An Efficient Cyclic Di-AMP Based Artificial Metalloribozyme for Enantioselective Diels–Alder Reactions

Article abstract of DOI:10.1002/ejoc.202000652

The diverse structures of nucleic acids as scaffolds have brought the significant advancement for DNA-based enantioselective catalysis, yet RNA-based enantioselective catalysis is lacking investigation. Herein, we report a small, natural RNA of cyclic di-AMP (c-di-AMP) and Cu²⁺ ions assemble into an artificial metalloribozyme (c-di-AMP·Cu²⁺), that could effectively catalyze the enantioselective Diels–Alder reactions with up to 80 percent ee. The enantioselective catalytic performance of c-di-AMP·Cu²⁺ has been studied by thorough investigations of different metal cofactors, c-di-AMP/Cu²⁺ molar ratios, additives, buffers and c-di-AMP analogues. In addition, the assembly of c-di-AMP·Cu²⁺ gives rise to 300-fold and 5-fold rate acceleration compared to the uncatalyzed reaction and Cu²⁺ ions, respectively. This work provides a simple and efficient strategy to construct the RNA-based catalysts that would expand the current nucleic acids-based catalysis and might hint the possible catalytic RNA in primordial chemistry.

Full text of DOI:10.1002/ejoc.202000652

European Journal of Organic Chemistry
Accepted Article
Accepted Article
Title: An Efficient Cyclic Di-AMP Based Artificial Metalloribozyme for
Enantioselective Diels–Alder Reactions
Authors: Qianqian Qi, Shuting Lv, Min Hao, Xingchen Dong, Youkun
Gu, Peizhe Wu, Wenyue Zhang, Yashao Chen, and
Changhao Wang
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.202000652
Link to VoR: https://doi.org/10.1002/ejoc.202000652
01/2020

10.1002/ejoc.202000652
European Journal of Organic Chemistry
FULL PAPER
An Efficient Cyclic Di-AMP Based Artificial Metalloribozyme for
Enantioselective Diels–Alder Reactions
Qianqian Qi, Shuting Lv, Min Hao, Xingchen Dong, Youkun Gu, Peizhe Wu, Wenyue Zhang, Yashao
Chen,* and Changhao Wang*[a]
[a]
Q. Qi, S. Lv, M. Hao, Y. Gu, P. Wu, W. Zhang, Prof. Y. S. Chen, Dr. C. Wang
Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education
School of Chemistry and Chemical Engineering, Shaanxi Normal University,
620 West Chang’an Avenue, 710119 Xi’an, China
E-mail: changhaowang@snnu.edu.cn
yschen@snnu.edu.cn
Supporting information for this article is given via a link at the end of the document.
Abstract: The diverse structures of nucleic acids as scaffolds
have brought the significant advancement for DNA-based
enantioselective catalysis, yet RNA-based enantioselective
catalysis is lack of investigation. Herein, we report that a small,
natural RNA of cyclic di-AMP (c-di-AMP) and Cu2+ ions
assemble into an artificial metalloribozyme (c-di-AMP·Cu2+) that
enantiomeric excess (ee) value over 90%.[10] Arseniyadis and
co-workers have recently designed an unnatural double-
stranded RNA-based hybrid catalyst for
a Friedel–Crafts
reaction with modest enantioselectivity.[11] Marek and Hennecke
demonstrated that DNA is a more effective scaffold than RNA to
recognize metal complexes to assemble into nuclei acid based
hybrid catalysts.[12] Very recently, our group has reported that an
artificial metalloribozyme of a cyclic dinucleotide termed cyclic
di-AMP (c-di-AMP) and copper(II) ions that could high-efficiently
catalyze enantioselective Friedel–Crafts reactions with up to 97%
ee and the structure of the artificial metalloribozyme was
proposed in a dimer form.[13]
could effectively catalyze the enantioselective Diels–Alder
reactions with up to 80% ee. The enantioselective catalytic
performance of c-di-AMP·Cu2+ has been studied by thorough
investigations of different metal cofactors, c-di-AMP/Cu2+ molar
ratios, additives, buffers and c-di-AMP analogues. In addition,
the assembly of c-di-AMP∙Cu2+ gives rise to a 300-fold and 5-fold
rate acceleration compared to the uncatalyzed reaction and Cu2+
ions, respectively. This work provides a simple and efficient
strategy to construct the RNA-based catalysts that would
expand the current nucleic acids-based catalysis and might hint
the possible catalytic RNA in primordial chemistry.
Cyclic dinucleotides (CDNs) are natural cyclic RNA molecules
of that serve as important second messengers and regulate
many cellular processes.[14] So far, five CDNs of c-di-GMP, c-di-
AMP, c-AMP-GMP, 2’3’-cGAMP and c-AMP-UMP have been
discovered in nature.[15] The CDNs have been identified to form
stable tertiary structures as evidenced by theoretical calculations
and experimental studies,[16] making them as promising scaffolds
to construct CDN-based hybrid catalysts. In this work, we report
that c-di-AMP and copper(II) ions are assembling into an
artificial metalloribozyme (c-di-AMP∙Cu2+) that could efficiently
catalyze the enantioselective D–A reactions (Scheme 1).
Introduction
In recent years, the natural chiral structures of nucleic acids
have attracted much attention of chemists to construct nucleic
acid-based hybrid catalysts for enantioselective catalysis. In
2005, Roelfes and Feringa reported the first DNA-based hybrid
catalyst composing of double-stranded DNA (dsDNA) and
achiral copper(II) complex that could modestly achieved an
enantioselective Diels–Alder (D–A) reaction.[1] This concept of
dsDNA-based asymmetric catalysis has been applied to several
Lewis acid catalysis[2] and organometallic catalysis[3] in aqueous
media. Recently, G-quadruplex DNA (G4DNA) that specifically
binds to metal ions or complexes has been reported to achieve
several enantioselective transformations.[4] Moreover, the
enantioselectivies of the G4DNA-based catalysis are largely
dependent on the tunable features of the G-quadruplex
4d, 5]
structures.[4b,
In addition, G-triplex DNA,[6] artificial
deoxyribozyme,[7] unnatural DNA[8] and guanosine-based
assembly[9] were also employed as scaffolds to construct nucleic
acid-based hybrid catalysts.
Compared to DNA, very few examples of RNA-based
enantioselective catalysis were reported owing to its comparably
lower stability. In 2000, Jӓschke and coworkers reported that a
synthetic 49-mer ribozyme could catalyze an enantioselective
Diels–Alder reaction between anthracene and maleimide with an
Scheme 1. Schematic representation of enantioselective Diels–Alder reactions
between aza-chalcones (1a-h) and cyclopentadiene (2) catalyzed by a c-di-
AMP based artificial metalloribozyme (c-di-AMP∙Cu2+).
1
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000652
European Journal of Organic Chemistry
FULL PAPER
Results and Discussion
nitrate to form the complexes. Compared to Cu2+ ions as
cofactors, the introduction of achiral ligand to c-di-AMP∙Cu2+
yields the reduced conversion, diastereoselectivity and
enantioselectivity to the corresponding D–A reactions (Table 1,
entries 6-9). Considering the chiral structures, the CD spectra of
c-di-AMP with different copper(II) complexes show the similar
typical peaks but varied intensity compared to c-di-AMP∙Cu2+
(Figure S2b). The above results show that the extra ligand
causes negative effect to c-di-AMP∙Cu2+ catalyzed D–A reaction
that is similar as previously reported.[13] The reduction of the
enantioselectivities might be ascribed to the presence of ligand
that causes the perturbation of the chiral microenvironment and
the hindrance between aza-chalcone substrate and Cu2+ ions.
In short, the above data demonstrate that Cu2+ ions are
The catalytic performance of c-di-AMP based metallo-RNA
with different metal cofactors
We selected an enantioselective D–A reaction of aza-chalcone
(1a) and cyclopentadiene (2) as a benchmark reaction to
examine the catalytic properties of c-di-AMP based metallo-RNA.
