Nucleoterpenes of thymidine and 2′-deoxyinosine: Synthons for a biomimetic lipophilization of oligonucleotides

Article abstract of DOI:10.1002/cbdv.201100338

2′-Deoxyinosine (1) and thymidine (7) were N-alkylated with geranyl and farnesyl moieties. These hydrophobic derivatives, 3a and 3b, and 9a and 9b, respectively, represent the first synthetic biomimetic nucleoterpenes and were subsequently 5′-protected an

Full text of DOI:10.1002/cbdv.201100338

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
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Nucleoterpenes of Thymidine and 2’-Deoxyinosine: Synthons for a
Biomimetic Lipophilization of Oligonucleotides
by Karl Kçstler, Emma Werz, Edith Malecki, Malayko Montilla-Martinez, and Helmut Rosemeyer*
Organic Chemistry I – Bioorganic Chemistry, Institute of Chemistry of New Materials, Fachbereich
Biologie/Chemie, University of Osnabrꢀck, Barbarastrasse 7, D-49076 Osnabrꢀck
In memoriam Prof. Dr. Friedrich Cramer, Gçttingen
2’-Deoxyinosine (1) and thymidine (7) were N-alkylated with geranyl and farnesyl moieties. These
hydrophobic derivatives, 3a and 3b, and 9a and 9b, respectively, represent the first synthetic biomimetic
nucleoterpenes and were subsequently 5’-protected and converted into the corresponding 3’-O-
phosphoramidites, 5a and 5b and 11a and 11b, respectively. The latter were used to prepare a series of
lipophilized oligonucleotide dodecamers, a part of which were additionally labelled with indocarbocya-
nine fluorescent dyes (Cy3 or Cy5), 18–23. The insertion of the lipooligonucleotides into, as well as
duplex formation at artificial lipid bilayers was studied by single-molecule fluorescence spectroscopy and
fluorescence microscopy.
1. Introduction. – With the discovery of gene silencing – also of human genes – by
short nucleic acids (siRNA) [1][2], a new therapeutic principle has evolved. This
culminated in the synthesis of the so-called antagomires, short modified oligomers
which are built-up from 2’-O-methyl-b-d-ribonucleosides, and which carry at both
termini several phosphorothioate internucleotide linkages as well as a cholesterol tag at
the 5’-end. Particularly, the latter modification renders the oligomer permeable for the
cell membrane [3]. However, it has been reported that already one cholesterol moiety
loads the oligomer synthesis, and its purification and handling with difficulties [4].
For these reasons, we have been searching for alternative methods for a less strong
and stepped lipophilization of oligonucleotides [5]. For this purpose, a series of base-
alkylated 2’-deoxyinosine- as well as 2’-deoxythymidine 2-cyanoethyl phosphorami-
dites, 5a and 5b, and 11a and 11b, respectively, were prepared and used for the
preparation of 5’-lipophilized oligonucleotides. Principally, these phosphoramidites can
be incorporated at each position of a growing nucleic acid chain by a conventional
solid-phase synthesis, so that the oligonucleotide can be hydrophobized at each
predetermined locus (Fig. 1).
The positioning of the lipophilic side chain was performed at a nucleobase atom
which is involved in a WatsonꢀCrick base pairing so that the resulting DNA building
blocks are pure hydrophobization tools with a basic nucleoside structure. As side
chains, acyclic mono- and sesquiterpenes, namely geranyl and farnesyl residues, were
chosen because such residues are used in post-translational prenylation of various
proteins in order to embed them within biological membranes [6].
ꢁ 2013 Verlag Helvetica Chimica Acta AG, Zꢀrich

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Fig. 1. Different positionings of nucleolipids within a nucleic acid
The only naturally-occurring meronucleoterpene known so far is avinosol and has
been isolated from the sponge Dysidea sp., near Papua New Guinea in 2006. This N(1)-
alkylated 2’-deoxyinosine derivative, carrying a benzene-1,4-diole and a sesquiterpene
residue, has been shown to possess promising anti-angiogenic and antimetastatic
properties [7].
2. Results and Discussion. – 2.1. Synthesis. For the preparation of N-geranylated and
N-farnesylated nucleoterpenes of 2’-deoxyinosine (1) and thymidine (7), respectively,
base-catalyzed alkylation in DMF with the corresponding terpenyl bromides was
chosen [8]. As it has been reported that, under such conditions, also O-alkylation of the
sugar OH groups as well as of the base occur as side reactions [9], we prepared first the
1,1,3,3-tetraisopropyldisiloxane-sugar derivatives of both 2’-deoxynucleosides, 2 and 8,
respectively. This so-called Markiewicz silyl clamp [10] can be easily introduced and
cleaved off with Bu4NF under mild conditions. Subsequent deprotonation of
compounds 2 and 8 was performed under various reaction conditions with respect to
solvent (DMF, MeCN) and base (NaH, K2CO3). In all cases, however, several reaction
products were formed upon alkylation. In the case of 2’-deoxyinosine, a solidꢀliquid
phase-transfer alkylation in the presence tris[(2-methoxyethoxy)ethyl]amine (MeCN,
K2CO3) was conducted but proved to be unsuccessful. The reason for the unsteady
alkylations of the silyl-protected educts might be either a shift of the silyl clamp [11] or
a silyl bridging of two nucleoside molecules.
As a consequence, both unprotected 2’-deoxynucleosides, 1 and 7, were alkylated
with geranyl bromide and farnesyl bromide (DMF, K2CO3). In the case of 2’-
deoxyinosine (1), a reaction time of 24 h at room temperature was sufficient for a

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
moderate yield (50–75%) of the products 3a and 3b, while for thymidine (7) 48 h and
408 were necessary to obtain the corresponding products 9a and 9b in comparable
yields. In Table 1, the lipophilicities of the thymidine derivatives in form of their
calculated log P values and their retention times in RP-18 HPLC are compiled.
Table 1. Calculated log P Values and Retention Times of Thymidine Derivatives in RP-18 HPLC (for
details, see Exper. Part)
Compound
log P Value
tR [min]
7
9a
9b
ꢀ1.11ꢁ0.49
4.57ꢁ0.61
6.60ꢁ0.64
1.89
3.34
7.85
Compounds 3a and 3b, and 9a and 9b represent, to our knowledge, the first
examples of synthetic nucleoterpenes. All of these compounds were characterized by
1H- and 13C-NMR, and UV spectroscopy as well as by elemental analyses. Moreover,
ESI mass spectra were recorded in the presence of 2% aqueous HCOOH. In all cases,
signals corresponding to the appropriate dimeric molecules, e.g., 6 and 12, were
detected. The tendency of dimerization is most pronounced in the case of the 2’-
deoxyinosine nucleoterpenes.
Of the thymidine nucleoterpenes, 9a and 9b, the farnesylated compound 9b
exhibited a stronger dimerization tendency than the geranylated derivative 9a (for an
example, see Fig. 2).
Scheme 1 displays a plausible mechanism for a dimerization of a farnesylated
nucleoterpene, showing only the formation of one conceivable product, namely an end-
to-end dimerization. It should be kept in mind, however, that also the formations of
other products, i.e., either an end-to-end reaction with an intermediary hydrid
rearrangement or non-end-to-end dimerization reaction, are possible.
To rule out that the synthesized nucleoterpenes, particularly the 2’-deoxyinosine
derivatives 3a and 3b, for which predominantly dimers were detected in their ESI mass
spectra, are principally dimeric molecules, as an example, a gel-permeation chroma-
tography (GPC) was performed (THF) with compound 3b in order to determine the
molecular weight of the product. Fig. 3 displays the elution profile of a typical
chromatographic run using a light-scattering detection.
This figure clearly shows that two peaks are detectable, one of which (484 g/mol)
corresponds to the calculated molar mass of compound 3b (456.6 g/mol). However, the
profile also exhibits the presence of high-molecular-weight aggregates with a mean
molecular weight of 1,571,000 g/mol, corresponding to the 3,440-fold of the molar mass
of the monomer. This result points to the occurrence of probably inverse micelles or
liposomes with the nucleoside residues forming an inner core, and the farnesyl residues
protruding into the solvent lattice.
Subsequently, the nucleoterpene 3b was labelled with two different dyes: i) with a
fluorenyl moiety (Fmoc) via a glycine spacer and ii) with Texas Red. Both reactions
were performed on a small scale. First, compound 3b was reacted at its 5’-OH group
with N-{[(9H-fluoren-9-yl)methoxy]carbonyl}glycine (13) by using a Steglich esterifi-
cation (DCC, DMAP) [12] (Scheme 2). TLC Analysis indicated the formation of three

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Fig. 2. ESI-MS of compound 3b (ionization with 2% aq. HCOOH)
products which could be separated by chromatography. All compounds were
1
characterized by H- and 13C-NMR as well as by UV/VIS spectroscopy. The fastest-
migrating compound was identified as the 3’,5’-difluorenylated derivative 14, and the
others were the 5’- and the 3’- labelled compounds, 15 and 16, respectively (Scheme 2).
1
Fig. 4 displays the H-NMR low-field regions of the three derivatives 14–16.
In the 1H-NMR regions depicted in Fig. 4, the Fmoc resonances of 14 exhibit double
the integral values as the signals of HꢀC(2) and HꢀC(8), establishing the double
labelling.
Next, compound 3b was coupled with sulforhodamin-101-sulfonyl chloride (Texas
Red). After extraction and silica gel chromatography, the product 17 was obtained as a
deep black, amorphous material. Fig. 5 displays the UV/VIS spectra of educts as well as
of the product.
The UV/VIS spectrum (Fig. 5) of the Texas Red-labelled derivative resembles
clearly the spectrum of the dye.

