Stereoselective synthesis of dithymidine phosphorothioates using xylose derivatives as chiral auxiliaries

Article abstract of DOI:10.1021/jo972318o

1,2-O-Cyclopentylidene-5-deoxy-5-isopropylamino-α-D-xylofuranose 1 and its enantiomer 11 were used as chiral auxiliaries to form, respectively, Sp and Rp dithymidine phosphorothioates 9 and 10 in 98% diastereomeric excess, using phosphoramidite methodology and 2-bromo-4,5-dicyanoimidazole as catalyst. The stereochemistry of the coupling reaction is discussed.

Full text of DOI:10.1021/jo972318o

J . Org. Chem. 1998, 63, 3647-3654
3647
Ster eoselective Syn th esis of Dith ym id in e P h osp h or oth ioa tes Usin g
Xylose Der iva tives a s Ch ir a l Au xilia r ies
Yi J in and George J ust*
Department of Chemistry, McGill University, Montreal, Canada H3A 2K6
Received December 29, 1997
1,2-O-Cyclopentylidene-5-deoxy-5-isopropylamino-R-D-xylofuranose 1 and its enantiomer 11 were
used as chiral auxiliaries to form, respectively, Sp and Rp dithymidine phosphorothioates 9 and
10 in 98% diastereomeric excess, using phosphoramidite methodology and 2-bromo-4,5-dicyanoimi-
dazole as catalyst. The stereochemistry of the coupling reaction is discussed.
Sch em e 1
In tr od u ction
The effect of oligonucleotide phosphorothioates (PS-
oligos) as antisense inhibitors of gene expression is well
recognized,1 and their therapeutic potential has been
demonstrated in clinical trials against AIDS and cancer.2
The PS-oligos currently employed in clinical studies and
biological evaluations are obtained as mixtures of 2n
diastereomers, where n is equal to the number of inter-
nucleotidic phosphorothioate linkages. To date only one
generally applicable stereoselective synthesis methodol-
ogy of PS-oligos has been described by Stec et al.3 It
unfortunately cannot be scaled up due to a difficult
chromatographic separation of the required chiral pre-
cursors. Therefore, there still exists a need to develop
practical stereoselective syntheses of PS-oligos.
In a preliminary communication,4 we have described
the usefulness of xylose derivative 1 and 11 as chiral
auxiliary for a stereocontrolled synthesis of phosphite
triesters and therefore of phosphorothioates. In what
follows, we give the full details of our work in this area.
Resu lts a n d Discu ssion
The known 1,2-O-cyclopentylidene-5-tosyl-R-D-xylo-
furanose was synthesized from D-xylose by modification
of a method described by Tronchet et al.5 The tosylate
function was then displaced with isopropylamine to
provide 1,2-O-cyclopentylidene-5-deoxy-5-isopropylamino-
R-D-xylofuranose 1. The overall yield for the transforma-
tion from D-xylose to 1 was ∼60%. Reaction of γ-amino
alcohol 1 with PCl3 and triethylamine in dry chloroform
provided phosphorochloridite 2. Its 31P NMR showed a
single peak at 148.42 ppm. By analogy with phosphora-
midite 4, it most likely has the Rp stereochemistry
depicted in Scheme 1. Reaction of the phosphorochlo-
ridite 2 with 5′-O-tert-butyldimethylsilylthymidine (T3′OH)
in dry chloroform gave a mixture of phosphoramidites 4
and 3 (31P NMR 129.8 and 138.7 ppm) in a ratio which
was time and temperature dependent. At -78 °C, the
two isomers were formed in a 3:2 ratio, whereas at rt,
the ratio was >10:1. After overnight heating at 50 °C,
the minor isomer 3 corresponding to the peak at 138.7
ppm was quantitatively isomerized to the thermodynami-
cally more stable isomer 4 (129.8 ppm). Both isomers
were configurationally stable after extraction and re-
moval of triethylammonium chloride and not separable
by flash chromatography.
(1) Uhlmann, E.; Peyman, A. Chem. Rev. 1990, 90, 543.
(2) Zon, G.; Stec, W. J . Phosphorothioate Oligonucleotides. In
Oligonucleotides Analogues: A Practical Approach; Eckstein, F. Ed.;
The Practical Approach Series; IRL Press: 1991, pp 87-108.
(3) (a) Stec, W. J .; Wilk, A. Angew. Chem., Int. Ed. Engl. 1994, 33,
709. (b) Stec, W. J .; Grajkowski, A.; Kobylanska, A.; Karwowski, B.;
Koziolkiewicz, M.; Misiura, K.; Okruszek, A.; Wilk, A.; Guga, P.;
Boczkowska, M. J . Am. Chem. Soc. 1995, 117, 12019.
(4) J in, Y.; Biancotto, G.; J ust, G. Tetrahedron Lett. 1996, 37, 973.
(5) Tronchet, J . M. J .; Zosimo-Landolfo, G.; Villedon-Denaide, F.;
Balkadjian, M.; Cabrini, D.; Barbalat-Rey, F. J . Carbohydr. Chem.
1990, 9(6), 823.
S0022-3263(97)02318-9 CCC: $15.00 © 1998 American Chemical Society
Published on Web 05/07/1998

3648 J . Org. Chem., Vol. 63, No. 11, 1998
J in and J ust
Sch em e 2
then underwent the coupling reaction with T5′OH to give
14 and 16 which were then sulfurized with Beaucage’s
reagent to give 15 and 17 (31P NMR 68.91 and 69.12
ppm). The diastereomeric ratio of 15 and 17 was 1:7
when the coupling reaction was performed at rt in
acetonitrile. When the coupling reaction was performed
at -15 °C in chloroform, the diastereomeric ratio of 15
and 17 was 1:70 as determined by 31P NMR. Only isomer
17 could be isolated by chromatography. After hydrolysis
of 15/17 with 70% TFA at rt, the phosphorothioate
dinucleosides 9 and 10 were obtained with an Sp:Rp
diastereomeric ratio identical to that of 15 to 17.
In summary, the use of chiral auxiliary 1 derived from
D-xylose leads to phosphorothioate dinucleoside 9 (Sp),
while use of the chiral auxiliary 11 derived from L-xylose
leads to phosphorothioate dinucleoside 10 (Rp).
The essential step in the synthesis of the phosphite
triester and therefore of phosphorothioate is the azole-
activated (tetrazole or, in our case, 2-bromo-4,5-dicy-
anoimidazole) coupling reaction of the phosphoramidite
with the free 5′-hydroxylfunction of thymidine. The
mechanism of this activation process still remains to be
clarified. The main question is whether azole serves only
as a proton donor yielding as primary product a proto-
nated nucleoside phosphoramidite or whether it will
attack as a nucleophile giving rise to the formation of an
azolide intermediate.9 In the first case, after protonation
of phosphoramidite, the free 5′-hydroxyl group of thymi-
dine (T5′OH) reacts with inversion of configuration at the
phosphorus center to give the desired phosphite triester.
The second assumption involves the displacement of the
amine function of the amidite by the azole after proto-
nation, followed by either displacement of the azole by
another azole leading to a diastereomeric mixture of
phosphorazolides or by displacement of the azole by an
alcohol which gives the desired phosphite triester. The
phosphite triesters are then sulfurized. The latter reac-
tion is known to proceed with retention of configuration.10
The stereochemistry of a reaction is one important clue
to the reaction mechanism. With structure assignments
for the intermediate phosphoramidites 3 and 4 being
somewhat ambiguous, we first tried to obtain crystals of
phosphoramidite 4 or of the corresponding thioate 4S (31P
NMR 67.5 ppm) or 3S (31P NMR 72.4 ppm)11 for X-ray
crystallographic structure determination. Neither of
them gave suitable crystals. The same was also true for
phosphoramidites 18a -d (31P NMR 129.0, 128.2, 125.3,
and 127.5 ppm, respectively), in which the exocyclic
thymidine was replaced by 2-propanol, cholesterol, and
p-nitrophenol, and for their sulfur derivatives 19a -d (31P
NMR 67.5, 66.6, 62.0, and 66.8 ppm) (Scheme 3).
The coupling reaction between phosphoramidite 4 and
3′-O-tert-butyldimethylsilyl thymidine (T5′OH) was first
carried out at room temperature in acetonitrile, using
2-bromo-4,5-dicyanoimidazole as catalyst. It provided
phosphite triesters 5 and 7 in a ratio of 6:1, as established
by 31P NMR (143.76 and 142.55 ppm for the major and
minor isomer, respectively). Without purification, the
mixture of triesters was sulfurized with Beaucage’s
reagent6 to give a 6:1 mixture of phosphorothioates 6 and
8, 31P NMR 68.23 and 68.43 ppm. When a similar
coupling reaction was carried out in CHCl3 at 0 °C for 4
h or at -15 °C for 6 h, the ratio of 5 to 7 increased to
40:1 and 68:1, respectively. Chromatography of the
reaction mixture obtained at -15 °C provided one isomer
of 6 only. After hydrolysis of 6/8 with 70% trifluoroacetic
acid at rt, the phosphorothioate dinucleosides 9 (31P NMR
55.70 ppm) and 10 (31P NMR 56.10 ppm) was obtained
in 85% yield with an Sp:Rp diastereomer ratio the same
as the ratio of 6 to 8. The configurations of the major
diastereomer 9 and minor diastereomer 10 were estab-
lished as Sp and Rp, respectively by snake venom
phosphodiesterase and P1 nuclease digestion and also by
HPLC analysis.7 It is opposite to the one we had
tentatively and erroneously assigned4 because the chemi-
cal shifts of 31P NMR for the phosphorothioate dinucleo-
sides 9 and 10 are opposite in CD3OD and D2O. In D2O,
the signal of 31P NMR corresponding to the Sp diastere-
omer occurs at higher field (31P NMR 55.70 ppm) than
that of the Rp diastereomer (31P NMR 56.10 ppm),8 while
in CD3OD the signal of 31P NMR corresponding to the
Sp diastereomer occurs at lower field (31P NMR 58.64
ppm) than that of the Rp diastereomer (31P NMR 58.57
ppm).
