A submicrogram-scale protocol for biomolecule-based PET imaging by rapid 6π-azaelectrocyclization:: Visualization of sialic acid dependent circulatory residence of glycoproteins
A submicrogram-scale protocol for biomolecule-based PET imaging by rapid 6π-azaelectrocyclization:: Visualization of sialic acid dependent circulatory residence of glycoproteins
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DOI：
10.1002/anie.200702989
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发表时间：
2008-01-01
影响因子：
16.6
通讯作者：
Fukase, Koichi
中科院分区：
文献类型：
--
作者：
Tanaka, Katsunori;Masuyama, Tatsuro;Fukase, Koichi
分子影像学是化学生物学、药物发现和诊断领域的重要课题。正电子发射断层扫描（PET）是一种广泛使用的非侵入性方法，可通过高成像对比度定量显示放射性示踪剂积聚的位置和水平。[1]2-脱氧-2-[18 F]氟代-d-葡萄糖（[18 F] FDG）已成为一种重要的基于小分子的PET示踪剂，尤其用于检测原发性和转移性癌症。目前的努力集中在开发基于生物分子的示踪剂，其由肽、单克隆抗体（mAb）和寡核苷酸组成。[1]通常，这些生物分子的PET成像通过在用合适的发射B+的金属进行放射性标记之前与金属螯合剂如dota（1，4，7，10-四氮杂环癸烷-1，4，7，10-四乙酸）或dtpa（二亚乙基三胺五乙酸）缀合来实现。螯合剂可以在肽的固相合成过程中引入，或者更直接地通过赖氨酸残基或N-末端氨基与dota-O-磺基琥珀酰亚胺酯的反应引入。[2]然而，后一种方法，使用活化dota酯，通常进行缓慢，需要多达几毫克的样品，以保持反应浓度高。由于PET实验只需要少量的示踪剂，而且重要的生物分子有时只能获得少量，因此亚微克级的缀合方法将大大扩展PET成像的适用性。在此，我们描述了亚微克级标记赖氨酸残基的快速6 β-氮杂电环化，这导致了一个有效的PET协议;实现了糖蛋白的唾液酸依赖性循环驻留的首次可视化。尽管已经报道了各种生物分子的标记方法，[3]仍然需要找到可以应用于各种缓冲溶液的有效、快速和温和的方案。最近，Katsumura及其同事报道了不饱和（E）-酯醛与伯胺（包括赖氨酸）在水中在5分钟内定量反应，从而在不可逆反应中提供1，2-二氢吡啶（方案1）。[4]这些反应通过中间体席夫碱（1-氮杂三烯）的平稳氮杂电环化进行，由于1-氮杂三烯系统内最高占据和最低未占据分子轨道（HOMO和LUMO）之间的有效相互作用，该反应被强烈加速;这些相互作用受到C4处的吸电子取代基和C6处的共轭作用的影响。[4d]受Katsumura的发现启发，我们设计并合成了一种新的dota标记探针dota-（E）-酯醛4a（方案2）。将（Z）-溴乙烯2和甘氨酸连接的（E）-锡烷1在[Pd 2（dba）3]（dba=反式，反式-二亚苄基丙酮）、P（2-呋喃基）3和LiCl存在下在DMF中加热至110 ℃，得到Stille偶联产物，[4d]随后用3 M盐酸处理，得到氨基醇3，两步产率为73%。
Molecular imaging is an important topic that has gained significant attention in the fields of chemical biology, drug discovery, and diagnosis. Positron emission tomography (PET) is a widely used, noninvasive method that quantitatively visualizes the locations and levels of radiotracer accumulation with high imaging contrast.[1] 2-deoxy-2-[18F] fluoro-d-glucose ([18F] FDG) has become an important small-molecule-based PET tracer used especially in the detection of primary and metastatic cancers. Current efforts focus on the development of biomolecule-based tracers, which are composed of peptides, monoclonal antibodies (mAbs), and oligonucleotides.[1] Generally, PET imaging of these biomolecules is achieved by conjugation to metalchelating agents, such as dota (1, 4, 7, 10-tetraazacyclodecane-1, 4, 7, 10-tetraacetic acid) or dtpa (diethylenetriaminepentaacetic acid), prior to radiolabeling with a suitable b+-emitting metal. The chelating agents can be introduced either during the solid-phase synthesis of peptides or more directly by the reaction of lysine residues or N-terminal amino groups with dota-O-sulfosuccinimidyl ester.[2] However, the latter method, using the activated dota ester, usually proceeds slowly and requires as much as a few milligrams of sample to keep the reaction concentrations high. Because PET experiments require only small amounts of the tracers, and because important biomolecules are sometimes obtained only in small amounts, a submicrogram-scale conjugation methodology would greatly expand the applicability of PET imaging. Herein, we describe the submicrogram-scale labeling of lysine residues by a rapid 6p-azaelectrocyclization, which led to an efficient PET protocol; a first visualization of sialic acid dependent circulatory residence of glycoproteins was realized.Although various labeling methods for biomolecules have been reported,[3] an efficient, rapid, and mild protocol that can be applied in various buffer solutions remains to be found. Recently, Katsumura and co-workers reported that unsaturated (E)-ester aldehydes quantitatively react with primary amines, including lysine, within five minutes in water, thus providing 1, 2-dihydropyridines in irreversible reactions (Scheme 1).[4] These reactions proceed by smooth azaelectrocyclization of the intermediary Schiff bases (1-azatrienes), which is strongly accelerated owing to the efficient interactions between the highest occupied and lowest unoccupied molecular orbitals (HOMO and LUMO) within the 1-azatriene systems; these interactions are favored by the electron-withdrawing substituent at C4 and the conjugation at C6.[4d] Inspired by Katsumura s findings, we designed and synthesized a new dota labeling probe, dota-(E)-ester aldehyde 4a (Scheme 2).(Z)-Vinyl bromide 2 and glycine-linked (E)-stannane 1 were heated to 1108C in the presence of [Pd2 (dba) 3](dba= trans, trans-dibenzylideneacetone), P (2-furyl) 3, and LiCl in DMF to provide the Stille coupling product,[4d] which was subsequently treated with 3m hydrochloric acid to give aminoalcohol 3 in 73% yield for two steps.