Improved synthesis of 2′-amino-2′-deoxyguanosine and its phosphoramidite

Article abstract of DOI:10.1016/j.bmc.2005.08.050

2′-Amino-2′-deoxynucleosides and oligonucleotides containing them have proven highly effective for an array of biochemical applications. The guanosine analogue and its phosphoramidite derivatives have been accessed previously from 2′-amino-2′-deoxyuridine

Full text of DOI:10.1016/j.bmc.2005.08.050

Bioorganic & Medicinal Chemistry 14 (2006) 705–713
Improved synthesis of 20-amino-20-deoxyguanosine and its
phosphoramidite
Qing Dai,a, Shirshendu K. Deb,b, James L. Houglandb and Joseph A. Piccirillia,b,
*
aHoward Hughes Medical Institute, The University of Chicago, 5841 S. Maryland Ave., MC 1028, Chicago, IL 60637, USA
bDepartment of Chemistry, Department of Biochemistry and Molecular Biology, The University of Chicago,
5841 S. Maryland Ave., MC 1028, Chicago, IL 60637, USA
Received 6 June 2005; revised 25 August 2005; accepted 26 August 2005
Available online 3 October 2005
Abstract—20-Amino-20-deoxynucleosides and oligonucleotides containing them have proven highly effective for an array of bio-
chemical applications. The guanosine analogue and its phosphoramidite derivatives have been accessed previously from 20-ami-
no-20-deoxyuridine by transglycosylation, but with limited overall efficiency and convenience. Using simple modifications of
known reaction types, we have developed useful protocols to obtain 20-amino-20-deoxyguanosine and two of its phosphoramidite
derivatives with greater convenience, fewer steps, and higher yields than reported previously. These phosphoramidites provide
effective synthons for the incorporation of 20-amino-20-deoxyguanosine into oligonucleotides.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
active site,13 thereby allowing identification of RNA
ligands to this metal ion.21 Additionally, GN forms
an integral component of an atomic mutation cycle de-
signed to explore the role of the 20-hydroxyl group in
hydrogen bonding.22,23 The literature contains two pre-
vious reports of 20-amino-20-deoxyguanosine phospho-
ramidites A and B (Fig. 1),24,25 both accessed from
uridine derivatives with limited overall efficiency.
Recently, Beigelman et al. demonstrated that 20-phtha-
limido protection of 20-aminouridine enhances the sta-
bility and coupling yields of the corresponding
phosphoramidites relative to 20-trifluoroacetamido pro-
tection.26 Subsequently, the corresponding 20-phthalim-
ido phosphoramidites of cytosine and adenosine were
prepared.27 Here, we extend this approach to 20-ami-
no-20-deoxyguanosine. The procedures developed offer
access to the nucleoside and the phosphoramidite in
fewer steps, and in greater yield than reported
previously.
20-Amino-20-deoxynucleosides and oligonucleotides
containing them have received much attention in recent
years, both as potential therapeutic agents1–4 and as
diagnostic and biochemical probes.5,6 As components
of antisense oligonucleotides,7–9 they have the potential
to improve drug efficacy by imparting resistance to
chemical and enzymatic degradation.10,11 In addition
to their therapeutic potential, the distinct physiochemi-
cal properties of 20-amino-20-deoxynucleosides render
them especially powerful tools for exploring RNA struc-
ture, function, and dynamics,12–19 particularly in defin-
ing the role and environment of specific 20-hydroxyl
groups within structural and catalytic RNA mole-
cules.13–18,20
Our interest in 20-amino-20-deoxyguanosine (GN) ema-
nates from its value in mechanistic investigations of the
group I intron, which catalyzes nucleotidyl transfer be-
tween an oligonucleotide substrate and guanosine. 20-
Amino-20-deoxyguanosine provides a direct probe for
one of the catalytic metal ions (Mc) at the ribozyme
2. Results and discussion
We first attempted to introduce a 20-phthalimido
group into the 20-a position of guanosine by SN2 dis-
placement of b-triflate from the 30,50-O-disilyl protect-
ed guanosine derivative (1) (Fig. 2),28 analogous to the
previously described preparations of 20-a-phthalimido
uridine, cytosine, and adenosine.27 However, treatment
Keywords: Transglycosylation; 20-Amino-20-deoxyguanosine; Phos-
phoramidite; Oligonucleotide.
*
Corresponding author. Tel.: +1 773 702 9312; fax: +1 773 702
0271; e-mail: jpicciri@uchicago.edu
These authors contributed equally to this work.
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.08.050

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Q. Dai et al. / Bioorg. Med. Chem. 14 (2006) 705–713
O
Onpe
A
B
N
N
N
N
NH
N
DMTrO
DMTrO
N
N
N
NHnpeoc
NMe
2
O
O
O
P
O
P
NHCOCF
NHnpeoc
Onpe
3
i-Pr
N
i-Pr
i-Pr
N
OCH CH CN
2
2
i-Pr
Figure 1. Previously synthesized phosphoramidites for the incorporation of 20-amino-20-deoxyguanosine into oligonucleotides. Benseler et al.
synthesized A in 12% yield from 20-trifluoroacetamido-20-deoxyuridine24 and Greiner synthesized B in 9% overall yield from 20-amino-20-deoxy-50-O-
(4,40-dimethoxytrityl)uridine.25 The abbreviation npeoc represents the [2-(4-nitrophenyl)ethoxy]carbonyl group, and npe represents the 2-(4-
nitrophenyl)ethyl group.
O
N
O
NH
NH
C H
15 31
N
N
O
N
N
N
O
NH
N
N
O
NH
NH
Me SiO
3
Me SiO
3
N
NH
C H
15 31
O
O
O
2
O
O
O
OTf
N
OH
O
OH
N
O
H
Si
O
O
10
9
1
Figure 2. Structures of compounds 1, 9, and 10.
of 1 with phthalimide in the presence of 1,8-diazabicy-
clo[5.4.0]undec-7-ene (DBU) gave no reaction. Instead,
we generated 20-phthalimidoguanosine by transglycosy-
lation from the known 20-deoxy-20-N-phthalimidouri-
dine intermediate 4a,26 analogous to the approach
described by Eckstein et al. for converting 20-amido-
20-deoxyuridine to the corresponding 20-amido-20-
deoxyguanosine.24,29
ded a mixture of N9 and N7-substituted guanosine
nucleosides (7 and 8) in 80% overall yield (N9/N7 = 4:1
as indicated by 1H NMR of the mixture). The high
polarity and poor solubility of these products rendered
them difficult to purify. To allow easier purification,
we isolated the transglycosylation reaction products be-
fore methanolic ammonia treatment, thereby retaining
the phthaloyl and palmitoyl groups.
