Synthesis and properties of phosphorylated 3′-O-β-D-ribofuranosyl-2′-deoxythymidine

Article abstract of DOI:10.1081/NCN-120022028

A strategy was developed for the synthesis of 3′-O-β-D-ribofuranosyl 2′-deoxythymidine derivatives using three different protecting groups, which allows the synthesis of a phosphoramidite building block for oligonucleotide synthesis. Likewise the 5′-O- an

Full text of DOI:10.1081/NCN-120022028

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
Synthesis and Properties of Phosphorylated 3′-O-β-
D-Ribofuranosyl-2′-deoxythymidine
Ekaterina V. Efimtseva , Alexandra A. Shelkunova , Sergey N. Mikhailov ,
Koen Nauwelaerts , Jef Rozenski , Eveline Lescrinier & Piet Herdewijn
To cite this article: Ekaterina V. Efimtseva , Alexandra A. Shelkunova , Sergey N. Mikhailov ,
Koen Nauwelaerts , Jef Rozenski , Eveline Lescrinier & Piet Herdewijn (2003) Synthesis and
Properties of Phosphorylated 3′-O-β-D-Ribofuranosyl-2′-deoxythymidine, Nucleosides,
Nucleotides and Nucleic Acids, 22:4, 359-371, DOI: 10.1081/NCN-120022028
To link to this article: http://dx.doi.org/10.1081/NCN-120022028
Published online: 07 Feb 2007.
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Date: 05 November 2015, At: 19:05

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, No. 4, pp. 359–371, 2003
Synthesis and Properties of Phosphorylated 30-O-b-D-
Ribofuranosyl-20-deoxythymidine
Ekaterina V. Efimtseva,1 Alexandra A. Shelkunova,1
Sergey N. Mikhailov,1 Koen Nauwelaerts,2
Jef Rozenski,2 Eveline Lescrinier,2
and Piet Herdewijn2,
*
1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences,
Moscow, Russia
2Rega Institute, Katholieke Universiteit Leuven, Leuven, Belgium
ABSTRACT
A strategy was developed for the synthesis of 30-O-b-d-ribofuranosyl 20-deoxy-
thymidine derivatives using three different protecting groups, which allows the
synthesis of a phosphoramidite building block for oligonucleotide synthesis. Like-
wise the 50-O- and 500-O-phosphorylated analogues were synthesized and their
conformation was determined using NMR spectroscopy.
Key Words: Disaccharide nucleosides; Synthesis; NMR.
1. INTRODUCTION
Recently a general method for the preparation of 20-O-b-d-ribofuranosylnuc-
leosides and some other disaccharide nucleosides and their incorporation into
*Correspondence: Piet Herdewijn, Rega Institute, Katholieke Universiteit Leuven, Minder-
broedersstraat 10, B-3000 Leuven, Belgium; Fax: +32-16337340; E-mail: piet.herdewijn@rega.
kuleuven.ac.be.
359
DOI: 10.1081/NCN-120022028
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

360
Efimtseva et al.
oligodeoxynucleotides (ONs) were developed.[1–4] Tm measurements have demon-
strated that modified ONs form stable duplexes with complementary RNA
(DTm ¼ 0ꢀC).[3] In order to determine the influence of this modification the solution
structure of the self-complementary decaribonucleotide 50-r(GCGXAUUCGC)-
30 (X-20-O-b-d-ribofuranosyadenosine residue, unit I) was studied using high-
resolution NMR spectroscopy and restrained molecular dynamics.[5] Incorporation
of such voluminous substituent into 20-O-position has no profound effect on the ther-
mal stability of the duplex. It was found that the duplex RNA maintains an A-type
helical geometry with extra 20-O-ribose moiety located in the minor groove.[5]
It should be also noted that 20-O-b-d-ribofuranosyladenosine was found in yeast
initiator tRNA in stem region of T domain.[6]
Oligodeoxynucleotides containing 20-O-b-d-ribofuranosyladenosine (unit I) were
prepared and used as modified primers in RNA-templated DNA synthesis catalyzed
by HIV reverse transcriptase.[7] It was shown that the additional 20-ribofuranose resi-
due in specific position (3–4) of primer prevents its elongation. The primer may be
elongated effectively if the modification is shifted further from 30-end.[7]
The presence of additional functionalities in ONs opens new possibilities for
postsynthetic functionalization. The cis-diol group in ONs containing 20-O-b-d-ribo-
furanosylnucleosides (unit I) may be readily oxidized by periodate to give reactive
dialdehyde groups (unit II). Such modified ONs were used successfully for the
affinity labeling of different enzymes of nucleic acids biosynthesis.[8,9]
Molecular modeling reveals that a disaccharide nucleoside with a ribose moiety
linked to the 30-position might be elongated in the 500 direction. Therefore, we
decided to prepare ONs containing 30-O-b-d-ribofuranosyl-20-deoxythymidine (unit
III) and to investigate their properties. In this manuscript we describe the synthesis
of the protected disaccharide nucleoside and its NMR analysis.
2. RESULTS AND DISCUSSION
30-O-b-d-Ribofuranosyl-20-deoxythymidine nucleosides can be prepared[10] by
condensation of blocked nucleoside 1 and 1-O-acetyl-2,3,5-tri-O-benzoyl-b-d-ribo-
furanose (2a) followed by deprotection (Sch. 1). However, this scheme is not useful

