Solid-phase synthesis of thermolytic DNA oligonucleotides functionalized with a single 4-hydroxy-1-butyl or 4-phosphato-/thiophosphato-1-butyl thiophosphate protecting group

Article abstract of DOI:10.1021/jo062087y

(Chemical Equation Presented) Several thermolytic CpG-containing DNA oligonucleotides analogous to 1 have been synthesized to serve as potential immunotherapeutic oligonucleotide prodrug formulations for the treatment of infectious diseases in animal models. Specifically, the CpG motif (GACGTT) of each DNA oligonucleotide has been functionalized with either the thermolabile 4-hydroxy-1-butyl or the 4-phosphato-/thiophosphato-1-butyl thiophosphate protecting group. This functionalization was achieved through incorporation of activated deoxyribonucleoside phosphoramidite 8b into the oligonucleotide chain during solid-phase synthesis and, optionally, through subsequent phosphorylation effected by phosphoramidite 9. Complete conversion of CpG ODNs hbu1555, psb1555, and pob1555 to CpG ODN 1555 (homologous to 2) occurred under elevated temperature conditions, thereby validating the function of these diastereomeric oligonucleotides as prodrugs in vitro. Noteworthy is the significant increase in solubility of CpG ODN psb1555 and CpG pob1555 in water when compared to that of neutral CpG ODN fma1555 (homologous to 1).

Full text of DOI:10.1021/jo062087y

Solid-Phase Synthesis of Thermolytic DNA Oligonucleotides
Functionalized with a Single 4-Hydroxy-1-butyl or 4-Phosphato-/
Thiophosphato-1-butyl Thiophosphate Protecting Group†
Andrzej Grajkowski,‡ Cristina Aus´ın,‡ Jon S. Kauffman,§ John Snyder,§ Sonja Hess,¶
John R. Lloyd,¶ and Serge L. Beaucage*,‡
DiVision of Therapeutic Proteins, Center for Drug EValuation and Research, Food and Drug
Administration, 8800 RockVille Pike, Bethesda, Maryland 20892, Lancaster Laboratories, 2425 New
Holland Pike, Lancaster, PennsylVania 17605-2425, and Laboratory of Bioorganic Chemistry, National
Institute of Diabetes and DigestiVe and Kidney Diseases, The National Institutes of Health,
9000 RockVille Pike, Bethesda, Maryland 20892
Serge.Beaucage@fda.hhs.goV
ReceiVed October 9, 2006
Several thermolytic CpG-containing DNA oligonucleotides analogous to 1 have been synthesized to serve
as potential immunotherapeutic oligonucleotide prodrug formulations for the treatment of infectious diseases
in animal models. Specifically, the CpG motif (GACGTT) of each DNA oligonucleotide has been
functionalized with either the thermolabile 4-hydroxy-1-butyl or the 4-phosphato-/thiophosphato-1-butyl
thiophosphate protecting group. This functionalization was achieved through incorporation of activated
deoxyribonucleoside phosphoramidite 8b into the oligonucleotide chain during solid-phase synthesis and,
optionally, through subsequent phosphorylation effected by phosphoramidite 9. Complete conversion of
CpG ODNs hbu1555, psb1555, and pob1555 to CpG ODN 1555 (homologous to 2) occurred under
elevated temperature conditions, thereby validating the function of these diastereomeric oligonucleotides
as prodrugs in vitro. Noteworthy is the significant increase in solubility of CpG ODN psb1555 and CpG
pob1555 in water when compared to that of neutral CpG ODN fma1555 (homologous to 1).
Introduction
phodiester groups of these biopolymers with acyloxymethyl,1
2-(S-acylthio)ethyl,2 and 2-/4-(acyloxy)benzyl3 protecting groups
or with groups derived from bis(hydroxymethyl)-1,3-dicarbonyl
Oligonucleotide prodrugs have attracted considerable attention
over the past decade in an effort to overcome problems
associated with the cellular uptake of antisense oligonucleotides
and the sensitivity of these biomolecules to ubiquitous nucleases
present in biological media and physiological environments. A
strategy to improve cellular permeation of negatively charged
oligonucleotide drugs entails temporarily masking the phos-
(1) Iyer, R. P.; Yu, D.; Agrawal, S. Bioorg. Chem. 1995, 23, 1-21.
(2) (a) Iyer, R. P.; Yu, D.; Agrawal, S. Bioorg. Med. Chem. Lett. 1994,
4, 2471-2476. (b) Barber, I.; Rayner, B.; Imbach, J.-L. Bioorg. Med. Chem.
Lett. 1995, 5, 563-568. (c) Tosquellas, G.; Alvarez, K.; Dell’Aquilla, C.;
Morvan, F.; Vasseur, J.-J.; Imbach, J.-L.; Rayner B. Nucleic Acids Res.
1998, 26, 2069-2074. (d) Bologna, J.-C.; Morvan, F.; Imbach, J.-L. Eur.
J. Org. Chem. 1999, 2353-2358. (e) Morvan, F.; Bre`s, J.-C.; Lefebvre, I.;
Vasseur, J.-J.; Pompon, A.; Imbach, J.-L. Nucleosides, Nucleotides &
Nucleic Acids 2001, 20, 1159-1163. (f) Morvan, F.; Bologna, J.-C.; Vive`s,
E.; Imbach, J.-L. Nucleosides, Nucleotides & Nucleic Acids 2001, 20, 1165-
1168. (g) Bologna, J.-C.; Vive`s, E.; Imbach, J.-L.; Morvan, F. Antisense &
Nucleic Acid Drug DeV. 2002, 12, 33-41. (h) Iyer, R. P.; Yu, D.; Agrawal,
S. Bioorg. Med. Chem. Lett. 1994, 4, 2471-2476.
† S.L.B. dedicates this paper to his doctoral mentor, Prof. Kelvin K. Ogilvie.
* To whom correspondence should be addressed. Tel: (301)-827-5162. Fax:
(301)-480-3256.
‡ Food and Drug Administration.
§ Lancaster Laboratories.
¶ The National Institutes of Health.
10.1021/jo062087y CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/11/2007
J. Org. Chem. 2007, 72, 805-815
805

Grajkowski et al.
treatment against specific viral infections.5a,b These observations
prompted us to design CpG ODN prodrugs exhibiting either a
shorter or longer half-time of thiophosphate deprotection relative
to that of CpG ODN 1 for the preparation of effective and long-
acting immunotherapeutic oligonucleotide formulations against
various infectious diseases in animal models. However, the
design of thermolytic oligonucleotide prodrugs poses a paradox
in that one must consider the criticality of oligonucleotide
solubility in biological media, which precludes the use of
lipophilic thiophosphate protecting groups, and of cellular
uptake, which is reportedly commensurate to the relative
lipophilicity of oligonucleotides.2f
SCHEME 1. Thermolytic Conversion of DNA
Oligonucleotide Prodrugs to Bioactive Therapeutic
Oligonucleotidesa
Given that CpG ODN fma1555 [d(GPS(FMA)CPS(FMA)TPS(FMA)
PS(FMA) GPS(FMA) APS(FMA)CPS(FMA)-GPS(FMA)TPS(FMA) TPS(FMA)
PS(FMA)GPS(FMA)CPS(FMA)GPS(FMA)T), where PS(FMA) stands
for the thermolytic 2-(N-formyl-N-methyl)aminoethyl phos-
phorothioate triester function] is functional as a prodrug in
mice,5a,b we propose to evaluate the biophysical implications
of replacing the thermolytic thiophosphate protecting group of
-
-
a B ) thymin-1-yl, cytosin-1-yl, adenin-9-yl, or guanin-9-yl.
A
A
compounds.4 Upon cellular uptake of these oligonucleotide
prodrugs, hydrolysis of the phosphodiester masking groups by
intracellular enzymes generates bioactive oligonucleotide drugs.
We recently reported a new class of DNA oligonucleotide
prodrugs, which did not require intracellular enzyme(s) for
prodrug-to-drug conversion.5a,b This class of oligonucleotide
prodrugs includes oligonucleoside phosphorothioates function-
alized with the thermolytic 2-(N-formyl-N-methyl)aminoethyl
group for thiophosphate protection (Scheme 1). These DNA
oligonucleotides exhibit the characteristics of oligonucleotide
prodrugs in that they are uncharged to facilitate cellular delivery
and are stable to hydrolytic nucleases. A distinctive feature of
this class of modified oligonucleotides is that only an aqueous
environment, maintained at a temperature of 37 °C or above,
is necessary for converting oligonucleoside 2-(N-formyl-N-
methyl)aminoethyl phosphorothioate triesters (1) to functionally
bioactive oligonucleoside phosphorothioate diesters (2) within
a reasonable period of time.5a,b
the CpG within the motif GPS(FMA)APS(FMA)CPS(FMA)GPS(FMA)
-
5c
T
T with thermolabile groups displaying slower or faster
PS(FMA)
deprotection kinetics than those of the FMA group. These new
heat-sensitive groups were designed to impart similar or better
solubility properties in biological media than those in the FMA
groups.6 The dinucleoside phosphorothioate derivatives 3-6
were prepared to serve as models for determining the thermolytic
deprotection kinetics of each thiophosphate protecting group.
