TRITIUM LABELLING OF DNA & RNA BY CHEMICAL & ENZYMATIC SYNTHESIS

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Tritiated ATP has been used as an essential component of several studies at the NTLF over the past few years. In initial studies [2-3H]-ATP of high specific activity was synthesized from 2-Bromo-ATP by heterogeneous tritiodehalogenation using tritium gas and PdO in aqueous solution. Availability of this labelled material allowed development of an enzymatic synthesis of highly tritiated RNA. This overall approach will be repeated and improved. Several synthetic efforts are underway to support the tritiated ATP project. Large quantities of 2-bromoadenosine have been prepared, ready for conversion to 2-bromo-ATP and subsequent use in RNA synthesis. Specific improvements over the past year have included the synthesis of 2-iodoadenosine as an alternative to 2-bromoadenosine. The two critical products share common TITLE: Tritium Labelling of DNA and RNA by Chemical and Enzymatic Synthesis (Continued) chemistry for all the previous synthetic steps. It remains to be seen whether the 2-iodoadenosine structure will survive the phosphorylation chemistry. Nevertheless, this precursor proved very useful for the labelling of [2-3H]-adenosine at high specific activity, for use in an adenosyl-cobalamin project. Similar chemistry was used recently to facilitate the production of [2-3H]-Deoxyadenosine for derivatization to the phosphoramidite, and subsequent incorporation into a DNA fragment. This overall process will also be repeated and improved. A particular improvement in the DNA sub-project involves moving away from chemical synthesis, and we will pursue the enzymatic synthesis of tritiated DNA by incorporation of tritiated dATP. To aid this approach we have embarked on a large scale synthesis of 2-Br-dATP to be used in an analogous tritiation reaction to 2-Br-ATP for RNA synthesis. We are also looking into synthesis of the analogous 2-iododeoxyadenosine compound, and its subsequent phosphorylation. The ability to synthesize specifically labelled RNA and DNA molecules will allow some very specific macromolecular NMR experiments to be conducted, addressing the interactions between DNA, proteins, RNA and other ligands. The importance of ribo- and deoxyribonucleoside-5'-triphosphates in biological systems as building units for RNA and DNA, have led to investigation for the development of a number of methods for their synthetic phosphorylation. Most of these methods involve the synthesis of the 5'-monophosphate of the nucleoside and its conversion to the triphosphate through a reactive intermediate or a barium salt derivative in two or more steps. Due to these lengthy, low yield and tedious methods a number of research groups have been exploring a one-pot, short and efficient synthetic method. Recently, a new method was reported for the conversion of nucleosides to nucleoside-5'-triphosphates in a convenient and quick route at relatively large scale, in which none of the functional groups sensitive to phosphorylation such as N-NH2 or secondary -OH need to be protected. This method appeared to be useful for the synthesis of a number of halogenated nucleosides as precursors for tritiation reactions in our RNA/DNA project. Through exploratory experiments we modified the method to work at mini-scale with adenosine and 2-deoxyadenosine for one-pot synthesis of ATP and dATP in good yield. A particularly important development was an HPLC method to monitor the monophosphorylation and triphosphorylation. Large differences in the rate of pyrophosphorylation were observed when comparing halogenated and non-halogenated bases, and between ribo and deoxyribomonophosphates. After purification, the nucleotides were converted to the tetrasodium salt and were finally analyzed by HPLC, 1H and 31P NMR, and mass spectrometry. The general procedure is as follows: Nucleoside (0.1-0.2 mmole) was pre-dried in a vacuum oven, dissolved in dried trimethyl phosphate, and the solution was kept at 0oC for 5 min. Freshly distilled phosphorus oxychloride was then added to the reaction vessel and the mixture was stirred at 0oC for 3 hrs. A solution of tributylammonium pyrophosphate in anhydrous DMF was separately made and kept at 0oC, after which dried tributylamine was added. This solution was combined with the nucleoside solution and was stirred for 1 min at 0oC. The reaction was then quenched with tributylammonium bicarbonate buffer and was stirred at 0oC for 3-6 hr. All solvents and volatiles were then removed under nitrogen gas to give a dry residue. The crude reaction was purified over DEAE Sephadex-25 using a linear gradient of 0 to 1M TEAB buffer. The appropriate fractions that showed a UV absorption at 260 nm for the triphosphate derivative were identified and combined for HPLC analysis of the nucleoside triphosphate. After the analysis, the residue was dried under nitrogen gas, and using a solution of anhydrous sodium perchlorate the tetrasodium salt was prepared for final HPLC, and 1H and 31P NMR analysis. We are in the process of applying this method to the synthesis of 2-halogenated nucleosides (2-bromoadenosine, 2-bromodeoxyadenosine and 2-iodoadenosine) and preparing the corresponding triphosphates as precursors for final heterogeneous catalytic dehalogenation with tritium gas to synthesize [2-3H]ATP and [2-3H]-dATP.