Mitsunobu reactions of 5-fluorouridine with the terpenols Phytol and Nerol: DNA building blocks for a biomimetic lipophilization of nucleic acids

Article abstract of DOI:10.1002/cbdv.201300107

The cancerostatic 5-fluorouridine (5-FUrd; 1) was sequentially sugar-protected by introduction of a 2′,3′-O-heptylidene ketal group (→2), followed by 5′-O-monomethoxytritylation (→3). This fully protected derivative was submitted to Mitsunobu reactions with either phytol ((Z and E)-isomer) or nerol ((Z)-isomer) to yield the nucleoterpenes 4a and 4b. Both were 5′-O-deprotected with 2% Cl₂CHCOOH in CH ₂Cl₂ to yield compounds 5a and 5b, respectively. These were converted to the 5′-O-cyanoethyl phosphoramidites 6a and 6b, respectively. Moreover, the 2′,3′-O-(1-nonyldecylidene) derivative, 7a, of 5-fluorouridine was resynthesized and labelled at C(5′) with an Eterneon-480 fluorophor (→7b). The resulting nucleolipid was studied with respect to its incorporation in an artificial bilayer, as well as to its aggregate formation. Additionally, two oligonucleotides carrying terminal phytol-alkylated 5-fluorouridine tags were prepared, one of which was studied concerning its incorporation in an artificial lipid bilayer. Copyright

Full text of DOI:10.1002/cbdv.201300107

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Mitsunobu Reactions of 5-Fluorouridine with the Terpenols Phytol and Nerol:
DNA Building Blocks for a Biomimetic Lipophilization of Nucleic Acids
by Edith Maleckia), Christine Kniesa), Emma Werza)b), and Helmut Rosemeyer*a)
a) Organic Materials Chemistry and Bioorganic Chemistry, Institute of Chemistry of New Materials,
University of Osnabrꢀck, Barbarastrasse. 7, D-49069 Osnabrꢀck
b) Ionovation GmbH, Westerbreite 7, D-49084 Osnabrꢀck
Dedicated to the memory of the late Prof. Dr. Dr. Eckard Schlimme
The cancerostatic 5-fluorouridine (5-FUrd; 1) was sequentially sugar-protected by introduction of a
2’,3’-O-heptylidene ketal group (!2), followed by 5’-O-monomethoxytritylation (! 3). This fully
protected derivative was submitted to Mitsunobu reactions with either phytol ((Z and E)-isomer) or
nerol ((Z)-isomer) to yield the nucleoterpenes 4a and 4b. Both were 5’-O-deprotected with 2%
Cl2CHCOOH in CH2Cl2 to yield compounds 5a and 5b, respectively. These were converted to the 5’-O-
cyanoethyl phosphoramidites 6a and 6b, respectively. Moreover, the 2’,3’-O-(1-nonyldecylidene)
derivative, 7a, of 5-fluorouridine was resynthesized and labelled at C(5’) with an Eterneon-480
fluorophorꢁ (! 7b). The resulting nucleolipid was studied with respect to its incorporation in an artificial
bilayer, as well as to its aggregate formation. Additionally, two oligonucleotides carrying terminal phytol-
alkylated 5-fluorouridine tags were prepared, one of which was studied concerning its incorporation in an
artificial lipid bilayer.
1. Introduction. – Post-biosynthetic lipophilization reactions of biomolecules such
as proteins and carbohydrates are inevitable in the chemistry of life [1]. Of particular
relevance in this concern is the recent discovery and biological characterization of
geranylated tRNA in bacteria [2], reflecting the chemodiversity of RNA constituents.
Also in biomedical chemistry, the lipophilization of compounds is of great importance.
One of the major drawbacks of many chemotherapeutics is their insufficient
penetration through cell membranes, as well as the crossing of the bloodꢀbrain barrier
due to their high hydrophilicity. This is particularly valid for antisense and antigene
oligonucleotides [3]. In the case of low-molecular-weight drugs, this kind of chemical
modification is heading for the fulfilment of ꢂLipinskiꢀs Rule of Fiveꢃ [4]. The rule
describes molecular properties relevant for a drugꢃs pharmacokinetics in the human
body, including their absorption, distribution, metabolism, and excretion, and it is of
high importance for drug development where a pharmacologically active lead structure
is optimized stepwise for increased activity and selectivity. One part of the rule
concerns the drugꢃs partition coefficient (log P between n-octan-1-ol and H2O) within
the range of ꢀ0.4 to þ5.5.
Meanwhile, synthesis and theranostic applications of nucleolipids and lipo-
oligonucleotides are established [5]. Herein, we describe the synthesis of lipophilic
derivatives of the cancerostatic nucleoside 5-fluorouridine (5-FUrd; nucleolipid), their
ꢄ 2013 Verlag Helvetica Chimica Acta AG, Zꢀrich

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conversion in 5’-O-(2-cyanoethyl)phosphoramidites, the appending of the nucleolipids
to oligonucleotides, and their incorporation in artificial lipid bilayers. For the
biomimetic lipophilization of 5-FUrd, we used phytol as well as nerol which were
linked to N(3) of the nucleoside applying Mitsunobu reaction conditions [6–15]. Nerol
is a monoterpene found in many essential oils such as lemongrass and hops. Phytol is –
inter alia – a constituent of chlorophyll with which the latter is embedded in the
thylakoid membranes of chloroplasts.
2. Results and Discussion. – 2.1. Synthesis of the Monomers. It has been found
recently that Mitsunobu reactions between an alcohol and a nucleoside leads only then
to a predominantly base-alkylated product, if the nucleoside is fully protected at the
glyconic moiety [6].
