2'-O-[2-[N,N-(dialkyl)aminooxy]ethyl]-modified antisense oligonucleotides.

Article abstract of DOI:10.1021/ol006555g

[structure] Oligonucleotides with two novel modifications, 2'-O-2-[N, N-(dimethyl)aminooxy]ethyl (2'-O-DMAOE) and 2'-O-2-[N, N-(diethyl)aminooxy]ethyl (2'-O-DEAOE), have been synthesized. These modifications exhibit high binding affinity to target RNA (and not to DNA) and enhance the nuclease stability of oligonucleotides considerably with t(1/2) > 24 h as a phosphodiester.

Full text of DOI:10.1021/ol006555g

ORGANIC
LETTERS
2000
Vol. 2, No. 25
3995-3998
2′-O-{2-[N,N-(Dialkyl)aminooxy]ethyl}-
Modified Antisense Oligonucleotides
Thazha P. Prakash, Muthiah Manoharan,* Andrew M. Kawasaki, Elena A. Lesnik,
Stephen R. Owens, and Guillermo Vasquez
Department of Medicinal Chemistry, Isis Pharmaceuticals Inc., 2292 Faraday AVenue,
Carlsbad, California 92008
mmanoharan@isisph.com
Received September 6, 2000
ABSTRACT
Oligonucleotides with two novel modifications, 2′-O-{2-[N,N-(dimethyl)aminooxy]ethyl} (2′-O-DMAOE) and 2′-O-{2-[N,N-(diethyl)aminooxy]ethyl}
(2′-O-DEAOE), have been synthesized. These modifications exhibit high binding affinity to target RNA (and not to DNA) and enhance the
nuclease stability of oligonucleotides considerably with t1/2 > 24 h as a phosphodiester.
An ideal antisense oligonucleotide should have high binding
affinity to the target RNA, high nuclease resistance, should
bind selectively to transport proteins, and should be cell
permeable in vivo.1 2′-O-Modified oligonucleotides2,3 used
with the “gapmer” technology2,3 have emerged as leading
second generation candidates for clinical applications. Among
the 2′-modifications reported in the literature, the 2′-O-(2-
methoxyethyl)4 modification, abbreviated as 2′-O-MOE,
offers a 2 °C increase in melting temperature (Tm) per
modification as a diester (2′-O-MOE/PO) compared to the
first generation 2′-oligodeoxyribonucleotide phosphorothioate
(2′-H/PS) compounds. This modification as a phosphodiester
linkage exhibits nuclease resistance (measured as the half-
life of the full-length oligonucleotide, t1/2) at approximately
the same level as a 2′-deoxyphosphorothioate modification.
To improve upon the 2′-O-MOE modification, we recently
reported5 the synthesis and properties of the 2′-O-(2-
aminooxyethyl) modification (2′-O-AOE), the pseudoisostere
of the 2′-O-MOE modification. Unfortunately, due to high
reactivity of 2′-O-AOE, several modified residues cannot be
conveniently incorporated into antisense oligonucleotides.
However, this modification is extremely valuable as a
conjugation site for various ligands.6
(1) (a) Crooke, S. T. In Basic Principles of Antisense Therapeutics.
Handbook of Experimental Pharmacology 131: Antisense Research and
Application; Crooke, S. T., Ed.; Springer: Berlin, 1998; pp 1-50. (b)
Phillips, M. I., Ed. Methods Enzymol. 2000, 313, 580; 2000, 314, 646
(Antisense Technology, Parts A and B).
(2) (a) Cook P. D. In Antisense Medicinal Chemistry. Antisense Research
and Application; Crooke, S. T., Ed.; Springer-Verlag: New York, 1998;
Vol. 131, pp 51-101. (b) Cook, P. D. Nucleosides Nucleotides 1999, 18,
1141-1162. (c) Prakash, T. P.; Manoharan, M.; Fraser, A. S.; Kawasaki,
A. M.; Lesnik, E. A.; Owens, S. R. Tetrahedron Lett. 2000, 41, 4855-
4859, and references cited.
Here we report the synthesis of two dialkyl derivatives of
2′-O-AOE, 2′-O-{2-[N,N-(dimethyl)aminooxy]ethyl} (2′-O-
DMAOE) and 2′-O-{2-[N,N-(diethyl)aminooxy]ethyl} (2′-
O-DEAOE) modified oligonucleotides (Figure 1), their
(3) (a) Manoharan, M. Biochim. Biophys. Acta 1999, 1489, 117-130.
(b) Monia, B. P.; Lesnik, E. A.; Gonzalez, C.; Lima, W. F.; Guinosso, C.
J.; Kawasaki, A. M.; Cook, P. D.; Freier, S. M. J. Biol. Chem. 1993, 268,
14514-14522.
(5) Kawasaki, A. M.; Casper, M. D.; Prakash, T. P.; Manalili, S.; Sasmor,
H.; Manoharan, M.; Cook, P. D. Tetrahedron Lett. 1999, 40, 661-664.
(6) Salo, H.; Virta, P.; Hakala, H.; Prakash, T. P.; Kawasaki, A. M.;
Manoharan, M.; Lonnberg, H. Bioconjugate Chem. 1999, 10, 815-823.
(4) Martin, P. HelV. Chim. Acta 1995, 78, 486-504.
10.1021/ol006555g CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/16/2000

