A double-headed nucleotide with two cytosines: DNA with condensed information and improved duplex stability

Article abstract of DOI:10.1016/j.bmcl.2019.01.002

Double-headed nucleotide monomers are capable of condensing the genetic information of DNA. Herein, a double-headed nucleotide with two cytosine bases (C_C) is constructed. The additional cytosine is connected through a methylene linker to the 2

Full text of DOI:10.1016/j.bmcl.2019.01.002

Accepted Manuscript
A double-headed nucleotide with two cytosines: DNA with condensed infor-
mation and improved duplex stability
Kasper Beck, Charlotte Reslow-Jacobsen, Mick Hornum, Christian Henriksen,
Poul Nielsen
PII:
S0960-894X(19)30002-2
https://doi.org/10.1016/j.bmcl.2019.01.002
BMCL 26239
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Accepted Date:
5 December 2018
2 January 2019
Please cite this article as: Beck, K., Reslow-Jacobsen, C., Hornum, M., Henriksen, C., Nielsen, P., A double-headed
nucleotide with two cytosines: DNA with condensed information and improved duplex stability, Bioorganic &
Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.01.002
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Graphical Abstract
A double-headed nucleotide with two
cytosines: DNA with condensed information
and improved duplex stability
Leave this area blank for abstract info.
Kasper Beck, Charlotte Reslow-Jacobsen, Mick Hornum, Christian
Henriksen and Poul Nielsen*
A double-headed nucleotide monomer with an additional cytosine
base in the 2´-position is prepared and found to behave as a
compressed dinucleotide thereby condensing the information in DNA
while also stabilizing the resulting duplex.

Bioorganic & Medicinal Chemistry Letters
A double-headed nucleotide with two cytosines: DNA with condensed
information and improved duplex stability
Kasper Beck, Charlotte Reslow-Jacobsen, Mick Hornum, Christian Henriksen and Poul Nielsen
aDepartment of Physics, Chemistry and Pharmacy, University of Southern Denmark, 5230 Odense M, Denmark
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Double-headed nucleotide monomers are capable of condensing the genetic information of
DNA. Herein, a double-headed nucleotide with two cytosine bases (CC) is constructed. The
additional cytosine is connected through a methylene linker to the 2´-position of arabinocytidine.
The nucleotide is incorporated into oligonucleotides and its effect on duplex stability is studied.
For single incorporations, a thermal stabilization of 4.0 °C is found as compared to the
unmodified duplex and it is shown that both nucleobases of CC participate in Watson-Crick base
pairing. In combination with the previously published UT monomer, it is also shown that
multiple incorporations are tolerated. For instance, a 16-mer sequence is targeted by a 13-mer
oligonucleotide by using one CC and two UT monomers without compromising the overall
duplex stability. Finally, the potential of double-headed nucleotides in triplex forming
oligonucleotides is studied, however, with the conclusion that the present design is not well-
suited for this function.
Keywords:
Nucleic acids;
Double-headed nucleotides;
Oligonucleotides;
Duplex;
Triplex
2009 Elsevier Ltd. All rights reserved.
———
 Prof. Poul Nielsen. Tel.: +45-6550-2565; e-mail: pouln@sdu.dk