Moreover, such a reaction requires a pKBHþ value of the nucleosidic educt between
0 and 11 [16]. In principle, 5-fluorouracil (5-FU; pKBHþ ¼8.0ꢁ0.1) as well as uracil
(pKBHþ ¼9.4ꢁ0.1) are, therefore, suitable for Mitsunobu alkylations. For these reasons,
we protected compound 1 first at the 2’,3’-OH groups by reaction with heptan-4-one in
the presence of (EtO)3CH and 4m HCl in 1,4-dioxane and obtained compound 2
(Scheme) [17]. This had been prepared before and possesses a suitably high acidic
stability at the ketal group (t¼130 min in 1n aq. HCl/MeCN 1:1 (v/v)). The latter was
then protected at the 5’-OH group with a (4-methoxyphenyl)diphenylmethyl (MMTs)
residue (! 3). This intermediate was next submitted to Mitsunobu alkylations with
either phytol or nerol (PPh3, diethyl azodicarboxylate (DEAD), THF, 08) to furnish
compounds 4a and 4b. Both were subsequently detritylated in 4% Cl2CHCOOH in
CH2Cl2 at room temperature for 10 min to afford compounds 5a and 5b which possess
significantly enhanced log P values compared to 5-FUrd (1: log P¼ ꢀ 1.34ꢁ0.46; 5a:
log P ¼ þ12.5ꢁ0.63; 5b: log P ¼ þ7.65ꢁ0.65). The latter were then phosphitylated to
give the phosphoramidites 6a and 6b, respectively.
Because it has been reported that Mitsunobu alkylations of nucleobases may occur
at the N- and the O-atoms of the heterocycle, the 13C-NMR spectra of the
corresponding O(2)-, the O(4)-, and of the N(3)-alkylated compounds were simulated
and compared with the recorded spectrum of the isolated main product (Table). As can
be seen from the Table, the selected 13C-NMR resonances of the recorded signals
coincide mostly with those of the N(3)-alkylated compounds 8 and 9.
Additionally, compound 7a was resynthesized, for which details of preparation will
be published in due course. This compound was fluorophore-labelled at 5’-OH with
Eterneon-480ꢁ via its N-hydroxysuccinimide ester (! 7b). The chemical structure of
the fluorescence dye has not been disclosed so far. The dye-labelled cancerostatic
nucleolipid was studied with respect to its incorporation in an artificial lipid bilayer
composed of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoethanolamine (POPE)/1-palmi-
toyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) 8 :2 (w/w) in decane (10 mg/ml) in a
set-up shown in Fig. 1 (see Exper. Part and [18][19] for details and for construction
plans of the device).
From Fig. 2, it can be seen that, upon injection of a dilute MeCN solution of
compound 7b into the cis compartment of the bilayer slide, the bilayer is torn at once.
As a result, compound 7b forms high molecular-weight aggregates (micelles) which
slowly mass at the Teflon-coated aperture annulus (Fig. 2,b–d).

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Scheme

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Table 1. 13C-NMR Chemical Shifts of Simulated as Well as of Recorded Spectra of Various Alkylated 5-
Fluorouridines
C-Atom Data from Simulated Spectraa)
5-FUrd
O(2)-Alkylated product O(4)-Alkylated product N(3)-Alkylated product
C(4)
C(5)
C(6)
C(2)
C(1’’)
158.2
140.2
128.4
150.8
–
168.8
140.0
124.5
155.5
55.9
160.4
121.0
102.2
155.1
57.8
162.9
140.2
128.4
151.4
38.8
C-Atom Data from Recorded Spectra
5-FUrd
Phytol-alkylated product 5a
Nerol-alkylated product 5b
C(4)
C(5)
C(6)
C(2)
C(1’’)
157.07/156.86
140.81/138.98
124.95/124.67
149.22
156.20/155.99
140.24/138.43
124.64/124.37
148.73
156.20/156.00
140.27/138.45
124.63/124.35
148.73
–
38.93
38.85
a) Data from nerol- and phytol-alkylated compounds are identical.
Fig. 1. a) Stage unit of the ꢁIonovation Explorerꢀ mounted on a standard inverted fluorescence microscope.
The computer controlled perfusion unit is a side board and is not shown. b) ꢁIonovation Bilayer Slideꢀ, 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.
Photon correlation spectroscopy (PCS) recordings with a light-scattering detection
at an angle of 908 (Fig. 3) give a Poisson-type size distribution of the resulting particles
with mean values between ca. 600 and 1200 nm. One exception (rept. 4) is probably due
to a dust particle. In a second experiment, a more concentrated solution of 7b in MeCN
was injected into the cis compartment of the bilayer slide. In this case, it was observed
that, during an incubation time of 5 min, the dye-labelled nucleolipid is immobilized
within the bilayer (Fig. 2,e and f). Fig. 4 shows the relative brightness intensities of the
bilayer before and after addition of compound 7b.

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Fig. 2. Insertion of compound 7b into an artificial bilayer. a) Z-Scan of an empty bilayer. b) Z-Scan after
injection of a dilute soln. of 7b in MeCN (1 ml) into the cis compartment of the slide and torn of the
bilayer. c) Z-Scan after 5 min of incubation. Slowly massing of aggregates at the Teflon annulus. d) Z-
Scan after further 5 min of incubation. Most of the aggregates are covering the Teflon annulus. e) Repeat
of the experiment as in a. Z-Scan of the empty bilayer. f) Z-Scan after injection of a MeCN soln. of 7b
(1 ml) to the cis compartment of the slide and 5 min of incubation.
