Design and Synthesis of Supramolecular Phosphatases Formed from a Bis(Zn2+-Cyclen) Complex, Barbital-Crown-K+ Conjugate and Cu2+ for the Catalytic Hydrolysis of Phosphate Monoester

Article abstract of DOI:10.1002/ejic.202001009

The development of artificial mimics of natural enzymes such as hydrolases and phosphatases is one of the great challenges in bioorganic and bioinorganic chemistry and related sciences. Supramolecular strategies are one of the useful methods to construct

Full text of DOI:10.1002/ejic.202001009

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Design and Synthesis of Supramolecular Phosphatases
Formed from a Bis(Zn2+-Cyclen) Complex, Barbital-Crown-
K+ Conjugate and Cu2+ for the Catalytic Hydrolysis of
Phosphate Monoester
Akib Bin Rahman,[a] Hirokazu Okamoto,[a] Yuya Miyazawa,[a] and Shin Aoki*[a, b, c]
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The development of artificial mimics of natural enzymes such as
hydrolases and phosphatases is one of the great challenges in
bioorganic and bioinorganic chemistry and related sciences.
Supramolecular strategies are one of the useful methods to
construct artificial catalysts as mimics of natural enzymes and to
understand their reaction mechanisms. Herein, we report on
the formation of amphiphilic supramolecular phosphatases by
the 2:2:2 self-assembly of a bis(Zn2+-cyclen) complex (cyclen=
1,4,7,10-teraazacyclododecane) containing a 2,2’-bipyridyl (bpy)
linker and one long alkyl chain (Zn2L3), 5,5-diethylbarbituric acid
(Bar) derivative functionalized with 1-aza-18-crown-6 ether and
Cu2+ in a two-phase solvent system (CHCl3/H2O). We hypothe-
sized that crown ether moiety of the Bar-crown ether conjugate
would form complexes with alkaline ions and other metal ions
such as Li+, Na+, K+, Rb+, Mg2+ and La3+ in organic phase to
mimic the Mg2+ found as the third metal ion in the active site
of alkaline phosphatase (AP). The results indicate that the
2:2:2:4 complexes of Zn2L3, a Bar block equipped with the 18-
crown-6 ether, Cu2+ and alkaline metal are constructed in a
two-phase solvent system. The resulting complexes have a
higher hydrolysis activity for mono(4-nitrophenyl)phosphate
(MNP) in the presence of K+ than that in the presence of Li+,
Na+, Rb+, Mg2+ and La3+ and a greater hydrolysis activity than
our previous supermolecules having no crown ether part,
suggesting that crown ether-K+ complex located in close
proximity to the Cu2(μ-OH)2 core contributes to the acceleration
of the MNP hydrolysis.
Introduction
AP.[3] These ions function not only to confer catalytic activity for
the hydrolysis of monoesters of phosphoric acid but also for
transphosphorylation reactions that proceed in the presence of
high concentrations of phosphorylation acceptors.[4]
A number of artificial phosphatases that mimic the active
center of metallophosphatases have been reported to date.[5]
However, only a very few of them function as catalysts for the
hydrolysis of a phosphate monoester such as mono(4-nitro-
The phosphorylation and dephosphorylation of proteins and
related biomolecules are essential regulatory reactions associ-
ated with signal transduction and numerous cellular functions.
