Modular Design of G-Quadruplex MetalloDNAzymes for Catalytic C-C Bond Formations with Switchable Enantioselectivity

Article abstract of DOI:10.1021/jacs.0c13251

Metal-binding DNA structures with catalytic function are receiving increasing interest. Although a number of metalloDNAzymes have been reported to be highly efficient, the exact coordination/position of their catalytic metal center is often unknown. Here, we present a new approach to rationally develop metalloDNAzymes for Lewis acid-catalyzed reactions such as enantioselective Michael additions. Our strategy relies on the predictable folding patterns of unimolecular DNA G-quadruplexes, combined with the concept of metal-mediated base-pairing. Transition-metal coordination environments were created in G-quadruplex loop regions, accessible by substrates. Therefore, protein-inspired imidazole ligandoside L was covalently incorporated into a series of G-rich DNA strands by solid-phase synthesis. Iterative rounds of DNA sequence design and catalytic assays allowed us to select tailored metalloDNAzymes giving high conversions and excellent enantioselectivities (≥99%). Based on their primary sequence, folding pattern, and metal coordination mode, valuable information on structure-activity relationships could be extracted. Variation of the number and position of ligand L within the sequence allowed us to control the formation of (S) and (R) enantiomeric reaction products, respectively.

Full text of DOI:10.1021/jacs.0c13251

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Article
Modular Design of G‑Quadruplex MetalloDNAzymes for Catalytic
C−C Bond Formations with Switchable Enantioselectivity
Philip M. Punt, Marie D. Langenberg, Okan Altan, and Guido H. Clever*
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ABSTRACT: Metal-binding DNA structures with catalytic
function are receiving increasing interest. Although a number of
metalloDNAzymes have been reported to be highly efficient, the
exact coordination/position of their catalytic metal center is often
unknown. Here, we present a new approach to rationally develop
metalloDNAzymes for Lewis acid-catalyzed reactions such as
enantioselective Michael additions. Our strategy relies on the
predictable folding patterns of unimolecular DNA G-quadruplexes,
combined with the concept of metal-mediated base-pairing.
Transition-metal coordination environments were created in G-
quadruplex loop regions, accessible by substrates. Therefore,
protein-inspired imidazole ligandoside L was covalently incorpo-
rated into a series of G-rich DNA strands by solid-phase synthesis.
Iterative rounds of DNA sequence design and catalytic assays allowed us to select tailored metalloDNAzymes giving high
conversions and excellent enantioselectivities (≥99%). Based on their primary sequence, folding pattern, and metal coordination
mode, valuable information on structure−activity relationships could be extracted. Variation of the number and position of ligand L
within the sequence allowed us to control the formation of (S) and (R) enantiomeric reaction products, respectively.
years, the scope of accepted substrates and reaction types
could be greatly enhanced.12−15
INTRODUCTION
■
Proteins have evolved over billions of years for countless
processes, including regulatory, structural, and catalytic
functions. Roughly half of the proteins need transition-metal
cations for their correct folding and function. Which purpose
this metal serves largely relies on its redox properties, spin
state, and Lewis acidity, fine-tuned by highly conserved
coordination environments.1
Owing to their high efficiencies and selectivities, metallo-
enzymes form the basis of numerous biotechnological
applications.2 The development of strategies to improve and
redesign metalloenzymes consisting of a protein scaffold and a
metal cofactor is a vibrant research field. For anchoring metal
cofactors inside an engineered protein, different covalent and
non-covalent strategies have been established: (a) coordination
of an unsaturated metal complex,3 (b) metal substitution,4 (c)
supramolecular anchoring, e.g., with a high-affinity tag,5 and
(d) covalent immobilization.6 In this way, a vast number of
artificial enzymes has been created to catalyze reactions such as
oxime formation, carbene transfer, cyclopropanation, imine
reduction, and ring-closing metathesis.5−9
With respect to transition-metal-containing oligonucleotides
