Superactivity of MOF-808 toward Peptide Bond Hydrolysis

Article abstract of DOI:10.1021/jacs.8b01902

MOF-808, a Zr(IV)-based metal-organic framework, has been proven to be a very effective heterogeneous catalyst for the hydrolysis of the peptide bond in a wide range of peptides and in hen egg white lysozyme protein. The kinetic experiments with a series of Gly-X dipeptides with varying nature of amino acid side chain have shown that MOF-808 exhibits selectivity depending on the size and chemical nature of the X side chain. Dipeptides with smaller or hydrophilic residues were hydrolyzed faster than those with bulky and hydrophobic residues that lack electron rich functionalities which could engage in favorable intermolecular interactions with the btc linkers. Detailed kinetic studies performed by ¹H NMR spectroscopy revealed that the rate of glycylglycine (Gly-Gly) hydrolysis at pD 7.4 and 60 °C was 2.69 × 10^-4 s^-1 (t_1/2 = 0.72 h), which is more than 4 orders of magnitude faster compared to the uncatalyzed reaction. Importantly, MOF-808 can be recycled several times without significantly compromising the catalytic activity. A detailed quantum-chemical study combined with experimental data allowed to unravel the role of the {Zr₆O₈} core of MOF-808 in accelerating Gly-Gly hydrolysis. A mechanism for the hydrolysis of Gly-Gly by MOF-808 is proposed in which Gly-Gly binds to two Zr(IV) centers of the {Zr₆O₈} core via the oxygen atom of the amide group and the N-terminus. The activity of MOF-808 was also demonstrated toward the hydrolysis of hen egg white lysozyme, a protein consisting of 129 amino acids. Selective fragmentation of the protein was observed with 55% yield after 25 h under physiological pH.

Full text of DOI:10.1021/jacs.8b01902

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Superactivity of MOF-808 towards peptide bond hydrolysis
Hong Giang T. Ly, Guangxia Fu, Aleksandar Kondinski, Bart Bueken, Dirk De Vos, and Tatjana N. Parac-Vogt
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b01902 • Publication Date (Web): 23 Apr 2018
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Journal of the American Chemical Society
Superactivity of MOF-808 towards peptide bond hydrolysis
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Hong Giang T. Ly,[a] Guangxia Fu,[b] Aleksandar Kondinski,[a] Bart Bueken,[b] Dirk De Vos,* [b]
and Tatjana N. Parac-Vogt* [a]
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[a] Laboratory of Bioinorganic Chemistry, Department of Chemistry, KU Leuven, Celestijnenlaan 200F, 3001 Leuven,
Belgium
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[b] Centre for Surface Chemistry and Catalysis, KU Leuven, Celestijnenlaan 200F, 3001 Leuven, Belgium
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ABSTRACT: MOF-808, a Zr(IV)-based metal-organic framework, has been proven to be a very effective heterogeneous
catalyst for the hydrolysis of the peptide bond in a wide range of peptides and in hen egg white lysozyme protein. The
kinetic experiments with a series of Gly-X dipeptides with varying nature of amino acid side chain have shown that MOF-
808 exhibits selectivity depending on the size and chemical nature of the X side chain. Dipeptides with smaller or hydro-
philic residues were hydrolyzed faster than those with bulky and hydrophobic residues that lack electron rich functionali-
ties which could engage in favourable intermolecular interactions with the btc linkers. Detailed kinetic studies performed
by 1H NMR spectroscopy revealed that the rate of glycylglycine (Gly-Gly) hydrolysis at pD 7.4 and 60 °C was 2.69 × 10-4 s-1
(t = 0.72 h), which is more than four orders of magnitude faster compared to the uncatalyzed reaction. Importantly,
½
MOF-808 can be recycled several times without significantly compromising the catalytic activity. A detailed quantum-
chemical study combined with experimental data allowed to unravel the role of the {Zr6O8} core of MOF-808 in accelerat-
ing Gly-Gly hydrolysis. A mechanism for the hydrolysis of Gly-Gly by MOF-808 is proposed in which Gly-Gly binds to two
Zr(IV) centers of the {Zr6O8} core via the oxygen atom of the amide group and the N-terminus. The activity of MOF-808
was also demonstrated towards the hydrolysis of hen egg white lysozyme, a protein consisting of 129 amino acids. Selec-
tive fragmentation of the protein was observed with 55% yield after 25 hours under physiological pH.
of metal-substituted polyoxometalate (POM) complexes.
We have shown that a series of Zr(IV)-substituted Lind-
qvist-, Keggin-, and Wells-Dawson-type POMs efficiently
hydrolyze unactivated peptide bonds in dipeptides and
oligopeptides.13 The superior reactivity of Zr(IV) over oth-
er metals including Mn(III), Fe(III), Co(II), Ni(II), Cu(III),
Y(III), La(III), Eu(III) or Yb(III) substituted in POMs has
been explained by the large coordination numbers, flexi-
ble geometries, oxophilicity and increased Lewis acidity of
Zr(IV).13a The reaction mechanism for peptide bond hy-
drolysis in these systems has been investigated in detail.
The reactions were significantly accelerated by formation
of hydrolytically active complexes in which the amine
nitrogen and amide oxygen atoms coordinate to Zr(IV),
polarizing the peptide bond and making it more suscepti-
ble for nucleophilic attack by water. In addition, the reac-
tions were also assisted by the presence of the C-terminal
carboxylate group which abstracts a proton from the at-
tacking water molecule, assisting the nucleophilic attack
of a solvent water molecule on the amide carbonyl car-
bon.14 The study of the catalytic activity of Zr(IV)-POMs
was expanded to the hydrolysis of proteins such as insulin
chain B, ovalbumin, hen egg white lysozyme, human se-
rum albumin, myoglobin and hemoglobin.15 Highly selec-
tive hydrolysis occurring mainly upstream from aspartate
residues was achieved. In addition to the direct binding to
the amide carbonyl oxygen, which results in activation of
the peptide bond towards hydrolysis, the negatively
charged Zr(IV)-substituted POMs were found to electro-
statically interact with positively charged patches on the
protein surface, resulting in selective hydrolysis of pro-
teins in mildly acidic and neutral media.
