Oxidative cleavage of ribofuranose 5-(α-hydroxyphosphonates): a route to erythrofuranose-based nucleoside phosphonic acids

Article abstract of DOI:10.1016/j.tet.2006.07.056

We report here an oxidative cleavage of (5R)- and (5S)-ribofuranosyl-5-C-phosphonate derivatives with periodate anion under both strong acidic and neutral conditions. In both cases, only (5R)-configured compound underwent the expected oxidation reaction a

Full text of DOI:10.1016/j.tet.2006.07.056

Tetrahedron 62 (2006) 9742–9750
Oxidative cleavage of ribofuranose 5-(a-hydroxyphosphonates):
a route to erythrofuranose-based nucleoside phosphonic acids
ˇ
Sarka Kralıkova, Milos Budesınsky, Ivana Tomeckova and Ivan Rosenberg
*
´
ˇ
ˇˇ´
ꢀ
ꢀ
ꢀ
ꢀ
ꢁ
ꢀ
´
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nam. 2,
166 10 Praha 6, Czech Republic
Received 21 November 2005; revised 4 July 2006; accepted 20 July 2006
Available online 17 August 2006
Abstract—We report here an oxidative cleavage of (5R)- and (5S)-ribofuranosyl-5-C-phosphonate derivatives with periodate anion under
both strong acidic and neutral conditions. In both cases, only (5R)-configured compound underwent the expected oxidation reaction and af-
forded the desired (4R)-erythrofuranosylphosphonate, whereas the second epimer, (5S)-ribofuranosyl-5-C-phosphonate did not provide the
corresponding (4S)-erythrofuranosylphosphonate derivative. This different behavior of epimers toward oxidative cleavage is an important
phenomenon. The obtained (4R)-erythrofuranosylphosphonate was used for the preparation of phosphonate mimic of adenosine 50-phosphate
via classical nucleosidation reaction. Condensation of the protected shortened AMP analogue with adenosine derivatives, however, provided
only the 20,50-linked ApA analogue. Study on hybridization of the modified 20-50 ApAwith polyU revealed its ability to form stable triplex-like
complex, similar to natural 20-50 r(ApA) and 30-50 r(ApA). NMR spectroscopy study showed that the erythrofuranose part of the phosphonate
nucleotide unit of modified 20-50 ApA was predominantly in the C20-endo conformation, which is characteristic for B-DNA.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
resembles the features of the earlier reported nucleoside
phosphonic acids 114,15 and 216,17 (Fig. 1), and the ribo ApA
dimer 18a with the isopolar, shortened phosphonate inter-
nucleotide linkage.
Structurally diverse isopolar phosphonate nucleotide ana-
logues containing a bridging P–C bond have attracted our at-
tention for many years.1–7 This class of compounds, known
as nucleoside phosphonic acids, represents a pool of poten-
tial antimetabolites exhibiting absolute stability against
phosphomonoesterase and nucleotidase cleavage.8 Some of
these compounds have already found clinical use as potent
antivirals.9–11 The search for novel nucleotide analogues
can reveal new biologically active compounds capable of
discriminating between cellular and viral or tumor enzymes
of nucleic acid metabolism, which could be used as antiviral
and/or anticancer agents. In addition, novel phosphonate
nucleotide analogues can be used for the construction of iso-
polar chimeric oligonucleotides containing nuclease-stable
internucleotide linkages,7 for instance, similar to already
prepared phosphonate 20,50-oligoadenylates, which are po-
tent, enzyme stable RNase L activators12 intended for RNase
L-mediated cleavage of pathogenic RNA.13
O
O
O
A
B
A
(HO)2P
(HO)2P
(HO)2P
O
O
O
HO OH
1
HO OH
2
HO OH
13
Figure 1.
2. Results and discussion
The strategy for the synthesis of 13 originated from the suit-
ably protected ribofuranosyl-5-C-phosphonate derivative
7a, the key compound obtained by a four-step synthesis
from 1,2:5,6-di-O-isopropylidene-a-D-allofuranose (3).18
Benzoylation of 3 afforded compound 4, which was selec-
tively deprotected in 60% aqueous acetic acid at 50 ꢀC to
give 5. Oxidation of the vicinal diol of 5 with sodium period-
ate in aqueous acetone resulted in the aldehyde 6. Subse-
quent addition of diethyl phosphite to aldehyde 6 in the
presence of triethylamine provided a 1:1 epimeric mixture
of (R)- and (S)-5-C-phosphonates 7a and 7b, respectively,
in an overall 54% yield (Scheme 1). The epimers were
Here we present the synthesis of the isopolar, nonisosteric
AMP analogue 13, a representative of a new class of modi-
fied nucleoside 50-phosphates, the structure of which
Keywords: AMP analogue; Nucleoside phosphonic acid; Sugar phospho-
nate; Periodate oxidation; NMR conformational analysis.
* Corresponding author. Tel.: +420 220 183 381; fax: +420 220 183 578;
e-mail: ivan@uochb.cas.cz
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.07.056

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S. Kralıkova et al. / Tetrahedron 62 (2006) 9742–9750
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´
9743
O
O
O
O
HO
HO
O
iii
~90 %
O
O
O
O
ii
i
O
O
O
O
O
O
O
O
~90 %
~100 %
HO
BzO
BzO
BzO
3
4
5
6
(EtO)2(O)P
H
OH
O
(EtO)2(O)P
OH
O
O CHO
OH
vi
v
H
OAc
OH
iv
O
(EtO)2(O)P
AcO OBz
14
OH
7c R=CHO
54 %
from 3
BzO
O
BzO
7b
7d R=H
H
OH
O
H
OH
(EtO)2(O)P
(EtO)2(O)P
O CHO
O
v
(EtO)2(O)P
O
O
OH
OH
BzO
7a
BzO
RO OBz
8
9a R=CHO
9b R=H
EtO
RO
O
O
HO
HO
O
O
ABz
A
A
P
P
(EtO)2(O)P
vi
(EtO)2(O)P
vii
O
O
OAc
ix
viii
73 %
from 7a
78 %
from 12b
AcO OBz
AcO OBz
HO OH
HO OH
12a R=Et
13
12b R=H (16 % from 10)
10
11
Scheme 1. (i) Benzoylcyanide, CH3CN, TEA; (ii) 60% aq acetic acid, 50 ꢀC, 3 h; (iii) NaIO4, 70% aq acetone, 0 ꢀC, 8 h; (iv) HP(O)(OEt)2, TEA, DCM, 80 ꢀC,
24 h; (v) (a) HIO4, 50% aq dioxane, 60 ꢀC, 3 days; (b) 0.1 M TEAB; (vi) Ac2O, DMAP, pyridine, 16 h, rt; (vii) silylated 6-N-benzoyladenine, SnCl4, CH3CN,
24 h , rt; (viii) 1 M NaOH, 48 h, rt; (ix) Me3SiBr, CH3CN, 24 h, rt. Yields of compounds 4, 5, and 6 were estimated from TLC (compounds were used for further
reaction step without purification).
separated by silica gel chromatography and subjected to sub-
sequent oxidative cleavage separately.
to depend on the time of oxidation of 7a, workup of 9a, and
conditions for acetylation of 9a to 10.
2.1. Oxidative cleavage of 7a (Scheme 1)
2.2. Oxidative cleavage of 7b (Scheme 1)
Hydrolysis of the 1,2-O-isopropylidene group of 7a and sub-
sequent oxidative cleavage of the C1–C2 bond, performed in
one step by aqueous periodic acid, led to an equilibrium mix-
ture of 8 and 9a in which the 3-O-formyl group protects the
acyclic form 8 (if present) from further oxidative cleavage of
the C4–C5 bond. We attempted to verify the presence of the
3-O-formyl group in the product (8 or 9a) but neither treat-
ment with 0.1 M TEAB in aqueous dioxane19 nor heating in
80% aqueous pyridine20 changed the mobility on TLC or RP
HPLC. It did not seem that the 3-O-formyl group in 8 could
survive in strongly acidic conditions during several days’
treatment with aqueous periodic acid. Thus, we concluded
that the nascent acyclic form 8 has to undergo a very fast cy-
clization reaction to furanose 9a. Then both the formyl de-
rivative 9a and the deformylated compound 9b are
resistant against oxidative cleavage by periodic acid. Since
the subsequent acetylation resulted in the expected acetyl
derivative 10 in good yield, the final product of oxidative
cleavage of 7a with periodic acid must have been predomi-
nantly the furanose 9b.
