Mechanistic studies in the synthesis of a series of thieno-expanded xanthosine and guanosine nucleosides

Article abstract of DOI:10.1016/j.tet.2008.09.011

During the synthetic pursuit of guanosine (tri-G) and xanthosine (tri-X) tricyclic nucleosides analogues, an interesting side product was discovered. In an effort to uncover the mechanistic factors leading to this result, a series of reaction conditions were investigated. It was found that by varying the conditions, the appearance of the side product could be controlled. In addition, the yield of the desired products could be manipulated to afford either a 50:50 mix of both tri-G and tri-X, or a majority of one or the other. To demonstrate the broad utility of the method, it was also adapted to the synthesis of guanosine and xanthosine from 5-amino-1-β-d-ribofuranosyl-4-imidazolecarboxyamide (AICAR). The mechanistic details surrounding the synthetic efforts are reported herein.

Full text of DOI:10.1016/j.tet.2008.09.011

Tetrahedron 64 (2008) 10791–10797
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Mechanistic studies in the synthesis of a series of thieno-expanded
xanthosine and guanosine nucleosides
*
Zhibo Zhang, Orrette R. Wauchope, Katherine L. Seley-Radtke
Department of Chemistry and Biochemistry, University of Maryland, Baltimore Co., Baltimore, MD 21250, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 June 2008
Received in revised form 25 August 2008
Accepted 3 September 2008
Available online 17 September 2008
During the synthetic pursuit of guanosine (tri-G) and xanthosine (tri-X) tricyclic nucleosides analogues,
an interesting side product was discovered. In an effort to uncover the mechanistic factors leading to this
result, a series of reaction conditions were investigated. It was found that by varying the conditions, the
appearance of the side product could be controlled. In addition, the yield of the desired products could be
manipulated to afford either a 50:50 mix of both tri-G and tri-X, or a majority of one or the other. To
demonstrate the broad utility of the method, it was also adapted to the synthesis of guanosine and
Keywords:
Tricyclic nucleosides
AICAR
xanthosine from 5-amino-1-b-D-ribofuranosyl-4-imidazolecarboxyamide (AICAR). The mechanistic de-
tails surrounding the synthetic efforts are reported herein.
Ó 2008 Elsevier Ltd. All rights reserved.
Xanthosine
Guanosine
Expanded nucleosides
1. Introduction
retaining the hydrogen bonding elements involved in recognition
and base pairing.
A number of laboratories,1–9 including ours,10–13 have designed
and synthesized various structurally unique unnatural nucleosides
to study nucleic acid structure and function. Expanding the ‘letters’
of the genetic alphabet beyond the five natural nucleosides would
allow further investigation of the requirements for base pairing and
stacking, helix stability and interactions, and recognition by en-
zyme systems such as polymerases or other nucleoside metabo-
lizing enzymes involved in biological processes. A number of design
approaches to realize this goal have been explored, including
pairings based on size or shape complementarity.14–18
In addition, molecular dynamics calculations have shown that
inclusion of the heteroaromatic spacer will increase the overall
aromaticity and polarizability for the base, which in turn, will result
in an increase in stacking effects, an important factor in stabiliza-
tion of the DNA helix.34–37
We have previously reported the design and synthesis of the
first three tricyclic thieno-expanded purine ribonucleosides11 and
their corresponding bases,13 tricyclic adenosine (tri-A), guanosine
(tri-G), and inosine (tri-I) shown in Figure 1. In parallel to the
studies in DNA,34 the tricyclic nucleosides were investigated in
A more traditional strategy relies on pairing up complementary
donor–acceptor patterns between unnatural bases.9,19–21 Recently,
use of expanded purine nucleosides such as Nelson Leonard’s22–24
lin-benzoadenosine has been explored, an approach we began to
pursue some time ago beginning with the synthesis of a series of
thieno-expanded tricyclic nucleosides.11,13,25,26
In contrast to Leonard’s linear system, which has been exten-
sively studied by Kool,2–4,27–29 and Matteucci’s extended cytidine
analogues30–33 the use of a heteroaromatic spacer ring provides
a number of advantages over the benzene spacer, including offering
forth a less dramatic expansion of the helix due to the curvature of
the base pairing, which contracts the helix width while still
* Corresponding author. Tel.: þ1 410 455 8684; fax: þ1 410 455 2608.
E-mail address: kseley@umbc.edu (K.L. Seley-Radtke).
Figure 1. Thieno-expanded nucleosides.
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.09.011

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Z. Zhang et al. / Tetrahedron 64 (2008) 10791–10797
several enzyme systems and not surprisingly, all three were rec-
ognized by nucleoside transporters, both in their original nucleo-
side form as well as the corresponding nucleobases.38 The extended
aromatic system resulted in an increase in recognition and uptake
over the natural nucleobases. In addition, the tricyclic nucleosides
were readily recognized, and in some cases, preferentially, by nu-
cleoside metabolizing enzymes such as guanosine fucose pyro-
phosphorylase39–41 and RNA dependent RNA polymerases, thus
providing impetus for expanding our drug design studies with
additional analogues.
the steps, no longer requiring tedious column chromatography,
since the products could be readily purified via recrystallization.
