Synthesis of 2′-amino-LNA: A new strategy

Article abstract of DOI:10.1039/b208864a

In this paper we present revised and significantly improved synthetic routes to 2′-amino-LNA (locked nucleic acid). The optimal route is convergent with the synthesis of LNA monomers ("2′-oxy-LNA") via a common intermediate obtained by a mild deacetylatio

Full text of DOI:10.1039/b208864a

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Synthesis of 2Ј-amino-LNA: a new strategy†
Christoph Rosenbohm,a Signe M. Christensen,a Mads D. Sørensen,b Daniel Sejer Pedersen,‡a
Lotte-Emilie Larsen,a,b Jesper Wengelc and Troels Koch*a
a Cureon A/S, Fruebjergvej 3, DK-2100 Copenhagen, Denmark. E-mail: tk@cureon.com;
Fax: ϩ45 7026 0097; Tel: ϩ45 7026 0096
b Department of Chemistry, University of Copenhagen, Universitetsparken 5,
DK-2100 Copenhagen, Denmark
c Nucleic Acid Center, Department of Chemistry, University of Southern Denmark,
Campusvej 55, DK-5230 Odense M, Denmark
Received 10th September 2002, Accepted 6th December 2002
First published as an Advance Article on the web 21st January 2003
In this paper we present revised and significantly improved synthetic routes to 2Ј-amino-LNA (locked nucleic
acid). The optimal route is convergent with the synthesis of LNA monomers (“2Ј-oxy-LNA”) via a common
intermediate obtained by a mild deacetylation for the liberation of the 2Ј-hydroxy group to give compound 23
without the concomitant ring closure that affords the 2Ј-oxy-LNA skeleton. After inversion of the stereochemistry
at C2Ј and triflate formation at the 2Ј-hydroxy group a new common intermediate 16 is obtained which gives easy
access to a range of other analogues exemplified by the introduction of a sulfur nucleophile leading to the 2Ј-thio-
LNA structure. After substitution of the triflate with azide a basic reduction affords the desired 2Ј-amino-LNA
structure, i.e., compound 18. This new synthesis strategy towards 2Ј-amino-LNA improves the overall yield
significantly and converges the syntheses of 2Ј-oxy-LNA and LNA analogues.
that they can be synthesised by efficient and convergent syn-
thesis strategies.
Introduction
The promising aspects of using modified analogues of natural
nucleic acids as therapeutic agents have stimulated much inter-
est in this field during recent years. LNA (locked nucleic acid)§
was introduced independently by Wengel1,2 and by Imanishi3
in 1998 as a novel class of conformationally restricted
oligonucleotide analogues.
It has been shown beyond any doubt that LNA is the single
nucleic acid modification that contributes the highest affinity
ever obtained by Watson–Crick hydrogen bonding.6 The 2Ј,4Ј-
linked bicyclic structure brings not only unprecedented affinity/
specificity of fully LNA modified oligonucleotides, but LNA
monomers can be mixed and act co-operatively with DNA and
RNA monomers and with most of the known nucleic acid
analogues such as phosphorothioates and 2Ј-O-alkyl modified
RNA.7 These unique properties position LNA as a central
player in nucleic acid chemistry.
The first LNA monomer was based on the 2Ј-OCH2-4Ј
bicyclic structure (LNA/2Ј-oxy-LNA). Later similar high
affinity/specificity functionalised LNA monomers such as
2Ј-NHCH2-4Ј, 2Ј-N(CH3)CH2-4Ј (2Ј-amino-LNA),5,8 and
2Ј-SCH2-4Ј (2Ј-thio-LNA)8,9 were synthesised. We anticipate
that such LNA analogues will have different pharmacological,
in particular pharmacokinetic, properties from the parent
2Ј-oxy-LNA. Having a broad range of biological properties
within a high affinity/specificity structure will provide the
necessary tools to design improved antisense compounds. It is
therefore of great importance for the use of LNA analogues
The first synthesis of an LNA nucleoside was performed by a
linear approach using uridine as starting material,10 but by
virtue of being a convergent synthesis an alternative route1,2,7,11
became the method of choice for the synthesis of LNA nucleo-
sides. 2Ј-Amino- and 2Ј-thio-LNA were originally synthesised
using slightly different intermediates from those used for the
2Ј-oxy-LNA monomers, but in this paper we demonstrate that
common intermediates at late stages can be used for synthesis
of both 2Ј-oxy-LNA, 2Ј-amino-LNA, and 2Ј-thio-LNA.
Results and discussion
During our implementation of the previously described syn-
thesis of 2Ј-amino-LNA monomers5,8 (Scheme 1) to the 100 g
scale synthesis we encountered several major problems. The
first reaction that required attention in the scale-up work was
the regioselective benzylation at C5 of compound 1.1 Working
in the 100 g scale the reaction yielded a mixture of compound 2,
the 4-C-benzyloxymethyl derivative, and the fully benzylated
derivative, even under optimised conditions. The maximum
yield of the desired compound 2 was 59% but with a general
yield of 45–50% compared to 71% on smaller scale.1 Further-
more, compound 2 could only be isolated through tedious
chromatography of closely eluting products.
The second key step in the original strategy5,8 that caused
problems during scale-up was the double nucleophilic substitu-
tion of the di-O-tosyl nucleoside 5 using benzylamine giving
nucleoside 6. Originally it was reported that on an 8 g scale the
reaction afforded 52% of nucleoside 6 and no side reaction was
reported.5 However, in our hands the reaction on a larger scale
(22 g) afforded a significant amount of a by-product identified
as the 2Ј-oxy-LNA derivative. One can envision the formation
of this by-product by substitution on the sulfur atom of the
2Ј-O-tosylate 5 resulting in a free hydroxy group that can make
a nucleophilic attack on the remaining 4Ј-C-tosyloxymethyl
functionality to afford the 2Ј-oxy-LNA structure. The desired
N-benzylated-2Ј-amino-LNA product 6 was obtained in only
15% yield together with 13% of the 2Ј-oxy-LNA by-product.
† Electronic supplementary information (ESI) available: further
experimental details and the structure of compound S11 (.pdb file). See
http://www.rsc.org/suppdata/ob/b2/b208864a/
‡ Present address: Cambridge University, University Chemical Labor-
atory, Lensfield Road, Cambridge, UK CB2 1EW.
§ LNA is defined as an oligonucleotide containing one or more 2Ј-O,4Ј-
C-methylene-β--ribofuranosyl nucleotide monomers (LNA mono-
mers).1,2 For clarity the term 2Ј-oxy-LNA is used herein for the parent
LNA and 2Ј-amino-LNA and 2Ј-thio-LNA refer to the analogues
where the 2Ј-oxygen has been substituted by respectively a nitrogen
atom or a sulfur atom.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 5 5 – 6 6 3
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The remaining material cannot be accounted for, presum-
ably due to decomposition by the strong base at elevated
temperature.
Yet another problem encountered was the debenzylation of
nucleoside 6 using ammonium formate and 10% Pd/C in
methanol. We found that the reaction proceeded very slowly
and literally never went to completion. Therefore, we always
found two products in the isolated material: one being the
desired product 7 and the other being one out of three
mono-benzylated compounds (LC–MS). Furthermore it
proved to be difficult to isolate product 7 from the mixture on a
larger scale.
Inspired both by the improvement in the large scale synthesis
of 2Ј-oxy-LNA11 and the synthesis of α--LNA12 we developed
a new strategy for the synthesis of 2Ј-amino-LNA monomers
avoiding the need for selectivity between the two hydroxy
groups of 3-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropyl-
idene-α--erythro-pentofuranose 1 (Scheme 2). Compound 1
was synthesised from 1,2:5,6-di-O-isopropylidene-α--gluco-
furanose according to the procedure of Youssefyeh et al.4
Initially we chose a synthetic route where the two primary
alcohols on the glucofuranose 1 were protected as benzoates.
However, this approach was not successful presumably due to
unfavorable interactions from the benzoyl groups in the critical
triflation and azide displacement steps (see supplementary
material†).
We identified the known trimesylate 1312 as a key compound
and achieved its synthesis by two different approaches outlined
in Schemes 2 and 3. On a small scale (200 mg starting material)
Scheme 3 Reagents and conditions: i) half sat. NH3 in MeOH;
ii) MsCl, pyridine.
