Dinuclear and trinuclear Zn(II) calix[4]arene complexes as models for hydrolytic metallo-enzymes. Synthesis and catalytic activity in phosphate diester transesterification

Article abstract of DOI:10.1021/jo982201f

Calix[4]arenes modified with two or three Zn(II)-2,6- bis(aminomethyl)pyridyl groups, 3-[Zn]₂ and 5-[Zn]₃, respectively, were investigated as models for dinuclear and trinuclear metallo-enzymes that catalyze the cleavage of phosphate

Full text of DOI:10.1021/jo982201f

3
896
J . Org. Chem. 1999, 64, 3896-3906
Din u clea r a n d Tr in u clea r Zn (II) Ca lix[4]a r en e Com p lexes a s
Mod els for Hyd r olytic Meta llo-En zym es. Syn th esis a n d Ca ta lytic
Activity in P h osp h a te Diester Tr a n sester ifica tion
†
†
‡
‡,§
Peter Molenveld, Wendy M. G. Stikvoort, Huub Kooijman, Anthony L. Spek,
,
†
,†
J ohan F. J . Engbersen,* and David N. Reinhoudt*
Laboratory of Supramolecular Chemistry and Technology, University of Twente, P.O. Box 217, 7500 AE
Enschede, The Netherlands, and the Bijvoet Center for Biomolecular Research, Crystal and Structural
Chemistry, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
Received November 3, 1998
Calix[4]arenes modified with two or three Zn(II)-2,6-bis(aminomethyl)pyridyl groups, 3-[Zn]
-[Zn] , respectively, were investigated as models for dinuclear and trinuclear metallo-enzymes
that catalyze the cleavage of phosphate diesters. Under neutral conditions, 0.48 mM of 3-[Zn]
causes a rate acceleration of 23 000 in the transesterification of the RNA model substrate
-hydroxyproyl-p-nitrophenyl phosphate (HPNP, 0.19 mM). Comparison with the activities of a
2
and
5
3
2
2
mononuclear complex 2-[Zn] and a reference complex lacking the calix[4]arene backbone 1-[Zn]
shows that the catalysis is due to cooperative action of the Zn(II) centers and indicates that
hydrophobic effects contribute to the catalysis. Saturation kinetics and pH variation studies
demonstrate that the high catalytic activity of the flexible complex 3-[Zn]
high substrate binding affinity, affording a Michaelis-Menten complex in which the substrate is
converted with a relatively moderate rate. A rigid analogue 4-[Zn] exhibits both a lower substrate
binding strength and a lower catalytic rate. This demonstrates the importance of a certain flexibility
between the cooperating catalytic centers. The trinuclear complex 5-[Zn] induces a rate acceleration
of 32 000 times, and shows a decreased substrate binding and an increased catalytic rate compared
to its dinuclear analogue 3-[Zn] . In a possible mechanism two Zn(II) ions activate the phosphoryl
group and another activates the â-hydroxyl group of HPNP.
2
originates from a very
2
3
2
In tr od u ction
Hydrolytic enzymes that cleave phosphate ester bonds
to stabilize the pentacoordinate phosphorus transition
state and possibly the leaving group by cooperative
action. A specific subclass of these metallo-hydrolases
possesses even a third metal ion in the active site.
Examples include phospholipase C and P1 nuclease
which use three Zn(II) ions to catalyze the hydrolytic
cleavage of phosphate diester bonds in phosphatidylcho-
line and in nucleotides such as RNA and DNA, respec-
often have in their active site two transition metal ions
such as Zn(II), Mg(II), Mn(II), Ni(II), or Fe(III) that act
cooperatively as Lewis acid sites in the catalytic process.1
The mode of catalysis of phosphate ester hydrolysis has
been studied with various synthetic model compounds
2
3,4,5
3b,6,7
in which two metal ions like Zn(II),
Cu(II),
Co-
1
8
9
tively. However, the function of a third metal ion in close
(
III), or lanthanides(III) are held apart by appropriate
1
0
proximity of a dinuclear metal cluster in enzymes is yet
not fully understood, and only one example of a biomi-
metic study has been reported.5
ligands. The metal ions are proposed to activate the
phosphate group and a nucleophilic water molecule and
*
To whom correspondence should be addressed.
Laboratory of Supramolecular Chemistry and Technology. E-
†
(7) (a) Wall, M.; Hynes, R. C.; Chin, J . Angew. Chem., Int. Ed. Engl.
1993, 32, 1633. (b) Young, M. J .; Chin, J . J . Am. Chem. Soc. 1995,
117, 10577. (c) Liu, S.; Hamilton, A. D. Bioorg. Med. Chem. Lett. 1997,
7, 1779. (d) Liu, S.; Luo, Z.; Hamilton, A. D. Angew. Chem., Int. Ed.
Engl. 1997, 36, 2678.
‡
Klablunde, T.; Krebs, B. Angew. Chem., Int. Ed. Engl. 1996, 35, 2024.
(8) (a) Williams, N. H.; Chin, J . J . Chem. Soc., Chem. Commun.
1996, 131. (b) Wahnon, D.; Lebuis, A.-M.; Chin, J . Angew. Chem., Int.
Ed. Engl. 1995, 34, 2412. (c) Vance, D. H.; Czarnik, A. W. J . Am. Chem.
Soc. 1993, 115, 12165. (d) Chung, Y.; Akkaya, E. U.; Venkatachalam,
T. K.; Czarnik, A. W. Tetrahedron Lett. 1990, 31, 5413. (e) Seog Seo,
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Olson, L. P. J . Am. Chem. Soc. 1996, 118, 9867.
(10) Artificial dinuclear metal complexes have also been reported
to promote the hydrolysis of amides, amino acid esters, and guani-
diniums: (a) Karet, G. B.; Kostic, N. M. Inorg. Chem. 1998, 37, 1021.
(b) Yamaguchi, K.; Koshino, S.; Akagi, F.; Suzuki, M.; Uehara, A.;
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P.; Tonellato, U. Eur. J . Org. Chem. 1998, 1143. (d) Frey, S. T.; Murthy,
N. N.; Weintraub, S. T.; Thompson, L. K.; Karlin, K. D. Inorg. Chem.
1997, 36, 956. (e) He, C.; Lippard, S. J . J . Am. Chem. Soc. 1998, 120,
105.
(
b) Wilcox, D. E. Chem. Rev. 1996, 96, 2435.
2) For a recent review, see Chin, J . Curr. Opin. Chem. Biol. 1997,
, 514.
3) (a) Breslow, R.; Singh, S. Bioorg. Chem. 1988, 16, 408. (b)
Clewley, R. G.; Slebocka-Tilk, H.; Brown, R. S. Inorg. Chim. Acta 1989,
(
1
(
1
1
57, 233. (c) Chapman, W. H., J r.; Breslow, R. J . Am. Chem. Soc. 1995,
17, 5462. (d) Yashiro, M.; Ishikubo, A.; Komiyama, M. J . Chem. Soc.,
Chem. Commun. 1995, 1793. (e) Koike, T.; Inoue, M.; Kimura, E.; Shiro,
M. J . Am. Chem. Soc. 1996, 118, 3091. (f) Bazzicalupi, C.; Bencini, A.;
Bianchi, A.; Fusi, V.; Giorgi, C.; Paoletti, P.; Valtancoli, B.; Zanchi, D.
Inorg. Chem. 1997, 36, 2784.
(
4) Preliminary results have been published: Molenveld, P.; Kap-
sabelis, S.; Engbersen, J . F. J .; Reinhoudt, D. N. J . Am. Chem. Soc.
997, 119, 2948.
5) Yashiro, M.; Ishikubo, A.; Komiyama, M. J . Chem. Soc., Chem.
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Reinhoudt, D. N. J . Am. Chem. Soc. 1998, 120, 6726.
1
(
(
1
0.1021/jo982201f CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/07/1999

Dinuclear and Trinuclear Zn(II) Calix[4]arene Complexes
Ch a r t 1
J . Org. Chem., Vol. 64, No. 11, 1999 3897
Ch a r t 2
In previous papers,4,6 we have shown that calix[4]-
arenes11 are suitable building blocks for the design of
mimics for dinuclear metallo-hydrolases and that high-
rate enhancements can be achieved in the catalytic
cleavage of phosphate diesters. The observed synergy in
catalysis by two Zn(II) or Cu(II) ions was attributed to
an enzyme-like dynamic binding of the substrate and
cooperative action is demonstrated by comparison of the
catalytic activity of dinuclear complex 3-[Zn] with mono-
nuclear complexes 2-[Zn] and 1-[Zn]. The importance of
flexibility in catalysis is demonstrated by comparison
2
with dinuclear complex 4-[Zn] which is rigidified by
modification of the calix[4]arene lower rim with a crown
ether bridge. In this way, the common interconversion
between calix[4]arene conformations with two diverged
(flattened) or parallel (pinched) opposing aromatic units,
via a symmetrical cone-shaped intermediate (Chart 2),
is inhibited.15 Furthermore, we have enlarged the di-
2
1
2,13
transition state,
tional changes of the flexible calix[4]arene backbone.
facilitated by low-energy conforma-
1
4-16
Here, we report a detailed study of the catalytic activity
of various calix[4]arene-based Zn(II) complexes in phos-
4
phate diester transesterification (Chart 1). The effect of
nuclear calix[4]arene system 3-[Zn]
Zn(II) center to trinuclear complex 5-[Zn]
2
with an additional
, mimicking the
3
(
11) (a) Gutsche, C. D. Calixarenes. In Monographs in Supramo-
active site of trinuclear metallo-hydrolases.
lecular Chemistry, Vol. 1; Stoddart, J . F., Ed.; The Royal Society of
Chemistry: Cambridge, England, 1989. (b) Vicens, J .; B o¨ hmer, V., Eds.;
Calixarenes: A Versatile Class of Macrocyclic Compounds; Kluwer
Academic Publishers: Dordrecht, 1991. (c) For recent reviews, see
B o¨ hmer, V. Angew. Chem., Int. Ed. Engl. 1995, 34, 713; Takeshita,
M.; Shinkai, S. Bull. Chem. Soc. J pn. 1995, 68, 1088.
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Chem., Int. Ed. Engl. 1996, 35, 1090. (d) Scheerder, J .; Vreekamp, R.
H.; Engbersen, J . F. J .; Verboom, W.; van Duynhoven, J . P. M.;
Reinhoudt, D. N. J . Org. Chem. 1996, 61, 3476. (e) Soi, A.; Bauer, W.;
Mauser, H.; Moll, C.; Hampel, F.; Hirsch, A. J . Chem. Soc., Perkin
Trans. 1998, 1471.
(12) (a) Koshland, D. E., J r. Proc. Natl. Acad. Sci. U.S.A. 1958, 44,
9
2
8. (b) Koshland, D. E., J r. Angew. Chem., Int. Ed. Engl. 1994, 33,
375. (c) J orgenson, W. L. Science 1991, 254, 954. (d) Fersht, A. Enzyme
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13) (a) Kirby, A. J . Angew. Chem., Int. Ed. Engl. 1996, 35, 706. (b)
1
(
Kirby, A. J . Acc. Chem. Res. 1997, 30, 290. (c) Sanders, J . K. M. Chem.
