Micellar Br?nsted Acid Mediated Synthesis of DNA-Tagged Heterocycles

Article abstract of DOI:10.1021/jacs.9b05696

The translation of well-established molecular biology methods such as genetic coding, selection, and DNA sequencing to combinatorial organic chemistry and compound identification has made extremely large compound collections, termed DNA-encoded libraries,

Full text of DOI:10.1021/jacs.9b05696

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Article
Micellar Brønsted Acid-Mediated Synthesis of DNA-Tagged Heterocycles
Mateja Klika Škopi#, Katharina Götte, Christian Gramse, Marvin Dieter, Sabrina
Pospich, Stefan Raunser, Ralf Weberskirch, and Andreas Brunschweiger
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 07 Jun 2019
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Micellar Brønsted Acid-Mediated Synthesis of DNA-Tagged
Heterocycles
M. Klika Škopić,a K. Götte,a C. Gramse,a M. Dieter,a S. Pospich,b S. Raunser,b R. Weberskirch,a* A.
Brunschweiger a*
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a) Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Straße 6, 44227 Dortmund, Germany;
b) Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany.
ABSTRACT: The translation of well-established molecular biology methods such as genetic coding, selection and DNA
sequencing to combinatorial organic chemistry and compound identification has made extremely large compound collections,
termed DNA-encoded libraries, accessible for drug screening. However, the reactivity of the DNA imposes limitations on the
choice of chemical methods for encoded library synthesis. For example, strongly acidic reaction conditions must be avoided
because they damage the DNA by depurination, i. e. the cleavage of purine bases from the oligomer. Application of micellar
catalysis holds much promise for encoded chemistry. Aqueous micellar dispersions enabled compound synthesis under often
appealingly mild conditions. Amphiphilic block copolymers covalently functionalized with sulfonic acid moieties in the lipophilic
portion assemble in water and locate the Brønsted catalyst in micelles. These acid nanoreactors enabled the reaction of DNA-
conjugated aldehydes to diverse substituted tetrahydroquinolines and aminoimidazopyridines by Povarov, and Groebke-Blackburn-
Bienaymé reactions, respectively, and the cleavage of tBoc protective groups from amines. The polymer micelle design was
successfully translated to the Cu/Bipyridine/TEMPO system mediating the oxidation of DNA-coupled alcohols to the
corresponding aldehydes. These results suggest a potentially broad applicability of polymer micelles for encoded chemistry.
INTRODUCTION
The last decade has witnessed the rise of DNA-encoded small
moleculs libraries (DELs) to a widely used small molecule
screening technology in academia and industry.1-6 Encoded
libraries are synthesized through split-and-pool combinatorial
routines encompassing alternated chemical compound
synthesis and DNA-tagging steps that endow the compound
with genetic information (Figure 1a).7-11 The technology
benefited from technical advances in molecular biology, i.e.
robust enzymatic concatenation of DNA barcode fragments,7-11
detection of minute DNA amounts by polymerase chain
reaction, and advanced DNA sequencing platforms to read and
quantitate genetic information. These genetic techniques allow
chemists to synthesize unprecedented numbers of compounds
as pools, and to identify bioactive compounds from these
pools by selection experiments on proteins and DNA barcode
sequencing (Figure 1b).1-6
While DNA tagging enables efficient handling of large
compound numbers for target-based screening, it imposes
Figure 1. The technology of DNA-encoded libraries (DELs). (a) Schematic
formidable
challenges
to
synthesis
methodology
presentation of combinatorial DNA-encoded chemistry. (b) Compounds
binding to a target protein of interest are identified by selection of DELs and
DNA barcode sequencing. (c) Structure of sulfonic acid-substituted block
copolymer 2a that localizes sulfonic acid moieties in polymer micelles. (d)
Application of 2a as micelle-based reaction system for DNA-compatible
synthesis of DNA-tagged target molecules.
development.12,13 To be encodable, chemistry must be
compatible with water as (co-)solvent, must afford the target
compound with high yields despite high starting material
dilution, should display a broad scope of ideally readily
available starting materials, must be operationally simple to be
compatible with multi-well plate reactor formats, and the
genetic information must be preserved under the reaction
conditions. The latter point requires pH values of above 3, and
avoidance of harsh reaction conditions, particularly in
combination with oxidizing agents and many Lewis acids.
DEL synthesis methods focused mostly on building block-
appending reactions, such as carbonyl chemistry,
nucleophilic substitution reactions, and Cu- and Pd- mediated
cross coupling reactions.14 Broadening the scope of encoded
chemistry has therefore been cited as particularly important to
advance the technology of DNA-encoded libraries.15,16 Lately,
modern photo redox chemistries have greatly expanded the
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moieties (pKa  -2.5) covalently connected to the lipophilic
polymer block. Here, we show that up to millimolar
concentrations of copolymer 2a were well tolerated by DNA
oligonucleotides, and enabled the reaction of DNA-conjugated
aldehydes to diverse substituted tetrahydroquinolines by
Povarov reaction (Figure 2) without detectable depurination.
