Impact of nature and length of linker incorporated in agonists on toll-like receptor 9-mediated immune responses

Article abstract of DOI:10.1021/jm100177p

Oligodeoxynucleotides containing unmethylated CpG motifs act as ligands of Toll-like receptor 9 (TLR9). We previously reported a novel class of TLR9 agonists, referred to as immune-modulatory oligonucleotides (IMOs), in which two 11-mers of the same seque

Full text of DOI:10.1021/jm100177p

3730 J. Med. Chem. 2010, 53, 3730–3738
DOI: 10.1021/jm100177p
Impact of Nature and Length of Linker Incorporated in Agonists on Toll-Like Receptor 9-Mediated
Immune Responses
Mallikarjuna Reddy Putta, Dong Yu, Lakshmi Bhagat, Daqing Wang, Fu-Gang Zhu, and Ekambar R. Kandimalla*
Idera Pharmaceuticals, Inc., 167 Sidney Street, Cambridge, Massachusetts 02139
Received February 10, 2010
Oligodeoxynucleotides containing unmethylated CpG motifs act as ligands of Toll-like receptor 9
(TLR9). We previously reported a novel class of TLR9 agonists, referred to as immune-modulatory
oligonucleotides (IMOs), in which two 11-mers of the same sequence are attached via their 30-ends
through a 1,2,3-propanetriol linker and contain a synthetic immune-stimulatory motif, Cp7-deaza-dG.
In the present study, we have examined the impact of length, nature, and stereochemistry of the linker
incorporated in agonists for TLR9 activation. The new linkers studied include (S)-(-)-1,2,4-butanetriol,
1,3,5-pentanetriol, cis,cis-1,3,5-cyclohexanetriol, cis,trans-1,3,5-cyclohexanetriol, 1,3,5-tris(2-hydroxy-
ethyl)isocyanurate, tetraethyleneglycol, and hexaethyleneglycol in place of 1,2,3-propanetriol linker.
Agonists with various linkers are studied for TLR9-mediated immune responses in HEK293 cells,
human cell-based assays, and in vivo in mice. Results of these studies suggest that C3-C5 linkers, 1,2,3-
propanetriol, (S)-(-)-1,2,4-butanetriol, or 1,3,5-pentanetriol, are optimal for stimulation of TLR9-
mediated immune responses. Rigid C3 linkers with different stereochemistry have little effect on
immune stimulation, while linkers longer than C5 reduced TLR9-mediated immune stimulation.
Introduction
of ligands results in the loss of immune-stimulatory acti-
vity.9-11 Furthermore, the presence of more than one accessible
50-end enhances the immune-stimulatory activity of TLR9
agonists.10 By combining novel structures and synthetic
immune-stimulatory motifs, we have created a portfolio of
agonists of TLR9 containing two 50-ends, referred to as
immune-modulatory oligonucleotides (IMOs).5,6,11-13 Cur-
rently, IMOs are being evaluated in clinical studies for the
treatment of cancers, infectious diseases, asthma, and allergies.5
A C3 linker, such as 1,2,3-propanetriol (commonly referred
to as glycerol throughout the paper), which approximates the
natural internucleotide distance, is commonly used in TLR9
agonits.6,11-13 We do not yet understand the effect of the
linker’s length, nature, and spatial orientation in the agonist
on TLR9-mediated immune responses. In the present study,
we have incorporated novel acyclic and cyclic linkers, (S)-(-)-
1,2,4-butanetriol, 1,3,5-pentanetriol, cis,cis-1,3,5-cyclohexane-
triol, cis,trans-1,3,5-cyclohexanetriol, 1,3,5-tris(2-hydroxyethyl)-
isocyanurate, tetraethyleneglycol, and hexaethyleneglycol
(Figure 1) into an agonist (Table 1). These novel TLR9
agonists were studied in cultures of TLR9-transfected HEK-
293 cells, human cell-based assays, and in vivo in mice to
demonstrate the impact of the linkers on TLR9-mediated
immune-stimulatory activity.
Bacterial and synthetic DNA containing unmethylated
CpGa motifs are ligands for Toll-like receptor (TLR) 9, a
receptor that belongs to a family of innate immune receptors
called TLRs.1 TLR9 is expressed intracellularly in the endo-
somal compartments of human B cells and plasmacytoid
dendritic cells (pDCs).2 TLR9 activation by its ligands results
in predominantly Th1-type immune responses.3,4 Modulation
of TLR9-mediated immune responses may prove useful in
treating various diseases, including cancers, allergies, asthma,
and infectious diseases and as an adjuvant with vaccines.
Using data from extensive structure-activity relationship
studies of agonists of TLR9, we have delineated structural
features in the pentose sugar, phosphate backbone, and
nucleobases of DNA and identified a number of synthetic
immune-stimulatory motifs that induce TLR9-mediated im-
mune responses.5,6 The sequences flanking the immune-
stimulatory motif7 and the presence of secondary structures
also play a role in TLR9-mediated immune stimulation.8 Our
studies have shown that an accessible 50-end is required for the
activity of TLR9 agonists and blocking the 50-end accessibility
*To whom correspondence should be addressed. Phone: 617-679-
5536. Fax: 617-679-5532. E-mail: ekandimalla@iderapharma.com.
aAbbreviations: CGE, capillary gel electrophoresis; CpG, deoxy-
cytidine-phosphate-deoxyguanosine; DCM, dichloromethane; DIC, N,
N0-diisopropylcarbodiimide; DMAP, 4-(dimethylamino)pyridine; DM-
TCl, 4,40-dimethoxytrityl chloride; ELISA, enzyme-linked immunosor-
bent assay; EtOAc, ethyl acetate; FBS, fetal bovine serum; IFN, inter-
feron; IMO, immune-modulatory oligonucleotide; LCAA-CPG, long
chain alkyl amine controlled pore glass; NF-κB, nuclear factor-κB;
PBMCs, peripheral blood mononuclear cells; TBAF, tetrabutylammo-
nium fluoride; SEAP, secreted alkaline phosphatase; TBDMS, t-butyl-
dimethylsilane; TEA, triethylamine; THF, tetrahydrofuran; TLR, Toll-
like receptor.
Results and Discussion
Design of TLR9 Agonists. TLR9 agonists containing two
11-mer identical sequences were attached via their 30-ends
through a non-nucleosidic linker such as 1,2,3-propanetriol
(glycerol; L1).6,11-13 To evaluate the effect of length, nature,
and spatial orientation of the linker on TLR9-mediated
immune-stimulatory activity, we designed and synthesized
r
pubs.acs.org/jmc
Published on Web 04/02/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3731
Figure 1. Structures of linkers used in the study.
