Supramolecular catalysis of orthoformate hydrolysis in basic solution: An enzyme-like mechanism

Article abstract of DOI:10.1021/ja802839v

A water-soluble self-assembled supramolecular host molecule catalyzes the hydrolysis of orthoformates in basic solution. Comparison of the rate constants of the catalyzed and uncatalyzed reactions for hydrolysis displays rate accelerations of up to 3900 for tri-n-propyl orthoformate. Kinetic analysis shows that the mechanism of hydrolysis with the supramolecular host obeys the Michaelis-Menten model. Mechanistic studies, including ¹³C-labeling experiments, revealed that the resting state of the catalytic system is the neutral substrate encapsulated in the host. Activation parameters for the k _cat step of the reaction revealed that upon substrate encapsulation in the assembly, the entropy of activation becomes more negative in contrast to the uncatalyzed reaction. Furthermore, solvent isotope effects reveal a normal k(H₂O)/k(D₂O) = 1.6, confirming an A-S_E2 mechanism in which proton transfer occurs in the rate-limiting step. This is in contrast with the A1 mechanism of the uncatalyzed reaction in which decomposition of the protonated substrate is rate-limiting.

Full text of DOI:10.1021/ja802839v

Published on Web 08/05/2008
Supramolecular Catalysis of Orthoformate Hydrolysis in Basic
Solution: An Enzyme-Like Mechanism
Michael D. Pluth, Robert G. Bergman,* and Kenneth N. Raymond*
Department of Chemistry, UniVersity of California, Berkeley, California, and DiVision of
Chemical Sciences, Lawrence Berkeley National Laboratory, Berkeley, California, 94720-1460
Received April 17, 2008; E-mail: rbergman@berkeley.edu; raymond@socrates.berkeley.edu
Abstract: A water-soluble self-assembled supramolecular host molecule catalyzes the hydrolysis of
orthoformates in basic solution. Comparison of the rate constants of the catalyzed and uncatalyzed reactions
for hydrolysis displays rate accelerations of up to 3900 for tri-n-propyl orthoformate. Kinetic analysis shows
that the mechanism of hydrolysis with the supramolecular host obeys the Michaelis-Menten model.
Mechanistic studies, including 13C-labeling experiments, revealed that the resting state of the catalytic
system is the neutral substrate encapsulated in the host. Activation parameters for the kcat step of the
reaction revealed that upon substrate encapsulation in the assembly, the entropy of activation becomes
more negative in contrast to the uncatalyzed reaction. Furthermore, solvent isotope effects reveal a normal
k(H2O)/k(D2O) ) 1.6, confirming an A-SE2 mechanism in which proton transfer occurs in the rate-limiting
step. This is in contrast with the A1 mechanism of the uncatalyzed reaction in which decomposition of the
protonated substrate is rate-limiting.
observed in biological systems.9 This suggests that transition-
state stabilization alone cannot explain the high reactivity of
Introduction
Enzymes are able to efficiently carry out a variety of chemical
transformations with extraordinary specificity and acceleration
over the uncatalyzed process, including the hydrolysis of
otherwise hydrolytically stable molecules. For example, DNases
are capable of hydrolyzing phosphate diester bonds in DNA
within seconds,1–3 while under the same conditions, in the
absence of enzyme, this hydrolysis has an estimated half-life
of 200 million years.4 Similarly, enzymatic peptidases are able
to cleave peptide bonds quite efficiently with rate accelerations
of up to 1010-1012 when compared to the background hydrolysis
under physiological conditions.5–7 Inspired by this enzyme-
mediated reactivity, synthetic chemists have sought to both
explain and emulate such reactivity in synthetic molecular
assemblies. Despite the intense study of enzymatic selectivity
and efficiency, a complete understanding of how enzymes are
able to achieve such heightened reactivity remains elusive. As
early as 1946, Linus Pauling suggested that enzymes must
preferentially recognize and stabilize the transition state over
the ground state of a reaction.8 However, Houk et al. recently
reported a survey of binding affinities in a wide variety of
enzyme-ligand, enzyme-transition-state, and synthetic host-
guest complexes and found that the average binding affinities
were insufficient to generate many of the rate accelerations
enzymes, but rather other forces must contribute to the activation
of substrate molecules. The nature and magnitude of these forces
remains an active area of research.10–15
Enzymes have evolved to create elaborate active sites often
containing precise arrangements of hydrogen-bonding networks,
electrostatic interactions, and functional group availability
specifically aimed at increasing the reactivity of bound sub-
strates. Although the development of synthetic host-guest
systems has not reached the level of enzyme specificity, the
characteristics of each synthetic assembly, such as the size,
shape, charge, and functional group availability, greatly influence
the guest-binding characteristics and have led to remarkable and
often unexpected reactivity.9,16–23 For example, the increased
local concentration upon encapsulation of substrates for bimo-
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A R T I C L E S
Pluth et al.
