DNA functionalization by dynamic chemistry

Article abstract of DOI:10.3762/bjoc.12.203

Dynamic combinatorial chemistry (DCC) is an attractive method to efficiently generate libraries of molecules from simpler building blocks by reversible reactions under thermodynamic control. Here we focus on the chemical modification of DNA oligonucleotid

Full text of DOI:10.3762/bjoc.12.203

DNA functionalization by dynamic chemistry
Zeynep Kanlidere, Oleg Jochim, Marta Cal and Ulf Diederichsen*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2016, 12, 2136–2144.
Institute of Organic and Biomolecular Chemistry, Georg-August
University Göttingen, Tammannstrasse 2, D-37077 Göttingen,
Germany
doi:10.3762/bjoc.12.203
Received: 03 June 2016
Accepted: 08 August 2016
Published: 06 October 2016
Email:
Ulf Diederichsen* - udieder@gwdg.de
Associate Editor: S. Flitsch
*
Corresponding author
©
2016 Kanlidere et al.; licensee Beilstein-Institut.
Keywords:
License and terms: see end of document.
base-pairing; base-pair mismatch; DNA functionalization; DNA
templates; dynamic combinatorial chemistry; D-threoninol based
scaffolds
Abstract
Dynamic combinatorial chemistry (DCC) is an attractive method to efficiently generate libraries of molecules from simpler build-
ing blocks by reversible reactions under thermodynamic control. Here we focus on the chemical modification of DNA oligonucleo-
tides with acyclic diol linkers and demonstrate their potential for the deoxyribonucleic acid functionalization and generation of
libraries of reversibly interconverting building blocks. The syntheses of phosphoramidite building blocks derived from D-threo-
ninol are presented in two variants with protected amino or thiol groups. The threoninol building blocks were successfully incorpo-
rated via automated solid-phase synthesis into 13mer oligonucleotides. The amino group containing phosphoramidite was used
together with complementary single-strand DNA templates that influenced the Watson–Crick base-pairing equilibrium in the mix-
ture with a set of aldehyde modified nucleobases. A significant fraction of all possible base-pair mismatches was obtained, whereas,
the highest selectivity (over 80%) was found for the guanine aldehyde templated by the complementary cytosine containing DNA.
The elevated occurrence of mismatches can be explained by increased backbone plasticity derived from the linear threoninol build-
ing block as a cyclic deoxyribose analogue.
Introduction
The well-defined duplex structure, self-assembling by base-pair applications requires the introduction of additional functional
recognition, and the accessibility by solid-phase synthesis make groups into its native structure [10,11]. Such chemically modi-
DNA oligonucleotides an ideal supramolecular scaffold in a fied oligonucleotides are useful intermediates for their subse-
wide field of applications [1,2]. In recent years oligonucleo- quent functionalization through post-synthetic protocols [11-
tides especially were applied to self-assembly into artificial 13]. Within a post-synthetic strategy, a nucleotide analog is
nanostructures [3-9]. Preparation of oligonucleotides for new modified with a reactive functional group, incorporated into
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oligonucleotides by standard solid–phase synthesis and reacted In case of an amine group on the backbone, a reversible imine
with the desired molecules on the oligonucleotide level. As exchange reaction with aldehyde modified nucleobases was per-
amines and thiols are among the widely used groups introduced formed (Figure 1a). In the presence of a thiol group on the
for the post-synthetic modifications, the acyclic threoninol backbone, a thioester exchange reaction with thioester modi-
linker (2-amino-1,3-butanediol) [14-21] constitutes an attrac- fied nucleobases was expected (Figure 1b).
tive choice for oligonucleotide functionalization. Threoninol
Results and Discussion
can be introduced in oligonucleotides via the corresponding
D-Threoninol-based building blocks: design
phosphoramidite generating a ribose-free abasic site on the
backbone that provides the amine group for later functionaliza- and synthesis
tion [22-28]. Similarly, a thiol functionality can be introduced Two D-threoninol-based phosphoramidite building blocks con-
by substitution of the amine group of threoninol and incorporat- taining orthogonally protected amine 4 or thiol 11 moieties
ed into the oligonucleotide backbone. The amine and thiol were successfully synthesized. As presented in Scheme 1 phos-
groups can be used for further oligonucleotide functionalization phoramidite 4 was obtained according to the procedures previ-
reacting these sites with functional molecules like metal ligands ously described in literature [14,23].
or fluorophores. Functional molecules of interest can be teth-
ered post-synthetically in an irreversible manner as amide or re- In order to obtain phosphoramidite 11 we have developed a syn-
versibly as imine or thioester.
thesis (Scheme 2) based on L-threonine as a starting material.
