Designing new isomorphic fluorescent nucleobase analogues: the thieno[3,2-d]pyrimidine core

Article abstract of DOI:10.1016/j.tet.2007.01.075

A convergent approach for a family of fluorescent nucleosides is described. It relies on thieno[3,2-d]pyrimidine-2,4(1H,3H)-dione that serves as a core heterocycle. This condensed pyrimidine is converted into an emissive pyrimidine nucleoside analogue by N-glycosylation and into an emissive purine nucleoside analogue by C-glycosidation at the thiophene's beta position. The design principles of this archetypical system are outlined together with the syntheses and photophysical properties of the resulting nucleosides.

Full text of DOI:10.1016/j.tet.2007.01.075

Tetrahedron 63 (2007) 3608–3614
Designing new isomorphic fluorescent nucleobase analogues:
the thieno[3,2-d]pyrimidine core
*
Yitzhak Tor, Susan Del Valle, David Jaramillo, Seergazhi G. Srivatsan,
Andro Rios and Haim Weizman
Department of Chemistry and Biochemistry, University of California, San Diego, MC 0358, La Jolla, CA 92093-0358, USA
Received 18 January 2007; revised 25 January 2007; accepted 25 January 2007
Available online 3 February 2007
Abstract—A convergent approach for a family of fluorescent nucleosides is described. It relies on thieno[3,2-d]pyrimidine-2,4(1H,3H)-dione
that serves as a core heterocycle. This condensed pyrimidine is converted into an emissive pyrimidine nucleoside analogue by N-glycosylation
and into an emissive purine nucleoside analogue by C-glycosidation at the thiophene’s beta position. The design principles of this archetypical
system are outlined together with the syntheses and photophysical properties of the resulting nucleosides.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
that such structures deviate from the natural bases and
cannot form canonical Watson–Crick base pairs, or be
accommodated within a native Watson–Crick duplex.6
A related approach involves condensing additional aro-
matic rings on the periphery of the heterocycle. Benzo-
[g]quinazoline-2,4-(1H,3H)-dione (BgQ), represents
a typical example (Fig. 1).9,10 More recent examples
include several structures of expanded, base-discrimi-
nating nucleosides, reported by Okamoto and Saito.11
(2) Conjugated base analogues: Linking the natural nucleo-
bases to fluorescent chromophores, typically via con-
jugating linkers (e.g., ethynyl), has been shown to
produce emissive nucleoside analogues (Fig. 2). Exam-
ples of attached chromophores include pyrene,12
phenanthroline,13 dimethylaminonaphthalene,14 anthra-
cene,15 and fluorene.16,17 Since the H-bonding face in
many of these derivatives remains intact and the chro-
mophore is projected into the major groove of a double
helix, these nucleobase analogues typically maintain the
hybridization properties of the parent nucleosides.
The core heterocycles commonly found in nucleic acids are
practically non-emissive.1 Numerous efforts to impart useful
photophysical features upon these pyrimidines and purines
have been reported;2 many of them are represented in this
issue of Tetrahedron’s Symposium-in-Print. The various
families of fluorescent nucleoside analogues can be broadly
classified into the following categories:
(1) Extended nucleobases: Inspired by the naturally occur-
ring and emissive tricyclic guanine derivatives such as
the ‘Y’ bases,3 Leonard has condensed an etheno bridge
over the H-bonding face of the purines and pyrimidines.4
Extending the conjugation of the natural bases in this
way resulted in modified nucleosides with favorable
fluorescence properties. One of the earliest examples is
1,N6-ethenoadenine (Fig. 1).5,6 Another approach, origi-
nally examined by Leonard,7 and recently adopted by
Kool,8 involves the expansion of the heterocycle’s size
by introducing aromatic rings between the pyrimidine
and the imidazole ring (e.g., Benzo-A, Fig. 1). Note
Figure 1. Fluorescent extended nucleobases. R stands for D-ribose or
20-deoxy-D-ribose.
* Corresponding author. Tel.: +1 858 534 6401; fax: +1 858 534 0202;
e-mail: ytor@ucsd.edu
Figure 2. Examples for conjugated base analogues where a fluorophore is
linked to a native heterocycle. R stands for D-ribose or 20-deoxy-D-ribose.
