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Ion-Induced Specificity Change in Polymer-Catalyzed Solvolyses of p-Nitrophenyl Alkanoates.

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Ion-Induced Specificity Change in
Polymer-Catalyzed Solvolyses of
p-Nitrophenyl Alkanoates**
Guang-Jia Wang and Wilmer K. Fife"
'
Electrostatic interactions are considered to be major contributors to protein structure and specificity of enzyme catalysis.[']
However, few investigations have explored the control of substrate specificity of enzymes with ions of different sizes and
charge densities. The control of substrate specificity in enzyme
catalysis by use of organic solvents is well established.[' -31 Aggregation of amphiphilic polymers in water and water/organic
solvent mixtures is well-known to lead to complex supramolecular structures with multiple morph~logies.[~~
Upon decreasing the number of generations, the aggregate morphology of
polystyrene with poly(propy1enimine) dendrimers in aqueous
solution has been shown to change from spherical micelles
through micellar rods to vesicles.[61The same morphological
changes have also been found for polystyrene-b-poly(acry1ic
acid) (PS-b-PAA) as poly(acry1ic acid) content de~reases.1~1
All
of these studiesc4-'I suggest that an increase in hydrophobic
effects of amphiphilic macromolecules upon an increase in ratio
of hydrophobic to hydrophilic monomers leads to such changes
of aggregate morphology. Consistent with the notion that ioninduced hydrophobic effects control aggregate morphology of
amphiphiles, the morphology of aggregates of PS-b-PAA,
polystyrene-b-poly(ethy1ene oxide) (PS-b-PEO), and PS-bpoly(4-vinylpyridinium methyl iodide) in water/organic solvent
mixtures can also be changed from spheres to rods, and to
vesicles by addition of salting-out agents such as NaCl and
CaCI, .r81
Polymers functionalized with the 4-(dialky1amino)pyridine
group have been regarded as useful model systems for investi- "1 We
gating the origins of enzymic efficiency and sele~tivity.[~
have reported that macromolecule 1 containing the 4-(dialkylamino)pyridine functionality and a bis(trimethy1ene)disiloxane
backbone as a nucleophilic catalyst exhibits enzyme-like substrate selectivity for the solvolysis of 2 in aqueous and methanol/
water solutions.[". 13, 14] To our knowledge, ion-induced substrate specificity changes have not been reported previously for
catalytic ester solvolysis.
catalysis. Macromolecule 1 is an amphiphilic polymer that contains distinct hydrophilic and hydrophobic rcgions, and it dynamically associates to form macromolecular aggregates by self'] The rate
assembly in aqueous or methanol/water
enhancements for 1-catalyzed solvolysis of 2 have been attributed to hydrophobic association between catalyst and substrate in
the reaction medium.[g-I3] We have investigated the solvolysis
of 2 (n = 2, 4, 6, 8, 10, 12, 14, 16, 18) catalyzed by 1 in different
reaction media composed of solvent and added salts at pH 8.0
and 30°C. Initial findings indicate that the solubility of 1 in
0 . 0 5 ~aqueous tris(hydroxymethy1)methylammonium (Tris)
buffer solution shows a more than tenfold increase over that in
pure water as a result of salting-in effects of tris(hydroxymethy1)methylammonium ion from the Tris buffer system.['5-'81 In 0.05 M aqueous phosphate or borate buffer solutions, the solubility of 1 is similar to that in pure water, and
aqueous solutions of 1 show appreciable turbidity when the
unitmolL-'.
concentration of 1is increased beyond 2.5 x
However, a solution of 1 at 2.5 x
unitmolL-' in 0 . 0 5 ~
aqueous Tris buffer solution remains clear even after standing
for prolonged periods. These results suggest that changes of
aggregate morphology of 1 at 2.5 x lo-' unitmolL-' from
vesicles to rods or spheres apparently accompany changes of
reaction medium from aqueous phosphate or borate to Tris
buffer solutions.['. "1
We have measured the pseudo-first-order rate constants for
1-catalyzed hydrolysis of 2 in 0.05 M aqueous Tris, phosphate, or
borate buffer solutions (Figure 1). Without 1, the hydrolysis
8.00
A
8.40
4.80
1.60
,
0.00
0
2
4
8
8
10
12
14
18
18
n-
1
2
( n = 2 , 4 , 6 , 8 , 10, 12,14, 16.18)
We present here ion-induced substrate specificity changes in
the 1-catalyzed solvolysis of 2 ( n = 2, 4, 6, 8, 10, 12, 14, 16, 18)
in aqueous and methanol/water solutions. These results encourage more precise modeling studies of the molecular origins of
catalytic efficiency and specificity in biological and chemical
[*I
[**I
Prof. W K. Fife, Dr. G. J. Wang
Department of Chemistry
Indiana University-Purdne University at Indianapolis
402 North Blackford Street, Indianapolis, IN 46202 (USA)
Fax: Int. code +(317)274-4701
e-mail: fife(&hem.iupui.edu
This work was supported by the Office of Naval Research.
