Catalytic Hydrolysis of Phosphate Monoester by Supramolecular Phosphatases Formed from a Monoalkylated Dizinc(II) Complex, Cyclic Diimide Units, and Copper(II) in Two-Phase Solvent System

Article abstract of DOI:10.1021/acs.inorgchem.8b03586

Design and synthesis of enzyme mimic with programmed molecular interaction among several building blocks including metal complexes and metal chelators is of intellectual and practical significance. The preparation of artificial enzymes that mimic the natu

Full text of DOI:10.1021/acs.inorgchem.8b03586

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Catalytic Hydrolysis of Phosphate Monoester by Supramolecular
Phosphatases Formed from a Monoalkylated Dizinc(II) Complex,
Cyclic Diimide Units, and Copper(II) in Two-Phase Solvent System
Akib Bin Rahman,† Hiroki Imafuku,† Yuya Miyazawa,† Ananda Kafle,§ Hideki Sakai,§ Yutaka Saga,†
and Shin Aoki*,†,∥,⊥
§
∥
†Faculty of Pharmaceutical Science, Faculty of Science and Technology, Division of Medical-Science-Engineering Cooperation,
⊥
Research Institute for Science and Technology, Imaging Frontier Center, Research Institute for Science and Technology, Tokyo
University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
S
* Supporting Information
ABSTRACT: Design and synthesis of enzyme mimic with
programmed molecular interaction among several building blocks
including metal complexes and metal chelators is of intellectual and
practical significance. The preparation of artificial enzymes that
mimic the natural enzymes such as hydrolases, phosphatases, etc.
remains a great challenge in the field of supramolecular chemistry.
Herein we report on the design and synthesis of asymmetric
(nonsymmetric) supermolecules by the 2:2:2 self-assembly of an
amphiphilic zinc(II)−cyclen complex containing a 2,2′-bipyridyl
linker and one long alkyl chain (Zn2L3), barbital analogues, and
Cu2+ as model compounds of an enzyme alkaline phosphatase that
catalyzes the hydrolysis of phosphate monoesters such as mono(4-
nitrophenyl)phosphate at neutral pH in two-phase solvent system (H2O/CHCl3) in pH 7.4 and 37 °C. Hydrolytic activity of
these complexes was found to be catalytic, and their catalytic turnover numbers are 3−4. The mechanistic studies based on the
UV/vis and emission spectra of the H2O and CHCl3 phases of the reaction mixtures suggest that the hydrophilicity/
hydrophobicity balance of the supramolecular catalysts is an important factor for catalytic activity.
Supramolecular strategies are useful for the construction of
three-dimensional and well-defined structures that can be
useful in molecular recognition, molecular sensing, molecular
storage, electronic devices, and catalytic reactions.6−8 In this
context, we previously reported on the formation of a
supramolecular complex 8 by the 2:2:2 assembly of dimeric
zinc(II) (Zn2+-cyclen) complexes containing a 2,2′-bipyridyl
(bpy) linker 1 (Zn2L1), a dianion of barbital (Bar2−) and its
analogues (4), and a copper ion (Cu2+) via 2:2 complexes of 1
with 4 (cyclen = 1,4,7,10-tetraazacyclododecane).9,10 It was
discovered that 8 possesses a Cu2(μ-OH)2 core that is
analogous to the active sites (Fe−Zn or Zn−Zn) of naturally
occurring dinuclear metalloenzymes such as AP. In addition,
these supramolecular complexes were found to accelerate the
hydrolysis of MNP at a neutral pH in single-phase aqueous
solution and in a two-phase solvent system (CHCl3/50 mM 4-
(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)).
Although 8 accelerates the hydrolysis of MNP, catalytic
turnover was not observed, possibly because the hydrolytic
activity of 8 is inhibited by HPO42−, an MNP hydrolysis
INTRODUCTION
■
The phosphorylation and dephosphorylation of proteins and
enzymes are important processes in intracellular regulation.
The dephosphorylation of phosphoserine and phosphothreo-
nine residues in proteins, the reverse of phosphorylation, by
protein kinase is catalyzed by protein phosphatases.1 For
example, alkaline phosphatases (APs) occur widely in nature
and are found in many organisms from bacteria to human. The
three-dimensional structure of AP indicates that APs are
homodimeric enzymes and that each catalytic site contains
three metal ions; that is, two Zn ions and one Mg ion are
required, not for only catalytic activity for the hydrolysis of
monoesters of phosphoric acid but also for transphosphor-
ylation reactions that proceed in the presence of large
concentrations of phosphorylation acceptors.2 The active
sites of phosphatases are similar in that they include dimetallic
diamond-shaped cores containing two metal cations, which are
coordinated by hydroxide ions and amino acids. Although
chemical models for hydrolases such as phosphatases have
been developed,3,4 artificial compounds that catalyze the
hydrolysis of phosphonic acid monoester such as mono(4-
nitrophenyl)phosphate (MNP) are rare, because phosphate
monoesters are less reactive than phosphate diesters and
triesters.4a,i,5
Received: December 24, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.8b03586
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Scheme 1. Formation of the 2:2:2 Supermolecules 8, 9, and 10 from Zn2L complexes (1−3), 5,5′-Diethylbarbital (Bar2−)
Derivatives (4a−f), and Cu2+ that Accelerate the Hydrolysis of MNP in a Single (H2O) and a Two-Phase Solvent System
(CHCl3/H2O)
Scheme 2. Proposed Scheme of Hydrolysis of MNP by Artificial Phosphatases 10 in a Two-Phase Solvent System
B
DOI: 10.1021/acs.inorgchem.8b03586
Inorg. Chem. XXXX, XXX, XXX−XXX

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Article
20 °C) were obtained from commercial sources: HEPES, pKa = 7.6.
Stock aqueous solutions of 20 mM MNP were prepared using
deionized and distilled water and stored at 0 °C prior to use. UV
spectra were recorded on a JASCO V-550 spectrophotometer,
product and an inhibitor of the overall reaction (product
inhibition).2,11
It is generally accepted that natural enzymes such as AP trap
(extract) the specific substrates from aqueous phase around the
enzymes into their hydrophobic catalytic pockets, accelerate
the specific and selective reactions (e.g., the hydrolysis of
phosphates in AP), and eventually release the products to
aqueous phase, so that their catalytic sites are regenerated to
accept the next substrate. On the basis of the structure of the
hydrophilic complex 8, as determined by X-ray crystal
structure analysis,9 a hydrophobic complex 9 (Scheme 1 and
Scheme 2) was designed for use in a two-phase solvent system
(CHCl3/H2O) to mimic the reaction sites and catalytic
mechanism of AP as mentioned above.10 It was expected that a
bis(Zn2+-cyclen) complex with two long alkyl chains 2
(Zn2L2), 4a and Cu2+ would form a 2:2:2 complex 9, which
would be more stable in organic solvents than in an aqueous
solution. It was hypothesized that Cu2+-bound OH− attacks
the phosphorus of MNP, which is achieved by the
coordination to the another Cu2+.12 It was also expected that
equipped with a temperature controller unit operating at 25
0.1
and 37 0.1 °C. IR spectra were recorded on a Perkin−Elmer ATR-
IR spectrum 100 at room temperature. Melting points were measured
by a Yanaco MP-J3Micro Melting Point apparatus without any
correction. 1H (300 MHz) and 13C (75 MHz) NMR spectra at 25
0.1 °C were recorded on a JEOL Always 300 spectrometer.
