Cooperative hydrolysis of aryl esters on functionalized membrane surfaces and in micellar solutions

Article abstract of DOI:10.1039/c4ob00247d

Catalytic hydrolysis of peptides, proteins, phosphates or carboxylate esters in nature is catalysed by enzymes, which are efficient, fast and selective. Most of the hydrolytic chemical catalysts published so far mimic the active site of enzymes and contain metal complexes and amino acid residues. Their synthesis can be laborious, while the hydrolytic activity is still limited compared to enzymes. We present an approach that uses fluid membranes of vesicles and micelles as a support for amphiphilic additives, which cooperatively cleave aryl ester bonds. The membrane anchored bis-Zn(ii)-complex 1 is hydrolytically active and hydrolyses fluorescein diacetate (FDA) with a second order rate constant (k₂) of 0.9 M^-1 s^-1. The hydrolytic activity is modulated by co-embedded membrane additives, bearing common amino acid side chain functional groups. With this approach, the hydrolytic activity of the system is enhanced up to 16 fold in comparison with cyclen 1 (k₂ = 14.7 M^-1 s^-1). DOPC and DSPC lipids form at room temperature fluid or gel phase membranes, respectively. Omitting the lipid, micellar solutions were obtained with hydrolytic activity reaching k₂ = 13.4 M^-1 s^-1. It is shown that cooperative hydrolysis is favoured in fluid membranes and micelles, allowing the active moieties to arrange freely. The embedding and dynamic self-assembly of membrane active components in fluid membranes and micelles provide facile access to hydrolytically active soft interfaces.

Full text of DOI:10.1039/c4ob00247d

Organic &
Biomolecular Chemistry
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Cooperative hydrolysis of aryl esters on
functionalized membrane surfaces and in micellar
solutions†
Cite this: Org. Biomol. Chem., 2014,
12, 3175
M. Poznik and B. König
Catalytic hydrolysis of peptides, proteins, phosphates or carboxylate esters in nature is catalysed by
enzymes, which are efficient, fast and selective. Most of the hydrolytic chemical catalysts published so far
mimic the active site of enzymes and contain metal complexes and amino acid residues. Their synthesis
can be laborious, while the hydrolytic activity is still limited compared to enzymes. We present an
approach that uses fluid membranes of vesicles and micelles as a support for amphiphilic additives, which
cooperatively cleave aryl ester bonds. The membrane anchored bis-Zn(II)-complex 1 is hydrolytically
active and hydrolyses fluorescein diacetate (FDA) with a second order rate constant (k2) of 0.9 M−1 s−1
.
The hydrolytic activity is modulated by co-embedded membrane additives, bearing common amino acid
side chain functional groups. With this approach, the hydrolytic activity of the system is enhanced up to
16 fold in comparison with cyclen 1 (k2 = 14.7 M−1 s−1). DOPC and DSPC lipids form at room temperature
fluid or gel phase membranes, respectively. Omitting the lipid, micellar solutions were obtained with
hydrolytic activity reaching k2 = 13.4 M−1 s−1. It is shown that cooperative hydrolysis is favoured in fluid
membranes and micelles, allowing the active moieties to arrange freely. The embedding and dynamic
self-assembly of membrane active components in fluid membranes and micelles provide facile access to
hydrolytically active soft interfaces.
Received 31st January 2014,
Accepted 14th March 2014
DOI: 10.1039/c4ob00247d
www.rsc.org/obc
perties of the enzyme active centre are the appropriate
distance and geometry of all participating groups, their
Introduction
Carboxyesters are ubiquitously found in nature. They are dynamics during the reaction, the microenvironment created
involved in many biological processes and are present in every- by the folded enzyme and the ability to bind the substrate.
