Nucleosidyl-O-methylphosphonates: A pool of monomers for modified oligonucleotides

Article abstract of DOI:10.1081/NCN-200033912

An unique set of 5′-O- and 3′-O-phosphonomethyl derivatives of four natural 2′-deoxyribonucleosides, 1-(2-deoxy-β-D-threo- pentofuranosyl)thymine, 5′-O- and 2′-O-phosphonomethyl derivatives of 1-(3-deoxy-β-D-erythro-pentofuranosyl)thymine, and 1-(3-deoxy-β-D- threo-pentofuranosyl)thymine has been synthesized as a pool of monomers for the synthesis of modified oligonucleotides. The phosphonate moiety was protected with 4-methoxy-1-oxido-2-pyridylmethyl ester group, serving also as an intramolecular catalyst in the coupling step.

Full text of DOI:10.1081/NCN-200033912

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Nucleosidyl‐O‐Methylphosphonates: A Pool of
Monomers for Modified Oligonucleotides
Dominik Rejman a , Milena Masojídková a & Ivan Rosenberg a
a Institute of Organic Chemistry and Biochemistry, Academy of Sciences, Flemingovo nam. 2,
166 10, Prague 6, Czech Republic
Published online: 01 Jun 2007.
To cite this article: Dominik Rejman , Milena Masojdkov & Ivan Rosenberg (2004) Nucleosidyl‐O‐Methylphosphonates: A Pool
of Monomers for Modified Oligonucleotides, Nucleosides, Nucleotides and Nucleic Acids, 23:11, 1683-1705, DOI: 10.1081/
NCN-200033912
To link to this article: http://dx.doi.org/10.1081/NCN-200033912
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 23, No. 11, pp. 1683–1705, 2004
Nucleosidyl-O-Methylphosphonates: A Pool of Monomers
for Modified Oligonucleotides
*
Dominik Rejman, Milena Masoj´ıdkova´, and Ivan Rosenberg
Institute of Organic Chemistry and Biochemistry, Academy of Sciences,
Prague 6, Czech Republic
ABSTRACT
An unique set of 5’-O- and 3’-O-phosphonomethyl derivatives of four natural 2’-
deoxyribonucleosides, 1-(2-deoxy-b-D-threo-pentofuranosyl)thymine, 5’-O- and 2’-O-
phosphonomethyl derivatives of 1-(3-deoxy-b-D-erythro-pentofuranosyl)thymine,
and 1-(3-deoxy-b-D-threo-pentofuranosyl)thymine has been synthesized as a pool
of monomers for the synthesis of modified oligonucleotides. The phosphonate moiety
was protected with 4-methoxy-1-oxido-2-pyridylmethyl ester group, serving also as an
intramolecular catalyst in the coupling step.
Key Words: Nucleoside phosphonates; P-C linkage; Nucleotide analogs;
Pentofuranosylthymine derivatives; Protection; Regioizomers; Non-isosteric
internucleotide linkage; Isopolar oligonucleotides.
INTRODUCTION
The solid-phase synthesis of isopolar phosphonate oligonucleotides employs a
modern phosphotriester methodology for incorporation of the nucleoside phosphonic acid
residues into the growing oligonucleotide chain.[1–4] The practical introduction of the
*
Correspondence: Ivan Rosenberg, Institute of Organic Chemistry and Biochemistry, Academy of
Sciences, Flemingovo nam. 2, 166 10 Prague 6, Czech Republic; E-mail: ivan@uochb.cas.cz.
1683
DOI: 10.1081/NCN-200033912
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com
Copyright D 2004 by Marcel Dekker, Inc.

1684
Rejman et al.
4-methoxy-1-oxido-2-picolyl (Pic) group for the protection of phosphate and phosphonate
moieties in nucleotides by Efimov[5] and Stawinski,[6] resp., enables both shortening the
coupling time and considerably increase the yield of the coupling step due to an ability of
the 4-methoxy-1-oxido-2-picolyl group to act as a powerful intramolecular catalyst on the
activation of the phosphorus moiety by arylsulfonyl chlorides.
Recently we reported the phosphotriester solid-phase synthesis and properties of
novel types of nuclease stable phosphonate oligonucleotides differing in the position of
the bridging methylene group inserted into the internucleotide linkage between ester
oxygen and phosphorus atom. Thus, two types regioisomeric phosphonate oligonucleo-
tides containing either 3’-O-P-CH2-O-5@ or 3’-O-CH2-P-O-5@ internucleotide linkage
were subjected to study on their hybridisation properties and ability to elicit RNase H
activity. Obtained encouraging results, mainly for the altering oligonucleotides
consisting of phosphonate and phosphodiester internucleotide bonds,[7] and also our
further study on oligonucleotide-based HIV-I integrase inhibitors[8] containing the 3’-O-
CH2-P-O-5@ internucleotide linkages in the cleavage site, prompted us to continue the
investigation in the area of phosphonate oligonucleotides. For a complex study on these
compounds, we have synthesized the protected nucleoside phosphonic acids derived
from four natural 2’-deoxynucleosides. Since there are two possible positions of an
extra methylene group in the internucleotide linkage, both nucleoside 5’- and
3’-phosphonates are highly desirable. Moreover it was decided, to extend this group
into thymine derivatives differing in configuration of its sugar moiety.
RESULTS AND DISCUSSION
Synthesis of 3’- and 5’-phosphonate monomers derived from 2-deoxy-b-D-erythro–
pentofuranosylderivatives of nucleobases (Schemes 1 and 2).
3’-O-2a–f and 5’-O-dimethoxytrityl-2’-deoxynucleosides 4a–e were used as
starting compounds for the synthesis of final phosphonate monomers 3g–l (Scheme 1)
and 5f–j (Scheme 2), respectively. Nucleosides 2a–f protected with the 3’-O-di-
methoxytrityl group were prepared in three steps including a regioselective silylation of
the 5’-hydroxyl of N-protected 2’-deoxynucleosides (1a, 1b,[9] 1c,[10] 1d,[10] 1e,[11,12]
1f,)[9] with tert-butyldiphenylsilyl chloride in pyridine, followed by the reaction of 3’-
hydroxyl with dimethoxytrityl chloride in DCM in the presence of DBU, according to
the procedure described for tritylation of secondary hydroxyl[13] and final treatment of
fully protected compounds with TBAF in THF. 5’-O-Dimethoxytrityl-2’-deoxynucleo-
sides 4a–e (Scheme 2) were prepared by the reaction of nucleosides 1a–e with
dimethoxytrityl chloride in pyridine.[14]
Dimethyl esters 3 and 5 (R1 = R2 = CH3), obtained from the reaction of alkoxides
generated in situ from dimethoxytrityl derivatives 2, 4 and sodium hydride with
dimethyl-tosyloxymethylphosphonate[15] (DTMP), were selectively demethylated in
60% aqueous pyridine to monoesters 3a–f, 5a–e, and these compounds were esterified
with 4-methoxy-1-oxido-2-pyridylmethanol (MOPM)[16] using 2-chloro-5,5-dimethyl-2-
oxido-1,3,2-dioxaphosphinane (CDOP) as a condensing agent and 4-methoxy-1-
oxidopyridine (MOP) as an O-nucleophilic catalyst.[11,12] Obtained mixed diesters
were treated with 60% aqueous pyridine to cleave off remaining methyl ester group to
afford final monomers 3g–l and 5f–j in 5’- and 3’-series, respectively.