Scheme 1 The previously published synthesis of 8. Reagents and
conditions: i) BnBr, NaH, DMF; ii) a) Ac2O, pyridine, b) 80% aq
AcOH, c) Ac2O, pyridine; iii) a) thymine, N,O-bis(trimethylsilyl)-
acetamide, TMS triflate, MeCN; b) NaOMe, MeOH; iv) TsCl, DMAP,
pyridine, CH2Cl2; v) BnNH2, heat; vi) 10% Pd/C, HCOONH4, MeOH;
vii) a) 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane, pyridine, b) MeI,
DBU, THF, CH2Cl2; c) TBAF, THF. 1 was prepared according to
Youssefyeh et al.4; steps i–iii according to Koshkin et al.1; steps iv to vii
according to Singh et al.5
compound 1 was converted to the tetraacetylated anomeric
mixture 10 by acidic hydrolysis and in situ acetylation using
acetic acid, acetic anhydride and sulfuric acid. The crude prod-
uct was subjected to a modified Vorbrüggen reaction13 using in
situ silylation of thymine and trimethylsilyl triflate-mediated
coupling to give the β-configured thymine nucleoside 11 in 91%.
Scheme 2 Reagents and conditions: i) Ac2O, pyridine; ii) AcOH, Ac2O, c. H2SO4 (100 : 10 : 0.1); iii) thymine, N,O-bis(trimethylsilyl)acetamide, TMS
triflate, MeCN; iv) sat. NH3 in MeOH; v) MsCl, pyridine, CH2Cl2; vi) DBU, MeCN; vii) acetone, 0.1 M H2SO4 (aq); viii) Tf2O, DMAP, pyridine,
CH2Cl2; ix) NaN3, DMF; x) NaN3, 15-crown-5, DMF, 80 ЊC; xi) PMe3, NaOH (aq), THF; xii) NaOBz, DMF; xiii) sat. NH3 in MeOH; xiv) 20%
Pd(OH)2/C, H2, abs EtOH.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 5 5 – 6 6 3
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When the same synthetic procedure was used on a larger scale
(5ϩ g starting material) the yield dropped and the formation of
an unidentified by-product was observed, presumably due to
the formation of an acetylated pyranose during the hydrolysis
and in situ acetylation step. The formation of the pyranose can
be avoided on large scale by performing the acetylation of the
two primary hydroxy groups of 1 to give compound 9 before
subjecting the compound to hydrolysis and in situ acetylation
yielding the tetraacetylated anomeric mixture 10. Deacetylation
of nucleoside 11 was performed using saturated methanolic
ammonia to give the nucleoside triol 12 in 84% yield. Sub-
sequent reaction with mesyl chloride in pyridine gave the
known trimesylate 1312 in 93% yield.
The trimesylate 13 is also readily available from the 2Ј-O-
acetyl dimesylated nucleoside 22 (Scheme 3) used in the
improved and recently published synthesis of 2Ј-oxy-LNA
nucleosides.11 This synthetic route has the great advantage of a
high degree of convergence with the 2Ј-oxy-LNA synthesis
making nucleoside 22 the common building block for both
2Ј-oxy-LNA and LNA nucleosides with other heteroatoms at
C2Ј, e.g. nitrogen or sulfur, via the triflate 16 (see Scheme 4).
of the azide but the reaction was too sluggish to be of practical
use (approx. 80% conversion after 7 days).
Reduction of the azide under modified Staudinger condi-
tions16,17 using trimethylphosphine and aqueous NaOH in
THF gave the primary amine which reacted directly with the
mesylate at C1Љ in an intramolecular SN2 reaction forming
nucleoside 18 with the desired 2-oxa-5-azabicyclo[2.2.1]heptane
skeleton.
Nucleophilic replacement of the mesylate on C5Ј of nucleo-
side 18 with benzoate gave nucleoside 19 which subsequently
could be hydrolysed in saturated methanolic ammonia giving
the 5Ј-hydroxy nucleoside 20. Subsequent reductive debenzyl-
ation with hydrogen and 20% Pd(OH)2/C in ethanol gave
nucleoside 21 in an overall yield of 95% from 18 and with all
analytical data identical to those previously reported.8 After
standard protection and phosphitylation nucleoside 21 can be
incorporated in oligonucleotides where the free secondary
amine will undoubtedly contribute with some interesting new
properties.
Interestingly, compound 16 is not only a useful synthon for
the synthesis of the 2Ј-amino-LNA skeleton but also a possible
precursor for a range of LNA analogues by reaction with
appropriate nucleophiles. As an example of the usefulness of
compound 16 the synthesis of the 2-oxa-5-thiabicyclo[2.2.1]-
heptane skeleton found in 2Ј-thio-LNA was accomplished by
a substitution reaction with potassium thioacetate in DMF
producing compound 24 (Scheme 4). Formation of the sulfur-
containing bicyclic nucleoside was achieved by hydrolysis of the
thioacetate with LiOH (aq) in THF to produce 25 in 47% yield
from 16. The conformation of 25 was confirmed by NOE dif-
ference NMR experiments showing an unusually high NOE
enhancement between H6 of the nucleobase and H3Ј (9.0%) as
expected due to the extreme north conformation adopted by the
nucleoside.10,18 The synthon 16 should therefore find many use-
ful applications for the preparation of LNA analogues in the
future.
Returning to the 2Ј-amino-LNA monomer synthesis, com-
pound 18 is ready for the introduction of different groups at
the “handle” that the secondary nitrogen atom provides,
e.g. different alkyl chains.
Because we are interested in studying the effects of 2Ј-amino-
LNA monomers when incorporated into oligonucleotides we
wanted to avoid bulky substituents in our initial studies. In addi-
tion we were looking for a substituent that would be stable
under the standard conditions employed in oligonucleotide syn-
thesis and purification. For these reasons we decided to develop
a new strategy for the synthesis of the known amidite 30 with a
N-methyl group.5
The method used for N-methylation (MeI, DBU) in the pre-
viously published synthesis of 2Ј-amino-LNA5 gave additional
methylation on the nucleobase when attempted on nucleoside
18. Eschweiler–Clarke conditions19,20 (formaldehyde in formic
acid) proved more efficient giving exclusively the desired 2Ј-N-
methyl derivative 26 in 90% yield after purification (Scheme 5).
The expected extreme north conformation of the bicyclic
nucleoside 26 was confirmed by NOE difference NMR experi-
ments showing an unusually high NOE enhancement between
H6 of the nucleobase and H3Ј (9.0%) just as observed for
nucleoside 25. Nucleophilic displacement of the mesylate on
C5Ј of nucleoside 26 with sodium benzoate followed by trans-
esterification with methoxide gave the 5Ј-hydroxy nucleoside 27
in 98% yield. Subsequent reductive debenzylation with hydro-
gen and 20% Pd(OH)2/C in acetic acid yielded nucleoside 28 in
97% yield. All analytical data for nucleoside 28 were identical
to those previously reported.5 4,4Ј-Dimethoxytritylation of the
5Ј-hydroxy group of 28 and phosphitylation of the 3Ј-hydroxy
group21 gave the amidite 305 in 90% yield ready for the auto-
mated incorporation into oligonucleotides. The DMT protected
thymine nucleoside 29 can be further converted into the
5-methylcytosine nucleoside 35 via the triazolation strategy22
Scheme 4 Reagents and conditions: i) KSAc, DMF; ii) LiOH (aq),
THF.
Deacetylation of nucleoside 22 was performed using half-
saturated methanolic ammonia producing the dimesylated
nucleoside 23 in quantitative yield. Subsequent reaction with
mesyl chloride in pyridine gave the trimesylate 13 in 96% on a
20 g scale.
When nucleoside 13 was reacted in neat benzylamine at
130 ЊC in a similar manner as described for the synthesis of
nucleoside 6 (see Scheme 1),5 formation of the 2Ј-benzylamino-
2Ј-deoxynucleoside with the desired 2-oxa-5-azabicyclo[2.2.1]-
heptane skeleton was not observed, but instead a compound in
which the nucleobase had been modified by attack of benzyl-
amine at C2 was obtained. To test if the desired 2,2Ј-anhydro
intermediate was formed during the reaction, nucleoside 13 was
refluxed with 1.1 equivalents of benzylamine in acetonitrile.
This in fact lead to the formation of 2,2Ј-anhydronucleoside 14
in 96% yield. However, it was not possible to achieve nucleo-
philic attack at C2Ј of 14 with either benzylamine or NaN3 in
excess of up to 10 equivalents. Even reaction of nucleoside 14 in
neat benzylamine at room temperature gave no reaction. The
temperature had to be raised to 130 ЊC before any reaction
would take place, and again only the undesired product where
the nucleobase had been affected was formed.
The 2,2Ј-anhydro intermediate 14 could also be synthesised
by treatment of 13 with 1.1 equivalents of DBU in anhydrous
acetonitrile. Opening of the 2,2Ј-anhydro intermediate by
refluxing it in a mixture of aqueous sulfuric acid (0.1 M)
and acetone (1 : 1 v/v) resulted in a clean reaction giving the
threo-configured nucleoside 15 in 98% yield.
Treatment of the threo-nucleoside 15 with trifluoromethane-
sulfonic anhydride, pyridine and DMAP in anhydrous di-
chloromethane at 0 ЊC gave the desired triflate 16 in 80%
yield after chromatography. However, a better overall yield
was obtained if the sensitive triflate was not purified prior to
the substitution reaction with NaN3 producing the desired
2Ј-azido-2Ј-deoxynucleoside 17 in 91% yield over the two steps.
