Synthesis and structure of novel conformationally constrained 1′,2′-azetidine-fused bicyclic pyrimidine nucleosides: Their incorporation into oligo-DNAs and thermal stability of the heteroduplexes

Article abstract of DOI:10.1021/jo052115x

The synthesis of novel 1′,2′-aminomethylene bridged (6-aza-2-oxabicyclo[3.2.0]heptane) "azetidine" pyrimidine nucleosides and their transformations to the corresponding phosphoramidite building blocks (20, 39, and 42) for automated solid-phase oligonucleo

Full text of DOI:10.1021/jo052115x

Synthesis and Structure of Novel Conformationally Constrained
1′,2′-Azetidine-Fused Bicyclic Pyrimidine Nucleosides: Their
Incorporation into Oligo-DNAs and Thermal Stability of the
Heteroduplexes
Dmytro Honcharenko,† Oommen P. Varghese,† Oleksandr Plashkevych, Jharna Barman, and
Jyoti Chattopadhyaya*
Department of Bioorganic Chemistry, Box 581, Biomedical Center, Uppsala UniVersity,
SE-75123 Uppsala, Sweden
jyoti@boc.uu.se
ReceiVed October 10, 2005
The synthesis of novel 1′,2′-aminomethylene bridged (6-aza-2-oxabicyclo[3.2.0]heptane) “azetidine”
pyrimidine nucleosides and their transformations to the corresponding phosphoramidite building blocks
(20, 39, and 42) for automated solid-phase oligonucleotide synthesis is reported. The novel bicyclo-
nucleoside “azetidine” monomers were synthesized by two different strategies starting from the known
sugar intermediate 6-O-benzyl-1,2:3,4-bis-O-isopropylidene-D-psicofuranose. Conformational analysis
performed by molecular modeling (ab initio and MD simulations) and NMR showed that the azetidine-
fused furanose sugar is locked in a North-East conformation with pseudorotational phase angle (P) in
the range of 44.5-53.8° and sugar puckering amplitude (φm) of 29.3-32.6° for the azetidine-modified
T, U, C, and 5-Me-C nucleosides. Thermal denaturation studies of azetidine-modified oligo-DNA/RNA
heteroduplexes show that the azetidine-fused nucleosides display improved binding affinities when
compared to that of previously synthesized North-East sugar constrained oxetane fused analogues.
Introduction
The substitution of the natural nucleoside blocks with the North-
type conformationally constrained units5,6 results in conforma-
tional preorganization of the modified single strand antisense
oligonucleotide (AON).5,6 The induced control of the geometry
in a single strand AON in turn dictates conformational
preorganization5-12 of the AON/RNA duplex to the rigid RNA/
RNA-type duplex,13-15 which can then modulate the strength
of the target RNA affinity as well as the RNase H promoted
cleavage efficiency of the target RNA strand in the hybrid
duplex, thereby facilitating the engineering of potential gene-
Conformationally constrained synthetic oligonucleotides hav-
ing furanose fused bicyclic and tricyclic carbohydrate moieties2,3
or modified pyranose derivative4 in the monomer nucleotide
units have been found to be useful for binding to the comple-
mentary mRNA, arresting its translation to protein products.
* To whom correspondence should be addressed. Fax: +46-18554495.
† Co-first authors.
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Biophys. Acta 1999, 1489, 167. (d) Meldgaard, M.; Wengel, J. J. Chem.
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10.1021/jo052115x CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/08/2005
J. Org. Chem. 2006, 71, 299-314
299

Honcharenko et al.
directed therapeutics and diagnostics.2,3,7 Recently, we have
synthesized novel 1′,2′-bridged oxetane nucleoside [1-(1′,3′-
anhydro-â-D-psicofuranosyl) nucleoside] in which the sugar
moiety is locked into a unique North-East type of conforma-
tion1,8 in the sugar pseudorotational cycle.31 The Tm of the
oxetane-modified-AON/RNA hybrid with fully natural phos-
phodiester backbone in both strands drops by ∼5-6 °C/
oxetane-T or ∼3 °C/oxetane-C modification, whereas the Tm
of oxetane-A and -G modified-AON/RNA hybrids8f do not show
any loss of stabilities compared to those of the native counter-
parts. It has been shown that any loss of the thermodynamic
stability of the heteroduplexes could, however, be regained by
the introduction of the appropriately tethered nontoxic DPPZ
(dipyridophenazine) group11,12 at the 3′-end, which also gives
additional stability against 3′-exonucleases similar to that of the
phosphorothioates AONs.1,8c The oxetane-modified appropri-
ately tethered 3′-DPPZ mixmer AON/RNA hybrids were found
to be excellent substrates for RNase H promoted cleavage.8 The
4 nt gap after each oxetane modification in the AON made this
gap resistant to RNase H promoted cleavage. However, the
RNase H promoted cleavage of the phosphodiester bonds
beyond the resistant 4 nt site in a mixmer with larger gaps (>4
nt) were very comparable to that of the native. The Michaelis-
Menten kinetics of the RNase H cleavage showed that Vmax and
Km increase with increase in the number (one to three) of T/C/A
modifications (with the exception of G, which is slightly lower
than that of the native) in the AONs, indicating higher catalytic
activity and lower enzyme binding affinity of the oxetane-
modified AON/RNA hybrids.1,8e In the â-D-LNA modified
AONs, on the other hand, requires a gap size of 8-10 nt in
order to get back the effective RNase H cleavage of the target.9
It is likely that use of a lengthy unmodified phosphodiester
oligonucleotide gaps in the gapmer AONs could be harmful in
view of their endonuclease susceptibility.10 The resistance to
the endonuclease promoted cleavage of these oxetane-modified
molecules was proportional to the number of the oxetane-
modified nucleotides per AON molecule: single modification
gave 2-fold protection to the cleavage, and double and triple
modification gave 4-fold protection compared to that of the
native phosphodiester oligonucleotide.1,8c
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1′,2′-Azetidine-Fused Bicyclic Pyrimidine Nucleosides
FIGURE 1. Chemical structures of the oxetane- and azetidine-modified pyrimidine nucleosides building blocks incorporated into oligo-DNA.
SCHEME 1a
a Reagents and conditions: (i) (a) persilylated thymine, trimethylsilyl trifluoromethanesulfonate, CH3CN, rt, 17 h, (b) persilylated uracil, trimethylsilyl
trifluoromethanesulfonate, CH3CN, rt, 17 h; (ii) 90% aq CF3COOH, rt, 30 min; (iii) MMTrCl, pyridine, rt, 20 h; (iv) 1,1′-thiocarbonyldiimidazole, toluene,
120 °C, 3 h; (v) benzyl bromide, NaH, CH3CN, 6 h; (vi) (a) 80% aq CH3COOH, rt, 24 h; (b) 1 N aq NaOH in ethanol-water, rt, overnight; (vii) Ac2O,
pyridine, rt, 3 h; (viii) 4-methoxybenzyl bromide, NaH, DMF, 6 h; (ix) sodium methoxide, MeOH, rt, 30 min; (x) methanesulfonyl chloride, pyridine, 4 °C,
overnight; (xi) potassium phthalimide, DMF, 110 °C, overnight; (xii) aq MeNH2, MeOH, rt, 4 h; (xiii) Et3N, pyridine, 90 °C, 48 h; (xiv) ceric ammonium
nitrate, CH3CN, H2O, rt, 3 h; (xv) phenoxyacetyl chloride, pyridine, rt, 3 h; (xvi) ammonium formate, 20% Pd(OH)2/C, methanol, reflux, 3 h; (xvii) DMTrCl,
pyridine, rt, overnight; (xviii) Ac2O, pyridine, rt, 28 h; (xix) (a) 2-chlorophenylphosphorodichloridate, 1,2,4-triazole, pyridine, rt, 6 h, (b) aq ammonia, rt,
overnight; (xx) NC(CH2)2OP(Cl)N(iPr)2, EtN(iPr)2, THF, rt, 2 h.
It has also been recently shown that the rationally designed
oxetane-T and -C-containing AONs effectively down-regulate
the proto-oncogene c-myb mRNA in the K562 human leukemia
cells12 in a manner that is very comparable to the gene silencing
efficiency of the phosphorothioate counterpart, thereby making
these oxetane-locked analogues potentially important for thera-
peutic use. Thus, we became interested to examine the properties
of 1′,2′-azetidine-fused AONs which are an isosteric analogue
of the oxetane-fused counterpart.
for two reasons. First, the endocyclic amino functionality of
the azetidine moiety could be utilized as a well-defined
conjugation site13 in a conformationally restricted azetidine
nucleoside, and thereby we can control the hydrophilic, hydro-
phobic, and steric requirements of a minor groove of the duplex.
Second, the amine-derivatized AONs have displayed increased
thermal affinities14 toward complementary native AONs pos-
sibly because of the introduction of positively charged moi-
eties at physiological pH and thus could influence partial
neutralization of the negatively charged phosphates in the
duplex.
The 1′,2′-azetidine-fused nucleoside (6-aza-2-oxabicyclo-
[3.2.0]heptane) analogues (Figure 1) appealed to us primarily
J. Org. Chem, Vol. 71, No. 1, 2006 301

Honcharenko et al.
SCHEME 2a
a Reagents and conditions: (i) 70% aq acetic acid, 80 °C, 5 h; (ii) 4-toluoyl chloride, pyridine, 0 °C, 4 h; (iii) Ac2O, pyridine, rt, 48 h; (iv) persilylated
uridine, TMSOTf, CH3CN, 40 °C, 2 h; (v) NaOEt, ethanol, 10 min; (vi) MMTrCl, pyridine, rt, overnight; (vii) diphenyl carbonate, DMF, 110 °C, 2 h; (viii)
benzyl bromide, NaH, CH3CN, overnight; (ix) 80% aq CH3COOH, rt, 48 h; (x) MsCl, pyridine, 0 °C, 1.5 h; (xi) NaN3, DMF, 100 °C, 60 h; (xii) aq NaOH,
rt, 2 h; (xiii) MsCl, pyridine, 0 °C, 24 h; (xiv) PMe3, THF, water, rt, 1 h; (xv) Et3N, pyridine, 90 °C, 48 h; (xvi) PACCl, pyridine, rt, 2.5 h; (xvii) BCl3,
CH2Cl2, -78 °C, 2 h, -20 °C 2 h; (xviii) DMTrCl, DMAP, pyridine, rt, 5 h; (xix) NC(CH2)2OP(Cl)N(iPr)2, EtN(iPr)2, THF, rt, 2 h; (xx) (a) Ac2O, pyridine,
rt, 24 h; (b) 2-chlorophenylphosphodichloridiate, 1,2,4-triazole, pyridine, rt, 6 h; (c) aq NH3, 5 °C, overnight; (xxi) (a) TMSCl, pyridine, 30 min; (b) isobutyryl
chloride, rt, 3 h.
We report here the synthesis of AONs incorporated with 1′,
2′-azetidine-fused nucleoside phosphoramidite building blocks
20, 39, and 42 with the desired 6-aza-2-oxabicyclo[3.2.0]heptane
skeleton (Schemes 1 and 2) as well as their incorporation into
AON/RNA heteroduplexes. A key step in the synthesis of these
conformationally restricted azetidine-C, -T, -U, and -5-Me-C
analogues appeared to be the nucleophilic displacement of a
3′-mesylate in 13 and 34 by intramolecular nucleophilic attack
of the 1′-amino group (see compounds 1, 2a, and 14 for
numbering in Scheme 1) leading to formation of the azetidine
ring. The NMR and computational structural studies are also
reported here, showing that by the introduction of the azetidine
modification the sugar ring appears conformationally locked in
the 3′-exo/4′-endo North-East conformation similar to that of
the oxetane-modified analogues.8f
desired â-anomer15 2a in 37% yield (R:â, 1:1). The â-config-
uration at anomeric C2′ was confirmed by 1D NOE difference
spectroscopy, which showed 2.3% NOE enhancement for H6-
H3′. After removing the 3′,4′-O-acetonide protection of 2a, using
90% aqueous trifluoroacetic acid at room temperature gave crude
3, which was subsequently treated with 4-monomethoxytrityl
(MMTr) chloride in dry pyridine to afford 4 in an overall 83%
(yield in two steps from 2a). Treatment of 4 with 1,1′-thio-
carbonyldiimidazole in dry toluene for 3 h at 120 °C gave the
2,3′-anhydro derivative16 5 in 75% yield, and some traces of
the starting material. Efforts to drive the reaction to completion
by keeping for a longer time resulted in the formation of some
undesired side products. The 4′-hydroxyl of 5 was protected
with a second benzyl group using benzyl bromide and sodium
hydride in dry acetonitrile to give 6a (81%). The 1′-O-MMTr
group from 6a was deprotected without any identifiable side
reactions by overnight treatment with 80% aqueous acetic acid
at room temperature to give crude 6b (>99% pure) which was
directly taken to the next step. Nucleophilic opening17 of the
2,3′-anhydro bridge in 6b by 1 N aqueous sodium hydroxide
in an ethanol-water mixture at room temperature gave the
D-fructo derivative 7 in 92% yield after two steps from 6a.
Acetylation of 7 with excess of acetic anhydride (20 equiv) in
Results and Discussion
Synthesis of 1′, 2′-Azetidine-Fused Nucleoside Phosphorami-
dite Building Block 20. For the preparation of the D-fructo
precursor 7 with â configuration, the 6′-O-benzyl-protected
psicofuranoside 1 was condensed with persilylated thymine
using trimethylsilyl triflate as the Lewis acid, which gave the
302 J. Org. Chem., Vol. 71, No. 1, 2006

