Dinuclear ZnDinuclear Zn2+ 2+ Catalysts as Biomimics of RNA Catalysts as Biomimics of RNA and DNA Phosphoryl Transfer Enzymes: and DNA Phosphoryl Transfer Enzymes:

Changing the Medium From Water to Alcohol Changing the Medium From Water to Alcohol Provides Large Rate Enhancements.Provides Large Rate Enhancements.

R. Stan Brown R. Stan Brown Department of ChemistryDepartment of Chemistry

Queen’s UniversityQueen’s UniversityKingston, OntarioKingston, OntarioCanada, K7L 3N6Canada, K7L 3N6

Acknowledgements: Canada Foundation for Innovation, Acknowledgements: Canada Foundation for Innovation, NSERC, United States Army Research Office, DTRA, NSERC, United States Army Research Office, DTRA, Canada Council for the Arts and Queen’s UniversityCanada Council for the Arts and Queen’s University

If You Want Fast Reactions, the Medium is the MessageIf You Want Fast Reactions, the Medium is the Message

IntroductionIntroduction

•Responsible for the storage of genetic information in all living systemsResponsible for the storage of genetic information in all living systems•Participation of the intramolecular OH in RNA provides large Participation of the intramolecular OH in RNA provides large rate acceleration that is not seen in cleavage of DNA. rate acceleration that is not seen in cleavage of DNA. • tt1/21/2 for RNA cleavage is ~110 years without catalyst at neutral for RNA cleavage is ~110 years without catalyst at neutral

pH while that for DNA is estimated at about 10pH while that for DNA is estimated at about 108 8 to 10to 1010 10 years. years.

Metal Containing Phosphodiesterases Metal Containing Phosphodiesterases (catalyze cleavage of phosphate diesters)(catalyze cleavage of phosphate diesters)

•(RO)PO(RO)PO22--(OR’) + H(OR’) + H22O O (RO)PO (RO)PO33

== + HOR’ + HOR’•Phospholipase CPhospholipase C•P1 nucleaseP1 nuclease

P OR'

O-

RO

O

Simplified models for phosphoryl Simplified models for phosphoryl transfer enzymestransfer enzymes

• Numerous researchers have looked at modeling the activity Numerous researchers have looked at modeling the activity with simple mono- or dinuclear complexes .with simple mono- or dinuclear complexes .• Where comparison is made, dinuclear complexes are better Where comparison is made, dinuclear complexes are better than mononuclear complexes.than mononuclear complexes.• None of these is None of these is veryvery good at catalyzing the reactions in good at catalyzing the reactions in water.water.• In fact, most of these are not much better than HOIn fact, most of these are not much better than HO-- in in promoting the reaction, and several are worse. promoting the reaction, and several are worse. •Notable exceptions: J. Chin, N. Williams, J. Richard and Notable exceptions: J. Chin, N. Williams, J. Richard and J.Morrow.J.Morrow.

Zn2+ Zn2+

O

H

-

This is a common motif for the This is a common motif for the dinuclear enzymes where the dinuclear enzymes where the catalyst provides Lewis activation of catalyst provides Lewis activation of the substrate, and a metal-bound OH the substrate, and a metal-bound OH that acts as a nucleophile or a base. that acts as a nucleophile or a base.

One important exception

N

N

N

N

N

N

Zn Zn

O-

2+ 2+

NH2

NH2NH2

H2N

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-

HO O P

O

O-

OAr

OAr = p-nitrophenoxy

HPNPP, an RNA model

Quote:

“ “ Enzyme active sites are non-aqueous, Enzyme active sites are non-aqueous, and the effective dielectric constants and the effective dielectric constants resemble those in organic solvents resemble those in organic solvents rather than that in water”.rather than that in water”.

((= H= H22O, 78; MeOH, 32.7; EtOH 24.6)O, 78; MeOH, 32.7; EtOH 24.6)

Cleland, Frey, Gerlt Cleland, Frey, Gerlt J. Biol. Chem. J. Biol. Chem. 1998, 1998, 273273, 25529., 25529.

