Diastereocontrolled synthesis of dinucleoside phosphorothioates using a novel class of activators, dialkyl(cyanomethyl)ammonium tetrafluoroborates

Article abstract of DOI:10.1021/ja017275e

A novel class of activators, dialkyl(cyanomethyl)ammonium tetrafluoroborates 1a-c, has been developed and applied to the condensations of diastereopure 5′-O-tert-butyldiphenylsilylthymidine 3′-cyclic phosphoramidites 3a-d with 3′-O-tert-butyldimethylsilyl

Full text of DOI:10.1021/ja017275e

Published on Web 04/11/2002
Diastereocontrolled Synthesis of Dinucleoside Phosphorothioates Using a
Novel Class of Activators, Dialkyl(cyanomethyl)ammonium Tetrafluoroborates
Natsuhisa Oka, Takeshi Wada,* and Kazuhiko Saigo*
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The UniVersity of Tokyo,
Bioscience Building 702, Kashiwa, Chiba 277-8562, Japan
Received October 10, 2001
Table 1. Condensations of Nucleoside 3′-Phosphoramidites 3a-d
with 4a in the Presence of 1a-c
Recent studies have shown that the properties of oligodeoxyri-
bonucleoside phosphorothioates (PS-ODNs) are affected by the
chirality of the phosphorus atoms.1 However, the PS-ODNs,
synthesized by the conventional phosphoramidite method,2 are
mixtures of diastereomers.3 Thus, stereoselective syntheses of PS-
ODNs have been extensively studied.4 Among them, Stec et al.4a
and Beaucage et al.4b have reported the fully P-stereocontrolled
synthesis of PS-ODNs. However, in their methods, the diastereopure
monomers had to be separated from a mixture of diastereomers by
troublesome column chromatography.
The phosphoramidite methods utilizing amino alcohols as chiral
auxiliaries have been reported in recent years.5 The advantage of
these methods is that the diastereopure monomers can be obtained
diastereoselectively from the appropriate enantiopure amino alco-
hols. Despite this advantage, the condensation reactions are more
or less nonstereospecific. The nonstereospecificity would be at-
tributed to the repetitive attack of a nucleophilic activator, such as
1H-tetrazole, to the phosphorus atom.
Under these circumstances, we tried to develop a new activator,
which did not generate a nucleophilic anion species, with the
expectation that such an activator would preferentially promote a
nucleophilic substitution at the phosphorus atom of a protonated
phosphoramidite by the hydroxy group of a nucleoside, while
depressing the repetitive attack of the activator to the phosphorus
atom, to give the corresponding dinucleoside phosphite without any
loss of the enantiopurity of the phosphorus atom. To develop such
an activator, the activator would have appropriate proton-donating
ability to the nitrogen of a phosphoramidite and contain a highly
less nucleophilic counteranion. On the basis of this consideration
we developed a new class of activators, dialkyl(cyanomethyl)-
ammonium tetrafluoroborates 1a-c. The activators 1a-c could be
easily obtained from the corresponding amines and tetrafluoroboric
acid etherate.
a R ) 5′-O-(tert-butyldiphenylsilyl)thymidin-3′-yl. b The chemical shifts
of 31P NMR are given in parentheses.
Scheme 1a
a Reagents and conditions: (i) 3′-O-(TBDMS)thymidine (4a), activating
agent 1a-c, CH3CN-CD3CN (4:1, v/v), room temperature; (ii) Ac2O,
pyridine; (iii) Beaucage reagent; (iv) DBU; (v) Et3N‚3HF.
thymidine (4a, 1 equiv) in the presence of 1a-c (2 equiv) in
CH3CN-CD3CN (4:1, v/v), and the reactions were monitored by
31P NMR (Table 1).The reaction time required for completion was
affected by the steric and electronic factors of the R2 group in the
oxazaphospholidine ring. The condensations of 3a,b (R2 ) H) with
4a proceeded quickly and completed within 5 min. On the other
hand, the condensations of 3c (R2 ) Me) and 3d (R2 ) Ph) with
4a required 15 min and 5 h, respectively, to complete. These results
indicate that it is important for a rapid condensation to diminish
the steric hindrance and electron-withdrawing character of the R2
group.
In the present study, we synthesized diastereopure nucleoside
3′-phosphoramidites 3 by using appropriate enantiopure amino
alcohols, (S)- and (R)-2-methylamino-1-phenylethanol6 (2a,b),
(1R,2S)-ephedrine (2c), and (1R,2S)-2-methylamino-1,2-diphenyl-
ethanol7 (2d) as the components of the oxazaphospholidine rings.
Compounds 3a-d were synthesized by applying the method in the
literature with some modifications.5a
Diastereopure nucleoside 3′-phosphoramidites 3a-d thus ob-
tained were allowed to condense with 3′-O-(tert-butyldimethylsilyl)-
* Corresponding author. E-mail: wada@k.u-tokyo.ac.jp.
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J. AM. CHEM. SOC. 2002, 124, 4962-4963
10.1021/ja017275e CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
Scheme 2a
two possible directions for the nucleophilic attack at the phosphorus
atom. The hydroxy group of a deoxyribonucleoside would prefer-
entially attack at the phosphorus atom from the backside of the
protonated nitrogen atom via an in-line mechanism with the
inversion of the P-configuration to give the corresponding phosphite
triester as a major product. On the contrary, the nucleophilic attack
of the hydroxy group from the front side of the protonated nitrogen