In the reported preparation of 4a,26 20-amino-20-deoxyur-
idine (2a) was treated with N-carbethoxyphthalimide, fol-
lowed by 4,40-dimethoxytrityl chloride (DMTr-Cl) to
allow purification as 3a. Subsequent trichloroacetic acid
treatment (TCA, 3% in acetonitrile) gave pure 4a. Consid-
ering that McGee et al. readily prepared 20-amino-50-O-
(4,40-dimethoxytrityl)-20-deoxyuridine (2b) in 68% yield
from commercially available 2,20-anhydrouridine, we
adopted a modified preparation of 4a, first treating 2b
with N-carbethoxyphthalimide to generate 3a, followed
by DMTr removal with 3% TCA (Scheme 1). Overnight
treatment of 2b with 1.2 equiv of N-carbethoxyphthali-
mide and triethylamine (Et3N) in tetrahydrofuran
(THF) produced 3a and ca. 10% 20-N-30-O-bisphthaloyl
by-product.26 Subsequent treatment of the reaction mix-
ture with methanol (MeOH)/Et3N converted the by-prod-
uct quantitatively to 3a. Detritylation of crude 3a with 3%
TCA generated 4a in 94% overall yield from 2b.
Depending on the work-up procedure, different prod-
ucts from the transglycosylation reaction were isolated.
If the reaction mixture was cooled to room temperature,
quenched with water (ca. 450 equiv), and extracted with
dichloromethane (CH2Cl2), we isolated mainly the gua-
nosine derivatives 9 and 10 (Fig. 2), each containing a
trimethylsilyl group at the 50-oxygen (as indicated by
1H NMR).30 Quenching with dilute hydrochloric acid
(HCl, 0.1 N, ca. 450 equiv) instead of water converted
9 and 10 to 5a and 6a,31 respectively, which usually were
contaminated with unreacted N2-palmitoylguanine fol-
lowing isolation. If we quenched the reaction with a
small amount of HCl (0.1 N, 8.0 equiv), most of the
unreacted N2-palmitoylguanine formed a precipitate,
thereby simplifying purification of 5a and 6a.
Using the above work-up procedure, the transglycosyla-
tion reaction of 4a with N2-palmitoylguanine gave 5a
and 6a in yields of 60% and 15%, respectively. Overnight
treatment of 5a with methanolic ammonia at 55 ꢁC re-
moved both the phthaloyl and palmitoyl groups com-
pletely, as estimated by thin-layer chromatography
(TLC). Considering that 20-amino-20-deoxyguanosine
(7) dissolves easily in water, we attempted to remove
2.1. Improved access to 20-amino-20-deoxyguanosine and
its phosphoramidite
Transglycosylation of 4a followed by methanolic ammo-
nia treatment according to a literature procedure29 affor-

Q. Dai et al. / Bioorg. Med. Chem. 14 (2006) 705–713
707
O
O
N
O
N
NH
NH
NH
N
O
O
O
RO
DMTrO
HO
O
O
O
ii
i
OH
OH
OH
NH
R
R
2
2a R=H
2b R=DMTr
4a R=NPhth
4b R=NHTFA
3a R=NPhth
3b R=NHTFA
O
N
NH C15H31
N
N
N
N
O
NH
NH
O
HO
HO
N
NH C15H31
O
O
O
iii
+
OH
R
OH
R
6a R=NPhth
6b R=NHTFA
5a R=NPhth
5b R=NHTFA
iv
O
iv
N
NH2
N
N
N
NH
NH2
NH
HO
N
HO
N
O
O
O
OH NH
2
OH NH
2
8
7
Scheme 1. Reagents and conditions: (i) For 3a, N-carbethoxyphthalimide, THF, Et3N/MeOH; for 3b, CF3C(O)SEt, MeOH, 84%. (ii) 3% TCA in
CH3CN; for 4a, 94% from 2b; for 4b, 92% from 3b. (iii) (a) N,O-Bis(trimethylsilyl) acetamide, N2-palmitoylguanine, reflux, 30 min; (b) trimethylsilyl
triflate, reflux, 3 h; (c) HCl (0.1 N); for 5a, 60%; for 6a, 15%; for 5b, 70%; for 6b, 12%. (iv) n-Butylamine, ethanol (EtOH), 55 ꢁC, 16 h; for 7 from 5a,
95%; for 8 from 6a, 93%.
the palmitoylamide and phthalamide by-products by
organic extraction to avoid the two ion-exchange col-
umns needed previously for purification of 7. After
removal of the solvents, the resulting residue was dis-
solved in water and extracted with CH2Cl2. The organic
phase contained only palmitoylamide, and the aqueous
phase contained 7 and phthalamide. To render the
organic extraction more effective, we increased the lipo-
philicity of the phthalamide using methylamine instead
of ammonia to deprotect 5a. However, the by-product,
N,N0-dimethyl phthalamide, still remained in the aque-
ous phase with 7. To increase the lipophilicity further,
we deprotected 5a with ethanolic n-butylamine (4:1)
at 55 ꢁC. Extraction removed both the N-butyl
palmitoylamide and N,N0-di-n-butyl-phthalamide by-
products to give crude 7, which was purified further
by recrystallization to give pure 7 in 95% yield. We
obtained the corresponding 7-(20-amino-20-deoxy-b-D-
ribofuranosyl)guanine (8) from 6a in the same way.
These procedures improve the overall yield of 20-ami-
no-20-deoxyguanosine from 24% to 36% starting from
uridine and eliminate the need for ion-exchange
purification.29
conversion to the phosphoramidite, necessitating repro-
tection of the exocyclic and ribofuranosyl amines.25,29 In
contrast, we transformed the transglycosylation product
5a directly to the phosphoramidite, assuming the palmi-
toyl group would offer suitable protection for the exocy-
clic amino group during solid-phase synthesis. This
simple modification allowed access to the 20-N-phthalo-
yl-20-deoxyguanosine phosphoramidite 12a in just two
steps (dimethoxytritylation and phosphitylation) follow-
ing transglycosylation (Scheme 2), resulting in signifi-
cantly greater yields for phosphoramidite 12a (41%
from 2b) compared to that for phosphoramidite A
(12% from 4b).