with rate-enhancing function, a major challenge remains
anchoring the catalytic center in a precise and predetermined
position. While proteins feature several amino acids to
coordinate or tether a metal complex, the four canonical
nucleobases of DNA offer only limited possibilities. As a
solution, chelate ligands were bound to DNA via covalent or
non-covalent interactions. In this way, peroxidase-like reactions
and ZnII- and CeIV-dependent site-specific RNA and DNA
cleavage were established.16−18 Roelfes and Feringa pioneered
the design of CuII complexes that bind to double-stranded
DNA for enantioselective transformations such as Diels−Alder
reactions, Michael additions, and Friedel−Crafts reac-
tions.19−23 In recent times, also DNA G-quadruplexes were
discovered for chiral catalysis, and a first example was reported
in 2010 by Moses et al. They showcased an enantioselective
Diels−Alder reaction by planar CuII chelates, non-covalently
Received: December 22, 2020
Published: February 25, 2021
In the past decades, a growing interest developed to expand
the concept of engineered or artificial metalloenzymes to
catalytically active oligonucleotides. In the context of single-
stranded DNA sequences that catalyze the cleavage or ligation
of other nucleic acids, the term “DNAzyme” (portmanteau of
“DNA” and “enzyme”) was coined in the 1990s.10,11 Over the
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stacked on a terminal G-quartet.24 Later, Wang and Li
demonstrated Friedel−Crafts reactions, cyclopropanations,
and other transformations. Most interestingly, they observed
a case of switchable enantioselectivity, depending on the
electrolyte composition, containing either Na+ or K+.25−29
Whereas previous approaches were based on non-covalently
̈
bound metal complexes, Jaschke et al. introduced covalently
connected bipyridine ligands into G-quadruplexes to catalyze
Michael additions in good to excellent enantiomeric excesses
(ee). Interestingly, enantioselectivity could be reversed by
changing the position.30
Although high efficiencies and selectivities were obtained in
these studies, the exact localization/orientation of the metal
center with respect to the oligonucleotide secondary structure
remained either out of direct control or even largely unknown,
making deeper systematic studies a challenging task. In
contrast, the field of “metal-mediated base-pairing”, describing
the incorporation of metal cations in precisely defined DNA
coordination environments by replacing hydrogen-bonded
base interactions, opened a new level of structural control
over such bio-artificial hybrids owing to its systematic
development in the past 20 years. Certain combinations of
canonical nucleobases as well as DNA-incorporated artificial
surrogates were shown to allow selective binding of metal
cations such as HgII, CuII, MnII, AgI, ZnII, and more,31−34
culminating in programmable mixed-metal wires with potential
application in nanotechnology.35
Recently, the concept was expanded from duplex DNA to
higher structures, such as three-way junctions,36 triplex DNA,37
and i-motifs.38 Our lab has focused on G-quadruplexes,39,40
assembling from guanine-rich sequences via Hoogsteen base-
pairing within π-stacked G-tetrads, stabilized by central cations,
usually Na+ or K+.41,42 We reported the first example of a
metal-mediated tetramolecular G-quadruplex in 2013.39 This
was later expanded to unimolecular G-quadruplexes, and the
concept was applied for the design of CuII-based EPR-distance
rulers and CuII-responsive DNAzymes with peroxidase
activity.43−46
Previously, we used unimolecular G-quadruplexes as
templates for the incorporation of glycol-based imidazole
ligandoside L to design tailored coordination environments for
different transition-metal cations (Figure 1).47,48 L was
covalently incorporated as a glycerol nucleic acid (GNA)
building block in both enantiomeric forms (R/S, LR/S) in the
DNA backbone by replacement of a G-tetrad or loop bases
(Figure 1). Here we show how the concept can be used for the
rational design and optimization of metalloDNAzymes
catalyzing a CuII-dependent Michael addition with tunable
enantioselectivity.49
Figure 1. (a) Synthesis of imidazole ligandoside L. Conditions: (1)
DMT-Cl, Et3N in CH2Cl2; (2) NaH in DMF, 40 °C; (3) CEDIP-Cl,
DIPEA in CH2Cl2, rt; (4) solid-phase DNA synthesis. Green,
adenosine; blue, thymidine; gray, guanosine; and red, L.