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

(Brown, Gibson (Brown, Gibson Can. J. Chem.Can. J. Chem. (2003), (2003), Inorg. ChemInorg. Chem (2006)) (2006))

This presentation has four partsThis presentation has four parts

• the cleavage of a series of 2-hydroxypropyl aryl the cleavage of a series of 2-hydroxypropyl aryl phosphates (RNA models) promoted by a dinuclear Zn(II) phosphates (RNA models) promoted by a dinuclear Zn(II) catalyst in methanol and ethanol;catalyst in methanol and ethanol;

• the question of stepwise vs. concerted cleavage of RNA the question of stepwise vs. concerted cleavage of RNA models when catalyzed by the di-Zn(II) catalyst;models when catalyzed by the di-Zn(II) catalyst;

• energetic considerations;energetic considerations;• the hydrolysis of a methyl aryl phosphate DNA model the hydrolysis of a methyl aryl phosphate DNA model

promoted by the same catalyst in ethanol.promoted by the same catalyst in ethanol.

OP

O

OO OH

Na+

XO

P

O

OOCH3

Na+

X

An Important Caveat about making An Important Caveat about making models based on X-ray structures of models based on X-ray structures of

enzyme active sitesenzyme active sites

This is the enzyme in ‘flight ‘ performing its catalytic taskThis is the enzyme in ‘flight ‘ performing its catalytic task

An Important Caveat about making An Important Caveat about making models based on X-ray structures of models based on X-ray structures of

enzyme active sitesenzyme active sites

This is what the crystallographer sees.This is what the crystallographer sees.

Substrates investigated; base reaction.

HO O P

O

O-

OAr

OAr = p-nitrophenoxy

HPNPP, an RNA model

MeOHO O

P

O O-

+

O-

CH3OH

O O H O O H

+

MeO-

(2.6x10-3) M-1s-1

MeO-

X

very slow

~6.9x10-7M-1s-1

P O

O-

H3CO P O

O-

H3CO

OP

O

OO OH

Na+

X

a X = 4-NO2b. X = 4-NO2, 3-CH3c. X = 3-NO2d. X = 4-Cle. X = 3-OCH3f. X = H, g. X = 4-OCH3

Complex investigated

Di-Zn(II) complex was reported before, and for Di-Zn(II) complex was reported before, and for hydrolysishydrolysis of of bisbis pp--nitrophenyl phosphate (nitrophenyl phosphate (in waterin water), it was found to be no more active than ), it was found to be no more active than the mono-Zn(II) complex the mono-Zn(II) complex (Kim and Lim, (Kim and Lim, Bull. Korean Chem. Soc., 1999Bull. Korean Chem. Soc., 1999))

N

N N

N

N N

H

H

H

H

N

N N

N

N N

H

H

H

Zn Zn

H2+ 2+

2 eq. Zn(OTf)2

1 eq. NaOR O

R

-

•In situIn situ treatment of ligand with 2 eq. of Zn(CF treatment of ligand with 2 eq. of Zn(CF33SOSO33--))2 2

and 1 eq. NaOR in methanol or ethanoland 1 eq. NaOR in methanol or ethanol• requires about 1 hour to fully form the catalystrequires about 1 hour to fully form the catalyst•This formulation sets the solution pH at 9.8 in methanolThis formulation sets the solution pH at 9.8 in methanol

Plot of log kPlot of log k2 2 (or k(or kcatcat/K/KMM) for cleavage of 2-) for cleavage of 2-

hydroxypropyl aryl phosphates promoted by catalysthydroxypropyl aryl phosphates promoted by catalyst

11 12 13 14 154

5

6

sspKa (phenol)

log

k2 o

r k c

at/K

M(M

-1s-1

)

N

N N

N

N N

H

H

H

Zn Zn

H2+ 2+

OH3C

-

-O P

O

O NO2

OHO

Relative to CHRelative to CH33OO- - reaction reaction

(k(kOMeOMe = 2.56 x 10 = 2.56 x 10-3-3 M M-1-1ss-1-1) )

catalyzed process gives an catalyzed process gives an acceleration of at least acceleration of at least 101088–fold.–fold.

