A catalyst for an acetal hydrolysis reaction from a dynamic combinatorial library

Article abstract of DOI:10.1039/b505316a

A transition-state analogue (TSA) for an acetal hydrolysis reaction was found to select and amplify a macrocycle from a dynamic combinatorial library (DCL) of disulfides in water. This host was able to accelerate the reaction by a factor of two; a similar value was progressively reached when the macrocycle was gradually produced in the course of the reaction. The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2005.

Full text of DOI:10.1039/b505316a

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L E T T E R
A catalyst for an acetal hydrolysis reaction from a dynamic
combinatorial library
Laurent Vial, Jeremy K. M. Sanders and Sijbren Otto*
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge,
UK CB2 1EW. E-mail: so230@cam.ac.uk; Fax: þ44 1223 336017; Tel: þ44 1223 336509
Received (in Durham, UK) 15th April 2005, Accepted 16th June 2005
First published as an Advance Article on the web 1st July 2005
A transition-state analogue (TSA) for an acetal hydrolysis
reaction was found to select and amplify a macrocycle from a
dynamic combinatorial library (DCL) of disulfides in water.
This host was able to accelerate the reaction by a factor of
two; a similar value was progressively reached when the macro-
cycle was gradually produced in the course of the reaction.
(reversible) disulfide exchange at pH 8.5 for 3–5 days, a
complex DCL was obtained (Fig. 1a). The same library pre-
pared in the presence of TSA 4 showed amplification of two
species (Fig. 1b), which were identified by mass spectrometry as
two diastereomeric trimers of building block 6;9 both of them
being macrocycles that exhibit an unfunctionalized aromatic
cavity of similar shape and size (Fig. 2). Starting from a biased
library made of only dithiol 6 and TSA 4, the trimers 8
constitute now more than 80% of the library material (Figs.
1c and d) and, after scale-up, we were able to isolate 40 mg of
the potential catalyst by preparative HPLC (Fig. 1e).
Dynamic combinatorial chemistry1 is gaining ground as an
attractive selection-based approach2 for the discovery of new
catalytic systems.3 In a DCL, all library members are con-
nected through a set of reversible reactions, which creates a
mixture that is under thermodynamic control. Introducing a
template that mimics the transition state of a chemical trans-
formation (a transition-state analogue or TSA) will shift the
equilibria in the direction of those receptors that bind the TSA
and, as a result, should have the potential of catalyzing the
corresponding reaction.
We recently became interested in the challenging idea of
using this strategy to produce an abiotic catalyst that mimics
glycosidase function, focusing on the hydrolysis of model
acetal 1. The rate determining step for this reaction involves
the cleavage of the heterocyclic carbon–oxygen bond of the
protonated acetal via transition state 2.4
The binding of TSA 4 with hosts 8 (as a mixture of
stereoisomers) was studied by isothermal titration microcalori-
metry and was found to be enthalpy driven (K ¼ 2.6 ꢀ 105
M
ꢁ1, DG1 ¼ ꢁ30.9 kJ molꢁ1 DH1 ¼ ꢁ24.6 kJ molꢁ1 and
TDS1 ¼ þ6.3 kJ molꢁ1), which is consistent with cation–p
interactions being the major contributor to binding.
The kinetics of the hydrolysis of 1 were studied in the
presence of 1.3 equivalents of hosts 8 in H2O–CH3CN 97 : 3
(v/v) under acidic conditions (pH 4.2) at 25 1C. The addition of
the small amount of acetonitrile was required to ensure solu-
bility of the starting material. The disappearance of 1 and the
formation of product 3 were monitored by HPLC at 300 nm.
