Department of Industrial Engineering, University of Padova, via Marzolo 9, 35131 Padova, Italy.
Molecules. 2012 Sep 13;17(9):11026-45. doi: 10.3390/molecules170911026.
Peptide Nucleic Acids (PNAs) linked to high molecular weight (MW) poly(ethylene oxide) (PEO) derivatives could be useful conjugates for the direct functionalisation of gold surfaces dedicated to Surface Plasmon Resonance (SPR)-based DNA sensing. However their use is hampered by the difficulty to obtain them through a convenient and economical route. In this work we compared three synthetic strategies to obtain PNA-high MW PEO conjugates composed of (a) a 15-mer PNA sequence as the probe complementary to genomic DNA of ]Mycobacterium tuberculosis, (b) a PEO moiety (2 or 5 KDa MW) and (c) a terminal trityl-protected thiol necessary (after acidic deprotection) for grafting to gold surfaces. The 15-mer PNA was obtained by solid-phase synthesis. Its amino terminal group was later condensed to bi-functional PEO derivatives (2 and 5 KDa MW) carrying a Trt-cysteine at one end and a carboxyl group at the other end. The reaction was carried out either in solution, using HATU or PyOxim as coupling agents, or through the solid-phase approach, with 49.6%, 100% and 5.2% yield, respectively. A differential solvent extraction strategy for product purification without the need for chromatography is described. The ability of the 5 KDa PEO conjugate to function as a probe for complementary DNA detection was demonstrated using a Grating-Coupling Surface Plasmon Resonance (GC-SPR) system. The optimized PEO conjugation and purification protocols are economical and simple enough to be reproduced also within laboratories that are not highly equipped for chemical synthesis.
聚乙二醇（PEO）衍生物与高分子量（MW）肽核酸（PNA）的连接物可以成为用于直接功能化金表面的有用缀合物，这些金表面专门用于基于表面等离子体共振（SPR）的 DNA 传感。然而，由于难以通过方便且经济的途径获得它们，因此它们的使用受到了阻碍。在这项工作中，我们比较了三种合成策略来获得由（a）与结核分枝杆菌基因组 DNA 互补的 15 聚体 PNA 序列，（b）PEO 部分（2 或 5 kDa MW）和（c）末端三苯甲基保护的硫醇组成的 PNA-高 MW PEO 缀合物，该硫醇在酸性脱保护后（c）有必要用于接枝到金表面。15 聚体 PNA 通过固相合成获得。然后，将其氨基末端基团缩合到双官能 PEO 衍生物（2 和 5 kDa MW）上，该衍生物的一端带有 Trt-半胱氨酸，另一端带有羧基。反应可以在溶液中进行，使用 HATU 或 PyOxim 作为偶联剂，也可以通过固相方法进行，产率分别为 49.6%、100%和 5.2%。描述了一种无需使用色谱法即可进行产物纯化的差溶剂萃取策略。使用光栅耦合表面等离子体共振（GC-SPR）系统证明了 5 kDa PEO 缀合物作为互补 DNA 检测探针的能力。优化的 PEO 缀合和纯化方案既经济又简单，即使在不具备高度化学合成设备的实验室中也可以复制。
