R. Stan Brown Department of Chemistry Queen’s University Kingston, Ontario Canada, K7L 3N6

1. Dinuclear Zn2+ Catalysts as Biomimics of RNA and DNA Phosphoryl Transfer Enzymes: Changing the Medium From Water to Alcohol Provides Large Rate Enhancements. If You Want Fast Reactions, the Medium is the Message R. Stan Brown Department of Chemistry Queen’s University Kingston, Ontario Canada, K7L 3N6 Acknowledgements: Canada Foundation for Innovation, NSERC, United States Army Research Office, DTRA, Canada Council for the Arts and Queen’s University

2. Introduction • Responsible for the storage of genetic information in all living systems • Participation of the intramolecular OH in RNA provides large rate acceleration that is not seen in cleavage of DNA. • t1/2 for RNA cleavage is ~110 years without catalyst at neutral pH while that for DNA is estimated at about 108 to 1010 years.

3. Metal Containing Phosphodiesterases (catalyze cleavage of phosphate diesters) • (RO)PO2-(OR’) + H2O  (RO)PO3= + HOR’ • Phospholipase C • P1 nuclease

4. Simplified models for phosphoryl transfer enzymes This is a common motif for the dinuclear enzymes where the catalyst provides Lewis activation of the substrate, and a metal-bound OH that acts as a nucleophile or a base. • Numerous researchers have looked at modeling the activity with simple mono- or dinuclear complexes . • Where comparison is made, dinuclear complexes are better than mononuclear complexes. • None of these is very good at catalyzing the reactions in water. • In fact, most of these are not much better than HO- in promoting the reaction, and several are worse. • Notable exceptions: J. Chin, N. Williams, J. Richard and J.Morrow.

5. One important exception Williams et al Angew. Chem. 2006 kcat /Km = 53 M-1s-1 ; kOH = 0.065 M-1s-1 ; catalyst is ~800 times better than HO-

6. Quote: “ Enzyme active sites are non-aqueous, and the effective dielectric constants resemble those in organic solvents rather than that in water”. (= H2O, 78; MeOH, 32.7; EtOH 24.6) Cleland, Frey, Gerlt J. Biol. Chem. 1998, 273, 25529.

7. This work • Since the reactivity of most of the available dinuclear complexes (investigated in water) is not very effective when compared with HO-, we investigated light alcohols like methanol and ethanol as solvents for the reactions; • requires controlling and measuring ‘pH’ in alcohol; • this is as simple as in water: take meter reading and add 2.24 for methanol or 2.54 for ethanol; • pKauto methanol = 16.77, neutral pH = 8.38; • pKauto ethanol = 19.11, neutral pH = 9.55. • (Brown, Gibson Can. J. Chem. (2003), Inorg. Chem (2006))

8. This presentation has four parts • the cleavage of a series of 2-hydroxypropyl aryl phosphates (RNA models) promoted by a dinuclear Zn(II) catalyst in methanol and ethanol; • the question of stepwise vs. concerted cleavage of RNA models when catalyzed by the di-Zn(II) catalyst; • energetic considerations; • the hydrolysis of a methyl aryl phosphate DNA model promoted by the same catalyst in ethanol.

9. An Important Caveat about making models based on X-ray structures of enzyme active sites This is the enzyme in ‘flight ‘ performing its catalytic task

10. An Important Caveat about making models based on X-ray structures of enzyme active sites This is what the crystallographer sees.

