Cleavage of models for RNA mediated by a diZn(II) complex of bis[1,4-N1,N1'(1,5,9-triazacyclododecanyl)]butane in methanol and ethanol

Article abstract of DOI:10.1139/V09-026

The cleavage of seven RNA model 2-hydroxypropyl aryl phosphates (1) catalyzed by a dinuclear Zn(II) complex bis[1,4-N1,N1'(1,5,9- triazacyclododecanyl)]butane (4) was studied in methanol and ethanol at 25 8C under pH controlled . The results are compared

Full text of DOI:10.1139/V09-026

640
Cleavage of models for RNA mediated by a
diZn(II) complex of bis[1,4-N1,N1’(1,5,9-
triazacyclododecanyl)]butane in methanol and
ethanol
C. Tony Liu, Stephanie A. Melnychuk, Chaomin Liu, Alexei A. Neverov, and R.
Stan Brown
Abstract: The cleavage of seven RNA model 2-hydroxypropyl aryl phosphates (1) catalyzed by a dinuclear Zn(II) complex
of bis[1,4-N1,N1’(1,5,9-triazacyclododecanyl)]butane (4) was studied in methanol and ethanol at 25 8C under pH controlled
conditions. The results are compared with what was reported earlier for the dinuclear Zn(II) complex of the lower homo-
logue bis[1,3-N1,N1’(1,5,9-triazacyclododecanyl)]propane (3). In methanol, the higher homologue exhibits saturation binding
with substrates having poor aryloxy leaving groups. With good leaving groups there is an observed linear dependence of
kobs versus complex concentration without saturation binding over the catalyst concentration range investigated. In ethanol,
strong saturation binding between the active form of the catalyst ((RO–):Zn(II)2:4) and all substrates is observed, the results
observed in both solvents being similar to what was reported for the lower (RO–):Zn(II)2:3 homologue. Energetics calcula-
tions are presented for the (RO–):Zn(II)2:4-catalyzed cleavage of each substrate in both solvents to assess the catalytic effi-
ciency via the DDG{ for catalyst binding a transition state comprising [RO–:1]{ or its kinetic equivalent.
Key words: dinuclear Zn(II) catalyst, RNA model cleavage, methanolysis, ethanolysis, kinetics, mechanism.
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ˆ ´ `
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Resume : Operant dans des conditions de pH controle, a 25 8C, en solution dans le methanol et l’ethanol, on a etudie la
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reaction de clivage de sept phosphates d’aryle et de 2-hydroxypropyle (1), des modeles de l’ARN, catalyse par un com-
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plexe dinucleaire du Zn(II) et le bis[1,4-N1,N1’(1,5,9-triazacyclododecanyl)]butane (4). On compare les resultats avec ceux
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rapportes anterieurement pour le complexe dinucleaire du Zn(II) l’homologue inferieur bis[1,3-N1,N1’(1,5,9-triazacyclode-
´
´
canyl)]propane (3). Dans le methanol, l’homologue superieur on observe qu’il y a saturation du site de fixation avec les
´ `
substrats pour lesquels l’habilite a partir des groupes aryloxy est faible. Avec de bons groupes partant, pour toute la plage
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de concentration de catalyseur utilisee dans ce travail, on observe une dependance lineaire de kobs en fonction de la
´
concentration du complexe. Dans l’ethanol, on observe une forte saturation des sites de fixation entre tous les substrats et
–
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la forme active du catalyseur [(RO ):Zn(II)2:4]; dans les deux solvants, les resultats sont similaires a ceux rapportes pour
–
{
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l’homologue inferieur [(RO ):Zn(II)2:3]. Afin d’evaluer l’efficacite catalytique par le biais du DDG de la fixation par le
–
{
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catalyseur d’un etat d’activation comportant [RO :1] ou son equivalent cinetique, on a effectue des calculs d’energie pour
–
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le clivage de chacun des substrats, catalyses par [(RO ):Zn(II)2:4].
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Mots-cles : catalyseur de Zn(II) dinucleaire, clivage d’un modele d’ARN, methanolyse, ethanolyse, cinetique, mecanisme.
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[Traduit par la Redaction]
3
˚
in close proximity (3–4 A apart). In an effort to understand
the origins of this exalted catalysis numerous researchers
have studied mono- and di-nuclear metal ion catalysts for
cleaving selected phosphate diesters.4–7 We have reported the
rapid cleavage of a series of activated RNA (1)8,9 and DNA
(2)8,10 models catalyzed by the dinuclear 3:Zn(II)2:(–OR)
complex in methanol and ethanol under SSpH controlled condi-
tions.11 As shown in Scheme 1, the catalyzed cleavage of 1
involves intramolecular participation of the 2-hydroxylpropyl
group to form a cyclic phosphate intermediate (5), which rap-
idly opens up to the two isomeric products, 6 and 7.12
Introduction
Phosphate diesters that form the backbone of RNA and
DNA are highly stable toward hydrolytic cleavage under
physiological conditions in the absence of enzymes or spe-
cific catalysts.1,2 However, with the assistance of enzymes,
the cleavage of these sorts of phosphate diesters can be accel-
erated by up to 1015–1017.1 Many naturally occurring phos-
phodiesterases have active sites containing two or more
metal ions such as Zn(II), Ca(II), Mg(II), Fe(III), and Mn(II)
Received 16 December 2008. Accepted 13 February 2009.
Published on the NRC Research Press Web site at
canjchem.nrc.ca on 23 April 2009.
C.T. Liu, S.A. Melnychuk, C. Liu, A.A. Neverov, and
R.S. Brown.1 Department of Chemistry, Queen’s University, 90
Bader Lane, Kingston, ON K7L 3N6, Canada.
1Corresponding author (e-mail: rsbrown@chem.queensu.ca).
Can. J. Chem. 87: 640–649 (2009)
doi:10.1139/V09-026
Published by NRC Research Press

Liu et al.
641
Scheme 1.
phenyl phosphate, 99.5%), methanol-d4 (D, 99.8%) and abso-
lute ethanol which was then de-gassed by bubbling Ar
through it for 1h prior to storing it under Ar. Freshly dispensed
degassed ethanol ([H2O], determined to be 0.028 0.007 mol/
L14,17 by Karl Fischer titration) was used for conducting kinetic
experiments. The sodium salts of the 2-hydroxylpropyl aryl
phosphates (1a–1g) were prepared as described in earlier re-
ports9,18,19 where the characterizations of the compounds can
be found. The dinuclear catalyst was prepared as 2.5 mmol/L
stock solutions by sequential addition of aliquots of stock sol-
utions of sodium alkoxide, 4, and Zn(CF3SO3)2 such that the
relative ratios were 1:1:2 in either solvent at 25 8C. The com-
plete formation of the complexes required 25 and 80 min in
methanol and ethanol (as monitored by the changed in cata-
lytic activity over time).
