A fluorescent component adjacent to the 3Ј-end of the anticodon of torula yeast tRNA Phe was isolated as the nucleoside wyosine, 1) and the structure of its base wye has been determined to be 1.1b,2) It has been proposed by comparison of the chemical properties and UV spectra of wyosine with those of model compounds that 3-b-D-ribofuranosylwye (2) is the most probable structure for this nucleoside.1b) However, Reese and Whittall have claimed that wyosine is unlikely to be a ribonucleoside on the basis of the rate studies on the hydrolysis of model compounds.3) As wyosine was isolated from the tRNA in only a minute quantity (0.13 A 260 unit 1b) ; estimated to be ca. 8 mg), precise identification of the position of glycosylation and the structure of the sugar moiety has to rest on chemical synthesis. We and other groups have already synthesized the target molecule 2.4) However, lack of a sample of wyosine has hampered its structural determination. As the exceptional susceptibility to hydrolysis of wyosine 1a) had been anticipated to aggravate the difficulties of its isolation, we carried out rate studies 4c,5) on the hydrolysis of 2, disclosing that 2 can be handled at pH 5-10 at 37°C without suffering a heavy loss in quantity. Being encouraged by this knowledge, we started the present investigation. A preliminary communication of a part of this work has been published. 6) In order to avoid difficulties presented by the instability and the poor solubility of 2 in an organic solvent such as CDCl 3 , we planned to identify the nucleoside through its more stable 7) 2Ј,3Ј,5Ј-O-triacetate. We preferred triacetyl-d 9 -2 (3) to triacetyl-2 in order to rule out a slim possibility that wyosine is an acetylated 2 by nature. An authentic sample of 3 was prepared in 93% yield by treatment of 2 4c) with Ac 2 Od 6 in pyridine at room temperature for 4 h. At the outset of this work we wished to determine a minimum amount of 3 required for obtaining a satisfactory 1 H-NMR spectrum by routine measurement with a 500 MHz instrument and found that 50 mg was enough to locate every proton of 3. Pretreatment of the solvent CDCl 3 with aluminum oxide was of prime importance for getting a good spectrum, otherwise poor reproducibility of the chemical shifts was obtained probably owing to a trace of DCl contaminated.Takemura's group obtained a mixture containing wyosine 5Ј-mononucleotide by digestion of the wyosine-containing hexanucleotide, which was obtained from torula yeast tRNAPhe by treatment with RNase T 1 /RNase A, with snake venom phosphodiesterase after removal of the 3Ј-terminal phosphate with alkaline phosphatase; the mixture was dephosphorylated with alkaline phosphatase, and then chromatographed two dimensionally on a cellulose plate.1) Later on, McCloskey's group isolated a minor nucleoside from unfractionated archaebacterial tRNA's by digestion using nuclease P 1 and alkaline phosphatase followed by reversed-phase HPLC, and they proposed its structure to be 7-methyl-2.
8)This procedure appeared more convenient to us. However...
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