day life. Esters of drugs are better transported through mem- Combining all such features in one organic molecule is chal-
branes with respect to free drugs and cleaved inside cells by lenging or even impossible and covalent enzyme models may
esterases. This is often used as a pro-drug concept in pharma- be incapable of adjusting the conformation for an efficient
ceutical sciences.1 Peptides, proteins, phosphates and carboxy hydrolysis.2,5,6
esters, respectively, are catalytically hydrolysed by natural
To investigate an alternative approach, we used functiona-
enzymes and many attempts to design artificial hydrolysing lized membrane surfaces of vesicles and micelles for the co-
enzymes have been reported over time, but their efficiency is operative hydrolysis of esters (Fig. 1) with zinc complex 1
still limited when compared to the natural models.2,3 Most
hydrolases contain metal ions such as zinc(II) (carboxypepti-
dase A, astacin) in their active centres.4 The stabilisation of the
transition state during hydrolysis occurs by hydrogen bonding
of the ester group to amino acid residues of the enzyme
protein backbone. Histidine, tyrosine and glutamine often par-
ticipate in the activation of the substrate. The crucial pro-
Institut für Organische Chemie, Universität Regensburg, Universitätsstraße 31,
D-93053 Regensburg, Germany. E-mail: burkhard.koenig@chemie.uni-regensburg.de;
Fax: +49 941 943 1717; Tel: +49 941 943 4575
†Electronic supplementary information (ESI) available: Experimental procedures
and compounds characterisation data, kinetic measurements data. See DOI:
10.1039/c4ob00247d
Fig. 1 Concept of the proposed membrane hydrolysis.
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Scheme 1 Amphiphilic bis-Zn-cyclen 1 used as a hydrolytic metal
center, fluorescein diacetate (FDA) used as a fluorescent probe and sup-
porting lipids for providing fluid (DOPC) and gel phase (DSPC)
membranes.
Fig. 2 Expected patch formation in different phospholipid membranes.
(Scheme 1) as a hydrolytically active metal center. This
complex is known to coordinate water molecules and due to its
Lewis acidity it produces hydroxide anions used for hydrolysis
reactions at neutral pH.7,8 The reaction rate was found to
increase dramatically once complex 1 was embedded in a lipid
membrane or in micelles.9 Embedded in membranes, complex
1 may also serve as a ligand for the molecular recognition of
phosphate anions.10
The effect of micellar catalysis of surfactant solutions has
been studied and described in detail in the literature.11,12 It
was discovered that positively charged surfactants such as tetra-
ammonium salts, amines, etc. embedded in micelles cleave
p-nitrophenol esters in buffered solutions at pH 7 with a high
efficiency.13–15 This effect is most likely caused by the high
local concentration of the polar functional groups on the
surface. Metallomicelles were also intensively studied for their
increased hydrolytic activity with respect to a homogeneous
solution.16,17
Fig. 3 Emission spectra of a CF (0.025 mM) and TAMRA (0.025 mM)
mixture in various lipids (λexc = 495 nm) and the mechanism of FRET.
The proposed cooperative catalytic effect by combining
embedded metal complexes and membrane additives on the
surface of a vesicular membrane is depicted in Fig. 1. Soft
interfaces are important for many biological processes and
have found use also in artificial systems.18 A potential advan-
tage of using a 2D functionalized membrane for hydrolysis is
the non-covalent assembly of the components in a two dimen-
sional fashion. In the fluid membrane of a vesicle, the hydro-
lytic active components may statistically arrange in distances
optimal for hydrolysis. This concept offers easy and facile
testing of different combinations of components and their
possible cooperative action. Micellar solutions of cyclen 1 and
membrane additives were also examined for their hydrolytic
activity. However, an advantage of the vesicular systems is that
their structure is better defined and the effects of different
membrane additives can be compared more easily.
dyes were embedded in DOPC and DSPC membranes, respect-
ively (Scheme 1). The unsaturated lipid DOPC forms a fluid
membrane at room temperature due to its transition tempera-
ture of −20 °C. DSPC yields a rigid gel phase membrane at
room temperature (transition temp. 55 °C). The FRET is
observed only in the fluid membrane of DOPC lipids, which
indicates partially mixed patch formation under these con-
ditions (Fig. 3).