Nucleosidyl-O-Methylphosphonates
1685
Scheme 1. i. TBDPSCI, pyridine; ii. DMTrCl, DBU, DCM; iii. TBAF, THF; iv. NaH, DMF,
TsOCH2P(O)(OCH3)2; v. 60% pyridine; vi. 4-methoxy-1-oxido-2-pyridylmethanol, 2-chloro-5,
5-dimethyl-2-oxido-1,3,2-dioxaphosphinane, pyridine; vii. 60% pyridine.
In the case, of the reaction of thymine nucleosides 2a and 4a, and also 9a, 15, 19,
23 and 27 with dimethyl tosyloxymethylphosphonate, the formation of substantial
amount of 3-N-methyl derivatives was observed. This seems to be the main limitation
of the use of dimethyl-tosyloxymethylphosphonate for introduction of O-phosphono-
methyl moiety on the 3-N-unprotected thymine derivatives because of very difficult
separation of 3-N-methyl and 3-N-unsubstituted derivatives.
Scheme 2. i. DMTrCl, pyridine; ii. NaH, DMF, TsOCH2P(O)(OCH3)2; iii. 60% pyridine;
iv. 4-methoxy-1-oxido-2-pyridylmethanol, 2-chloro-5,5-dimethyl-2-oxido-1,3,2-dioxaphosphinane,
pyridine; v. 60% pyridine.

1686
Rejman et al.
At the same end of this work we found that diisopropyl tosyloxymethylphosph-
onate[17] is much advantageous reagent than DTMP. This agent does not alkylate
thymine and 2-N-protected guanine moieties at the 3-N and 1-N positions, respectively.
Resulting diisopropyl ester is stable under workup and chromatography, and the
removal of both diisopropyl ester groups is accomplish using bromotrimethylsilane. Re-
esterification of free phosphonate moiety with 4-methoxy-1-oxido-2-pyridylmethanol in
the presence of DCC yielded mostly monoester 3g and 5f and traces of diester that was
transformed into monoester by treatment with aqueous pyridine. This improved method
will be published afterwards in the work dealing with different structures.
Another difficulty was observed during the alkylation of guanine derivative 2e with
dimethyl tosyloxymethylphosphonate in the presence of sodium hydride. In this case,
the 6-O-[2-(4-nitrophenyl)ethyl] protecting group of compound 2e underwent very fast
b-elimination reaction (interestingly, under the same reaction conditions, the 6-O-[2-(4-
nitrophenylthio)ethyl] protecting group in compounds 2f and 4e was stable). Therefore,
the phosphonate 3 (B = 2-N-isobutyrylguanine) was isolated as dimethyl ester
(R1 = R2 = Me), and this compound was protected again with 2-(4-nitrophenyl)ethyl
group using 2-(4-nitrophenyl)ethanol, triphenylphosphine and DEAD.
Synthesis of 5’-phosphonate monomer derived from 1-(2-deoxy-b-D-threo-
pentofuranosyl)thymine (Scheme 3).
Sodium salt of 2,3’-anhydrothymidine (6), generated in situ with sodium hydride at
À30°C was condensed with DTMP in DMF. Also in this case, the alkylation was
accompanied by the formation of unwanted 3-N-methyl derivative. Obtained dimethyl
ester 7 was hydrolysed in 0.5 M aqueous sodium hydroxide giving, after
dimethoxytritylation of the 3’-hydroxy group with dimethoxytrityl chloride in pyridine
and in presence of silver triflate,[18] monomethyl ester 8a. Following esterification with
4-methoxy-1-oxido-2-pyridylmethanol[16] led to mixed diester 8 (R1 = CH3; R2 = Pic),
which was selectively hydrolysed, in aqueous pyridine giving 5’-monomer 8b.
Synthesis of 3’-phosphonate monomer derived from 1-(2-deoxy-b-D-threo-
pentofuranosyl)thymine (Scheme 4).
5’-Hydroxyl group of 2,3’-anhydrothymidine (6) was tritylated using dimethoxy-
trityl chloride in pyridine and the anhydro ring was subsequently opened in 0.5 M
sodium hydroxide in aqueous pyridine. Obtained 1-(2-deoxy-5-O-dimethoxytrityl-b-D-
threo-pentofuranosyl)thymine was alkylated with DTMP as described above. In this
case, desired product 10a was obtained only in 21% yield and unwanted 3-N-
Scheme 3. i. NaH, DMF, TsOCH2P(O)(OCH3)2; ii. NaOH; iii. DMTrCl, TfOAg, pyridine;
iv. 4-methoxy-1-oxido-2-pyridylmethanol, 2-chloro-5,5-dimethyl-2-oxido-1,3,2-dioxaphosphinane,
pyridine; iv. 60% aq. pyridine.