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.01.075

Y. Tor et al. / Tetrahedron 63 (2007) 3608–3614
3609
Figure 3. G and A pteridine-based analogues. R stands for D-ribose or
20-deoxy-D-ribose.
(3) Pteridine as purine analogues: The pteridines represent
fluorescent heterocycles that contain two condensed six
membered rings (Fig. 3).2c,18 Some of these very useful
probes possess a H-bonding face that is identical to the
purines found in DNA and RNA. Very little variation
in emission wavelengths is observed among these
intensely emissive derivatives.
Figure 5. Isomorphic fluorescent purine and pyrimidine bases.
(4) Aromatic hydrocarbon and chromophoric base ana-
logues: In these novel structures, the natural heterocycle
these otherwise extremely useful reporter oligonucleotides
do not respond well to some aminoglycosides, particularly
neomycin B, one of the most potent A-site binders. We
have demonstrated that a fluorescent furan-modified U
residue can be incorporated into an A-site construct and
used effectively to monitor the binding of neomycin B.29
This example illustrates the fundamental challenge arti-
culated above and highlights the need for the development
of new emissive nucleosides, in particular isosteric ana-
logues of the naturally occurring pyrimidines and purines.
Here we disclose a convergent approach for a family of fluo-
rescent nucleosides under investigation in our laboratory. It
relies on identifying an emissive condensed pyrimidine
core and converting it into fluorescent pyrimidine and purine
nucleosides. The design of an archetypical system, as well as
its synthesis and photophysical characterization is outlined
in this contribution.
is replaced with
a fluorescent aromatic residue
(Fig. 4).19–21 These analogues can be accommodated
within oligonucleotides although they do not form
canonical base pairs.
(5) Isomorphic base analogues: These fluorescent base
analogues closely resemble the corresponding natural
nucleobases with respect to their overall dimensions,
hydrogen bonding face and ability to form isostructural
Watson–Crick base pairs (Fig. 5). Possibly the most
utilized nucleobase is 2-aminopurine (2-AP), a highly
emissive isosteric adenosine analogue.22 The exquisite
sensitivity of 2-AP to its microenvironment has found
numerous applications in biochemical studies and assay
development.23,24 5-Methylpyrimidin-2-one (m5K)25
and pyrrolo-C26 have been introduced as isomorphic
pyrimidine analogues.27,28
Despite this diversity in structures and photophysical proper-
ties, the implementation of fluorescent nucleoside analogues
in biophysical assays remains challenging, as there is no sin-
gle analogue that can be universally employed. For example,
in a recent study we compared ligand binding to fluorescent
bacterial A-site RNA constructs.29 Aminoglycoside anti-
biotics, a family of naturally occurring highly cationic
bactericidal agents, target this autonomous RNA fold, also
known as the ribosomal decoding site. Antibiotics discovery
assays have been developed, where 2-AP replaces key adeno-
sine residues in this small RNA sequence.30,31 Intriguingly,
2. Results and discussion
2.1. Design
Examination of the photophysical properties of simple con-
densed conjugated heterocycles such as benzofuran and
benzothiophene reveals useful fluorescent properties.32 Com-
paring the absorption and emission spectra of benzene and
thiophene to that of the corresponding benzo[b]thiophene,
clearly illustrates the favorable features of the condensed sys-
tem (Fig. 6). While benzene and thiophene have relatively
weak absorption bands above 250 nm and no apparent emis-
sion under these conditions, the ‘hybrid’ molecule, benzo[b]-
thiophene, is significantly more emissive (Fig. 6).
Using this as a platform, we explored families of fluorescent
nucleobases where thieno[3,2-d]pyrimidine-2,4(1H,3H)-
dione (1) serves as the core heterocycle (Fig. 7). Glycosyl-
ation with D-ribose (or 20-deoxy-D-ribose) at N-1 is expected
to provide an expanded and emissive pyrimidine nucleoside
analogue A, while C-glycosidation at the thiophene’s beta
position is anticipated to provide a viable route to a fluores-
cent purine nucleoside analogue B (Fig. 7). Elaboration of
functional groups can then expand A and B to include vari-
ous isosteric pyrimidine and purine analogues that mimic the
naturally occurring nucleosides as schematically shown in
Figure 7.