lecular reactions has been exploited for enhanced reactivity
inside of synthetic hosts. By preorganizing substrates, supramo-
lecular assemblies are able to catalyze cycloadditions or
pericyclic reactions such as Diels-Alder reactions.24–26 In
addition to often large rate accelerations, encapsulation in
synthetic host molecules can alter the reactivity of substrates
to produce selectivity otherwise not observed in solution.27–30
Synthetic chemists have also used assemblies that preferen-
tially encapsulate charged guests to try to emulate the hydrolytic
efficiency of enzymes. Other supramolecular systems have
demonstrated the ability of the interior cavity or periphery of
the host to shift the pKa of encapsulated guests by up to 2 pKa
units,31–34 and we have shown shifts of up to 4.5 pKa units.35
This type of stabilization should also affect the transition states
of reactions which have high-energy protonated species on the
reaction coordinate. Assemblies able to concentrate solvent
molecules inside of the host cavity have been used to accelerate
the alcoholysis of alkyl halides with marked size selectivity.36
Natural cyclodextrins, such as ꢀ-cyclodextrin, have been used
to catalyze the hydrolysis of acetals at neutral pH presumably
by exploitation of the hydrogen-bonding network around the
periphery of the host.37 Similarly, functionalized synthetic
cyclodextrins have been used in the hydrolysis of glycosides at
physiological pH with sizable rate accelerations over the
background reaction.38–40 The reactivity which has been achieved
to date in synthetic molecular hosts exemplifies how simple and
defined local environments are able to alter substrate reactivity.
Over the past decade, Raymond and co-workers have used
the strategy of self-assembly to develop tetrahedral supramo-
lecular assemblies comprised of the stoichiometry M4L6 (M )
GaIII (1), AlIII, InIII, FeIII, TiIV, or GeIV, L ) N,N′-bis(2,3-
dihydroxybenzoyl)-1,5-diaminonaphthalene) (Figure 1).41,42
While the 12- overall charge of 1 imparts water solubility, the
naphthalene walls of the assembly provide a hydrophobic
interior cavity, able to encapsulate guests, which is isolated from
Figure 1. (Left) Schematic representation of 1 with only one ligand shown
for clarity. (Right) A space-filling model of 1 as viewed down the 2-fold
axis defined by the naphthalene-based ligand.
the bulk aqueous solution. A wide variety of small neutral and
monocationic guests including aliphatic hydrocarbons,43 pro-
tonated guests,44 simple organic cations,45 and reactive orga-
nometallic complexes46–48 have been encapsulated in 1. Analysis
of the mechanism for guest exchange revealed that 1 stays intact
during the guest exchange process and that the apertures along
the 3-fold axis of 1 dilate to allow for guest ingress and egress.45
Using 1 to mediate the reactivity of organometallic guests, both
stoichiometric and catalytic reactions have been carried out
inside of 1 with both size- and stereoselectivity.46–48 Further-
more, 1 has been used as a catalyst for the sigmatropic
rearrangement of enammonium cations.49 We have recently
reported the ability of 1 to greatly favor the protonated form of
encapsulated substrates such as amines, with pKa shifts of up
to 4.5 pKa units, and have exploited this stabilization for the
catalytic hydrolysis of orthoformates and acetals in basic
solution.44,50 Herein we expand upon our initial report of
orthoformate hydrolysis to include a detailed study of the
mechanism of hydrolysis in 1.
Results and Discussion
(23) Koblenz, T. S.; Wassenaar, J.; Reek, J. N. H. Chem. Soc. ReV. 2008,
37, 247–262.
Reactivity of Neutral Substrates. After our initial report of
the reactivity of cationic substrates in the [3,3] sigmatropic
rearrangement of enammonium cations, we hoped to expand
the catalytic potential of 1 by searching for reactions involving
neutral substrates that could be catalyzed by 1. With the
knowledge that both neutral and protonated guests could enter
1, and that 1 is able to shift the basicities of encapsulated guests,
reactions were sought in which high-energy protonated species
could be stabilized upon encapsulation. Many types of acid-
catalyzed reactions proceed through mechanisms involving high-
energy cationic species which potentially can be stabilized by
encapsulation in 1. Ideally, the final product of the host-mediated
hydrolysis would either be more weakly bound than the substrate
(24) Kang, J. M.; Hilmersson, G.; Santamaria, J.; Rebek, J., Jr J. Am. Chem.
Soc. 1998, 120, 3650–3656.
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B 2006, 110, 5132–5138.
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2007, 129, 12094–12095.
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2002, 124, 254–263.
