Stereocontrolled synthesis of Oligoribonucleoside phosphorothioates by an oxazaphospholidine approach

Article abstract of DOI:10.1021/ol802910k

Oligoribonucleoside phosphorothioates (PS-ORNs) stereodefined at the phosphorus atoms were synthesized on solid support. Thermal denaturating experiments of the resultant PS-ORNs showed that a backbone consisting of (Sp)-PS-linkages as well as stereorando

Full text of DOI:10.1021/ol802910k

ORGANIC
LETTERS
2009
Vol. 11, No. 4
967-970
Stereocontrolled Synthesis of
Oligoribonucleoside Phosphorothioates
by an Oxazaphospholidine Approach
Natsuhisa Oka, Tomoaki Kondo, Satoshi Fujiwara, Yukihiro Maizuru, 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
wada@k.u-tokyo.ac.jp
Received December 17, 2008
ABSTRACT
Oligoribonucleoside phosphorothioates (PS-ORNs) stereodefined at the phosphorus atoms were synthesized on solid support. Thermal denaturating
experiments of the resultant PS-ORNs showed that a backbone consisting of (Sp)-PS-linkages as well as stereorandom PS-linkages had an unexpectedly
large destabilizing effect on a PS-ORN-ORN duplex, whereas a backbone consisting of (Rp)-PS-linkages slightly stabilized a duplex.
RNA-mediated gene silencing systems, particularly RNA
interference,1 have been widely used as powerful tools
for gene knockdown because efficient gene silencing can
be performed with comparative ease by using relatively
short synthetic oligoribonucleotides (ORNs),2 and their
therapeutic applications, as well as the elucidation of the
silencing mechanism, are current topics of great interest.3
Chemically synthesized ORNs with appropriate modifica-
tions are requisite for these purposes as probes for the
mechanistic elucidation or as drug candidates with en-
hanced stability to nucleases.3 Oligoribonucleoside phos-
phorothioates (PS-ORNs) are one of the most frequently
used analogue probes of the functions of internucleotidic
linkages. Such molecules can also potentially serve as drug
candidates with improved stability to nucleases and cell
membrane permeability.3,4
One of the most important features of PS-ORNs is the
chirality of their phosphorus atoms. PS-ORNs work as probes
or drugs through interactions with chiral biomolecules (e.g.,
nucleic acids, proteins). Therefore, their properties and
functions as probes or drugs are theoretically dependent on
the configurations of the phosphorus atoms, and elucidation
of the dependence is crucial for their applications. However,
only a limited number of studies on this subject have been
conducted due to the unavailability of stereodefined PS-
ORNs.5-8 Attempts to develop a method to synthesize
stereodefined PS-ORNs to date have only succeeded in the
synthesis of diastereopure 2-3mers6a,b and all-(Rp)-PS-ORN
4-12mers with moderate stereoselectivity.6c,d Chromato-
graphic separations of ORNs containing internucleotidic
(3) (a) Tomari, Y.; Zamore, P. D. Genes DeV. 2005, 19, 517–529. (b)
Bumcrot, D.; Manoharan, M.; Koteliansky, V.; Sah, D. W. Y. Nat. Chem.
Biol. 2006, 2, 711–719
.
(1) Fire, A.; Xu, S.; Montgomery, M. K.; Kostas, S. A.; Driver, S. E.;
Mello, C. C. Nature 1998, 391, 806–811.
(4) (a) Parrish, S.; Fleenor, J.; Xu, S.; Mello, C.; Fire, A. Mol. Cell
2000, 6, 1077–1087. (b) Mescalchin, A.; Detzer, A.; Wecke, M.; Overhoff,
M.; Wuensche, W.; Sczakiel, G. Expert Opin. Biol. Ther. 2007, 7, 1531–
(2) Elbashir, S. M.; Harborth, J.; Lendeckel, W.; Yalcin, A.; Weber,
K.; Tuschl, T. Nature 2001, 411, 494–498.
1538
.
10.1021/ol802910k CCC: $40.75
Published on Web 01/21/2009
2009 American Chemical Society

phosphorothioate diester linkages (PS-linkages)7 or dimer
building blocks to synthesize ORNs with stereodefined PS-
linkages8 have also been reported, though ORNs containing
consecutive stereodefined PS-linkages or fully modified
stereodefined PS-ORNs are unavailable.
tives as monomer units. The synthetic cycle is shown in
Scheme 1. The oxazaphospholidine monomers (Rp)- or (Sp)-
Scheme 1. Synthesis Cycle for Stereodefined PS-ORNs
To overcome this limitation, we developed a method to
synthesize stereodefined PS-ORNs by using ribonucleoside
3′-O-oxazaphospholidine derivatives as monomer units. As
a result, we found that the hybridization affinity of PS-ORNs
to the complementary ORNs was highly dependent on the
configurations of their phosphorus atoms. The results of the
study are described in this paper.