C-di-AMP has been demonstrated to fold into highly ordered
structure as evidenced by the circular dichroism (CD) spectrum
(Figure S1). Of note, the folded c-di-AMP alone slightly
promotes the D–A reaction to provide a conversion of 13%, an
endo/exo of 92:8 and an enantioselectivity of 18% ee (Table 1,
entry 1). These results suggest that c-di-AMP in a tertiary
scaffold shows weak activity to yield the chiral product which is
similar as G4DNA based catalysis[4b, 4c] and c-di-AMP catalyzed
Friedel-Crafts reaction.[13] Once the copper(II) nitrate was added
to c-di-AMP, the conversion of the corresponding D–A reaction
remarkably improves to nearly 90% and the enantioselectivity
increases to 61% ee (Table 1, entry 2), indicating that copper(II)
ions are effectively interacting with c-di-AMP to assemble into a
c-di-AMP based artificial metalloribozyme (c-di-AMP∙Cu2+).
When altering different copper(II) salts as metal cofactors, the
corresponding D–A reactions show insignificant changes of the
endo/exo and the ee values (Table 1, entries 2-5) and the c-di-
AMP∙Cu2+ using Cu(OTf)2 as metal cofactor provides an
excellent conversion of 98% and 65% ee (Table 1, entry 5). In
addition, the CD spectra display the similar profiles of c-di-AMP
in the presence of copper(II) salts with different anions (Figure
S2a). These results indicate that the Cu2+ ions are efficient metal
cofactors to assist c-di-AMP to exert the enantioselective
catalytic performance and the c-di-AMP∙Cu2+ shows an
independent behavior with the counter-anions of copper(II) salts.
Furthermore, different copper(II) complexes were investigated
as metal cofactors for the c-di-AMP based artificial
metalloribozyme. Four achiral ligands of bipyridine (bpy), 4,4’-
dimethylbipyridine (dmbpy), phenanthroline (phen) and
terpyridine (tpy) were employed to coordinate with copper(II)
specifically binding to c-di-AMP to form
a
potent
metalloribozyme. From the recent report of c-di-AMP∙Cu2+of the
catalyzed Friedel-Crafts reaction,[13] the structure of c-di-
AMP∙Cu2+ is depicted in a dimeric form where two c-di-AMP are
stacking intercrossingly and the Cu2+ ions are sitting in the
center of an adenine-adenine plane (Scheme 1). For the binding
affinity
of
Cu2+
ions
in
adenine-containing
nucleotides/nucleosides, the N7 atom in the purine moiety and
the phosphates have been proved as the preferred binding
sites.[17] The coordination structures of N7-Cu2+ and P(O)-Cu2+
would yield a macro-chelate formation.[18] Based on the structure
of c-di-AMP, the highly specific bonding of Cu2+ ions is possibly
ascribed to the presence of two N7 atoms and the phosphate
groups in the c-di-AMP dimer.
The molar ratio effect of c-di-AMP and Cu2+
For the assembly of c-di-AMP∙Cu2+, c-di-AMP mainly contributes
80
(a)
65%
60
54%
40%
38%
40
31%
Table 1. Enantioselective Diels–Alder reaction catalyzed by c-di-AMP in the
presence of different metal cofactors.[a]
20
0
10:1
5:1
2:1
1:1
1:2
c-di-AMP/Cu2+
12
6
(b)
Entry Metal cofactor
Conv. (%)
endo/exo
92:8
ee (3a%, endo)
1
2
3
4
5
6
7
8
9
none
13
89
85
80
98
67
69
57
56
18
61
64
65
65
37
40
55
49
0
Cu(NO3)2
CuSO4
94:6
c-di-AMP:Cu2+
92:8
-6
10:1
5:1
2:1
1:1
1:2
CuCl2
94:6
Cu(OTf)2
Cu(bpy)
Cu(dmbpy)
Cu(phen)
Cu(tpy)
93:7
-12
-18
89:11
87:13
91:9
240
260
280
300
320
Wavelength (nm)
91:9
Figure 1. (a) The ee of 3a (endo) that was produced by c-di-AMP∙Cu2+
catalyzed Diels-Alder reactions with different molar ratios. Reaction conditions:
1a (1 μmol), 2 (250 μmol), c-di-AMP (250 μM), Cu(OTf)2 (25-500 μM), MES
buffer (500 μL, 20 mM, pH 5.5), 4 oC, 24 h. (b) CD spectra of c-di-AMP (250
μM) in the presence of Cu(OTf)2 at different concentrations (25-500 μM).
[a] Reaction conditions: 1a (2 μmol), 2 (250 μmol), c-di-AMP (250 μM), metal
cofactor (50 μM), MES buffer (500 μL, 20 mM, pH 5.5), 4 oC, 24 h. Achiral
ligands: bpy, bipyridine; dmbpy, 4,4’-dimethylbipyridine; phen, phenanthroline;
tpy, terpyridine.
2
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000652
European Journal of Organic Chemistry
FULL PAPER
to the chiral induction and the Cu2+ ions are the catalytic species. The effect of additives
Therefore, the molar ratio between c-di-AMP and Cu2+ ions is a
In nature, the structures of nucleic acids are sensitive to the
presence of additive metal ions such as alkaline metal ions (Na+,
K+) and alkaline earth metal ions (Mg2+). Furthermore, the
presence of Na+ or K+ has been demonstrated to cause great
effect in G4DNA-based enantioselective catalysis.[5] Compared
to c-di-AMP∙Cu2+ catalyzed benchmark D–A reaction without
additive metal ions, the addition of 10 mM Na+, K+ or Mg2+
causes negligible effect to the corresponding enantioselectivities
yet results in slight decrement of the conversions (Figure 2a).
These results indicate that the tertiary structure of c-di-AMP is
independent on the additional metal ion which is different from
the G-quadruplex structures. In addition, the presence of
key factor to determine the enantioselective activity of c-di-
AMP∙Cu2+. We investigated different molar ratios of c-di-
AMP/Cu2+ with a fixed concentration of c-di-AMP at 250 μM.
Compared to the c-di-AMP promoted benchmark reaction (Table
1, entry 1), the addition of a small amount of Cu2+ ions (25 μM)
could increase the conversion from 13% to 83% and enhance
the ee from 18% to 40% with c-di-AMP/Cu2+ = 10:1 (Figure 1a,
Table S1). When the molar ratio of c-di-AMP/Cu2+ is at 5:1, the
corresponding D–A reaction provides an approximately full
conversion and an enhanced ee of 65% (Figure 2). Further
increasing the molar ratios of c-di-AMP/Cu2+, the conversions of
the corresponding D–A reactions show no significant changes
(Table S1) yet the ee values of 3a are gradually decreasing
(Figure 1a). These results indicate that the coordination between
c-di-AMP and Cu2+ ions is feasible at the molar ratio of 5:1.
Continue increasing the amount of Cu2+ ions will enhance the
racemic reaction, thus resulting in a decreased ee value.
Furthermore, the chiral structures of c-di-AMP/Cu2+ with various
molar ratios were characterized. Although c-di-AMP/Cu2+ with
different molar ratios display the similar CD profiles, the intensity
of c-di-AMP/Cu2+ at 5:1 is the highest (Figure 1b), indicating that
c-di-AMP/Cu2+ at 5:1 forms a more compact structure. Taken
together, the molar ratio of c-di-AMP/Cu2+ causes great effect to
c-di-AMP∙Cu2+ catalyzed D-A reaction and the c-di-AMP/Cu2+ at
5:1 exhibits the best catalytic performance.
+
ammonium ions (NH4 ) shows the similar effect as the above
metal ions (Figure 2a). Compared to the c-di-AMP∙Cu2+ without
any additive, the corresponding CD spectra of c-di-AMP∙Cu2+
with different additives show the similar representative peaks
and the intensities (Figure 2b), suggesting that the additives
cause negligible effect to the chiral structure of c-di-AMP∙Cu2+.
This might be the possible reason that the corresponding c-di-
AMP∙Cu2+ catalyzed D–A reactions give the similar activities and
enantioselectivities in the presence of different additives.
