Bioinspired Catalysis: Self-Assembly of a Protein and DNA as a Catalyst for the Aldol Reaction in Aqueous Media

Article abstract of DOI:10.1055/s-0036-1591854

An interesting bioinspired catalyst formed from readily available DNA and a protein through electrostatic interaction in situ proved to be efficient in catalyzing aldol reactions under mild conditions in water. By using a self-assembling catalytic system

Full text of DOI:10.1055/s-0036-1591854

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© Georg Thieme Verlag Stuttgart · New York
2017, 28, A–F
letter
A
en
H. Liu et al.
Letter
Synlett
Bioinspired Catalysis: Self-Assembly of a Protein and DNA as a
Catalyst for the Aldol Reaction in Aqueous Media
Hongxin Liua,b
Guangxun Li*a,b
CHO
Ying-wei Wangc
Shiqi Zhangc
Zhuo Tang*a,b
OH
O
R
Ar
O
DNA
+
R
Ar
protamine
a Natural Products Research Center, Chengdu Institution of Biology, Chinese
Academy of Science, Chengdu, Sichuan 610041, P. R. of China
b University of Chinese Academy of Sciences, 100049, P. R. of China
c College of Chemical Engineering, Sichuan University, Chengdu, Sichuan
610041, P. R. of China
protein
buffer
14 examples
61–96% yield
13–56% ee
tangzhuo@cib.ac.cn
ligx@cib.ac.cn
Received: 08.09.2017
Accepted after revision: 09.11.2017
Published online: 20.12.2017
plexes into a chiral DNA scaffold, a variety of asymmetric
catalytic reactions have been developed, and have achieved
good enantioselectivities.23–28 In contrast, and to the best of
our knowledge, no metal-free DNA complex has been suc-
cessfully employed in promoting asymmetric transforma-
tions. Inspired by the simple DNA-catalyzed racemic aldol
reaction in aqueous conditions (Scheme 1c),29 we became
interested in exploring novel metal-free DNA complexes as
catalysts for asymmetric reactions.
Here, we report our preliminary results on the DNA–
protamine complex catalyzed asymmetric aldol reaction
under aqueous conditions to give the desired products in
high yields and moderate enantioselectivities (Scheme 1e).
Importantly, the present results indicated the catalytic po-
tential of the self-assembly of DNA with a protein in asym-
metric transformations.
Because the sugar phosphate ester backbone of DNA is
negatively charged in aqueous phases, we speculated that a
positively charged moiety might bind to DNA through elec-
trostatic interactions in aqueous solution to form an inter-
esting catalytic system. For example, basic amino acids,
such as L-lysine or L-arginine,30–34 are positively charged
through protonation in aqueous solution under neutral or
acidic pH conditions, which might enable them to bind to
DNA molecules through electrostatic interactions. Impor-
tantly, the free amino group of the DNA complex can acti-
vate a ketone donor, thereby acting as an aldolase mimic.
To verify our hypothesis, commercial DNA, derived from
salmon sperm, and various amino acids were tested in a
model aldol reaction of 2-nitrobenzaldehyde (1a) with ace-
tone (2a) in a phosphate buffer (pH = 7.5) (Table 1). DNA
alone afforded only a trace amount of the product (Table 1,
entry 1), whereas L-lysine alone provided product 3a in 45%
yield, but with extremely low enantioselectivity (3% ee; en-
try 2). However, when a DNA–L-lysine complex was em-
DOI: 10.1055/s-0036-1591854; Art ID: st-2017-w0674-l
Abstract An interesting bioinspired catalyst formed from readily avail-
able DNA and a protein through electrostatic interaction in situ proved
to be efficient in catalyzing aldol reactions under mild conditions in wa-
ter. By using a self-assembling catalytic system formed from protamine
and DNA, aldol adducts were obtained with high yields and moderate
enantioselectivities. Preliminary experiments demonstrated that the
chirality of the DNA could be effectively transferred to the reaction
product through the bound molecules or proteins.
Key words self-assembly, protamine, DNA, electrostatic interaction,
bioinspired catalysis, aldol reaction
The key principles and concepts of biological systems
have always inspired chemists to develop new chemical re-
actions and catalytic processes1–3 in so-called ‘biomimetic
chemistry’.4–9 For example, the aldol reaction, which is fre-
quently induced by aldolases in biological systems,10–13 has
also been developed as an important organic reaction in or-
ganic synthesis. The aldolase-catalyzed mechanism via an
enamine or enol transition state (Scheme 1a)13 has become
more and more popular, and has encouraged numerous
successes in antibody catalysis and organocatalysis.14 De-
spite these achievements, mimicking the behavior of en-
zymes by employing other ‘biomolecules’ is still an attrac-
tive target, but has rarely been studied. In this field, small
peptides have been successfully used as catalysts in aque-
ous media by Gong and co-workers (Scheme 1b).15 Further-
more, peptides have also been reported to serve as catalysts
in some other transformations.16–21 However, stable and
easily prepared DNA has rarely been employed in organic
transformations. Since the report in 2005 by Feringa’s
group, DNA-based asymmetric catalysis has been devel-
oped (Scheme 1d).22 By embedding transition-metal com-
© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–F

