Baranowski Marek R, Nowicka Anna, Rydzik Anna M, Warminski Marcin, Kasprzyk Renata, Wojtczak Blazej A, Wojcik Jacek, Claridge Timothy D W, Kowalska Joanna, Jemielity Jacek
†Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland.
§Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097 Warsaw, Poland.
J Org Chem. 2015 Apr 17;80(8):3982-97. doi: 10.1021/acs.joc.5b00337. Epub 2015 Apr 8.
To broaden the scope of existing methods based on (19)F nucleotide labeling, we developed a new method for the synthesis of fluorophosphate (oligo)nucleotide analogues containing an O to F substitution at the terminal position of the (oligo)phosphate moiety and evaluated them as tools for (19)F NMR studies. Using three efficient and comprehensive synthetic approaches based on phosphorimidazolide chemistry and tetra-n-butylammonium fluoride, fluoromonophosphate, or fluorophosphate imidazolide as fluorine sources, we prepared over 30 fluorophosphate-containing nucleotides, varying in nucleobase type (A, G, C, U, m(7)G), phosphate chain length (from mono to tetra), and presence of additional phosphate modifications (thio, borano, imido, methylene). Using fluorophosphate imidazolide as fluorophosphorylating reagent for 5'-phosphorylated oligos we also synthesized oligonucleotide 5'-(2-fluorodiphosphates), which are potentially useful as (19)F NMR hybridization probes. The compounds were characterized by (19)F NMR and evaluated as (19)F NMR molecular probes. We found that fluorophosphate nucleotide analogues can be used to monitor activity of enzymes with various specificities and metal ion requirements, including human DcpS enzyme, a therapeutic target for spinal muscular atrophy. The compounds can also serve as reporter ligands for protein binding studies, as exemplified by studying interaction of fluorophosphate mRNA cap analogues with eukaryotic translation initiation factor (eIF4E).
为了拓宽基于（19）F核苷酸标记的现有方法的范围，我们开发了一种合成氟代磷酸（寡）核苷酸类似物的新方法，该类似物在（寡）磷酸部分的末端位置含有O到F的取代，并将它们评估为（19）F NMR研究的工具。我们使用了三种基于磷酰咪唑化学和四正丁基氟化铵、氟代单磷酸或氟代磷酸咪唑作为氟源的高效且全面的合成方法，制备了30多种含氟代磷酸的核苷酸，它们在核碱基类型（A、G、C、U、m（7）G）、磷酸链长度（从单磷酸到四磷酸）以及是否存在额外的磷酸修饰（硫代、硼代、亚氨基、亚甲基）方面各不相同。使用氟代磷酸咪唑作为5'-磷酸化寡核苷酸的氟代磷酰化试剂，我们还合成了寡核苷酸5'-(2-氟代二磷酸)，它们有可能用作（19）F NMR杂交探针。这些化合物通过（19）F NMR进行了表征，并被评估为（19）F NMR分子探针。我们发现氟代磷酸核苷酸类似物可用于监测具有各种特异性和金属离子需求的酶的活性，包括人类DcpS酶，它是脊髓性肌萎缩症的治疗靶点。这些化合物还可以作为蛋白质结合研究的报告配体，例如研究氟代磷酸mRNA帽类似物与真核翻译起始因子（eIF4E）的相互作用。