Bis[1,4-N1,N1’(1,5,9-triazacyclododecanyl)]butane (4)
To a solution of 1,5,7-triazabicyclo[4,4,0]dec-5-ene20
(2.50 g, 13.8 mmol) in 50 mL of dry acetonitrile, 1.48 g
(6.9 mmol) of 1,4-dibromobutane was added. The reaction
mixture was heated at reflux for 3 d and the resulting off-
white solid was filtered and washed with dry acetonitrile.
The crude product was then mixed with 6 N aqueous HCl
(20 mL) and heated to reflux for 48 h before the majority
of the solvent was removed in vacuo. Absolute ethanol was
added to the residue to induce precipitation. The white pre-
cipitates were washed four times with 8 mL portions of ab-
solute ethanol and then dried in vacuo after which they were
recrystallized from 1:9 aqueous ethanol to afford the HCl
salt of 4 as a white solid in 67% overall yield. Conversion
of the HCl salt into the free amine (4) was accomplished by
stirring 897 mg (1.48 mmol) in 30 mL of water along with
excess 5 N NaOH for 2 h at ambient temperature, followed
by extracting the solution with four 25 mL portions of
CHCl3. The combined CHCl3 extracts were dried over
MgSO4 and filtered before the solvent was removed in va-
cuo affording pure 4 as a nearly colorless oil in 78% yield
Although 3:Zn(II)2 is reported to exhibit poor catalysis of
the cleavage of bis-p-nitrophenyl phosphate in water,13 it is
dramatically different in methanol9 and ethanol14 where, as
an alkoxy form (3a, 3b), it accelerates the cleavages of 1a–
1g by 1011 to 1014 relative to the background alkoxide-facili-
tated reactions at near neutral SSpH values and 25 8C. The
origin of the very large enhancement of activity obtained by
an apparently simple change in solvent is uncertain at this
time, but is probably related to a variety of factors including
a reduced polarity and dielectric constant (dielectric con-
stant, e = 78, 31.5, and 24.3 for water, methanol, and etha-
nol, respectively15) and overall solvation changes that
enhance the interaction between the oppositely charged cata-
lyst and substrate in the ground state and increasingly so in
the transition state for the catalyzed reaction.
Herein, we report on a slightly more flexible Zn(II)2 com-
plex of ligand 4, which is the C4 higher homologue of 3. To
shed light on how the additional flexibility of the higher ho-
mologue might affect the catalysis under SSpH controlled con-
ditions, studies of the 4:Zn(II)2-catalyzed transesterification
of 1a–1g in methanol and ethanol were undertaken to com-
pare with what was seen earlier with 3:Zn(II)2. As will be
1
(456 mg). H NMR (400 MHz, CD3OD, 25 8C) d: 2.80 (t,
8H, J = 6.0 Hz, HNCH2CH2CH2NH), 2.75 (t, 8H, J =
6.0 Hz, HNCH2(CH2)2N(CH2)2), 2.59 (t, 8H, J = 6.0 Hz,
N(CH2)2CH2), 2.46 (m, 4H, N(CH2)2CH2), 1.70 (m, 12H,
HNCH2CH2CH2N), 1.49 (m, 4H, CH2CH2CH2CH2). 13C NMR
(100 MHz, D2O, 25 8C) d: 53.2, 51.8, 48.8, 47.3, 25.4, 25.2,
23.2. HRMS (EI-TOF) m/z: calcd. for (C22H48N6): 396.3940;
found: 396.3943 (M+).
shown, 4:Zn(II)2 accelerates the cleavage reactions of 1a–1g
S
by around 1011 to 1012 (at pH 11 9.5 and 25 8C) in methanol
S
S
and a factor of 1015 to 1016 (at pH 9.0 and 25 8C) in ethanol
S
relative to the background alkoxide-promoted reactions in
Methods
the respective solvents.16
1H NMR and 31P NMR spectra were determined at 400
+
+
and 162.04 MHz. The CH3OH2 and CH3CH2OH2 concen-
trations were determined potentiometrically with a combina-
tion glass electrode (Radiometer model # XC100-111-120-
161) calibrated with standard aqueous buffers (pH = 4.00
and 10.00) as described previously.11 The SSpH values in
methanol and ethanol were determined by subtracting the
correction constants of –2.2411a,11b and –2.54,11c respec-
tively, from the electrode readings. The autoprotolysis con-
stants are 10–16.77 (mol/L)2 for methanol and 10–19.1 (mol/L)2
for ethanol. The kinetic experiments were conducted under
buffer-free conditions of low but variable ionic strength deter-
mined by the catalyst concentration since the presence of buf-
fer and excess salts that might be used to keep ionic strength
Experimental
Materials
Anhydrous methanol (99.8%), sodium methoxide (0.50 mol/L
solution in methanol, titrated against N/50 certified standard
aqueous HCl solution and found to be 0.49 mol/L), sodium
ethoxide (21 wt. % solution in denatured ethanol, titrated
against N/50 standard aqueous HCl solution and found to be
2.68 mol/L), Zn(CF3SO3)2, and tetrabutylammonium trifluor-
omethanesulfonate (99%) were used without further purifica-
tion. HClO4 (70% aqueous solution, titrated to be 11.40 mol/
L) was used as supplied as was Paraoxon1 (diethyl p-nitro-
Published by NRC Research Press

642
Can. J. Chem. Vol. 87, 2009
S
max corr
obs
constant is observed to inhibit the catalysis, probably due to
binding of the anions.
Fig. 1. Two pH/log k
profiles for the reaction of 4:Zn(II)2:
S
(–OCH2CH3) and 1b, as well as 4:Zn(II)2:(–OCH3) with 1g conducted
at 25 8C in ethanol and methanol. The lines through the data points
in the indicated boxes are computed on the basis of a bell-
Stopped-flow kinetics
S
shaped pH/rate profile. (See text and ref. 14).