The rate of hydrolysis obeyed first-order kinetics with kcat
9.94 ꢀ 10ꢁ4 minꢁ1 (n in Fig. 3). We have also monitored the
¼
We reasoned that ammonium salt 4 should be a suitable
TSA as it resembles transition state 2 in terms of shape and
charge distribution5 and it is also a promising template for the
amplification of macrocyclic hosts from DCLs made from
dithiols 5–7 in water. Our previous studies have demonstrated
that such building blocks can engage in hydrophobic and
cation–p interactions with non-polar ammonium salts.6 More-
over, the disulfide exchange chemistry in which these building
blocks can participate is complementary to the acetal hydro-
lysis conditions: disulfide bonds undergo rapid exchange in the
presence of a catalytic amount of thiolate anion in water at pH
7–9 (DCLs conditions), whereas the bonds—and therefore the
library members—are kinetically stable under acidic conditions
(pH o 5, hydrolysis conditions).7
Fig. 1 HPLC analyses (310 nm) of the DCLs made from a) dithiols
5–7 in the absence of template; b) dithiols 5–7 in the presence of TSA 4;
c) dithiol 6 in the absence of template; d) dithiol 6 in the presence of
TSA 4. e) HPLC analysis (310 nm) after scale-up and purification of 8
from trace d). HPLC analyses were carried out using a 250 ꢀ 4.6 mm
Waters Symmetry C18 column with a gradient of 5–95% CH3CN (0.1%
TFA) in H2O (0.1% TFA). Peaks due to TSA 4 and receptors 8 (as a
mixture of stereoisomers) are shown in green and red, respectively.
When equimolar amounts of building blocks 5–78 were
submitted to (irreversible) air oxidation and simultaneous
T h i s j o u r n a l i s & T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 5
N e w J . C h e m . , 2 0 0 5 , 2 9 , 1 0 0 1 – 1 0 0 3
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protonation pre-equilibrium more towards 1 ꢂ H1, whether
they actually stabilize the transition state 2, or whether they
act through a combination of these effects. The only modest
acceleration is almost certainly a consequence of the lack of
functional groups that can participate in the catalytic process.
Our research efforts are now directed towards the development
of a new generation of building blocks that contain such
functional groups. Given that we have met with promising
degrees of success in our two first attempts to use dynamic
combinatorial chemistry for catalyst development (this study
and ref. 3), we are optimistic that dynamic libraries can be
developed into a practical method for catalyst discovery in the
near future.
Fig. 2 Energy-minimized structures (PM3 level, Spartan00410) of
trimeric hosts 8. Left: minor diastereoisomer and right: major dia-
stereoisomer. Colour code used for labelling atoms: C ¼ grey; H ¼
white; O ¼ red; S ¼ yellow.
Experimental
kinetics of hydrolysis of 1 at pH 4.2 in the absence of hosts 8,
giving k0 of 4.70 ꢀ 10ꢁ4 minꢁ1 (J in Fig 3). Comparison of the
values of kcat and k0 indicates that hosts 8 accelerate the
hydrolysis of 1 by a factor of 2.1.
Acetal 1 and TSA 4 were obtained using the procedures
described in refs. 13 and 14, respectively.
In a typical DCL experiment, the dithiols (10 mM overall)
were suspended in water (1–10 mL) and a NaOH solution
(1.0 M; 1 equiv. with respect to the number of carboxylic acid
groups) was added. After the dithiols had dissolved, the pH
value was adjusted to 8.5. Where appropriate, the TSA 4
(5 mM) was added and the mixtures were allowed to oxidize
and equilibrate for 3–5 days by stirring in an open vial
(evaporated water was replenished every day).
The equilibrium constant (K), enthalpy (DH1) and entropy
(TDS1) of binding of TSA 4 in hosts 8 at 298 K were
determined using isothermal titration microcalorimetry. Guest
solutions (0.91 mM) were titrated into host solutions (0.10
mM, mixture of stereoisomers), all prepared in 10 mM borate
buffer (pH 9.0).
In a typical kinetic experiment, the hydrolysis reaction was
initiated by the injection of 30 mL of 1 (33.3 mM in CH3CN) in
a thermostatted cell initially containing 970 mL of a solution of
either 8 (as a mixture of stereoisomers; 1.34 mM; 1.3 equiv.) or
6 (4.02 mM; 3.9 equiv.) in citrate buffer (10 mM, 970 mL, pH ¼
4.2) at 25 1C. Reactions were monitored by analysing 5 mL
aliquots by HPLC. The pseudo-first-order rate constants were
determined from the slopes of semi-logarithmic plots of [1]
against time.