Table 1. Screening of Different G-Quadruplex-Forming
a
Sequences with 0−6 Counts of L
sequence
CuII only
N-Me-imidazole (4 equiv)
LS (4 equiv)
L
conversion (%)
ee (%)
−
−
4
0
2
3
4
6
23 ( 1)
30 ( 1)
24 ( 3)
10 ( 1)
28 ( 2)
28 ( 6)
10 ( 1)
0
0
0
1 (S) ( 3)
5 (S) ( 7)
31 (S) ( 1)
31 (R) ( 1)
25 (R) ( 2)
0
htel22
htelLS A
2
htelLS A
3
htelLS A
4
htelLS
6
a
Conditions: 120 μM G-quadruplex, 100 μM CuSO4, 10 mM HEPES
pH 8, 100 mM KCl, 100 mM DMM, 1 mM MA1, and 1% v/v
DMSO, 5 °C, 3 d. All experiments were performed in duplicate. The
reported error is the standard deviation from two experiments.
details).36 Indeed, CuII complexes of htelLS A and htelLS A
2
3
were found to catalyze the reaction as compared to unmodified
htel22, however, showing low conversions and enantio-
selectivities. Pleasingly, opposite product enantiomers were
formed (htelLS A: 31% ee (S); htelLS A: 31% ee (R)),
RESULTS AND DISCUSSION
■
Sequence Design and Initial Screen. Our design
followed two considerations. First, a coordinatively unsaturated
CuII center was deemed necessary to enable substrate
activation, limiting the number of L to ≤3. Second, proximity
of all incorporated ligands was crucial to enable CuII chelation.
Based on the sequence of the human telomeric repeat htel22
(AGG GTT AGG GTT AGG GTT AGG G; L replaces
2
3
illustrating the potential to design tailored metalloDNAzymes
for accessing both enantiomers without having to reverse
stereochemistry in the DNA backbone. To test whether
coordinatively unsaturated CuII was mandatory, we screened a
series of sequences containing ≥4 counts of LS, indeed all
showing poor conversion and ee values.
Altered Sequences for Optimized Catalytic Perform-
ance. Encouraged by these results, a series of sequences with
two or three LS in loops 1 and 3 was synthesized, keeping a
constant loop length of three bases. Catalytic studies unveiled
crucial structure−function relationships for certain groups of
underlined nucleotides), we designed sequences htelLS A and
2
htelLS A with the S-enantiomer of L (LS) incorporated into
3
loops 1 and 3 (Figure 1, Supporting Information (SI) Table
S2). To test our concept, we performed Michael additions of
acceptor MA1 with dimethyl malonate (DMM, see Table 1 for
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sequences. One group containing three LS, one in loop 1 and
two in loop 3, separated by one base (htelLS B, htelLS H-M),
Circular Dichroism (CD) Studies. To shed light on the
influence of the electrolyte, G-quadruplexes htelLS D and
3
3
3
was identified to efficiently catalyze the Michael addition with
enantioselectivities of up to 96% ee (R) and conversions >80%
htelLS G were investigated by CD spectroscopy (Figure 2). In
2
(Table 2 and SI). The best-performing sequence, htelLS B
3
Table 2. Conversion and Enantioselectivities of Different G-
a
Quadruplex Sequences
LS@
loop 1
LS@
loop 3
conversion
(%)
sequence
salt
ee (%)
htelLS F
2
2
2
2
2
2
1
1
1
1
1
1
0
0
0
0
0
0
2
2
2
2
2
2
KCl
KCl
KCl
NaCl
NaCl
NaCl
KCl
KCl
KCl
75 ( 2)
69 ( 4)
73 ( 1)
78 ( 7)
61 ( 2)
67 ( 2)
92 ( 8)
80 ( 7)
97 ( 2)
45 ( 2)
20 ( 1)
22 ( 7)
46 (S) ( 1)
29 (R) ( 1)
4 (S) ( 1)
2
htelLS G
2
htelLS H
2
htelLS F
71 (S) ( 2)
67 (S) ( 1)
61 (S) ( 1)
96 (R) ( 3)
91 (R) ( 1)
91 (R) ( 3)
55 (R) ( 1)
68 (R) ( 1)
52 (R) ( 4)
2
Figure 2. CD spectra of htelLS D (left) and htelLS G (right) in the
3
2
htelLS G
absence (black) and presence (red) of CuII. Conditions: 2 μM G-
quadruplex, 2 μM CuSO4, 10 mM HEPES pH 8, 100 mM K/NaCl.
