Solid-phase synthesis of stereoregular oligodeoxyribonucleoside phosphorothioates using bicyclic oxazaphospholidine derivatives as monomer units

Article abstract of DOI:10.1021/ja805780u

Nucleoside 3′-O-bicylic oxazaphospholidine derivatives were designed as monomer units for a solid-phase synthesis of stereoregular oligodeoxyribonucleoside phosphorothioates (PS-ODNs). The trans-isomers of appropriately designed nucleoside 3′-O-bicyclic oxazaphospholidine derivatives were generated exclusively by the reaction between the 3′-OH of the corresponding protected nucleosides and 2-chloro-1,3,2-oxazaphospholidine derivatives. The resultant trans-oxazaphospholidine isomers were configurationally stable, and their diastereopurity was not impaired by epimerization in the presence of an acidic activator during the condensation on a solid support. As a result, the formation of both (Rp)- and (Sp)-phosphorothioate internucleotide linkages by using the oxazaphospholidine monomers and the acidic activator proceeded without any loss of diastereopurity (diastereoselectivity ≥99:1). In addition, ab initio molecular orbital calculations showed that the epimerization of oxazaphospholidine derivatives was most likely to proceed via an edge inversion process that was accelerated by N-protonation. The calculations rationalized not only the relations between the ring structure and the configurational stability of the oxazaphospholidines observed in this study but also the observations reported in the literature that the inversion of tricoordinated organophosphorus compounds were accelerated by acids or nucleophiles.

Full text of DOI:10.1021/ja805780u

Published on Web 11/04/2008
Solid-Phase Synthesis of Stereoregular
Oligodeoxyribonucleoside Phosphorothioates Using Bicyclic
Oxazaphospholidine Derivatives as Monomer Units
Natsuhisa Oka, Mika Yamamoto, Terutoshi Sato, and Takeshi Wada*
Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The UniVersity
of Tokyo, Bioscience Building 702, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan
Received July 24, 2008; E-mail: wada@k.u-tokyo.ac.jp
Abstract: Nucleoside 3′-O-bicylic oxazaphospholidine derivatives were designed as monomer units for a
solid-phase synthesis of stereoregular oligodeoxyribonucleoside phosphorothioates (PS-ODNs). The trans-
isomers of appropriately designed nucleoside 3′-O-bicyclic oxazaphospholidine derivatives were generated
exclusively by the reaction between the 3′-OH of the corresponding protected nucleosides and 2-chloro-
1,3,2-oxazaphospholidine derivatives. The resultant trans-oxazaphospholidine isomers were configurationally
stable, and their diastereopurity was not impaired by epimerization in the presence of an acidic activator
during the condensation on a solid support. As a result, the formation of both (Rp)- and (Sp)-phosphorothioate
internucleotide linkages by using the oxazaphospholidine monomers and the acidic activator proceeded
without any loss of diastereopurity (diastereoselectivity g99:1). In addition, ab initio molecular orbital
calculations showed that the epimerization of oxazaphospholidine derivatives was most likely to proceed
via an edge inversion process that was accelerated by N-protonation. The calculations rationalized not
only the relations between the ring structure and the configurational stability of the oxazaphospholidines
observed in this study but also the observations reported in the literature that the inversion of tricoordinated
organophosphorus compounds were accelerated by acids or nucleophiles.
Introduction
having stereodefined PS-linkages would be useful to elucidate
the biosynthetic pathway and functions of the naturally occurring
Modification of oligodeoxyribonucleotides (ODNs) with
phosphorothioate diester linkages (PS-linkages), in which one
of the nonbridging oxygen atoms of natural phosphate diester
linkages (PO-linkages) is replaced with a sulfur atom, has been
widely used to create functional ODNs, such as probes for
enzymatic reactions and nucleic acid drugs with improved
nuclease stability and membrane permeability.1,2 One of the
most important features of the PS-linkage is its chirality.2,3 Since
the targets of the above-mentioned functional ODNs are chiral
in most cases (e.g., enzymes, DNA, RNA, etc.), stereodefined
PS-linkages are indispensable to create sophisticated functional
ODNs.
In addition, the existence of PS-linkages in some bacterial
genomic DNA has been revealed very recently.4 It has been
known that the genomic DNA from Streptomyces liVidans DNA
degradation phenotype undergoes Tris-dependent degradation
during electrophoresis,5 and a series of works confirmed that
the phenomenon was attributed to an (Rp)-PS-linkage at a
specific site of the DNA.4,6 It is expected that synthetic ODNs
PS-linkages.
However, access to stereoregular PS-ODNs is still severely
limited despite the considerable efforts by many research
groups.7-16 Currently, these ODNs are available via the
oxathiaphospholane method,3,8a-c in which nucleoside 3′-O-
(2-thio-1,3,2-oxathiaphospholane) derivatives are used as mono-
mers, though the method has some drawbacks, such as the time-
consuming chromatographic isolation of the diastereopure
monomers from ca. 1:1 mixtures of diastereomers, relatively
(6) (a) Dyson, P.; Evans, M. Nucleic Acids Res. 1998, 26, 1248–1253.