The structure, subcellular localization and stability of such
molecules can have a significant impact on the biological
activity of such proteins. Phosphorylation is accomplished via a
kinase action, as exemplified by tyrosine kinases which are
related to signal transduction in living cells.[1] Indeed, several
molecular targeted drugs that target tyrosine kinases in cancer
cells and inhibit their action have been developed for the
treatment of cancer.[2] At the same time, dephosphorylation via
phosphatases is a vital switching-on and -off mechanism in cell
signaling. Dephosphorylation is promoted by protein phospha-
tases such as alkaline phosphatase (AP), which contains two
zinc ions (Zn2+) and one magnesium (Mg2+) ion in its active
center, as revealed by the X-ray crystal structure analysis of
phenyl) phosphate (MNP),
a typical model substrate of
phosphate hydrolysis. Hence, dephosphorylation by artificial
catalysts that mimic protein phosphatases remains a great
challenge because phosphate monoesters are generally less
reactive than phosphate diesters and triesters.[6] The synthesis
of artificial enzyme models that are constructed by covalent
bonds typically requires long and tedious synthetic routes
especially for their functionalization, which is one of the
drawbacks of such artificial systems.[7]
A supramolecular strategy that utilizes the self-assembly of
artificial molecular building blocks equipped with appropriate
functionalities could be a powerful approach to overcome the
aforementioned drawbacks.[8–10] In this context, we previously
reported on the formation of the supramolecular complex 8a,
by the 2:2:2 assembly of a bis(Zn2+-cyclen) complex (cyclen=
1,4,7,10-tetraazacyclododecane) containing the 2,2'-bipyridyl
(bpy) linker 1 (Zn2L1), a dianion of barbital 4a (Bar), and a
copper(II) ion in an aqueous solution (Scheme 1a).[11] The
complex is stabilized mainly by coordination bonds between
imide anions of the Bar units and Zn2+ ions and hydrogen
bonds between the NH protons of Bar units and the NH groups
[a] A. B. Rahman, H. Okamoto, Y. Miyazawa, Prof. Dr. S. Aoki
Faculty of Pharmaceutical Science, Tokyo University of Science,
2641 Yamazaki, Noda, Chiba 278-8510, Japan
E-mail: shinaoki@rs.tus.ac.jp
[b] Prof. Dr. S. Aoki
Research Institute for Science and Technology,
Tokyo University of Science,
2641 Yamazaki, Noda, Chiba 278-8510, Japan
[c] Prof. Dr. S. Aoki
Research Institute for Biomedical Sciences,
Tokyo University of Science,
2641 Yamazaki, Noda, Chiba 278-8510, Japan
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Scheme 1. (a) Formation of the 2:2:2 supramolecular complexes 8–10 by the self-assembly of Zn2L complexes (1–3), 5,5-diethylbarbital (Bar) derivatives (4a–
c) and Cu2+ in an aqueous solution at neutral pH or in a two-phase solvent system (CHCl3/H2O) that was used in our previous study. (b) Proposed scheme for
the formation of the 2:2:2 supramolecular complexes 14a–f by the self-assembly of 3 (Zn2L3), a Bar derivative having 1-aza-18-crown-6 ether (12) and Cu2+ in
the presence of other metals (e.g. Li+, Na+, K+, Rb+, Mg2+ and La3+) in a two-phase solvent system (CHCl3/H2O).
of cyclen, as disclosed by an X-ray crystal structure analysis. It
was discovered that 8a contains a Cu2(μ-OH)2 core, which
resembles the active centers of metallophosphatases such as
AP, and that one of the ethyl groups of the Bar unit is located
in close proximity to the Cu2(μ-OH)2 core. More importantly, 8a
was found to accelerate the hydrolysis of MNP, although the
yield was low, possibly due to product inhibition by inorganic
phosphate (HPO42À ), a byproduct of the reaction.
Scheme 2a is a schematic presentation of the hydrophobic
active site of AP, in which the substrate (MNP) is activated by
the two Zn2+ ions and then undergoes hydrolysis. To mimic this
situation, we constructed the hydrophobic supramolecular
complexes 9a–c and the amphiphilic supramolecular com-
plexes 10a–c (Scheme 1a) by the 2:2:2 assembly of a bis(Zn2+
-cyclen) complex containing two long alkyl chains 2 (Zn2L2)[12]
and a complex that contained one long alkyl chain 3 (Zn2L3),[13]
respectively, with the functionalized Bar derivatives and Cu2+ in
a two-phase solvent system (CHCl3/H2O). It was expected that
the hydrophobic or amphiphilic 2:2:2 complexes 9 or 10 would
be formed mainly in the organic layer, and that product
2À
inhibition by HPO4 could be reduced, since the hydrophilic
2À
HPO4 would be released into the aqueous layer with the
Cu2(μ-OH)2 core being regenerated. Indeed, 9 and 10 were both
found to accelerate the hydrolysis of MNP and their catalytic
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decided to add the third Lewis acid to the Cu2(μ-OH)2 active
sites of our previous supramolecular phosphatases, as shown in
Scheme 2b.
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It is well known that crown ethers and their derivatives are
capable of forming complexes with hard metal cations such as
alkaline metals, alkaline earth metals and lanthanides.[14–16]
Therefore, the side chains of the Bar units were functionalized
with 1-aza-18-crown-6 ether in this work (12 in Scheme 1b),
because one of the two ethyl groups of each Bar unit in 8 is
located in close proximity to the Cu2(μ-OH)2 catalytic site,[11] as
described above. Since it is also known that the complexes of
crown ethers with alkali metal ions are more stable in organic
solvents than in an aqueous phase, we hypothesized that
alkaline and alkaline earth metal ions such as Li+, Na+, K+, Rb+
and Mg2+[16f–h] and lanthanide ions[16i–k] contained in the
aqueous buffer solution would be extracted into the organic
layer by the complexation with the crown ether moiety of the
Bar unit in the supramolecular complexes (Scheme 3). We
expected that these alkaline (and alkaline earth) metals would
assist in the activation of MNP, resulting in a more efficient
hydrolysis, as predicted in Scheme 2b. In this paper, we report
on the construction of 2:2:2 supramolecular complexes (14a–f
in Scheme 1b and Scheme 3) from 3 (Zn2L3) with Bar derivative
equipped with 1-aza-18-crown-6 ether (12) and Cu2+ in the
presence of additional metals (e.g. Li+, Na+, K+, Rb+, Mg2+ and
La3+). To the best of our knowledge, model systems containing
such a complicated and sophisticated architecture have not
been previously reported.