Beginning with 6 (Scheme 1), conversion to the key bicyclic in-
termediate 11 was accomplished in five steps in a 16% overall yield.
In that regard, xanthosine (Fig. 2) is a naturally occurring purine
nucleoside that has attracted some interest in recent years due to
the ability to act as a universal base.42,43 Xanthosine offers forth
a Watson–Crick ‘acceptor–donor–acceptor’ pairing pattern, thus
can advantageously pair with a number of unnatural nucleosides.
One notable example is Benner’s diaminopyrimidine pairing with
xanthosine.44–49 This nonstandard pairing was recently shown to
be utilized with satisfactory fidelity by variants of HIV reverse
transcriptase for synthesis of duplex DNA.44 This finding is highly
encouraging to those pursuing expansion of the genetic alphabet,
since this is the first example of DNA containing base pairs with
alternative hydrogen bonding patterns that was efficiently
amplified by PCR.44 Spurred on by those findings, we employed our
tricyclic scaffold to produce the corresponding expanded tricyclic
xanthosine (tri-X, 4) and the corresponding 20-deoxyxanthosine 5,
shown in Figure 2. In the process of this synthetic effort, an
unexpected side product was discovered, thus prompting a mech-
anistic study of the formation of these analogues.
Scheme 1. Reagents and conditions: (a) (i) EtMgBr, THF, 4 h; (ii) anhydrous DMF; (b)
pyrimidine, hydroxylamine hydrochloride, anhydrous EtOH, reflux; (c) Ac2O, reflux; (d)
NH2C(O)CH2SH, K2CO3, DMF, 60 ꢀC; (e) NaOEt, EtOH.
At this point the ring closure was undertaken using a one-pot,
four-step procedure detailed in Scheme 2 to give both benzyl-
protected tri-G (12) and a small amount of tri-X (13).11,54 Next,
increasing the ratio of sodium hydroxide to substrate in step 1
successfully improved the ratio of tri-G to tri-X (entry 2 in Table 1)
but surprisingly, this also resulted in the formation of a new com-
pound not previously observed. Following characterization by NMR
and elemental analysis, this product proved to be a methoxy-
substituted nucleoside (14, Fig. 3). This was quite unexpected, be-
cause to our knowledge, no similar methoxy side product had ever
been reported in the literature for these reactions, thus we sought
to further investigate the cause of this result.
Figure 2. Xanthosine and thieno-expanded xanthosine nucleosides.
Scheme 2. Reagents and conditions: (a) (i) NaOH, MeOH, rt; (ii) CS2, MeOH, 145 ꢀC,
18 h; (iii) H2O2, MeOH, 0 ꢀC, 2 h; (iv) NH3, MeOH, 125 ꢀC, 18 h.
2. Results and discussion
2.1. Manipulation of the ring closure reaction
The most straightforward explanation could be attributed to
a methoxy anion acting as a nucleophile due to the increase in base
present, however, this seemed unlikely given the intermediates
proposed in the literature for similar reactions. It should be noted
that in contrast to the literature reports,52,53 which utilize NaOH
instead of NaOMe in the final step, this was not possible with our
compounds due to lack of solubility in aqueous solution as a result
of the benzyl protecting groups, which were not present in the
literature examples. In an effort to more closely follow those pro-
cedures, removal of the benzyl groups at the bicyclic stage was
carried out, however, the yield for the cyclization reaction for the
deprotected bicyclic intermediate (step 1) was unsatisfactory, thus
this route was not pursued further.
Speculating that the nucleophilic attack by the OMe could not be
occurring in the first step, but most likely in the third or fourth step
when the leaving groups would be more favorable, we returned to
the benzyl-protected bicyclic intermediate 11. It was rationalized
that by significantly increasing the amount of MeOH used in the last
step, it would effectively decrease the overall concentration of the
NaOH still present in the reaction mixture, thus lowering the
concentration of the OMe anion. This indeed proved to be the case;
In reviewing the literature, only a few routes appeared to be
available to obtain xanthosine nucleosides directly, and both were
plagued with extremely poor yields, ranging from a low of 8% to
a moderate yield of 47%.50–53 Interestingly, a number of literature
routes to guanosine have noted xanthosine as a minor side product,
however, none of these reports evaluated the mechanistic details
that would result in successfully obtaining various ratios of the
products. Indeed, during the course of scaling up our synthesis of
the tri-G analogue 2, we also noted that a small amount of tricyclic
xanthosine was formed. Speculating that it might be possible to
obtain both 12 and 13 in more satisfactory quantities by altering the
reaction conditions, or by manipulating the conditions such that
either the xanthosine or the guanosine would be obtained as the
major product, we began a mechanistic investigation, since either
scenario would prove useful to us and the synthetic community.