Figure 4. Fluorescent aromatic residues replace the heterocyclic bases. R
stands for 20-deoxy-D-ribose; some of these derivatives have the a configura-
tion at the anomeric center.

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Y. Tor et al. / Tetrahedron 63 (2007) 3608–3614
Scheme 1. Synthesis of thienopyrimidine-based N-nucleosides 2 and 3. Re-
agents and conditions: (a) KOCN, AcOH/H2O (80%); (b) (i) Me3SiCl,
Me3SiNHSiMe3, (ii) 1-O-acetyl-2,3,5-tribenzoyl-D-ribofuranoside, CH2Cl2,
58% for two steps, (iii) NH3, MeOH, 76%; (c) (i) imidazole, 1,3-
dichloro-1,1,3,3-tetraisopropyldisiloxane, 72%, (ii) PhCSCl, DMAP,
CH3CN followed by AIBN, TTMSS, dioxane, 71%, (iii) TBAF, THF, 71%.
Figure 6. Absorption and emission spectra of benzothiophene (red) in com-
parison to thiophene (blue) and benzene (green); all taken in hexanes
(1ꢀ10ꢁ6 M).
2.2. Synthesis
1 with bromine in acetic acid provided the substituted deriv-
ative 4 in almost quantitative yield. Treatment with POCl3
yielded the dichloro derivative 5. Note this reaction has to
be carefully monitored to avoid over chlorination. Derivative
5 was then converted into the dimethoxy derivative 6 using
sodium methoxide in dry methanol. Palladium mediated
coupling with the known glycal, 1,2-dehydro-3-O-(t-butyl-
diphenylsilyl)-5-hydroxymethyl-furan, gave the Heck-
coupled product, which underwent desilylation under the
reaction conditions to provide the keto derivative 7. Stereo-
specific reduction using sodium triacetoxyborohydride then
yielded 8. Final de-methylation of the heterocycle using NaI
in acetic acid gave the desired C-nucleoside 9 (Scheme 2;
see Section 4).
Not unexpectedly, nucleosides containing the thienopyr-
imidine core were previously prepared and examined for
potential antiviral activity,33 although their photophysical
characteristics have not been reported. As illustrated in
Scheme 1, constructing the parent heterocycle is accom-
plished by treating the commercially available methyl
3-aminothiophene-2-carboxylate with potassium cyanate.
The resulting thienopyrimidine can be converted into ribo-
nucleoside 2 using typical N-glycosylation conditions to
exclusively provide the b-anomer (Scheme 1).33 Barton-
type deoxygenation of 2 to yield the corresponding 20-deoxy
derivative 3 turned out to be somewhat challenging, but was
ultimately achieved using tris(trimethylsily)silane (TTMSS)
instead of tri-n-butyltinhydride (Scheme 1; see Section 4).
The synthesis of the purine mimic 9 was accomplished as
shown in Scheme 2. Bromination of the parent heterocycle
Figure 7. General design of new emissive nucleosides based on the
thieno[3,2-d]pyrimidine-2,4(1H,3H)-dione core (1). Glycosylation at N-1
is expected to provide an expanded pyrimidine analogue A, while C-glyco-
sidation at the thiophene’s beta position is anticipated to provide a purine
analogue B. Elaboration of functional groups can then expand A and B to
include various isosteric pyrimidine and purine analogues that mimic the
naturally occurring nucleosides.
Scheme 2. Synthesis of thienopyrimidine-based C-nucleoside 9. Reagents
and conditions: (a) Br2, AcOH, 97%; (b) POCl3, N,N-dimethylaniline,
64%; (c) NaOMe, MeOH, 97%; (d) Pd(OAc)2, Bu4NCl, NaHCO3, 1,2-
dehydro-3-O-(t-butyldiphenylsilyl)-5-hydroxymethyl-furan, DMF, 61%;
(e) NaB(OAc)3H, ACN/AcOH 88%; (f) NaI, AcOH, 74%.