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Scheme 1
Fig. 3. Elution profile of a gel-permeation chromatography of compound 3b in THF

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
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Scheme 2
2.2. Oligonucleotides. The phosphoramidites 5b and 11a, and 11b were used to
prepare a series of lipophilized oligonucleotides, and their insertion into artificial lipid
bilayers was studied [13–19]. The oligonucleotides synthesized and characterized by
MALDI-TOF-MS are compiled in Table 2.
The oligonucleotides 18–20 contain – besides a nucleoterpene (i.e., 3b or 9a, and
9b) – an indocarbocyanine dye in the 5’-(n–1) position which was introduced via its

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Fig. 4. 1H-NMR Low-field region of compounds 14–16 (from left to right)
Fig. 5. UV/VIS Spectrum of compound 17 as well as of the starting materials, Texas Red, and 3b

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Table 2. Sequences and MALDI-TOF Data of Oligonucleotides
Oligonucleotide (sequence, formula No, abbreviation)
[MþH]þ (calc.) [MþH]þ (found)
5’-d(3b-Cy3-TAG GTC AAT ACT)-3’
5’-d(9b-Cy3-TAG GTC AAT, ACT)-3’
5’-d(9a-Cy3-TAG GTC AAT, ACT)-3’
5’-d(9b-TAG GTC AAT, ACT)-3’
5’-d(9b-ATC CAG TTA TGA)-3’
5’-d(Cy5-AGT ATT GAC CTA)-3’
18
19
20
21
22
23
KK1
EW1
EW2
EW3
EW4
EW5
4671.6
4660.6
4592.5
4153.0
4153.0
4178.1
4671.1
4659.5
4590.5
4152.3
4152.0
4178.4
phosphoramidite. The oligomers 21 and 22 carry the thymidine terpene 9b at the 5’-end,
while the oligomer 23 carries a Cy5 fluorophore label and is complementary to the
oligonucleotide 21 in an antiparallel strand orientation, but not to 22.
First, the insertion of the oligonucleotides 18–20 (i.e., KK1, EW1, and EW2, resp.)
was tested at artificial bilayer membranes composed of 1-palmitoyl-2-oleyl-sn-glycero-
3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocho-
line (POPC; 8 :2 (w/w)) in decane (10 mg/ml) in a set-up shown in Fig. 6 (see Exper.
Part and [20][21] for the detailed construction). From Figs. 7 and 8, it can be clearly
seen that all Cy3-labelled lipo-oligonucleotides are inserted into the lipid bilayer, but to
a different extent and with different stabilities towards perfusion. It is obvious that the
oligomer carrying the N(3)-geranyl-thymidine nucleoterpene was inserted to the
highest extent, but is washed out by one perfusion already to ca. 50%. The oligomers
carrying farnesylated nucleosides at their 5’-end are significantly more stable towards
perfusion.
Next, we studied the duplex formation between bilayer-immobilized lipo-oligonu-
cleotides 21 and 22 (i.e., EW3 and EW4, resp.) with a Cy5-labelled oligomer 23 (EW5)
which is complementary only to 21 (EW3) but not to 22 (EW4). Fluorescence
microscopy (cf. Figs. 9 and 10 and Fig. 11) clearly evidence duplex formation as
expected for 21·23 but not for 22·23 [20].
Furthermore, the diffusion times (tD [ms]) of the duplex 21·23 were measured,
both without and in the presence of an artificial bilayer (Table 3). For the
determination of the free diffusion times, the corresponding oligomer duplex solution
(50 nm) was diluted so that there was only a single fluorescent molecule in the confocal
measuring volume (ca. 1 fl). Each measurement was repeated ten times for 30 s. To
determine the diffusion times of the lipophilized oligonucleotide duplex 21·23 in the
presence of a bilayer, two measuring positions, one above (in solution but in close
proximity to the bilayer) and one within the bilayer were chosen. Each measurement
was performed i) by recording reference data of a stable, blank bilayer, ii) after
formation of the oligonucleotide duplex and a subsequent 30-min incubation, followed
by recording of the data, iii) recording further data after perfusion of the Bilayer Slide.
The results compiled in Table 3 indicated that the diffusion of oligomer 23 as well as of
the duplex 21·23 was fast. However, the broad diffusion-time distribution of the duplex
21·23 indicated aggregate formation of heterogeneous size. In the close proximity of a
stable bilayer, the diffusion time increased approximately by a factor of 10. Probably,
the molecules aggregate, and the aggregates interact partly with the bilayer. The

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Fig. 6. Experimental setup: a) schematic drawing of the laser scanning microscope, the optical transparent
microfluidic bilayer slide, and the lipid bilayer with incorporated double-tailed nucleolipids. The bilayer
slide encloses two microfluidic channels (cis and trans) which are separated by a thin medical-grade
polytetrafluoroethylene (PTFE) foil. This foil hosts a central 100-mm aperture which is located 120 mm
above the coverslip and thus within the working distance of high NA (numeric apeture) objectives. It is
the only connection between the trans and cis channel. When a lipid soln. is painted across the aperture a
bilayer is formed spontaneously. Electrodes in the cis and trans channels allow an online monitoring of
the bilayer integrity as well as electrophysiological recordings. b) Stage unit of the ꢀIonovation Explorerꢁ
mounted on a standard inverted fluorescence microscope. The computer controlled perfusion unit is a side
board and is not shown. c) ꢀIonovation Bilayer Slideꢁ, a disposable, optical transparent microfluidic
sample carrier with perfusion capabilities. The ꢂBilayer Portꢃ gives direct access to the lipid bilayer, while
both sides of the bilayer can be perfused via the cis and trans channel. Calibration wells allow optical
control experiments when needed.
diffusion time of the bilayer-immobilized DNA duplex increased further by a factor of
10.
Further studies with other types of lipophilic nucleoside phosphoramidites, their
incorporation into oligonucleotides, and their insertion into and transfer through
artificial bilayer membranes are in progress.
The authors gratefully acknowledge financial support by the B. Braun AG (D-Melsungen) as well as
the Bundesministerium fꢀr Wirtschaft (FKZ: KF 2369401 S B9 and FKZ 2369501 S B9). We are grateful
to Prof. Dr. Richard Wagner (Biophysics) and Prof. Dr. Uwe Beginn (Organic Chemistry I, Organic
Materials Chemistry), both University of Osnabrꢀck, for excellent laboratory facilities, and all members
of the Ionovation GmbH, D-Osnabrꢀck, for valuable discussions and support. We also thank Mrs.
Marianne Gather Steckhan for NMR spectra, Dr. Stefan Walter for the HR-ESI mass spectra, Mrs. Anja

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
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Fig. 7. Protocol of the insertion of the oligonucleotides 18–20 into an artificial bilayer. a) z-Scan of an
empty bilayer; both channels (cis and trans) were perfused (30 s, 1.1 ml/min, each). b) Pixel-resolved 3D
scan of an empty bilayer. c) z-Scan after addition of 18 (4 ml, 50 nm) to the cis channel and 25 min of
incubation. d) z-Scan after first perfusion of the cis compartment (60 s, 1.1 ml/min). e) Tilted 3D view
onto the bilayer, filled with 18, after first perfusion. f) Pixel-resolved 3D scan of the bilayer, filled with 18,
after first perfusion. g) z-Scan after second perfusion of the cis compartment (60 s, 1.1 min/ml). h) Pixel-
resolved 3D scan of the bilayer, filled with 18, after second perfusion. i) z-Scan of the bilayer after
addition of 19 (4 ml, 50 nm) to the cis channel and 25 min of incubation. j) z-Scan of the bilayer after first
perfusion of the cis compartment (60 s, 1.1 ml/min). k) z-Scan of the bilayer after second perfusion of the
cis compartment (60 s, 1.1 min/ml). l) z-Scan of the bilayer after third perfusion of the cis compartment
(60 s, 1.1 min/ml). m) Tilted 3D view onto the empty bilayer. n) z-Scan of the bilayer after addition of 20
(4 ml, 50 nm) to the cis channel and 25 min of incubation. o) z-Scan of the bilayer, filled with 20, after first
perfusion of the cis compartment (60 s, 1.1 ml/min). p) z-Scan of the bilayer after second perfusion of the
cis compartment (60 s, 1.1 min/ml).

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Fig. 7 (cont.)
Schuster for elemental analyses, Mrs. Sandra Schwidtke for gel-permeation chromatography, and Mrs.
Petra Bçsel for the formula drawings.

