Synthesis of 2′O-[2-[(N,N-dimethylamino)oxy]ethyl] modified nucleosides and oligonucleotides

Article abstract of DOI:10.1021/jo0103975

A versatile synthetic route has been developed for the synthesis of 2′-O-[2-[(N,N-dimethylamino)oxy]ethyl] (abbreviated as 2′-O-DMAOE) modified purine and pyrimidine nucleosides and their corresponding nucleoside phosphoramidites and solid supports. To synthesize 2′-O-n DMAOE purine nucleosides, the key intermediate B (Scheme 1) was obtained from the 2′-O-allyl purine nucleosides (13a and 15) via oxidative cleavage of the carbon-carbon bond to the corresponding aldehydes followed by reduction. To synthesize pyrimidine nucleosides, opening the 2,2′-anhydro-5-methyluridine 5 with the borate ester of ethylene glycol gave the key intermediate B. The 2′-O-(2hydroxyethyl) nucleosides were converted, in excellent yield, by a regioselective Mitsunobu reaction, to the corresponding 2′-O-[2-[(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)oxy]ethyl] nucleosides (18, 19, and 20). These compounds were subsequently deprotected and converted into the 2′-O-[2[(methyleneamino)oxy]ethyl] derivatives (22, 23, and 24). Reduction and a second reductive amination with formaldehyde yielded the corresponding 2′-O-[2-[(N,N-dimethylamino)oxy]ethyl] nucleosides (25, 26, and 27). These nucleosides were converted to their 3′-O-phosphoramidites and controlled-pore glass solid supports in excellent overall yield. Using these monomers, modified oligonucleotides containing pyrimidine and purine bases were synthesized with phosphodiester, phosphorothioate, and both linkages (phosphorothioate and phosphodiester) present in the same oligonucleotide as a chimera in high yields. The oligonucleotides were characterized by HPLC, capillary gel electrophoresis, and ESMS. The effect of this modification on the affinity of the oligonucleotides for complementary RNA and on nuclease stability was evaluated. The 2′-O-DMAOE modification enhanced the binding affinity of the oligonucleotides for the complementary RNA (and not for DNA). The modified oligonucleotides that possessed the phosphodiester backbone demonstrated excellent resistance to nuclease with t_1/2 > 24 h.

Full text of DOI:10.1021/jo0103975

J . Org. Chem. 2002, 67, 357-369
357
Syn th esis of 2′-O-[2-[(N,N-Dim eth yla m in o)oxy]eth yl] Mod ified
Nu cleosid es a n d Oligon u cleotid es
Thazha P. Prakash, Andrew M. Kawasaki, Allister S. Fraser, Guillermo Vasquez, and
Muthiah Manoharan*
Department of Medicinal Chemistry, Isis Pharmaceuticals Inc., 2292 Faraday Avenue,
Carlsbad, California 92008
mmanoharan@isisph.com
Received April 17, 2001
A versatile synthetic route has been developed for the synthesis of 2′-O-[2-[(N,N-dimethylamino)-
oxy]ethyl] (abbreviated as 2′-O-DMAOE) modified purine and pyrimidine nucleosides and their
corresponding nucleoside phosphoramidites and solid supports. To synthesize 2′-O-DMAOE purine
nucleosides, the key intermediate B (Scheme 1) was obtained from the 2′-O-allyl purine nucleosides
(13a and 15) via oxidative cleavage of the carbon-carbon bond to the corresponding aldehydes
followed by reduction. To synthesize pyrimidine nucleosides, opening the 2,2′-anhydro-5-methyl-
uridine 5 with the borate ester of ethylene glycol gave the key intermediate B. The 2′-O-(2-
hydroxyethyl) nucleosides were converted, in excellent yield, by a regioselective Mitsunobu reaction,
to the corresponding 2′-O-[2-[(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)oxy]ethyl] nucleosides (18, 19,
and 20). These compounds were subsequently deprotected and converted into the 2′-O-[2-
[(methyleneamino)oxy]ethyl] derivatives (22, 23, and 24). Reduction and a second reductive
amination with formaldehyde yielded the corresponding 2′-O-[2-[(N,N-dimethylamino)oxy]ethyl]
nucleosides (25, 26, and 27). These nucleosides were converted to their 3′-O-phosphoramidites and
controlled-pore glass solid supports in excellent overall yield. Using these monomers, modified
oligonucleotides containing pyrimidine and purine bases were synthesized with phosphodiester,
phosphorothioate, and both linkages (phosphorothioate and phosphodiester) present in the same
oligonucleotide as a chimera in high yields. The oligonucleotides were characterized by HPLC,
capillary gel electrophoresis, and ESMS. The effect of this modification on the affinity of the
oligonucleotides for complementary RNA and on nuclease stability was evaluated. The 2′-O-DMAOE
modification enhanced the binding affinity of the oligonucleotides for the complementary RNA (and
not for DNA). The modified oligonucleotides that possessed the phosphodiester backbone demon-
strated excellent resistance to nuclease with t1/2 > 24 h.
In tr od u ction
achieve these goals, structural analogues of nucleic acids
with modified heterocycle, sugar, and phosphodiester
backbone moieties have been synthesized.5 Some of the
most successful analogues resulted from modification of
the sugar at the 2′-position.5 The 2′-O-modified oligo-
nucleotides used with the “gapmer” technology6 have
emerged as the leading second generation candidates for
clinical applications. Gapmer oligonucleotides have one
or two 2′-O-modified regions and a 2′-deoxyoligonucleo-
tide phosphorothioate region that allows RNase H diges-
tion of mRNA. Among the various 2′-O-modified oligo-
nucleotides reported in the literature, the 2′-O-(2-
methoxyethyl)7 modified oligonucleotides (2′-O-MOE)
offer a 2 °C increase in melting temperature (Tm) per
modification as a diester (2′-O-MOE/PdO) compared to
In antisense therapeutic technology, synthetic oligo-
nucleotides that base-pair with messenger RNA encoding
a disease-related protein specifically inhibit the synthesis
of that protein.1 Antisense oligonucleotides have demon-
strated effective inhibition of many viral and cellular
gene products, both in vitro and in vivo.2 The first
successful drug to emerge from this technology was ISIS-
2922, “Vitravene,” a 2′-deoxyoligonucleotide phosphoro-
thioate.3
An ideal antisense oligonucleotide should have high
binding affinity for the target RNA, should be resistant
to nuclease digestion, should bind selectively to transport
proteins, and should be cell permeable in vivo.4 To
* Author for correspondence.
(4) (a) Cook P. D. Antisense Medicinal Chemistry; Crooke, S. T., Ed.;
Springer-Verlag: New York, 1998; Antisense Research and Applica-
tion, 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 therein.
(1) (a) Crooke, S. T. In Basic Principles of Antisense Therapeutics;
Crooke, S. T., Ed.; Antisense Research and Application, Vol. 131,
Springer: Berlin, 1998, pp 1-50. (b) Phillips, M. I. Ed.; Methods
Enzymol. 2000, 313, 580; 314, 646: Antisense Technology, Parts A and
B. (c) Crooke, S. T.; Lebleu, B. Antisense Research and Applications;
CRC Press Inc.: Boca Raton, FL, 1993. (d) Mesmaeker, A. D.; Ha¨ener,
R.; Martin, P.; Moser, H. E. Acc. Chem. Res. 1995, 28, 36-374.
(2) Applied Antisense Oligonucleotide Technology; Stein, C. A.;
Krieg, A. M., Eds.; Wiley-Liss, Inc.: New York, 1998.
(5) Cook, P. D. Annu. Rep. Med. Chem. 1998, 33, 313-325.
(6) 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.
(3) Manoharan M. Biochim. Biophys. Acta 1999, 1489, 117-130.
(7) Martin, P. Helv. Chim. Acta 1995, 78, 486-504.
10.1021/jo0103975 CCC: $22.00 © 2002 American Chemical Society
Published on Web 12/28/2001

358 J . Org. Chem., Vol. 67, No. 2, 2002
Prakash et al.
Ch a r t 1. Novel 2′-Ca r boh yd r a te Mod ifica tion s
Sch em e 1
Ch a r t 2. Gen er a l Str u ctu r e of 2′-O-DMAOE
P h osp h or a m id ites
the 2′-deoxyphosphorothioate (2′-H/PdS) compounds.
This modification with a phosphodiester linkage exhibits
resistance to snake venom phosphodiesterase (measured
as the half-life of disappearance of the full-length oligo-
nucleotide, t1/2) at approximately the same level as a 2′-
deoxyoligonucleotide phosphorothioate. Our recent8 syn-
thesis of the 2′-O-[2-[(N,N-dimethylamino)oxy]ethyl]-5-
methyluridine modified oligonucleotides was an attempt
to improve upon the 2′-O-MOE modification.
Resu lts a n d Discu ssion
Syn th esis of 2′-O-Mod ified Nu cleosid e P h osp h or -
a m id ites a n d Solid Su p p or ts. The 2′-O-DMAOE nu-
cleosides and their phosphoramidites synthesized in this
study are shown in Chart 2. A general synthetic strategy
planned for the syntheses of these amidites (Chart 2) is
described in Scheme 1. The 2′-O-(2-hydroxyethyl) deriva-
tive (B, Scheme 1) was a convenient intermediate for the
following reasons. First, this intermediate could be con-
verted to an aminooxy derivative using the Mitsunobu12
reaction. Second, the resulting, highly reactive aminooxy
derivative could be converted into a methyleneaminooxy
derivative, reduced to the monomethyl compound, and
reductively alkylated with formaldehyde to give the
dimethylaminooxy compound.13 To synthesize pyrimidine
nucleosides, B was obtained from the 2,2′-anhydro-5-
methyluridine (5) via ring opening of the anhydro nucleo-
side by ethylene glycol following a method developed by
Ross et al.14 For the purine nucleosides, preferential 2′-
O-alkylation gave the 2′-O-allyl derivatives (A, Scheme
1), which were converted to the 2′-O-(2-hydroxyethyl)
derivatives. These 2′-modified nucleosides were then
converted into the target 2′-O-DMAOE nucleosides and
their corresponding phosphoramidites (Schemes 2-8).
This new 2′-O-modification (abbreviated as 2′-O-
DMAOE) was designed to improve the antisense proper-
ties of the 2′-O-MOE in three ways. First, we envisaged
that this modification would maintain the C3-endo sugar
pucker shown by the 2′-O-MOE modification10 thus
retaining the “gauche effect.”9 This gauche effect results
in preorganization of the 2′-O-MOE antisense single
strand required for the enhanced hybridization to the
RNA target compared to the 2′-deoxy compound. Second,
these dialkyl modifications would be more lipophilic than
the 2′-O-(2-aminoethyl) (abbreviated as 2′-O-AOE)11 and
2′-O-MOE modifications, a characteristic affecting the
protein binding and cellular permeation properties of
oligonucleotides. Finally, the increased steric size would
improve the nuclease resistance even more than that
observed for the 2′-O-MOE and 2′-O-AOE modifications
(Chart 1).
In this report, we describe the synthesis of all four basic
building blocks, namely the 2′-O-DMAOE modified nu-
cleosides containing the nucleobases A, 5MeU, G, and 5MeC
as well as their 3′-phosphoramidites and their solid
supports. We describe the syntheses of fully modified 2′-
O-DMAOE oligonucleotides with these amidites. In ad-
dition, we studied the hybridization of the fully modified
2′-O-DMAOE oligonucleotides with complementary RNA
and DNA. The stability of 2′-O-DMAOE oligonucleotides
to snake venom phosphodiesterase (SVPD) was also
investigated.
The synthesis of 2′-O-(2-hydroxyethyl)-5-methyluridine
(7) is described in Scheme 2. The 5′-hydroxy group of 2,2′-
anhydro-5-methyluridine (5) was silylated with tert-
butylchlorodiphenylsilane (TBDPSCl) in pyridine to pro-
duce 6 in good yield. The tert-butyldiphenylsilyl (TBDPS)
group was used in preference to other silyl protecting
groups15 because of its high stability under acidic condi-
tions. The product was next subjected to the ring opening
(8) Prakash, T. P.; Manoharan, M.; Kawasaki, A. M.; Lesnik, E. A.;
Owens, S. R.; Vasquez, G. Org. Lett. 2000, 2, 3995-3998.
(9) Wolfe, S. Acc. Chem. Res. 1972, 5, 102-111.
(10) Teplova, M.; Minasov, G.; Tereshko, V.; Inamati, G. B.; Cook,
P. D.; Manoharan, M.; Egli, M. Nature Struct. Biol. 1999, 6, 535-
539.
(11) Kawasaki, A. M.; Casper, M. D.; Prakash, T. P.; Manalili, S.;
Sasmor, H.; Manoharan, M.; Cook, P. D. Tetrahedron Lett. 1999, 40,
661-664.
(12) Mitsunobu, O.; Kimura, J .; Fujisawa, Y. Bull. Chem. Soc. J pn.
1972, 45, 245-247. (b) Montero, J . -L.; Criton, M.; Dewynter, G.-F.;
Imbach, J .-L. Tetrahedron Lett. 1991, 32, 5357-5358.
(13) (a) Perbost, M.; Hoshiko, T.; Morvan, F.; Swayze, E.; Griffey,
R. H.; Sanghvi, Y. S. J . Org. Chem. 1995, 60, 5150-5156. (b) Swayze,
E. E.; Sanghvi, Y. S. Synlett 1997, 839-841.
(14) Ross, B. S.; Springer, R. H.; Tortorici, Z.; Dimock, S. Nucleosides
Nucleotides 1997, 16, 1641-1643.
(15) Nelson, T. D.; Crouch, R. D. Synthesis 1996, 9, 1031-1069.

