2′-Deoxyimmunosine: Stereoselective synthesis, base pairing and duplex stability of oligonucleotides containing 8-oxo-7-thiaguanine

Article abstract of DOI:10.1039/b718500f

Oligonucleotides containing 7-thia-8-oxoguanine represent a new class of molecules in which sulfur replaces the 7-nitrogen of a purine base. The monomeric 7-thia-8-oxoguanine 2′-deoxyribonucleoside (2′- deoxyimmunosine, 4) was prepared by nucleobase anion glycosylation in a regio- and stereoselective way employing5-{[(di-n-butylamino)methylidene]amino} thiazolo[4,5-d]pyrimidine-2,7(3H,6H)-dione (18) and 1-chloro-2-deoxy-3,5-di-O-p- toluoyl-α-d-erythro-pentofuranose (6). The nucleoside was converted into the phosphoramidite 22 and oligonucleotides were prepared by solid-phase synthesis. Oligonucleotide duplexes containing the 4-dC base pair show a similar stability as those containing the dG-dC motif. Thus the sterically demanding sulfur and the additional 8-oxo group are well accommodated in the major groove of DNA. As expected, compound 4 does not form a Hoogsteen pair, as reported for 8-oxo-2′-deoxyguanosine. Compared to 2′-deoxyguanosine, 2′-deoxyimmunosine shows a better mismatch discrimination in Watson-Crick base pairs. The Royal Society of Chemistry.

Full text of DOI:10.1039/b718500f

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
2ꢀ-Deoxyimmunosine: stereoselective synthesis, base pairing and duplex
stability of oligonucleotides containing 8-oxo-7-thiaguanine†
Frank Seela*a,b and Xin Minga,b
Received 29th November 2007, Accepted 23rd January 2008
First published as an Advance Article on the web 7th March 2008
DOI: 10.1039/b718500f
Oligonucleotides containing 7-thia-8-oxoguanine represent a new class of molecules in which sulfur
replaces the 7-nitrogen of a purine base. The monomeric 7-thia-8-oxoguanine 2ꢀ-deoxyribonucleoside
(2ꢀ-deoxyimmunosine, 4) was prepared by nucleobase anion glycosylation in a regio- and stereoselective
way employing 5-{[(di-n-butylamino)methylidene]amino}thiazolo[4,5-d]pyrimidine-2,7(3H,6H)-dione
(18) and 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-a-D-erythro-pentofuranose (6). The nucleoside was
converted into the phosphoramidite 22 and oligonucleotides were prepared by solid-phase synthesis.
Oligonucleotide duplexes containing the 4–dC base pair show a similar stability as those containing the
dG–dC motif. Thus the sterically demanding sulfur and the additional 8-oxo group are well
accommodated in the major groove of DNA. As expected, compound 4 does not form a Hoogsteen
pair, as reported for 8-oxo-2ꢀ-deoxyguanosine. Compared to 2ꢀ-deoxyguanosine, 2ꢀ-deoxyimmunosine
shows a better mismatch discrimination in Watson–Crick base pairs.
dihydro-8-oxo-2ꢀ-deoxyguanosine (OxodG),19,20 7-methyl-8-oxo-
Introduction
2ꢀ-deoxyguanosine21 and 7-deaza-2ꢀ-deoxyguanosine (3b)11,22,23
The development of clinically useful agents that restore and
have been described. However, very little is known about the
synthesis of 2ꢀ-deoxyimmunosine (4) as well as of oligonu-
enhance the ability of the human immune system to ward off
infections or other invasion challenges has become a major
cleotides incorporating the 8-oxo-7-thiaguanine as nucleobase.
Herein, we wish to report on the stereoselective synthesis of 2ꢀ-
deoxyimmunosine, its protection and conversion into a phos-
phoramidite building block as well as its incorporation into
oligo-2ꢀ-deoxyribonucleotides. The base pairing properties of 2ꢀ-
deoxyimmunosine will be studied. This is the first report on
oligonucleotides containing a sulfur atom in the five-membered
ring of the nucleobase.
objective of current pharmaceutical research efforts. The AIDS
epidemic and the need for adjuvant therapy to boost the immune
system of the elderly and cancer patients has brought the area of
immunopotentiation into focus.1–4 Many different types of com-
pounds have been demonstrated to possess immune stimulatory
activity. Among them are the class of nucleosides5–9 as well as
oligonucleotides with particular sequence motifs.10,11
Among the monomeric nucleosides, guanosine analogues such
as 7-thia-8-oxoguanosine (1, immunosine, TOG, isatoribine) and
its analogues,12–15 7-allyl-8-oxoguanosine (2b, loxoribine)16 or 7-
deazaguanosine (3a)17 (purine numbering is used throughout
this paper) and its 2ꢀ-deoxy derivative (3b)11 (Fig. 1) possess
in vivo activity against a variety of DNA and RNA viruses.
Immunosine exhibits a stimulatory effect on both cellular and
humoral components of the immune response; the observed
antiviral effect has been attributed primarily to the induction
of a-interferon.15 Recently, it was found that immunosine and
other guanosine analogues activate immune cells via Toll-like
receptor 7 (TLR7).18 Also, oligonucleotides can activate the
immune system, and the CpG motif was found to generate such
an activity in nucleic acids.10 Oligonucleotides incorporating 7,8-
Fig. 1 Structures of nucleosides 1–4.
Results and discussion
aLaboratory of Bioorganic Chemistry and Chemical Biology, Center
for Nanotechnology, Heisenbergstrasse 11, 48149, Mu¨nster, Germany.
E-mail: Frank.Seela@uni-osnabrueck.de, seela@uni-muenster.de;Web:
www.seela.net; Fax: +49-(0)251-53406-587; Tel: +49-(0)251-53406-500;
Tel: +49-(0)173-7250-297
1. Synthesis of 2ꢀ-deoxyimmunosine (4)
Two synthetic routes were taken into consideration for the 2ꢀ-
deoxyimmunosine (4) synthesis. The first one is a convergent
approach which involves the nucleobase 5 and the halogenose
6 as starting materials, while the other route requires deoxy-
genation of the ribonucleoside immunosine (1). The Barton
deoxygenation is the more lengthy protocol used for the ri-
bonucleoside immunosine (1). Reports for the synthesis of 4
bLaboratorium fu¨r Organische und Bioorganische Chemie, Institut fu¨r
Chemie, Universita¨t Osnabru¨ck, Barbarastrasse 7, 49069, Osnabru¨ck,
Germany
† Electronic supplementary information (ESI) available: pKa titration
profiles of compound 5; RP-HPLC profiles of hydrolysis mixtures. See
DOI: 10.1039/b718500f
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already exist. In 1991 Cottam and Robins reported that 2ꢀ-
deoxyimmunosine can be synthesized by the fusion procedure. 5-
Aminothiazolo[4,5-d]pyrimidin-2,7(3H,6H)-dione (5)24 was sily-
lated with hexamethyldisilazane, and the silylated intermediate
was fused with 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-a-D-erythro-
pentofuranose (6) to yield an anomeric mixture of the protected
2ꢀ-deoxyribonucleosides in 14% yield.25 After deprotection, the
anomeric mixture was separated by crystallization from EtOH to
give nucleoside 4. However, no proof of the anomeric configuration
was given. The separation of the anomers is tedious and the
unambiguous configurational assignment on the basis of 1H-
NMR data is difficult. In a second route the McCombie–Barton
deoxygenation of the ribonucleoside was employed (Z. So¨zen,
Thesis, Ulm, 2001). However, the overall yield is low (3.6%).
Next, we initiated studies to use the nucleobase anion gly-
cosylation, paying particular attention to the stereoselectivity
of the glycosylation reaction. Normally, this protocol forms 2ꢀ-
deoxyribonucleosides in a stereoselective way but shows draw-
backs when the nucleobase is not fully soluble in the reaction mix-
ture. Our first synthetic attempt towards the 2ꢀ-deoxyimmunosine
synthesis made use of the non-protected nucleobase. The 5-
aminothiazolo[4,5-d]pyrimidin-2,7(3H,6H)-dione (5) was pre-
pared in 5 steps from 2,4-diamino-5-hydroxypyrimidine according
to Baker and Chatfield.24 Treatment of the nucleobase with
halogenose 6 in DMF in the presence of sodium hydride (room
temperature) afforded a mixture (29% yield) of the protected
nucleosides. Although compound 5 contains two lactam functions,
the reaction mixture was tentatively assigned to the anomeric
compounds 7/8. Deblocking with 0.2 M NaOCH3 in MeOH gave
an anomeric mixture of the nucleosides 4/9 in a 2 : 1 ratio, which
could not be separated by flash column (FC) chromatography.