(a)100
Conversion
ee (3a%, endo)
80
60
40
20
0
100
80
60
40
20
0
Conversion
ee (3a, endo)
(a)
0
10
50
100
500
1000
NaCl concentration (mM)
30
(b)
15
0
MgCl2
none
NaCl
NH4Cl
KCl
(b)
6
0
0 mM
NaCl
10 mM
50 mM
100 mM
500 mM
1000 mM
-15
-30
none
NaCI
KCI
-6
240
260
280
300
320
NH4CI
Wavelength (nm)
-12
-18
MgCI2
Figure 3. (a) c-di-AMP∙Cu2+ catalyzed Diels–Alder reaction with Na+ ions at
varied concentrations (0-1000 mM) and (b) the corresponding CD spectra.
Reaction condition: 1a (2 μmol), 2 (250 μmol), c-di-AMP (250 μM), Cu(OTf)2
(50 μM), MES buffer (500 μL, 20 mM, pH 5.5), 4 oC, 24 h.
240
260
280
300
320
Wavelength (nm)
Figure 2. (a) c-di-AMP∙Cu2+ catalyzed Diels–Alder reaction with different
additives at 10 mM and (b) the corresponding CD spectra. Reaction conditions:
1a (2 μmol), 2 (250 μmol), c-di-AMP (250 μM), Cu(OTf)2 (50 μM), additive
(MCl or MCl2, 10 mM), MES buffer (500 μL, 20 mM, pH 5.5), 4 oC, 24 h.
We further examined the effect of Na+ ions with different
concentrations. Compared to c-di-AMP∙Cu2+ without any additive,
the conversions of 1a are gradually decreasing when Na+ ions
are continuously added (Figure 3a). The ee values of 3a (endo)
3
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000652
European Journal of Organic Chemistry
FULL PAPER
remain almost unchanged when the concentrations of Na+ ions
are below 100 mM, however, further increasing the amount of
Na+ ions causes an obvious decrement of the enantioselectivity
(Figure 3a). In the corresponding CD spectra, the structures of
c-di-AMP·Cu2+ change from compact state to a loose form when
the amount of Na+ ions is increasing (Figure 3b). The reduction
of reactivity and enantioselectivity to c-di-AMP∙Cu2+ catalyzed
D–A reactions might be ascribed to the comparable unstable
structure in high concentration of Na+ ions, which shows the
similar phenomena as described in G4DNA-based catalysis[4b, 4c,
5] and c-di-AMP based catalysis.[13] In short, the enantioselective
catalytic properties of c-di-AMP∙Cu2+ is less dependent on the
additive species but largely dependent on their concentrations.
The effect of c-di-AMP analogues
Different c-di-AMP analogues were employed to examine the
catalytic performance of CDN-based artificial metalloribozyme.
With the assistance of Cu2+ ions as metal cofactors, the
replacement of c-di-AMP to c-di-GMP causes a sharp decrease
of the corresponding ee from 65% to 4% (Figure 4a). The
distinct CD spectra of c-di-AMP and c-di-GMP might be the
possible reason for the decreased enantioselectivity (Figure 4b).
The other CDN of either c-di-CMP or c-di-UMP with different
structures provides racemic 3a in Cu2+-involved D–A reaction
(Figure 4). Compared to c-di-AMP, the c-AMP-GMP with only
one substitution of one GMP still gives rise to a racemic D–A
product (Figure 4a), which shows the different phenomenon as
described in c-di-AMP based Friedel-Crafts reaction.[13] The
possible reason might be attributed to the different reaction
mechanisms between these two reactions. In addition, the non-
structured 3’,5’-cyclic AMP (cAMP), AMP and adenine in the
presence of Cu2+ ions provide the racemic products (Figure 4).
Briefly, the c-di-AMP analogue experiments indicate that Cu2+
ions can specifically bind to c-di-AMP to form an efficient
metalloribozyme as described previously.[13]
The effect of buffers
To optimize the buffer solution for the c-di-AMP∙Cu2+ catalyzed
D–A reaction, the effect of buffers was investigated with different
species and pH values. For 4-morpholineethanesulfonic acid
(MES) buffer at pH 4.5, the corresponding D–A reaction gives
rise to 80% conversion and 53% ee (Table 2, entry 1). With
increasing the pH value of MES buffer to 5.5, the conversion is
increased to 98% and the ee value is up to 65% (Table 2, entry
2). However, further increment of the pH to 6.5 causes the
reduced conversion and ee to the corresponding D–A reaction
(Table 2, entry 3). The possible reason is ascribed to the
protonation of c-di-AMP that may affect the interaction between
c-di-AMP and Cu2+ ions. The similar phenomenon was also
observed in the 4-morpholinepropanesulfonic acid (MOPS)
buffer. With increasing the pH from 5.5 to 7.4 in MOPS buffer,
the conversions of the corresponding reactions are gradually
decreasing (Table 2, entries 4-7). The ee values of 3a remain
unchanged at pH 5.5 and 6.5, yet further increasing the pH
provides the reduced enantioselectivities (Table 2, entries 4-7).
(a) 75
65%
60
45
30
15
4%
In
addition,
phosphate
buffer
saline
(PBS)
and
<1%
<1%
P
<1%
P
<1%
<1%
<1%
ne
0
tris(hydroxymethyl)aminomethane (Tris) buffers at pH 7.4
generate the low conversions and ee values, which is
comparable as that in MOPS buffer (Table 2, entry 7 vs. entries
8,9). These results indicate that high pH values cause negative
effect to the c-di-AMP∙Cu2+ catalyzed D–A reaction and MES
buffer at pH 5.5 is screened as the optimal reaction medium.
P
P
P
P
P
M
M
M
ni
ade
A
-A
-CM
c-di
-UM
-GM
c-di
-GM
cA
P
c-di
M
c-di
c-A
c-di-AMP
c-di-GMP
c-di-CMP
c-di-UMP
c-AMP-GMP
Adenine
AMP
(b)10
5
0
Table 2. Enantioselective Diels–Alder reaction catalyzed by c-di-AMP∙Cu2+ in
different buffers.[a]
cAMP
Entry
Buffer
MES
pH
4.5
5.5
6.5
5.5
6.5
7.0
7.4
7.4
7.4
Conv. (%)
endo/exo
91:9
ee (3a%, endo)
1
2
3
4
5
6
7
8
9
80
98
69
89
81
50
18
9
53
65
21
57
58
29
22
19
5
-5
93:7
-10
82:18
94:6
MOPS
240
260
280
300
320
Wavelength (nm)
94:6
91:9
Figure 4. (a) The enantioselectivities of 3a (endo) that were produced by Cu2+
in the presence of different c-di-AMP analogues. Reaction condition: 1a (2
μmol), 2 (250 μmol), c-di-AMP analogue (250 μM), Cu(OTf)2 (50 μM), MES
buffer (500 μL, 20 mM, pH 5.5), 4 oC, 24 h. (b) CD spectra of different c-di-
AMP analogues (250 μM) and Cu(OTf)2 (50 μM) in MES buffer (20 mM, pH
5.5).
92:8
PBS
Tris
83:17
75:25
8
[a] Reaction conditions: 1a (2 μmol), 2 (250 μmol), c-di-AMP (250 μM),
Cu(OTf)2 (50 μM), buffer (500 μL, 20 mM), 4 oC, 24 h.
4
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000652
European Journal of Organic Chemistry
FULL PAPER
Kinetic study of the c-di-AMP·Cu2+
Table 4. Substrate scope of the c-di-AMP·Cu2+ catalyzed Diels–Alder
reactions.[a]
In order to study the kinetic behavior of the c-di-AMP based
metalloribozyme, we measured the apparent second-order rate
constant (kapp) of the c-di-AMP∙Cu2+ catalyzed benchmark D–A
reaction. Compared to the uncatalyzed reaction, the Cu2+ ions
provide a 65-fold rate acceleration (Table 3, entry 1 vs. entry 2).
