2′-O-lysylaminohexyladenosine modified oligonucleotides

Article abstract of DOI:10.1007/s00706-010-0318-0

Development of therapeutically active oligonucleotides for sequence-specific gene knockdown relies on chemical modifications that confer high stability and target affinity and ideally enable cellular uptake. 2′-O-Lysylaminohexyluridine-containing antisense and siRNA oligonucleotides have been shown to be well suited for gene knockdown. They are highly resistant to enzymatic degradation while having good affinity for the targeted RNA strand and efficiently down-regulate their target in cell culture tumor models. The 2′-O-lysylaminohexyl modification was expanded to adenosine nucleosides. The corresponding phosphoramidite building block was prepared in a straightforward procedure comprising six steps starting from adenosine. After 2′-O-alkylation with N-(6-bromohexyl)phthalimide and removal of the N-protecting group, the protected lysine was specifically attached to the alkylamino group. Incorporation of 2′-O- lysylaminohexyladenosine nucleotides in a test sequence confirmed that the cationic chains lead only to minor duplex destabilization and do not disturb the duplex structure. Results further emphasize the advantageous properties of 2′-O-lysylaminohexyl modified oligonucleotides for therapeutic applications. Springer-Verlag 2010.

Full text of DOI:10.1007/s00706-010-0318-0

Monatsh Chem (2010) 141:809–815
DOI 10.1007/s00706-010-0318-0
ORIGINAL PAPER
20-O-Lysylaminohexyladenosine modified oligonucleotides
•
Johannes Winkler Benedikt Giessrigl
•
•
Clemens Novak Ernst Urban Christian R. Noe
•
Received: 3 December 2009 / Accepted: 2 May 2010 / Published online: 9 June 2010
Ó Springer-Verlag 2010
Abstract Development of therapeutically active oligo-
nucleotides for sequence-specific gene knockdown relies
on chemical modifications that confer high stability and
target affinity and ideally enable cellular uptake. 20-O-
Lysylaminohexyluridine-containing antisense and siRNA
oligonucleotides have been shown to be well suited for
gene knockdown. They are highly resistant to enzymatic
degradation while having good affinity for the targeted
RNA strand and efficiently down-regulate their target in
cell culture tumor models. The 20-O-lysylaminohexyl
modification was expanded to adenosine nucleosides. The
corresponding phosphoramidite building block was pre-
pared in a straightforward procedure comprising six
steps starting from adenosine. After 20-O-alkylation
with N-(6-bromohexyl)phthalimide and removal of the
N-protecting group, the protected lysine was specifically
attached to the alkylamino group. Incorporation of 20-O-
lysylaminohexyladenosine nucleotides in a test sequence
confirmed that the cationic chains lead only to minor
duplex destabilization and do not disturb the duplex
structure. Results further emphasize the advantageous
properties of 20-O-lysylaminohexyl modified oligonucleo-
tides for therapeutic applications.
Introduction
Although therapeutic gene silencing by nucleic acid
analogues has been regarded as one of the great expecta-
tions in drug research for several decades, only one
antisense oligonucleotide, fomivirsen, and one ribozyme
oligonucleotide, pegaptanib, have been granted approval.
Application of both drugs by injection into the eye reveals
the still very limited therapeutic options. Apart from that,
there is a rather long history of unsuccessful clinical trials
of antisense oligonucleotides. Their use has been hampered
by several factors, for example poor bioavailability,
insufficient stability, substantial toxicity, insufficient tar-
geting, and off-target effects, which are, at least in part,
caused by the chemical modifications used.
Design of antisense drugs started with work on chemical
modifications of the lead nucleic acid structure, which itself
lacks sufficient stability against enzymatic degradation [1, 2].