Angew. Chem. I n f . Ed. Engl. 1997, 36, No. 13/14
Figure 1. Pseudo-first-order rate constants (&) for the hydrolysis of 2 ( n = 2.4,6,
8, 10, 12, 14, 16, 5 . 0 ~ 1 0 ~ ~ ~ ) c a t a lbyyz2e. 5d ~ l O - ~ u n i t m o l L -lasafunction
'
of alkanoate chain length n in 0.0501 aqueous buffer solution at pH 8.0 and 30°C:
0 : Tns buffer, 7 : phosphate buffer, m : borate buffer.
rates of 2 (n = 2, 4, 6, 8, 10, 12, 14, 16) are very slow and no
substrate selectivity is obtained in aqueous Tris, phosphate, or
borate buffer solutions. In the presence of 1the hydrolysis rates
of 2 (n = 2, 4, 6, 8, 10, 12, 14, 16) are also very slow and differ
from each other by only small factors in aqueous phosphate or
borate buffer solutions, indicating that no appreciable catalysis
is occurring in these systems. Increasing the alkanoate chain
length in 2 causes small decreases in the hydrolysis rate and no
substrate specificity is found for these systems (Table 1) in conformity with results from aqueous buffer solutions that contain
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Table 1 Summary of the effect of added salt concetration on the substrate specificity of I-catalyzed solvolysis of 2 ( n = 2, 4, 6 , 8, 10, 12, 14, 16, 18) in aqueous and
methanoliwater solutions at 30 'C
Reaction medium
Salt
C [MI
Substrate specificity
aqueous solntion[a]
Tris buffer
phosphate buffer
borate buffer
0.05
0.05
0.05
2 (n = 6 )
no
no
methanol/water[b]
NaCl
NaCl
NaCl
NaCl
NaCl
NaCl
0.00
0.01
0.10
0.25
0.50
1.00
2(n=14)
2 ( n = 14)
2 ( n = 14)
2 (n = 12)
2 ( n = 12)
2 ( n = 12)
Id] 1 ( 2 . 5 ~ 1 0 ~ ~ u n i t r n o l L ~ ~ ) . p H 8 .10( .5[.b0 J~ 1 0 ~ ~ u n i t m o l L - ' ) , r n e t h a n o l /
aqueous buffer ( l : l , vjv; 0.05 M H,PO;/HPO:-),
pH 8.0.
no catalyst. However, in aqueous Tris buffer solution the rates
for 1-catalyzed hydrolysis of 2 (n = 2, 4, 6, 8) are much faster
than those in aqueous phosphate or borate buffer solutions. Of
added importance, macromolecule 1 demonstrates the same
substrate preference for 2 (PZ = 6) that is observed with cholesterol esterase for the same hydrolysis reaction.[201Under conditions where tenfold excess substrate is used in kinetic studies, the
catalytic effectiveness of 1 is maintained to complete reaction.