2.2. Lipo-oligonucleotides: Synthesis and Bilayer Incorporation. The phosphorami-
dite 6a was used to prepare the following oligonucleotides with an appending
nucleolipid 5a:
5’-d(5a-Cy5-TAG GTC AAT ACT)-3’
5’-d(5a-TAG GTC AAT ACT)-3’
3’-d(ATC CAG TTA TGA)-5’
(8)
(9)
(10)
The cyanine-5-labelled oligomer 8 was used to study the efficiency of lipid bilayer
incorporation with respect to its stability (Fig. 5).

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Fig. 3. Graphical presentation of PCS measurements (intensity vs. particle size) of 7b-particles (see:
Exper. Part). The curve of rept. 4 is probably due to a dust particle.
Fig. 4. Relative bilayer brightness intensity before and after addition of compound 7b
As can be seen from Fig. 5, the oligomer 8 carrying a 5’-phytol residue is highly
resistant against perfusion of the cis compartment of the bilayer slide; after five
perfusions (30 s each, with 1 ml/min of 250 nm KCl, 10 mm MOPS/Tris, pH 7.0;
incubation time between two consecutive perfusions, 5 min), only ca. 20% of 8 had
been washed out.
The oligomer 9 was prepared in advance in order to study the duplex formation
between this lipo-oligonucleotide and its complementary strand 10 at the lipid
bilayerꢀwater phase boundary layer using SYBRꢁ Green as intercalating fluorescent

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
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Fig. 5. Bilayer brightness [%] after incorporation of the oligomer 8 in the lipid bilayer as a function of the
perfusion number (for details, see Exper. Part)
dye. The results of these experiments will be compared with analogous ones on
oligonucleotides carrying other types of lipids at their 5’-end and will be published later.
Such studies will be of importance for the optimization of our novel nucleic-acid
biosensor technology [18]. Moreover, the properties of hybrid molecules as those
presented here might be of interest regarding recent reports on similar nucleoterpenes
found in nature [20][21].
The authors gratefully acknowledge financial support by the BBraun AG (D-Melsungen) as well as
the Bundesministerium fꢂr Wirtschaft (FKZ: KF 2369401 S B9 and FKZ 2369501 S B9). We thank 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. Moreover, we thank Mrs.
Marianne Gather Steckhan for NMR spectra, Mrs. Anja Schuster for elemental analyses, and Mrs. Petra
Bçsel for the formula drawings. Last but not least, we thank Mrs. Malayko Montilla Martinez for her help
with the PCS recordings.
Experimental Part
General. All chemicals were purchased from SigmaꢀAldrich (D-Deisenhofen) or from TCIꢀEurope
(B-Zwijndrecht). Solvents were of laboratory grade and were distilled before use. 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: AMX-500 spectrometer (Bruker, D-
Rheinstetten); 1H: 500.14, 13C: 125.76, and 31P: 101.3 MHz, chemical shifts, d in ppm rel. to TMS as
internal standard for 1H and 13C, and external 85% H3PO4; J values in Hz. Elemental analyses (C, H, N):
VarioMICRO instrument (Fa. Elementar, D-Hanau). Particle size distribution of 7b particles were
determined by photon correlation spectroscopy (PCS) with an N5 Submicro Particle Size Analyzer
(Beckman Coulter, D-Krefeld). Light-scattering detection was performed at 90 deg. 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 characterized (MALDI-TOF MS) by Eurogentec (Eurogentec S.A., Liege Science Park, B-
Seraing).

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Incorporation of Compound 7b in an Artificial Bilayer. The incorporation of 7b in an artificial bilayer
was performed at a lipid mixture 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoethanolamine (POPE)/1-
palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) 8 :2 (w/w; 10 mg/ml of decane). For the
preparation of the horizontal bilayers, planar slides (Ionovation GmbH, D-Osnabrꢀck) were used.
These slides contain chambers for cis and trans compartments, as well as electrode access (see Fig. 1). The
main body of the slides consists of PTFE (Teflon) foil (thickness, 25 mm) with an aperture of ca. 100 mm
diameter. This foil separates the chamber into the cis and trans compartments which are only connected
by the aperture. After filling of the chamber with buffer (250 mm KCl, 10 mm MOPS/Tris; pH 7), the cis
and trans compartments were linked with Ag/AgCl electrodes embedded in agarose/3m KCl. Then, a
soln. of POPC/POPE (0.2 ml) was applied onto the aperture of the PTFE foil using a Hamilton syringe
(Hamilton, CH-Bonaduz). A small Faraday cage shielded the bilayer and the electrodes from HF-
electrical noise. Next, a bilayer was made-up automatically using a perfusion system (Bilayer Explorer
V01, Ionovation GmbH, D-Osnabrꢀck). The formation of a stable bilayer was monitored both optically,
using a laser scanning microscope (Insight Cell 3D, Evotec Technologies GmbH, D-Hamburg), as well as
electrically by capacity measurements. When a stable bilayer had been obtained (capacity, 50–75 pF),
the corresponding soln. was injected into the cis compartment of the chip. During an incubation time of
25 min, the intactness of the bilayer was electro-physiologically controlled using a headstage EPC 10
USB with a patch clamp amplifier (software, Patchmaster, HEKA Elektronik Dr. Schulze GmbH, D-
Lambrecht). The following optical pictures of fluorescence fluctuations were obtained with a confocal
laser scanning microscope (Insight Cell 3D, Evotec Technologies GmbH, D-Hamburg), equipped with a
He-Ne laser (543 nm), and with an emitting laser diode (635 nm), a 40ꢂ H2O-immersion objective
(UApo 340, 40ꢂ ; NA, 1.15, Olympus, Tokyo), and an Avalanche photodiode detector (SPCM-AQR-13-
FC, Perkin-Elmer Optoelectronics, Fremont, USA). Fluorescence irradiation was obtained with a laser
power of 200ꢁ5 mW. 2D and 3D scans were performed by scanning the confocal spot in XY direction
with a rotating beam scanner and movement of the objective in Z direction. The movement in both
directions was piezo-controlled which allows a nm-precise positioning. For the 2D pictures (z-scans;
Figs. 3 and 5) the confocal plane was moved in 100-nm steps. Diffusion coefficients were determined by
photon correlation spectroscopy (PCS) and are compiled in the Table.