The scope of micellar Brønsted acid catalysis was
choice of building block-appending reactions for encoded
chemistry.17 Heterocyclic structures, i.e. small ring systems
containing heteroatoms, are well represented in the chemical
space of bioactive molecules.18-24 A recent analysis of FDA-
approved drugs found that with 59 % the majority of them
contain a nitrogen heterocycle, underpinning the importance of
heterocyclic chemistry in drug research.25 Research efforts
towards encoded heterocyclic chemistry have resulted to date
in only a few synthesis methods. These are cycloadditions
such as the (hetero) Diels-Alder and 3+2 cycloaddition
reactions,26 and condensation and substitution reactions of
functionalized starting materials.14 Brønsted acids are essential
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subsequently
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the
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aminoimidazopyridines by Groebke-Blackburn-Bienaymé
(GBB) reaction and the removal of tBoc-protective groups
from amines. Finally, we could translate the copolymer
micelle design to a Cu(I)/TEMPO catalyst for the selective
oxidation of DNA-coupled alcohols to the corresponding
aldehydes.
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catalysts in organic chemistry, enabling
a plethora of
reactions, among them many that give heterocycles from
simple starting materials.27 However, the application of protic
acid catalysis in encoded compound synthesis entails the risk
of DNA depurination, i.e. the loss of purine bases from the
oligomer by purine N7 protonation and subsequent cleavage of
the acetal-like ribosidic linkage (see DNA-1 incubated at
different pH values in Figure S1, supplementary
information).28 Protic acid catalysis for encoded compound
synthesis is currently restricted to barcoding with more stable
peptide nucleic acids or dedicated DNA-barcoding
strategies.29-31
RESULTS AND DISCUSSION
Investigation of the Povarov reaction to DNA-
tetrahydroquinoline conjugates under homogeneous
reaction conditions. Tetrahydroquinolines are frequently
occurring scaffolds among bioactive molecules. They can be
found as central structures in natural products, a very recently
disclosed inhibitor of CRISPR-Cas9, and clinical candidates
(4-6, Figure 2a).49-51 The Povarov reaction, a formal aza-Diels-
Alder reaction, affords in one pot diverse tetrahydroquinolines
11 from readily available aldehydes 7, anilines 8, and electron-
rich olefins 10 (Figure 2b).52-64
Compartmentation of reaction media is an intriguing principle
in chemistry with many applications. It can be effected by
amphiphilic, soap-like molecules, among them block
copolymers.32-37 Above a certain concentration called critical
micelle concentration (CMC) amphiphiles associate in water
to nanometer-sized micelles that are characterized by a
lipophilic core and a hydrophilic corona (Figure 1c).38,39 These
oil-in-water (o/w) micelles can serve as microheterogeneous
systems to solubilize hydrophobic chemicals in water.
Micellar dispersions have repeatedly been shown to accelerate
reaction rates by concentrating reactants in nanometer-sized,
confined reaction vessels and/or by altering their reactivity in
the micellar environment, often resulting in appealingly mild
reaction
conditions.34,40,41
Second-generation
micellar
nanoreactor designs linked catalysts covalently to the
lipophilic portion of the amphiphile preventing catalyst
partitioning between phases.42-45 A few times, micellar systems
have been used to react DNA to target conjugates. For
instance, amino-linker modified DNA was coupled to water-
insoluble carboxylic acids by a means of a micellar system,
and micelles with a corona composed of DNA strands have
been designed for DNA-templated chemistry.46,47 A micellar
catalyst system composed of a lipidated dopamine oxidizing
DNAzyme and a lapidated dopamine binding DNA aptamer
was shown to accelerate dopamine oxidation by concentrating
the substrate close to the catalytic site.48 We hypothesized that
a micellar reaction system designed to direct a catalyst into the
lipophilic core and to minimize its phase distribution might
enable Brønsted acid catalysis for the selective reaction of
DNA-conjugated starting materials. It benefits from the
inaccessibility of the catalytic site to water-soluble DNA and
from the typically low catalyst concentrations employed in
micellar catalysis. To this end, we explored the sulfonic acid-
substituted, amphiphilic copolymer 2a (Figure 1c, Figures S2-
S5). In water it forms already at low micromolar
concentrations micellar nanoreactors containing sulfonic acid
Figure 2. The tetrahydroquinoline is a frequently occurring heterocyclic core
structure in bioactive compounds. (a) Natural product virantmycin 4; inhibitor
of CRISPR-Cas9 5, and clinical candidate 6. (b) The Povarov three-
component reaction gives straightforward excess to the characteristic
tetrahydroquinoline core 11 displayed by compounds 5 and 6.