A major step forward seemed to be achieved by replacement
of one of the oxygen atoms of the phosphate bridge by a sulfur
atom, which resulted in good plasma stability while only
mildly interfering with base pairing. These phosphorothioate
oligonucleotides are now referred to as first-generation anti-
sense drugs. So far, most antisense agents used in late-stage
clinical trials belong to this group. The clinical history of this
class of drug candidates has revealed their drawbacks. The
best example is oblimersen (Genasense, G3139). The largest
study ever conducted for advanced melanoma using a com-
bination of oblimersen and dacarbazine compared with
dacarbazine monotreatment failed to reach the primary end
point, overall survival [3]. In addition to its rational effect,
several reports in recent years have linked oblimersen to
unspecific or off-target effects [4–6]. A proteomic analysis of
the effect of oblimersen on human melanoma cells has
revealed several proteins that were down-regulated, with
Keywords Oligonucleotides ꢀ Zwitterions ꢀ
Circular dichroism ꢀ Thermal denaturation ꢀ Alkylation
J. Winkler ꢀ B. Giessrigl ꢀ C. Novak ꢀ E. Urban ꢀ C. R. Noe (&)
Department of Medicinal Chemistry, University of Vienna,
Vienna, Austria
e-mail: christian.noe@univie.ac.at
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J. Winkler et al.
many of them being involved in apoptosis resistance and
cancer progression [7]. Additionally, a number of glycolytic
enzymes were found to be down-regulated by oblimersen
treatment, indicating a partial reversal of the Warburg effect.
Using control oligonucleotides it was shown that the effect is
indeed caused by the phosphorothioate backbone modifica-
tion. As a matter of fact, it can be stated that the chemical
modification used in more than half of all clinical studies over
many years is directly associated with unwanted effects. In
future, the phosphorothioate modification should only be
used in selected cases, e.g. if induction of apoptosis is an
immediate therapeutic objective.
(7) (Scheme 1) proceeded analogously to the respective
uridine compound [18] with adjustments being made with
regard to the exocyclic amino function of adenosine. After
tritylation of adenosine, 20-alkylation was achieved with NaI
and 6-phthalimidohexyl chloride [9]. The phthalimido group
was selected because of the easy and orthogonal deprotec-
tion by hydrazinolysis. Coupling of the lysyl moiety,
protected as bis(trifluoroacetyl)amide, was performed in
high yield with the classical peptide coupling reagents
dicyclohexyl carbodiimide (DCC) and 1H-hydroxybenzo-
triazole (HOBt) and proceeded selectively at the alkyl amine
without, at this stage, the need for protection of the aromatic
amine of the nucleobase. Therefore, N6-protection for
automated oligonucleotide synthesis can conveniently be
carried out after 20-O-sidechain attachment. Finally, after
N-benzoylation and 30-phosphitylation according to stan-
dard procedures, the phosphoroamidite building block 7
suitable for solid phase oligonucleotide synthesis was pre-
pared in a total yield of 13%.
As a consequence of this situation there is a heavy demand
for appropriate chemical modifications to enable the design
of effective nucleic acid drugs. The 20-O-alkyl modification
of oligonucleotides has been shown to be effective [8, 9]. The
compounds impair duplex formation only weakly, and sig-
nificantly increased plasma stability does not impede
activation of the effector enzyme RNAseH. They have
superior in vitro characteristics and have been shown to be
devoid of the phosphorothioate-related off-target effects.
20-O-Alkyl-modified compounds are therefore classified as
second-generation antisense drugs. In the meantime—
aiming at a third generation—more rigorously modified
compounds have also been prepared on the basis of
replacement of the furanose ring by other moieties, with
peptide nucleic acids [10], morpholino oligonucleotides [11,
12], and locked nucleic acids (LNA) [13] as the most
promising modifications. The advent of siRNA a decade ago
[14] drew much of the attention away from the slowly
developing antisense field. However, in the meantime it has
become clear that—despite their different molecular effec-
tors—the close structural relationship of antisense and
siRNA results in the same hurdles in drug design [15–17].
Our early work on modification of oligonucleotides at the
20-O-position included design and studies of zwitterionic
compounds [2, 18–22]. With this group of compounds,
particularly promising results were obtained by introduction
of a lysine building block at the 20-position of uridine. Solid-
phase oligomerization was shown to result in highly stable
antisense and siRNA oligonucleotides with good base-
pairing properties and excellent target down-regulating
properties in vitro [18, 19]. Building on the promising results
obtained with the 20-O-lysylaminohexyluridine nucleoside,
we report the expansion of this chemistry to the purine
nucleoside adenosine.
The modified adenosine phosphoroamidite 7 was sub-
sequently successfully incorporated into 20-desoxyoli-
gonucleotides using the standard coupling reagent
4,5-dicyanoimidazole (DCI) with a coupling time of 15 min.
To assess the effect on oligonucleotide structure and target
affinity, an increasing number of lysylaminohexyl-modified
adenosine nucleosides was incorporated into a model
dodecamer sequence (Table 1).
