A simplified and efficient route to 2′-O, 4′-C-methylene-linked bicyclic ribonucleosides (locked nucleic acid)

Article abstract of DOI:10.1021/jo010732p

A novel efficient method for the synthesis of locked nucleic acid (LNA) monomers is described. The LNA 5′,3′-diols containing thymine, 4-N-acetyl- and 4-N-benzoylcytosine, 6-N-benzoyladenine, and 2-N-isobutyrylguanine as nucleobases were prepared via convergent syntheses. The method is based on the use of the common sugar intermediate 1,2-di-O-acetyl-3-O-benzyl-4-C-methanesulfonoxymethyl- 5-O-methanesulfonyl-D-erythro-pentofuranose (8) that easily can be prepared from D-glucose in multigram scale. Four different nucleobases were stereoselectively coupled to 8 using a modified Vorbrueggen procedure to give the corresponding 4′-C-branched nucleoside derivatives. Subsequent ring closing furnished the protected LNA nucleosides. The 5′-O-mesyl groups were efficiently displaced by nucleophilic substitution using sodium benzoate. Saponification of the 5′-benzoates followed by catalytic removal of the 3′-O-benzyl groups afforded the free LNA diols. The exocyclic amino groups of adenosine and cytidine were selectively acylated to give 4-N-acetyl- or 4-N-benzoyl-LNA-C and 6-N-benzoyl-LNA-A. The isobutyryl group of guanine was retained during the preparation of 2-N-isobutyryl-LNA-G. The LNA-T diol and base-protected LNA diols can be directly converted into LNA-phosphoramidites for automated chemical synthesis of LNA containing oligonucleotides.

Full text of DOI:10.1021/jo010732p

8504
J . Org. Chem. 2001, 66, 8504-8512
A Sim p lified a n d Efficien t Rou te to 2′-O, 4′-C-Meth ylen e-Lin k ed
Bicyclic Ribon u cleosid es (Lock ed Nu cleic Acid )
Alexei A. Koshkin,* J ef Fensholdt,† Henrik M. Pfundheller, and Christian Lomholt
Exiqon A/ S, Department of Chemistry, Bygstubben 9, DK-2950 Vedbæk, Denmark
koshkin@exiqon.com
Received J uly 20, 2001
A novel efficient method for the synthesis of locked nucleic acid (LNA) monomers is described. The
LNA 5′,3′-diols containing thymine, 4-N-acetyl- and 4-N-benzoylcytosine, 6-N-benzoyladenine, and
2-N-isobutyrylguanine as nucleobases were prepared via convergent syntheses. The method is based
on the use of the common sugar intermediate 1,2-di-O-acetyl-3-O-benzyl-4-C-methanesulfonoxy-
methyl-5-O-methanesulfonyl-D-erythro-pentofuranose (8) that easily can be prepared from D-glucose
in multigram scale. Four different nucleobases were stereoselectively coupled to 8 using a modified
Vorbru¨ggen procedure to give the corresponding 4′-C-branched nucleoside derivatives. Subsequent
ring closing furnished the protected LNA nucleosides. The 5′-O-mesyl groups were efficiently
displaced by nucleophilic substitution using sodium benzoate. Saponification of the 5′-benzoates
followed by catalytic removal of the 3′-O-benzyl groups afforded the free LNA diols. The exocyclic
amino groups of adenosine and cytidine were selectively acylated to give 4-N-acetyl- or 4-N-benzoyl-
LNA-C and 6-N-benzoyl-LNA-A. The isobutyryl group of guanine was retained during the
preparation of 2-N-isobutyryl-LNA-G. The LNA-T diol and base-protected LNA diols can be directly
converted into LNA-phosphoramidites for automated chemical synthesis of LNA containing
oligonucleotides.
In tr od u ction
In recent years, much effort has been put into the
design and synthesis of modified oligonucleotides (ONs)
with the aim of obtaining strong recognition of comple-
mentary DNA and/or RNA sequences.1-3 Among others,
conformationally restricted ONs containing bi- and tri-
cyclic carbohydrate-modified nucleotides have been stud-
ied with a number of these showing increased stabilities
of duplexes with complementary nucleic acids, as com-
pared to the corresponding unmodified duplexes.3
F igu r e 1. Chemical structure of a LNA nucleotide. The
pentofuranose moiety is effectively locked in an N-type (north
type) conformation. B ) nucleobase.
ONs containing 2′-O,4′-C-methylene-linked bicyclic
ribonucleosides termed LNA (locked nucleic acid, Figure
1) were introduced a few years ago4,5 showing unprec-
edented thermal stabilities toward complementary DNA
and RNA with excellent mismatch discrimination.4 In
addition, LNA is fully compatible with conventional DNA/
RNA chemistry. Thus, fully or partly modified LNA ONs
can efficiently be oligomerized using commercially avail-
able automatic DNA synthesizers. The very high thermal
stabilities of duplexes containing LNA were attributed
to the fixed N-type [3′-endo/3E] conformation of the
bicyclic carbohydrate units.6 The N-conformation of the
LNA monomers in ONs has been verified by NMR6a-d as
well as X-ray crystallography.6e
On the basis of the exciting properties of LNA, a large
variety of applications in molecular diagnostics including
gene array analysis can be envisioned.7 In fact, 5′-
anthraquinone conjugated 8-mer LNAs have been photo-
chemically immobilized in microtiter plates for use in
† Present address: Leo Pharmaceutical Products, Industriparken
55, DK-2750 Ballerup, Denmark.
(1) (a) Uhlmann, E.; Peyman, A. Chem. Rev. 1990, 90, 4. (b)
Beaucage, S. L.; Iyer, R. P. Tetrahedron 1993, 49, 6123. (c) Freier, S.
M., Altmann, K.-H. Nucleic Acids Res. 1997, 1337.
(2) (a) Herdewijn, P. Liebigs Ann. Chem. 1996, 1337. (b) Egli, M.
Antisense Nucleic Acid Drug Dev. 1998, 8, 123.
(3) (a) Tarkoy, M.; Bolli, M.; Schweizer, B.; Leumann, C. Helv. Chim.
Acta 1993, 76, 481. (b) Altmann, K.-H.; Kesselring, R.; Francotte, E.;
Rihs, G. Tetrahedron Lett. 1994, 35, 2331. (c) Christensen, N. K.;
Petersen, M.; Nielsen, P.; J acobsen, J . P.; Olsen, C. E.; Wengel, J . J .
Am. Chem. Soc. 1998, 120, 5458. (d) Steffens, R.; Leumann, C. J . J .
Am. Chem. Soc. 1997, 119, 11548.
(4) (a) Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J . Chem.
Commun. 1998, 455. (b) Koshkin, A. A.; Singh, S. K.; Nielsen, P.;
Rajwanshi, V. K.; Kumar, R.; Meldgaard, M.; Olsen, C. E.; Wengel, J .
Tetrahedron 1998, 54, 3607. (c) Obika, S.; Nanbu, D.; Hari, Y.; Andoh,
J .; Morio, K.; Doi, T.; Imanishi, T. Tetrahedron Lett. 1998, 39, 5401.
(d) Koshkin, A. A.; Nielsen, P.; Meldgaard, M.; Rajwanshi, V. K.; Singh,
S. K.; Wengel, J . J . Am. Chem. Soc. 1998, 120, 13252. (e) Wengel, J .
Acc. Chem. Res. 1999, 32, 301.
(6) (a) Nielsen, C. B.; Singh, S. K.; Wengel, J .; J acobsen, J . P. J .
Biomol. Struct. Dyn. 1999, 17, 175. (b) Nielsen, K. E.; Singh, S. K.;
Wengel, J .; J acobsen, J . P. Bioconjugate Chem. 2000, 11, 228. (c)
Petersen, M.; Nielsen, C. B.; Nielsen, K. E.; J ensen, G. A.; Bondens-
gaard, K.; Singh, S. K.; Rajwanshi, V. K.; Koshkin, A. A.; Dahl, B. M.;
Wengel, J .; J acobsen, J . P. J . Mol. Recognit. 2000, 13, 44. (d)
Bondensgaard, K.; Petersen, M.; Singh, S. K.; Rajwanshi, V. K.; Kumar,
R.; Wengel, J .; J acobsen, J . P. Chem.-Eur. J . 2000, 6, 2687. (e) Egli,
M.; Minasov, G.; Teplova, M.; Kumar, R.; Wengel, J . Chem. Commun.
2001, 651-652.
(5) Termed so by us. Different name (2′, 4′-BNA) was recently
introduced (Obika, S.; Hari, Y.; Sugimoto, T.; Sekiguchi, M.; Imanishi,
T. Tetrahedron Lett. 2000, 41, 8923).
(7) (a) Braasch, D. A.; Corey, D. R. Chem. Biol. 2001, 8, 1. (b)
www.exiqon.com.
10.1021/jo010732p CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/15/2001

2′-O, 4′-C-Methylene-Linked Ribonucleosides
J . Org. Chem., Vol. 66, No. 25, 2001 8505
Sch em e 1a
diagnostic genotyping including, for example, the detec-
tion of the prothrombic mutation factor V Leiden8 and
the detection of the apolipoprotein B3500 polymorphism
causing hypercholesterolemia.7b Furthermore, LNA ONs
have shown very promising properties as antisense
therapeutics. Thus, short sequences of LNA exhibited
potent antisense activity in a G-protein coupled receptor
assay system in living rats without detectable toxic
reactions and were not readily degraded in blood serum
and cell extracts.9
Although a few novel LNA-like nucleoside analogues
were reported to be already synthesized after introduc-
tion of LNA,10 the production of the 2′-O,4′-C-methylene-
ribonucleosides has until now not been a routine proce-
dure. As a result of our continued endeavors for optimiz-
ing the synthesis of 2′-O, 4′-C-methylene-linked nucleo-
sides, we hereby present an improved convergent synthesis
of the LNA monomers.
a
Reagents: (i) NaH, BnBr, DMF; (ii) Ac2O (for 3A) or TsCl (for
For the synthesis of LNA monomers, two strategies can
be envisioned: a linear strategy using nucleosides as the
starting material or a convergent strategy where an
appropriate modified glycosyl donor is synthesized and
then coupled to the nucleobase affording the modified
nucleoside. Taking advantage of the former strategy,
Obika et al. were the first to report the synthesis of
LNA-U and LNA-C nucleosides with uridine as the
starting material.11 We have used a similar strategy for
the synthesis of LNA-A.12 Despite some advantages such
as a relatively short chain of chemical transformations
and high availability of the starting RNA nucleosides,
the linear strategy has turned out not to be effective in
terms of scaling up possibilities. A key reaction in the
synthetic pathway is the introduction of the additional
oxymethyl group into the 4′-C-position of the protected
RNA nucleosides. This can be carried out via oxidation
of the 5′-OH group to the corresponding aldehyde fol-
lowed by a crossed aldol condensation with formaldehyde
and consecutive reduction. All methods reported so far
for preparation of 5′-aldehydonucleosides from natural
ribonucleosides required handling of complicated reaction
mixtures which proved to be difficult to work up, and fine-
tuning of the reaction conditions were complicated due
to low stability of the intermediates. Moreover, the
obtained results were very dependent on the nature of
the nucleosides, and no universal method was developed.