2
htelLS H
2
htelLS B
3
htelLS D
Na-containing solution, both sequences show the typical CD
signature of an antiparallel G-quadruplex with a positive
Cotton effect around ∼295 nm. Addition of CuII had only
minor effects on the CD signature. In contrast, with KCl used
3
htelLS J
3
htelLS B
NaCl
NaCl
NaCl
3
htelLS D
3
htelLS J
as electrolyte, the CD signature of copper-free htelLS D has a
3
3
a
For further data, see the SI. Conditions: 120 μM G-quadruplex, 100
significantly different appearance, showing a striking similarity
to the spectrum of a G-quadruplex in a (3 + 1) topology with
two G-tetrads.50,51 Here, addition of CuII leads to quite
dramatic CD spectral changes. In accordance with our
previously reported observations for a related pyridine-
functionalized G-quadruplex,43 we propose a copper-induced
structural change toward a more pronounced antiparallel
μM CuSO4, 10 mM HEPES pH 8, 100 mM KCl/NaCl, 100 mM
DMM, 1 mM MA1, and 1% v/v DMSO, 5 °C, 3 d. All experiments
were performed in duplicate. The reported error is the standard
deviation from two experiments.
(96% ee (R)), contained besides LS only thymine in loops 1
topology with three G-tetrads to be the reason. For htelLS D,
and 3, while less active quadruplexes htelLS H-M contained
3
3
this can be actually expected, since metal coordination to three
ligands in two opposite loops should template the formation of
one adenine in loop 1 or 3, suggesting that the formation of
A‑T base pairs within the loop has a negative influence on the
catalytic fidelity.
an antiparallel topology. For htelLS G, a similar transformation
2
Interestingly, with respect to htelLS B, changing the position
of the CD spectrum is observed, albeit not in the same
3
magnitude. Upon addition of CuII, sequence htelLS D hence
of T18 and LS19 in loop 3 to LS18 and T19 led to an inversion of
3
the enantioselectivity in htelLS C (57% ee (S)), while changing
shows a strong preference to fold into the same antiparallel G-
quadruplex, in both NaCl and KCl solution, leading to
formation of the same enantiomer (R) in the Michael addition.
When comparing the obtained enantioselectivities for both
3
the position of LS17 and T18 to LS18 and T17 as in htelLS D had
3
only a minor influence on the enantioselectivity (91% ee (R)).
More drastic was the effect when LS was incorporated twice in
loop 1 and once in loop 3 (htelLS A, htelLS E, and htelLS F),
sequences, however, it is noticeable that htelLS G forms the
3
3
3
2
leading to poor conversions (<30%) and enantioselectivities
(<31% ee). Sequences with only two counts of LS were
generally showing poor ee values <50%, although for some
(S) enantiomer in the presence of NaCl (similar to other
strands containing both ligands in loop 1) but the (R) product
in KCl solution. We suggest two possible explanations: either
sequences, including htelLS F−J, good to excellent conversions
htelLS G, containing both ligands in the same loop, does not
2
2
fully convert into an antiparallel topology upon CuII
coordination (as there is no further ligand to bridge to in
another loop), or opposing enantioselectivities in the observed
cases result from two distinctive antiparallel folding motifs, one
of basket type, with parallel orientation of loops 1 and 3, and
the other of chair type, with antiparallel loop orientation.
Overall, the results suggest that sequences with three ligands
adopt rather fixed topologies, templated by the chelated CuII
cations, while structures of quadruplexes with only two ligands
(i.e., when situated in the same loop) are under only weak
control of the rather loosely coordinated metal cation and thus
more prone to changes caused by the overall sequence and
electrolyte used. This hypothesis could be further supported by
melting curve analysis and a series of native ESI-MS
experiments, demonstrating that sequences in which CuII
cations bridge ligands in opposite loops possess significantly
higher gas-phase stabilities of their cation-coordinated, folded
between 69 and 97% were observed (Table 2, SI Table S7).
Role of Electrolyte. To our surprise, this dramatically
changed when NaCl instead of KCl was used as electrolyte.