S
The rates for the catalyzed transesterification of 1a–1g
were determined by following the appearance of the phe-
nolic products using a stopped-flow reaction analyzer ther-
mostatted at 25.0
0.1 8C. The production of the phenol
was monitored at the following wavelengths: 1a, 320 nm;
1b, 323 nm; 1c, 340 nm; 1d, 284 nm; 1e, 282 nm; 1f,
280 nm; and 1g, 292 nm. Stock solutions (2.5 mmol/L) of
4:Zn(II)2:(–OCH3) in methanol and 4:Zn(II)2:(–OCH2CH3) in
ethanol were prepared in oven-dried vials that were capped
and sealed with Parafilm1. These were used to prepare cata-
lyst solutions of concentrations ranging from 0.34 mmol/L £
4:Zn(II)2:(–OCH3)
£
2.06 mmol/L in methanol and
0.06 mmol/L £ 4:Zn(II)2:(–OCH2CH3) £ 0.28 mmol/L in etha-
nol. The catalyst solutions were loaded into one of the two
syringes in the stopped-flow analyzer while a solution con-
taining 0.1 mmol/L of substrate 1a–1g in either methanol or
ethanol was loaded into the other. The final concentrations
of catalyst after mixing in the cell of the stopped-flow ana-
lyzer were 0.17 mmol/L £ 4:Zn(II)2:(–OCH3) £ 1.03 mmol/L
in methanol and 0.03 mmol/L £ 4:Zn(II)2:(–OCH2CH3) £
0.14 mmol/L in ethanol while the final concentration of 1a–
1g was 0.05 mmol/L in either alcohol. At each catalyst con-
centration at least five kinetic runs were recorded and the
average values were used for the kobs for the appearance of
the product. The SSpKa values of the corresponding phenol
leaving groups of 1a–1g in methanol9 and ethanol14 have
been reported.
at the end of the reactions and assumed to have been set im-
S
mediately on mixing. The pH/log rate constant profile plot
S
is presented in Fig. 1 with the line through the data being
derived from a fit of the solid squares data to a process that
depended on two ionizable groups having SSpKa values of
8.48 and 10.38 (see the Supplementary data).
Methanol
S
S
The pH/log rate constant profile for the cleavage of 1g
catalyzed by 4:Zn(II)2 in methanol was conducted as
described above. The final concentrations of triflate-free
4:Zn(II)2 and 1g after mixing in the reaction chamber of the
stopped-flow analyzer were 6.4 ꢀ 10–4 mol/L (after triflate
correction) and 5 ꢀ 10–5 mol/L, respectively. The kinetic
data, shown in Fig. 1, were fitted to a process depending on
two ionizable groups having SSpKa values of 9.38 and 9.80.
The degree of triflate inhibition on the catalysis in methanol
was determined from a kinetic experiment that involved
monitoring the cleavage of paraoxon (0.05 mmol/L) mediated
by 0.43 mmol/L of 4:Zn(II)2:(–OCH3) in the presence of
added tetrabutylammonium triflate so that the final [triflate
anion]total ranged between 2.0 and 8.0 mmol/L. The effect of
[triflate anion] on the catalyzed reactions in ethanol was deter-
mined by following the change in reactivity of 0.08 mmol/L
4:Zn(II)2:(–OCH2CH3) mediating the cleavage of 1a
(0.05 mmol/L) in ethanol with varying tetrabutylammonium
Results
4:Zn(II)2:(–OCH3)-catalyzed cyclization of 1a–1g in
methanol
–
triflate concentration so that the final [CF3SO3 ]total ranged be-
As is the case with 3:Zn(II)2-catalyzed methanolysis reac-
tions, triflate anion inhibits the catalysis afforded by
4:Zn(II)2 (inhibition constant, Ki = 7.2 0.9 mmol/L; see
the Supplementary data). The raw kinetic data are accord-
ingly corrected for triflate inhibition to determine the con-
centration of free catalyst, [4:Zn(II)2:(–OCH3)]free. The
detailed methodology for these treatments can be found in
earlier publications8,9 and is applied specifically to this sys-
tem in the Supplementary data.
Unfortunately, the kinetic data we have for the catalyzed
reactions with substrates 1 are available only over a
0.2 mmol/L < [4:Zn(II)2:(–OCH3)]free < 0.75 mmol/L range
due to the limited solubility of the catalyst (2.3 mmol/L in
one drive syringe of the stopped-flow reaction analyzer is
diluted by a factor of 2 on mixing) and to its relatively high
triflate inhibition constant. Due to these limitations, fitting
tween 0.3 and 3.4 mmol/L.
Kinetic titration of 4:Zn(II)2 for reactions of 1g in
methanol and 1b in ethanol
Ethanol
Varying amounts of NaOCH2CH3 or HClO4 stock solu-
tions (5 mmol/L) in ethanol were added to solutions contain-
ing 1.6 ꢀ 10–4 mol/L of a preformed 4:Zn(II)2:(–OCH2CH3)
complex in ethanol, which had been prepared 80 min in ad-
vance to allow for complete formation of the complex. After
the introduction of the additional acid or base, the mixture
was allowed to stand for 15 min to equilibrate (independent
experiments showed that this time was optimum to stabilize
the kinetic activity) and then loaded into one syringe of the
stopped-flow analyzer while a 1.0 ꢀ 10–4 mol/L solution of
1b in ethanol was loaded into the other. The final concentra-
of the data to any given model has a larger error than was
8–10
tions after mixing were 8 ꢀ 10–5 mol/L of 4:Zn(II)2 and 5 ꢀ
the case with [3:Zn(II)2:(–OCH3)]free
.
Nevertheless, the
S
S
10–5 mol/L of 1b in ethanol. The pH values were measured
following limited observations and analysis can be made.
Published by NRC Research Press

Liu et al.
643
Fig.2. A plot of kobs vs [4:Zn(II)2:(–OCH3)]free for the catalyzed
Fig.3. A plot of kobs vs [4:Zn(II)2:(–OCH3)]free for the catalyzed
methanolysis of 1a (5 ꢀ 10–5 mol/L) determined from the rate of
methanolysis of 1g (5 ꢀ 10–5 mol/L) determined from the rate of
S
S
S
S
appearance of product phenol at 320 nm, pH 9.5 0.2, and 25.0
appearance of product phenol at 295 nm, pH 9.5 0.2, and 25.0
0.1 8C. Linear regression of the data gives a second order rate con-
stant (k2) of (2.08 0.04) ꢀ 104 (mol/L)–1s–1 with an intercept of A
= 0.12 0.02 mmol/L, r2 = 0.998.