(b) Boybek, A.; Ray, T. D.; Evans, M. C.; Dyson, P. J. Nucleic Acids
Res. 1998, 26, 3364–3371. (c) Zhou, X.; He, X.; Liang, J.; Li, A.;
Xu, T.; Kieser, T.; Helmann, J. D.; Deng, Z. Mol. Microbiol. 2005,
57, 1428–1438. (d) He, X.; Ou, H.-Y.; Yu, Q.; Zhou, X.; Wu, J.; Liang,
J.; Zhang, W.; Rajakumar, K.; Deng, Z. Mol. Microbiol. 2007, 65,
1034–1048. (e) Liang, J.; Wang, Z.; He, X.; Li, J.; Zhou, X.; Deng,
Z. Nucleic Acids Res. 2007, 35, 2944–2954.
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Nucleic Acids Res. 1991, 19, 5883–5888. (b) Stec, W. J.; Grajkowski,
A.; Kobylan´ska, A.; Karwowski, B.; Koziołkiewicz, M.; Misiura, K.;
Okruszek, A.; Wilk, A.; Guga, P.; Boczkowska, M. J. Am. Chem.
Soc. 1995, 117, 12019–12029. (c) Stec, W. J.; Karwowski, B.;
Boczkowska, M.; Guga, P.; Koziołkiewicz, M.; Sochacki, M.; Wiec-
zorek, M. W.; Błaszczyk, J. J. Am. Chem. Soc. 1998, 120, 7156–
7167. (d) Nawrot, B.; Re¸bowska, B.; Cies´lin´ska, K.; Stec, W. J.
Tetrahedron Lett. 2005, 46, 6641–6644. (e) Wozniak, L. A.; Go´ra,
M.; Bukowiecka-Matusiak, M.; Mourgues, S.; Pratviel, G; Meunier,
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Eckstein, F. Antisense Nucleic Acids Drug DeV. 2000, 10, 117–121.
(2) Guga, P. Curr. Top. Med. Chem. 2007, 7, 695–713.
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722.
(4) Wang, L.; Chen, S.; Xu, T.; Taghizadeh, K.; Wishnok, J. S.; Zhou,
X.; You, D.; Deng, Z.; Dedon, P. C. Nat. Chem. Biol. 2007, 3, 709–
710.
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J. AM. CHEM. SOC. 2008, 130, 16031–16037 16031

A R T I C L E S
Oka et al.
Scheme 1. Synthesis of Thymidine 3′-O-Oxazaphospholidine
low efficiency of the internucleotidic bond formation under
strongly basic conditions, and requirement of another set of four
nucleoside 3′-O-(2-oxo-1,3,2-oxathiaphospholane) monomers for
the synthesis of stereoregular phosphate/phosphorothioate chi-
meric ODNs (PO/PS-chimeric ODNs) due to the incompatibility
of the method with the conventional phosphoramidite method.8c
Under these circumstances, we have developed a method to
synthesize stereoregular PS-ODNs using nucleoside 3′-O-(1,3,2-
oxazaphospholidine) derivatives11 as monomers, which were
stereoselectively synthesized from enantiopure 1,2-amino al-
cohols and a less-nucleophilic acidic activator and applied to a
manual solid-phase synthesis.9 The method has some advan-
tages, such as the diastereoselective formation of the monomers
due to the chirality of the 1,2-amino alcohols and rapid
condensation under mild acidic conditions. In addition, since
the method consists of steps analogous to those of the
conventional phosphoramidite method [(i) condensation pro-
moted with a mild acidic activator; (ii) capping by acylating
reagents; (iii) oxidation/sulfurization; (iv) 5′-O-detritylation],
stereoregular PO/PS-chimeric ODNs may also be synthesized
by switching the commonly used ꢀ-cyanoethyl phosphoramid-
ites/oxazaphospholidines and/or oxidation/sulfurization. How-
ever, a non-negligible loss of diastereopurity during the
condensation reactions was observed (up to 5-6% per cycle).9b
The partial loss of the diastereopurity can be attributed to
the epimerization of the oxazaphospholidine monomers in the
presence of the acidic activator. Though epimerization of
tricoordinate phosphoramidite derivatives is usually negligible
under neutral conditions at ambient temperatures, it is known
to be accelerated under acidic conditions and/or at higher
temperatures.17 Since the contact of the oxazaphospholidine
monomers with the acidic activator prior to the reaction with
the 5′-OH of nucleosides on a solid support is inevitable for a
solid-phase synthesis, an oxazaphospholidine monomer that
would not epimerize even in the presence of the acidic activator
is required. The oxazaphospholidine derivative also has to fulfill
other requirements for the synthesis of stereoregular PS-ODNs,
such as the high diastereoselectivity of their synthesis, repetitive
condensations with high efficiency, and the removal of the 1,2-
amino alcohol moiety from the resultant PS-linkages without
any loss of diastereopurity. In this paper, we describe the
development of nucleoside 3′-O-bicyclic oxazaphospholidine
derivatives that fulfill all of these requirements and their
applications to the synthesis of stereoregular PS-ODNs on an
automated DNA synthesizer.
Derivatives 3a-6a
phospholidine derivatives (2a-d) (Scheme 1).9 Crude 2-chloro-
1,3,2-oxazaphospholidine derivatives 2a-d were synthesized
from the corresponding 1,2-amino alcohols (7a-d) as previously
reported and used without further purification.9,18 We have
reported that the stereochemistry of the reaction is kinetically
controlled under these conditions and one of the two P-
diastereomers of the oxazaphospholidine derivatives is prefer-
entially generated from the (Rp)- or (Sp)-2-chloro-1,3,2-
oxazaphospholidine derivative, which are rapidly epimerized
between each other due to repetitive nucleophilic attacks of Cl-.
