2'-modified-2-thiothymidine oligonucleotides.

Article abstract of DOI:10.1021/ol0348607

[reaction: see text] Oligonucleotides with novel modifications, 2'-O-[2-(methoxy)ethyl]-2-thiothymidine and 2'-deoxy-2'-fluoro-2-thiothymidine, have been synthesized. These modified oligonucleotides exhibited very high thermal stability when hybridized wi

Full text of DOI:10.1021/ol0348607

ORGANIC
LETTERS
2003
Vol. 5, No. 17
3005-3008
2′-Modified-2-thiothymidine
Oligonucleotides
Kallanthottathil G. Rajeev, Thazha P. Prakash, and Muthiah Manoharan*
Department of Medicinal Chemistry, Isis Pharmaceuticals, Inc.,
Carlsbad, California 92008
mmanoharan@alnylam.com
Received May 18, 2003
ABSTRACT
Oligonucleotides with novel modifications, 2′-O-[2-(methoxy)ethyl]-2-thiothymidine and 2′-deoxy-2′-fluoro-2-thiothymidine, have been synthesized.
These modified oligonucleotides exhibited very high thermal stability when hybridized with complementary RNA. 2-O-(2-Methoxy)ethyl-2-
thiothymidine modified oligonucleotide phosphodiesters showed enhanced resistance toward nucleases (t1/2 > 24 h), but 2′-deoxy-2′-fluoro-
2-thiothymidine modified oligonucleotide phosphodiesters showed limited stablity to nucleotytic degradation.
Oligonucleotides that form stable duplexes with RNA and
that are resistant to digestion by nucleases are needed for
antisense applications.1 In addition, to be therapeutically
useful, the synthesis of the modifications and of the oligo-
nucleotides must be straightforward and economical. 2-Thio-
uridine residues are known to adopt a C3′-endo sugar pucker2
and oligonucleotides with this modification form thermo-
dynamically stable duplexes with complementary RNA.3
Furthermore, due to steric hindrance and the weaker H-
bonding ability of sulfur relative to oxygen, 2-thiouridine
destabilizes U-G wobble2a and U-2-amino-A base pairs4
compared to uridine. As a result, a 2-thiouridine exhibits
excellent hybridization specificity. Recently, Sekine et al.
have reported the synthesis of 2′-O-methyl-2-thiouridine
modified oligonucleotides5 and have demonstrated better
selectivity of 2′-O-methyl-2-thiouridine for adenine than
guanine relative to unmodified uridine.
The 2′-O-[2-(methoxy)ethyl] (2′-O-MOE)-modified gap-
mer oligonucleotides have emerged as a promising choice
for antisense applications.1,6 2′-O-MOE-modified nucleotides
prefer to adopt the C3′-endo sugar pucker favorable for RNA
binding, due to an extended gauche effect and hydration
involving the 2′-O-MOE side chain.6c Another 2′-modifica-
tion that favors a C3′-endo sugar pucker and A-type confor-
mation when hybridized with RNA is 2′-deoxy-2′-fluoro (2′-
F).7 Though the 2′-deoxy-2′-fluoro ribo modification containing
oligonucleotides favors A-form helices while binding to
(1) (a) Cook P. D. Antisense Research and Application. In Antisense
Medicnal Chemistry; Crooke, S. T., Ed.; Springer-Verlag: New York, 1998;
Vol. 131, pp 51-101. (b) Manoharan, M. Biochim. Biophys. Acta 1999,
1489, 117-130.
(2) (a) Testa, S. M.; Disney, M. D.; Turner, D. H.; Kierzek, R.
Biochemistry 1999, 38, 16655-16662. (b) Sierzputowska-Gracz, H.;
Sochacka, E.; Malkiewicz, A.; Kuo, K.; Gehrke, C. W.; Agris, P. F. J. Am.
Chem. Soc. 1987, 109, 7171-7177. (c) Kawai, G.; Yamamoto, Y.;
Kamimura, T.; Masegi, T.; Sekine, M.; Hata, T.; Iimori, T.; Watanabe, T.;
Miyazawa, T.; Yokoyama, S. Biochemistry 1992, 31, 1040-1046.
(3) Kumar, R. K.; Davis, D. R. Nucleic Acids Res. 1997, 25, 1272-
1280.
(5) Shohda, K.; Okamoto, I.; Wada, T.; Seio, K.; Sekine, M. Bioorg.
Med. Chem. Lett. 2000, 10, 1795-1798.
(6) (a) Martin, P. HelV. Chim. Acta 1995, 78, 486-504. (b) Monia, B.
