Solid-phase synthesis of positively charged deoxynucleic guanidine (DNG) oligonucleotide incorporating 7-deazaguanine bases

Article abstract of DOI:10.1016/j.bmc.2006.05.074

DNG nucleotides represent a positively charged DNA analog in which the negatively charged phosphodiester linkages of DNA are replaced by positively charged guanidinium linkages. We report herein the synthesis of 3′-end, middle, and 5′-end monomers require

Full text of DOI:10.1016/j.bmc.2006.05.074

Bioorganic & Medicinal Chemistry 14 (2006) 7333–7346
Solid-phase synthesis of positively charged deoxynucleic guanidine
(DNG) oligonucleotide incorporating 7-deazaguanine bases
Moti L. Jain and Thomas C. Bruice*
Department of Chemistry and Biochemistry, University Of California, Santa Barbara, CA 93106, USA
Received 22 April 2006; revised 31 May 2006; accepted 31 May 2006
Available online 30 August 2006
Abstract—DNG nucleotides represent a positively charged DNA analog in which the negatively charged phosphodiester linkages of
DNA are replaced by positively charged guanidinium linkages. We report herein the synthesis of 30-end, middle, and 50-end mono-
mers required for the synthesis of a DNG sequence in which the natural guanine base is replaced by 7-deazaguanine (c7G). 7-Deaza-
guanine nucleobase was chosen because of their unique glycoside bond stability and their ability to prevent G-quartet formation. A
facile and high yield two-step synthesis of xylo-7-deazaguanine 7, a key intermediate for introducing 30-amino functionality, is car-
ried out under Mitsunobu conditions. Subsequently, the 30-Fmoc-protected thiourea monomers 13 and 19 were prepared from 7 via
their corresponding 30-amino-7-deazaguanines 11 and 18, respectively. The smooth coupling of these thiourea monomers with
monomethoxytrityl (MMTr)-protected 30-end monomer 25, prepared from 5, occurred on solid phase in 30 ! 50 direction. The
resultant trimeric HO-c7Ggc7Ggc7G-OH (1) has been designed to be included into DNA using standard DNA synthesis technology.
The combination of C-c7G base pairing and electrostatic association of phosphodiester and guanidinium backbone allows the small
synthesized DNG trimer 1 to form 1:1 complex with DNA-C pentamer.
ꢀ 2006 Published by Elsevier Ltd.
1. Introduction
they form G-quartets, especially in the presence of
monovalent cations via inter- or intrastrand H-bondings
(Fig. 1a). This potentially restricts the effectiveness of
purine-rich TFOs as antigene agents.4,5 G-rich oligonu-
cleotides have also been shown to have a strong anti-
proliferative activity against a number of cancer cell
lines.6 In view of these findings, it is important to design
potential therapeutic TFOs that do not form G-quar-
tets. Among other modifications, the replacement of
guanosine N-7 with carbon creates 7-deazaguanosine
(c7G). c7G nucleosides have gained extensive attention
because some of them, such as queuosine7 and cadeguo-
mycin,8–10 exhibits a broad spectrum of biological and
chemotherapeutic activities. It has been demonstrated
that the removal of this H-bond acceptor (N-7) elimi-
nates the ability of ODNs to form G-quartets by remov-
ing Hoogsteen H-bonds between the 2-amino and N-7
nitrogen,11,12 while retaining the H-bond donor and
acceptor pattern required for the formation of c7G-C
duplexes13,14 and c7G:G-C triplets15 (Fig. 1b). However,
the binding study of these duplexes13,14 and triplexes15
of the ODN substituting c7G in place of dG showed a
substantial decrease in binding affinity compared to
dG, due to altered p-electron system of pyrrolo[2,3-d]py-
rimidine nucleobase which affects base stacking and
hydrogen bonding.
The artificial control of gene expression by synthetic oli-
gonucleotides, either at translational (antisense) or at a
transcriptional (antigene) level, is a long-standing dream
in human therapy and biotechnology. There has been
steady progress in antisense technology, which is evident
from the fact that one antisense drug, Fomivirsen, is
currently on the market to treat cytomegalovirus
(CMV) retinitis, while several others are at different
stages of clinical trial for treatment of cancer/and or
viral diseases.1,2 In contrast, there is no antigene
oligodeoxynucleotide (ODN) in clinical trials, although
targeting the gene dsDNA is a more logical approach
as there are only two copies of genes in the eukaryotes
that are to be targeted forming a triple helix, instead
of thousands of copies of m-RNA in the antisense
approach. It has been demonstrated that the best triplex
forming oligonucleotides (TFOs) are those that contain
polypurine stretches, especially G-rich oligonucleotides.3
However, the major concern with G-rich TFOs is that
Keywords: Deoxynucleic guanidine (DNG); 7-Deazaguanine; Triplex
forming oligonucleotide (TFO); G-quatrets.
*
Corresponding author. Tel.: +1 805 893 2044; fax: +1 805 893
2229; e-mail: tcbruice@chem.ucsb.edu
0968-0896/$ - see front matter ꢀ 2006 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2006.05.074

7334
M. L. Jain, T. C. Bruice / Bioorg. Med. Chem. 14 (2006) 7333–7346
H
N
dR
N
dR
N
A
B
N
O
H
N
H
H
N
N
N
Rd N
N
N
N
H
N
H
O
H
H
H
O
N
N
N
H
H
K
O
N
dR
N H
N
H N
H
H
O
N
N
N
H
O
O
H
N
N
N
H
N
N
N dR
H
dR
N
N
N
H
dR
Figure 1. (A). Structure of a G-tetrad showing Hoogsteen H-bonds involving N-7 of G; (B) reverse Hoogsteen triplet interactions of 7-deazaguanine
with G–C base pair, dR signifies 20-deoxyribose.
To overcome the challenges in designing an ideal anti-
sense/antigene ODN having not only high binding affin-
ity but also maintaining base pair fidelity, resistance to
degradation by nucleases, and improved cell permeabili-
ty, several approaches have been reported. These include
backbone modification, for example, phosphorothio-
late,16 phosphonates,17 carbamates,18 methylene meth-
ylimino,19 and locked nucleic acids;20 or replacing the
entire sugar phosphodiester backbone such as in the case
of peptide nucleic acid (PNA),21 phosphonic ester nucleic
acid (PHONA),22 or nucleic acid analog peptide
(NAAP).23 Recently it has been shown that the introduc-
tion of positively charged groups at multiple sites in the
backbone,24–27 sugar28,29 or base30,31 greatly enhances
the duplex and triplex stability.32 Our investigation in
this area has resulted in the discovery of positively
charged guanidinium linkages replacing the negatively
charged phosphodiester linkages.33 As expected, these
deoxynucleic guanidines (DNGs) bind strongly to the
complementary DNA/RNA sequences with high affinity
and fidelity because the repulsive electrostatic effects of
natural duplex DNA are replaced by electrostatic attrac-
tion. DNG linkages have been shown to be resistant to
nucleases.34 It is also possible that the positive charge
of the DNG backbone may improve the cell permeability
through electrostatic attraction of the negatively charged
phosphate groups on the cell surface. Our earlier studies
have demonstrated that thimidinyl DNG oligomers have
a high affinity for and bind only with complementary
poly(dA) in a 2:1 stoichiometry without forming any
complexes with poly(dG), poly(dC) or poly(dT).33c Sim-
ilarly, adenyl DNG33b and cytidinyl DNG35 bind with
poly(dT) and poly(dG) in 1:2 and 1:1 stoichiometry,
respectively, with high affinity. Indeed, poly(dC)
DNG–poly(dG) DNA duplex is 1000 time more stable
than poly(dC–dG) duplex.35 We have also reported a
DNG chimera having mixed thymidinyl and guanyl
bases.36 However, we were unable to prepare DNG with
only guanyl bases. Therefore, we chose to incorporate
c7G bases in place of guanine because of their unique gly-
cosidic bond stability,37,38 stability toward enzymatic
hydrolysis,39 and most importantly, their ability to pre-
vent G-quartet formation.
stranded DNA, as well as general improved antisense/
antigene properties. Herein, we describe the preparation
of monomer building blocks and solid-phase synthesis
of DNG oligomer 1 incorporating c7G bases (Fig. 2).
A convenient and new method for the synthesis of
xylo-7-deazaguanine is also presented.
2. Results and discussion
2.1. Synthesis of monomers
The xylo-7-deazaguanine 7, required to prepare building
blocks 13 and 19 for the solid-phase synthesis of posi-
tively charged DNG oligonucleotide with guanidinium
linkages, was prepared as shown in Scheme 1. The pro-
tected 7-deazaguanine was prepared from 30,50-bis-O-(4-
chlorobenzoyl-20-deoxy-a-D-ribofuranosyl chloride (2)
instead of Hoffer’s a-chloro sugar synthon reported ear-
lier,40 as 2 is more stable and readily crystallizes from
the solvent.41 Treatment of the sodium salt of 2-ami-
no-4-chloropyrrolo[2,3-d]pyrimidine42 with 2 in acetoni-
trile underwent regio- and stereospecific glycosylation to
afford exclusively the desired b-anomer of the protected
3050-bis-O-benzoyl-4-chloro-7-deazanucleoside 3 in high
yield (78%). Hydrolysis of the benzoyl groups with
simultaneous nucleophilic substitution of the 4-chloro
group of 3 was initially carried out using sodium meth-
oxide in refluxing methanol, affording 4 in 70% yield.
O
NH
N
N
HO
NH2
O
O
NH
HN
NH
H2N
N
N
NH2
O
O
NH
HN
NH
NH2
H2N
N
N
O
A logical choice was to combine one of our best back-
bone modifications (i.e. DNG) with an appropriate
modified guanine base (i.e. c7G) to create a ODN which
can exhibit higher affinity toward single or double
OH
1
Figure 2. DNG containing c7G bases.