Eur. J . 1998, 4, 1378. (d) Rowan, S. J .; Sanders, J . K. M. Curr. Opin.
Chem. Biol. 1997, 1, 483. (e) Eblinger, F.; Schneider, H.-J . Angew.
Chem., Int. Ed. Engl. 1998, 37, 826.
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Ungaro, R. J . Org. Chem. 1995, 60, 1454.
(16) Arduini, A. Fanni, S.; Manfredi, G.; Pochini, A.; Ungaro, R.;
Sicuri, A. R.; Ugozzoli, F. J . Org. Chem. 1995, 60, 1448.

3
898 J . Org. Chem., Vol. 64, No. 11, 1999
Molenveld et al.
Sch em e 1. Syn th esis of a Ca lix[4]a r en e-Ba sed Tr in u clea tin g 2,6-Bis(a m in om eth yl)P yr id yl Liga n d a
(
a) Hexamethylenetetraamine, TFA, 55%; (b) 33% MeNH2 in EtOH, H2, 10% Pd-C, 96%; (c) 2-bromomethyl-6-hydroxymethylpyridine,
K2CO3, THF, 65%; (d) SOCl2, CH2Cl2; (e) 33% Me2NH in EtOH, 77% over two steps.
Resu lts a n d Discu ssion 4
3
CHCl , the aldehydes in the triformyl derivative 7 were
introduced by a Duff reaction using hexamethylenetet-
raamine in trifluoroacetic acid. This method gives a
In this paper, we describe the synthesis of the ligands
-5, the formation of the corresponding Zn(II) complexes,
19
1
higher yield and is less laborious than the reported
and their catalytic activity in the cleavage of the phos-
phate diester bond of an RNA model substrate. The rate
of phosphate diester transesterification was studied as
a function of the Zn(II) concentration, the pH of the
reaction medium, and the substrate concentration, re-
spectively. The catalytic activity of the investigated
16
procedure with SnCl
4
and Cl
2
CHOMe in CHCl
3
.
The
aldehyde functionalities were converted in high yield to
secondary amines (8) by reductive amination using
methylamine under hydrogenation conditions. Originally,
the characterization of the crude reductive amination
product of the diformyl derivative 10 was hampered by
complexes follows the order 5-[Zn]
2-[Zn] > 1-[Zn] and is explained in terms of cooperat-
3
> 3-[Zn]
2
> 4-[Zn]
2
1
the presence of broad peaks in the H NMR spectrum.
>
Since this aminomethyl calix[4]arene (12) could not be
purified by common methods such as crystallization or
chromatography, it was temporary derivatized to the Boc-
protected amine (11). Purification and deprotection with
ivity between the Zn(II) centers with respect to substrate
binding and conversion.
Syn th esis. The reference 2,6-bis(aminomethyl)pyri-
dine ligand 1 was synthesized by an improved literature
procedure17 in which 2,6-bis(chloromethyl)pyridine was
reacted with excess of dimethylamine, giving the product
in quantitative yield. The flexible calix[4]arene ligands
trifluoroacetic acid yielded the aminomethyl calix[4]arene
1
(
12) in 74%. The H NMR spectrum of 12‚2TFA showed
3
for the six protons of the two N-CH groups one singlet
at 2.40 ppm, whereas in the free amine 12 these protons
appear as two singlets at 2.62 and 2.26 ppm integrating
for 4.5 and 1.5 protons, respectively. The same holds for
the benzylic protons of 12 appearing as two singlets at
2
, 3, and 5 are substituted at the lower rim with four
1
8
ethoxyethyl groups. These ethoxyethyl groups prevent
inversion of the aromatic units through the annulus of
the macrocycle and bring the calix[4]arene skeleton in a
rapid equilibrium between cone and flattened/pinched
cone conformations (Chart 2). In contrast, the rigidified
calix[4]arene ligand 4 was at the lower rim modified with
a crown ether bridge. This modification inhibits inter-
conversion between two flattened/pinched cone conforma-
tions and fixes the calix[4]arene skeleton in a conforma-
tion in which the upper rim-functionalized aromatic
nuclei diverge from the cavity (Chart 1).15
3
.46 and 3.40 ppm, which indicates a hindered rotation
of the two N-CH groups in 12. This phenomenon was
3
not observed for the tri-, mono-, and rigidified disubsti-
tuted aminomethyl calix[4]arenes 8, 17, and 20 of which
the crude reductive amination product was pure enough
and could be well characterized. The 2-hydroxymethylpy-
ridyl moieties in 9 were introduced by reacting the
secondary amine 8 with exactly 3 equiv of 2-bromo-
20
methyl-6-hydroxymethylpyridine, avoiding in that way
The stepwise synthesis of the 2,6-bis(aminomethyl)-
pyridyl ligand system in calix[4]arenes 2-5 was started
from the formyl derivatives, as is depicted for the
trinucleating ligand 5 in Scheme 1. Whereas for the
synthesis of the mono- and disubstituted analogues 2, 3,
overalkylation of the resulting tertiary amines. Conver-
sion of the hydroxyl groups in 9 to the chlorides proceeded
easily with SOCl affording the tris(2-chloromethylpyri-
2
dine) derivative as a hydrochloride in almost quantitative
yield. The corresponding free amine appeared to be
and 4 the aldehyde functionalities were introduced by
Gross formylation6
,16
4 2
using SnCl and Cl CHOMe in
(19) (a) Smith, W. E. J . Org. Chem. 1972, 37, 3973. (b) Linnane, P.;
J ames, T. D.; Shinkai, S. J . Chem. Soc., Chem. Commun. 1995, 1997.
(c) Dondoni, A.; Marra, A.; Scherrmann, M.-C.; Casnati, A.; Sansone,
F.; Ungaro, R. Chem. Eur. J . 1997, 3, 1774.
(20) Newcomb, M.; Timko, J . M.; Walba, D. M.; Cram, D. J . J . Am.
Chem. Soc. 1977, 99, 6392.
(
17) Markies, B. A.; Wijkens, P.; Boersma, J .; Kooijman, H.; Veld-
man, N.; Spek, A. L.; van Koten, G. Organometallics 1994, 13, 3244.
18) Arduini, A.; Casnati, A.; Fabbi, M.; Minari, P.; Pochini, A.;
Sicuri, A. R.; Ungaro, R. Supramol. Chem. 1993, 1, 235.
(

Dinuclear and Trinuclear Zn(II) Calix[4]arene Complexes
J . Org. Chem., Vol. 64, No. 11, 1999 3899
Ta ble 1. P r oton a tion Con sta n ts of Liga n d 1 a n d th e
Cor r esp on d in g Zn (II) a n d Cu (II) Com p lexes Deter m in ed
a
by P oten tiom etr ic p H Titr a tion s
log Kab
log K1
log K2
log K3 (M)
log K4 (MOH2)
log K5 (MOH2)
1
1-[Zn]c
1-[Cu]d
7.50 ( 0.04
8.32 ( 0.02
5.56 ( 0.02
7.94 ( 0.1
9.63 ( 0.09
12.20 ( 0.04
8.11 ( 0.09
11.59 ( 0.19
a
b
In 0.1 M KNO3 in 35% EtOH/H2O (v/v), at 20 °C. K1 ) [H21]/
+
+
[
[
(
H1][H ]; K2 ) [H1]/[1][H ]; K3 ) [1M]/[1][M]; K4 ) [1M(H2O)n]/
1M(OH)(H2O)n-1][H ]; K5
H2O)n-2][H ]. M ) Zn(II). M ) Cu(II).
+
)
[1M(OH)(H2O)n-1]/[1M(OH)2-
+
c
d
unstable and was therefore not isolated and character-
ized. Hence, the tris(2-chloromethylpyridine) hydrochlo-
ride was immediately reacted with excess of dimethyl-
amine yielding the trinucleating ligand 5 in 77%. This
highly polar and basic compound could easily be purified
by chromatography using aluminum oxide.
F igu r e 1. Experimental titration curves for ligand 1‚3HNO
in 0.1 M KNO in 35% EtOH/H O at 20 °C, in the absence (a)
3
3
2
and in the presence of 1 equiv Zn(NO
respectively.
3 2 3 2
) (b) or Cu(NO ) (c),
of 1-[Zn] (105.6 M-1) and 1-[Cu] (1012.2 M-1) and the pK
s
a
of the metal-bound water molecules are in the same
range as those found for similar 2,6-bis(aminomethyl)-
Sp ectr op h otom etr ic Titr a tion s. The addition of Zn-
(ClO
4 2 3
) to the ligands in 50% CH CN/20 mM HEPES pH
3
b,25
pyridine metal complexes in the literature.
slightly lower pK of 1-[Zn]OH compared to 1-[Cu]OH
might be due to a different coordination number,
The
7
.0, the conditions for the catalysis experiments (vide
2
1
a
2
2
infra), results in an increase in UV absorbance of the
2
5c,26
or
pyridine groups at λ ) 266 nm with isosbestic points at
due to the lower stability of 1-[Zn] as is observed for the
pK s of a series of triamine complexes with decreasing
stability constants.
2
54 and 282 nm in the overlap spectra at different
a
concentrations of Zn(ClO
4 2
) . Upon titration of the ligands
2
7
(
1 and 2, 0.2 mM; 3, 4, and 5, 0.05 mM) with Zn(ClO ) ,
4 2
Groves2 and Coates28 have already shown that small
increases in hydrophobicity can effect a dramatic increase
in the acidity of a metal-bound water molecule. Recently,
5c
the increase in absorbance shows saturation curves
according to the formation of mononuclear complexes
1
-[Zn] and 2-[Zn], dinuclear complexes 3-[Zn]
and trinuclear complex 5-[Zn] . Fitting of the titration
curves by a nonlinear least-squares method yielded
2 2
and 4-[Zn] ,
6
we have reported that the proximity of a hydrophobic
3
2
2
a
calix[4]arene surface lowers the pK of a Cu(II) complex,
5
-1
mimicking in this way the hydrophobic cavity of metallo-
enzymes.28 This effect is also expected in the Zn(II) and
Cu(II) complexes of calix[4]arenes 2-5 (vide infra). Also,
association constants larger than 10 M for binding of
Zn(II) to the 2,6-bis(aminomethyl)pyridyl groups. The
titration curve for 5-[Zn]
5% of the Zn(II) ions are bound during the catalysis
studies.
3
demonstrates that more than
the presence of 50% CH
for the catalysis experiments may increase the acidity
of the metal-bound water molecules to a pK value lower
than 7.9 or 8.1 which were determined for 1-[Zn] and
-[Cu] in 35% EtOH.24 In conclusion, the spectrophoto-
3
CN in the reaction mixtures used
8
a
P oten tiom etr ic Titr a tion s. To understand the pH
behavior of the metal complexes in catalysis, we have
determined the protonation constants of the mononucle-
ating ligand 1 and the deprotonation constants of the
water molecules ligated to the corresponding complexes
1
metric and potentiometric titrations reveal the formation
of sufficiently stable Zn(II) and Cu(II) complexes which
possess a metal-bound hydroxide around neutral condi-
tions.