Incorporation of the Oligonucleotide 8 in an Artificial Bilayer. The incorporation of the
oligonucleotide 8 (concentration, 50 nm) in an artificial bilayer was principally performed as described
above for compound 7b. The laser irradiation of the cyanine-5-labelled oligomer was performed at
635 nm with an irradiation power of 200 mW. Perfusions were conducted for 30 s each, with 250 nm KCl,
10 mm MOPS/Tris: pH 7.0 buffer. Consecutive perfusions of the cis compartment of the bilayer slide were
interrupted by incubation periods of 5 min.
5-Fluoro-1-[(3aR,4R,6R,6aR)-tetrahydro-6-(hydroxymethyl)-2,2-dipropylfuro[3,4-d][1,3]dioxol-4-
yl]pyrimidine-2,4(1H,3H)-dione (2) [17]. Anh. 5-fluorouridine (5-FUrd; 1; 2 g, 7.64 mmol) was dissolved
in dry DMF (30 ml). After addition of heptan-4-one (2.2 ml; 15.28 mmol), (EtO)3CH (2 ml;
11.46 mmol), and 4m HCl in 1,4-dioxane (6.8 ml), the mixture was stirred for 22 h at r.t. The raw
product was partitioned between 350 ml of CH2Cl2 and 350 ml of conc. aq. NaHCO3, and extracted. The
org. layer was washed with H2O (350 ml), dried (Na2SO4), filtered, and evaporated to dryness. Residual
DMF was removed at 408 in high vacuum. The resulting yellowish oil was crystallized from CH2Cl2 at 48
overnight, and the crystals were again dried at 408 in high vacuum. Yield: 1.88 g, (69%). Colorless
needles. M.p. 1558. Rf (CHCl3/MeOH 9 :1) 0.45. 1H-NMR ((D6)DMSO): 11.83 (s, HꢀN(3)); 8.16 (d,
3J(6,F)¼5.0, HꢀC(6)); 5.84 (s, HꢀC(1’)); 5.14 (t, 3J¼7.5, HOꢀC(5’)); 4.90–4.89 (m, HꢀC(2’)); 4.77–4.75
2
(m, HꢀC(3’)); 4.11–4.09 (m, HꢀC(4’)); 3.63–3.55 (m, J¼ ꢀ10.0, CH2(5’)); 1.68–1.64 (m, CH2(a’));
1.53–1.50 (m, CH2(a)); 1.45–1.37 (m, CH2(b’)); 1.33–1.24 (m, CH2(b)); 0.91 (t, 3J¼5.0, Me(g’)); 0.87 (t,
3J¼5.0, Me(g)). 13C-NMR ((D6)DMSO): 157.0 (d, 2J(4,F)¼ ꢀ26.28, C(4)); 149.0 (C(2)); 139.8 (d,
2
1J(5,F)¼230.49, C(5)); 125.9 (d, J(6,F)¼ ꢀ34.70, C(6)); 116.4 (OꢀCꢀO); 91.1 (C(4’)); 86.68 (C(1’));
83.9 (C(2’)); 80.5 (C(3’)); 61.2 (C(5’)); 38.7 (C(a’), C(a)); 16.9 (C(b’)); 16.3 (C(b)); 14.1 (C(g’)); 14.0
(C(g)).
5-Fluoro-1-[(3aR,4R,6R,6aR)-tetrahydro-6-{[(4-methoxyphenyl)(diphenyl)methoxy]methyl}-2,2-
dipropylfuro[3,4-d][1,3]dioxol-4-yl]pyrimidine-2,4(1H,3H)-dione (3). Compound 2 (0.5 g, 1.4 mmol)
was evaporated trice from anh. pyridine and then dissolved in anh. pyridine (4 ml). Then, (4-

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
2217
methoxyphenyl)(diphenyl)methyl chloride (MMTrCl; 0.53 g, 1.67 mmol) was added under N2. The
mixture was stirred for 18 h at r.t., and the reaction was then quenched by addition of MeOH (3.5 ml).