P.; Lesnik, E. A.; Gonzalez, C.; Lima, W. F.; McGee, D.; Guinosso, C. J.;
Kawasaki, A. M.; Cook, P. D.; Freier, S. M. J. Biol. Chem. 1993, 268,
14514-14522. (c) Teplova, M.; Minasov, G.; Tereshko, V.; Inamati, G.
B.; Cook, P. D.; Manoharan, M.; Egli, M. Nat. Struct. Biol. 1999, 6, 535-
539.
(4) Kutyavin, I. V.; Rhinehart, R. L.; Lukhtanov, E. A.; Gorn, V. V.;
Meyer, R. B., Jr.; Gamper, H. B., Jr. Biochemistry 1996, 35, 11170-11176.
10.1021/ol0348607 CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/02/2003

complementary nucleic acids, its resistance to nucleolytic
degradation was very poor.7a On the other hand, the 2′-O-
MOE side chain provided better resistance to nucleolytic
degradation compared to the corresponding unmodified 2′-
deoxy sugar-containing oligonucleotides.
Scheme 1a
We envisioned that oligonucleotides modified with 2-thio-
uridine and a 5-methyl substituent along with the use of either
2′-O-MOE (1a, Figure 1) or 2′-F (1b, Figure 1) sugar
Figure 1. 2′-O-MOE-2-thiothymidine (1a) and 2′-deoxy-2′-F-2-
thiothymidine (1b).
modifications would significantly improve the hybridization
affinity toward RNA relative to an unmodified DNA oligo-
nucleotide and the use of 2′-O-MOE should provide resis-
tance to nucleolytic degradation.
Here we report the facile synthesis of 5′-O-DMT-2′-O-
MOE-5-methyl-2-thiouridine-3′-phosphoramidite (7a), 5′-O-
DMT-2′-deoxy-2′-fluoro-5-methyl-2-thiouridine-3′-phosphor-
amidite (7b), and the corresponding solid supports (9a and
9b) (Scheme 1). Also reported are incorporation of these
novel nucleoside modifications into oligonucleotides, thermal
stability of the modified oligonucleotide duplexes with
complementary DNA and RNA, and resistance to snake
venom phosphodiesterase (SVPD) digestion relative to
unmodified DNA.
a Reagents and conditions: (i) (a) HOAc/H2O (4:1), rt, (b) MsCl/
Py-dichloromethane (1:1), -20 °C; (ii) NaHCO3 (2.5 mol equiv)/
EtOH, reflux, 36 h; (iii) DMT-Cl, DMAP/Py, rt; (iv) for 6a: H2S,
TMG (10 mol equiv)/Py, rt, 72 h; for 6b: H2S, Py, 60 °C, 72 h;
(v) 2-cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopro-
pylammonium tetrazolide, CH3CN, rt; (vi) succinic anhydride, 1,2-
dichloroethane, DMAP, (C2H5)3N, rt; (vii) 2-(1H-benzotriazole-1-
yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4-methylmor-
pholine, DMF, aminoalkyl controlled pore glass (CPG), rt.
Syntheses of phosphoramidites 7a and 7b are shown in
Scheme 1. Compound 3a was prepared from 5′-O-DMT-2′-
O-MOE-thymidine according to standard procedures.8 The
methanesulfonate 3a was refluxed in absolute ethanol in the
presence of 2.5 molar equiv of anhydrous NaHCO3 under
argon atmosphere for 36 h to afford 2′-O-MOE-2-O-
ethylthymidine (4a) in 75% yield.9 Reaction of compound
4a with DMT-Cl in anhydrous pyridine in the presence of
DMAP afforded 5′-O-DMT-2′-O-MOE-2-O-ethylthymidine
(5a) in 90% yield. Treatment of compound 5a with H2S and
1,1,3,3-tetramethylguanidine (TMG) in anhydrous pyridine
gave 5′-O-DMT-2′-O-MOE-5-methyl-2-thiouridine (6a) in
85% yield.10 Flash column chromatography with a 1:1
mixture of ethyl acetate and hexane was used to remove
TMG-H2S salt contamination from the product. Phosphity-
lation of compound 6a gave the desired phosphoramidite 7a
in over 90% yield.11
Compound 3b was prepared in quantitative yield from
compound 2b according to standard procedures.8 Treatment
of compound 3b with anhydrous NaHCO3 in ethanol under
reflux gave a mixture of products with close Rf values, not
observed when 4a was synthesized from compound 3a. This
may be due to side reactions such as 2,2′-anhydro formation
in the presence of NaHCO3 and subsequent ring opening at
elevated temperature in ethanol.12 The mixture of products
was treated with DMT-Cl in pyridine in the presence of a
catalytic amount of DMAP to obtain compound 5b in 33%
over-all isolated yield from compound 3b. Compound 5b
was heated with saturated H2S in anhydrous pyridine at 60
°C for 72 h to afford compound 6b in 53% yield. TMG was
excluded to avoid the possible formation of the 2,2′-anhydro
(7) (a) Kawasaki, A. M.; Casper, M. D.; Freier, S. M.; Lesnik, E. A.;
Zounes, M. C.; Cummins, L. L.; Gonzalez, C.; Cook, P. D. J. Med. Chem.
