Reaction of N3-benzoyl-3′,5′-O-(di-tertbutylsilanediyl)uridine with hindered electrophiles: Intermolecular N3 to 2′-O protecting group transfer

Article abstract of DOI:10.1081/NCN-120015728

The compound N³-benzoyl-3′,5′-O-(di-tert-butylsilanediyl)uridine 2 was alkylated with various alkyl iodides in CH3CN in the presence of base. Normal 2′-O-alkylated products were obtained with methyl or benzyl iodide. If hindered alkyl iodides w

Full text of DOI:10.1081/NCN-120015728

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REACTION OF N3-BENZOYL-3′,5′-O-(DI-TERT-
BUTYLSILANEDIYL)URIDINE WITH HINDERED
ELECTROPHILES: INTERMOLECULAR N3 To 2′-O
PROTECTING GROUP TRANSFER
Lei Zhu a , Osvaldo dos Santos a , Nadrian C. Seeman a & James W. Canary a
a Department of Chemistry , New York University , New York, NY, 10003, U.S.A.
Published online: 17 Aug 2006.
To cite this article: Lei Zhu , Osvaldo dos Santos , Nadrian C. Seeman & James W. Canary (2002) REACTION OF N3-
BENZOYL-3′,5′-O-(DI-TERT-BUTYLSILANEDIYL)URIDINE WITH HINDERED ELECTROPHILES: INTERMOLECULAR N3 To 2′-O
PROTECTING GROUP TRANSFER, Nucleosides, Nucleotides and Nucleic Acids, 21:10, 723-735, DOI: 10.1081/NCN-120015728
To link to this article: http://dx.doi.org/10.1081/NCN-120015728
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MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016
©2002 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.
NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 21, No. 10, pp. 723–735, 2002
REACTION OF N3-BENZOYL-30,50-O-(DI-TERT-
BUTYLSILANEDIYL)URIDINE WITH HINDERED
ELECTROPHILES: INTERMOLECULAR N3 TO
20-O PROTECTING GROUP TRANSFER
Lei Zhu, Osvaldo dos Santos, Nadrian C. Seeman,* and
James W. Canary*,{
Department of Chemistry, New York University,
New York, NY 10003
ABSTRACT
The compound N3-benzoyl-30,50-O-(di-tert-butylsilanediyl)uridine 2 was
alkylated with various alkyl iodides in CH3CN in the presence of base.
Normal 20-O-alkylated products were obtained with methyl or benzyl
iodide. If hindered alkyl iodides with b-branching such as 2-ethylbutyl
iodide were used as electrophiles under the same conditions, N3-alkyl-20-
O-benzoyl uridine derivatives were produced. This unexpected transfor-
mation is usually dormant with reactive alkylating agents, but expressed
with sterically hindered, less reactive electrophiles. This unwanted reac-
tion gives isomeric products whose spectra differ in only subtle ways from
target compounds.
Key Words: Alkylation reaction; Protecting group; 20-O-Alkyl ribo-
nucleosides
*Corresponding authors. Fax: (212) 260-7905
{E-mail: james.canary@nyu.edu
723
DOI: 10.1081=NCN-120015728
Copyright # 2002 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

724
ZHU ET AL.