INTRODUCTION
In the quest for new efficient and selective artificial
proteases, which are of great importance for protein
structure analysis, protein engineering, and target-specific
protein-cleaving drug design,1 efforts have been directed
to using metal ions or metal complexes as Lewis-acid cat-
alysts for cleaving proteins into suitable fragments with
restored carboxylic and amine functional groups. Howev-
er, this is a challenging task due to the extreme inertness
of the peptide bond, which exhibits an estimated half-life
of up to 350 years under physiological pH and tempera-
ture.2 Certain salts of lanthanides and transition metals
can enhance the rate of peptide bond hydrolysis at neu-
tral pH.3 However, the main limitation of such approach
is the formation of gels at neutral and basic pH condi-
tions, which is often observed for many transition metal
and lanthanide ions, especially for Ce(IV) and Zr(IV). Ef-
forts to avoid gel formation have been made by adding
organic ligands to form metal complexes.4 Several metal
complexes of Ce(IV),5 Zr(IV),4 Co(III),6 Cu(II),7 Zn(II),3, 8
Pd(II),9 and Ni(II)10 have been reported to promote the
hydrolysis of amide bonds in a wide range of peptides and
proteins. Complexes of Cu(II) have been shown to hydro-
lyze the X-Ser(Thr) bond in X-Ser(Thr)-His sequences,
but their reactivity was lost at physiological conditions
due to the formation of strong metal-amide nitrogen
bonds resulting in catalytically inactive complexes.11
Moreover, complexes of Pt(II) and Pd(II) only selectively
hydrolyzed at Met and His residues at low pH values, typ-
ically below pH 2.9b, 12
In our efforts towards developing novel artificial pepti-
dases, we have recently focused our attention on the use
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Page 2 of 14
Despite the remarkable ability of Zr(IV)-substituted equilibria in solution, often resulting in formation of cata-
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POMs to cleave peptide bonds, catalyst recycling and
product purification remain problematic. The catalysts
often cause formation of precipitates in the reaction mix-
ture, thereby preventing the use of mass spectrometric
techniques to identify fragmentation patterns. Further-
more, the Zr(IV)-substituted POMs undergo complex
lytically inactive dimers. Therefore, the development of
heterogeneous catalysts that are able to hydrolyze peptide
bonds and can be easily removed from the reaction mix-
tures and recycled, would greatly advance the potential of
using artificial metalloenzymes in the fields of proteomics
and biotechnology.
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Figure 1. Combined ball-and-stick and polyhedral representation of MOF-808 (a), its functional [Zr6O4(OH)4] cluster sand-
wiched between formate and btc ligands (b) and the primary pore (c). Color code: Zr = blue, O = red, C = black, {ZrO8} = yellow
polyhedra. Hydrogen atoms have been omitted for clarity.
Metal–organic frameworks (MOFs) are a class of hybrid
inorganic–organic microporous crystalline materials,
which consist of metal ions with bridging organic
linkers.16 The appropriate combination of organic linkers
and metal clusters can result in an almost infinite number
of MOFs containing catalytically active sites located in-
side the porous framework with controlled pore sizes.17
Owing to their fascinating structures and unique proper-
ties such as nanoscale porosity, high surface area, good
thermal stability, and uniformly structured cavities,
MOFs hold great potential for a wide range of applica-
tions, especially in catalysis.18 The incorporation of Lewis
acidic Zr(IV) ions into hybrid frameworks forms various
Zr-carboxylate MOFs such as UiO-66, MOF-808, and
NU-1000.19 The Lewis-acid catalytic activity of these Zr-
MOFs has been exploited e.g. for the hydrolysis of phos-
phate ester bonds and the citronellal cyclization.20 These
studies showed excellent catalytic performance of Zr-
MOFs; however, to the best of our knowledge there is
only one recent report in which hydrolysis of a protein
has been achieved in the presence of a MOF, viz. HKUST-
1 or [Cu3(btc)2](btc= benzene-1,3,5-tricarboxylate).21 How-
ever, the authors reported that in the presence of a pro-
tein some of Cu(II) leached out into the solution suggest-
ing a limited stability of HKUST-1 under condition used
for protein hydrolysis.
interconnected by six btc linkers binding in trigonal an-
tiprism fashion (Figure 1b). The resulting MOF-808
framework features the spn topology and exhibits pores
with a diameter 18 Å, which can allow the permeation of
oligopeptides in the pores of the framework (Figure 1c).19c,
23
The remaining sites of the eight-coordinated Zr(IV)
centers are covered with terminal formate ligands that
exhibit relative lability, easily leading to generation of
Lewis acidic sites. These have been utilized for catalytic
hydrolysis of the notorious nerve agent simulant dimethyl
4-nitrophenyl phosphate (DMNP).20b In addition, Klem-
pere and coworkers have reported that the formate lig-
ands can be selectively displaced from MOF-808 by sul-
fate moieties to generate its sulfated analogue which is
catalytically active in various acid-catalyzed reactions
including Friedel−Crafts acylation, esterification, and
isomerization.24
The high stability of the {Zr6O8}-connector brick and its
coverage with formate ligands were our first motivation
to investigate in detail the reactivity of MOF-808 towards
the hydrolysis of peptide bonds in a series of dipeptides.
The effects of pH, temperature, and inhibitors on the hy-
drolysis rate are investigated. In addition, the effects of
the amino acid side chains on the hydrolysis of dipeptides
containing a glycine (Gly) residue at the N-terminus (Gly-
X) by MOF-808 are explored. Due to the highly stable
molecular Keplerate which is a promising candidate to
remain intact during the catalytic process, highly oxophi-
lic sites which can be used to coordinate a dipeptide moi-
ety as well as the unsaturated coordination of the Zr(IV)
ion, we envisioned that MOF-808 could be a stable and
superior catalyst for peptide bond hydrolysis. Insights
into the preferable binding modes that allow activation of
the peptide hydrolysis were obtained by molecular mod-
eling and DFT calculations. Finally, the ability of MOF-
808 to selectively cleave peptide bonds in a protein under
One of the {Zr6O8}-cluster-based MOFs that has recent-
ly
received
much
attention
is
MOF-808
([Zr6O4(OH)4(btc)2(HCOO)6] (btc
=
benzene-1,3,5-
tricarboxylate).22 Similarly to other MOFs based on the
{Zr6O&} cluster, MOF-808 shows excellent chemical sta-
bility, but is thermally more labile due to the lower clus-
ter connectivity as compared to for instance UiO-66. The
crystal structure of MOF-808 (Figure 1a) exhibits ideally
octahedral {Zr6O8} cluster-type building blocks that are
tetraprotonated (i.e. {Zr6(μ3-O)4(μ3-OH)4}), capped and
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neutral pH has been also demonstrated.
Journal of the American Chemical Society
The hydrolysis of HEWL (0.02 mM) by MOF-808 (3.1
1
2
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4
mg) was performed in phosphate buffer (pH 7.4). Samples
were stirred and incubated at 37 °C and 60 °C and aliquots
were taken at different time increments and analyzed by
SDS-PAGE.
