Development of DNA Metalloenzymes Using a Rational Design Approach and Application in the Asymmetric Diels-Alder Reaction

Article abstract of DOI:10.1021/acscatal.5b01046

We report here DNA metalloenzymes that catalyzed the asymmetric Diels-Alder reactions with high conversion, excellent endo/exo selectivities, and enantioselectivities up to -97% ee. Their catalytic-pocket architectures were organized using a rational desi

Full text of DOI:10.1021/acscatal.5b01046

Subscriber access provided by UNIV OF MISSISSIPPI
Letter
Development of DNA Metalloenzymes Using a Rational Design
Approach and Application in the Asymmetric Diels–Alder Reaction
Soyoung Park, Izumi Okamura, Sohei Sakashita, Ji Hye Yum, Acharya Chiranjit, Li Gao, and Hiroshi Sugiyama
ACS Catal., Just Accepted Manuscript • Publication Date (Web): 10 Jul 2015
Downloaded from http://pubs.acs.org on July 11, 2015
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Catalysis is published by the American Chemical Society. 1155 Sixteenth Street
N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 15
ACS Catalysis
1
2
3
4
5
6
7
8
9
Development of DNA Metalloenzymes Using a Rational
Design Approach and Application in the Asymmetric
Diels–Alder Reaction
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Soyoung Park,*, 1 Izumi Okamura, 1Sohei Sakashita,1 Ji Hye Yum,3
Acharya Chiranjit,4 Li Gao,5 and Hiroshi Sugiyama*,1,2
1Department of Chemistry, Graduate School of Science, Kyoto University,
Kitashirakawaꢀoiwakecho, Sakyoꢀku, Kyoto 606ꢀ8502, Japan
2Institute for Integrated CellꢀMaterial Sciences (iCeMS), Kyoto University,
Yoshidaꢀushinomiyacho, Sakyoꢀku, Kyoto 606ꢀ8501, Japan
3Department of Chemistry, Kyungpook National University, 80 Daehakro, Bukgu,
Daegu, 702ꢀ701, Korea
4Department of Chemistry, CSIRꢀIndian Institute of Chemical Biology, Raja S. C.
Mullick Road, Jadavpur, Kolkata 700ꢀ032, India
5Department für Chemie, LudwigꢀMaximiliansꢀUniversität, Butenandtstrasse 5ꢀ13,
81377 München, Germany
*Corresponding author: Dr. Soyoung Park, Prof. Dr. Hiroshi Sugiyama
Tel.: (+)81ꢀ75ꢀ753ꢀ4002; Fax: (+)81ꢀ75ꢀ753ꢀ3670
Eꢀmail: oleesy@kuchem.kyotoꢀu.ac.jp, hs@kuchem.kyotoꢀu.ac.jp (H.S.)
1
ACS Paragon Plus Environment

ACS Catalysis
Page 2 of 15
1
2
3
4
5
6
Received Date: (to be automatically inserted after your manuscript is accepted if required
7
8
according to the journal that you are submitting your paper to)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Abstract
We report here DNA metalloenzymes that catalyzed the asymmetric Diels–Alder
reactions with high conversion, excellent endo/exo selectivities, and enantioselectivities
up to –97% ee. Their catalyticꢀpocket architectures were organized using a rational
design strategy based on the Cu(II) ion, the composition of nucleobases, and the
incorporation of flexible linkers. Without using the mirror image of BꢀDNA, DNA
metalloenzymes afforded the opposite enantiomer of the Diels–Alder product compared
with those obtained using a supramolecular Cu(II)–dmbpy/st–DNA catalyst system.
Furthermore, we devised DNA metalloenzymes without the incorporation of an artificial
binding ligand and successfully performed Diels–Alder carbon–carbon bondꢀforming
reactions. This study provides a new perspective on the catalytic repertoire of nucleic
acids in the realm of proteinꢀdominated metalloenzymes.
KEYWORDS
DNA, Metalloenzyme, asymmetric catalysis, DielsꢀAlder reaction, Cu(II) ion, ligand
freedom, enantiomeric preference
2
ACS Paragon Plus Environment