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Results and Discussion
Proposed Structure of 14c
Prior to the synthesis of 12, the three-dimensional structure of
14c (formed from 3, 12, Cu2+ and K+) was speculated using
“Biovia Discovery 2020 client” software based on the crystal
structure of 8a[11] (Figure 1a, in which Cu2+-bound H2O
molecules are omitted for clarity). As shown in Figure 1b, in
which two C22 alkyl groups are assumed to be oriented in the
same direction (so called cis structure), it was expected that two
of the four azacrown ether-K+ complex units of 14c would be
located close to its Cu2(μ-OH)2 site by choosing a C3 linker
between the Bar and the azacrown units.
Scheme 2. (a) Schematic presentation of the active center of AP containing
two Zn2+ ions and one Mg2+ ion. (b) Hypothesis for mimicking the active
center of AP, where two Cu2+ ions are present in the active site and other
metals as Lewis acidic sites would be introduced into the close proximity to
the Cu2(μ-OH)2 site, in the organic layer of the two-phase solvent system.
Synthesis of the Barbital-18-Crown-6 Conjugate 12
turnover numbers (CTNs) were 2~4 (CTN=2~2.7 for 9b–c and
3~4 for 10a).[12,13] One of most important findings was that the
hydrolysis of MNP by 9 and 10 in the two-phase solvent system
obeyed Michaelis-Menten kinetics, suggesting that these reac-
tion systems consisting of a supramolecular complex and two-
phase solvent system mimic the active sites of AP reasonably
well.
The barbital derivative functionalized with 1-aza-18-crown-6
ether 12 was synthesized as shown in Scheme 4. Compound 18
was synthesized from diethyl malonate 11 and 15 via 16 and
17, according to our previous papers[12,13] and was reacted with
1-aza-18-crown-6 ether to afford 12.
It is also known that the active site of AP contains Mg2+, as
the third Lewis acidic ion, which generates Mg2+-bound OHÀ
ion that activate Ser (ser102) residue.[3,4i] In this work, we
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Scheme 3. Proposed scheme for the hydrolysis of MNP by amphiphilic supramolecular complexes (14a–f) having a crown ether-alkaline (alkaline earth) metal
complex formed from 3 (Zn2L3), 12, Cu2+ and additional metal ions for the hydrolysis of MNP in a two-phase solvent system.
Figure 1. (a) X-ray crystal structure of 8a[11] and (b) proposed structure of 14c (assuming a cis form, in which two alkyl groups are orientated to the same side).
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Scheme 4. Synthesis of the barbital-18-crown-6 conjugate 12 as a building
unit for supramolecular complexes 14a–f.
Figure 2. (a) UV/Vis titrations of 3 (40 μM) with 12 in the presence of an
excess amount of Na+ (0.05 M) in DMSO/50 mM HEPES (pH 7.4 with I=0.1
°
(NaNO3)) (4/96) at 37 C. (b) UV/Vis titration of 3 (40 μM) upon the addition
of 12 in the presence of an excess amount of K+ (0.05 M) in DMSO/50 mM
Complexation Behavior of 3 (Zn2L3) with 12and Cu2+ as
Studied by UV/Vis Titration in the Presence of an Excess
Amount of Na+ or K+
°
HEPES (pH 7.4 with I=0.1 (KNO3)) (4/96) at 37 C.
The formation of the 2:2 supramolecular complex 13 from the
bis(Zn2+-cyclen) complex 3 (Zn2L3) and 12 was predicted in
Scheme 1b and checked by the UV/Vis titration of 3 with 12 in
the presence of an excess amount of Na+ or K+. Because the
titration of 3 in the two-phase solvent system (CHCl3/50 mM
supermolecules 14b and 14c at μM order concentrations in the
presence of Na+ or K+ in DMSO/50 mM HEPES buffer (pH 7.4
[17]
°
with I=0.1 (NaNO3 or KNO3)) (4/96) at 37 C.