Circular dichroism (CD) spectrometry is a sensitive
method for determining secondary structures of oligonu-
cleotides in solution. Duplexes of adenosine–thymidine
homomers give a pronounced CD spectrum, in which
subtle changes caused by chemical modifications can be
detected. Gradual substitution of desoxyadenosine against
20-O-lysylaminohexyladenosine resulted only in minor
quantitative changes in the CD spectrum (Fig. 1) without
alteration of the curve shape, indicating that the original
duplex structure prevailed. Incorporation of up to four
modifications (12) caused only minor changes of the CD
spectra, whereas lower band intensities in the far-UV
region and loss of the characteristic double peak of A/T
homomer duplexes at 260 nm were observed for 14, with
the anionic phosphate backbone fully neutralized. This
indicates a less defined duplex structure or incomplete
hybridization to the counter strand, possibly caused by
unspecific intrastrand interactions of 14.
The duplex stabilities as determined by recording tran-
sition temperatures showed a gradual decrease with
increasing number of cationic nucleotides (Fig. 2),whereas
substitution of the first two 50-terminal nucleosides results
in a relatively pronounced effect on the transition tem-
perature and thus on duplex stability. No further negative
effect was observed when up to five nucleosides were
modified. An even higher number of cationic modifications
Results and discussion
The preparation of N-benzoyl-20-O-[N2,N6-bis(trifluoroace-
tyl)lysyl]aminohexyl-50-O-(4,40-dimethoxytrityl)adenosine
30-O-[O-(2-cyanoethyl)-N,N0-diisopropylphosphoramidite]
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20-O-Lysylaminohexyladenosine modified oligonucleotides
811
Scheme 1
Table 1 Sequences and duplex transition temperatures (Tm SEM)
of oligonucleotides
Compound No. Sequence (50–30)
Tm (°C)
8
AAAAAAAAAAAA
34.7 0.4
31.4 0.2
25.9 0.9
26.3 0.6
25.4 0.7
27.5 0.4
18.3 0.7
16.6 0.3
78.7 0.5
9
A*AAAAAAAAAAA
10
11
12
13
14
15
16
17
A*A*AAAAAAAAAA
A*A*A*AAAAAAAAA
A*A*A*A*AAAAAAAA
A*A*A*A*A*AAAAAAA
A*A*A*A*A*A*AAAAAA
A*A*A*A*A*A*A*AAAAA
TCTCCCAGCGTGCGCCAT
U*CU*CCCA*GCGU*GCGCCA*T 66.6 0.6
Fig. 1 Circular dichroism spectra of duplexes of dA12–dT12 homo-
mers with 20-O-lysylaminohexyl modifications: squares, 8;
rhombuses, 12; triangles, 14
* 20-O-Lysylaminohexyl modified nucleotide; others are 20-
desoxynucleotides
123

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J. Winkler et al.
Experimental
Reagents were purchased from Sigma-Aldrich in standard
quality and used without purification. To prepare anhy-
drous solvents tetrahydrofuran (THF) was heated under
reflux with metallic sodium until the indicator benzophe-
none turned blue, and then distilled, and dichloromethane
and pyridine were distilled from calcium hydride. NMR
spectra were recorded on a 200-MHz Bruker Spectrospin or
a 500-MHz Bruker Advance. Shifts are reported relative to
the solvent peak (CHCl3 in CDCl3: d = 7.26 and 77.00).
All reactions were monitored by thin-layer chromatogra-
phy (TLC) using silica gel 60-F254 precoated aluminum
plates from Merck. Compounds were visualized by UV-
deletion or using anisaldehyde–sulfuric acid. Column
chromatography was performed with Merck silica gel 60
which was impregnated with diluted triethylamine before
use. Elemental analyses (C, H, N) were conducted at the
Microanalytical Laboratory of the University of Vienna
using the 2400 CHN Elemental Analyzer (Perkin–Elmer);
their results were in good agreement ( 0.3%) with the
calculated values.
Fig. 2 Transition temperatures (Tm) of dA12–dT12 duplexes with the
indicated number of 20-O-lysylaminohexyladenosine nucleosides
led to a significant decrease in transition temperatures.
