Metalloglycosidase Mimics: Oxidative Cleavage of Saccharides Promoted by Multinuclear Copper Complexes under Physiological Conditions

Article abstract of DOI:10.1021/acs.inorgchem.0c01193

Degradation of saccharides is relevant to the design of catalytic therapeutics, the production of biofuels, inhibition of biofilms, as well as other applications in chemical biology. Herein, we report the design of multinuclear Cu complexes that enable cl

Full text of DOI:10.1021/acs.inorgchem.0c01193

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Metalloglycosidase Mimics: Oxidative Cleavage of Saccharides
Promoted by Multinuclear Copper Complexes under Physiological
Conditions
Zhen Yu,† Zechariah Thompson,† Shelby L. Behnke, Kevin D. Fenk, Derrick Huang, Hannah S. Shafaat,
and J. A. Cowan*
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ABSTRACT: Degradation of saccharides is relevant to the design of catalytic therapeutics, the production of biofuels, inhibition of
biofilms, as well as other applications in chemical biology. Herein, we report the design of multinuclear Cu complexes that enable
cleavage of saccharides under physiological conditions. Reactivity studies with para-nitrophenyl (pNP)-conjugated carbohydrates
show that dinuclear Cu complexes exhibit a synergistic effect and promote faster and more robust cleavage of saccharide substrates,
relative to the mononuclear Cu complex, while no further enhancement is observed for the tetranuclear Cu complex. The use of
scavengers for reactive oxygen species confirms that saccharide cleavage is promoted by the formation of superoxide and hydroxyl
radicals through CuII/I redox chemistry, similar to that observed for native copper-containing lytic polysaccharide monooxygenases
(LMPOs). Differences in selectivity for di- and tetranuclear Cu complexes are modest. However, these are the first reported small
multinuclear Cu complexes that show selectivity and reactivity against mono- and disaccharide substrates and form a basis for further
development of metalloglycosidases for applications in chemical biology.
accharides play a vital role in multiple biological processes,
CuI chemistry in recent studies; however, a nonphysiological
condition, such as a pH of 10.5, is usually required to facilitate
efficient glycosidase activity of these mononuclear copper
complexes.23 Herein, we report novel copper complexes
derived from the Cu-binding ligand, di(2-picolyl)amine,24
which promotes CuII/CuI redox activity under physiological
conditions (Figure 1). In addition to a mononuclear copper
complex, 1a, two dinuclear Cu complexes, 2 and 3, were
designed by use of a xylylenediamine linker, as well as a
tetranuclear copper complex, 4, that contains a diaminobenzi-
dine core to tether four copper centers. These multinuclear Cu
complexes demonstrate synergy in reactivity.25
Glycosidase activity was evaluated by use of para-
nitrophenyl(pNP)-derivatized glucose and galactose mono-
and disaccharides as model substrates (Figure S1), with
hydrogen peroxide and ascorbate as physiologically relevant
coreagents that stimulate redox activity. Following cleavage of
these substrates, the release of p-nitrophenolate results in a
distinct absorbance band that is used to monitor the reaction
progress. In the presence and absence of both ascorbate and
peroxide, but without a catalyst, no cleavage was observed
(Figure S2 and Table S1). In the presence of a catalyst and
ascorbate/dioxygen, the activity levels are ∼10%, reflecting a
1
2
Sincluding cell adhesion, cell recognition, and the host
immune response,3 while also serving a vital role in bacterial
cell walls4 and post-translational modification in eukaryotic
cells.5 Accordingly, catalysts that promote cleavage of the
glycosidic linkage under physiological conditions may be
developed into pharmaceutically active agents and as chemical
tools to investigate cellular function and pathways.3,6
Degradation of saccharides from biomass can also produce
biofuels and other saccharide derivatives for use in food or
synthetic industries.7 Conventional approaches for these
reactions include biomass hydrolysis that requires harsh acidic
conditions,8 while high temperatures and pressures are also
usually required.9 Enzymatic degradation is another option;
however, reaction condition compatibility, product inhibition,
and cost are all obstacles that have yet to be overcome.7,10,11
For these reasons there is increasing interest in the
development of milder and more efficient conditions to
promote biomass degradation.