After 10 min, an ice-cold aq. 5% NaHCO3 soln. was added, and the mixture was extracted three times
with CH2Cl2 (80 ml each). The combined org. layers were dried for 30 min (Na2SO4), filtered,
evaporated, and dried in high vacuum to yield a colorless foam. Column chromatography (CC) of the
residue (SiO2 60 H, column: 10ꢂ14 cm; CH2Cl2/MeOH 97:3) afforded one main zone from which
compound 3 (0.8 g, 91%) was isolated. Colorless foam. Rf (CH2Cl2/MeOH 97:3) 0.60. UV (MeOH): 270
(9.000). 1H-NMR ((D6)DMSO): 11.86 (s, HꢀN(3)); 8.05 (d, 3J(6,F)¼4.0, HꢀC(6)); 7.39–7.19 (m,
2 HꢀC(3’’), 4 HꢀC(8’’), 4 HꢀC(9’’), 2 HꢀC(10’’)); 6.87 (d, 3J¼9.0, 2 HꢀC(4’’)); 5.80 (s, HꢀC(1’)); 4.99–
4.97 (m, HꢀC(2’)); 4.66–4.64 (m, HꢀC(3’)); 4.17–4.14 (m, HꢀC(4’)); 3.74 (s, MeO); 3.34–3.31 (m, 2J¼
ꢀ10.25, 1 H, CH2(5’)); 3.14–3.12 (m, 2J¼ ꢀ5.25, 1 H, CH2(5’)); 1.66–1.63 (m, CH2(a’)); 1.50–1.47 (m,
CH2(a)); 1.43–1.36 (m, CH2(b’)); 1.28–1.21 (m, CH2(b)); 0.91 (t, 3J¼7.5, Me(g’)); 0.85 (t, 3J¼7.5,
2
Me(g)). 13C-NMR ((D6)DMSO): 158.2 (C(5’’)); 157.0 (d, J(4,F)¼ ꢀ26.28, C(4)); 148.8 (C(2)); 144.1
1
(C(7’’)); 139.9 (d, J(5,F)¼231.5, C(5)); 134.7 (C(2’’)); 129.9–127.4 (m, C(3’’), C(8’’), C(9’’), C(10’’));
126.8 (d, 2J(6,F)¼ ꢀ5.40, C(6)); 116.8 (OꢀCꢀO); 113.1 (C(4’’)); 91.9 (C(1’’)); 85.9 (C(4’)); 85.6 (C(1’));
83.6 (C(2’)); 80.7 (C(3’)); 64.1 (C(5’)); 54.9 (C(6’’)); 38.7 (C(a’)); 38.6 (C(a)); 16.9 (C(b’)); 16.3 (C(b));
14.1 (C(g’)); 14.0 (C(g)). Anal. calc. for C36H29FN2O7 (630.70): C 68.56, H 6.23, N 4.44; found: C 68.85, H
6.03, N 4.23.
5-Fluoro-1-[(3aR,4R,6R,6aR)-tetrahydro-6-{[(4-methoxyphenyl)(diphenyl)methoxy]methyl}-2,2-
dipropylfuro[3,4-d][1,3]dioxol-4-yl]-3-[(7R,11R)-3,7,11,15-tetramethylhexadec-2-en-1-yl]pyrimidine-
2,4(1H,3H)-dione (4a, (E)þ(Z)). Compound 3 (1 g, 1.59 mmol) was dissolved in anh. THF (10.6 ml).
After addition of phytol (0.61 ml, 1.59 mmol) and Ph3P (0.62 g, 2.38 mmol), the mixture was stirred for
5 min at r.t. under N2 and with exclusion of light. Then, the mixture was cooled to 08, and a 40% soln. of
diethyl azodicarboxylate (DEAD) in toluene (0.69 ml; 2.38 mmol) was added dropwise within 1 min.
After further 5 min of stirring at 08, the mixture was allowed to warm to r.t., and stirring was continued
for 2 h. After evaporation of the solvent in high vacuum (458), the residue was purified by repeated CC
(SiO2, 1st column: 2ꢂ25 cm; AcOEt/petroleum ether (PE) 1:13; 2nd column: 2ꢂ18 cm; AcOEt/PE,
15 :75; each solvent with 1% of Et3N). Yield 1.1 g (76%). Colorless foam. Rf (AcOEt/PE 1:7) 0.55 and
0.60 (E)- and (Z)-isomers, resp.). UV (MeOH): 270 (9,000). 1H-NMR ((D6)DMSO): 8.16 (d, 3J(6cis,F)¼
3
6.5, HcisꢀC(6)); 8.25 (d, J(6,F)¼6.5, HꢀC(6)(E)); 7.38–7.21 (m, 2 HꢀC(3’’), 4 HꢀC(8’’), 4 HꢀC(9’’),
2 HꢀC(10’’)); 6.84 (d, 3J¼9.0, 2 HꢀC(4’’)); 5.84 (s, HꢀC(1’)); 4.99–4.97 (m, HꢀC(2’), HꢀC(2’’’)); 4.67–
4.63 (m, HꢀC(3’)); 4.36 (d, 3J¼5.0, HꢀC(1’’’)(Z)); 4.33 (d, 3J¼5.0, HꢀC(1’’’)(E)); 4.21–4.18 (m,
HꢀC(4’)); 3.72 (s, Me(6’’)); 3.35–3.32 (m, HaꢀC(5’)); 3.15–3.09 (m, HbꢀC(5’)); 2.07 (t, 3J¼7.5,
HꢀC(4’’’)(Z)); 1.87 (t, 3J¼7.5, HꢀC(4’’’)(E)); 1.67 (s, Me(20’’’)); 1.66–1.63 (m, CH2(a’)); 1.51–1.43 (m,
CH2(a), HꢀC(15’’’)); 1.42–1.30 (m, CH2(b’), CH2(5’’’), HꢀC(7’’’), HꢀC(11’’’)); 1.28–0.98 (m, CH2(b),
CH2(6’’’), CH2(8’’’), CH2(9’’’), CH2(10’’’), CH2(12’’’), CH2(13’’’), CH2(14’’’)); 0.91 (t, 3J¼7.0, Me(g’)); 0.84
3
(t, J¼7.0, Me(g)); 0.83–0.78 (m, Me(16’’’), Me(17’’’), Me(18’’’), Me(19’’’)). 13C-NMR ((D6)DMSO):
2
4
158.2 (C(5’’)); 156.1 (d, J(4,F)¼ ꢀ25.78, C(4)); 148.6 (d, J(2,F)¼6.28, C(2)); 144.0 (C(7’’)); 139.4 (d,
1J(5,F)¼229.8, C(5)); 139.8 (s, C(3’’’)(Z)); 139.6 (s, C(3’’’)(E)); 134.7 (C(2’’)); 129.9–126.7 (m, C(3’’),
C(8’’), C(9’’), C(10’’)); 125.6 (d, 2J(6,F)¼ ꢀ32.82, C(6)); 117.8 (C(2’’’)); 116.7 (OꢀCꢀO); 113.1 (C(4’’));
93.2 (C(4’)); 86.3 (C(1’’)); 86.0 (C(1’)); 83.7 (C(2’)); 80.8 (C(3’)); 64.1 (C(5’)); 54.9 (C(6’’)); 39.0 (C(1’’’));
38.7 (C(a’)); 38.5 (C(a)); 36.7–36.5 (m, C(6’’’), C(8’’’), C(10’’’), C(12’’’)); 35.8, 35.7 (2s, C(7’’’), C(11’’’));
27.3 (C(15’’’)); 24.3 (C(5’’’)); 24.0 (C(9’’’)); 23.6 (C(13’’’)); 22.4, 22.3 (2s, C(16’’’), C(17’’’)); 19.5, 19.4 (2s,
C(18’’’), C(19’’’)); 16.9(C(b’)); 16.3 (C(b)); 15.9 (C(20’’’)); 14.1 (C(g’)); 14.0 (C(g)). Anal. calc for
C56H77FN2O7 (909.22): C 73.98, H 8.54, N 3.08; found: C 73.75, H 8.57, N 2.73.