Similar to the results with 20-O-lysylaminohexyluridine
nucleosides [18], the decrease on the transition tempera-
ture amounted to 1.4 °C per modification when five
neighboring cationic chains were present. Most of the
destabilization can be attributed to the lower thermal sta-
bility of DNA/RNA hybrids. Substitution of dA by rA
alone contributes to an overall decrease in melting tem-
perature of 1.9 °C [18, 23]. This confirms the assumption
that with isolated lysylaminohexyl chains the adverse
effect on the counter-strand affinity, probably caused by
the high steric bulk, overcomes positive effects of inter-
strand amine–phosphate interactions [19]. Several
adjacent modifications seem to be necessary for effective
interstrand charge interactions. The sharp decline in
duplex stability of 14 and 15, which was also reflected in
CD data, can be attributed to charge interactions, which
occur if the overall charge of the strand approaches
neutrality.
Incorporation of 20-O-lysylaminohexyladenosine and
uridine nucleotides in a phosphodiester oligonucleotide
isosequential to oblimersen (Table 1), a clinically relevant
antisense oligonucleotide, confirmed that the reduction in
melting temperature is higher for isolated modifications
(2.4 °C per modification). The observed effect for five
lysylalkyl chains is very similar to that of a full phosp-
horothioate backbone, the modification of oblimersen. This
demonstrates that the reduction in counter-strand affinity is
acceptable for clinical applications, especially because it is
associated with higher stability against enzymatic degrada-
tion and better pharmacokinetics [18].
50-O-(4,40-Dimethoxytrityl)-20-O-(6-phthalimido-
hexyl)adenosine (2, C45H46N6O8)
50-O-(4,40-Dimethoxytrityl)adenosine [24] (1, 2.5 g,
4.39 mmol) was dissolved in 45 cm3 DMF under an argon
atmosphere and cooled in an ice bath. Sodium hydride
(130 mg, 95% dispersion, 5.27 mmol) was added in
portions. After intensive stirring for 30 min, 130 mg
sodium iodide (0.88 mmol) and 3.4 g N-(6-bromohexyl)-
phthalimide (10.97 mmol) dissolved in 5 cm3 DMF were
added. The solution was warmed to room temperature and
stirred for a total of 67 h. The organic solvent was removed
and the residue dissolved in ethyl acetate. The organic
phase was washed successively with water, bicarbonate,
and brine. After solvent removal in vacuo, the residue was
purified on silica gel with dichloromethane–triethylamine
9:1 as mobile phase. The corresponding fractions were
washed with bicarbonate and dried, affording 1.27 g 2 as a
white foam (36%).
1H NMR (200 MHz, CDCl3): d = 8.27 (s, 1H, H-8),
8.04 (s, 1H, H-2), 7.84–7.80 (m, 2H, Phth H-4,7), 7.69–
7.65 (m, 2H, Phth H-5,6), 7.43–7.23 (m, 9H, Ar-H, amide-
H), 6.81 (d, 4H, Ar-H), 6.12–6.10 (m, 1H, H-10), 4.49 (m,
2H, H-20, H-30), 4.24–4.22 (ddd, 1H, H-40), 3.78 (s, 6H,
2OCH3), 3.71–3.43 (m, 6H, H-50, alkyl-H-1, alkyl-H-6),
1.67–1.26 (m, 8H, alkyl-H) ppm; 13C-NMR (50 MHz,
CDCl3): d = 168.42 (s, Phth: C-1, C-3), 158.46 (s, Ar-4),
155.49 (s, C-6), 149.46 (s, C-4), 144.52 (Ar-C-1), 135.67,
135.59 (Ar-C-1), 133.81 (d, Ar-C), 132.01 (s, Ar-C),
130.05 (Ar-C), 128.12 (Ar-C), 127.82 (Ar-C), 126.85 (Ar-
C), 123.12 (Ar-C), 120.00 (Ar-C), 113.10 (Ar-C), 86.84
In conclusion, the 20-O-lysylaminohexyl technology was
expanded to adenosine. The respective building block for
oligonucleotide synthesis was successfully prepared in
good yield. Biophysical experiments further confirmed the
usefulness of this type of modification in nucleic acid drug
design.
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20-O-Lysylaminohexyladenosine modified oligonucleotides
813
(C-10), 86.47 (Ar-C), 83.83 (C-40), 81.57 (C-20), 71.07
(OCH2), 69.82 (C-30), 63.03 (C-50), 55.15 (OCH3), 37.65
(NCH2), 29.34, 28.29, 26.37, 25.32 (CH2) ppm; ESI MS:
m/z = 800.0 (M?).
