Assembly of the active center of organophosphorus hydrolase in metal-organic frameworks: Via rational combination of functional ligands

Article abstract of DOI:10.1039/c7cc06270b

Different from popular mimics of bimetallic nuclear centers bridged by a hydroxide, a total coordination sphere of the active center of organophosphorus hydrolase was assembled in metal-organic frameworks by rational design and combination of ligands, which resulted in efficient destruction of nerve agent stimulants without a base as a co-catalyst.

Full text of DOI:10.1039/c7cc06270b

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc3N@C2n (2n 68 and 80). Synthesis of the
first methanofullerene derivatives of Sc N@D5h-C80
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Assembly of the active center of organophosphorus hydrolase in
metal–organic frameworks via rational combination of functional
ligands
Received 00th January 20xx,
Accepted 00th January 20xx
Mengfan Xia,†a Caixia Zhuo,†a Xuejuan Ma,a Xiaohong Zhang,a Huaming Sun,b Quanguo
Zhai,∗b Yaodong Zhang∗a
DOI: 10.1039/x0xx00000x
www.rsc.org/
Different from popular mimics of bimetallic nuclear center bridged
by a hydroxide, total coordination sphere of the active center of
organophosphorus hydrolase was assembled in metal–organic
frameworks by rational design and combined ligands,
which resulted in efficient destruction of nerve agent stimulants
without a base as co-catalyst.
Acute toxicity of organophosphorus nerve agents that contain
phosphonate ester bonds is associated with their capability to
bind to acetylcholinesterase. This bond causes neuromuscular
paralysis and organ failure.1 Recent abuse of nerve agents,2
especially in military events in Syria and assassination in
Malaysia, resulted in development of effective strategies for
rapid detection and destruction of these chemical warfare
agents (CWAs)3 (Fig. 1a).
In nature, organophosphorus hydrolase (OPH, EC 3.1.8.1)
decomposes phosphate ester bonds with high activity.4 OPH
active sites comprise a pair of Zn2+, bridged by a hydroxo
ligand, one Zn2+(α) center with His55 (Pseudomonas diminuta,
Protein Data Bank (PDB) code 1HZY5), His57 and Asp301, and
another Zn2+(β) with His201 and His230 (Fig. 1b ; ESI,† Fig. S1).
A plausible catalytic cycle of OPH based on results in
experimental and computational chemistry exhibits synergistic
effects of bimetal nuclear center and amino acid residues.6
However, high cost and fragile nature of enzymes hinder their
industrial applications.1,3 The structure of an enzyme–product
complex reveals the critical role of terminal hydroxide
nucleophiles in the mechanisms of bacterial OPH and other
Fig.1 (a) Molecular structure of three organophosphates. (b) Coordination environment
of bimetallic active site region for OPH. Extraction from structure of OPH (PDB code
1HZY) using Autodock 4.2. For clarity, bridged hydroxide and carboxylated lysine are
not shown. (c) Structural formula of ligands of BDIB and BPTC.
binuclear
zinc
enzymes.7
Binding
of
paraoxon
(organophosphate, a nerve agent stimulant) with OPH (1HZY)
was docked using Autodock 4.2,8 and the lowest binding
energy measured −4.49 kcal/mol (ESI,†Fig. S1b).
a.Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, Key
Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education,
School of Chemistry and Chemical Engineering
Shaanxi Normal University
Changan West Road 620, 710119, Xi’an, China
E-mail: ydzhang@snnu.edu.cn; Fax: +86-29-81530727;Tel: +86-29-81530726
b.Key Laboratory of Macromolecular Science of Shaanxi Province, Key Laboratory of
Applied Surface and Colloid Chemistry, Ministry of Education
E-mail: zhaiqg@snnu.edu.cn
† These authors contributed equally to this work.
Electronic Supplementary Information (ESI) available.See DOI: 10.1039/x0xx00000x
A wide variety of enzymes mimic catalytically degraded
CWAs, including many coordination complexes,9 porous
organic polymers,10 metal oxides,11 and metal–organic
frameworks(MOFs).12 Most of these artificial enzymes with
high OPH-like activity originate from mimicry of bimetallic Zn2+
center. Structural motif of binuclear metal center was
detected in the amidohydrolase superfamily of enzymes,
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which include urease, adenosine deaminase, atrazine imidazole group of histidine and construct the bimetallic
chlorohydrolase, and dihydroorotase.13 However, urease and center (Fig. 1c, see Supporting Information for synthesis and
OPH do not catalyze degradation of organophosphates and characterization details, ESI,† Scheme S1 and Fig. S2-S5). The
urea, respectively.14 Synergistic effects of functional groups carboxyl groups of biphenyl-3,3’,5,5’-tetracarboxylic acid
(i.e., His55, His57, His201, His230, Asp301, Lys169, and (BPTC) might mimic the function of carboxyl group in Asp.