On the other hand, treatment of 7b with periodic acid to ob-
tain 14 caused a total decomposition of compound 7b with
the formation of a very complex mixture of derivatives. Ap-
plication of a two-step procedure20 using 90% aqueous TFA
for hydrolysis of 1,2-O-isopropylidene group in 7b in the first
step, and then, after removal of TFA, sodium periodate oxi-
dative cleavage in the second step also led to a total decom-
position of 7b. Under the same conditions, the epimer 7a
reacted as smoothly as with periodic acid alone and follow-
ing acetylation provided a comparable yield of product 10.
The nature of the very different reactivities of 7a and 7b to-
ward periodic acid (or TFA–sodium periodate20) is not quite
clear. The explanation for this phenomenon could perhaps be
seen in the equilibrium between acyclic structure 7c and its
furanose form, which can be shifted in favor of the acyclic
compound 7c. Under such conditions, hydrolysis of the
3-O-formyl group in the acyclic structure 7c leads to 7d
and this compound, bearing a C3–C4 vicinal diol, is imme-
diately cleaved by periodic acid (or sodium periodate)
(Scheme 2). It can be speculated that the cleavage of the
O-formyl group could proceed in an oxidative manner via
the extremely labile carbonic acid monoester (R-OCOOH)
but, more likely, groupings like P]O or hydrated C1
aldehyde CH(OH)2 in 7c could facilitate the formyl group
hydrolysis via intramolecular participation due to a favorable
conformation (Scheme 2). Similarly, the different reactivity
of epimers 7a and 7b toward acetolysis was described re-
cently; whereas the epimer 7a provided the expected
Note: the transformation of compound 9b to 10, which we
performed several times has resulted in several cases in
pure b-anomer of 10 (3-O-acetyl-2-O-benzoyl) but in sev-
eral cases also in an anomeric mixture of both 3-O-acetyl-
2-O-benzoyl and 2-O-acetyl-3-O-benzoyl regioisomers. We
believe that each of these isomers afforded, after nucleosida-
tion and deprotection, the identical product. The formation
of the regioisomers (migration of the benzoyl group) seems

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S. Kralıkova et al. / Tetrahedron 62 (2006) 9742–9750
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9744
product, the second epimer 7b afforded a mixture of com-
pounds.21
acetyl derivatives of 17, we found by NMR for regioisomers
at the beginning of the condensation and after 48 h of stand-
ing in pyridine solution practically identical ratios (31:69
and 29:71, respectively). Since we still reasoned that 30-50
dimer 18b was not obtained due to both low yield of conden-
sation and relatively low content of 20-O-acetyl regioisomer
in the mixture of acetyl derivatives 17, we prepared com-
pounds 19a and 19b in which the 20-hydroxyl was protected
with nonmigrating, alkali-stable tetrahydropyranyl and 1-(2-
chloroethoxy)ethyl groups, respectively, and condensed
them with 16. Surprisingly, also in this case we did not obtain
the expected 30-50 isomer 18b. Neither higher concentration
of the reactants nor the presence of DMAP gave rise to the
product 18b. Concerning the formation of the phosphonate
anhydride 20 as the primary product in the DCC-induced
condensation, and its further activation by DCC, it can be
concluded that the DCC-activated anhydride 20 cannot be
attacked, due to a steric hindrance, by the 30-hydroxyl of
compounds 19a and 19b. We did not find any other reason-
able explanation for this phenomenon.
-
EtO OEt
IO4
P
O
O
(EtO)2(O)P
H
OH
O CH
(EtO)2(O)P
H
OH
OH
H
O
CH
O
HO
OH
OH
OH
OH
OH
OH
BzO
BzO
BzO
7c
7c
7d
Scheme 2. Proposed mechanisms of periodate degradation of (S)-configured
5-hydroxyphosphonate 7c.
Concerning the different reactivity of 7a and 7b toward
periodate oxidation, the separation of epimers 7a and 7b
does not seem to be, from a practical point of view, too
advantageous since the yield of 10, when the reaction was
carried out with an epimeric mixture, varied in the range
of 38–51% whereas with pure epimer 7a, it was 65–73%.
The nucleosidation reaction of the sugar phosphonate 10
with silylated 6-N-benzoyladenine in the presence of
SnCl4 provided a mixture of products in which the expected
derivative 11 was not recognized. Therefore, the mixture
was treated with concentrated aqueous ammonia to remove
acyl-protecting groups, and the adenine-containing com-
pounds were desalted on Dowex 50. A mixture of diethyl-
and monoethyl ester 12a and 12b was obtained as the only
products in a low yield of 13–20%. Diester 12a was con-
verted to the monoester 12b by treatment with dilute sodium
hydroxide and compound 12b was isolated on Dowex 1 by
a gradient elution with acetic acid in water. With respect to
the low yield, the nucleosidation reaction, a weak point of
the synthesis, still remains open for improvement. Replace-
ment of tin tetrachloride by trimethylsilyl triflate in the
nucleosidation reaction did not provide any of the expected
products 12a and 12b. Compound 12b was transformed into
the free phosphonic acid 13 by treatment with bromotri-
methylsilane in the presence of 2,6-lutidine to prevent poten-
tial furanose ring cleavage. Ion exchange chromatography
on Dowex 1 afforded the pure free phosphonic acid 13.
2.3. Conformational features of dimer 18a
Strong NOE contacts between H-8 of the adenine, and H-20
and H-30 of the furanose ring in both nucleoside residues
indicate the preferred anti conformation of both adenine
moieties. The only interresidual NOE contact was observed
between H-20and H-8 that is commonly used for sequential
assignment in oligonucleotides. From the vicinal proton cou-
plings in the furanose rings (Table 2), a generalized Karplus–
Haasnoot equation,26 and the two-state pseudorotation
model we could establish for dimer 18a (Scheme 3) in aque-
ous solution a similar population of C30-endo and C20-endo
form (ratioz58:42) for ‘upper’ furanose ring (residue A),
whilst for the ‘lower’ furanose ring (residue B) the C20-
endo form significantly prevails (ratio C30-endo:C20-endoz
14:86). For comparison, in natural ribo ApA dimers (for
NMR data see Tables 1–3) we estimated the ratios of
C30-endo:C20-endo form to be approximately 45:55
for ‘upper’ ring and 55:45 for ‘lower’ ring in the case of
20-50 r(ApA), and z58:42 for both rings in 30-50 r(ApA).
The dimer 18a differs from natural ribo ApA dimers mainly
in the preference for the C20-endo conformation in the
‘lower’ ring with a phosphonate group in position C40.
Since compound 13 is a ‘shortened’ nucleotide analogue, its
incorporation into an oligonucleotide chain was considered
to be very interesting with respect to hybridization properties
of the modified chain. Shortening of the internucleotide link-
age renders the chain with reduced number of degrees of
freedom, which could positively influence hybridization
ability as it was found recently for another type of shortened
linkage.22 For the study of changes induced by the presence
of a modified internucleotide linkage, the dimers, as the
shortest oligonucleotides capable of base-stacking and
base-pairing, have been employed as model compounds.23,24
For the synthesis of modified ApA dimers 18a and 18b, the
monoester 12b was benzoylated by N-benzoyltetrazole25 to
yield a mixture of fully and partially benzoylated phospho-
nates 15a and 15b, which was resolved by silica gel chroma-
tography. Derivative 15a was converted into the protected
phosphonic acid 16 by bromotrimethylsilane treatment,
and this compound was condensed with a mixture of
20(30)-O-acetyl derivatives 17 in pyridine using DCC.
