Aniline-terminated DNA catalyzes rapid DNA-hydrazone formation at physiological pH

Article abstract of DOI:10.1039/c4cc00292j

We report the effect of DNA-templation on aniline-catalyzed N-acylhydrazone formation. The reaction occurs in both two-component and three-component systems. Through systematic catalyst modifications, we are able to increase the efficiency of the DNA-temp

Full text of DOI:10.1039/c4cc00292j

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Aniline-terminated DNA catalyzes rapid
DNA–hydrazone formation at physiological pH†
Cite this: Chem. Commun., 2014,
50, 3831
Dylan W. Domaille* and Jennifer N. Cha*
Received 13th January 2014,
Accepted 21st February 2014
DOI: 10.1039/c4cc00292j
www.rsc.org/chemcomm
We report the effect of DNA-templation on aniline-catalyzed effect a proline-mediated aldol reaction between a hybridized alde-
N-acylhydrazone formation. The reaction occurs in both two- hyde–DNA and an untethered aldehyde at a rate more than 170-fold
component and three-component systems. Through systematic above that of the uncatalyzed reaction.27
catalyst modifications, we are able to increase the efficiency of
The successful integration of DNA-templation with sequence-
the DNA-templated variant to 85-fold above that of the uncata- specific DNA–hydrazone formation may allow us to realize a sensitive
lyzed reaction at physiological pH.
and catalytic system for the detection of bacterial ribosomal RNA or
provide a system that increases the efficiency of DNA–hydrazone
The reaction of a hydrazine or an alkoxyamine with an aldehyde to formation for bioconjugation. We reasoned that we could use a DNA
form the corresponding hydrazone or oxime is a commonly used templating strategy to organize an aniline nucleophilic catalyst in
bioconjugation reaction, as it benefits from a high equilibrium close proximity to an acyl hydrazine nucleophile. Because this
constant and shows good bioorthogonality and compatibility with approach would increase the effective molarity of the organocatalyst,
endogenous cellular functionality.1–5 Recent studies have shown that we could avoid using high concentrations of an exogenous nucleo-
aniline and related compounds can significantly increase the rate of philic catalyst. We postulated that a direct consequence of the
product formation over that of the uncatalyzed variant.6–8 Mechan- favorable placement of the organocatalyst would be an increase in
istically, aniline acts as a nucleophilic catalyst and reacts with the the rate of hydrazone formation between the acyl hydrazine-
aldehyde to form an aniline Schiff base intermediate in the rate derivatized DNA and an untethered aldehyde. Herein, we report
limiting step;6,9 however, high concentrations of aniline (10–100 mM) our studies directed toward extending DNA-templated synthesis to
are required to realize a significant catalytic effect, and such a high the formation of DNA–N-acylhydrazones.
concentration precludes the use of this in certain biological applica-
tions, though biocompatible aniline-derivatives have recently been an aniline organocatalyst in close proximity to an acyl hydrazine, we
reported.10 synthesized DNA (A15) with a 30-aniline group (A15-aniline) and
In order to probe the effect of using DNA hybridization to direct
DNA-templated synthesis is a powerful method that has found monitored its ability to increase the rate of hydrazone formation
applications in a diverse range of fields, from the construction of when hybridized to a complementary strand of DNA bearing a 50-acyl
complex molecules11–14 to the identification of novel chemical reac- hydrazine group (T15-acyl hydrazine) (Fig. 1). We used 4-nitro-
tions15 to the detection of biomolecules.12,16–24 Typically, DNA- benzaldehyde as the aldehyde substrate because the absorption
templation relies on DNA hybridization to bring two functionalized and extinction coefficient of the resulting hydrazone are suitable
DNA strands together: the resulting increase in local concentration
prompts the rapid and selective reaction between the two functional
groups, effectively ligating the DNA strands together.25,26 However,
few reports have focused on DNA-templated organocatalysis with
untethered reagents, the notable exception being a report from Tang
et al., in which it was demonstrated that prolinamide–DNA could
Fig. 1 T15-acyl hydrazine DNA (green) hybridizes to A15-aniline DNA
(blue), and the high local concentration of aniline enhances the rate of
acyl hydrazone formation. If 4-nitrobenzaldehyde is the aldehyde sub-
strate, the resulting hydrazone formation can be monitored by UV-visible
spectroscopy in a region distinct from the large DNA absorption.