Page 3 of 15
ACS Catalysis
1
2
3
4
5
6
7
8
9
In nature, a variety of metalloenzymes catalyze biologically essential chemical
reactions under mild reaction conditions with high efficiency, definitive substrate
specificity, and excellent enantioselectivity, beyond the performance of synthetic
catalysts. Taking inspiration from the superior characteristics of metalloenzymes, the
development of artificial metalloenzymes that combine metalꢀassisted catalysis with a
chiral biomacromolecule has received attention as an attractive research concept for
developing environmentally friendly and efficient catalysts for the synthesis of
enantiomerically pure compounds.1 Among the biomolecules that are used as the chiral
scaffold, DNA is a notable chiral source because of its many advantageous properties,
such as its unique helical chirality and chemical stability, and the diverse
threeꢀdimensional structures created by the simple fourꢀletter alphabet. In 2005, Feringa
and Roelfes developed the novel concept of DNAꢀbased asymmetric catalysis with a
metal complex, DNA, and a binding ligand, and reported Diels–Alder reactions.2 Since
then, a series of DNA metalloenzymes have been developed and applied successfully to
the important carbon–carbon or carbon–heteroatom bondꢀforming reactions, such as the
Michael additions, Friedel–Crafts alkylations, synꢀhydrations, and fluorination.3,4
Regarding the structural diversity of the DNA, Gꢀquadruplex DNA metalloenzymes
have been developed and investigated based on the relationship between the
conformation of the quadruplex DNA and the catalytic reaction.5 In addition,
remarkable approaches have been reported to control the enantiomeric preference of the
DNAꢀbased asymmetric catalysis. Roelfes and coworkers reported that the
enantioselectivity of the products in the Diels–Alder and Friedel–Crafts alkylation
reactions could be controlled by changing the denticity of the ligand coordinated to the
Cu(II) ion.6 Smietana and Arseniyadis demonstrated that the leftꢀhanded DNAꢀbased
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
catalyst made from Lꢀnucleic acids gave rise to the opposite enantiomeric outcome in
the Cu(II)ꢀcatalyzed Friedel–Crafts alkylation and Michael addition reactions compared
with the natural rightꢀhanded DNA.7
Very recently, we devised artificial DNA metalloenzymes with intrastrand
bipyridine ligands via direct incorporation into the phosphate backbone, and performed
asymmetric intramolecular Friedel–Crafts alkylation to extend our understanding of
DNAꢀbased asymmetric catalysis.8 In a previous study, we identified the critical factors
that affect the reactivity and enantioselectivity of DNAꢀbased asymmetric catalysis,
3
ACS Paragon Plus Environment