The structural assignment for the phosphoramidites
3 and 4 was therefore made as described by Huang et
al. and others.12-15 They showed that in bicyclic six-
membered ring phosphites the 1,3,2-dioxaphosphorinane
In a parallel run, L-xylose was transformed to the
amino alcohol 11. In a series of reactions identical to
those described (Scheme 2), 11 was converted via phos-
phorochloridite 12 (31P NMR 148.75 ppm) to phosphora-
midite 13 (31P NMR 129.34 ppm). Phosphoramidite 13
(9) Berner, S.; Muhlegger, K.; Seliger, H. Nucleic Acids Res. 1989,
17, 853.
(10) (a) Fujii, M.; Ozaki, K.; Sekine, M. and Hata,T. Tetrahedron
1987, 43, 3395-3407. (b) Mikolajczyk, M. J . Chem. Soc., Chem.
Commun. 1969, 1221.
(11) Although the phosphoramidites 3 and 4 are not separable, the
phosphorothioamidates 3S and 4S can be separated by a chromatog-
raphy method.
(12) Huang, Y.; Yu, J .; Bentrude, W. G. J . Org. Chem. 1995, 60,
4767-4773.
(13) (a) Hermans, R. J . M.; Buck, J . M. Phosphorus Sulfur 1987,
31, 255. (b) Nelson, K. A.; Sopchik, A. E.; Bentrude, W. G. J . Am. Chem.
Soc. 1983, 105, 7752.
(6) (a) Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett. 1981,
22, 1859-62. (b) Iyer, R. P.; Egan, W.; Regan, J . B.; Beaucage, S. L. J .
Am. Chem. Soc. 1990, 112, 1253-54. (c) For a review, see: Beaucage,
S. L., Iyer, R. P. Tetrahedron 1992, 48, 2223-2311.
(7) The snake venom phosphodiesterase and P1 nuclease digestion
and HPLC analysis were carried out at ISIS Pharmaceuticals (Carls-
bad, CA) by Dr. M. Manoharan.
(8) (a)Uznanski, B.; Niewiarowski, W.; Stec, W. J . Tetrahedron Lett.
1982, 23, 4289-4292. (b). Lesnikowski, Z. J .; J aworska, M. M.
Tetrahedron Lett. 1989, 30, 3821-3824.

Synthesis of Dinulceoside Phosphorothioates
J . Org. Chem., Vol. 63, No. 11, 1998 3649
Ta ble 2. Selected 1H NMR Cou p lin g Con sta n tsa
Sch em e 3
comp J H3′′-H4′′ J H3′′-P J H4′′-H5′′a J H4′′-H5′′e J H5′′a-P J H5′′e-P J H3′-P
4
3
4S
3S
2.4
3.9
2.4
4.1
1.0
3.2
2.4
5.3
2.4
7.3
2.4
6.6
2.4
7.3
2.4
6.4
2.4
4.7
3.2
7.1
6.1
20
19
10.5
9.0
10
12
10
aSolvent: CDCl3. The protons of the branched-chain sugar units
at the 3′-end of the molecule are denoted by H′ and the protons of
the sugar at the chiral auxiliary are denoted by H′′.
Sch em e 5
Sch em e 4
Ta ble 1. Selected 13C NMR Cou p lin g Con sta n tsa
comp
J C3′-P
J C2′′-P
J C3′′-P
J C4′′-P
J C5′′-P
J CHN-P
3S
4S
3
4.6
5.5
10
12
11
4.6
4.6
6.3
9.2
2
1.8
4.6
2.7
1.8
5.5
7.3
3.7
3.2
6.4
6.4
37.5
36.6
3
4
19.2
3.7
Coupling constants J PNCH have not been found to be
useful in conformational analyses of three-coordinate
phosphorus-containing heterocyclic six-membered rings.12,14
The data observed for conformer 4a are similar to those
found in the previously studied three-coordinate 1,3,2-
dioxa- and oxazaphosphorinanes that populate analogous
chair conformations with substituents attached axially
to phosphorus. Thus the small values of 3J H3′′-P (1.0 Hz),
3J H3′′-H4′′ (2.4 Hz),3J H4′′-H5a′′ (2.4 Hz),3J H4′′-H5e′′ (2.4 Hz),
aSolvent: CDCl3. The carbons of the branched-chain sugar units
at the 3′-end of the molecule are denoted by C′ and the carbons of
the sugar at the chiral auxiliary are denoted by C′′.
ring preferentially adopts a chair conformation with the
exocyclic OR attached to the phosphorus group in an axial
position. The thermodynamically less stable isomers,
however, showed a preference for a twist conformation
of the dioxaphosphorinane ring in which the OR group
occupies a pseudoaxial position12 instead of a chair
conformation in which the OR group would occupy an
equatorial position. By analogy, we suggest that the
thermodynamically more stable phosphoramidite 4 has
the thymidine residue at the axial position of the six-
membered ring, which has a chair conformation (Scheme
4). This is based on the 13C NMR data: the large 2J
coupling constant between the C3′ of thymidine and
phosphorus of 19.2 Hz, the analogous coupling of 3.7 Hz
for C-O-P of the oxazaphosphorinane carbon, and the
small coupling constant of 1.8 Hz for J C4′′-P in isomer 4
(Table 1).
3
and J H5a′′-P (2.4 Hz) recorded are characteristic of gauche
HCCH and HCOP arrangements, while the large value
of 3J H5e′′-P (7.1 Hz) observed is typical of the antiperipla-
nar HCOP geometry (Table 2).
The conformation of 4 was also confirmed by a 2D NOE
experiment in which H5′′a showed NOE with the H3′′. The
2D NOE experiment also showed a cross-peak between
H3′′ and H4′ which tells us that H3′′ and OT3′ should be
on the same side of the six-membered ring. No detectable
NOE’s with H2′′/H5′′a or H1′′/H5′′a were observed.
Using a similar line of reasoning, twist conformation
3c is the most likely conformation for 3 (Scheme 5).
When we used a 3:2 mixture of 4 and 3 to carry out
the coupling reaction with thymidine derivative T3′OH
under the same coupling reaction conditions as for
diastereomer 4, we obtained the phosphite triesters 5 and
7 in a ratio of 3:2. Treatment with Beaucage’s reagent
gave a 3:2 mixture of phosphorothioates 6 and 8 (31P
NMR 68.23 and 68.43 ppm). We can therefore conclude
that the two isomers gave reversed diastereoselectivity
in the coupling reaction, with 3 giving a phosphorothioate
with the Rp configuration. We also observed that the
Therefore, there are only two possible conformers, 4a
(Sp) and 4b (Rp) for the isomer 4 (Scheme 4), of which
4a should be strongly favored because of the far smaller
1,3-diaxial interactions of the P-OT3′ bond.
The conformational analyses of the six-membered rings
3
3
were also based on the values of J POCH and J HCCH
.
(14) Huang, Y.; Arif, A. M.; Bentrude, W. G. J . Org. Chem. 1993,
58, 6235.
(15) Van Heeswijk, W. A. R.; Goedhart, J . B.; Vliegenthart, J . F. G.
Carbohyd. Res. 1977, 58, 337-344.

3650 J . Org. Chem., Vol. 63, No. 11, 1998
J in and J ust
dimethoxytritylthymidine, we realized that the reaction
conditions were too acidic, resulting in hydrolysis of the
dimethoxytrityl group. The same was true when the
thymidine was substituted at 5′ with a monomethoxytri-
tyl function. A trityl group appeared to be stable but is
not suitable for automated DNA synthesis. We therefore
investigated other less acidic catalysts to see if 2-bromo-
4,5-dicyanoimidazole could be replaced without loss of
stereoselectivity of the reaction.
First, we tried to examine the pKa effect of catalyst.
Since 1H-tetrazole (pKa ) 4.8) is a commonly used
efficient activator for the coupling reactions,9 we tried
using it in our coupling reaction. 31P NMR showed the
coupling reaction to be very slow and without good
stereoselectivity. We also tried other catalysts with pKa
> 4.8, such as 4,5-dicyanoimidazole (pKa ) 5.2), 2-benzyl-
4,5-dicyanoimidazole (pKa ) 5.3), 2-bromo-4,5-diethyl-
carboxylimidazole (pKa ) 6.4), and 2-nitroimidazole (pKa
) 6.4). The 31P NMR showed that the coupling reactions
were very slow and without stereoselectivity. Then we
tried using a catalyst with pKa < 4.8. Benzimidazolium
triflate (pKa ) 4.5) and p-nitrophenyltetrazole (pKa ) 3.7)
have been reported to be better activators than 1H-
tetrazole,18 but the coupling reaction was slow and
without stereoselectivity in our system.