Now, sequences htelLS B, htelLS D, and htelLS H-M, each
3
3
3
containing three LS, suffered a strong decrease of both
conversion (≤65%) and ee (≤68% (R)). Instead, a group of
three G-quadruplexes (htelLS F−H) with two LS in loop 1 was
2
now found to catalyze the formation of the opposite (S)
enantiomer with fair ee (61−71%) and conversions of 61−
78%. Further sequences, also containing two LS, but both in
loop 3 or one each in loops 1 and 3, were still poorly
performing. The only exception was htelLS I, with two LS in
2
loop 3, showing a modest conversion of 43% with 44% ee (S)
in the presence of NaCl electrolyte. Although these values were
not very high, a most interesting observation was made when
KCl was used in the reaction with htelLS I, which showed a
2
switch of enantioselectivity to the (R) enantiomer (71% ee)
with a high conversion of 97%.
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forms than those containing all ligands in the same loop
(Figures S38−S42).
90 (S)) as compared to htelLS D and htelLS G (91% (R) and
3 2
67% (S)). The best-performing sequence htelLR D was
3
Comparison of Ligand Stereochemistry. Next, we
examined how the stereo configuration of L affects the
catalytic performance. Up to this point, only LS was studied.
Ligand LR was then incorporated into the previously best-
performing sequences, resulting in diastereomeric G-quad-
ruplexes whose performances are listed in Table 3. No general
analyzed by preliminary molecular dynamics (MD) simulations
(Figure 3a,b), suggesting that neighboring nucleotides are
shielding one face of the substrate for nucleophilic attack, thus
giving rise to the observed enantioselectivity.
Kinetic Investigations. After identifying the most efficient
sequences in terms of enantioselectivity, we investigated the
kinetics of the Michael addition. For this study, sequences
htelLR D, htelLR H, htelLR G, and htelLR F were chosen, as
Table 3. Influence of Ligand Configuration on Conversion
3
3
2
2
a
they show the best conversions. The concentration was
reduced to 12 μM G-quadruplex and 10 μM CuII,
corresponding to 1% active DNAzyme. To determine v0 and
kcat, the onset of product formation was plotted against the
time and fitted via linear regression, and v0 was determined.
Dividing v0 by the concentration of the active DNAzyme gave
kcat.
and Enantioselectivity
sequence
LR
electrolyte
conversion (%)
ee [%]
htelLR2G
htelLR2H
htelLR2F
htelLR3D
htelLR3B
htelLR3J
htelLR3H
2
2
2
3
3
3
3
NaCl
NaCl
NaCl
KCl
KCl
KCl
92 ( 7)
93 ( 1)
95 ( 4)
94 ( 7)
97 ( 1)
96 ( 4)
95 ( 1)
90 (S) ( 1)
56 (S) ( 1)
86 (S) ( 1)
≥99 (R) ( 1)
84 (R) ( 1)
85 (R) ( 1)
68 (R) ( 1)
While for CuII alone a reaction rate v0 = 2 μM h−1 was
determined, the rate significantly increased in the presence of
htelLR H and htelLR G to v0 = 4 and 9 μM h−1, respectively
KCl
3
2
a
(Figure 4). For htelLR D, an even further increase was
Conditions: 120 μM G-quadruplex, 100 μM CuSO4, 10 mM HEPES
3
pH 8, 100 mM electrolyte, 100 mM DMM, 1 mM MA1, and 1% v/v
DMSO, 5 °C, 3 d. The reported error is the standard deviation from
two experiments.
influence on enantioselectivity was observed, as the same
trends for the LR-containing sequences were observed as with
LS. G-quadruplexes containing three ligands, one of them in
loop 1 and two in loop 3, favored the formation of the (R)
enantiomer (Figure 3d), while G-quadruplexes with two
ligands in loop 1 favored the formation of the (S) enantiomer
(Figure 3c). Two sequences (htelLR D: AGG GTLR TGG
3
GTT AGG GTLRLRGG G and htelLR G: AGG GTLRLRGG
2
GTT AGG GTT AGG G) were identified with even further
improved conversions and enantioselectivities (≥99% (R) and
Figure 4. Time-dependent onset of product formation for Cu(II)
complexes of htelLR D, htelLR H, htelLR G, htelLR F and free CuII.
3
3
2
2
Every data point is the average of two independent experiments.
