Use of a Novel 5′‐Regioselective Phosphitylating Reagent for One‐Pot Synthesis of Nucleoside 5′‐Triphosphates from Unprotected Nucleosides
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- Table of Contents
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- Literature Cited
Abstract
5??Triphosphates are building blocks for enzymatic synthesis of DNA and RNA. This unit presents a protocol for convenient synthesis of 2??deoxyribo? and ribonucleoside 5??triphosphates (dNTPs and NTPs) from any natural or modified base. This one?pot synthesis can also be employed to prepare triphosphate analogs with a sulfur or selenium atom in place of a non?bridging oxygen atom of the ??phosphate. These S? or Se?modified dNTPs and NTPs can be used to prepare diastereomerically pure phosphorothioate or phosphoroselenoate nucleic acids. Even without extensive purification, the dNTPs or NTPs synthesized by this method are of high quality and can be used directly in DNA polymerization or RNA transcription. Synthesis and purification of the 5??triphosphates, as well as analysis and confirmation of natural and sulfur? or selenium?modified nucleic acids, are described in this protocol unit. Curr. Protoc. Nucleic Acid Chem. 52:1.30.1?1.30.21. © 2013 by John Wiley & Sons, Inc.
Keywords: nucleoside 5??triphosphate; sulfur modification; selenium modification; phosphorothioate; phosphoroselenoate; diastereomer
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PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Basic Protocol 1: One‐Pot Synthesis of Native Nucleoside 5′‐Triphosphates
- Basic Protocol 2: One‐Pot Synthesis of Nucleoside 5′‐(α‐P‐Thio)Triphosphates
- Basic Protocol 3: One‐Pot Synthesis of Nucleoside 5′‐(α‐P‐Seleno)Triphosphates
- Commentary
- Literature Cited
- Figures
- Tables
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Basic Protocol 1: One‐Pot Synthesis of Native Nucleoside 5′‐Triphosphates
Materials
Basic Protocol 2: One‐Pot Synthesis of Nucleoside 5′‐(α‐P‐Thio)Triphosphates
Materials
Basic Protocol 3: One‐Pot Synthesis of Nucleoside 5′‐(α‐P‐Seleno)Triphosphates
Materials
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Figure 1.30.1 General scheme for one‐pot synthesis of natural and sulfur‐ or selenium‐modified 5′‐triphosphates from unprotected nucleosides. Abbreviations: DMF, N , N ‐dimethylformamide; TBA, tributylamine. View Image -
Figure 1.30.2 Synthesis of native 2′‐deoxyribo‐ and ribonucleoside 5′‐triphosphates (8 O and 9 O ) utilizing iodine as the oxidizing agent. View Image -
Figure 1.30.3 RP‐HPLC profile of crude chemically synthesized thymidine 5′‐triphosphate (TTP, 8t O ). The crude sample can be analyzed on a Welchrom C18 (or Ultisil C18) reversed‐phase column (4.6 × 250 mm) measured at 260 nm at a flow of 1.0 mL/min using a linear gradient of 0% to 40% buffer B in 20 min. Buffer A: 20 mM triethylammonium acetate (TEAA, pH 7.1); buffer B: 50% acetonitrile in buffer A. Retention times for crude 5′‐TTP and 3′‐TTP after NaCl/ethanol precipitation = 19.4 and 20.7 min, respectively. 3′‐TTP was compared and characterized with a 3′‐TTP standard. View Image -
Figure 1.30.4 RP‐HPLC profiles of chemically synthesized and commercial TTP. (a ) Synthesized 5′‐dTTP after NaCl‐ethanol precipitation and RP‐HPLC purification (retention time: 19.8 min); (b ) 5′‐dTTP standard (retention time: 19.4 min); (c ) co‐injection of a and b (retention time: 19.4 min). View Image -
Figure 1.30.5 Synthesis of nucleoside 5′‐(α‐ P ‐thio)triphosphates utilizing sulfurizing reagent II as the oxidizing agent. View Image -