F igu r e 1. The mechanism of the coupling reaction (imid )
2-bromo-4,5-dicyanoimidazole or its anion).
Sch em e 6
Since increasing the size of the substituents of imida-
zole seemed to give higher stereoselectivity,19 we syn-
thesized 2-iodo-4,5-dicyanoimidazole and found that it did
not give better stereoselectivity than 2-bromo-4,5-dicy-
anoimidazole. Also it is not a good catalyst choice
because of its high acidity.
Con clu sion
We have shown that diastereomerically pure cyclic
phosphoramidites 4 and 13 obtained without chromato-
graphic purification can lead diastereoselectively to Sp
and Rp dinucleoside phosphorothioates 9 and 10, respec-
tively. The conditions employed here were suitable for
solution synthesis but are for the time being not ap-
plicable to solid phase synthesis. The stereochemistry
of the coupling reaction suggested involves only one
inversion at phosphorus in the case of 1,2-O-cyclopen-
tylidene-5-deoxy-5-isopropylamino-R-D-xylofuranose 1 or
1,2-O-cyclopentylidene-5-deoxy-5-isopropylamino-R-L-xy-
lofuranose 11 derived phosphoramidites, if our analysis
of the conformers of the intermediate phosphoramidites
is correct. It should however be noticed that a closely
related system described in Scheme 6 gives the opposite
stereochemistry.
thermodynamically less favored diastereomer 3 is con-
siderably more reactive than diastereomer 4.
Analysis of the stereochemistry of phosphoramidite 4
(Sp) and phosphorothioate 9 (Sp) indicates that the
transformation involves one inversion only for this
particular system (Figure 1). This excludes the double
inversion postulated by Stec and Zon16 and by Seliger et
al.9 Indeed, when bromoacetic acid (pKa ) 2.86) was used
as activator in the coupling reaction, the same phosphite
triesters 5 and 7 were obtained in a ratio of 4:1. This is
to be compared to a 6:1 ratio when 2-bromo-4,5-dicy-
anoimidazole was used as catalyst, using identical reac-
tion conditions. This change in reaction path is particu-
larly puzzling because the use of a closely related chiral
auxiliary 20 (Scheme 6) gave the opposite Rp stereo-
chemistry.17 If the mechanism we suggested here is
general, the phosphoramidite 21 formed from chiral
auxiliary 20 should have opposite configuration (Rp).
Exp er im en ta l Section
Ma ter ia ls a n d Meth od s. NMR spectra were recorded at
500 MHz for 1H NMR, 125 MHz for 13C NMR, and 202 MHz
for 31P NMR.20 Chloroform was distilled from P2O5; triethy-
lamine and acetonitrile were distilled from CaH2. Tetrazole
and Beaucage’s reagent were gifts from Isis Pharmaceuticals.
All reactions involving trivalent phosphorus compounds were
performed under dry argon.
Effect of th e Acid ity of th e Ca ta lyst on th e
Ster eoselectivity a n d Ra te of th e Rea ction . When
trying to apply the 2-bromo-4,5-dicyanoimidazole-cata-
lyzed coupling reaction of the phosphoramidite to 5′-
1,2-O-Cyclop en t ylid en e-5-d eoxy-5-isop r op yla m in o-r-
D-xylofu r a n ose (1). A solution of 1,2-O-cyclopentylidene-5-
(18) (a) Froehler, B. C.; Matteucci, M. D. Tetrahedron Lett. 1983,
24, 3171. (b) Pon, R. T. Tetrahedron Lett. 1987, 28, 3643. (c) Hayakawa,
Y.; Kataoka, M.; Noyori, R. J . Org. Chem. 1996, 61, 7996.
(19) Xin, Z.; J ust, G. Tetrahedron Lett. 1996, 37, 969.
(16) Stec, W. J .; Zon, G. Tetrahedron Lett. 1984, 25, 5279.
(17) Marsault, E.; J ust, G. Tetrahedron 1997, 53, 16945-16958.

Synthesis of Dinulceoside Phosphorothioates
J . Org. Chem., Vol. 63, No. 11, 1998 3651
tosyl-R-D-xylofuranose (2.64 g, 7.1 mmol) in isopropylamine
(15 mL) was stirred at 55 °C overnight in a pressure bottle.
The solvent was removed by rotary evaporator, and the
remaining yellow syrup was taken up with chloroform, washed
with saturated NaHCO3 and brine, and dried over MgSO4.
After removal of the solvent, the mixture was chromato-
graphed on silica gel (ethyl acetate-3% triethylamine) to
(C-1′′), 86.44 (d, J ) 2.8 Hz, C-4′), 84.83 (C-1′), 84.82 (d, J )
7.3 Hz, C-2′′), 73.75 (d, J ) 19.2 Hz, C-3′), 73.07 (d, J ) 1.8
Hz, C-4′′), 71.85 (d, J ) 3.7 Hz, C-3′′), 63.21 (C-5′), 50.0 (d, J
) 36.6 Hz, NCH), 40.28 (d, J ) 5.5 Hz, C-2′), 36.92, 36.24
(CH2CCH2), 36.12 (d, J ) 3.2 Hz, C-5′′), 25.94 (SiCCH3), 23.49,
22.80 (CH2CH2CCH2CH2), 22.03 (d, J ) 9.2 Hz, CH3CHN),
21.70 (d, J ) 6.4 Hz, CH3CHN), 18.36 (SiCCH3), 12.52
(CH3CdC), -5.37, -5.44 (Me2Si); 31P NMR (CDCl3) δ 129.80;
HRMS (FAB, glycerol) m/e calcd for C29H49N3O9PSi [MH+]
642.2973, found 642.2976.
furnish 1.33 g of white solid 1 (73%): mp 44-45 °C; [R]20
)
D
31.06 (c ) 2, CHCl3); 1H NMR (CDCl3) δ 8.0 (b, NH), 5.90 (1H,
d, J ) 3.9 Hz, H-1), 4.38 (1H, d, J ) 3.9 Hz, H-2), 4.27 (1H, m,
H-3), 4.20 (1H, m, H-4), 3.36-2.92 (2H, ABX, 2 × H-5), 2.74-
2.70 (1H, septet, NCH), 1.95-1.63 (8H, m, cyclopentylidene
protons), 1.04-1.03 (6H, dd, Me2CH); 13C NMR (CDCl3) δ
121.06 (OCO), 104.82 (C-1), 86.14 (C-2), 78.30 (C-3), 77.07 (C-
4), 48.64 (NCH), 45.90 (C-5), 36.85, 36.32 (CH2CCH2), 23.51,
22.84 (CH2CH2CCH2CH2), 22.64 (CH3CHN), 22.34 (CH3CHN);
HRMS (EI) m/e calcd for C13H23NO4 [M+] 257.1627, found
257.1630.
Oxa za p h osp h or in a n e (3). The reaction procedure and
workup were analogous to those of 4 except the reaction was
done at -78 °C and workup was done at 0 °C. After
purification with silica gel, a mixture of 3 and 4 was obtained
in a ratio of 2:3. The NMR spectrum of 3 was obtained by
subtracting the spectrum of 4 from the spectrum of the
mixtrure of 3 and 4: 1H NMR (CDCl3) δ 8.19 (1H, bs, NH),
7.45 (1H, d, J ) 1.4 Hz, H-6), 6.34 (1H, dd, J H1′-H2a′ ) 5.1 Hz,
J H1′-H2b′ ) 8.3 Hz, H-1′), 5.97 (1H, d, J H1′′-H2′′ ) 3.9 Hz, H-1′′),
4.71, 4.68 (1H, ddt, J H3′-4′ ) J H3′-2a′ ) 2.2 Hz, J H3′-2b′ ) 6.1
Hz, J H3′-P ) 9.0 Hz, H-3′), 4.56 (1H, d, J H1′′-H2′′ ) 3.9 Hz, H-2′′),
4.09 (1H, dd, J H3′′-H4′′ ) 3.4 Hz, J 3′′-P ) 3.2 Hz, H-3′′), 4.51
(1H, dt, J H4′′-H3′′ ) 3.9 Hz, J H4′′-H5a′′ ) J H4′′-H5e′′ ) 7.3 Hz, H-4′′),
4.04 (1H, q, J H4′-H3′ ) J H4′-H5a′ ) J H4′-H5b′ ) 2.2 Hz, H-4′), 3.86-
3.73 (2H, ABX, 2 × H-5′), 3.50 (1H, d.septet, J NCH-CH3 ) 6.8
Hz, J NCH-P ) 10.7 Hz, NCH), 3.21 (1H, ddd, J H5e′′-H4′′ ) 7.8
Hz, J H5e′′-H5b′′ ) 13.7 Hz, J H5e′′-P ) 6.1 Hz, H-5e′′), 3.08 (1H,
ddd, J H5a′′-H4′′ ) 6.9 Hz, J H5a′′-H5e′′ ) 13.7 Hz, J H5a′-P ) 4.7 Hz,
H-5a′′), 2.37 (1H, m, H-2′), 2.02 (1H, m, H-2′), 1.88 (3H, d, J )
1.5 Hz, MeCdC), 1.97-1.62 (8H, m, cyclopentylidene protons),
1.13-1.10 (6H, m, Me2CH), 0.89 (9H, s, t-BuSi), 0.08 (6H, s,
Me2Si); 13C NMR (CDCl3) δ 163.57 (C-4), 150.08 (C-2), 135.33
(C-6), 121.42 (OCO), 110.78 (C-5), 105.76 (C-1′′), 86.78 (d, J )
3.7 Hz, C-4′), 84.80 (C-1′), 84.53 (d, J ) 4.6 Hz, C-2′′), 73.03
(d, J ) 10 Hz, C-3′), 78.72 (d, J ) 2.7 Hz, C-4′′), 75.82 (d, J )
2.0 Hz, C-3′′), 63.06 (C-5′) 48.16 (d, J ) 37.5 Hz, NCH), 40.30
(d, J ) 4.5 Hz, C-2′), 37.03, 36.62 (CH2CCH2), 38.91 (d, J )
3.7 Hz, C-5′′), 25.90 (SiCMe3), 23.39, 23.09 (CH2CH2CCH2CH2),
22.58 (d, J ) 8.0 Hz, CH3CHN), 22.29 (d, J ) 3.7 Hz, CH3-
CHN) 18.33 (SiCMe3), 12.51 (CH3CdC), -5.35, -5.47 (Me2-
Si); 31P NMR (CDCl3) δ 138.70.