Nevertheless, from the 13C NMR spectrum it was apparent that
two nucleosides are formed, which was confirmed by the HPLC
profile resulting in two peaks in a ratio of 2 : 1 (63% : 30%).
The almost identical 13C NMR chemical shifts observed for the
nucleobase moieties made the formation of anomers with nitrogen-
9 as the glycosylation site more likely over that of regioisomers with
sugar moieties in the six-membered and the five-membered rings
(Scheme 1).
the glycosylation of 5 with halogenose 6. Unfortunately, the
nucleobase 5 is poorly soluble in this solvent, and so we increased
the nucleobase lipophilicity by protecting the amino group of
5 with the lipophilic (N,N-dimethylamino)methylidene residue.
For this, compound 5 was treated with N,N-dimethylformamide
dimethyl acetal to yield the derivative 10. Glycosylation of the
protected nucleobase 10 with halogenose 6 in MeCN with sodium
hydride as base (room temperature) resulted in a 55% yield of
protected reaction products still forming a mixture of anomers
11/12. Deprotection of the sugar moiety was performed with
the mixture of 11/12 (in 0.1 M NaOMe/MeOH), yielding the
nucleosides 13/14. Neither the sugar-protected intermediates
11/12 nor the amidine-protected nucleosides 13/14 could be
separated on preparative scale. However, as it was found that the
5ꢀ-DMT derivatives are separable by column chromatography, the
13/14 mixture was treated with DMT-Cl, affording a mixture of
15/16 from which the single anomers 15 and 16 were isolated
in a pure form by flash chromatography. Detritylation of 15
yielded 13, while under the same conditions 16 gave 14. The free
nucleosides 4 and 9 were isolated after treatment of compounds
13 and 14 with saturated NH3/CH3OH for 3 days at room tem-
perature. Reaction of compound 15 with chloro(2-cyanoethoxy)-
N,N-diisopropylaminophosphine afforded the phosphoramidite
17 (Scheme 2).
Despite the improved glycosylation yield and the successful
separation of anomers it was difficult to run the glycosylation
reaction to completion. The yield could not be improved by using
an excess of more than 1.3 equivalents of the chloro sugar 6,
as side products were formed. A further increase of the sugar
halide led to the introduction of two sugar residues (one at
each lactam moiety). This results from the two immunosine
lactam deprotonation sites with pKa values of 7.2 and 10.0
(ESI†). As the separation of the DMT compounds was tedious—
the material became partially deprotected during the chromato-
graphic work-up process—we choose the more lipophilic N,N-
dibutylaminomethylidene protecting group instead. This group
has already been successfully employed for the protection of
7-deaza-2ꢀ-deoxyisoguanosine28 and 5-methylisocytosine.29 The
reaction of 5 with N,N-dibutylaminoformamide dimethyl acetal30
gave the amidine in 75% yield. Glycosylation of 18 with the
chloro sugar 6 in MeCN at room temperature in the pres-
ence of powdered KOH and TDA-1 tris(3,6-dioxaheptyl)amine
resulted in a 65% yield of the protected nucleosides as an
anomeric mixture (in a ratio of 5 : 1), which was separable
by FC to yield compound 19. This was deprotected under
mildly alkaline conditions (0.1 M NaOMe/MeOH) to afford the
amidine 20, which upon treatment with concentrated ammonium
hydroxide gave 2ꢀ-deoxyimmunosine (4). Compound 20 was
converted into the 4,4ꢀ-dimethoxytrityl derivative 21. Reaction
with chloro(2-cyanoethoxy)-N,N-diisopropylaminophosphine af-
forded the phosphoramidite 22 (Scheme 3).
2. Physical characterization of 2ꢀ-deoxyimmunosine and its
derivatives
Scheme 1 Reagents and conditions: (i) NaH, DMF, r.t.; (ii) 0.2 M
NaOCH3, 2 h, r.t.
All compounds were characterized by 1H- and 13C-NMR spectra
as well as by elemental analysis. 31P-NMR spectra were taken for
the phosphoramidites 17 and 22 (see Tables 2–4 and Experimental
As the formation of anomeric glycosylation mixtures had al-
ready been observed when the anion glycosylation was performed
in DMF26,27 but not in MeCN, this solvent was used next for
1
section). H NMR data are displayed in Table 2, and 13C NMR
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Scheme 2 Reagents and conditions: (i) NaH, MeCN, r.t.; (ii) 0.1 M NaOCH3, 2 h, r.t.; (iii) DMT-Cl, pyridine, 1 h, r.t.; (iv) 1% dichloroacetic acid, 20 min,
r.t.; (v) saturated NH3/CH3OH, 72 h, r.t.; (vi) 2-(cyanoethyl)diisopropylphosphoramido chloridite, 15 min, r.t.
Scheme 3 Reagents and conditions: (i) KOH, TDA-1 tris(3,6-dioxaheptyl)amine, MeCN, 30 min, r.t.; (ii) 0.1 M NaOCH3, 2 h, r.t.; (iii) 25% aq. NH3,
72 h, r.t.; (iv) DMT-Cl, pyridine, 1 h, r.t.; (v) 2-(cyanoethyl)diisopropylphosphoramido chloridite, 15 min, r.t.
chemical shifts are compiled in Table 3. Although the anomeric
2ꢀ-deoxyimmunosines showed noticeable changes of the 13C NMR
chemical shifts of the sugar moieties, no unambiguous assignment
was possible from these data. Also, NOE difference spectra could
not be used for identification as no proton is present in the 8-
position, and thus a single-crystal X-ray analysis was performed.31
The crystal structure of compound 4 is shown in Fig. 2. In the
solid state, compound 4 adopts the syn conformation with a
torsion angle of v = 61.0◦. This is different from most of the
other 2ꢀ-deoxyribonucleosides, which adopt an anti conformation.
The sugar ring has an N-conformation (3ꢀ-endo-4ꢀ-exo, 3T4).
The conformation around the C4ꢀ–C5ꢀ bond is in the +ap
(trans) orientation. The syn conformation is typical for most
of the 8-substituted guanine ribonucleosides, with a C2ꢀ-endo
pucker and a (+) gauche conformation (+sc) around the C4ꢀ–C5ꢀ
bond.32–35 Examples are 8-bromo-,33 8-chloro-,34 and 7-methyl-8-
thioxoguanosines.36 Some compounds such as 7-deaza-8-methyl-
2ꢀ-deoxyguanosine37 display an anti conformation in the solid state,
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3
Table 1 JH,H coupling constants of the sugar moiety and conformer
population of nucleoside 4
Pseudorotational
parameters
H positions
3JH,H/Hz
1ꢀ,2ꢀ
1ꢀ,2ꢀꢀ
2ꢀ,3ꢀ
2ꢀꢀ,3ꢀ
3ꢀ,4ꢀ
7.06
7.42
7.30
3.92
3.19
%N
27
73
154
23
0.416
%S
PS/deg
WS/deg
Rms
with the sugar in the S-conformation and a trans orientation of
the C4ꢀ–C5ꢀ bond.
An N-conformation of the nucleoside would lead to un-
favourable effects during base pairing; the conformation was
also studied in aqueous solution with the aid of the PSEUROT
program (version 6.3).38 A minimization of the differences between
the experimental and calculation coupling is accomplished by a
nonlinear Newton–Raphson minimization, the quality of the fit
being expressed by the root-mean-square (rms) difference. This
procedure presupposes the existence of a two state N/S equi-
librium. The input contained the following coupling constants:
3J(H1ꢀ,H2ꢀ), J(H1ꢀ,H2ꢀꢀ), J(H2ꢀ,H3ꢀ), J(H2ꢀꢀ,H3ꢀ), J(H3ꢀ,H4ꢀ).
During the interactions either the puckering parameters (P and
W) of the minor conformer (N) or the puckering amplitudes of
both conformers were constrained. The coupling constants and
the pseudo-rotational parameters are shown in Table 1. The sugar
Fig. 2 Single crystal X-ray structure of 2ꢀ-deoxyimmunosine.
moiety of compound 4 shows an S-confomer population of 73%,
which is in line with that of other 2ꢀ-deoxyribonucleosides.