Alternatively, a convergent strategy for the synthesis
of LNA monomers was explored by Wengel and co-
workers.4a,b Using the previously described 3-O-benzyl-
4-C-hydroxymethyl-1,2-O-isopropylidene-R-D-erythro-pento-
furanose (1)13a (Scheme 1) as a starting material, LNA
monomers with all the natural nucleobases were syn-
thesized and incorporated into DNA.4a,b Importantly, 1
3B), pyridine; (iii) (a) 80% AcOH, (b) Ac2O, pyridine; (iv) nucleo-
base, BSA, TMSOTf, MeCN or 1,2-dichloroethane; (v) (a) MeONa,
MeOH, (b) TsCl, pyridine, (c) NaH, DMF; (vi) (a) NH3, methanol,
(b) NaH, DMF; (vii) debenzylation. B ) nucleobase.
can readily be synthesized in multigram scale from
D-glucose.13 However, the use of 1 was complicated by
the presence of two diastereotopic hydroxymethyl groups.
Even though the regioselective benzylation of 1 to 2 has
been described,4a,b,14 it was impossible for us to obtain
the reported 71% yield when performing the reaction on
a larger scale. Additionally, all attempts to increase the
regioselectivity by varying the reaction conditions (e.g.,
temperature and concentration) failed as well, as did the
use of the dibutyltin oxide method.15 Thus, chromato-
graphic purification of the reaction mixture remained
necessary. Acetylation of 2 followed by acetolysis and
subsequent acetylation afforded 3A that was used as a
glycosyl donor for coupling to the appropriate protected
nucleobases to give the 4′-C-branched nucleoside deriva-
tives 4A. Deacetylation with sodium methoxide and
monotosylation followed by a ring closing reaction gave
the protected LNA nucleosides 5. The free diols 6 were
finally obtained after debenzylation of 5.
Later, Wengel reported12 an improvement for this
procedure, taking advantage of another glycosyl donor.
Using the coupling sugar 3B (Scheme 1) containing tosyl
instead of acetyl on the 4-C-hydroxymethyl group, the
nucleoside 4B could be obtained. In contrast to 4A, 4B
was converted into protected LNA nucleoside 5 via a
simple two-step ring closing procedure, which was per-
formed by deacetylation with methanolic ammonia fol-
lowed by reaction with sodium hydride in DMF. However,
this method was only exemplified by the synthesis of the
LNA-T nucleoside.
(8) Ørum, H.; J akobsen, M. H.; Koch, T.; Vuust, J .; Borre, M. B.
Clin. Chem. 1999, 45, 1898.
To synthesize LNA monomers on a larger scale, the
synthetic pathway based on the selective benzylation of
1 is not ideal. In this paper, we present a new strategy
for synthesis of LNA monomers based on a new di-O-
mesylated glycosyl donor. Using this strategy, LNA
(9) (a) Wahlestedt, C.; Salmi, P.; Good, L.; Kela, J .; J ohnsson, T.;
Ho¨kfelt, T.; Broberger, C.; Porreca, F.; Lai, J .; Ren, K.; Ossipov, M.;
Koshkin, A.; J akobsen, N.; Skouv, J .; Ørum, H.; J acobsen, M. H.;
Wengel, J . Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5633.
(10) (a) Singh, S. K.; Kumar, R.; Wengel, J . J . Org. Chem. 1998, 63,
6078. (b) Wang, G.; Gunic, E.; Girardet, J .-L.; Stoisavljevic, V. Bioorg.
Med. Chem. Lett. 1999, 9, 1147. (c) Rajwanshi, V. K.; Kumar, R.; Kofod-
Hansen, M.; Wengel, J . J . Chem. Soc., Perkin Trans. 1 1999, 1407.
(11) Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.; In, Y.; Ishida, T.;
Imanishi, T. Tetrahedron Lett. 1997, 38, 8735. The detailed synthetic
procedures published recently: Obika, S.; Uneda, T.; Sugimoto, T.;
Nanbu, D.; Minami, T.; Doi, T.; Imanishi, T. Bioorg. Med. Chem. 2001,
9, 1001.
(13) (a)Youssefyeh, R. D.; Verheyden, J . P. H.; Moffatt, J . G. J . Org.
Chem. 1979, 44, 1301. (b) Leland, D. L.; Kotick, M. P. Carbohydr. Res.
1974, 38, 32. (c) Surzhykov, S. A.; Krayevsky, A. A. Nucleosides
Nucleotides 1994, 13, 2283.
(14) Waga, T.; Nishizaki, T.; Miyakawa, I.; Ohuri, H.; Meguro, H.
Biosci. Biotechnol. Biochem. 1993, 57, 1433.
(12) Koshkin, A. A.; Rajwanshi, V. K.; Wengel, J . Tetrahedron Lett.
1998, 39, 4381.
(15) Pfundheller, H. M.; Bryld, T.; Olsen, C. E.; Wengel, J . Helv.
Chim. Acta 2000, 83, 128.

8506 J . Org. Chem., Vol. 66, No. 25, 2001
Koshkin et al.
Sch em e 2a
a
Reagents: (i) MsCl, pyridine; (ii) (a) 80% TFA; (b) Ac2O, pyridine; (iii) AcOH, Ac2O, concd H2SO4; (iv) nucleobase, BSA, TMSOTf,
MeCN or 1,2-dichloroethane; (v) NaOH or LiOH, THF or 1,4-dioxane (+NH4OH for 10F ); (vi) NaOBz, DMF; (vii) NaOH, THF or EtOH/
pyridine; (viii) NH4OH, MeNH2, MeOH; (ix) 20% Pd(OH)2/C, HCO2NH4, MeOH; (x) 10% Pd/C, HCO2H, MeOH; (xi) NH4OH, (xii) (a) BzCl,
pyridine; (b) NaOH, EtOH/pyridine.
nucleosides with the four natural nucleobases thymine,
adenine, cytosine, and guanine have effectively been
synthesized.
smoothly, and no traces of side products from 5′-O-
demesylation or reaction(s) with the nucleobase were
detected. Recently, an analogous method was used for
the ring closing procedure during the synthesis of the 3′-
azido LNA-T nucleoside using a methanolic solution of
potassium carbonate.17 To deprotect the 5′-hydroxy group,
we attempted a direct hydrolysis of mesylate 10A. The
consecutive heating of the same reaction mixture that
was used for the ring closing reaction resulted in hy-
drolysis (or nucleophilic substitution) of the 5′-O-mes-
ylate, and nucleoside 12A was isolated in 78% yield.
However, removal of the 5′-O-mesyl group using this
method was time-consuming as it was necessary to reflux
the reaction mixture for 1-2 days. Furthermore, the
reaction was accompanied by the formation of a number
of side products. We therefore investigated nucleophilic
displacement of the mesyl group by sodium benzoate.18
Thus, after treatment of 10A with sodium benzoate in
DMF, the benzoylated nucleoside 11A was easily ob-
tained after crystallization from ethanol in 86% yield. It
is worth noticing that the 5′-mesylate likely can be
substituted by other benzoate-like nucleophilic reagents,
allowing the synthesis of other 5′-modified LNA nucleo-
sides, for example, LNA triphosphates. Saponification of
the 5′-O-benzoyl ester of 11A afforded the 3′-O-benzylated
LNA-T nucleoside 12A in 91% yield. Subsequent catalytic
removal of the 3′-O-benzyl group afforded the desired
unprotected LNA-T nucleoside 6A as a crystalline prod-
uct in 83% yield (Scheme 2).
Resu lts a n d Discu ssion
On the basis of the convergent strategy published by
Wengel and co-workers (Scheme 1),4b,12 we developed a
more efficient method for the synthesis of all four LNA
nucleosides. The LNA nucleosides containing thymine,
Bz-protected adenine, Bz- or Ac-protected cytosine, and
isobutyryl-protected guanine were synthesized. The nu-
cleobase protecting groups of the LNA nucleosides were
chosen to make the monomers fully compatible with
chemical oligomerization based on the phosphite triester
methodology.
To overcome the early chromatographic purification
step and essentially increase the overall yields of the
syntheses, we were interested in replacing the benzyl
group on the 5-hydroxy functionality. Encouraged by the
positive result obtained with the glycosyl donor 3B, we
envisioned that it would be possible to convert the
furanose 1 into 1,2-di-O-acetyl-3-O-benzyl-4-C-methane-
sulfonoxymethyl-5-O-methanesulfonyl-D-erythro-pento-
furanose 8 (Scheme 2) and use this as a universal glycosyl
donor. Indeed, permesylation of the ribofuranose 1 gave
the di-O-mesylated compound 7, and subsequent acetoly-
sis and acetylation in a one-pot procedure afforded the
anomeric mixture 8 in nearly quantitative yield without
any chromatographic purification.
The anomeric mixture 8 was then used as a common
glycosyl donor for coupling reactions with protected
nucleobases by the method of Vorbru¨ggen and co-work-
ers.16 Thus, stereoselective reaction of 8 with silylated
thymine afforded nucleoside 9A in 88% yield after
chromatographic purification. The compound was sub-
sequently converted into the 3′,5′-di-O-protected LNA-T
nucleoside 10A in a one-pot deacetylation and ring
closing procedure after treatment of 9A with aqueous
sodium hydroxide. This fast conversion proceeded very
Following the principle of a convergent synthesis
strategy, analogous synthetic procedures were used for
the synthesis of the other LNA monomers. The cytidine
derivative 9B was furnished in 82% yield via stereo-
selective coupling of 8 with 4-N-acetylcytosine. Treatment
of 9B with sodium hydroxide resulted in a 2′-O-depro-
tection and ring closing reaction in a similar way as
described for 9A. Additionally, due to the lability of the
4-N-acetyl group of 9B, the reaction was allowed to
proceed until complete nucleobase deprotection was
(17) Obika, S.; Andoh, J .; Sugimoto, T.; Miyashita, K.; Imanishi, T.
Tetrahedron Lett. 1999, 40, 6465.
(18) Codington, J . F.; Fecher, R.; Fox, J . J . J . Am. Chem. Soc. 1960,
82, 2794.
