Substrate structure governs maximum rate of catalysis exerted by cyclodextrin oxidase chemzymes

Article abstract of DOI:10.1007/s10847-010-9774-8

Selectively modified α-and β-cyclodextrin ketones or aldehydes act as artificial oxidases on a variety of small lipophilic substrates. The structure of the substrate is a highly important factor governing how effectively the oxidation reaction can be catalyzed. Amino acid-type substrates were not prone to catalysis, which yields new information about the limits of CD catalysis. Aniline showed some non-quantifiable catalysis, but for quinones and benzyl alcohols no net catalysis was detected. For aminophenol oxidation, o-aminophenols are far better substrates than p-aminophenols. The CD-catalyzed reaction follows Michaelis-Menten kinetics, involves CD cavity binding of the substrate and substrate recognition, and thus encompasses many of the hallmarks of natural enzymatic catalysis. Strong binding of the cooxidant H ₂O₂ to the CD catalytic carbonyl group is a prerequisite for the subsequent oxidation of the substrate and in accordance with this, the binding of H₂O₂ to β-CD dialdehyde was shown to be strong (K_d = 1.4 mM). β-CD 6^A,6^D-diketone which binds H₂O₂ weaker than an aldehyde was accordingly a less efficient oxidase. The wide range of substrates applicable to CD chemzyme catalysis brings about optimism for future scopes of synthetic biology.

Full text of DOI:10.1007/s10847-010-9774-8

J Incl Phenom Macrocycl Chem (2011) 69:417–423
DOI 10.1007/s10847-010-9774-8
ORIGINAL ARTICLE
Substrate structure governs maximum rate of catalysis exerted
by cyclodextrin oxidase chemzymes
•
Jeannette Bjerre Mikael Bols
Received: 30 October 2009 / Accepted: 16 March 2010 / Published online: 21 April 2010
Ó Springer Science+Business Media B.V. 2010
Abstract Selectively modified a- and b-cyclodextrin
ketones or aldehydes act as artificial oxidases on a variety
of small lipophilic substrates. The structure of the substrate
is a highly important factor governing how effectively the
oxidation reaction can be catalyzed. Amino acid-type
substrates were not prone to catalysis, which yields new
information about the limits of CD catalysis. Aniline
showed some non-quantifiable catalysis, but for quinones
and benzyl alcohols no net catalysis was detected. For
aminophenol oxidation, o-aminophenols are far better
substrates than p-aminophenols. The CD-catalyzed reac-
tion follows Michaelis–Menten kinetics, involves CD
cavity binding of the substrate and substrate recognition,
and thus encompasses many of the hallmarks of natural
enzymatic catalysis. Strong binding of the cooxidant H2O2
to the CD catalytic carbonyl group is a prerequisite for the
subsequent oxidation of the substrate and in accordance
with this, the binding of H2O2 to b-CD dialdehyde was
shown to be strong (Kd = 1.4 mM). b-CD 6A,6D-diketone
which binds H2O2 weaker than an aldehyde was accord-
ingly a less efficient oxidase. The wide range of substrates
applicable to CD chemzyme catalysis brings about opti-
mism for future scopes of synthetic biology.
Abbreviations
CD
Km
Kd
Cyclodextrin
Michaelis–Menten constant
Dissociation constant
kcat Catalyzed reaction rate
kuncat Uncatalyzed reaction rate
Introduction
Oxidases are an interesting class of enzymes; crucial
components in the vast majority of biological processes
and natural systems. The specialized natural oxidases
respond to the need of oxidation of distinct substrates
having widely different chemical design and structure
[1, 2]. Mimicking oxidase activity in an artificial enzyme
has been the goal of our research in which the cyclodextrin
macromolecule is used as a supramolecular host [3].
Modified cyclodextrins functioning as macromolecular
ensembles in enzymatic catalysis have been studied before,
commonly for esterase activity [4–6], but also for oxidation
[7–9]. CDs are cyclic a(1-4)glucopyranosides, i.e., circular
structures comprised of 6 (for a-CD) or 7 (b-CD)
alphabetically denominated glucose units which form a
cone-shaped macrostructure with a primary hydroxyl rim
(narrower) and secondary hydroxyl rim (wider). The non-
polar cavity can selectively bind small lipophilic com-
pounds whilst the polar CD exterior affords the inclusion
complex to be water-soluble, making the CD an attractive
structure to base the design of a biomimetic artificial
enzyme upon [10]. By selective organic synthetic manip-
ulations, the CD is equipped with catalytic groups which
react with the substrates that bind inside the CD cavity,
Keywords Supramolecular chemistry ꢀ Artificial
enzyme ꢀ Oxidase chemzyme ꢀ Cyclodextrin catalysis ꢀ
Synthetic biology ꢀ Substrate specificity
This work was presented at the First European Cyclodextrin
Conference, Aalborg, Denmark, 11–13th of October 2009.