conditions with Ph3P, N-hydroxyphthalimide, and diethyl-
azodicarboxylate (DEAD) in THF, gave the phthalimido
derivative 4 in 86% yield. Deprotection of the phthalimido
group with N-methylhydrazine (NMH) at 0 °C gave the
aminooxy derivative that was used without purification for
the next step (Scheme 2). Treatment of the aminooxy
Figure 1.
Scheme 2a
affinity toward target RNA, and nuclease stability. These
two modifications were selected for the following reasons:
First, we envisaged that these modifications may retain the
“gauche effect” 7 within the side chain maintaining the C3′-
endo sugar pucker shown by the 2′-O-MOE modification.8
This should maintain the stable hybridization to the RNA
target observed with the 2′-O-MOE modification. Second,
these dialkyl modifications would be more lipophilic than
the 2′-O-AOE and 2′-O-MOE modifications, a property that
affects the protein binding and cellular permeation capabili-
ties of antisense oligonucleotides.
For initial evaluations, we synthesized 2′-O-DMAOE and
2′-O-DEAOE modified 5-methyluridine phosphoramidites
(13 and 14) and incorporated these phosphoramidites into
oligonucleotides using a DNA synthesizer. The phosphor-
amidites 13 and 14 were synthesized according to Schemes
1-3. The 5′-hydroxy group of 2,2′-anhydro-5-methyluridine
1
was silylated with tert-butyldiphenylsilyl chloride
(TBDPSCl) in pyridine (Py) to yield 2 in 75% yield (Scheme
1).9 Product 2 was then subjected to ring opening10 with a
a (a) For 5, 1. N-methylhydrazine, CH2Cl2, -10 to 0 °C; 2.
MeOH, HCHO; for 6, 1. N-methylhydrazine, CH2Cl2, -10 to 0
°C; 2. MeOH, CH3CHO; (b) for 7, 1. 1 M PPTS in MeOH,
NaCNBH3; 2. HCHO, 1 M PPTS in MeOH, NaCNBH3; for 8, 1.
1 M PPTS in MeOH, NaCNBH3; 2. CH3CHO, 1 M PPTS in MeOH,
NaCNBH3; (c) TEA‚3HF, TEA, THF; (d) DMTCl, Py, DMAP, rt.
Scheme 1a
compound with formaldehyde or acetaldehyde in methanol
at room temperature gave the formaldoximino derivative 5
(78% yield) or the acetaldoximino derivative 6 (55.5% yield).
Reduction of 5 gave the monomethylaminooxy derivative
which was then treated with formaldehyde under reductive
conditions to give the dimethylaminooxy derivative 7 in 80%
yield.
Reduction of compound 6 gave the monoethylaminooxy
derivative which was treated with acetaldehyde to give the
diethylaminooxy derivative 8 in 75% isolated yield. Com-
pounds 7 and 8 were then desilylated with triethylamine
trihydrogenfluoride (TEA‚3HF) and triethylamine (TEA) in
THF to give 9 (92% yield) and 10 (74% yield), respectively.
a (a) TBDPSCl, Py, rt; (b) BH3‚THF, ethylene glycol, 150 °C;
(c) Ph3P, N-hydroxyphthalimide, DEAD.
(7) Wolfe, S. Acc. Chem. Res. 1972, 5, 102-111.
(8) Teplova, M.; Minasov, G.; Tereshko, V.; Inamati, G. B.; Cook, P.
D.; Manoharan, M.; Egli, M. Nat. Struct. Biol. 1999, 6, 535-539.
(9) Nelson, T. D.; Crouch, R. D. Synthesis 1996, 1031-1069.
(10) Ross, B. S.; Springer, R. H.; Tortorici, Z.; Dimock, S. Nucleosides
Nucleotides 1997, 16, 1641-1647.
(11) (a) Mitsunobu, O.; Kimura, J.; Fujisawa, Y. Bull. Chem. Soc. Jpn.
1972, 45, 245-247. (b) Mitsunobu, O. Synthesis 1981, 1-28.
borate ester generated from ethylene glycol and borane in
THF to provide 2′-O-(2-hydroxyethyl) nucleoside 3 in 50%
yield. This simple procedure gave the intermediate for the
construction of the 2′-O-DMAOE and 2′-O-DEAOE pyr-
imidine nucleosides. Compound 3, treated under Mitsunobu11
3996
Org. Lett., Vol. 2, No. 25, 2000