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Ed. 2007, 119, 8741–8743.
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2007, 129, 11459–11467.
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7919.
(36) Ryu, E.-H.; Cho, H. K.; Zhao, Y. Org. Lett. 2007, 9, 5147–5150.
(37) Krishnaveni, N. S.; Surendra, K.; Reddy, M. A.; Nageswar, Y. V. D.;
Rao, K. R. J. Org. Chem. 2003, 68, 2018.
(46) Leung, D. H.; Bergman, R. G.; Raymond, K. N. J. Am. Chem. Soc.
2006, 128, 9781–9797.
(38) Ortega-Caballero, F.; Rousseau, C.; Christensen, B.; Petersen, T. E.;
Bols, M. J. Am. Chem. Soc. 2005, 127, 3238–3239.
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(40) Bjerre, J.; Nielsen, E. H.; Bols, M. Eur. J. Org. Chem. 2008, 4, 745–
752.
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2007, 129, 2746–2747.
(48) Leung, D. H.; Fiedler, D.; Bergman, R. G.; Raymond, K. N. Angew.
Chem., Int. Ed. 2004, 43, 963–966.
(49) Fiedler, D.; van Halbeek, H.; Bergman, R. G.; Raymond, K. N. J. Am.
Chem. Soc. 2006, 128, 10240–10252.
(41) Caulder, D. L.; Powers, R. E.; Parac, T. N.; Raymond, K. N. Angew.
Chem., Int. Ed. 1998, 37, 1840–1843.
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9
11424 J. AM. CHEM. SOC. VOL. 130, NO. 34, 2008

Supramolecular Catalysis of Orthoformate Hydrolysis
A R T I C L E S
Figure 2. Scope of orthoformates investigated in 1.
or be able to undergo further reaction in solution to prohibit
further encapsulation, in order to allow for catalytic turnover.
An ideal class of hydrolysis reaction is the acid-catalyzed
hydrolysis of orthoformates, which are stable in neutral or basic
solution but can be hydrolyzed in acidic media to the corre-
sponding formate ester. The mechanism of acid-catalyzed
orthoformate hydrolysis is a well-understood process, proceeding
by an A-1 mechanism in which the neutral orthoformate is in
rapid pre-equilibrium with the protonated substrate followed by
its rate-limiting decomposition.51–53 The study of orthoformate
hydrolysis has contributed to the mechanistic understanding of
hydrolysis reactions in general and was fundamental in the
formation of the Brønsted theory of acids almost a century ago.54
One advantage of investigating orthoformate hydrolysis by 1
in basic solution is that the formate ester product generated from
the acid-catalyzed hydrolysis is quickly hydrolyzed to formate
anion by base. The anionic formate product is not re-
encapsulated in the highly anionic 1, thereby facilitating catalysis
and abating product inhibition.
Substrate Scope. In order to test the hypothesis that ortho-
formates could be catalytically hydrolyzed by 1, triethyl
orthoformate was added to 1 in basic solution. After mild
heating, the formation of formate anion and 3 equiv of ethanol
was observed. In probing the scope of this reaction, a number
of small orthoformates were introduced to 1 at pH 11 and 50
°C. Small orthoformates able to fit in 1 were readily hydrolyzed,
whereas larger substrates could not enter 1 and remained
unreacted (Figure 2). The observed size selectivity is consistent
with the finite volume of the cavity of 1. By blocking the interior
of 1 with the strongly binding guest NEt4+, (Ka ) 104.55(5)
M-1),45 the catalysis was inhibited. This suggests that the
interior cavity of 1 is essential in the mediated hydrolysis of
orthoformates.
1
Figure 3. Comparison of the H NMR resonances of 1 after the addition
of various substrates. Substrates that undergo hydrolysis in 1 greatly perturb
1
the H NMR shifts of 1, whereas unreactive substrates do not.
Tripentyl orthoformate, however, which is too large to enter 1
and is not catalytically hydrolyzed, does not appreciably perturb
the H NMR spectrum of the assembly (Figure 3). In order to
confirm that the shift in the H NMR resonances is not due to
1
1
interaction of the orthoformates with the exterior of 1, triethyl
orthoformate was added to an aqueous solution of [NEt4 ⊂
1]11-. The 1H NMR resonances corresponding to 1 did not shift
upon substrate addition, suggesting that the orthoformate is not
interacting appreciably with the exterior of 1.
Mechanistic Considerations. The presence of a fast pre-
equilibrium involving guest exchange is analogous to the
Michaelis-Menten mechanism in which substrate binding is a
fast equilibrium before the rate-limiting step of the reaction. In
order to solidify this analogy, Michaelis-Menten kinetics were
explored using 3 as the substrate. When the substrate concentra-
tion was increased while maintaining constant pH and concen-
tration of 1, substrate saturation was observed that was consistent
with the Michaelis-Menten formalism (Figure 4a).