To design a configurationally stable nucleoside 3′-O-oxaza-
phospholidine derivative, we focused on proline-derived bicyclic
oxazaphospholidine rings. The simplest one [Figure 1, (Rp)-
4a] has been reported to generate only the trans-isomer of the
nucleoside 3′-O-oxazaphospholidine,11b,c and we also obtained
the trans-isomer almost exclusively (trans:cis ) 98:2) under
the kinetic conditions.19 However, the diastereomer ratio of
(Rp)-4a was decreased to 93:7 by treatment with 2 equiv of an
acidic activator, which was used to activate the oxazaphospho-
lidine monomers [N-(cyanomethyl)pyrrolidinium triflate (CMPT)
8],9 in CH3CN-CD3CN (4:1, v/v) for 4 h at rt, though the
epimerization was significantly suppressed compared to that of
the monocyclic oxazaphospholidine derivative (Rp)-3a9 (trans:
cis ) 58:42 under the same conditions). On the basis of the
results, we designed new bicyclic oxazaphospholidine deriva-
Results and Discussion
Design and Synthesis of Configurationally Stable Nucleoside
3′-O-Bicyclic Oxazaphospholidine Derivatives. Thymidine 3′-O-
oxazaphospholidine derivatives (3a-6a) were synthesized by
the reaction between the 3′-OH of 5′-O-(dimethoxytrityl)thy-
midine [5′-O-(DMTr)thymidine, 1a] and 2-chloro-1,3,2-oxaza-
(13) (a) Hyodo, M.; Ando, H.; Nishitani, H.; Hattori, A.; Hayakawa, H.;
Kataoka, M.; Hayakawa, Y. Eur. J. Org. Chem. 2005, 5216–5223.
(b) Hayakawa, Y.; Hirabayashi, Y.; Hyodo, M.; Yamashita, S.;
Matsunami, T.; Cui, D.-M.; Kawai, R.; Kodama, H. Eur. J. Org. Chem.
2006, 3834–3844.
(14) Almer, H.; Szabo, T.; Stawinski, J. Chem. Commun. 2004, 290–291.
(15) (a) Wada, T.; Kobayashi, N.; Mori, T.; Sekine, M. Nucleosides
Nucleotides 1998, 17, 351–364. (b) Seio, K.; Kumura, K.; Bologna,
J.-C.; Sekine, M. J. Org. Chem. 2003, 68, 3849–3859.
(16) Lesnikowski, Z. J.; Jaworska, M. M. Tetrahedron Lett. 1989, 30, 3821–
3824.
(10) Wilk, A.; Grajkowski, A.; Phillips, L. R.; Beaucage, S. L. J. Am. Chem.
Soc. 2000, 122, 2149–2156.
(11) (a) Iyer, R. P.; Yu, D.; Ho, N.-H.; Tan, W.; Agrawal, S. Tetrahedron:
Asymmetry 1995, 6, 1051–1054. (b) Iyer, R. P.; Guo, M.-J.; Yu, D.;
Agrawal, S. Tetrahedron Lett. 1998, 39, 2491–2494. (c) Yu, D.;
Kandimalla, E. R.; Roskey, A.; Zhao, Q.; Chen, L.; Chen, J.; Agrawal,
S. Bioorg. Med. Chem. 2000, 8, 275–284.
(17) (a) Mikołajczyk, M. Pure Appl. Chem. 1980, 52, 959–972. (b) Nielsen,
J.; Dahl, O. J. Chem. Soc., Perkin Trans. 2 1984, 553–558.
(18) The corresponding 1,2-amino alcohols (7a-d) and PCl3 (1 equiv) were
reacted in toluene in the presence of N-methylmorpholine (2 equiv)
for 30 min at 0 °C to room temperature. The mixture was filtered
under argon to remove the resultant N-methylmorpholine hydrochloride
and concentrated to dryness to afford crude 2a-d, which was used
without further purification.
(12) (a) Marsault, E.; Just, G. Tetrahedron 1997, 53, 16945–16958. (b)
Jin, Y.; Just, G. J. Org. Chem. 1998, 63, 3647–3654. (c) Lu, Y.; Just,
G. Tetrahedron Lett. 2000, 41, 9223–9227. (d) Wang, J.-C.; Just, G.
Tetrahedron Lett. 1997, 38, 3797–3800. (e) Lu, Y.; Just, G. Angew.
Chem., Int. Ed. 2000, 39, 4521–4524.
(19) See Supporting Information.
