Terpyridine-Cu(II) targeting human telomeric DNA to produce highly stereospecific G-quadruplex DNA metalloenzyme

Article abstract of DOI:10.1039/c5sc01381j

The cofactors commonly involved in natural enzymes have provided the inspiration for numerous advances in the creation of artificial metalloenzymes. Nevertheless, to design an appropriate cofactor for a given biomolecular scaffold or vice versa remains a challenge in developing efficient catalysts in biochemistry. Herein, we extend the idea of G-quadruplex-targeting anticancer drug design to construct a G-quadruplex DNA metalloenzyme. We found that a series of terpyridine-Cu(ii) complexes (CuLn) can serve as excellent cofactors to dock with human telemetric G-quadruplex DNA. The resulting G-quadruplex DNA metalloenzyme utilising CuL1 catalyzes an enantioselective Diels-Alder reaction with enantioselectivity of >99% enantiomeric excess and about 73-fold rate acceleration compared to CuL1 alone. The terpyridine-Cu(ii) complex cofactors demonstrate dual functions, both as an active site to perform catalysis and as a structural regulator to promote the folding of human telemetric G-quadruplex DNA towards excellent catalysts.

Full text of DOI:10.1039/c5sc01381j

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Terpyridine-Cu(II) Targeting Human Telomeric DNA to Produce
Highly Stereospecific G-quadruplex DNA Metalloenzyme†
Yinghao Li,ab Mingpan Cheng,ab Jingya Hao,ab Changhao Wang,a Guoqing Jia*a and Can Li*a
Received 00th January 20xx,
Accepted 00th January 20xx
The cofactors commonly involved in natural enzymes have provided the inspiration for numerous advances in the creation
of artificial metalloenzymes. Nevertheless, to design an appropriate cofactor for a given biomolecular scaffold or vice versa
remains a challenge in developing efficient catalysts in biochemistry. Herein, we extend the idea of G-quadruplex-targeting
anticancer drug design to construct a G-quadruplex DNA metalloenzyme. We found that a family of terpyridine-Cu(II)
complex can serve as an excellent cofactor to dock with human telemetric G-quadruplex DNA. The resulting G-quadruplex
DNA metalloenzyme catalyzes an enantioselective Diels-Alder reaction with enantioselectivity over 99% enantiomeric
excess and about 73-fold rate acceleration compared to terpyridine-Cu(II) complex alone. The terpyridine-Cu(II) complex
cofactor demonstrates dual functions, both as an active site to perform catalysis and as a structural regulator to promote
the folding of human telemetric G-quadruplex DNA towards excellent catalysts.
DOI: 10.1039/x0xx00000x
www.rsc.org/
Considerable efforts have been made to develop cofactors for
G-quadruplex DNA, including a Cu(II)-bipyridine complex and
its analogs by Moses et al.,32 Cu(II) ions by this group,33-36 and
Introduction
Enzymes, the catalysts of biological systems, can mediate a
wide range of chemical reactions with high catalytic rates and
perfect regio-/stereoselectivity.1,2 Cofactors usually play a
crucial role in this process.1 Metal complexes are one
important type of cofactor that occurs in many natural
proteins to mediate various reactions.3 In a biomimetic spirit,
catalytic metal complexes have also been widely used as
cofactors to combine covalently or non-covalently with
biomacromolecules, such as proteins, apo-enzymes and
antibodies, to construct artificial metalloenzymes.4-8 In
particular, the chiral feature of biomacromolecules makes
artificial metalloenzymes an ideal catalyst for enantioselective
reactions.9-12
Cu(II) cationic porphyrin by Hennecke et al.37 However, these
G-quadruplex metalloenzymes only provide modest
enantioselectivity for enantioselective reactions, which is most
possibly due to the case that these cofactors cannot fit well
with the G-quadruplex DNA binding pocket, and consequently
the resulting catalytic centers are not able to provide suitable
interactions with prochiral substrates to promote efficient
enantioselectivity. Therefore, to develop a suitable cofactor
with the best fit to the binding pocket provided by G-
quadruplex DNA is a key issue in constructing efficient artificial
G-quadruplex metalloenzymes.