Conditions: 12 μM G-quadruplex, 10 μM CuSO4, 10 mM HEPES pH
8, 100 mM KCl (three LR) or NaCl (two LR), 100 mM DMM, 1 mM
MA1, and 1% v/v DMSO, 5 °C.
observed (v0 = 15 μM h−1), but most remarkably, htelLR F
2
showed a reaction rate of v0 = 40 μM h−1, a 20-fold increase
compared to that obtained with CuII alone.
To explain these findings, we took a deeper look at the loop
compositions. For htelLR D (v0 = 15 μM h−1) and htelLR H
3
3
(v0 = 4 μM h−1), only the latter quadruplex contains an
adenine in loop 1, likely to interact with a thymine in loop 2 to
form a loose A‑T base pair. Such a hydrogen-bonded
interaction across the cavity created by the loops may hinder
substrate access to the catalytic metal site and hence be
responsible for the diminished reaction rate. This explanation
might also be valid for sequences htelLR F (v0 = 40 μM h−1)
2
and htelLR G (v0 = 9 μM h−1) where, most interestingly, the
Figure 3. (a) MD simulation of htelLR D in the presence of CuII and
2
3
only difference is an inverted sequence of loop 1 in htelLR F
substrate (red). (b) Zoom-in on the CuII site (for details, see the SI)
and scheme of G-quadruplex properties that contribute to an
enrichment of either the (c) (S) or (d) (R) enantiomer.
2
(LRLRT) as compared to htelLR G (TLRLR). We speculate that
2
only htelLR G can form an A‑T base pair with an adenine in
2
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the oppositely arranged loop 3, again leading to deceleration of
the catalytic reaction. Notably, the assumptions made herein
based on kinetic data are in good agreement with the above-
mentioned trends for conversion and enantioselectivity
dependent on the contained adenines and thymines. Such
indications for base-pairing within the loops should count as
important considerations for the optimization of sequences in
future studies.
Table 4. Conversion and Enantioselectivities for Michael
Acceptors MA1−7
a
Next, we wondered whether our system shows Michaelis−
Menten-type kinetics, a prominent feature of natural enzymes.
To accurately analyze this, we made sure that substrate MA1
was completely dissolved, allowing us to investigate a series of
CuII-htelLR D-catalyzed Michael addition experiments with
3
sequence
electrolyte substrate conversion (%)
ee (%)
increasing substrate concentrations (2.5−45 μM). For this
purpose, 10% DMSO was added, and the temperature was
increased to 25 °C (hence, the obtained reaction rates cannot
be directly compared to the values described above). The
reaction was followed by the change of absorption at 336 nm,
considering that only MA1 is contributing to the absorption at
this wavelength. From the slope, reaction rate v was calculated,
plotted against the substrate starting concentration, and fitted
using a typical Michaelis−Menten kinetics model (see SI for
details). A value of KM = 35.2 μM (vmax = 8.2 nM min−1) was
htelLR2G
htelLR2G
htelLR2G
htelLR2G
htelLR2G
htelLR2G
htelLR2G
NaCl
NaCl
NaCl
NaCl
NaCl
NaCl
NaCl
MA1
MA2
MA3
MA4
MA5
MA6
MA7
92 ( 7)
17 ( 1)
94 ( 3)
67 ( 4)
73 ( 5)
94 ( 2)
98 ( 1)
90 (S) ( 1)
77 (+) ( 1)
81 (+) ( 1)
95 (+) ( 1)
90 (+) ( 2)
89 (+) ( 2)
71 (+) ( 3)
#
htelLR3D
htelLR3D
htelLR3D
htelLR3D
htelLR3D
htelLR3D
htelLR3D
KCl
KCl
KCl
KCl
KCl
KCl
KCl
MA1
MA2
MA3
MA4
MA5
MA6
MA7
94 ( 7)
34 ( 2)
90 ( 8)
74 ( 8)
59 ( 9)
99 ( 1)
97 ( 3)
≥99 (R) ( 1)
75 (−) ( 3)
98 (−) ( 1)
86 (−) ( 6)
98 (−) ( 1)
99 (−) ( 1)
90 (−) ( 1)
determined for the reaction catalyzed by CuII-htelLR D (SI,
3
#
Figure S28a, black curve), while no Michaelis−Menten
behavior could be observed for the reaction catalyzed by free
CuII cations.
