A general synthetic approach for the synthesis of "Nand 170-doubly labelled pyrimidine nucleosides is described. The I5N isotopes in uridine and the I7O isotope in the urea-derived carbonyl group of uridine and cytidine originate from (15N2)['70]urea (5) which was synthesized from "NH,CI, thiophosgene (I), and H,['70]. The third "N isotope of cytidine in 4-position stems from the substitution of the 1,2,4-triazole moiety of ('SN3[02-170]uridine derivative 8a/b with "NH,OH. Hydrolysis of the same key intermediate 8a/b with Na[170]H/H2[170] introduced the second I7O isotope into the 4-position of uridine. The "Nand I70-NMR spectra of the target compounds 12 and 14 in phosphate-buffered H,O serve as references for heteronuclear NMR spectra of labelled RNA fragments.
The 5-methyl('5N2)[02,04-1702Juridine (= (15N2)[02,04-'70,]ribosylthymine; 15) was synthesized and analyzed by I5N-and 170-NMR spectroscopy. (I5N,)Urea was condensed with 2,3-dibromo-2-methylpropanoyl chloride (3) and cyclized to form ("N,)thymine (5) After glycosidation, the "0 isotopes were introduced in two separate steps: hydrolytic ring opening of 2,5'-anhydro derivative 9 and hydrolysis of 3-nitro-1H-l,2,4-triazole derivative 12 with labelled water in the presence of a strong base. The "N-and I70-NMR spectra (Fig.) of 15 in phosphate-buffered water serve as references for heteronuclear NMR spectra of labelled RNA fragments.The chemical shifts of both "N and I7O nuclei are known to be sensitive to the formation of H-bonds. When incorporated into the bases of RNA, these nuclei might serve as a monitoring device for folding and unfolding processes. Doubly labelled 5-methyluridine ( = ribosylthymine) was chosen for a first analysis by "N-and I70-NMR spectroscopy on the nucleoside level (see also following paper). Protection of the 2 -and 3'-OH groups of 7 (-+ 8) and cyclization of the 5'-OH group with one base carbonyl group yields 2,5'-anhydro derivative 9. The first I7O isotope is introduced at C(2) through the hydrolysis of 9 with a Na[170]H/H2['70] solution giving the doubly labelled nucleoside derivative 10 [3]. Compound 10 is monomethoxytritylated to 11. The introduction of the second l7O isotope at C(4) is accomplished in three in situ steps to avoid loss of material owing to unnecessary purification procedures. The introduction of a leaving group, 3-nitro-lH-l,2,4-triazole [4], proceeds efficiently (+ 12), as judged by TLC and 'H-NMR. Subsequent hydrolysis with less than 2 equiv. of H,[I70] yields compound 13. Detritylation furnishes isopropylidene derivative 14 in 68 YO overall yield over those steps. Final deprotection with CF,COOH affords the target compound 15.Nucleoside 15 ('rT) is characterized by mass, and I7O-and I5N-NMR spectroscopy (Fig.). Comparison of the mass spectra of nucleosides 7, 10, and 15 reveals that the isotope-substitution efficiencies are 92% (34 atom-% "0) for the first and 80% (29.7
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