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Fig. 8. Relative bilayer brightness as a function of the perfusion number
Experimental Part
General. All chemicals were purchased from Sigma-Aldrich (D-Deisenhofen) or from TCI – Europe
(B-Zwijndrecht). Solvents were of laboratory grade and were distilled before use. Column chromatog-
raphy (CC) and flash chromatography (FC, 0.5 bar) were performed on silica gel 60. TLC: aluminum
sheets, silica gel 60 F254; 0.2 mm layer (Merck, Germany). M.p. Bꢂchi SMP-20, uncorrected. UV Spectra:
Cary 1E spectrophotometer (Varian, D-Darmstadt). NMR Spectra (incl. 1H-DOSY spectra): AMX-500
1
spectrometer (Bruker, D-Rheinstetten); H: 500.14, 13C: 125.76, and 31P: 101.3 MHz; chemical shifts in
ppm rel. to TMS as internal standard for 1H and 13C nuclei, and external 85% H3PO4; J values in Hz. ESI-
MS: Bruker Daltronics Esquire HCT instrument (Bruker Daltronics, D-Leipzig); ionization performed
with a 2% aq. HCOOH soln. Elemental analyses (C, H, N) of crystallized compounds: VarioMICRO
instrument (Fa. Elementar, D-Hanau). Gel-permeation chromatography (GPC): three columns with a
light-scattering detector (Dawn Helios) and an RI detector (Optilab rEX, Wyatt), the results were
evaluated and displayed with the program ASTRA 5.3.4, version 14. log P Values were calculated using
the program suite ChemSketch (version 12.0, provided by Advanced Chemistry Developments Inc.;
Toronto, Canada; http://www.acdlabs.com). Oligonucleotides were synthesized, purified, and charac-
terized (MALDI-TOF-MS) by Eurogentec (Eurogentec S. A., Liege Science Park, B-Seraing).
Oligonucleotide Incorporation in Artificial Bilayers [20]. The incorporation of the oligonucleotides
18–22 in artificial lipid bilayers was performed using a lipid mixture of 1-palmitoyl-2-oleyl-sn-glycero-3-
phosphoethanolamine (POPE) and 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC; 8 :2
(w/w); 10 mg/ml of decane). The horizontal bilayers were produced automatically within the ꢂBilayer
Slidesꢃ using an ꢂIonovation Explorerꢃ (Ionovation GmbH, D-Osnabrꢀck). After pre-filling with buffer
(250 mm KCl, 10 mm MOPS/Tris, pH 7), the ꢂBilayer Slideꢃ was inserted into the stage unit of the
ꢂIonovation Explorerꢃ. The Ag/AgCl electrodes were mounted, and, after addition of 0.2 ml of POPE /
POPC to the cis compartment, the automated bilayer production was started. The ꢂIonovation Explorerꢃ
uses a modified painting technique, where the airꢀwater interface paints the lipid across the aperture.

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Fig. 9. Chronological protocol of duplex formation of the oligonucleotide EW3 with the complementary,
Cy3-labelled oligomer EW5 at an artificial lipid bilayerꢀaq. buffer boundary, followed by a perfusion. a)
z-Scan of an empty bilayer. b) Tilted 3D view on an empty bilayer. c) Pixel-resolved 3D scan on an empty
bilayer. d) z-Scan after addition of the oligomer EW3 (6 ml, 500 nm) to the cis compartment and 60 min
of incubation. e) z-Scan after addition of the Cy5-labelled oligomer (6 ml, 50 nm) to the cis compartment
and 60 min of incubation. f) z-Scan after perfusion of the cis compartment (30 s, 1.1 ml/min). g) Tilted
3D view on the bilayer as in (f). h) Pixel-resolved 3D scan of the bilayer as in (f).

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
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Fig. 10. Chronological control experiment with the non-complementary oligonucleotides EW4 and EW5.
a) z-Scan of an empty bilayer. b) Tilted 3D view on an empty bilayer. c) z-Scan after addition of the
oligomer EW4 (6 ml, 500 nm) to the cis compartment, and 50 min of incubation and perfusion (30 s,
1.1 ml/min). d) z-Scan after addition of the Cy5-labelled oligonucleotide (6 ml, 50 nm) to the cis
compartment and 40 min of incubation. e) z-Scan after perfusion of the cis compartment (30 s, 1.1 ml/
min). f) z-Scan after further incubation for 20 min and two perfusions of the cis compartment (60 s,
1.1 ml/min, each).
The bilayer formation was monitored via capacitance measurements. When a stable bilayer was
established (< 50 pF), the corresponding oligonucleotide soln. was injected into the cis compartment of
the ꢂBilayer Slideꢃ. During the incubation time of 25 min, the bilayer integrity was monitored by the
ꢂIonovation Explorerꢃ through continuous capacitance measurements.
A confocal laser scanning microscope (Insight Cell 3D, Evotec Technologies GmbH, D-Hamburg),
equipped with a 635-nm emitting laser diode (LDH-P-635, PicoQuant GmbH, D-Berlin), a 40ꢂH2O-
immersion objective (UApo 340, 40ꢂ ; NA (numeric aperture) 1.15, Olympus, Tokyo, Japan), and an
Avalanche photodiode detector (SPCM-AQR-13-FC, Perkin-Elmer Optoelectronics, Fremont, CA,
USA), was used for the optical measurements. Fluorescence irradiation was obtained with an excitation
laser power of 200ꢁ5 mW. 2D- and 3D scans were performed by scanning the confocal laser spot in x,y
direction with a rotating beam scanner and movement of the objective in Z direction. The movement in

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Fig. 11. Relative bilayer brightness
Table 3. Diffusion times (tD [ms]) of 21·23 without and in the Presence of a Lipid Bilayer. Location: 1,
bilayer; 2, solution in close proximity to the bilayer.
Position
tD [ms]
23
Diffusion (in solution without bilayer)
0.24ꢁ0.1
0.12ꢁ0.1
26.6ꢁ2.0
2.39ꢁ0.3
21·23
21·23
21·23
1
2
all directions was piezo-controlled which allows a nm-precise positioning. For the 2D pictures (z-scans;
Figs. 7, 9, and 10, resp.), the confocal plane was moved in 100-nm steps.
From the fluorescence signals of single molecules which pass the laser spot, the diffusion constants
can be calculated by means of fluorescence correlation analysis. The diffusion times of the fluorescent
oligonucleotides within and in the proximity of the bilayer were measured at overall five different
positions above, below, and within the layer (Fig. 6,a). At each point five 30-s measurements were
conducted. In summary, each measurement protocol consisted of: i) a reference scan of the stable
(empty) bilayer; ii) addition of the sample with 30-min of incubation, followed by a scan series; iii)
additional scan series, each after a 1st and 2nd perfusion (60 s each).
Subsequently, the cyanine-5-labelled oligonucleotide 23 (50 nm, 6 ml) was injected into the cis
compartment of the ꢂBilayer Slidesꢃ containing either membrane-bound 21 or membrane-bound 22. After