In order to further elucidate the mechanism of catalysis, the
overall rate law was determined. Working under saturation
conditions, i.e., pseudo-zeroth-order in substrate, kinetic analysis
showed that the reaction was first-order in both [H+] and [1]
and both substrate consumption and product formation are
pseudo-zeroth-order as expected (Figure 5). In the stoichiometric
reaction, the dependence of the rate on substrate concentration
was found to be first-order. The combined kinetic data yielded
the overall rate law, rate ) k[H+][substrate][1], which at
saturation reduces to rate ) k′[H+][1]. In the Michaelis-Menten
kinetic analysis, kcat ) k2[H+].
From the data discussed above, we propose that the neutral
substrate enters 1 to form a host-guest complex, leading to
the observed substrate saturation. On the basis of the ability
of 1 to encapsulate neutral hydrophobic guests, we surmised
that this was the resting state of the catalytic cycle (vide
infra). The encapsulated substrate then undergoes protonation
followed by two successive hydrolysis steps, liberating 2
equiv of the corresponding alcohol and affording the proto-
nated formate ester. Finally, the protonated formate ester is
ejected from 1 and further hydrolyzed by base in solution to
formate ion. It should be noted that any of the steps after
the initial acid-catalyzed first step could be catalyzed by either
acid or base, so if at any point the substrate escaped the
Typically, upon encapsulation, the NMR resonances of the
encapsulated guest are shifted upfield by 2-3 ppm due to the
magnetic anisotropy of the naphthalene walls of 1. However,
during the course of catalysis, no new upfield resonances
corresponding to the encapsulated substrate were observed.
1
Under the catalytic conditions, the H NMR resonances corre-
sponding to the substrate in solution were significantly broad-
+
ened but could be sharpened by addition of 1 equiv of NEt4
.
These data suggest that the encapsulated and free orthoformates
are exchanging quickly on the NMR time scale. Analysis of
1
the six H NMR resonances corresponding to T-symmetric 1
provided additional information. Upon addition of small ortho-
1
formates to 1, the T-symmetry of 1 is maintained, but the H
NMR resonances shift, thereby suggesting an interaction of the
orthoformate with 1. All of the orthoformates that undergo
1-catalyzed hydrolysis shift the 1H NMR resonances of 1.
(51) Bell, R. P. The Proton in Chemistry, 2nd ed.; Cornell University Press:
Ithica, NY, 1973.
(52) Cordes, E. H.; Bull, H. G. Chem. ReV. 1974, 74, 581.
(53) Cordes, E. H. Prog. Phys. Org. Chem. 1967, 4, 1.
(54) Bronsted, J. N.; Wynne-Jones, W. F. K. Trans. Faraday Soc. 1929,
25, 59.
9
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A R T I C L E S
Pluth et al.
Figure 6. Proposed mechanism for hydrolysis of orthoformates in 1.
Figure 4. Rate dependence on [3] with catalytic 1 in H2O pH ) 11.0, 100
mM K2CO3, 50 °C. Substrate saturation (a) is observed. The corresponding
Lineweaver-Burke plot (b). Modified from ref 50.
Figure 7. Solvent dependence on the initial rate of hydrolysis for 2.
substrate. Each of the individual steps are examined further
(vide infra), and all are consistent with the proposed
mechanism.
Investigation of Steps in the Catalytic Cycle. The initial pre-
equilibrium forming the host-guest complex with encapsulated
neutral substrate is presumably driven by the hydrophobic effect
in which desolvation plays an important driving force for the
encapsulation process. If this is the case, the addition of
organic solvents to an aqueous solution of 1 is expected to
retard the reaction by disfavoring the initial encapsulation
of the substrate.55 In order to probe this hypothesis, a number
of organic solvents (25% by volume) such as MeOH-d3,
DMSO-d6, DMF-d7, or dioxane-d8 were added to an aqueous
solution of 1 under the catalytic conditions. In all cases, the
addition of organic solvents greatly reduced the rate of
product formation (Figure 7).
Figure 5. Substrate consumption and product formation are pseudo-zeroth-
Having established the importance of neutral substrate
encapsulation in the catalytic cycle, the resting state of the
catalysis was probed. Although the encapsulated guests could
not be directly observed under catalytic conditions, greatly
increasing the concentration of 1 to a near-saturated (80-100
mM) solution followed by addition of a stoichiometric amount
of 3 allowed for observation of the 1:1 host-guest complex [3
order in the saturation regime of the catalyzed reaction. Modified from ref
50.
confines of 1, it would be quickly converted to product by
base in solution. The overall reaction scheme outlined in
Figure 6 parallels enzymatic Michaelis-Menten kinetics as
evidenced by the initial pre-equilibrium involving exchange
of the substrate followed by the first-order rate-limiting step
involving proton transfer from protonated water to the
1
⊂ 1]12- by H NMR. In order to probe the identity of the
(55) Breslow, R. Acc. Chem. Res. 1991, 24, 159–164.
