Deoxynucleic guanidine: Synthesis and incorporation of purine nucleosides into positively charged DNG oligonucleotides

Article abstract of DOI:10.1016/j.bmc.2003.12.043

The synthesis of purine nucleosides capable of making the guanidinium linkage is described for the first time starting from the corresponding 2′-deoxynucleosides. The positively charged mixed base DNG oligomer containing guanine was synthesized on solid-p

Full text of DOI:10.1016/j.bmc.2003.12.043

Bioorganic & Medicinal Chemistry 12 (2004) 1475–1481
Deoxynucleic guanidine:
synthesis and incorporation of purine nucleosides into positively
charged DNG oligonucleotides
Hemavathi Challa and Thomas C. Bruice*
Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, USA
Received 4 December 2003; accepted 18December 2003
Abstract—The synthesis of purine nucleosides capable of making the guanidinium linkage is described for the first time starting
from the corresponding 20-deoxynucleosides. The positively charged mixed base DNG oligomer containing guanine was synthesized
on solid-phase using CPG as support from 30 to 50 direction using the precursor building block nucleosides.
# 2004 Elsevier Ltd. All rights reserved.
1. Introduction
It is logical to design a complementary oligonucleotide
with net positive charge so as to enhance the binding
affinity to the negatively charged natural DNA sense
oligonucleotide. Examples of positively charged oligo-
nucleotides include modifications made to the base,
sugar ring or backbone linkage.12ꢀ14 Prior reports from
this laboratory have demonstrated that replacement of
the internucleoside phosphate linkages with positively
charged guanidinium group renders an achiral oligonu-
cleotide with enhanced binding affinity to DNA
oligonucleotides.14ꢀ21 These molecules, termed as deoxy-
nucleic guanidines (DNG, Fig. 1) bind to com-
plementary DNA sequences with high affinity without
compromising the specificity of binding. For example, a
DNG thymidyl pentamer sequence (T5-DNG) binds in
a 2:1 stoichiometry with complementary adenyl DNA
oligomers, while the DNG adenyl oligomers form 1:2
complexes with the thymidyl DNA oligomers. Mis-
match studies of DNG sequences resulted in lowering of
melting values, a measure of binding affinity, suggesting
weakened DNA association. Also, it was demonstrated
that T5-DNG does not bind to poly dG, poly dC or
poly T but binds only to poly dA indicative of its fidelity
of binding. The guanidinium linkage has been shown to
be resistant to nucleases19 and it is possible that the
positive charges of the DNG backbone may improve
cell permeability through electrostatic attraction of the
oligomer to the negatively charged phosphate groups on
the cell surface.
The approval of ISIS fomivirsen (VitraveneTM, mar-
keted by Novartis Ophthalmics) as an antisense drug for
the treatment of HIV-associated CMV retinitis is very
promising for the field of antisense therapy. Several
groups are in active pursuit of oligonucleotide analogues
capable of arresting cellular processes at the translational
and transcriptional levels by binding to complementary
RNA or DNA target sequence.1ꢀ3 Unraveling the
human genome has been an added motivation for dis-
covery of therapies at the molecular level. With the
concurrent advances in molecular diagnostic techniques,
several companies are developing a huge database of
possible target sequences for any particular disease. The
recurring concerns in designing an efficient antisense/
antigene oligonucleotide are: (1) higher binding affinity
to the complementary sequence while maintaining fidelity
of recognition, (2) resistance to degradation by cellular
nucleases, and (3) improved cell membrane permeability.
Innumerable oligonucleotide analogues have been
reported in the past decade, for example, phosphoro-
thioate,4 phosphonates,5 carbamates,6 methylenemethyl-
imino,7 locked nucleic acids8 where the backbone
linkages have been modified. Another approach is
where the entire sugar-phosphodiester backbone has
been replaced to give PNA,9 PHONA10 or PNAA.11
Keywords: Purines; DNG; Positively charged; Nucleic acid; Guanidinium.
* Corresponding author. Tel.: +1-805-893-2044; fax: +1-805-893-
2229; e-mail: tcbruice@bioorganic.ucsb.edu
In order to fully realize the potential of the promising
properties of DNG, it is important to be able to synthesize
0968-0896/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.12.043

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H. Challa, T. C. Bruice / Bioorg. Med. Chem. 12 (2004) 1475–1481
Figure 1.
DNG sequences containing all four purine and pyr-
imidine bases. This would allow versatility in choice of
target sequences in determining the efficacy of DNG as
an antisense agent. Thus far, there have been several
reports on sequences containing guanidinium linkages
involving thymidyl monomers or DNG/DNA,20 DNG/
PNA,21 DNmt/DNA19 chimera. More recently, DNG
sequences containing both adenyl and thymidyl bases
have been reported.18 In this report we present, for the
first time, the synthesis of guanyl base for incorporation
into DNG sequences to give rise to mixed base DNG
oligomers using the standard solid-phase chemistry. An
alternative method for the synthesis of adenyl base
compatible with the standard solid-phase synthesis of
DNG is also described.