3-[(2Z)-3,7-Dimethylocta-2,6-dien-1-yl]-5-fluorotetrahydro-1-[(3aR,4R,6R,6aR)-6-{[(4-methoxy-
phenyl)(diphenyl)methoxy]methyl}-2,2-dipropylfuro[3,4-d][1,3]dioxol-4-yl]pyrimidine-2,4(1H,3H)-di-
one (4b). Compound 3 (1 g, 1.59 mmol) was dissolved in anh. THF (11 ml) and reacted with nerol
(0.28 ml, 1.59 mmol), Ph3P (0.62 g, 2.38 mmol), and DEAD (40% in toluene, 0.69 ml, 2.38 mmol) as
described for 4a. The raw product was purified by CC (SiO2 60, 2ꢂ30 cm; AcOEt/PE 1:13, containing
1% of Et3N). From the main zone, compound 4b (0.98 g, 79%) was isolated. Colorless oil. Rf (AcOEt/PE
1:7) 0.42. UV (MeOH): 270 (11,500). 1H-NMR ((D6)DMSO): 8.15 (d, 3J(6,F)¼6.0, HꢀC(6)); 7.38–7.20
3
(m, 2 HꢀC(3’’), 4 HꢀC(8’’), 4 HꢀC(9’’), 2 HꢀC(10’’)); 6.85 (d, J¼9.0, 2 HꢀC(4’’)); 5.84 (s, HꢀC(1’));

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
3
3
5.12 (t, J¼6.0, HꢀC(2’’’)); 5.01, 5.00 (2s, HꢀC(2’), HꢀC(6’’’)); 4.66 (t, J¼4.5, HꢀC(3’)); 4.30 (pseudo-
3
quint., J¼7.5, CH2(1’’’)); 4.22–4.20 (m, HꢀC(4’)); 3.73 (s, MeO); 3.35–3.31 (m, HaꢀC(5’)); 3.13–3.11
(m, HbꢀC(5’)); 2.14–2.05 (m, CH2(4’’’), CH2(5’’’)); 1.67–1.59 (m, CH2(a’), Me(8’’’), Me(9’’’), Me(10’’’));
1.50–1.46 (m, CH2(a)); 1.43–1.41 (m, CH2(b’)); 1.27–1.22 (m, CH2(b)); 0.92 (t, 3J¼7.0, Me(g’)); 0.87 (t,
3J¼7.0, Me(g)). 13C-NMR ((D6)DMSO): 158.2 (C(3’’)); 156.1 (d, 2J (4,F)¼ ꢀ25.09, C(4)); 148.6
4
1
(C(7’’)); 144.1 (d, J(2,F)¼25.09, (C(2)); 148.6 (C(7’’)); 144.1 (d, J(5,F)¼229.6, C(5)); 139.4 (C(3’’’));
134.7 (C(2’’)); 131.1 (C(7’’’)); 129.9–126.8 (C(3’’), C(8’’), C(9’’), C(10’’)); 125.7 (d, 2J(6,F)¼32.57, C(6));
123.8 (C(2’’’)); 118.8 (C(6’’’)); 116.7 (OꢀCꢀO); 113.1 (C(4’’)); 93.4 (C(4’)); 86.2 (C(1’’)); 86.0 (C(1’)); 83.8
(C(2’)); 80.9 (C(3’)); 64.2 (C(5’)); 54.9 (C(6’’)); 38.9 (C(1’’’)); 38.7 (C(a’)); 38.5 (C(a)); 31.6 (C(4’’’)); 26.3
(C(5’’’)); 25.4 (C(9’’’)); 22.9 (C(10’’’)); 17.4 (C(8’’’)); 16.9 (C(b’)); 16.2 (C(b)); 14.0 (C(g’), C(g)). Anal.
calc. for C46H55FN2O7 ·0.5 C6H12 (809.02): C 72.75, H 7.60, N 3.46; found: C 72.48, H 7.43, N 3.28.
