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Mar 26, 1991 - Rate limiting P-O(5') bond cleavage of RNA fragment: ab initio molecular orbital calculations on the base-catalyzed hydrolysis of phosphate.

Nucleic Acids Research, Vol. 19, No. 10 2747

.::j 1991 Oxford University Press

Rate limiting P-O(5') bond cleavage of RNA fragment: ab initio molecular orbital calculations on the base-catalyzed hydrolysis of phosphate Kazunari Taira, Tadafumi Uchimaru1, Kazutoshi Tanabel, Masami Uebayasi and Satoshi Nishikawa Fermentation Research Institute and 'National Chemical Laboratory for Industry, Agency of Industrial Science & Technology, MITI, Tsukuba Science City 305, Japan Received December 28, 1990; Revised and Accepted March 26, 1991

ABSTRACT In order to examine the energetics in base-catalyzed hydrolysis of RNA, a tentative pentacoordinated intermediate (1) has been characterized by molecular orbital calculations. Ab initio studies at the level of 3-21 G * indicate that, under the Cs symmetry restricted conditions, the P-0(2) bond possessing antiperiplanar (app) lone pair electrons (Ip) on the equatorial oxygen (0(3)) can be cleaved with almost no barrier (TS1 transition state; 0.08 kcal mol-1), from the pentacoordinated intermediate (3) of base-catalyzed hydrolysis of phosphate, compared to the P-0(5) bond (TS2 transition state; 28.9 kcal mol-1) which lacks app Ip assistance from 0(3). The dianionic intermediate, however, loses the f11 transition state thus its property as an intermediate when the Cs restriction is removed. The analysis of the entire potential energy surface enables us to conclude that, in a related system examined by Lim and Karplus ((1990) J. Am. Chem. Soc., 112, 5872 - 5873) for attack by OH - on ethylene phosphate monoanion, the TS1 transition state had also been lost and thus no intermediate had been found. These results further support our earlier conclusions (Taira et al. (1990) Protein Engineering, 3, 691 - 701) of rate limiting transition state possessing extended P-0(5') bond breaking character (the 7S2 transition state) in the base-catalyzed hydrolysis of RNA (see also references 6 and 7). Finally, although the lack of 2',3'-migration of phosphate moieties in basic condition appears to be in accord with the short-lived intermediate, it really does not prove the absence of the intermediate. The detail will be discussed in the text.

INTRODUCTION We have previously carried out ab initio molecular orbital calculations to characterize the metastable phosphoranes, whose properties are not easily elucidated experimentally (1). The RNA cleaving reaction proceeded via a cyclic pentacoordinated

intermediate (1-4) and the energy profile calculated with the minimal basis set indicated that, if the pentacoordinated intermediate were dianion, it is intrinsically more difficult to cleave the P-0(5') bond under base-catalyzed conditions, as shown in Figure 2(a). Under these conditions, even if 2'-hydroxide productively attacks phosphorus to form the metastable pentacoordinated intermediate (0, only a small fraction of the dianionic intermediate can actually complete the cleavage reaction of the P-0(5') bond under basic conditions (rapid equilibrium). Because of the cyclic nature of the intermediate, the P-0(2') bond is uniquely oriented with respect to the lone paired electrons (ip) on the equatorial 0(3') oxygens (see Figure 1 and also the general reaction at the bottom of Figure 2(a)). Moreover, the cyclic intermediate may have ring strain which can be relieved only by breaking the P-0(2') bond. Thus the cause of the energy difference in Figure 2(a) between P-0(2') versus P-0(5') bond cleavages could be explained by combination of stereoelectronic effects and ring strain. In order to eliminate or at least minimize the effect of the ring strain on the model compound (D) of the cyclic RNA intermediate (0), we have now carried out similar calculations with a larger basis set on a much simpler acyclic counterpart utilizing dianionic trihydroxyphosphorane (3) as an acyclic model compound. It is also noteworthy that Lim and Karplus independently worked on a similar dianionic cyclic phosphorane in which the axial methyl group of our model compound (2) is replaced by a hydrogen and they concluded that, when a higher basis set is utilized, no stable pentacoordinated intermediate can exist (5). Our present result is in accord with theirs, in that the acyclic intermediate (0) exists only under Cs constrained conditions. Moreover, we now find that with higher theory the first transition state of 2'-OH attack (TS1) is stabilized relative to the second 5'-OR expulsion step (TS2). Thus, pentacoordinated dianionic intermediates are very reactive if they exist (6,7), and in some cases they do not have a finite life-time. Whichever the case (stepwise or concerted reaction), the actual transition state Qf a base-catalyzed hydrolysis of RNA apparendy looks like TS2 of Figure 2(a) as expected from the condition of rapid equilibrium (1).