0.1 8C. Fitting the data to the expression given in eq. [S1] of the
max
cat
Supplementary data gives k
(1.9 0.3) ꢀ 10–4 mol/L, and an x-intercept of A = 0.20
= (5.2 0.4) ꢀ 10–1 s–1, KM
=
0.01 mmol/L, r2 = 0.986.
The more reactive substrates (1a–1d) exhibited a linear
relationship between kobs versus [4:Zn(II)2:(–OCH3)]free for
the catalyzed reactions, whereas the less reactive ones
(1e–1g) showed apparent saturation over that same concen-
tration range. Shown in Figs. 2 and 3 are kobs versus
[4:Zn(II)2:(–OCH3)]free plots for the catalyzed transesterifi-
cations of 1a and 1g. It was earlier observed that the kobs
versus [3:Zn(II)2:(–OCH3)]free plots displayed a prominent
x-intercept that was attributed to a dissociation of Zn(II)
away from the catalytic complex at very low concentra-
tions,8–10 and the data in Figs. 2 and 3 show the same
trend with [4:Zn(II)2:(–OCH3)]free. The kinetic kobs versus
catalyst concentration data for substrates 1a–1c are linear
(as are those for 1d, which may have a perceptible down-
ward curvature but are analyzed as being linear) over the
concentration range investigated. However, these data for
1e–1g show a definite downward curvature suggestive of
saturation binding.
[–OCH2CH3] is in unity (see the Supplementary data). Sec-
ond, the inhibition constant of triflate anion on the cataly-
sis afforded by 4:Zn(II)2:(–OCH2CH3) in ethanol is found
to be Ki = 1.21 mmol/L (see the Supplementary data) sug-
gesting that triflate inhibition is even stronger in ethanol
than in methanol. However, the triflate inhibition observed
with 4:Zn(II)2:(–OCH2CH3) is not as significant as that for
the 3:Zn(II)2:(–OCH2CH3) system in ethanol (Ki = 0.36
0.02 mmol/L).14 Third, as is the case with the 3:Zn(II)2 sys-
tem in ethanol,14 triflate is an uncompetitive21 inhibitor of
the 4:Zn(II)2:(–OCH2CH3)-catalyzed reactions. Thus, the raw
kinetic data (kuobncsorr) were corrected for triflate inhibition us-
ing eqs. [1] and [2] as described14 to yield kocobrsr values.
½1ꢁ
½4:ZnðIIÞ2:ðꢂOCH2CH3Þꢁfree
ðKiÞ½4:ZnðIIÞ2:ðꢂOCH2CH3Þꢁtotal
¼
Ki þ ½ꢂOTfꢁ
The saturation kinetics observed for each of 1e–1g were
fit by nonlinear least-squares (NLLSQ) treatment to a uni-
versal binding equation presented in earlier reports,8–10,14
which is applicable to both weak and strong binding cases
(this is given as eq. [S1] of the Supplementary data, which
uncorr
obs
k
ðKi þ ½ꢂOTfꢁÞ
corr
obs
½2ꢁ
k
¼
Ki
corr
obs
The
k
values obtained as
a
function of
[4:Zn(II)2:(–OCH2CH3)] in ethanol follow saturation be-
haviour with a strong binding for the entire series of 1a–1g
as is exemplified by the plot shown in Fig. 4 for 1a. We
have not used buffers due to the inhibitory effect of their
also describes the mathematical treatment to account for the
max
cat
x-intercept). In Table 1, the best fit KM and k
constants
for the [4:Zn(II)2:(–OCH3)]free catalyzed transesterification
of 1e–1g are presented, along with the experimentally ob-
served second order rate constants (kc2at) for substrates 1a–
1d derived from the linear regression of the rate constant
versus [4:Zn(II)2:(–OCH3)]free data.
associated anions, so it is important to note that the solu-
S
S
tion pH decreases slightly as the ratio of catalyst concentra-
tion/[1] increases up to the point where it finally stabilizes at
a reasonably constant value of 9.0 0.3 (measured after the
stopped-flow reaction) at the plateau region, where the first
4:Zn(II)2:(–OCH2CH3)-catalyzed cyclization of 1a–1g in
ethanol
order rate constant of the catalyzed reaction, corrected for
max corr
cat
triflate inhibition is defined as k
.
The rates of the reactions of 1a–1g as a function of in-
creasing [4:Zn(II)2:(–OCH2CH3)] exhibit saturation kinetics
with very strong binding (see the Supplementary data), sim-
ilar to what was observed previously with 3:Zn(II)2 in etha-
nol.14 A few key points are notable. First, a kinetic study
where the [–OCH2CH3] is varied shows that the rate for the
cyclization of 1a is highest when the ratio of [4:Zn(II)2]/
Fitting of the kinetic data to the universal binding equa-
tion and iteratively varying the 4:Zn(II)2:(–OCH2CH3):1 dis-
sociation constant (KM) until the goodness-of-fit is
maximized14 allows estimation of upper limits for the KM
max corr
cat
(10–7.5 mol/L (3.2 ꢀ 10–8 mol/L)) and k
values for
catalytic cleavage of the bound substrates given in Table 2.
Published by NRC Research Press

644
Can. J. Chem. Vol. 87, 2009
Table 1. Kinetic constants (maximum rate constants (kmcaatx), Michaelis–Menten constants (KM), and second-
order rate constants (k2 or kmcaatx/KM)) for the cleavage of 1a–1g mediated by [4:Zn(II)2:(–OCH3)]free in anhy-
S
S
drous methanol at pH 9.5 0.2 and 25.0 0.1 8C.
max
cat
S
kc2at
k
KM (mol/L)
(ꢀ104)a
Accel. pH 9.5
S
S
S
Subs
1a
1b
1c
1d
1e
Phenol pKa
((mol/L)–1s–1 ꢀ 10–3)
(s–1 ꢀ 10)a
(ꢀ10–11
)
11.30
11.50
12.49
13.59
13.93
14.33
14.77
20.8 0.4
15.1 0.7
13.8 0.8
2.6 0.1
2.3 1.3d
4.0 1.0d
2.7 0.5d
NA
NA
NA
NA
9.5 1.9
7.2 0.7
5.2 0.4
NA
1.5b
NA
NA
NA
4.8 3.1
1.8 0.4
1.9 0.3
5.3b
3.8b
9.4c
10.5c
11.1c
16.2c
1f
1g
max
cat
aKM and k
values were not determined for the faster reacting substrates (1a–1d).
bAcceleration in terms of 1 mmol/L catalyst reacting with the observed second order rate constant relative to the
methoxide reaction at SSpH 9.5.
cAcceleration in terms of kmcaatx relative to the methoxide reaction at SpH 9.5 where [–OCH3] = 5.37 Â 10–8 mol/L.