1′,2′-Azetidine-Fused Bicyclic Pyrimidine Nucleosides
dry pyridine afforded 1′,3′-O-bisacetyl nucleoside 8 in 98%
yield. The N-3 of thymine base in 8 was protected with
4-methoxybenzyl (PMB)18,19 to give 9 (80%) in order to avoid
1′,2-anhydro formation during nucleophilic displacement of 1′-
mesylate in subsequent steps. Deacetylation of 9 (94%) using
sodium methoxide in methanol followed by mesylation at C1′
and C3′ hydroxyls in dry pyridine at 4 °C gave one major
product, the desired 1′,3′-bismesyl product 11a [1H NMR
(CDCl3): δ 2.89 for 1′-OMs and 2.29 for 3′-OMs] along with
the minor component, the 3′-mesyl product with a free 1′-OH
group 11b [1H NMR (CDCl3): δ 2.29 for 3′-OMs]. Since the
bismesyl product was found to be unstable during column
chromatography, we treated the crude reaction products 11a/
11b with an excess of potassium phthalimide in dry DMF at
110 °C overnight20 to afford 1′-phthalimido-3′-O-Ms-derivative
12 in 77% yield (after two steps from 10; the starting 11b was
recovered in 7% yield). The selective displacement of 1′-mesy-
late from 11a to yield 12 keeping the 3′-mesylate group of 11a
intact was possible because the 3′-carbon is sterically more
hindered in D-fructo sugar compared to that of the D-arabino
analogue. Further experimental evidence of the 3′-center to be
more sterically hindered than C1′ is provided by the selective
displacement of 1′-mesylate in 30 by azide, resulting in 31 which
has the 2,3′-anhydro bond left intact (Scheme 2).
strategy for the synthesis of 1′,2′-azetidine-fused uridine (39)
and cytidine (42) phosphoramidite solid-phase oligodeoxynucle-
otide synthesis building blocks (Scheme 2) due to low yields
of the desired â-anomer 24 in the coupling of the persilylated
uracil with the 6-O-benzyl-1,2:3,4-di-O-isopropylidene-â-D-
psicofurnose (1), as shown in Scheme 1, which gave a complex
anomeric mixture (R:â, 1:0.8), from which only 23% of the
â-anomer could be isolated. By utilizing 2-O-acetyl-6-O-benzyl-
1,3,4-tri-O-(4-toluoyl)-D-psicofuranose 23 as a coupling precur-
sor, the desired â-anomer 24 was obtained in 65% yield from
an anomeric mixture in which the â-anomer was the predomi-
nant constituent (R:â, 1:9) (Scheme 2). The â-configuration at
the anomeric C2′ center was confirmed by 1D NOE difference
spectroscopy, which showed 1.9% enhancement for H6-H3′.
Another advantage of this modified synthetic scheme, as shown
in Scheme 2, was keeping the 2, 3′-anhydro bond intact until
the 1′-mesylate in 30 was displaced with the azido group to
give 31, thereby avoiding a key nucleobase protection step at
N-3 used earlier in Scheme 1. Thus the opening of the 2,3′-
anhydro bond in 31 gave a direct entry to the desired â-D-fructo
configured nucleoside 32 by bypassing acetylation/deacetylation
and thyminyl N-3 protection/deprotection steps (in 8 f 10/15
in Scheme 1).
The psico-sugar 1 was treated with 70% aqueous acetic acid
at 80 °C for 5 h followed by 4-toluoylation using 4-toluoyl
chloride in dry pyridine to afford 22 as an anomeric mixture
(∼1:1) in 75% yield. Acetylation of 22 in dry pyridine with a
20-fold excess of acetic anhydride in 48 h at room temperature
gave 23 (90%) as an anomeric mixture. The coupling between
23 and the uracil nucleobase using Vorbruggen-type reaction25,27
utilizing trimethylsilyltriflate as Lewis acid and N,O-bis-
(trimethylsilyl)acetamide as silylating agent afforded 24 in 65%
yield. Complete detoluoylation of nucleoside 24 was ac-
complished with 1 M NaOEt in ethanol to give intermediate
25, which was subsequently monomethoxytritylated as for 4
and gave 26 in 75% yield in two steps from 24. The use of
1,1′-thiocarbonyldiimidazole16 in toluene at 120 °C to obtain
the anhydro derivative 27, as used in Scheme 1, gave some
undesired side products. This problem was circumvented by
treating 26 with diphenyl carbonate28 and NaHCO3 in DMF at
110 °C and afforded 27 in 91% yield. Benzylation of the 4′-
hydroxyl of 27 went smoothly to give 28 (85%), from which
1′-O-MMTr was deprotected with 80% aqueous acetic acid to
give 29 (79%) along with D-fructo-nucleoside as a byproduct
(11%). Compound 29 upon treatment with Ms-Cl (3 equiv) in
dry pyridine at 0 °C for 1.5 h gave the expected 1′-O-mesylated
product 30 (89%) along with traces of starting material, while
any prolongation of the reaction time in an attempt to drive the
reaction to completion reduced the yield considerably. Displace-
ment of 1′-mesylate of 30 with NaN3 in DMF at 100 °C went
smoothly with no traces of any side reactions to give 31 (87%).
Nucleophilic opening of the 2,3′-anhydro bond in 31 with 1 M
NaOH in THF gave the desired D-fructo-nucleoside 32 with 85%
yield along with traces of nonnucleosidic sugar impurity. The
nucleoside 32 was mesylated at C3′ with Ms-Cl in dry pyridine
at 0 °C, which furnished a key precursor 33 in 90% yield.
Reduction of the azide was carried out under Staudinger’s
conditions29 (PMe3, THF, and H2O), which afforded 34 in 90%
yield.
The deprotection of 1′-phthalimido group from 12 by aqueous
methylamine in methanol at room temperature21 gave the
corresponding 1′-amino derivative 13 (93%). Multiple attempts
to perform azetidine cyclization at ambient temperature by
intramolecular nucleophilic attack of the 1′-amino group with
concomitant demesylation at C3′ were carried out under variety
of conditions, such as 1 M NaOH in dioxane, NaOEt in ethanol,
or 1 M NaHMDS in THF, all of which proved to be
unsuccessful except for a mixture of Et3N and pyridine at 90
°C for 48 h, which gave the azetidine-modified nucleoside 14
in 66% yield. Deprotection of N3-PMB group was performed
using ceric ammonium nitrate in aqueous acetonitrile (1:9, v/v)18
at room temperature, giving 15 in 62% yield. Phenoxyacetyl
(PAC) protection was employed to protect the azetidine-nitrogen
to avoid N-branching during the solid-phase oligonucleotide
synthesis using the phosphoramidite approach.22 Other protecting
groups such as acetyl and trifluoroacetyl were not useful in our
case as acetyl deprotection was sluggish, whereas trifluoroacetyl
group was found to be unstable. The PAC-protected nucleoside
was obtained by treating 15 with PAC-Cl in pyridine, which
1
afforded a mixture of diastereomers 16 (by H and 13C NMR)
in 86% yield as a result of chiral tervalent azetidine-nitrogen
atom,23 which was also found for the nitrogen-derivatives of
the LNA analogues.24 Debenzylation of 16 using ammonium
formate and 20% Pd(OH)2/C25 gave 17 in 92% yield. Treatment
of 17 with 4,4′-dimethoxytrityl (DMTr) chloride in pyridine
afforded 18a in 73% yield. The 4′-hydroxyl of DMTr-protected
nucleoside 18a was acetylated and the product 18b was
converted into the 5-methylcytosine derivative via the C-4-
triazolyl intermediate,26 where thymine was initially activated
by conversion into the 1,2,4-triazole derivative, which was
subsequently displaced with ammonia along with deacetylation
afforded 19 (70%). Phosphitylation of 18a with 2-cyanoethyl-
N,N-diisopropylphosphoramidochloridite8b gave the desired
amidite 20 in 86% yield [31P NMR (CDCl3): δ 154.7, 150.7,
149.9, 148.7] for the solid-phase synthesis.
Cyclization of 34 by intramolecular nucleophilic attack of
the 1′-amino group with concomitant demesylation at C3′, as
performed in Scheme 1 (Et3N and pyridine at 90 °C), gave
Synthesis of 1′,2′-Azetidine-Fused Phosphoramidite Build-
ing Blocks 39 and 42. We have, however, envisaged a different
J. Org. Chem, Vol. 71, No. 1, 2006 303

Honcharenko et al.
TABLE 1. Sequences Synthesized and Thermal Denaturation Studies toward Complementary RNA Targets and Comparison with
Oxetane-Modified Sequencesa
a Tm values were measured as the maximum of the first derivative of the melting curve (A260 vs temperature) recorded in medium salt buffer (60 mM
Tris-HCl at pH 7.5, 60 mM KCl, 0.8 mM MgCl2, and 2 mM DTT) with temperature range 20-70 °C using 1 µM concentrations of the two complementary
strands; ∆Tm ) Tm relative to native duplex; ∆Tm1 ) Tm relative to oxetane-modified duplex; “nd” ) not determined. Results of 2a-5a and 10a were taken
from refs 8c and 9.
fused-azetidine-modified uracil nucleoside 35 (63%). Protection
of the azetidine ring using PAC-Cl in dry pyridine, as used for
15, gave 36 in 84% yield. This compound was found to be a
mixture of diastereomers by 1H and 13C spectroscopy as it was
previously found for 16. Debenzylation of 36 with 20% Pd-
(OH)2/C, ammonium formate,25 and 20% Pd(OH)2/C, H2, in
ethanol did not work satisfactorily in our hands, but the reaction
proceeded to completion very smoothly with 1 M BCl35r in CH2-
Cl2 at -78 °C. The crude product obtained was selectively
dimethoxytritylated to 38 (85% in two steps from 36) followed
by phosphitylation in the same way as was done for 20 furnished
the desired phosphoramidite building block 39 (93%) as a
mixture of four diastereomers [31P NMR (CDCl3): δ 154.6,
150.6, 149.9, and 149.2]. The 6′-O-DMTr-protected uracil
nucleoside 38 was acetylated using acetic anhydride in pyridine,
and the product 38a obtained was converted into the cytosine
nucleoside 40 (70%) using the procedure used for 19 (Scheme
1).26 This nucleoside was transiently protected by trimethylsi-
lylation followed by treatment with isobutyryl chloride in
pyridine and subsequent desilylation to afford 41 in 83% yield.
Compound 41 thus obtained was transformed to its correspond-
ing phosphoramidite 42 (92%) [31P NMR (CDCl3): δ 154.4,
150.6, 149.6, 149.0].
were spectroscopically pure and their properties are documented
in the Experimental Section. Structure determinations of these
compounds were based upon 1H NMR, COSY, HETCORR, and
NOE experiments as well as by MALDI-TOF mass spectrom-
etry.
Thermal Stabilities (Tm) of Azetidine-Modified AON/RNA
Hybrids. The thermostability of the azetidine-modified AONs
and their oxetane counterparts measured at pH 7.5 are shown
in Table 1. Single azetidine-T incorporation enhances the Tm
of the AON/RNA duplex by ∼1 °C compared to the isosequen-
tial oxetane-T modified hybrid duplex. However aze-C incor-
poration enhanced the Tm by ∼2 °C with respect to oxetane-
C-modified AON/RNA duplexes. Aze-U incorporation did not
improve the target binding affinity in comparison with aze-T
modified hybrid duplexes. Although the moderate improvement
in binding affinity of azetidine-modified AONs at pH 7.5 was
encouraging, we argued that lowering of the pH to 6 might
enhance the target affinity even further owing to the efficient
protonation of the azetidine-nitrogen (pKa ) 6.07 for 3′,5′-
bisethyl phosphate of azetidine-locked T) leading to partial
neutralization of the negative charge of the phosphate backbone.
To check this possibility we have measured the Tm of azetidine-
modified AON/RNA hybrids at pH 6 (Tables 2). Surprisingly,
there was no significant Tm increment observed at this pH
compared to the one at pH 7.5. This suggests that the protonation
of azetidine-nitrogen does not lead to effective phosphate charge
Phosphoramidites 20, 39, and 42 were incorporated at
different positions of mixed 15-mer AON sequences as depicted
in Table 1. Their structural integrity was confirmed by mass
measurements by MALDI-TOF spectroscopy. All compounds
304 J. Org. Chem., Vol. 71, No. 1, 2006

1′,2′-Azetidine-Fused Bicyclic Pyrimidine Nucleosides
3
3
3
FIGURE 2. Experimental JH-3′,H-4′ and JH-4′,H-5′ vicinal proton coupling constants (Hz) (panel A) and experimental JH-4′,H-5′. vs calculated
pseudorotational phase angle P (panel B) for the oxetane-modified T and C and azetidine-modified T, C, 5-Me-C, and U nucleosides compared to
the theoretical dependences (colored lines) of the 3JH,H calculated for 0 e P e 360° employing a generalized Karplus-type equation (P1 ) 13.24°,
P2 ) -0.91°, P3 ) 0, P4 ) 0.53°, P5 ) -2.41°, P6 ) 15.5°) developed by Altona et al.31 at fixed puckering amplitudes of 30° (blue dashed line),
35° (red dot-dashed line), 40° (black line), and 45° (black dotted line). The experimental coupling constants for the oxetane- and azetidine-
1
modified nucleosides were obtained through 600 MHz H NMR spectra (DMSO-d6 + CD3OD) at 298 K.
TABLE 2. Thermal Denaturation Studies at 110 and 40 mM [Na+]
endocyclic vicinal proton-proton coupling constant, thus deny-
pH 6, 110 mM [Na+]a
pH 6, 40 mM [Na+]b
ing straightforward approach to determine sugar conformation
3
from three related endocyclic JH,H of the sugar moiety.
c
c
AON
Tm
∆Tm
Tm
37
32
32
31.5
30
29.5
31.5
33
31.5
36
36
∆Tm
However, the conformation adopted by the pentose in the
bicyclic azetidine-modified nucleosides (azetidine-modified T,
U, C, and 5-Me-C, Figure 1), can still be estimated with high
accuracy from the only remaining experimental 3JH-3′,H-4′ and
3JH-4′,H-5′ (Figure 2) coupling constants. These estimations can
further be verified by direct theoretical modeling using a
generalized Karplus-type equation developed by Altona et
1
2
3
4
5
6
7
8
43.5
39
39.5
39.5
39
39.5
39.5
39
-4.5
-4.0
-4.0
-4.5
-4.0
-4.0
-4.5
-4.0
-1.5
-1.5
-5.0
-5.0
-5.5
-7.0
-7.5
-5.5
-4.0
-5.5
-1.0
-1.0
3
al.,31,32 which sets empirical rules relating JH,H′ coupling
9
10
11
39
42
42
constants of pentose with values of the respective torsion angles.
A combination of the 3JH-2′,H-3′ and 3JH-3′,H-4′ provides a good
quantitative measure of the pseudorotational phase angle (see
Figure 2) except for the region of relative ambiguity when
simultaneously the 3JH-2′,H-3′ constant is in the range of 3 to 5
a 0.1 mM EDTA, 100 mM sodium chloride, 10 mM sodium phosphate,
pH 6.0. b 0.1 mM EDTA, 30 mM sodium chloride, 10 mM sodium
phosphate, pH 6.0. c ∆Tm is melting temperature of the azetidine-modified
duplex relative to its native counterpart.
3
Hz while the JH-3′,H-4′ is in the range of 5-6 Hz.
3
3
The JH-3′,H-4′ and JH-4′,H-5′ experimental NMR coupling
constants of the azetidine-modified T, U C, and 5-Me-C
neutralization, presumably because both entities are far apart
from each other.
nucleosides (Figure 2, panel A) are in the ranges 5.6-5.7 and
3
To check the role of electrostatics in RNA binding affinity
of azetidine-modified AONs, we have determined the Tm values
at high (100 mM) and low (40 mM) salt concentration at pH 6
(Table 2). In these conditions the thermostability of the AON/
RNA hybrids increases at higher salt concentration but decreases
at the lower salt concentration, which means that the intramo-
lecular electrostatics-modulated stabilization is negligible com-
pared to the external salt effect.30
Conformational Analysis of the Azetidine-Fused Sugar
Moiety Based on Experimental 3JH,H Coupling Data. Fusion
of the azetidine ring into sugar moiety removes the H1′ proton
from the native pentose and, correspondingly, the important
8.2-8.4 Hz (Table 3), respectively, compared to JH-3′,H-4′
)
3
3.9 Hz and JH-4′,H-5′ ) 8.5 Hz observed for the oxetane-
modified T and C.10 Comparison of the estimated JH-3′,H-4′
3
vs 3JH-4′,H-5′ dependence calculated using the standard Karplus
equation31 with the experimentally obtained proton coupling
constants suggests that the sugar conformations of the azetidine-
C, -T, -U, and -5-Me-C nucleosides is of the North-type (Figure
2, panel A) with its pseudorotational phase angle (P) being in
a range of 55-75°, which is apparently much higher compared
(32) Altona, C.; Sundaralingam, M. J. Am. Chem. Soc. 1972, 94, 8205
and references therein.
J. Org. Chem, Vol. 71, No. 1, 2006 305

