Synthesis of D-Altritol Nucleosides with a 3′ -O-Tert-Butyldimethylsilyl Protecting Group

Article abstract of DOI:10.1081/NCN-120028338

Four D-altritol nucleosides with a 3′-O-tert- butyldimethylsilyl protecting group are synthesized (base moieties are adenine, guanine, thymine and 5-methylcytosine). The nucleosides are obtained by ring opening reaction of 1,5:2,3-dianhydro-4,6-O-benzylid

Full text of DOI:10.1081/NCN-120028338

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Synthesis of D‐Altritol Nucleosides with a
3′‐O‐Tert‐Butyldimethylsilyl Protecting Group
Michael Abramov a , Arnaud Marchand a , Agnes Calleja‐Marchand a & Piet Herdewijn a
a Laboratory for Medicinal Chemistry, Rega Institute for Medical Research,
Minderbroedersstraat 10, B‐3000, Leuven, Belgium
Available online: 01 Jun 2007
To cite this article: Michael Abramov, Arnaud Marchand, Agnes Calleja‐Marchand & Piet Herdewijn (2004): Synthesis of
D‐Altritol Nucleosides with a 3′‐O‐Tert‐Butyldimethylsilyl Protecting Group , Nucleosides, Nucleotides and Nucleic Acids,
23:1-2, 439-455
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 23, Nos. 1 & 2, pp. 439–455, 2004
Synthesis of D-Altritol Nucleosides with
a 3’-O-Tert-Butyldimethylsilyl
Protecting Groupy
Michael Abramov, Arnaud Marchand, Agnes Calleja-Marchand,
*
and Piet Herdewijn
Laboratory for Medicinal Chemistry, Rega Institute for Medical Research,
Leuven, Belgium
ABSTRACT
Four D-altritol nucleosides with a 3’-O-tert-butyldimethylsilyl protecting group are
synthesized (base moieties are adenine, guanine, thymine and 5-methylcytosine). The
nucleosides are obtained by ring opening reaction of 1,5:2,3-dianhydro-4,6-O-
benzylidene-D-allitol. Optimal reaction circumstances (NaH, LiH, DBU, phase
transfer, microwave irridation) for the introduction of the heterocycles are base-
specific. For the introduction of the 3’-O-silyl protecting group, long reaction times
and several equivalents of tert-butyldimethylsilyl chloride are needed.
Key Words: D-altritol nucleosides; 3’-O-tert-butyldimethylsilyl protecting group;
1,5:2,3-dianhydro-4,6-O-benzylidene-D-allitol.
yIn honor and celebration of the 70th birthday of Professor Leroy B. Townsend.
*
Correspondence: Piet Herdewijn, Laboratory for Medicinal Chemistry, Rega Institute for Medical
Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium; E-mail: piet.herdewijn@rega.
kuleuven.ac.be.
439
DOI: 10.1081/NCN-120028338
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com
Copyright D 2004 by Marcel Dekker, Inc.

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INTRODUCTION
D-altritol Nucleic Acids (ANA) are oligonucleotides with a phosphorylated D-altritol
backbone and nucleobases introduced in the 2-position of the hexitol ring. ANA hybridize
strongly and sequence selective with RNA in an antiparallel way.[1] These oli-
gonucleotides were previously synthesized from 3-O-benzoyl-2-deoxy-4-O-[N,N-diiso-
propyl(2-cyanoethyl)]phosphoramidite-6-O-monomethoxytrityl-D-altritol building
blocks.[1,2] The disadvantage of this approach is the instability of the 3-O-benzoyl group
due to migration reaction of the benzoyl protecting group from the 3-position to the 4-
position. This migration reaction is very fast due to the axial orientation of the 3-OH
group and the equatorial orientation of the 4-OH group. A similar migration reaction
(from the 2’-OH to the 3’-OH group) in the case of ribonucleosides[3] was one of the
reasons for the introduction of the tert-butyldimethylsilyl protecting group for the 2’-OH
function in RNA synthesis.[4] The introduction of a tert-butyldimethylsilyl group in an
axially oriented hydroxyl group of a pyranosyl nucleoside is, however, more difficult than
the introduction of the same protecting group on a secondary hydroxyl group of a
ribofuranose nucleoside. More vigorous reaction conditions are needed. Here we describe
the synthesis of four new building blocks, with a 3-O-tert-butyldimethylsilyl protecting
group, that will be used for ANA synthesis.
CHEMISTRY
The easiest way to obtain D-altritol nucleosides, is by epoxide opening of 1,5:2,3-
dianhydro-4,6-O-benzylidene-D-allitol using nucleobases. This sugar compound can be
prepared from commercially available tetraacetyl-a-D-bromoglucose in 5 steps (54%
overall yield).[5] The advantage of this approach is that a D-altritol nucleoside is
obtained with a free 3’-OH group and a protected 4’-OH and 6’-OH group, which avoid
problems with the regioselective introduction of a protecting group in the 3’-position.
Different conditions were evaluated for the nucleophilic opening of the epoxide by the
salts of nucleobases. As well classical sodium and lithium salts, as a more soft base
(DBU) and new technologies (phase transfer catalyst using tricaprylylmethylammonium
chloride or microwave assisted synthesis) were explored.
Reaction of the sodium salt of adenine 1 (3 equiv.) with 1,5:2,3-dianhydro-4,6-O-
benzylidene-D-allitol in DMF at 120°C for 18 h. yielded 70% of 2-(adenin-9-yl)-1,5-
anhydro-4,6-O-benzylidene-2-deoxy-D-altro-hexitol 2.[2] When using the lithium salt of
adenine in the presence of 12-crown-4-ether the reaction could be speeded up (1 h.) and
somewhat higher yields (80%) were obtained (Scheme 1). The use of DBU as base or a
phase transfer catalyst or microwave circumstances didn’t improve the yield (see
further, for the observed differences when using the 2-amino-6-chloropurine base).
Monobenzoylation at the N6-position of the adenine base was carried out with benzoyl
chloride using the transient protection strategy.[6] Previously, we reported the
difficulties obtained for introduction of a tert-butyldimethylsilyl protecting group in
the 3-O-position of 1,5-anhydro-4,6-O-benzylidene-2-deoxy-D-altro-hexitol nucleosides
(base moiety in the 2-position).[2] Due to steric hindrance, low amounts of materials
were obtained. In order to force the reaction to completion, we tried more vigorous
reaction conditions using a 5-fold excess of tert-butyldimethylsilyl chloride and a
7-fold excess of imidazole (both reagents were added in three portions) and a reaction

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441
Scheme 1. Synthesis of 1,5-anhydro-2-(N5-benzoyladenin-9-yl)-3-O-tert-butyldimethylsilyl-2-
deoxy-6-O-monomethoxytrityl-D-altro-hexitol.
time of 38 h at 70°C in DMF as solvent. In these circumstances, 1,5-anhydro-4,6-O-
benzylidene-2-(N6-benzoyladenin-9-yl)-3-O-tert-butyldimethylsilyl-2-deoxy-D-altro-
hexitol 4 was obtained in 68% yield. Removal of the benzylidene protecting group
could be done with trifluoroacetic acid in dichloromethane without migration of the
3-O-silyl protecting group. Likewise, the 6-O-monomethoxytrityl group can be
Scheme 2. Synthesis of 1,5-anhydro-2-deoxy-3-O-tert-butyldimethylsilyl-6-O-monomethoxytri-
tyl-2-(thymlin-1-yl)-D-altro-hexitol.

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Scheme 3. Synthesis of 1,5-anhydro-2-(N4-benzoyl-5-methylcytosin-1-yl)-3-O-tert-butyldi-
methylsilyl-2-deoxy-6-O-monomethoxytrityl-D-altro-hexitol.
introduced under common reaction circumstances (pyridine, room temperature) without
(3’!4’) tert-butyldimethylsilyl migration, which was difficult to perform using a
3-O-benzoyl protecting group.[2] Overall, this reaction scheme offers a better yield of the
individual steps and the migration problems of the protecting group could be avoided.
The compound with the thymine and 5-methylcytosine base is described here for
the first time (Schemes 2 and 3). The sodium salt of thymine 7 was reacted with
1,5:2,3-dianhydro-4,6-O-benzylidene-D-altritol in DMF at 130°C for 18 h yielding 55%
of 1,5-anhydro-4,6-O-benzylidene-2-deoxy-2-(thymin-1-yl)-D-altro-hexitol 8. Introduc-
tion of the 3-O-tert-butyldimethylsilyl protecting group was carried out with tert-
butyldimethylsilyl chloride and imidazole in DMF (yield 88%). After removal of the
benzylidene protecting group (90% yield), the primary hydroxyl group was protected
with a monomethoxytrityl group (92% yield). These reactions occur without any
problems dealing with protecting group migration from the 3’-O-axial position to the
Table 1. Nucleophilic opening of epoxide in the presence of nucleoside bases in DMF.a
Aliquat 336
c
K2CO3
NaH
DBU
MW irradiation
Guanine
–
0
0
10
iso-Butyrylguanine
2-Amino-6-chloropurine
Thymine
–
traces
70b
0
–
traces
35
75
45
55
86
70
45b
75
96
70
Uracil
Adenine
–
55
–
50
aFor conditions, see experimental section.
bHMPA.
cAliquat 336/K2CO3. tricaprylylmethylammonium chloride.

