Synthesis and ¹⁵N‐ and ¹⁷O‐NMR Spectra of 5‐Methyl(¹⁵N₂)[O²,O⁴‐¹⁷O₂]uridine (= (¹⁵N₂)[O²,O⁴‐¹⁷O₂]Ribosylthymine)

Abstract

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

Chemical Synthesis of 13C and 15N Labeled Nucleosides

“…If 17 O labeling is requested, the oxygen label at 2-CO can be introduced by carrying out the condensation of malic acid with 17 O labeled urea. Vorbruggen condensation followed by introduction of a good leaving group (tetrazole, nitrotriazole) at C-4 allows one to synthesize 2-17 O labeled cytosine 45 as well as 2,4-17 O uridine 92 , depending whether 15 NH 3 or 17 OH 2 is used in the substitution reaction.…”

Synthesis of (15N2)[17O]Urea, (15N2)[O2, O4‐17O2]Uridine, and (15N3)[O2‐17O]Cytidine

“…Na2S04 was added and stirring continued for another 30 min at r.t. The suspension was filtered, the precipitates washed with Et20 (5 x 20 ml), and the filtrate carefully evaporated ( (6). A mechanically stirred mixture of 5 (2.895 g, 45.97 mmol), polyphosphoric acid (80 g) and freshly distilled propynoic acid (3.1 ml, 3.53 g. 50.4 mmol) was heated to 85".…”

“…Activation of unlabelled 7, i.e., u-7, with 3-nitro-1H-l,2,4-triazole and diphenyl phosphorochloridate, a method that was so efficient for the corresponding activation of thymine derivatives [6] [ 151, merely resulted in the formation of (2',3',5'-tri-O-benzoyluridin-04-yl) 3-nitro-lH-1,2,4-triazolyl phenyl phosphate (30% yield), a dead-end product with respect to further substitutions at 04. In contrast, the relatively stable crystalline key intermediate u-8a/b was obtained from u-7 in 94% yield using phosphoryltris(lH-l,2,4-triazole) as activator [16].…”

“…During our attempts to hydrolyze u-8a/b with a minimal amount of water, we observed once more a marked difference between the reactivity of an activated thymine derivative (a 4-dehydroxy-5-methy1-4-(3-nitro-lH-1,2,4-triazolyI)uridine derivative [6])…”