Nucleosidyl-O-Methylphosphonates
1687
Scheme 4. i. DMTrCl, pyridine; ii. NaOH, H2O, pyridine; iii. NaH, DMF, TsOCH2P(O)(OCH3)2;
iv. 60% aq. pyridine; v. 4-methoxy-1-oxido-2-pyridylmethanol, 2-chloro-5,5-dimethyl-2-oxido-
1,3,2-dioxaphosphinane, pyridine; vi. 60% aq. pyridine.
methylderivative (a little higher Rf on TLC) in 18% yield. Product 10a was further
transformed into 3’-phosphonate monomer 10b as mentioned above.
Synthesis of 2’- and 5’-phosphonate monomers derived from 1-(3-deoxy-b-D-
erythro-pentofuranosyl)thymine (14) (Scheme 5).
Acetolysis of 3-deoxy-1,2-O-isopropyliden-5-O-methoxycarbonyl-b-D-erytro-pen-
tofuranose (11a), prepared by radical deoxygenation[19] of 1,2-O-isopropyliden-5-O-
methoxycarbonyl-b-D-xylo-pentofuranose[20,21] (R. Liboska, unpublished results) (11)
via its immidazoylthiocarbonyl derivative, in acetic anhydride-acetic acid-sulphuric
acid mixture led to the precursor 12. Nucleosidation reaction of this compound with
2,4-O-bis-trimethylsilylthymine in acetonitrile under tin tetrachloride catalysis afforded
1-(2-O-acetyl-3-deoxy-5-O-methoxycarbonyl-b-D-erythro-pentofuranosyl)thymine[22,23]
(13). This compound was used as a starting material for the synthesis of both key
dimethoxytrityl derivatives 15 and 19. 2’-O-Acetyl and 5’-O-methoxycarbonyl groups
were removed from nucleoside 13 by its heating with Dowex 1 (OHÀ form) in ethanol.
Scheme 5. i. Bisimidazoylthiocarbonate, Bu3SnH, toluene; ii. Ac2O, AcOH, DCM, H2SO4; iii.
2,4-O-bistrimethylsilylthymine, SnCl4, acetonitrile; iv. Dowex 1 OHÀ, methanol; v. DMTrCl,
pyridine; vi. 60% pyridine; vii. 4-methoxy-1-oxido-2-pyridylmethanol, 2-chloro-5,5-dimethyl-2-
oxido-1,3,2-dioxaphosphinane, pyridine; viii. 60% pyridine; ix. HCl, methanol; x. DMTrCl, DBU,
DCM; xi. aq. NH3.

1688
Rejman et al.
Scheme 6. i. MsCl, pyridine; ii. Dowex 50 OHÀ, methanol; iii. DMTrCl, pyridine; iv. NaH, DMF,
TsOCH2P(O)(OCH3)2; v. 60% pyridine; vi. 4-methoxy-1-oxido-2-pyridylmethanol, 2-chloro-5,5-
dimethyl-2-oxido-1,3,2-dioxaphosphinane, pyridine; vii. 60% pyridine; viii. TBDPSCI, pyridine;
ix. DMTRCI, DBU, pyridine; x. TBAF, THF.
Obtained nucleoside 14 was dimethoxytritylated at the 5’-position by currently used
procedure giving 5’-protected compound 15.
The synthesis of the 3’-O-DMTr derivative 19 started also from precursor 13. Its
selective deacetylation by treatment with 1M hydrogen chloride in methanol afforded in
a high yield 3’-deoxy-5’-O-methoxycarbonylthymidine (17), and this compound was
dimethoxytritylated using dimethoxytrityl chloride and DBU in DCM. 2’-O-Dimethoxy-
trityl derivative 19 was obtained by removal of methoxycarbonyl group in meth-
anolic ammonia.
Both protected nucleoside derivatives 15 and 19 were transformed into phos-
phonate monomers 16b and 20b employing described procedure.
Synthesis of 2’- and 5’-phosphonate monomers derived from 1-(3-deoxy-b-D-threo-
pentofuranosyl)thymine (22) (Scheme 6).
Inversion of configuration on 2’ carbon atom of compound 17 was carried out via
anhydro intermediate 21 obtained by cyclisation of 2’-O-mesylderivative of compound
17. The anhydro ring of compound 21 was opened by treatment with Dowex 1 (OHÀ
form), and simultaneously the 5’-O-methoxycarbonyl group was cleaved off giving nu-
cleoside 22.[24] This compound was transformed into both 5’-O-dimethoxytrityl de-
rivative 23[25] and 2’-O-dimethoxytrityl derivative 27. The compound 27 was obtained
as described for compound 2. Dimethoxytrityl derivatives 23 and 27 were converted
into 5’-phosphonate 28b and 2’-phosphonate 28b, respectively, as described above.
EXPERIMENTAL
Unless stated otherwise, all used solvents were anhydrous. Final products were
lyophilized from dioxane or benzene, and dried over phosphorus pentoxide at 40°C
and 13 Pa. TLC was performed on silica gel pre-coated aluminium plates Silufol UV
254 foils (Kavalier Glassworks, Votice, Czech Republic), and compounds detected by
UV light (254 nm), by heating (detection of dimethoxytrityl group; orange color), and
by spraying with 1% solution of 4-(4-nitrobenzyl)pyridine in ethanol followed by
heating and treating with gaseous ammonia (blue color of mono- and diesters of

Nucleosidyl-O-Methylphosphonates
1689
phosphonic acid). Preparative column chromatography was carried out on silica gel
(40–60 mm; Fluka) neutralized with triethylamine (1 ml/100 g), and elution was
performed at the flow rate of 40 ml/min. The following solvent systems were used for
TLC and preparative chromatography: toluene-ethyl acetate 4:1 (T1), 1:1 (T2); toluene-
acetone 1:1 (T3); chloroform-ethanol 9:1 (C1); ethyl acetate-acetone-ethanol-water
4:1:1:1 (H1), 12:2:2:1 (H3). The concentrations of solvent systems are stated in volume
percents (%, v/v). Mass spectra were recorded on ZAB-EQ (VG Analytical) instrument,
using FAB (ionization with Xe, accelerating voltage 8 kV). Glycerol and thioglycerol
were used as matrices. NMR spectra were measured on Varian Unity 500 instrument
(1H at 500 MHz, 13C at 125.7 MHz) in deuterated chloroform or hexadeuteriodime-
thylsulfoxide (DMSO-d6).
General Methods
Method A. Preparation of methyl esters 3a–f, 5a–e, 10a, 16a, 20a, 24a, 28a
(Tables 1 and 2).
Sodium hydride (60% suspension in mineral oil; 3 equiv) was added at À30°C, under
argon atmosphere, to a solution of protected nucleoside and DTMP (1.1 equiv) in DMF
(5 ml/mmol of nucleoside). The suspension was vigorously stirred at r.t. for 3–16 h until
the starting nucleoside disappeared (TLC in H-3). The reaction mixture was then cooled
to À30°C, and carefuly neutralised with glacial acetic acid (2 equiv) in DMF (1 ml/
mmol). The temperature was increased to r.t., and the solution was concentrated at 40°C
in vacuo (13 Pa). The residue was dissolved in chloroform (50 ml/mmol) and the solution
was washed with 1M solution of sodium hydrogen carbonate. Organic layer was dried
over sodium sulfate, evaporated, and the crude product was treated in 60% aqueous
pyridine (30 ml/mmol) at 50°C for 16 h. After evaporation, the crude monomethyl ester
was dissolved in chloroform, and washed with 1M aqueous TEAB to remove methyl-
pyridinium cation. Organic layer was dried over sodium sulfate, evaporated, and the
desired product was obtained by purification on silica gel column using a linear gradient
from ethyl acetate to solvent system H3 followed by H3 to solvent system H1.
Method B. Synthesis of 4-methoxy-1-oxido-2-pyridylmethyl esters 3g–l, 5f–e,
8b, 10b, 16b, 20b, 24b, 28b (Tables 3 and 4).
Table 1. Alkylation of 3’-O-dimethoxytritylnucleosides.
DMTr-Nucleoside
mmol
2a 5.45 10
DMTOMPa
NaH
mmol
DMF
(ml)
Conditions
(Temp./time)
Yield
(%)
g
g
mmol
g
Product
3.24
0.9
11
3
1.22
0.26
0.46
0.94
0.6
30
6.6
50
15
20
40
25
30
20°C/24 h
10°C/3 h
20°C/24 h
20°C/20 h
20°C/24 h
10°C/4 h
37
35
72
50
5.7
18
3a
3b
3c
3d
3e
3f
2b 1.7
2c 2.4
2d 5.14
2e 3.94
2.2
3.8
7.8
5
1.24
2.53
1.62
4.2
8.6
5.5
4
11.4
23.4
15
2f
2.69
3.28 1.17
0.4
10
aDimethyl tosyloxymethylphosphonate.