INTRODUCTION
Antisense agents are oligonucleotide analogs capable of inhibiting
certain points of genetic information flow via base pair interactions with
DNA or mRNA. These molecules have been explored extensively because
of their potential as therapeutic agents, as well as their increasing use as
biotechnological tools.[1] To achieve the desired interaction with target
fragments, antisense oligonucleotides have to be able to penetrate cellular
membranes, resist degradation by nucleases and bind strongly with target
oligonucleotides.[1] Several strategies have been developed to achieve these
goals. There are early reports in which nucleobases have been modified to
increase the nuclease resistance but at the expense of selectivity.[2] More
recently, the polyanionic backbone of traditional antisense nucleotides has
been recognized as a disadvantage in intracellular uptake and the cause of
limited affinity to targeted natural oligonucleotide fragments.[3] The
negatively charged structure is also sensitive to nucleases.[3] Replacement
of the nucleotide backbone has led to PNA (peptide nucleic acid) tech-
nology and more radical structures such as positively charged guanidinium
linkages.[3–5] Currently there are also groups developing oligonucleotides
with 30-30-internucleotide linkages at the end of the sequence, to achieve
resistance to exonucleases.[6] A very large number of modified species have
been produced and characterized by the anti-sense and gene therapy
enterprises.[7]
In ribonucleotides, functionalization of the free 20-OH provides the
locus for another strategy to design antisense agents.[8] It should be pos-
sible to modify the 20-O position with a variety of functional entities, such
as positively charged groups, fluorescent probes and other species. Our
interest in the synthesis of 20-O-alkyl ribonucleoside derivatives relates in
part to DNA nanotechnology.[9–11] One of our projects would require
branched, highly functionalized groups within the alkyl group such as in
compound 1 (Fig. 1a). An initial approach to the synthesis of this com-
pound necessitated the alkylation of a ribosyl 20-OH with a sec-butyl
group. However, published alkylations have been achieved only with
relatively reactive electrophiles: for example, methyl iodide has been
used frequently to methylate the ribosyl 20-OH in reaction with silver
oxide,[12–14] sodium hydride[15] or 2-tert-butylimino-2-diethylamino-1,3-
dimethyl-perhydro-1,3,2-diazaphosphorine (BEMP).[16] Reactive benzyl
and allyl halide derivatives[17–20] often have been incorporated into oli-
gonucleotides via 20-O-alkylation as antisense groups or fluorescent
probes.[18] Protecting group agents such as alkoxymethyl halides[21] and
alkyl bromoacetates[22–24] also have worked well, owing to their high
reactivities. Treating 2,20-anhydrouridine with boron alkoxides has pro-
vided an alternative access to 20-O-alkyluridine with less reactive electro-
philes,[25,26] but high temperature ( > 100ꢀC) required in this reaction

N3 TO 20-O PROTECTING GROUP TRANSFER
725
Figure 1. a) Reactions of alkyl iodides with N3-benzoyl-30,50-O-(di-tert-butylsilanediyl)-
uridine; b) Synthesis of iodide 3d; c) Synthesis of compounds 15.
limited the types of functional groups which could be incorporated in the
alkyl groups. Ribosyl 20-OH also has been alkylated with primary halides
without branching under various base and solvent systems,[23] but alky-
lation by more hindered electrophiles, such as one with b-substitution, has
not been reported.

726
ZHU ET AL.
RESULTS AND DISCUSSION
In our study, Sproat’s approach[16] was carried out to access 20-O-
alkylated uridine derivatives because of its satisfying yields with various
alkyl halides as well as its mild conditions which were compatible with the
functional groups we wished to incorporate in the alkyl halides. We
investigated the alkylation of 20-OH of a base-protected nucleoside, N3-
benzoyl-30,50-O-(di-tert-butylsilanediyl)uridine 2, by four alkyl iodides (3a–
3d) shown in Fig. 1a. The benzoyl protecting group was chosen to prevent
N3-alkylation of uridine.[14,17,21,27] It could be incorporated selectively into
the N3 position by a phase-transfer reaction because of the difference between
the acidity of the uracil moiety and that of the ribosyl 20-OH.[28] It would be
removed easily at the deprotection stage of automated oligonucleotide
synthesis.
Compound 2 and alkyl halide were dissolved in CH3CN, followed by
the non-nucleophilic base BEMP. The 20-O-alkylation by methyl and benzyl
iodides proceeded uneventfully with respectable yields to give compounds 4
and 5. With the much less reactive 2-ethylbutyl iodide (3c) or 3d (Fig. 1b),
1
superficial analysis of the H NMR data seemed to indicate 20-O-alkylation.
However, upon closer inspection, we were surprised to find N3-alkylated
products 6 and 7 had been obtained exclusively in ꢁ50% yield in which the
benzoyl group had transferred to the 20-O. Products of 20-O-alkylation were
not detected. The elimination of hydrogen iodide from 3d was the major side
reaction which compromised the yield of 7. The alkylated nucleosides were
1
characterized by 1H=13C NMR and FAB-MS (Table 1). All of the H NMR
peaks were assigned through the use of 2D NMR. The 1.21 and 1.33 ppm
downfield shifts of 20-H in compounds 6 and 7 respectively from 4.47 ppm in
compound 2 after alkylation indicated the presence of strongly deshielding
Table 1. Characterization of N3- vs. 20-O-Alkyl and Acyl Derivatives of Uridine
2
4
5
6
7
12
20-H=ppma
4.47
4.00
168.6
4.2 ꢁ 4.6
5.68
165.5
5.80
165.1
5.68
165.8
C¼O=ppmb 168.7
168.8
FAB-MS
(m=z)
489.6
(MHþ) (MNaþ)
525.8e 471.3 (C)
377.2 (B)
487.2d (A)
377.0c (B)
579.3 (MHþ) 451.3 (C)
1024.3d (MHþ) 489.2c (MHþ)
573.3 (MHþ)
a1H NMR=CDCl3.
