Study on reactivity and protection of the α-hydroxyphosphonate moiety in 5′-nucleotide analogues: Formation of the 3′-O-P-C(OH)-C4′ internucleotide linkage

Article abstract of DOI:10.1081/NCN-120021432

The recently described epimeric nucleosidyl-5′-C-phosphonates (α-hydroxyphos-phonates) represent novel nucleotide analogues that can be incorporated into chimeric oligonucleotides by the phosphotriester condensation method. In order to prepare suitable protected monomer(s) we have studied condensation reaction between protected 2′-deoxythymidine and 2′-deoxythymidinyl-5′-C-phosphonate, both as model compounds, in dependence on the nature of the 5′-hydroxyl protecting group. We have found that the O-acetyl group is unstable in the presence of TPSCl or MSNT used as condensing agents for activation of the phosphorus moiety. This instability negatively influences the scope of the condensation process. On the other hand, introduction of the O-methoxycarbonyl group gave excellent results. The O-methoxycarbonyl group does not participate in the condensation process, and its quantitative introduction into the nucleotide analogues is accomplished using a novel acylating agent, methoxycarbonyl tetrazole.

Full text of DOI:10.1081/NCN-120021432

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Study on Reactivity and Protection of the α-
Hydroxyphosphonate Moiety in 5′-Nucleotide
Analogues: Formation of the 3′-O-P-C(OH)-C4′
Internucleotide Linkage
Šárka Králíková a , Milena Masojídková a , Miloš Buděšínský a & Ivan Rosenberg a b
a Institute of Organic Chemistry and Biochemistry , Academy of Sciences , Prague, Czech
Republic
b Institute of Organic Chemistry and Biochemistry , Academy of Sciences , Flemingovo nám
2, 166 10, Prague 6, Czech Republic
Published online: 07 Feb 2007.
To cite this article: Šárka Králíková , Milena Masojídková , Miloš Buděšínský & Ivan Rosenberg (2003) Study on Reactivity
and Protection of the α-Hydroxyphosphonate Moiety in 5′-Nucleotide Analogues: Formation of the 3′-O-P-C(OH)-C4′
Internucleotide Linkage, Nucleosides, Nucleotides and Nucleic Acids, 22:3, 329-347, DOI: 10.1081/NCN-120021432
To link to this article: http://dx.doi.org/10.1081/NCN-120021432
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, No. 3, pp. 329–347, 2003
Study on Reactivity and Protection of the
a-Hydroxyphosphonate Moiety in 50-
Nucleotide Analogues: Formation
of the 30-O-P-C(OH)-C40
Internucleotide Linkage
ˇ
Sarka Kralıkova, Milena Masojıdkova, Milos Budesınsky,
´
´ ´
´
´
´
and Ivan Rosenberg
ˇ
ˇˇ´
´
*
Institute of Organic Chemistry and Biochemistry, Academy of Sciences,
Prague, Czech Republic
ABSTRACT
The recently described epimeric nucleosidyl-50-C-phosphonates (a-hydroxyphos-
phonates) represent novel nucleotide analogues that can be incorporated into chi-
meric oligonucleotides by the phosphotriester condensation method. In order to
prepare suitable protected monomer(s) we have studied condensation reaction
between protected 20-deoxythymidine and 20-deoxythymidinyl-50-C-phosphonate,
both as model compounds, in dependence on the nature of the 50-hydroxyl pro-
tecting group. We have found that the O-acetyl group is unstable in the presence
of TPSCl or MSNT used as condensing agents for activation of the phosphorus
moiety. This instability negatively influences the scope of the condensation pro-
cess. On the other hand, introduction of the O-methoxycarbonyl group gave
excellent results. The O-methoxycarbonyl group does not participate in the
*Correspondence: Ivan Rosenberg, Institute of Organic Chemistry and Biochemistry, Acad-
´
emy of Sciences, Flemingovo nam 2, 166 10 Prague 6, Czech Republic; E-mail: ivan@uochb.
cas.cz.
329
DOI: 10.1081/NCN-120021432
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

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Kralıkova et al.
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330
condensation process, and its quantitative introduction into the nucleotide analo-
gues is accomplished using a novel acylating agent, methoxycarbonyl tetrazole.
Key Words: Nucleotides; Oligonucleotides; a-hydroxyphosphonates; Acylation;
Protecting groups.
INTRODUCTION
Isopolar phosphonate-based nucleotide analogues containing a bridging P-C
bond instead on the ester P-O linkage have attracted attention for many years.[1–10]
Recently we reported the synthesis of a novel type of isopolar 50-nucleotide analo-
gues[9] related to nucleoside 50-monophosphates. These compounds with an a-hydro-
xyphosphonate moiety located on the 50-carbon of the 20-deoxyribo-nucleoside and
ribonucleoside ring exist as epimeric pairs. Their simple, high-yield synthesis via
nucleophilic addition of various phosphorous acid esters to nucleoside 50-aldehydes
provides a convenient route to the use of these compounds, after suitable protection,
as monomers for the oligonucleotide construction.
This work describes the formation of dinucleoside phoshonates by triester-like
condensation of the protected 20-deoxythymidinyl-50-C-phosphonate with nucleoside
dependent on the nature of the O-protecting group of a-hydroxyphosphonate moi-
ety. The configuration of the formed dimers containing a chiral 30-O-P(HO)-500 inter-
nucleotide linkage was assigned from NMR spectra. In addition, the chemical
stability and enzymatic resistance of the modified internucleotide linkage is briefly
discussed.
RESULTS AND DISCUSSION
The preparation of dinucleoside monophosphate analogues by reaction of the
protected nucleoside 50-phosphonate with nucleoside by the phosphotriester metho-
dology must involve, as the first step, the protection of the hydroxyl group of the
50-a-hydroxyphosphonate moiety (Sch. 1).
Thus, treatment of dimethyl ester[9] 1 with acetic anhydride in pyridine followed
by hydrolysis of one methyl ester group of the diester 2a in 60% aqueous pyridine
resulted in quantitative yield of the 50-O-acetyl derivative 3a. The condensation of
this compound with an excess of 50-O-dimethoxytritylthymidine (7a) using mesity-
lenesulfonyl nitrotriazolide[11] (MSNT) in the presence of 4-methoxypyridine-1-oxide
(MPNO) as a nucleophilic catalyst[12] afforded, after demethylation in aqueous pyr-
idine and removal of DMTr group in 80% acetic acid, dimer 8c as a mixture of epi-
mers (9:91) in a 70% yield (related to 3a). In contrast to the literature data[12] the
reaction proceeded extremely slowly and prolongation of the reaction time led only
to detritylation and decomposition of the formed 8a. Neither pre-activation of the
P-component 3a with MSNT nor the use of 2-chloro-5,5-dimethyl-2-oxo-1,3,2-di-
oxaphosphinance (CDOP)[13] instead of MSNT increased the yield. The use of 2,4,6-
triisopropylbenzenesulfonyl chloride (TPSCl) as the condensing agent has led to a
very complex mixture of products. A similar reaction of phosphonate 3a with the

a-Hydroxyphosphonate Moiety in 50-Nucleotide Analogues
331
Scheme 1.
50-O-tert-butyldiphenylsilythymidine (7b) resulted in dimer 8d that was converted to
derivative 8e (31 : 69 mixture of epimers) in a 75% yield. In the case of condensation
of ester 3a with nucleoside 7a, we isolated considerable amounts of 30-O-acetyl deri-
vatice 9 and 2,30-anhydrothymidine derivative 10.
In order to increase the rate and yield of esterification of the phosphonate moi-
ety with the nucleoside we prepared 4-methoxy-l-oxido-2-picolyl ester 5a that should
enhance the condensation rate via intramolecular catalysis.[13] Thus, monomethyl
ester 3a was esterified with 4-methoxy-l-oxido-2-pyridinemethanol (MOP) using
CDOP as condensing agent and MPNO as catalyst.[12] Demethylation of the mixed
diester 4a in 60% aqueous pyridine afforded two epimeric pairs, determined by NMR
as compound 5a (41:59; 92%) and its deacetylated derivative 5b (37:63; 8%) in overall
50% yield. Beside esters 5a, 5b we also isolated the 50-O-acetylated phosphonic acid 6
and 4-methoxy-l-oxido-2-picolyl acetate (11). The condensation of the picolyl ester
5a with 7b in pyridine using TPSCl proceeded also very slowly to afford dimer 8f
in a low yield.