With the exception of the 3-hydroxy-1-propyl group of dinucle-
otide 3, the thiophosphate protecting groups of dinucleotides
4-6 are expected to follow an intramolecular cyclodeesterifi-
cation pathway7 under thermolytic conditions at near-neutral
pH.8
Thus, when synthetic single-stranded DNA oligonucleoside
phosphorothioates containing unmethylated CpG motifs (CpG
ODNs) are functionalized with 2-(N-formyl-N-methyl)amino-
ethyl thiophosphate protecting groups and administered to mice,
an immunostimulatory response similar to that produced with
traditional CpG ODN phosphodiesters in terms of the number
of cells secreting cytokines, chemokines, and immunoglobulins
is generated.5a,b However, a delay in the induction of these
immunostimulatory events is observed, consistent with the
thermolytic conversion half-time of 2-(N-formyl-N-methyl)-
aminoethyl thiophosphate triesters (t1/2 ) 73 h at 37 °C) to the
biologically active phosphorothioate diesters. A noteworthy
outcome of these findings relates to the co-administration of
CpG ODN produgs of type 1 and conventional CpG ODN of
type 2 in mice, which widened the timetable of therapeutic
We now report the preparation of deoxyribonucleoside
phosphoramidites 7a and 8a-d,9 which are required for the
synthesis of all model dinucleotides and the solid-phase as-
sembly of the DNA oligonucleotides shown in Table 1 of the
Supporting Information (SI). The usefulness of the phosphityl-
ating reagent, bis[S-(4,4′-dimethoxtrityl)-2-mercaptoethyl]-N,N-
diisopropylphosphoramidite (9),10 in the preparation of, mini-
mally, negatively charged CPG ODN prodrugs is also reported.
(3) (a) Iyer, R. P.; Yu, D.; Devlin, T.; Ho, N -H.; Agrawal, S. Bioorg.
Med. Chem. Lett. 1996, 6, 1917-1922. (b) Iyer, R. P.; Ho, N.-H.; Yu, D.;
Agrawal, S. Bioorg. Med. Chem. Lett. 1997, 7, 871-876.
(4) (a) Ora, M.; Ma¨ki, E.; Poija¨rvi, P.; Neuvonen, K.; Oivanen, M.;
Lo¨nnberg, H. J. Chem. Soc., Perkin Trans. 2 2001, 881-885. (b) Poija¨rvi,
P.; Ma¨ki, E.; Tomperi, J.; Ora, M.; Oivanen, M.; Lo¨nnberg, H. HelV. Chim.
Acta 2002, 85, 1869-1876. (c) Poija¨rvi, P.; Oivanen, M.; Lo¨nnberg, H.
Lett. Org. Chem. 2004, 1, 183-188. (d) Poija¨rvi, P.; Heinonen, P.; Virta,
P.; Lo¨nnberg, H. Bioconjugate Chem. 2005, 16, 1564-1571.
(5) (a) Grajkowski, A.; Pedras-Vasconcelos, J.; Wang, V.; Aus´ın, C.;
Hess, S.; Verthelyi, D.; Beaucage, S. L. Nucleic Acids Res. 2005, 33, 3550-
3560. (b) Grajkowski, A.; Pedras-Vasconcelos, J.; Aus´ın, C.; Verthelyi, D.;
Beaucage, S. L. Ann. N.Y. Acad. Sci. 2005, 1058, 26-38. (c) The
(6) The maximum concentration of CpG ODN fma1555 obtainable in
water is ∼1 mM.
(7) (a) Wilk, A.; Srinivasachar, K.; Beaucage, S. L. J. Org. Chem. 1997,
62, 6712-6713. (b) Wilk, A.; Grajkowski, A.; Phillips, L. R.; Beaucage,
S. L. J. Org. Chem. 1999, 64, 7515-7522. (c) Wilk, A.; Grajkowski, A.;
Srinivasachar, K.; Beaucage, S. L. Antisense & Nucleic Acid Drug DeV.
1999, 9, 361-366. (d) Wilk, A.; Grajkowski, A.; Chmielewski, M. K.;
Phillips, L. R.; Beaucage, S. L. In Current Protocols in Nucleic Acid
Chemistry; Beaucage, S. L., Bergstrom, D. E., Glick, G. D., Jones, R. A.,
Eds.; John Wiley & Sons: New York, 2001; Vol. I, pp 2.7.1-2.7.12.
C
C
C
PS(FMA)GPS(FMA) sequence within the 5′-proximal GPS(FMA)APS(FMA)-
PS(FMA)GPS(FMA)TPS(FMA)T motif is critical for bioactivity in vivo. Replacing
PS(FMA)GPS(FMA) of the motif with GPS(FMA)GPS(FMA) (ODN fma1556) results
in complete suppression of the immunostimulatory properties of CpG ODN
fma1555 in mice (see ref 5a).
806 J. Org. Chem., Vol. 72, No. 3, 2007

Thermolytic Thiophosphate Protecting Groups
Results and Discussion
(20×) of cold hexane. The phosphoramidite precipitate that
ensued was dissolved in C6H6 (∼10 mL/g), and the resulting
solution was freeze-dried under high vacuum over an extended
period of time (16-24 h) to give triethylamine-free 7a or 8a-
d.11
A prerequisite to the synthesis of dinucleoside phosphotri-
esters 3 and 4, and that of the CpG DNA oligonucleotides, is
the preparation of deoxyribonucleoside phosphoramidites 7a and
8a,b. Specifically, 1,3-propanediol or 1,4-butanediol was con-
densed with an equimolar amount of levulinic acid in the
presence of N,N′-dicyclohexylcarbodiimide (DCC) to afford the
levulinylated alcohol 10 or 11 in yields averaging 46% after
silica gel chromatography. The reaction of 10 or 11 with an
equimolar amount of bis(N,N-diisopropylamino)chlorophosphine
and excess i-Pr2NEt in dry CH2Cl2 gave the corresponding
phosphorodiamidite 12 or 13, which was purified by silica gel
chromatography and isolated in yields of ca. 70%.
The solid-phase-linked dinucleoside thiophosphate triesters
14 and 15 were prepared on a 0.2 µmol scale by adding a
solution of phosphoramidite 7a or 8a and 1H-tetrazole in dry
MeCN to a controlled-pore glass support (CPG) functionalized
with thymidine. A sulfuration reaction effected by 0.05 M 3H-
1,2-benzodithiol-3-one-1,1-dioxide12 in MeCN completed the
preparation of 14 or 15. Sequential treatment of the solid-phase-
linked dinucleotide 14 or 15 with (i) 0.5 M hydrazine hydrate
in pyridine/acetic acid (3:2 v/v) to remove the levulinyl group,
(ii) 3% trichloroacetic acid (TCA) in CH2Cl2 to cleave the 4,4′-
dimethoxytrityl (DMTr) group, and (iii) pressurized MeNH2 gas
(∼2.5 bar) for 3 min gave the hydroxyalkylated dinucleoside
thiophosphate triester 3 or 4. Reversed-phase high-performance
liquid chromatography (RP-HPLC) analysis of the crude di-
nucleotide revealed that the purity of 3 or 4 was ∼97% (data
shown in Charts 1 and 2 of the SI). Parenthetically, a solution-
phase approach to the preparation of the 4-hydroxy-1-butyl
phosphate analogue of 4 was reported in the literature more
than 25 years ago.13
The thermostability of each thiophosphate protecting group
was then evaluated using RP-HPLC-purified 3 and 4. On the
basis of our earlier findings in regard to the thermolytic
properties of various phosphate/thiophosphate protecting groups,8
it is anticipated that the 4-hydroxy-1-butyl thiophosphate
protecting group of 4 would undergo intramolecular cyclodees-
terification7 under elevated temperature conditions to liberate
the dinucleoside phosphorothioate diester TPST with the con-
comitant formation of tetrahydrofuran (Scheme 2). It is also
anticipated that the 3-hydroxy-1-propyl thiophosphate protecting
group of 3 would not elicit significant thermolytic cyclodees-
terification considering the unfavorable formation of trimeth-
ylene oxide (Scheme 2). As predicted, when RP-HPLC-purified
dinucleotide 4 was heated in 1X phosphate-buffered saline (PBS,
pH 7.2) at 90 °C, a clean and quantitative conversion to the
parent dinucleoside phosphorothioate diester TPST was achieved
Dry N-protected 5′-O-DMTr-2′-deoxyribonucleosides were
reacted with phosphorodiamidite 12 or 13 and 1H-tetrazole in
anhydrous MeCN over a period of 3 h. While the preparation
of 7a or 8a-c was complete under these conditions, complete
formation of 8d was accomplished within 16 h. The crude
deoxyribonucleoside phosphoramidites were purified by silica
gel chromatography employing an eluent containing Et3N to
neutralize the inherent acidity of silica gel, which otherwise
would cause hydrolysis and partial dedimethoxytritylation of
the phosphoramidite monomers during purification. It is,
however, critically important to remove residual Et3N from the
purified phosphoramidite monomers to prevent reduced coupling
efficiency caused by neutralization of 1H-tetrazole while
performing solid-phase oligonucleotide synthesis. Removal of
the remaining Et3N from purified 7a or 8a-d was achieved by
dissolving the phosphoramidite in a minimum amount of dry
C6H6 (∼5 mL/g) and by adding the solution to a large volume
(8) (a) Wilk, A.; Grajkowski, A.; Phillips, L. R.; Beaucage, S. L. J. Am.
Chem. Soc. 2000, 122, 2149-2156. (b) Grajkowski, A.; Wilk, A.;
Chmielewski, M. K.; Phillips, L. R.; Beaucage, S. L. Org. Lett. 2001, 3,
1287-1290. (c) Wilk, A.; Chmielewski, M. K.; Grajkowski, A.; Phillips,
L. R.; Beaucage, S. L. Tetrahedron Lett. 2001, 42, 5635-5639. (d) Wilk,
A.; Chmielewski, M. K.; Grajkowski, A.; Phillips, L. R.; Beaucage, S. L.