°
HEPES (pH 7.4, I=0.1 (NaNO3 or KNO3)) with 12 at 37 C were
Hydrolysis of MNP by 2:2:2 Complexes in a Two-Phase
Solvent System
unsuccessful due to the low solubility of 3 in aqueous solution,
the UV/Vis titrations were successfully carried out in DMSO/
50 mM HEPES (pH 7.4 with I=0.1 (NaNO3 or KNO3)) (4/96) at
The hydrolysis of MNP (100 μM) by 14 (20 μM in the total
solution including the aqueous phase and the CHCl3 phase),
which was formed by the self-assembly of 3, 12, and Cu2+, was
conducted in a two-phase solvent system (CHCl3/50 mM HEPES
+
37 C. In these experiments, Na and K+ were added to the
°
buffer at high concentration (0.05 M) to check their effect on
the complexation of 3 with 12. As shown in Figure 2a and
Figure 2b, 3 (40 μM) has absorption maxima (λmax) at 287 nm,
which increased upon the addition of 12 and reached a plateau
at a 1:1 ratio. It should be noted that the complexation of 3
(and 12) with K+ and Na+ is supposed to be very weak in a
polar solvent system such as DMSO/H2O (4/96). These results
suggest that the 2:2 complexation of 3 and 12 is not negligibly
affected by K+ and Na+ under these conditions.
°
buffer (pH 7.4) with I=0.1 (NaNO3 or KNO3)) (2/8) at 37 C. In
such system, it is very likely that Na+ and K+ are extracted from
the aqueous phase to the organic layer by complexation with
the azacrown ether moiety of 12 to form 14 (Scheme 1a and
Scheme 3). As shown in Figure 4a, the hydrolysis of MNP
(100 μM) in the presence of 10a and 14c (20 μM) proceeded to
a similar extent and the supermolecule 14a–b (with Li+ and
Na+) showed a somewhat lower activity than those of 10a and
14c (with K+). Next, the hydrolysis of MNP by 10a, 14a, 14b
and 14c (20 μM) was carried out at [MNP]=1000 μM. As shown
in Figure 4b, 14c, whose crown ether parts are supposed to be
complexed with K+ in the organic phase, exhibited a higher
The addition of Cu2+ to 13b and 13c (formed from 3 and
12 in a 1:1 ratio in the presence of Na+ and K+, respectively)
induced a red shift from 287 nm to 307 nm, which reached a
plateau at [13b or c]:[Cu2+]=1:2, as shown in Figure 3a and
Figure 3b, suggesting the quantitative formation of the 2:2:2
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14d are similar to that of our previously reported complex 10a
(CTN~4).
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Michaelis-Menten Kinetics for the Hydrolysis of MNP by 14a-f
in the Two-Phase Solvent System
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The results for the hydrolysis of MNP by the supramolecular
phosphatases at [MNP]=100, 200, 500, 800, and 1000 μM (in
the total solution) were analyzed based on Michaelis-Menten
kinetics, as described in our previous reports.[10r,11–13] The
reciprocal values for the concentrations of MNP and the rate of
hydrolysis (Vo) in the total solution of the two-phase solvent
system were plotted to calculate Vmax (the maximum velocity for
the formation of NP from MNP catalyzed by 14, μM/min) and Km
(Michaelis constant, μM). Based on the Lineweaver-Burk plots
shown in Figure 7, the Km, Vmax and kcat (rate constants for the
catalytic conversion of the substrate into the product, as
defined by eq. (1))[10r] values for the supramolecular phospha-
tases were determined as summarized in Table 1. These values
clearly imply that 14c has the highest Vmax ((7.9�0.4)×10À 2 μM/
min), kcat ((4.0�0.2)×10À 3 minÀ 1) and CTN (4.7) among 14a–f
(see also Figure 4b), possibly due to the positive effect of the
crown-K+ complex unit on MNP hydrolysis.
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Figure 3. (a) UV/Vis titrations of 13b (from 3+12+Na+) (20 μM) upon the
addition of Cu2+ in DMSO/50 mM HEPES buffer (pH 7.4 with I=0.1 (NaNO3))
+
°
(4/96) at 37 C. (b) UV/Vis titrations of 13c (from 3+12+K ) (20 μM) upon
the addition of Cu2+ in DMSO/50 mM HEPES buffer (pH 7.4 with I=0.1
°
(KNO3)) (4/96) at 37 C.
