New approach to the synthesis of Oligo(nucleoside methanephosphonate)s

Article abstract of DOI:10.1021/jo951333v

A new method of preparation of 5′-O-DMT-(N-protected) nucleoside 3′-O-(methanephosphonofluoridates) 2 and their phosphonylating properties toward alcohols, in particular the 5′-hydroxyl groups of nucleosides (or nucleotides), is presented. Preparation of dinucleoside (37prime;,5′)-methanephosphonates 3 in solution and solid-phase synthesis of Me-Oligo 6 demonstrate the potential use of 2 as monomers.

Full text of DOI:10.1021/jo951333v

J . Org. Chem. 1996, 61, 879-881
879
New Ap p r oa ch to th e Syn th esis of Oligo(n u cleosid e
m eth a n ep h osp h on a te)s1
Lucyna Woz´niak, J arosław Pyzowski, and Wojciech J . Stec*
Polish Academy of Sciences, Centre of Molecular and Macromolecular Studies, Department of Bioorganic
Chemistry, Sienkiewicza 112, Ło´dz´ 90-363, Poland
Received J uly 21, 1995X
A new method of preparation of 5′-O-DMT-(N-protected) nucleoside 3′-O-(methanephosphonofluo-
ridates) 2 and their phosphonylating properties toward alcohols, in particular the 5′-hydroxyl groups
of nucleosides (or nucleotides), is presented. Preparation of dinucleoside (3′,5′)-methanephospho-
nates 3 in solution and solid-phase synthesis of Me-Oligo 6 demonstrate the potential use of 2 as
monomers.
Sch em e 1
In the course of our studies on the synthesis and
reactivity of 5′-O-DMT-nucleoside 3′-O-(Se-methyl meth-
anephosphonoselenolates) 12 we have found that they are
readily converted to 5′-O-DMT-protected nucleoside 3′-
O-methanephosphonofluoridates (2, B ) Thy, AdeBz
,
CytBz, Guaibu) in quantitative yield by treatment at room
temperature with a 20% molar excess of aqueous AgF
(Scheme 1). This conversion, contrary to our expectations
based upon stereochemical results of reactions of P-chiral
phosphoryl aziridates with pyridine-x(HF)n complex,3
but in agreement with results of other studies,4,5 is not
stereospecific.
Starting from any single diastereoisomer [RP]-1 or [SP]-
1, examination of this reaction by means of 31P NMR and
19F NMR established that complete disappearance of the
signals for 1 was accompanied by the appearance of a
nearly 1:1 ratio of a pair of dublets characteristic for the
diastereomeric mixture 2. This reaction was complete
within 15 min. Dilution of the reaction mixture with
chloroform and washing of the resulting solution with
brine leaves a practically pure diastereomeric mixture
of 2 in the organic phase. The identity of 2 has been also
proved by FAB MS analysis.
Ta ble 1. Syn th esis a n d P h ysica l P r op er ties of
5′-O-DMT-(N-p r otected )-n u cleosid e
3′-O-(m eth a n ep h osp h on oflu or id a tes) (2)
substrate 1
product 2
J 1
diast FAB
P-F
31P
J 1
31P
P-Se
B′ NMR: δa (Hz) NMR: δa (Hz) ratio MS yieldb
T
49.7
50.3
50.6
50.2
430
430
431
432
30.7
30.6
30.3
30.2
31.3
30.9
30.6
30.3
1060 1/1c 623.4
1059
96
95
90
95
CBz
GiBu
ABz
1060 1/1
1059
712.2
718.2
736.2
1058 1/1
1060
1057 1/1
1059
It was found that 2 (B ) Thy) diluted in EtOH in the
presence of 10 mol equiv of DBU provides quantitatively
5′-O-DMT-thymidine 3′-O-(O-ethyl methanephosphonate)
(4). The identity of 4 has been proved by MS analysis
a
b
All 31P NMR measurements in CDCl3. Isolated by means of
precipitation (see Experimental Section). c When the reaction was
carried out at -40 °C (THF-MeCN), observed diastereomeric ratio
of 2 was 3/2.
