Ammonium acetate buffer

Methanol (pro-analysis), Folin–Ciocalteu phenol reagent, aquadest, aquabidest, GA, ammonium acetate buffer, CuCl₂, K₂S₂O₄, ammonium acetate buffer, neocuproine, AlCl₃, FeCl₃, HCl, and quercetin were obtained from Merck-Millipore (Darmstadt, Germany). Trolox, ABTS, sodium carbonate, glacial acetate acid, and DPPH were obtained from Sigma-Aldrich (St. Louis, MO, USA). 2,4,6-tripydyl-s-triazine (TPTZ) and acetic acid were obtained from Sisco Research Laboratories Pvt. Ltd. (Maharashtra, India).

Ultrapure and metal-free buffers for radiosynthesis were prepared using high-quality Milli-Q water and pretreated with Chelex 100 resin sodium form (Sigma-Aldrich, St. Louis, MO, USA). The compound of PSMA-617 used herein was synthesized by the method as published by Benesova et al. [43 (link)]. Ammonium acetate buffer (0.2 M, pH 5.5, Merck, Darmstadt, Germany) in an amount of 80 μL was added in a LoBind Eppendorf tube containing 5 nmol of PSMA-617 in Milli-Q water (1 nmol/μL). After the addition of ¹⁷⁷Lu (5 μL, 25 MBq, IRT-T Nuclear Research Reactor of Tomsk Polytechnic University), the reaction mixture was vortexed and incubated for 30 min at 80 °C. Radiochemical yield and purity were determined by using radio-iTLC and radio-HPLC methods. The iTLC method used glass-fiber sheets (Agilent Technologies, Inc., Folsom, CA, USA) eluted in 0.2 M citric acid with a pH of 2.0. Performing radio-iTLC analysis in this system provides retention of the ¹⁷⁷Lu-labeled PSMA ligand molecules at the point of application, while free ¹⁷⁷Lu³⁺ ions migrate with the solvent front. The HPLC technique employed for [¹⁷⁷Lu]Lu-PSMA-617 was identical to that used to analyze [¹²³I]PSMA-p-IB.

DOTA-ST8950 (Figure 1) was custom-made by PolyPeptide (San Diego, CA, USA). DOTA-NOC (Figure 1) was synthesized by standard Fmoc-solid-phase peptide synthesis, purified by preparative RP-HPLC and characterized by ESI-MS and analytical RP-HPLC (Figure 1). ^natLu-DOTA-ST8950 and ^natLu-DOTA-NOC were synthesized after incubation of the DOTA-conjugates with a 2.5-fold excess of ^natLuCl₃ × 6H₂O (Sigma Aldrich, St. Louis, MO, USA) in ammonium acetate buffer (Sigma Aldrich, St. Louis, MO, USA), 0.4 M, pH 5 at 95 °C for 30 min. Free metal ions were eliminated via SepPak C-18 cartridge (Waters), preconditioned with methanol (Merck, Darmstadt, Germany) and water. The reaction mixture was loaded and the free ^natLu was eluted with water while the metallated peptides were eluted with ethanol, evaporated to dryness, redissolved in water and lyophilized. The ¹⁷⁷Lu-labeled conjugates were synthesized by dissolving 5–10 μg (3–6 nmol) of the DOTA-conjugates in 250 μL of sodium acetate buffer (0.4 M, pH 5.0) followed by incubation with [¹⁷⁷Lu]LuCl₃ (10–200 MBq, depending on the planned experiment) for 30 min at 95 °C. The stability of [¹⁷⁷Lu]Lu-DOTA-ST8950 under two different storage conditions, room temperature (RT) and at 4 °C, was evaluated over 24 h after synthesis.