New method for the preparation of 3'- and 2'-O-phosphoramidites of 2'- and 3'-difluoromethyluridine derivatives.

Article abstract of DOI:10.1081/NCN-120006526

Hydrogenation of 2'-deoxy-2'-difluoromethylene-5'-O-dimethoxytrityluridine (1) and 3'-deoxy-3'-difluoromethylene-5'-O-dimethoxytrityluridine (7), gave the corresponding 2'- and 3'-difluoromethyluridine derivatives 2a and 8a. Detritylation of compounds 2a,

Full text of DOI:10.1081/NCN-120006526

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
NEW METHOD FOR THE PREPARATION OF 3′-
AND 2′-O-PHOSPHORAMIDITES OF 2′- AND 3′-
DIFLUOROMETHYLURIDINE DERIVATIVES
Pawel J. Serafinowski & Catherine A. Brown
To cite this article: Pawel J. Serafinowski & Catherine A. Brown (2002) NEW METHOD FOR THE
PREPARATION OF 3′- AND 2′-O-PHOSPHORAMIDITES OF 2′- AND 3′-DIFLUOROMETHYLURIDINE
DERIVATIVES, Nucleosides, Nucleotides and Nucleic Acids, 21:1, 1-13, DOI: 10.1081/
NCN-120006526
To link to this article: http://dx.doi.org/10.1081/NCN-120006526
Published online: 17 Aug 2006.
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Date: 05 November 2015, At: 23:38

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 21(1), 1–13 (2002)
NEW METHOD FOR THE PREPARATION
OF 30- AND 20-O-PHOSPHORAMIDITES OF
20- AND 30-DIFLUOROMETHYLURIDINE
DERIVATIVES
Pawel J. Serafinowski* and Catherine A. Brown
CRC Centre for Cancer Therapeutics at the Institute of Cancer
Research, 15 Cotswold Road, Sutton,
Surrey, SM2 5NG, UK
ABSTRACT
Hydrogenation of 20-deoxy-20-difluoromethylene-50-O-dimethoxytritylur-
idine (1) and 30-deoxy-30-difluoromethylene-50-O-dimethoxytrityluridine (7),
gave the corresponding 20- and 30-difluoromethyluridine derivatives 2a and
8a. Detritylation of compounds 2a, 2b and 8a, 8b resulted in the formation
of 1-(2-deoxy-2-C-difluoromethyl-b-D-arabino-pentofuranosyl)uracil (3a)
and 1-(3-deoxy-3-C-difluoromethyl-b-D-xylo-pento furanosyl)- uracil (9a)
as well as corresponding minor isomers 3b and 9b. Compounds 3a and
3b were also obtained from 20-deoxy-20-difluoromethylene-30,50-O-(tetra-
isopropyldisiloxane-1,3-diyl)uridine (4). Finally, phosphitylation of 2a and
8a provided the title 20- and 30-O-phosphoramidites 6 and 10.
INTRODUCTION
Various structural modifications to the carbohydrate moiety have been
found to markedly enhance key properties of antisense oligonucleotides1,2. In
particular, the 20- and 30-O-alkyl sugar modifications had been studied
extensively but there was considerably less work on 20- and 30-C-alkyl
*Corresponding author. E-mail: p.serafinowski@icr.ac.uk
1
Copyright # 2002 by Marcel Dekker, Inc.
www.dekker.com

2
SERAFINOWSKI AND BROWN
modifications. Although 20-C-alkyl modified oligodeoxynucleotides have
shown reduced binding affinity for complementary DNA and RNA, they
exhibited increased stability against nucleases with dramatic increases being
seen with 20-C-methyl, methoxymethyl and phenyl groups3. Owing to the
shortcomings of existing synthetic methods, however, oligonucleotides
modified with electronegative 20-trifluoromethyl and 20-fluoromethyl sub-
stituents were investigated to only a limited extent while 20- or 30-difluor-
omethyl modifications were not investigated at all3,4. The difluoromethyl
group has been proposed as an isosteric and isopolar replacement of a
hydroxyl group5–7. The electronegativity of this group is likely to cause the
sugar ring to adopt the 30-endo conformation which is the preferred con-
formation of RNA in DNA=RNA hybrids8. Unlike CF3 substituents, the
CF2H groups can act as a hydrogen bond donor, which may be related to the
enhanced biological activity of the CF2H compound over its CF3 counter-
part9. Thus, ‘‘RNA like’’ hydrogen bonding could result in oligonucleotides
with higher binding affinity for target RNA10. Fluorine has also been iden-
tified as improving lipophilicity and hence may aid the distribution of sub-
sequent modified oligonucleotides within an organism5. Until recently,
the synthesis of 20-deoxy-20-difluoromethyl nucleosides remained
unreported11–13. Dunkel and Reither14 described the preparation of
20-C-a-difluoromethylarauridine via the addition of difluoromethyl-
phenylsulfone to a suitably protected 20-oxouridine derivative15. Deoxy-
genation of the unreactive, tertiary 20-hydroxyl function resulting from this
addition, was, however, not carried out presumably due to problems with its
derivatisation. Similar difficulties were encountered by us during the
attempted synthesis of 30-difluoromethylene-30-deoxythymidine using the
same approach16. Some 30-deoxy-30-C-difluoromethyl nucleosides were
obtained by the treatment of corresponding 20,50-di-O-protected-30-C-formyl
nucleosides with (diethylamino)sulphur trifluoride (DAST)17 but the synth-
eses of the required 30-C-formyl nucleosides are low yielding and some crucial
steps lack stereoselectivity18–21. In view of this, it was of interest to investigate
the synthesis of 20- and 30-C-difluoromethyl substituted nucleoside precursors
and subsequently examine the properties of oligonucleotides modified with 20-
and 30-C-difluoromethyl groups. It was thought that the catalytic hydro-
genation of the 20- and 30-difluoromethylene substituted nucleosides such as
20-deoxy-20-difluoromethylene-50-O-dimethoxytrityluridine and 30-deoxy-30-
difluoromethylene-50-O-dimethoxytrityluridine reported by us previou-
sly22,23, would provide a viable route towards 20- and 30-C-difluoromethyl
substituted nucleosides which in turn could be phosphitylated to furnish the
corresponding phosphoramidites, 6 and 10, required for the automated
synthesis of the target, modified oligonucleotides.

