Study on the diastereoselective synthesis of dithymidine phosphorothioates through a D-xylose derived chiral auxiliary and development of a novel catalyst

Article abstract of DOI:10.1016/S0040-4039(00)01678-6

1,2-Di-O-isopropylidene-3-C-cyanoethyl-5-deoxy-5-isopropylamino-D-xylofuranos e 16a was synthesized. The use of 16a as a chiral auxiliary led to the diastereoselective formation of dithymidine phosphorothioate. The chiral auxiliary was easily removed by treatment with concentrated ammonia. 2-Mesityl-4,5-dicyanoimidazole displays higher diastereoselectivity than the 2-bromo-4,5-dicyanoimidazole in the coupling reaction. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01678-6

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 9223–9227
Study on the diastereoselective synthesis of dithymidine
phosphorothioates through a -xylose derived chiral
D
auxiliary and development of a novel catalyst
Yixin Lu and George Just*
Department of Chemistry, McGill University, Montreal, Quebec, Canada H3A 2K6
Received 17 May 2000; revised 19 September 2000; accepted 19 September 2000
Abstract
1,2-Di-O-isopropylidene-3-C-cyanoethyl-5-deoxy-5-isopropylamino-
D
-xylofuranose 16a was synthe-
sized. The use of 16a as a chiral auxiliary led to the diastereoselective formation of dithymidine
phosphorothioate. The chiral auxiliary was easily removed by treatment with concentrated ammonia.
2-Mesityl-4,5-dicyanoimidazole displays higher diastereoselectivity than the 2-bromo-4,5-dicyanoimida-
zole in the coupling reaction. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: chiral auxiliaries; diastereoselection; nucleotides; phosphorothioates.
Oligonucleotide phosphorothioates (PS-Oligos) have been one of the most advanced antisense
agents.1 The PS-Oligos currently used in clinical trials and biological studies are mixtures of
diastereomers. The most useful method to address the stereoselective synthesis of PS-Oligos to
date is the oxathiaphospholane approach developed by Stec and coworkers.2 However, it suffers
from the tedious and expensive chromatographic separation of the chiral precursors.
We reported3 the use of xylose derivatives as chiral auxiliaries for the stereoselective synthesis
of phosphorothioates (A) (Scheme 1). It had been found that the reaction proceeded with
highest stereoselectivity when 2-bromo-4,5-dicyanoimidazole was used as a catalyst, and the
coupling reaction was carried out at low temperature. The usual catalyst, tetrazole, gave no
selectivity. The removal of the chiral auxiliary at the end of the synthesis was achieved by
treatment with 70% aqueous trifluoroacetic acid, which seemed to be too harsh if bases other
than thymidine are used. We wanted to modify the chiral auxiliary so that it could be easily
removed under mild conditions. Chiral auxiliaries of type B or C seemed to be good candidates.
An SN1 type displacement or b-elimination should allow for their easy removal. However,
phosphite triesters have been shown to be reasonably good leaving groups in b-eliminations4 or
* Corresponding author. Tel: (514) 398-6232; fax: (514) 398-3797; e-mail: just@chemistry.mcgill.ca
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01678-6

