Synthesis and biophysical evaluation of 2′,4′-Constrained 2′O-Methoxyethyl and 2′,4′-Constrained 2′O-Ethyl nucleic acid analogues

Article abstract of DOI:10.1021/jo902560f

"Chemical Equation Presented" We have recently shown that combining the structural elements of 2′O-methoxyethyl (MOE) and locked nucleic acid (LNA) nucleosides yielded a series of nucleoside modifications (cMOE, 2′,4′-constrained MOE; cEt, 2′,4′-constrain

Full text of DOI:10.1021/jo902560f

pubs.acs.org/joc
Synthesis and Biophysical Evaluation of 20,40-Constrained
20O-Methoxyethyl and 20,40-Constrained 20O-Ethyl Nucleic Acid
Analogues
Punit P. Seth,*,† Guillermo Vasquez,† Charles A. Allerson,† Andres Berdeja,‡
Hans Gaus,‡ Garth A. Kinberger,† Thazha P. Prakash,† Michael T. Migawa,†
Balkrishen Bhat,§ and Eric E. Swayze†
†Department of Medicinal Chemistry and ‡Department of Structural Biology, Isis Pharmaceuticals,
1896 Rutherford Road, Carlsbad, California 92008. §Current address: Regulus Therapeutics,
1896 Rutherford Road, Carlsbad, CA 92008.
pseth@isisph.com
Received December 2, 2009
We have recently shown that combining the structural elements of 20O-methoxyethyl (MOE)
and locked nucleic acid (LNA) nucleosides yielded a series of nucleoside modifications (cMOE,
20,40-constrained MOE; cEt, 20,40-constrained ethyl) that display improved potency over MOE and an
improved therapeutic index relative to that of LNA antisense oligonucleotides. In this report we
present details regarding the synthesis of the cMOE and cEt nucleoside phosphoramidites and the
biophysical evaluation of oligonucleotides containing these nucleoside modifications. The synthesis of
the cMOE and cEt nucleoside phosphoramidites was efficiently accomplished starting from inexpen-
sive commercially available diacetone allofuranose. The synthesis features the use of a seldom used
2-naphthylmethyl protecting group that provides crystalline intermediates during the synthesis and can
be cleanly deprotected under mild conditions. The synthesis was greatly facilitated by the crystallinity
of a key mono-TBDPS-protected diol intermediate. In the case of the cEt nucleosides, the introduction
of the methyl group in either configuration was accomplished in a stereoselective manner. Ring closure
of the 20-hydroxyl group onto a secondary mesylate leaving group with clean inversion of stereochem-
istry was achieved under surprisingly mild conditions. For the S-cEt modification, the synthesis of all
four (thymine, 5-methylcytosine, adenine, and guanine) nucleobase-modified phosphoramidites was
accomplished on a multigram scale. Biophysical evaluation of the cMOE- and cEt-containing
oligonucleotides revealed that they possess hybridization and mismatch discrimination attributes
similar to those of LNA but greatly improved resistance to exonuclease digestion.
Introduction
generation antisense oligonucleotides (ASOs), which typi-
cally have a central DNA region of 8-14 nucleotides,
flanked on the 50 and 30 ends with five to two 20-O-methoxy-
ethyl (MOE, 2) residues.2 The above “gapmer” design
Antisense drug discovery technology represents a power-
ful method to modulate gene expression in animals.1 The
most advanced oligonucleotides in the clinic are second
(2) Monia, B. P.; Lesnik, E. A.; Gonzalez, C.; Lima, W. F.; McGee, D.;
Guinosso, C. J.; Kawasaki, A. M.; Cook, P. D.; Freier, S. M. J. Biol. Chem.
1993, 268, 14514–14522.
(1) Antisense Drug Technology: Principles, Strategies, and Applications,
2nd ed.; Crooke, S. T., Ed.; CRC Press: Boca Raton, 2007.
DOI: 10.1021/jo902560f
r
Published on Web 02/08/2010
J. Org. Chem. 2010, 75, 1569–1581 1569
2010 American Chemical Society

Seth et al.
JOCArticle
FIGURE 2. Structures of LNA and other related conformationally
restricted 20-40 bridged nucleic acids (BNA) reported in theliterature.
FIGURE 1. Design of constrained methoxyethyl (R-cMOE 5 and
S-cMOE 6) and constrained ethyl (R-cEt 7 and S-cEt 8) nucleic acids.
the LNA scaffold will provide additional modifications that
have a superior biological profile as compared to that of
LNA. As such, since the first report of LNA by Imanishi7-9
and by Wengel,10-14 a number of analogues have been
reported in the literature (Figure 2). Some of the analogues
contain subtle structural changes such as replacing the 20-
oxygen atom with nitrogen or sulfur to provide 20-thio LNA
1015 and 20-amino LNA 9,16,17 respectively. Other ana-
logues differ based on the size and composition of the
20-40 bridging substituent. Examples of such analogues
include ENA 11,18 aza ENA 12,19 carbo ENA 13,20,21 14,22
PrNA 15,18 BNACOC 1623 and BNANC 17.24,25 Recently,
supports RNase H mediated degradation of target mRNA
due to the central DNA region. At the same time, the
flanking MOE residues increase hybridization to comple-
mentary mRNA and further stabilize the oligonucleotide
toward enzymatic degradation. There are currently almost
20 second generation ASOs in human clinical trials for a
variety of disease indications including hypercholesteremia,
diabetes, and cancer.
We have shown that replacing MOE nucleosides in second
generation ASOs with locked nucleic acid (LNA) 4 increases
the potency of some ASOs in animals. However, this was
sometimes accompanied with an increased risk for hepato-
toxicity.3 We hypothesized that replacing LNA with novel
nucleoside monomers that combine the structural elements
of MOE and LNA might mitigate the toxicity of LNA while
maintaining potency. To this end we designed and prepared
nucleoside analogues 5 (R-constrained MOE, R-cMOE) and
6 (S-constrained MOE, S-cMOE), 7 (R-constrained ethyl, R-
cEt), and 8 (S-constrained ethyl, S-cEt) where the 20O-alkyl
group was constrained back to the 40 position of the furanose
ring (Figure 1).4 The 20-40 constraint enforces a N-type
sugar pucker of the furanose ring and thereby improves
hybridization with complementary RNA.5 Biological eva-
luation of the cMOE and cEt containing ASOs showed that
the S-cEt-modified ASOs displayed activity comparable to
that of LNA with no elevations in liver transaminase levels
after 3 weeks of dosing.6
(7) Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.-I.; In, Y.; Ishida, T.;
Imanishi, T. Tetrahedron Lett. 1997, 38, 8735–8738.
(8) Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J.-I.; Morio, K.-I.; Doi, T.;
Imanishi, T. Tetrahedron Lett. 1998, 39, 5401–5404.
(9) Imanishi, T.; Obika, S. J. Synth. Org. Chem. Jpn. 1999, 57, 969–980.
(10) Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J. Chem. Commun.
1998, 455–456.
(11) Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K.; Kumar,
R.; Meldgaard, M.; Olsen, C. E.; Wengel, J. Tetrahedron 1998, 54, 3607–
3630.
(12) Singh, S. K.; Wengel, J. Chem. Commun. 1998, 1247–1248.
(13) Koshkin, A. A.; Rajwanshi, V. K.; Wengel, J. Tetrahedron Lett.
1998, 39, 4381–4384.
(14) Koshkin, A. A.; Nielsen, P.; Meldgaard, M.; Rajwanshi, V. K.;
Singh, S. K.; Wengel, J. J. Am. Chem. Soc. 1998, 120, 13252–13253.
(15) Kumar, R.; Singh, S. K.; Koshkin, A. A.; Rajwanshi, V. K.;
Meldgaard, M.; Wengel, J. Bioorg. Med. Chem. Lett. 1998, 8, 2219–2222.
(16) Singh, S. K.; Kumar, R.; Wengel, J. J. Org. Chem. 1998, 63, 6078–
6079.
(17) Singh, S. K.; Kumar, R.; Wengel, J. J. Org. Chem. 1998, 63, 10035–
10039.
(18) Morita, K.; Takagi, M.; Hasegawa, C.; Kaneko, M.; Tsutsumi, S.;
Sone, J.; Ishikawa, T.; Imanishi, T.; Koizumi, M. Bioorg. Med. Chem. 2003,
11, 2211–2226.
(19) Varghese, O. P.; Barman, J.; Pathmasiri, W.; Plashkevych, O.;
Honcharenko, D.; Chattopadhyaya, J. J. Am. Chem. Soc. 2006, 128,
15173–87.
(20) Albk, N.; Petersen, M.; Nielsen, P. J. Org. Chem. 2006, 71, 7731–
7740.
Thus small structural changes on the LNA scaffold appear
to have a significant impact on the biological profile of
oligonucleotides containing these modifications. It is con-
ceivable that further structure activity relationship studies on
(3) Swayze, E. E.; Siwkowski, A. M.; Wancewicz, E. V.; Migawa, M. T.;
Wyrzykiewicz, T. K.; Hung, G.; Monia, B. P.; Bennett, C. F. Nucleic Acids
Res. 2007, 35, 687–700.
(4) Seth, P. P.; Siwkowski, A.; Allerson, C. R.; Vasquez, G.; Lee, S.;
Prakash, T. P.; Kinberger, G.; Migawa, M. T.; Gaus, H.; Bhat, B.; Swayze,
E. E. Nucleic Acids Symp. Ser. 2008, 553–554.
(21) Kumar, S.; Hansen, M. H.; Albk, N.; Steffansen, S. I.; Petersen, M.;
Nielsen, P. J. Org. Chem. 2009, 74, 6756–6769.
(22) Wang, G.; Gunic, E.; Girardet, J.-L.; Stoisavljevic, V. Bioorg. Med.
Chem. Lett. 1999, 9, 1147–1150.
(23) Hari, Y.; Obika, S.; Ohnishi, R.; Eguchi, K.; Osaki, T.; Ohishi, H.;
Imanishi, T. Bioorg. Med. Chem. 2006, 14, 1029–1038.
(24) Miyashita, K.; Rahman, S. M.; Seki, S.; Obika, S.; Imanishi, T.
Chem. Commun. 2007, 3765–3767.
(25) AbdurRahman, S. M.; Seki, S.; Obika, S.; Yoshikawa, H.;
Miyashita, K.; Imanishi, T. J. Am. Chem. Soc. 2008, 130, 4886–4896.
(5) Wengel, J. Acc. Chem. Res. 1999, 32, 301–310.
(6) Seth, P. P.; Siwkowski, A.; Allerson, C. R.; Vasquez, G.; Lee, S.;
Prakash, T. P.; Wancewicz, E. V.; Witchell, D.; Swayze, E. E. J. Med. Chem.
2009, 52, 10–13.
1570 J. Org. Chem. Vol. 75, No. 5, 2010

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SCHEME 1. Problems with 30O-Bn Protecting Group Strategy
FIGURE 3. Retrosynthetic analysis for cMOE and cEt nucleic
acids.
Chattopadhyaya reported the synthesis of substituted car-
bocyclic LNA and ENA analogues 18 via an intramolecular
radical cyclization approach,26-28 while Nielsen29 and we30
reported the synthesis of a (R)-6-hydroxymethyl-substituted
LNA analogue 19. With the exception of thio-LNA 10 and
BNANC 17, all other analogues reduce thermal stability for
complementary nucleic acids relative to LNA. A majority of
the analogues reported thus far are also significantly more
difficult to synthesize, especially in quantities required
to carry out meaningful animal experiments, as compared
to LNA.
In this report, we describe the detailed synthesis and
biophysical characterization of the cMOE- and cEt-modified
oligonucleotides. We show that these modifications exhibit
similar thermal stability and mismatch discrimination but
increased nuclease stability as compared to those of LNA.
