Comparative assessment of structure and reactivity of wyosine by chemistry, spectroscopy and ab initio calculations

Abstract

The hypermodified fluorescent Y-nucleosides (Wyosine, Ia) and its 7-substituted congeners occur naturally adjacent to the 3′-end of the anticodon loop in yeast phenylalanine transfer RNA (tRNA Phe). The biological importance of Wyosine is emphasised by the fact that the removal of the aglycone (Y- or wye-base) from Y-nucleoside in tRNA Phe by mild acidic treatment causes the loss of its codon recognition property required for protein biosynthesis. In this work, we have performed detailed ab initio calculations on several isomerically methylated Y-base analogs modelling the unique N4-methylation site of Y-base and the N5-methylated site of the N4-desmethyl counterpart. These results have been compared with our earlier spectroscopic and chemical studies on Wyosine ( 1a) (refs. 1,3), which can be summarized as follows: (i) HF/6-31G ** calculations on model compounds 4 and 5 show that the former is preferred by 12.8 ( vacuo), 10.3 (ε =8.9) and 9.9 (ε = 78.3) kcal/mol which is in excellent agreement with our facile Lewis acid promoted isomerization of 1b (RH, R′Ac) into its thermodynamically preferred N 1 isomer ( 2) (ref. 1) (ii) N3,N5-dimethyl derivative 6 exhibits an energetic preference (HF/6-31G ** level) of 12.0 kcal/mol over the N3,N4-isomer 5, which is consistent with the chemical methylation studies of 4-desmethylwyosine ( 3a) which gives N5-methyl-4-desmethylwyosine ( 3b) as the dominant product whereas wyosine ( 1b) is formed only in 3% yield (ref 2). (iii) Relative HF/6-31G ** energies show that the first protonation site is N5 for both N3,N4-dimethyl analog 8 and its N1,N4 isomer 10 (N5-H + species is preferred by 1.2 - 3.0 and 9.3 - 16.4 kcal/mol over N1-H + counterparts 9 and 11), which is in excellent agreement with our 15N-NMR titration studies with CF 3COOH (ref. 1). (iv) The examination of N 1-H ( 14)⇌ N 3-H ( 15) tautomerism has shown that N 1-H tautomer 14 is energetically preferred by 5.8 to 8.8 kcal/mol which is consistent with our studies based on 13C-NMR (ref. 4) and fluorescence decay time measurements (ref. 6) of wye-base ( 14). These studies suggest that the wyosine is a derivative of the thermodynamically unfavoured N 3-H tautomer ( 15). The biological consequence of the thermodynamically unstable natural wyosine is the extreme lability of its glycosyl bond under mild acidic conditions, which thus constitutes a switch for the deactivation of the codon function of tRNA Phe. This finding of the inherent thermodynamic unstablility of natural wyosine also suggests that guanosine is most probably the building block for the biosynthesis of wyosine.