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
55
an equilibration time of 60 min, the cis channel was perfused repeatedly for 30 s (1.1 ml/min), and the
bilayers were inspected by confocal fluorescence microscopy.
RP-18 HPLC. RP-18 HPLC was carried out with a 250ꢂ4 mm RP-18 column (Merck, Germany) on
a Merck-Hitachi HPLC apparatus with one pump (Model 655A-12) connected with a proportioning
valve, a variable wavelength monitor (Model 655 A), a controller (Model L-5000), and an integrator
(Model D-2000). Solvent: MeCN/0.1m Et3NHþAcꢀO (35 :65 (v/v), pH 7.0).
1,9-Dihydro-9-[(6aR,8R,9aS)-tetrahydro-2,2,4,4-tetra(propan-2-yl)-6H-furo[3,2-f][1,3,5,2,4]trioxa-
disilocin-8-yl]-6H-purin-6-one (2). Anh. 2’-deoxyinosine (1; 1.01 g, 4 mmol) was suspended in dry
pyridine (40 ml), and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (1.39 g; 4.4 mmol) was added under
moisture exclusion. After stirring for 24 h at r.t., the solvent was evaporated, and the residue was
partitioned between AcOEt and H2O (80 ml, 1:1 (v/v)). The org. layer was washed twice with 1m HCl
(80 ml), followed by H2O, conc. aq. HaHCO3, and brine (80 ml, each). After drying (Na2SO4, 1 h) the
solvent was evaporated, and the residue was chromatographed (column: 6ꢂ10 cm, CHCl3/MeOH 9 :1
(v/v)). From the main zone, 2 (1.96 g, 99%) was isolated. Colorless amorphous material. M.p. 2108. Rf
(CHCl3/MeOH 9 :1 (v/v)) 0.56. UV (MeOH): 244 (12,250); e260, 7,500. 1H-NMR ((D6)DMSO): 12.33 (s,
NH); 8.18 (s, HꢀC(2)); 7.96 (s, HꢀC(8)); 6.27 (dd, J(1’,2’a)¼8.0, J(1’,2b’ )¼3.0, HꢀC(1’)); 4.94 (ddd,
J(3’,2’a)¼J(3’,2a’ )¼J(3’,4’)¼7.5, HꢀC(3’)); 3.92–3.91 (m, CH2(5’)); 3.93–3.80 (m, HꢀC(4’)); 2.80
(ABdd, JAB ¼ꢀ 13.0, J(2’b,1’)¼7.5, J(2b’ ,3’)¼3.0, HbꢀC(2’)); 2.60–2.53 (m, HaꢀC(2’)); 1.08–1.01 (m,
4 Me2CH). 13C-NMR ((D6)DMSO): 156.5 (C(6)); 147.4 (C(4)); 145.5 (C(2)); 138.7 (C(8)); 124.7 (C(5));
84.5 (C(1’)); 82.1 (C(4’)); 71.2 (C(3’)); 62.4 (C(5’)); 38.7 (C(2’)); 17.1 (8 Me); 12.33 (4 CH). Anal. calc. for
C22H38N4O5Si2 (494.732): C 53.41, H 7.74, N 11.32; found: C 53.21, H 7.78, N 11.23.
2’-Deoxy-1-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]inosine (3b). Anh. 1 (1.01 g, 4 mmol)
was suspended in anh., amine-free DMF and heated on a H2O bath (558). Then, anh. K2CO3 (1.44 g,
10.4 mmol) was added, and the mixture was stirred for 10 min. After cooling to r.t., farnesyl bromide
(1.32 g, 4.4 mmol) was added dropwise under N2. After stirring for 24 h at r.t., the solvent was
evaporated, and the residue was dried in high vacuum. Chromatography (column: 6ꢂ14 cm; CHCl3/
MeOH 9 :1 (v/v)) gave one main zone from which after evaporation of the solvent 3b (1.28 g, 74%) was
isolated. Amorphous solid. Rf (CHCl3/MeOH 95 :5, (v/v)) 0.42. log P: 3.40ꢁ0.94. UV (MeOH): 250
(10,150); e260 7,000. 1H-NMR ((D6)DMSO): 8.34 (s, HꢀC(2)); 8.30 (s, HꢀC(8)); 6.28 (dd, J(1’,2’a)¼
J(1’,2’b)¼7.0, HꢀC(1’)); 5.28 (t, J(2’’,1’’)¼7.5, HꢀC(2’’)); 5.35 (d, J(OH3’,3’)¼7.5, HOꢀC(3’)); 5.03–4.98
(m, HꢀC(6’’), HꢀC(10’’)); 4.91 (t, J(OH5’,5’)¼6.5, HOꢀC(5’)); 4.60 (d, J(1’’,2’’)¼7.5, CH2(1’’)); 4.38
(ddd, J(3’,2b)¼9.0, J(3’,2a)¼J(3’,4’)¼3.5, HꢀC(3’)); 3.87–3.85 (m, HꢀC(4’)); 3.60–3.49 (m, HꢀC(5’));
2.63–2.58 (m, HbꢀC(2’)); 2.28 (ABdd, JAB ¼ꢀ 13.5, J(2’a,1’)¼6.5, J(2a’ ,3’)¼3.5, HaꢀC(2’)); 2.05–2.02 (m,
CH2(8’’)); 2.00–1.97 (m, CH2(9’’)); 1.94–1.92 (m, CH2(5’’)); 1.87–1.84 (m, CH2(4’’)); 1.77 (s, Me(13’’));
1.60 (s, Me(12’’)); 1.512, 1.507 (2s, Me(14’’), Me(15’’)). 13C-NMR ((D6)DMSO): 155.7 (C(6)); 148.0
(C(4)); 147.0 (C(2)); 140.1 (C(3’’)); 139.0 (C(8)); 134.7 (C(7’’)); 130.6 (C(11’’)); 124.0 (C(6’’)); 123.8
(C(5)); 123.4 (C(10’’)); 119.4 (C2’’)); 88.0 (C(1’)); 83.6 (C(4’)); 70.7 (C(3’)); 61.6 (C(5’)); 43.2 (C(1’’));
39.5 (C(2’)); 39.1 (C(8’’)); 38.8 (C(4’’); 26.1 (C(5’’)); 25.6 (C(12’’)); 25.4 (C(9’’)); 17.5 (C(15’’)); 16.2
(C(14’’)); 15.8 (C(13’’)). Anal. calc. for C25H36N4O4 (456.58): C 65.76, H 7.95, N 1227; found: C 65.42, H
8.06, N 12.04.
2’-Deoxy-1-[(2E)-3,7-dimethylocta-2,6-dien-1-yl]inosine (3a). Compound 3a was prepared and
worked up from 1 (1.01 g, 4 mmol) and geranyl bromide (0.96 g, 4.4 mmol) as described for 3b. Yield:
0.80 g (51%). Rf (CHCl3/MeOH 95 :5 (v/v)) 0.41. UV (MeOH): 250 (10,450); e260 7,800. log P: 1.37ꢁ0.93.
1H-NMR (CDCl3): 8.15 (s, HꢀC(2)); 8.01 (s, HꢀC(8)); 6.39 (dd, J(1’,2’b)¼7.5, J(1’,2’a)¼6.5, HꢀC(1’));
5.37–5.32 (m, HꢀC(2’’)); 5.08 (t, J(OH3’,3’), HOꢀC(3’)); 4.80 (t, J(6’’,5’’)¼6.5, HꢀC(6’’)); 4.70 (d,
J(1’’,2’’)¼6.5, CH2(1’’)); 4.20–4.19 (m, HꢀC(3’)); 4.01–3.89 (m, HꢀC(4’), CH2(5’)); 2.86–2.80 (m,
HbꢀC(2’)); 2.46 (ABdd, JAB ¼ꢀ 13.5, J(2’a,1’)¼5.5, J(2a’ ,3’)¼3.0, HaꢀC(2’)); 2.14–2.08 (m, CH2(5’’),
CH2(4’’)); 1.83 (s, Me(9’’)); 1.70 (s, Me(8’’)); 1.62 (Me(10’’)). 13C-NMR (CDCl3): 156.2 (C(6)); 148.5
(C(4)); 147.5 (C(2)); 140.6 (C(3’’)); 139.5 (C(8)); 131.5 (C(7’’)); 124.3 (C(6’’)); 124.2 (C(5)); 119.9 (C2’’));
88.4 (C(1’)); 84.1 (C(4’)); 71.1 (C(3’)); 62.1 (C(5’)); 43.7 (C(1’’)); 39.7 (C(2’)); 39.3 (C(8’’)); 39.0 (C(4’’);
26.2 (C(5’’)); 25.8 (C(8’’)); 18.0 (C(14’’)); 16.7 (C(13’’)). Anal. calc. for C20H28N4O4 (388.46): C 61.84, H
7.27, N 14.42; found: C 61.72, H 7.31, N 14.31.