S
dThe k2 values for 1a–1d are the experimentally observed values from the slopes of the kobs versus [4:Zn(II)2:(–OCH3)]free
plots. The k2 values in italics are computed from kmcaatx/KM.
Fig. 4. A plot of kobs versus [4:Zn(II)2:(–OCH2CH3)]total for the cat-
file shown in Fig. 1 for the cleavage of 1b catalyzed by
4:Zn(II)2 in ethanol can be constructed as described in the
S
alyzed cleavage of 1a (5 ꢀ 10–5 mol/L) at 323 nm, pH 9.0 0.3,
S
and 25.0 0.1 8C. Fitting of the data to the universal binding ex-
pression given in the Supplementary data gives kmcaatx = 22.4 0.5 s–1
and an intercept, A = (2.5 0.1) ꢀ 10–5 mol/L. The raw kinetic data
Experimental above. The plot exhibits a broad plateau from
S
S
.pH 7.9–10 and a precipitous drop in rate constant at higher
and lower values. The line through the data was derived
from a fit of the solid squares data to a kinetic process that
depended on two ionizable groups14 having first and second
–
have been corrected for OTf inhibition as described in the text.
S
S
.pK values of 8.48 0.02 and 10.38 0.03. Only the data
a
in the shaded box were used for the fitting because of the
S
S
rapid fall-off in the rate constant at lower and higher pH
values, where the 4:Zn(II)2 complex that is substantially de-
void of alkoxide or has more than one alkoxide apparently
decomposes during the timescale of the experiment.
S
S
In an analogous way, the kinetic pKa values were deter-
mined for 4:Zn(II)2 in methanol from the reaction of
4:Zn(II)2:(–OCH3) with 1g conducted at 25 8C in methanol
as described in the Experimental above. The fit of the bell-
shaped data set enclosed in the dotted box of Fig. 1 gave
S
S
two pK values of 9.38 0.07 and 9.80 0.06.
a
Discussion
General considerations
Shown in Fig. 5 is a Brønsted plot of log (kmcaatx corr) for the
Earlier studies of the cyclization of 1a–1g promoted by
3:Zn(II)2(–OR) in methanol9 and ethanol14 were interpreted
in terms of the general process illustrated in Scheme 1 in-
volving at least two binding events, followed by one or
more chemical steps that released the observed phenol prod-
uct of the reaction. The binding steps were proposed to in-
volve phosphate diester association with one metal followed
by a rearrangement where it becomes doubly activated via
binding to the second metal ion. The chemical step was con-
sidered to involve internal deprotonation of the 2-hydroxyl
group followed by cyclization with expulsion of the aryloxy
leaving group. The major difference between methanol and
ethanol was the very much stronger binding of the anionic
alkoxide and substrate to the complex in ethanol. Thus, for
the process in Scheme 2, the first substrate binding step (k1/
k–1) is very much larger in ethanol. The findings with
4:Zn(II)2 in the present study are consistent with this general
scheme.
S
catalyzed reactions versus the SpKa values14 of the corre-
sponding phenols in ethanol. The two lines through the data
are from a linear regression and a nonlinear regression as
described in the legend of Fig. 5.
S
Determination of kinetic pK values for formation of
S
a
4:Zn(II)2:(–OR) in methanol and ethanol
In the absence of any buffer, the pH values depend on
S
S
the catalytic system itself and were generally found to be
corr
obs
9.0
0.3 in the plateau region of the k
versus
[4:Zn(II)2:(–OCH2CH3)]total plots. Unfortunately, as was the
case with 3:Zn(II)2, the complexes with 4:Zn(II)2 require re-
S
S
equilibration when the pH is altered so it is not possible to
undertake potentiometric titrations of the complexes in ei-
ther methanol or ethanol due to the time required for the
catalyst to reach equilibrium following addition of each ali-
S
S
max corr
obs
quot of acid or base. However, the kinetic pH/k
pro-
Published by NRC Research Press

Liu et al.
645
Table 2. Maximum catalytic rate constant (kmcaatx corr), for the 4:Zn(II)2:(–OCH2CH3)-catalyzed
reactions, second order rate constants for the ethoxide-promoted reactions (k–2OEt),14 and accel-
S
S
erations of cleavage provided for bound 1a–1g at pH 9.0 in ethanol at 25 0.1 8C.
max corr
cat
catalyzed reactions
22.4 0.5
10.9 0.2
SSpKa
phenol
k
(s–1) for the
k–2OEt ((mol/L)–1s–1) for Acceleration by
S
a
Subs
1a
1b
1c
1d
1e
the ethoxide reactions
(2.43 0.09)ꢀ10–4
(8.3 0.4)ꢀ10–5
(9.7 0.7)ꢀ10–6
(6.3 0.3)ꢀ10–7
3.7ꢀ10–7 c
catalyst at pH 9.0b
S
12.05
12.39
13.60
14.83
15.15
15.60
15.92
1.2ꢀ1015
1.7ꢀ1015
9.5ꢀ1015
3.2ꢀ1016
1.7ꢀ1016
2.8ꢀ1016
4.2ꢀ1016
7.3 0.3
1.60 0.05
0.50 0.02
0.31 0.01
0.25 0.01
1f
1g
1.4ꢀ10–7 c
7.4ꢀ10–8 c
akmcaatx values are determined as described in text by fitting the kccaotrr vs [4:Zn(II)2:(–OCH2CH3)] data to
eq. [S1], Supplementary data.
bAcceleration was calculated at SSpH 9.0 in ethanol, which corresponds to 10(–19.1 + 9.0) mol/L of ethoxide. For
example, for substrate 1c the acceleration = 7.3 s–1/[(10–10.1 mol/L) Â (9.7 Â 10–6 (mol/L)–1 s–1)] = 9.5 Â 1015.
ck–2OEt values for the base promoted reactions of the less reactive substrates (1e–1g) were estimated from
the linear regression equation for the Brønsted plot in Fig. 1 of ref. 14.