b13C NMR=CDCl3.
cESI-MS.
dDimethoxytrityl derivative.

N3 TO 20-O PROTECTING GROUP TRANSFER
727
Figure 2. FAB mass spectrometry fragmentation patterns.
20-O-acyl groups. Alkyl groups would not induce such drastic shifts of 20-H,
as observed in compounds 4 and 5, where no deshielding effect was observed.
The approximate 3-ppm shifts of the benzoyl carbonyl carbon in 13C NMR
spectra of 6 and 7 also indicated the transfer of benzoyl group to a different
chemical environment. FAB-MS data (Table 1) further confirmed the
structures by characteristic fragmentation resulting from loss of the base. In
Fig. þ2, fragment A represents the base portion of the molecule (noted as
BH2 by McCloskey),[29] establishing the connection between R2 and N3 in
compound 7 (Table 1), and fragment B confirms the identity of 20-O-sub-
stituents in compound 6 (Table 1).[29] Compounds 8 and 9 in Fig. 3 were
obtained by desilating compound 7 with HF-pyridine.[30] In compound 8,
1
both 20-H and anomeric 10-H were downfield shifted relative to 9 in the H
NMR spectra. 2D NMR and deuterium exchange experiments confirmed
that 8=9 were isomers generated from 20,30-O-migration, which was pre-
cedented for ribosyl 20-O-acyl groups.[31] This migration event provided
further evidence for the structure of compound 7. Debenzoylations were
performed on compounds 4 and 6 to give compounds 10 and 11 respectively
(Fig. 1a). Compared with 4 (20-H, 4.00 ppm), the chemical shift of 20-H in 10
(20-H, 4.00 ppm) did not change upon debenzoylation while in compound 11
(20-H, 4.31 ppm), the 20-H shifted 1.37 ppm further upfield compared to 6 (20-
H, 5.68 ppm); these data indicated clearly that the benzoyl group was ori-
ginally connected to the 20-O in 6.
A control experiment was performed in which 2 was treated with
BEMP in the absence of alkyl halides. A substantial amount of 2 was
transformed after 3 days into another species, which was identified as com-
pound 12 (Fig. 1a). The presence of N3-H in 12 was evidenced by the ‘‘W’’
1
shaped long range coupling (4JNH-H5 ¼ 2.0 Hz) in H NMR between N3-H

728
ZHU ET AL.
Figure 3. Ribose 20-O-benzoyl migration in compound 7.
and C5-H on the uracil moiety. The structure of 12 was established by the
data presented in Table 1. In this reaction, the ribosyl 20-OH (pKa ꢁ 13)[32]
is deprotonated by BEMP in solution. In the absence of a strong electrophile,
intermolecular nucleophilic substitution occurs, during which the benzoyl
group transfers to 20-O. Uracil N3-H (pKa ꢁ 9)[28] is more acidic than 20-OH
which renders the anionic uracil more stable than ribose 20-alkoxide. The
1
intermolecular nature of the transformation is further supported by the H
NMR spectrum of the crude reaction product in the formation of 7 which
contained peaks consistent with structure of N3-benzoyl-30,50-O-(di-tert-
butylsilanediyl)-20-O-benzoyluridine (13). The 20-O-alkylation and benzoyl
transfer are competing transformations so that whichever holds the kinetic
advantage will result. With hindered alkylating agents, the benzoyl transfer
occurs, and the freed N3-position of the uracil moiety is alkylated. We sug-
gest that this pathway is always available in base-catalyzed alkylating sys-
tems with electrophilic protecting groups, but it has not been reported
previously because of the high reactivities of commonly used alkylating
agents.[17,20,21,33]
Finally, a base-protecting group immune to nucleophilic attack should
enable alkylation of ribosyl hydroxyl(s) with a broader range of alkylating
agents. The benzyloxymethyl group was chosen to protect the uracil-N3 to
give compound 14 as the precursor for the desired 20-O-alkylation. Reaction
of 14 with the simplest b-substituted alkyl halide, 1-methylpropyl iodide,
gave compound 15 (Fig. 1c), representing a successful alkylation with b-

N3 TO 20-O PROTECTING GROUP TRANSFER
729
substitution on the ribosyl-20-OH. The poor yield of this reaction resulted
from competing elimination of b-substituted iodides inherently compromised
the applicability of this approach to alkylation of ribosyl-20-OH with steri-
cally hindered entities.