Table 1. Sequences, MALD-TOF Mass Spectral, and HPLC Purity Data of TLR9 Agonists Containing Different Linkers
mol wtb
purity (% FLP)c
(IE-HPLC)
agonist
11
12
sequencea
calcd
obsd
50-TCTGTCRTTCT-L1-TCTTRCTGTCT-50
50-TCTGTCRTTCT-L2-TCTTRCTGTCT-50
50-TCTGTCRTTCT-L3-TCTTRCTGTCT-50
50-TCTGTCRTTCT-L4-TCTTRCTGTCT-50
50-TCTGTCRTTCT-L5-TCTTRCTGTCT-50
50-TCTGTCRTTCT-L6-TCTTRCTGTCT-50
50-TCTGTCRTTCT-L7-TCTTRCTGTCT-50
50-TCTGTCRTTCT-L8-TCTTRCTGTCT-50
50-ACACACCAACT-L1-TCAACCACACA-50
7146
7160
7174
7186
7186
7315
7248
7336
7078
7141
7167
7178
7187
7187
7315
7258
7341
7065
96
98
98
96
98
98
95
95
96
13
14
15
16
17
18
Control 10
a R = 7-deaza-dG; L1 = 1,2,3-propanetriol (glycerol); L2 = (S)-(-)-1,2,4-butanetriol; L3 = 1,3,5-pentanetriol; L4 = cis,cis-1,3,5-cyclohexanetriol;
L5 = cis,trans-1,3,5-cyclohexanetriol; L6 = 1,3,5-tris(2-hydroxyethyl)isocyanurate; L7 = tetraethyleneglycol; L8 = hexaethyleneglycol. b Molecular
weight of compounds as calculated (calcd) and determined (obsd) by MALDI-TOF mass spectral analysis. c FLP = full length product; IE-HPLC =
anion-exchange HPLC; similar purities were also observed by capillary gel electrophoresis (CGE).
novel acyclic and cyclic linkers. As shown in Figure 1,
flexible, rigid, and hydrophilic linkers (S)-(-)-1,2,4-butane-
triol (L2), 1,3,5-pentanetriol (L3), cis,cis-1,3,5-cyclohexane-
triol (L4), cis,trans-1,3,5-cyclohexanetriol (L5), 1,3,5-tris-
(2-hydroxyethyl)isocyanurate (L6), tetraethyleneglycol (L7),
and hexaethyleneglycol (L8) were investigated in the present
study. The C3 linker (L1) approximates the internucleotide
distance found in the deoxyribose/ribose unit of a nucleotide.
The acyclic C4 (L2) and C5 (L3) linkers are one and two carbon
atoms longer than the C3 linker, respectively. The cyclic linkers
L4 and L5 have spacing similar to that of the C3 linker (L1) but
provide more rigid structures with different orientations of the
two attaching groups. Longer linkers with a more rigid central
core (L6) and hydrophilic nature (L7 and L8) were also
incorporated into agonists to evaluate the distance required
between two 50-ends for optimal stimulation of TLR9-
mediated immune responses.
Synthesis of Linkers and Solid Support Derivatization.
Solid-support functionalized linkers were required in order
to incorporate the new linkers into agonists. The long chain
alkyl amine-controlled pore glass (LCAA-CPG) derivatized
linkers were synthesized starting from corresponding triols
1a-c,e as shown in Scheme 1. (S)-(-)-1,2,4-Butanetriol (1a),
cis,cis-1,3,5-cyclohexanetriol (1c), and 1,3,5-tris(2-hydro-
xyethyl)isocyanurate (1e) were obtained from a commercial
source, and 1,3,5-pentanetriol (1b) was prepared by LiAlH4
reduction of diethyl 3-hydroxy glutarate.14 Selective protec-
tion of two hydroxyls of triol 1a-c,e with 4,40-dimethoxy-
trityl chloride (DMTCl) in the presence of 4-(dimethyl-
amino)pyridine (DMAP) afforded bis-DMT alcohols
2a-c,e in good yields. Bis-DMT cis,trans-1,3,5-cyclohexane-
triol (2d) was prepared from cis,cis-1,3,5-cyclohexanetriol
(1c) as shown in Scheme 1. To make trans-configuration of
the two strands of the agonist, it was critical to achieve trans-
configuration specifically at the DMT-protected hydroxyls
of cyclohexanetriol. The configuration at one of the hydro-
xyls of 1c was inverted by the Mitsunobu reaction, by
orthogonally protecting the remaining hydroxyls, to achieve
the cis,trans configuration (Scheme 1). The cis,cis-triol 1c
was selectively converted into mono t-butyldimethylsilane
(TBDMS)-ether 5 in high yield (∼94%) in the presence of a
mixture of NEt3 and NaH as the bases, which tremendously
reduces the formation of bis and tris-TBDMS ethers.15,16
The second hydroxyl of 5 was protected with DMTCl, to
produce 6, and the configuration at the third hydroxyl was
inverted by the Mitsunobu reaction using benzoic acid to
obtain 7 with the desired cis,trans-configuration. Debenzoyla-
tion of 7 with K2CO3 in methanol produced 8, which
was subsequently reacted with DMTCl in the presence of
DMAP to produce fully protected cis,trans-triol 9. TBDMS

3732 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Putta et al.
Scheme 1a
a Reagents and conditions: (a) DMTCl, DMAP, pyridine, 0 °C-rt; (b) TBDMSCl, Et3N, NaH, THF, rt; (c) PPh3, benzoic acid, DEAD; (d) K2CO3,
MeOH, rt; (e) TBAF, THF; (f) succinic anhydride, DMAP, pyridine; (g) LCAA-CPG, DMAP, DIC, pyridine, acetonitrile.
was removed using tetrabutylammonium fluoride (TBAF)
in tetrahydrofuran (THF) to obtain bis-DMT protected
cis,trans-1,3,5-cyclohexanetriol 2d. To facilitate the loading
of the linker on to the solid support, the third hydroxyl of
each linker was converted into its succinate 3a-e in high
yields. The succinates 3a-e were then attached to an amino-
functionalized solid support in the presence of N,N0-diiso-
propylcarbodiimide (DIC)/DMAP in pyridine/acetonitrile
mixture, producing linker-derivatized solid supports 4a-e.
We found a 50 μmol/g loading to be ideal for enhanced
nucleotide couplings and improved final product yields.
Hydrophilic tetraethyleneglycol and hexaethyleneglycol
linkers were procured from a commercial source as phos-
phoramidites.