Once c-di-AMP and Cu2+ ions assemble into a c-di-AMP·Cu2+
metalloribozyme, the corresponding reaction rate improves
about 300-fold compared to the uncatalyzed reaction and 5-fold
compared to the bare Cu2+ ions (Table 3, entry 3). These results
suggest that the presence of c-di-AMP not only contributes to
the chiral induction but also provides a rate enhancement to the
Cu2+-involved D–A reaction. Furthermore, the rate acceleration
effect of c-di-AMP·Cu2+ catalyzed D–A reaction is accordance
with the phenomenon in c-di-AMP based Friedel-Crafts
reaction,[13] resulting in an assumption of the possible synergistic
effect between c-di-AMP and Cu2+ ions.
Aza-
chalcone
Conv.
(%)
endo
/exo
ee
(%, endo)
Entry
1[b]
R
1a
1b
1c
99
85
59
93:7
97:3
96:4
65
45
55
2
3
4
1d
42
95:5
23
5
6
1e
1f
84
90
94:6
6
5
Table 3. The apparent second-order rate constants for the uncatalyzed and
catalyzed Diels-Alder reactions.[a]
Entry
Catalyst
none
kapp (M-1 s-1)
krel
1
90:10
1
2
3
(1.2
(7.8
(3.8



0.2) x 10-4
0.4) x 10-3
0.5) x 10-2
7
8
1g
1h
99
97
97:3
96:4
80
80
Cu2+
65
317
c-di-AMP·Cu2+
[a] Reaction conditions: 1a (50 μM), 2 (5 mM), c-di-AMP (250 μM), Cu(OTf)2
(50 μM), MES buffer (2000 μL, 20 mM, pH 5.5), 4 oC. The rate acceleration
[a] Reaction conditions: 1 (2 μmol), 2 (250 μmol), c-di-AMP (250 μM),
Cu(OTf)2 (50 μM), MES buffer (1 mL, 20 mM, pH 5.5), 4 oC, 3 d. [b] Reaction
time 24 h.
effect krel = kapp,cat/kapp,uncat
.
Substrate scope
and electronic effect to the enantioselective catalytic
performance of c-di-AMP∙Cu2+.
In order to examine the substrate scope of the c-di-AMP∙Cu2+
catalyzed D–A reactions, aza-chalcones with different
substitutes were investigated. Compared to 1a with R = Ph,
either electro-donating or electro-withdrawing substitutions on
the phenyl group provides reduced conversions and
enantioselectivities (Table 4, entry 1 vs. entries 2-6), indicating
that the substitutions in aza-chalcones cause negative effect to
the enantioselective catalytic performance of c-di-AMP∙Cu2+.
When the aza-chalcones bearing the electro-donating
substitutions such as 4-Me and 4-OMe on the phenyl group (1b
and 1c), the corresponding D–A reactions provides the
Conclusion
In summary, we found that
a c-di-AMP based artificial
metalloribozyme could efficiently catalyze enantioselective D–A
reactions with modest to good enantioselectivities. The
enantioselective activity of c-di-AMP∙Cu2+ is largely dependent
on the metal cofactor, the molar ratio of c-di-AMP/Cu2+, the
additives and the buffers. In addition, a plausible synergistic
effect between c-di-AMP and Cu2+ ions gives rise to a modest
rate enhancement compared to Cu2+ ions. This work provides
another example of CDN-based enantioselective catalysis and
we anticipate that this work would promote the further
exploration on RNA-based enantioselective catalysis and the
possible catalytic roles of CDNs in early chemical evolution.
significant
reduction
of
the
conversions
and
the
enantioselectivities (Table 4, entry 1 vs. entries 2-3). Once
altering the methoxy substitution from 4’-position to 2’-position
on the phenyl group (1c and 1d), a further decreased conversion
and enantioselectivity were obtained (Table 4, entry 3 vs. entry
4), indicating that c-di-AMP∙Cu2+ exhibits an obvious steric effect
to the aza-chalcone substrates. Compared to 1a, the aza-
chalcones with electro-withdrawing substituted phenyl group (1e
and 1f) results in a sharp decrement of the ee values to nearly
racemic products, although the conversions remain almost
unchanged (Table 4, entry 1 vs. entries 5-6). Intriguingly, aza-
chalcones with the moieties of furyl (1g) and thienyl group (1h)
Experimental Section
General information
The cyclic dinucleotides of c-di-AMP, c-di-GMP, c-di-CMP, c-di-UMP and
c-AMP-GMP were chemically synthesized using commercial
phosphoramidites as starting materials according to the references.[16f, 19]
3’,5’-Cyclic AMP, AMP and adenine were purchased from Sangon
(Shanghai, China). The metal salts, achiral ligands and buffers were
obtained from commercial sources and used without further purification.
Water used was distilled and deionized using a Milli-Q A10 water
show
nearly
quantitative
conversions
and
good
enantioselectivities up to 80% ee (Table 4, entries 7-8). The
above results suggest that the c-di-AMP∙Cu2+ is eligible for a
series of aza-chalcone substrates in D–A reactions and the
substitution in aza-chalcone substrates shows significant steric
5
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000652
European Journal of Organic Chemistry
FULL PAPER
purification system. The aza-chalcones (1a-h) were synthesized followed
(E)-3-(4-nitrophenyl)-1-(pyridin-2-yl)prop-2-en-1-one (1f): 1H NMR
(400 MHz, CDCl3) δ 8.72 (dd, J = 3.5, 1.1 Hz, 1H), 8.39 (d, J = 16.1 Hz,
1H), 8.28 – 8.20 (m, 2H), 8.16 (dd, J = 7.8, 0.7 Hz, 1H), 7.85 (ddd, J =
17.6, 8.2, 6.7 Hz, 4H), 7.50 (ddd, J = 7.5, 4.7, 1.1 Hz, 1H). 13C NMR (100
MHz, CDCl3) δ 188.88 (s), 153.57 (s), 148.97 (s), 148.52 (s), 141.25 (d, J
= 11.1 Hz), 137.19 (s), 129.23 (s), 127.33 (s), 124.82 (s), 124.10 (s),
123.05 (s). HRMS (ESI) calcd. For [C14H10N2O3]∙Na+ (M+Na)+: m/z
277.0584, found 277.0573.
(E)-3-(furan-2-yl)-1-(pyridin-2-yl)prop-2-en-1-one (1g): 1H NMR (600
MHz, CDCl3) δ 8.82 – 8.67 (m, 1H), 8.14 (dd, J = 18.8, 11.8 Hz, 2H), 7.85
(td, J = 7.7, 1.7 Hz, 1H), 7.69 (d, J = 15.8 Hz, 1H), 7.53 (d, J = 1.1 Hz,
1H), 7.46 (ddd, J = 7.5, 4.7, 1.0 Hz, 1H), 6.76 (d, J = 3.4 Hz, 1H), 6.50
(dd, J = 3.3, 1.7 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 189.32 (s),
154.22 (s), 152.12 (s), 148.92 (s), 145.12 (s), 136.96 (s), 130.68 (s),
126.81 (s), 122.81 (s), 118.75 (s), 116.21 (s), 112.65 (s). HRMS (ESI)
calcd. For [C14H10N2O3]∙Na+ (M+Na)+: m/z 222.0525, found 222.0524.
(E)-1-(pyridin-2-yl)-3-(thiophen-2-yl)prop-2-en-1-one (1h): 1H NMR
(600 MHz, CDCl3) δ 8.73 (d, J = 4.6 Hz, 1H), 8.16 (d, J = 7.8 Hz, 1H),
8.05 (s, 2H), 7.85 (td, J = 7.7, 1.5 Hz, 1H), 7.49 – 7.44 (m, 1H), 7.41 (dd,
J = 13.3, 4.3 Hz, 2H), 7.08 (dd, J = 4.7, 3.9 Hz, 1H). 13C NMR (150 MHz,
CDCl3) δ 189.12 (s), 154.16 (s), 148.87 (s), 140.90 (s), 137.14 (s),
137.00 (s), 132.16 (s), 129.21 (s), 128.29 (s), 126.86 (s), 122.85 (s),
119.87 (s). HRMS (ESI) calcd. For [C12H9NOS]∙Na+ (M+Na)+: m/z
238.0297, found 238.0293.
by the reference.[20]
CD spectra were collected on
a Chirascan circular dichroism
spectrometer (Applied Photophysics Ltd, UK) from 230 to 320 nm using a
o
10 mm quartz cell with a scanning speed of 100 nm/min at 4 C. UV-Vis
spectra were measured on a HITACHI U-3900 spectrophotometer from
190 nm to 800 nm. The Diels-Alder reactions were analyzed by a chiral
HPLC (Shimadzu Prominence-i LC-2030) using hexane and iso-propanol
as eluents. Proton nuclear magnetic resonance (1H NMR) and carbon
nuclear magnetic resonance (13C NMR) spectra were recorded on Bruker
AV 600 and 400 MHz spectrometers using residue solvent peaks as an
internal standard. High-resolution mass spectra (HRMS) were collected
on a Bruker maxis system.