1
1
-[Zn] and 1-[Cu] by potentiometric pH titrations (Table
2
3
). The titrations were performed in 0.1 M KNO
3
in 35%
2
4
X-r a y Cr ysta llogr a p h y. To demonstrate the mode of
Zn(II) binding by a 2,6-bis(aminomethyl)pyridyl ligand,
EtOH/H
2
O. Titration of 1‚3HNO
3
in the absence (Figure
1
(
a) and in the presence of equimolar amounts of Zn(NO
b), or Cu(NO (c), with 3 equiv base show titration
curves corresponding to the formation of 1-[Zn] and
-[Cu] in the pH regions 5.5-7.2 and 3.6-6.6, respec-
3 2
)
2
9
we have prepared a crystalline Zn(II) complex of 1. Slow
diffusion of Et O to an equimolar solution of 1 and Zn-
O in CH CN afforded crystals of (1)Zn-
O, suitable for X-ray structure analysis.
3 2
)
2
(
(
CH
CH
3
3
COO)
COO)
2
‚2H
‚H
2
3
1
2
2
tively. Upon further titration of the complexes with base,
an additional acidic group, which must be a metal-bound
In the solid state, the Zn(II) center is coordinated by the
water molecule, deprotonates with an apparent pK
a
of
(
25) (a) Kady, I. O.; Tan, B. Tetrahedron Lett. 1995, 36, 4031. (b)
7
.9 for 1-[Zn] and 8.1 for 1-[Cu]. The stability constants
Kady, I. O.; Tan, B.; Ho, Z.; Scarborough, T. J . Chem. Soc., Chem.
Commun. 1995, 1137. (c) Groves, J . T.; Olson, J . R. Inorg. Chem. 1985,
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Trans. 1996, 419.
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Chem. 1996, 35, 5540.
(
21) Because the calix[4]arene Zn(II) complexes are insoluble in pure
water, we selected CH CN as a cosolvent. The pH discussed in the
text refers to the pH of the aqueous portion of the reaction mixture
before dilution with CH CN up to 50% (v/v). For this method, see also
Gellman, S. H.; Petter, R.; Breslow, R. J . Am. Chem. Soc. 1986, 108,
288.
22) de Boer, J . J . A.; Reinhoudt, D. N.; Harkema, S.; van Hummel,
G. J .; de J ong, F. J . Am. Chem. Soc. 1982, 104, 4073.
23) Gans, P.; Sabatini, A.; Vacca, A. J . Chem. Soc., Dalton Trans.
985, 1195.
24) Besides titrations in 0.1 M KNO
also performed titrations in 0.1 M NaClO
Although these different conditions show almost equal titration curves
the pK determinations were calculated from the titrations in 35%
EtOH since possible hydration of CH CN may give irreproducible
results. See: Chin, J . Acc. Chem. Res. 1991, 24, 145.
3
3
2
(
(
1
(
3
in 35% EtOH/H
2
O we have
CN/H O.
4
in 50% CH
3
2
(28) Coates, J . H.; Gentle, G. J .; Lincoln, S. F. Nature 1974, 249,
773.
(29) Unfortunately, we were not successful in growing suitable
crystals of the calix[4]arene Zn(II) complexes.
a
3

3
900 J . Org. Chem., Vol. 64, No. 11, 1999
Molenveld et al.
three nitrogens of the tridentate ligand 1 and by two
oxygens of two monodentate acetates. The τ-descriptor30
for the complex is 0.36, meaning that the coordination
geometry around Zn(II) is intermediate between the
idealized square pyramidal (τ ) 0) and trigonal-bipyra-
midal (τ ) 1) extremes. The asymmetric unit contains
besides one Zn(II) complex also one water molecule. This
water molecule donates hydrogen bonds to a coordinating
(O20) and a noncoordinating (O18) acetate oxygen, form-
ing an infinite chain of hydrogen-bonded molecules
parallel to the a-axis. Presumably, this hydrogen bonding
causes the difference in acetate-Zn(II) coordination angles
and distances.
3 2
The X-ray structure of (1)Zn(CH COO) demonstrates
that the 2,6-bis(aminomethyl)pyridyl ligand occupies in
the solid state, and presumably also in solution, three of
five available coordination sites on Zn(II). This indicates
that two cis-oriented coordination sites on each Zn(II)
2 2 3
center in 1-[Zn], 2-[Zn], 3-[Zn] , 4-[Zn] , and 5-[Zn] are
available for the binding of a substrate and a water
molecule in the catalysis. Crystalline Cu(II) complexes
of 1 display an almost idealized square pyramidal
geometry.31
F igu r e 2. X-ray crystal structure of (1)Zn(CH
3
COO)
2
2
‚H O
drawn as anisotropic displacement ellipsoid plot (at 50%
probability level). Hydrogen atoms and the water solvate
molecule are omitted for clarity.
Ca ta lysis. The catalytic activity of the complexes
1
-[Zn], 2-[Zn], 3-[Zn]
situ by adding stoichiometric amounts of Zn(ClO
ligands, was studied toward the transesterification of the
2
, 4-[Zn]
2
, and 5-[Zn]
3
, generated in
pared with the dinuclear complex 3-[Zn]
2
the third
4 2
)
to the
Zn(II) center in 5-[Zn] effects a 40% higher rate en-
3
hancement in HPNP cleavage.
Since dinuclear Cu(II) complexes have been reported
as efficient catalysts in phosphate diester cleavage, we
3
2
RNA model substrate 2-hydroxypropyl-p-nitrophenyl
phosphate33 (HPNP, Chart 1) in 50% CH
CN/20 mM
6,7
3
aqueous buffer at 25 °C.21 The complexes showed good
pseudo-first-order kinetics. The initial rate constants for
both the catalyzed (kobs) and uncatalyzed (kuncat) reactions
were calculated from the increase in absorbance at λ )
have also measured the catalytic activities of 3-[Cu]
-[Cu] . However, these complexes show only a very low
activity similar to the activity of the mononuclear Zn(II)
complex 2-[Zn]. Since the pK of a Cu(II)-bound water
molecule in 1-[Cu] was found to be in the same range as
the pK of a Zn(II)-bound water in 1-[Zn], it is not
2
and
5
3
a
4
00 nm due to the release of p-nitrophenolate (Table 2).
4
In a preliminary communication, we have already
reported the enormous rate acceleration,34 compared to
the uncatalyzed reaction, of a factor 23 000 that is
a
expected that the dramatic rate differences between the
calix[4]arene Cu(II) and Zn(II) complexes is only caused
by differences in protonation state at a specific pH. The
lack of efficient catalytic power of the Cu(II) complexes
may be explained by the different Cu(II) coordination
geometry (see X-ray Crystallography) disfavoring coop-
erative action in catalysis and by the known low binding
affinity of Cu(II) to anionic phosphate ligands.
Tu r n over Ca ta lysis. In experiments with the sub-
strate HPNP present in 4-fold excess over the catalyst
2
induced by 0.48 mM of the dinuclear complex 3-[Zn] at
pH 7.0.21 Comparison with the catalytic activities of
mononuclear complexes 1-[Zn] and 2-[Zn], which are a
factor 300 and 50 less active, respectively, shows that
the high rate acceleration is due to synergetic action of
the two Zn(II) centers in 3-[Zn]
2
. Furthermore, the fact
6,35
that the mononuclear calix[4]arene complex 2-[Zn] is a
factor 6 more active than reference complex 1-[Zn],
lacking the calix[4]arene backbone, indicates that hy-
drophobic effects play a role in the catalytic process.
3
2 3
-[Zn] or 5-[Zn] , we observed turnover conversion to
completion. The loss of activity during the course of a
multiple turnover reaction was determined by (i) mea-
suring the initial rate of a mixture of equimolar amounts
HPNP and catalyst; (ii) continuing the reaction to total
conversion; (iii) adding a new equivalent of HPNP; (iv)
measuring the initial rate, up to conversion of 4 equiv of
HPNP. Whereas these experiments showed for the di-
The conformationally rigid dinuclear complex 4-[Zn]
cleaves HPNP also by the cooperative action of the Zn-
II) centers, but is a factor 8 less efficient than 3-[Zn]
The trinuclear complex appeared to be the most efficient
catalyst; at 0.48 mM of 5-[Zn] , at pH 7.0, a rate
2
(
2
.
3
acceleration of 32 000 times was observed. This corre-
sponds to a reduction of the half-life from approximately
3
nuclear complex 3-[Zn]
each catalytic cycle, the trinuclear complex 5-[Zn]
0% of its activity after each turnover. Product inhibition
2
only 10% loss of activity after
00 days for the uncatalyzed reaction to 13 min. Com-
3
loses
4
(
30) Addison, A. W.; Rao, T. N.; Reedijk, J .; van Rijn, J .; Verschoor,
G. C. J . Chem. Soc., Dalton Trans. 1984, 1349.
31) Unfortunately, we were not successful in growing suitable
crystals of (1)Cu(CH COO) . A search on (derivatives of) 1-[Cu(II)] in
the Cambridge Structural Database afforded limited information on
X-ray structures of (1)CuBr and (1)Cu(SCN) : Day, V. W.; Fredrich,
M. F.; Dabestani, S.; Bryan, P. S. Am. Cryst. Assoc., Ser. 2 1977, 5,
is most probably the reason for the loss of activity since
the addition of equimolar amounts of diethyl phosphate
to a mixture of catalyst and HPNP also caused a decrease
(
3
2
in rate of approximately 10% for 3-[Zn]
-[Zn] . This indicates an enhanced binding of the cyclic
phosphate diester product to the Zn(II) centers in 5-[Zn]
compared to those in 3-[Zn]
2
and 40% for
2
2
5
3
2
3.
3
(32) Perreault, D. M.; Anslyn, E. V. Angew. Chem., Int. Ed. Engl.
2
.
1
997, 36, 433.
(
33) Brown, D. M.; Usher, D. A. J . Chem. Soc. 1965, 6558.
(34) For a discussion about rate enhancements, see Morrow, J . R.;
(35) Martell, A. E.; Smith, R. M., Eds.; Critical Stability Constants;
Trogler, W. C. Inorg. Chem. 1988, 27, 3387.
Plenum: New York, 1974; Vol. 2.

Dinuclear and Trinuclear Zn(II) Calix[4]arene Complexes
J . Org. Chem., Vol. 64, No. 11, 1999 3901
Ta ble 2. Obser ved P seu d o-F ir st-Or d er Ra te Con sta n ts
a n d Rela tive Ra te Acceler a tion s for th e
substrate complex and conversion of the substrate within
this complex. Efficient binding of the substrate to the
catalyst requires the displacement of one or more Zn(II)-
bound Lewis bases, e.g., weakly bound water molecules
at low pH and tightly bound hydroxide ions at high pH.
Moreover, the bound hydroxides lower the catalysts
substrate binding affinity by diminishing the Lewis
acidity of the Zn(II) centers. On the other hand, conver-
sion of the bound substrate by transesterification requires
a general base. This can be buffer ions at low pH or
hydroxide ions, either bound to Zn(II) or free in solution,
at higher pH. Reference complex 1-[Zn], lacking the calix-
Tr a n sester ifica tion of HP NP Ca ta lyzed by Zn (II) a n d
a
Cu (II) Com p lexes
relative
rate acceleration
kobs × 10- (s-1
4
)
catalyst
noneb
2.7 × 10
-4
1
170
900
c
1
2
3
3
4
5
5
-[Zn]
-[Zn]
-[Zn]2
0.045
0.25
6.26
0.18
0.82
8.55
0.16
c
d
23 000
700
3000
32 000
600
d
-[Cu]2
d
-[Zn]2
-[Zn]3
-[Cu]3
d
d
[
4]arene backbone, shows a sigmoidal pH-rate profile
a
In 50% CH3CN/20 mM HEPES buffer pH 7.0, at 25 °C.