(16) (a) Vorbru¨ggen, H.; Krolikiewicz, K.; Bennua, B. Chem. Ber.
1981, 114, 1234. (b) Vorbru¨ggen, H.; Ho¨fle, G. Chem. Ber. 1981, 114,
1256. (c) Vorbru¨gen, H. Acta Biochim. Pol. 1996, 43, 25.

2′-O, 4′-C-Methylene-Linked Ribonucleosides
J . Org. Chem., Vol. 66, No. 25, 2001 8507
other purine nucleobases.16b The long reaction time was
explained by slow conversion of the kinetically favored
N-3 and N-7 products into the thermodynamically favor-
able N-9 isomer. Following this method, the isomerically
pure adenosine nucleoside 9E was isolated by column
chromatography in 68% yield after refluxing the ano-
meric mixture 8 and 6-N-benzoyl adenine for 29 h under
Vorbru¨ggen coupling conditions. The deacetylation and
ring closing reaction steps were performed as described
for the pyrimidine derivatives 9A and 9B. Thus, the
bicyclic nucleoside 10E was isolated in 78% yield from
9E after treatment with aqueous alkali. Both sodium and
lithium hydroxide were successfully used to promote the
reaction. Since all our attempts to develop a method for
selective O-debenzylation of the LNA-A nucleosides in
the presence of the 6-N-benzoyl group failed, we alter-
natively attempted to remove the 6-N-benzoyl group with
ammonium hydroxide during the cyclization of 9E giving
derivative 10F in 78% yield. The 5′-O-benzoyl nucleosides
11E and 11F were obtained via benzoate displacement
of 5′-mesylates 10E and 10F in 88% and 84% yield,
respectively. The free diol 6F was obtained in 78% yield
after total deprotection of 11E using a mixture of am-
monia and methylamine in methanol followed by cata-
lytic transfer hydrogenation. On the contrary, derivative
11F was first debenzylated, and the crude product
subsequently reacted with a 5-fold excess of benzoyl
chloride to give perbenzoylated LNA-A nucleoside. The
base-protected diol 6E was finally obtained in 75% yield
after selective saponification of the 3′- and 5′-benzoates
with sodium hydroxide (Scheme 2).
The most problematic chemistry encountered during
the convergent synthesis of nucleosides with all the
naturally occurring nucleic bases usually occurs with
guanine. Coupling of guanine-type bases with sugar
derivatives produces an isomeric mixture of N-9/N-7
nucleosides even when the reaction is performed under
thermodynamic control. Additionally, the two isomers are
often difficult to separate.21-23 Furthermore, the ratio of
produced isomers frequently depends on reaction condi-
tions and the nature of protecting groups on the nucleo-
bases and the glycosyl donors. In the first published
synthesis of the LNA-G nucleoside (Scheme 1),4b the
formation of three isomers (ratio 25:5:2) was detected
during the coupling of 2-N-isobutyrylguanine with sugar
derivative 3A. Our attempts to couple 2-N-isobutyryl-
guanine with the new coupling sugar 8 resulted in the
formation of a mixture of two isomers (ratio ca. 9:1).
Further investigations demonstrated that replacement
of 2-N-isobutyrylguanine by 2-N-acetylguanine in the
coupling reaction with 8 did not improve the regioselec-
tivity as the corresponding two isomers were obtained
in a ratio of ca. 1:1. Furthermore, the glycosidation of
2-N-isobutyrylguanine with 1,2-di-O-benzoyl-3-O-benzyl-
4-C-methanesulfonoxymethyl-5-O-methanesulfonyl-D-
erythro-pentofuranose gave a mixture with an isomeric
ratio of N-9/N-7 ) 6:1 (data not shown). Another method
reported for increasing the ratio of N-9/N-7 isomers in
the glycosylation of guanines is constriction of the
guanine system into “6-enolate” derivatives.23 For that
purpose, several 6-O-protecting groups of guanine, such
as diphenylcarbamoyl,23 benzyl,24 and p-nitrophenyl-
Sch em e 3
accomplished, thus giving 10C, which could be isolated
as a white solid material in 87% yield. Nucleophilic
displacement of the 5′-mesylate group with benzoate was
performed by treatment of 10C with sodium benzoate in
DMF at 100 °C and afforded the benzoylated nucleoside
11C in 71% yield. Similar reactions of the mesyloxy
groups have been reported by Kellogg et al.19 taking
advantage of cesium acetate rather than sodium benz-
oate. However, a similar nucleophilic substitution of the
mesylate 10C by the acetate was unsuccessful in our
hands. To obtain the fully unprotected LNA-C nucleoside
6C, the 5′-O-benzoyl-3′-O-benzyl-LNA-C nucleoside 11C
was subjected to a one-pot procedure involving catalytic
hydrogenation and debenzoylation reactions. Thus, treat-
ment of 11C with ammonium formate and Pd(OH)2/C in
refluxing methanol followed by treatment with am-
monium hydroxide afforded the free nucleoside 6C as a
white solid material in 77% yield (Scheme 2). To use this
in ON synthesis, the exocyclic amine needed to be
protected. This can be done selectively by treatment of
the unprotected nucleoside with acetic anhydride in
DMF.20 Thus, 6C was treated with 1.2 equiv of acetic
anhydride in DMF overnight giving the N-acetylated
derivative 6B after crystallization from methanol in 65%
yield (Scheme 3). For the synthesis of 4-N-benzoyl LNA-C
6D, nucleoside 6C was reacted with 2 equiv of benzoic
anhydride in anhydrous pyridine affording a mixture of
mono-, di-, and tribenzoylated nucleosides, which subse-
quently was partially hydrolyzed after addition of sodium
hydroxide to the reaction mixture. Following this reaction
sequence, the 4-N-benzoylated LNA-C nucleoside 6D was
obtained in 93% yield as a crystalline product (Scheme
3).
It is commonly known that the two major products
formed during a Vorbru¨ggen coupling reaction between
a glycosyl donor and a properly protected purine are the
N-7 and the N-9 isomers, where the N-9 isomer is the
naturally occurring isomer. The latter isomer is the
thermodynamic product of the reaction, and hence pro-
longed reaction time and elevated temperature can
increase the yield of this desired isomer.16 Thus, reactions
between silylated purines and 8 were performed under
conditions favoring the thermodynamic products. It has
previously been shown16b,21 that the coupling reactions
of 6-N-benzoyl adenine with different glycosyl donors
have to be conducted for longer time than in cases of
(19) Kruizinga, W. H.; Strijtveen, B.; Kellogg, R. M. J . Org. Chem.
1981, 46, 4321.
(20) Bhat, V.; Ugarkar, B. G.; Sayeed, V. A.; Grim, K.; Kosora, N.;
Domenico, P. A.; Stoker, E. Nucleosides Nucleotides 1989, 8, 179.
(21) Miyaki, M.; Shimizu, B. Chem. Pharm. Bull. 1970, 18, 1446.
(22) Garner, P.; Ramakanth, S. J . Org. Chem. 1988, 53, 1294.
(23) Robins, M. J .; Zou, R.; Guo, Z.; Wnuk, S. J . Org. Chem. 1996,
61, 9207 and references therein.
(24) Martin, P. Helv. Chim. Acta 1995, 78, 486.

8508 J . Org. Chem., Vol. 66, No. 25, 2001
Koshkin et al.
ethyl25 protective groups were suggested. However, cou-
pling reactions of 8 with 6-O-benzyl-2-N-isobutyryl-
guanine or 2-N-acetyl-6-O-diphenylcarbamoylguanine
under standard conditions did not furnish the desired N-9
products in satisfactory yields (data not shown). Thus,
in this specific case, the best results were obtained when
the coupling was performed with 8 and N-2-isobutyryl
guanine, and this method was chosen for the synthesis
of the LNA-G monomer.
The mixture of isomers containing ca. 90% of 9G was
obtained from 8 in 84% yield and used in the next step.
An isomerically pure compound was obtained after the
ring closing reaction, and hence 10G was obtained as the
pure N-9 isomer in 85% yield after chromatographic
purification. The nucleoside derivative 10G was then
converted into the diol 6G via nucleophilic replacement
of the mesyl group with benzoyl and a final deprotection
of the 5′- and 3′-hydroxy groups generally proceeding as
described for the other LNA nucleosides. However, some
additional precautions for the handling of the LNA-G
nucleoside were required due to the presence of the
relatively labile 2-N-isobutyryl group. In fact, we had to
use an EtOH/pyridine mixture as a solvent during
treatment of 11G with aqueous NaOH for 5′-hydroxyl
deprotection. Second, a mixture of Pd/C + HCO2H was
used for the final removal of the 3′-O-benzyl group from
12G. The use of formic acid as a hydrogen donor in the
reaction prevented removal of the 2-N-isobutyryl group,
which took place when ammonium formate was used as
the H donor (Scheme 2).
points were determined on a Bu¨chi B-540 melting point
apparatus and are uncorrected. All 1H NMR spectra were
recorded at 400 MHz; 13C NMR spectra were recorded at 62.9
MHz for 11A, 10C, 9G, and 11G and at 100.6 MHz for all other
compounds. The values for chemical shifts are reported in ppm
relative to tetramethylsilane (TMS) as the internal standard.
Matrix-assisted laser desorption ionization-time of flight
(MALDI-TOF) mass spectra were recorded in positive ion
mode on a Voyager-DE PRO spectrometer from PerSeptive
Biosystems using 2,5-dihydroxybenzoic acid as matrix. Ele-
mental analyses were performed at The Microanalytical
Laboratory, Department of Chemistry, University of Copen-
hagen.
3-O-Ben zyl-4-C-m eth a n esu lfon oxym eth yl-5-m eth a n e-
su lfon yl-1,2-O-isop r op ylid en e-r-D-er yth r o-p en t ofu r a n -
ose (7). A solution of 3-O-benzyl-4-C-hydroxymethyl-1,2-O-
isopropylidene-R-D-erythro-pentofuranose 113a (11.1 g, 35.8
mmol) in anhyd pyridine (30 mL) was cooled in an ice bath,
and MsCl (8.3 mL, 107.2 mmol) was added. The mixture was
stirred for 1 h at room temperature, diluted with Et2O (200
mL), and washed with H2O (3 × 200 mL). The organic layer
was dried (Na2SO4), concentrated under reduced pressure,
coevaporated with toluene (2 × 100 mL), and dried in vacuo
to yield 16.4 g (98%) of compound 7 as a white solid material.
