Nucleosides with 5′-fixed lipid groups - Synthesis and anchoring in lipid membranes

Article abstract of DOI:10.1002/ejoc.200700521

Nucleosides were synthesized bearing one or two lipophilic groups at the 5′-position. The lipophilic substituents can be fixed at a 5′-amino group or at the 5′-phosphate moiety. Selected examples of these lipophilic nucleosides are shown by solid-state NMR spectroscopy to anchor in lipid double layers. Wiley-VCH Verlag GmbH & Co. KGaA, 2007.

Full text of DOI:10.1002/ejoc.200700521

FULL PAPER
DOI: 10.1002/ejoc.200700521
Nucleosides with 5Ј-Fixed Lipid Groups – Synthesis and Anchoring in Lipid
Membranes
Nicolai Brodersen,[a] Jun Li,[a] Oliver Kaczmarek,[a] Andreas Bunge,[b] Ludwig Löser,[b]
Daniel Huster,[b] Andreas Herrmann,[c] and Jürgen Liebscher*[a]
Dedicated to Prof. Dr. Horst Hartmann on the occasion of his 70th birthday
Keywords: Nucleosides / Amphiphiles / Lipophilicity / Membrane anchoring / Synthesis
Nucleosides were synthesized bearing one or two lipophilic
groups at the 5Ј-position. The lipophilic substituents can be
fixed at a 5Ј-amino group or at the 5Ј-phosphate moiety. Se-
lected examples of these lipophilic nucleosides are shown by
solid-state NMR spectroscopy to anchor in lipid double lay-
ers.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2007)
pid membranes has also been used in chip technology, when
both the chip surface covered by a lipid double layer and a
functionalized vesicle are established with complementary
lipophilic oligonucleotide units.[18] Lipophilic nucleosides
and nucleotides exhibit antiviral activities[4,19] and lipo-
philic nucleoside-5Ј-phosphate as diesters show antitumour
activity[20–23] (L1210 murine leukemia, murine ascitic carci-
noma II) or can inhibit thymidine-5Ј-diphosphate-glucose-
4,6-dehydratase.[24] The lipophilic moiety is believed to im-
prove the activity because of improved membrane passage
and thus cellular uptake. Other very important features of
lipophilic oligonucleotides are the ability to permeate cell
membranes causing antiviral activity[25] and the property of
facilitating membrane passage of siRNA, which was
achieved by the introduction of lipid anchors, such as chole-
steryl or dialkylaminophenyl groups into the 5Јand 3Ј-end
of the oligonucleotide chain.[26,27]
Introduction
Lipophilic nucleosides and oligonucleotides have re-
ceived broad interest. Because of their amphiphilic proper-
ties they can form interesting supramolecular structures,
such as monolayer films, micelles and/or vesicles,[1–8] or-
ganogels[5] or multilamellar layers.[5,8] Normally their nu-
cleobases are exposed to the aqueous phase, and they po-
tentially can form complementary Watson–Crick base
pairs.[8–10] The lipophilic substituent can also act as an an-
chor for fixing lipophilic nucleosides or oligonucleotides in
biocompatible lipid membranes, again by exposing the nu-
cleotides to the aqueous phase.[11–14] Recently we could
show that nucleosides with lipid moieties linked to the nu-
cleobase anchor in giant unilamellar vesicles (GUVs) and
large unilamellar vesicles (LUVs) composed of unilamellar
phospholipid bilayers.[15] Oligonucleotides with two of such
lipophilic nucleotides incorporated into the nucleotide
chain anchor in both, GUVs and LUVs and were shown to
form double-strand DNAs with complementary oligonu-
cleotides in solution.[16,17] For biotechnological applica-
tions, these lipophilic nucleosides can be enriched in lipid
domains of distinct physical properties, for example, in li-
quid-disordered domains. The property of anchoring in li-
In continuation of our studies on lipophilic nucleosides
and oligonucleotides, we report here the synthesis of new
lipophilic nucleosides 1, where one or two lipophilic moie-
ties are connected to the 5Ј-position as phosphate (X =
PO4) or as amine (X = NR).
[a] Institute of Chemistry, Humboldt University Berlin,
Brook-Taylor-Str.2, 12489 Berlin, Germany
E-mail: liebscher@chemie.hu-berlin.de
[b] Junior Research Group “Structural Biology of Membrane Pro-
teins”, Institute of Biotechnology, Martin-Luther University
Halle-Wittenberg,
Kurt-Mothes-Str. 3, 06120 Halle, Germany
[c] Institute of Biology/Biophysics, Humboldt University Berlin,
Invalidenstr. 42, 10115 Berlin, Germany
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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Nucleosides with 5Ј-Fixed Lipid Groups
Nucleoside-5Ј-phosphate triesters were rarely reported.
O,O-Bis(n-butyl)thymidine 5Ј-phosphate contains only
short alkyl groups at the phosphate.[28] Nucleoside phos-
phates with two O-alkyl groups up to n-C16H33 were re-
ported; however, only in the arabinose nucleoside series.[29]
Results
Synthesis of Lipophilic Nucleosides
Several methods were reported to synthesize phosphoric
acid triester with one nucleoside as a constituent. Thus, 5-
fluoro-2Ј-deoxyuridine was transformed into a phosphoric
acid triester by reaction with a phosphoric acid diester chlo-
ride derived from a steroid and 2-chlorophenol.[24] This
method was also used in the synthesis of dinucleotide,
wherein the bridging phosphate is a triester.[30] The phos-
phoramidite method is a versatile method to introduce
phosphates as diesters into alcohols to establish phosphoric
acid triesters via intermediate phosphites. In this way, lipo-
philic phosphates like cholesteryl phosphates or long-chain
glycerol ether phosphates were transferred as well. When
nucleosides serve as substrates, the phosphate moiety can
be introduced into several positions, position 5Ј in-
cluded.[19,31–33] This method seemed to provide promising
access to lipophilic nucleoside 5Ј-phosphate triesters 6.
The synthesis of starting phosphoramidites 3 was ap-
proached by adopting a procedure where N,N-diisopro-
pylphosphoramidic dichloride derived from PCl3 was
treated with the corresponding alcohols in the presence of
triethylamine in cyclohexane.[34] It turned out that the re-
ported procedure working at 0 °C was only useful for
alcohols up to C6, whereas alcohols between C6 and C12 or
C16 and C22 required higher temperatures (50–60 or 80 °C,
respectively) (Table 1). Because of the sensitivity of lipo-
philic products 3, treatment with water was omitted during
work up. Without prior purification, phosphoramidites 3
were treated with known 3Ј-acetyl-2Ј-deoxyuridine 2[35] in
dichloromethane in the presence of tetrazole. Resulting
phosphites 4 were oxidized in situ with m-chloroperbenzoic
acid to afford high yields of lipophilic phosphates 5
(Scheme 1, Table 2). As shown for dihexadecyl ester 5e,
highly selective deprotection of the 3Ј-position to nucleotide
diester 6 is possible by treatment with potassium cyanide in
methanol at room temperature by adopting a procedure
used for the deacetylation of sugars.[36]
Scheme 1.
Table 2. Lipophilic nucleotide phosphate dialkyl esters 5.