kcat ¼ Vmax=½Catalyst�
(1)
The hydrolysis of MNP by 14b and 14c in the presence of
2À
activity than 10a and 14a–b. It was also found that the
reactivity of 10a in the presence of 10 eq. (against 10a) of 1-
aza-18-crown-6 ether was almost same as that of 10a alone in
the two-phase solvent system containing an excess amount of
KNO3 (0.05 M) (Figure 4b). Therefore, we concluded that higher
MNP hydrolysis reactivity of 14c than other supramolecular
complexes is due to the intramolecular effect of the crown-K+
units of 14c. As shown in Figure 4c, the hydrolysis of MNP by
14c (20 μM) at [MNP]=100, 200, 500, 800 and 1000 μM
(namely, at [14c]/[MNP]=20%, 10%, 4%, 2.5% and 2%) gives
more than 20 μM of the product (indicated with a dashed
straight line in Figure 4c), suggesting that 14c functions as a
catalyst for the hydrolysis of MNP.
The hydrolysis of MNP (100 μM) by supermolecules 14d–f
(20 μM) in the presence of Rb+, Mg2+ and La3+ (14d, 14e and
14f, respectively) was also carried out (RbNO3, Mg(OH)2 and
La(NO3)3 were used as the metal sources) at [MNP]=100, 200,
500, 800 and 1000 μM and typical results at [MNP]=1000 μM
are summarized in Figure 5. Once again, the higher activity of
K+-14 complex (14c) can be explained by higher stability and
selectivity of the 18-crown-6 ether-K+ complex unit than those
with other metals in the organic layer.[15]
HPO4 was examined in order to determine the Ki values
(inhibitory constants) of HPO42À , as listed in Table 1, because it
is well described that natural AP (competitive inhibition)[18] and
our artificial AP models (competitive or mixed type
inhibition)[11–13] are inhibited by HPO42À . The Ki values for 14b
and 14c were determined to be ca. 40 μM and ca. 97 μM
(entries 6 and 7), respectively, possibly in a mixed-type manner.
It should be mentioned that the Ki value of 14c (ca. 97 μM) is
greater than those of 8a, 9a, and 10a (ca. 15 μM, ca. 36 μM
and ca. 80 μM, respectively), indicating that the K+ ion would
assist the release of inorganic phosphate from the Cu2(μ-OH)2
core, although its mechanism is yet to be studied.
We previously reported that the Km/Ki values are good
parameters to evaluate the catalytic activity of the
supramolecular phosphatases (smaller Km/Ki value is more
favorable).[13] For example, the Km/Ki values (4.8~5.2) for 10a
and 14b–c (entries 4, 6 and 7 in Table 1) are close to that for AP
(ca. 2.3) (entry 1 in Table 1), and smaller than those for 8a (ca.
27) and 9a (ca. 36), whose CTN are 0.4~1 (entries 1 and 3). The
Vmax and CTNs of 14e (with Mg2+) and 14f (with La3+) for the
hydrolysis of MNP were found to be smaller than those of 14c
(entries 9 and 10 in Table 1). These data allowed us to conclude
that 14c having crown-K+ complex has highest phosphatase
activity among supramolecular complexes tested in this study.
Although there are several reports on the complexation of
crown ether derivatives with alkaline earth metals (e.g., Mg2+
and Ca2+) and lanthanides (e.g., Y3+, La3+, Eu3+, etc.),[16] the CTN
values of 14e and 14f, which possibly contain Mg2+ and La3+
,
were nearly same as or lower than that of 14c (K+ complex). As
displayed in Figure 6, the CTN value for 14c is 4.7 at [MNP]=
1000 μM, while the CTNs of 14a is lower and those of 14b and
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Conclusion
alkaline metals and other metals in the reaction medium by
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means of the self-assembly with 3 (Zn2L3) and Cu2+ in a two-
phase solvent system to mimic the structure of the active site of
AP. The hydrolysis of MNP by the supramolecular complexes
14a–f follows Michaelis-Menten kinetics in the two-phase
We report on the design, and synthesis of Bar building block
functionalized with 1-aza-18-crown ether moiety (12) for the
construction of supramolecular phosphatases 14 containing
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Figure 4. (a) Hydrolysis (%) of MNP (100 μM in total solution) by 14a (20 μM) in CHCl3/50 mM HEPES (pH 7.4 with I=0.1 (LiNO3)), 14b (20 μM) in CHCl3/50 mM
HEPES (pH 7.4 with I=0.1 (NaNO3)), 14c (20 μM) in CHCl3/50 mM HEPES (pH 7.4 with I=0.1 (KNO3)) and 10a (20 μM). (b) Hydrolysis (%) of MNP (1000 μM in
total solution) by 10a (20 μM), 10a (20 μM)+1-aza-18-crown-6 ether (200 μM)) in 50 mM HEPES buffer (pH 7.4 with I=0.1 (KNO3)), 14a (20 μM) in CHCl3/
50 mM HEPES (pH 7.4 with I=0.1 (LiNO3)), 14b (20 μM) in CHCl3/50 mM HEPES (pH 7.4 with I=0.1 (NaNO3)), and 14c (20 μM) CHCl3/50 mM HEPES (pH 7.4 with
I=0.1 (KNO3)). (c) Change in the concentration of 4-NP, which is produced by the hydrolysis of MNP promoted by 14c (20 μM) at [MNP]=100 μM, 200 μM,
500 μM, 800 μM and 1000 μM (solid line with open squares) and change in the concentration of 4-NP at [MNP]=1000 μM produced by 10a (20 μM) (dashed
line with filled triangles) in CHCl3/50 mM HEPES (pH 7.4 with I=0.1 (KNO3)).