Phosphorylated 30-O-b-D-Ribofuranosyl-dT
361
Scheme 1.
for incorporation of this nucleoside in oligonucleotides as no selective deprotection
of the 500 position is possible. Very recently the blocked sugar namely 1,2,3-tri-
O-benzoyl-5-O-phenoxyacetyl-d-ribofuranose (2b) was successfully used for the
preparation of tRNA minor nucleoside O-b-d-ribofuranosyl-(100-20)-guanosine-
500-O-phosphate.[11] Condensation of nucleoside 1 with sugar 2b in the presence of
tin tetrachloride proceeded stereospecifically with the formation of b-anomer 3 in
65% yield. After desilylation, disaccharide nucleoside 5 was monomethoxytritylated.
Selective removing of phenoxyacetyl group with 0.1 M NaHCO3 in methanol gave 6
in good overall yield. Nucleoside 6 was then converted to the corresponding phos-
phoramidite 7 (Sch. 1).
Preparation of Model Compounds for NMR Studies
Two model phosphorylated 30-O-b-d-ribofuranosyl-20-deoxythymidines (10 and
12) were prepared for NMR studies. For the preparation of 500-phosphate 10
we decided to use sugar phosphate synthon (8), which was recently used for the

362
Efimtseva et al.
Scheme 2.
synthesis of 50-nucleotides.[12] Condensation with 1 gave 9 in 35%. After deblocking,
10 was obtained in overall good yield (Sch. 2).
The other nucleotide 12 was prepared according to the Sch. 3 by phosphory-
lation of 11.[10]
The structures of the obtained compounds were proved by mass- and NMR
spectroscopy. Most of the chemical shifts and coupling constants may be calculated
directly from NMR spectra. In some cases comparison with the published spectra of
disaccharide nucleosides and 1H-13C correlation and COSY spectra were used for the
assignment.
Scheme 3.

Phosphorylated 30-O-b-D-Ribofuranosyl-dT
363
Table 1. Measured chemical shifts (ppm) in the ribose and nucleoside moieties.
Compound 12 (rTP)
Compound 10 (PrT)
Nucleus
Ribose moiety
Nucleoside moiety
Ribose moiety
Nucleoside moiety
H10
5.22 d
4.16 dd
6.36 dd
2.58 ddd
2.44 ddd
4.64 ddd
4.34 ddd
4.11 dd
4.11 dd
7.839e
5.17 d
4.17 dd
6.30 dd
2.61 ddd
2.49 ddd
4.51 ddd
4.17 ddd
3.87 dd
3.81 dd
7.669e
H20
H200
H30
H40
H50a
H50b
H6
4.30 dd
4.11 ddd
3.90 dd
3.74 dd
–
4.37 dd
4.19 ddd
4.09 ddd
3.98 ddd
–
HMe
–
1.95 d
–
1.93 d
C10
C20
C30
C40
C50
C2
C4
C5
C6
CMe-5
106.00 s
84.58 s
36.83 s
77.77 s
106.34 s
74.35 s
70.56 s
81.67 dc
65.52 dd
85.27 s
37.03 s
76.98 s
84.30 s
61.13 s
166.46 s
151.04 s
111.37 s
137.63 s
11.46 s
74.01 s
70.17 s
82.30 s
62.24 s
83.30 da
63.94 db
166.03 s
151.23 s
111.25 s
136.93 s
11.10 s
–
–
–
–
–
P
1.99
1.46
–
a
0
JC4 -P ¼ 7.8 Hz.
b
0
JC5 -P ¼ 5.1 Hz.
c
0
JC4 -P ¼ 8.2 Hz.
d
0
JC5 -P ¼ 5.3 Hz.
eJ ¼ 1.2 Hz.
Conformational Study
The conformation of a five membered ring can be completely described by two
independent parameters: phase angle and puckering amplitude. Since the puckering
amplitude always stays within a narrow range and the pseudorotational angle can
adopt values ranging from 0 to 360 degrees, the different conformations are mostly
visualized using the pseudorotational wheel.
It is widely accepted that furanose rings, which are flexible entities, will show a high-
speed equilibrium between two low energy conformations in solution. One of those
conformations is situated in the northern hemisphere of the pseudorotational wheel
(the so called N conformer) and the other in the southern hemisphere (the S conformer).
A complete description of this system now requires five independent parameters.
Four parameters describing the conformers and one parameter describing the frac-
tion of one of the conformers.
The two conformers and their relative abundance can be resolved by NMR
3
spectroscopy, through measurement of the JH,H coupling constants. Because

