Synthesis and properties of DNA oligonucleotides with a zwitterionic backbone structure

Article abstract of DOI:10.1039/c4cc06371f

The nucleosyl amino acid (NAA)-modification of oligonucleotides is introduced, which enables the preparation of oligonucleotides with zwitterionic backbone structures. It is demonstrated that partially zwitterionic NAA-modified DNA oligonucleotides are capable of duplex formation with native polyanionic counterstrands and show retained sensitivity towards base-pairing mismatches. This journal is

Full text of DOI:10.1039/c4cc06371f

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Synthesis and properties of DNA oligonucleotides
with a zwitterionic backbone structure†
Cite this: Chem. Commun., 2014,
50, 13742
Boris Schmidtgall,ab Anatol P. Spork,b Falk Wachowius,c Claudia Hobartnerc and
¨
Christian Ducho*ad
Received 14th August 2014,
Accepted 8th September 2014
DOI: 10.1039/c4cc06371f
www.rsc.org/chemcomm
The nucleosyl amino acid (NAA)-modification of oligonucleotides is
The apparent conclusion to overcome such problems arising
introduced, which enables the preparation of oligonucleotides with from electroneutral backbone structures is the introduction of
zwitterionic backbone structures. It is demonstrated that partially positively charged moieties into the backbone, thus resulting in
zwitterionic NAA-modified DNA oligonucleotides are capable of zwitterionic structures. Several examples for the attachment of
duplex formation with native polyanionic counterstrands and show positively charged units, often linked to the 20-hydroxy group or
retained sensitivity towards base-pairing mismatches.
the nucleobase, have been reported,13–15 but in these cases,
the phosphate-derived backbone has been kept intact. Thus,
Oligonucleotides represent promising biomedical agents for even though some of the resultant oligonucleotide analogues
antigene, antisense or RNA interference applications.1 However, showed promising properties, this strategy leads to densely
due to their high polarity hindering cellular uptake and their charged oligomers as the positive charge is an additional
limited stability towards nucleases, chemical or enzymatic feature in these cases. An alternative approach can be pursued
modifications of nucleic acid structures2,3 are an essential by replacing some of the phosphate moieties with positively
prerequisite for their biomedical application.
charged groups as linker units,16 thus retaining the overall
One structural feature of nucleic acids which has attracted number of charges in the oligomer. Consequently, a nucleic
significant interest is their polyanionic backbone resulting acid analogue with such a ‘real’ zwitterionic backbone may be
from the linking phosphate diesters.4 The polyanionic nature expected to be less polar than the aforementioned congeners
of nucleic acids largely contributes to their limited biomedical with attached cationic units.
applicability, and hence, several chemical modifications have
Due to electrostatic attraction, the presence of alternating
aimed at the manipulation of the backbone’s charge pattern. charge patterns in zwitterionic backbones may harm funda-
Thus, a significant number of artificial electroneutral inter- mental properties of nucleic acids though. Benner has hypothe-
nucleotide linkages such as sulfone,5 triazole6 or amide sised that a uniform charge pattern in the backbone is not only
moieties7–10 have been studied. Peptide nucleic acid (PNA) crucial to prevent longer nucleic acid strands from folding, but
represents probably the most striking example of an electro- that the repeating anionic motifs are also important for the
neutral nucleic acid analogue.11,12 However, fully electroneutral molecular recognition of two native nucleic acid strands with
nucleic acid analogues often show low water solubility and are complementary base sequences.17,18 It is therefore of major
prone to aggregate formation.
importance to study whether oligonucleotides with zwitterionic
backbones can still form stable duplex structures and retain
base pairing fidelity.
a University of Paderborn, Department of Chemistry, Warburger Str. 100,
Overall, only few positively charged artificial internucleotide
linkages have been reported,16 and consequently, oligonucleo-
tides with zwitterionic backbones have not been extensively
studied yet. Bruice et al. have replaced the phosphate unit with
either a guanidine19 or with an S-methylthiourea20 group. The
use of these moieties for the preparation of partially zwitterionic
oligonucleotides (‘chimera’16) was only studied with few oligo-
nucleotides though, and the results were not fully conclusive.21,22
Letsinger et al. have developed phosphoramidate linkages in
33 098 Paderborn, Germany
b
¨
Georg-August-University Gottingen, Department of Chemistry, Institute of Organic
¨
and Biomolecular Chemistry, Tammannstr. 2, 37 077 Gottingen, Germany
c Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11,
¨
37 077 Gottingen, Germany
d Saarland University, Department of Pharmacy, Pharmaceutical and Medicinal
¨
Chemistry, Campus C2 3, 66 123 Saarbrucken, Germany.