Our original route11,13,26 began with ribosylated 4,5-dibromo-
imidazole, however, more recently12 a switch to the 4,5-diiodo
substituted imidazole intermediate 6 shown in Scheme 1 was
made, since it provided a much more facile workup for several of

Z. Zhang et al. / Tetrahedron 64 (2008) 10791–10797
10793
Table 1
Ring closure conditions and product ratioa
Entry Substrate/NaOH Temperature Reaction time Volume of
Product ratio
12/14/13
equivalents
(step 1)
(ꢀC) (step 2) (h) (step 2)
MeOH
(mL) (step 4)
1
2
3
4
5
6
7
8
9
1:5
1:6
1:10
1:40
1:5
1:5
1:5
145
145
145
145
180
145
145
145
145
18
18
18
18
6
18
18
18
18
80
80
80
80
80
160
250
250
250
3:0:1
3:1:1
2:4:3
2:7:3
3:2:1
2:1:2
1:0:1
Figure 4. Ring closure reaction intermediates.
1:6
1:8
1:Trace:1
2:1:2
such as guanosine and xanthosine, as well as an important player in
the field of universal nucleosides. Using the conditions in Table 1,
entry 7, AICAR (17, Scheme 3) was converted to a 1:1 ratio of xan-
thosine (19) and guanosine (20) (40% for each), thus confirming the
practicality of this method and the ability to manipulate the re-
action conditions to deliver both nucleosides in good yield. It
should be noted that the TBDMS group was utilized in place of the
benzyl protecting group, since AICAR was obtained commercially,
and use of the benzyl group would have required a protracted
protection–deprotection strategy, since AICAR contains labile
amino groups in addition to the ribose hydroxyls.
a
Based on an average of at least five separate reactions for each set of conditions.
an initial increase of MeOH from 80 mL to 160 mL improved the
ratio of 12 to 13, and significantly decreased the formation of the
OMe product 14. Further dilution with 250 mL of MeOH produced
the desired affect, and resulted in a 1:1 ratio of 12 to 13 with no
formation of the tri-OMe side product 14. To test this further, the
amount NaOH was increased by 1 equiv (entry 8 in Table 1), and
only a trace of tri-OMe was found, while increasing it even further
resulting in the reappearance of the tri-OMe product 14, thus
supporting our theory.
To substantiate this hypothesis, each step of the one pot reaction
mixture was monitored closely by mass spectrometry. In the sec-
ond step, following the addition of CS2, mass spectrometry analysis
showed three major components, one with a mass of 649.1 [MþNa]
C33H29N4NaO5S2 corresponding to intermediate 15 (Fig. 4), the
other with a mass of 633.2 [MþNa] C33H29N4NaO6S corresponding
to the tri-X 13, and a third corresponding to the mass of the gly-
cosidic bond cleaved sugar moiety, coincidentally, with a methoxy
group present, however, no mass corresponding to the C2-OMe
side product 14.
After addition of H2O2 in the next step, TLC showed a new
product had formed and mass spectrometry indicated that this was
the oxidized sulfonic acid product 16 (Fig. 4), however, it was not
until after addition of the NH3 in the final step that the tri-OMe side
product 14 began to appear. This observation supports the premise
that the side product was not forming until the last step, despite the
fact that the MeOH and NaOH are introduced in the first step of the
one pot procedure. The OMe anion present in the reaction mixture
is obviously in competition with the ammonia as a nucleophile,
however, the other intermediates formed in previous steps are not
reactive enough to undergo nucleophilic substitution by the OMe
anion. Once the sulfonic acid moiety has formed, however, it
presents an excellent leaving group that is then attacked by both
the OMe and ammonia. Indeed, increasing the amount of NaOH
added in the first step leads to the formation of the OMe byproduct
14 as the major product, but only after formation of the –SO3Na
adduct (16), thus confirming our mechanistic hypothesis for the
formation of the methoxy analogue.
Scheme 3. Reagents and conditions: (a) TBDMSCl, imidazole, DMF; (b) (i) NaOH,
MeOH, rt; (ii) CS2, MeOH, 145 ꢀC, 18 h; (iii) H2O2, MeOH, 0 ꢀC, 2 h; (iv) NH3, MeOH,
125 ꢀC, 18 h.
Finally, our efforts turned to conversion of 4 to the requisite
20-deoxy tri-X (5). Deprotection of the OBn groups also fortuitously
deblocked the 2-OMe group resulting in an excellent yield of 4
(Scheme 4).
Mechanistic questions answered, we moved on to test the utility
of the ring closure conditions for more standard nucleosides. AICAR
is a key intermediate in the synthesis of a number of nucleosides
Figure 3. 2-Methoxy-substituted thieno-expanded xanthosine.
Scheme 4. Reagents and conditions: (a) THF$BF3, CH2Cl2, rt, 24 h.

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Z. Zhang et al. / Tetrahedron 64 (2008) 10791–10797
Next, using the well-known Barton deoxygenation pro-
4.2. New procedures in the synthesis of bicyclic
intermediate 11
cedures,55,56 bis-silyl protection of the 30- and 50-hydroxyl groups
(Scheme 5), followed by treatment with phenyl chloro-
thionoformate to give the thio-ester resulted in 22. Treatment with
AIBN and tributyl tin hydride removed the 20-hydroxyl group to
give 23. Subsequent deblocking of the silyl protecting group with
standard conditions then provided 5 in a 9.5% overall yield in four
steps from 4.