RSB RSB et alet al JACS 2007, JACS 2007, 129129, 16239, 16239

Subst. + cat cat:Subst. PKM

kcat

275,000 M275,000 M-1-1ss-1 -1 for for pp--NONO22 derivative derivative

slopeslope

slope = -1.1slope = -1.1

Di-Zn(II)-catalyst rapidly cleaves RNA models Di-Zn(II)-catalyst rapidly cleaves RNA models in ethanol as wellin ethanol as well

• General observations in ethanol:General observations in ethanol:• dielectric constant of ethanol (24.6) is smaller than dielectric constant of ethanol (24.6) is smaller than

methanol (32.7);methanol (32.7);• binding of anionic phosphate to di-Zn(II) catalyst is binding of anionic phosphate to di-Zn(II) catalyst is

much stronger in ethanol than methanol (Kmuch stronger in ethanol than methanol (Kdisdis is at is at

least 300-times smaller in ethanol);least 300-times smaller in ethanol);

• all the plots of kall the plots of kobsobs vs. [catalyst] exhibit unusual vs. [catalyst] exhibit unusual

looking saturation kinetic profiles.looking saturation kinetic profiles.

Subst. + cat cat:Subst. PKdis

kcat

Plot of kPlot of kobsobs vs. [di-Zn(II)-catalyst] for the cleavage of vs. [di-Zn(II)-catalyst] for the cleavage of pp--

methoxy phosphate diester (5 x 10methoxy phosphate diester (5 x 10-5 -5 M), 25 M), 25 ooC in ethanolC in ethanol

0 2.5×10-5 5.0×10-5 7.5×10-5 1.0×10-40

1

2

3

[cat:(-OCH2CH3)], (M)

k ob

s,

(s-1

)

N

N N

N

N N

H

H

H

Zn Zn

H2+ 2+

O

CH2CH3

-+ -O P

O

O

OCH3

OHO

cat:subst

cat:prod + HOAr

kcatKdis

kkcat cat = 2.67 s= 2.67 s-1-1

KKdisdis ~ 3.2 x 10 ~ 3.2 x 10-7 -7 MM

RSB RSB et al et al JACS 2008, JACS 2008, 130130, 16711, 16711

This value is an estimate This value is an estimate based on fitting .based on fitting .

Plot of log kPlot of log kcatcat vs. pKa of leaving phenol for vs. pKa of leaving phenol for

cleavage of RNA models in ethanolcleavage of RNA models in ethanol

11.5 12.5 13.5 14.5 15.5 16.50.0

0.5

1.0

1.5

2.0

2.5

ss pKa of phenol in ethanol

log

(kc

at)

, s-1

Slope= -1.12Slope= -1.12

Slope= ~0Slope= ~0

The break in the plot is consistent with a mechanism where there The break in the plot is consistent with a mechanism where there is a change in rate limiting step from binding (good leaving is a change in rate limiting step from binding (good leaving groups) to P-OAr cleavage (poor leaving groups)groups) to P-OAr cleavage (poor leaving groups)

OP

O

OO OH

Na+

X

a X = 4-NO2b. X = 4-NO2, 3-CH3c. X = 3-NO2d. X = 4-Cle. X = 3-OCH3f. X = H, g. X = 4-OCH3

X-Ray Diffraction structure of di Zn(II) hydroxide complex

1-Zn(II)2:(-OH) (CF3SO3-)3(HOCH3)

Zn-Zn dist 3.67 Å

View without triflate anions and View without triflate anions and methanol solvatemethanol solvate

N

N N N N

N

Cu Cu(CF3SO3)2

HO

PO OO O

N

N N N N

N

Cu Cu(CF3SO3)3

HO

OHH

O O

POO -

Phosphate binding to Cu(II) analogues

Proposed mechanism

This mechanism fits all available data so far:1. 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 group2. This is the case with fast reacting substrates with good leaving groups