We also prepared the corresponding 20-deoxy-20-N-tri-
fluoroacetyl guanosine phosphoramidite 12b by a similar
route (Schemes 1 and 2), again retaining the palmitoyl
group following transglycosylation. We converted 2b to
the corresponding trifluoroacetamide 3b in 84% yield
using S-ethyl trifluorothioacetate. TCA-induced removal
of the DMTr group afforded 20-trifluoroacetamido-20-
deoxyuridine 4b in 92% yield. Transglycosylation of 4b
with N2-palmitoylguanine gave N2-palmitoyl-20-trifluo-
roacetamido-20-deoxyguanosine 5b in 53% yield. We
converted 5b to a suitably protected phosphoramidite
using consecutive 50-dimethoxyltritylation and phosph-
In previous reports, complete deprotection of the trans-
glycosylation product to the parent nucleoside preceded

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Q. Dai et al. / Bioorg. Med. Chem. 14 (2006) 705–713
O
O
N
O
NH
NH
N
N
O
NH
N
DMTrO
C15H31
N
i
HO
O
N
NH
C15H31
O
OH
R
OH
R
11a R=NPhth
5a NPhth
5b R=NHTFA
11b R=NHTFA
O
N
O
N
N
NH
NH
DMTrO
C
15H31
O
ii
O
P
R
i-Pr
N
OCH CH CN
2
2
i-Pr
12a R=NPhth
12b R=NHTFA
Scheme 2. Reagents and conditions: (i) DMTr-Cl, pyridine, 16 h; for 11a, 82%; for 11b, 80%. (ii) 2-Cyanoethyl N,N-(diisopropylchloro)phospho-
ramidite, 1-methylimidozale, N,N-diisopropylethylamine; for 12a, 88%; for 12b, 75%.
Table 1. Comparative overview of the syntheses of 20-amino-20-deoxyguanosine and its phosphoramidites
Target molecule
Starting material (SM)
Steps
Overall
yield (%)
Notes
References
20-Amino-20-
deoxyguanosine
(7)
50-O-DMTr-20-amino-20-
deoxyuridine (2b)
4
4
54
29
SM prepared from 2,20-anhydrouridine
in 68% yield
Current work
29
20-Azido-20-deoxyuridine
SM prepared from uridine in 50% yield;
two ion-exchange purifications needed
SM prepared in 29% yield (see above)
Many synthetic steps; low overall yield
Purification following glycosylation
eliminates synthetic steps
Phosphoramidite A
Phosphoramidite B
Phosphoramidite 12a
7
4
10
5
21
6
24
25
50-O-DMTr-2,20-anhydrouridine
2b
41
Current work
Phosphoramidite 12b
2b
5
38
Same as for 12a
Current work
itylation reactions. This approach gives the 20-TFA pro-
tected phosphoramidite 12b in 38% overall yield from 2b,
whereas the previous method provided 20-TFA protected
phosphoramidite A (Fig. 1) in 21% overall yield from 7
(Table 1).
not been reported for the previously synthesized 20-
amino-20-deoxyguanosine phosphoramidites, A and B
(Fig. 1). Following standard oligonucleotide deprotec-
tion conditions (55% NH4OH/EtOH, 24 h; TBAF),
0
the MALDI mass spectrum of CUCG2 -NH2A gave
the expected peak at 1528 Da, consistent with the cal-
culated molecular weight (1529 Da), and demonstrating
full deprotection of 20-N-phthaloyl and palmitoyl
groups.
2.2. Oligonucleotide synthesis
We tested the suitability of phosphoramidites 12a and
12b for incorporating 20-aminoguanosine into oligonu-
0
cleotides of the sequence CUCG2 -NH2A. Using a
To establish further the integrity of the modified oligo-
0
nucleotide CUCG2 -NH2A, we examined its electropho-
10 min coupling time, we found that 12a and 12b cou-
pled with roughly equal efficiency based on the release
of DMTr cation (ꢀ40% relative to commercial guano-
sine phosphoramidite), although we made no attempts
to optimize this. Apparently, 20-phthaloyl protection
gives no advantage over 20-TFA protection during
the coupling of these phosphoramidites, in contrast to
previous observations for the corresponding phospho-
ramidites of 20-amino-20-deoxyuridine.26 Optimization
of coupling conditions may be necessary before any
differences become apparent. Coupling yields have
retic mobility upon exposure to T1 nuclease and the
electrophilic reagent, sulfosuccinimidyl-6-(biotinamido)
hexanoate (NHS).16 The oligonucleotides CUC-
G2 -NH2A and CUCGA were 50-radiolabeled with 32P-
0
phosphate ( ) using 32P-ATP and T4 polynucleotide ki-
*
nase (New England Biolabs) and purified by non-dena-
turing polyacrylamide gel electrophoresis (PAGE). As
expected, the control oligonucleotide, *CUCGA, is
cleaved in the presence of ribonuclease T1, which cuts
after guanosine residues in single-stranded RNA

Q. Dai et al. / Bioorg. Med. Chem. 14 (2006) 705–713
709
otherwise noted. All reactions using air-sensitive or
moisture-sensitive reagents were carried out under an ar-
1
gon atmosphere. H, 19F, and 13C NMR spectra were
recorded on Bruker 500 or Bruker 400 MHz NMR spec-
trometers. 1H chemical shifts are reported in d (ppm) rel-
ative to tetramethylsilane. 19F chemical shifts are
reported in d (ppm) relative to an external standard of
trifluoroacetic acid in CDCl3. 31P chemical shifts are
reported in d (ppm) relative to an external standard of
85% aqueous H3PO4. High-resolution mass spectra were
obtained from the Department of Chemistry, University
of California at Riverside. Merck silica gel (9385 grade,
230–400 mesh, 60A, Aldrich) was used for column chro-
matography. Silica gel on glass with fluorescent indica-
tor (Sigma) was used for TLC.
4.1.1. 50-O-(4,40-Dimethoxytrityl)-20-trifluoroacetamido-
20-deoxyuridine (3b). To a solution of 50-O-(4,40-dimeth-
oxytrityl)-20-amino-20-deoxyuridine
(2b)
(531 mg,
Figure 3. Biochemical reactivity profile of an oligonucleotide contain-
ing 20-amino-20-deoxyguanosine, CUCGNA. The control oligonucleo-
tide CUCGA (right panel) contains only unmodified ribonucleotides.
Both samples were 50-radiolabeled with 32P-phosphate. Input (lanes 1
and 4) indicates unreacted sample. T1 (lanes 2 and 5) contains samples
incubated with ribonuclease T1 (37 ꢁC, 20 min); NHS (lanes 3 and 6)
contains samples incubated with sulfosuccinimidyl-6-(biotinamido)
hexanoate (37 ꢁC, 1 h).