The double-helical structure of DNA self-assembles through
the formation of predictable and highly specific Watson-Crick
base pairs. The DNA duplex, as well as the more complex DNA
triplex, provides a brilliant platform for the design of therapeutics
based on supramolecular chemistry, e.g. anti-sense
oligonucleotides, nucleic acid aptamers or triplex forming
oligonucleotides.1-3 With the convenient automated synthesis of
DNA, it is easy to introduce and study the effect of chemically
procedure from Lemaire et. al.21 Stereoselective conversion of 2
to the 2´(S)-spiroepoxide 3 was achieved using trimethyl-
sulfoxonium iodide.13,22 The epoxide was opened by uracil in a
N1-regioselective manner to afford the protected double-headed
nucleoside 4. This regioselectivity was confirmed by the presence
of a 3JCH coupling between the protons of the 2´-methylene linker
1
and both C2 and C6 of the newly introduced uracil in the H,13C-
HMBC NMR spectrum of 4. Hereafter, the two uracils were
converted to protected cytosines in a three-step one-pot synthesis
of 5 with a decent yield of 55%. First, O4 of uracil was activated
by tosylation, then replaced by a nucleophilic attack from
ammonia at the C4-position, and finally the newly introduced
amino group was protected as a benzoyl amide. This procedure
was originally reported by Du et. al.23 for a single uracil-to-
modified nucleotides on these systems, and
a focus on
modifications that improve or introduce new properties to the
oligonucleotides has emerged.4,5
In this context, an interesting modification is the addition of a
second nucleobase to the nucleotide skeleton – the so-called
double-headed nucleotide.6 We have previously reported the
synthesis and recognition properties of several double-headed
nucleotides with the additional nucleobase attached to the 5´-, 2´-
or 5-position through various linkers,7-17 while others have used
the 4´-position or the 2´-N of amino-LNA as attachment
points.18,19 For the dUB design (Fig. 1) with the additional
nucleobase attached via a methylene linker to the 2´-position of a
2´-deoxynucleotide, it has been shown that both the natural and
the additional nucleobase participate in Watson-Crick base
pairing.10,11 More recently, we have examined a similar design
with an arabino-configuration (UB, Fig. 1), with a significantly
easier synthesis and little or no reduction in the recognition
potential as compared to dUB.13 For this design, the full set of
uridine-based double-headed nucleotides have been reported
comprising UT, UC, UG and UA.14 In practice, these double-
headed nucleotides behave like compressed dinucleotides, and
consequently, the information of two nucleotides can be
Figure 1. Double-headed nucleotides. T = thymin-1-yl, C = cytosin-1-yl,
G = guanin-9-yl, A = adenin-9-yl.
cytosine conversion. Removal of the silyl protecting group was
effectively achieved using triethylamine trihydrofluoride to give
the double-headed nucleoside 6, which was readily reprotected at
the 5´-O-position by treatment with 4,4´-dimethoxytrityl
chloride, affording 7. Finally, phosphitylation at the 3´-O-
position was accomplished to give the fully protected and
activated phosphoramidite 8.
condensed to
a single double-headed nucleotide, thereby
reducing the molecular size and the number of phosphates in a
given oligonucleotide. We expect this to form the basis for
improved nucleic acid based therapeutics.
In this study we introduce the first cytidine-based double-
headed nucleotide CC (Fig. 1) with two cytosines. With this
expansion, we are for the first time able to examine the
recognition properties of double-headed nucleotides binding only
through G:C base pairs. Together with UT, this new CC monomer
provides an excellent basis for evaluating the potential of double-
headed nucleotides in triplex-forming oligonucleotides (TFOs).
These two monomers were therefore incorporated into a standard
homo-pyrimidine TFO sequence20 at various positions resulting
in a selection of modified oligonucleotides with both single and
multiple incorporations of double-headed nucleotides. The
recognition potential of these strands in both duplexes and
triplexes is presented.
Phosphoramidite 8 was then used in standard solid-phase
DNA-synthesis in order to introduce the modified nucleotide CC
into oligonucleotides. In parallel, the corresponding phosphor-
amidite of UT was synthesized as previously reported.13 Standard
conditions were used in the DNA-synthesis, but with
4,5-dicyanoimidazole as activator and extended coupling times
(15 min) for both modified phosphoramidites. Treatment with
concentrated ammonia at room temperature (24 h) was used for
deprotection and cleavage from the solid-support. All
oligonucleotides were purified using reversed phase HPLC, and
MALDI-TOF MS and ion-exchange chromatography analysis
were used to confirm the identity and determine purity,
respectively.
The synthesis of the double-headed nucleoside is shown in
Scheme 1. Starting from uridine 1, the protected ketone 2 was
easily prepared using the optimized and chromatography-free
5’
3’ 5’
3’ 5’
3’
G C
C G
T A
C G
A T
C G
T A
C G
C G
T A
C G
C G
C G
A T
G C
C G
T A
C G
A T
C G
T A
C X
C G
T A
C G
C G
C G
A T
G C
C G
T A
C G
A T
C G
T A
C G
C Y
T A
C G
C G
C G
A T
A
B
C
55.5
X
Y
G
C
A
T
59.5
42.0
46.5
45.5
G
C
A
T
59.5
41.0
42.5
43.5
A
B
C
Figure 2. Structures of studied DNA duplexes and their melting
temperaturesa (Tm,°C). (A) 14-mer reference duplex. (B + C) Duplexes used
to study match and mismatch properties of the two cytosines in CC (marked
in red). a Melting temperatures were determined in a buffer containing
2.5 mM Na2HPO4, 5.0 mM NaH2PO4, 100 mM NaCl and 0.1 mM EDTA at
pH 7.0 using 1.5 μM concentrations of both strands.