-O

P

O

O NO2

O

HO

-O

P

O

O NO2

O

HO

CH3

k1

k-1

Zn

Zn

Zn

Zn

Zn

Zn

k2

k-2

kcat

P

O-

O

O

PO-

O

O

R'O

+ P O-

O

O

R'OP

O-

O

O

R'O + HOR'

+O-

R RZn

Zn

O-

RO-

O-

OHOH

O-

Xfast

Proposed mechanism

This mechanism fits all available data so far:1. 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.2. This is the case with a slow reacting substrates with poor leaving groups

-O

P

O

O Cl

O

HO

-O

P

O

O

O

HO

NO2

-O

P

O

O H

O

HO

k1

k-1

Zn

Zn

Zn

Zn

Zn

Zn

k2

k-2

kcat

P

O-

O

O

PO-

O

O

R'O

+ P O-

O

O

R'OP

O-

O

O

R'O + HOR'

+O-

R RZn

Zn

O-

RO-

O-

OHOH

O-

How much faster is the catalyzed How much faster is the catalyzed reaction than the ethoxide reaction at the reaction than the ethoxide reaction at the

pH where the catalytic reaction is run?pH where the catalytic reaction is run?

OP

O

OO OHX

x kcat (s-

1)kOMe (s-

1)kcat/kOMe

4-NO2 168 2.4 e-13 7 e14

3-NO2 139 9.7 e-15 1.4 e16

4-Cl 36 6.3 e-16 5.7 e16

3-OMe 14.5 3.7 e-16 4 e16

H 4.5 1.4 e-16 3 e16

4-OMe 2.7 7.4 e-17 3.5 e16

At pH =10, At pH =10, [ethoxide] = 10[ethoxide] = 10-9-9 M M

N

N N

N

N N

H

H

H

Zn Zn

H2+ 2+

O

CH2CH3

-

2. Mechanistic Details:2. Mechanistic Details:Is the actual cleavage process step-wise, Is the actual cleavage process step-wise,

or concerted?or concerted?

Zn

Zn

P

O

O

O

P

O-

O

O

O

+

Zn

Zn

O-

H3CO-

O-

X

Zn

ZnP

O-

O

O

O

O

X

kcat

---

kp

+HO X

slow fastCH3OH

Available data for substrates with aryloxy leaving Available data for substrates with aryloxy leaving groups where chemical cleavage is rate limiting are groups where chemical cleavage is rate limiting are consistent with:consistent with:

1. cleavage is a result of a two-step process with a 1. cleavage is a result of a two-step process with a rate limiting formation of a five-coordinate rate limiting formation of a five-coordinate intermediate, (kintermediate, (kcatcat), followed by fast loss of the ), followed by fast loss of the

aryloxy group; oraryloxy group; or

Mechanistic Details:Mechanistic Details:Is the kIs the kcatcat term for the actual cleavage term for the actual cleavage

process step-wise, or concerted?process step-wise, or concerted?

Available data for substrates Available data for substrates with aryloxy leaving with aryloxy leaving groups where chemical cleavage is rate limiting groups where chemical cleavage is rate limiting are are consistent with:consistent with:

1. 1. kkcatcat is a two step process with the rate limiting step being is a two step process with the rate limiting step being

formation of a five-coordinate intermediate (kformation of a five-coordinate intermediate (kcatcat), followed ), followed

by fast loss of the aryloxy group; orby fast loss of the aryloxy group; or

2.2. kkcatcat is concerted with addition occurring simultaneously is concerted with addition occurring simultaneously

with departure of the aryloxy leaving group with departure of the aryloxy leaving group

Zn

Zn

P

O

O

O

P

O-

O

O

O

+

Zn

Zn

O-

H3CO-

O-

X

Zn

ZnP

O-

O

O

O

O

X

kcat

---

+HO X

CH3OH

Is there a break in the Brønsted plot as Is there a break in the Brønsted plot as the leaving group gets poorer?the leaving group gets poorer?