Figure 2. Activation of HEK293 cells expressing mouse TLR9 with
agonists 11-18 and control compound 10 at 1 and 10 μg/mL. Data
shown are of one representative experiment of three independent
experiments.
Synthesis of TLR9 Agonists. All TLR9 agonists (10-18)
used in this study were oligodeoxynucleotides with phos-
phorothioate internucleotide linkages. To evaluate the effect
of the linker on TLR9-mediated immune-stimulatory acti-
vity, glycerol linker agonist 11 was substituted with L2-L8
(Figure 1 and Table 1). Agonists 12-16 with L2-L6 linkers
were synthesized on an automated DNA synthesizer10-12
using solid supports derivatized with bis-DMT linkers 4a-e.
Although the agonists 17 and 18 with L7 and L8 linkers
contained identical sequences, they were synthesized in a
linear synthetic strategy11 utilizing commercially available
linker phosphoramidites. The sequence preceding the linker
was synthesized using a 50-dT solid-support and 50-phos-
phoramidites; regular 30-phosphoramidites were used after
the linker coupling to obtain the agonist with two 50-ends.
Compound 10 was used as a negative control that had the
same length and structure as other agonists but lacked an
immune-stimulatory dinucleotide motif. All compounds
were synthesized as described in Experimental Section,
characterized by MALDI/TOF mass spectral analysis for
their sequence integrity and by capillary gel electrophoresis
and IE-HPLC for purity (Table 1).
were performed in HEK293 cells stably expressing mouse
TLR9 to evaluate the impact of linkers on TLR9 agonist
activity. As shown in Figure 2, agonists with different linkers
induced a dose-dependent activation of NF-κB greater than
that of control compound 10. Although there was no signi-
ficant difference in NF-κB activation at a 10 μg/mL concen-
tration of agonists, the difference was apparent at 1 μg/mL
(Figure 2). Agonists 11-15 with C3-C5 linkers produced
similar levels of NF-κB activation at 1 μg/mL. The ability of
agonists 16-18 to activate NF-κB with the increased length
of the linker was decreased compared with 11 (Figure 2). As
shown in Figure 2, cyclic C3 linkers (agonists 14 and 15)
produced similar levels of NF-κB activation at 10 μg/mL, but
the cis,cis-linker (agonist 14) produced slightly higher acti-
vation than did the cis,trans-linker (agonist 15) at 1 μg/mL,
suggesting that the nature of the linker plays a role in TLR9-
mediated immune responses. The variation in NF-κB activa-
tion by agonists with different length linkers suggests that the
linker’s length or the distance between the two 50-ends plays a
role in TLR9-mediated immune stimulation.
Induction of Cytokine Secretion in Mouse Spleen Cell
Cultures by TLR9 Agonists Containing Different Linkers.
To further understand the impact of linkers on TLR9-
mediated immune-stimulatory activity, we evaluated the
Activation of TLR9 in HEK293 Cells by Agonists Contain-
ing Different Linkers. Agonists containing a CpR dinucleo-
tide activate the immune system through TLR9.6 Studies

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3733
Figure 3. Dose-dependent (A) IL-12 and (B) IL-6 induction by agonists 11-18 and control compound 10 in C57BL/6 mouse spleen cell
cultures. Spleen cells were cultured in the presence of agonists at 0.1, 0.3, 1, 3, and 10 μg/mL concentrations for 24 h as described in the
Experimental Section, and the levels of secreted IL-12 and IL-6 in culture supernatants were measured by ELISA. Each value is an average of
three replicate wells, and the results are of one representative experiment of at least two independent experiments.
and chemokine production in human PBMCs to further elu-
cidate the impact of linkers on the ability of agonists to induce
TLR9-mediated immune stimulation. Multiple cytokine and
chemokine profiles were analyzed using the Luminex multiplex
assay. All agonists, containing different linkers, induced pro-
duction of a number of cytokines/chemokines at higher
levels than did control compound 10, suggesting that these
linkers are tolerated by human TLR9. However, variation in
cytokine/chemokine induction profiles (Figure 4 and Support-
ing Information) with different linkers suggests that the linker
plays a role in TLR9-mediated immune stimulation. At
2 μg/mL, agonists containing both cyclic and acyclic C3-C5
linkers (agonists 11-15) induced similar levels of IL-6, but
agonists 17 and 18 containing tetra or hexaethyleneglycol
linkers induced significantly lower IL-6 levels. Conversely,
agonists containing other linkers induced comparable levels
of IL-12 at 2 μg/mL. Agonist 16 induced low levels of IL-12
and IL-6 (data not shown). These results provide evidence that
the length of linkers plays a role in TLR9-mediated cytokine/
chemokine induction and that this induction by agonists can
be modulated by incorporating the appropriate linker.
Figure 4. Cytokine induction profiles of agonists 11-15, 17, 18,
and control compound 10 containing different linkers in human
PBMC cultures. PBMCs were stimulated with 2 and 10 μg/mL
agonists for 24 h, and the supernatants were collected and cytokine
levels were measured by Luminex multiplex assay. The data shown
are for one donor and are representative of three independent
experiments.
Human B Cell Activation by TLR9 Agonists Containing
Different Linkers. We examined human B cell proliferation
induced by TLR9 agonists containing different linkers to
evaluate the effect of the linker on TLR9-mediated B cell
activation. All agonists induced dose-dependent B-cell pro-
liferation compared with control compound 10 (Figure 5).
Similar to previous observations, agonists 17 and 18 contain-
ing tetra and hexaethyleneglycol linkers, respectively, in-
duced slightly lower levels of B cell proliferation than did
agonists containing other linkers (Figure 5). The B cell
proliferative ability of agonist 16 was similar to that of 17
and 18 (data not shown). Agonists containing either acyclic
or cyclic C3-C5 linkers induced almost identical levels of B
cell proliferation. These results suggest that TLR9 recognizes
synthetic agonists containing new linkers to different extents
and that the different agonists induce distinct B cell re-
sponses; specifically, C5 and cis,trans- linkers are more
favorable for TLR9-mediated B cell proliferation.
agonist activity in C57BL/6 mouse spleen cell cultures. In
general, all agonists with different linkers induced dose-
dependent IL-12 and IL-6 production (Figure 3). Similar
to the results observed in HEK293 cell assays, the linkers
affected cytokine induction by agonists in mouse spleen cell
cultures. Incorporation of longer linkers, such as tetra or
hexaethyleneglycol (17 and 18), significantly reduced IL-6
and IL-12 production compared with agonists containing
other linkers (11-16). Agonists 14 and 15, containing cis,cis-
and cis,trans-linkers, respectively, induced both IL-12 and
IL-6 to a similar extent. However, these agonists induced
slightly lower levels of IL-12 and slightly higher levels of IL-6
compared with agonists containing linear C3-C5 linkers
(agonists 11-13). These results suggest that the linker plays a
role in TLR9-mediated cytokine induction and the ability of
the agonist to induce cytokines can be modulated by incor-
porating linkers with appropriate length and nature.