Deprotection in concentrated ammonia and chromatography
on Dowex 1 in acetate form afforded only the 20-50 regio-
isomer 18a in a low yield (15%). No 30-50 regioisomer 18b
was isolated. Concerning the ratio of the 20-O- and 30-O-
Concerning the conformational features of the prepared di-
mer 18a, we have subjected it to a study of hybridization
properties with polyU to compare them with those of natural
20-50 r(ApA) and 30-50 r(ApA) isomers. The hypochromic
effect of the complexes [18a*polyU] at various ratios of
18a and polyU was measured at 260 nm in the presence
of Mg2+ ions at 3 ꢀC, and at total base concentration of
0.15 mM. The obtained mixing curve exhibited a local min-
imum at 1A:2U ratio, indicating triplex-like complex forma-
tion (curve not shown). The same stoichiometry was found
for both natural ApA dimers. The thermal characteristics
of the complex [polyU*18a*polyU] exhibited only single
transition profile suggesting direct dissociation of pseudo
triplex structure into individual strands. Shortening of the
internucleotide linkage in ApA analogue 18a does not
influence the complex stability in comparison with natural
phosphodiester linkage. Under experimental conditions,
the Tm value of the complex is 15 ꢀC and lies very close

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S. Kralıkova et al. / Tetrahedron 62 (2006) 9742–9750
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9745
O
P
O
P
HO
A
O
EtO
HO
HO
HO
B
ABz
residue A
O
O
i
ii
iii
12b
HO
O
P
O
O
BzO
ABz
OBz
BzO
OBz
A
HO
15a B=ABz
15b B=A
16
residue B
iv
TBDPSO
O
HO
A
OH
18a
O
O
H, Ac
17
CEE ... 1-(2-chloroethoxy)ethyl
HO
O
80% 3'-OAc
ABz
BzO
OBz
HO
TBDPSO
O
O
P
O
OH
O
O
ABz
A
P
O
O
HO
Bz O
P
O
A
HO
OR
OH
19a R=THP
19b R=CEE
20
BzO
OBz
18b
HO
OH
Scheme 3. (i) N-Benzoyltetrazole, DMAP, DMF, 55 ꢀC; (ii) Me3SiBr, CH3CN, 24 h, rt; (iii) (a) 16, DCC, pyridine, 5 days, rt; (b) 1 M TBAF in THF, 16 h, rt;
(c) concd aq ammonia, 48 h, rt; (iv) (a) 16 and 17a or 17b, DCC, pyridine, 5 days, rt; (b) 1 M TBAF in THF, 16 h, rt; (c) concd aq ammonia, 48 h, rt; (d) aq 0.1 M
HCl, 16 h, rt.
Table 1. Proton NMR chemical shifts
Compd
Solv.
H-10
H-20
H-30
H-40
H-50
H-500
H-2
H-8
Other protons
13
7a
D2O+NaOD
DMSO
5.81
5.91
4.75
4.88
4.33
5.20
4.08
4.63
—
4.16
—
—
8.67
—
8.22
—
—
50-OH: 6.10; 2ꢁOEt: 3.95 (4H),
1.15 (3H), 1.09 (3H); i-Pr:
1.41 (3H), 1.26 (3H); Bz: 8.01 (2H),
7.65 (1H), 7.54 (1H)
7b
10
DMSO
DMSO
5.90
6.27
4.90
5.53
4.96
5.69
4.43
4.57
3.99
—
—
—
—
—
—
—
50-OH: 5.85; 2ꢁOEt: 4.05 (4H),
1.22 (3H), 1.21 (3H); i-Pr:
1.42 (3H), 1.26 (3H); Bz: 8.02
(2H), 7.70 (1H), 7.56 (1H)
2ꢁOAc: 2.10 (3H), 1.96 (3H);
Bz: 8.03 (2H), 7.71 (1H),
7.57 (2H); 2ꢁOEt: 4.09 (4H),
1.25 (3H), 1.245 (3H)
2ꢁOAc: 2.15 (3H), 2.01 (3H); Bz:
8.04 (2H), 7.62 (1H), 7.48 (2H);
2ꢁOEt: 4.20 (4H), 1.35 (6H)
NH2: 7.83; 2ꢁOH: 5.69 and 5.61;
OEt: 3.93 (2H), 1.16 (3H)
OEt: 3.98 (2H), 1.24 (3H)
—
CDCl3
DMSO
6.34
5.99
5.64
4.62
5.90
4.34
4.44
4.15
—
—
—
—
—
—
12b
8.50
8.24
D2O
D2O
6.11
6.17
5.60
5.79
5.92
4.79
5.16
4.41
4.62
4.53
4.60
4.61
4.49
4.60
4.46
4.30
4.24
4.26
4.31
4.32
—
3.90
—
3.85
4.32
—
3.77
—
3.7
4.14
8.53
7.72
8.11
7.86
8.01
8.18
8.06
8.21
8.19
8.13
18a residue A
18a residue B
20-50 ApA residue A
20-50 ApA residue B
—
—
—
D2O
Table 2. Coupling constants of protons
Compd
Solv.
Proton–proton
Proton–phosphorus
10,20
20,30
30,40
40,50
40,500
50,500
10,P
20,P
30,P
40,P
50,P
13
7a
7b
10
D2O
7.4
3.9
3.8
1.2
1.4
7.6
6.7
4.1
6.9
3.5
4.1
5.3
5.6
5.1
4.6
4.7
4.6
5.1
5.3
4.8
5.0
5.1
2.2
7.6
8.5
7.6
8.0
1.6
3.1
5.5
2.4
5.0
5.4
—
—
—
—
—
—
—
—
3.2
—
3.6
3.8
—
—
—
—
—
—
—
13.1
—
13.1
12.2
7.2
6.2
3.9
2.7
1.0
0.9
3.5
4.3
DMSO
DMSO
DMSO
CDCl3
DMSO
D2O
D2O
D2O
D2O
D2O
2.4
2.7
—
—
—
—
2.2
—
13.0
13.0
13.8
14.2
6.1
12b
2.0
1.2
7.8
18a residue A
18a residue B
20-50 ApA residue A
20-50 ApA residue B
9.3
7.4
9.0
5.4
2.5
2.5
3.8

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to the Tm values of both 20-50 and 30-50 natural ribo (ApA)
dimers, which are 13 and 16 ꢀC, respectively.23
3. Conclusion
Only (R)-epimer of the erythrofuranosyl-4-C-phosphonate
has been prepared via periodic acid-mediated oxidative
cleavage of an epimeric mixture of the pentofuranosyl-5-
C-phosphonate derivatives. We proposed a mechanism for
explaining different reactivities of starting (5R)- and (5S)-
pentofuranosylphosphonates toward oxidation. The useful-
ness of the prepared sugar phosphonate derivative for the
synthesis of novel nucleoside phosphonic acids was proved
at the nucleosidation reaction with protected adenine giving
an isopolar, nonisosteric AMP analogue. Besides, the corre-
sponding 20-50-linked diadenosine monophosphate analogue
was prepared and its hybridization properties were evalu-
ated. NMR spectroscopy study of the dimer showed that
the sugar part of the ribo-configured phosphonate nucleotide
unit was predominantly in the C20-endo conformation,
which is characteristic for B-DNA and not for RNA. The ob-
tained results suggest that a thorough study on the synthesis
of nucleoside phosphonic acids with various nucleobases,
their 20- and 30-deoxy derivatives, and also their incorpora-
tion into longer oligonucleotides seems to be fully justified
and highly desirable.