1690
Rejman et al.
Table 2. Alkylation of 5’-O-dimethoxytritylnucleosides.
DMTr-Nucleoside
DMTOMPa
mmol
3.24 11
NaH
mmol
DMF
(ml)
Conditions
(temp./time)
Yield
(%)
g
mmol
g
g
Product
4a 5.5
4b 4.5
10
1.22
0.68
1.22
30
17
50
50
50
30
50
20°C/20 h
20°C/24 h
20°C/20 h
20°C/24 h
10°C/3 h
50
51
38
60
14
5a
5b
5c
5d
5e
6.2
9.7
2
3.24 11
7
4c 6.13
4d 3.84
30
18
5.84 1.9
7.2 2.75
6.43 0.7
9.36 0.86
4e
5.9
21.6
aDimethyl tosyloxymethylphosphonate.
A mixture of monomethyl ester, obtained according to Method A, 4-methoxy-1-
oxido-2-pyridylmethanol (1.1 eq.), and 4-methoxy-1-oxido-2-pyridine (3 eq.) dried by
co-evaporation with pyridine was dissolved in pyridine (10 ml/mmol of methyl ester),
and the solution was treated with 2-chloro-5,5-dimethyl-2-oxido-1,3,2-dioxaphosphi-
nane (CDOP) (3 eq.) under argon atmosphere and vigorous stirring at r.t. for 6–16 h
(TLC in H1). An excess of CDOP was decomposed by addition of small amount of
water, and the reaction mixture was concentrated in vacuo at low temperature in a bath.
The residue was partitioned between chloroform and 1M TEAB, organic layer was
evaporated, and the residue was treated in 60% aqueous pyridine (ꢀ30 ml/mmol) at
50–60°C for 16 h. After evaporation of the reaction mixture, the residue was par-
titioned between chloroform and 1M TEAB. The organic layer was evaporated, and
crude product was purified on silica gel, using a linear gradient of solvent system H3 in
ethyl acetate followed by gradient of solvent system H1 in H3.
4-Methoxy-1-oxido-2-pyridylmethyl 2-deoxy-3-O-dimethoxytrityl-1-(thymin-1-
yl)-b-D-erythro-pentofuranos-5-yloxymethylphosphonate (3g). Compound 3g was
prepared according to Method B (Table 5) from monomethyl ester 3a obtained
according to Method A (Table 3).
Table 3. Synthesis of 4-methoxy-1-oxido-2-pyridylmethyl esters of protected nucleosid-5’-
phosphonic acids.
Methyl ester
mmol
MOPMa
mmol
MOPb
mmol
CDOPc
Py Temp./time Yield
mmol (ml) (°C/min) (%) Product
g
g
g
g
3a 1.34 1.78 0.3 1.96 0.7
3b 0.71 0.84 0.15 0.84 0.3
5.4 1
2.3 0.5
5.4
2.3
25
15
20
120
3
20/60
20/60
20/30
20/120
20/60
20/90
87
64
27
31
89
64
3g
3h
3i
3c 1.99 2.36 0.4 2.6
3d 6.93 8 1.37 8.8
0.9
3
7.1 1.31 7.1
24 4.5 24
3j
3e 0.09 0.09 0.02 0.1
3f 0.58 0.61 0.11 0.7
0.03 0.27 0.05 0.27
0.26 2.1 0.39 2.1
3k
3l
10
a4-methoxy-1-oxido-2-pyridylmethanol.
b4-methoxy-1-oxidopyridine.
c2-chloro-5,5-dimethyl-2-oxido-1,3,2-dioxaphosphinane.