bimetallic Zn2+) in the active center of OPH are essential for These two ligands were rationally combined and thus resulted
their high catalytic efficiency. Therefore, the presence of the in a novel MOF, Zn(BDIB)0.5(BPTC)0.5·3DMF (SNNU-101, DMF =
electron-rich nucleophile, such as dialkylaminopyridine dimethylformamide), which exhibits a three-dimensional (3D)
(DAAP), was required for the efficient hydrolysis of CWAs using framework (Fig. 2a, and see Supporting Information for the
artificial OPH, which mimicked the binuclear zinc center structure of SNNU-101, ESI,† Fig. S6 and S7) with permanent
12d
bridged by a hydroxide.11b,
The coordinative attachment porosity and realizes degradation of nerve agent stimulants
with supernucleophilic DAAP, such as dimethylaminopyridine (such as paraoxon) by a convenient and efficient pathway
(DMAP), or addition of bases, such as N-methylmorpholine without using bases as co-reactants.
(NMM)11b and N-ethylmorpholine (NEM),12a,12b,12e as co-
SNNU-101 was readily synthesized by solvothermal
catalyst (Table 1) is necessary to enhance hydrolytic activity of reaction of ZnSO4·7H2O, BDIB, BPTC, and DMF at 65 °C for 72 h
these kinds of artificial enzymes to activate metal-bound water (see Supporting Information). Single-crystal X-ray diffraction
molecules by polarization,11b which is similar to the roles of (XRD) analysis showed that SNNU-101 crystallizes in a
histidines as general bases in OPH catalysis.15 Thus, practical monoclinic system called C2/c space group17 (see Supporting
applications (i.e., gas filters, protective clothing, and suits) of Information for the detailed crystallographic data and the
these materials still confront many issues.3
selected bond distances and angles, Table S1- S3). The
In this study, we focused on systematic assembly of asymmetric unit of SNNU-101 contains one Zn atom, half BDIB,
functional groups of OPH active sites in MOFs. 1,3- half BPTC, and three DMF molecules. Each Zn center connects
Bis(diimidazole-2-ylhydroxymethyl)benzene
(BDIB)
was two BPTC and one BDIB, and each BPTC links four Zn centers
designed, synthesized and characterized to model the (Fig. 2b).
Overall, zinc ions and BPTC can be regarded as 3- and 4-
connected nodes, respectively. Subsequently, SNNU-101
Table
biomimetic catalysts.
1 Comparison of phosphate ester degradation by OPH and several
displayed
a
(3,4)-connected 3D network with
a
Catalyst Co-Catalyst[a]
Conditions
Activity[c]
Ref.
(62·82·102)(62·8)2 topology. 1D channels with dimensions of
[b]
SNNU-
101
×
DENP, pH
9.0, 25 °C
Vmax=0.75
This
µM min−1
,
work
Km = 2.5 mM,
TOF= 0.058 s−1
Vmax=0.37
Crystal
1
×
DENP, pH
9.0, 25 °C
This
µM min−1
,
work
Km = 3.1 mM,
TOF= 0.014 s−1
kcat= 2100 s−1
,
16
OPH
×
DENP, pH
9.0, 25 °C
kcat/Km=4×107
M−1 s−1
12d
11b
MIL-101
DMAP[d]
DENP, pH
10.0, R.T.
DENP, pH
10.0, 45 °C
t1/2 = 5.0 h
CeO2
NPs
0.45 M
NMM
Vmax = 1.56
mM min−1
,
Km = 15.78 mM
TOF = 1.5 s−1
1/2 = 45 min
12a
12b
12e
UiO-66
0.45 M NEM
0.45 M NEM
0.40 M NEM
DMNP, pH
10.0, 60 °C
DMNP, pH
10.0, R.T.
,
t
NU-
1000
MOF-
808
t1/2 = 15 min,
TOF = 0.06 s−1
t1/2 = 0.5 min,
TOF = 1.4 s−1
DMNP, R.T.
[a]
N-methylmorpholine:
NMM;
N-ethylmorpholine:
NEM.