We also describe a new orthogonal protecting group strategy
that greatly facilitates the synthesis of this class of molecules
and should find general application for the synthesis of other
LNA analogues.
cMOE and cEt nucleoside analogues. Access to diastereo-
merically pure cMOE and cEt nucleoside analogues hinged
upon identifying methods to prepare nucleoside 21 in a
stereodefined manner. We envisaged a strategy where sec-
ondary alcohol 22 could be prepared from aldehyde 23 by the
stereoselective addition of an appropriate organometallic
reagent. The overall topology of the sugar in 23 and the
presence of a neighboring co-ordination site were expected to
provide the appropriate elements of stereocontrol during
addition of the organometallic reagent. Lastly, it was crucial
to identify a robust protecting group strategy that could
differentiate the two primary alcohols in 24 on a multigram
scale.
At first we chose to differentiate the two primary hydro-
xyls in diol 25 using the benzyl protecting group strategy that
has been utilized for the synthesis of LNA and related
molecules in the literature7,11 (Scheme 1). However, separa-
tion of the desired benzyl intermediate 26 from its regio-
isomers was found to be cumbersome, especially on a large
scale. Nonetheless, we were able to elaborate diol 25 to the
cyclized nucleoside intermediate 27.30 However, removal of
the 30O-benzyl protecting group in 27 proved to be difficult
and irreproducible. Use of Pd/C under mild reaction condi-
tions resulted in very slow and erratic removal of the benzyl
group and often required multiple catalyst changes to drive
the reaction to completion. Use of more forcing conditions
(ammonium formate, palladium hydroxide) resulted in par-
tial hydrogenolysis of the uridine nucleobase, which could
not be easily separated from the desired product 28.32 Use of
boron trichloride resulted in the formation of significant
amounts of the anhydro nucleoside 29.30 Presumably, pre-
sence of the methyl substituent in 27 results in increased ring
strain, which in turn activates the cyclic ether to cleave under
strong Lewis acid conditions. To circumvent the problems
associated with removal of the 30O-benzyl group, we decided
to replace the benzyl group with a 2-naphthylmethyl (abbrev.
naphthyl or Nap) protecting group, which possesses similar
acid stability as a benzyl group but can be cleaved under mild
Results and Discussion
The general strategy used to prepare LNA typically in-
volves a cycloetherification reaction between the 20-hydroxyl
and an appropriate leaving group at the 40 position of the
nucleoside furanose ring system.7,11,13,31 For our purpose,
we decided to implement a similar strategy to prepare the
cMOE and cEt analogues (Figure 3). However, to the best of
our knowledge, there were no reports that described the
stereoselective cyclization of the 20-hydroxyl onto a second-
ary leaving group required to generate the 6-substituted-2,5-
dioxa-bicyclo[2.2.1]heptane ring system present in the
(26) Srivastava, P.; Barman, J.; Pathmasiri, W.; Plashkevych, O.;
Wenska, M.; Chattopadhyaya, J. J. Am. Chem. Soc. 2007, 129, 8362–8379.
(27) Zhou, C.; Liu, Y.; Andaloussi, M.; Badgujar, N.; Plashkevych, O.;
Chattopadhyaya, J. J. Org. Chem. 2009, 74, 118–134.
(28) Xu, J.; Liu, Y.; Dupouy, C.; Chattopadhyaya, J. J. Org. Chem. 2009,
74, 6534–6554.
(29) Enderlin, G.; Nielsen, P. J. Org. Chem. 2008, 73, 6891–6894.
(30) Seth, P. P.; Swayze, E. E. U.S. Patent 7399845, 2008.
(31) Koshkin, A. A.; Fensholdt, J.; Pfundheller, H. M.; Lomholt, C.
J. Org. Chem. 2001, 66, 8504–8512.
(32) Kumar, S. T.; Kumar, P.; Sharma, P. K.; Hrdlicka, P. J. Tetrahedron
Lett. 2008, 49, 7168–7170.
J. Org. Chem. Vol. 75, No. 5, 2010 1571

Seth et al.
SCHEME 3. Introduction of Methoxymethyl and Methyl
Group for Synthesis of cMOE and cEt Nucleoside Analogues
JOCArticle
SCHEME 2. Synthesis of Key Mono-TBDPS Diol 32 from
Diacetone Allofuranose
oxidative conditions using 2,3-dichloro-4,5-dicyano quinone
(DDQ)33 or by hydrogenolysis.34
Synthesis of the cMOE and cEt nucleosides commenced
with protecting the 30-hydroxyl of commercially available
diacetone allofuranose with 2-bromomethyl naphthalene to
provide 30O-naphthyl diacetone allofuranose 30 (Scheme 2).
The use of the naphthyl protecting group yielded immediate
dividends as, unlike 30O-benzyl-protected diacetone allofur-
anose, 30 could be cleanly precipitated from the reaction
mixture in essentially quantitative yield. Selective cleavage of
the 5,6-acetonide with aqueous acetic acid, followed by
oxidative cleavage of the resulting diol with sodium period-
ate and subsequent aldol/Cannizzaro reaction35 provided
diol 31 in excellent yield (66% over 3 steps).
Once again, the naphthyl protecting strategy proved ad-
vantageous as the diol 31 was cleanly isolated from the
reaction mixture by precipitation. The next step in the
synthesis involved selective protection of the diol 31. Ex-
ploratory reactions using tert-butyldimethylsilyl protection
provided a mixture of the mono- and di-TBS-substituted
sugars, which could be separated by chromatography. In
contrast, use of the TBDPS protecting group provided only a
mixture of the two mono-protected sugars 32 (60%) and 33
(20%). No di-TBDPS-protected sugar was formed under the
reaction conditions employed, presumably due to the in-
creased steric bulk of the TBDPS group. This proved to be
extremely beneficial as it permitted using excess TBDPSCl to
drive the reaction to completion. Another unexpected out-
come of the TBDPS protecting strategy was that the major
and desired product 32 was crystalline (33 is an oil that slowly
solidifies upon refrigeration) and could be isolated from the
crude mixture by crystallization.36
Armed with a method that allowed for the preparation
of the differentiated diol 32 on a kilogram scale, we explored
the possibility of introducing the substituent group
(methoxymethyl for cMOE and methyl for cEt) in a stereo-
selective fashion (Scheme 3). Oxidation of the primary
alcohol under Swern conditions37 followed by addition of
the methoxymethyl magnesium bromide38 provided sugars
34a and 34b as an inseparable mixture. It was very important
to control the temperature during the preparation and use of
MeOCH2MgBr. This reagent was found to be unstable at
temperatures above -20 °C, and the reaction to form the
organometallic reagent did not proceed very efficiently at
temperatures below -25 °C. Nonetheless, multigram quan-
tities of alcohols 34a,b (95% from 32) could be prepared
reproducibly by carefully controlling the reaction conditions
for Grignard formation. Even though alcohols 34a,b could
not be separated at this stage we decided to move ahead with
the hope of separating the mixture at a later stage in the
synthesis.
For the cEt analogues, addition of MeMgBr in the pre-
sence of cerium chloride provided a separable mixture of
alcohols 35 and 36 in excellent yield and diastereoselectivity
(35:36 ∼10:1). Since we needed access to both methyl stereo-
isomers, the major alcohol 35 from the Grignard addition
was converted to 36 by oxidation to the corresponding
ketone followed by reduction with lithium borohydride
(36:35 >15:1). The absolute stereochemistry of the alcohols
35 and 36 was not established until after cyclization to the
(33) Xia, J.; Abbas, S. A.; Locke, R. D.; Piskorz, C. F.; Alderfer, J. L.;
Matta, K. L. Tetrahedron Lett. 2000, 41, 169–173.
(34) Gaunt, M. J.; Yu, J.; Spencer, J. B. J. Org. Chem. 1998, 63, 4172–
4173.
(35) Youssefyeh, R. D.; Verheyden, J. P. H.; Moffatt, J. G. J. Org. Chem.
1979, 44, 1301–1309.
(37) Mancuso, A. J.; Huang, S.-L.; Swern, D. J. Org. Chem. 1978, 43,
2480–2482.
(36) Bhat, B.; Xia, J.; Seth, P. P.; Vasquez, G.; Migawa, M. T.; Allerson,
C.; Prakash, T. P.; Swayze, E. E. U.S. Patent 7569686, 2009.
(38) Kim, M.; Grzeszczyk, B.; Zamojski, A. Tetrahedron 2000, 56, 9319–
9337.
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SCHEME 4. Synthesis of R- and S-cMOE and R- and S-cEt Uridine Phosphoramidites
R-cEt 41 and S-cEt 42 nucleosides, respectively, assuming a
SN2 inversion of the secondary leaving group during the ring-
closure reaction. The stereoselectivity observed during addi-
tion of the methyl and the hydride group can be explained by
means of a chelate-controlled transition state where attack of
the nucleophile takes place away from the bulky TBDPS
group. The poor stereoselectivity observed during addition
of methoxymethylmagnesium bromide could be attributed
to the differences in the size, reactivity, and ability to
coordinate metal ions for this reagent.
Mesylation of the secondary alcohol in sugar intermedi-
ates 34a,b, 35, and 36 followed by acetolysis of the 1,2-iso-
propylidene group provided diacetates 37a,b, 38, and 39, res-
pectively, as an anomeric mixture in good yields (Scheme 4).
Installation of the nucleobase in a stereoselective fashion
was accomplished by means of a modified Vorbruggen
condensation reaction with persilylated uracil.39-41 The
nucleoside mixture was not purified at this stage but treated
with potassium carbonate in methanol to deprotect the 20O-
acetyl group. To our delight, these conditions resulted in
removal of the 20O-acetyl protecting group as well as cycliza-
tion onto the secondary mesylate to provide the cyclized
nucleosides 40a,b, 41, and 42, respectively, in excellent yields.
Treatment of the mixture of R- and S-cMOE nucleosides
40a,b with DDQ resulted in clean removal of the 30O-Nap
protecting group to provide nucleosides 43a and 43b as a
separable mixture. The more polar compound was identified
to be the R-cMOE nucleoside 43a, while the less polar spot
was the S-cMOE nucleoside 43b. Presumably, the methoxy-
methyl group in the S-cMOE nucleoside 43b is capable of
FIGURE 4. Structural elucidation of R-cMOE 43a, S-cMOE 43b,
R-cEt 44, and S-cEt 45.
forming an intramolecular H-bond with the liberated 30-
hydroxyl group, causing this nucleoside to be less polar than
the R-cMOE isomer 43a (Figure 4). Deprotection of the 30O-
Nap group in nucleosides 41 and 42 proceeded smoothly to
yield nucleosides 44 and 45, respectively, in high yield.
Removal of the 50O-TBDPS protecting group in nucleo-
sides 43a, 43b, 44, and 45 using buffered triethylamine
trihydrofluoride42 provided nucleosides 46-49, respectively.
Further protection of the 50-hydroxyl group with dimethox-
ytrityl chloride in pyridine provided nucleosides 50-53 in
excellent yields (90-99%). A phosphitylation reaction43,44
(42) Bhat, B.; Swayze, E. E.; Wheeler, P.; Dimock, S.; Perbost, M.;
Sanghvi, Y. S. J. Org. Chem. 1996, 61, 8186–8199.
€
(39) Vorbruggen, H.; Krolikiewicz, K.; Barbel, B. Chem. Ber. 1981, 114,
1234–1255.
(40) Vorbruggen, H.; Barbel, B. Chem. Ber. 1981, 114, 1279–1286.
(43) Sinha, N. D.; Biernat, J.; McManus, J.; Koster, H. Nucleic Acids Res.
1984, 12, 4539–4557.
(44) Prakash, T. P.; Johnston, J. F.; Graham, M. J.; Condon, T. P.;
Manoharan, M. Nucleic Acids Res. 2004, 32, 828–33.
€
€
(41) Vorbruggen, H.; Holfe, G. Chem. Ber. 1981, 114, 1256–1268.