Through the lens of molecular biology, G-quadruplex DNAs
are attractive targets for the development of anticancer
drugs.38,39 Small molecules capable of inducing and stabilizing
G-quadruplex DNA structure in human telomeres and
oncogene-promoter regions have shown therapeutic
intervention in cancer.40-43 It is reported that a family of
terpyridine-metal complexes shows good affinity and high
selectivity for human telomeric G-quadruplex DNA.44,45
Inspired by this specific recognition, in this work we have
employed terpyridine-Cu(II) complexes (CuLn) as cofactors to
DNA has served as a new biological scaffold in constructing
artificial metalloenzymes for enantioselective catalysis.12-14
Roefles and Feringa et al. found the cofactor Cu(II)-(4,4
′-
dimethyl-2,2 -dipyridyl) could be most compatible with
′
double-stranded DNA (dsDNA).15 The resulting artificial dsDNA
metalloenzyme proceeds with excellent enantioselectivity in a
series of enantioselective reactions.16-26 Recently, G-
quadruplex DNA, due to its diverse and tunable chiral
structures,27-31 has become another candidate in constructing
DNA metalloenzymes for enantioselective reactions.
construct
human
telomeric
G-quadruplex
DNA
metalloenzymes. Our study shows that the resulting human
telomeric G-quadruplex DNA-CuL1 can afford excellent
catalytic function in a Diels-Alder reaction with both high
enantioselectivity over 99% enantiomeric excess (ee) and
about 73-fold rate increase compared to CuL1 alone. To the
best of our knowledge, this is the most efficient G-quadruplex
DNA metalloenzyme for Diels-Alder reaction reported so far.
Circular dichroism (CD) spectroscopic analysis, UV melting
a.State Key Laboratory of Catalysis, Dalian Institute of Chemical Physics, Chinese
Academy of Sciences, Dalian 116023, China. E-mail: canli@dicp.ac.cn;
gqjia@dicp.ac.cn.
b.University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing, 100049,
China.
†
Electronic Supplementary Information (ESI) available: Experimental details,
supplementary figures and tables, 1H-NMR spectra and HPLC traces. See DOI:
10.1039/x0xx00000x
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experiment and isothermal titration calorimetry (ITC) further
indicate that the terpyridine-Cu(II) cofactor not only functions
as a catalytic center but also plays an important role in
inducing and stabilizing human telemetric G-quadruplex DNA
with a catalytically active three-dimensional structure. Based
on this proof-of-concept experiment, it is envisioned that
much broader G-quadruplex metalloenzymes could be found
by exploring G-quadruplex-targeting ligand pool.
Results
Terpyridine-Cu(II) complex targeting G-quadruplex DNA to
produce artificial metalloenzyme
Inspired by the G-quadruplex DNA targeting nature of
terpyridine-metal complexes, we synthesized the terpyridine-
Cu(II) complex (CuL1) and employed CuL1 to directly interact
with a human telomeric DNA sequence (HT21) (Fig. 1a). CD
spectroscopic analysis show that, upon interaction of CuL1
with HT21, an obvious spectral change in the region of 220-310
nm is observed, accompanied by a new CD band ranging from
310-370 nm (Fig. 1b). The spectral region of 310-370 nm
originates from the electric transition of the terpyridine-Cu(II)
core.46 Therefore, the new CD band (310-370 nm) can be
readily ascribed to the induced circular dichroism (ICD) signal
of CuL1 imposed by the chiral surroundings of HT21, which is
also direct evidence of the CuL1-HT21 association. The CD
spectrum of 220-310 nm is usually indicative of the G-
quadruplex DNA structure.29-31 However, it is complicated in
this system by the overlapping absorption band of CuL1. To aid
in understanding the CD results, UV melting experiments were
performed (Fig. 1c). The melting temperature (Tm) of HT21
increases by approximately 13 °C upon interaction with CuL1,
which clearly indicates that the conformation of HT21 is
significantly stabilized by the HT21-CuL1 complex formation. In
this respect, the CD spectral change in the region of 220-310
nm contains much information on the structural transition of
HT21. Moreover, the increased Tm clearly indicates that this
structural transition occurs from a looser state to a more
stable G-quadruplex DNA conformation. This deduction is
further supported by the ITC experiment (Fig. 1d and 1e),
which can determine the molecular nature of the binding
interaction from a thermodynamic perspective.47 The binding
isotherms reveal a complex binding process and can be best fit
Fig.