4. Experimental
4.1. General
Unless stated otherwise, the solvents were concentrated at
40 ꢀC using a rotary evaporator. The products were dried
over phosphorus pentoxide at 40–50 ꢀC and 13 Pa. The
course of the reactions was followed by TLC on silica gel
Merck and Fluka UV 254 foils and the products were visu-
alized by UV monitoring. Preparative column chromato-
graphy (PLC) was performed on silica gel (40–60 mm,
Fluka) whereby the amount of sorbent used was 20–40 times
the weight of the mixture separated. Elution was performed
at the linear flow rate of 2–4 cm minꢂ1. For TLC and PLC
runs, the following solvent systems were used (v/v): toluene–
ethyl acetate 1:1 (T), chloroform–ethanol 9:1 (C), ethyl
acetate–acetone–ethanol–water 4:1:1:1 (H1) and 12:2:2:1
(H3), 2-propanol–concentrated aqueous ammonia–water
7:1:2 (IPAW). The HPLC analyses were performed on a
LC 5000 liquid chromatograph (INGOS, Czech Republic)
using Luna C18 5 mm reverse phase column (4.6ꢁ150 mm;
Phenomenex), by gradient of methanol in 0.1 M triethyl-
ammonium acetate buffer. Mass spectra (m/z) were recorded
on ZAB-EQ (VG Analytical) instrument, using FAB in
both positive and negative modes (ionization by Xe, acceler-
ating voltage 8 kV) with glycerol–thioglycerol (3:1) and
2-hydroxyethyldisulfide as matrices. 1H and 13C NMR
spectra were measured on Varian Unity 500 instrument (1H
at 500 MHz, 13C at 125.7 MHz) in DMSO (referenced to
the solvent signals dH¼2.50 and dC¼39.7), CDCl3 (refer-
enced to TMS), and D2O (referenced to DSS). Signals in
1
the H NMR spectra were assigned to protons on the basis
of chemical shifts, observed multiplicities, and homonuclear
2D-COSYexperiments. Chemical shifts and interaction con-
stants were obtained from a first-order spectra analysis as

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9747
read from expanded records. The exchange of hydroxyl
protons for deuterium was carried out using several drops of
tetradeuterioacetic acid. Assignments of signals in 13C NMR
spectra were accomplished on the basis of J-modulated
spectra (APT) enabling to discriminate between C, CH,
CH2, and CH3 signals, and in some cases confirmed by 1H,
J¼7.6, 3.9, 2.4 Hz, H-40), 4.16 (1H, J¼13.0, 2.4 Hz, H-50),
3.95 (4H, q, J¼7.0 Hz, 2ꢁOCH2), 1.41 (3H, s, CH3), 1.26
(3H, s, CH3), 1.15 (3H, t, J¼7.0 Hz, OCH2CH3), 1.09
(3H, t, J¼7.0 Hz, OCH2CH3); dC (125.7 MHz, DMSO)
165.22, 133.82, 129.70 (2), 129.57, 128.97 (2), 112.57,
104.79, 79.52 (d, J¼12.7 Hz), 78.00, 71.30, 66.32 (d,
J¼165.0 Hz), 62.48 (d, J¼6.8 Hz), 62.21 (d, J¼6.8 Hz),
27.19, 26.94, 16.44 (d, J¼5.9 Hz), 16.38 (d, J¼4.4 Hz).
Compound 7b: dH (500 MHz, DMSO) 8.02 (2H, m, ortho-
Ar-H), 7.70 (1H, m, para-Ar-H), 7.56 (2H, meta-Ar-H),
5.85 (1H, br s, OH), 5.90 (1H, d, J¼3.8 Hz, H-10), 4.96
(1H, dd, J¼8.5, 5.1 Hz, H-30), 4.90 (1H, dd, J¼5.1,
3.8 Hz, H-20), 4.43 (1H, dt, J¼8.5, 2.7, 2.7 Hz, H-40), 3.99
(1H, J¼13.0, 2.7 Hz, H-50), 4.05 (4H, q, J¼7.0 Hz,
2ꢁOCH2), 1.42 (3H, s, CH3), 1.26 (3H, s, CH3), 1.22 (3H,
t, J¼7.0 Hz, OCH2CH3), 1.21 (3H, t, J¼7.0 Hz, OCH2CH3);
dC (125.7 MHz, DMSO) 165.48, 134.16, 129.82 (2), 129.35,
129.25 (2), 112.68, 104.79, 78.20 (d, J¼2.4 Hz), 77.15,
72.24 (d, J¼9.8 Hz), 68.68 (d, J¼6.8 Hz), 62.10 (d,
J¼6.8 Hz), 27.06, 26.91, 16.69 (d, J¼5.9 Hz), 16.62 (d,
J¼5.9 Hz). For 1H and 13C NMR data see Tables 1–3.
1
13C-correlated HMQC spectra. The H and 13C NMR data
are summarized in Tables 1–3.
4.2. (5RS)-3-O-Benzoyl-1,2-O-isopropylidene-D-ribo-
furanos-5-C-ylphosphonate (7a, 7b)
The 1,2:5,6-di-O-isopropylidene-a-D-allofuranose18
3
(13.01 g, 50 mmol) was treated with benzoylcyanide
(7.21 g, 55 mmol) and triethylamine (0.695 mL, 5 mmol)
in acetonitrile (60 mL) at 0 ꢀC under exclusion of moisture
(TLC in T-1) overnight. The reaction mixture was quenched
by addition of methanol (5 mL) and the solvent was evapo-
rated in vacuo. The residue was dissolved in chloroform and
the organic layer was washed several times with water. After
evaporation of solvent, the benzoyl derivative 4 was treated
with 60% aqueous acetic acid (600 mL) at 50 ꢀC for 3 h
(TLC in T-1 and C-1). The acid was evaporated in vacuo,
the residue was taken into chloroform (300 mL), and the
organic layer was washed with saturated aqueous solution
of NaCl (250 mL). The layers were separated by centrifuga-
tion, the organic layer was washed with water (4ꢁ250 mL),
dried over anhydrous Na2SO4, and evaporated. The residue
was dissolved in 70% aqueous acetone (650 mL) and to
this solution, saturated aqueous solution of sodium periodate
(12.84 g, 60 mmol) was added at 0 ꢀC. Resulting solution
was stirred for 8 h at rt (TLC in C-1) and, after cooling the
mixture in an ice bath, the suspension was filtered through
Celite. Filtration cake was washed with acetone, and the
combined filtrates were evaporated. The residue was re-dis-
solved in acetone (200 mL) and the rest of sodium iodate
was filtered off. Crude aldehyde 6 was co-distilled with
dry toluene, dissolved in dichloromethane (50 mL), and
treated with diethyl phosphite (7.74 mL, 60 mmol) and tri-
ethylamine (2.78 mL, 20 mmol) at 80 ꢀC for 24 h (TLC in
T-1 and C-1). The solvent was evaporated in vacuo and the
crude epimeric phosphonates 7a and 7b were purified on
silica gel using elution with a linear gradient of ethyl acetate
in toluene. Yield: 1.27 g (5%) of 7a, 1.82 g (7%) of 7b, and
10.72 g (42%) of the mixture of 7a and 7b. HR-FAB calcd
for C19H28O9P (M+H)+: 431.1471; found: 431.1470. Com-
pound 7a: nmax (KBr) 3430 (w, br, OH), 3243 (m, OH),