J. Org. Chem. Vol. 75, No. 5, 2010 1573

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SCHEME 5. Synthesis of Guanine-, Adenine-, and Thymine-
Modified S-cEt Nucleosides
SCHEME 6. Synthesis of Guanine-, Adenine-, and Thymine-
Modified S-cEt Phosphoramidites
(Scheme 5).31 A Vorbruggen reaction of silylated 6-chloro-
2-aminopurine with diacetate 39 in refluxing dichloroethane
provided the nucleoside, which was not purified but treated
with the sodium salt of 3-hydroxypropionitrile (20O-acetate
deprotection, ring closure, SNAr displacement of chloro
followed by β elimination of acrylonitrile) to provide the
cyclized nucleoside 58 in good yield (82% over 2 steps).
Protection of the exocyclic amino group as the isobutyryl
amide provided nucleoside 59 (71%). Similarly, a Vorbrug-
gen reaction of silylated N-benzoyl adenine with diacetate
39 provided the nucleoside, which was not purified but
directly subjected to the cyclization conditions (K2CO3,
MeOH) to provide the cyclized product 60 in good yield
(73% from 48). Because of partial deprotection of the N-
benzoyl group during the cyclization, the reaction was
allowed to continue until the benzoyl group was completely
deprotected. The cyclized product 60 was then isolated by
crystallization or by silica gel chromatography and reacted
with benzoyl chloride to provide the N-benzoyl nucleoside
61 (79%). Synthesis of the thymine-modified S-cEt nucleo-
side 62 was accomplished using procedures analogous for
the preparation of the S-cEt uridine nucleoside 42 (91%
over 2 steps).
The glycosylation reaction of activated anomeric sugars
using silylated purine heterocycles is known to give mixtures
of nucleosides arising from reaction at N7 and N9 positions
on the purine heterocycle. Typically, the regioisomers equili-
brate to the more stable N9 isomer after prolonged heating.
Interestingly, during the glycosylation reaction of acetate 39
with 6-chloro-2-aminopurine and N-benzoyl adenine, only a
single major N9 isomer was detected after 2 h of reflux in
dichloroethane (by LCMS and TLC analysis).
Conversion of nucleosides 59, 61, and 62 to the corre-
sponding phosphoramidites 72, 73, and 74 was carried out as
shown in Scheme 6. Removal of the 30O-naphthyl group
using DDQ followed by removal of the 50O-TBDPS protect-
ing group using triethylamine trihydrofluoride yielded nu-
cleosides 66, 67, and 68, respectively, in excellent yield.
of the 50O-DMT-protected nucleosides provided phosphor-
amidites 54-57, respectively, in excellent yield after chro-
matography (83-96%).
The stereochemistry of the methoxymethyl group in the
cMOE nucleosides 43a and 43b and of the methyl group in
the cEt nucleosides 44 and 45 was elucidated by NOESY
spectroscopy. For all of the nucleosides, the H10, H20, and
H30 proton resonances appear as singlets (J ≈ 0 Hz), which is
consistent with the sugar pucker being locked in the C30-endo
conformation.7 The stereochemistry of the methoxymethyl
groups in 43a and 43b was further confirmed by NOESY
spectroscopy where a crosspeak between the H10 and the
methylene group of the methoxymethyl substituent was seen
for nucleoside 43a and a crosspeak between the H10 and H60
was seen in case of nucleoside 43b. Similarly, a NOESY
crosspeak was seen between the axial methyl group and H10
for 44 and between the H60 and H10 for 45. In addition, the
structure of the fully deprotected S-cEt uridine nucleoside 49
was unambiguously assigned by X-ray crystallography
(Supporting Information).
To be able to practice antisense drug discovery with a
nucleoside modification in a meaningful manner, it is abso-
lutely essential to have access to multigram quantities of the
pyrimidine and purine nucleobase-modified phosphorami-
dites. Moreover, we also wanted to investigate if the Nap
protecting strategy was compatible with the synthesis of purine
nucleosides. As a result, we prepared the purine (adenine and
guanine) and the 5-methyl pyrimidine (thymine and 5-methyl
cytosine) phosphoramidites of the S-cEt modification for
further evaluation in antisense drug discovery applications.
Synthesis of the purine phosphoramidites was carried
out using slight modifications of the procedures previously
described for the preparation of LNA purine analogues
1574 J. Org. Chem. Vol. 75, No. 5, 2010

Seth et al.
JOCArticle
SCHEME 7. Synthesis of 5-Methylcytosine-Modified S-cEt
Phosphoramidite
TABLE 1. Thermal Stability Measurements of R- and S-cMOE and R-
and S-cEt Modifications with RNA Complement
b
sequence
(50-30)a
Tm
(oC) (oC)/mod.
ΔTm
oligomer modification
A1
A2
A3
A4
A5
A6
A7
A8
DNA
d(GCGTTTTTTGCT) 45.6
d(GCGTTUTTTGCT) 50.2
d(GCGTTUTTTGCT) 50.4
d(GCGTTUTTTGCT) 50.3
d(GCGTTUTTTGCT) 50.1
d(GCGTTTTTTGCT) 51.7
d(GCGTTUTTTGCT) 50.1
d(GCGTTTTTTGCT) 50.7
0
R-cMOE
S-cMOE
R-cEt
S-cEt
S-cEt
þ4.6
þ4.8
þ4.7
þ4.6
þ6.1
þ4.6
þ5.1
LNA
LNA
aSequence and composition of ASOs; base code is A = adenine, T =
thymine, G = guanine, C = cytosine, U = uracil; upper case letters
indicate deoxynucleotide monomers, and bold letters indicate BNA
monomers; all internucleosidic linkages are phosphodiester. bTm values
were measured in 10 mM sodium phosphate buffer (pH 7.2) containing
100 mM NaCl and 0.1 mM EDTA. Sequence of RNA complement
50-r(AGCAAAAAACGC)-30; each Tm value reflects the average of at
least three measurements.
Reaction with dimethoxytrityl chloride gave the 50O-DMT-
protected nucleosides 69 (85%), 70 (91%), and 71 (70%),
respectively. A phosphitylation reaction provided the phos-
phoramidites 72 (83%), 73 (95%), and 74 (98%) in excellent
yield.
Lastly, synthesis of the 5-methyl cytosine S-cEt amidite 76
was accomplished starting from the 50O-DMT-protected
nucleoside 71. In situ transient protection of the 30-hydroxyl
group as the trimethylsilyl ether, followed by triazolation,
displacement with aqueous ammonia and protection of the
amino group as the benzoyl amide provided nucleoside 75 in
good yield (86% over 3 steps).45,46 A phosphitylation reac-
tion provided the desired 5-Me-cytosine-modified S-cEt
phosphoramidite 76 (77%) (Scheme 7).
Evaluation of cMOE- and cEt-Modified Oligonucleotides
in Thermal Denaturation Experiments. All of the nucleoside
phosphoramidites were incorporated into oligonucleotides
using standard automated phosphoramidite chemistry
(Supporting Information). The modified oligomers were
evaluated in thermal stability and in nuclease resistance
assays. We first evaluated the R- and S-cMOE and R- and
S-cEt modifications in an oligonucleotide sequence that has
been previously used to measure thermal stability of LNA-
containing oligonucleotides9 (Table 1). In this sequence the
R-and S-cMOE and R- and S-cEt oligomers (A2-A5)
showed thermal stability similar to that of LNA oligomer
A7 (Δ Tm ∼4.5 °C/mod.), indicating that substitution in
either configuration at the 6 position of the 2,5-dioxabicyclo
ring system is tolerated and does not interfere with hybridi-
zation. This observation is important since it suggests that
it should be possible to introduce conjugates and/or fluor-
escent tags by further derivatization of the 6-substituent
group,29,30 either in the minor groove or along the edges
of the major groove (based on the configuration of the
substituent) at site-specific locations within an oligonucleo-
tide without disrupting hybridization. Introduction of the
5-methyl substituent (change from uracil to thymine) on the
nucleobase in LNA resulted in a slight increase in thermal
stability (þ0.6 °C/mod. A7 vs A8), whereas the effect of the
5-Me substituent on thermal stability was much more pro-
nounced for the S-cEt oligomer A6 (þ1.5 °C/mod. A5 vs A6).
We also examined the ability of oligonucleotides A1-A8
to discriminate between mismatched base pairs using RNA
complements with a single base mismatch (Table 2) as
reported previously by Imanishi.25 In general, the mismatch
discrimination properties of all the modifications evaluated
were similar to that observed for the natural DNA/RNA
base pairs (A1, -4 °C for T-G, -13 °C for T-C, and -14 °C
for T-U mismatched pairs). Oligonucleotides A2-A3 (R-
and S-cMOE), A4 and A5 (R- and S-cEt), and A7 (LNA)
containing the uracil nucleobase exhibited excellent
mismatch discrimination for the U-C (-14 °C) and U-U
(-13 °C) mismatched pairs and good mismatch discrimina-
tion (-5 to -6 °C) for the U-G mismatch pair. For oligo-
nucleotides A6 (S-cEt) and A8 (LNA) containing the
thymine nucleobase, slightly improved mismatch discrimi-
nation properties (-6 °C for T-G, -16 °C for T-C, and
-14 °C for T-U) were observed.
Nuclease Stability of R-cEt, S-cEt, LNA, and MOE Oli-
gonucleotides. Oligonucleotides containing the R- and S-cEt
modifications were evaluated in nuclease stability experi-
ments (Figure 5). In one set of experiments, 10-mer poly T
DNA oligomers (A09-A13) with two modified residues at
the 30 end were digested with snake venom phosphodiester-
ase (SVPD) (Figure 5). Significant increase in stability
toward exonuclease digestion was seen for the R- and S-
cEt oligonucleotides A12 and A13 as compared to LNA A11,
MOE, A10 and DNA A09 oligonucleotides. For both the
R-and S-cEt oligonucleotides A12 and A13, >70% intact
oligomer was seen after 1290 min. In contrast the MOE
(A10) and LNA (A11) oligonucleotides were almost
completely degraded at this time point. This result is con-
sistent with recent reports where substitution on the 20-40
bridging substituent in the BNA scaffold was reported to
increase stability of oligonucleotides toward exonuclease
digestion.25,26
Conclusion
In conclusion, we have shown that the cMOE/cEt mod-
ifications exhibit similar thermal stability and mismatch
discrimination but increased nuclease stability as compared
to those of LNA. The synthesis features the use of a seldom
used 2-naphthyl protecting group that provides crystalline
intermediates during the synthesis and can be cleanly
(45) Divakar, K. J.; Reese, C. B. J. Chem. Soc., Perkin Trans. 1 1982,
1171–1176.
(46) Bhat, V.; Ugarkar, B. G.; Sayeed, V. A.; Grimm, K.; Kosora, N.;
Domenico, P. A.; Stocker, E. Nucleosides, Nucleotides Nucleic Acids 1989, 8,
179–183.
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Seth et al.