EXPERIMENTAL SECTION
Materials
N,N-Dimethylformamide
(DMF),
1,3,5-
benzenetricarboxylic acid (H3btc), formic acid, acetic ac-
id, anhydrous methanol, anhydrous acetone, chloroform,
acetonitrile, ethanol and diethyl ether were obtained from
Acros Organics. D2O, DCl, NaOD, ZrOCl2·8H2O, dipep-
tides, hen egg white lysozyme (HEWL), dicarboxylic acids
(oxalic, malonic, succinic, glutaric, adipic, and malic ac-
id), and citric acid were purchased from Sigma-Aldrich.
All chemicals were purchased as pure reagent grades and
used without further purification.
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Electrophoresis
SDS-PAGE was performed on a 4% (w/v) polyacryla-
mide gel in 0.5 M Tris-HCl buffer pH 6.8 stacking gel and
a 16% (w/v) polyacrylamide in 1.5 M Tris-HCl buffer pH
8.8 resolving gel. Samples (15 µL) were supplemented with
5 µL of sample buffer and heated at 100 °C for 5 min, fol-
lowed by loading 10 µL of the resulting solution on the
gel. Page Ruler unstained low range protein ladder was
used as a molecular mass standard. An OmniPAGE elec-
trophoretic cell was combined with an EV243 power sup-
ply (both produced by Consort, Turnhout, Belgium). Ex-
periments were performed at 200 V for 1.5 h.
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Synthesis of MOF-808 and molecular Zr6-clusters
MOF-808 was prepared solvothermally by heating solu-
tions containing zirconium salt, the acid form of the or-
ganic linker and formic acid as modulating agent, as de-
scribed in literature with some modifications.22 Typically,
H3btc (3.75 mmol, 0.786 g) and ZrOCl2·8H2O (3.75 mmol,
1.209 g) were mixed in DMF (150 mL) and formic acid (150
mL). The mixture was placed in a closed 1-L Schott
DURAN bottle, which was heated at 130 °C for 48 h. Fol-
lowing synthesis, the obtained white powder was washed
by DMF twice per day for three days, followed by washing
with methanol twice per day for three days and collected
by centrifugation. The MOF-808 was activated at 150 °C
for 20 h before performing experiments. The zirconium
benzoate (Zr6(OH)4O4(OBz)12(PrOH)·4BzOH) and zirco-
nium methacrylate (Zr6(OH)4O4(OMc)12(PrOH)·3McOH)
clusters were synthesized following the procedures de-
scribed in literature.25
Gel image analysis
Proteins in SDS-PAGE gels were visualized with silver
staining and an image of each gel was taken using a Gel
Doc EZ Imager (Bio-Rad, Hercules, CA). The band per-
centage of the fragments was determined with the Bio-
Rad Image Lab software.
Adsorption studies
The adsorption was carried out at pD 7.4 and room
temperature. To solid samples of MOF-808 (3.1 mg, 2.0
µmol) in glass vials was added D2O; these mixtures were
stirred at room temperature for 30 min to finely disperse
the MOF particles. To the suspensions were then added
40 mM Gly-Gly to have 1 mL-mixtures containing 2.0
µmol of MOF-808 and 0.5 to 50.0 µmol of Gly-Gly. The
mixtures were adjusted to pD 7.4 with NaOD and were
kept at room temperature. After a certain time, the mix-
tures were centrifuged to remove the MOF before record-
ing 1H NMR spectra.
Peptide bond hydrolysis studies
The hydrolyses were performed at pD 7.4 and 60 °C. To
a solid sample of MOF-808 (3.1 mg, 2.0 µmol) in a glass
vial was added 950 µL of D2O; this was stirred at room
temperature for 30 min to finely disperse the MOF parti-
cles. To the suspension was then added peptide (50 µL of
40 mM peptide, 2.0 µmol of peptide). The reaction mix-
ture was adjusted to pD 7.4 with NaOD and was kept at
60 °C. After different time increments, the reaction mix-
ture was centrifuged at 15000 rpm for 20 min to remove
the MOF. To the solution was added 3-(trimethylsilyl)
propionic-2,2,3,3-d4 acid sodium salt (TMSP-d4) before
recording 1H NMR spectra. The effect of MOF-808
amounts ranging from 0.5 µmol to 10.0 µmol on the hy-
drolysis of 2.0 mM Gly-Gly was studied in a similar way.
Recycling experiments
In order to test the recyclability of MOF-808 as a cata-
lyst for peptide bond hydrolysis, Gly-Gly hydrolysis was
repeated five times starting from one batch of catalyst in a
glass vial. After each run, the vial was centrifuged to sepa-
rate the solution containing the unreacted substrate Gly-
Gly and the hydrolyzed products (Gly and cyclic Gly-Gly).
H2O was added to the vial and stirred for 1 h and subse-
quently centrifuged to remove H2O. This process was re-
peated four times. Next, the material was stirred in an
organic solvent (methanol, acetone, chloroform, acetoni-
trile, ethanol, diethyl ether, or N,N-dimethylformamide)
for 1 day to exchange water. This process was repeated
twice and the used MOF-808 was air dried and activated
at 150 °C for 20 h. Subsequently, the catalysis was repeat-
ed as described above.
To study the effect of mono-, di- and tricarboxylic acids
as inhibitors on the hydrolysis of Gly-Gly by MOF-808 at
pD 7.4 and 60 °C, reaction mixtures containing 3.1 mg of
MOF-808, 2.0 µmol of Gly-Gly, and 10.0 µmol of mono-
carboxylic acids or 5.0 µmol of the other acids were ad-
justed to pD 7.4 with NaOD and kept at 60 °C for 2h.
Measurements
1
The observed reaction rate constants were obtained by
fitting peptide concentrations to a first-order decay func-
tion.
Solution H NMR spectra were recorded on a Bruker
Avance 400 spectrometer and TMSP-d4 was used as an
internal reference. Powder X-ray Diffraction (PXRD) was
performed on a STOE STADI P Combi instrument in De-
bye-Scherrer geometry (Cu Kα1) using an IP position-
Protein hydrolysis studies
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Page 4 of 14
sensitive detector (2θ = 0-60°; Δ2θ = 0.03°). Fourier trans- hydrolysis of Gly-Gly at 60 °C and pD 7.4 was obtained
1
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3
4
5
6
7
8
form infrared spectra (FTIR) were recorded on a Bruker
Vertex 70 ATR-FTIR spectrometer with the RAMII Raman
module, equipped with a 1064 nm laser source. Scanning
electron microscope (SEM) images were recorded with a
Philips XL30 FEG. Before analysis, the samples were coat-
ed with 5 nm of Pt. Inductively Coupled Plasma-Optical
Emission Spectrometry (ICP-OES) was performed on a
Perkin Elmer optical emission spectrometry Optima 8300
instrument. N2 physisorption isotherm was measured on a
Micromeritics 3Flex Surface Analyzer at 77 K. Prior to
analysis, the sample was degassed for 12 h under vacuum
at 100 ℃.
