Synthesis of 3′-S-phosphorothiolate oligonucleotides for their potential use in RNA interference

Article abstract of DOI:10.1080/15257770701490753

The potency of RNA interference (RNAi) undoubtedly can be improved through chemical modifications to the small interfering RNAs (siRNA). By incorporation of the 3′-S-phosphorothiolate modification into strands of RNA, it is hoped that specific regions of a siRNA duplex can be stabilised to enhance the target binding affinity of a selected antisense strand into the activated RNA-induced silencing complex (RISC*). Oligonucleotides composed entirely of this modification are desirable so unconventional 5′ → 3′synthesis is investigated, with initial solution-phase testing proving successful. The phosphoroamidite monomer required for solid-phase synthesis has also been produced. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770701490753

Nucleosides, Nucleotides, and Nucleic Acids, 26:709–712, 2007
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701490753
ꢀ
SYNTHESIS OF 3 -S-PHOSPHOROTHIOLATE OLIGONUCLEOTIDES
FOR THEIR POTENTIAL USE IN RNA INTERFERENCE
James W. Gaynor, John Brazier, and Rick Cosstick
2
Department of Chemistry,
University of Liverpool, Liverpool, United Kingdom
2
The potency of RNA interference (RNAi) undoubtedly can be improved through chemical modifi-
ꢁ
cations to the small interfering RNAs (siRNA). By incorporation of the 3 -S-phosphorothiolate mod-
ification into strands of RNA, it is hoped that specific regions of a siRNA duplex can be stabilised
to enhance the target binding affinity of a selected antisense strand into the activated RNA-induced
silencing complex (RISC*). Oligonucleotides composed entirely of this modification are desirable so
ꢁ
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unconventional 5 → 3 synthesis is investigated, with initial solution-phase testing proving suc-
cessful. The phosphoroamidite monomer required for solid-phase synthesis has also been produced.
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Keywords RNA Interference; siRNA; phosphorothiolate; reverse synthesis; 5 -3
INTRODUCTION
RNA interference (RNAi) was discovered in 1998 by Fire and Mello
[1]
et al.,
and is a potent method for gene knockout with potential use
in therapeutics. During the RNAi process, the double-stranded small
interfering RNAs (siRNAs) are incorporated into an RNA-induced silencing
∗
complex (RISC), which is activated (RISC ) by ATP-driven unwinding of
the siRNA. It is established that the strand selected for assembly into RISC*
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is the one whose 5 -end is less tightly paired to its complement. Various
chemical modifications have been investigated in the siRNA duplex to
improve the potency of RNAi.[
2]
The sugar conformations of DNA and RNA differ, with RNA preferring
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the C3 -endo (north) conformer and DNA the C2 -endo (south) conformer.
1
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H NMR studies of the 3 -S-phosphorothiolate linkage (Figure 1) by Beevers
et al. has shown there is a shift to the north pucker of the sugar directly
attached to the sulphur atom. Hence, the 3 -S-phosphorothiolate modifi-
[3]
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cation is an excellent mimic of natural RNA. It also has been observed that
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the deoxyribose pucker of the 3 -S-phosphorothiolate actually populates
[4]
the north conformation to a greater extent than natural RNA. For these
Address correspondence to J. Gaynor, Department of Chemistry, Crown Street, Liverpool,
Merseyside L69 7ZD, England. E-mail: j.w.gaynor@liverpool.ac.uk
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J. W. Gaynor et al.
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FIGURE 1 3 -S-phosphorothiolate linkage.
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reasons, it is expected that integration of the 3 -S-phosphorothiolate linkage
into RNA will enhance duplex stability of a modified RNA strand to its
complement.
RESULTS AND DISCUSSION
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We are currently investigating the incorporation of 3 -S-phos-
phorothiolate analogues into the phosphodiester backbone of RNA to im-
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prove efficacy of RNAi. For the incorporation of the 3 -S-phosphorothiolate
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modification, the phosphorothioamidite of 2 -deoxyuridine has been syn-
thesised (A, Scheme 1). This currently is being integrated into the RNA
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sequence 5 -GCGU GCG-3 (U10). Once purified, UV thermal melting
10
(
T ) studies of the RNA:RNA duplexes containing the modified U10 se-
m
quence and its complement will be measured, with an increase in Tm ex-
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pected. Stabilisation of specific regions of a siRNA duplex using the 3 -
S-phosphorothiolate could then be used to direct the required antisense
∗
strand into the RISC complex of a specific gene target.
SCHEME 1 i) DMT-Cl (1.1eq), pyridine, RT, 3h; ii) DIAD (1.5eq), PPh3 (1.5eq), EtOAc, RT,
◦
overnight; iii) NaSCOPh (10eq), DMF, 110 C, 3h; iv) 10M NaOH in EtOH, then KH2PO4;
v) P(NPr2)2(OCH2CH2CN), Tetrazole, CH2Cl2, RT, overnight.