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Kralıkova et al.
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332
Scheme 2. Proposed mechanism of transacetylation during activation of phosphonate moiety.
The presence of compound 6 in the reaction mixture was a surprise because in
aqueous pyridine we have never observed[14] simultaneous hydrolysis of both the
methyl and picolyl ester groups of compound 12 lacking a-acetoxy moiety. Only
the methyl group was always cleaved off with high selectivity and resulted mono-
picolyl ester was quite stable when heated in aqueous pyridine. Concerning these
facts it is clear that the a-acetoxy group must play a crucial role in the hydrolysis
of the picolyl ester group of diester 4a. On the other hand, the unexpected formation
of acetyl derivatives 9 and 11 could be explained by one of possible transacetylation
mechanism as is proposed in Scheme 2. Possible formation of five-membered acetyl-
oxonium intermediate would not be surprising because a similar intermediate is sup-
posed to be formed at the activation of 1,2-di-O-acetyl derivatives of furanoses with
Lewis acids during nucleosidation reaction.
To clarify more the participation of a-acetoxy group at the activation of the
phosphorus moiety in compound 3a or 5a and transacetylation reaction, we pre-
pared the simplest a-acetoxyphosphonates 14a and 14b differing in the nature of
the acetoxy group (primary vs. secondary) (Sch. 3). Whereas the condensation of
14a with 7a using MSNT-MPNO[12] afforded epimeric nucleoside phosphonates 15
(50:50) in a high yield, a different result was obtained with the ‘‘secondary’’ acetoxy
derivative 14b. From the reaction mixture we isolated 30-O-acetylated product 9
and its 30-epimer, the 1-(3-O-acetyl-2-deoxy-5-O-dimethoxytrityl-b-D-threo-pento-
furanosyl)thymine. Both these compounds were formed as the products of the
transacetylation reaction (cf. Sch. 2) from the nucleoside 7a and MSNT (the
30-epimer via 2,20-anhydro nucleoside). Although these findings confirmed our
above-mentioned results, we still are not able to fully explain the nature of different
Scheme 3.

a-Hydroxyphosphonate Moiety in 50-Nucleotide Analogues
333
reactivities of primary (14a) and secondary (3a, 4a, 5a, 14b) a-acetoxyphosphonates
in the condensation reactions. We already observed differences in reactivity of
other types of a-hydroxyphosphonate derivatives. Thus, a-tosyloxyphosphonates
bearing a secondary tosyloxy group did not undergo nucleophilic substitution of
tosyloxy group with alkoxide anion, whereas a tosyloxymethylphosphonate
bearing primary tosyloxy group did.[15]
In the light of these findings we turned our attention towards other types of
groups to protect the 50-hydroxy group of diester 1 (Sch. 1). Thus, the 50-O-tetra-
hydropyranyl derivative 3b was prepared in 67% yield by treatment of 1 with dihy-
dropyran and pyridinium 4-toluenesulfonate in dichloromethane followed by partial
demethylation of formed 2b in 60% aqueous pyridine. However, the use of the
obtained 50-O-tetrahydropyranyl derivative 3b instead of the 50-O-acetyl derivative
3a in the condensation reaction with nucleoside 7b under standard conditions[12]
did not improve the yield of the dimer 8h. After a partial deprotection of 8b we
afforded epimeric dimers 8i (33:67) in a moderate yield of 52%. Similarly, the con-
densation of phosphonate 3b with nucleoside 7a afforded dimer 8j. Its partial depro-
tection gave epimeric dimers 8k (20:80; 43%). The tetrahydropyranyl group in dimer
8h was removed by treatment with 0.02 M HCl in aqueous dioxane overnight,
whereas the cleavage of the same group in dimer 8j required treatment in 0.2 M
HCl for several days.
As another type of group for protection of the 50-hydroxyphosphonate moiety,
we examined the tert-butyldimethylsilyl group. The reaction of dimethyl phospho-
nate 16 (Sch. 4) with an excess of tert-butylchlorodimethylsilane in the presence of
imidazole in dimethylformamide proceeded very slowly. Neither heating at 40ꢀC
nor addition of DBU accelerated the silylation reaction. The expected 50-O-tert-
butyldimethylsilyl derivative 17 was obtained in overall 38% yield as an epimeric
mixture (17:83) whose epimeric composition differed from that in the starting com-
pound 16 (10:90). Recovery of the major epimer of 16 from the reaction mixture sug-
gests that the minor epimer was silylated by the bulky tert-butyldimethylsilyl group
faster than the major one. These findings are in a good agreement with the results
obtained from conformational analysis of single epimers 16 performed in our labora-
tory earlier.[9] The silyl derivative 17 was subjected to partial demethylation in 60%
aqueous pyridine to give the monoester 18a. This demethylation, accompanied by
complete and very fast hydrolysis of the 50-O-tert-butyldimethylsilyl group, afforded
Scheme 4.

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Kralıkova et al.
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334
compound 18b instead of silyl derivative 18a. This unexpected hydrolysis of the silyl
group seem to be accelerated by the presence of the phosphorus moiety; a migration
of the silyl group from secondary 50-hydroxyl to the hydroxyl of the phosphorus
moiety and its fast hydrolysis as ester group is very likely the driving force of this
hydrolytic process.
In our search for the protecting groups of the 50-hydroxyl of the 50-hydroxyphos-
phonate moiety we also examined the methoxycarbonyl group that could be easily
attached to the hydroxyl and would not participate during activation of the phos-
phorus moiety. Thus, the treatment of phosphonate 1 with a large excess of MCC
and DMAP in pyridine at 50ꢀC for 6 d afforded the 50-O-methoxycarbonyl derivative
3c in a moderate yield (54%; 32% of 1 recovered). As a side reaction, we also observed
partial demethylation. A comparison of the epimer ratio (determined by NMR spec-
troscopy) in the product 3c (36:64), starting phosphonate 1 (22:78) and the recovered
compound 1 (8:92) showed again the reaction preference for the minor isomer.
A crucial improvement of the methoxycarbonylation was achieved using N-methoxy-
carbonyl tetrazole[16] (MCT), a novel acylating reagent prepared in analogy to the
known N-benzoyl tetrazole.[17,18] Thus, the treatment of phosphonate 1 with MCT
(2 equiv) in tetrahydrofuran containing DMAP led to complete consumption of the
starting compound 1 within 5 min at rt. The workup and isolation afforded
dimethyl phosphonate 2c (27:73; 71%) and monomethyl ester 3c (24%). Conversion
of 2c to 3c in 60% aqueous pyridine afforded the monoester 3c in overall 95%
yield. The condensation of the 50-O-methoxycarbonyl derivative 3c with nucleoside
7b under standard conditions[12] smoothly afforded the fully protected dimer 8l that
was not isolated but immediately subjected to the removal of the methyl ester
group in aqueous pyridine as well as the methoxycarbonly group in a mixture
methanol-Et3N-water overnight. The partially deprotected dimer 8i was obtained
in excellent (93%) yield. Also the transformation of the 50-O-methoxycarbonyl deri-
vative 3c into the picolyl ester 4b according to the standard procedure[13] provided
this compound in 96% yield.
Although both the acetyl and tetrahydropyranyl protecting groups of the
a-hydroxyphosphonate moiety are acceptable for the synthesis of very short oligo-
nucleotides in solution, they are not suitable for the synthesis of longer oligonucleo-
tides on the solid phase where the interference of the protecting group must not
affected the efficiency of coupling steps. We found that the methoxycarbonyl group
is the group of choice because of its quantitative introduction and no participation
during condensation step using triester method. Solid phase synthesis of longer oli-
gonucleotides composed of nucleoside 50-hydroxyphosphonates will be the subject of
our further investigation.
Concerning the chemical stability of the 30-O-P-(HO)-50 internucleotide linkage
in dimer 8g, this compound is quite stable under standard deprotection conditions
used in the solid phase synthesis of oligonucleotides (37% aqueous ammonia;
55ꢀC; 16 h) and in 0.05 M aqueous NaOH for several days as determined by analy-
tical reversed-phase HPLC. Complete resistance of the modified internucleotide link-
age in both individual epimers 8g against nucleases was observed in an L-1210 cell
free extract under conditions leading to complete cleavage of the natural dimer TpT.
Determination of configuration at the C50 carbon atom is closely connected with
the conformation around the C40-C50 bond. Small vicinal couplings J(H40,H50) and