9
11426 J. AM. CHEM. SOC. VOL. 130, NO. 34, 2008

Supramolecular Catalysis of Orthoformate Hydrolysis
A R T I C L E S
1
Figure 8. Calculated chemical shifts relative to 2 and experimental JCH
couplings for possible resting-state species for the catalysis in 1.
encapsulated species, 13C-labeled H13C(OEt)3 (13C-3) was used
as the substrate. This allowed for differentiation between
different possible resting states in the reaction based on the
observed 13C{1H} chemical shift and the 1JCH coupling constant.
Possible resting states in the catalytic cycle could include the
neutral orthoformate (3), protonated orthoformate (H+-3),
hemiorthocarboxylate (10), oxonium (11), or the formate ester
product (12). In order to compare predicted 13C{1H} shifts
corresponding to the different possible resting states the
magnetic tensors were calculated using Gaussian 0356 at the
B3LYP/6-31++G(d,p) level of theory using the gauge-
independent atomic orbital (GIAO) method (Figure 8) for the
1
model complex 2.57 The JCH coupling constants reported in
Figure 8 were measured from authentic 13C-labeled materials.
Under stoichiometric conditions at high concentration, the
13C{1H} NMR resonance corresponding to 13C-3 was shifted
from 113.5 to 110.7 ppm. This modest 2.8 ppm shift is consistent
with the typical range of NMR shifts upon encapsulation in 1.
The 13C{1H} chemical shift of 110.7 ppm, as compared to the
113.5 ppm of unencapsulated 13C-3 in D2O, is most consistent
with the neutral substrate as the encapsulated guest. The
observed chemical shift eliminates the oxonium and formate
ester as possibilities for the resting state but does not exclude
the possibility of 10. However, in the generation of the resting
state, no production of ethanol was observed. This eliminates
10 as a possible resting state. Furthermore, hemiorthocarboxylate
10 would be extremely sensitive to either acid or base and would
be quickly degraded upon ejection from 1. On the basis of the
known fast pre-equilibrium involving substrate exchange, 10
can be further eliminated as a resting-state possibility. In order
Figure 9. (a) 13C NMR spectrum of 13C-3 with K4(NEt4)7[NEt4 ⊂ 1] in
D2O (top), and 13C NMR spectrum of [13C-3 ⊂ 1]12- in D2O (bottom). (b)
1H-13C{1H} HSQC NMR spectrum showing correlation between the C-H
(*) of encapsulated 13C-3.
drolysis reactions, including orthoformate hydrolysis.51–53 Eyring
analysis of kcat values determined at different temperatures were
used to determine the activation parameters for the catalyzed
1
to compare the JCH coupling constants of the encapsulated
reaction: ∆Gq
) 22(2) kcal/mol, ∆Hq ) 21(1) kcal/mol,
species and the free orthoformate, an equivalent sample was
298K
+
and ∆Sq ) -5(1) cal/mol K. Additionally, measurement of kcat
in normal and deuterated water at pH (or pD) 11.0 revealed
kcat(D2O) ) 5.1 × 10-3 s-1 and kcat(H2O) ) 8.1 × 10-3 s-1
thereby affording the normal solvent isotope effect of 1.6.
In general, the acid-catalyzed hydrolysis of orthoformates is
thought to proceed through an A-1 mechanism where protona-
tion of the substrate is fast and reversible, and the rate-limiting
step is the decomposition of the protonated substrate.51–53 These
reactions are generally characterized by a positive ∆Sq in the
range of 6-10 cal/mol K.58,59 Similarly, since proton transfer
is not involved in the rate-limiting step, the observed solvent
isotope effect is due to the effect of deuteration on the pre-
equilibrium and generally ranges from 0.25 to 0.5. The solvent
isotope effect for the hydrolysis of triethyl orthoformate in
aqueous solution ranges from 0.37 to 0.47 depending on the
temperature and experimental details.59–61 However, neither the
observed activation parameters nor solvent isotope effects for
prepared but with 8 equiv of NEt4 to block the interior of 1.
1
A coupling constant in the 13C NMR spectrum of JCH ) 186
Hz was observed, compared with 1JCH ) 184 Hz for the resting
state of the encapsulated orthoformate (Figure 9a). This confirms
that the labeled carbon did not change hybridization and that
the orthoformate is still intact upon encapsulation. Additionally,
a 2D HSQC 1H-13C{1H} experiment on the encapsulated
species revealed a cross peak between the 13C{1H} signal and
the formyl C-H proton at the expected chemical shift (Figure
9b).