Impact of Linkers on In Vivo Activity of TLR9 Agonists.
Agonists with CpR dinucleotides induce production of
serum cytokines in vivo. Therefore, immune-stimulatory pro-
files of TLR9 agonists containing new linkers were studied in
C57BL/6 mice at a dose of 1 mg/kg. Agonists were administered
subcutaneously (sc), and serum IL-12 levels were measured
Activation of Human PBMCs by TLR9 Agonists Contain-
ing Different Linkers. We measured agonist-induced cytokine

3734 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Putta et al.
showed that an accessible 50-end of oligonucleotide is required
for TLR9-mediated immune responses.9,10,12,13 Moreover,
TLR9 agonists consisting of two 50-ends bind to TLR9
strongly17 and induce rapid and greater TLR9-mediated
immune responses compared with agonists containing single
50-end.9,10,12,13 Recent studies have shown that TLR9 exists in
dimer form and the binding of agonists to the receptor brings
conformational changes in the endodomain of the receptor
leading to the recruitment of adoptor molecules and the
subsequent activation of immune signaling pathways.18 Non-
CpG oligonucleotides also bind to TLR9 but are not capable
of bringing conformational changes in the endodomain, failing
to activate immune signaling pathways.18 The results of the
present studies suggest that TLR9 agonists containing two
50-ends effectively bind to the two monomeric units of TLR9
and stabilize dimerization of the receptor leading to potent
induction of TLR9-mediated immune responses. This notion
is further supported by the observation that various levels of
immune responses are induced by agonists containing different
lengths and natures of linkers.
Figure 5. B-cell proliferation induced by agonists 11-15, 17, 18,
and control compound 10 containing different linkers. Human
B-cells isolated from PBMCs obtained from healthy human volun-
teers were stimulated with agonists at various concentrations and
[3H]-thymidine uptake was determined by scintillation counting and
the data are shown as counts per minute (cpm). The data shown are
mean ( SD of four donors.
Conclusions
We have synthesized solid-support functionalized novel
non-nucleoside linkers and successfully incorporated the
new linkers into TLR9 agonists. The new compounds have
been systematically studied both in vitro and in vivo to
understand the impact of length and nature of the linker
present in agonists on TLR9-mediated immune stimulation of
agonists. Compounds with C3-C5 linkers and rigid linkers
with different stereochemistry have minimal impact on TLR9
agonist-induced immune stimulation. Incorporation of longer
and more flexible linkers, such as cyanurate, tetra, and hex-
aethyleneglycol linkers, significantly reduced the immune-
stimulatory activity of agonists. These results demonstrate
that the length and nature of the linker in TLR9 agonists play
a role in the recognition of TLR9 and subsequent TLR9-
mediated signaling, resulting in varying immune response
profiles. The ability to modulate TLR9-mediated immune
responses in a desired fashion using different linkers allows
researchers to design novel TLR9 agonists for treating specific
disease indications as required.
Figure 6. IL-12 secretion in C57BL/6 mice induced by agonists
11-18 and control compound 10 following sc administration at a
dose of 1 mg/kg. Blood was collected 2 h after agonist administra-
tion and IL-12 in the serum was determined by ELISA as descri-
bed in Experimental Section. Each value is an average of three
mice ( SD.
by ELISA in blood samples collected 2 h after administration
of the agonist. As shown in Figure 6, all agonists studied,
except 18, induced higher levels of IL-12 than did the control
compound 10. IL-12 levels induced by these agonists varied
widely, suggesting that linkers play a role in TLR9-mediated
immune responses. The agonists that exhibited higher activ-
ity in mouse spleen cell culture assays also induced higher
levels of IL-12 in vivo (Figure 6). Agonist 13 containing a C5
linker induced slightly lower levels of IL-12 than did agonist
11 containing a C3 linker. Similarly, agonist 14 containing a
cis,cis-linker induced slightly higher levels of IL-12 than did
agonist 15 containing a cis,trans-linker (Figure 6). Agonist
containing cyanurate linker (agonist 16) induced lower levels
of IL-12 than C5- linker containing agonist 12. Consistent
with in vitro studies, incorporation of tetra (agonist 17) or
hexaethyleneglycol (agonist 18) linkers greatly reduced the in
vivo activity of the agonist, suggesting that linkers longer
than five carbon atoms are not optimal for TLR9-mediated
immune responses. These in vivo results demonstrate that
the length and nature of the linker play a role in TLR9
recognition and subsequent immune stimulation.
Experimental Section
Chemistry. General Methods. Unless otherwise noted, all
commercially available starting materials, reagents, and sol-
vents of anhydrous quality were obtained from Sigma-Aldrich
(St. Louis, MO) and used without further purification. Thin
layer chromatography (TLC) was performed on silica gel 60
F254 coated on aluminum sheets and visualized by UV light or by
a 5% phosphomolybdic acid (PMA) solution. Flash column
chromatography was performed on silica gel 60 (mesh size
0.040-0.063 mm and 230-400 mesh ASTM) which was ob-
tained from EMD Chemicals (Gibbstown, NJ), and the required
solvents were purchased from J. T. Baker (Phillipsburg, NJ).
NMR spectra were performed on Varian 400 MHz Unity Inova
instrument. Chemical shifts (δ) are in ppm relative to TMS and
the coupling constants (J) are in Hz. HRMS (ESþ/TOF) was
performed on the Micromass LCT-TOF mass spectrometer.
Long chain alkyl amine controlled pore glass (LCAA-CPG;
˚
120-200 mesh, 500 A, 90-120 μmol/g NH2 groups) was
obtained from Millipore (Lincoln Park, NJ).
General Procedure: Synthesis of Bis-DMT Protected Linkers
2a-c,e. Triol 1a-c and e (20 mmol) and DMAP (250 mg,
2 mmol) were dissolved in dry pyridine (100 mL), cooled to
0 °C, and maintained under argon atmosphere. DMTCl (14.91 g,
The results presented here suggest that dCp7-deaza-dG
dinucleotide motif is recognized by TLR9 leading to potent
TLR9-mediated immune responses. Our previous studies

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3735
44 mmol) in pyridine (150 mL) was added dropwise to the above
stirring solution. After the addition, the reaction mixture was
allowed to reach room temperature (∼4 h) and continued stirring
for overnight. TLC [hexanes/ethyl acetate (EtOAc) 3:1 contain-
ing 0.5% triethylamine(TEA)] indicated completion of the reac-
tion. Pyridine was evaporated to dryness, residue was dissolved in
EtOAc (500 mL) and washed successively with saturated NH4Cl
solution (500 mL) and brine (500 mL) solution. EtOAc layer was
dried over anhydrous MgSO4 and rotoevaporated to dryness.
