Lc forte r

¹H and ¹³C NMR experiments were performed on a Bruker AM 400, using different solvents (CD₃OD and DMSO-d₆). The internal standard was tetramethylsilane (TMS) (Andover, MA, USA). HRESI mass spectra were obtained on a qToF mass spectrometer (qToF Premier^TM, Waters Corp., Milford, MA, USA). The UPLC system, equipped with a binary solvent delivery system, an autosampler, and a UV detector, was also from Waters Corp. Separations were carried out on a medium pressure liquid chromatography (MPLC) system (LC-forte/R; YMC Co., Ltd., Kyoto, Japan), where reversed-phase (RP) cartridges purchased from YMC were employed. For purification, preparative high-performance liquid chromatography (HPLC) was performed using a Gilson HPLC system (Gilson, Inc., Middleton, WI, USA) with a YMC octadecyl-functionalized silica gel-AQ column (250 mm × 20 mm, i.d. 5 μm). A Multi-Mode Microplate Reader SpectraMax M2 (Molecular Devices, Sunnyvale, CA, USA) was used for the enzymatic assays.

To quantify the ligated peptides, RP-HPLC was performed using a Vydac C18 analytical column (218TP54; 150 mm × 4.6 mm, 5 μm C18 column; Separation Group, Hesperia, CA, USA) on a 1260 Infinity system (Agilent Technology, Santa Clara, CA, USA). TFA (0.05% v/v) was added to buffer A (DW) and buffer B (acetonitrile). The flow rate was 1 mL/min, and the injection volume was 5 μL. The elution gradient was 13–28% B over 35 min. Absorbance was measured at 210 and 280 nm. Chemically synthesized peptides (purity of >90%) were analyzed as the standard.
The final ligated peptide was purified using preparative RP-HPLC with a Vydac C18 preparative column (218TP1022; 250 mm × 22 mm, 10 μm) on a multiple preparative HPLC system (LC-forte/R; YMC, Kyoto, Japan). UV/Vis detection was performed at 210 and 230/280 nm and a flow rate of 10 mL/min. The gradient elution was set as follows: 0–50 min (20–35% B); 50.01–51 min (35–90% B); 51.01–56 min (90% B); 56.01–65 min (20% B). Fractions were collected, and those containing ligated peptides were lyophilized using a lyophilizer (FD-8512, ilShinBioBase, Dongducheon, Republic of Korea) for 3 d. The condenser temperature was maintained at –85 °C, with a pressure of 5 mTorr. The lyophilized polymers were collected and stored at –80 °C until use.

To synthesize A2E, all-trans-retinal (100 mg, 352 μmol), ethanolamine (9.5 mg, 155 μmol) in ethanol (3 mL), and acetic acid (9.3 µL, 155 μmol) were mixed by vigorous vortexing for 2 min. The mixture was incubated for 48 h under dark conditions. Subsequently, the mixture was concentrated at 20 °C using a nitrogen evaporator (Biotage, TurboVap-LV, USA). An A2E sample was separated and purified using silica gel and HPLC. Separation on a Sep-Pak C18 cartridge (WAT023635, Waters, Milford, MA, USA) was performed using a step gradient elution with 5:95 MeOH/CH₂Cl₂, 5:95 MeOH/CH₂Cl₂, and 8:92:0.001 MeOH/CH₂Cl₂/TFA. Additionally, the fractions were further purified using a multiple preparative HPLC (LC-forte/R, YMC Co., Kyoto, Japan) with a YMC-Triart Prep C18 column (250 mm × 10.0 mm, 10 μm). The purity of the A2E was analyzed using the gradient HPLC method with a YMC-Triart C18 column (4.6 mm × 250 mm, 5 µm) (Supplementary Figure S1B).

SP was fractionated using a Prep-HPLC instrument (LC-Forte/R, YMC, Kyoto, Japan) equipped with an ODS C18 column (250 × 15 mm, 20 μm) [8 (link)]. The solvent system consisted of DW (solvent A) and MeOH (solvent B) at a 10 mL/min flow. The elution program was as follows: 0% B for 5 min and 30% B for 30 min for a total run of 30 min. All fractions were concentrated using a rotary vacuum evaporator (Eyela) to remove MeOH. Concentrates were freeze-dried and stored in a deep freezer (−50 °C) until use.

The ¹H and ¹³C-NMR spectra were recorded on a JNM-ECZ600R FT-NMR spectrometer (JEOL, Tokyo, Japan), using CD₃OD with tetramethylsilane as the internal standard (Andover, MA, USA). Melting points (mp) were measured on a Thomas Scientific Capillary Melting Point Apparatus (Swedesboro, NJ, USA) and were uncorrected. The IR spectra were recorded on a Perkin Elmer Frontier FT-IR spectrophotometer (PerkinElmer, Waltham, MA, USA). ESI-HR mass spectra were obtained on a quadrupole time of flight mass spectrometer (Xevo G2-S QTOF, Waters Corp., Milford, MA, USA). The UPLC system, equipped with a binary solvent delivery system, an auto-sampler, and a UV detector, was also obtained from Waters Corp. Separation was performed using a medium-pressure liquid chromatography (MPLC) system (LC-forte/R; YMC Co., Ltd., Kyoto, Japan) with reversed-phase (RP) cartridges. Purification was carried out on a recycling HPLC system (LC-200 NEXT; Japan Analytical Industry Co., Ltd., Tokyo, Japan), with which a GS-310 (500 mm × 20 mm, i.d. 20 µm) column purchased from Japan Analytical Industry (JAI) was employed. Enzymatic assays were carried out on a SpectraMax Multi-Mode Microplate Reader M2 (Molecular Devices, San Jose, CA, USA).

The ¹H NMR and ¹³C NMR (500 MHz) spectra were measured on an Avance III 500 (Bruker, Rheinstetten, Germany) at the Center for Instrumental Analysis, Kyungpook National University (KNU). A prep-HPLC system was run on an LC-forte/R (YMC, Kyoto, Japan) for the purification of compounds. High-resolution FAB-MS spectra were conducted at the Korea Basic Science Institute (KBSI) using a JMS-700 (Jeol, Tokyo, Japan) to confirm the identity of the compounds. ICP-MS (PerkinElmer Inc., Norwalk, CT, USA) was used for a quantitative analysis of the Gd complex. The phantom and in vivo studies were carried out by Signa Architect 3.0 T System (127.8 MHz, GE Healthcare, Milwaukee, WI, USA) and 9.4 T BioSpec 94/20 (400 MHz, Bruker, Germany). Absorbance was measured using SpectraMax^® i3 (Molecular Devices, San Jose, CA, USA) and Lambda 950 (PerkinElmer Inc., Waltham, MA, USA). Fluorescence data were obtained with the F-7000 (Hitachi, Tokyo, Japan). Chemiluminescence western imaging system was run on Amersham™ ImageQuant™ 800 (Cytiva, Amersham, UK).