We have reported that a proline-derived bicyclic oxaza-
phospholidine ring affords the corresponding trans-deoxyri-
bonucleoside 3′-O-oxazaphospholidines with diastereomer
ratio (dr) of >99:1. The resultant oxazaphospholidines were
successfully applied in the synthesis of stereodefined oli-
godeoxyribonucleoside phosphorothioates (PS-ODNs) with
dr of >99:1 for each PS-linkage.9 Similarly, diastereopure
trans-ribonucleoside 3′-O-oxazaphospholidine monomers
[(Rp)- and (Sp)-3a-d] for PS-ORNs were stereoselectively
synthesized from 2′-O-TBDMS-protected ribonucleosides
1a-d and 2-chloro-1,3,2-oxazaphospholidines L- and D-2,
which were derived from L- and D-proline, respectively,9 in
modest to good yields (Table 1). trans-(Rp)- and (Sp)-3a-d
3a-d were allowed to react with the 5′-OH of a nucleoside
anchored to a highly cross-linked polystyrene (HCP)11 or a
controlled pore glass (CPG) via a usual succinate linker to
afford phosphite intermediates 4. The phosphites were then
sulfurized with N,N′-dimethylthiuram disulfide (DTD),12 and
unreacted 5′-OH and the secondary amino group derived
from the chiral auxiliary were acylated with trifluoroacetyl-
imidazole (CF3COIm).9 It was confirmed that CF3COIm
capped the remaining 5′-OH quantitatively without causing
any observable desulfurization.13 Then the 5′-O-DMTr group
was removed by treatment with 3% dichloroacetic acid/
CH2Cl2. The cycle was repeated to synthesize the protected
PS-ORNs, which were then deprotected and cleaved from
the support by treatment with concd NH3-EtOH (3:1, v/v)
at rt. The resultant 2′-O-TBDMS-protected PS-ORNs were
desilylated with TBAF.
Table 1. Synthesis of Ribonucleoside 3′-O-Oxazaphospholidine
Monomers 3a-d
a
entry
BPRO
(Rp)- or (Sp)-3
yield (%)
trans/cis
1
2
3
4
5
6
7
8
Ur
(Rp)-3a
(Rp)-3b
(Rp)-3c
(Rp)-3d
(Sp)-3a
(Sp)-3b
(Sp)-3c
(Sp)-3d
51
47
51
41
75
70
72
46
>99:1
>99:1
>99:1
>99:1
>99:1
>99:1
>99:1
>99:1
Cyac
Adac
Guce,pac
Ur
First, (Sp)- and (Rp)-dinucleoside phosphorothioates [(Sp)-
Cyac
and (Rp)-7a-d]6 were synthesized by using the cycle.
Adac
Guce,pac
(6) (a) Lesnikowski, Z. J. Nucleosides Nucleotides 1992, 11, 1621–1638.
(b) Sierzchala, A.; Okruszek, A.; Stec, W. J. J. Org. Chem. 1996, 61, 6713–
6716. (c) Almer, H.; Stawinski, J.; Stro¨mberg, R. J. Chem. Soc., Chem.
Commun. 1994, 1459–1460. (d) Almer, H.; Stawinski, J.; Stro¨mberg, R.
Nucleic Acids Res. 1996, 24, 3811–3820.
a BPRO ) protected nucleobase; Ur ) uracil-1-yl; Cyac ) N4-acetyl-
cytosin-1-yl; Adac ) N6-acetyladenin-9-yl; Guce,pac ) O6-cyanoethyl-
N2-phenoxyacetylguanin-9-yl.
(7) Slim, G.; Gait, M. J. Nucleic Acids Res. 1991, 19, 1183–1188.
(8) Yano, J.; Ohgi, T.; Ueda, T. WO2006/043521-A1, CAN: 144:
450874.
were configurationally assigned based on the 2JPC values of
their oxazaphospholidine rings.9,10
Stereodefined PS-ORNs were manually synthesized
on solid supports using the oxazaphospholidine deriva-
(9) Oka, N.; Yamamoto, M.; Sato, T.; Wada, T. J. Am. Chem. Soc. 2008,
130, 16031–16037.