Nonlipophilic 5Ј-amino-5Ј-deoxynucleosides can be syn-
thesized by various methods, such as the Mitsunobu reac-
tion of nucleosides with phthalimides,[37,38] nosylamides,[39]
phosphorazidate,[40] nitric acid[41] and N-Boc-O-Cbz-hy-
droxyamine,[42] Appel substitution of 5Ј-OH by azide in the
presence of triphenylphosphane/tetrabromomethane[43–45]
and nucleophilic substitution at nucleoside 5-tosylates or
mesylates with azide[46–50] or amines.[38,51–57] We used the
latter method to synthesize lipophilic 5Ј-amino-5Јdeoxynu-
cleosides 8 by reaction with long-chain primary or second-
ary amines. Tosylate 7 is known and was obtained from
uridine by threefold TBS-protection, selective 5Ј-deprotec-
tion[58] and 5Ј-tosylation.[59] Interestingly, during column
chromatographic purification on silica, a small amount of
unreacted 7 underwent transformation into the isomer 9,
where the tosyl group was found at the pyrimidine ring.
Substitution of the tosyloxy group of uridine derivative 7
by primary or secondary long-chain amines in THF failed.
However, by heating under neat conditions and by using an
excess amount of amine as the solvent or in a melt was
successful. Deprotection of resulting 5Ј-amino-5Ј-deoxyuri-
Table 1. O,O-Dialkyl-N,N-(diisopropyl)phosphoramidites 3.
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J. Liebscher et al.
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dines 8 to aminouridines 10 was possible by treatment with
In general, the isolated yields of unprotected pure 5Ј-
TBAF in THF. As shown in the synthesis of products 10c amino-5Ј-deoxyuridines 10 are low, because several chro-
and 10d, a one-pot procedure starting from 7 is also pos- matographic purifications of these amphiphilic materials
sible by omitting the isolation of silylated substitution prod- were necessary.
uct 8. The 5Ј-amino substituent in former product 10c not
In order to increase the hydrophilic property, 5Ј-diocta-
only provides lipophilic properties but can serve as a fluo- decylamino-5Ј-deoxyurdine 8b was methylated to afford
rescence marker at the same time (Table 3).
amphiphilic quaternary ammonium salt 11 after deprotec-
tion. We also succeeded to synthesize 5Ј-amino-5Ј-deoxyuri-
dine 12 with two lipophilic groups, where the 5Ј-amino
group lacks basicity, by first introducing octadecylamine
and then by acylating the resulting 5Ј-octadecylamino5Ј-de-
oxyuridine with palmitoyl chloride (Scheme 2).
Table 3. Lipophilic 5Ј-amino-5Ј-deoxyuridines 8 and 10.
It can be expected that the extension of the methodology
use to synthesize 5Ј-amino5Ј-deoxynucleosides from 5Ј-sul-
fonated nucleosides and amines to nucleosides other than
uridine will face problems, because such nucleoside-5Ј-sul-
fonates tend to undergo intramolecular substitution of the
sulfonate by the nucleobase under basic conditions.[60,61]
Therefore, an alternative route was followed by first intro-
ducing the amino group into the sugar and finally establish-
ing the nucleoside by N-glycoside formation as shown in
Scheme 3. Known 1Ј-methyl-2Ј,3Ј-isopropylidene-5Ј-tosylri-
bose 13 was treated with neat N,N-dioctadecylamine at
90 °C to afford 5Ј-dioctadecylamino-5Ј-deoxyribose glyco-
side 14. In order to obtain the β-anomer in the final N-
glycoside formation, an acyl group had to be introduced
into the 2Ј-postion of glycoside 14. Deprotection and acety-
lation with acetic anhydride gave 1Ј,2Ј,3Ј-triacetyl-5Ј-
amino-5Ј-deoxyribose 15 in low yield. Transformation into
5Ј-amino-5Јdeoxyadenosine 16 was achieved by treatment
[a] Over two steps.
Scheme 2.
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Scheme 3.
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Nucleosides with 5Ј-Fixed Lipid Groups
with adenine in the presence of tin tetrachloride in dichloro-
methane. Final deacetylation with ammonia in methanol
gave anticipated 5Ј-(N,N-dioctadecanyl)amino-5Ј-deoxy-
adenosine 17. This methodology has not been applied to
5Ј-amino-5Ј-deoxyribosides before, but only to ribosides
with the azido group masked as an amino functionality in
the 5Ј-position.[62–64]
Anchoring in Lipid Membranes
We used solid-state NMR spectroscopy to investigate the
incorporation of lipophilic nucleosides 10b, 11 and 17 into
lipid membranes. The hydrophilic head groups of these nu-
cleosides are uridine or adenine. The membrane anchorage
of these units was achieved by lipophilic groups that are
connected to the 5Ј-position of the ribose as amino moieties
or as a quaternary ammonium salt.
Figure 1. 31P NMR spectra of pure POPC membranes (D) and of
POPC in the presence of 11 (20 mol-%) (A), 10b (B) and 17 (C).
All measurements were carried out at a water content of 40 wt.-%
and a temperature of 30 °C. The dotted lines represent the best-fit
simulations of the spectra.
To study the alterations in the general membrane mor-
phology and structure after incorporation of lipophilic nu-
2
cleosides 10b, 11 and 17, 31P and H NMR spectroscopic
experiments were carried out.
The 31P NMR spectra of POPC membranes in the pres-
ence 20 mol-% of each of the three compounds are shown
in Figure 1.[65] From the shape of the NMR spectra, infor-
mation about the structural and dynamic properties of the
phospholipid head groups could be obtained. In the pres-
ence of the three lipophilic nucleosides, the POPC mem-
branes remained in the lamellar liquid crystalline mem-
brane phase, as indicated by the very similar spectra com-
pared to the axially symmetric powder pattern for the pure
lipid membrane. The chemical shift difference between the
low- and the high-field edges of the 31P NMR spectra is
termed chemical shift anisotropy and is related to the orien-
tation and dynamics of the phosphate group of the lipid. A
small decrease in the 31P NMR chemical shift anisotropy
of the lipid head group can be observed in the presence of
10b and 17, whereas the spectrum of 11 is slightly broad-
ened. The small deviations indicate only negligible changes
in the chemical environment of the lipid phosphate groups.
Quantitative values for the 31P NMR chemical shift anisot-
ropy were obtained by using best-fit simulations and are
listed in Table 4.
Table 4. 31P NMR chemical shift anisotropy values, average order
parameters and chain extent of POPC membranes in the presence
of lipophilic nucleosides 10b, 11 and 17.
Membrane system
∆σ [ppm]
Sav
͗L*C͘ [Å]
POPC
45.2
46.5
47.7
42.9
0.154
0.165
0.209
0.157
11.48
11.84
13.04
11.60
POPC/10b
POPC/11
POPC/17
To investigate the influence of the lipophilic nucleosides
on lipid chain packing properties in the membranes, 2H
NMR spectra of chain-perdeuterated [D31]POPC in the
presence of substances 10b, 11 and 17 were conducted.[66]
Figure 2 shows the smoothed order parameter profiles de-
rived from the 2H NMR spectra as a function of the carbon
atom position.
The carbon segments are numbered starting at the car-
bonyl group of the palmitoyl chain of the [D31]POPC mole-
cule. Small order parameter differences relative to pure lipid
Figure 2. Smoothed order parameter profiles determined from so-
lid-state H NMR experiments of sn-1 chain perdeuterated [D31]-
POPC (o) and 1:4 (mol/mol) mixtures of 10b/[D31]POPC (᭹), 11/
[D31]POPC (᭿) and 17/[D31]POPC membranes (᭢) at a tempera-
ture of 303 K and a water content of 40 wt.-%.