Nucleosidyl-O-Methylphosphonates
1691
Table 4. Synthesis of 4-methoxy-1-oxido-2-pyridylmethyl esters of protected nucleosid-3’-
phosphonic acids.
Methyl ester
mmol
MOPMa
mmol
MOPb
mmol
CDOPc
Py Temp./time Yield
mmol (ml) (°C/min) (%) Product
g
g
g
g
5a 0.81 1.07 0.21 1.37 0.47 3.72 0.69 3.72 15
5b 2.98 3.18 0.6 3.5 1.2 9.54 1.8 9.54 40
5c 3.6 4.37 0.74 4.19 1.67 13.15 2.43 13.15 40
5d 3.04 3.5
5e 0.97 1.02 0.2
20/50
20/60
20/60
20/50
20/40
70
81
40
52
39
5f
5g
5h
5i
0.6
3.85 1.3 10.5 1.9 10.5
1.23 0.4 3.1 0.58 3.1
50
15
5j
a4-methoxy-1-oxido-2-pyridylmethanol.
b4-methoxy-1-oxidopyridine.
c2-chloro-5,5-dimethyl-2-oxido-1,3,2-dioxaphosphinane.
HR FAB calcd for C39H41N3O12P 774.2428 (M-H)À, found 774.2460.
NMR: Tables 5, 6, and 7.
4-Methoxy-1-oxido-2-pyridylmethyl 2-deoxy-3-O-dimethoxytrityl-1-(4-O-(2-(4-
nitrophenylthio)ethyl)thymin-1-yl)-b-D-erythro-pentofuranos-5-yloxymethyl-phos-
phonate (3h). Compound 3h was prepared according to Method B (Table 5) from
monomethyl ester 3b obtained according to Method A (Table 3).
HR FAB calcd for C47H48N4O14PS 955.2625 (M-H)À, found 955.2545.
NMR: Tables 5, 6, and 7.
4-Methoxy-1-oxido-2-pyridylmethyl 2-deoxy-3-O-dimethoxytrityl-1-(4-N-ben-
zoylcytosin-1-yl)-b-D-erythro-pentofuranos-5-yloxymethylphosphonate (3i). Com-
pound 3i was prepared according to Method B (Table 5) from monomethyl ester 3c
obtained according to Method A (Table 3).
HR FAB calcd for C45H44N4O12P 863.2693 (M-H)À, found 863.2693.
NMR: Tables 5, 6, and 7.
4-Methoxy-1-oxido-2-pyridylmethyl 2-deoxy-3-O-dimethoxytrityl-1-(6-N-ben-
zoyladenin-9-yl)-b-D-erythro-pentofuranos-5-yloxymethylphosphonate (3j). Com-
pound 3j was prepared according to Method B (Table 5) from monomethyl ester 3d
obtained according to Method A (Table 3).
HR FAB calcd for C46H44N6O11P 887.2806 (M-H)À, found 887.2760.
NMR: Tables 5, 6, and 7.
4-Methoxy-1-oxido-2-pyridylmethyl 2-deoxy-3-O-dimethoxytrityl-1-(2-N-iso-
butyryl-6-O-(2-(4-nitrophenyl)ethyl)guanin-9-yl)-b-D-erythro-pentofuranos-5-yloxy-
methylphosphonate (3k). Compound 3k was prepared according to Method B
(Table 5) from monomethyl ester 3e. Dimethoxytrityl derivative 2e (3.94 g; 5 mmol)
was treated with DTMP and sodium hydride according to Method A (Table 3). The
reaction mixture was neutralized by glacial acetic acid at low temperature and
concentrated in vacuo at 40°C. Residue in chloroform was applied on silica gel column

1692
Rejman et al.

Nucleosidyl-O-Methylphosphonates
Table 6. Interaction constants for Table 5.
1693
1’2’
1’2@
2’2@
2’3’
2@3’
3’4’
4’5’
4’5@
5’5@
P-CH2
3a
3b
3c
3d
3e
3f
9.8
9.5
7.5
9.3
9.5
9.6
9.5
9.3
8.4
9.2
9.5
9.8
5.4
5.5
5.4
5.5
5.6
5.9
5.6
5.6
5.5
5.5
5.8
5.8
5.4
5.6
6.8
6.7
13.9
13.7
14.2
13.9
13.9
13.9
13.2
13.2
13.4
13.4
13.2
13.4
13.6
13.6
5.6
5.5
5.6
5.6
5.0
5.1
5.2
5.5
5.6
5.4
5.4
5.5
5.4
5.4
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.7
1.5
1.0
1.0
6.8
6.5
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.7
1.5
1.0
1.0
5.0
5.0
2.7
2.8
2.4
2.9
3.2
3.0
2.5
2.3
2.6
3.0
4.0
3.2
4.5
4.5
3.4
3.0
3.2
3.1
3.4
4.0
3.0
3.7
3.6
4.0
4.5
4.3
5.4
4.5
10.7
10.5
10.7
10.7
10.5
10.5
10.5
10.6
10.5
10.8
10.5
10.6
10.7
–
8.3
8.7
8.1
8.1
8.5
8.2
8.3
8.8
8.6
8.6
8.2
9.0
8.6
8.6
8.8
9.3
8.5
–
9.2
8.8
–
3g
3h
3i
8.8
8.8
9.2
–
3j
3k
3l
9.0
8.8
–
8a
8b
and phosphonate 3 (B = 2-N-isobutyrylguanine; R1 = R2 = CH3) 0.4 g (0.525 mmol;
10.5%) eluted with a linear gradient of ethanol in chloroform (up to 10%). This
compound, 2-(4-nitrophenyl)ethanol (0.3 g; 1.84 mmol) and triphenylphosphine (0.525
g; 2 mmol) were dried by co-evaporation with dioxane, and this mixture treated with
DEAD (0.32 ml; 1.84 mmol) in dioxane (3 ml) at r.t. overnight. The solution was
concentrated in vacuo, the residue was partitioned between chloroform and 1M sodium
hydrogencarbonate, organic layer was evaporated and crude product was heated in 60%
aqueous pyridine (ꢀ 30 ml/mmol) at 50–60°C for 16 h. After evaporation, the residue
was partitioned between chloroform and 1M TEAB, organic layer was evaporated and
crude product was purified on silica gel, using a linear gradient of solvent system H3 in
ethyl acetate followed by a gradient of solvent system H1 in H3. Obtained monomethyl
ester 3e was then converted into the 4-methoxy-1-oxido-2-pyridylmethyl ester 3k
according to Method B.
HR FAB calcd for C51H54N7O14PNa 1042.3363 (M + Na)+, found 1042.3364.
NMR: Tables 5, 6, and 7.
4-Methoxy-1-oxido-2-pyridylmethyl 2-deoxy-3-O-dimethoxytrityl-1-(2-N-iso-
butyryl-6-O-(2-(4-nitrophenylthio)ethyl)guanin-9-yl)-b-D-erythro-pentofuranos-5-
yloxymethylphosphonate (3l). Compound 3l was prepared according to Method B
(Table 5) from monomethyl ester 3f obtained according to Method A (Table 3).
HR FAB calcd for C51H53N7O14PS 1050.3109 (M-H)À, found 1050.3095.
NMR: Tables 5, 6, and 7.
4-Methoxy-1-oxido-2-pyridylmethyl 2-deoxy-5-O-dimethoxytrityl-1-(thymin-1-
yl)-b-D-erythro-pentofuranos-3-yloxymethylphosphonate (5f). Compound 5f was
prepared according to Method B (Table 8) from monomethyl ester 5a obtained
according to Method A (Table 4).
HR FAB calcd for C45H58N4O12P 877.3789 (M + Et3N + H)+, found 877.3788.
NMR: Tables 8, 9, and 10.