20- AND 30-DIFLUOROMETHYLURIDINE PHOSPHORAMIDITES
3
RESULTS AND DISCUSSION
20-Deoxy-20-difluoromethylene-50-O-dimethoxytrityluridine (1) and
30-deoxy-30-difluoromethylene-50-O-dimethoxytrityluridine (7), required as
starting materials for the hydrogenation step, were prepared from the
corresponding 20- and 30-oxonucleosides following the procedure reported by
us earlier22. Compounds 1 and 7 were hydrogenated in the presence of
palladium on activated carbon (10% Pd) to give the corresponding 20-deoxy-
20-difluoromethyluridine derivatives 2a and 2b as well as 30-deoxy-30-difluor-
omethyluridine derivatives 8a and 8b, respectively. In each case the reaction
showed high regioselectivity for the S isomers, 2a and 8a, with the R isomers,
2b and 8b, being formed only in a small amount. This was thought to be a
result of attack by the catalyst on the least hindered a-face. Neither pair of
diastereoisomers could be resolved at this stage but both the major isomers,
2a and 8a, were obtained pure by column chromatography on silicagel in 73%
and 69% yield, respectively (Scheme). Removal of the dimethoxytrityl group
was carried out only on the crude mixtures of 2a and 2b as well as 8a and 8b
using 80% aqueous acetic acid. Thus, detritylation of the crudecompound 2a
contaminated with 2b afforded 1-(2-deoxy-2-C-difluoromethyl-b-D-arabino-
pentofuranosyl)uracil [(20S)-20-deoxy-20-C-difluoromethyluridine] (3a) and
the corresponding 20R isomer, 1-(2-deoxy-2-C-difluoromethyl-b-D-ribo-pen-
tofuranosyl)uracil [(20R)-20-deoxy-20-C-difluoromethyluridine] (3b), in the
ratio of six to one. Similarly, detritylation of 8a contaminated with 8b gave
1-(3-deoxy-3-C-difluoromethyl-b-D-xylo-pentofuranosyl)uracil
deoxy-30-C-difluoromethyluridine] (9a) and the corresponding 30R isomer,
1-(3-deoxy-3-C-difluoromethyl-b-D-ribo-pentofuranosyl)-uracil
[(30R)-30-
[(30S)-30-
deoxy-30-C-difluoromethyluridine] (9b), in the ratio of eight to one. Each pair
of diastereoisomers could be resolved at this stage by preparative HPLC. The
isolated isomers were fully characterised by their MS and NMR spectra. 1H-
NMR of 3a, 3b, 9a and 9b showed a characteristic triplet of doublets at
around 6 ppm corresponding to the CF2H protons. 19F-NMR was consistent
with the presence of two diastereotopic fluorine atoms. Thus, 3a displayed a
doublet of doublets at À 117 ppm and a doublet of doublets at À 121 ppm
whereas compounds 3b, 9a and 9b showed two doublets of doublets of
doublets each, between À 112 and À 123 ppm. These findings are in agree-
ment with available literature data19,24. Configurations at the 20-carbon and
30-carbon for compounds 3a, 3b, 9a and 9b were assigned by 2D NOESY
studies which is discussed in detail in the experimental section.
Compounds 3a and 3b were also obtained via a different route star-
ting from 20-deoxy-20-difluoromethylene-30,50-O-(tetraisopropyldisiloxane-
1,3-diyl)uridine (4). 20-Deoxy-20-difluoromethylene-30,50-O-(tetraisopropyl-

4
SERAFINOWSKI AND BROWN
Scheme. Synthesis of 30- and 20-O-Phosphoramidites of 20- and 30-Difluoromethyluridine (i)
10% palladium on activated carbon, EtOH; (ii) 80% triethylamine trihydrofluoride, THF; (iii)
80% aqueous acetic acid; (iv) dimethoxytrityl chloride, pyridine; (v) diisopropyl-ammo-
niumtetrazolide, NCCH2CH2OP(NiPr2)2, CH2Cl2.
disiloxane-1,3-diyl)uridine (4) was synthesised as described earlier22 and
was subsequently hydrogenated in an atmosphere of hydrogen
over 10% palladium on charcoal to give (20S)-20-difluoromethyl-30,50-O-
(tetraisopropyldisiloxane-1,3-diyl)uridine (5a) and the corresponding 20R
isomer 5b. Compounds 5a and 5b were readily separated by column