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3
-S-Phosphorothiolate Oligonucleotides
711
Standard solid-phase oligonucleotide synthesis generally occurs in a
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3
→5 manner. However, there is a limitation to the number of 3 -S-
phosphorothiolate modifications which can be incorporated into oligonu-
cleotides using this method due to the reduced reactivity of the phospho-
rothioamidite. Conversely, oligonucleotides can be very efficiently synthe-
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sised in the reverse direction (5 →3 ) by using monomers functionalised
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[5]
with a 5 -amidite and a 3 -DMT protected hydroxyl group. Application of
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this approach to the 3 -S-phosphorothiolate linkage involves nucleophilic
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attack of a 3 -thiol on a reactive 5 -O-phosphoroamidite.
Initial tests were successfully undertaken using solution-phase condi-
tions by synthesising an unmodified TT dimer (E, Scheme 2,). For the syn-
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thesis of a 3 -S-phosphorothiolate modification, the UsT dimer (F, Scheme
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2
) was successfully prepared from the 3 -thiol of uridine (C, Scheme 3)
and 5 -amidite of thymidine (D, Scheme 4) using the same conditions for
the TT dimer. For application for solid-phase synthesis, the appropriate 5 -
phosphoroamidite monomer with a 3 -DMT-protected thiol was synthesised
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successfully in a reasonable yield (G, Scheme 5). Initial solid phase synthesis
is currently ongoing and it is hoped that coupling yields will be enhanced so
that oligonucleotides composed entirely of the phosphorothiolates can be
synthesised.
SCHEME 2 ix) a. Tetrazole, MeCN, Under Argon; b. 4% (w/v) solution of I2 in pyridine/water/THF
◦
(
1:1:8); c. NH3, 50 C, Overnight; d. NEt3.3HF, RT, 24 h.
SCHEME 3 vi) TBDPS-Cl or TBDMS-Cl (1.1eq), Imidazole, DMF, RT, overnight; ii-iv) See Scheme 1.
SCHEME 4 vii) a. DMT-Cl (1.1eq), pyridine, RT, 3h; b. TBDMS-Cl, Imidazole, DMF, RT, 2h; viii) 80%
AcOH, RT, overnight; v) See Scheme 1.

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J. W. Gaynor et al.
SCHEME 5 ix) a. Tetrazole, MeCN, Under Argon; b. 4% (w/v) solution of I2 in pyride/water/THF
◦
(
1:1:8); c, NH3, 50 C, Overnight; d. NHt3.3HF, RT, 24H.
CONCLUSIONS
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The phosphorothioamidite of 2 -deoxyuridine has been synthesised in
moderate yield and is being incorporated into strands of RNA. It is expected
to result in increased binding affinity in an RNA/RNA duplex. Stabilisation
ꢁ
of specific regions of a siRNA duplex using the 3 -S-phosphorothiolate could
∗
then be used to direct the required antisense strand into the RISC of a spe-
cific gene target. Incorporation of the 3 -S-phosphorothiolate modification
into TT and UsT dimers via solution-phase 5 →3 reverse synthesis was suc-
cessfully achieved together with the phosphoroamidite monomer required
for solid-phase synthesis.
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REFERENCES
1.
Fire, A.; Xu, S.Q.; Montgomery, M.K.; Kostas, S.A.; Driver, S.E.; Mello, C.C. Potent and specific
genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998, 391, 806–
811.
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Manoharan, M. RNA interference and chemically modified small interfering RNAs. Curr. Opin.
Chem. Biol, 2004, 8, 570–579.
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3
Beevers, A.P.G.; Fettes, K.J.; O Neil, I.A.; Roberts, S.M.; Arnold, J.R.P.; Cosstick, R.; Fisher, J. Probing
ꢁ
the effect of a 3 -S-phosphorothiolate link on the conformation of a DNA: RNA hybrid; implications
for antisense drug design. Chem. Comm. 2002, 1458–1459.
4
.
.
Beevers, A.P.G.; Witch, E.M.; Jones, B.; Cosstick, R.; Arnold, J.R.P.; Fisher, J. Conformational analysis
of 3 -S-PO3-linked ribo- and deoxyribodinucleoside monophosphates. Magn. Res. Chem. 1999, 37,
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8
14–820.
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5
Wagner, T.; Pfleiderer, W. Synthesis of 2 -deoxyribonucleoside 5 -phosphoramidites: New building
blocks for the inverse (5 -3 )-oligonucleotide approach. Helv. Chimica Acta 2000, 83, 2023–2035.
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