S
Fig. 5. Brønsted plot of log (kmcaatx corr) vs the pKa values of the
Scheme 3. Thermodynamic cycle for assessing the binding energy
of catalyst 4:Zn(II)2 to a hypothetical transition state comprising
alkoxide and substrate.
S
corresponding phenol leaving groups for the 4:Zn(II)2:(–OCH2CH3)-
catalyzed cleavage of 1a–1g in absolute ethanol at 25 8C. The line
was constructed by NLLSQ fitting all the data to an expression
max corr
cat
k
= k1k2 / (k–1 + k2) = C1C210(b1+b2)pKa/(C–110b–1pK
+
a
C210b2pKa).22 The two fitted b values are –0.76 and –0.10, r2 =
0.9768, with a point of transition at pKa = 14.3. The linear
S
S
regression of the data (dotted line) has a gradient of –0.50 0.05,
r2 = 0.9537 (7 data).
for 4:Zn(II)2 than 3:Zn(II)2, the concentration range over
which the former’s catalytic activity could be investigated
was limited, which introduces considerable error into the
max
cat
calculated k
and KM values given in Table 1. Neverthe-
less, it appears that 4:Zn(II)2:(–OCH3) is slightly less cata-
lytically active than its lower homologue and binds slightly
weaker with phosphates 1e–1g than does 3:Zn(II)2:(–OCH3)
in methanol, although the differences for substrates where
saturation behaviour is observed are not large. When com-
paring the maximum rate constants (kmcaatx) for the bound sub-
strates 1e–1g, a given reaction promoted by 3:Zn(II)2 is
about 3–5 times faster than that with 4:Zn(II)2. In this case,
the shift from a rate-limiting binding process (for 1a–1c) to
a rate-limiting chemical process (for 1e–1g) seems to occur
around substrate 1d (see the Supplementary data).
Scheme 2. R’ = CH3 or CH2CH3
The Brønsted plot (not shown) of the observed k2 or com-
puted kmcaatx/KM values for the entire series of substrates
gives a poorly defined linear regression, log(k2) = –(0.28
S
0.05) pK + (7.41 0.70) (r2 = 0.84, 7 data). This can be
S
a
4:Zn(II)2:(–OCH3)-promoted cleavage of 1a–1g in
methanol
compared with what was found for the 3:Zn(II)2:(–OCH3)
max
cat
S
S
reaction, log k
= (–0.97 0.05) pKa + (14.36 0.57),
and with the regression of the CH3O–-promoted reaction,
The kobs versus catalyst concentration profiles for cata-
lyzed cleavage of phosphates 1a–1g of both 3:Zn(II)2 and
4:Zn(II)2 complexes show the same trend of being linear for
substrates containing good leaving groups (1a–1c, e.g., Fig.
2) and downward curved for substrates with poorer leaving
groups (1e–1g, e.g., Fig. 3). Unfortunately, due to the com-
bination of poorer solubility and a greater triflate inhibition
log(k–2OMe) = (–0.72 0.08) pK + (5.36 1.08).9 In the
S
S
a
case of the C3 catalyst, the larger negative blg is consistent
with a rate limiting step where there substantial cleavage
of the P–OAr bond. The apparent reduced value of the blg
observed for the catalyzed reaction promoted by
4:Zn(II)2:(–OCH3) indicates a more modest dependence on
Published by NRC Research Press

646
Can. J. Chem. Vol. 87, 2009
– OR
Table 3. Summary of the (kmcaatx/KM/k2 constants and the computed free energies for the formation
of catalytic complexes (DGBind – DGM), the free energies of activation for kmcaatx (DGc{at), and the
free energies of stabilization of the RO–:1 transition state through its binding to 4:Zn(II)2 (DDG{stab
)
as in the reaction of 4:Zn(II)2:(–OR) with substrates 1a–1g at 25 8C in methanol (M) and ethanol
(E).
DGBind – DGM
DG{cat
DG{Non
DDG{stab
(kcal/mol)
– OR
Subs
kmcaatx/KM/k2
(kcal/mol)b
(kcal/mol)c
(kcal/mol)c
1a (M)d
1a (E)
1b (M)d
1b (E)
1c (M)d
1c (E)
1d (M)d
1d (E)
1e (M)
1e (E)
1f (M)
1f (E)
8.0ꢀ106
2.9ꢀ1012
2.8ꢀ107
4.2ꢀ1012
2.1ꢀ107
2.4ꢀ1013
5.0ꢀ107
8.0ꢀ1013
1.4ꢀ108
4.3ꢀ1013
3.3ꢀ108
7.1ꢀ1013
4.5ꢀ108
1.1ꢀ1014
20.9
22.3
21.9
23.0
21.7
24.2
23.3
25.9
23.9
26.2
24.1
26.8
24.5
27.1
–19.5e
–31.5
–20.2e
–31.7
–20.0e
–32.7
–20.6e
–33.4
–21.1
–33.1
–21.7
–33.3
–21.9
–33.6
–24.7
–24.7
–24.7
15.6
16.0
16.2
–24.7
–14.6
–24.7
–15.1
–24.7
–15.1
–24.7
17.1
17.4
17.8
17.6
18.1
17.8
18.2
1g M)
1g (E)
aKinetic first SpKa in methanol determined as in the Experimental section to be 10–9.38; KWmethanol = 10–16.77; (Ka/
Kw)M = 2.5 Â 107; DGBMienOdH = –10.1 kcal/mol. Kinetic first SSpKa in ethanol is 10–8.48;; KWethanol = 10–19.1; (Ka/Kw)E =
4.16 Â 1010; DGBEtiOndH = –14.5 kcal/mol.
S
bComputed as (DGBind – DGM) = –RTln((Ka/Kw)/KM).