Cyclic P h osp h or och lor id ite (2). To a solution of freshly
distilled phosphorus trichloride (96 mL, 1,1 mmol) in 2.5 mL
of anhydrous chloroform stirred at 0 °C under Ar was added
a mixture of 1 (257 mg, 1.0 mol) and triethylamine (278 mL,
2.2 mmol) in 3.5 mL of anhydrous CHCl3. The mixture was
heated at 50 °C, and the reaction was followed by 31P NMR
until a single peak was obtained. The product was not isolated
and was directly used in the following step: 1H NMR (CDCl3)
δ 5.75 (1H, d, J ) 3.9 Hz, H-1), 4.51 (1H, m, H-3), 4.37 (1H, d,
J ) 3.9 Hz, H-2), 4.19-4.17 (1H, m, H-4), 3.42-3.34 (1H,
septet, NCH), 3.33-3.00 (2H, ABX, 2 × H-5), 1.81-1.51 (8H,
m, cyclopentylidene protons), 1.05 (6H, d, J ) 6.8 Hz, Me2-
CH); 13C NMR (CDCl3) δ 121.48 (OCO), 104.26 (C-1), 83.85
(d, J ) 3.7 Hz, C-2), 73.01 (d, J ) 6.4 Hz, C-3), 72.35 (C-4),
50.28 (d, J ) 35.7 Hz, C-5), 36.93 (d, J ) 5.5 Hz, NCH), 36.62,
35.91 (CH2CCH2), 23.22, 22.50 (CH2CH2CCH2CH2), 20.84 (d,
J ) 12.8 Hz, CH3CHN), 19.62 (d, J ) 5.5 Hz, CH3CHN); 31P
NMR (CDCl3) δ 148.42.
Oxa za p h osp h or in a n e (4). The solution of 2 was cooled
to 0 °C under Ar; then a solution of T3′OH (356 mg, 1.0 mmol)
in 4.5 mL of anhydrous CHCl3 and triethylamine (140 mL,
1.1 mmol) was added slowly. The mixture was heated to 50
°C until a single peak corresponding to a new product was
found in the 31P NMR spectrum. The solution was taken up
in ethyl acetate (prewashed with a saturated solution of
NaHCO3), washed with saturated NaHCO3, and dried over
MgSO4. The crude product was flash chromatographyed on a
silica gel column (hexane-ethyl acetate-triethylamine ) 5:3:
P h osp h or oth ioa tes (6/8). A mixture of phosphoramidite
4 (15 mg, 0.0234 mmol), T5′OH (10 mg, 1.2 equiv) and 2-bromo-
4,5-dicyanoimidazole (9.2 mg, 2.0 equiv) was dried at high
vacuum overnight. Anhydrous acetonitrile (0.6 mL) was added
at 0 °C under Ar. The solid dissolved instantly. The reaction
was warmed to rt, and its course was followed by 31P NMR.
Within 5 min, the peak corresponding to the phosphoramidite
had disappeared and two new peaks corresponding to 5 and 7
(143.76 ppm, 142.55 ppm ) 6:1) appeared: MS (FAB, ni-
trobenzyl alcohol) [M + H+] calcd for C45H77N5PO14Si2 phos-
phite triester 998, found 998.4. This product was used in the
following sulfurization without isolation. To this solution was
added Beaucage’s reagent (5.6 mg, 1.2 equiv) in 140 µL of
acetonitrile (0.2 M). Instantaneously the 31P NMR showed
another two peaks corresponding to 6 and 8 (68.23 ppm, 68.43
ppm ) 6:1), while the peaks corresponding to the starting
material disappeared. The solvent was evaporated, and the
mixture was redissolved in ethyl acetate, washed with satu-
rated NaHCO3 and water, and dried over MgSO4. The crude
product was purified on a silica gel column (ethyl acetate:
methanol ) 95:5) to give 22 mg of a white solid (91%). When
a similar reaction was carried out at 0 °C for 4 h and at -15
°C for 6 h, using chloroform as the solvent, the ratio of 6 and
8 increased to 40:1 and 68:1, respectively. Chromatography
of the reaction mixture obtained at -15 °C only provided one
2) to furnish 510 mg of 4 as a white foam (80%): mp 68-70
1
°C; [R]20 ) +62.91 (c ) 0.5, CHCl3); H NMR (CDCl3) δ 8.19
D
(1H, bs, NH), 7.47 (1H, d, J H6-CH3CdC ) 1.4 Hz, H-6), 6.34-
6.31 (1H, dd, J H1′-H2a′ ) 5.6 Hz, J H1′-H2b′ ) 8.6 Hz, H-1′), 5.89
(1H, d, J H1′′-H2′′ ) 3.9 Hz, H-1′′), 4.57-4.54 (1H, ddt, J H3′-H4′
)
J H3′-H2a′ ) 2.2 Hz, J H3′-H2b′ ) 6.6 Hz, J H3′-P ) 10.5 Hz, H-3′),
4.41 (1H, d, J H1′′-H2′′ ) 3.9 Hz, H-2′′), 4.35 (1H, dd, J H3′′-H4′′
2.4 Hz, J H3′′-P ) 1.0 Hz, H-3′′), 4.17 (1H, q, J H4′′-H3′′ ) J H4′′-H5a′′
)
) J H4′′-H5e′′ ) 2.4 Hz, H-4′′), 4.06 (1H, q, J H4′-H3′ ) J H4′-H5a′
)
J H4′-H5b′ ) 2.2 Hz, H-4′), 3.90-3.77 (2H, ABX, 2 × H-5′), 3.47-
3.41 (1H, d septet, J NCH-CH3 ) 6.6 Hz, J NCH-P ) 11.7 Hz, NCH),
3.47-3.44 (1H, dt, J H5a′′-H5e′′ ) 14.2 Hz, J H5a′′-H4′′ ) J H5a′′-P
)
2.4 Hz, H-5a′′), 3.06-3.01 (1H, ddd, J H5e′′-H4′′ ) 2.4 Hz,
J H5e′′-H5a′′ ) 14.2 Hz, J H5e′′-P ) 7.1 Hz, H-5e′′), 2.39-2.35 (1H,
ddd, J H2a′-H1′ ) 5.9 Hz, J H2a′-H3′ ) 2.2 Hz, J H2a′-H2b′ ) 13.7 Hz,
H-2′), 2.11-2.06 (1H, ddd, J H2b′-H1′ ) 8.4 Hz, J H2b′-H3′ ) 6.6
Hz, J H2b′-H2a′ ) 14 Hz, H-2′), 1.90 (3H, d, J ) 1.5 Hz, MeCdC),
1.97-1.62 (8H, m, cyclopentylidene protons), 1.13-1.10 (6H,
dd, J ) 6.8 Hz, J ) 7.8 Hz, Me2CH), 0.91 (9H, s, t-BuSi), 0.10
(6H, d, J ) 1.5 Hz, Me2Si); 13C NMR (CDCl3) δ 163.38 (C-4),
150.10 (C-2), 135.24 (C-6), 121.46 (OCO), 110.94 (C-5), 104.63
5
isomer (6). 1H NMR (CDCl3) δ 7.46 (1H, d, J ) 1.5 Hz, H-6),
3
7.23 (1H, d, J ) 1.5 Hz, H-6), 6.37-6.35 (1H, dd, J ) 5.4 Hz,
5
3
J ) 9.3 Hz, H-1′), 6.18 (1H, t, J ) 6.4 Hz, H-1′), 5.87 (1H, d,
5
(20) 1H and 13C NMR chemical shifts are expressed in ppm relative
to the internal tetramethylsilane (TMS). Positive 31P NMR chemical
shifts are expressed in ppm downfield from external 85% H3PO4 with
proton-decoupling ({1H}). 1H NMR peak assignments for all the
compounds were derived from 2D COSY and NOE (compounds 3, 4,
3S, 4S, 13) experiments. 13C NMR peak assignments for all compounds
are derived from 2D HMQC experiments.