Zn

Zn

P

O

O

O

P

O-

O

O

LgO

+

Zn

Zn

O-

H3CO-

O-

Zn

ZnP

O-

O

O

LgO

Okc

---

kp

+

CH3OHk-c

LgOH

11.5 14.0 16.5 19.0-5

-4

-3

-2

-1

0

1

2

3

4

sspKa(HOLg)

log

(kc

at s

-1)

q.p.q.p.

• q.p. is the quasi-q.p. is the quasi-symmetrical point where symmetrical point where there is equal likelihood for there is equal likelihood for two leaving groups to two leaving groups to depart.depart.

• with worse leaving with worse leaving groups the plot should groups the plot should break downward showing a break downward showing a stronger dependence on stronger dependence on the leaving group.the leaving group.

βlg = -0.88±0.14

There is no break in Brønsted plot at the There is no break in Brønsted plot at the quasi-symmetrical pointquasi-symmetrical point

11 13 15 17 19-6

-4

-2

0

2

4

s

6a 6b

6c

3q.p.

spKaLG

log

k ca

t (s-1

)

HO

H3C

O P

O

O-

OR

6a R = CF3CH2

6b R = CFH2CH2

6c R = CH3CH2

3 R = CH3

Because there is no break at the ‘quasi-symmetrical point’ there is no 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 kevidence for a change in rate-limiting step for the chemical cleavage kcat cat

term, so the displacement reaction is suggested to be concerted.term, so the displacement reaction is suggested to be concerted.

Slope = -0.81 ± 0.03

3. Energetics calculations for catalyzed reaction: general considerations

S + Cat

S:cat

(S:cat)

S

E

P + cat

reaction coordinate

• 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

Ggs binding

GTS binding

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

RO- + Sub + 1-Zn(II)2

GNon

GBind

1-Zn(II)2:(-OR) + Sub GM

1-Zn(II)2:(-OR):Sub

[RO-:Sub]+ 1-Zn(II)2

Gcat

Gstab

Gcat1-Zn(II)2:(OCH3)

[1-Zn(II)2:(-OR):Sub]

(Michaelis complex)

OP

O

OO OH

Na+

X

a X = 4-NO2b. X = 4-NO2, 3-CH3c. X = 3-NO2d. X = 4-Cle. X = 3-OCH3f. X = H, g. X = 4-OCH3

Sub

Go (

kca

l/mol

)

0

5

10

15

20

25

-5

-10

-15

-20

1:Zn(II)2

+ [CH3O-:Sub]

1 :Zn(II)2

+ CH3O-

+ Sub

1:Zn(II)2:(-OCH3) + Sub

21.7

1:Zn(II)2:(-OCH3): Sub

14.3

-10.0

-4.4

21.8k2

-OMe

kcat/KM10.0

[1:Zn(II)2:(-OCH3): Sub]

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

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

1:Zn(II)2

+ [CH3O-:Sd]

1:Zn(II)2

+ CH3O-

+ Sd

1:Zn(II)2:(-OCH3) + Sd

21.7

1:Zn(II)2:(-OCH3): Sd

14.3

-10.0

-4.4

21.8k2

-OMe

[1:Zn(II)2:(-OCH3): Sd]

Go (kcal/mol)

0

5

10

15

20

25

-5

-10

-15

-20

1:Zn(II)2

+ [CH3CH2O-:Sd]

1:Zn(II)2

+ -OCH2CH3

+ Sd

1:Zn(II)2:(-OCH2CH3) + Sd

24.2

1:Zn(II)2:(OCH2CH3):Sd

14.5

-16.2

-8.9

34.8

(k2-OEt)

[1:Zn(II)2:(-OCH2CH3):Sd]

30

(kcatmax)

-25

(kcatmax.)