E-mail: christian.ducho@uni-saarland.de
† Electronic supplementary information (ESI) available: Synthesis of phosphor-
amidites and oligonucleotides, NMR spectra, analytical data of oligonucleotides,
Tm data and CD spectra of oligonucleotide duplexes. See DOI: 10.1039/c4cc06371f which positively charged groups (namely amines) were connected
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Fig. 1 Design concept of nucleosyl amino acid (NAA)-modified oligonucleotides.
to the backbone via alkyl linkers.23 They could thus obtain even The obtained ‘dimeric’ phosphoramidites were used for the auto-
fully zwitterionic DNA, but due to the conformational flexibility of mated synthesis of 24 different DNA oligonucleotides bearing one,
the positively charged moieties, one cannot rule out interactions two or four NAA-modifications at various positions and thus having
with the phosphate groups which would be less likely if the partially zwitterionic backbone structures. After oligonucleotide
positively charged moieties were more rigidly fixed at the inter- synthesis and base-mediated cleavage from the solid support, the
nucleotide linkage sites.
desired full-length products were purified and isolated. The fully
We have now conceived a novel artificial internucleotide characterised modified oligonucleotides (see ESI†) are classified
linkage which enables the preparation of zwitterionic nucleic into structural types of sequences 6, 7 and 8 (Fig. 2). All modified
acid analogues. Two structural motifs contributed to the design
of this new internucleotide unit (Fig. 1): (i) established amide
internucleotide linkages such as 1 and 27–10 and (ii) the nucleo-
side moiety of muraymycin nucleoside antibiotics (e.g. muray-
mycin A5 3) and its 50-deoxy analogues 4. Muraymycins
represent a promising class of natural products with anti-
microbial activity.24 In the course of our synthetic investigations
on muraymycins,25–27 we have previously reported on the efficient
stereoselective preparation of protected forms of 4 and have
coined the term nucleosyl amino acids (NAAs) for these types of
compounds.28,29 However, if one merges the structural principles
of 4 with an amide internucleotide bridge of type 1, an oligo-
nucleotide modification of type 5 (‘NAA-modification’) is obtained.
The amino group of the NAA-modification is expected to be
positively charged at physiological pH values. The configuration
of the stereocenter within the NAA-linkage is anticipated to
influence the spatial orientation of this cationic motif. In
contrast to Letsinger’s approach (vide supra), the NAA-motif
provides a positive charge in a conformationally more fixed
position which is in immediate vicinity to the position of
the negative charge in native phosphate linkages. The NAA-
modification therefore enables the introduction of positive
charges at specific sites of an otherwise phosphate-linked
oligoanionic nucleic acid, thus representing a novel system
providing (partially) zwitterionic DNA analogues.
For the automated solid-phase synthesis of stereoisomerically
pure NAA-modified oligonucleotides, ‘dimeric’ phosphoramidite Fig. 2 Stability of duplexes formed with NAA-modified DNA oligonucleotides
of types 6 (A) as well as 7 and 8 (B). Note that 8 (f and g in B) is self-
complementary. Sites of NAA-modifications = x, all other linkages were native
building blocks containing the protected NAA-motif with either
(60S)- or (60R)-configuration were prepared (see ESI†). We decided to
phosphates. Complementary strand: native DNA or native RNA with fully
matching sequences (for 6 and 7). Values in the diagram: alterations of duplex
initiate our studies with the introduction of NAA-modifications at
T–T motifs. Analogous NAA-modified X–T motifs (with X = A, C, G)
will then later be accessible using our synthetic route (see ESI†). without NAA-modifications. For full data see Tables S3 to S7 (ESI†).
Tm values per NAA-modification (DTm/mod) in comparison to native duplexes
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oligonucleotides formed duplexes with complementary DNA and
RNA oligonucleotides at physiologically relevant salt concentrations.
The stability of these duplexes was studied by UV-monitored thermal
denaturation providing melting temperatures (Tm values).
Modified DNA oligonucleotides of type 6 were designed in
order to test if the distance between two NAA-modifications
within a strand exerts an influence on the structure and
stability of nucleic acid duplexes (Fig. 2A). The results indicate
that all duplexes containing one or more NAA-modifications in
one strand were moderately destabilised in comparison to their
unmodified congeners. NAA-modified DNA–RNA hybrids were
destabilised more strongly (B2 to 4 1C per mod) than DNA/DNA
duplexes (B0.5 to 2 1C per mod). Also, a slight difference in
thermal stability (B1 1C per mod) between duplexes containing
the (60S)-NAA- and the (60R)-NAA-modification was observed in
nearly all experiments. However, no evidence was found for a
relationship between the distance of two NAA-modifications
(in the case of sequences b–d, Fig. 2A) and the duplex stability.
