2'-O-[2-(amino)-2-oxoethyl] oligonucleotides.

Article abstract of DOI:10.1021/ol027131k

[structure: see text] Oligonucleotides with novel modifications, 2'-O-[2-(amino)-2-oxoethyl] (2'-O-NAc), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMAc), 2'-O-[2-(dimethylamino)-2-oxoethyl] (2'-O-DMAc), and 2'-O-[2-[[2-(dimethylamino)ethyl]amino]-2-oxoethyl

Full text of DOI:10.1021/ol027131k

ORGANIC
LETTERS
2003
Vol. 5, No. 4
403-406
2′-O-[2-(Amino)-2-oxoethyl]
Oligonucleotides
Thazha P. Prakash, Andrew M. Kawasaki, Elena A. Lesnik,
Stephen R. Owens, and Muthiah Manoharan*
Department of Medicinal Chemistry, Isis Pharmaceuticals Inc., 2292 Faraday AVenue,
Carlsbad, California 92008
mmanoharan@isisph.com
Received October 17, 2002
ABSTRACT
Oligonucleotides with novel modifications, 2′-O-[2-(amino)-2-oxoethyl] (2′-O-NAc), 2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMAc), 2′-O-[2-
(dimethylamino)-2-oxoethyl] (2′-O-DMAc), and 2′-O-[2-[[2-(dimethylamino)ethyl]amino]-2-oxoethyl] (2′-O-DMAEAc), have been synthesized. These
modified oligonucleotides exhibit high binding affinity to complementary RNA (and not to DNA) and considerably enhance the nuclease
stability of oligonucleotides with t1/2 > 24 h.
Antisense oligonucleotides modified at the 2′-O-position of
the sugar at one or both termini (gapmers)1,2 have emerged
as leading second-generation candidates for clinical applica-
tions. Chemical modifications such as 2′-O-methoxyethyl (2′-
O-MOE) improve the metabolic stability, cellular absorption,
and protein binding properties of oligonucleotides.2
To improve the antisense properties of 2′-O-MOE-modi-
fied oligonucleotides, we designed oligonucleotides with
novel 2′-O-modifications, including 2′-O-[2-(methylamino)-
2-oxoethyl] (2′-O-NMAc), 2′-O-[2-(dimethylamino)-2-oxo-
ethyl] (2′-O-DMAc), and 2′-O-[2-[[2-(dimethylamino)ethyl]-
amino]-2-oxoethyl] (2′-O-DMAEAc), as shown in Figure 1.
Among them, the parent 2′-O-[2-(amino)-2-oxoethyl]3 (2′-
O-NAc) modification has been synthesized and studied by
Sproat et al. We postulated that the nucleosides containing
Figure 1.
these modifications should retain the “gauche effect”4 and
thus the C3′-endo sugar pucker characteristic of the 2′-O-
MOE nucleosides.5 Consequently, oligonucleotides contain-
ing these modified nucleoside residues should display the
enhanced hybridization to RNA targets observed with the
2′-O-MOE modified oligonucleotides. In addition, 2′-O-
NMAc, 2′-O-DMAc, and 2′-O-DMEAc oligonucleotides
should be more lipophilic than the corresponding 2′-O-MOE
counterparts, and thus may have better protein binding and
cellular permeation properties. Here we report the synthesis,
(1) (a) Cook P. D. In Antisense Medicnal Chemistry; Crooke, S. T., Ed.;
Antisense Research and Application, Vol. 131; Springer-Verlag: New York,
1998; 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 therein.
(2) (a) Martin, P. HelV. Chim. Acta 1995, 78, 486-504 (b) Manoharan,
M. Biochim. Biophys. Acta 1999, 1489, 117-130. (c) 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.
(3) Grotli, M.; Beijer, B.; Sproat, B. Tetrahedron 1999, 55, 4299-4314.
(4) Wolfe, S. Acc. Chem. Res. 1972, 5, 102-111.
(5) Teplova, M.; Minasov, G.; Tereshko, V.; Inamati, G. B.; Cook, P.
D.; Manoharan, M.; Egli, M. Nat. Struct. Biol. 1999, 6, 535-539.
10.1021/ol027131k CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/23/2003