Modified Nucleosides and Oligonucleotides
J . Org. Chem., Vol. 67, No. 2, 2002 359
Sch em e 2a
Sch em e 3a
a
(i) TBDPSCl, pyridine, DMAP, rt; (ii) BH3.THF, ethylene
glycol, 150 °C.
a
reaction14 with the borate ester generated from ethylene
glycol and borane in THF to obtain the 2′-O-(2-hydroxy-
ethyl) nucleoside 7 in 50% yield.
(i) TMSCl, pyridine, BzCl, NH4OH, H2O; (ii) TBDPSCl,
CH2Cl2, DMAP, triethylamine, rt.
protection conditions17 to obtain 12a and 12b (Scheme
3, 62%) and for the subsequent regioselective silylation
at the 5′-position with TBDPSCl in pyridine. It is
noteworthy that after silylation of 12a and 12b, the 2′-
O-allyl and 3′-O-allyl adenosine derivatives 13a and 13b,
respectively, could be separated by flash silica gel column
chromatography to afford the required 2′-O-allyl isomer
13a . The two isomers were identified by 2D 1H-1H COSY
NMR experiments.
The same approach could not be used for the synthesis
of the 2′-O-allyl guanosine 14 because of the well-known
susceptibility of guanosines to electrophilic attack at the
O-6 and N-2 positions of the purine ring. Indeed, direct
alkylation of the guanosine with alkyl halides in the
presence of NaH in DMF gave inseparable mixtures of
products.16g In contrast, 2-aminoadenosine, which might
be considered as a convenient precursor of guanosine,18
smoothly underwent alkylation with allyl bromide to give
the 2′-O- and 3′-O-isomers 11a and 11b in a ratio of 3:1,
respectively, in a total yield of 70%. In the next step
depicted in Scheme 4, the mixture of 11a and 11b was
treated with adenosine deaminase in aqueous buffer at
pH 7.5 as reported by Robins et al.18 and McGee et al.18
Under these conditions, 11a was selectively converted to
14, which precipitated from the reaction mixture to give
the product in more than 90% isomeric purity, while the
unreacted 11b remained in solution. Compound 14 was
subsequently reacted with TBDPS-Cl in DMF in the
presence of imidazole to furnish compound 15 in 72%
yield.
Sproat16f and co-workers have carried out 2′-O-alkyl-
ation with allyl bromide using 3′,5′-bis-protected nucleo-
sides. We attempted the direct alkylation of the unpro-
tected nucleoside at the 2′-O-position (pKa of 2′-OH of
adenosine ) 12.14)16g since this material is quite
inexpensive.16d,16e Alkylation of adenosine with allyl
bromide in the presence of NaH gave a mixture of the 2′
and 3′-O-allyl isomers (10a and 10b, in a ratio of 3:1) in
66% isolated yield. However, separation of the two
isomers (the 2′-O- and 3′-O-alkylation products) at this
step using silica gel column chromatography proved
difficult. Hence, we decided to use the mixture of 2′-O-
and 3′-O- alkylated adenosines (10a ,b) for the benzoyl-
ation of the exocyclic amino group under transient
The 2′-O-allyl derivatives 13a and 15 were converted
to the 2′-O-(2-hydroxyethyl) derivatives 16 and 17,
respectively (Scheme 5). Dihydroxylation of 13a and 15
with OsO4 and 4-methylmorpholine N-oxide afforded the
corresponding vicinal-diols.19 The dihydroxylation reac-
(16) (a) Christensen, L. F.; Broom, A. D. J . Org. Chem. 1972, 37,
3398-33401. (b) Takaku, H.; Kamaike, K. Chem. Lett. 1982, 2, 189-
192. (c) Yano, J .; Kan, L. S.; Ts’o, P. O. Biochim. Biophys. Acta 1980,
629, 178-183. (d) Guinosso, C. J .; Woke, G. D.; Frier, S.; Martin, J .
F.; Ecker, D. J .; Mirabelli, C. K.; Crooke, S. T.; Cook, P. D. Nucleosides
Nucleotides 1991, 10, 259-262. (e) Manoharan, M.; Guinosso, C. J .;
Cook, P. D. Tetrahedron Lett. 1991, 32, 7171-7174. (f) Sproat, B. S.;
Iribarren, A. M.; Garcia, R. G.; Beijer, B. Nucleic Acids Res. 1991, 19,
733-738. (g) Velikyan, I.; Acharya, S.; Trifonova, A.; Fo¨ldesi, A.;
Chattopadhyaya, J . J . Am. Chem. Soc. 2001, 123, 2893-2894.
(17) Ti, G. S.; Gaffney, B. L.; J ones, R. A. J . Am. Chem. Soc. 1982,
104, 1316-1319.
(18) (a) Robins, M. J .; Hanske, F.; Beriner, S. E. Can. J . Chem. 1981,
59, 3360-3364. (b)McGee, D. P. C.; Cook, P. D.; Guinosso, C. J . US
PCT 92-918362, 1994. (c) Robins, M. J .; Zou, R.; Hansske, F.; Wnuk,
S. F. Can. J . Chem. 1997, 75, 762-767. (d)Yokozeki, K.; Tsuji, T. J .
Mol. Catal. B: Enzymol. 2000, 10, 207-213.

360 J . Org. Chem., Vol. 67, No. 2, 2002
Prakash et al.
Sch em e 4a
Sch em e 6a
a
(i) Adenosine deaminase, 100 mM sodium phosphate, pH 7.5,
rt; (ii) DMF, TBDPSCl, imidazole, rt.
a
(i) Ph3P, N-hydroxyphthalimide, THF, DED, rt; (ii) pyridine,
Sch em e 5a
isobutyryl chloride.
Sch em e 7a
a
(i) (a) 4-Methylmorpholine N-oxide, OsO4, dioxane for 13a and
CH2Cl2/(CH3)2CO for 15; (b) NaIO4 on SiO2, CH2Cl2; (c) 1 M
pyridinium p-toluenesulfonate (PPTS) in MeOH, NaBH3CN, rt.
tion of 2′-O-allyl adenosine 13a was carried out in
dioxane. However, the 2′-O-allyl guanosine derivative 15
was not soluble in dioxane. Hence an alternative solvent
system containing CH2Cl2/(CH3)2CO was used for the
osmylation of 15 to afford the corresponding vicinal-diol
which was cleaved in the presence of NaIO4 adsorbed on
silica in CH2Cl2 to obtain the corresponding aldehyde.20
Attempts to reduce the aldehyde with NaBH3CN in acetic
acid failed to give the expected products. However,
reaction with NaBH3CN and 1 M pyridinium p-toluene-
sulfonate (PPTS) in MeOH gave the alcohols in high
yield. The reduction of the aldehyde with NaBH3CN in
1 M PPTS in MeOH afforded the 2′-O-(2-hydroxyethyl)
derivatives 16 and 17 (72-78%). It appears that 1 M
PPTS in MeOH produces ideal conditions21 for the
reduction of the above-mentioned aldehydes.
Treatment of 7, 16 and 17 under Mitsunobu12 condi-
tions with N-hydroxyphthalimide, Ph3P, and DEAD in
THF gave the phthalimido derivatives 18, 19 and 20 in
69-92% yields. The exocyclic amino group of the gua-
nosine derivative 20 was protected with isobutyryl
chloride in anhydrous pyridine (Scheme 6) to afford the
N2-isobutyryl derivative 21 in 65% isolated yield.
a
(i) (a) CH2Cl2, N-methylhydrazine (b) MeOH, HCHO, rt; (ii)
(a) 1 M PPTS in MeOH, NaBH3CN, rt; (b) 1 M PPTS in MeOH,
HCHO, NaBH3CN, rt; (iii) triethylamine trihydrofluoride, THF,
(C2H5)3N, rt; (iv) 4,4′-dimethoxytrityl chloride, pyridine, DMAP,
rt; (v) 2-cyanoethyl tetraisopropylphosphorodiamidite, N,N-diiso-
propylammonium tetrazolide, CH3CN, rt.
fication, the aminooxy derivatives were converted into
the methyleneaminooxy derivatives 22, 23, and 24 using
formaldehyde in MeOH. Reduction of the compounds 22,
23, and 24 with NaBH3CN in 1 M PPTS in MeOH gave
the monomethylaminooxy derivative, which was then
treated with formaldehyde under reductive conditions to
give the dimethylaminooxy derivatives 25, 26, and 27 in
80-96% yield. Reduction of the methyleneaminooxy
derivatives 22, 23, and 24 proceeded smoothly13b and was
complete in 2 h at ambient temperature. Desilylation of
25, 26, and 27 with triethylamine trihydrofluoride and
triethylamine in THF gave compounds 28, 29, and 30 in
good yield. These nucleosides were then selectively
protected at the 5′-position by reaction with 4,4′-
The phthalimido group in compounds 18, 19, and 21
(Scheme 7) was deprotected with N-methylhydrazine13a
(NMH) at 0 °C to give the aminooxy derivatives. The use
of NMH for deprotection of the phthalimido group13a
resulted in a thick precipitate of 1,2-dihydro-4-hydroxy-
2-methyl-1-oxophthalazine that was readily separated by
filtration. Excess NMH was easily removed from the
reaction mixture by evaporation. Without further puri-
(19) Van Rheenen, V.; Kelly, R. C.; Cha, D. Y. Tetrahedron Lett.
1976, 23, 1973-1976.
(20) Zhong, Y.-Li.; Shing, T. K. M. J . Org. Chem. 1997, 62, 2622-
2624.
(21) Borch, F. R.; Bernstein, M. D.; Durst, H. D. J . Am. Chem. Soc.
1971, 93, 2897-2904.

Modified Nucleosides and Oligonucleotides
J . Org. Chem., Vol. 67, No. 2, 2002 361
Sch em e 8a
Sch em e 9a
a
(i) Succinic anhydride, 1,2-dichloroethane, DMAP, (C2H5)3N,
60 °C; (ii) 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium-
tetrafluoroborate (TBTU), 4-methylmorpholine, DMF, aminoalkyl
controlled pore glass (CPG), rt.
on to the aminoalkyl controlled pore glass (CPG, Scheme
9) according to the standard synthetic procedure24 to yield
the functionalized solid supports 43-46 (55-60 µmol/
g).
Syn th esis of 2′-O-DMAOE Mod ified Oligon u cleo-
tid es. The oligonucleotides 48, 50, 52, 54, 56, 58, and
59 (Table 1) were synthesized on a solid-phase DNA
synthesizer using the phosphoramidites 1, 2, 3, and 4. A
0.1 M solution of the phosphoramidites in anhydrous
acetonitrile was used for the synthesis. Oxidation of the
internucleosidic phosphite to the phosphate was carried
out using 1-S-(+)-(10-camphorsulfonyl)oxaziridine (CSO).25
3-H-1,2-Benzodithiol-3-one 1,1-dioxide (the Beaucage
reagent)26 was used as the sulfur-transfer agent for the
synthesis of oligonucleotide phosphorothioates. The over-
all coupling efficiency of all modified phosphoramidites
was more than 97%. Oligonucleotides were deprotected
with concentrated aqueous ammonium hydroxide. After
deprotection, all the 5′-DMTr on oligonucleotides were
isolated by reverse phase HPLC. When the 5′-terminal
DMTr group was removed, the oligonucleotides 48, 50,
52, 54, 56, 58, and 59 were desalted and characterized
by electrospray MS. Their purity was confirmed by HPLC
and capillary gel electrophoresis.
Eva lu a tion of Hybr id iza tion of th e 2′-O-DMAOE
Mod ified Oligon u cleotid es to Com p lem en ta r y RNA
a n d DNA by Th er m a l Den a tu r a tion Stu d ies. A series
of oligonucleotides with the 2′-O-DMAOE modification
were used to investigate the effect of this modification
on hybridization affinity for complementary RNA and
DNA. Melting temperatures (Tm) of the modified oligo-
nucleotides with target RNA were compared to those of
the heteroduplexes formed with unmodified DNA oligo-
nucleotides (Table 1). Free energies of duplex formation
∆G° were obtained from UV absorbance vs temperature
curves assuming a two-state model with a linear sloping
baseline.28 These results are summarized in Table 1.
a
Bz ) benzoyl; (i) TBDPSCl, DMF, imidazole, 60 °C; (ii) 1,2,4-
triazole, POCl3, (C2H5)3N, CH3CN; (iii) DMF, benzoic anhydride,
rt; (iv) triethylamine trihydrofluoride, (C2H5)3N, THF, rt; (v)
DMTr-Cl, pyridine, DMAP, rt; (vi) diisopropylammonim tetra-
zolide, 2-cyanoethyl tetraisopropylphosphorodiamidite, CH3CN, rt.
dimethoxytrityl chloride (DMTCl) and a catalytic amount
of DMAP in pyridine to yield the products 31 (78% yield),
32 (75% yield), and 33 (91% yield). Phosphitylation of
compounds 31-33 at the 3′-position with 2-cyanoethyl
N,N,N′N′-tetraisopropylphosphorodiamidite in the pres-
ence of N,N-diisopropylamine tetrazolide in CH3CN af-
forded the phosphoramidite building blocks 1-3 in 52-
86% yield.
The synthesis of the 5-methylcytidine phosphoramidite
4 is illustrated in Scheme 8. Compound 25 was silylated
at the 3′-O- position with TBDPSCl and imidazole in
DMF at 60 °C to yield 34 in 85% isolated yield. Com-
pound 34 was converted into the triazole derivative with
1,2,4-triazole, POCl3, and Et3N.22 Subsequent treatment
with aqueous NH3:dioxane (1:1) at ambient temperature
for 14 h gave the 5-methylcytidine analogue 35 in 94%
yield. The exocyclic amino group was protected with a
benzoyl group23 to afford 36 (87% yield), followed by
removal of the silyl group to give 37 (97% yield). The
dimethoxytritylation of 37 at the 5′-O-position yielded 38
(72% yield). Finally, phosphitylation at the 3′-O-position
gave the phosphoramidite 4 (85% yield) in good overall
yield.
(24) (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.
(25) Manoharan, M.; Lu, Y.; Casper,M. D.; J ust, G. Org. Lett. 2000,
2, 243-246.
Compounds 31, 32, 33, and 38 were also converted into
the 3′-O-succinyl derivatives 39-42 (Scheme 9) and loaded
(26) Iyer, R. P.; Phillips, L. R.; Egan, W.; Regan, J . B.; Beaucage, S.
L. J . Org. Chem. 1990, 55, 4693-4699.
(22) Divakar, K. J .; Reese, C. B. J . Chem. Soc., Perkin Trans. 1 1982,
5, 1171-1176.
(27) Freier, S. M.; Altmann, K.-H. Nucleic Acids Res. 1997, 25,
4429-4443.
(23) Bhat, V.; Ugarkar, B. G.; Sayeed, V. A.; Grimm, K.; Kosora,
N.; Domenico, P. A.; Stocker, E. Nucleosides Nucleotides 1989, 8, 179-
183.
(28) Petersheim, M.; Turner, D. H. Biochemistry 1983, 22, 256-
263.