Following the nucleophilic displacement with azide, com-
pound 17 was converted to the amine by reduction with hydro-
gen and Pd/C or Pd(OH)2/C. Unfortunately, these conditions
also induced partial deprotection of the 3Ј-O-benzyl group. The
use of Lindlar’s catalyst14,15 resulted in the exclusive reduction
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Scheme 5 Reagents and conditions: i) CH2O, HCO2H; ii) a) NaOBz, b) MeONa, DMF; iii) 20% Pd(OH)2/C, H2, AcOH; iv) DMT–Cl, pyridine;
v) NC(CH2)2OP(N(iPr)2)2, 4,5-dicyanoimidazole, MeCN, CH2Cl2; vi) Ac2O, pyridine; vii) Et3N, 1,2,4-triazole, POCl3, MeCN; viii) 1 : 1 MeCN,
sat. aq NH3; ix) BzCl, pyridine; x) LiOH (aq), THF.
where thymine is initially activated by conversion into the
triazole 32 and subsequently substituted with ammonia to give
the 5-methylcytosine nucleoside 33. Followed by protection in
the usual manner and phosphitylation of the 3Ј-OH nucleoside
33 was converted to the 5-methyl cytosine phosphoramidite 36
ready for oligonucleotide synthesis.
Chromatography (DCVC) was performed according to the
published procedure.23 1H, 13C, 19F, and 31P NMR spectra
were recorded at respectively 400 MHz, 100 MHz, 376 MHz,
and 121 MHz with solvents as internal standard (δH: CDCl3
7.26 ppm, DMSO-d6 2.50; δC: CDCl3 77.0 ppm, DMSO-d6 39.4
ppm). 31P NMR was run with 85% H3PO4 as external standard.
J values are given in Hz. Assignments of NMR spectra are
based on 2D spectra and follow the standard carbohydrate/
nucleoside nomenclature (the carbon atom of the 4Ј-C-sub-
stituent is numbered C1Љ) even though the systematic com-
pound names of the bicyclic nucleoside derivatives are given
according to the von Baeyer nomenclature. Crude compounds
were used without further purification if they were ≥95% pure
by TLC and HPLC-MS (RP C18 column, UV detection).
Elemental analyses were obtained from the University of
Copenhagen, Microanalytical Department. X-Ray Crystallo-
graphic Data of nucleoside S11 were obtained from Cambridge
University Chemical Laboratory X-Ray Department. CCDC
reference number 198987. See http://www.rsc.org/suppdata/ob/
b2/b208864a/ for crystallographic files in .cif or other electronic
format.
Conclusion
We have developed a synthetic method using cheap and com-
mercially available reagents in scalable reactions with a mini-
mum use of chromatography. This gives access to sufficient
amounts of both 2Ј-amino-LNA thymine and 2Ј-amino-LNA
5-methyl cytosine phosphoramidites as well as other LNA
phosphoramidites needed for the automated synthesis of, e.g.,
2Ј-amino-LNA oligomers and in depth evaluation of their in
vitro and in vivo properties.
Furthermore, by the presence of common intermediates at
late stages in the synthesis of both 2Ј-oxy-LNA, 2Ј-amino-
LNA, and 2Ј-thio-LNA monomers, the convergence between
these has been increased significantly. In addition this new
strategy provides a stable triflate as an intermediate that is a
potential synthon for a wide variety of LNA analogues when
reacted with appropriate nucleophiles.
1-(2,5-Di-O-acetyl-4-C-acetyloxymethyl-3-O-benzyl-ꢀ-D-
erythro-pentofuranosyl)thymine (11)
In vitro and in vivo evaluations of 2Ј-amino-LNA containing
oligonucleotides are currently in progress and will be published
in due course.
To a stirred solution of 3-O-benzyl-4-C-hydroxymethyl-1,2-O-
isopropylidene-α--erythro-pentofuranose 14 (200 mg, 0.64
mmol) in acetic acid (3.69 mL, 64.4 mmol) at 0 ЊC was
added acetic anhydride (0.61 mL, 6.44 mmol) and conc H2SO4
(0.34 µL, 6.44 µmol). After 25 min the reaction mixture was
allowed to warm to rt. Stirring was continued for 2 h after
which the mixture was poured into ice cooled sat. aq NaHCO3
(150 mL). The solution was extracted with dichloromethane
(2 × 150 mL), and the combined organic phases were washed
with sat. aq NaHCO3 (2 × 100 mL), dried (Na2SO4), filtered
and evaporated to dryness in vacuo to give the crude anomeric
mixture 10 as a colorless liquid (258 mg, 0.59 mmol). The liquid
(246 mg, 0.56 mmol) was dissolved in anhydrous acetonitrile
(5 mL) with stirring. Thymine (144 mg, 1.14 mmol) and
Experimental
For reactions conducted under anhydrous conditions glassware
was dried overnight in an oven at 150 ЊC and was allowed to
cool in a desiccator over anhydrous KOH. Anhydrous reactions
were carried out under an atmosphere of argon. Solvents were
HPLC grade, of which DMF, pyridine, acetonitrile and di-
chloromethane were dried over molecular sieves (4 Å from
Grace Davison) and THF was freshly distilled from Na–benzo-
phenone to a water content below 20 ppm. TLC was run on
Merck silica 60 F254 aluminium sheets. Dry Column Vacuum
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N,O-bis(trimethylsilyl)acetamide (0.99 mL, 4.00 mmol) were
added, and the mixture was refluxed for 1.5 h and then cooled
to 0 ЊC. Trimethylsilyl triflate (0.23 mL, 1.25 mmol) was added
dropwise during 5 min and the mixture was heated to 80 ЊC for
3.5 h. The reaction mixture was allowed to cool to rt, and ice
cooled sat. aq NaHCO3 (10 mL) was added. Extraction was
performed with dichloromethane (2 × 20 mL), and the com-
bined organic phases were washed successively with sat. aq
NaHCO3 (2 × 20 mL) and brine (20 mL), dried (Na2SO4), fil-
tered and evaporated to dryness in vacuo. The residue was puri-
fied by flash chromatography (0–1% MeOH in dichloromethane
v/v) to give nucleoside 11 (259 mg, 91%) as a white solid
added. The product slowly precipitated from the reaction
mixture. After 2 h the reaction was completed and concen-
trated in vacuo to facilitate precipitation. The reaction mix-
ture was cooled to Ϫ20 ЊC and the product collected by fil-
tration to afford nucleoside 14 (7.64 g, 91%) as a white solid
1
material. FAB-MS m/z found 517.0 ([MH]ϩ, calcd 517.1); H
NMR (DMSO-d6) δ 7.79 (d, J = 1.3, 1H, H6), 7.45–7.32 (m,
5H, Ph), 6.40 (d, J = 6.0, 1H, H1Ј), 5.60 (dd, J = 6.1, 2.8, 1H,
H2Ј), 4.82 (d, J = 11.5, 1H, CH2Ph), 4.70 (d, J = 11.5, 1H,
CH2Ph), 4.51 (d, J = 2.8, 1H, H3Ј), 4.43 (d, J = 10.6, 1H),
4.36 (d, J = 6.2, 1H), 4.33 (d, J = 5.9, 1H), 4.25 (d, J = 11.0,
1H) (H5Ј, H1Љ), 3.22 (s, 3H, Ms), 3.16 (s, 3H, Ms), 1.80
(s, J = 1.1, 3H, CH3); 13C NMR (DMSO-d6) δ 171.5 (C4), 159.1
(C2), 136.9, 132.1, 128.5, 128.1, 127.9 (C6, Ph), 117.1 (C5),
89.1 (C1Ј), 86.1 (C2Ј), 85.4 (C4Ј), 83.7 (C3Ј), 72.4 (CH2Ph),
68.6, 68.0 (C5Ј, C1Љ), 36.9, 36.8 (Ms), 13.6 (CH3); Anal. calcd
for C20H24N2O10S2: C, 46.5; H, 4.7; N, 5.4. Found: C, 46.6; H,
4.8; N, 5.3%.
1
material. FAB-MS m/z found 505.0 ([MH]ϩ, calcd 505.2); H
NMR (CDCl3) δ 9.93 (s, 1H, NH), 7.37–7.28 (m, 5H, Ph), 7.09
(d, J = 0.9, 1H, H6), 5.79 (d, J = 3.5, 1H, H1Ј), 5.53 (dd, J = 6.3,
3.7, 1H, H2Ј), 4.64–4.08 (m, 7H, CH2Ph, H3Ј, H5Јa, H5Јb,
H1Љa, H1Љb), 2.11 (s, 3H, CH3C(O)), 2.10 (s, 3H, CH3C(O)),
2.07 (s, 3H, CH3C(O)), 1.91 (s, 3H, CH3); 13C NMR (CDCl3)
δ 170.4, 169.9, 163.9, 149.9 (CH3C(O), C2, C4), 137.1, 136.8,
128.3, 128.0, 127.8 (C6, Ph), 111.0 (C5), 90.6 (C1Ј), 84.2 (C4Ј),
77.0 (C3Ј), 74.2 (CH2Ph), 73.7 (C2Ј), 63.6, 62.2 (C5Ј, C1Љ), 20.6,
20.5 (CH3C(O)), 12.3 (CH3).