Honcharenko et al.
TABLE 3. Sugar Moiety Conformational Parameters (ν0-ν4 Torsions, Pseudorotational Phase Angle P, Puckering Amplitude Om) and Related
3
3
Experimental and Calculated JH-3′,H-4′ and JH-4′,H-5′ Vicinal Proton NMR Coupling Constants of the Azetidine-C, -T, -U, and -5-Me-C
Nucleosidesa
sugar conformational
parameters
azetidine-T
azetidine-U
azetidine-C
azetidine-5-Me-C
ν0: C5′-O5′-C2′-C3′
ν1: O5′-C2′-C3′-C4′
ν2: C2′-C3′-C4′-C5′
ν3: C3′-C4′-C5′-O5′
ν4: C4′-C5′-O5′-C2′
phase angle P
-13.22° (-6.4 ( 12.2°)
-6.16° (-8.9 ( 6.3°)
20.94° (19.3 ( 11.9°)
-28.96° (-24.6 ( 18.1°)
27.18° (19.8 ( 19.4°)
44.45° (37.2 ( 27.0°)
29.33° (25.1 ( 18.2°)
-19.33° (-5.3 ( 12.5°)
-1.39° (-9.9 ( 6.5°)
19.21° (19.8 ( 11.1°)
-30.90° (-24.2 ( 17.1°)
33.06° (18.9 ( 18.0°)
53.84° (33.6 ( 31.7°)
32.56° (25.5 ( 16.5°)
-13.91° (-5.8 ( 12.3°)
-5.83° (-9.0 ( 6.6°)
21.03° (18.9 ( 11.7°)
-29.45° (-23.7 ( 17.7°)
27.97° (18.8 ( 18.1°)
45.36° (35.9 ( 29.6°)
29.93° (24.5 ( 17.6°)
-19.25° (-6.81 ( 11.4°)
-1.73° (-10.2 ( 6.5°)
19.64° (20.9 ( 9.8°)
-31.32° (-25.8 ( 14.9°)
32.32° (20.5 ( 15.8°)
53.33° (32.4 ( 29.7°)
32.88° (27.0 ( 13.9°)
puckering amplitude φm
vicinal proton coupling
constants
azetidine-T
azetidine-U
azetidine-C
azetidine-5-Me-C
H3′-C3′-C4′-H4′ calc
3JH-3′,H-4′ calc, Hz
3JH-3′,H-4′ exptl, Hz
error, Hz
35.22
5.68
5.60
0.08
32.86
5.95
5.69
0.26
35.48
5.65
33.39
5.89
5.60
0.29
5.69
-0.04
H4′-C4′-C5′-H5′ calc
3JH-4′,H-5′ calc, Hz
3JH-4′,H-5′ exptl, Hz
error, Hz
-152.98
8.54
-155.54
8.83
-153.38
8.58
-155.82
8.86
8.31
8.25
8.22
8.31
0.23
0.58
0.36
0.55
sugar conformational
parameters
oxetane-C
oxetane-T
ν0: C5′-O5′-C2′-C3′
ν1: O5′-C2′-C3′-C4′
ν2: C2′-C3′-C4′-C5′
ν3: C3′-C4′-C5′-O5′
ν4: C4′-C5′-O5′-C2′
phase angle P
-15.20° (-9.7 ( 8.5°)
-8.57° (-10.7 ( 5.9°)
26.34° (25.0 ( 6.7°)
-35.76° (-32.0 ( 9.5°)
32.66° (26.7 ( 10.5°)
42.79° (35.3 ( 18.2°)
36.55° (32.3 ( 8.7°)
-14.30° (-10.5 ( 9.1°)
-8.89° (-10.2 ( 6.2°)
26.03° (24.9 ( 7.5°)
-34.88° (-32.4 ( 10.7°)
31.52° (27.5 ( 11.6°)
41.87° (36.6 ( 20.7°)
35.60° (32.9 ( 9.5°)
puckering amplitude, φm
a Corresponding sugar conformation parameters of the oxetane-modified C and T10 are also show for comparison. Theoretical values were calculated
using optimized parameters for the standard Karplus equation30 without electronegativity corrections: (3JH′,H′ ) A cos2 φ + B cos φ + C, A ) 7.76, B )
-1.10, C ) 1.4) and corresponding H3′-C3′-C4′-H4′ and H4′-C4′-C5′-H5′ torsions from (a) ab initio 6-31G** Hartree-Fock optimized by GAUSSIAN
9835 molecular geometries and (b) from unrestrained 2 ns MD simulation by Amber 736 averaged in 1.5-2 ns time interval of the respective simulations
(shown in parentheses).
to 39.8° < P < 42.8° observed for the oxetane-modified
nucleosides.8f However, only preliminary conclusions can be
drawn at this point as the theoretical dependences by standard
Karplus equation31 shown in Figure 2A provide only rough
estimation. Further analysis of the sugar conformation in
azetidine pyrimidines is provided in the next section discussing
conformational parameters obtained from the ab initio and
molecular dynamics (MD) simulations.
The dynamics of proton exchange with water, which has
direct effect on the observed vicinal proton constants, can be
used as a measure of proton exposure to the solvent. As the
3
3JH-3′,H-4′ and JH-4′,H-5′ of the azetidine-modified C, T, U,
(33) (a) Obika S.; Nanbu D., Hari Y., Morio, K.; In, Y.; Ishida, T.;
Imanishi, T. Tetrahedron Lett. 1997, 38, 8735. (b) Koshkin, A. A.; Singh,
S. K.; Nielson, P., Rajwanshi, V. K.; Kumar R.; Meldgaard, M.; Wengel,
J. Tetrahedron 1998, 54, 3607. (c).Wengel, J. Acc. Chem. Res. 1999, 32,
301.
FIGURE 3. Empirical standard and generalized Karplus dependencies37
(34) Ravn, J.; Nielsen, P. J. Chem. Soc., Perkin Trans. 1 2001, 985.
3
compared to the experimental JH,H constants (Hz) for the H-3′,H-4′
(35) Tarko¨y, M.; Bolli, M.; Schweizer, B.; Leumann, C. HelV. Chim.
and H-4′,H-5′ proton-proton interactions in the azetidine-modified
Acta 1993, 76, 481.
pyrimidine nucleosides plotted against the values of the H3′-C3′-
C4′-H4′ and H4′-C4′-C5′-H5′ torsions from the 6-31G** ab initio
calculated molecular structures of the azetidine-modified C, T, U, and
5-Me-C nucleosides.
(36) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,
M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, Jr., J. A.;
Stratmann, R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels, A.
D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi,
M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.;
Ochterski, J.; Petersson, G.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick,
D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.;
Ortiz, J. V.; Baboul, A. G.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz,
P.; Komaromi, I.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-
Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe,
M.; Gill, P. M. W.; Johnson, B. G.; Chen, W.; Wong, M. W.; Andres, J.
L.; Head-Gordon, M.; Replogle E. S.; Pople, J. A. GAUSSIAN 98, Revision
A.6; Gaussian, Inc.: Pittsburgh, PA, 1998.
and 5-Me-C have been found to be temperature-independent,
the 1H NMR data suggest that the sugar moiety of the azetidine-
modified nucleosides is locked in the North-East conforma-
tion,33a-c,34,35 as found for the oxetane-modified nucleosides.8f
Theoretical Conformational Analysis (ab Initio and MD
Simulations) of Azetidine-Fused Nucleosides. Conformational
306 J. Org. Chem., Vol. 71, No. 1, 2006

1′,2′-Azetidine-Fused Bicyclic Pyrimidine Nucleosides
FIGURE 4. Superimposed molecular 3D ab initio and MD (10 snapshots taken from 1.5 to 2 ns time interval of respective MD trajectories)
structures of the oxetane-C and -T (left panels A and B) and azetidine-C, -T, -5-Me-C, and -U (right panels C and D) shown in two projections
(see included axis schemes): (1) from the top (columns A and C) and (2) vertically rotated by 90° around the y-axis (columns B and D).
analysis of the azetidine-modified C, T, U, and 5-Me-C
nucleosides has been also performed on the basis of (a) ab initio
gas-phase geometry optimizations using 6-31G** Hartree-Fock
in the Gaussian 9836 program package and (b) results of 2 ns
AMBER 737 molecular dynamics (MD) simulations in the
explicit aqueous medium to probe a conformational hyperspace
available for these systems (see details in Experimental section).
3
3JH-3′,H-4′ and JH-4′,H-5′ coupling constants have been back-
calculated from the ab initio obtained torsions employing
standard and generalized Karplus equations31,32 and compared
with the experimental ones (Figure 3, Table 3).
(i) Sugar Pucker Conformation. Contrary to the rough
estimation of the sugar pucker from the 3JH-3′,H-4′ vs 3JH-4′,H-5′
dependence (Figure 2, panel A), which showed North-type
but much closer to the East than to the North sugar conforma-
tion, both ab initio and MD obtained geometries have demon-
strated that the sugar in the azetidine-modified pyrimidine
(37) Case, D. A.; Pearlman, D. A.; Caldwell, J. W.; Cheatham, T. E.,
III; Wang, J., Ross, W. S.; Simmerling, C. L.; Darden, T. A.; Merz, K. M.;
Stanton, R. V.; Cheng, A. L.; Vincent, J. J.; Crow-ley, M.; Tsui, V.; Gohlke,
H.; Radmer, R. J.; Duan, Y.; Pitera, J.; Massova, I.; Seibel, G. L.; Singh,
U. C.; Weiner, P. K.; Kollman, P. A. AMBER 7, University of California,
San Francisco, 2002.
J. Org. Chem, Vol. 71, No. 1, 2006 307

