Proline-modified DNA as catalyst of the aldol reaction

Article abstract of DOI:10.1002/anie.200701416

(Chemical Equation Presented) Unbound reactant: Proline-modified DNA acts as catalyst of the intramolecular aldol reaction between a complementary aldehyde and free non-tethered ketones (see scheme). The results of these studies extend the methodological repertoire of DNA-templated reactions.

Full text of DOI:10.1002/anie.200701416

Angewandte
Chemie
DOI: 10.1002/anie.200701416
DNA Catalysis
Proline-Modified DNA as Catalyst of the Aldol Reaction**
Zhuo Tang and Andreas Marx*
Dedicated to Professor Peter Welzel on the occasion of his 70th birthday
DNA-templated synthesis has emerged as a powerful tool to
steer reactivity by modulating natureꢀs approach to increase
the effective molarity of the reactants.[1] By making recourse
to the inherently selective duplex formation properties of
cognate DNA strands, small molecule reactants are brought
to close proximity increasing the effective molarity and
thereby significantly accelerating chemical reactions. Several
applications of this approach have been developed such as
compound discovery from synthetic libraries, reaction dis-
covery, and nucleic acid sensing.[1,2] Additionally, these kinds
of reactions were suggested to serve as model systems
demonstrating how nucleic acids can catalyze chemical
reactions and thus, implicating their role as catalysts in an
early stage in the history of life on earth.[3,4]
enamine intermediate. We reckoned that a proline attached
to one DNA strand might act as a catalyst for the cross-aldol
reaction between an aldehyde tethered to a complementary
DNA sequence and a non-tethered ketone. It should be noted
that aqueous buffer conditions required for most DNA-
templated reactions still represent a challenge for organo-
catalysis.[8] Though there are two reports about DNA-
templated aldol reactions,[4,9] our approach differs concep-
tually due to the covalent attachment of the catalyst and a
reaction partner that is not bound to DNA.
We first synthesized the oligonucleotide template 1, which
bears a conjugated aldehyde at its 3’-terminus, and a
complementary strand 2, which contains a proline moiety at
its 5’-terminus (Figure 1).[10] Subsequently, we tested the
All of the depicted approaches have in common that the
two reactants are covalently linked to two DNA or PNA
strands that are subsequently allowed to react by self-
hybridization or alignment through hybridization with a
third nucleic acid strand. This design results in covalent
interconnection of the two modified oligonucleotides in most
cases with only few exceptions.[1,2,5] Along these lines, a DNA-
templated ester hydrolysis promoted by a CuII catalyst was
reported as well as a DNA-templated Staudinger ligation
reaction.[5e,f] In these cases a non-tethered reagent (i.e. water)
reacted with a DNA-tethered reagent by DNA-templation.
We intended to expand the scope of DNA-templated syn-
thesis using non-tethered reagents. Herein we report on a new
approach that enables the cross-aldol reaction of a DNA-
tethered aldehyde with free non-tethered ketones. Our
approach is based on the covalent attachment of a catalyst
to an oligonucleotide complementary to a DNA-tethered
aldehyde component.
Recently, the field of organocatalysis has gained much
attention in the chemical research community.[6] Numerous
small organic compounds were found to achieve remarkably
selective and efficient transformations. As one of the most
important components of organocatalysts, proline[7a–b] and its
derivatives[7c–f] have been shown to possess the extraordinary
ability to catalyze for example, the aldol reaction via an
Figure 1. a) DNA-templated cross-aldol reaction catalyzed by prolina-
mide 2. b) A) HPLC analysis of the reaction mixture of 1 and 2 in
100 mm phosphate buffer (pH 7.2) without addition of acetone. B)
HPLC analysis of the reaction between 1 and acetone catalyzed by 2
after 4 h in 100 mm phosphate buffer (pH 7.2) at 258C. Phosphate
buffer: Na2HPO4/NaH2PO4. The reaction was performed in aqueous
buffer/acetone (v/v 4:1).