5-Fluoro-1-[(3aR,4R,6R,6aR)-tetrahydro-6-(hydroxymethyl)-2,2-dipropylfuro[3,4-d][1,3]dioxol-4-
yl]-3-[(7R,11R)-3,7,11,15-tetramethylhexadec-2-en-1-yl]pyrimidine-2,4(1H,3H)-dione (5a, (E)þ(Z)).
Compound 4a ((E)þ(Z); 200 mg, 0.22 mmol) was dissolved in CH2Cl2 (4.5 ml). Then, 4.5 ml of a 4%
soln. of Cl2CHCOOH in CH2Cl2 was added dropwise. The mixture was stirred for 10 min at r.t. and then
washed with H2O, until the aq. phase became neutral. The layers were separated by centrifugation, and
the org. phase was evaporated to dryness. The residue was dissolved in a small volume of AcOEt,
adsorbed to a small amount of SiO2 subjected to CC (SiO2, 2ꢂ15 cm; AcOEt/PE, 1:4) to yield, from the
main zone, 5a (87 mg, 62%). Rf (AcOEt/PE 1:4) 0.41. UV (MeOH): 270 (10,600). 1H-NMR
((D6)DMSO): 8.23 (d, 3J(6,F)¼6.5, HꢀC(6)); 5.89 (s, HꢀC(1’)); 5.18–5.11 (m, HꢀC(5’), HꢀC(2’’));
4.90–4.88 (m, HꢀC(2’), HꢀC(2’’)); 4.78–4.76 (m, HꢀC(3’)); 4.40 (d, 3J¼5.0, HꢀC(1’’)(Z)); 4.39 (d, 3J¼
5.0, HꢀC(1’’)(E)); 4.15–4.14 (m, HꢀC(4’)); 3.62–3.60 (m, HaꢀC(5’)); 3.59–3.57 (m, HbꢀC(5’)); 2.12 (t,
3J¼7.5, HꢀC(4’’)(Z)); 1.92 (t, 3J¼7.5, HꢀC(4’’)(E)); 1.71 (s, Me(20’’)); 1.68–1.65 (m, CH2(a’)); 1.53–1.47
(m, CH2(a), HꢀC(15’’)); 1.46–1.31 (m, CH2(b’), CH2(5’’), HꢀC(7’’), HꢀC(11’’)); 1.29–1.04 (m, CH2(b),
CH2(6’’), CH2(8’’), CH2(9’’), CH2(10’’), CH2(12’’), CH2(13’’), CH2(14’’)); 0.91 (t, 3J¼7.5, Me(g’)); 0.86 (t,
3J¼7.5, Me(g)); 0.85–0.80 (m, Me(16’’), Me(17’’), Me(18’’), Me(19’’)). 13C-NMR ((D6)DMSO): 157.9 (d,
2J(4,F)¼ ꢀ46.38, C(4)); 148.7 (d, 4J(2,F)¼3.77, C(2)); 139.3 (d, 1J(5,F)¼228.75, C(5)); 140.0 (s,
C(3’’)(Z)); 139.6 (s, C(3’’)(E)); 124.5 (d, 2J(6,F)¼ ꢀ34.58, C(6)); 118.7 (s, C(2’’)(Z)); 118.0 (s,
C(2’’)(E)); 116.4 (OꢀCꢀO); 92.1 (C(4’)); 86.9 (C(1’)); 84.1 (C(2’)); 80.5 (C(3’)); 61.2 (C(5’)); 38.7
(C(1’’)); 38.6 (C(a’)); 38.5 (C(a)); 36.6–36.5 (m, C(6’’), C(8’’), C(10’’), C(12’’)); 35.8, 35.7 (2s, C(7’’),
C(11’’)); 27.3 (C(15’’)); 24.2 (C(5’’)); 24.2 (C(9’’)); 24.0 (C(13’’)); 22.4, 22.3 (2s, C(16’’), C(17’’)); 19.50,
19.45 (2s, C(18’’), C(19’’)); 16.9 (C(b’)); 16.2 (C(b)); 15.9 (C(20’’)); 14.1 (C(g’)); 14.0 (C(g)). Anal. calc.
for C36H61FN2O6 (636.88): C 67.89, H 9.65, N 4.40; found: C 67.61, H 9.79, N 4.29. log P ¼ þ12.5ꢁ0.63.
3-[(2Z)-3,7-Dimethylocta-2,6-dien-1-yl]-5-fluoro-1-[(3aR,4R,6R,6aR)-tetrahydro-6-(hydroxy-
methyl)-2,2-dipropylfuro[3,4-d][1,3]dioxol-4-yl]pyrimidine-2,4(1H,3H)-dione (5b). Compound 4b
(1.25 g, 1.63 mmol) was detritylated and purified as described for 5a. CC (SiO2, 2ꢂ7.5 cm, AcOEt/PE
1:4) gave one main zone from which 5b (0.528 g, 66%) was obtained. Colourless oil. Rf (AcOEt/PE 1:4)
0.25. UV (MeOH): 270 (9900). 1H-NMR ((D6)DMSO): 8.23 (d, 3J(6,F)¼7.0, HꢀC(6)); 5.90 (s,
HꢀC(1’)); 5.18–5.13 (m, HꢀC(2’), HꢀC(2’’), HꢀC(6’’)); 4.90–4.88 (m, HOꢀC(5’)); 4.77–4.75 (m,
HꢀC(3)); 4.40 (d, 3J¼7.0, CH2(1’’)); 4.15 (dt, 3J¼3.5, HꢀC(4’)); 3.62–3.57 (m, CH2(5’)); 2.18–2.06 (m,
CH2(4’’), CH2(5’’)); 1.68–1.66 (m, CH2(a’), Me(9’’), Me(10’’)); 1.59 (s, Me(8’’)); 1.53–1.49 (m, CH2(a));
1.44–1.39 (m, CH2(b’)); 1.30–1.52 (m, CH2(b)); 0.93 (t, 3J¼7.0, Me(g’)); 0.91 (t, 3J¼7.0, Me(g)).