which has been studied before,15,17,20 having single, double or
triple incorporations of either CC, UT or both in exchange for the
5’
3’
5’
3’
5’
5’
3’ 5’
3’ 5’
3’ 5’
3’
3’
T A
T A
T A
T A
C G
T A
T A
U A
T A
C G
C G
C G
C G
C G
C G
T A
T A
T A
T A
T A
C G
T A
T A
T A
T A
C G
C G
C G
C G
C G
C G
T A
T A
T A
T A
T A
C G
T A
T A
U A
T A
C G
C G
C G
C G
C G
C G
T A
T A
T A
T A
T A
C G
U A
T A
T A
T A
C G
C G
C G
C G
C G
C G
T A
T A
T A
T A
T A
C G
T A
T A
T A
T A
C G
C G
C G
C G
C G
C G
T A
T A
T A
T A
T A
C G
U A
T A
T A
T A
C G
C G
C G
C G
C G
C G
T A
T A
T A
U A
T A
C G
T A
U A
T A
T A
C G
C G
C G
C G
C G
C G
T A
D6
D1
D2
D3
D4
D5
D7
47.0
48.5
45.5
44.5
53.0
50.0
47.5
(–1.5)
(–3.0)
(–4.0)
(+4.5)
(+1.5)
(–1.0)
Figure 3. Structures of studied DNA duplexes, their melting temperaturesa
(Tm, °C) and the differences in melting temperatures (∆Tm, °C) relative to
duplex D1. a See caption of Fig. 2.
corresponding dinucleotides (Fig. 3). First, each strand was
hybridised with the 16-mer complimentary homo-purine strand,
giving the unmodified duplex (Fig. 3, entry D1) and the six
modified duplexes (entries D2–D7). Looking first at the single-
modified duplexes, there is a clear correlation between the found
stabilizing effect of CC of 4.5 °C (entry D4) and the previously
found 4.0 °C in the aforementioned mixed sequence (Fig. 2).
Likewise, the thermal destabilization of –3.0 °C and –4.0 °C
(entries D2 and D3) found for a single incorporation of UT is in
accordance with the published –3.5 °C for its incorporation into a
13-mer mixed sequence.13 From these results it is apparent that
the thermal stability change provided by the double-headed
Scheme 1. Reagents and conditions: (a) i. TIPDSCl2, imidazole, CH2Cl2,
ii. TEMPO, PhI(OAc)2, AcOH, CH2Cl2, 71% (ref. 21); (b) NaH, (CH3)3SOI,
THF, DMSO, 84% (ref. 22); (c) NaH, uracil, DMF, 71%; (d) i. TsCl, DMAP,
Et3N, CH3CN, ii. NH4OH, iii. BzCl, pyridine, 55%; (e) Et3N·3HF, THF, 88%;
(f) DMTrCl, pyridine, 86%; (g) NC(CH2)2OP(N(i-Pr)2)Cl, (i-Pr)2NEt, DCE,
66%. DMTr = 4,4´-dimethoxytrityl.
At first, we studied the effect of a single incorporation of the
CC monomer by exchanging the central 5´-CC dinucleotide of
our standard mixed 14-mer sequence with the CC monomer,
giving a modified 13-mer (Fig. 2). This strand was hybridized
with the complementary 14-mer sequence and the melting
temperature (Tm) was obtained from the maximum of the first
derivative of the UV (A260 nm) melting curve. It was found that the
introduction of CC caused a substantial increase in thermal
stability of 4.0 °C as compared to the unmodified duplex (Fig. 2,
59.5 °C compared to 55.5 °C, respectively). Hence, the ability of
CC to function as a dinucleotide is well-founded, and the CC
monomer is even better tolerated in the DNA duplex than its
uridine-based counterparts (UT, UC, UG and UA) showing ∆Tm‘s
ranging from +1.0 °C to –3.5 °C in similar 14-mer duplexes.14
A mismatch study was made, targeting not only the additional
2´-attached nucleobase of CC, but also the “natural” 1´-attached
nucleobase. When placing a mismatch nucleotide opposite to the
latter nucleobase, a considerable decrease of 13.0–17.5 °C was
observed in the melting temperature of the duplex (Fig. 2B). For
the additional cytosine, the mismatch discrimination is even
slightly better in each of the three mismatches with decreases in
melting temperatures of 16.0–18.5 °C (Fig. 2C). Altogether, this
shows that the mismatch discrimination of CC is excellent, and it
confirms that also the additional cytosine base actually takes part
in Watson-Crick base pairing with the opposite guanine.
nucleotides has only
a minor dependence on the local
environment in the form of nucleotide sequence. It should be
noticed, however, that the A:U base pair of UT is compared
directly with an unmodified A:T base pair, and the lack of a
methyl group might add to the decrease in stability.24
Hereafter, duplexes with multiple incorporations were studied
(Fig. 3, entries D5–D7). The melting temperatures of these
duplexes can approximately be explained by accumulating the
isolated effect of each incorporation. Specifically, the ∆Tm for the
doubly modified duplex D5 (+1.5 °C) only differs by 0.5 °C from
the sum of the corresponding ∆Tm’s for the single UT
incorporation D3 (–3.0 °C) and the single CC incorporation D4
(+4.0 °C), and so the thermal effects of the double-headed
nucleotides seem to stack. A limitation to this trend arises when
the double-headed nucleotides are incorporated consecutively. In
this case a thermal destabilization of –3.0 °C is observed,
compared to having a gap of three canonical nucleotides (D6
compared to D5). This might be explained by the geometry of the
double-headed nucleotides demanding some degree of
adjustment of neighbouring nucleotides. In duplex D7, a total of
three double-headed nucleotides were incorporated, and even
with gaps of only two canonical nucleotides, the change in
melting temperature (–1.0 °C) was still roughly the sum of two
single UT and a single CC incorporation. D7 is essentially
comprised of a 13-mer modified strand that recognizes a 16-mer
complementary strand.
To get further insight into the effect of double-headed
nucleotides on both duplex and triplex stability, a series of six
modified oligonucleotides were synthesized. All of these were
based on the same 16-mer homo-pyrimidine TFO sequence,