20- AND 30-DIFLUOROMETHYLURIDINE PHOSPHORAMIDITES
5
chromatography on silica gel. The hydrogenation was rather low yielding,
45% overall yield, and took seventy two hours to complete but resulted in a
more favourable, 3:2, ratio of 20S=20R isomers. It was of particular impor-
tance in view of the hydrogenation of compound 1 leading to the mixture of
20S=20R isomers in the ratio of 6:1 and paved the way for future preparation
of the target phosphoramidite 6 having the 20-difluoromethyl group in the R
configuration. The observed stereoselectivity was found to be similar to that
observed by Cicero and co-workers during the hydrogenation of 20-deoxy-20-
methylene-30,50-O-(tetraisopropyldisiloxane-1,3-diyl)uridine25. Subsequent
desilylation of compounds 5a and 5b with triethylamine trihydrofluoride26
gave 1-(2-deoxy-2-C-difluoromethyl-b-D-arabino-pentofuranosyl)uracil (3a)
and 1-(2-deoxy-2-C-difluoromethyl-b-D-ribo-pentofuranosyl)uracil (3b),
respectively, in nearly quantitative yields. 1-(2-Deoxy-2-C-difluoromethyl-b-
D-arabino-pentofuranosyl)uracil (3a) was tritylated with dimethoxytrityl
chloride in pyridine to give 1-(2-deoxy-2-C-difluoromethyl-5-O-dimethoxy-
trityl-b-D-arabino-pentofuranosyl)uracil (2a) (Scheme) thus providing an
alternative route for the preparation of compound 2a required for the
synthesis of the target phosphoramidite 6.
The methodology for the synthesis of 20-deoxy-20-difluoromethyl
nucleosides and 30-deoxy-30-difluoromethyl nucleosides described in this
report combines four relatively simple and high yielding steps of oxidation,
difluoromethylenation, desilylation and catalytic hydrogenation. This route
is considerably simpler and more efficient than the methods suggested so far
for similar compounds and would appear to be the method of choice for a
large scale preparation of such derivatives.
Compounds 2a and 8a were phosphitylated with 2-cyanoethyl-bis-dii-
sopropylamino-phosphine in the presence of diisopropylammonium tetra-
zolide27 to give the corresponding phosphoramidites 6 and 10 in 70 and 85%
respectively. The compounds were characterised by means of their MS and
NMR. The 31P-NMR confirmed the formation of two pairs of diastereo-
isomers. Each compound showed two singlets of roughly equal intensity
between À 150 and À 153 ppm. The new phosphoramidite monomers are
currently being used for the preparation of 20- and 30-C-difluoromethylated
oligonucleotides having (30–50) or (20–50)-internucleotide linkages and the
results will be published elsewhere11.
EXPERIMENTAL
Melting points were determined on a Reichert micro hot stage appa-
ratus and are uncorrected. UV spectra were measured in 95% ethanol with a
Pye-Unicam SP-8-150 UV-vis spectrophotometer. 1H and 19F-NMR spectra,
were recorded at 250 MHz using a Bruker WH-250 spectrometer with TMS
or CFCl3 as internal standards. The protons of 20-OH, 30-OH, 50-OH, and

6
SERAFINOWSKI AND BROWN
1
NHCO were exchangeable with D2O. 13C-NMR spectra with H decoupling
were recorded at 100 MHz using a Bruker AMX400. Unless otherwise
indicated, DMSO-d6 was used as the solvent. Phase sensitive NOESY was
run at 400 MHz on a Bruker AMX-400 using the Bruker software package
with D1 ¼ 1.47 and D8 ¼ 0.5 sec. 31P-NMR spectra were recorded at
400 MHz on a Bruker AMX-400 spectrometer with 85% aqueous H3PO4 as
an external standard. Mass spectra were obtained on a VG ZAB-SE spec-
trometer with FAB ionisation. Accurate masses were determined with
MNOBA þ Na as the matrix. HPTLC was run on Merck Kieselgel 60F254
analytical plates in the following systems: A CH2Cl2=EtOAc (4:1), B
CH2Cl2=EtOH (19:1), C CHCl3=MeOH (9:1), D Hexane=Acetone (6:4), E
CH2Cl2=EtOAc (1:1), Merck Kieselgel 60H was used for short column
chromatography.
Reverse phase HPLC was performed using a Waters chromatography
system with a variable wavelength detector set at 254 nm and 280 nm. Col-
umns Apex WP ODS (250610 mm id, 7 m, 300 A) used for analytical and
preparative scales were supplied by Jones Chromatography. The mobile
phases were A 0.05 M aqueous [Et3NH]þ [CH3COO]À and B MeCN. Sol-
vent removal was performed in vacuo at 30–40ꢀC. Other solvents used in
reactions were purchased anhydrous from Aldrich. Solvents for chromato-
graphy were BDH GPR grade reagents.
20-Deoxy-20-difluoromethylene-50-O-dimethoxytrityluridine (1), 30-
deoxy-30-difluoromethylene-50-O-dimethoxytrityluridine (7) and 20-deoxy-20-
difluoromethylene-30,50-O-(tetraisopropyl-disiloaxane-1,3-diyl)uridine
(4)
were prepared as described earlier (22). Palladium on activated carbon (10%
Pd) was purchased from Aldrich. Triethylamine trihydrofluoride was pur-
chased from Fluka.
Hydrogenation of 20-Deoxy-20-difluoromethylene-50-O-dimethoxytrityl-
uridine(1) and 30-Deoxy-30-difluoromethylene-50-O-dimethoxytrityluridine (7)
(General Procedure). To a solution of 20-deoxy-20-difluoromethylene-50-O-
dimethoxytrityluridine (1) or 30-deoxy-30-difluoromethylene-50-O-dimethoxy-
trityluridine (7) (0.58 g, 1 mmol) in dry ethanol (80 mL) 10% palladium on
activated carbon (0.58 g for 1 and 0.29 g for 7) was added. The apparatus
was evacuated and flushed with hydrogen three times. Each solution was then
stirred at rt under an atmosphere of hydrogen for 7 h (1) or 18 h (7). The
apparatus was evacuated and flushed with argon three times, the catalyst was
filtered off (glass microfibre filter) and the filtrate was concentrated in vacuo.
Each colourless residue was dissolved in dry dichloromethane (3 mL) and
chromatographed on silicagel eluting with CH2Cl2=EtOH (96:4) for 2a or
CH2Cl2=EtOH (97:3) for 8a.
1-(2-Deoxy-2-C-difluoromethyl-5-O-dimethoxytrityl-b-D-arabino-pento-
furanosyl)-uracil [(20S)-20-deoxy-20-C-difluoromethyl-50-O-dimethoxytrityluri-