1-(3-O-Benzyl-5-O-methanesulfonyl-4-C-methanesulfonyloxy-
methyl-ꢀ-D-threo-pentofuranosyl)thymine (15)
Nucleoside 14 (3.70 g, 7.16 mmol) was suspended in a mixture
of acetone (160 mL) and aq H2SO4 (0.1 M, 160 mL). The mix-
ture was refluxed overnight with stirring. After cooling to rt a
white solid precipitated. The volume was reduced to approx.
half in vacuo and a white solid was isolated by filtration. The
solid was washed thoroughly with water and dried in vacuo to
give nucleoside 15 (3.77 g, 98%) as a white solid. FAB-MS
1-(3-O-Benzyl-4-C-hydroxymethyl-ꢀ-D-erythro-pentofuranosyl)-
thymine (12)
Nucleoside 11 (149 mg, 0.30 mmol) was dissolved in a sat. solu-
tion of NH3 in MeOH (15 mL). The mixture was stirred over-
night at rt in a sealed flask and evaporated to dryness under
reduced pressure. The residue was dissolved in EtOAc (30 mL)
and washed with water (10 mL). The aqueous phase was
extracted with EtOAc (30 mL) and the combined organic
phases were coevaporated to dryness with acetonitrile (2 ×
10 mL) under reduced pressure. The residue was purified by
DCVC (1–4% MeOH in dichloromethane v/v), affording
nucleoside 12 (93 mg, 84%) as a viscous liquid. Rf = 0.32 (10%
MeOH in EtOAc, v/v); FAB-MS m/z found 379.0 ([MH]ϩ,
calcd 379.1); 1H NMR (DMSO-d6) δ 11.29 (br s, 1H, NH), 7.73
(d, J = 1.3, 1H, H6), 7.40–7.26 (m, 5H, Ph), 5.90 (d, J = 6.2, 1H,
H1Ј), 5.51 (d, J = 7.5, 1H, OH), 5.18 (t, J = 5.0, 1H, OH), 4.86
(t, J = 5.49, 1H, OH), 4.81 (d, J = 11.7, 1H), 4.56 (d, J = 11.7,
1H), 4.36 (q, J = 6.3, 1H, H2Ј), 4.08 (d, J = 5.5, 1H, H3Ј), 3.60–
1
m/z found 535.0 ([MH]ϩ, calcd 535.1); H NMR (DMSO-d6)
δ 11.35 (s, 1H, NH), 7.41–7.32 (m, 6H, H6, Ph), 6.20 (d,
J = 5.0, 1H, H1Ј), 6.10 (d, J = 4.8, 1H, 2Ј-OH), 4.77 (d, J = 11.9,
1H, CH2Ph), 4.67 (d, J = 11.9, 1H, CH2Ph), 4.56 (d, J = 10.6,
1H), 4.50–4.41 (m, 3H), 4.32 (d, J = 10.6, 1H), 4.16 (d,
J = 3.7, 1H, H3Ј), 3.25 (s, 3H, Ms), 3.20 (s, 3H, Ms), 1.79 (s, 3H,
CH3); 13C NMR (DMSO-d6) δ 163.9 (C4), 150.6 (C2), 137.8,
137.6, 128.4, 127.9, 127.7 (C6, Ph), 108.2 (C5), 84.8 (C1Ј),
84.3 (C3Ј), 81.7 (C4Ј), 73.3 (C2Ј), 72.3 (CH2Ph), 68.1, 67.6
(C5Ј, C1Љ), 37.0, 36.8 (Ms), 12.2 (CH3); Anal. calcd for
C20H26N2O11S2: C, 44.9; H, 4.9; N, 5.2. Found: C, 44.5; H,
4.8; N, 5.1%.
3.50 (m, 4H) (H5Ј, H1Љ, CH2Ph), 1.79 (d, J = 1.1, 3H, CH3); 13
C
1-(3-O-Benzyl-5-O-methanesulfonyl-4-C-methanesulfonyloxy-
methyl-2-O-trifluoromethanesulfonyl-ꢀ-D-threo-pentofuranosyl)-
thymine (16)
NMR (DMSO-d6) δ 163.6 (C4), 150.7 (C2), 138.6, 136.3, 128.0,
127.2 (C6, Ph), 109.3 (C5), 87.7, 87.5 (C1Ј, C4Ј), 78.5 (C3Ј),
73.3 (C2Ј), 72.7, 62.8, 61.3 (C5Ј, C1Љ, CH2Ph), 12.2 (CH3);
Anal. calcd for C18H22N2O7ؒ0.25 H2O: C, 56.5; H, 5.9; N, 7.3.
Found: C, 56.5; H, 5.9; N, 7.0%.
Nucleoside 15 (300 mg, 0.56 mmol) was dissolved in anhydrous
pyridine (2 × 5 mL) and concentrated in vacuo to remove
water traces. The compound was dissolved in a mixture of
anhydrous dichloromethane (20 mL) and anhydrous pyridine
(0.45 mL, 5.60 mmol) followed by the addition of DMAP
(274 mg, 2.24 mmol). After cooling to 0 ЊC trifluoro-
methanesulfonic anhydride (0.19 mL, 1.12 mmol) was
added dropwise during 30 min. The reaction mixture was
stirred for an additional 1.5 h and poured into ice cooled sat. aq
NaHCO3 (20 mL). The organic phase was separated and
washed successively with aq HCl (1 M, 2 × 20 mL) and sat. aq
NaHCO3 (2 × 20 mL), dried (Na2SO4), filtered and evaporated
in vacuo. The residue was purified by DCVC (0–100% EtOAc in
n-heptane v/v) yielding nucleoside 16 (302 mg, 80%) as a white
foam. FAB-MS m/z found 667.0 ([MH]ϩ, calcd 667.0); 1H
NMR (DMSO-d6) δ 11.62 (br s, 1H, NH), 7.51 (s, 1H, H6),
7.40–7.33 (m, 5H, Ph), 6.45 (br s, 1H, H1Ј), 5.91 (t, J = 6.0, 1H,
H2Ј), 4.97 (d, J = 5.7, 1H, H3Ј), 4.82–4.36 (m, 6H, CH2Ph,
H5Јa, H5Јb, H1Љa, H1Љb), 3.30 (s, 3H, Ms), 3.24 (s, 3H, Ms),
1.81 (s, 3H, CH3); 13C NMR (DMSO-d6) δ 163.3 (C4), 150.0
(C2), 136.5, 128.3, 128.0, 127.8 (C6, Ph), 117.6 (q, J = 320,
CF3), 110.1 (C5), 88.0 (C1Ј), 81.7, 81.0 (C3Ј, C4Ј), 73.1
(CH2Ph), 68.0, 67.6 (C5Ј, C1Љ), 36.7, 36.6 (Ms), 11.8 (CH3);
Anal. calcd for C21H25F3N2O13S3: C, 37.8; H, 3.8; N, 4.2. Found:
C, 38.1; H, 3.8; N, 4.1%.
1-(3-O-Benzyl-2,5-di-O-methanesulfonyl-4-C-(methanesulfonyl-
oxymethyl)-ꢀ-D-erythro-pentofuranosyl)thymine (13)
Nucleoside 12 (0.83 g, 3.2 mmol) was dissolved in anhydrous
pyridine (20 mL) and cooled to 0 ЊC with stirring. Methane-
sulfonyl chloride (0.85 mL, 11 mmol) was added dropwise and
the temperature was allowed to reach 15 ЊC over 3 h. The reac-
tion was quenched with sat. aq NaHCO3 (50 mL) and trans-
ferred to a separatory funnel with brine (50 mL) and EtOAc
(100 mL). The phases were separated and the aqueous phase
extracted with EtOAc (2 x 50 mL). The combined organic
phases were extracted with brine (100 mL), dried (Na2SO4),
filtered and evaporated in vacuo to give a viscous yellow liquid.