J. Org. Chem. 2002, 67, 6430-6438. (e) Wilk, A.; Chmielewski, M. K.;
Grajkowski, A.; Phillips, L. R.; Beaucage, S. L. In Current Protocols in
Nucleic Acid Chemistry; Beaucage, S. L., Bergstrom, D. E., Glick, G. D.,
Jones, R. A., Eds.; John Wiley & Sons: New York, 2002; Vol. I, pp 3.9.1-
3.9.16. (f) Cies´lak, J.; Beaucage, S. L. J. Org. Chem. 2003, 68, 10123-
10129. (g) Cies´lak, J.; Grajkowski, A.; Livengood, V.; Beaucage, S. L.
J. Org. Chem. 2004, 69, 2509-2515.
(11) Pure deoxyribonucleoside phosphoramidites 7a and 8a-d were
characterized by 31P NMR spectroscopy and by high-resolution mass
spectrometry. 31P NMR spectra of 7a and 8a-d are presented in the SI.
(12) (a) Beaucage, S. L.; Iyer, R. P.; Egan, W.; Regan, J. B. Ann. N.Y.
Acad. Sci. 1990, 616, 483-485. (b) Iyer, R. P.; Egan, W.; Regan, J. B.;
Beaucage, S. L. J. Am. Chem. Soc. 1990, 112, 1253-1254. (c) Iyer, R. P.;
Phillips, L. R.; Egan, W.; Regan, J. B.; Beaucage, S. L. J. Org. Chem.
1990, 55, 4693-4699. (d) Regan, J. B.; Phillips, L. R.; Beaucage, S. L.
Org. Prep. Proced. Int. 1992, 24, 488-492.
(9) Even though only 7a, 8a, and 8b are required for the present work,
phosphoramidites 8c,d were also prepared and characterized for use in
experiments that are beyond the scope of this report.
(10) Aus´ın, C.; Grajkowski, A.; Cies´lak, J.; Beaucage, S. L. Org. Lett.
2005, 7, 4201-4204.
(13) Ogilvie, K. K.; Beaucage, S. L. Nucleic Acids Res. 1979, 7, 805-
824.
J. Org. Chem, Vol. 72, No. 3, 2007 807

Grajkowski et al.
SCHEME 2. Thermolytic Cyclodeesterification of the
Hydroxyalkylated Thiophosphate Protecting Groups from
Dinucleotides 3 and 4 under Near-Neutral Conditionsa
heated in DMSO-d6/D2O (5:1 v/v) for 4 h at 90 °C showed
signals at 1.7 and 3.5 ppm that were identical to those obtained
from commercial THF in terms of chemical shifts and signal
multiplicity (data shown in Chart 3 of the SI). Signals at 7.1-
7.4 ppm corresponded to those of diphenyl phosphate. This
assignment was further supported by 31P NMR analysis of the
deprotection reaction, which clearly showed the conversion of
16 (δP -12 ppm) to diphenyl phosphate (δP -13 ppm) (data
presented in Chart 4 of the SI).
Given that the 3-hydroxy-1-propyl group for thiophosphate
protection in 3 is relatively stable under thermolytic conditions,
we rationalized that phosphorylation/thiophosphorylation of this
protecting group would considerably enhance its thermolability.
In order to test the validity of this rationale, hydrazinolysis of
the levulinylated solid-phase-linked dinucleoside phosphorothio-
ate triester 14 was carried out over a period of 10 min, without
cleaving the dinucleotide from the support,15 to give 17 (Scheme
3). Phosphitylation of 17 using activated 9 in excess, followed
by sulfuration with 0.1 M 3H-1,2-benzodithiol-3-one-1,1-dioxide
in MeCN, produced 19. Removal of all of the DMTr groups
was effected under acidic conditions (3% TCA in CH2Cl2) over
a period of 15 min. Treatment of the detritylated solid-phase-
linked dinucleotide with an aqueous solution of DL-dithiothreitol
(DTT) and Et3N was necessary to reduce the amount of side
products produced during elimination of the 2-mercaptoethyl
groups. Release of the thiophosphatoalkylated dinucleotide 5b
from the support was achieved upon brief exposure (3 min) to
pressurized methylamine gas. RP-HPLC analysis of the crude
dinucleotide confirmed the efficiency of reagent 9 in the
preparation of 5b, as less than 2% of unreacted dinucleotide 3
was detected. However, the chromatographic profile of RP-
HPLC-purified 5b hinted to its thermolability, given that TPST
(<10%) was also present in spite of the mildness of the
conditions under which the analysis was performed.16 RP-
HPLC-purified 5b was characterized by 31P NMR spectroscopy
and MALDI-TOF mass spectrometry (data shown in the SI).
As anticipated, the 3-thiophosphato-1-propyl group was easily
cleaved from 5b in PBS (pH 7.2) to produce TPST; the half-
time of the thermolytic deprotection reaction was 156 min at
37 °C (see Table 2 of the SI). Such relatively rapid deprotection
kinetics would be of limited value in the development of long-
acting oligonucleotide prodrug formulations. Consequently,
oligonucleotides functionalized with 3-thiophosphato-1-propyl
groups for thiophosphate protection were not given further
consideration as potential oligonucleotide prodrugs. Oligonucle-
otides functionalized with 3-phosphato-1-propyl groups for
thiophosphate protection were also not considered for the
development of thermolytic oligonucleotide prodrugs, as ubiq-
uitous phosphatases might convert 3-phosphato-1-propyl groups
a Keys: PBS, 1X phosphate-buffered saline.
within 6 h.14 A reference sample of TPST exhibited a RP-HPLC
retention time (tR ) 20.5 and 21.1 min) identical to that of TPST
generated from the thermolytic deprotection of 4.14 Thermolytic
cleavage of the 4-hydroxy-1-butyl group from 4 in PBS (pH
7.2) occurred with a half-time of 47 min at 90 °C or 168 h at
37 °C (see Table 2 of the SI). In accordance with our
expectations, heating the RP-HPLC-purified dinucleotide 3 in
PBS (pH 7.2) for 6 h at 90 °C resulted in the production of less
than 5% TPST, as determined by RP-HPLC analysis of the
reaction mixture. Several unidentified products were detected
under these conditions, accounting for less than 20% of the total
peak area (data shown in Chart 2 of the SI). Aside from these
unidentified products, which were generated presumably through
cleavage of the internucleotidic linkage, most of the dinucleoside
triester 3 (ca. 80%) was left unreacted.
To confirm whether thermal cleavage of the 4-hydroxy-1-
butyl group from 4 followed the typical cyclodeesterification
pathway, a simpler model was used to monitor the formation
1
of THF by H NMR spectroscopy during the process. Specif-
ically, the reaction of 1,4-butanediol with diphenyl chlorophos-
phate and i-Pr2NEt in dry C6H6 afforded the 4-hydroxy-1-butyl
ester of diphenylphosphate (16) in a yield of 77% after
purification on silica gel. 1H NMR analysis of pure 16 that was
(15) (a) Iwai, S.; Ohtsuka, E. Tetrahedron Lett. 1988, 29, 5383-5386.
(b) Iwai, S.; Ohtsuka, E. Nucleic Acids Res. 1988, 16, 9443-9456. (c) Ueno,
Y.; Shibata, A.; Matsuda, A.; Kitade, Y. Bioconjugate Chem. 2003, 14,
684-689.
(16) RP-HPLC chromatogram of the dinucleotide is shown in Chart 13A
of the SI.
(14) RP-HPLC chromatograms of the thermolytic conversion of 4 to TPST
are shown in Chart 1 of the SI.
808 J. Org. Chem., Vol. 72, No. 3, 2007

Thermolytic Thiophosphate Protecting Groups
SCHEME 3. Solid-Phase Synthesis of the Phosphato-/
Thiophosphatoalkylated Dinucleoside Thiophosphate
Triesters 5b and 6a-ba
not detected during the preparation of 6a, which was reflective
of the “harder” nucleophilic character of the phosphate mo-
noesters relative to that of the thiophosphate monoesters in
inducing cyclodeesterification reactions. RP-HPLC-purified 6a
and 6b were characterized by 31P NMR spectroscopy and
MALDI-TOF mass spectrometry (Experimental Section, see
also Charts 5 and 7 of the SI). The purified dinucleotide 6a
was dissolved in PBS (pH 7.2) and heated at elevated temper-
atures to induce thermolytic cleavage of the 4-phosphato-1-butyl
group and production of TPST. The thermolytic thiophosphate
deprotection of 6a proceeded slowly with a half-time of 305
min at 90 °C or 45 days at 37 °C (see Table 2 and Chart 12 of
the SI). Such slow deprotection kinetics raise doubts on the
usefulness of the 4-phosphato-1-butyl group in the development
of thermolytic oligonucleotide prodrugs. However, the dinucle-
otide 6a served as a substrate for human alkaline phosphatase
in vitro as it was quantitatively dephosphorylated to its
4-hydroxybutylated derivative 4 within 2 h at 37 °C (data shown
in Chart 8B of the SI). Thus, oligonucleotides functionalized
with the 4-phosphato-1-butyl group for phosphate/thiophosphate
protection may, theoretically, be dephosphorylated in vivo by
intracellular phosphatases to the corresponding 4-hydroxybu-
tylated oligonucleotide prodrugs and, then, may proceed through
the thermolytic prodrug-to-drug conversion process. This po-
tential phosphatase-dependent approach to the generation of
thermolytic oligonucleotide prodrugs in situ is interesting and
deserves further investigations.