1694
Rejman et al.

Nucleosidyl-O-Methylphosphonates
1695

1696
Rejman et al.
Table 9. Interaction constants for Table 8.
1’2’
1’2@
2’2@
2’3’
2@3’
3’4’
4’5’
4’5@
5’5@
P-CH2
5a
5b
5c
8.3
7.9
6.6
7.4
6.6
8.4
7.7
6.3
7.6
7.2
2.4
2.7
5.9
6.1
6.1
6.3
6.2
5.9
6.2
6.3
6.3
6.5
8.7
8.8
13.7
13.7
13.9
13.9
13.8
13.7
13.6
13.9
13.9
13.7
15.1
14.9
6.1
6.2
6.0
6.1
6.5
6.0
6.0
6.3
6.0
6.0
1.0
1.0
2.4
2.2
3.4
3.2
4.4
2.0
2.0
3.5
2.8
2.5
5.3
5.5
3.0
3.0
3.0
2.9
3.2
2.5
2.5
3.0
2.6
3.0
3.5
4.4
4.4
4.5
4.6
5.8
6.0
3.9
3.5
4.7
6.0
6.3
7.6
6.7
3.2
3.0
3.4
4.6
3.2
3.1
2.8
3.4
4.8
5.0
3.4
4.6
10.5
10.5
10.5
10.3
10.5
10.5
10.3
10.5
10.2
10.2
10.2
10.0
9.0
9.4
8.8
8.3
9.0
9.0
9.0
8.7
8.6
8.4
9.8
9.6
8.8
9.2
8.8
–
5d
5e
–
5f
–
5g
5h
5i
–
–
–
5j
–
10a
10b
9.1
8.5
2-Deoxy-5-O-dimethoxytrityl-1-(4-O-(2-(4-nitrophenylthio)ethyl)thymin-1-yl)-b-
D-erythro-pentofuranos-3-yloxymethyl-phosphonate (5g). Compound 5g was pre-
pared according to Method B (Table 8) from monomethyl ester 5b obtained according
to Method A (Table 4).
HR FAB calcd for C47H48N4O14PS 955.2625 (M-H)À, found 955.2500.
NMR: Tables 8, 9, and 10.
4-Methoxy-1-oxido-2-pyridylmethyl 2-deoxy-5-O-dimethoxytrityl-1-(4-N-ben-
zoylcytosin-1-yl)-b-D-erythro-pentofuranos-3-yloxymethylphosphonate (5h). Com-
pound 5h was prepared according to Method B (Table 8) from monomethyl ester 5c
obtained according to Method A (Table 4).
HR FAB calcd for C45H46N4O12P 865.2850 (M + H)À, found 865.2849.
NMR: Tables 8, 9, and 10.
4-Methoxy-1-oxido-2-pyridylmethyl 2-deoxy-5-O-dimethoxytrityl-1-(6-N-ben-
zoyladenin-9-yl)-b-D-erythro-pentofuranos-3-yloxymethylphosphonate (5i). Com-
pound 5i was prepared according to Method B (Table 8) from monomethyl ester 5d
obtained according to Method A (Table 4).
HR FAB calcd for C52H61N6O11P 990.4167 (M + Et3N + H)+, found 990.4335.
NMR: Tables 8, 9, and 10.
4-Methoxy-1-oxido-2-pyridylmethyl 2-deoxy-5-O-dimethoxytrityl-1-(2-N-iso-
butyryl-6-O-(2-(4-nitrophenylthio)ethyl)guanin-9-yl)-b-D-erythro-pentofuranos-3-
yloxymethylphosphonate (5j). Compound 5j was prepared according to Method B
(Table 8) from monomethyl ester 5e obtained according to Method A (Table 4).
HR FAB calcd for C51H53N7O14PS 1050.3109 (M-H)À, found 1050.2983.
NMR: Tables 8, 9, and 10.
4-Methoxy-1-oxido-2-pyridylmethyl 2-deoxy-3-O-dimethoxytrityl-1-(thymin-1-
yl)-b-D-threo-pentofuranos-5-yloxymethylphosphonate (8b). A mixture of 2,3’-
anhydrothymidine (6) (4.36 g; 19.45 mmol) and dimethyl tosyloxymethylphosphonate

Nucleosidyl-O-Methylphosphonates
1697

1698
Rejman et al.
(6.5 g, 22 mmol) dried by co-evaporation with DMF was treated under vigorous stirring
with sodium hydride (60% suspension in mineral oil; 2.34 g; 58.5 mmol) in DMF
(100 ml) at À15°C for 3 h, and then at r.t. overnight (TLC in H1). The reaction mixture
was cooled to À10°C, carefully neutralized with glacial acetic acid (2.4 ml; 40 mmol),
and DMF was evaporated at 40°C (13 Pa). The residue was partitioned between
chloroform and 1M aqueous sodium hydrogencarbonate. The organic phase was
concentrated in vacuo, oily residue dissolved in 1M aqueous solution of sodium
hydroxide (100 ml), and the solution stirred at r.t. for 2 h. The solution was neutralized
to pH 7 with Dowex 50 (H+ cycle), the resin was filtered off, and obtained solution was
concentrated. The residue was co-evaporated several times with pyridine, dissolved in
pyridine (100 ml), and treated with dimethoxytrityl chloride (8.1 g; 24 mmol) and
silver triflate (6.2 g; 24 mmol) at r.t. for 16 h. The reaction was quenched by addition
of small amount of methanol, and the solution was concentrated. Compound 8a was
obtained by silica gel chromatography using a linear gradient of solvent system H3 in
ethyl acetate followed by a linear gradient of solvent system H1 in H3. Yield 3.66 g
(4.86 mmol; 25%) of 8a. This compound was transformed into 4-methoxy-1-oxido-2-
pyridylmethyl ester 8b according to Method B. Compound 8b was obtained in 42%
yield (1.81 g; 2.064 mmol).
HR FAB calcd for C39H41N3O12P 774.2428 (M-H)À, found 774.2402.
NMR: Tables 5, 6, and 7.
4-Methoxy-1-oxido-2-pyridylmethyl 2-deoxy-5-O-dimethoxytrityl-1-(thymin-1-
yl)-b-D-threo-pentofuranos-3-yloxymethylphosphonate (10b). Dimethoxytrityl
chloride (23 g; 68 mmol) was added in six portions during 6 h to a stirred solution of
thymidine (15 g; 62 mmol) in pyridine (150 ml), and the solution was set aside for
o
24 h. Mesyl chloride (6.24 ml; 80.60mmol) was added at 0 C, the reaction mixture
was stirred at r.t. for 2 h and then diluted with 2M aqueous sodium hydroxide
(300 ml). Obtained dark solution was stirred 2 d at r.t., neutralized to pH 7 with
Dowex 50 (H+ form), and after filtration, extracted with chloroform. Organic layer was
concentrated in vacuo and residue purified on silica gel using a linear gradient of
acetone in toluene to afford pure compound 9a in 99% yield (33.5 g; 61.5 mmol).
This compound 9a (11 g; 20 mmol) was alkylated with DTMP to the methylester
10a according to Method A in 21% yield (2.7 g; 4.12 mmol). The obtained compound
10a, was further transformed to monomer 10b according to Method B in 60% yield
(2.18 g; 2.49 mmol).
HR FAB calcd for C39H41N3O12P 774.2428 (M-H)À, found 774.2431.
NMR: Tables 8, 9, and 10.
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-b-D-erythro-pentofuranosyl)thy-
mine (13). A solution of 1,2-O-isopropyliden-5-O-methoxycarbonyl-D-xylofuranose
(11) (55 g; 221 mmol) and bis-imidazoylthiocarbonate (237 g; 465 mmol) in DCM
(1000 ml) was stirred under argon atmosphere at r.t. for 48 h. The reaction mixture was
concentrated to a half volume, extracted first with 1 M HCl (2x500 ml) and then with
saturated aqueous solution of sodium bicarbonate (2 Â 500 ml). The organic layer was
evaporated, the residue was dissolved in diethyl ether (300 ml), filtered through celite
and filtrate was evaporated. Yellow solid was dissolved in toluene (2200 ml) and the
solution was heated to reflux. The solution of tributyltin hydride (98 ml; 332 mmol) in