1
Construction of human telomeric G-quadruplex DNA metalloenzyme. (a)
Schematic diagram of molecular recognition between terpyridine-Cu(II) complex (CuL1)
and human telomeric DNA sequence (HT21: 5′-(GGGTTA)3GGG)-3′). (b) CD spectra. (c)
UV melting curves monitored by UV absorption at 295 nm. (d, e) Isothermal titration
calorimetry (ITC) experiment. Raw ITC data is shown in panel (d); Plots of integrated
calorimetric data after control subtraction (solid square), fitting curve (solid line) with a
three-event binding model, and corresponding thermodynamic parameters are
included in panel (e).
Upon confirming that CuL1 can directly induce and stabilize
the HT21 G-quadruplex structure to form a stable HT21-CuL1
complex, we next select the Diels-Alder reaction between aza-
chalcone (1a) and cyclopentadiene (2) as a model to test the
catalytic performance of HT21-CuL1. As shown in Table 1,
HT21-CuL1 exhibits much higher catalytic performance than
CuL1 (entry 2 vs. entry 1), and catalyzes the reaction with 92%
conversion (turnover number (TON)
=
9.2), high
diastereoselectivity of product 3a (endo/exo of 97:3) and 90%
ee of the major endo isomer in 24 hours (entry 2). Although
these catalytic data were modest compared with the results
with a dsDNA-based metalloenzyme,15 this result gives the
best enantiomeric outcome concerning G-quadruplex DNA-
based catalysis. This finding thus proves that CuL1 can act as
an effective cofactor for the construction of a human telomeric
G-quadruplex DNA metalloenzyme. More interestingly,
combining the structural analysis and catalytic data, it is
obvious that the CuL1 cofactor not only serves as a catalytic
center but also shows structural regulation of human
telomeric G-quadruplex DNA to promote excellent catalysis.
by a three-event model (Fig. 1e and Table S1†
).48,49 The high-
affinity binding site allows the interaction of one CuL1
molecule with HT21 by an affinity constant of approximately
108 M-1. Thermodynamic parameters further show that this
strong association process is driven by a highly favorable
enthalpy and a less unfavorable entropic contribution. The
favorable enthalpy can be linked to either Hoogsteen
hydrogen bond formation or association between CuL1 and
HT21, which contributes to the structural regulation of HT21
from a looser state to a more stable G-quadruplex DNA
conformation, whereas the unfavorable entropy change is
mainly ascribed to decreased freedom from both the compact
G-quadruplex DNA structure and the bound state of CuL1.50
Structural optimization of G-quadruplex DNA in artificial
metalloenzyme
In both natural and artificial metalloenzymes, subtle
modifications of the secondary coordination sphere of the
metal center usually exert a substantial influence on the
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Table
1 Diels-Alder reaction catalyzed by human telomeric G-quadruplex DNA
metalloenzyme
endo
exo
O
∗
∗
∗
O
N
N
∗
∗
∗
HT G4-DNA
CuL1
∗
∗
+
+
O
N
1a
2
3a
DNA
ee (%,
endo)b
0
Entry
M+
Conv.(%)a endo/exob
Sequence (5′→3′)
1c
-
-
-
42
92
71/29
97/3
HT21:
2
90
(GGGTTA)3GGG
Conformation stabilized by monovalent cations
3
4
5
Na+
K+
54
48
99
89/11
88/12
99/1
54
49
94
HT21:
(GGGTTA)3GGG
+
NH4
Flanking base variants
A-HT21:
A(GGGTTA)3GGG
TA-HT21:
+
6
7
NH4
NH4
NH4
NH4
NH4
NH4
97
96
98
97
95
95
98/2
98/2
98/2
97/3
96/4
96/4
90
94
93
88
81
73
+
+
+
+
+
TA(GGGTTA)3GGG
TTA-HT21:
8
TTA(GGGTTA)3GGG
HT21-T:
9
(GGGTTA)3GGGT
HT21-TT:
10
11
(GGGTTA)3GGGTT
HT21-TTA:
Fig. 2 CD spectra of HT21 in the presence of different monovalent cations and their
corresponding metalloenzyme. (a) 50 mM NaCl; (b) 150 mM KCl; (c) 30 mM NH4Cl (DNA
strand concentration, 5 µM; CuL1, 10 µM; MOPS buffer, 20 mM, pH 6.5).