Screen of Substrate Scope. To show the generality of the
approach, the two sequences featuring the highest observed
enantioselectivities, htelLR G and htelLR D, were investigated
a
Conditions: 120 μM DNA, 100 μM CuSO4, 10 mM HEPES pH 8,
100 mM electrolyte, 100 mM DMM, 1 mM substrate, and 1% v/v
DMSO, 5 °C, 3 d. All experiments were performed in duplicate. The
reported error is the standard deviation from two experiments. 4% v/
2
3
for transforming a wider scope of substrates, involving
electron-rich and -poor as well as bulky Michael acceptors
(Table 4). Substrate MA2, with a nitro group in the para
position, was shown to poorly react, and conversions of only
17% (htelLR G) and 34% (htelLR D) were observed. Both the
#
v DMSO.
CONCLUSIONS
2
3
■
A new modular approach for the rational design of CuII-
dependent metalloDNAzymes was introduced. The modifica-
tion of htel22 with imidazole-based ligandoside L led to the
design of highly efficient CuII-based catalysts for the enantio-
selective Michael addition. Iterative rounds of screening and
sequence design unveiled crucial structure−function relation-
ships, enabling the optimization of DNAzymes for obtaining
both enantiomeric products. For the (S) enantiomer, two
ligands have to be incorporated in loop 1, while for the (R)
enantiomer, one ligand is placed in loop 1 and two in loop 3.
Besides, the electrolyte composition was also found to control
enantioselectivity.
very low solubility of substrate MA2 and electron-withdrawing
character of the −NO2 group could be responsible for this
result. A deactivating effect was also observed for substrate
MA5, carrying another electron-withdrawing substituent
(−CF3), for which low conversions of 73% (htelLR G) and
2
59% (htelLR D) were obtained, but with good enantio-
3
selectivities of 90% (+) (htelLR G) and 98% (−) (htelLR D).
2
3
In agreement with these results, electron-rich substrates MA3
(4-OCH3) and MA6 (4-CH3) yielded higher conversions,
especially for substrate MA6, with conversions of >94% for
both sequences and excellent enantioselectivities of up to 99%
(−). A bulky tert-butyl group in MA4 resulted in a lower
Interestingly, the stereoconfiguration of the ligand itself (LS
reactivity, but interestingly, with htelLR G, the best enantio-
2
and LR) had only a minor impact, although htelLR G (92%
2
selectivity of 95% (+) was observed among all substrates. For
MA7, where N-methyl-imidazole was replaced with pyridine,
excellent conversions of >97% for both sequences were
obtained, but also a decrease of their enantioselectivities to
71% (+) (htelLR G) and 90% (−) (htelLR D). Interestingly,
conv., 90% ee (S)) and htelLR D (94% conv., ≥99% ee (R))
3
were identified as the best-performing sequences. The general
applicability of the approach was shown for a larger substrate
scope, including ones with electron-poor and -rich as well as
bulky substituents and a pyridine. For most of the investigated
substrates, good to excellent conversions with high enantio-
selectivities were observed, although electron-withdrawing
substituents led to lower reactivities. Kinetic studies identified
2
3
for all investigated substrates, again htelLR G enriched the first-
2
eluting (+) enantiomer, while htelLR D enriched the (−)
3
enantiomer. Although the absolute stereoconfiguration was not
individually determined for products resulting from MA2−7,
this suggests that the G-quadruplex properties governing the
formation of (R) or (S) enantiomers with MA1 are the same
for all other investigated substrates.
htelLR F as the most efficient DNAzyme in the presence of
2
CuII cations, accelerating the reaction 20-fold compared to that
with free CuII. For htelLR D, Michaelis−Menten kinetics were
3
elucidated, showing vmax = 8.2 nM min−1 with a corresponding
3559
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J. Am. Chem. Soc. 2021, 143, 3555−3561

Journal of the American Chemical Society
pubs.acs.org/JACS
Article
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KM = 35.2 μM. The herein introduced system serves as a
versatile platform for the development of novel metallo-
DNAzymes, enabling the systematic optimization of catalysts
for a variety of reactions. Required oligonucleotides contain a
synthetically simple modification and are readily accessible via
automated DNA synthesis.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c13251.