The liquid was dissolved in a mixture of dichloromethane and
toluene and evaporated in vacuo to give nucleoside 13 (1.48 g,
93%) as a white foam. Analytical data were identical to those
previously published.12
2,2Ј-Anhydro-1-(3-O-benzyl-5-O-methanesulfonyl-4-C-methane-
sulfonyloxymethyl-ꢀ-D-threo-pentofuranosyl)thymine (14)
Nucleoside 13 (10 g, 16.3 mmol) was dissolved in anhydrous
acetonitrile (100 mL) and DBU (2.69 mL, 18.0 mmol) was
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 5 5 – 6 6 3
659

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1-(2-Azido-3-O-benzyl-2-deoxy-5-O-methanesulfonyl-4-C-
(methanesulfonyloxymethyl)-ꢀ-D-erythro-pentofuranosyl)-
thymine (17)
Ph), 108.3 (C5), 88.4 (C1Ј), 85.6 (C4Ј), 76.3 (C3Ј), 70.9, 66.6
(CH2Ph, C5Ј), 59.4 (C2Ј), 50.1 (C1Љ), 36.9 (Ms), 12.3 (CH3);
Anal. calcd for C19H23N3O7S: C, 52.1; H, 5.3; N, 9.6. Found: C,
52.0; H, 5.2; N, 9.2%.
Method A. To a solution of nucleoside 16 (215 mg, 0.32
mmol) in anhydrous DMF (10 mL), NaN3 (23 mg, 0.35 mmol)
and 15-crown-5 (64 µL, 0.32 mmol) were added. The mixture
was stirred at 80 ЊC for 1 h and then cooled to rt whereupon
water (20 mL) was added. The solution was extracted with
EtOAc (50 mL) and the organic phase was washed with sat. aq
NaHCO3 (2 × 20 mL), dried (Na2SO4), filtered and evaporated
to dryness in vacuo. The residue was purified by DCVC
(50–100% EtOAc in n-heptane v/v) yielding nucleoside 17 (164
mg, 91% from 16) as a white foam. Analytical data were identi-
cal to those reported below.
(1R,3R,4R,7S )-1-Benzoyloxymethyl-7-benzyloxy-3-(thymin-1-
yl)-2-oxa-5-azabicyclo[2.2.1]heptane (19)
Compound 18 (950 mg, 2.17 mmol) was dissolved in anhydrous
DMF (20 mL) and sodium benzoate (626 mg, 4.34 mmol) was
added. The reaction mixture was heated to 80 ЊC for 5.5 h and
then cooled to rt. The reaction was diluted with dichloro-
methane (50 mL) and washed with brine (2 × 50 mL). The
combined aqueous phases were extracted with dichloro-
methane (50 mL). The combined organic phases were dried
(Na2SO4) and concentrated under reduced pressure. Purifi-
cation by DCVC (id 2.2 cm, 0–10% MeOH in dichloromethane
v/v, 0.5% increments, 10 mL fractions) afforded nucleoside 19
(980 mg, 97%) as a white solid. Rf = 0.34 (10% MeOH in
dichloromethane, v/v); FAB-MS m/z found 464.3 ([MH]ϩ,
Method B. A solution of nucleoside 15 (5.35 g, 10 mmol) in
anhydrous dichloromethane (300 mL) was cooled to 0 ЊC.
Anhydrous pyridine (8.08 mL, 100 mmol) and DMAP (4.89 g,
40 mmol) were added followed by the dropwise addition of
trifluoromethanesulfonic anhydride (3.3 mL, 20 mmol).
After 2 h at 0 ЊC the reaction was quenched by the addition of
ice cold sat. aq NaHCO3 (200 mL) and the reaction mixture
was transferred to a separatory funnel. The phases were
separated and the aq phase was extracted with dichloromethane
(200 mL). The combined organic phases were washed with aq
HCl (1.0 M, 2 × 300 mL) and sat. aq NaHCO3 (300 mL),
dried (Na2SO4), filtered and concentrated in vacuo to give a
white solid. The solid was dissolved in anhydrous DMF
(300 mL) and NaN3 (1.86 g, 30 mmol) was added. After stirring
at rt for 4 h brine (300 mL) was added and the mixture was
transferred to a separatory funnel. The aqueous phase was
extracted with dichloromethane (3 × 200 mL) and the com-
bined organic phases were dried (Na2SO4), filtered and concen-
trated in vacuo yielding a yellow residue that was purified by
DCVC (id 5 cm, 25–100% EtOAc in n-heptane v/v, 5%
increments, 100 mL fractions) affording nucleoside 17 (5.1 g,
91% from 15) as a white solid. ESI-MS m/z found 560.0
([MH]ϩ, calcd 560.1); 1H NMR (CDCl3) δ 9.30 (br s, 1H, NH),
7.40–7.34 (m, 5H, Ph), 7.15 (d, J = 1.3, 1H, H6), 5.69 (d, J = 4.8,
1H, H1Ј), 4.80 (d, J = 11.2, 1H), 4.66–4.63 (m, 2H), 4.56 (d,
J = 11.7, 1H), 4.47 (dd, J = 6.6, 4.8, 1H, H2Ј), 4.40 (d, J = 10.8,
1H), 4.32 (d, J = 7.7, 1H, H3Ј), 4.29 (d, J = 8.6, 1H) (CH2Ph,
H5Ј, H1Љ), 3.03 (s, 3H, Ms), 2.99 (s, 3H, Ms), 1.92 (d, J = 1.1,
3H, CH3); 13C NMR (CDCl3) δ 163.4 (C4), 150.0 (C2), 136.9,
136.0, 128.7, 128.5, 128.2 (C6, Ph), 112.5 (C5), 90.8 (C1Ј), 83.7
(C4Ј), 78.8 (C3Ј), 74.9 (CH2Ph), 68.1, 67.5 (C5Ј, C1Љ), 60.3
(C2Ј), 37.5 (Ms), 12.1 (CH3); Anal. calcd for C20H25N5O10S2ؒ
0.33 EtOAc: C, 43.5; H, 4.7; N, 11.9. Found: C, 43.7; H, 4.5; N,
12.2%.
1
calcd 464.5); H-NMR (DMSO-d6) δ 11.39 (br s, 1H, NH),
7.91 (dd, J = 8.2, 1.3, 2H, Bz), 7.66 (m, 1H, Bz), 7.51
(m, 2H, Bz), 7.33–7.22 (m, 6H, Ph, H6), 5.46 (s, 1H, H1Ј), 4.78
(d, J = 12.6, 1H, H5Јa), 4.65 (d, J = 11.9, 1H, CH2Ph), 4.59
(d, J = 12.6, 1H, H5Јb), 4.52 (d, J = 11.7, 1H, CH2Ph), 3.92 (s,
1H, H3Ј), 3.74 (s, 1H, H2Ј), 3.16 (d, J = 10.1, 1H, H1Љa), 2.87 (d,
J = 10.1, 1H, H1Љb), 1.44 (d, J = 0.9, 3H, CH3); 13C NMR
(DMSO-d6) δ 165.1 (PhC(O)), 163.7 (C4), 149.9 (C2), 137.7,
134.0, 133.5, 129.1, 128.8, 128.0, 127.6, 127.5 (C6, Ph), 108.1
(C5), 88.4 (C1Ј), 86.1 (C4Ј), 76.1 (C3Ј), 70.7 (CH2Ph), 60.6
(C5Ј), 59.4 (C2Ј), 50.3 (C1Љ), 11.9 (CH3); Anal. calcd for
C25H25N3O6ؒ0.33H2O: C, 63.9; H, 5.5; N, 9.0. Found: C, 63.8;
H, 5.3; N, 9.0%.
(1R,3R,4R,7S )-7-Benzyloxy-1-hydroxymethyl-3-(thymin-1-yl)-
2-oxa-5-azabicyclo[2.2.1]heptane (20)
Nucleoside 19 (1.12 g, 2.41 mmol) was dissolved in a saturated
solution of NH3 in MeOH (200 mL). The mixture was stirred
for 30 h at rt in a sealed flask and evaporated to dryness under
reduced pressure. Purification by DCVC (id 3.5 cm, 0–10%
MeOH in dichloromethane v/v, 0.5% increments, 30 mL
fractions) afforded nucleoside 20 (862 mg, 99%) as a white
solid. Rf = 0.20 (10% MeOH in dichloromethane, v/v); FAB-MS
1
m/z found 360.1 ([MH]ϩ, calcd 360.4); H-NMR (DMSO-d6)
δ 11.30 (br s, 1H, NH), 7.70 (d, J = 1.1, 1H, H6), 7.33–7.24
(m, 5H, Ph), 5.39 (s, 1H, H1Ј), 4.56 (d, J = 11.7, 1H, CH2Ph),
4.50 (d, J = 11.7, 1H, CH2Ph), 3.83 (s, 1H, H3Ј), 3.76
(d, J = 12.8, 1H, H5Јa), 3.71 (d, J = 12.8, 1H, H5Јb), 3.57
(s, 1H, H2Ј), 2.96 (d, J = 9.7, 1H, H1Љa), 2.64 (d, J = 9.9,
1H, H1Љb), 1.76 (s, 3H, CH3); 13C NMR (DMSO-d6) δ 163.9
(C4), 150.0 (C2), 138.1, 135.3, 128.1, 127.5, 127.4 (C6, Ph),
107.7 (C5), 88.9 (C4Ј), 88.0 (C1Ј), 75.9 (C3Ј), 70.8 (CH2Ph),
59.3, (C2Ј), 57.1 (C5Ј), 50.0 (C1Љ), 12.5 (CH3); Anal. calcd for
C18H21N3O5ؒ0.5H2O: C, 58.7; H, 6.0; N, 11.4. Found: C, 58.1;
H, 6.0; N, 11.9%.