As nothing is known about the 13C-NMR chemical shift
assignment of 7-thia-8-oxoguanine nucleosides, we assigned all
signals on the basis of gated-decoupled 1H/13C-NMR spectra
as well as DEPT-135 spectra. The gated-decoupled spectrum
of C8 and C4 of 2ꢀ-deoxyimmunosine (4) shows three bond
3
3
3
3
3
couplings with the anomeric proton of C1ꢀ with J(C8,H1ꢀ) =
6.9 Hz and 3J(C4,H1ꢀ) = 2.6 Hz. The anomeric compound
Table 2 1H-NMR chemical shifts (d) of 5-aminothiazolo[4,5-d]pyrimidine derivativesa
Cpd
NH
NH2
N
H1ꢀ
5ꢀ-OH
3ꢀ-OH
H3ꢀ
H4ꢀ
H5ꢀ
H2ꢀb
H2ꢀa
N-CH3
=
CH
4 (b)
11.22
11.24
11.93
11.92
11.90
11.84
11.97
11.89
11.82
6.92
6.89
—
—
—
—
—
—
—
—
—
6.24
6.11
6.36
6.21
6.46
6.33
6.50
6.34
6.44
5.17
5.17
5.23
5.47
—
—
—
4.66
4.68
4.68
4.69
5.27
5.48
—
4.31
3.58
4.31
3.61
4.32
4.32
5.78
4.33
4.30
3.70
3.36
3.69
3.40
3.86
4.15
4.50
3.69
3.86
3.53
4.07
3.58
4.15
3.72
3.22
4.50
3.48
3.85
2.90
2.74
2.88
2.85
2.78
2.72
3.40
2.91
2.81
1.98
2.40
2.06
2.50
2.15
2.56
2.57
2.06
2.16
9 (a)
13 (b)
14 (a)
15 (b)
16 (a)
19 (b)
20 (b)
21 (b)
8.65
8.92
8.64
8.78
8.68
8.62
8.60
3.16, 3.05
3.03, 2.90
2.38, 2.34
5.21
—
4.67
5.22
a Measured in DMSO-d6 at 25 ◦C.
Table 3 13C-NMR chemical shifts (d) of 5-aminothiazolo[4,5-d]pyrimidine derivativesa
C2b
C5c
C4b
C5b
C6b
C7c
C8b
C2c
C3ac
C7ac
C1ꢀ
C2ꢀ
C3ꢀ
C4ꢀ
C5ꢀ
N
CH
=
Cpd
1
155.5
155.6
155.4
155.7
157.2
159.4
159.4
159.3
159.3
159.6
159.4
159.4
159.5
154.3
154.3
154.1
156.5
154.1
152.9
153.1
152.9
152.9
154.1
152.7
152.9
152.9
85.8
85.8
85.8
87.6
92.1
90.6
90.6
90.6
90.6
92.1
90.9
90.7
90.8
156.5
156.7
156.5
157.2
159.6
157.3
157.4
157.4
157.4
157.2
157.4
157.4
157.4
169.2
169.1
169.4
171.2
169.9
168.7
169.4
168.7
169.3
169.8
169.2
168.9
168.9
88.8
83.0
81.9
—
70.4
35.2
35.6
—
69.6
71.1
70.1
—
84.8
87.5
85.6
—
62.1
62.2
61.0
—
—
—
—
—
4 (b)
9 (a)
5
10
—
—
—
—
—
—
13 (b)
14 (a)
15 (b)
16 (a)
18
82.5
82.2
82.5
82.5
—
82.1
82.4
82.5
36.0
35.9
36.8
35.9
—
33.8
36.1
36.9
70.6
70.3
70.8
71.2
—
74.0
70.6
70.9
87.3
86.0
85.3
85.4
—
82.1
87.3
85.3
61.9
61.4
64.5
64.6
—
63.5
61.9
64.5
158.9
159.5
—
—
—
158.5
—
—
19 (b)
20 (b)
21 (b)
a Measured in DMSO-d6 at 25 ◦C. b Purine numbering. c Systematic numbering.
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Table 4 JC,H values (in Hz) of 5-aminothiazolo[4,5-d]pyrimidine
Table 5 UV maxima and extinction coefficients of compound 4 and
derivativesa
related derivativesa
4
9
13
14
Compound
kmax/nm
e
C8,H1ꢀ
C4,H1ꢀ
C1ꢀ,H1ꢀ
C1ꢀ,H2ꢀ
C2ꢀ,H2ꢀ
C3ꢀ,H3ꢀ
C4ꢀ,H4ꢀ
C5ꢀ,H5ꢀ
6.9
2.6
165.5
—
132.8
149.9
146.2
139.5
6.1
2.8
165.6
6.8
133.4
143.7
146.6
138.5
6.5
3.2
165.7
—
132.7
147.8
145.2
140.0
4.4
4.4
166.4
6.8
135.3
142.7
147.1
138.6
4
9
247
302
248
302
255
259
246
293
8300
9100
8300
9200
12 900
13 400
12 600
9700
dG43
3b (c7dG)44
8-oxodG42
a Measured in DMSO-d6 at 25 ◦C; purine numbering used.
a Measured in H2O.
9 shows similar values (Table 4). The C5 signal appears as a
singlet with d = 85.8 ppm. The signals C2 and C6 could not
be assigned unambiguously. Thus, the deuterium isotope upfield
shift approach was used39,40 to assign the carbon-2 signal, which is
directly connected with the amino group. In DMSO solution in the
presence of a H2O–D2O mixture it shows two singlets, the original
one and one which is shifted 50 ppb upfield. Consequently, C2 was
assigned to the 155.6 ppm signal and C6 to the 156.7 ppm signal.
For the sugar portion of compound 4, the following coupling
coupling yields of the phosphoramidites were always higher than
95%. The oligonucleotides were deprotected by incubation with
a 25% aqueous NH3 solution at room temperature for 24 h. The
oligonucleotides were purified before and after detritylation by
reverse-phase HPLC.
As the thiazole system is prone to degradation, harsh alkaline
condition had to be avoided. Ammonia treatment at 60 ◦C led
to the formation of side products and gave very little of the
target oligonucleotide. Thus, the stability of the nucleoside 4 was
studied in aqueous ammonia. As shown in Fig. 4a, compound 4
is partially degraded under standard oligonucleotide deprotection
conditions (25% aqueous NH3, 60 ◦C, 16 h). However, at room
constants were observed: J(C1ꢀ,H1ꢀ) = 165.5 Hz, J(C3ꢀ,H3ꢀ) =
149.9 Hz and 1J(C4ꢀ,H4ꢀ) = 146.2 Hz. The large coupling constant
of C1ꢀ is indicative of the anomeric carbon.41 The coupling
constants of 1J(C1ꢀ,H1ꢀ), 1J(C2ꢀ,H2ꢀ), 1J(C4ꢀ,H4ꢀ) and 1J(C5ꢀ,H5ꢀ)
of the a-D compound 9 are very similar to the b-D compound 4,
except for 1J(C3ꢀ,H3ꢀ), which is 6 Hz less than that of compound 4
(Table 4). The largest difference between the aand b compounds is
that the a-D compounds 9 and 14 show 3J(C1ꢀ,H2ꢀ) = 6.8 Hz, while
the b-D compounds 4 and 13 do not show this coupling (Table 4).
This is indicative of the anomeric assignment. The signals of C5ꢀ
and C2ꢀ are triplets. For compounds 15, 16 and 21 with DMT-
residues on the 5ꢀ-OH, the chemical shifts show characteristic
values from about 62.0 ppm to 64.5 ppm, while the C4ꢀ shift varies
from ∼87.3 ppm to 85.3 ppm.
1
1
The UV data of compound 4 and other dG derivatives are
summarized in Table 5. The replacement of nitrogen-7 by sulfur
changes the UV-spectrum significantly (Table 5 and Fig. 3). The
UV maxima of compound 4 show a strong red shift compared
to that with a nitrogen at the identical position: 8-oxo-2ꢀ-
deoxyguanosine (oxodG) (kmax = 246 nm, 293 nm)42 vs. 4 (kmax
=
246 nm, 302 nm). Compound 4 also shows a lower pKa value of
deprotonation compared to 2ꢀ-deoxyguanosine or its derivatives:
4 (pKa = 8.2), oxodG (pKa = 8.6),42 dG (pKa = 9.4)43 and c7dG
(pKa = 10.2).44
3. Synthesis and base pairing of oligonucleotides containing
2ꢀ-deoxyimmunosine (4)
3.1 Oligonucleotide synthesis and characterization. Solid-
phase oligonucleotide synthesis was performed employing the
phosphoramidites 17 or 22 protected with a dialkylaminoalkyli-
dene protecting group on an ABI 392-08 automated DNA
synthesizer with controlled pored glass (CPG-500) serving as
solid phase. Base labile (tert-butylphenoxy)acetyl (tac) groups
were chosen for amino protection in the cases of dA, dG and
dC. The syntheses followed the standard protocol.45 The coupling
time for the phosphoramidite 17 or 22 was 10 minutes and the
Fig. 3 UV spectra of (a) 2ꢀ-deoxyimmunosine (4) and (b) 7-deaza-
2ꢀ-deoxyguanosine (3b) in water.