(GGGTTA)3GGGTTA
a Determined for the crude product by HPLC analysis on a chiral stationary phase
(Supplementary Methods Note 5), Reproducible within ±2%. b Determined by
was selected to stabilize HT21 G-quadruplex DNA, the HT21-
+
NH4 -CuL1 metalloenzyme demonstrated
c
chiral-phase HPLC. Reproducible within ±2%. Cu(L1)(NO3)2 alone as catalyst.
a
substantial
Reaction conditions: 1a (1 mM), 2 (10 uL, 260 mM), human telomeric G-
quadruplex DNA (50 μM), Cu(L1)(NO3)2 (100 μM), NaCl (50 mM) or KCl (150 mM)
or NH4Cl (30 mM), MOPS buffer (0.5 mL, 20 mM, pH 6.5), 4 oC, 24h.
increase in catalytic activity compared with HT21-CuL1,
showing 99% conversion (TON
diastereoselectivity (endo/exo
=
9.9) and excellent
99:1), and the
of
enantioselectivity for the major endo product 3a increased to
94% ee (Table 1, entry 5). For a comparison of intrinsic
catalytic activity, we performed an enzymatic dynamics study.
catalytic function of the metalloenzyme.12,51 For the HT21-
CuL1 metalloenzyme, this phenomenon provides an
opportunity to improve the catalytic performance by varying
the human telomeric G-quadruplex DNA structure. For this
+
The Michaelis-Menten kinetics showed that the HT21-NH4 -
CuL1 metalloenzyme reaches a catalytic efficiency (kcat/KM) of
36±4 M-1s-1 (kcat = 0.0015±0.0001 s-1, KM = 41±3 µM), which is
approximately 73-fold more active than CuL1 (0.49±0.06 M-1s-
+
purpose, monovalent cations M+ (Na+/K+/NH4 ) were
introduced to induce HT21 to form a G-quadruplex structure in
advance,52 and then CuL1 was added to construct an artificial
human telomeric G-quadruplex DNA metalloenzyme,
represented by HT21-M+-CuL1.
1) (Fig. S1
†). The rate acceleration in this system is comparable
+
to dsDNA-based catalysis.53 At the same time, HT21-NH4 -CuL1
metalloenzyme shows 10-fold rate increase compared to
The CD spectra of HT21-Na+-CuL1 (Fig. 2a) and HT21-K+-CuL1
(Fig. 2b) indicate that CuL1 has less structural regulation for
HT21 G-quadruplex DNA (220-310 nm), and the ICD signal for
HT21-CuL1 (Fig. S1†). Based on the above results, it is
concluded that NH4+ ion can function as an efficient chaperone
for the HT21 G-quadruplex DNA metalloenzyme to create a
favorable modification of HT21 G-quadruplex DNA structure,
which can help the CuL1 cofactor to fit well with the binding
pocket within HT21 G-quadruplex DNA and consequently
promote the Diels-Alder reaction.
+
CuL1 (310-370 nm) is also obscure. However, for HT21-NH4 -
CuL1, substantial structural change of HT21 G-quadruplex DNA
(220-310 nm) and a clear ICD signal (310-370 nm) are observed
(Fig. 2c). In addition, HT21-M+-CuL1 shows distinct catalytic
behaviors for the Diels-Alder reaction compared with HT21-
CuL1 (Table 1, entries 3-5 vs. entry 2). HT21-Na+-CuL1 shows
an obvious decrease in both catalytic activity (conv. = 54%,
TON = 5.4) and enantioselectivity (54% ee) (Table 1, entry 3). A
similar case holds for HT21-K+-CuL1, with 48% conversion (TON
Under optimized experimental conditions in NH4+ media, we
further modified the HT21 G-quadruplex structure by
introducing 1∼3 flanking bases at the 5’ or 3’ end. These two
positions were selected because the binding of terpyridine-
Cu(II) complex with human telomeric G-quadruplex DNA
+
= 4.8) and 49% ee (Table 1, entry 4). Surprisingly, when NH4
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Structural optimization of cofactor structure in artificial
metalloenzyme
Having validated the optimization of the second coordination
sphere around Cu(II) by tuning the G-quadruplex structures,
we further optimized the CuLn cofactor by tuning the
terpyridine ligand, namely the first coordination sphere
around CuII (Table 2). It is found that high catalytic activity and
high enantioselectivity are not specific to CuL1 as a cofactor
but are also obtained for other terpyridine-Cu(II) complexes
possessing aminoalkyl side chains (CuLn, n=2-4) (Table 2,
entries 1-4). In contrast, when the side chain (R) was changed
+
from an aminoalkyl to a methyl group (CuL5), the HT21-NH4 -
CuL5 metalloenzyme showed substantial declines in both
catalytic activity (conv.