(1R,3R,4R,7S )-7-Benzyloxy-1-methanesulfonyloxymethyl-3-
(thymin-1-yl)-2-oxa-5-azabicyclo[2.2.1]heptane (18)
To a solution of 17 (5.83 g, 10.4 mmol) in THF (300 mL) at rt,
aq NaOH (2.0 M, 104 mL, 208 mmol) and PMe3 in THF (1.0
M, 20.8 mL, 20.8 mmol) were added with stirring. After 8 h the
THF was partly removed under reduced pressure. Brine (200
mL) and EtOAc (300 mL) was added and the phases were sep-
arated. The aqueous phase was extracted with EtOAc (2 × 300
mL) and dichloromethane (2 × 300 mL). The combined organic
phases were dried (Na2SO4), filtered and concentrated in vacuo
to give nucleoside 18 (4.22 g, 93%) as a white solid. Rf = 0.15
(10% MeOH in EtOAc, v/v); ESI-MS m/z found 438.0 ([MH]ϩ,
calcd 438.1); 1H NMR (DMSO-d6) δ 11.33 (br s, 1H, NH), 7.46
(s, 1H, H6), 7.36–7.27 (m, 5H, Ph), 5.44 (s, 1H, H1Ј), 4.67
(d, J = 11.7, 1H), 4.59 (d, J = 11.5, 1H), 4.56 (d, J = 11.9, 1H),
4.52 (d, J = 11.7, 1H) (H5Ј, CH2Ph), 3.84 (s, 1H, H3Ј), 3.65
(s, 1H, H2Ј), 3.26 (s, 3H, Ms), 3.06 (d, J = 10.1, 1H, H1Љa), 2.78
(d, J = 9.9, 1H, H1Љb), 1.77 (s, 3H, CH3); 13C NMR (DMSO-d6)
δ 163.9 (C4), 150.1 (C2), 137.9, 134.7, 128.2, 127.7, 127.6 (C6,
(1R,3R,4R,7S )-7-Hydroxy-1-hydroxymethyl-3-(thymin-1-yl)-2-
oxa-5-azabicyclo[2.2.1]heptane (21)
Nucleoside 20 (675 mg, 1.87 mmol) was dissolved in absolute
ethanol (30 mL). Pd(OH)2 and charcoal (20% moist, 200 mg)
was added and the reaction flask was evacuated and filled with
hydrogen gas several times. The reaction was stirred vigorously
under an atmosphere of hydrogen gas overnight. The catalyst
was removed by filtration through a plug of Celite. The Celite
was washed thoroughly with hot methanol (250 mL). The sol-
vents were removed in vacuo yielding nucleoside 21 (500 mg,
99%) as a white solid. Rf = 0.08 (20% MeOH in dichloro-
methane, v/v). All analytical data were identical to those
previously reported.8
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 5 5 – 6 6 3
660

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1-(3-O-Benzyl-5-O-methanesulfonyl-4-C-methanesulfonyloxy-
methyl-ꢀ-D-erythro-pentofuranosyl)thymine (23)
4.61 (d, J = 11.7, 1H), 4.60 (d, J = 11.7, 1H), 4.56 (d, J = 11.5,
1H) (H5Ј, CH2Ph), 4.20 (d, J = 1.8, 1H, H3Ј), 4.00 (d, J = 2.0,
1H, H2Ј), 3.29 (s, 3H, Ms), 3.02 (d, J = 10.6, 1H, H1Љa), 2.90 (d,
J = 10.4, 1H, H1Љb), 1.78 (s, 3H, CH3); 13C NMR (DMSO-d6)
δ 163.9 (C4), 150.1 (C2), 137.5, 134.1, 128.3, 127.7 (C6, Ph),
108.3 (C5), 90.5 (C1Ј), 86.6 (C4Ј), 76.9 (C3Ј), 70.9, 66.8 (C5Ј,
CH2Ph), 49.5 (C2Ј), 36.8 (Ms), 35.1 (C1Љ), 12.3 (CH3); Anal.
calcd for C19H22N2O7S2ؒ0.33 EtOAc: C, 50.5; H, 5.1; N, 5.8.
Found: C, 50.8; H, 5.1; N, 5.8%.
Nucleoside 2211 (30 g, 52 mmol) was dissolved in MeOH
(600 mL), and the solution was cooled to 0 ЊC. Freshly prepared
sat. methanolic ammonia (600 mL) was added, and the temper-
ature was allowed to reach rt. After 5 h at rt the reaction was
quenched with glacial acetic acid (50 mL) and transferred to a
beaker, where it was neutralised with sat. aq NaHCO3. EtOAc
(900 mL) and brine (500 mL) was added and the phases were
separated. The aqueous phase was extracted with EtOAc
(3 × 500 mL) and the combined organic phases were
washed with sat. aq NaHCO3 (500 mL) and brine (500 mL).
The organic phase was dried (Na2SO4), filtered and the sol-
vent removed in vacuo to afford 23 (27 g, 97%) as a white foam.
Rf = 0.33 (100% EtOAc); ESI-MS m/z found 557.0 ([MNa]ϩ,
(1R,3R,4R,7S )-7-Benzyloxy-1-methanesulfonyloxymethyl-5-
methyl-3-(thymin-1-yl)-2-oxa-5-azabicyclo[2.2.1]heptane (26)
To a solution of 18 (4.22 g, 9.64 mmol) in formic acid (20 mL)
formaldehyde (37% aq solution, 20 mL) was added with stirring
and the reaction mixture was heated to 80 ЊC. After 1 h the
reaction was diluted with EtOAc (150 mL) and quenched by
carefully pouring it into sat. aq NaHCO3 (100 mL). The phases
were separated and the organic phase was washed with sat. aq
NaHCO3 (4 × 100 mL). The combined aqueous phases were
extracted with dichloromethane (2 × 200 mL). The combined
organic phases were dried (Na2SO4), filtered and concen-
trated under reduced pressure. Purification by DCVC (id 6 cm,
0–15% MeOH in EtOAc v/v, 1% increments, 100 mL fractions)
afforded nucleoside 26 (3.89 g, 90%) as an off-white solid.
Rf = 0.30 (10% MeOH in EtOAc, v/v); ESI-MS m/z found 452.1
1
calcd 557.1); H NMR (CDCl3) δ 10.21 (br s, 1H, NH), 7.33–
7.25 (m, 6H, Ph, H6), 5.77 (d, J = 3.9, 1H, H1Ј), 4.84 (d,
J = 11.4, 1H, H3Ј), 4.59–4.57 (m, 3H), 4.42–4.37 (m, 3H), 4.26–
4.19 (m, 2H) (H2Ј, H2Љ, H5Љ, CH2Ph, OH), 2.98 (s, 3H, CH3),
2.76 (s, 3H, CH3), 1.80 (s, 3H, CH3); 13C NMR (CDCl3) δ 162.5
(C4), 151.0 (C2), 136.7 (Ph), 136.2 (C6), 128.5, 128.3, 128.2
(Ph), 111.3 (C5), 92.1 (C1Ј), 84.0 (C4Ј), 77.7 (C3Ј), 74.1, 73.5
(C2Ј, CH2Ph), 68.6, 68.3 (C5Ј, C1Љ), 37.2, 37.1 (Ms), 12.0
(CH3); Anal. calcd for C20H26N2O11S2: C, 44.9; H, 4.9; N, 5.2.
Found: C, 45.0; H, 4.7; N, 5.1%.
1
([MH]ϩ, calcd 452.1); H NMR (DMSO-d6) δ 11.34 (br s, 1H,
NH), 7.43 (s, 1H, H6), 7.34–7.28 (m, 5H, Ph), 5.58 (s, 1H, H1Ј),
4.67 (m, 4H, H5Ј, CH2Ph), 3.88 (s, 1H, H3Ј), 3.58 (s, 1H, H2Ј),
3.27 (s, 3H, Ms), 2.98 (d, J = 9.7, 1H, H1Љa), 2.76 (d, J = 9.7, 1H,
H1Љb), 2.57 (s, 3H, NCH3), 1.76 (s, 3H, CH3); 13C NMR
(DMSO-d6) δ 163.9 (C4), 149.9 (C2), 137.6 (Ph), 134.6 (C6),
128.3, 127.7 (Ph), 108.4 (C5), 86.1 (C1Ј), 85.3 (C4Ј), 77.3 (C3Ј),
71.0, 66.3 (CH2Ph, C5Ј), 64.9 (C2Ј), 58.7 (C1Љ), 40.8 (NCH3),
36.9 (Ms), 12.3 (CH3); Anal. calcd for C20H25N3O7Sؒ0.25 H2O:
C, 52.7; H, 5.6; N, 9.1. Found: C, 52.9; H, 5.6; N, 8.9%.