Nucleosidyl-O-Methylphosphonates
1699
toluene (500 ml) was added to the gently refluxing solution during 6 h, and the reaction
mixture was refluxed for additional 2 h (TLC in S1). The reaction mixture was
concentrated and residue was partitioned between acetonitrile and hexane at 40°C. The
acetonitrile layer was evaporated, and crude product 11a was purified on silica gel
using a linear gradient of ethyl acetate in toluene giving 59% (30.3 g; 130.5 mmol) of
11a. Concentrated sulfuric acid (2.42 ml) was added to a mixture of 11a (14.05 g; 60.5
mmol), acetic anhydride (25 ml), and acetic acid (15 ml) in DCM (180 ml) at 0°C. The
reaction mixture was stirred at r.t. overnight. Anhydrous sodium acetate (8.2 g) was
added, and the suspension was stirred vigorously for 30 min. The solution was
concentrated at first in the vacuum of the water pump followed by oil pump. The
residue was partitioned between chloroform and water. The organic layer was washed
with saturated solution of potassium chloride, dried with sodium sulfate, evaporated,
and finally co-evaporated with xylene (3 Â 50 ml) and DCM (3 Â 50 ml). Diacetyl
derivative 12 was obtained in 64% yield (10.62 g; 38.45 mmol).
A solution of 12 (18.6 g; 67.33 mmol) in acetonitrile (400 ml) was added through a
teflon tubing via argon pressure to crystalline 2,4-O-bis(trimethylsilyl)thymine (18.2 g;
67.33 mmol). After complete dissolution of thymine derivative, anhydrous tin
tetrachloride (16 ml; 135 mmol) was added. The reaction mixture was stirred at r.t.
for 20 h. Pyridine (45 ml; 550 mmol) was added and the mixture was concentrated in
vacuo. The residue was diluted with chloroform, and resulting suspension was filtered
through celite. The filtrate was evaporated, and pure compound 13 was obtained in
40% yield (9.32 g; 27.23 mmol) by chromatography on silica gel using a linear
gradient of ethyl acetate in toluene.
HR FAB calcd for C14H19N2O8 343.1141 (M + H)+, found 343.1210.
1H NMR: 1.78 (d, 3H, J(CH3,6) = 1.2, CH3); 2.05 (s, 3H, OAc); 2.06 (ddd, 1H,
J (3@, 2’) = 2.2, J (3@, 4’) = 6.2, J (3@, 3’) = 13.9, H-3@); 2.27 (ddd, 1H, J (3’,
2’) = 6.9, J (3’,4’) = 8.9, J (3’, 3@) = 13.9, H-3); 3.72 (s, 3H, OCH3); 4.24 (dd, 1H, J
(5’, 4’) = 5.6, J (5’, 5@) = 11.9, H-5’); 3.36 (dd, 1H, J (5@, 4’) = 2.8, J (5@, 5’) = 11.9,
H-5@); 4.39 (m, 1H, H-4’); 5.24 (dt, 1H, J (3’, 1’) ꢀ J (2’, 3@) = 2.3, J (2’, 3’) = 6.9,
H-2’); 5.80 (d, 1H, J (1’, 2’) = 2.5, H-1’); 7.46 (brq, 1H, J (6, CH3) = 1.2, H-6); 11.38
(s, 1H, NH).
13C NMR: 12.24 (CH3); 20.90 (CH3); 32.36 (C-3’); 55.11 (OCH3); 68.15 (C-5’);
76.97, 2C (C-2’ a C-4’); 90.14 (C-1’); 110.00 (C-5); 136.74 (C-6); 150.42 (C-2); 155.25
(OC(O)O); 163.95 (C-4); 170.03 (C=O).
4-Methoxy-1-oxido-2-pyridylmethyl 3-deoxy-5-O-dimethoxytrityl-1-(thymin-
1yl)-b-D-erythro-pentofuranos-2-yloxymethylphosphonate (16b). Dowex 1 (OHÀ
form) was added to a solution of compound 13 (3 g; 8.76 mmol) in methanol (200 ml).
The suspension was stirred at r.t. for 24 h, neutralized with acetic acid to pH 7 and
filtered off. The filtrate was evaporated, and the residue was co-evaporated several
times with water (50 ml) and ethanol (50 ml) to remove traces of acetic acid, and
finally dried with pyridine (2 Â 50 ml). Obtained nucleoside 14 in pyridine (60 ml)
was treated with dimethoxytrityl chloride (4.8 g; 8.76 mmol) which was added in 10
potions during 6 h. The reaction mixture was left at r.t. overnight, concentrated, and
dimethoxytrityl derivative 15 was purified on silica gel using a linear gradient of
ethanol in chloroform giving 63% yield (3 g; 5.5 mmol) of 15. The compound 15 (3 g;
5.5 mmol) was transformed to methylester 16a in 66% yield (2.79 g; 3.64 mmol)