The purified dinucleotide 6b is, unlike 6a, a poor substrate
for human alkaline phosphatase, as dethiophosphorylation to
dinucleotide 4 occurred to the extent of ∼15% within 30 h at
37 °C (data shown in Chart 9B of the SI). However, given the
“soft” nucleophilic character of thiophosphate monoesters,
thermolytic cleavage of the 4-thiophosphato-1-butyl thiophos-
phate protecting group from dinucleotide 6b occurred smoothly;
the half-time of the deprotection reaction was 13 min at 90 °C
or 30 h at 37 °C (see Table 2 and Chart 14 of the SI). As
discussed above, the thermolytic thiophosphate deprotection of
5b and that of dinucleotide 6b proceeded, presumably via a
cyclodeesterification mechanism leading to the generation of
TPST and the concomitant formation of the respective oxathia-
phosphorinane and oxathiaphosphepane derivatives 22 and 23.
To confirm the identity of cyclodeesterification product 23
a Reagents and conditions: (i) 0.5 M NH2NH2‚H2O in C5H5N/AcOH
(3:2 v/v), 10 min; (ii) 9, 0.45 M 1H-tetrazole/MeCN, 3 min; (iii) 0.1 M
3H-1,2-benzodithiol-3-one-1,1-dioxide/MeCN, 2 min, or 0.1 M ethyl(m-
ethyl)dioxirane/CH2Cl2, 1 min; (iv) 3% TCA/CH2Cl2, 15 min (v), 1.2%
(w/v) DTT/H2O containing 5% (v/v) Et3N, 60 min; (vi) MeNH2 gas (∼2.5
bar), 3 min.
to thermolytically stable 3-hydroxy-1-propyl groups in vivo
(vide infra).17
The solid-phase preparation of dinucleotides 20 and 21 was
achieved in a manner identical to that of dinucleotide 19, with
the exception of using a solution of 0.1 M ethyl(methyl)-
dioxirane in CH2Cl218 as an oxidant in the synthesis of 20. When
employing 9 as a phosphitylating reagent, the use of this oxidant
instead of iodine or tert-butyl hydroperoxide produced higher
yields of the phosphate monoesters.10 Conversion of 20 and 21
to 6a and 6b, respectively, proceeded under conditions identical
to those described for the conversion of 19 to 5b. Consistent
with a slower thermolytic cyclodeesterification process, the
formation of TPST (∼2%) during the preparation of 6b was
significantly less than that produced during the preparation of
5b (see Charts 5 and 6 of the SI). The formation of TPST was
by means of spectroscopic methods, the structurally simpler
thiophosphatoalkylated phosphate triester 26 (Scheme 4) was
prepared. Typically, the reaction of O,O-diphenyl-O-(4-hydroxy-
1-butyl)phosphate (16) with 2-chloro-1,3,2-benzodioxaphos-
phorin-4-one, as described for the preparation of deoxyribonu-
cleoside 3′H-phosphonates,19 gave the H-phosphonate monoester
24 in a yield of 51% after silica gel chromatography. Condensa-
tion of 24 with trimethylsilyl chloride and Et3N in dry pyridine20
produced the bistrimethylsilyl phosphite triester derivative 25,
which, without workup, was treated with elemental sulfur to
(17) This statement is substantiated by the rapid and homologous
conversion of dinucleotide 6a to the hydroxybutylated dinucleotide 4 upon
reaction with human alkaline phosphatase at 37 °C.
(18) Kataoka, M.; Hattori, A.; Okino, S.; Hyodo, M.; Asano, M.; Kawai,
R.; Hayakawa, Y. Org. Lett. 2001, 3, 815-818.
(19) Stawinski, J.; Stro¨mberg, R. In Current Protocols in Nucleic Acid
Chemistry; Beaucage, S. L., Bergstrom, D. E., Glick, G. D., Jones, R. A.,
Eds.; John Wiley & Sons: New York, 2001; pp 2.6.1-2.6.15 and references
therein.
(20) Hata, T.; Sekine, M. J. Am. Chem. Soc. 1974, 96, 7363-7364.
J. Org. Chem, Vol. 72, No. 3, 2007 809

Grajkowski et al.
SCHEME 4. Preparation of the 4-Thiophosphato-1-butyl
Phosphotriester 26 and Evaluation of Its Thermolytic
Cyclodeesterification Side Productsa
SCHEME 5. Alternate Synthesis of the
Oxathiaphosphepane Derivative 23a
a Reagents and conditions: (i) 4-mercaptobutan-1-ol, Et3N, 3 h, 25 °C;
(ii) 2-chloro-1,3,2-benzodioxaphosphorin-4-one, 1,2-dichloroethane, 16 h,
5 °C; (iii) 0.3 M TEAB, pH 7.5; (iv) silica gel chromatography [CH2Cl2/
MeOH (5f12% v/v)]; (v) Me3SiCl/Et3N, C5H5N, 5 h, 25 °C; (vi) MeCN/
H2O (99:1 v/v), 30 min, 25 °C.
SCHEME 6. Preparation of the O-(4-Mercaptobut-1-yl)
a Reagents and conditions: (i) 2-chloro-1,3,2-benzodioxaphosphorin-4-
one/dioxane, 1 h, 5f25 °C; (ii) 1 M triethylammonium bicarbonate (TEAB),
pH 7.5; (iii) silica gel chromatography [CHCl3/MeOH (9:1 v/v)]; (iv)
Me3SiCl/Et3N, C5H5N, 4 h, 25 °C; (v) S8, 16 h, 25 °C, RP-HPLC
purification; (vi) DMSO-d6/D2O (5:1 v/v), 6 h,90 °C.
Phosphate Monoester 27a
give 26 in a yield of ca. 90% on the basis of 31P NMR analysis
of the reaction products. RP-HPLC-purified 26 was then heated
to 90 °C in DMSO-d6/D2O (5:1 v/v) for 6 h to induce
cyclodeesterification of the 4-thiophosphato-1-butyl phosphate
protecting group. 31P NMR analysis of the thermolytic cy-
clodeesterification reaction revealed the formation of the oxa-
thiaphosphepane derivative 23 (δP ∼25 ppm) along with that
of diphenyl phosphate (δP ∼ -10 ppm). The formation of the
most likely 4-mercaptobut-1-yl phosphate monoester (27, δP ∼0
ppm) was also detected, presumably as a result of the sensitivity
of 23 to ring-opening hydrolysis under thermolytic conditions
(data shown in Chart 10 of the SI).
To validate the spectral characteristics of 23, the compound
was prepared using an alternate route (Scheme 5). Specifically,
the reaction of 2,2′-dithiodipyridine with 4-mercaptobutan-1-
ol gave 4-(2-pyridyldithio)butan-1-ol (28) in a yield of 39%.
Phosphonylation of purified 28 with 2-chloro-1,3,2-benzodiox-
aphosphorin-4-one was achieved under conditions similar to
those described in the literature for a different dithioalcohol.21
Purification of the crude H-phosphonate derivative by silica gel
chromatography afforded pure 29 in a yield of 61%. Silylation
of 29 led to the silylated phosphite intermediate 30, which
underwent intramolecular cyclization to afford the oxathiaphos-
phepane 23 after hydrolytic workup. 31P NMR analysis of crude
23 in DMSO-d6/D2O (5:1 v/v) revealed a signal exhibiting a
chemical shift (δP 24.5 ppm, Chart 10C of the SI) identical to
that obtained from the cyclodeesterification of 26 (Chart 10B
of the SI), which was unambiguously demonstrated through a
spiking experiment (data shown in Chart 11 of the SI).
Due to the inherent instability of the oxathiaphosphepane 23,
its characterization was difficult. Indeed, 31P NMR analysis of
a Reagents and conditions: (i) CH3ONa, MeOH, 16 h, reflux; (ii) Dowex
50WX4-100 hydrogen form; (iii) DMTrCl, pyridine, 14 h, 25 °C; (iv)
LiAlH4,THF,2h,0°C;(v)silicagelchromatography;(vi)i-Pr2NP(OCH2CH2CN)2,
1H-tetrazole, MeCN, 2 h, 25 °C; (vii) 0.2 M I2 in THF/Pyr/H2O, 5 min, 25
°C; (viii) concd NH4OH/dioxane (5:1 v/v), 8 h, 55 °C; (ix) 0.9% TFA in
CHCl3/MeOH (15:2 v/v), 2 min, 25 °C.
crude 23 that was stored in DMSO-d6/D2O (5:1 v/v) at -20 °C
for 4 days tentatively revealed the formation (ca. 50%) of
4-mercaptobut-1-yl phosphate monoester (27, Scheme 4 and
Chart 11B of the SI), which exhibited a 31P NMR chemical
shift (δP 0.47 ppm) identical to that of the thermolytic side
product obtained from the cyclodeesterification of 26.
To corroborate the identity of 27, its synthesis was achieved
as shown in Scheme 6. Commercial γ-thiobutyrolactone (31)
was refluxed with an equimolar amount of sodium methoxide
in absolute methanol to provide methyl-4-mercaptopropionate
(32) in 80% yield. Reaction of crude 32 with 4,4-dimethoxytrityl
chloride in dry pyridine gave the S-DMTr derivative 33, which
was subsequently reduced to the corresponding alcohol 34 by
(21) Lartia, R.; Asseline, U. Tetrahedron Lett. 2004, 45, 5949-5952.