a-Hydroxyphosphonate Moiety in 50-Nucleotide Analogues
335
Figure 1. Preferred conformations of 50-(S) and 50-(R) isomers of compound 8g.
J(P,H40) observed in the 50-diastereoisomers of compound 8g (2.1–3.0 Hz, see
Table 2) indicate that they adopt the preferred conformation (A) and (B) with a
gauche orientation of atoms H40=H50 and P=H40. Distinguishing between 50-(R)
and 50-(S)-isomers is then possible on the basis of J(P,C30) couplings. The large value
J(P,C30) ¼ 11.7 Hz in the major isomer corresponds to the 50-(S) configuration
(conformer A) with trans-orientation P=C30 while J(P,C30)ꢁ0 Hz in the minor isomer
corresponds to the 50-(R) configuratoin (conformer B) with gauche-P=C30 orienta-
tion. We should also notice a large difference observed between geminal couplings
J(P,C40) in 50-diastereoisomers (ꢁ0 Hz in 50 (S)-isomer in comparison with 10.2 Hz
in 50(R)-isomer) probably due to a different orientation of phosphorus and the
oxygen atom O40. Configuration at C50 in compounds 8c, 8e, 8j and 8k follow from
their chemical correlations with diastereoisomers of 8g.
The observed NOE contacts of thymine proton H-6 with furanose ring protons
H-10,H-20 and H-30 indicate a preferred anti-orientation of both thymine rings in dia-
stereoisomers (S)-8g and (R)-8g. Vicinal coupling constants together with a two-state
model and generalized Karplus relation[19] have been used for estimation of confor-
mation behaviour of furanose rings. It was found that the equilibrium between C30-
endo and C20-endo forms in diasteroisomers (S)-8g and (R)-8g is shifted significantly
towards the C20-endo form for 50-residues (ca 15:85 and 25:75 in (S)-8g and (R)-8g)
but much less for 30-residues (ca 45:55 and 35:65 in (S)-8g and (R)-8g). The only
slightly different sets of vicinal coupling constants indicate a similar conformational
behaviour for other epimers 8.
EXPERIMENTAL
Unless stated otherwise, the solvents were evaporated at 40ꢀC and 2 kPa and the
products were dried over phosphorus pentoxide at 50–70ꢀC and 13 Pa. The course of
the reaction was checked by TLC on silica gel Silufol UV 254 foils (Kavalier Glass-
works, Votice, Czech Republic) and the products were detected both by UV moni-
toring and by spraying with a 1% ethanolic solution of 4-(4-nitro-benzyl)pyridine
[after short heating and exposing to ammonia vapours the product (dialkyl phospho-
nate) affords an intense blue spot]. Preparative column chromatography (PLC) was
carried out on silica gel (40–60 mm; Fluka); the amount of adsorbent was 20–40 times
the weight of the separated mixture, elution rate 40 mL=min. PLC and TLC were

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Kralıkova et al.
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carried out in the following solvent systems (v=v): chloroform–ethanol 9:1 (Cl);
ethyl acetate-acetone-ethanol-water 4:1:1:1 (H1); 2-propanol–concentrated aqueous
ammonia–water 7:1:2 (I, for charged compounds); TI – 50% EtOAc-toluene, T2 –
20% EtOAc-toluene. Preparative reversed-phase chromatography (PRPC) was
carried out on a spherical octadecyl silica column (25 ꢂ 300 mm, 20–40 mm, IOCB
Prague); compounds were eluted with a linear gradient of methanol in water at
10 mL=min. Chromatography on DEAE-Sephadex A-25 was performed with linear
gradient of 0-0.2 M triethylammonium hydrogen carbonate (TEAB) in water. HPLC
analysis was performed on a column of reverse phase (4.6 ꢂ 150 mm) Nucleosil 100-5
C18 (Macharey-Nagel), either isocratically at various concentrations of methanol in
0.1 M triethylammonium acetate (TEAA) or by gradient of methanol in the same
buffer. The electrophoresis was made on Whatman No. 1 in 0.1 M TEAB (pH 7.5)
at 20 V=cm. UV spectra were taken on PYE-Unicam SP 8000 spectrophotometer
in water or in a methanol-water mixture (1:1, v=v) at pH 2, pH 7, and pH 12. Mass
spectra (m=z) were recorded on ZAB-EQ (VG Analytical) instrument, using FAB
(ionisation by Xe, accelerating voltage 8 kV) technique with glycerol and thioglycerol
as matrices. NMR spectra were measured on a Varian UNITY-500 spectrometer
13
(1H at 500 MHz; C at 125.7 MHz frequency) in d6-DMSO and=or D2O at 20ꢀC.
The signals were referenced either to solvent signal (converted to d scale using rela-
tions dH(DMSO) ¼ 2.50 and dC(DMSO) ¼ 39.7 ppm) or to DSS (in D2O). Proton
2D-COSY spectra were used for the structural assignment of coupled protons and
2D-ROESY spectra for detection of the NOE contacts. Carbon-13 chemical shifts
and coupling constants J(C,P) were obtained from broadband proton-decoupled
spectra using standard and attached proton test pulse sequences.
Method A: Condensation of the P- and OH-components by MSNT in the Presence
of MPNO. A mixture of triethylammonium salt of 50-O-protected phosphonate
monoester (1 mmol), 50-O-protected thymidine (2 mmol) and MPNO (5 mmol),
pre-dried by co-evaporation with toluene and dichloromethane (2 ꢂ 20 mL), was
treated in dichloromethane (12 mL) with MSNT (6 mmol) at rt under exclusion of
moisture. After 40 min (TLC in Cl and H1) the reaction mixture was quenched with
2M-TEAB (10 mL), stirred for 15 min, then diluted with chloroform and the organic
layer was washed with 1M-TEAB (3 ꢂ 200 mL) and dried over anhydrous Na2SO4.
The product was purified on silica gel by elution with gradient of 1–10% of ethanol in
chloroform; triethylamine (5 mL per 100 g of silica gel) was added to the suspension
of silica gel when dimethoxytrityl derivatives were isolated.
Dimethyl-30-O-tert-butyldiphenylsilyl-20-deoxy-50-O-methoxycarbonylthymidinyl-
50-C-phosphonate (2c). To a stirred solution of sublimed tetrazole (700 mg,
10 mmol) and Et3N (1.39 mL, 10 mmol) in THF (30 mL) was added dropwise, under
an argon atmosphere, methoxycarbonyl chloride (0.77 mL, 10 mL). After 90 min the
suspension was filtered under argon and a part of the methoxycarbonyl tetrazole
solution (8 mL, 2.65 mmol) was added under stirring to a mixture of a-hydroxy-
phosphonate (1) (R=S 34=66) (390 mg, 0.66 mmol) and DMAP (324 mg, 2.65 mmol)
dissolved in THF (2 mL). After 15 min (TLC in Cl and H1) the reaction mixture was
quenched by addition of methanol (0.2 mol) and the solvent evaporated in vacuo.
The product was purified on silica gel by elution with linear gradient of ethanol in