362 J . Org. Chem., Vol. 67, No. 2, 2002
Prakash et al.
Ta ble 1. Effect of th e 2′-O-DMOE Mod ifica tion on Du p lex Sta bility a ga in st Com p lem en ta r y RNA Ta r gets
oligo
no.
Tm
°C
∆Tm/modification
∆G°
37
sequence
°C
kcal mol-1
47
48
49
50
51
52
53
54
55
56
57
58
5′ ToCoCoAoGoGoToGoToCoCoGoCoAoToC 3′
5′ T*oCoCoAoGoGoT*oGoT*oCoCoGoCoAoT*oC 3′
5′ GoCoGoToToToToToToToToToToGoCoG 3′
62.3
-14.87
-17.82
-13.00
-17.77
-14.80
-23.70
-13.20
-20.1
66.78
48.30
62.90
58.20
69.80
53.70
65.80
59.70
86.50
51.20
78.00
1.12
1.46
1.16
1.21
1.34
1.34
5′GoCoGoT*oT*oT*oT*oT*oT*oT*oT*oT*oT*oGoCoG 3′
5′ToToC§oToC§sGsC§sTsGsGsTsGsAsGsToToToC§oA 3′
5′ T*oT*oC*oT*oC*sGsC§sTsGsGsTsGsAsGsT*oT*oT*oC*oA∇ 3′
5′ TsTsC§sTsC§sGsC§sTsGsGsTsGsAsGsTsTsTsC§sA 3′
5′ T*sT*sC*sT*sC*sGsC§sTsGsGsTsGsAsGsT*s T*s T*sC*sA∇ 3′
5′ ToC§oToGoAoGoToAoGoC§oAoGoAoGoGoAoGo C§oToC§ 3′
5′ T*oC*oT*oG*oA*oG*oT*oA*oG*oC*oA*oG*oA*oG*oG*oA*oG*oC*oT*oC* 3′
5′ TsC§sTsGsAsGsTsAsGsC§sAsGsAsGsGsAsGsC§s TsC§ 3′
5′ T*sC*sT*sG*sA*sG*sT*sA*sG*sC*sA*sG*s A*s G*sG*sA*sG*sC*sT*sC* 3′
-14.7
-24.3
-11.7
-19.20
a
T* ) 2′-O-{2-[(N,N-dimethylamino)oxy]ethyl}-5-methyluridine, A* )2′-O-{2-[(N,N-dimethylamino)oxy]ethyl}adenosine, G* )2′-O-
{2-[(N,N-dimethylamino)oxy]ethyl}guanosine, C* ) 2′-O-{2-[(N,N-dimethylamino)oxy]ethyl}-5-methylcytidine, A∇ ) 2′-O-[2-(methoxy)-
ethyl]adenosine,C§ ) 5-methylcytidine s ) phosphorothioate backbone, o ) phosphodiester backbone.
Ta ble 2. Tm Da ta for th e Oligon u cleotid e Con ta in in g
2′-O-DMAOE Mod ifica tion a ga in st Com p lem en ta r y DNA
Ta r get
no. of
modifications
Tm
(°C)
∆Tm
(°C)
∆Tm
(°C)/modification
oligo no.
DNA parent
54.2
50
10
45.4 -8.8
-0.88
Substitution with 2′-O-DMAOE nucleosides led to an
increase in the melting temperatures relative to unmodi-
fied oligonucleotides. A Tm enhancement of 1.12 °C per
substitution was observed when modified bases were not
contiguous as in the oligonucleotide 48 (Table 1). In the
oligonucleotide 50 with 10 adjacent substitutions, the Tm
enhancement was 1.46 °C/substitution. The dependence
of the stability of the RNA heteroduplexes with 2′-O-
DMAOE modified oligonucleotides on the base composi-
tion and the placement of the modification were verified
by studying the hybridization affinity of the gapmer3,6
oligonucleotides 52 and 54 and the uniformly modified
oligonucleotides 56 and 58 to the complementary RNA.
The gapmer with a mixed backbone (oligonucleotide 52)
showed a Tm enhancement of 1.16 °C/substitution whereas
the gapmer 54 with a full phosphorothioate backbone
showed a Tm enhancement of 1.21 °C/substitution. The
uniformly modified oligonucleotides 56 and 58 showed
an average Tm of 1.34 °C/substitution. These results
suggest that the Tm enhancement due to 2′-O-DMAOE
is sequence independent. In contrast to the increase in
the Tm observed with RNA, the hybridization of the
oligonucleotide 50 with complementary DNA led to
duplexes less stable than those formed with unmodified
DNA oligonucleotides (-0.9 °C per modification, Table
2). The favorable stabilization per 2′-O-DMAOE modifi-
cation corresponds to a net stabilization of more than 2
°C per modification compared to the 2′-deoxyoligonucleo-
tide phosphorothioate, taking into account the fact that
the 2′-deoxyoligonucleotide phosphorothioate forms a
duplex with complementary RNA destabilized by ap-
proximately -0.8 °C/modification.27
F igu r e 1. Relative nuclease resistance of the 2′-O-DMAOE
oligonucleotide versus the 2′-O-propylamino, 2′-O-MOE, 2′-O-
propyl and 2′-deoxy oligonucleotides. T* represents 2′-modified
5MeU. The modifications were placed at the 3′ end of the
sequence 59 TTT TTT TTT TTT TTT T*T*T* T* and digested
with snake venom phosphodiesterase.
internucleosidic linkages were phosphodiesters. Figure
1 shows the relative nuclease stability of the modified
oligonucleotides compared to the unmodified DNA oligo-
nucleotide, 2′-O-MOE, 2′-O-propyl, and 2′-O-aminopropyl
modified oligonucleotides of the same sequence. The half-
life of disappearance of the full length 2′-O-DMAOE
modified oligonucleotide was more than 24 h. The nu-
clease stability of the 2′-O-DMAOE capped oligonucleo-
tide was higher than that of the 2′-O-MOE, 2′-O- propyl
and the 2′-deoxy modified oligonucleotides. Only the 2′-
O-aminopropyl modified oligonucleotide was more stable
than the 2′-O-DMAOE oligonucleotide. The amino group
in the 2′-O-aminopropyl modification is protonated under
physiological conditions (pKa ) 9) and leads to enhanced
nuclease resistance. The observed order of nuclease
stability is consistent with the reported data.30 The
relative nuclease stability of the modified oligonucleotides
is in the following order: 2′-O-AP > 2′-O-DMAOE > 2′-
O-MOE > 2′-O-Pr > 2′-deoxy.
In conclusion, efficient synthetic routes have been
developed for all four 2′-O-DMAOE modified nucleosides,
Stability of 2′-O-DMAOE Oligon u cleotides Again st
Sn a k e Ven om P h osp h od iester a se. To evaluate the
resistance of the 2′-O-DMAOE oligonucleotides toward
nucleases, the oligonucleotide 59 was treated with snake
venom phosphodiesterase (SVPD).29 The modifications in
the oligonucleotide 59 were placed at the 3′-end and all
(30) (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) Manoharan,
M.; Prakash, T. P.; Barber-Peoc’h, I.; Bhat, B.; Vasquez, G.; Ross, B.
S.; Cook, P. D. J . Org. Chem. 1999, 64, 6468-6472. (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.
(29) 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.

Modified Nucleosides and Oligonucleotides
J . Org. Chem., Vol. 67, No. 2, 2002 363
N,N-d iisop r op yl]p h osp h or a m id ite (2). Compound 32 (1.20
g, 1.6 mmol) was coevaporated with toluene (5 mL). The
residue obtained was mixed with N,N-diisopropylamine tet-
razolide (0.27 g, 1.6 mmol) and dried over P2O5 in a vacuum
overnight at 40 °C. The mixture was dissolved in anhydrous
CH3CN (8.2 mL) and 2-cyanoethyl N,N,N′N′-tetraisopropyl-
phosphorodiamidite (2.0 mL, 6.3 mmol) was added dropwise.
The reaction mixture was stirred at room temperature under
an argon atmosphere for 4 h. The reaction was monitored by
TLC (ethyl acetate:hexane, 8:2 containing 0.5% pyridine). The
solvent was removed under reduced pressure, the resulting
residue was dissolved in ethyl acetate (75 mL) and washed
with 5% aqueous NaHCO3 (2 × 40 mL). The organic phase
was dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The residue was purified by flash silica gel
column chromatography (ethyl acetate:hexane, 8:2 containing
0.5% pyridine) to afford 2 (1.23 g, 81%): 31P NMR (162 MHz,
CDCl3) δ 151.14, 150.72; LRMS (FAB) m/z 1093 (M + Cs)+,
HRMS (FAB) Calcd for C51H62N8O9P+ 961.4377; found 961.4360.
5′-O-(4,4′-Dim e t h oxyt r it yl)-2′-O-{2-[(N ,N -d im e t h yl-
a m in o)oxy]et h yl}-N2-isobu t yr ylgu a n osin e-3′-[(2-cya n o-
eth yl)-N,N-d iisop r op yl]p h osp h or a m id ite (3). Compound
33 (1.32 g, 1.8 mmol) was dissolved in CH2Cl2 (18 mL), and
diisopropylamine tetrazolide (0.15 g, 0.9 mmol) and 2-cyano-
ethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (0.63 mL,
2.0 mmol) were added. After 2 h at ambient temperature, an
additional amount of 2-cyanoethyl-N,N,N′,N′-tetraisopropyl-
phosphorodiamidite (0.21 mL, 0.7 mmol) was added and the
solution was stirred for 9 h. The solvent was evaporated under
reduced pressure to a volume of 5 mL. The resulting solution
was purified by flash column chromatography using silica gel
[pretreated with 1% triethylamine] eluting with CH2Cl2-
acetone-triethylamine, 60:40:1, followed by CH2Cl2-acetone-
(C2H5)3N, 30:70:1, to provide the title compound 3 as a white
foam (0.87 g, 52%): 31P NMR (81 MHz, CDCl3) δ 150.7, 150.2.
HRMS (FAB) Calcd for C48H63N8O10PNa+ 965.4302, found
965.4341.
their phosphoramidite building blocks, and solid supports
with traditional protecting groups at the exocyclic amino
groups. The methods described herein provide general
procedures for functionalization of all four nucleosides
at the 2′-position. The use of the 2′-O-DMAOE building
blocks in oligonucleotide synthesis resulted in high
coupling efficiency. We have shown the use of these
phosphoramidites and solid supports for the syntheses
of oligonucleotides with phosphorothioate, phosphodi-
ester, and oligonucleotide-containing phosphodiester and
phosphorothioate backbones. These modified oligonucleo-
tides showed RNA-selective, high binding affinity, and
excellent nuclease stability and make them ideal candi-
dates for further evaluation for antisense drug develop-
ment. While this report was in preparation, we also
synthesized oligonucleotide gapmers with 2′-O-DMAOE
modifications at the wings and fully modified oligo-
nucleotides containing 2′-O-DMAOE modifications and
evaluated their ability to suppress gene expression in
several biological targets. In the gapmer format, oligo-
nucleotides with 2′-O-DMAOE wings are able to activate
RNase H and have been shown to suppress gene expres-
sion. The oligonucleotides having the 2′-O-DMAOE modi-
fication throughout the sequence are able to inhibit
translation by the non RNase H mediated mode of action.
These results will be reported elsewhere.
Exp er im en ta l Section
Gen er a l P r oced u r es. 2,2′-Anhydrothymidine was pur-
chased from Ajinomoto (Tokyo, J apan). Anhydrous MeCN
(water content < 0.001%), standard phosphoramidites and
ancillary reagents for oligonucleotide synthesis were purchased
from PE Biosystems (Foster City, CA). All other reagents and
anhydrous solvents were purchased from Aldrich and used
without further purification. Adenosine deaminase was pur-
chased from Reliable Biopharmaceuticals, St. Louis, MO. Flash
chromatography was performed using silica gel 60 (35-75 µm,
EM Science). Thin-layer chromatography was performed on
precoated plates (silica gel 60 F254 from EM Science) and
visualized with UV light and spraying with a solution of
p-anisaldehyde (6 mL), H2SO4 (8.3 mL), and CH3COOH (2.5
mL) in C2H5OH (227 mL) followed by charring. 1H NMR
spectra were referenced using internal standard (CH3)4Si and
31P NMR spectra using external standard 85% H3PO4. Micro-
analyses were performed by Quantitative Technologies Inc.,
NJ . Mass spectra were recorded by Mass Consortium, San
Diego, CA and the College of Chemistry, University of Cali-
fornia, Berkeley, CA.
5′-O-(4,4′-Dim e t h oxyt r it yl)-2′-O-{2-[(N ,N -d im e t h yl-
am in o)oxy]eth yl}-5-m eth ylu r idin e-3′-[(2-cyan oeth yl)-N,N-
d iisop r op yl]p h osp h or a m id ite (1). Compound 31 (3.9 g, 6.0
mmol) was coevaporated with toluene (20 mL). To the residue,
N,N-diisopropylammonium tetrazolide (0.97 g, 5.7 mmol) was
added and the mixture was dried over P2O5 in a vacuum
overnight at 40 °C. The reaction mixture was dissolved in
anhydrous CH3CN (28.4 mL) and 2-cyanoethyl N,N,N′,N′-
tetraisopropylphosphorodiamidite (7.3 mL, 23.00 mmol) was
added. The reaction mixture was stirred at ambient temper-
ature for 4 h under an argon atmosphere. The progress of the
reaction was monitored by TLC (hexane:ethyl acetate 1:1). The
solvent was removed under reduced pressure and the residue
was dissolved in ethyl acetate (150 mL) and washed with 5%
aqueous NaHCO3 (75 mL) and brine (75 mL). The ethyl acetate
layer was dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The resulting residue was chromato-
graphed (ethyl acetate as eluent) to afford 1 as a white foam
(4.39 g, 86%): 31P NMR (81 MHz, CDCl3) δ 150.73; HRMS
(FAB) Calcd for C44H58N5O10PNa+ 870.3819, found 870.3796.
N6-Ben zoyl-5′-O-(4,4′-d im et h oxyt r it yl)-2′-O-{2-[(N,N-
d im eth yla m in o)oxy]eth yl}a d en osin e-3′-[(2-cya n oeth yl)-
N4-Ben zoyl-5′-O-(4,4′-d im et h oxyt r it yl)-2′-O-{2-[(N,N-
dim eth ylam in o)oxy]eth yl}-5-m eth ylcytidin e-3′-[(2-cyan o-
eth yl)-N,N-d iisop r op yl]p h osp h or a m id ite (4). Compound
38 (2.74 g, 3.7 mmol) was coevaporated with toluene (20 mL).
It was then mixed with diisopropylamine tetrazolide (0.63 g,
3.7 mmol) and dried over P2O5 in a vacuum at 40 °C overnight.
The resulting mixture was dissolved in anhydrous CH3CN
(18.3 mL) and 2-cyanoethyl N,N,N′,N′-tetraisopropylphos-
phorodiamidite (4.64 mL, 14.6 mmol) was added. The reaction
mixture was stirred at room temperature for 4 h under an
argon atmosphere, followed by removal of the solvent in a
vacuum. Ethyl acetate (100 mL) was added to the residue, and
it was washed with 5% aqueous NaHCO3 (50 mL) and brine
(50 mL). The organic phase was dried over anhydrous Na2SO4
and concentrated under reduced pressure. The resulting
residue was purified by flash silica gel column chromatography
and eluted with ethyl acetate:hexane:pyridine, 60:39:1, to yield
4 (2.96 g, 85% yield) as a white foam: 31P NMR (81 MHz,
CDCl3) δ 150.83; MS (FAB) m/z 949 [M - H]-; HRMS (FAB)
Calcd for C51H63N6O10PCs+ 1083.3398, found 1083.3440.
5′-O-(ter t-Bu t yld ip h en ylsilyl)-2,2′-a n h yd r o-5-m et h yl-
u r id in e (6). 2,2′-Anhydro-5-methyluridine 5 (100.0 g, 416.5
mmol) and DMAP (0.66 g, 5.4 mmol) were dissolved in
anhydrous pyridine (500 mL) at room temperature under an
argon atmosphere with mechanical stirring. tert-Butyldi-
phenylchlorosilane (119 mL, 458.0 mmol) was added. The
reaction was stirred for 16 h at ambient temperature. TLC
(Rf 0.22, ethyl acetate) indicated a complete reaction. The
solution was concentrated under reduced pressure to a thick
oil. This was partitioned between CH2Cl2 (1 L), saturated
NaHCO3 (2 × 1L), and brine (1 L). The organic layer was dried
over Na2SO4 and concentrated under reduced pressure to a
thick oil. The oil was dissolved in a mixture of ethyl acetate
and diethyl ether (1:1, 600 mL), and the solution was cooled
to -10 °C. A crystalline product obtained was collected by
filtration, washed with diethyl ether (3 × 200 mL), and dried
(40 °C, 1 mmHg, 24 h) to afford 6 (149 g, 74.8%) as a white
1
solid: mp 87-88 °C; Rf 0.14 (5% MeOH in CH2Cl2); H NMR