810 J. Org. Chem., Vol. 72, No. 3, 2007

Thermolytic Thiophosphate Protecting Groups
treatment with LAH in anhydrous THF. The alcohol 34 was
purified by silica gel chromatography and phosphitylated upon
reaction with bis(2-cyanoethyl)-N,N-diisopropylphosphoramidite
and 1H-tetrazole in MeCN. After aqueous workup, the phosphite
ester 35 was isolated by precipitation from hexane. The
phosphite 35 was then converted to the phosphate triester 36
via oxidation with 0.2 M iodine in pyridine/THF/H2O.22 The
crude phosphate triester 36 was dissolved in a concd NH4OH/
dioxane (5:1 v/v) mixture and heated at 55 °C until â-elimination
of the 2-cyanoethyl phosphate protecting groups was complete.
Following the removal of excess NH4OH, the crude phosphate
monoester was precipitated from diethyl ether and, then,
lyophilized from dioxane. The lyophilisate was dissolved in
∼1% TFA in CHCl3/MeOH to remove the S-(4,4′-dimethoxy-
trityl)group, and crude 27 was isolated by precipitation from
diethyl ether. The 31P NMR spectrum of 27 obtained from 36
was identical to that of 27 produced from the cyclodeesterifi-
cation of 26 (Scheme 4, Charts 10 and 11 of the SI). These
results rigorously confirmed the thermolytic conversion of
dinucleotide prodrug model 6b to its phosphorothioate diester
TPST through a cyclodeesterification pathway.
The information obtained collectively from the dinucleotide
prodrug models in terms of prodrug-to-drug conversion under
thermolytic conditions prompted us to initiate the solid-phase
synthesis of oligonucleotide CpG ODN hbu1555, which was
performed by employing deoxyribonucleoside phosphoramidites
functionalized with the 2-(N-formyl-N-methyl)aminoethyl group
for phosphorus protection and phosphoramidite 8b under
conditions identical to those used for the preparation of CpG
ODN fma1555.5a The average coupling efficiency of these
phosphoramidites was 98-99%, as determined by colorimetric
measurements of the dimethoxytrityl cation after each chain
elongation step. Upon completion of the synthesis in the “trityl-
on” mode, the solid-phase-linked oligonucleotide was first
subjected to hydrazinolysis to remove the levulinyl group from
the thermolabile oligonucleotide.23 Then, pressurized ammonia
gas was used to deprotect the nucleobases and release the
oligonucleotide from the support.24 The crude 5′-DMTr oligo-
nucleotide was purified by RP-HPLC and was then treated with
aqueous 80% AcOH to cleave the 5′-DMTr group. Purification
of the dedimethoxytritylated oligonucleotide by RP-HPLC gave
pure CpG ODN hbu1555, which was characterized satisfactorily
by ESI-TOF mass spectrometry (see Table 1 of the SI).
The solid-phase synthesis of oligonucleotide CpG ODN
pob1555 and CpG ODN psb1555 was achieved differently than
that described for CpG ODN hbu1555 to enable an efficient
chromatographic separation of the modified oligonucleotides
from their respective failures sequences. Although the solid-
phase synthesis of CpG ODN pob1555 and CpG ODN psb1555
was accomplished initially, in a manner identical to that of CpG
ODN hbu1555, it was stopped temporarily at the penultimate
chain elongation cycle to perform hydrazinolysis of the levulinyl
group protecting the single internucleotidic 4-hydroxybutyl
thiophosphate triester function. The resulting solid-phase-linked
4-hydroxybutylated oligonucleotide was then reacted with
phosphoramidite 9 in the presence of 1H-tetrazole. This coupling
reaction was repeated once more, and the newly formed
phosphite triester was oxidized with either 3H-1,2-benzodithiol-
3-one-1,1-dioxide or ethyl(methyl)dioxirane to produce the
respective 4-thiophosphato- or 4-phosphato-1-butylated oligo-
nucleotide. After execution of a standard capping reaction,
complete 5′-O- and S-dedimethoxytritylation was achieved by
acidification with 3% TCA in CH2Cl2, which was immediately
followed by an aqueous treatment with DTT and Et3N to
mitigate the formation of side products. The final chain
elongation cycle was then resumed to complete the oligonucle-
otide assembly. The terminal 5′-DMTr group was left intact to
facilitate oligonucleotide purification.
Nucleobase deprotection and RP-HPLC purification of oli-
gonucleotides CpG ODN psb1555 and CpG ODN pob1555 were
effected under conditions identical to those described above for
CpG ODN hbu1555. The recovery of CpG ODN psb1555 and
CpG ODN pob1555 was lower than that of CpG ODN hbu1555,
thus indicating that steric factors may presumably be responsible
for the less than optimal solid-phase phosphorylation (∼85%)
of the 4-hydroxybutyl thiophosphate protecting group effected
by phosphoramidite 9. This limitation is currently being
investigated, and a solution to this shortcoming will be com-
municated later.
Desalted CpG ODN pob1555 and CpG ODN psb1555 were
characterized by ESI-TOF mass spectrometry (see Table 1 of
the SI). Conversion of diastereomeric RP-HPLC-purified CpG
ODN hbu1555, CpG ODN psb1555, and CpG ODN pob1555
to CpG ODN 15555a,b was accomplished by heating to 90 °C
in PBS (pH 7.2) over a period of 6, 4, and 16 h, respectively.
Analysis of each thermolytic deprotection reaction by poly-
acrylamide gel electrophoresis (PAGE) under denaturing condi-
tions revealed, as expected, a band exhibiting an electrophoretic
mobility identical to that of diastereomeric CpG ODN 1555 (data
shown in Chart 18 of the SI). The thermolytic conversion of
CpG ODN pob1555 to CpG ODN 1555 occurred with some
internucleotidic bond cleavage; two faint fast-moving bands
were observed by PAGE analysis. These findings were expected
given that 2′-deoxyadenosine depurinates to the extent of less
than 3% when heated in PBS (pH 7.2) over a period of 16 h at
90 °C (data shown in Chart 19 of the SI).
All analytical methods used confirmed the identity of each
of the modified oligonucleotides and their abilities to function
as prodrugs, in vitro, through clean thermolytic conversion to
CpG ODN 1555. Noteworthy is the increased solubility of CpG
ODN pob1555 and CpG ODN psb1555 relative to that of CpG
ODN fma1555 in aqueous media. This attribute was assessed
by independent reconstitution of equal amounts of either
lyophilized CpG ODN pob1555 or CpG ODN psb1555 and CpG
ODN fma1555 in a limited but equal volume of water. The
concentration of each reconstituted oligonucleotide was deter-
mined by UV spectrophotometry. When compared with each
other, the concentration of reconstituted CpG ODN pob1555
or CpG ODN psb1555 was either 49 or 25% higher than that
of CpG ODN fma1555. These findings suggest that the
incorporation of a phosphate/thiophosphate monoester into an
otherwise uncharged oligonucleotide may be sufficient to
enhance aqueous solubility without significantly altering cellular
uptake.
(22) (a) Letsinger, R. L.; Lunsford, W. B. J. Am. Chem. Soc. 1976, 98,
3655-3661. (b) Letsinger, R. L.; Finnan, J. L.; Heavner, G. A.; Lunsford,
W. B. J. Am. Chem. Soc. 1975, 97, 3278-3279.
The resistance of oligonucleotide prodrugs CpG ODN
hbu1555, CpG ODN pob1555, and CpG ODN psb1555 to
nucleases was evaluated by exposing each of the prodrugs to
snake venom phosphodiesterase (SVP) and S1 nuclease over a
(23) Only hydrazinolysis was effective in the cleavage of the levulinyl
group within an acceptable period of time at ∼25 °C.
(24) Boal, J. H.; Wilk, A.; Harindranath, N.; Max, E. E.; Kempe, T.;
Beaucage, S. L. Nucleic Acids Res. 1996, 24, 3115-3117.
J. Org. Chem, Vol. 72, No. 3, 2007 811

Grajkowski et al.
silica gel chromatography, 4-hydroxy-1-butyl levulinate (11) was
isolated as a pure oil (8.02 g, 42.6 mmol) in 43% yield: 1H NMR
(300 MHz, DMSO-d6) δ 4.40 (b, 1H), 3.99 (t, J ) 6.6 Hz, 2H),
3.40 (t, J ) 6.2 Hz, 2H), 2.70 (t, J ) 6.6 Hz, 2H), 2.45 (t, J ) 6.6
Hz, 2H), 2.1 (s, 3H), 1.58 (m, 2H), 1.44 (m, 2H); 13C NMR (75
MHz, DMSO-d6) δ 206.6, 172.2, 63.8, 60.2, 37.3, 29.3, 28.7, 27.6,
24.9.
period of 24 h at 37 °C. RP-HPLC analysis of the hydrolysates
showed that the stability of all three oligonucleotide prodrugs
to SVP and S1 nuclease was similar to that of CpG ODN
fma1555, which was used as a negative control. Less than 2
and 4% degradation products were detected from the respective
SVP and S1 digests. Under similar conditions, unmodified DNA
oligonucleotides were completely digested within 15 min.25
These results provide assurance that these three oligonucleotide
prodrugs will maintain structural integrity while being tested
as immunotherapeutic formulations in animal models for
extended periods of time.
To summarize, we have designed and prepared thermolytic
CpG oligonucleotide prodrugs, each of which has a CpG motif
functionalized with a thiophosphate protecting group exhibiting
an increased or decreased thermolability relative to that of the
previously studied 2-(N-formyl-N-methyl)aminoethyl thiophos-
phate protecting group. Although CpG ODN hbu1555 and CpG
ODN psb1555 will not require intracellular enzymes for
conversion to the bioactive CpG ODN 1555, the oligonucleotide
prodrug CpG ODN pob1555 shall benefit from intracellular
phosphatase activity for faster conversion to CpG ODN 1555
through the thermolabile intermediate CpG ODN hbu1555. Such
a two-stage conversion process to CpG ODN 1555 may provide
an additional degree of control on prodrug-to-drug conversion.
