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Synthesis of nucleoside methylphosphonothioates
Tetrahedron printed in
Letters,Vol.28,No.28,pp Great Britain
SYNTHESIS
OF NUCLEOSIDE
Wolfgang Department
of Chemistry
oo40-4039/87 $3.00 + .OO Pergamon Journals Ltd.
3205-3208,1987
METHYLPHOSPHONOTHIOATES
K.-D. Brill and Marvin
and Biochemistry,
University
H. Caruthers
of Colorado,
Boulder,
CO 80309-0215
USA
linkDeoxydinucleotide methylphosphonothioates containing 5 '-5' and 3'-5' internucleotide ages were synthesized regioselectively from nucleosides and methylphosphonothioic dichloride by The diastereomers were resolved by hplc. successive displacement of chlorine atoms. Recent cleotide
advances
analogs
in nucleic
having
chiral phosphorus
that are both interesting Because
these features much current
cations, facile
synthetic
easily
ment of methods
useful
(2).
linkages
field has focused
primarily
because
these analogs
nucleoside
and dlnucleotide
chloride
especially
diastereomers
derivatives
from 1 via transient
methyldisilazanide
proved
protection
mediated
valuable
7) of 1.
of
the more inter-
are the alkylphosare chiral and do not toward
the develop-
(1) and 5'-0-trityl-N3-anisoylprimarily
Compound
of the 3'-hydroxyl
and +butyldimethylsilylchloride and fluoride
Among
directed
of 10 were more easily
(compound
appli-
alkylphosphonothioates.
used for this study were 5'-0-critylthymidine The latter compound
compounds.
on the development
of polynucleotides
Here we report our research
oligonu-
properties
occurring
and medicinal
of this group of compounds.
derivatives
of several
and biochemical
naturally
for a wide range of biochemical
phosphorus
for synthesizing
ally than the corresponding
anisoyl
internucleotide
in the polynucleotide
solution.
O,O-dialkylmethylphosphonothioate
flask procedure
have led to the development
from the corresponding
for the preparation
in aqueous
Nucleosides thymidine
research
and alkylphosphonates2
phonothioatesl racemize
and different
may prove useful
routes
esting and potentially
acid chemistry
because
resolved
the nucleoside chromatographic-
2 was synthesized
group3 with
in THF (1 hr), subsequent
lithium addition
in a one hexaof
desilylation. 4
s=b-oRz CA, 7: Rl=H, R2=-CHzCHzCN 8 : R,=H,&=-(CH&CH3
ck 5:R,=H 6:R,=Anrsoyl Initial earlier
attempts
investigations
with O-cyanoethyl-
to synthesize
dialkylmethylphosphonothioates
with 0-alkylmethylphosphonochloridothioates.5
or 0-n-octylmethylphosphonochloridothioates
3205
(7-10) were based upon When 1 and 2 were reacted
6-g in pyridine
at room tempera-
3206
ture, the yields formed.
of 7, 8, and 10 were very low and several uncharacterized
The major
limitation
of this approach
0-alkylmethylphosphonochloridothioates isolated
pure for subsequent
Other initial earlier
reports
ates.9t10
during
attempts
to synthesize
on the synthesis
plate.
(12 hr).
lability
in the presence
stituents
could be selectively
were prompted displaced
research
by alkoxy groups.5
5 (31P NNR 6 97.0) or 6, (3lP NMR 6 97.3). methylphosphonothioate primary
alcohol
interest
the total integral
formed
(56%).19
Extension
A regioselective preparation
of 5.
and anhydrous Thymidine continue
dichloride
synthesis
yield*O
strategy
pyridine
into pentane.
in anhydrous
The reaction
mixture
Of considerable
of 8 and 10 could be sepa-
react
of N-methylimidazole
(159 pl, 2 mmol)
showed one peak at 87.1 ppm having When this phosphonothioating
result
pyridine
used to synthesize
The first step was
(5 ml) were allowed
twice with water
with
1 (242 mg, 0.5 mmol), to react for 17 hr.
(2 ml) was added and the reaction
was extracted
11 was
(2 ml) and 20 ml CH2C12.
9.
(223 mg. 1.5 mmol).
in dry CH2CL2
reagent
that the symmetri-
(987 mg, 4 mmol) was reacted
in anhydrous
for
85% of
(12 ml) for 48 hr, only compound
led to the unexpected
dichloride
This reagent was syn-
(247 Pl, 2 mmol) with methylphosphono-
when thymidine
pyridine
inter-
using either procedure.16-18
components.
was therefore
(162 ~1, 2 mmol)
of 6 molar equivalents
of 7, 8, and 10 via flash column chroma-
in pyridine
(149 mg, 1 mmol)
Methylphosphonothioic
(986 mg, 4 mmol) (3 hr).
mixture
of this observation
cal 12 was formed in quantitative methylphosphonothioic
(1 or 2, and anhy-
to form the 3'-O-bis-
Addition
bulky model compound.