Membrane additives (Scheme 2) were designed to support
the hydrolysis reaction resembling functional groups that are
present in the active centers of hydrolytic enzymes, e.g. imida-
zole, phenol, acids or amides. Micellar hydrolytic activity has
been reported previously for amines, oximes and histidine.21,22
The series was extended to cover a larger range of acidic,
neutral, basic and cationic functional groups. Amphiphiles are
grouped in Scheme 2 according to increasing pKa values of the
corresponding non-amphiphilic molecules (see ESI† for
reported literature values).
The membrane active amphiphilic molecules are, most
likely, not randomly distributed in the membrane, but form
clusters and patches. Complete phase separation or limited
mobility in the membrane might inhibit the cooperative action
(Fig. 2).19
To support this concept a well-described FRET pair of two
amphiphilic fluorescent dyes was co-embedded in membranes
(Fig. 3).20 The amphiphilic dye TAMRA exhibits in close proxi-
mity to amphiphilic carboxyfluorescein CF an emission at
580 nm after FRET, if irradiated at 495 nm. Both amphiphilic
Results and discussion
Vesicular hydrolysis
The hydrolytic activity was monitored using fluorescein di-
acetate (FDA, Scheme 1), a substrate for membrane esterases,
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Table 1 Hydrolytic activity of vesicular systemsa
DOPC
DSPC
kobs
k0obs
kobs
k0obs
(10−5 s−1
)
(10−5 s−1
)
(10−5 s−1
)
(10−5 s−1
)
Zn-cyclen 1 [5 mol%]
0.26
—
2.14
—
With a membrane additive [10 mol%]
A1
A2
A3
A4
A5
A6
A7
A8
0.07
0.9
0.3
2.4
0.4
0.4
0.5
0.4
4.0
4.4
0.01
0.03
0.02
0.03
0.03
0.02
0.04
0.05
0.5
0.6
2.1
2.5
2.2
1.9
2.6
1.4
1.9
21.6
6.0
9.3
4.2
5.0
5.3
4.2
3.3
3.9
0.01
0.02
0.02
0.01
0.02
0.02
0.02
0.02
0.8
0.3
1.2
0.03
0.1
A9
A10
A11
A12
A13
A14
A15
A16
A17
0.9
Scheme 2 Membrane additives investigated for cooperative hydrolysis
in their expected protonation state at pH 7.4, grouped by pKa values of
the functional group.
11.9
3.3
0.05
4.0
9.8
2.5
0.9
10.9
17.2
7.6
0.08
0.05
0.04
0.03
1.6
0.9
0.05
0.03
often used for evaluating the viability of cell samples.23,24 FDA
is a non-fluorescent diaryl ester whose fluorescence is trig-
gered by the cleavage of its ester groups. The often used
p-nitrophenol esters are more labile towards hydrolysis and
their photometric determination is less sensitive than the
measurement of fluorescence.
a kobs: 10 mol% of amphiphiles (0.05 mM) and 5 mol% of cyclen 1
(0.025 mM) relative to the lipids in DOPC and DSPC membranes; ko0b
hydrolytic rates for systems without bis-Zn-cyclen 1.
:
All measurements were performed under pseudo first-
order-reaction conditions and the rate constant of hydrolysis
(kobs) was derived from the slope of the increased fluorescein
concentration over 20 min (s−1) and from the initial FDA con-
centration. The concentration of the released fluorescein was
determined using a recorded calibration curve in vesicular
solutions not exceeding its linear region (see ESI†). All
reported membrane additives were tested in vesicular mem-
branes prepared by sonication above their transition tempera-
ture from both DOPC and DSPC in the presence of bis-Zn-
cyclen 1. To properly examine the hydrolysis, the best compo-
sition of the membrane was found to be 5 mol% of 1, 10 mol%
of the respective membrane additive and 85 mol% of phos-
pholipid. Hydrolytic activity was determined also for vesicles
containing only the zinc cyclen complex 1 (5 mol%) or exclu-
sively non-metal ion containing membrane additives (10 mol%).