0.974 mmol) in methanol (6 mL) was added S-ethyl tri-
fluorothioacetate (194 lL, 1.46 mmol, 1.5 equiv). The
solution was stirred at room temperature for 1 h and a
white solid precipitated. TLC showed quantitative con-
version of the starting material. After the mixture was
concentrated to dryness, the residue was purified by
silica gel chromatography, eluting with 4% MeOH in
CH2Cl2 containing 0.2% Et3N, to give 3b (525 mg,
84%) as a white amorphous solid. Analytical data agree
with those previously reported.11
*
0
*
(Fig. 2, lane 5), but CUCG2 -NH2A resists T1 digestion
0
(Fig. 3, lane 2). Conversely, CUCG2 -NH2A reacts with
1H NMR (400.1 MHz) (CD3CN) d: 7.34 (d, J = 8.2 Hz,
1H), 7.17 (d, J = 7.2 Hz, 2H), 7.04 (m, 6H), 7.03 (m,
1H), 6.60 (dd, J = 8.1, 0.8 Hz, 4H), 5.77 (d, J = 7.8 Hz,
1H), 5.14 (d, J = 8.1 Hz, 1H), 4.43 (m, 1H), 4.15 (m,
1H), 3.84 (m, 1H), 3.14 (dd, J = 7.2, 3.6 Hz, 1H), 3.02
(dd, J = 7.9, 2.9 Hz, 1H). 13C NMR (100.6 MHz)
(CD3CN) d: 162.9, 158.8, 157.3 (q, J = 37.8 Hz), 150.7,
144.7, 140.0, 135.5, 135.3, 132.5, 130.1, 128.0, 127.0,
117.4, 113.2, 108.4, 103.8, 102.2, 86.8, 86.0, 85.1, 70.9,
70.1, 66.8, 63.4, 56.0, 54.9. 19F NMR (376.5 MHz)
NHS as indicated by retarded gel mobility (Fig. 2, lane
*
3), whereas CUCGA exhibits no reaction (Fig. 2, lane
6). These results demonstrate the viability of phospho-
ramidites 12a and 12b for the incorporation of 20-ami-
no-20-deoxyguanosine into RNA.
3. Conclusions
We have synthesized 9-(N2-palmitoyl-20-phthalimido-
20-deoxy-b-D-ribofuranosyl)guanine (5a) and 9-(N2-pal-
mitoyl-20-trifluoroacetamido-20-deoxy-b-D-ribofurano-
syl)guanine (5b) via transglycosylation from 20-
phthalimido and 20-trifluoroacetamido-20-deoxyuridine,
respectively. Retention of the amine-protecting groups
following these transglycosylation reactions allowed
direct access to the corresponding phosphoramidites
in two steps and simplified access to the nucleoside it-
self, resulting in greater yields than reported previous-
ly (refer to Table 1). Although these phosphoramidites
carry the unconventional palmitoyl protecting group,
both couple efficiently during solid-phase synthesis
and undergo quantitative deprotection afterwards, en-
abling facile access to oligonucleotides containing
20-amino-20-deoxyguanosine.
(CD3CN) d: ꢁ0.16 ppm. HRMS calcd
for
C32H30F3N3O8, [MNa+] 664.1871 (calcd), 664.1877
(found).
4.1.2. 20-N-Phthalamido-20-deoxyuridine (4a). To a solu-
tion of 20-amino-50-O-(4,40-dimethoxytrityl)-20-deoxyur-
idine (2b) (1.16 g, 2.13 mmol) in THF (30 mL) was
added N-carbethoxyphthalimide (559 mg, 2.56 mmol,
1.2 equiv). The solution was stirred overnight at room
temperature. Et3N (1 mL) and MeOH (1 mL) were then
added. After stirring for another hour, the mixture was
concentrated to dryness. The residue was treated with
TCA (3% in CH3CN) for 1 h and MeOH (1 mL) was
added. After concentrating the mixture to dryness, the
residue was purified by silica gel chromatography, elut-
ing with 5–10% MeOH in CH2Cl2, to give 4a (747 mg,
94%) as a white foam. 1H NMR (500.1 MHz) (CD3CN)
d: 7.87 (m, 2H), 7.83 (m, 2H), 7.69 (d, J = 8.0 Hz, 1H),
6.64 (d, J = 5.5 Hz, 4H), 5.64 (d, J = 8.5 Hz, 1H), 4.89
(dd, J = 8.0, 5.5 Hz, 1H), 4.44 (dd, J = 8.0, 6.1 Hz,
1H), 4.19 (m, 1H), 3.85 (dd, J = 12.3, 2.6 Hz, 1H),
3.70 (dd, J = 12.3, 3.4 Hz, 1H). 13C NMR
(125.8 MHz) (CD3CN) d: 168.2, 162.9, 151.0, 134.5,
4. Experimental
4.1. Materials and methods
All reagents and anhydrous solvents were purchased
from Aldrich; other solvents were from Fisher unless

710
Q. Dai et al. / Bioorg. Med. Chem. 14 (2006) 705–713
1
131.6, 123.1, 117.3, 101.8, 86.2, 85.9, 68.5, 61.0, 56.8.
HRMS calcd for C17H15N3O7, [MH+] 374.0988 (calcd),
374.0999 (found).
For 5a: H NMR (500.1 MHz) (DMSO-d6) d: 12.11 (s,
1H), 11.63 (s, 1H), 8.32 (s, 1H), 7.87 (m, 4H), 6.85 (d,
J = 5.9 Hz, 1H), 5.70 (d, J = 4.8 Hz, 1H), 5.18 (dd,
J = 7.9, 6.0 Hz, 1H), 4.98 (t, J = 5.5 Hz, 1H), 4.52
(dd, J = 11.7, 6.2 Hz, 1H), 4.15 (m, 1H), 3.72 (m,
1H), 3.56 (m, 1H), 2.45 (t, J = 7.3 Hz, 2H), 1.57 (t,
J = 6.6 Hz, 2H), 1.21 (m, 24H), 0.84 (t, J = 7.1 Hz,
3H). 13C NMR (125.8 MHz) (DMSO-d6) d: 177.2,
168.9, 155.6, 149.5, 148.9, 139.1, 135.5, 132.2, 124.1,
121.2, 87.1, 82.9, 69.3, 62.4, 57.4, 36.8, 32.2, 29.9,
29.8, 29.7, 29.6, 29.3, 25.2, 23.0, 14.8. HRMS calcd
for C34H46N6O7, [MNa+] 673.3320 (calcd), 673.3336
(found).