EXPERIMENTAL SECTION
General Methods
1
CH3CN was dried by refluxing with CaH2. H NMR and 13C NMR
were recorded on Varian-Gemini-200 or 300 MHz spectrometers. MALDI-
TOF mass spectra were measured with a Kratos MALDI-I spectrometer
using the matrix a-cyano-4-hydroxycinnamic acid. FAB mass spectra were
measured using the matrix NBA=CHCl3. ESI-MS spectra were measured at
the New York University Protein Analysis Facility, Skirball Institute of
Biomolecular Medicine, NYU School of Medicine. Elemental analyses were
performed by the Complete Analysis Laboratories, Inc.
N3-Benzoyl-30,50-O-(di-tert-butylsilanediyl)-20-O-methyluridine (4). N3-
Benzoyl-30,50-O-(di-tert-butylsilanediyl)uridine 2 (122 mg, 0.25 mmol) was
dissolved in CH3CN (0.34 mL) under argon and cooled in an ice bath. BEMP
(109 mL, 0.375 mmol) was added followed immediately by methyl iodide
(15.6 mL, 0.25 mmol) with stirring and exclusion of moisture. Solvent was
removed under vacuum after compound 2 was consumed as evidenced by
TLC. Extractive isolation with CH2Cl2 and water gave a residue that was
submitted to silica chromatography (CH2Cl2 to 10% ethyl acetate in CH2Cl2)
to give 89.1 mg of product as a glass. The yield was 71%. IR (NaCl) 1747 (s),
1
1708 (s), 1678 (s) cmꢂ 1; H NMR (300 MHz, CDCl3) d 8.0–7.5 (m, 5H,
ArH), 7.39 (d, 1H, J ¼ 9.0 Hz, 6-H), 5.87 (d, 1H, J ¼ 9.0 Hz, 5-H), 5.69 (s, 1H,
10-H), 4.50 (m, 1H, 40-H), 4.13 (m, 1H, 30H), 3.99 (3H, 20, 50, 500-H), 3.60 (s,
3H), 1.08 (s, 9H), 1.04 (s, 9H); 13C NMR (75.5 MHz, CDCl3) d 168.6, 162.0,
139.4, 135.2, 130.7, 129.4, 102.7, 92.2, 82.7, 75.0, 67.6, 59.4, 32.0, 27.6, 27.3,
22.8, 20.6; MS (MALDI-TOF) calcd (MþNaþ) 525.6, found 525.8.
N3-Benzoyl-30,50-O-(di-tert-butylsilanediyl)-20-O-benzyluridine (5).
Co mpound 5 was prepared from 20-O-alkylation of N3-benzoyl-30,50-O-(di-
tert-butylsilanediyl)uridine 2 with benzyl iodide according to the procedure
used for the preparation of compound 4 above. The crude product was
chromatographed on silica gel (19=1 CHCl3=EtOAc) to yield 80 mg (68%) of
1
pure product. H NMR (200 MHz, CDCl3) d 7.9–7.2 (m, 11H, ArH, 6-H),
5.84 (d, 1H, J ¼ 8.2 Hz, 5-H), 5.81 (s, 1H, 10-H), 4.93 (d, 1H, J ¼ 12.0 Hz),
4.81 (d, 1H, J ¼ 12.0 Hz), 4.51 (m, 1H, 40-H), 4.23 (m, 1H, 30-H), 4.16–3.96
(m, 3H, 20, 50, 500-H), 1.14 (s, 9H), 1.10 (s, 9H); 13C NMR (50 MHz, CDCl3) d

730
ZHU ET AL.