2
membranes are observed for 10b and 17, which indicates nificantly increased, which indicates that the large pertur-
that the molecules are well incorporated into the membrane bation of the membrane packing properties may be due to
without large alterations in the phospholipids packing den- the electrostatic interaction of the positive charge with the
sity. In contrast, the chain order of [D31]POPC in the pres- negatively charged phosphate group of the phospholipids or
ence of positively charged ammonium compound 11 is sig- a by a change in the lipid head group orientation. Further,
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J. Liebscher et al.
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because of the Born repulsion of the charge from the hydro- termination of the distribution of the sugar/base moiety in
phobic membrane surface, the head group of the nucleoside the membrane. To this end, intermolecular cross peaks be-
is likely to be repelled from this surface. This can also be tween the phospholipids and the nucleoside were analyzed.
seen from the average order parameter, which is largest for The intermolecular cross peak volumes were calculated at
the mixture of POPC/11 and translates into the longest various mixing times, and the corresponding NOE build-up
chain length of the lipid matrix (Table 4).
curves were fitted to the standard spin pair model to obtain
The preferential location of the lipophilic nucleosides cross relaxation rates σij.
within the membrane was investigated by two dimensional
1H NOESY NMR spectroscopy under magic-angle spin- contact probabilities between interacting molecular seg-
ning conditions.[67] This technique further allowed the de- ments,[67] spatial information can be obtained to find the
localization of the lipophilic nucleosides in the lipid mem-
brane.
Because the intermolecular σij values are proportional to
The nucleoside moieties of 10b, 11 and 17 were broadly
distributed along the bilayer as illustrated in Figure 3 due
to the thermal disorder within the membrane. The highest
probability of contacts (highest cross relaxation rate) and
therefore the most probable localization of the hydrophilic
nucleoside moieties could be observed in the lipid–water
interface region of the membranes. The probability distribu-
tion of localization of the 6-H, 5-H and 1Ј-H protons of
positively charged 11 is significantly shifted to the head-
group region of the phospholipids relative to 10b, which
2
may also explain the more ordered membrane seen by H
NMR spectroscopy. This effect is more pronounced for the
polar sugar moiety where the localization of the less-polar
uracil base indicates a backfolding of the nucleobase
towards the hydrophobic core of the membrane.
Conclusions
A number of new amphiphilic nucleosides bearing one
or two lipophilic groups at the 5Ј-position were synthesized.
Reaction of 3Ј-acetyl-2Ј-deoxyuridine with various long-
chain dialkyl phosphoramidites and subsequent oxidation
with m-chloroperbenzoic acid afforded 2Ј-deoxyuridine-5Ј-
phosphate triesters 5 and 6, whereas the reaction of 2Ј,3Ј-
diprotected uridine-5Ј-tosylate with long-chain alkylamines
afforded 5Ј-amino-5Ј-deoxyuridines 10. In an alternative
way, dioctadecylamine was first introduced into a protected
ribose 5Ј-tosylate and finally N-glycosylated with adenine.
Two lipophilic groups could be introduced into uridine by
reaction of the 5Ј-tosylate with N-octadecylamine and sub-
sequent acylation with palmitoyl chloride. 5Ј-NЈ,NЈ-Diocta-
decylamino-5Ј-deoxyuridine could be quaternized by
methyl iodide to afford an amphiphilic nucleotide with
more pronounced polar properties. The latter as well as
nonquaternized 5Ј-amino-5Ј-deoxynucleosides anchor in
the phospholipid membranes with their lipophilic alkyl
Figure 3. 1H NOESY NMR cross-relaxation rates (s–1) between
chains. As determined by 2H-, 31P- and 1H MAS NMR
molecular segments of the lipophilic nucleosides and segments of
spectroscopy, this anchoring does not disturb the structure
POPC molecules in the membrane as a function of the coordinates
of the membrane, and the polar sugar moieties are oriented
in the interphase between the polar choline phosphate head
groups of the membrane and water. The sugar/base moiety
of the nucleosides is localized in the lipid water interface of
the membrane, whereas the lipophilic chains insert into the
hydrophobic membrane interior.
obtained from a molecular dynamics simulation of pure POPC
membranes.[68] Top: Distribution of the 6-H proton of the uracil
moiety of 10b (᭹) and 11 (᭿) and the 8-H proton of the adenine
nucleobase of 17 (᭢) with respect to the membrane normal; middle:
localization of the 5-H proton of the uracil nucleobase of 10b (᭹)
and 11 (᭿) and bottom: distribution of the 1Ј-H proton of the
ribose moiety of 10b (᭹) and 11 (᭿) within the POPC matrix.
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Nucleosides with 5Ј-Fixed Lipid Groups
+ CH) ppm. 13C NMR (75.5 MHz, CDCl3): δ = 13.8 (CH3), 19.1
(CH2), 24.5 (CH3), 24.6 (CH3), 33.3 (CH2), 33.4 (CH2), 42.6 (CH),
42.7 (CH), 63.0 (CH2O) ppm. 31P NMR (242.8 MHz, CDCl3): δ =
145.54, 139.79 ppm.
Experimental Section
1
General Remarks: H and 13C NMR spectra were recorded at 300
and 75.5 MHz, respectively, with a Bruker AC 300 in CDCl3 with
TMS as an internal standard. POPC (1-[D31]palmitoyl-2-oleoyl-sn-
glycero-3-phosphocholine) and [D31]POPC were purchased from
Avanti Polar Lipids (Alabaster, AL) and used without further puri-
General Procedure for the Synthesis of O,O-Dialkyl-O-(3Ј-O-acetyl-
2Ј-deoxyuridin-5Ј-yl)phosphates 5a–h: O,O-Dialkyl-N,N-diisopro-
pylphosphoramidite 3a–h (0.40 mmol) and 3Ј-O-acetyl-2Ј-deoxyur-
idine 2 (0.20 mmol) were dissolved in a tetrazole solution (~0.45 
in MeCN, 2 mL) and dry DCM (2 mL). The suspension was stirred
at room temperature for 24 h. The mixture was cooled to 0 °C and
m-CPBA (138 mg, 0.80 mmol) was added. After stirring for 1 h at
0 °C and 2 h at room temperature, the precipitate was filtered off
and washed with DCM (5ϫ5 mL). The combined filtrates were
concentrated and dried in vacuo, and crude materials 5a–h were
submitted to silica gel column chromatography (DCM/EtOAc, 5:3).
2
1
fication. For H-, 31P- and H NOESY NMR measurements, mix-
tures of phospholipids and lipophilic nucleosides were prepared in
a chloroform/methanol mixture (1/1). The solvent was removed by
rotary evaporation, and the resulting lipid film was redissolved in
cyclohexane and lyophilized overnight to obtain a fluffy powder.