1
Tetramethylsilane (TMS) was used as internal reference for H and
13C NMR measurements by CDCl3, deuterated dimethyl sulfoxide
(DMSO-d6) or CD3OD. Mass spectra were recorded on a JEOL JMS-
SX102A and Varian 910-MS. Elemental analyses were performed on a
PerkinElmer CHN 2400 series II CHNS/O analyzer at the Research
Institute for Science and Technology, Tokyo University of Science.
Thin-layer chromatographies (TLC) and silica gel column chroma-
tographies were performed using Merck Silica gel 60 F254 TLC plate
or Fuji Silysia Chemical CHROMATOREX NH-TLC PLATE, and
Fuji Silysia Chemical FL-100D or Fuji Silysia Chemical CHROMA-
TOREX NH Chromatography Silica Gel, respectively.
Synthesis of 1-Docosyl-4,10-bis(tert-butyloxycarbonyl)-1,4,7,10-
tetraazacyclododecane (16). A mixture of 1-bromodocosane 14
(0.63 g, 1.62 mmol) and sodium iodide (0.24 g, 1.62 mmol) in
acetone (6.5 mL) and hexane (6.5 mL) was heated at reflux for 12 h
under an argon atmosphere. On the completion of reaction, the
insoluble compounds were removed by filtration and washed with
hexane (10 mL). The filtrate was concentrated under reduced
pressure to give 15 (0.71 g) as a colorless solid, which was used in the
next step without further purification. To a solution of 1,7-bis(tert-
butyloxycarbonyl)-1,4,7,10-tetraazacyclododecane 13 (0.55 g, 1.5
mmol) and potassium carbonate (0.2 g, 1.5 mmol) in MeCN (14
mL) and CHCl3 (6 mL), 15 (0.71 g) in CHCl3 (10 mL) was slowly
added over a period of 15 min. The reaction mixture was stirred at
room temperature for 3 d under argon atmosphere. After insoluble
inorganic salts were removed by filtration, the filtrate was evaporated
under reduced pressure. The remaining residue was purified by NH
silica gel column chromatography (CHCl3/MeOH = 100/1) to give
16 as a colorless oil (0.30 g, 29% yield). IR (attenuated total
reflectance (ATR)) cm−1: 3225, 2923, 2853, 1694, 1459, 1407, 1365,
2−
product inhibition by HPO4 could be avoided, since the
2−
hydrophilic HPO4 would be released into the aqueous layer
as it was formed, thus permitting the Cu2(μ-OH)2 center to be
regenerated like catalytic reactions by natural enzymes
(Scheme 2). Namely, we attempted at mimicking the catalytic
function of AP not only by the structures of supramolecular
complexes but also by the selection of appropriate solvent
systems. Very interestingly, the hydrolysis of MNP by 9
followed Michaelis−Menten kinetics even in the two-phase
solvent system containing CHCl3 and H2O. However,
negligible catalytic turnover was observed, possibly due to
low efficiency in the extraction of MNP by hydrophobic
complex 9 toward the organic phase and/or the slow release of
2−
HPO4 into the aqueous phase.
To address the aforementioned problems, we designed and
synthesized a series of new supramolecular complexes by the
self-assembly of a nonsymmetric amphiphilic dinuclear Zn2+−
cyclen complex containing one long alkyl chain 3 (Zn2L3) with
Bar2− derivatives and Cu2+ as artificial phosphatases (10 in
Scheme 1) that function in two-phase solvent system (CHCl3/
H2O), in this work. We expected that the active sites, Cu2(μ-
OH)2, of 10 would be localized in closer proximity to the
organic−water interface than 9, and hence product inhibition
by the inorganic phosphate would be reduced by its release
into the aqueous layer (Scheme 2). Moreover, the side chains
of the Bar units were functionalized, because one of the two
ethyl groups of each Bar unit in 8 are located in close proximity
to the Cu2(μ-OH)2 catalytic site (11 in a dashed box of
Scheme 2), as confirmed by an X-ray crystal structure analysis
of 8.
1
1246, 1160, 861, 754. H NMR (300 MHz, CDCl3/TMS) δ: 3.40−
3.25 (m, 8H), 2.85−2.62 (m, 8H), 2.53−2.47 (m, 2H), 1.45 (s, 18H),
1.25 (m, 40H), 0.88 (t, 3H, J = 6.6 Hz). 13C NMR (75 MHz, CDCl3/
TMS) δ: 156.1, 155.8, 79.6, 79.4, 54.3, 53.7, 52.9, 50.3, 50.1, 49.8,
49.5, 49.2, 48.6, 48.4, 47.7, 47.4, 31.9, 29.8, 29.6, 29.6, 29.3, 28.4,
22.6, 14.1. Fast atom bombardment mass spectrometry (FAB-MS) m/
z: 681.6256 (Calcd for C40H81N4O4 [M + H]+: 681.6258).
Tris(tert-butyl)-10-((5′-(bromomethyl)-[2,2′-bipyridin]-5-yl)-
methyl)-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate (19).
A mixture of 1213 (0.46 g, 0.98 mmol), 5,5′-bis(bromomethyl)-2,2′-
bipyridine 18 (0.40 g, 1.23 mmol), and sodium carbonate (0.59 g, 5.3
mmol) in MeCN (50 mL) was refluxed for 12 h under an argon
atmosphere. After the reaction was completed, the mixture was
evaporated and suspended in CHCl3 (30 mL). The organic layer was
washed with H2O (20 mL × 3) and brine, dried over Na2SO4, and
filtered, and the filtrate was evaporated under reduced pressure. The
resulting residue was purified by silica gel column chromatography
(hexane/AcOEt = 1/1) to give 19 as a pale brown oil (0.19 g, 25%
yield). IR (ATR) cm−1: 2975, 1683, 1596, 1552, 1463, 1414, 1364,
1248, 1153, 979, 752. 1H NMR (300 MHz, CDCl3/TMS) δ: 8.68 (d,
1H, J = 3 Hz), 8.54 (d, 1H, J = 0.9 Hz), 8.37 (t, 2H, J = 8.4 Hz), 7.85
(dd, 1H, J = 9 Hz, 2.1 Hz), 7.76 (dd, 1H, J = 6 Hz, 2.4 Hz, 2.1 Hz),
4.54 (s, 2H), 3.83 (s, 2H), 3.60−3.35 (m, 12H), 2.69 (brs, 4H), 1.48
(m, 27H). 13C NMR (75 MHz, CDCl3/TMS) δ: 155.8, 154.6, 137.5,
133.5, 120.9, 120.7, 79.6, 79.4, 55.4, 53.9, 50.0, 47.7, 29.6, 28.6, 28.4.
FAB-MS m/z: 733.3281 (Calcd for C35H54BrN6O6 [M + H]+:
733.3283).