ACS Catalysis
Page 4 of 15
1
2
3
4
5
6
7
8
such as the counter base of metal complexes, neighboring bases in the active site, and
disposition of the active site. Based on these observations, we established a systematic
strategy for the development of DNA metalloenzymes via the rational creation of an
active site. Herein, we report the development and application of copperꢀcontaining
DNA metalloenzymes for asymmetric Diels–Alder reactions.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In our previous study, we found that cytosine exhibited distinct features compared
with other bases (A, T, and G) in the determination of the catalytic capability of the
Cu(II)ꢀbipyridine–DNA conjugate catalyst. For instance, the DNAꢀbased hybrid
catalysts produced by the combination of an intrastrand bipyridine ligand and cytosine
as a counter base afforded the best result regarding the cyclized product, with a yield of
71% and enantioselectivity of 84% ee in intramolecular Friedel–Crafts alkylations.8
Therefore, we have focused on cytosine at the active site for the creation of the
coordination environment, and designed a DNA metalloenzyme via the direct
incorporation of flexible linkers to control the size of the catalytic site. In this regard,
we introduced a triethylene glycol linker into the DNA phosphate backbone and
generated a variety of Cu(II)–DNA–triethylene glycol conjugates.9
Oligo(ethylene glycol)s are used widely as linkers or spacers because they are
inexpensive, water soluble, and commercially available in a broad range of
molecularꢀweight distributions. The phosphoramidites of triethylene glycol linkers were
readily synthesized by phosphitylation and incorporated into the DNA oligonucleotides
by automated solidꢀphase synthesis (see SI, Scheme S1). We generated DNA
metalloenzymes based on 13ꢀmer DNA oligonucleotides, 5′–d(GCATGXEYACGGT)–
3′/5′–d(ACCGTXCYCATGC)–3′, where E = a triethylene glycol linker.10 The
asymmetric Diels–Alder reaction between azaꢀchalcone 1a and cyclopentadiene 2 was
performed to prove the catalytic activity of Cu(II)–DNA–triethylene glycol conjugates.
The results are summarized in Table 1. Under the reaction conditions (20 mM MOPS
buffer at pH 6.5, 3 mol% of Cu(NO3)2, and 4 mol% of DNA), the present Cu(II)–DNA–
ethylene glycol conjugates afforded the Diels–Alder adduct with significant conversion,
endo/exo selectivity, and a promising enantioselectivity (entry 1 in Table 1). We
examined the neighboring base pairs of the cytosine and the triethylene glycol linker. As
4
ACS Paragon Plus Environment

Page 5 of 15
ACS Catalysis
1
2
3
4
5
6
7
8
shown in entries 1–6 in Table 1, we found that the enantioselectivities were influenced
greatly by the neighboring base pairs of the cytosine and the triethylene glycol linker.
For example, the DNA metalloenzyme comprising ODN3/ODN12 (–CEC–/–GCG–)
and the Cu(II) ion gave the product with –13% ee, whereas the DNA metalloenzyme
comprising ODN5/ODN14 (–TET–/–ACA–) and the Cu(II) ion had a markedly
increased enantioselectivity, up to –81%.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To our great delight, the DNA metalloenzyme generated by the combination of
cytosine and the neighboring base pair –AET– afforded the Diels–Alder adduct with
high conversion (99%) and excellent enantioselectivity (–94% ee), together with very
high endo/exo selectivity (entry 6 in Table 1). Subsequently, we replaced the triethylene
glycol linker with more hydrophobic alkyl linkers. After considering the alkyl chain
length, octyl and propyl linkers, such as ODN7 and ODN8, were introduced into the
DNA strand and investigated. As shown in entries 7 and 8, the DNA metalloenzymes
that included alkyl linkers also led the completion of Diels–Alder reactions successfully
and gave the desired products with high conversions and excellent endo/exo selectivities
and enantioselectivities.
When we performed the Diels–Alder reaction using a DNA metalloenzyme with a
propyl linker, the corresponding product was obtained up to –97% ee. The control
experiment using ODN9/ODN15 in the presence of Cu(II) ions resulted in low
selectivities, which suggests that the security of the space via the introduction of
ethylene glycol or alkyl linkers is important for affording high enantioselectivity (entry
9 in Table 1). When the reaction was performed using a single DNA–ethylene glycol
conjugate strand, ODN6, and Cu(II) ions, very low enantioselectivity was obtained (see
entry 10 in Table 1). The results of this experiment indicate that the doubleꢀhelical
structure of the Cu(II)–DNA–triethylene glycol conjugates is important for inducing
enantioselectivity in the asymmetric Diels–Alder reaction.
5
ACS Paragon Plus Environment