M. L. Jain, T. C. Bruice / Bioorg. Med. Chem. 14 (2006) 7333–7346
7335
Cl
N
Cl
RO
RO
N
NH2
N
b
O
N
a
O
+
(93%)
Cl
N
H
(78%)
N
NH2
OR
OR
2
3
R = 4-chlorobenzoyl
R = 4-chlorobenzoyl
OCH3
OCH3
OCH3
N
N
N
N
N
O
O
HO
N
RO
d
N
HO
N
NH2
OR
N
N
N
O
H
H
O
c
(70%)
O
(77%)
OH
OH
6: R = 4-Nitrobenzoyl
7: R = OH
e
4
5
(85%)
Scheme 1. Reagents and conditions: (a) NaH, CH3CN, rt, 3 h; (b) Dowex 550 A (OH) anion exchange resin, MeOH, reflux, 2 h; (c) TMSCl, pyridine,
rt, 0.5 h; (iPrCO)O2, pyridine, rt, 6 h; (d) 4-Nitrobenzoic acid, DIAD, PPh3, THF, rt, 4 h; (e) NH3/MeOH, rt, 1 h.
However, a clean and quantitative conversion occurred
when 3 was refluxed with Dowex 550A (OH) anion-ex-
change resin in methanol for 2 h. To our knowledge, this
is the first example of using this resin for one-pot simul-
taneous debenzoylation and substitution of 4-chloro
group to introduce methoxy functionality. The amino
function of 4 was masked as the corresponding isobutyr-
ic amide 5 in one step using a transient protection strat-
egy43 instead of using a two-step procedure reported
earlier.44 Attention was then focused on the stereospecif-
ic inversion of the 30-hydroxy group to prepare xylo-
isomer 7, a key intermediate to introduce amino
functionality at the 30-position. In pyrimidine nucleo-
sides, inversion of the stereochemistry at 30-position
can be easily achieved via anhydride formation resulting
from the intramolecular attack of the pyrimidine C2
oxygen onto the 30-carbon functionalized with a suitable
leaving group.45 Since there is no anhydride formation
with purines, the strategy involved the reaction of 5 with
4-nitrobenzoic acid under Mitsunobu conditions.46,47
The electron withdrawing p-nitro functionality of the
benzoic acid not only leads to complete stereo selective
inversion to afford exclusively the xylo-diester 6 in excel-
lent yield (77%), but also its subsequent removal with
ammonia was very clean. The overall yield of this two-
step conversion for preparing xylo-isomer 7 from 5
was 68%. This provides a facile and high yielding two
step route to prepare the xylo-intermediate from its
corresponding threo-isomer, for introducing 30-azido
or amino functionality, in comparison to lengthy or
low yielding routes available to prepare 20,30-dideoxy-
30-amino guanine.48–51
azide in DMF at 90 ꢁC, affording diazido derivative 9.
Our initial approach for removal of methoxy group of
9 using aqueous NaOH or TMSCl/NaI resulted in poor
yields (65–70%) of 10. However, a clean and quantita-
tive conversion occurred by heating 9 with sodium thio-
cresolate in DMF at 90 ꢁC for 1 h, instead of using
carcinogenic phosphorus triamide solvent as previously
reported.52 Catalytic hydrogenation of diazide 10 with
activated Pd/C yielded corresponding diamine 11 in
quantitative yields. For regioselective tritylation of 50-
NH2 function of diamine 11, the best results were ob-
tained by dropwise addition of a cooled solution of
monomethoxytrityl chloride (MMTrCl) in CH2Cl2 dur-
ing 1 h to a cooled solution of 11 and diethyl amine at
ꢀ40 ꢁC in CH2Cl2, to afford the desired 50-N-tritylamino
derivative 12 in 68% yield. A small amount of bis-trity-
lated derivative (<5%) formed, which was easily separat-
ed by silica gel column chromatography. Finally, the
30-amino group was protected using fluorenylmethyl-
oxycarbonyl isothiocyanate (Fmoc-NCS)53 to afford
the desired thiourea building block 13 in high yields.
The capping building block 19, also synthesized from 20-
deoxy-30xylo-7-deazaguanosine 7, is shown in Scheme 3.
Tritylation of 50-OH with MMTrCl in pyridine followed
by mesylation afforded the 50-monomethoxytritylated-
30mesyl derivative 15. Nucleophilic substitution of 15
with lithium azide occurred smoothly to afford the cor-
responding azide 16. Deprotection of the methoxy group
using sodium thiocresolate, followed by catalytic hydro-
genation using Pd/C, and protection of the amino func-
tionality with Fmoc-NCS (as discussed for preparing 13
from 10) afforded the desired capping thiourea mono-
mer 19.
The elongating monomer, 30,50-protected diamino 13,
was prepared from 7 as shown in Scheme 2. Mesylation
of 7 with MsCl in pyridine afforded 30,50-dimesylate 8
which underwent nucleophilic substitution with lithium
The loading monomer 25 was prepared from 5 as
shown in Scheme 4. The regioselective 50 mesylation

7336
M. L. Jain, T. C. Bruice / Bioorg. Med. Chem. 14 (2006) 7333–7346
OCH3
OCH3
N
N
O
N
N
O
HO
N
MsO
N
OH
N
H
N
OMs
O
H
O
b
a
(88%)
(80%)
8
7
OCH3
O
N
N
N
O
NH
O
N3
N3
N
N
N
H
N
H
c
O
O
d
(96%)
(98%)
N3
N3
9
10
O
N
O
N
NH
NH
O
O
N
MMTrHN
N
RHN
N
N
f
H
H
O
O
(76%)
NH
NH2
S
11. : R = H
12. : R = MMTr
NH
Fmoc
e
13
(68%)
Scheme 2. Reagents and conditions: (a) MsCl, DMAP, pyridine, rt, 15 h; (b) LiN3, DMF, 90 ꢁC, 2 h; (c) sodium thiocresolate, DMF, 90 ꢁC, 1 h; (d)
H2, Pd/C, EtOH, rt, 5 h; (e) MMTCl, Et2NH, CH2Cl2, ꢀ40 ꢁC to rt; (f) Fmoc-NCS, CH2Cl2, rt, 2 h.
of 5 was carried out using MsCl in pyridine at 0 ꢁC.
Subsequent conversion to azide 21 using lithium azide,
followed by catalytic hydrogenation over Pd/C, gave
corresponding 50-amino derivative 22. Tritylation of
the amino function by dropwise addition of MMTrCl
in pyridine at 0 ꢁC followed by subsequent removal
of the methoxy group with sodium thiocresolate oc-
curred smoothly in high yield to give 24. Esterification
of the 30-OH with succinic anhydride afforded the
loading monomer 25.
2.2. Solid-phase synthesis of DNG (1)
Solid-phase synthesis of DNG trimer was performed on
CPG support from 30 ! 50 direction, as in the standard
DNA synthesis. A typical solid-phase synthesis is out-
lined in Scheme 5. Upon deblocking of the acid labile
MMTr group, the 50-amino functionality becomes avail-
able to couple with incoming precursor for the forma-
tion of a guanidinium linkage. The coupling reaction
was accomplished in the presence of HgCl2/TEA, where-
by the 30-Fmoc-protected thiourea of the incoming pre-
cursor 13 is converted into an activated carbodiimide
intermediate via abstraction of the sulfur atom on thio-
urea by Hg (II). The carbodiimide intermediate then re-
acts with 50-NH2 of the unmasked CPG loaded
monomer to provide a guanidinium linkage.33d The
HgS precipitate formed during the reaction was re-
moved by demercuration using 20% thiophenol in
DMF. After coupling, the unreacted 50-amino sites were
blocked by capping, rendering them inert toward further
chain elongation. The terminal 50-MMTr was then
deprotected and the coupling yield was determined to
be 80% by UV analysis. The coupling cycle was then
repeated using capping monomer 19. After final cou-
pling, the capping and deprotection steps were omitted
to simplify the crude product purification. The resulting
trimer 50-c7Ggc7Ggc7G*-30 was removed from CPG
using methanolic ammonia at 60 ꢁC. The Fmoc-protect-
The triethylammonium salt of 25 underwent excellent
coupling with the amino function of long-chain alkyl
amino controlled pore glass (LCAA-CPG) in presence
of 4-(4,6-dimethoxy-1,3,5-traiazin-2-yl)-4-methylmorph-
olinium chloride (DMTMM)54 to afford monomer load-
ed CPG 26. In comparison to the frequently used
approach of activating the acid group by 4-nitrophenol
using DCC and subsequent loading on CPG reported
earlier for DNG synthesis33 which is tedious, time-con-
suming, and often gives lower loading yields (25–
30 lmol/g loading after first coupling), the present meth-
od is facile, clean, and gave excellent loading (51 lmol/g)
on CPG within 1 h (Scheme 4). The loading yield was
determined spectrophotometrically from the amount of
MMT cation released upon treatment with 4% dichloro-
acetic acid (DCA) solution in CH2Cl2. After loading, the
unprotected sites were capped with Ac2O/TEA.