Two three-dimensionality inducing sp3-C-C bonds are formed
in the course of tetrahydroquinoline synthesis. This, together
with the broad reactant scope makes the reaction highly
attractive for screening library synthesis.50,58 We investigated a
translation of published, homogenous reaction conditions for
the Povarov reaction to a DNA-tagged format. The DNA-
aldehyde conjugate 3a (Figure S6), serving as a model
substrate for a DNA-encoded compound, was reacted with
aniline 8a, and the olefin 10a screening different equivalents
of reactants, co-solvent systems and protic acid catalysts.55
Acid concentrations in a DNA-compatible range failed to yield
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the target DNA tetrahydroquinoline conjugate DNA-12a in analysed by HPLC and mass spectrometry. More complex
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detectable amounts (Figure 3, Table S3, Figures S7, S8). The
Lewis acids Yb(III), Sc(III), Ceric ammonium nitrate (CAN),
and Ce(III) were previously shown to catalyse the Povarov
reaction, but failed under several tested reaction conditions to
yield the DNA-coupled heterocycle, too (Table S3, Figures
S9-12).59-64 Thus, none of the evaluated homogeneous
reactions based on Brønsted acid or Lewis acid catalysis met
under the evaluated reaction conditions the requirement for
encodable chemistry to afford the target product at high yields
and preserve DNA integrity at the same time. These results
prompted us to explore whether we could harness the
aforementioned attractive features of micellar catalysis to
enable the Povarov reaction on DNA-linked substrates.
reactions call for comparison of exemplary “on-DNA”
reactions with authentic samples for confirmation. We isolated
product DNA-12a, and co-injected it with an authentic sample
ref-DNA-12a into an HPLC-column to confirm the micellar
Povarov reaction (Figure 3, Figure S19). Likewise, a DNA-
conjugated quinoline reference molecule ref-DNA-13a
matched the retention time of the side-product, supporting the
hypothesized heterocycle aromatization as side reaction during
micellar catalysis (Figure 3, Figure S19).65 Analysis of the
reaction kinetics in a range between 25-60 °C revealed a clear
temperature dependency of the micellar reaction. DNA-3a was
almost quantitatively converted to the tetrahydroquinoline
DNA-12a, at 40 °C after 6 h, at 50 °C after 4 h, and at 60 °C
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Design of a micelle-based acid catalyst. We aimed at
amphiphiles that form oil-in-water micelles, locate a catalyst
in the micellar environment, and associate at very low
concentrations to minimize the fraction of catalysts free to
interact with DNA in the water phase. Block copolymer 2a
meets all of these demands. It is composed of hydrophilic
poly(N,N-dimethylacrylamide) and lipophilic poly(n-butyl
acrylate), the latter was statistically copolymerized with a
sulfonic acid-substituted monomer.[4a] Above the CMC that
was determined to be approximately 1 µM, block copolymer
2a formed micellar aggregates in aqueous medium. Based on
the block copolymer structure it can be assumed that the
sulfonic acid moieties are located in the hydrophobic core and
the interface to the hydrophilic shell (Figure 1c). According to
DLS measurements block copolymer 2a formed micelles with
a diameter of circa 20 nm in water (Figure S2). Electron
microscopic images confirmed the DLS measurements and
revealed formation of uniform, spherical structures (Figure 5a,
Figure S5).
Development of the micelle-mediated Povarov reaction.
Polymer 2a was incubated at different concentrations with the
DNA-aldehyde conjugate 3a for 18 h at room temperature.
The DNA tolerated concentrations of up to 0.5 mM and up to
200 equivalents of the polymer (Figure 1d and Table S4,
Figure S13). Some DNA degradation by depurination (approx.
30 %) occurred only at 2 mM polymer concentration and a
very high, 500fold excess of the polymer versus DNA. Having
established a concentration range at which DNA can be
incubated with the acid catalyst, we systematically explored a
micelle-mediated variant of the Povarov reaction with the
14mer DNA-aldehyde conjugate DNA-3a, aniline 8a, and
olefin 10a to the target tetrahydroquinoline conjugate DNA-
12a (Figure 3). A first series of experiments investigated the
effect of copolymer concentration on product formation. Low,
sub-CMC concentrations failed to yield the target compound.
To our delight, at higher copolymer concentrations of 0.5 mM
and 50-100fold excess versus DNA the aldehyde DNA-3a was
after 2 h (Figure 4, Table S7, Figures S20-23).
Figure 3. Comparison of homogeneous and micellar acid-mediated reactions
of DNA-aldehyde DNA-3a to DNA-tetrahydroquinoline DNA-12a. (a) Scheme
of the Povarov reaction yielding DNA-12a and an oxidized side product DNA-
13a under the conditions of micellar catalysis. (b) HPLC traces of an
attempted homogeneous Povarov reaction (upper left-hand trace), the
Povarov reaction mediated by block copolymer 2a at 25 °C after 6 and 18 h
(upper right-hand and middle left-hand traces), the micelle-mediated reaction
upon addition of 5 % of ethyl acetate as co-solvent (middle left-hand trace),
and reference molecules ref-DNA-12a and ref-DNA-13a (lower traces).
almost
completely
converted
to
the
desired
tetrahydroquinoline
DNA-12a
without
detectable
depurination. HPLC analysis showed formation of a late
eluting major product with a mass matching the target DNA-
12a, accompanied by a minor side-product eluting slightly
before the main peak, which was later confirmed to be caused
by oxidative heterocycle aromatization (Figure 3, Scheme S6,
Table S6, Figures S16-S18).63 DNA conjugates are routinely
Prolonging reaction times at elevated temperatures led to
notable DNA degradation and increased formation of the
oxidized side product (Figure S22). Electron microscopic
images were taken of different reagent mixtures to gain insight
into the micelle-mediated reaction. Addition of either DNA-3a
or olefin 10a to copolymer 2a did not change the overall
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morphology of the micelles (Figure 5b,c, Figure S5).
failed to yield the product (Figure S28).34,40,41 These
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However, addition of DNA-3a, and both aniline 8a and olefin
10a at concentrations optimized for Povarov reaction to block
copolymer 2a had a pronounced impact on micelle shape.
