Synthesis and characterization of imidazole-substituted calix[4]arenes as simple enzyme-mimics with acyltransferase activity

Article abstract of DOI:10.1016/S0040-4039(01)01688-4

Calix[4]arenes which bear imidazole groups at different positions at the wide rim act as catalysts for enzyme-like release of p-nitrophenol from p-nitrophenyl esters in buffered solution.

Full text of DOI:10.1016/S0040-4039(01)01688-4

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 7837–7840
Synthesis and characterization of imidazole-substituted
calix[4]arenes as simple enzyme-mimics with
acyltransferase activity
Gu¨nter Dospil and Ju¨rgen Schatz*
Division of Organic Chemistry I, University of Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany
Received 16 August 2001; accepted 10 September 2001
Abstract—Calix[4]arenes which bear imidazole groups at different positions at the wide rim act as catalysts for enzyme-like release
of p-nitrophenol from p-nitrophenyl esters in buffered solution. © 2001 Elsevier Science Ltd. All rights reserved.
To mimic the action and efficiency of natural enzymes
was and still is a major challenge for supramolecular
chemistry. Such enzyme mimics usually consist of a
hydrophobic cavity and suitable catalytic substituents
preorganized to enable catalysis of an organic transfor-
mation.1–3 Cyclodextrins4,5 or cyclophanes6 bearing
thiazolium7 or imidazole moieties8 as well as metal
complexes as reactive sites have found widespread
application as artificial enzymes. Both bond-breaking
such as hydrolytic reactions8–10 as well as bond-forming
Scheme 1. Synthesis of calix[4]arenes 3–8; (i) AlCl3, toluene, rt, 69–98%; (ii) EtOH/H2O, NaOH, 3 days, 80°C, 94–98%; (iii)
CH2(NMe2)2, THF/HOAc, 48 h, rt, 92–95%; (iv) 1. MeI, DMSO, 1 h, rt; 2. imidazole, 3 h, 80°C, 70–90% (R=H), 72–85%
(R=Bz).
* Corresponding author. Tel.: +49-731-502-3153; fax: +49-731-502-2803; e-mail: juergen.schatz@chemie.uni-ulm.de
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01688-4