9224
OT5'
S
P OT3'
H
N
O
O
O
O
R1
R3
R2
A: R1 = H, R2, R3 = CH2CH2CH2CH2
B: R1 = R2 = R3 =CH3
C: R1 =CH2CN, R2 = R3 =CH3
T3'OH: 5'-O-TBDMS- thymidine
T5'OH: 3'-O-TBDMS- thymidine
Scheme 1.
when an appropriate neighboring group can assist in their departure.5 It was therefore not clear
at the outset of this work whether intermediates such as 17, 18 or 19 had the required stability.
To construct chiral auxiliary type B, ketone 3, obtained from 1,2-di-O-isopropylidene- -xylo-
D
furanose 1 in two steps,6 was used as a starting material. A reaction of 3 with methyl magnesium
bromide gave monotrityl diol 4. Removal of the trityl group, followed by selective tosylation
and the displacement of the tosylate with isopropylamine afforded the trans-aminoalcohol 7
(Scheme 2).
TrO
RO
O
ii
O
OH
(98%)
O
O
O
O
O
3
1 R = H
2 R = Tr
i
iii (83%)
(90%)
vii
R
O
N
P
O
O
O
HO
Cl
O
O
O
4 R = OTr
5 R = OH
6 R = OTs
(85%)
(95%)
(73%)
iv
v
vi
8
7 R = NH(i-Pr)
Scheme 2. (i) TrCl, Et3N, CH2Cl2; (ii) DMSO, Ac2O, rt; (iii) CH3MgBr, THF, 0°C to rt; (iv) AcOH, MeOH, reflux;
(v) TsCl, pyridine, 0°C; (vi) i-PrNH2, 55°C; (vii) PCl3, Et3N, CH2Cl2
We then proceeded to see whether cyclic phosphoramidite could be formed efficiently. It
should be noted that similar trans-fused cyclic sulfides have been obtained efficiently and in high
yield.7 Trans-aminoalcohol 7 in dichloromethane was treated with phosphorus trichloride in the
presence of triethylamine. 31P NMR spectroscopy revealed several peaks ranging from 10 to 220
ppm after the addition. The equilibration of the mixture at 50°C for 2 days failed to produce
any major product. We concluded from this experiment that it is necessary to have hydroxy and
amino groups cis to each other in the chiral auxiliary in order to have an efficient six-membered
ring formation.

9225
The chiral auxiliary of type C was synthesized as illustrated in Scheme 3.
D
-Xylose derivative
1 was first protected regioselectively at the 5-position as its silyloxy ether 9, and then oxidized
to ketone 10. Wittig reaction of 10 with diethyl cyanomethylphosphonate afforded two isomers
of alkenes in a 5:1 ratio, and these were separated by flash chromatography. The major isomer
11a was deprotected to give 12a, which was treated with N-benzyltrimethylammonium hydrox-
ide (triton-B) and tert-butyl hydroperoxide (TBHP) to furnish epoxide 13a in which the epoxide
oxygen had been formed by reaction at the b-face of the molecule. Samarium diiodide-mediated8
reduction of the a,b-epoxy nitrile was unsuccessful, and only starting material was recovered.
The selective reduction of the epoxide with lithium borohydride in diglyme gave cis-diol 14a,
whose configuration was confirmed by 2D-NOESY. Swern oxidation of diol 14a followed by
reductive amination with isopropylamine afforded the desired g-aminoalcohol 16a.
RO
RO
TBSO
O
OH
O
O
ii
iii
O
O
(96%)
(98%)
(80%)
O
H
O
O
O
O
X
10
1 R = H
9 R = TBS
i
a X = CN, b X = H
11 R = TBS
12 R = H
iv (60%)
v (72%)
R
O
OH
HO
O
vi
O
O
(59%)
O
O
O
X
X
a X = CN, b X = H
13a X = CN
13b X = H
14 R = CH2OH
15 R = CHO
16 R = CH2NH(i-Pr)
vii
viii
(77%)
Scheme 3. (i) TBDMSCl, imidazole, DMF; (ii) CrO3, pyridine, Ac2O, rt; (iii) a: CNCH2P(O)(OEt)2, NaH, THF, 0°C
to rt; b: CH3PPh3Br, NaH, THF, 0°C to rt; (iv) TBAF, 0°C; (v) a: Triton-B, TBHP, THF; b: MCPBA, CH2Cl2; (vi)
a: LiBH4, diglyme; b: LiAlH4; (vii) (COCl)2, DMSO, CH2Cl2; (viii) i-PrNH2, Na(OAc)3BH, DCE, rt
The synthesis of phosphoramidites and phosphorothioates is depicted in Scheme 4. g-
Aminoalcohol 16a was treated with phosphorus trichloride in the presence of triethylamine. 31P
NMR spectroscopy indicated that a major peak at 147.6 ppm was formed immediately, which
was assigned to 17a. Without isolation, the reaction mixture was treated with 5%-O-TBDMS-
thymidine (T3%OH)9 in dichloromethane. A single isomer of 18a with 31P NMR signal at 133.1
ppm appeared. Purification furnished pure phosphoramidite 18a. The coupling reaction between
18a and 3%-O-TBDMS-thymidine (T5%OH) was then carried out, using 2-bromo-4,5-dicyanoimi-
dazole as the catalyst. Phosphite triesters 19a were obtained, as established by 31P NMR
spectroscopy (138.5:137.5 ppm=1:6). Without purification, phosphite triesters were sulfurized10
to give phosphorothioates 20a with 31P NMR signals at 59.05 and 59.13 ppm in the same ratio.
After treatment with concentrated aqueous ammonia at room temperature for 5 min, the 31P
NMR resonances shifted to 57.3 and 56.8 ppm (1:6). The silyl protecting groups were removed