1-(3-O-Benzyl-2,5-di-O-methanesulfonyl-4-C-(methane-
sulfonyloxymethyl)-ꢀ-D-erythro-pentofuranosyl)thymine (13)
Nucleoside 23 (20 g, 37 mmol) was dissolved in anhydrous
dichloromethane (100 mL) and anhydrous pyridine (100 mL)
was added. The solution was cooled to 0 ЊC and methane-
sulfonyl chloride (4.4 mL, 56 mmol) was added dropwise. After
2 h the reaction was quenched with sat. aq NaHCO3 (200 mL),
and the phases were separated. The aq phase was extracted with
dichloromethane (2 × 150 mL), and the combined organic
phases were washed with aq HCl (1 M, 2 × 200 mL), sat. aq
NaHCO3 (2 × 250 mL) and brine (250 mL). The organic phase
was dried (Na2SO4), filtered and the solvent was removed
in vacuo. The crude product was co-evaporated with toluene
affording 13 (22 g, 96%) as a white foam. Rf = 0.41 (100%
EtOAc); ESI-MS m/z found 635.0 ([MNa]ϩ, calcd 635.1). All
analytical data were identical to those previously reported.12
(1R,3R,4R,7S )-7-Benzyloxy-1-hydroxymethyl-5-methyl-3-
(thymin-1-yl)-2-oxa-5-azabicyclo[2.2.1]heptane (27)
Compound 26 (3.00 g, 6.64 mmol) was dissolved in anhydrous
DMF (30 mL) and sodium benzoate (1.93 g, 13.3 mmol) was
added. The reaction mixture was heated to 100 ЊC for 7 h and
then cooled to rt. Sodium methoxide (1.44 g, 26.6 mmol) was
added and after 1 h the reaction was diluted with dichloro-
methane (100 mL) and washed with brine (2 × 100 mL). The
combined aq phases were extracted with dichloromethane (3 ×
100 mL) and diethyl ether (3 × 100 mL). The combined organic
phases were dried (Na2SO4) and concentrated under reduced
pressure. Purification by DCVC (id 4 cm, 0–10% MeOH
in dichloromethane v/v, 0.5% increments, 50 mL fractions)
afforded nucleoside 27 (2.42 g, 98%) as a white solid. Rf = 0.19
(7% MeOH in dichloromethane, v/v); ESI-MS m/z found 374.1
([MH]ϩ, calcd 374.2), 408.1, 410.1 ([MCl]Ϫ, calcd 408.1, 410.1);
1H-NMR (DMSO-d6) δ 11.31 (br s, 1H, NH), 7.67 (d, J = 1.1,
1H, H6), 7.33–7.24 (m, 5H, Ph), 5.52 (s, 1H, H1Ј), 5.24 (br s,
1H, 5Ј-OH), 4.56 (q, J = 11.3, 2H, CH2Ph), 3.87 (s, 1H, H3Ј),
3.72 (br s, 2H, H5Ј), 3.47 (s, 1H, H2Ј), 2.85 (d, J = 9.5, 1H,
H1Љa), 2.61 (d, J = 9.5, 1H, H1Љb), 2.55 (s, 3H, NCH3), 1.75 (d,
J = 1.1, 3H, CH3); 13C NMR (DMSO-d6) δ 163.8 (C4), 149.8
(C2), 137.8 (Ph), 135.1 (C6), 128.0, 127.3 (Ph), 107.8 (C5), 89.4
(C1Ј), 84.9 (C4Ј), 76.8 (C3Ј), 70.8 (CH2Ph), 64.7 (C5Ј), 58.8,
56.9 (C2Ј, C1Љ), 40.9 (NCH3), 12.4 (CH3); Anal. calcd for
C19H23N3O5 ؒ0.25H2O: C, 60.4; H, 6.3; N, 11.1. Found: C, 60.3;
H, 6.5; N, 10.8%.
(1R,3R,4R,7S )-7-Benzyloxy-1-methanesulfonyloxymethyl-3-
(thymin-1-yl)-2-oxa-5-thiabicyclo[2.2.1]heptane (25)
Nucleoside 16 (0.10 g, 0.17 mmol) was dissolved in anhydrous
DMF (1 mL) and potassium thioacetate (25 mg, 0.22 mmol)
was added. The reaction was stirred at ambient temperature for
5 h and transferred to a separatory funnel with brine (10 mL).
The aq phase was extracted with dichloromethane (3 × 10 mL)
and the combined organic phases were dried (Na2SO4), filtered
and evaporated in vacuo to give a yellow liquid. The crude
product 24 was dissolved in THF (2 mL) and LiOHؒH2O (35
mg in 1 mL water, 0.84 mmol) was added. After 20 min the
reaction was complete and quenched by the addition of glacial
acetic acid (0.5 mL). The THF was removed in vacuo and the
residue dissolved in dichloromethane (10 mL) and extracted
with sat. aq NaHCO3 (2 × 10 mL). The aqueous phases were
extracted with dichloromethane (10 mL). The combined
organic phases were dried (Na2SO4), filtered and evaporated
in vacuo to give a yellow liquid that was purified by DCVC
(id 1 cm, 0–80% EtOAc in n-heptane v/v, 2.5% increments,
10 mL fractions). Fractions containing nucleoside 25 were
combined and evaporated in vacuo to afford a white powder (36
mg, 47% from 16). Rf = 0.38 (80% EtOAc in n-heptane, v/v);
ESI-MS m/z found 455.0 ([MH]ϩ, calcd 455.1); 1H NMR
(DMSO-d6) δ 11.38 (br s, 1H, NH), 7.50 (d, J = 1.1, 1H, H6),
7.36–7.27 (m, 5H, Ph), 5.77 (s, 1H, H1Ј), 4.68 (d, J = 11.7, 1H),
(1R,3R,4R,7S )-7-Hydroxy-1-hydroxymethyl-5-methyl-3-
(thymin-1-yl)-2-oxa-5-azabicyclo[2.2.1]heptane (28)
Compound 27 (2.60 g, 6.64 mmol) was dissolved in glacial
acetic acid (50 mL) and the reaction flask was evacuated and
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661

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filled with argon several times. Pd(OH)2 on charcoal (20%
moist, 200 mg) was added and the reaction flask was evacuated
and filled with hydrogen gas several times. The reaction was
stirred vigorously under an atmosphere of hydrogen gas for 8 h.
The catalyst was removed by filtration through a plug of Celite.
The Celite was washed thoroughly with hot methanol (200
mL). The solvents were removed in vacuo. The residue was dis-
solved in water (10 mL) and lyophilised yielding the acetate salt
of nucleoside 28 (2.10 g, 97%) as off-white flakes. Rf = 0.11
(0.5% Et3N, 10% MeOH, 89.5% EtOAc, v/v/v); ESI-MS m/z
found 284.1 ([MH]ϩ, calcd 284.1). All analytical data were
identical to those previously reported.5
15 min the reaction mixture was allowed to reach room tem-
perature. The resulting yellow slurry was stirred under argon at
ambient temperature. After 4.5 h the reaction mixture was
poured into a slurry of sat. aq NaHCO3 (50 mL) and ice and
extracted with EtOAc (3 × 25 mL). The combined organic
phases were washed with brine (100 ml) and dried (Na2SO4).
Filtration and evaporation in vacuo afforded the triazole deriv-
ative 32 as a pink foam which was immidiately dissolved in
anhydrous MeCN (50 ml) and sat. aq NH4OH (50 mL) was
added. After stirring for 16 h solid NaCl was added until the
phases separated. The aqueous phase was extrated with EtOAc
(3 × 50 mL) and the combined organic phases dried (Na2SO4),
filtered and evaporated to give nucleoside 33 as an off-white
solid. The product was dissolved in anhydrous pyridine (50 mL)
and benzoyl chloride (0.87 mL, 7.5 mmol) was added. The reac-
tion was stirred for 3 h under argon and then concentrated in
vacuo. The residue was diluted with EtOAc (100 mL) and
extracted with sat. aq NaHCO3 (100 mL). The phases were
separated and the aqueous phase extracted with EtOAc (2 ×
100 ml). The combined organic phases were washed with brine
(200 ml) and dried (Na2SO4). Filtration and evaporation of the
organic phase produced a clear oil that was dissolved in THF
(100 mL). LiOH (aq, 1.0 M, 25 mL) was added and the
reaction was stirred for 2 h. The reaction mixture was trans-
ferred to a separatory funnel with EtOAc (100 mL) and
brine (100 mL) and extracted with EtOAc (2 × 100 ml). The
combined organic phases were washed with brine (200 ml)
and dried (Na2SO4). Filtration and evaporation in vacuo
gave a yellow foam that was purified by DCVC (id 4 cm,
50–100% EtOAc, n-heptane v/v (the column was pretreated
with 1% Et3N in heptane v/v), 5% increments, 100 mL frac-
tions)) affording nucleoside 35 (1.12 g, 65%) as a white solid.