Scheme 1. Reagents and conditions: (a) benzoyl chloride, anhydrous
pyridine, rt; (b) trifluoromethanesulfonic anhydride, 10% v/v pyridine
in DCM, ꢀ35 ꢁC to rt; (c) aqueous NaOH (2M), 5:4:1 dioxane/
methanol/water, 0 ꢁC.
was not undertaken due to the literature precedence of
reportedly low yields and laborious procedures.22
Treatment of the 30-OH-20-deoxyxylonucleosides (4, 13)
with methanesulfonyl chloride in pyridine at room
temperature for 4 h afforded the 30,50-disulfonates (5, 4)
(Scheme 2).23 These compounds were then transformed
into their respective 30,50-diazido derivatives (6, 15)
using LiN3 as nucleophile at 90 ꢁC in DMF. After pur-
ification by silica gel chromatography, the 30,50-diazido
compounds were reduced by catalytic hydrogenation
with palladium over carbon to the respective 30,50-di-
amino-20-deoxynucleosides (7, 16). Selective protection
of the 50-amino group with the acid-labile Mmt group,
followed by protection of the 30-amino group with base-
labile Fmoc group yielded the final precursors (9 and
18) that can be used for solid-phase synthesis of DNG.
2. Results and discussion
The building blocks for the solid-phase synthesis of
positively charged DNG oligonucleotides with guanidi-
nium linkages, instead of the natural phosphodiester
linkages of the DNA backbone, were synthesized as
shown in Schemes 1–3. The key repetitive unit, 9/18,
was synthesized starting from 20-deoxyguanosine or 20-
deoxyadenosine respectively. The first step in the synth-
esis involves inversion of configuration at the 30-position
to give the 20-deoxy-threo isomer (Scheme 1). This was
achieved by adaptation of the previously reported proce-
dure by Herdewijn and Van Aerschot.22 The 50-benzoyl
purine nucleosides, with the exocyclic amino function-
alities of the bases protected by isobutyryl for guanine
(2) and benzoyl for adenine (11), were treated with 1.5
equiv of trifluoromethanesulfonic anhydride in di-
chloromethane containing 10% of anhydrous pyridine
to activate the 30-hydroxyl group followed by addition
of water to cleave the cyclic hemi-orthoester inter-
mediate, formed by the attack of 50-O-carbonyl group
on the activated 30-position, to yield the 30-O-benzoate
product (3, 12). The 30-O-benzoate was then quantita-
tively converted to the 30-OH-20-deoxyxylonucleosides
(4, 13) by selective alkaline hydrolysis. The synthesis of
13 was previously achieved starting from adenosine.18
An analogous synthesis of 4 starting from guanosine
The analogous thymidine precursor, 23, was also prepared
via the 30,50-diamino24 route (Scheme 3), thus reducing
the number of synthetic steps from the previously
reported15ꢀ17 procedures. Treatment of thymidine with
methanesulfonyl chloride afforded the disulfonate (20),
which upon reflux under alkaline conditions yielded the
anhydronucleoside (21). The 30,50-diamino derivative
(22) was obtained by treatment of 21 with LiN3 fol-
lowed by catalytic hydrogenation in presence of 10%
palladium over carbon. Selective protection of first the
50-amino and then the 30-amino groups of 22 with
MmtCl and Fmoc-isothiocyanate, respectively, yielded
the desired thymidine repetitive precursor unit (23).
Solid-phase synthesis of DNG tetramer mixed base
sequence, 50-TgGgTgT-30, was performed on CPG sup-
port from 30 to 50 direction as in the standard DNA
solid-phase synthesis. The 50-methoxytrityl protected
thymidine monomer required for loading onto the
LCAA-CPG support was synthesized as previously

H. Challa, T. C. Bruice / Bioorg. Med. Chem. 12 (2004) 1475–1481
1477
Scheme 2. Reagents and conditions: (a) methanesulfonyl chloride, anhydrous pyridine, rt, 4 h; (b) LiN3, anhydrous DMF, 90 ꢁC, 4 h; (c) H2, 10%
Pd/C, EtOH, rt, 6 h; (d) Mmt-Cl, Et3N, DCM, ꢀ30 ꢁC to rt; (e) Fmoc-NCS, DCM, rt.