We have also measured the effects of salting-out agent NaCl
on substrate specificity for 1-catalyzed solvolysis of 2 in 1:1
(v/v) methanol/water solution (Figure 2). Without NaCl,
water solution. Apparently the aggregate morphology of 1 in
1 :1 (v/v) methanol/water solution changes from spheres or rods
to vesicles as the concentration of NaCl is increased from 0 to
1 . 0 0 ~ . [19]~ .We suggest that salting-out effects of NaCl induce
changes of aggregate morphology of 1in 1 : 1 (v/v) methanoljwater solution from spheres or rods to vesicles,r'5-181while salting-in effects of the tris(hydroxymethyl)methylamrnonium ion
induce changes of aggregate morphology of 1 in aqueous solution from vesicles to rods or sphere^.['^-'^' This is based on
observations that added salts change aggregate morphology of
small-molecule and macromolecular amphiphiles.[** 1, 22]
Recently Zhang and Eisenberg have reported that the stretching of the hydrophobic chains in macromolecular amphiphiles is
greatest in spherical micellar aggregates and decreases as the
aggregate morphology changes from spheres to rods, and decreases further as vesicles are formed.['* 191 The spherical aggregates tend to provide the strongest hydrophobic binding for
lipophilic substrates in the reaction medium. We suggest that the
changes of substrate specificity induced by NaCl may be attributed to changes in morphology of aggregates of 1 in 1 :1 (v/v)
methanol/water solution. Changes of aggregate morphology of
1 from spheres to rods or vesicles should accompany decreased
hydrophobic binding for the substrates and decreased dependence of formation of complexes involving 1and 2 on the hydrophobicity of the substrate. The substrate specificity change from
2 ( n = 14) to 2 (n = 12) may result from NaCl driving a sphere
to rod transition in aggregate morphology of 1leading to hydrophobic binding that is optimum for 2 (n = 12) in 1 : l (v/v)
methanol/water solution. This may be the origin of the molecular discrimination described by the term hydrophobic interactions at active sites of enzymes and catalysts for biological and
chemical reactions. The behavior of 1 in aqueous Tris buffer
solution may also be ascribed to spherical aggregates of 1
formed in response to the salting-in effects of tris(hydroxymethy1)methylammonium ion that contribute to efficient reaction within 1.2 complexes for 2 (n = 6) in aqueous solution.
These results provide the first instance in which the substrate
specificity of catalytic ester solvolysis is seen to respond to the
structural characteristics of aggregates of catalyst in the reaction
medium, and illustrate a new approach to the control of chemical reactivity and aggregate morphology of amphiphilic macromolecules that can be useful in understanding the fundamental
basis of controlling substrate specificity at the molecular level in
biological and chemical catalysis. The kinetic behavior of our
model system is quite similar to that of natural acyl-transfer
enzymes such as chymotrypsin and cholesterol esterase for
transfer of structural elements in cellular processes.
Experimental Section
Figure 2. Effects of NaCl concentration on pseudo-first-order rate constants (k,J
for the solvolysis of 2 (n = 6, 8, 10, 32, 14, 16, 18, 5 . 0 ~1 0 - ' ~ ) catalyzed by
5.0 x
u n i t m o l l - ' 1 as a function of alkanoate chain length n in methanol/
aqueous buffer ( I :1, v/v; 0 . 0 5 ~H,PO;/HPO ?-, pH 8.0) solution at 30'C: a: no
NaCI; A : 0 2 5 NaCI,
~
m: 1.00~
NaCl.
macromolecule 1 exhibits substrate specificity for 2 (n = 14).
The SOlVOlYSlS rate for 2 (n =lo, 12, 14, 16) decreases as the
concentration of NaCl is increased from 0 to 1.OOM. Surprising-
Kinetic measurements: The cuvette was tilled with 2.5 mL of a fresh solution containing catalyst in 0 . 0 5 aqueous
~
buffer solutions at pH 8.0 and the solution was
equilibrated for 10 min at 30 "C in the thermostated cell compartment of a HewlettPackard Model 8450 spectrophotometer. The fresh catalyst solutions containing
sodium chloride were prepared in methanoljaqueous buffer (1 :l. vjv, 0 . 0 5 ~
H,PO,/HPO:-,
pH 8.0) solution. A stock solution (5 pL j ofp-nitrophenyl alkanoates (2.5 x ~ O - ' M ) in dioxane was added by microsyringe. The reaction mixture
was quickly mixed by shaking and the absorbance at 400 nm was recorded as a
function of time. The reactions were performed for four-to-five half-lives and the
pseudo-first-order rate constants (kobJ were obtained as slopes of plots of In[A,/
( A , - A,)] against time, where A , and A , are the absorbance at infinite time and
time 1. respectively. The first-order rate constants (k,,,) represent the average of
three runs and experimental error is less than 5%.
ly, the substrate specificity is found to change from 2 (n = 14) to
2 (n = 12) when the concentration of NaCl is increased beyond
0.25 M (Table 1). Furthermore, we find that as the concentration
, solutions of 1 at
of NaCl is increased to more than 1 . 0 0 ~ the
5.0 x
unitmolL-' become opalescent in 1 :1 methanol/
1544
8 VCH Verla~.~~eselischaft
nibH, 0.69451
Wernheim, 1997
Received: January 28, 1997 [Z 10046IEI
German version: Angew. Chem. 1997, 109. 1520-1523
Keywords: aggregates
solvolysis
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homogeneous catalysis
-
Polymers
Angew. Chem. h17. Ed. Engl. 1997,36, NO. 13/14
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[15]
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