364
Efimtseva et al.
Table 2. Measured J coupling constants (Hz) in the ribose and nucleoside moieties.
Compound 12 (rTP)
Compound 10 (PrT)
Nucleus
Ribose moiety
Nucleoside moiety
Ribose moiety
Nucleoside moiety
0
JH1 -H2
0
JH1 -H2
0
JH2 -H3
00
JH2 -H3
0
1.5
5.0
8.2
5.7
5.6
3.2
1.6
4.5
7.8
6.2
5.4
3.5
00
0
0
00
JH2 -H2
0
ꢁ13.9
ꢁ14.1
0
JH3 -H4
0
7.8
6.4
2.3
4.3
6.7
6.0
2.7
4.1
0
JH4 -H5 a
0
0
JH4 -H5 b
0
3.0
ꢁ12.6
4.3
4.9
ꢁ12.6
4.7
ꢁ12.4
0
0
JH5 a-H5 b
0
JP-H5 a
0
JP-H5 b
5.2
5.2
4.8
5.9
the equilibrium, mentioned above, can be considered as fast on NMR time scale,
average coupling constants and average chemical shifts will be observed. Assum-
ing the two state model, described above, it is possible to find the conformations
3
and their populations that best fit the experimentally observed JH,H coupling
constants.[13,14]
A 2D TOCSY experiment[15,16] was performed for the assignment of all signals
(Table 1). The presence of a phosphate group was confirmed by 31P spectroscopy
and its correct attachment to the molecule was evident from characteristic downfield
shift of H50a and H50b and their couplings with phosphorous (JH,P). The correct
glycosidic linkage of C10 of the ribose moiety with C30 of the nucleoside moiety could
be confirmed by a NOE-contact between H10 of the ribose and H30 of the nucleoside.
Table 3. Ring conformers of the ribose and nucleoside moieties.
Compound 12 (rTP)
Compound 10 (PrT)
Ribose moiety
Nucleoside moiety
Ribose moiety
Nucleoside moiety
a
PN
Nb
18
3E
0.89
162
2E
ꢁ20.2
2E
0.26
160
2T3
2.2
3T2
0.87
178
2T3
2.4
3T2
0.28
171
2T3
c
XN
a
PS
Sb
c
XS
RMSDd
0.11
0.151
0.74
0.125
0.13
0.130
0.72
0.098
aPhase angle of pseudorotation (degrees).
bType of conformer.
cMolar fraction of the conformer (%).
dRoot Mean Square Deviation. The puckering amplitude was kept 38 degrees in all cases.

Phosphorylated 30-O-b-D-Ribofuranosyl-dT
365
A high-resolution 1D 1H spectrum was used to measure the coupling constants.
3
The JH,H coupling constants of both ribose moieties, measured from spectra
recorded at 292 K, are presented in Table 2. Analysis of these data using Pseurot
6.2,[17] generated the results as shown in Table 3.
It was shown that the additional ribose moiety of both compounds will appear
predominantly in the N conformation, which we could expect because of stereo
electronic effects due to the 20OH.[18] The deoxyribose rings of the nucleoside prefer
the S conformer (74% and 72%) as could be expected.
The presence of a phosphate group on the C50 of the deoxyribose and ribose
moieties causes some conformational changes. The phosphate causes a slight shift
towards the S conformer. (deoxyribose 0.72 vs. 0.74 by 50-O-phosphorylation; ribose
0.11 vs. 0.13 after 500-O-phosphorylation) and a change of pseudorotational angle of
the N conformer (deoxyribose ꢁ20.2 vs. 2.4; ribose: 18 vs. 2.2). In the deoxyribose
moiety, where the effect is more pronounced, the phosphate group is then located
closer to the heterocyclic base.
To probe the O- and N-glycosidic bond populations, NOESY-spectra were
recorded with 150, 300 and 500 ms mixing times respectively. No relevant data could
be obtained, most likely due to fast rotation around these bonds in a fast equilibrium
between several low energy conformations in solution. To obtain more experimental
information on the relative populations of these bond conformations, experiments
on a 13C labeled sample will be necessary.
0
00
00 0 0
Observation of intermediate values of JH5 -P H5 -P JH5 -H4 and JC5 -P indicate free
J
rotation around the C40-C50 and C50-O50 bonds. This can also be confirmed by the
identical chemical shifts of H50 and H500 in the nucleoside moiety of rTP.
CONCLUSIONS
An efficient method is developed for the synthesis of protected 30-O-b-ribofura-
nosyl-20-deoxythymidine, which will allow the incorporation of this disaccharide
nucleoside in oligonucleotides. NMR analysis reveals that phosphorylation of the
disaccharide nucleoside in the 50-O-position or in the 500-O-position leads to a shift
in the pseudo rotational phase angle of about 20ꢀ.
EXPERIMENTAL PART
General
Column chromatography (CC): silica gel (0.06–0.20 mm). TLC: Kieselgel 260 F
(Merck); eluents: CHCl3 (A); CHCl3=MeOH 98:2 (B); CHCl3=MeOH 95:5 (C); iso-
propanol=NH4OH=H2O 7:1:2 (D); detection by UV light. NMR Spectra: Bruker
AMX 400 and Varian Unity 500 NMR spectrometers; at 300 K; chemical shifts d
in ppm were measured relative to the solvent signals (1H and 13C) and relative exter-
nal ref. ¼ H4PO3 (capil.) (31P); coupling constants J in Hz. The signals were assigned
using double resonance techniques and COSY experiments. Mass spectrometry and
exact mass measurements: quadruple=orthogonal-acceleration time-of-flight tandem