Samples were hydrated with 40 wt.-% water and equilibrated by 10
freeze–thaw cycles and gentle centrifugation. The liposome disper-
sion were transferred into 4-mm high-resolution MAS rotors fitted
with spherical Kel-F inserts for liquid samples or into 5-mm glass
vials for static 2H NMR experiments. 31P NMR spectra were re-
corded with a Bruker DRX 600 NMR spectrometer at a resonance
frequency of 242.8 MHz for 31P by using a Hahn-echo pulse se-
quence with a 90° pulse length of 7 µs, a Hahn-echo delay of 50 µs,
a spectral width of 100 kHz, and a recycle delay of 4 s. Continuous-
wave proton decoupling was applied during signal acquisition.
Spectral simulations of the 31P NMR line shape were carried out
to obtain the chemical shift anisotropy (∆σ) by using a program
written in Mathcad 2001 (MathSoft Engineering & Education Inc.,
O,O-Dibutyl-O-(3Ј-O-acetyl-2Ј-deoxyuridin-5Ј-yl)phosphate
(5a):
Yield: 80 mg (87%). 1H NMR (300 MHz, [D6]DMSO): δ = 0.68
(m, 6 H, CH3), 1.12 (m, 4 H, CH2), 1.37 (m, 4 H, CH2), 1.87 (s, 3
H, CH3), 2.15 (m, 2 H, CH2Ј), 3.76 (q, J = 6.0 Hz, 4 H, CH2), 3.96
(m, 3 H, CH4Ј, CH25Ј), 5.00 (s, 1 H, CH3Ј), 5.44, 5.47 (d, J =
9.0 Hz, 1 H, CH5), 5.96 (t, J = 7.2 Hz, 1 H, CH1Ј), 7.47, 7.50 (d,
J = 9.0 Hz, 1 H, CH6), 11.23 (s, 1 H, NH) ppm. 13C NMR
(75.5 MHz, CDCl3): δ = 13.5 (CH3), 18.2 (CH2), 20.9 (CH3), 31.7
(CH2), 31.8 (CH2), 35.9 (C2Ј), 66.6, 66.7 (C5Ј), 67.1 (CH2O), 67.2
(CH2O), 73.6 (C3Ј), 82.0, 82.1 (C1Ј), 84.6 (C4Ј), 103.0 (C5), 139.6
(C6), 150.4 (C2), 163.3 (C4), 170.5 (O =CCH3) ppm. 31P NMR
(242.8 MHz, CDCl3): δ = 0.26 ppm. MS (ESI): m/z = 462.31.
2
Cambridge, MA). H NMR spectra were recorded with a Bruker
Avance400 NMR spectrometer at
a resonance frequency of
61.5 MHz for 2H by using a solids probe with a 5 mm solenoid
coil. The 2H NMR spectra were accumulated by using the quadru-
polar echo sequence and a relaxation delay of 1 s. The two 3.1 µs
2
O,O-Dihexadecyl-O-(3Ј-O-acetyl-2Ј-deoxyuridin-5Ј-yl)phosphate
π/2 pulses were separated by a 60 µs delay. H NMR spectra were
1
(5e): Yield: 120 mg (75%). H NMR (300 MHz, CDCl3): δ = 0.76
depaked and order parameters for each methylene group in the
chain were determined as described.[68] 1H MAS NMR spectra
were acquired at a spinning speed of 6009 Hz with a Bruker DRX
600 NMR spectrometer by using a 4-mm HR MAS probe. Typical
(t, J = 6.8 Hz, 6 H, CH3), 1.13 (m, 52 H, CH2), 1.56 (m, 4 H, CH2),
1.99 (s, 3 H, CH3O), 2.01–2.37 (m, 2 H, CH2Ј), 3.94 (q, J = 6.9 Hz,
4 H, CH2O), 4.06 (m, 1 H, CH4Ј), 4.17 (m, 2 H, CH25Ј), 5.16, 5.18
(d, J = 6.0 Hz, 1 H, CH3Ј), 5.66 (dd, J = 9.0 Hz, J = 3.0 Hz, 1 H,
CH5), 6.26 (q, J = 4.9 Hz, 1 H, CH1Ј), 7.58, 7.60 (d, J = 6.0 Hz,
1 H, CH6), 9.24 (s, 1 H, NH) ppm. 13C NMR (75.5 MHz, CDCl3):
δ = 14.1 (CH3), 20.9 (CH3), 22.7 (CH2), 25.4 (CH2), 29.1 (CH2),
29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 29.7 (CH2), 30.3 (CH2), 30.3
(CH2), 31.9 (CH2), 37.6 (C2Ј), 66.8, 66.9 (C5Ј), 68.3 (CH2O), 68.4
(CH2O), 74.5 (C3Ј), 83.1, 83.2 (C1Ј), 84.8 (C4Ј), 103.1 (C5), 139.4
(C6), 150.4 (C2), 163.0 (C4), 170.5 (O =CCH3) ppm. 31P NMR
(242.8 MHz, CDCl3): δ = –0.15 ppm. HRMS: calcd. for
C43H79N2O9P 799.55 [M + H]+; found 799.67.
2
π/2 pulse lengths were 9 µs. A H lock was used for field stability.
1
Two-dimensional H MAS NOESY spectra were acquired at vari-
ous mixing times (between 1 and 600 ms). The dwell time of the
indirect dimension was set equal to one rotor period to avoid fold-
ing of spinning sidebands into the centre band region of the 2D
NOESY spectra. Typically, between 400 and 500 data points were
acquired in the indirect dimension with 32 scans per increment at
a relaxation delay of 3.5 s. The volumes of the diagonal and cross
peaks were integrated by using the Bruker XWINNMR software
package. NOE build-up curves were fitted to the spin-pair model
obtaining cross relaxation rates. All spectra were acquired at a tem-
perature of 30 °C. Silica gel (0.04–0.063 mm, Merck) was used for
preparative column chromatography. Starting materials 3,[34] 7[58,59]
and 13[26] were synthesized according to literature procedures. All
the other materials were purchased from commercial suppliers.
O,O-Dihexadecyl-O-(2Ј-deoxyuridin-5Ј-yl)phosphate (6): Nucleo-
side-5Ј-phosphate 5e (130 mg, 0.16 mmol) and KCN (20 mg,
0.31 mmol) were dissolved in MeOH (5 mL) at room temperature
and stirred for 4 h. After evaporation of the solvent, the residue
was submitted to silica gel column chromatography (DCM/MeOH,
20:1; Rf = 0.4). Product 6 was obtained as a white solid. Yield:
121 mg (98 %). 1H NMR (300 MHz, CDCl3): δ = 0.85 (t, J =
6.8 Hz, 6 H, CH3), 1.23 (m, 52 H, CH2), 1.65 (m, 4 H, CH2), 2.13,
2.41 (m, 2 H, CH2Ј), 3.91 (m, 1 H, CH4Ј), 4.03 (q, J = 7.2 Hz, 4
H, CH2O), 4.23 (m, 2 H, CH25Ј), 4.47 (m, 1 H, CH3Ј), 5.70, 5.73
(d, J = 9.0 Hz, 1 H, CH5), 6.28 (t, J = 6.3 Hz, 1 H, CH1Ј), 7.58,
7.61 (d, J = 9.0 Hz, 1 H, CH6), 9.36 (s, 1 H, NH) ppm. 13CNMR
(75.5 MHz, CDCl3): δ = 14.1 (CH3), 22.7 (CH2), 25.4 (CH2), 29.1
(CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 29.7 (CH2), 30.2 (CH2),
30.3 (CH2), 31.9 (CH2), 40.4 (C2Ј), 66.4 (C5Ј), 68.4 (CH2O), 68.5
(CH2O), 70.7 (C3Ј), 84.8, 84.9 (C4Ј), 85.1 (C1Ј), 102.6 (C5), 139.7
(C6), 150.4 (C2), 163.2 (C4) ppm. 31P NMR (242.8 MHz, CDCl3):
Representative Procedure for the Preparation of Phosphoramidites 3.