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Solid-Phase Synthesis of Stereoregular PS-ODNs
A R T I C L E S
Table 1. Synthesis of Nucleoside 3′-O-Oxazaphospholidine
Monomers
entry
oxazaphospholidine
isolated yield (%)
trans:cis
1
2
3
4
5
6
7
8
(Sp)-Th
(Sp)-5a
(Sp)-5b
(Sp)-5c
(Sp)-5d
(Rp)-5a
(Rp)-5b
(Rp)-5c
(Rp)-5d
54
50
58
45
51
52
43
44
>99:1
>99:1
>99:1
>99:1
>99:1
>99:1
>99:1
>99:1
(Sp)-Cyac
(Sp)-Addmf
(Sp)-Guce,pac
(Rp)-Th
(Rp)-Cyac
(Rp)-Addmf
(Rp)-Guce,pac
Figure 1. Ratios of trans:cis and time course of epimerization of thymidine
3′-O-oxazaphospholidine derivatives 3a-6a. Notes: (a) RO ) 5′-O-
(DMTr)thymidin-3′-yl. (b) Diastereomer ratios of 3a-6a after being treated
by 2 equiv of CMPT in CH3CN-CD3CN (4:1, v/v) at room temperature
for 4 h (31P NMR). (c) Diastereomer ratios of (2S,4R,5R)-6a and (2R,4S,5S)-
6a were 80:20 and 90:10, respectively.
epimerization in the presence of CMPT 8 decreased in the
following order: (Rp)-3a > 6a > (Rp)-4a > (Rp)-5a as shown
in Figure 1. To rationalize these results, we carried out ab initio
molecular orbital (MO) calculations.
tives having a phenyl group at the 5-position [(Rp)-5a, 6a20]
and carried out their synthesis under the same conditions to find
that both of these oxazaphospholidine rings gave the trans-
isomers of the thymidine 3′-O-oxazaphospholidines, exclusively.
In addition, the epimerization in the presence of CMPT was
almost completely suppressed for (Rp)-5a, whereas 10-20%
cis-isomers were generated for 6a. These results showed that
the newly designed bicyclic oxazaphospholidine ring structure
of (Rp)-5a generated the trans-isomer of the corresponding
thymidine 3′-O-oxazaphospholidine derivative exclusively with-
out being contaminated by the cis-isomer and also has virtually
complete configurational stability, thus fulfilling two of the four
requirements for the synthesis of stereoregular PS-ODNs
mentioned above. By using the oxazaphospholidine ring, (Rp)-
and (Sp)-nucleoside 3′-O-oxazaphospholidine monomers cor-
responding to the four kinds of nucleobases [(Rp)- and (Sp)-
5a-d] were synthesized under the same conditions. The trans-
isomers were generated exclusively in all of the cases and
isolated in modest yields (Table 1).21
The mechanism of inversion process at the phosphorus atoms
of tricoordinate organophosphorus compounds has not been fully
clarified.22-24 In particular, little is known about the mechanism
of the inversion process of phosphorus-containing chiral het-
erocyclic compounds, despite the fact that these compounds have
been widely used to synthesize optically pure organophosphorus
compounds such as phosphine-based ligands25 and biologically
active compounds.2,3,7-13,24,26 It has been reported that nucleo-
philic species (e.g., amines, water, Cl-) and acids (e.g., amine
hydrochlorides) accelerate the inversion process, and their
repetitive nucleophilic attacks to the phosphorus atom are
proposed to be involved.17,26 However, it cannot rationalize the
fact that the inversion is also accelerated in the case that the
nucleophilic species and the leaving group at the phosphorus
(22) (a) Dixon, D. A.; Arduengo, A. J., III; Fukunaga, T. J. Am. Chem.
Soc. 1986, 108, 2461–2462. (b) Arduengo, A. J., III; Dixon, D. A.;
Roe, D. C. J. Am. Chem. Soc. 1986, 108, 6821–6823. (c) Dixon, D. A.;
Arduengo, A. J., III J. Am. Chem. Soc. 1987, 109, 338–341. (d) Creve,
S.; Nguyen, M. T. J. Phys. Chem. A 1998, 102, 6549–6557.
(23) (a) Baechler, R. D.; Mislow, K. J. Am. Chem. Soc. 1970, 92, 3090–
3093. (b) Baechler, R. D.; Mislow, K. J. Am. Chem. Soc. 1971, 93,
773–774.
As described above, the proline-derived trans-nucleoside 3′-
O-bicyclic oxazaphospholidine derivatives (Rp)-4a,(Rp)-5a, and
6a had markedly increased configurational stability compared
to the monocyclic (Rp)-3a, and the rate of trans to cis
(24) Kolodiazhnyi, O. I. Tetrahedron: Asymmetry 1998, 9, 1279–1332.
(25) (a) Cros, P.; Buono, G.; Peiffer, G.; Denis, D.; Mortreux, A.; Petit, F.
New J. Chem. 1987, 11, 573–579. (b) Arzoumanian, H.; Buono, G.;
Choukrad, M.; Petrignani, J.-F. Organometallics 1988, 7, 59–62. (c)
Juge´, S.; Genet, J. P. Tetrahedron Lett. 1989, 30, 2783–2786. (d)
Polosukhin, A. I.; Bondarev, O. G.; Lyubimov, S. E.; Shiryaev, A. A.;
Petrovskii, P. V.; Lysenko, K. A.; Gavrilov, K. N. Russ. J. Coord.
Chem. 2001, 27, 591–597. (e) Hoge, G. J. Am. Chem. Soc. 2004, 126,
9920–9921. (f) Juge´, S. Phosphorus Sulfur Silicon 2008, 183, 233–
248.
