Tetrahedron Vol. 48. No. 4. pp. 695-718. 1992 Rited in Great Britain omo-4020192 $3.00+.00 @ 1992 Pexgamon Press plc Assessment of Competing 2’--+5’ versus 3’-+5’ Stackings in Solution Structure of Branched-RNA by lH- & 31P-NMR Spectroscopy Christian Sund, Peter Agback, Leo H. Koole, Anders Sandstriim & Jyoti Chattopadhyaya* Jkpartmenfof Bioorganic Chemistry, Box 581, Biomedical Center, University of Uppsala, S-751 23 Uppsala, Sweden (Received in UK 18 November 1991) Abstract: Preparation offive novel phosphorylaled derivatives of adenosine. i.e. adenosine 2’.3’-bis(elhylphosphale) (II). adenosine 2’3’-bis(phosphale) (13). adenosine 2’.3’.S’-rris(erhylphosphare) (15). adenosine 2’,.5’- bis(ethylphosphate) (17), and adenosine 3’5’bis(erhylphosphare) (19) is reported. These compounds, along wirh methyl B-D-ribofuranosyl-bis-2’,3’-ethylphosphate (9). were used as reference sysrems for 31P and IH-NMR conformational studies on rhe branched RNA structures 20 - 30. Cornpout& II, 13,15,17. and 19 preserve rhe essential srructural elements of the branch point adenosine. while the in@ amolecular base sracking interacdons are removed. The 31P-NMR chemical shi/s of 20 - 30, referenced against 9, II, 13. or 15. show a pattern that is generally con&rent with our previous resultsfrom variable temperature 31P-NMR experiments. The &a indicate that the contribution of gg around the P-03’(<) and P-OS’ (a) bonds is significantly greater for the 2*-phosphate group than for the 3’-phosphategroup. These results point towards preferential 2’4 rather than 3’4’ base stackedstructuresin all of these syntheticmodels of the lariat. This is especially the casefor the branched trimer20 and the peniamer 27 which are pan of a naturallyoccurring lariw strucuue. Note that the srrongest2’-ts’ stacking is however found in the unnatural trimers 22 and 23 in which the 2’4inked residue is a pyrimidine nucleodde. Compounds II, 13. and 15 were also used 10 calibrate the ‘H-NMR oligomerizarionshifis of ihe H2 prorons of the branch-point adenosine. These data show a consistency withthe results from variable temperature ‘H-NMR experiments, as well as withthe results of 31P-NMR experiments. The results obtained withthe series of compounds 20 (A~p~!$ ) 26a (UA2’p5’G 3.p5.U). 27 (A?$$), 28 (CL’Ayp;$, 29 (CVA2j$-U;;). and 30 (CCUA2$;;fUj;) are Of special interest since these sIrucuues are constituents of rhe narurally occurring lariat in the excised inlron in Group II splicing of bl I of Yeast mitochondria. Qualitatively, the present IH- and 31P-NMR data on 26a, 27,28,29, and 30 show 2’+5’ base stacking of an ituermediale strength: 2’4’ base slacking is substanrially strongerfor trimer 20 and penmner 27. This difference is ascribed to Ihe S-conformadonal transmissioneffect owing 10 the presence of a1 least one nucleodde upstream of rhe branch-point. S-Conformational transmission appears IO weaken the 2’-*5’ stacking at the expense of some 3’+5’ stacking. The experimental data on the conformadon of 20 (A yp$ r3’P and ‘H chemical shifts, vicinal ‘H-IH. IH- 31P, and 13C31P coupling constants) formed the basis for a series of AMBER molecular mechanics calculations.These molecular modellingstudies allowedus to conch& thatgg conformation in the I’-phosphate group is primarily g‘([),g- (a). This isfound LO be the only conformationthat gives 2’4 base stackingas evidenr in rhe temperature &pet&n1 chemical shif and in the oligomerization shift smdies. Modelling studiesjiuthermore showed hv~ energetically possible Cand a torsions for the J ’-phosphate group (g-(&g-(u) and g-(&(a)). The present use of reference compounds 9, II, 13. IS, 17, and 19 has led to a refined and partially revised concept for rhe conformational description of oligomeric branched RNAs. The tertiary structure of nucleic acids is stabilized to a large extent by vertical base-base interactions, usually referred to as base stacking.’ Base stacking probably originates from dipole-dipole interactions 695