Several micelles aggregated to large conglomerates, and a
substantial fraction of micelles was transformed from a
spherical to a rod-like shape (Figure 5d, Figure S5).
experiments underpinned the need for immobilization of the
catalytic moiety in the micelle. Next, we investigated the
placement of the catalyst moiety. Locating the catalyst group
in the hydrophilic part of the amphiphile has been reported for
interfacial catalyst application and was realized with block
copolymer 2c.66 Two batches of this block copolymer with
slightly differing dp were compared head-to-head with block
copolymer 2a. Both block copolymers 2a and 2c showed
similar DNA-degrading activity at room temperature (Tables
S4,S5, Figures S13,S14). Block copolymer 2c mediated the
reaction of DNA-3a with aniline 8a and olefin 10a with
comparable, if slightly less product formation rates at room
temperature and 40 °C, respectively (Figures S29, S30).
However, at higher temperatures the catalytic behaviour of
block copolymer 2c differed notably from block copolymer 2a
(Figure 6, Table S11, Figures S31, S32). In contrast to catalyst
2a which showed a clear temperature-dependent catalytic
activity, block copolymer 2c yielded reproducibly less DNA-
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Figure 4. Kinetics of the block copolymer 2a-mediated reaction of aldehyde
DNA-3a to tetrahydroquinoline DNA-12a as depicted in Figure 3. Longer
reaction times at elevated temperature decreased conversion to target DNA-
12a due to DNA degradation. The conversions were estimated by HPLC
analysis of the crude reaction mixture, missing percentage to 100 %: either
DNA-3a or degraded DNA, and side products.
DLS measurements of this complex reactant mixture indicated
swelling of the micelles from a diameter of approx. 20 nm to
approx. 100 nm and supported the EM images (Figure S1).
Thus, both EM images and DLS measurements are in line with
our hypothesis that micelles composed of block copolymer 2a
accommodate organic reactants concentrating them in a
confined nanoreactor for subsequent reaction. Water-insoluble
starting materials often require co-solvents or biphasic solvent
systems to obtain synthetically useful concentrations. Ethyl
acetate,
CH2Cl2,
1,2-dichlorethane,
THF,
and
hexafluoroisopropanol, added at 5 vol % to the micellar
dispersion, were compatible with the reaction, whereas
addition of MeOH, DMSO, and DMF led to increased side
product formation (Figure 3, Table S9, Figure S25). The
robustness of a catalyst impacts its potential for application.
Polymer synthesis is inherently less controllable than small
organic molecule synthesis. Differences in the dp (degree of
polymerization) may occur and lead to batch-to-batch
differences in the polymer size. Fortunately, the micellar
Povarov reaction could be reproduced faithfully with two
batches of block copolymer 2a that differed in the dp (Table
S10, Figure S26). We then explored the requirements of the
micellar reaction. Exclusion of any single component of the
reaction precluded tetrahydroquinoline formation (Figure
S27). Two additional polymers were designed to study the
impact of catalyst design on the Povarov reaction: block
copolymer 2b without covalently linked sulfonic acid served
as a control for catalyst immobilization and block copolymer
2c directed the sulfonic acid into the hydrophilic corona. As
expected, block copolymer 2b failed to mediate synthesis of
DNA-12a due to the absence of a catalytically active moiety.
Addition of methane sulfonic acid or the more lipophilic para-
toluenesulfonic acid to a dispersion of block copolymer 2b
following a well-established concept in micellar catalysis
12a at 50 °C than at 40 °C at any time point.
Figure 5. Structural characterization of polymer 2a and reactant mixtures by
transmission electron microscopy (TEM). (a) TEM image of polymer 2a from
an aqueous 0.25 mM polymer solution. (b) TEM image of DNA conjugate
DNA-3a and 50 equivalents of polymer 2a. (c) TEM image of polymer 2a and
160 equivalents of 3,4-dihydropyran 10a. (d) TEM image of DNA-3a, 50
equivalents of block copolymer 2a, 8000 equivalents of aniline 8a, and 8000
equivalents of 3,4-dihydropyran 10a. Enlarged and further images are shown
in Figure S5.