J. Bjerre ꢀ M. Bols (&)
Department of Chemistry, University of Copenhagen,
Copenhagen, Denmark
e-mail: bols@kemi.ku.dk
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Fig. 1 Proposed catalytic cycle
for oxidation of o-aminophenol
by CD aldehydes
Determination of catalysis rate by UV
thereby effecting catalysis of the conversion of substrate to
product [11, 12]. Most of the CDs previously found to pos-
sess oxidase activity contain primary hydroxyl rim ketone
bridges as the catalytic entity, but recently it was noticed that
CDs equipped with aldehyde groups on the primary hydro-
xyl rim also catalyze oxidation of amongst others a large
range of aromatic amine substrates [13] (Fig. 1).
Each assay was performed on 8 samples (1 mL each) of the
appropriate substrate at different concentrations in 190 mM
phosphate buffer, pH 7.0 containing 72 mM H2O2, and
either the CD catalyst added (0.04–0.50 mM), or only
phosphate buffer and H2O2 as control. The reactions were
followed at 25 °C ( 1 °C) using UV-absorption at
400 nm, and were typically monitored for 30 min for
aminophenols and other substrates, and 5 h for benzyl
alcohols. Each assay was typically performed three times
for validation. At the end of each assay, the phosphate
buffered reactions were extracted with CH2Cl2, the com-
bined organic layer was dried (MgSO4), filtered, and con-
centrated in vacuo. The residues containing oxidation
products from respectively the catalyzed and non-catalyzed
reaction fractions were analyzed individually by electro-
spray mass spectrometry (ES-MS). For calculations of
kinetic parameters, velocities were determined as the slope
of the progress curve of each reaction. Uncatalyzed
velocities were obtained from a series of separate experi-
ments. Catalyzed velocities were calculated by subtracting
the uncatalyzed rate from the total rate of the appropriate
cyclodextrin containing sample. The catalyzed velocities
were used to construct Hanes plots ([S]/V vs. [S]) to ensure
that the reaction followed Michaelis–Menten kinetics. In
that case Km and Vmax were determined using least square
non-linear regression fitting to the Vmax vs. [S] curve. For
non-linear regression fitting, the NIST programme
Dataplot was employed [14]. kcat was calculated as
The catalyzed reaction follows enzyme-characteristic
Michaelis–Menten kinetics and employs hydrogen peroxide
as a cooxidant. The binding of hydrogen peroxide to the
catalytic CD aldehyde groups activates these for subsequent
oxidation of the amine group in the bound substrate.
A crucial factor in the catalytic context is substrate selec-
tivity, as the structural composition of the substrate sets the
boundaries for how effectively the reaction can be catalyzed
by the CD chemzyme. A wide range of new substrates
tested afford information of the realms of CD catalysis.
Materials and methods
General
Solvents were distilled under anhydrous conditions. All
reagents were used as purchased without further purifica-
tion. Evaporation was carried out in a rotary evaporator at
1
40 °C under reduced pressure. H NMR experiments were
carried out with a Varian Mercury 300 instrument. Catal-
ysis assays were performed using a Spectronic Genesys 5
spectrophotometer.
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419
Scheme 1 Examples of selective syntheses of some permethylated CD aldehydes
Vmax/[cyclodextrin]. kuncat was determined as the slope
from a plot of Vuncat vs. [S]. The following extinction
sides of the CD primary hydroxyl rim [15]. Chemical
manipulation specifically of these groups is possible by the
use of a 6A,6D (or just 6A, depending on reaction conditions)
selective de-O-benzylation step [16, 17], illustrated in
Scheme 1 where it forms part of the synthesis route of some
of the permethylated CD aldehydes recently studied [18].
coefficients (25 °C, pH 7.0, k = 400 nm) were determined
and used: 2-amino-phenoxazin-3-one, 0.42 mM-1 cm-1
;
N,N0-bis-(p-hydroxyphenyl)-2-hydroxy-5-amino-1,4-benzo-
quinone diimine, 1.59 mM-1 cm-1; 4-nitro-3-methylphenol,
5.51 mM-1 cm-1
;
2-nitro-4-methylphenol, 1.06 mM-1
cm-1; 2-nitro-3-methylphenol, 0.68 mM-1 cm-1; 2-nitro-
5-methylphenol, 2.26 mM-1 cm-1
azobenzene, 7.00
Enzyme kinetics
;
mM-1 cm-1; azoxybenzene, 7.00 mM-1 cm-1
.