20- AND 30-DIFLUOROMETHYLURIDINE PHOSPHORAMIDITES
7
dine] (2a). Yield: 0.42 g (73%); Rf 0.56 (B) 0.33 (D); colourless glass;
1H-NMR d 3.16 (m, 1H, H-20), 3.27 (m, 2H, H-50, H-500), 3.74 (s, 6H,
OCH3), 3.86 (m, 1H, H-40), 4.44 (m, 1H, H-30), 5.27 (d, 1H, H-5,
J ¼ 8.17 Hz), 5.73 (m, 1H, 30-OH), 6.08 (t of d, 1H, CF2H, JHF ¼ 50.5 Hz,
JHH ¼ 4.72 Hz), 6.27 (d, 1H, H-10, J ¼ 7.50 Hz), 6.85–7.31 (m, 13H, trityl),
7.72 (d, 1H, H-6, J ¼ 8.17 Hz), 11.36 (s, 1H, NH); 19F-NMR d-132.3 (dd,
2F, JHgemF ¼ 49.6 Hz, JFF ¼ 74.9 Hz); observed FAB MS 579.1960, [C31H30-
F2N2O7-H]À requires 579.1943.
1-(3-Deoxy-3-C-difluoromethyl-5-O-dimethoxytrityl-b-D-xylo-pentofura-
nosyl)uracil [(30S)-30-deoxy-30-C-difluoromethyl-50-O-dimethoxytrityluridine]
(8a). Yield: 0.403 g (69.4%); Rf 0.52 (B), 0.29 (D); white solid, mp
1
ꢀ
105–107 C; H-NMR d 2.92 (m, 1H, H-30), 3.24 (m, 2H, H-50, H-500), 3.71 (s,
6H, OCH3), 4.30 (m, 1H, H-20), 4.45 (m, 1H, H-40), 5.52 (d, 1H, H-5,
J ¼ 8.10 Hz), 5.74 (d, 1H, H-10, J ¼ 3.40 Hz), 5.86 (d, 1H, 20-OH,
J ¼ 5.57 Hz), 6.14 (t of d, 1H, CF2H, JHF ¼ 61.5 Hz, JHH ¼ 5.86 Hz ), 6.85–
7.42 (m, 13H, trityl), 7.55 (d, 1H, H-6, J ¼ 8.10 Hz), 11.34 (s, 1H, NH); 19F-
NMR d À 112.80 1F d (JFF ¼ 267 Hz) of d (JHgemF ¼ 46.4 Hz) of d
0
(JH3 F ¼ 13.56 Hz), À 118.17 1F d (JFF ¼ 267 Hz) of d (JHgemF ¼ 55.4 Hz) of
d (JH3 F ¼ 16.4 Hz); observed ES MS 603.1940, [C31H30F2N2O7 þ Na]þ re-
0
quires 603.1919.
Detritylation of Crude 1-(2-Deoxy-2-C-difluoromethyl-5-O-dimethoxy-
trityl-b-D-arabino-pentofuranosyl)uracil (2a=2b) or 1-(3-Deoxy-3-C-di- fluor-
omethyl-5-O-dimethoxytrityl-b-D-xylo-pentofuranosyl)uracil (8a=8b) (General
Procedure). Crude 1-(2-deoxy-2-C-difluoromethyl-5-O-dim- ethoxytrityl-b-
D-arabino-pentofuranosyl)uracil (2a=2b) or 1-(3-deoxy-3-C-difluoromethyl-
5-O-dimethoxytrityl-b-D-xylo-pentofuranosyl)uracil
(8a=8b)
(0.58 g,
1 mmol) was dissolved in 80% aqueous acetic acid (10 mL) and the solution
was stirred at rt for 30 min. The solvent was removed in vacuo, each residue
was coevaporated with toluene (2620 mL) and partitioned between
CHCl3=H2O (1:4, 50 mL). Each aqueous layer was extracted with chloro-
form (4620 mL) and concentrated in vacuo. Each colourless residue was
dissolved in methanol (10 mL), silicagel (0.5 g) was added, and the suspen-
sion was concentrated in vacuo. Each residue was treated with
CHCl3=MeOH (97.5:2.5) (5 mL) and the slurry was chromatographed on
silicagel eluting with CHCl3=MeOH (92.5:7.5) to give isomeric mixtures
3a=3b and 9a=9b as colourless froths. Each froth was dissolved in water
(2 mL) and freeze dried to give compounds 3a=3b and 9a=9b as white
powders. Analysis of 3a=3b by reverse phase HPLC (gradient elution; 5% B–
60% B over 26 min) showed the main product at retention time of 7.7 min
and a minor component at 8.35 min. Preparative HPLC followed by freeze
drying resulted in compounds 3a and the isomeric 3b. Both the compounds
were found to be > 98% pure by analytical reverse-phase HPLC. Analysis of