11. Substrates investigated; base reaction.

12. Complex investigated • In situ treatment of ligand with 2 eq. of Zn(CF3SO3-)2 and 1 eq. NaOR in methanol or ethanol • requires about 1 hour to fully form the catalyst • This formulation sets the solution pH at 9.8 in methanol Di-Zn(II) complex was reported before, and for hydrolysis of bisp-nitrophenyl phosphate (in water), it was found to be no more active than the mono-Zn(II) complex (Kim and Lim, Bull. Korean Chem. Soc., 1999)

13. Plot of log k2 (or kcat/KM) for cleavage of 2-hydroxypropyl aryl phosphates promoted by catalyst 275,000 M-1s-1 for p-NO2 derivative slope= 0.0 Relative to CH3O- reaction (kOMe = 2.56 x 10-3 M-1s-1) catalyzed process gives an acceleration of at least 108–fold. slope = -1.1 RSB et al JACS 2007, 129, 16239

14. Di-Zn(II)-catalyst rapidly cleaves RNA models in ethanol as well • General observations in ethanol: • dielectric constant of ethanol (24.6) is smaller than methanol (32.7); • binding of anionic phosphate to di-Zn(II) catalyst is much stronger in ethanol than methanol (Kdis is at least 300-times smaller in ethanol); • all the plots of kobs vs. [catalyst] exhibit unusual looking saturation kinetic profiles.

15. Plot of kobs vs. [di-Zn(II)-catalyst] for the cleavage of p-methoxy phosphate diester (5 x 10-5 M), 25 oC in ethanol kcat = 2.67 s-1 Kdis ~ 3.2 x 10-7 M This value is an estimate based on fitting . RSB et al JACS 2008, 130, 16711

16. Plot of log kcat vs. pKa of leaving phenol for cleavage of RNA models in ethanol Slope= ~0 Slope= -1.12 The break in the plot is consistent with a mechanism where there is a change in rate limiting step from binding (good leaving groups) to P-OAr cleavage (poor leaving groups)

17. X-Ray Diffraction structure of di Zn(II) hydroxide complex Zn-Zn dist 3.67 Å 1-Zn(II)2:(-OH) (CF3SO3-)3(HOCH3)

18. View without triflate anions and methanol solvate

19. Phosphate binding to Cu(II) analogues

20. Proposed mechanism This mechanism fits all available data so far: If kcat > k-2, every time the bis-coordinated phosphate is formed it immediately breaks down to product, so a binding step is rate limiting and there is very little rate dependence on the nature of the aryloxy group This is the case with fast reacting substrates with good leaving groups

21. Proposed mechanism This mechanism fits all available data so far: If kcat < k-2, all the binding steps are at equilibrium and the rate limiting step is the chemical cleavage of the substrate that shows a large dependence on the nature of the aryloxy leaving group. This is the case with a slow reacting substrates with poor leaving groups

22. How much faster is the catalyzed reaction than the ethoxide reaction at the pH where the catalytic reaction is run? At pH =10, [ethoxide] = 10-9 M

23. 2. Mechanistic Details:Is the actual cleavage process step-wise, or concerted? Available data for substrates with aryloxy leaving groups where chemical cleavage is rate limiting are consistent with: 1. cleavage is a result of a two-step process with a rate limiting formation of a five-coordinate intermediate, (kcat), followed by fast loss of the aryloxy group; or

24. Mechanistic Details:Is the kcat term for the actual cleavage process step-wise, or concerted? Available data for substrates with aryloxy leaving groups where chemical cleavage is rate limiting are consistent with: 1. kcat is a two step process with the rate limiting step being formation of a five-coordinate intermediate (kcat), followed by fast loss of the aryloxy group; or 2. kcat is concerted with addition occurring simultaneously with departure of the aryloxy leaving group

25. Is there a break in the Brønsted plot as the leaving group gets poorer? • q.p. is the quasi-symmetrical point where there is equal likelihood for two leaving groups to depart. • with worse leaving groups the plot should break downward showing a stronger dependence on the leaving group. βlg = -0.88±0.14 q.p.

26. There is no break in Brønsted plot at the quasi-symmetrical point Slope = -0.81 ± 0.03 Because there is no break at the ‘quasi-symmetrical point’ there is no evidence for a change in rate-limiting step for the chemical cleavage kcat term, so the displacement reaction is suggested to be concerted.

