Soukchareun S, Haralambidis J, Tregear G
The Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria 3052, Australia.
Bioconjug Chem. 1998 Jul-Aug;9(4):466-75. doi: 10.1021/bc980004h.
The chemical modification of antisense oligodeoxynucleotides (ODNs) by conjugating synthetic peptides of known membranotropic activities to the 3' and/or 5' terminus of ODNs may serve two functions that are important for increasing their bioavailability by protecting the ODNs from exonuclease digestion and facilitated delivery into cells. We have previously reported the preparation of ODN-peptide conjugates by the total synthesis approach. However, by such technology the preparation of ODN-peptide conjugates in amounts sufficient for in vitro functional analysis is at present limited to the syntheses of peptides containing residues without acidolytic deprotection. Requisite to the alternative method of site-specific conjugation, the segment coupling approach is the derivatization of an ODN with a nucleophilic moiety. In this paper, we describe a novel method of functionalizing synthetic ODNs by incorporating S-thiobutyl-protected Nalpha-Fmoc-cysteine to aminopropyl-functionalized CPG by standard Nalpha-Fmoc SPPS methodology. The derivatized solid support can be used to synthesize an ODN of any sequence by the phosphoramidite chemistry, and the removal of the S-thiobutyl side chain function can be conveniently affected by the standard amminolytic deprotection of ODNs containing 1 M DTT. The purified cysteine-derivatized ODN was shown to react specifically and efficiently with two types of synthetic peptides corresponding to regions within the glycoprotein (gp) of HIV that have been shown to have membranotropic activities: a 18 residue maleimide-derivatized Tat peptide of the transactivator (tat) of HIV and a 22 residue peptide corresponding to the carboxyl terminus of gp41(Ca-gp41).
通过将已知具有膜亲和活性的合成肽与反义寡脱氧核苷酸（ODN）的3'和/或5'末端偶联来对其进行化学修饰，可能具有两种重要功能，即通过保护ODN免受核酸外切酶消化并促进其进入细胞来提高其生物利用度。我们之前报道了通过全合成方法制备ODN-肽缀合物。然而，利用这种技术，目前能够制备出足以用于体外功能分析的ODN-肽缀合物的量，仅限于合成不含酸解去保护残基的肽。对于位点特异性偶联的替代方法而言，片段偶联方法需要用亲核部分对ODN进行衍生化。在本文中，我们描述了一种通过标准的Nα-Fmoc固相肽合成（SPPS）方法，将S-硫代丁基保护的Nα-Fmoc-半胱氨酸掺入氨丙基功能化的CPG中，从而对合成ODN进行功能化的新方法。这种衍生化的固相载体可用于通过亚磷酰胺化学合成任何序列的ODN，并且通过含有1 M二硫苏糖醇（DTT）的ODN的标准氨解去保护，可以方便地实现S-硫代丁基侧链功能的去除。纯化后的半胱氨酸衍生化ODN显示出能与两种合成肽特异性且高效地反应，这两种合成肽对应于已被证明具有膜亲和活性的HIV糖蛋白（gp）内的区域：一种是HIV反式激活因子（tat）的18个残基的马来酰亚胺衍生化Tat肽，另一种是对应于gp41羧基末端的22个残基的肽（Ca-gp41）。