3072 (w, C–H), 2986 (m, CH3), 2876 (w, CH3), 1251 (s,
P]O), 1219 (m), 1206 (m), 1131 (s, COCOC), 1032 (vs,
POC), 1480 (w, OCH2CH3), 1602 (w), 1584 (w), 1492
(w), 1452 (w), 1731 (m, C]O), 1724 (s, C]O), 1275 (s,
C]O), 1217 (s, C(CH3)2), 1098 (m, COCOC), 1002 (m),
688 (w) cmꢂ1. Compound 7b: nmax (KBr) 3506 (w, br,
OH), 3272 (m, OH), 3066 (w, C–H), 2992 (m, CH3), 2871
(w, CH3), 1256 (s, P]O), 1236 (s, P]O), 1219 (m), 1206
(m), 1152 (s, COCOC), 1033 (vs, POC), 1601 (w), 1583
(w), 1492 (w), 1452 (w), 1731 (m, C]O), 1720 (s,
C]O), 1711 (s, C]O), 1282 (s, C]O), 1100 (s, COCOC),
999 (m), 692 (w) cmꢂ1. Compound 7a: dH (500 MHz,
DMSO) 8.01 (2H, m, ortho-Ar-H), 7.65 (1H, m, para-
Ar-H), 7.54 (2H, meta-Ar-H), 6.10 (1H, br s, OH), 5.91
(1H, d, J¼3.9 Hz, H-10), 5.20 (1H, dd, J¼7.6, 5.6 Hz,
H-30), 4.88 (1H, dd, J¼5.6, 3.9 Hz, H-20), 4.63 (1H, ddd,
4.3. (4R)-Diethyl-[1,3-di-O-acetyl-2-O-benzoyl-D-
erythrofuranos-4-yl]phosphonate (10)
Periodic acid dihydrate (0.99 g, 4.35 mmol) was added to a
solution of phosphonate 7a (1.25 g, 2.9 mmol) in 50% aque-
ous dioxane (20 mL), and the reaction mixture was heated at
60 ꢀC in the dark for 1–3 days (TLC in C-1). The solution was
treated with Dowex 1ꢁ2 in acetate form (10 mL) under stir-
ring for 10 min to remove periodate and iodate anions, the
resin was filtered off, washed with 50% dioxane (30 mL),
and the combined filtrates were evaporated. The residue
was co-distilled several times with water to remove acetic
acid, and finally treated with 0.1 M TEAB (30 mL) for
20 min. The aqueous solution was evaporated to dryness,
the crude 9b was co-distilled with methanol (3ꢁ50 mL),
dried with pyridine (3ꢁ10 mL), and treated with acetic anhy-
dride (1.21 mL, 12.8 mmol) and DMAP (20 mg) in pyridine
(12 mL) overnight (TLC in C-1). The reaction mixture was
quenched by addition of water (1 mL) at 0 ꢀC, the solvent
was evaporated in vacuo, and the crude product 10 was puri-
fied on silica gel byelution with a lineargradient of acetone in
toluene. Yield: 940 mg (73%, yellow oil) of 10 (b-anomer).
HR-FAB calcd for C19H26O10P (M+H)+: 445.1264; found:
445.1273. dH (500 MHz, CDCl3) 8.04 (2H, m, ortho-Ar-H),
7.62 (1H, m, para-Ar-H), 7.48 (2H, meta-Ar-H), 6.34 (1H,
d, J¼1.4 Hz, H-10), 5.90 (1H, ddd, J¼14.2, 8.0, 4.7 Hz,
H-30), 5.64 (1H, dd, J¼4.7, 1.4 Hz, H-20), 4.44 (1H, dt,
J¼8.0, 0.9 Hz, H-40), 4.20 (4H, q, J¼7.0 Hz, 2ꢁOCH2),
2.15 (3H, s, OAc), 2.01 (3H, s, OAc), 1.35 (6H, t,
J¼7.0 Hz, 2ꢁOCH2CH3); dC (125.7 MHz, CDCl3) 169.21,
169.06, 164.89, 133.72, 129.79 (2), 128.71, 128.58 (2),
98.31, 75.41, 74.11, 70.51, 64.57 (d, J¼5.9 Hz), 63.41 (d,
1
J¼5.9 Hz), 16.40 (2C, d, J¼5.9 Hz), 20.91, 20.33. For H
and 13C NMR data see Tables 1–3.
4.4. (4R)-Ethyl-[1-(adenin-9-yl)-1-deoxy-b-D-erythro-
furanos-4-yl]phosphonate (12b)
6-N-Benzoyladenine (598 mg, 2.5 mmol) in hexamethyl-
disilazane (HMDS) (25 mL, 118 mmol) was refluxed in the
presence of chlorotrimethylsilane (2.5 mL, 20 mmol) under

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stirring and exclusion of moisture for 10 h. Volatiles were re-
moved under reduced pressure and the resulting oil was co-
distilled with xylene (2ꢁ15 mL) and acetonitrile (2ꢁ15 mL)
to remove traces of HMDS and xylene, respectively. A solu-
tion of diethyl phosphonate 10 (940 mg, 2.1 mmol) (dried by
co-distillation with toluene and acetonitrile) in acetonitrile
(6 mL) was added via septum under argon to the silylated ad-
enine derivative followed by the addition of tin tetrachloride
(0.24 mL, 2 mmol). The mixture was kept at rt for 24 h (TLC
in C-1, H-1). The reaction mixture was then diluted with
pyridine (1 mL) and toluene (20 mL) and stirred overnight.
The thick suspension (precipitated complex of tin tetrachlo-
ride with pyridine) was filtered through Celite, washed with
chloroform, and the solvents were removed under dimin-
ished pressure. The residue was treated with aqueous 1 M
NaOH (50 mL) at rt for 48 h to remove the acyl-protecting
groups and one ethyl ester group from 12a (TLC in C-1,
H-1). The solution was neutralized by addition of Dowex
50 (H+), the monoethyl ester 12b was eluted with 2.5% aque-
ous ammonia, and the crude product 12b was purified on
Dowex 1ꢁ2 (CH3COOꢂ) column (elution with a linear gra-
dient of 0–0.5 M aqueous acetic acid). Yield: 160 mg (16%,
related to 7a) of ethyl phosphonate 12b. HR-FAB calcd for
C11H17N5O6P (M+H)+: 346.0917; found: 346.0913. dH
(500 MHz, DMSO) 8.50 (1H, s, H-2), 8.24 (1H, s, H-8),
5.69 (1H, br s, OH), 5.61 (1H, s, OH), 5.99 (1H, dd, J¼7.6,
2.0 Hz, H-10), 4.62 (1H, dd, J¼7.6, 4.6 Hz, H-20), 4.34
(1H, ddd, J¼6.1, 4.6, 1.6 Hz, H-30), 4.15 (1H, dd, J¼3.5,
1.6 Hz, H-40), 3.93 (2H, q, J¼7.0, 2ꢁOCH2), 1.16 (3H, t,
J¼7.0 Hz, OCH2CH3); dC (125.7 MHz, DMSO) 156.48
(2), 151.32, 140.26, 119.25, 87.32, 81.38 (d, J¼155.3 Hz),
74.53, 71.15 (d, J¼6.8 Hz), 61.22 (d, J¼6.3 Hz), 16.71 (d,
J¼4.9 Hz). For 1H and 13C NMR data see Tables 1–3.
(1 mL, 7.86 mmol) in DCM (50 mL) in the presence of tol-
uenesulfonic acid hydrate (0.19 g, 1 mmol) at rt for 2 h. The
resulting clear solution was washed with saturated aqueous
solution of sodium hydrogen carbonate, the organic layer
was dried over sodium sulfate, and evaporated. The residue
was treated with 80% aqueous acetic acid (50 mL) for
30 min, the solution was concentrated in vacuo, and crude
17 was purified on a silica gel column in chloroform–10%
ethanol. Yield: 2 g (62%; white foam) of 17 (a mixture of
20-O-Ac and 30-O-Ac regioisomers in a ratio 3:7 assigned
from NMR). HR-FAB calcd for C35H38N5O6Si (M+H)+:
652.2591; found: 652.2581.