1
and by 31P and H NMR spectroscopy. In the light of this
observation, nucleoside methanephosphonofluoridates 2
have been employed for the phosphonylation of 3′-O-
protected nucleosides 5. Reaction of 2 with 5 in aceto-
nitrile solution, performed in the presence of 10-15 equiv
of DBU, is usually accomplished within 15-20 min, with
almost exclusive formation of dinucleoside (3′,5′)-metha-
nephosphonates 3. Diastereomeric ratios of 3 obtained,
according to Scheme 2, and physicochemical character-
istics of products are presented in Table 2.
Sch em e 2
Further simplification of this approach has been
achieved reacting 1 with 5 (B ) Thy) under anhydrous
conditions in the presence of triethylamine trihydrofluo-
ride and DBU. In this case, conversion of nucleoside 3′-
O-(Se-methyl methanephosphonoselenolate)s 1 into 2 and
their condensation with 5 leads with reasonable yields
to dinucleoside (3′,5′)-methanephosphonates 3 in a one-
pot reaction proceeding at room temperature. Indepen-
dently, triethylamine trihydrofluoride (1 M) in dioxane
was used as fluorinating agent for the conversion 1 f 2.
It was established that under the reaction conditions (3-
fold molar excess of Et3N.3HF, MeCN) but without DBU,
X Abstract published in Advance ACS Abstracts, J anuary 1, 1996.
(1) This paper is dedicated to Professor Colin Reese on the occasion
of his 65th birthday.
(2) Wozniak, L. W.; Pyzowski, J .; Wieczorek, M.: Stec, W. J . J . Org.
Chem. 1994, 59, 5843.
(3) Misiura, K.; Silverton, J . V. S.; Stec, W. J . J . Org. Chem. 1985,
50, 1815.
(4) (a) Corriu, R. J . P.; Dutheil, J .-P.; Lanneau, G. F. J . Chem. Soc.,
Chem. Commun. 1981, 101. (b) Corriu, R. J . P.; Dutheil, J .-P.; Lanneau,
G. F. J . Am. Chem. Soc. 1984, 106, 1060.
(5) Misiura, K.; Pietrasik, D.; Stec, W. J . J . Chem. Soc., Chem.
Commun. 1995, 613.
0022-3263/96/1961-0879$12.00/0 © 1996 American Chemical Society

880 J . Org. Chem., Vol. 61, No. 3, 1996
Woz´niak et al.
Ta ble 2. Syn th esis a n d P r op er ties of Din u cleosid e
Ta ble 3. Syn th etic P r otocol for th e Au tom a ted
Solid -P h a se Syn th esis of Me-Oligos Usin g Mon om er s 2
(3′,5′)-Meth a n ep h osp h on a tes 3
product 3
volume
(mL)
time
(s)
step
1
reagent or solvent
purpose
substrates
31P
NMR: δa ratiob fast/slow
diastereom
HPLC tR
(min)
FAB
MS
c
2
5
(a) dichloroacetic acid
in CH2Cl2
(b) acetonitrile
(a) DBU activated 2
in CH3CNa
(b) methylene chloride
(c) acetonitrile
(a) DMAP/Ac2O/lutidine
in THF
2.3
detritylation
50
Gibu
T
32.47
32.75
32.67
32.89
32.60
32.94
32.39
33.15
32.23
32.55
31.79
32.22
32.43
32.67
32.04
33.03
31.34
31.71
32.63
33.15
32.43
32.79
32.04
33.46
32.23
32.71
32.57
32.94
32.39
32.62
32.10
33.29
55
45
44
56
11.28
12.20
13.45
982.4
1071.4
1167.6
1077.4
976.4
7.0
0.6
150
600
2
coupling
CBz
ABz
GiBz
T
3.33
7.0
0.33
wash
wash
capping
200
150
20
47(61)d
53(39)
50
13.22
13.79
9.68
3
50
55
45
45
55
55
45
57
43
54
46
54
46
59
41
58
42
52
48
49
51
57
43
54
9.86
(b) acetonitrile
7.0
wash
150
CBz
13.16
13.79
12.09
13.09
14.54
15.54
10.74
11.05
14.08
14.36
12.81
13.23
13.74
14.49
9.47
a
For the 1 µmol scale, a mixture of 450 µL of 0.5 M DBU in
CH3CN and 150 µL of 0.1 M 2 in CH3CN was used.