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TABLE 2. Mismatch discrimination of DNA, S-cEt and LNA versus mismatched RNA complements
Tmb (ΔTm = Tm(mismatch) - Tm(match)) (oC)
oligomer
sequence (50-30)a
X = A (match)
G
C
U
A1 (DNA)
A2 (R-cMOE)
A3 (S-cMOE)
A4 (R-cEt)
A5 (S-cEt)
A6 (S-cEt)
A7 (LNA)
d(GCGTTTTTTGCT)
d(GCGTTUTTTGCT)
d(GCGTTUTTTGCT)
d(GCGTTUTTTGCT)
d(GCGTTUTTTGCT)
d(GCGTTTTTTGCT)
d(GCGTTUTTTGCT)
d(GCGTTTTTTGCT)
45.6
50.2
50.4
50.3
50.1
51.7
50.1
50.7
41.5 (-4.1)
44.0 (-6.2)
44.4 (-6.2)
43.9 (-6.2)
45.6 (-4.5)
45.6 (-5.1)
45.0 (-5.1)
46.0 (-5.7)
32.3 (-13.1)
35.3 (-14.9)
36.1 (-14.3)
35.8 (-14.4)
36.2 (-13.9)
34.1 (-16.1)
35.2 (-14.9)
35.8 (-16.0)
31.8 (-13.8)
37.1 (-13.1)
37.2 (-13.2)
36.3 (-14.0)
36.5 (-13.6)
37.2 (-13.5)
37.2 (-12.9)
37.4 (-14.3)
A8 (LNA)
aSequence and composition of ASOs; base code is A = adenine, T = thymine, G = guanine, C = cytosine, U = uracil; upper case letters indicate
deoxynucleotide monomers, and bold letters indicate BNA monomers; all internucleosidic linkages are phosphodiester. bTm values were measured in
10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl and 0.1 mM EDTA. Sequence of RNA complement 50-r(AGCAAXAAACGC)-30.
δ: 7.93-7.78 (m, 4 H), 7.60-7.45 (m, 3 H), 5.74 (d, J = 3.6 Hz, 1
H), 4.94 (d, J = 11.9 Hz, 1 H), 4.77 (d, J = 11.9 Hz, 1 H),
4.66-4.54 (m, 1 H), 4.46-4.30 (m, 1 H), 4.24-4.12 (m, 1 H),
4.08-3.88 (m, 3 H), 1.61 (s, 3 H), 1.38 (br s, 9 H). 13C NMR (75
MHz, CDCl3) δ: 135.0, 133.2, 133.2, 128.3, 127.9, 127.7, 127.1,
126.2, 126.1, 126.1, 113.0, 109.7, 103.9, 78.2, 78.0, 77.8, 74.9,
72.4, 65.2, 26.9, 26.7, 26.3, 25.2. ESI-MS m/z: [M þ Na]þ found
423.1, calcd 423.1886. Anal. Calcd for C23H28O6.0.25 H2O: C,
68.21; H, 7.09. Found: C, 68.21; H, 6.92.
30 obtained above was dissolved in glacial acetic acid (2.2 L)
and H2O (0.74 L). The reaction was stirred at rt for 16 h after
which ∼70% of the solvent was evaporated under reduced
pressure on a rotary evaporator. The residue was dissolved in
FIGURE 5. R- and S-cEt-modified oligonucleotides show im-
proved stability to SVPD as compared to LNA, MOE and DNA
oligonucleotides. Sequence used for evaluation was TTTTT-
TTTTTXX; upper case letters denotes deoxynucleotide residues
while XX denote modified nucleosides; all internucleosidic linkages
are phosphodiester. A09, XX = DNA; A10, XX = MOE T; A11,
XX = LNA U; A12, XX = R-cEt U; A13, XX = S-cEt U.
EtOAc (1 L) and the organic layer was washed with water (1 L),
saturated sodium bicarbonate, brine, dried (Na2SO4) and con-
centrated to provide crude 3-O-(2-naphthyl)-1,2-O-isopropyli-
dene-R-D-allofuranose as an oil, which was used without any
further purification. 1H NMR (300 MHz, CDCl3) δ: 7.90-7.74
(m, 4 H), 7.55-7.41 (m, 3 H), 5.71 (d, J = 3.4 Hz, 1 H), 4.89 (d,
J = 11.5 Hz, 1 H), 4.69 (d, J = 11.5 Hz, 1 H), 4.61-4.48 (m, 1
H), 4.12 (dd, J = 3.1 Hz, 8.8, 1 H), 4.06-3.88 (m, 2 H), 3.68 (br s,
2 H), 2.97 (br s, 1 H), 2.81 (br s, 1 H), 1.60 (s, 3 H), 1.34 (s, 3 H).
13C NMR (75 MHz, CDCl3) δ: 134.4, 133.2, 128.4, 127.9, 127.7,
127.2, 126.3, 126.2, 126.0, 113.2, 104.2, 79.1, 77.4, 77.3, 77.0,
72.3, 71.1, 63.1, 26.8, 26.6. ESI-MS m/z: [M þ Na]þ found 383.0,
calcd 383.1573.
A solution of sodium periodate (107 g, 500 mmol) in water
(3 L) was added to a solution of the crude diol obtained above in
1,4-dioxane (1.5 L). After stirring at rt for 1 h, the reaction was
extracted with EtOAc (1.5 L) and the organic layer was sequen-
tially washed with water, brine, dried (Na2SO4) and concen-
trated to provide the crude aldehyde (and hydrate) as an oil,
which was used without any further purification.
Sodium hydroxide solution (2N, 600 mL) was added to a cold
(0 °C) solution of crude aldehyde from above and formaldehyde
(250 mL, 35% aqueous solution) in THF:H2O (1:1, 1 L) and the
mixture was stirred at rt for 72 h. The reaction was extracted
with ethyl acetate and the organic layer was washed with water,
brine, dried (Na2SO4) and concentrated. After roughly 80%
of the solvent was removed (a suspension is formed), hexanes
(300 mL) was added to the suspension which was allowed to
stand for 16 h at rt. The white solid was collected by filtration
and resuspended in a mixture of water and hexanes and stirred
vigorously for 2-3 h. The solid was collected by filtration and
dried under reduced pressure for 16 h to provide diol 31 (124 g,
66% from 1,2:5,6-Di-O-isopropylidene-R-D-allofuranose) as a
white solid. 1H NMR (300 MHz, CDCl3) δ: 7.84 (m, 4 H), 7.50
(m, 3 H), 5.76 (d, J = 3.8 Hz, 1 H), 4.96 (d, J = 11.9 Hz, 1 H),
4.74 (d, J = 11.9 Hz, 1 H), 4.71-4.59 (m, 1 H), 4.26 (d, J = 5.3
Hz, 1 H), 3.96 (br s, 2 H), 3.80 (d, J = 11.9 Hz, 1 H), 3.61 (m, 1
H), 2.40 (m, 1 H), 1.89 (br s, 1 H), 1.66 (s, 3 H), 1.34 (s, 3 H). 13C
NMR (75 MHz, CDCl3) δ: 134.7, 133.2, 133.2, 128.5, 127.9,
deprotected under mild conditions. The new orthogonal
protecting group strategy described in this report will greatly
facilitate the synthesis of this class of molecules and should
find general application for the synthesis of other LNA
analogues. With the exception of LNA, the cMOE/cEt
analogues are the only class of BNA modifications where
the synthesis of the nucleoside phosphoramidites is scaleable
enough to permit meaningful evaluation of these modifica-
tions in animal experiments. We are currently evaluating the
cMOE and cEt modifications for their ability to modulate
gene expression using RNaseH and other antisense mecha-
nisms as well as in diagnostic applications. The results from
these experiments will be reported in due course.
Experimental Section
4-C-Hydroxymethyl-1,2-O-isopropylidene-3-O-(2-naphthyl)-r-
D-ribofuranose (31). Sodium hydride (29.0 g, 727.0 mmol) was
added carefully in portions (3-4 g) over 45 min to a cold (0 °C)
solution of commercially available 1,2:5,6-di-O-isopropylidene-
R-D-allofuranose (135.0 g, 519.0 mmol) and 2-bromomethyl
naphthalene (126 g, 570 mmol) in DMF (500 mL). After stirring
for 1 h, the reaction was very carefully quenched with H2O and
then poured onto crushed ice, and the white solid was collected by
filtration. The solid was resuspended in a biphasic mixture of
water and hexanes (500 mL each), and the suspension was
stirred vigorously for 45 min. The solid was collected by filtra-
tion and then dried under reduced pressure for 16 h to provide
3-O-(2-naphthyl)-1,2:5,6-di-O-isopropylidene-R-D-allofuranose
30 (206 g, 99%) as a white solid. 1H NMR (300 MHz, CDCl3)
1576 J. Org. Chem. Vol. 75, No. 5, 2010

Seth et al.
JOCArticle
127.8, 126.9, 126.4, 126.2, 125.6, 113.6, 104.5, 86.3, 78.5, 78.4,
72.9, 64.3, 63.3, 26.6, 25.9. ESI-MS m/z: [M þ Na]þ found 383.1,
calcd 383.1573. Anal. Calcd for C20H24O6: C, 66.65; H, 6.71.
Found: C, 66.64; H, 6.62.
THF (5 mL), and the reaction was cooled to -20 °C. A few
drops of neat methoxymethyl bromide were added to initiate the
reaction. After a few minutes, a solution of methoxymethyl
bromide (9.33 mL, 102.8 mmol) in THF (12 mL) was added (1
mL every 15 min via a syringe) to the reaction over approxi-
mately 3 h. The temperature of the external bath was carefully
maintained between -20 and -25 °C during the addition. A
small volume of dry THF (5 mL) was added intermittently (over
3 h) to the reaction to facilitate stirring. After the addition of the
bromide was complete, the reaction was stirred at -25 °C for
100 min, and a solution of crude aldehyde (from above) in THF
(30 mL) was added. After stirring at -20 °C for 45 min, no
starting aldehyde was detected by TLC analysis. The reaction
was carefully quenched with a solution of saturated ammonium
chloride and diluted with ethyl acetate. The organic layer was
washed with 5% HCl, a saturated solution of sodium bicarbo-
nate, and brine, dried (Na2SO4), and concentrated to provide
alcohols 34a,b (∼1:1). The reaction was repeated a second time
using identical conditions as described above with similar
results. The crude products from both reactions were combined
and purified by chromatography (silica gel, eluting with 15% to
30% ethyl acetate in hexanes) to provide alcohols 34a,b (24.4 g,
95% from 32) as a viscous oil. 1H NMR (300 MHz, CDCl3) δ:
7.90-7.74 (m, 8 H), 7.64-7.44 (m, 14 H), 7.44-7.25 (m, 12 H),
5.95-5.78 (m, 2 H), 5.02 (m, 2 H), 4.75 (m, 2 H), 4.72-4.51 (m, 5
H), 4.36-4.25 (m, 1 H), 3.90 (d, J = 11.3 Hz, 1 H), 3.74 (m, 3 H),
3.62-3.51 (m, 1 H), 3.45 (d, J = 10.9 Hz, 1 H), 3.39-3.20 (m, 4
H), 3.28 (s, 3 H), 3.23 (s, 3 H), 3.13 (s, 1 H), 1.69 (s, 3 H), 1.64 (s, 3
H), 1.39 (s, 6 H), 0.92 (s, 9 H), 0.91 (s, 9 H). 13C NMR (75 MHz,
CDCl3) δ: 135.5, 135.5, 134.7, 134.4, 133.2, 133.1, 133.0, 133.0,
132.8, 132.7, 129.8, 129.7, 129.6, 128.6, 128.4, 127.9, 127.9,
127.7, 127.6, 127.1, 126.8, 126.3, 126.3, 126.2, 126.1, 125.6,
125.6, 114.4, 113.9, 105.0, 104.8, 89.6, 88.0, 79.5, 79.3, 78.1,
78.1, 73.2, 73.0, 72.8, 72.5, 72.3, 70.8, 64.6, 63.5, 59.0, 58.8, 27.0,
26.9, 26.7, 26.6, 19.1, 19.1. ESI-MS m/z: [M þ Na]þ found 665.3,
calcd 665.3013.
5-O-(tert-Butyldiphenylsilyl)-4-C-(1S-hydroxyethyl)-1,2-O-
isopropylidene-3-O-(2-naphthyl)-r-D-ribofuranose (35) and 5-O-
(tert-Butyldiphenylsilyl)-4-C-(1R-hydroxyethyl)-1,2-O-isopropyli-
dene-3-O-(2-naphthyl)-R-D-ribofuranose (36). Dimethylsulfoxide
(10.8 mL, 152.0 mmol) was added dropwise to a cold (-78 °C)
solution of oxalyl chloride (6.7 mL, 76.0 mmol) in dichloro-
methane (400 mL). After stirring for 30 min, a solution of alcohol
32 (34.2 g, 56.4 mmol) in dichloromethane (40 mL) was added to
the reaction. The stirring was continued for 45 min at -78 °C and
triethylamine (31.4 mL, 224.0 mmol) was added to the reaction.