a-Hydroxyphosphonate Moiety in 50-Nucleotide Analogues
337
chloroform. Yield 303 mg (71%, white foam) of 2c (R=S 27=73). HR FAB calcd for
C30H40N2O10Psi 647.2190 (M þ H)þ, found 647.2197. 1H NMR (DMSO, 500 MHz)
2c-(R): d 11.37 (1H, bs, NH), 7.58 (1H, q, J(6,CH3) ¼ 1.2 Hz, H-6), 7.60 (4H, m,
o-Ar-H), 7.50 (2H, m, p-Ar-H), 7.46 (4H, m, m-Ar-H), 6.30 (1H, dd, J(10,20) ¼ 9.1,
J(10,200) ¼ 5.5 Hz, H-10), 5.16 (1H, dd, J(50,40) ¼ 6.0, J(50,P) ¼ 10.0 Hz, H-50), 4.49
(1H, dt, J(30,200) ¼ J(30,40) ¼ 2.1, J(30,20) ¼ 5.6 Hz, H-30), 4.23 (1H, m, H-40), 4.10
(3H, s, COOCH3), 3.58 and 3.55 (2 ꢂ 3H), 2 ꢂ d, J(OCH3,P) ¼ 10.8 Hz,
P(OCH3)2), 2.08 (1H, ddd, J(20,10) ¼ 9.1, J(20,200) ¼ 13.5, J(20,30) ¼ 5.6 Hz, H-20),
1.98 (1H, ddd, J(200,10) ¼ 5.5 J(200,20) ¼ 13.5, J(200,30) ¼ 2.1 Hz, H-200), 1.73 (3H, d,
J(CH3,6) ¼ 1.2 Hz, CH3), 1.03 (9H, s, t-But). 2c-(S): d 11.35 (1H, bs, NH), 7.35
(1H, q, J(6,CH3) ¼ 1.2 Hz, H-6)), 7.60 (4H, m, o-Ar-H), 7.50 (2H, m, p-Ar-H),
7.46 (4H, m, m-Ar-H), 6.34 (1H, dd, J(10,20) ¼ 8.3, J(10,200) ¼ 5.8 Hz, H-10), 4.85
(1H, dd, J(50,40) ¼ 4.3, J(50,P) ¼ 11.5 Hz, H-50), 4.46 (1H, ddd, J(30,200) ¼ 2.7,
J(30,20) ¼ 5.5, J(30,40) ¼ 2.3 Hz, H-30), 4.24 (1H, m, H-40), 3.71 (3H, s, COOCH3),
3.59 and 3.57 (2 ꢂ 3H, 2 ꢂ d, J(OCH3,P) ¼ 10.8 Hz, P(OCH3)2), 2.17 (1H, ddd,
J(200,10) ¼ 5.8, J(200,20) ¼ 13.6, J(200,30) ¼ 2.7 Hz, H-200), 2.01 (1H, ddd, J(20,10) ¼ 8.3,
J(200,20) ¼ 13.6, J(20,30) ¼ 5.5 Hz, H-20), 1.71 (3H, d, J(CH3,6) ¼ 1.2 Hz, CH3), 1.04
(9H, s, t-But).
Methyl-50-O-acetyl-30-O-tert-butyldiphenylsilyl-20-deoxythymidinyl-50-C-phospho-
nate (3a). a-Hydroxyphosphonate 1 (R=S 20=80)(1.353 g, 2.30 mmol) was treated
with acetic anhydride (1.08 mL, 11.50 mmol) in pyridine (20 mL) in the presence of
DMAP (50 mg, 0.41 mmol) at rt under exclusion of moisture until the starting com-
pound disappeared (18 h; TLC in Cl and H1). Water (13.3 mL) was added to obtain
a final concentration of pyridine equal 60%, and the mixture was heated at 50ꢀC
overnight. The solution was concentrated in vacuo, the residue co-distilled with
ethanol (4 ꢂ 20 mL) and treated with Dowex 50ꢂ2 (Et3NHþ form) in 75% aqueous
ethanol to remove N-methylpyridinium cations. The crude product was then purified
by PRPC. Yield 1.535 g (93%, white foam) of triethylammonium salt of 3a (R=S
20=80). HR FAB calcd for C29H37N2NaO9Psi 639.1904 (M þ Na)þ, found
1
639.1846. H NMR (DMSO, 500 MHz) 3a-(R): d 11.27 (1H, bs, NH), 10.07 (1H,
bs, P-OH), 7.80 (1H, bs, H-6), 7.65-7.59 (4H, m, Ar-H), 7.50-7.41 (6H, m, Ar-H),
6.21 (1H, dd, J(10,20) ¼ 8.7, J(10,200) ¼ 5.4 Hz, H-10), 4.99 (1H, dd, J(50,40) ¼ 4.2,
J(50,P) ¼ 11.7 Hz, H-50), 4.71 (1H, m, H-30), 4.31 (1H, ddd, J(40,30) ¼ 1.2,
J(40,50) ¼ 4.2, J(40,P) ¼ 13.4 Hz, H-40), 3.30 (3H, d, J(P,OCH) ¼ 10.0 Hz, P-OCH3),
2.00 (1H, m, H-200), 1.95 (1H, m, H-20), 1.90 (3H, s, OAc), 1.71 (3H, d, J(CH3, H-
6) ¼ 1.0 Hz, CH3), 1.03 (9H, s, t-But). 3a-(S): d 11.27 (1H, bs, NH), 10.07 (1H, bs,
P-OH), 7.92 (1H, q, J(6,CH3) ¼ 1.0 Hz, H-6), 7.65-7.59 (4H, m, Ar-H), 7.50-7.41
(6H, m, Ar-H), 6.25 (1H, dd, J(10,20) ¼ 8.7,J(10,200) ¼ 5.4 Hz, H-10), 4.85 (1H, dd,
J(50,40) ¼ 4.2, J(50,P) ¼ 11.5 Hz, H-50), 4.67 (1H, m, H-30), 4.27 (1H, ddd,
J(40,30) ¼ 1.5, J(40,50) ¼ 4.2, J(40,P) ¼ 8.1 Hz, H-40), 3.33 (3H, d, J(P,OCH) ¼ 10.2 Hz,
Hz, P-OCH3), 2.02 (1H, ddd, J(200,10) ¼ 5.4, J(200,20) ¼ 13.2, J(200,30) ¼ 5.0 Hz, H-200),
1.93 (1H, ddd, J(20,10) ¼ 8.7, J(20,20) ¼ 13.2, J(20,30) ¼ 5.0 Hz, H-20), 1.92 (3H, s,
OAc), 1.72 (3H, d, J(CH3,6) ¼ 1.0 Hz, CH3), 1.03 (9H, s, t-But).
Methyl-30-O-tert-butyldiphenylsilyl-20-deoxy-50-O-tetrahydropyranylthymidinyl-50-
C-phosphonate (3b). a-Hydroxyphosphonate 1 (R=S 25=75) (708 mg, 1.20 mmol)