Synthesis of aza-chalcones and their racemic products
Synthesis of aza-chalcones (1a-h). For 1a-d: 2-acetylpyridine (8.25
mmol) and the appropriate benzaldehyde (8.25 mmol) were introduced in
50 mL of distilled water at 4 °C after stirring for 0.5 h. Then, 5 mL of
sodium hydroxide solution (10 wt%) was added. After stirring for another
0.5 h and left the mixture to stand overnight at 4 °C. The reaction mixture
was filtered and washed by water to provide the crude products with
>90% yields. The pure products were obtained by recrystallization in
ethanol. For 1e-h: To a stirred solution of 0.5 mL of 10% aqueous sodium
hydroxide and the appropriate aldehyde (8.25 mmol) in 10 mL of ethanol,
2-acetylpyridine (8.25 mmol) was added dropwise after stirring for 1 h.
After being stirred for another 2 h at 0 °C, the reaction mixture was
filtered and nearly pure product was obtained with >90% yields.
(E)-3-phenyl-1-(pyridin-2-yl)prop-2-en-1-one (1a): 1H NMR (600 MHz,
CDCl3) δ 8.75 (s, 1H), 8.31 (d, J = 16.0 Hz, 1H), 8.19 (d, J = 7.7 Hz, 1H),
7.94 (d, J = 16.0 Hz, 1H), 7.87 (td, J = 7.7, 1.5 Hz, 1H), 7.73 (dd, J = 6.8,
2.3 Hz, 2H), 7.58 – 7.34 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 189.48
(s), 154.23 (s), 148.84 (s), 144.81 (s), 137.04 (s), 135.18 (s), 130.57 (s),
128.86 (d, J = 3.4 Hz), 126.91 (s), 122.95 (s), 120.92 (s). HRMS (ESI)
calcd. For [C14H11NO]∙Na+ (M+Na)+: m/z 232.0733, found 232.0731.
(E)-1-(pyridin-2-yl)-3-p-tolylprop-2-en-1-one (1b): 1H NMR (400 MHz,
CDCl3) δ 8.75 – 8.66 (m, 1H), 8.24 (d, J = 16.0 Hz, 1H), 8.16 (d, J = 7.9
Hz, 1H), 7.91 (d, J = 16.0 Hz, 1H), 7.82 (td, J = 7.7, 1.7 Hz, 1H), 7.60 (d,
J = 8.1 Hz, 2H), 7.43 (ddd, J = 7.5, 4.8, 1.1 Hz, 1H), 7.18 (d, J = 8.0 Hz,
2H), 2.35 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 189.44 (s), 154.34 (s),
148.82 (s), 144.85 (s), 141.09 (s), 136.96 (s), 132.45 (s), 129.63 (s),
128.88 (s), 126.79 (s), 122.86 (s), 119.86 (s), 21.55 (s). HRMS (ESI)
calcd. For [C15H13NO]∙Na+ (M+Na)+: m/z 246.0889, found 246.0884.
(E)-3-(4-methoxyphenyl)-1-(pyridin-2-yl)prop-2-en-1-one (1c): 1H
NMR (400 MHz, CDCl3) δ 8.78 – 8.70 (m, 1H), 8.19 (dd, J = 11.9, 4.0 Hz,
2H), 7.89 (ddd, J = 13.2, 9.4, 8.8 Hz, 2H), 7.70 (d, J = 8.7 Hz, 2H), 7.48
(ddd, J = 7.5, 4.8, 1.1 Hz, 1H), 6.94 (d, J = 8.7 Hz, 2H), 3.86 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ 189.31 (s), 161.77 (s), 148.72 (s), 144.78 (s),
137.07 (s), 130.68 (s), 128.00 (s), 126.73 (s), 122.92 (s), 118.55 (s),
114.35 (s), 55.41 (s). HRMS (ESI) calcd. For [C15H13NO2]∙Na+ (M+Na)+:
m/z 262.0838, found 262.0831.
(E)-3-(2-methoxyphenyl)-1-(pyridin-2-yl)prop-2-en-1-one (1d): 1H
NMR (600 MHz, CDCl3) δ 8.74 (s, 1H), 8.32 (q, J = 16.2 Hz, 2H), 8.18 (d,
J = 7.8 Hz, 1H), 7.86 (td, J = 7.7, 1.6 Hz, 1H), 7.79 (dd, J = 7.7, 1.4 Hz,
1H), 7.47 (dd, J = 6.8, 5.0 Hz, 1H), 7.44 – 7.33 (m, 1H), 6.99 (t, J = 7.5
Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 3.91 (s, 3H). 13C NMR (150 MHz,
CDCl3) δ 189.79 (s), 158.91 (s), 154.55 (s), 148.82 (s), 139.99 (s),
136.97 (s), 131.92 (s), 128.84 (s), 126.71 (s), 124.22 (s), 122.93 (s),
121.11 (s), 120.67 (s), 111.19 (s), 55.59 (s). HRMS (ESI) calcd. For
[C15H13NO2]∙Na+ (M+Na)+: m/z 262.0838, found 262.0833.
(E)-3-(4-chlorophenyl)-1-(pyridin-2-yl)prop-2-en-1-one (1e): 1H NMR
(400 MHz, CDCl3) δ 8.73 (d, J = 4.6 Hz, 1H), 8.27 (d, J = 16.1 Hz, 1H),
8.18 (d, J = 7.9 Hz, 1H), 7.87 (dd, J = 8.7, 7.3 Hz, 2H), 7.65 (d, J = 8.4
Hz, 2H), 7.54 – 7.46 (m, 1H), 7.38 (d, J = 8.4 Hz, 2H). 13C NMR (100
MHz, CDCl3) δ 189.29 (s), 154.06 (s), 148.88 (s), 143.19 (s), 137.07 (s),
136.43 (s), 133.68 (s), 129.96 (s), 129.16 (s), 127.01 (s), 122.96 (s),
121.36 (s). HRMS (ESI) calcd. For [C14H10NOCl]∙Na+ (M+Na)+: m/z
266.0343, found 266.0336.
Synthesis of the racemates (3a-h). To a stirred solution of Cu(OTf)2 (0.11
mmol) in H2O (5 mL), aza-chalcone 1 (0.19 mmol) in CH3CN and
cyclopentadiene (1 mL) were added. The reaction mixture was stirred at
room temperature until the completion of the reaction as monitored by
TLC. The reaction mixture was extracted with ethyl acetate (3 x 15 mL)
and the combined organic layer was dried over anhydrous Na2SO4. The
solvent was removed under reduced pressure and the crude residue was
purified by flash chromatography on silica gel (0–30% EtOAc/petrol
ether) to afford the pure product.
(3-Phenylbicyclo[2.2.1]hept-5-en-2-yl)(pyridin-2-yl)methanone (3a):
1H NMR (400 MHz, CDCl3) δ 8.78 – 8.59 (m, 1H), 8.11 – 7.97 (m, 1H),
7.79 (td, J = 7.7, 1.7 Hz, 1H), 7.42 (ddd, J = 7.6, 4.8, 1.3 Hz, 1H), 7.30
(ddd, J = 13.2, 8.3, 1.3 Hz, 4H), 7.17 (ddd, J = 8.5, 2.6, 1.3 Hz, 1H), 6.51
(dd, J = 5.6, 3.2 Hz, 1H), 5.85 (dd, J = 5.6, 2.8 Hz, 1H), 4.56 (dd, J = 5.2,
3.4 Hz, 1H), 3.64 – 3.43 (m, 2H), 3.10 (d, J = 1.4 Hz, 1H), 2.09 (d, J = 8.6
Hz, 1H), 1.68 – 1.55 (m, 1H). 13C NMR (100 MHz, CDCl3) δ 201.17 (s),
153.68 (s), 148.99 (s), 144.75 (s), 139.53 (s), 136.95 (s), 132.99 (s),
128.51 (s), 127.77 (s), 127.02 (s), 125.96 (s), 122.29 (s), 54.40 (s), 49.47
(s), 48.89 (s), 48.36 (s), 45.69 (s). HRMS (ESI) calcd. For
[C19H17NO]∙Na+ (M+Na)+: m/z 298.1202, found 298.1199.
