL/DGU-20 A3R/CTO-20AC, Shimadzu, Kyoto, Japan) equipped with an SB-804 HQ column (Shodex, Tokyo, Japan) and a refractive index (RI) detector (RID-20A, Shimadzu, Kyoto, Japan), using Tris–HCl buffer (10 mM) containing 100 mM NaCl as an eluent at a flow rate of 0.7 mL min⁻¹ at 25 °C. ReadyCal-Kit polyethylene glycol (PEG) (PSS GmbH, Mainz, Germany) was used as the calibration standard. S-Trityl-l-cysteine acrylamide (S-Tri-Cys-AAm) was prepared according to a previous report.^{28 (link)}

Chiminord (Milan, Italy) and Aldrich Chemical (Milan, Italy) purchased all chemicals. Solvents were reagent grade. Unless otherwise stated, all commercial reagents were used without further purification. Organic solutions were dried over anhydrous sodium sulphate. A thin layer chromatography (TLC) system was used for routine monitoring of the course of reactions and confirming the purity of analytical samples. Detection of spots was performed using UV light. Merck silica gel, 230–400 mesh, was used for chromatography. Flash chromatography was performed using Isolera one instrument (Biotage, Uppsala, Sweden) using Silicagel column. Melting points are not “corrected” and were measured with a Buchi M-560 instrument (Buchi instruments, Flawil, Switzerland). NMR spectra were recorded on JEOL JNM-ECZR (400 MHz, Tokyo, Japan) instruments (Figures S1–S42, Supporting Information) using CDCl₃ or DMSO-d₆ as solvent; chemical shifts are reported as δ (ppm) and signals were characterized as s (singlet), d (doublet), t (triplet), n t (near triplet), q (quartet), m (multiplet), br s (broad signal); J are reported in Hz.
Elemental analysis was determined with an elemental analyzer EA 1110 (Fison-Instruments, Milan, Italy) and the purity of all synthesized compounds was >95%; products are considered pure when the difference between calculated and found values is ± than 0.4.