364 J . Org. Chem., Vol. 67, No. 2, 2002
Prakash et al.
(200 MHz, DMSO-d6) δ 0.90 (s, 9H), 1.76 (s, 3H), 3.40 (dd, J
) 6.9 and 4.44 Hz, 1H), 3.57 (dd, J ) 4.78 and 6.52 Hz, 1H),
4.17 (m, 1H), 4.41 (t, J ) 3.2 Hz, 1H), 5.23 (d, J ) 5.82 Hz,
1H), 5.98 (d, J ) 4.66 Hz, 1H), 6.31 (d, J ) 5.7 Hz, 1H), 7.35-
7.6 (m, 10H), 7.79 (s, 1H); 13C NMR (50 MHz, CDCl3) δ 13.87,
19.19, 26.66, 63.02, 75.13, 87.89, 89.37, 90.07, 118.85, 127.83,
129.93, 131.30, 132.99, 135.46, 159.49, 173.04; MS (MALDI)
m/z 501.2 [M + Na]+.
atmosphere. Benzoyl chloride (29.3 mL, 252.3 mmol) was
added dropwise. The reaction mixture was brought to room
temperature and stirred for 4 h and then cooled to 0 °C in an
ice bath. Water (100.0 mL) was added, and the reaction
mixture was stirred for 30 min at 0 °C. Aqueous NH3 (30 wt
%, 100.0 mL) was added, and stirring was continued for an
additional 1 h at 0 °C. The solvent was evaporated and the
residue partitioned between water and ether. The product
precipitated out as an oil, which was further purified by silica
gel column chromatography (5% MeOH in CH2Cl2), to yield
12a ,b as a white foam (12.61 g, 62%): 1H NMR (200 MHz,
DMSO-d6) δ 3.45-3.77 (m, 2H), 3.95-4.22 (m, 3H), 4.38 (br s,
1H), 4.57 (t, J ) 5.38 Hz, 1H), 5.00-5.42 (m, 3H), 5.6-6.00
(m, 1H), 6.08 (d, J ) 5.86 Hz, 1H), 6.21 (d, J ) 5.76 Hz, 1H),
7.4-7.71 (m, 3H), 8.07 (d, J ) 8.1 Hz, 2H), 8.74 (s, 1H), 8.76
(s, 1H), 8.79 (s, 1H); HRMS (MALDI) Calcd for C20H21N5O5-
Na+ 434.1435, found 434.1450.
5′-O-(ter t-Bu tyld ip h en ylsilyl)-2′-O-(2-h yd r oxyeth yl)-5-
m eth ylu r id in e (7). To a 2 L stainless steel pressure reactor
containing borane in tetrahydrofuran (1.0 M, 622 mL), ethyl-
ene glycol (350 mL) was added cautiously with stirring until
the evolution of hydrogen gas subsided. Compound 6 (149 g,
311.6 mmol) and NaHCO3 (0.074 g, 0.88 mmol) were added
with stirring. The reactor was sealed and heated in an oil bath
at internal temperature of 160 °C for 16 h (pressure < 100
psi). The reaction vessel was cooled to ambient temperature
and opened. TLC (Rf 0.67 for the desired product and Rf 0.82
for the thymine arabinofuranoside side product, ethyl acetate)
indicated about 70% conversion to the product. To avoid
additional side product formation, the reaction was stopped
and the solution concentrated under reduced pressure (10 to
1 mmHg) in a warm water bath (40-100 °C) with more
extreme conditions used to remove the ethylene glycol. The
residue was purified by column chromatography (2 kg silica
gel, ethyl acetate:hexanes gradient 1:1 to 4:1). The appropriate
fractions were combined and concentrated in a vacuum to yield
7 as white crisp foam (84 g, 50%), contaminated starting
material (17.4 g), and pure reusable starting material 20 g.
Based on consumed starting material, the yield, was 58%: Rf
2′/3′-O-Allyl-N6-ben zoyl-5′-O-(ter t-bu tyld ip h en ylsilyl)-
a d en osin e (13a ,b). Compound 12a ,b (11.17 g, 27.2 mmol) was
dried over P2O5 in a vacuum at 40 °C and then dissolved in
anhydrous CH2Cl2 (56 mL). DMAP (0.34 g, 2.8 mmol), triethyl-
amine (23.8 mL, 167.0 mmol), and tert-butyldiphenylsilyl
chloride (14.5 mL, 55.7 mmol) were added. The reaction was
stirred vigorously for 12 h and found to be complete by TLC
(ethyl acetate: hexane 1:1). It was diluted with CH2Cl2 (50
mL) and washed with water (3 × 60 mL). The organic phase
was dried over anhydrous Na2SO4 and concentrated to dryness.
The 2′-O-allyl derivative 13a and 3′-O-allyl derivative 13b
were separated by flash silica gel column chromatography
(ethyl acetate: hexane 1:1). The pure 2′-O-allyl isomer 13a
was isolated in 50% yield (8.85 g) as a white foam: Rf 0.35
(ethyl acetate: hexane, 1:1); 1H NMR (200 MHz, DMSO-d6) δ
0.97 (s, 9H), 3.78-4.23 (m, 5H), 4.52 (q, J ) 5.26 and 4.82 Hz,
1H), 4.65 (t, 5.1 Hz, 1H), 5.04-5.22 (m, 2H), 5.41 (d, J ) 5.92
Hz, 1H), 5.74-5.91 (m, 1H), 6.2 (d, J ) 5 Hz, 1H), 7.31-7.69
1
0.22 (5% MeOH in CH2Cl2); H NMR (200 MHz, DMSO-d6) δ
1.04 (s, 9H), 1.45 (s, 3 H), 3.55 (m, 4H), 3.78-4.07 (m, 4H),
4.26 (m, 1 H,), 4.76 (t, J ) 5.26 Hz, 1H), 5.15 (d, J ) 5.7 Hz,
2H), 5.92 (d, J ) 5.38 Hz, 1H), 7.39-7.68 (m, 11H), 11.42 (s,
1H); 13C NMR (50 MHz, CDCl3) δ 11.85, 19.39, 27.07, 61.41,
63.39, 69.02, 72.21, 82.68, 84.73, 86.70, 111.52, 127.94, 130.01,
132.27, 133.02, 135.01, 135.18, 135.48, 151.07, 164.12; MS
(API-ES) 539.1 [M - H]-; HRMS (MALDI) Calcd for C28H36N2-
O7SiNa+ 563.2184, found 563.2173. Anal. Calcd for C28H36N2O7-
Si: C, 62.20; H, 6.71; N, 5.18. Found: C, 61.91; H, 6.66; N,
5.15.
(m, 13H), 8.04 (d, J ) 6.9 Hz, 2H), 8.6 (s, 1H), 8.66 (s, 1H); 13
C
NMR (50 MHz, CDCl3) δ 19.49, 27.22, 63.53, 69.64, 72.18,
81.32, 85.45, 87.28, 119.1, 123.5, 128.09, 129.00, 130.17, 132,
88, 133.42, 134.08, 135.83, 141.63, 149.8, 151.69, 152.99,
164.88; MS (FAB) m/z 673 [M + Na]+; HRMS (MALDI) Calcd
for C36H39N5O5SiNa+ 672.2613, found 672.2430.
2′/3′-O-Allyl Ad en osin e (10a ,b). Adenosine 8 (20.00 g, 74.8
mmol) was dried over P2O5 in a vacuum at 40 °C overnight
and then suspended in anhydrous DMF (380 mL). To this was
added NaH (3.00 g, 74.8 mmol, 60% dispersion in mineral oil),
and the reaction mixture was stirred at room temperature for
10 min. Then allyl bromide (7.2 mL, 83.2 mmol) was added
dropwise and the stirring continued at room temperature for
additional 18 h. DMF was removed in a vacuum, and the
residue was triturated with ethyl acetate (100 mL). The ethyl
acetate layer was decanted, and the solid obtained was
dissolved in MeOH, adsorbed on silica gel, and purified by flash
silica gel column chromatography (10% MeOH in CH2Cl2) to
yield a mixture of 10a and 10b in 66% yield (15.19 g): 1H NMR
(200 MHz, DMSO-d6) δ 3.45-3.91 (m, 2H), 3.95-4.2 (m, 3H),
4.29 (q, J ) 4.76 Hz, and 3.04 Hz, 1H), 4.48 (t, J ) 5.36 Hz,
1H), 4.97-5.4 (m, 3H), 5.41-5.52 (m, 1H), 5.69-5.9 (m, 1H),
6.01 (d, J ) 6.14 Hz, 1H), 7.35 (s, 2H), 8.13 (s, 1H), 8.34 (s,
overlapping with peak at 8.37), 8.37 (s, 1H); HRMS (FAB)
2′-O-Allylgu a n osin e (14). A mixture of 2′- and 3′-O-allyl
2-aminoadenosine 11a ,b (20.00 g, 62.1 mmol) was suspended
in 100 mM sodium phosphate buffer (640 mL, pH 7.5) and
adenosine deaminase (1 g, 2-5 U/mg) was added. The result-
ing solution was stirred very slowly for 60 h, keeping the
reaction vessel open to the atmosphere. The reaction mixture
was then cooled in an ice bath for 1 h and the precipitate
obtained was filtered and dried over P2O5 in a vacuum to yield
14 as a white powder (13.92 g, 69.6% yield): Rf 0.19 (10%
1
MeOH in CH2Cl2); H NMR (200 MHz, DMSO-d6) δ 3.56 (m,
2H), 3.86-4.2 (m, 3H), 4.26 (m, 2H), 5.08 (m, 2H), 5.2 (d, J )
4.34 Hz, 1H), 5.72-5.9 (m, 1H), 5.82 (d, J ) 5.48 Hz, 1H), 6.46
(br s, 2H), 7.95 (s, 1H), 10.64 (s, 1H); 13C NMR (50 MHz,
DMSO-d6) δ 61.34, 69.05, 70.34, 80.82, 84.84, 85.87, 116.79,
134.79, 135.41, 151.23, 153.80, 156.82; MS (FAB) m/z 324 [M
+ H]+; HRMS (MALDI) Calcd for C13H17N5O5Na+ 346.1122,
found 346.1155.
2′-O-Allyl-3′,5′-b is(ter t-b u t yld ip h en ylsilyl)gu a n osin e
(15). 2′-O-Allylguanosine 14 (6.00 g, 18.6 mmol) was mixed
with imidazole (10.18 g, 149.5 mmol) and dried over P2O5
under reduced pressure overnight. To this mixture was added
anhydrous DMF (50 mL), and the reaction mixture was stirred
at room temperature for 10 min. tert-Butyldiphenylsilyl chlo-
ride (19.5 mL, 74.8 mmol) was added, and the reaction mixture
was stirred at room-temperature overnight under an argon
atmosphere. DMF was evaporated under reduced pressure to
obtain an oil, which was dissolved in ethyl acetate (100 mL)
and washed with water (2 × 75 mL). The organic phase was
dried over anhydrous Na2SO4 and concentrated to dryness
under reduced pressure. The residue obtained was purified by
flash silica gel column chromatography and eluted with 5%
MeOH in CH2Cl2 to yield 15 (10.84 g, 72% yield) as a white
+
Calcd for C13H18N5O4 308.1359, found 308.1360.
2′/3′-O-Allyl-2,6-d ia m in op u r in e Ribosid e (11a ,b). From
2,6-diaminopurine riboside 9 (30.00 g, 106.4 mmol), DMF (540
mL), sodium hydride (4.26 g, 106.4 mmol, 60% dispersion in
mineral oil), and allyl bromide (10.2 mL, 117.9 mmol), a
mixture of 11a and 11b (26.38 g, 77%) was obtained according
to the procedure described for the synthesis of compounds
+
10a ,b: HRMS (MALDI) Calcd for C13H19N6O4 323.1619,
found 323.1468.
2′/3′-O-Allyl-N6-ben zoyla d en osin e (12a ,b). A mixture of
compounds 10a ,b (15.19 g, 49.4 mmol) was dried over P2O5 in
a vacuum overnight at 40 °C. It was then dissolved in
anhydrous pyridine (494.4 mL) and protected from moisture.
The reaction mixture was cooled to 0 °C in an ice bath.
Trimethylchlorosilane (32 mL, 252.3 mmol) was added at 0
°C, and the reaction mixture was stirred for 1 h under an argon
1
foam: Rf ) 0.32 (5% MeOH in CH2Cl2); H NMR (200 MHz,
DMSO-d6) δ 0.89 (s, 9H), 1.06 (s, 9H), 3.43 (m, 1H), 3.62 (dd,