OP

O

OO OHX

d. X = 4-ClS

Di-Zn(II)Di-Zn(II)22 complex also catalyzes cleavage complex also catalyzes cleavage

of DNA models in methanol and ethanolof DNA models in methanol and ethanol

6.0 8.5 11.0 13.5 16.0 18.5-7.5

-5.0

-2.5

0.0

2.5

5.0

spKa (phenol)s

(CH3O)2PO2-

= -0.60

= -0.57

log

k2

or lo

g k

cat/K

M (

M-1

s-1)

H3CO P

O

O-

OAr

a. 2-chloro-4-nitrophenylb. 2, 4, 5-trichlorophenylc. 4-nitrophenyld. 3-nitrophenyle. 4-chlorophenylf. 3-methoxyphenylg. phenylh. 4-methoxyphenyl

Na+

diZn(II) complex catalyzeddiZn(II) complex catalyzed

--OCHOCH33 catalyzed catalyzed

N

N N

N

N N

H

H

H

Zn Zn

H2+ 2+

OH3C

-

101088

Dielectric constants (Dielectric constants (єє): water (78) > ethanol (24.3)): water (78) > ethanol (24.3)

4. Catalyzed hydrolysis of a phosphate diester 4. Catalyzed hydrolysis of a phosphate diester in ethanolin ethanol

0 2.5×10-5 5.0×10-5 7.5×10-5 1.0×10-4 1.2×10-4 1.5×10-40

1.0×10-3

2.0×10-3

[Catalyst], (M)

k ob

s,(s

-1)

O P

O

OMe

O

Cl

O2N

Kd ≤ 3.2 x 10Kd ≤ 3.2 x 10-7-7 M Mkkcatcat = 1.47x10 = 1.47x10-3-3 s s1 1 (t(t1/21/2 = 7.8 min) = 7.8 min) in ethanol at pH 7.9in ethanol at pH 7.9

N

N N

N

N N

H

H

H

Zn Zn

H2+ 2+

O

CH2CH3

-

5 x 105 x 10-5 -5 M substrateM substrate

Catalyzed reaction is 10Catalyzed reaction is 1014 14 times faster than the ethoxide reaction at that pHtimes faster than the ethoxide reaction at that pH

3.75 3.70 3.65 3.60 3.55 3.50

0.00

0.01

0.02

0.03

0.04

0.05

3.17

3.62

3.65

3.66

3.69

Reaction Products with small amount of waterReaction Products with small amount of water

1H NMR

O P

O

OMe

O

Cl

O2N

SM Products?7 mM of H2ORO P

O

OMe

O

HO P

O

OMe

O

Hydrolysis

EtO P

O

OMe

O

Ethanolysis

3.80 3.75 3.70 3.65 3.60 3.55 3.50

0.00

0.01

0.02

0.03

0.04

0.05

3.00

3.64

3.67

3.67

3.70

1H NMR

Reaction Products with larger amount of waterReaction Products with larger amount of water

O P

O

OMe

O

Cl

O2N

Products?

0.5 M of H2O

EtO P

O

OMe

OHydrolysis

Ethanolysis

HO P

O

OMe

O

RO P

O

OMe

O

0.0 0.5 1.0 1.5 2.00

25

50

75

100

[H2O], M

% H

ydro

lysi

s p

rod

uct

kcatP

O-

OArOMeO

+Zn

ZnO-R

Kb

PO-

OArO

MeOZn

Zn

RO-w

kcat

CH3OPO32-

CH3OPO2OEt-

ER=H, Et

(Ethanolysis)

Hydrolysis in Ethanol as a Function of [HHydrolysis in Ethanol as a Function of [H22O]O]

28 mM H28 mM H22O gives 46% O gives 46%

hydrolysis and 54% hydrolysis and 54% ethanolysisethanolysis

2.5 mM each of catalyst and phosphate2.5 mM each of catalyst and phosphate

2.1 M H2.1 M H22O (3.8 vol %) givesO (3.8 vol %) gives

93% hydrolysis and 7% 93% hydrolysis and 7% ethanolysisethanolysis

0.00 0.25 0.50 0.75 1.000

1.0×10-4

2.0×10-4

3.0×10-4

4.0×10-4

5.0×10-4

6.0×10-4

7.0×10-4

[H2O], M

k ob

s,

s-1

Effect of water on the rate of the reactionEffect of water on the rate of the reaction

• Rate depression vs. [water] probably due to change in medium.Rate depression vs. [water] probably due to change in medium.