Figure 1.30.6 RP‐HPLC profiles of chemically synthesized uridine 5′‐(α‐ P ‐thio)triphosphate (UTPαS, 9u S ). (a ) Commercial UTP (retention time: 14.1 min); (b ) crude synthesized UTPαS following NaCl/ethanol precipitation, showing diastereomers (retention times: 17.5 and 18.3 min); (c ) HPLC‐purified diastereomer of UTPαS (peak I, S p isomer, retention time: 15.6 min); (d ) HPLC‐purified diastereomer of UTPαS (peak II, R p isomer, retention time: 16.6 min); (e ) co‐injection of resolved UTP and UTPαS peaks I and II (retention times: 14.2, 15.4, and 16.3 min, respectively). View Image -
Figure 1.30.7 High‐resolution HPLC profiles of commercial and synthesized 5′‐dGTPαS. (a ) Commercial 5′‐dGTPαS showing resolution of S p and R p diastereomers (retention times: 14.9 and 15.5 min, respectively); (b ) crude synthesized 5′‐dGTPαS following NaCl/ethanol precipitation, showing only the S p isomer (retention time: 14.7 min); (c ) co‐injection of a and b showing enhanced peak I over peak II (retention times: 14.8 and 15.3 min, respectively). View Image -
Figure 1.30.8 Synthesis of nucleoside 5′‐(α‐ P ‐seleno)triphosphates utilizing BTSe as the oxidizing agent. View Image -
Figure 1.30.9 RP‐HPLC profiles of commercial GTP and chemically synthesized guanosine 5′‐(α‐ P ‐seleno)triphosphates (GTPαSE, 9g Se ) showing resolution of the diastereomers. (a ) Synthesized GTPαSe (peak I, S p isomer, retention time: 11.5 min); (b ) synthesized GTPαSe (peak II, R p isomer, retention time: 12.1 min); (c ) co‐injection of a, b, and commercial GTP (retention time: 10.0 min). View Image -
Figure 1.30.10 Results of PS‐DNA synthesis from crude dNTPαS. (A ) Primer and template sequences used in polymerization. (B ) Primer extension reaction using chemically synthesized dNTPαS, commercial dNTPs, and Klenow fragment exo(−) (Kf−). Primer was 5′‐end‐labeled using [γ‐32 P]ATP and polynucleotide kinase. Polymerization reactions were carried out with primer (3.5 µM), template (5 µM), all dNTPs and dNTPαS (0.1 mM each, final concentration), and Kf− (0.015 µL per µL reaction) at 37°C for 1 hr. Reactions were analyzed by 19% polyacrylamide gel electrophoresis. Lanes: (1) 5′‐32 P‐labled primer; (2) primer and all dNTPs, but no Kf−; (3) positive control: primer, template, all commercial dNTPs, and Kf−; (4, 6, 8, 10) negative controls: same as lane 3 without dATP, dCTP, dGTP, and TTP, respectively; (5, 7, 9, 11) same as lanes 4, 6, 8, 10 with addition of crude synthesized dATPαS, dCTPαS, dGTPαS, and TTPαS (S‐dA, S‐dC, S‐dG, S‐T), respectively. View Image -
Figure 1.30.11 Results of PS‐RNA synthesis from crude NTPαS. (A ) Primer and template sequences used in transcription. (B ) Transcription reaction using chemically synthesized NTPαS, commercial NTPs, and T7 RNA polymerase. Transcribed RNAs were bodily‐labeled by [α‐32 P]ATP during transcription. Transcription reactions are performed with promoter strand and template (1.0 µM, each), all NTPs and NTPαS (1.0 mM each), and RNA polymerase (0.1 µL per µL reaction) at 37°C for 2 hr. Reactions were analyzed by 19% polyacrylamide gel electrophoresis. Lanes: (1) promoter strand (5′‐32 P‐labeled as marker); (2): promoter strand, template, [α‐32 P]ATP, and all NTPs without RNA polymerase; (3) promoter strand, template, [α‐32 P]ATP, and all NTPs with RNA polymerase; (4, 6, 8, 10) same as lane 3 without ATP, CTP, GTP, and UTP, respectively; (5, 7, 9, 11) same as lanes 4, 6, 8, 10 with addition of crude synthesized ATPαS, CTPαS, GTPαS, and UTPαS (S‐A, S‐C, S‐G, S‐U), respectively; (12) same as lane 9 using commercial GTPαS (S‐undefined). View Image
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