The measurements indicate which species is mainly
responsible for the hydrolysis. All data are summarized in
Table 1 (values sorted according to Scheme 2). Bis-Zn-cyclen 1
embedded in the membrane showed a higher activity towards
hydrolysis than a non-amphiphilic solution of 1 (data not
shown, see ESI†), which confirms previous observations for bis-
(4-nitrophenyl) phosphate.9 Non-amphiphilic models of mem-
brane additives were also tested in hydrolysis, but were found
inactive (see ESI†). We found that 1 is much more active in
rigid DSPC- than in fluid DOPC-membranes. Compounds A9,
A10, A11, A13, A14 and A15, respectively, exhibit high rates of
hydrolysis in the absence of 1 in the membrane, often exceed-
ing the rate of sole cyclen 1. These compounds have functional
groups that have been previously reported to be hydrolytically
active in micellar solutions.
Fig. 4 Comparison of the hydrolytic effects in selected vesicle
compositions.
Only palmitic acid A1 caused a notable drop in hydrolytic
activity. The coordination of its carboxylate group to bis-Zn-
cyclen 1 may provide a rational explanation for the inhibition.
Amphiphiles which are expected to be neutral at the investi-
gated pH = 7.4, A7, A8, A3, A5 and A6, respectively (for DSPC
also A2, A4), induced only small changes in the hydrolytic
activity. High-polar and basic membrane additives exhibited
the highest hydrolytic activity in combination with cyclen 1.
A comparison of hydrolytic rates of membranes containing
only cyclen 1 or its combination with a membrane additive
(Fig. 4) clearly indicates cooperative effects.
Fig. 4 shows that selected membrane additives (A4, A9, A10,
A11, A13, A14, A15, A16, and A17) are hydrolytically more
active in fluid membranes. With the exception of A9 a larger
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increase of the hydrolytic rate is observed in DOPC membranes
than in DSPC. The effect is most pronounced for membrane
additives that are only hydrolytically active in combination
with complex 1, such as oxime A4 (ninefold higher activity),
phenol A2 (threefold) and guanidinium A17 (threefold). A
rationale behind this may be the higher dynamics of
embedded amphiphiles in the membrane. All membrane addi-
tives that increase the hydrolytic activity of complex 1 have
some common structural features. All compounds bear a N–O
or H–O bond and are at pH 7 either neutral with a pKa value
close to 7 (imidazole: 6.95, phenol: 8.05) or are protonated
(amines, guanidinium). Protonated membrane additives A
with more hydrogen atoms tend to accelerate the hydrolysis
more: the addition of the primary octadecylamine A14 induced
a more than 10 times faster hydrolysis compared to the tertiary
amine A12 in DOPC.
The hydrolytic effect of micellar solutions with polar head
groups is often credited to the high local concentration of
positive charge on the surface, therefore creating a local high
concentration of hydroxide ions, but the example of trimethyl-
ammonium salt A17 shows that this effect is only small in
comparison with cations generated by protonation (amines,
guanidinium). The observations indicate that the rate enhan-
cing effect of some membrane additives may have its origin in
creation of hydrogen bonds, which stabilize the transition
state of the substrate hydrolysis reaction.
Fig. 5 Second order kinetics for DSPC vesicles and micelles (left) and
saturation kinetics for DOPC (right) containing cyclen 1.
first-order kinetic rate constants were plotted against the
overall cyclen 1 concentration in the solutions (Fig. 5). The
reaction mechanism with embedded cyclen 1 for DSPC and
micelles tends to be different from that for DOPC membranes.
Two kinetic models were used: second-order kinetics were
observed in the case of DSPC vesicles and micelles with kobs
linearly dependent on the concentration of cyclen 1 in solu-
tion. In contrast, DOPC vesicles show a Michaelis–Menten type
behaviour of saturation kinetics. Apparently, a complex of a
substrate and a catalyst is formed, which then undergoes the
hydrolysis reaction. The kinetic data were non-linearly fitted
using eqn (1) according to previously published methods.25
kcatccyclen
kobs
¼
ð1Þ
KM þ ccyclen
KM describes the stability of the catalyst–substrate complex
and kcat the rate of its decomposition to a product and a
catalyst.