=
37%, TON
=
3.7) and
enantioselectivity (18% ee) (Table 2, entry 5). With further
+
removal of the aromatic ring (CuL6), the HT21-NH4 -CuL6
Fig. 3 Inversion of global chirality of G-quadruplex DNA metalloenzyme. (a) Natural D-
DNA building block for HT21 and its enantiomer for L-HT21. (b) The Diels-Alder reaction
(1a and 2) catalyzed by HT21 and L-HT21 G-quadruplex DNA metalloenzymes. (c) CD
metalloenzyme led to only 25% conversion (TON = 2.5) and a
complete inhibition of enantioselectivity (Table 2, entry 6).
These catalytic data show that both the aminoalkyl side chain
and the aromatic ring are key components for the terpyridine-
Cu(II) cofactor to construct an efficient human telomeric G-
quadruplex DNA metalloenzyme.
+
spectra of HT21 and L-HT21 in NH4 media (30 mM NH4Cl). (d) CD spectra of HT21 G-
quadruplex DNA metalloenzyme and L-HT21 G-quadruplex DNA metalloenzyme.
suggests that the aromatic surface around the copper could
interact with human telomeric G-quadruplex DNA by
π-π
stacking on the external G-quartet.44 Therefore, the flanking
bases at the 5’ or 3’ end are also expected to modify the
To clarify the underlying reason, we further focused on
structural understanding of the HT21-NH4 + -CuLn
metalloenzymes. We first used CD spectroscopy to detect the
+
microenvironment of the CuL1 cofactor in the HT21-NH4 -CuL1
+
interaction of HT21-NH4 with terpyridine-Cu(II) complexes
metalloenzyme. For the Diels-Alder reaction between 1a and
possessing different side chains (Fig. 4). In comparison with
+
HT21-NH4 itself, all test terpyridine-Cu(II) cofactors
2, these derivative human telomeric G-quadruplex DNA
metalloenzymes present an interesting effect on the
enantioselectivity (Table 1, entries 6-11). The sequences with
flanking bases at the 5’-end have little effect on
demonstrate structural regulation on the HT21 G-quadruplex
DNA structure (220-310 nm), but the degree of structural
change is strongly dependent upon the type of side chain (R).
At the same time, the ICD signals of the terpyridine-Cu(II)
cofactor (310-370 nm) imposed by HT21 G-quadruplex DNA
+
enantioselectivity (90-94% ee) compared with HT21-NH4 -CuL1
(Table 1, entries 6-8 vs. entry 5). In contrast, a negative effect
on enantioselectivity can be observed when the flanking bases
are at the 3’-end (Table 1, entries 9-11 vs. entry 5). These data
clearly indicate that the CuL1 cofactor is more sensitive to the
Table 2 The ligand effect of terpyridine-Cu(II) cofactor on the catalytic function
of human telomeric G-quadruplex DNA metalloenzyme
3’-end of HT21 G-quadruplex DNA, and
a crowded
microenvironment at the 3’-end is not favorable for high
enantioselectivity.