Pyridin-2-yl(3-p-tolylbicyclo[2.2.1]hept-5-en-2-yl)methanone (3b): 1H
NMR (400 MHz, CDCl3) δ 8.68 (ddd, J = 4.7, 1.6, 0.8 Hz, 1H), 8.03 –
7.96 (m, 1H), 7.81 (td, J = 7.7, 1.7 Hz, 1H), 7.45 (ddd, J = 7.5, 4.8, 1.2 Hz,
1H), 7.22 (d, J = 8.1 Hz, 2H), 7.09 (d, J = 8.0 Hz, 2H), 6.49 (dd, J = 5.5,
3.1 Hz, 1H), 5.82 (dd, J = 5.6, 2.8 Hz, 1H), 4.52 (dd, J = 5.2, 3.4 Hz, 1H),
3.54 (s, 1H), 3.41 (d, J = 4.5 Hz, 1H), 3.05 (d, J = 1.4 Hz, 1H), 2.29 (d, J
= 9.0 Hz, 3H), 2.07 (d, J = 8.4 Hz, 1H), 1.63 – 1.54 (m, 1H). 13C NMR
(100 MHz, CDCl3) δ 201.18 (s), 153.66 (s), 148.85 (s), 141.55 (s), 139.46
(s), 136.82 (s), 135.30 (s), 132.77 (s), 129.05 (s), 127.56 (s), 126.84 (s),
122.18 (s), 54.18 (s), 49.61 (s), 48.72 (s), 48.21 (s), 45.31 (s), 20.90 (s).
HRMS (ESI) calcd. For [C20H19NO]∙Na+ (M+Na)+: m/z 312.1359, found
312.1354.
(3-(4-Methoxyphenyl)bicyclo[2.2.1]hept-5-en-2-yl)(pyridin-2-
yl)methanone (3c): 1H NMR (400 MHz, CDCl3) δ 8.68 (dd, J = 4.7, 0.7
Hz, 1H), 8.00 (d, J = 7.8 Hz, 1H), 7.81 (td, J = 7.7, 1.7 Hz, 1H), 7.45 (ddd,
J = 7.5, 4.8, 1.2 Hz, 1H), 7.24 (d, J = 8.6 Hz, 2H), 6.88 – 6.77 (m, 2H),
6.48 (dd, J = 5.5, 3.2 Hz, 1H), 5.82 (dd, J = 5.6, 2.7 Hz, 1H), 4.49 (dd, J =
5.1, 3.4 Hz, 1H), 3.76 (d, J = 7.6 Hz, 3H), 3.53 (s, 1H), 3.38 (d, J = 5.0 Hz,
1H), 3.02 (s, 1H), 2.06 (d, J = 8.4 Hz, 1H), 1.74 – 1.51 (m, 2H), 1.26 (s,
1H). 13C NMR (100 MHz, CDCl3) δ 201.21 (s), 157.77 (s), 153.65 (s),
148.85 (s), 139.45 (s), 136.76 (d, J = 15.3 Hz), 132.74 (s), 128.55 (s),
126.85 (s), 122.18 (s), 113.79 (s), 55.28 (s), 54.30 (s), 49.68 (s), 48.69
(s), 48.17 (s), 44.93 (s). HRMS (ESI) calcd. For [C20H19NO2]∙Na+
(M+Na)+: m/z 328.1308, found 328.1303.
6
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000652
European Journal of Organic Chemistry
FULL PAPER
(3-(2-Methoxyphenyl)bicyclo[2.2.1]hept-5-en-2-yl)(pyridin-2-
yl)methanone (3d): 1H NMR (400 MHz, CDCl3) δ 8.63 (d, J = 4.5 Hz),
8.09 (d, J = 7.8 Hz), 7.82 (tt, J = 8.3, 4.1 Hz), 7.48 – 7.38 (m), 7.34 (dd, J
= 10.2, 6.4 Hz), 7.20 – 7.11 (m), 6.93 (t, J = 7.3 Hz), 6.71 (t, J = 8.9 Hz),
6.48 (dd, J = 5.5, 3.3 Hz), 5.92 (dd, J = 5.6, 2.8 Hz), 4.37 (dd, J = 5.1, 3.4
Hz), 3.42 (s), 3.32 (d, J = 10.5 Hz), 3.31 – 3.24 (m), 1.95 (d, J = 8.3 Hz),
1.61 (dd, J = 8.3, 1.7 Hz). 13C NMR (100 MHz, CDCl3) δ 200.83 (s),
157.73 (s), 154.06 (s), 148.92 (s), 138.03 (s), 136.86 (s), 133.86 (s),
133.60 (s), 126.72 (d, J = 12.9 Hz), 125.87 (s), 122.00 (s), 120.28 (s),
109.84 (s), 54.66 (s), 52.11 (s), 48.36 (d, J = 15.5 Hz), 46.39 (s), 41.74
(s), 29.81 (s). HRMS (ESI) calcd. For [C20H19NO2]∙Na+ (M+Na)+: m/z
328.1308, found 328.1301.
are averaged over two individual experiments. The endo/exo and the ee
were determined by the chiral HPLC with the reproducibility of ±5%. The
conversions of 1b-h were roughly estimated form the crude 1H-NMR with
the reproducibility of ±10%. The conversion of 1a was calculated from the
HPLC with the reproducibility of ±5% by the following equation as
described previously.[4b]
Conversion of 1a (%) = A3a/(A3a + A1a/f)
where A1a and A3a are the HPLC areas of 1a and 3a, respectively. f is the
correction factor of 0.595 from a fitting curve (Figure S5).
Kinetic study
(3-(4-Chlorophenyl)bicyclo[2.2.1]hept-5-en-2-yl)(pyridin-2-
The kinetic studies of c-di-AMP·Cu2+ catalyzed Diels-Alder reactions
were evaluated upon the initial rate Vinit and the apparent second-order
rate constant kapp as described in the literature.[20] The Vinit of the Diels-
yl)methanone (3e): 1H NMR (400 MHz, CDCl3) δ 8.60 (d, J = 4.6 Hz,
1H), 7.93 (d, J = 7.8 Hz, 1H), 7.76 (t, J = 7.7 Hz, 1H), 7.43 – 7.32 (m, 1H),
7.18 (d, J = 10.5 Hz, 5H), 6.41 (d, J = 3.8 Hz, 1H), 5.82 – 5.69 (m, 1H),
4.45 – 4.33 (m, 1H), 3.47 (s, 1H), 3.33 (d, J = 4.9 Hz, 1H), 2.98 (s, 1H),
1.94 (d, J = 8.5 Hz, 1H), 1.70 – 1.62 (m, 1H). 13C NMR (100 MHz, CDCl3)
δ 148.87 (s), 139.25 (s), 136.91 (s), 133.00 (s), 128.96 (s), 128.42 (s),
127.68 – 127.15 (m), 122.23 (s), 54.45 (s), 49.27 (s), 48.71 (s), 48.16 (s),
45.10 (s), 29.70 (s), 13.72 (s). HRMS (ESI) calcd. For [C19H16NOCl]∙Na+
(M+Na)+: m/z 332.0813, found 332.0806.