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Fig. 5 Reverse-phase HPLC profiles (column 250 × 4 mm, RP-18) of
the hydrolysis mixture of 4 in 0.5 M HCl at room temperature after (a)
50 min, (b) 19 h. Buffer: 5% MeCN in 0.1 M (Et3NH)OAc, pH = 7.0,
1.0 mL min−1. The profile was measured at 302 nm.
Fig. 4 Reverse-phase HPLC profiles (column 250 × 4 mm, RP-18) of
4 after treatment with 25% aqueous NH3: (a) at 60 ◦C for 16 h, (b) at
room temperature for 24 h. HPLC elution buffer: 5% MeCN in 0.1 M
(Et3NH)OAc, pH = 7.0, 1.0 mL min−1. The profile was measured at 302 nm.
oligonucleotides containing 2ꢀ-deoxyimmunosine 4 was studied.
Two oligonucleotides, 5ꢀ-d(TAG GTC AAT ACT) (36) and 3ꢀ-
d(ATC CAG TTA TGA) (27) were used as reference compounds.46
Nucleoside 4 was used to replace dG residues to form the
new oligonucleotides 43, 44, 46 and 47. The self-complementary
oligonucleotide 45 leading to six modifications in the duplex
structure was also synthesized. The oligonucleotides were char-
acterized by MALDI-TOF mass spectra; the masses were in good
agreement with the calculated values (Table 7 in the experimental
part). The oligonucleotides 45 and 47 were hydrolyzed with snake-
venom phosphodiesterase followed by alkaline phosphatase47 to
yield the free nucleosides, and the digest was separated by RP-18
HPLC (Fig. 6). According to the HPLC profiles of the enzymatic
degradation, the composition of oligonucleotides was proved.
In the first series of experiments, the nucleoside 4 was used as
an analogue of dG. Table 6 shows the Tm values when nucleoside
4 was placed opposite to dC. The incorporation of one 4–dC
base pair resulted in no change of the Tm values (DTm = 0 ◦C)
compared to the standard duplex 36·27. Incorporation of two
or three 4–dC base pairs also resulted in no significant changes
temperature (24 h) 2ꢀ-deoxyimmunosine is rather stable, leading
to only very little degradation (Fig. 4b). In contrast, the depro-
tection of monomeric amidine-protected nucleosides 13 and 20 in
25% aqueous NH3 solution at room temperature required 72 h
for completion (see Experimental section). For oligonucleotide
deprotection in 25% aqueous NH3 at room temperature a reaction
time of 24 h was sufficient.
In order to circumvent possible problems occurring during the
acid-catalyzed detritylation in the oligonucleotide synthesis, the
stability of compound 4 was also studied under acidic conditions.
Nucleoside 4 was treated with 0.1 M HCl at room temperature
for 10 h and the reaction was followed by RP18-HPLC. After
10 h treatment, almost no change was observed (data not shown).
Then, the reaction was performed under stronger acidic conditions
(0.5 M HCl) at room temperature. While compound 4 was not
significantly hydrolyzed within 50 min (Fig. 5a), half of the educt
was degraded after 19 h by glycosylic bond hydrolysis, to form the
immunosine base 5 (Fig. 5b). For comparison, the glycosylic bond
stability was also studied for dG and 7-deaza-2ꢀ-deoxyguanosine
(c7Gd). According to the HPLC profiles (ESI†), the glycosylic
bond of 2ꢀ-deoxyimmunosine (4) is more stable than that of dG
but less stable than that of 7-deaza-2ꢀ-deoxyguanosine (c7Gd).
Thus, no degradation of 2ꢀ-deoxyimmunosine is expected during
oligonucleotide synthesis.
◦
in the stability (DTm = 1–2 C). Duplexes containing central 4–
dC pairs (47·27) or separated central 4–dC pairs (36·46) exhibited
similar duplex stabilities, with DTm = 0.5 ◦C per modification. The
complete replacement of all dG residues was performed on the self-
complementary duplex d(4–C)3, and the stability was compared
to the standard duplex d(G–C)3. Here the Tm value of duplex
melting also decreased by only 0.5 ◦C per modification. Thus, the
3.2 Base-pairing and duplex stability of oligonucleotides con-
taining 2ꢀ-deoxyimmunosine (4). Next, the base pairing of
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Table 6
T
m values and thermodynamic data of duplex melting of oligonucleotides with regular and base-modified nucleosides (4 = 2ꢀ-deoxyimmunosine).a
Duplex
No.
Tm/◦C
DTm/◦C b
DG310/kcal mol−1
5ꢀ-d(TAG GTC AAT ACT)49
3ꢀ-d(ATC CAG TTA TGA)
5ꢀ-d(TAG GTC AAT ACT)
3ꢀ-d(ATC CA4 TTA TGA)
5ꢀ-d(TAG GTC AAT ACT)
3ꢀ-d(ATC CAG TTA T4A)
5ꢀ-d(TAG GTC AAT ACT)
3ꢀ-d(ATC CA4 TTA T4A)
5ꢀ-d(TA4 4TC AAT ACT)
3ꢀ-d(ATC CAG TTA TGA)
5ꢀ-d(TA4 4TC AAT ACT)
3ꢀ-d(ATC CA4 TTA TGA)
5ꢀ-d(TA4 4TC AAT ACT)
3ꢀ-d(ATC CAG TTA T4A)
5ꢀ-d(TA4 4TC AAT ACT)
3ꢀ-d(ATC CA4 TTA T4A)
36
27
36
43
36
44
36
46
47
27
47
43
47
44
47
46
50
50
50
49
49
48
49
47
—
−11.6
−11.3
−11.7
−11.1
−11.0
−10.7
−11.0
−10.2
0
0
−0.5
−0.5
−1
−0.5
−1
5ꢀ-d(GCGCGC)50
3ꢀ-d(CGCGCG)
5ꢀ-d(4C4C4C)
46
42
—
−8.2
−8.1
45
45
−0.5
3ꢀ-d(C4C4C4)
5ꢀ-d(TAG GTA AAT ACT)
3ꢀ-d(ATC CAG TTA TGA)
5ꢀ-d(TAG GTA AAT ACT)
3ꢀ-d(ATC CA4 TTA TGA)
5ꢀ-d(TAG GTT AAT ACT)
3ꢀ-d(ATC CAG TTA TGA)
5ꢀ-d(TAG GTT AAT ACT)
3ꢀ-d(ATC CA4 TTA TGA)
5ꢀ-d(TAG GTG AAT ACT)
3ꢀ-d(ATC CAG TTA TGA)
5ꢀ-d(TAG GTGAAT ACT)
3ꢀ-d(ATC CA4 TTA TGA)
37
27
37
43
38
27
38
43
39
27
39
43
35
31
36
36
33
27
−15
−19
−14
−14
−17
−23
−7.3
−6.8
−7.7
−7.7
−7.1
−6.1
a Measured in 1 M NaCl, 100 mM MgCl2 and 60 mM Na-cacodylate (pH 7.0) with 5 lM single-strand concentration. b Refers to the contribution of the
modified residues divided by the number of replacements.
nucleoside 4 forms a strong base pair with dC, which has a similar
stability to that of dG–dC. The sulfur-containing compound
thioguanine (thioG) decreases the DNA stability significantly
when incorporated as a 2ꢀ-deoxynucleoside, resulting from spatial
requirement of the sulfur and its low H-bonding character. The
and dG). Hybridization experiments were performed; the duplex
melting data are shown in Table 6. As expected, compound 4
forms a weaker interaction with adenine, which is different from
oxodG.19,20 One incorporation opposite to dA (37·43) reduces
the Tm by −19 ◦C, opposite to dT (38·43) by −14 ◦C, and
◦
T
m of one thioG–C base pair in a non-self-complementary 13-mer
opposite to dG (39·43) by −23 C. Regarding these results, 2ꢀ-
duplex is 6 ◦C lower than that of the G–C base pair.48
deoxyimmunosine shows an excellent discrimination against all
canonical nucleosides. It does not form a Hoogsteen pair due to the
absence of a nitrogen at the 7-position (as observed for 7-deaza-2ꢀ-
deoxyguanosine23,51), and is different to 8-oxo-2ꢀ-deoxyguanosine,
which forms a rather stable base pair with dA.19,20 Compound
4 shows a better discrimination than dG when incorporated
opposite to dA. Although compound 4 forms a syn conformation
in the solid state, the conformation within the B-DNA duplex has
to be anti. From the high stability of the 4–dC base pair, it can
be concluded that due to solid-state forces, no conclusion can be
drawn regarding the base pairing from the single crystal X-ray
structure of a nucleoside, as has been discussed for 8-substituted
purine nucleosides such as 8-bromo-2ꢀ-deoxyguanosine.52
It is reported that 8-oxodG can pair with dA in the syn
conformation thereby forming a Hoogsteen base pair.20 Thus, it
was of interest to investigate the base-pairing properties of 2ꢀ-
deoxyimmunosine with the other canonical nucleosides (dA, dT
Table 7 Molecular masses of oligonucleotides determined by MALDI-
TOF mass spectrometrya
Oligonucleotides
[M]+ (calc.)