M. L. Jain, T. C. Bruice / Bioorg. Med. Chem. 14 (2006) 7333–7346
7337
OCH3
OCH3
N
N
N
N
O
O
HO
N
MMTrO
N
OH
OR
N
N
c
H
H
O
O
a
(73%)
(82%)
7
14. : R = H
b
15. : R = Ms
(75%)
OCH3
O
N
N
NH
O
O
MMTrO
N
N
O
MMTrO
N
N
N
e
H
H
d
O
(94%)
(88%)
N3
N3
17
16
O
O
NH
NH
O
O
MMTrO
N
MMTrO
N
O
N
N
N
N
H
H
O
f
(78%)
NH2
HN
S
18
Fmoc NH
19
Scheme 3. Reagents and conditions: (a) MMTrCl, pyridine, rt, 18 h; (b) MsCl, pyridine, rt, 16 h; (c) LiN3, DMF, 90 ꢁC, 2 h; (d) sodium
thiocresolate, DMF, 90 ꢁC, 1 h; (e) H2, Pd/C, EtOH, rt, 6 h; (f) Fmoc-NCS, CH2Cl2, rt, 1.5 h.
ing groups on guanidinium linkages and isobutyryl
groups on exocyclic deazaguanine bases were also re-
moved simultaneously to afford MMTr-protected
DNG 29 (Scheme 5).
indicates that DNG 1 binds to complimentary penta-
meric DNA-C5 in 1:1 ratio, consistent with the forma-
tion of a Watson–Crick base paired duplex.
The crude DNG 29 was purified on reverse-phase HPLC
(altech C8 column) using 100 mM triethylammonium
acetate (TEAA) buffer (pH 7.0) as solvent A with a
gradient of 5% ! 95% CH3CN as solvent B in 40 min.
The trityl group on the 50-terminus of the oligomer
29 was then deprotected with 4% DCA in CH2Cl2
solution and precipitated with excess ether. The precip-
itated product was centrifuged, dried, and purified by
reverse-phase HPLC to afford desired detritylated
trimer 1.
3. Conclusions
The first stepwise solid-phase synthesis of a dual
modified ODN with both a positively charged guanidini-
um backbone and incorporating 7-deazaguanine bases
has been accomplished. The trimeric DNG oligomer
30-OH-c7Ggc7Ggc7G-OH-50 (1) was synthesized in
30 ! 50 direction on CPG solid support, comparable
with standard DNA solid-phase synthesis. The synthesis
of orthogonally protected precursor deazaguanyl mono-
mers and a novel facile method for the inversion of 30-
OH functionality to prepare xylo-7-deazaguanine using
4-nitrobenzoic acid under Mitsunobu conditions are de-
scribed. The synthesized DNG trimer ODN binds to
cytidinyl-pentamer in 1:1 ratio. The synthesis of mono-
mers 13 and 19, as well as DNG 1 having a free hydroxyl
groups at both 30 and 50 ends described here can allow
us to prepare chimera DNA-O-c7Gg-(c7Gg)n-O-DNA
or DNA-O-c7Gg-Ag-Tg-Cg-G-O-DNA of desired
length. The synthesis of these chimeras is currently
underway to fully explore the Watson–Crick base pair-
ing for duplex and triplex formation, and their antisense
and antigene properties.
2.3. Binding studies of DNG 1
The binding stoichiometry of the trimeric DNG 1 with
complimentary pentameric cytidine DNA (DNA-C5)
was determined by the method of continuous variation55
to generate mixing curves of the absorbance versus mole
fraction of DNG and DNA (Fig. 3). This method is
based on the assumption that a decrease in absorbance
is proportional to the number of base pairs hydrogen
bonded between the interacting species. Increasing mole
fraction of DNA-C5 to DNG 1 lowered the UV absor-
bance at 260 nm. The inflection point at 50% DNA-C5

7338
M. L. Jain, T. C. Bruice / Bioorg. Med. Chem. 14 (2006) 7333–7346
OCH3
OCH3
N
N
N
N
O
O
R
N
HO
N
N
N
c
O
H
a
O
H
(93%)
(73%)
OH
OH
5
20. R = OMs
21. R = N3
b
(81%)
OCH3
O
N
NH
N
N
O
O
N
O
N
MMTrNH
RNH
N
H
N
f
O
e
H
(90%)
(85%)
OH
OH
22. R = H
24
d
23. R = MMTr
(73%)
O
N
O
NH
NH
O
O
MMTrNH
N
N
O
MMTrNH
N
N
N
g
H
O
H
O
O
CPG
O
O
N
OH
H
O
O
26
25
Scheme 4. Reagents and conditions: (a) MsCl, pyridine, 0 ꢁC to rt, 14 h; (b) LiN3, DMF, 90 ꢁC, 2 h; (c) H2, Pd/C, EtOH, rt, 5 h; (d) MMTrCl,
pyridine, 0 ꢁC to rt 18 h; (e) sodium thiocresolate, DMF, 90 ꢁC, 1 h; (f) succinic anhydride, Et3N, CH2Cl2, 3 h; (g) LCAA-CPG, DMTMM, MeOH,
1 h.
4. Experimental
4.2. Synthesis: 2-amino-4-chloro-7-[20-deoxy-30,50-di-O-
(4-chlorobenzoyl)-b-D-erythro-pentofuranosyl]-7H-pyr-
rolo[2,3-d]pyrimidine (3)
4.1. Materials
All anhydrous solvents and reagents were purchased
from Aldrich and used without further purification.
HPLC grade triethylammonium acetate buffer was
purchased from Fluka and LCAA-CPG (500 ,
80–120 mesh size) was purchased from Sigma. All
1H and 13C spectra were recorded on 400 and
500 MHz Varian instruments as indicated, using
CDCl3 or DMSO-d6 as solvents, and chemical shifts
are reported in d ppm. TLC was carried out on silica
gel (Kieselgel 60 F254 glass-backed commercial plates)
and visualized by UV light. All reactions were per-
formed under nitrogen unless otherwise indicated.
Hydrogenation was carried out with a par hydro-
genator equipped with a 500 mL hydrogenation vessel.
Reverse-phase HPLC was performed on a Hewlett
Packard 1050 system equipped with a quaternary sol-
vent delivery system, UV detector set at 260 nm, and
an Altech macrosphere C8 RP semiprep column
(10 · 250 mm).
To a suspension of sodium hydride (60% emulsion in
oil, 1.70 g, 42.5 mmol) in dry acetonitrile (100 mL)
was added 2-amino-4-chloropyrrolo[2,3-d]pyrimidine
(6.50 g, 38.6 mmol) at rt. After 1 h, 1-chloro-20deoxy-
3050-di-O-(4-chlorobenzoyl)-a-D-erythro-pentofuranose
(2) (18.0 g, 46.4 mmol) was added to the reaction mix-
ture and stirred further for 3 h. The reaction mixture
was filtered and solvent was removed under vacuum.
The residue was partitioned in CH2Cl2 (250 mL) and
water (50 mL), and organic phase was dried (anhy-
drous Na2SO4). The residue obtained after removal
of the solvent was chromatographed on silica gel
(EtOAc/Hexanes 1:4) to afford 3 (15.5 g, 78% yield)
as a white foam. 1H NMR (400 MHz, CDCl3): d
2.63–2.69 (m, 1H, H-20), 2.93–2.99 (m, 1H, H-20),
4.55–4.63 (m, 2H, H-40 and H-50), 4.75–4.80 (m, 1H,
H-50), 5.09 (s, 2H, NH2), 5.76 (m, 1H, H-30), 6.41
(d, J = 3.6 Hz, 1H, H-5), 6.53–6.56 (dd, J = 6.0,
8.4 Hz, 1H, H-10), 6.98 (d, J = 3.6 Hz, 1H, H-6),

M. L. Jain, T. C. Bruice / Bioorg. Med. Chem. 14 (2006) 7333–7346
7339
O
O
NH
O
NH
O
MMTrNH
N
N
MMTrNH
N
N
N
N
O
H
O
H
a, b
a, b
c
O
c
O
NH
13
CPG
19
NH
O
Fmoc
N
N
N
N
26
N
H
O
O
CPG
27
O
N
O
N
NH
NH
O
MMTrO
N
MMTrO
N
N
NH2
O
H
O
O
O
NH
NH
NH
NH
O
Fmoc
N
H2N
N
HN
N
HN
N
N
N
NH2
O
H
O
d
O
O
NH
NH
N
NH
NH
O
Fmoc
N
H2N
N
N
N
N
N
N
NH2
O
H
O
O
OH
29
CPG
28
Scheme 5. Reagents and conditions: (a) Capping: (CH3CO)2O, TEA, DMF, 10 min; (b) Deprotection: 4% DCA in CH2Cl2, 1 min; (c) Coupling:
HgCl2, TEA, DMF, 3 h, then 20% PhSH in DMF, 1 min; (d) NH4OH, 60 ꢁC, 12 h.
calcd for C25H19N4O5Cl3(M+H)+ 561.0493. Found
564.0520.
0.65
0.6
4.3. 2-Amino-7-(20-deoxy-b-D-erythro-pentofuranosyl)-4-
0.55
methoxy-7H-pyrrolo-[2,3-d]pyrimidine (4)40
0.5
Dowex 550A anion exchange resin (41.0 g) was added to a
0.45
0.4
solution of 3 (10.2 g, 18.2 mmol) in anhydrous MeOH
(100 mL) and the reaction mixture was heated to reflux
for 2 h until all the starting material was consumed, as
observed from TLC. The suspension was filtered, washed
with hot MeOH and combined filtrates were concentrat-
ed under vacuum. The residue was triturated with
0
20
40
60
80
100
% of DNA C-5
Figure 3. Job plot illustrating 1:1 binding of DNG 1 to DNA C-5.
Total oligomer concentration was 4 lmol and buffer contained
100 mM [NaCl], 10 mM [NaHPO4], adjusted to pH 7.1.
1
ether to afford 4 (4.71 g, 93%) as white solid. H NMR
(400 MHz, DMSO-d6): d 2.04–2.12 (m, 1H, H-20), 2.37–
2.42 (m, 1H, H-20), 3.48–3.52 (m, 2H, H-50). 3.72–3.77
(m, 1H, H-40), 3.91 (s, 3H, OCH3), 4.28–4.31 (m, 1H,
H-30), 4.95 (t, J = 5.4 Hz, 1H, OH), 5.23 (d, J = 3.6 Hz,
1H, OH), 6.23 (br s, 2H, NH2), 6.26 (d, J = 4.0 Hz, 1H,
H-5), 6.41 (dd, J = 6.0, 8.4 Hz, 1H, H-10), 7.10 (d,
J = 4.0 Hz, 1H, H-6); HRMS (ESI) m/z calcd for
C12H16N4O4 (M+H)+ 281.1244. Found 281.1249.
7.36–7.47 (m, 4H, ArH), 7.92–8.02 (m, 4H, ArH);
13C NMR (500 MHz, CDCl3): d 36.8, 64.3, 95.4,
81.6, 84.2, 101.4, 111.3, 122.5, 127.6, 127.8, 128.7,
128.8, 128.9, 129.0, 130.9, 131.0, 131.1, 139.8, 140.1,
152.9, 153.5, 158.6, 165.0, 165.34; HRMS (ESI) m/z