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Figure 5. Hydrolysis (%) of MNP (1000 μM in the total solution) by 14c (20 μM) in the presence of 0.05 M K+ in 50 mM HEPES (pH 7.4 with I=0.1 (KNO3), 14d
(20 μM) in the presence of 0.05 M Rb+ in 50 mM HEPES (pH 7.4 with I=0.1 (RbNO3), 14e (20 μM) and 14f (20 μM) in 50 mM HEPES (pH 7.4 with I=0.1
2+
(NaNO3)) at 37 C, 4 eq. of Mg(OH)2 and La(NO3)3 was added in the respective solution as source of Mg and La3+
.
°
a two-phase solvent system. These findings should be useful for
the design of more efficient catalysts with reference to
biochemistry including enzymatic reactions.
Experimental Section
General Information
All reagents and solvents were of the highest commercial quality
and were used without further purification, unless otherwise noted.
N,N-Dimethylformamide (DMF) was obtained by distillation from
calcium hydride. MNP was purchased from Nacalai Tesque (Japan).
All aqueous solutions were prepared using deionized and distilled
Figure 6. Comparison of catalytic turnover numbers (CTNs) of 8a, 9a, 10a,
10b, 10c and 14a–f (20 μM in total solution) at [MNP]=1000 μM in CHCl3/
50 mM HEPES buffer (pH 7.4) with I=0.1 (LiNO3, NaNO3, KNO3 or RbNO3) (2/
°
water. The Good’s buffer reagents (Dojindo, pKa at 20 C) were
obtained from commercial sources: HEPES (2-(4-(2-hydroxyethyl)-1-
piperazinyl))ethanesulfonic acid, pKa =7.6). For the measurement of
UV/Vis spectra, CHCl3 and DMSO were purchased from Nacalai
Tesque (Japan). UV/Vis spectra were recorded on a JASCO V-550 or
JASCO V-630 spectrophotometer with quartz cuvettes (path length:
10 mm). IR spectra were recorded on a Perkin-Elmer attenuated
°
8) at 37 C.
solvent system in this study. The values of Km/Ki ratio of 14b–c
(4.9–5.2) are also comparable to our previously reported
catalytic complex 10a (4.8). In comparison with the MNP
hydrolysis by 9 and 10 in the two-phase system, 14c comprised
of two Cu2+ ions and possibly one K+ ion in its active site
proved to be more efficient in terms of a faster and higher rate
of catalysis than 9 and 10 that contained no crown ether unit.
We therefore conclude that the unprecedented 2:2:2:4
complexes of the bis(Zn2+-cyclen) complex 3 (Zn2L3), the Bar
total reflectance (ATR)-IR spectrometer 100 at room temperature.
13
1H- (300 MHz) and CÀ (100 MHz) NMR spectra at 25�0.1 C were
°
recorded on a JEOL Always 300 spectrometer. Mass spectra were
recorded on a JEOL JMS-700 and Varian 910-MS spectrometer.
Thin-layer chromatography (TLC) and alumina gel column chroma-
tography was performed using Merck Silica gel 60 F254 plate and
Wako pure chemical Alumina Gel for chromatography, respectively.
5,5-Bis(3-(1,4,7,10,13-pentaoxa-16-azacyclooctadecan-16-yl)
propyl)-pyrimidine2,4,6-(1H,3H,5H)-trione (12)
building block equipped with the 18-crown-6 ether (12), Cu2+
,
and alkaline metal cations such as K+ are constructed in the
two-phase solvent system (CHCl3/H2O) and that the catalytic
phosphatase activity is improved by the functionalization of Bar
unit (eventually, functionalization of the supramolecular phos-
phatase) with the crown ether-K+ complex as a mimic of the
Mg2+ in the catalytic site of AP. We believe that this type of
functionalization of supramolecular complexes opens a strategy
for assessing their catalytic activity for the hydrolysis of MNP in
To a solution of 1-aza-18-crown-6 ether (0.1 g, 0.38 mmol) and
triethylamine (0.04 g, 0.38 mmol) in distilled DMF (0.6 ml), com-
pound 18[12b] (0.1 g, 0.17 mmol) was added at room temperature.