Honcharenko et al.
nucleosides assumes North-East conformation (44° < P < 54°,
29° < φm < 33° from ab initio results) similar to that of the
oxetane-modified T and C nucleosides8f with P being within
2° of the corresponding value for the oxetane-C and T (Table
3) and puckering amplitude (φm) being ∼7° lower compared to
the oxetane-T and -C values. As expected, lower electronega-
tivity of nitrogen atom compared to the oxygen led to about
0.02 Å increase in C2′-C3′ and C3′-C4′ bonds and weakened
restraining power of the azetidine moiety compared to the
oxetane. This resulted in broader conformational subspace
available for the azetidine-modified sugar moiety, which has
been reflected in higher by ∼10° amplitude of the P variation
during MD trajectories (compare average P ) 34.8 ( 29.5°,
φm ) 34.8 ( 29.5° for the azetidine-modified pyrimidine series
with corresponding P ) 36.0 ( 19.7°, φm ) 32.6 ( 9.1° for
the oxetanes). Although for the azetidine-U and -5-Me-C the
ab initio obtained sugar puckers both the P and φm were found
to be ∼10° higher than that of the azetidine-T and -C, the
apparent gas phase conformational preference disappeared in
the MD dynamic model (Table 3) resulting in very close
corresponding average values of P and φm and similar variations
along the trajectories for all azetidine-modified pyrimidines in
question.
Conclusions
Fused azetidine derivatives of T, U, and C phosphoramidites
building blocks were synthesized from 6-O-benzyl-1,2:3,4-bis-
O-isopropylidene-D-psicofuranose and incorporated into 15 mer
AONs. The thermal denaturation studies revealed that the
azetidine-modified AONs showed improved target affinity when
compared to the isosequential oxetane-modified AON/RNA
hybrids. The molecular structures of these monomer units have
been studied by means of high-field NMR and theoretical ab
initio and MD simulations. The combined experimental NMR
data and theoreticalsimulations show that azetidine modification
leads sugar moiety into locked North-East conformation with
the puckering amplitudes and phase angles similar to those
observed in the class of oxetane-modified nucleosides.8f How-
ever, the relatively less electronegative nature of the nitrogen
atom compared to the oxygen atom decreases the strength of
restrains introduced by the azetidine moiety on the sugar
conformation, and although it leads to very similar average sugar
conformation, the dynamics of the sugar pucker in the azetidine-
modified nucleoside increases P fluctuation amplitude by almost
30%. Broader conformational subspace available for the aze-
tidine modification can potentially be a decisive factor for the
in vivo and in vitro applications, making these oligos better
substrate for a selected target. Further work is in progress.
(ii) RMSd Differences. RMS differences of the sugar atoms
along MD trajectories during simulations of azetidine-modified
nucleosides have been found to be 0.09 ( 0.07 Å (0.32 ( 0.14
Å for all heavy atoms), 0.09 ( 0.05 Å (0.38 ( 0.15 Å), 0.09 (
0.03 Å (0.34 ( 0.10 Å), and 0.11 ( 0.06 Å (0.40 ( 0.12 Å)
for the azetidine-C, -T, -U and -5-Me-C respectively. The RMS
difference in positioning of the sugar atoms in the ab initio
optimized geometries of these nucleosides has only been 0.01
Å. Figure 4 compares superimposed ab initio and MD (10
snapshots) structures of the oxetane-C and -T (left panels A
and B) and azetidine-C, -T, -5-Me-C, and -U (right panels C
and D) nucleosides. The major movements have been observed
for the aglycon part of the azetidine-modified nucleosides similar
to conclusions drawn for the oxetane-modified nucleosides.10
However, significantly more dynamics can be seen for the
backbone atoms and the azetidine moiety (up to 0.5 Å)
compared to the oxetane-modified nucleosides. However, overall
sugar conformation is very similar in azetidine and oxetane-
modified nucleosides (sugar atoms RMSd ) 0.06 Å).
Experimental Section
General experimental methods are given in Supporting Informa-
tion. All NMR data are given in δ scale.
1-[6-O-Benzyl-3,4-O-isopropylidene-â-D-psicofuranosyl]thy-
mine (2a). Thymine (7.1 g, 56.4 mmol) was suspended in
hexamethyldisilazane (140 mL); trimethylchlorosilane (13.9 mL)
was added and stirred at 120 °C under a nitrogen atmosphere for
16 h. The volatile material was evaporated and the residue was
dried for 20 min. Sugar 1 (14.1 g, 40.3 mmol) dissolved in dry
acetonitrile was added to the persilylated nucleobase. The mixture
was cooled in an ice bath and trimethylsilyl trifluoromethane-
sulfonate (9 mL, 52.3 mmol) was added dropwise under nitrogen
atmosphere. After being stirred for 1 h the reaction was warmed to
room temperature and stirred for 17 h. Saturated aqueous NH4Cl
was added with stirring for additional 30 min, followed by filtration.
To this residue was added saturated aqueous NaHCO3 and the
mixture was extracted with CH2Cl2 (4 times). The organic phase
was dried over MgSO4, filtered, and evaporated. The resultant oil
was chromatographed using 0 to 3% MeOH in CH2Cl2 as eluent
to furnish 2a (6.23 g, 14.9 mmol, 37%). Rf ) 0.45 (CH2Cl2/CH3-
OH 92:8 v/v); MALDI-TOF m/z [M - H]- found 417.1, calcd
(iii) Coupling Constants. The empirical Karplus equation31,32
provided us means to back-calculate the 3JH,H vicinal coupling
constants from the ab initio geometries. Theoretical values
calculated using optimized parameters for the standard Karplus
equation31 without electronegativity corrections (3JH′,H′ ) A cos2
φ + B cos φ + C; A ) 7.76, B ) -1.10, C ) 1.4) applied to
ab initio calculated H3′-C3′-C4′-H4′ and H4′-C4′-C5′-
H5′ torsions appeared to provide somewhat better agreement
1
417.1; H NMR (270 MHz, CDCl3) 7.56 (d, J ) 1.11 Hz, 1H,
H-6), 7.34-7.24 (m, 3H, benzyl), 7.15-7.11 (m, 2H, benzyl), 5.30
(d, JH-3′,
) 5.94 Hz, 1H, H-3′), 4.75 (dd, JH-4′,
) 0.87
H-5′
H-4′
Hz, 1H, H-4′), 4.63 (m, 1H, H-5′), 4.43 (d, Jgem ) 11.63 Hz, 1H,
CH2Ph), 4.32 (d, 1H, CH2Ph), 4.25 (d, Jgem ) 12.25 Hz, 1H, H-1′),
3.80 (d, 1H, H-1′′), 3.66-3.51 (m, 2H, H-6′, H-6′′), 2.86 (br s,
1H, OH), 1.78 (s, 3H, CH3, thymine), 1.56 (s, 3H, CH3, isopropyl),
1.35 (s, 3H, CH3, isopropyl); 13C NMR (67.9 MHz, CDCl3) 164.4
(C-4), 150.1 (C-2), 138.0 (C-6), 136.7, 128.4, 128.0, 127.0, 112.9
(C-5), 107.7, 101.1 (C-2′), 86.1 (C-3′), 84.6 (C-5′), 82.0 (C-4′),
73.5 (CH2Ph), 70.4 (C-6′), 64.5 (C-1′), 25.7 (CH3, isopropyl), 24.3
(CH3, isopropyl), 12.4 (CH3, thymine).
3
3
with the experimental JH-3′,H-4′ and JH-4′,H-5′ values than
obtained employing the modified Karplus equation32 (P1 ) 7.76,
P2 ) 1.1, P3 ) 1.4, P4 ) P5 ) P6 ) 0). Maximum absolute
3
3
error of the calculated JH-3′,H-4′ and JH-4′,H-5′ constants has
been 0.58 Hz compared to experimental value (Table 3, Figure
3
3). The experimental values of JH-4′,H-5′ for the azetidine-
1-[6-O-Benzyl-1-O-(4-methoxytrityl)-â-D-psicofuranosyl]thy-
mine (4). Compound 2a (6.23 g, 14.9 mmol) was stirred with 75
mL of 90% aqueous CF3COOH at room temperature for 30 min.
The reaction mixture was evaporated to give 3. The crude reaction
mixture was coevaporated with pyridine (4 times) and dissolved
in 125 mL of the same solvent, and 4-methoxytrityl chloride (5.29
modified nucleosides fit well with the corresponding values of
the oxetane-C and -T (Figure 2, panel B) while the difference
3
in the experimental JH-3′,H-4′ directly derived from the H3′-
C3′-C4′-H4′ torsions explains the ∼7° decrease in the
puckering amplitude of the azetidine-modified pyrimidines.
308 J. Org. Chem., Vol. 71, No. 1, 2006

1′,2′-Azetidine-Fused Bicyclic Pyrimidine Nucleosides
g, 17.15 mmol) was added and stirred at room temperature for 20
h under nitrogen atmosphere. The reaction mixture was poured into
saturated aqueous NaHCO3 and extracted with CH2Cl2 (3 times).
The organic phase was dried over MgSO4, filtered, and evaporated.
The residue was coevaporated with toluene (4 times) and CH2Cl2
(3 times) followed by purification by silica gel column chroma-
tography using 0 to 3% MeOH in CH2Cl2 as eluent to afford 4
(8.1 g, 12.46 mmol, 83% after 2 steps). Rf ) 0.35 (CH2Cl2/CH3-
OH 95:5 v/v); MALDI-TOF m/z [M + Na]+ found 673.1, calcd
1-[4,6-O-Benzyl-â-D-fructofuranosyl]thymine (7). Compound
6a (5.47 g, 7.57 mmol) was dissolved in 220 mL of 80% aqueous
acetic acid and stirred at ambient temperature for 24 h. The acid
was removed under reduced pressure, coevaporated with toluene
(4 times) and CH2Cl2 (3 times) to give 6b. The product obtained
without further purification was dissolved in 70 mL of EtOH-
H2O solution (v/v 1:1), and 13 mL of 1 N NaOH was added,
followed by dropwise addition of dioxane till the solution becomes
homogeneous. The mixture was stirred overnight, then poured into
saturated aqueous NaHCO3, and extracted with CH2Cl2 (4 times).
The organic phase was dried over MgSO4, filtered, evaporated, and
coevaporated with toluene (4 times) and CH2Cl2 (3 times). The
residue was subjected to column chromatography using 0 to 5%
MeOH in CH2Cl2 as eluent to give 7 (3.26 g, 6.96 mmol, 92%
after two steps). Rf ) 0.37 (CH2Cl2/CH3OH 92:8 v/v); MALDI-
TOF m/z [M - H]- found 467.1, calcd 467.1; 1H NMR (270 MHz,
CDCl3) 7.75 (s, 1H, H-6), 7.39-7.20 (m, 10H, benzyl), 4.72-4.62
(m, 2H, 1 × CH2Ph, H-3′), 4.57-4.46 (m, 3H, 2 × CH2Ph, 1 ×
CH2Ph), 4.41 (m, 1H, H-5′), 4.29-4.13 (m, 2H, H-1′, OH), 4.04-
3.93 (m, 2H, H-4′, H-1′′), 3.70-3.54 (m, 2H, H-6′, H-6′′), 1.80 (s,
3H, CH3, thymine); 13C NMR (67.9 MHz, CDCl3) 164.8 (C-4),
151.0 (C-2), 138.2 (C-6), 136.9, 136.8, 128.5, 128.1, 127.9, 127.7,
127.6, 108.9 (C-5), 101.2 (C-2′), 85.1 (C-4′), 83.6 (C-5′), 76.0 (C-
3′), 73.5 (CH2Ph), 71.7 (CH2Ph), 69.3 (C-6′), 63.8 (C-1′), 12.4 (CH3,
thymine).
1
673.2; H NMR (270 MHz, CDCl3) 8.13 (br s, 1H, NH), 7.74 (d,
J ) 1.11, Hz, 1H, H-6), 7.37-7.10 (m, 17H, MMTr, benzyl), 6.77
(d, 2H, MMTr), 4.60 (dd, JH-4′, H-5′ ) 2.85 Hz, JH-3′, H-4′ ) 5.20
Hz, 1H, H-4′), 4.51-4.42 (m, 2H, CH2Ph, H-5′), 4.33 (d, Jgem
)
11.50 Hz, 1H, CH2Ph), 4.30 (d, 1H, H-3′), 3.76 (s, 3H, OCH3),
3.66-3.52 (m, 3H, H-1′, H-6′, H-6′′), 3.45 (d, J ) 10.39, Hz, 1H,
H-1′′), 2.86 (s, 1H, OH), 1.92 (s, 3H, CH3, thymine); 13C NMR
(67.9 MHz, CDCl3) 163.4 (C-4), 158.5, 151.0 (C-2), 143.9, 143.7,
138.7 (C-6), 137.1, 134.9, 130.1, 128.3, 128.2, 128.2, 127.7, 127.2,
126.9, 113.0 (C-5), 108.4, 99.4 (C-2′), 86.7, 85.7 (C-5′), 77.9 (C-
4′), 74.0 (C-3′), 73.6 (CH2Ph), 70.4 (C-6′), 63.5 (C-1′), 55.1 (OCH3),
12.3 (CH3, thymine).
2,3′-Anhydro-1-[6-O-benzyl-1-O-(4-methoxytrityl)-â-D-fructo-
furanosyl]thymine (5). 1,1′-Thiocarbonyldiimidazole (2.73 g, 15.33
mmol) was added to the stirred solution of compound 4 (8.1 g,
12.46 mmol) in dry toluene (120 mL). The reaction mixture was
heated at 120 °C for 3 h under nitrogen atmosphere. Solvent was
evaporated, the residue was dissolved in CH2Cl2, and saturated
aqueous NaHCO3 was added and extracted with CH2Cl2 (4 times).
The organic phase was dried over MgSO4, filtered, and evaporated.
The residue was purified by column chromatography using 0 to
3% MeOH in CH2Cl2 as eluent to afford 5 (5.9 g, 9.33 mmol, 75%).
Rf ) 0.47 (CH2Cl2/CH3OH 92:8 v/v); MALDI-TOF m/z [M - H]-
found 631.1, calcd 631.2; 1H NMR (270 MHz, CDCl3) 7.37-7.07
(m, 17H, MMTr, benzyl), 6.80 (d, 2H, MMTr), 6.69 (d, J ) 1.24,
Hz, 1H, H-6), 5.38 (d, JH-3′, H-4′) 1.36 Hz, 1H, H-3′), 4.62 (br s,
1H, H-4′), 4.36-4.30 (m, 3H, CH2Ph, H-5′), 3.74 (s, 3H, OCH3),
3.68 (d, Jgem ) 10.39 Hz, 1H, H-1′), 3.60 (d, 1H, H-1′′), 3.38 (dd,
Jgem ) 10.52 Hz, JH-5′, H-6′ ) 3.46 Hz, 1H, H-6′), 3.26 (dd, JH-5′,
1-[1,3-O-Acetyl-4,6-O-benzyl-â-D-fructofuranosyl]thymine (8).
See Supporting Information (SI) page S3 for details.
1-[1,3-O-Acetyl-4,6-O-benzyl-â-D-fructofuranosyl]3-N-(4-meth-
oxybenzyl)thymine (9). Compound 8 (3.3 g, 5.98 mmol) was
dissolved in 70 mL of dry DMF and cooled to 0 °C and sodium
hydride (0.216 g, 9 mmol) was added. The reaction mixture was
stirred at 0 °C for 1 h under nitrogen atmosphere. To this solution
was added 4-methoxybenzyl chloride (PMBCl) (1.08 mL, 7.96
mmol), and the mixture was allowed to warm slowly to room
temperature and stirred for 6 h. The reaction was quenched by
saturated aqueous NaHCO3 and extracted with CH2Cl2 (3 times).
The organic phase was dried over MgSO4, filtered, and concentrated
in vacuo. The residue was purified by column chromatography using
0 to 20% ethyl acetate in cyclohexane as eluent to furnish 9 (3.2 g,
4.76 mmol, 80%). Rf ) 0.71 (CH2Cl2/CH3OH 98:2 v/v); MALDI-
TOF m/z [M - H]- found 671.1, calcd 671.2; 1H NMR (270 MHz,
CDCl3) 7.57 (s, 1H, H-6), 7.45-7.16 (m, 12H, benzyl, PMB), 6.80
(d, 2H, PMB), 5.67 (s, H-3′), 5.19 (d, Jgem ) 13.36 Hz, 1H, PMB),
4.88-4.80 (m, 2H, 1 × CH2Ph, 1 × PMB), 4.79-4.68 (ABq, Jgem
) 12.00 Hz, 2H, H-1′, H-1′′), 4.62 (d, 1H, CH2Ph), 4.53-4.37 (m,
3H, 2 × CH2Ph, H-5′), 3.82 (d, JH-4′, H-5′ ) 2.60 Hz, 1H, H-4′),
3.75 (s, 3H, OCH3), 3.52 (dd, Jgem ) 10.39 Hz, JH-5′, H-6′ ) 5.81
Hz, 1H, H-6′), 3.42 (dd, JH-5′, H-6′′ ) 5.57 Hz 1H, H-6′′), 1.99 (s,
3H, CH3, O-acetyl), 1.93 (s, 3H, CH3, thymine), 1.32 (s, 3H, CH3,
O-acetyl); 13C NMR (67.9 MHz, CDCl3) 169.9 (CdO), 168.7 (Cd
O), 163.6 (C-4), 159.0, 149.9 (C-2), 137.4, 137.0, 134.8 (C-6),
130.8, 128.3, 127.8, 127.7, 127.4, 113.5, 108.4 (C-5), 96.7 (C-2′),
83.4 (C-5′), 82.6 (C-4′), 76.9 (C-3′), 73.1 (CH2Ph), 71.6 (CH2Ph),
68.9 (C-6′), 62.2 (C-1′), 55.1 (OCH3), 43.5 (CH2, PMB), 20.5 (CH3,
O-acetyl), 19.8 (CH3, O-acetyl), 13.3 (CH3, thymine).
H
-6′′
) 5.07 Hz, 1H, H-6′′), 1.88 (s, 3H, CH3, thymine); 13C NMR
(67.9 MHz, CDCl3) 172.9 (C-4), 159.8 (C-2), 158.8, 143.0, 142.7,
137.0, 133.9, 130.2, 129.2 (C-6), 128.3, 128.1, 128.0, 127.7, 127.7,
127.3, 118.2 (C-5), 113.3, 99.8 (C-2′), 90.1 (C-3′), 87.6 (C-5′), 87.2,
76.9 (C-4′), 73.4 (CH2Ph), 69.0 (C-6′), 62.0 (C-1′), 55.1 (OCH3,
MMTr), 14.0. (CH3, thymine).
2,3′-Anhydro-1-[4,6-O-benzyl-1-O-(4-methoxytrityl)-â-D-fructo-
furanosyl]thymine (6a). Compound 5 (5.9 g, 9.33 mmol) was
dissolved in 95 mL of dry acetonitrile and cooled to 0 °C and
sodium hydride (0.336 g, 14 mmol) was added. The reaction mixture
was stirred at 0 °C for 20 min under nitrogen atmosphere. To this
solution was added benzyl bromide (1.44 mL, 12.135 mmol), and
the mixture was allowed to warm slowly to room temperature and
stirred for 6 h. The reaction was quenched with saturated aqueous
NaHCO3 and extracted with CH2Cl2 (3 times). The organic phase
was dried over MgSO4, filtered, and concentrated in vacuo. The
residue was purified by column chromatography (eluent 0 to 3%
MeOH in CH2Cl2) to give 6a (5.47 g, 7.57 mmol, 81%). Rf ) 0.39
(CH2Cl2/CH3OH 95:5 v/v); MALDI-TOF m/z [M - H]- found
1-[4,6-O-Benzyl-â-D-fructofuranosyl]-3-N-(4-methoxybenzyl)-
thymine (10). See SI page S3 for details.
1
721.1, calcd 721.2; H NMR (270 MHz, CDCl3) 7.37-7.11 (m,
1-[4,6-O-Benzyl-3-O-methanesulfonyl-1-phthalimido-â-D-fructo-
furanosyl]-3-N-(4-methoxybenzyl)thymine (12). Compound 10
(1.0 g, 1.7 mmol) was coevaporated with dry pyridine (3 times)
and dissolved in 50 mL of the same solvent, the solution was cooled
to 0 °C, and methanesulfonyl chloride (0.8 mL, 10.22 mmol) was
added dropwise. The reaction mixture was stirred at 0 °C for 1 h
and then at 4 °C overnight. The crude reaction mixture was poured
into ice-cooled saturated aqueous NaHCO3 and extracted with CH2-
Cl2 (3 times). The organic phase was dried over MgSO4, filtered,
and evaporated to give 11a along with traces of 11b. The residue
was coevaporated with toluene (3 times) and kept for drying on an
oil pump for 20 min. Crude compound 11a was dissolved in 50
22H, MMTr, benzyl), 6.81 (d, 2H, MMTr), 6.65 (d, J ) 1.24 Hz,
1H, H-6) 5.13 (s, 1H, H-3′), 4.63 (d, Jgem ) 11.50 Hz, 1H, CH2-
Ph), 4.54 (d, 1H, CH2Ph), 4.41-4.26 (m, 4H, CH2Ph, H-4′, H-5′),
3.77 (s, 3H, CH3, MMTr), 3.58 (ABq, Jgem ) 10.64 Hz, 2H, H-1′,
H-1′′), 3.33-3.20 (ddd, Jgem ) 10.52 Hz, JH-5′,
) 4.58 Hz,
H-6′
2H, H-6′, H-6′′), 1.91 (s, 3H, CH3, thymine); 13C NMR (67.9 MHz,
CDCl3) 172.3 (C-4), 159.6 (C-2), 158.8, 142.9, 142.7, 136.7, 136.1,
133.9, 130.2, 128.6 (C-6), 128.2, 128.0, 127.9, 127.8, 127.6, 127.3,
118.5 (C-5), 113.3, 100.1 (C-2′), 87.3 (C-3′), 87.1, 85.9 (C-5′), 83.8
(C-4′), 73.5 (CH2Ph), 72.2 (CH2Ph), 68.7 (C-6′), 61.7 (C-1′), 55.1
(OCH3), 14.1 (CH3, thymine).
J. Org. Chem, Vol. 71, No. 1, 2006 309