L-Threonine was converted to bromo-derivative 6 by a diazoti-
Recent advances in dynamic combinatorial chemistry [29-40] zation reaction using sodium nitrite followed by potassium bro-
have enabled the utilization of presynthesized oligomers with mide substitution under overall retention of configuration due to
abasic sites on the backbone for the addition of individual double inversion. Next, the subsequent reduction of carboxylic
monomeric nucleobases and consider the synthesis of new acid 6 to alcohol 7 was achieved by borane dimethyl sulfide
oligonucleotide analogues possessing different backbone (BMS) under dry conditions. The reaction between alcohol 7
topologies [41]. Ghadiri et al. employed this approach for an en- and 3-mercaptopropanenitrile (8) resulted in substitution of
zyme-free synthesis of an oligonucleotide analogue with a bromine to the thiol group and finally the introduction of the
peptide backbone carrying nucleobases on its amino acid side thiol functional group. The 3-mercaptopropanenitrile (8) was
chains [42] while Bradley et al. used the backbone of a peptide synthesized separately in two steps from acrylonitrile or the
nucleic acid (PNA) with abasic sites which gives a reactive sec- 3-chloropropanenitrile according to the previously described
ondary amine for reversible attachment of aldehyde modified procedure [48-50]. Next, the obtained compound 9 containing
nucleobases [43]. Moreover, the DNA template-directed selec- two hydroxy groups and the cyanoethyl protected thiol group
tion of one nucleobase from the reaction mixture with the amine was converted into the phosphoramidite being compatible with
or thiol functional group was investigated [44-47].
conditions of solid-phase oligonucleotide synthesis. The DMTr
protecting group was incorporated and the conversion of the
In our studies, dynamic chemistry is applied for post-synthetic secondary alcohol 10 to phosphoramidite 11 was performed.
functionalization of the threoninol based modified oligonucleo- The base-labile cyanoethyl group [51,52] is known to be resis-
tides in a reversible manner. Here we synthesized the phosphor- tant under synthesis conditions for the preparation of the phos-
amidite building blocks derived from D-threoninol which phoramidite building block and for solid-phase oligonucleotide
contain protected amine or thiol groups. These building blocks synthesis [49,53].
are used for later incorporation into oligonucleotides via solid-
Building blocks compatibility with solid-phase
phase synthesis. Using these modified oligonucleotides and
single strand DNA templates, we generated the libraries of re- synthesis of DNA single strands
versibly interconverting building blocks – dynamic combinato- Phosphoramidites 4 and 11 were introduced at position seven
rial libraries (DCL) (Figure 1). The abasic strand and its com- of 13mer oligonucleotides ON1 and ON2 applying automated
plementary template strand are spontaneously assembled into a solid-phase synthesis (Table 1 and Supporting Information
double helix through Watson–Crick base-pairing and the in- File 1, Table S1). The last step in the oligonucleotide
coming nucleobase monomer benefits from the hydrogen bond- synthesis involved the deprotection of the amine group
ing recognition by the respective nucleobase in the template using ammonium hydroxide at 55 °C. The Fmoc protecting
strand. The reversible attachment generates a dynamic system group of oligonucleotide ON1 was removed, however, the
that enables the combinatorial screening of the best bound cyanoethyl group as a base-labile protecting group of the thiol
nucleobase by allowing a rapid and continuous exchange be- was not removed quantitatively from the oligonucleotide
tween the threoninol site and the set of nucleobase monomers. ON2 [54-58].
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Beilstein J. Org. Chem. 2016, 12, 2136–2144.
Figure 1: Template-directed dynamic chemistry assay for the attachment of modified nucleobase monomers to an abasic backbone. a) Reversible
imine exchange reaction. b) Reversible thioester exchange reaction.