Scheme 3. Reagents and conditions: (a) methanesulfonyl chloride, anhydrous pyridine, rt, 4 h; (b) aqueous NaOH (2M), EtOH, reflux, 1 h; (c) LiN3,
anhydrous DMF, 90 ꢁC, 4 h; (d) H2, 10% Pd/C, ethanol, rt, 4 h; (e) Mmt-Cl, Et3N, DCM, ꢀ30 ꢁC to rt; (f) Fmoc-NCS, DCM, rt; (g) methane-
sulfonyl chloride, Et3N, DCM, 0 ꢁC; (h) LiN3, anhydrous DMF, 90 ꢁC, 4 h; (i) H2, 10% Pd/C, EtOH, rt, 6 h; (j) Mmt-Cl, Et3N, DMAP, anhydrous
pyridine, rt; (k) succinic anhydride, DMAP, pyridine; (l) 4-nitrophenol, DCC, anhydrous pyridine, dioxane, rt; (m) CPG, Et3N, anhydrous DMF, rt.
Scheme 4. Activation and coupling reaction.
reported and loaded onto the CPG as its 30-succinyl
ester using standard protocols (Scheme 3).25 After
loading, the unreacted sites were capped and the loading
yield was determined spectrophotometrically from the
amount of Mmt cation released. A typical solid-phase
synthesis cycle is illustrated in Scheme 5. Upon
deblocking the acid-labile trityl group of the growing
chain, the 50-amino functionality is now available to
couple with the incoming precursor for the formation of
the guanidinium linkage. The coupling reaction is
accomplished in the presence of HgCl2 and TEA
(Scheme 4), whereby the 30-Fmoc protected thiourea of
the incoming precursor is converted into an activated
carbodiimide intermediate through the abstraction of the

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H. Challa, T. C. Bruice / Bioorg. Med. Chem. 12 (2004) 1475–1481
Scheme 5. Solid phase synthesis (30 to 50) cycle for DNG oligos on CPG support.
sulfur atom on the thiourea by mercury (II). The carbo-
diimide intermediate then reacts with the unmasked 50-
NH2 of the growing chain to yield an Fmoc protected
guanidinium linkage. This Fmoc protecting group
remains on the guanidinium linkage until the end of the
synthesis when it is easily deprotected during cleavage
of the oligomer from the CPG support. The HgS pre-
cipitate formed during the coupling reaction is removed
by demercuration using 20% thiophenol in DMF.
Then, capping with acetic anhydride and TEA to ensure
inertness towards subsequent chain elongation reactions
blocks the unreacted 50-NH2 sites. This completes one
cycle of the synthesis. The coupling efficiency at the end
of each cycle can be monitored by colorimetric analysis
of the Mmt-cation released at the acid deblock step. The
coupling yield in each cycle was 90–95%, thus the
overall yield of the 50-protected DNG mixed base tetra-
mer is expected to be ꢂ80%. After the final coupling
reaction, the Mmt protecting group was retained on the
oligomer to simplify the purification of the crude product.
The Mmt-on DNG oligomer was then cleaved off the
CPG support using methanolic ammonia solution at
60ꢁC for 16 h, when the Fmoc protection on the guanidi-
nium linkage and the isobutyryl groups on the exocyclic
guanine base were also deprotected simultaneously. The
crude product containing the desired oligomer along
with the failure sequences was purified on reverse-phase
HPLC (Altech C8column) using 100 mM TEAA buffer
as solvent A with a gradient of 5% to 95% CH3CN as
solvent B in 30 min. The HPLC purified trityl-on oligo-
mer was then detritylated using 1 mL of 80% acetic acid
(1 h, rt), lyophilized, redissolved in water, and further
purified by RP-HPLC. The final detritylated and HPLC
purified oligomer was analyzed by mass spectrometry
(ESI) to be the desired tetramer: m/z=1061.80, 1083.83
(M+H/Na)+; calculated for C43H59N21O12(M+H)+
1062.07. Thus, the precursor building block 9 was suc-
cessfully assembled into mixed base DNG oligomer.
3. Conclusion
The first stepwise solid-phase synthesis of DNG oligo-
mer containing guanine purine nucleoside has been
accomplished. The tetrameric DNG mixed base oligo-
mer 30-OH-TgTgGgT-NH2-50 was synthesized in 30 to 50
direction with average coupling yields of 95% on CPG
solid support as used for DNA solid-phase synthesis.
The orthogonally protected precursor guanyl monomer
with acid-labile 50-amino and base-labile 30-amino func-
tionalities is described for the first time and the analo-
gous adenyl precusor synthesis is also detailed, both
starting from the 20-deoxy purine nucleosides. The
described methods enable the synthesis of DNG mixed
base sequences for evaluating their interactions with
specific DNA target sequences.
4. Experimental
4.1. General
All 1H and 13C spectra were recorded on 400 and
100 MHz Varian instrument respectively, using CDCl3
or DMSO-d6 or CD3OD as solvent. Chemical shifts are
reported in d (ppm) as s (singlet), d (doublet), t (triplet),
q (quartet), m (multiplet), or br (broad). TLC was
monitored on aluminum-backed silica gel 60 (F254) 0.25
mm plates (Merck) with fluorescent indicator. Column
chromatography was performed using silica gel (particle
size 63–200 A, Selecto Scientific, Georgia, USA).