Department of Chemical and Biological Engineering, University of Colorado
Boulder, CO 80303, USA. E-mail: Dylan.Domaille@colorado.edu,
Jennifer.Cha@colorado.edu
† Electronic supplementary information (ESI) available: Synthetic procedures and
molecular characterization. See DOI: 10.1039/c4cc00292j
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Table 1 Rates for hydrazone formation between T15 acyl hydrazine DNA
and 4-nitrobenzaldehyde in the absence and presence of DNA–anilinea
pH
kuncat (sꢀ1
)
kcat (sꢀ1
)
kcat/kuncat
4.5
6.0
7.0
1.64(2) ꢂ 10ꢀ3
1.04(5) ꢂ 10ꢀ4
3.91(9) ꢂ 10ꢀ5
1.23(9) ꢂ 10ꢀ2
5.80(4) ꢂ 10ꢀ4
1.22(1) ꢂ 10ꢀ4
7.5
5.6
3.1
a
All spectroscopic measurements were carried out in phosphate buffer
(50 mM) containing NaCl (150 mM) with T15 acyl hydrazine DNA
(5.0 mM), 4-nitrobenzaldehyde (500 mM), and in the case of DNA-
templated organocatalysis, A15-aniline DNA (5.0 mM).
for monitoring the rate of product formation by UV-visible spectro-
scopy (e340 = 13 500 Mꢀ1 cmꢀ1).28
The kinetics of the reaction were measured at different pH values
and in the presence and absence of complementary A15-aniline
under pseudo-first order conditions (5.0 mM DNA, 500 mM aldehyde).
We observed a pH dependence on the rate of hydrazone formation,
in line with previous mechanistic investigations of hydrazone for-
mation.6,29 The rate was slower at pH 7.0 (kobs = 3.91 ꢁ 0.09 ꢂ
10ꢀ5
s
ꢀ1) than at pH 6.0 (kobs = 1.04 ꢁ 0.05 ꢂ 10ꢀ4
s
ꢀ1), and the
reaction at pH 4.5 was the fastest (kobs = 1.64 ꢁ 0.02 ꢂ 10ꢀ2 sꢀ1
)
(Table 1; Fig. S1A–C, blue traces, ESI†). Upon the addition of
1.0 equivalent of A15-aniline, we saw a significant rate enhancement
at each pH value (Table 1; Fig. S1A–C, red traces, ESI†). Whereas the
reaction at pH 4.5 was ca. 80% complete in roughly 20 minutes in the
absence of A15-aniline, in the presence of 1 equivalent of A15-aniline,
the reaction was 100% complete within 5 minutes. Likewise, at
pH 6.0, the reaction was ca. 30% complete after one hour, whereas
the hydrazone conjugation was 490% complete in the presence of
A15-aniline at the same timepoint. The corresponding rate enhance-
ment factors range from a 3-fold rate enhancement at pH 7.0 to a
7.5-fold rate enhancement at pH 4.5 (Table 1; Fig. S1D, ESI†). Control
experiments established that the Schiff base intermediate formed
between aniline and 4-nitrobenzaldehyde contributes a negligible
amount of signal to the measured absorbance (Fig. S2, ESI†).
Additional control experiments with complementary alkyl amine-
terminated DNA and with non-complementary A15-aniline did not
produce rate constants significantly different from the uncatalyzed
variant (Fig. S3, ESI†). Thus, both the aniline moiety and the correct
basepairing are necessary for the rate enhancement of hydrazone
formation.