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443
4’-O-equatorial position. The crucial reaction is the epoxide opening using the
pyrimidine base. The approach we followed here was the same as previously used for
the synthesis of the uracil congener.[2] Yields were higher with the uracil base (88%)
than with the thymine base (55%). Later on, we found out that the reaction using DBU
instead of NaH or the use of microwave irridation, gives higher yields of the
pyrimidine nucleosides (Table 1) i.e. 75% and 96% respectively (which was not the
case with the adenine base).
1,5-Anhydroxy-4,6-O-benzylidene-3-O-tert-butyldimethylsilyl-2-deoxy-2-(thymin-
1-yl)-D-altro-hexitol 19 was used as starting material for the synthesis of the protected
5-methylcytosine congener (Scheme 3). The method used is 1,2,4-triazolyl activation of
the 4-position of the thymine base, followed by substitution with ammonia (65%
yield).[7] The N4-position is protected with a benzoyl group, followed by benzylidene
removal and 6-O-monomethoxytritylation. For all cases investigated, it seems that the
conversion of the uracil/thymine base in the cytosine/methylcytosine base is a better
way (higher yield) to obtain the 4-aminopyrimidine nucleosides than the direct opening
reaction of the epoxide ring with the salts of the respective nucleobases (cytosine and
5-methylcytosine).
As could be expected, the guanine nucleoside is a particular case. Previously, we
described the epoxide opening using the sodium salt of 2-amino-6-chloropurine in DMF
Scheme 4. Synthesis of 1,5-anhydro-3-O-tert-butyldimethylsilyl-2-(N2-iso-butylguanin-9-yl)-2-
deoxy-6-O-monomethoxytrityl-D-altro-hexitol.

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in 40% yield (Scheme 4). Besides the major product, two side compounds were
identified, i.e. the N-7-substituted compound and the bis-purinyl nucleoside. The same
reaction using the lithium salt of N2-acetyl-2-amino-6-[2-(trimethylsilyl)ethoxy)purine]
afforded the protected guanine nucleoside in 45% yield (after deacetylation).[2] These
results are unsatisfactory for large scale synthesis of the altritol nucleosides. The reaction
in the presence of aliquat 336/K2 CO3 in DMF gives 45% of the described compound
together with three side compounds. The additional side compound, observed here, is the
N-9-substituted 2-amino-4-dimethylaminopurine nucleoside (Scheme 4). By utilizing the
same phase transfer catalyst but in HMPA as solvent, side product formation could be
avoided and the desired compound was obtained in 70% yield. Reactions with related
bases (guanine and N2-iso-butyrylguanine) did not lead to the condensation product. The
6-chloro-2-aminopurine base was converted into the guanine base, followed by transient
protection procedure, to introduce the isobutyryl group and 3-O-silylation. After removal
of the benzylidene group, the primary hydroxyl group was protected with mono-
methoxytrityl chloride. All the deprotection and protection reactions could be carried out
in satisfactory yields (77%–92%).
CONCLUSION
The four building blocks for the synthesis of altritol nucleic acids were obtained
from 1,5:2,3-dianhydro-4,6-O-benzylidene-D-allitol. The optimal circumstances for ring
opening of the oxirane is base specific. Uracil and thymine are used as DBU salt, or
introduced by microwave irridation. Adenine is introduced as lithium salt, but the
sodium and DBU salt of adenine base are useful alternatives. For the introduction of
the 2-amino-6-chloropurine base, phase transfer conditions and HMPA as solvent is the
method of choice. Introduction of the 3-O-tert-butyldimethylsilyl protecting group
needs more vigorous reaction conditions than described for ribofuranose nucleosides.
These building blocks will be used for oligonucleotide synthesis after conversion in
the phosphoramidites.
EXPERIMENTAL SECTION
Tetra-O-acetyl-a-D-bromoglucose was provided by Fluka; adenine, cytosine,
guanine and uracil were from ACROS. All other chemicals were provided by Aldrich
or ACROS and were of the highest quality. 1H NMR and 13C NMR spectra were
determined with a 200 MHz Varian Gemini apparatus with tetramethylsilane as internal
standard for the 1H NMR spectra (s = singlet, d = doublet, dd = double doublet,
t = triplet, br s = broad signal, br d = broad doublet, m = multiplet) and the solvent
signal DMSO-d6 (39.6 ppm) or CDCl3 (76.9 ppm) for the 13C NMR spectra. For some
products a Varian Unity-500 spectrometer (500 MHz for 1H) was used. Coupling
constant values were derived by first-order spectral analysis. Exact mass measurements
were performed on a quadrupole/orthogonal acceleration time-of-flight tandem mass
spectrometer (qTOF2, Micromass, Manchester, UK) equiped with a standard
electrospray ionization interface. Precoated Machery-Nagel Alugram SILG/UV254
plates were used for TLC, and the spots were examined with UV light and sulfuric

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445
acid/anisaldehyde spray. Column chromatography was performed on ACROS silica gel
(0.060–0.200 mm or 0.035–0.060 mm). Anhydrous solvents were obtained as follows:
dichloromethane was stored over calcium hydride, refluxed and distilled. Pyridine was
refluxed over potassium hydroxide pellets and distilled. Dimethylformamide was dried
˚
over 4 A activated molecular sieves. HMPA was dried by azeotropic distilation using
toluene. Absolute methanol was refluxed overnight over magnesium iodide and
distilled. Methanolic ammonia was prepared by bubbling NH3 gas through absolute
methanol at 0°C and was stored at ꢀ 20°C. All microwave irradiation experiments
were carried out in a dedicated CEM-Discover monomode microwave apparatus,[8]
operating at a frequency of 2.45 GHz with continuous irradiation power from 0 to
300 W utilizing the standard absorbance level (300 W maximum power). The reactions
were carried out in 10 mL glass tubes, sealed with an aluminum/Teflon crimp top,
which can be exposed to 250°C and 20 bar internal pressure. The temperature was
measured with an IR sensor on the outer surface of the process vial. After the
irradiation period, the reaction vessel was cooled rapidly (60–120 s) to ambient
temperature by gas jet cooling.
2-(Adenin-9-yl)-1,5-anhydro-4,6-O-benzylidene-2-deoxy-D-altro-hexitol
(2). Lithium hydride (0.716 g, 90.00 mmol) was added to a stirred suspension of
adenine (12.57 g, 93.00 mmol) and 12-crown-4 ether (0.145 ml, 0.90 mmol) in
anhydrous DMF (560 ml) at room temperature under nitrogen. The mixture was stirred
at 120°C for 1.5 hours (clear solution) and the epoxide (7.03 g, 30.00 mmol) was
added. The stirring was continued for 1 hour at 120°C and the reaction mixture was
cooled and evaporated to dryness. The residue was dissolved in ethyl acetate (1000 ml),
and the organic layer was washed with a saturated solution of sodium hydrogencarbo-
nate (3 ꢁ 1000 ml) and brine (1000 ml). The organic layer was dried over sodium
sulfate, filtered and evaporated to dryness. The residue was dissolved in a mixture of
dichloromethane and methanol (1/1: 75 ml) and precipitated with hexane. Two crops
were obtained, collected and precipitated one more time to give the title compound
2 as a pure white solid (6.78 g, 61%). The filtrate can be purified on silica gel
chromatography using a stepwise gradient of methanol (2–5%) in dichloromethane to
afford 2.11 g of pure material (total yield: 80%). Physical and chemical data are in
accord with the literature.[2]
HRMS calcd for C18H20N5O4(MH) + 370,1515, found 370,1507.
1-5-Anhydro-4,6-O-benzylidene-2-(N6-benzoyladenin-9-yl)-2-deoxy-D-altro-hex-
itol (3). Trimethylsilyl chloride (2.57 ml, 20 mmol) was added dropwise to a cold
suspension (0°C) of 2 (3.69 g, 10 mmol) in dry pyridine (50 ml) under nitrogen. The
reaction mixture was stirred at room temperature, for 1 hour, and cooled at 0°C. Then,
benzoyl chloride (5.81 ml, 50 mmol) was added dropwise, and the reaction mixture was
stirred at room temperature, for 2 hours. The solution was cooled in an ice-bath, and
water (10 ml) was added, followed 5 min later by 10 ml of aqueous ammonia (25%).
The reaction mixture was stirred at room temperature for 30 minutes and the volatiles
were removed. The residue was dissolved in ethyl acetate (150 ml) and the organic
layer was extracted with a saturated solution of sodium hydrogencarbonate (3 ꢁ 150 ml).
The solvent was evaporated and the residue was dissolved in THF (50 ml). Then, a 1M
TBAF solution in THF was added (10 ml). After 5 min stirring at room temperature,