(Figure 4). Table S1 summarizes the observed rate con-
stants for the hydrolysis of Gly-Gly by several Zr(IV)-
POMs (Figure S1), as reported in our previous studies.13a,
13c, 13e
These studies revealed that Zr(IV)-POM based cata-
lysts exhibit only a single active Zr(IV) center that can
facilitate the peptide bond hydrolysis. Remarkably, Gly-
Gly hydrolysis in the presence of MOF-808 was enhanced
by nearly three orders of magnitude compared to the re-
actions catalyzed by Zr(IV)-POMs under the same exper-
imental conditions. Moreover, a rate constant of 7.4 × 10-9
s-1, corresponding to a half-life of 3 years, was estimated
for the uncatalyzed hydrolysis of Gly-Gly at pD 7.2 and 60
°C.2 This indicates a rate enhancement of 3.6 × 104 times
when MOF-808 was used. In addition, in the presence of
10-fold excess of Gly-Gly (20.0 µmol) in comparison to
MOF-808 (2.0 µmol) a complete hydrolysis of the peptide
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Computational details
Density functional theory (DFT) calculations were car-
ried out with the Amsterdam Density Functional program
(ADF2016).26 Numerical integration was performed using
Becke grid integration.27 Geometry optimization was con-
ducted using GGA Becke exchange28 plus the Perdew 86
correlation29 (BP) functional and all-electron Slater basis
sets of triple-ζ quality with one polarization function
(TZP).30 Scalar relativistic effects were accounted for using
the Zeroth-Order Regular Approximation (ZORA).31 Solv-
ation effects were introduced using the COnductor-like
Screening MOdel (COSMO) with the default parameters
for water (ε = 78.39; solvent radius = 1.93 Å) where sol-
vent-excluding surface (SES) correction was included.32
Next to geometry optimizations at BP86/TZP/ZORA-
Scalar/COSMO, single-point calculations using the hybrid
B3LYP functional29 were performed to calculate different
molecular properties (bonding energies, frontier molecu-
lar orbitals and molecular electrostatic potentials) of the
different binding modes.
bond was obtained with a rate constant of 4.83 × 10-5 s-1 (t
½
= 3.98 h), indicating that the reaction is truly catalytic. To
test the heterogeneity, the reaction was stopped after 2 h;
MOF-808 was removed via centrifugation and the homo-
geneous solution was allowed to react further. The per-
centage of Gly-Gly hydrolyzed before and after the re-
moval of the catalyst is presented in Figure 5. As can be
seen, after removing MOF-808 from the reaction mixture,
no additional Gly-Gly hydrolysis was observed. This indi-
cates that the outstanding catalytic activity of MOF-808
towards peptide bond hydrolysis is exclusively associated
with catalytically active sites of the solid MOF-808. Im-
portantly, MOF-808 could be recycled and reused for the
hydrolysis of Gly-Gly for five runs by using ethanol or
methanol for washing and exchanging water before the
activation process (Figure S2). The reactivity of the indi-
vidual Zr6O4(OH)4 cluster core towards peptide bond hy-
drolysis was also tested. Benzoate- and methacrylate-
capped Zr6 clusters were synthesized and used to study
the cleavage of Gly-Gly under identical experimental con-
ditions as for MOF-808.25 However, no hydrolysis of Gly-
Gly was observed after 5 h, likely due to the coordinative-
ly saturated environment around each Zr(IV) center. The
hydrolysis of 2.0 µmol of Gly-Gly by 2.0 µmol of ZrCl4 at
pD 7.4 and 60 °C was also studied. A much slower hydrol-
ysis was obtained with an observed rate constant of 5.55 ×
10-7 s-1, which corresponds to a half-life of 14.4 days.
RESULTS AND DISCUSSION
Hydrolysis of Gly-Gly
Gly-Gly was chosen as a model dipeptide as its hydroly-
1
sis can be easily followed by means of H NMR spectros-
copy. A mixture containing 2.0 µmol of Gly-Gly and 2.0
µmol of MOF-808 was reacted at 60 °C and pD 7.4. After
different time increments the fractions of both the dipep-
tide and hydrolyzed product were determined (Figure 2).
Similar to the reaction in the presence of Zr-POMs13a, 13c
the by-product, cyclic Gly-Gly (cGly-Gly), formed by the
intramolecular condensation between the carboxylate and
the amino group was observed during the course of the
hydrolytic reaction (Scheme 1). In the absence of MOF-
808, neither Gly nor cGly-Gly were observed after 7
months at pD 7.4 and 60 °C. This indicates that MOF-808
catalyzed both the hydrolysis and cyclization processes.
Some other artificial metallopeptidases based on Ce(IV)
and Zr(IV) have been previously reported to promote the
formation of cyclic dipeptide products which were not
The hydrolysis of 2.0 mM Gly-Gly was further studied
in the presence of different amounts of MOF-808 ranging
from 0.5 µmol to 10.0 µmol. The observed rate constants
for the hydrolysis of Gly-Gly were plotted as a function of
concentration of MOF-808 (i.e. concertation of {Zr6O8}
clusters) and fitted to a pseudo-Michaelis-Menten model
(equation [1]) as shown in Figure 6. 33
13a, 13c
directly hydrolyzed into individual amino acids.5,
Figure 3 shows the percentage of Gly-Gly, cGly-Gly, and
Gly as a function of reaction time. The data show that the
fraction of cGly-Gly reached a maximum of approximately
10 % after 0.5 h reaction and then gradually decreased. An
where kobs is the observed first-order reaction rate con-
stant, kmax is the maximal observed rate constant and KM is
the Michaelis constant which represents the concentration of
MOF-808 for which kobs equals kmax/2. A maximal rate con-
observed rate constant of 2.69 × 10-4 s-1(t = 0.72 h) for the
stant kmax of 11.0 × 10-4 s-1 (t = 10.5 min) and a Michaelis
½
½
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constant KM of 5.42 mM were calculated for the hydrolysis
1
of Gly-Gly by MOF-808 at pD 7.4 and 60 °C.
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Figure 3. Percentage of Gly-Gly ( ), c(Gly-Gly) ( ), and
Gly ( ) as a function of reaction time for the hydrolysis of
2.0 µmol of Gly-Gly by 2.0 µmol of MOF-808 at pD 7.4
and 60 °C.