B
H. Liu et al.
Letter
Synlett
N
Main mechanism:
R2
OH
O
O
O
OH
aldolase
R2
+
a)
R1
R2
R1
H
R2
Ph
Previous works:
Ph
O
O
H
N
OMe
N
H
N
H
NH
O
O
b)
Ph
Pro-Phe-Phe-Phe-OMe
OH
O
OH
O
O
O
OH
+
OH
R
R
R
R
H
20% cat. THF/H2O 0 °C
OH
minor
68–88% yields
84–96% ee
OH
O
DNA
CHO
O
c)
R
+
H2O
40–60 °C
48–80% yields
e) Our strategy using a novel catalyst
to promote the aldol reaction
substrate
CHO
O
ligand
metal
DNA
R
Ar
O
HO
biomacromolecule
self-assembly
?
+
chiral product
Ar
R
aqueous
d)
Scheme 1 The development of the aldol reaction and our strategy
ployed as a catalyst, a slightly increased stereoselectivity
was observed (8% ee; entry 3). This increase in enantiose-
lectivity also occurred when a DNA–L-arginine complex
was used in the same reaction (12% ee; entry 5), indicating
a transfer of chirality from DNA to the aldol product. Next,
we used a synthetic peptide containing two Trp-Thr-Lys
tripeptide units as the catalyst. This peptide has been
shown to bind efficiently with DNA.35 Unfortunately, the
catalytic effect was very poor (entries 6 and 7). When pro-
tamine was screened, to our delight the hybrid catalytic
system efficiently catalyzed the aldol reaction with better
enantioselectivity (24%, entry 9) than that produced by pro-
tamine alone (14% ee; entry 8). Our remaining preliminary
investigations and optimization studies focused on screen-
ing various buffers. The buffer species was found to affect
the enantioselectivity of the hybrid catalyst catalyzed reac-
tion (entries 9–12), and 2-[1-(2-hydroxyethyl)piperazin-1-
ium-4-yl]ethanesulfonate (Hepes) buffer was identified as
the best choice (entry 12). Furthermore, we found that the
pH had a significant influence on the yield and enantiose-
lectivity (entries 12–16). The enantioselectivity reached
33% ee at a pH of 6.5 (entry 15). The pH plays an important
role in the catalytic role of protamine; a lower pH value led
to an obvious decrease in the yield of the product, indicat-
ing that a decrease in the basicity of protamine results in a
reduction in its reactivity. However, changing the pH had
little effect on the enantioselectivity of the product (entries
17–19). In an attempt to improve the yield, we examined
the effect of the ratio of protamine to DNA (see Supporting
Information), and we found that a 1:1 mass ratio was the
best choice (entry 15; 46% yield, 33% ee). Finally, the best
results, a 71% yield and 32% ee, were achieved by increasing
the amount of acetone to 0.3 mL and extending the reaction
time to two days at room temperature (entry 22).
© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–F