J ) 3.9 Hz, H-1′′), 5.14 (1H, dd, J ) 5.4 Hz, J ) 9.3 Hz, H-
3′), 4.84 (1H, dd, J ) 2.9 Hz, J ) 12.2 Hz, H-3′′), 4.59 (1H, d,
3
J ) 3.9 Hz, H-2′′), 4.35 (m, 1H, H-4′′), 4.26-4.20 (4H, m, H-
3
5
3
5
3′, 2 × H-5′, H-4′), 4.00 (1H, m, H-4′), 3.86 (2H, m, 2 × H-
5′), 2.83 (2H, m, 2 × H-5′′), 2.77 (1H, septet, NCH), 2.45 (1H,
m, 5H-2′), 2.25 (2H, m, 2 × 3H-2′), 2.15 (1H, m, 5H-2′), 1.92

3652 J . Org. Chem., Vol. 63, No. 11, 1998
J in and J ust
(3H, d, J ) 1.0 Hz, 3CH3CdC), 1.90 (3H, d, J ) 1.0 Hz,
5CH3CdC), 1.97-1.61 (8H, m, cyclopentylidene protons), 1.02
(6H, dd, J ) 5.9 Hz, Me2CHN), 0.92 (9H, s, 3t-BuSi), 0.87 (9H,
(CH3CdC), -5.39, -5.45 (Me2Si); 31P NMR (CDCl3) δ 129.34;
HRMS (FAB, glycerol) m/e calcd for C29H49N3O9PSi [MH+]
642.2973, found 642.2976.
5
s, t-BuSi), 0.13 (6H, s, Me2Si), 0.07 (6H, s, Me2Si); 13C NMR
P h osp h or oth ioa tes (15 a n d 17). A mixture of 13 (15 mg,
0.0234 mmol), T5′OH (10 mg, 1.2 equiv), and 4,5-dicyano-2-
bromo-imidazole (9.17 mg, 2.0 equiv) was dried in high vacuum
overnight. Anhydrous acetonitrile (0.6 mL) was added at 0
°C under Ar. The solid was dissolved instantly. The reaction
was followed by 31P NMR. Within 5 min, the peak correspond-
ing to the phosphoramidite at 129.34 ppm was replaced by
two new peaks corresponding to phosphite triesters 14 and
16 (142.14 ppm:140.67 ppm ) 1:7). To this solution was added
Beaucage’s reagent (5.6 mg, 1.2 equiv) in 140 µL of acetonitrile
(0.2 M) Instantaneously the 31P NMR showed another two
peaks corresponding to 15 and 17 (67.44 ppm:68.60 ppm )
1:7). The workup and purificatin step is the same as that of
6/8 to give 23 mg of white solid 15/17 (91%). When a similar
reaction was carried out at -15 °C for 6 h, using chloroform
as the solvent, the ratio of 15 and 17 increased to 1:70. After
purification, only one product (17) was obtained: 1H NMR
(CDCl3) δ 163.97, 163.66 (5C-4, 3C-4), 150.39, 150.04 (5C-2, 3C-
2), 135.58, 134.49 (5C-6, 3C-6), 121.77 (OCO), 111.26, 111.04
3
5
(5C-5, C-5), 104.00 (C-1′′), 85.57 (d, J ) 6.4 Hz, C-4′), 85.48
(3C-1′), 84.65 (d, J ) 9.2 Hz, 3C-4′), 84.39 (5C-1′), 83.47 (C-2′′),
5
80.70 (d, J ) 4.6 Hz, C-3′′), 80.68 (d, J ) 4.6 Hz, C-3′), 79.00
(d, J ) 7.3 Hz, 3C-3′), 71.49 (C-4′′), 67.66, 67.52 (d, J ) 4.6
Hz, C-5′), 63.47 (5C-5′), 48.93 (NCH), 45.23 (C-5′′), 40.37 (3C-
3
2′), 39.12 (d, J ) 6.4 Hz, 5C-2′), 37.06, 36.16 (CH2CCH2), 25.88,
25.60 (5SiCMe3, 3SiCMe3), 23.52, 22.85 (CH2CH2CCH2CH2),
22.65 22.39 (NCHMe2), 18.28, 17.81 (5SiCMe3, 3SiCMe3), 12.51,
12.46 (5CdCCH3, 3CdCCH3), -4.66, -4.83, -5.40, -5.46 (5-
SiMe2, 3SiMe2); 31P NMR (CDCl3) δ 68.23; HRMS (FAB,
glycerol-CsI) m/e calcd for C45H77N5O14PSSi2 [MH+] 1030.4460,
found 1030.4464.
P h osp h or oth ioa te Dith ym id in e (9). The protected phos-
phorothioate dinucleoside 6 (14 mg, 0.0136 mmol) was dis-
solved in 1.0 mL of 70% TFA-H2O at 0 °C and allowed to stir
at rt for 2 h. Evaporation of the solvent and coevaporation
with methanol furnished a white solid which was purified by
chromatography to give 7.2 mg of white solid 9 (94%): 1H NMR
(CD3OD) δ 7.87 (s, 1H, 5H-6), 7.85 (1H, s, 3H-6), 6.36-6.33 (1H,
5
(CDCl3) δ 7.46 (d, J ) 1.5 Hz, 1H, H-6), 7.27 (d, J ) 1.5 Hz,
3
5
1H, H-6), 6.34-6.31 (dd, 1H, J ) 5.2 Hz, J ) 9.3 Hz, H-1′),
3
6.12-6.09 (t, 1H, J ) 6.6 Hz, H-1′), 5.86 (d, J ) 3.4 Hz, 1H,
5
H-1′′), 5.16 (dd, 1H, J ) 5.6 Hz, J ) 9.8 Hz, H-3′), 4.82 (dd,
J ) 2.7 Hz, J ) 11.7 Hz, 1H, H-3′′), 4.55 (d, J ) 3.9 Hz, 1H,
5
3
dd, J ) 6.4 Hz, J ) 7.8 Hz, H-1′), 6.30-6.27 (1H, dd, J ) 6.4
H-2′′), 4.39-4.37 (m, 2H, H-3′, H-4′′), 4.25-4.21 (m, 3H, 2 ×
Hz, J ) 7.3 Hz 3H-1′), 5.06-5.03 (1H, m, 5H-3′), 4.53-5.52 (1H,
5H-5′, 5H-4′), 3.98 (m, 1H, 3H-4′), 3.91-3.85 (m, 2H, 2 × H-
5
m, 3H-3′), 4.21-4.06 (4H, m, 5H-4′, 2 × 3H-5′, 3H-4′), 3.84-
5′), 2.84 (d, J ) 6.4 Hz, 2H, 2 × H-5′′), 2.81 (m, 1H, NCH),
5
5
3
3.80 (2H, m, 2 × H-5′), 2.50-2.46 (1H, m, H-2′), 2.31-2.24
2.52-2.47 (m, 1H, 5H-2′), 2.25-2.23 (m, 2H, 2 × H-2′), 2.08-
(2H, m, 2 × 3H-2′), 2.21-2.17 (1H, m, 5H-2′), 1.96 (3H, s,
-
2.02 (m, 1H, H-2′), 1.91 (d, 3H, J ) 1.0 Hz, CH3CdC), 1.89
(d, 3H, J ) 1.0 Hz, 5CH3CdC), 1.92-1.65 (m, 8H, cyclopen-
tylidene protons), 1.05 (d, J ) 5.9 Hz, 6H, Me2CHN), 0.90 (s,
9H, 3t-BuSi), 0.86 (s, 9H, 5t-BuSi), 0.11 (s, 6H, Me2Si), 0.05 (s,
6H, Me2Si); 13C NMR (CDCl3) δ 163.80, 163.59 (5C-4, 3C-4),
150.33, 150.15 (5C-2, 3C-2), 136.13, 134.64 (5C-6, 3C-6), 122.03
(OCO), 111.43, 111.11 (5C-5, 3C-5), 104.28 (C-1′′), 86.29 (3C-
1′), 85.93 (d, J ) 6.4 Hz, 5C-4′), 84.78 (d, J ) 10 Hz, 3C-4′),
84.69 (5C-1′), 83.59 (C-2′′), 80.83 (d, J ) 5.5 Hz, C-3′′), 80.68
(d, J ) 4.6 Hz, 5C-3′), 79.00 (d, J ) 8.2 Hz, 3C-3′), 71.30 (C-
4′′), 67.36 (d, J ) 5.5 Hz, 3C-5′), 63.37 (5C-5′), 48.91 (NCH),
3
5
3
CH3CdC), 1.87 (3H, s, 5CH3CdC); 31P NMR (D2O) δ 55.70;
HRMS (FAB, glycerol) m/e calcd for C20H28N4O11PS [MH+]
563.11347, found 563.11350. HPLC showed only a single peak.