affinity toward target RNA, and nuclease stability of the 2′-
O-NAc analogues.
The phosphoramidite 5 and the solid support 7 were
synthesized as described in Scheme 1. Compound 1 was
solid support 7 was used for 11. To avoid transamination
during the treatment with amines, dCAc phosphoramidite was
used for incorporation of the dC residues.8 Oxidation of the
internucleoside phosphite groups was carried out with 1-S-
(+)-(10-camphorsulfonyl)oxaziridine.9
The 2′-O-modified oligonucleotides 12-23 were synthe-
sized by using solid support-bound oligonucleotides 8-11
that contain the chemically reactive methyl ester function.
Upon completion of the oligonucleotide synthesis, the
modifications were introduced and the oligonucleotides were
deprotected by treating the solid support-bound material with
an appropriate amine as shown in Figure 2.10 The solid
Scheme 1a
Figure 2.
support-bound oligonucleotides 8-11 were treated with 50%
N,N-dimethylethylenediamine in EtOH (to yield 12-15,
Table 1), or 40% aqueous methylamine (to yield 16-19,
Table 1), or saturated methanolic ammonia (to yield 20-
23, Table 1). Reactions were allowed to proceed at room
temperature for 24 h. Under these conditions, the methyl ester
functions were converted to the corresponding amides in high
yields (Figure 2). Simultaneously, the base and phosphate
protecting groups were removed and the oligonucleotides
were cleaved from solid support. The oligonucleotides
bearing 2′-O-DMAEAc, 2′-O-NMAc, and 2′-O-NAc modi-
fications (12-15, 16-19, and 20-23, respectively) were
purified by reverse-phase HPLC and characterized by ES
MS. The purity of the compounds 12-23 was assessed by
HPLC and capillary gel electrophoresis.
In contrast to these results, treatment of the solid supports
8-11 with 40% aqueous dimethylamine for 3 days at room
temperature gave only 50-60% of the desired oligonucle-
otides 27-30. We attributed the low yield to the widely
known, low reactivity of secondary amines toward ester
functions. Furthermore, the deprotection at elevated temper-
ature led to complex mixtures of products.
a Reagents and conditions: (i) Methyl 2-bromoacetate, NaH,
DMF, -40 °C to room temperature. (ii) (a) H2 30 psi, 10% Pd on
carbon, MeOH; (b) triethylamine, MeOH. (iii) DMTCl, DMAP,
Py. (iv) 2-Cyanoethyl-N,N-diisopropylaminochlorophosphite, di-
isopropylethylamine, CH2Cl2, rt. (v) succinic anhydride, 1,2-
dichloroethane, DMAP, (C2H5)3N, rt. (vi) 2-(1H-benzotriazole-1-
yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4-methyl
morpholine, DMF, aminoalkyl controlled pore glass (CPG), rt.
alkylated with methyl 2-bromoacetate to give 2′- and 3′-
regio-isomers 2a and 2b in a ratio of 9:1.6 The compound
2a was isolated by silica gel chromatography and converted
to 5 and 7 according to standard procedures.7
Oligonucleotides 8-11 (Table 1) were assembled on the
solid support with use of the phosphoramidite 5 and the
standard phosphoramidites for incorporation of A, T, and G
residues. Commercial solid supports were used for 8-10 and
Table 1. Oligonucleotides with 2′-Modificationsa
(6) Cook, P. D.; Manoharan, M.; Guinosso, C. J. PCT International
Application, Isis Pharmaceuticals, Inc., 1995.
(7) Prakash, T. P.; Kawasaki, A. M.; Fraser, A. S.; Vasquez, G.;
Manoharan, M. J. Org. Chem. 2002, 67, 357-369.
(8) Reddy, M. P.; Farooqui, F.; Hanna, N. B. Tetrahedron Lett 1996,
37, 8691-8694.
(9) Manoharan, M.; Lu, Y.; Casper, M. D.; Just, G. Org. Lett. 2000, 2,
243-246. Although CSO has been used for oxidation of internucleosidic
phosphite groups, other conventional oxidizing agents such as tert-butyl
hydroperoxide in acetonitrile or iodine in tetrahydrofuran, pyridine, and
water may also be used.
(10) (a) Keller, T. H.; Haner, R. HelV. Chim. Acta 1993, 76, 884-892.
(b) Keller, T. H.; Haner, R. Nucleic Acids Res. 1993, 21, 4499-4505.
a All oligonucleotides were phosphodiesters. 8-11: t ) 2′-O-(2-methoxy-
2-oxoethyl)-5-methyluridine. 12-15: t ) 2′-O-DMAEAc-5-methyluridine.
16-19: t ) 2′-O-NMAc-5-methyluridine. 20-23: t ) 2′-O-NAc-5-
methyluridine. 27-30: t ) 2′-O-DMAc-5-methyluridine.
404
Org. Lett., Vol. 5, No. 4, 2003

