Simple cyclodextrin aldehydes as excellent artificial oxidases

Article abstract of DOI:10.1007/s10847-010-9771-y

Cyclodextrin based oxidases, with a ketone as functional group are well known as good artificial enzyme mimics (Fenger et al. Org Biomol Chem 7:933-943; Marinescu and Bols Angew Chem Int Ed 45:4590-4593; Bjerre et al. Eur J Org Chem 704-710; Marinescu et al. J Am Chem Soc 127:17578-17579). We here report a series of modified cyclodextrins, having aldehydes as functional groups. The aldehyde based artificial enzymes have, in most cases, better catalysis than the ketones, because of their powerful covalent binding of hydrogen peroxide. Among the modified cyclodextrins studied are mono and di aldehydes on the 6 positions, with or without methylated hydroxyl groups. The aldehyde functionality was also introduced close to the secondary side, by attaching ethoxy-2-al or propoxy-3-al to the 2 position. The modified cyclodextrins showed excellent enzymatic activity towards oxidation of different aminophenols, and 4-methoxy benzyl alcohol with hydrogen peroxide as a stoichiometric oxidant. Rate enhancements up to 4,600 were achieved for oxidation of 4-methoxy benzyl alcohol, where as oxidation of amines gave rate enhancements up to 3,400. The artificial oxidases catalyses oxidations under enzymatic conditions (water, pH 7, 25 °C), following Michaelis-Menten kinetics. To confirm the enzyme activity, inhibition studies with sodium naphthalene-2-sulfonate were carried out. These studies showed competitive inhibition of the enzymes, verifying the cyclodextrins enzyme like character.

Full text of DOI:10.1007/s10847-010-9771-y

J Incl Phenom Macrocycl Chem (2011) 69:397–402
DOI 10.1007/s10847-010-9771-y
ORIGINAL ARTICLE
Simple cyclodextrin aldehydes as excellent artificial oxidases
•
Thomas Hauch Fenger Mikael Bols
Received: 30 October 2009 / Accepted: 16 March 2010 / Published online: 30 March 2010
Ó Springer Science+Business Media B.V. 2010
Abstract Cyclodextrin based oxidases, with a ketone as
functional group are well known as good artificial enzyme
mimics (Fenger et al. Org Biomol Chem 7:933–943; Ma-
rinescu and Bols Angew Chem Int Ed 45:4590–4593;
Bjerre et al. Eur J Org Chem 704–710; Marinescu et al. J
Am Chem Soc 127:17578–17579). We here report a series
of modified cyclodextrins, having aldehydes as functional
groups. The aldehyde based artificial enzymes have, in
most cases, better catalysis than the ketones, because of
their powerful covalent binding of hydrogen peroxide.
Among the modified cyclodextrins studied are mono and di
aldehydes on the 6 positions, with or without methylated
hydroxyl groups. The aldehyde functionality was also
introduced close to the secondary side, by attaching ethoxy-
2-al or propoxy-3-al to the 2 position. The modified
cyclodextrins showed excellent enzymatic activity towards
oxidation of different aminophenols, and 4-methoxy benzyl
alcohol with hydrogen peroxide as a stoichiometric oxidant.
Rate enhancements up to 4,600 were achieved for oxidation
of 4-methoxy benzyl alcohol, where as oxidation of amines
gave rate enhancements up to 3,400. The artificial oxidases
catalyses oxidations under enzymatic conditions (water, pH
7, 25 °C), following Michaelis–Menten kinetics. To con-
firm the enzyme activity, inhibition studies with sodium
naphthalene-2-sulfonate were carried out. These studies
showed competitive inhibition of the enzymes, verifying the
cyclodextrins enzyme like character.
Introduction
Enzymes are very fascinating, catalysing reactions in bio-
logical systems that would take millions of years, in only
seconds. One of the highest rate enhancements achieved is
1017 for OMP decarboxylase [5]. To mimic enzymes with
the same rate enhancements would have a great impact on
economy as well as the environment.
Catalysis of chemical reactions by artificial enzymes are
indeed helpful, in the sense that they can catalyse under
native enzyme conditions, leading a way into green
chemistry, and being environmentally friendly by avoiding
various organic solvents and chemicals.
To synthesise an artificial enzyme one needs a template
capable of binding a given class of compounds. Here the
cyclodextrins abilities of forming inclusion complexes and
ease of chemical modification, makes the cyclodextrin an
obvious template choice [6] (Fig. 1).