Remarkably, DNA/DNA duplexes with sequences of type e did
not display a more pronounced destabilisation although e
contained four consecutive NAA-motifs, i.e. a zwitterionic back-
Fig. 3 Destabilisation of DNA/DNA duplexes with one NAA-modified
strand caused by base-pairing mismatches. Depicted sequences formed
duplexes with DNA complements bearing mismatches at the indicated
position (opposite of red T). Sequences A and F: unmodified native
references without NAA-modifications. Values in the diagram: alterations
of duplex Tm values resulting from the presence of the mismatch, i.e. Tm
values of mismatch-containing duplexes in comparison to similar duplexes
with fully matching sequences (DTm (mismatch–match)). For full data see
Table S8 (ESI†).
bone segment spanning over ten base pairs (with the charge in the A-form of the DNA–RNA hybrid, thus leading to a greater
pattern ꢀ/+/ꢀ/+/ꢀ/+/ꢀ/+/ꢀ). Interestingly, the DNA/DNA duplex loss of thermal stability in the case of DNA/RNA duplexes.
containing the (60R)-isomer of sequence e showed a DTm/mod However, this also implies that conformational effects can over-
value of only ꢀ0.5 1C, indicating that such a significantly rule effects resulting from the charge pattern of the backbone,
modified DNA structure can display nearly the same melting thus questioning the supposedly crucial significance of the
stability as its native congener. For the discussion of all charges for the overall properties of nucleic acids (vide supra).
reported Tm values, it should be taken into account that the
Another aspect of interest was the base-pairing specificity of
experimental precision of Tm values is estimated to be around the modified nucleic acids. Thus, some of the NAA-modified
ꢁ1 1C,30 translating into DTm/mod values, for example, as low oligonucleotides were chosen for melting temperature experi-
as ꢁ0.25 1C for duplexes containing four NAA-modifications.
ments with DNA complements containing a mismatched base
In a second set of experiments, several sequences of structural in close proximity to the modification (Fig. 3). In this context,
type 7 were studied to investigate the correlation between the we compared the Tm values of duplexes composed of NAA-
position of the modification and the duplex stability. In general, it modified oligonucleotides B–E and G, H (Fig. 3) and a native
was found that internal NAA-modifications cause a stronger counterstrand having one mismatched base to corresponding
destabilisation compared to those in proximity to the 30- or the duplexes derived from unmodified reference oligonucleotides
50-end (Fig. 2B). As for sequences 6, the decrease in duplex stability A and F, respectively. In general, a pronounced destabilisation
was more pronounced in the case of DNA/RNA duplexes and a of the duplexes resulting from the mismatch was retained in
similar effect of the NAA-configuration was observed. In the case all NAA-modified duplexes. It can thus be concluded that the
of sequence e (Fig. 2B) bearing four NAA-modifications (in mismatch recognition of nucleic acids remains nearly untouched
pronounced distance to each other though), no extraordinary drop by the presence of NAA-modifications in the backbone regardless
in duplex stability was found. In addition, self-complementary of their number or position. The sensitivity towards base-pairing
palindromic sequences 8 were used to study the self-pairing mismatches is not overruled by the presence of positive charges in
between two NAA-modified DNA strands. It was thus demon- the NAA-modified backbone and the supposedly resulting partial
strated that two strands bearing the NAA-modification are able electrostatic attraction.
to form duplexes with only a moderate decrease in stability
compared to the native congeners (DTm B 0 to ꢀ2 1C).
We also examined the properties of NAA-modified duplexes
under various ionic strengths. Melting temperature experi-
Overall, the results depicted in Fig. 2 indicate a moderate ments at elevated concentrations of sodium chloride were
destabilisation of DNA/DNA and DNA/RNA duplexes due to the performed (see Tables S9 to S12, ESI†). Native oligoanionic
presence of NAA-modifications. DNA/DNA duplexes proved to DNA/DNA duplexes show increased Tm values at higher sodium
be less destabilised by NAA-modifications than DNA–RNA chloride concentrations. Using their approach employing alkyl-
hybrids. The reason for these effects might be the rigid peptide linked positively charged moieties (vide supra), Letsinger et al.
bond which fixes the torsion angle of the z-bond, giving rise to a have reported that a fully zwitterionic nonameric oligo-dT
conformation that is less favourable for base pairing and stacking analogue formed a duplex with poly-dA with a Tm value inde-
than in the case of native phosphate linkages. Apparently, the pendent of ionic strength, i.e. sodium chloride concentration.23
NAA-modification is better accommodated in a B-form helix than In the case of the partially zwitterionic NAA-modified DNA/DNA
13744 | Chem. Commun., 2014, 50, 13742--13745
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duplexes reported in this study, the increase of Tm values as a (FCI, Sachkostenzuschuss) and the Max Planck Society for
function of sodium chloride concentration was slightly less financial support. B.S. is grateful for a doctoral fellowship of
pronounced than for the respective native reference duplexes the Studienstiftung des deutschen Volkes.
(Tables S9 and S11, ESI†). This may reflect the partially zwitter-
Notes and references
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all NAA-modified duplexes and of unmodified reference
duplexes were recorded. All DNA/RNA and DNA/DNA duplexes
showed the characteristic features of A- and B-form helices,
respectively, and no significant differences between NAA-
modified and unmodified native nucleic acids were observed
(see ESI†).
In summary, typical chemical properties of nucleic acids are
retained in NAA-modified DNA oligonucleotides. Despite their
partial zwitterionic backbone, they are capable of forming
helical duplexes (though moderate destabilisation occurs) and
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This journal is ©The Royal Society of Chemistry 2014
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