4.2.1. 2,3-Dibenzyloxy-5-benzyloxymethyl-1-[(5-iodo-4-
carbaldehyde)imidazole-3-yl]- -ribofuranose (7)
b-D
Compound 6 (28.47 g, 39.41 mmol) was dissolved in anhydrous
THF (300 mL), then EtMgBr (12.88 mL, 3 M solution) was added
dropwise under N2. The reaction mixture was stirred for 5 h at
room temperature. Anhydrous DMF (20 mL) was added and the
reaction mixture stirred overnight, at which point the solvent was
removed under vacuum. Saturated NH4Cl (200 mL) was added, and
the reaction mixture extracted with CH2Cl2 (3ꢁ500 mL). The or-
ganic layers were combined, washed with brine, dried over anhy-
drous MgSO4, and the solvent removed under vacuum to give
a yellow syrup. The crude syrup was purified via column chroma-
tography eluting with hexanes/EtOAc (5:1) to give 16.1 g of 7 as
a white solid (60%), mp 160–161.5 ꢀC. 1H NMR (400 MHz, CDCl3):
9.70 (s, 1H), 8.63 (s, 1H), 7.25–7.31 (m, 15H), 6.45 (d, 1H, J¼4.0 Hz),
4.36–5.01 (m, 6H), 4.27–4.30 (m, 1H), 4.20–4.22 (m, 1H), 3.90–3.99
(m, 2H), 3.63–3.66 (m,1H); 13C NMR (100 MHz, CDCl3): 181.0, 143.5,
137.5, 137.4, 137.3, 128.8, 128.5, 128.3, 128.1, 128.0, 127.8, 102.5, 89.8,
80.9, 80.1, 74.3, 73.5, 72.7, 72.3, 67.0; ESI-MS calcd for C30H29IN2
-
NaO5 [MþNa]þ 647.11, found 647.2; HR-FAB calcd for C30H30IN2O5
Scheme 5. Reagents and conditions: (a) 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane,
pyridine, rt, 24 h; (b) phenyl chlorothionoformate, DMAP, rt, 24 h; (c) AIBN, Bu3SnH,
toluene, reflux, 6 h; (d) 1 M TBAF, THF, rt, 4 h.
[MþH]þ 625.11995, found 625.12032.
4.2.2. 2,3-Dibenzyloxy-5-benzyloxymethyl-1-[(5-iodo-4-
carbaldehydeoxime)imidazole-3-yl]-b-D-ribofuranose (8)
3. Summary
Compound 7 (22.8 g, 36.5 mmol) was dissolved in anhydrous
EtOH (300 mL) and hydroxyamine hydrochloride (3.10 g,
44.15 mmol) was added, followed by addition of pyridine (3 mL).
The reaction mixture was refluxed for 2.5 h, the solvent removed
under vacuum, H2O (200 mL) added, and the reaction mixture
extracted with CH2Cl2 (3ꢁ500 mL). The organic phases were
combined, dried over anhydrous MgSO4, then evaporated to afford
23.0 g of 8 (quantitative) as a white solid, which was used directly
without further purification, mp 158 ꢀC. 1H NMR (400 MHz, CDCl3):
8.27 (s, 1H), 8.07 (s, 1H), 7.25–7.31 (m, 15H), 6.43 (d, 1H, J¼1.8 Hz),
4.47–4.70 (m, 6H), 4.31–4.33 (m, 1H), 4.18–4.23 (m, 1H), 3.99–4.03
(m, 1H), 3.83 (dd, J¼2.3, 11.0 Hz, 1H), 3.56 (dd, J¼2.3, 11.0 Hz, 1H);
13C NMR (100 MHz, CDCl3): 142.0, 140.6, 138.1, 137.5, 128.7, 128.6,
128.1, 128.0, 127.9, 127.7, 90.1, 81.2, 81.1, 75.3, 73.5, 73.1, 72.9, 67.6;
HR-FAB calcd for C30H31IN3O5 [MþH]þ 640.13085, found 640.13096.
In summary, reaction conditions were developed to provide
a facile route to realize both guanosine and xanthosine analogues in
a 1:1 ratio in excellent yield. In addition, two new tricyclic xan-
thosine analogues were synthesized and a mechanistic puzzle for
the formation of an unexpected, but potentially interesting, side
product was unraveled. Current efforts are focused on the synthesis
of the triphosphate and phosphoramidite of 5, for use in studying
the unique hydrogen bonding properties of xanthosine analogues,
as well as their recognition by biological significant systems. The
details of those studies will be reported in due time as they become
available.