Herein, we report on the self-assembly of 3, the function-
alized Bar units, and Cu2+ to form 10 and the catalytic activity
of 10 for the MNP hydrolysis in a two-phase solvent system.
Mechanistic aspects of its catalytic activity are discussed based
on the UV/vis and emission spectra of the supramolecular
complexes in H2O and CHCl3 layers of the reaction mixtures.
EXPERIMENTAL PROCEDURES
■
General Information. All reagents and solvents were of the
highest commercial quality and were used without further purification,
unless otherwise noted. Anhydrous dimethylformamide (DMF) was
obtained by distillation from calcium hydride. Anhydrous CCl4 was
obtained by distillation. The Good’s buffer reagents (Dojindo, pKa at
1,4,7-Tris(tert-butoxycarbonyl)-10-((5′-((4,10-bis(tert-butoxycar-
bonyl)-7-docosyl-1,4,7,10-tetraazacyclododecan-1-yl)methyl)-
[2,2′-bipyridin]-5-yl)methyl)-1,4,7,10-tetraazacyclododecane (20).
C
DOI: 10.1021/acs.inorgchem.8b03586
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A mixture of 19 (0.20 g, 0.27 mmol), 16 (0.26 g, 0.38 mmol), and
sodium carbonate (0.17 g, 1.6 mmol) in MeCN (10 mL) and CHCl3
(10 mL) was heated at reflux temperature for 16 h under an argon
atmosphere. After the reaction mixture was evaporated, the resulting
residue was suspended in CHCl3 (50 mL) and was then washed with
H2O (20 mL × 3) and brine, dried over Na2SO4, filtered, and
evaporated under reduced pressure. The resulting residue was purified
by silica gel column chromatography (CHCl3/MeOH = 80/1) to give
20 as a yellow amorphous/solid (0.15 g, 28% yield). IR (ATR) cm−1:
2923, 2853, 1687, 1595, 1551, 1458, 1411, 1364, 1247, 1152, 858,
751. 1H NMR (300 MHz, CDCl3/TMS) δ: 8.53 (d, 2H, J = 6.3 Hz),
8.31 (d, 2H, J = 7.5 Hz), 7.80 (d, 1H, J = 7.2 Hz), 7.74 (dd, 1H, J =
9.0 Hz, J = 1.5 Hz,), 3.83 (s, 2H), 3.71 (s, 2H), 3.61 (s, 4H), 3.39 (m,
14H), 2.65 (m, 12H), 2.41 (t, 2H, J = 7.2 Hz), 1.48−1.43 (m, 45H),
1.26 (s, 42H), 0.88 (t, 3H, J = 6.6 Hz). 13C NMR (75 MHz, CDCl3/
TMS) δ: 155.8, 155.4, 150.5, 138.6, 135.3, 120.6, 120.5, 56.0, 55.5,
53.9, 50.0, 47.8, 45.6, 31.9, 29.7, 29.4, 28.7, 28.5, 27.8, 22.7, 14.1. ESI-
MS m/z: 1334.0204 (Calcd. for C75H133N10O10 [M + H]+:
1334.0201).
1-((5′-((1,4,7,10-Tetraazacyclododecan-1-yl)methyl)-[2,2′-bipyri-
din]-5-yl)methyl)-7-docosyl-1,4,7,10-tetraazacyclododecane·9HBr·
2H2O (21). To a solution of 20 (0.1 g, 75 μmol) in MeOH (3 mL)
and CHCl3 (3 mL), 47% HBr (2 mL) was slowly added at 0 °C. The
resulting mixture was stirred at room temperature for 24 h and then
concentrated under reduced pressure. The resulting residue was
recrystallized from 47% HBr and MeOH to give 21 as a brown solid
(0.1 g, 89% yield). IR (ATR) cm−1: 3385, 2929, 2850, 2608, 1599,
1570, 1547, 1436, 1067, 931, 754, 722. 1H NMR (300 MHz, DMSO-
d6/TMS) δ: 8.73 (s, 2H), 8.46 (d, 2H, J = 8.1 Hz), 8.10 (t, 2H, J =
11.1 Hz), 3.89 (d, 4H, J = 7.8 Hz), 3.22−3.13 (m, 16H), 2.88−2.77
(m, 16H), 1.46 (brs, 2H), 1.23 (s, 40H), 0.85 (t, 3H, J = 6.3 Hz). 13C
NMR data could not be obtained because of the low solubility of this
material. Electrospray ionization mass spectrometry (ESI-MS) m/z:
833.7571 (Calcd for C50H93N10 [M + H]+: 833.7579). Anal. Calcd.
for C50H105Br9N10O2: C, 37.59; H, 6.62; N, 8.77. Found: C, 37.36; H,
6.46; N, 8.42%.
3·4ClO4·2H2O. The pH of an aqueous mixture (5 mL) of 21 as the
HBr salt (60 mg) was adjusted to 12 with a 2 N NaOH aqueous
solution, and the resulting solution was extracted with CHCl3 (20 mL
× 5). The combined organic layer was washed with brine, dried over
Na2SO4, filtered, and concentrated under reduced pressure. The
resulting deprotonated 21 (39 mg, 46 μmol) was dissolved in a
mixture of MeOH (4 mL) and CHCl3 (3 mL), to which a solution of
Zn(ClO4)2·6H2O (35 mg, 92 μmol) in H2O was added. The solution
was stirred at 70 °C for 36 h. After the solvent was evaporated, the
residue was crystallized from EtOH to give 3 as a pink solid (61 mg,
94% yield). mp >200 °C. IR (ATR) cm−1: 3529, 3285, 2922, 2852,
1480, 1067, 958, 928, 853, 748, 720, 620. 1H NMR (300 MHz,
DMSO-d6/TMS) δ: 8.70 (s, 2H), 8.44 (d, 2H, J = 8.1 Hz), 8.00 (d,
2H, J = 7.8 Hz), 4.76−4.49 (m, 4H), 3.95 (s, 4H), 2.95−2.89 (m,
4H), 2.78−2.68 (m, 24H), 1.53 (brs, 2H), 1.23 (s, 40H), 0.85 (t, 3H,
J = 6.3 Hz) ppm. 13C NMR data could not be obtained because of the
low solubility of this material. FAB-MS m/z: 579.4 (Calcd for
C50H92Cl2N10O8 [M-2ClO4]2+: 579.2525). Anal. Calcd. for
C50H96Cl4N10O18Zn2: C, 42.96; H, 6.92; N, 10.02. Found: C,
42.81; H, 6.74; N, 9.75%.
7.33−7.23 (m, 10H), 4.51 (s, 4H), 4.17 (q, 4H, J = 6.9 Hz), 4.01 (s,
4H), 1.20 (t, 4H, J = 7.2 Hz). 13C NMR (75 MHz, CDCl3/TMS) δ:
168.2, 138.1, 127.4, 127.3, 73.2, 68.0, 61.3, 59.4, 13.9. FAB-MS m/z:
401.1963 (Calcd. for C23H28O6 [M + H]+: 401.1959).