DMAP:
dimethylaminopyridine; [b] DENP: diethyl 4-nitrophenylphosphate; DMNP:
dimethyl 4-nitrophenylphosphate; CHES: 2-(cyclohexylamino)ethanesulfonic acid;
R.T.: room temperature. [c] Various reports on degradation of phosphate ester
using OPH mimics have been provided.3 [d] Coordinatively attaching MIL-101
with DMAP ligand. However, optimal conditions of different catalysts differ. To
date, various index, such as, kcat, t1/2, TOF, and/or V0, are used for evaluation of
catalytical activity. Catalytic efficiency of synthesized OPH mimic was evaluated
according to that of natural OPH.
Fig.2 (a) 3D porous network of SNNU-101. (b) Coordination environment of Zn2+ in
SNNU-101 (symmetry codes: #1 −x + 1, y, −z + 3/2). (c) PXRD data for SNNU-101
simulated pattern of precatalysis and in comparison with postcatalysis.
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approximately 15 Å × 10 Å were observed along the c-axis
directions. Purity and stability of SNNU-101 were confirmed
using powder XRD (PXRD) (Fig. 2c). Thermal gravimetric
analysis also demonstrated high thermal stability of SNNU-101
(ESI,† Fig. S8).
Given the risk in handling highly toxic CWAs, nerve agent
stimulants are essential tools for evaluating phosphate ester
decomposition.3,12b,18 Catalytic efficacy of SNNU-101 for
destruction of phosphate ester bonds was tested using a less
toxic
stimulant
called
paraoxon
(diethyl
4-
nitrophenylphosphate12a,12b (Fig. 3a). However, the catalytic
efficacy may be different to that of the hydrolysis of other
nerve agents. Degree of decomposition of paraoxon can be
evaluated by measuring formation of p-nitrophenoxide at 400
nm at room temperature (Fig. 3b and 3c). No appreciable
spontaneous hydrolysis of paraoxon was observed under
identical reaction conditions without SNNU-101. Base
conditions benefits substrate hydrolysis (ESI,† Fig. S9 and S10).
Given the practical use, pH = 9.0 was selected to investigate
catalytic efficiency. Steady-state kinetics of hydrolysis was
investigated, whereas SNNU-101 (1.0 mg/mL) was dispersed in
20 mM 2-(cyclohexylamino) ethanesulfonic acid (CHES) buffer
(pH = 9.0) without addition of base as co-reactants (detailed in
Supporting Information). In this case, a typical Michaelis–
Menten plot was obtained when concentration of paraoxon
solution was increased from 1 mM to 7.5 mM (ESI,† Fig. S11).
The straight line in the Lineweaver–Burk plot (Fig. 3d) suggests
that SNNU-101 follows an enzyme-like kinetics. Maximum
initial velocity (Vmax) of the SNNU-101 (1.0 mg/mL)-catalyzed
hydrolysis reaction of paraoxon measured 0.75 µM/min.
Michaelis constant Km reached 2.5 mM . Turnover frequency
value (TOF) totaled 0.058 S−1. The ordered channels or cavities
in MOFs can offer the hydrophobic confined environment
similar to natural enzymes and provide a highly density of
substrate accessible active catalytic sites. This catalysis level
surpassed efficiency of OPH mimics based on zinc(II) complex19
or molecularly imprinted polymer.20 Compared with several
reported catalysts, such as UiO-66,12a NU-1000,12b and
vacancy-engineered nanoceria (i.e., CeO2 nanoparticles)11b
(Table 1), SNNU-101 functions less efficiently. However, during
activation, all these catalytic reactions require addition of
Fig.3 (a) Hydrolysis reaction of paraoxon catalyzed by SNNU-101. (b) UV–Vis spectra of
the product p-nitrophenolate over time with SNNU-101. (c) Hydrolysis of paraoxon
catalyzed by SNNU-101. (d) Lineweaver–Burk plot of kinetics data of paraoxon
hydrolysis catalyzed by SNNU-101 (1.0 mg/mL SNNU-101 in 20 mM CHES, pH = 9.0
buffer solution at 25 °C; a–h= 0.5, 1, 2, 3, 4, 5, 17, and 29 h, respectively).