– OR
cComputed from DGc{at = –RTln(kmcaatx/(kT/h)) or DGN{ on = –RTln(k2 /(kT/h)) from the Eyring equation where
(kT/h) = 6 Â 1012 s–1 at 298 K.
dkcmaatx and KM values for substrates 1e–1g from Table 1 and 2; k2 values for substrates 1a–1d from Table 1
determined as gradients of the kobs vs [4:Zn(II)2:(–OCH3)]free plots. k2–OMe and k2–OEt values from refs. 9 and 14.
eDDG{ computed from application of kinetic and equilibrium constants to eqs. [3] and [4].
stab
the nature of the leaving group, and is consistent with the
mechanism shown in Scheme 1 where there is relatively
little cleavage of the P–OAr bond in the cases where the
chemical steps are rate limiting. This could be indicative
of a stepwise cleavage reaction where there is little change
in the P–OAr bonding in a rate limiting cyclization step
forming a phosphorane intermediate or alternatively to a
concerted process with an early transition state having a
component of the intramolecular rearrangement. Possibly
due to the increased flexibility of the longer linker chain,
this rearrangement process is slower for 4:Zn(II)2:(–OCH3)
than 3:Zn(II)2:(–OCH3) as evidenced by the k2 values
(Table 1) for 1a and 1b of 21 000 and 15 000 (mol/L)–1s–1,
respectively, which are 13 times lower than observed for
those same substrates when reacted with 3:Zn(II)2:(–OCH3)
(275 000 and 194 000 (mol/L)–1s–1, respectively9).
as the dissociation constant for the 4:Zn(II)2:(–OCH2CH3):1
Michaelis complex, is *10–7.5 mol/L (3.2 ꢀ 10–8 mol/L).
S
The Brønsted plot of log(kmcaatx corr) versus the pKa of the
S
corresponding phenols of 1a–1g is shown in Fig. 5. Due to
max corr
cat
the rather scattered nature of the k
data, we cannot
distinguish with certainty whether the plot is linear (dotted
line suggestive of a single step being rate limiting with blg
= 0.5) or a downward curving line suggestive of a change
max corr
cat
in rate limiting step for the k
term. The latter would
be consistent with what was proposed for the 3:Zn(II)2:(–
OCH2CH3) system in ethanol where there was a transition
from substrate binding being rate limiting for substrates
with good leaving groups, and chemical cleavage being rate
limiting for substrates with poorer leaving groups.14 The line
through the data in Fig. 5 is obtained by NLLSQ fitting to
the equation given in the caption22 depending on the two
b values of –0.76 0.33 for poor leaving groups, and –0.1
0.69 for good leaving groups. Although the parameters,
particularly the latter one, are poorly defined in the fit, the
blg value of –0.1 suggests that the rate limiting step for
this process does not involve much change in the POAr
bonding as would expected for a process such as depicted
in Scheme 1 involving a rate limiting rearrangement step.14
The data also support the contention that the rearrange-
ment process for 4:Zn(II)2:(–OCH2CH3) is about 10 times
4:Zn(II)2:(–OCH2CH3)-promoted cleavage of 1a–1g in
ethanol
The findings for 4:Zn(II)2-promoted cleavage of 1a–1g in
ethanol are very similar to those found for 3:Zn(II)2 in etha-
nol.14 Following correction of the kinetic data for triflate in-
corr
obs
hibition (as described in ref. 14) the plots of k
versus
[4:Zn(II)2:(–OCH2CH3)]total for all substrates showed very
strong binding behavior illustrated in Fig. 4. Due to the strong
binding, exact determination of the KM values is not possible,
so an iterative procedure was used14 where the KM was held
constant, but varied until the goodness-of-fit was optimized.
This upper limit for the KM value for all substrates, defined
slower than that for 3:Zn(II)2:(–OCH2CH3) since the
max corr
cat
respective k
values of substrates 1a and 1b are 22 and
11 s–1 for the C4 catalyst and 168 and 133 s–1 with the C3 cata-
lyst, respectively.
Published by NRC Research Press

Liu et al.
647
Energetics of 4:Zn(II)2:(–OR) catalyzed cleavages of 1a–
1g in methanol and ethanol
tributes –11.0 to –13.2 kcal/mol in methanol and
approximately –17 to –19 kcal/mol in ethanol, with the
slowest reacting substrates having the more negative val-
ues. These numbers give the energetic reduction of the cat-
alyzed apparent second order rate constant for the 4:Zn(II)2
promoted reactions relative to the alkoxide reactions.
One can quantify the catalytic efficiency by calculating
the difference in energy of a transition state containing only
[RO–:1], and one where the latter is bound to the catalyst23
to form a hypothetical transition state [4:Zn(II)2:(–OR):1]{
or its kinetic equivalent [4:Zn(II)2:(–1)]{. Presented in
Scheme 3 is a thermodynamic cycle involving both the alk-
oxide reaction and the 4:Zn(II)2:(–OR) promoted reactions of
1a–1g. Equation [3] provides the free energy difference
(DDGs{tab) between [4:Zn(II)2:(–OR):1]{ relative to [RO–:1]{.
In Scheme 2, DGBind, DG{cat and DGN{ on are the respective
free energies for (1)
Conclusions
The rate constants and energetics for the cyclizations of
1a–1g promoted by 4:Zn(II)2 found in this study invite com-
parison with what was found recently for the same cycliza-
tion reactions catalyzed by 3:Zn(II)2 in both methanol9 and
ethanol.14 In methanol, 4:Zn(II)2 is somewhat more fragile
than 3:Zn(II)2 as is judged from the kinetic stability as a
function of time. Whereas 3:Zn(II)2 retains good activity for
nearly one day, the activity of 4:Zn(II)2 diminishes after
about 10 h. Additionally, 4:Zn(II)2 suffers from a somewhat
reduced solubility in methanol and a larger triflate ion inhib-
ition, which reduces the overall concentration range over
which the study can be conducted. Although we have deter-
mined that 4:Zn(II)2 can promote cleavage of the DNA
models 2 with appreciable rate accelerations, the above tech-
nical factors prevent determination of meaningful binding
and kcat constants for the entire series that was investigated
with 3:Zn(II)2 in methanol.8,10
¼
¼
¼
½3ꢁ
DDGstab ¼ ðDGBind ꢂ DGM þ DGcatÞ ꢂ DGNon
ꢀ
ðkcat=KMÞðKa=KwÞ
¼ ꢂRTln
ꢁ
ꢂOR
k2
À
Á
¼
¼4:ZnðIIÞ2:ðꢂORÞ
¼
½4ꢁ
DDGstab ¼ DGBind þ DGcat
ꢂ DGNon
ꢀ
ꢁ
ðk2obsÞðKa=KwÞ
¼ ꢂRTln
k2ꢂOR
methoxide binding to 4:Zn(II)2, (2) the free energy of acti-
vation for unimolecular reaction of the 4:Zn(II)2:(–OR):1
complex or its kinetic equivalent, and (3) the free energy of
activation for the reaction of alkoxide with 1. DGM is the
free energy for association of 1 and 4:Zn(II)2:(–OR) com-
puted from the Michealis constant for each substrate in
either solvent. In eq. [4] is the free energy calculation speci-
fic for the reactions with 1a–1d in methanol that show only
a first order dependence on [4:Zn(II)2:(–OCH3)]free through-
out the concentration range investigated. The various terms
in eqs. [3] and [4] are described in earlier publications.9,14
Ka/Kw can be readily shown to be formally equivalent to
the binding constant of alkoxide to the dinuclear complex
4:Zn(II)2 to form 4:Zn(II)2:(–OR). Ka refers to the first acid
In spite of these difficulties, it appears that the overall
mechanism by which 4:Zn(II)2 cleaves 1 is similar to what
we proposed to be operative for 3:Zn(II)2. From the avail-
able data in Table 1 above and the comparable data in Ta-
max
cat
ble 4 of ref. 9, the k
and KM terms for both catalysts are
similar for substrates where the chemical steps are rate lim-
iting (1e–1g), although the binding of the C4 catalyst for
these slower reacting substrates is slightly weaker. On the
other hand, the apparent second order rate constants for
those substrates that do not show saturation kinetics, and
where the rate limiting step is proposed to be a nonchemical
one, such as substrate binding and translocation (1a–1d), is
about one order of magnitude less than was seen for
3:Zn(II)2. Perhaps this is a consequence of the flexibility of
the system, which resists the putative intramolecular rear-
rangement to reposition the first bound substrate complex
into a doubly-activated complex as shown in Scheme 1.