Y. Tor et al. / Tetrahedron 63 (2007) 3608–3614
Table 1. Photophysical data of nucleoside 2a
3611
b
lmax (nm)
c
Irel
Solvent
lem (nm)
Water
292
294
294
296
351
350
344
345
1.7
1.3
1.2
1.0
Methanol
Acetonitrile
Ethyl acetate
Conditions for absorption and emission spectra: 50 and 5.0ꢀ10ꢁ6 M,
a
respectively.
b
c
The lowest energy maximum is given. Note, the nucleoside shows another
intense absorption at higher energies, at around 263–270 nm.
Relative emission intensity with respect to intensity in ethyl acetate.
Figure 8. Absorption (5.0ꢀ10ꢁ5 M) and emission (5.0ꢀ10ꢁ6 M) spectra of
nucleoside 2 in water (red), methanol (blue), acetonitrile (green), and ethyl
acetate (black). Stock solutions for absorption and emission spectra
contained 5% and 0.5% DMSO, respectively.
2.3. Photophysical characteristics
Absorption spectra of the free ribonucleoside 2 show, in ad-
dition to a high energy transition at around 265 nm, a longer
wavelength absorption at ca. 300 nm. Note that, while the
former shows slight sensitivity to solvent polarity, the latter,
lower energy absorption band is practically insensitive to
changes in medium polarity (Fig. 8).34 Excitation at the
lower energy band results in a relatively strong emission
centered at around 350 nm (Fig. 8). Quantum yield measure-
ments in water, using tryptophan as a standard, reveal a
moderate value of 0.058. Unlike many other modified
nucleosides, the emission of 2 is relatively insensitive to
solvent polarity. A slight bathochromic shift (ca. 6 nm)
and a moderate hyperchromic effect (1.7 fold) are observed
when the emission in ethyl acetate is compared to the emis-
sion in water (Figs. 8 and 9 and Table 1). Steady-state Stern–
Volmer titrations of nucleoside 2 with the native nucleobase
monophosphates, show significant quenching by GMP, CMP
and TMP and marginal quenching by AMP (Fig. 10). The
Figure 10. Steady-state Stern–Volmer plot for the titration of nucleoside 2
with AMP, GMP, CMP, and TMP. Quenching constant, Ksv for GMP, CMP,
and TMP were determined to be 7.72ꢀ10ꢁ3 Mꢁ1, 4.83ꢀ10ꢁ3 Mꢁ1, and
4.56ꢀ10ꢁ3 Mꢁ1, respectively.
20-deoxy nucleoside 3 shows analogous photophysical
features.35
The photophysical characteristics of the C-nucleoside 9 are
similar to the corresponding N-nucleoside 2, with small
differences in absorption and emission maxima (Fig. 11
and Table 2). The emission quantum yield for 9 is, however,
Figure 11. Absorption (5.0ꢀ10ꢁ5 M) and emission spectra (5.0ꢀ10ꢁ6 M)
of nucleoside 9 in water (red), methanol (blue), acetonitrile (green), and
ethyl acetate (black). Stock solutions for absorption and emission spectra
contained 5% and 0.5% DMSO, respectively.
Figure 9. Emission energy plotted against ET(30), a microscopic solvent polarity
scale. Note the trend for the lower emission energy in more polar solvent.

3612
Y. Tor et al. / Tetrahedron 63 (2007) 3608–3614
Table 2. Photophysical data of nucleoside 9a
were measured at the UCSD Mass Spectrometry Facility.
b
lmax (nm)
c
UV–vis spectra were recorded on a Hewlett Packard 8452
A Diode Array Spectrophotometer. Emission spectra were
recorded using Perkin Elmer LS 50B and FluoroMax-3,
Horiba Jobin Yvon luminescence spectrometer.