2748 Nucleic Acids Research, Vol. 19, No. 10

METHODS Ab initio calculations were carried out with Gaussian 86 program (8) on FACOM M780 computer. The reaction profile for the cleavage steps of axial hydroxyl groups of the trihydroxyphosphorane (i) was calculated with 3-21G* basis set and the results assuming Cs symmetry are shown in Figure 2(b). This reaction profile represents an attack of a hydroxide (0(2)H-) on phosphate monoanion, followed by a departure of a hydroxide (0(5)H-) without equatorial hydroxyl group (0(3A) rotation. The transition states for the cleavage of these two axial groups were located with Cs symmetry because; (1) computational time limits and (2) to evaluate, at a higher basis set, the magnitude of the stereoelectronic effect without invoking ring strain. Without the Cs restriction, the second transition state (TS2) would not be located since the equatorial hydroxyl group (0(3)H) could rotate to form the lower TSI transition state. The computer graphics of HOMOs and LUMOs of the model compound 3 were obtained by means of AVS Chemistry Viewer on TITAN 3000 at Kubota Computer, Inc.

RESULTS The Cs restricted reaction profile for the base-catalyzed hydrolysis of phosphate (3 in Figure 1) is calculated at the level of 3-21G* molecular orbital (MO) theory and the results are shown in Figure 2(b). The hydrolysis reaction proceeds via an intermediate 3, depicted in Figure 1. The total energies (a.u.) of the Cs intermediate and transition state structures (TSI and TS2) are -713.00214, -713.00201, and -712.95601, respectively. The imaginary frequencies relevant to the axial P-O(2) and P-0(5) bond cleavages are 170.4i and 1 10.7i cm-1, respectively. The motion of the atoms in the normal modes corresponding to these imaginary frequencies firmly connects the reactants and the products. In the Figure 1 and also in the following discussion we intentionally use valence bond terminology within MO framework because most of the researchers in the nucleic acids field, we believe, are more familiar with the valence bond pictures. Therefore, sp3 hybridized lone pair electrons (lp) on the 3'-oxygens are included in Figure 1. In the STO-3G calculation of the cyclic model compound 2, the ribose moiety of the RNA intermediate 1 is omitted (Figure 2(a)) (1). With respect to the orientation of lp on the equatorial 3'-oxygen, the 0(2) of the acyclic model compound 3 is equivalent to the 2'-oxygen of 1 and consequently to the 0(2) of 2. Similarly, the 0(5) of 3 represents 5'-oxygen. Although the orientation of 0(2)H in 3 differs from that of the corresponding O(2)-methylene bond in 2, since we are interested in pinpointing the cause of the energy difference between the P-0(2) and the P-O(5) bond cleavages as depicted in Figure 2(a), we intentionally avoided potential steric interaction between the hydrogens on °2 and 03 of the model compound 3. The orientation of the axial O(2)-H bond is expected to have little influence on the overall energy. The reaction profile of Figure 2(b) indicates that the axial hydroxyl group (0(2)H) located on the same side of the hydrogen of the equatorial hydroxyl group (0(3)H) (cisconformation) is cleaved much readily (TS] transition state) than the opposite side hydroxyl group (0(5)H) (trans-conformation; TS2 transition state). In fact, there is almost no barrier for the P-0(2) bond cleavage (see the expanded region of the insert in Figure




barrier heights


for the




0.08 (TSI) and 28.9 kcal/mol (TS2),




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