168.8, 162.2, 149.2, 139.5, 138.2, 135.6, 131.9, 131.0, 129.7, 128.7, 128.1,
128.0, 102.8, 92.3, 80.9, 76.9, 75.4, 73.2, 67.9, 28.0, 27.7, 23.4, 21.0; MS
(FAB) calcd (MþHþ) 579.24, found 579.34.
N3-(2-Ethylbutyl)-30,50-O-(di-tert-butylsilanediyl)-20-O-benzoyluridine (6).
N3-Benzoyl-30,50-O-(di-tert-butylsilanediyl)uridine 2 (245 mg, 0.5 mmol) and
1-bromo-2-ethylbutane (140 mL, 1.0 mmol) were dissolved in CH3CN
(1.0 mL) with n-Bu4NI (37 mg, 0.1 mmol) as catalyst. BEMP (217 mL,
0.75 mmol) was added dropwise at –20ꢀC. The solution was stirred for 72 h
and then poured onto ice (25 g). Extractive isolation was performed with
CH2Cl2=hexanes (1=3) and water. The organic layer was dried over Na2SO4
and the solvent was removed by reduced pressure. The residue was chro-
matographed with hexanes=ethyl acetate (from 10=1 to 10=3) to give 6
1
(124 mg, 43% yield) as a glass. H NMR (300 MHz, CDCl3) d 8.1–7.4 (m,
5H, ArH), 7.18 (d, 1H, J ¼ 8.0 Hz, 6-H), 5.90 (s, 1H, 10-H), 5.81 (d, 1H,
J ¼ 8.0 Hz, 5-H), 5.68 (d, 1H, J ¼ 5.6 Hz, 20-H), 4.50 (m, 1H, 40-H), 4.30 (m,
1H, 30-H), 4.08 (m, 2H, 50, 500-H), 3.85 (m, 2H), 1.75 (m, 1H), 1.30 (m, 4H),
1.08 (s, 9H), 0.80 (m, 15H); 13C NMR (75.5 MHz, CDCl3) d 165.5, 163.0,
151.1, 138.4, 133.9, 130.4, 129.7, 128.8, 103.2, 92.8, 75.9, 75.6, 75.4, 67.8,
45.3, 39.3, 27.9, 27.5, 24.0, 23.9, 23.3, 20.7, 11.2, 11.1; MS (FAB) calcd
(MþHþ) 573.29, found 573.23; Anal. calcd for C30H44N2O7Si: C, 62.91; H,
7.91; N, 4.89. Found: C, 63.17; H, 7.90; N, 4.70.
Compound 7. Compound 7 was prepared by treating N3-benzoyl-30,50-
O-(di-tert-butylsilanediyl)uridine 2 with iodide 3d according to the procedure
used for preparation of compound 6 above to give 7 as a white solid in 40 ꢁ
55% yield. IR (NaCl) 1770 (m), 1713 (s), 1670 (m) cmꢂ 1 1H NMR
;
(200 MHz, CDCl3) d 8.1–7.3 (14H, ArH, 6-H), 5.95 (s, 1H, 10-H), 5.85–5.81
(2H, 5-H, 20-H), 4.53–4.00 (6H, 30, 40, 50, 500-H, CH2), 3.70 (m, 4H), 2.10 (m,
1H), 1.79 (m, 4H), 1.10 (s, 9H), 0.80 (s, 9H); 13C NMR (50 MHz, CDCl3) d
168.6, 168.5, 165.1, 163.1, 151.2, 138.4, 134.1, 134.0, 133.7, 132.7, 130.4,
128.8, 123.7, 123.6, 102.9, 92.5, 76.9, 75.9, 75.7, 75.3, 67.8, 45.4, 36.3, 32.6,
31.5, 31.4, 28.2, 28.0, 27.4, 23.4, 20.7; MS (MALDI-TOF) calcd (MþNaþ)
886.0, found 886.3; Anal. calcd for C45H50N4O11Si: C, 61.19; H, 5.58; N,
6.15. Found: C, 60.96; H, 5.34; N, 6.00.
Compounds 8 and 9. HF.pyridine (100 mL, 3.84 mmol) was carefully
diluted with pyridine (0.51 mL) and then added dropwise to a solution of 7
(86.3 mg, 0.10 mmol) in THF (0.6 mL) at 0ꢀC. The mixture was warmed to
room temperature, stirred for 5 min, and then quenched with 5% NaHCO3.
