Dual recognition of a C-G pyrimidine-purine inversion site: Synthesis and binding properties of triplex forming oligonucleotides containing 2′-aminoethoxy-5-methyl-1H-pyrimidin-2-one ribonucleosides

Article abstract of DOI:10.1016/S0040-4039(03)01135-3

The synthesis of a new ribonucleoside analogue, which combines two modifications, namely a 2′-aminoethoxy side-chain on the ribose and a 5-methyl-1H-pyrimidin-2-one (^4HT) unit as a base replacement, is presented. This building block was incorporated into triplex forming oligonucleotides and the binding properties to CG inversion sites in DNA duplex targets were studied. The data clearly show that the ^4HT base selectively recognizes the CG base-pair, while the aminoethoxy chain adds to the overall stability of the triple helix.

Full text of DOI:10.1016/S0040-4039(03)01135-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 5065–5068
Dual recognition of a C–G pyrimidine–purine inversion site:
synthesis and binding properties of triplex forming
oligonucleotides containing
2%-aminoethoxy-5-methyl-1H-pyrimidin-2-one ribonucleosides
Sabrina Buchini and Christian J. Leumann*
Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland
Received 9 April 2003; revised 22 April 2003; accepted 5 May 2003
Abstract—The synthesis of a new ribonucleoside analogue, which combines two modifications, namely a 2%-aminoethoxy
side-chain on the ribose and a 5-methyl-1H-pyrimidin-2-one (4HT) unit as a base replacement, is presented. This building block
was incorporated into triplex forming oligonucleotides and the binding properties to CG inversion sites in DNA duplex targets
were studied. The data clearly show that the 4HT base selectively recognizes the CG base-pair, while the aminoethoxy chain adds
to the overall stability of the triple helix. © 2003 Elsevier Science Ltd. All rights reserved.
Specific recognition of double stranded DNA by triplex
forming oligonucleotides (TFO) has been a field of
intense investigations over recent years.1,2 With such
TFOs gene expression can be controlled on the level of
transcription, which is of considerable interest for bio-
technology and medicine.3,4 Two triplex binding motifs
are known in which the TFOs bind within the major
groove of DNA either in a parallel or an antiparallel
fashion to the purine strand of the target duplex.
Sequence recognition is restricted to homopurine–
homopyrimidine sequence tracts, and a general recogni-
tion code for any given DNA sequence is still elusive.
Oligonucleotide modifications investigated to overcome
this limitation included backbone modifications and the
use of new bases designed to bind a whole Watson–
Crick base pair, or to selectively recognize a pyrimidine
base.5 Since pyrimidine bases only present one free
hydrogen bonding site within the major groove, their
recognition is generally hampered by low affinity and
low selectivity with respect to the bidentate recognition
of purine bases.
tional H-bond with the assistance of a non-conven-
tional CꢀHꢀO hydrogen bond.6,7 The general
importance of such unconventional hydrogen bonds in
(bio)molecular recognition has been demonstrated
before.8,9
We found that CG recognition was highly selective,
albeit at a lower level of affinity compared to a conven-
tional pu–pu–py triplet. In order to increase affinity we
decided to combine the 4HT base with the 2%-
aminoethoxy ribose modification which has been shown
before to increase DNA target affinity of TFOs by
additional salt bridge formation between the proto-
We recently reported on the selective recognition of a
CG base pair within the parallel DNA triple-helical
binding motif by the base 5-methyl-1H-pyrimidin-2-one
(
4HT, Fig. 1) in the context of deoxyribo-TFOs.6 This
base was designed to recognize cytosine via one conven-
* Corresponding author. Tel.: +41-31-631-4355; fax: +41-31-631-3422;
e-mail: leumann@ioc.unibe.ch
Figure 1. 4HT-CG triplet in the parallel triplex binding motif.
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)01135-3