Table 2. Tm Data for the Oligonucleotides Hybridized to Complementary RNAa
12, 16, 20, 27
13, 17, 21, 28
∆Tm/unit
14, 18, 22, 29
∆Tm/unit
t
Tm
∆Tm/unit
Tm
Tm
thymidine
62.3
67.8
69.2
69.5
67.1
48.3
50.0
60.0
61.0
60.8
61.8
62.1
66.0
66.9
66.2
2′-O-DMAEAc-5-methyluridine
2′-O-NMAc-5-methyluridine
2′-O-NAc-5-methyluridine
2′-O-DMAc-5-methyluridine
1.4
1.7
1.8
1.2
0.2
1.2
1.3
1.3
0.1
1.0
1.3
1.1
a Oligonucleotides 12, 16, 20, 27: 5′ d(tCC AGG tGt CCG CAt C) 3′. Oligonucleotides 13, 17, 21, 28: 5′ d(GCG ttt ttt ttt tGC G) 3′. Oligonucleotides
14, 18, 22, 29: 5′ d(CTC GTA Ctt ttC CGG TCC) 3′. Tm values were assessed in 100 mM Na+, 10 mM phosphate, 0.1 mM EDTA, pH 7 at 260 nm, with
4 µM oligonucleotides and 4 µM complementary length matched RNA. Experimental error did not exceed (0.5 °C.
In view of these facts, an alternative approach was taken
for the synthesis of the 2′-O-DMAc modified oligonucle-
otides. The nucleoside 4 was treated with 2 M dimethylamine
in THF for 18 h at room temperature to give 24 in 95%
isolated yield (Scheme 2). This was then converted into a
3′-phosphoramidite 25 and a solid support 26 in a conven-
tional manner.
and 29, demonstrated a duplex stabilization of 1.1-1.3 and
1.9-2.1 °C per modification as compared to the correspond-
ing 2′-deoxy phosphodiester oligonucleotides (PO) and their
phosphorothioate (PS) analogues,11 respectively. Oligonucle-
otides 16, 20, and 27 with four dispersed modifications
stabilized the duplex with RNA by 1.2-1.8 and 2-2.6 °C,
compared to the corresponding PO and PS analogues,
respectively.11 These values are slightly better than the Tm
enhancements observed for 2′-OMe and 2′-O-MOE modi-
fications.
Scheme 2 a
The oligonucleotides 12, 13, and 14 bearing cationic
dimethylamino groups demonstrated a significant dependence
of the melting temperatures (Tm) on the distance between
the modified nucleoside residues. Compounds 13 and 14 with
consecutively placed dimethylamino groups showed only a
moderate increase in Tm (0.1-0.2 °C per modification
compared to the PO control). In contrast, a Tm enhancement
of 1.4 °C per modification was observed for oligonucleotide
12 where dimethylamino groups were dispersed. These
observations are in agreement with the reported hybridization
profile of oligonucleotides bearing 2′-O-aminopropyl and
homologous groups.12,1c
a Reagents and conditions: (i) 2 M dimethylamine in THF, rt.
(ii) 2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite, N,N-
diisopropylammonium tetrazolide, CH3CN, rt. (iii) (a) succinic
anhydride, ClCH2CH2Cl, DMAP, (C2H5)3N, rt; (b) TBTU, 4-me-
thylmorpholine, DMF, amino alkyl CPG, rt.
Comparison of the Tm values of 2′-O-NAc oligonucleotides
20-22 with their 2′-O-NMAc (16-18) and 2′-O-DMAc
analogues (27-29) demonstrated no significant difference
in their thermal stability. This suggested that the addition of
the steric bulk in the 2′-O-NMAc and 2′-O-DMAc oligo-
nucleotides did not affect their hybridization affinity to RNA.
The oligonucleotides 27-30 were assembled on the solid
support as described above for 8-11 except that the
phosphoramidite 25 and the solid support 26 were used
instead of 5 and 7. The solid support-bound material was
deprotected with methylamine (4% solution) in aqueous
ammonium hydroxide8 at room temperature for 24 h to give
27-30 with no major side products observed by ES MS
analysis of the crude material. The oligonucleotides obtained
were purified and characterized as described above. In
contrast, deprotection of 27-30 with concentrated aqueous
ammonium hydroxide at elevated temperature (55 °C, 6 h)
led to mixtures of the products where the dimethylamino
group in the 2′-O-DMAc side chain was partially displaced
with the amino group as evidenced by ES MS.
Specificity of hybridization of several 2′-modified oligo-
nucleotides including 2′-O-MOE against complementary
RNA reported in the literature was similar to that of
unmodified DNA.2a,11 We expect the specificity of hybridiza-
tion of the novel 2′-modified oligonucleotides described in
this Letter to be similar to that of unmodified DNA as well.
In contrast to results with complementary RNA, hybridiza-
tion with the complementary DNA led to duplexes less stable
than those formed with the corresponding PO controls (Table
3). These results demonstrated an RNA-selective hybridiza-
(11) Freier, S. M.; Altmann, K.-H. Nucleic Acids Res. 1997, 25, 4429-
4443.
(12) Griffey, R. H.; Monia, B. P.; Cummins, L. L.; Freier, S.; Greig, M.
J.; Guinosso, C. J.; Lesnik, E.; Manalili, S. M.; Mohan, V.; Owens, S.;
Ross, B. R.; Sasmor, H.; Wancewicz, E.; Weiler, K.; Wheeler, P. D.; Cook,
P. D. J. Med. Chem. 1996, 39, 5100-5109.
Hybridization of the modified oligonucleotides with
complementary RNA (Table 2) was studied. The oligonucle-
otides with consecutive modifications, 17, 18, 21, 22, 28,
Org. Lett., Vol. 5, No. 4, 2003
405