Reversed phase HPLC was performed on a Hewlett
Packard 1050 system equipped with a quaternary solvent
delivery system, a UV detector set at 260 nm, and an

H. Challa, T. C. Bruice / Bioorg. Med. Chem. 12 (2004) 1475–1481
1479
Altech Macrosphere C8RP semiprep column (7 m,
10ꢃ250 mm). All solvents were purchased dry from
Aldrich.
sitting over DCM for 1 h crystallized the desired pro-
duct. The product was collected by filtration and dried
on vacuum to give 2.67 g of 3 (45% yield) as a white
1
solid. H NMR (400 MHz, DMSO-d6) d 12.0 (s, 1H),
4.1.1. Synthesis of N2-isobutyryl-30-N-(9-fluorenylmethyl-
oxycarbonylamino)-50 -N-(4-monomethoxytritylamino)-
11.7 (s, 1H), 8.1 (s, 1H), 7.5–7.8 (m, 5H), 6.2 (dd, 1H),
5.6 (t, 1H), 4.9 (t, 1H), 4.3 (m, 1H); 3.7–3.8(m, 2H), 3.0
(m, 1H), 2.7–2.8(m, 2H), 1.1 (d, 6H). Anal. calcd for
C21H23N5O6: C, 57.1; H, 5.3; N, 15.9. Found: C, 56.9;
H, 5.4; N, 15.5. m/z (HRESI-MS) 442.1711, calcd for
C21H23N5O6 (M+H)+ 442.1726.
20,30-dideoxyguanosine,
9.
N2-Isobutyryl-20-deoxy-
guanosine (1).26 20-Deoxyguanosine (5.705 g, 20 mmol)
was coevaporated with anhydrous pyridine and then
suspended in 200 mL anhydrous pyridine. Trimethyl-
chlorosilane (13 mL, 100 mmol) was added slowly to the
suspension cooled in an ice-bath. After 30 min, iso-
butyric anhydride (17 mL, 100 mmol) was added drop-
wise and the reaction mixture was stirred for 2.5 h at
room temperature. The reaction mixture was then chil-
led in an ice-bath, 40 mL of cold water was added and
let stir for 15 min. Concentrated aqueous NH4OH was
added, let stir for another 30 min and rotovapped to
give an oil with salts. Water was added until all salts
dissolved and washed once with equal volume of ether.
The product crystallizes immediately in the aqueous
layer, which was filtered and dried on vacuum until
constant weight to give 5.575 g of 1 (82% yield) as a
white solid. 1H NMR (400 MHz, DMSO-d6) d 11.4–12.2
(s, br, 2H), 8.2 (s, 1H), 6.2 (t, 1H), 5.3 (d, 1H), 4.9 (t,
1H), 4.3 (m, 1H), 3.8(m, 1H), 3.4–3.6 (m, 2H), 2.7 (m,
1H), 2.6 (m, 1H), 2.2 (m, 1H), 1.1 (d, 6H). Anal. calcd
for C14H19N5O5: C, 49.8; H, 5.7; N, 20.8. Found: C,
49.2; H, 5.8; N, 19.7.
4.1.4. N2-isobutyryl-20-deoxyxyloguanosine (4). To a
solution of 3 (1.34g, 3.02 mmol) in a mixture of diox-
ane/methanol/water (50 mL, 5:4:1 v/v/v), cooled in an
ice-bath, were added 7 mL of aqueous NaOH (2M) and
let stir at 0 ꢁC for 40 min. The reaction mixture was then
neutralized with Dowex 50W-X8pyridinium H +-form
cation exchange resin (Fluka). Once the solution was
neutral (ꢂ15 min), the resin was filtered and the filtrate
was rotovapped to a thick paste which upon trituration
with DCM gave 1.00g of 4 (98% yield) as an off-white
solid after drying on vacuum. 1H NMR (400 MHz,
DMSO-d6) d 12.0 (s, 1H), 11.7 (s, 1H), 8.2 (s, 1H), 6.1
(dd, 1H), 5.3 (d, 1H), 4.7 (t, 1H), 4.5 (m, 1H), 3.9 (m,
1H), 3.5–3.7 (m, 2H), 2.6–2.8(m, 2H), 2.2 (m, 1H), 1.1
(d, 6H). Anal. calcd for C14H19N5O5: C, 49.8; H, 5.7; N,
20.8. Found: C, 49.8; H, 5.7; N, 20.4. m/z (HRESI-MS)
338.1463, calcd for C14H19N5O5 (M+H)+ 338.1464.