R =
N
R
O
L1
N
O
The above data strongly suggest that the conformation of
L4
N
N
N
O
O
HT21 G-quadruplex DNA plays
a dominant role in the
N
N
Cu2+
L2
L3
N
CH3
enantioselective control. Therefore, based on the high level of
+
enantioselectivity afforded by HT21-NH4 -CuL1, we further
CuL6
N
N
Cu2+
O
L5
CuLn
considered whether we could selectively access either
enantiomer in the Diels-Alder reaction by tuning the HT21 G-
quadruplex DNA conformation. To this end, mutation was
performed by replacing the naturally occurring D-DNA with L-
DNA (Fig. 3a). CD spectra show that L-HT21 forms a G-
quadruplex structure that is a mirror image of natural HT21
(Fig. 3c). More importantly, upon assembly with CuL1, the
ee (%,
endo)b
94
Entry
Ligands
Conv.(%)a
endo/exob
1
2
3
4
5
6
L1
L2
L3
L4
L5
L6
99
99
99
95
37
25
99/1
98/2
93
97/3
92
98/2
93
+
81/19
68/32
18
resulting L-HT21-NH4 -CuL1 metalloenzyme also presents the
+
mirror image structure of natural HT21-NH4 -CuL1 (Fig. 3d). For
6
+
, L-HT21-NH4 -CuL1 can
a Determined for the crude product by HPLC analysis on a chiral stationary phase
(Supplementary Methods Note 5), Reproducible within ±2%. b Determined by
chiral-phase HPLC. Reproducible within ±2%. Reaction conditions: 1a (1 mM), 2
(10 uL, 260 mM), HT21 (50 μM), Cu(Ln)(NO3)2 (100 μM), NH4Cl (30 mM), MOPS
buffer (0.5 mL, 20 mM, pH 6.5), 4 oC, 24h.
the Diels-Alder reaction of 1a and
2
successfully switch the absolute configuration of the major
product 3a from 94% ee to -92% ee (Fig. 3b).
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Table 3 The binding thermodynamic parameters of different cofactors (CuLn) to human telomeric G-quadruplex DNA in 30 mM NH4Cl at 298K
High-affinity binding site
Low-affinity binding site
Cofactor
a
a
a
a
a
a
Ka1 (M-1)
n1
ΔH1
-TΔS1
-14.3
ΔG1
Ka2 (M-1)
n2
ΔH2
-TΔS2
ΔG2
CuL1b
CuL2b
CuL3b
CuL4b
CuL5c
CuL6d
(1.7±0.2)E7
(1.7±0.3)E7
(1.7±0.3)E7
(1.7±0.3)E7
(2.0±0.5)E5
-
1.0
1.1
1.1
1.2
2.5
-
4.4±0.1
4.2±0.3
6.7±0.2
4.2±0.1
-1.1±0.1
-
-9.9
-9.8
-9.9
-9.9
-7.2
-
(6.6±0.5)E5
(1.0±0.1)E6
(1.1±0.1)E6
(5.8±0.6)E5
-
5.0
5.8
6.8
5.4
-
-2.2±0.1
-2.1±0.1
-1.5±0.1
-2.5±0.1
-
-5.7
-6.1
-6.8
-5.3
-
-7.9
-8.2
-8.3
-7.8
-
-14.0
-16.6
-14.1
-6.1
-
-
-
-
-
-
a Units are kcal•mol-1. b The data were obtained by the two-event binding model. c The data were obtained by the one-event binding model. d The affinity between the
reactants is too low, neither the affinity nor the enthalpy of binding can be reliably determined.
+
also vary with different side chains. In detail, HT21-NH4 -CuLn ICD signal is also obscured (Fig. 4c). The ICD signal in the
(n=1-4) metalloenzymes with terpyridine-Cu(II) cofactors wavelength region 310-370 nm originates from the electric
possessing aminoalkyl side chains show similar CD spectral transition of the terpyridine-Cu(II) core, which is common to all
features, with clear structural regulation of the HT21 G- six CuLn cofactors. Thus, the distinct amplitude of the ICD
+
quadruplex DNA (220-310 nm) accompanied by the signal in HT21-NH4 -CuLn indicates a different molecular
appearance of a strong ICD signal of the CuLn cofactors (Fig. orientation of the terpyridine-Cu(II) chromophore relative to
4a). Upon changing the cofactor to CuL5 (Fig. 4b), the the surrounding nucleobases,54 which gives direct evidence of
+
structural transition of HT21 G-quadruplex DNA is also different recognition between CuLn and HT21-NH4 G-
+
observed but shows a distinct CD feature from the HT21-NH4 - quadruplex DNA. More interestingly, a positive correlation
CuLn (n=1-4) metalloenzyme. Moreover, HT21-NH4+-CuL5 between catalytic performance and amplitude of ICD signal
+
exhibits a smaller magnitude of ICD signal compared with indicates that the catalytic function of HT21-NH4 -CuLn
HT21-NH4+-CuLn (n=1-4). Upon changing to CuL6, the metalloenzyme is strongly dependent on the molecular
structural transition of HT21 becomes less notable, and the recognition between CuLn and HT21 G-quadruplex DNA.