4. Experimental
4.1. General
4.2.3. 2,3-Dibenzyloxy-5-benzyloxymethyl-1-[(5-iodo-4-
carbonitrile)imidazole-3-yl]-b-D-ribofuranose (9)
All chemicals were obtained from commercial sources and used
without further purification unless otherwise noted. Anhydrous
DMF, MeOH, DMSO, and toluene were purchased from Fisher Sci-
entific. Anhydrous THF, acetone, CH2Cl2, CH3CN, and ether were
obtained using a solvent purification system (mBraun Labmaster
130). Melting points are uncorrected. NMR solvents were pur-
chased from Cambridge Isotope Laboratories (Andover, MA). All 1H
and 13C NMR spectra were obtained on a JEOL ECX 400 MHz NMR,
operated at 400 and 100 MHz, respectively, and referenced to in-
ternal tetramethylsilane (TMS) at 0.0 ppm. The spin multiplicities
are indicated by the symbols s (singlet), d (doublet), dd (doublet of
doublets), t (triplet), q (quartet), m (multiplet), and br (broad).
Reactions were monitored by thin-layer chromatography (TLC)
using 0.25 mm Whatman Diamond silica gel 60-F254 precoated
plates. Column chromatography was performed using silica gel
Compound 8 (22.0 g, 34.5 mmol) was suspended in anhydrous
acetic anhydride (300 mL), refluxed for 3 h, at which point the
acetic anhydride was removed under reduced pressure to afford
a crude brown syrup. Following purification by silica gel chroma-
tography eluting with hexanes/EtOAc (6:1), 16.6 g of 9 was obtained
as a white solid (85%). 1H NMR (400 MHz, CDCl3): 7.84 (s, 1H), 7.22–
7.39 (m, 15H), 5.87 (d, 1H, J¼4.6 Hz), 4.47–4.70 (m, 6H), 4.38–4.40
(m, 1H), 4.14–4.20 (m, 2H), 3.78 (dd, 1H, J¼3.2, 10.5 Hz), 3.60 (dd,
1H, J¼3.2, 10.5 Hz); 13C NMR (100 MHz, CDCl3): 139.0, 137.3, 137.3,
136.7, 128.8, 128.7, 128.3, 128.1, 110.1, 103.9, 89.7, 82.9, 80.7, 75.5,
73.7, 72.8, 72.6, 68.6, 63.9; HR-FAB calcd for C30H29IN3O4 [MþH]þ
622.12028, found 622.12037.
4.3. Ring closure procedures
(63–200 m) from Dynamic Adsorbtions Inc. (Norcross, GA), and
eluted with the indicated solvent system. Yields refer to chro-
matographically and spectroscopically (1H and 13C NMR) homo-
geneous materials. Mass spectra were recorded at the Johns
Hopkins Mass Spectrometry Facility. Elemental analyses were
recorded at Atlantic Microlabs, Inc. (Norcross, GA).
4.3.1. 2,3-Dibenzyloxy-5-benzyloxymethyl-1-[(2-amino-imidazo-
[40,50:4,5]-thieno-[3,2-d]pyrimidin-3-yl-7-one)]-
b-
D
-ribofuranose
(12), 2,3-dibenzyloxy-5-benzyloxymethyl-1-[(5-hydroxylimidazo-
[40,50:4,5]-thieno-[3,2-d]-pyrimidin-3-yl-7-one)]-
-ribofuranose
(13), and 2,3-dibenzyloxy-5-benzyloxymethyl-1-[(5-methyl-
b-D

Z. Zhang et al. / Tetrahedron 64 (2008) 10791–10797
10795
oxyimidazo-[40,50:4,5]-thieno-[3,2-d]-pyrimidin-3-yl-7-one)]-
ribofuranose (14)
In a steel reaction vessel, compound 11 (993 mg, 1.7 mmol) was
dissolved in 40 mL anhydrous MeOH, NaOH (680 mg, 17 mmol)
added and the mixture stirred at room temperature for 30 min until
b
-D-
removed under reduced pressure to give a crude yellow syrup,
which was purified via column chromatography eluting with hex-
ane/EtOAc (2:1) to give 18 as a colorless syrup (450 mg, 66.6%). 1H
NMR (400 MHz, CDCl3): 7.53 (s, 1H), 5.48 (d, 1H, J¼5.96 Hz), 4.42 (s,
2H, NH2), 4.26–4.25 (m, 1H), 4.17–4.16 (m, 1H), 4.06–4.03 (m, 1H),
3.88–3.84 (m, 1H), 3.78–3.75 (m, 1H), 0.91 (s, 9H), 0.89 (s, 9H), 0.83
(s, 9H), 0.87 (s, 3H), 0.82 (s, 3H), 0.80 (s, 3H), 0.77 (s, 3H), 0.06 (s,
3H), ꢂ0.11 (s, 3H); 13C NMR (100 MHz, CDCl3): 159.3, 137.4, 132.0,
112.9, 89.9, 85.8, 82.8, 72.1, 62.6, 26.1, 25.9, 25.8,18.5,18.1,17.9, ꢂ4.6,
ꢂ5.3, ꢂ5.4; ESI-MS calcd for C27H56N4O5Si3þH: 601.01, found:
601.03.