ACS Catalysis
Page 6 of 15
1
2
3
4
5
6
7
8
9
Table 1. Asymmetric Diels–Alder reaction catalyzed by DNA metalloenzymes
containing Cu(II) and a triethylene glycol (alkyl) linker.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
O
N
N
DNA (4 mol%l), Cu(NO3)2 (3 mol%)
MOPS buffer, 5 °C, 3 days
+
+
O
N
1a
2
3a
(
endo
)
3a (exo)
O
O
3K
8K
E
entrya
DNA sequences
ee (%)
endo/exo conversion (%)
5'-GCATGGECACGGT-3' (ODN1)
3'-CGTACCCGTGCCA-5' (ODN10)
−36
14/1
10/1
16/1
22/1
20/1
99
57
75
70
99
1
2
5'-GCATGGEGACGGT-3' (ODN2)
3'-CGTACCCCTGCCA-5' (ODN11)
−41
−13
−58
−81
5'-GCATGCECACGGT-3' (ODN3)
3'-CGTACGCGTGCCA-5' (ODN12)
3
5'-GCATGAEAACGGT-3' (ODN4)
3'-CGTACTCTTGCCA-5' (ODN13)
4
5'-GCATGTETACGGT-3' (ODN5)
3'-CGTACACATGCCA-5' (ODN14)
5
5'-GCATGAETACGGT-3' (ODN6)
3'-CGTACTCATGCCA-5' (ODN15)
6b
−94
−87
−97
>40/1
36/1
99
95
98
5'-GCATGA8KTACGGT-3' (ODN7)
3'-CGTACTCATGCCA-5' (ODN15)
7b
8b
5'-GCATGA3KTACGGT-3' (ODN8)
3'-CGTACTCATGCCA-5' (ODN15)
>40/1
5'-GCATGAGTACGGT-3' (ODN9)
3'-CGTACTCATGCCA-5' (ODN15)
9
10
a
−13
−10
8/1
5/1
82
87
5'-GCATGAETACGGT-3' (ODN6)
Experiments were carried out using 3.3 mM azaꢀchalcone, 80 mM cyclopentadiene,
6
ACS Paragon Plus Environment

Page 7 of 15
ACS Catalysis
1
2
3
4
5
6
0.13 mM DNA, and 0.1 mM Cu(NO3)2 at 5 °C in 20 mM MOPS buffer (pH 6.5) for 3
7
8
9
days. The conversion and enantioselectivities were determined by chiral HPLC analysis.
b
Results represent the average value of more than two experiments. The
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
enantioselectivities were reproducible within ± 5%. The yields were reproducible within
± 10%.
The enantioselectivity of the Diels–Alder products was determined by chiral HPLC
based on the literature.2,4j,6 Interestingly, we found that the present DNA metalloenzyme
afforded the opposite enantiomeric outcome compared with the previously reported
Cu(II)–dmbpy/st–DNA catalytic system.4a,13 In addition, the circular dichroism spectra
of the DNA duplex formed by ODN6/ODN15 or ODN8/ODN15 in the presence of the
Cu(II) complex indicated a typical right-handed BꢀDNA structure (see SI). This result
clearly demonstrates that the enantiomeric preference of the reaction could be changed
readily via the introduction of simple linear linkers, such as an ethylene glycol linker
and an alkyl chain linker, to the DNA phosphate backbone; i.e., the present study
suggests a new alternative: that the enantioselectivity could be reversed completely by
perturbing only the catalytic site in the DNA duplex, without an overall change in
helical chirality or in the employment of special ligands.6,7
To confirm the catalytic ability of the present DNA metalloenzymes for asymmetric
Diels–Alder reactions, the scope of dienophiles with various functional groups was
investigated under optimized reaction conditions (Table 2). Using a Cu(II)–DNA–
triethylene glycol conjugate, dienophiles containing the substituted phenyl group with
electronꢀwithdrawing or ꢀdonating substituents gave the corresponding product in
almost full conversion with high enantioselectivity (entries 1–3 in Table 2). In the case
of a dienophile including a pꢀnitro group on the phenyl ring, a Cu(II)–DNA–propyl
linker conjugate was used for the reaction, to avoid the electrostatic repulsion between
the nitro group and the triethylene glycol linker.11 Instead of 2ꢀacyl pyridyl, the use of
the 2ꢀacyl imidazole group also led to a high product enantioselectivity (–88% ee, entry
5 in Table 2). In all cases examined, DNA metalloenzymes provided the opposite
enantiomers of the Diels–Alder product compared with those obtained using a
supramolecular Cu(II)–dmbpy/st–DNA catalyst system.
7
ACS Paragon Plus Environment