C
H. Liu et al.
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Table 1 Screening of the Effects of Reaction Conditionsa
Entry Catalyst
Buffer
pH
8.0
Yieldb (%) eec (%)
13
14
15
16
17
18
19
20h
21i
22j
DNA + protamine
Hepes
Hepes
Hepes
Hepes
Hepes
Hepes
Hepes
Hepes
Hepes
Hepes
85
55
46
26
74
68
58
20
60
71
22
30
33
31
16
16
17
28
25
32
NO2
NO2
OH
O
O
cat. 10%
CHO
DNA + protamine
DNA + protamine
DNA + protamine
protamine
7.0
6.5
6.0
7.5
7.0
6.5
6.5
6.5
6.5
+
buffer, room temperature
3a
2a
1a
cat.
H2N
NH
O
O
protamine
protamine
H2N
N
OH
OH
H
NH2
NH2
protamine
L-lysine
L-arginine
NH2
NH2
DNA + protamine
DNA + protamine
DNA + protamine
HN
NH
O
O
O
O
H
H
N
H
H
N
N
N
a Reaction conditions: 1 (0.1 mmol), acetone (2; 0.2 mL), catalyst (10 wt% of
1), 20mM buffer (2 mL), r.t., 1 d.
b Isolated yield.
N
N
N
N
H
O
H
H
H
O
O
O
OH
H2N
O
OH
c Determined by chiral HPLC.
Peptides: (Trp-Thr-Lys)2-Lys-NH2
d Phosphate buffer
e Synthetic peptide containing two Trp-Thr-Lys tripeptide units.
f 3-(N-Morpholino)propanesulfonic acid
g H2NC(CH2OH)3
Entry Catalyst
Buffer
pH
7.5
Yieldb (%) eec (%)
h Protamine/DNA =1:1.5 (by mass).
i Protamine/DNA =1:0.5 (by mass).
j Acetone (0.3 mL) for 2 d at r.t.
1
DNA
PBd
PB
trace
45
17
94
85
37
10
89
76
66
54
65
–
2
L-lysine
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
3
3
DNA + L-lysine
L-arginine
PB
8
4
PB
4
The hybrid catalytic system of DNA and protamine effi-
ciently catalyzed the reaction with better enantioselectivi-
ties. Protamine is an extremely important substance in
medicine and genetics, and it contains an abundance of
positively charged amino acids, particularly arginine. Con-
sequently, it binds efficiently with DNA. In fact, the DNA
that we used is folded into thousands of toroid-shaped
structures in the sperm cell during spermatogenesis, and
each toroid contains about 50,000 bases (Scheme 2).36–39
5
DNA + L-arginine
Peptidee
PB
12
0
6
PB
7
DNA+ peptides
protamine
PB
3
8
PB
14
24
27
19
28
9
DNA + protamine
DNA + protamine
DNA + protamine
DNA + protamine
PB
10
11
12
MOPSf
Trisg
Hepes
Electrostatic
interactions
O
NH
H2N
NH2
O
COOH
OH
N
H
H2N
H2N
protein
(protamine)
Formation of protein
N
NH2
H
rich
arginine-
O
HN
NH
rich
polypetide
OH
DNA
H2N
lysine NH2
DNA
protein
(protamine)
Spermatogenesis
Folded into
a toroid
A sperm cell can contain
up to 50,000 toroids
Formation of
spermatid
protecting the paternal genetic message
increase in sperm hydrodynamics
Each toroid containing
about 50 kilobases
Scheme 2 The role of protamine in spermatogenesis
© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–F