1993, 36, 831-841. (b) Ikeda, H.; Fernandez, R.; Wilk, A.; Barchi, J. J.,
Jr.; Huang, X.; Marquez, V. E. Nucleic Acids Res. 1998, 26, 2237-2244.
(8) See Supporting Information for details.
(9) Huang, J. J.; Ragouzeos, A.; Rideout, J. L. J. Heterocycl. Chem. 1995,
32, 691-695.
(10) Reese, C. B.; Varaprasad, C. V. N. S. J. Chem. Soc., Perkin Trans.
1 1994, 189-195.
(11) Prakash, T. P.; Kawasaki, A. M.; Fraser, A. S.; Vasquez, G.;
Manoharan, M. J. Org. Chem. 2002, 67, 357-369.
(12) Krug, A.; Oretskaya, T. S.; Volkov, E. M.; Cech, D.; Shabarova,
Z. A.; Rosenthal, A. Nucleosides Nucleotides 1989, 8, 1473-1483.
3006
Org. Lett., Vol. 5, No. 17, 2003

derivative of 6b. At room temperature the thiolation reaction
of compound 5b with H2S in anhydrous pyridine was very
slow. Phosphitylation of compound 6b gave compound 7b
in 60% yield. Compounds 6a and 6b were converted into
3-O-succinyl derivatives, 8a and 8b, and loaded on to amino
alkyl controlled pore glass (CPG, Scheme 1) to obtain
supports 9a (loading capacity: 56.96 µmol/g) and 9b
(loading capacity: 58.31 µmol/g).11
Commercially available phosphoramidites were used for
oligonucleotide synthesis unless otherwise specified. Phos-
phoramidite 7a was incorporated into oligonucleotides 10,
17 (Table 1), and 23 (Figure 2a).8 Oxidation of the inter-
Table 1. Effect of the 2′-O-MOE-2-thiothymidine (X),
2′-Deoxy-2′-fluoro-2-thiothymidine (Y),
2′-Deoxy-2-thiothymidine (Z), 2′-O-MOE-thymidine (t), and
2′-Deoxy-2′-flourouridine (u) Modifications on Stability of
Duplexes with Complementary RNA
seq
no.
Tm,8
∆Tm,16
(°C)/unit
sequence 5′-3′
°C
10
11
12
13
14
15
16
17
18
19
20
21
22
d(XCCAGGXGXCCGCAXC)
d(YCCAGGYGYCCGCAYC)
d(ZCCAGGZGZCCGCAZC)
d(tCCAGGtGtCCGCAtC)
d(TCCAGGTGTCCGCATC)
d(uCCAGGuGuCCGCAuC)17
d(UCCAGGUGUCCGCAUC)17
d(GCGXXXXXXXXXXGCG)
d(GCGZZZZZZZZZZGCG)
d(GCGttttttttttGCG)
74.1
77.3
65.8
66.0
62.4
64.8
60.0
82.9
65.4
60.5
48.5
76.9
61.8
+2.9
+3.7
+0.9
+0.9
+0.6
-0.6
+3.4
+1.7
+1.2
Figure 2. The disappearance of oligonucleotides 23-27 in the
presence of SVPD as a function of time: 23, 5′-T15X4-3′ (X ) 2′-
O-MOE-2-thiothymidine); 24, 5′-T15t4-3′ (t ) 2′-O-MOE-thymi-
dine); 25, T20; 26, 5′-T15Y4-3′ (Y ) 2′-deoxy-2′-fluoro-2-
thiothymidine); 27, 5′-T15Z4-3′ (Z ) 2′-deoxy-2-thiothymidine);
each point is an average of two experiments.8
d(GCGTTTTTTTTTTGCG)
d(CTCGTACYYYYCCGGTCC)
d(CTCGTACTTTTCCGGTCC)
+3.8
freshly prepared 0.05 M K2CO3 in methanol to complete the
deprotection.15 HPLC purification, detritylation, and desalting
gave 11, 21 (5-10% isolated yield), and 26 (30% isolated
yield).
nucleotide phosphite to the phosphate was carried out with
tert-butylhydroperoxide/acetonitrile/water (10:87:3) with a
10 min oxidation wait period.13 The oligonucleotides were
characterized by ES-MS, capillary gel electrophoresis, and
HPLC. Oligonucleotides containing 2′-deoxy-2-thiothymi-
dine (12 and 18, Table 1, and 27, Figure 2b) were synthesized
according to the same protocols with use of commercial
phosphoramidite 7c obtained from Glen Research Inc.