1
mp 108-110 °C (MeOH). H NMR (CDCl3): δ 7.38-7.26 (m,
5H), 5.79 (d, J ) 4.0 Hz, 1H), 4.88 (d, J ) 11.9 Hz, 1H), 4.77
(d, J ) 11.5 Hz, 1H), 4.65 (dd, J ) 5.1 and 3.8 Hz, 1H), 4.57
(d, J ) 11.5, 1H), 4.42 (d, J ) 12.0 Hz, 1H), 4.32 (d, J ) 11.0
Hz, 1H), 4.19 (d, J ) 5.1 Hz, 1H), 4.15 (d, J ) 11.1 Hz, 1H),
3.08 (s, 3H), 2.98 (s, 3H), 1.69 (s, 3H), 1.34 (s, 3H). 13C NMR
(CDCl3): δ 136.5, 128.5, 128.3, 128.0, 113.9, 104.4, 83.1, 78.3,
77.7, 72.7, 69.3, 68.5, 37.9, 37.3, 26.1, 25.5. MALDI-MS m/z:
489.3 [M + Na]+. Anal. Calcd for C18H26O10S2: C, 46.34; H,
5.62. Found: C, 46.33; H, 5.52.
1,2-Di-O-a ce t yl-3-O-b e n zyl-4-C-m e t h a n e su lfon oxy-
m eth yl-5-O-m eth an esu lfon yl-D-er yth r o-pen tofu r an ose (8).
Meth od 1. Ac2O (28 mL, 297 mmol) and concd H2SO4 (28 µL)
were added to a solution of compound 7 (18.6 g, 39.9 mmol) in
AcOH (280 mL), and the mixture was stirred overnight at room
temperature. More concd H2SO4 (5 µL) was added, and the
reaction was continued for 24 h. H2O (200 mL) was added,
and the mixture was stirred for 3 h and washed twice with
CH2Cl2 (250 and 100 mL). The combined organic layers were
washed with saturated NaHCO3 (4 × 200 mL), dried (Na2SO4),
and concentrated under reduced pressure to give compound 8
(19.7 g, 97%) as a colorless syrup (two anomers; ratio ∼1:5).
Meth od 2. A solution of compound 7 (16 g, 34.3 mmol) in
80% aqueous trifluoroacetic acid (100 mL) was stirred at room
temperature for 1 h. The solvents were removed under reduced
pressure, and the residue was dissolved in dichloromethane
(200 mL) and washed with saturated NaHCO3 (2 × 200 mL).
The organic layer was dried (Na2SO4) and concentrated under
reduced pressure to give a colorless oily intermediate. The
intermediate was coevaporated with anhyd pyridine (2 × 50
mL), dissolved in anhyd pyridine, and treated with Ac2O (12
mL, 127 mmol) overnight. The reaction mixture was quenched
by addition of saturated NaHCO3 (250 mL) and washed with
EtOAc (2 × 200 mL). The organic layers were combined,
washed with brine (150 mL), dried (Na2SO4), and concentrated
under reduced pressure to yield compound 8 (15.9 g, 91%) as
Con clu sion
Following the universal convergent scheme as de-
scribed above, it was possible to efficiently synthesize
LNA monomers with all four naturally occurring nucleo-
bases. These can be converted into their corresponding
phosphoramidites to be used in LNA synthesis as previ-
ously described.4b,11 The method introduced herein has
three key points which make it considerably more useful
in comparison to the procedures reported so far: (i) no
regioselective benzylation reaction is needed, and the
easily produced di-O-mesyl furanose 8 is used as a
glycosyl donor, (ii) simplified deacetylation and ring
closing of nucleosides 9A, 9B, 9E, and 9G in aqueous
solutions, and (iii) effective substitution of 5′-mesylates
in all LNA nucleosides by sodium benzoate. The hereby
reported results exemplify the power of the convergent
strategy, and this strategy can likewise be applied for
the syntheses of LNA monomers containing other nucleo-
bases or heterocycles. The synthesis of LNA monomers
with other base moieties is currently being investigated
and will be presented elsewhere.
1
a mixture of two stereoisomers (ratio ∼1:7 by H NMR). The
product was pure by NMR spectra and was used in the next
step without further purification. The analytical pure sample
was obtained by silica gel column chromatography (20-50%
v/v EtOAc/hexane). 13C NMR (CDCl3, main isomer): δ 169.1,
168.6, 136.3, 128.5-128.0 (m), 97.2, 82.6, 78.6, 73.9, 73.3, 68.8,
68.4, 37.5, 37.3, 20.8, 20.5. MALDI-MS m/z: 533.7 [M + Na]+.
Anal. Calcd for C19H26O12S2: C, 44.70; H, 5.13. Found: C,
44.80; H, 5.06.
Exp er im en ta l Section
Gen er a l P r oced u r es. All chemicals were obtained from
commercial suppliers and were used without additional puri-
fication. An atmosphere of nitrogen was applied when reac-
tions were conducted in anhydrous solvents. Column chroma-
tography was performed using Silica gel 60 (0.063-0.200 mm)
from Merck, and HPLC was performed using a column Prep
Nova-Pak HR Silica 60 (6 µm, 30 × 300 mm) from Waters.
After column chromatography, appropriate fractions were
pooled out and concentrated under reduced pressure. Melting
1-(2-O-Acetyl-3-O-ben zyl-4-C-m eth an esu lfon oxym eth yl-
5-O-m et h a n esu lfon yl-â-D-er yth r o-p en t ofu r a n osyl)t h y-
m in e (9A). N,O-Bis(trimethylsilyl)acetamide (BSA) (70 mL,
283 mmol) was added to a mixture of 8 (52 g, 102 mmol) and
thymine (16 g, 127 mmol) in anhyd MeCN (250 mL). The
reaction mixture was refluxed for 1 h to get a clear solution.
(25) J enny, T. F.; Schneider, K. C.; Benner, S. A. Nucleosides
Nucleotides 1992, 11, 1257.

2′-O, 4′-C-Methylene-Linked Ribonucleosides
J . Org. Chem., Vol. 66, No. 25, 2001 8509
Trimethylsilyl triflate (TMSOTf) (24 mL, 133 mmol) was
added, and refluxing was continued further for 4 h. The
solution was cooled to room temperature, diluted with CH2Cl2
(200 mL), and washed with saturated NaHCO3 (2 × 250 mL).
The organic layer was dried (Na2SO4), concentrated under
reduced pressure, and the residue was purified by silica gel
column chromatography (0-2% v/v MeOH/CH2Cl2) to give 9A
(51.6 g, 88%) as a white solid material. 1H NMR (CDCl3): δ
9.33 (br s, 1H), 7.40-7.28 (m, 5H), 7.08 (d, J ) 1.2 Hz, 1H),
5.71 (d, J ) 3.3 Hz, 1H), 5.58 (dd, J ) 6.4 and 3.3 Hz, 1H),
4.70 (d, J ) 6.4 Hz, 1H), 4.60 (d, J ) 10.8 Hz, 1H), 4.55 (d, J
) 10.8 Hz, 1H), 4.53 (d, J ) 11.7 Hz, 1H), 4.38 (d, J ) 10.8
Hz, 1H), 4.34 (d, J ) 10.8 Hz, 1H), 4.32 (d, J ) 11.7 Hz, 1H),
3.02 (s, 3H), 3.00 (s, 3H), 2.11 (s, 3H), 1.92 (d, J ) 1.1 Hz,
3H). 13C NMR (CDCl3): δ 170.0, 163.7, 150.1, 137.9, 136.6,
128.6, 128.5, 128.4, 111.8, 92.4, 84.0, 77.9, 74.8, 73.7, 68.4, 67.5,
37.7, 37.6, 20.7, 12.6. MALDI-MS m/z: 598.8 [M + Na]+. Anal.
Calcd for C22H28N2O12S2: C, 45.83; H, 4.89; N, 4.86. Found:
C, 45.80; H, 4.83; N, 4.65.
saturated NaHCO3 (2 × 100 mL). The aqueous layers were
combined and washed with dichloromethane (100 mL). The
combined organic layers were dried (Na2SO4), concentrated,
and purified by silica gel HPLC (2-3% v/v MeOH/CH2Cl2) to
give 12A (0.74 g, 91%) as a white solid material. 1H NMR
(CDCl3): δ 9.28 (br s, 1H), 7.45 (d, J ) 1.1 Hz, 1H), 7.38-7.22
(m, 5H), 5.66 (s, 1H), 4.67 (d, J ) 11.6 Hz, 1H), 4.56 (d, J )
11.7 Hz, 1H), 4.54 (s, 1H), 4.05 (d, J ) 7.9 Hz, 1H), 4.01 (d, J
) 12.5 Hz, 1H), 3.96 (s, 1H), 3.95 (d, J ) 12.6 Hz, 1H), 3.83
(d, J ) 7.9 Hz, 1H), 1.88 (d, J ) 1.1 Hz, 3H). 13C NMR
(CDCl3): δ 163.9, 149.8, 137.0, 134.7, 128.5, 128.2, 127.8, 110.3,
88.2, 87.3, 76.9, 75.9, 72.3, 72.0, 57.6, 12.7. MALDI-MS m/z:
383.3 [M + Na]+. Anal. Calcd for C18H20N2O6‚1/5 H2O: C, 59.40;
H, 5.65; N, 7.70. Found: C, 59.33; H, 5.41; N, 7.68.
(1S,3R,4R,7S)-7-Hyd r oxy-1-h yd r oxym eth yl-3-(th ym in -
1-yl)-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (6A). A mixture of
compound 12A (3 g, 8.24 mmol), 20% Pd(OH)2/C (1.5 g), and
HCO2NH4 (1.6 g, 25.4 mmol) was suspended in MeOH (20 mL).
After refluxing the mixture for 10 min, the catalyst was filtered
off and washed with MeOH. The combined filtrates were
concentrated to a white solid residue. Crystallization from 15%
MeOH/CH2Cl2 (v/v) gave 6A (1.9 g, 83%) as a white solid
material. mp 196-198 °C. 1H NMR (DMSO-d6): δ 11.32 (br s,
1H), 7.60 (d, J ) 1.1 Hz, 1H), 5.68 (d, J ) 4.1 Hz, 1H), 5.38 (s,
1H), 5.20 (t, J ) 5.6 Hz, 1H), 4.09 (s, 1H), 3.89 (d, J ) 4.0 Hz,
1H), 3.80 (d, J ) 7.8 Hz, 1H), 3.74 (d, J ) 5.5 Hz, 2H), 3.61 (d,
J ) 7.8 Hz, 1H), 1.75 (d, J ) 1.1 Hz, 3H). 13C NMR (DMSO-
d6): δ 164.1, 150.1, 135.1, 108.6, 89.0, 86.5, 79.1, 71.2, 68.9,
56.2, 12.6. MALDI-MS m/z: 293.2 [M + Na]+. Anal. Calcd for
(1S,3R,4R,7S)-7-Ben zyloxy-1-m eth an esu lfon oxym eth yl-
3-(th ym in -1-yl)-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (10A). To
a solution of compound 9A (44 g, 76.3 mmol) in 1,4-dioxane/
H2O (1/1 v/v; 200 mL) was added 2 M NaOH (200 mL). The
mixture was stirred for 1 h at room temperature, diluted with
saturated NaHCO3 (200 mL), and extracted with CH2Cl2 (2 ×
200 mL). The combined organic layers were dried (Na2SO4),
concentrated under reduced pressure, and the residue was
purified by silica gel column chromatography (1-3% v/v
MeOH/CH2Cl2) to give 10A (31.5 g, 94%) as a white solid
1
material. H NMR (CDCl3): δ 9.24 (br s, 1H), 7.41-7.22 (m,
C
11H14N2O6‚1/2 H2O: C, 47.31; H, 5.41; N, 10.03. Found: C,
47.39; H, 5.16; N, 10.01.