When the mixture of protected phosphorothioates 6/8 was
hydrolyzed in the same reaction condition, the phosphorothio-
ate dinucleosides 9/10 were obtained with an Sp:Rp diastere-
omer ratio the same as the ratio of 6 to 8.
1,2-O-Cyclop en t ylid en e-5-d eoxy-5-isop r op yla m in o-r-
L-xylofu r a n ose (11). Using the same procedure as described
for 1, 11 was obtained in
a
72% yield from
5
1,2-O-cyclopentylidene-5-tosyl-R-L-xylofuranose: mp 39-41 °C;
45.18 (C-5′′), 40.35 (3C-2′), 39.11 (d, J ) 3.7 Hz, C-2′), 37.14,
[R]20 ) -31.37 (C ) 2, CHCl3); 1H NMR (CDCl3) δ 8.0 (bs,
36.23 (CH2CCH2), 25.88, 25.65 (5SiCMe3, 3SiCMe3), 23.55,
22.89 (CH2CH2CCH2CH2), 22.80, 22.55 (NCHMe2), 18.28,
17.86 (5SiCMe3, 3SiCMe3), 12.52, 12.49 (3CdCCH3, 5CdCCH3),
-4.66, -4.83, -5.40, -5.46 (5SiMe2, 3SiMe2); 31P NMR (CDCl3)
D
NH), 5.90 (1H, d, J ) 3.9 Hz, H-1), 4.38 (1H, d, J ) 3.9 Hz,
H-2), 4.27 (1H, m, H-3), 4.20 (1H, m, H-4), 3.36-2.92 (2H, ABX,
2 × H-5), 2.74-2.70 (1H, septet, NCH), 1.94-1.62 (8H, m,
cyclopentylidene protons), 1.03-1.01 (6H, dd, J ) 2.4 Hz, J )
6.4 Hz, Me2CH); 13C NMR (CDCl3) δ 121.01 (OCO), 104.79
(C-1), 86.10 (C-2), 78.26 (C-3), 77.04 (C-4), 48.62 (NCH), 45.87
(C-5), 36.81, 36.28 (CH2CCH2), 23.49, 22.81 (CH2CH2CCH2CH2),
22.62 (CH3CHN), 22.32 (CH3CHN); HRMS (EI) m/e calcd for
δ 68.60; HRMS (FAB, glycerol-CsI) m/e calcd for C45H77N5O14
-
PSSi2 [MH+] 1030.4460, found 1030.4464.
P h osp h or oth ioa te Dith ym id in e (10). The protected
phosphorothioate dinucleoside 17 (15 mg, 0.014 mmol) was
dissolved in 1.0 mL of 70% TFA-H2O at 0 °C and allowed to
stir at rt for 2 h. Evaporation of the solvent and coevaporation
with methanol furnished a white solid which was purified by
chromatography to give 7.3 mg of white solid 10 (94%): 1H
C
13H23NO4 [M+] 257.16270, found 257.16250.
Cyclic p h osp h or och lor id ite (12) was obtained by the
procedure as described for 2. The product was not isolated,
and it was used directly in the following step. 12: 31P NMR
(CDCl3) δ 148.75.
5
3
NMR (CD3OD) δ 7.91 (s, 1H, H-6), 7.86 (s, 1H, H-6), 6.36-
5
6.33 (dd, J ) 6.4 Hz, J ) 7.8 Hz, 1H, H-1′), 6.29-6.26 (dd, J
3
5
Oxa za p h osp h or a n in e (13) was obtained from 12 by the
) 5.9 Hz, J ) 7.8 Hz, H-1′), 5.08-5.05 (m, 1H, H-3′), 4.52-
3 5
procedure described for 4; mp 99-101 °C; [R]20 ) -72.00 (c
5.51 (m, 1H, H-3′), 4.21 (m, 1H, H-4′), 4.14-4.11 (m, 2H, 2
D
) 0.5, CHCl3); H NMR (CDCl3) δ 8.77 (bs, 1H, NH), 7.46 (s,
×
3H-5′), 4.04 (m, 1H, 3H-4′), 3.86-3.79 (m, 2H, 2 × 5H-5′),
1
5
1H, H-6), 6.33-6.30 (dd, J ) 5.9 Hz, J ) 7.8 Hz, 1H, H-1′),
5.88 (d, J ) 3.9 Hz, 1H, H-1′′), 4.56-4.53 (m, 1H, H-3′), 4.43
(d, J ) 3.9 Hz, 1H, H-2′′), 4.35 (m, 1H, H-3′′), 4.18 (q, J ) 2.0
Hz, 1H, H-4′′), 4.05 (m, 1H, H-4′), 3.90-3.76 (ABX, 2H, 2 ×
H-5′), 3.45-3.42 (m, 2H, H-5′′, NCH), 3.03-2.99 (m, 1H, H-5′′),
2.38-2.35 (m, 1H, H-2′), 2.12-2.06 (m, 1H, H-2′), 1.89 (s, 3H,
MeCdC), 1.96-1.62 (m, 8H, cyclopentylidene protons), 1.11-
1.08 (dd, 6H, J ) 6.4 Hz, J ) 11.2 Hz, Me2CH), 0.90 (s, 9H,
t-BuSi), 0.09 (d, J ) 2.0 Hz, 6H, Me2Si); 13C NMR (CDCl3) δ
163.74 (C-4), 150.31 (C-2), 135.18 (C-6), 121.44 (OCO), 110.93
(C-5), 104.59 (C-1′′), 86.59 (d, J ) 5.5 Hz, C-4′), 84.74 (d, J )
2.8 Hz, C-2′′), 84.70 (C-1′), 73.96 (d, J ) 17.4 Hz, C-3′), 73.06
(d, J ) 1.8 Hz, C-4′′), 71.80 (d, J ) 4.6 Hz, C-3′′), 62.98 (C-5′),
49.91 (d, J ) 36.6 Hz, NCH), 39.93 (d, J ) 2.8 Hz, C-2′), 36.86,
36.19 (CH2CCH2), 36.09 (d, J ) 3.1 Hz, C-5′′), 25.92 (SiCMe3),
23.45, 22.76 (CH2CH2CCH2CH2), 21.95 (d, J ) 9.2 Hz, CH3-
CHN), 21.66 (d, J ) 6.4 Hz, CH3CHN), 18.35 (SiCMe3), 12.52
2.48-2.44 (m, 1H, 3H-2′), 2.31-2.24 (m, 2H, 2 × H-2′), 2.23-
2.16 (m, 1H, 3H-2′), 1.97 (s, 3H, 3CH3CdC), 1.87 (s, 3H,
5CH3CdC); 31P NMR (D2O) δ 56.10; HRMS (FAB, glycerol) m/e
calcd for C20H28N4O11PS [MH+] 563.11347, found 563.11350.
P h osp h or oth ioa m id a te (4S). To a solution of phosphora-
midite 4 (46 mg, 0.075 mmol) in dry methanechloride (1 mL)
was added Beaucage’s reagent (1.2 equiv) at room tempera-
ture. The reaction mixture was stirred for 5 min. Afer flash
chromatography (ethyl acetate:hexane ) 1:2), white solid
phosphorothioamidate 4S was obtained in quantitative yield:
1H NMR (CDCl3) δ 8.94 (br, 1H, NH), 7.51 (d, 1H, J ) 1.2 Hz,
H-6), 6.36-6.33 (dd, J H1′-H2a′ ) 5.1 Hz, J H1′-H2b′ ) 9.3 Hz, 1H,
H-1′), 5.94 (d, J H1′′-H2′′ ) 3.9 Hz, 1H, H-1′′), 5.05-5.02 (dd,
J H3′-H2′ ) 5.6 Hz, J H3′-P ) 10 Hz, 1H, H-3′), 4.67 (t, J H3′′-H4′′
J H3′′-P ) 2.4 Hz, 1H, H-3′′), 4.57 (d, J H2′′-H1′′ ) 3.9 Hz, 1H,
H-2′′), 4.30-4.29 (pentet, J H4′′-H3′′ ) J H4′′-H5a′′ ) J H4′′-H5e′′
J H4′′-P ) 2.4 Hz, 1H, H-4′′), 4.27 (m, 1H, H-4′), 4.25 (septet,
)
)

Synthesis of Dinulceoside Phosphorothioates
J . Org. Chem., Vol. 63, No. 11, 1998 3653
1H, CHN), 3.38 (AB, 2H, 2 × H-5′), 3.46-3.39 (ddd, J H5e′′-H4′′
) 2.7 Hz, J H5e′′-H5a′′ ) 14.3 Hz, J H5e′′-P ) 20 Hz, 1H, H-5e′′),
3.35-3.30 (dt, J H5a′′-H5e′′ ) 14.3 Hz, J H5a′′-H4′′ ) J H5a′′-P ) 3.2
Hz, 1H, H-5a′′), 2.41 (m, 1H, H-2a′), 2.09 (m, 1H, H-2b′), 1.89
(d, J ) 1.5 Hz, 3H, CH3CdC), 1.96-1.62 (m, 8H, cyclopentyl-
idene protons), 1.11 (d, J ) 6.8 Hz, 3H, CH3CHN), 1.05 (d, J
) 6.8 Hz, 3H, CH3CHN), 0.90 (s, 9H, t-BuSi), 0.11 (s, 6H, Me2-
Si); 13C NMR (CDCl3) δ 163.81 (C-4), 150.53 (C-2), 134.92 (C-
6), 121.91 (OCO), 111.23 (C-5), 104.68 (C-1′′), 85.91 (d, J )
1.8 Hz, C-4′), 84.80 (C-1′), 84.00 (d, J ) 11.0 Hz, C-2′′), 80.95
(d, J ) 9.2 Hz, C-3′′), 78.25 (d, J ) 5.5 Hz, C-3′), 72.60 (d, J )
4.6 Hz, C-4′′), 63.55 (C-5′), 47.90 (d, J ) 6.4 Hz, CHN), 39.40
(d, J ) 7.3 Hz, C-5′′), 39.25 (C-2′), 36.85, 36.20 (CH2CCH2),
25.86 (SiCMe3), 23.35, 22.78 (CH2CH2CCH2CH2), 20.60 (d, J
) 6.4 Hz, NCHMe), 20.20 (d, J ) 2.7 Hz, NCHMe), 18.25
(SiCMe3), 12.44 (CdCCH3), -5.47, -5.50 (SiMe2); 31P NMR
(CDCl3) δ 67.54; HRMS (FAB, glycerol) m/e calcd for C29H49N3O9-
PSSi [MH+] 674.2693, found 674.2696.