The modified CDs were tested for catalysis of aromatic
amine oxidation on a range of structurally different small
molecule substrates, in phosphate buffer at pH 7.0 and
25 °C. The catalysis was monitored by measuring the
absorbance of the oxidized products by UV/Vis at 400 nm
[19, 20]. The assays were performed both with and without
CD added, and the net catalysis was obtained by sub-
tracting the non-catalyzed reaction rates from the cata-
lyzed. Via calculations based on the Michaelis–Menten rate
law equation, kinetic parameters such as Michaelis-Menten
constant (Km) reflecting the strength of substrate binding to
the CD, uncatalyzed (kuncat), and catalyzed (kcat) rate
constants were obtained. Thus, the reactions followed the
rate Scheme 2.
Investigation of epoxidation of styrene
To a solution of styrene (10.4 mg, 0.10 mmol) and 6A,6D-
dioxo-2A–G,3A–G,6B–C,E–G-nonadecakis-O-methyl-b-cyclo-
dextrin (5.6 mg, 0.004 mmol) in H2O (1.0 mL) at 0 °C
were added oxone (KHSO5, 30.6 mg) and NaHCO3
(16.8 mg) every 10 min over an 1 h time period. In total,
3 eq. (183.6 mg) of oxone and 12 eq. (100.8 mg) of
NaHCO3 were added. Finally, water was added, the
aqueous layer was extracted with CH2Cl2, the organic layer
was dried over MgSO4, filtered, and concentrated in vacuo.
1H NMR (CDCl3) of the residue was recorded. Similar
control experiments were also performed, with ommitance
of either CD or oxone; confirming that no catalysis takes
place without the dual presence of both these agents.
Chemzyme trends
Some general trends for recently tested CD chemzymes
1–4 are evident from Table 1. The strength of catalysis is
roughly proportional to the number of catalytic groups
present in the CD; the dialdehyde 2 is in most cases about
Results and discussion
Designing CDs as macromolecular catalysts
k1
kcat
The substrates in this paper were all tested with selectively
modified aldehyde- and ketone CDs as supramolecular oxi-
dation catalysts. Most of the artificial oxidases studied in the
Bols group have originated from selective modifications of
the 6A,6D-positions, creating catalytic entities on opposite
CD
S
CD
P
+
+
CD-S
k-1
Scheme 2 Michaelis–Menten kinetics; conversion of substrate (S) to
product (P)
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J Incl Phenom Macrocycl Chem (2011) 69:417–423
Table 1 Influence of substrate structure and CD chemzyme identity on catalysis
Non-catalyzed substrates
H2N
OH
OH
OH
OH
OH
OH
OH
OH
NH2
HO
COOH
NH2
HO
COOH
NH2
O
NH2
OH
HO
OH H2N
COOH
O
Non-catalyzed substrates were tested with:
Illustration to the right: substrates that were not subject to CD catalysis
twice as efficient as the monoaldehyde 3. Even in methanol
2 displays some catalysis, whereas in ethanol catalysis is
absent. This is presumably a result of the inclusion of the
solvent in the CD cavity, excluding a catalytically pro-
ductive binding mode of the substrate. The a-CD dialde-
hyde 1 performs slightly better or comparable to the b-CD
dialdehyde 2. In general, good catalysis seems to be
associated with weaker binding (high Km). This may imply
that a strong and unproductive substrate binding impedes
catalysis.
hydroxyl group this dramatically lowers catalysis for both
o- and p-aminophenols. If the amino group is placed meta
to the hydroxyl group, no catalysis can be measured.
Catalysis of p-aminophenol oxidation is impeded some-
what by methyl substitution in the substrate, whereas
for o-aminophenol the presence of a methyl group para to
the hydroxyl group is well-tolerated. These findings are
summarized graphically in Fig. 2.