a clear solution was obtained. CS2 (615 mL, 10.2 mmol) was added,
the vessel sealed, and the reaction heated in an oil bath at 145 ꢀC for
18 h. The vessel was cooled, and the solvent removed under re-
duced pressure to give an orange solid to which MeOH (80 mL) was
added. The mixture was cooled to 0 ꢀC and 7.5 mL H2O2 added. This
mixture was stirred at 0 ꢀC for 2 h, then transferred to a steel re-
action vessel, cooled to 0 ꢀC, and NH3 bubbled into the solution for
20 min. The reaction vessel was sealed and heated in oil bath at
120 ꢀC for 18 h. The vessel was cooled, the reaction mixture evap-
orated to dryness under vacuum to give a yellow solid. The crude
mixture was purified by chromatography eluting with hexanes/
EtOAc (1:1) to afford 424.4 mg of 14 as a white solid (40%), mp
182 ꢀC. 1H NMR (400 MHz, CDCl3): 8.22 (s, 1H), 7.22–7.34 (m, 10H),
7.05–7.16 (m, 5H), 6.41 (d, 1H, J¼4.6 Hz), 4.62–4.70 (m, 3H), 4.50–
4.58 (m, 3H), 4.41–4.44 (m, 1H), 4.25–4.28 (m, 1H), 4.08–4.13 (m,
1H), 3.80–3.82 (m, 1H), 3.78 (s, 3H), 3.65 (dd, 1H J¼3.6, 10.5 Hz); 13C
NMR (100 MHz, CDCl3): 161.5, 156.4, 151.6, 144.4, 143.1, 137.5, 137.2,
128.6, 128.0, 127.9, 115.6, 88.9, 82.1, 80.1, 75.6, 73.6, 72.5, 72.4, 68.9,
60.5, 55.3. ESI-MS calcd for C34H32N4O6S: 624.2, found 625.4
[Mþ1]þ. Anal. Calcd for C34H32N4O6S: C 65.37, H 5.16, N 8.97, S 5.13.
Found C 65.22, H 4.86, N 9.04, S 5.19. Further elution with hexanes/
EtOAc (1:2) afforded 207.5 mg of 13 as a white foam (20%). 1H NMR
(400 MHz, CDCl3): 9.59 (s, 1H), 9.20 (s, 1H), 7.89 (s, 1H), 6.94–7.34
(m, 15H), 5.80 (d, 1H, J¼8.2 Hz), 4.99–5.06 (m, 1H), 4.41–4.64 (m,
4H), 4.24–4.27 (m, 2H), 3.89–3.92 (m, 1H), 3.77–3.83 (m, 2H), 3.36–
3.39 (dd, 1H, J¼1.8, 11.0 Hz); 13C NMR (100 MHz, CDCl3): 159.9,
152.4, 151.0, 144.3, 137.1, 135.9, 128.8, 128.7, 128.4, 128.3, 122.0,
110.3,100.0, 88.0, 84.4, 80.3, 75.1, 73.6, 73.1, 72.9, 67.8; HR-FAB calcd
for C33H31N4O6S [MþH]þ 611.19643, found 611.19878. Further elu-
tion with hexanes/EtOAc (1:3) provided 12 as a white foam
(310.65 mg, 30%) whose spectral data agreed with the literature
values.11
4.3.4. Synthesis of TBDMS-protected xanthosine (19) and
guanosine (20)
In a steel reaction vessel, 18 (150 mg, 0.25 mmol) was dis-
solved in 10 mL anhydrous MeOH, NaOH (100 mg, 2.5 mmol)
added, and the mixture stirred at room temperature for 30 min
until a clear solution was obtained. CS2 (90 mL, 1.5 mmol) was
then added and the steel vessel sealed and heated at 145 ꢀC for
18 h. The vessel was cooled, and the solvent removed under
reduced pressure to give an orange solid, to which MeOH
(10 mL) was added. The mixture was then cooled to 0 ꢀC, 1.1 mL
H2O2 added, and the mixture stirred at 0 ꢀC for 2 h. Following
transfer to a steel reaction vessel, the mixture was again cooled
to 0 ꢀC and NH3 bubbled into the solution for 20 min. After
saturation with NH3, the vessel was sealed and heated at 120 ꢀC
for 18 h. The bomb was cooled and the reaction mixture evap-
orated to dryness to give a crude yellow solid, which was pu-
rified by column chromatography eluting with hexanes/EtOAc
(2:1) to afford 19 (62.4 mg, 40%) as a colorless syrup. 1H NMR
(400 MHz, CDCl3): 12.45 (s, 1H), 10.25 (s, 1H), 7.45 (s, 1H), 5.67
(d, 1H, J¼5.6 Hz), 4.31–4.33 (m, 1H), 4.20–4.22 (m, 1H), 4.04–4.11
(m, 2H), 4.01–4.03 (m, 1H), 0.96 (s, 9H), 0.94 (s, 9H), 0.92 (s,
9H), 0.90 (s, 3H), 0.87 (s, 3H), 0.85 (s, 3H), 0.79 (s, 3H), 0.09 (s,
3H), ꢂ0.47 (s, 3H); 13C NMR (100 MHz, CDCl3): 160.1, 152.2,
142.7, 130.9, 122.5, 110.1, 91.2, 83.0, 75.6, 69.5, 60.9, 29.8, 25.8,
22.8, 17.3, 17.2, 17.0, ꢂ4.6, ꢂ5.4, ꢂ5.6; ESI-MS calcd for