366
Efimtseva et al.
mass spectrometer (Q-Tof-2, Micromass, Manchester, UK) equipped with a standard
electrospray ionization (ESI) interface.
NMR Spectra
Samples of 10 and 12 were prepared with 6 mg of both compounds, dissolved in
250 mL D2O.pD was adjusted to 7.2. Spectra were recorded using a Varian 500 Unity
spectrometer at 292 K. Chemical shifts were measured relative to the water signal.
The coupling constants were measured in Hz. The signals were assigned using double
resonance techniques and TOCSY-experiments.[16,17] The TOCSY consisted of 2048
data points in t2 and 256 increments in t1. The data were apodized with a shifted sine-
bell square function in both dimensions and processed to a 2K ꢂ 1K matrix.
Analysis of the couplings was performed with Pseurot 6.2.[17] This program uses
the Altona-Sundaralingam formalism[13,14] to describe the ring puckering and a gen-
eralized Karplus equation developed by Diez et al. in collaboration with Donders
et al.[20] to describe the relation between coupling and dihedral angle. RMS was
minimized, using a Newton Raphson Minimization.
NOESY-spectra[21] were recorded with 150, 300 and 500 ms mixing times respec-
tively. The NOESY consisted of 2048 data points in t2 and 256 increments in t1. The
data were apodized with a shifted sine-bell square function in both dimensions and
processed to a 2K ꢂ 1K matrix.
1-[5-O-tert-Butyldiphenylsilyl-3-O-(2,3-di-O-benzoyl-5-O-phenoxyacetyl-b-d-
ribofuranosyl)-2-deoxy-b-d-ribofuranosyl]thymine (3). To a cooled solution (0ꢀC)
under nitrogen of 1,2,3-tri-O-benzoyl-5-O-phenoxyacetyl-d-ribofuranose (2) (1.15g,
1.93 mmol) in 1,2-dichloroethane (20 mL), SnCL4 (0.27 mL, 2.32 mmol) was added
and the solution was kept at 0ꢀC for 10 min. After addition of 50-O-tert-butyl-
diphenylsilyl-20-deoxythymidine (1) (774 mg, 1.61 mmol), the resulting solution was
kept at 0ꢀC for 1.5 h. The reaction mixture was diluted with CHCl3 (20 mL), 10%
aqueous solution of sodium bicarbonate (20 mL) was added and the suspension
was stirred at 20ꢀC for 20 min. The suspension was filtered through Hyflo Super
Cel, the organic layer was separated, washed with water (20 mL), dried over Na2SO4
and evaporated to dryness. The residue was purified by CC (silica gel 50 g). The col-
umn was washed with system A and then elution with system 0.5% MeOH=CHCl3
gave 3 as a foam. Yield 1.0 g (65%). Rf 0.51 (C). ESI MS (pos.): 955.3470
([C53H54N2O13Si þ H]þ; calc. 955.3473).
1H NMR (CDCl3): 8.63 brs (1H, NH), 8.01–7.89 m (4H, Bz), 7.67–7.23 m (19H,
Bz, Ph, H-6), 6.95 t (1H, J ¼ 7.5, Ph), 6.90 d (2H, J ¼ 7.8, Ph), 6.34 dd (1H,
0
J1 ,2 a ¼ 5.6, J1 ,2 b ¼ 8.1, H-10 Thd), 5.67 dd (1H, J3 ,2 ¼ 5.0, J3 ,4 ¼ 6.8, H-3 Rib),
0
0
0
0
0
0
0
0
5.59 d (1H, H-20 Rib), 5.29 s (1H, H-10 Rib), 4.71 s (2H, PhOCH2), 4.66–4.51 m
(4H, H-30 Thd, H-40,50a,50b Rib), 4.12 m (1H, H-40 Thd), 3.96 dd (1H, J5 a,4 ¼ 3.1,
0
0
J5 a,5 b ¼ ꢁ11.5, H-50a Thd), 3.84 dd (1H, J5 b,4 ¼ 2.8, H-5 b Thd), 2.66 ddd (1H,
0
0
0
0
0
J2 a,3 ¼ 1.9, J2 a,2 b ¼ ꢁ13.7, H-20a Thd), 2.18 ddd (1H, J2 b,3 ¼ 6.5, H-2 b Thd),
0
0
0
0
0
0
0
1.63 d (3H, J5,6 ¼ 1.2, Me-5), 1.08 s (9H, t-Bu).
13C NMR (CDCl3): 168.70, 165.23, 165.11 (C ¼ O), 163.46 (C-4), 157.79 (Ph),
155.04 (C-2), 135.52 (C-6), 135.33, 133.26, 133.48, 130.11, 129.77, 129.74, 129.51,