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and dihydropyran (0.38 mL, 4.20 mmol) were treated in dichloromethane (12 mL)
with pyridinium 4-toluensulfonate (301 mg, 1.20 mmol) at rt under stirring and
exclusion of moisture. Further portions of dihydropyran (4 ꢂ 0.2 mL) and 0.5 M
solution of pyridinium 4-toluensulfonate in dichloromethane (2 ꢂ 2.4 mL) were
added within next 4 d (TLC in Cl). The reaction mixture was diluted by aqueous
2M-TEAB (2 mL), stirred for 10 min then diluted with chloroform (100 mL) and
extracted with 1M-TEAB (3 ꢂ 20 mL). The organic layer was dried over anhydrous
Na2SO4 and evaporated. The residue was purified by silica gel chromatography
using a linear gradient of ethanol in chloroform and the resulting protected dimethyl
phosphonate was treated with 60% aqueous pyridine (20 mL) at 50ꢀC for 48 h (TLC
in C1 and H1). The solution was concentrated, the residue co-distilled with ethanol
(4 ꢂ 20 mL), and treated with Dowex 50 ꢂ 2 (Et3NHþ form) in 75% aqueous ethanol
to remove N-methylpyridinium cations. The product was finally purified by PRPC.
Yield 606 mg (67%, white foam) of triethylammonium salt of 3b (ratio of epimers
was not determined). HR FAB calcd for C38H59N3O9PSi 760.3758
(M þ Et3N þ H)þ, found 760.3876.
Methyl-30-O-tert-butyldiphenylsilyl-20-deoxy-50-O-methoxycarbonylthymidinyl-50-C-
phosphonate (3c). a-Hydroxyphosphonate 1 (R=S 22=78) (441 mg, 0.75 mmol) was
treated with methoxycarbonyl chloride (0.40 mL, 5.25 mmol) in pyridine (7 mL) in
the presence of DMAP (183 mg, 1.50 mmol) at rt under stirring and exclusion of
moisture for 3 d (TLC in Cl and H1). Then, further portions of methoxycarbonyl
chloride (0.40 mL, 5.25 mmol) and DMAP (92 mg, 0.75 mmol) were added and the
reaction mixture was heated at 50 ꢀC for the next 2 d. The reaction mixture was
quenched by addition of water (1 mL), and the product was partioned between
1 M TEAB (20 mL) and chloroform (100 mL). The organic layer was washed with
1M TEAB (3 ꢂ 20 mL) and dried over anhydrous Na2SO4. The product was purified
on silica gel by elution with linear gradient of H3 in ethyl acetate followed by H1 in
H3). Yield 289 mg (54%, white foam) of pyridinium salt of 3c (R=S 36=64). HR FAB
1
calcd for C29H38N2O10PSi 633.2033 (M þ H)þ, found 633.1987. H NMR (DMSO,
500 MHz) 3c-(R): d 11.25 (1H, bs, NH), 7.96 (1H, bs, H-6), 7.60 (4H, m, Ar-H), 7.40
(6H, m, Ar-H), 6.21 (1H, dd, J(10,20) ¼ 8.5, J(10,200) ¼ 5.5 Hz, H-10), 4.85 (1H, dd,
J(50,40) ¼ 4.0, J(50,P) ¼ 11.0 Hz, H-50), 4.70 (1H, m, H-30), 4.35 (1H, m, H-40), 3.66
(3H, s, OCH3), 3.32 (3H, d, J(OCH,P) ¼ 10.0 Hz, P-OCH3), 2.00 (1H, m, H-200),
1.85 (1H, m, H-20), 1.71 (3H, bs, J(CH3,6) ¼ 1.0 Hz, CH3), 1.02 (9H, s, t-But). 3c-
(S): d 11.25 (1H, bs, NH), 7.92 (1H, bs, H-6), 7.60 (4H, m, Ar-H), 7.40 (6H, m,
Ar-H), 6.24 (1H, dd, J(10,20) ¼ 8.2, J(10,200) ¼ 5.6 Hz, H-10), 4.73 (1H, dd,
J(50,40) ¼ 4.5, J(50,P) ¼ 11.8 Hz, H-50), 4.62 (1H, m, H-30), 4.37 (1H, ddd,
J(40,30) ¼ 2.1, J(40,50) ¼ 4.5, J(40,P) ¼ 6.0 Hz, H-40), 3.64 (3H, s, OCH3), 3.37 (3H, d,
J(OCH,P) ¼ 10.3 Hz, P-OCH3), 2.00 (1H, ddd, J(200,10) ¼ 5.6, J(200,20) ¼ 13.2,
J(200,30) ¼ 2.6 Hz, H-200), 1.82 (1H, ddd, J(20,10) ¼ 8.2, J(200,20) ¼ 13.2, J(20,30) ¼ 5.2 Hz,
Hz, H-20), 1.70 (3H, d, J(CH3,6) ¼ 1.0 Hz, CH3), 1.02 (9H, s, t-But).
(4-Methoxy-1-oxido-2-picolyl)-50-O-acetyl-30-O-tert-butyldiphenylsilyl-20-deoxythymi-
dinyl-50-C-phosphonate (5a). To a solution of triethylammonium salt of monoester
3a (R=S 25=75) (310 mg, 0.43 mmol), 4-methoxy-1-oxido-2-pyridinemethanol
(74 mg, 0.48 mmol) and MPNO (162 mg, 1.29 mmol) (co-evaporated several times

a-Hydroxyphosphonate Moiety in 50-Nucleotide Analogues
339
with pyridine) in pyridine (4.5 mL) was added at rt and under exclusion of moist-
ure CDOP (239 mg, 1.29 mmol). After 2 h (TLC in C1 and H1) the reaction mix-
ture was diluted with saturated aqueous solution of NaHCO3 (10 mL) and
chloroform (50 mL), the layers were separated, after shaking, by centrifugation
and the organic layer was washed with solution of NaHCO3 (4 ꢂ 10 mL) and dried
over anhydrous Na2SO4. The solvent was evaporated and the residue was treated
with 60% aqueous pyridine (15 mL) at 50ꢀC for 24 h. The solution was concen-
trated in vacuo, the residue co-distilled with ethanol (4 ꢂ 20 mL) and treated with
Dowex 50 ꢂ 2 (Et3NHþ form) in 75% aqueous ethanol to remove N-methylpyridi-
nium cations. The product was purified by PRPC. Yield 210 mg (58%, white foam)
of triethylammonium salt of 5a (R=S 41=59). HR FAB calcd for C35H43N3O11PSi
1
740.2405 (M þ H)þ, found 740.2476. H NMR (DMSO, 500 MHz) 5a-(R): d 11.20
(1H, bs, NH), 10.05 (1H, bs, P-OH), 7.73 (1H, bs, H-6), 8.16 (1H, d, J ¼ 7.3 Hz,
Ar-H-pic), 7.05 (1H, d, J ¼ 3.5 Hz, Ar-H-pic), 6.97 (1H, dd, J ¼ 7.3 and 3.5 Hz,
Ar-h-pic), 7.61 (4H, m Ar-H), 7.42 (6H, m, Ar-H), 6.20 (1H, dd, J(10,20) ¼ 8.5,
J(10,200) ¼ 5.5 Hz, H-10), 4.98 (1H, dd, J(50,40) ¼ 4.5, J(50,P) ¼ 12.4 Hz, H-50), 4.77
(1H, m, H-30), 4.49 (1H, m, H-40), 3.65 (3H, s, OCH3), 1.95 (1H, m, H-200), 1.90
(3H, s, OAc), 1.85 (1H, m, H-20), 1.69 (3H, d, J(CH3,6) ¼ 1.2 Hz, CH3), 1.02
(9H, s, t-But). 5a-(S): d 11.20 (1H, bs, NH), 10.05 (1H, bs, P-OH), 7.82 (1H, bs,
H-6), 8.14 (1H, d, J ¼ 7.1 Hz, Ar-H-pic), 7.02 (1H, d, J ¼ 3.5 Hz, Ar-H-pic), 6.93
(1H, dd, J ¼ 7.1 and 3.5 Hz, Ar-H-pic), 7.61 (4H, m, Ar-H), 7.42(6H, m, Ar-H),
6.23 (1H, dd, J(10,20) ¼ 8.4, J(10,200) ¼ 5.6 Hz, H-10), 4.99 (1H, dd, J(50,40) ¼ 4.4,
J(50,P) ¼ 12.0 Hz, H-50), 4.92 (2H, d, J(OCH,P) ¼ 8.2 Hz, P-OCH2-pic), 4.63 (1H,
m, H-30), 4.41 (1H, ddd, J(40,30) ¼ 1.6, J(40,50) ¼ 4.4, J(40,P) ¼ 6.2 Hz, H-40), 3.67
(3H, s, OCH3), 1.98 (1H, ddd, J(200,10) ¼ 5.6, J(200,20) ¼ 13.2, J(200,30) ¼ 2.0 Hz, H-
200), 1.89 (3H, s, OAc), 1.86 (1H, m, H-20), 1.72 (3H, d, J(CH3,6) ¼ 1.0 Hz, CH3),
1.02 (9H, s, t-But).
(4-Methoxy-1-oxido-2-picolyl)-30-O-tert-butyldiphenylsilyl-20-deoxy-50-O-methoxy-
carbonylthymidinyl-50-C-phosphonate (5c). Esterification of the monester 3c (R=S
25=75) (280 mg of pyridinium salt, 0.39 mmol) by 4-methoxy-1-oxido-2-pyridine-
methanol (67 mg, 0.43 mmol) in the presence of MPNO (148 mg, 1.18 mmol) and
CDOP (218 mg, 1.18 mmol) as the condensing agent was performed in pyridine
(4 mL) for 60 min according to the procedure described for compound 5a. The crude
product was purified on silica gel by elution with linear gradient of H3 in ethyl acet-
ate followed by H1 in H3). Yield 324 mg (96%, white foam) of triethylammonium
salt of 5c (R=S 41=59). HR FAB calcd for C35H42N3NaO12PSi 778.2173 (M þ Na)þ,
found 778.1893. 1H NMR (DMSO, 500 MHz) 5c-(R): d 11.20 (1H, bs, NH, 8.11 (1H,
d, J ¼ 7.3Hz, Ar-H-pic), 8.00 (1H, bs, H-6), 7.60 (4H, M, Ar-H), 7.40 (6H, m, Ar-H),
6.99 (1H, d, J ¼ 3.5 Hz, Ar-H-pic), 6.89 (1H, dd, J ¼ 7.3 and 3.5 Hz, Ar-H-pic), 6.23
(1H, dd, J(10,20) ¼ 8.5, J(10,200) ¼ 6.0 Hz, H-10), 4.95-4.68 (4H, m, H-30, H-50 and P-
OCH2-pic), 4.47 (1H, m, H-40), 3.77 and 3.58 (2 ꢂ 3H, 2 ꢂ s, 2 ꢂ OCH3), 1.02 (9H,
s, t-But). 5c-(S): d 11.20 (1H, bs, NH), 8.08 (1H, d, J ¼ 7.1 Hz, Ar-H-pic), 7.90
(1H, bs, H-6), 7.60 (4H, m. Ar-H), 7.40 (6H, m, Ar-H), 6.98 (1H, d, J ¼ 3.5 Hz,
Ar-H-pic), 6.93 (1H, dd, J ¼ 7.1 and 3.5 Hz, Ar-H-pic), 6.26 (1H, t, J(10,200) ¼
J(10,200) ¼ 7.0 Hz, H-10), 4.95 - 4.68 (4H, m, H-30, H-50 and P-OCH2-pic), 4.36