C28H54N4O6Si3þH: 627.3, found: 627.3. Further elution with
hexane/EtOAc (1:3) gave 20 (62.3 mg, 40%). 1H NMR (400 MHz,
CDCl3): 12.5 (s, 1H), 8.5 (s, 1H), 8.08 (s, 1H), 6.06 (d, 1H,
J¼4.6 Hz), 4.79–4.88 (m, 1H), 4.15–4.29 (m, 2H), 3.97–4.01 (m,
1H), 3.71–3.74 (m, 1H), 3.40–3.41 (m, 1H), 0.95 (s, 9H), 0.93 (s,
9H), 0.91 (s, 9H), 0.90 (s, 3H), (s, 3H), 0.88 (s, 3H), 0.85 (s, 3H),
0.77 (s, 3H), 0.09 (s, 3H), ꢂ0.48 (s, 3H); 13C NMR (100 MHz,
CDCl3): 157.4, 137.6, 112.8, 89.89, 85.81, 82.9, 72.1, 62.6, 29.1,
26.2, 25.9, 18.8, 18.5, 17.7, ꢂ4.6, ꢂ5.3, ꢂ5.5; ESI-MS calcd for
C28H55N5O5Si3þH: 626.02, found: 626.3.
4.3.2. 2,3-Dibenzyloxy-5-benzyloxymethyl-1-[(2-amino-imidazo-
[40,50:4,5]-thieno-[3,2-d]-pyrimidin-3-yl-7-one)]-
b-D-ribofuranose
(12) and 2,3-dibenzyloxy-5-benzyloxymethyl-1-[(5-hydroxyl-
imidazo-[40,50:4,5]-thieno-[3,2-d]-pyrimidin-3-yl-7-one)]-
b
-D-ribofuranose (13)
In a steel reaction vessel, 11 (1.35 g, 2.31 mmol) was dissolved in
40 mL anhydrous MeOH and NaOH (462 mg, 11.55 mmol) added.
This mixture was stirred at room temperature for 30 min until
a clear solution was obtained, then CS2 (836 mL, 13.87 mmol) was
added and the vessel sealed and heated in an oil bath at 145 ꢀC for
18 h. The solvent was removed under reduced pressure to obtain an
orange solid, and 80 mL MeOH added. The reaction mixture was
cooled to 0 ꢀC, 7.5 mL H2O2 added, and the mixture stirred at 0 ꢀC
for 2 h. The mixture was transferred to a steel reaction vessel, di-
luted with MeOH (250 mL), cooled to 0 ꢀC, and NH3 bubbled in for
20 min. The vessel was sealed and heated in oil bath at 120 ꢀC for
18 h. The vessel was cooled, the solvents removed under vacuum to
give a yellow solid. The crude compound was purified by chroma-
tography eluting with hexanes/EtOAc (1:2) to afford 12 as white
foam (634.67 mg, 45%). As before, further elution with hexanes/
EtOAc (1:3) gave 13 (633.51 mg, 45%). Spectral data was identical to
the previous entries.
4.4. Deprotection procedures
4.4.1. 1-[(5-Hydroxylimidazo[40,50:4,5]thieno[3,2-d]pyrimidin-3-yl-
7-one)]-
Compound 13 (200 mg, 0.328 mmol) was dissolved in anhy-
drous CH2Cl2 (10 mL), and BF3$OEt2 (586.4 L, 2.82 mmol) and EtSH
b-D-ribofuranose (4)
m
(2.82 mL, 37.53 mmol) were added, and the solution stirred at room
temperature for 2 days. After evaporation of the solvent and excess
reagents, the residue was dissolved in 40 mL H2O, and washed with
CH2Cl2 (3ꢁ75 mL). The water phase was evaporated to dryness to
give a yellow syrup, which was dissolved in H2O/MeOH (1:1), then
cooled to 4 ꢀC, and a white solid precipitated. The solid was filtered,
washed with small portions of cold H2O and MeOH, and dried
under vacuum to give 98.27 mg of a hygroscopic white solid (88%)
(Note: compound 14 can be deprotected in an analogous manner to
afford 4 in similar yields). 1H NMR (400 MHz, DMSO-d6): 11.34 (s,
1H) 11.26 (s, 1H), 8.44 (s, 1H), 5.90 (d, 1H, J¼7.3 Hz), 4.80–5.40 (br,
3H), 4.02–4.08 (m, 2H), 3.90–3.97 (m, 1H), 3.60–3.69 (m, 2H); 13C
4.3.3. Synthesis of TBDMS-protected AICAR (18)
To commercially obtained AICAR (17) (300 mg, 1.12 mmol) dis-
solved in 5 mL anhydrous DMF was added imidazole (613.4 mg,
8.96 mmol) and TBDMSCl (676.1 mg, 4.48 mmol). The reaction so-
lution was stirred at room temperature for 2 days. The DMF was

10796
Z. Zhang et al. / Tetrahedron 64 (2008) 10791–10797
NMR (100 MHz, DMSO-d6): 160.4, 151.7, 150.4, 146.0, 131.6, 122.8,
108.8, 89.5, 86.7, 76.1, 70.2, 61.2; HR-FAB calcd for C12H13N4O6S
[MþH]þ 341.05558, found 341.05614. Anal. Calcd for C12H12N4O6S
(1.2H2O) C, 39.82; H, 4.01; N, 15.48; S, 8.86. Found: C, 39.91; H, 4.17;
N, 15.65; S, 8.88.