in the presence
(986 mg, 4 mmol)
sub-
this was shown using O-o.u-dimethy-E-cyano-
a sterically
value of all 31P containing
deoxynucleoside
that the alkoxymethylchloridothioates
Initially
Inves-
(2 ml) gave (4hr) 7, 8 or 10 in 70-76%
3,3-dimethyl-3-hydroxypropionitrile
was reacted with thymidine
dichloride.
that the chlorine
(224 mg, 1.5 mmol)
that the diastereomers
was the observation
31P NHR of the reaction
two hours at 35°C.
pyridine
of 7 were not resolved
(223 mg, 1.5 mmol)
Protected
Dimerization
and precipitation
with deoxynucleosides.
by reacting
oxidato the
for 17 hr in order to obtain quantita-
of the diastereomers
phosphonochloridothioate,
thioic dichloride
due presumably
indicating
was not observed.
in anhydrous
It was observed
tic.
the diastereomers
regioselectively ethylmethyl thesized
separability
(5 m1)15
derivative
yield after flash column chromatography
Of particular
tic
and subsequent
products
dichloride
tively either
or preparative
Formation
on an analytical
using methylphosphonothioic
by earlier
(161 pl, 2 mmol) in dry CH2CL2
rated whereas
Dowex-50WX-4
into pentane.l3
with cyanoethanol,
to decomposition
drouspyridine
tography
of 1 g anhydrous
was not observed
0.5 mmol) was first treated with methylphosphonothioic
est was the relative
intermedi-
heat and transesterification.l0,14
with this reagent
of the appropriate
of
of 1 mmol of 1 with bis
and precipitation
condensation
of 7-10 were completed
tigations
(thymidilyl)
could not be
via phosphonamidite
by condensation
v/v/v)
sulfur in CS2 leads mainly
syntheses
The compounds
were
of
7-10 were based upon modifications
methylphosphonite
of 3 with tetraxole,lO
of 4 to moisture,
Successful
instability
The yield of 3 was 68% after flash chromatography
65:30:5,
3'-3' dinucleoside
Activation
work-up.
methylphosphonates
11 (1.5 mmol)
and acetonitrile
tion with elemental
compounds
(3) was prepared
(toluene:ethylacetate:triethylamine; of a symmetrical
distillative
of nucleoside
(N,N-diisopropylamino)methylphosphine form)l*
side-products
to be the thermal
reactions.
The methylphosphonamidite
(pyridinium
appeared
allowed
to
to remove 12. The organic
3207
NC CH, C(CH,),-OCH3
HC)
HO
II
HO \
’
6H
12 layer was concentrated ated from side-products The diastereomers overall
isolated
having
deoxynucleosides
and starting
material
of 9 were then separated
a mixture
by reversed
of diastereomers,
phase tic (acetone:
by reverse phase hplc (CH3CN:H20;
was fraction-
H20; 3:7, v/v). 3:2, v/v) with an
yield of 56% from 1, and characterized.2l
These results thioates
to a dry foam and the product,
outline
a pathway
for synthesizing
a 3'-5' internucleotide followed
linkage.
by their incorporation
thymidine
Extension
dinucleotide
of this synthetic
nethylphosphonoroute to the other
into DNA should now be possible.
Acknowledgements We wish to thank P. Sadecky and R. Barkley for the FAB mass spectral analysis and M. Ashley for recording the COSY NMR spectra. This work was supported by NIH (GM25680). This is paper XX Pauer XIX is H. H. Caruthers. R. Kierzek and J.-Y Tann in Phosphorus on Nucleotide Chemistrv. Chemistry Directed To&ds B>ology (W. J. Stec, Ed.) Elsevier, Amsterdam, in prgss. References 1. 2. 3.
4. 5. 6.
7. 8. 9. 10. 11.