Phosphorylated 30-O-b-D-Ribofuranosyl-dT
367
128.51, 128.40, 127.99, 127.95 (Bz, Ph), 121.74, 114.76 (Ph), 111.06 (C-5), 105.16 (C-
10 Rib), 84.66 (C-10 Thd, C-40 Thd), 79.32 (C-30 Thd), 78.62 (C-40, Rib), 75.73 (C-20,
Rib), 71.88 (C-30, Rib), 65.29, 65.01 (C-50 Thd, C-50 Rib), 63.95 (CH2), 38.61 (C20
Thd), 26.98 (Me3), 19.31 (SiCMe3), 12.04 (Me-5).
1-[3-O-(2,3-Di-O-benzoyl-5-O-phenoxyacetyl-b-d-ribofuranosyl)-2-deoxy-b-d-
ribofuranosyl]thymine (4). To a solution of 3 (1.7 g, 1.78 mmol) in tetrahydrofuran
(4 mL), a solution of tetrabutylammonium fluoride trihydrate (674 mg, 2.14 mmol)
in tetrahydrofuran (4 mL) was added. After 20 min at 20ꢀC water (2 mL) and
Dowex-50 (Naþ-form) (2 mL) were added and the mixture was stirred for 20 min
at 20ꢀC. The resin was filtered off and washed with ethyl acetate (100 mL). The fil-
trate was washed with water (2 ꢂ 30 mL). The organic layer was dried over
Na2SO4, evaporated to dryness and applied to CC (silica gel (30 g). The column
was washed with system A, and eluted with system B to give 4 as a foam. Yield
950 mg (75%). Rf 0.25 (C). ESI MS (pos.): 717.2297 ([C37H36N2O13 þ H]þ; calc.
717.2296).
1H NMR (CDCl3): 8.87 brs (1H, NH), 8.01–7.89 m (4H, Bz), 7.61–7.25 m (9H,
Bz, Ph, H-6), 6.97 t (1H, J ¼ 7.4, Ph), 6.89 d (2H, J ¼ 8.1, Ph), 6.02 t (1H,
0
J1 ,2 a ¼ J1 ,2 b ¼ 6.9, H-10 Thd), 5.66 dd (1H, J3 ,2 ¼ 5.0, J3 ,4 ¼ 6.5, H-3 Rib), 5.60
0
0
0
0
0
0
0
0
d (1H, H-20 Rib), 5.37 s (1H, H-10 Rib), 4.72 s (2H, PhOCH2), 4.65–4.51 m (4H,
H-30 Thd, H-40, 50a, 50b Rib), 4.12 m (1H, H-40 Thd), 3.91 dd (1H, J5 a,4 ¼ 2.8,
0
0
J5 a,5 b ¼ ꢁ12.1, H-50a Thd), 3.77 dd (1H, J5 b,4 ¼ 2.8, H-5 b Thd), 2.67 brs (1H,
HO-50), 2.52 m (2H, H-20a,20b Thd), 1.87 d (3H, J5,6 ¼ 1.2, Me-5).
13C NMR (CDCl3): 168.80, 165.35, 165.26 (C ¼ O), 163.58 (C-4), 157.71 (Ph),
150.28 (C-2), 137.65 (C-6), 133.63, 133.56, 129.79, 129.75, 129.56, 129.01, 128.78,
128.54, 128.44 (Bz, Ph), 121.87, 114.68 (Ph), 110.93 (C-5), 105.48 (C-10 Rib), 88.11
(C-10 Thd), 85.00 (C-40 Thd), 79.50 (C-30 Thd), 78.84 (C-40, Rib), 75.73 (C-20,
Rib), 71.91 (C-30, Rib), 65.25, 65.05 (C-50 Thd, C-50 Rib), 62.55 (CH2), 37.61 (C20
Thd), 12.34 (Me-5).
0
0
0
0
0
1-[5-O-Monomethoxytrityl-3-O-(2,3-di-O-benzoyl-b-d-ribofuranosyl)-2-deoxy-b-
d-ribofuranosyl]thymine (6). Following co-evaporation with anhydrous pyridine,
nucleoside 4 (890 mg, 1.24 mmol) was dissolved in pyridine (15 mL) and mono-
methoxytrityl chloride (460 mg, 1.49 mmol) was added. The mixture was kept for
16 h at 20ꢀC. MeOH (0.2 mL) was added and after 30 min the mixture was con-
centrated. The residue was dissolved in CHCl3 (70 mL), washed with 10% aqueous
solution of sodium bicarbonate (20 mL) and water (2 ꢂ 20 mL). The organic layer
was dried over Na2SO4, evaporated in vacuo, co-evaporated with toluene
(2 ꢂ 10 mL) to give 1-[5-O-monomethoxytrityl-3-O-(2,3-di-O-benzoyl-5-O-phenoxya-
cetyl-b-d-ribofuranosyl)-2-deoxy-b-d-ribofuranosyl]thymine (5). Rf 0.53 (C). ESI MS
(pos.): 1011.3301 ([C57H52N2O14 þ Na]þ; calc. 1011.3316).
1H NMR (CDCl3): 8.62 brs (1H, NH), 8.02–7.87 m (4H, Bz), 7.61–7.19 m (21H,
Bz, Ph, H-6), 6.97–6.82 m (5H, Ph), 6.36 dd (1H, J1 ,2 a ¼ 5.9, J1 ,2 b ¼ 7.8, H-10 Thd),
0
0
0
0
0
0
0
0
0
0
5.65 dd (1H, J3 ,2 ¼ 5.0, J3 ,4 ¼ 6.8, H-3 Rib), 5.57 d (1H, H-2 Rib), 5.27 s (1H,
H-10 Rib), 4.70 s (2H, PhOCH2), 4.64–4.50 m (4H, H-30 Thd, H-40, 50a, 50b Rib),
4.15 m (1H, H-40 Thd), 3.75 s (3H, OMe), 3.50 dd (1H, J5 a,4 ¼ 3.4, J5 a,5 b
¼
¼
0
0
0
0
0
ꢁ10.6,H-50a Thd), 3.35 dd (1H, J5 b,4 ¼ 2.8, H-5 b Thd), 2.66 ddd (1H, J2 a,3
0
0
0
0

