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{"title":"3′-O-Caged 2′-Deoxynucleoside Triphosphates for Light-Mediated, Enzyme-Catalyzed, Template-Independent DNA Synthesis","authors":"Anu Stella Mathews, Haikang Yang, Carlo Montemagno","doi":"10.1002/cpnc.41","DOIUrl":null,"url":null,"abstract":"<p>Synthesis, purification, and characterization of 3′-<i>O</i>-caged 2′-deoxyribonucleoside triphosphates (dNTPs), namely 3′-<i>O</i>-(2-nitrobenzyl)-2′-deoxy ribonucleoside triphosphates (NB-dNTPs) and 3′-<i>O</i>-(4,5-dimethoxy-2-nitrobenzyl)-2′-deoxy ribonucleoside triphosphates (DMNB-dNTPs), are discussed in detail. A total of eight 3′-<i>O</i>-caged dNTPs are synthesized with specific protocols depending on the nitrogenous base on the first carbon, i.e., adenine, guanine, thymine, and cytosine, as well as the photo-cleavable group, i.e, 2-nitrobenzyl and 4,5- dimethoxy-2-nitrobenzyl, to be attached in the 3′-<i>O</i> position. The purification of the synthesized compounds is done using ion-exchange and flash chromatography; this is followed by structural confirmation by nuclear magnetic resonance (NMR) and mass spectroscopy (MS). The efficiency of the designed compounds is tested by conducting and evaluating UV-cleaving experiments at 365 nm with proton NMR and LC-MS curves. Finally, the application of the 3′-<i>O</i>-cagged dNTPs in template-independent, enzyme-catalyzed, photo-mediated oligonucleotide synthesis is demonstrated. © 2017 by John Wiley & Sons, Inc.</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.41","citationCount":"10","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current Protocols in Nucleic Acid Chemistry","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cpnc.41","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"Chemistry","Score":null,"Total":0}
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Abstract
Synthesis, purification, and characterization of 3′-O -caged 2′-deoxyribonucleoside triphosphates (dNTPs), namely 3′-O -(2-nitrobenzyl)-2′-deoxy ribonucleoside triphosphates (NB-dNTPs) and 3′-O -(4,5-dimethoxy-2-nitrobenzyl)-2′-deoxy ribonucleoside triphosphates (DMNB-dNTPs), are discussed in detail. A total of eight 3′-O -caged dNTPs are synthesized with specific protocols depending on the nitrogenous base on the first carbon, i.e., adenine, guanine, thymine, and cytosine, as well as the photo-cleavable group, i.e, 2-nitrobenzyl and 4,5- dimethoxy-2-nitrobenzyl, to be attached in the 3′-O position. The purification of the synthesized compounds is done using ion-exchange and flash chromatography; this is followed by structural confirmation by nuclear magnetic resonance (NMR) and mass spectroscopy (MS). The efficiency of the designed compounds is tested by conducting and evaluating UV-cleaving experiments at 365 nm with proton NMR and LC-MS curves. Finally, the application of the 3′-O -cagged dNTPs in template-independent, enzyme-catalyzed, photo-mediated oligonucleotide synthesis is demonstrated. © 2017 by John Wiley & Sons, Inc.
3 ' -O-Caged 2 ' -脱氧核苷三磷酸光介导,酶催化,模板非依赖性DNA合成
详细讨论了3 ' - o笼型2 ' -脱氧核糖核苷三磷酸(dNTPs),即3 ' - o -(2-硝基苄基)-2 ' -脱氧核糖核苷三磷酸(NB-dNTPs)和3 ' - o -(4,5-二甲氧基-2-硝基苄基)-2 ' -脱氧核糖核苷三磷酸(DMNB-dNTPs)的合成、纯化和表征。根据第一个碳上的含氮碱基,即腺嘌呤、鸟嘌呤、胸腺嘧啶和胞嘧啶,以及附着在3′- o位置的光可切割基团,即2-硝基苯基和4,5-二甲氧基-2-硝基苯基,以特定的方案合成了总共8个3′- o笼型dNTPs。合成化合物的纯化采用离子交换和闪蒸色谱法;其次是核磁共振(NMR)和质谱(MS)的结构确认。通过质子核磁共振和LC-MS曲线在365 nm处进行紫外切割实验,并对所设计化合物的效率进行了测试。最后,展示了3 ' - o- cage dNTPs在模板不依赖、酶催化、光介导的寡核苷酸合成中的应用。©2017 by John Wiley &儿子,Inc。
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