8
SERAFINOWSKI AND BROWN
9a=9b by reverse phase HPLC (gradient elution; 5% B–60% B over 26 min)
showed the main product at retention time of 7.62 min and a minor com-
ponent at 8.23 min. Preparative HPLC followed by freeze drying resulted in
compounds 9a and the isomeric 9b. Both the compounds were found to be
> 98% pure by analytical reverse-phase HPLC.
1-(2-Deoxy-2-C-difluoromethyl-b-D-arabino-pentofuranosyl)uracil [(20S)-
20-deoxy-20-C-difluoromethyluridine] (3a). Yield: 0.14 g (51.4%); Rf 0.14
1
ꢀ
(C); white solid, mp 172–176 C; H-NMR d 3.02 (m, 1H, H-20), 3.63 (m, 3H,
H-40, H-50, H-500), 4. 33 (t, 1H, H-30 J ¼ 7.69 Hz), 4.95 (bs, 1H, 50-OH), 5.61
(d, 1H, H-6, J ¼ 8.12 Hz), 5.83 (d, 1H, 30-OH, J ¼ 7.25 Hz), 6.02 (t of d, 1H,
CF2H, JHF ¼ 45.6 Hz, JHH ¼ 4.47 Hz ), 6.21 (d, 1H, H-10, J ¼ 7.75 Hz), 7.84
(d, 1H, H-6, J ¼ 8.12 Hz), 11.41 (bs, 1H, NH); 13C NMR d 51.60 (t,
JC-F ¼ 19 Hz, C-20), 57.96 (C-50), 66.31 (C-30), 81.04 (C-10), 83.84 (C-40),
100.44 (C-5), 114.54 (t, JC-F ¼ 240 Hz, CF2H), 140.18 (C-6), 149.31 (C-2),
162.13 (C-4); 19F-NMR
d
À 117.17 (1F,
d
(JFF ¼ 292Hz) of
d
(JHgemF ¼ 54.6 Hz), À 121.46 (1F, d (JFF ¼ 292 Hz) of d (JHgemF ¼ 53.53 Hz);
UV ꢀmax259 nm "max 6468, ꢀmin 229 nm "min 1564; Observed FAB MS
279.0770, [C10H12F2N2O5 þ H]þ requires 279.0793.
1-(2-Deoxy-2-C-difluoromethyl-b-D-ribo-pentofuranosyl)uracil [(20R)-20-
deoxy-20-C-difluoromethyluridine] (3b). Yield 0.024 g (8.6%); Rf 0.16 (C);
1
colourless foam; H-NMR d 2.89 (m, 1H, H-20), 3.59 (m, 2H, H-50, H-500),
3.85 (m, 1H, H-40), 4.36 (d, 1H, H-30, J ¼ 4.48 Hz), 5.12 (bs, 1H, 50-OH), 5.69
(d, 1H, H-5, J ¼ 8.13 Hz), 5.79 (bs, 1H, 30-OH), 6.33 (d, 1H, H-10,
J ¼ 8.53 Hz), 6.19 (t of d, 1H, CF2H, JHF ¼ 55.4 Hz, JHH ¼ 6.80 Hz), 7.85 (d,
1H, H-6, J ¼ 8.13 Hz), 11.32 (bs, 1H, NH); 19F-NMR d À 114.58 (1F, d
0
(JFF ¼ 295 Hz) of d (JHgemF ¼ 54.1 Hz) of d (JH2 F ¼ 10.0 Hz), À 123.51 (1F,
0
d (JFF ¼ 295 Hz) of d (JHgemF ¼ 55.9 Hz) of d (JH2 F ¼ 14.3 Hz); UV ꢀmax
260 nm "max 7426, ꢀmin 230 nm "min 1098; Observed FAB MS 279.0702,
[C10H12F2N2O5þH]þrequires 279.0793.
1-(3-Deoxy-3-C-difluoromethyl-b-D-xylo-pentofuranosyl)uracil [(30S)-30-
deoxy-30-C-difluoromethyluridine] (9a). Yield: 0.211 g (76%); Rf 0.16 (C);
1
colourless glass; H-NMR d 2.87 (m, 1H, H-30), 3.57 (m, 2H, H-50, H-500),
4.30 (m, 2H, H-20, H-40), 5.32 (bs, 1H, 50-OH), 5.71 (m, 3H, H-10, 20-OH, H-
5), 6.31 (t of d, 1H, CF2H, JHF ¼ 49.08 Hz, JHH ¼ 6.88 Hz ), 7.84 (d, 1H, H-6,
13
J ¼ 8.16 Hz), 11.34 (bs, 1H, NH); C NMR d 45.66 (C-20), 49.38 (t, JC-
¼ 19.5 Hz, C-30), 60.58 (C-50), 76.90 (C-40), 87.96 (C-10), 102.40 (C-5),
F
116.96 (t, JC-F ¼ 251 Hz, CF2H), 140.69 (C-6), 150.92 (C-2), 163.03 (C-4);
19F-NMR d À 112.08 1F d (JFF ¼ 294 Hz) of d (JHgemF ¼ 55.55 Hz) of d
0
(JH3 F ¼ 11.57 Hz), À 116.75 1F d (JFF ¼ 294 Hz) of d (JHgemF ¼ 56.3 Hz) of
0
d (JH3 F ¼ 15.08 Hz); UV ꢀmax 260 nm "max 8778 ꢀmin 229 nm "min 3436;
Observed ES MS 279.0802, [C10H12F2N2O5 þ H]þ requires 279.0793.

