Arar K, Aubertin A M, Roche A C, Monsigny M, Mayer R
Laboratoire de Biochimie des Glycoconjugués, CNRS, Orléans, France.
Bioconjug Chem. 1995 Sep-Oct;6(5):573-7. doi: 10.1021/bc00035a011.
Antisense oligonucleotides represent an interesting tool for selective inhibition of gene expression. In order to direct oligonucleotides to specific compartments within the cell, we have investigated the possibility of coupling them to a signal peptide Lys-Asp-Glu-Leu (KDEL). This sequence should be able to convey oligonucleotides to the endoplasmic reticulum and from there to the cytosol and the nucleus where their targets are located. On this basis we prepared peptide-oligonucleotide conjugates by coupling, in a single step, a Nalpha-bromoacetyl peptide with an oligonucleotide bearing a thiol group, through a thioether bond. This paper deals with the definition of the optimal pH and temperature conditions leading to an efficient synthesis of peptide-oligonucleotide conjugates: the reaction was quantitative at pH 7.5 within few hours. This method was first set up using a 5',3'-modified dodecanucleotide and a (bromoacetyl)pentapeptide as a conjugation model. Then a 5',3'-modified pentacosanucleotide, complementary to the translation initiation region of the gag mRNA of HIV, was coupled to a (bromoacetyl)dodecapeptide containing a KDEL signal sequence. The anti-HIV activity of the pentacosanucleotide was compared with that of pentacosanucleotide-dodecapeptide conjugates linked through either a thioether bond or a disulfide bridge. The conjugate with a thioether bond has a higher antiviral activity than the peptide-free oligonucleotide and the conjugate linked via a disulfide bond.
反义寡核苷酸是一种用于选择性抑制基因表达的有趣工具。为了将寡核苷酸导向细胞内的特定区域，我们研究了将它们与信号肽赖氨酸-天冬氨酸-谷氨酸-亮氨酸（KDEL）偶联的可能性。该序列应能够将寡核苷酸转运至内质网，然后从内质网转运至胞质溶胶和其靶标所在的细胞核。在此基础上，我们通过硫醚键将Nα-溴乙酰化肽与带有巯基的寡核苷酸一步偶联，制备了肽-寡核苷酸缀合物。本文探讨了实现肽-寡核苷酸缀合物高效合成的最佳pH和温度条件的确定：该反应在pH 7.5时几小时内即可定量完成。该方法首先使用一种5',3'-修饰的十二聚核苷酸和一种（溴乙酰基）五肽作为偶联模型建立起来。然后，将与HIV gag mRNA翻译起始区域互补的5',3'-修饰的二十五聚核苷酸与含有KDEL信号序列的（溴乙酰基）十二肽偶联。将二十五聚核苷酸的抗HIV活性与通过硫醚键或二硫键连接的二十五聚核苷酸-十二肽缀合物的抗HIV活性进行了比较。通过硫醚键连接的缀合物比无肽寡核苷酸和通过二硫键连接的缀合物具有更高的抗病毒活性。