Micellar hydrolysis
Kinetic measurements of micellar solutions were done under
the same conditions as those described for the vesicular solu-
tions. The concentration of every component was kept con-
stant and only the lipids were omitted in the preparation step.
The concentrations of amphiphiles were exceeding their cmc
values (cmc for cyclen 1 is less than 0.01 mM, see ESI†). Fluo-
rescence is strongly quenched in the co-micellar solutions and
the hydrolysis was therefore monitored by absorption spec-
troscopy. Pseudo first-order-reaction rate constants of hydro-
lysis (kobs) were calculated from the slope of the increased
fluorescein absorption band (λmax = 505 nm) over 3 min
(M s−1) divided by the initial FDA concentration (see ESI†).
For micellar hydrolysis only the membrane additives A9,
A11, and A14 were tested, because they gave the best results in
vesicular systems. An enhancement of the hydrolytic activity
was observed for micelles of cyclen 1 with respect to the vesicu-
lar solution. In contrast, the membrane additives without the
lipid were less active (A11, A14) or insoluble (A9). Co-micellar
solutions gave high catalytic activity towards hydrolysis in
all three tested systems exceeding the activity of vesicles. An
average 3-fold increase of activity in comparison with micellar
cyclen 1 solution without a membrane additive implies
the existence of co-micelles and cooperative effects in the
hydrolysis.
In a fluid DOPC membrane, Zn-cyclen 1 shows saturation
kinetics even without the presence of a membrane additive,
but in a rigid DSPC membrane and in micelles, the hydrolysis
rate increases linearly with the Zn-cyclen 1 concentration.
The kinetics of functionalized membranes with the highest
hydrolytic activity consisting of cyclen 1 and membrane addi-
tives A4, A14, A9 or A11 were recorded and fitted to linear and
non-linear (1) models. The derived constants are summarized
in Table 2 (for data evaluation, see ESI†). For a better compari-
son of second order rate constants and reactions following a
Michaelis–Menten kinetics, the initial slope was calculated by
dividing kcat by KM (k′ ). For most of the functionalized DOPC
2
membranes, non-linear fitting gave better results as they fol-
lowed not a single kinetic model, whereas rigid DSPC systems
Table 2 Hydrolytic activity of micellar systemsa
kobs
k0obs
(10−5 s−1
)
(10−5 s−1
)
Zn-cyclen 1
8.6
—
With an additive
A9
A11
A14
34.6
30.7
22.4
n.d.
0.5
1.7
Mechanism and kinetics
a kobs: rate of the system with cyclen 1 (0.025 mM) and membrane
additives (0.05 mM), k0obs: micellar solutions of membrane additives.
The same concentration as that for vesicular measurements is used.
For mechanistic studies, various concentrations of vesicular
and micellar solutions were used and the observed pseudo-
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Table 3 Second order rate constantsa
kcat
KM
k′ (kcat/KM)
k2
2
(10−5 s−1
)
(10−5 M)
(M−1 s−1
)
(M−1 s−1
)
DOPC
DSPC
Only cyclen 1 (5%)
0.3
10.4
—
40.9
44.2
0.7
8.4
—
9.3
3.0
0.5
1.2
—
4.4
14.7
—
—
1.6
—
—
5% 1 + 10% A4
5% 1 + 10% A9
5% 1 + 10% A11
5% 1 + 10% A14
Only cyclen 1 (5%)
5% 1 + 10% A4
5% 1 + 10% A9
5% 1 + 10% A11
5% 1 + 10% A14
—
—
—
—
—
—
—
—
2.7
—
—
—
—
4.1
0.9
0.9
8.4
3.7
—
11.1
Micelles
Only cyclen 1
1 + A9
1 + A11
—
—
—
—
—
—
—
—
—
—
—
—
3.2
13.4
12.9
7.8
1 + A14
a Only best performing systems were selected.
Table 4 Fluorescein diesters
Namepr.