Honcharenko et al.
mL of dry DMF, and potassium phthalimide (1.89 g, 10.2 mmol)
was added followed by stirring at 110 °C overnight under nitrogen
atmosphere. The reaction mixture was poured into saturated aqueous
NaHCO3 and extracted with CH2Cl2 (3 times). The organic phase
was dried over MgSO4, filtered, and evaporated. The residue was
coevaporated with toluene (5 times) and CH2Cl2 (3 times). The
mixture was purified by silica gel column chromatography using 0
to 30% ethyl acetate in cyclohexane as eluent to afford 12 (1.04 g,
1.3 mmol, 77% after two steps and recovered 11b, 7%). Rf ) 0.69
(CH2Cl2/CH3OH 98:2v/v); MALDI-TOF m/z [M - H]- found
94.1 (C-2′), 82.2 (C-5′), 78.9 (C-4′), 73.4 (CH2Ph), 72.8 (CH2Ph),
70.1 (C-6′), 65.2 (C-3′), 55.2 (C-1′), 55.1 (OCH3), 43.5 (CH2, PMB),
13.0 (CH3, thymine).
(1R,3R,4S,5R)-3-Benzyloxymethyl-4-benzyloxy-1-(thymine-1-
yl)-6-aza-2-oxabicyclo[3.2.0]heptane (15). Compound 14 (0.38 g,
0.668 mmol) was dissolved in 7.6 mL CH3CN-H2O mixture (v/v
9:1), ceric ammonium nitrate (1.36 g, 2.48 mmol) was added, and
the reaction mixture was stirred at room temperature for 3 h.
Saturated aqueous NaHCO3 was added, and the mixture was stirred
for an additional 20 min, then filtered and extracted with CH2Cl2
(3 times). The extract was dried over MgSO4, filtered, and
evaporated. The residue was purified by column chromatography
using 0 to 5% MeOH in CH2Cl2 as eluent to give 15 (0.187 g,
0.416 mmol, 62%). Rf ) 0.35 (CH2Cl2/CH3OH 92:8 v/v); MALDI-
TOF m/z [M - H]- found 448.1, calcd 448.1; 1H NMR (270 MHz,
CDCl3) 8.55 (br s, 1H, NH), 7.41-7.24 (m, 10H, benzyl), 6.81 (d,
J ) 0.99 Hz, 1H, H-6), 4.72 (d, JH-3′, H-4′ ) 5.32 Hz, 1H, H-3′),
4.61-4.50 (ABq, 2H, CH2Ph), 4.49 (s, 2H, CH2Ph), 4.47-4.39
(m, 1H, H-5′), 4.22 (dd, JH-4′, H-5′ ) 7.79 Hz, 1H, H-4′), 4.11 (d,
1
794.0, calcd 794.2; H NMR (270 MHz, CDCl3) 7.91-7.70 (m,
4H, phthalimido), 7.41-7.24 (m, 13H, benzyl, 2 × PMB, H-6),
6.81 (d, 2H, PMB), 5.56-5.47 (m, 2H, 1 × PMB, H-3′), 4.84-
4.71 (m, 4H, 2 × CH2Ph, 1 × PMB, H-5′), 4.65 (d, Jgem ) 14.23
Hz, 2H, H-1′), 4.53-4.45 (m, 3H, 2 × CH2Ph, H-1′′), 4.35 (d,
JH-4
) 2.35 Hz, 1H, H-4′), 3.77 (s, 3H, OCH3), 3.51 (ddd,
H-5′
Jgem′, ) 10.27 Hz, JH-5′, H-6′ ) 6.19 Hz, JH-5′, H-6′′ ) 5.94 Hz, 2H,
H-6′, H-6′′), 2.27 (s, 3H, CH3, O-mesyl), 1.77 (s, 3H, CH3, thymine);
13C NMR (67.9 MHz, CDCl3) 167.8 (CdO), 163.7 (C-4), 158.9,
150.6 (C-2), 137.5, 136.9, 134.1, 133.7 (C-6), 131.5, 130.1, 129.5,
128.3, 127.8, 127.6, 123.4, 113.6, 109.4 (C-5), 96.7 (C-2′), 83.8
(C-4′), 83.4 (C-3′), 83.1 (C-5′), 73.1 (CH2Ph), 72.1 (CH2Ph), 69.3
(C-6′), 55.2 (OCH3), 44.1 (CH2, PMB), 38.5 (C-1′), 37.4 (CH3,
O-mesyl), 13.2 (CH3, thymine).
Jgem ) 10.39 Hz, 1H, H-1′), 3.86 (d, 1H, H-1′′), 3.71 (ddd, Jgem
)
10.64 Hz, JH-5′, H-6′ ) 2.47 Hz, JH-5′, H-6′′ ) 6.80 Hz, 2H, H-6′,
H-6′′), 1.88 (s, 3H, CH3, thymine); 13C NMR (67.9 MHz, CDCl3)
163.5 (C-4), 149.0 (C-2), 137.7, 137.2, 135.8 (C-6), 128.4, 128.3,
127.9, 127.7, 111.0 (C-5), 93.5 (C-2′), 82.1 (C-5′), 78.7 (C-4′), 73.4
(CH2Ph), 72.8 (CH2Ph), 70.2 (C-6′), 65.1 (C-3′), 55.0 (C-1′), 12.2
(CH3, thymine).
1-[1-Amino-4,6-O-benzyl-3-O-methanesulfonyl-â-D-fructo-
furanosyl]-3-N-(4-methoxybenzyl)thymine (13). Compound 12
(0.8 g, 1.01 mmol) was dissolved in 10 mL of methanol, 50 mL of
40% aqueous methylamine was added, followed by dropwise
addition of methanol till the solution becomes homogeneous. The
reaction mixture was stirred at room temperature for 4 h, then was
poured into saturated aqueous NaHCO3, and extracted with CH2-
Cl2 (4 times). The organic phase was dried over MgSO4, filtered,
and evaporated. The residue was coevaporated with toluene (3
times) and CH2Cl2 (3 times). The mixture was purified by silica
gel column chromatography using 0 to 5% MeOH in CH2Cl2 as
eluent gave 13 (0.62 g, 0.93 mmol, 93%). Rf ) 0.55 (CH2Cl2/CH3-
OH 92:8 v/v); MALDI-TOF m/z [M - H]- found 664.1, calcd
(1R,3R,4S,5R)-3-Benzyloxymethyl-4-benzyloxy-6-N-phenoxy-
acetyl-1-(thymine-1-yl)-6-aza-2-oxabicyclo[3.2.0]heptane (16).
Compound 15 (0.32 g, 0.71 mmol) was coevaporated with dry
pyridine (3 times) and dissolved in 12 mL of the same solvent.
Phenoxyacetyl chloride (0.13 mL, 0.93 mmol) was added dropwise
and stirred at room temperature for 3 h. The reaction mixture was
poured into ice-cooled saturated aqueous NaHCO3 and extracted
with CH2Cl2 (3 times). The organic phase was dried over MgSO4,
filtered, and evaporated. The residue was coevaporated with toluene
(4 times) and CH2Cl2 (3 times) followed by purification by silica
gel column chromatography using 0 to 3% MeOH in CH2Cl2 as
eluent to afford 16 (0.36 g, 0.62 mmol, 86%). Rf ) 0.53 (CH2Cl2/
CH3OH 92:8 v/v); MALDI-TOF m/z [M - H]- found 582.1, calcd
582.2; 13C NMR (67.9 MHz, CDCl3) 169.6, 163.3, 157.1, 149.0,
137.6, 129.6, 128.3, 127.6, 121.9, 121.5, 114.2, 103.2, 111.7, 90.6,
89.7, 81.6, 78.0, 73.9, 73.3, 73.0, 70.7, 68.9, 68.1, 67.8, 67.3, 61.1,
57.5, 12.2.
1
664.2; H NMR (270 MHz, CDCl3) 7.65 (d, J ) 1.24 Hz, 1H,
H-6), 7.39-7.25 (m, 12H, benzyl, PMB), 6.80 (d, 2H, PMB), 5.32
(d, Jgem ) 14.47 Hz 1H, 1 × PMB), 5.24 (s, 1H, H-3′), 4.80-4.70
(m, 2H, 1 × CH2Ph, 1 × PMB), 4.57 (d, Jgem ) 11.75 Hz, 1H, 1
× CH2Ph), 4.52 (s, 2H, CH2Ph), 4.36-4.29 (m, 1H, H-5′), 4.26
(d, JH-4′,
) 2.10 Hz, 1H, H-4′), 3.76 (s, 3H, OCH3), 3.64-
H-5′
3.47 (m, 3H, H-1′, H-6′, H-6′′), 3.27 (d, Jgem ) 14.23 Hz, 1H, H-1′′),
2.29 (s, 3H, CH3, O-mesyl), 1.96 (s, 3H, CH3, thymine); 13C NMR
(67.9 MHz, CDCl3) 163.6 (C-4), 159.0, 150.0 (C-2), 137.4, 136.8,
135.5 (C-6), 130.2, 129.3, 128.4, 128.0, 127.8, 127.6, 113.7, 108.6
(C-5), 99.0 (C-2′), 83.9 (C-4′), 83.5 (C-5′), 83.1 (C-3′), 73.4 (CH2-
Ph), 72.3 (CH2Ph), 69.6 (C-6′), 55.2 (OCH3), 43.9 (CH2, PMB),
37.5 (CH3, O-mesyl), 13.5 (CH3, thymine).
(1R,3R,4S,5R)-3,4-Hydroxy-6-N-phenoxyacetyl-1-(thymine-1-
yl)-6-aza-2-oxabicyclo[3.2.0]heptane (17). Compound 16 (0.03 g,
0.051 mmol) was dissolved in 1.5 mL of methanol, and Pd(OH)2
on charcoal (20% moist, 0.01 g) and ammonium formate (0.042 g,
0.66 mmol) were added to the solution of nucleoside. The resulting
suspension was heated under reflux for 3 h. The reaction mixture
was filtered through silica gel bed and washed with hot methanol.
The filtrate was concentrated to dryness under reduced pressure
and purified by silica gel column chromatography using 0 to 10%
MeOH in CH2Cl2 as eluent to afford 17 (0.019 g, 0.047 mmol,
92%). Rf ) 0.28 (CH2Cl2/CH3OH 90:10 v/v); MALDI-TOF m/z
[M - H]-, found 402.0, calcd 402.1; 13C NMR (67.9 MHz, CD3-
OD): 172.6, 166.7, 159.9, 159.4, 151.6, 138.5, 131.0, 130.8, 123.1,
122.7, 116.0, 112.3, 91.4, 85.5, 85.2, 73.8, 73.0, 71.7, 71.2, 68.0,
66.5, 62.7, 62.4, 59.7, 12.5.
(1R,3R,4S,5R)-3-Benzyloxymethyl-4-benzyloxy-1-(3-N-(4-meth-
oxybenzyl)thymine-1-yl)-6-aza-2-oxabicyclo[3.2.0]heptane (14).
Compound 13 (2.45 g, 3.68 mmol) was dissolved in 55 mL of
pyridine, 2.6 mL of triethylamine was added and heated at 90 °C
for 48 h under nitrogen atmosphere. The solvent was removed under
reduced pressure. The residue was coevaporated with toluene (3
times) and CH2Cl2 (3 times) and subjected to column chromatog-
raphy using 0 to 5% MeOH in CH2Cl2 as eluent to give 14 (1.38
g, 2.42 mmol, 66%). Rf ) 0.43 (CH2Cl2/CH3OH 92:8 v/v); MALDI-
TOF m/z [M - H]- found 568.1, calcd 568.2; 1H NMR (270 MHz,
CDCl3) 7.44-7.25 (m, 12H, benzyl, 2 × PMB), 6.84-6.75 (m,
3H, 2 × PMB, H-6), 5.06-4.93 (ABq, Jgem ) 13.73 Hz, 2H, PMB),
4.69 (d, JH-3′, H-4′ ) 5.32 Hz, 1H, H-3′), 4.59-4.38 (m, 5H, 2 ×
(1R,3R,4S,5R)-3-(4,4′-Dimethoxytrityloxymethyl)-4-hydroxy-
6-N-phenoxyacetyl-1-(thymine-1-yl)-6-aza-2-oxabicyclo[3.2.0]-
heptane (18a). See SI page S4 for details.
(1R,3R,4S,5R)-3-(4,4′-Dimethoxytrityloxymethyl)-4-hydroxy-
1-(5-methylcytosin-1-yl)-6-N-phenoxyacetyl-6-aza-2-oxabicyclo-
[3.2.0]heptane (19). Compound 18a (0.35 g, 0.496 mmol) was
dissolved in 10 mL of dry pyridine, and acetic anhydride (0.47
mL, 4.96 mmol) was added and stirred at room temperature for 28
h. Then reaction was quenched with methanol (0.95 mL) and stirred
overnight at room temperature. Saturated aqueous NaHCO3 was
added to the reaction mixture and extracted with CH2Cl2 (3 times).
CH2Ph, 2 × CH2Ph, H-5′), 4.30 (dd, JH-4′,
) 7.67 Hz, 1H,
H-5′
H-4′), 4.08 (d, Jgem ) 10.39 Hz, 1H, H-1′), 3.86 (d, 1H, H-1′′),
3.81-3.73 (m, 4H, OCH3, H-6′), 3.67 (dd, Jgem ) 10.70 Hz, JH-5′,
H
) 6.43 Hz, 1H, H-6′′), 1.88 (s, 3H, CH3, thymine); 13C NMR
-6′′
(67.9 MHz, CDCl3) 163.3 (C-4), 159.0, 149.8 (C-2), 137.8, 137.3,
133.7 (C-6), 130.5, 128.4, 128.2, 127.9, 127.6, 113.6, 110.4 (C-5),
310 J. Org. Chem., Vol. 71, No. 1, 2006