56
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
5’-O-[Bis(4-methoxyphenyl)(phenyl)methyl]-2’-deoxy-1-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-tri-
en-1-yl]inosine (4b). Compound 3b (0.46 g, 1.0 mmol) was co-evaporated twice from dry pyridine (1 ml,
each) and then dissolved in anh. pyridine (5 ml). After addition of 1,1’-[chloro(phenyl)methanediyl]-
bis(4-methoxybenzene) (0.40 g, 1.15 mmol), the mixture was stirred for 24 h at r.t. under N2. Then, the
reaction was quenched by addition of MeOH (3 ml). After addition of aq. 5% NaHCO3 (30 ml), the aq.
phase was extracted three times with CH2Cl2 (30 ml each), and the combined org. layers were dried
(Na2SO4, 1 h) and filtered. Chromatography (column: 6ꢂ10 cm; CHCl3/MeOH 96 :4 (v/v)) gave one
main zone from which compound 4b (0.46 g, 61%) was isolated. Slightly yellowish glass. M.p. 688. Rf
1
(CHCl3/MeOH 96 :4 (v/v)) 0.13. UV (MeOH): 235 (38,200); e260 14,150. log P: 9.81ꢁ0.96. H-NMR
((D6)DMSO): 8.23 (s, HꢀC(2)); 8.16 (s, HꢀC(8)); 7.32–7.31 (m, HꢀC(4) and HꢀC(2,6) of Ph); 7.23–7.17
(m, 2 HꢀC(2,6) of 4-MeOꢀC6H4, HꢀC(3,5) of Ph); 6.82–6.78 (m, 2 HꢀC(3,5) of 4-MeOꢀC6H4); 6.31
(dd, J(1’,2’a)¼J(1’,2’b)¼6.5, HꢀC(1’)); 5.33 (br. s, HOꢀC(3’)); 5.24 (t, J(2’’,1’’)¼7.0, HꢀC(2’’)); 5.02 (t,
J(10’’,9’’)¼6.0, HꢀC(10’’)); 4.98 (t, J(6’’,5’’)¼6.5, HꢀC(6’’)); 4.63–4.55 (m, HꢀC(1’’)); 4.40 (br. s,
HꢀC(3’)); 4.00–3.97 (m, HꢀC(4’)); 3.71 (s, 2 MeO); 3.20–3.12 (m, CH2(5’)); 2.74 (ABdd, JAB ¼ꢀ 13.5,
J(2’b,1’)¼J(2’b,3’)¼6.0, HbꢀC(2’)); 2.32 (ABdd, JAB ¼ꢀ 13.5, J(2’a,1’)¼5.5, J(2a’ ,3’)¼6.5, HaꢀC(2’)); 2.05
(t, J(8’’,9’’)¼6.5, CH2(8’’)); 2.00 (t, J(9’’,8’’)¼6.5, CH2(9’’)); 1.93 (t, J(5’’,4’’)¼7.5, CH2(5’’)); 1.85 (t,
J(4’’,5’’)¼7.5, CH2(4’’)); 1.77 (s, Me(13’’)); 1.59 (s, Me(12’’)); 1.50 (s, Me(14’’), Me(15’’)). 13C-NMR
((D6)DMSO): 157.9 (2 MeOꢀC); 155.7 (C(6)); 147.7 (C(2)); 146.9 (C(4)); 144.7 (C(1) of Ph); 140.0
(C(3’’)); 139.1 (C(8)); 135.5 (2 C(1) of 4-MeOꢀC6H4); 134.6 (C(7’’)); 130.5 (C(11’’)); 129.5 (2 C(2,6) of 4-
MeOꢀC6H4); 129.5 (C(3,5) of Ph); 127.6 (C(2,6) of Ph); 126.5 (C(4) of Ph); 123.9 (C(6’’)); 123.9
(C(10’’)); 123.4 (C(5)); 119.3 (C(2’’)); 113.0 (2 C(3,5) of 4-MeOꢀC6H4); 85.9 (CAr3); 85.4 (C(1’)); 83.4
(C(4’)); 70.5 (C(3’)); 64.0 (C(5’)); 54.9 (2 MeO); 43.1 (C(2’)); 39.0 (C(8’’)); 38.9 (C(4’’)); 38.8 (C(1’’));
26.0 (C(9’’)); 25.6 (C(5’’)); 25.3 (C(12’’)); 17.4 (C(15’’)); 16.1 (C(14’’)); 15.7 (C(13’’)). Anal. calc. for
C46H54N4O6 (758.94): C 72.80, H 7.17, N 7.38; found: C 72.53, H 7.14, N 7.27.
5’-O-[Bis(4-methoxyphenyl)(phenyl)methyl]-2’-deoxy-1-[(2E)-3,7-dimethylocta-2,6-dien-1-yl]ino-
sine (4a). Compound 4a was prepared from 3a (0.39 g, 1.0 mmol) and worked up as described for 4b.
Yield: 0.48 g (69%). Colorless glass. M.p.: 748. Rf (CHCl3/MeOH 96 :4, v/v) 0.19. UV (MeOH): 235
(32,820); e260 12,791. log P: 7.77ꢁ0.94. 1H-NMR ((D6)DMSO): 8.25 (s, HꢀC(2)); 8.18 (s, HꢀC(8)); 7.28–
2.22 (m, HꢀC(4) and HꢀC(2,6) of Ph); 2.28–7.20 (m, 2 HꢀC(2,6) of 4-MeOꢀC6H4, HꢀC(3,5) of Ph);
6.82–6.79 (2 HꢀC(3,5) of 4-MeOꢀC6H4); 6.32 (dd, J(1’,2’a)¼J(1’,2b’ )¼6.5, HꢀC(1’)); 5.46–5.34 (m,
HOꢀC(3’)); 5.24 (t, J(2’’,1’’)¼6.5, HꢀC(2’’)); 5.01 (t, J(6’’,5’’)¼6.0, HꢀC(6’’)); 4.61–4.59 (m, HꢀC(1’’));
4.41–4.40 (m, HꢀC(3’)); 3.99–3.98 (m, HꢀC(4’)); 3.72–3.71 (m, 2 MeO); 3.21–3.12 (m, HꢀC(5’)); 2.75
(ABdd, JAB ¼ꢀ 13.0, J(2’b,1’)¼J(2’b,3’)¼6.5, HbꢀC(2’)); 2.34 (ABdd, JAB ¼ꢀ 13.0, J(2a’ ,1’)¼6.0, J(2a’ ,3’)¼
6.5, HaꢀC(2’)); 2.04–1.99 (m, CH2(5’’), CH2(4’’)); 1.77 (s, Me(9’’)); 1.57 (s, Me(8’’)); 1.51 (s, Me(10’’)).
13C-NMR ((D6)DMSO): 158.0 (2 MeOꢀC); 155.7 (C(6)); 147.7 (C(2)); 147.0 (C(4)); 144.7 (C(1) of Ph);
140.1 (C(3’’)); 139.1 (C(8)); 135.5 (2 C(1) of 4-MeOꢀC6H4); 130.9 (C(11’’)); 129.56 (2 C(2,6) of 4-
MeOꢀC6H4); 129.55 (C(3,5) of Ph); 127.7 (C(2,6) of Ph); 126.5 (C(4) of Ph); 124.0 (C(6’’)); 123.6 (C(5));
119.3 (C(2’’)); 113.0 (2 C(3,5) of 4-MeOꢀC6H4); 85.9 (CAr3); 85.4 (C(1’)); 83.4 (C(4’)); 70.5 (C(3’)); 64.0
(C(5’)); 54.9 (2 MeO); 43.1 (C(2’)); 38.7 (C(4’’)); 38.7 (C(1’’)); 25.7 (C(5’’)); 25.3 (C(8’’)); 17.4 (C(14’’));
16.1 (C(13’’)). Anal. calc. for C41H46N4O6 (690.827): C 71.28, H 6.71, N 8.11; found: C 70.94, H 6.67, N 7.93.
5’-O-[Bis(4-methoxyphenyl)(phenyl)methyl]-3’-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phos-
phanyl}-2’-deoxy-1-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]inosine (5b). Compound 4b
(100 mg, 0.13 mmol) was co-evaporated twice with CH2Cl2 and then dissolved in CH2Cl2 (5 ml). After
addition of EtNiPr2 (42 ml, 0.24 mmol) and (chloro)(2-cyanoethoxy)(diisopropylamino)phosphine
(¼2-cyanoethyl dipropan-2-ylphosphoramidochloridoite; 52 ml, 0.24 mmol), the mixture was stirred
for 15 min (!) at r.t. under N2. Then, ice-cold 5% aq. NaHCO3 was added (4 ml), and the mixture was
extracted three times with CH2Cl2 (8 ml, each). The combined org. layers were dried (Na2SO4, 10 min),
filtered, and the solvent was evaporated (<258). FC (column: 2ꢂ8 cm; CH2Cl2/acetone 8 :2 (v/v)) gave
5b (98 mg, 78%). Amorphous material. Rf (diastereoisomers; CH2Cl2/acetone 8 :2 (v/v)) 0.64, 0.76. log
P:12.86ꢁ1.11. 31P-NMR (CDCl3):148.9; 149.0.
5’-O-[Bis(4-methoxyphenyl)(phenyl)methyl]-3’-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phos-
phanyl}-2’-deoxy-1-[(2E)-3,7-dimethylocta-2,6-dien-1-yl]inosine (5a). Compound 5a was prepared and
worked up from 4a (100 mg, 0.14 mmol) as described for 5b. Yield: 79 mg (61%). Amorphous material