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Abramov et al.
the reaction mixture was evaporated to dryness, dissolved in dichloromethane (100 ml)
and washed with water (3 ꢁ 100 ml). The organic layer was dried over sodium sulfate,
filtered and evaporated to dryness. The residue was subjected to silica gel column
chromatography using a mixture of ethyl acetate/n-hexane (1/9) as eluent, to afford the
title compound 3 as a white solid (3.72 g, 79%).
1H-NMR (CDCl3, 500 MHz) d 3.64 (1H, dd, J = 2.6 Hz, J = 9.7 Hz, 4’-H); 3.74
(1H,t, J = 10.4 Hz, 6’ax-H); 4.17 (1H, dd, J = 5.1 Hz, J = 10.0 Hz, 5’-H); 4.21 (1H, br
d, J = 14.4 Hz, 1’ax-H); 4.42 (1H, dd, J = 4.9 Hz, J = 10.5 Hz, 6’eq-H); 4.48–4.52
(2H, m, 3’-H and 1’eq-H); 4.87 (1H, br s, 2’-H); 5.51 (1H, s, PhCH); 7.26–7.60 (8H, m,
H arom); 8.05 (2H, d, J = 7.5 Hz, II ortoBz); 8.55 (1H, s, 8-H); 8.74 (1H, s, 2-H); 9.46
(1H, br. s, NH). 13C-NMR (CDCl3, 125 MHz) d 555.36 (2’-C); 64.99 (1’-C); 66.21
(3’-C); 67.12 (5’-C); 68.91 (6’-C); 76.63 (4’-C); 102.15 (PhCH); 122.68 (5-C); 125.97–
136.74 (8 ꢁ C arom), 141.96 (8-C); 149.76 (4-C); 152.12 (2-C); 152.68 (6-C);
164.87 (CONH).
HRMS calcd for C25H24N5O5(MH)+ 474,1777, found 474.1774.
1,5-Anhydro-2-(N6-benzoyladenin-9-yl)-4,6-O-benzylidene-3-O-tert-butyldi-
methylsilyl-2-deoxy-D-altro-hexitol (4). Tert-butydimethylsilyl chloride (1.81 g,
12 mmol) was added to a solution of 3 (1.89 g, 4 mmol) and imidazole (1.36 g, 20 mmol)
in dry DMF (20 ml). The reaction mixture was stirred at 70°C, under nitrogen. After
15 hours, imidazole (0.272 g; 4 mmol) and TBDMSCl (0.603 g; 4 mmol) were added and
stirring was continued for 15 hours. Then, imidazole (0.272 g; 4 mmol) and TBDMSCl
(0.603 g; 4 mmol) were added and stirring was continued for 8 hours at 70°C. DMF was
removed under reduced pressure and the residue was dissolved in dichloromethane (70 ml)
and washed with water (2 ꢁ 70 ml). The organic layer was dried over sodium sulfate,
filtered, and evaporated to dryness. The brownish residue was subjected to a silica gel
column chromatography using a stepwise gradient of methanol (0–0.5%) in dichloro-
methane, to afford the title compound 4 as a white foam (1.48 g, 68%).
1H-NMR (CDCl3, 500 MHz) d 0.13 (3H, s, CH3 Si); 0.28 (3H, s, CH3 Si); 0.99
(9H, s, (CH3)3C); 3.55 (1H, dd, J = 2.5 Hz, J = 9.6 Hz, 4’-H); 3.73 (1H, t, J = 10.5 Hz,
6’ax-H); 4.16–4.21 (1H, m, 5’-H); 4.29 (1H, br d, J = 13.2 Hz, 1’ax-H); 4.39 (1H, dd,
J = 5.1 Hz, J = 10.5 Hz, 6’eq-H); 4.51 (1H, t, J = 2.9 Hz, 3’-H); 4.57 (1H, dd, J =
2.93 Hz, J = 13.2 Hz, 1’eq-H); 4.68 (1H, t, J = 2.7 Hz, 2’-H); 5.45 (1H, s, PhCH);
7.26–7.61 (8H, m, H arom); 8.06 (2H, d, J = 7.0 Hz, H ortoBz); 8.59 (1H, s, 8-H);
8.79 (1H, s, 2-H); 9.33 (1H, br s, NH).
13C-NMR (CDCl3, 125 MHz) d54.90 (CH3Si); ꢀ 4.63 (CH3Si); 18.08 ((CH3)3C);
25.77 ((CH3)3C); 56.84 (2’-C); 64.74 (1’-C); 66.95 (5’-C); 66.99 (3’-C); 69.00 (6’-C);
76.63 (4’-C); 102.31 (PhCH); 122.72 (5-C); 126.15-137.19 (11 ꢁ C arom), 141.99
(8-C); 149.61 (4-C); 152.52 (2-C); 152.70 (6-C); 164.80 (CONH).
HRMS calcd for C31H38N5O5Si (MH)+ 588.2642, found 588.2645.
1,5-Anhydro-2-(N6-benzoyladenin-9-yl)-3-O-tert-butyldimethylsilyl-2-deoxy-D-
altro-hexitol (5). The compound 4 (6.23 g, 10.60 mmol) was dissolved in dichloro-
methane (112 ml) and TFA was added (48 ml). After 1 hour stirring at room
temperature, the volatiles were removed under reduced pressure and the residue
was purified on silica gel column chromatography using a stepwise gradient of
methanol (0–4%) in dichloromethane to afford the title compound 5 as a white solid
(3.14 g, 74%).