(20) The oxazaphospholidine 6a was synthesized as a mixture of two
isomers having either a (2S,4R,5R)- or a (2R,4S,5S)-oxazaphospholi-
dine ring by using racemic 1,2-amino alcohol 7d. The 1,2-amino
alcohols 7c,d were synthesized according to the procedures reported
in the literature. The details are given in Supporting Information. (a)
Bejjani, J.; Chemla, F.; Audouin, M. J. Org. Chem. 2003, 68, 9747–
9752. (b) Meyers, A. I.; Ten Hoeve, W. J. Am. Chem. Soc. 1980,
102, 7125–7126.
(21) The nucleoside 3′-O-oxazaphospholidine derivatives 5a-d were
partially decomposed on silica gel during chromatography, resulting
in modest isolated yields. The isolated 5a-d were stable for at least
1 year under proper storage conditions (at-30 °C in a glass vial flushed
with an inert gas).
(26) (a) Kinas, R.; Pankiewicz, K.; Stec, W. J. Bull. Acad. Pol. Sci. 1975,
23, 981–984. (b) Kawashima, T.; Kroshefsky, R. D.; Kok, R. A.;
Verkade, J. G. J. Org. Chem. 1978, 43, 1111–1114. (c) Hall, C. R.;
Inch, T. D. Phosphorus Sulfur 1979, 7, 171–184.
9
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A R T I C L E S
Oka et al.
Scheme 2. Solution-Phase Synthesis of Dithymidine
Phosphorothioates (Rp)- and (Sp)-13a
Figure 2. Edge inversion process.
atom are different from each other. In addition, CMPT consisting
of less nucleophilic components also accelerated the epimer-
ization of the oxazaphospholidines as above. Therefore, elucida-
tion of the epimerization mechanism of the oxazaphospholidine
derivatives observed in this study would shed light on the
unsolved inversion mechanism of tricoordinated organophos-
phorus compounds.
It has been calculated that the inversion of phosphine
derivatives, such as PH3, proceeds through a classical trigonal-
planar transition state (vertex inversion) and those having
electronegative substituents, such as PF3, invert through a
T-shaped transition state (edge inversion, Figure 2).22a As the
first step of our MO calculations, the transition states of a model
structure of (Rp)-3a having a methyl group in the place of the
5′-O-(DMTr)thymidin-3′-yl group and its N3-protonated form
were calculated at the HF/6-31G* level. Two transition states
were obtained for both the nonprotonated and the protonated
forms, and all of these had a T-shaped structure,19 indicating
that the epimerization of the oxazaphospholidine derivatives
proceeds through an edge inversion process. It is reasonable
because a planar trigonal transition state has a theoretical
O-P-N bond angle of ca. 120°, which would be significantly
less stable for the oxazaphospholidines than the T-shaped one
due to the existence of the five-membered oxazaphospholidine
ring. The two of these transition states have the O1 and the O2
atoms at the apical positions, and the other two have the N3
and the O2 atoms at the apical positions. Since electron-
withdrawing substituents at the apical positions stabilize the
T-shaped transition states,22c the smallest activation energy is
obtained for the one in which the extended P-N bond (2.90
Å) by N3-protonation occupies the apical position.19 The MO
calculations thus showed that N3-protonation of the oxazaphos-
pholidine ring extended the P-N bond to accelerate an edge
inversion process through a T-shaped transition state in which
the P-NH+ bond occupies the apical position.
The important thing here is that it has been shown both
theoretically and experimentally that the edge inversion barrier
at pnictogens is significantly reduced by coordination of
nucleophilic species to the unoccupied orbital in the T-shaped
transition state and the stabilization effect does not exist in a
vertex inversion process.27 Therefore, an edge inversion process
is also supported by the reported acceleration of inversion by
nucleophiles, especially those different from the leaving group
on the phosphorus atom. In addition, another reported accelera-
tion effect by acids is probably due to not only the coordination
of the nucleophilic counteranion (e.g., Cl-) but also the
extension of the P-N bond by N-protonation.
dine structures (4a, 5a, 6a) to obtain the activation energy of
the edge inversion process to find that the calculated energy
values increased in the following order: (Rp)-3a < 6a < (Rp)-
4a < (Rp)-5a, which was consistent with the order of
configurational stability obtained in this study.19 Though the
possibility of stabilization by nucleophiles cannot be excluded,
the calculations showed that the epimerization of the oxaza-
phospholidine derivatives in the presence of less-nucleophilic
CMPT is most likely to proceed via an edge process, which is
accelerated by the extension of the P-N bond by N3-protonation,
and the edge inversion energy barrier for those with a bicyclic
oxazaphospholidine ring, especially (Rp)-5a, is significantly
larger than that for the monocyclic one, resulting in the
configurational stability.
Solution-Phase Synthesis of (Sp)- and (Rp)-Dithymidine
Phosphorothioates. The oxazaphospholidine derivative (Rp)-
5a with the best configurational stability was then applied to
the stereocontrolled synthesis of internucleotidic PS-linkages.
First, dithymidine phosphorothioate was synthesized in solution-
phase by using (Rp)-5a as the monomer unit (Scheme 2). The
oxazaphospholidine (Rp)-5a was allowed to condense with a
3′-O-protected thymidine in the presence of CMPT 8 to afford
a dithymidine phosphite intermediate (9) stereospecifically.
Acetylation of the protonated secondary amino group of 9 and
Our MO calculations thus rationalized the reported observa-
tion on the inversion of tricoordinated phosphorus compounds.