7340
M. L. Jain, T. C. Bruice / Bioorg. Med. Chem. 14 (2006) 7333–7346
4.4. 7-(20-deoxy-b-D-erythro-pentofuranosyl)-2-isobuty-
rylamino-4-methoxy-7H-pyrrolo[2,3-d]pyrimidine (5)44
residue was chromatographed on silica gel (EtOAc/
MeOH 1:5) to give desired product (2.0 g, 85%) as white
foam. 1H NMR (400 MHz, CDCl3):
d 1.08 (d,
TMSCl (7.53 g, 69.3 mmol) was added dropwise to a
solution of 4 (2.48 g, 8.86 mmol) in anhydrous pyridine
(50 mL). After stirring for 0.5 h, isobutyric anhydride
(7.34 g, 46.4 mmol) was added to the reaction mixture
and let stirred for 6 h at rt. The reaction was quenched
by addition of water (40 mL) and NH4OH (10 mL)
and held for 0.5 h. The oil obtained after removal of
the volatiles under vacuum was diluted with CH2Cl2
(150 mL) and washed with water (50 mL). The organ-
ic-phase dried (Na2SO4), concentrated under vacuum,
and the residue was chromatographed over silica gel
J = 6.8 Hz, 6H, (CH3)2), 2.07–2.12 (m, 1H, H-20),
2.71–2.78 (m, 1H, H-20), 2.85–2.90 (m, 1H, CH), 3.56–
3.62 (m, 1H, H-50), 3.68–3.74 (m, 1H, H-50), 3.82–3.86
(m, 1H, H-40), 4.02 (s, 3H, OCH3), 4.30–4.34 (m, 1H,
H-30), 4.63 (t, J = 6.0 Hz, 1H, OH), 5.33 (d,
J = 4.0 Hz, 1H, OH), 6.42 (dd, J = 2.8, 8.4 Hz, 1H, H-
10), 6.46 (d, J = 3.6 Hz, 1H, H-5), 7.64 (d, J = 3.6 Hz,
1H, H-6), 10.20 (s, 1H, NH); 13C NMR (500 MHz,
CDCl3): d 19.9, 19.9, 34.8, 41.4, 54.1, 60.5, 69.6, 82.1,
85.0, 99.4, 101.6, 124.9, 152.0, 152.8, 163.0, 175.8;
HRMS (ESI) m/z calcd for C16H22N4O5 (M+Na)+
373.1482. Found 373.1467.
1
(MeOH/EtOAc 1:9) to afford 5 (2.17 g, 70% yield). H
NMR (400 MHz, DMSO-d6): d 1.09 (d, J = 6.8 Hz,
6H, (CH3)2), 2.13–2.19 (m, 1H, H-20), 2.45–2.52 (m,
1H, H-20 partially merged with solvent peak), 2.85–
2.92 (m, 1H, CH), 3.47–3.57 (m, 2H, H-50), 3.76–3.82
(m, 1H, H-40), 4.02 (s, 3H, OCH3), 4.32–4.37 (m, 1H,
H-30), 4.91 (t, J = 5.2 Hz, 1H, OH), 5.28 (d,
J = 3.6 Hz, 1H, OH), 6.48 (d, J = 3.6 Hz, 1H, H-5),
6.53 (dd, J = 6.0, 8.4 Hz, 1H, H-10), 7.48 (d,
J = 3.6 Hz, 1H, H-6), 10.24 (s, 1H, NH); 13C NMR
(500 MHz, DMSO-d6): d 19.4, 19.4, 34.2, 39.0–40.0
(one signal merged with solvent peaks), 53.5, 61.9,
71.0, 82.4, 87.2, 99.2, 101.2, 123.0, 151.6, 152.5, 162.4,
175.0; HRMS (ESI) m/z calcd for C16H22N4O5
(M+Na)+ 373.1482. Found 373.1501.
4.7. 7-[20-Deoxy-30,50-di-O-mesyl-b-D-threo-pentofurano-
syl]-2-isobutyrylamino-4-methoxy-7H-pyrrolo[2,3-d]py-
rimidine (8)
To a solution of 7 (2.15 g, 6.5 mmol) in anhydrous pyr-
idine (30 mL) were added MsCl (2.40 mL, 3.52 g,
30.8 mmol) and DMAP (0.21 g, 1.71 mmol) at rt. The
reaction mixture was quenched with MeOH after stir-
ring for 15 h and concentrated. The residue was dis-
solved in CH2Cl2 (150 mL) and washed with water (2·
50 mL). The organic layer was dried (Na2SO4), concen-
trated under vacuum, and the residue was purified by sil-
ica gel chromatography (EtOAc/hexanes 1:5) to afford 8
as a white foam (2.72 g, 88%). 1H NMR (400 MHz,
CDCl3): d 1.28 (d, J = 6.8 Hz, 6H, (CH3)2), 2.84–2.90
(m, 1 H, H-20) 2.95–3.00 (m, 1H, H-20), 3.05–3.08 (m,
4H, SO2CH3, and CH), 3.13 (s, 3H, SO2CH3), 4.07 (s,
3H, OCH3), 4.37–4.42 (m, 1H, H-40), 4.47–4.56 (m,
2H, H-50) 5.39–5.42 (m, 1H, H-30), 6.54 (d, J = 3.6 Hz,
1H, H-5), 6.64 (dd, J = 4.0, 8.0 Hz, 1H, H-10), 7.21 (d,
J = 3.6 Hz, 1H, H-6), 7.80 (br s, 1H, NH); 13C NMR
(500 MHz, CDCl3): 19.5, 19.5, 35.8, 37.8, 38.8, 39.0,
54.1, 66.2, 78.2, 78.5, 82.5, 101.1, 102.3, 122.1, 151.8,
153.0, 163.6, 176.6; HRMS (ESI) m/z calcd for
C18H26N4O9 S2 (M+H)+ 507.1213. Found 507.1229.
4.5. 7-[20-Deoxy-30,50-di-O-(4-nitrobenzoyloxy)-b-D-
threo-pentofuranosyl]-2-isobutyrylamino-4-methoxy-7H-
pyrrolo[2,3-d]pyrimidine (6)
To a solution of 5 (3.40 g, 9.71 mmol) in anhydrous THF
(50 mL) were added PPh3 (7.67 g, 29.2 mmol) and DIAD
(5.9 g, 29.4 mmol) at rt. 4-Nitrobenzoic acid (4.90 g,
29.3 mmol) was added to the reaction mixture after
20 min and the reaction mixture was stirred further for
4 h. The solvent was removed under vacuum and the res-
idue was chromatographed over silica gel (EtOAc/Hex-
1
anes 1:5) to afford 6 (4.76 g, 77%) as a yellow foam. H
NMR (CDCl3): d 1.27 (d, J = 6.8 Hz, 3H, CH3), 1.29
(d, J = 6.8 Hz, 3H, CH3), 2.92–3.03 (m, 1H, H-20),
3.03–3.11 (m, 2H, CH and H-20), 4.04 (s, 3H, OCH3),
4.62–4.66 (m, 1H, H-40), 4.74 (d, J = 5.6 Hz, 2H, H-50),
5.92 (t, J = 4.0 Hz, 1H, H-30), 6.52 (d, J = 3.6 Hz, 1H,
H-5), 6.63 (dd, J = 3.2, 7.6 Hz, 1H, H-10), 7.23 (d,
J = 3.6 Hz, 1H, H-6), 7.75 (s, 1H NH), 8.08 (d,
J = 8.4 Hz, 2H, ArH), 8.16 (d, J = 8.4 Hz, 2H, ArH),
8.26 (dd, J = 3.6, 8.4 Hz, 4H, ArH); 13C NMR
(500 MHz, CDCl3): d 19.5, 19.5, 36.2, 39.0, 54.0, 63.5,
74.3, 79.4, 83.5, 100.4, 102.5, 121.6, 123.7, 123.9, 128.3,
130.9, 131.0, 132.4, 134.5, 134.8, 150.8, 151.0, 151.6,
152.6, 163.5, 163.8, 164.4, 174.8; HRMS (ESI) m/z calcd
for C30H28N6O11 (M+H)+ 649.1888. Found 649.1917.
4.8. 7-[30,50-Diazido-2030-dideoxy-b-D-erythro-pentofur-
anosyl]-2-isobutyrylamino-4-methoxy-7H-pyrrolo[2,3-
d]pyrimidine (9)
Lithium azide (2.60 g, 54.1 mmol) was added to a solu-
tion of 8 (2.70 g, 5.34 mmol) in anhydrous DMF
(20 mL). The reaction mixture was heated at 90 ꢁC until
all the starting material was consumed (2 h). The solvent
was evaporated under vacuum and the residue was chro-
matographed over silica gel (EtOAc/hexanes 2:3) to af-
ford 9 as a white foam (1.70 g, 80%). 1H NMR
(400 MHz, CDCl3): d 1.28 (d, J = 6.8 Hz, 3H, CH3),
1.30 (d, J = 6.8 Hz, 3H, CH3), 2.44–2.51 (m, 1H, H-
20), 3.02–3.10 (m, 2H, H-20, and CH), 3.60 (dd,
J = 4.0, 13.2 Hz, 1H, H-50), 3.69 (dd, J = 4.0, 13.2 Hz,
1H, H-50), 3.98–4.02 (m, 1H, H-40), 4.05 (s, 3H,
OCH3), 4.88 (br s, 1H, H-30), 6.30 (dd, J = 5.6, 7.2 Hz,
1H, H-10), 6.47 (d, J = 3.6 Hz, 1H, H-5), 7.03 (d,
J = 3.6 Hz, 1H, H-6), 7.80 (br s, 1H, NH); 13C NMR
(500 MHz, CDCl3): 19.5, 19.6, 36.2, 37.1, 52.3, 54.0,
61.5, 82.7, 85.1, 100.1, 103.1, 123.6, 151.3, 152.3,
4.6. 7-[20-Deoxy-b-D-threo-pentofuranosyl]-2-isobuty-
rylamino-4-methoxy-7H-pyrrolo[2,3-d]pyrimidine (7)
A suspension of 6 (4.4 g, 6.79 mmol) in methanolic
ammonia (40 mL) was stirred for 1 h at rt. The homoge-
neous solution was concentrated under vacuum and the