In order to establish whether this acceleration is due to the
reaction taking place in the hydrophobic cavity of receptors 8,
we studied the hydrolysis of 1 in the presence of 3.9 equivalents
of 6. Unexpectedly, the rate of the reaction in the presence of 6
turned out to increase progressively with time (B in Fig. 3).
The study of the related HPLC traces revealed that, in the
course of the hydrolysis of acetal 1, dithiol 6 is completely
converted to the corresponding disulfide trimers 8 (Fig. 3,
traces a–f).11 Trimer formation most likely occurs under
kinetic control, since under the acidic conditions of the hydro-
lysis experiments disulfide exchange is extremely slow.7,12
During the first 15% conversion, when the amount of hosts 8
is still insignificant, the rate constant for the reaction (obtained
from the slope of the dashed line in Fig. 3) is 4.82 ꢀ 10ꢁ4
minꢁ1, which is—within experimental error—the same as the
value for k0. Control experiments on a solution of 6 revealed
that the production of receptor 8 is independent of the presence
of 1 or its hydrolysis products, indicating that this process is
not templated.
The absence of any catalytic activity due to 6, as well as the
acceleration of the reaction following the formation of the
macrocyclic species, demonstrate that the cavities of receptors
8 are responsible for acceleration of the hydrolysis of acetal 1,
probably through stabilization of the transient positive charge
that develops during the reaction. We have been unable to
ascertain whether hosts 8 accelerate the reaction by shifting the
Acknowledgements
We are grateful to Ana Belenguer for help with the HPLC
analyses and to EPSRC, Marie Curie Intra-European Fellow-
ships (MEIF-CT-2003-501648) and the Royal Society for
financial support.
References
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Fig. 3 Left: HPLC analyses (300 nm) of the hydrolysis experiments of
1 in the presence of 6 (3.9 equiv.) after a) 1.4; b) 4.9; c) 10.1; d) 24.0; e)
31.3; f) 47.6; g) 55.2; h) 76.6 and i) 97.3 hours. HPLC analyses were
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mixture of CH3CN–H2O (80 : 20). Acetal 1, product 3 and hosts 8 are
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mic plots of the concentration of 1 versus time, t, at pH 4.2 in the
absence of any catalyst (J); in presence of 3.9 equiv. of 6 (B); and 1.3
equiv. of 8 (n). The dashed line represents the tangent of the curve for
the reaction in the presence of 6 at t ¼ 0.
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Building blocks 5 and 6 were obtained as reported in ref. 6. Dithiol
7 has been prepared from 3,7-dihydroxy-2-naphthoic acid using
analogous procedures.
an opposite configuration compared to the other two). From the
relative integration of the 1H-NMR signals it is possible to assign
the C2-symmetry to the major signal in HPLC (ratio of B2 : 1 in
agreement with a statistical distribution).
10 Spartan004, Wavefunction, Inc., Irvine, CA, USA.
11 The two diastereomers overlap in the HPLC conditions used for
the kinetic experiments.
9
As 6 was synthesized as a racemic mixture of two enantiomers (see
ref. 6), the 1H-NMR spectrum of 8 revealed 16 signals: 4 belonging
to a diastereoisomer of D3-symmetry (where every monomer has
the same configuration [i.e., (9R,10R) or (9S,10S)] and 12 belong-
ing to a diastereoisomer of C2-symmetry (where one monomer has
12 The concentration of thiolate anion has been estimated at 20 mM
using the Henderson–Hasselbach equation (pKa of thiols B6.6).
13 F. E. Romesberg, M. E. Flanagan, T. Uno and P. G. Schultz,
J. Am. Chem. Soc., 1998, 120, 5160.
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N e w J . C h e m . , 2 0 0 5 , 2 9 , 1 0 0 1 – 1 0 0 3
1003