atom would give rise to the product with the retention of the
P-configuration. The later process is, however, inherently inter-
rupted because of the steric hindrance of the N-methyl group. Thus,
the dithymidine phosphorothioate would be diastereoselectively
obtained through an in-line mechanism.
a Reagents and conditions: (i) activating agent 1a, CH3CN-CD3CN (4:
1, v/v), room temperature, <5 min.
Table 2. Condensations of 5′-O-(DMTr)nucleoside
3′-Phosphoramidites 8a-d with 4 in the Presence of 1a
1
2
In summary, a new class of activators, dialkyl(cyanomethyl)-
ammonium tetrafluoroborates 1, were found to be effective for the
stereospecific internucleotidic bond formation of diastereopure
nucleoside 3′-cyclic phosphoramidites 3a-d and 8a-d with
deoxyribonucleosides. It was also found that both the rate of
condensation and diastereoselectivity were affected by the steric
and electronic factors of the substituent at the 4-position of the
oxazaphospholidine ring in 3a-d. The solid-phase synthesis of
P-stereodefined PS-ODNs by the present approach is now in
progress.
phosphoramidite
B
B
phosphite
Rp-9:Sp-9a
8a
8b
8c
Th
Th
Th
Th
9a
9b
9c
>99:1 (139.6)
AdBz
CyBz
GuPa
Th
98:2 (139.4, 138.8)
>99:1 (138.9)
8db
8a
Th
9db
9e
98:2 (141.2, 139.6)
98:2 (142.5, 142.3)
AdBz
a The chemical shifts of 31P NMR are given in parentheses. b GuPa
2-N-phenylacetylguanin-9-yl.
)
The diastereoselectivity of the condensations varied with the
activators; in the cases of 1a,b, the condensations resulted in good
to excellent diastereoselectivity, especially, the condensation of 3a
with 4a in the presence of 1a gave only one diastereoisomer of the
corresponding phosphite 5a.9 The result is in contrast with the fact
that the condensation proceeded very slowly with low diastereo-
selectivity when a conventional activator, 1H-tetrazole, was used
in place of 1. The diastereoselectivity changed dramatically, when
1c was used; 1c rather promoted the formation of the other
diastereomer, although the reason is still unclear.
The resultant phosphites 5 were subjected to sulfurize by the
Beaucage reagent.10 The sulfurization of 5d proceeded smoothly
by using the Beaucage reagent. In the cases of 5a-c, however, the
formation of some unidentified byproducts was detected by 31P
NMR analyses; these byproducts would arise from some reaction
of the secondary amino groups in 5a-c with the Beaucage reagent
and/or its residue. On the basis of the consideration, the amino
groups were acetylated before sulfurization. This acetylation
eliminated the undesired side reaction.
After sulfurization, the chiral auxiliary in 6a-c was removed
by treatment with 10 equiv of 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) at 50 °C to give 5′-O- and 3′-O-silylated dithymidine
phosphorothioate within 30 min.11,12 Finally, the 5′-O- and 3′-O-
silyl groups were removed by treatment with Et3N‚3HF,13 and
purification by reverse-phase column chromatography gave fully
deprotected dithymidine phosphorothioate (7) in good total yield.14
The reverse-phase HPLC analysis showed that the diastereomer
ratio was preserved during the deprotection steps; the phospho-
rothioate 7, obtained from 3a upon activating with 1a, was almost
diastereopure (Rp:Sp ) 99:1).9,15
Next, the most effective activating agent 1a was applied to the
reactions of 5′-O-(DMTr)nucleoside 3′-phosphoramidites (8a-d)
with nucleosides 4a,b (Scheme 2). It was noteworthy that no
cleavage of the 5′-O-DMTr group during the reactions was detected
by TLC analyses. In all cases, the 31P NMR analyses showed that
the reactions proceeded quickly with excellent diastereoselectivity
(Table 2).9
Acknowledgment. This work was supported by Grants from
the Ministry of Education, Science, Sports, Culture and Technology,
Japan.
Supporting Information Available: 1H, 13C, and 31P NMR spectra
of 3a-d, 7 and 8a-d; 31P NMR spectra of the mixtures obtained by
the reaction of 3a with 4a in the presence of 1a or 1c, and those of
8a-d with 4a,b in the presence of 1a, reverse-phase HPLC chromato-
grams of 7, the optimized geometry and the LUMO of 10 (PDF). This
material is available free of charge via the Internet at http://pubs.acs.org.
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(11) The chiral auxiliary of 6d, which was not acetylated, was removed within
4 h.
(12) We presumed that the oxygen atom of the enolate, generated from the
N-acetyl group, attacked the benzyl carbon in 6a,b to release the chiral
auxiliary as 2-methylene-3-methyl-5-phenyl-1,3-oxazolidine.
(13) Gasparutto, D.; Livache, T.; Bazin, H.; Duplaa, A.-M.; Guy, A.; Khorlin,
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out for N-protonated (2S,5R)-2-methoxy-3-methyl-5-phenyl-1,3,2-
oxazaphospholidine (10) as a model of N-protonated cyclic phos-
phoramidite intermediates.9 The optimized geometry and the LUMO
of 10, obtained at the HF/6-31G* level, indicate that there exist
(14) Isolated total yields of 7 were 74 and 68% from 3a and 3b, respectively
(5 steps).
(15) (Rp)-TpsT eluted faster than (Sp)-TpsT in a reverse-phase HPLC. Wada,
T.; Kobayashi, N.; Mori, T.; Sekine, M. Nucleosides Nucleotides 1998,
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