Thermal denaturation studies of duplexes formed between
oligonucleotides 10-22 and complementary RNA are sum-
marized in Table 1. Incorporation of 2′-O-MOE-2-thiothy-
midine (10 and 17, Table 1) provided a melting temperature
(Tm) enhancement of 2.9-3.4 and 2.0-2.2 °C per modifica-
tion compared to 2′-deoxy oligonucleotides (14 and 20) and
2′-O-MOE oligonucleotides (13 and 19), respectively. The
Tm enhancement observed with oligonucleotides containing
singly modified 2′-deoxy-2-thiothymidine (12 and 18) or 2′-
O-MOE-thymidine (13 and 19) hybridized to complementary
RNA was between 0.9 and 1.7 °C per modification when
compared to the 2′-deoxyoligonucleotides (14 and 20).
Earlier studies showed that both 2′-deoxy-2-thiothymidine2
and 2′-O-MOE-thymidine6c favor the C3′-endo sugar con-
formation in the oligonucleotides and thus contribute to the
stability of duplexes formed with complementary RNA.
Replacement of thymidine with 2-thiothymidine, due to the
The synthesis of oligonucleotides 11, 21 (Table 1), and
26 (Figure 2b) was first attempted with phosphoramidite 7b
and standard phosphoramidites of A, G, and C. However,
deprotection with aqueous ammonia gave a complex mixture
of products as evident from ES-MS analysis. Therefore,
2-cyanoethyl phosphoramidites and solid supports with N6-
phenoxyacetyl-A, N2-(4-isopropyl-phenoxyacetyl)-G, and N4-
acetyl-C (Glen Research Inc.) were used for the synthesis.
The coupling and oxidation protocols were the same as those
used for the synthesis of oligonucleotide 10. The solid-
support bound oligonucleotides were treated with 1 M
piperidine in anhydrous acetonitrile, to remove the cyanoethyl
protection from the phosphate,14 and subsequently with
(14) Guzaev, A. P.; Manoharan, M. J. Org. Chem. 2001, 66, 1798-
1804.
(15) Kuijpers, W. H.; Huskens, J.; Koole, L H.; van Boeckel, C. A.
Nucleic Acids Res. 1990, 18, 5197-5205.
(13) Kumar, R. K.; Davis, D. R. J. Org. Chem. 1995, 60, 7726-7727.
Org. Lett., Vol. 5, No. 17, 2003
3007

intrinsic C3′-endo conformational preference of the later, does
not allow binding of human RNase H to RNA-DNA hybrid
containing 2-thiothymidine in the DNA part.18 Thus the
advantage of using 2-thiothymidine (as 2′-deoxy) is lost in
antisense applications due to its inability to recruit RNase
H. This makes the combination of a 2′-modification and the
2-thiothymidine preferred. When these two modifications
were combined in one nucleoside residue, as in 2′-O-MOE-
2-thiothymidine, the Tm enhancement for the duplex formed
with complementary RNA was additive.
The oligonucleotide with 2′-deoxy-2′-fluorouridine (15),
which lacks a 5-methyl group relative to 2′-fluoro-thymidine,
stabilized the duplex by 0.6 °C per modification compared
to DNA control 14.17 The loss in thermal stability due to
the lack of a methyl group was 0.6 °C per modification
(16),17 and therefore, one would expect an increase of about
1.2 °C per modification for an oligonucleotide modified with
2′-deoxy-2′-fluorothymidine. The oligonucleotides 11 and 21
containing the 2′-deoxy-2′-fluoro-2-thiothymidine increased
thermal stability of duplex with complementary RNA up to
3.8 °C per modification (Table 1) compared to the unmodi-
fied controls (14 and 22). These results indicate the syner-
gistic stereoelectronic effect of 2-thio and 2′-O-MOE or 2′-F
modifications on enhancement of thermal stability against
complementary RNA. This is in contrast to the 2′-O-methyl-
2-thiouridine modification where no additive effect on
thermal stability was observed.5
These data suggest an RNA selective preorganization and
hybridization of the 2′-O-MOE-2-thiothymidine and 2′-
deoxy-2-thiothymidine modified oligonucleotides.
