Griffey R H, Monia B P, Cummins L L, Freier S, Greig M J, Guinosso C J, Lesnik E, Manalili S M, Mohan V, Owens S, Ross B R, Sasmor H, Wancewicz E, Weiler K, Wheeler P D, Cook P D
Isis Pharmaceuticals, Carlsbad, California 92008, USA.
J Med Chem. 1996 Dec 20;39(26):5100-9. doi: 10.1021/jm950937o.
Oligonucleotides containing 2'-O-aminopropyl-substituted RNA have been synthesized. The 2'-O-(aminopropyl)adenosine (APA), 2'-O-(aminopropyl)cytidine (APC), 2'-O-(aminopropyl)-guanosine (APG), and 2'-O-(aminopropyl)uridine (APU) have been prepared in high yield from the ribonucleoside, protected, and incorporated into an oligonucleotide using conventional phosphoramidite chemistry. Molecular dynamics studies of a dinucleotide in water demonstrates that a short alkylamine located off the 2'-oxygen of ribonucleotides alters the sugar pucker of the nucleoside but does not form a tight ion pair with the proximate phosphate. A 5-mer with the sequence ACTUC has been characterized using NMR. As predicted from the modeling results, the sugar pucker of the APU moiety is shifted toward a C3'-endo geometry. In addition, the primary amine rotates freely and is not bound electrostatically to any phosphate group, as evidenced by the different sign of the NOE between sugar proton resonances and the signals from the propylamine chain. Incorporation of aminopropyl nucleoside residues into point-substituted and fully modified oligomers does not decrease the affinity for complementary RNA compared to 2'-O-alkyl substituents of the same length. However, two APU residues placed at the 3'-terminus of an oligomer gives a 100-fold increase in resistance to exonuclease degradation, which is greater than observed for phosphorothioate oligomers. These structural and biophysical characteristics make the 2'-O-aminopropyl group a leading choice for incorporation into antisense therapeutics. A 20-mer phosphorothioate oligonucleotide capped with two phosphodiester aminopropyl nucleotides targeted against C-raf mRNA has been transfected into cells via electroporation. This oligonucleotide has 5-10-fold greater activity than the control phosphorothioate for reducing the abundance of C-raf mRNA and protein.
已合成了含有2'-O-氨丙基取代RNA的寡核苷酸。已从核糖核苷高产率制备了2'-O-(氨丙基)腺苷(APA)、2'-O-(氨丙基)胞苷(APC)、2'-O-(氨丙基)鸟苷(APG)和2'-O-(氨丙基)尿苷(APU)，进行了保护，并使用传统的亚磷酰胺化学方法将其掺入寡核苷酸中。对水中二核苷酸的分子动力学研究表明，位于核糖核苷酸2'-氧原子上的短链烷基胺改变了核苷的糖环构象，但未与相邻的磷酸形成紧密的离子对。已使用核磁共振对序列为ACTUC的5聚体进行了表征。正如从建模结果预测的那样，APU部分的糖环构象向C3'-内型几何结构偏移。此外，伯胺自由旋转，未静电结合到任何磷酸基团上，糖质子共振与丙胺链信号之间的NOE具有不同的符号就证明了这一点。与相同长度的2'-O-烷基取代基相比，将氨丙基核苷残基掺入点取代和完全修饰的寡聚物中不会降低对互补RNA的亲和力。然而，将两个APU残基置于寡聚物的3'-末端会使对外切核酸酶降解的抗性增加100倍，这比硫代磷酸酯寡聚物观察到的抗性更大。这些结构和生物物理特性使2'-O-氨丙基基团成为掺入反义治疗剂的首选。一种用两个磷酸二酯氨丙基核苷酸封端的靶向C-raf mRNA的20聚体硫代磷酸酯寡核苷酸已通过电穿孔转染到细胞中。该寡核苷酸在降低C-raf mRNA和蛋白质丰度方面比对照硫代磷酸酯具有高5-10倍的活性。