D
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To verify the formation of a self-assembled catalyst
through electrostatic interaction between protamine and
DNA, we conducted gel-mobility-shift assay experiments
through agarose gel electrophoresis to investigate the nega-
tively charged DNA molecules (Figure 1A: for details, see
Supporting Information). A DNA band was clearly detected
on the gel (Lane 1; Figure 1A). Once mixed with protamine,
the DNA molecules remained in the loading slot, confirm-
ing that a DNA–protein complex had formed, thereby mark-
edly decreasing the mobility of DNA on the gel (Lane 2, Fig-
ure 1A). In comparison, when bovine serum albumin (BSA),
which does not interact with DNA, was used in the same
experiment, the DNA moved freely on the gel. Moreover,
the fluorescence intensity of DNA stained by ethidium bro-
mide (EB) showed an obvious decrease when protamine
was added, whereas no fluorescence change occurred on
addition of BSA (Figure 1B). Those results confirm the pres-
ence of a strong interaction between DNA and protamine,
which is consistent with the conclusions of other research
groups.39–40
After elucidating the composition and catalytic mecha-
nism of the self-assembled catalyst, we set out to explore
the substrate generality of this reaction under the opti-
mized reaction conditions. Various aromatic aldehydes gave
the desired products in good yields and with moderate en-
antioselectivities (Scheme 3). The position of substituents
on the aromatic ring of the aldehyde affected the enantio-
selectivity; for example, a nitro group in the ortho-position
of 3a led to a higher enantioselectivity (33% ee) than did the
corresponding group in the meta- (3b, 16% ee) or the para-
position (3c, 16% ee). In addition, polysubstituted aromatic
aldehydes gave products with better enantioselectivity,
especially substrates with substituents in both the 2- and
6-positions (3f, 55% ee; 3g, 42%). We then evaluated the use
of various N-heterocyclic aromatic aldehydes as substrates.
Most of the reactions were completed within two days un-
der the standard reaction conditions and gave products in
excellent yields (83–96%) and moderate enantioselectivities
(3h–m; 13–35% ee). For example, acridine-9-carbaldehyde
reacted efficiently to afford the corresponding product 3h
in 83% yield with 35% ee. Pyridinecarbaldehydes and quino-
linecarbaldehydes also reacted efficiently to afford the de-
sired product 3i–m in excellent yields (93–96%) and 13–
27% ee. We also carried out the reactions of aldehydes 1a,
1f, and 1g on a 0.5 mmol scale and, to our delight, we ob-
tained products 3a, 3f, and 3g in 62–75% yield and 31–56%
ee.
Encouraged by these results, we attempted to expand
the generality of the reaction with regard to the ketone.
However, the desired products were not obtained in the
presence of straight-chain aliphatic ketones (2-butanone or
2-heptanone) or aromatic ketones (acetophenone) with
various aromatic aldehydes under the standard conditions.
Next, we examined the reactions of cyclohexanone and cy-
clopentanone under the optimized conditions. By screening
various aldehydes, we found that cyclopentanone under-
went an aldol reaction with pentafluorobenzaldehyde
when the hybrid self-assembled system was employed as
the catalyst, and gave the corresponding product 3n with
relatively moderate enantioselectivity (43% ee) and high
diastereoselectivity (dr > 20:1).
In summary, an electrostatic interaction was successful-
ly employed in the construction of new DNA-based cata-
lysts, endowing the DNA molecule with new and improved
catalytic ability. With the self-assembled catalytic system
formed from protamine and DNA, aldol adducts were ob-
tained in high yields and moderate enantioselectivities in
aqueous medium under mild conditions.41 Preliminary ex-
periments demonstrated that the chirality of the DNA could
be effectively transferred to the reaction product through
the bound protein. We believe that the present study con-
siderably advances the concept of DNA-based catalysis and
provides valuable information for researchers in this field.
Figure 1 (A) Gel-mobility-shift assay. Lane 1: DNA only; Lane 2: DNA +
protamine; Lane 3: DNA + BSA; Lane 4: protamine; Lane 5: BSA. (B) Flu-
orescence spectra (excited at 366 nm) of various DNA samples.
These primary researches proved that a novel hybrid
self-assembled catalytic system consisting of DNA and pro-
tamine was formed through electrostatic interactions be-
tween the negatively charged DNA and positively charged
protamine.
© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–F