1′,2′-Azetidine-Fused Bicyclic Pyrimidine Nucleosides
The organic phase was dried over MgSO4, filtered, and evaporated
under reduced pressure to give 18b. The residue was coevaporated
with dry pyridine (3 times) and dissolved in 5 mL of the same
solvent. To this solution was added 1,2,4-triazole (0.342 g, 4.96
mmol), and the mixture was cooled to 0 °C. 2-Chlorophenylphos-
phodichloridate (0.41 mL, 2.48 mmol) was added dropwise to the
reaction mixture under nitrogen atmosphere. After being stirred at
0 °C for 30 min the reaction mixture was warmed to room
temperature and stirred for 6 h. Aqueous ammonia (32%, 10 mL)
was added, and the reaction mixture was stirred at room temperature
overnight. The resulting solution was treated with saturated aqueous
NaHCO3 and extracted with CH2Cl2 (4 times). The organic phase
was dried over MgSO4, filtered, and evaporated. The residue was
purified by silica gel column chromatography using 0 to 5% MeOH
in CH2Cl2 as eluent to give 19 (0.245 g, 0.348 mmol, 70%). Rf )
0.37 (CH2Cl2/CH3OH 92:8 v/v); MALDI-TOF m/z [M - H]- found
703.2, calcd 703.2; 13C NMR (67.9 MHz, CDCl3) 170.5, 169.7,
165.5, 158.3, 157.7, 157.1, 155.2, 144.6, 137.9, 137.7, 135.7, 129.9,
129.6, 129.2, 127.9, 127.6, 126.6, 121.8, 121.0, 114.5, 114.2, 112.9,
103.3, 103.1, 89.8, 89.6, 86.0, 85.9, 83.1, 81.5, 72.8, 72.4, 71.6,
70.9, 67.1, 66.0, 63.2, 62.6, 62.1, 55.0, 12.9.
(1R,3R,4S,5R)-4-(2-Cyanoethoxy(diisopropylamino)-phosphin-
oxy)-3-(4,4′-dimethoxytrityloxy-methyl)-6-N-phenoxyacetyl-1-
(thymine-1-yl)-6-aza-2-oxabicyclo[3.2.0]heptane (20). Compound
18a (0.054 g, 0.0765 mmol) was dissolved in 0.8 mL of dry THF,
diisopropylethylamine (0.066 mL, 0.382 mmol) was added at 0 °C
under nitrogen atmosphere, and the reaction mixture was stirred
for 15 min. To this solution was added 2-cyanoethyl-N,N-diiso-
propylphosphoramidochloridite (0.034 mL, 0.153 mmol)dropwise,
the mixture was stirred at this temperature for 20 min, and thereafter
the reaction was warmed to room temperature and stirred for 2 h.
The reaction was quenched with methanol (0.3 mL) and continued
stirring for another 30 min. The reaction mixture was poured into
saturated aqueous NaHCO3 and extracted with CH2Cl2 (3 times).
The organic phase was dried over MgSO4, filtered, and evaporated.
The crude residue was purified by silica gel column chromatography
using 0 to 1.5% MeOH in CH2Cl2 as eluent containing 0.5% Et3N
to afford 20 (0.059 g, 0.065 mmol, 86%). Rf ) 0.55 (CH2Cl2/CH3-
OH 92:8 v/v); MALDI-TOF m/z [M - H]- found 904.2, calcd
904.3; 31P NMR (67.9 MHz, CDCl3) 154.7, 150.7, 149.9, 148.6.
6-O-Benzyl-1,3,4-tri-O-(4-toluoyl)-D-psicofuranose (22). The
sugar 1 (7 g, 20 mmol) was refluxed with 70% aqueous CH3COOH
(120 mL) at 80 °C for 5 h. The reaction mixture was cooled, acid
was evaporated, and residue was coevaporated with water (5 times)
to give compound 21. The crude product was coevaporated with
dry pyridine (3 times), dissolved in dichloromethane/pyridine (7:
1, v/v, 100 mL), at 0 °C, under nitrogen atmosphere, 4-toluoyl
chloride (8.2 mL, 62 mmol) was added dropwise, and the reaction
was stirred for 4 h at this temperature. The reaction mixture was
poured into saturated aqueous NaHCO3 solution and extracted with
CH2Cl2 (3 times). The organic phase was dried over MgSO4,
filtered, evaporated, and coevaporated with toluene (3 times) and
CH2Cl2 (3 times). The residue was purified by column chroma-
tography (eluent ethyl acetate 0-30% in cyclohexane), which
afforded 22 (9.4 g, 15 mmol, 75% after two steps) as a mixture of
anomers. Rf ) 0.57 (cyclohexane/EtOAc 65:35 v/v); MALDI-TOF
m/z [M + Na]+ found 647.0, calcd 647.2; 13C NMR (125.7 MHz,
CDCl3) 171.2, 166.4, 166.1, 165.5, 165.4, 164.9, 164.8, 148.7,
144.3, 144.2, 144.1, 144.0, 143.7, 143.6, 137.6, 136.9, 136.8, 130.1,
129.8,129.7, 129.6, 129.2, 129.1, 129.0, 128.9, 128.5, 128.4, 128.0,
127.9, 127.6, 127.4, 126.7, 126.7, 126.4, 126.2, 126.1, 124.0, 104.3,
102.0, 81.8, 81.6, 76.5, 73.8, 73.5, 72.6, 72.4, 71.9, 69.8, 69.4, 65.5,
64.9, 21.7, 21.6, 21.5.
NaHCO3 solution and extracted with CH2Cl2. The organic phase
was dried over MgSO4, filtered, evaporated, and coevaporated with
toluene (3 times). Column chromatography (gradient 0-30% ethyl
acetate in cyclohexane) afforded 23 as an anomeric mixture (9 g,
13.5 mmol, 90%). Rf ) 0.61 (cyclohexane/EtOAc 65:35 v/v);
MALDI-TOF m/z [M + Na]+ found 689.0, calcd 689.2; 13C NMR
(125.7 MHz, CDCl3) 171.3, 168.8, 168.7, 165.6, 165.5, 165.4,
164.7, 164.6, 148.8, 144.4, 144.2, 144.1, 143.9, 143.7, 143.6, 137.7,
137.6, 136.8, 130.2, 129.8, 129.7, 129.6, 129.2, 129.1, 129.0, 128.9,
128.4, 128.3, 127.8, 127.7, 127.6, 127.4, 126.7, 126.6, 126.2, 126.1,
108.5, 106.5, 83.8, 82.3, 74.9, 73.6, 73.5, 72.9, 71.4, 70.8, 68.9,
68.8, 63.3, 61.3, 21.8, 21.7, 21.6, 21.5.
1-[1,3,4-O-(4-Toluoyl)-6-O-benzyl-â-D-psicofuranosyl]uracil
(24). The sugar 23 (6.6 g, 10 mmol) and uracil (1.3 g, 12 mmol)
were dissolved in dry acetonitrile (100 mL). N,O-Bis(trimethylsilyl)-
acetamide (4.95 mL, 20 mmol) was added and the reaction was
heated at 90 °C for 1.5 h (the reaction becomes clear solution) under
nitrogen atmosphere. The reaction mixture was cooled to room
temperature; trimethylsilyl trifluoromethanesulfonate (1 mL, 5.5
mmol) was added and again heated at 40 °C for 2 h under nitrogen
atmosphere. After cooling the reaction mixture was poured into
saturated aqueous NaHCO3 solution and extracted with CH2Cl2 (3
times). The organic phase was dried over MgSO4, filtered, and
evaporated. The crude residue after column chromatography (0 to
2% MeOH in CH2Cl2) afforded 24 (R:â, 1:9; 4.65 g, 6.5 mmol,
65%). Rf ) 0.57 (CH2Cl2/CH3OH 96:4 v/v); MALDI-TOF m/z [M
1
+ Na]+ found 741.6, calcd 741.2; H NMR (270 MHz, CDCl3)
8.67 (br s, 1H, NH), 7.91 (d, JH-5, H-6 ) 8.16 Hz, 1H, H-6), 7.87-
7.76 (m, 6H, 4-toluoyl), 7.37-7.25 (m, 5H, benzyl), 7.19-7.12
(m, 6H, 4-toluoyl), 6.42 (d, JH-3′, H-4′ ) 5.44 Hz, 1H, H-3′), 5.83
(dd, JH-4′,
) 2.85 Hz, 1H, H-4′), 5.14 (d, Jgem ) 11.75 Hz,
H-5′
1H, H-1′), 4.99 (d, 1H, H′′), 4.71 (m, 1H, H-5′), 4.55 (d, Jgem
)
11.26 Hz, 1H, CH2Ph), 4.40 (d,1H, CH2Ph), 3.80-3.70 (m, 2H,
H-6′, H-6′′), 2.37 (s, 6H, CH3, 4-toluoyl), 2.36 (s, 3H, CH3,
4-toluoyl); 13C NMR (67.9 MHz, CDCl3) 165.55 (CdO), 165.5
(CdO), 164.4 (CdO), 163.6 (C-4), 149.8 (C-2), 144.4, 144.2, 141.2
(C-6), 136.5, 129.8, 129.3, 128.7, 127.6, 100.4 (C-5), 97.5 (C-2′),
84.4 (C-5′), 76.8 (C-3′), 74.0 (CH2Ph), 73.6 (C-4′), 69.0 (C-6′),
64.4 (C-1′), 21.7 (CH3, 4-Toluoyl).
1-[6-O-Benzyl-1-O-(4-monomethoxytrityl)-â-D-psicofuranosyl]-
uracil (26). See SI page S4 for details.
2′,3-Anhydro-1-[6-O-benzyl-1-O-(4-monomethoxytrityl)-â-D-
fructofuranosyl]uracil (27). Compound 26 (3.1 g 4.8 mmol) was
dissolved in dry DMF (20 mL), and diphenyl carbonate (1.13 g,
5.28 mmol) and NaHCO3 (806 mg, 9.6 mmol) were added. The
mixture was heated at 110 °C for 2 h under nitrogen atmosphere.
The solvent was evaporated under reduced pressure, the residue
was dissolved in CH2Cl2, and brine was added and extracted with
CH2Cl2 (3 times). The organic phase was dried (MgSO4), filtered,
and concentrated under reduced pressure. The residue was purified
using column chromatography (eluent 0 to 3% MeOH in CH2Cl2),
which afforded 27 (2.35 g, 3.8 mmol, 91%). Rf ) 0.3 (CH2Cl2/
CH3OH 96:4 v/v); MALDI-TOF m/z [M + H]+ found 619.5, calcd
1
619.2; H NMR (270 MHz, CDCl3) 7.36-7.07 (m, 17H, MMTr,
Benzyl), 6.90 (d, JH-6,
) 7.55 Hz, 1H, H-6), 6.79 (d, 2H,
H-5
MMTr), 6.07 (br s, 1H, OH-4′), 5.92 (d, JH-5, H-6 ) 7.55 Hz, 1H,
H-5), 5.41 (s, 1H, H-3′), 4.65 (br s, 1H, H-4′), 4.36 (m, 1H, H-5′),
4.27 (ABq, Jgem ) 12.49 Hz, 2H, CH2Ph), 3.76 (s, 3H, OCH3,
MMTr), 3.65 (ABq, Jgem ) 10.64 Hz, 2H, H-1′, H-1′′), 3.36-3.19
(ddd, Jgem ) 10.89 Hz, JH-5′, H-6′ ) 3.22 Hz, 2H, H-6′, H-6′′); 13
C
NMR (67.9 MHz, CDCl3) 172.7 (C-4), 160.4 (C-2), 158.7, 143.0,
142.7, 137.0, 133.9 (C-6), 133.7, 130.1, 128.2, 128.1, 128.03, 127.9,
127.7, 127.2, 113.3, 109.3(C-5), 99,9 (C-2′), 90.5 (C-3′), 88.4 (C-
5′), 87.1, 76.4(C-4′), 73.4 (CH2Ph), 69.9 (C-6′), 62.2 (C-1′), 55.1
(OCH3).
2-O-Acetyl-6-O-benzyl-1,3,4-tri-O-(4-toluoyl)-D-psicofura-
nose (23). The sugar 22 (9.4 g, 15 mmol) after coevaporation with
pyridine (3 times), was dissolved in 75 mL of this same solvent
and treated with acetic anhydride (28 mL, 300 mmol). The reaction
mixture was stirred at room temperature for 48 h under nitrogen
atmosphere. The reaction mixture was poured into saturated aqueous
2′,3-Anhydro-1-[4,6-O-benzyl-1-O-(4-monomethoxytrityl)-â-
D-fructofuranosyl]uracil (28). Compound 27 (2.3 g, 3.8 mmol)
was dissolved in 40 mL of dry CH3CN and cooled to 0 °C, NaH
(140 mg, 3.45 mmol) was added, and the mixture was stirred for
J. Org. Chem, Vol. 71, No. 1, 2006 311