6H), 5.68 (s, 1H), 4.66 (d, J ) 11.5 Hz, 1H), 4.61 (s, 1H), 4.59
(d, J ) 12.1 Hz, 1H), 4.56 (d, J ) 11.5 Hz, 1H), 4.52 (d, J )
12.1 Hz, 1H), 4.08 (d, J ) 7.9 Hz, 1H), 3.93 (s, 1H), 3.87 (d, J
) 7.9 Hz, 1H), 3.08 (s, 3H), 1.93 (s, 3H). 13C NMR (CDCl3): δ
163.6, 149.6, 136.3, 134.0, 128.4, 128.2, 127.8, 110.7, 87.5, 85.5,
76.6, 75.9, 72.3, 71.5, 64.0, 37.8, 12.4. MALDI-MS m/z: 439.7
[M + H]+. Anal. Calcd for C19H22N2O8S‚1/6 H2O: C, 51.70; H,
5.10; N, 6.35. Found: C, 51.73; H, 5.11; N, 6.45.
1-(2-O-Acetyl-3-O-ben zyl-4-C-m eth an esu lfon oxym eth yl-
5-O-m et h a n esu lfon yl-â-D-er yth r o-p en t ofu r a n osyl)-4-N-
a cetylcytosin e (9B). BSA (23 mL, 93.0 mmol) was added to
a suspension of compound 8 (24 g, 47.0 mmol) and 4-N-
acetylcytosine (11 g, 71.8 mmol) in anhyd MeCN (300 mL).
The mixture was refluxed for 1 h and cooled to room temper-
ature. TMSOTf (10 mL, 55.3 mmol) was added dropwise, and
the resulting mixture was refluxed for 3 h and stirred
overnight at room temperature. EtOAc (250 mL) was added,
and the solution was washed with saturated NaHCO3 (2 ×
300 mL) and brine (250 mL). The organic phase was dried
(Na2SO4) and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (1-
4% v/v MeOH/CH2Cl2) to give 9B (23.5 g, 82%) as a white solid
(1S,3R,4R,7S)-1-Ben zoyloxym eth yl-7-ben zyloxy-3-(th y-
m in -1-yl)-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (11A). NaOBz
(5.4 g, 37.5 mmol) was added to a solution of compound 10A
(8.2 g, 18.7 mmol) in anhyd DMF (400 mL). The mixture was
stirred for 5 h at 100 °C, cooled to room temperature, and
filtrated. The solvent was evaporated under reduced pressure,
and the residue was suspended in EtOAc (150 mL), washed
with H2O (3 × 100 mL), dried (Na2SO4), and concentrated to
a solid residue. Crystallization from EtOH afforded 11A (7.55
g, 86%) as a white solid material. mp 164-166 °C. 1H NMR
(CDCl3): δ 8.78 (br s, 1H), 7.94 (m, 2H), 7.61 (m, 1H), 7.45
(m, 2H), 7.30-7.20 (m, 6H), 5.63 (s, 1H), 4.83 (d, J ) 12.6 Hz,
1H), 4.73 (d, J ) 11.9 Hz, 1H), 4.66 (s, 1H), 4.56 (d, J ) 12.6
Hz, 1H), 4.52 (d, J ) 11.9 Hz, 1H), 4.18 (d, J ) 7.7 Hz, 1H),
3.97 (d, J ) 7.8, 1H), 3.91 (s, 1H), 1.58 (s, 3H). 13C NMR
(CDCl3): δ 165.5, 163.4, 149.5, 136.4, 133.7, 133.6, 129.5, 129.1,
128.6, 128.5, 128.2, 127.8, 110.3, 87.5, 86.2, 76.6, 75.7, 72.1,
72.0, 58.9, 12.1. MALDI-MS m/z: 487.3 [M + Na]+. Anal. Calcd
for C25H24N2O7‚1/4H2O: C, 64.03; H, 5.26; N, 5.97. Found: C,
63.88; H, 5.18; N, 5.95.
(1S,3R,4R,7S)-7-Ben zyloxy-1-h ydr oxym eth yl-3-(th ym in -
1-yl)-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (12A). Meth od 1. H2O
(50 mL) and 2 M NaOH (200 mL) were added to a solution of
compound 10A (22 g, 50 mmol) in 1,4-dioxane (200 mL). The
reaction mixture was refluxed for 24 h, cooled to room
temperature, and neutralized with AcOH (25 mL). Saturated
NaHCO3 (200 mL) was added, and the mixture was washed
with CH2Cl2 (2 × 200 mL). Organic layers were combined,
dried (Na2SO4), and concentrated under reduced pressure.
Purification by silica gel column chromatography (1-3% v/v
MeOH/CH2Cl2) afforded 12A (14.1 g, 78%) as a white solid
material.
1
material. H NMR (CDCl3): δ 9.79 (br s, 1H), 7.73 (d, J ) 7.5
Hz, 1H), 7.41 (d, J ) 7.5 Hz, 1H), 7.37-7.27 (m, 5H), 5.76-
5.72 (m, 2H), 4.78 (d, J ) 5.7 Hz, 1H), 4.60-4.53 (m, 3H), 4.45
(d, J ) 10.6 Hz, 1H), 4.41 (d, J ) 10.7 Hz, 1H), 4.37 (d, J )
11.5 Hz, 1H), 3.01 (s, 3H), 3.00 (s, 3H), 2.26 (s, 3H), 2.12 (s,
3H). 13C NMR (CDCl3): δ 170.9, 169.8, 163.4, 154.6, 146.6,
136.7, 128.6, 128.4, 128.3, 97.0, 94.4, 84.7, 77.9, 74.4, 73.9, 68.1,
67.8, 37.6, 37.5, 24.9, 20.8. MALDI-MS m/z: 626.5 [M + Na]+.
Anal. Calcd for C23H29N3O12S2‚1/5 H2O: C, 45.49; H, 4.88; N,
6.92. Found: C, 45.54; H, 4.91; N, 6.65.
(1S,3R,4R,7S)-7-Ben zyloxy-1-m eth an esu lfon oxym eth yl-
3-(cytosin -1-yl)-2,5- d ioxa bicyclo[2.2.1]h ep ta n e (10C). Nu-
cleoside 9B (22.5 g, 37.1 mmol) was dissolved in THF/MeOH
(2:1 v/v, 300 mL), and 1 M NaOH (200 mL) was added to the
solution. After stirring for 1 h, AcOH (10 mL) was added, and
the mixture was concentrated under reduced pressure to ca.
1/2 of its volume and cooled in an ice bath. The precipitate was
filtered off, washed with ice-cold H2O, and dried in vacuo.
Purification by silica gel column chromatography (2-5% v/v
MeOH/CH2Cl2) afforded 10C (14.1 g, 87%) as a white solid
material. 1H NMR (CD3OD): δ 7.70 (d, J ) 7.7 Hz, 1H), 7.32-
7.28 (m, 5H), 5.89 (d, J ) 7.6 Hz, 1H), 5.63 (s, 1H), 4.67 (d, J
) 12.1 Hz, 1H), 4.62 (d, J ) 11.5 Hz, 1H), 4.61 (d, J ) 12.1
Hz, 1H), 4.56 (d, J ) 11.6 Hz, 1H), 4.52 (s, 1H), 4.06 (d, J )
7.9 Hz, 1H), 3.92 (s, 1H), 3.89 (d, J ) 8.0 Hz, 1H), 3.15 (s,
3H). 13C NMR (CD3OD): δ 167.9, 157.7, 141.1, 138.6, 129.6,
129.4, 129.3, 95.9, 89.6, 87.0, 78.3, 77.3, 73.3, 73.0, 66.2, 37.5.
MALDI-MS m/z: 446.4 [M + Na]+. Anal. Calcd for C18H21N3O7S‚
5/6 H2O: C, 49.31; H, 5.17; N, 9.52. Found: C, 49.31; H, 5.12;
N, 9.43.
Meth od 2. To a solution of compound 11A (1.05 g, 2.24
mmol) in THF/H2O (1:1 v/v; 16 mL) was added 2 M NaOH (4
mL). After stirring for 1 h, AcOH (1 mL) was added, and the
solvents were removed under reduced pressure. The residue
was suspended in dichloromethane (100 mL) and washed with

8510 J . Org. Chem., Vol. 66, No. 25, 2001
Koshkin et al.
(1S,3R,4R,7S)-1-Ben zoyloxym eth yl-7-ben zyloxy-3-(cy-
tosin -1-yl)-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (11C). A mix-
ture of compound 10C (8.5 g, 19.4 mmol) and NaOBz (5.79 g,
40.2 mmol) was suspended in anhyd DMF (300 mL), stirred
for 5 h at 100 °C, and cooled to room temperature. The solution
was filtrated, and the solvent was removed under reduced
pressure. The residue was redissolved in CH2Cl2 (150 mL),
washed with saturated NaHCO3 (2 × 200 mL), dried (Na2SO4),
and concentrated. The residue was crystallized from EtOH to
give 11C (6.2 g, 71%) as a white solid material. mp 199-200
°C. The mother liquor was concentrated under reduced pres-
sure, and the residue was purified by silica gel HPLC (2-4%
v/v MeOH/CH2Cl2) to give 1.9 g more of 11C (total yield 93%).