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(1H, m, H-40), 3.76 and 3.59 (2 ꢂ 3H, 2 ꢂ s, 2 ꢂ OCH3), 1.90 and 1.64 (2 ꢂ 1H, 2 ꢂ m,
H-20 and H-200), 1.65 (3H, bs, CH3), 1.02 (9H, s, t-But).
(20,30-Dideoxythymidin-30-yl)-500-O-acetyl-300-O-tert-butyldiphenylsilyl-200-deoxythymi-
dinyl-500-C-phosphonate (8c). Triethylammonium salt of monoester 3a (R=S 18=82)
(275 mg, 0.38 mmol) and 50-O-dimethoxytritylthymidine (7a) (416 mg, 0.76 mmol)
were condensed according to Method A. After extraction, the solvent was evaporated
and the residue treated with 60% aqueous pyridine (20 mL) at 50ꢀC for 20 h.
The solution was concentrated in vacuo, the residue co-distilled with ethanol
(4 ꢂ 20 mL) and treated with Dowex 50 ꢂ 2 (pyridinium form) in 70% aqueous etha-
nol to remove N-methylpyridinium cations. Because double chromatography on
silica gel (using linear gradient of H3 in ethyl acetate followed by H1 in H3) did
not afford pure dimer 8b, the crude product (461 mg) was detritylated in 80% acetic
acid (100 mL) for 30 min. The acid was evaporated at low temperature, the residue
co-distilled three times with water and the dimer 8c purified on a silica gel in the sol-
vent system mentioned above. Yield 240 mg (76%, white foam) of 8c (R=S 9=91). HR
1
FAB calcd for C38H47N4NaO13PSi 849.2544 (M þ Na)þ, found 849.2381. H NMR
data – see Table 1, 2.
(50-O-tert-Butyldiphenylsilyl-20,30-dideoxythymidin-30-yl)-500-O-acetyl-300-O-tert-butyl-
diphenylsilyl-200-deoxythymidinyl-500-C-phosphonate (8e). Triethylammonium salt of
monoester 3a (R=S 20=80) (448 mg, 0.62 mmol) and 50-O-tert-butyldiphenylsilylthy-
midine (7b) (600 mg, 1.25 mmol) were condensed according to Method A. After
extraction of the product and evaporation of the solvent, the residue was treated
with 60% aqueous pyridine (20 mL) at 50ꢀC for 17 h. The solution was concentrated
in vacuo, the residue co-distilled with ethanol (4 ꢂ 20 mL) and treated with Dowex
50 ꢂ 2 (pyridinium form) in ethanol to remove N-methyl pyridinium cations. Chro-
matography on silica gel by elution with linear gradient of H3 in ethyl acetate
followed by H1 in H3 afforded 542 mg (75%, white foam) of triethylammonium
salt of 8e (R=S 31=69). HR FAB calcd for C54H65N4NaO13PSi2 1087.3722 (M þ
1
Na)þ, found 1087.3633. H NMR data – see Table 1, 2.
(50-O-tert-Butyldiphenylsilyl-20,30-dideoxythymidin-30-yl)-4-methoxyl-1-oxido-2-
picolyl)-500-O-acetyl-300-O-tert-butyldiphenylsilyl-200-deoxythymidinyl-500C-phospho-
nate (8f). Triethylammonium salt of picolyl ester 5a (R=S 41=59) (200 mg,
0.24 mmol) and 50-O-tert-butyldiphenylsilylthymidine (7b) (180 mg, 0.36 mmol),
pre-dried by co-evaporation with pyridine (2 ꢂ 15 mL), was treated with TPSCl
(218 mg, 0.72 mmol) in pyridine (4 mL) under exclusion of moisture at rt. After
70 min (TLC in H1), the reaction mixture was cooled in an ice bath, quenched by
addition of water (1 mL), stirred for 5 min and then partitioned between saturated
solution of NaHCO3 (20 mL) and chloroform (100 mL). The layers were separated
by centrifugation; the organic part was washed several times with saturated solution
of NaHCO3, dried over anhydrous Na2SO4 and finally evaporated. The residue was
purified by silica gel chromatography; elution with linear gradient of ethanol in
chloroform. Yield 106 mg (35% white form) of 8f (R=S 48=52). HR FAB calcd for
C61H73N5O15PSi2 1202.4379 (M þ H)þ, found 1202.4352.