group with a highly symmetric structure. A binuclear metal
center with two Zn(II) ions is bridged by two ligands. Each
Zn(II) ion includes coordinatively unsaturated metal sites,
rendering this binuclear Zn(II) complex a Lewis acid and
allowing nucleophilic attack by certain nucleophiles.12d, 21
Notably, the zinc center in the SNNU-101 structure is
coordinated with two imidazole groups of BDIB and two
carboxyl groups of BPTC. In OPH, two Zn2+ ions are also
coordinated with two imidazole groups of histidine residues
and carboxyl groups of Asp or Lys, respectively. Interestingly,
one carboxyl group of BPTC forms a pseudo-coordination bond
(~2.30 Å) with Zn(II) ion in SNNU-101 (Fig. 4a). The side-chain
carboxylate of Asp301 is within hydrogen-bonding distance
from the bridging hydroxide in the active site and shuttle
protons from the active site to bulk solvent during substrate
turnover.22 However, five zinc atoms in the structure of Crystal
1
are coordinated. In this structure, each zinc atom is
coordinated with four imidazole groups and one sulfate ligand
(Fig. 4b; ESI,† Fig. S12). Thus, coordination environment of Zn2+
in SNNU-101 is more similar to that in OPH. Catalytic activity of
SNNU-101 is 47.6 times (initial rate) faster than that of Crystal
general bases, such as NEM (0.45 M)12a,12b and NMM (0.45 M)
1
( Table 1 ; ESI,† Fig. S15 - S18). These results indicate that
11b
,
which play the activation role similar to that of histidine in
OPH.15 Otherwise, catalytic efficiency becomes considerably
lower. Addition of NMM (0.45 M) can also increase
decomposition rate of paraoxon. However, enhanced catalytic
efficiency of SNNU-101 was considerably lower than those of
vacancy-engineered nanoceria11b and UiO-6612a because the
imidazole groups of BDIB possibly play a similar role.
construction of coordination environment of two metal ions is
highly important for catalytic efficiency on mimicry of OPH.
In the active center of natural OPH, two Zn2+ are bridged by
To illustrate the mechanism of catalysis, we synthesized
another complex crystal (Crystal
1 = Zn2(SO4)2(BDIB)2·6H2O)
that only uses BDIB as ligand. Crystal 117 (see Supporting
Information for the detailed crystallographic data and the
selected bond distances and angles, ESI,† Fig. S12-S14, Table
S4-S6) was synthesized using solvothermal reaction of
ZnSO4·7H2O, BDIB, and DMF at 80 °C for 48 h. The compound
crystallizes in monoclinic crystal system with P21/n space
F
ig.4 Coordination environment and structure of Zn2+ in (a) SNNU-101 and (b)
Crystal 1.
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OH− linkage, and bond length (or distance) for Znα…Znβ, Znα–
OH–, and Znβ–OH– measures 3.40, 2.00, and ~1.99 Å,
respectively.5-6, 23.Distances of Zr…Zr in zirconium-based MOFs
structures,24 such as UiO-66,12a UiO-67,25 MOF-808,12e and NU-
100012b, total 3.53, 3.53, 3.56, and 3.53 Å, respectively, and are
very close to the distance (3.40 Å) of Znα…Znβ in natural OPH.
Interestingly, all these zirconium-based MOFs exhibit high
catalytic activity. On the one hand, from the perspective of
structure–activity relationship, Zr…Zr distance in the combined
structure and hydrolysis mechanism of natural OPH favor
synergistic catalysis. On the other hand, from the crystal
structure analysis, although SNNU-101 effectively mimics
coordination environment of Znα and Znβ in the active center
of natural OPH through BDIB and BPTC, bimetallic Zn…Zn
distance reached 7.70 Å (Fig. 4a) in SNNU-101 structure, and
this condition is detrimental to the formation of hydroxyl
bridge and synergistic effects of the two metal ions. Zn…Zn
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levels than SNNU-101. When Zn…Zn distance in SNNU-101
structure is further optimized to mimic Znα…Znβ distance
(3.40 Å) in the center of natural OPH, then the bridged
hydroxo ligand may form more easily. Thus, biomimetic MOFs
are expected to be synthesized with high catalytic activity.
In conclusion, SNNU-101 is an active biomimetic catalyst
for paraoxon degradation without a base as co-catalyst (but
increased degradation occurs at higher pH). SNNU-101
performance can be attributed to functionally mimic
coordination environment and structure of binuclear metal
center of natural OPH. The paper also demonstrated the
importance of synergistic effects of functional groups on
catalytic efficiency. A new ligand was designed to modulate
Zn…Zn distance and to construct a bridged hydroxide in SNNU-
101 to further develop destruction of CWAs.
,
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(SNNU-101)
and
CCDC
1062340
(Zn2(SO4)2·(BDIB)2·6H2O) contain the crystallographic data,
which can be obtained free of charge from The Cambridge
Crystallographic Data Centre.
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This work was supported by the National Natural Science
Foundation of China (21275097 and 21671126), Fundamental
Research Fund for the Central Universities (GK201602010 and
GK201701003) and the 111 Project(B14041) of China.
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