1H NMR (CDCl3): δ 7.95 (m, 2H), 7.66-7.47 (m, 4H), 7.26-
7.22 (m, 5H), 5.76 (d, J ) 7.5 Hz, 1H), 5.69 (s, 1H), 4.76 (d, J
) 12.5 Hz, 1H), 4.75 (s, 1H), 4.66 (d, J ) 11.6 Hz, 1H), 4.61
(d, J ) 12.5 Hz, 1H), 4.45 (d, J ) 11.6 Hz, 1H), 4.18 (d, J )
7.7 Hz, 1H), 3.97 (d, J ) 7.7 Hz, 1H), 3.90 (s, 1H). 13C NMR
(CDCl3): δ 165.8, 165.5, 155.2, 139.2, 136.5, 133.5, 129.4, 129.1,
128.5, 128.4, 128.0, 127.6, 94.6, 88.0, 85.7, 76.5, 75.9, 72.0, 71.9,
59.5. MALDI-MS m/z: 472.6 [M + Na]+. Anal. Calcd for
9-(2-O-Acetyl-3-O-ben zyl-4-C-m eth an esu lfon oxym eth yl-
5-O-m et h a n esu lfon yl-â-D-er yth r o-p en t ofu r a n osyl)-6-N-
ben zoyla d en in e (9E). To a suspension of compound 8 (19.6
g, 38.4 mmol) and 6-N-benzoyladenine (11.02 g, 46.1 mmol)
in anhyd 1,2-dichloroethane (175 mL) was added BSA (25.1
mL, 101 mmol). The mixture was refluxed for 1 h and cooled
to room temperature. TMSOTf (13.9 mL, 76.8 mmol) was
added, and the solution was refluxed for 5 h, stirred at room
temperature overnight, and refluxed further for 24 h. The
reaction mixture was poured into ice-cold saturated NaHCO3
(200 mL), stirred for 0.5 h, and filtrated. The phases were
separated, and the organic phase was washed with saturated
NaHCO3 (3 × 150 mL), dried (Na2SO4), and concentrated
under reduced pressure. Purification by silica gel column
chromatography (1-1.5% v/v MeOH/CH2Cl2) afforded 9E (18.1
1
g, 68%) as a slightly yellow solid material. H NMR (CDCl3):
δ 8.76 (s, 1H), 8.12 (s, 1H), 8.02 (m, 2H), 7.61 (m, 1H), 7.51
(m, 2H), 7.40-7.34 (m, 5H), 6.23 (d, J ) 3.5 Hz, 1H), 6.08 (dd,
J ) 5.9 and 3.5 Hz, 1H), 5.12 (d, J ) 6.0 Hz, 1H), 4.68 (d, J )
11.1 Hz, 1H), 4.67 (d, J ) 11.6 Hz, 1H), 4.64 (d, J ) 11.0 Hz,
1H), 4.44 (d, J ) 10.8 Hz, 1H), 4.39 (d, J ) 11.7 Hz, 1H), 4.36
(d, J ) 11.0 Hz, 1H), 3.03 (s, 3H), 2.87 (s, 3H), 2.13 (s, 3H).
13C NMR (CDCl3): δ 169.5, 164.5, 152.5, 150.9, 149.7, 142.4,
136.3, 133.2, 132.8, 128.7, 128.5, 128.4, 128.3, 128.2, 128.0,
127.9, 127.8, 123.5, 87.8, 84.1, 77.3, 74.6, 73.4, 67.3, 67.2, 37.6,
37.3, 20.5. MALDI-MS m/z: 712.4 [M + Na]+.
(1S,3R,4R,7S)-3-(6-N-Ben zoyladen in e-9-yl)-7-ben zyloxy-
1-m e t h a n e su lfon yloxym e t h yl-2,5-d ioxa b icyclo[2.2.1]-
h ep ta n e (10E). Compound 9E (17.9 g, 26.0 mmol) was
dissolved in a mixture of THF (160 mL) and H2O (110 mL).
LiOH‚H2O (5.5 g, 131 mmol) was added, and the mixture was
stirred for 3.5 h at room temperature. The solution was
neutralized with AcOH (6 mL) to give a precipitate. The
precipitate was filtered off and washed with water to give 10E
(11.6 g, 78%) as a white solid material. 1H NMR (CDCl3): δ
8.72 (s, 1H), 8.15 (s, 1H), 8.02 (m, 2H), 7.63-7.59 (m, 1H),
7.54-7.50 (m, 2H), 7.32-7.27 (m, 5H), 6.10 (s, 1H), 4.95 (s,
1H), 4.67 (d, J ) 11.7 Hz, 1H), 4.62 (d, J ) 12.0 Hz, 1H), 4.60
(d, J ) 11.7 Hz, 1H), 4.57 (d, J ) 11.7 Hz, 1H), 4.33 (s, 1H),
4.21 (d, J ) 7.9 Hz, 1H), 4.01 (d, J ) 7.9 Hz, 1H), 3.04 (s, 3H).
13C NMR (CDCl3): δ 164.2, 152.5, 150.6, 149.5, 140.5, 136.4,
133.3, 132.8, 128.7, 128.4, 128.2, 127.8, 127.7, 123.3, 86.7, 85.3,
77.4, 76.8, 72.5, 72.2, 64.1, 37.7. MALDI-MS m/z: 575.0 [M +
Na]+. Anal. Calcd for C26H25N5O7S‚H2O: C, 54.83; H, 4.78; N,
12.30. Found: C, 54.89; H, 4.85; N, 12.42.
C
24H23N3O6: C, 64.14; H, 5.16; N, 9.35. Found: C, 64.07; H,
4.96; N, 9.27.
(1S,3R,4R,7S)-7-Hyd r oxy-1-h yd r oxym eth yl-3-(cytosin -
1-yl)-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (6C). A mixture of
compound 11C (6.44 g, 14.3 mmol) and 20% Pd(OH)2/C (3 g)
was suspended in MeOH (100 mL), and HCO2NH4 (6 g, 95.1
mmol) was added. The mixture was refluxed for 1 h, diluted
with concd NH4OH (50 mL), and stirred overnight at 50 °C.
The catalyst was filtered off and washed with boiling H2O. The
combined filtrates were concentrated under reduced pressure
to a solid residue and crystallized from H2O/EtOH (1:1 v/v) to
give 6C (2.95 g, 77%) as a white solid material. mp 275-278
°C (dec). 1H NMR (DMSO-d6): δ 7.74 (d, J ) 7.5 Hz, 1H), 7.15
(br s, 1H), 7.19 (br s, 1H), 5.75 (d, J ) 7.5 Hz, 1H), 5.39 (s,
1H), 4.09 (s, 1H), 3.82 (d, J ) 7.8 Hz, 1H), 3.81 (s, 1H), 3.75
(s, 2H), 3.63 (d, J ) 7.8 Hz, 1H). 13C NMR (DMSO-d6): δ 165.9,
154.8, 139.9, 93.4, 88.6, 87.0, 79.1, 71.1, 68.5, 56.3. MALDI-
MS m/z: 256.0 [M + H]+. Anal. Calcd for C10H13N3O5‚11/16
H2O: C, 44.88; H, 5.37; N, 15.70. Found: C, 44.78; H, 5.10; N,
15.45.
(1S,3R,4R,7S)-3-(4-N-Acet ylcyt osin -1-yl)-7-h yd r oxy-1-
h yd r oxym eth yl-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (6B). To
a solution of compound 6C (0.42 g, 1.57 mmol) in anhyd DMF
(20 mL) was added Ac2O (0.18 mL, 1.9 mmol). The mixture
was stirred at room temperature overnight, and the solvent
was removed under reduced pressure. Crystallization from
MeOH gave 6B (0.32 g, 65%) as a white solid material. mp
223-225 °C (dec). 1H NMR (DMSO-d6): δ 10.88 (br s, 1H),
8.17 (d, J ) 7.5 Hz, 1H), 7.24 (d, J ) 7.5 Hz, 1H), 5.67 (d, J )
4.0 Hz, 1H), 5.47 (s, 1H), 5.14 (t, J ) 5.7 Hz, 1H), 4.20 (s, 1H),
3.86 (d, J ) 7.8 Hz, 1H), 3.82 (d, J ) 4.0 Hz, 1H), 3.80-3.76
(m, 2H), 3.68 (d, J ) 7.9 Hz, 1H). 13C NMR (DMSO-d6): δ
171.4, 162.9, 154.5, 144.6, 95.5, 89.5, 87.7, 78.9, 71.4, 68.8, 56.6,
24.7. MALDI-MS m/z: 297.9 [M + H]+. Anal. Calcd for
(1S,3R,4R,7S)-3-(6-N-Ben zoyla d en in e-9-yl)-1-ben zoyl-
oxym et h yl-7-b en zyloxy-2,5-d ioxa b icyclo[2.2.1]h ep t a n
(11E). Compound 10E (11.5 g, 20.2 mmol) was dissolved in
anhyd DMF (450 mL). NaOBz (5.40 g, 37.4 mmol) was added,
and the mixture was stirred at 90 °C for 7 h, cooled to room
temperature, filtrated, concentrated under reduced pressure,
and coevaporated with MeCN. The residue was redissolved
in CH2Cl2 (150 mL), washed with saturated NaHCO3 (3 × 100
mL) and brine (100 mL), dried (Na2SO4), and concentrated
under reduced pressure. Crystallization from H2O/EtOH (1:1
v/v) gave 11E (10.6 g, 88%) as a white solid material. mp 112-
C
12H15N3O6‚1/3 H2O: C, 47.52; H, 5.21; N, 13.86. Found: C,
1
47.69; H, 5.11; N, 13.69.
115 °C. H NMR (DMSO-d6): δ 11.2 (br s, 1H), 8.72 (s, 1H),
(1S,3R,4R,7S)-3-(4-N-Ben zoylcyt osin -1-yl)-7-h yd r oxy-
1-h yd r oxym eth yl-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (6D). To
a suspension of compound 6C (3.85 g, 14.4 mmol) in anhyd
pyridine (50 mL) was added Bz2O (6.7 g, 29.6 mmol). The
mixture was stirred overnight, diluted with EtOH (50 mL),
and 2 M NaOH (75 mL) was added. After stirring for 1 h,
AcOH (10 mL) was added to the solution, and the solvents were
removed under reduced pressure. The residue was crystallized
from H2O to give 6D (4.88 g, 93%) as a white solid material.
mp 221-223 (dec). 1H NMR (DMSO-d6): δ 11.26 (br s, 1H),
8.25 (d, J ) 7.5 Hz, 1H), 7.99 (m, 2H), 7.65-7.50 (m, 3H), 7.42
(d, J ) 7.5 Hz, 1H), 5.68 (d, J ) 4.2 Hz, 1H), 5.53 (s, 1H), 5.17
(t, J ) 5.7 Hz, 1H), 4.24 (s, 1H), 3.89-3.79 (m, 4H), 3.71 (d, J
) 7.7 Hz, 1H). 13C NMR (DMSO-d6): δ 167.7, 163.6, 154.4,
144.6, 133.5, 133.1, 128.8, 96.0, 89.3, 87.5, 78.7, 71.2, 68.5, 56.3.