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G. Dospil, J. Schatz / Tetrahedron Letters 42 (2001) 7837–7840
of calixcrowns13–15 is possible. Furthermore, very effec-
tive dinuclear metallo-phosphodiesterase models based
on calix[4]arenes have been examined recently.16–18
Here we want to present the synthesis and characteriza-
tion of simple metal-free enzyme mimics with trans-
acyltransferase activity based on calix[4]arenes (Scheme
1).
reactions (e.g. benzoin condensation reactions7) can be
enhanced by macrocyclic catalysts. As expected, the
observed rate accelerations depend strongly on the
active site used for catalysis. Usually systems based on
catalytic metal complexes exhibit higher rate enhance-
ments compared to metal-free models. Cooperativity of
functional groups is important for the catalytic proper-
ties of the supramolecular enzyme mimic. In particular
imidazole functionalities were often used as an acid–
base couple or nucleophile, which can enhance
hydrolytic processes or Aldol-type condensation reac-
tions.8 Recently, calix[n]arenes11 have also come into
focus as platforms for enzyme mimics. Catalysis of ester
hydrolysis by deshielding of the substrate12 by complex-
ation in the hydrophobic pocket, or barium complexes
The synthesis of imidazole substituted calix[4]arenes
3–8 as possible acid–base catalysts starting from p-tert-
butylcalix[4]arene (1) was possible in high overall yields
by a five- or four-step procedure for the catalysts 3, 5,
7 and 4, 6, 8, respectively. Selective acylation of 1 with
benzoyl chloride by adapted procedures19–22 gave rise to
calix[4]arenes 2 bearing ester functionalities at positions
25,26,27-, 25,27- (distal), or 25, 26- (proximal) at the
lower rim of the macrocycle.
Exploiting the difference in reactivity of the free phenol
units and the phenol esters, tert-butyl groups could be
removed selectively by treating the compounds with
AlCl3 in toluene as an acceptor. Now, the introduction
of the imidazole moiety succeeded following the p-
quinonemethide route19 already established for the tet-
raimidazolylmethylcalix[4]arene.23 This protocol yielded
mono-imidazole 4 and distal substituted calix[4]arene 6
in the partial cone, and macrocycle 8 in an alternate
conformation. As expected, hydrolysis of the esters was
possible by heating the calixarenes for 3 days at 80°C
with an excess of NaOH in EtOH/H2O solution.20
Selective removal of the tert-butyl groups and introduc-
tion of the heterocycle as described before gave calix-
arenes 3, 5, and 7 in the cone conformation, as proven
by 13C absorptions of 32–33 ppm for the methylene
bridges which are in agreement with the de Mendoza
rule.24
Scheme 2. Hydrolysis of p-nitrophenyl benzoate (PNB) and
acetate (PNA) catalysed by macrocycles 3–8.
The initial rate (w0ꢀd(cPNB)/dt) for the release of p-
nitrophenol from p-nitrophenyl benzoate (PNB) was
determined by UV/vis spectroscopy at umax 410 and 450
nm, respectively (errors 55%, cf. Scheme 2 and Fig. 1).
Initial rates were measured to avoid interfering effects
such as product inhibition at higher rates of product
formation. In an aqueous reaction medium (Table 1)
the initial release of PNB was slightly increased by the
addition of methyl imidazole (9) used for comparison.
Figure 1. Initial release of p-nitrophenol from p-nitrophenyl
acetate (3.98 mmol L−1) in MeOH/MeCN (9:1) catalysed by
calixarene 5 (ꢀ, 1.03 mmol L−1) and without added macrocy-
cle (2) monitored by UV/vis spectroscopy (u=410 nm; T=
28°C).
Attachment of one nucleophilic group onto the upper
rim of the calix[4]arene resulted in a further increase of
the initial reaction rate. The macrocyclic skeleton
improved the hydrolysis by 13% compared with the
non-macrocyclic catalyst and by 52% towards the blank
hydrolysis. Addition of diimidazole calix[4]arene 5
bearing the catalytic groups in a distal arrangement at
a similar effective concentration of imidazole units dou-
bled the initial reaction rate indicating some kind of
cooperativity of the catalytic sites. At the chosen pH
6.3 roughly 50% of the imidazole units are protonated8
and therefore both imidazole groups can act as an
acid–base catalysts for the ester hydrolysis in aqueous
solution.
Table 1. Initial rate (w0) for the release of p-nitrophenol
from p-nitrophenyl benzoate (NPB) in MeOH/aq. buffer
pH 6.3 (70:30) with addition of various catalysts
(c (NPB)=95.0×10−3 mol L−1; T=30.0°C)
Cat.
c (cat)a (10−3 mol
c (NPB)/c (cat) w0 (10−10 mol
L−1
)
L−1 s−1
)
–
9
3
5
0.00
66.0
47.0
66.0
0.00
0.69
0.51
0.69
46.4
62.6
70.7
91.2
a Per imidazole unit.

G. Dospil, J. Schatz / Tetrahedron Letters 42 (2001) 7837–7840
7839
substrate. Fig. 2 summarizes the dependence of w0 for
the release of nitrophenyl from NPA from the added
buffer.
Both with and without added catalyst, a bell-shaped
profile typical for an acid–base catalysis could be
observed. At the optimum buffer composition,
calix[4]arene 8 increased the initial rate by a factor of 4.
This acceleration compared to the reaction without
added macrocycle was even higher when moving away
from optimum conditions. In this case, the hydrolysis
was accelerated about 9–11 times.
Addition of the parent p-tert-butylcalix[4]arene (1)
bearing no catalytic groups did not affect the reaction
rate (Table 2). A possible host–guest inclusion of the
NPA in the cavity of the calixarene does neither sup-
port nor interfere with the hydrolytic process. Methyl
imidazole (9) and mono-substituted calix[4]arenes 3 and
4 exhibited only a slight improvement. In the organic
reaction medium used now, the solvent can compete
efficiently with the substrate and the productive host–
guest binding is less favoured as compared to the
situation in aqueous solution.
Figure 2. Initial rate (w0) for the release of p-nitrophenol from
p-nitrophenyl acetate in MeOH/MeCN (9:1) catalysed by
calixarene 8 (2) and without added macrocycle (ꢀ) as a
function of added buffer (c (8)=1.03×10−3 mol L−1
c (NPA)=13.2×10−3 mol L−1; T=28°C).
,
Table 2. Initial rate (w0) for the release of p-nitrophenol
from p-nitrophenyl acetate (NPA) in MeCN/MeOH
(90:10) with addition of various catalysts (c (cat.)=2.05×
10−3 mol L−1 per imidazole unit, c (NPA)=13.2×10−3
mol L−1; buffer: c (iPr2EtNH+)=20.3×10−3 mol L−1
c (iPr2EtN)=63.8×10−3 mol L−1; T=28.0°C)
,
Surprisingly,
proximal
and
distal
substituted
calix[4]arenes 5–8 improved the hydrolysis in a similar
way; the acylated species (6, 8) exhibited higher cata-
lytic ability than calixarenes 5 and 7, bearing only
hydroxyl groups at the lower rim. This result is in
favour of a mechanism for the catalysis involving the
imidazole groups of the calixarenes and not acylation at
the lower rim as observed for a barium complex of a
calixcrown.15 This is also supported by time-dependent
NMR studies which gave no indication for an acylation
at the lower rim and accumulation of such an interme-
diate above a 3–5% level in the reaction mixture.
Cat.
w0 (10−10 mol L−1 s−1
)
w0 rel
–
1
9
3
4
5
6
7
8
21.5
19.6
38.1
29.9
40.8
55.3
68.7
51.2
84.8
ꢀ1.0
0.9
1.8
1.4
1.9
2.6
3.2
2.4
3.9
Assuming a nucleophilic assistance of the imidazole
moieties of the calixarenes, semi-empirical (PM3)
optimization25 of such intermediates for the ester
hydrolysis (Fig. 3) suggests that both calix[4]arene 5
and 7 is flexible enough to adopt a conformation
suitable for the productive interaction with NPA and
Owing to the limited solubility especially of p-tert-
butylcalix[4]arene (1) needed for comparison, the
hydrolysis was also studied in an organic solvent. In
this case, p-nitrophenyl acetate (NPA) was used as
Figure 3. Molecular structures of the assumed intermediates for the ester hydrolysis of NPA catalysed by calix[4]arenes 5 (left,
Erel=0.0 kcal mol−1) and 7 (right, Erel=0.6 kcal mol−1) obtained by semi-empirical geometry optimization (PM3).25