Preparative analysis
Aminophenol structure impact on catalysis
The oxidation of o-aminophenol catalyzed by b-CD 6A,6D-
dialdehyde 2 was also examined in a preparative manner in
order to attempt to quantify the bulk of the oxidation
products. The quantitative assays were made analogous to
the standard kinetic assays, but in a larger scale; ending
with standard organic synthesis work-up; extraction of the
aqueous layer with CH2Cl2 and NMR analysis of the
organic residue components. The dual solubility of
The catalysis data in Table 1 reveal that the structural
composition of the aminophenol substrate governs how
effectively the reaction can be catalyzed, a trend persisting
regardless of the nature of the CD catalyst utilized.
O-aminophenols are far better substrates than p-amino-
phenols. However, if a methyl group is placed meta to the
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J Incl Phenom Macrocycl Chem (2011) 69:417–423
421
inherent instability. p-nitrosophenol was the only readily
available intermediate, in the form of a wetted matrix
suspension though, and it was tested for catalysis with
b-CD 6A,6D-dialdehyde 2. The matrix seemed to disturb
the catalysis and UV-readings excessively, and another
obstacle in this sense was that nitrosophenol absorbs at the
same wavelength as the oxidation product. Though the
hope was to be able to measure a difference in intensity of
the measured UV-absorption for the reaction, the instability
and formulation of the substrate made the direct obtain-
ment of finite data under catalysis assay conditions non-
straightforward. The search for alternative methods to
monitor the details of these reaction mechanisms more
closely will continue to be of interest.
para-aminophenol
worksb estw ithout
CH3-substitution
AC H3 groupp laced
meta to theO H
decreasesc atalysis
ortho-relationship
is favorable
NH2
OH
NH2
OH
CH3 placed
meta to OH
notf avorable
If NH2 is meta to
OH,n oc atalysis
is detected
CH3 placed
para to OH
well-tolerated
Fig. 2 General structural trends affecting how effectively the ami-
nophenol-type substrate can be catalyzed by the CD chemzyme
permethylated b-CD 6A,6D-dialdehyde 2 in both phosphate
buffer and organic phase complicated the spectra signifi-
cantly and made obtainment of unambiguous data difficult.
This technique would however be beneficial in case of non-
organic soluble catalysts, and provides as such an inter-
esting alternative to analysis of oxidation products.
Probing the limits of CD enzymatic catalysis
Some substrates were not subject to catalysis (depicted
right of Table 1). These yield valuable information about
the limits of CD catalysis. Several are amino acids and
hence catalysis of amino acid oxidation does not seem to be
an easy achievement with CDs. In three out of four cases
for the carboxylic acid-containing substrates, the carbox-
ylic acid entity or substituent is placed meta to the OH
which, as illustrated in Fig. 2, is generally not beneficial for
catalysis. Having a carboxylic acid group in the substrate
has significant steric as well as electronic impacts on the
aminophenol substrate, and the catalytically productive
binding of substrate to CD could be hampered by this. The
mimicking of natural amino acid oxidases might be feasi-
ble if based upon another supramolecular structure, how-
ever. Benzyl alcohols have previously acted as oxidizable
substrates for bridged ketone CD chemzymes [22], but the
CDs 2 and 4 show distinct substrate discrimination against
these; thus, no catalysis is observed for neither
2-hydroxyphenol nor 1-phenylethanol. The o-aminophe-
noloxidase GriF has been shown to catalyze the oxidation
of pyrocatechol with a kcat of 12 s-1, and a Km of 3.5 mM.
Rate-determining step
The oxidation of aminophenols is a process involving
several steps; it has been confirmed that the biomimetic
CDs catalyze the conversion of starting material into
product, but it would be of interest to find out exactly
which step is the rate-determining and therefore the one
prone to CD catalysis. Scheme 3 illustrates the oxidation
steps that follow from the initial hydroxylamine oxidation
product (see Fig. 1 for its formation), in case of both
o-aminophenol and the methyl-substituted aminophenols
[21].
By measuring the CD catalysis of some of these indi-
vidual intermediates, it might be possible to pinpoint
exactly which step is subject to CD catalysis, which would
be of great interest for optimizing the future chemical
design of the CD chemzymes. Efforts were however
hampered by the lack of commercial availability of most of
these compounds and related analogs; probably due to their
Scheme 3 Intermediates in
oxidation of aminophenols with
CDs and H2O2
NH2
OH
HO
NH
NH
N
O
NH2
O
OH
O
[ox.]
H2O
HO
NH
O
O
O
N
N
[ox.]
[ox.]