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Strikingly, neither batch of block copolymer 2c mediated the Scope of the micelle-mediated Povarov reaction. The
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Povarov reaction to DNA-12a at 60 °C, rather we found
significant DNA degradation after 4 h reaction time (one batch
tested). The reason for this behaviour is probably related to the
fact that polymer micelles tend to be less aggregated at higher
micellar Povarov reaction was established with a 5’-C(6)-
aminolinker-conjugated aldehyde DNA-3a (Figure 3). This
aldehyde was coupled via a longer and more hydrophilic 5’-
terminal PEG(4)-aminolinker, and via a more lipophilic 5’-
C(12)-aminolinker to the DNA oligomer. Neither linker had
an impact on the reaction result (Table S12, Figure S34). A
single-stranded 21mer DNA aldehyde conjugate gave the
target DNA-tetrahydroquinoline conjugate as well (Table S13,
Figure S35). The scope of the three-component reaction was
then explored with a range of reagents (Figure 7a). One
intriguing aspect of this reaction is the possibility to synthesize
different heterocyclic cores through the choice of olefin 10
(Figure 7b). The 3,4-dihydropyran furnished a tricyclic 6-6-6
ring system DNA-12, while N-Boc-protected pyrroline 10b
and 2,3-dihydrofuran 10c gave 6-6-5 ring systems DNA-14
and -15 (Figure S36, S37). Finally, ethyl vinyl ether 10d gave
the 6-6 bicycle quinoline DNA-16 after heterocycle oxidation
and elimination of the ethyloxy-substituent (Figure 7b and
Figures S38, S39).[59-61] Next, we tested the aniline scope with
20 diverse substituted anilines 8a-t that were reacted with
DNA-3a and either 3,4-dihydropyran 10a or N-Boc-protected
pyrroline 10b (23 anilines, 8a-z). Ten of these reactions are
shown in Figure 7c, the remainder can be found in the
extended Tables S14 and S15, and in Figures S40-S44. No
product was formed with 2,6-disubstituted aniline 8b, which
would only allow for addition of an electron-rich olefin 10 to
an imine 9 (Figure 2b). Aniline 8c gave the target product
DNA-12c with a 45 % conversion and DNA-14c with more
than 90 % conversion as determined by HPLC analysis of the
crude reaction mixtures. Ortho-substituted anilines such as
8d/m, rather poorly represented in literature on the Povarov
reaction, gave the products DNA-12d/m at conversions of 90
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%
and 55 %, respectively. However, the analogous
tetrahydroquinolines DNA-14d/m were obtained both with
>90 % conversions. Meta-substituted anilines 8e-g which can
furnish two regioisomers were tested as well. These starting
materials gave high conversions to both target heterocycles
DNA-12 and DNA-14 in most cases. A notable exception was
the highly polar sulfonamide 8s that reacted poorly to DNA-
12s but afforded DNA-14s with an excellent conversion.
Finally, para-substituted anilines 8h-j gave mixed results in
the reac tion to tetrahydroquinoline DNA-12. For instance,
para-alkyl, and -phenyl substituents were well tolerated, also
4-aminobenzoic acid 8i gave rise to the target conjugate DNA-
12i at more than 90 % conversion, whereas para-bromoaniline
8j gave DNA-12j only with a conversion of 35 %. The
micelle-mediated Povarov reaction with N-Boc-pyrroline 10b
and all para-substituted anilines allowed for synthesis of
tetrahydroquinolines DNA-14 at conversions exceeding 90 %.
Overall, the micelle-mediated Povarov reaction to DNA-
tetrahydroquinolines DNA-14 was higher yielding than the
micelle-mediated reaction to DNA-tetrahydroquinolines DNA-
12. Out of 20 anilines that were tested, 7 anilines afforded the
tetrahydroquinoline conjugates DNA-12 at conversions of 90
%, whereas 21 out of 23 anilines gave rise to
tetrahydroquinolines DNA-14 with full consumption of the
aldehyde. The different lipophilicity of olefins 10a and 10b
could plausibly explain the differential reactivity of these two
Figure 6. Comparison of the catalytic activity of block copolymers 2a and 2c.
(a) Structure of block copolymer 2c. (b) Reaction scheme of the micelle-
mediated Povarov reaction to DNA-12a/13a. (c) Head-to-head comparison of
reactions mediated by block copolymers 2a and 2c. Block copolymer 2a is
more efficacious than 2c at elevated temperatures. In striking contrast to
catalyst 2a block copolymer 2c even failed to mediate the Povarov reaction
at 60 °C.
temperatures due to increasing dynamics of the polymer
chain.67At temperatures of 50 °C and 60 °C, block copolymer
2c assembled the reactants and the acid catalyst less efficiently
or not at all in the manner required for product formation.
Finally, a 69mer single stranded DNA was incubated with the
micellar catalyst 2a under the conditions used in the reaction
to compounds DNA-12, precipitated, and amplified by PCR.
The yield of the PCR product was comparable to a control
experiment without catalyst 2a (Figure S33).
olefins in
a
micellar environment. Higher effective
concentrations of the more lipophilic N-Boc-protected
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catalysis with high yields. Only ortho-substituted aldehyde DNA-3e gave
partial conversion to the product (boxed peak in the MALDI spectrum).
pyrroline 10b might be reached in the micelle core. The
significant difference in reactivity between olefins 10a and
10b in the micelle-mediated Povarov reaction prompted us to
compare the kinetics of both reactions. Indeed, the reaction to
DNA-14a proceeded much faster than to DNA-12a (Table
S16, Figure S45). Aldehyde
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DNA-3a
was
almost
completely
converted
to
tetrahydroquinoline DNA-14a after 2 h at room temperature,
and after 1 h at 40 °C. Of note, the reactions to DNA-14 did
not produce oxidized side products. The micellar reaction
system shall be applied in combinatorial encoded library
synthesis. In this application it must provide target products
with high yields from complex mixtures of starting materials.
We simulated a library synthesis with eight carboxaldehydes
DNA-3a-h representing different substitution patterns (ortho,
meta, para, electron-rich and -poor, heterocyclic) that were
mixed to two partly redundant pools of five compounds each.