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1H-NMR (CDCl3, 500 MHz) d 0.20 (3H, s, CH3Si); 0.36 (3H, s, CH3Si); 0.98 (9H,
s, (CH3)3C); 3.67–3.73 (2H, m, 4’-H and 5’-H); 3.77 (1H, dd, J = 3.7 Hz, J = 12.2 Hz,
6’ax-H); 3.87 (1H, br d, J = 11.2 Hz, 6’eq-H); 4.21 (1H, br d, J = 12.5 Hz, 1’ax-H);
4.34–4.37 (2H, m, 3’-H and 1’eq-H); 4.72 (1H, br s, 2’-H); 7.46–7.49 (2H, m, H
arom); 7.54–7.57 (1H, m, H arom); 8.00–8.03 (2H, m, H arom); 8.65 (1H, s, 8-H);
8.77 (1H, s, 2-H); 9.76 (1H, br. s, NH).
13C-NMR (CDCl3, 125 MHz) d-4.85 (CH3Si); -4.54 (CH3Si);18.01 ((CH3)3C);
25.79 ((CH3)3C); 55.90 (2’-C); 61.94 (1’-C); 64.02 (5’-C); 64.25 (3’-C); 69.29 (6’-C);
77.13 (4’-C); 121.98 (5-C); 128.02–133.77 (4 ꢁ C arom); 142.25 (8-C); 149.66 (4-C);
152.21 (2-C); 152.77 (6-C); 165.22 (CONH).
HRMS calcd for C24H34N5O5Si (MH)ꢀ 500.2329, found 500.2324.
1,5-Anhydro-2-(N6-benzoyladenin-9-yl)-3-O-tert-butyldimethylsilyl-2-deoxy-6-
O-monomethoxytrityl-D-altro-hexitol (6). Monomethoxytrityl chloride (1.94 g,
6.20 mmol) was added to a stirred solution of 5 (2.82 g, 5.64 mmol) in dry
pyridine (30 ml) at room temperature under nitrogen. After 16 hours, methanol (3 ml)
and solid sodium hydrogencarbonate were added. The volatiles were removed and
the residue was dissolved in ethyl acetate (100 ml). The organic phase was washed
with a 5% solution of sodium hydrogencarbonate (2 ꢁ 150 ml), dried over anhydrous
sodium sulfate, evaporated and coevaporated with toluene. The residue was subjected
to silica gel column chromatography using a mixture of ethyl acetate/hexane (9/1),
(containing 0.2% triethylamine) to afford the title compound 6 as a white solid (4.00
g, 92%).
1H-NMR (CDCl3, 500 MHz) d 0.18 (3H, s, CH3Si); 0.32 (3H, s, CH3Si); 0.97 (9H,
s, (CH3)3C); 1.94 (1H, d, J = 8.5 Hz, 4’-OH); 3.38–3.46 (1H, m, 4’-H); 3.49 (1H, dd,
J = 2.7 Hz, J = 10.3 Hz, 5’-H); 3.72–3.90 (5H, m, 6’ax-H, 6’cq-H and CH3O); 4.26–
4.36 (3H, m, 1’ax-H, 3’-H and 1’eq-H); 4.75 (1H, m, 2’-H); 6.84 (2H, d, J = 9.0 Hz, H
arom); 7.23–7.63 (15H, m, H arom); 8.03 (2H, d, J = 7.0 Hz, H arom); 8.70 (1H, s, 8-H);
8.81 (1H, s,2-H); 9.11 (1H, br s, NH).
13C-NMR (CDCl3, 125 MHz) d-4.85 (CH3Si); ꢀ 4.57 (CH3Si);18.00 ((CH3)3C);
25.79 ((CH3)3C); 55.18 (CH3O); 55.77 (2’-C); 63.44 (1’-C); 63.97 (5’-C); 65.23 (3’-C);
69.19 (6’-C); 76.39 (4’-C); 86.62 (CPh3); 113.18 (Cmeta-PhOCH3); 122.43 (5-C);
126.94-135.56 (9 ꢁ C arom), 142.67 (8-C); 144.25 (C arom); 149.47 (4-C); 152.25
(2-C); 152.77 (6-C); 158.59 (C arom); 164.47 (CONH).
HRMS calcd for C44H50N5O6Si (MH)+ 772.3530, found 772.3519.
1.5-Anhydro-4,6-O-benzylidene-2-deoxy-2-(thymin-1-yl)-D-altro-hexitol (8).
a) A mixture of thymine (6.31 g, 50.00 mmol) and sodium hydride 60% in
mineral oil (1.80 g, 48.00 mmol) in anhydrous DMF (200 ml) was stirred at
90°C for 30 minutes under nitrogen. Then, the mixture was cooled down to
60°C and the epoxide (4.69 g, 20.00 mmol) was added. The temperature was
raised up to 130°C and stirring was continued for 18 hours. The reaction
mixture was then cooled and evaporated to dryness. The pink residue was
dissolved in ethyl acetate (250 ml), and the organic layer was washed with a
saturated solution of sodium hydrogencarbonate (3 ꢁ 250 ml). The organic
layer was dried over sodium sulfate, filtered and evaporated to dryness. The
crude material was subjected to silica gel column chromatography, using a

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stepwise gradient of methanol (1–2.5%) in dichloromethane to afford the title
compound (8) after crystallization from toluene (3.98 g, 55%).
b) A mixture of epoxide (3,04 g, 13 mmol), thymine (3.78 g, 30 mmol), and
DBU (6.0 mL, 39 mmol) in dry DMF (50 mL) was stirred at 80–90 C under
argon for 3 h. The reaction was monitored with TLC. When starting epoxide
has disappeared the reaction mixture was cooled, the volatiles were removed,
and the residue was powdered into ice-water (250 mL). The pH was adjusted
to neutral with 1N HCl at 0°C and solid formed was filtered off, washed with
water, and dissolved in dichloromethane (50 mL). The solution was dried
over magnesium sulfate, concentrated and crude material was purified by
column chromatography on silica gel using 2% MeOH in dichloromethane.
Yield 3.5 g (75%).
1H-NMR (CDCl3, 500 MHz) d 1.93 (3H, s, CH3); 3.64 (1H, dd, J = 2.9 Hz,
J = 9.7 Hz, 4’-H); 3.69 (1H, t, J = 10.5 Hz, 6’ax-H); 3.88 (1H, br d, J = 13.7 Hz, 1’ax-
H); 4.11 (1H, td, J = 5.1 Hz, J = 10.0 Hz, 5’-H); 4.30-4.38 (3H, m, 1’eq-H, 3’-H and
6’eq-H); 4.42 (2H, m, 2’-H and 3’-OH); 5.57 (1H, s, PhCH); 7.29–7.33 (3H, m, H
arom); 7.43–7.45 (2H, m, H arom); 7.77 (1H, s, 6-H); 10.36 (1H, br s, NH).
13C-NMR (CDCl3, 125 MHz) d52.69 (CH3); 57.01 (2’-C); 63.91 (1’-C); 65.76
(3’-C); 66.07 (5’-C); 68.91 (6’-C); 76.60 (4’-C); 102.06 (PhCH); 111.34 (5-C);
126.13–137.03 (4 ꢁ C arom), 137.74 (6-C); 151.45 (2-C); 164.04 (4-C).
HMRS calcd for C18H21N2O6 (MH)+ 361.1399, found 361.1408.
1,5-Anhydro-4,6-O-benzylidene-3-O-tert-butyldimethylsylil-2 deoxy-2-(thymin-
1-yl)-D-altro-hexitol (9). Tert-butyldimethylsilyl chloride (9.04 g, 60 mmol) was
added to a solution of 8 (7.21 g, 20 mmol) and imidazole (6.68 g, 100 mmol) in dry
DMF (60 ml). The reaction mixture was stirred at 70°C, under nitrogen. After 15 hours,
1 equivalent of imidazole (1.36 g) and 1 equivalent of TBDMSCl (3.00 g) were added
and stirring was continued for 24 hours. The DMF was removed under reduced
pressure and the residue was dissolved in ethylacetate (100 ml) and washed with brine
(2 ꢁ 100 ml). The organic layer was dried over sodium sulfate, filtered, and evaporated
to dryness. The white solid was subjected to silica gel column chromatography using a
stepwise gradient of methanol (0–2%) in dichloromethane, to afford the title compound
9 as a white foam (3.88 g, 88%).
1H-NMR (CDCl3, 500 MHz) d 0.08 (3H, s, CH3Si); 0.18 (3H, s, CH3Si); 0.94 (9H,
s, (CH3)3C); 1.98 (3H, s, CH3); 3.56 (1H, dd, J = 2.2 Hz, J = 9.5Hz, 4’-H); 3.71 (1H,
t, J = 10.4 Hz, 6’ax-H); 4.02 (1H, br d, J = 13.4 Hz, 1’ax-H); 4.11 (1H, td, J = 5.1 Hz,
J = 10.0 Hz, 5’-H); 4.27–4.29 (2H, m, 2’-H and 3’-H); 4.35 (1H, dd, J = 5.1 Hz,
J = 10.3 Hz, 6’eq-H); 4.39 (1H, dd, J = 2.9 Hz, J = 13.4 Hz, 1’eq-H); 5.55 (1H, s,
PhCH); 7.33–7.35 (3H, m, H arom); 7.43–7.45 (2H, m, H arom); 7.86 (1H, s, 6-H);
9.00 (1H, br. s, NH).
13C-NMR (CDCl3, 125 MHz) d-5.0 1 (CH3Si); -4.74 (CH3Si); 12.69 (CH3); 18.03
((CH3)3C); 25.73 ((CH3)3C); 57.85 (2’-C); 64.00 (1’-C); 66.22 (5’-C); 66.77 (3’-C);
68.98 (6’-C); 76.95 (4’-C); 102.32 (PhCH); 110.96 (5-C); 126.18-137.27 (4 ꢁ C arom),
137.84 (6-C); 150.95 (2-C); 163.65 (4-C).
HMRS calcd for C24H35N2O6Si (MH)+ 475.2264, found 475.2263.