368
Efimtseva et al.
0
2.5, J2 a,2 b ¼ ꢁ13.7, H-20a Thd), 2.32 ddd (1H, J2 b,3 ¼ 6.5, H-2 b Thd), 1.48 d (3H,
0
0
0
0
J5,6 ¼ 1.2, Me-5).
13C NMR (CDCl3): 168.71, 165.33, 165.22 (C ¼ O), 163.50 (C-4), 158.86, 157.76
(Ph), 150.07 (C-2), 137.62 (C-6), 135.45, 134.85, 133.62, 133.56, 130,32, 129.78,
129.73, 129.55, 129.21, 128.52, 128.39, 127.99, 127.84, 127.27 (Bz, Ph), 121.73,
114.75, 113.33 (Ph), 111.07 (C-5), 105.05 (C-10 Rib), 87.28 (C-O), 84.99 (C-10
Thd), 83.72 (C-40 Thd), 79.49 (C-30 Thd), 78.67 (C-40, Rib), 75.72 (C-20, Rib),
71.89 (C-30, Rib), 65.24, 65.03 (C-50 Thd, C-50 Rib), 63.48 (CH2), 55.19 (OMe)
38.71 (C20 Thd), 12.35 (Me-5).
Nucleoside 5, without further purification, was dissolved in MeOH (5 mL),
0.1 M NaHCO3 in MeOH (15 mL) was added. A solution was kept for 40 min at
20ꢀC. Following neutralization with 10% acetic acid in MeOH, the mixture was con-
centrated in vacuo to dryness and the residue was partitioned between ethyl acetate
(100 mL) and 10% aqueous solution of sodium bicarbonate (30 mL), the organic
layer was washed with water (30 mL), dried over Na2SO4 and evaporated to dryness.
The residue was applied to CC (silica gel 30 g). The column was washed with system
A and then eluted with system B affording 6 as a foam. Yield 650 mg (61%). Rf 0.27
(C). ESI MS (pos.): 877.2923 ([C49H46N2O12 þ Na]þ; calc. 877.2948).
1H NMR (CDCl3): 8.69 brs (1H, NH), 8.00–7.88 m (4H, Bz), 7.65–7.23 m (19H,
Bz, Ph, H-6), 6.85 d (2H, J ¼ 8.7, Ph), 6.38 dd (1H, J1 ,2 a ¼ 5.3, J1 ,2 b ¼ 8.7, H-10
0
0
0
0
00
0
0
0
0
Thd), 5.72 dd (1H, J3 ,2 ¼ 5.0, J3 ,4 ¼ 7.2, H-3 Rib), 5.60 d (1H, H-2 Rib), 5.30 s
(1H, H-10 Rib), 4.50 m (2H, H-30 Thd, H-40 Rib), 4.49 m (1H, H-40 Thd), 04.01 dd
(1H, J5 a,4 ¼ 3.1, J5 a,5 b ¼ ꢁ12.1, H-50a Rib), 3.88 dd (1H, J5 b,4 ¼ 5.0, H-5 b Rib),
0
0
0
0
0
0
3.77 s (3H, OMe), 3.53 dd (1H, J5 a,4 ¼ 3.1, J5 a,5 b ¼ ꢁ10.6, H-50a Thd), 3.37 dd
0
0
0
0
0
(1H, J5 b,4 ¼ 2.8, H-5 b Thd), 3.01 ddd (1H, J2 a,3 ¼ 1.6, J2 a,2 b ¼ ꢁ13.4, H-20a
0
0
0
0
0
0
0
0
0
Thd), 2.23 ddd (1H, J2 b,3 ¼ 5.6, H-2 b Thd), 1.49 d (3H, J5,6 ¼ 1.0, Me-5).
13C NMR (CDCl3): 165.54, 165.21 (C ¼ O), 163.77 (C-4), 158.85 (Ph), 150.75
(C-2), 135.37 (C-6), 133.44, 133.30, 130.33, 129.74, 129.70, 129.22, 129.07, 128.45,
128.36, 128.32, 127.99, 127.26 (Bz, Ph), 113.33 (Ph), 111.40 (C-5), 105.58 (C-10
Rib), 87.32 (C-O), 85.04 (C-10 Thd), 84.26 (C-40 Thd), 82.36 (C-30 Thd), 80.29
(C-40, Rib), 76.16 (C-20, Rib), 71.45 (C-30, Rib), 63.65, 62.78 (C-50 Thd, C-50 Rib),
55.19 (OMe) 38.82 (C20 Thd), 11.84 (Me-5).
50-O-Monomethoxytrityl-30-O-[5-O-(P-b-cyanoethyl-N,N-diisopropylaminophos-
phinyl)-2,3-di-O-benzoyl-b-d-ribofuranosyl]-thymidine (7). The monomethoxytri-
tylated derivative 6 (822 mg, 0.96 mmol) was dissolved in 6 mL dichloromethane
under argon and diisopropylethylamine (0.5 mL, 2.88 mmol) and 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite (535 mL, 2.4 mmol) were added and the
solution was stirred for 60 min when TLC indicated complete reaction. Water
(3 mL) was added, the solution was stirred for 10 min and partitioned between
CH2Cl2 (60 mL) and aqueous NaHCO3(40 mL). The organic phase was washed with
aqueous sodium chloride (3 ꢂ 40 mL) and the aqueous phases were back extracted
with CH2Cl2 (30 mL). Evaporation of the organics left an oil which was flash purified
on 45 g of silica gel (hexane: acetone: TEA, 66:33:1) to afford the product as a foam
after coevaporation with dichloromethane. Dissolution in 3 mL of dichloromethane
and double precipitation in 100 mL cold (ꢁ70ꢀC) hexane afforded 840 mg
(0.83 mmol, 83%) of the title product 7 as a white powder. Rf (hexane: acetone:

Phosphorylated 30-O-b-D-Ribofuranosyl-dT
369
TEA 49:49:2): 0.43. C58H64N4O13P1 calcd. 1055.4207, found 1055.4204 [M þ H]þ.
31P NMR (ppm, external ref. ¼ H3PO4 capil.) 149.86, 149.18
1-[5-O-tert-Butyldiphenylsilyl-3-O-(2,3-di-O-acetyl-5-O-bis(p-nitrophenylethyl)-
phosphorylyl-b-d-ribofuranosyl)-2-deoxy-b-d-ribofuranosyl]thymine (9). Following
the procedure for the preparation of 3, condensation of 1,2,3-tri-O-acetyl-5-O-
bis(p-nitrophenylethyl)phosphorylyl-d-ribofuranose (8) (300 mg, 0.46 mmol) in the
presence of tin tetrachloride (0.064 mL, 0.55 mmol) with nucleoside 1 (250 mg,
0.52 mmol) for 4 h at 0ꢀC gave 9 as a foam. Yield 169 mg (35%). Rf 0.37 (C).
1H NMR (CDCl3): 9.18 brs (1H, NH), 8.14 m (4H, Ph), 7.67–7.37 m (15H, Ph,
H-6), 6.22 dd (1H, J1 ,2 a ¼ 5.9, J1 ,2 b ¼ 8.1, H-10 Thd), 5.29 dd (1H, J3 ,2 ¼ 5.0,
0
0
0
0
0
0
0
0
0
0
0
0
0
J3 ,4 ¼ 5.9, H-3 Rib), 5.13 dd (1H, J2 ,1 ¼ 1.5, H-2 Rib), 5.07 d (1H, H-1 Rib),
4.47 m (1H, H-30 Thd), 4.23 m (5H, H-40 Rib, CH2O), 4.08 m (2H, H-50a,50b Rib),
4.01 m (1H, H-40 Thd), 3.93 dd (1H, J5 a,4 ¼ 3.4, J5 a,5 b ¼ ꢁ11.5, H-50a Thd), 3.81
0
0
0
0
0
0
0
0
0
dd (1H, J5 b,4 ¼ 2.5, H-5 b Thd), 3.05 (4H, CH2), 2.55 ddd (1H, J2 a,3 ¼ 1.9,
J2 a,2 b ¼ ꢁ13.7, H-20a Thd), 2.14 m (1H, H-20b Thd, Ac), 2.12 s (3H, Ac), 2.06 s
0
0
(3H, Ac), 1.60 d (3H, J5,6 ¼ 1.1, Me-5), 1.09 s (9H, t-Bu).
13C NMR (CDCl3): 169.54, 169.40 (C¼O), 163.63 (C-4), 150.14 (C-2), 144.85,
144.89 (Ph), 135.49, 135.29 (Ph), 135.06 (C-6), 132.42, 130.12, 130.07, 129.86,
127.98, 127.95 (Bz, Ph), 123.65, 114.76 (Ph), 111.10 (C-5), 105.00 (C-10 Rib), 84.68
(C-10 Thd), 84.60 (C-40 Thd), 79.74 d (C-40 Rib, J(C,P) ¼ 7.9), 78.73 (C-30 Thd),
74.83 (C-20 Rib), 70.69 (C-30 Rib), 67.30 d (CH2O, J(C,P) ¼ 5.1), 67.11 d (C-50
Rib, J(C,P) ¼ 5.6), 63.82 (C-50 Thd,), 38.61 (C20 Thd), 36.28, 36.21 (CH2), 26.96
(Me3), 20.42, 20.40 (Ac), 19.30 (SiCMe3), 11.99 (Me-5). 31P NMR (161.98 MHz)
(CDCl3): ꢁ3.61.
O-b-d-Ribofuranosyl-(100-30)-thymidine-500-O-phosphate (10). A solution of tri-
ester 9 (155 mg, 0.144 mmol) in 0.5 M DBU in dry pyridine (15 mL, 7.5 mmol) was
stored for 24 h at 20ꢀC, neutralized with acetic acid (0.43 mL, 7.5 mmol), and evapo-
rated. The residue was dissolved in 5 M ammonia in methanol (5 mL) and kept for 2
days at 20ꢀC and then concentrated in vacuo. The residue was dissolved in 0.5 M
tetrabutylammonium fluoride in tetrahydrofuran (10 mL) and kept for 16 h at
20ꢀC, and evaporated. The residue was dissolved in water (50 mL) and washed with
CHCl3 (2 ꢂ 15 mL). The aqueous layer was concentrated in vacuo and diluted wiꢁth
water (50 mL) and then applied to a column of DEAE-cellulose (200 mL, HCO3
-
form). The column was washed with water (500 mL), a 0.05 M solution of NH4HCO3
and eluted with 0.1 M solution of NH4HCO3. The UV-absorbing fractions were
combined, evaporated in vacuo, and coevaporated with water (5 ꢂ 10 mL). The resi-
due was dissolved in water (2 mL), applied to a column of Dowex 50 (Naþ-form)
(2 mL), eluted with water and freeze-dried. Monophosphate 10 was obtained as its
sodium salt. Yield 40 mg (58%). Rf 0.15 (D). ESI MS (pag.): 477.0877 [M þ Na]þ
UV (pH 1): lmax 267 nm (e 9100); (pH 7): lmax 267 nm (e 9000); (pH 13): lmax
266 nm (e 7000).
O-b-d-Ribofuranosyl-(100-30)-thymidine-50-O-phosphate (12). A mixture of 1-[3-
O-(2,3,5-tri-O-benzoyl-b-d-ribofuranosyl)-2-deoxy-b-d-ribofuranosyl]thymine (11)
(686 mg, 1 mmol) and a 3 mL 1 M solution of b-cyanoethyl phosphate in pyridine