Both pools were reacted with aniline 8a and pyrroline 10b.
Mass spectrometric analysis of the product mixtures showed
full conversion of five DNA-conjugated aldehydes. The ortho-
substituted aldehyde 3e gave ca. 30% conversion and in case
of two aldehydes 3g-h we found reduced conversion to
products due to partial aldehyde oxidation in aqueous solution
(Figures 7d, S46-S54).
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Expanding the scope of micellar acid catalysis: Gröbke-
Blackburn-Bienaymé reaction and tBoc protective group
removal. Similar to the Povarov reaction, the Gröbke-
Blackburn-Bienaymé (GBB) reaction is catalyzed either by
Brønsted or Lewis acids.68,69 A micellar variant of the GBB
reaction gave access to DNA-coupled 3-aminoimidazo[1,2-
a]pyridines DNA-19 from aldehyde DNA-3a, 2-
aminopyridines 17, and isocyanides 18 (Figure 8a). First
efforts towards DNA-tagged 3-aminoimidazo[1,2-a]pyridines
DNA-19 involved reaction conditions that were optimized for
the synthesis of DNA-tetrahydroquinoline conjugates.
However, under these conditions only 30 % of DNA-3a was
converted to the product DNA-19a (Table S17, Figure S55).
Prolonged reaction times increased formation of DNA-19a to
55 % after 36 h, and 75 % after 54 h. At 40 °C the starting
material was almost quantitatively converted to the target
heterocycle (Table S17, Figure S56). Ethanol, acetonitrile, and
ethyl acetate added at 5 vol% to the reaction mixture did not
have any impact on the formation of DNA-19a (Table S18,
Figure S57). The micellar GBB reaction was sensitive to
positioning of the catalyst within the micelle, as the block
copolymer 2c-mediated reaction gave the target 3-
aminoimidazo[1,2-a]pyridine conjugate (Figure S58) with
only 40 % conversion. A small scope of the GBB reaction was
explored with different 2-aminopyridines 17 and isocyanides
18 (Table S19, Figure S59). DNA-3a was reacted with 2-
aminopyridine 17b and cyclohexyl isocyanide 18a to target
DNA-19b with 60 % conversion, the 5-ethyl substituted 2-
aminopyridine 17c gave DNA-19c with a similar 70 %
conversion, while the functionalized 5-(N-tBoc-aminomethyl)-
2-aminopyridine gave only 30 % of DNA-19d. DNA-3a was
then reacted with either 2-amino-5-methylpyridine 17a and
tert-butyl isocyanide 18b, or 2-amino-5-methylpyridine 17a
and benzyl isocyanide 18c. The two isocyanides gave DNA-3-
aminoimidazo[1,2-a]pyridine conjugates DNA-19e/f with 50
% and 70 % conversions, respectively (Table S19, Figure
S59). Last, benzyl isocyanide 18c was successfully reacted
with DNA-3a and 2-aminopyridine 17b or 2-amino-5-
Figure 7. Scope of the micellar Povarov reaction to DNA-tetrahydroquinoline
conjugates. (a) Reaction scheme. (b) Different core heterocycles are
accessible depending on the olefin 10. (c) A broad scope of anilines 8
furnished the target heterocycles. The conversion rates were estimated by
HPLC. (d) Simulating an encoded library synthesis scenario, a pool of DNA-
aldehyde conjugates was converted to tetrahydroquinolines by micellar
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ethylpyridine 17c, furnishing DNA-19g/h with 90 % and 50 % operation in encoded library synthesis. In the literature, this
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conversion, respectively. Thus, block copolymer 2a facilitated
the synthesis of diverse DNA-tagged 3-aminoimidazo[1,2-
a]pyridines by Gröbke-Blackburn-Bienaymé reaction.
protective group is removed by heating encoded libraries at
pH 9.4 for prolonged time.14 A micellar dispersion of block
copolymer 2a could remove the tBoc protective group from
the tBoc-glycine conjugate DNA-20. However, the reaction
was accompanied by severe DNA damage. A systematic
investigation in micellar deprotection of the tBoc-glycine
conjugate DNA-20 could not arrive at conditions that
separated removal of the protective group from depurination in
a satisfactory manner (Figure 8b, Table S20, Figures S61-
S64). It has been reported that Mg(II) salts can decrease the
rate of depurination in acidic buffers.70 Addition of increasing
amounts of MgCl2 to the micellar dispersion of block
copolymer 2a and tBoc-glycine conjugate DNA-20 indeed
supressed depurination to an acceptable level of ca. 10 %
while yielding 40 % of the deprotected glycine conjugate
(Table S21, Figures S65-S67). These results were reproduced
with the tBoc-protected tetrahydroquinoline conjugate DNA-
14a (Figure S69). Removal of the tBoc group from DNA-14a
was followed by amide coupling as would be done in an
obvious library synthesis scenario leading to compounds that
are closely related to the chemotype of the CRISPR-Cas9
inhibitor 5 (Figure S70).