Honcharenko et al.
15 min under nitrogen atmosphere. To this turbid solution was
added benzyl bromide (0.54 mL, 4.56 mmol), and the mixture was
allowed to warm slowly to room temperature. It was then stirred
overnight under nitrogen atmosphere. The solvent was removed
under reduced pressure; saturated aqueous NaHCO3 was added and
extracted with CH2Cl2 (3 times). The organic phase was dried using
MgSO4, filtered, and concentrated in vacuo. The residue was
purified by column chromatography (eluent 0 to 3% MeOH in CH2-
Cl2) to give 28 (2.28 g, 3.23 mmol, 85%). Rf ) 0.38 (CH2Cl2/CH3-
OH 96:4 v/v); MALDI-TOF m/z [M + H]+ found 709.6, calcd
was dried over MgSO4, filtered, and concentrated under reduced
pressure. The residue was purified using column chromatography
(eluent 0 to 2.5% MeOH in CH2Cl2) to give 31 (4.01 g, 8.7 mmol,
87%). Rf ) 0.33 (CH2Cl2/CH3OH 95:5 v/v); MALDI-TOF m/z [M
+ Na]+ found 484.7, calcd 484.1; IR (KBr) ν 2100 cm-1 (strong);
1H NMR (270 MHz, CDCl3) 7.41 (d, JH6-H5 ) 7.55 Hz, 1H, H-6),
7.33-7.26 (m, 8H, benzyl), 7.18-7.14 (m, 2H, benzyl), 6.05 (d,
JH-5, -6 ) 7.55 Hz, 1H, H-5), 5.12 (s, 1H, H-3′), 4.65-4.50 (ABq,
H
Jgem ) 11.75 Hz, 2H, 2 × CH2Ph), 4.48 (m, 1H, H-5′), 4.38 (d,
Jgem ) 12.37 Hz, CH2Ph), 4.28 (d, JH-4′, H-5′ ) 1.61 Hz, 1H, H-4′),
4.21 (d, 1H, CH2Ph), 3.87 (ABq, Jgem ) 13.36 Hz), 3.34-3.18
1
709.2; H NMR (270 MHz, CDCl3) 7.33-7.12 (m, 22H, MMTr,
benzyl), 6.90 (d, JH-6,
) 8.29 Hz, 1H, H-6), 6.79 (d, 2H,
(ddd, Jgem ) 10.64 Hz, JH-5′, H-6′ ) 3.46 Hz, 2H, H-6′, H-6′′); 13
C
H-5
MMTr), 5.99 (d, JH-5, H-6 ) 8.16 Hz, 1H, H-5), 5.08 (s, 1H, H-3′),
4.52 (ABq, Jgem ) 11.75 Hz, 2H, CH2Ph), 4.47-4.22 (m, 4H, CH2-
Ph, H-4′, H-5′), 3.76 (s, 3H, CH3, MMTr), 3.58 (ABq, Jgem ) 10.64
Hz, 2H, H-1′, H-1′′), 3.32-3.20 (ddd, Jgem ) 10.51 Hz, JH-5′, H-6′
) 4.08 Hz, 2H, H-6′, H-6′′); 13C NMR (67.9 MHz, CDCl3) 171.7
(C-4), 159.9 (C-2), 158.7, 142.8, 142.7, 136.6, 136.0, 133.8, 132.5
(C-6), 130.0, 128.5, 128.2, 128.1, 128.0, 127.9, 127.6, 127.2 113.3,
109.8(C-5), 99.9 (C-2′), 87.5(C-3′), 87.1, 86.1 (C-5′), 83.7 (C-4′),
73.5 (CH2Ph), 72.1(CH2Ph), 68.5 (C-6′), 61.9 (C-1′), 55.1 (OCH3).
2′,3-Anhydro-1-[4,6-O-benzyl-â-D-fructofuranosyl]uridine (29).
Compound 28 (2.2 g, 3.2 mmol) was dissolved in 22 mL of 80%
aqueous acetic acid and stirred at room temperature for 48 h. The
acid was removed under vacuum and coevaporated with toluene 4
times and CH2Cl2 (3 times). The residue was subjected to column
chromatography (eluent 0 to 3% MeOH in CH2Cl2) to give 29 (1.1
g, 2.5 mmol, 79%). Rf ) 0.27 (CH2Cl2/CH3OH 95:5 v/v); MALDI-
TOF m/z [M + 2H]+ found 438.7, calcd 438.1; 1H NMR (270 MHz,
CDCl3) 7.36-7.26 (m, 9H, benzyl, H-6), 7.12-7.09 (m, 2H,
benzyl), 5.89 (d, JH-5, H-6 ) 7.42 Hz, 1H, H-5), 5.45 (s, 1H, H-3′),
4.70 (d, Jgem ) 11.75 Hz, CH2Ph), 4.52 (d, Jgem ) 11.75 Hz, CH2-
Ph), 4.43-4.40 (m, 1H, H-5′), 4.33-4.17 (m, Jgem ) 12.25 Hz,
CH2Ph, 4H, CH2Ph, H-1′, H-4′), 3.86 (d, JH-1′, H-1′′ ) 13.24 Hz,
NMR (67.9 MHz, CDCl3) 171.4 (C-4), 159.6 (C-2), 136.4, 135.9,
132.9 (C-6), 128.4, 128.1, 128.0, 127.9, 127.7, 127.6, 110.4 (C-5),
99.4 (C-2′), 87.4 (C-3′), 86.8 (C-5′), 83.9 (C-4′), 73.3 (CH2Ph),
72.0 (CH2Ph), 68.2 (C-6′), 52.6 (C-1′).
1-[1-Azido-4,6-O-benzyl-1-deoxy-â-D-fructofuranosyl]uracil
(32). To a solution of compound 31 (4 g, 8.7 mmol) in 50% aqueous
ethanol was added distilled THF to dissolve the compound
completely, followed by 1 M NaOH (15.5 mL). The reaction
mixture was stirred for 2 h and then concentrated in vacuo. The
residue was dissolved in CH2Cl2 and washed with brine. The
aqueous phase obtained was extracted 3 times with CH2Cl2. The
combined organic phase was dried over MgSO4, filtered, and
evaporated under reduced pressure. It was then subjected to column
chromatography (eluent 0 to 3% MeOH in CH2Cl2), which afforded
32 (3.54 g, 7.39 mmol, 85%). Rf ) 0.41 (CH2Cl2/CH3OH 95:5 v/v);
MALDI-TOF m/z [M + Na]+ found 502.6, calcd 502.1; 1H NMR
(270 MHz, CDCl3) 7.80 (d, JH-6, H-5 ) 8.29 Hz, 1H, H-6), 7.40-
7.22 (m, 10H, benzyl), 5.66 (d, JH-5,
) 8.29 Hz, 1H, H-5),
H-6
4.69-4.43 (m, 6H, 2 × CH2Ph, 2 × CH2Ph, H-3′, H-5′), 4.04 (br
s, 1H, H-4′), 3.99 (d, Jgem ) 13.11 Hz, 1H, H-1′), 3.84 (d, 1H,
H-1′′), 3.72-3.54 (ddd, Jgem ) 10.39 Hz, JH-5′,
) 3.46 Hz,
H-6′
2H, H-6′, H-6′′); 13C NMR (67.9 MHz, CDCl3) 164.4 (C-4), 150.7
(C-2), 141.8 (C-6), 137.0, 136.7, 128.7, 128.0, 127.9, 127.7, 127.6,
101.1 (C-5), 100.9 (C-2′), 85.3 (C-4′), 84.4 (C-5′), 76.7 (C-3′), 73.7
(benzyl), 72.1 (benzyl), 69.4 (C-6′), 53.6 (C-1′).
1H, H-1′′), 3.30-3.17 (ddd, Jgem ) 10.52 Hz, JH-5′,
) 3.59
H-6′
Hz, 2H, H-6′, H-6′′); 13C NMR (67.9 MHz, CDCl3) 173.3 (C-4),
160.6 (C-2), 136.5, 136.1, 134.4 (C-6), 128.4, 128.2, 128.1, 127.9,
127.8, 108.8 (C-5), 102.2 (C-2′), 87.6 (C-3′), 86.5 (C-5′), 86.6 (C-
4′), 73.5 (CH2Ph), 71.9 (CH2Ph), 68.6 (C-6′), 61.5 (C-1′).
1-[1-Azido-4,6-O-benzyl-3-O-methanesulfonyl-1-deoxy-â-D-
fructofuranosyl]uracil (33). See SI page S5 for details.
2′,3-Anhydro-1-[4,6-O-benzyl-1-O-methanesulfonyl-â-D-fructo-
furanosyl]uracil (30). Compound 29 (5 g, 11.4 mmol) was
coevaporated with dry pyridine (3 times) and dissolved in 55 mL
of the same solvent. The reaction mixture was cooled in an ice
bath, and methanesulfonyl chloride (2.66 mL, 34.2 mmol) was
added dropwise and stirred under nitrogen atmosphere for 1.5 h.
The reaction was quenched by saturated aqueous NaHCO3 and
extracted with CH2Cl2 (3 times). The organic phase was dried over
MgSO4, filtered, concentrated in vacuo, and coevaporated with
toluene (3 times). The residue was purified using column chroma-
tography (eluent 0 to 3% MeOH in CH2Cl2) to give 30 (5.21 g,
10.1 mmol, 89%). Rf ) 0.35 (CH2Cl2/CH3OH 95:5 v/v); MALDI-
TOF m/z [M + H]+ found 515.5, calcd 515.1; 1H NMR (270 MHz,
CDCl3) 7.36-7.26 (m, 9H, benzyl, H-6), 7.18-7.14 (m, 2H,
benzyl), 6.08 (d, JH-5, H-6 ) 7.55 Hz, 1H, H-5), 5.21 (s, 1H, H-3′),
1-[1-Amino-4,6-O-benzyl-3-O-methanesulfonyl-1-deoxy-â-D-
fructofuranosyl]uracil (34). To a solution of 33 (3.7 g, 6.65 mmol)
in 100 mL of THF and 33 mL of water was added 1 M PMe3 in
THF (13.3 mL, 13.3 mmol), and the mixture was stirred at room
temperature for 1 h. To this solution was added brine, and the
mixture was extracted with CH2Cl2 (5 times). The organic layer
was dried over MgSO4, filtered, and concentrated under reduced
pressure. The resulting oil was purified using column chromatog-
raphy (eluent 0 to 5% MeOH in CH2Cl2) to give 34 (3.17 g, 5.98
mmol, 90%). Rf ) 0.63 (CH2Cl2/CH3OH 85:15 v/v); MALDI-TOF
1
m/z [M + Na]+ found 554.4, calcd 554.1; H NMR (270 MHz,
CDCl3) 7.78 (d, JH-6,
) 8.29 Hz, 1H, H-6), 7.34-7.25 (m,
H-5
10H, benzyl), 5.63 (d, JH-5, H-6 ) 8.29 Hz, 1H, H-5), 5.29 (s, 1H,
H-3′), 4.77 (d, Jgem ) 11.88 Hz, 1H, CH2Ph), 4.61 (d, 1H, CH2-
Ph), 4.51(s, 2H, 2 × CH2Ph), 4.37-4.32 (m, 1H, H-5′), 4.28 (d,
4.69-4.46 (m, 5H, 2 × CH2Ph, H-1′, H-1′′, H-5′), 4.41 (d, Jgem
)
J
) 2.23 Hz), 3.63-3.49 (m, 3H, H-6′, H-6′′, H-1′), 3.30
H-4′, H-5′
12.37 Hz, 1H, CH2Ph,), 4.30 (ABq, JH-5′,H-4′ ) 1.48 Hz), 4.22 (d,
1H, CH2Ph), 3.36-3.20 (ddd, Jgem ) 10.64 Hz, JH-5′, H-6′ ) 3.59
Hz, 2H, H-6′, H-6′′), 3.03 (s, 3H, CH3, O-mesyl); 13C NMR (67.9
MHz, CDCl3) 171.4 (C-4), 159.9 (C-2), 136.4, 135.8, 132.6 (C-6),
128.5, 128.3, 128.1, 127.9, 110.3 (C-5), 98.3 (C-2′), 87.5 (C-5′),
87.1 (C-3′), 83.8 (C-4′), 73.5 (CH2Ph), 72.3 (CH2Ph), 68.2 (C-
6′), 65.0 (C-1′), 38.0 (O-mesyl).
2′,3-Anhydro-1-[1-azido-4,6-O-benzyl-1-deoxy-â-D-fructofura-
nosyl]uracil (31). Compound 30 (5.2 g, 10 mmol) was dissolved
in 60 mL of dry DMF, NaN3 (3.25 g, 50 mmol) was added, and
the reaction was heated at 100 °C for 60 h under nitrogen
atmosphere. The solvent was removed in vacuo, and the residue
was diluted with CH2Cl2 and washed with brine. The aqueous phase
was extracted with CH2Cl2 (3 times). The combined organic phase
(d, JH-1′, H-1′′ ) 14.23 Hz, 1H, H-1′′), 2.95 (s, 3H, CH3, O-mesyl);
13C NMR (67.9 MHz, CDCl3) 163.9 (C-4), 149.7 (C-2), 141.6 (C-
6), 137.2, 136.6, 128.4, 128.0, 127.8, 127.7, 100.8 (C-5), 98.9 (C-
2′), 83.6 (C-5′), 83.5 (C-4′), 83.0 (C-3′), 73.3 (benzyl), 72.2
(benzyl), 69.3 (C-6′), 44.0 (C-1′), 38.3 (O-mesyl).
(1R,3R,4S,5R)-3-(Benzyloxymethyl)-4-(benzyloxy)-1-(uracil-
1-yl)-6-aza-2-oxabicyclo[3.2.0]heptane (35). Compound 34 (3.17
g, 5.98 mmol) was dissolved in 90 mL pyridine, 4 mL triethylamine
was added, and the mixture was heated at 90 °C for 48 h under
nitrogen atmosphere. The solvent was removed under reduced
pressure and coevaporated with toluene (3 times). The residue
obtained was purified by column chromatography (eluent 0 to 5%
MeOH in CH2Cl2) to give 35 (1.64 g, 3.76 mmol, 63%). Rf ) 0.71
(CH2Cl2/CH3OH 85:15 v/v); MALDI-TOF m/z [M + 2H]+ found
312 J. Org. Chem., Vol. 71, No. 1, 2006