5,5-Bis(benzyloxymethyl)barbituric acid (4b). Diethyl 2,2-bis-
((benzyloxy)methyl)malonate 23 (0.11 g, 0.27 mmol) in freshly
distilled DMF (1.5 mL) was added dropwise to a slurry of NaH (27
mg, 1.1 mmol, 55 wt % dispersion in oil) and urea (0.12 g, 1.9 mmol)
in freshly distilled DMF (3 mL) at 0 °C. The mixture was stirred at
100 °C for 24 h. After it cooled to room temperature, water was
added to the mixture, which was then extracted with Et2O. The
combined organic layers were washed with brine and dried over
Na2SO4. The solvents were evaporated, and the resulting oil was
purified by silica gel chromatography (CHCl3) to afford 4b as a
colorless solid (16 mg, 16%). mp 165−168 °C. IR (ATR) cm−1: 3417,
1
3222, 1751, 1696, 1425, 1341, 1228, 1089, 835, 732, 614, 493. H
NMR (300 MHz, CDCl3/TMS) δ: 7.34−7.16 (m, 10H), 4.45 (s,
4H), 3.76 (s, 4H). 13C NMR (75 MHz, CDCl3/TMS) δ: 170.5,
148.6, 136.7, 128.5, 128.0, 127.5, 73.7, 71.1, 58.8. FAB-MS m/z:
369.1447 (Calcd. for C23H28O6 [M + H]+: 369.1445). Anal. Calcd.
for C23H28O6: C, 65.21; H, 5.47; N, 7.60. Found: C, 65.19; H, 5.29;
N, 7.39%.
Hydrolysis of MNP. The hydrolysis reaction of MNP was
performed in CHCl3/50 mM HEPES buffer (pH 7.4 with I = 0.1
(NaNO3)) (2/8) (total volume 3.0 mL) in the presence of 10a−f
(final concentration: 20 μM in the total solution), and all the
hydrolysis reactions were triplicated. Stock solutions of barbital
derivatives (4a−f) (6.0 mM in CHCl3), Cu(ClO4)2·6H2O (6.0 mM
in H2O), HEPES (100 mM in H2O, pH 7.4 with I = 0.2 (NaNO3)),
and MNP (20 mM in H2O) were prepared, respectively. Prior to the
MNP hydrolysis, the reaction mixtures of 4a−f (or 3) and Cu2+ in
CHCl3/50 mM HEPES buffer (pH 7.4 with I = 0.1 (NaNO3)) were
incubated at 37 °C for 1 d using shaking water bath (shaking speed:
150 rpm) (PersonalH incubator) to form 10a−f in situ. After this, a
given volume (15 to ∼150 μL) of an aqueous solution of 20 mM
MNP was added to start the hydrolysis of MNP, and all the hydrolysis
experiments were performed at 37 °C using shaking water bath
(shaking speed: 150 rpm). The aqueous and organic layers of the
reaction mixtures were separated by centrifugation (3000 rpm × 10
min at 25 0.1 °C) at a given time (from day 0 up to day 7), and the
UV/vis absorption spectra of the aqueous layer were measured to
determine the concentrations of 4-nitrophenol (4-NP) (the ε400 value
of NP is 1.35 × 104 M−1·cm−1 at pH 7.4 in aqueous solution), a
hydrolysis product from MNP, to calculate the yields of MNP
hydrolysis ([NP produced in the presence of the supermolecule] −
[4-NP produced in the absence of the supermolecule]). The partition
ratio of 4-NP (79% in aqueous solution) was used for calculation of
yields in CHCl3/H2O system based on our previous paper.10
RESULTS AND DISCUSSION
■
Synthesis of a Dimeric Zn2+ Complex Containing One
Long Alkyl Chain. A bis(Zn2+−cyclen) containing a bpy unit
and one long alkyl chain (in one cyclen unit) 3 (Zn2L3) was
synthesized as shown in Scheme 3. Cyclen was reacted with di-
tert-butyldicarbonate (Boc2O) and N-(tert-butoxycarbonyl)-
succinimide (Boc-OSu) to give 3-Boc-cyclen 1213 and 2-Boc-
cyclen 13,14 respectively, according to the previously reported
procedures. The monoalkylation of 13 with 1-iododocosane
15, prepared from 1-bromodocosane 14, afforded 16. The
alkylation of 5,5′-bis(bromomethyl)-2,2′-bipyridine 188,15,16
obtained from 5,5′-dimethyl-2,2′-bipyridine 17 with 12 in the
presence of Na2CO3 gave 19. After 19 reacted with 16 to
produce 20, the Boc protecting groups were removed by the
treatment with aqueous HBr, giving 21 as a HBr salt. The acid-
free 21 was obtained by extracting with CHCl3 from 2 N
NaOH and then reacted with 2 equiv of Zn(ClO4)2·6H2O to
afford 3 (Zn2L3).
Diethyl 2,2-bis((benzyloxy)methyl)malonate (23). To a slurry of
NaH (55 mg, 2.3 mmol, 55 wt % dispersion in oil) in distilled DMF
(6 mL), diethyl melonate (22) in DMF (6 mL) was added, and the
resulting solution was stirred for 30 min at 0 °C. Then
((chloromethoxy)methyl)benzene dissolved in distilled DMF (0.3 g,
1.92 mmol, purity >90%) was slowly added dropwise to the
suspension. The resulting mixture was refluxed overnight under an
argon atmosphere. After it cooled to room temperature, the mixture
was neutralized with water and extracted with Et2O. The organic layer
was then washed with brine and dried over Na2SO4 and concentrated
under reduced pressure. Residue was purified by silica gel
chromatography (hexane/AcOEt = 30/1) to afford 23 as colorless
oil (0.12 g, 37%). IR (ATR) cm−1: 3463, 2859, 1730, 1454, 1301,
1
1204, 1075, 751, 692, 482. H NMR (300 MHz, CDCl3/TMS) δ:
D
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Scheme 3. Synthesis of 3 (Zn2L3)
Scheme 4. Structures of the 2:2:2 Complexes 10a−f Formed
from 7a−f (2:2 Complexes of 3 and 4a−f) and Cu2+
Supporting Information), indicating that the 1:1 complexation
of 3 and Cu2+, which is different from the 1:2 complexation of
7a and Cu2+.
Hydrolysis of MNP by 2:2:2 Complexes in Two-Phase
Solvent Systems. The hydrolysis of MNP (100 μM) was
performed in CHCl3/50 mM HEPES buffer (pH 7.4 with I =
0.1 (NaNO3)) (2/8) in the presence of 10a (prepared from 3,
Bar2− 4a and Cu2+, [10a] = 20 μM in the total solution
including organic and aqueous layers) at 37 °C. As shown in
Figure S5 in Supporting Information (UV/vis spectral change
of the aqueous layers of the reaction mixtures during the MNP
hydrolysis producing 4-NP, which has strong absorbance at
400 nm) and Figure 1a, 10a (20 μM in the total solution)
accelerated the hydrolysis of MNP, and its initial rate was
higher than the analogous reaction promoted by 9 (20 μM) in
the same solvent system. Interestingly, we also found that the
hydrolysis by 10a proceeded in ca. 35% yield. Negligible
acceleration was observed in the presence of 3 alone, Bar2−
alone, and Cu2+ alone under the same conditions. The
hydrolysis of MNP by the 3 + Cu2+ complex was also
accelerated, albeit in a noncatalytic manner. Figure 1b displays
that the MNP hydrolysis by 10a at [MNP] = 100, 200, 500,
and 1000 μM and [10a] = 20 μM (namely, at [10a]/[MNP] =
20%, 10%, 4% and 2%) affords more than 20 μM of 4-NP
(indicated with a dashed line in Figure 1b), suggesting that 10a
functions as a catalyst for the hydrolysis of MNP.