ACS Catalysis
Page 8 of 15
1
2
3
4
5
6
7
8
Table 2. Substrate scope of the asymmetric Diels–Alder reaction by DNA
metalloenzymes.
O
R2
9
O
DNA (4 mol%), Cu(NO3)2 (3 mol%)
MOPS buffer, 5 °C, 3 days
R1
+
+
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
R1
R2
R1
3b−f (endo)
R2
3b−f (exo)
O
1b−f
2
1b: R1= 2-pyridyl, R2= p-MeOC6H4
1c: R1= 2-pyridyl, R2= o-ClC6H4
1d: R1= 2-pyridyl, R2= p-BrC6H4
1e: R1= 2-pyridyl, R2= p-NO2C6H4
1f: R1= 2-(1-methylimidazolyl), R2= C6H5
entrya
substrate
ee (%)
endo/exo
conversion (%)
1b
1
−92
−97
−86
−96
−88
>40/1
>40/1
25/1
99
99
99
99
99
1c
1d
1e
1f
2
3
4b
5
>40/1
21/1
a
Experiments were carried out using 3.3 mM azaꢀchalcone, 80 mM cyclopentadiene,
0.13 mM DNA (ODN6/ODN15), and 0.1 mM Cu(NO3)2 at 5 °C in 20 mM MOPS
buffer (pH 6.5) for 3 days. The conversion and enantioselectivities were determined by
chiral HPLC analysis. b ODN8/ODN15 was used in this experiment.
The results observed here clearly indicate that DNA metalloenzymes could perform
the asymmetric Diels–Alder reaction successfully, even if they had no binding ligand
for Cu(II) ions, such as bipyridine or phenanthroline. Therefore, we devised DNA
metalloenzymes comprising three oligonucleotides and Cu(II) ions. To generate ample
space for the active site, we excluded the counter base for cytosine. The template
oligonucleotide strand (15ꢀmer) with cytosine in the center was hybridized with two
kinds of complementary oligonucleotides (7ꢀmers) in the presence of Cu(II) ions, and
gave rise to DNA metalloenzymes. The catalytic ability of these DNA metalloenzymes
was investigated in the asymmetric Diels–Alder reaction. Surprisingly, as shown in
Table 3, the designed DNA metalloenzymes afforded the Diels–Alder adducts in almost
8
ACS Paragon Plus Environment

Page 9 of 15
ACS Catalysis
1
2
3
4
5
6
7
8
full conversion with very high enantioselectivities. Regarding the neighboring base
pairs of the cytosine, similar to Cu(II)–DNA–ethylene glycol (alkyl linker) conjugates, a
neighboring A–T pair gave higher enantioselectivity (–64% ee, entry 1 in Table 3) than
did a C–G pair (–17% ee, entry 2 in Table 3) in the present reaction. Considering the
proximity to cytosine, the presence of a 3′ꢀ or 5′ꢀend phosphate group on the short
complementary oligonucleotides increased the enantioselectivity of the Diels–Alder
product. As shown in entry 5 in Table 3, a DNA metalloenzyme containing 5′ꢀ and
3′ꢀend phosphorylated complementary oligonucleotides afforded the Diels–Alder
product with –83% ee. Furthermore, DNA metalloenzyme containing an abasic site
opposite the counter base cytosine exhibited high enantioselectivity up to –96% (entry 6
in Table 3).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Li and colleagues reported enantioselective Friedel–Crafts alkylations using a
human telomeric G4DNA metalloenzyme that was assembled using G4DNA and Cu(II)
ions without additional ligands (up to 75% ee).5c In this study, we demonstrated clearly
that DNA metalloenzymes comprising a native DNA duplex and Cu(II) ions without any
artificial binding ligand can function as the asymmetric catalyst for the Diels–Alder
reaction to give almost full conversion, high enantioselectivity, and an excellent
endo/exo ratio. At this stage the exact location of the Cu(II) ion remained distinctly
unproven, however, earlier studies have reported Cu(II)ꢀcytosine (or cytidine) binding
mode based on N(3) preference.12 Also, the examined DNA metalloenzyme libraries
suggest that the combination of cytosine and copper plays an important role in the
creation of the catalytic pocket to induce high enantioselectivity in the Diels–Alder
reaction (Figure S2 and S3 in SI). Considering the correlation between
enantioselectivity and the structure of DNA metalloenzymes, the presence of triethylene
glycol (alkyl) linkers or phosphate groups on the complementary strand corresponding
to the template strand containing cytosine might provide the steric conformation to
induce high enantioselectivity of the product (Figure S8). Further studies are under way
to explore the present DNA metalloenzymes in greater detail.
9
ACS Paragon Plus Environment