(10) Hyodo, M.; Ando, H.; Nishitani, H.; Hattori, A.; Hayakawa, H.;
Kataoka, M.; Hayakawa, Y. Eur. J. Org. Chem. 2005, 5216–5223.
(11) McCollum, C.; Andrus, A. Tetrahedron Lett. 1991, 32, 4069–4072.
(12) Song, Q.; Wang, Z.; Sanghvi, Y. S. Nucleosides Nucleotides Nucleic
Acids 2003, 22, 629–633.
(5) (a) Burgers, P. M. J.; Eckstein, F. Biochemistry 1979, 18, 592–596.
(b) Almer, H.; Stawinski, J.; Stro¨mberg, R.; Thelin, M. J. Org. Chem. 1992,
57, 6163–6169. (c) Oivanen, M.; Ora, M.; Almer, H.; Stro¨mberg, R.;
Lo¨nnberg, H. J. Org. Chem. 1995, 60, 5620–5627.
(13) (a) Hel´ınski, J.; Skrzypczy´nski, 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.
968
Org. Lett., Vol. 11, No. 4, 2009

N-(Cyanomethyl)pyrrolidinium triflate (CMPT, 6),14 an
acidic activator with extremely low nucleophilicity, was used
to activate the monomers. Reversed-phase HPLC (RP-HPLC)
analysis of the resultant 2mers showed that the yields
(96-98%) and diastereoselectivity (>99:1) were sufficient
for the synthesis of oligomers (Table 2).15
steric hindrance of the 2′-O-TBDMS groups. Use of a CPG
in the place of an HCP did not improve the results (entries
2 and 3).
To solve the problem of low coupling efficiency, we turned
our attention to the use of nucleophilic azoles, which have
been widely used as activators in the phosphoramidite
chemistry.17 We employed 5-benzylthio-1H-tetrazole (BTT
10),18 4,5-dicyanoimidazole (DCI, 11),19 and benzimidazo-
lium triflate (BIT, 8)20 to synthesize all-(Rp)-ASGSCSU 15
and found that the coupling efficiency was greatly improved
in all of these cases (entries 6-8). In particular, BIT 8 gave
15 with sufficient average coupling efficiency for the
synthesis of long oligomers (entry 8). The RP-HPLC profiles
of the resultant all-(Rp)-2′-O-TBDMS-protected ASGSCSU
4mers showed that the ratios of diastereoisomers obtained
by using 8, 10, or 11 were as high as that obtained by using
CMPT 6.15 To examine the diastereoselectivity of the
synthesis of each PS-linkage promoted by azole-type activa-
tors, (Sp)- and (Rp)-USU were synthesized by using BIT 8.
An RP-HPLC analysis showed that (Sp)- and (Rp)-2′-O-
TBDMS-USU were synthesized with dr of >99:1 (97% yield)
and 96:4 (98% yield), respectively.
Table 2. Synthesis of Dinucleoside Phosphorothioates 7a-da
entry monomer
productb
yield (%)c Rp/Spc
1
2
3
4
5
6
7
8
(Rp)-3a
(Rp)-3b
(Rp)-3c
(Rp)-3d
(Sp)-3a
(Sp)-3b
(Sp)-3c
(Sp)-3d
(Sp)-USU (Sp)-7a
98
97
98
97
98
98
96
98
>1:99
>1:99
>1:99
>1:99
>99:1
>99:1
>99:1
>99:1
(Sp)-CSU
(Sp)-ASU
(Sp)-7b
(Sp)-7c
(Sp)-GSU (Sp)-7d
(Rp)-USU (Rp)-7a
(Rp)-CSU (Rp)-7b
(Rp)-ASU (Rp)-7c
(Rp)-GSU (Rp)-7d
a CMPT 6 was used as activator. b Subscript “S” ) PS-linkage.
c Determined by RP-HPLC.