M. L. Jain, T. C. Bruice / Bioorg. Med. Chem. 14 (2006) 7333–7346
7341
163.6, 175.8; HRMS (ESI) m/z calcd for C16H20N10O3
CH), 2.49–2.59 (m, 2H, H-50), 3.70–3.81 (m, 5H, H-30,
H-40 and OCH3), 6.27 (dd, J = 4.8, 6.8 Hz, 1H, H-10),
6.60 (d, J = 3.2 Hz, 1H, H-5), 6.62 (d, J = 3.2 Hz, 1H,
H-6), 6.77 (d, J = 8.8 Hz, 2H, ArH), 7.17–7.31 (m,
10H, ArH), 7.41 (d, J = 8.8 Hz, 2H, ArH), 8.89 (br s,
2H, NH2), 10.29, (br s, 1H, NH); 13C NMR
(500 MHz, CDCl3,): d 19.1, 19.2, 36.6, 41.2, 45.9, 53.6,
55.4, 70.3, 82.6, 86.9, 104.3, 105.4, 113.3, 113.4, 119.0,
126.6, 128.1, 128.7, 130.0, 137.8, 146.0, 146.4, 147.5,
158.1, 158.1, 178.8. HRMS (ESI) m/z calcd for
C35H38N6O4 (M+H)+ 607.3027. Found 607.3054.
(M+H)+ 401.1792. Found 401.1807.
4.9. 7-[30,50-Diazido-2030-dideoxy-b-D-erythro-pentofur-
anosyl]-2-isobutyrylamino-3,7-dihydro-4H-pyrrolo[2,3-
d]pyrimidin-4-one (10)
Sodium thiocresolate (1.84 g, 12.6 mmol) was added to a
solution of 9 (0.75 g, 1.87 mmol) in anhydrous DMF
(15.0 mL) and the reaction mixture was heated at
90 ꢁC for 1 h. The solvent was removed under vacuum
and the residue was chromatographed over silica gel
(EtOAc/hexanes 3:2) to give 10 as a yellow foam
4.12. 7-[30-N-(9-fluorenylmethoxycarbonylamino)-50-N-
(4-monomethoxytritylamino)-20,30-dideoxy-b-D-pentofur-
anosyl]-2-isobutyrylamino-3,7-dihydro-4H-pyrrolo[2,3-
d]pyrimidin-4-one (13)
1
(0.70 g, 96%). H NMR (400 MHz, CDCl3): d 1.25 (d,
J = 6.8 Hz, 3H, CH3), 1.27 (d, J = 6.8 Hz, 3H, CH3),
2.41–2.47 (m, 1H, H-20), 2.58–2.71 (m, 2H, H-20 and
CH), 3.48 (dd, J = 4.0, 13.2 Hz, 1H, H-50), 3.64 (dd,
J = 4.0, 13.2 Hz, 1H, H-50), 3.97–4.01 (m, 1H, H-40),
4.31–4.36 (m, 1H, H-30), 6.34 (t, J = 6.4 Hz, 1H, H-10),
6.71 (d, J = 3.6 Hz, 1H, H-5), 6.95 (d, J = 3.6 Hz, 1H,
H-6), 8.16 (br s, 1H, NH), 11.76 (br s, 1H, NH); 13C
NMR (500 MHz, CDCl3): 19.2, 19.2, 36.7, 37.5, 52.3,
61.2, 82.2, 83.1, 104.9, 105.8, 118.8, 146.4, 147.6,
157.9, 178.4; HRMS (ESI) m/z calcd for C15H18N10O3
(M+H)+ 387.1636. Found 387.1619.
A solution of Fmoc-NCS (0.29 g, 1.04 mmol) in CH2Cl2
(5 mL) was added to a solution of 12 (0.43 g 0.70 mmol)
in anhydrous CH2Cl2 (50 mL) and stirred for 2 h at rt.
The reaction mixture was concentrated and the crude
product was purified by ether precipitation to give 13
(0.48 g, 76% yield) as an off-white solid. 1H NMR
(400 MHz, CDCl3): d 1.10 (d, J = 7.2 Hz, 3H, CH3),
1.18 (d, J = 7.2 Hz, 3H, CH3), 2.32–2.49 (m, 5H, H-20,
CH, and H-50), 3.20–3.26 (m, 1H, H-30), 3.74 (s, 3H,
OCH3), 4.02–4.06 (m, 1H, H-40), 4.25 (t, J = 6.4 Hz,
1H, OCH2CH), 4.54 (d, J = 6.8 Hz, 2H, OCH2), 6.05
(dd, J = 2.8, 8.4 Hz, 1H, H-10), 6.18–6.24 (m, 1H,
NH), 6.34 (d, J = 3.6 Hz, 1H, H-5), 6.74 (d,
J = 8.8 Hz, 2H, ArH), 6.77 (d, J = 3.6 Hz, 1H, H-6),
7.10–7.57 (m, 18H, ArH), 7.79 (d, J = 8.0 Hz, 2H,
ArH), 8.27 (br s, 1H, NH), 8.59 (s, 1H, NH), 9.85 (br
s, 1H, NH), 11.70 (br s, 1H, NH); 13C NMR
(500 MHz, CDCl3): d 19.0, 19.1, 36.8, 37.2, 44.2, 46.6,
55.4, 56.4, 68.7, 70.0, 83.3, 85.1, 103.9, 106.6, 113.2,
120.5, 121.9, 124.9, 126.5, 127.5, 128.0, 128.2, 128.4,
128.6, 129.9, 137.9, 141.6, 142.8, 142.9, 145.8, 146.0,
146.1, 146.7, 152.8, 157.9, 158.0, 178.3, 179.7; HRMS
(ESI) m/z calcd for C51H49N7O6S (M+Na)+ 910.3357.
Found 910.3314.
4.10. 7-[30,50-Diamino-20,30-dideoxy-b-D-erythro-pento-
furanosyl]-2-isobutyrylamino-3,7-dihydro-4H-pyr-
rolo[2,3-d]pyrimidin-4-one (11)
To a solution of 10 (0.44 g, 1.14 mmol) in absolute
EtOH (50 mL), 10% Pd/C (0.10 g) was added and the
suspension was shaken under hydrogen gas (45 psi) for
5 h. The reaction mixture was filtered through a pad
of Celite and washed with ethanol (2· 5 mL). Combined
filtrate was concentrated under vacuum and the residue
was evaporated twice with CH2Cl2 to afford 11 (0.39 g,
98%) as pale yellow solid. 1H NMR (400 MHz,
DMSO-d6): d 1.11 (d, J = 6.8 Hz. 6H, (CH3)2), 2.11
(m, 1H, H-20) 2.36 (m, 1H, H-20), 2.76 (m, 3H, H-50,
and CH), 3.51 (m, 2H, H-40, and H-30), 6.34 (t,
J = 6.8 Hz, 1H, H-10), 6.48 (d, J = 3.6 Hz, 1H, H-5),
7.20 (d, J = 3.6 Hz, 1H, H-6), 8.26 (s, 1H, NH); 13C
NMR (500 MHz, DMSO-d6): d 18.9, 18.9, 34.7, 40.5,
43.8, 52.6, 81.9, 87.7, 102.9, 104.0, 119.6, 146.8, 147.3,
156.7, 180.0; HRMS (ESI) m/z calcd for C15H22N6O3
(M+H)+ 335.1826. Found 335.1834.
4.13. 7-[20-Deoxy-50-O-(4-monomethoxytrityl)-b-D-threo-
pentofuranosyl]-2-isobutyrylamino-4-methoxy-7H-pyr-
rolo[2,3-d]pyrimidine (14)
MMTrCl (1.27 g, 4.10 mmol) was added dropwise to a
solution of 13 (1.25;g, 3.57 mmol) in anhydrous pyridine
(50 mL). After 18 h, the reaction mixture was quenched
with MeOH (5 mL) and concentrated under vacuum.
The residue was purified by silica gel chromatography
(EtOAc/hexanes 4:1) to afford 14 (1.82 g, 82%). 1H
NMR (400 MHz, CDCl3): d 1.24 (d, J = 6.8 Hz, 3H,
CH3), 1.26 (d, J = 6.8 Hz, 3H, CH3), 2.54–2.59 (m,
1H, H-20) 2.72–2.80 (m, 1H, H-20), 3.05 (br s, 1H,
CH), 3.51–3.55 (m, 1H, H-50), 3.58–3.62 (m, 1H,
H-50), 3.78 (s, 3H, MMTr-OCH3), 4.0–4.06 (m, 1H,
H-40), 4.07 (s, 3H, OCH3), 4.42–4.50 (m, 1H, H-30),
4.92 (d, J = 3.2 Hz, 1H, OH), 6.19 (dd, J = 3.2, 9.2 Hz,
1H, H-10), 6.43 (d, J = 3.6 Hz, 1H, H-5), 6.79 (d,
J = 8.8 Hz, 2H, ArH), 7.16–7.45 (m, 11H, H-6, and
ArH), 7.45 (d, J = 7.2 Hz, 2H, ArH) 7.64 (s, 1H, NH).
13C NMR (500 MHz, CDCl3): d 19.5, 19.6, 35.9, 40.6,
4.11. 7-[30-Amino-50-N-(4-monomethoxytritylamino)-
20,30-dideoxy-b-D-erythro-pentofuranosyl]-2-isobutyryl-
amino-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (12)
A solution of MMTrCl (0.47 g, 1.52 mmol) in CH2Cl2
(10.0 mL) was added dropwise over 1 h to a cooled
(ꢀ40 ꢁC) suspension of 11 (0.50 g, 1.43 mmol) and
diethylamine (0.3 mL, 2.88 mmol) in CH2Cl2 (35 mL).
The reaction mixture was warmed to room temperature
after 1 h and quenched with MeOH (5 mL). The solvent
was removed under vacuum and the residue was chro-
matographed (MeOH/EtOAc 1:20) to afford 12
(0.59 g, 68%). 1H NMR (CDCl3):
d
J = 6.8 Hz, 3H, CH3), 1.20 (d, J = 6.8 Hz, 3H, CH3),
1.18 (d,
2.10–2.17 (m, 1H, H-20), 2.29–2.42 (m, 2H, H-20 and