Modified Nucleosides and Oligonucleotides
J . Org. Chem., Vol. 67, No. 2, 2002 365
J ) 3.52 and 7.82 Hz, 1H), 3.74 (d, J ) 4.06 Hz, 2H), 4.1 (m,
1H), 4. 22 (m, 1H), 4. 47 (m, 1H), 5.00 (d, J ) 12.82 Hz, 2H),
5.63 (m, 1H), 5.97 (d, J ) 6.62 Hz, 1H), 6.45 (br s, 2H), 7.31-
7.71 (m, 21H), 10.66 (s, 1H); 13C NMR (50 MHz, CDCl3) δ 19.15,
19.33, 26.90, 26.98, 63.43, 71.24, 71.42, 80.41, 85.53, 117.40,
117.64, 127.59, 127.65, 127.77, 129.55, 129.85, 129.95, 132.59,
132.85, 133.12, 133.37, 133.82, 134.79, 135.41, 135.53, 135.83,
136.04, 151.77, 153.45, 159.13; MS (FAB) m/z 822 [M + Na]+,
HRMS (MALDI) Calcd for C45H53N5O5Si2Na+ 822.3477, found
822.3455. Anal. Calcd for C45H53N5O5Si2 ‚H2O: C, 66.06, H,
6.78; N, 8.56. Found: C, 66.38; H, 6.60; N, 8.65.
ethyl acetate layer was dried over anhydrous Na2SO4 and
evaporated to dryness under reduced pressure. The residue
obtained was purified by flash silica gel column chromatog-
raphy and eluted with 10% MeOH in CH2Cl2 to afford 17 (6.46
g, 72% yield): Rf ) 0.46 (10% MeOH in CH2Cl2); 1H NMR (200
MHz, DMSO-d6) δ 0.89 (s, 9H), 1.07 (s, 9H), 3.15-3.46 (m, 5H),
3.62 (dd, J ) 3.2 and 8.3 Hz, J ) 1H), 4.11 (br s, 1H), 4.28 (m,
1H), 4.5 (br s, 1H), 4.56 (t, J ) 5.14 Hz, 1H), 5.97 (d, J ) 6.4
Hz, 1H), 6.48 (br s, 2H), 7.33-7.74 (m, 21H), 10.69 (s, 1H);
13C NMR (50 MHz, DMSO-d6) δ 18.74, 19.06, 26.67, 26.90,
60.02, 63.74, 71.57, 71.84, 80.88, 84.13, 85.12, 116.88, 127.31,
127.94, 130.05, 132.4, 132.6, 133.01, 134.67, 135.09, 135.49,
135.68, 142.73, 147.30, 151.53, 153.96, 156.84; HRMS (MALDI)
Calcd for C44H53N5O6Si2Na+ 826.3427, found 826.3411.
N6-Ben zoyl-5′-O-(ter t-b u t yld ip h en ylsilyl)-2′-O-(2-h y-
d r oxyeth yl)a d en osin e (16). Compound 13a (5.50 g, 8.5
mmol) and 4-methylmorpholine N-oxide (1.43 g, 12.2 mmol)
were dissolved in dioxane (45.4 mL). OsO4 (4% aqueous
solution, 2.0 mL, 0.31 mmol) was added. The reaction mixture
was protected from light and stirred for 3 h. Reaction was
monitored by TLC (5% MeOH in CH2Cl2). The reaction mixture
was diluted with ethyl acetate (100 mL) and washed with
water (1 × 50 mL). The ethyl acetate layer was dried over
anhydrous Na2SO4 and evaporated to afford dihydroxylated
product (5.9 g), Rf 0.17 (5% MeOH in CH2Cl2). The crude
dihydroxylated compound (5.59 g, 8.17 mmol) was dissolved
in anhydrous CH2Cl2 (40.4 mL). To this solution, NaIO4
adsorbed on silica gel (16.34 g, 2 g/mmol, prepared as reported
in ref 20) was added and stirred at ambient temperature for
30 min. The reaction was monitored by TLC (5% MeOH in
CH2Cl2), and the silica gel was filtered and washed thoroughly
with CH2Cl2. The combined organic phase was concentrated
under reduced pressure to yield the aldehyde (5.60 g), Rf 0.3
(5% MeOH in CH2Cl2). The crude aldehyde (5.55 g, 8.5 mmol)
was dissolved in a solution of 1 M pyridinium p-toluene-
sulfonate (PPTS) in anhydrous MeOH (85 mL). The reaction
mixture was protected from moisture. NaBH3CN (1.08 g, 17.3
mmol) was added, and the reaction mixture was stirred at
ambient temperature for 8 h, diluted with ethyl acetate (150
mL), and washed with 5% aqueous NaHCO3 (75 mL) and brine
(75 mL). The ethyl acetate layer was dried (Na2SO4) and
concentrated under reduced pressure. The residue obtained
was purified by flash column chromatography (silica gel, 5%
MeOH in CH2Cl2) to afford 16 (4.31 g, 78%): Rf 0.21 (5% MeOH
in CH2Cl2); 1H NMR (200 MHz, DMSO-d6) δ 0.98 (s, 9H), 3.55
(m, 4H), 3.87 (m, 2H), 4.09 (m, 1H), 4.55 (m, 1H), 4.73 (m,
2H), 5.30 (d, J ) 5.54 Hz, 1H), 6.20 (d, J ) 4.9 Hz, 1H), 7.3-
8.1 (m, 15H), 8.62 (s, 1H), 8.68 (s, 1H),11.23 (br s, 1H); 13C
NMR (50 MHz, CDCl3) δ 19.24, 26.96, 61.52, 63.37, 69.44,
72.47, 82.76, 85.29, 87.19, 123.40, 127.57, 127.88, 128.37,
128.82, 129.97, 132.58, 132.76, 133.01, 133.48, 133.71, 135.47,
135.59; MS (FAB) m/z 654 (M + H)+; HRMS (MALDI) Calcd
for C35H39N5O6SiNa+ 676.2562, found 676.2586.
2′-O-(2-h yd r oxyeth yl)-3′,5′-bis(ter t-bu tyld ip h en ylsilyl)-
gu a n osin e (17). Compound 15 (9.00 g, 11.3 mmol) was
dissolved in a mixture of CH2Cl2 (80 mL) and acetone (50 mL).
To the clear solution obtained, 4-methylmorpholine-N-oxide
(1.89 g, 16.1 mmol) was added. The reaction flask was
protected from light. OsO4 (2.7 mL, 4 wt % aqueous solution,
0.4 mmol) was added, and the reaction mixture was stirred at
room temperature for 6 h. The reaction mixture was concen-
trated to half of its volume, and ethyl acetate (50 mL) was
added. It was then washed with water (30 mL) and brine (30
mL). The ethyl acetate layer was dried over anhydrous Na2SO4
and concentrated to dryness under reduced pressure. The
residue obtained was dissolved in CH2Cl2, and NaIO4, adsorbed
on silica gel (22.60 g, 2 g/mmol), was added and the reaction
mixture stirred for 30 min at room temperature. The reaction
mixture was filtered, and the silica gel was washed thoroughly
with CH2Cl2. The combined CH2Cl2 layer was evaporated to
dryness under reduced pressure. The residue was then dis-
solved in 1 M PPTS in anhydrous MeOH (99.5 mL). The
reaction mixture was protected from moisture by passing a
stream of argon gas through the reaction vessel. To the clear
solution, NaBH3CN (1.14 g, 18.1 mmol) was added and stirred
at room temperature for 4 h. Aqueous NaHCO3 (5%, 50 mL)
was added to the reaction mixture slowly, and the resulting
mixture was extracted with ethyl acetate (2 × 50 mL). The
5′-O-(ter t-Bu tyld ip h en ylsilyl)-2′-O-[2-[(1,3-d ih yd r o-1,3-
d ioxo-2H-isoin d ol-2-yl)oxy]eth yl]-5-m eth ylu r id in e (18).
Compound 7 (20.00 g, 37.0 mmol) was mixed with triphenyl-
phosphine (11.65 g, 44.4 mmol) and N-hydroxyphthalimide
(7.24 g, 44.4 mol). It was dried over P2O5 under vacuum at 40
°C overnight. The reaction mixture was flushed with argon,
and anhydrous THF (370 mL) was added to afford a clear
solution. DEAD (7.00 mL, 44.36 mmol) was added dropwise
to the reaction mixture. The rate of addition was maintained
such that the resulting deep red coloration had just discharged
before the next drop was added. After completion of the
addition, the reaction mixture was stirred at room temperature
for 4 h. The solvent was removed under reduced pressure. The
residue obtained was purified by flash silica gel column
chromatography and eluted with ethyl acetate:hexane (60:40),
to afford 18 (21.82 g, 86%) as a white foam: Rf 0.56 (ethyl
1
acetate:hexane, 60:40); H NMR (400 MHz, DMSO-d6) δ 1.01
(s, 9H) 1.44 (s, 3H), 3.81-3.94 (m, 5H), 4.09 (t, J ) 5.6 Hz,
1H), 4.25 (m, 1H), 4.31 (br s, 2H), 5.18 (d, J ) 5.6 Hz, 1H),
5.90 (d, J ) 6 Hz, 1H), 7.34-7.46 (m, 6H), 7.6-7.65 (m, 5H),
7.82 (m, 4H), 11.32 (s, 1H); 13C NMR (50 MHz, CDCl3) δ 11.8,
19.40, 26.99, 62.62, 68.36, 68.56, 77.64, 83.04, 84.14, 87.50,
110.93, 123.59, 127.86, 129.89, 132.45, 134.59, 134.89, 135.17,
135.44, 150.50, 163.63, 163.97; MS (FAB) m/z 684 [M - H]-;
HRMS (MALDI) Calcd for C36H39N3O9SiNa+ 708.2348, found
708.2325.
N6-Ben zoyl-5′-O-(ter t-bu tyld ip h en ylsilyl)-2′-O-[2-[(1,3-
d ih yd r o-1,3-d ioxo-2H -isoin d ol-2-yl)oxy]e t h yl]a d e n o-
sin e (19). From compound 16 (3.22 g, 4.9 mmol), triphenyl-
phosphine (1.55 g, 5.9 mmol), N-hydroxyphthalimide (0.96 g,
5.9 mmol), anhydrous THF (49.2 mL), and DEAD (0.93 mL,
5.9 mmol), compound 19 (3.60 g, 91.5%) was synthesized
according to the procedure used for the synthesis of compound
18: Rf 0.27 (ethyl acetate: hexane, 7: 3); 1H NMR (200 MHz,
DMSO-d6) δ 0.96 (s, 9H), 3.7-4.0 (m, 5H), 4.31 (m, 2H), 4.55
(m, 1H), 4.76 (m, 1H), 5.32 (d, J ) 5.4 Hz, 1H), 6.14 (d, J )
5.0 Hz, 1H), 7.3-8.1 (m, 19H), 8.58 (s, 1H), 8.60 (s, 1H); 13C
NMR (50 MHz, CDCl3) δ 19.64, 27.38, 63.86, 69.53, 69.91,
77.96, 83.13, 85.56, 87.72, 124.01, 128.8, 129.14, 130.18,
132.13, 132.37, 132.57, 132.91, 133.34, 133.58, 134.37, 134.98,
135.99, 142.20, 149.98, 151.90, 152.98, 163.89, 164.76; MS
(FAB) m/z 821 (M + Na)+; HRMS (MALDI) Calcd for
C43H42N6O8SiNa+ 821.2726, found 821.2742.
3′,5′-O-Bis-(ter t-bu tyld ip h en ylsilyl)-2′-O-[2-[(1,3-d ih y-
d r o-1,3-d ioxo-2H-isoin d ol-2-yl)oxy]eth yl]gu a n osin e (20).
From compound 17 (3.67 g, 4.6 mmol), triphenylphosphine
(1.39 g, 5.3 mmol), N-hydroxyphthalimide (0.87 g, 5.3 mmol),
anhydrous THF (46 mL), and DEAD (0.83 mL, 5.3 mmol),
compound 20 was isolated as a foam (2.98 g, 69%) according
to the procedure used for the synthesis of compound 18.
Compound 20 was purified using flash silica gel column
chromatography eluting with ethyl acetate, 100%, then ethyl
acetate:MeOH, 93:7: 1H NMR (200 MHz, DMSO-d6) δ 0.85
(s, 9H), 0.99 (s, 9H), 3.5-3.8 (m, 3H), 3.95 (m, 2H), 4.11 (m,
2H), 4.24 (m, 1H), 4.47 (m, 1H), 5.94 (d, J ) 5.7 Hz, 1H), 6.41
(br s, 2H), 7.3-7.8 (m, 25H), 10.62 (br s, 1H); 13C NMR (50
MHz, CDCl3) δ 19.1, 19.3, 26.9, 63.2, 68.4, 70.9, 77.2, 81.6,
84.7, 86.0, 123.5, 127.7, 127.8, 128.6, 129.8, 129.9, 130.0, 132.6,
132.9, 133.0, 133.1, 134.5, 135.4, 135.5, 135.7, 135.9, 151.5,
153.3, 159.2, 163.4; HRMS [FAB] calcd for C52H56N6O8Si2Na+