By virtue of incorporating a phosphate/thiophosphate monoester
function into the oligonucleotide chain, the solubility of CpG
ODN psb1555 and CpG ODN pob1555 in water has increased
significantly relative to that of CpG ODN fma1555. These
findings thus foster the chemical development of novel,
minimally charged, oligonucleotide prodrugs.
O-[3-(2,4-Dioxopent-1-yl)oxy-1-propyl]-N,N,N′,N′-tetraisopro-
pylphosphorodiamidite (12). To a stirred solution of 3-hydroxy-
1-propyl levulinate (10, 0.87 g, 4.99 mmol) and i-Pr2NEt (4.15 g,
34.6 mmol) in dry CH2Cl2 was added bis(N,N-diisopropylamino)-
chlorophosphine (1.35 g, 5.06 mmol). Progress of the reaction was
monitored by 31P NMR spectroscopy, which showed complete
conversion of bis(N,N-diisopropylamino)chlorophosphine (δP 135.5)
to the phosphorodiamidite 12 (δP 122.0) within 2 h at ∼25 °C.
The suspension was filtered off, and the filtrate was evaporated to
an oil under reduced pressure. The crude phosphorodiamidite was
dissolved in a minimal volume of C6H6/Et3N (9:1 v/v); the solution
was added to the top of a chromatography column packed with
silica gel (∼30 g) that was equilibrated in C6H6/Et3N (9:1 v/v).
The column was eluted using the equilibration solvent, and fractions
containing the product were identified by 31P NMR spectroscopy.
Fractions containing the product were collected together and
rotoevaporated under low pressure to give an oil: Yield 75% (1.52
1
g, 3.75 mmol); H NMR (300 MHz, C6D6) δ 4.23 (t, J ) 6.6 Hz,
2H), 3.60 (dt, J ) 6.2 Hz, JHP ) 7.1 Hz, 2H), 3.51 (sept, J ) 6.8
Hz, 2H), 3.47 (sept, J ) 6.8 Hz, 2H), 2.36 (t, J ) 6.5 Hz, 2H),
2.17 (t, J ) 6.5 Hz, 2H), 1.80 (m, 2H), 1.62 (s, 3H), 1.21 (d, J )
6.8 Hz, 12H), 1.17 (d, J ) 6.8 Hz, 12H); 13C NMR (75 MHz,
2
C6D6) δ 204.4, 172.3, 61.9, 61.0 (d, JCP ) 21.5 Hz), 44.8, 44.6,
3
37.6, 31.1 (d, JCP ) 8.4 Hz), 29.1, 28.1, 24.7, 24.6, 24.1, 24.0;
31P NMR (121 MHz, C6D6) δ 123.5.
O-[4-(2,4-Dioxopent-1-yl)oxy-1-butyl]-N,N,N′,N′-tetraisopro-
pylphosphorodiamidite (13). The preparation of this phosphoro-
diamidite was performed employing 4-hydroxy-1-butyl levulinate
(11, 15.0 mmol) under conditions identical to those used for the
synthesis and purification of 12: Yield 72% (4.52 g, 10.8 mmol);
1H NMR (300 MHz, C6D6) δ 4.04 (t, J ) 6.4 Hz, 2H), 3.54 (dt,
J ) 5.9 Hz, JHP ) 7.4 Hz, 2H), 3.53 (sept, J ) 6.8 Hz, 2H), 3.49
(sept, J ) 6.8 Hz, 2H), 2.35 (t, J ) 6.5 Hz, 2H), 2.15 (t, J ) 6.5
Hz, 2H), 1.61 (s, 3H), 1.60 (m, 4H), 1.23 (d, J ) 6.8 Hz, 12H),
1.19 (d, J ) 6.8 Hz, 12H); 13C NMR (75 MHz, C6D6) 204.5, 172.3,
64.5, 64.2 (d, 2JCP ) 21.5 Hz), 44.7, 44.6, 37.6, 29.1, 28.4 (d, 3JCP
) 8.4 Hz), 28.1, 26.0, 24.8, 24.7, 24.1, 24.0; 31P NMR (121 MHz,
C6D6) δ 122.8.
Studies assessing cellular uptake and biological activity of
diastereomeric CpG ODNs hbu1555, psb1555, and pob1555 in
animal models are ongoing, and the results of these studies will
be reported elsewhere in due course. Other thermolytic oligo-
nucleotide prodrugs are currently being synthesized in our
laboratories to provide increasingly effective heat-sensitive
therapeutic oligonucleotide formulations against infectious
diseases and certain types of cancer in animals.
Experimental Section
3-Hydroxy-1-propyl Levulinate (10). To a stirred solution of
1,3-propanediol (7.61 g, 100 mmol), levulinic acid (11.6 g, 100
mmol), and 4-dimethylaminopyridine (500 mg) in 1,4-dioxane (60
mL) was added DCC (20 g, 97 mmol), portionwise, over a period
of 2 h at ∼25 °C. The reaction mixture was allowed to stir overnight
under these conditions. The N,N′-dicyclohexylurea precipitate was
filtered off and washed with 1,4-dioxane (20 mL). The filtrates
were collected and evaporated to an oil under reduced pressure.
The oily material was dissolved in a minimum volume of CHCl3-
MeOH (96:4 v/v), and the solution was added to the top of a column
packed with silica gel (∼150 g), pre-equilibrated in CHCl3-MeOH
(96:4 v/v). The column was then eluted employing the equilibration
solvent to give pure 3-hydroxy-1-propyl levulinate (10) in 49% yield
(8.51 g, 48.8 mmol): 1H NMR (300 MHz, DMSO-d6) δ 4.05 (t,
J ) 6.4 Hz, 2H), 3.45 (t, J ) 6.1 Hz, 2H), 2.70 (t, J ) 6.4 Hz,
2H), 2.45 (t, J ) 6.4 Hz, 2H), 2.10 (s, 3H), 1.7 (tt, J ) 6.1, 6.4 Hz,
2H); 13C NMR (75 MHz, DMSO-d6) δ 206.8, 172.3, 61.3, 57.2,
37.4, 31.5, 29.4, 27.6.
General Procedure for the Preparation of Deoxyribonucleo-
side Phosphoramidites 7a and 8a-d. Suitably protected 2′-
deoxyribonucleosides (1.0 mmol) were dried by coevaporation with
anhydrous pyridine (2 × 5 mL) and dry toluene (5 mL) under
reduced pressure. The foamy nucleoside was then dissolved in
anhydrous MeCN (5 mL), and phosphorodiamidite 12 or 13 (1.0
mmol) was added by syringe, under a positive pressure of argon.
Sublimed 1H-tetrazole (70 mg, 1.0 mmol) was added to the stirred
solution, portionwise, over a period of 1 h. The reaction mixture
was allowed to stir at ∼25 °C for 3 h26 and was then rotoevaporated
to a foam under vacuum. The material was dissolved in a minimal
volume of C6H6/Et3N (9:1 v/v). The solution was added to the top
of a chromatography column packed with silica gel (∼30 g) that
was equilibrated in C6H6/Et3N (9:1 v/v). The column was eluted
employing the equilibration solvent, and fractions containing the
product were identified by TLC. These fractions were pooled
together and rotoevaporated under low pressure to a white foam.
The material was dissolved in dry C6H6 (∼5 mL), and the solution
was added to cold (-10 °C) hexanes (100 mL). The precipitate
was isolated by decanting off the hexanes and was dissolved by
adding dry C6H6 (10 mL). The resulting solution was frozen in a
4-Hydroxy-1-butyl Levulinate (11). This compound was pre-
pared from 1,4-butanediol and levulinic acid in a manner identical
to that of 10 in terms of scale and purification conditions. Following
(25) Koga, M.; Moore, M. F.; Beaucage, S. L. J. Org. Chem. 1991, 56,
3757-3759.
(26) To ensure optimal formation of 8d, the reaction time was extended
to 16 h at ∼25 °C.
812 J. Org. Chem., Vol. 72, No. 3, 2007

Thermolytic Thiophosphate Protecting Groups
dry ice-EtOH bath and was then lyophilized under high vacuum
to give 7a or 8a-d as white powders in yields ranging from 70 to
90%.
5′-O-(4,4′-Dimethoxytrityl)-3′-O-[(N,N-diisopropylamino)(3-
(2,4-dioxopent-1-yl)oxy-1-propyloxy)]phosphinyl-2′-deoxythymi-
dine (7a): 31P NMR (121 MHz, C6H6) δ 148.8, 148.4; APESI-
HRMS calcd for C45H59N3O11P (M + H)+, 848.3887; found,
848.3883.
5′-O-(4,4′-Dimethoxytrityl)-3′-O-[(N,N-diisopropylamino)(4-
(2,4-dioxopent-1-yl)oxy-1-butyloxy)]phosphinyl-2′-deoxythymi-
dine (8a): 31P NMR (121 MHz, C6H6) δ 148.3, 147.8; APESI-
HRMS calcd for C46H61N3O11P (M + H)+, 862.4044; found,
862.4032.
conditions identical to those used for the kinetic study performed
at 90 °C. The RP-HPLC retention time (tR) of 4 and its thermolytic
thiophosphate deprotection half-time (t1/2) at both 37 and 90 °C
are reported in Table 2 of the SI.