The residue was purified on silica gel flash column chromato-
graphy using hexane/EtOAc (3:1) containing 0.5% TEA to give
bis-DMT alcohol 2a-c,e as a white foam.
added in portions to a stirring solution of bis-DMT alcohol 2
(5 mmol) and DMAP (0.61 g, 5 mmol) in dry pyridine (50 mL) at
room temperature. The reaction mixture was stirred for over-
night and rotoevaporated to dryness. The residue was dissolved
in DCM (250 mL) and successively washed with ice cold 10%
citric acid solution (2 ꢀ 250 mL) and water (250 mL). The DCM
layer was dried over anhydrous MgSO4, concentrated to 50 mL
volume using rotoevaporator, and purified on silica gel flash
column chromatography using 0 f 2% methanol in DCM
containing 0.5% TEA. Pure product was white foam obtained
as triethylammonium salt of succinate.
Compound 3a (3.28 g, 81%). 1H NMR (CDCl3, 400 MHz):
Compound 2a (13.08 g, 92%). 1H NMR (CDCl3, 400 MHz):
δ 1.65-1.89 (m, 2H, -CH(OH)CH2CH2-), 2.75 (bs, 1H,OH),
3.05-3.20 (m, 1H, -CH(OH)CH2CH2-), 3.24-3.42 (m, 1H,
-CH(OH)CH2CH2-), 3.47-3.71 (m, 1H, -CH(OH)CH2-
CH2-), 3.82 (d, 12H, -OCH3), 3.87-4.17 (m, 2H, -CH2CH-
(OH)CH2CH2-), 6.80-6.87 (m, 8H, Ar-H), 7.16-7.45 (m,
18H, Ar-H). 13C NMR (CDCl3, 100 MHz): δ 33.30, 35.00,
55.59, 61.37, 62.03, 67.00, 67.72, 70.12, 71.76, 72.12, 81.76, 86.29,
86.66, 127.00, 128.10, 128.18, 129.46, 130.00, 130.29, 131.35,
136.44, 138.08, 145.00, 145.29, 145.34, 147.66, 158.71, 158.86,
158.96, 159.32. HRMS (ESI/TOFþ) calcd for C46H46O7 (M þ
Na)þ 733.3141, found 733.3133.
δ 1.32 (t, 9H, J = 7.6,-N(CH2CH3)3), 1.92 (m, 2H,
-CHCH2CH2-), 2.94-3.22 (m, 10H, -CH2CHCH2CH2-
and -N(CH2CH3)3), 3.75 (d, 12H, -OCH3), 5.21-5.31 (m,
1H, -CH2CHCH2CH2-), 6.76 (m, 8H, Ar-H), 7.16-7.40 (m,
18H, Ar-H). 13C NMR (CDCl3, 100 MHz): δ 8.89, 30.71, 31.82,
45.50, 55.49, 59.71, 64.81, 71.20, 86.19, 113.36, 126.91, 128.00,
128.06, 128.45, 130.34, 136.36, 136.68, 145.32, 145.41, 158.60,
158.65, 172.86, 177.03. HRMS (ESI/TOFþ) calcd for
C50H50O10 (MþNa)þ 833.3302, found 833.3312.
Compound 3b (3.42 g, 83%). 1H NMR (CDCl3, 400 MHz):
δ 1.20 (t, J = 7.6, 9H, -N(CH2CH3)3), 1.80-1.85 (m, 4H,
-CH2CHCH2-), 2.40 (s, 4H, -COCH2CH2CO-), 2.90 (q,
6H, -N(CH2CH3)3), 3.03-3.11 (m, 4H, 1 and 5-CH2-), 3.78
(s, 12H, -OCH3), 5.20-5.26 (m, 1H, -CH-), 6.81 (d, J=8.8,
8H, Ar-H), 7.17-7.42 (m, 18H, Ar-H). 13C NMR (CDCl3, 100
MHz): δ 9.09, 30.94, 31.77, 34.45, 45.08, 52.92, 55.32, 59.73,
69.73, 86.00, 113.12, 126.71, 127.85, 128.32, 130.11, 136.54,
145.22, 158.40, 173.07, and 177.71. HRMS (ESI/TOFþ) calcd
for C51H52O10 (M þ Na)þ 847.3458, found 847.3480.
Compound 2b (10.44 g, 72%). 1H NMR (CDCl3, 400 MHz):
δ 1.65-1.81 (m, 4H, -CH2CHCH2-), 3.16-3.31 (m, 4H,
DMTO-CH2-), 3.78 (s, 12H, -OCH3), 3.95-4.01 (m, 1H,
-CH-), 6.81 (d, J = 8.8, 8H, Ar-H), 7.17-7.42 (m, 18H,
Ar-H). 13C NMR (CDCl3, 100 MHz): δ 37.22, 55.38, 61.81,
69.58, 86.54, 113.27, 126.88, 128.01, 128.26, 130.15, 136.44,
145.16, 158.55.
Compound 3c19 (3.26 g, 78%). 1H NMR (CDCl3, 400 MHz):
δ 1.17 (t, J = 7.6, 9H, -N(CH2CH3)3), 1.17-1.25 (m, 2H,
4-CH2-), 1.36-1.51 (m, 4H, 2 and 6-CH2-), 2.37-2.47 (m,
4H, -COCH2CH2CO-), 2.86 (q, 6H, -N(CH2CH3)3),
2.99-3.10 (m, 2H, 3 and 5-CH-), 3.78 (d, 12H, -OCH3),
3.98-4.08 (m, 1H, 1-CH-), 6.76 (dd, J = 8.8, 8H, Ar-H),
7.15-7.37 (m, 18H, Ar-H). 13C NMR (CDCl3, 100 MHz):
δ 9.38, 31.16, 31.96, 39.02, 41.76, 45.18, 55.37, 67.68, 86.24,
113.12, 126.79, 127.77, 128.42, 130.33, 130.38, 137.15, 137.26,
146.28, 158.54, 172.58, and 178.16. HRMS (ESI/TOFþ) calcd
for C52H52O10 (M þ Na)þ 859.3458, found 859.3434.