1
Figure 2. H NMR spectra recorded at different reaction
times during the hydrolysis of 2.0 µmol of Gly-Gly by 2.0
µmol of MOF-808 at pD 7.4 and 60 °C.
Figure 4. ln[Gly-Gly] as a function of time for the reac-
tion between 2.0 µmol of Gly-Gly and 2.0 µmol of MOF-
808 at pD 7.4 and 60 °C.
Scheme 1. Hydrolysis of Gly-Gly and its cyclization to
cGly-Gly by MOF-808.
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Figure 5. Hydrolysis of 2.0 µmol of Gly-Gly in the pres-
Page 6 of 14
1
2
ence of 2.0 µmol of MOF-808 ( ) before and ( ) after the
removal of MOF-808 (pD 7.4 and 60 °C).
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The effect of pD on Gly-Gly hydrolysis in the presence
of MOF-808 was investigated in the pD range from 4.6 to
9.8 at 60 °C. The conversion of Gly-Gly after 4 h in the
reaction mixtures containing 2.0 µmol of Gly-Gly and 2.0
µmol of MOF-808 at different pD values is presented in
Figure S3. As can be seen from this figure, the reaction
was fast over a wide pD range with the fastest reaction
occurring under physiological pH. In addition, a slight
decrease in the reaction rate at higher pD value was ob-
served. In order to check the effect of pD on the stability
of MOF-808 structure, PXRD of the MOF-808 samples
after the reaction were recorded (Figure S4). The changes
in PXRD that were observed from pD 8.4 onwards could
be a result of partial instability of MOF-808 in alkaline
conditions, resulting in a slight decrease in catalytic activ-
ity.34 At all lower pH values, the PXRD patterns give no
evidence for any structure degradation.
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Figure 6. Influence of the amounts of MOF-808 on the
rate constant for the hydrolysis of 2.0 mM Gly-Gly at pD
7.4 and 60 °C.
Analysis of the Gly-Gly adsorption in MOF-808
The sizes of the primary cages of MOF-808 (ca. 18 Å)35
are sufficiently large to accommodate dipeptide species.
In order to gain insight into the ability of MOF-808 to
capture peptides, the adsorption of Gly-Gly on MOF-808
was studied at pD 7.4 and room temperature. To avoid
the hydrolysis of Gly-Gly by MOF-808, the adsorption was
studied within 30 min at room temperature. The adsorp-
tion of 2.0 µmol of Gly-Gly on 2.0 µmol of MOF-808 as a
function of time is shown in Figure S8. The result shows
that Gly-Gly was quickly adsorbed and the adsorption of
Gly-Gly was at equilibrium after 10 min stirring at room
temperature. In order to study the capacity of MOF-808
for Gly-Gly adsorption, mixtures containing 2.0 µmol of
MOF-808 and 0.5-50.0 µmol of Gly-Gly were stirred for 30
min at pD 7.4 and room temperature. As can be seen in
Figure S9, the amount of Gly-Gly adsorbed on the solid
phase of MOF-808 increased with an increase of the ini-
tial concentration of Gly-Gly. A saturation adsorption
capacity of 1.15 mmol of Gly-Gly per g of MOF-808 (0.65
mmol of Zr6O8 cluster) was obtained.
The effect of temperature on the hydrolytic activity of
MOF-808 at pD 7.4 was investigated as well. Increasing
the reaction temperature from 37 °C to 80 °C resulted in a
25-fold increase of the reaction rate constant and a de-
crease of the half-life from 4.4 h to 0.17 h. When the data
in Figure S5 were fitted to the Arrhenius equation, an ex-
perimental activation energy parameter (Ea) of 69 ± 1 kJ
mol-1 was obtained (Figure S6). This value is significantly
higher than what would be expected if the reaction kinet-
ics were determined by a slow diffusion of the dipeptide
reactant through the MOF-808 micropores. Comparison
of the activation energy of the reaction in the presence of
the MOF to the non-catalyzed reaction is difficult due to
the extremely slow rate of the latter. However, one can
compare with the Ea values that were previously deter-
mined for Gly-Gly hydrolysis in the presence of Zr(IV)-
Keggin and Wells-Dawson POMs, and which were in the
range of 90 - 91 kJ mol−1.13a, 13c This indicates that in the
presence of MOF-808 the activation energy for the hy-
drolysis of Gly-Gly is lowered by ca. 22 kJ mol−1. Linear
fitting of ln(kobs/T) as a function of 1/T (Figure S7) allows
for the calculation of the enthalpy of activation, ∆H# = 66
± 1 kJ mol−1, and entropy of activation, ∆S# = −116 ± 1 J
mol−1 K−1. The Gibbs energy of activation (∆G#) for the
hydrolysis of Gly-Gly at 37 °C is 105 ± 1 kJ mol-1. The nega-
tive activation entropy obtained is most likely the result
of the coordination of the dipeptides to the Zr sites.
Hydrolysis of Gly-Gly-NH2 and Gly-Gly-Ala
In order to get more insight in the binding mode of
Gly-Gly to MOF-808, the hydrolysis of 2.0 µmol of Gly-
Gly-Ala and Gly-Gly-NH2 in the presence of 2.0 µmol of
MOF-808 was investigated at pD 7.4 and 60 °C. An over-
view of the chemical structures of these substrates is pre-
sented in Table 1.
1
As can be seen from the H NMR spectra of Gly-Gly-Ala
hydrolyzed by MOF-808 (Figure S10), after 5 minutes at
60 °C Gly (3.56 ppm), Ala (1.45−1.47 ppm), and Gly-Ala
(1.35−1.37 ppm) were all observed. The immediate for-
mation of these products indicates that the hydrolysis of
the N-terminal peptide bond occurred simultaneously
with the hydrolysis of the C-terminal peptide bond in this
tripeptide. After 30 minutes of reaction, the signals of the
methyl group of Ala-Gly at 1.53−1.55 ppm were clearly
seen. The formation of Ala-Gly is a result of ring-opening
of c(Gly-Ala) which is the cyclisation product of Gly-Ala
(Figure s11) formed from Gly-Ala, obtained during the
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hydrolysis of Gly-Gly-Ala.13g The percentage of the hydro-
ence of the carboxylic acid inhibitors causes a decrease in
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lyzed product Gly as a function of time is presented in
Figure S12. A rate constant kobs of 2.06 × 10-4 s-1 (t1/2 = 0.93
h) was obtained for the formation of Gly. However, the
rate constant of Ala formation cannot be calculated from
the NMR spectra due to the overlap of the methyl group
in Ala and c(Gly-Ala).
the reaction rate for the hydrolysis of Gly-Gly. The
strongest inhibition effect (ca. 90 %) was observed in the
presence of acids with short aliphatic chain lengths while
the dicarboxylic acids with longer aliphatic chains caused
a rate decrease of approximately 72 %. These nonreactive
carboxylic acids are known to act as multidentate ligands
for many metal ions,36 and therefore, the observed effect
of reduced Gly-Gly hydrolysis in the presence of these
inhibitors is likely due to a competitive coordination of
Zr(IV) to the carboxyl and hydroxyl groups. Notably, the
inhibition effect of dicarboxylic acids is ca. two-fold
stronger than that of monocarboxylic acids (Figure S14).