a-Hydroxyphosphonate Moiety in 50-Nucleotide Analogues
341
(20,30-Dideoxythymidin-30-yl)-200-deoxythymidinyl-500-C-phosphonate (8g). Triethyl-
ammonium salt of 8e (R=S 31=69) (500 mg, 0.43 mmol) was treated with a mixture of
methanol-concentrated aqueous ammonia (2:3; 50 mL) at rt overnight (TLC
in H1) and then, after evaporation of the solvents and co-distillation of the residue with
ethanol (3 ꢂ 20 mL) and toluene (2 ꢂ 10 mL), with 0.5 M tetra-n-butylammonium
fluoride in THF (5 mL, 2.5 mmol). The mixture was set aside for 20 h at rt under exclu-
sion of moisture (TLC in H1 and I). After evaporation of the solvent and treatment
with Dowex 50 ꢂ 2 (Et3NHþ form) in 50% aqueous ethanol to remove tetra-n-butyl-
ammonium cations, the deprotected product was purified on DEAE-Sephadex.
Freeze-drying from water afforded 177 mg (85%, white foam) of epimer (S)-8g and
50 mg (72%, white foam) of epimer (R)-8g. HR FAB calcd for C26H43N5O12P
1
648.2646 (M þ Et3N þ H)þ, found 648.2682. H and 13C NMR data – see Table 1
and 2.
(50-O-tert-Butyldiphenylsilyl-20,30-dideoxythymidin-30-yl)-300-O-tert-butyldiphenyl-
silyl-200-deoxythymidinyl-500-C-phosphonate (8i). Procedure 1: Triethylammonium
salt of phosphonate monoester 3b (ratio of epimers not determined) (300 mg,
0.39 mmol) and 50-O-tert-butyldiphenylsilylthymidine (7b) (375 mg, 0.78 mmol) were
condensed according to Method A. Yield 227 mg (52%) of 8h. HR FAB calcd for
C58H73N4NaO13PSi2 1143.4348 (M þ Na)þ, found 1143.4457. This dimer 8h was
treated with 60% aqueous pyridine (20 mL) at 50ꢀC overnight to remove the methyl
ester protecting group (TLC in C1 and H1). The solution was concentrated in vacuo,
the residue co-distilled with ethanol (4 ꢂ 20 mL) and treated with Dowex 50 ꢂ 2
(Et3NHþ form) in 70% aqueous ethanol to remove N-methylpyridinium cations.
The residue was, after evaporation, treated with 0.02 M hydrochloric acid in 50%
aqueous dioxane (50 mL) at rt for 1 d (TLC in H1). The reaction mixture was
neutralized with Et3N and the solvent was evaporated. The product was purified
by PRPC. Yield 175 mg (40%, white foam) of triethylammonium salt of 8i (R=S
33=67). HR FAB calcd for C52H63N4NaO12PSi2 1045.3616 (M þ Na)þ, found
1045.3514. 1H NMR data – see Table 1 and 2.
Procedure 2: Pyridinium salt of phosphonate monoester 3c (R=S 36=64) (191 mg,
0.27 mmol) and 50-O-tert-butyldiphenylsilylthymidine (7b) (260 mg, 0.54 mmol) were
condensed according Method A for 20 min. Isolation on silica gel afforded both fully
protected dimer 8a and demethylated product 8b. Both products were combined and
treated with methanol-Et3N-water mixture (2:5:5; 12 mL) overnight. Under these
conditions, both methyl and methoxycarbonyl group were removed. The product
was purified by PRPC. Yield 281 mg (93%, white foam) of triethylammonium salt
of 8i (R=S 38=62).
(20,30-Dideoxythymidin-30-yl)-300-O-tert-butyldiphenylsilyl-200-deoxythymidinyl-500-
C-phosphonate (8k). Triethylammonium salt of phosphonate monoester 3b
(epimeric ratio not determined) (250 mg, 0.38 mmol) and 50-O-dimethoxytritylthymi-
dine (7a) (416 mg, 0.76 mmol) were condensed according to Method A. After extrac-
tion and evaporation, the residue was treated with 60% aqueous pyridine (20 mL) at
50ꢀC overnight. The solution was concentrated in vacuo, the residue co-distilled with
ethanol (4 ꢂ 20 mL) and treated with 0.2 M HCl in 50% aqueous dioxane (50 mL) at
rt for 3 d (TLC in H1). The reaction mixture was neutralized with Et3N and the

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a-Hydroxyphosphonate Moiety in 50-Nucleotide Analogues
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product was purified by PRPC. Yield 145 mg (43%, white foam) of triethylammo-
nium salt of 8k (R=S 20=80). HR FAB calcd for C36H45N4NaO12PSi 807.2439
1
(M þ Na)þ, found 807.2438. H NMR data – see Table 1, 2.
(20-Deoxy-50-O-dimethoxytrityl-30-thymidinyl)-(methyl)-acetoxymethylphospho-
nate (15). Dimethyl hydroxymethylphosphonate (1.27 g, 9.20 mmol), pre-dried by
co-distillation with pyridine (2 ꢂ 15 mL), was treated with acetic anhydride
(1.72 mL, 18.20 mmol) in pyridine (20 mL) at rt under exclusion of moisture until
the starting compound disappeared (1 d; TLC in H3). The reaction mixture was
diluted with water (13.3 mL) to obtain final concentration of pyridine 60%, heated
at 50ꢀC for 11 h and then concentrated in vacuo. The residue was co-distilled with
ethanol (4 ꢂ 20 mL) and treated with Dowex 50 ꢂ 2 (Hþ form) in water to remove
N-methylpyridinium cations and traces of pyridine. The suspension was filtered,
the filtrate evaporated and the residue co-distilled several times with water to remove
the acetic acid and then with ethanol and toluene. A solution of the acetoxy deri-
vative 14a in dichloromethane (50 mL) and triethylamine (1.25 mL, 9 mmol) was
evaporated and the residue dried in vacuo. Triethylammonium salt of phosphonate
monoester 14a (538 mg, 2 mmol) and 50-O-dimethoxytritylthymidine (7a) (1.30 g,
2.4 mmol) were condensed for 3 h and worked up according to Method A. Yield
622 mg (45%, light yellow foam) of 15 (R=S 50=50). HR FAB calcd for
C35H39N2NaO11P 717.2189 (M þ Na)þ, found 717.2233. 15-(R,S): 1H NMR
(DMSO, 500 MHz) d 11.39 (1H, bs, NH), 7.50 (1H, bs, H-6), 7.40 - 7.20 (9H, m,
Ar-H), 6.89 (4H, m, Ar-H), 6.22 and 6.21 (1H, bs, 2 ꢂ t, J(10,20) ¼ J(10,200) ¼ 6.9 Hz,
H-10), 5.14 (1H, m, H-30), 4.47 and 4.44 (2H, 2 ꢂ d, J(CH,P) ¼ 8.5 Hz, P-CH2),
4.14 (1H, m, H-40), 3.74 (3H, s, OCH3), 3.69 and 3.62 (3H, 2 ꢂ d,
J(OCH,P) ¼ 11.0 Hz, P-OCH3), 3.27 (2H, m, H-50 and H-500), 2.45 (2H, m, H-20
amd H-200), 2.06 and 2.00 (3H, 2 ꢂ s, OAc), 1.46 (3H, bs, CH3).
Dimethyl-30,50di-O-tert-butylmethylsilyl-20-deoxythymidinyl-50-C-phosphonate
(17). a-Hydroxyphosphonate (16) (R=S 10=90) (613 mg, 1.32 mmol) and imidazole
(449 mg, 6.60 mmol), pre-dried by co-distillation with DMF (2 ꢂ 20 mL), were trea-
ted with tert-butylchlorodimethylsilane (239 mg, 1.58 mmol) in DMF (10 mL) at rt
under exclusion of moisture. After 3 d, a further portion of tert-butylchlorodimethyl-
silane (478 mg, 3.16 mmol) was added and the reaction mixture was heated at 40ꢀC
for 2 d (TLC in C1). The reaction mixture was quenched with methanol (1 mL), eva-
porated and the residue partitioned between 1M-TEAB (20 mL) and chloroform
(100 mL). The organic layer was washed with 1M-TEAB (3 ꢂ 20 mL) and dried over
anhydrous MgSO4. The product was purified on silica gel (elution with linear gra-
dient of ethanol in chloroform). Yield 290 mg (38%,white foam) of 17 (R=S
17=83). HR FAB calcd for C24H48N2O8PSi2 579.2687 (M þ H)þ, found 579.2613.
1H NMR (DMSO, 500 MHz) 17-(R): d 11.38 (1H, s, NH), 7.66 (1H, q,
J(6,CH3) ¼ 1.2 Hz, H-6), 6.19 (1H, dd, J(10,200) ¼ 6.6,J(10, 20) ¼ 9.5 Hz, H-10), 4.56
(1H, dt, J(30,20) ¼ J(30,4) ¼ 4.0 Hz, J(30,200) ¼ 6.6 Hz, H-30), 4.20 (1H, dd,
J(50,40) ¼ 4.6, J(50,P) ¼ 9.3 Hz, H-50), 3.85 (1H, dt, J(4030) ¼ J(40,50) ¼ 4.3 Hz,
J(40,P) ¼ 19.3 Hz, H-40), 3.64 (3H, d, J(P,OCH) ¼ 10.5 Hz, P-OCH3), 3.62 (3H, d,
J(P,OCH) ¼ 10.5 Hz, P-OCH3), 2.31 (1H, dt, J(200,10) ¼ J(200,30) ¼ 6.6 Hz, J(200,20) ¼
14.2 Hz, H-200), 2.08 (1H, m, H-20), 1.78 (3H, d, J(CH3,H-6) ¼ 1.2 Hz, CH3), 0.88