27. 3. Energetics calculations for catalyzed reaction: general considerations GTSbinding Ggsbinding • If charge is neutralized in the T.S., then a reduced dielectric medium may accelerate the reaction, but only if the stabilization of the catalyzed T.S. is greater than the stabilization of the S:cat complex on binding. • leads to notion that catalysts must bind the TS better than ground state

28. Can we quantify the binding of the catalyst to the TS? Energetics calculations for catalyzed reaction

29. Energetics calculations for catalyzed reaction: standard state of 1 M, Sub = 4-Chlorophenyl hydroxypropyl phosphate • Activation energy for methoxide promoted reaction is 21.7 kcal/mol • Experimental results show that the binding of methoxide and Sub to the catalyst releases -14.4 kcal/mol. • Activation energy for cleavage of bound phosphate from Michaelis complex is 14.3 kcal/mol • Catalyst binds TS for the reaction by -21.8 kcal/mol

30. Comparison of energetics calculations for catalyzed cleavage for substrate Sd in methanol and ethanol

31. Di-Zn(II)2 complex also catalyzes cleavage of DNA models in methanol and ethanol diZn(II) complex catalyzed 108 -OCH3 catalyzed

32. 4. Catalyzed hydrolysis of a phosphate diester in ethanol Dielectric constants (є): water (78) > ethanol (24.3) 5 x 10-5 M substrate Kd ≤ 3.2 x 10-7 M kcat = 1.47x10-3 s1 (t1/2 = 7.8 min)in ethanol at pH 7.9 Catalyzed reaction is 1014 times faster than the ethoxide reaction at that pH

33. Reaction Products with small amount of water 1H NMR 7 mM of H2O SM Products? Ethanolysis Hydrolysis

34. Reaction Products with larger amount of water 1H NMR Products? 0.5 M of H2O Ethanolysis Hydrolysis

35. Hydrolysis in Ethanol as a Function of [H2O] 2.5 mM each of catalyst and phosphate 2.1 M H2O (3.8 vol %) gives 93% hydrolysis and 7% ethanolysis (Ethanolysis) 28 mM H2O gives 46% hydrolysis and 54% ethanolysis

36. Effect of water on the rate of the reaction • Rate depression vs. [water] probably due to change in medium.

37. Catalyst selects for water in the presence of ethanol solvent • at 28 mM water in ethanol, about ½ of the reaction product comes from hydrolysis; • since the concentration of water is about 1630 times less than the concentration of ethanol (17.17 M), the catalyst shows a large selectivity for water; • at a concentration of 28 mM, one can compute that the hydrolysis process promoted by the catalyst is accelerated by about 1014 relative to the k2 reaction for the ethoxide reaction. Liu, Neverov, Brown, J. Am. Chem. Soc. 2008, 130, 13870

38. Conclusions • The combination of a dinuclear catalyst AND a low dielectric constant medium effect produced by methanol and ethanol solvents produces an extremely active system for the cleavage of phosphate diesters; • Experimental results show that for the entire series of substituted aryl hydroxypropyl phosphate (RNA) models, that the catalyst binds the transition state by some 21 to 23 kcal/mol for substrates with poor and good aryloxy leaving groups in methanol, and 33-36 kcal/mol in ethanol; • The catalysis is very effective; cleavage of the substrate in the Michaelis complex (the kcat term) is about 1012 fold greater than the background reaction in methanol and up to 1017 fold greater in ethanol; • The available data indicate that the cleavage reaction for the phosphate ester, when it is bound by the catalyst, is probably concerted;

39. Conclusions In ethanol, the catalytic system selects for small amounts of water to produce hydrolytic products from a phosphate diester; perhaps this suggests a more general phenomenon whereby one can achieve large rate accelerations for metal ion promoted hydrolytic processes in alcohol solvents.

40. Acknowledgements Graduate Students Stephanie A. Melnychuk C. Tony Liu Mark A. Mohamed Chris I Maxwell Undergraduates Christopher White PDF and RA Zhong-Lin Lu Alexei A. Neverov Wing Yin Tsang David R. Edwards Chaomin Liu Funding: Natural Sciences and Engineering Research Council of Canada Killam Foundation of the Canada Council for the Arts US Army; Defense Threat Reduction Agency