4.1.5. N2-Isobutyryl-30,50-dimesyl-20,30-dideoxyguanosine
(5). To a solution of 4 (1.00g, 2.96 mmol) in anhydrous
pyridine was added neat methanesulfonyl chloride (0.9
mL, 11.83 mmol) dropwise over 15 min. The reaction
mixture was stirred at room temperature for 4 h, quen-
ched with MeOH and rotovapped to a thick oily paste.
The paste was redissolved in ethyl acetate/water and the
product extracted into ethyl acetate. The combined
organic layers were dried over anhydrous Na2SO4, fil-
tered, rotovapped to give 1.32 g of 5 (90% yield) as a
pale wheatish brittle foam. 1H NMR (400 MHz,
DMSO-d6) d 12.1 (s, 1H), 11.7 (s, 1H), 8.0 (s, 1H), 6.2
(dd, 1H), 5.5 (m, 1H), 4.5–4.6 (m, 3H), 3.29 (s, 3H), 3.21
(s, 3H), 3.1 (m, 1H), 2.7–2.8(m, 2H), 1.1 (d, 6H).
Anal. calcd for C16H23N5O9S2: C, 38.9; H, 4.7; N,
14.2. Found: C, 38.6; H, 4.8; N, 14.0. m/z (HRESI-
MS) 494.1000, calcd for C16H23N5O9S2 (M+H)+
494.1015.
4.1.2. N2-Isobutyryl-50-O-benzoyl-20-deoxyguanosine (2).27
To a suspension of 1 (5.04g, 14.9 mmol) in anhydrous
pyridine (40 mL) was added dropwise a solution of
benzoyl chloride (1.76 mL, 15.2 mmol) in 10 mL anhy-
drous pyridine over 4 h. After completion of addition,
the reaction mixture was allowed to stir for 1 h and then
quenched with water and rotovapped to an oil. The oil
was dissolved in 100 mL DCM and washed with water
until the emulsions began to solidify. At this point, the
DCM layer was stored under fresh water for 30 min
when the product crystallized out. The product was
collected by filtration and dried on vacuum to give 5.33
g of 2 (81% yield) as a white solid. 1H NMR (400 MHz,
DMSO-d6) d 12.1 (s, 1H), 11.6 (s, 1H), 8.2 (s, 1H), 7.5–
7.9 (m, 5H), 6.3 (t, 1H), 5.6 (d, 1H), 4.4–4.6 (m, 3H), 4.1
(m, 1H), 2.7–2.8(m, 2H), 2.4 (m, 1H), 1.1 (d, 6H). Anal.
calcd for C21H23N5O6: C, 57.1; H, 5.3; N, 15.9. Found:
C, 55.8; H, 5.3; N, 14.8. m/z (HRESI-MS) 442.1743,
calcd for C21H23N5O6 (M+H)+ 442.1726.
4.1.6. N2-Isobutyryl-30,50-diazido-20,30-dideoxyguanosine
(6). To a solution of 5 (1.83 g, 3.7 mmol) in anhydrous
DMF (50 mL) was added LiN3 (3.41 g, 69.7 mmol). The
reaction mixture was stirred at 90 ꢁC until all the start-
ing material was consumed as shown by TLC (ꢂ4 h).
The reaction mixture was filtered and rotovapped to
give a brown oil. The oil was purified by silica gel col-
umn chromatography using 4:1 EtOAc/CHCl3 to give
0.84 g of 6 (60% yield) as brittle foam. 1H NMR
(400 MHz, DMSO-d6) d 12.1 (s, 1H), 11.6 (s, 1H), 8.2 (s,
1H), 6.2 (s, 1H), 4.6 (m, 1H), 4.0 (m, 1H), 3.6 (m, 2H),
3.0 (m, 1H), 2.7 (m, 1H), 2.6 (m, 1H), 1.1 (d, 6H). Anal.
calcd for C14H17N11O3: C, 43.4; H, 4.4; N, 39.8. Found:
C, 43.6; H, 4.3; N, 38.9. m/z (HRESI-MS) 388.1600,
calcd for C14H17N11O3 (M+H)+ 388.1594.