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1,5-Anhydro-3-O-tert-butyldimethylsilyl-2-deoxy-2-(thymin-1-yl)-D-altro-hexitol
(10). The compound 9 (2.37 g, 5.00 mmol) was dissolved in dichloromethane (52.5 ml)
and TFA was added (22.5 ml). After 1 hour stirring at room temperature, the volatiles were
removed under reduced pressure (water bath < 30°C) and the residue was purified on silica
gel column chromatography using a stepwise gradient of methanol (2–4%) in
dichloromethane to afford the title compound 10 as a white solid (1.74 g, 90%).
1H-NMR (CDCl3, 500 MHz) d 0.18 (3H, s, CH3Si); 0.28 (3H, s, CH3Si); 0.95 (9H,
s,(CH3)3C); 1.91 (3H, s, CH3); 2.11 (2H, br s, 6’-OH and 4’-OH); 3.61 (1H,dd, J =
2.9 Hz, J = 9.8 Hz, 4’-H); 3.71 (1H, ddd, J = 2.9 Hz, J = 5.2 Hz, J = 9.6 Hz, 5’-H);
3.85 (1H, dd, J = 5.4 Hz, J = 11.9 Hz, 6’ax-H); 3.96 (1H, dd, J = 2.8 Hz, J = 11.8 Hz,
6’eq-H); 4.06 (1H, br d, J = 13.4 Hz, 1’ax-H); 4.14 (1H, t, J = 2.9 Hz, 3’-H); 4.23 (1H,
dd, J = 3.4 Hz, J = 13.4 Hz, 1’eq-H); 4.36 (1H, td, J = 3.4 Hz, J = 13.4 Hz, 2’-H); 7.91
(1H, s, 6-H); 9.13 (1H, br. s, NH).
13C-NMR (CDCl3, 125 MHz) d-5.09 (CH3Si); ꢀ 4.57 (CH3Si);12.63 (CH3); 17.98
((CH3)3C); 25.75 ((CH3)3C); 56.45 (2’-C); 63.00 (6’-C); 63.23 (1’-C); 65.26 (4’-C);
69.14 (3’-C); 76.37 (5’-C); 110.95 (5-C); 138.33 (6-C); 151.21 (2-C); 163.69 (4-C).
HMRS calcd for C17H31N2O6Si (MH)+ 387.1951, found 387.1956.
1,5-Anhydro-3-O-tert-butyldimethylsilyl-2 deoxy-6-O-monomethoxytrityl-2-
(thymin-1-yl)-D-altro-hexitol (11). Monomethoxytrityl chloride (2.36 g, 7.64 mmol)
was added to a stirred solution of 10 (2.69 g, 6.95 mmol) in dry pyridine (35 ml) at
room temperature under nitrogen. After 2 hours, methanol (5 ml) and solid sodium
hydrogencarbonate were added. The volatiles were removed and the residue was
dissolved in dichloromethane (150 ml). The organic phase was washed with a 5%
solution of sodium hydrogencarbonate (2 ꢁ 100 ml), dried over anhydrous sodium
sulfate, evaporated and coevaporated with toluene. The residue was subjected to silica
gel column chromatography using a stepwise gradient of ethylacetate (20–40%) in
hexane (containing 0.2% triethylamine), to afford the title compound 11 as a white
solid (4.23 g, 92%).
1H-NMR (CDCl3, 500 MHz) d 0.16 (3H, s, CH3Si); 0.27 (3H, s, CH3Si); 0.92 (9H,
s, (CH3)3C); 1.78 (3H, s, CH3); 1.85 (1H, br s, 4’-OH); 3.34 (1H, dd, J = 3.7 Hz,
J = 10.3 Hz, 4’-H); 3.44 (1H, dd, J = 2.2 Hz, J = 10.3 Hz, 5’-H); 3.67 (1H, td,
J = 2.8 Hz, J = 9.3 Hz, 6’ax-H); 3.79 (3H, s, OC H3); 3.97 (1H, dd, J = 2.9 Hz,
J = 9.6 Hz, 6’eq-H); 4.05 (1H, br d, J = 13.4 Hz, 1’ax-H); 4.18–4.22 (2H, m, 3’-H and
1’eq-H); 4.36 (1H, t, J = 2.4 Hz, 2’-H); 6.83 (2H, d, J = 9.0 Hz, H arom); 7.22–7.48
(12H, m, H arom); 8.16 (1H, s, 6-H); 8.79 (1H, br. s, NH).
13C-NMR (CDCl3, 125 MHz) d-5.06 (CH3Si); -4.64 (CH3Si);12.81 (CH3); 17.97
((CH3)3C); 25.76 ((CH3)3C); 55.19 (OCH3); 56.53 (2’-C); 63.07 (1’-C); 63.48 (6’-C);
64.88 (4’-C); 69.27 (3’-C); 75.79 (5’-C); 86.36 (CPh3); 110.87 (5-C); 113.13 (Cmeta-
PhOCH3); 126.94–135.46 (5 ꢁ C arom), 138.65 (6-C); 144.38 (2 ꢁ C arom); 151.16
(2-C); 158.59 (C arom); 163.62 (4-C).
HMRS calcd for C37H46N2O7SiNa (MNa)+ 681.2972, found 681.2996.
1,5-Anhydro-4,6-O-benzylidene-3-O-tert-butyldimethylsilyl-2 deoxy-2-(5-
methylcytosin-1-yl)-D-altro-hexitol (12). Phosphorous oxychloride freshly distilled
(0.700 ml, 7.5 mmol) was added to a stirred solution of 1,2,4-1H-triazole (1.73 g,
25.0 mmol) in dry pyridine (17.5 ml) under nitrogen. After 20 minutes stirring, 9

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Abramov et al.
(1.19 g, 2.50 mmol) was added, and stirring was continued for 3 hours. The volatiles
were removed and the residue was coevaporated with toluene (2 times) and partitioned
between water and ethyl acetate (strong emulsion). The organic layer was washed with
water (2 times), brine (2 times), and evaporated to dryness to afford a yellow foam.
This crude intermediate was dissolved in dioxane (17 ml), and 8 ml aqueous ammonia
(25%) were added. After 45 minutes stirring, the volatiles were evaporated and the
solid was coevaporated with toluene (2 times). The residue was subjected to silica gel
column chromatography, using a stepwise gradient of methanol (2–4%) in
dichloromethane, to afford the title compound 12 as a white powder (0.770 g, 65%).
1H-NMR (CDCl3, 500 MHz) d 0.08 (3H, s, CH3Si); 0.22 (3H, s, CH3Si); 0.94 (9H,
s, (CH3)3C); 1.97 (3H, s, CH3); 3.53 (1H, dd, J = 2.4 Hz, J = 9.5 Hz, 4’-H); 3.69 (1H,
t, J = 10.4 Hz, 6’ax-H); 4.01 (1H, br d, J = 13.7 Hz, 1’ax-H); 4.11 (1H, td, J = 5.1 Hz,
J = 10.0 Hz, 5’-H); 4.34 (1H, dd, J = 5.1 Hz, J = 10.3 Hz, 6’eq-H); 4.36–4.41 (3H, m,
3’-H, 1’ax-H and 2’-H); 5.52 (1H, s, PhCH); 7.32–7.36 (3H, m, H arom); 7.42–7.45
(2H, m, H arom); 7.88 (1H, s, 6-H).
13C-NMR (CDCl3, 125 MHz) d-4.80 (CH3Si); -4.74 (CH3Si); 13.51 (CH3); 18.04
((CH3)3C); 25.80 ((CH3)3C); 58.24 (2’-C); 64.49 (1’-C); 66.19 (5’-C); 66.51 (3’-C);
69.08 (6’-C); 76.97 (4’-C); 101.60 (5-C); 102.21 (PhCH); 126.18–137.44 (4 ꢁ C
arom), 140.76 (6-C); 156.32 (2-C); 165.48 (4-C).
HMRS calcd for C24H36N3O5Si (MH)+ 474.2424, found 474.2421.
1,5-Anhydro-4,6-O-benzylidene-2-(N4-benzoyl-5-methylcytosin-1-yl)-3-O-tert-
butyldimethylsylil-2 deoxy-D-altro-hexitol (13). Benzoyl chloride (3.20 ml,
27.55 mmol) was added to a stirred solution of 12 (2.61 g, 5.51 mmol) in dry
pyridine (40 ml) at 0°C under nitrogen. The reaction mixture was stirred at room
temperature overnight and the pyridine was removed. The residue was suspended in
dichloromethane (50 ml) and the organic phase was washed with a saturated solution of
sodium hydrogencarbonate (2 ꢁ 50 ml). Aqueous ammonia (25%), 20 ml was added at
0°C to the organic phase, and the mixture was stirred at room temperature for 1 hour.
The volatiles were removed and the crude material was subjected to silica gel column
chromatography using a mixture of hexane/ethyl acetate (7/3) as eluent, to afford the
title compound 13(1.83 g, 92%).
1H-NMR (CDCl3, 500 MHz) d 0.09 (3H, s, CH3Si); 0.20 (3H, s, CH3Si); 0.94 (9H,
s, (CH3)3C); 2.16 (3H, br d, J = 1.0 Hz, CH3); 3.59 (1H, dd, J = 2.4 Hz, J = 9.5 Hz, 4’-H);
3.73 (1H, t, J = 10.4 Hz, 6’ax-H); 4.07 (1H, br d, J = 13.7 Hz, 1’ax-H); 4.13 (1H, td,
J = 5.1 Hz, J = 10.0 Hz, 5’-H); 4.31 (1H, t, J = 2.4 Hz, 2’-H); 4.35 (1H, t, J = 2.9 Hz,
3’-H); 4.37 (1H, dd, J = 5.5 Hz, J = 10.5 Hz, 6’eq-H); 4.43 (1H, dd, J = 3.4 Hz,
J = 13.4 Hz, 1’eq-H); 5.55 (1H, s, PhCH); 7.32–7.35 (3H, m, H arom); 7.42–7.45 (4H,
m, H arom); 7.51–7.54 (1H, m, H arom); 8.02 (1H, s, 6-H); 8.31–8.33 (2H, m, H arom).
13C-NMR (CDCl3, 125 MHz) d-5.01 (CH3Si); -4.72 (CH3Si); 13.89 (CH3); 18.04
((CH3)3C); 25.74 ((CH3)3C); 58.32 (2’-C); 64.00 (1’-C); 66.27 (5’-C); 66.60 (3’-C);
69.00 (6’-C); 76.74 (4’-C); 102.32 (PhCH); 112.00 (5-C); 126.17–137.25 (8 ꢁ C
arom), 139.13 (6-C); 148.57 (2-C); 159.65 (4-C); 179.65 (PhCO).
HMRS calcd for C31H40N3O6Si (MH)4 578.2686, found 578.2687.
1,5-Anhydro-2-(N4-benzoyl-5-methylcytosin-1-yl)-3-O-tert-butyldimethylsilyl-2-
deoxy-D-altro-hexitol (14). Compound 13 (1.83 g, 3.17 mmol) was dissolved in
dichloromethane (34 ml) and cooled to 0°C. Trifluoroacetic acid (15 ml) was then