Therefore the model was applied to the other oxazaphospholi-
(27) (a) Dixon, D. A.; Arduengo, A. J. Int. J. Quantum Chem., Symp. 1988,
22, 85–98. (b) Yamamoto, Y.; Chen, X.; Akiba, K.-y. J. Am. Chem.
Soc. 1992, 114, 7906–7907. (c) Yamamoto, Y.; Chen, X.; Kojima,
S.; Ohdoi, K.; Kitano, M.; Doi, Y.; Akiba, K.-y. J. Am. Chem. Soc.
1995, 117, 3922–3932.
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Solid-Phase Synthesis of Stereoregular PS-ODNs
A R T I C L E S
Scheme 3. Synthesis Cycle for Stereoregular PS-ODNs
Table 2. Synthesis of dinucleoside phosphorothioates (Rp)- and
(Sp)-13a-d
entry
dNSTa
yield (%)b
Rp:Spb
1
2
3
4
5
6
7
8
(Rp)-TST
(Rp)-13a
(Rp)-13b
(Rp)-13c
(Rp)-13d
(Sp)-13a
(Sp)-13b
(Sp)-13c
(Sp)-13d
98
98
98
96
97
99
98
96
>99:1
>99:1
>99:1
>99:1
>1:99
1:99
(Rp)-dCST
(Rp)-dAST
(Rp)-dGST
(Sp)-TST
(Sp)-dCST
(Sp)-dAST
(Sp)-dGST
>1:99
1:99
a Subscript “S” ) PS-linkage. b Yield and diastereomer ratio of
products were determined by RP-HPLC.
Table 3. Synthesis of Stereoregular PS-ODNs 17-21.
coupling
yield (%)b
isolated
yield (%)c
no.
oligonucleotidea
17 all-(Rp)-TSTSTSTSTSTSTSTSTST
18 all-(Sp)-TSTSTSTSTSTSTSTSTST
19 (Rp,Rp,Rp,Rp,Rp,Sp,Sp)-TSTSTSTSTSTSTST
20 all-(Rp)-d[CSASGSTSCSASGSTSCSASGST]
21 all-(Sp)-d[CSASGSTSCSASGSTSCSASGST]
99
98
98
96
95
34
32
30
16
12
a Subscript “S”
)
PS-linkage. b Average coupling yield was
estimated by DMTr+ assay. c Isolated by RP-HPLC.
condensation, capping, and the following sulfurization by a cost-
effective sulfurizing reagent N,N′-dimethylthiuram disulfide
(DTD)30 and 5′-O-detritylation. After the chain elongation by
repeating the cycle, the protected PS-ODNs on the support were
deprotected and cleaved from the support by treatment with
concentrated NH3. The N-trifluoroacetylated chiral auxiliary was
removed without observable strand cleavage, though the com-
plete removal required a long treatment as the oligomers
lengthened (15-48 h). First, (Rp)- and (Sp)-dinucleoside
phosphorothioates were synthesized by this method to find that
the formation of PS-linkages was successfully conducted with
excellent yields and diastereoselectivity (Table 2, 96-99%
yields, dr g99:1). Stereoregular PS-ODNs 8-12mers (17-21,
Table 3) were prepared efficiently by using the cycle and isolated
by RP-HPLC (Table 3, Figures 3 and 4). The PS-ODNs were
characterized by MALDI-TOF-MS, and their diastereopurity
was confirmed by enzymatic digestion.19 The HPLC profiles
shown in Figures 3 and 4 showed that all the PS-ODNs were
produced with excellent diastereoselectivity. Thus, the configu-
rationally stable bicyclic oxazaphospholidine monomers proved
to be useful for the synthesis of stereodefined (Rp)- and (Sp)-
PS-linkages at the oligomer level.
the following sulfurization of the phosphorus atom afforded a
fully protected dithymidine phosphorothioate triester (11). Two
31P NMR signals were observed for 10 and 11 due to the
existence of the acetamide rotamers.9,10 Deprotection of the PS-
linkage of 11 required a long treatment with concentrated
ammonia at an elevated temperature but proceeded without loss
of diastereopurity to afford a 3′,5′-O-protected dithymidine
phosphorothioate [(Sp)-12] in an excellent yield. The desired
fully deprotected dithymidine phosphorothioate (Sp)-13a was
obtained in an excellent yield by 3′,5′-O-detritylation under usual
acidic conditions. The configuration and diastereopurity of 13a
was confirmed by RP-HPLC.19 Diastereopure (Rp)-13a was also
obtained in an excellent yield from (Sp)-5a. Thus, the newly
designed bicyclic oxazaphospholidine monomers gave both
(Rp)- and (Sp)-diastereopure PS-linkages in solution phase
efficiently.
Automated Solid-Phase Synthesis of Stereoregular PS-ODNs.