55.1 (OCH3), 39.3, 39.1 (NCH2), 31.7–22.1 (CH2) ppm;
ESI MS: m/z = 990.1 (M?).
N-Benzoyl-20-O-[N2,N6-bis(trifluoroacetyl)lysyl]amino-
hexyl-50-O-(4,40-dimethoxytrityl)adenosine
(5, C54H58F6N8O10)
20-O-Aminohexyl-N-benzoyl-50-O-(4,40-dimethoxytri-
tyl)adenosine (3, C44H48N6O7)
20-O-Lysylaminohexyladenosine derivative 4 (312 mg,
0.317 mmol) was dissolved under argon in 6 cm3 of a
mixture of pyridine and dichloromethane (5:1). Chlorotri-
methylsilane (13 mg, 1.2 mmol) was added dropwise while
stirring constantly. After 30 min, 95 mg benzoyl chloride
(0.671 mmol) was added and the resulting mixture was
stirred at r.t. under argon for 16 h. After addition of 5 cm3
5% NaHCO3 solution and evaporation of the organic
solvent, the residue was dissolved in dichloromethane and
extracted with NaHCO3 solution. After drying and evap-
oration of the organic phase, the residue was dissolved in
3 cm3 THF, and 0.5 cm3 of a 1 M solution of tetrabutyl-
ammonium fluoride in THF was added. After stirring at r.t.
for 20 h, dichloromethane was added. After repeated
extraction with NaHCO3, the organic phase was dried
under reduced pressure. The residue was chromatographed
using a mixture of dichloromethane and methanol (98:2) as
mobile phase. The fractions containing the product were
pooled and evaporated to dryness to give 280 mg 5 as a
white foam (0.284 mmol, 89%).
1H NMR (500 MHz, CDCl3): d = 8.49 (s, 1H, H-8),
8.18 (s, 1H, H-2), 7.45–7.12 (m, 18H, Ar-H, amide-H),
6.68 (d, 4H, Ar-H), 6.12 (d, 1H, H-10), 4.56–4.44 (m, 2H,
H-20, H-30), 4.21 (d, 1H, H-40), 3.64 (s, 6H, OCH3), 3.50–
2.92 (m, 9H, H-50, alkyl-H-2, alkyl-H-6, lysyl-H-2, lysyl-
H-6), 1.67–0.93 (m, 14H, alkyl-CH2, lysyl-CH2) ppm; 13C
NMR (125 MHz, CDCl3): d = 172.6 (benzoylamide),
169.7 (lysylamide), 158.5 (Ar-C-4), 155.3 (C-6), 149.6
(C-4), 144.4 (Ar-C-1), 135.5 (Ar-C-1), 133.7 (Ar-C-1),
133.2 (Ar-C-2-6), 130.0 (Ar-C-2,6), 128.3, 127.7, 126.9
(Ar-C-2,3,4,5,6), 113.1 (Ar-C-3,5), 86.6 (C-10), 86.5
(CPh3), 84.2 (C-40), 81.7 (C-20), 70.9 (OCH2), 69.9
(C-30), 63.1 (C-50), 55.1 (OCH3), 39.5, 39.1 (NCH2),
31.9–21.9 (CH2) ppm; ESI MS: m/z = 1094.1 (M?).
Alkylated adenosine derivative 2 (450 mg, 0.563 mmol)
was dissolved in 10 cm3 tetrahydrofuran. After addition
of 250 mm3 hydrazine hydrate, the reaction mixture was
heated to reflux under an argon atmosphere for 4 h.
After cooling, NaHCO3–K2CO3 buffer (1 M, pH 9,
5 cm3) was added. After extraction with dichlorometh-
ane, drying of the organic phase, and evaporation,
340 mg (0.508 mmol, 90%) of a white foam of 3
resulted.