MALDI-MS m/z: 360.5 [M + H]+. Anal. Calcd for C17H17N3O6‚
1/6 H2O: C, 56.35; H, 4.82; N, 11.60. Found: C, 56.37; H, 4.68;
N, 11.62.
8.48 (s, 1H), 8.06 (m, 2H), 7.94 (m, 2H), 7.66 (m, 2H), 7.54 (m,
4H), 7.36-7.26 (m, 5H), 6.11 (s, 1H), 4.97 (s, 1H), 4.82 (s, 2H),
4.77 (s, 1H), 4.75 (d, J ) 12.4 Hz, 1H), 4.69 (d, J ) 11.9 Hz,
1H), 4.19 (d, J ) 8.0 Hz, 1H), 4.07 (d, J ) 7.9 Hz, 1H). 13C
NMR (DMSO-d6): δ 165.6, 165.3, 151.8, 151.6, 150.5, 141.9,
137.7, 133.7, 133.6, 132.5, 129.3, 129.1, 128.9, 128.6, 128.5,
128.3, 127.7, 127.6, 125.7, 85.9, 85.3, 77.9, 77.1, 72.0, 71.3, 60.6.
MALDI-MS m/z: 600.7 [M + Na]+. Anal. Calcd for C32H27N5O6‚
H2O: C, 64.53; H, 4.91; N, 11.76. Found: C, 64.77; H, 5.01; N,
11.85.
(1S,3R,4R,7S)-3-(Ad en in -9-yl)-7-ben zyloxy-1-h yd r oxy-
m eth yl-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (12F ). Compound
11E (15.5 g, 26.8 mmol) was suspended in a mixture of MeOH
(150 mL) and concd NH4OH (200 mL). The solution was stirred
for 2 days at room temperature, whereafter MeNH2 (40%, 20
mL) was added, and the mixture was stirred overnight. The
precipitate was filtered off, dried in vacuo, and crystallized
from EtOH to give 12F (8.55 g, 86%) as a white solid material.

2′-O, 4′-C-Methylene-Linked Ribonucleosides
J . Org. Chem., Vol. 66, No. 25, 2001 8511
mp 218-219.5 °C. 1H NMR (DMSO-d6): δ 8.18 (s, 1H), 8.14
(s, 1H), 7.33-7.30 (m, 5H), 5.97 (s, 1H), 5.17 (t, J ) 5.8 Hz,
1H), 4.73 (s, 1H), 4.63 (s, 2H), 4.35 (s, 1H), 3.95 (d, J ) 7.9
Hz, 1H), 3.84-3.81 (m, 3H). 13C NMR (DMSO-d6): δ 156.1,
152.8, 148.6, 138.0, 137.9, 128.3, 127.7, 127.6, 119.1, 88.0, 85.4,
77.3, 77.0, 72.1, 71.3, 56.8. MALDI-MS m/z: 370.7 [M + H]+.
Anal. Calcd for C18H19N5O4‚1/3 H2O: C, 57.59; H, 5.28; N, 18.66.
Found: C, 57.58; H, 5.40; N, 18.43.
(1S,3R,4R,7S)-3-(Ad en in -9-yl)-7-h yd r oxy-1-h yd r oxy-
m eth yl-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (6F ). To a suspen-
sion of compound 12F (3.69 g, 10.0 mmol) in EtOH (50 mL)
were added 20% Pd(OH)2/C (1 g) and HCO2NH4 (3.2 g, 50.4
mmol). The solution was refluxed for 3 h, and more HCO2NH4
(1 g, 15.9 mmol) was added. After 2 h, the hot solution was
filtrated through a Celite pad, and Celite was washed with
boiling EtOH/H2O (200 mL). Evaporation of the combined
filtrates gave 6F (2.54 g, 91%) as white crystals. mp 266-268
°C (dec). 1H NMR (DMSO-d6): δ 8.22 (s, 1H), 8.15 (s, 1H), 7.30
(br s, 2H), 5.89 (s, 1H), 5.68 (d, J ) 4.2 Hz, 1H), 5.05 (t, J )
5.8 Hz, 1H), 4.41 (s, 1H), 4.25 (d, J ) 3.7 Hz, 1H), 3.92 (d, J
) 7.8 Hz, 1H), 3.82 (m, 2H), 3.76 (d, J ) 7.9, 2H). 13C NMR
(DMSO-d6): δ 156.1, 152.8, 148.5, 137.9, 119.1, 88.6, 85.4, 79.3,
71.5, 70.0, 56.8. MALDI-MS m/z: 280.6 [M + H]+ Anal. Calcd
for C11H13N5O4‚1/8 H2O: C, 46.93; H, 4.74; N, 24.88. Found:
C, 47.04; H, 4.56; N, 24.74.
dropwise, and the mixture was stirred for 20 h at room
temperature. The mixture was diluted with EtOAc (200 mL),
washed with saturated NaHCO3 (3 × 200 mL), dried (Na2SO4),
and concentrated under reduced pressure. The oily residue was
dissolved in pyridine/EtOH (1:1 v/v, 200 mL), and 2 M NaOH
(50 mL) was added. After 15 min, AcOH (9 mL) was added,
and the solvents were removed under reduced pressure. The
residue was suspended in 20% EtOH/CH2Cl2 and filtrated
through a short silica gel column. The filtrate was concentrated
to a yellow solid residue and purified by silica gel HPLC (5-
15% v/v MeOH/CH2Cl2) to give 6E (3.75 g, 75%) as a white
solid material. 1H NMR (CD3OD): δ 8.73 (s, 1H), 8.57 (s, 1H),
8.11 (m, 2H), 7.69 (m, 1H), 7.59 (m, 2H), 6.16 (s, 1H), 4.67 (s,
1H), 4.42 (s, 1H), 4.12 (d, J ) 8.0 Hz, 1H), 4.01 (s, 2H), 3.95
(d, J ) 7.9 Hz, 1H). Dichloromethane (s, 5.53 ppm) was
detected as an impurity. 13C NMR (CD3OD): δ 168.1, 153.3,
152.4, 151.1, 143.0, 134.9, 134.0, 129.8, 129.5, 125.3, 90.4, 87.8,
81.0, 73.0, 71.6, 58.3. MALDI-MS m/z: 406.5 [M + Na]+. Anal.
Calcd for C18H17N5O5‚7/24 CH2Cl2: C, 53.83; H, 4.34; N, 17.16.
Found: C, 53.81; H, 4.39; N, 17.04.
9-(2-O-Acet yl-3-O-b en zyl-4-C-m et h a n esu lfoxym et h yl-
5-O-m et h a n esu lfon yl-â-D-er yth r o-p en t ofu r a n osyl)-2-N-
isobu tyr ylgu a n in e (9G). To a suspension of 8 (18.5 g, 36.3
mmol) and 2-N-isobutyrylguanine (9.15 g, 41.4 mmol) in anhyd
1,2-dichloroethane (150 mL) was added BSA (30 mL, 122
mmol). The mixture was refluxed for 1.5 h, and TMSOTf (13.5
mL, 74.3 mmol) was added. After refluxing further for 2 h,
the reaction mixture was cooled to room temperature, diluted
with CH2Cl2 (200 mL), washed with saturated NaHCO3 (2 ×
200 mL), dried (Na2SO4), and concentrated under reduced
pressure. The residue was purified by silica gel column
chromatography (1-2% v/v MeOH/CH2Cl2) to give a mixture
of two isomers (20.6 g, 84%) as a white solid material (ratio
1:10 by 1H NMR). The mixture was used in the next step
without separation of isomers. For the main isomer assigned
(1S,3R,4R,7S)-3-(Ad en in -9-yl)-7-ben zyloxy-1-m eth a n e-
su lfon oxym eth yl-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (10F ).
To a solution of 9E (2.5 g, 3.6 mmol) in 1,4-dioxane (20 mL)
was added concd NH4OH (20 mL). The solution was stirred at
room temperature overnight and diluted with 2 M NaOH (5
mL). After 30 min, the solvents were removed under reduced
pressure, and the residue was resuspended in CH2Cl2 (100
mL), washed with saturated NaHCO3 (100 mL), dried (Na2SO4),
and concentrated under reduced pressure. Purification by silica
gel column chromatography (2-5% v/v MeOH/CH2Cl2) gave
10F (1.26 g, 78%) as a white solid material. 1H NMR (CDCl3):
δ 8.30 (s, 1H), 7.90 (s, 1H), 7.31-7.27 (m, 5H), 6.04 (s, 1H),
4.93 (s, 1H), 4.68 (d, J ) 11.7 Hz, 1H), 4.60 (d, J ) 11.7, 1H),
4.59 (d, J ) 11.7 Hz, 1H), 4.57 (d, J ) 11.9 Hz, 1H), 4.35 (s,
1H), 4.19 (d, J ) 7.9 Hz, 1H), 4.02 (d, J ) 7.9 Hz, 1H), 3.03 (s,
3H). 13C NMR (CDCl3): δ 155.4, 152.9, 148.6, 138.0, 136.4,
128.4, 128.2, 127.8, 119.7, 86.6, 85.1, 77.5, 76.8, 72.4, 72.2, 64.4,
37.7. MALDI-MS m/z: 470.4 [M + Na]+. Anal. Calcd for
1
as 9G, H NMR (CDCl3): δ 12.22 (br s, 1H), 9.34 (br s, 1H),
7.76 (s, 1H), 7.40-7.30 (m, 5H), 6.03 (d, J ) 3.9 Hz, 1H), 5.76
(dd, J ) 6.0 and 3.9 Hz, 1H), 5.08 (d, J ) 6.0 Hz, 1H), 4.91 (d,
J ) 10.5 Hz, 1H), 4.67 (d, J ) 10.9), 4.61 (d, J ) 11.1 Hz, 2H),
4.49 (d, J ) 10.5 Hz, 1H), 4.39 (d, J ) 11.0 Hz, 1H), 4.32 (d,
J ) 11.7 Hz, 1H), 3.14 (s, 3H), 3.02 (s, 3H), 2.70 (m, 1H), 2.09
(s, 3H) 1.24 (m, 6H). 13C NMR (CDCl3): δ 179.3, 169.7, 154.7,
148.2, 147.1, 138.8, 136.5, 128.5, 128.3, 128.1, 121.5, 88.7, 84.4,
78.1, 74.7, 74.2, 67.9, 67.3, 37.6, 37.5, 36.1, 20.5, 18.9, 18.8.
MALDI-MS m/z: 694.4 [M + Na]+. Anal. Calcd for C26H33N5-
C
19H21N5O6S‚1/8 H2O: C, 50.75; H, 4.76; N, 15.57. Found: C,
50.64; H, 4.51; N, 15.52.