The susceptibility of oligonucleotides containing novel
modifications to nuclease digestion was evaluated by using
a standard assay with SVPD.19 The enzymatic hydrolysis of
phosphodiester oligonucleotides 23-27, capped at the 3′-
terminus with four modified nucleoside residues, was evalu-
ated. The plot of the time-dependent disappearance of the
novel oligonucleotides and their 2′-deoxy and 2′-O-MOE
analogues is shown in Figure 2. The half-life of the
oligonucleotide with 2′-O-MOE-2-thiothymidine modifica-
tion (23) was over 24 h and showed better resistance to
SVPD than the 2′-O-MOE-thymidine modified oligonucle-
otide (24, Figure 2A). However, the 2′-deoxy-2′-fluoro-2-
thiothymidine (26) and the 2′-deoxy-2-thiothymidine (27)
modified oligonucleotides were degraded as rapidly as the
2′-deoxy-oligonucleotide (half-lives between 1.5 and 5 min,
Figure 2B). Thus the replacement of thymine with 2-thio-
thymine did not improve the nuclease resistance of oligo-
nucleotides. On the other hand, when the 2′-O-MOE-2-
thiothymidine (Figure 2A) and the 2′-deoxy-2-thiothymidine
modification (Figure 2B) are compared, it is clear that a
combination of 2′-O-MOE ribose sugar and 2-thio-5-me-
thyluracil in a modified pyrimidine nucleoside provides
enhanced nuclease resistance compared to the corresponding
singly modified nucleoside when incorporated into oligo-
nucleotides..
In conclusion, facile syntheses of 2′-O-MOE-2-thiothy-
midine and 2′-F-2-thiothymidine and their incorporation into
oligonucleotides were demonstrated. The additive effect of
the 2′-O-MOE or 2′-F and the 2-thiothymine modifications
on thermal stability suggests that there is a synergistic
stereoelectronic effect of sugar and base modifications in
preorganizing the modified oligonucleotides to form an
A-type helix. High binding affinity, nuclease stability, and
expected favorable pharmacokinetic properties (due to sulfur
and 2′-O-MOE modifications) warrant further evaluation of
2′-O-MOE-2-thiothymidine modified oligonucleotides for
antisense applications. The remarkably high binding affinity
of 2′-F-2-thiothymidine makes it a good candidate for
hybridization-dependent nontherapeutic applications as well.
Unlike the 2′-O-MOE-thymidine oligonucleotide that
destabilizes a duplex with complementary DNA (19, Table
2) compared to the control oligonucleotide 20, both 2′-O-
Table 2. Effect of the 2′-O-MOE-2-thiothymidine (X),
2′-Deoxy-2-thiothymidine (Z), and 2′-O-MOE-thymidine (t)
Modifications on Stability of Duplexes with Complementary
DNA
seq
no.
Tm,8
∆Tm,
°C/modification
modification
°C
17
18
19
20
X
Z
t
73.50
62.00
42.40
54.20
+1.9
+0.8
-1.2
parent DNA
Acknowledgment. We thank Dr. B. S. Ross for helpful
discussions, Dr. E. A. Lesnik for Tm studies, and S. Owens
for help with the nuclease stability assay.
MOE-2-thiothymidine (17, Table 2) and 2′-deoxy-2-thio-
thymidine (18, Table 2) oligonucleotides stabilize the
duplexes with complementary DNA. However, when com-
pared to the duplex with RNA (17 and 18, ∆Tm ) 3.4 and
1.7 °C per modification, respectively, Table 1), the stabiliza-
tion of the duplex with DNA (17 and 18, ∆Tm ) 1.9 and
0.8 °C per modification, respectively, Table 2) was less.
Supporting Information Available: Experimental pro-
cedures and spectroscopic data for compounds 2a-8a, 2b-
8b, 10, 11, 17, 21, 23, 26, and 27; procedures used for Tm
and nuclease studies. This material is available free of charge
via the Internet at http://pubs.acs.org.
OL0348607
(16) ∆Tm per modification with respect to thymidine.
(17) Freier, S. M.; Altmann, K.-H. Nucleic Acids Res. 1997, 25, 4429-
4443.
(19) Cummins, L. L.; Owens, S. R.; Risen, L. M.; Lesnik, E. A.; Freier,
S. M.; McGee, D.; Guinosso, C. J.; Cook, P. D. Nucleic Acids Res. 1995,
23, 2019-2024.
(18) Unpublished results (Isis Pharmaceuticals Inc).
3008
Org. Lett., Vol. 5, No. 17, 2003