Compound 2c19 (8.54 g, 58%). 1H NMR (CDCl3, 400 MHz):
δ 1.04-1.13 (m, 3H, 2,4 and 6-CH2-), 1.24-1.28 (m, 3H, 2,4
and 6-CH2-), 1.66 (d, 1H, 1-OH), 2.84-2.93 (m, 1H, -CH-
OH), 3.10-3.18 (m, 2H, 3 and 5-CH-), 3.78 (d, 12H, -OCH3),
6.78 (d, J = 8.8, 8H, Ar-H), 7.16-7.42 (m, 18H, Ar-H). 13C
NMR (CDCl3, 100 MHz): δ 41.42, 42.67, 55.37, 66.16, 67.91,
86.28, 113.12, 126.83, 127.81, 128.53, 130.41, 137.39, 146.29,
158.53. HRMS (ESI/TOFþ) calcd for C48H48O7 (M þ Na)þ
759.3298, found 759.3335.
Compound 2e20 (9.0 g, 52%). 1H NMR (CDCl3, 400 MHz):
δ 2.73 (bs, 1H, -OH), 3.36-3.44 (m, 2H, -NCH2CH2OH),
3.76-3.89 (m, 4H, -NCH2CH2ODMT), 4.07 (m, 2H,
-NCH2CH2OH), 4.13 (m, 4H, -NCH2CH2ODMT), 3.80 (s,
12H, -OCH3), 6.74-6.86 (m, 8H, Ar-H), 7.15-7.39 (m, 18H,
Ar-H).
Compound 3d (3.31 g, 79%). 1H NMR (CDCl3, 400 MHz):
δ 0.72-0.99 (m, 3H, 3HCHax), 1.20t, J = 7.2, 9H, -N(CH2-
CH3)3), 1.24 (m, 1H, HCHeq), 1.47 (m, 1H, HCHeq), 1.76 (m,
1H, HCHeq), 2.40-2.49 (m, 4H, -COCH2CH2CO-), 2.95 (q,
6H, -N(CH2CH3)3), 3.70 (d, J=8.2, 6H, 2ꢀ -OCH3), 3.76 (s,
6H, 2ꢀ -OCH3), 3.85 (m, 1H, HCODMT), 4.08 (m, 1H,
HCOCO-), 4.79 (m, 1H, HCODMT), 6.73 (m, J = 8.3, 4H,
Ar-H), 6.79 (dd, J=8.8 and 3.3, 4H, Ar-H), 7.14-7.27 (m, 12H,
Ar-H), 7.35 (dd, 4H, J=8.8 and 3.8, Ar-H), 7.46 (d, 2H, J=7.2,
Ar-H). 13C NMR (100 MHz, CDCl3): δ 8.9, 30.14, 45.15, 55.44,
68.05, 69.12, 86.44, 86.66, 113.24, 126.73, 126.85, 127.86, 128.37,
128.61, 130.48, 130.64, 137.00, 137.39, 137.48, 146.15, 146.55,
and 158.61. HRMS (ESI/TOFþ) calcd for C52H52O10 (M þ
Na)þ 859.3458, found 859.3456.
trans-1,3-Bis-(4,4-dimethoxytrityloxy)-cis-5-hydroxy-cyclo-
hexane (2d). To a solution of 9 (4.25 g, 5 mmol) in THF (20 mL)
at 0 °C was added TBAF (1 M in THF, 20 mL, 20 mmol) over
5 min, and stirring was continued at room temperature for 6 h.
The reaction was diluted with dichloromethane (DCM; 100 mL)
and quenched with saturated NH4Cl (100 mL) solution. The
organic layer was separated, and the aqueous layer was ex-
tracted with DCM (2 ꢀ 100 mL). The combined organic phase
was dried over anhydrous MgSO4 and concentrated. Flash
column chromatography of the residue on silica gel using 5:1
hexanes/EtOAc mixture containing 0.5% TEA gave pure 2d
(3.34 g, 91%) as a white solid. 1H NMR (400 MHz, CDCl3): δ
0.90-1.04 (m, 3H, 3HCHax) 1.26 (m, 1H, HCHeq), 1.35-1.45
(m, 3H, 2HCHeq and HC-OH), 2.14 (m, 1H, HCOH), 3.73 (d,
J=4, 12H, 4ꢀ -OCH3), 4.03 (m, 1H, HCODMT), 4.12 (m, 1H,
HCODMT), 6.80 (m, 8H, ArH), 7.14-7.30 (m, 14H, ArH), 7.36
(dd, 4H, J=7 and 1.2, ArH). 13C NMR (100 MHz, CDCl3): δ
40.37, 40.45, 55.37, 59.54, 67.19, 67.34, 69.87, 113.16, 113.25,
113.38, 126.85, 126.93, 127.82, 127.94, 128.06, 128.48, 128.61,
129.34, 130.54, 136.93, 137.07, 137.39, 137.48, 146.06, 146.41,
158.62, and 158.69. HRMS (ESI/TOFþ) calcd for C48H48O7
(MþNa)þ 759.3298, found 759.3280.
Compound 3e (4.44 g, 92%). 1H NMR (CDCl3, 400 MHz):
δ 1.41 (t, J = 7.6, 9H, -N(CH2CH3)3), 2.47-2.68 (m, 4H,
-COCH2CH2CO-), 3.10 (q, 6H, -N(CH2CH3)3), 3.75-3.89
(m, 4H, -NCH2CH2ODMT), 3.80 (s, 12H, -OCH3), 4.10-
4.35 (m, 6H, NCH2CH2ODMT and NCH2CH2O-), 4.40-4.51
(m, 2H, NCH2CH2O-), 6.83 (d, (m, 8H, Ar-H), 7.14-7.31 (m,
18H, Ar-H).