■
sı
Synthetic procedures, NMR, MS, UV−vis, CD, and
HPLC data, and MD simulations, including Figures S1−
S47 and Tables S1−S15 (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Guido H. Clever − Faculty of Chemistry and Chemical
Biology, TU Dortmund University, 44227 Dortmund,
Germany; orcid.org/0000-0001-8458-3060;
Email: guido.clever@tu-dortmund.de
(17) Golub, E.; Albada, H. B.; Liao, W.-C.; Biniuri, Y.; Willner, I.
Nucleoapzymes: Hemin/G-Quadruplex DNAzyme−Aptamer Binding
Site Conjugates with Superior Enzyme-like Catalytic Functions. J. Am.
Chem. Soc. 2016, 138, 164.
(18) Huang, P. J.; Rochambeau, D.; Sleiman, H. F.; Liu, J. Target
Self-Enhanced Selectivity in Metal-Specific DNAzymes. Angew. Chem.
2020, 132, 3601.
(19) Marek, J. J.; Singh, R. P.; Heuer, A.; Hennecke, U.
Enantioselective Catalysis by Using Short, Structurally Defined
DNA Hairpins as Scaffold for Hybrid Catalysts. Chem. - Eur. J.
2017, 23, 6004.
(20) Boersma, A. J.; Feringa, B. L.; Roelfes, G. Enantioselective
Friedel−Crafts Reactions in Water Using a DNA-Based Catalyst.
Angew. Chem., Int. Ed. 2009, 48, 3346.
Authors
Philip M. Punt − Faculty of Chemistry and Chemical Biology,
TU Dortmund University, 44227 Dortmund, Germany
Marie D. Langenberg − Faculty of Chemistry and Chemical
Biology, TU Dortmund University, 44227 Dortmund,
Germany
Okan Altan − Faculty of Chemistry and Chemical Biology, TU
Dortmund University, 44227 Dortmund, Germany
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c13251
Notes
(21) Roelfes, G.; Feringa, B. L. DNA-Based Asymmetric Catalysis.
Angew. Chem., Int. Ed. 2005, 44, 3230.
The authors declare no competing financial interest.
(22) Park, S.; Matsui, H.; Fukumoto, K.; Yum, J. H.; Sugiyama, H.
Histidine-Conjugated DNA as a Biomolecular Depot for Metal Ions.
RSC Adv. 2020, 10, 9717.
(23) Mansot, J.; Lauberteaux, J.; Lebrun, A.; Mauduit, M.; Vasseur,
J.-J.; Marcia de Figueiredo, R.; Arseniyadis, S.; Campagne, J.-M.;
Smietana, M. DNA-Based Asymmetric Inverse Electron-Demand
Hetero-Diels−Alder. Chem. - Eur. J. 2020, 26, 3519.
(24) Roe, S.; Ritson, D. J.; Garner, T.; Searle, M.; Moses, J. E.
Tuneable DNA-based Asymmetric Catalysis using a G-quadruplex
Supramolecular Assembly. Chem. Commun. 2010, 46, 4309.
(25) Wang, C.; Jia, G.; Zhou, J.; Li, Y.; Liu, Y.; Lu, S.; Li, C.
Enantioselective Diels−Alder Reactions with G-Quadruplex DNA-
Based Catalysts. Angew. Chem., Int. Ed. 2012, 51, 9352.
(26) Hao, J.; Miao, W.; Cheng, Y.; Lu, S.; Jia, G.; Li, C.
Enantioselective Olefin Cyclopropanation with G-Quadruplex DNA-
Based Biocatalysts. ACS Catal. 2020, 10, 6561.
(27) Wang, C.; Li, Y.; Jia, G.; Liu, Y.; Lu, S.; Li, C. Enantioselective
Friedel−Crafts Reactions in Water Catalyzed by a Human Telomeric
G-quadruplex DNA Metalloenzyme. Chem. Commun. 2012, 48, 6232.
(28) Wang, C.; Jia, G.; Li, Y.; Zhang, S.; Li, C. Na + /K + Switch of
Enantioselectivity in G-quadruplex DNA-based Catalysis. Chem.
Commun. 2013, 49, 11161.
ACKNOWLEDGMENTS
■
Funded by the Deutsche Forschungsgemeinschaft (DFG,
German Research Foundation) under Germany’s Excellence
Strategy, EXC 2033-390677874-RESOLV.
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