CBz
ABz
Gibu
T
1065.4
1161.7
1071.6
887.4
Ta ble 4. Oligo-Me 6 p r ep a r ed fr om 2 via th e
Solid -P h a se Syn th esis
average
HPLC
sequence
yield (trityl assay)
data (tR)a (min)
T
TT
TTT
TTTT
TCCTG
TCCTT
92%
78%
83%
81%
87%
7.50
8.80
9.60
9.60
9.35
CBz
ABz
Gibu
T
976.4
1072.7
982.5
a
ODS HYPERSIL; gradient 5-40% MeCN in 0.1 M TEAB, pH
) 7.0, flow ) 1.5 mL/min, t ) 15 min).
9.92
ABz
12.69
13.63
14.95
15.31
16.74
17.03
10.87
11.30
1000.5
1089.6
1185.8
1095.4
CBz
ABz
Gibu
In the light of recent reports by Arnold9 and Reynolds
et al.10 demonstrating that chimeric oligonucleotides
containing [RP]-methanephosphonates linked to phos-
phates or phosphorothioates at alternate positions are
promising tools for an antisense mRNA strategy,11 an easy
access to 2, which can be produced in solution in bulk
quantities, represents a new source of dinucleoside (3,5)-
methanephosphonates for a dimer-block synthesis of
aforementioned chimeras. Also, in the context of re-
ported results it has to be mentioned that ionic nucleoside
3′-O-phosphorofluoridates have been used for the syn-
thesis of an oligonucleotide phosphate link by von Tiger-
strom and Smith in the early 1970’s.12 Our studies on
the “Revisited von Tigerstrom-Smith Approach” as ap-
plied to preparation of Me-oligos indicate that, in the
presence of DBU, nucleoside 3′-O-methanephosphono-
fluoridates 2 are very efficient, as expected,13 phospho-
nylating agents toward oxygen nucleophiles, including
nucleosides, and can effectively be employed for the
preparation of dinucleoside (3′,5′)-methanephosphonates.
46
a
b
NMR data for fully protected dimers. Diastereomeric ratio
determined by means of integrated signals in 31P NMR spectra.
c HPLC data; deprotected dimers, ODS-HYPERSIL, 5 µm, 25 cm;
conditions 4-40% MeCN/0.1 M TEAB, flow 1.5 mL/min, 1.5%/min.
d
HPLC integration, fully deprotected GPMeA (in brackets).
exclusive formation of 2 was observed (31P NMR δ 30.8,
30.65, J P-F ) 1060 Hz for both diastereomers, as
1
measured in C6D6/MeCN).
Since DBU-resistant solid supports with anchored
“starter” nucleoside are used in our laboratory for the
synthesis of oligo(nucleoside phosphorothioate)s6 and
oligo(nucleoside phosphorodithioate)s7 we have used 5
linked to LCA-CPG via a succinyl-sarcosinoyl linker8 for
solid-phase synthesis of oligo(nucleoside methanephos-
phonate)s 6. The protocol used for automated synthesis
of 6 is shown in Table 3, and compounds 6 obtained by
this method are listed in Table 4.
Very recently, the reaction of 2 with 5′-O-silylated
nucleosides performed in the presence of potassium
fluoride, has been presented as a new approach to the
synthesis of 3.14 Since the preparation of starting 1 is
relatively simple, and their stability seems to be higher
It has to be mentioned that a step-yield of condensation
via methanephosphonofluoridates is >92%, as measured
by trityl assay, and preparative yield of isolated dimer
T
MeT by means of HPLC reaches 90%.
The pentamer TCCTG, formerly prepared as homo-
(9) Arnold, L., J r. First International Antisense Conference of J apan,
Kyoto, Abstract OP-31 (1994).
(10) Reynolds, M. A.; Schwartz, D. A.; Riley, T. A.; Vaghefi, M. M.;
Hogrefe, R. I.; J aeger, J . A.; Lebedev, A.; Arnold, L. J ., J r. First
International Antisense Conference of J apan, Kyoto, Abstract P1-06
(1994).
(11) Hogrefe, R. I.; Reynolds, M. A.; Vaghefi, M. M.; Young, K. M.;
Riley, T. A.; Klem, R. E.; Arnold, L. J ., J r. In Synthetic Chemistry of
Oligonucleotides and Their Analogs, Agrawal, S., Ed.; Humana Press:
Totowa, NJ , 1993; pp 116-143.
chiral [all-RP]- and [all-SP]-constructs,2 is presently being
used in attempts at separation of the diastereomeric
mixture (16 isomers) by chiral HPLC column (data not
shown). Experiments toward improvement of the yield
of oligo(nucleoside methanephosphonate)s prepared by
the method presented are in progress.