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
57
(diastereoisomers; CH2Cl2/acetone 8 :2 (v/v)) Rf 0.64, 0.76. log P: 10.82ꢁ1.10. 31P-NMR (CDCl3):148.9;
149.0.
5-Methyl-1-[(6aR,8R,9aS)-tetrahydro-2,2,4,4-tetra(propan-2-yl)-6H-furo[3,2-f][1,3,5,2,4]trioxadisi-
locin-8-yl]pyrimidine-2,4(1H,3H)-dione (8). Anh. thymidine (7; 0.242 g, 1 mmol) was dissolved in dry
pyridine (10 ml), and 1,3-dichloro-1,1,3,3-tetra(propan-2-yl)disiloxane (0.347 g, 1.1 mmol) was added.
The mixture was stirred for 24 h at r.t. After evaporation of the solvent, the residue was partitioned
between AcOEt and H2O (80 ml; 1:1 (v/v)). The org. layer was washed twice with cold 1m aq. HCl and
H2O (20 ml, each), followed by sat. aq. NaHCO3 and brine. After drying (anh. Na2SO4) and filtration, the
soln. was evaporated to dryness. Chromatography (column: 2ꢂ10 cm, CHCl3/MeOH 9 :1 (v/v)) gave,
after evaporation of the main zone, 8 (0.476 g, 98%). Colorless solid. M.p. 1748. Rf (Si2O; CHCl3/MeOH
9 :1 (v/v)) 0.9. UV(MeOH): 265 (12.040). 1H-NMR ((D6)DMSO): 11.33 (s, NH); 7.40 (d, J(Me,6)¼1.0,
HꢀC(6)); 6.00 (dd, J(1’,2’a)¼J(1’,2b’ )¼5.0, HꢀC(1’)); 4.56 (ddd, J(3’,2a’ )¼J(3’,2b’ )¼J(3’,4’)¼7.5,
HꢀC(3’)); 3.99 (ABd, JAB ¼ꢀ 12.2, J(5b’ ,4’)¼5.5, HbꢀC(5’)); 3.93 (ABd, JAB ¼ꢀ 12.0, J(5a’ ,4’)¼3.25,
HaꢀC(5’)); 3.70 (ddd, J(4’,3’)¼7.5, J(4’,5b’ )¼5.5, J(4’,5a’ )¼3.25, HꢀC(4’)); 2.44–2.39 (m, HbꢀC(2’));
2.33–2.27 (m, HaꢀC(2’)); 1.76 (d, J(Me,6)¼0.5, Me); 1.09–0.99 (m, 4 Me2CH). 13C-NMR:
((D6)DMSO): 163.7 (C(4)); 150.1 (C(2)); 136.2 (C(6)); 109.3 (C(5)); 84.2 (C(1’)); 83.2 (C(4’)); 70.3
(C(3’)); 61.7 (C(5’)); 38.5 (C(2’)); 17.3, 17.2, 17.2, 17.1, 17.0, 16.9, 16.8, 17.3–16.8 (4 Me2CH); 12.7–12.0
(4 Me2CH); 12.1 (Me). Anal. calc. for C22H40N2O6Si2 (484.73): C 54.51, H 8.32, N 5.78; found: C 54.54, H
8.21, N 5.68.
3,4-Dihydro-2’-deoxy-3-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]thymidine (9b). Anh. 7
(0.97 g, 4 mmol) was dissolved in amine-free, anh. DMF (20 ml), and dry K2CO3 (1.2 g, 10.4 mmol)
was added. Then, (E,E)-farnesyl bromide (0.87 ml, 4.4 mmol) was added dropwise within 10 min under
N2, and the mixture was stirred for 48 h at 408. After filtration, the mixture was then partitioned between
CH2Cl2 and H2O (100 ml; 1:1 (v/v)), the org. layer was separated and dried (anh. Na2SO4). After
filtration and evaporation of the solvent, the residue was dried in high vacuum. Subsequent gradient
chromatography (column: 6ꢂ10 cm; 1. CH2Cl2/MeOH 95 :5 (v/v); 2. CH2Cl2/MeOH 9 :1 (v/v)) gave,
after evaporation of the main zone, 9b (0.76 g, 44%). Rf (CH2Cl2/MeOH 9 :1 (v/v)) 0.5; Rf (Si2O; CH2Cl2/
1
MeOH 95 :5 (v/v)) 0.3. UV (MeOH): 266 (9,660). log P¼6.60ꢁ0.64. H-NMR ((D6)DMSO): 7.75 (d,
J(Me,6)¼1.26, HꢀC(6)); 6.20 (dd, J(1’,2a’ )¼J(1’,2b’ )¼7.0, HꢀC(1’)); 5.20 (d, J(OH3’ ,3’)¼4.5,
HOꢀC(3’)); 5.10 (t, J(2’’,1’’)¼6.5, HꢀC(2’’)); 5.05–5.01 (m, (HꢀC(6’’), HꢀC(10’’)); 5.00 (dd,
J(OH5’,5’b)¼J(OH5’,5a’ )¼5.2, HOꢀC(5’)); 4.39 (d, J(1’’,2’’)¼7.0, CH2(1’’)); 4.24 (ddd, J(4’,3’)¼3.8,
J(4’,5’b)¼4.0, J(4’,5a’ )¼4.0, HꢀC(4’)); 3.78 (ddd, J(3’,2a’ )¼J(3’,2’b)¼J(3’,4’)¼3.8, HꢀC(3’)); 3.60 (ABdd,
JAB ¼ꢀ 12.0, J(5’b,4’)¼4.0, J(5b’ ,OH5’)¼5.2, HbꢀC(5’)); 3.57–3.53 (m, HaꢀC(5’)); 2.04–1.86 (m,
(CH2(4’’), CH2(5’’), CH2(8’’), CH2(9’’)); 1.81 (d, J(Me,6¼1.0, CH3)); 1.74 (s, Me(13’’)); 1.63 (s,
Me(12’’)); 1.55 (s, Me(14’’)); 1.52 (s, Me(15’’)). 13C-NMR ((D6) DMSO): 162.4 (C(4)); 150.2 (C(2)); 138.7
(C(3’’); 134.7 (C(6)); 134.5 (C(7’’)); 130.6 (C(11’’)); 124.1 (C(6’’)); 123.6 (C(10’’)); 119.0 (C(2’’)); 108.5
(C(5)); 87.4 (C(1’)); 84.8 (C(4’)); 70.3 (C(3’)); 61.2 (C(5’)); 40.1 (C(2’)); 39.2 (C(1’’)); 38.9 (C(8’’)); 38.6
(C(4’’)); 26.2 (C(5’’)); 25.7 (C(9’’)); 25.4 (C(12’’)); 17.5 (C(15’’)); 16.1 (C(14’’)); 15.8 (C(13’’)); 12.8 (Me).
Anal. calc. for C25H38N2O5 (446.58): C 67.24, H 8.58, N 6.27; found: C 67.39, H 8.56, N 5.90.
2’-Deoxy-3-[(2E)-3,7-dimethylocta-2,6-dien-1-yl]-3,4-dihydrothymidine (9a). Anh. 7 (0.97 g,
4 mmol) was reacted and worked up with geranyl bromide (0.87 ml, 4.4 mmol) as described for 9b.
Chromatography (Si2O, column: 6ꢂ10 cm; CH2Cl2/MeOH 9 :1 (v/v)) gave, after evaporation of the main
zone, 9a (0.86 g, 2.27 mmol). Yellowish amorphous solid. Rf (CH2Cl2/MeOH 9 :1, v/v) 0.4. UV (MeOH):
266 (7,579). log P: 4.57ꢁ0.61. 1H-NMR ((D6)DMSO): 7.75 (d, J(Me,6)¼1.3, HꢀC(6)); 6.21 (dd,
J(1’,2’a)¼J(1’,2b’ )¼7.0, HꢀC(1’)); 5.20 (d, J(OH3’,3’)¼4.5, HOꢀC(3’)); 5.11 (t, J(2’’,1’’)¼6.75, HꢀC(2’’));
5.04–5.01 (m, HꢀC(6’’)); 4.99 (dd, J(OH5’,5b’ )¼J(OH5’,5’a)¼5.2, HOꢀC(5’)); 4.39 (d, J(1’’,2’’)¼6.5,
CH2(1’’)); 4.24 (ddd, J(4’,3’)¼3.5, J(4’,5b’ )¼J(4’,5a’ )¼4.0, HꢀC(4’)); 3.78 (ddd, J(3’,2a’ )¼J(3’,2’b)¼
J(3’,4’)¼3.8, HꢀC(3’)); 3.61 (ABdd, JAB ¼ꢀ 12.0, J(5’b,4’))¼J(5b’ ,OH5’)¼4.0, HbꢀC(5’)); 3.55 (ABdd,
JAB ¼ꢀ 12.0, J(5’a,4’)¼4.0, J(5a’ ,OH5’)¼5.0, HaꢀC(5’)); 2.11 (ABdd, JAB ¼ꢀ 12.0, J(2a’ ,1’)¼7.0, J(2a’ ,3’)¼
4.8, HaꢀC(2’)); 2.09 (ABdd, JAB ¼ꢀ 12.0, J(2b’ ,1’)¼7.0, J(2’b,3’)¼4.8, HbꢀC(2’)); 2.02–1.99 (m, CH2(4’’));
1.95–1.93 (m, CH2(5’’)); 1.82 (d, J(Me, 6¼1.0, Me)); 1.74 (s, Me(9’’)); 1.61 (s, Me(8’’)); 1.53 (s, Me(10’’)).
13C-NMR ((D6)DMSO): 162.3 (C(4)); 150.2 (C(2)); 138.7 (C(3’’)); 134.6 (C(6)); 130.8 (C(7’’)); 123.7
(C(6’’)); 118.8 (C(2’’)); 108.4 (C(5)); 87.3 (C(1’)); 84.7 (C(4’)); 70.2 (C(3’)); 61.2 (C(5’)); 40.1 (C(2’)); 39.4