4.1.3. 20-Trifluoroacetamido-20-deoxyuridine (4b). 50-O-
(4,40-Dimethoxytrityl)-20-trifluoroacetamido-20-deox-
yuridine (3b) (4.10 g, 6.39 mmol) was treated with 3%
TCA in CH3CN for 15 min and then MeOH (1 mL)
was added. The reaction mixture was concentrated to
dryness. The residue was purified by silica gel chroma-
tography, eluting with 8–12% MeOH in CH2Cl2, to give
6 (1.99 g, 92%) as a white foam. Analytical data agree
with those previously reported.29
1H NMR (500.1 MHz) (CD3CN) d: 9.84 (s, 1H), 7.93 (d,
J = 8.1 Hz, 1H), 7.84 (d, J = 8.1 Hz, 1H), 6.03 (d,
J = 7.9 Hz, 1H), 5.05 (d, J = 8.1 Hz, 1H), 4.55 (m,
1H), 4.33 (m, 1H), 4.08 (m, 1H), 3.72 (m, 2H). 13C
NMR (125.8 MHz) (CD3CN) d: 163.7, 157.3 (q,
J = 37.4 Hz), 151.0, 140.9, 117.3, 115.7 (q, J = 288 Hz),
102.3, 86.7 (d, J = 14.7 Hz), 70.3, 61.6, 55.9, 48.9, 46.6.
19F NMR (470.5 MHz) (CD3CN) d: ꢁ0.20 ppm. HRMS
calcd for C11H12F3N3O6, [MH+] 340.0756 (calcd),
340.0753 (found).
For 6a: H NMR (500.1 MHz) (DMSO-d6) d: 12.17 (s,
1
1H), 11.57 (s, 1H), 8.47 (s, 1H), 7.87 (m, 4H), 6.86 (d,
J = 5.3 Hz, 1H), 5.65 (d, J = 5.2 Hz, 1H), 5.01 (dd,
J = 9.5, 5.4 Hz, 1H), 4.90 (t, J = 5.6 Hz, 1H), 4.47
(dd, J = 12.4, 6.9 Hz, 1H), 4.17 (m, 1H), 3.74 (m,
1H), 3.58 (m, 1H), 2.40 (t, J = 7.3 Hz, 2H), 1.53 (t,
J = 6.7 Hz, 2H), 1.21 (m, 24H), 0.81 (t, J = 6.7 Hz,
3H). 13C NMR (125.8 MHz) (DMSO-d6) d: 176.6,
168.3, 158.9, 152.6, 147.8, 135.0, 131.7, 123.6, 110.7,
86.8, 86.0, 68.1, 61.8, 58.2, 36.2, 31.7, 29.4, 29.3,
29.2, 29.1, 29.0, 28.7, 22.5, 14.3. HRMS calcd For
C34H46N6O7, [MNa+] 673.3320 (calcd), 673.3330
(found).
4.1.4. ÔOne-potÕ synthesis from 2b. To a solution of 20-
amino-50-O-(4,40-dimethoxytrityl)-20-deoxyuridine (2b)
(1.12 g, 2.06 mmol) in MeOH (6 mL) was added S-ethyl
trifluorothioacetate (0.54 mL, 4.12 mmol, 2 equiv). The
solution was stirred at room temperature for 1 h and a
white solid precipitated. TLC showed that starting
material was converted quantitatively to product. The
solvent was removed under vacuum to give crude 50-
O-(4,40-dimethoxytrityl)-20-trifluoroacetamido-20-deox-
yuridine as white foam. This foam was treated with 3%
TCA in CH3CN. After stirring for 15 min, TLC indicat-
ed that the intermediate was converted to product com-
pletely. MeOH (1 mL) was added, and the reaction
mixture was concentrated to dryness. The residue was
purified by silica gel chromatography, eluting with 8–
12% MeOH in CH2Cl2, to give 4b (0.627 g, 90%) as a
white foam.
4.1.6. 9-(N2-Palmitoyl-20-trifluoroacetamido-20-deoxy-b-
D-ribofuranosyl)guanine (5b) and 7-(N2-palmitoyl-20-
trifluoroacetamido-20-deoxy-b-D-ribofuranosyl)guanine
(6b). 20-Trifluoroacetamido-20-deoxyuridine(4b) (276 mg,
0.813 mmol) was added to a pressure tube (15 mL) con-
taining dry CH3CN (5 mL) and N,O-bis(trimethylsilyl)
acetamide (1.68 mL, 6.92 mmol, 8.6 equiv) under argon.
To this homogeneous solution was added N2-palmitoyl-
guanine (570 mg, 1.47 mmol, 1.8 equiv). The resulting
suspension was heated to reflux for 30 min, and the
reaction mixture became a clear solution. Trimethylsilyl
triflate (157 lL, 1.04 mmol, 1.3 equiv) was added, and
the solution was refluxed for an additional 3 h. The
solution was cooled to room temperature, aqueous
HCl (0.1 N, 653 lL) was added, and a white solid pre-
cipitated. The solid was filtered and washed with
CH3CN. The solvent of the combined filtrate was evap-
orated to dryness, and the residue was purified by silica
gel chromatography, eluting with 8–12% MeOH in
CH2Cl2, to give 5b (351 mg, 70%) and 6b (60.1 mg,
12%) as white solids.
4.1.5. 9-(N2-Palmitoyl-20-phthalimido-20-deoxy-b-D-ribo-
furanosyl)guanine (5a) and 7-(N2-palmitoyl-20-phthalimi-
do-20-deoxy-b-D-ribofuranosyl)guanine (6a). 20-N-Phtha-
loylamido-20-deoxyuridine (4a) (464 mg, 1.25 mmol)
was added to a pressure tube (15 mL) containing dry
CH3CN (10 mL) and N,O-bis(trimethylsilyl) acetamide
(2.57 mL, 10.6 mmol, 8.6 equiv) under argon. To this
homogeneous solution was added N2-palmitoylguanine
(873 mg, 2.25 mmol, 1.8 equiv). The resulting suspen-
sion was heated to reflux for 30 min, and the reaction
mixture became a clear solution. Trimethylsilyl triflate
(241 lL, 1.59 mmol, 1.3 equiv) was added, and the solu-
tion was refluxed for an additional 3 h. The solution was
cooled to room temperature, aqueous HCl (0.1 N,
0.1 mL) was added, and a white solid precipitated. The
solid was filtered and washed with CH3CN. The solvent
of the combined filtrate was evaporated to dryness, and
the residue was purified by silica gel chromatography,
eluting with 8–12% MeOH in CH2Cl2, to give 5a
(487 mg, 60%) and 6a (122 mg, 15%) as white solids.