The fact that azole-type activators also afforded PS-ORNs
with high diastereoselectivity is rather surprising because
highly nucleophilic azole-type activators are known to
epimerize diastereopure acyclic phosphoramidites as well as
monocyclic oxazaphospholidines by repetitive nucleophilic
attacks upon the chiral phosphorus atoms, resulting in the
formation of phosphite triesters of low diastereopurity,14,16,21
whereas less-nucleophilic CMPT 6 is considered to activate
oxazaphospholidines only by N-protonation, without affecting
the chiral phosphorus atoms.14 Although the mechanism of
the highly diastereoselective formation of phosphites pro-
moted by azoles is still not clear, it may be attributed to the
configurational stability of the proline-derived bicyclic ox-
azaphospholidine ring structure.9
All-(Rp)-[US]9U 16 was then synthesized by using BIT 8
or N-(phenyl)imidazolium triflate (PhIMT) 9, which has been
reported as one of the best activators for the synthesis of
unmodified ORNs.20b We found that PhIMT 9 afforded 16
with excellent coupling efficiency (entry 10). All-(Sp)-[US]9U
(17) was also efficiently synthesized by using PhIMT 9 (entry
11). Although oligomers containing phosphodiester linkages
were observed as major byproducts12 due to incomplete
manual sulfurization, the desired all-(Rp)- and all-(Sp)-[US]9U
were generated in good yields and diastereoselectivity.15
After desilylation by TBAF, 16 and 17 were isolated by RP-
HPLC in 6 and 11% yields, respectively, and identified by
MALDI-TOF-MS.15
Given this result, we investigated the synthesis of stereo-
defined PS-ORNs. All-(Sp)- and all-(Rp)-[US]3U and ASG-
SCSU 4mers (Table 3, 12-15) were synthesized by using
Table 3. Synthesis of Stereodefined PS-ORNs 12-17a
entry
oligonucleotideb
activator coupling yield (%)c
1
2
3
4
5
6
7
8
all-(Sp)-[US]3U 12
all-(Rp)-[US]3U 13
all-(Rp)-[US]3U 13
all-(Sp)-ASGSCSU 14
all-(Rp)-ASGSCSU 15 CMPT 6
all-(Rp)-ASGSCSU 15 BTT 10
all-(Rp)-ASGSCSU 15 DCI 11
all-(Rp)-ASGSCSU 15 BIT 8
CMPT 6
CMPT 6
CMPT 6
CMPT 6
94
90
88
90
67
84
90
97
92
97
99
9
10
11
all-(Rp)-[US]9U 16
all-(Rp)-[US]9U 16
all-(Sp)-[US]9U 17
BIT 8
PhIMT 9
PhIMT 9
a HCP was used in entries 1 and 2, and CPG was used in entries 3-11.
b Subscript “S” ) PS-linkage. c Average coupling yields were determined
by RP-HPLC for 4mers 12-15, and by DMTr+ assay for 10mers 16, 17.
CMPT 6. However, an RP-HPLC analysis of the resultant
2′-O-TBDMS-protected 4mers showed that the average
coupling yields were not sufficient for the synthesis of long
oligomers (67-94%, entries 1-5). The coupling efficiency
for (Rp)-PS-linkages was lower than that for (Sp)-PS-
linkages; the difference in coupling efficiency between the
(Rp)- and (Sp)-monomers could be attributed to the chirality
of ribose.16 The lower coupling yields for the synthesis of
PS-ORNs compared to those for PS-ODNs9 are due to the
Stability of the resultant all-(Rp)- and all-(Sp)-[US]9U (16,
17) to snake venom phosphodiesterase (svPDE)22 and
(17) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1992, 48, 2223–2311.
(18) Welz, R.; Mu¨ller, S. Tetrahedron Lett. 2002, 43, 795–797.
(19) Vargeese, C.; Cater, J.; Yegge, J.; Krivjansky, S.; Settle, A.; Kropp,
E.; Peterson, K.; Pieken, W. Nucleic Acids Res. 1998, 26, 1046–1050.
(20) (a) Hayakawa, Y.; Kataoka, M.; Noyori, R. J. Org. Chem. 1996,
61, 7996–7997. (b) Hayakawa, Y.; Kawai, R.; Hirata, A.; Sugimoto, J.;
Kataoka, M.; Sakakura, A.; Hirose, M.; Noyori, R. J. Am. Chem. Soc. 2001,
34, 8165–8176.
(14) Oka, N.; Wada, T.; Saigo, K. J. Am. Chem. Soc. 2003, 125, 8307–
8317.
(15) See the Supporting Information.
(16) Yu, D.; Kandimalla, E. R.; Roskey, A.; Zhao, Q.; Chen, L.; Chen,
J.; Agrawal, S. Bioorg. Med. Chem. 2000, 8, 275–284.
(21) Stec, W. J.; Zon, G. Tetrahedron Lett. 1984, 25, 5279–5282.