Materials and methods
Chemicals
All chemicals were provided by Sigma–Aldrich and used
without further purification. Phosphate buffer solutions with
different pH were prepared and used for kinetic studies.
Solvents were distilled under anhydrous conditions.
Keywords Cyclodextrin ꢀ Artificial enzyme ꢀ Oxidation ꢀ
Inhibition ꢀ Catalysis ꢀ Inclusion
Enzyme kinetics
All enzyme kinetics were measured by UV–Vis on a
Thermo Scientific Evolution 600. The reaction velocities
(V) were measured from seven samples with a fixed
enzyme concentration (0.17 mM) and increasing substrate
T. H. Fenger (&) ꢀ M. Bols
Department of Chemistry, University of Copenhagen,
Copenhagen, Denmark
e-mail: fenger@kiku.dk
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concentration, giving a Dixon plot, from which the inhibition
constant Ki easily could be determined [8].
Results and discussion
Various simple cyclodextrin aldehydes were synthesised
and tested for their ability to oxidize aromatic amines,
benzylic alcohols, hydroquinone and catechol. Furthermore
a series of inhibition studies were carried out to verify their
properties as artificial enzymes.
Synthesis
Fig. 1 a and b cyclodextrin, represented as a cone structure, capable
of binding small lipophilic molecules
Among the aldehydes synthesised, were the series of four a
and b cyclodextrin mono (6A) and dialdehydes (6A, D), as
shown in Scheme 1 (1–4). The monool and diol perben-
zylated a/b cyclodextrins were synthesised by the selective
debenzylation by DIBAL-H as reported earlier by Pearce
and Sinay¨ [9] followed by oxidation and subsequent deb-
enzylation as reported by Fenger et al. [10]. The series of
the four permethylated a/b cyclodextrin mono- and dial-
dehydes has also been synthesised [10].
The 6A-oxo-a-permethylcyclodextrin 5 was synthesised
analogously to a procedure by Chen et al. [10, 11]. Starting
by mono protection of a-cyclodextrin on the 6 position,
then subsequent methylation, removal of the protecting
group and finally oxidation to compound 5 (Scheme 2).
To study the effect of aldehydes on the secondary side of
the cyclodextrins, chemically modification on the 2A posi-
tion was done by allylation, methylation, hydroboration/
concentrations ([S] = 1–10 mM) in a phosphate buffer
solution (0.9 mM, pH 7) containing hydrogen peroxide
(64 mM) at 25 °C. The uncatalysed reactions (kuncat) were
measured in the same way, but having buffer instead of
enzyme. To be sure that the reactions followed Michaelis–
Menten kinetics, Wolf-Hanes plots were made ([S]/V vs.
[S]). Enzyme rate calculations were done by using least-
square nonlinear regression using Data plot, fitting to the
Michaelis–Menten equation determinating Vmax and KM.
kcat can be calculated from Vmax and the cyclodextrin
concentration [7].
k1
E þ S ꢀ ES ꢁ! E þ P
KM ¼ ðk2 þ kcatÞ=k1
k2
kcat
Inhibition studies were done by having a fixed
enzyme concentration and varying substrate and inhibitor
Scheme 1 Synthesis of the four
primary side cyclodextrin
aldehydes
Scheme 2 Synthesis of
permethylated primary side
aldehyde 5
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J Incl Phenom Macrocycl Chem (2011) 69:397–402
399
oxidation [10, 12], to give the two methylated b-cyclodex-
trins with either an 2-acetaldehyde or a 3-propanal group as
shown in Scheme 3.
2-aminophenol, 4-aminophenol, hydroquinone, catechol
and 4-methoxybenzyl alcohol.
For oxidation of 2-aminophenol the observed product is
compound 8 [13] and for oxidation of 4-aminophenol the
observed product is compound 9 [14] (Scheme 5). The
4-methoxybenzyl alcohol is oxidized to the corresponding
aldehyde.
Substrate oxidation with carbonyl and hydrogen
peroxide, a mechanistic insight
Perhydrate oxidations of amines and alcohols are well
known [1–4]. The believed mechanism for the oxidation is
shown in Scheme 4. (a) Hydrogen peroxide attacks the
carbonyl forming the active perhydrate. (b) An inclusion
complex forms from the activated artificial enzyme and
substrate, and reaction takes place. (c–d) The bound
product leaves the cavity, and the unactivated enzyme is
ready for yet another catalytic cycle.