Having established the resting state of the catalytic system,
the next step was to probe the nature of the transition state in
order to further characterize the rate-limiting step. This was
accomplished by measuring the activation parameters and the
solvent isotope effect, k(H2O)/k(D2O), for the kcat step of the
reaction. Both of these kinetic parameters have been used
extensively to characterize the transition states of many hy-
(56) Frisch, M. J.; et al. Gaussian 03, revision D.01; Gaussian, Inc.:
Wallingford, CT, 2004.
(57) Trimethyl orthoformate was chosen as a model substrate due to the
limited degrees of freedom when compared to triethyl orthoformate.
(58) Koskikullio, J.; Whalley, E. Trans. Faraday Soc. 1959, 66, 809.
(59) Brescia, F.; LaMer, V. K. J. Am. Chem. Soc. 1940, 62, 612.
(60) Horiiel, J. C.; Butler, J. A. V. J. Chem. Soc. 1936, 1361.
(61) Brescia, F.; LaMer, V. K. J. Am. Chem. Soc. 1938, 60, 1962.
9
J. AM. CHEM. SOC. VOL. 130, NO. 34, 2008 11427

A R T I C L E S
Pluth et al.
previously shown that hydroxide, which would be a more potent
nucleophile than water in the attack of the protonated substrate,
is unable to approach the exterior of highly charged 1 in aqueous
solution.49 It should be noted that, after the first hydrolysis step,
all of the other steps can be either acid- or base-catalyzed, so
ejection of the substrate from 1 after the initial hydrolysis step
would quickly lead to formation of the formate product.
Although alkyl orthoformates generally proceed through an
A-1 hydrolysis mechanism, orthoformates that are able to more
greatly stabilize the carbocation intermediates during the course
of the reaction, such as triphenyl orthoformate, are thought to
proceed through an A-SE2 mechanism.62 In general, as the
stability of the carbocations on the reaction coordinate increases,
the transition state is moved closer to the reactants and the
magnitude of the solvent isotope effect decreases.52,63,64 Since
the highly anionic 1 is able to greatly stabilize cationic guest
molecules, the observed shift in mechanism from A-1 to A-SE2
upon encapsulation may be a direct effect of the preference of
monocationic guests to be encapsulated in 1.
On the basis of these more detailed kinetic data, the complete
catalytic cycle for the hydrolysis reaction is shown in Figure 6.
The substrate enters empty 1 to generate the resting state of the
catalytic cycle. When H3O+ enters the cavity of 1, rate-limiting
proton transfer occurs to generate the protonated substrate which
is then quickly attacked by water inside of 1. After this initial
hydrolysis step, subsequent hydrolyses can occur inside of 1 to
form the formate ester or the intermediate could be ejected into
basic solution thereby also forming the formate ester. Upon
entering basic solution, the formate ester is quickly hydrolyzed
by base to form formate anion and the empty assembly is
regenerated.
Analysis of the Rate Law. On the basis of the mechanism
outlined for the hydrolysis of orthoformates in 1 under alkaline
conditions, the overall reaction can be described as in eq 1 where
the initial pre-equilibrium is defined by k1 and k-1 and is
followed by the rate-limiting k2 proton-transfer step (the kcat
measured from Michaelis-Menten kinetics). By applying
steady-state analysis to the [S ⊂ 1] resting state, the complete
rate law can be derived (eq 2).
Figure 10. Mechanisms and transition states for A-1, A-2, and A-SE2
orthoformate hydrolysis. Orthoformate hydrolysis in 1 likely occurs through
the A-SE2 transition state.
the hydrolysis of 3 catalyzed by 1 coincide with the kinetic
parameters for the background orthoformate hydrolysis reaction.
This suggests that the reaction occurring in 1 proceeds through
a transition state and rate-limiting step that are different from
those of the background reaction.
The observed entropy of activation for catalysis in 1 of -5(1)
cal/mol K is not consistent with the A-1 mechanism known for
orthoformate hydrolysis. The negative activation entropy sug-
gests that the transition state is more ordered than the ground
state, suggesting a mechanism in which either attack of the
protonated substrate is rate-limiting (A-2 mechanism) or in
which proton transfer from the acid (likely H3O+) is rate-limiting
(A-SE2 mechanism).51 The solvent isotope effects can be used
to differentiate between these two possibilities. For A-2 hy-
drolysis mechanisms, the nucleophilic attack of solvent on the
protonated species is rate-limiting and leads to large negative
entropies of activation which lead to solvent isotope effects
closer to unity than an A-1 mechanism and generally ranging
from 0.55-0.8.51–53 In A-SE2 hydrolysis mechanisms, the
entropies of activation are also negative but proton transfer is
rate-limiting. This is evidenced by solvent isotope effects
greater than unity due to the difference in zero-point energy
between O-H and O-D bonds. These values are typically
approximately 1.7 but can range from 1.3 to 4.0. For the
hydrolysis catalyzed by 1, the solvent isotope effect value
of 1.6 is consistent with an A-SE2 mechanism for hydrolysis
in which proton transfer is occurring in the rate-limiting step
leading to a transition state as shown in Figure 10.