Complexation Behavior of 3 with Bar2− and Cu2+ As
Studied by UV/Vis Titrations. Because 3 was not soluble in
either organic or aqueous solutions, UV/vis titrations of 3 with
barbital (Bar2−) and its derivatives 4a−4f and Cu(ClO4)2·
6H2O were performed in dimethyl sulfoxide (DMSO)/50 mM
HEPES buffer (pH 7.4, with I = 0.1 (NaNO3) (4/96) at 37 °C
(Scheme 4). The slight increase in the absorption of 3 (20
μM) at 287 nm was observed upon the addition of Bar2−,
reaching a plateau at a 1:1 ratio, as shown in the inset of Figure
S2 in Supporting Information, suggesting that 2:2 complex-
ation of 3 with Bar2− had occurred.
UV/Vis titrations of 7a (10 μM) with Cu2+ in DMSO/50
mM HEPES buffer (pH 7.4 with I = 0.1 (NaNO3) (4/96))
were conducted at 37 °C. Figure S3 in Supporting Information
shows a red shift in the absorption maxima of 7a from ca. 287
to ca. 307 nm reaching a plateau at [Cu2+]/[7a] = 2.0 (the
inset of Figure S3 in Supporting Information), indicating that
1:2 complexation of 7a and Cu2+ had occurred. It was also
indicative of the quantitative formation of 10a at the
micromolar order.
In the proposed structure of 10c (formed from 3, 4c, and
Cu2+) generated by Mercury, Ver: 3.10 software based on the
crystal structure of 8 (Figure 2a, in which Cu2+-bound H2O
molecules are omitted for clarity), it was assumed that the
benzyl groups of 4b−e units in 10b−e are located close to the
Cu2(μ-OH)2 sites and provide a hydrophobic environment for
the substrate as shown in Figure 2b (the C22 alkyl groups are
oriented to the same direction (a so-called cis conformer) in
UV/Vis titrations of 3 (Zn2L3 itself) (40 μM) with Cu2+ in
DMSO/50 mM HEPES buffer (pH 7.4 with I = 0.1 (NaNO3)
(4/96)) were also performed at 37 °C for the comparison with
the UV/vis titrations of 7a with Cu2+ described above. As
shown in Figure S4 in Supporting Information, the UV/vis
spectra exhibited a red-shift from ca. 287 to ca. 308 nm, which
reached a plateau at [Cu2+]/[3] = 1.0 (the inset of Figure S4 in
E
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Scheme 5. Synthesis of Functionalized Barbital Derivatives
4b−f
Figure 1. (a) Hydrolysis (%) of MNP (100 μM in the total solution)
2+
■
○
●
by 10a (20 μM) ( ), 3 + Cu (40 μM) ( ), 9a (20 μM) ( ), 3 (40
2+
□
△
▲
μM) ( ), 4a (40 μM) ( ), and Cu only (40 μM) ( ) in CHCl3/
50 mM HEPES buffer (pH 7.4 with I = 0.1 (NaNO3)) (2/8) at 37
°C. (b) Concentrations of 4-NP, which is produced by the MNP
hydrolysis, at [MNP] = 100 μM ( ), 200 μM ( ), 500 μM ( ), and
1000 μM ( ), promoted by 10a (20 μM) in CHCl3/50 mM HEPES
■
●
▲
○
buffer (pH 7.4 with I = 0.1 (NaNO3)) (2/8) at 37 °C. The initial
concentrations of MNP and supramolecular complexes were
determined in the total solution of the two-phase solvent systems.
A dashed line in Figure 1b indicates that the concentration of 10a is
20 μM.
respectively (only 4b was newly synthesized in this work).9,13
Deprotection of the benzyl groups17 of 4c afforded 4f having
hydroxylethyl groups.
Hydrolysis of MNP by 2:2:2 Complexes Formed
Reacting 3 (Zn2L3) with Barbital Derivatives and Cu2+
and Their Phosphate Hydrolysis Activity in Two-Phase
Solvent Systems. Supramolecular complexes 10a−f were
prepared in situ from 3 (Zn2L3), Bar derivatives 4a−f, and
Cu2+, and their MNP hydrolysis activity was then evaluated in
CHCl3/50 mM HEPES buffer (pH 7.4 with I = 0.1 (NaNO3))
(2/8) at [MNP] = 100 μM and [10a−f] = 20 μM (in total
solution) at 37 °C. As shown in Figure 3a, it was found that
10b−f hydrolyzed MNP in over 20% yield, similar to 10a. The
hydrolysis of MNP by 10a−f was also performed at [MNP] =
1000 μM and [10] = 20 μM (2% equivalent to MNP) (Figure
3b). As summarized in Figure 4, catalytic turnover numbers
(CTN) for 10a−f were determined to be higher than 3, while
our previous supramolecular complexes 8 and 9 negligibly
functioned as catalysts.
Michaelis−Menten Kinetics for Hydrolysis of MNP by
10a−f in Two-Phase Solvent Systems. The kinetics of the
hydrolysis of MNP by 9 and 10a−f was studied by the reaction
at [MNP] = 100, 200, 500, 800, and 1000 μM (in total
CHCl3/H2O solvent) for 24 h at 37 °C. In a Lineweaver−Burk
plot (Figure S6 in Supporting Information), the concentrations
of MNP and the produced NP in the total solution of the two-
phase solvent systems were used for the calculation (V0 =
initial hydrolysis rate), from which the approximate Vmax (the
Figure 2. (a) X-ray crystal structure of 89 and (b) proposed structure
of the amphiphilic supermolecule 10c (assuming a cis form, in which
two alkyl groups are hypothesized to be orientated to the same side).
this structure, and there must also be trans form, as indicated in
Scheme 4).