General Procedure: Preparation of Bis-DMT Linker Loaded
Controlled-Pore Glass Solid Supports 4a-e. A solution of
succinate 3 (1 mmol), DMAP (0.4 g, 3.3 mmol) and DIC
(5 mL) in 1:6 mixture of pyridine/acetonitrile (105 mL) was
added to controlled-pore glass solid support (25 g), and the
slurry was shaken for 24 h at room temperature. Solution was
filtered off, and the solid support was washed with acetonitrile
General Procedure: Synthesis of Bis-DMT Protected Linker
Succinates 3a-e. Succinic anhydride (0.75 g, 7.5 mmol) was

3736 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Putta et al.
containing 5% pyridine (2 ꢀ 100 mL) followed by acetonitrile
(3 ꢀ 250 mL). Cap A (acetic anhydride in pyridine/THF, 89 mL)
and Cap B (1-methylimidazole in THF, 100 mL) solutions were
added to solid support and shaken for 4 h. Solutions filtered off
and capping reaction was repeated one more time. Finally, solid
support was washed thoroughly with acetonitrile containing 5%
pyridine, followed by acetonitrile and dried under high vacuum
for overnight to get dry solid supports 4a-d. Loading was
determined by treating a known amount of linker derivatized
solid support with 3% trichloroacetic acid in DCM and assayed
DMT content by measuring absorbance at 498 nm.
rotoevaporated, and the residue was dried under vacuum. The
residue was dissolved in DCM (250 mL), and insoluble material
was filtered off. The DCM solution was washed with brine
(250 mL) and dried over anhydrous MgSO4. Solvent was rotoeva-
porated to dryness, and the resulting residue was purified on silica
gel flash column chromatography using 9:1 hexanes/EtOAc con-
taining 0.5% TEA to afford pure product 8(3.79 g, 96%) as a white
1
solid. H NMR (400 MHz, CDCl3): δ -0.05 (d, 6H, J = 2.8,
Me2Si), 0.83 (s, 9H, TBDMS tert-butyl), 1.24-1.35 (m, 3H,
3HCHax), 1.66 (m, 1H, HCHeq), 1.83 (m, 1H, HCHeq), 3.67-
3.75 (m, 1H, HC-OTBDMS), 3.78 (s, 6H, 2ꢀ -OCH3), 3.81-3.89
(m, 1H, HC-ODMT), 4.04 (bs, 1H, HC-OH), 6.81 (d, 4H, J=8.8
Ar-H), 7.16-7.28 (m, 3H, Ar-H), 7.40 (dd, 4H, J=8.8 and 2.2, Ar-
H), 7.51 (m, 2H, Ar-H). 13C NMR (100 MHz, CDCl3): δ -4.61,
-4.45, 0.21, 11.73, 18.36, 26.08, 40.36, 41.64, 44.29, 46.42, 55.39,
66.00, 67.19, 67.26, 86.40, 113.14, 126.89, 127.85, 128.63, 129.34,
130.59, 137.58, 137.61, 146.40, and 158.66. HRMS (ESI/TOFþ)
calcd for C33H44O5Si (M þ Na)þ 571.2856, found 571.2851.
trans-1,3-bis-(4,4-Dimethoxytrityloxy)-cis-5-(tert-butyldimethyl-
silyloxy)-cyclohexane (9). DMTCl (3.42 g, 10.1 mmol) in dry
pyridine (50 mL) was added dropwise to a stirring solution of 8
(3.7 g, 6.74 mmol) and DMAP (1.23 g, 10.1 mmol) in dry pyridine
(50 mL) at room temperature. After stirring for 24 h at room
temperature, pyridine was rotoevaporated to dryness. The residue
was dissolved in DCM (500 mL) and washed successively with
NH4Cl (500 mL), brine (500 mL), and water (500 mL). The organic
layer was dried over anhydrous MgSO4, rotoevaporated, and the
residue was purified by silica gel flash column chromatography
using 0f10% gradient of EtOAc in hexanes containing 0.5% TEA
to get pure compound 9 (4.7 g, 82%) as a white solid. 1H NMR
(400 MHz, CDCl3): δ -0.18 (d, J = 11.6, 6H, Me2Si), 0.73 (s,
9H, TBDMS tert-butyl), 1.12-1.28 (m, 3H, 3HCHax) 1.71 (m,
1H, HCHeq), 1.83-2.01 (m, 1H, HCHeq), 3.56-3.65 (m, 1H,
HCODMT), 3.86 (m, 1H, HC-OTBDMS), 4.04-4.14 (m, 1H,
HCODMT), 3.71 (d, J=7.2, 6H, 2ꢀ -OCH3), 3.78 (d, J=2, 6H,
2ꢀ -OCH3), 6.74 (d, 4H, J=7.7, Ar-H), 6.81 (dd, 4H, J=9.2 and
1.8, Ar-H), 7.12-7.30 (m, 12H, Ar-H), 7.43 (dd, 4H, J=6.5 and
2.2, Ar-H), 7.53 (d, 2H, J=7.6, Ar-H). 13C NMR (100 MHz,
CDCl3): δ -4.42, 0.22, 14.42, 18.23, 26.08, 39.96, 40.18, 44.24,
55.29, 55.37, 66.61, 68.08, 68.78, 86.30, 86.40, 113.10, 113.13,
113.38, 126.74, 126.79, 127.79, 128.50, 128.70, 129.34, 130.41,
130.75, 137.20, 137.33, 137.62, 137.72, 146.21, 146.74, and
158.55. HRMS (ESI/TOFþ) calcd for C54H62O7Si (M þ Na)þ
873.4163, found 873.4138.
(1S,3R,5S)-1-Hydroxy-3-(4,4-dimethoxytrityloxy)-5-(tert-butyl-
dimethylsilanyloxy)-cyclohexane (6). DMTCl (9.83 g, 29 mmol)
in dry pyridine (100 mL) was added dropwise to a stirring
solution of cis-diol 515,16 (6.66 g, 27 mmol) and DMAP
(3.54 g, 29 mmol) in dry pyridine (100 mL) at 0 °C. After the
addition, reaction mixture allowed to slowly reach room tem-
perature (∼4 h), and stirring was continued overnight. TLC
(hexanes/EtOAc 2:1 mixture containing 0.5% TEA) indicated
the completion of the reaction and pyridine was rotoevaporated
to dryness. The residue was dissolved in DCM (500 mL) and
washed successively with saturated aqueous NH4Cl solution
(500 mL), brine (500 mL), and water (500 mL). The organic layer
was dried over anhydrous MgSO4 and rotoevaporated to dry-
ness. The residue was purified on silica gel flash column chro-
matography using hexanes/EtOAc 3:1 mixture containing 0.5%
TEA. Pure compound 6 was obtained as a pale-yellow solid
(6.37 g, 43%). 1H NMR (400 MHz, CDCl3): δ -0.90 (d, J=8.8,
6H, Me2Si), 0.80 (s, 9H, TBDMS tert-butyl), 1.15-1.36 (m, 5H,
3HCHax, HCHeq, HCOH), 1.83 (m, 1H, HCHeq), 2.01 (m, 1H,
HCHeq), 3.18-3.26 (m, 1H, HCODMT), 3.29-3.45 (m, 2H,
HC-OTBDMS and HC-OH), 3.79 (s, 6H, 2ꢀ -OCH3), 6.81 (dd,
4H, J=8.8 and 2.0, Ar-H), 7.14-7.32 (m, Ar-H), 7.39 (dd, 4H,
J=8.8 and 4.4, Ar-H), 7.50 (d, 2H, J=7.2, Ar-H). 13C NMR
(100 MHz, CDCl3): δ -4.68, -4.54, 18.28, 25.99, 43.03, 43.07,
44.84, 55.37, 66.34, 66.64, 67.88, 86.44, 113.22, 126.93, 127.91,
128.53, 129.41, 130.46, 130.54, 137.30, 137.49, 146.30, and
158.68. HRMS (ESI/TOFþ) calcd for C33H44O5Si (M þ Na)þ
571.2856, found 571.2849.