4.09 (1H, m, H-4′), 3.91-3.79 (2H, ABX, 2 × H-5′), 3.49-3.47
(1H, m, H-5a′′), 3.47-3.43 (1H, septet, NCH), 3.08-3.03 (1H,
m, H-5e′′), 2.41-2.37 (1H, m, H-2′), 2.13-2.07 (1H, m, H-2′),
1.92 (3H, d, J ) 1.0 Hz, MeCdC), 1.49 (3H, s, CH3CCH3), 1.31
(3H, s, CH3CCH3), 1.14-1.11 (6H, m, Me2CH), 0.93 (9H, s,
t-BuSi), 0.12 (6H, d, J ) 1.5 Hz, Me2Si); 13C NMR (CDCl3) δ
164.38 (C-4), 150.50 (C-2), 135.24 (C-6), 111.61 (OCO), 110.94
(C-5), 104.78 (C-1′′), 86.34 (C-4′), 84.73 (C-1′), 84.82 (C-2′′),
73.64 (d, J ) 18.1 Hz, C-3′), 72.32 (C-4′′), 71.77 (C-3′′), 63.11
(C-5′), 49.92 (d, J ) 36.6 Hz, NCH), 40.24 (C-2′), 36.01 (C-5′′),
26.62, 26.09 (Me2C), 25.94 (SiCCH3), 22.03, 21.80 (Me2CHN),
18.27 (SiCCH3), 12.46 (CH3CdC), -5.37, -5.44 (Me2Si); 31P
NMR (CDCl3) δ 129.0; MS (FAB, nitrobenzyl alcohol) m/e
[MH+] 616.
Oxa za p h osp h or in a n e (19a ). Using the procedure de-
scribed for 4S, 19a was obtained in quantitative yield: 1H
NMR (CDCl3) δ 9.02 (1H, bs, NH), 7.52 (1H, d, J H6-CH3CdC
1.0 Hz, H-6), 6.36-6.33 (1H, dd, J H1′-H2a′ ) 5.1 Hz, J H1′-H2b′
)
)
P h osp h or oth ioa m id a te (3S). To a solution of a mixture
of phosphoramidite 3 and 4 in a ratio of 2:3 (45 mg, 0.075
mmol) in dry chloroform (1.0 mL) was added Beaucage’s
reagent (1.2 equiv) at room temperature. The reaction mixture
was stirred for 5 min. After flash chromatography (ethyl
acetate:hexane ) 1:3), the slow eluent (25 mg) is 4S according
to 31P NMR (67.54 ppm), while the fast eluent (18 mg) is 3S
by 31P NMR (72.35 ppm): 1H NMR (CDCl3) δ 8.41 (br,, 1H,
9.0 Hz, H-1′), 5.96 (1H, d, J H1′′-H2′′ ) 3.7 Hz, H-1′′), 5.03 (1H,
dd, J ) 5.6 Hz, J ) 10.0 Hz, H-3′), 4.66 (1H, m, H-3′′), 4.51
(1H, d, J H1′′-H2′′ ) 3.9 Hz, H-2′′), 4.27-4.23 (3H, m, H-4′, H-4′′,
NCH), 3.92-3.86 (2H, m, 2 × H-5′′), 3.47-3.32 (2H, m, 2 ×
H-5′′), 2.43-2.39 (1H, m, H-2′), 2.12-2.06 (1H, m, H-2′), 1.89
(3H, s, MeCdC), 1.47 (3H, s, CH3CCH3), 1.29 (3H, s, CH3CCH3),
1.10 (3H, d, J ) 6.8 Hz, Me2CH), 1.05 (3H, d, J ) 6.8 Hz,
Me2CH), 0.90 (9H, s, t-BuSi), 0.11 (6H, s, Me2Si); 13C NMR
(CDCl3) δ 163.65 (C-4), 150.41 (C-2), 134.97 (C-6), 112.32
(OCO), 111.25 (C-5), 104.94 (C-1′′), 85.96 (d, J ) 2.8 Hz, C-4′),
84.85 (C-1′), 84.12 (d, J ) 11.0 Hz, C-2′′), 80.95 (d, J ) 9.2 Hz,
C-3′′), 78.29 (d, J ) 5.5 Hz, C-3′), 72.45 (d, J ) 4.6 Hz, C-4′′),
63.61 (C-5′), 48.00 (d, J ) 6.4 Hz, NCH), 39.46 (d, J ) 7.3 Hz,
C-5′′), 39.23 (C-2′), 26.64, 26.60 (Me2C), 25.90 (SiCCH3),
20.65(d, J ) 5.5 Hz, CH3CHN), 20.47 (d, J ) 2.8 Hz, CH3-
CHN), 18.30 (SiCCH3), 12.49 (CH3CdC), -5.42, -5.45 (Me2-
Si); 31P NMR (CDCl3) δ 67.50; HRMS (FAB, glycerol): m/e
calcd. for C27H47N3O9PSSi [MH+]: 648.25422, found 648.25399.
Oxa za p h osp h or in a n e (18b). By the same procedure as
described for 18a , 18b was obtained with yield of 60%. 1H
NMR (CDCl3) δ 5.93 (1H, d, J ) 3.7 Hz, H-1), 4.51 (1H, d, J )
3.7 Hz, H-2), 4.41 (1H, m, H-3), 4.17-4.13 (2H, m, H-4, OCH),
3.49-3.46 (1H, m, H-5), 3.40 (1H, septet, NCH),), 3.01-2.99
(1H, m, H-5), 1.47 (3H, s, CCH3), 1.30 (3H, s, CCH3), 1.23 (6H,
m, OCHMe2), 1.10 (6H, m, NCHMe2); 13C NMR (CDCl3) δ
111.48 (OCO), 104.91 (C-1), 84.94 (d, J ) 2.8 Hz, C-2), 73.48
(C-4), 71.15 (d, J ) 2.8 Hz, C-3), 67.14 (d, J ) 18.3, OCH),
49.74 (d, J ) 35.7 Hz, NCH), 36.24 (d, J ) 3.7 Hz, C-5), 26.68,
26.19 (Me2C), 24.56 (d, J ) 4.6 Hz, OCHCH3), 24.33 (d, J )
2.7 Hz, OCHCH3), 21.91 (d, J ) 10.1 Hz, NCHCH3), 21.39 (d,
J ) 6.4 Hz, NCHCH3); 31P NMR (CDCl3) δ 128.18.