(Figure 4) with the inflection point at pH 7.5, which is
b
c
Measured from a 2.0 mM HPNP solution. Catalyst concentra-
tion 0.98 mM. Catalyst concentration 0.48 mM.
in agreement with the pK
metric titrations. The activity of the mononuclear calix-
4]arene analogue 2-[Zn] increases strongly from pH 6.8-
.4 with a kinetic pK of 7.1 and decreases slowly to 80%
a
determined by the potentio-
d
[
7
a
of the optimum activity at pH 8.2. Apparently, the
strength of substrate binding plays a crucial role in the
catalysis and is for the calix[4]arene-based complexes
efficient around neutral pH.4
Su bstr a te Bin d in g a n d Con ver sion . The HPNP
binding affinities of the calix[4]arene Zn(II) catalysts
were studied by measuring the rate of transesterification
as a function of HPNP concentration at a fixed catalyst
concentration. This results in saturation curves due to
the formation of a catalyst-substrate complex with a
stability constant Kass and conversion of the substrate
within this complex with a catalytic rate constant kcat
4 2
F igu r e 3. Dependence of kobs on the amount of Zn(ClO ) with
respect to dinucleating ligand 3 [(0) 0.48 mM] and trinucle-
ating ligand 5 [(b) 0.48 mM] for the cleavage of HPNP (0.19
(Figure 5). The kinetic data for this Michaelis-Menten
process were obtained by analyzing the experimental
data with Eady-Hofstee plots and nonlinear least-
squares curve fitting22 and are summarized in Table 3.
Previously, we have reported that the HPNP binding
3
mM) in 50% CH CN/20 mM HEPES buffer pH 7.0, at 25 °C.
Effect of Zn (II) Con cen tr a tion . To demonstrate
cooperative catalytic action of the two Zn(II) centers in
2
affinity of dinuclear calix[4]arene 3-[Zn] is extremely
3
2 2 3
-[Zn] and 4-[Zn] and the three Zn(II) centers in 5-[Zn] ,
high in the pH region 7.0-7.6 and decreases strongly at
we have determined the rate as a function of the ratio
ligand:[Zn]. This shows for 3 and 4 an optimum at 2 equiv
4
more alkaline pH. At pH 7.0, the binding constant and
the catalytic rate constant are a factor 70 and 40 smaller,
respectively, for the mononuclear complex 2-[Zn]. This
demonstrates the cooperative catalytic action of the Zn-
of Zn(ClO
Figure 3). The decrease in rate that is observed at high
concentrations of Zn(ClO can be explained by competi-
4 2 4 2
) and for 5 an optimum at 3 equiv of Zn(ClO )
(
4 2
)
(
II) centers in the flexible calix[4]arene 3-[Zn]
2
. The
tive binding of HPNP to free Zn(II) in solution. These
studies confirm that the substrate HPNP is indeed
cleaved by 3-[Zn] , 4-[Zn] , and 5-[Zn] and not by the
2 2 3
ligands or by free Zn(II) ions, which was already apparent
from the spectrophotometric titrations of the ligands with
rigidified complex 4-[Zn] is a factor 8 less efficient in
2
both HPNP binding (Kass) and conversion (kcat), which
shows the importance of a certain flexibility in the
6
,10c,12,13
cooperatingZn(II)centers duringthecatalyticprocess.
The 6 times higher activity and the slightly different pH
behavior of the mononuclear calix[4]arene complex 2-[Zn]
over the reference complex 1-[Zn] points to an enhanced
binding of the substrate, probably due to favorable
hydrophobic interactions with the calix[4]arene surface.
4 2
Zn(ClO ) .
Effect of p H. The pH-rate profiles for the transes-
terification of HPNP catalyzed by the dinuclear and
trinuclear complexes are bell shaped in the pH region
4
6
7
.0-8.0 with the optimum for 3-[Zn]
.4 and for 5-[Zn]
2
and 4-[Zn]
2
at pH
3
The trinuclear complex 5-[Zn] shows saturation kinet-
3
at pH 7.0 (Figure 4). For the dinuclear
and 4-[Zn] , the optimum activity is
reached by acid-base transitions with kinetic pK s of
approximately 7.1 and 7.8, whereas for the trinuclear
complex 5-[Zn] , these pK s are approximately 6.5 and
.5. According to the potentiometric titrations, these two
complexes 3-[Zn]
2
2
ics, but the rate of transesterification decreases at high
HPNP concentrations (Figure 5). This decrease is prob-
ably caused by inhibition due to binding of more than
one molecule of HPNP to this catalyst. Analysis of the
experimental data at low HPNP concentrations indicates
a binding affinity which is 45 times lower than for the
a
3
a
7
acid-base transitions must correspond to the subsequent
deprotonation of two water molecules bound to the
Zn(II) centers. This indicates that the catalytically most
receptor 3-[Zn]
2
. However, the catalytic power (kcat) of
5-[Zn] is 3 times larger, as may be expected on the basis
3
active form of calix[4]arene complexes 3-[Zn]
and 5-[Zn] possesses only one Zn(II)-bound hydroxide
ion. Bell-shaped pH-rate profiles at low concentrations
2
, 4-[Zn]
2
,
of three catalytic Zn(II) centers which are in close
proximity to each other. Apparently, there is a kinetic
barrier for complexation of the substrate, probably caused
by steric hindrance by the three 2,6-bis(aminomethyl)-
pyridyl groups. Once the substrate is bound, it is rapidly
3
3
c,4,6,7b-d
of phosphate diester substrates
can result from
opposing pH effects on the formation of a catalyst-

3
902 J . Org. Chem., Vol. 64, No. 11, 1999
Molenveld et al.
F igu r e 4. Dependence of kobs on pH for the transesterification of HPNP (0.19 mM) catalyzed by 1-[Zn] (0.98 mM, left) and 5-[Zn]
0.48 mM, right) in 50% CH CN/20 mM buffer, at 25 °C. Buffers: pH 6.0-7.0, MES; pH 7.0-8.2, HEPES; pH 8.2-8.6, EPPS.
3
(
3
3
F igu r e 5. Saturation kinetics curves for the transesterification of HPNP in 50% CH CN/20 mM HEPES pH 7.0, at 25 °C and
-
1
-4 -1
0
5
.48 mM catalyst. (Left) 4-[Zn]
2
; the experimental data points are fitted with Kass ) 7000 M and kcat ) 0.95 × 10
s . (Right)
-
1
-[Zn] ; the experimental data points are fitted to a 1:1 binding equation at low concentrations of HPNP with Kass ) 1200 M
3
-
4
-1
and kcat ) 24 × 10
s
(obtained by means of an Eady-Hofstee plot).
Ta ble 3. Kin etic Da ta for th e Tr a n sester ifica tion of
HP NP Ca ta lyzed by Ca lix[4]a r en e Zn (II) Com p lexesa
coordination, one Zn(II) center activating the phosphoryl
group and the other activating the â-hydroxyl group,
b
kcatc,d × 10
-4
Kassc × 10
2
Kme
(mM)
k2
subsequently followed by general base-promoted cycliza-
catalyst
(M- s-1
1
)
(s
-1
)
(M
-1
)
tion.
3c,7c
2
Also 4-[Zn] shows a distinct cooperativity in the
2
3
4
5
-[Zn]
0.015
43
0.68
2.9
0.19
7.7
0.95
7.5
550
70
12
1.3
catalytic cleavage of HPNP. Whereas from CPK models
the two-point binding mode seems possible for the
-[Zn]2
-[Zn]2
-[Zn]3
0.018
0.14
0.83
rigidified complex 4-[Zn]
Zn(II) center in 4-[Zn] delivers a bound hydroxide that
can deprotonate HPNP (not shown).
In the trinuclear complex 5-[Zn] , three Zn(II) centers
are cooperatively involved in the catalysis. The enhanced
kcat in HPNP transesterification indicates double Lewis
2
, it is more likely that one
24
2
a
b
In 50% CH3CN/20 mM HEPES pH 7.0, at 25 °C. Calculated
c
d
by k2 ) kcat/Km. Determined by curve fitting. Determined by
means of an Eady-Hofstee plot. Calculated by Km ) 1/Kass.
e
3
converted to the products, and the products are slowly
released as was pointed out by the turnover experiments.
Mech a n ism of Ca ta lysis. A generally proposed mech-
anism for phosphate ester cleavage catalyzed by two
metal ions concerns double Lewis acid activation by
bridging of the phosphoryl group between the two metal
centers to form a catalyst-substrate complex.2,3c-f,7,8
Subsequent in-line nucleophilic attack by either a (metal-
bound) hydroxide ion or a substrate â-hydroxyl group
results in leaving group expulsion through a pentacoor-
dinate trigonal bipyramidal transition state. In contrast
to transesterification of the RNA model substrate HPNP,
acid activation of the phosphoryl group by two neighbor-
ing Zn(II) centers (Figure 6). The third Zn(II) center
situated at further distance can possibly facilitate depro-
tonation of the â-hydroxyl group of HPNP, either by
Lewis acid activation similar to catalysis by 3-[Zn] or
2
via a bound hydroxide ion.
Con clu sion s
The transesterification of the RNA model substrate
HPNP is efficiently catalyzed by dinuclear and trinuclear
calix[4]arene complexes, exhibiting rate accelerations
over the uncatalyzed reaction of a factor 23 000 and
2
dinuclear complex 3-[Zn] is hardly active in the hydroly-
sis of diethyl p-nitrophenyl phosphate, ethyl p-nitrophe-
nyl phosphate, and p-nitrophenyl phosphate. As is known
that double Lewis acid activation of the phosphoryl group
induces cleavage of these substrates, it is unlikely that
the phosphoryl group of HPNP bridges the two Zn(II)
centers in 3-[Zn]
ciled with the mechanism shown in Figure 6. Herein,
catalyst 3-[Zn] is proposed to bind HPNP by two-point
32 000, at 0.48 mM 3-[Zn]
high catalytic activity of dinuclear complex 3-[Zn]
2
and 5-[Zn]
3
, respectively. The
results
2
from a very high substrate binding affinity (Kass), afford-
ing a Michaelis-Menten complex in which the substrate
is subsequently converted with a relatively moderate rate
(kcat). Generally, binding interactions of a substrate and
a transition state with the active site of an enzyme
2
. The experimental data can be recon-
4
2

Dinuclear and Trinuclear Zn(II) Calix[4]arene Complexes
J . Org. Chem., Vol. 64, No. 11, 1999 3903
Exp er im en ta l Section
Gen er a l In for m a tion . THF was freshly distilled from
sodium/benzophenone, CH Cl from CaCl , and SOCl from
2 2 2 2
linseed oil. Other solvents and chemicals were of reagent grade
and were used as received from commercial sources. Column
chromatography was performed with silica gel (SiO
2
, 0.040-
0
.063 mm, 230-400 mesh). Melting points are uncorrected.