1H NMR (200 MHz, CDCl3): d = 8.31 (s, 1H, H-8),
8.01 (s, 1H, H-2), 7.42–7.21 (m, 9H, Ar-H), 6.74 (d, 4H,
Ar-H), 6.12 (d, 1H, H-10), 4.51-4.42 (m, 2H, H-20, H-30),
4.23 (d, 1H, H-40), 3.51 (s, 6H, OCH3), 3.67–3.31 (m, 6H,
H-50, alkyl-H-2, alkyl-H-6), 1.51–1.19 (m, 8H, alkyl-CH2)
ppm; 13C NMR (50 MHz, CDCl3): d = 158.2 (Ar-C-4),
155.5 (C-6), 152.7 (C-2), 149.3 (C-4), 144.3 (Ar-C-1),
135.5 (Ar-C-1), 131.4-126.8 (Ar-C-2,3,4,5,6), 113.0 (Ar-C-
3,5), 86.5 (C-10), 86.4 (CPh3), 83.7 (C-40), 81.3 (C-20), 70.8
(OCH2), 69.6 (C-30), 62.9 (C-50), 55.0 (OCH3), 41.5
(NCH2), 29.2–25.3 (4 CH2) ppm; ESI MS: m/z = 670.0
(M?).
20-O-[N2,N6-bis(trifluoroacetyl)lysyl]aminohexyl-50-O-
(4,40-dimethoxytrityl)adenosine (4, C47H54F6N8O9)
20-O-Aminohexyladenosine derivative
3
(250 mg,
0.318 mmol) was dissolved in 20 cm3 dry THF. 1H-1-
Hydroxybenzotriazole (50 mg), 45 mg diisopropylcar-
bodiimide, 106 mg N2,N6-bis(trifluoroacetyl)lysine (0.313
mmol), and 4 mg 4-dimethylaminopyridine were added
sequentially. The reaction mixture was stirred at r.t. for
20 h. After solvent evaporation, the residue was chromato-
graphed on a silica column with dichloromethane–
methanol 97:3 as mobile phase. The fractions containing
the product (Rf = 0.32) were pooled and evaporated to
give 239 mg 4 as a white foam (0.242 mmol, 76%).
2-Cyanoethoxybis(diisopropylamino)phosphan (6)
Phosphorus trichloride (10 g, 73 mmol) was dissolved in
125 cm3 dry acetonitrile and stirred under an argon
atmosphere. Diisopropylamine (45 g, 445 mmol) was
added dropwise over a period of 1 h. After addition of
125 cm3 hexane, 5.2 g hydroxypropionitrile (73 mmol)
was added dropwise over a period of 30 min. After stirring
for 1 h, the solution was filtered and the liquid phases
separated. The acetonitrile phase was washed three times
with fresh n-hexane, and the combined n-hexane phase was
1H NMR (500 MHz, CDCl3): d = 8.24 (s, 1H, H-8),
8.06 (s, 1H, H-2), 7.42–7.22 (m, 12H, Ar-H, amide-H),
6.80 (d, 4H, Ar-H), 6.14 (d, 1H, H-10), 4.57–4.46 (m, 2H,
H-20, H-30), 4.25 (d, 1H, H-40), 3.76 (s, 6H, OCH3), 3.67–
3.17 (m, 9H, H-50, alkyl-H-2, alkyl-H-6, lysyl-H-2, lysyl-
H-6), 1.89–1.26 (m, 14H, alkyl-CH2, lysyl-CH2) ppm; 13C
NMR (125 MHz, CDCl3): d = 170.1 (lysylamide), 158.5
(Ar-C-4), 155.5 (C-6), 149.5 (C-4), 144.5 (Ar-C-1), 135.6
(Ar-C-1), 130.0 (Ar-C-2,6), 128.1, 127.8, 126.9 (Ar-C-
2,3,4,5,6), 113.1 (Ar-C-3,5), 86.6 (C-10), 86.5 (CPh3), 84.2
(C-40), 81.7 (C-20), 70.9 (OCH2), 69.9 (C-30), 63.1 (C-50),
1
dried to give 9.2 g of a yellow oil (7, 42%). H, 13C, and
31P NMR spectra were found to be identical with reports in
the literature [25].
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J. Winkler et al.