4.1.3. N2-Isobutyryl-30-O-benzoyl-20,30-dideoxyguanosine
(3). A solution of 2 (6.10g, 13.82 mmol) dissolved in
anhydrous 10% v/v pyridine in DCM (100 mL) was
cooled to ꢀ35 ꢁC in an isopropanol/dry ice bath. Tri-
fluoromethanesulfonic anhydride (3.5 mL, 20.73 mmol)
was added slowly while maintaining the reaction temp-
erature at ꢀ35 ꢁC. After completion of addition, the
reaction mixture was allowed to warm to 0 ꢁC, let stir
for 45 min, quenched with 3 mL water and let stir
overnight at room temperature. The reaction mixture
was then rotovapped to an oil, redissolved in ethyl ace-
tate and washed twice with equal volumes of water. The
organic layer was rotovapped to a paste, which upon

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H. Challa, T. C. Bruice / Bioorg. Med. Chem. 12 (2004) 1475–1481
4.1.7. N2-isobutyryl-30,50-diamino-20,30-dideoxyguanosine
(7). To a solution of 6 (0.84g, 2.17 mmol) in absolute
EtOH (100 mL) was added 100 mg of Pd/C (10% wet)
and hydrogenated at 45 PSI for 6 h. The reaction mix-
ture was filtered over a pad of Celite and the filtrate was
rotovapped and coevaporated with DCM to give 0.71 g
1H), 2.4 (m, 1H). Anal. calcd for C24H21N5O5: C, 62.7;
H, 4.6; N, 15.2. Found: C, 62.5; H, 4.6; N, 15.1. m/z
(HRESI-MS) 460.1628, calcd for C24H22N5O5 (M+H)+
460.1621.
4.1.12. N6-Benzoyl-30-O-benzoyl-20,30-dideoxyadenosine
(12). 1H NMR (400 MHz, DMSO-d6) d 11.2 (s, br,
1H), 8.62 (s, 1H), 8.58 (s, 1H), 7.5–8.0 (m, 10H), 6.5 (dd,
1H), 5.7 (m, 1H), 5.0 (t, 1H), 4.4 (m, 1H), 3.8(m, 2H),
3.1 (m, 1H), 3.0 (m, 1H). Anal. calcd for C24H21N5O5:
C, 62.7; H, 4.6; N, 15.2. Found: C, 62.8; H, 4.6; N, 14.8.
m/z (HRESI-MS) 460.1631, calcd for C24H22N5O5
(M+H)+ 460.1621.
1
of 7 (97% yield) as a pale yellowish solid. H NMR
(400 MHz, DMSO-d6) d 8.2 (s, 1H), 6.2 (m, 1H), 3.5 (m,
3H), 2.7–2.8(m, 2H), 2.2 (m, 1H), 1.1 (d, 6H), 1.0 (m,
1H). Anal. calcd for C14H21N7O3: C, 50.1; H, 6.3; N,
29.2. Found: C, 50.3; H, 6.2; N, 29.1. m/z (HRESI-MS)
336.1769, calcd for C14H21N7O3 (M+H)+ 336.1784.
4.1.8. N2-Isobutyryl-30-amino-50-N-(4-monomethoxytri-
tylamino)-20,30-dideoxyguanosine (8). To a suspension of
7 (690 mg, 2.06 mmol) in DCM was added triethyl-
amine (0.6 mL, 4.11 mmol) and cooled to ꢀ30 ꢁC in an
isopropanol/dry ice bath. A solution of Mmt-Cl in
DCM (635 mg, 2.06 mmol) was added slowly and the
reaction mixtured was allowed to warm up to room
temperature. The reaction was quenched with MeOH
and rotovapped to an oil. The oil was purified by silica
gel column chromatography using EtOAc with 0–30%
4.1.13. N6-Benzoyl-20-deoxyxyloadenosine (13). 1H NMR
(400 MHz, DMSO-d6) d 11.2 (s, br, 1H), 8.75 (s, 1H),
8.65 (s, 1H), 8.0 (d, 1H), 7.5–7.6 (m, 4H), 6.4 (dd, 1H),
5.6 (s, br, 1H), 4.7 (s, br, 1H), 4.4 (m, 1H), 4.0 (m, 1H),
3.6–3.7 (m, 2H), 2.8(m, 1H), 2.3 (m, 1H). Anal. calcd
for C17H17N5O4: C, 57.5; H, 4.8; N, 19.7. Found: C,
56.8; H, 4.8; N, 18.7. m/z (HRESI-MS) 356.1365, calcd
for C17H18N5O4 (M+H)+ 356.1359.
1
MeOH gradient to give 793 mg of 8 (63% yield). H
4.2. Solid phase synthesis
NMR (400 MHz, CD3OD-d6) d 12.1 (s, 1H), 11.6 (s,
1H), 7.1–7.5 (m, 12H), 6.8(d, 2H), 6.1 (dd, 1H), 5.4 (t,
1H), 4.1 (m, 1H), 3.8(s, 3H), 3.0 (m, 1H), 2.6–2.8(m,
2H), 2.1 (m, 1H), 1.8(m, 1H), 1.4 (m, 1H), 1.2 (d, 6H).
Anal. calcd for C34H37N7O4: C, 67.2; H, 6.1; N, 16.1.
Found: C, 66.5; H, 6.1; N, 15.7. m/z (HRESI-MS)
608.3005, calcd for C34H37N7O4 (M+H)+ 608.2985.