E
H. Liu et al.
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O
OH
O
(protamine: DNA = 1:1) 10%
Ar CHO
Ar
buffer pH = 6.5
20 mM, 2 d
2
1
3
NO2
O
O
O2N
O
NO2
O
O2N
O2N
OH
OH
OH
OH
3a
3b
3c
3d
71% yield, 33% ee
(73% yield, 31% ee)a
73% yield,
16% ee
76% yield,
16% ee
85% yield,
21% ee
F
F
OH
O
NO2
O
Cl
OH
Cl
O
F
F
OH
F
F
3f
3g
3e
61% yield, 55% ee
(62% yield, 56% ee)a
77% yield, 42% ee
(75% yield, 41% ee)a
82% yield,
21% ee
O
HO
HO
O
N
OH
O
O
N
N
3h
3i
3j
83% yield,
35% ee
95% yield,
13% ee
96% yield,
27% ee
HO
F
F
OH
HO
HO
O
F
O
O
N
N
F
F
N
3k
3l
3m
95% yield,
3n
96% yield,
93% yield,
66% yield, dr > 20:1;
20% ee
21% ee
21% ee
30% ee, 43% ee
Scheme 3 Substrate scope of various aldehydes and ketones. Reaction conditions: 1 (0.1 mmol), 2 (0.3 mL), protamine–DNA catalyst (10 mass% of 1),
Hepes buffer (2 mL), r.t., 2 d. Isolated yield of products are reported. The enantioselectivity was determined by chiral HPLC. a Reactions carried out on a
0.5 mmol scale.
Work to develop other self-assembled DNA catalysts
through this strategy and to employ them in new reactions
is underway in our laboratory.
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M.; Charvet, A.; Dalstein, V. M. D.; Jauseau, R.; Hiatt-Gipson, G.
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We are grateful to the National Natural Science Foundation of China
(21402188) and the Sichuan Province Natural Science Foundation of
China (2017JY0056).
N
oaitn
a
l
N
a
utarl
S
ecin
c
e
F
o
u
n
d
oaitn
of
C
h
n
i
a
2(
1
4
0
2
1
8
8)
Supporting Information
Supporting information for this article is available online at
https://doi.org/10.1055/s-0036-1591854.
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© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–F

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A mixture of protamine (1.5 mg) and DNA (1.5 mg) in 20 mM
Hepes buffer (2 mL) at r.t. was stirred with a magnetic stirrer for
1 h. Aldehyde 1 (0.1 mmol) and acetone (0.3 ml) were added,
and the resulting mixture was stirred for 2 d at r.t. until the
reaction was complete (TLC). The mixture was extracted with
EtOAc (3 × 2 mL), and then the combined organic extracts were
washed with brine (5 mL), dried (Na2SO4), and filtered. The
solvent was removed under reduced pressure, and the residue
was purified by flash column chromatography [silica gel, PE–
EtOAc (5:1 to 3:1)].
4-Hydroxy-4-(2-nitrophenyl)butan-2-one (3a)
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faint yellow solid; yield: 14.8 mg (71%; 33% ee); mp 58–60 °C
(Lit.42 59–61 °C); HPLC: Chiralpak AS-H (hexane–i-PrOH
(80:20); flow rate: 1 mL/min, λ = 254 nm): t1 = 8.9 min; t2 = 11.3
min. 1H NMR (400 MHz, CDCl3): δ = 7.95 (dd, J = 8.2, 1.0, 1 H),
7.89 (d, J = 7.5, 1 H), 7.71–7.62 (m, 1 H), 7.50–7.36 (m, 1 H), 5.67
(dd, J = 9.4, 1.9, 1 H), 3.74 (s, 1 H), 3.12 (dd, J = 17.8, 2.0, 1 H),
2.72 (dd, J = 17.8, 9.5, 1 H), 2.23 (s, 3 H). 13C NMR (101 MHz,
CDCl3): δ = 208.95, 147.22, 138.52, 133.95, 128.40, 128.29,
124.56, 65.73, 51.18, 30.57.
(36) Balhorn, R. Genome Biol. 2007, 8, 227.
(42) Lei, M.; Xia, S.; Wang, J. F.; Ge, Z. M.; Cheng, T. M.; Li, R. T.
Chirality. 2010, 22, 580.
© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–F