Solvent
lem (nm)
Irel
Water
294
294
296
296
351
351
345
343
1.4
1.5
1.3
1.0
Methanol
Acetonitrile
Ethyl acetate
4.2. Synthesis of nucleoside 2
Conditions for absorption and emission spectra: 50 and 5.0ꢀ10ꢁ6 M,
a
respectively.
b
c
The parent heterocycle 1 was synthesized from methyl
3-aminothiophene-2-carboxylate and potassium cyanate
and was condensed with 1-O-acetyl-2,3,5-tri-O-benzoyl-
D-ribofuranose to afford 1-(b-D-ribofuranosyl)thieno[3,2-
d]pyrimidine-2,4-dione (2) as reported.33
The lowest energy maximum is given. Note, the nucleoside shows another
intense absorption at higher energies, at around 262–270 nm.
Relative emission intensity with respect to intensity in ethyl acetate.
slightly lower (0.037) compared to the value obtained for 2.
As with 2, the emission of 9 is relatively insensitive to sol-
vent polarity. A slight bathochromic shift (ca. 8 nm) and
a very modest hyperchromic effect (1.4 fold) are observed
when the emission in ethyl acetate is compared to the emis-
sion in water (Fig. 11, Table 2). These results confirm our
working hypothesis and illustrate that emissive pyrimidine
and purine analogues can indeed be generated from the
thienopyrimidine core 1.
4.3. Synthesis of nucleoside 3
4.3.1. 1-(2-Deoxy-b-D-erythropentosyl)thieno[3,2-d]-
pyrimidine-2,4-dione (3). Imidazole (1.8 g, 26.6 mmol)
and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (1.8 mL,
6.66 mmol) were added successively to a solution of the
deprotected ribonucleoside 2 (2 g, 6.66 mmol) in anhydrous
DMF (27 mL) under an inert atmosphere. The mixture was
stirred at room temperature for 1 h, and partitioned between
H2O and CHCl3. The organic layer was washed with saturated
NaHCO3 solution followed by H2O, dried over Na2SO4, fil-
tered, and concentrated to give a clear syrup, which was puri-
fied by flash chromatography (10–50% EtOAc/ hexanes) to
give 2.6 g of a white foam (72% yield). (TLC CH2Cl2:CH3OH
98:2, Rf¼0.29);IR(KBr) cmꢁ1: 3600–3440 (OH), 1710–1660
(CO), 1490, 1390, 1110, 1050; 1H NMR (DMSO-d6): d 11.62
(1H, NH), 8.12 (d, 1H, H-6, J¼5.4 Hz), 7.37 (d, 1H, H-7,
J¼5.4 Hz), 6.15 (1H, OH), 5.81 (d, 1H, H-10), 5.17 (m, 1H,
H-20), 4.68 (m, 1H, H-30), 4.54 (m, 1H, H-40), 3.97 (m, 2H,
CH2-50), 1.05 (s, 24H, CH3); 13C NMR (DMSO-d6):
d 157.5, 150.3, 145.5, 135.4, 117.6, 113.5, 92.6, 80.9, 71.4,
70.2, 61.4, 17.0; ESI-MS m/z calcd for C23H38N2O7SSi2
[M–H]ꢁ 541, found 541.
3. Summary
As a proof of concept, we have demonstrated the synthesis
of emissive nucleosides, all derived from a single hetero-
cycle, thieno[3,2-d]pyrimidine-2,4(1H,3H)-dione. N-glyco-
sylation provided an expanded pyrimidine analogue,
whereas C-glycosidation at the thiophene’s beta position
generated a C-nucleoside that can be viewed as an isosteric
purine mimic. Rewardingly, all nucleosides were found to
be fluorescent with emission centered at around 350 nm
and quantum efficiency between 4 and 6%. Further func-
tional group elaboration is anticipated to generate additional
emissive derivatives that can mimic the natural nucleobases
and contribute to the growing repertoire of biophysically
useful and minimally perturbing fluorescent nucleoside
analogues.