Kinetic Analysis of the Thermolytic Cleavage of the 3-Hy-
droxy-1-propyl Thiophosphate Protecting Group from 3. This
analysis was achieved at 90 ( 2 °C under conditions identical to
those described for the thermolytic cleavage of the 4-hydroxy-1-
butyl thiophosphate protecting group. RP-HPLC analysis of the
deprotection reaction indicated that the 3-hydroxy-1-propyl group
was thermolytically cleaved from 3 to the extent of less than 5%
over a period of 6 h at 90 °C. Under these conditions, unreacted
dinucleoside phosphotriester 3 and unidentified side products
accounted for ∼80% and less than 20% of the total peak area of
the chromatogram, respectively.
N4-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-3′-O-[(N,N-diisopro-
pylamino)(4-(2,4-dioxopent-1-yl)oxy-1-butyloxy)]phosphinyl-2′-
deoxycytidine (8b): 31P NMR (121 MHz, C6H6) δ 148.3, 148.2;
APESI-HRMS calcd for C52H64N4O11P (M + H)+, 951.4309;
found, 951.4303.
Preparation of the Thiophosphatoalkylated Dinucleoside
Thiophosphate Triesters 5b and 6b. A commercial synthesis
column packed with succinyl LCAA-CPG, functionalized with 5′-
O-DMTr-2′-deoxythymidine (0.2 µmol), was treated with 3% TCA
in CH2Cl2 for 2 min. After carefully washing the support with CH2-
Cl2 and MeCN, the coupling reaction was carried out by agitating
the support in a solution composed of phosphoramidite 7a or 8a
(30 µmol) and 0.45 M 1H-tetrazole in MeCN (300 µL) for 10 min.
The synthesis column was flushed with MeCN, and the support
was then treated with 0.05 M 3H-1,2-benzodithiol-3-one-1,1-dioxide
in MeCN (1 mL) for 3 min. Unused oxidant was washed out of
the support with MeCN, affording the dinucleoside phosphotriesters
14 or 15. The levulinyl group was then cleaved from 14 or 15 by
treatment with 0.5 M NH2NH2‚H2O in pyridine/acetic acid (3:2
v/v, 3 mL) for 10 min at ∼25 °C. Following extensive washing of
the CPG support 17 or 18 with CH2Cl2 and MeCN, a coupling
reaction was achieved using phosphoramidite 9 (27 mg, 30 µmol)
activated with 0.45 M 1H-tetrazole in MeCN (300 µL) over a period
of 3 min. The synthesis column was rinsed out with MeCN, and
the support was treated with 0.05 M 3H-1,2-benzodithiol-3-one-
1,1-dioxide in MeCN (1 mL) for 2 min. Excess oxidant was washed
away from the support with MeCN to give the dinucleotides 19 or
21. Removal of all of the DMTr groups was effected by slowly
flushing 3% TCA in CH2Cl2 through the synthesis column over a
period of 15 min. The support was then washed with CH2Cl2 and
MeCN followed by treatment with a solution of 1.2% (w/v) DTT
in H2O (1 mL) containing 5% (v/v) Et3N over a period of 60 min.
After extensive washing of the support with MeCN, the synthesis
column was exposed to pressurized methylamine gas (∼2.5 bar)
for 3 min to release the crude thiophosphatoalkylated dinucleoside
thiophosphate triesters 5b or 6b. Each dinucleotide was purified
by RP-HPLC under chromatographic conditions identical to those
described for the thermolytic cleavage of the 4-hydroxy-1-butyl
thiophosphate protecting group from 4. The RP-HPLC retention
time of diastereomeric 5b or 6b under these conditions is reported
in Table 2 of the SI. Thymidylyl-(3′f5′)-thymidine Thiophos-
phate 3-Thiophosphato-1-propyl Ester (5b): 31P NMR (121
MHz, D2O) δ 67.9, 67.7, 57.4, 57.3, 49.7, 11.8; +MALDI-TOF
MS calcd for C23H34N4O14P2S2 (M + 2H)+, 716.10; found, 716.10.
Thymidylyl-(3′f5′)-thymidine Thiophosphate 4-Thiophosphato-
1-butyl Ester (6b): 31P NMR (121 MHz, H2O) δ 67.1, 66.9, 51.9;
-MALDI-TOF MS calcd for C24H35N4O14P2S2 (M - H)-, 729.63;
found, 729.66.
N6-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-3′-O-[(N,N-diisopro-
pylamino) (4-(2,4-dioxopent-1-yl)oxy-1-butyloxy)]phosphinyl-2′-
deoxyadenosine (8c): 31P NMR (121 MHz, C6H6) δ 148.0;
APESI-HRMS calcd for C53H64N6O10P (M + H)+, 975.4422;
found, 975.4418.
N2-Isobutyryl-5′-O-(4,4′-dimethoxytrityl)-3′-O-[(N,N-diisopro-
pylamino)(4-(2,4-dioxopent-1-yl)oxy-1-butyloxy)]phosphinyl-2′-
deoxyguanosine (8d): 31P NMR (121 MHz, C6H6) δ 148.2, 147.9;
APESI-HRMS calcd for C50H66N5O11P (M + H)+, 957.4527;
found, 957.4519.
Preparation of the Dinucleoside Thiophosphate Triesters 3
and 4. A commercial synthesis column packed with succinyl long-
chain alkylamine controlled-pore glass (LCAA-CPG) functionalized
with 5′-O-DMTr-2′-deoxythymidine (0.2 µmol) was treated with
3% TCA in CH2Cl2 for 2 min. After washing the support extensively
with CH2Cl2 and MeCN, the coupling reaction was performed by
shaking the support in a solution composed of phosphoramidite 7a
or 8a (30 µmol) and 0.45 M 1H-tetrazole in MeCN (300 µL) for
10 min. The synthesis column was rinsed out with MeCN, and the
support was mixed with 0.05 M 3H-1,2-benzodithiol-3-one-1,1-
dioxide in MeCN (1 mL) for 2 min. The solid support was then
washed with MeCN and treated with 0.5 M NH2NH2‚H2O in
pyridine/acetic acid (3:2 v/v, 3 mL) for 10 min to remove the
levulinyl group. Following extensive washing of the CPG support
with CH2Cl2 and MeCN, removal of the 5′-DMTr group was
effected under acidic conditions (3% TCA in CH2Cl2) over a period
of 2 min. After carefully washing the support with CH2Cl2, the
synthesis column was exposed to pressurized methylamine gas for
3 min to release the dinucleoside thiophosphate triester 3 or 4. The
purity of the crude dinucleoside triesters was about 97% as
determined by RP-HPLC (data shown in Charts 1A and 2A of the
SI). Thymidylyl-(3′f5′)-thymidine Thiophosphate 3-Hydroxy-
1-propyl Ester (3): +MALDI-TOF MS calcd for for C23H32N4-
NaO12PS (M + Na)+, 642.14; found. 642.64. Thymidylyl-(3′f5′)-
thymidineThiophosphate4-Hydroxy-1-butylEster(4):+MALDI-
TOF MS calcd for C24H35N4O12PS (M)+, 634.17; found, 634.72.
Kinetic Analysis of the Thermolytic Cleavage of the 4-Hy-
droxy-1-butyl Thiophosphate Protecting Group from 4. RP-
HPLC-purified 4 (∼10 OD260) was dissolved in PBS (pH 7.2, 500
µL) and heated to 90 ( 2 °C. Aliquots of the deprotection reaction
were taken out, periodically, over a period of 6 h. Each aliquot
was analyzed by RP-HPLC using a 5 µm Supelcosil LC-18S column
(25 cm × 4.6 mm) under the following conditions: starting from
0.1 M triethylammonium acetate (TEAA), pH 7.0, a linear gradient
of 1% MeCN/min was pumped at a flow rate of 1 mL/min for 40
min.
Preparation of the Phosphatoalkylated Dinucleoside Thio-
phosphate Triester 6a. Dinucleotide 6a was prepared in a manner
identical to that described for the preparation of dinucleotides 5b
and 6b with the following exception: upon phosphitylation of 18
with 9, a solution of 0.1 M ethyl(methyl)dioxirane in CH2Cl2,
instead of 3H-1,2-benzodithiol-3-one-1,1-dioxide in MeCN, was
used over a period of 1 min to give the dinucleotide 20. The RP-
HPLC purification of dinucleotide 6a was done under conditions
identical to those described for the purification of 5b and 6b. The
RP-HPLC retention time of 6a under such chromatographic
conditions is reported in Table 2 of the SI. Thymidylyl-(3′f5′)-
The thermolytic cleavage of the 4-hydroxy-1-butyl thiophosphate
protecting group was also investigated at 37 ( 2 °C using ∼20
OD260 of RP-HPLC-purified 4 in PBS (pH 7.2, 1 mL). Aliquots of
the deprotection reaction were retained, periodically, over a period
of 200 h and were analyzed by RP-HPLC under chromatographic
J. Org. Chem, Vol. 72, No. 3, 2007 813

Grajkowski et al.
thymidine Thiophosphate 4-Phosphato-1-butyl Ester (6a): 31P
NMR (121 MHz, H2O) δ 68.0, 1.8; +MALDI-TOF MS calcd for
C24H35N4O15P2SNa (M + H + Na)+, 736.12; found, 736.39
Kinetic Analysis of the Thermolytic Cleavage of the Thio-
phosphatoalkyl and Phosphatoalkyl Thiophosphate Protecting
Groups from 5b, 6b, and 6a. This analysis was carried out at 37
( 2 °C under conditions identical to those described for the
thermolytic cleavage of the 4-hydroxy-1-butyl thiophosphate pro-
tecting group from dinucleotide 4. Aliquots of the deprotection
reaction were taken out periodically over a period of 200 h in the
case of 5b and 6b or until the deprotection reaction was 50%
complete in the case of 6a. The thermolytic thiophosphate depro-
tection half-times of dinucleotides 5b, 6b, and 6a are reported in
Table 2 of the SI.