9226
to give dithymidine phosphorothioates 21 (56.8:56.6 ppm=1:6). The minor and major isomers
of 21 were assigned as Rp and Sp, by comparison of 31P NMR and HPLC data.11
OT5'
N
P OT3'
R
P
iii
O O
i
N
H
O
O
16
O
O
O
O
X
X
a X =CN, b X =H
17 R = Cl
18 R = OT3'
(96%)
19 a X =CN, b X =H
iv
ii
OT5'
S
P OT3'
T
HO
T
HO
O
O
N
H
O
O
v
+
O
P
O
P
O
(82%)
O
X
T
O
T
O
O
O
O
S
O
S
20a X =CN, b X =H
(61%)
OH
21 (Sp)
OH
21 (Rp)
(major)
Scheme 4. (i) PCl3, Et3N, CH2Cl2, 0°C to rt; (ii) T3%OH, Et3N, CH2Cl2, 0°C to rt; (iii) 1.1 equiv. T5%OH, 1.5 equiv.
2-bromo-4,5-dicyanoimidazole, CDCl3, 0°C; (iv) Beaucage’s reagent; (v) concd ammonia, rt, TBAF
Chiral auxiliary 16b was synthesized in a similar manner, and transformed to B, using the
procedure described to transform cyanoethyl derivative 16a to 20a (C). However, we were not
able to remove the chiral auxiliary to form free phosphorothioate 21.
The stereoselectivity of the coupling reaction is lower than that of the similar system.3
Different stereochemical outcomes might suggest different coupling mechanisms. When a
precursor of type A was used in the coupling reaction,3 the coupling mechanism we suggested
was the single protonation mechanism in which the catalyst only served as a proton donor. This
is in agreement with the fact that the different sized catalysts give similar selectivities. To verify
whether reactions reported here follow the same mechanism, we synthesized the more sterically
hindered catalyst 24 (Scheme 5). Commercially available 22 was first diazotized to give 23, which
was then refluxed in mesitylene to give 24.12 When 24 was used as the activator in the coupling
reaction of 20b, the same phosphite triesters were obtained in a 15:1 ratio, as compared to a 6:1
ratio when 2-bromo-4,5-dicyanoimidazole was used as a catalyst under the same conditions.
NC
NC
N
i
NC
NC
N
NC
NC
N
ii
NH2
N N
N
H
N
N
H
(70%)
(100%)
23
22
24
Scheme 5. (i) NaNO2, HCl; (ii) mesitylene

9227
This preliminary result suggests that catalysts not only serve as proton donors, but may also
participate in the coupling reactions in our system. Catalyst 24 has a pKa value similar to
tetrazole, and will therefore probably be compatible with the dimethoxytrityl group used in
conventional DNA synthesis.
In summary, cyclic phosphoramidite 18a incorporating a tertiary alcohol was stable enough to
be isolated, and allows for the stereoselective synthesis of Sp dithymidine phosphorothioate 21.
The chiral auxiliary was removed rapidly by reaction with concentrated ammonia at room
temperature. With the removable chiral auxiliary and less acidic catalyst in hand, we are
currently optimizing our system and adapting the methodology for routine oligonucleotide
synthesis. It is not clear whether the substituted imidazole-based catalyst functions as an acid
catalyst only, or as both an acid catalyst and a nucleophile.3b
Acknowledgements
We wish to thank the Natural Science and Engineering Research Council of Canada and
Dalton Laboratories for generous financial support.
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conditions: Phenomenex C8 column; solvent A: water; solvent B: acetonitrile; flow rate: 1.5 ml min−1; 3% B
increases linearly to 7% for the first 30 minutes, then increases to 40% during the next 20 minutes. Retention
times of Sp and Rp isomers are 14.62 and 10.04 minutes, respectively.
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