370
Efimtseva et al.
was evaporated in vacuo and dried by co-evaporations with dry pyridine
(2 ꢂ 10 mL). The residue was dissolved in pyridine (10 mL), N,N0-dicyclohexylcarbo-
diimide (1,03 g, 5 mmol) was added and the mixture was stored at 20ꢀC for 3 days.
After addition of water (10 mL), the precipitated dicyclohexyl urea was filtered off
and washed with of 20% aqueous pyridine (20 mL). The combined filtrates were
washed with CHCl3 (2 ꢂ 50 mL), concentrated in vacuo, evaporated with toluene
(3 ꢂ 10 mL) and applied on a column of silica gel (30 g). The column was washed
with system B (300 mL) and then eluted with CHCl3=MeOH=Et3N 95:5:5. Fractions
containing the product were collected and evaporated in vacuo to dryness. The resi-
due was dissolved in 5 M ammonia in methanol (10 mL) and kept for 3 days at 20ꢀC
and then concentrated in vacuo to dryness. The residue was partitioned between
CHCl3 (15 mL) and water (30 mL), the water layer was washed with chloroform
(2 ꢂ 15 mL). The aqueous layer was concentrated in vacuo. The residue was dis-
solved in 1N NaOH (5 mL) and kept for 20 min at 20ꢀC, then 10% aqueous acetic
acid was added to pH 7.0. The solution was diluted with water (30 mL) and then
applied to a column of DEAE-cellulose (200 mL, HCO3ꢁ-form). The column was
washed with water (500 mL), a 0.05 M solution of NH4HCO3 and eluted with
0.1 M solution of NH4HCO3. The UV-absorbing fractions were combined, evapo-
rated in vacuo, and coevaporated with water (5ꢂ10 mL). The residue was dissolved
in 20 mL of water and freeze-dried. Monophosphate 12 was obtained as its ammo-
nium salt. Yield 177 mg (37%). Rf 0.15 (D).
ESI MS (pos.): 477.0896 [M þ Na]þ.
UV (pH 1): l
267 nm (e 9200); (pH 7): lmax 267 nm (e 9100); (pH 13): lmax
max
267 nm (e 7100).
ACKNOWLEDGMENT
This work has been supported by a grant from K. U. Leuven (GOA) and
RFOR. Koen Nauwelaerts is a research Associate of the FWO.
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Received October 29, 2002
Accepted February 4, 2003