[*] Dr. Z. Tang, Prof. Dr. A. Marx
Department of Chemistry
catalytic ability of the prolinamide 2 in the reaction between
acetone and 1 initially at pH 7.2 in an aqueous buffer. The
reactions were analyzed by denaturing HPLC[11] at 508C to
obtain the required resolution. The signals were assigned to
the respective entities by ESI-MS analysis.[10] Much to our
delight we discovered that the desired product of a cross-aldol
reaction of the DNA-tethered aldehyde 1 and acetone is
formed in high yields (Figure 1). The disappearance of the
signal for 1 was observed as well as the appearance of signals
University of Konstanz
Universitätsstrasse 10, 78457 Konstanz (Germany)
Fax: (+49)7531-88-5140
E-mail: andreas.marx@uni-konstanz.de
[**] We gratefully acknowledge the award of a fellowship to Z.T. by the
Alexander von Humboldt Foundation.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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for two new reaction products. One was identified by ESI-MS
indicating that the efficiency of 6 to promote the reaction is
considerably lower. Comparison of the initial rates revealed
that the reaction promoted by 2 (243 ” 10À11m sÀ1) is about
170-times faster than the one promoted by 6 (1.43 ”
10À11m sÀ1).[10] Even diluting the reactants 1 and 2 by tenfold
resulted in the same yield (Table 1, entry 4). Poor efficiency
was observed when the by-product 4 was employed to
catalyze the reaction (Table 1, entry 5). In contrast to the
high proficiency of the oligonucleotide conjugate 2 to
promote the reaction, non-DNA-conjugated prolineamide
was a poor catalyst of this reaction (Table 1, entries 6 and 7).
Next we investigated the scope of the reaction. We found
that DMSO is the best cosolvent for ketones insoluble in
water. Moderate to high yields were obtained when DNA-
templated prolinamide 2 was used to catalyze the cross-aldol
reaction between oligonucleotide 1 and different ketones
(Figure 3). Thus, employing the DNA-templated prolinamide
2, different cross-aldol reactions can be performed starting
from DNA-conjugated aldehyde 1. However, the initial rates
were lower than the one observed for the reaction with
acetone.
to be the desired product 3. The by-product is suggested to be
4, which can be explained by condensation of 2 with
acetone.[12]
Encouraged by these initial findings we further optimized
the reaction conditions and found pH 7.0–8.0 in 200 mm
phosphate buffer to be best suited for our approach. To
verify that the observed results are indeed caused by proline
catalysis we investigated the formation of product 3 upon
addition of several oligonucleotide constructs in a time-
dependent manner (Figure 2, Table 1). When unmodified
Figure 2. a) Catalysis of a cross-aldol reaction by different DNA
constructs. b) Time-dependent formation of reaction products cata-
lyzed by various DNA constructs.
Figure 3. Cross-aldol products of 1 and different ketones catalyzed by
2. The concentration of oligonucleotides is 3 mm in 100 mm phosphate
buffer (pH 8.0) and 30:10:3 (v/v/v) of aqueous phase/DMSO/ketone.
Initial rates and yields are given.
Table 1: Cross-aldol reaction catalyzed by different catalysts.
Entry
Catalyst[a]
t [h]
Yield 3 [%]
Only a few examples are reported in which catalytic
turnover could be achieved in DNA-templated reactions.[13]
We expected that the herein depicted reaction is highly
unlikely to proceed with catalytic turnover when substoichio-
metric amounts of 2 are used because of the formation of the
bicyclic imidazolidinone 4 that inactivates the catalyst
(Table 1, entry 5). To address this shortcoming, modified
oligonucleotide 7 was synthesized. Construct 7 comprises two
proline moieties connected through a peptide bond. The
formation of an imidazolidinone should be suppressed due to
the absence of an amide proton in the g-position to the
N atom involved in enamine formation. Indeed, this was
achieved without formation of significant amounts of by-
product when 1 was allowed to react with acetone in the
presence of 7 (Figure 4).