58
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
(C(4’’)); 39.1 (C(1’’)); 25.8 (C(5’’)); 25.3 (C(8’’)); 17.4 (C(10’’)); 16.1 (C(9’’)); 12.8 (Me). Anal. calc. for
C20H30N2O5 (378.46): C 63.47, H 7.99, N 7.40; found: C 63.26, H 7.98, N 7.19.
5’-O-[Bis(4-methoxyphenyl)(phenyl)methyl]-2’-deoxy-3-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-tri-
en-1-yl]-3,4-dihydrothymidine (10b). Compound 9b (0.45 g, 1 mmol) was co-evaporated twice with anh.
pyridine (1 ml, each) and then dissolved in anh. pyridine (5 ml). 1,1’-[Chloro(phenyl)methanediyl]bis(4-
methoxybenzene) (0.39 g, 1.15 mmol) was added under N2, and the mixture was stirred for 24 h at r.t.
Then, the reaction was quenched by addition of MeOH (3 ml). After 10 min, ice-cold 5% aq. NaHCO3
was added, and the soln. was extracted with CH2Cl2. The org. layer was dried (Na2SO4), filtered, and the
solvent was evaporated. The residue was dried in high vacuum until a yellowish foam formed.
Chromatography (column: 6ꢂ10 cm; CH2Cl2/MeOH 99 :1 (v/v)) gave, after evaporation of the main
zone, 10b (0. 48 g, 65%). Yellowish glass. Rf (CH2Cl2/MeOH 99 :1 (v/v)) 0.4. UV (MeOH): 232 (26,900),
268 (13,400). log P: 12.17ꢁ0.64. 1H-NMR ((D6)DMSO): 7.54 (d, J(Me,6)¼0.9, HꢀC(6)); 7.38 (d, J(2Ph
/
6Ph,3Ph/5Ph)¼7.25, HꢀC(2,6) of Ph); 7.30 (t, J(4Ph,3Ph/5Ph)¼7.4, HꢀC(4) of Ph); 7.26–7.24 (m, HꢀC(2,6) of
2 4-MeOꢀC6H4, HꢀC(3,5) of Ph); 6.88 (d, J(34MeOPh/54MeOPh,24MeOPh/64MeOPh)¼8.2, HꢀC(3,5) of 2 4-
MeOꢀC6H4); 6.24 (dd, J(1’,2’a)¼J(1’,2b’ )¼6.62, HꢀC(1’)); 5.30 (d, J(OH3’,3’)¼4.4, HOꢀC(3’)); 5.12–
5.10 (m, (HꢀC(2’’)); 5.04–5.02 (m, HꢀC(6’’), HꢀC(4) of Ph); 4.40 (d, J(1’’,2’’)¼5.0, CH2(1’’)); 4.33–4.31
(m, HꢀC(4’)); 3.92–3.88 (m, HꢀC(3’)); 3.73 (s, 2 MeO); 3.25–3.17 (m, CH2(5’)); 2.26–2.17 (m,
CH2(2’)); 2.03–2.00 (m, CH2(5’’)); 1.99–1.93 (m, CH2(8’’), CH2(9’’)); 1.91–2.88 (m, CH2(4’’)); 1.74 (s,
Me(5)); 1.61 (s, Me(13’’)); 1.53 (s, Me(12’’)); 1.52 (s, Me(14’’)); 1.49 (s, Me(15’’)). 13C-NMR
((D6)DMSO): 161.2 (C(4)); 157.1 (2 MeOꢀC); 149.1 (C(2)); 143.6 (C(1) of Ph); 137.7 (C(3’’)); 134.4
(2 C(1) of 4-MeOꢀC6H4); 133.4 (C(6)); 129.5 (C(7’’)); 128.6 (C(2,6) of 4-MeOꢀC6H4); 126.8 (C(3,5) of
Ph); 126.6 (C(2,6) of Ph); 125.7 (C(4) of Ph); 123.0 (C(6’’)); 122.5 (C(10’’)); 117.8 (C(2’’)); 112.2 (2 C(3,5)
of 4-MeOꢀC6H4); 107.7 (C(5)); 84.8 (Ar3C); 84.5 (C(1’)); 83.7 (C(4’)); 69.4 (C(3’)); 62.6 (C(5’)); 54.0 (2
MeO); 39.1 (C(2’)); 37.8 (C(4’’)); 37.6 (C(1’’)); 25.1 (C(5’’)); 24.6 (C(9’’)); 25.3 (C(8’’)); 16.4 (C(15’’));
15.1 (C(14’’)); 14.7 (C(13’’)); 11.3 (Me). Anal. calc. for C46H56N2O7 (748.95): C 73.77, H 7.54, N 3.74;
found: C 73.39, H 7.38, N 3.74.
5’-O-[Bis(4-methoxyphenyl)(phenyl)methyl]-2’-deoxy-3-[(2E)-3,7-dimethylocta-2,6-dien-1-yl]-3,4-
dihydrothymidine (10a). Compound 9a (0.38 g, 1 mmol) was dried with anh. pyridine, reacted with 1,1’-
[Chloro(phenyl)methanediyl]bis(4-methoxybenzene) (0.39 g, 1.15 mmol), and worked up as described
for 10b. Yield: 0.39 g (57%) of 10a. Yellowish foam. Rf (CH2Cl2/MeOH 99 :1 (v/v)) 0.57. UV (MeOH):
231 (24,770), 268 (12,200). log P: 10.14ꢁ0.61. 1H-NMR ((D6)DMSO): 7.55 (d, J(Me,6)¼0.95, HꢀC(6));
7.38 (d, J(2Ph/6Ph,3Ph/5Ph)¼7.25, HꢀC(2,6) of Ph); 7.31 (t, J(4Ph,3Ph/5Ph)¼7.4, HꢀC(4) of Ph); 7.26–7.24 (m,
HꢀC(2,6) of 2 4-MeOꢀC6H4, HꢀC(3,5) of Ph); 6.89 (d, J(34MeOPh/54MeOPh,24MeOPh/64MeOPh)¼8.9, HꢀC(3,5)
of 2 4-MeOꢀC6H4); 6.25 (dd, J(1’,2’a)¼J(1’,2b’ )¼6.8, HꢀC(1’)); 5.30 (d, J(OH3’,3’)¼4.4, HOꢀC(3’));
5.13–5.10 (m, HꢀC(2’’)); 5.04–5.01 (m, HꢀC(6’’)); 4.40 (d, J(1’’,2’’)¼5.0, CH2(1’’)); 4.34–4.31 (m,
HꢀC(4’)); 3.90 (ddd, J(3’,2’a)¼J(3’,2b’ )¼J(3’,4’)¼3.9, HꢀC(3’)); 3.74 (s, 2 MeO); 3.24–3.18 (m,
CH2(5’)); 2.28–2.16 (m, CH2(2’)); 2.03–1.99 (m, CH2(5’’)); 1.95–1.91 (m, CH2(4’’)); 1.74 (s, Me(5)); 1.60
(s, Me(9’’)); 1.53 (s, Me(8’’)); 1.50 (s, Me(10’’)). 13C-NMR (D6)DMSO): 162.3 (C(4)); 158.1 (2 MeOꢀC);
150.1 (C(2)); 144.6 (C(1) of Ph); 144.6 (C(3’’)); 135.4 (2 C(1) of 4-MeOꢀC6H4); 134.3 (C(6)); 130.8
(C(7’’)); 129.7 (2 C(2,6) of 4-MeOꢀC6H4); 127.8 (C(3,5) of Ph); 127.6 (C(2,6) of Ph); 126.7 (C(4) of Ph);
123.8 (C(6’’)); 118.8 (C(2’’)); 113.2 (2 C(3,5) of 4-MeOꢀC6H4); 108.7 (C(5)); 85.8 (CAr3); 85.5 (C(1’));
84.7 (C(4’)); 70.4 (C(3’)); 63.6 (C(5’)); 55.0 (2 MeO); 40.1 (C(2’)); 30.9 (C(4’’)); 38.6 (C(1’’)); 25.8
(C(5’’)); 25.3 (C(8’’)); 17.4 (C(10’’)); 16.1 (C(9’’)); 12.3 (Me). Anal. calc. for C41H48N2O7 (680.83): C
72.33, H 7.11, N 4.11; found: C 72.16, H 7.00, N 3.84.
5’-O-[Bis(4-methoxyphenyl)(phenyl)methyl]-3’-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phos-
phanyl}-2’-deoxy-3-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]-3,4-dihydrothymidine (11b).
Compound 10b (0.32 g, 0.3 mmol) was co-evaporated twice from dry CH2Cl2 and dissolved in CH2Cl2
(15 ml). Then, EtNiPr2 (126 ml, 0.72 mmol) and (chloro)(2-cyanoethoxy)(diisopropylamino)phosphine
(156 ml, 0.72 mmol) were added under N2. The mixture was stirred for 20 min at r.t., and the reaction was
then quenched by addition of ice-cold 5% aq. NaHCO3 (12 ml). The raw product was extracted with
CH2Cl2, and the soln. was dried (Na2SO4) for 2 min, filtered, and evaporated to dryness (bath temp.:
>258), followed by drying in high vacuum for 5 min. FC (column: 2ꢂ10 cm; CH2Cl2/acetone 8 :2 (v/v),
with 8 drops of Et3N per l, total time of chromatography >15 min) afforded 11b (0.27 g, 67%). Colorless.