°
The reaction mixture was stirred at 70 C for 6 h under an argon
atmosphere. After evaporating the reaction mixture to dryness, the
resulting residue was purified by alumina gel column chromatog-
raphy (CHCl3/MeOH=25/1) to give 12 as a colorless oil. IR (ATR)
1
cmÀ 1: 2860, 1723, 1698, 1408, 1350, 1109, 943, 838, 499. H-NMR
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Figure 7. Lineweaver-Burk plots for the hydrolysis of MNP catalyzed by 14a (closed diamond with solid line), 14b (closed square with dashed line), 14c (open
squares with solid line), 14d (gray circles with solid line), 14e (closed circles with solid line), 14f (open circles with dashed line), and 10a (closed triangles with
dashed line).
Table 1. Typical kinetic parameters for the hydrolysis of MNP by 8a and AP in a single-phase solvent system (10 mM HEPES buffer (pH 7.4) with I=0.1
°
(NaNO3)) at 37 C, and by 9a, 10a and 14a–f in a two-phase solvent system (CHCl3/50 mM HEPES buffer (pH 7.4) with I=0.1 (LiNO3, NaNO3 or KNO3 or
°
RbNO3) (2/8)) at 37 C.
[b]
Entry
1
Cat.[a]
Mn+
-
V
max [μMminÀ 1
]
K
m [μM]
k
cat [minÀ 1
]
Ki [μM]
Km/Ki
CTN[c]
0.4
8a[d]
(8.9�0.2)×10À 2 [d]
1.3�0.1[e]
(4.1�0.3)×102 [d]
7�4[e]
(8.9�0.2)×10À 4 [d]
(2.4�0.2)×103[e]
(7.0�2.0)×10À 4[g]
(3.4�0.2)×10À 3[h]
ca.15
ca. 27
(mixed-type)
3�1
2
3
4
AP[d]
9a[g]
-
-
-
ca. 2.3
ca. 36
>103[f]
1.0
(competitive)
ca.15
(1.4�0.4)×10À 2[g]
(6.8�0.3)×10À 2[h]
(5.4�0.5)×102[g]
(3.8�0.2)×102[h]
(competitive)
10a[h]
ca.80[h]
ca. 4.8 [h]
~4
(mixed-type)
n.d[m]
ca.40
(mixed-type)
ca.97
5
6
14a[i]
14b[j]
Li+
(2.2�0.3)×10À 2
90�6
(1.1�0.2)×10À 3
n.d.[m]
ca. 4.9
2.4
3.9
Na+
(4.4�0.2)×10À 2
(2.3�0.3)×102
(2.2�0.1)×10À 3
7
14c[k]
K+
(7.9�0.4)×10À 2
(5.9�0.3)×102
(4.0�0.2)×10À 3
ca. 5.2
4.7
(mixed-type)
8
9
10
14d[l]
14e[j]
14f[j]
Rb+
(3.5�0.2)×10À 2
(3.3�0.4)×10À 2
(5.5�0.3)×10À 2
94�2
(1.8�0.1)×10À 3
(1.7�0.2)×10À 3
(2.8�0.2)×10À 3
n.d.[m]
n.d.[m]
n.d.[m]
n.d.[m]
4
4.3
4.2
Mg2+
Ln3+
51�3
n.d.[m]
(2.2�0.2)×102
n.d.[m]
[a] Cat=catalyst. [b] Calculated by eq. (1) in the text. [c] Catalytic turnover numbers determined at [MNP]=1000 μM (see Figure 6). [d] From ref. 11 (in a
single aqueous solution (10 mM HEPES, pH 7.4 with I=0.1 (NaNO3)). [e] From ref. 10r (in a single aqueous solution (10 mM HEPES, pH 7.4 with I=0.1 (NaNO3)).
[f] Calculated from data in ref. 10r. [g] From ref. 12a. [h] From ref. 13. [i] Determined in CHCl3/50 mM HEPES (pH 7.4 with I=0.1 (LiNO3)) (2/8). [j] Determined in
CHCl3/50 mM HEPES (pH 7.4 with I=0.1 (NaNO3)) (2/8). [k] Determined in CHCl3/50 mM HEPES (pH 7.4 with I=0.1 (KNO3)) (2/8). [l] Determined in CHCl3/
50 mM HEPES (pH 7.4 with I=0.1 (RbNO3)) (2/8). [m] Not determined.