20- AND 30-DIFLUOROMETHYLURIDINE PHOSPHORAMIDITES
9
1-(3-Deoxy-3-C-difluoromethyl-b-D-ribo-pentofuranosyl)uracil [(30R)-30-
deoxy-30-C-difluoromethyluridine] (9b). Yield: 0.025 g (9%); Rf 0.20 (C);
colourless glass; 1H-NMR d 2.73 (m, 1H, H-30), 3.52 (m, 1H, H-50), 3.75 (m,
1H, H-500), 4.34 (m, 3H, H-20, H-40, 50-OH), 5.61 (d, 1H, J ¼ 8.24 Hz, H-5)
5.68 (bs, 2H, 30-OH, H-10), 6.20 (t, JHF ¼ 56.1 Hz of d JHH ¼ 5.28 Hz, 1H,
CF2H), 7.98 (d, 1H, H-6, J ¼ 8.24 Hz), 11.30 (bs, 1H, NH); 19F-NMR
0
À 116.03 1F d (JFF ¼ 290 Hz) of d (JHgemF ¼ 55.6 Hz) of d (JH3 F ¼ 10.06 Hz),
0
À 122.70 1F d (JFF ¼ 290 Hz) of d (JHgemF ¼ 57.1 Hz) of d (JH3 F ¼ 18.7 Hz);
UV ꢀmax 262 nm "max 8885 ꢀmin 230 nm "min 2020; Observed FAB MS
301.0600, [C10H12F2N2O5þNa]þ requires 301.0612. The stereochemistry of
the products was confirmed by a series of 2D NOESY experiments.
3a – 2D NOESY showed a cross peak between H-20 and H-40 and at the same
time the lack of a cross peak between H-20 and H-50. There was also a cross
peak between H-6 and CF2H. This is only possible if the H-20 proton is on
the a face.
3b – 2D NOESY showed a cross peak between H-20 and H-5 and at the same
time the lack of a cross peak between H-20 and H-40. This is only possible if
the H-20 proton is on the b face.
9a – 2D NOESY showed a cross peak between between H-50 and CF2H and
at the same time the lack of a cross peak between H-30 and H-50. There was
also a cross peak between H-6 and CF2H. This is only possible if the H-30
proton is on the a face.
9b – 2D NOESY showed a cross peak between H-40 and CF2H and at the
same time the lack of a cross peak between H-50 and CF2H. There was also a
cross peak between H-30 and H-6. This is only possible if the H-30 proton is
on the b face.
Hydrogenation of 20-Deoxy-20-difluoromethylene-30, 50-O-(tetraisopropyl-
disiloxane-1,3-diyl)uridine (4). To a solution of 20-deoxy-20-difluoro- me-
thylene-30,50-O-(tetraisopropyldisiloxane-1,3-diyl)uridine
(4)
(0.22 g,
0.42 mmol) in dry ethanol (35 mL), 10% palladium on activated carbon
(0.10 g) was added. The apparatus was evacuated and flushed with hydrogen
three times. The solution was then stirred at rt under an atmosphere of hy-
drogen for 72 h. The apparatus was evacuated and flushed with argon three
times, the catalyst was filtered off (glass microfibre filter) and the filtrate was
concentrated in vacuo. The colourless residue was dissolved in dry di-
chloromethane (3 mL) and chromatographed on silicagel eluting with
CH2Cl2=EtOAc (4:1).
1-[2-Deoxy-2-C-difluoromethyl-b-D-arabino-pentofuranosyl-3,5-O-(tetra-
isopropyl-disiloxane-1,3-diyl)]uracil [(20S)-20-deoxy-20-C-difluoromethyl-30,50-
O-(tetraisopropyldisiloxane-1,3-diyl)uridine] (5a). Yield: 0.06 g (27%); Rf
1
0.55 (A); colourless foam; H-NMR d 0.83–1.13 (m, 24H, iPr), 3.29 (m, 1H,

10
SERAFINOWSKI AND BROWN
H-20), 3.50 (m, 4H, iPr), 3.29 (m, 1H, H-20), 3.84 (m, 1H, H-40), 3.93–4.10 (m,
2H, H-50, H-500), 4.65 (m, 1H, H-30), 5.60 (d, 1H, H-5, J ¼ 7.92 Hz), 5.97 (t of
d, 1H, CF2H, JHF ¼ 54.32 Hz, JHH ¼ 3.22 Hz), 6.21 (d, 1H, H-10,
J ¼ 7.82 Hz), 7.52 (d, 1H, H-6, J ¼ 7.92 Hz), 11.41 (s, 1H, NH); 19F-NMR d
À 117.10 (1F, d of m), À 122.10 (1F, d of m); Observed ES MS 543.2124,
[C22H38F2N2O6Si2þNa] þ requires 543.2134.
1-[2-Deoxy-2-C-difluoromethyl-b-D-ribo-pentofuranosyl-3,5-O-(tetraiso-
propyldisiloxane-1,3-diyl)]uracil [(20R)-20-deoxy-20-C-difluoromethyl-30,50-O-
(tetraiso-propyldisiloxane-1,3-diyl) uridine] (5b). Yield: 0.04 g (18%); Rf
1
0.43 (A); colourless foam; H-NMR d 0.81–1.12 (m, 24H, iPr), 3.31 (m, 1H,
H-20), 3.68 (m, 4H, iPr), 3.77–4.11 (m, 3H, H-40, H-50, H-500), 4.76 (m, 1H, H-
30), 5.66 (d, 1H, H-5, J ¼ 8.10Hz), 6.07 (d, 1H, H-10, J ¼ 5.43 Hz), 6.26 (t of d,
1H, CF2H, JHF ¼ 54.60 Hz, JHH ¼ 4.13 Hz ), 7.65 (d, 1H, H-6, J ¼ 8.10 Hz),
11.38 (s, 1H, NH); 19F-NMR d À 117.85 (1F, d (JFF ¼ 306 Hz) of d
0
(JHgemF ¼ 57.75 Hz) of d (JH2 F ¼ 11.0 Hz) , À 123.56 (1F d (JFF ¼ 307 Hz) of
0
d (JHgemF ¼ 58.88 Hz) of d (JH2 F ¼ 22.6 Hz); Observed ES MS 543.2120,
[C22H38F2N2O6Si2þNa]þ requires 543.2134.
Desilylation of 1-[2-Deoxy-2-C-difluoromethyl-b-D-arabino-pento fur-
ano- syl-3,5-O-(tetraisopropyldisiloxane-1,3-diyl)]uracil (5a) and 1-[2-Deoxy-
2-C-difluoromethyl-b-D-ribo-pentofuranosyl-3,5-O-(tetraisopropyldisil-
oxane-1,3-diyl)]uracil (5b). Compounds 5a (0.06 g,0.11 mmol) and 5b
(0.04 g, 0.08 mmol) were dissolved in THF (3 mL) for 5a and 2 mL for 5b.
Triethylamine trihydrofluoride 98% (0.2 mL, 0.2 g, 12.3 mmol) (5a) or
(0.13 mL, 0.13 g, 8.2 mmol) (5b) was added and each solution was stirred at
rt for 2 h. The solvent was removed in vacuo and the organic layer was
washed with 5% aqueous sodium bicarbonate (10 mL), water (10 mL), brine
(10 mL), dried over anhydrous sodium sulphate and concentrated in vacuo
to give compounds 3a and 3b.
1-(2-Deoxy-2-C-difluoromethyl-b-D-arabino-pentofuranosyl)uracil
(3a). Yield: 0.031 g (96%).
1-(2-Deoxy-2-C-difluoromethyl-b-D-ribo-pentofuranosyl)uracil
(3b). Yield: 0.019 g (90%). The spectroscopic and analytical data were
consistent with those quoted above.
Dimethoxytritylation of 1-(2-Deoxy-2-C-difluoromethyl-b-D-arabino-
pentofuranosyl)uracil (3a). Compound 3a (0.03 g, 0.1 mmol) and di-
methoxytrityl chloride (0.041 g, 0.12 mmol) were dissolved in dry pyridine
ꢀ
(1.5 mL) and the solution was stirred at 50 C for 4 h and then at rt for 18 h.
The solvent was removed in vacuo and the residue was partitioned between
CHCl3=sat. aq. NaHCO3 (2:1, 20 mL), the organic layer was washed with