Enzymatic Dephosphorylation of Dinucleotide 6a Catalyzed
by Human Alkaline Phosphatase. To a solution of RP-HPLC-
purified dinucleotide 6a (3 OD260) in 10 mM glycine buffer (pH
10.4, 200 µL) was added human alkaline phosphatase (1 U, 15
µL). The solution was incubated at 37 ( 2 °C for 2 h. An aliquot
was analyzed by RP-HPLC under conditions identical to those used
for the purification of dinucleotides 5b and 6a,b. The analysis
revealed complete dephosphorylation of 6a (tR ) 27.8 min) to
dinucleotide 4 (tR ) 30.5 min). When an identical experiment was
carried out with the dinucleotide 6b, less than 5 and 15%
dethiophosphorylation to dinucleotide 4 was detected after an
incubation time of 2 and 30 h, respectively.
Solid-Phase Oligonucleotide Synthesis. Solid-phase synthesis
of the modified oligonucleotide CpG ODN hbu1555 was performed
on a 1 µmol scale and under conditions identical to those described
earlier for the preparation of CpG ODN fma15555a and CpG ODN
1555. An additional glass bottle containing the phosphoramidite
8b as a 0.1 M solution in dry MeCN was connected to the DNA/
RNA synthesizer. The phosphoramidite 8b was numerically labeled
in the programmed alphabetical DNA sequence and recognized as
such during execution of the automated DNA chain extension.
Upon completion of the DNA chain assembly, the 5′-DMTr solid-
phase-linked oligonucleotide was treated with 0.5 M NH2NH2‚H2O
in pyridine/AcOH (3:2 v/v) by pushing the solution (3 mL) through
the synthesis column, over a period of 10 min, with a 3 mL syringe.
The excess reagent was discarded, and the column was rinsed out,
consecutively, with CH2Cl2 and MeCN (10 mL each). Residual
solvent was eliminated from the support by placing the synthesis
column under reduced pressure for ∼3 min.
(10 mL). The final chain elongation cycle was completed using
the DNA/RNA synthesizer, leaving on the terminal 5′-DMTr group.
The synthesis columns were then placed under reduced pressure
for ∼3 min to remove residual solvent.
Oligonucleotide Deprotection and Purification. The solid-
phase-linked 5′-DMTr oligonucleotides corresponding to CpG ODN
hbu1555, CpG ODN psb1555, and CpG ODN pob1555 were
deprotected under conditions identical to those reported for the
deprotection of CpG ODN fma1555.5a Specifically, the columns
were placed into a stainless steel pressure vessel and exposed to
pressurized ammonia (10 bar at 25 °C) for 12 h. Upon removal of
residual ammonia from the pressure container, the oligonucleotides
were eluted off their respective synthesis column with 40% MeCN
in 0.1 M TEAA, pH 7.0, (1 mL). Purification of each oligonucle-
otide was accomplished by RP-HPLC in the “trityl-on” mode and
then in the “trityl-off” mode, as described for the purification of
CpG ODN fma1555.5a The product peaks were collected, and the
eluates were evaporated using a stream of air without heating. The
concentration of CpG ODN hbu1555, CpG ODN psb1555, and CpG
ODN pob1555 was determined by UV spectrophotometry at 260
nm upon reconstitution of the purified oligonucleotides in ddH2O
(1 mL). The recovered yields of CpG ODN hbu1555, CpG ODN
psb1555, and CpG ODN pob1555 were 71, 55, and 59 OD260 units,
respectively. The pure oligonucleotides were stored frozen at
-20 °C.
Oligonucleotide Characterization. RP-HPLC analysis of puri-
fied CpG ODN hbu1555, CpG ODN psb1555, and CpG ODN
pob1555 (7 OD260 each) was achieved using an analytical 5 µm
Supelcosil LC-18S column (4.6 mm × 25 cm) under the following
conditions: starting from 0.1 M TEAA (pH 7.0), a linear gradient
of 1% MeCN/min was pumped at a flow rate of 1 mL/min for 40
min and then held isocratically for 20 min. RP-HPLC profiles of
the purified oligonucleotides are shown in Charts 15-17 of the
SI. RP-HPLC retention times of the purified oligonucleotides are
listed in Table 1 of the SI. Samples of the purified oligonucleotides
were characterized by ESI-TOF MS operating in the positive-ion
mode. The results of these analyses are presented in Table 1 of
the SI.
Functional characterization of CpG ODN hbu1555, CpG ODN
psb1555, and CpG ODN pob1555 consisted of converting these
oligonucleotides to CpG ODN 15555a upon heating at 90 °C in 1X
PBS (pH 7.2) for 6, 4, and 16 h, respectively. RP-HPLC and PAGE
analysis of the thermolytically deprotected oligonucleotides revealed
complete conversion to CpG ODN 1555 (data shown in Charts 15-
18 of the SI).
Determination of Relative Oligonucleotide Solubility in
Water. In duplicate experiments, lyophilized CpG ODN pob1555
or CpG ODN psb1555 (20 OD260) and CpG ODN fma1555 (20
OD260) were suspended in H2O (50 µL). Each suspension was
vigorously agitated using a vortexer, at full speed, for 30 s. Each
suspension was sedimented by microcentrifugation at 16 000g for
30 s. The concentration of each oligonucleotide in 5 µL of each
supernatant was determined by UV spectrophotometry at 260 nm
and averaged 1.00 OD for CpG ODN fma1555, 1.49 OD for CpG
ODN pob1555, and 1.25 O.D for CpG ODN psb1555. Thus, the
concentration of CpG ODN pob1555 or that of CpG ODN psb1555
is 49%, or 25% higher than that of CpG ODN fma1555 in water.
Solid-phase synthesis of oligonucleotides CpG ODN pob1555
and CpG ODN psb1555 was achieved initially under conditions
identical to those used for the preparation of CpG ODN hbu1555
but was interrupted at the second to last chain elongation cycle,
leaving the terminal 5′-DMTr group intact. Hydrazinolysis of the
levulinyl group was carried out identically as described for CpG
ODN hbu1555. A solution of activated phosphoramidite 9 (0.2 M
in 0.45 M 1H-tetrazole/MeCN, 0.3 mL) was then syringed back
and forth through each of the synthesis columns for 3 min. The
synthesis columns were purged with dry MeCN (10 mL), and the
phosphitylation step was performed again. The support of one of
the two columns was reacted with 0.05 M 3H-1,2-benzodithiol-3-
one-1,1-dioxide in MeCN (1 mL) for 2 min, whereas the support
of the other column was treated with 0.1 M ethyl(methyl)dioxirane
in CH2Cl2 (1 mL) for 1 min. The oxidant was then flushed out of
the columns with MeCN (10 mL), and an equal volume (0.5 mL)
of commercial Cap A and Cap B reagents was syringed back and
forth through each of the synthesis columns for 3 min. The capping
reagents were expelled out of each column with MeCN (10 mL).
The DMTr groups were then cleaved by pushing 3% TCA in CH2-
Cl2 (15 mL) through each column over a period of 15 min to achieve
complete S-dedimethoxytritylation. Residual acid was washed out
of each column with CH2Cl2 (10 mL) and MeCN (3 mL). A solution
of 1.2% (w/v) DTT/H2O containing 5% (v/v) Et3N (3 mL) was
syringed through the columns over a period of 60 min. The synthesis
columns were rinsed out with H2O (10 mL) and then with MeCN
Acknowledgment. This research is supported, in part, by
an appointment to the Postgraduate Research Participation
Program at the Center for Drug Evaluation and Research
administered by the Oak Ridge Institute for Science and
Education through an interagency agreement between the U.S.
Department of Energy and the U.S. Food and Drug Administra-
tion.
Supporting Information Available: Experimental Section
including materials and methods; synthesis and characterization of
814 J. Org. Chem., Vol. 72, No. 3, 2007

Thermolytic Thiophosphate Protecting Groups
16, 23, 24, 26-29, and 32-36; generation of THF during thermal
cyclodeesterification of 16; stability of CpG ODN hbu1555, CpG
ODN psb1555, and CpG ODN pob1555 to snake venom phos-
phodiesterase and S1 nuclease; 1H NMR spectra of 10-13 and 26;
13C NMR spectra of 10-13, 26, 29, 35, and 36; 31P NMR spectra
of 5b, 6a-b, 7a, 8a-d, 12, 13, 23, 24, 26-27, 29, 35, and 36; 1H
and 31P NMR analysis of the thermolytic cyclodeesterification of
16; RP-HPLC analysis of the thermolytic thiophosphate deprotection
of 3, 4, 5b, 6a, and 6b; RP-HPLC analysis of the digestion of 6a
and 6b catalyzed by human alkaline phosphatase; RP-HPLC
analysis of RP-HPLC-purified CpG ODN hbu1555, CpG ODN
psb1555, CpG ODN pob1555, and their respective thermolytic
conversion to CpG ODN 1555; polyacrylamide gel analysis of the
thermolytic conversion of CpG ODN hbu1555, CpG ODN psb1555,
and CpG ODN pob1555 to CpG ODN 1555; RP-HPLC analysis
of heat-induced depurination of 2′-deoxyadenosine.· This material
is available free of charge via the Internet at http://pubs.acs.org.
JO062087Y
J. Org. Chem, Vol. 72, No. 3, 2007 815