O,O-Dibutyl-N,N-diisopropylphosphoramidite (3a): N,N-diisopro-
pylphosphoramidite dichloride[34] (1.01 g, 5.00 mmol) was dis-
solved in hexane (6 mL) under an atmosphere of argon. The solu-
tion was cooled to –50 °C and a solution of n-BuOH (740 mg,
10.0 mmol) and DIPEA (1.9 g, 15.0 mmol) in hexane (4 mL) was
added. The solution was stirred and the temperature slowly rose
to 20 °C. Further stirring at room temperature for 2 h led to the
completion of the reaction. The precipitate was filtered off, and the
filtrate was concentrated in vacuo. Compound 3a was obtained as
a colourless oil. Yield: 1.08 g (78%). 1H NMR (300 MHz, CDCl3):
δ = 0.84 (t, J = 7.4 Hz, 6 H, CH3), 1.09, 1.11 (d, J = 6.0 Hz, 12 H,
CH3), 1.30 (m, 4 H, CH2), 1.53 (m, 4 H, CH2), 3.50 (m, 6 H, CH2O δ = 0.19 ppm.
Eur. J. Org. Chem. 2007, 6060–6069
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6065

J. Liebscher et al.
FULL PAPER
General Procedure for the Synthesis of 5Ј-Amino-2Ј,3Ј-[Bis(tert-bu-
tyldimethylsilyl)]-5Ј-deoxyuridines 8 and 5Ј-Amino-5Ј-deoxyuridines
10a–d: 5Ј-Tosyl-2Ј3Ј-[bis(tert-butyldimethylsilyl)]uridine 7 (1 equiv.)
was dissolved in neat amine HNR1R2 (11–30 equiv.) at 80–90 °C
under an atmosphere of argon. The solution was kept at that tem-
perature for 3.5–28 h under vigorous stirring. As TLC indicated
the final progress of the reaction, the melt was solidified at room
temperature and pulverized (except 8d). The material was sus-
pended in Et2O and stored at –20 °C for 1 h. The solid material
was filtered off and washed with cold Et2O. The filtrates were com-
bined, and the solvent evaporated. The residue was submitted to
silica gel column chromatography. In the case of 8c,d the filtrates
were evaporated, and the residues treated with TBAF (1  in THF)
without purification. Compounds 8a,b were isolated, characterized
and deprotected with TBAF afterwards (r.t., 30 min). Raw materi-
als 10a–d were purified by silica gel column chromatography.
(m, 1 H, -CH-OH, 3Ј), 5.69 (d, J = 7.5 Hz,1 H, CH5), 5.77 (s, 1
H, -CH-, 1Ј), 7.62 (d, J = 7.5 Hz, 1 H, CH6) ppm. 13C NMR
(75.5 MHz, CDCl3): δ = 14.19 (-CH2-CH3), 22.74 (-CH2-), 27.07
(-CH2-), 29.36 (-CH2-), 29.41 (-CH2-), 29.64 (-CH2-), 29.71
(-CH2-), 29.76 (-CH2-), 31.98 (-CH2-), 49.33 (-CH2-), 49.74
(CH25Ј), 71.27 (CH3Ј), 73.97 (CH2Ј), 80.54 (CH4Ј), 91.3795
(CH1Ј), 102.49 (CH5), 142.03 (CH6), 150.35 (C2), 163.16 (C4)
+
ppm. HRMS: calcd. for C27H50N3O5 496.3745; found 496.3751.
5Ј-Dioctadecylamino-5Ј-deoxyuridine (10b): Compound 8b (130 mg,
0.13 mmol), THF (2 mL), TBAF (1  in THF; 1 mL, 1 mmol).
Column chromatography MeOH/CHCl3, 1:7; Rf = 0.2. Yield:
40 mg (40 %). Colourless solid, m.p. 53–55 °C. 1 H NMR
(300 MHz, CDCl3): δ = 0.87 (t, J = 6.8 Hz, 6 H, -CH2-CH3), 1.25
(s, 60 H, -CH2-,), 1.45 (s, 4 H, -NH-CH2-CH2-), 2.56 (s, 4 H, -NH-
CH2-CH2-), 2.82 (m, 2 H, -CH25Ј), 4.00 (m, J = 6.0 Hz, 1 H, -CH-
OH, 4Ј), 4.10 (m, J = 6.0 Hz, 1 H, -CH-OH, 2Ј), 4.21 (s, 1 H,
-CH-OH, 3Ј), 5.71 (d, J = 8.3 Hz,1 H, CH5), 5.75 (d, J = 1.8 Hz,1
H, -CH-, 1Ј), 7.71 (d, J = 8.3 Hz, 1 H, CH6) ppm. 13C NMR
(75.5 MHz, CDCl3): δ = 14.27 (-CH2-CH3), 22.83 (-CH2-), 26.37
(-CH2-), 27.62 (-CH2-), 29.40 (-CH2-), 29.51 (-CH2-), 29.76
(-CH2-), 29.81 (-CH2-), 29.87 (-CH2-), 32.02 (-CH2-), 54.82
(-CH2-), 56.06 (CH25Ј), 72.21 (CH3Ј), 74.85 (CH2Ј), 81.59 (CH4Ј),
5Ј-Octadecylamino-2Ј,3Ј-[bis(tert-butyldimethylsilyl)]-5Ј-deoxy-
uridine (8a): Starting material 7 (150 mg, 0.24 mmol), octadecyl-
amine (1.90 g, 7.0 mmol, 29 equiv.), 80 °C, 3.5 h. Column
chromatography EtOAc, Rf = 0.3. Yield: 110 mg (63%). Colourless
1
oil. H NMR (300 MHz, CDCl3): δ = 0.06 (s, 9 H, Si-CH3), 0.09
(s, 3 H, Si-CH3), 0.86 (s, 3 H, -CH2-CH3), 0.87 [s, 9 H, Si-C-
(CH3)3], 0.89 [s, 9 H, Si-C(CH3)3], 1.23 (s, 30 H, -CH2-,), 1.47 (s, 2 92.05 (CH1Ј), 102.40 (CH5), 140.84 (CH6), 151.06 (C2), 163.94
+
H, -NH-CH2-CH2-), 2.63 (t, J = 6.8 Hz, 2 H, -NH-CH2-CH2-), (C4) ppm. HRMS: calcd. for C45H86N3O5 748.6562; found
2.86 (m, 2 H, -CH25Ј), 3.88 (m, 1 H, -CH-OH, 4Ј), 4.13 (m, 1 H, 748.6563.