Fig. 2 (A) Underivatized DNA organizes the DNA-catalyst (blue strand) in
close proximity to the DNA-acyl hydrazine (green strand) to template the
organocatalytic reaction. (B) DNA-anchored aniline organocatalyst deri-
vatives and their associated catalytic rate enhancements compared to that
of the uncatalyzed variant. kc/ku refers to the ratio of the catalyzed reaction
(kc) to that of the uncatalyzed reaction (ku). (C) Time-course UV-visible
spectroscopy study that shows the DNA-templated formation of the
DNA–hydrazone between
a
DNA–50-acyl hydrazine (2.2 mM) and
4-nitrobenzaldehyde (500 mM) with a complementary DNA template
(2.5 mM) in the absence (‘‘w/o’’) or the presence of the indicated DNA-
anchored organocatalyst (2.2 mM, 1.0 equiv.). Spectra were collected in
PBS, pH 7.4 by monitoring the signal at 340 nm. Data were fitted to a first
order reaction to obtain the apparent rate constants.
Encouraged by these results, we reasoned that it may be possible
to use an underivatized DNA template to organize the DNA-reagents
in an arrangement shown in Fig. 2A. In this case, an unmodified
30-mer DNA oligonucleotide that is complementary to both the
In our first-round screen for more efficient catalysts, we examined
15-mer DNA–aniline and 12-mer DNA–acyl hydrazine effectively the effect of regioisomeric positioning of the amino group on the rate
anchors the catalyst in close proximity to the nucleophile. This of DNA–hydrazone formation. To this end, DNA was elaborated with
arrangement should allow the aniline to form a Schiff base the amino group in either a meta or ortho position with respect to the
intermediate with an exogenous aldehyde, and the high local amide linkage to the DNA; this provided 20 and 30, respectively
concentration of DNA acyl hydrazine should facilitate rapid (Fig. 2A). As expected, the 2-amino derivative showed poor catalytic
DNA–hydrazone formation. However, when we employed DNA– efficacy, most likely owing to steric congestion around the organoca-
aniline in this system, we saw that the DNA–aniline only talytic amino group. However, we were pleased to see that the 30
increased the rate of DNA–hydrazone formation by a little more derivative was capable of enhancing the rate of hydrazone formation
than 2-fold compared to that of the uncatalyzed reaction to 13-fold above that of the uncatalyzed reaction (kobs = 1.7 ꢁ 0.2 ꢂ
(Fig. 2B, 10). Thus, we initiated a systematic search to find 10ꢀ4
more efficient DNA-anchored organocatalysts.
s
ꢀ1), an improvement of more than 5-fold above that of the
initial catalyst, 10 (Fig. 2B, 30).
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DNA–hydrazone formation when directly hybridized to a complemen-
tary DNA bearing a 50-acyl hydrazine. Further, our data indicate that
this rate enhancement persists when the reagents are organized by a
third oligonucleotide. A systematic search of more efficient DNA-
anchored aniline derivatives revealed an exceptionally efficient catalyst
that is capable of increasing the rate of DNA–hydrazone formation to
more than 85-fold above that of the uncatalyzed reaction. Our
approach obviates the need for acidic pH conditions, eliminates high
concentrations of added organocatalysts, and synthesizes the product
in a highly DNA-sequence dependent manner. Taken together, these
characteristics suggest that this DNA-templated variant of organo-
catalytic hydrazone formation should find extensive application in the
fields of bioassay development and stimuli-responsive materials.
This work was supported by an NSF Career Award (DMR-1056808)
and the Office of Naval Research (Award Number N00014-09-01-
0258). We are greatly indebted to Dr Subhadeep Roy (Caruthers lab)
for help with analytical and preparative HPLC. We also thank Dr
Jeremy Balsbaugh for help with ESI-MS analysis and Kirsten Fitch
(Goodwin lab) for assisting with IR data collection.
Fig. 3 Proposed mechanism of DNA-templated hydrazone formation.
Hybridization of the DNA-reagents to the DNA template organizes the
organocatalyst in close proximity to the nucleophilic acyl hydrazine.
Aniline-Schiff base formation activates the aldehyde for subsequent
nucleophilic attack by the DNA–acyl hydrazine. Collapse of the tetrahedral
intermediate produces the DNA–N-acyl hydrazone and results in an
absorbance increase.
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Chem. Commun., 2014, 50, 3831--3833 | 3833