NH), 7.52 (d, 1H, J ) 1.2 Hz, H-6), 6.36-6.33 (dd, J H1′-H2′a
)
5.3 Hz, J H1′-H2′b ) 9.3 Hz, 1H, H-1′), 6.00 (d, 1H, J H1′′-H2′′ ) 4
Hz, H-1′′), 5.20-5.17 (ddd, 1H, J H3′-H2′ ) 5.6 Hz, J H3′-4′ ) 1
Hz, J H3′-P ) 10 Hz, H-3′), 4.65-4.63 (dd, 1H, J H3′′-H4′′) 4.1
Hz, J H3′′-H2′′ ) 5.3 Hz, H-3′′), 4.63 (d, 1H, J H2′′-H1′′ ) 4 Hz, H-2′′),
4.56-4.52 (ddd, 1H, J H4′′-H3′′ ) 4.1 Hz, J H4′′-H5a′′ ) J H4′′-H5b′′
)
6.6 Hz, H-4′′), 4.16 (q, 1H, J H4′-H3′ ) J H4′-H5a′ ) J H4′-H5b′ ) 1
Hz, H-4′), 3.95 (septet, 1H, CHN), 3.88 (d, 2H, 2 × H-5′), 3.45-
3.37 (ddd, 1H, J H5a′′-H4′′ ) 6.4 Hz, J H5a′′-H5b′′ ) 14, J H5a′′-P ) 19
Hz, H-5a′′), 3.13-3.07 (ddd, 1H, J H5b′′-H4′′ ) 6.5 Hz, J H5b′′-H5a′′
) 14 Hz, J 5b′′-P ) 12 Hz, H-5b′′), 2.49-2.46 (dd, 1H, J H2a′-H2b′
) 14 Hz, J H2a′-H3′ ) 5.4 Hz, H-2a′), 2.10-2.04 (ddd, 1H,
J H2b′-H2a′ ) 15 Hz, J H2b′-H1′ ) 9 Hz, J H2b′-H3′ ) 5.3 Hz, H-2b′),
1.89 (d, 3H, J ) 1 Hz, CH3CdC), 1.96-1.62 (m, 8H, cyclopen-
tylidene protons), 1.11 (d, 3H J ) 6.8 Hz, CH3CHN), 1.07 (d,
3H, J ) 6.8 Hz, CH3CHN), 0.91 (s, 9H, t-BuSi), 0.12 (s, 6H,
Me2Si); 13C NMR (CDCl3) δ 163.43 (C-4), 150.13 (C-2), 135.07
(C-6), 121.87 (OCO), 111.15 (C-5), 105.65 (C-1′′), 86.25 (d, J )
6.4 Hz, C-4′), 84.72 (C-1′), 83.97 (d, J ) 12 Hz, C-2′′), 80.83 (d,
J ) 6.3 Hz, C-3′′), 79.13 (d, J ) 4.6 Hz, C-3′), 77.80 (d, J ) 1.8
Hz, C-4′′), 63.46 (C-5′), 46.90 (d, J ) 6.4 Hz, CNH), 40.74 (C-
2′), 39.36 (d, J ) 5.5 Hz, C-5′′), 37.04, 36.62 (CH2CCH2), 25.93
(SiCMe3), 23.37, 23.11 (CH2CH2CCH2CH2), 20.97 (d, J ) 2.7
Hz, NCHMe), 20.57 (d, J ) 4.6 Hz, NCHMe), 18.34 (SiCMe3),
12.49 (CdCCH3), -5.39 (SiMe2); HRMS (FAB, glycerol) m/e
calcd for C29H49N3O9PSSi [MH+] 674.2693, found 674.2696.
Syn th esis of 18a -d a n d 19a -d . 1,2-O-Isop r op ylid en e-
5-d eoxy-5-isop r op yla m in o-r-D-xylofu r a n ose. Using the
procedure described for 1, 1,2-O-isopropylidene-5-deoxy-5-
isopropylamino-R-D-xylofuranose was obtained in 80% yield
from 1,2-O-isopropylidene-5-tosyl-R-L-xylofuranose: 1H NMR
(CDCl3) δ 8.0 (bs, NH), 5.91 (1H, d, J ) 3.9 Hz, H-1), 4.44
(1H, d, J ) 3.9 Hz, H-2), 4.25-4.18 (2H, m, H-3, H-4), 3.39-
2.90 (2H, ABX, 2 × H-5), 2.79-2.66 (1H, septet, NCH), 1.44
(3H, s, CCH3), 1.03 (6H, d, J ) 6.4 Hz, Me2CH); 13C NMR
(CDCl3) δ 111.43 (OCO), 105.12 (C-1), 86.14 (C-2), 78.23 (C-
3), 76.91 (C-4), 48.84 (NCH), 45.90 (C-5), 26.81, 26.17
(CH3CCH3), 22.64 (CH3CHN), 22.34 (CH3CHN); MS (CI) m/e
232 ([M + H+], 100%).
P h osp h or oth ioa m id a te (19b). By the same procedure as
described for 4S, 19b was obtained in quantitative yield. 1H
NMR (CDCl3) δ 5.97 (1H, d, J ) 3.7 Hz, H-1), 4.71-4.66 (1H,
m, OCH), 4.65 (1H, d, J ) 3.7 Hz, H-2),4.60 (1H, m, H-3), 4.28
(1H, septet, NCH), 4.24 (1H, m, H-4), 3.42-3.32 (2H, m, 2 ×
H-5), 1.47 (3H, s, CCH3), 1.31 (3H, d, J ) 6.4 Hz, OCHCH3),
1.30(3H, s, CCH3), 1.27 (3H, d, J ) 6.1 Hz, OCHCH3), 1.10
(3H, d, J ) 6.6 Hz, NCHCH3), 1.04 (3H, d, J ) 6.4 Hz,
NCHCH3) 13C NMR (CDCl3) δ 112.08 (OCO), 105.04 (C-1),
84.20 (d, J ) 11.0 Hz, C-2), 80.33 (d, J ) 9.1 Hz, C-3), 71.94
(d, J ) 4.6 Hz, C-4), 71.87 (d, J ) 6.4 Hz, OCH), 47.60 (d, J )
6.4 Hz, NCH), 39.20 (C-5), 26.68, 26.20 (Me2C), 23.59 (d, J )
6.4 Hz, OCHCH3), 23.40 (d, J ) 3.7 Hz, OCHCH3), 20.69 (d,
J ) 7.3 Hz, NCHCH3), 20.10 (d, J ) 1.8 Hz, NCHCH3); 31P
NMR (CDCl3) δ 67.08; HRMS (FAB, glycerol): m/e calcd. for
C
14H27NO5PS [MH+]: 352.13485, found 352.13476.
Cyclic p h osp h or och lor id ite was obtained by the proce-
dure described for 2. The product was not isolated and it was
directly used in the following step: 31P NMR (CDCl3) δ 148.31.
Oxa za p h osp h or in a n e (18a ). Using the procedure de-
scribed for 4, 18a was obtained from the above cyclic phos-
phorochloridite in 84% yield: 1H NMR (CDCl3) δ 8.19 (1H, bs,
NH), 7.50 (1H, d, J H6-CH3CdC ) 1.0 Hz, H-6), 6.35-6.33 (1H,
dd, J H1′-H2a′ ) 5.4 Hz, J H1′-H2b′ ) 8.3 Hz, H-1′), 5.94 (1H, d,
J H1′′-H2′′ ) 3.9 Hz, H-1′′), 4.60-4.57 (1H, m, H-3′), 4.51 (1H, d,
J H1′′-H2′′ ) 3.9 Hz, H-2′′), 4.36 (1H, m, H-3′′), 4.18 (1H, m, H-4′′),
Oxa za p h osp h or in a n e (18c). Using the procedure de-
scribed for 18a , 18c was obtained with a yield of 78%: 31P
NMR (CDCl3) δ 125.31.
P h osp h or oth ioa m id a te (19c). By the procedure de-
scribed for 19a , 19c was obtained in quantitative yield: 1H
NMR (CDCl3) δ 8.24-7.34 (4H, AA′BB′, aromatic protons), 6.02
(1H, d, J ) 3.4 Hz, H-1), 4.71 (2H, m, H-2, H-3), 4.41 (1H,
septet, NCH), 4.30 (1H, m, H-4), 3.60-3.43 (2H, m, 2 × H-5),
1.48 (3H, s, CCH3), 1.31 (3H, s, CCH3), 1.16 (3H, d, J ) 6.6
Hz, NCHCH3), 1.07 (3H, d, J ) 6.4 Hz, NCHCH3); 13C NMR

3654 J . Org. Chem., Vol. 63, No. 11, 1998
J in and J ust
(CDCl3) δ 156.11, 144.39, 125.57, 120.77, 120.73 (aromatic
carbons), 112.42 (OCO), 104.90 (C-1), 84.12 (d, J ) 11.0 Hz,
C-2), 81.43 (d, J ) 10.1 Hz, C-3), 71.90 (d, J ) 5.5 Hz, C-4),
48.43 (d, J ) 7.3 Hz, NCH), 39.38 (C-5), 26.58, 26.15 (Me2C),
20.86 (d, J ) 6.4 Hz, NCHCH3), 20.26 (d, J ) 2.8 Hz,
NCHCH3); 31P NMR (CDCl3) δ 61.58; HRMS (FAB, glycerol)
m/e calcd for C17H24N2O7PS [MH+] 431.10426, found 431.10419.
Ack n ow led gm en t. We thank the Natural Science
and Engineering Research Council of Canada and ISIS
Pharmaceuticals, Carlsbad, CA, for generous financial
support and Dr. Orval Mamer, McGill University Bio-
medical Mass Spectrometry Unit, for the measurement
of mass spectra. We also thank Dr. Eric Marsault for
his valuable comments on the paper. Y.J . thanks
McGill University for an Alexander McFee Memorial
fellowship, an FCAR fellowship, a J . W. McConnell
McGill Major fellowship, and a Beijing Memorial McGill
fellowship.
Oxa za p h osp h or in a n e (18d ). By the procedure described
for 18a , 18d was synthesized and its 31P NMR showed one
peak at 127.50 ppm, but we cannot purify the product by
chromatography. Therefore, the reaction mixture was used
in the next step.
Su p p or tin g In for m a tion Ava ila ble: Synthetic details for
1 and 11 and 1H and 13C NMR spectra for all compounds (35
pages). This material is contained in libraries on microfiche,
immediately follows this article in the microfilm version of the
journal, and can be ordered from the ACS; see any current
masthead page for ordering information.
P h osp h or oth ioa m id a te (19d ). By the procedure de-
scribed for 19a , 19c was synthesized and its 31P NMR showed
one peak at 66.8 ppm. Unfortunately we also cannot purify
the product by chromatography.
J O972318O