This assumption allowed us to synthesize the functionalized
barbital derivatives 4b−f that are shown in Scheme 5. The
2,2′-disubstituted malonates 23−26 were prepared by the
dialkylation of diethyl malonate 22, and successive cyclization
with urea afforded the corresponding barbital derivatives 4b−f,
F
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maximum rate in the NP production from MNP) of 10a−f was
calculated to be (6.8 0.3) × 10−2, (3.4 0.3) × 10−2, (5.9
0.2) × 10−2, (2.0 0.3) × 10−1, (2.8 0.2) × 10−2, and (2.8
0.1) × 10−2 μM/min, respectively, as summarized in Table
1. The Km (Michaelis constant) values for 10a−f were
determined to be (3.8 0.2) × 102, (4.4 0.3) × 102, (1.4
0.3) × 102, (1.6 0.1) × 103, (1.7 0.3) × 102, and (1.2
0.2) × 102 μM, respectively. Their kcat values defined by eq 1
are also listed in Table 1.
kcat = Vmax/Km
(1)
The Km values in Table 1 are compared with the Kd values of
4
Cu2+ complexes 27 (CuL ) reported by Esteban-Gomez et
́
al.18a,b and 28 (CuL5) synthesized by Delgado et al. with
phosphates (Scheme 6). For instance, the complexation
constant (log Ks) of 27 (CuL4) with inorganic phosphate
18
c
−
(H2PO4 ) in DMSO was reported to be 5.1, from which the
Kd value was calculated to be ca. 80 μM.18a The log Ks value of
28 (CuL5) with H2PO4− in a 1:1 mixture of MeOH/H2O was
reported to be 4.1, meaning that the Kd value for the Cu2L5−
H2PO4 is 87 μM.18 We consider that Km values of 10a−f
determined in a two-phase solvent system (CHCl3/H2O), as
listed in the Table 1, are almost compatible to the Kd values of
−
c
Figure 3. Time course for the hydrolysis (%) of MNP at [MNP] =
100 μM (a) and [MNP] = 1000 μM (b) by 9 and 10a−f (20 μM in
total solution) in CHCl3/50 mM HEPES buffer (pH 7.4 with I = 0.1
(NaNO3)) (2/8) at 37 °C. The initial concentrations of MNP and
supramolecular complexes were determined in the total solution of
the two-phase solvent system.
−
27 and 28 with H2PO4 determined in organic solution or a
mixture of MeOH/H2O (single phase).
Scheme 6. Structures of Cu2+ Complexes Reported by
Esteban-Gomez et al. and Delgado et al.18
18a
c
́
It is known that HPO42−, a hydrolysis product of MNP,
inhibits AP (product inhibition).2,11 In Figure 3a,b, it was
indicated that 10-catalyzed MNP hydrolysis rate becomes
slower as the reaction proceeds, possibly because of the
Figure 4. Comparison of CTN of 8, 9, and 10a−f at [MNP] = 1000
μM and [8, 9, or 10] = 20 μM (in the total solution) in CHCl3/50
mM HEPES buffer (pH 7.4 with I = 0.1 (NaNO3)) (2/8) at 37 °C.
Table 1. Kinetic Parameters (Vmax, Km, kcat and Ki of Inorganic Phosphate) for Hydrolysis of MNP by 8, 9, 10a−f and AP
Vmax (1 μM·min−1
)
b
Km (μM)
kcat (min−1
)
Ki (μM)
Km/Ki
a
c
b
c
b
b
8
9
(8.9 0.2) × 10−2
(1.4 0.4) × 10−2
(6.8 0.3) × 10−2
(3.4 0.3) × 10−2
(5.9 0.2) × 10−2
(2.0 0.3) × 10−1
(2.8 0.2) × 10−2
(2.8 0.1) × 10−2
(4.1 0.3) × 102
(5.4 0.5) × 102
(3.8 0.2) × 102
(4.4 0.3) × 102
(1.4 0.3) × 102
(1.6 0.1) × 103
(1.7 0.3) × 102
(2.2 0.2) × 10−4
(2.7 0.4) × 10−5
(1.8 0.2) × 10−4
(7.8 1) × 10−5
(4.5 0.9) × 10−4
(1.3 0.2) × 10−4
(1.7 0.4) × 10−4
(2.5 0.5) × 10−4
ca. 15 (mixed-type)
ca. 27
ca. 36
ca. 4.8
ca. 4.5
c
ca. 15 (competitive)
ca. 80 (mixed-type)
ca. 98 (mixed-type)
d
d
10a
10b
10c
10d
10e
d
e
e
nd
nd
nd
nd
nd
nd
nd
nd
d
e
e
d
e
e
d
e
e
10f
AP
(1.2 0.2) × 102
f
f
f
f
f
1.3 0.1
7
4
(2.4 0.2) × 103
3
1 (competitive)
ca. 2.3
a
b
c
d
From ref 9 (in a single aqueous phase). Determined in a single aqueous solution (10 mM HEPES buffer, pH 7.4). From ref 10. Data of 10a−f
e
f
were obtained in CHCl3/50 mM HEPES buffer (pH 7.4) (2/8). Not determined. From ref 5g.
G
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●
Figure 5. Hydrolysis of MNP at [MNP] = 100 μM in total solution by 10a ( ) (20 μM) as control, 10a with excess (5 equiv) of 3 (200 μM) from
2+
■
○
▲
day 1 (dashed curve with ) and 5 equiv of (4a + Cu ) (200 μM) in the same vial from day 3 ( ), 3 alone ( ) (40 μM), 10a with excess (5
equiv) of 4a ( ) (200 μM), 10a with excess (5 equiv) of Cu ( ) (200 μM) in CHCl3/50 mM HEPES buffer (pH 7.4) with I = 0.1 (NaNO3)
(2/8) at 37 °C.
2+
□
△
Scheme 7. Our Hypothesis for the Inhibition of MNP Hydrolysis by 10a upon the Addition of an Excess Amount of 3
product inhibition. Therefore, MNP hydrolysis by 10a and 10b
would accelerate the hydrolysis of phosphate esters and other
ester substrates. As shown in Figure 5, the addition of an excess
amount of 3 1 d after starting the hydrolysis of MNP (100
μM) by 10a (20 μM) caused a considerable inhibition in the
hydrolysis (dashed curve with open circles). To overcome this
inhibitory effect, an excess amount of 4a and Cu2+ was added
on day 3 in the same vial, and this addition restarted the MNP
hydrolysis (plain curve with closed triangles). We conclude
that 3 likely binds to MNP at the CHCl3/H2O interface,
resulting in the inhibition of MNP hydrolysis by 10a, as
presented in Scheme 7. The addition of excess amount of
4a(open triangles) or Cu2+ (open squares) to 10a had no
effect on the hydrolysis of MNP, as shown in Figure 5.
Hydrolysis of Bis(4-nitrophenyl)phosphate (BNP) by
2:2:2 Complexes in Two-Phase Solvent Systems. The
hydrolysis of BNP (200 μM, 500 μM, 800 μM, and 1 mM) was
also performed in CHCl3/50 mM HEPES buffer (pH 7.4 with
I = 0.1 (NaNO3)) (2/8) in the presence of 10a (prepared
from 3, Bar2− 4a, and Cu2+) ([10a] = 20 μM in the total
solution including organic and aqueous layers) at 37 °C
(Figure 6). In this case, the hydrolysis reaction proceeded in
ca. 24% yield catalyzed by 10a (when [BNP] = 1 mM and
[10a] = 20 μM (indicated with a dashed line in Figure 6) and,
hence, [10a]/[BNP] = 0.02), indicating that it catalyzes
hydrolysis of BNP (CTN is up to ca. 12), as well.