The oxidation of hydroquinone to p-benzoquinone was
more complex than firstly anticipated. Following the
reaction for p-benzoquinone at 246 or 425 nm, did not
work, since the hydroquinone and p-benzoquinone can join
to make a quinhydrone charge transfer complex. Following
the reaction of the quinhydrone charge transfer complex,
did not work either, possibly due to subsequent decompo-
sition of p-benzoquinone and their by the quinhydrone
charge transfer complex [15].
Catalysis
Instead the reaction was followed at 289 nm, measuring
conversion of hydroquinone. By following the reaction at
this wavelength, the enzyme concentration should be kept
low due to the n ? p* absorbance of the carbonyl group
on the enzyme. Also the hydrogen peroxide concentration
As mentioned earlier the reactions were studied by UV–Vis
by increase in absorption of a given product, depending
on the substrate. Among the substrates tested were
Scheme 3 Synthesis of the
secondary side aldehydes
6 and 7
Scheme 4 Proposed enzymatic
catalytic cycle for oxidation,
with cyclodextrin aldehydes and
hydrogen peroxide
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J Incl Phenom Macrocycl Chem (2011) 69:397–402
change at 276 nm, but a rise at 245 nm. Hence the catechol
oxidation was abandoned.
The kinetic amine oxidation results of compound 1–7
are shown in Table 1 and the kinetic results for oxidation
of 4-methoxy benzyl alcohol in Table 2.
For oxidation of 2-aminophenol a linear relationship is
observed between the number of aldehyde groups present
and the rate observed, as seen by the pairs 1–2 and 3–4.
Between compound 1 and 3 (a and b cyclodextrin
respectively) there is not a difference, the same is also seen
for compound 2 and 4. Compound 6 is quite interesting, it
has only got one aldehyde group, but is just as effective as
the primary dialdehydes, this pattern is almost the same for
compound 5. The KM value gives an indication of the
binding of the substrate, for compound 7 the KM value is
quite high and the kcat is quite low, possibly indicating
catalysis outside the cavity of the cyclodextrin, because of
the long tethered aldehyde.
Scheme 5 Oxidation products of 2-aminophenol and 4-aminophenol
should be low, because of a possible further oxidation of
p-benzoquinone to 2,5-dihydroxy-p-benzoquinone [16]
(Scheme 6).
The highest rate enhancement (kcat/kuncat) achieved for
oxidation of hydroquinone was 1,030, with a KM of
1.04 mM, by compound 2. The experiment was done in a
phosphate buffered solution at pH 7, 25 °C, 1 h, with a
hydrogen peroxide concentration of 6.4 mM. The enzyme
concentration was 5.27E-5 M, and the substrate concen-
tration between 0.1 and 0.9 mM.
Oxidation of 4-aminophenol shows almost the same
trend as for 2-aminophenol oxidation with compound 1–4,
the more aldehyde groups the higher the activity. The
permethylated 6A a cyclodextrin 5 showed a drop in
intensity compared to the non methylated analog. Com-
pound 6 was again, surprisingly, the best artificial enzyme,
with a very high kcat compared to the other compounds.
For oxidation of 4-methoxy benzyl alcohol, compound 5
was the best, with an impressive kcat/kuncat of 4,600.
Oxidation of catechol leads to the unstable o-quinone.
A scan of the background reaction was done, at a cat-
echol concentration of 0.94 mM, and hydrogen peroxide of
6.40 mM. No conversion of catechol was observed at
276 nm. This was not expected as the catechol should be
oxidized to o-quinone in the presence of hydrogen peroxide
[17]. An enzyme screening of compounds 2–7 under the
same conditions, for 2‘ h did not show any noticeable
Scheme 6 Oxidation of
hydroquinone
Table 1 Oxidation of 2-aminophenol and 4-aminophenol
Enzyme
2-Aminophenol
4-Aminophenol
kcat (10-5 s-1
)
KM (mM)
k
cat/kuncat
kcat (10-5 s-1
)
KM (mM)
kcat/kuncat
1
2
3
4
5
6
7
597 86
1131 98
684 97
19.6 3.0
26.2 2.3
18.4 2.7
28.9 3.0
23.9 0.8
23.7 2.0
51.5 33.6
1426 229
2700 303
1632 258
3432 427
2419 182
2919 242
1443 947
102 59
283 67
390 37
442 33
47.3 4.7
2438 464
632 803
19.2 11.1
42.8 6.6
54.7 9.4
48.3 4.2
15.3 2.1
93.8 19.3
103 131
142 84
395 66
544 109
616 79
66 10
1438 147
1013 25
1223 52
605 395
3401 740
881 1123
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J Incl Phenom Macrocycl Chem (2011) 69:397–402
401
Surprisingly the secondary side aldehyde 6 was not better at
oxidizing benzylic alcohols than the primary side aldehydes.
would either reconfirm or reject the statement. To verify
the enzymatic activity, a series of inhibitors were tried.