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added, and the reaction mixture was stirred at room temperature for 45 min. Solvents
were then removed and the residue was coevaporated with toluene (water bath < 30°C).
The crude material was subjected to silica gel column chromatography, using a
stepwise gradient of methanol (0–2%) in dichloromethane, to afford the title compound
14 as a white foam (1.05 g, 67%), which was directly used for the next step because of
its poor stability.
1,5-anhydro-2-(N4-benzoyl-5-methylcytosin-1-yl)-3-O-tert-butyldimethylsilyl-2-
deoxy-6-O-monomethoxytrityl-D-altro-hexitol (15). Monomethoxytrityl chloride
(0.46 g, 1.42 mmol) was added to a solution of 14 (0.63 g, 1.29 mmol) in dry
pyridine (6.45 ml) at room temperature under nitrogen. After 16 hours, methanol (1 ml)
and solid sodium hydrogencarbonate were added. The volatiles were removed and the
residue was dissolved in dichloromethane (50 ml). The organic phase was washed with
a 5% solution of sodium hydrogencarbonate (2 ꢁ 50 ml), dried over anhydrous sodium
sulfate, evaporated and coevaporated with toluene. The residue was subjected to silica
gel column chromatography using a stepwise gradient of ethyl acetate (0–10%) in
hexane (containing 0.2% triethylamine), to afford the title compound 15 as a white
solid (0.74 g, 75%).
1H-NMR (CDCl3, 500 MHz) d 0.16 (3H, s, CH3Si); 0.28 (3H, s, CH3Si); 0.93 (9H,
s, (CH3)3C); 1.97 (3H, s, CH3); 3.37 (2H, dd, J = 3.4 Hz, J = 10.5 Hz, 4’-H and 4’-
OH); 3.46 (1H, dd, J = 2.2 Hz, J = 10.4 Hz, 5’-H); 3.69 (1H, td, J = 2.8 Hz, J = 9.8 Hz,
6’ax-H); 3.79 (3H, s, OCH3); 3.98 (1H, dd, J-2.2 Hz, J = 9.0 Hz, 6’eq-H); 4.11 (1H, br d,
J = 13.4 Hz, 1’ax-H); 4.20–4.24 (2H, m, 3’-H and 1’eq-H); 4.43 (1H, t, J = 2.4 Hz, 2’-H);
6.84 (2H, d, J = 9.0 Hz, H arom); 7.21–7.54 (12H, m, H arom); 8.30–8.33 (3H, m, H
aromand 6-H).
13C-NMR (CDCl3, 125 MHz) d ꢀ 5.03 L (CH3Si); -4.63 (CH3Si); 13.85 (CH3);
18.00 ((CH3)3C); 25.78 ((CH3)3C); 55.21 (OCH3); 57.06 (2’-C); 63.05 (1’-C); 63.55
(6’-C); 64.87 (4’-C); 69.27 (3’-C); 75.86 (5’-C); 86.39 (CPh3); 111.94 (5-C); 113.16
(Cmeta-PhOCH3); 126.95–137.26 (10 ꢁ C arom), 140.04 (6-C); 144.38 (2 ꢁ C
arom); 148.80 (2-C); 158.62 (C arom); 159.77 (4-C); 179.65 (PhCO).
HMRS calcd for C44H52N3O7Si (MH)+ 762.3574, found 762.3576.
2-(2-Amino-6-chloropurin-9-yl)-1,5-anhydro-4,6-O-benzylidene-2-deoxy-D-
altro-hexitol (17).
a) A mixture of epoxide (304 mg, 1.3 mmol), 2-amino-6-chloropurin (450 mg,
2.65 mmol), aliquat 336 (343 mg, 1.3 mmol), and potassium carbonate
(360 mg, 2,6 mmol) in dry HMPA (5 mL) was stirred at 80–90 C under argon
for 3 h. The reaction was monitored with TLC and, when starting epoxide has
disappeared, the mixture was cooled immediately and powdered into ice-water
(50 mL). The suspension was stirred for 1 h, solid was filtered off, washed
with water, and dissolved in dichloromethane (50 mL). Solution was dried
over magnesium sulfate, evaporated to dryness and crude material was purified
by column chromatography on silica gel using 2% MeOH in dichloromethane.
Yield 335 mg (64%).
b) General procedure for purine nucleosides: a mixture of purine base (1.5 mmol),
epoxide (0.5 mmol), and DBU (0.5 mL) dry DMF (1.5 mL) in a 10 mL glass