1
13
H NMR and C NMR spectra were recorded in CDCl
3
with
Me Si as internal standard. FAB-MS spectra were recorded
4
with m-NBA as a matrix. Tetrakis(2-ethoxyethoxy)calix[4]-
arene 6,18 diformyl-tetrakis(2-ethoxyethoxy)calix[4]arene 10,
6,16
1
6
formyl-tetrakis(2-ethoxyethoxy)calix[4]arene 16, diformyl-di-
1
5
n-propoxycalix[4]arene-crown-3 20, and 2-bromomethyl-6-
hydroxymethylpyridine20 were synthesized according to lit-
erature procedures. The pH meter used for adjustment of
buffered solutions was calibrated daily. UV-vis spectra were
measured with a diode array spectrophotometer equipped with
a thermostated cuvette holder (7 cuvettes, 1.0 cm path length)
and a sample transport accessory.
2
,6-Bis[(d im eth yla m in o)m eth yl]p yr id in e (1). This com-
1
7
pound was synthesized by an improved literature procedure.
A solution of 2,6-pyridine dimethanol (3.00 g, 21.6 mmol) in
SOCl (100 mL) was stirred for 3 h, and subsequently
2
concentrated under reduced pressure. The residue was dis-
solved in EtOH (20 mL), added to a stirred 33% dimethylamine
solution in EtOH (100 mL), and stirred for 18 h. The solution
was concentrated in vacuo, the residue was taken up in EtOAc/
saturated Na
phase was extracted with EtOAc (4 × 100 mL). The combined
organic phases were dried over K CO , filtered, and concen-
2 3
CO solution (100/100 mL), and the aqueous
2
3
trated in vacuo to give the pure product as a yellow liquid (4.18
g, 100%) with the same analytical data as reported in the
literature.
5
,11,17-Tr ifor m yl-25,26,27,28-tetr a kis(2-eth oxyeth oxy)-
ca lix[4]a r en e (7). To a solution of tetrakis(2-ethoxyethoxy)-
calix[4]arene 618 (1.00 g, 1.40 mmol) in trifluoroacetic acid (30
mL) was added hexamethylenetetraamine (3.14 g, 22.4 mmol).
The reaction mixture was refluxed for 18 h, cooled to room
temperature, and poured out in ice water (150 mL). The
F igu r e 6. Schematic representations of possible mechanisms
for HPNP cleavage catalyzed by 3-[Zn] and 5-[Zn] .
2 3
involve an induced fit, in which the enzyme undergoes
conformational changes.12,13,36 A similar dynamic process
might explain the enzyme-like substrate-binding affinity
aqueous suspension was extracted with CH
after which the combined organic phases were washed with
saturated Na CO solution (75 mL) and water (75 mL). The
2
Cl
2
(3 × 50 mL),
2
3
of the flexible enzyme model 3-[Zn]
2
, since the rigidified
solution was dried with MgSO
was evaporated under reduced pressure. The residue was
purified by column chromatography (CH Cl /EtOAc, 9/1) to
give the product as a colorless oil (610 mg, 55%). The analytical
4
and filtered, and the solvent
2
analogue 4-[Zn] demonstrates a lower binding strength
as well as a lower catalytic rate.4 Also in the hydrolysis
,6
2
2
of amino acid esters catalyzed by dinuclear Cu(II) com-
1
6
data were the same as reported in the literature.
,11,17-Tr is(m eth yla m in om eth yl)-25,26,27,28-tetr a k is-
2-eth oxyeth oxy)ca lix[4]a r en e (8). Triformyl calix[4]arene
1
0c
plexes, the group of Tonellato concluded that flexibility
of the catalyst is better than rigidity.13 The presence of
a third Zn(II) center in close proximity of the dinuclear
5
(
7 (510 mg, 0.641 mmol) was dissolved in a 33% methylamine
solution in EtOH (50 mL), after which 10% Pd-C (50 mg) was
added. The reaction mixture was stirred for 16 h in a hydrogen
atmosphere, filtered over Hyflo, and evaporated till dryness.
Zn(II) cluster, e.g., complex 5-[Zn]
substrate affinity and an increased catalytic rate. This
implies that 5-[Zn] binds the pentacoordinate phospho-
rus transition state better than 3-[Zn] . Participation of
3
, results in a decreased
3
The residue was taken up in saturated Na
containing 5% of EDTA (40 mL) and was extracted with
CH Cl
(3 × 40 mL). The combined organic phases were dried
over K CO and evaporated to dryness to give the crude
2 3
CO solution
2
three Zn(II) ions in the catalytic cleavage of phosphate
esters was already proposed for the trinuclear enzymes
2
2
2
3
1
phospholipase C and P1 nuclease, and the efficiency is
secondary amine (620 mg, 96%), which was pure enough for
1
further modification. H NMR (CDCl , 250 MHz): δ (ppm) 6.73
3
now confirmed by using artificial trinuclear Zn(II) com-
plexes.5
(s, 4 H), 6.43-6.51 (m, 3 H), 6.40 (s, 2 H), 4.48 and 3.12 (2 AB
q, 8 H J ) 13.2 Hz), 4.13 (t, 4 H, J ) 5.7 Hz), 4.05 (t, 4 H, J
In conclusion, we have shown that calix[4]arenes
functionalized with Lewis acidic Zn(II) centers can mimic
properties of natural metallo-phosphodiesterases. For the
design of artificial enzymes with other catalytic functions,
calix[4]arenes might be suitable building blocks since
multiple catalytic groups can be preorganized dynami-
cally, similar to the amino acid residues and cofactors in
the polypeptide backbone of enzymes.
)
5.0 Hz), 3.88-3.83 (m, 8 H), 3.59-3.51 (m, 8 H), 3.49 (s, 4
H), 3.30 (s, 2 H), 2.36 (s, 6 H), 2.19 (s, 3 H), 1.24-1.16 (m, 12
H). 13C NMR (CDCl
, 250 MHz): δ (ppm) 155.8, 155.7, 154.7,
135.3, 135.2, 134.4, 134.2, 133.5, 128.2, 128.1, 127.9, 127.7,
3
1
3
22.2, 121.9, 73.3, 72.9, 69.6, 66.3, 66.2, 55.8, 55.5, 35.9, 35.8,
+
0.8, 15.3 (2×). FAB-MS: m/z 842.4 ([M + H] , calcd 842.5).
5
,11,17-Tr is[((6-h yd r oxym et h yl)p yr id in -2-yl-m et h yl)-
N-m et h yla m in om et h yl]-25,26,27,28-t et r a k is(2-et h oxy-
eth oxy)ca lix[4]a r en e (9). A mixture of calix[4]arene 8 (510
2 3
mg, 0.641 mmol), K CO (265 mg, 1.92 mmol), and 2-bromom-
2
0
ethyl-6-hydroxymethylpyridine (389 mg, 1.92 mmol) was
stirred for 2 days in THF (30 mL). The solvent was removed
under reduced pressure, the residue was taken up in EtOAc/
(
36) For a recent report concerning guest-induced structural changes
in artificial receptors, see Hayashi, T.; Asai, T.; Borgmeier, F. M.;
Hokazono, H.; Ogoshi, H. Chem. Eur. J . 1998, 4, 1266.

3
904 J . Org. Chem., Vol. 64, No. 11, 1999
Molenveld et al.
saturated Na
was extracted with EtOAc (3 × 50 mL). The combined organic
phases were dried over Na SO , filtered and concentrated in
vacuo. The residue was purified by column chromatography
CH Cl /MeOH/Et N, 90/9/1) to give 9 as a colorless oil (500
mg, 65%). H NMR (CDCl , 250 MHz): δ (ppm) 7.76-7.54 (m,
2
CO
3
solution (50/50 mL), and the aqueous phase
C
58
H
82
N
2
O
12: C, 69.71; H, 8.27; N, 2.80. Found: C, 69.80; H,
8.43; N, 2.74.
2
4
5,17-Bis(m et h yla m in om et h yl)-25,26,27,28-t et r a k is(2-
eth oxyeth oxy)ca lix[4]a r en e (12). The Boc protected amine
11 (6.23 g, 6.23 mmol) was dissolved in CH Cl (300 mL) after
2 2
which trifluoroacetic acid (15 mL, excess) was added. The
reaction mixture was stirred for 16 h and evaporated till
dryness. The residue was dissolved in a minimum amount of
(
2
2
3
1
3
3
H), 7.25-7.12 (m, 6 H), 6.76 (s, 2 H), 6.73 (s, 2 H), 6.53 (s, 2
H), 6.52 (d, 2 H, J ) 7.3 Hz), 6.26 (t, 2 H, J ) 7.3 Hz), 5.33 (br
s, 3 H), 4.73 (s, 6 H), 4.48 and 3.13 (AB q, 8 H, J ) 13.0 Hz),
CH
2
Cl
2
, and hexane was added until a precipitate formed.
COOH‚H O as a sticky solid material
(3.25 g, 41%). H NMR (CDCl , 250 MHz): δ (ppm) 12.10 (br
4
3
6
.18-4.08 (m, 8 H), 3.90-3.83 (m, 8 H), 3.60-3.50 (m, 8 H),
Filtration gave 12‚4CF
3
2
1
.43 (s, 4 H), 3.24 (s, 2 H), 3.10 (s, 4 H), 3.07 (s, 2 H), 2.06 (s,
3
H), 1.73 (s, 3 H), 1.25-1.17 (m, 12 H). 13C NMR (CDCl
, 250
s, 4 H), 8.18 (br s, 4 H), 6.95 (d, 4 H, J ) 7.2 Hz), 6.81 (t, 2 H,
J ) 7.2 Hz), 6.56 (s, 4 H), 4.51 and 3.16 (AB q, 8 H, J ) 13.0
Hz), 4.32 (t, 4 H, J ) 6.1 Hz), 4.01-3.94 (m, 8 H), 3.78 (t, 4 H,
J ) 4.7 Hz), 3.67 (br s, 4 H), 3.62-3.50 (m, 8 H), 2.40 (s, 6 H),
1.23 (t, 6 H, J ) 7.0 Hz), 1.19 (t, 6 H, J ) 7.0 Hz). C NMR
(CDCl , 250 MHz): δ (ppm) 161.0, 160.4, 156.4, 156.3, 135.3,
135.2, 129.4, 128.8, 124.0, 123.2, 117.8, 113.2, 74.2, 72.2, 69.5,
3
MHz): δ (ppm) 159.4, 159.3, 158.2, 155.7, 155.1, 137.2, 135.0
(
1
4
2×), 134.6, 134.3, 131.5, 129.2, 129.0, 127.9, 121.4, 121.2,
19.3, 119.1, 73.3, 73.0, 69.7, 66.4, 66.3, 64.7, 64.4, 62.0, 61.1,
6.+0, 42.3, 41.7, 30.8, 15.3, 8.8. FAB-MS: m/z, 1205.6 ([M +
1
3
H] , calcd 1205.7).