In light of the ready availability of tBoc-protected building
blocks, removal of this protective group is an important
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Oxidation of DNA-coupled benzyl alcohol by a micellar
Cu/bipyridine/TEMPO catalyst system. Functional group
transformations are rarely described in encoded
chemistry.11,14,71,72 The selective transformation of alcohols to
the corresponding aldehydes and ketones represents a key
transformation in modern organic chemistry that has yet to be
demonstrated on DNA-coupled substrates. Oxidation reactions
may damage DNA by 8-oxoA or 8-oxoG formation. These
DNA lesions cause increased depurination rates and
misreading of the code by DNA polymerases.73 Alcohols are
conveniently oxidized by a catalyst system consisting of
copper ions, the stable radical TEMPO and atmospheric
oxygen.74 We attempted to oxidize a DNA-coupled benzyl
alcohol under the published homogeneous reaction conditions
but obtained only starting materials under conditions that did
not damage DNA. Recently, we have developed a micellar
catalytic approach for the Cu(I)/TEMPO based aerobic
oxidation of alcohols in aqueous medium.42 Two amphiphilic
block copolymers 24a,b functionalized with either one or two
bipyridine ligands in the lipophilic portion were designed to
immobilize copper(I) in the micelle (Figures 8c, S71-S75). In
analogy to a homogeneous catalyst system,74 the oxidation of
DNA-benzyl alcohol 23 was investigated in the presence of
micelles formed by block copolymers 24a,b, CuBr, and
TEMPO as the N-oxyl source. At lower catalyst loadings (5-
10 equivalents versus DNA) and in contrast to the
homogeneous conditions (Figure S77) the micellar system
showed
a successful time- and micelle concentration-
dependent oxidation of both DNA-coupled benzyl alcohols
and an aliphatic alcohol to the corresponding aldehydes with
no detectable formation of 8-oxopurines or over-oxidation to
the carboxylic (Figure 8d, Table S24, Figures S78-S82).
Indeed, we were able to synthesize a DNA tetrahydroquinoline
conjugate from a DNA-conjugated alcohol by two consecutive
micelle-mediated reactions: the product of micellar alcohol
oxidation was isolated and reacted to the heterocycle by
micellar Povarov reaction (Figure S79).
Figure 8. Expanding the scope of micellar catalysis. (a) Block copolymer 2a-
mediated Gröbke-Blackburn-Bienaymé reaction to 3-aminoimidazo[1,2-
α]pyridines DNA-19. (b) Removal of a tBoc protective group required addition
of MgCl2 to protect DNA against depurination. (c) A block copolymer-based
micellar system for the Cu(I)/bipyridine/TEMPO catalyst. (d) Comparison of
the homogeneous Cu(I)/bipyridine/TEMPO system with the micelle-based
variant for the oxidation of a DNA-tagged benzyl alcohol to the corresponding
benzaldehyde.
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added MgCl2 (0.5 µmol, 1000 eq.), dissolved in 1 µL of water
CONCLUSION
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and copolymer 2a (12.5 nmol, 25 eq.) dissolved in 8 µL of
water. The reaction mixture was filled with distilled water to a
volume of 50 μL giving a final concentration of 0.25 mM of
copolymer 2a. The reaction mixture was shaken at 50 °C for
4 h. After that, 70 µL of water was added and the reaction
mixture was extracted with ethyl acetate. The aqueous solution
was evaporated, the residue was re-dissolved in 40 µL of
water, and the DNA-tBoc deprotected conjugates DNA-20 and
DNA-22a were analyzed by RP-HPLC and MALDI-MS.
Reactions mediated by low pH are challenging to translate to a
DNA-tagged format due to the risk of DNA degradation by
depurination. In this study, we explored sulfonic acid catalysts
based on amphiphilic block copolymers (2a, Figure 1c) for the
reaction of DNA-coupled starting materials. The block
copolymers aggregated in water at low, micromolar
concentration and localized a sulfonic acid in nanometer-sized
oil-in-water micelles. These acidic nanoreactors facilitated
three reactions to DNA-tagged target molecules: Povarov and
GBB reactions to diverse substituted heterocycles and the
removal of a tBoc protective group from amines. Both
heterocycle-forming three-component reactions showed a
broad scope of simple, readily available starting materials and
proceeded without detectable DNA degradation. A micelle-
mediated high-yielding synthesis of pools of DNA-conjugated
tetrahydroquinolines suggested the utility of our catalytic
system for encoded library synthesis. The block copolymer
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Block copolymer 24-mediated oxidation of DNA-alcohol to
DNA-aldehyde conjugates.
