Pyrrolidino-DNA

Article abstract of DOI:10.1081/NCN-120022832

We synthesized pyrrolidino-C-nucleosides, incorporated them into oligodeoxynucleotides and investigated their pairing properties. The thermal duplex and triplex stabilities were measured. While triplex formation is destabilized in the case of pyrrolidino-

Full text of DOI:10.1081/NCN-120022832

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Pyrrolidino-DNA
Adrian Häberli a , Alain Mayer a b & Christian J. Leumann a
a Department of Chemistry and Biochemistry , University of Bern , Bern, Switzerland
b Department of Chemistry and Biochemistry , University of Bern , Freiestrasse 3, CH-3012,
Bern, Switzerland
Published online: 31 Aug 2006.
To cite this article: Adrian Häberli , Alain Mayer & Christian J. Leumann (2003) Pyrrolidino-DNA, Nucleosides, Nucleotides and
Nucleic Acids, 22:5-8, 1187-1189, DOI: 10.1081/NCN-120022832
To link to this article: http://dx.doi.org/10.1081/NCN-120022832
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 1187–1189, 2003
Pyrrolidino-DNA
*
Adrian Haberli, Alain Mayer, and Christian J. Leumann
¨
Department of Chemistry and Biochemistry, University of Bern,
Bern, Switzerland
ABSTRACT
We synthesized pyrrolidino-C-nucleosides, incorporated them into oligodeoxy-
nucleotides and investigated their pairing properties. The thermal duplex and
triplex stabilities were measured. While triplex formation is destabilized in the
case of pyrrolidino-pseudo-U and -T, pyrrolidino-pseudo-iso-C leads to an
increase of the Tm value for third strand dissociation. Duplexes are destabilized
with all pyrrolidino-C-nucleosides.
Triplex formation between a triplex forming oligonucleotide (TFO) and a target
DNA duplex can modulate gene expression on the level of transcription and is thus
of interest as a tool in molecular biology and in human therapy (antigene therapy).
Although TFOs bind with high specificity, their affinity is usually much weaker than
that of the underlying DNA duplex. In part this is thought to be due to the charge
repulsion resulting from bringing together three polyanionic DNA strands. Several
studies have shown, that the introduction of positive charges in the sugar-phosphate
backbone or at the bases can enhance triplex stability.[1] We reasoned that exchange
of the 40-oxygen by nitrogen in a nucleoside unit of a TFO would position a positive
*Correspondence: Alain Mayer, Department of Chemistry and Biochemistry, University of
Bern, Freiestrasse 3, CH-3012 Bern, Switzerland; E-mail: mayer@ioc.unibe.ch.
1187
DOI: 10.1081/NCN-120022832
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

¨
Haberli, Mayer, and Leumann
1188
Scheme 1. (a) Pd(OAc)2, AsPh3, Bu3N, DMF, 65ꢁC, 20 h, (58%); (b) Pd(OAc)2, AsPh3,
N,N-diisopropylethylamine, DMF, 80ꢁC, 22 h, (63%); (c) AcOH, TBAF, THF, ꢀ15ꢁC ! rt,
37 h; (d) NaB(OAc)3H, AcOH, MeCN, ꢀ15ꢁC ! rt, 20 min, (87%); (e) HFꢂpyridine, MeCN,
rt, 6 h; (f) NaB(OAc)3H, AcOH, MeCN, ꢀ15ꢁC ! rt, 5 h, (79%); (g) BSA, CH3I, CH2Cl2,
rt, 70 h, (77%); (h) H2, Pd=C, MeOH, rt, 4 to 7 h, (quant.); (i) Fmoc-OSu, THF, dioxane,
5% NaHCO3 soln., rt, 3 h, (65–91%); (j) DMT-Cl, pyridine, rt, 2 h, (65%); (k) (iPr2N)
(NCCH2CH2O) PCl, iPr2NEt, THF, rt, 2 h, (71–88%).
charge next to a phosphate unit of the duplex target, thus enhancing triplex stability.
We therefore embarked on the synthesis of the pyrrolidino nucleosides depicted in
Sch. 1.
Starting from trans-3-hydroxy-L-proline (1), CBz-protected enamine 2 was
obtained in 7 steps in 52% overall yield.[2] The next steps were a palladium-mediated
Heck coupling leading to 5 and 6. The silyl ethers were then cleaved, and the
obtained keto groups were stereoselectively reduced in one pot, to give nucleosides
7 and 9. At this stage, CBz-protected pyrrolidino pseudo-U 7 can be converted into
its N-10-methyl derivative 8. The pyrrolidine ring nitrogens in 7–9 were then repro-
tected with the Fmoc group which is compatible with standard oligonucleotide
synthesis. Tritylation of the primary alcohol followed by reaction with the corre-
sponding chloro phosphite reagent lead to building blocks 13a–c. The same pathway
was used as a new synthesis for pseudo-iso-C whereas pseudo-U is commercially
available.
The different building blocks were incorporated into 15-mer oligonucleotides by
standard oligonucleotide synthesis. The melting temperatures (Tm) of the modified
oligonucleotides were measured and compared to the Tm of the natural sequence.
The Tm values for triplex dissociation from UV-melting curve experiments of the
strands containing pyrrolidino-pseudo-U and its N-10-methyl derivative units are sig-
nificantly lower compared to the natural oligomer.[3] We find relative destabilizations
of triplex formation by ca. ꢀ13 to ꢀ1ꢁC per mod. in a strongly sequence dependent
mode. Both sugar and base modifications contribute to the destabilization. In the
case of pyrrolidino-pseudo-iso-C (Y), the protonated pyrrolidine unit leads to a