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tube, which was tightly sealed with an aluminum/Teflon crimp, was irradiated
at 150 W, 110°C with gas jet cooling for 15 min in a CEM-Discover mono-
mode microwave apparatus. The vessel was cooled down to 60°C and the
residue was powdered into ice-water (10 mL). The pH was adjusted to neutral
with 1N HCl at 0°C and solid formed was filtered off, washed with water, and
dissolved in dichloromethane (50 mL). The solution was dried over
magnesium sulfate, concentrated and crude material was purified by column
chromatography on silica gel (35% yield).
1H NMR: (DMSO-d6, 500 MHz, d): 3.69 (1H, dd, J = 2.4 Hz, J = 9.5 Hz, 4’-H);
3.80 (1H, t, J = 10.2 Hz, 6’ax-H), 4.00 (1H, td, J = 5.1 Hz, J = 10.2 Hz, 5’-
H); 4.23–4.30 (4H, m, 1’eq-H, 1’ax-H, 3’-H, and 6’eq-H); 4.46 (1H, br s, 2’-
H); 5.64 (1H, s, PhCH); 5.88 (1H, d, J = 4.5 Hz, 3’-OH), 6.98 (2H, s, NH2);
7.32–7.34 (3H, m, H arom); 7.38–7.40 (2H, m, H arom); 8.23 (1H, s, 8–11).
13C NMR: (DMSO-d6, 125 MHz, d): 55.87 (2’-C); 64.29 (1’-C); 64.77 (3’-C);
66.46 (5’-C); 68.13 (6’-C); 76.27 (4’-C); 101.06 (PhCH); 123.02 (5-C);
126.41-137.75 (4 ꢁ C arom); 141.37 (8-C); 149.73 (2-C); 154.26 (4-C);
159.93 (6-C).
1
HRMS: calcd for C18H19ClN5O4(M + H) 404.1125, found 404.1119.
c) When epoxidation of 2-amino-6-chloropurin was carried out in DMF the
formation of next by-products have been observed.
2-(2-Amino-6-N,N-dimethylaminopurin-9-yl)-1,5-anhydro-4,6-O-benzylidene-2-
deoxy-D-altro-hexitol. Yield 25% (18). 1H NMR: (DMSO-d6, 500 MHz, d): 3.57 (6H,
br s, N(CH3)2); 3.67 (1H, dd, J = 2.4 Hz, J = 9.5 Hz, 4’-H); 3.77 (1H, t, J = 10.2 Hz,
6’ax-H), 3.99 (1H, td, J = 5.1 Hz, J = 10.2 Hz, 5’-H); 4.16–4.29 (4H, m, 1’eq-H, 1’ax-H,
3’-H, and 6’eq-H); 4.39 (1H, br s, 2’-H); 5.64 (1H, s, PhCH); 5.80 (1H, br s, 3’-OH), 5.88
(2H, s, NH2); 7.32–7.34 (3H, m, H arom); 7.40–7.42 (2H, m, H arom); 7.92 (1H, s,8-H).
13C NMR: (DMSO-d6, 125 MHz, d): 37.70 (N(CH3)2); 55.16 (2’-C); 64.59 (1’-C);
64.96 (3’-C); 66.34 (5’-C); 68.21 (6’-C); 76.42 (4’-C); 101.06 (PhCH); 113.24 (5-C);
126.41–137.75 (4 ꢁ C arom); 134.74 (8-C); 152.87 (4-C); 154.88 (4-C) and 159.66 (2-C
and 6-C).
HRMS: calcd for C20H25N6O4(M + H)+ 413.1937, found 413.1916.
2-(2-Amino-6-chloropurin-7-yl)-1,5-anhydro-4,6-O-benzylidene-2-deoxy-D-
1
altro-hexitol (19). Yield 5%. 1H NMR: H NMR: (DMSO-d6, 500 MHz, d): 3.62
(1H, dd, J = 2.4 Hz, J = 9.5 Hz, 4’-H); 3.78 (1H, t, J = 10.2 Hz, 6’ax-H), 3.99 (1H, td,
J = 5.1 Hz, J = 10.2 Hz, 5’-H); 4.24–4.38 (4H, m, 1’eq-H, 1’ax-H, 3’-H, and 6’eq-H);
4.74 (1H, br s, 2’-H); 5.61 (1H, s, PhCH); 6.00 (1H, d, J = 4.4 Hz, 3’-OH), 6.65 (2H, s,
NH2); 7.32–7.34 (3H, m, H arom); 7.38–7.40 (2H, m, H arom); 8.46 (1H, s, 8-H).
13C NMR: (DMSO-d6, 125 MHz, d): 57.90 (2’-C); 64.45 (1’-C); 66.23 (3’-C);
66.50 (5’-C); 68.11 (6’-C); 75.69 (4’-C); 101.00 (PhCH); 115.22 (5-C); 126.35–137.77
(4 ꢁ C arom); 142.49 (8-C); 147.54 (2-C); 160.11 (6-C); 163.86 (4-C).
HRMS: calcd for C18H19ClN5O4(M + H)+ 404.1125, found 404.1119.
[2-Amino-6-(2-amino-6-chloropurin)purin-9-yl]-1,5-anhydro-4,6-O-benzyli-
dene-2-deoxy-D-altro-hexitol (20). Yield 5%. 1H NMR: (DMSO-d6, 500 MHz,

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D-Altritol Nucleosides with a 3’-O-TBDMS Group
453
d): 3.72 (1H, dd, J = 2.4 Hz, J = 9.5 Hz, 4’-H); 3.83 (1H, t, J = 10.2 Hz, 6’ax-H), 4.02
(1H, td, J = 5.1 Hz, J = 10.2 Hz, 5’-H); 4.25–4.32 (4H, m, 1’eq-H, 1’ax-H, 3’-H, and
6’eq-H); 4.56 (1H, br s, 2’-H); 5.67 (1H, s, PHCH); 5.92 (1H, br s, 3’-OH); 6.98 (2H, s,
NH2); 7.05 (2H, s, NH2); 7.32–7.35 (3H, m, H arom); 7.39–7.43 (2H, m, H arom);
8.37 (1H, s, 8@-H); 8.63 (1H, s, 8-H).
13C NMR: (DMSO-d6, 125 MHz, d): 55.75 (2’-C); 64.42 (1’-C); 64.87 (3’-C);
66.47 (5’-C); 68.08 (6’-C); 76.31 (4’-C); 101.07 (PhCH); 118.86; 123.35; 126.33-137.75
(4 ꢁ C arom); 142.03; 145.42; 150.14; 153.83; 155.96; 160.08; 160.41; 160.55.
HRMS: calcd for C23H22N10O4 (M + H)+ 537.1513, found 537.1514.
1,5-Anhydro-4,6-O-benzylidene-2-deoxy-2-(guanin-9-yl)-D-altro-hexitol
(21). A suspension of 2-(2-amino-6-chloropurin-9-yl)-1,5-anhydro-4,6-O-benzylidene-
2-deoxy-D-altro-hexitol (2.5 g, 6.2 mmol) and DABCO (200 mg) in 1 N NaOH (100 ml)
was stirred at 90 C for 2 h. The resulting clear colorless solution was cooled with ice-
bath. The pH was adjusted to neutral with 1N HCl at 0 C and solid formed was filtered
off, washed with water and dried under reduced pressure. Yield 2.2 g (92% of 21) (2).
1,5-Anhydro-4,6-O-benzylidene-2-(N2-iso-butyrylguanin-9-yl)-2-deoxy-D-altro-
hexitol (22). 1,5-Anhydro-4,6-O-benzylidene-2-deoxy-(guanin-9-yl)-D-altro-hexitol
(3.4 g, 8.8 mmol) was co-evaporated with with pyridine (2 ꢁ 100 ml) and to a
resulting suspension in pyridine (150 mL) was added TMSCl (5.5 mL) dropwise at 0°C
under argon. The resulting clear solution was stirred overnight at room temperature,
after that extra TMSCl (8 mL) was added and stirring was continued for 8 h. iso-
Butyric anhydride (6 mL) was added and reaction mixture was stirred for 24 h. Ice-
water (20 mL) was added at 0°C followed by 25% NH3(20 mL) and resulting mixture
was stirred for 2 h and evaporated under reduced pressure. The residue was extracted
with ethyl acetate, washed with water, dried over magnesium sulfate and evaporated to
dryness. Semisolid was dissolved in THF (30 mL) and 1N NBu4F (15 mL) was added
dropwise at 0°C to the resulting solution. After 30 min, water (50 mL) was added and
THF was removed under reduced pressure and cooled. Solid was filtered off, dried and
purified by silica gel column chromatography, using a stepwise gradient of methanol
(2–4%) in dichloromethane. Yield 3.1 g (77%).
1H NMR: (CDCl3, 500 MHz, d): 1.22 (3H, d, J = 6.8 Hz, CH3CH); 1.27 (3H,
d, J = 6.8 Hz, CH3CH); 2.79 (1H, dt, J = 7.1 Hz, J = 13.6 Hz, (CH3)2CH); 3.59
(1H, dd, J = 1.8 Hz, J = 9.5 Hz, 4’-H); 3.69 (1H, t, J = 10.5, Hz, 6’ax-H), 4.09
(1H, td, J = 5.1 Hz, J = 10.2 Hz, 5’-H); 4.21 (1H, br d, J = 13.0, 1’ax-H); 4.32
(1H, dd, J = 4.8 Hz, J = 10.2 Hz, 6’eq-H); 4.40–4.50 (3H, m, 1’eq-H, 1’ax-H, 3’-H
and 2’-H); 4.79 (1H, br s, 3’-OH), 5.48 (1H, s, PhCH); 7.22–7.23 (3H, m, H arom);
7.32–7.34 (2H, m, H arom); 8.09 (1H, s, 8-H); 10.46 (1H, br s, NH); 12.23 (1H,
br s, NH).
13C NMR: (CDCl3, 125 MHz, d): 18.91 (CH3)2CH); 19.02 (CH3)2CH); 36.15
(CH3)2CH); 55.78 (2’-C); 64.71 (1’-C); 66.03 (3’-C); 66.86 (5’-C); 68.85 (6’-C); 76.75
(4’-C); 101.95 (PhCH); 119.89 (5-C); 125.97–136.97 (4 ꢁ C arom); 138.11 (8-C);
148.12 (4-C); 149.15 (2-C); 155.91 (6-C); 180.00 (C = O).
HRMS: calcd for C22H26N5O6(M + H)+ 456.1882, found 456.1887.
1,5-Anhydro-4,6-O-benzylidene-3-O-tert-butyldimethylsilyl-2-(N2-iso-butyryl-
guanin-9-yl)-2-deoxy-D-altro-hexitol (23). A mixture of 1,5-Anhydro-4,6-O-benzyli-