To a solution of block
copolymer 24b (500 nmol) in 190 µL of dry acetonitrile was
added CuBr (1 µmol, 2.0 eq.) dissolved in 10 µL of dry
acetonitrile. The reaction mixture was stirred at room
temperature under argon atmosphere for 30 minutes. The
solvent was evaporated in vacuo, and the micelle catalyst 24
was dissolved in 500 µL of distilled water. To a solution of
micelle catalyst 24 (40 nmol, 10 eq.) in distilled water were
added N-methylimidazole (80 nmol, 20 eq.), dissolved in 1 µL
of distilled water, and TEMPO (40 nmol, 10 eq.), dissolved in
1 µL of distilled water. The reagent mixture was incubated at
room temperature for 5 minutes and DNA-alcohol conjugate
DNA-23 (4 nmol) was added. The reaction mixture was filled
with distilled water to a volume of 40 μL giving a final
concentration of 1.0 mM of catalyst 24. The reaction mixture
was shaken at room temperature for 22 hours. After that, 70
µL of water was added and the reaction mixture was
thoroughly extracted with ethyl acetate. The aqueous solution
was evaporated, and the residue was re-dissolved in 100 µL of
water for analysis by RP-HPLC and by MALDI-MS.
catalyst design was subsequently extended to
a
Cu(I)/bipyridine/TEMPO reagent. It allowed for the oxidation
of both a DNA-coupled benzylic and aliphatic alcohol to the
corresponding aldehyde without detectable oxidative DNA
damage such as 8-oxoG formation. The two micellar systems
demonstrated in this study hint at a potentially broad scope of
designed micellar catalysts for DNA-encoded compound
synthesis. Future research directions in this field could
comprise investigations in the compatibility of block
copolymer-based catalysis with different biomacromolecules;
further micelle-based catalysts for synthesis of DNA-tagged
target molecules; and the use of micellar catalysis in different
DEL formats - DNA-recorded as well as DNA-templated
chemistry - for screening library synthesis.
EXPERIMENTAL
ASSOCIATED CONTENT
Block copolymer 2a-mediated Povarov reaction. To an
aqueous solution of DNA-aldehyde conjugate DNA-3
(500 pmol) were added an aniline 8 (4 µmol, 8000 eq.),
dissolved in 2.5 µL of ethyl acetate, an olefine 10 (4 µmol,
8000 eq.), and block copolymer 2a (25 nmol, 50 eq.) dissolved
in 16 µL of distilled water. The reaction mixture was filled
with water to a volume of 50 μL giving a final concentration
of 0.5 mM of block copolymer 2a. The reaction mixture was
shaken at 25 °C for 18 h. After that, 70 µL of water was added
and the reaction mixture was thoroughly extracted with ethyl
acetate (at least 6 x 400 μL). The aqueous solution was
evaporated, the residue was re-dissolved in 40 µL of water for
analysis by RP-HPLC and by MALDI-MS.
Supporting Information contains protocols for the synthesis of
DNA conjugates, HPLC traces and MALDI MS spectra of DNA
conjugates; synthesis and characterization of reference molecules
and amphiphilic block copolymers; and detailed synthesis
protocols for the micelle-mediated reactions. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* andreas.brunschweiger@tu-dortmund.de; ralf.weberskirch@tu-
dortmund.de
Author Contributions
Block
copolymer
2a-mediated
Gröbke-Blackburn-
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Bienaymé reaction. The protocol for the GBB reaction
included the following changes compared to the Povarov
reaction: A 2-aminopyridine 17 (4 µmol, 8000 eq.) was
dissolved in 2.5 µL of ethanol, the isocyanide 18 (4 µmol,
8000 eq.) was directly pipetted into the reaction mixture, and
the reaction mixtures were shaken at 40 °C for 54 h. The crude
reactions were extracted seven times with each 500 µL of
ethyl acetate prior solvent evaporation and compound analysis.
Funding Sources
This work was supported by the EFRE-NRW-funded Drug
Discovery Hub Dortmund (DDHD), and the Mercator Research
Center Ruhr (MERCUR) Grant Pr-2016-0010.
ACKNOWLEDGMENT
Block copolymer 2a-mediated cleavage of tBoc protective
group from amines. To an aqueous solution of DNA-tBoc
protected conjugate DNA-21 or DNA-14a (500 pmol) were
We thank Prof. Rehage (TU Dortmund) for DLS measurements.
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guided radical-based synthesis of C(sp3)-C(sp3) linkages on DNA. Proc. Nat.
Elias Klarhorst (TU Dortmund) gave support for the synthesis of
Acad. Sci. USA 2018, 115, E6404-E6410. b) Phelan, J. P.; Lang, S. B.; Sim,
J.; Berritt, S.; Peat, A. J.; Billings, K.; Fan, L.; Molander, G. A. Open-Air
Alkylation Reactions in Photoredox-Catalyzed DNA-Encoded Library
Synthesis. J. Am. Chem. Soc. 2019, 141, 3723-3732; c) Kölmel, D. K.;
Loach, R. P.; Knauber, T.; Flanagan, M. E. Employing Photoredox Catalysis
for DNA-Encoded Chemistry: Decarboxylative Alkylation of α-Amino Acids.
ChemMedChem 2018, 13, 2159-2165.
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DNA conjugates.
ABBREVIATIONS
CMC, critical micellar concentration; DEL, DNA-encoded
library; dp, degree of polymerization; GBB, Gröbke-Blackburn-
Bienaymé; TEM, transmission electron microscopy.
(18) Schreiber, S. L. Target-Oriented and Diversity-Oriented Organic
Synthesis in Drug Discovery. Science 2000, 287, 1964−1969.
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System for Chemoselective Aerobic Oxidation of Primary Alcohols. J. Am.
Chem. Soc., 2011, 133, 16901-16910.
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SYNOPSIS TOC
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stat
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O
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H2O
X
*
R
*
*
polymer micelles
immobilized sulfonic acid
R
N
H
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