In addition, the inhibition trends observed here are simi-
lar to those in the presence of Zr-POMs indicating that
both carboxylate groups of diacids act in chelating with
the {Zr6O8} core.13b, c, 13e
The hydrolysis progress of Gly-Gly-NH2 in the presence
of MOF-808 is shown in Figure S13. After 5 minutes of
reaction, Gly was still below the limit of detection. Since
the signals of Gly-Gly and Gly-NH2 are overlapped, it is
impossible to identify which bond was preferentially
cleaved. However, a rate constant for the formation of Gly
out of this peptide (kobs= 0.55 × 10-4 s-1, corresponding to a
half-life of 3.5 h) could be calculated. This shows that the
conversion of Gly-Gly-NH2 to Gly is more than three
times slower than that of Gly-Gly-Ala. Obviously, when
the peptides are blocked at the C-terminal end the reac-
tion rates are lower than for the unblocked peptides.
From these results we can conclude that the terminal car-
boxylate group is directly involved in the acceleration of
the hydrolysis process when MOF-embedded Zr centers
are used.
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Table 1. Observed rate constants for the formation of
Gly from the hydrolysis of Gly-Gly-Ala and Gly-Gly-
NH2 by MOF-808 at pD 7.4 and 60 °C.
Substrate
104kobs (s-1)
t1/2 (h)
Inhibition studies
Further insight into the coordination of Gly-Gly to MOF-
808 was obtained by following the hydrolysis of Gly-Gly
by MOF-808 in the presence of several nonreactive car-
boxylic acids (Figure 7). These inhibitors have multiple
carboxylic groups which can coordinate to the oxophilic
Zr(IV), thus blocking the access of the substrate.
2.06
0.93
Gly-Gly-Ala
0.55
3.50
Gly-Gly-NH2
Figure 7. Chemical structures of the examined inhibitors
Figure 8 provides an overview of the conversion of Gly-
Gly in the presence of the inhibitors. In all cases the pres-
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Figure 8. Conversion of Gly-Gly after 2 h in the reactions Stability of MOF-808
Page 8 of 14
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containing 2.0 µmol of Gly-Gly, 2.0 µmol of MOF-808, and
5.0 µmol of the inhibitor at pD 7.4 and 60 °C.
Figure S17a represents the N2 physisorption isotherm
for the MOF-808 material used in our experiments. The
isotherm features two distinct steps in N2 uptake, repre-
senting adsorption in the different cages of MOF-808. As
shown by the calculated pore size distribution (Figure
17b), indeed both the small tetrahedral cage of ~7-10 Å in
size and the much larger adamantine-type cage (18.4 Å)
are resolved. Using the BET method, a specific surface
area of 1820 m²/g was calculated. The t-plot calculation
revealed a micropore area of 1333 m²/g and an external
surface area of 487 m²/g.
Hydrolysis of Gly-X dipeptides
The catalytic activity of MOF-808 towards dipeptides
containing different X amino acid side chains at the C-
terminus was examined at pD 7.4 and 60 °C. Considering
the potential intermolecular interactions between the btc
linkers and dipeptides and the entrance in the MOF-808
pores, we selected a group of dipeptides that differ in
terms of size, hydrophobicity index37 and additional func-
tionalities. The conversion of the selected dipeptides in
presence of MOF-808 over 2 h was plotted as a function
of the side chain volume (Figure 9). Our results show that
the catalytic hydrolysis of neutral or highly hydrophilic
dipeptides is highly dependent on the volume of the ami-
no acid side chains. Thus, their conversion decreased with
an increase in the side chain bulkiness: Gly-Gly > Gly-Ala
> Gly-Asp > Gly-Asn > Gly-Lys ≈ Gly-Arg. This is a logical
outcome as steric hindrance can hinder the access of the
amide carbonyl oxygen atom to the active {Zr6O8} core
and can potentially limit the diffusion of the dipeptides in
the pores of MOF-808. Gly-X dipeptides with clearly hy-
drophobic X residues (e.g. Val, Leu, Ile) are less reactive
for the hydrolytic conversion than dipeptides with neutral
or hydrophilic X residues. Indeed, these residues (group
II) lack electron rich functionalities and therefore cannot
engage in favourable intermolecular interactions with the
btc linkers, while their pronounced hydrophobicity im-
pacts the interaction with the active core and the diffu-
sion into the MOF-808 pores. On the other hand, reac-
tion rates are higher than expected for Met and Phe
(group I): their hydrophobic residues contain electron
rich thiol and aromatic functionalities which can facilitate
intermolecular thiol-aromatic bonding38 or π-π stacking
respectively.
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The stability of MOF-808 under the experimental con-
ditions was examined by several complementary tech-
niques including 1H NMR spectroscopy, ICP, FT-IR,
PXRD, and SEM for the samples before and after the cata-
lytic process. The abortion of the Gly-Gly hydrolysis di-
rectly after the removal of MOF-808 was the first indica-
tion that MOF-808 is not leaching Zr(IV) ions or {Zr6O8}
cores, which could also facilitate the peptide hydrolysis,
into solution. In order to confirm this, the reaction mix-
ture containing 2.0 µmol of Gly-Gly and 2.0 µmol of MOF-
808 at pD 7.4 was centrifuged after 2 h reaction at 60 °C
to remove MOF-808. The solution after catalyst removal
was used to measure ICP. The result shows that Zr(IV)
1
was below the detection limit (20 ppb). In addition, H
NMR spectra of the acid digested MOFs show that negli-
gible linker had leached from the framework after the
reaction. Moreover, FT-IR spectra and PXRD patterns of
MOF-808 before and after reaction were recorded and are
shown in Figure S15 and Figure S16. The resemblance of
FT-IR spectra and PXRD patterns before and after the
reaction further corroborates the stability of MOF-808
under the experimental conditions, even after the second
reaction cycle. Moreover, from SEM images of MOF-808
taken before and after reactions (Figure S17c), it is evident
that the MOF-808 crystals retain their octahedral mor-
phology, demonstrating that the bulk MOF-808 exhibits a
remarkable stability over the catalytic process.