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Abramov et al.
dene-2-(N2-iso-butyrylguanin-9-yl)-2-deoxy-D-altro-hexitol (2.4 g, 5.2 mmol) and
pyridine (150 mL) was evaporated up to 50 mL and imidazole (2.8 g, 40 mmol) and
TBDMSCl (3.1 g, 20 mmol) was added under argon at room temperature. The reaction
mixture was stirred at 70°C for 8 h. Imidazole (1.4 g 20 mmol) and TBDMSCl (1.5 g,
10 mmol) was added and stirring was continued for 18h. The last operation was
repeated and resulting solution was powdered into 5% NaHCO3(250 ml). White
semisolid was collected, dissolved in ethyl acetate, washed with 5% NaHCO3, water,
dried over magnesium sulfate and solvent was removed under reduced pressure. The
title compound was purified by column chromatography on silica gel using 2% MeOH
in dichloromethane. Yield 2.7 g (91%).
1H NMR: (CDCl3, 500 MHz, d): 0.10 (3H, s, CH3Si); 0.18 (3H, s, CH3Si); 0.96 (3H,
s, (CH3)3Si); 1.26 (3H, d, J = 6.2 Hz, CH3CH); 1.27 (3H, d, J = 6.2 Hz, CH3CH); 2.69
(1H, dt, J = 7.1 Hz, J = 13.6 Hz, (CH3)2CH); 3.57 (1H, dd, J = 1.8 Hz, J = 9.5 Hz, 4’-H);
3.72 (1H, t, J = 10.5, Hz, 6’ax-H), 4.09 (1H, td, J = 5.1 Hz, J = 10.2 Hz, 5’-H); 4.21 (1H,
br d, J = 13.0, 1’ax-H); 4.33–4.36 (3H, m, 3’-H, 6’eq-H and 2’-H); 4.45 (1H, dd,
J = 5.1 Hz, J = 2.8 Hz 1’eq-H), 5.46 (1H, s, PhCH); 7.31–7.32 (3H, m, H arom); 7.39–
7.41 (2H, m, H arom); 8.09 (1H, s, 8-H); 8.17 (1H, br s, NH); 11.95 (1H, br s, NH).
13C NMR: (CDCl3, 125 MHz, d): ꢀ 4.91 (CH3Si); -4.47 (CH3Si); 18.11 (CH3)3C);
18.79 (CH3)2CH); 19.00 (CH3)2CH); 36.39 (CH3)2CH); 56.73 (2’-C); 64.65 (1’-C);
66.78 (3’-C); 67.48 (5’-C); 68.95 (6’-C); 76.75 (4’-C); 102.31 (PhCH); 120.84 (5-C);
126.16-137.22 (4 ꢁ C arom); 137.93 (8-C); 147.37 (4-C); 148.43 (2-C); 155.43 (6-C);
178.10 (C = O).
HRMS: calcd for C28H40N5O6Si (M-H)+ 570.2747, found 570.2733.
1,5-Anhydro-3-O-tert-butyldimethylsilyl-2-(N2-iso-butyrylguanin-9-yl)-2-deoxy-
6-D-altro-hexitol (24). To a solution of 1,5-Anhydro-3-O-tert-butyldimethylsilyl-2-
(N2-iso-butyrylguanin-9-yl)-2-deoxy-D-altro-hexitol (3.2 g, 5.6 mmol) in dichloro-
methane (60 mL), TFA (15 mL) was added dropwise at 0°C and reaction mixture was
stirred for 15 min and co-evaporated with toluene (water bath < 30°C). The crude
product was purified by column chromatography on silica gel using using stepwise
gradient of methanol (2–4%) in dichloromethane containing triethylamine (0.2%). First
fraction contained 1.6 g of recycled starting compound. Second fraction was collected,
evaporated to dryness and the crude product was used directly on the next step.
1,5-Anhydro-3-O-tert-butyldimethylsilyl-2-(N2-iso-butyrylguanin-9-yl)-2-deoxy-
6-O-monomethoxytrityl-D-altro-hexitol (25). A solution of crude 1,5-anhydro-3-O-
tert-butyldimethylsilyl-2-(N2-iso-butyrylguanin-9-yl)-2-deoxy-6-O-monomethoxytrilyl-
D-altro-hexitol in pyridine (60 mL) was evaporated up to 20 mL and MMTrCl (1.0g,
3.25 mmol) was added under argon at room temperature. After 6 h methanol (5 mL)
and solid sodium hydrocarbonate were added. The volatiles were removed, the residue
was coevaporated with toluene (2 ꢁ 20 mL) and the title product was purified by
column chromatography on silica gel using stepwise gradient of methanol (1–2%) in
dichloromethane containing triethylamine (0.2%). Yield 1.8 g (85% on reacted 1,5-
anhydro-4,6-O-benzylidene-O-tert-butyldimethylsilyl-(iso-butyrylguanin-9-yl)-2-deoxy-
D-altro-hexitol).
1H NMR: (CDCl3, 500 MHz, d): 0.12 (3H, s, CH3Si); 0.16 (3H, s, CH3Si); 0.91
(3H, s, (CH3)3Si); 1.15 (3H, d, J = 6.9 Hz, CH3CH); 1.19 (3H, d, J = 6.9 Hz, CH3CH);
2.07 (1H, br s, 4’-OH); 2.60 (1H, m, (CH3)2CH); 3.35 (1H, m, 6’ax-H); 3.46 (1H, d,

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D-Altritol Nucleosides with a 3’-O-TBDMS Group
455
J = 10.0, 6’eq-H); 3.67 (2H, m,5’-H and 4’-H); 3.72 (3H, s, CH3O); 4.15–4.20 (2H, m,
3’-H, 1’eq-H); 4.21 (1H, br d, J = 13.0, 1’ax-H); 4.45 (1H, m, 2’-H); 6.83 (2H, d,
J = 8.8, H arom);7.18–7.48 (12H, m, H arom); 8.22 (1H, s, 8-H); 8.74 (1H, br s, NH);
12.04 (1H, br s, NH).
13C NMR: (CDCl3, 125 MHz, d): ꢀ 4.75 (br, CH3Si); 17.92 (CH3)3C); 18.76
(CH3)2CH); 18.94 (CH3)2CH); 36.18 (CH3)2CH); 55.16 (CH3O); 55.61 (2’-C); 63.89
(1’-C); 64.22 (6’-C); 65.82 (4’-C); 69.86 (3’-C); 76.25 (5’-C); 86.71 (Ph3C); 113.16
(Cmeta-PhOCH3); 120.74 (5-C); 126.94-135.32 (5 ꢁ C arom); 138.70 (8-C); 147.35
(4-C); 144.25 (C arom); 144.27 (C arom); 148.57 (2-C); 155.62 (6-C); 158.58 (C
arom); 178.61 (C = O). HRMS: calcd for C41H52N5O7Si (M + H) 1754.3635, found
754.3634.
ACKNOWLEDGMENTS
This research was financially supported by grants from the Katholicke Universiteit
Leuven (GOA 2002/13) and IWT (GBOU). We thank C. Biernaux for editorial help.
REFERENCES
1. Allart, B.; Khan, K.; Rosemeyer, H.; Schepers, G.; Hendrix, C.; Rothenbacher, K.;
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hydridisation properties, stability, and initial structural analysis. Chem. Eur. J. 1999,
5, 2424–2431.
2. Allart, B.; Busson, R.; Rozenski, J.; Van Aerschot, A.; Herdewijn, P. Synthesis of
protected D-altritol nucleosides as building blocks for oligonucleotide synthesis.
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3. Reese, C.B.; Trentham, D.R. Acyl migration in ribonucleoside derivatives.
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7. Divakar, K.J.; Reese, C.B. 4-(1,2,4-Triazol-1-yl)-and 4-(3-nitro-1,2,4-triazol-1-
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Received July 15, 2003
Accepted October 31, 2003

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