1
2
3
4
5
6
7
5
6
24
24
4
4
24
4
<5
40
92
92
<5
6
2
2[b]
4
prolinamide[c]
prolinamide[d]
4
15
[a] Conditions: The concentration of oligonucleotides is 3 mm in 100 mm
phosphate buffer (pH 7.2) and the ratio of aqueous buffer/acetone is 5:1
(v/v). The reactions were incubated at 258C. [b] The concentration of
oligonucleotides is 0.3 mm. [c] The concentration of prolinamide is
0.3 mm. [d] The concentration of prolinamide is 3.0 mm.
oligonucleotide 5, which is complementary to 1, was used, the
formation of the desired reaction product 3 was below the
detection limit within 24 h (Table 1, entry 1). Interestingly, the
DNA-linked primary amine 6 could also promote the cross-
aldol reaction in about 40% yield after 24 h (Table 1, entry 2).
Nevertheless, the proline-modified DNA 2 gave 3 in 92%
yield already after 4 h (Figure 2 and Table 1, entry 3),
The initial rate (41.2 ” 10À11m sÀ1) of the reaction pro-
moted by 7 was lower than the one promoted by 2 but
nevertheless, afforded the desired product in excellent yield
(Table 2, entry 1). We next investigated whether 7 was able to
promote the reaction in substoichiometric amounts, and
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catalysts loading was 10 mol% and the reaction was cycled 25
times the yield was increased compared to the outcome of the
reaction when isothermal reaction conditions were applied
(see Table 2, entries 2–4 and Table 3, entry 2). This yield
could be increased to 71% when the number of cycles was
increased (Table 3, entry 2). Notably, when the reaction was
conducted under isothermal conditions at 808C for 1 h in the
absence of 7 only a poor yield of 14% was achieved. Thus, this
rules out that the higher yield observed in the cycled reactions
derives from higher reaction times at the increased reaction
temperature. The catalyst loading could be further decreased
to 5 mol% and yet turnover could still be detected resulting in
significant product formation.
In summary, we show that a proline tethered to a DNA
strand can efficiently catalyze the cross-aldol reaction of a
complementary DNA–aldehyde conjugate with non-tethered
ketones. The developed system is able to catalyze the aldol
reaction between a DNA-tethered aldehyde and several
ketones thereby tolerating DMSO as co-solvent in cases
where water-insoluble ketones were employed. Interestingly,
through optimization of the proline catalyst design a species
was derived that was able to achieve catalytic turnover. The
turnover numbers could be increased by cycling the ambient
temperature between the reaction temperature and the DNA
duplex denaturation temperature. Our finding that DNA-
tethered prolines even catalyze intermolecular aldol reactions
between tethered and non-tethered reactants adds extends
the methodological repertoire of DNA-templated reactions.
Figure 4. a) DNA-templated cross-aldol reaction between 1 and ace-
tone catalyzed by prolinamide 7. b) Denaturing HPLC analysis of
reaction products.
Table 2: Cross-aldol reaction catalyzed by oligonucleotide 7.
entry
7 [mol%][a]
t [h]
Yield [%]
1
2
3
4
100
10
10
24
24
48
72
93
26
34
41
10
[a] The concentration of oligonucleotide 1 is 3 mm in 100 mm phosphate
buffer (pH 7.3) at 258C. The ratio of aqueous buffer/acetone is 5:1 (v/v).
Received: April 2, 2007
Revised: June 16, 2007
Published online: August 17, 2007
found that it could (Table 2, entries 2–4). With 10 mol% of 7,
the reaction between oligonucleotide 1 and acetone gave the
desired product in 41% yield after 72 h.
Keywords: aldol reaction · DNA-templated synthesis ·
One can envision that the low turnover results from the
formation of a stable duplex of the catalyst 7 and the reaction
product 3 and subsequent sluggish strand exchange of 3 with
reactant 1. We envisaged that strand exchange might be
promoted by cycling the ambient temperature between 258C
(reaction temperature) and 808C (duplex denaturation tem-
perature), since the melting temperature (Tm) for the strand
comprising 1 and 7 is 638C.[10] When annealing by cooling
from 808C to 258C, unreacted 1 will initially (when present in
higher concentration than product 3) have an increased
probability to anneal to catalyst 2. Thus, product formation
should be favored until the system reaches equilibrium.
Indeed we found that the turnover numbers can be increased
significantly by temperature cycling (Table 3). When the
.
organocatalysis · prolinamide
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Entry
7 [mol%][a]
Cycles
Yield [%]
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4
5
10
10
5
5
0
25
50
25
50
50
56
71
41
53
14
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Communications
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