7840
G. Dospil, J. Schatz / Tetrahedron Letters 42 (2001) 7837–7840
catalysis stems mainly from cooperative action of the
functional groups attached at the wide rim of the
calix[4]arene skeleton. In agreement with the kinetic
data, the intermediate derived for the calixarene 5 is
slightly (−0.6 kcal mol−1) more stable than the corre-
sponding intermediate calculated for calixarene 7.
10. Diederich, F.; Chao, I. Recl. Trav. Chim. Pays-Bas 1993,
112, 335–338.
11. Mandolini, L.; Ungaro, R.; Eds. Calixarenes in Action;
Imperial College Press: London, 2000.
12. Shinkai, S.; Shirahama, Y.; Tsubaki, T.; Manabe, O. J.
Am. Chem. Soc. 1989, 111, 5477–5478.
13. Cacciapaglia, R.; Casnati, A.; Mandolini, L.; Ungaro, R.
J. Am. Chem. Soc. 1992, 114, 10956–10958.
14. Cacciapaglia, R.; Mandolini, L. Chem. Soc. Rev. 1993,
221–231.
In summary, imidazole substituted calix[4]arenes are
easily accessible acid–base catalysts with acyltransferase
activity which show rate enhancements up to a factor of
10, as tested for the release of p-nitrophenol from
p-nitrophenolates.
15. Cacciapaglia, R.; Mandolini, L.; Arnecke, R.; Bo¨hmer,
V.; Vogt, W. J. Chem. Soc., Perkin Trans. 2 1998, 419–
423.
Supplementary material
16. Molenveld, P.; Stikvoort, W. M. G.; Kooijman, H.; Spek,
A. L.; Engbersen, J. F. J.; Reinhoudt, D. N. J. Org.
Chem. 1999, 64, 3896–3906.
Details about geometry optimization of the intermedi-
ates of calixarenes 5 and 7 with NPA are available as
Supplementary Material.
17. Molenveld, P.; Engbersen, J. F. J.; Reinhoudt, D. N. J.
Org. Chem. 1999, 64, 6337–6341.
18. Molenveld, P.; Engbersen, J. F. J.; Reinhoudt, D. N.
Chem. Soc. Rev. 2000, 29, 75–86.
19. Gutsche, D. C. Calixarenes Revisited; Royal Society of
Acknowledgements
Chemistry: Cambridge, 1998.
20. Berthalon, S.; Regnouf-de-Vains, J.-B.; Lamartine, R.
Synth. Commun. 1996, 26, 3103–3108.
21. Dalbavie, J.-O.; Regnouf-de-Vains, J.-B.; Lamartine, R.;
Lecocq, S.; Perrin, M. Eur. J. Inorg. Chem. 2000, 683–
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This research was supported by the Fonds der chemis-
chen Industrie and the Deutschen Forschungsgemein-
schaft. Generous support by Professor Dr. G. Maas is
gratefully acknowledged.
22. Gutsche, D. C.; Kanamathareddy, S. J. Org. Chem. 1995,
60, 6070–6075.
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