(1R,3R,5R)-1-Benzoyloxy-3-(4,4-dimethoxytrityloxy)-5-(tert-
butyldimethylsilyloxy)-cyclohexane (7). To a solution of com-
pound 6 (4.4 g, 8 mmol) and triphenylphosphine (2.62 g,
10 mmol) in anhydrous THF (75 mL) was add benzoic acid
(1.22 g, 10 mmol) in one portion and maintained under nitrogen
atmosphere. The solution was cooled to -10 °C in an ice-salt
mixture with stirring, and diisopropyl azodicarboxylate (2.42 g,
12 mmol) in anhydrous THF (25 mL) was added dropwise over a
period of 30 min while maintaining the temperature well below
0 °C. The reaction mixture was stirred until it reaches gradually
to room temperature in about 5 h. Then solvent was rotoeva-
poratedunder vacuum and the residuewas purified on a silicagel
flash column chromatography using 9:1 hexanes/EtOAc con-
taining 0.5% TEA to afford pure product 7 (4.91 g, 94%) as
white solid. 1H NMR (CDCl3): δ -0.03 (d, 6H, J=1.2, Me2Si),
0.83 (s, 9H, TBDMS tert-butyl), 1.34-1.52 (m, 4H, 3HCHax and
1HCHeq), 1.94-2.05 (m, 2H, 2HCHeq), 3.72 (d, 6H, J=6.6, 2ꢀ
-OCH3), 3.67-3.82 (m, 2H, HC-ODMT and HC-OTBDMS),
5.24 (t, 1H, HC-OBz), 6.73 (t, 4H, J=8.8 Ar-H), 7.14-7.23 (m,
Ar-H), 7.36 (dd, 4H, J=8.8 and 6.0, Ar-H), 7.45 (m, 4H, Ar-H),
7.57 (m, 1H, Ar-H), 7.80 (d, 2H, J = 8.2, Ar-H). 13C NMR
(100 MHz, CDCl3): δ -4.56, -4.42, 18.35, 26.04, 37.32, 39.30,
44.11, 55.33, 66.46, 67.62, 70.69, 86.54, 113.17, 126.87, 127.84,
128.48, 128.52, 129.57, 130.40, 130.47, 130.80, 133.00, 137.31,
146.31 158.62 and 165.39. HRMS (ESI/TOFþ) calcd for
C40H48O6Si (M þ Na)þ 675.3118, found 675.3099.
Synthesis of TLR9 Agonists 10-18. All agonists were synthe-
sized on a 2-10 μmol scale, as DMT-off, using β-cyanoethyl-
phosphoramidite chemistry9,10,21,22 on a MerMade 6 synthesizer
(Bioautomation, Inc., Plano, TX). Unmodified 30-phosphora-
midites were obtained from Glen Research (Sterling, VA).
Glycerol loaded solid support, 7-deaza-dG, ethylene glycol
linker phosphoramidites, and all 50-phosphoramidites were
obtained from ChemGenes Corporation (Wilmington, MA).
Beaucage reagent was used as an oxidizing agent to obtain the
phosphorothioate backbone modification.23 A modified cou-
pling protocol recommended by the supplier was used for
7-deaza-dG and 50-phosphoramidites incorporation. After the
synthesis, oligodeoxynucleotides were cleaved from the solid
support and deprotected using standard protocols. Crude oligo-
deoxynucleotides were purified on preparative anion-exchange
HPLC (Waters Delta 600 system) equipped with Source 15Q
(GE Healthcare Biosciences-AB, Uppsala, Sweden) column
(250 mm ꢀ 20 cm). The mobile phases used were (A) 25 mM
Tris-HCl, pH 7.5 buffer containing 20% ACN and (B) 25 mM
Tris-HCl, pH 7.5 buffer containing 20% ACN and 2 M NaCl.
After purification, oligodeoxynucleotides were desalted on
C18 reversed phase-HPLC column and dialyzed against
United States Pharmacopeia-quality sterile water for irrigation
(B. Braun Medical, Inc., USA). All oligodeoxynucleotides
synthesized were lyophilized, reconstituted in distilled water,
and the concentrations were determined by measuring UV
(1R,3R,5S)-1-Hydroxy-3-(4,4-dimethoxytrityloxy)-5-(tert-butyl-
dimethylsilyloxy)-cyclohexane (8). To a solution of compound 7
(4.7 g, 7.2 mmol) in anhydrous methanol (75 mL) was added
anhydrous potassium carbonate (1.1 g, 8 mmol), and the reaction
mixture was stirred at room temperature for 24 h. Methanol was

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3737
absorbance at 260 nm. The purity of TLR9 agonists synthesized
was found to be g95% and the rest being shorter by one or
two nucleotides (n - 1 and n - 2) as determined by analytical
anion-exchange HPLC (DNA Pack100 column), capillary gel
electrophoresis (P/ACE MDQ system, Beckman Coulter, Inc.,
Brea, CA) and denaturing PAGE. The sequence integrity was
characterized by MALDI-TOF mass spectrometry (Micro MX,
Waters Co., USA). All of the compounds were tested for
endotoxin by Limulus assay (Bio-Whittaker), and the endotoxin
levels were found to be <0.5 EU/mL.
is available free of charge via the Internet at http://pubs.
acs.org.
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in 96-well plates at a concentration of 5 ꢀ 106 cells/mL. The
agonists dissolved in phosphate-buffered saline (PBS) were
added to the cell cultures at a final concentration of 2 or
10 μg/mL. The cells were then incubated at 37 °C for 24 h.
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purified from human PBMCs as described previously12 were
stimulated with different concentrations of agonists for 56 h,
then pulsed with 0.75 μCi of [3H]-thymidine and harvested 16 h
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out in accordance with Idera’s IACUC-approved animal protocols
and guidelines. Female C57BL/6 mice (n=3) were injected sc
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Acknowledgment. We thank Dr. Sudhir Agrawal for sup-
port, encouragement, and suggestions.
Supporting Information Available: Table showing cytokine
and chemokine induction in human PBMCs by agonists of
TLR9 containing different lengths of linkers. This material

3738 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Putta et al.
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