[M]+ (found)
5ꢀ-d(AGT ATT 4AC CTA) (43)
5ꢀ-d(A4T ATT GAC CTA) (44)
5ꢀ-d(4C4C4C) (45)
3677
3677
1892
3709
3709
3677
3677
1892
3709
3709
5ꢀ-d(A4T ATT 4AC CTA) (46)
5ꢀ-d(TA4 4TC AAT ACT) (47)
Fig. 8 displays the three-dimensional models of 8-oxo-7-
thiaguanine, the nucleobase of 2ꢀ-deoxyimmunosine (4), the re-
lated 7-deazaguanine as well as 8-oxoguanine and guanine. From
Fig. 8, it is obvious that the spatial requirements of the nucleobases
a 4 = 2ꢀ-deoxyimmunosine.
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Fig. 7 Base pair motifs of 2ꢀ-deoxyimmunosine (4) and 8-oxoGd with dC
or dA.
Fig. 6 RP-18 HPLC-elution profile of the enzymatic digests of the
oligonucleotides (a) 5ꢀ-d(4C4 C4C) (45) and (b) 5ꢀ-d(TA4 4TC AAT ACT)
(47). Buffer: 5% MeCN in 0.1 M (Et3NH)OAc, pH = 7.0, 1.0 mL min−1
.
The profile was measured at 247 nm.
are different. The space-demanding sulfur atom within the five-
membered ring of 4 (Fig. 8A) shows a similar size to the hydrogen-
bearing 7-carbon in 7-deazaguanine (Fig. 8B). The latter fits
nicely in the B-DNA duplex and is well accepted in the form
of a nucleoside triphosphate by many DNA-polymerases.53 Also,
the 8-oxo group of compound 4 (base pair motif I in Fig. 7)
is well accommodated in a duplex structure, as is demonstrated
by the stable base pairs formed with dC. The corresponding 8-
oxoguanine (Fig. 8C, base pair motif II in Fig. 7) has a slightly
negative effect on the base pair stability compared to that of
compound 4.19,20 These observations are in line with findings by
our laboratory and by others that 7-substituents of moderate size
located within the major groove of B-DNA have no or only a
little influence on the duplex stability compared to modifications
occurring in the minor groove.54 The better base discrimination
of 2ꢀ-deoxyimmunosine compared to 8-oxo-2ꢀ-deoxyguanosine
results from the absence of a 7-hydrogen. This 7-hydrogen can
act as hydrogen bond donator while 2ꢀ-deoxyimmunosine cannot.
The 8-oxo groups have no noticeable negative effect on the duplex
stability, as is demonstrated for oligonucleotides containing the
immunosine base or 8-oxoguanine. Moreover, the 8-oxo group
can act as hydrogen bond acceptor, and binds water molecules or
other ligands in the major groove of DNA.
Fig. 8 Molecular models of (A) the immunosine base, (B) 7-deazagua-
nine, (C) 8-oxoguanine and (D) guanine.
Conclusion
The stereoselective and regioselective nucleobase anion glyco-
sylation of 5-amino-thiazolo[4,5-d]pyrimidine-2,7(3H,6H)-dione
has many difficulties because of its bad solubility and the
presence of two “lactam functions” which are both prone to
glycosylation. Amino group protection with a lipophilic amidine
residue resulted in the base derivatives 10 and 18, which could
be glycosylated with 1-chloro-2-deoxy-3,5-di-O-(p-toluoyl)-a-D-
erythro-pentofuranose (6) in a regioselective and stereoselective
way. This selectivity results from the more acidic (and nucleophilic)
character of the five-membered ring lactam and the bulky protect-
ing groups of the nucleobase derivative 18. Compared to dG, the
nucleoside 4 is more labile under alkaline conditions but more
stable in an acidic medium. Compound 4 has the same capability
to form a strong base pair with dC and no base pair with dA;
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it cannot form “dG” quartets. The molecular shape models of
the 2ꢀ-deoxyimmunosine base and related 7-modified derivatives
are shown in Fig. 8. Compared to guanine and 7-deazaguanine,
the sulfur atom of the immunosine nucleobase is more space
demanding. However, the spatial requirements are similar to that
of 8-oxoguanine.
5-{[(Di-n-butylamino)methylidene]amino}thiazolo[4,5-d]pyri-
midine-2,7(3H,6H)-dione (18). Compound 5 (1.00 g, 5.43 mmol)
was treated with N,N-dimethyllformamide dibutyl acetal30
(4.0 cm3) in the same way as described for 10 at 40 ◦C. The reaction
mixture was evaporated to dryness. The residue was purified by
FC (silica gel, column, 15 × 5.5 cm, CH2Cl2–CH3OH 100 : 1)
to yield compound 18 (1.32 g, 75%) as colorless solid (Found:
C, 52.11; H, 6.54; N, 21.69. C14H21N5O2S requires C, 51.99; H,
6.54; N, 21.65%); TLC (silica gel, CH2Cl2–CH3OH 40 : 1): Rf
0.16; UV/Vis: kmax(MeOH)/nm: 319 (e/dm3 mol−1 cm−1 23 600),
290 (16 700), 249 (16 000); dH (250 MHz; DMSO-d6; Me4Si) 12.16
Oligonucleotides containing 4 in place of dG within GpC
motives have the potential to act as immunostimulatory agents.
Experimental
=
(1H, s, H–N(3), 11.71 (1H, s, H–N(6)), 8.49 (1H, s, N CH–C(2)),
General
3.49–3.35 (4H, m, 2 × NCH2), 1.54 (4H, m, 2 × CH2), 1.27 (4H,
m, 2 × CH2) and 0.90 (6H, t, J 7.0, 2 × CH3).
All chemicals were purchased from Acros, or Sigma-Aldrich
(Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). Solvents
were of laboratory grade. TLC: Aluminium sheets, silica gel 60
F254 (0.2 mm, VWR International, Darmstadt, Germany). Flash
column chromatography (FC): silica gel 60 (VWR International,
Darmstadt, Germany) at 0.4 bar. UV Spectra: U-3200 UV-vis
spectrometer (Hitachi, Japan). Reverse-phase HPLC was carried
out on a 250 × 10 mm RP-18 LiChrosorb column (Merck) with
a Merck-Hitachi HPLC pump (model L-7100) connected with
a variable wavelength monitor (model L-7400). NMR spectra:
Avance-DPX-250, Avance-DPX-300 spectrometer (Bruker, Rhe-
instetten, Germany); d values in ppm relative to Me4Si as internal
standard (1H and 13C) or external 85% H3PO4 (31P), J values in Hz.
Melting points were determined by a Linstro¨m apparatus and are
notcorrected. Element analyses were performed byMikroanalytis-
ches Laboratorium Beller, Go¨ttingen, Germany. MALDI-TOF
mass spectra were recorded with an Applied Biosystems Voyager
DE PRO spectrometer with 3-hydroxypicolinic acid (3-HPA) as a
matrix.
Glycosylation of 5-aminothiazolo[4,5-d]pyrimidine-2,7(3H,6H)-
dione with 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-a-D-erythro-pento-
furanose (6). To a solution of 5 (300 mg, 1.63 mmol) in
anhydrous DMF (10 cm3), NaH (60% suspended in oil, 75 mg,
1.87 mmol) was added under stirring. Stirring was continued at r.t.
for 15 min; then 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-a-D-erythro-
pentofuranose (6)55 (951 mg, 2.45 mmol) was added, and stirring
was continued for 30 min. The mixture was filtered; the filtrate
was evaporated to dryness and the residue was subjected to FC
(silica gel, column 10 × 5.5 cm, CH2Cl2–MeOH 100 : 1) to yield
an anomeric mixture of 7/8 (254 mg, 29%) as yellowish foam of
5-amino-3-[2-deoxy-3,5-di-O-(p-toluoyl)-D-erythro-pentofuran-
osyl]thiazolo[4,5-d]pyrimidine-2,7(3H,6H)-diones (7/8): TLC
(silica gel, CH2Cl2–MeOH 20 : 1, one spot): Rf = 0.20; HPLC:
two peaks, the ratio is 2 : 1.