2−
in the presence of HPO4 was performed (at the initial
[HPO42−] = 0, 10, and 50 μM). The Lineweaver−Burk plot in
2−
Figure S7 in Supporting Information suggests that [HPO4
]
exhibits mixed-type inhibition,19 and its Ki values against 10a
and 10b were estimated to be ca. 80 and ca. 98 μM,
respectively (Table 1).20 The complex 10f showed the smallest
Km value, which was ca. 3.4−4.5 times smaller than those of 8
and 9, respectively. On the other hand, the Ki values of
2−
HPO4 against 10a and 10b are ca. 5−6 times higher than
those against 8 and 9, and the Km/Ki ratios of 10a and 10b are
4.5−4.8, which are much smaller than those of 8 and 9 (27−
36) and are almost comparable to that of AP (2.3). These
2−
kinetic data suggest that 10a−f have lower affinity to HPO4
than that of 9, possibly due to their closer localization to the
aqueous−organic interface, resulting in more efficient release
of HPO42−, as we expected in Scheme 2.
Inhibitory Effect of 3 (Zn2L3) on the MNP Hydrolysis.
As mentioned above (Figure 1a), the activity of 3 (Zn2L3)
itself in the MNP hydrolysis was negligible. During our
experiments, we found that the addition of a slight excess of
Zn2L3 against Bar2− (4a) unit and Cu2+ resulted in a decrease
in the MNP hydrolysis rates. Therefore, we suspected that the
presence of Zn2L3 had an inhibitory effect on the hydrolysis of
MNP, although it is normally predicted that Zn2+ complexes
H
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for 3 (Figure 7b), indicating that 1 remains in the aqueous
layer, 2 (soluble in CHCl3) is distributed mainly in the CHCl3
layer, and 3 (insoluble in both water and CHCl3) is located
mainly at the aqueous−organic interface. Similarly, the data
shown in Figure 7c,d suggest that 5 and 6 are present in the
same location as were 1 and 2 and that small amounts of 3
moved to the CHCl3 layer by complexation with Bar2− (by the
formation of 7a). The addition of 100 μM of Cu2+ to the
solutions of 5, 6, and 7a resulted in the emission in all the
layers being reduced, as shown in Figure 7f, possibly because
the emission of the bpy unit in supramolecular complexes is
quenched by Cu2+.
For a more detailed analysis, UV/vis absorption and
emission spectra of the aqueous and organic layers were
measured after these two layers were separated by
centrifugation. As shown in Figure 8a, UV/vis absorption
spectra of aqueous layers for Zn2L (1, 2, and 3) exhibit
absorption maxima in the 280−320 nm region, and the
concentration of 1 (Zn2L1) was found to be much higher than
those (dashed curve) of 2 (Zn2L2) (blue curve) and 3 (red
curve). On the contrary, the concentration of 2 was highest in
CHCl3 layer, and the concentration of 3 (Zn2L3, depicted in a
red bold line) was between those of 1 and 2 (Figure 8b).
Similar behavior was observed for the 2:2 complexes, 5, 6, and
7a (Figure 8c,d). The addition of Cu2+ to solutions of 5, 6, and
7a exhibited similar result in the UV/vis spectra of both
aqueous and organic layers, as shown in Figure 8e,f,
respectively.
□
▲
Figure 6. Hydrolysis of BNP at [BNP] = 1000 μM ( ), 800 μM ( ),
●
○
500 μM ( ), and 200 μM ( ) by 10a (20 μM) in CHCl3/50 mM
HEPES buffer (pH 7.4 with I = 0.1 (NaNO3)) (2/8) at 37 °C. A
dashed line indicates the concentration of 10a.
Location of Complexes 8, 9, and 10a in the Two-
Phase Solvent System, As Determined from UV/Vis and
Emission Spectra of H2O and CHCl3 Phases. To elucidate
how and why 10a−f function as catalysts for the hydrolysis of
MNP and BNP, the location of the supramolecular complexes
in the two-phase solvent system was examined. Namely, Zn2L
alone (1, 2, 3), 2:2 complex of Zn2L and Bar2− (5, 6, 7a), and
2:2:2 complex of Zn2L, Bar2−, and Cu2+ (8, 9, 10a) were
prepared in CHCl3/50 mM HEPES (pH 7.4, I = 0.1
(NaNO3)) (1/1), shaken vigorously, and then centrifuged
(2000 rpm × 10 min) at room temperature.
For comparison, UV/vis absorption and emission spectra of
nonfunctionalized bpy were obtained in both aqueous and
CHCl3 solutions.21 Since bpy is not soluble in H2O, a stock
solution of bpy in DMSO was prepared and diluted with H2O
or CHCl3, respectively, for the measurement. From the UV/vis
spectral curves of bpy in the H2O and CHCl3 layers (Figure
S8a,b in Supporting Information), the absorption coefficients
(ε) of bpy in H2O and CHCl3 layers were calculated to be ca.
6.9 × 103 and ca. 7.7 × 103 M−1 cm−1, respectively. Since 1−3
and 5−7a contain two bpy units, we estimated the distribution
(%) (Table 2) of each complex (5, 6, and 7a) in each phase
using equations derived from the Beer−Lambert law (eqs 2
and 3 in Scheme S2 in Supporting Information). As
summarized in Table 2, the finding indicates that more than
98% of 5 remains in the H2O layer and that most (>99%) of 6
is located in the CHCl3 layer. In contrast, 7a is distributed in
both the H2O layer (ca. 35%) and the CHCl3 layer (ca. 65%).
Figure 9 shows the emission spectra of 1−3 and 5−7 in the
H2O and CHCl3 layers (emission spectra of 8−10a that
include Cu2+ are not shown, because these emissions are
quenched by Cu2+). Note that 6 has emission maxima at ca.
330 and ca. 480 nm in the CHCl3 layer (a blue curve) and that
7a has broad emission curve from 320 to 550 nm (a red curve)
in Figure 9d. For comparison, Figure S8c,d in Supporting
Information shows fluorescent emission maxima of bpy in H2O
and CHCl3 layers at 330 and 370 nm, respectively, not around
500 nm, indicating that emission of 7a at ∼500 nm is due to
the complexation of 3 with barbital (4a).
Figure 7a,b shows the pictures of Zn2L (1, 2, and 3) (100
μM) under sunlight and under irradiation at 365 nm,
respectively. Although the difference between a control sample
that contains no Zn2+ complex (Ctrl) and 1 under sunlight
(Figure 7a) was negligible, a strong emission in CHCl3 layer
was observed for 2 and at the interface of CHCl3/H2O layers
Figure 7. Pictures of mixtures of Zn2L and their complexes with Bar
and Cu2+ in CHCl3/50 mM HEPES (pH 7.4 with I = 0.1 (NaNO3))
(50/50)). Upper layer is the aqueous layer, and the lower one is
CHCl3. Each sample was incubated for 12 h at 37 °C with rigorous
stirring and centrifuged (2000 rpm × 10 min). (a, b) Vials of control
that include only solvents (Ctrl), 1, 2, and 3 (100 μM) from left
under sunlight (a) and irradiation at 365 nm (b). (c, d) Vials of
control containing only solvents (Ctrl), 5, 6, and 7a (50 μM) from
left under sunlight (c) and irradiation (d) at 365 nm. (e, f) Vials of
control (Ctrl), 8, 9, and 10a (50 μM) from left under sunlight (e) and
irradiation at 365 nm (f).
