1260 infinity

HPLC-purified peptide 3 was dissolved in PBS buffer (pH=8.0) to a final concentration of 50 μM. FITC (Thermo Fisher) was dissolved in DMSO (10 mg/mL) and added slowly to the peptide solution to a final concentration of 250 μM. The reaction was incubated for 18 h at 25 °C. After centrifugation at 16,100 × g for 10 min, the supernatant was purified on an Agilent 1260 Infinity HPLC system with a Phenomenex Luna C18 column (250 mm × 4.6 mm, 10 μm particle size, 100 Å pore size). Solvent A was 0.1% TFA in H₂O and solvent B was 80% MeCN/H₂O containing 0.086% TFA. An elution gradient from 0% solvent B to 100% solvent B over 45 min at 1 mL/min was used and FITC labeled XY3-3 core peptide eluted at 66-67% solvent B. PTAP (GGPEPTAPPEE) peptide containing two additional N-terminal Gly residues (synthesized by United Biosynthesis) was labelled with FITC and purified as described above. The FITC labelled PTAP peptide eluted at 48-49% solvent B.

Five milligrams of the peptide (1.87 µmol) was dissolved in 0.5 mL of DMF and mixed with 2.1 mg of DOTA-NHS-ester (2.79 µmol in 0.5 mL of DMF). The pH of the reaction mixture was adjusted to 7 using diisopropylethylamine (DIPEA). Product was separated on a reversed phase-high performance liquid chromatography (RP-HPLC) system (Varian ProStar) with an Agilent Technology 1260 Infinity photodiode array detector using a semipreparative C-18 Luna column (5 mm, 10 × 250 mm Phenomenex) and a gradient elution starting with 98% H₂O (0.1% TFA) and 2% MeOH (0.1% TFA) reaching 90% of MeOH in 65 min at a flow rate of 2 mL/min. The desired POL-D at ~41.25 min of elution time was collected and evaporated. The resulting residue was dissolved in deionized water and lyophilized, yielding 3.85 mg (1.3 µmol) of the product as a white powder (yield: 61.3%), which was used for further experiments. To confirm modification, the resulting conjugate was analyzed by mass spectrometry. Theoretical chemical formula, C₁₁₁H₁₆₆N₃₆O₂₇S₂; exact mass, 2499.22; molecular weight, 2500.86; observed m/z 2498.47, (M + 1)⁺¹; 1250.06, (M + 2)⁺²/2; 833.76, (M + 3)⁺³/ 3 (Figure S1, panels A–C, of the Supporting Information).

In order to characterize AldeRed 588-A produced upon hydrolysis, AldeRed 588 aldehyde diethyl acetal 5 (0.5 mg in 300 µL) was mixed with 300 µL of 2N HCl and incubated for 30 min at room temperature. The reaction mixture was then diluted with 2 mL of water. The clear purple solution was injected into the HPLC. The purification of AldeRed-588A was performed using an Agilent 1260 infinity preparative HPLC system equipped with a Phenomenex Luna C18, 10 micron column and a flow rate of 10 mL/min. The desired product eluted at 3.3 min with acetonitrile/water (40/60) and collected. (The solvent front eluted at 1.1 min.) The collected fraction was frozen at −78°C immediately and lyophilized to dryness. Approximately 0.4 mg of AldeRed-588A was obtained as a dark powder. HRESI-MS C₁₈H₁₈BF₂N₄O₂ calcd 371.1485; found: 371.1489 (Supplementary Fig. 14).