This conclusion based on CD data is further supported by ITC
data (Table 3, Fig. S2† and Fig. S3†). For terpyridine-Cu(II)
complexes possessing aminoalkyl side chains (CuLn, n=1-4), all
binding isotherms are similar and reveal obvious two-event
binding process. The high-affinity binding process, with a
binding constant (Ka1) of 1.7×
107 M-1, is the result of a highly
favorable entropy change and a smaller unfavorable enthalpy
change. The low-affinity binding process displays a binding
constant (Ka2) of 5.8× ×
105 ~1.1 106 M-1, which is 16~30 times
lower than the Ka1. In contrast to the high-affinity binding
process, this low-affinity binding is favored by both entropy
+
and enthalpy changes. The titration of CuL5 into HT21-NH4
displays a relatively simple binding isotherm. A binding
constant (Ka) of 2.0×
105 M-1 is estimated by fitting a single
binding event model, which is of the same order of magnitude
+
as the low-affinity binding of CuLn (n=1-4) to HT21-NH4 G-
quadruplex DNA. Furthermore, the molecular recognition
+
process between CuL5 and HT21-NH4 G-quadruplex DNA also
shows a similar thermodynamic signature to the low-affinity
+
binding of CuLn (n=1-4) to HT21-NH4 G-quadruplex DNA (Fig.
S3†
). In contrast, the binding isotherm of CuL6 to HT21-NH4+ is
essentially the same as the background calorimetric titration of
+
MOPS buffer to HT21-NH4 , suggesting that weak binding
+
between CuL6 and HT21-NH4 occurs, and neither the binding
constant nor the thermodynamic parameters can be reliably
determined. Thus, based on the different energetic
characteristics, the ITC results clearly show three types of
molecular recognition processes, and correspondingly, these
three distinct interaction processes give the binding affinity
Fig. 4 CD spectra of human telomeric G-quadruplex DNA metalloenzymes with different
+
terpyridine-Cu(II) cofactors compared with HT21 itself in NH4 media. (a) CuLn (n=1-4);
(b) CuL5; (c) CuL6 (DNA strand concentration, 5 µM; NH4Cl, 30 mM; CuLn, 10 µM;
MOPS buffer, 20 mM, pH 6.5).
+
order of CuLn (n=1-4)>CuL5>CuL6 to HT21-NH4 G-quadruplex
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Fig. 5 Substrate scope for human telomeric G-quadruplex DNA metalloenzyme. Reaction conditions: 1 or 4 (1 mM), 2 (10 μL), HT21 (50 μM), CuL1 (100 μM), NH4Cl (30 mM), MOPS
buffer (20 mM, pH 6.5), 4 °C, 24h. All data are averaged over two experiments and reproducible within ±2%. Conversion measured by 1H-NMR analysis of the crude product. The
endo/exo ratios and ee (for the endo isomer) are determined by chiral-phase HPLC. The absolute configuration of 5a was obtained by referring 16.
DNA. This sequence is consistent with not only the amplitude Small molecule targeting of G-quadruplex DNA represents an
of the ICD signal in the CD experiment discussed above but attractive approach to anticancer drug design.40-43 This
also the ꢀTm values in UV melting experiments, where CuLn recognition process is analogous to the assembly of an artificial
(n=1-4) increases the melting temperature of HT21-NH4 most enzyme between a catalytic cofactor and a biomolecular
+
significantly (ꢀTm ≈ 20
and CuL6 ( Tm ≈ 5
°
C), followed by CuL5 (
ꢀ
Tm ≈ 10
°
C) scaffold.10-12 Following this idea, we have found an excellent G-
quadruplex metalloenzyme through molecular recognition
ꢀ
°
C) (Fig. S4†
).