(12) von Tigerstrom, R.; Smith, M. Science 1970, 167, 1266.
(13) (a) Horner, L.; Flemming, H.-W. Phosphorus Sulfur Relat. Elem.
1984, 19, 345. (b) Horner, L.; Gehring, R. Phosphorus Sulfur Relat.
Elem. 1982, 12, 295. (c) Watanabe, Y.; Hyodo, N.; Ozaki, S. Tetrahedron
Lett. 1988, 29, 5763.
(14) Pirrung, M. C.; Chidambaram, N. Abstracts of Papers; 209th
ACS National Meeting, 1995, Abstract CARB 053.
(6) Stec, W. J .; Grajkowski, A.; Koziołkiewicz, M.; Uznan´ski, B. Nucl.
Acids Res. 1991, 19, 5883.
(7) Okruszek, A.; Sierzchała, A.; Sochacki, M.; Stec, W. J . Tetrahe-
dron Lett. 1992, 33, 7585.
(8) Brown, T.; Pritchard, C. E.; Turner, G.; Salisbury, S. A. J . Chem.
Soc., Chem. Commun., 1989, 891.

Synthesis of Oligo(nucleoside methanephosphonate)s
J . Org. Chem., Vol. 61, No. 3, 1996 881
than that of nucleoside methanephosphonamidites,15 the
methodology presented here can be considered as an
alternative to the widely applied phosphonamidite
method.16
sid e 3′-O-Met h a n ep h osp h on oflu or id a t es (2) w it h 3′-O-
P r otected Nu cleosid es 5. 2 (0.05 mmol, 2.5 mol equiv) and
5 (0.02 mmol, 1 equiv) were dissolved in MeCN (2.5 mL),
followed by addition of DBU (10-15 mol equiv). The progress
of condensation was followed by TLC. After the reaction was
complete (usually 15-20 min), the reaction mixture was
diluted with CHCl3 (20 mL) and washed twice with 0.1 M citric
acid (10 mL). After drying the organic phase over anhydrous
MgSO4, concentration, and precipitation from petroleum ether,
the crude product was analyzed by means of 31P NMR.
Purification of dimers 3 as well as separation of diastereomers
were performed by means of silica gel column chromatography
[Kiesegel 60 or 60H, eluent system: chloroform-methanol (0-
5%)]. Yields of 3 varied from 95% for TPMeT to 80% for GPMeA.
Removal of base-labile protective groups and detritylation were
performed as described earlier.2
P r ep a r a t ion of 5′-O-DMT t h ym id ylyl (3′,5′)-3′-O-
Acetylth ym id in e 3′-m eth a n ep h osp h on a te (3) via Activa -
tion of 1 w ith Tr ieth yla m in e Tr ih yd r oflu or id e a n d DBU.
1 (45 mg, 0.065 mmol)) and 5 (14 mg, 0.05 mmol) were
dissolved in MeCN (1 mL) with DBU (0.05 mL) added to this
solution, followed by triethylamine trihydrofluoride (0.025 mL,
1 M in dioxane). After 10 min the reaction was complete
(HPTLC assay). The reaction mixture was diluted with CHCl3
(10 mL), washed twice with 0.1 M citric acid, dried with
MgSO4, and concentrated. 31P NMR spectrum confirmed the
formation of dimer TPMeT, δ 32.07 and 31.80 in the ratio [SP]:
[RP] ) 46:54, with total yield 70%.
Solid -P h a se Syn th esis. Solid-phase experiments were
carried out on an Applied Biosystems 391 PCR-Mate Synthe-
sizer, using protocol described in Table 3. Columns (1 µmol)
were prepared using CPG (controlled pore glass) support with
the succinyl-sarcosinoyl linkers with a loading: for 5′-O-DMT-
NiBu-Gua 32.12 µmol/g; for 5′-O-DMT-Thy - 29.38 µmol/g.17
Prepared compounds 6 are listed in Table 4. Their release
from support and deprotection was performed as follows: the
solid support was transferred to the Eppendorf tube and
treated with a solution of NH3/H2O:EtOH:MeCN (10:45:45) (0.5
mL) for 30 min. Solvents were evaporated to dryness, followed
by addition of 1:1 mixture of EtOH and ethylenediamine (0.5
mL). After 6-10 h the solution was separated from solid CPG,
concentrated to dryness, and dissolved in EtOH:H2O (1:1), pH
) 7 (adjusted). Products 6 were purified by means of HPLC
(data included in Table 4).