These mechanistic parameters suggest that protonated water
is able to enter 1 and react with the encapsulated orthoformate.
After the rate-limiting proton transfer, the bound water molecule
is poised to act as a nucleophile for attack on the protonated
substrate. Alternatively, if multiple water molecules are con-
comitantly encapsulated with the substrate in the cavity of 1,
any of these could act as a nucleophile after the proton-transfer
step in the hydrolysis reaction. The observed activation param-
eters and solvent isotope effect suggest that at least the initial
hydrolysis step following rate-limiting protonation is occurring
inside of 1. If, for example, after protonation in 1, the substrate
were to egress from 1 into bulk solution, the observed solvent
isotope effect would be greater than unity. Similarly, we have
+
[
H
]
k1
k2
S + 1hS 1 f P + 1
(1)
(2)
k-1
+
[
]
[ ]
[ ]
k1k2 S H 1 tot
rate )
+
[
[ ]
k-1 + k1 S + k2 H
]
On the basis of the derived rate law, the reaction should be
first-order in the concentration of substrate, proton, and 1, all
of which are experimentally observed. Similarly, the reaction
should show saturation behavior in both substrate and proton
concentration. However, during the experimental determination
of the rate law, only substrate saturation is observed. This
implies that k1[S] . k2[H+] which is consistent with the low
concentration of [H+] present under the reaction conditions.
Although no saturation behavior was observed in [H+] from
pH 13 to pH 8, it is possible that, at much higher concentrations
of [H+], saturation behavior could be observed. However, 1 itself
is not stable in acidic media due to protonation of the catechol
oxygen atoms. Nonetheless, the derived rate law is consistent
(62) Price, M. L.; Adams, J.; Lagenaur, C.; Cordes, E. H. J. Org. Chem.
1969, 34, 22–25.
(63) Hammond, G. S. J. Am. Chem. Soc. 1955, 77, 334–338.
(64) Thornton, E. R. J. Am. Chem. Soc. 1967, 89, 2915–2927.
9
11428 J. AM. CHEM. SOC. VOL. 130, NO. 34, 2008

Supramolecular Catalysis of Orthoformate Hydrolysis
A R T I C L E S
Table 1. Tabulation of Kinetic Parameters for Hydrolysis of Orthoformates in 1 at 50 °C, pH ) 11.0
substrate
Vmax (M s-1
)
KM (mM)
kcat (s-1
)
kuncat (s-1
)
k
cat/KM (M-1 s-1
)
(kcat/KM)/kuncat (M-1
)
kcat/kuncat
2
3
4
5
1.3 × 10-5
1.8 × 10-5
4.0 × 10-5
9.2 × 10-6
24.0
21.5
19.6
7.69
5.7 × 10-3
8.1 × 10-3
1.8 × 10-2
3.9 × 10-3
3.7 × 10-5
1.4 × 10-5
4.6 × 10-6
4.3 × 10-6
0.238
0.275
0.918
0.502
6.4 × 103
1.9 × 104
2.0 × 105
1.2 × 105
150
560
3900
890
with the observed kinetic data since under the reaction conditions
it can be further reduced to eq 3.
a detailed kinetic analysis of the mechanism of catalysis. The initial
encapsulation of the neutral substrate is driven by the hydrophobic
effect as shown by mixed solvent studies and 13C-labeling
experiments. Upon encapsulation, the observed entropy of activa-
tion and solvent isotope effect suggest that the initial step of the
hydrolysis proceeds through an A-SE2 mechanism which is
different from the A-1 mechanism for orthoformate hydrolysis in
bulk solution. Kinetic analysis of the rate constants of the catalyzed
and uncatalyzed reactions show that the assembly is able to
efficiently catalyze the hydrolysis, with rate accelerations of over
103, which are among the highest catalytic rate accelerations that
have been observed in synthetic supramolecular systems.