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S. Buchini, C. J. Leumann / Tetrahedron Letters 44 (2003) 5065–5068
Scheme 1. Reagents and conditions: (a) HCl 2% (w/w), MeOH, 3.5 h, rt, 63%; (b) TIPS-Cl (1.2 equiv.), pyridine, 1 h, rt, 83%; (c)
BrCH2CO2Me (5 equiv.), NaH (2.2 equiv.), DMF, 10 h, −10°C–rt, 77%; (d) LiBH4 (4 equiv.), THF/MeOH (4/1), 1 h, 0°C, 94%;
(e) Ph3P (1.4 equiv.), LiN3 (4 equiv.), CBr4 (1.4 equiv.), DMF, 9 h, 0°C–rt; (f) TsCl (2 equiv.), NEt3 (2.1 equiv.), DMAP (0.1
equiv.), CH2Cl2, overnight, rt, 93%; (g) LiN3 (2 equiv.), DMF, 1.5 h, 90°C, 96%; (h) Lindlar catalyst (50%), H2, THF, overnight,
rt, quantitative; (i) Et3N (1 equiv.), CF3CO2Et, 4 h, rt, 89%; (j) Ac2O/AcOH/H2SO4, 14 h, rt, 91%; (k) (11, 1.05 equiv.), BSA (2.6
equiv.), SnCl4 (1 equiv.), CH3CN, 2 h, 0°C, 55%; (l) Na2CO3 anhyd. (10% w/w), MeOH, 30 min, rt, 90%; (m) DMT-Cl (2 equiv.),
pyridine, 5 h, rt, 75%; (n) iPr2NEt (3 equiv.), [(iPr2N)(NCCH2CH2O)P]Cl (1.5 equiv.), THF, 2 h, rt, 75%.
phoramidite 1 was then successfully obtained by stan-
dard procedures. Thus, intermediate 814 may be a useful
candidate for the introduction of other bases of
interest.
nated amino function of the side-chain and a phosphate
group of the target duplex.10
Here we report on the synthesis of compound 1
(Scheme 1) and on the pairing properties of
oligodeoxynucleotides containing this nucleoside
analogue.
Oligonucleotides were prepared on a DNA synthesizer
using standard phosphoramidite chemistry (Table 1).15
Sequence 12a corresponds to the control containing
thymine, sequence 12b has previously been studied in
our laboratories and differs from sequence 12c only by
the missing 2%-aminoethoxy chain. Modified oligonucleo-
tide 12c containing building block 1 was initially
deprotected using standard conditions: NH3 33%, 55°C,
16 h. However, under those conditions strand cleavage
occurred at the site of base modification (HPLC and
ESI-MS control), indicating instability of the base 4HT
Methyl b- -ribofuranoside 3 was synthesized starting
D
from ribose, and was subsequently transformed into the
TIPS protected methyl riboside 4. Alkylation of 4 at the
2%-hydroxyl function with methyl bromoacetate, fol-
lowed by reduction of the methyl ester, tosylation and
substitution by azide yielded the azidoethyl derivative
6. Direct azidation of 5 via Mitsunobu reaction proved
to be unsuccessful. After reduction of the azide and
protection of the resulting amine (“7) the coupling
with the base 11 that was synthesised in two steps
starting from propionaldehyde diethylacetal 10 (Scheme
2),12,13 was attempted.
However, formation of the corresponding nucleoside
could not be observed. The sugar was therefore con-
verted into the triacetate 8 in order to facilitate the
nucleosidation step.11 Using 8 the nucleosidation could
be achieved under Vorbru¨ggen conditions in 55% yield.
Only the b-anomeric nucleoside 9 was isolated. Phos-
Scheme 2. Reagents and conditions: (a) (i) POCl3 (2.2 equiv.),
DMF (2.4 equiv.), 2 h, 70°C, (ii) H2O, overnight, K2CO3,
36%; (b) (i) Na (2.3 equiv.), urea (1.2 equiv.), EtOH, 2 days,
reflux, (ii) HCl 25% (pH 2), 1 day, reflux, 50%.