ACS Catalysis
Page 10 of 15
1
2
3
4
5
6
7
8
Table 3. Investigation of DNA metalloenzymes for the asymmetric Diels–Alder
reaction.
O
O
9
N
N
DNA (4 mol%), Cu(NO3)2 (3 mol%)
MOPS buffer, 5 °C, 1 day
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
+
O
N
1a
2
3a (endo)
3a (exo)
DNA sequences
conversion
(%)
entrya
ee (%)
−20
endo/exo
15/1
(short strands 3’ to 5’/template 5’ to 3’)
GTGGCAC GGTACGA
75
98
1
CACCGTGCCCATGCT
2b
GTGGCAT AGTACGA
−64
25/1
CACCGTACTCATGCT
GTGGCATpAGTACGA
3b,c
4b,c
5b,c
−74
−80
−83
33/1
29/1
29/1
95
96
99
CACCGTACTCATGCT
GTGGCATp AGTACGA
CACCGTACTCATGCT
GTGGCATp pAGTACGA
CACCGTACTCATGCT
GTGGCATFAGTACGA
CACCGTACTCATGCT
6b,d
−96
−6
32/1
14/1
99
91
7
CACCGTACTCATGCT
a
Experiments were carried out using 3.3 mM azaꢀchalcone, 80 mM cyclopentadiene,
0.13 mM DNA, and 0.1 mM Cu(NO3)2 at 5 °C in 20 mM MOPS buffer (pH 6.5) for 1
day. The conversion and enantioselectivities were determined by chiral HPLC analysis.
b
Results represent the average value of more than two experiments. The
enantioselectivities were reproducible within ± 5%. The yields were reproducible within
± 10%. The Tp means that the T at the 5′ position contains a phosphate group. The A
c
p
means that the at the 3′ position contains phosphate group.
d5′ꢀOꢀDimethoxytritylꢀ1′,2′ꢀdideoxyriboseꢀ3′ꢀ[(2ꢀcyanoethyl)ꢀ(N,Nꢀdiisopropyl)]ꢀ
A
a
phosphoramidite was used as a dSpacer (F).
10
ACS Paragon Plus Environment

Page 11 of 15
ACS Catalysis
1
2
3
4
5
6
7
8
9
In conclusion, we developed Cu(II)ꢀcontaining DNA metalloenzymes via the
direct introduction of flexible linkers and successfully applied them in the Diels–Alder
carbonꢀcarbon bondꢀforming reactions. The devised DNA metalloenzymes afforded
Diels–Alder products with high conversion, excellent endo/exo selectivities, and
enantioselectivities up to –97% ee. In addition, we established a systematic strategy for
the development of ligand-free DNA metalloenzymes via the construction of an active
site. In prebiotic world, the role of DNA has been out of the spotlight due to the lack of
catalytic ability, however our finding is provocative of curiosity; DNA with a dangling
base may function as a catalyst through metal binding. Further investigations of the
application of DNA metalloenzymes are currently ongoing in our laboratory.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ASSOCIATED CONTENT
Supporting Information
Synthetic routes of triethylene glycol and alkyl linkers, characterization data of new
compounds, HPLC data, ESIꢀTOFꢀMass data and CD spectra of oligonucleotides. This
material is available free of charge via the Internet at http://pubs.acs.org.
Note
The authors declare no competing financial interest.
11
ACS Paragon Plus Environment