1
For 5b: H NMR (400.1 MHz) (DMSO-d6) d: 12.12 (s,
1H), 11.66 (s, 1H), 9.58 (d, J = 6.5 Hz, 1H), 8.18 (s,
1H), 6.06 (d, J = 8.2 Hz, 1H), 5.82 (d, J = 4.4 Hz, 1H),
5.05 (m, 2H), 4.36 (m, 1H), 3.96 (m, 1H), 3.62 (m,
1H), 3.55 (m, 1H), 2.48 (t, J = 7.4 Hz, 2H), 1.58 (t,
J = 6.8 Hz, 2H), 1.22 (m, 24H), 0.85 (t, J = 7.1 Hz,
3H). 13C NMR (125.8 MHz) (DMSO-d6) d: 176.8,
157.1 (q, J = 36.5 Hz), 155.2, 149.4, 148.4, 138.3,
120.9, 116.0 (q, J = 288 Hz), 86.9, 84.1, 69.5, 61.6,
55.4, 36.3, 31.6, 31.0, 29.4, 29.3, 29.2, 29.0, 28.7, 24.7,
22.4, 14.3. 19F NMR (376.5 MHz) (DMSO-d6) d: 2.25.
HRMS calcd for C28H43F3N6O6, [MH+] 617.3274
(calcd), 617.3279 (found).

Q. Dai et al. / Bioorg. Med. Chem. 14 (2006) 705–713
711
For 6b: 1H NMR (400.1 MHz) (DMSO-d6) d: 12.22 (br,
1H), 11.58 (br, 1H), 9.47 (br, 1H,), 8.43 (s, 1H), 6.27 (d,
J = 7.9 Hz, 1H), 5.82 (br, 1H), 5.03 (t, J = 5.4 Hz, 1H),
4.90 (t, J = 6.6 Hz, 1H), 4.31 (m, 1H), 3.99 (m, 1H),
3.66 (m, 1H), 3.56 (m, 1H), 2.41 (t, J = 7.3 Hz, 2H),
1.55 (t, J = 6.8 Hz, 2H), 1.22 (m, 24H), 0.81 (t,
J = 6.8 Hz, 3H). 13C NMR (100.6 MHz) (DMSO-d6) d:
176.6, 158.6, 157.0 (q, J = 38.4 Hz), 152.6, 147.8,
144.1, 116.0 (q, J = 288 Hz), 111.2, 87.1, 86.9, 69.5,
61.6, 57.2, 31.7, 29.4, 29.3, 29.2, 29.1, 28.7, 24.8, 22.9,
14.5. 19F NMR (376.5 MHz) (DMSO-d6) d: 2.07.
HRMS calcd for C28H43F3N6O6, [MH+] 617.3274
(calcd), 617.3269 (found).
residue was purified by silica gel chromatography,
eluting with 5–7% MeOH in CH2Cl2, to give 9
(361 mg, 40%) and 10 (90.1 mg, 10%) as white solids.
1
For 9: H NMR (500.1 MHz) (CDCl3) d: 9.71 (br, 1H),
9.63 (br, 1H), 7.85 (s, 1H), 7.82 (m, 2H), 7.66 (m, 2H),
7.18 (d, J = 7.0 Hz, 1H), 5.31 (t, J = 6.9 Hz, 1H), 4.89
(d, j = 7.6 Hz, 1H), 4.68 (dd, J = 6.4, 3.6 Hz, 1H), 4.29
(m, 1H), 4.02 (d, J = 12.0 Hz, 1H), 3.74 (t, J = 11.2 Hz,
1H), 2.47 (m, 2H), 1.63 (t, J = 6.7 Hz, 2H), 1.25 (m,
24H), 0.85 (t, J = 6.8 Hz, 3H), ꢁ0.09 (s, 9H). 13C NMR
(125.8 MHz) (CD3Cl) d: 175.3, 168.3, 155.3, 147.6,
138.9, 134.3, 131.3, 123.3, 122.1, 99.6, 87.1, 83.8, 70.6,
61.8, 57.9, 37.0, 31.8, 29.6, 29.5, 29.4, 29.3, 29.2, 28.9,
24.6, 22.6, 14.0, ꢁ0.4. HRMS calcd for C37H54N6O7Si,
[MNa+] 745.3716 (calcd), 745.3724 (found).
4.1.7.
9-(20-Amino-20-deoxy-b-D-ribofuranosyl)guanine
(7). To a pressure tube (15 mL) was added a solution
of 5a (100 mg, 154 lmol) in anhydrous EtOH (4 mL)
followed by n-butylamine (1 mL). The mixture was heat-
ed overnight at 55 ꢁC. The solution was cooled to room
temperature, transferred to a flask, and evaporated to
dryness. Water (40 mL) was added, and the resulting
solution was extracted with CH2Cl2 (3 · 40 mL). The
aqueous phase was evaporated to dryness, and the
resulting solid was rinsed with acetone and recrystallized
from water to give 7 (41.1 mg, 95%) as a white solid. 1H
NMR (500.1 MHz) (DMSO-d6): 3.49 (m, 2H), 3.55 (m,
1H), 3.72 (m, 1H), 3.96 (m, 1H), 5.07 (br, 1H), 5.47
(d, J = 5 Hz, 1H), 5.48 (d, J = 8.5 Hz, 1H), 6.54 (br,
2H), 7.90 (s, 1H). 13C NMR (100.6 MHz) (DMSO-d6)
d: 156.8, 153.8, 151.7, 135.7, 116.8, 87.1, 86.3, 71.6,
61.9, 57.4. d: HRMS calcd for C10H14N6O4, [MH]+
283.1155 (calcd), 283.1165 (found).
1
For 10: H NMR (500.1 MHz) (CDCl3) d: 10.18 (br,
1H), 8.16 (s, 1H), 7.82 (m, 2H), 7.72 (m, 2H), 7.15 (d,
J = 6.9 Hz, 1H), 5.20 (t, J = 7.3 Hz, 1H), 4.89 (dd,
j = 7.5, 4.8 Hz, 1H), 4.40 (d, J = 8.8 Hz, 1H), 4.32 (m,
1H), 4.02 (m, 1H), 3.78 (m, 1H), 2.57 (m, 2H), 1.68 (t,
J = 7.1 Hz, 2H), 1.23 (m, 24H), 0.86 (t, J = 7.0 Hz,
3H), ꢁ0.09 (s, 9H). 13C NMR (125.8 MHz) (CD3Cl) d:
175.8, 168.5, 158.9, 152.7, 147.8, 144.5, 134.3, 131.5,
123.3, 110.8, 87.5, 85.3, 68.8, 61.0, 59.5, 37.1, 31.8,
29.6, 29.5, 29.4, 29.2, 28.9, 24.7, 22.6, 14.0, ꢁ0.5. HRMS
calcd for C34H46N6O7, [MNa+] 745.3715 (calcd),
745.3720 (found).