1′,2′-Azetidine-Fused Bicyclic Pyrimidine Nucleosides
1
437.7, calcd 437.1; H NMR (270 MHz, CDCl3) 7.32-7.26 (m,
(1R,3R,4S,5R)-1-(Cytosin-1-yl)-3-(4,4′-dimethoxytrityloxy-
methyl)-4-hydroxy-6-N-(phenoxyacetyl)-6-aza-2-oxabicyclo-
[3.2.0]heptane (40). Compound 38 (0.92 g, 1.33 mmol) was
dissolved in dry pyridine(13 mL), and acetic anhydride (1.25 mL,
13.3 mmol) was added and stirred at ambient temperature for 24
h. Thereafter the reaction was quenched with MeOH (1 mL) and
stirred overnight at room temperature. To this reaction mixture was
added saturated aqueous NaHCO3, and the mixture was extracted
with CH2Cl2 (3 times). The organic phase was dried (MgSO4),
filtered, and evaporated under reduced pressure to get 38a. To this
residue was added 1,2,4-triazole (0.91 g, 13.3 mmol) and coevapo-
rated with dry pyridine (3 times), and 13 mL of the same solvent
was added and cooled to 0 °C. To the resulting clear solution was
added 2-chlorophenylphosphodichloridate (1.1 mL, 6.65 mmol)
under nitrogen atmosphere. After 30 min the reaction was allowed
to reach room temperature and stirred at this temperature for 5 h.
Thereafter aqueous NH3 (15 mL 32%aqueous NH3) was added,
and the reaction was cooled to 5 °C and kept overnight at this
temperature. The resulting solution was extracted with CH2Cl2 (5
times) and the organic phase was dried (MgSO4), filtered, and
evaporated under reduced pressure. Purification by column chro-
matography (eluent 0 to 5% MeOH in CH2Cl2) afforded 40 (640
g, 0.93 mmol, 70%). Rf ) 0.73 (CH2Cl2/CH3OH 85:15 v/v);
MALDI-TOF m/z [M + Na]+ found 713.6, calcd 713.2; 13C NMR
(125.7 MHz, CDCl3) 170.6, 169.6, 165.8, 158.4, 157.2, 154.8,
144.7, 144.6, 141.0, 140.8, 135.8, 130.1, 130.0, 129.8, 129.7, 129.6,
128.2, 128.1, 127.8, 127.7, 126.8, 122.1, 121.6, 114.5, 114.2, 113.1,
95.3, 90.3, 89.9, 86.1, 83.6, 81.7, 73.2, 72.7, 72.0, 71.6, 67.5, 66.7,
63.5, 62.7, 62.4, 55.2.
10H, benzyl), 6.98 (d, JH-6, H-5 ) 8.04 Hz, 1H, H-6), 5.65 (d, JH-5,
H-6 ) 8.04 Hz, 1H, H-5), 4.71 (d, JH-3′, H-4′ ) 5.44 Hz, 1H, H-3′),
4.58-4.41 (m, 5H, 2 × CH2Ph, 2 × CH2Ph, H-5′), 4.22 (dd, JH-3′,
H
-4′
) 5.44 Hz, JH-4′, H-5′ ) 7.67 Hz, 1H, H-4′), 4.12 (d, Jgem
)
)
10.52 Hz, 1H, H-1′), 3.85 (d, 1H, H-1′′), 3.76-3.62 (ddd, Jgem
10.64 Hz, JH-5′, H-6′ ) 2.60 Hz, 2H, H-6′, H-6′′); 13C NMR (67.9
MHz, CDCl3) 163.5 (C-4), 149.2 (C-2), 139.9 (C-6), 128.3, 128.2,
127.9, 127.6, 102.4 (C-5), 93.5 (C-2′), 82.2 (C-5′), 78.5 (C-4′), 73.3
(benzyl), 72.7 (benzyl), 70.1 (C-6′), 65.0 (C-3′), 54.6 (C-1′).
(1R,3R,4S,5R)-3-(Benzyloxymethyl)-4-(benzyloxy)-6-N-(phen-
oxyacetyl)-1-(uracil-1-yl)-6-aza-2-oxabicyclo[3.2.0]heptane (36).
Compound 35 (1.64 g, 3.76 mmol) was coevaporated with dry
pyridine (3 times) and dissolved in 37 mL of the same solvent,
and phenoxyacetyl chloride (0.67 mL, 4.88 mmol) was added
dropwise and stirred at room temperature for 2.5 h. The reaction
was quenched by saturated aqueous NaHCO3 and extracted with
CH2Cl2 (3 times). The residue was purified by column chroma-
tography (eluent 0 to 3% MeOH in CH2Cl2) to give 36 (1.8 g, 3.15
mmol, 84%). Rf ) 0.39 (CH2Cl2/CH3OH 95:5 v/v); MALDI-TOF
m/z [M + H]+ found 570.5, calcd 570.2; 13C NMR (67.9 MHz,
CDCl3) 169.9, 169.0, 163.1, 157.4, 157.3, 149.2, 139.7, 137.7,
137.6, 136.9, 129.8, 128.4, 127.8, 127.6, 122.0, 121.7, 114.4, 103.3,
103.2, 91.0, 90.1, 82.0, 81.8, 78.2, 73.8, 73.5, 73.2, 71.0, 69.0, 68.4,
67.9, 67.4, 61.3, 57.7, 29.7.
(1R,3R,4S,5R)-3-(4,4′-Dimethoxytrityloxymethyl)-4-hydroxy-
6-N-(phenoxyacetyl)-1-(uracil-1-yl)-6-aza-2-oxabicyclo[3.2.0]-
heptane (38). Compound 36 (1.8 g, 3.15 mmol) was dissolved in
anhydrous CH2Cl2 (31 mL) at -78 °C and BCl3 (26.5 mL, 1 M
solution in CH2Cl2) was added dropwise under nitrogen atmosphere.
The reaction mixture was kept at this temperature for 2 h and then
slowly warmed to -20 °C and stirred at this temperature for 2 h.
MeOH (30 mL) was added and the reaction was slowly warmed to
room temperature and stirred for 10 min. The solvent was removed
under reduced pressure and coevaporated with MeOH (3 times) to
give 37. The residue without further purification was coevaporated
with dry pyridine (3 times) and dissolved in 30 mL of the same
solvent. To this reaction mixture were added DMTrCl (1.38 g, 4
mmol) and DMAP (385 mg, 3.15 mmol), and the mixture was
stirred at room temperature for 5 h. The reaction was quenched
with saturated aqueous NaHCO3 solution and extracted with CH2-
Cl2 (3 times). The organic phase was dried over MgSO4, filtered,
concentrated in vacuo, and purified by column chromatography to
give 38 (1.85 g, 2.67 mmol, 85%). Rf ) 0.37 (CH2Cl2/CH3OH 95:5
v/v); MALDI-TOF m/z [M + Na]+ found 714.6, calcd 714.2; 13C
NMR (125.7 MHz, CDCl3) 170.5, 169.1, 162.5, 162.6, 158.4, 158.5,
157.3, 157.1, 149.0, 144.6, 144.4, 139.5, 135.7, 135.6, 130.1, 130.0,
129.9, 129.8, 129.0, 128.2, 128.1, 128.0, 127.8, 127.7, 126.9, 126.8,
122.3, 122.1, 114.4, 114.1, 113.1, 113.0, 103.3, 103.2, 90.1, 89.5,
86.5, 86.2, 84.1, 82.2, 73.1, 72.5, 71.7, 71.6, 67.7, 67.4, 62.9, 62.5,
62.2, 58.0, 55.1, 53.4.
(1R,3R,4S,5R)-4-(2-Cyanoethoxy(diisopropylamino)-phosphin-
oxy)-3-(4,4′-dimethoxytrityloxymethyl)-6-N-(phenoxyacetyl)-1-
(uracil-1-yl)-6-aza-2-oxabicyclo[3.2.0]heptane (39). Compound 38
(0.92 g, 1.33 mmol) was dissolved in 13 mL of dry THF, and
diisopropylethylamine (1.35 mL, 7.75 mmol) was added at 0 °C
followed by 2-cyanoethyl N,N-diisopropylphosphoramidochloridite.
After 30 min the reaction was warmed to room temperature and
stirred for 2 h. MeOH (0.5 mL) was added and stirring was
continued for 5 min. Thereafter saturated aqueous NaHCO3 was
added and extracted with freshly distilled CH2Cl2 (3 times). The
organic phase was dried over MgSO4, filtered, and concentrated
under reduced pressure. The crude residue was purified by silica
gel column chromatography using 40% to 100% CH2Cl2 in
cyclohexane containing 1% Et3N as the eluent, which afforded 39
(1.1 g, 1.23 mmol, 93%) as a mixture of four isomers. Rf ) 0.3
(CH2Cl2/CH3OH 96:4 v/v); MALDI-TOF m/z [M + H]+ found
892.9, calcd 892.3;31P NMR (109.4 MHz, CDCl3) 154.6, 150.6,
149.9, 149.2.
(1R,3R,4S,5R)-3-(4,4′-Dimethoxytrityloxymethyl)-4-hydroxy-
1-(4-N-isobutyrylcytosin-1-yl)-6-N-(phenoxyacetyl)-6-aza-2-
oxabicyclo[3.2.0]heptane (41). Compound 40 (640 mg, 0.93 mmol)
was dried by coevaporating with dry pyridine (3 times) and
suspended in 15 mL of the same solvent. To this solution was added
trimethylchlorosilane (0.58 mL, 4.65 mmol) under nitrogen atmo-
sphere, and the mixture was stirred for 30 min. Thereafter isobutyryl
chloride (0.2 mL, 1.86 mmol) was added and stirred for 3 h. The
reaction was quenched by adding MeOH (0.5 mL) and stirred
overnight. Saturated aqueous NaHCO3 was added to the reaction
and extracted with CH2Cl2 (3 times); the organic layer was collected,
dried (MgSO4), filtered, and evaporated under reduced pressure.
The residue obtained was purified by column chromatography
(eluent 0 to 2% MeOH in CH2Cl2), which afforded 41 (585 mg,
0.77 mmol, 83%). Rf ) 0.45 (CH2Cl2/CH3OH 95:5 v/v); MALDI-
TOF m/z [M + H]+ found 761.7, calcd 761.3; 13C NMR (150.9
MHz, CDCl3) 176.9, 170.7, 169.6, 162.9, 162.9, 158.5, 158.5, 157.2,
154.2, 144.6, 144.3, 135.7, 135.6, 130.1, 130.0, 129.9, 129.8, 129.7,
129.6, 128.2, 128.1, 128.0, 127.8, 126.8, 122.1, 121.7, 114.5, 114.3,
114.2, 113.1, 97.1, 96.9, 90.9, 90.3, 86.2, 84.2, 82.5, 73.2, 72.5,
71.9, 71.6, 67.5, 66.9, 63.1, 62.5, 62.1, 58.2, 55.2, 36.7, 29.6, 19.0,
18.9.
(1R,3R,4S,5R)-4-(2-Cyanoethoxy(diisopropylamino)-phosphin-
oxy)-3-(4,4′-dimethoxytrityloxymethyl)-1-(4-N-isobutyrylcytosin-1-
yl)-6-N-(phenoxyacetyl)-6-aza-2-oxabicyclo[3.2.0]heptane (42).
See SI page S6 for details.
Theoretical Calculations. The structural parameters of the
azetidine- and oxetane-modified nucleosides and conformational
hyperspace available for the compounds have been determined by
the ab initio geometry optimizations followed by 2 ns molecular
dynamics (MD) simulations. The geometry optimizations of the
modified nucleosides have been carried out by the GAUSSIAN 98
program package36 at the Hartree-Fock level using 6-31G** basis
set. The atomic charges and optimized geometries of azetidine-
modified C, T, U, and 5-Me-C nucleosides were then used in
AMBER37 force field parameters employed in the MD simulations.
The protocol of this MD simulations is based on Cheathan-
J. Org. Chem, Vol. 71, No. 1, 2006 313

Honcharenko et al.
Kollman’s38 procedure employing modified version of Amber 1994
force field as it is implemented in AMBER 7 program package.37
The TIP3P water model was used to introduce explicit solvent
molecules in the MD calculations. Periodic boxes containing 1048,
1125, 1084, and 1122 water molecules were created around
azetidine-modified C, T, U, and 5-Me-C, respectively, extending
10.0 Å from these molecules in three dimensions.
program (RIGHT), and Philip Morris USA Inc. is gratefully
acknowledged.
Supporting Information Available: General experimental
methods and experimental conditions for the preparation of
compounds 8, 10, 18a, 26, 33, and 42; 13C NMR spectra of
compounds 2a, 4, 5, 6a, 7-10, 12-17, 18a, 19, 22-24, 26-36,
38, 40,and 41; 31P NMR spectra of compounds 20, 39, and 42;
Cartesian coordinates of azetidine-C, -T, -U, and -5-Me-C nucleo-
sides. This material is available free of charge via the Internet at
http://pubs.acs.org.
Acknowledgment. Generous financial support from the
Swedish Natural Science Research Council (Vetenskapsrådet),
the Stiftelsen fo¨r Strategisk Forskning, EU 6th Framework
(38) Cheatham, T. E., III; Kollman, P. A. J. Am. Chem. Soc. 1997, 119,
4805.
JO052115X
314 J. Org. Chem., Vol. 71, No. 1, 2006