The reaction was stirred at -78 °C for 15 min, after which the ice
bath was removed and the reaction was allowed to gradually
warm over 45 min. The reaction was diluted with dichloro-
methane, and the organic phase was sequentially washed with
5% aqueous HCl, saturated sodium bicarbonate, and brine, dried
(Na2SO4), and concentrated to provide the aldehyde, which was
used without any further purification.
5-O-(tert-Butyldiphenylsilyl)-4-C-hydroxymethyl-1,2-O-isopro-
pylidene-3-O-(2-naphthyl)-r-D-ribofuranose (32) and 4-C-(tert-
Butyldiphenylsilyloxymethyl)-1,2-O-isopropylidene-3-O-(2-na-
phthyl)-R-D-ribofuranose (33). tert-Butyldiphenylsilyl chloride
(78.2 mL, 305 mmol) was added to a cold (0 °C) solution of diol
31 (100 g, 278 mmol) and triethylamine (42.8 mL, 305 mmol) in
dichloromethane(500 mL). After stirringatrtfor 4 d, the reaction
was diluted with dichloromethane, sequentially washed with 5%
HCl, saturated sodium bicarbonate, and brine, dried (Na2SO4)
and concentrated under reduced pressure to provide a thick oil.
Hexanes (150 mL) was added to the oil, and the mixture was
sonicated to induce crystallization. After standing for 1 h, the
white solid was collected by filtration and washed with 5% ether
in hexanes to yield diol 32 (100 g total yield; 80 g first crop, 20 g
second crop, 60%) as a white solid. Purification of the residual oil
by chromatography (silica gel, eluting with 20% to 40% ethyl
acetate in hexanes) provided diol 33 (32.5 g, 20%) as a viscous oil.
32: 1H NMR (300 MHz, CDCl3) δ: 7.90-7.26 (m, 17 H), 5.81
(d, J = 3.6 Hz, 1 H), 4.97 (d, J = 11.9 Hz, 1 H), 4.77-4.63 (m,
2 H), 4.48 (d, J = 5.3 Hz, 1 H), 3.96-3.63 (m, 4 H), 2.51-2.35
(m, 1 H), 1.67 (s, 3 H), 1.38 (s, 3 H), 0.92 (s, 9 H). 13C NMR (75
MHz, CDCl3) δ: 135.6, 135.5, 134.8, 133.2, 133.2, 133.0, 129.7,
129.7, 128.5, 127.9, 127.8, 127.7, 127.7, 126.9, 126.3, 126.2,
125.6, 113.8, 104.5, 87.5, 79.2, 78.0, 72.8, 65.5, 63.3, 27.0, 26.8,
26.3, 19.2. ESI-MS m/z: [M þ Na]þ found 621.1, calcd 621.2751.
Anal. Calcd for C36H42O6Si: C, 72.21; H, 7.07. Found: C, 72.17;
H, 7.06.
33: 1H NMR (300 MHz, CDCl3) δ: 7.88-7.65 (m, 8 H),
7.52-7.31 (m, 9 H), 5.70 (d, J = 3.8 Hz, 1 H), 4.84 (d, J = 12.2
Hz, 1 H), 4.77 (d, J = 12.1 Hz, 1 H), 4.63-4.51 (m, 1 H),
4.30-4.16 (m, 2 H), 4.16-3.99 (m, 2 H), 3.73-3.57 (m, 1 H),
2.05-1.94 (m, 1 H), 1.25 (br s, 6 H), 1.05 (s, 9 H). 13C NMR (75
MHz, CDCl3) δ: 135.9, 135.8, 135.2, 133.3, 133.2, 133.1, 129.6,
128.2, 127.9, 127.7, 127.7, 126.8, 126.1, 126.0, 125.8, 113.2,
104.0, 87.3, 78.9, 77.9, 72.7, 65.2, 64.6, 26.9, 26.4, 25.9, 19.3.
ESI-MS m/z: [M þ Na]þ found 621.1, calcd 621.2751. Anal.
Calcd for C36H42O6Si.0.5 H2O: C, 71.20; H, 7.14. Found: C,
71.21; H, 7.12.
5- O-(tert-Butyldiphenylsilyl)-4-C-(1-hydroxy-2-methoxyethyl)-
1,2-O-isopropylidene-3-O-(2-naphthyl)-r-D-ribofuranose (34a,b).
Dimethylsulfoxide (3.5 mL, 50.0 mmol) was added dropwise to
a cold (-78 °C) solution of oxalyl chloride (2.2 mL, 25.0 mmol) in
dichloromethane(130 mL). After stirringfor 30 min, a solutionof
diol 32 (12 g, 16.7 mmol) in dichloromethane (30 mL) was added
to the reaction. The stirring was continued for 45 min at -78 °C,
and triethylamine (10.5 mL, 75.0 mmol) was added to the
reaction. The reaction was stirred at -78 °C for 15 min, after
which the ice bath was removed and the reaction was allowed to
gradually warm over 45 min. The reaction was then poured into
CH2Cl2, and the organic phase was sequentially washed with 5%
aqueous HCl, saturated NaHCO3, and brine, dried (Na2SO4),
and concentrated under reduced pressure to provide the alde-
hyde, which was used without any further purification. 1H NMR
(300 MHz, CDCl3) δ: 9.93(s, 1 H), 7.94-7.72 (m, 4 H), 7.63-7.26
(m, 13 H), 5.85 (d, J = 3.2 Hz, 1 H), 4.88 (d, J = 12.2, 1 H), 4.81
(d, J = 12.4 Hz, 1 H), 4.72-4.61 (m, 1 H), 4.62-4.52 (m, 1 H),
3.87 (d, J = 11.3Hz, 1 H), 3.82 (d, J = 11.5 Hz, 1 H), 1.64(s, 3 H),
1.37 (s, 3 H), 0.92 (s, 9 H). 13C NMR (75 MHz, CDCl3) δ: 200.2,
135.6, 135.5, 134.5, 133.2, 133.2, 132.8, 132.6, 129.9, 129.8, 128.5,
127.9, 127.8, 127.8, 127.0, 126.3, 126.2, 125.7, 114.2, 105.0, 90.7,
79.1, 78.7, 73.0, 63.2, 26.7, 26.2, 19.2. ESI-MS m/z: [M þ Na]þ
found 619.1, calcd 619.2594.
A suspension of cerium(III) chloride (9.2 g, 37.5 mmol) in
THF (400 mL) was stirred at rt for 60 min. The reaction was
cooled in an ice bath, and methyl magnesium bromide (75.0 mL
of a 1.0 M solution in THF) was added over 5 min. After stirring
at 0 °C for 90 min, the reaction was cooled to -78 °C, and a
solution of crude aldehyde from above in THF (75 mL) was
added to the reaction. After 3 h at -78 °C, the reaction was
gradually warmed to rt and carefully quenched with saturated
ammonium chloride. The reaction was diluted with ethyl acet-
ate, and the organic layer was sequentially washed with 5% HCl,
saturated sodium bicarbonate, and brine, dried (Na2SO4), and
concentrated. Purification by column chromatography (silica
gel, eluting with 10% to 30% ethyl acetate in hexanes) provided
pure alcohol 35 (7.4 g, 21% from 32) and a mixture of alcohols
35 and 36 (26.3 g, 76% from 32, 35:36 = 10:1) as viscous oils.
A mixture of magnesium turnings (2.5 g, 102.8 mmol) and
mercury(II) chloride (93 mg, 0.34 mmol) was covered with dry
J. Org. Chem. Vol. 75, No. 5, 2010 1577

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JOCArticle
35: 1H NMR (300 MHz, CDCl3) δ: 7.89-7.79 (m, 4 H),
7.65-7.26 (m, 13 H), 5.84 (d, J = 3.6 Hz, 1 H), 5.05 (d, J = 11.5
Hz, 1 H), 4.83-4.53 (m, 4 H), 3.91 (d, J = 11.1 Hz, 1 H), 3.84 (d,
J = 11.1 Hz, 1 H), 3.36 (s, 1 H), 1.63 (s, 3 H), 1.39 (s, 3 H), 1.10
(d, J = 6.6 Hz, 3 H), 0.91 (s, 9 H). 13C NMR (75 MHz, CDCl3) δ:
135.6, 135.5, 134.4, 133.3, 133.3, 133.2, 133.1, 129.7, 129.7,
128.7, 128.0, 127.8, 127.7, 127.7, 127.2, 126.4, 126.3, 125.7,
113.8, 104.8, 88.6, 79.4, 78.3, 73.0, 68.8, 62.4, 27.1, 26.8, 26.7,
19.2, 16.1. HRMS (ESI-FT), Calcd for C37H44O6SiNa,
635.2799; Found 635.2800. ESI-MS m/z: [M þ Na]þ found
635.2. Anal. Calcd for C37H44O6Si.1 H2O: C, 70.44; H, 7.35.
Found: C, 70.73; H, 7.11.
dried (Na2SO4), and concentrated to provide the mesylate,
which was used without any further purification.
Concentrated sulfuric acid (6-10 drops) was added to a
solution of crude mesylate (1 equiv) from above, acetic acid,
and acetic anhydride (5:1, 0.3 M). After stirring for 3 h at rt, the
solvent was removed under reduced pressure, and the residual
oil was diluted with ethyl acetate. The organic layer was washed
with water, saturated sodium bicarbonate (until pH >10), and
brine, dried (Na2SO4), and concentrated. Purification by col-
umn chromatography (silica gel, eluting with 20% to 30% ethyl
acetate in hexanes) provided an anomeric mixture of diacetates.
1,2-Di-O-acetyl-5-O-(tert-butyldiphenylsilyl)-4-C-(1-metha-
nesulfonyloxy-2-methoxyethyl)-3-O-(2-naphthyl)-r-D-ribofur-
anose (37a,b). Prepared from alcohol 34a,b (11.0 g, 17.1 mmol)
to provide an anomeric mixture of anomeric diacetates 37a,b
(10.0 g, 77%) as a viscous oil. ESI-MS m/z: [M þ Na]þ found
787.2, calcd 787.2687. Anal. Calcd for C40H48O11SSi.1 H2O:
C, 61.36; H, 6.44. Found: C, 61.08; H, 6.15.
1,2-Di-O-acetyl-5-O-(tert-butyldiphenylsilyl)-4-C-(1S-methanesul-
fonyloxyethyl)-3-O-(2-naphthyl)-r-D-ribofuranose (38). Prepared
from alcohol 35 (7.4 g, 12.0 mmol) using the general procedure
described above to provide anomeric diacetates 38 (7.7 g, 89%) as
a viscous oil. 1H NMR for major anomer (300 MHz, CDCl3) δ:
7.84-7.35 (m, 17 H), 6.22 (s, 1 H), 5.37 (d, J = 5.3 Hz, 1 H),
5.34-5.22 (m, 1 H), 4.77-4.58 (m, 2 H), 4.42 (d, J = 5.5 Hz, 1 H),
3.83(d, 1 H, J = 10.9 Hz), 3.74 (d, J =10.9Hz, 1 H), 2.79 (s, 3 H),
2.11 (s, 3 H), 1.78 (s, 3 H), 1.56 (d, J = 4.1 Hz, 3 H), 1.06 (s, 9 H).