The silyl protected ribonucleoside (800 mg, 1.48 mmol) and
DMAP (558 mg, 4.6 mmol) were flushed with argon and
dissolved in anhydrous CH3CN (48 mL). Phenylthionyl-
chloride (307 mL, 2.21 mmol) was added to the mixture
under argon, and the reaction mixture was stirred at room
temperature for 4 h. The mixture was concentrated, taken
up in CH2Cl2, washed with ice water, 0.5 M HCl, then
with ice water again. The organic layer was dried over
Na2SO4, and concentrated to produce a tan foam. The
foam was dried under vacuum and freeze-pump-thawed
several times. AIBN (73 mg, 0.44 mmol) was added to the
flask and the mixture was dissolved in anhydrous dioxane
(15 mL) and transferred to a three-neck flask that was fitted
with an addition funnel and condenser. TTMSS (1.2 mL,
3.84 mmol) was added dropwise, and the mixture was heated
to 100 ꢂC. After 2 h, the dioxane was removed under
reduced pressure to give an oil, which was purified over a
silica column (0–1% MeOH/CH2Cl2) to give 552 mg of
4. Experimental
4.1. General considerations
All chemicals were obtained commercially and used without
further purification. O-(t-Butyldiphenylsilyl)-5-hydroxy-
methyl-furan was synthesized from thymidine according to
a reported procedure.36 To increase the reproducibility of
the procedure, we have introduced an additional purification
step. Specifically, in the last base-induced elimination step,
the silylated intermediate was first passed through a short sil-
ica gel column using chloroform as an eluent, before being
subjected to potassium carbonate in MeOH. Reactions
were monitored by thin layer chromatography (TLC) using
0.25 mm thick pre-coated UV sensitive silica gel plates.
Compounds were visualized with short-wave ultraviolet
light at 254 nm and/or by immersion in staining solution
(cerric ammonium molybdate). Flash chromatography was
carried out using Kieselgel 60 (230–400 mesh). NMR
spectra were recorded on a Varian Mercury 300 MHz or
Varian Mercury 400 MHz spectrometers. Mass spectra
a
yellow foam (71% yield). (TLC EtOAc/hexanes
1
Rf¼0.41). H NMR (CDCl3): d 8.03 (d, 1H, H-6), 7.71 (d,
1H, H-7, J¼5.37 Hz), 7.24 (d, 1H, H-6, J¼5.37 Hz), 6.50
(s, 1H, H-10), 4.84 (m, 1H, H-30), 4.04 (m, 1H, H-40), 3.82
(m, 2H, CH2-50), 2.53 (m, 2H, CH2-20), 1.10 (s, 24H,
CH3); 13C NMR (CDCl3): d 157.2, 149.9, 145.8, 134.9,
118.1, 94.9, 80.5, 74.6, 69.5, 60.9, 27.9, 17.6; ESI-MS m/z

Y. Tor et al. / Tetrahedron 63 (2007) 3608–3614
3613
calcd for C23H38N2O6SSi2 [M+H]+ 527, found 527,
[M+Na]+ calcd 549, found 549.
4.4.4. 2,4-Dimethoxy-7-(b-D-glycero-pentofuran-30-ulos-
10yl)thieno[3,2-d]pyrimidine (7). Compound 6 (0.11 g,
0.4 mmol) and tetrabutylammonium chloride (0.81 g,
2.9 mmol) were co-evaporated with CH3CN and dried under
vacuum for 1 h. Separately, 1,2-dehydro-3-O-(t-butyl-
diphenylsilyl)-5-hydroxymethyl-furan (0.27 g, 0.8 mmol;
see Section 4.1) was also co-evaporated with CH3CN and
dried under vacuum. Compound 4/Bu4NCl was dissolved
in dry DMF (5 mL) and purged under argon for 5 min.
