Artificial enzymes based on cyclodextrin with phenol as the catalytic group

Article abstract of DOI:10.1016/j.tetlet.2012.07.050

β-Cyclodextrin containing one or two N-linked ortho-N- acetylaminophenols has been synthesized and tested for their properties as artificial glycosidases. Four different para-nitrophenyl glycosides were used as substrates and k_cat/k_uncat

Full text of DOI:10.1016/j.tetlet.2012.07.050

Tetrahedron Letters 53 (2012) 5023–5026
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Tetrahedron Letters
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Artificial enzymes based on cyclodextrin with phenol as the catalytic group
⇑
⇑
Emil Lindbäck, You Zhou, Christian M. Pedersen , Mikael Bols
Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
b-Cyclodextrin containing one or two N-linked ortho-N-acetylaminophenols has been synthesized and
tested for their properties as artificial glycosidases. Four different para-nitrophenyl glycosides were used
as substrates and kcat/kuncat values of up to 381 were found for the di-functionalized products, whereas
the mono derivative showed up to 47 times rate enhancement. The optimum pH for the catalysis was
found to be approximately pH 8 and the influence of phosphate concentration was investigated.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 13 May 2012
Revised 21 June 2012
Accepted 11 July 2012
Available online 17 July 2012
Keywords:
Chemzymes
Glycosidase
Cyclodextrin
Catalysis
Enzymes are outstanding in their ability to catalyze reactions
with rate enhancements1 of up to 1019. Significant interest has
been devoted to understanding and copying nature’s machines,
and ‘artificial enzymes’ have become an important research area.2
One of the model systems for artificial enzymes is based on cyclo-
a higher probability that the substrate is bound in a manner where
the functional group is close to the reaction site. The importance of
the catalytical group orientation7 for catalysis and pH tolerance8
has earlier been investigated, and the need for a system which
can be fine-tuned to a certain pKa would greatly improve the future
development of chemzymes. In this communication, we present
the first cyclodextrin based artificial enzyme based on amido
phenols.
2-Acetaminophenol was chosen as the catalytic group due to its
ready introduction in the cyclodextrin scaffold by reductive amina-
tion of the known aldehyde 19 to give the N-linked amino phenol,
which upon acetylation would give the target chemzymes 4 and 8
in a comparatively few steps (Fig. 1). The perbenzylated b-cyclo-
dextrin aldehyde 1 was therefore treated with 2-aminophenol in
1,2-dichloroethane (DCE) and sodium triacetoxyborane as the
reductant and acetic acid as the catalyst (Scheme 1). The reaction
mixture was degassed and kept in the dark to avoid oxidation of
the aminophenol. When TLC showed complete conversion of the
starting material, pyridine and acetic anhydride (Ac2O) were added
immediately, which gave 2 in 72% yield (2 steps). Selective O-
deacetylation was conducted using LiOH in aqueous THF followed
by neutralization using HCl (1 M in water) to give amide 3. This
product was hydrogenolyzed using Pd/C/H2(g) in 2-methoxyetha-
nol and TFA as the acid catalyst to give the desired mono-function-
alized cyclodextrin 4 in 90% yield from 3 (Scheme 1).
dextrins, which are cyclic oligomers consisting of 1,4-a-linked d-
glucopyranosides, which results in an apolar cavity and a polar
exterior, making it water soluble. The development in this specific
area has moved from the unmodified (or randomly functionalized)
cyclodextrin which can be used as an additive to dissolve organic
compounds, to specifically modified artificial enzymes, chem-
zymes.3 With improvements in methods for selective protecting
group manipulations it has been possible to design structures
which resemble the active site in an enzyme and to synthesize bet-
ter and more sophisticated artificial enzymes. One group of en-
zymes of particular interest to us is the glycosidases, which are
responsible for cleavage of the anomeric bond in various sub-
strates. It is important for the improvements in biomass degrada-
tion as well as medicinal chemistry to understand these
processes and the scope of chemzymes in this area should be
investigated.
Our group has been interested in cyclodextrin-based glycosi-
dases.4 One advantage of these chemzymes is their broader sub-
strate recognition compared with very specific natural enzymes.5
It has been demonstrated that not only carboxylates, but also var-
ious other functional groups are able to function as glycosidases.6
Bis-functionalized chemzymes normally exceed mono-functional-
ized chemzymes in rate acceleration, which can be explained by
The bis-functionalized cyclodextrin 8 was synthesized in a sim-
ilar way using the known8 perbenzylated b-cyclodextrin dialde-
hyde 5 as the starting material (Scheme 2). Reductive amination
followed by acetylation gave 6 in 34% yield. Selective cleavage of
the phenol acetates gave 7 in 86% yield, which was subjected to
Pd/C-mediated hydrogenolysis of the benzyl protective groups to
give the bis-functionalized product 8 in 91% yield.10
⇑
Corresponding authors. Tel.: +45 35320160; fax: +45 35320214.
E-mail addresses: bols@chem.ku.dk, bols@kemi.ku.dk (M. Bols).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.07.050