Alder reaction between 1a and
2 was determined on a UV-Vis
spectrometer (Agilent Cary 3500) by monitoring the disappearance of the
absorption of 1a at 326 nm. To a 3 mL quartz cuvette (1 cm path length),
a small magnet was placed followed by the addition of an aqueous
solution of MES buffer (2 mL, 20 mM, pH 5.5) containing c-di-AMP (250
o
μM) and Cu(OTf)2 (50 μM). After stirring for 30 min at 4 C, a mixture of
1a (10 μL of 10 mM 1a in CH3CN, 0.1 μmol) and freshly distilled
cyclopentadiene 2 (10 μL of 1.0 M 2 in CH3CN, 10 μmol) was added and
the UV absorbance at 326 nm was started to collect every 20 seconds
with continuous stirring at 4 oC. The Vinit was calculated by the following
equation:
(3-(4-Nitrophenyl)bicyclo[2.2.1]hept-5-en-2-yl)(pyridin-2-
yl)methanone (3f): 1H NMR (400 MHz, CDCl3) δ 8.69 – 8.63 (m, 1H),
8.13 (d, J = 8.6 Hz, 2H), 8.01 (d, J = 7.8 Hz, 1H), 7.83 (dd, J = 10.9, 4.5
Hz, 1H), 7.51 – 7.40 (m, 3H), 6.48 (dd, J = 5.4, 3.3 Hz, 1H), 5.87 (dd, J =
5.5, 2.7 Hz, 1H), 4.53 – 4.41 (m, 1H), 3.60 (s, 1H), 3.53 (d, J = 5.1 Hz,
1H), 3.12 (s, 1H), 2.00 (d, J = 8.6 Hz, 1H), 1.67 (d, J = 8.4 Hz, 2H). 13C
NMR (100 MHz, CDCl3) δ 200.39 (s), 153.24 (s), 152.81 (s), 148.92 (s),
138.97 (s), 137.01 (s), 133.43 (s), 128.37 (s), 127.18 (s), 123.64 (s),
122.32 (s), 54.71 (s), 48.86 (d, J = 19.0 Hz), 48.20 (s), 45.76 (s). HRMS
(ESI) calcd. For [C19H16N2O3]∙Na+ (M+Na)+: m/z 343.1053, found
343.1044.
Vinit = d[A1a]/dt·(d·(ε1a - ε3a))-1
where d[A1a]/dt is the slope of the UV absorbance of 1a at 326 nm vs.
time during the initial conversion below 15%, d is the path length of the
cuvette, ε1a and ε3a are the molar extinction coefficients of 1a and 3a,
respectively.
The apparent second-order rate constant kapp was determined based
on the Vinit using the following equation:
(3-(Furan-2-yl)bicyclo[2.2.1]hept-5-en-2-yl)(pyridin-2-yl)methanone
(3g): 1H NMR (400 MHz, CDCl3) δ 8.78 – 8.63 (m), 8.05 – 7.94 (m), 7.88
– 7.76 (m), 7.47 (ddd, J = 7.5, 4.8, 1.2 Hz), 7.36 – 7.27 (m), 6.43 (ddd, J
= 18.2, 5.5, 3.2 Hz), 6.27 (dd, J = 3.1, 1.9 Hz), 6.10 (t, J = 4.4 Hz), 5.79
(dd, J = 5.6, 2.8 Hz), 4.59 (dd, J = 4.9, 3.6 Hz), 3.53 (s), 3.39 (d, J = 4.3
Hz), 3.07 (d, J = 1.3 Hz), 2.04 (d, J = 8.3 Hz), 1.61 – 1.48 (m). 13C NMR
(100 MHz, CDCl3) δ 200.45 (s), 158.18 (s), 153.46 (s), 148.99 (s), 141.16
(s), 138.46 (s), 136.98 (s), 132.90 (s), 127.08 (s), 122.35 (s), 110.13 (s),
104.89 (s), 52.22 (s), 49.22 (s), 48.66 (s), 48.33 (s), 39.75 (s), 29.81 (s).
HRMS (ESI) calcd. For [C19H16N2O3]∙Na+ (M+Na)+: m/z 288.0995, found
288.0984.
kapp = Vinit/([1a]0·[2]0)
where [1a]0 and [2]0 are the initial concentrations of 1a and 2,
respectively.
The rate acceleration effect krel was estimated by the ratio of
kapp,cat/kapp,uncat
,
krel = kapp,cat/kapp,uncat
where kapp,cat and kapp,uncat are the apparent second-order rate constants
with and without catalyst, respectively.
Pyridin-2-yl(3-(thiophen-2-yl)bicyclo[2.2.1]hept-5-en-2-yl)methanone
(3h): 1H NMR (400 MHz, CDCl3) δ 8.70 (d, J = 4.3 Hz), 8.00 (d, J = 7.9
Hz), 7.82 (td, J = 7.7, 1.7 Hz), 7.46 (ddd, J = 7.6, 4.9, 1.2 Hz), 7.15 –
7.08 (m), 6.96 – 6.86 (m), 6.45 (dd, J = 5.5, 3.2 Hz), 5.80 (dd, J = 5.6, 2.8
Hz), 4.58 (dd, J = 4.9, 3.5 Hz), 3.70 – 3.51 (m), 3.06 (s), 2.12 (d, J = 8.7
Hz), 1.64 (dd, J = 8.7, 1.7 Hz). 13C NMR (100 MHz, CDCl3) δ 200.50 (s),
153.46 (s), 149.06 (d, J = 9.5 Hz), 138.77 (s), 136.94 (s), 132.77 (s),
127.08 (s), 126.75 (s), 123.78 (s), 123.07 (s), 122.31 (s), 55.77 (s), 51.60
Acknowledgements
This work was financially supported by the National Natural
Science Foundation of China (21703132, 21773149, 21273142),
the Natural Science Foundation of Shaanxi Province of China
(2019JQ161) and the Fundamental Research Funds for the
Central Universities (GK201802001).
(s), 48.66 (d,
J = 3.6 Hz), 41.67 (s). HRMS (ESI) calcd. For
[C17H15NOS]∙Na+ (M+Na)+: m/z 304.0767, found 304.0761.
Typical procedure
Keywords: metalloribozyme • c-di-AMP
• enantioselective
To a 10 mL vial, a stock solution of c-di-AMP (final conc. 250 μM) and a
freshly prepared solution of Cu(OTf)2 (final conc. 50 μM) were
successively added to a MES buffer (20 mM, pH 5.5) to make a total
volume of 500 μL. After stirred for 30 min at 4 °C, a thoroughly mixed
solution of aza-chalcone 1 (10 μL of 0.1 M stock solution in CH3CN or
DMSO, 2 μmol) and freshly distilled cyclopentadiene 2 (21 μL, 250 μmol)
were added immediately. After the mixture was stirred for 3 days (1a for
24 h) at 4 °C, the aqueous media was extracted by diethyl ether (3 × 2
mL) and flushed on a short gel column. The combined organic solvent
was removed under reduced pressure and the residue was directly
analyzed by chiral HPLC with the eluents of hexane and isopropanol
using Daicel chiralpak columns (OD, ODH, AD, 250 × 4.6 mm). All data
catalysis Diels-Alder reaction primordial chemistry
•
•
[1]
[2]
G. Roelfes, B. L. Feringa, Angew. Chem. Int. Ed. 2005, 44, 3230-
3232; Angew. Chem. 2005, 117, 3294-3296.
a) D. Coquiere, B. L. Feringa, G. Roelfes, Angew. Chem. Int. Ed.
2007, 46, 9308-9311; Angew. Chem. 2007, 119, 9468-9671; b) N.
Shibata, H. Yasui, S. Nakamura, T. Toru, Synlett 2007, 1153-
1157; c) E. W. Dijk, B. L. Feringa, G. Roelfes, Tetrahedron-
Asymmetry 2008, 19, 2374-2377; d) A. J. Boersma, B. L. Feringa,
G. Roelfes, Angew. Chem. Int. Ed. 2009, 48, 3346-3348; Angew.
Chem. 2009, 121, 4490-4493; e) A. J. Boersma, D. Coquiere, D.
7
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000652
European Journal of Organic Chemistry
FULL PAPER
Geerdink, F. Rosati, B. L. Feringa, G. Roelfes, Nat. Chem. 2010
,
,
8, 726-729; e) B. Wang, Z. Wang, U. Javornik, Z. Xi, J. Plavec,
Sci. Rep. 2017, 7, 16550; f) C. Wang, M. Sinn, J. Stifel, A. C.
