Coupling strategies for the synthesis of Peptide-oligonucleotide conjugates for patterned synthetic biomineralization.
Coupling strategies for the synthesis of Peptide-oligonucleotide conjugates for patterned synthetic biomineralization.
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DOI：
10.4061/2011/926595
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发表时间：
2011
影响因子：
2.3
通讯作者：
Labean TH
中科院分区：
文献类型：
--
作者：
Carter JD;Labean TH
这项工作描述了将DNA寡核苷酸的自组装行为与多肽的分子识别能力相结合的多肽-寡核苷酸结合物的制备策略。这些合成包括固相片段偶联反应和固相片段偶联策略，其中寡核苷酸被固定在DEAE Sepharose.评价了四种偶联剂的产率，两种试剂在水中，1-乙基-3-(3-二甲氨基丙基)碳二亚胺盐酸盐和二甲基亚胺(4-(4，6-dimethoxy[1，3，5]triazin-2-yl)-4-methyl-morpholinium氯化物中，两种在二甲基甲酰胺、PYBOP(苯并三唑-1-氧基)三吡咯烷六氟磷酸盐和HBTU(邻苯并三氮唑-N，N，N‘，N’-四甲基铀酸六氟磷酸盐)中，寡核苷酸片段要么在溶液中，要么固定在DEAE上。这些偶联策略依赖于与肽C末端反应的寡核苷酸上无保护的5‘氨基连接物。该多肽是从一个组合文库中挑选出来的，具有与金结合的行为，长12个氨基酸，带有一个N-末端的乙酰帽。凝胶电泳法和质谱法证实了偶联物的形成，原子力显微镜证实了多肽部分的分子识别功能。溶液相产率优于固相得率。EDC的固相法和固相法的产率最高，分别为95%和24%，而以DMF为基础的试剂PyBOP和HBTU的产率较低，回收率较低。所有可回收的偶联物都显示了金纳米颗粒的模板能力。
This work describes preparation strategies for peptide-oligonucleotide conjugates that combine the self-assembling behavior of DNA oligonucleotides with the molecular recognition capabilities of peptides. The syntheses include a solution-phase fragment coupling reaction and a solid-phase fragment coupling strategy where the oligonucleotide has been immobilized on DEAE Sepharose. The yield of four coupling reagents is evaluated, two reagents in water, EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and DMTMM (4-(4,6-dimethoxy[1,3,5]triazin-2-yl)-4-methyl-morpholinium chloride), and two in dimethylformamide (DMF), PyBOP ((Benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate) and HBTU (O-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate), while the oligonucleotide fragment is either in solution or immobilized on DEAE. These coupling strategies rely on an unprotected 5′ amino linker on the oligonucleotide reacting with the peptide C-terminus. The peptide, selected from a combinatorial library for its gold-binding behavior, was 12 amino acids long with an N-terminus acetyl cap. Formation of the conjugates was confirmed by gel electrophoresis and mass spectrometry while molecular recognition functionality of the peptide portion was verified using atomic force microscopy. Solution-phase yields were superior to their solid-phase counterparts. EDC resulted in the highest yield for both solution-phase (95%) and solid-phase strategies (24%), while the DMF-based reagents, PyBOP and HBTU, resulted in low yields with reduced recovery. All recoverable conjugates demonstrated gold nanoparticle templating capability.