366 J . Org. Chem., Vol. 67, No. 2, 2002
Prakash et al.
971.3582, found: 971.3596. Anal. Calcd for C52H56N6O8Si2
H2O: C, 64.57; H, 6.04; N, 8.69. Found: C, 64.75, H, 5.85, N,
8.50.
of methylhydrazine (0.03 mL, 0.5 mmol) was added. The
reaction mixture was stirred another hour at 5 °C, and the
solids were filtered and washed three times with cold CH2Cl2.
The filtrate was diluted with CH2Cl2 (200 mL) and washed
with water (2 × 100 mL) and brine (100 mL). The organic
phase was dried with MgSO4, and the solvent was evaporated
in a vacuum to give a foam which was further dried in a
vacuum for 1 h. The deprotected crude product was dissolved
in anhydrous MeOH (10 mL) and formaldehyde (0.41 mL, 20
wt % solution in H2O, 2.7 mmol) was added at ambient
temperature. After stirring for 12 h, the solvent was evapo-
rated in a vacuum to give the oxime as the crude foam 24,
which was used for the next step without purification.
3′,5′-O-Bis-(t er t -b u t yld ip h e n ylsilyl)-2′-O-[2-[(1,3-d i-
h yd r o-1,3-d ioxo-2H -isoin d ol-2-yl)oxy]et h yl]-N 2-isob u -
tyr ylgu a n osin e (21). Compound 20 (3.66 g, 3.9 mmol) was
dissolved in anhydrous pyridine (40 mL), and the solution was
cooled to 5 °C in an ice bath. Isobutyryl chloride (0.81 mL, 7.7
mmol) was added dropwise. The reaction mixture was allowed
to warm to 25 °C. After 2h, an additional amount of isobutyryl
chloride (0.40 mL, 3.8 mmol) was added at 25 °C. After 1 h,
the solvent was evaporated under reduced pressure at 30 °C
to give a foam which was dissolved in ethyl acetate (150 mL)
to give a fine suspension. The suspension was washed with
water (2 × 100 mL) and brine (100 mL), and the organic layer
was separated and dried over MgSO4. The solvent was
evaporated in a vacuum to furnish a foam which was purified
by flash silica gel column chromatography eluting with CH2Cl2:
MeOH, 94:6, to afford the title compound as a white foam (2.57
g, 65%): 1H NMR (200 MHz, CDCl3) δ 1.02 (s, 9H), 1.05 (s,
9H), 1.26 (d, J ) 3.1 Hz, 3H), 1.30 (d, J ) 3.2 Hz, 3H), 2.67
(m, 1H), 3.32 (m, 1H), 3.60 (m, 2H), 3.83 (m, 3H), 4.24 (m,
2H), 4.46 (m, 1H), 5.93 (d, J ) 3.3 Hz, 1H), 7.8-7.2 (m, 25H),
8.73 (s, 1H), 11.97 (br s, 1H); HRMS [FAB] calcd for C56H63N6O9-
5′-O-(ter t-Bu t yld ip h en ylsilyl)-2′-O-[2-[(N,N-d im et h yl-
am in o)oxy]eth yl]-5-m eth ylu r idin e (25). Compound 22 (7.71
g, 13.6 mmol) was dissolved in a solution of 1 M PPTS in
anhydrous MeOH (136 mL). The reaction mixture was cooled
to 10 °C in an ice bath, and NaBH3CN (1.71 g, 27.2 mmol)
was added. The reaction mixture was stirred for 10 min at 10
°C and then allowed to come to room temperature. The reaction
mixture was stirred at room temperature for an additional 2
h and monitored by TLC (5% MeOH in CH2Cl2). The reaction
mixture was concentrated to half of its volume, diluted with
ethyl acetate (300 mL), and washed with water (150 mL),
aqueous NaHCO3 solution (5%, 200 mL), and brine (200 mL).
The organic phase was dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The residue obtained
was dissolved in a solution of 1 M PPTS in MeOH (136 mL).
Formaldehyde (2.3 mL, 20 wt % solution in water, 15.0 mmol)
was added, and the reaction mixture was stirred at room
temperature for 10 min. The reaction mixture was cooled to
10 °C in an ice bath, NaBH3CN (1.71 g, 27.2 mmol) was added,
and the reaction mixture stirred at 10 °C for 10 min. The
reaction mixture was allowed to come to ambient temperature
and stirred for 2 h. It was then concentrated to half of its
volume and diluted with ethyl acetate (300 mL). The organic
phase was washed with water (240 mL), aqueous NaHCO3 (5%,
250 mL), and brine (250 mL). The ethyl acetate layer was dried
over anhydrous Na2SO4 and concentrated under reduced
pressure. The residue obtained was purified by flash silica gel
column chromatography and eluted with 5% MeOH in CH2Cl2
to yield compound 25 in 80% yield (6.34 g): Rf 0.35 (5% MeOH
in CH2Cl2); 1H NMR (200 MHz, DMSO-d6) δ 1.03 (s, 9H), 1.44
(s, 3H), 2.43 (s, 6H), 3.6-4.1 (m, 8H), 4.25 (q, J ) 4.4 Hz, 1H),
5.22 (d, J ) 5.48 Hz, 1H), 5.94 (d, J ) 5.92 Hz, 1H), 7.3-7.7
(m, 11H), 11.41 (s, 1H); 13C (50 MHz, CDCl3) δ 11.65, 19.47,
27.12, 47.53, 63.39, 68.99, 70.14, 70.73, 82.95, 84.66, 87.34,
111.14, 127.68, 127.97, 130.1, 134.62, 135.27, 135.54, 150.47,
163.87; MS (FAB) m/z 584 [M + H]+; HRMS (FAB) Calcd for
+
Si2 1019.4195, found: 1019.4213.
5′-O-(t er t -Bu t yld ip h e n ylsilyl)-2′-O-[2-[(m e t h yle n e -
am in o)oxy]eth yl]-5-m eth ylu r idin e (22). Compound 18 (12.67
g, 18.5 mmol) was dissolved in anhydrous CH2Cl2 ((185 mL).
To this was added methylhydrazine (1.3 mL, 24.4 mmol)
dropwise at -10 °C. The reaction mixture was stirred at -10
to 0 °C for 1 h. The white precipitate formed was filtered and
washed with ice cold CH2Cl2 (200 mL). The combined CH2Cl2
layer was concentrated to afford the 5′-O-(tert-butyldiphenyl-
silyl)-2′-O-[2-(aminooxy)ethyl]-5-methyluridine, which was then
dissolved in MeOH (277 mL). Formaldehyde (3.1 mL, 20 wt %
solution in water, 20.3 mmol) was added, and the mixture was
stirred at room temperature for 5 h. The solvent was removed
in a vacuum and the residue was purified by silica gel column
chromatography and eluted with 5% MeOH in CH2Cl2 to afford
22 in 74% isolated yield (7.71 g) as a white foam: Rf 0.32 (5%
1
MeOH in CH2Cl2); H NMR (200 MHz, DMSO-d6) δ 1.03 (s,
9H), 1.45 (s, 3H), 3.6-4.08 (m, 6H), 4.13 (t, J ) 4.3 Hz, 2H),
4.23 (q, J ) 5.38 and 4.76 Hz, 1H), 5.20 (d, J ) 5.92 Hz, 1H),
5.90 (d, J ) 5.42 Hz, 1H), 6.54 (d, J ) 7.7 Hz, 1H), 6.99 (d, J
) 7.64 Hz, 1H), 7.37-7.67 (m, 11H), 11.39 (s, 1H); 13C NMR
(50 MHz, DMSO-d6) δ 11. 84, 19.41, 27.01, 62.96, 68.57, 70.01,
72.61, 82.67, 84.33, 87.14, 111.12, 127.90, 129.98, 132.37,
133.1, 134.93, 135.18, 135.44, 137.96, 150.50, 164.02; MS (ES)
m/z 590 [M + Na]+; HRMS (MALDI) Calcd for C29H37N3O7-
SiNa+ 590.2293, found 590.2304. Anal. Calcd for C29H37N3O7-
Si: C, 61.351; H, 6.57; N, 7.40. Found C, 60.53; H, 6.52; N,
7.27.
C
30H42N3O7Si+ 584.2792 found 584.2795.
N6-Ben zoyl-5′-O-(ter t-bu tyld ip h en ylsilyl)-2′-O-{2-[(N,N-
d im eth yla m in o)oxy]eth yl)a d en osin e (26). From compound
23 (2.2 g, 3.2 mmol) in a solution of 1 M PPTS in MeOH (32
mL), NaBH3CN (0.41 g, 6.5 mmol), formaldehyde (0.55 mL,
20 wt % solution in water, 3.6 mmol), and NaBH3CN (0.41 g,
6.5 mmol), compound 26 was obtained according to the
procedure used for the synthesis of compound 25 in 81.8% yield
N 6-B e n zo y l-5′-O -(t er t -b u t y ld ip h e n y ls ily l)-2′-O -{2-
[(m eth ylen ea m in o)oxy]eth yl)a d en osin e (23). From com-
pound 19 (3.50 g, 4.4 mmol) in CH2Cl2 (44 mL), N-methyl-
hydrazine (0.28 mL, 5.3 mmol), MeOH (65.7 mL), and form-
aldehyde (0.73 mL, 20 wt % solution in water, 4.8 mmol),
compound 23 was obtained according to the procedure used
for the synthesis of compound 22 in 83% yield (2.47 g): Rf 0.37
(5% MeOH in CH2Cl2);1H NMR (200 MHz, CDCl3) δ 1.11 (s,
9H), 3.87 (m, 2H), 4.07 (m, 2H), 4.26 (m, 3H), 4.59 (m, 2H),
6.24 (d, J ) 4.0 Hz, 1H), 6.46 (d, J ) 7.9 Hz, 1H), 7.02 (d, J )
7.9 Hz, 1H), 7.3-8.1 (m, 15H), 8.31 (s, 1H), 8.78 (s, 1H), 9.15
(br s, 1H); 13C NMR (50 MHz, CDCl3) δ 19.29, 27.01, 63.41,
69.36, 70.18, 72.73, 82.52, 85.22, 87.17, 123.68, 127.86, 128.36,
128.73, 129.94, 131.90, 132.01, 132.65, 133.04, 133.84, 135.52,
135.63, 138.02, 141.67, 149.69, 151.59, 152.71, 164.66; MS
(FAB) m/z 681 (M + H)+; HRMS (MALDI) Calcd for C36H40N6O6-
SiNa+ 703.2671, found 703.2640.
1
(1.90 g): Rf 0.29 (5% MeOH in CH2Cl2); H NMR (200 MHz,
CDCl3) δ 1.11 (s, 9H), 2.62 (s, 6H), 3.72 (m, 1H), 3.86 (m, 2H),
3.93 (m, 1H), 4.07 (m, 2H), 4.21 (m, 1H), 4.30 (m, 1H), 4.55
(m, 2H), 6.23 (d, J ) 3.7 Hz, 1H), 7.4-8.1 (m, 15H), 8.31 (s,
1H), 8.78 (s, 1H), 9.09 (br s, 1H); 13C NMR (50 MHz, CDCl3) δ
19.30, 27.01, 47.53, 63.53, 69.53, 70.13, 70.90, 82.66, 85.39,
87.23, 127.83, 128.34, 128.5, 128.57, 128.82, 129.89, 131.84,
132.01, 132.65, 135.63, 141.70, 149.59, 152.74; MS (FAB) m/z
719 (M + Na)+. Anal. Calcd for C37H44N6O6Si: C, 63.77; H,
6.36; N, 12.06. Found: C, 63.03; H, 6.31; N, 12.01.
3′,5′-O-Bis-(ter t-b u t yld ip h en ylsilyl)-2′-O-{2-[(N,N-d i-
m et h yla m in o)oxy]et h yl}-N2-isob u t yr ylgu a n osin e (27).
Compound 24 (crude product) was dissolved in anhydrous
MeOH (24 mL). Pyridinium p-toluenesulfonate (6.03 g, 24.0
mmol) was added to the solution, which was then cooled to 5
°C in an ice bath, after which NaBH3CN (0.31 g, 4.9 mmol)
was added. The reaction mixture was allowed to warm to
ambient temperature, and after 2 h ethyl acetate (200 mL)
3′,5′-O-Bis-(ter t-b u t yld ip h en ylsilyl)-2′-O-{2-[(m et h yl-
en ea m in o)oxy]eth yl)-N2-isobu tyr ylgu a n osin e (24). Com-
pound 21 (2.5 g, 2.5 mmol) was dissolved in anhydrous CH2Cl2
(25 mL), the solution was cooled to 5 °C in an ice bath, and
methylhydrazine (0.14 mL, 2.6 mmol) was added dropwise.
After the reaction stirred for 1 h at 5 °C, an additional amount

Modified Nucleosides and Oligonucleotides
J . Org. Chem., Vol. 67, No. 2, 2002 367
was added. The organic layer was washed with H2O (100 mL)
and brine (2 × 100 mL) and dried with MgSO4. The solvent
was evaporated under reduced pressure to give the N-methyl-
aminooxyethyl derivative as a crude foam. This mixture was
then added to 1 M PPTS in MeOH (24 mL), and formaldehyde
(0.41 mL, 20 wt % solution in H2O, 2.7 mmol) was added. The
reaction mixture was stirred at room temperature for 10 min
and then cooled to 5 °C in an ice bath. To this was added
NaBH3CN (0.31 g, 4.9 mmol), and the reaction mixture was
allowed to warm to room temperature. After 2 h it was
concentrated to half of its volume and diluted with ethyl
acetate (100 mL) and then washed with 5% aqueous NaHCO3
(75 mL) and brine (75 mL). The organic phase was dried over
anhydrous Na2SO4 and concentrated. The residue obtained
was purified by flash column chromatography using silica gel
and CH2Cl2-MeOH, 92:7.5, to provide the title compound 27
as a solid (2.15 g, 96%): 1H NMR (200 MHz, CDCl3) δ 0.98 (s,
9H), 1.11 (s, 9H), 1.25 (d, J ) 6.8 Hz, 6H), 2.43 (s, 6H), 3.36
(m, 3H), 3.64 (m, 3H), 4.10 (m, 2H), 4.49 (m, 1H), 6.04 (d, J )
6.4 Hz, 1H), 7.2-7.8 (m, 21H), 8.12 (s, 1H), 11.9 (br s, 1H);
13C NMR (50 MHz, CDCl3) δ 18.84, 19.08, 19.24, 26.85, 36.32,
47.47, 63.43, 68.96, 69.87, 71.23, 82.06, 85.54, 127.63, 127.76,
129.88, 132.54, 132.78, 133.08, 135.41, 135.49, 136.67, 136.28,
136.79, 147.23, 148.08, 155.41, 178.13; HRMS (FAB) calcd for
tography and CH2Cl2:MeOH, 85:15, to afford the title com-
pound 30 as a foam (1.35 g), which was contaminated with
(C2H5)3N‚3HF (2.7 mol equiv): 1H NMR (400 MHz, DMSO-
d6) δ 2.34 (s, 6H), 2.75 (m, 1H), 3.40-3.73 (m, 6H), 3.93 (m,
1H), 4.27 (m, 1H), 4.40 (m, 1H), 5.09 (m, 1H), 5.19 (d, J ) 4.4
Hz, 1H), 5.88 (d, J ) 6.8 Hz, 1H), 8.28 (s, 1H);. 13C NMR (100
MHz, CD3OD) δ 19.44, 36.99, 47.87, 62.52, 70.73, 70.82, 71.65,
84.34, 87.19, 87.82, 121.20, 139.86, 150.00, 150.60, 157.53,
181.73; HRMS (FAB) calcd for C18H28N6O7Na+ 463.1917; found
463.1901.
5′-O-(4,4′-Dim e t h oxyt r it yl)-2′-O-[2-[(N ,N -d im e t h yl-
a m in o)oxy]eth yl]-5-m eth ylu r id in e (31). Compound 28 (2.7
g, 7.8 mmol) was mixed with DMAP (0.20, 1.6 mmol) and dried
over P2O5 in a vacuum overnight at 40 °C. It was then
coevaporated with anhydrous pyridine (20 mL). The residue
obtained was dissolved in anhydrous pyridine (18 mL), and
4,4′-dimethoxytrityl chloride (3.18 g, 9.4 mmol) was added. The
reaction mixture was stirred at room temperature for 6 h
under an argon atmosphere. Pyridine was removed under
reduced pressure, and the resulting residue was purified by
flash silica gel column chromatography and eluted with 10%
MeOH in CH2Cl2 (containing 0.5% pyridine) to afford 31 in
1
78% yield (3.94 g): Rf 0.44 (10% MeOH in CH2Cl2); H NMR
(200 MHz, CDCl3) δ 1.35 (s, 3H), 2.60 (s, 6H), 3.43 (dd, J )
2.72, 8.2 Hz, 1H), 3.57 (dd, J ) 1.86 Hz, 9.06 Hz, 1H), 3.7-
3.89 (m, 3H), 3.80 (s, 6H), 4.03-4.19 (m, 4H), 4. 45 (q, J )
5.38 Hz, 7.36 Hz, 1H), 5.99 (d, J ) 3.2 Hz, 1H), 6.84 (d, J )
8.84 Hz, 4H), 7.2-7.44 (m, 9H), 7.68(s, 1H), 8.22 (s, 1H); 13C
(50 MHz, CDCl3) δ 11.75, 47.54, 55.23, 62.42, 69.23, 70.10,
70.76, 83.05, 83.67, 86.91, 87.87, 110.92, 113.34, 123.69,
127.10, 127.95, 128.28, 130.17, 135.37, 135.50, 144.45, 149.76,
150.45, 156.81, 163.95; MS (FAB) m/z 648 (M + H)+, MS (ES)
m/z 646 [M - H], HRMS (MALDI) Calcd for C35H41N3O9Na+
670.2735, found 670.2731. Anal. Calcd for C35H41N3O9: C,
64.90; H, 6.38; N, 6.49. Found: C, 64.48, H, 6.41; N, 6.41.
N6-Ben zoyl-5′-O-(4,4′-d im et h oxyt r it yl)-2′-O-[2-[(N,N-
d im eth yla m in o)oxy]eth yl]a d en osin e (32). From compound
29 (1.00 g, 2.3 mmol), DMAP (0.05 g, 0.41 mmol), anhydrous
pyridine (5 mL), and 4,4′-dimethoxytrityl chloride (0.88 g, 2.6
mmol), compound 32 was obtained in 75% yield (1.24 g)
according to the procedure described for compound 31, except
that the reaction mixture was stirred for 8 h at room temper-
ature under an argon atmosphere: Rf 0.20 (5% MeOH in
C
C
50H64N6O7Si2Cs+ 1049.3429; found 1049.3475. Anal. Calcd for
50H64N6O7Si2: C, 65.47; H, 7.03; N, 9.16; found: C, 65.29, H,
6.95, N, 9.09.
2′-O-{2-[(N,N-d im eth yla m in o)oxy]eth yl}-5-m eth ylu r i-
d in e (28). Triethylamine trihydrofluoride (8.7 mL, 53.2 mmol)
was dissolved in anhydrous THF (106 mL), and triethylamine
(3.70 mL, 26.6 mmol) was added. This mixture was then added
to compound 25 (6.20 g, 10.6 mmol), and the resulting mixture
was stirred at room temperature for 24 h. The reaction was
monitored by TLC (5% MeOH in CH2Cl2). The solvent was
removed under reduced pressure. The residue obtained was
purified by flash silica gel column chromatography and eluted
with 10% MeOH in CH2Cl2 to afford compound 28 in 93% yield
1
(3.39 g): Rf 0.27 (5% MeOH in CH2Cl2); H NMR (200 MHz,
DMSO-d6) δ 1.76 (s, 3H), 2.43 (s, 6H), 3.54-3.66 (br s, 6H),
3.84 (d, J ) 3.3 Hz, 1H), 3.95 (t, J ) 5.1 Hz, 1H), 4.09 (q, J )
4.4 and 4.88 Hz, 1H), 5.08 (d, J ) 5.74 Hz, 1H), 5.17 (t, J )
4.88 Hz, 1H), 5.86 (d, J ) 5.48 Hz, 1H), 7.78 (s, 1H); 13C NMR
(50 MHz, CDCl3) δ 12.61, 47.66, 61.6, 69.24, 70.32, 70.88,
82.25, 85.51, 90.20, 110.85, 138.01, 150.93, 164.63; MS (FAB)
1
CH2Cl2); H NMR (400 MHz, CDCl3) δ 2.61 (s, 6H), 3.47 (m,
+
m/z 346 (M + H)+; HRMS (FAB) Calcd for C14H24O7N3
346.1614, found 346.1614.
2H), 3.79 (s, 6H), 3.86 (m, 3H), 4.06 (m, 1H), 4.28 (m, 2H),
4.49 (m, 1H), 4.70 (m, 1H), 6.21 (d, J ) 4.5 Hz, 1H), 6.8-8.1
(m, 18H), 8.24 (s, 1H), 8.75 (s, 1H), 9.07 (br s, 1H); 13C NMR
(100 MHz, CDCl3) δ 47.42, 55.09, 63.11, 69.95, 70.81, 82.29,
84.26, 86.46, 87.19, 113.06, 123.46, 126.82, 127.82, 128.07,
128.64, 129.97, 132.58, 133.57, 135.54, 141.81, 144.42, 149.52,
151.46, 152.52, 158.44, 164.71; MS (FAB) m/z 759 (M - H)-;
N6-Ben zoyl-2′-O-{2-[(N,N-d im et h yla m in o)oxy]et h yl)-
a d en osin e (29). From compound 26 (1.8 g, 2.6 mmol),
triethylamine trihydrofluoride (4.1 mL, 25.0 mmol), anhydrous
THF (25.0 mL), and triethylamine (1.74 mL, 12.5 mmol),
compound 29 was synthesized according to the procedure used
for the synthesis of compound 28 in 89% yield (1.00 g): Rf 0.40
(10% MeOH in CH2Cl2); 1H NMR (200 MHz, DMSO-d6) δ 2.34
(s, 6H), 3.66 (m, 6H), 4.02 (m, 1H), 4.35 (m, 1H), 4.62 (m, 1H),
5.17 (m, 1H), 5.27 (d, J ) 5.36 Hz, 1H), 6.16 (d, J ) 6.0 Hz,
1H,) 7.5-7.7 (m, 5H), 8.75 (s, 1H), 8.76 (s, 1H); 13C NMR (50
MHz, CDCl3) δ 47.40, 63.16, 70.03, 70.79, 82.26, 88.06, 89.59,
124.54, 127.93, 128.74, 132.81, 133.37, 143.31, 150.34, 150.54,
151.95, 164.78; LRMS (FAB) m/z 481 [M + Na]+; HRMS
HRMS (FAB) Calcd for
C
42H44N6O8Cs+ 893.2275, found
893.2252. Anal. Calcd for C42H44N6O8•H2O: C, 64.77; H, 5.95;
N, 10.79. Found: C, 64.71; H, 5.65; N, 10.71.
5′-O-(4,4′-Dim e t h oxyt r it yl)-2′-O-[2-[(N ,N -d im e t h yl-
a m in o)oxy]eth yl]-N2-isobu tyr ylgu a n osin e (33). From com-
pound 30 (0.85 g, 1.9 mmol), anhydrous pyridine (20 mL),
DMAP (0.05 g, 0.4 mmol), and 4,4′-dimethoxytrityl chloride
(1.08 g, 3.2 mmol), compound 33 was synthesized according
to the procedure described for 31 in 81% yield (1.37 g) after
flash silica gel column chromatography using silica (pretreated
with 1% NEt3) eluting with CH2Cl2:MeOH:NEt3, 90:10:1, to
afford the title compound as a foam: 1H NMR (400 MHz,
CDCl3) δ 0.88 (d, 3H), 0.97 (d, 3H), 2.18 (m, 1H), 2.56 (s, 6H),
3.25 (m, 1H), 3.48 (m, 1H), 3.75 (s, 3H), 3.76 (s, 3H), 3.80 (m,
2H), 4.22 (m, 1H), 4.49 (m, 1H), 4.71 (m, 1H), 5.94 (d, J ) 5.9
Hz, 1H), 6.7-7.5 (m, 14), 7.87 (s, 1H); 13C NMR (100 MHz,
CDCl3) δ 18.5, 35.7, 47.3, 55.1, 63.5, 69.5, 69.9, 70.5, 81.8, 84.2,
86.2, 86.6, 113.1, 121.8, 126.9, 127.9, 129.9, 135.5, 135.8, 138.3,
144.7, 147.7, 148.3,156.0, 158.5, 178.8; HRMS (FAB) calcd for
+
(MALDI) Calcd for C21H27N6O6 459.1992, found 459.2001.
2′-O-{2-[(N ,N -Dim e t h yla m in o)oxy]e t h yl}-N 2-isob u -
tyr ylgu a n osin e (30). Triethylamine trihydrofluoride (7.56
mL, 46.4 mmol) was added to anhydrous THF (40 mL) followed
by addition of triethylamine (3.24 mL, 23.2 mmol). Compound
27 (2.13 g, 2.3 mmol) was added, and after 24 h the solvent
was evaporated in a vacuum to give a gelatin which was
dissolved in n-BuOH (100 mL). Saturated NaHCO3 was added
slowly until evolution of the gas ceased and the pH reached 7.
The organic phase was separated and washed with saturated
NaHCO3 (50 mL) and brine (50 mL), and then the aqueous
washes were combined and extracted with n-BuOH (3 × 15
mL). The solvent was evaporated in a vacuum at 50 °C to give
an oil, which was dissolved in MeOH and adsorbed onto silica
gel (10 g) by evaporation of the solvent. The product adsorbed
on silica gel was purified by flash silica gel column chroma-
C
C
39H46N6O9Na+ 765.3224; found 765.3215. Anal. Calcd for
39H46N6O9: C, 63.06; H, 6.24; N, 11.31. Found: C, 62.61, H,
6.26, N, 11.10.
5′,3′-O-Bis(ter t-b u t yld ip h e n ylsilyl)-2′-O-{2-[(N,N -d i-
m eth yla m in o)oxy]eth yl)-5-m eth ylu r id in e (34). Compound