Pyrrolidino-DNA
1189
Table 1. Tm values (ꢁC) determined from UV melting experiments (l ¼ 260 nm). Duplex or
triplex concentration ¼ 1.2 mM; buffer: 140 mM KCl, 7 mM NaH2PO4, 0.5 mM MgCl2; pH for
duplex: 7.0.
Tm (triplex)a DTm=mod Tm (duplex)b DTm=mod
Sequence
[ꢁC]
[ꢁC]
[ꢁC]
[ꢁC]
50-TTTTTCTCTCTCTCT-30
43.1
45.5
23.0
29.7
30.1
53.0
37.1
28.0
16.4
13.5
10.0
46.9
27.9
38.3
28.3
0
45.2
39.1
0
50-TTTTTCTYTCTCTCT-30, pH 6.0
50-TTTTTCTYTCTCTCT-30, pH 7.0
50-TTTTTCTQTCTCTCT-30, pH 6.0
50-TTTTTCTQTCTCTCT-30, pH 7.0
50-TTTTTYTYTYTYTYT-30, pH 6.0
50-TTTTTYTYTYTYTYT-30, pH 8.0
50-TTTTTYTYTYTYTYT-30, pH 9.0
50-TTTTTQTQTQTQTQT-30, pH 6.0
50-TTTTTQTQTQTQTQT-30, pH 8.0
50-TTTTTQTQTQTQTQT-30, pH 9.0
50-TTTTTYTCTYTCTCT-30, pH 6.0
50-TTTTTYTCTYTCTCT-30, pH 7.0
50-TTTTTQTCTQTCTCT-30, pH 6.0
50-TTTTTQTCTQTCTCT-30, pH 7.0
þ2.4
ꢀ20.1
ꢀ13.4
ꢀ13.0
þ2.0
ꢀ1.2
ꢀ3.0
ꢀ5.3
ꢀ5.9
ꢀ6.6
þ1.9
ꢀ7.6
ꢀ2.4
ꢀ7.4
ꢀ6.1
43
ꢀ2.2
ꢀ5.5
17.8
24.6
ꢀ4.1
33.1
37.6
ꢀ6.1
ꢀ3.8
aTarget duplex: 50-CGTAAAAAGAGAGAGAGATCG-30=50-CGATCTCTCTCTCTTT-
TTAGC-30.
bAntiparallel complementary DNA strand: 50-AGAGAGAGAGAAAAA-30.
stabilization of the triplex by ca. 2ꢁC per mod. at pH 6 (Table 1). Since the oligonu-
cleotides containing pseudo-iso-C (Q) give less stable triplexes, we can conclude that
the sugar modification is responsible for the stabilization in the latter case. The dif-
ferential behavior between the pyrrolidino-C-nucleosides containing different bases
is not clear at this point. For all the oligonucleotides containing pyrrolidino-C-
nucleosides, the Tm of the hybrid duplex is lower than that of the unmodified duplex.
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Husken, D.; Natt, F.; Wolf, R.M.; Altmann, K.-H.; Martin, P.; Moser, H.E.
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Angew. Chem. Int. Ed. 1998, 37, 1288–1291; c) Blommers, M.J.; Natt, F.;
Jahnke, W.; Cuenoud, B. Biochemistry 1998, 37, 17,714–17,725.
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3. Ha¨berli, A.; Leumann, C.J. Org. Lett. 2002, 4 (19), 3275–3278.