7342
M. L. Jain, T. C. Bruice / Bioorg. Med. Chem. 14 (2006) 7333–7346
54.14, 55.40, 62.6, 71.5, 82.8, 85.3, 87.0, 99.2, 103.4,
113.4, 125.5, 126.4, 127.4, 128.0, 128.1, 128.6, 129.4,
130.6, 135.6, 144.5, 147.3, 151.1, 151.6, 158.7, 163.5,
176.2; HRMS (ESI) m/z calcd for C36H38N4O6
(M+Na)+ 645.2683. Found 645.2665.
4.16. 7-[30-Azido-20,30-dideoxy-50-O-(4-monomethoxytri-
tyl)-b-D-erythro-pentofuranosyl]-2-isobutyrylamino-3,7-
dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (17)
Sodium thiocresolate (0.72 g, 4.98 mmol) was added to a
solution of 16 (1.01 g, 1.54 mmol) in anhydrous DMF
(5 mL) and the reaction mixture was heated at 90 ꢁC un-
til all starting material was consumed, as shown by TLC
(1 h). The solvent was removed under vacuum and the
residue was chromatographed over silica gel (EtOAc/
hexanes 3:2) to give 17 as a white foam (0.90 g, 88%).
1H NMR (400 MHz, CDCl3): d 1.14 (d, J = 6.8 Hz,
3H, CH3), 1.21 (d, J = 6.8 Hz, 3H, CH3), 2.29–2.42
(m, 2H, H-20 and CH), 2.67–2.74 (m, 1H, H-20), 3.26
(dd, J = 3.6, 10.4 Hz, 1H, H-50), 3.38 (dd, J = 3.6,
10.4 Hz, 1H, H-50), 3.79 (s, 3H, MMTr-OCH3), 4.05
(q, J = 4.0, 8.0 Hz, 1H, H-40), 4.37–4.41 (m, 1H, H-30),
6.30 (t, J = 7.2 Hz, 1H, H-10), 6.62 (d, J = 3.2 Hz, 1H,
H-5), 6.82 (d, J = 8.4 Hz, 2H, ArH), 6.88 (d,
J = 3.6 Hz, 1H, H-6), 7.17–7.33 (m, 10H, ArH), 7.42
(d, J = 7.2 Hz, 2H, ArH), 7.87 (br s, 1H, NH), 11.68
(br s, 1H, NH). 13C NMR (500 MHz, CDCl3): 19.1,
19.1, 36.7, 37.6, 55.5, 61.5, 63.6, 83.2, 83.5, 87.1, 104.4,
105.9, 113.4, 119.4, 127.4, 128.0, 128.1, 128.2, 128.5,
129.4, 130.6, 135.2, 144.1, 144.2, 146.2, 147.6, 158.0,
159.0, 178.4; HRMS (ESI) m/z calcd for C35H35N7O5
(M+Na)+ 656.2591. Found 656.2586.
4.14. 7-[20-Deoxy-30-O-mesyl-50-O-(4-monomethoxytri-
tyl)-b-D-threo-pentofuranosyl]-2-isobutyryl-4-methoxy-
7H-pyrrolo[2,3-d]pyrimidine (15)
MsCl (0.81 g, 7.05 mmol) was added to a mixture of 14
(1.75 g, 2.81 mmol) and DMAP (1.80 g, 14.1 mmol) in
anhydrous pyridine at 0 ꢁC. After 10 min, reaction mix-
ture was allowed to warm to rt and stirred for 16 h. The
reaction mixture was partitioned between water and
CH2Cl2. Organic phase was washed with water, dried
(Na2SO4), and concentrated. The residue was purified
on silica gel (EtOAc/hexanes 3:2) to afford 15 (1.52 g,
75%). 1H NMR (400 MHz, CDCl3):
d 1.27 (d,
J = 6.8 Hz, 6H, (CH3)2), 2.70–2.75 (m, 2H, H-20), 2.78
(s, 3H, SO2CH3), 3.22 (br s, 1H, CH), 3.29–3.33 (m,
1H, H-50), 3.63–3.67 (m, 1H, H-50), 3.80 (s, 3H,
MMTr-OCH3), 4.05 (s, 3H, OCH3), 4.22–4.27 (m, 1H,
H-40), 5.41–5.44 (m, 1H, H-30), 6.46 (d, J = 3.6 Hz,
1H, H-5), 6.52 (dd, J = 3.2, 8.8 Hz, 1H, H-10), 6.84 (m,
2H, ArH), 7.17 (d, J = 3.6 Hz, 1H, H-6), 7.22–7.32 (m,
10H, ArH), 7.41 (d, J = 7.2 Hz, 2H, ArH), 7.82 (s, 1H,
NH); 13C NMR (500 MHz, CDCl3): d 19.5, 19.6, 35.8,
38.7, 39.9, 54.1, 55.5, 60.9, 79.6, 80.7, 81.9, 87.3, 100.7,
102.2, 113.4, 122.4, 127.4, 128.0, 128.1, 128.2, 128.4,
128.5, 130.4, 135.1, 143.9, 144.0, 151.7, 153.0, 158.9,
163.5, 175.8; HRMS (ESI) m/z calcd C37H40N4O8S
(M+H)+ 701.2639. Found 701.2625.
4.17. N2-Isobutyryl-7-[30-amino-2030-dideoxy-50-O-(4-
monomethoxytrityl)-b-D-erythro-pentofuranosyl]-3,7-
dihydro-4H-pyrrolo[2,3-d]pyrmidin-4-one
To a solution of 17 (0.78 g, 1.2 mmol) in EtOH (125 mL)
was added Pd/C (0.14 g, 10% wet), and the suspension
was shaken over hydrogen gas (45 psi) for 6 h. The sus-
pension was filtered through a pad of Celite and filtrate
was concentrated to an oil. The residue was evaporated
with CH2Cl2 to afford 18 as a pale yellow foam (0.70 g,
4.15. 7-[30-Azido-50-O-(4-monomethoxytrityl)-20,30-dide-
oxy–b-D-erythro-pentofuranosyl]-2-isobutyrylamino-4-
methoxy-7H-pyrrolo[2,3-d]pyrimidine (16)
1
To a solution of 15 (1.40 g, 2.0 mmol) in anhydrous
DMF (20 mL) was added lithium azide (0.5 g,
10.4 mmol) and the reaction mixture was heated at
90 ꢁC until all starting material was consumed (2 h).
The solvent was removed under vacuum and the residue
was taken in CH2Cl2 and filtered. The filtrate was con-
centrated and the residue was chromatographed over sil-
ica gel (EtOAc/hexanes 2:5) to afford the desired
product 16 (0.93 g, 73%) as a white foam. 1H NMR
(400 MHz, CDCl3): d 1.25 (d, J = 6.8 Hz, 6H, (CH3)2),
2.44–2.50 (m, 1H, H-20), 2.72–2.80 (m, 1H, H-20), 3.20
(m, 1H, CH), 3.32 (dd, J = 4.0, 10.4 Hz, 1H, H-50),
3.39 (dd, J = 4.0, 10.4 Hz, 1H, H-50), 3.79 (s, 3H,
MMTr-OCH3), 4.0–4.04 (m, 1H, H-40), 4.07 (s, 3H,
OCH3), 4.51–4.56 (m, 1H, H-30), 6.44 (d, J = 3.6 Hz,
1H, H-5), 6.48 (t, J = 6.4 Hz, 1H, H-10), 6.80 (d,
J = 8.8 Hz, 2H, ArH), 7.09 (d, J = 3.6 Hz, 1H, H-6),
7.20–7.31 (m, 10H, ArH), 7.41 (d, J = 7.2 Hz, 2H,
ArH), 7.73 (br s, 1H, NH). 13C NMR (500 MHz,
CDCl3): 19.5, 19.5, 35.5, 37.8, 54.1, 55.4, 61.2, 63.5,
83.2, 83.7, 87.0, 100.2, 102.5, 113.4, 122.4, 127.2,
127.4, 128.0, 128.1, 128.5, 128.6, 129.4, 130.6, 135.3,
144.2, 144.3, 151.63, 152.6, 158.8, 163.5, 176.2; HRMS
(ESI) m/z calcd for C36H37N7O5 (M+H)+ 648.2928.
Found 648.2936.
94%). H NMR (400 MHz, CDCl3): d 1.22 (dd, J = 4.8,
6.8 Hz, 6H, CH3)2), 2.17–2.22 (m, 1H, H-20), 2.46–2.55
(m, 2H, H-20 and CH), 3.30–3.38 (m, 2H, H-50), 3.74–
3.82 (m, 5H, H-30, H-40 and MMTr-OCH3), 6.35 (dd,
J = 4.4, 6.8 Hz, 1H, H-10), 6.60 (d, J = 3.6 Hz, 1H, H-
5), 6.80 (d, J = 8.8 Hz, 2H, ArH), 6.86 (d, J = 3.6 Hz,
1H, H-6), 7.20–7.31 (m, 10H, ArH), 7.41 (d, J = 7.2 Hz,
2H, ArH), 8.18 (br s, 1H, NH), 11.84 (s, 1H, NH); 13C
NMR (500 MHz, CDCl3): d 19.2, 19.2, 36.7, 41.1, 53.0,
55.4, 64.2, 82.9, 86.1, 86.5, 104.1, 105.6, 113.3, 119.3,
127.3, 128.1, 128.6, 130.6, 135.4, 144.3, 146.1, 147.4,
158.0, 158.8, 178.4; HRMS (ESI) m/z calcd for
C35H37N5O5 (M+H)+ 608.2867. Found 608.2866.
4.18. 7-[20Deoxy-30-N-(9-fluorenylmethoxycarbonylami-
no)-50-O-(4-monomethoxytrityl)-b-D-erythro-pentofur-
anosyl]-2-isobutyrylamino-3,7-dihydro-4H-pyrrolo[2,3-
d]pyrimidin-4-one (19)
A solution of Fmoc-NCS (0.24 g, 0.85 mmol) in CH2Cl2
(10 mL) was added to a solution of 18 (0.41 g, 0.68 mmol)
in CH2Cl2 (50 mL), and stirred for 1.5 h. The reaction
mixture was concentrated and the residue upon precipita-
tion with ether afforded the desired product (0.47 g, 78%).
1H NMR (400 MHz, CDCl3): d 1.17 (d, J = 6.8 Hz, 3H,