Catalyst selects for water in the presence Catalyst selects for water in the presence of ethanol solventof ethanol solvent

• at 28 mM water in ethanol, about ½ of the reaction product at 28 mM water in ethanol, about ½ of the reaction product comes from hydrolysis;comes from hydrolysis;

• since the concentration of water is about 1630 times less than since the concentration of water is about 1630 times less than the concentration of ethanol (17.17 M), the catalyst shows a the concentration of ethanol (17.17 M), the catalyst shows a large selectivity for water;large selectivity for water;

• at a concentration of 28 mM, one can compute that the at a concentration of 28 mM, one can compute that the hydrolysis process promoted by the catalyst is accelerated by hydrolysis process promoted by the catalyst is accelerated by about 10about 101414 relative to the k relative to the k22 reaction for the ethoxide reaction. reaction for the ethoxide reaction.

Liu, Neverov, Brown, Liu, Neverov, Brown,

J. Am. Chem. Soc. J. Am. Chem. Soc. 2008, 2008, 130130, 13870, 13870

Conclusions• The combination of a dinuclear catalyst The combination of a dinuclear catalyst AND AND a low dielectric constant a low dielectric constant medium effect produced by methanol and ethanol solvents produces an medium effect produced by methanol and ethanol solvents produces an extremely active system for the cleavage of phosphate diesters;extremely active system for the cleavage of phosphate diesters;

• Experimental results show that for the entire series of substituted aryl Experimental results show that for the entire series of substituted aryl hydroxypropyl phosphate (RNA) models, that the catalyst binds the hydroxypropyl phosphate (RNA) models, that the catalyst binds the transition state by some 21 to 23 kcal/mol for substrates with poor and 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;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 The catalysis is very effective; cleavage of the substrate in the Michaelis complex (the kcomplex (the kcatcat term) is about 10 term) is about 1012 12 fold greater than the background fold greater than the background

reaction in methanol and up to 10reaction in methanol and up to 101717 fold greater in ethanol; fold greater in ethanol;

•The available data indicate that the cleavage reaction for the phosphate The available data indicate that the cleavage reaction for the phosphate ester, when it is bound by the catalyst, is probably concerted;ester, when it is bound by the catalyst, is probably concerted;

Conclusions In ethanol, the catalytic system selects for small amounts of water In ethanol, the catalytic system selects for small amounts of water to produce hydrolytic products from a phosphate diester; to produce hydrolytic products from a phosphate diester;

perhaps this suggests a more general phenomenon whereby one perhaps this suggests a more general phenomenon whereby one can achieve large rate accelerations for metal ion promoted can achieve large rate accelerations for metal ion promoted hydrolytic processes in alcohol solvents.hydrolytic processes in alcohol solvents.

AcknowledgementsAcknowledgementsPDF and RAPDF and RA

Zhong-Lin LuZhong-Lin Lu

Alexei A. NeverovAlexei A. Neverov

Wing Yin TsangWing Yin Tsang

David R. EdwardsDavid R. Edwards

Chaomin LiuChaomin Liu

Graduate StudentsGraduate Students

Stephanie A. MelnychukStephanie A. Melnychuk

C. Tony LiuC. Tony Liu

Mark A. MohamedMark A. Mohamed

Chris I MaxwellChris I Maxwell

UndergraduatesUndergraduates

Christopher WhiteChristopher WhiteFunding:Funding:

Natural Sciences and Engineering Research Council of CanadaNatural Sciences and Engineering Research Council of Canada

Killam Foundation of the Canada Council for the ArtsKillam Foundation of the Canada Council for the Arts

US Army; Defense Threat Reduction AgencyUS Army; Defense Threat Reduction Agency