Exp er im en ta l Section
Warning! Biological properties of nucleoside 3′-O-metha-
nephosphonofluoridates have not been evaluated. Therefore,
use caution to avoid skin contact in handling these compounds.
Chemical shifts (δ) are reported relative to TMS (1H) and
85% H3PO4 (31P) as external standards. Positive chemical shift
values are assigned for compounds resonating at lower fields
than standards. High-pressure liquid chromatography was
run on a gradient system, equipped with reverse-phase column
(ODS Hypersil, 5 µm, 25 cm, 4.6 mm). The solvent system
used: 0.1 M triethylammonium bicarbonate (TAEB) at pH )
7.0 (A), and 40% MeCN in 0.1 M TEAB (B). Column chroma-
tography and HPTLC analyses were performed on silica gel
(240-400 mesh) and silica gel precoated F254 plates (E. Merck,
Inc.), respectively. Solvents and reagents were purified ac-
cording to standard laboratory techniques and stored under
Ar.
AgF (25% weight in water) was purchased from POCH,
Poland. Triethylamine trihydrofluoride was purchased from
Aldrich Ltd. 1, 2, and 5 were dried before condensation by
means of coevaporation with pyridine and left under high
vacuum overnight. Condensation reactions were performed
under dry argon.
Gen er a l P r oced u r e for P r ep a r a t ion of 5′-O-DMT-
n u cleosid e 3′-O-Meth a n ep h osp h on oflu or id a tes 2. Into a
stirred solution of 12 (0.1mmol) in acetonitrile (MeCN) (5 mL)
a solution of AgF (0.125 mmol, 25% in water) was added in
one portion. The reaction was performed at ambient temper-
ature. Stirring was continued for 5 min, followed by dilution
of the reaction mixture with CHCl3 (25 mL) and extraction of
the resulting mixture with brine (twice, 10 mL). The organic
fraction was dried with MgSO4, concentrated to dryness,
redissolved in a small volume of dry CHCl3, and precipitated
from petroleum ether (bp 40-60 °C). Pure 2 were stored as
white powders for several weeks. Yields and analytical data
are collected in Table 1.
Rea ction betw een 5′-O-DMT-Th ym id in e 3′-O-Meth a -
n ep h osp h on oflu or id a te (2) a n d Eth a n ol. Into a solution
of 2 (31 mg, 0.05 mmol) in freshly distilled EtOH (0.2 mL) a
solution of DBU in MeCN (10% v/v, 0.2 mL) was added. The
31P NMR spectrum, recorded after 10 min, confirmed formation
of an exclusive product 4 as a mixture of diastereomers: δ
32.01 (42%) and 31.83 (58%) (EtOH/C6D6). After short column
chromatography (2% EtOH in CHCl3), 4 was analyzed by MS:
FAB [M - H]- 649.5. Yield 27 mg (85%).
Ack n ow led gm en t. This project was in-part finan-
cially assisted by Genta, Inc., San Diego, CA. Authors
are indebted to Professor G. M. Blackburn of the
University of Sheffield for critical reading of the manu-
script, valuable comments, and suggested amendments.
J O951333V
Gen er a l P r oced u r e for Cou p lin g of 5′-O-DMT-Nu cleo-
(15) J ager, A.; Engels, J . Tetrahedron Lett. 1984, 25, 1437.
(16) Miller, P. S.; Ts’o, P. O. P.; Hogrefe, R. I.; Reynolds, M. A.;
Arnold, L. J ., J r. Antisense Research and Applications, Crooke, S. T.,
Lebleu, B., Ed., CRC Press, Inc.: Boca Raton, FL, 1993; pp 189-203.
(17) Stec, W. J .; Lesnikowski, Z. J . Invited Chapter for Oligonucle-
otide Synthesis Protocols; Agrawal, S., Ed. In the Series Methods in
Molecular Biology; Humana Press, Inc.: Totowa, NJ , 1993; pp 285-
314.