Extractive isolation was performed with CH2Cl2=water. The organic layer
was collected, washed with 5% NaHCO3, and dried over sodium sulfate.
After evaporating most of the solvents under vacuum, the residue was co-
evaporated with CH3CN repeatedly to remove pyridine and by-product di-

N3 TO 20-O PROTECTING GROUP TRANSFER
731
tert-butyldifluorosilane (bp. 109ꢀC) to obtain 8 (70 mg) in almost quantitative
yield. Compound 8 was highly reactive and could not be purified further.
Compound 9 was obtained after 8 was chromatographed on silica gel
1
(CH2Cl2=ethylacetate) with 50 ꢁ 60% isolated yield. 8: H NMR (300 MHz,
CDCl3) d 8.0–7.3 (14H, ArH, 6-H), 6.15 (d, 1H, J ¼ 5.4 Hz, 10-H), 5.76 (d,
1H, J ¼ 9.0 Hz, 5-H), 5.67 (dd, 1H, J ¼ 6.0, 5.4 Hz, 20-H), 4.77 (dd, 1H,
J ¼ 6.0, 6.0 Hz, 30-H), 4.21 (dd, 1H, J ¼ 6.0, 3.0 Hz, 40-H), 4.05 (dd, 1H,
J ¼ 9.0, 3.0 Hz, 50-H), 4.00 (d, 2H, J ¼ 7.0 Hz), 3.89 (dd, J ¼ 9.0, 3.0 Hz, 500-H),
1
3.73 (m, 4H), 2.05 (m, 1H), 1.76 (m, 4H); H NMR (200 MHz, DMSO-d6) d
8.1–7.4 (14H, ArH, 6-H), 6.15 (d, 1H, J ¼ 5.4 Hz, 10-H), 5.74 (d, 1H,
J ¼ 9.0 Hz, 5-H), 5.64 (br, s, 1H, OH, D2O exchangeable), 5.38 (dd, 1H,
J ¼ 6.0, 6.0 Hz, 20-H), 5.30 (br, s, 1H, OH, D2O exchangeable), 4.40 (br, m,
1H, 30-H, D2O exchangeable to triplet), 4.05 (dd, 1H, J ¼ 6.0, 3.0 Hz, 40-H),
3.90–3.65 (4H, 50, 500-H, CH2), 3.58 (m, 4H), 1.85 (m, 1H), 1.63 (m, 4H); 13C
NMR (75.5 MHz CDCl3) d 168.1, 166.1, 163.0, 151.6, 139.5, 133.9, 132.6,
130.2, 128.8, 123.4, 102.6, 91.3, 85.0, 70.0, 62.0, 45.0, 36.1, 32.6, 31.1, 31.0;
MS (ESI) calcd (MþHþ) 723.222, found 723.218. 9: IR(NaCl) 1770 (m),
1710 (s), 1660 (m) cmꢂ 1; 1H NMR (300 MHz, CDCl3) d 8.1–7.4 (14H, ArH,
6-H), 5.86 (d, 1H, J ¼ 5.4 Hz, 10-H), 5.77 (d, 1H, J ¼ 9.0 Hz, 5-H), 5.60 (dd,
1H, J ¼ 5.4, 1.5 Hz, 30-H), 5.00 (br, s, 1H, 20-H, CD3OD exchangeable to
triplet), 4.48 (br, s, 1H, OH, CD3OD exchangeable), 4.38 (m, 1H, 40-H), 4.01
(d, 2H, J ¼ 7.0 Hz), 3.95 (2H, 50, 500-H), 3.74 (m, 4H), 3.41 (br, s, 1H, OH,
CD3OD exchangeable), 2.07 (m, 1H), 1.78 (m, 4H); 13C NMR (75.5 MHz,
CDCl3) d 168.7, 166.3, 163.0, 152.1, 140.0, 134.1, 133.9, 133.7, 132.5, 130.2,
129.9, 128.8, 123.5, 102.5, 93.9, 84.2, 73.8, 72.9, 62.6, 53.5, 45.3, 36.2, 32.0,
31.2; MS (MALDI-TOF) calcd (MþNaþ) 745.7, found 746.1.