1
4.6.1. H NMR (pyridine-d5). Compound 17, minor re-
gioisomer (20-O-Ac): 8.87 (1H, s, H-8), 8.77 (1H, s, H-2);
8.23 (2H, m), 7.75 (1H, m), 7.35 (2H, m, CO-C6H5),
7.75 (4H, m), 7.30–7.40 (6H, m, Si(C6H5)2), 6.725
(1H, d, J(10,20)¼4.1 Hz, H-10), 6.32 (1H, dd, J(20,10)¼
4.1 Hz, J(20,30)¼5.5 Hz, H-20), 5.35 (1H, dd, J(30,20)¼
5.5 Hz, J(30,40)¼5.0 Hz, H-30), 4.56 (1H, ddd,
J(40,30)¼5.0 Hz, J(40,50)¼3.2 Hz, J(40,500)¼4.4 Hz, H-40),
4.25 (1H, dd, J(50,500)¼11.6 Hz, J(50,40)¼3.2 Hz, H-50),
4.11 (1H, dd, J(500,50)¼11.6 Hz, J(500,40)¼4.4 Hz, H-500),
1.98 (3H, s, 20-OAc), 0.99 (9H, s, C(CH3)3).
Compound 17, major regioisomer (30-O-Ac): 8.83 (1H, s,
H-8), 8.78 (1H, s, H-2), 8.24 (2H, m), 7.75 (1H, m), 7.35
(2H, m, CO-C6H5), 7.75 (4H, m), 7.30–7.40 (6H, m,
Si(C6H5)2), 6.625 (1H, d, J(10,20)¼6.0 Hz, H-10), 5.58 (1H,
dd, J(20,10)¼6.0 Hz, J(20,30)¼5.5 Hz, H-20), 5.94 (1H, dd,
J(30,20)¼5.5 Hz, J(30,40)¼3.9 Hz, H-30), 4.59 (1H, q,
J(40,30)¼3.9 Hz, J(40,50)¼4.0 Hz, J(40,500)¼4.5 Hz, H-40),
4.18 (1H, dd, J(50,500)¼11.5 Hz, J(50,40)¼4.0 Hz, H-50),
4.06 (1H, dd, J(500,50)¼11.5 Hz, J(500,40)¼4.5 Hz, H-500),
2.06 (3H, s, 20-OAc), 1.02 (9H, s, C(CH3)3).
4.5. (4R)-[1-(Adenin-9-yl)-1-deoxy-b-D-erythrofuranos-
4-yl]phosphonic acid (13)
4.6.2. 13C NMR (pyridine-d5). Compound 17, minor
regioisomer (20-O-Ac): 169.72 (CO(Ac)), 167.40 (N-CO),
151.95 (C-2), 151.67 (C-4), 151.17 (C-6), 142.35 (C-8),
125.52 (C-5), 137.40–127.40 (3ꢁC6H5), 86.78 (C-10),
85.11 (C-40); 76.05 (C-20), 68.74 (C-30), 63.53 (C-50),
20.10 (CH3(Ac)), 18.76 and 26.32 (Si-C(CH3)3).
Carefully dried phosphonate 12b (142 mg, 0.41 mmol) was
treated with bromotrimethylsilane (0.32 mL, 2.46 mmol)
and 2,6-lutidine (0.29 mL, 2.46 mmol) in acetonitrile (5 mL)
at rt for 20 h (TLC in C-1, H-1). The reaction mixture was
concentrated in vacuo, 2 M TEAB (2 mL) was added, and
the resulting solution was evaporated to dryness. The crude
phosphonic acid 13 was purified on Dowex 1ꢁ2 (CH3COOꢂ
form) column (elution with a linear gradient of 0–0.5 M aque-
ous acetic acid). Conversion into the sodium salt on Dowex
50ꢁ2 (Na+) and freeze-drying from water afforded 101 mg
(78%) of phosphonic acid 13. HR-FAB calcd for
C9H11N5O6P (MꢂH)ꢂ: 316.0447; found: 316.0443; nmax
(KBr) 3407 (s, vbr, OH), 3184 (s, vbr, OH), 2800–2200
(m–w, P-OH), 1691 (vs, NH+3), 1647 (m, NH2), 1506 (w),
1419 (m), 1330 (w), 1222 (m, br), 1161 (m, br), 1128 (s, br),
785 (w), 721 (w), 643 (w) cmꢂ1. Compound 13: dH
(500 MHz, D2O+NaOD) 8.67 (1H, s, H-2), 8.22 (1H, s, H-8),
5.81 (1H, d, J¼7.4 Hz, H-10), 4.75 (1H, dd, J¼7.4, 5.3 Hz,
H-20), 4.33 (1H, ddd, J¼7.2, 5.3, 2.2 Hz, H-30), 4.08 (1H, dd,
J¼6.2, 2.2 Hz, H-40). For 1H NMR data see Tables 1 and 2.
Compound 17, major regioisomer (30-O-Ac): 169.86
(CO(Ac)), 167.40 (N-CO), 152.47 (C-4), 151.95 (C-2),
151.17 (C-6), 142.02 (C-8), 125.52 (C-5), 137.40–127.40
(3ꢁC6H5), 88.66 (C-10), 82.78 (C-40), 72.51 (C-20), 72.88
(C-30), 63.85 (C-50), 20.27 (CH3(Ac)), 18.76 and 26.36
(Si-C(CH3)3).
4.7. Adenosin-20-yl-(4R)-[1-(adenin-9-yl)-1-deoxy-b-D-
erythrofuranos-4-yl]phosphonate (18a)
A mixture of phosphonate 12b (391 mg, 1.13 mmol), aque-
ous 1.89 M tetraethylammonium hydroxide (0.598 mL,
1.13 mmol), and ethanol (10 mL) was evaporated to dryness,
and the residue was dried by co-distillation with ethanol
(2ꢁ15 mL) and DMF (2ꢁ10 mL). Resulting tetraethyl-
ammonium salt of the phosphonate 12b was treated with
benzoyltetrazole (1.18 g, 6.8 mmol) in DMF (10 mL) in the
presence of DMAP (0.83 g, 6.8 mmol) at 55 ꢀC under exclu-
sion of moisture until the starting compound disappeared
(ca. 24 h, TLC in H-1). The reaction mixture was quenched
4.6. 20(30)-O-Acetyl-6-N-benzoyl-50-O-tert-butyldiphenyl-
silyladenosine (17)
6-N-Benzoyl-50-O-tert-butyldiphenylsilyladenosine1 (3 g,
4.92 mmol) was treated at rt with trimethyl orthoacetate

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by addition of methanol (0.5 mL) and the solvent was evapo-
rated in vacuo. The residue was treated with 60% aqueous
pyridine overnight to destroy the mixed anhydride. The
solvent was evaporated in vacuo, and the residue was
partitioned between chloroform (150 mL) and 1 M TEAB
(3ꢁ100 mL). The organic layer was dried over anhydrous
Na2SO4 and the crude product 15a was purified on silica
gel (elution with a linear gradient of H-3 in ethyl acetate fol-
lowed by H-1 in H-3). Yield: 299 mg (40%) of 15a and
141 mg (24%) of 15b. HR-FAB calcd for C32H29N5O9P
(M+H)+: 658.1703; found: 658.1704.
J¼5.4, 2.4 Hz, H-40 (res. B)), 4.24 (1H, ddd, J¼5.5, 3.2,
2.2 Hz, H-40 (res. A)), 3.90 (1H, dd, J¼13.1, 2.2 Hz, H-50
´
(res. A)), 3.77 (1H, dd, J¼13.1, 3.2 Hz, H-500 (res. A)); dC
(125.7 MHz, D2O) 158.12, 157.47, 155.60, 154.66,
151.06, 150.33, 143.64, 141.91, 120.86, 120.77, 91.32 (d,
J¼6.4 Hz), 88.76 (d, J¼3.9 Hz), 87.61, 84.88 (d,
J¼157.2 Hz), 81.16 (d, J¼6.3 Hz), 79.07 (d, J¼2.9 Hz),
1
73.56 (d, J¼7.3 Hz), 73.27, 63.77. For H and 13C NMR
data see Tables 1–3.