3
5
,11,17-Tr is[((6-N,N-d im eth yla m in om eth yl)p yr id in -2-
yl-m eth yl)-N-m eth yla m in o-m eth yl]-25,26,27,28-tetr a k is-
66.5, 66.4, 52.4, 32.4, 30.7, 15.1, 15.0. FAB-MS: m/z 999.3 ([M
+
(
[
2-eth oxyeth oxy)ca lix[4]a r en e (5). To a solution of calix-
4]arene 9 (500 mg, 0.415 mmol) in CH Cl /toluene (40 mL/5
(0.91 mL, 12.4 mmol), and the solution
+ H] , calcd 999.5). Anal. calcd for C48
H
66
N
2
O
8
‚4CF COOH‚
3
2
2
H
2
O: C, 52.09; H, 5.78; N, 2.17. Found: C, 52.02; H, 5.78; N,
2.18. The mother liquor was concentrated in vacuo, taken up
in saturated Na CO solution (200 mL) and extracted with
(3 × 100 mL). The combined organic phases were dried
CO and filtered, and the solvent was evaporated under
mL) was added SOCl
2
was stirred for 4 h. The solvent was removed under reduced
pressure to give the crude tris(2-chloromethylpyridine) hydro-
chloride. The tris(2-chloromethylpyridine) hydrochloride was
2
3
CH
over K
2
Cl
2
2
3
dissolved in CH
Me NH solution in EtOH (30 mL). The mixture was stirred
for 16 h and concentrated in vacuo. Saturated Na CO solution
40 mL) and CH Cl (40 mL) was added, the aqueous layer
was extracted with CH Cl
(3 × 40 mL), and the combined
organic phases were dried over K CO . Filtration and evapora-
tion of the solvent gave the crude product which was purified
by column chromatography (basic Al , CH Cl /MeOH, 99/1)
to give 5 as a colorless oil (410 mg, 77%). H NMR (CDCl
2
Cl
2
(20 mL) and was added dropwise to a 33%
reduced pressure to give the free amine 12 as a colorless oil
2
(2.89 g, 58%), pure enough for further modification (overall
1
2
3
yield 99%). H NMR (CDCl
3
, 250 MHz): δ (ppm) 6.68 (s, 2 H),
(
2
2
6.51 (s, 4 H), 6.63-6.49 (m, 4 H), 4.48 and 3.12 (AB q, 8 H, J
) 13.3 Hz), 4.13 (t, 4 H, J ) 5.8 Hz), 4.08 (t, 4 H, J ) 5.8 Hz),
3.87-3.81 (m, 8 H), 3.59-3.49 (m, 8 H), 3.46 and 3.40 (s, 4
H), 2.62 and 2.26 (s, 6 H), 1.51 (br s, 2 H), 1.26-1.14 (m, 12
H).
2
2
2
3
2
O
3
2
1
2
3
,
5,17-Bis[((6-h yd r oxym et h yl)p yr id in -2-yl-m et h yl)-N-
m e t h y la m in o m e t h y l]-25,26,27,28-t e t r a k is (2-e t h o x y -
eth oxy)ca lix[4]a r en e (13). A mixture of calix[4]arene 12
2
7
7
50 MHz): δ (ppm) 7.56 (t, 2 H, J ) 7.7 Hz), 7.43 (t, 1 H, J )
.6 Hz), 7.30 (d, 2 H, J ) 7.7 Hz), 7.18 (d, 2 H, J ) 7.7 Hz),
.11 (d, 1 H, J ) 7.6 Hz), 6.96 (d, 1 H, J ) 7.6 Hz), 6.89 (s, 2
(2.26 g, 2.83 mmol), K
momethyl-6-hydroxymethylpyridine (1.15 g, 5.67 mmol) was
stirred for 16 h in CH CN (150 mL). The solvent was removed
under reduced pressure, the residue was taken up in saturated
Na CO solution (125 mL) and the aqueous phase was ex-
tracted with EtOAc (3 × 100 mL). The combined organic
phases were dried over K CO , filtered, and concentrated in
vacuo. The residue was purified by column chromatography
(CH Cl /MeOH, 9/1) to give 13 as a colorless oil (1.89 g, 64%).
H NMR (CDCl , 250 MHz): δ (ppm) 7.62 (t, 2 H, J ) 7.6 Hz),
2 3
CO (783 mg, 5.67 mmol), and 2-bro-
2
0
H), 6.83 (s, 2 H), 6.30 (s, 2 H), 6.18 (d, 2 H, J ) 7.5 Hz), 5.99
(
4
t, 1 H, J ) 7.5 Hz), 4.40 and 3.07 (2 AB q, 8 H, J ) 13.1 Hz),
3
.15 (t, 4 H, J ) 6.2 Hz), 3.90 (t, 4 H, J ) 4.7 Hz), 3.82 (t, 4 H,
J ) 6.2 Hz), 3.73 (t, 4 H, J ) 4.7 Hz), 3.57 (s, 4 H), 3.50 (s, 4
H), 3.45 (s, 2 H), 3.53-3.39 (m, 8 H), 3.34 (s, 4 H), 3.17 (s, 2
H), 2.89 (s, 2 H), 2.20 (s, 12 H), 2.17 (s, 6 H), 2.10 (s, 6 H),
2
3
2
3
1
.69 (s, 3 H), 1.15 (t, 6 H, J ) 7.0 Hz), 1.09 (t, 6 H, J ) 7.0
13
Hz). C NMR (CDCl
3
, 250 MHz): δ (ppm) 159.5, 159.3, 158.3,
2
2
1
1
1
1
4
57.9, 156.3, 155.1, 154.2, 136.7, 136.4, 135.7, 135.6, 133.8,
33.5, 132.2, 132.1, 129.3, 129.2, 128.2, 127.6, 122.1, 121.1,
21.0, 73.6, 72.5, 69.6, 66.4, 66.2, 65.9, 63.2, 63.0, 61.8, 61.5,
3
7.17 (d, 2 H, J ) 7.6 Hz), 7.10 (d, 2 H, J ) 7.6 Hz), 6.74 (d, 4
H, J ) 7.3 Hz), 6.63 (t, 2 H, J ) 7.3 Hz), 6.50 (s, 4 H), 4.76 (s,
4 H), 4.48 and 3.13 (AB q, 8 H, J ) 13.1 Hz), 4.19 (t, 4 H, J )
6.0 Hz), 4.05 (t, 4 H, J ) 5.4 Hz), 3.90 (t, 4 H, J ) 6.0 Hz),
3.82 (t, 4 H, J ) 5.4 Hz), 3.54 (q, 8 H, J ) 7.0 Hz), 3.33 (s, 4
5.7, 45.6, 42.3, 42.2, 30.8, 15.3 (2×). FAB-MS: m/z 1285.3
+
([M] , calcd 1285.8).
5
,17-Bis[(N-Boc-N-m et h yl)a m in om et h yl]-25,26,27,28-
1
3
tetr a k is(2-eth oxyeth oxy)ca lix[4]a r en e (11). Diformyl-tet-
H), 2.81 (s, 4 H), 1.99 (s, 6 H), 1.20 (t, 12 H, J ) 7.0 Hz).
C
6
,16
rakis(2-ethoxyethoxy)calix[4]arene 10
(6.43 g, 8.36 mmol)
NMR (CDCl , 250 MHz): δ (ppm) 158.6, 156.4, 137.2, 135.4,
134.2, 128.7, 128.2, 122.2, 121.7, 119.5, 73.4, 72.9, 69.7, 66.3,
3
was dissolved in a 33% methylamine solution in EtOH (300
mL) after which 10% Pd-C (2.00 g) was added. The reaction
mixture was stirred for 16 h in a hydrogen atmosphere, filtered
over Hyflo, and evaporated till dryness to give the crude
secondary amine. This was dissolved in MeOH (300 mL) after
64.8, 62.2, 60.7, 42.2, 30.8, 15.3 (2×). FAB-MS: m/z 1039.8
-
+
([M-H] , calcd 1039.6), 1042.1 ([M + H] , calcd 1041.6).
5,17-Bis[((6-N,N-d im et h yla m in om et h yl)p yr id in -2-yl-
m eth yl)-N-m eth yla m in o-m eth yl]-25,26,27,28-tetr a k is(2-
eth oxyeth oxy)ca lix[4]a r en e (3). To a solution of calix[4]-
2 3
which (Boc) O (9.13 g, 41.8 mmol) and Et N (5.80 mL, 41.8
mmol) were added. The reaction mixture was stirred for 20 h
and subsequently evaporated till dryness. The residue was
arene 13 (640 mg, 0.644 mmol) in CH
2 2
Cl (60 mL) was added
SOCl (0.47 mL, 6.4 mmol), and the solution was stirred for 5
2
taken up in CH
2
Cl
2
(250 mL), washed with 0.1 M HCl (2 ×
h. The solvent was removed under reduced pressure to give
the bis(2-chloromethylpyridine) 14 as the bis(hydrochloride)
(694 mg, 100%), which was pure enough for further modifica-
tion. An analytical sample of 14, free from hydrochloric acid,
2
00 mL) and water (200 mL), and dried with MgSO
4
. The
mixture was filtered and the solvent was evaporated under
reduced pressure. The crude product was purified by column
chromatography (CH
2
Cl
oil (6.23 g, 74%). H NMR (CDCl
br s, 4 H), 6.44 (br s, 6 H), 4.49 and 3.11 (AB q, 8 H, J ) 13.3
Hz), 4.19-4.04 (m, 12 H), 3.81-3.88 (m, 8 H), 3.55 (q, 4 H, J
2
/EtOAc, 9/1) to give 11 as a colorless
was obtained by extraction from saturated NaHCO
with CH Cl , subsequently followed by drying over Na
filtering, and concentration in vacuo. H NMR (CDCl
3
solution
SO
, 250
1
3
, 250 MHz): δ (ppm) 6.68
2
2
2
4
,
1
(
3
MHz): δ (ppm) 7.68 (t, 2 H, J ) 7.7 Hz), 7.38 (d, 2 H, J ) 7.8
Hz), 7.33 (d, 2 H, J ) 7.7 Hz), 6.82 (s, 4 H), 6.42-6.31 (m, 6
H), 4.66 (s, 4 H), 4.47 and 3.13 (AB q, 8 H, J ) 13.3 Hz), 4.17
(t, 4 H, J ) 6.1 Hz), 4.04 (t, 4 H, J ) 5.5 Hz), 3.81-3.88 (m, 8
H), 3.57 (s, 4 H), 3.56 (q, 4 H, J ) 7.0 Hz), 3.52 (q, 4 H, J )
)
7.0 Hz), 3.53 (q, 4 H, J ) 7.1 Hz), 2.70 (br s, 6 H), 1.50 (s,
1
8 H), 1.22 (t, 6 H, J ) 7.0 Hz), 1.19 (t, 6 H, J ) 7.1 Hz). 13
C
NMR (CDCl
3
, 250 MHz): δ (ppm) 156.2, 155.6, 135.5, 134.3,
1
3
9
31.2, 127.9, 122.4, 79.4, 73.4, 72.9, 69.7, 69.6, 66.4, 66.3, 66.2,
3.5, 30.9, 28.5, 15.3. FAB-MS: m/z 999.6 ([M + H] , calcd
99.6), 897.8 ([M-t-BuOCO] , calcd 897.5). Anal. calcd for
+
7.0 Hz), 3.32 (s, 4 H), 2.16 (s, 6 H), 1.22 (t, 6 H, J ) 7.0 Hz),
+
13
1.17 (t, 6 H, J ) 7.0 Hz). C NMR (CDCl
3
, 250 MHz): δ (ppm)

Dinuclear and Trinuclear Zn(II) Calix[4]arene Complexes
J . Org. Chem., Vol. 64, No. 11, 1999 3905
1
1
6
59.8, 156.1, 155.7, 137.3, 135.3, 134.4, 134.3, 133.3, 131.9,
29.1, 128.8, 127.8, 122.2, 120.8, 73.4, 72.8, 69.6, 66.4, 66.2,
H), 4.08-4.02 (m, 4 H), 3.91-3.80 (m, 8 H), 3.59-3.50 (m, 8
H), 3.33 (s, 4 H), 3.23 (s, 2 H), 2.01 (s, 3 H), 1.25-1.14 (m, 12
2.7, 61.7, 46.9, 46.5, 42.5, 30.8, 15.3. FAB-MS: m/z 1042.3
H). 13C NMR (CDCl
3
, 250 MHz): δ (ppm) 158.6, 158.2, 156.6,
+
([M-Cl] , calcd 1041.5). To a solution of calix[4]arene bis-
155.9, 155.1, 137.0, 135.4, 134.5, 134.3, 131.6, 128.6, 128.3
(2×), 128.0, 122.2, 122.1, 121.6, 118.4, 73.4, 72.9, 69.7, 66.4,
66.3, 64.0, 62.2, 61.3, 42.5, 30.9, 15.3. FAB-MS: m/z 877.4 ([M
(
hydrochloride) 14 (694 mg, 0.644 mmol) in CH
NH‚HCl (1.04 g, 12.8 mmol) and K
2.8 mmol). The mixture was stirred for 16 h and concentrated
in vacuo. Saturated Na CO solution (40 mL) was added, the
aqueous layer was extracted with CH Cl
(3 × 40 mL), and
the combined organic phases were dried over K CO . Filtration
and evaporation of the solvent gave the crude product which
was purified by column chromatography (basic Al , CH Cl
MeOH, 99/1) to give 3 as a colorless oil (460 mg, 65%). H NMR
CDCl , 250 MHz): δ (ppm) 7.64 (t, 2 H, J ) 7.7 Hz), 7.36 (d,
H, J ) 7.7 Hz), 7.25 (d, 2 H, J ) 6.5 Hz), 6.88 (s, 4 H), 6.32
s, 6 H), 4.47 and 3.13 (AB q, 8 H, J ) 13.3 Hz), 4.19 (t, 4 H,
J ) 6.1 Hz), 4.01 (t, 4 H, J ) 5.3 Hz), 3.80-3.89 (m, 8 H), 3.62
s, 4 H), 3.58 (s, 4 H), 3.51 (q, 8 H, J ) 7.0 Hz), 3.38 (s, 4 H),
3
CN (60 mL)
was added Me
1
2
2
CO (1.77,
3
+
+ H] , calcd 877.5).