Table 3. Tm Data for the Oligonucleotidesa 13, 17, 21, and 28
Hybridized to the Complementary DNA
∆Tm (°C)/
t
Tm (°C)
∆Tm (°C)
modification
thymidine (parent)
2′-O-DMAEAc-5-
methyluridine (13)
2′-O-NMAc-5-
48.3
41.5
-6.8
-8.4
-6.9
-2.6
-0.7
-0.8
-0.7
-0.3
39.9
41.4
45.7
methyluridine (17)
2′-O-NAc-5-
methyluridine (21)
2′-O-DMAc-5-
methyluridine (28)
a 5′-d(GCGt10GCG); Tm values were assessed in 100 mM Na+, 10 mM
phosphate, 0.1 mM EDTA, pH 7 at 260 nm, with 4 µM oligonucleotides
and 4 µM complementary length matched DNA. Experimental error did
not exceed (0.5 °C.
Figure 3. The disappearance of oligonucleotides 15, 19, 23, 30,
31, and 32 in the presence of SVPD as a function of time. 31:
5′-T15 t4-3′ (t ) 2′-O-MOE-5-methyluridine).; 32: T20. 5′-32P labeled
oligonucleotides were digested with SVPD (5 × 10-3 U mL-1) in
50 mM Tris-HCl buffer at pH 8.5, containing 72 mM NaCl and 14
mM MgCl2 at 37 °C.
tion of the 2′-O-NAc-, 2′-O-NMAc-, 2′-O-DMAc-, and 2′-
O-DMAEAc-modified oligonucleotides.
To evaluate the metabolic stability of the novel oligo-
nucleotides, digestion with snake venom phosphodiesterase
(SVPD) was monitored as described previously.13 The
enzymatic hydrolysis of compounds 15, 19, 23, and 30,
phosphodiester oligonucleotides capped at the 3′-terminus
with four 2′-O-modified nucleoside residues, was evaluated.
The plot of the time-dependent disappearance of the novel
oligonucleotides and their 2′-deoxy and 2′-O-MOE analogues
is shown in Figure 3. The enzymatic stability of the 2′-O-
modified oligonucleotides studied decreased in the following
order 2′-O-DMAEAc > 2′-O-NMAc > 2′-O-DMAc > 2′-
O-NAc > 2′-O-MOE . 2′-deoxy. All the novel modifica-
tions imparted significant stability. In particular, the resis-
tance of the 2′-O-DMAEAc compound 15 against nucleolytic
degradation is worth noticing.
affinity to complementary RNA and nuclease stability. Of
the modified oligonucleotides described in this Letter, 2′-
O-NMAc and 2′-O-DMAEAc oligonucleotides were the most
synthetically feasible and have been selected for further
biological evaluation.
Supporting Information Available: Experimental pro-
cedures and 1H, 13C NMR, and mass spectral data for
compounds 2-4, 6, 24, and 26 and 31P NMR and mass
spectral data for 5 and 25; experimental procedure, ES MS
data, and HPLC retention times for oligonucleotides 12-23
and 27-30; procedures used for the Tm and nuclease studies.
This material is available free of charge via the Internet at
http://pubs.acs.org.
In conclusion, we have synthesized three novel types of
2′-modified oligonucleotides that showed excellent binding
(13) 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.
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Org. Lett., Vol. 5, No. 4, 2003