4.1.10.
phthalimido-20-deoxyguanosine (11a). N2-Palmitoyl-20-
N-phthalimido-20-deoxyguanosine
(5a) (400 mg,
50-O-(4,40-Dimethoxytrityl)-N2-palmitoyl-20-
4.1.8.
7-(20-Amino-20-deoxy-b-D-ribofuranosyl)guanine
0.614 mmol) was dissolved in pyridine (5 mL), and
4,40-dimethoxytrityl chloride (0.263 g, 0.740 mmol,
1.2 equiv) was added while the solution was stirring.
After being stirred overnight at room temperature, the
reaction was quenched with MeOH (1 mL), stirred for
an additional 5 min, and then evaporated to dryness.
The residue was dissolved in CH2Cl2 and washed con-
secutively with 5% sodium bicarbonate, water, and
brine. The organic layer was dried over sodium sulfate
and concentrated. The residue was purified by silica
gel chromatography, eluting with 2–5% MeOH in
CH2Cl2 containing 0.2% Et3N, to give 11a (480 mg,
(8). Using the procedure described for the preparation
of 7, 8 (20.2 mg, 93%) was obtained from 5b as a white
solid. 1H NMR (400.1 MHz) (DMSO-d6) d: 8.24 (s, 1H),
6.20 (s, 2H), 5.76 (d, J = 8.0 Hz, 1H), 5.40 (br, 1H), 5.01
(m, 1H), 3.99 (m, 1H), 3.89 (m, 1H), 3.66 (m, 2H), 3.51
(m, 1H). 13C NMR (100.6 MHz) (DMSO-d6) d: 161.5,
155.2, 153.6, 143.3, 108.7, 90.9, 87.1, 71.7, 62.5, 59.5.
HRMS calcd for C10H14N6O4, [MH]+ 283.1155 (calcd),
283.1167 (found).
4.1.9. 9-(N2-Palmitoyl-50-O-trimethylsilyl-20-phthalimi-
do-20-deoxy-b-D-ribofuranosyl)guanine (9) and 7-(N2-pal-
mitoyl-50-O-trimethylsilyl-20-phthalimido-20-deoxy-b-D-
ribofuranosyl)guanine (10). 20-N-Phthalimido-20-deox-
yuridine (4a) (464 mg, 1.25 mmol) was added to a pres-
sure tube (15 mL) containing dry CH3CN (10 mL) and
1
82%) as a pale yellow foam. H NMR (CD2Cl2): 11.97
(br, 1H), 9.33 (br, 1H), 9.47 (br, 1H), 7.46 (dd,
J = 5.4, 3.1 Hz, 2H), 7.36 (m, 2H), 7.18 (m, 2H), 6.89–
7.04 (m, 8H), 6.43 (m, 4H), 5.29 (m, 1H), 5.06 (br,
1H), 4.82 (m, 1H), 4.18 (m, 1H), 3.41 (s, 6H), 3.12 (m,
2H), 2.05 (m, 2H), 1.30 (m, 2H), 1.02 (m, 24H), 0.62
(t, J = 6.8 Hz, 3H). 13C NMR (125.8 MHz) (CD2Cl2)
d: 175.8, 168.5, 158.5, 155.5, 148.3, 147.5, 145.0, 139.1,
136.1, 135.8, 134.2, 132.4, 131.5, 130.1, 130.0, 129.0,
128.2, 127.4, 127.6, 126.7, 123.3, 121.4, 113.0, 112.9,
86.2, 86.1, 69.7, 64.5, 56.8, 55.2, 55.1, 36.8, 31.9, 29.8,
29.7, 29.6, 29.5, 29.4, 29.1, 24.3, 22.7, 13.9. HRMS calcd
for C55H64N6O9, [MNa]+ 975.4624 (calcd), 975.4627
(found).
N,O-bis(trimethylsilyl)
acetamide
(2.57 mL,
10.6 mmol, 8.6 equiv) under argon. To this homoge-
neous solution was added N2-palmitoylguanine
(873 mg, 2.25 mmol, 1.8 equiv). The resulting suspen-
sion was heated to reflux for 30 min, and the reaction
mixture became a clear solution. Trimethylsilyl triflate
(241 lL, 1.59 mmol, 1.3 equiv) was added, and the
solution was refluxed for an additional 3 h. After the
reaction solution was cooled to room temperature,
CH2Cl2 (50 mL) and water (50 mL) were added. The
aqueous layer was extracted with dichloromethane
(50 mL). The organic phases were combined, dried
over sodium sulfate, and evaporated to dryness. The
4.1.11. 50-O-(4,40-Dimethoxytrityl)-N2-palmitoyl-20-tri-
fluoroacetamido-20-deoxyguanosine (11b). N2-Palmitoyl-
20-trifluoroacetamido-20-deoxguanosine (5b) (220 mg,

712
Q. Dai et al. / Bioorg. Med. Chem. 14 (2006) 705–713
0.375 mmol) was dissolved in pyridine (3 mL), and 4,40-
dimethoxytrityl chloride (450 mg, 0.714 mmol,
dissolved in ethyl acetate and washed with 5% aqueous
sodium carbonate and brine, dried over sodium sulfate,
and concentrated. The residue was purified by silica gel
chromatography, eluting with 12% acetone in CH2Cl2
containing 0.2% Et3N, to give 12b (59.3 mg, 75%) as a
colorless oil. 31P NMR (202.5 MHz) (CD3CN) d: 150.6
and 150.4 ppm. 19F NMR (470.5 MHz) (CD3CN) d:
1.2 equiv) was added while stirring the solution. After
being stirred overnight at room temperature, the reac-
tion mixture was diluted with MeOH (1 mL) and stirred
for an additional 5 min. The reaction mixture was con-
centrated to dryness under vacuum. Water was added
to the resulting residue, and the mixture was extracted
with CH2Cl2. The organic phase was washed consecu-
tively with 5% sodium bicarbonate, water, and brine,
and then dried over sodium sulfate. After the organic
phase was concentrated to dryness, the residue was puri-
fied by silica gel chromatography, eluting with 4%
MeOH in CH2Cl2 containing 0.2% Et3N, to give 11b
ꢁ0.17
and
ꢁ0.28 ppm.
HRMS
calcd
for
C58H78F3N8O9P, [MNa]+ 1141.5474 (calcd), 1141.5434
(found).