2
3
5-[(6-N,N-Dim eth yla m in om eth yl)p yr id in -2-yl-m eth yl)-
N -m e t h y l a m i n o m e t h y l ] -2 5 , 2 6 , 2 7 , 2 8 -t e t r a k i s ( 2 -
eth oxyeth oxy)ca lix[4]a r en e (2). This compound was syn-
thesized according to the synthesis of the bis-derivative 3. The
2
2
2
3
2
O
3
2
2
/
crude product was purified by column chromatography (basic
1
1
Al
2
O
3
, CH
2
Cl
2
) to give 2 as a colorless oil (83%). H NMR
(
2
(
3
(CDCl
3
, 250 MHz): δ (ppm) 7.63 (t, 1 H, J ) 7.7 Hz), 7.24 (d,
1 H, J ) 7.7 Hz), 7.14 (d, 1 H, J ) 7.6 Hz), 6.74-6.58 (m, 8
H), 6.43-6.37 (m, 2 H), 6.31-6.25 (m, 1 H), 4.51 and 3.15 (AB
q, 8 H, J ) 13.3 Hz), 4.49 and 3.12 (AB q, 8 H, J ) 13.2 Hz),
4.19-4.14 (m, 4 H), 4.09-4.03 (m, 4 H), 3.90-3.82 (m, 8 H),
(
2
6
1
1
4
.29 (s, 12 H), 2.18 (s, 6 H), 1.23 (t, 6 H, J ) 7.0 Hz), 1.17 (t,
3.58-3.50 (m, 10 H), 3.37 (s, 2 H), 3.21 (s, 2 H), 2.27 (s, 6 H),
1
3
13
H, J ) 7.0 Hz). C NMR (CDCl
3
, 250 MHz): δ (ppm) 159.3,
3
2.05 (s, 3 H), 1.25-1.15 (m, 12 H). C NMR (CDCl , 250
58.0, 156.4, 155.3, 136.8, 135.6, 134.0, 132.0, 129.3, 127.7,
22.2, 121.2, 73.5, 72.7, 69.6, 66.4, 66.2, 65.7, 63.0, 61.8, 45.6,
MHz): δ (ppm) 159.6, 158.0, 156.5, 155.9, 155.1, 136.6, 135.3
(2×), 134.6, 134.3, 132.3, 128.5, 128.3, 128.2, 128.0, 122.2,
122.1, 121.3, 121.0, 73.3, 72.9, 69.7, 66.4, 66.3, 65.9, 62.9, 61.6,
+
2.4, 30.8, 15.3. FAB-MS: m/z 1096.5 ([M + H] , calcd 1095.7).
+
5
-[(N -Boc-N -m e t h yl)a m in om e t h yl]-25,26,27,28-t e t -
45.7, 42.6, 31.5, 30.8, 15.3. FAB-MS, m/z 904.5 ([M + H] , calcd
r a k is(2-eth oxyeth oxy)ca lix[4]a r en e (15). This compound
904.5).
was synthesized according to the synthesis of the bis-derivative
11,23-Bis(m eth ylam in om eth yl)-26,28-di-n -pr opoxycalix-
[4]a r en e-25,27-cr ow n -3 (19). This compound was synthe-
sized according to the synthesis of the tris-derivative 8,
1
1
1, starting from formyl-tetrakis(2-ethoxyethoxy)calix[4]arene
1
6
6. The crude product was purified by column chromatog-
Cl /EtOAc, 95/5) to give 15 as a colorless oil (83%).
H NMR (CDCl , 250 MHz): δ (ppm) 6.71-6.61 (m, 6 H), 6.49
1
5
raphy (CH
2
2
starting from diformyl-di-n-propoxycalix[4]arene-crown-3 20.
The crude reaction product was pure enough for further
1
3
1
(
br s, 3 H), 6.36 (br s, 2 H), 4.50 and 3.14 (AB q, 4 H, J ) 13.3
3
modification (yield 94%). H NMR (CDCl , 400 MHz): δ (ppm)
Hz), 4.49 and 3.11 (AB q, 4 H, J ) 13.3 Hz), 4.20-4.15 (m, 4
H), 4.08-4.04 (m, 4 H), 3.98 (br s, 2 H), 3.90-3.80 (m, 8 H),
3
9
7.08 (s, 4 H), 6.17 (t, 2 H, J ) 7.6 Hz), 5.98 (d, 4 H, J ) 7.6
Hz), 4.35 and 3.16 (AB q, 8 H, J ) 13.5 Hz), 4.20 (t, 4 H, J )
4.0 Hz), 3.99 (t, 4 H, J ) 4.0 Hz), 3.76 (s, 4 H), 3.66 (t, 4 H, J
) 6.8 Hz), 2.51 (s, 6 H), 1.88-1.79 (m, 4 H), 1.57 (br s, 2 H),
.60-3.50 (m, 8 H), 2.50 and 2.31 (br s, 1 H, 2 H), 1.46 (br s,
13
H), 1.24-1.17 (m, 12 H). C NMR (CDCl , 250 MHz): δ
3
(ppm) 156.7, 155.9, 155.2, 135.4, 134.6, 134.5, 131.2, 128.3,
1.09 (t, 6 H, J ) 7.0 Hz). 13C NMR (CDCl
, 250 MHz): δ (ppm)
3
1
3
8
28.0, 127.4, 122.3, 122.1, 79.2, 73.4, 73.0, 69.7, 66.4, 66.3, 33.3,
0.9, 28.5, 15.4, 15.3. FAB-MS: m/z 856.4 ([M + H] , calcd
56.5).
157.1, 154.3, 136.4, 133.2, 132.6, 128.6, 126.6, 121.6, 76.3, 72.6,
+
+
69.7, 55.4, 35.6, 30.3, 23.0, 10.4. FAB-MS: m/z 664.3 ([M] ,
+
calcd 664.4), 634.5 ([M - NHCH
3
] , calcd 634.4).
5
-(Meth yla m in om eth yl)-25,26,27,28-tetr a k is(2-eth oxy-
11,23-Bis[((6-h yd r oxym et h yl)p yr id in -2-yl-m et h yl)-N-
m eth ylam in om eth yl]-26,28-dipr opoxycalix[4]ar en e-25,27-
cr ow n -3 (21). This compound was synthesized and purified
eth oxy)-ca lix[4]a r en e (17). This compound was synthesized
according to the synthesis of the bis-derivative 12. After
stirring in trifluoroacetic acid, the reaction mixture was
according to the synthesis and purification of the tris-deriva-
1
concentrated in vacuo to give crude 17‚3CF
which was pure enough for further modification. 1H NMR
CDCl , 250 MHz): δ (ppm) 11.68 (br s, 2 H), 8.08 (br s, 2 H),
3
COOH (99%),
tive 9 (yield 70%). H NMR (CDCl
3
, 250 MHz): δ (ppm) 7.64
(t, 2 H, J ) 7.7 Hz), 7.46 (d, 2 H, J ) 7.0 Hz), 7.18-7.12 (m,
6 H), 6.37-5.93 (m, 6 H), 4.75 (s, 4 H), 4.36 and 3.17 (AB q, 8
H, J ) 13.5 Hz), 4.19 (br s, 4 H), 4.01 (br s, 4 H), 3.75 (s, 4 H),
3.62 (s, 4 H), 2.33 (s, 6 H), 1.83 (m, 4 H), 1.09 (t, 6 H, J ) 7.4
(
3
6
4
4
3
.88-6.71 (m, 5 H), 6.47-6.39 (m, 4 H), 4.50 and 3.15 (AB q,
H, J ) 13.5 Hz), 4.48 and 3.13 (AB q, 4 H, J ) 13.3 Hz),
.25-4.19 (m, 4 H), 4.07-4.00 (m, 4 H), 3.91-3.80 (m, 8 H),
Hz). 13C NMR (CDCl
, 250 MHz): δ (ppm) 158.2, 157.7, 154.8,
3
.62-3.51 (m, 10 H), 2.18 (s, 3 H), 1.25-1.03 (m, 12 H). 13
C
137.2, 137.0, 136.8, 133.1, 130.1, 127.1, 122.1, 121.5, 118.6,
76.9, 73.1, 70.2, 64.0, 62.8, 61.9, 53.5, 42.6, 30.8, 23.5, 11.0.