+
[
]
[ ]
[ ]
[ ][ ]
k1k2 S H 1 tot k1kcat S 1 tot
rate )
)
(3)
[ ]
k-1 + k1 S
[ ]
k-1 + k1 S
Rate Acceleration. In order to compare the rate acceleration
of the catalyzed over the uncatalyzed reaction, the kcat rate
constants from Michaelis-Menten studies were compared to
the background reaction for hydrolysis (Table 1). This analysis
revealed sizable acceleration in all cases with the largest
acceleration being 3900 in the case of 4.65 Further analysis of
the Michaelis-Menten parameters revealed a number of trends.
Assuming a fast pre-equilibrium for guest encapsulation with
respect to kcat, then KM ∼ Kd, which allows for comparison of
the binding affinities of different substrates in 1. As the
hydrophobicity of the orthoformate increases, the binding affinity
of the substrate increases, consistent with the hydrophobic effect
driving encapsulation. Of the substrates investigated, the two
propyl isomers (4 and 5) show the highest affinity for 1, with
triisopropyl orthoformate being more tightly bound that tri-n-
propyl orthoformate. Although 4 and 5 have similar sizes,
encapsulation of 4 in 1 results in a greater loss of conformational
freedom than encapsulation of the more compact 5. The
enhanced binding of 5 may explain why the rate acceleration
is attenuated when compared to 4. Comparison of kcat/KM for
the different substrates, often referred to as the specificity factor,
allows for comparison of the second-order proportionality
constant for the rate of conversion of preformed host-substrate
complex to product, thereby providing a comparison of how
efficiently different substrates can compete for the active site
of 1. As the size of the substrate increases, the specificity
increases to a maximum for 4 and then decreases for 5. For
substrates such as 4, a balance between hydrophobicity and steric
size makes it an ideal fit for 1, and this is reflected by the highest
specificity factor and the highest rate acceleration in comparison
to the background reaction. The catalytic proficiency ((kcat/KM)/
kuncat) for different substrates is a good measure of how
encapsulation affects the transition-state stabilization with
respect to the uncatalyzed reaction. For substrates 2-4, as the
alkyl chain lengthens, the catalytic proficiency increases,
suggesting that a more optimal fit for the transition state in 1 is
achieved for the larger substrates. Although substrates 4 and 5
have similar catalytic proficiencies, 4 shows a much higher rate
acceleration, which is likely due to the greater stabilization of
encapsulated 5. This is consistent with the notion that both
ground-state and transition-state effects can play an active role
in the catalysis inside of the supramolecular host.
Experimental Section
General Procedures. All NMR spectra were obtained using an
AV-500 MHz spectrometer at the indicated frequencies. The temper-
ature of all variable temperature NMR experiments was calibrated with
an ethylene glycol standard. NMR spectra measured in H2O were
obtained using the Watergate solvent suppression sequence.
Materials. Orthoformates were either purchased from a commercial
supplier or prepared by alcoholysis of triethyl orthoformate and
fractionally distilled through a 12 in. Vigreux column followed by
distillation over powdered 3Å molecular sieves. All orthoformates were
stored under N2 until use. Carbon-13-labeled triethylorthoformate was
purchased from Cambridge Isotope Laboratories and used as received.
The host assembly K12[Ga4L6] was prepared as described in the
literature and precipitated with either acetone or ether.
General Procedure for Kinetics Runs. In a N2-filled glovebox,
1 was weighed into a 20 mL scintillation vial at which point H2O
buffered to the desired pH and an internal standard (DMSO) were
added. The stock solution was divided into NMR tubes in 500 µL
aliquots. All kinetic runs with 1 were performed in the probe by
allowing the NMR tube to equilibrate at the desired temperature
for 10 min at which point the tube was ejected, the substrate injected
to the tube, and the tube reinserted into the NMR instrument. All
Michaelis-MentenkineticdatawerefitdirectlytotheMichaelis-Menten
equation using nonlinear least-squares refinement.
Acknowledgment. We thank Dr. Dennis Leung, Dr. Gojko Lalic,
and Courtney Hastings for helpful discussions and Dr. Herman van
Halbeek and Rudi Nunlist for NMR assistance. This work was
supported by the Director, Office of Science, Office of Basic Energy
Sciences, and the Division of Chemical Sciences, Geosciences, and
Biosciences of the U.S. Department of Energy at LBNL under Contract
No. DE-AC02-05CH11231 and an NSF predoctoral fellowship to
M.D.P.
Conclusions
In summary, we have expanded our study of catalytic hydrolysis
of orthoformates mediated by a supramolecular assembly to include
Supporting Information Available: Kinetic data, Eyring plots,
full citation for ref 56. This material is available free of charge
via the Internet at http://pubs.acs.org.
(65) The low water solubility of tributyl orthoformate and triisobutyl
orthoformate prohibited saturation studies for Michaelis-Menten
analysis.
JA802839V
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J. AM. CHEM. SOC. VOL. 130, NO. 34, 2008 11429