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
59
Stored at ꢀ208. Rf (diasteoisomers; CH2Cl2/acetone 8 :2, v/v) 0.87, 0.97. 31P-NMR (CDCl3): 149.1 (PR);
148.5 (PS).
5’-O-[Bis(4-methoxyphenyl)(phenyl)methyl]-3’-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phos-
phanyl}-2’-deoxy-3-[(2E)-3,7-dimethylocta-2,6-dien-1-yl]-3,4-dihydrothymidine (11a). Compound 11a
was prepared from 10a (0.2 g, 0.3 mmol) as described for 11b. Yield: 0.25 g (97%). Colorless foam. Rf
(diastereoisomers; CH2Cl2/acetone 8 :2 (v/v)) 0.84, 0.94. 31P-NMR (CDCl3): 149.0 (PR); 148.5 (PS).
Small-Scale Coupling of Compound 3b with i) N-[(9H-Fluoren-9-ylmethoxy)carbonyl]glycine (13)
to Yield Compounds 14–16 and ii) Sulforhodamin 101 Sulfonyl Chloride to Yield Compound 17.
i) Compound 13 (65.4 mg, 0.22 mmol) was dissolved in CH2Cl2 (20 ml) and 4-(dimethylamino)pyr-
idine (DMAP; 5 mg) and 3b (100 mg, 0.22 mmol) were added. The mixture was cooled to 08, and N,N’-
dicyclohexylcarbodiimide (DCC; 45.5 mg, 0.22 mmol) in CH2Cl2 (2 ml) were added dropwise. After
5 min, the mixture was allowed to warm to r.t., and stirring was continued overnight. Then, further
portions of 13, DMAP, and DCC (30 mol-%, each) were added, and stirring was continued. After a total
reaction time of 48 h, the suspension was filtered, and the filtrate was evaporated to dryness.
Chromatography (column: 2ꢂ15 cm; CH2Cl2/acetone 6 :4 (v/v)) afforded three main zones from which
the following fluorene-labelled nucleolipids were obtained upon evaporation of the solvent.
(2R,3S,5R)-2-{[({[(9H-Fluoren-9-ylmethoxy)carbonyl]amino}acetyl)oxy]methyl}-5-{6-oxo-1-
[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]-1,6-dihydro-9H-purin-9-yl}tetrahydrofuran-3-yl
{[(9H-Fluoren-9-ylmethoxy)carbonyl]amino}acetate (14). Rf (CHCl3/MeOH 96 :4 (v/v)) 0.56. UV
(MeOH): 263 (45,200); e260 44,200. log P: 11.67ꢁ1.22. 1H-NMR (CDCl3): 7.92 (s, HꢀC(2)); 7.88 (s,
HꢀC(8)); 7.77–7.73 (m, HꢀC(4,5) of 2 Fmoc); 7.59 (d, J(1Fmoc/8Fmoc,2Fmoc/7Fmoc), HꢀC(1,8) of 2 Fmoc);
7.41–7.36 (m, HꢀC(3,6) of 2 Fmoc); 7.32–7.27 (m, HꢀC(2,7) of 2 Fmoc); 6.23 (dd, J(1’,2’a)¼J(1’,2b’ )¼7.0,
HꢀC(1’)); 5.67 (br. s, NH); 5.60–5.59 (m, HꢀC(3’)); 5.52 (br. s, NH); 5.33–5.31 (m, HꢀC(2’’)); 5.08–5.06
(m, HꢀC(6’’), HꢀC(10’’)); 4.70–4.60 (m, HꢀC(1’’)); 4.44–4.40 (m, CH2 of 2 Fmoc, CH2(5’)); 4.33–4.32
(m, HꢀC(4’)); 4.26–4.20 (m, HꢀC(9) of 2 Fmoc); 4.04–4.01 (m, CH2 of Gly); 3.06–3.01 (m, HbꢀC(2’));
2.55–2.53 (m, HaꢀC(2’)); 2.10–2.04 (m, CH2(8’’), CH2(9’’), CH2(5’’)); 1.95–1.92 (m, CH2(4’’)); 1.80 (s,
Me(13’’)); 1.60 (s, Me(12’’)); 1.59 (s, Me(14’’)); 1.27 (s, Me(15’’)). 13C-NMR ((D6)DMSO): 169.9 (CO of
Gly); 156.5 (CO of Fmoc); 155.6 (C(6)); 148.1 (C(2)); 147.1 (C(4)); 143.7 (C(8a,9a) of Fmoc); 140.7
(C(4a,4b) of Fmoc); 140.1 (C(3’’)); 139.0 (C(8)); 134.6 (C(7’’)); 130.5 (C(11’’)); 127.5 (C(3,6) of Fmoc);
127.0 (C(4,5) of Fmoc); 125.1 (C(2,7) of Fmoc); 124.0 (C(6’’)); 123.4 (C(10’’)); 120.0 (C(5)); 119.9 (C(1,8)
of Fmoc); 119.3 (C(2’’)); 83.3 (C(1’); 81.5 (C(4’)); 74.7 (C(3’)); 65.8 (CH2 of Fmoc); 55.8 (C(5’)); 47.4
(C(9) of Fmoc); 46.5 (CH2 of Gly); 43.3 (C(1’’)); 39.0 (C(2’)); 38.7 (C(8’’)); 35.9 (C(4’’)); 26.0 (C(5’’));
25.3 (C(12’’)); 24.4 (C(9’’)); 17.4 (C(15’’)); 16.2 (C(14’’)); 15.7 (C(13’’)).
2’-Deoxy-5’-O-{N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}-1-[(2E,6E)-3,7,11-trimethyldodeca-
2,6,10-trien-1-yl]inosine (15). Rf (CHCl3/MeOH 96 :4, v/v) 0.31. UV (MeOH): 263 (27,800); e260 27,100.
log P: 7.57ꢁ1.15. 1H-NMR ((D6)DMSO): 8.31 (s, HꢀC(2)); 8.29 (s, HꢀC(8)); 7.88 (d, J(4Fmoc/5Fmoc,3Fmoc
/
6Fmoc)¼7.5, HꢀC(4,5) of Fmoc); 7.71 (d, J(1Fmoc/8Fmoc,2Fmoc/7Fmoc)¼7.5, HꢀC(1,8) of Fmoc); 7.41 (dd,
J(3Fmoc/6Fmoc,2Fmoc/7Fmoc)¼J(3Fmoc/6Fmoc,4Fmoc/5Fmoc)¼8.0, HꢀC(3,6) of Fmoc); 7.33 (dd, J(2Fmoc/7Fmoc,3Fmoc
/
6Fmoc)¼J(2Fmoc/7Fmoc,1Fmoc/8Fmoc)¼8.0, HꢀC(2,7) of Fmoc); 6.29 (dd, J(1’,2’b)¼8.5, J(1’,2a’ )¼7.0,
HꢀC(1’)); 5.40 (d, J(OH3’,3’)¼5.5, HꢀC(3’)); 5.26 (t, J(2’’,1’’)¼7.0, HꢀC(2’’)); 5.01 (t, J(6’’,5’’)¼7.0,
HꢀC(6’’)); 5.04–4.99 (m, HꢀC(10’’)); 4.60 (d, J(1’’,2’’)¼6.5, HꢀC(1’’)); 4.34 (d, J(CH2Fmoc,9Fmoc)¼7.0,
HꢀCH2 of Fmoc); 4.27–4.24 (m, HꢀC(4’)); 4.08–7.07 (m, HꢀC(9) of Fmoc); 3.85 (d, J(CH2Gly,NH)¼6.0,
CH2 of Gly); 3.66–3.55 (m, CH2(5’)); 2.88 (ABdd, JAB ¼13.0, J(2’b,3’)¼6.0, J(2’b,3’)¼8.0, HbꢀC(2’)); 2.34
(mc (superimposed by the solvent signals, HaꢀC(2’)); 2.06 (t, J(8’’,9’’)¼7.5, CH2(8’’)); 1.99 (t, J(9’’,8’’)¼
7.5, CH2(9’’)); 1.93 (t, J(5’’,4’’)¼7.5, CH2(5’’)); 1.86 (t, J(4’’,5’’)¼7.5, CH2(4’’)); 1.78 (s, Me(13’’)); 1.60 (s,
Me(12’’)); 1.52 (s, Me(14’’)); 1.51 (s, Me(15’’)). 13C-NMR ((D6)DMSO): 170.0 (CO of Gly); 156.4 (CO of
Fmoc); 155.7 (C(6)); 148.0 (C(2)); 147.0 (C(4)); 143.7 (C(8a,9a) of Fmoc); 140.6 (C(4a,4b) of Fmoc);
140.0 (C(3’’)); 139.0 (C(8)); 134.6 (C(7’’)); 130.5 (C(11’’)); 127.5 (C(3,6) of Fmoc); 127.0 (C(4,5) of
Fmoc); 125.1 (C(2,7) of Fmoc); 124.0 (C(6’’)); 123.8 (C(10’’)); 123.4 (C(5)); 120.0 (C(1,8) of Fmoc);
119.3 (C(2’’)); 84.1 (C(1’)); 83.3 (C(4’)); 70.4 (C(3’)); 65.8 (CH2 of Fmoc); 64.0 (C(5’)); 46.5 (C(9) of
Fmoc); 43.2 (CH2 of Gly); 42.0 (C(1’’)); 39.9 (C(2’)); 39.7 (C(8’’)); 39.6 (C(4’’)); 26.0 (C(5’’)); 25.5
(C(9’’)); 25.3 (C(12’’)); 17.4 (C(15’’)); 16.1 (C(14’’)); 15.7 (C(13’’)).

60
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
2’-Deoxy-3’-O-{N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}-1-[(2E,6E)-3,7,11-trimethyldodeca-
2,6,10-trien-1-yl]inosine (16). Rf (CHCl3/MeOH 96 :4, v/v) 0.20. UV (MeOH): 263 (27,730); e260 27,100.
log P: 7.73ꢁ0.98. 1H-NMR (CDCl3): 8.03 (s, HꢀC(2)); 7.96 (s, HꢀC(8)); 7.75 (d, J(4Fmoc/5Fmoc,3Fmoc
/
6
Fmoc)¼7.5, HꢀC(4,5) of Fmoc); 7.58 (d, J(1Fmoc/8Fmoc,2Fmoc/7Fmoc)¼7.5, HꢀC(1,8) of Fmoc); 7.38 (dd,
J(3Fmoc/6Fmoc,2Fmoc/7Fmoc)¼J(3Fmoc/6Fmoc,4Fmoc/5Fmoc)¼7.5, HꢀC(3,6) of Fmoc); 7.30–7.27 (m, HꢀC(2,7) of
Fmoc); 6.34 (dd, J(1’,2’b)¼J(1’,2’ )¼6.5, HꢀC(1’)); 5.74–5.73 (m, HOꢀC(3’)); 5.32 (t, J(2’’,1’’)¼6.5,
a
HꢀC(2’’)); 5.08–5.07 (m, HꢀC(10’’)), HꢀC(6’’)); 4.76–4.75 (m, HꢀC(3’)); 4.67–4.64 (m, HꢀC(1’’));
4.44–4.39 (m, CH2 of Fmoc); 4.22–4.20 (m, HꢀC(4’), HꢀC(9) of Fmoc); 4.00–3.99 (m, CH2 of Gly);
3.78–3.75 (m, CH2(5’)); 3.60 (br. s, NH); 2.81–2.78 (m, HbꢀC(2’)); 2.55–2.54 (m, HaꢀC(2’)); 2.18–2.03
(m, CH2(8’’), CH2(9’’), CH2(5’’)); 1.99–1.96 (m, CH2(4’’)); 1.80 (s, Me(13’’)); 1.68 (s, Me(12’’)); 1.60 (s,
Me(14’’)); 1.59 (s, Me(15’’)). 13C-NMR ((D6)DMSO): 169.7 (CO of Gly); 156.5 (CO of Fmoc); 155.6
(C(6)); 148.0 (C(2)); 147.0 (C(4)); 143.7 (C(8a,9a) of Fmoc); 140.7 (C(4a,4b) of Fmoc); 140.0 (C(3’’));
138.8 (C(8)); 134.6 (C(7’’)); 130.5 (C(11’’)); 127.5 (C(3,6) of Fmoc); 127.2 (C(4,5) of Fmoc); 125.1 (C(2,7)
of Fmoc); 124.0 (C(6’’)); 123.8 (C(10’’)); 121.3 (C(5)); 120.0 (C(1,8) of Fmoc); 119.3 (C(2’’)); 85.1 (C(1’);
83.5 (C(4’)); 75.5 (C(3’)); 65.8 (CH2 of Fmoc); 61.4 (C(5’)); 46.5 (C(9) of Fmoc); 43.3 (CH2 of Gly); 42.3
(C(1’’)); 39.0 (C(2’)); 38.7 (C(8’’)); 36.8 (C(4’’)); 26.0 (C(5’’)); 24.5 (C(9’’)); 25.3 (C(12’’)); 17.4 (C(15’’));
16.1 (C(14’’)); 15.7 (C(13’’)).
ii) 2’-Deoxy-5’-O-{[4-(2,3,6,7,12,13,16,17-octahydro-1H,5H,11H,15H-pyrido[3,2,1-ij]quinolizi-
no[1’,9’:6,7,8]chromeno[2,3-f]quinolin-4-ium-9-yl)-3-sulfonatophenyl]sulfonyl}-1-[(2E,6E)-3,7,11-trime-
thyldodeca-2,6,10-trien-1-yl]inosine (17). Compound 3b (2.74 mg, 6.0 mmol) was dissolved in anh.
pyridine (3 ml), and sulforhodamin 101 sulfonyl chloride (2.5 mg, 4.0 mmol) was added. The mixture was
stirred under N2 for 48 h. Then, H2O (10 ml) was added, and the mixture was extracted once with CH2Cl2.
The aq. layer was separated and evaporated to dryness. CC (column: 2ꢂ10 cm; CH2Cl2/MeOH 1:1 (v/
v)) gave, after evaporation of the solvent, 17. Black, amorphous material. Rf (CHCl3/MeOH 96 :4, (v/v))
0.56. UV (MeOH): 252 (18,200); e260 16,400.
Oligonucleotides. The synthesis of the oligonucleotides 18–23 and their MS analysis were performed
`
by Eurogentec S.A., Liege Science Park, Belgium.
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Received October 19, 2011