ACS Catalysis
Page 12 of 15
1
2
3
4
5
6
7
ACKNOWLEDGMENT
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
We express sincere thanks for a GrantꢀinꢀAid Priority Research from Japan Society for
the Promotion of Science (JSPS) and the grant from the WPI program (iCeMS, Kyoto
University).
REFERENCES
[1] For reviews, see: a) B. G. Davis, Curr. Opin. Biotechnol. 2003, 14, 379–386; b) C.
M. Thomas, T. R. Ward, Chem. Soc. Rev. 2005, 34, 337–346; c) R. Krämer, Angew.
Chem. Int. Ed. 2006, 45, 858–860; d) Y. Lu, Angew. Chem. Int. Ed. 2006, 45, 5588–
5601; e) C. Letondor, T. R. Ward, ChemBioChem 2006, 7, 1845–1852; f) M. D.
Mihovilovic, J. Chem. Technol. Biotechnol. 2007, 82, 1067–1071.
[2] G. Roelfes, B. L. Feringa. Angew. Chem. Int. Ed. 2005, 44, 3230–3232.
[3] For reviews, see: a) G. Roelfes. Mol. BioSyst. 2007, 3, 126–135; b) A. J. Boersma,
R. P. Megens, B. L. Feringa, G. Roelfes. Chem. Soc. Rev, 2010, 39, 2083–2092; c) S. K.
Silverman. Angew. Chem. Int. Ed. 2010, 49, 7180–7201; d) S. Park, H. Sugiyama.
Angew. Chem. Int. Ed. 2010, 49, 3870–3878; e) S. Park, H. Sugiyama. Molecules 2012,
17, 12792–12803.
[4] a) G. Roelfes, A. J. Boersma, B. L. Feringa. Chem. Commun. 2006, 635–637; b) D.
Coquière, B. L. Feringa, G. Roelfes. Angew. Chem. Int. Ed. 2007, 46, 9308–9311; c) A.
J. Boersma, B. L. Feringa, G. Roelfes. Org. Lett. 2007, 9, 3647–3650; d) N. Shibata, H.
Yasui, S. Nakamura, T. Toru. T. Syn. Lett. 2007, 1153–1157; e) A. J. Boersma, B. L.
12
ACS Paragon Plus Environment