30,50-O-(di-tert-butylsilanediyl)-20-O-methyluridine (10). Compound 4
(10 mg) was dissolved in methanol (1.0 mL) and treated with concentrated
ammonia (29% in water, 2.0 mL) at room temperature for 4 h. Methanol was
removed by vacuum and extraction was performed with CH2Cl2 and 5%
NaHCO3. The organic layer was washed with 5% NaHCO3 and dried over
Na2SO4. Compound 10 (7.9 mg) was obtained in almost quantitative yield
1
after evaporation of solvent. H NMR (300 MHz, CDCl3) d 9.16 (d, 1H,
2.1 Hz, NH), 7.26 (d, 1H, 8.2 Hz, 6-H), 5.76 (dd, 1H, J ¼ 8.2, 2.1 Hz, 5-H),
5.68 (s, 1H, 10-H), 4.48 (m, 1H, 40-H), 4.11 (m, 1H, 30-H), 3.95 (3H, 20, 50, 500-
H), 3.62 (s, 3H), 1.08 (s, 9H), 1.02 (s, 9H); 13C NMR (75.5 MHz, CDCl3) d
163.3, 149.8, 139.9, 102.8, 91.6, 82.5, 82.3, 74.7, 67.5, 59.4, 29.9, 27.5, 27.2,
23.0, 20.5; MS (MALDI-TOF) calcd (MþHþ) 399.5, found 399.4.
N3-(2-ethylbutyl)-30,50-O-(di-tert-butylsilanediyl)uridine
(11). Com-
pound 6 was debenzoylated according to the procedure used for the pre-
paration of compound 10 above to give compound 11 in almost quantitative

732
ZHU ET AL.
1
yield. H NMR (300 MHz, CDCl3) d 7.18 (d, 1H, J ¼ 8.1 Hz, 6-H), 5.75 (d,
1H, J ¼ 8.1 Hz, 5-H), 4.47 (m, 1H, 40-H), 4.30 (d, 1H, J ¼ 4.0 Hz, 20-H), 4.03
(3H, 30, 50, 500-H), 3.82 (m, 2H), 2.88 (s, 1H), 1.77 (m, 1H), 1.29 (m, 4H), 1.08
(s, 9H), 1.02 (s, 9H), 0.87 (m, 6H); 13C NMR (75.5 MHz, CDCl3) d 163.0,
150.8, 138.1, 102.4, 94.2, 76.1, 74.7, 73.9, 73.7, 76.5, 44.8, 38.8, 31.8, 27.5,
27.4, 23.4, 22.9, 20.6, 14.3, 10.7; MS (MALDI-TOF) calcd (MþHþ) 469.7,
found 469.8.
20-O-Benzoyl-30,50-(di-tert-butylsilanediyl)uridine
(12). N3-Benzoyl-
30,50-O-(di-tert-butylsilanediyl)uridine 2 (49 mg, 0.1 mmol) in CH3CN
(0.15 mL), was treated with BEMP (44 mL, 0.15 mmol), at ambient tem-
perature under argon for 3 days. The reaction mixture was poured onto ice
(25 g). The crude product was obtained by extractive isolation
(CH2Cl2=water) and was purified by silica chromatography (hexanes=ethyl
acetate) to give 29 mg of product as a white solid. The yield was 60%. IR
1
(NaBr) 1720–1700 (br, s) cmꢂ 1; H NMR (200 MHz, CDCl3) d 8.69 (d, 1H,
J ¼ 2.0 Hz, NH), 8.15–7.40 (m, 5H, ArH), 7.27 (d, 1H, J ¼ 8.0 Hz, 6-H), 5.81
(dd, 1H, J ¼ 8.0, 2.0 Hz, 5-H), 5.76 (s, 1H, 10-H), 5.68 (d, 1H, J ¼ 6.0 Hz, 20-
H), 4.52–4.41 (m, 2H, 30, 40-H), 4.08 (m, 2H, 50, 500-H), 1.08 (s, 9H), 0.82 (s,
9H); 13C NMR (50 MHz, CDCl3) d 165.8, 163.0, 149.8, 141.6, 134.1, 130.4,
129.5, 128.9, 103.6, 93.2, 75.7, 75.6, 75.3, 67.7, 27.9, 27.5, 23.4, 20.8; MS
(ESI) calcd (MþHþ) 489.2, found 489.2.