Previously, we have developed catalyst complexes that target
DNA,12,13 RNA,14 proteins,15,16 and other antimicrobial
targets,17 especially metallopeptides that selectively promote
cleavage of L-fucose, a carbohydrate that commonly decorates
cell surfaces.18 The CuII/CuI couple is ubiquitous in copper-
containing enzymes in biological systems, especially lytic
polysaccharide monooxygenases (LPMOs) that degrade
polymeric carbohydrates via reactive oxygen species
(ROS).19−22 We were therefore motivated to develop new
metalloglycosidase mimics promoting efficient saccharide
cleavage. Some mononuclear copper complexes have been
developed to promote cleavage of saccharides through CuII/
Received: April 22, 2020
https://dx.doi.org/10.1021/acs.inorgchem.0c01193
© XXXX American Chemical Society
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Figure 1. Metal complexes used in these studies.
Table 1. Michaelis−Menten Parameters for Saccharide Cleavage
Cu complexes
saccharides
KM (mM)
kcat (min−1
)
k )
cat/KM (M−1 min−1
1a
pNP-α-D-glucose
pNP-β-D-glucose
pNP-α-D-galactose
pNP-β-D-galactose
pNP-β-D-cellobiose
pNP-β-D-lactose
pNP-α-D-glucose
pNP-β-D-glucose
pNP-α-D-galactose
pNP-β-D-galactose
pNP-β-D-cellobiose
pNP-β-D-Lactose
pNP-α-D-glucose
pNP-β-D-glucose
pNP-α-D-galactose
pNP-β-D-galactose
pNP-β-D-cellobiose
pNP-β-D-lactose
pNP-α-D-glucose
pNP-β-D-glucose
pNP-α-D-galactose
pNP-β-D-galactose
pNP-β-D-cellobiose
pNP-β-D-lactose
1.04 0.19
0.42 0.030
0.54 0.109
1.92 0.69
1.18 0.2
1.61 0.45
1.41 0.11
0.93 0.090
1.4 0.34
1.10 0.20
0.51 0.05
0.64 0.09
1.20 0.25
0.90 0.335
1.4 0.29
0.41 0.070
0.52 0.06
0.82 0.12
0.88 0.173
1.27 0.01
0.66 0.065
0.61 0.077
0.72 0.07
0.28 0.04
0.41 0.07
0.42 0.07
0.30 0.09
0.57 0.08
1.1 0.05
1.4 0.2
15.3 4.8
5.4 1.1
9.7 0.04
3.0 0.84
7.4 0.2
10.0 0.2
9.9 1.1
6.5 0.35
10.3 1.1
3.6 0.2
6.7 0.2
9.8 0.4
6.6 1.0
12.4 0.54
7.5 0.36
15.6 0.7
7.0 0.2
400 100
990 180
610 220
310 120
900 160
1000 310
10800 3500
7100 1600
8230 1900
2740 910
14500 1500
15700 2200
8760 2100
9757 3700
7630 1700
9910 1800
13000 1500
11800 1800
8400 2100
9800 430
11600 1300
16200 2200
9500 960
20600 3000
2
3
4
5.8 0.2
requirement to generate peroxide by an electron donor to
promote robust Fenton-type chemistry. All complexes
displayed Michaelis−Menten profiles, demonstrating forma-
tion of a prereaction complex with the catalyst. Complex 1a
yielded a kcat in the range of 0.3 to 0.56 min−1 for the four
monosaccharides and around 2-fold higher for the disaccharide
substrates. The kcat for Cu complexes with two copper centers,
2 and 3, increased up to 37-fold depending on the substrates
(Table 1, Figures S5 and S6). The significant increase in kcat
for the dinuclear Cu complexes most likely reflects synergism
between the copper centers in promoting the formation of
ROS or facilitate substrate binding. Alternatively, enhanced
reactivity may reflect slight shifts in the reduction potentials
between the multinuclear complexes in aqueous solutions,
although previous reports on multinuclear di(2-picolyl)amine
scaffolds only show minor change.26 Interestingly, further
addition of two more copper centers to yield a tetranuclear
copper complex exhibited only a marginal change in kcat values.
This may be due to steric hindrance or suboptimal orientation
of metal centers toward the substrate.
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Figure 2. Cleavage of pNP β-D-glucose inhibited by ROS scavengers. Cleavage rates were measured in the absence (control, normalized to 100%)
or the presence of 1% (v/v) DMSO, 10% (v/v) D2O, 5 mM KI, or 10 U SOD.