R
log Pc
FDAa
CH3
CH2CH3
Ph
iPr
(CH2)2CH3
tBu
3.20
4.22
4.41
4.93
5.24
5.75
9.31
17.47
F-C2a
F-Phb
F-iPrb
F-C3b
F-tBub
F-C7b
F-C15b
(CH2)6CH3
(CH2)14CH3
a Prepared from the acid chloride. b Prepared from the acid anhydride.
c Values obtained from ACDlabs software.
showed mostly linear correlations. The membrane additive A9
behaves differently from the other functionalized lipids and
shows a linear second-order kinetics also in the DOPC.
Embedded amine A14 leads to a saturation kinetics also in
DSPC vesicles. Micelles of cyclen 1 and membrane additives
showed the best hydrolytic activity (Table 3).
Fig. 6 Hydrolysis of fluorescein esters (0.02 mM) spontaneously at pH
To study the effect of lipophilicity and steric hindrance of
the ester group on the hydrolysis rate, a series of fluorescein
esters was prepared (Table 4). All these compounds were tested
under the same conditions as those previously described and
pseudo-first order hydrolytic rates were recorded for the three
best performing membrane compositions with DSPC and
DOPC lipids (Tables 1 and 2).
Fig. 6 shows that the trends in hydrolysis for different
esters remain the same in different vesicular systems (for data,
see ESI†); the molecular structure of the ester does not signifi-
cantly affect the mechanism. The reactivity of the different
esters is not affected by the membrane fluidity (DOPC vs.
DSPC). The highest hydrolytic activity was reached for FDA, the
least lipophilic ester (Fig. 6). With increasing lipophilicity of
the esters, the hydrolytic activity decreases. The lowest rates of
10.15 and by functionalized DSPC membranes at pH 7.4.
t
hydrolysis were recorded for phenyl or Bu esters (F-Ph, F-tBu),
which are more stable due to steric hindrance.
The trend in vesicular catalytic hydrolysis of the esters
follows the reactivity for spontaneous hydrolysis. The stabiliz-
ing effect of increasing lipophilicity is less pronounced for
reactions in vesicular solutions.
Comparison with hydrolytic enzymes
A direct comparison of kinetic data from the literature can be
challenging due to different substrates and variations in the
reaction conditions. Therefore commercially available hydro-
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edly. Their assembly has to be dynamic, as in DOPC mem-
Table 5 Comparison of enzymatic and vesicular hydrolysis of FDA
branes or micelles, to gain a cooperative hydrolytic effect.
k′ (kcat/KM)
2
kcat
KM
(10−4 (mg
(10−5 s−1
)
(10−1 mg ml−1
)
ml ) )
−1 −1 s−1
Porcine liver esterase
Lipase Candida rugosa
DOPC 5% 1 + 10% A14
68.7
73.2
44.2
0.08
0.4
0.2
858
166
391
Notes and references
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11 R. A. Moss and W. L. Sunshine, J. Org. Chem., 1974, 39,
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lytic enzymes were used under identical conditions to bench-
mark the hydrolytic rate of the functionalized vesicles. Porcine
liver esterase and the lipase Candida rugosa, typical enzymes
used in organic synthesis, were selected. Kinetic data were
derived using the same non-linear fit as that for vesicles to
obtain kcat and KM. Since the enzymes are produced by extrac-
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enzymes and vesicles. For vesicles the weight of cyclen
complex 1, membrane additive A and the lipid was considered
(see ESI† for data).
DOPC membranes functionalized with cyclen 1 and amine
A14 reach the activity of the two purchased enzymes (Table 5).
However, the catalytic activity of the enzyme active site still
remains much higher when compared to cyclen complex 1 and
membrane additives, as the vesicles contain many hydrolytic
active sites on their surface, while each enzyme has only one
active centre.
12 F. Mancin, P. Scrimin, P. Tecilla and U. Tonellato, Coord.
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Conclusions
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hydrolytic activity up to 25 fold in comparison with complex 1 Chem. Rev., 2009, 253, 2166–2177.
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temperature lead to more pronounced cooperative action of 730–735.
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3180 | Org. Biomol. Chem., 2014, 12, 3175–3180
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