Glycosylation of 10 with 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-a-
D-erythro-pentofuranose (6). To the solution of compound 10
(500 mg, 2.09 mmol) in dry CH3CN (200 cm3), NaH (60%
suspended in oil, 92 mg, 2.30 mmol) was added under stirring
at r.t. Stirring was continued at r.t. for 15 min, then 1-chloro-
2-deoxy-3,5-di-O-p-toluoyl-a-D-erythro-pentofuranose (894 mg,
2.30 mmol) was added in portions within 20 min. The stirring
was continued for another 30 min. Insoluble material was filtered
off and the filtrate was evaporated to dryness. The residue was
applied to a FC (silica gel, column 10 × 5.5 cm, CH2Cl2–CH3OH
100 : 1) to yield an anomeric mixture of 11/12 as yellowish solid
(680 mg, 55%).
Melting curves were measured with a Cary-100 Bio UV/Vis
spectrophotometer (Varian, Australia) equipped with a Cary
thermo-electrical controller. The calculation of thermodynamic
data was performed with the program MeltWin (version 3.0) using
the curve fitting of the melting profiles according to a two-state
model.
5-Aminothiazolo[4,5-d]pyrimidine-2,7(3H,6H)-dione (5). Com-
pound 5 was prepared as described by Baker and Chatfield.24,25
dH (250 MHz; DMSO-d6; Me4Si) 12.09 (1H, s, NH), 10.99 (1H, s,
NH) and 6.77 (2H, s, NH2).
5-{[(Dimethylamino)methylidene]amino}thiazolo[4,5-d ]pyri-
3-[2-Deoxy-3,5-di-O-(p-toluoyl)-D-erythro-pentofuranosyl]-5-
{[(dimethylamino)methylidene]amino}thiazolo[4,5-d]pyrimidine-2,
7(3H,6H)-dione (11/12). TLC (silica gel, CH2Cl2–MeOH 40 :
1, one spot): Rf 0.22; HPLC: two peaks, the ratio is 2 : 1. dH
(250 MHz; DMSO-d6; Me4Si) taken from the mixture: Isomer I:
8.69 (NH) and 5.79 (q, J 5.7 Hz, H–C(3ꢀ)); Isomer II: 8.64 (NH)
and 5.51 (q, J 7.7, H–C(3ꢀ)).
midine-2,7(3H,6H)-dione (10). To
a
suspension of 5-
aminothiazolo[4,5-d]pyrimidine-2,7(3H,6H)-dione (5) (1.0 g,
5.43 mmol) in MeOH (30 cm3), N,N-dimethylformamide
dimethyl acetal (6.0 cm3, 44.9 mmol) was added and stirred at
r.t. for 4 h, resulting in a clear solution. The reaction mixture
was adsorbed onto silica gel 60 (2.0 g), and applied on the top
of a silica gel column (10 × 5.5 cm). Elution with CH2Cl2–
MeOH–triethylamine (20 : 1 : 0.2) yielded compound 10 (0.91 g,
70%) as a yellowish solid (Found: C, 40.06; H, 3.35; N, 29.11.
C8H9N5O2S requires C, 40.16; H, 3.79; N, 29.27%); TLC (silica
gel, CH2Cl2–MeOH 20 : 1) Rf 0.31; UV/Vis: kmax(MeOH)/nm
317 (e/dm3 mol−1 cm−1 19 100), 289 (sh, 14 600), 246 (14 800) and
212 (17 200); dH (250 MHz; DMSO-d6; Me4Si) 12.19 (1H, s, NH),
3-(2-Deoxy-D-erythro-pentofuranosyl)-5-{[(dimethylamino)-
methylidene]amino}thiazolo[4,5-d]pyrimidine-2,7(3H,6H)-dione
(13/14). The anomeric mixture of 11/12 (1.2 g, 2.03 mmol) was
dissolved in 0.1 M NaOCH3/MeOH (40 cm3) and was stirred at
r.t. for 2 h. The reaction mixture was adsorbed onto silica gel 60
(1.6 g), and applied on the top of a silica gel column (10 × 5.5 cm).
Elution with CH2Cl2–MeOH 10 : 1 afforded a mixture of 13/14 as
a colorless solid (592 mg, 82%). TLC (silica gel, CH2Cl2–CH3OH
=
11.75 (1H, s, NH), 8.50 (1H, s, N CH), 3.15 (3H, s, NCH3) and
3.04 (3H, s, NCH3).
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10 : 1): Rf 0.26; dH (250 MHz; DMSO-d6; Me4Si) taken from the
OH–C(5ꢀ)), 4.68 (1H, t, J 5.7, OH–C(3ꢀ)), 4.31 (1H, m, H–C(3ꢀ)),
3.69 (1H, m, H–C(4ꢀ)), 3.58, 3.62–3.39 (2H, m, H–C(5ꢀ)), 3.20
(3H, s, H–NCH3), 3.06 (3H, s, H–NCH3), 2.88 (1H, m, H–C(2ꢀ))
and 2.06 (1H, m, H–C(2ꢀ)).
=
mixture: Isomer I: 11.93 (s, NH), 8.65 (s, H–N CH) and 6.36 (t,
ꢀ
=
J 6.9, H–C(1 )); Isomer II: 11.92 (s, NH), 8.92 (s, H–N CH) and
6.21 (t, J 7.6, H–C(1ꢀ)).
Dimethoxytritylation of the anomeric mixture 13/14. The mix-
ture of nucleosides 13/14 (500 mg, 1.41 mmol) was co-evaporated
with pyridine (3 × 10 cm3) and then dissolved in pyridine (20 cm3).
The solution was treated with 4,4ꢀ-dimethoxytriphenylmethyl
chloride (713 mg, 2.11 mmol) at room temperature for 1 h, MeOH
(3 cm3) was added, and stirring was continued for 10 minutes.
The solution was concentrated to half of the volume, and
CH2Cl2 (70 cm3) was added. The organic layer was washed with
aqueous NaHCO3 (5%, 50 cm3), dried over Na2SO4, filtered and
evaporated, and the residue was applied to FC (silica gel, column
20 × 5 cm, CH2Cl2–acetone 4 : 1).
3-[2-Deoxy-5-O-(4,4ꢀ -dimethoxytriphenylmethyl)-b-D-erythro-
pentofuranosyl]-5-{[(dimethylamino)methylidene]amino}thiazolo-
[4,5-d]pyrimidine-2,7(3H,6H)-dione (15). From the slower mi-
grating zone, compound 15 was isolated (497 mg, 54%) as a
colorless foam (Found: C, 62.20; H, 5.30; N, 10.46. C34H35N5O7S
requires C, 62.09; H, 5.36; N, 10.65%); TLC (silica gel,
CH2Cl2–acetone 4 : 1): Rf 0.22; UV/Vis: kmax (MeOH)/nm 319
(e/dm3 mol−1 cm−1 18 800), 282 (15 000) and 235 (28 800); dH
3-(2-Deoxy-a-D-erythro-pentofuranosyl)-5-{[(dimethylamino)-
methylidene]amino}thiazolo[4,5-d]pyrimidine-2,7(3H,6H)-dione
(14). Compound 16 (810 mg, 1.23 mmol) was treated as de-
scribed for compound 15. Purification by FC (silica gel, column
10 × 5 cm, CH2Cl2–CH3OH 10 : 1) gave a colorless solid. Crys-
tallization from EtOH afforded 14 (319 mg, 73%) as a colorless
solid (Found: C, 43.79; H, 4.85; N, 19.60. C13H17N5O5S requires
C, 43.94; H, 4.82; N, 19.71%); TLC (silica gel, CH2Cl2–CH3OH
10 : 1): Rf 0.26; UV/Vis: kmax (MeOH)/nm 319 (e/dm3 mol−1 cm−1
20 000), 291 (sh, 14 600), 247 (14 100) and 216 nm (16 700); dH
(250 MHz; DMSO-d6; Me4Si): 11.92 (1H, s, NH), 8.92 (1H, s,
ꢀ
=
N CH), 6.21 (1H, t, J 7.5, H–C(1 )), 5.47 (1H, d, J 4.0, OH–
C(5ꢀ)), 4.69 (1H, t, J 5.44, OH–C(3ꢀ)), 4.15 (2H, m, H–C(5ꢀ)), 3.61
(1H, m, H–C(3ꢀ)), 3.40 (1H, m, H–C(4ꢀ)), 3.17 (3H, s, NCH3),
3.06 (3H, s, NCH3), 2.88–2.83 (1H, m, H–C(2ꢀ)-b) and 2.50 (H, m,
H–C(2ꢀ)-a).