Next, the method was applied to the solid-phase synthesis of
stereoregular PS-ODNs on a DNA synthesizer. The synthesis
cycle is shown in Scheme 3. The monomers 5a-d were allowed
to condense with the 5′-OH of a nucleoside anchored to a highly
cross-linked polystyrene (HCP)28 (14) in the presence of CMPT
8 to afford a phosphite intermediate (15). Since we found that
the removal of N-acetylated chiral auxiliaries from PS-linkages
by treatment with conc NH3 caused partial strand cleavage at
an oligomer level, N-trifluoroacetylimidazole (CF3COIm) was
used to cap the secondary amino group of 15 and any unreacted
5′-OH on the support. No desulfurization was observed under
the capping conditions.29 It was also confirmed that any of the
5′-OH remaining on the solid-support were quantitatively capped
under the conditions. The synthesis cycle consists of the
Conclusion
In summary, the nucleoside 3′-O-bicyclic oxazaphospho-
lidines designed as monomer units to synthesize stereodefined
PS-internucleotide linkages were generated with complete
diastereoselectivity and were also configurationally stable even
in the presence of an acidic activator. Automated synthesis of
PS-linkages by using the monomers proceeded without any loss
of diastereopurity, and stereoregular PS-ODNs were also
synthesized efficiently. The method should facilitate the syn-
thesis of stereoregular PS-ODNs and may also become useful
to provide stereoregular PO/PS-chimeric ODNs since the method
uses a series of reactions similar to those for the conventional
phosphoramidite method. In addition, ab initio MO calculations
(28) McCollum, C.; Andrus, A. Tetrahedron Lett. 1991, 32, 4069–4072.
(29) In contrast, serious desulfurization was observed when (CF3CO)2O
was used in the place of CF3COIm as reported in the literature. (a)
Helin´ski, J.; Skrzypczyn´ski, Z.; Wasiak, J.; Michalski, J. Tetrahedron
Lett. 1990, 31, 4081–4084. (b) Bruzik, K. S.; Stec, W. J. J. Org. Chem.
1990, 55, 6131–6135.
(30) Song, Q.; Wang, Z.; Sanghvi, Y. S. Nucleosides Nucleotides Nucleic
Acids 2003, 22, 629–633.
9
J. AM. CHEM. SOC. VOL. 130, NO. 47, 2008 16035

A R T I C L E S
Oka et al.
Figure 3. Reversed-phase HPLC profiles of PS-T10: (A) crude all-(Rp)-
PS-T10 17; (B) purified 17; (C) crude all-(Sp)-PS-T10 18; (D) purified 18.
RP-HPLC was performed with a linear gradient of 0-20% acetonitrile in
0.1 M ammonium acetate buffer (pH 7.0) at 50 °C for 90 min at a rate of
0.5 mL/min.
Figure 4. Reversed-phase HPLC profiles of PS-d[CAGT]3: (A) crude all-
(Rp)-PS-d[CAGT]3 20; (B) purified 20; (C) crude all-(Sp)-PS-d[CAGT]3
21; (D) purified 21. RP-HPLC was performed with a linear gradient of
0-20% acetonitrile in 0.1 M ammonium acetate buffer (pH 7.0) at 50 °C
for 90 min at a rate of 0.5 mL/min.
showed that the epimerization of the oxazaphospholidines was
most likely to proceed via an edge inversion process. The
calculations rationalized the observations reported in the
literature that the epimerization of tricoordinated organophos-
phorus compounds was accelerated by acids or nucleophiles.
Though the effect of nucleophiles or coordinating solvents was
not included in the calculations in this study because we consider
the effect of N-protonation dominates, such calculations can also
be carried out easily. Such calculations may be useful for the
design of tricoordinated organophosphorus compounds, such as
chiral ligands, because practical configurational stability of these
compounds in organic solvents or in the presence of a small
amount of impurities can be predicted.
by repeated co-evaporations with dry pyridine and dry toluene and
dissolved in freshly distilled THF (6.0 mL) under argon. Et3N (1.39
mL, 10 mmol) and a 0.5 M solution of the corresponding crude
(4S,5R)-2-chloro-1,3,2-oxazaphospholidine [(4S,5R)-2c], which was
prepared from an L-proline-derived 1,2-amino alcohol (RR,2S)-7c,
in freshly distilled THF (6.0 mL, 3.0 mmol) were successively
added dropwise to the solution at -78 °C with stirring. The mixture
was stirred for 30 min at room temperature, poured into an ice-
cold saturated NaHCO3 aqueous solution (100 mL), and extracted
with CHCl3 (100 mL). The organic layer was washed with ice-
cold saturated NaHCO3 aqueous solutions (2 × 100 mL), and the
combined aqueous layers were back-extracted with CHCl3 (100
mL). The combined organic layers were dried over Na2SO4, filtered,
and concentrated to dryness under reduced pressure. The resultant
crude (Rp)-5a-d was purified by silica gel column chromatography.
The purification conditions, isolated yields, and characterizing data
are given in Supporting Information. The monomers (Sp)-5a-d
were also synthesized according to the same method by using the
protected nucleosides (1a-d) and (4R,5S)-2-chloro-1,3,2-oxaza-
phospholidine derivative [(4R,5S)-2c], which was prepared from
the D-proline-derived amino alcohol (RS,2R)-7c.
Experimental Section
Ab Initio MO Calculations. The calculations were carried out
using the Spartan’0431 on a Dell Inc. PRECISION 650 workstation.
Geometry optimizations and single-point energy calculations were
carried out at the HF/6-31G* level.