(300 MHz, CD3OD/TMS) δ: 3.64–3.57 (m, 40H), 2.75(t, 8H, J=5.5H),
2.52 (t, 4H, J=7.2H), 1.92–1.87 (m, 4H), 1.44–1.33 (m, 4H). 13C-NMR
(75 MHz, CD3OD/TMS) δ: 175.8, 152.5, 71.7, 71.6, 71.3, 71.2, 70.1,
56.6, 56.5, 54.9, 37.6, 23.5. HRMS (ESI-MS) m/z: 735.43 (Calcd. for
C34H62N4O13 [M+H]+: 735.43).
CHCl3/50 mM HEPES (pH 7.4) with I=0.1 (RbNO3) (2/8) (for 14d) at
°
37 C. Stock solutions of 12 (6.0 mM in H2O), and Cu(ClO4)2 ·6H2O
(6 mM in H2O) were used for the preparation of sample solutions of
14 in CHCl3/50 mM HEPES (pH 7.4) with I=0.1 (LiNO3, NaNO3, KNO3,
or RbNO3) (2/8) (total volume=3.0 mL). A stock aqueous solution of
MNP (20 mM) was used for the hydrolysis reaction. Prior to the
hydrolysis of MNP in the two-phase solvent system, the reaction
mixtures of 3, 12, and Cu2+ in CHCl3/50 mM HEPES (pH 7.4) with I=
0.1 (LiNO3, NaNO3, KNO3, or RbNO3) (2/8) were incubated overnight
Hydrolysis of MNP
The hydrolysis of MNP was carried out in CHCl3/50 mM HEPES
(pH 7.4) with I=0.1 (NaNO3) (2/8) (for 14b, 14e and 14f). For the
MNP hydrolysis with 14e and 14f, 14e was prepared at [Mg(OH)2]-
=80 μM and [NaNO3]=0.05 M and 14f was prepared at [La(NO3)3]-
=80 μM and [NaNO3]=0.05 M, due to low solubility of Mg2+ and
La3+ ions. The hydrolysis of MNP was carried out in CHCl3/50 mM
HEPES (pH 7.4) with I=0.1 (LiNO3) (2/8) (for 14a) and in CHCl3/
50 mM HEPES (pH 7.4) with I=0.1 (KNO3) (2/8) (for 14c) and in
°
at 37 C using a shaking heat controlled incubator (shaking speed:
150 rpm) (BioShaker BR-23FP, TAITEC, Japan) to form 14a–f in situ,
and the aqueous solution of MNP was then added. All of the
hydrolysis experiments were performed in triplicate. The yields of
the hydrolysis product from MNP in the presence of the
supramolecular complexes were calculated based on the increase
in the absorption of the released 4-nitrophenol (NP) at 400 nm ([NP
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produced in the presence of the supramolecular complex]-[NP
produced in the absence of the supramolecular complex]) (ɛ400
value of NP is 1.35×104 MÀ 1 ·cmÀ 1 at pH 7.4)[10r] in aqueous layer.
The partition ratio of NP (79% in aqueous solution), which had
been determined in a previous report[12,13] was used for the
calculation of the yields in the CHCl3/H2O system.
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This work was supported by grants-in-aid from the Ministry of
Education, Culture, Sports, Science and Technology (MEXT) of
Japan (Nos. 16 K10396, 17 K08225, 18F18412, and 20 K05712 for
S.A.), research grant from Uehara Memorial Foundation, research
grant from Tokyo Ohka Foundation for the Promotion of Science
and Technology, Kanagawa, Japan and research grant from
Tokyo Biochemical Research foundation, Tokyo, Japan and the
TUS (Tokyo University of Science) grants for President’s Research
Promotion. We appreciate the aid of Mrs. Fukiko Hasegawa
(Faculty of Pharmaceutical Sciences, Tokyo University of Science),
Mrs. Noriko Sawabe (Faculty of Pharmaceutical Sciences, Tokyo
University of Science), and Dr. Yayoi Yoshimura (Faculty of
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Conflict of Interest
The authors declare no conflict of interest.
Keywords: Supramolecular catalysts · Hydrolysis · Phosphate
monoesters · Phosphatase · Zinc · Copper · Potassium
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Manuscript received: November 2, 2020
Revised manuscript received: January 5, 2021
Accepted manuscript online: January 11, 2021
Eur. J. Inorg. Chem. 2021, 1213–1223
www.eurjic.org
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