S. Buchini, C. J. Leumann / Tetrahedron Letters 44 (2003) 5065–5068
5067
Table 1. Modified and natural TFOs as well as target
duplexes investigated
Table 3. Tm (°C) data of third strand dissociation from
UV-melting curves (260 nm)a
Tm triplex (°C)
17/20
18/21
19/22
19.6
6.9
–
b
a c(triplex)=1.6 mM in 10 mM Na-cacodylate, 100 mM NaCl, 0.25
mM spermine, pH 6.5.
b No Tm detectable.
aminoethoxy side-chain in 12c leads to an increase in
thermal stability of about 1.5°C/mod., relative to 12b,
without changing the selectivity. This indicates that at
pH 6.5 the protonated aminoethoxy side-chain can
interact specifically with a nearby phosphate group of
the double helix much in the same way as reported
before.16
Triplex formation was also investigated at different pH
values (Table 2). The data shows that there is no
change in target selectivity for TFOs 12b and 12c
within the pH range investigated (pH 6.5–7.4). At pH
7.0 the stability of the triplex 12c/15 is still higher than
the stability of 12b/15, while at pH 7.4 the two triplexes
12b/15 and 12c/15 are almost equally stable. This could
either be due to incomplete protonation of the
aminoethoxy side-chain, or reflect changes in the sugar
conformation (preferred 2%-endo conformation for the
deoxyribo- and 3%-endo conformation for the ribo-
nucleoside) of the two 4HT nucleotide units, that may
be masked at low pH.
In order to explore the limits of this approach we
investigated triplex formation of TFOs containing 1
with dsDNA targets containing multiple CG interrup-
tions (Table 1, 17–22). The TFOs 17 and 18, containing
two and three units of 1, were found to still form
triplexes with their respective targets 20 and 21 respec-
tively at pH 6.5 (Table 3), however with significantly
reduced Tm compared to TFOs targeted to single CG
inversions.
towards extended exposure to conc. NH3 at 55°C.
Successful deprotection conditions were: NH3 33%, rt, 2
h or MeNH2 (40% aqueous solution), rt, 3 h.
In a first series of experiments, the binding efficiency of
the TFOs 12a–c to the target duplexes 13–16 was
assessed by UV-melting curve analysis. Inspection of
the Tm-data (Table 2) clearly shows that there is a
preference for 4HT-CG base-triple formation with both
12b and 12c. The loss of the carbonyl oxygen O(4) of
thymine in 4HT is responsible for the altered base-pair
selectivity. Moreover the introduction of the 2%-
The formation of a triplex with five 1-CG triplets, as in
19–22 could not be observed, even by decreasing the
pH to 5.5.
In order to further extend TFO binding also to dsDNA
targets as e.g. 22, we will prepare and investigate TFOs
Table 2. Tm (°C) data of third strand dissociation from UV-melting curves (260 nm)a
pH
12a
12b
12c
13 (A/T) 14 (T/A) 15 (C/G9 16 (G/C) 13 (A/T) 14 (T/A) 15 (C/G) 16 (G/C) 13 (A/T) 14 (T/A) 15 (C/G) 16 (G/C)
6.5
7.0
7.4
61.0
45.0
38.4
29.3
21.1
13.8
38.0
29.7
22.6
32.3
23.3
17.1
27.4
19.0
13.8
29.7
17.0
13.3
38.9
30.1
23.9
29.5
18.2
12.5
29.8
21.1
14.8
27.6
20.0
13.2
40.5
31.4
24.2
31.1
20.4
12.6
a c(triplex)=1.6 mM in 10 mM Na-cacodylate, 100 mM NaCl, 0.25 mM spermine, pH 6.5. UV-melting curves were recorded with a temperature
gradient of 0.5°C/min. Data were obtained from the first derivative of the melting curve.

5068
S. Buchini, C. J. Leumann / Tetrahedron Letters 44 (2003) 5065–5068
containing
modified.
1
that are uniformly 2%-aminoethoxy
8. Wahl, M. C.; Sundaralingam, M. TIBS 1997, 22, 97–102.
9. Desiraju, G. R. Acc. Chem. Res. 1996, 29, 441–449.
10. Cuenoud, B.; Casset, F.; Hu¨sken, D.; Natt, F.; Wolf, R.
M.; Altmann, K. H.; Martin, P.; Moser, H. E. Angew.
Chem., Int. Ed. 1998, 37, 1288–1291.
Acknowledgements
11. Sollogoub, M.; Dominguez, B.; Fox, K. R.; Brown, T.
Chem. Commun. 2000, 23, 2315–2316.
12. Nair, V.; Vietti, D. E.; Cooper, C. S. J. Am. Chem. Soc.
1981, 103, 3030–3036.
The authors wish to thank the Swiss National Science
Foundation (Grant No. 20-63582.00) for financial sup-
port of this work.
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