Rf = 0.56 (EtOAc); ESI-MS m/z found 689.3 ([MH]ϩ, calcd.
(1R,3R,4R,7S )-1-(4,4Ј-Dimethoxytrityloxymethyl)-7-hydroxy-
5-methyl-3-(thymin-1-yl)-2-oxa-5-azabicyclo[2.2.1]heptane (29)
Compound 28 (2.00 g, 5.83 mmol) was dissolved in anhydrous
pyridine (2 × 50 mL) and concentrated in vacuo. The nucleoside
was dissolved in anhydrous pyridine (50 mL) and 4,4Ј-di-
methoxytrityl chloride (2.96 g, 8.74 mmol) was added and the
reaction was stirred at rt for 9 h. The reaction was concentrated
to half volume in vacuo and the residue was diluted with EtOAc
(100 mL). The organic phase was washed with sat. aq NaHCO3
(3 × 100 mL) and brine (100 mL), dried (Na2SO4), filtered and
concentrated under reduced pressure. Purification by DCVC
(id 4 cm, 0–10% MeOH in EtOAc ϩ 0.5% Et3N v/v, 0.5%
increments, 50 mL fractions) afforded nucleoside 29 (3.13 g,
92%) as an off-white solid. Rf = 0.38 (0.5% Et3N, 10% MeOH,
89.5% EtOAc, v/v/v); ESI-MS m/z found 586.2 ([MH]ϩ, calcd
586.2). All analytical data were identical to those previously
reported.5
(1R,3R,4R,7S )-7-(2-Cyanoethoxy(diisopropylamino)-
phosphinoxy)-1-(4,4Ј-dimethoxytrityloxymethyl)-5-methyl-3-
(thymin-1-yl)-2-oxa-5-azabicyclo[2.2.1]heptane (30)
1
689.3); H NMR (DMSO-d6) δ 8.16 (s, 2H, Bz), 7.86 (s, 1H,
H6), 7.61–7.44 (m, 5H, Bz, DMT), 7.36–7.24 (m, 7H, Bz,
DMT), 6.92 (dd, 4H, J = 9.0, 2.4, DMT), 5.64 (s, 1H, H1Ј), 5.41
(d, J = 5.3, 1H, H3Ј), 4.14 (d, J = 5.3, 1H, H2Ј), 5.64 (s, 1H,
H1Ј), 3.75 (s, 6H, OCH3), 3.39 (d, J = 10.8, 1H, H5Ј), 3.28 (d,
J = 10.8 Hz, 1H, H5Ј), 2.89 (d, J = 9.5, 1H, H1Љ), 2.59 (s, 3H,
NCH3), 2.58 (d, J = 9.2, 1H, H1Љ), 1.73 (s, 3H, CH3); 13C NMR
(DMSO-d6) δ 178.2 (PhC(O)), 160.3 (C4), 158.2 (Ph), 147.0
(C2), 144.8 (Ph), 137.4 (C6), 135.4, 135.2, 132.5, 129.9, 129.3,
128.4, 128.0, 127.7, 126.9, 113.3 (Ph), 108.6 (C5), 88.9 (C1Ј),
85.7 (C4Ј), 85.0 (Ph), 70.5 (C3Ј), 67.0 (C5Ј), 59.6, 58.6 (C2Ј,
C1Љ), 55.1 (OCH3), 40.1 (NCH3), 14.1 (CH3); Anal. calcd. for
C40H40N4O7: C, 69.7; H, 5.9; N, 8.1. Found: C, 69.5; H, 5.9; N,
7.7%.
Compound 29 (500 mg, 0.85 mmol) was dissolved in
anhydrous dichloromethane (4 mL) and 4,5-dicyanoimidazole
in MeCN (1.0 M, 0.59 mL, 0.59 mmol) was added at ambient
temperature with stirring. 2-Cyanoethyl N,N,NЈ,NЈ-tetra-
isopropylphosphorodiamidite (0.27 mL, 0.85 mmol) was
added dropwise to the reaction mixture. After 2 h the reaction
was diluted with dichloromethane (10 mL) and transferred
to a separatory funnel and extracted with sat. aq NaHCO3
(2 × 15 mL) and brine (15 mL). The combined aq phases
were extracted with dichloromethane (10 mL). The organic
phases were pooled and dried (Na2SO4). After filtration the
organic phase was evaporated in vacuo to give nucleoside
30 as a slightly yellow foam (660 mg, 98% yield). Rf = 0.56
(0.5% Et3N, 10% MeOH, 89.5% EtOAc, v/v/v); ESI-MS m/z
found 786.3 ([MH]ϩ, calcd 786.4). 31P NMR (CDCl3) δ 149.8,
149.6.5
(1R,3R,4R,7S )-1-(4,4Ј-Dimethoxytrityloxymethyl)-7-(2-cyano-
ethoxy(diisopropylamino) phosphinoxy)-5-methyl-3-(4-benzoyl-
5-methylcytosin-1-yl)-2-oxa-5-azabicyclo[2.2.1]heptane (36)
Compound 35 (0.50 g, 0.73 mmol) was dissolved in anhydrous
dichloromethane (10 mL) and 4,5-dicyanoimidazole in MeCN
(1.0 M, 0.51 mL, 0.51 mmol) was added at ambient temper-
ature with stirring. 2-Cyanoethyl N,N,NЈ,NЈ-tetraisopropyl-
phosphorodiamidite (0.23 mL, 0.74 mmol) was added dropwise
to the reaction mixture. After 2 h the reaction was diluted with
dichloromethane (20 mL) and transferred to a separatory fun-
nel and extracted with sat. aq NaHCO3 (2 × 30 mL) and brine
(30 mL). The combined aqueous phases were extracted with
dichloromethane (30 mL). The organic phases were pooled and
dried (Na2SO4). After filtration the organic phase was evapor-
ated in vacuo to give a yellow foam. Purification by DCVC
(id 4 cm, 0–100% EtOAc, n-heptane ϩ 0.5% Et3N v/v/v (the
column was pretreated with 1% Et3N in heptane v/v), 5%
increments, 50 mL fractions) afforded nucleoside 36 (0.58 g,
92%) as a white solid. Rf = 0.67 (20% heptane, 79.5% EtOAc,
0.5% Et3N, v/v/v); ESI-MS m/z found 889.2 ([MH]ϩ, calcd
889.4); 31P NMR (DMSO-d6) δ 148.4, 147.4
(1R,3R,4R,7S )-1-(4,4Ј-Dimethoxytrityloxymethyl)-7-hydroxy-
5-methyl-3-(4-benzoyl-5-methylcytosin-1-yl)-2-oxa-5-aza-
bicyclo[2.2.1]heptane (35)
Compound 29 (1.5 g, 2.5 mmol) was dissolved in anhydrous
pyridine (25 mL). Acetic anhydride (2.4 mL, 25 mmol) was
added and the reaction stirred for 24 h at ambient temperature.
The reaction was quenched with water (25 mL) and extracted
with EtOAc (2 × 25 ml). The combined organic phases were
washed with sat. aq NaHCO3 (2 × 50 ml), brine (50 ml), and
dried (Na2SO4). The organic phase was filtered and evaporated
in vacuo to give compound 31 as a white foam. Residual
water was removed from the crude product by evaporation
from anhydrous MeCN. The product was then dissolved in
anhydrous MeCN (50 ml) and Et3N (3.5 mL, 25.3 mmol) was
added followed by 1,2,4-triazole (1.75 g, 25 mmol). The reac-
tion mixture was cooled on an ice bath and POCl3 (0.48 mL,
5.0 mmol) was added dropwise to give a white slurry. After
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662

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9 R. Kumar, S. K. Singh, A. A. Koshkin, V. K. Rajwanshi,
M. Meldgaard and J. Wengel, Bioorg. Med. Chem. Lett., 1998, 8,
2219–2222.
10 S. Obika, D. Nanbu, Y. Hari, J. A. K. Morio, Y. In, T. Ishida and
T. Imanishi, Tetrahedron Lett., 1997, 38, 8735–8738.
11 A. A. Koshkin, J. Fensholdt, H. M. Pfundheller and C. Lomholt,
J. Org. Chem., 2001, 66, 8504–8512.
Acknowledgements
The help and assistance of Dr J. E. Davies of the Cambridge
University Chemical Laboratory X-Ray department is grate-
fully acknowledged.
12 A. E. Håkansson, A. A. Koshkin, M. D. Sørensen and J. Wengel,
J. Org. Chem., 2000, 65, 5161–5166.
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