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Kralıkova et al.
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346
(9H, s, t-But), 0.86 (9H, s, t-But), 0.11 (3H, s, SiCH3), 0.10 (3H, s, SiCH3). 17-(S):
11.38 (1H, s, NH), 7.76 (1H, q, J(6,CH3) ¼ 1.2 Hz, H-6), 6.17 (1H, dd,
J(10,20) ¼ 9.3, J(10,200) ¼ 5.4 Hz, H-10), 4.43 (1H, dd, J(50,40) ¼ 3.2, J(50,P) ¼ 10.7 Hz,
H-50), 4.43 (1H, bd, J(30,200) ¼ J(30,40) ¼ 1.0, J(30,20) ¼ 5.0 Hz, H-30), 4.10 (1H, bt,
J(40,50) ¼ J(40,P) ¼ 3.2 Hz, H-40), 3.68 (3H, d, J(P,OCH) ¼ 10.5 Hz, P-OCH3), 3.63
(3H, d, J(P,OCH) ¼ 10.5 Hz, P-OCH3), 2.11 (1H, bdd, J(200,10) ¼ 5.4, J(200,30) ¼
1.0 Hz, J(200,20) ¼ 13.2 Hz, H-200), 2.02 (1H, ddd, J(20,30) ¼ 4.9, J(20,10) ¼ 9.3, J(20,200) ¼
13.2 Hz, H-20), 1.77 (3H, d, J(CH3,H-6) ¼ 1.2 Hz, CH3), 0.88 (9H, s, t-But), 0.87 (9H,
s, t-But), 0.16 (3H, s, SiCH3), 0.11 (3H, s, SiCH3)
ACKNOWLEDGMENTS
The work was supported by grant No. 203=01=1166 (G.A. of the Czech Repub-
lic) and No. A4055101 (Acad. Sci., Czech Rep.) under research project Z4 055 905 of
the Czech Republic. The authors are indebted to the staff of the MS department of
this Institute (Dr. K. Ubik, Head) for measurements of mass spectra.
REFERENCES
´
´
´
1. Otmar, M.; Rosenberg, I.; Masojıdkova, M.; Holy, A. [5-(Adenin-9-yl)-
5-deoxy-L-pentofuranosyl]phosphonates - a novel type of nucleotide analogues
related to HPMPA. 1. Derivatives of L-arabino, D-arabino, 2-deoxy-L-erythro
and 2-deoxy-L-threo configuration. Collect. Czech. Chem. Commun. 1993a,
58, 2159–2179.
´
´
´
2. Otmar, M.; Rosenberg, I.; Masojıdkova, M.; Holy, A. [5-(Adenin-9-yl)-
5-deoxy-L-pentofuranozyl]phosphonates - a novel type of nucleotide analogues
related to HPMPA. 2. Synthesis of L-ribo and L-xylo configurated analogues
by recyclisation of 5-diethylphosphono-1,2-O-isopropylidene-5-O-mesyl-D-
pentofuranoses under acidic conditions. Collect. Czech. Chem. Commun.
1993b, 58, 2180–2196.
´
´
3. Liboska, R.; Masojıdkova, M.; Rosenberg, I. Carbocyclic phosphonate-based
nucleotide analogs related to PMEA I. Racemic trans-configured derivatives.
Collect. Czech. Chem. Commun. 1996a, 61, 313–332.
´
´
4. Liboska, R.; Masojıdkova, M.; Rosenberg, I. Carbocyclic phosphonate-based
nucleotide analogs related to PMEA II. Recemic cis-configured derivatives.
Collect. Czech. Chem. Commun. 1996b, 61, 778–790.
´
´
´
ˇˇ´
´
5. Endova, M.; Masojıdkova, M.; Budesınsky, M.; Rosenberg, I. Synthesis of
1-phosphonoalkylidene and -arylidene derivatives of nucleosides. Tetrahedron
Lett. 1996, 37, 3497–3500.
0
0
´
´
´
ˇˇ´
´
6. Endova, M.; Masojıdkova, M.; Budesınsky, M.; Rosenberg, I. 2 ,3 -O-Phos-
phonoalkylidene derivatives of ribonucleosides: Synthesis and reactivity. Tet-
rahedron 1998a, 54, 11,151–11,186.

a-Hydroxyphosphonate Moiety in 50-Nucleotide Analogues
347
0
0
´
´
´
ˇˇ´
´
7. Endova, M.; Masojıdkova, M.; Budesınsky, M.; Rosenberg, I. 3 ,5 -O-Phos-
phonoalkylidene derivatives of 1-(2-deoxy-b-D-threo-pentofuranosyl)thymine:
Synthesis and rectivity. Tetrahedron 1998b, 54, 11,187–11,208.
ˇ
´ ´
´
ˇˇ´
´
´
´
8. Kralıkova, S.; Budesınsky, M.; Masojıdkova, M.; Rosenberg, I. Efficient
synthesis of nucleoside 30-C-phosphonates: Reactivity of geminal hydroxyl
phosphonate moiety. Nucleosides, Nucleotides & Nucleic Acids 2000a, 19
(7), 1159–1183.
ˇ
´ ´
´
ˇˇ´
´
´
´
9. Kralıkova, S.; Budesınsky, M.; Masojıdkova, M.; Rosenberg, I. Geminal
hydroxy phosphonate derivatives of nucleosides: A novel class of nucleoside
50-monophosphate analogs. Tetrahedron Lett. 2000b, 41, 955–958.
ˇ
´ ´
´
ˇˇ´
´
´
´
´
10. Kralıkova, S.; Budesınsky, M.; Masojıdkova, M.; Kavenova, I.; Rosenberg, I.
Synthesis of a-hydroxyphosphonate analogs of ApA and UV spectroscopy
study on their complexes with poly(U). Collect. Czech. Chem. Commun, Sym-
posium Series 1999, 2, 89–91.
11. Reese, C.B.; Ubasawa, A. Nature of side-reactions in oligonucleotide synthesis
involving arenesulphonyl derivatives of 3-nitro-1,2,4-triazole and related con-
densing agents. Nucleic Acids, Symposium Serie 1980, 7, 5–21.
12. Efimov, V.A.; Reverdatto, S.V.; Chakhmakhcheva, O.G. New effective method
for the synthesis of oligonucleotides via phosphotriester intermediates. Nucleic
Acids Research 1982, 10, 6675–6694.
´
13. Szabo, T.; Kers, A.; Stawinski, J. A new approach to the synthesis of the
50-deoxy-50-methylphosphonate linked thymidine oligonucleotide analogues.
Nucleic Acids Research 1995, 23, 893–900.
14. Rejman, D. PhD Thesis, Inst. Organ. Chem. & Biochem. AS, Prague 2001.
Synthesis and properties of isopolar nonisoteric phosphonate oligonucleotides
containing the 30-O-P-C-O-50 and 30-O-C-P-O-50 internucleotide linkages.
15. Pacꢀes, O. Diploma Work, Charles University, Faculty of Natural Sciences,
Prague 2000. Synthesis and properties of modified dinucleoside monophos-
phates.
16. Begtrup, M.; Larsen, P. Alkylation, acylation and silylation of azoles. Acta
Chem. Scand. 1990, 44, 1050–1057.
17. Stawinski, J.; Hozumi, T.; Narang, S.A. Benzoyltetrazole: A mild benzoylating
reagent for nucleosides. J. Chem. Soc. Chem. Commun. 1976, 243.
18. Bhat, B.; Sanghvi, S. A mild and highly selective N-benzoylation of cytosine
and adenine bases in nucleosides with N-benzoyltetrazole. Tetrahedron Lett.
1997, 38, 8811–8814.
19. de Leeuw, F.A.A.M.; Altona, C. Conformational analysis of b-D-ribo,
b-D-deoxyribo, b-D-arabino, b-D-xylo, and b-D-lyxo-nucleosides from
proton-proton coupling constants. J. Chem. Soc., Perkin Trans 2 1982,
375–384.
Received January 22, 2003
Accepted January 27, 2003