M. L. Jain, T. C. Bruice / Bioorg. Med. Chem. 14 (2006) 7333–7346
7343
CH3), 1.20 (d, J = 6.8 Hz, 3H, CH3), 2.31–2.44 (m, 3H, H-
20 and CH), 3.14–3.24 (m, 1H, H-30), 3.32–3.42 (m, 2H, H-
50), 3.74 (s, 3H, OCH3), 4.04–4.08 (m, 1H, H-40), 4.23 (t,
J = 6.8 Hz, 1H, OCH2CH), 4.45–4.55 (m, 2H, OCH2),
5.70–5.76 (m, 1H, NH), 6.12 (dd, J = 4.0, 7.6 Hz, 1H,
H-10), 6.60, (d, J = 3.2 Hz, 1H, H-5), 6.76 (d,
J = 8.8 Hz, 2H, ArH), 6.84 (d, J = 3.2 Hz, 1H, H-6),
7.15–7.56 (m, 18H, ArH), 7.80 (d, J = 7.6 Hz, 2H,
ArH), 8.12 (s, 1H, NH), 8.36 (s, 1H, NH), 9.88 (br s,
1H, NH), 11.64 (s, 1H, NH); 13C NMR (500 MHz,
CDCl3): 19.1, 19.2, 36.8, 37.4, 46.6, 55.4, 57.2, 64.2,
68.69, 82.8, 84.8, 87.0, 104.0, 106.3, 113.2, 120.5, 121.3,
125.0, 127.2, 127.5, 128.0, 128.1, 128.4, 128.6, 130.6,
135.3, 141.5, 142.9, 144.2, 145.8, 146.9, 152.8, 158.0,
158.8, 178.2, 179.5; HRMS (ESI) m/z calcd for
C51H48N6O7S (M+Na)+ 911.3197, found 911.3241.
176.1; HRMS (ESI) m/z calcd for C16H21N7O4 (M+H)+
376.1727. Found 376.1727.
4.21. 7-[50-Amino-20-deoxy-b-D-erythro-pentofuranosyl]-
2-isobutyrylamino-4-methoxy-7H-pyrrolo[2,3-d]pyrimi-
dine (22)
Pd/C (10% wet, 0.30 g) was added to a solution of 21
(1.25 g, 3.20 mmol) in anhydrous EtOH (20 mL) and
hydrogenated at 45 psi for 5 h. The reaction mixture
was filtered through a pad of Celite and washed with
ethanol (25 mL). The solvent was removed from the
combined filtrates and the residue was evaporated twice
with CH2Cl2 to afford 22 as a yellow solid (1.08 g, 93%).
1H NMR (400 MHz, DMSO-d6): d 1.10 (d, J = 6.8 Hz,
6H, (CH3)2), 2.15–2.20 (m, 1H, H-20), 2.49–2.56 (m,
1H, H-20, partially merged with solvent peak), 2.64–
2.74, (m, 2H, H-50), 2.84–2.92 (m, 1H, CH), 3.68–3.74
(m, 1H, H-40), 4.02 (s, 3H, OCH3), 4.31–4.35 (m, 1H,
H-30), 5.30 (br s, 1H, OH), 6.48 (d, J = 3.6 Hz, 1H, H-
5), 6.50 (t, J = 8.4 Hz, 1H, H-10, partially merged with
H-5 peak), 7.46 (d, J = 3.6 Hz, 1H, H-6), 10.24 (s, 1H,
NH). 13C NMR (500 MHz, DMSO-d6): d 19.4, 19.4,
34.3, 39.0–40.0 (one signal merged with solvent peaks),
44.1, 53.5, 71.2, 82.3, 87.8, 99.2, 101.2, 123.1, 151.6,
152.5, 162.5, 175.1; HRMS (ESI) m/z calcd for
C16H23N5O4 (M+H)+ 350.1822. Found 350.1826.
4.19. 7-[20-Deoxy-50-O-mesyl-b-D-erythro-pentofurano-
syl)-2-isobutyrylamino-4-methoxy-7H-pyrrolo[2,3-d]py-
rimidine (20)
MsCl (0.90 g, 7.82 mmol) was added dropwise to a
cooled solution (0 ꢁC) of 5 (2.50 g, 7.14 mmol) in anhy-
drous pyridine (50 mL). The reaction mixture was
warmed to rt and let stirred for 14 h. The residue ob-
tained after removing the solvent under vacuum was
diluted with CH2Cl2 (150 mL), washed with water
(40 mL), and dried (Na2SO4). The solvent was removed
under vacuum and the residue was chromatographed
over silica gel (EtOAc/hexanes 4:1—MeOH/EtOAc
1:20) to afford 20 as a white foam (2.22 g, 73% yield).
1H NMR (400 MHz, CDCl3): d 1.28 (d, J = 7.2 Hz,
6H, (CH3)2), 2.46–2.52 (m, 2H, H-20), 2.82–2.89 (m,
1H, CH), 2,95 (s, 3H, OSO2CH3), 3.54 (br s, 1H, OH),
4.05 (s, 3H, OCH3), 4.28–4.31 (m, 1H, H-30), 4.50–
4.54 (m, 2H, H-50), 4.80–4.86 (m, 1H, H-40), 6.49 (d,
J = 3.6 Hz, 1H, H-5), 6.72 (t, J = 6.8 Hz, 1H, H-10),
7.10 (d, J = 3.6 Hz, 1H, H-6), 7.89 (s, 1H, NH); 13C
NMR (CDCl3,): d 19.7,19.7, 36.6, 37.5, 39.7, 54.1,
70.2, 72.1, 84.3, 84.8, 100.3, 103.2, 123.2, 151.3, 152.7,
163.5, 176.0; HRMS (ESI) m/z calcd for C17H24N4O7S
(M+Na)+ 451.1257. Found 451.1242.
4.22. 7-[20-Deoxy-50-N-(4-monomethoxytritylamino)-b-D-
erythro-pentofuranosyl]-2-isobutyrylamino-4-methoxy-
7H-pyrrolo[2,3-d]pyrimidine (23)
MMTrCl (0.93 g, 3.01 mmol) was added dropwise to a
cooled solution (0 ꢁC) of 22 (1.01 g, 2.75 mmol) in anhy-
drous pyridine over 15 min. and reaction mixture was al-
lowed to warm to rt and stirred for 18 h. The solvent
was removed under vacuum and the residue was taken
in CH2Cl2 (150 mL), washed with water (40 mL), dried
(Na2SO4), and concentrated to an oil. The crude prod-
uct was purified by silica gel chromatography (EtOAc/
hexanes 4:1) to afford 23 as a yellow foam (1.27 g,
73%). 1H NMR (400 MHz, CDCl3):
d 1.25 (d,
J = 6.8 Hz, 6H, (CH3)2), 2.30–2.37 (m, 1H, H-20),
2.40–2.50 (m, 2H, H-50), 2.54–2.60 (m, 1H, H-20),
2.85–2.90 (m, 1H, CH), 3.77 (s, 3H, MMTr-OCH3),
4.05 (s, 3H, OCH3), 4.08–4.14 (m, 1H, H-40), 4.58–
4.62 (m, 1H, H-30), 6.43 (d, J = 3.6 Hz, 1H, H-5), 6.64
(t, J = 6.8 Hz, 1H, H-10), 6.73 (d, J = 3.6 Hz, 1H, H-
6), 6.79 (d, J = 8.8 Hz, 2H, ArH), 7.31 (m, 11H, ArH
a nd NH), 7.43 (d, J = 8.0 Hz, 2H, ArH), 7.80 (s, 1H,
NH); 13C NMR (CDCl3): d 19.5, 19.6, 35.8, 40.5, 46.3,
54.0, 55.4, 70.3, 73.5, 83.2, 86.1, 100.3, 102.5, 113.3,
122.0, 126.5, 128.1, 128.7, 130.0, 140.0, 146,1, 151.5,
152.8, 158.1, 163.5; HRMS (ESI) m/z calcd for
C36H39N5O5 (M+H)+ 622.3023, found 622.3028.
4.20. 7-[50-azido-20-deoxy-b-D-erythro-pentofuranosyl]-2-
isobutyrylamino-4-methoxy-7H-pyrrolo[2,3-d]pyrimidine
(21)
Lithium azide (1.20 g, 20.8 mmol) was added to a solution
of 20 (2.10 g, 4.90 mmol) in anhydrous DMF (20 mL) and
the reaction mixture was heated at 90 ꢁC for 2 h. The sol-
vent was removed under vacuum and the residue was tak-
en in CH2Cl2 (75 mL) and filtered. The filtrate was
concentrated and the residue was purified by silica gel
chromatography (MeOH/EA 1:9) to afford 21 as a white
foam (1.54 g, 81%). 1H NMR (400 MHz, CDCl3): d 1.28
(d, J = 6.8 Hz, 6H, (CH3)2), 2.47–2.52 (m, 1H, H-20),
2.60–2.67 (m, 1H, H-20), 2.95 (br s, 1H, CH), 3.58–3.68
(m, 2H, H-50), 4.03 (s, 3H, OCH3), 4.20–4.24 (m, 1H, H-
40), 4.50 (br s, 1H, OH) 4.63–4.66 (m, 1H, H-30), 6.50 (d,
J = 3.6 Hz, 1H, H-5), 6.83 (t, J = 6.8 Hz, 1H, H-10), 7.19
(d, J = 3.6 Hz, 1H, H-6), 8.01 (s, 1H, NH); 13C NMR
(500 MHz, CDCl3,): d 19.6, 19.6, 37.5, 40.3, 52.9, 54.0,
72.6, 83.9, 85.3, 100.2, 102.9, 122.6, 151.1, 152.7, 163.4,
4.23. 7-[20-Deoxy-50-N-(4-monomethoxytritylamino)-b-D-
erythro-pentofuranosyl]-2-isobutyrylamino-3,7-dihydro-
4H-pyrrolo[2,3- d]pyrimidin-4-one (24)
To a solution of 23 (0.70 g, 1.1 mmol) in anhydrous
DMF (10.0 mL) was added sodium thiocresolate
(0.80 g, 5.48 mmol), and reaction mixture was heated