HO
HO
CH3
CH3
HO
CH3
H2O
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GriF also catalyzes the oxidation of o-aminophenol with a
kcat of 20 s-1, and a Km of 19 mM, and even seems to be
able to combine in the oxidation process, these two sub-
strates, affording a combined dimerization product [23].
Inspired by this, quinone substrates were investigated for
CD catalysis. Under the assay conditions employed, no
catalysis was to be found for either hydroquinone or
pyrocatechol, which illustrates a marked difference in
substrate compatibility between GriF and CD chemzymes.
Fig. 3 Catalytic rate vs. [H2O2] for 2-catalyzed oxidation of o-
aminophenol b-CD diketone 4 has previously been shown to catalyze
the epoxidation of styrene, with 100% conversion of the substrate
within 1 h [26, 27] (Scheme 5)
Catalysis of aniline oxidation
Aniline is an interesting substrate, having an aromatic
amine group reminiscent of the majority of substrates tes-
ted, but devoid of any hydroxyl groups. In earlier work, a
non-quantifiable catalysis of aniline was noted with
bridged ketone CDs [24]. With b-CD dialdehyde 2, aniline
again showed some non-quantifiable catalysis (Scheme 4).
The collective data suggest, that in order to achieve a
significant degree of catalysis, the substrates should pref-
erentially originate from phenol-type structures.
O
O
A
D
CH3
H3C
β
O
(OH)19
Oxone, NaHCO3
H2O, 1 hr, 0 oC
Scheme 5 b-CD 6A,6D-dimethylketone-catalyzed epoxidation of
styrene
Reactivity differences between CD ketones
and aldehydes
1H NMR and the conversion of substrate can be effectively
monitored. The permethylated b-CD dialdehyde 2 was
recently tested for catalysis of styrene epoxidation, with
oxone as a cooxidant. 1H NMR showed no catalysis, which
is possibly due to the reactive aldehyde group in the CD
itself being oxidized to acid by oxone and hence not being
able to engage in catalysis, like the ketone CD is [28]. This
is yet another illustrative example of the chemical differ-
ences in CD aldehydes and ketones as oxidation catalysts.
With this knowledge at hand, the customized design
of CD-based biomimetic catalysts for specialized purposes
can be further expanded. In the future, the goal of our
artificial enzyme research will be the synthesis of even
more potent macromolecular CD chemzymes for various
applications in catalysis and green chemistry.
Substrate design is obviously very important for catalysis,
albeit other factors are of interest as well; Table 1 reveals
that diketone 4 is a poor chemzyme compared to the CD
aldehydes. This can be attributed to the fact that ketones
bind H2O2 less efficiently than aldehydes [25]. To measure
the binding of H2O2 to CD dialdehyde 2, the catalysis
of o-aminophenol oxidation was studied at different con-
centrations of H2O2 with 2 (Fig. 3). The hyperbolic curve
shows that at Vmax = 50%, Kd = 1.4 mM for binding of
H2O2. This strong binding of the cooxidant peroxide cor-
relates with the dialdehyde 2 being a potent oxidation
catalyst.
In contrast to the previously studied unprotected CD
chemzymes tested for styrene epoxidation, the permethy-
lated CD aldehydes have dual solubility, merging both with
the aqueous layer and the organic phase upon work-up.
Still, it is possible to distinguish starting material clearly in
Conclusion
A diverse range of new substrates tested for CD-effected
catalysis of oxidation afford new information of the
boundaries of CD chemzyme catalysis. Selectively modi-
fied a- and b-CD ketones or aldehydes catalyze the oxi-
dation of different aminophenol substrates, with o-
aminophenol being the optimal substrate. Interestingly, the
oxidation of aniline was also catalyzed. Amino acid-type,
benzyl alcohol or quinone substrates were beyond the
realms of CD catalysis. The binding of the cooxidant H2O2
to CD dialdehyde is strong (Kd = 1.4 mM) and the less
O
O
H
H
N
N
N
β
NH2
(OMe)19
190 mM phosphate buffer, pH 7.0
o
N
O
72 mM H O , 25
C
2
2
Scheme 4 b-CD 6A,6D-dialdehyde-catalyzed oxidation of aniline
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epoxidation, whereas the aldehyde is uneffective for this
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potentials of CD catalysis, which fosters optimism for
future applications of CD chemzymes in synthetic biology.
Acknowledgments We thank The Lundbeck Foundation for finan-
cial support.
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