General Procedure for the Synthesis of the Nucleoside
3′-O-Oxazaphospholidine Monomers (Rp)-5a-d. The appropri-
ately protected 2′-deoxyribonucleoside (1a-d, 2.0 mmol) was dried
(Rp)-5a. Crude (Rp)-5a obtained as above was purified by silica
gel column chromatography [3.5 × 6 cm, 30 g of silica gel,
hexane-ethyl acetate-triethylamine (20:80:2, v/v/v)]. The fractions
containing (Rp)-5a were combined, washed with a saturated
NaHCO3 aqueous solution (500 mL), dried over Na2SO4, filtered,
(31) Kong, J. et al. Spartan’04; Wavefunction, Inc.: Irvine, CA; J. Comput.
Chem. 2000, 21, 1532-1548.
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16036 J. AM. CHEM. SOC. VOL. 130, NO. 47, 2008

Solid-Phase Synthesis of Stereoregular PS-ODNs
A R T I C L E S
and concentrated to dryness under reduced pressure to afford (Rp)-
5a (0.772 g, 1.0 mmol, 51%) as a white foam. IR (KBr) 3412,
3182, 2953, 1690, 1607, 1582, 1509, 1465, 1366, 1252, 1177, 1154,
1076, 1034, 962, 915, 829, 793, 756, 706, 584, 559 cm-1. 1H NMR
(300 MHz, CDCl3) δ 9.46 (brs, 1H), 7.59 (s, 1H), 7.40-7.19 (m,
14H), 6.79 (d, J ) 8.1 Hz, 4H), 6.44 (dd, J ) 7.1, 6.0 Hz, 1H),
5.71 (d, J ) 6.3 Hz, 1H), 4.93 (m, 1H), 4.12 (m, 1H), 3.81 (m,
1H), 3.75 (s, 6H), 3.56 (m, 1H), 3.47 (dd, J ) 10.6, 2.4 Hz, 1H),
3.37 (dd, J ) 10.6, 2.3 Hz, 1H), 3.16 (m, 1H), 2.58 (ddd, J )
13.6, 6.0, 2.7 Hz, 1H), 2.37 (ddd, J ) 13.6, 7.1, 6.8 Hz, 1H), 1.63
(m, 2H), 1.41 (s, 3H), 1.19-1.12 (m, 1H), 0.99-0.85 (m, 1H).
13C NMR (75 MHz, CDCl3) δ 163.9, 158.3, 158.3, 150.3, 144.0,
137.8 (d, 3JPC ) 4.1 Hz), 135.3, 135.1, 135.0, 129.8, 129.8, 127.9,
127.9, 127.6, 127.2, 126.8, 125.1, 112.9, 110.9, 86.6, 85.2 (d, 3JPC
) 2.0 Hz), 84.4, 82.1 (d, 2JPC ) 9.5 Hz), 73.0 (d, 2JPC ) 12.8 Hz),
67.1 (d, 2JPC ) 3.2 Hz), 62.7, 55.0, 47.0 (d, 2JPC ) 34.7 Hz), 39.9
were combined and diluted with a 0.1 M p-toluenesulfonic acid
monohydrate solution in CH3CN to make a 50.0 mL solution for
every cycle to perform the DMTr+ assay. Average coupling yields
were 95-99%. After the synthesis, the HCP was dried in vacuo
and treated with a 25% NH3 aqueous solution-EtOH (5:1, v/v) (1
mL) for 15-48 h at 55 °C. The mixture was cooled to room
temperature, and the HCP was removed by membrane filtration.
The filtrate was concentrated to dryness under reduced pressure.
The residue was dissolved in H2O (3 mL), washed with Et2O (3 ×
3 mL), and concentrated to dryness under reduced pressure. The
purification and/or analysis of the resultant crude PS-ODNs were
performed by using RP-HPLC with a linear gradient of acetonitrile
(0-15%/45 min for the dimers 13a-d or 0-20%/90 min for the
oligomers 17-21) in 0.1 M ammonium acetate buffer (pH 7.0) at
50 °C at a rate of 0.5 mL/min.
3
3
(d, JPC ) 4.6 Hz), 28.0, 25.8 (d, JPC ) 3.4 Hz), 11.7. 31P NMR
Acknowledgment. We thank Professor Kazuhiko Saigo (Uni-
versity of Tokyo) for helpful suggestions. This research was
supported by grants from MEXT Japan and Futaba Electronics
Memorial Foundation (to N.O.).
(121 MHz, CDCl3)
δ 155.2. FAB-HRMS: m/z calcd for
C42H45N3O8P+ [(M + H)+] 750.2939, found 750.2976.
Automated Solid-Phase Synthesis of Stereoregular PS-ODNs
[(Rp)- and (Sp)-13a-d, 17-21]. The automated solid-phase
synthesis of stereoregular PS-ODNs was performed on an Expedite
8909 Nucleic Acid Synthesis System (Applied Biosystems) ac-
cording to the cycle shown in Supporting Information. The detailed
protocol for the Expedite is also given in Supporting Information.
All the PS-ODNs were synthesized using a thymidine-loaded highly
cross-linked polystyrene (HCP) (0.5 µmol, 30 µmol/g, succinate
linker) The fractions of the detritylation and the following washings
Supporting Information Available: Experimental procedures,
spectral data, and HPLC profiles and the full ref 30. This
material is available free of charge via the Internet at http://
pubs.acs.org.
JA805780U
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J. AM. CHEM. SOC. VOL. 130, NO. 47, 2008 16037