4.2.1. Loading. The 50-monomethoxytritylamino-50-
deoxythymidine monomer was loaded onto commer-
cially available LCAA-CPG (500 A, 80–120 mesh size)
as its succinyl ester using standard protocols.25 The
efficiency of loading was determined to be 32 mmol/g by
treating an aliquot of thymidine-loaded CPG with a
solution of 4% DCA in DCM and assaying the trityl
cation released by UV spectroscopy. Then the unreacted
amino groups on the CPG were capped with acetic
anhydride and triethylamine to prevent side reactions.
The T-loaded CPG was stored at 5 ꢁC.
4.1.1.9. N2-Isobutyryl-30-N-(9-fluorenylmethyloxycarbonyl-
amino)-50-N-(4-monomethoxytritylamino)-20,30-dideoxy-
guanosine (9). To a solution of 8 (340 mg, 559 mmol) in
DCM (40 mL) was added a solution of Fmoc-NCS in
DCM (157 mg, 559 mmol) and let stir for 1 h. The reac-
tion mixture was rotovapped and the crude product
purified by ether precipitation to give 397 mg of 9 (80%
yield) as an off-white solid. 1H NMR (400 MHz,
CD3OD-d6) d 12.0 (s, 1H), 11.4 (s, 1H), 11.6 (s, 1H),
7.1–7.9 (m, 20H), 6.8(d, 2H), 6.0 (dd, 1H), 4.4 (m, 2H),
4.3 (t, 1H), 4.0 (m, 1H), 3.7 (s, 3H), 3.1 (m, 1H), 2.6–2.8
(m, 2H), 2.0 (m, 1H), 1.6 (m, 1H), 1.3 (m, 1H), 1.2 (d,
6H). Anal. calcd for C50H48N8O6S: C, 67.6; H, 5.4; N,
12.6. Found: C, 66.9; H, 5.3; N, 12.4. m/z (HRESI-MS)
889.3521, calcd for C50H48N8O6S (M+H)+ 889.3496.
4.2.2. Deblocking. An aliquot of 100 mg (4 mmol scale)
of the T-loaded CPG was placed in a fritted peptide-
synthesis vessel and treated with a deblock solution (4%
DCA in DCM) while collecting the solution into a 10 mL
volumetric flask as it drips through the frit by gravity. The
beads were treated with the deblock solution until no more
yellow coloration was apparent (2 min). The beads were
then washed with DCM and neutralized with 1% TEA in
DMF. The filterate collected in the volumetric flask was
assayed for the released monomethoxytrityl cation.
4.1.10. Synthesis of N6-benzoyl-30-N-(9-fluorenylmethyl-
oxycarbonylamino)-50 -N-(4-monomethoxytritylamino)-
20,30-dideoxyadenosine (18). N6-Benzoyl-20-deoxyadenosine
(10). 1H NMR (400 MHz, DMSO-d6) d 11.2 (s, br,
1H), 8.75 (s, 1H), 8.65 (s, 1H), 8.0 (d, 2H), 7.5–7.6 (m,
3H), 6.5 (t, 1H), 5.4 (d, 1H), 5.0 (t, 1H), 4.4 (m, 1H), 3.9
(m, 1H), 3.5–3.6 (m, 2H), 2.8(m, 1H), 2.3 (m, 1H).
Anal. calcd for C17H17N5O4: C, 57.5; H, 4.8; N, 19.7.
Found: C, 56.7; H, 4.9; N, 18.9. m/z (HRESI-MS)
356.1365, calcd for C17H18N5O4 (M+H)+ 356. 1359.
4.2.3. Coupling. To the exposed 50-amino group on the
CPG was added 5 equiv of the fully-protected 9 or 23
dissolved in 1 mL of DMF. Then 10 equiv of HgCl2 and
TEA, dissolved separately in 0.5 mL DMF each, were
added simultaneously to the reaction vessel when a
cloudy yellow-white precipitate was observed. The
reaction mixture was agitated for 1 h, the supernatant
filtered, beads washed with DMF until no more pre-
cipitate was visible, washed with 20% thiophenol in
DMF until the beads were clear, washed with copious
amounts of DMF and then the coupling reaction was
repeated two more times in the same manner.
4.1.11. N6-Benzoyl-50-O-benzoyl-20-deoxyadenosine (11).
1H NMR (400 MHz, DMSO-d6) d 11.2 (s, br, 1H), 8.7
(s, 1H), 8.6 (s, 1H), 7.5–8.0 (m, 10H), 6.5 (t, 1H), 5.6 (d,
1H), 4.7 (m, 1H), 4.4–4.6 (m, 2H), 4.2 (m, 1H), 3.0 (m,
4.2.4. Capping. The unreacted sites were capped by
addition of 1 mL of 100 mM acetic anhydride in DMF

H. Challa, T. C. Bruice / Bioorg. Med. Chem. 12 (2004) 1475–1481
1481
and 1 mL of 200 mM TEA in DMF (5 minꢃ2). The
beads were washed copiously with DMF and DCM and
then dried on high vacuum.