4.5.4. 1-[(5-Hydroxylimidazo[40,50:4,5]thieno[3,2-d]pyrimidin-3-yl-
7-one)]- -2-deoxyribofuranose 3,5-diol (5)
Compound 23 (86 mg, 0.15 mmol) was dissolved in 10 mL an-
hydrous THF, then 1 M TBAF in THF (680 L) was added. The solu-
b-D
m
tion was stirred at room temperature for 4 h, the solvent was
evaporated to provide a pale-brown syrup. Purification with silica
gel chromatography eluting with hexanes/EtOAc (1:2) gave 30 mg
of 5 as a hygroscopic white solid (61%). 1H NMR (400 MHz, DMF-
d7): 11.03 (s,1H),10.94 (s,1H), 8.4 (s,1H), 6.25–6.29 (m,1H), 5.54 (br
s, 1H), 5.25 (br s, 1H), 4.32 (br s, 1H), 3.82–3.83 (m, 1H), 3.50–3.63
(m, 2H); 13C NMR (100 MHz, DMF-d7): 160.2, 151.6, 131.7, 127.7,
113.9, 111.2, 108.7, 88.5, 86.9, 70.5, 61.3, 42.7; HR-FAB calcd for
C12H13N4O5S [MþH]þ 325.06067, found 325.06021. Anal. Calcd for
C12H12N4O5S (1.65H2O) C, 40.71; H, 4.36; N, 15.83; S, 9.06. Found: C,
40.77; H, 3.99; N, 15.56; S, 8.89.
4.5. Synthesis of the 20-deoxy thieno-expanded xanthosine
nucleoside
4.5.1. 1-[(5-Hydroxylimidazo[40,50:4,5]thieno[3,2-d]pyrimidin-3-yl-
b-D
7-one)]-10- -ribofuranoside-30,50-O-
(tetraisobutyrylsilicoane)ether (21)
To a solution of 4 (292 mg, 0.86 mmol) dissolved in 12 mL
anhydrous pyridine stirring under N2 was added 1,3-dichloro-
1,1,3,3-tetraisopropylsiloxane (293 ml, 0.93 mmol). This solution
was stirred at room temperature for 24 h. The solvent was then
evaporated under reduced pressure, and an off-white solid
obtained, which was dissolved in CH2Cl2 (20 mL), washed with
water (10 mL), then saturated NaHCO3 (10 mL). The organic phases
were combined, dried over anhydrous MgSO4, and evaporated to
dryness. The residue was purified via column chromatography
eluting with hexanes/EtOAc (1:1) to give 300 mg of 21 as a white
foam (60%). 1H NMR (400 MHz, DMF-d7): 11.34 (s, 1H), 8.48 (s, 1H),
7.99 (s, 1H), 6.36 (d, 1H, J¼6.0 Hz), 4.76–4.78 (m, 1H), 4.41–4.45 (m,
1H), 4.27–4.30 (m, 1H), 4.18–4.22 (m, 1H), 4.04–4.07 (m, 1H), 0.93–
1.06 (m, 28H); 13C NMR (100 MHz, DMF-d7): 160.2, 151.5, 150.0,
131.5, 108.8, 91.8, 90.3, 83.3, 75.7, 74.3, 70.7, 60.5, 18.7, 18.0, 17.3, 17.1,
16.3, 16.0, 15.5, 14.2, 14.1, 13.7, 13.6, 13.3, 12.9, 12.7; ESI-MS calcd for
C24H38N4O7SSi2 [Mþ1]þ 583.2, found 583.1; HR-FAB calcd for
C24H39N4O7SSi2 [MþH]þ 583.2478, found 583.2478.
Acknowledgements
We are grateful to the National Institutes of Health (GM073645)
for support. We would also like to thank Joshua Sadler for editorial
help, and Dr. Pasupathy Krishnamoorthy for his efforts in the large
scale synthesis of starting materials.
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was refluxed for 6 h, and the solvent was evaporated under vacuum
to give a yellow syrup. The crude syrup was purified via chroma-
tography eluting with hexanes/EtOAc (1:1) to give 50 mg of 23
(52%). 1H NMR (400 MHz, DMF-d7): 11.60 (s,1H), 8.67 (s,1H), 8.17 (s,
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