The effect of organic solvents on the process of washing
MOF-808 after use was studied. Direct activation of
MOF-808 after washing with water compromises the
MOF structure due to the high surface tension of water,
yielding a poor activity.39 Therefore, several organic sol-
vents were used to wash and exchange water before acti-
vation at high temperature to avoid damage to the MOF
structure. As can be seen in Figure 10, the Gly-Gly hydrol-
ysis activity for the second reaction cycle was recovered
when acetonitrile, methanol or ethanol were used before
the activation process. PXRD indicated that MOF-808
treated with methanol after the second reaction cycle
remained unchanged (Figure S16).
Figure 9. Conversion after 2 h at pD 7.4 and 60 °C for the
hydrolysis of 2.0 µmol of Gly-X dipeptides in the presence of
2.0 µmol of MOF-808 as a function of the volume of amino
acid X.
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HOMO and a Zr-dominated LUMO (d-atomic like orbit-
1
2
als) resembling the oxo and metal band respectively typi-
cal for extended solids.41
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The replacement of the single formate ligand with sin-
gle deprotonated Gly-Gly− (= NH2CH2CONHCH2COO−)
leads to a [Zr6O8(HCOO)11(Gly-Gly)]4− model core. We
have investigated three different binding mode configura-
tions: a) mode 1 featuring Gly-Gly− binding via the two O
atoms of the terminal carboxylate ligand; b) mode 2 fea-
turing Gly-Gly− interaction with the {Zr6O8} core via two
O atoms deriving from the carboxylate and from the am-
ide functionality; c) mode 3 featuring binding via O atom
from the amide group and N atom of the N-terminus.
Relative to mode 3, mode 1 and mode 2 lay 55.3 kJ·mol-1
and 5.5 kJ·mol-1 higher in bonding energy respectively
(Table S3). The small difference in relative bonding ener-
gy especially between modes 2 and 3 suggests that the
binding modes have similar stability and therefore they
are in a close competition with each other. The energy
and the character of the HOMO in binding modes 1, 2 and
3 is similar and comparable to that of [Zr6O8(HCOO)12]4−,
which suggests that the Gly-Gly does not alter the elec-
tronic structure of the {Zr6O8} core (Figure 11). However,
depending on the binding mode, the character and the
energy of the LUMO are severely altered. In the case of
mode 1 where the binding of Gly-Gly occurs via the car-
boxylate moiety, the LUMO is virtually similar to that of
[Zr6O8(HCOO)12]4−. In binding mode 2, the LUMO is pre-
dominantly distributed over the Zr-centers, while minor
electron distribution occurs over the N and C centers of
the bound Gly-Gly moiety. Finally, mode 3 features a
LUMO predominantly distributed over the C and N atoms
of the peptide bond depicting p-atomic like orbitals ar-
ranged in an antibonding fashion along the peptide bond.
Population of this orbital will consequently destabilize
the dipeptide bond. Further, the LUMO in mode 3 is low-
er in energy than the LUMOs in mode 1 and mode 2. This
implies that upon binding in mode 3, the unoccupied an-
tibonding orbital coming from the Gly-Gly molecules is
effectively lowered.
Figure 10. Conversion of Gly-Gly after 2 h at 60 °C for the
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first (black) and second (purple) reaction cycles. Different
organic solvents were used to wash and exchange water be-
fore activation at 150 °C.
Atomistic insights into the Gly-Gly dipeptide bond
activation
In order to design a suitable model that can provide at-
omistic insights into the dipeptide bond activation, we
take into account two experimental observations: i) MOF-
808 exhibits remarkable stability during the catalytic pro-
cess; ii) the catalytic efficiency is dependent on the resi-
due-linker interactions. These two observations, includ-
ing the recent theoretical studies on MOF-808 cores by
Morris and coworkers,40 support our belief that the
{Zr6O8} core solely provides catalytic sites while the btc-
peptide residue interactions fine-tune the process effi-
ciency. Following this, the loss of a carboxylate bearing
moiety, most likely formate, exposes a pair of electro-
philic Zr(IV) centres that defines the catalytically active
site for the peptide hydrolysis process.
The small Gly-Gly molecules can interact directly with
the {Zr6O8} cores without developing significant linker-
substrate interactions that affect the interaction geome-
try. This provides us with an advantage to virtually re-
place all btc ligands by single formate ligands and thus to
focus solely on the changes of the electronic structure as a
function of the interaction geometry. The influence of
protonation of the {Zr6O8} cores was evaluated by consid-
ering
non-protonated
and
tetraprotonated
{Zr6O4(OH)4}ores.
Geometry optimizations of our first model
[Zr6O8(HCOO)12]4− at BP86 level provided a good approx-
imation of the relevant bond lengths (Zr−O and C−C) and
interatomic (Zr···Zr) distances especially when compared
to those experimentally obtained for MOF-808 (Table S2).
Single point calculation of the [Zr6O8(HCOO)12]4− model
at B3LYP level shows a high energy gap (ca. 5.7 eV) be-
tween the highest occupied molecular orbital (HOMO)
and the lowest unoccupied molecular orbital (LUMO)
(Figure 11). This is an expected feature for fully-oxidized
Zr(IV) based materials that exhibit an O-dominated
Figure 11. Frontier molecular orbitals HOMO (bottom row)
and LUMO (top row) plotted over ball-and-stick representa-
tions of the [Zr6O8(HCOO)12]4− cluster and the three binding
modes of [Zr6O8(HCOO)11(gly-gly)]4− model systems. Colour
code: Zr = light blue, O = red, C = black and H = white.
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Decoration of [Zr6O8(HCOO)12]4− with four protons ar-
Page 10 of 14
presence of MOF-808 already occurred after 1 h incuba-
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ranged in a tetrahedral fashion around the O atoms of
{Zr6O8} leads to a [Zr6(OH)4O4(HCOO)12] model that can
be subject to further formate ligand replacement with
Gly-Gly− ligands. Considering the three binding modes
(mode 1-3) of [Zr6O8(HCOO)11(Gly-Gly)]4−, six analogous
neutral isomers can respectively be constructed for
[Zr6(OH)4O4(HCOO)11(Gly-Gly)] which differ with respect
to the relative disposition of the dipeptide moiety to the
four protons of the {Zr6O8} core (Figure S18). Our optimi-
zations and single point calculations have shown that the
six binding modes are differing within 26 kJ·mol-1 (Table
S4). As the four protons bind directly to the {Zr6O8} core,
all of the six modes of [Zr6(OH)4O4(HCOO)11(Gly-Gly)]
exhibit Zr-based LUMO’s. The unoccupied MOs that ex-
hibit antibonding interactions along the peptide bond
appear lowest in energy in 3a and 3b and respectively lay
at -1.8 eV and -1.5 eV on the energy scale. (Figure S19).