20- AND 30-DIFLUOROMETHYLURIDINE PHOSPHORAMIDITES
11
H2O (265 mL) and brine (10 mL), dried (Na2SO4) and concentrated in
vacuo. The residue was dissolved in CH2Cl2 (2 mL) and chromatographed on
silicagel, eluting with CH2Cl2=EtOH (24:1), to give compound 2a as a col-
ourless glass.
1-(2-Deoxy-2-C-difluoromethyl-5-O-dimethoxytrityl-b-D-arabino-pento-
furanosyl)uracil (2a).
Yield: 0.045 g (72%). The spectroscopic and analytical data were con-
sistent with those quoted above.
Phosphitylation of 1-[2-Deoxy-2-C-difluoromethyl-5-O-dimethoxytrityl-b-D-
arabino-pentofuranosyl]uracil (2a) and 1-(3-Deoxy-3-C-difluoromethyl-5-O-dimeth-
oxytrityl-b-D-xylo-pentofuranosyl]uracil (8a) (General Procedure). Compounds
2a or 8a (0.58 g, 1 mmol) and bis-diisopro- pylammonium tetrazolide (0.180 g,
1.05 mmol) were dissolved in dry dichloromethane (10 mL) and the solution was
stirred at rt under argon. 2-Cyanoethoxy-bis-diisopropylaminophosphine (0.37 g,
0.4 mL, 1.2 mmol) in dry dichloromethane (2.5 mL) was added and the mixture
was stirred at rt under argon for 8 h. The reaction was quenched by the addition of
dry dichloromethane (40 mL). The organic layer was washed with 5% aqueous
sodium bicarbonate (10 mL), water (10 mL), brine (10 mL), dried over anhy-
drous sodium sulphate and concentrated in vacuo to give compounds 6 and 10.
1-{2-Deoxy-2-C-difluoromethyl-5-O-dimethoxytrityl-3-O-[(2-cyanoethyl-
N,N-diisopropyl)phosphoramidite]-b-D-arabino-pentofuranosyl}uracil {(20S)-
20-deoxy-20-C-difluoromethyl-50-O-dimethoxytrityluridine-30-O-[(2-cyanoethyl-
N,N-diisopropyl)phosphoramidite]}(6). Yield: 0.55 g (70%); Rf 0.49, 0.36
1
two diasteroisomers (A); colourless glass; H-NMR d 1.05–1.14 (m, 14H,
iPr), 2.59 (m, 1H, H-20), 2.72 (t, 2H, J ¼ 5.50 Hz, OCH2CH2CN), 3.45 (m,
2H, iPr), 3.51 (m, 2H, H-50, H-500), 3. 73 (s, 6H, OCH3), 4.04 (m, 3H,
OCH2CH2CN, H-40), 4.67 (m, 1H, H-30), 5.31 (d, 1H, H-5, J ¼ 9.13 Hz), 6.10
t (JHF ¼ 56 Hz) of m, 1H, CF2H), 6.30 (m, 1H, H-10), 6.87–7.42 (m, 13H,
trityl), 7.65 (2d, unresolved, H-6), 11.39 (bs, 1H, NH); 19F-NMR d À 117.9
(d of m, 1F, CF2H ), À 122.2 (d of m, 1F, CF2H ); 31P-NMR d À 150.18 (s),
À 150.64 (s); Observed FAB MS 779.3050, [C40H47F2N4O8P-H] requires
779.3021.
1-{3-Deoxy-3-C-difluoromethyl-5-O-dimethoxytrityl-2-O-[(2-cyanoethyl-
N,N-diisopropyl)phosphoramidite]-b-D-xylo-pentofuranosyl}uracil {(30S)-30-
deoxy-30-C-difluoromethyl-50-O-dimethoxytrityluridine-20-O-[(2-cyanoethyl-
N,N-diisopropyl)-phosphoramidite]}(10). Yield: 0.764 g(98%); Rf 0.44 (A);
white froth, mp indef; 1H-NMR d (1.21, m, 14H, iPr), 2.88 (t, 2H,
J ¼ 6.82 Hz, OCH2CH2CN), 2.99 (m, 1H, H-30), 3.48(m, 4H, H-50, H-500, iPr),
3.71 (s, 6H, OCH3), 4.04 (m, 3H, OCH2CH2CN, H-40), 4.51 (m, 1H, H-20),
5.59 (d, 1H, H-5, J ¼ 8.11 Hz), 5.89 (d, 1H, H-10, J ¼ 5.62 Hz), 6.18 t