Acknowledgments
1
(262 mg, 80%) as a white foam. H NMR (500.1 MHz)
Q.D. was a research associate and J.A.P. is an Investiga-
tor of the Howard Hughes Medical Institute. J.L.H. was
supported in part by the Predoctoral Training Program
at the Interface of Chemistry and Biology (2 T32
GM008720-06) at the University of Chicago. We thank
Dr. N. Li for coupling experiments and members of the
Piccirilli lab for helpful discussions and comments on
the manuscript.
(CD2Cl2) d: 12.29 (br, 1H), 9.60 (br, 1H), 8.36 (br,
1H), 7.97 (s, 1H), 7.18-7.45 (m, 9H), 6.78 (m, 4H),
6.17 (br, 1H), 5.33 (br, 1H), 4.87 (br, 1H), 4.22 (br,
1H), 3.72 (s, 6H), 3.44 (m, 2H), 2.32 (br, 2H), 1.58 (br,
2H), 1.25 (m, 24H), 0.90 (t, J = 7 Hz, 3H). 13C NMR
(125.8 MHz) (CD2Cl2) d: 176.0, 167.5, 158.6, 155.8,
149.3, 147.8, 144.6, 138.5, 135.6, 135.4, 134.9, 132.4,
130.8, 130.0, 129.9, 129.0, 128.6, 128.0, 127.9, 127.8,
127.7, 127.6, 127.0, 126.8, 120.3, 115.7 (q, J = 287 Hz),
113.0, 86.6, 85.6, 67.8, 63.9, 55.1, 46.3, 38.7, 36.8, 31.8,
30.3, 29.6, 29.5, 29.4, 29.3, 28.9, 24.4, 23.7, 22.9, 22.6,
13.8, 10.7. 8.8. 19F NMR (376.5 MHz) (CD2Cl2) d:
0.36. HRMS calcd for C49H61F3N6O8, [MNa]+
941.4395 (calcd), 941.4366 (found).
References and notes
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4.1.12. 50-O-(4,40-Dimethoxytrityl)-N2-palmitoyl-20-N-
phthalimido-20-deoxyguanosine 30-O-(2-cyanoethyl-N,N-
diisopropyl)phosphoramidite (12a). 50-O-(4,40-Dimeth-
oxytrityl)-N2-palmitoyl-20-trifluoroacetamido-20-deoxy-
guanosine (11a) (80.0 mg, 83.9 lmol) was dissolved in
dry CH2Cl2 (5 mL) and 1-methylimidazole (3.40 mg,
41.5 lmol).
N,N-Diisopropylethylamine
(78.0 mg,
0.420 mmol) was added to the stirring solution followed
by 2-cyanoethyl N,N-(diisopropylchloro)-phosphorami-
dite (80.3 mg, 0.336 mmol). After being stirred at room
temperature for 1 h, the reaction mixture was concen-
trated to dryness. The residue was then dissolved in eth-
yl acetate and washed with 5% aqueous sodium
carbonate and brine, dried over sodium sulfate, and
concentrated. The residue was purified by silica gel chro-
matography, eluting with 10–12% acetone in CH2Cl2
containing 0.2% Et3N, to give 12a (85.1 mg, 88%) as a
white foam. 31P NMR (202.5 MHz) (CD3CN) 152.0
and 150.2 ppm. HRMS calcd for C64H81N8O10P,
[MNa]+1175.5706 (calcd), 1 175.5694 (found).
4.1.13. 50-O-(4,40-Dimethoxytrityl)-N2-palmitoyl-20-tri-
fluoroacetamido-20-deoxyguanosine 30-O-(2-cyanoethyl-
N,N-diisopropyl)phosphoramidite
(12b).
50-O-(4,40-
Dimethoxytrityl)-N2-palmitoyl-20-trifluoroacetamido-
20-deoxyguanosine (11b) (65.0 mg, 70.7 lmmol) was dis-
solved in dry CH2Cl2 (5 mL) and 1-methylimidozale
(2.90 mg,
35.0 lmol).
N,N-Diisopropylethylamine
(67.0 mg, 0.360 mmol) was added to the stirring solution
followed by 2-cyanoethyl N,N-(diisopropylchloro)phos-
phoramidite (67.0 mg, 283 lmol, 4.0 equiv). After being
stirred at room temperature for 1 h, the reaction mixture
was concentrated to dryness. The residue was then

Q. Dai et al. / Bioorg. Med. Chem. 14 (2006) 705–713
713
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K.; Beigelman, L. Bioorg. Med. Chem. Lett. 2002, 12,
3345.
28. Schwans, J. P.; Cortez, C. N.; Olvera, J. M.; Piccirilli, J. A.
J. Am. Chem. Soc. 2003, 125, 10012.
reside on O6. The 1H NMR spectrum (CD3Cl) of 5a
contains a D2O exchangeable doublet and triplet corre-
sponding to the 30- and 50-hydroxyl groups, respectively.
The 1H NMR spectrum (CD3Cl) of 9 contains the doublet
but not the triplet, suggesting that the trimethylsilyl group
resides on the 50-oxygen.
31. The structure of 5a was confirmed by comparison of its 1H
NMR spectrum to the literature data (see Ref. 29). The
1
structure of 6a was assigned on the basis of its H NMR
and 13C NMR spectra. According to the literature
(Chenon, M.-T.; Pugmire, R. J.; Grant, D. M. Panzica,
R. P.; Townsend, L. B. J. Am. Chem. Soc. 1975, 97, 4627;
Li, N.-S, Piccirilli, J. A. Synthesis, in press), the chemical
shift of the purine C5 carbon may be used to distinguish
the N-9 and N-7 isomers of guanosine derivatives. For the
N-9 isomer, the chemical shift of C5 usually occurs near
120 ppm (DMSO-d6), whereas for the N-7 isomer, the C5
resonance usually occurs near 110 ppm. Consistent with
this trend, the C5 carbon of 5a resonates at 121.2 ppm
(DMSO-d6). For 6a, the C5 carbon resonates at 110.7 ppm
(DMSO-d6), consistent with the N-7 configuration.
29. Imazawa, M.; Eckstein, F. J. Org. Chem. 1979, 44, 2039.
30. We assigned the location of the trimethylsilyl group in 9
and 10 by a comparison of 1H NMR and 13C NMR
1
spectra for compounds 5a, 6a, 9, and 10. The H NMR
spectrum of 9 contains two broad, D2O exchangeable
peaks at 9.71 and 9.63 ppm corresponding to the NH
protons, thereby excluding the possibility that the trim-
ethylsilyl group is bonded to the purine base. The C6
carbon resonance occurs at nearly the same 13C chemical
shift in compounds 9 (155.3 ppm) and 5a (155.6 ppm),
further suggesting that the trimethylsilyl group does not