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E. Lindbäck et al. / Tetrahedron Letters 53 (2012) 5023–5026
Scheme 2. Synthesis of the artificial glycosidase 8.
+
+
Figure 1. Artificial glycosidases 4 and 8 and substrates 9–12.
+
+
+
+
Figure 2. Hanes plot (S/V in mM/Unit vs substrate concentration in mM) for the
hydrolysis of 10 catalyzed by 8 in the presence of no (Â) or the competitive
inhibitor c-pentanol (+). The Ki for c-pentanol is 14.3 mM.
Table 1
Kinetic constants for the conversion of substrates in phosphate buffer at 59 °C. The
catalyst concentration was varied from 0.2 to 0.3 mM. E means enzyme and S means
substrate
E
S
[PO4À] (mM)
pH
kcat (107 sÀ1
)
KM(mM)
kcat/kuncat
8
8
8
8
8
8
8
8
8
8
8
8
4
4
4
4
9
9
9
9
9
9
9
9
9
10
11
12
9
500
500
500
500
50
100
250
500
500
500
500
500
500
500
500
500
5.9
6.8
7.1
7.4
8.0
8.0
8.0
8.0
8.9
8.0
8.0
8.0
7.4
8.0
8.0
8.0
28.9
25.8
30.7
88.0
18.0
23.2
46.5
83.4
47.7
64.2
78.5
69.6
10.3
10.4
10.1
12.6
0.59
2.86
2.53
6.57
2.25
3.08
3.86
5.91
4.30
3.04
2.04
3.86
4.58
1.62
4.42
0.93
29
90
45
268
96
76
202
381
112
358
290
121
31
Scheme 1. Synthesis of the artificial glycosidase 4.
With the two model enzymes 4 and 8 in hand their catalytic
properties were studied in the hydrolysis of 4-nitrophenyl glyco-
sides 9–12 in phosphate buffer at 59 °C and pH 5.9–8.0. The exper-
iments were based on monitoring the formation of 4-nitrophenol
spectrophotometrically.11
Under these conditions, native b-cyclodextrin has no effect on
the hydrolysis, but there is a spontaneous background reaction,
which must be subtracted. Thus experiments were performed at
different substrate concentrations (1–8 mM) with and without
the presence of 4 or 8 (0.2–0.3 mM). After subtraction of the uncat-
alyzed (i.e., background) reactions from the catalyzed (i.e., cyclo-
dextrin containing reactions), the net catalyzed rate was obtained
for each substrate concentration. These data were used to create
a Hanes plot (Fig. 2) from which KM and Vmax were determined.12
The values shown in Table 1 are for many different conditions.
We see that 4 and 8 displayed enzyme-like catalysis with sub-
9
10
11
47
56
47
strates 9–12 with kcat between 10À6 sÀ1 and 10À5 sÀ1, and KM from
0.5 to 6.6 mM. We also found that the rate acceleration (kcat/kuncat
performed by 4 or 8 varied from 29 to 381. Compared to previous
artificial glycosidase catalysts this means that phenols 4 and 8 are
less potent than cyanohydrins8 have about the same effectiveness
as the carboxylates6a and are more effective than aldehyde hy-
drates,8 fluorinated alcohols,6b sulfates,6a and phosphates.13
)