It was previously reported that the emission of bpy in silica
gel glass is at ca. 400 and ca. 450 nm.22 It was also reported
that the emission of bpy is at ca. 400 and ca. 450 nm in EtOH
at high concentration (0.1 M), while only one emission
maxima was observed at ca. 400 nm at a concentration of 3
mM. Such a dual emission of bpy at ca. 400 and ca. 450 nm
I
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Figure 8. UV/Vis absorption spectra of aqueous and CHCl3 layers separated from the two-phase solvent system including Zn2L, 4a, and Cu2+
([Zn2L] = [Bar2− (4a)] = [Cu2+] = 100 μM in the initial two-phase solvent system) at 37 °C. (a, b) UV/Vis absorption spectra of 1, 2, and 3 in
H2O layer (a) and CHCl3 layer (b). (c, d) UV/Vis spectra of 5, 6, and 7a in H2O layer (c) and CHCl3 layer (d). (e, f) UV/Vis absorption spectra
of 8, 9, and 10a in H2O layer (e) and CHCl3 layer (f).
Table 2. Approximate Distribution (%) of 5, 6, and 7a in the
H2O Layer and the CHCl3 Layer after Separation of Two-
Phase Solvent Systems [5, 6, 7a] = 50 μM in a Mixture of
CHCl3/H2O Estimated by UV/Vis Spectra of These
Complexes in H2O and CHCl3, As Shown in Figure 8
Complex
in H2O layer (%)
in CHC13 layer (%)
5
6
7a
>98%
<1%
ca. 35%
<2%
>99%
ca. 65%
can be explained by the excimer formation among several bpy
molecules in silica gel and in EtOH at high concentrations.
In our work, hydrophilic 1 (100 μM) exhibited an emission
maximum at ca. 390 nm with a small shoulder at ca. 340 nm,
and 5 (50 μM formed from 100 μM 1 and 100 μM 4a)
exhibits mainly two emission maxima at ca. 380 and at ca. 500
nm in a single aqueous phase, as shown in Figure S9 in
Supporting Information.23 As described in the Introduction,
2:2 complexes of Zn2L and Bar2− (5, 6, and 7a) include two
bpy units that are arranged in a parallel manner due to the π−π
stacking interaction, which possibly induce an excimer
emission at ca. 500 nm, as shown in Figure 9c,d. These facts
support the formation and distribution of 7a in H2O and
CHCl3 layers, as shown in Figure 7d and 9c,d. It is also
suggested that 2 and 3 themselves are present in the form of
aggregates in CHCl3 layers, which exhibit excimer emission
(Figure 9b).
Figure 9. (a, b) Emission spectra of 1, 2, and 3 in aqueous layer (a)
and in CHCl3 layer (b) (correspond to samples of Figure 8a,b,
respectively) at 25 °C. (c, d) Emission spectra of 5, 6, and 7a in
aqueous layer (c) and CHCl3 layer (d) at 25 °C (correspond to
samples of Figure 8c,d, respectively) (excitation at 293 nm) (a.u. is
arbitrary unit).
from the organic layer to the aqueous layer also appear to be
important factors.
These facts allow us to conclude that the supramolecular
catalyst 10 is located in both the aqueous layer and organic
layer in these systems, and its hydrophobicity/hydrophilicity
balance is a key factor for the catalytic hydrolysis of MNP. The
efficient extraction of MNP from the aqueous layer to the
organic layer and the successful release of inorganic phosphate
CONCLUSION
■
In conclusion, we report on the formation of supramolecular
complexes 10 by the 2:2:2 self-assembly of 3, functionalized
Bar2− units (4a−f), and Cu2+ as well as their catalytic activity
J
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Notes
for the hydrolysis of a phosphate monoester, MNP. The
findings indicate that the hydrolysis of MNP by 10a−f (20 μM
in the total solution, 0.2−0.02 equiv vs MNP) in a two-phase
solvent system proceeds catalytically. Moreover, the hydrolysis
of MNP by these supramolecular complexes obeys Michaelis−
Menten kinetics in two-phase solvent systems, and the
calculated Km values for 10a−f are smaller as well as Ki of
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants-in-aid from the Ministry of
Education, Culture, Sports, Science and Technology (MEXT)
of Japan (No. 17K0826 for Y.S. and Nos. 24640156,
15K00408, 16K10396, and 17K08225 for S.A.) and “Academic
Frontiers” project for private universities: matching funds from
MEXT, and the Tokyo University of Science (TUS) fund for
strategic research areas. We wish to acknowledge Ms. T.
Matsuo (Research Institute for Science and Technology, TUS)
for the elemental analysis. We appreciate the aid of Mrs. F.
Hasegawa (Faculty of Pharmaceutical Sciences, TUS) and Mrs.
N. Sawabe (Faculty of Pharmaceutical Sciences, TUS) for
2−
HPO4 values are higher than those for 8 and 9. In addition,
the overall CTNs of 10a−f are ∼3−4, and their Km/Ki values
(4.5−4.8) are close to that of AP (2.3) and much smaller than
those of noncatalytic supramolecular complexes 8 and 9 (27−
36). To the best of our knowledge, this is the first example of
the formation of artificial catalysts by the self-assembly of three
components (3, Bar2− blocks (4a−f) and Cu2+) (or four
components, i.e., Zn2+, 21 (L3), 4a−f, and Cu2+) for the
catalytic hydrolysis of a phosphate monoester, MNP, in two-
phase solvent system. This work along with our previous works
and mechanistic studies based on the distribution of
supramolecular complexes suggest that the hydrophilicity/
hydrophobicity balance of the complexes is important for the
catalytic hydrolysis of phosphate monoester. The results
reported here should be useful in the future design of stable,
biologically active, and biocompatible supramolecular com-
plexes.
It is well-established that catalytic reactions using phase
transfer catalysts in two-phase solvent systems (liquid−liquid,
liquid−solid, etc.) are powerful methods for organic synthesis,
especially for asymmetric synthesis using chiral phase-transfer
catalysts.24 However, kinetic aspects and detailed mechanisms
of these reactions are yet to be studied. The information
described in this manuscript might be important in terms of
understanding the mechanism of the catalytic activity of
natural enzymes such as AP and that of artificial catalysts
including chiral phase-transfer catalysts.
One of other remaining questions in this work is the
stereochemistry of 10a−f. In this work, we considered that
10a−f can exist in both a cis form, in which their two C22 alkyl
side chains are oriented in the same direction, and in a trans
form, in which the two C22 alkyl groups are oriented in the
opposite directions, in the reaction mixtures (Scheme 1,
Scheme 4, and Figure 2b). The design and synthesis of new
Zn2+ complexes that can be used to answer this question as
well as the design and synthesis of Bar2− units equipped with
functional groups for achieving higher NMP hydrolysis activity
are currently underway.
1
measurement of mass spectra and H NMR spectra.
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.8b03586.
Possible mechanism of MNP hydrolysis, pH rate profile,
UV/vis titration data, UV/vis spectra, Liveweaver−
Burke plots, estimated distributions of 5, 6, 7a, 8, 9, and
10a (PDF)
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: shinaoki@rs.noda.tus.ac.jp. Phone: +81-4-7121-3670.
ORCID
Shin Aoki: 0000-0002-4287-6487
K
DOI: 10.1021/acs.inorgchem.8b03586
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