8. Orum, H.; Wengel, J. Curr. Opin. Mol. Therap. 2001, 3,
239.
9. Uhlmann, E.; Peyman, A.; Breiphol, G.; Will, D. W.
Angew. Chem., Int. Ed. Engl. 1998, 37, 2796.
10. Peyman, A.; Uhlmann, E.; Wagner, K.; Augustin, S.;
Breiphol, G.; Will, D. W.; Schafer, A.; Wallmeier, H.
Angew. Chem., Int. Ed. Engl. 1996, 35, 2636.
11. Fujii, M.; Yoshida, K.; Hidaka, J. Bioorg. Med. Chem.
Lett. 1997, 7, 637.
4.2.5. Elongation. The deblocking/coupling/capping
steps were repeated two more times with the appro-
priate elongation monomer to give the desired tetramer
DNG sequence on the LCAA-CPG. The final coupling
reaction, the capping and deblocking steps were skipped
to allow the trityl group to remain on the oligomer.
12. Ueno, Y.; Mikawa, M.; Matsuda, A. Bioconjugate Chem.
1998, 9, 33.
13. Skibo, E. B.; Xing, C. Biochemistry 1998, 37, 15199.
14. Dempcy, R. O.; Almarsson, O.; Bruice, T. C. Proc. Natl.
Acad. Sci. U.S.A. 1994, 91, 6097.
15. Dempcy, R. O.; Browne, K. A.; Bruice, T. C. Proc. Natl.
Acad. Sci. U.S.A. 1995, 92, 6097.
16. Dempcy, R. O.; Browne, K. A.; Bruice, T. C. J. Am.
Chem. Soc. 1995, 117, 6140.
17. Linkletter, B. A.; Szabo, I. E.; Bruice, T. C. J. Am. Chem.
Soc. 1999, 121, 3888.
4.2.6. Cleavage and deprotection. The desired tetramer
DNG sequence was cleaved from the CPG support by
treating with methanolic ammonia at 60 ꢁC for 16 h.
Simultaneously, the isobutyryl protecting groups on the
G-bases were also deprotected. The solution now con-
taining the desired tetramer was filtered and lyophilized
to yield a white residue of the crude product mixture.
18. Linkletter, B. A.; Szabo, I. E.; Bruice, T. C. Nucleic Acids
Res. 2001, 29, 2370.
Acknowledgements
19. Challa, H.; Bruice, T. C. Bioorg. Med. Chem. Lett. 2001,
11, 2423.
20. Barawkar, D. A.; Bruice, T. C. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 11047.
21. Barawkar, D. A.; Kwok, Y.; Bruice, T. W.; Bruice, T. C.
J. Am. Chem. Soc. 2000, 122, 5244.
This research was supported by grants from the
National Institute of Health.
22. Herdewijn, P.; Van Aerschot, A. Tetrahedron Lett. 1989,
30, 855.
References and notes
23. Herdewijn, P.; Balzarini, J.; Pauwels, R.; Janssen, G.; Van
Aerschot, A.; de Clercq, E. Nucleosides & Nucleotides
1989, 8, 1231.
1. Stephenson, M. L.; Zamecnik, P. C. Proc. Natl. Acad. Sci.
U.S.A. 1978, 75, 285.
2. Crooke, S. T. Methods Enzymology, Vol. 313; Academic
Press: New York, 2000; pp 3–45.
24. Lavandera, I.; Fernandez, S.; Ferrero, M.; Gotor, V. J.
Org. Chem. 2001, 66, 4079.
3. Uhlmann, E. Curr. Opin. Drug Discov. Dev. 2000, 3, 203.
4. Stein, C. A. Chemistry and Biology 1996, 3, 319.
5. Levis, J. T.; Butler, W. O.; Tseng, B. Y.; Ts’o, P. O. P.
Antisense Res. Dev. 1995, 5, 251.
25. Atkinson, T.; Smith, M. Oligonucleotide Synthesis: A
Practical Approach; Oxford University Press: New York,
1990; p 35.
26. Jones, R. A. Oligonucleotide Synthesis:
A Practical
6. Waldner, A.; De Mesmaeker, A.; Lebreton, J.; Fritsch,
V.; Wolf, R. M. Synlett. 1994, 57.
Approach; Oxford University Press: New York, 1990;
p 23.
7. Morvan, F.; Sanghvi, Y. S.; Perbost, M.; Vasseur, J.-J.;
Bellon, L. J. Am. Chem. Soc. 1996, 118, 255.
27. Nishino, S.; Yamamoto, H.; Nagato, Y.; Ishido, Y.
Nucleosides & Nucleotides 1986, 5, 159.