5-Amino-3-(2-deoxy-b-D-erythro-pentofuranosyl)thiazolo[4,5-
d]pyrimidine-2,7-(3H,6H)-dione (4). A solution of compound
13 (300 mg, 0.84 mmol) in saturated NH3–CH3OH (CH3OH
◦
(250 MHz; DMSO-d6; Me4Si) 11.90 (1H, s, H–N), 8.64 (1H, s,
saturated with NH3 at 0 C, 50 cm3) was stirred at room
ꢀ
=
N CH), 7.37–6.78 (15H, m, arom. H), 6.46 (1H, m, H–C(1 )),
temperature for 72 h in a sealed bottle. The reaction mixture was
adsorbed onto silica gel 60 (1.0 g), and applied to the top of a
silica gel column (10 × 5 cm). Elution with CH2Cl2–CH3OH 6 :
1 afforded 4 (229 mg, 91%) as colorless solid (Found: C, 39.94;
H, 4.00; N, 18.70. requires C10H12N4O5S C, 40.00; H, 4.03; N,
18.66%); Crystallization from EtOH afforded colorless crystals;
m.p. 178◦C (decomp.); TLC (silica gel, CH2Cl2–MeOH 6 : 1): Rf
0.23; UV/Vis: kmax (MeOH)/nm 302 (e/dm3 mol−1 cm−1 8800), 246
(8500) and 216 (25 900); dH (250 MHz; DMSO-d6; Me4Si) 11.22
(s, 1H, NH), 6.92 (s, 2H, NH2), 6.24 (1H, t, J 7.1, H–C(1ꢀ)), 5.17
(1H, d, J 3.6, OH–C(5ꢀ)), 4.66 (1H, m, OH–C(3ꢀ)), 4.31 (1H, d, J
2.3, H–C(3ꢀ)), 3.70 (1H, m, H–C(4ꢀ)), 3.53 (2H, m, H–C(5ꢀ)), 2.90
(1H, m, H–C(2ꢀ)) and 1.98 (m, 1H, H–C(2ꢀ)).
5.27 (1H, d, J 4.7, OH–C(3ꢀ), 4.32 (1H, m, H–C(3ꢀ)), 3.86 (1H, m,
H–C(4ꢀ)), 3.72 (8H, m, H–C(5ꢀ), 2 × OCH3), 3.16 (3H, s, N–CH3),
3.05 (3H, s, N–CH3), 2.78 (1H, m, H–C(2ꢀ)-b) and 2.15 (m, 1H,
H–C(2ꢀ)-a).
3-(2-Deoxy-5-O-(4,4ꢀ -dimethoxytriphenylmethyl)-a-D-erythro-
pentofuranosyl)-5-{[(dimethylamino)methylidene]amino}thiazolo-
[4,5-d]pyrimidine-2,7(3H,6H)-dione (16). The faster migrating
zone yielded compound 16 (253 mg, 26%) as a colorless foam
(Found: C, 62.23; H, 5.29; N, 10.47. C34H35N5O7S requires C,
62.09; H, 5.36; N, 10.65%); TLC (silica gel, CH2Cl2–acetone 4 : 1):
Rf 0.38; UV/Vis: kmax(MeOH)/nm 319 (e/dm3 mol−1 cm−1 18 100),
283 (13 700) and 235 (26 900); dH (250 MHz; DMSO-d6; Me4Si)
=
11.84 (1H, s, H–N), 8.78 (1H, s, N CH), 7.41–6.86 (15H, m, arom.
5-Amino-3-(2-deoxy-a-D-erythro-pentofuranosyl)thiazolo[4,5-
d]pyrimidine-2,7-(3H,6H)-dione (9). Compound 14 (300 mg,
0.85 mmol) was treated as described for 4. FC (silica gel, column
10 × 5 cm, CH2Cl2–CH3OH 6 : 1) afforded compound 9 (225 mg,
88%) as a white solid (Found: C, 39.92; H, 3.93; N, 18.49.
requires C10H12N4O5S C, 40.00, H, 4.03, N, 18.66%); TLC (silica
gel, CH2Cl2–MeOH 6 : 1): Rf 0.23; UV/Vis kmax(MeOH)/nm
302 (e/dm3 mol−1 cm−1 9600), 247 (9100) and 217 (28 100); dH
(250 MHz; DMSO-d6; Me4Si) 11.24 (1H, s, H–N), 6.89 (2H, s,
NH2), 6.11 (1H, t, J 7.4, H–C(1ꢀ)), 5.17 (1H, d, J 4.8 Hz, OH–
C(5ꢀ)), 4.68 (1H, t, J 5.1 Hz, OH–C(3ꢀ)), 4.07 (2H, m, H–C(5ꢀ)),
3.58 (1H, m, H–C(3ꢀ)), 3.36 (1H, m, H–C(4ꢀ)), 2.74 (1H, m, H–
C(2ꢀ)) and 2.46–2.35 (m, 1H, H–C(2ꢀ)).
H), 6.33 (1H, t, J 7.5, H–C(1ꢀ)), 5.48 (1H, d, J 5.0, OH–C(3ꢀ), 4.32
(1H, m, H–C(3ꢀ)), 4.15 (1H, m, H–C(4ꢀ), 3.72 (6H, s, 2 × OCH3),
3.22 (2H, m, H–C(5ꢀ), 3.03 (3H, s, N–CH3), 2.90 (3H, s, N–CH3),
2.72 (1H, m, H–C(2ꢀ)-b) and 2.56 (1H, m, H–C(2ꢀ)-a).
3-(2-Deoxy-b-D-erythro-pentofuranosyl)-5-{[(dimethylamino)-
methylidene]amino}thiazolo[4,5-d]pyrimidine-2,7(3H,6H)-dione
(13). Compound 15 (320 mg, 0.49 mmol) was dissolved in 1%
trichloroacetic acid in CH2Cl2 (40 cm3). The solution was stirred
at r.t. for 20 min, and then neutralized with triethylamine. The
reaction mixture was adsorbed onto silica gel 60 (1.0 g), and
applied to the top of a silica gel column (10 × 5.5 cm). Elution
with CH2Cl2–CH3OH (10 : 1) gave a white solid. Crystallization
from EtOH afforded compound 13 (127 mg, 73%) as colorless
needles (Found: C, 43.90; H, 4.9◦0; N, 19.66. C13H17N5O5S C, 43.94;
H, 4.82, N, 19.71%); m.p. 196 C (from EtOH); TLC (silica gel,
CH2Cl2–CH3OH 10 : 1): Rf 0.26; UV/Vis: kmax (MeOH)/nm 318
(e/dm3 mol−1 cm−1 19 300), 290 (sh, 13 900), 246 (13 300) and 215
3-[2-Deoxy-5-O-(4,4ꢀ -dimethoxytriphenylmethyl)-b-D-erythro-
pentofuranosyl]-5-{[(dimethylamino)methylidene]amino}thiazolo-
[4,5-d]pyrimidine-2,7(3H,6H )-dione 3ꢀ -(2-cyanoethyl)diisopro-
pylphosphoramidite (17). To the solution of compound 15
(350 mg, 0.53 mmol) in dry CH2Cl2 (10 cm3), iPr2EtN (0.17 cm3,
0.80 mmol) and (2-cyanoethyl)diisopropylphosphoramido chlo-
ridite (0.26 cm3, 1.17 mmol) were added. The solution was stirred
(17 100); dH (250 MHz; DMSO-d6; Me4Si) 11.93 (1H, s, NH), 8.65
(1H, s, N CH), 6.36 (1H, t, J 6.9, H–C(1 )), 5.23 (1H, d, J 4.6,
ꢀ
=
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at r.t. for 10 min. The solution was diluted with CH2Cl2 (40 cm3)
and washed with 5% aq. NaHCO3 solution. The mixture was
extracted with CH2Cl2 (2 × 20 cm3), and the combined organic
phase dried (Na2SO4), filtered, and evaporated. The residue was
purified by FC (column 15 × 5 cm, CH2Cl2–acetone 9 : 1) to afford
a colorless foam. This was dissolved in CH2Cl2 (3 cm3) and added
gradually to stirring cyclohexane (80 cm3) cooled to −30 ◦C. The
precipitate was isolated by filtration and the powder was dried
under vacuum, yielding 17 as a colorless foam (360 mg, 80%).
TLC (silica gel, CH2Cl2–acetone 4 : 1): Rf 0.32; dP (250 MHz;
CDCl3; H3PO4): 150.0, 149.8 ppm.
top of a silica gel column (8 × 5 cm). Elution with CH2Cl2–CH3OH
(5 : 1) afforded a colorless solid. Crystallization from EtOH
afforded colorless crystals of 4 (132 mg, 76%). Analytical data
were the same as that for compound 4 obtained from compound
13.