Org. Lett., Vol. 11, No. 4, 2009
969

nuclease P1 (nP1)23 was investigated. These enzymes have
been used for configurational assignment of PS-ODNs,
whereas only a limited number of experiments have been
reported for stereodefined PS-ORNs.5-8 According to these
reports, svPDE is (Rp)-specific at least for dinucleoside
phosphorothioates5,6 and ORNs containing PS-linkages,7
whereas nP1 is not always completely (Sp)-specific and
hydrolyzes (Rp)-PS-linkages at a much slower rate than that
for the (Sp)-isomers depending on the nucleobases6b and
lengths24 of ORNs. All-(Rp)- and all-(Sp)-[US]9U (16, 17)
were incubated with svPDE or nP1 over 16 h under standard
conditions. All-(Rp)-[US]9U (16) and all-(Sp)-[US]9U (17)
were completely hydrolyzed by svPDE and nP1, respectively,
whereas 53% of 16 and 23% of 17 were hydrolyzed by nP1
and svPDE, respectively.15 The results indicate that at least
47% of 16 and 77% of 17 were stereochemically homoge-
neous. However, considering that they were completely
hydrolyzed by nP1 and svPDE, respectively, and hydrolysis
by nP1 may not be completely (Sp)-specific, diastereopurity
of these PS-ORNs is likely to be higher than these values.
Next, the effects of the P-configurations of PS-ORNs
on duplex stability were investigated. Thermal denatur-
ating experiments were conducted for the duplexes of all-
(Rp)-[US]9U (16), all-(Sp)-[US]9U (17), stereorandom
[US]9U (18), and natural [UO]9U (19) with the comple-
mentary [AO]9A (20) (subscript “O” ) phosphodiester
linkage). The resultant melting curves showed that the
thermal stability of the duplexes was significantly affected
by the configurations of their phosphorus atoms (Figure
1). The Tm value of all-(Rp)-[US]9U 16-[AO]9A 20 was
28.9 °C (red), which was slightly higher than that of the
natural counterpart [UO]9U 19-[AO]9A 20 (25.7 °C,
purple). In sharp contrast, no distinct Tm value was
observed above 4 °C for the duplex of all-(Sp)-[US]9U 17
with 20 (blue). In addition, a duplex of stereorandom
[US]9U 18 with 20 had a low Tm value (10.3 °C, green).
Thus, the results showed that a backbone consisting of
(Rp)-PS-linkages slightly stabilizes a PS-ORN-ORN
duplex, whereas a backbone consisting of (Sp)-PS-linkages
or stereorandom PS-linkages has a great destabilizing
effect on the duplex.25
Figure 1. UV melting curves of [US]9U-[AO]9A duplexes.
In conclusion, we have succeeded in the first synthesis of
stereodefined PS-ORNs by the oxazaphospholidine method
and demonstrated that the thermal stability of PS-ORN-ORN
duplexes is significantly affected by the configurations of
the phosphorus atoms of PS-ORNs. In particular, the large
destabilizing effect of (Sp)-PS-linkages including those in a
stereorandom PS-backbone on PS-ORN-ORN duplexes is
notable because nonstereoregular PS-linkages have been
widely used to modify ORNs. In contrast, stereodefined (Rp)-
linkages would be useful to modify ORNs without compro-
mising their binding affinity to the complementary RNA
sequences.
Acknowledgment. We thank Professor Kazuhiko Saigo
(University of Tokyo) for helpful suggestions. This research
was supported by grants from the Ministry of Education,
Culture, Sports, Science, and Technology (MEXT) Japan and
the New Energy and Industrial Technology Development
Organization (NEDO) Japan for its Functionalized RNA
Project.
Supporting Information Available: Experimental details,
NMR spectra, and RP-HPLC profiles. This material is
available free of charge via the Internet at http://pubs.acs.org.
(22) (a) Burgers, P. M. J.; Eckstein, F. Proc. Natl. Acad. Sci. U.S.A.
1978, 75, 4798–4801. (b) Bryant, F. R.; Benkovic, S. J. Biochemistry 1979,
18, 2825–2829.
OL802910K
(23) Potter, B. V. L.; Connolly, B. A.; Eckstein, F. Biochemistry 1983,
22, 1369–1377.
(25) Reference 8 has described that ORNs having an all-(Rp)- or all-
(Sp)-PO/PS-alternating backbone, which were synthesized by using dimer
building blocks, had higher and lower affinity to the complementary ORN,
respectively, compared to the unmodified ORN counterpart.
(24) Griffiths, A. D.; Potter, B. V. L.; Eperon, I. C. Nucleic Acids Res.
1987, 15, 4145–4162.
970
Org. Lett., Vol. 11, No. 4, 2009