In ethanol, both catalysts display very strong binding for
which only upper limits can be set for the dissociation of
the Michaelis complex (10–7.5 and 10–6.5 mol/L, respectively,
for 4:Zn(II)2 and 3:Zn(II)2). The apparent greater binding
constant for the C4 complex relative to the C3 complex prob-
ably cannot be considered significant given that the determi-
nation of the KM values in each case gave only an upper
limit for the dissociation constant. When comparing the data
obtained with the C4 catalyst in this study with that disclosed
in Table 3 of ref.14 for the C3 catalyst, the (DGBind – DGM)
term, which refers to the composite energy of binding of the
ethoxide and substrate to the catalyst, is nearly the same for
each catalyst (only about *0.4 kcal/mol less for the C3 com-
plex than the C4 complex). This results from an apparent
stronger binding of the phosphate substrate to the C4 catalyst
dissociation constant of 4:Zn(II)2:(HOR), determined from
S
the kinetic pKa studies above as 10–8.48 in ethanol and 10–
S
9.38
in methanol, and Kw is the autoprotolysis constant for
– OR
ethanol (10–19.1) and methanol (10–16.77). Finally, k2 is the
second order rate constant for the methoxide9 and ethoxide14
reactions of each substrate.
– OR
– OR
Listed in Table 3 are the kcat/KM/k2 (or k42:Zn(II) :OMe/k2
)
2
constants and computed DGBind, DGM, DGcat, DGNon, and
DDG{stab values for the various processes described in
Scheme 2 for the complex and alkoxide catalyzed and reac-
tions of 1; all values were computed at the standard state of
1 mol/L and 25 8C. The first ratio provides the acceleration
in second order rate constant for the complex catalyzed re-
actions relative to the alkoxide reactions, while the various
DG values, when inserted into eqs. [3] and [4], provide the
free energy of stabilization (DDG{stab) of the complex cata-
lyzed reaction relative to the methoxide reaction. Of these
constants, the Ka/Kw terms contributes –10.1 and
–
14.5 kcal/mol while the (DGBind – DGM) term contributes
approximately –15 and –24.5 kcal/mol in methanol and
ethanol, respectively, to the DDG{stab for the series. This
means that in the less polar solvent, the binding of both
alkoxide and substrate to the 4:Zn(II)2 complex is more ex-
ergonic. In the two solvents, the kcat/KM/k–2OMe term con-
compensated by weaker binding of ethoxide. What is inter-
max
cat
esting is the k
values for the various substrates are about
5–20 times less for the C4 complex relative to the C3 com-
Published by NRC Research Press

648
Can. J. Chem. Vol. 87, 2009
Tonellato, U. Chem. Commun. (Camb.) 2006, 2540;
(c) Morrow, J. R.; Iranzo, O. Curr. Opin. Chem. Biol. 2004,
8, 192 doi:10.1016/j.cbpa.2004.02.006. PMID:15062781.; (d)
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that this stems from a less tightly bound substrate/catalyst
complex at the transition state for the C4 catalyzed process,
perhaps suggesting that flexibility, while required for the ini-
tial strong catalyst:substrate binding, has a deleterious effect
on the transition state for the catalyzed reaction.
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Supplementary data
Tables of original pseudofirst order rate constants for the
cleavage of 1a–1g as a function of [4:Zn(II)2:(–OR)] in
methanol and ethanol; Figures plotting the latter; informa-
S
S
max
obs
(6) Yatsimirsky, A. K. Coord. Chem. Rev. 2005, 249, 1997.
doi:10.1016/j.ccr.2005.04.016.
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profiles to
determine the kinetic first and second SSpKa values for the
complex in both solvents. Supplementary data for this article
are available on the journal Web site (canjchem.nrc.ca) or
may be purchased from the Depository of Unpublished
Data, Document Delivery, CISTI, National Research Coun-
cil Canada, Ottawa, ON K1A 0R6, Canada. DUD 3908. For
more information on obtaining material, refer to cisti-icist.
nrc-cnrc.gc.ca/cms/unpub_e.shtml.
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Acknowledgements
The authors gratefully acknowledge the financial assis-
tance of the Natural Sciences and Engineering Research
Council of Canada (NSERC), the Canada Council of the
Arts through the award of a Killam Research fellowship to
RSB, and the Canada Foundation for Innovation (CFI). This
project received support from the Defense Threat Reduction
Agency - Joint Science and Technology Office, Basic and
Supporting Science Division, Grant # HDTRA-08-1-0046.
In addition, CTL thanks NSERC for a Postgraduate Scholar-
ship (PGS D-2).
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C. J.; Melnychuk, S. A.; Brown, R. S. J. Am. Chem. Soc.
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S
´
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