4.8. 6-N-Benzoyl-50-O-tert-butyldiphenylsilyl-20-O-
(tetrahydropyran-2-yl)adenosine (19a)
4.7.1. Preparation of protected phosphonic acid 16. The
carefully dried phosphonate 15b (265 mg, 0.4 mmol) was
treated with bromotrimethylsilane (0.26 mL, 2.0 mmol)
and 2,6-lutidine (0.23 mL, 2.0 mmol) in acetonitrile (4 mL)
at rt for 48 h (TLC in H-1). The reaction mixture was con-
centrated in vacuo, 2 M TEAB (2 mL) was added, the solu-
tion was evaporated, and the product 16 was purified by
reversed-phase chromatography (elution with a linear gradi-
ent of methanol in water). Yield: 190 mg (75%) of triethyl-
ammonium salt of 16. HR-FAB calcd for C30H25N5O9P
(M+H)+: 630.1390; found: 630.1383.
6-N-Benzoyl-20-O-(tetrahydropyran-2-yl)adenosine27 (0.456 g,
1 mmol) was treated with tert-butylchlorodiphenylsilane
(0.3 mL, 1.15 mmol) in pyridine (3 mL) at rt for 48 h
(TLC in C-1). The reaction mixture was quenched by addi-
tion of methanol (0.2 mL) followed by triethylamine
(0.2 mL, 1.4 mmol), the resulting suspension was diluted
with ethyl acetate (20 mL), filtered, the precipitate was
washed with ethyl acetate, and the combined filtrates were
concentrated in vacuo. The residue was applied on a silica
gel column in chloroform and the compound was eluted
with a linear gradient of ethanol in chloroform (0/10%).
Yield: 0.645 g (93%, white foam) of 19a (a mixture of dia-
stereoisomers). HR-FAB calcd for C38H44N5O6Si (M+H)+:
694.3061; found: 694.3052. Compound 19a: dH (500 MHz,
DMSO) mixture of two diastereoisomers in the ratiow1:1;
most of the signals are doubled: 11.20 (2H, br s, NH),
8.655 (1H, s), 8.65 (1H), 8.60 (1H, s), 8.59 (1H, s, H-2
and H-8), 8.05 (4H, m), 7.70–7.50 (13H, m), 7.50–7.30
(13H, m, 2ꢁC6H5 (TBDPS) and C6H5 (N-Bz)), 6.235 (1H,
d, J¼5.6 Hz), 6.23 (1H, d, J¼6.0 Hz, H-10), 5.41 (1H, d, J¼
5.5 Hz), 5.26 (1H, d, J¼5.8 Hz, 30-OH), 4.96 (1H, t, J¼
5.5 Hz), 4.95 (1H, t, J¼5.1 Hz, H-20), 4.76 (1H, dd, J¼3.7,
2.7 Hz), 4.66 (1H, t, J¼3.5 Hz), 3.40 (2H, m), 3.28 (1H,
m), 3.13 (1H, m), 1.90–1.20 (12H, m, OTHP), 4.52 (1H,
m), 4.51 (1H, m, H-30), 4.12 (1H, m), 4.115 (1H, m, H-40),
3.96 (1H, dd), 3.955 (1H, dd, J¼11.2, 3.5 Hz, H-50a), 3.82
(1H, dd), 3.815 (1H, dd, J¼11.2, 4.5 Hz, H-50b), 0.98
(18H, s, t-Bu).
4.7.2. Preparation of dimer 18a. A mixture of triethyl-
ammonium salt of the phosphonic acid 16 (190 mg,
0.3 mmol) and 20(30)-O-acetyl-6-N-benzoyl-50-O-tert-butyl-
diphenylsilyladenosine (17) (254 mg, 0.4 mmol) was treated
with DCC (619 mg, 3 mmol) in pyridine (4 mL) at rt over-
night and then concentrated to a thick oil that was set aside
at rt for 5 days (disappearance of the starting phosphonic
acid 16, TLC in H-1). Reaction mixture was diluted with
60% aqueous pyridine (10 mL) and, after 2 h of standing,
concentrated in vacuo. The residue was co-distilled with eth-
anol (2ꢁ20 mL) and toluene (2ꢁ10 mL), and finally treated,
under stirring and exclusion of moisture, with 0.5 M TBAF
in tetrahydrofuran (5 mL, 2.5 mmol) at rt for 16 h (TLC in
H-1). The reaction mixture was diluted with concentrated
aqueous ammonia (50 mL), the suspension was stirred at rt
for 3 days, and then evaporated to dryness. The residue in
50% aqueous ethanol (20 mL) was deionized (removal of
tetra-n-butylammonium cations) on Dowex 50ꢁ2 (Et3NH+),
the resin was filtered off, washed with aqueous ethanol, and
the combined filtrates were concentrated. The residue was
suspended in 3% aqueous ammonia (50 mL) under sonica-
tion, dicyclohexylurea was filtered off, the filtrate was
evaporated, and the crude dimer 18a was purified on Dowex
1ꢁ2 (CH3COOꢂ) column (elution with a linear gradient of
aqueous 0–0.5 M acetic acid). Conversion into the sodium
salt on Dowex 50ꢁ2 (Na+) and freeze-drying from water
afforded 26 mg (15%) of 20,50-isomer 18a. HR-FAB calcd
for C19H24N10O9P (M+H)+: 567.1466; found: 567.1469.
nmax (KBr) 3401 (s, br, OH), 3192 (br, OH), 1646 (vs,
NH2), 1602 (m), 1578 (m), 1507 (w), 1420 (w), 1333 (m),
1216 (m, br, P]O), 1068 (s, br, C-OH), 797 (w), 725 (w),
648 (w), 550 (w, br, POC) cmꢂ1. Compound 18a: dH
(500 MHz, D2O) 8.21 (1H, s, H-8 (res. B)), 8.11 (1H, s,
H-2 (res. B)), 8.06 (1H, s, H-8 (res.A)), 7.72 (1H, s, H-2
(res.A)), 6.17 (1H, d, J¼4.1 Hz, H-10 (res. A)), 5.60 (1H,
d, J¼6.9 Hz, H-10 (res. B)), 5.16 (1H, ddd, J¼9.3, 5.3,
4.1 Hz, H-20 (res. A)), 4.61 (1H, dd, J¼5.5, 5.3 Hz, H-30
(res. A)), 4.49 (1H, ddd, J¼7.4, 4.8, 2.4 Hz, H-30 (res. B)),
4.41 (1H, dd, J¼6.9, 4.8 Hz, H-20 (res. B)), 4.26 (1H, dd,
4.9. 6-N-Benzoyl-50-O-tert-butyldiphenylsilyl-20-O-[1-(2-
chloroethoxy)ethyl]adenosine (19b)
6-N-Benzoyl-20-O-[1-(2-chloroethoxy)ethyl]adenosine28
(0.487 g, 1 mmol) was treated with tert-butylchlorodiphe-
nylsilane (0.3 mL, 1.15 mmol) in pyridine (3 mL) at rt for
48 h (TLC in C-1). The workup of the reaction mixture
was performed as described for 19a. Yield: 0.609 g (85%,
white foam) of 19b (a mixture of diastereoisomers). HR-
FAB calcd for C37H43ClN5O6Si (M+H)+: 716.2671; found:
716.2679. Compound 19b: dH (500 MHz, DMSO) mixture
of two diastereoisomers in the ratiow1.7:1. Major diastereo-
isomer: 11.25 (1H, br s, NH), 8.68 (1H, s), 8.62 (1H, H-2 and
H-8), 8.05 (2H, m), 7.70–7.30 (13H, m, 2ꢁC6H5 (TBDPS)
and C6H5 (N-Bz)), 6.193 (1H, d, J¼5.6 Hz, H-10), 5.38
(1H, d, J¼5.6 Hz, 30-OH), 4.935 (1H, br t, J¼5.2 Hz,
H-20), 4.88 (1H, q, J¼5.4 Hz), 3.70–3.40 (4H, m), 1.24
(3H, d, J¼5.4 Hz, 20-O-CH(CH3)-CH2CH2Cl), 4.47 (1H,
br q, H-30), 4.12 (1H, br q, H-40), 3.987 (dd, 1H, J¼11.5,
4.1 Hz, H-50a), 3.847 (1H, dd, J¼11.5, 4.9 Hz, H-50b),
0.97 (9H, s, t-Bu). Minor diastereoisomer: 11.25 (1H, br s,

ˇ
S. Kralıkova et al. / Tetrahedron 62 (2006) 9742–9750
´ ´
´
9750
10. Holꢀy, A.; Rosenberg, I. Collect. Czech. Chem. Commun. 1987,
52, 2801–2809.