Unoccupied MOs with antibonding character are also
present in the isomeric modes 1a and 1b; however they lay
above -0.5 eV on the energy scale. This shows that even
tion. After 25 h reaction ca. 55% HEWL was hydrolyzed
(Table S5), resulting in fragment bands with molecular
weights (MWs) of approximately 12.2, 10.7, 8.2, and 6.4
kDa. The MWs of the fragments suggest that the HEWL
was most likely selectively hydrolyzed at 3 peptide bonds.
The fragments at 8.2 and 6.4 kDa are likely originating
from one cleavage site as their added MW is close to the
MW of the intact HEWL. The shorter fragments that re-
sult from the cleavage at the second and the third site
(that produced larger fragment at 12.2 and 10.7 kDa) un-
fortunately could not be observed as short polypeptides
tend to leak from the SDS PAGE gel. The cleavage of
HEWL under physiological pH and temperature was also
tested (Figure S21). At 37 °C the bands appeared at the
same position in SDS-PAGE gel as observed at 60 °C, indi-
cating the presence of the fragments having the same
molecular weight. The control experiments showed that
hydrolysis of HEWL was not observed in the absence of
MOF-808 at 37 °C or even at 60 °C (Figure S21 and Figure
S22), confirming the catalytic role of MOF-808 in protein
hydrolysis. Analysis of the cleavage sites and the mecha-
nism of HEWL hydrolysis by MOF-808 are currently un-
derway. Nevertheless, these results already demonstrate
the unprecedented ability of MOF-808 to catalyze hydrol-
ysis of extremely stable peptide bonds in proteins. Two
possible hypotheses can be put forward for the interac-
tion between the HEWL protein and MOF-808. While in
theory HEWL is too large (3.5 x 2.5 x 4.5 nm³) to diffuse
through the pore windows of MOF-808 (18.4 Å) to access
the clusters located in the interior of the framework, pre-
vious work by Chen et al. has shown that partial unfold-
ing of the protein structure can enable diffusion into the
pores of a MOF, as exemplified by the uptake of Cyt c in
Tb-mesoMOF.42 Alternatively, an initial hydrolysis step
can occur on the outer surface of the octahedral MOF-808
crystals, which are terminated with (111) facets. While the
exact surface termination of MOF-808 is not known at
present, the (111) lattice plane cuts halfway through the
large pores of the MOF-808 framework, which likely re-
sults in half-spherical pockets on the surface of the crys-
tals. These are of similar size as the smallest dimension of
the HEWL protein (~1.8 nm vs ~2.5 nm) and could pro-
vide several interaction sites between Zr-nodes and the
protein (Figure s23). For the crystals of MOF-808 pre-
pared in this manuscript, approximately 1% of the clusters
are exposed on the surface. Once an initial cleavage has
occurred, it is possible that these fragments diffuse into
the crystal to undergo further hydrolysis.
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under
a
tetraprotonated scenario (i.e. having
{Zr6O4(OH)4} cores), Gly-Gly binding to the two Zr(IV)
centers via the oxygen atom from the amide group at the
N-terminus remains the most attractive coordination
mode that can facilitate the peptide bond activation and
hydrolysis.
The above discussed calculations show that the degree
of protonation does not affect the relative stability order
between the different binding modes, however it does
affects the energy difference in each individual model set.
In this context it can be very difficult to accurately calcu-
late and predict a reaction mechanism for the hydrolysis
of dipeptides and polypeptides by MOF 808. However,
the preservation of the bonding energy trends and the
calculated relative bonding energy ranges for both proto-
nated and unprotonated scenarios, hint that in aqueous
media different modes of Gly-Gly are competing with one
another to access the catalytic site of {Zr6O8} cores. Such
competition is only further augmented in the presence of
inhibitors (vide supra). From the different bonding
modes, only mode 3 can successfully promote the peptide
bond activation and hydrolysis, while similar relevance of
this type of binding mode has been outlined in our recent
work focusing on the interaction and hydrolysis of dipep-
tides with Zr(IV) substituted Keggin polyanions.14
Hydrolysis of Hen Egg White Lysozyme (HEWL) by
MOF-808
The reactivity of MOF-808 was further tested on
HEWL, a 14.3 kDa protein containing 129 amino acids.
HEWL was incubated with MOF-808 at 60 °C and subse-
quently reaction aliquots were taken at different time
intervals and analyzed by SDS-PAGE to evaluate the pro-
gressive hydrolysis of HEWL in the presence of MOF-808.
Figure 12 shows the SDS-PAGE image of HEWL hydroly-
sis. The appearance of new bands with lower molecular
weights during the course of the reaction indicates that
selective HEWL hydrolysis occurred. As can be seen in
Figure 12 and Figure S20, the hydrolysis of HEWL in the
Direct comparison between these results and the previ-
ously reported hydrolysis of bovine serum album (BSA) by
HKUST-1 (or [Cu3(btc)2] (btc
=
benzene-1,3,5-
tricarboxylate))21 is not straightforward due to different
structures of these MOFs and different reaction condi-
tions and protein substrates used in these studies. How-
ever, in order to observe efficient BSA hydrolysis, alkaline
pH conditions of up to pH = 9 were needed in the previ-
ous study. This also resulted in leaching of Cu(II) from
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H.G.T.L. and B. B thank the FWO (Research Foundation –
the framework, suggesting limited stability of HKUST-1
1
2
3
4
5
6
7
8
under the conditions used for BSA hydrolysis.
Flanders) for the postdoctoral fellowship. T.N.P.V. thanks
KU Leuven for the financial support under project
OT/13/060. T.N.P.V and D.D.V. thank FWO for the
financial support under project G095017N. A.K. thanks
FWO for the postdoctoral fellowship and the Excellence
Initiative of the German Federal Ministry of Education
and Research (StUpPD_270-17). G. F. thanks the CSC for
fellowships.
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Figure 12. Silver stained SDS-PAGE gel of HEWL hydrolysis
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ASSOCIATED CONTENT
Supporting Information
Supporting Information Available: Kinetic data for the
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Corresponding Authors
*tatjana.vogt@kuleuven.be
*dirk.devos@kuleuven.be
ACKNOWLEDGEMENT
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