1700
Rejman et al.

Nucleosidyl-O-Methylphosphonates
Table 12. Interaction constants for Table 11.
1701
1’2’
2’3’
2’3@
3’3@
3’4’
3@4’
4’5’
4’5@
5’5@
P-CH2
20a
20b
16c
16b
28a
28b
92d
24a
24b
4.6
4.0
3.6
3.0
1.0
1.0
1.0
7.1
7.2
6.8
7.7
6.3
6.9
5.5
5.5
5.5
8.5
8.7
4.0
3.8
4.0
3.0
1.0
1.0
1.0
7.1
7.2
13.4
13.4
13.6
13.4
13.2
13.2
13.2
n*
6.8
7.7
6.8
6.9
5.5
5.5
5.5
n*
4.8
6.7
5.0
4.8
5.2
5.0
5.0
n*
2.8
2.7
2.8
3.3
4.0
4.0
3.0
3.2
2.6
6.0
5.0
6.4
4.4
4.6
4.5
4.0
4.5
4.5
10.6
10.7
10.7
10.8
10.7
10.7
10.5
11.0
11.0
8.6
8.2
8.8
8.4
9.6
10.0
9.8
8.3
8.3
–
–
9.0
8.4
9.0
10.0
9.8
8.5
–
n*
n*
n*
*Unclear multiplet—unable to determine interaction constant.
according to Method A, and final picolyl ester 16b was prepared from 16a according
to Method B in 31% yield (1.01 g; 1.134 mmol).
HR FAB calcd for C39H41N3O12P 774.2428 (M-H)À, found 774.2408.
NMR: Tables 11, 12, and 13.
4-Methoxy-1-oxido-2-pyridylmethyl 3-deoxy-2-O-dimethoxytrityl-1-(thymin-1-
yl)-b-D-erythro-pentofuranos-5-yloxymethylphosphonate (20b). A solution of com-
pound 13 (3 g; 8.76 mmol) in 1M HCl in methanol (220 ml) was left at r.t. for 3 h and
evaporated. Compound 17 was obtained after chromatohraphy on silica gel using a
linear gradient of ethanol in chloroform in 71% yield (1.96 g; 6.24 mmol).
The mixture of 17 (1.96 g; 6.24 mmol), DBU (1.4 ml; 9 mmol), and
dimethoxytritylchloride (3 g; 9 mmol) in DCM (20 ml) was stirred ar r.t. for 48 h.
The reaction was quenched with methanol (5 ml), the solution was evaporated, and the
residue was partitioned between saturated aqueous solution of citric acid and
chloroform. Dimethoxytrityl derivative 18 was purified on a silica gel using a linear
gradient of acetone in toluene. Compound 18 was dissolved in methanol, the solution
was saturated with gaseous ammonia at 15°C, and left at r.t. for 24 h. After evap-
orating methanol, obtained dimethoxyderivative 19 was purified by chromatography on
silica gel using a linear gradient of acetone in toluene to obtain 38% yield (1.3 g;
2.39 mmol).
The compound 19 (1.3 g; 2.39 mmol) was transformed to the methylester 20a in
21% yield (0.64 g; 0.834 mmol) according to Method A, and this compound to picolyl
ester 20b in 81% yield (0.526 g; 0.678 mmol) by Method B.
HR FAB calcd for C39H41N3O12P 774.2428 (M-H)À, found 774.2407.
NMR: Tables 11, 12, and 13.
4-Methoxy-1-oxido-2-pyridylmethyl 3-deoxy-5-O-dimethoxytrityl-1-(thymin-1-
yl)-b-D-threo-pentofuranos-2-yloxymethylphosphonate (24b). Dimethoxytrityl
chloride (2 g; 6 mmol) was added in 8 portions during 6 h to a solution of compound
22[24] (1.4 g; 5.4 mmol) in pyridine (25 ml). The reaction mixture was stirred at r.t.
overnight, quenched with methanol (0.5 ml), and evaporated. The residue was parti-
tioned between chloroform and 1M sodium hydrogencarbonate. 5’-O-Dimethoxytrityl

1702
Rejman et al.

Nucleosidyl-O-Methylphosphonates
1703
derivative 23 was obtained by purification on silica gel using a linear gradient of
acetone in toluene in 99% yield (3 g; 5.37 mmol).
Compound 23 (0.56 g; 1 mmol) was transformed to the methylester 24a in 73%
yield (0.56 g; 0.73 mmol) according to Method A. Compound 24a afforded a final
monomer 24a by Method B in 44% yield (0.39 g; 0.44 mmol).
HR FAB calcd for C45H58N3O12P 877.3789 (M + H + TEA)+, found 877.3715.
NMR: Tables 11, 12, and 13.
4-Methoxy-1-oxido-2-pyridylmethyl 3-deoxy-2-O-dimethoxytrityl-1-(thymin-1-
yl)-b-D-threo-pentofuranos-5-yloxymethylphosphonate (28b). Tert-butyldiphenyl-
silyl chloride (1.73 ml; 6.74) was added to a solution of nucleoside 22[25] (1.57 g; 6.48
mmol) in pyridine (25 ml). The reaction mixture was stirred at r.t. for 48 h, quenched
with methanol (0.5 ml), and evaporated. The residue was partitioned between
chloroform and 1M sodium hydrogencarbonate. The organic layer was dried with
sodium sulfate, evaporated, and dissolved in DCM (20 ml). DBU (1.21 ml; 8 mmol)
and dimethoxytrityl chloride (2.73 g; 8 mmol) were added to the solution, and the
mixture was stirred at r.t. for 24 h, quenched with methanol (0.5 ml), and evaporated.
The residue was partitioned between chloroform and 10% aqueous citric acid. The
organic layer was dried with sodium sulfate, evaporated, the residue was co-evaporated
with toluene (50 ml), THF (50 ml), and treated with 0.678 M TBAF in THF (30 ml) at
r.t. for 16 h. Dowex 50 (Et3NH+ form) (30 ml) and methanol (50 ml) were added, and,
after short stirring, the suspension was filtered, and the filtrate was evaporated. 2’-O-
Dimethoxytrityl derivative 27 was obtained by purification on silica gel using a linear
gradient of acetone in toluene in 17% yield (0.62 g; 1.1 mmol). Compound 27 (0.62 g;
1.1 mmol) was transformed to the methylester 28a in 42% yield (0.35 g; 0.46 mmol)
according to Method A, and this compound afforded a final monomer 28b in 80%
yield (0.33 g; 0.37 mmol) by Method B. HR FAB calcd for C45H58N3O12P 877.3789
(M + H + TEA)+, found 877.38610.
NMR: Tables 11, 12, and 13.
ACKNOWLEDGMENTS
Support by grants #203/01/1166 (GA, Czech Rep.) and #A4055101 (GA Acad.
Sci., Czech Rep.) under research project Z4055905 is gratefully acknowledged. The
authors are indebted to the staff of the MS department of this Institute for mea-
surements of mass spectra.
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Received February 1, 2004
Accepted June 7, 2004