The inhibitors tried were: cyclopentanol, sodium p-tol-
uenesulfonate and sodium naphthalene-2-sulfonate. The
inhibition experiments were tried for both oxidation of
2-aminophenol and 4-aminophenol. For inhibition experi-
ments with cyclopentanol there was a problem with solu-
bility, a drop in the rate of 4-aminophenol oxidation with
compound 6 was thus observed.
Inhibition studies
Even though the kinetic studies confirmed that the cyclo-
dextrins had enzyme like character, by following the
Michaelis–Menten kinetics, a simple inhibition study
Table 2 4-Methoxy benzyl alcohol oxidation
Competitive inhibition was observed for oxidation of
2-aminophenol with sodium naphthalene-2-sulfonate as
inhibitor. The Ki values of the cyclodextrins were all
around 100 mM, and shown in Table 3 [18] (Figs. 2, 3).
Inhibition with sodium p-toluenesulfonate gave higher
Ki values than with sodium naphthalene-2-sulfonate.
Enzyme
4-Methoxy benzyl alcohol
kcat (10-5 s-1
)
KM (mM)
kcat/kuncat
5
6
0.66 0.25
0.13 0.02
36.7 16.2
0.24 0.75
4642 1737
882 82
Fig. 2 Dixonplotofcompound1
Fig. 3 Dixonplotofcompound4
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J Incl Phenom Macrocycl Chem (2011) 69:397–402
5. Miller, B.G., Wolfenden, B.: Catalytic proficiency: the unusual
case of OMP decarboxylase. Annu. Rev. Biochem. 71, 847–885
(2002)
Table 3 Ki values determined from Dixon plots, with sodium
naphthalene-2-sulfonate as inhibitor
Enzyme
1
3
4
5
6. Murakami, Y., Kikuchi, J., Hisaeda, Y., Hayashida, O.: Artificial
enzymes. Chem. Rev. 96, 721–758 (1996)
7. Michaelis, L., Menten, M.L.: The kinetics of the inversion effect.
Biochem. Zeitschrift. 49, 333–369 (1913)
Ki (mM)
107
105
115
140
8. Dixon, M.: The determination of enzyme inhibitor constants.
Biochem. J. 55, 170–171 (1974)
Conclusion
9. Pearce, A., Sinay¨, P.: Diisobutylaluminium promoted regiose-
lective de-O-benzylation of perbenzylated cyclodextrins: a pow-
erful new strategy for the preparation of selectively modified
cyclodextrins. Angew. Chem. Int. Ed. 39, 3610–3612 (2000)
10. Fenger, T.H., Bjerre, J., Bols, M.: Cyclodextrin aldehydes are
oxidase mimics. ChemBioChem. 10, 2494–2503 (2009)
11. Chen, Z., Bradshaw, J.S., Lee, M.L.: A convenient synthesis of
mono-6-hydroxy permethylated b-cyclodextrin via tert butyli-
dimethylsilylation. Tetrahedron Lett. 37, 6831–6834 (1996)
12. Yi, G., Bradshaw, J.S., Rossiter, B.E., Malik, A., Li, W., Pet-
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Cyclodextrin aldehydes are indeed simple and effective
artificial oxidases. Compound 6 was the most versatile
enzyme capable of catalysing both 2-and 4-aminophenol
with great rate enhancements, higher than previously
reported [1–4]. Oxidation of 4-methoxy benzyl alcohol was
quite good for compound 5, but not that impressive for
compound 6. The enzyme like properties of the cyclo-
dextrin aldehydes were further proven by inhibition studies
with various inhibitors.
13. Horvath, T., Kaizer, J., Speier, G.: Functional phenoxazinone
synthase models kinetic studies on the copper-catalyzed oxy-
genation of 2-aminophenol. J. Mol. Catal. A. 215, 9–15 (2004)
14. Brown, K.C., Corbett, J.F.: Benzoquinone imines. Part 16. Oxi-
dation of p-aminophenol in aqueous solution. J. Chem. Soc.
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Acknowledgements We thank the Lundbeck Foundation for
financial support.
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