Heiler, A. Sommershof, J. S. Hartig, J. Am. Chem. Soc. 2017, 139,
16154-16160.
2, 991-995; f) R. P. Megens, G. Roelfes, Chem. Commun. 2012
48, 6366-6368; g) Y. Li, C. Wang, G. Jia, S. Lu, C. Li,
Tetrahedron 2013, 69, 6585-6590; h) A. Garcia-Fernandez, R. P.
Megens, L. Villarino, G. Roelfes, J. Am. Chem. Soc. 2016, 138,
16308-16314.
[17] a) P. Puig, A. Terron, V. Moreno, Inorg. Chim. Acta 1986, 123,
87-90; b) R. Cini, G. Giorgi, Inorg. Chim. Acta 1987, 137, 87-90; c)
P. Zhou, R. Shi, J.-F. Yao, C.-F. Sheng, H. Li, Coord. Chem. Rev.
2015, 292, 107-143.
[3]
[4]
a) P. Fournier, R. Fiammengo, A. Jaeschke, Angew. Chem. Int.
Ed. 2009, 48, 4426-4429; Angew. Chem. 2009, 121, 4490-4493;
b) J. Oelerich, G. Roelfes, Chem. Sci. 2013, 4, 2013-2017; c) A.
Rioz-Martinez, J. Oelerich, N. Segaud, G. Roelfes, Angew. Chem.
Int. Ed. 2016, 55, 14136-14140; Angew. Chem. 2016, 128,
14342-14346.
[18] H. Sigel, R. Griesser, Chem. Soc. Rev. 2005, 34, 875-900.
[19] B. L. Gaffney, E. Veliath, J. W. Zhao, R. A. Jones, Org. Lett. 2010
,
12, 3269-3271.
[20] S. Otto, F. Bertoncin, J. Engberts, J. Am. Chem. Soc. 1996, 118,
a) S. Roe, D. J. Ritson, T. Garner, M. Searle, J. E. Moses, Chem.
Commun. 2010, 46, 4309-4311; b) C. Wang, G. Jia, J. Zhou, Y. Li,
Y. Liu, S. Lu, C. Li, Angew. Chem. Int. Ed. 2012, 51, 9352-9355;
Angew. Chem. 2012, 124, 9486-9489; c) C. Wang, Y. Li, G. Jia, Y.
Liu, S. Lu, C. Li, Chem. Commun. 2012, 48, 6232-6234; d) M.
Wilking, U. Hennecke, Org. Biomol. Chem. 2013, 11, 6940-6945;
7702-7707.
e) Y. Li, G. Jia, C. Wang, M. Cheng, C. Li, ChemBioChem 2015
,
,
16, 618-624; f) S. Dey, A. Jaeschke, Angew. Chem. Int. Ed. 2015
54, 11279-11282; Angew. Chem. 2015, 127, 11432-11436; g) Y.
Li, M. Cheng, J. Hao, C. Wang, G. Jia, C. Li, Chem. Sci. 2015, 6,
5578-5585; h) M. Cheng, Y. Li, J. Zhou, G. Jia, S.-M. Lu, Y. Yang,
C. Li, Chem. Commun. 2016, 52, 9644-9647; i) S. Dey, C. L.
Ruehl, A. Jaeschke, Chem. Eur. J. 2017, 23, 12162-12170.
C. Wang, G. Jia, Y. Li, S. Zhang, C. Li, Chem. Commun. 2013, 49,
11161-11163.
[5]
[6]
[7]
[8]
X. Xu, W. Mao, F. Lin, J. Hu, Z. He, X. Weng, C.-J. Wang, X.
Zhou, Catal. Commun. 2016, 74, 16-18.
M. Chandra, S. K. Silverman, J. Am. Chem. Soc. 2008, 130,
2936-2937.
a) S. Park, L. Zheng, S. Kumakiri, S. Sakashita, H. Otomo, K.
Ikehata, H. Sugiyama, ACS Catal. 2014, 4, 4070-4073; b) M. Su,
M. Tomas-Gamasa, T. Carell, Chem. Sci. 2015, 6, 632-638; c) J.
J. Marek, R. P. Singh, A. Heuer, U. Hennecke, Chem. Eur. J.
2017, 23, 6004-6008.
[9]
J. Bai, X. Sun, H. Wang, C. Li, R. Qiao, J. Org. Chem. 2020, 85,
2010-2018.
[10] B. Seelig, S. Keiper, F. Stuhlmann, A. Jaschke, Angew. Chem. Int.
Ed. 2000, 39, 4576-4579; Angew. Chem. 2000, 112, 4764-4768.
[11] N. Duchemin, E. Benedetti, L. Bethge, S. Vonhoff, S. Klussmann,
J.-J. Vasseur, J. Cossy, M. Smietana, S. Arseniyadis, Chem.
Commun. 2016, 52, 8604-8607.
[12] J. J. Marek, U. Hennecke, Chem. Eur. J. 2017, 23, 6009-6013.
[13] C. Wang, M. Hao, Q. Qi, J. Dang, X. Dong, S. Lv, L. Xiong, H.
Gao, G. Jia, Y. Chen, J. S. Hartig, C. Li, Angew. Chem. Int. Ed.
2020, 59, 3444-3447; Angew. Chem. 2020, 132, 3472-3477.
[14] a) R. Hengge, Nat. Rev. Microbiol. 2009, 7, 263-273; b) R. M.
Corrigan, A. Gruendling, Nat. Rev. Microbiol. 2013, 11, 513-524; c)
U. Romling, M. Y. Galperin, M. Gomelsky, Microbiol. Mol. Biol.
Rev. 2013, 77, 1-52.
[15] a) P. Ross, H. Weinhouse, Y. Aloni, D. Michaeli, P.
Weinbergerohana, R. Mayer, S. Braun, E. Devroom, G. A.
Vandermarel, J. H. Vanboom, M. Benziman, Nature 1987, 325,
279-281; b) G. Witte, S. Hartung, K. Buttner, K. P. Hopfner, Mol.
Cell 2008, 30, 167-178; c) B. W. Davies, R. W. Bogard, T. S.
Young, J. J. Mekalanos, Cell 2012, 149, 358-370; d) P. Gao, M.
Ascano, Y. Wu, W. Barchet, B. L. Gaffney, T. Zillinger, A. A.
Serganov, Y. Liu, R. A. Jones, G. Hartmann, T. Tuschl, D. J. Patel,
Cell 2013, 153, 1094-1107; e) A. T. Whiteley, J. B. Eaglesham, C.
C. d. O. Mann, B. R. Morehouse, B. Lowey, E. A. Nieminen, O.
Danilchanka, D. S. King, A. S. Y. Lee, J. J. Mekalanos, P. J.
Kranzusch, Nature 2019, 567, 194-199.
[16] a) Z. Y. Zhang, S. Kim, B. L. Gaffney, R. A. Jones, J. Am. Chem.
Soc. 2006, 128, 7015-7024; b) S. Nakayama, I. Kelsey, J. Wang,
K. Roelofs, B. Stefane, Y. Luo, V. T. Lee, H. O. Sintim, J. Am.
Chem. Soc. 2011, 133, 4856-4864; c) S. Nakayama, I. Kelsey, J.
Wang, H. O. Sintim, Chem. Commun. 2011, 47, 4766-4768; d) S.
Nakayama, K. Roelofs, V. T. Lee, H. O. Sintim, Mol. Biosyst. 2012
,
8
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000652
European Journal of Organic Chemistry
FULL PAPER
Entry for the Table of Contents
c-di-AMP based enantioselective catalysis: A small cyclic RNA of c-di-AMP and Cu2+ ions could assemble into an
artificial metalloribozyme that is able to catalyze the enantioselective Diels Alder reactions with up to 80% ee. The
–
presence of c-di-AMP in the metalloribozyme not only achieves the chirality transfer but also gives rise to a significant rate
enhancement.
9
This article is protected by copyright. All rights reserved.