Molecular Tracer and Imaging Core Facility, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, USA.
Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, USA.
Bioorg Med Chem. 2019 Oct 1;27(19):115041. doi: 10.1016/j.bmc.2019.08.002. Epub 2019 Aug 5.
The efficient radiosynthesis of biomolecules utilizing minute quantities of maleimide substrate is important for availability of novel peptide molecular imaging agents. We evaluated both 3-F-fluoropropane-1-thiol and 2-(2-(2-(2-F-fluoroethoxy)ethoxy)ethoxy)ethane-1-thiol (F-fluoro-PEG thiol) as prosthetic groups for radiolabeling under physiological conditions. The precursor employed a benzoate for protection of the thiol and an arylsulfonate leaving group. The radiofluorination was fully automated on an Eckert & Ziegler synthesis system using standard Kryptofix/KCO conditions. In order to minimize the amount of biological molecule required for subsequent conjugation, the intermediates, S-(3-F-fluoropropyl) benzothioate and F-fluoro-PEG benzothioate, were purified by HPLC. The intermediates were isolated from the HPLC in yields of 37-47% and 28-35%, respectively, and retrieved from eluate using solid phase extraction. Treatment of the benzothioates with sodium methoxide followed by acetic acid provided the free thiols. The desired maleimide substrate in acetonitrile or phosphate buffer was then added and incubated at room temperature for 15 min. The final radiolabeled bioconjugate was purified on a separate HPLC or NAP-5 column. Maleimides utilized for the coupling reaction included phenyl maleimide, an Evans Blue maleimide derivative, a dimeric RGDfK maleimide (E[c(RGDfK)]), two aptamer maleimides, and PSMA maleimide derivative. Isolated radiochemical yields (non-decay corrected) of maleimide addition products based on starting F-fluoride ranged from 6 to 22% in a synthesis time of about 90 min. F-thiol prosthetic groups were further tested in vivo by conjugation to E[c(RGDfK)] maleimide in a U87MG xenograft model. PET studies demonstrated similar tumor accumulation of both prosthetic groups. F-fluoro-PEG-S-E[c(RGDfK)] displayed a somewhat favorable pharmacokinetics compared to F-fluoropropyl-S-E[c(RGDfK)]. Bone uptake was low for both indicating in vivo stability.
利用微量马来酰亚胺底物高效合成生物分子对于新型肽分子成像剂的可用性非常重要。我们评估了 3-F-氟丙硫醇和 2-(2-(2-(2-氟乙氧基)乙氧基)乙氧基)乙烷-1-硫醇（F-氟-PEG 硫醇）作为在生理条件下进行放射性标记的前体。所使用的前体采用苯甲酸酯保护巯基和芳基磺酸酯离去基团。放射性氟标记在 Eckert & Ziegler 合成系统上完全自动化进行，使用标准的 Kryptofix/KCO 条件。为了尽量减少后续缀合所需的生物分子的量，对中间体 S-(3-F-氟丙基)苯硫酯和 F-氟-PEG 苯硫酯进行 HPLC 纯化。中间体分别以 37-47%和 28-35%的产率从 HPLC 中分离出来，并使用固相萃取从洗脱液中回收。用甲醇钠处理苯硫酯，然后用乙酸处理，得到游离巯基。然后将所需的马来酰亚胺底物加入到乙腈或磷酸盐缓冲液中，在室温下孵育 15 分钟。最后，将放射性标记的生物缀合物在单独的 HPLC 或 NAP-5 柱上进行纯化。用于偶联反应的马来酰亚胺包括苯马来酰亚胺、 Evans Blue 马来酰亚胺衍生物、二聚 RGDfK 马来酰亚胺（E[c（RGDfK）]）、两种适体马来酰亚胺和 PSMA 马来酰亚胺衍生物。基于起始 F-氟化物的马来酰亚胺加成产物的分离放射性化学产率（未校正衰变）在约 90 分钟的合成时间内为 6-22%。F-硫醇前体通过与 U87MG 异种移植模型中的 E[c（RGDfK）]马来酰亚胺缀合进一步在体内进行测试。PET 研究表明，两种前体的肿瘤积聚相似。与 F-氟丙基-S-E[c（RGDfK）]相比，F-氟-PEG-S-E[c（RGDfK）]显示出更好的药代动力学特性。两种都显示出低的骨骼摄取，表明体内稳定性。