Page 13 of 15
ACS Catalysis
1
2
3
4
5
6
7
8
Feringa, G. Roelfes. Angew. Chem. Int. Ed. 2009, 48, 3346–3348; f) P. Fournier, R.
Fiammengo, A. Jäschke, Angew. Chem. Int. Ed. 2009, 48, 4426–4429; g) A. J.
Boersma, D. Coquière, D. Greerdink, F. Rosati, B. L. Feringa, G. Roelfes. Nature
Chem. 2010, 2, 991–995; h) S. Park, K. Ikehata, R. Watabe, Y. Hidaka, A. Rajendran,
H. Sugiyama. Chem. Commun. 2012, 48, 10398–10400. i) J. Oelerich, G. Roelfes.
Chem. Sci. 2013, 4, 2013–2017; j) S. Park, K. Ikehata, H. Sugiyama. Biomaterials
Science 2013, 1, 1034–1036; k) G. P. Petrova, Z. Ke, S. Park, H. Sugiyama, K.
Morokuma. Chem. Phys. Lett. 2014, 600, 87–95; l) E. Benedetti, N. Duchemin, L.
Bethge, S. Vonhoff, A. Klussmann, J.ꢀJ. Vasseur, J. Cossy, M. Smietana, S.
Arseniyadis. Chem. Commun. 2015, 51, 6076–6079.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[5] a) S. Roe, D. J. Ritson, T. Garner, M. Searle, J. E. Moses. Chem. Commun. 2010,
46, 4309−4311; b) C. Wang, Y. Li, G. Jia, Y. Liu, S. Lu, C. Li. Chem. Commun. 2012,
48, 6232–6234; c) C. Wang, G. Jia, J. Zhou, Y. Li, Y. Liu, S. Lu, C. Li. Angew. Chem.
Int. Ed. 2012, 51, 9352–9355; d) M. Wilking, U. Hennecke. Org. Biomol. Chem. 2013,
11, 6940−6945.
[6] A. J. Boersma, B. de Bruin, B. L. Feringa, G. Roelfes. Chem. Commun. 2012, 48,
2394–2396.
[7] J. Wang, E. Benedetti, L. Bethge, S. Vonhoff, A. Klussmann, J.ꢀJ. Vasseur, J.
Cossy, M. Smietana, S. Arseniyadis. Angew. Chem. Int. Ed. 2013, 52, 11546–11549.
[8] S. Park, L. Zheng, S. Kumakiri, S. Sakashita, H. Otomo, K. Ikehata, H. Sugiyama.
ACS Catalysis 2014, 4, 4070–4073.
13
ACS Paragon Plus Environment

ACS Catalysis
Page 14 of 15
1
2
3
4
5
6
7
8
[9] a) U. Jakobsen, K. Rohr, S. Vogel. Nucleosides, Nucleotides, and Nucleic Acids,
2007, 26, 1419–1422; b) N. S. Oltra, G. Roelfes. Chem. Commun. 2008, 6039–6041; c)
L. Gjonaj, G. Roelfes. ChemCatChem, 2013, 5, 1718–1721.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[10] Complementary strands of ODN6 containing natural bases (A, T, and G) were also
examined to investigate the counter base effect. The DNA metalloenzymes by the
combination of triethylene glycol and a counter base A, T, or G afforded lower
enantioselectivities of product compared with a counter base C (entries 1ꢀ3 in Table
S2).
[11] In the case of dienophile including a pꢀnitro group on the phenyl ring, a decrease in
enantioselectivity was observed using a Cu(II)–DNA–triethylene glycol conjugate (99%
conversion, endo/exo = 18/1, –59% ee).
[12] a) D. Tran Qui, E, Palacios. Acta Cryst. 1990, C46, 1220–1223. b) I.
Samasundaram, M. K. Kommiya, M. Palaniandavar. J. Chem. Soc., Dalton Trans. 1991,
2083–2089; c) A. GarcíaꢀRaso, J. J. Fiol, B. Adrover, V. Moreno, E. Molins, I. Mata. J.
Chem. Soc., Dalton Trans. 1998, 1031–1036; d) A. GarcíaꢀRaso, J. J. Fiol, B. Adrover,
A. Caubet, E. Espinosa, I. Mata. E. Molins, Polyhedron 2002, 21, 1197–1201; e) K.ꢀH.
Cho, S.ꢀW. Joo. Bull. Korean Chem. Soc. 2008, 29, 69–75; f) D. Krilov, V, Gomzi, J. N.
Herak. Spectrochimica Acta Part A. 2008, 70, 1238–1242.
[13] The control experiment using a Cu(II)ꢀdmbpy/ODN6/ODN15 system afforded the
DielsꢀAlder product with –87% ee, endo/exo ratio of 36:1 and 97% conversion.
14
ACS Paragon Plus Environment

Page 15 of 15
ACS Catalysis
1
2
3
4
5
6
7
8
O
N
+
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
T
A
Ra onal
design
O
N
+
C
O
G
N
Up to e.e.= −97%
endo/exo= >40/1
conv.=99%
Ligand-free !
DNA metalloenzyme
Chiral products
15
ACS Paragon Plus Environment