Pd(OAc)2 (0.03 g, 0.1 mmol) and NaHCO3 (0.05 g,
0.7 mmol) were added followed by the furan in DMF
(4 mL). The solution was heated at 40 ꢂC for 36 h, cooled,
and the solvent was removed. The residue was purified by
flash chromatography (50% EtOAc/hexane), yielding 7
(0.07 g, 61%) as a yellow solid. 1H NMR (400 MHz,
CDCl3): d 7.79 (s, 1H), 5.50 (q, 1H, H-10, J¼4.8 Hz,
J¼11.2 Hz), 4.88 (d, 1H, OH, J¼7.2 Hz), 4.14 (s, 3H),
4.09 (t, 1H, H-40, J¼2.8 Hz), 4.01 (s, 3H), 3.96 (s, 2H, H-
50), 3.21 (q, 1H, H-20, J¼6.8 Hz, J¼18 Hz), 2.81 (dd, 1H,
H-20, J¼6 Hz); 13C NMR (100 MHz, CD3CN): d 214.1,
166.0, 163.9, 160.6, 135.2, 133.0, 112.7, 82.5, 73.2, 61.9,
55.0, 54.4, 43.1; ESI-MS m/z calcd for C13H14N2O5S
[M+Na]+ 333.06, found 333.03.
The siloxane protected nucleoside (500 mg, 0.95 mmol) was
dissolved in anhydrous THF (19 mL) and treated with a solu-
tion of tetrabutylammonium fluoride in THF (1 M, 1.9 mL).
The reaction mixture was stirred at room temperature for 1 h
and then concentrated to give the crude product. The result-
ing oil was purified by flash chromatography (5–10%
CH3OH/CH2Cl2) to give 242 mg of a white solid (71%
yield). This compound was crystallized from water. (TLC
CH2Cl2:CH3OH 9:1, Rf¼0.20); IR (KBr) cmꢁ1: 3500–
3420 (OH), 3180 (NH), 1675 (CO), 1490, 1420, 1090,
1
1010; H NMR (DMSO-d6): d 11.52 (1H, NH), 8.06 (d,
1H, H-6), 7.67 (d, 1H, H-7), 6.52 (d, 1H, H-10), 5.00–5.24
(OH), 4.63 (m, 1H, H-30), 3.73 (m, 1H, H-40), 3.65 (m, 2H,
CH2-50), 2.42 (m, 2H, H-20); 13C NMR (DMSO-d6):
d 157.7, 150.7, 144.2, 134.7, 119.4, 114.0, 86.7, 83.7,
69.4, 60.6, 37.2; ESI-MS m/z calcd for C11H12N2O5S
[M–H]ꢁ 283, found 283. An X-ray crystal structure further
confirmed the structure and the anoneric configuration of
this nucleoside.
4.4. Synthesis of nucleoside 9
4.4.5. 10-b-[7-(2,4-Dimethoxythieno[3,2-d]pyrimidine)]-
20-deoxyribofuranose (8). Sodium triacetoxyborohydride
(0.11 g, 0.5 mmol) was added to a solution of 7 (0.12 g,
0.4 mmol) in CH3CN/AcOH (1:1, 20 mL) at –15 ꢂC and
the mixture stirred for 1 h. It was warmed to room tempera-
ture, the solvent removed, and the residue purified by flash
chromatography (5% MeOH/EtOAc), to afford 8 (0.11 g,
88%) as a yellow solid. 1H NMR (400 MHz, CDCl3):
d 7.70 (s, 1H), 5.43 (q, 1H, H-10, J¼5.6 Hz, J¼11.2 Hz),
4.67 (d, 1H, H-30, J¼5.2 Hz), 4.12 (s, 3H), 4.01 (s, 3H),
3.94 (d, 1H, H-50, J¼12 Hz), 3.77 (d, 1H, H-50, J¼12 Hz),
2.74 (m, 1H, H-20), 2.09 (m, 1H, H-20), 2.05 (m, 1H,
H-40); 13C NMR (100 MHz, CDCl3): d 165.6, 163.3,
160.3, 134.7, 132.8, 112.9, 94.2, 88.7, 75.0, 63.8, 55.3,
54.3, 41.8; ESI-MS m/z calcd for C13H16N2O5S [M+Na]+
335.08, found 335.07.
4.4.1. 7-Bromothieno[3,2-d]pyrimidine-2,4(1H,3H)-
dione (4). To compound 1 (1.0 g, 5.9 mmol) in AcOH
(40 mL) was added bromine (0.91 mL, 17.8 mmol) and the
mixture was kept at 110 ꢂC inside a pressure tube for 48 h.