13C NMR for anomeric mixture (75 MHz, CDCl3) δ: 169.7,
169.0, 135.6, 134.4, 133.1, 133.0, 132.6, 130.0, 129.9, 128.4, 128.0,
127.9, 127.8, 127.7, 126.8, 126.3, 126.2, 125.5, 98.2, 88.2, 78.9,
78.8, 74.2, 74.1, 65.6, 38.8, 26.9, 20.9, 20.8, 19.4, 17.0. ESI-MS m/
z: [M þ Na]þ found 757.1, calcd 757.2581.
36: 1H NMR (300 MHz, CDCl3) δ: 7.88-7.78 (m, 4 H),
7.61-7.27 (m, 13 H), 5.87 (d, J = 3.6 Hz, 1 H), 4.96 (d, J = 12.1
Hz, 1 H), 4.74 (t, 1 H), 4.66 (d, J = 12.1 Hz, 1 H), 4.54 (d, J = 5.3
Hz, 1 H), 4.32-4.18 (m, 1 H), 3.69 (d, J = 10.7 Hz, 1 H), 3.52 (d,
J = 10.7 Hz, 1 H), 3.12 (s, 1 H), 1.69 (s, 3 H), 1.39 (s, 3 H), 1.11
(d, J = 6.4 Hz, 3 H), 0.90 (s, 9 H). 13C NMR (75 MHz, CDCl3) δ:
135.5, 134.8, 133.2, 133.2, 132.9, 132.8, 129.8, 129.7, 128.4,
127.9, 127.7, 126.9, 126.3, 126.1, 125.7, 114.3, 104.5, 90.4,
79.6, 78.1, 72.8, 67.1, 64.6, 26.9, 26.7, 19.1, 17.0. ESI-MS m/z:
[M þ Na]þ found 635.2, calcd 635.2907. Anal. Calcd for
C37H44O6Si.0.5 H2O: C, 71.46; H, 7.29. Found: C, 71.81; H,
7.55.
Preparation of Alcohol 36 Using Lithium Borohydride.
Dimethylsulfoxide (37.9 mL, 489.0 mmol) was added dropwise
to a cold (-78 °C) solution of oxalyl chloride (21.4 mL, 244.0
mmol) in dichloromethane (800 mL). After stirring for 30 min, a
solution of alcohol 35 (100.0 g, 163.0 mmol) in dichloromethane
(200 mL) was added to the reaction. The stirring was continued
for 45 min at -78 °C, and triethylamine (102.0 mL, 726.0 mmol)
was added to the reaction. The reaction was stirred at -78 °C for
15 min, after which the ice bath was removed and the reaction
was allowed to gradually warm over 45 min. The reaction was
diluted with dichloromethane, and the organic phase was se-
quentially washed with 10% citric acid solution, saturated
sodium bicarbonate, and brine, dried (Na2SO4), and concen-
trated to provide the crude ketone, which was used without any
further purification. 1H NMR (300 MHz, CDCl3) δ: 7.82-7.27
(m, 17 H), 6.06 (d, J = 4.1 Hz, 1 H), 4.88 (d, J = 12.1 Hz, 1 H),
4.82 (d, J = 4.5 Hz, 1 H), 4.66 (d, J = 12.1 Hz, 1 H), 4.24 (d, J =
5.5 Hz, 1 H), 3.89 (d, J = 10.9 Hz, 1 H), 3.70 (d, J = 10.7 Hz, 1
H), 2.38 (s, 3 H), 1.63 (s, 3 H), 1.43 (s, 3 H), 0.97 (s, 9 H). 13C
NMR (75 MHz, CDCl3) δ: 207.8, 135.5, 134.9, 133.2, 133.0,
132.5, 132.4, 129.9, 129.8, 128.2, 127.9, 127.8, 127.7, 126.6,
126.1, 125.9, 125.7, 114.8, 106.7, 95.9, 80.9, 79.9, 73.4, 67.6,
28.7, 27.5, 26.8, 26.6, 19.1. ESI-MS m/z: [M þ Na]þ found 633.1,
calcd 633.2751.
A solution of lithium borohydride (122.0 mL of a 2 M
solution in THF, 244 mmol) was added dropwise over 30 min
to a cold (-78 °C) solution of the ketone (163 mmol) from above
in methanol (500 mL). After the addition was complete, the
cooling bath was removed, and the reaction was stirred for 2 h.
The reaction was then cooled in an ice bath and carefully
quenched using saturated NH4Cl solution and diluted with
ethyl acetate. The organic layer was separated and sequentially
washed with water, saturated sodium bicarbonate, and brine,
dried (Na2SO4), and concentrated. Purification by column
chromatography (silica gel, eluting with 30% ethyl acetate in
hexanes) provided alcohol 36 (97.2 g, 95%, 36:35 > 15:1) as a
viscous oil.
General Procedure for Mesylation and Acetolysis of 1,2-Acet-
onide. Methanesulfonyl chloride (1.4 equiv) was added dropwise
over 30 min to a cold (0 °C) solution of alcohol (1 equiv),
triethylamine (1.8 equiv), and DMAP (0.14 equiv) in dichloro-
methane (0.5 M). After stirring for 3 to 6 h, the reaction was
diluted with chloroform. The organic layer was sequentially
washed with 5% HCl, saturated sodium bicarbonate, and brine,
1,2-Di-O-acetyl-5-O-(tert-butyldiphenylsilyl)-4-C-(1R-metha-
nesulfonyloxyethyl)-3-O-(2-naphthyl)-r-D-ribofuranose (39). Pre-
pared from alcohol 36 (16.9 g, 27.6 mmol) using the general
procedure described above to provide anomeric diacetates 39
(18.2 g, 90%) as a viscous oil. 1H NMR for major anomer (300
MHz, CDCl3) δ:7.85-7.41 (m, 17 H), 6.21 (s, 1 H), 5.57-5.45 (m,
1 H), 5.36-5.23 (m, 1 H), 4.76 (d, J = 11.3 Hz, 1 H), 4.70 - 4.60
(m, 1H), 3.92-3.71 (m, 3 H), 3.01 (s, 3 H), 2.14 (s, 3 H), 1.78 (s, 3
H), 1.43 (d, J =6.6 Hz, 3 H), 1.05 (s, 9 H). 13C NMRfor anomeric
mixture (75 MHz, CDCl3) δ: 170.6, 169.9, 169.4, 135.7, 135.6,
135.6, 135.5, 135.0, 134.1, 133.2, 132.8, 132.4, 132.2, 130.1, 130.0,
129.9, 128.5, 128.1, 127.9, 127.9, 127.8, 127.8, 127.7, 126.9, 126.4,
126.3, 125.8, 125.5, 125.1, 97.8, 94.1, 90.4, 88.2, 81.7, 81.1, 77.9,
77.7, 74.8, 74.5, 74.2, 73.2, 62.8, 38.6, 38.4, 26.8, 21.3, 20.8, 20.5,
19.3, 19.1, 18.3, 18.1. ESI-MS m/z: [M þ Na]þ found 757.1, calcd
757.2581. Anal. Calcd for C39H46O10SSi.0.25 H2O: C, 63.35; H,
6.33. Found: C, 63.32; H, 6.26.
General Procedure for Vorbruggen Reaction and Ring Closure.
N,O-Bis(trimethylsilyl)acetamide (4 equiv) was added to a sus-
pension of anomeric diacetates (1 equiv) and uracil (2 equiv) in
acetonitrile (0.3M). After heating at 40 °C for 15 min to get a
clear solution, the reaction was cooled in an ice bath and tri-
methylsilyltriflate (1.5 equiv) was added. After refluxing for 2 h
the reaction was cooled to room temperature and poured into
ethyl acetate. The organic layer was washed with half-saturated
sodium bicarbonate solution and brine, dried (Na2SO4), and
concentrated to provide the corresponding nucleoside mixture,
which was used without any further purification.
Potassium carbonate (3 equiv) was added to a solution of
crude nucleoside from above in methanol (0.1 M). After stirring
at room temperature for 16 h, the reaction was concentrated
under reduced pressure. The residue was dissolved in ethyl
acetate, and the organic layer was washed with water and brine,
dried (Na2SO4), and concentrated. Purification by column
chromatography (silica gel, eluting with 5% to 7.5% acetone
in chloroform) provided the cyclized nucleoside.
1578 J. Org. Chem. Vol. 75, No. 5, 2010

Seth et al.
(1S,3R,4R,6R,7S)-1-(tert-Butyldiphenylsilyloxymethyl)-6-metho-
JOCArticle
H), 5.52 (d, J = 8.3 Hz, 1 H), 4.40 (s, 1 H), 4.32 (d, J = 5.1 Hz, 1
H), 4.18 (t, J = 6.3 Hz, 1 H), 4.07 (d, J = 12.2 Hz, 1 H), 3.95 (d,
J = 12.4, 1 H), 3.56 (d, J = 6.4 Hz, 2 H), 3.27 (s, 3 H), 3.21-3.13
(m, 1 H), 1.09 (s, 9 H). 13C NMR (75 MHz, CDCl3) δ: 163.5,
149.9, 139.2, 135.6, 135.4, 132.7, 132.3, 130.2, 130.1, 128.0,
101.7, 89.2, 86.4, 79.0, 77.9, 71.2, 70.4, 59.2, 58.4, 26.8, 19.3.
HRMS (ESI-FT) Calcd for C28H35N2O7Si, 539.2208; Found
539.2206. ESI-MS m/z: [M þ Na]þ found 539.2.
xymethyl-7-(2-naphthyloxy)-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]-
heptane and (1S,3R,4R,6S,7S)-1-(tert-Butyldiphenylsilyloxymethyl)-
6-methoxymethyl-7-(2-naphthyloxy)-3-(uracil-1-yl)-2,5-dioxabicyclo-
[2.2.1]heptane (40a,b). Prepared from diacetates 37a,b (10.0 g, 13.0
mmol) using the general procedure described above to provide
1
nucleosides 40a,b (7.8 g, 89% from 36a,b) as a white solid. H
NMR (300 MHz, CDCl3) δ: 8.58 (br s, 2 H), 7.94-7.27 (m, 36 H),
5.70-5.58 (m, 2 H), 5.48-5.31 (m, 2 H), 4.94-4.58 (m, 5 H), 4.50
(s, 1 H), 4.35-4.26 (m, 1 H), 4.24 (s, 1 H), 4.16-3.92 (m, 8 H),
3.61-3.47 (m, 5 H), 3.26 (s, 3 H), 3.18 (s, 3 H), 1.07 (s, 18 H). 13C
NMR (75 MHz, CDCl3) δ: 162.8, 149.6, 149.5, 139.1, 139.0,
135.6, 135.5, 135.3, 134.0, 133.8, 133.2, 132.7, 132.2, 130.1, 130.1,
128.5, 128.5, 128.0, 127.9, 127.8, 127.8, 127.1, 126.8, 126.5, 126.4,
126.3, 125.7, 125.6, 101.7, 101.6, 89.2, 89.1, 86.9, 86.7, 82.9, 78.6,
77.2, 76.7, 76.3, 75.9, 72.7, 72.6, 71.0, 70.1, 59.3, 58.9, 58.5, 26.8,
19.3. ESI-MS m/z: [M þ H]þ found 679.2, calcd 679.2761.
(1S,3R,4R,6R,7S)-1-(tert-Butyldiphenylsilyloxymethyl)-6-methyl-
7-(2-naphthyloxy)-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (41).