The α- and β-glycosidic linkages appear to play an important
role in defining relative kcat for Cu complexes 2, 3, and 4, but
not 1a. The Cu complexes 2 and 3 exhibit higher kcat values
toward cleavage of α substrates relative to β substrates, while 4
promotes faster cleavage of β substrates than α substrates. The
absence of this trend for copper complex 1a indicates that the
preference for a specific structural isomer over another may
require two copper centers in close proximity. The copper
centers of 4 may be positioned in a different orientation from
Cu complexes 2 and 3, which in turn results in an opposite
preference for glycosidic linkage, or the steric effect of
benzidine may undermine cleavage of the α isomer.
All Cu complexes exhibit similar KM values, ranging from 0.5
to 1 mM for monosaccharides, and as low as 0.28 mM for Cu
complex 4 toward pNP-lactose (Figure S1), reflecting a weak
interaction between the copper complex and saccharide
substrate. The incorporation of phenyl or diphenyl rings into
the Cu complexes does not improve binding affinity to the
substrate, relative to the mononuclear analogue 1a, indicating
either the absence of π−π stacking between the p-nitrophenyl
ring and aromatic linker of the Cu complexes or favorable π-
bonding with the axial C−H bonds of the saccharide moiety.27
A slight increase in catalytic efficiency is observed for Cu
complex 2 toward α- and β-galactose, and for Cu complex 4 for
lactose over cellobiose. These results suggest that the
appropriate linking of multiple copper centers can improve
cleavage selectivity against a specific structural isomer;
however, a more effective target recognition domain would
be necessary for use as a molecular probe.
that robust activity is observed using ascorbic acid and
hydrogen peroxide (potentials of −66 mV and 380 mV vs
NHE respectively),28,29 it is not expected that our synthetic
modifications would cause further deviations outside of this
electrochemical potential window. We were therefore inter-
ested in exploring if redox cooperativity can account for the
drastic increase in catalytic activity, which was assessed using
solution-phase electrochemistry. A single, irreversible reduc-
tion process is observed in the redox-inactive zinc control
complex 1b that is attributed to a ligand-centered transition,
with a cathodic peak at −794 mV and an anodic peak at −882
mV (Figure S3). In addition to the ligand-based peak, a
pronounced electrochemical signal is observed for the
mononuclear copper complex 1a with a midpoint potential
of −749 mV, while smaller signals seen at −872 and −1214
mV are attributed to impurities or oligomerization products.30
The dinuclear copper compounds 2 and 3 each also show
three quasi-reversible couples, with peaks shifted slightly from
each other and from the monometallic species (Figure S3 and
Table S2). The redox transitions of the tetranuclear compound
4 exhibit greater irreversibility, as indicated by a greater
separation between the cathodic and anodic peaks. This
suggests that there is little electronic communication between
the two dimer units, as otherwise additional peaks would be
expected. The quasi-reversible nature of compounds 2 and 3
may contribute to the enhanced reactivity over the monomer,
and the irreversibility of 4 may contribute to the absence of
enhanced reactivity over the dinuclear complexes.
To explore the mechanism of saccharide cleavage, ROS
scavengers or enhancers were added to reaction mixtures.
