Table 1. ES-MS Analysis of Oligonucleotides with 2′-O-DMAEOE Modification and the Effect of 2′-O-DMAEOE and 2′-O-MOE
Modifications on Duplex Stability with Complementary RNAa
ES MS
c
c
c
no.
sequence
calcd
found
Tm, °C
∆Tm, °C
∆Tm /unit
7
8
9
0
1
2
3
4
5′d(GCGTTTTTTTTTTGCG)3′ (parent)
48.3
59.8
59.6
62.3
66.7
65.5
1
1 1 1 1 1 1 1 1 1
5′d(GCGT T T T T T T T T T GCG)3′
5′d(GCGT*T*T*T*T*T*T*T*T*T*GCG)3′
5′d(TCCAGGTGTCCGCATC)3′ (parent)
11.5
11.3
1.2
1.1
6186.83
6187.94
1
1
1
1
1
1
1
1
1
5′d(T CCAGGT GT CCGCAT C)3′
5′d(T*CCAGGT*GT*CCGCAT*C)3′
5′ TTTTTTTTTTTTTTTT*T*T*T*3′
5′d(GCGTAT*ACGC)3′
4.4
3.2
1.1
0.8
5357.90
6542.20b
3159.23
5354.54
6542.62
3158.23
a
T1 ) 2′-O-MOE-5-methyluridine, T* ) 2′-O-DMAEOE-5-methyluridine. b DMT-on. c Tm values were assessed in 100 mM NaCl, 10 mM phosphate
buffer, 0.1 mM EDTA, pH 7, at 260 nm, and 4 µM oligonucleotides and 4 µM complementary length matched RNA. Standard deviation did not exceed (0.5
C.
°
deoxyphosphorothioate (2′-H/PS) compounds. This modifi-
cation with a PdO linkage exhibits nuclease resistance
synthesized by using phosphoramidite 4 and solid support 5
and the standard phosphoramidites for incorporation of A,
T, G, and C residues. Oxidation of the internucleosidic
phosphite groups was carried out with 1-S-(+)-(10-cam-
phorsulfonyl)oxaziridine. (see Supporting Information for
details.)
(measured as the half-life of the full-length oligonucleotide,
t
1/2) at approximately the same level as a 2′-deoxyphospho-
9
rothioate modification. The 2′-O-AP modification and its
homologue 2′-O-DMAP exhibit exceptional nuclease resis-
tance (t1/2 8-fold better than 2′-deoxyphosphorothioate com-
pounds) due to the cationic alkyl chain, but have only
moderate affinity for target RNA. To improve upon these
modifications, we designed and synthesized the 2′-O-[2-[2-
Hybridization of the modified oligonucleotides 9 and 12
to complementary RNA and DNA was next studied. Oligo-
nucleotides 9 and 12 demonstrated a duplex stabilization of
1
.1 and 0.8 °C per modification as compared to the DNA
(
N,N-dimethylamino)ethoxy]ethyl] (2′-O-DMAEOE) modi-
analogue (Table 1) and 1.9 and 1.6 °C compared to DNA/
8
fication. This modification combines the advantages of the
10
PdS oligonucleotides. There is no significant difference
gauche effect (as in 2′-O-MOE) and the charge effect (as in
in T
DMAEOE (9 and 12) and those modified with 2′-O-MOE
8 and 11). This suggests that the addition of the steric
m
values between oligonucleotides modified with 2′-O-
2′-O-AP). Moreover, 2′-O-DMAEOE oligonucleotides can
be expected to be more lipophilic than the 2′-O-MOE
analogues, a property affecting protein binding and cellular
permeation of oligonucleotides.
(
bulk in the 2′-O-DMAEOE modified oligonucleotides
does not result in a substantial destabilization of hybridiza-
tion with RNA. In contrast, hybridization of 9 with com-
plementary DNA led to a duplex less stable than those
formed with unmodified DNA oligonucleotides (0.42 °C
destabilization per unit of modification). These findings
suggest that preorganization of the 2′-O-DMAEOE oligo-
nucleotide results in a preference for formation of a duplex
with RNA.
2
′-O-DMAEOE-5-methyluridine-3′-phosphoramidite 4
and solid support 5 were synthesized as described in Scheme
1. Oligonucleotides 9, 12, 13, and 14 (Table 1) were
Scheme 1a
To evaluate the stability of 2′-O-DMAEOE oligonucle-
otides against nucleases, a T19 PdO oligonucleotide 13 with
(5) (a) 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. (b) Prakash, T. P.; Manoharan,
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Lett. 2000, 41, 4855-4859. (c) Teplova, M.; Wallace, S. T.; Tereshko, V.;
Minasov, G.; Symons, A. M.; Cook, P. D.; Manoharan, M.; Egli, M. Proc.
Natl. Acad. Sci. U.S.A. 1999, 96, 14240-14245.
(
6) Eliel, E. L.; Wilen, S. H.; Mander, L. N. Stereochemistry of Organic
Compounds; Wiley-Interscience: New York, 1994.
7) Teplova, M.; Minasov, G.; Tereshko, V.; Inamati, G. B.; Cook, P.
D.; Manoharan, M.; Egli, M. Nature Struct. Biol. 1999, 6, 535-539.
8) (a) Manoharan, M.; Cook, P. D. US Patent Isis Pharmaceuticals, Inc.,
USA, 2000. (b) Prhavc, M.; Prakash, T. P.; Egli, M.; Manoharan, M.; Ross,
B. S.; Song, Q. Abstr. Pap. Am. Chem. Soc. 2001, 221, CARB-095.
(
a
Reagents and conditions: (a) BH ‚THF, 2-[2-(N,N-dimethy-
3
(
lamino)ethoxy]ethanol, 150 °C. (b) DMTCl, Py, DMAP, rt. (c) N,N-
Diisopropylammonium tetrazolide, (2-cyanoethyl)-N,N,N′,N′-tet-
raisopropylphosphorodiamidite, CH
NEt , (CH Cl) , DMAP, rt; (ii) 2-(1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium tetrafluoroborate (TBTU), DMF, CPG, rt.
3
CN, rt. (d) (i) Succinic anhydride,
(
9) Manoharan, M.; Lu, Y.; Casper, M. D.; Just, G. Org. Lett. 2000, 2,
3
2
2
2
43-246.
(10) Freier, S. M.; Altmann, K.-H. Nucleic Acids Res. 1997, 25, 4429-
4
443.
2018
Org. Lett., Vol. 5, No. 12, 2003