E. Lindbäck et al. / Tetrahedron Letters 53 (2012) 5023–5026
5025
Cyclopentanol was found to act as a competitive inhibitor of the
reaction giving a Ki value of 14.3 mM (Fig. 2). This confirmed that
binding to the cyclodextrin cavity was necessary for catalysis.
The kcat was found to show linear dependence on the phosphate
concentration (Fig. 3). Similar observations were earlier reported
for cyclodextrin carboxylates where it was suggested that phos-
phate participated in the catalysis.14 In this case the plot suggests
that a minor portion of the catalysis is independent since the graph
shows some catalysis at zero phosphate concentration (Fig. 3).
The dependence of pH of the catalysis (kcat) by 8 on substrate 9
is shown in Figure 4. The data fit a crude bell-shaped pH curve fol-
lowing pKas of 7.2 and 8.8 that is the catalysis increases as an acid
with pKa 7.2 is deprotonated and decreases as an acid with pKa 8.8
is deprotonated. The low value is identical with the pKa of the sec-
ond deprotonation of phosphoric acid while the high value is rea-
sonable for the phenol (2-hydroxyacetanilide pKa = 9.3) meaning
that the best catalysis is obtained when the phenol is protonated
Scheme 3. Mechanism for the glycosidase activity of 4 or 8 that fits the data.
independent of the phosphate and a plausible mechanism for the
phosphate independent catalysis is the mechanism of Scheme 3
but with water as the nucleophile. Though it was not specifically
shown in this work that glucose or glucose phosphate was also
formed this has been shown for other artificial glycosidases and
it is a logical assumption that this is also the case here. In any case
the reaction performs turnover without any visible drop in activity
which indicates that glycosylation of the catalyst is not taking
place.
In conclusion, we have shown that cyclodextrin functionalized
with 2-acetamidophenols can function as artificial glycosidases.
The presence of two catalytic groups enhances the rate accelera-
tion with a factor of about 8 compared with the mono-functional-
ized derivative. kcat/kuncat values of up to 381 make this model
system very promising for further development of artificial
glycosidases.
2À
and HPO4 is present.
A comparison of catalysts 4 and 8 (Table 1) revealed that mono-
functionalized 4 is a 6 to 8 fold poorer catalyst than di-functional-
ized 8. This could reflect that in 8 there is a higher probability that
the bound nitrophenyl glycoside is close to the phenol: since the
cyclodextrin conus is symmetrical the substrate can presumably
bind in orientations having the oxygen lone pair pointing toward
or away from the phenol.
There is no major difference in kcat between the substrates 9–12
(Fig. 1) for either catalyst. There is a little larger variation in the KM
value between the substrate but nothing major. This all fits with
the fact that the variation in the substrate structure has taken place
outside the binding site and therefore does not significantly influ-
ence catalysis. The best rate enhancement was obtained with 4-
Acknowledgements
We thank the Danish Research Council for natural science (FNU)
and the Lundbeck foundation for support.
nitrophenyl b-D-glucopyranoside 9 (381 times).
The above observations (Figs. 2–4) are in accordance with a ma-
jor mechanism where (1) the phenol acts as a protonator that is,
general acid catalysis, (2) monohydrogen phosphate (and possibly
phosphate) acts as a nucleophile and (3) the nitrophenyl group of
the substrate is bound in the cyclodextrin cavity prior to catalysis
(Scheme 3). Figure 3 shows that a minor portion of the catalysis is
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.07.
050.
References and notes
1. Wolfenden, R. Acc. Chem. Res. 2001, 34, 938–945.
2. Kirby, A. J.; Hollfelder, F. From Enzyme Models to Model Enzymes, RSC Cambridge
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3. Reviewed in (a) Marinescu, L.; Bols, M. Curr. Org. Chem. 2010, 14, 1380–1398;
(b) Bjerre, J.; Rousseau, C.; Marinescu, L.; Bols, M. Appl. Microbiol. Biotechnol.
2008, 81, 1–11; (c) Breslow, R. in: Breslow, R.; Dong, S. D, ‘‘Artificial Enzymes’’,
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4. Marinescu, L.; Bols, M. Trends Glycosci. Glycotechnol. 2009, 21, 309–323.
5. Bjerre, J.; Nielsen, E. H.; Bols, M. Eur. J. Org. Chem. 2008, 745–752.
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Figure 3. Dependancy of the hydrolysis of 9 catalyzed by 8 on the phosphate
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10. NMR spectroscopy of 7 and 8 was complicated by the appearance of two sets of
rotamers due to the amides.
11. Catalysis experiments: The experiments were carried out on a spectrometer
Spectronic Genesys 5 by Milton Roy and a Thermo Scientific Evolution 600. The
artificial enzymes were dissolved in water (2.0 or 3.0 mM in stock solution).
Substrates were dissolved in phosphate buffer with suitable concentration and
pH. Each assay was performed on 8 or 14, 1 mL samples, with increasing
substrate concentration, 2–8 mM. The chemzyme concentration in each
sample was 0.2–0.3 mM. As
a control, water was added instead of the
artificial enzyme. The hydrolysis was monitored for 6–18 h, at 59 °C at 400 nm.
Velocities were determined as the slope of the progress curve of each reaction.
The velocities of uncatalyzed reactions were obtained directly from the control
samples, those of catalyzed reactions were calculated by subtracting the
uncatalyzed rate from the total rate of the appropriate CD-containing sample.
The Vcat values were used to construct a Hanes-Plot ([S]/V v s [S]) from which
Figure 4. Dependence of kcat on pH in a 500 mM phosphate buffer. The data fit to a
curve constructed from two pKa’s of 7.2 and 8.8.

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E. Lindbäck et al. / Tetrahedron Letters 53 (2012) 5023–5026
Km and Vmax were determined. kcat were calculated as Vmax/[CD]. kuncat was
13. López, Ó.; Bols, M. Tetrahedron 2008, 64, 7587–7593.
14. Rousseau, C.; Caballero, F. O.; Nordstrøm, L. U.; Christensen, B.; Pedersen, T. E.;
Bols, M. Chem. Eur. J. 2005, 5094–5101.
determined as the slope of Vuncat versus [S]. The inhibition experiments were
made by adding c-pentanol (22.04 mM) to a catalyzed sample.
12. See Supplementary data for kinetic details.