368 J . Org. Chem., Vol. 67, No. 2, 2002
Prakash et al.
25 (7.1 g, 12.2 mmol) was dissolved in anhydrous DMF (74
mL). To this were added imidazole (11.3 g, 165.7 mmol) and
tert-butyldiphenylsilyl chloride (21.60 mL, 82.9 mmol), and the
reaction mixture was stirred for 14 h at 60 °C. The reaction
mixture was allowed to come to room temperature before being
poured into water (300 mL). The resulting mixture was
extracted with ethyl acetate (2 × 100 mL). The organic phase
was dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The residue was purified by flash silica gel
column chromatography and eluted with ethyl acetate:hexane
(1:1) to yield 34 (8.54 g, 85%): Rf 0.42 (ethyl acetate:hexane,
133.02, 133.13, 133.47, 135.21, 135.45, 135.83, 136.09, 136.43,
137.22, 147.99, 159.6, 179.59; MS (ES), m/z 924.39 [M - H]-,
HRMS (MALDI) Calcd for C53H64N4O7Si2Na+ 947.4206, found
947.4066.
N4-Ben zoyl-2′-O-{2-[(N,N-d im eth yla m in o)oxy]eth yl}-5-
m eth ylcytid in e (37). Compound 36 (5 g, 5.4 mmol) was dried
over P2O5 under high vacuum. In a 100 mL round-bottom flask,
triethylamine trihydrofluoride (8.82 mL, 54.0 mmol) was
dissolved in anhydrous THF (39 mL). Triethylamine (3.8 mL,
27 mmol) was added to this solution, and the mixture was
quickly poured onto compound 36. The resulting mixture was
stirred at room temperature overnight. The solvent was
removed in a vacuum and the residue dissolved in ethyl acetate
(100 mL). The organic phase was washed with 5% aqueous
NaHCO3 (80 mL) and brine (80 mL). The ethyl acetate layer
was dried over anhydrous Na2SO4 and concentrated in a
vacuum under reduced pressure. The resulting residue was
dissolved in CH2Cl2 (20 mL) and added dropwise into vigor-
ously stirring hexane (600 mL). The precipitate formed was
collected by filtration to afford 37 (2.36 g, 97%) as a white
solid: Rf 0.25 (5% MeOH in CH2Cl2); 1H NMR (200 MHz,
DMSO-d6) δ 2.00 (s, 3H), 2.45 (s, 6H), 3.7 (m, 6H), 3.90 (m,
1H), 4.00 (t, J ) 4.5 Hz, 1H), 4.14 (q, t ) 5.4 Hz, 1H), 5.09 (d,
J ) 5.86 Hz, 1H), 5.28 (t, J ) 4.76 Hz, 1H), 5.86 (d, J ) 3.42
Hz, 1H), 7.52 (m, 3H), 8.19 (m, 3H), 12.98 (s, 1H); 13C NMR
(50 MHz, CDCl3) δ 13.53, 47.50, 61.66, 69.08, 70.08, 70.92,
81.80, 85.50, 91.52, 111.81, 128.12, 129.88, 132.53, 136.94,
139.00, 148.12, 159.60, 179.53; HRMS (FAB) Calcd for
1
1:1); H NMR (400 MHz, CDCl3) δ 0.99 (s, 9H), 1.09 (s, 9H),
1.43 (s, 3H), 2.5 (s, 6H), 3.27 (dd, J ) 2 and 9.6 Hz, 1H), 3.43
(m, 1H), 3.56 (m, 1H), 3.68 (m, 2H), 3.78 (m, 2H), 4.00 (d, J )
2.4 Hz, 1H), 4.39 (m, 1H), 6.27 (d, J ) 6.8 Hz, 1H), 7.28-7.76
(m, 21H); 13C NMR (50 MHz, CDCl3) δ 11.71, 18.95, 19.33,
26.54, 26.86, 47.55, 63.62, 68.69, 70.26, 71.54, 81.67, 85.32,
85.86, 111.131, 127.56, 127.88, 129.48, 129.83, 129.93, 132.08,
132.92, 133.36, 134.77, 135.07, 135.36, 135.76, 136.02; HRMS
(MALDI) Calcd for C46H60N3O7Si2+ 822.3964, found 822.3938.
3′,5′-O-Bis(ter t-b u t yld ip h e n ylsilyl)-2′-O-{2-[(N,N -d i-
m eth yla m in o)oxy]eth yl}-5-m eth ylcytid in e (35). A suspen-
sion of 1,2,4-triazole (17.61 g, 255 mmol) in anhydrous CH3CN
(98 mL) was cooled in an ice bath for 5 to 10 min under an
argon atmosphere. To this, a cold suspension of POCl3 (5.6 mL,
60 mmol) was added slowly over 10 min and stirring continued
for an additional 5 min. Triethylamine (41.8 mL, 300 mmol)
was added slowly over 30 min, keeping the bath temperature
around 0-2 °C. The reaction mixture was stirred at 0-2 °C
for an additional 30 min. Compound 34 (6.16 g, 7.5 mmol) was
added in anhydrous CH3CN (45.8 mL) in one portion and
stirred for 10 min. The reaction mixture was removed from
the ice bath and stirred at room temperature for 4 h. It was
next concentrated to one-third of its volume, diluted with ethyl
acetate (300 mL), and washed with water (2 × 200 mL) and
brine (200 mL). The organic phase was dried over anhydrous
Na2SO4 and concentrated under reduced pressure. The result-
ing residue was dissolved in a solution of aqueous NH3 (24
mL, 30 wt % solution) and dioxane (118 mL). The reaction
mixture was stirred at room-temperature overnight in a
pressure bottle. The solvent was removed in a vacuum, and
the resulting residue was purified by flash silica gel column
chromatography and eluted with 5% MeOH in CH2Cl2 to yield
35 (5.79 g, 94%) as a white foam: Rf 0.27 (5% MeOH in
C
C
21H28N4O7Na+ 471.1856, found 471.1861. Anal. Calcd for
21H28N4O7: C, 56.24; H, 6.29; N, 12.49. Found: C, 56.01; H,
6.27; N, 12.37.
N4-Ben zoyl-5′-O-(4,4′-d im et h oxyt r it yl)-2′-O-{2-[(N,N-
d im eth yla m in o)oxy]eth yl}-5-m eth ylcytid in e (38). Com-
pound 37 (2.30 g, 5.1 mmol) was dried over P2O5 in a vacuum
overnight. It was then coevaporated with anhydrous pyridine
(10 mL), and the residue obtained was dissolved in anhydrous
pyridine (14 mL). To this was added DMAP (0.063 g, 0.5
mmol), and the solution was stirred at room temperature
under an argon atmosphere for 4 h. Pyridine was removed in
a vacuum, and the residue obtained was dissolved in ethyl
acetate (125 mL) and washed with 5% aqueous NaHCO3 (75
mL) and brine (75 mL). The ethyl acetate layer was dried over
anhydrous Na2SO4 and concentrated to dryness under reduced
pressure. The residue obtained was purified by flash silica gel
column chromatography and eluted with ethyl acetate:hexane:
pyridine 60:39:1 to yield compound 38 (2.77 g, 72%): Rf 0.38
1
CH2Cl2); H NMR (200 MHz, DMSO-d6) δ 0.9 (s, 9H), 1.02 (s,
9H), 1.38 (s, 3H), 2.37 (s, 6H), 3.19 (m, 1H), 3.37 (m, 1H), 3.50
(br s, 3H), 3.69 (m, 2H), 3.96 (br s, 1H), 4.34 (br s, 1H), 6.14
(d, J ) 5.98 Hz, 1H), 6.83 (br s, 1H), 7.17 (s, 1H), 7.32-7.68
(m, 21H); 13C NMR (50 MHz, CDCl3) δ 12.31, 19.26, 19.43,
26.87, 27.03, 47.67, 63.39, 68.31, 70.61, 71.24, 82.21, 84.04,
87.06, 101.89, 127.56, 127.7, 127,82, 129.82, 132.48. 133.1,
133.29, 133.42, 135.22, 135.41, 135.79, 136.04, 137.62, 155.83,
165.69; MS (ES) m/z 821.3 [M + H]+, HRMS (MALDI) Calcd
for C46H60N4O6Si2Na+ 843.3944, found 843.3967.
1
(ethyl acetate:hexane; 60:40); H NMR (200 MHz, DMSO-d6)
δ 1.61 (s, 3H), 2.48 (s, 6H), 3.25-3.4 (m, 2H), 3.77 (br s, 10H),
4.09 (m, 2H), 4.3 (m, 1H), 5.23 (d, J ) 6.46 Hz, 1H), 5.9 (d, J
) 3.42 Hz, 1H), 6.94 d, J ) 8.8 Hz, 4H), 7.22-7.66 (m, 12H),
7.83 (s, 1H), 8.17 (d, J ) 6.84 Hz, 2H), 12.96 (s, 1H); 13 C NMR
(50 MHz, CDCl3) δ 12.79, 47.52, 55.21, 61.97, 68.8, 69.94,
71.03, 83.23, 83.65, 86.81, 88.30, 111.85, 113.25, 127.09,
128.03, 128.18, 129.83, 130.14, 132.36, 135.29, 136.58, 137.13,
144.37, 148.03, 158.79, 159.77, 179.59; HRMS (FAB) Calcd for
N4-Ben zoyl-3′,5′-O-bis(ter t-bu tyld ip h en ylsilyl)-2′-O-{2-
[(N,N-d im eth yla m in o)oxy]eth yl}-5-m eth ylcytid in e (36).
Compound 35 (5.79 g, 7.1 mmol) was dissolved in anhydrous
DMF (18.1 mL). Benzoic anhydride (2.45 g, 10.9 mmol) was
added, and the reaction mixture was stirred at room-temper-
ature overnight. Water (500 mL) was added, and the reaction
mixture was extracted with ethyl acetate (2 × 150 mL). The
C
C
42H46N4O9Cs+ 883.2319, found 883.2351. Anal. Calcd for
42H46N4O9: C, 67.19; H, 6.18; N, 7.46. Found: C, 66.96; H,
6.12; N, 7.37.
5′-O-(4,4′-Dim e t h oxyt r it yl)-2′-O-{2-[(N ,N -d im e t h yl-
a m in o)oxy]et h yl}-5-m et h ylu r id in e-3′-O-su ccin a t e (39).
The nucleoside 31 (1.00 g, 1.6 mmol) was mixed with succinic
anhydride (0.24 g, 2.4 mmol) and DMAP (0.10 g, 0.8 mmol)
and dried in a vacuum at 40 °C overnight. The mixture was
dissolved in anhydrous ClCH2-CH2Cl (3.90 mL), (C2H5)3N
(0.43 mL, 3.1 mmol) was added and the solution stirred at
room temperature under argon atmosphere for 4 h. It was then
diluted with CH2Cl2 (50 mL) and washed with ice cold aqueous
citric acid (10 wt %, 50 mL) and water (50 mL). The organic
phase was dried over anhydrous Na2SO4 and concentrated to
dryness. The residue obtained was purified by flash silica gel
column chromatography and eluted with 10% MeOH in CH2Cl2
to afford compound 39 in 82% isolated yield (0.95 g): Rf 0.48
(10% MeOH in CH2Cl2); 1H NMR (200 MHz, DMSO-d6) δ 1.45
(s, 3H), 2.40 (s, 6H), 2.57 (m, 4H), 3.16-3.38 (m, 2H), 3.61 (m,
4H), 3.74 (s, 6H), 4.15 (m, 1H), 4.36 (m, 1H), 5.29 (m, 1H),
organic phase was washed with
a saturated solution of
NaHCO3 in water (150 mL) and brine (150 mL). The ethyl
acetate layer was dried over anhydrous Na2SO4 and concen-
trated in vacuum. The residue obtained was purified by flash
silica gel column chromatography and eluted with 5% MeOH
in CH2Cl2 to yield 36 (5.68 g, 87%) as a white foam: Rf 0.64,
1
(5% MeOH in CH2Cl2); H NMR (200 MHz, DMSO-d6) δ 0.92
(s, 9H), 1.04 (s, 9H), 1.59 (s, 3H), 2.37 (s, 6H), 3.27 (m, 1H),
3.41-3.6 (m, 4H), 3.77 (d, J ) 12.22 Hz, 1H), 3.90 (t, J ) 5.86
Hz, 1H), 4.08 (br s, 1H), 4.4 (t, J ) 4.14 Hz, 1H), 6.08 (d, J )
5.76 Hz, 1H), 7.33-7.75 (m, 24H), 8.12 (d, J ) 6.72 Hz, 2H),
12.59 (br s, 1H); 13C NMR (50 MHz, CDCl3) δ 12.82, 19.31,
19.44, 26.94, 27.08, 47.66, 63.69, 68.83, 70.33, 71.65, 82.03,
85.49, 86.47, 112.11, 127.69, 127.97, 129.90, 132.26, 132.38.