NMR (CDCl , 250 MHz): δ (ppm) 156.9, 156.6, 155.8, 135.8,
3
+
1
1
3
35.6, 135.1, 134.6, 134.1, 129.6, 128.7, 128.5, 127.9, 123.5,
22.6, 121.9, 73.6, 73.5, 72.9, 69.6, 66.4, 66.3, 51.9, 31.4, 31.1,
FAB-MS: m/z 907.5 ([M + H] , calcd 907.5).
11,23-Bis[(6-N,N-d im et h yla m in om et h yl)p yr id in -2-yl-
m e t h y l)-N -m e t h y la m i n o m e t h y l]-26,28-d i p r o p o x y -
ca lix[4]a r en e-25,27-cr ow n -3 (4). This compound was syn-
thesized according to the synthesis of the tetrakis(2-ethoxy-
ethoxy) derivative 5. The crude product was purified by column
+
0.8, 30.7, 15.3. FAB-MS: m/z 756.2 ([M + H] , calcd 756.4).
The free amine 17 was obtained by extraction with CH
2
Cl
2
from saturated Na
2
CO
3
solution, followed by drying over K
2
-
1
CO
3
. H NMR (CDCl
3
, 250 MHz): δ (ppm) 6.47-6.67 (m, 11
H), 4.50 and 3.13 (AB q, 4 H, J ) 13.3 Hz), 4.48 and 3.11 (AB
q, 4 H, J ) 13.3 Hz), 4.06-4.15 (m, 8 H), 3.83-3.87 (m, 8 H),
chromatography (basic Al
2
O
3
, CH
2 2
Cl ) to give 4 as a colorless
1
oil (62%). H NMR (CDCl
3
, 400 MHz): δ (ppm) 7.67 (t, 2 H, J
3
1
1
1
3
7
.50-3.58 (m, 8 H), 3.38 (s, 2 H), 2.26 (s, 3 H), 1.40 (br s, 1 H),
) 7.7 Hz), 7.49-7.45 (m, 2 H), 7.29 (d, 2 H, J ) 7.2 Hz), 7.17
(s, 4 H), 6.13-5.94 (m, 6 H), 4.37 and 3.18 (AB q, 8 H, J )
13.7 Hz), 4.19 (br s, 4 H), 4.02 (br s, 4 H), 3.77 (s, 4 H), 3.68 (t,
1
3
.12-1.26 (m, 12 H). C NMR (CDCl , 250 MHz): δ (ppm)
3
56.4, 156.3, 155.3, 135.2, 135.1, 135.0, 134.8, 133.3, 128.3,
28.2, 128.1, 128.0, 122.2, 121.9, 73.2, 73.1, 69.7, 66.4, 55.6,
5.8, 31.5, 30.9, 15.3. FAB-MS: m/z 756.9 ([M + H] , calcd
56.4).
4 H, J ) 6.6 Hz), 3.61 (s, 8 H), 2.31 (s, 18 H), 1.96-1.82 (m, 4
+
13
H), 1.11 (t, 6 H, J ) 7.2 Hz). C NMR (CDCl , 250 MHz): δ
3
(ppm) 158.9, 157.7, 157.1, 155.8, 154.3, 136.3, 136.2, 132.6,
129.5, 126.5, 121.5, 120.7, 120.6, 76.3, 72.6, 69.7, 65.4, 62.7,
61.4, 45.1, 42.1, 30.3, 23.0, 10.4. FAB-MS: m/z 961.7 ([M +
5
-[(6-H yd r oxym et h yl)p yr id in -2-yl-m et h yl)-N-m et h yl-
a m in om e t h yl]-25,26,27,28-t e t r a k is(2-e t h oxye t h oxy)-
ca lix[4]a r en e (18). This compound was synthesized according
to the synthesis of the bis-derivative 13. The crude product
+
H] , calcd 961.6).
Sp ectr op h otom etr ic Titr a tion s. To a cuvette containing
was purified by column chromatography (CH
to give 18 as a colorless oil (65%). H NMR (CDCl
δ (ppm) 7.64 (t, 1 H, J ) 7.7 Hz), 7.11 (d, 2 H, J ) 7.7 Hz),
2
Cl
2
/MeOH, 9/1)
, 250 MHz):
3
2 mL of 50% CH CN/20 mM aqueous HEPES solution pH 7.0
1
21
3
(v/v, see kinetics), was added 8 µL of a 50 mM stock solution
of ligand 1 or 2 in EtOH (2 µL 50 mM for 3, 4, and 5). The
increase in absorbance at λ ) 266 nm at 25 °C by the ligand
6
6
.77-6.60 (m, 6 H), 6.55 (s, 2 H), 6.43-6.35 (m, 2 H), 6.30-
.24 (m, 1 H), 4.72 (s, 2 H), 4.50 and 3.15 (AB q, 8 H, J ) 13.3
3
-1
-1
3
-1
-1
1 or 2 (ꢀ266: 1, 4.0 × 10 M cm ; 2, 4.9 × 10 M cm ; 3,
3
-1
-1
3
-1
-1
3
-1
Hz), 4.48 and 3.11 (AB q, 8 H, J ) 13.2 Hz), 4.21-4.16 (m, 4
5.9 × 10 M cm ; 4, 6.0 × 10 M cm ; 5, 12 × 10 M

3
906 J . Org. Chem., Vol. 64, No. 11, 1999
Molenveld et al.
cm-1) was followed upon the stepwise addition of 2 µL of 50
Hydrogen atom coordinates were included as parameters in
the refinement. A final difference Fourier showed no residual
mM Zn(ClO ‚6H O in water up to 3 equiv of Zn(ClO ‚6 H
6 equiv for 3, 4, and 5). The concentration of the ligands before
titration was 0.2 mM for 1 and 2, and 0.05 mM for 3, 4, and
, respectively. The absorbance was corrected for both dilution
upon titration and for the weak absorbance of free Zn(ClO
4
)
2
2
4
)
2
2
O
-
3
(
density outside -0.61 and 0.31 e Å
Kin etics. Solutions for spectrophotometric titrations and
kinetic measurements were made by adding CH CN (spectro-
.
5
3
4
)
2
photometric grade) up to 50% (v/v) to a 20 mM aqueous buffer
solution adjusted with NaOH to the desired pH. Buffers (MES,
pH 6.0-7.0; HEPES, pH 7.0-8.2; EPPS, pH 8.2-8.6) were
obtained from commercial sources and used without further
purification in deionized distilled water. HPNP was prepared
according to the literature.33 Stock solutions were freshly
prepared before performing the kinetic measurements. In a
typical experiment, the ligand 5 (20 µL, 50 mM in EtOH) and
-1
-1
(ꢀ266 ) 70 M cm ). Stability constants for Zn(II) complexation
22
were estimated by nonlinear least-squares fitting of the
titration curves to a 1:1 model using the absorption end values
at 266 nm.
P oten tiom etr ic p H Titr a tion s. Solutions (50 mL) of
ligand 1 (0.4 mM) in 0.1 M KNO
acidified with HNO (1.2 mM), were titrated under N
C in the presence and absence of 1 equiv of Zn(NO
NO
3 2
in 35% EtOH/H O (v/v),
3
2
at 20.0
or Cu-
°
(
3
)
2
Zn(ClO
4
)
2
‚6H
2
O (60 µL, 50 mM in water) were added to a
CN/20 mM buffer solution
3
)
2
, respectively. The titrations were carried out at fixed
cuvette containing 2 mL of 50% CH
3
titrant increments of 50 µL NaOH solution (0.020 M). The
(v/v) and thermostated at 25 °C. After a couple of minutes
equilibration time, HPNP (4 µL, 100 mM in water) was injected
and the increase in UV absorption at λ ) 400 nm due to the
release of p-nitrophenolate was recorded every 30 s. Final
concentrations were 0.98 mM for complexes 1-[Zn] and 2-[Zn],
titration apparatus was calibrated with appropriate buffers
immediately before use and checked by the pK
of acetic acid. Three independent titrations were always made
for pK determinations. For calculation of deprotonation
a
determination
a
constants and Zn(II)/Cu(II) association constants from the
titration data a multiparameter curve fitting program based
on SUPERQUAD was used. The resulting equilibrium con-
2 2 3
and 0.48 mM for complexes 3-[Zn] , 4-[Zn] , and 5-[Zn] , 0.19
mM in HPNP, and 10 mM in buffer. All solutions remained
clear during the time of the kinetic measurements. In the
absence of ligand precipitation of polymeric Zn(II) hydroxide
took place. The observed pseudo-first-order rate constants kobs
2
3
stants could be interpreted as conditional constants at I ) 0.1
M. The value for K
()[H ][OH ]) at 20 °C was 10-14.38.
+
-
w
-
1
X-r a y Cr ysta llogr a p h y. To a solution of 1 (100 mg, 0.517
mmol) and Zn(CH COO) ‚2 H O (113 mg, 0.517 mmol) in
acetonitrile (2 mL) was added Et O (5 mL). The obtained white
crystals were dissolved in acetonitrile (2 mL) and recrystalized
by slow vapor diffusion of Et O to the solution, yielding crystals
of (1)Zn(CH COO) suitable for X-ray crystal structure analy-
sis. Crystal data for (1)Zn(CH COO) ‚H Zn‚
O, M
) 394.79, colorless, block-shaped crystal (0.2 × 0.2 ×
.3 mm), monoclinic, space group P2 /c with a ) 9.0875(15), b
16.725(3), c ) 14.255(2) Å, â ) 120.11(8)°, V ) 1874.2(16)
(s ) were calculated with the extinction coefficient of p-
nitrophenolate at λ ) 400 nm by an initial slope method (<4%
conversion). All rate constants were obtained by averaging
three kinetic measurements. The pseudo-first-order rate con-
3
2
2
2
-
1
2
stants for uncatalyzed reactions (kuncat, s ) were measured
from a 2.0 mM HPNP solution by following the increase in
absorbance at 400 nm due to the release of p-nitrophenolate
for 1 week.
3
2
3
2
2 25 3 4
O: C15H N O
H
2
r
0
1
)
Ack n ow led gm en t. This work was supported in part
(A.L.S.) by the council for Chemical Sciences of The
Netherlands Organization for Scientific Research (CW-
NWO).
3
-3
-1
Å , Z ) 4, D
reflections measured, 4293 independent, Rint ) 0.0461, (1.5°
θ < 28.5°, T ) 150 K, MoKR radiation, graphite monochro-
x
) 1.399 g cm , µ(MoKR) ) 13.4 cm , 27 324
<
mator, λ ) 0.710 73 Å), on a Nonius κ-CCD diffractometer on
rotating anode. Data were corrected for Lp effects but not for
absorption. The structure was solved by automated direct
Su p p or tin g In for m a tion Ava ila ble: Further details of
the X-ray structure determination, including atomic coordi-
nates, bond lengths and angles and thermal parameters, and
2
methods (SHELXS86). Refinement F was carried out by full-
matrix least-squares techniques (SHELXL-97) for 230 param-
eters; no observance criterion was applied during refinement.
1
H NMR spectra of all new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
Refinement converged at a final wR
2 1
value of 0.0831, R )
0
.0344 [for 3610 reflections with I > 2σ(I)], S ) 1.025.
J O982201F