A Mechanistic Study in Methanol: Cleavage of RNA Models and Highly Stable Phosphodiesters with Dinuclear Zn(II) Complexes

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Melnychuk, Stephanie
phosphodiesters , methanol , dinuclear metal catalysis
Phosphoryl transfer reactions are vital to life. In response to the slow intrinsic rates of phosphoryl transfer, Nature has evolved a series of enzymes designed to accelerate these reactions and allow them to occur at biologically relevant rates. These metallo-enzymes are largely characterized by bi- or tri-nuclear active sites with effective dielectric constants that more closely resemble those of organic solvents than water. This project was designed to better understand the mechanisms by which metallo-enzymes cleave phosphodiesters with poor leaving groups. The stability of the phosphodiester is central to the storage of genetic information in DNA and RNA. The cleavage of a series of more reactive RNA models, 2-hydroxylpropyl aryl phosphates 1a-g, catalyzed by a dinuclear Zn(II)2 complex of 53 in methanol was explored. A solution of 53:Zn(II)2:(-OCH3) was observed to accelerate the decomposition of 1a-g with rates that were 10^11-10^12-fold greater than the methoxidepromoted reaction at ss pH 9.47, approaching rate accelerations achieved by natural enzymes. The remarkable activity of 53:Zn(II)2:(-OCH3) and 36:Zn(II)2:(-OCH3) towards the cleavage of 1a-g probed the study of the decomposition of diribonucleotides(3'->€™ 5')UpU and (3'->€™ 5'€™)ApC in methanol. The 53:Zn(II)2:(-OCH3)- and 36:Zn(II)2:(-OCH3)-catalyzed decomposition of UpU achieved k2 values of 1.21 ± 0.17 and (7.04 ± 0.99) x 10^-2 M^-1s^-1. The reactivity of ApC in the presence of these systems was unimpressive, however Zn(II) ions in ethanol resulted in the isomerization of 3'->€™ 5'€™)ApC to (2'->™ 5'€™)ApC providing support for the existence of a pentacoordinate phosphorane intermediate. The pentacoordinate phosphorane was further explored through the reaction of 36:Zn(II)2:(-OCH3) with the cyclic phosphate 58 and 2-hydroxylpropyl methyl phosphate (59). In the presence of 36:Zn(II)2:(-OCH3) the rate of isomerization of 59/59a (kobs = (4.7 ± 0.5) x 10^-3 s^-1) exceeded that of expulsion of the methoxy group (kobs = 1.62 x 10^-3 s^-1), thus confirming the existence of a pentacoordinate phosphorane intermediate (60)and providing support for a two-step phosphodiester cleavage reaction. The catalytic efficiency of 36:Zn(II)2:(-OCH3) towards the cleavage of stable phosphodiesters probed its application towards the decomposition of dimethyl phosphate (2) in methanol-d4. The exchange of OCH3 for OCD3 occurred with kcatmax = (2.27 ± 0.03) x 10^-6 s^-1.
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