Scheme 1: Synthesis of the phosphoramidite building block 4 [23]. DMTr: dimethoxytrityl group, Fmoc: 9-fluorenylmethylcarbonyl, DMAP: 4-dimethyl-
aminopyridine, DIEA: N,N-diisopropylethylamine.
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Beilstein J. Org. Chem. 2016, 12, 2136–2144.
Scheme 2: Synthesis of the phosphoramidite building block 11.
At the beginning we determined the conversion of ON1 with
the respective nucleobase aldehydes in presence of the comple-
mentary DNA template strand (Table 1). The ON1 oligonucleo-
tide was allowed to react with only one nucleobase in order to
produce the individual product (Supporting Information File 1,
Figure S1, Table S2). A reaction between GCHO and ON1 in
the presence of TC was accomplished with complete conver-
sion of ON1 into the guanine incorporated product (ON1+G).
The reactions with cytosine (CCHO) as well as adenine (ACHO)
aldehydes gave similar yields, however, with lower conversion
level of ON1 compared to the previous case. Finally, lowest
conversion was observed for the reaction of ON1 with the TA
template and the thymine aldehyde (TCHO) (Supporting Infor-
mation File 1, Table S3).
Table 1: Sequences of modified oligonucleotide and templates used in
this work.
Strand
Sequence (5’-3’)
ON1
ON2
TCb
TGb
TTb
CGCTATXTATCGCa
GCGATAYATAGCGa
GCGATACATAGCGGTTc
GCGATAGATAGCGGTT
GCGATATATAGCGGTT
GCGATAAATAGCGGTT
TAb
aX and Y represent the abasic site with amino or thiol group, respec-
tively. bHere, the capital letter index of T stands for the cytosine,
guanine, thymine or adenine nucleobase positioning opposite to the
amine on ON1. cNucleobases that are opposite to the abasic site of
the modified strand are shown in bold letters. The GTT sequence at
the 3’-terminus was added to facilitate separation by HPLC.
The composition of libraries: base-pairing
Dynamic template-driven assembly of double
strand libraries
selectivity
To determine the influence of the DNA template on nucleobase
Oligonucleotide ON1 was used for further investigations incorporation into the strands at abasic site through imine
towards dynamic libraries of double strand DNA constructs. attachment to the amine group, four reactions were carried out
Oligonucleotide ON1 was used with the deprotected amine under identical conditions (pH 6, 20 mM phosphate buffer), but
group in the reaction of forming the imine bond between ON1 differing in the applied template strand. The 13mer oligonucleo-
and four nucleobase aldehydes (GCHO, CCHO, ACHO, TCHO) tide ON1 was mixed with one of the four complementary tem-
in the presence of complementary template strands TC, TG, TT plate strands (TC, TG, TT or TA, Table 1) in a 1:1 molar ratio.
or TA (Table 1). The respective template strand should control All four nucleobase aldehydes (GCHO, CCHO, ACHO, TCHO)
the incorporation of the corresponding nucleobase reversibly were added in excess amount and in equimolar concentrations.
linked as imine (Figure 2). The structures of four nucleobase
aldehydes were shown in Figure 3. These compounds were Sodium cyanoborohydride (NaBH3CN) was used for irre-
synthesized according to the procedures described previously versible conversion of the imine products obtained in equilib-
[
59-61].
rium into respective amines (Figure 2), thereby enabling the
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Beilstein J. Org. Chem. 2016, 12, 2136–2144.
Figure 2: Initial building blocks of a dynamic combinatorial library, a library of all possible imine products and a library of amine products after
reduction.
Figure 3: Set of aldehyde-modified nucleobases used in dynamic chemistry assay.
isolation and analysis of the library derived from oligonucleo- Figure 4 all possible products were eluted as a mixture separat-
tide ON1. Anion exchange high-performance chromatography ed by anion exchange HPLC at 80 °C; under these conditions
was used for the analysis of the final reaction mixture. The reac- dissociation of obtained oligonucleotide double strands is provi-
tion mixtures were composed of six oligonucleotides: the unre- ded. Well separated signals correspond to the temple strands
acted initial strand ON1, one of its complementary strands TC, and starting oligonucleotide ON1. The obtained four new
TG, TT or TA and the four possible product strands ON1+G, strands (ON1+C, ON1+G, ON1+T, ON1+A) were eluted with
ON1+C, ON1+A, ON1+T (Figure 4). HPLC separation of the similar retention time and broad elution profiles. Therefore, the
four possible products in the same reaction mixture was chal- product containing fractions were subjected to a second HPLC
lenging because the lengths of the starting sequence purification step applying basic conditions (pH 12) to separate
CGCTATXTATCGC (ON1) and the product sequences these compounds (Figure 5). At high pH deprotonation of
(
ON1+G, ON1+C, ON1+A, ON1+T) were identical differing guanine and thymine allow better separation. As indicated by
only by one nucleobase in the central position X. As shown in the elution profiles in Figure 5, the template strands significant-
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Beilstein J. Org. Chem. 2016, 12, 2136–2144.