13C-NMR ((D6)DMSO): 156.1 (d, 2J(4,F)¼ ꢀ25.65, C(4)); 148.7 (C(2)); 139.4 (d, 2J(5,F)¼228.76,
1
C(5)); 139.5 (C(3’’)); 131.1 (C(7’’)); 124.5 (d, J(6,F)¼34.8, C(6)); 123.8 (C(2’’)); 118.9 (C(6’’)); 116.4
(OꢀCꢀO); 92.1 (C(4’)); 86.9 (C(1’)); 84.1 (C(2’)); 80.5 (C(3’)); 61.2 (C(5’)); 38.7 (C(a’)); 38.6 (C(a));
31.6 (C(4’’)); 25.9 (C(5’’)); 25.4 (C(9’’)); 22.9 (C(10’’)); 17.4 (C(8’’)); 16.9 (C(b’)); 16.2 (C(b)); 14.02
(C(g’)); 13.97 (C(g)). Anal. calc. for C26H39FN2O6 (494.60): C 63.14, H 7.95, N 5.66; found: C 63.08, H
8.13, N 5.69. log P ¼ þ7.65ꢁ0.65.
2-Cyanoethyl [(3aR,4R,6R,6aR)-6-{5-Fluoro-2,4-dioxo-3-[(7R,11R)-3,4-dihydro-3,7,11,15-tetrame-
thylhexadec-2-en-1-yl]pyrimidin-1(2H)-yl}-2,2-dipropyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl]methyl
Di(propan-2-yl)phosphoramidite (6a, (E)þ(Z)). Compound 5a (E)þ(Z); (0.2 g, 0.314 mmol) was
evaporated three times from anh. CH2Cl2 and then dissolved in anh. CH2Cl2 (12 ml). Thereupon, EtNiPr2
(Hꢂnigꢃs base; 101.5 ml, 0.597 mmol) and chloro(2-cyanoethyl)(diisopropyl)phosphine (126 ml,

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
2219
0.565 mmol) were added under N2. The mixture was stirred at r.t. for exactly 15 min. After addition of an
ice-cold 5% aq. NaHCO3 soln. (10 ml), the mixture was extracted three times with CH2Cl2 (5 ml each).
The combined org. layers were dried (Na2SO4) for 1 min under N2 and with cooling. After filtration, the
soln. was evaporated to dryness. The residue was subjected to flash chromatography (0.5 bar, SiO2, 2ꢂ
10 cm; CH2Cl2/acetone 85 :15) within ca. 20 min. Evaporation of the main zone afforded 6a (0.178 g,
68%). Colorless oil. Rf (CH2Cl2/acetone, 85 :15): 0.96. 31P-NMR (CDCl3): 149.93; 149.75.
2-Cyanoethyl [(3aR,4R,6R,6aR)-6-{3-[(2Z)-3,7-Dimethylocta-2,6-dien-1-yl]-5-fluoro-3,4-dihydro-
2,4-dioxopyrimidin-1(2H)-yl}-2,2-dipropyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl]methyl Di(propan-2-
yl)phosphoramidite (6b). Compound 5b (0.2 g, 0.405 mmol) was phosphitylated and purified as
described for 6a to give 6b. Yield: 255 mg (91%). Colorless oil. Rf (CH2Cl2/acetone, 85 :15): 0.96. 31P-
NMR (CDCl3): 149.84; 149.66.
5-Fluoro-1-[(3aR,4R,6aR)-tetrahydro-6-(hydroxymethyl)-2,2-dinonylfuro[3,4-d][1,3]dioxol-4-yl]-
pyrimidine-2,4(1H,3H)-dione (7a) [17]. Anh. 1 (1.0 g, 3.82 mmol) was dissolved in anh. DMF, and
nonadecan-10-one (2.16 g, 7.64 mmol) was added. After addition of (EtO)3CH (1.0 g, 5.73 mmol) and 4m
HCl in 1,4-dioxane (3.4 ml), the mixture was stirred for 48 h at r.t. Then, the mixture was partitioned
between CH2Cl2 (175 ml) and a sat. aq. NaHCO3 soln. The org. layer was washed three times with H2O
(25 ml, each). Both aq. phases were back-extracted with CH2Cl2. All combined org. layers were
evaporated to dryness and dried overnight in high vacuum. The residue was chromatographed twice
(SiO2, 6.5ꢂ12 cm; CH2Cl2/MeOH 95 :5). Evaporation of the corresponding main zone afforded 7a
(1.38 g, 68%). Colorless oil. Rf (CH2Cl2/MeOH 95 :5) 0.56. All anal. data were identical to those reported
in [17].
Small-Scale Labelling of Compound 7a with the N-Hydroxysuccinimide Ester of Eterneon 480ꢁ (!
7b). Eterneon 480ꢃ (5 mg, 0.0095 mmol) and 7a (5.3 mg, 0.0095 mmol) were both dissolved in MeCN
(1.5 ml, each). The soln. of 7a was added dropwise to the fluorophore soln. under N2 and under exclusion
of light within 5 min. The mixture was stirred for 26 h at r.t. The product was purified by CC (SiO2, 2ꢂ
19 cm; CH2Cl2/MeOH 99 :1) to afford 7b. Deep red solid. Rf (CH2Cl2/MeOH 99 :1) 0.5.
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Received April 2, 2013