Table 1. Characterization of Oligonucleotides Containing 2′-O-DMAOE-5-methyluridine (T*) and 2′-O-DEAOE-5-methyluridine (T§)
Modifications
ES MS
no.
ISIS no.
sequence
calcd
found
HPLC tR, min
19
20
21
22
23
24
17963
17961
20444
32390
32391
32388
5′GCG T*T*T* T*T*T* T*T*T* T*GC G 3′
5′T*CC AGG T*GT* CCG CAT* C 3′
5′TTT TTT TTT TTT TTT T*T*T* T* 3′
5′GCG T§T§ T§ T§ T§T§ T§ T§ T§ T§GC G 3′
5′ T§CC AGG T§G T§ CCG CAT§ C 3′
5′TTT TTT TTT TTT TTT T§T§T§ T§ 3′
5906.4
5246.4
6131.1
6187.6
5194.4
6243.2
5905.6
5245.6
6130.0
6188.1
5193.6
6242.9
26.2
23.6
25.4
32.4
25.5
29.9
a Waters, C-4, 3.9 × 300 mm, A ) 50 mM triethylammonium acetate, B ) acetonitrile, 5 to 60% B in 55 min, flow 1.5 mL min-1, λ ) 260 nm.
Compounds 9 and 10 were converted into 5′-O-dimethoxy-
trityl derivatives 11 and 12 (80 and 87% yields, respectively)
and then phosphytilated to yield compounds 13 (75% yield)
and 14 (70% yield).12 Compounds 11 and 12 were also
converted into 3′-O- succinyl derivatives (15 and 16) and
loaded on to amino alkyl controlled pore glass (CPG, Scheme
3) according to the standard synthetic procedure13 to yield
functionalized solid supports 17 (55 µmol/g loading) and 18
(57 µmol/g loading), respectively (Scheme 3).
Hybridization properties of the modified oligonucleotides
19, 20, 22, and 23 against complementary RNA and DNA
were studied (Tables 2 and 3). When RNA (Table 2) was
Table 2. Tm Data for the Oligonucleotides Containing
2′-O-DMAOE and 2′-O-DEAOE Modifications against
Complementary RNA
no. of
Tm
∆Tm
∆Tm (°C)/
oligo no.
substituents
(°C)
(°C)
modification
parent DNA1
19
22
parent DNA2
20
23
48.3
62.9
63.3
62.3
66.83
65.5
Scheme 3a
10
10
14.6
15.0
1.46
1.50
4
4
4.5
4.2
1.12
1.05
used as a target, oligonucleotides 19 and 22, which contained
10 consecutive modifications, demonstrated a duplex stabi-
lization of 1.5 °C per modification as compared to the
unmodified DNA analogue and 2.3 °C per modification as
compared to the phosphorothioate, respectively.16 Similarly,
oligonucleotides 20 and 23, which contained 4 dispersed
modifications, stabilized the duplex with RNA by 1.1 and
(13) (a) Kumar, P.; Sharma, A. K.; Sharma, P.; Garg, B. S.; Gupta, K.
C. Nucleosides Nucleotides 1996, 15, 879-888. (b) TBTU-mediated
synthesis of functionalized CPG synthesis: Bayer, E.; Bleicher, K.; Maier,
M. A.; Z. Naturforsch. 1995, 50b, 1096-1100.
(14) 0.1 M solution of amidites 13 and 14 in anhydrous acetonitrile was
used for the synthesis of the modified oligonucleotides. For incorporation
of 13 and 14, phosphoramidite solutions were delivered in two portions,
each followed by a 5 min coupling wait time. All other steps in the protocol
supplied by Millipore were used without modification except for the
oxidation step.15 The coupling efficiencies were more than 97%. After
completion of the synthesis, CPG was suspended in aqueous ammonium
hydroxide (30 wt %) and kept at room temperature for 2 h. The CPG was
filtered and the filtrate was heated at 55 °C for 6 h to complete the removal
of all protecting groups. Crude oligonucleotides were purified by high
performance liquid chromatography (HPLC, Waters, C-4, 7.8 × 300 mm,
A ) 50 mM triethylammonium acetate, pH ) 7, B ) acetonitrile, 5 to
60% B in 55 min, flow 2.5 mL min-1, λ ) 260 nm). Detritylation with
aqueous 80% acetic acid followed by desalting gave 2′-modified oligo-
nucleotides. Oligonucleotides were analyzed by HPLC, CGE, and mass
spectroscopy. The isolated yields for modified oligonucleotides were 30-
40%. These yields are similar to those obtained during unmodified DNA
oligomer synthesis.
a (a) N,N-Diisopropylamine tetrazolide, (2-cyanoethyl)-N,N,N′,N′-
tetraisopropylphophoramidite, CH3CN, rt; (b) succinic anhydride,
TEA, ClCH2-CH2Cl, DMAP, rt; (c) TBTU, DMF, 4-methylmor-
pholine, CPG, rt.
Oligonucleotides listed in Table 1 were synthesized using
these building blocks.14 Oxidation of the internucleotide
phosphite to phosphate was carried out using CSO [1-S-(+)-
(10-camphorsulfonyl)oxaziridine].15
(15) See: Manoharan, M.; Lu, Y.; Casper, M. D.; Just, G. Org. Lett.
2000, 2, 243-246 and references cited.
(16) The 2′-deoxyphosphorothioates cause -0.8 °C/modification in RNA
hybridization compared to DNA. See ref 17.
(12) All compounds were characterized by 1H, 13C, and, where appropri-
ate, by 31P NMR and mass spectroscopy.
Org. Lett., Vol. 2, No. 25, 2000
3997

