J. CHEM. SOC. PERKIN TRANS. 2 1994 327 Models for Enzyme-catalysed Phosphate Transfer: Comparisons of Reactivity towards a Neighbouring Hydroxy Group for Phosphodiester Anions and Acids. General Base Catalysis of the Cyclisation of a Hydroxyalkyl Phosphate Triester Anthony J. Chandler,8 Florian Hollfelder,a Anthony J. Kirby,*Sa Fiona O'Carroll and Roger Stromberg *pb a University Chemical Laboratory, Cambridge, UK CB2 IEW Sweden Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S- 106 9 1 Stockholm, Methylation of phenyl 1,2-isopropy~idene-~-~-xy~ofuranose 3'-phosphate increases the rate of intramolecular cyclisation by a factor of over lo5. This confirms previous estimates of the effect of protonation on the reactivity of phosphate diesters towards a neighbouring hydroxy group, which depend on the correct assignment of kinetically equivalent mechanisms, and makes available reliable data on the magnitude of the effect for reactions catalysed by a range of general acids and bases. General base catalysis is characterised for the intramolecular cyclisation of one diastereoisomer (7b) of methyl phenyl 12- isopropyl idene-P-~-xylofuranose3'-phosphate triester: the Brsnsted P is 0.65 and catalysis is enhanced by the proximity of the positive centres of suitable diamine monocations. Breslow'.2 has suggested that the initial step of the ribo- nuclease-catalysed hydrolysis of RNA may involve attack by the 2'-hydroxy group on the protonated phosphate diester group, with assistance by the imidazole group of an active-site histidine acting as a general base. RO, RO, Im* ?--oxB + ROH O x 0 0, OH 7\30 0-0 cz-o%B Ro OH There is no doubt that protonation of the exceedingly unreactive phosphate diester anion will increase its reactivity towards nucleophilic attack, but it is not a simple matter to quantify the rate factor involved. In recent work3 on the hydrolysis of a methyl phosphate diester 1 undergoing efficient intramolecular nucleophilic attack by phenolate oxygen, we identified separate reactions of the monoanion 1 and the dianion 2 to which we assigned similar mechanisms, involving nucleophilic attack by the phenolate anion on the neutral phosphoric acid and the phosphate anion. The data thus allowed very precise estimates of the rate enhancement due to protonation of the diester anion, of 5.5 x lo' for the uncatalysed reaction (HA = H20), and 8.1 x lo4 for the reaction catalysed by the imidazolium cation. These figures compare well with a similar estimate available from results for a system more closely related to the nucleotide s t r u c t ~ r e , ~ but are much larger than those suggested by earlier work,' discussed below, on the relative reactivity of similar di- and tri- esters. Furthermore, as is always the case when kinetically equivalent mechanisms differ only in the position of a proton in the transition state, the new estimates depend absolutely on the correctness of the assignment of mechanism. The kinetic ambiguity disappears if the proton is replaced by an alkyl group, so we have compared the rate of the intramolecular cyclisation of a model hydroxyalkyl phosphate diester anion with that for the corresponding methyl phosphate triester, where the proton of the diester acid is replaced by a methyl group. The study also allows us to characterise general base HA OMe 2 catalysis of the reaction of the triester, and thus, in principle, of the diester acid. The earlier work ' showed that the relative rates of reaction with external nucleophiles of triesters and the corresponding diesters depend strongly on the charge on the nucleophile. For the displacement of 2,4-dinitrophenolate from the phosphate phosphorus of the esters 3 and 4 the ratio is less than 100 for neutral nucleophiles (26 for water, 2-40 for pyridines), but several thousand for anions (4000 for phosphate, nearly 5000 for fluoride).' This suggested that the electrostatic repulsion Ar = 2,4dinitrophenyl 4 involved in the anion-anion reaction is at least as important as the difference in the intrinsic electrophilicity of the two phosphorus centres. However, phosphate-transfer reactions which do not depend on metal ions generally involve general base catalysed attack by hydroxy oxygen. This makes the degree of charge development at the attacking oxygen, and thus the potential electrostatic repulsion for attack on the