(1S,3R,4R,7S)-3-(Ad en in -9-yl)-7-b en zyloxy-1-b en zoyl-
oxym eth yl-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (11F ). NaOBz
(5.36 g, 37.2 mmol) was added to a solution of compound 10F
(8.3 g, 18.6 mmol) in anhyd DMF (250 mL). The mixture was
stirred at 100 °C for 4 h, cooled to room temperature, filtrated,
and concentrated under reduced pressure. The residue was
suspended in EtOAc (200 mL), washed with saturated NaH-
CO3 (3 × 200 mL), dried (Na2SO4), and concentrated under
reduced pressure. Crystallization from EtOH gave 11F (7.4
g, 84%) as a white solid material. mp 169-171 °C. 1H NMR
(CDCl3): δ 8.31 (s, 1H), 7.96 (m, 2H), 7.84 (s, 1H), 7.57 (m,
1H), 7.43 (m, 2H), 7.28-7.21 (m, 5H), 6.23 (br s, 2H), 6.05 (s,
1H), 4.96 (s, 1H), 4.81 (d, J ) 12.6 Hz, 1H), 4.70 (d, J ) 11.8
Hz, 1H), 4.65 (d, J ) 12.6 Hz, 1H), 4.55 (d, J ) 11.9 Hz, 1H),
4.35 (s, 1H), 4.28 (d, J ) 7.9 Hz, 1H), 4.10 (d, J ) 7.9 Hz, 1H).
13C NMR (CDCl3): δ 165.7, 155.5, 153.0, 148.6, 137.6, 136.5,
133.3, 129.4, 129.1, 128.4, 128.3, 128.0, 127.7, 119.9, 86.7, 85.6,
77.4, 76.8, 72.5, 72.2, 59.8. MALDI-MS m/z: 496.6 [M + Na]+.
Anal. Calcd for C25H23N5O5: C, 63.42; H, 4.90; N, 14.79.
Found: C, 63.21; H, 4.93; N, 14.84.
(1S,3R,4R,7S)-3-(6-N-Ben zoyla d en in -9-yl)-7-h yd r oxy-1-
h yd r oxym eth yl-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (6E). A
mixture of compound 11F (5.8 g, 12.2 mmol) and 20%
Pd(OH)2/C (3 g) was suspended in MeOH/1,4-dioxane (9:1, 150
mL), and HCO2NH4 (8 g, 126.9 mmol) was added. The mixture
was refluxed overnight, cooled to room temperature, and
filtrated through a Celite pad. The filtrate was concentrated
under reduced pressure, coevaporated with anhyd pyridine (2
× 50 mL), dissolved in anhyd pyridine (100 mL), and cooled
in an ice bath. Benzoyl chloride (7.1 mL, 61.5 mmol) was added
O
12S2‚1/3 H2O: C, 46.08; H, 5.01; N, 10.33. Found: C, 46.10;
H, 4.98; N, 10.27.
(1S,3R,4R,7S)-7-Ben zyloxy-1-m eth an esu lfon oxym eth yl-
3-(2-N -isob u t yr ylgu a n in -9-yl)-2,5-d ioxa b icyclo[2.2.1]-
h ep ta n e (10G). To a solution of compound 9G (10.2 g, 15.1
mmol) in THF (100 mL) was added 1 M NaOH (100 mL), and
the mixture was stirred at 0 °C for 1 h. Acetic acid (6 mL) was
added, and the solution was concentrated to ca. 1/2 of its volume
under reduced pressure. The formed precipitate was filtered
off, washed with water, and dried in vacuo. Purification by
silica gel column chromatography (0.8-3.5% v/v MeOH/
CH2Cl2) gave 10G (6.9 g, 85%) as a white solid material. 1H
NMR (CDCl3): δ 12.14 (br s, 1H), 9.51 (br s, 1H), 7.77 (s, 1H),
7.30-7.26 (m, 5H), 5.84 (s, 1H), 4.67 (d, J ) 11.5 Hz, 1H),
4.63 (d, J ) 12.0 Hz, 1H), 4.62 (s, 1H), 4.62 (d, J ) 11.5 Hz,
1H), 4.56 (d, J ) 11.9 Hz, 1H), 4.50 (s, 1H), 4.12 (d, J ) 8.0,
1H), 3.93 (d, J ) 7.9 Hz, 1H), 3.06 (s, 3H), 2.78 (m, 1H), 1.26
(m, 6H). 13C NMR (CDCl3): δ 179.3, 155.3, 147.7, 147.3, 136.7,
136.3, 128.3, 128.0, 127.7, 121.2, 86.8, 85.3, 77.5, 76.9, 72.3,
72.0, 64.6, 37.5, 36.0, 18.9, 18.8. MALDI-MS m/z: 556.8 [M +
Na]+. Anal. Calcd for C23H27N5O8S‚1/3 H2O: C, 51.20; H, 5.17;
N, 13.00. Found: C, 51.02; H, 5.09; N, 13.08.
(1S,3R,4R,7S)-7-Ben zyloxy-1-ben zoyloxym eth yl-3-(2-N-
isob u t yr ylgu a n in -9-yl)-2,5-d ioxa b icyclo[2.2.1]h ep t a n e
(11G). A mixture of compound 10G (12.5 g, 23.4 mmol) and
NaOBz (6.8 g, 47.2 mmol) was suspended in anhyd DMF (25
mL) and was stirred for 2 h at 100 °C and overnight at 80 °C.
The mixture was cooled to room temperature, filtrated, and
concentrated under reduced pressure. The residue was redis-
solved in EtOAc (200 mL) and washed with saturated NaHCO3

8512 J . Org. Chem., Vol. 66, No. 25, 2001
Koshkin et al.
(2 × 200 mL) and H2O (200 mL). The organic phase was dried
(Na2SO4) and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography
(0.9-3.5% v/v MeOH/CH2Cl2) to give 11G (12.1 g, 92%) as a
white solid material. 1H NMR (CDCl3): δ 12.12 (br s, 1H), 9.30
(br s, 1H), 7.92 (m, 2H), 7.72 (s, 1H), 7.57 (m, 1H), 7.42 (m,
2H), 7.24-7.20 (m, 5H), 5.81 (s, 1H), 4.80 (d, J ) 12.6 Hz,
1H), 4.66 (s, 1H), 4.64 (d, J ) 12.0 Hz, 1H), 4.61 (d, J ) 12.7
Hz, 1H), 4.21 (d, J ) 8.1 Hz, 1H), 4.20 (s, 1H), 4.00 (d, J ) 7.9
Hz, 1H), 2.77 (m, 1H), 1.27 (m, 6H). 13C NMR (CDCl3): δ 179.3,
165.6, 155.2, 148.0, 147.1, 136.4, 135.4, 133.3, 129.3, 128.9,
128.4, 128.3, 127.9, 127.5, 120.9, 86.2, 85.5, 76.9, 72.3, 72.1,
59.4, 35.9, 18.8. MALDI-MS m/z: 582.5 [M + Na]+. Anal. Calcd
for C29H29N5O7‚1/4 H2O: C, 61.75; H, 5.27; N, 12.41. Found:
C, 61.80; H, 5.33; N, 12.31.
Anal. Calcd for C22H25N5O6: C, 58.02; H, 5.53; N, 15.38.
Found: C, 57.91; H, 5.35; N, 15.24.
(1S,3R,4R,7S)-7-Hyd r oxy-1-h yd r oxym eth yl-3-(2-N-iso-
bu tyr ylgu a n in -9-yl)-2,5-d ioxa bicyclo[2.2.1]h ep ta n e (6G).
To a solution of compound 12G (5.8 g, 12.7 mmol) in MeOH
(50 mL) were added 10% Pd/C (2 g) and HCO2H (3 mL, 79.5
mmol). The mixture was refluxed for 5 h, cooled to room
temperature, and filtrated through a Celite pad. The filtrate
was concentrated under reduced pressure to afford a glasslike
residue. Crystallization from H2O gave 6G (3.9 g, 80%) as a
white solid material. mp 196-199 °C. 1H NMR (DMSO-d6): δ
7.80 (s, 1H), 5.51 (s, 1H), 5.44 (br s, 1H), 4.77 (br s, 1H), 4.12
(s, 1H), 3.88 (s, 1H), 3.65 (d, J ) 7.7 Hz, 1H), 3.53 (m, 2H),
3.47 (d, J ) 7.7 Hz, 1H), 2.50 (m, 1H), 0.84 (m, 6H). 13C NMR
(DMSO-d6): δ 180.3, 154.8, 148.3, 147.6, 136.4, 120.6, 88.8,
85.3, 79.3, 71.6, 69.8, 56.7, 34.8, 19.0, 18.9. MALDI-MS m/z:
388.4 [M + Na]+. Anal. Calcd for C15H19N5O6‚H2O: C, 47.00;
H, 5.52; N, 18.27. Found: C, 46.90; H, 5.30; N, 18.37.
(1S,3R,4R,7S)-7-Ben zyloxy-1-h yd r oxym et h yl-3-(2-N-
isob u t yr ylgu a n in -9-yl)-2,5-d ioxa b icyclo[2.2.1]h ep t a n e
(12G). To a solution of compound 11G (8.2 g, 14.5 mmol) in
EtOH/pyridine (8:1 v/v, 450 mL) was added 2 M NaOH (15.5
mL), and the mixture was stirred for 30 min at room temper-
ature. AcOH (25 mL) was added, and the solvents were
removed under reduced pressure. The residue was crystallized
from H2O/EtOH (1:1 v/v) to give 12G (5.8 g, 88%) as a white
Ack n ow led gm en t. Fruitful discussions with Prof.
J. Wengel and Dr. M. Meldgaard are gratefully acknowl-
edged.
1
solid material. mp 231-233 °C. H NMR (DMSO-d6): δ 8.05
(s, 1H), 7.33-7.26 (m, 5H), 5.85 (s, 1H), 5.17 (t, J ) 5.4 Hz,
1H), 4.69 (s, 1H), 4.64 (s, 2H), 4.23 (s, 1H), 3.95 (d, J ) 7.9
Hz, 1H), 3.83 (m, 2H), 3.80 (d, J ) 8.0 Hz, 1H), 2.78 (m, 1H),
1.12 (m, 6H). 13C NMR (DMSO-d6): δ 180.2, 154.8, 148.2,
147.7, 137.9, 136.3, 128.3, 127.6, 127.5, 120.5, 88.2, 85.2, 76.9,
72.1, 71.3, 56.7, 34.8, 18.9. MALDI-MS m/z: 478.5 [M + Na]+.
Su p p or tin g In for m a tion Ava ila ble: Copies of 13C NMR
spectra for all reported compounds and copies of 1H NMR
spectra for 8, 6E, and 9E. This material is available free of
charge via Internet at http://pubs.acs.org.
J O010732P