-CH-OH, 2Ј), 4.21 (t, J = 3.8 Hz,1 H, -CH-OH, 3Ј), 5.65 (d, J =
5Ј-(9,9-Dioctadecyl-9H-fluorene-2-ylamino)-5Ј-deoxyuridine (10c):
Compound 7 (200 mg, 0.32 mmol), 2-amino-9,9-dioctadecyl-9H-
fluorene (2.4 g, 3.5 mmol, 11 equiv.), 80–90 °C, 28 h. One-pot pro-
cedure: for deprotection THF (18 mL) and TBAF (1  in THF;
9 mL, 9 mmol) was added, and the mixture was kept at room tem-
perature for 50 min. Column chromatography EtOAc/cyclohexane,
1:9; Rf = 0.1. Yield: 123 mg (42%). Yellow oil. 1H NMR (300 MHz,
CDCl3): δ = 0.61 (s, 4 H, -CH2-), 0.86 (t, J = 6.8 Hz, 6 H,-CH2-
CH3), 1.33–0.93 (m, 60 H, -CH2-,), 1.86 (m, 4 H, -N-CH2-CH2-),
3.76–3.38 (m, 2 H,-CH25Ј), 4.19 (m, 1 H, -CH-OH, 4Ј), 4.23 (m, 1
H, -CH-OH, 2Ј), 4.28 (m, 1 H, -CH-OH, 3Ј), 5.47 (s, 1 H, -CH-,
1Ј), 5.80 (s, 1 H, CH5), 6.63 (m, 2 H, CHar), 7.28–7.10 (m, 3 H,
CHar),7.46 (d, J = 8.0 Hz, 1 H, CHar), 7.47 (d, J = 8.3 Hz, 1 H,
CHar), 7.52 (d, J = 7.3 Hz, 1 H, CHar) ppm. 13C NMR (75.5 MHz,
CDCl3): δ = 14.27 (-CH2-CH3), 22.84 (-CH2-), 23.97 (-CH2-), 29.52
(-CH2-), 29.77 (-CH2-), 29.83 (-CH2-), 29.88 (-CH2-), 30.28
(-CH2-), 32.07 (-CH2-),40.72 (-CH2-), 46.09 (CH25Ј), 54.95
(-CHar-), 70.78 (CH3Ј), 74.71 (CH2Ј), 83.30 (CH4Ј), 91.41 (CH1Ј),
3.4 Hz,1 H, -CH-, 1Ј), 5.70 (d, J = 8.3 Hz,1 H, CH5), 7.74 (d, J =
8.3 Hz,1 H, CH6) ppm. 13C NMR (75.5 MHz, CDCl3): δ = –4.75
(Si-CH3), –4.73 (Si-CH3), –4.43 (Si-CH3), –4.11 (Si-CH3), 14.23
(-CH2-CH3), 18.08 [-C(CH3)3], 18.15 [-C(CH3)3], 22.79 (-CH2-),
25.88 [-C(CH3)3], 25.93 [-C(CH3)3], 27.43 (-CH2-), 29.49 (-CH2-),
29.69 (-CH2-), 29.75 (-CH2-), 29.77 (-CH2-), 29.80 (-CH2-), 30.25
(-CH2-), 32.02 (-CH2-), 50.56 (CH25Ј), 72.51 (CH3Ј), 75.26 (CH2Ј),
83.15 (CH4Ј), 91.37 (CH1Ј), 102.04 (CH5), 141.11 (CH6), 150.38
+
(C2), 163.87 (C4) ppm. HRMS: calcd. for C39H78N3O5Si2
724.5475; found 724.5475.
5Ј-Dioctadecylamino-2Ј,3Ј-[bis(tert-butyldimethylsilyl)]-5Ј-deoxyuri-
dine (8b): Starting material 7 (180 mg, 0.29 mmol), dioctadecylamine
(2.10 g, 4.0 mmol, 14 equiv.), 80 °C, 12 h. Column chromatography
EtOAc/cyclohexane, 1:4; Rf = 0.3. Yield: 230 mg (82%). Colourless
1
oil. H NMR (300 MHz, CDCl3): δ = 0.06 (s, 3 H, Si-CH3), 0.07
(s, 6 H, Si-CH3), 0.09 (s, 3 H, Si-CH3), 0.87 (t, J = 6.8 Hz, 6 H,
-CH2-CH3), 0.88 [s, 9 H, Si-C(CH3)3], 0.90 [s, 9 H, Si-C(CH3)3],
1.24 (s, 60 H, -CH2-,), 1.42 (s, 4 H, -N-CH2-CH2-), 2.47 (m, 4 H, 102.41 (CH5), 107.55 (-Car-), 112.18 (-Car-), 118.44 (-Car-), 120.62
-N-CH2-CH2-), 2.64 (m, 2 H, -CH25Ј), 3.81 (m, 1 H, -CH-OH, 4Ј), (-Car-), 122.73 (-Car-), 125.43 (-Car-), 126.75 (-Car-), 132.21 (-Car-),
4.10 (m, 1 H, -CH-OH, 2Ј), 4.21 (t, J = 3.8 Hz,1 H, -CH-OH, 3Ј), 140.52 (CH6), 141.53 (-Car-), 147.82 (-Car-), 149.76 (-Car-), 151.36
5.68 (d, J = 3.8 Hz,1 H, -CH-, 1Ј), 5.72 (d, J = 8.3 Hz,1 H, CH5), (C2), 152.89(-Car-),163.92 (C4) ppm. UV (0.13 mmol L–1 in
7.56 (d, J = 8.3 Hz, 1 H, CH6) ppm. 13C NMR (75.5 MHz, CHCl3): λ (ε, L mol–1 cm–1) = 300 (26970). HRMS: calcd. for
+
CDCl3): δ = –4.67 (Si-CH3), –4.34 (Si-CH3), –4.00 (Si-CH3), 14.25 C58H94N3O5 912.7188; found 912.7188.
(-CH2-CH3), 18.12 [-C(CH3)3], 22.82 (-CH2-), 25.94 [-C(CH3)3],
[5Ј-(N-Methyl)dioctadecylammonio-5Ј-deoxyuridine]iodide (11):
Compound 8b (182 mg, 0.19 mmol) was dissolved in dry Et2O
(1 mL) and MeI (160 µL, 2.57 mmol) was added under an atmo-
sphere of argon. The solution was stirred at room temperature for
72 h. For deprotection, Et2O (17 mL), MeOH (6 mL) and ammo-
nium fluoride (500 mg, 13.5 mmol) were added, and the suspension
was stirred at 45 °C for 3 h and at room temperature for 48 h. After
TLC indicated the final progress of the deprotection, MeOH
(10 mL), Et2O (3 mL) and water (20 mL) were added, and the re-
sulting white precipitate was filtered off, washed with water and
27.07 (-CH2-), 27.66 (-CH2-), 29.79 (-CH2-), 29.84 (-CH2-), 32.05
(-CH2-), 55.11 (-CH2-), 56.39 (CH25Ј), 73.41 (CH3Ј), 74.64 (CH2Ј),
83.08 (CH4Ј), 91.08 (CH1Ј), 102.05 (CH5), 140.87 (CH6), 150.23
(C2), 163.63 (C4) ppm.