1.9 °C, compared to phosphodiester and phosphorothioate
analogues, respectively.17 Comparison of the Tm values of
the oligonucleotides modified with 2′-O-DMAOE (19 and
20) with those modified with 2′-O-DEAOE (22 and 23)
demonstrates no significant difference between the two
substituents. This suggests that the addition of the steric bulk
in the 2′-O-DEAOE modified oligonucleotides does not result
in any substantial destabilization of hybridization with RNA.
Furthermore, even with the increased bulk, the novel
modifications demonstrate a palpable increase in binding
affinity compared to the 2′-O-MOE modification.4,17
In contrast, hybridization of 19 and 22 with complementary
DNA led to duplexes less stable than those formed with
unmodified DNA oligonucleotides (-0.9 and -1.1 °C per
modification, respectively, as shown in Table 3). These
Table 3. Tm Data for the Oligonucleotides Containing
2′-O-DMAOE and 2′-O-DEAOE Modifications against
Complementary DNA
Figure 2. SVPD assay demonstrating relative nuclease resistance
of 2′-O-DMAOE and 2′-O-DEAOE capped oligonucleotides TTT
TTT TTT TTT TTT T*T*T* T*.
no. of
Tm
∆Tm
∆Tm (°C)/
oligo no.
substituents
(°C)
(°C)
modification
parent DNA1
19
22
54.2
45.4
42.9
higher than that of the 2′-O-MOE oligonucleotide. Further-
more, we compared the relative lipophilicity of these two
modifications. As expected, 2′-O- DEAOE is more lipophilic
than 2′-O-DMAOE as assessed by reverse phase HPLC
retention times (Table 1).
10
10
-8.8
-11.3
-0.88
-1.13
results demonstrate a high, RNA-selective hybridization of
In conclusion, we have synthesized two novel types of
2′-modified oligonucleotides that showed high binding
affinity to complementary RNA and high nuclease stability.
High binding affinity to RNA, favorable lipophilicity, and
high nuclease resistance makes these modifications ideal
candidates for further evaluation for antisense drug develop-
ment. Evaluation of other antisense properties such as in vivo
pharmacology is in progress.
the 2′-O-DMAOE and 2′-O-DEAOE oligonucleotides.
To evaluate the stability of 2′-O-DMAOE and 2′-O-
DEAOE oligonucleotides against nucleases, phosphodiester
oligonucleotides 21 and 24 were synthesized and digested
with snake venom phosphodiesterase (SVPD).18 The modi-
fications were placed at the 3′-terminal ends. Figure 2 shows
the relative nuclease stability of these modified oligonucleo-
tides compared to unmodified DNA oligonucleotide of the
same sequence. Half-lives of modified oligonucleotides were
more than 24 h. The ethyl analogue has a longer lifetime
than the methyl analogue. The nuclease stability of 2′-O-
DMAOE and 2′-O-DEAOE capped oligonucleotides was
Acknowledgment. We are grateful to Dr. P. Dan Cook
for his impetus and support in the design of the modifications
described in this paper. We thank Drs. Bruce Ross and Bal
Bhat for helpful discussions.
Supporting Information Available: Analytical data for
compounds 11-14. This material is available free of charge
via the Internet at http://pubs.acs.org.
(17) Freier, S. M.; Altmann, K.-H. Nucleic Acids Res. 1997, 25, 4429-
4443.
(18) 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.
OL006555G
3998
Org. Lett., Vol. 2, No. 25, 2000