N3-Benzoylxymethyl-30,50-O-(di-tert-butylsilanediyl)uridine (14). The
compounds 30,50-O-(di-tert-butylsilanediyl)uridine (1.92 g, 5.0 mmol) and
tetrabutylammonium iodide (3.7 g, 10 mmol) were dissolved in CH2Cl2
(20 mL) under argon. DIPEA ( ꢁ 2mL, 10 mmol) was introduced by syringe
and the solution was stirred for 15 min followed by addition of benzyl
chloromethyl ether (693 mL, 5 mmol) dropwise. The solution was stirred for
3 h before being partitioned between CH2Cl2 and water. The organic layer
was washed with water 3 times and dried over Na2SO4. The solvent was
removed under vacuum without heating. The residue was subjected to silica
chromatography, eluting with hexanes=ethyl acetate (up to 10=3) to give 1.8 g
of white solid (71% yield). 1H NMR (300 MHz, CDCl3) d 7.39–7.25 (m, 5H,
ArH), 7.17 (d, 1H, J ¼ 8.2 Hz, 6-H), 5.76 (d, 1H, J ¼ 8.2 Hz, 5-H), 5.59 (s, 1H,
10-H), 5.49 (s, 2H), 4.70 (s, 2H), 4.48 (d, 1H, J ¼ 3.0 Hz, 40-H), 4.33 (d, 1H,
J ¼ 3.0 Hz, 20-H), 4.12 (m, 1H, 30-H), 4.01 (m, 2H, 50-H), 2.71 (s, 1H, OH),
1.09 (s, 9H), 1.04 (s, 9H); 13C NMR (75.5 MHz, CDCl3) d 162.6, 150.7, 139.5,
138.1, 128.5, 127.9, 102.4, 94.8, 76.1, 74.9, 73.6, 72.6, 70.5, 67.4, 27.6, 27.4,
22.9, 20.6; MS (MALDI-TOF) calcd (MþNaþ) 527.6, found 527.1.
N3-Benzoylxymethyl-30,50-O-(di-tert-butylsilanediyl)-20-O-(2-methylpro-
pyl)-uridine (15). Compound 14 (252 mg, 0.5 mmol) and 1-iodo-2-methyl-
propane (230 mL, 2 mmol) were dissolved in CH3CN (2.0 mL). BEMP (579

N3 TO 20-O PROTECTING GROUP TRANSFER
733
mL, 2 mmol) was added dropwise in situ. The solution was stirred for 48 h
before being poured onto ice. Extractive isolation was performed with
CH2Cl2 and water. The organic layer was dried over Na2SO4 and solvent was
removed at reduced pressure without heating. The residue was chromato-
graphed with hexanes=ethyl acetate (up to 4=1) to give compound 15 (24 mg,
9% yield). 1H NMR (200 MHz, CDCl3) d 7.38–7.27 (ArH, 5H), 7.21 (d, 1H,
J ¼ 8.2 Hz, 6-H), 5.74 (d, 1H, J ¼ 8.2 Hz, 5-H), 5.70 (s, 1H, 10-H), 5.50 (s, 2H),
4.70 (s, 2H), 4.50 (m, 1H, 40-H), 4.19 (m, 1H, 30-H), 4.02–3.80 (3H, 20, 50-H),
3.57 (m, 2H), 1.92 (m, 1H), 1.07 (s, 9H), 1.03 (s, 9H), 0.96 (d, 6H, J ¼ 6.4 Hz);
13C NMR (50 MHz, CDCl3) d 162.9, 150.8, 138.4, 138.1, 128.8, 128.1, 102.3,
92.0, 81.7, 75.2, 72.9, 70.8, 68.0, 29.5, 27.9, 27.6, 23.4, 21.0, 19.9; MS
(MALDI-TOF) calcd (MþNaþ) 583.7, found 583.8.
ACKNOWLEDGMENTS
We thank the National Science Foundation, the National Institute of
General Medical Sciences, DARPA, the USAF Information Directorate
Based in Rome, New York, and the New York University Technology
Transfer Fund for support of this research. FAB mass spectra were obtained
at the Michigan State University Mass Spectrometry Facility. ESI mass
spectra were obtained at the New York University Protein Analysis Facility,
Skirball Institute of Biomolecular Medicine, NYU School of Medicine.
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Received May 24, 2002
Accepted July 19, 2002