Oligonucleotide charge reversal: 2′-O-lysylaminohexyl modified oligonucleotides

Article abstract of DOI:10.1080/15257770701507978

A novel cationic building nucleoside building block designed for antisense and siRNA oligonucleotides is presented. Protected L-lysine was coupled to 2′-O-aminohexyluridine and the resulting nucleoside was phosphitylated for automated oligonucleotide synt

Full text of DOI:10.1080/15257770701507978

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Oligonucleotide Charge Reversal:
2′-O-Lysylaminohexyl Modified
Oligonucleotides
Johannes Winkler a & Christian R. Noe a
a Department of Medicinal Chemistry , University of Vienna , Vienna,
Austria
Published online: 10 Dec 2007.
To cite this article: Johannes Winkler & Christian R. Noe (2007) Oligonucleotide Charge Reversal: 2′-
O-Lysylaminohexyl Modified Oligonucleotides, Nucleosides, Nucleotides and Nucleic Acids, 26:8-9,
939-942, DOI: 10.1080/15257770701507978
To link to this article: http://dx.doi.org/10.1080/15257770701507978
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Nucleosides, Nucleotides, and Nucleic Acids, 26:939–942, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701507978
OLIGONUCLEOTIDE CHARGE REVERSAL: 2ꢀ-O-LYSYLAMINOHEXYL
MODIFIED OLIGONUCLEOTIDES
Johannes Winkler and Christian R. Noe
Department of Medicinal Chemistry,
2
University of Vienna, Vienna, Austria
A novel cationic building nucleoside building block designed for antisense and siRNA oligonu-
2
cleotides is presented. Protected L-lysine was coupled to 2ꢁ-O-aminohexyluridine and the resulting
nucleoside was phosphitylated for automated oligonucleotide synthesis. An increasing number of
these 2ꢁ-O-lysylaminohexyl nucleosides lowered the melting temperature of desoxy-thymidine homod-
odecamers, but the decrease was lower than that for DNA/RNA hybrids. Incubation with an ex-
onuclease showed the exceptionally high resistance against enzymatic degradation. CD spectrometry
revealed a gradual transition towards an A-type oligonucleotide structure. Based on these data, the
cationic building block is particularly suited for gapmer antisense as well as siRNA oligonucleotides.
Keywords Antisense oligonucleotides; siRNA; cellular uptake; CD spectrometry
Gene silencing mediated by antisense oligonucleotides (ASO) or short
interfering RNA (siRNA) has become an important technique in scientific
research in the last decade. Attempts to exploit it for therapeutic purposes
have so far been hampered by poor pharmacokinetic and pharmacody-
namic properties as well as off-target effects caused by the chemical struc-
tures of oligonucleotides used in clinical trials.
In particular, the polyanionic nature of oligonucleotides all but prevents
their uptake into living cells. Charge reduction or neutralization by zwitte-
rionic nucleotide building blocks can give positive effects not only on cellu-
lar uptake, but also on target affinity.[1,2] Conjugating cationic peptides to
oligonucleotides has been shown to improve cellular uptake.[3,4] We present
a cationic building block that results in a charge reversal, as its incorpora-
tion in an oligonucleotide adds a positive charge instead of the negative one
of a wild-type nucleoside.
The synthesis of the cationic uridine building block 5 proceeds in two
steps from protected 2ꢁ-O-aminohexyl uridine[1] (Figure 1). A trifluoro
Address correspondence to Johannes Winkler, Department of Medicinal Chemistry, Univer-
sity of Vienna, Centre of Pharmacy, Room 2E 362, Althanstrasse 14, A-1090 Wien, Austria. E-mail:
johannes.winkler@univie.ac.at
939

940
J. Winkler and C. R. Noe
FIGURE 1 Synthesis scheme of 2ꢁ-O-lysylaminohexyl building block 5.
acetate protected L-lysine building block, prepared from L-lysine using tri-
fluoro acetate, is coupled to the free amino group in excellent yield using
DIC as catalyst. After phosphitylation, the building block can be used in
standard oligonucleotide synthesis when extended coupling times are em-
ployed.
For an evaluation of the biophysical parameters of charge reversed
oligonucleotides, desoxy-thymidine homododecamers incorporating an in-
creasing number of building block 5 were used. The decrease in target affin-
ity as determined by transition temperatures (Tm) and free energy values
(ꢀG) was lower than expected for dT-rU hybrids, indicating an increase in
target affinity caused by the cationic lysine chains (Table 1). The calculated
values were determined by a nearest neighbor model and take into account
the poorer hybridization affinity of rU compared to dT. Data show that a few
terminal cationic nucleotides are tolerated very well, whereas a pronounced
influence on duplex stability was observed with several modifications spread
over the entire oligonucleotide strand.