In connection with the catalytic data and the above between terpyridine-Cu(II) and human telomeric G-quadruplex
characterizations (CD, UV melting, and ITC), we thus concluded DNA, although the precise nature of the interaction is not well
that the molecular recognition between HT21 G-quadruplex known. This modular assembly method allows rationally
DNA and terpyridine-Cu(II) plays an essential role in the tunable enantioselectivity of the Diels-Alder reaction. The G-
+
catalytic function of the HT21-NH4 -CuLn metalloenzyme. In quadruplex DNA scaffold can be modulated by the addition of
fact, the high affinity of CuLn (n=1-4) for HT21 G-quadruplex cation, modification of flanking bases at the 3’-end, and even
DNA can be easily understood as the interplay of two effects: inversion of global chirality by replacing natural D-DNA with
1) the π-π stacking interactions between the terpyridine and unnatural L-DNA. Modification of the substitute in the
the G-quartet; 2) the electrostatic interaction between the terpyridine ligand can also change the binding affinity between
positive charge of the aminoalkyl group and the negative G-quadruplex DNA and the terpyridine-Cu(II) cofactor, which
charge of the phosphate backbones.44,55
alters the microenvironment of the active site. Based on this
proof-of-concept experiment, we expect this approach to
provide a general way to find a broad variety of G-quadruplex
metalloenzymes by screening G-quadruplex-targeting ligand
pools.
Substrate Scope
+
With the excellent HT21-NH4 -CuL1 metalloenzyme in hand,
we further tested the substrate specificity (Fig. 5). The HT21-
+
NH4 -CuL1 metalloenzyme was found to be an active catalyst
for all the tested aza-chalcone substrates (1b-f). In general, the
introduction of a bulkier R2 group has a positive influence on
the enantioselectivity. In particular, R2 with a nitro group
afforded up to 99% ee (3e). Furthermore, high activity and
enantioselectivity are also observed for the other type of
dienophiles possessing acyl-imidazol groups (4a-e). A nearly
pure isomers (> 99% ee) are obtained for 4-MeO and 4-Br (5c
and 5e).
Conclusions
In conclusion, we have designed
a
G-quadruplex
metalloenzyme that can afford both high catalytic activity and
high enantioselectivity (up to 99% ee) for the Diels-Alder
reaction. The success of this artificial metalloenzyme can be
ascribed to the G-quadruplex DNA-targeting nature of
terpyridine-Cu(II). The terpyridine-Cu(II) cofactor is found to
play dual roles as both an active site for chemical catalysis and
a structural regulator for the folding of the catalytic DNA
structure. A positive correlation between the affinity of
Discussion
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23 L. Gjonaj and G. Roelfes, ChemCatChem, 2013,
24 H. Zhao and K. Shen, RSC Adv., 2014, , 54051.
25 E. Benedetti, N. Duchemin, L. Bethge, S. Vonhoff, S.
Klussmann, J.-J. Vasseur, J. Cossy, M. Smietana and S.
Arseniyadis, Chem. Commun., 2015, 51, 6076.
26 A. Draksharapu, A. J. Boersma, M. Leising, A. Meetsma, W. R.
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27 S. Burge, G. N. Parkinson, P. Hazel, A. K. Todd and S. Neidle,
Nucleic Acids Res., 2006, 34, 5402.
5, 1718.
terpyridine-Cu(II) for HT21 and the enantioselectivity of the
Diels-Alder reaction is observed, which further confirms the
importance of molecular recognition in constructing artificial
G-quadruplex DNA metalloenzymes. Our work paves a new
way to design G-quadruplex-based DNAzymes by learning from
G-quadruplex-targeting drug design.
4
28 J. Dai, M. Carver and D. Yang, Biochimie, 2008, 90, 1172.
29 J. Kypr, I. Kejnovská, D. Renčiuk and M. Vorlíčková, Nucleic
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31 S. Masiero, R. Trotta, S. Pieraccini, S. De Tito, R. Perone, A.
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We thank Prof. Yan Liu, Dr. Jun Zhou, Dr. Shengmei Lu and Dr.
Jun Li for their helpful discussions. This work is financially
supported by National Natural Science Foundation of China
(grant numbers 31000392, 21173213).
Randazzo and G. P. Spada, Org. Biomol. Chem., 2010,
2683.
8,
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