N-Benzoyl-20-O-[N2,N6-bis(trifluoroacetyl)lysyl]amino-
hexyl-50-O-(4,40-dimethoxytrityl)adenosine
30-O-[O-(2-cyanoethyl)-N,N-diisopropylphos-
14: calc. 5154.8, found 5154.5; 15: calc. 5398.1, found
5398.5; 17: calc. 6535.6, found 6535.3.
phoramidite](7, C63H75F6N10O11P)
Circular dichroism and transition temperature
N -Benzoyl-20-O-[N2,N6-bis(trifluoroacetyl)lysyl]aminohe-
xyl-50-O-(4,40-dimethoxytrityl)adenosine (5, 120 mg, 0.122
mmol) was dissolved under moisture-free conditions under
an argon atmosphere in 5 cm3 dry dichloromethane. After
the addition of 9 mg 1H-tetrazole and 36.5 mg 2-cyano-
ethoxy-bis(diisopropylamino)phosphane (6), the mixture
was stirred at r.t. for 16 h. After washing with NaHCO3
solution, the organic phase was evaporated to dryness and
the residue was purified on a silica column with dichlo-
romethane–methanol (98:2) as mobile phase. Drying of
the respective fractions gave 85 mg of a white foam
(0.072 mmol, 59%).
1H NMR (500 MHz, CDCl3): d = 8.59 (s, 1H, H-8),
8.30 (s, 1H, H-2), 7.43–7.19 (m, 18H, Ar-H, amide-H),
6.78 (d, 4H, Ar-H), 6.18 (d, 1H, H-10), 4.58–4.47 (m, 2H,
H-20, H-30), 4.23 (d, 1H, H-40), 3.73 (s, 6H, OCH3),
3.66–3.16 (m, 13H, H-50, alkyl-H-2, alkyl-H-6, lysyl-H-2,
lysyl-H-6, isopropyl-CH, cyanoethyl-CH2), 2.74 (t, 2H,
NC-CH2), 1.70–1.24 (m, 26H, alkyl-CH2, lysyl-CH2,
isopropyl-CH3) ppm; 13C NMR (125 MHz, CDCl3): d =
172.4 (benzoylamide), 169.9 (lysylamide), 158.4 (Ar-C-4),
155.6 (C-6), 149.9 (C-4), 144.3 (Ar-C-1), 135.5 (Ar-C-1),
133.7 (Ar-C-1), 133.0 (Ar-C-2-6), 129.9 (Ar-C-2,6), 128.6,
127.5, 126.8 (Ar-C-2,3,4,5,6), 113.1 (Ar-C-3,5), 86.9
(C-10), 86.4 (CPh3), 84.3 (C-40), 81.3 (C-20), 70.9
(OCH2), 69.6 (C-30), 63.0 (C-50), 55.0 (OCH3), 46.0
(cyanoethyl-OCH2), 45.2 (isopropyl-CH), 39.5, 39.2, 39.1
(cyanoethyl-CH2, alkyl-NCH2), 31.9–21.9 (CH2), 22.7
(isopropyl-CH3) ppm; ESI MS: m/z = 1293.7 (M?).
Concentrations of purified and desalted oligonucleotides
were determined by UV measurement at 260 nm. Molar
extinction coefficents were calculated by addition of
nucleotides (184,800 cm-1 M-1 for adenosine dodeca-
mers, 105,600 cm-1 M-1 for thymidine dodecamers). CD
spectra were recorded on a Jasco J-810 spectropolarimeter.
Oligonucleotides were diluted to a concentration of 9 lM
in a solution of 0.15 M NaCl and 0.01 M Tris–HCl
(pH 7.0) of a total volume of 200 mm3. Complementary
strands were hybridized for 5 min at 80 °C, then slowly
cooled to room temperature to ensure duplex formation.
Measurements were conducted in a quartz cuvette with a
path length of 1 mm, and the wavelength range was set to
320–200 nm with a scanning speed of 50 nm min-1. For
determination of the transition temperature the duplex
solution was heated from 0 to 80 °C at 50 °C h-1
.
Reported denaturation temperatures are means from trip-
licate experiments.
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Oligonucleotide synthesis
DNA-based oligonucleotides were prepared on a Polygen
10-column DNA synthesizer on 0.2 mM scale using stan-
dard synthetic procedures. 20-O-Lysylaminohexyl-modified
nucleotides were coupled using extended coupling times of
15 min. Oligonucleotides were prepared in DMT-off mode
and cleaved from the solid support by use of concentrated
ammonia (1 h at r.t.). Deprotection was achieved by
heating the resulting solution to 55 °C for 18 h. Ammonia
was removed in vacuo and the residue was redissolved in
0.5 cm3 water. Crude oligonucleotide products were
desalted using Sephadex G-25 (GE Life Sciences, UK).
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ToF-MS on a Kratos Seq using 3-hydroxypicolinic acid as
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´
´
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123