Oligonucleotide Charge Reversal
941
TABLE 1 Sequences and properties of oligonucleotides 7–13. U∗: 2ꢁ-O-Lysylaminohexyl-uridine
Sequence
Tm meas. Tm calc. ꢀꢀG meas. ꢀꢀG calc. MW calc. MW found
5ꢁ-TTT TTT TTT TTT-3ꢁ
34,7
33,6
32,4
30,4
28,3
26,2
17,6
9,5
31,0
25,1
22,7
20,6
18,6
16,8
19,6
17,7
0,0
0,0
0,0
1,4
3,8
3,9
0,0
1,7
5,0
8,4
11,8
15,1
3577,4
3817,7
4047,0
4276,3
4505,6
4735,0
4276,3
4505,6
3576,3
7 5ꢁ-U*TT TTT TTT TTT-3ꢁ
3816,0
4048,0
4276,0
4505,6
4734,7
4282,0
4507,0
8 5ꢁ-U*U*T TTT TTT TTT-3ꢁ
9 5ꢁ-U*U*U* TTT TTT TTT-3ꢁ
10 5ꢁ-U*U*U* U*TT TTT TTT-3ꢁ
11 5ꢁ-U*U*U* U*U*T TTT TTT-3ꢁ
12 5ꢁ-TTU* TTT U*TT TU*T-3ꢁ
13 5ꢁ-TU*T TU*T TU*T TU*T-3ꢁ
MALDI analyses confirmed the successful incorporation of lysine amino-
hexyl uridine into oligonucleotides. Neither transacetylation of lysine amino
groups during synthesis nor cleavage of the amide bond during standard
ammonia deprotection occurred.
Incubation of 7 with 5ꢁ-exonuclease revealed the excellent stability
of charge reversed oligonucleotides (Figure 2). Only one modified nu-
cleotide leads to practically complete resistance against enzymatic degrada-
tion. Lysylaminohexyl oligonucleotides are clearly superior to aminohexyl
FIGURE 2 HPLC of 7 (top) and T12 (bottom) after 30 minutes of incubation with phosphodiesterase.

942
J. Winkler and C. R. Noe
FIGURE 3 CD spectra of duplexes of 7–13 with dA12
.
modified ones, where three terminal modifications are required for a simi-
lar effect.[1]
CD spectra of duplexes with wild-type DNA sense showed a conversion
towards an A-type oligonucleotide secondary structure with increasing num-
ber of modified building blocks (Figure 3). The characteristic double peak
of dA12-dT12 gradually disappears. In addition, the oligonucleotides with the
highest number of cationic nucleotides exhibit a strong negative band in the
far UV region, a characteristic of an A-type structure, often associated with
RNA.
In conclusion, 2ꢁ-O-lysylaminohexyl modified oligonucleotides exhibit
auspicious biophysical characteristics. In particular, the gapmer approach
promises a positive effect on target affinity and sufficient stability against
degrading enzymes at the same time.
REFERENCES
1. Noe, C.R.; Winkler, J.; Urban, E.; Gilbert, M.; Haberhauer, G.; Brunar, H. Zwitterionic oligonu-
cleotides: A study on binding properties of 2ꢁ-O-aminohexyl modifications. Nucleot. Nucleot. Nucl.
2005, 24, 1167–1185.
2. Winkler, J.; Urban, E.; Noe, C.R. Oligonucleotides conjugated to short lysine chains. Bioconjug. Chem.
2005, 16, 1038–1044.
3. Fraley, A.W.; Pons, B.; Dalkara, D.; Nullans, G.; Behr, J.P.; Zuber, G. Cationic oligonucleotide-peptide
conjugates with aggregating properties enter efficiently into cells while maintaining hybridization
properties and enzymatic recognition. J. Am. Chem Soc. 2006, 128, 10763–10771.
4. Sazani, P.; Kang, S.H.; Maier, M.A.; Wei, C.; Dillman, J.; Summerton, J.; Manoharan, M.; Kole, R.
Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs. Nucleic Acids Res.
2001, 29, 3965–3974.