Modified Nucleosides and Oligonucleotides
J . Org. Chem., Vol. 67, No. 2, 2002 369
Ta ble 3. HP LC a n d Ma ss Sp ectr a l An a lysis of th e
2′-O-DMAOE Oligon u cleotid es Used for Tm An a lysis
5.87 (d, J ) 6.24 Hz, 1H), 6.91 (d, J ) 8.78 Hz, 4H), 7.23-
7.40 (m, 9H), 7.51 (s, 1H), 11.47 (s, 1H), 12.25 (br s, 1H); 13C
NMR (50 MHz, CDCl3) δ 11.75, 29.47, 47.36, 55.29, 62.36,
69.74, 70.32, 70.95, 80.60, 81.31, 87.12, 111.373, 113.38,
127.29, 128.10, 130.18, 135.19, 135.52, 144.14, 150.60, 158.84,
164.29, 171.72, 175.35; MS (FAB) m/z 748 [M + H]+; HRMS
mass
oligo.
no.
HPLC retention
time, mina
calcd
found
48
50
52
54
56
58
5246.36
5906.36
7018.43
7148.16
8300.50
8605.56
5245.60
5905.60
7017.69
7147.15
8301.67
8605.50
23.58
26.21
27.95
29.99
27.93
31.54
+
(FAB) Calcd for C39H46N3O12 748.3082 found 748.3074.
5′-O-(4,4′-Dim et h oxyt r it yl)-N6-b en zoyl-2′-O-{2-[(N,N-
d im eth yla m in o)oxy]eth yl}a d en osin e-3′-O-su ccin a te (40).
From compound 32 (2.5 g, 3.3 mmol), succinic anhydride (0.49
g, 4.9 mmol), DMAP (0.31 g, 2.6 mmol), triethylamine (0.92
mL, 6.6 mmol), and 1,2-dichloroethane (9.13 mL), compound
40 was isolated in 85% yield (2.4 g), according to the procedure
used for the synthesis of compound 39. Compound 40 was
purified by flash silica gel column chromatography (10% MeOH
in CH2Cl2): Rf 0.11 (5% MeOH in CH2Cl2); 1H NMR (200 MHz,
CDCl3) δ 2.48 (s, 6H), 2.71 (m, 4H), 3.39-3.88 (m, 6H), 3.77
(s, 6H), 4.36 (m, 1H), 5.03 (m, 1H), 5.53 (m, 1H), 6.18 (d, J )
6.2 Hz, 1H), 6.81 (m, 4H), 7.17-7.64 (m, 12H), 8.04 (d, J )
6.6 Hz, 2H), 8.21 (s, 1H), 8.67 (s, 1H), 9.31 (br s, 1H); 13C NMR
(50 MHz, CDCl3) δ 28.95, 47.01, 54.95, 62.79, 69.65, 69.92,
71.32, 79.67, 82.14, 86.60, 113.00, 123.55, 126.80, 127.69,
127.90, 128.08, 128.40, 129.82, 132.54, 133.08, 135.19, 141.97,
144.10, 149.73, 151.90, 158.38, 165.09, 171.21, 175.35; MS (ES)
a
Water C-4, 3.9 × 300 mm, A ) 50 mM triethylammonium
acetate, pH 7, B ) acetonitrile, 5 to 60% B in 55 min, flow 1.5 mL
min-1, λ ) 260 nm.
steps in the protocol supplied by the manufacturer were used
without modification. Oxidation of the internucleotide phos-
phite to the phosphate was carried out using CSO {[1-S-(+)-
(10-camphorsulfonyl)oxaziridine], 0.5 M in acetonitrile}. The
Beaucage reagent (0.1 M in acetonitrile) was used as a
sulfurizing agent. The coupling efficiencies were more than
97%. After completion of the synthesis, the solid support was
suspended in aqueous ammonium hydroxide (30 wt %) and
kept at room temperature for 2 h. The solid support 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, C-4 column, Waters, 7.8 × 300 mm, A ) 100 mM
ammonium acetate, B ) acetonitrile, 5-60% of 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 oligonucleo-
tides in 30-40% isolated yield. The oligonucleotides were
characterized by ESMS, and their purity was assessed by
HPLC (Table 3) and capillary gel electrophoresis.
Tm An a lysis. The thermal stability of the duplexes formed
by oligonucleotides with the 2′-O-DMAOE modification and
complementary RNA was studied by measuring the UV
absorbance versus temperature curves as described previ-
ously.31 Each sample contained 100 mM Na+, 10 mm phos-
phate, 0.1 mM EDTA, 4 µM oligonucleotides, and 4 µM
complementary length matched RNA. Each Tm reported was
an average of two experiments. ∆Tm per modification was
calculated by subtracting the Tm of the unmodified DNA-RNA
parent duplex and dividing by the number of modified residues
in the sequence.
m/z 899 [M + K]+; HRMS (FAB) Calcd for C46H49N6O11
861.3459 found 861.3458.
+
5′-O-(4,4′-Dim eth oxytr ityl)-N2-isobu tyr yl-2′-O-{2-[(N,N-
d im eth yla m in o)oxy]eth yl}gu a n osin e-3′-O-su ccin a te (41).
From compound 33 (0.6 g, 0.8 mmol), succinic anhydride (0.16
g, 1.6 mmol), DMAP (0.1 g, 0.8 mmol), triethylamine (0.23 mL,
1.6 mmol), and CH2ClCH2Cl (3 mL), compound 41 was isolated
in 97% yield (0.66 g), according to the procedure used for the
synthesis of compound 39. Compound 41 was purified by flash
silica gel column chromatography (10% MeOH in CH2Cl2): Rf
1
0.19 (5% MeOH in CH2Cl2; H NMR (200 MHz, DMSO-d6) δ
1.12 (d, J ) 5.46 Hz, 6H), 2.29 (s, 6H), 2.51-2.65 (m, 4H),
2.74 (m, 1H), 3.18 (dd, J ) 3.4 and 7.2 Hz, 1H), 3.42 (m, 5H),
3.72 (s, 6H), 4.18 (br s, 1H), 4.83 (m, 1H), 5.30 (m, 1H), 5.92
(d, J ) 7.4 Hz, 1H), 6.82 (m, 4), 7.11-7.4 (m, 9H), 8.15 (s, 1H),
11.58 (s, 1H), 12.08 (s, 1H); 13C NMR (50 MHz, CDCl3) δ 18.36,
29.01, 29.20, 35.68, 46.87, 54.95, 63.00, 69.65, 69.88, 71.07,
79.39, 81.91, 86.31, 113.00, 121.34, 126.89, 127.53, 129.75,
135.14, 135.44, 138.46, 144.39, 147.47, 148.2, 155.29, 158.46,
171.38, 174.54, 178.88; MS (API-ES) m/z 841.3 [M - H]-;
HRMS (FAB) Calcd for C43H51N6O12+ 843.3565 found 843.3575.
N 4 -B e n z o y l -2 ′-O -{2 -[(N ,N -d i m e t h y l a m i n o )o x y ]-
eth yl}-5′-O-(4,4′-d im eth oxytr ityl)-5-m eth ylcytid in e-3′-O-
su ccin a te (42). From compound 38 (0.50 g, 0.7 mmol),
succinic anhydride (0.10 g, 1.0 mmol), DMAP (0.04 g, 0.34
mmol), (C2H5)3N (0.19 mL, 1.34 mmol), and 1,2-dichloroethane
(2.5 mL), compound 42 was isolated in 90% yield (0.51 g),
according to the procedure used for the synthesis of compound
39. Compound 42 was purified by flash silica gel column
chromatography (10% MeOH in CH2Cl2): Rf 0.27 (10% MeOH
in CH2Cl2); 1H NMR (200 MHz, CDCl3) δ 1.57 (s, 3H), 2.66 (s,
6H), 2.5-2.8 (m, 4H), 3.42 (dd, J ) 2.22 and 8.8 Hz, 1H), 3.63
(dd, J ) 2.24 and 8.78 Hz, 1H), 3. 82 (s, 6 H) 3.69-4.02 (m,
4H), 4.34 (m, 2H), 5.36 (t, J ) 5.36 Hz, 1H), 6.11 (d, J ) 4.18
Hz, 1H), 6.87 (d, J ) 8.3 Hz, 4H), 7.27-7.55(m, 12H), 7.80 (s,
1H), 8.31 (d, J ) 6.68 Hz, 2 H); 13C NMR (50 MHz, CDCl3) δ
12.85, 29.53, 29.84, 47.19, 55.28, 61.98, 69.56, 70.00, 70. 58,
80.78, 81.29, 87.21, 87.69, 112.37, 113.36, 127.27, 128.10,
128.17, 129.91, 130.14, 132.47, 135.11, 136.35, 137.06, 144.10,
148.02, 158.80, 159.59, 171.59, 174.06, 179.58; MS (API-ES+)
m/z 873 [M + Na]+, MS (API-ES-) 849 [M-H]-; HRMS (FAB)
Nu clea se Sta bility Assa y. The nuclease stability of the
2′-modified oligonucleotides was evaluated using snake venom
phosphodiesterase as described previously.29
Ack n ow led gm en t. We thank our colleagues M.
Casper for CPG derivatization of some of the succinates,
Dr. E. A. Lesnik for the Tm analysis, S. R. Owens for
the nuclease stability assay, Dr. P. D. Cook for his
enthusastic support of this research, and Nancy Meskan
for her assistance in preparing this manuscript. We are
grateful to Drs. B. Ross, B. Bhat, A. Guzaev, and
Professor M. E. J ung (UCLA) for helpful discussions and
valuable comments.
Su p p or tin g In for m a tion Ava ila ble: Copies of the 1H
NMR, 13C NMR, and mass spectra of compounds 7, 13, 15-
17, 25-33, 35, 37, and 38. 31P NMR spectra and mass spectra
of 1-4. ESMS data and HPLC profiles for oligonucleotides 48,
50, 52, 54, 56, and 58. This material is available free of charge
via the Internet at http://pubs.acs.org.
+
Calcd for C46H51N4O12 851.3503 found 851.3492.
Oligon u cleotid e Syn th esis. A 0.1 M solution of the
amidites 1, 2, 3, and 4 in anhydrous acetonitrile was used for
the synthesis of modified oligonucleotides. The oligonucleotides
were synthesized on functionalized controlled pore glass (CPG)
on an automated solid-phase DNA synthesizer. CPG 43, 44,
45, and 46 functionalized with 2′-O-DMAOE modified nucleo-
sides were used wherever necessary. For incorporation of 1,
2, 3, and 4, phosphoramidite solutions were delivered in two
portions, each followed by a 5 min coupling wait time. All other
J O0103975
(31) (a) Monia, B. P.; J ohnston, J . J .; Ecker, D. J .; Zounesli, M. A.;
Lima, W. F.; Freier, S. M. J . Biol. Chem. 1992, 267, 19954-19962. (b)
Lesnik, E. A.; Guinosso, C. J .; Kawasaki, A. M.; Sasmor, H.; Zounes,
M.; Cummins, L. L.; Ecker, D. J .; Cook, P. D.; Freier, S. M. Biochem-
istry 1993, 32, 7832-7838. (c) Lesnik, E. A.; Freier, S. M. Biochemistry
1998, 37, 6991-6997.