7344
M. L. Jain, T. C. Bruice / Bioorg. Med. Chem. 14 (2006) 7333–7346
at 90 ꢁC for 1 h. The solvent was removed under vacu-
um and the residue was chromatographed over silica
gel (MeOH/EtOAc 1:20) to afford the desired product
py. The unreacted amino groups on the CPG were
capped with acetic anhydride and triethyl amine to pre-
vent side reactions. The dzaG loaded CPG was stored at
4 ꢁC.
(0.68 g, 85%). 1H NMR (CDCl3):
d
1.18 (d,
J = 6.8 Hz, 6H, (CH3)2), 2.25–2.35 (m, 2H, H-50),
2.40–2.46 (m, 1H, H-20), 2.51–2.56 (m, 1H, H-20), 2.68
(br s, 1H, CH), 3.77 (s, 3H, MMTr–OCH3), 4.10–4.14
(m, 1H, H-30), 4.40–4.46 (m, 1H, H-40), 5.58 (br s, 1H,
OH), 6.39 (t, J = 6.8 Hz, 1H, H-10), 6.49 (d,
J = 3.6 Hz, 1H, H-5), 6.60 (d, J = 3.6 Hz, 1H, H-6),
6.80 (d, J = 8.8 Hz, 2H, ArH), 7.25–7.34 (m, 11H,
ArH, and NH), 7.40 (d, J = 8.4 Hz, 2H, ArH) 8.44 (s,
1H, NH), 11.80 (br s, 1H, NH); 13C NMR (500 MHz,
CDCl3): 19.1, 19.2, 36.6, 40.3, 46.6, 55.4, 70.3, 73.6,
83.0, 86.5, 104.6, 105.5, 113.4, 118.7, 126.7, 128.1,
128.6, 130.0, 137.7, 145.8, 145.9, 146.4, 147.9, 158.1,
158.2, 178.9; HRMS (ESI) m/z calcd for C35H37N5O5
(M+H)+ 608.2867, found 608.2884.
5.2. Deblocking
An aliquot of 100 mg (5 lmol scale) of 26 was placed
in a screw-capped vial with a coarse frit and stopcock,
and treated with a deblock solution (4% DCA solu-
tion in CH2Cl2) while collecting the solution into a
25 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 appar-
ent. The beads were then washed thoroughly with
CH2Cl2 and neutralized with 1% TEA in DMF. The
filtrate was collected in the volumetric flask and as-
sayed for the released monomethoxytrityl cation to
determine the loading yield.
4.24. 7-[20-Deoxy-50-N-(4-monomethoxytritylamino)-
30O-succinyl-b-D-erythro-pentofuranosyl]-2-isobutyryl-
amino-3,7-dihydro-4H-pyrrolo[2,3- d]pyrimidin-4-one (25)
5.3. Coupling
A solution of fully protected 13 (5 equiv in 1 mL DMF)
was added to the deblocked resin followed by simulta-
neous addition of HgCl2 (10 equiv in 0.5 mL DMF)
and TEA (10 equiv in 0.5 mL DMF) to the reaction ves-
sel, upon which a cloudy yellow-white precipitate was
formed. The reaction mixture was agitated for 3 h, the
supernatant was filtered. The beads were washed with
20% thiophenol in DMF until the beads were clear,
and washed with copious amounts of DMF to afford
27. The coupling was repeated two more times to opti-
mize the coupling yields.
Succinic anhydride (0.062 g, 0.62 mmol) was added to a
mixture of 24 (0.25 g, 0.42 mmol) and triethylamine
(0.18 mL, 1.29 mmol) in anhydrous CH2Cl2 (10 mL)
and stirred for 3 h. The reaction mixture was concentrat-
ed under vacuum and the residue was diluted with
CH2Cl2 (100 mL) and washed with water. The organic
phase was dried (Na2SO4), and concentrated to a white
foam. It was re-dissolved in CH2Cl2 and the product
was precipitated out with excess hexanes to afford 25
as a white solid (0.26 g, 90%). 1H NMR (400 MHz,
CDCl3): d 1.19 (d, J = 6.8 Hz, 6H, (CH3)2), 2.24-2.81
(m, 9H, H-20, H-50, CH, CO (CH2)2), 3.76 (s, 3H,
OCH3), 4.23–4.27 (m, 1H, H-40), 5.24–5.30 (m, 1H, H-
30), 6.20 (dd, J = 4.4, 8.4 Hz, 1H, H-10), 6.51 (d,
J = 3.6 Hz, 1H, H-5), 6.58(d, J = 3.6 Hz, 1H, H-6),
6.78 (d, J = 8.8 Hz, 2H, ArH), 7.41 (m, 11H, ArH,
and NH), 7.43 (d, J = 7.6 Hz, 2H, ArH), 9.60 (s, 1H,
NH), 11.92 (br s, 1H, NH). 13C NMR (500 MHz,
CDCl3,): d 19.1, 19.2, 30.0, 30.1, 36.4, 37.6, 46.2, 55.4,
70.3, 76.4, 83.5, 83.6, 104.5, 105.4, 113.4, 113.5, 118.7,
126.7, 127.4, 128.0, 128.1, 128.6, 129.4, 129.9, 137.6,
145.8, 147.0, 147.3, 158.0, 158.2, 172.2, 176.5, 179.4;
HRMS (ESI) m/z calcd for C39H41N5O8 (M-H)ꢀ
706.2882. Found 706.2878.
5.4. Capping
The unreacted sites on CPG were capped by the addi-
tion of a solution of acetic anhydride (1 mL, 100 mM
in DMF) and TEA (1 mL, 200 mM in DMF). The reac-
tion mixture was agitated for 10 min, filtered, washed
thoroughly with DMF, CH2Cl2, and then dried under
high vacuum.
5.5. Elongation
The coupling yield of the reaction was calculated to
be 80% from the trityl release assay. The coupling
was repeated with monomer 19 to give the desired
protected DNG trimer 28 on the LCAA-CPG. The
capping and deblocking steps were omitted to allow
the MMTr group to remain on the 50-terminus of
the DNG.
5. Solid-phase synthesis of 50-MMTr-protected DNG (29)
5.1. Loading
Triethylammonium salt of 50-monomethoxytritylamino-
30-succinate 25 (0.075 g, 0.107 mmol) was loaded onto
commercially available LCAA-CPG (0.50 g) by shaking
with DMTMM (0.030 g, 0.108 mmol) min MeOH for
1 h. The suspension was then filtered; the beads were
washed with copious amounts of MeOH, CH2Cl2, and
dried. The efficiency of loading was determined to be
51.36 lmol/g by treating an aliquot of 7-deazaguanine
loaded CPG 26 with a solution of 4% DCA in CH2Cl2
and assaying the trityl cation released by UV spectrosco-
5.6. Cleavage and deprotection
The CPG was transferred to a pressure-resistant vial,
and methanolic ammonia solution (5 mL) was added.
The vial was sealed and heated at 60 ꢁC for 12 h. The
isobutyryl and Fmoc-protecting groups were concur-
rently cleaved under these conditions. After cooling,
the volatiles were removed by vacuum centrifugation,
yielding a white residue containing the crude MMTr-
protected DNG trimer 29.

M. L. Jain, T. C. Bruice / Bioorg. Med. Chem. 14 (2006) 7333–7346
7345
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6. HPLC purification of 29 and 1
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The crude trityl-on product 29 was purified on reverse-
phase HPLC using 5% ! 95% gradient of acetonitrile
in 100 mM TEAA buffer (pH 7.0), and characterized
by ESI ESI/TOF+ analysis which exhibited peaks at
m/z 1117.48 (M+H) and 559.24 (M+2H), calcd
1117.47 (M+H) and 559.24 (M+2H) for C55H60N18O9.
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7. Stoichiometry of the binding
Job plot analysis55 was performed to measure the stoi-
chiometry of association. To accommodate the differ-
ence in strand lengths of DNG 1 and DNA-C5
oligomer, the concentration of nucleotide solutions
was determined by using the extinction coefficients
(per mole of the nucleotide). The absorbance at
260 nm was measured for samples containing a constant
concentration of 4 lM oligonucleotides, varying be-
tween 0 and 100% DNA-C5 with DNG 1 making up
the remainder. All experiments were conducted in buffer
containing 100 mM [NaCl] and 10 mM NaHPO4 adjust-
ed to pH 7.1. The solutions were heated to 90 ꢁC for
5 min. and allowed to cool to rt slowly before being
stored at 4 ꢁC overnight. The inflection point at 50%
of DNA-C5 in the plot indicates the DNG 1 binds to
pentameric cytidine strand with 1:1 stoichiometry to
form a Watson–Crick base paired duplex.
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Acknowledgment
31. Ueno, Y.; Mikawa, M.; Matsuda, A. Bioconjugate Chem.
1998, 9, 33.
This research was supported by a grant (5R370KU9171)
from National Institute of Health.
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Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2006.05.074.
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