The solution was cooled; the solvent removed under vacuum
and cold water was added. The precipitate was filtered,
washed with water, and dried under vacuum to give 4
(1.43 g, 97%) as a white solid. 1H NMR (400 MHz,
DMSO-d6): d 11.54 (s, 1H), 11.42 (s, 1H), 8.22 (s, 1H);
13C NMR (100 MHz, DMSO-d6): d 158.4, 151.5, 144.4,
132.9, 111.6, 99.0; ESI-MS m/z calcd for C6H4BrN2O2S
[M+H]+ 246.91, found 247.04.
4.4.2. 7-Bromo-2,4-dichlorothieno[3,2-d]pyrimidine (5).
To compound 4 (2.2 g, 8.9 mmol) and POCl3 (8.2 mL,
89 mmol) was added N,N-diethylaniline (5.7 mL,
35.6 mmol) and the solution was refluxed for 3 h. It was
cooled and volatiles were removed under vacuum. The
residue was diluted with CHCl3 and washed with ice-cold
H2O (twice). The organic layers were dried (Na2SO4), the
solvent removed, and the residue purified by flash chromato-
graphy (5% EtOAc/hexane). The product 5 (1.63 g, 64%)
was isolated as a white solid. 1H NMR (400 MHz,
CDCl3): d 8.10 (s, 1H); 13C NMR (100 MHz, CDCl3):
d 159.9, 157.3, 156.1, 135.4, 128.5, 109.4; ESI-MS m/z calcd
for C6H2BrCl2N2S [M+H]+ 282.84, found 283.05.
4.4.6. 10-b-[7-(Thieno[3,2-d]pyrimidine-2,4-dione)]-
20-deoxyribofuranose (9). To compound
8 (0.02 g,
0.06 mmol) in AcOH (2 mL) was added NaI (0.05 g,
0.3 mmol) and the reaction stirred at 60 ꢂC for 2 h. The
solution was cooled, the solvent removed, and the residue
diluted with EtOAc. It was washed with NaHCO3, the
organic layer dried (Na2SO4), and concentrated. The residue
was purified by flash chromatography (10% MeOH/
CH2Cl2), to give 9 (0.01 g, 74%) as a white solid. 1H NMR
(400 MHz, DMSO-d6): d 11.28 (s, 2H), 7.98 (s, 1H), 5.81
(s, 1H, OH-50), 5.22 (dd, 1H, H-10, J¼3.6 Hz, J¼10 Hz),
5.13 (d, 1H, OH-30, J¼3.2 Hz), 4.26 (m, 1H, H-30), 3.85
(m, 1H, H-40), 3.60 (m, 2H, H-50), 1.98 (m, 2H, H-20); 13C
NMR (100 MHz, DMSO-d6): d 159.0, 151.4, 144.0, 131.6,
131.5, 112.5, 87.7, 75.2, 72.9, 61.8, 41.8; ESI-MS m/z calcd
for C11H13N2O5S [M+H]+ 285.05, found 285.08.
4.4.3. 7-Bromo-2,4-dimethoxythieno[3,2-d]pyrimidine
(6). To compound 5 (0.27 g, 0.9 mmol) in dry MeOH
(30 mL) was added NaOMe (0.20 g, 3.8 mmol) and the mix-
ture refluxed for 22 h. The reaction was cooled, HCl (1 M,
1.9 mL) added, and extracted with CH2Cl2. The organic
layers were dried (Na2SO4) and the solvent removed to yield
6 (0.25 g, 97%) as a white solid. 1H NMR (400 MHz,
CDCl3): d 7.75 (s, 1H), 4.13 (s, 3H), 4.09 (s, 3H); 13C
NMR (100 MHz, CDCl3): d 165.9, 164.5, 159.5, 130.9,
111.0, 108.4, 55.1, 54.5; ESI-MS m/z calcd for
C8H8BrN2O2S [M+H]+ 274.94, found 275.04.
Acknowledgements
We are grateful to the National Institutes of Health
(GM069773) for support.

3614
Y. Tor et al. / Tetrahedron 63 (2007) 3608–3614
19. (a) Ren, R. X.-F.; Chaudhuri, N. C.; Paris, P. L.; Rumney,
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