Prepared from anomeric diacetates 38 (7.5 g, 10.2 mmol) using the
general procedure to provide nucleoside 41 (5.3 g, 81%) as a white
solid. 1H NMR (300 MHz, CDCl3) δ: 8.74 (br s, 1 H), 7.91 (d, J =
8.3 Hz, 1 H), 7.87-7.28 (m, 17 H), 5.65 (s, 1 H), 5.43 (d, J=8.1Hz,1
H), 4.83 (d, J = 11.5 Hz, 1 H), 4.75 (d, J = 11.5 Hz, 1 H), 4.49 (s,
1H), 4.38-4.27 (m, 1 H), 4.20 (s, 1 H), 3.93-3.88 (m, 2 H), 1.19 (d,
J= 6.4 Hz, 3 H), 1.07 (s, 9 H). 13C NMR (75 MHz, CDCl3) δ:162.9,
149.6, 139.2, 135.6, 135.3, 134.1, 133.2, 132.5, 132.0, 130.2, 130.1,
128.5, 128.0, 127.9, 127.8, 126.9, 126.4, 126.3, 125.7, 101.6, 88.8, 86.7,
76.5, 76.3, 72.6, 58.4, 26.8, 19.3, 13.8. HRMS (ESI-FT), Calcd for
C38H41N2O6Si 649.2728; found 649.2738. ESI-MS m/z: [M þ H]þ
found 649.2. Anal. Calcd for C38H40N2O6Si.0.5 H2O: C, 69.38; H,
6.28; N, 4.26. Found: C, 69.01; H, 6.19; N, 4.26.
(1S,3R,4R,6S,7S)-1-(tert-Butyldiphenylsilyloxymethyl)-6-methyl-
7-(2-naphthyloxy)-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (42).
Prepared from anomeric diacetates 39 (12.3 g, 16.7 mmol) using the
general procedure to provide nucleoside 42 (9.2 g, 85%) as a white
solid. 1H NMR (300 MHz, CDCl3) δ:9.20(brs, 1H), 7.84-7.31 (m,
18 H), 5.66 (s, 1 H), 5.43 (d, J = 7.9 Hz, 1 H), 4.82 (d, J = 11.3 Hz, 1
H), 4.72 (s, 1H), 4.71-4.61 (m, 1 H), 4.14-3.97 (m, 4 H), 1.26 (d,
J= 6.6 Hz, 3 H), 1.07 (s, 9 H). 13C NMR (75 MHz, CDCl3) δ:163.2,
149.7, 139.1, 135.5, 135.3, 134.0, 133.1, 132.6, 132.1, 130.1, 130.0,
128.4, 127.9, 127.8, 127.7, 126.9, 126.4, 126.3, 125.7, 101.5, 89.3, 87.1,
81.1, 76.7, 76.5, 72.6, 58.7, 26.7, 19.3, 16.3. ESI-MS m/z: [M þ H]þ
found 649.2, calcd 649.2656. Anal. Calcd for C38H40N2O6Si.0.25
H2O: C, 69.86; H, 6.25; N, 4.29. Found: C, 69.61; H, 6.17; N, 4.22.
General Procedure for Deprotection of 30O-Nap Group Using
2,3-Dichloro-4,5-dicyano Quinone (DDQ). DDQ (2-2.5 equiv)
was added to a solution of nucleoside (1 equiv) in dichloro-
methane and water (20:1, 0.1 M). The biphasic reaction was
stirred at room temperature for 16 h, after which the solvent was
evaporated under reduced pressure and the residue was parti-
tioned between ethyl acetate and water. The organic layer was
sequentially washed with sodium bisulfite, saturated sodium
bicarbonate, and brine, dried (Na2SO4), and concentrated.
Purification by column chromatography (silica gel, eluting
10% to 30% acetone in chloroform) provided the 30OH nucleo-
side.
43b: 1H NMR (300 MHz, CDCl3) δ: 9.20 (br s, 1 H),
7.81-7.61 (m, 4 H), 7.65 (d, J = 8.1 Hz, 1H) 7.55-7.35 (m, 6
H), 5.64 (s, 1 H), 5.60 (d, J = 8.1 Hz, 1 H), 5.07 (d, J = 10.7 Hz, 1
H), 4.43 (s, 1 H), 4.18-3.94 (m, 4 H), 3.66 (dd, J = 11.5, 2.8 Hz,
1 H), 3.52 (d, J = 11.3 Hz, 1 H), 3.35 (s, 3 H), 1.09 (s, 9 H). 13C
NMR (75 MHz, CDCl3) δ: 163.2, 149.7, 139.0, 135.8, 135.4,
132.7, 132.1, 130.1, 130.1, 128.0, 127.9, 101.8, 90.1, 86.4, 82.8,
80.3, 70.5, 70.1, 59.5, 59.2, 26.7, 19.3. HRMS (ESI-FT) Calcd
for C28H35N2O7Si, 539.2208; Found 539.2209. ESI-MS m/z: [M
þ Na]þ found 539.2.
(1S,3R,4R,6R,7S)-1-(tert-Butyldiphenylsilyloxymethyl)-7-hy-
droxy-6-methyl-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (44).
Prepared from nucleoside 41 (5.3 g, 8.2 mmol) using the general
procedure to provide nucleoside 44 (4.15 g, 99%) as a crisp foam.
1H NMR (300 MHz, CDCl3) δ: 9.31 (br s, 1 H), 7.92 (d, J = 8.1
Hz, 1 H), 7.77-7.64 (m, 4 H), 7.54-7.35 (m, 6 H), 5.60 (s, 1 H),
5.53 (d, J = 8.1 Hz, 1 H), 4.35 (s, 1 H), 4.30 (d, J = 5.1 Hz, 1 H),
4.25-4.16 (m, 1 H), 3.93-3.86 (m, 2 H), 2.91 (m, 1 H), 1.20 (d, J =
6.2 Hz, 3 H), 1.09 (s, 9 H). 13C NMR (75 MHz, CDCl3) δ: 163.4,
149.8, 139.2, 135.6, 135.4, 132.5, 132.2, 130.2, 130.1, 128.0, 101.7,
88.9, 86.3, 79.0, 75.8, 71.3, 58.4, 26.8, 19.3, 13.9. ESI-MS m/z: [M þ
H]þ found 509.1, calcd 509.2030.
(1S,3R,4R,6S,7S)-1-(tert-Butyldiphenylsilyloxymethyl)-7-hy-
droxy-6-methyl-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (45).
Prepared from nucleoside 42 (9.2 g, 14.2 mmol) using the general
procedure to provide nucleoside 45 (6.2 g, 86%) as a crisp foam. 1H
NMR (300 MHz, CDCl3) δ: 9.67 (br s, 1 H), 7.80-7.65 (m, 5 H),
7.52-7.36 (m, 6 H), 5.59 (s, 1 H), 5.49 (d, J = 8.1 Hz, 1 H), 4.56 (s,
1 H), 4.22-3.96 (m, 4 H), 3.13 (m, 1 H), 1.28 (d, J = 6.8 Hz, 3 H),
1.09 (s, 9 H). 13C NMR (75 MHz, CDCl3) δ: 163.7, 149.9, 139.3,
135.6, 135.4, 132.7, 132.3, 130.1, 130.1, 128.0, 101.6, 89.4, 86.7,
81.1, 79.7, 70.8, 58.9, 26.8, 19.3, 16.3. ESI-MS m/z: [M þ H]þ
found 509.1, calcd 509.2030.
General Procedure for Removing 50O-TBDPS Protecting
Group. Triethylamine trihydrofluoride (6 equiv) was added to
a solution of nucleoside (1 equiv) and triethylamine (2.5 equiv)
in THF (0.1 M), and the reaction was stirred at rt for 1-2 days.
The solvent was removed under reduced pressure, and the thick
oil was purified by chromatography (silica gel, eluting with 5%
to 12.5% methanol in chloroform) provided the corresponding
deprotected nucleoside.
(1S,3R,4R,6R,7S)-7-Hydroxy-1-hydroxymethyl-6-methoxy-
methyl-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (46). Pre-
pared from nucleoside 43a (6.7 g, 12.0 mmol) using the general
procedure described above to provide 46 (3.7 g, 99%). 1H
NMR (300 MHz, DMSO-d6) δ: 11.36 (s, 1 H), 7.79 (d, J = 8.1
Hz, 1 H), 5.73 (d, J = 4.1 Hz, 1H), 5.64 (d, J = 8.1 Hz, 1 H),
5.40 (s, 1 H), 5.20 (br s, 1 H), 4.10 (s, 1H), 4.02-3.94 (m, 2 H),
3.75 (d, J = 3.8 Hz, 1 H), 3.71-3.54 (m, 2 H), 3.46 (dd, J = 6.4
Hz, 10.0, 1 H), 3.28 (s, 3 H). 13C NMR (75 MHz, DMSO-d6) δ:
163.2, 149.9, 139.0, 100.8, 88.8, 85.8, 78.6, 77.6, 70.5, 69.8, 58.5,
55.2. ESI-MS m/z: [M þ H]þ found 301.1, calcd 301.0958.
Anal. Calcd for C12H16N2O7.0.11 CHCl3: C, 46.39; H, 5.18; N,
8.93. Found: C, 46.17; H, 5.22; N, 8.77.
(1S,3R,4R,6S,7S)-7-Hydroxy-1-hydroxymethyl-6-methoxy-
methyl-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (47). Pre-
pared from nucleoside 43b (6.4 g, 12.5 mmol) using the general
procedure described above to provide 47 (3.5 g, 99%). 1H
NMR (300 MHz, DMSO-d6) δ: 11.42 (br s, 1 H), 7.80 (d, J =
8.1 Hz, 1 H), 5.73 (br s, 1 H), 5.68 (d, J = 8.1 Hz, 1 H), 5.47 (s, 1
(1S,3R,4R,6R,7S)-1-(tert-Butyldiphenylsilyloxymethyl)-7-hy-
droxy-6-methoxymethyl-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane
(43a) and (1S,3R,4R,6S,7S)-1-(tert-Butyldiphenylsilyloxymethyl)-7-
hydroxy-6-methoxymethyl-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]-
heptane (43b). Prepared from nucleosides 40a,b (16.8 g, 24.9
mmol) using the general procedure to provide nucleosides 43a
(6.4 g, 50%, Rf = 0.15, 25% acetone in chloroform) and 43b
(6.7 g, 48%, Rf = 0.22, 25% acetone in chloroform) as crisp
foams, respectively.
43a: 1H NMR (300 MHz, CDCl3) δ: 9.44 (s, 1 H), 7.91 (d, J =
8.3 Hz, 1 H), 7.77-7.64 (m, 4 H), 7.54-7.35 (m, 6 H), 5.58 (s, 1
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Seth et al.
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H), 5.22 (br s, 1 H), 4.28 (s, 1 H), 4.02-3.91 (m, 2 H), 3.83 (br s,
2 H), 3.59 (d, J = 5.5 Hz, 2 H), 3.30 (s, 3 H). 13C NMR (75
MHz, DMSO-d6) δ: 163.3, 150.0, 139.2, 100.8, 89.0, 86.1, 82.8,
79.2, 71.0, 68.9, 58.2, 55.8. ESI-MS m/z: [M þ H]þ found 301.1,
calcd 301.0958.
(1S,3R,4R,6R,7S)-7-Hydroxy-1-hydroxymethyl-6-methyl-
3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (48). Prepared from
nucleoside 44 (4.2 g, 8.2 mmol) using the general procedure
described above to provide 48 (2.1 g, 92%). 1H NMR (300
MHz, DMSO-d6) δ: 11.35 (br s, 1 H), 7.81 (d, J = 8.1 Hz,
1 H), 5.71-5.59 (m, 2 H), 5.38 (s, 1 H), 5.16 (t, J = 5.3 Hz, 1 H),
4.21-4.06(m, 1 H), 4.05 (s, 1H), 3.99 (d, J = 4.1Hz, 1 H), 3.62 (d,
J = 5.1 Hz, 2 H), 1.14 (d, 3 H). 13C NMR (75 MHz, DMSO-d6) δ:
163.1, 149.8, 138.9, 100.6, 88.4, 85.6, 78.4, 75.0, 69.7, 55.1, 13.8.
ESI-MS m/z: [M þ H]þ found 271.0, calcd 271.0872. Anal. Calcd
for C11H14N2O6.0.5 H2O: C, 47.31; H, 5.41; N, 10.03. Found: C,
47.01; H, 5.29; N, 9.70.