Since peroxide can be an intermediate generated by Cu
complexes, only ascorbate was used to facilitate copper
The reduction potential of 1a has previously been reported
as 230 mV vs RHE,26 while other similar di- and trinuclear
di(2-picolyl)amine compounds shift by 10 to 20 mV.26 Given
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reduction in order to unambiguously determine the identity of
ROS produced. D2O, which extends the lifetime of singlet
oxygen, exhibited no significant enhancement of cleavage rate
(<10%). The addition of either 1% DMSO or 5 mM KI
significantly decreased the cleavage rate of β-D-glucose by
(>90%), indicating that a hydroxyl radical and peroxide is
formed during the cleavage reaction, respectively, which is
independent of nuclearity (Figure 2a). These observations are
in line with cleavage of DNA via a hydroxyl radical
mechanism31 by previously reported coumarin-di(2-picolyl)-
amine complexes.32 Superoxide dismutase, which converts
superoxide (O2*−) to molecular oxygen and hydrogen
peroxide, was also added to determine if superoxide was a
likely candidate for saccharide cleavage. No significant
inhibition in the mononuclear compound 1a was observed;
however, for all other compounds tested, regardless of
nuclearity, the relative activity was diminished by approx-
imately 40−50%. These results suggest that hydroxyl radicals
are produced by Fenton-like chemistry, and facilitate hydrogen
abstraction from the saccharide ring, promoting cleavage and
further degradation (Figure 2b), similar to the mechanisms
employed by copper-containing LPMOs to degrade their
native substrates.22
While robust chemistry is observed, it was of interest to
determine if this was due to freely diffusible ROS or if it
resulted from close proximity of the Cu complex to the
saccharide substrate, consistent with prereaction Michaelis
complex formation, or a metal-associated mechanism. There-
fore, the rate of formation of freely diffusible ROS was
determined for each Cu complex using a CELLROX probe,
which only fluoresces in the presence of ROS. Results are
shown in (Table S3) with relatively low levels of diffusible
ROS produced in the presence of hydrogen peroxide, while
even less was observed in its absence. More interestingly, the
level of diffusible ROS generated is quite low (<1%) relative to
the amount of pNP liberated from the pNP-saccharide
(Figures S2 and S4−S7 and Tables S1 and S3). This result
is not surprising given the relatively short half-life of ROS in
solution33−35 and is consistent with the proposed reaction
mechanism.
natural enzymes at neutral pH. These findings will be applied
to the design of novel multinuclear Cu complexes for
saccharide cleavage in future studies.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.0c01193.
■
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Materials and instruments, Tables S1−S3, Figures S1−
S7 (PDF)
AUTHOR INFORMATION
Corresponding Author
■
J. A. Cowan − Department of Chemistry and Biochemistry, The
Ohio State University, Columbus, Ohio 43210, United States;
orcid.org/0000-0002-4686-6825; Phone: 614 292 2703;
Email: cowan.2@osu.edu; Fax: 614 292 1685
Authors
Zhen Yu − Department of Chemistry and Biochemistry, The
Ohio State University, Columbus, Ohio 43210, United States;
orcid.org/0000-0003-2092-9960
Zechariah Thompson − Department of Chemistry and
Biochemistry, The Ohio State University, Columbus, Ohio
43210, United States
Shelby L. Behnke − Department of Chemistry and
Biochemistry, The Ohio State University, Columbus, Ohio
43210, United States
Kevin D. Fenk − Department of Chemistry and Biochemistry,
The Ohio State University, Columbus, Ohio 43210, United
States
Derrick Huang − Department of Chemistry and Biochemistry,
The Ohio State University, Columbus, Ohio 43210, United
States
Hannah S. Shafaat − Department of Chemistry and
Biochemistry, The Ohio State University, Columbus, Ohio
43210, United States; orcid.org/0000-0003-0793-4650
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.inorgchem.0c01193
In summary, we have reported the design and character-
ization of multinuclear Cu complexes that promote cleavage of
saccharides under physiological conditions. Robust saccharide
cleavage by these Cu complexes requires the formation of ROS
through CuII/CuI redox chemistry, as peroxide and hydroxyl
radicals are the main ROSs participating in saccharide
degradation, while superoxide is also generated and used as a
cosubstrate. All Cu complexes exhibit similar KM values in the
mid-micromolar to low millimolar range, reflecting relatively
weak interaction. A synergistic effect on saccharide cleavage
was observed for compounds 2 and 3 with two copper centers,
while there is no further enhancement for complex 4.
Electrochemical characterization shows that the multinuclear
compounds lack redox cooperativity, however, there does
appear to be a synergistic effect in the rate of cleavage with
regard to having two copper centers as opposed to one. While
other natural glycosidases36−38 exhibit enhanced activity
relative to these Cu complexes, natural enzymes lose activity
at an exponential rate above pH 6.0, while activity is quite
robust for Cu complexes at physiological pH. This robust
activity at physiological pH could be beneficial depending
upon the application and further development of these
metalloglycosidases could result in activity that surpasses
Author Contributions
†These authors have contributed equally to this work
Funding
This work was supported by grants from the National Science
Foundation [CHE-1800239] to J.A.C. H.S.S. and S.L.B.
acknowledge support from the DOE Basic Energy Sciences
Physical Biosciences program (SC0018020). Both Z.Y. and
Z.T. acknowledge support from the Pelotonia Fellowship
Program.
Notes
The authors declare no competing financial interest.
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