12
SERAFINOWSKI AND BROWN
(JHF ¼ 48.5 Hz) of m, 1H, CF2H), 6.82–7.45 (m, 13H, trityl), 7.55 (d, 1H, H-
6, J ¼ 8.11 Hz), 11.34 (s, 1H, NH); 19F-NMR d À 112.58 - ( À 114.86) (m, 1F,
CF2H ) À 117.11 - ( À 118.99) (m, 1F, CF2H ); 31P-NMR d À 152.502 (s),
À 152.409 (s); Observed FAB MS 779.3063, [C40H47F2N4O8P-H] requires
779.3021.
ACKNOWLEDGMENTS
These investigations were supported by the Cancer Research Campaign.
We thank Mrs. Jane Hawkes of the University of London Intercollegiate
Research Service for 13C-NMR spectra, as well as NOESY and COESY
studies. We also thank Mr. Mike Cocksedge of the University of London
Intercollegiate Research Service for mass spectra.
REFERENCES
1. Cook, P.D. Annual Reports in Medicinal Chemistry 1999, 33, 313–325.
2. Manoharan, M.; Kawasaki, A.M.; Prakash, T.P.; Fraser, M.P.; Inamati, G.P.;
Casper, M.D.; Cook, P.D. Nucleosides & Nucleotides 1999, 18 (6&7), 1737–
1746.
3. Schmit, C.; Beviere, M.O.; De Mesmaeker, A.; Altman, K.H. Bioorg. & Med.
Chem. Lett. 1994, 4, 1969–1976.
4. a) Schmit, C. Synlett 1994, 238–240; b) Schmit, C. Synlett 1994, 241–242.
5. Ternansky, R.J.; Hertel, L.W. In Organo£uorine Compounds in Medicinal
Chemistry and Biomedical Applications; Filler, R., Kobayashi Y., Yagupolskii,
L.M., Eds.; Elsevier: Amsterdam, 1993; 23–73.
6. Blackburn, M.; Eckstein, F.; Kent D.E.; Perre, T.D. Nucleosides & Nucleotides
1985, 4 (1&2), 165–167.
7. Houlton, J.S.; Motherwell, W.B.; Ross, B.C.; Tozer, M.J.; Williams, D.J.; Sla-
win, A.M.Z. Tetrahedron 1993, 49, 8087–8106.
8. Egli, M. Angew. Chem. Int. Ed. Engl. 1996, 35, 1895–1909.
9. Erickson, J.A.; McLoughlin, J.I. J. Org. Chem. 1995, 60, 1626–1631.
10. Egli, M. Adv. Enz. Regul. 1998, 38, 181–203.
11. Preliminary account of this work was presented at 14th International Round
Table, Nucleosides, Nucleotides
&
Their Biological Applications,
San Francisco, 10–14th September, 2000. This presentation will appear in a
special conference issue of Nucleosides, Nucleotides & Nucleic Acids: Serafinows-
ki, P.J., Brown, C.A., Barnes, C.L., Synthesis of Some Fluoroalkyl Substituted
Nucleosides and Oligonucleotides. Nucleosides, Nucleotides & Nucleic Acids
2001, in press.
12. After completion of this manuscript, a paper appeared, ref. 13, in which the
synthesis of compounds 3a and 3b as well as 9a and 9b was described using
different methods than those presented in this manuscript. Thus, an ‘‘insepar-
able’’ mixture of compounds 3a and 3b was obtained starting from 20-deoxy-20-

20- AND 30-DIFLUOROMETHYLURIDINE PHOSPHORAMIDITES
13
difluoromethylene-30, 50-O-(tetraisopropyldisiloxa-ne-1,3-diyl)uridine (4)(22).
The crucial hydrogenation step, however, was performed on the deprotected
20-deoxy-20-difluoromethyleneuridine. Similarly, an ‘‘inseparable’’ mixture of
compounds 9a and 9b was obtained starting from 30-deoxy-30-difluoromethy-
lene-20, 50-O-bis-t-butyldimethylsilyl uridine (22). Again, the hydrogenation
step was performed on the deprotected 30-deoxy-30-difluoromethyleneuridine.
Eventually, compound 3a and its a-anomer were obtained via a multistep ela-
boration of a carbohydrate precursor and its subsequent condensation with si-
lylated uracil. Comparison of the spectroscopic data is difficult since the NMR
spectra were recorded in different solvents.
13. Marcotte, S.; Gerard, B.; Pannencoucke X.; Feasson, C.; Quirion, J-C. Synthe-
sis 2001, 929–933.
14. Dunkel, M.; Reither, V. Bioorg. & Med. Chem. Lett. 1999, 9, 787–792.
15. Sabol, J.S.; Mc Carthy, J.R. Tetrahedron Lett. 1992, 33, 3101–3104.
16. Serafinowski, P.J.; Barnes, C.L. Synthesis 1997, 225–228.
17. Bamford, M.J.; Coe, P.L.; Walker, R.T. J. Med. Chem. 1990, 33, 2488–2494.
18. Yoshimura, J. Adv. Carb. Chem. Biochem. 1984, 42, 69–74.
19. Bamford, M.J.; Coe, P.L.; Walker, R.T. J. Med. Chem. 1990, 33, 2494–2501.
20. Vaseur, J.J.; Debart, F.; Sanghvi, Y.; Cook, P.D. J. Am. Chem. Soc. 1992, 114,
4006–4008.
21. Sanghvi, Y.S.; Bharadwaj, R.; Debart, F.; De Mesmaeker, A. Synthesis, 1994,
1163–1166.
22. Serafinowski, P.J.; Barnes, C.L. Tetrahedron 1996, 52, 7929–7938.
23. Serafinowski, P.J.; Brown, C.A. Tetrahedron 2000, 56, 333–339.
24. North, M. In Principles and Applications of Stereochemistry; Stanley Thornes
(Publishers) Ltd: Cheltenham, 1998; 142–156.
25. Cicero, D.O.; Neuner, P.J.S.; Franzese, O.; D’Onofrio, C.D.; Irribarren, A.M.
Bioorg. & Med. Chem. Lett. 1994, 4, 861–866.
26. Westman, E.; Stromberg, R. Nucleic Acids Research, 1994, 22, 2430–2431.
27. Caruthers, M.H.; Barone, A.D.; Beaucage, S.L.; Dodds, D.R.; Fisher, E.F.;
Mc Bride, L.J.; Matteucci, M.; Stabinsky, Z.; Tang, J.Y. Methods in Enzymol-
ogy 1987, 154, 287–315.
Received June 15, 2001
Accepted October 25, 2001