Figure 4: Representative HPLC chromatograms for the mixture containing ON1 and four nucleobases in the presence of a DNA-template: a) TC;
b) TG; c) TT; d) TA. The chromatograms were performed after reductive amination of the samples (for more details see Supporting Information File 1).
ly affect the composition of the dynamic library. The control ent HPLC conditions. In all cases, incorporation of GCHO in the
experiment lacking the template provided a nearly equal distri- presence of template TC was obtained with clear preference.
bution of oligonucleotides (results are not given here).
In case of incorporation of the thymine aldehyde the TA con-
The highest selectivity of more than 80% was obtained for the taining template was not effective by supporting the expected
incorporation of GCHO (Figure 5a) with the complementary ON1+T product as it would have been supported by the A·T
template strand (TC). The Watson–Crick base-pairing with base pair formation (Figure 5d). Moreover, this dynamic library
three hydrogen bonds together with a high-stacking contribu- is even dominated by the two A·C and A·G mismatches indicat-
tion of purine nucleobases seems to be beneficial. The selec- ing a highly flexible arrangement of the incoming nucleobases.
tivity for the incorporation of the other aldehydes is significant- In general, the low template directed selectivity for incorpora-
ly lower (20–40%). Especially for CCHO with the complemen- tion of individual nucleobases is likely due to the higher flexi-
tary guanine (TG) template nucleobase incorporation was not bility in the backbone derived from threoninol units. The canon-
supported by Watson–Crick G·C base-pairing (Figure 5b). The ical Watson–Crick A·T and C·G base pairs are most energeti-
templating reactions were repeated four times applying differ- cally favorable, while other purine–purine (like A·A, G·G)
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Beilstein J. Org. Chem. 2016, 12, 2136–2144.
Figure 5: Representative HPLC chromatograms obtained using elevated pH, for samples collected from first purification step (in Figure 4 marked with
dashed line). The samples were collected from the reaction of ON1 and four nucleobases in the presence of a DNA-template: a) TC; b) TG; c) TT;
d) TA. The chromatograms were obtained after reductive amination of the samples (for more details see Supporting Information File 1).
mismatches are less frequent than T·G and C·A ones. These modified with the threoninol deoxyribose analogous by solid
results indicate that the selectivity of base pairing is not only phase synthesis. As proof of principle the 13mer oligonucleo-
driven by the number and strength of hydrogen bonds formed tide containing a threoninol derived amine functionality was
between two bases, but also by the backbone plasticity provid- submitted to dynamic combinatorial library (DCL) studies for
ing the frame for this interaction.
DNA template directed nucleobase incorporation. Whereas a
significant preference for the incorporation of the guanine unit
directed by a complementary cytosine was found, linkage of the
Conclusion
The efficient synthesis and DNA incorporation of two D-threo- other nucleobases was not at all selective and it seems likely
ninol based phosphoramidite building blocks with orthogonally that the high flexibility of the threoninol as a deoxyribose ana-
protected amine or thiol functional groups was described. logue does not allow better selection. Due to difficulties in
Therefore, DNA analogues were presented that can be cova- deprotection of the thiol group, oligonucleotides with threo-
lently functionalized by imine or thioester formation. In prin- ninol derived thiol functionality are still under investigation as
ciple this concept allows dynamic DNA functionalization with well as the simultaneous functionalization of DNA oligonucleo-
all kind of functional or recognition units at positions that were tides at various positions with different kind of functional units.
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Acknowledgements
Support from the State of Lower Saxony (International Ph.D.
program Catalysis for Sustainable Synthesis, CaSuS) is grate-
fully acknowledged.
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