5Ј-Octadecylamino-5Ј-deoxyuridine (10a): Compound 8a (267 mg,
0.37 mmol), THF (2 mL), TBAF (1  in THF; 1 mL, 1 mmol,
2.7 equiv.). Column chromatography MeOH/CHCl3, 1:30 to 1:10;
1
Rf = 0.1. Yield: 41 mg (22%). Colourless oil. H NMR (300 MHz,
CDCl3): δ = 0.85 (t, J = 6.8 Hz, 3 H, -CH2-CH3), 1.23 (s, 30 H,
-CH2-,), 1.56 (s, 2 H, -NH-CH2-CH2-), 2.76 (s, 2 H, -NH-CH2- submitted to silica gel chromatography (MeOH/CHCl3, 1:9; Rf =
CH2-), 3.08 (m, 2 H, -CH25Ј), 4.16 (m, 2 H, -CH-OH, 2Ј4Ј), 4.27 0.2). Product 11 was obtained as a pale-yellow oil. Yield: 51 mg
6066
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© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 6060–6069

Nucleosides with 5Ј-Fixed Lipid Groups
1
(30%). H NMR (300 MHz, CDCl3): δ = 0.88 (t, J = 6.8 Hz, 6 H,
β-D-1Ј,2Ј,3Ј-Tri-O-acetyl-5Ј-deoxy-5Ј-dioctadecylribofuranose (15):
-CH2-CH3), 1.25 (s, 60 H, -CH2-,), 1.74 (s, 4 H, -N-CH2-CH2-), 14 (950 mg, 1.3 mmol) was dissolved in acetic anhydride (8.5 mL,
3.31 (s, 3 H, -N-CH3), 3.46 (s, 4 H, -N-CH2-CH2-), 4.23 (m, 2 H, 90 mmol), AcOH (6.5 mL) and CHCl3 (1.5 mL). Sulfuric acid (con-
-CH25Ј), 4.45 (s, 1 H, -CH-OH, 4Ј), 4.67 (s, 1 H, -CH-OH, 2Ј), 4.83 centrated, 0.67 mL) was added, and the suspension was stirred at
(s, 1 H, -CH-OH, 3Ј), 5.79 (s, 1 H, CH5), 5.88 (s, 1 H, -CH-, 1Ј),
room temperature for 3.5 h. The orange solution was kept in the
7.91 (s, 1 H, CH6), 10.26 (s, 1 H, -NH-, 3) ppm. 13C NMR refrigerator at –24 °C overnight and then brought to room tem-
(75.5 MHz, CDCl3): δ = 14.12 (-CH2-CH3), 22.69 (-CH2-), 26.32
(-CH2-), 29.24 (-CH2-), 29.38 (-CH2-), 29.52 (-CH2-), 29.62
(-CH2-), 29.69 (-CH2-), 29.78 (-CH2-), 31.93 (-CH2-), 46.31
perature after 16 h. The reaction mixture was quenched with
NaOAc (1.8 g, 22 mmol). EtOH (25 mL) was added and evapo-
rated, and the cycle was repeated three times. The residue was dried
(-CH25Ј-), 50.11 (-N-CH3), 71.20 (CH3Ј), 72.53 (CH2Ј), 72.62 at 10 mbar (45 °C) for 50 min. CHCl3 (20 mL) was added, and the
(CH4Ј), 77.28 (CH1Ј), 102.82 (CH5), 130.92 (CH6) ppm. HRMS: organic phase was washed with tris buffer solution (1 , 3 mL) and
+
calcd. for C46H88N3O5 762.6719; found 762.6719.
brine (30 mL). The organic phase was separated and dried with
MgSO4. The filtrate was concentrated and purified by silica gel
chromatography (MeOH/DCM, 1:50; Rf = 0.3). Product 15 was
obtained as a yellow oil. Yield: 140 mg (14%). The major fractions
gave N-acetyldioctadecylamine as a fragmentation product (30%).
1H NMR (300 MHz, CDCl3): δ = 0.88 (t, J = 6.8 Hz, 6 H, -CH2-
CH3), 1.25 (s, 60 H, -CH2-,), 1.39 (m, 4 H, -N-CH2-CH2-), 2.05 (s,
3 H, -CO-CH3), 2.08 (s, 3 H, -CO-CH3), 2.11 (s, 3 H, -CO-CH3),
2.45 (m, 4 H, -N-CH2-CH2-), 2.66 (m, J = 7.4 Hz, 2 H, -CH25Ј),
4.29 (t, J = 6.6 Hz, 1 H, -CH-OH, 4Ј), 5.26 (m, 2 H, -CH-OH,
2Ј3Ј), 6.13 (s, 1 H, -CH-, 1Ј) ppm. 13C NMR (75.5 MHz, CDCl3):
δ = 14.10 (-CH2-CH3), 20.50 (-CO-CH3-), 21.05 (-CO-CH3-), 22.68
(-CH2-), 27.39 (-CH2-), 29.36 (-CH2-), 29.70 (-CH2-), 31.92
(-CH2-), 54.69 (-CH2-), 57.09 (CH25Ј), 72.73 (CH2Ј), 74.19 (CH3Ј),
80.57 (CH4Ј), 98.45 (CH1Ј), 169.08 (CO-CH3), 169.46 (CO-CH3),
169.70 (CO-CH3) ppm.
2Ј,3Ј-[Bis(tert-butyldimethylsilyl)]-5Ј-deoxy-3-(N-palmitoyl)-5Ј-(N-
palmitoyl-N-octylamino)uridine (12): Compound 8a (178 mg,
0.25 mmol) was dissolved in dry Et2O (10 mL) and DIPEA (60 µL,
0.35 mmol) was added. Under vigorous stirring, palmitoyl chloride
(100 µL, 0.33 mmol) was slowly added to the solution. TLC indi-
cated the final progress of the reaction after 10 min. The white
precipitate was filtered off, and the filtrate was concentrated in
vacuo. The resulting residue was submitted to silica gel column
chromatography (EtOAc/cyclohexane, 1:12; Rf = 0.1). The product
was obtained as a colourless oil. Yield: 103 mg (35%). 1H NMR
(300 MHz, CDCl3): δ = 0.02 (s, 3 H, -Si-CH3), 0.05 (s, 3 H, -Si-
CH3), 0.06 (s, 3 H, -Si-CH3), 0.07 (s, 3 H, -Si-CH3), 0.92–0.81 [m,
27 H, -C(CH3)3 and -CH2-CH3], 1.24 (s, 78 H,-CH2-), 1.58 (m, 4
H, -CO-CH2-CH2- and -NH-CH2-CH2-), 1.71 (m, 2 H, -CO-CH2-
CH2-), 2.29 (t, J = 7.2 Hz, 2 H, -CO-CH2-), 2.77 (t, J = 7.2 Hz, 2
H,-CO-CH2-), 3.26 (t, J = 6.8 Hz, 2 H, -NH-CH2-), 3.81–3.35 (m,
2 H, CH2-5Ј), 3.89 (m, 1 H, -CH-OH, 4Ј), 4.09 (m, 1 H, -CH-OH,
2Ј), 4.27 (m, 1 H, -CH-OH, 3Ј), 5.76 (d, J = 6.0 Hz, 1 H, -CH-,
1Ј), 5.79 (d, J = 8.3 Hz, 1 H, CH5), 7.64 (d, J = 8.3 Hz, 1 H, CH6)
ppm. 13C NMR (75.5 MHz, CDCl3): δ = –4.75 (-Si-CH3), –4.68
(-Si-CH3), –4.55 (-Si-CH3), –4.36 (-Si-CH3), 14.22 (-CH2-CH3),
18.01 [-C(CH3)3], 18.10 [-C(CH3)3], 22.79 (-CH2-), 23.50 (-CH2-),
25.63 (-CH2-), 25.81 [-C(CH3)3], 25.84 [-C(CH3)3], 26.92 (-CH2-),