(1S,3R,4R,6S,7S)-7-Hydroxy-1-hydroxymethyl-6-methyl-
3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (49). Prepared from
nucleoside 45 (6.2 g, 12.2 mmol) using the general procedure
described above to provide 49 (3.3 g, 99%). 1H NMR (300 MHz,
DMSO-d6) δ: 11.36 (s, 1 H), 7.73 (d, J = 8.1Hz, 1 H), 5.62 (d, J =
8.1 Hz, 1 H), 5.55 (d, J = 3.8 Hz, 1 H), 5.38 (s, 1 H), 5.11 (t, J =
5.4 Hz, 1 H), 4.19 (s, 1 H), 3.94 (m, 1 H), 3.86 (d, J = 3.8 Hz, 1 H),
3.80 (d, J = 5.3 Hz, 2 H), 1.22 (d, 3 H). 13C NMR (75 MHz,
DMSO-d6) δ: 163.1, 149.9, 139.2, 100.7, 89.0, 86.1, 80.2, 79.2,
69.6, 55.9, 16.2. HRMS (ESI-FT), Calcd for C11H14N2O6Na,
293.0744; Found 293.0745. ESI-MS m/z: [M þ H]þ found 271.0.
Anal. Calcd for C11H14N2O6.0.25 CH3OH: C, 48.56; H, 5.43; N,
10.07. Found: C, 48.71; H, 5.06; N, 9.92.
General Procedure for 50O-DMT Protection. 4,40-Dimethoxy-
trityl chloride (1.5 equiv) was added to a solution of nucleoside
(1 equiv) in pyridine (0.2 M). After stirring at rt for 16 h, the
reaction was quenched with methanol and diluted with ethyl
acetate. The organic layer was washed with water and brine,
dried (Na2SO4), and concentrated. Purification by chromato-
graphy (silica gel, eluting with 10% to 20% acetone in
dichloromethane) provided the corresponding 50O-DMT-
protected nucleoside.
(1S,3R,4R,6R,7S)-1-(4,40-Dimethoxytrityloxymethyl)-7-hy-
droxy-6-methoxymethyl-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]-
heptane (50). Prepared from nucleoside 46 (3.7 g, 12.5 mmol)
using the general procedure described above to provide 50
(6.7 g, 90%). 1H NMR (300 MHz, CDCl3) δ: 9.11 (br s, 1 H),
8.01 (d, J = 8.1 Hz, 1 H), 7.45-7.24 (m, 9 H), 6.86 (d, J = 8.5
Hz, 4 H), 5.66-5.51 (m, 2 H), 4.46-4.31 (m, 2 H), 4.23-4.08
(m, 1 H), 3.79 (s, 6 H), 3.70-3.40 (m, 4 H), 3.24 (s, 3 H), 2.69 (d,
J = 4.7 Hz, 1 H). 13C NMR (75 MHz, CDCl3) δ: 163.3, 158.7,
149.7, 144.4, 139.4, 135.3, 130.1, 130.0, 128.0, 127.2, 113.3,
101.8, 88.2, 87.0, 86.4, 78.9, 78.2, 71.6, 70.3, 59.2, 57.4, 55.3.
ESI-MS m/z: [M þ Na]þ found 625.1, calcd 625.2264.
(1S,3R,4R,6S,7S)-1-(4,40-Dimethoxytrityloxymethyl)-7-hy-
droxy-6-methoxymethyl-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]-
heptane (51). Prepared from nucleoside 47 (3.5 g, 12.0 mmol)
using the general procedure described above to provide 51
(6.5 g, 91%). 1H NMR (300 MHz, CDCl3) δ: 8.69 (br s, 1 H),
7.70 (d, J = 8.3 Hz, 1 H), 7.50-7.24 (m, 9 H), 6.86 (d, J = 8.7
Hz, 4 H), 5.75-5.60 (m, 2 H), 5.05 (d, J = 10.9 Hz, 1 H), 4.43 (s,
1 H), 4.17 (s, 1 H), 3.97 (d, J = 10.9 Hz, 1 H), 3.80 (s, 6 H),
3.75-3.58 (m, 2 H), 3.41 (m, 2 H), 3.22 (s, 3 H). 13C NMR (75
MHz, CDCl3) δ: 162.8, 158.7, 149.6, 144.3, 138.9, 135.5, 135.1,
130.0, 128.0, 128.0, 127.1, 113.3, 113.3, 101.9, 89.2, 86.6, 86.6,
83.1, 80.3, 71.1, 70.0, 59.2, 58.9, 55.3. ESI-MS m/z: [M þ Na]þ
found 625.1, calcd 625.2264.
(300 MHz, CDCl3) δ: 9.03 (br s, 1 H), 8.03 (d, J = 8.1 Hz, 1 H),
7.47-7.25 (m, 9 H), 6.94-6.76 (m, 4 H), 5.69-5.52 (m, 2 H),
4.42-4.27 (m, 2 H), 4.16 (m, 1 H), 3.80 (s, 6 H), 3.51-3.34 (m,
2 H), 2.49 (d, J = 5.7 Hz, 1 H), 1.17 (d, 3 H). 13C NMR (75 MHz,
CDCl3) δ: 163.3, 158.8, 149.7, 144.4, 139.4, 135.2, 135.2, 130.0,
130.0, 128.0, 128.0, 127.2, 113.3, 101.7, 88.1, 87.0, 86.4, 78.9, 76.0,
71.7, 60.4, 57.5, 55.3, 13.8. 293.0745. HRMS (ESI-FT): calcd for
C32H32N2O8Na 595.2051; found 595.2048. ESI-MS m/z: [M þ
Na]þ found 595.1.
(1S,3R,4R,6S,7S)-1-(4,40-Dimethoxytrityloxymethyl)-7-hy-
droxy-6-methyl-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (53).
Prepared from nucleoside 49 (3.3 g, 12.2 mmol) using the general
procedure described above to provide 53 (7.0 g, 99%). 1H NMR
(300 MHz, CDCl3) δ: 9.53-9.17 (br s, 1 H), 7.86 (d, J = 8.1 Hz,
1 H), 7.50-7.26 (m, 9 H), 6.85 (d, J = 8.7 Hz, 4 H), 5.69-5.56 (m,
2 H), 4.58 (s, 1 H), 4.22 (d, J = 5.3 Hz, 1 H), 4.19-4.07 (m, 1 H),
3.78 (s, 6 H), 3.58 (d, J = 10.9 Hz, 1 H), 3.53 (d, J = 11.1 Hz, 1 H),
1.18 (d, J = 6.8 Hz, 3 H). 13C NMR (75 MHz, CDCl3) δ: 163.5,
158.7, 149.8, 144.5, 139.4, 135.4, 135.2, 130.1, 130.0, 128.0, 127.1,
113.3, 113.2, 101.8, 88.5, 86.8, 86.8, 81.2, 79.6, 71.3, 58.2, 55.2, 16.4.
HRMS (ESI-FT): calcd for C32H32N2O8Na 595.2051; found
595.2050. ESI-MS m/z: [M þ Na]þ found 595.1.
General Procedure for Uridine Phosphoramidite Synthesis.
2-Cyanoethyl tetraisopropylphosphordiamidite (1.5 equiv)
was added to a solution of nucleoside (1 equiv), tetrazole
(0.8 equiv), and N-methylimidazole (0.25 equiv) in DMF
(0.2 M). After stirring at room temperature for 8 h the reaction
was diluted EtOAc, and the organic layer was washed with
brine, dried (Na2SO4), and concentrated. Purification by chro-
matography (silica gel, eluting with 50% to 60% ethyl acetate in
hexanes) provided the corresponding phosphoramidite as a
white foam.
(1S,3R,4R,6R,7S)-7-[2-Cyanoethoxy(diisopropylamino)phos-
phinoxy]-1-(4,40-dimethoxytrityloxymethyl)-6-methoxymethyl-
3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (54). Prepared from
nucleoside 50 (2.0 g, 3.3 mmol) using the general procedure
described above to provide 54 (2.54 g, 96%). 31P NMR
(121 MHz, CDCl3) δ: 149.8, 149.5. HRMS (ESI-FT): calcd
for C42H52N4O10P 803.3416; found 803.3421. ESI-MS m/z:
[M þ H]þ found 803.1. Anal. Calcd for C42H51N4O10P.0.25
H2O:C, 62.48; H, 6.43; N, 6.94. Found: C, 62.55; H, 6.21; N, 6.85.
(1S,3R,4R,6S,7S)-7-[2-Cyanoethoxy(diisopropylamino)phos-
phinoxy]-1-(4,40-dimethoxytrityloxymethyl)-6-methoxymethyl-
3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (55). Prepared
from nucleoside 51 (2.0 g, 3.3 mmol) using the general procedure
described above to provide 55 (2.55 g, 96%). 31P NMR (121 MHz,
CDCl3) δ: 150.0, 149.8. HRMS (ESI-FT): calcd for C42H52N4O10P
803.3416; found 803.3426. ESI-MS m/z: [M þ H]þ found 803.2.
Anal. Calcd for C42H51N4O10P.0.5 H2O: C, 62.13; H, 6.46; N, 6.90.
Found: C, 62.46; H, 6.18; N, 6.72.
(1S,3R,4R,6R,7S)-7-[2-Cyanoethoxy(diisopropylamino)phos-
phinoxy]-1-(4,40-dimethoxytrityloxymethyl)-6-methyl-3-(uracil-
1-yl)-2,5-dioxabicyclo[2.2.1]heptane (56). Prepared fromnucleo-
side 52 (2.1 g, 3.7 mmol) using the general procedure described
above to provide 56 (2.7 g, 96%). 31P NMR (121 MHz, CDCl3) δ:
149.8, 149.2. HRMS (ESI-FT): calcd for C41H50N4O9P 773.3310;
found773.3314. ESI-MSm/z: [M þ H]þ found773.2. Anal. Calcd
for C41H49N4O9P.0.25H2O: C, 63.35; H, 6.42; N, 7.21. Found: C,
63.02; H, 6.37; N, 7.06.
(1S,3R,4R,6S,7S)-7-[2-Cyanoethoxy(diisopropylamino)phos-
phinoxy]-1-(4,40-dimethoxytrityloxymethyl)-6-methyl-3-(uracil-
1-yl)-2,5-dioxabicyclo[2.2.1]heptane (57). Prepared from nu-
cleoside 53 (3.0 g, 5.2 mmol) using the general procedure
described above to provide 57 (3.4 g, 83%). 31P NMR
(121 MHz, CDCl3) δ: 149.6, 149.4. HRMS (ESI-FT): calcd
for C41H50N4O9P 773.3310; found 773.3315. ESI-MS m/z:
[M þ H]þ found 773.2. Anal. Calcd for C41H49N4O9P.0.5 H2O:
C, 62.99; H, 6.45; N, 7.17. Found: C, 62.77; H, 6.60; N, 7.06.
(1S,3R,4R,6R,7S)-1-(4,40-Dimethoxytrityloxymethyl)-7-hy-
droxy-6-methyl-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (52).
Prepared from nucleoside 48 (2.1 g, 7.5 mmol) using the general
procedure described above to provide 52 (4.3 g, 99%). 1H NMR
1580 J. Org. Chem. Vol. 75, No. 5, 2010

Seth et al.
Acknowledgment. The authors thank Prof. Michael Jung
for many useful suggestions.
JOCArticle
for all phosphoramidites; protocols for oligonucleotide
synthesis and purification, thermal denaturation, and nuclease
stability experiments; analytical data for oligonucleotides
and X-ray crystal structure and CIF file for 49. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Supporting Information Available: General experimental
1
procedures for synthesis of 58-76; H and 13C NMR spectra
and analytical data for all new compounds; 31P NMR spectra
J. Org. Chem. Vol. 75, No. 5, 2010 1581