NH), 8.64 (1H, s), 8.62 (1H, H-2 and H-8), 8.05 (2H, m),
7.70–7.30 (13H, m, 2ꢁC6H5 (TBDPS) and C6H5 (N-Bz)),
6.186 (1H, d, J¼5.1 Hz, H-10), 5.44 (1H, d, J¼6.0 Hz,
30-OH), 4.949 (1H, br t, J¼5.1 Hz, H-20), 4.55 (1H, br q,
H-30), 4.24 (1H, q, J¼5.4 Hz), 3.70–3.40 (4H, m), and
1.19 (3H, d, J¼5.4 Hz, (20-O-CH(CH3)-CH2CH2Cl)), 4.10
(1H, br q, H-40), 3.989 (1H, dd, J¼11.5, 3.9 Hz, H-50a),
3.837 (1H, dd, J¼11.5, 5.1 Hz, H-50b), 0.99 (18H, s, t-Bu).
ˇ
´
ꢀ
ꢀ
ꢀ
ꢀ
11. Holy, A.; Gunter, J.; Dvorakova, H.; Masojıdkova, M.; Andrei,
G.; Snoeck, R.; Balzarini, J.; De Clercq, E. J. Med. Chem.
1999, 42, 2064–2068.
´
12. Pav, O.; Protivınska, E.; Pressova, M.; Collinsova, M.; Jiracek,
J.; Snasel, J.; Masojıdkova, M.; Budesınsky, M.; Rosenberg, I.
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢁ
ˇ
ꢀ
´
ˇˇ´
ꢀ
ꢀ
J. Med. Chem. 2006, 49, 3955–3962.
13. Xu, Z.; Kuang, M.; Okicki, J. R.; Cramer, H.; Chaudhary, N.
Antiviral Res. 2004, 61, 195–206.
Acknowledgements
14. Parikh, J. R.; Wolf, M. E.; Burger, A. J. Am. Chem. Soc. 1957,
79, 2778–2781.
15. Raju, N.; Smee, D. F.; Robins, R. K.; Vaghefi, M. M. J. Med.
Chem. 1989, 32, 1307–1313.
Support by grant nos. 203/04/P273, 203/05/0827, and 202/
05/0628 (Czech Science Foundation), and under Research
project Z40550506 is gratefully acknowledged. Authors
ˇ
´
ꢁ
´
are indebted to Dr. Zdenek Tocık of this Institute for valuable
discussion, and the staff of the Department of organic anal-
ysis of this Institute for measurements of HR mass and IR
spectra, and elemental analysis.
ꢀ
ꢀ
16. Otmar, M.; Rosenberg, I.; Masojıdkova, M.; Holy, A. Collect.
Czech. Chem. Commun. 1993, 58, 2159–2179.
´
Czech. Chem. Commun. 1993, 58, 2180–2196.
ꢀ
17. Otmar, M.; Rosenberg, I.; Masojıdkova, M.; Holy, A. Collect.
ꢀ
18. Specha, M. Helv. Chim. Acta 1993, 76, 1832–1846.
19. Smrt, J. Collect. Czech. Chem. Commun. 1982, 47, 2157–2169.
20. Kofoed, T.; Pedersen, E. B. Nucleosides Nucleotides 1995, 14,
References and notes
345.
ˇ
´
ˇˇ´
ꢀ
ꢀ
ꢀ
1. Endova, M.; Masojıdkova, M.; Budesınsky, M.; Rosenberg, I.
´
ˇˇ´
´
21. Kralıkova, S.; Budesınsky, M.; Masojıdkova, M.; Rosenberg, I.
ꢀ
Tetrahedron 2006, 62, 4917–4932.
ꢀ
ꢀ
ꢀ
Tetrahedron 1998, 54, 11151–11186.
´
Tetrahedron 1998, 54, 11187–11208.
ˇ
ꢀ
ꢀ
ˇˇ´
ꢀ
2. Endova, M.; Masojıdkova, M.; Budesınsky, M.; Rosenberg, I.
´
ˇˇ´
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
22. Kralıkova, S.; Budesınsky, M.; Novotna, M.; Pav, O.; Liboska,
R.; Rosenberg, I. 16th International Round Table on Nucleo-
sides, Nucleotides and Nucleic Acids, Minneapolis, Minnesota,
USA. September 12–16. In Abstract Book; 2004; p 229.
ˇ
3. Kralıkova, S.; Budesınsky, M.; Masojıdkova, M.; Rosenberg, I.
ꢀ ´
Nucleosides Nucleotides Nucleic Acids 2000, 19, 1159–1183.
ꢀ
ˇˇ´
ꢀ
´
ꢀ
ˇ
4. Kralıkova, S.; Budesınsky, M.; Masojıdkova, M.; Rosenberg, I.
´
Tetrahedron Lett. 2000, 41, 955–958.
ˇˇ´
´
ꢀ
ꢀ
ꢀ
ꢀ
´
ꢁ
ꢀ
ꢀ
23. Pressova, M.; Endova, M.; Tocık, Z.; Liboska, R.; Rosenberg, I.
Bioorg. Med. Chem. Lett. 1998, 8, 1225–1230.
ˇ
24. Hanus, J.; Barvık, I.; Ruszova-Chmelova, K.; Stepanek, J.;
´
5. Rejman, D.; Masojıdkova, M.; De Clercq, E.; Rosenberg, I.
Nucleosides Nucleotides Nucleic Acids 2001, 20, 1497–1522.
ꢀ
ˇ
Turpin, P.-Y.; Bok, J.; Rosenberg, I.; Petrova-Endova, M.
´
ꢀ
ꢀ
ˇ ꢀ
ꢀ
ꢀ
ˇ
ꢀ ´
ꢀ
´
ꢀ
ˇˇ´
ꢀ
6. Kralıkova, S.; Masojıdkova, M.; Budesınsky, M.; Rosenberg, I.
Nucleosides Nucleotides Nucleic Acids 2003, 22, 329–347.
7. Rosenberg, I. Frontiers in Nucleosides and Nucleic Acids;
Schinazi, R. F., Liotta, D. C., Eds.; IHL: Tucker, GA, 2005;
pp 519–548.
Nucleic Acids Res. 2001, 29, 5182–5194.
25. Bhat, B.; Sanghvi, Y. S. Tetrahedron Lett. 1997, 38, 8811–
8814.
26. Haasnoot, C. A. G.; de Leeuw, F. A. A. M.; de Leeuw, H. P. M.;
Altona, C. Org. Magn. Reson. 1981, 15, 43–52.
27. Markiewicz, W. T.; Biala, E.; Kierzek, R. Bull. Pol. Acad. Sci.,
Chem. 1984, 32, 433–451.
8. Scheit, K. H. Nucleotide Analogues. Synthesis and Biological
Function; Wiley-Interscience: New York, NY, 1980.
9. De Clercq, E.; Holꢀy, A.; Rosenberg, I.; Sakuma, T.; Balzarini,
J.; Maudgal, P. C. Nature 1986, 323, 464–467.
28. Sakatsume, O.; Yamaguchi, T.; Ishikawa, M.; Hirao, I.; Miura,
K.; Takaku, H. Tetrahedron 1991, 47, 8717–8728.