3-(2-Deoxy-5-O-(4,4ꢀ -dimethoxytriphenylmethyl)-b-D-erythro-
pentofuranosyl)-5-{[(di-n-butylamino)methylidene]amino}thia-
zolo[4,5-d]pyrimidine-2,7(3H,6H)-dione (21). Compound 20
(220 mg, 0.50 mmol) was co-evaporated with pyridine (3 × 10 cm3)
and then dissolved in pyridine (15 cm3). This solution was treated
with 4,4ꢀ-dimethoxytriphenylmethyl chloride (270 mg, 0.79 mmol)
at room temperature for 1 hour, MeOH (3 cm3) was added, and
stirring continued for 10 minutes. The solution was concentrated
to half of the volume, and CH2Cl2 (50 cm3) was added. The organic
layer was washed with aqueous NaHCO3 (5%, 50 cm3), and the
organic phase was dried over Na2SO4, filtered and evaporated. The
residue was purified by FC (column 20 × 5 cm, CH2Cl2–acetone
20 : 1), yielding 21 (220 mg, 60%) as a white foam (Found: C,
64.28; H, 6.32; N, 9.40. C40H47N5O7S requires C 64.76, H 6.39, N
9.44%); TLC (silica gel, CH2Cl2–acetone 4 : 1): Rf 0.38; UV/Vis:
kmax(MeOH)/nm 322 (e/dm3 mol−1 cm−1 21 300), 283 (15 700), 235
(28 500); dH (250 MHz; DMSO-d6; Me4Si) 11.82 (1H, s, H–N), 8.60
Glycosylation of 18 with 2-deoxy-3,5-di-O-(p-toluoyl)-a-D-
erythro-pentofuranose chloride (6). To a solution of compound
18 (580 mg, 1.79 mmol) in MeCN (200 cm3), KOH (626 mg,
8.94 mmol) and TDA-1 tris(3,6-dioxaheptyl)amine (0.2 cm3,
0.60 mmol) was added and the mixture was stirred at r.t for
20 min. Then 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-a-D-erythro-
pentofuranose (765 mg, 1.97 mmol) was added in portions over
10 min, stirring was continued for another 20 min, and the reaction
mixture was filtered and evaporated to dryness. The residue was
purified by FC (silica gel, column 15 × 5.5 cm, CH2Cl2–MeOH
200 : 1).
=
(1H, s, CH N), 7.42–6.69 (13H, m, arom H), 6.44 (1H, q, J 4.1,
3-[2-Deoxy-3,5-di-O-(p-toluoyl)-b-D-erythro-pentofuranosyl)]-5-
{[(di-n-butylamino)methylidene]amino}thiazolo[4,5-d]pyrimidine-
2,7(3H,6H)-dione (19). From the main zone, compound 19 was
obtained as a colorless foam (629 mg, 55%) (Found: C, 62.56; H,
6.28; N, 9.98. C35H41N5O7S requires C, 62.20, H, 6.12, N, 10.36%);
TLC (silica gel, CH2Cl2–MeOH 40 : 1): Rf 0.34; UV/Vis: kmax
(MeOH)/nm 323 (e/dm3 mol−1 cm−1 22 200), 284 (14 600) and
242 (41 700); dH (250 MHz; DMSO-d6; Me4Si) 11.97 (1H, s, HN),
H–C(1ꢀ)), 5.22 (1H, m, OH–C(3ꢀ)), 4.30 (1H, m, H–C(3ꢀ)), 3.86
(1H, m, H–C(4ꢀ)), 3.71 (6H, s, 2 × OCH3), 3.50–3.18 (6H, m,
H–C(5ꢀ), 2 × N–CH2), 2.81 (1H, m, H–C(2ꢀ)), 2.16 (1H, m, H–
C(2ꢀ)), 1.58–1.50 (4H, m, 2CH2), 1.36–1.17 (4H, m, 2 × CH2) and
0.96–0.8 (6H, m, 2 × CH3).
3-(2-Deoxy-5-O-(4,4ꢀ -dimethoxytriphenylmethyl)-b-D-erythro-
pentofuranosyl-5-{[(di-n-butylamino)methylidene]amino}thia-
=
8.68 (1H, s, CH N), 7.95–7.75 (4H, m, arom. H), 7.39–7.21 (4H,
zolo[4,5-d]pyrimidine-2,7(3H,6H)-dione
3ꢀ-(2-cyanoethyl)diiso-
m, arom. H), 6.50 (1H, q, J 4.6, H–C(1ꢀ)), 5.78 (1H, m, H–C(3ꢀ)),
4.58–4.35 (3H, m, H–C(5ꢀ), H–C(4ꢀ)), 3.46–3.34 (4H, m, NCH2),
2.38 (3H, s, CH3), 2.34 (3H, s, CH3), 1.54–1.06 (8H, m, 4 × CH2,),
0.88 (3H, t, J 7.3, CH3) and 0.76 (3H, t, J 7.3, CH3).
propylphosphoramidite (22). To the solution of compound 21
(160 mg, 0.22 mmol) in dry CH2Cl2 (10 cm3), (iPr)2EtN (0.060 cm3,
0.33 mmol) and (2-cyanoethyl)diisopropylphosphoramido
chloridite (0.074 cm3, 0.33 mmol) were added. The solution was
stirred at r.t. for 15 min. The solution was diluted with CH2Cl2
(40 cm3) and washed with 5% aq. NaHCO3 solution. The mixture
was extracted with CH2Cl2 (2 × 20 cm3). The combined organic
phase was dried (Na2SO4), filtered, and evaporated. The residue
was purified by FC (column 15 × 5 cm, CH2Cl2–acetone 15 : 1) to
afford a white foam. The foamy residue was dissolved in CH2Cl2
(3 cm3) and added gradually to stirring cyclohexane (80 cm3)
cooled to −30 ◦C. The precipitate was isolated by filtration
and the resulting powder was dried under vacuum, yielding
compound 22 as a colorless foam (161 mg, 80%). TLC (silica
gel, CH2Cl2–acetone 4 : 1): Rf 0.5; dP (250 MHz; CDCl3; H3PO4)
150.0, 149.8.
3-(2-Deoxy-b-D-erythro-pentofuranosyl)-5-{[(di-n-butylamino)-
methylidene] amino}thiazolo[4,5-d]pyrimidine-2,7(3H,6H)-dione
(20). Compound 19 (380 mg, 0.56 mmol) was dissolved in 0.1 M
NaOCH3 (30 cm3) and stirred at r.t. for 4 h. The solid residue,
obtained after evaporation, was purified by FC (silica gel, column
10 × 5 cm, CH2Cl2–CH3OH 20 : 1) to yield a colorless solid,
which was crystallized from ethyl acetate to give 20 (200 mg,
81%) as a colorless solid (Found: C, 52.00; H, 6.59; N, 15.75.
C19H29N5O5S requires C, 51.92, H, 6.65, N, 15.93%); TLC (silica
gel, CH2Cl2–MeOH 20 : 1): Rf 0.2; UV/Vis kmax (MeOH)/nm
321 (e/dm3 mol−1 cm−1 23 400), 292 (15 400), 250 (13 900) and 216
(17 000); dH (250 MHz; DMSO-d6; Me4Si) 11.89 (1H, s, H–N), 8.62
ꢀ
=
(1H, s, CH N), 6.34 (1H, t, J 6.9, H–C(1 )), 5.21 (1H, d, J 4.6,
OH–C(5ꢀ)), 4.67 (1H, t, J 5.7, OH–C(3ꢀ)), 4.33 (1H, m, H–C(3ꢀ)),
3.69 (1H, m, H–C(4ꢀ)), 3.62–3.41 (6H, m, H–C(5ꢀ), 2 × N–CH2),
2.91 (1H, m, H–C(2ꢀ)), 2.06 (1H, m, H–C(2ꢀ)), 1.58 (4H, m, 2 ×
CH2), 1.28 (4H, m, 2 × CH2) and 0.91 (6H, t, J 7.2, 2 × CH3).
Acknowledgements
We thank Dipl.-Ing. Simone Budow for the measurement of the
NMR spectra, Dipl.-Chem. Nhaˆt Quang Tran for the oligonu-
cleotide synthesis and Dr T. Koch, Roche Diagnostics GmbH,
for the MALDI-TOF mass spectra. We also thank Dr Peter
Leonard for a useful discussion. Financial support from Roche
Diagnostics GmbH, Coley Pharmaceuticals and ChemBiotech
(Mu¨nster, Germany) is gratefully acknowledged.
5-Amino-3-(2-deoxy-b-D-erythro-pentofuranosyl)thiazolo[4,5-
d]pyrimidine-2,7-(3H,6H)-dione (4). Compound 19 (256 mg,
0.58 mmol) was dissolved in concentrated ammonia solution
(30 cm3) and stirred at room temperature for 72 h. The reaction
mixture was adsorbed onto silica gel 60 (1.0 g), and applied to the
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27 A. J. Hubbard, A. S. Jones and R. T. Walker, Nucleic Acids Res., 1984,
12, 6827–6837.
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