Finally, the modified oligonucleotides were evaluated for their
potential as triplex-forming oligonucleotides (TFOs).
modified strand (being in fact a 13-mer) recognizes a 16-mer
complementary strand with a drop of just 1.0 °C in thermal
stability as compared to the unmodified duplex. The potential of
CC in triplex-forming oligonucleotides has been evaluated as
well, but clearly, the present design is not well-suited for this
function. From these results, it can be concluded that CC is able
to condense the information of two deoxycytidines to a single
double-headed nucleotide, while it at the same time is improving
the thermal stability of the duplex. This is a truly unique set of
properties that may have future potential in the development of
nucleic acid based therapeutics.
Polypyrimidine oligonucleotides already studied in duplexes
(Fig. 3, entries D2–D5 and D7) were hybridized with a standard
29-mer target DNA duplex15,20 in a high salt buffer at pH 6, and
the melting temperatures of the resulting triplexes were
measured. Triplex formation, however, was only observed for
one of the modified sequences having a single CC incorporation
(Fig. 4). Even this triplex was considerably destabilized by the
modification, showing a decrease in melting temperature of 7.5
°C compared to the unmodified triplex (24.5 °C). Consequently,
it seems that the design of the presented double-headed
nucleotides is not optimal for accommodation in triplexes.
Probably neither the constrained double-headed nucleotide
structure nor the target duplex allows for the necessary
adjustment of the geometry.
Acknowledgments
This work was supported by grants from the Danish Council
for Independent Research | Natural Sciences.
In summary, we have shown some systematic effects on
duplex stability of the different double-headed nucleotides. The
incorporation of UT destabilizes the duplex by –3.5 °C,14 whereas
the CC monomer shows a stabilizing effect of 4.0 °C. In
comparison, the UA monomer gave a destabilization of –2.5 °C,
whereas UC and UG showed minor effects on duplex stability of
+1.0 °C and 0.0 °C, respectively, in similar sequences.14 Even
though G:C pairs are always stronger than A:T and A:U pairs, the
present results demonstrate that G:C base pairs provide a
relatively larger stabilizing effect in the condensed DNA
provided by the double-headed nucleotides. The reason might be
found in the special geometry, and a speculation could be that the
third hydrogen bond of the G:C pair thermodynamically drives
the neighbouring base-pairs into a more fixed geometry. Further
studies are needed to enlighten this observation.
Supplementary Material
Electronic supplementary material contains experimental
details including synthetic procedures, selected NMR spectra,
procedures for oligonucleotide synthesis and hybridization
studies, as well as MS data for modified oligonucleotides.
References and notes
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(2)
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8 steps from
commercially available uridine, and thereafter protected and
activated for oligonucleotide synthesis. A selection of modified
oligonucleotides has been prepared containing this CC monomer
and/or the corresponding uridine-based UT monomer. The
hybridization studies showed that CC is very well-accommodated
in double-stranded DNA, and moreover, the mismatch
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5’
T T T T C T T T T C C C C C C T
5’
C C A C T T T T T A A A A G A A A A G G G G G G A C T G G
G G T G A A A A A T T T T C T T T T C C C C C C T G A C C
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3’
17.0 (–7.5)
Figure 4. Structure of a selected triplex, its melting temperaturea (Tm, °C) and
the difference in melting temperature (∆Tm, °C) relative to the unmodified
triplex. a Melting temperatures were determined in a buffer containing 10 mM
sodium cacodylate, 150 mM NaCl and 10 mM MgCl2 at pH 6.0 using 1.5 μM
concentrations of the TFOs and 1.0 mM concentrations of each strands of the
target DNA-duplex. In all experiments the melting temperature of the duplex
was found to be 68 °C.
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