28.72 (-CH2-), 28.92 (-CH2-), 29.38 (-CH2-), 29.47 (-CH2-), 29.53
(-CH2-), 29.57 (-CH2-), 29.63 (-CH2-), 29.70 (-CH2-), 32.03
(-CH2-), 33.12 (-CH2-), 40.67 (-CH2-), 46.81 (-CH2-), 47.81
(CH25Ј), 73.74 (CH3Ј), 74.17 (CH2Ј), 82.47 (CH4Ј), 90.22 (CH1Ј),
102.66 (CH5), 140.81 (CH6), 149.03 (C2), 161.71 (C4), 173.97
(-CH2-CO-N-), 176.03 (-CH2-CO-N-) ppm. HRMS: calcd. for
2Ј,3Ј-Di-O-Acetyl-5Ј-deoxy-5Ј-dioctadecyladenosine (16): Com-
pound 15 (140 mg, 0.18 mmol) and adenine (190 mg, 1.44 mmol,
8 equiv.) were dissolved in dry MeCN (6 mL) and dry DCM
(5 mL). SnCl4 (0.36 mL, 3.1 mmol, 17 equiv.) was added to the sus-
pension, and the mixture was stirred under an atmosphere of argon
at room temperature for 20 h. The solvent was evaporated and
DCM (20 mL), water (20 mL) and triethylamine (2 mL) were
added. The organic phase was separated and washed with brine
(10 mL). The aqueous phases were extracted with DCM, and the
combined organic phase was evaporated to dryness. The residue
was treated with toluene and evaporated, and the cycle was re-
peated three times. The crude material was purified by silica gel
chromatography (MeOH/DCM, 1:14 + Et3N, 1 %; Rf = 0.3). Prod-
1
uct 15 was obtained as a brown oil. Yield: 70 mg (45%). H NMR
(300 MHz, CDCl3): δ = 0.89 (t, J = 6.9 Hz, 6 H, -CH2-CH3), 1.26
(s, 60 H, -CH2-,), 1.42 (m, 4 H, -N-CH2-CH2-), 2.07 (s, 3 H, -CO-
CH3), 2.14 (s, 3 H, -CO-CH3), 2.48 (m, 4 H, -N-CH2-CH2-), 2.84
(m, 2 H, -CH25Ј), 4.30 (m, 1 H, -CH-OH, 4Ј), 5.57 (m, J = 5.1 Hz,
1 H, -CH-OH, 3Ј), 5.92 (m, J = 5.5 Hz, 1 H, -CH-OH, 2Ј), 6.14
(d, J = 5.5 Hz,1 H, -CH-, 1Ј), 8.01 (s, 1 H, CH2), 8.36 (s, 1 H,
CH8) ppm. 13C NMR (75.5 MHz, CDCl3): δ = 14.12 (-CH2-CH3),
20.45 (-CO-CH3-), 20.61 (-CO-CH3-), 22.69 (-CH2-), 27.45
(-CH2-), 29.37 (-CH2-), 29.71 (-CH2-), 31.93 (-CH2-), 55.05
(-CH2-), 55.96 (CH25Ј), 72.06 (CH3Ј), 72.92 (CH2Ј), 81.69 (CH4Ј),
85.99 (CH1Ј), 120.04 (C5), 149.79 (C4), 153.27 (CH2), 155.49 (C6),
169.43 (CO-CH3), 169.64 (CO-CH3) ppm. HRMS: calcd. for
+
C71H138N3O7Si2 1201.0068; found 1201.0085.
5Ј-Dioctadecyl-5Ј-deoxy-2Ј,3Ј-O-isopropylidene-1-O-methyl-β-D-
ribofuranose (14): Starting material 13 (400 mg, 1.10 mmol) was
dissolved in a melt of dioctadecylamine (1.26 g, 2.4 mmol) and
stirred at 90 °C for 2 d and 5 h. When TLC indicated the final
progress of the reaction, CHCl3 (40 mL) was added, and the solu-
tion was quickly cooled to room temperature. Silica gel was added,
and the solvent was evaporated. The powder was submitted to silica
gel column chromatography (EtOAc/cyclohexane, 1:4; Rf = 0.8).
1
Product 14 was obtained as a yellow oil. Yield: 500 mg (59%). H
NMR (300 MHz, CDCl3): δ = 0.87 (t, J = 6.6 Hz, 6 H, -CH2-CH3),
1.26 (s, 60 H, -CH2-,), 1.30 [s, 3 H, C(CH3)2], 1.40 (s, 4 H, -N-CH2-
CH2-), 1.47 [s, 3 H, C(CH3)2], 2.39 (m, J = 6.4 Hz, 4 H, -N-CH2-
CH2-), 2.47 (m, J = 7.5 Hz, 2 H, -CH2-,5Ј), 3.30 (s, 3 H, -O-CH3),
4.20 (t, J = 8.1 Hz, 1 H, -CH-OH, 4Ј), 4.55 (d, J = 6.0 Hz, 1 H,
-CH-OH, 2Ј), 4.67 (d, J = 5.9 Hz, 1 H, -CH-OH, 3Ј), 4.92 (s, 1 H,
-CH-, 1Ј) ppm. 13C NMR (75.5 MHz, CDCl3): δ = 14.08 (-CH2-
+
C50H91N6O5 855.7045; found 855.7037.
5Ј-Dioctadecyl-5Ј-deoxyadenosine (17): Compound 16 (300 mg,
0.35 mmol) was dissolved in DCM (12 mL) and MeOH (15 mL).
The solution was cooled under an atmosphere of argon to –72 °C
and gaseous ammonia was condensed (approximately 40 mL). The
solution was kept at –70 °C for 1 h and then the temperature was
CH3), 22.68 (-CH2-), 24.95 [C(CH3)2], 26.45 [C(CH3)2], 27.03 gradually raised to 20 °C overnight. When ammonia finally disap-
(-CH2-), 27.45 (-CH2-), 29.39 (-CH2-), 29.73 (-CH2-), 31.94 peared, argon was led through the equipment and the solvent was
(-CH2-), 54.58 (-CH2-), 54.72 (O-CH3), 57.55 (CH25Ј), 83.20 evaporated. The crude material was purified by silica gel
(CH3Ј), 85.24 (CH2Ј4Ј), 109.55 (CH1Ј), 111.90 [C(CH3)2] ppm. chromatography (MeOH/CHCl3, 1:5; Rf = 0.3). Product 17 was
Eur. J. Org. Chem. 2007, 6060–6069
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6067

J. Liebscher et al.
FULL PAPER
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obtained as a colourless solid. M.p. 60–61 °C. Yield: 98 mg (36%).
1H NMR (300 MHz, CDCl3): δ = 0.89 (t, J = 6.8 Hz, 6 H, -CH2-
CH3), 1.26 (s, 60 H, -CH2-,), 1.45 (m, J = 6.2 Hz,4 H, -N-CH2-
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90.23 (CH1Ј), 119.90 (C5), 149.00 (C4), 152.64 (CH2), 155.55 (C6)
+
ppm. HRMS: calcd. for C46H87N6O3 771.6834; found 771.6833.
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Received: June 6, 2007
Published Online: October 23, 2007
Eur. J. Org. Chem. 2007, 6060–6069
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6069