R. L. Hilderbrand in The Role of Phosphonates in Living Systems, C.R.C. Press, Inc., Boca Raton, FL (1982). P. S. Miller, M. P. Reddy. A. Murakami, K.R. Blake, S-B Lin, and C.H. Agric, Biochemistry . . . . 25, 5092-5097 (1986) and references cited therein. H.-J. Fritz, W.-B. Frommer, W. Kramer, and W. Werr in Cheml'cal and Enzymatic Synthesis of Gene Fragments (eds. H. G. Gassen and A. Lang) p. 43, Verlag Chemie, Weinheim, Germany (1982). F. W. Adamiak, N. 2. Barciszewska, E. Biala, K. Grzeskowiak, R. Kierzek, A. Kraszewski. W. T. Markiewicz and W. Wiewiorowski, Nucleic Acids Res. 3, 3397-4015 (1976). F. Hoffmann, D. Wadsworth and H. Weiss, J. Am. Chem. Sot. 80. 3945-3948 (1958). 3lP NMR and 1~ NMR for all compounds were recorded in CDC13 unless otherwise specified. For 31P NMR, 85% H3P04 was the external reference. UV spectra were recorded in dichloromethane. Rf values refer to separation on silica gel tic plates unless otherwise specified. All isomer fractionation6 on hplc were performed using a 250 mm X 10 mm Econosil C-18 column from Altech Applied Science. 0-B-Cyanoeth lmethylphosphonochloridothioate. Mass Spectrum, 183 CM+), 148 (N-Cl)+; 31P NMR 6 97.7; YH NMR 6 4.4 (M, a-CH2), 2.9 ( 8-CH2), 1.9-25 (ABX, 3, P-CH3). ?*H NMR:6 3.95 (d, o-CH2 of n-octyl), 1.8-1.1 0-FOctylmethylphosphonochloridothioate. (m, CH2 of n-octyl, P-CH3), 0.88 (CH3 of n-octyl). M. A. Dorman, S. A. Noble, L. J. McBride and M. H. Caruthers. Tetrahedron 40, 95-102 (1984). A. Jiiger and J. Engels. Tetrahedron Lett 25, 1437-1440 (1984). Bis (N,N-diisopropylamino)methylphosphine. 31P NMR 6 131.6; 1H NMR 6 4.5 (He, CH(CH3)2), 3.3 (d JHP * 18H2, p-CH3). 1.4 (d, CH3)2).
Letters,Vol.28,No.28,pp Great Britain
SYNTHESIS
OF NUCLEOSIDE
Wolfgang Department
of Chemistry
oo40-4039/87 $3.00 + .OO Pergamon Journals Ltd.
3205-3208,1987
METHYLPHOSPHONOTHIOATES
K.-D. Brill and Marvin
and Biochemistry,
University
H. Caruthers
of Colorado,
Boulder,
CO 80309-0215
USA
linkDeoxydinucleotide methylphosphonothioates containing 5 '-5' and 3'-5' internucleotide ages were synthesized regioselectively from nucleosides and methylphosphonothioic dichloride by The diastereomers were resolved by hplc. successive displacement of chlorine atoms. Recent cleotide
advances
analogs
in nucleic
having
chiral phosphorus
that are both interesting Because
these features much current
cations, facile
synthetic
easily
ment of methods
useful
(2).
linkages
field has focused
primarily
because
these analogs
nucleoside
and dlnucleotide
chloride
especially
diastereomers
derivatives
from 1 via transient
methyldisilazanide
proved
protection
mediated
valuable
7) of 1.
of
the more inter-
are the alkylphosare chiral and do not toward
the develop-
(1) and 5'-0-trityl-N3-anisoylprimarily
Compound
of the 3'-hydroxyl
and +butyldimethylsilylchloride and fluoride
Among
directed
of 10 were more easily
(compound
appli-
alkylphosphonothioates.
used for this study were 5'-0-critylthymidine The latter compound
compounds.
on the development
of polynucleotides
Here we report our research
oligonu-
properties
occurring
and medicinal
of this group of compounds.
derivatives
of several
and biochemical
naturally
for a wide range of biochemical
phosphorus
for synthesizing
ally than the corresponding
anisoyl
internucleotide
in the polynucleotide
solution.
O,O-dialkylmethylphosphonothioate
flask procedure
have led to the development
from the corresponding
for the preparation
in aqueous
Nucleosides thymidine
research
and alkylphosphonates2
phonothioatesl racemize
and different
may prove useful
routes
esting and potentially
acid chemistry
because
resolved
the nucleoside chromatographic-
2 was synthesized
group3 with
in THF (1 hr), subsequent
lithium addition
in a one hexaof
desilylation. 4
s=b-oRz CA, 7: Rl=H, R2=-CHzCHzCN 8 : R,=H,&=-(CH&CH3
ck 5:R,=H 6:R,=Anrsoyl Initial earlier
attempts
investigations
with O-cyanoethyl-
to synthesize
dialkylmethylphosphonothioates
with 0-alkylmethylphosphonochloridothioates.5
or 0-n-octylmethylphosphonochloridothioates
3205
(7-10) were based upon When 1 and 2 were reacted
6-g in pyridine
at room tempera-
3206
ture, the yields formed.
of 7, 8, and 10 were very low and several uncharacterized
The major
limitation
of this approach
0-alkylmethylphosphonochloridothioates isolated
pure for subsequent
Other initial earlier
reports
ates.9t10
during
attempts
to synthesize
on the synthesis
plate.
(12 hr).
lability
in the presence
stituents
could be selectively
were prompted displaced
research
by alkoxy groups.5
5 (31P NNR 6 97.0) or 6, (3lP NMR 6 97.3). methylphosphonothioate primary
alcohol
interest
the total integral
formed
(56%).19
Extension
A regioselective preparation
of 5.
and anhydrous Thymidine continue
dichloride
synthesis
yield*O
strategy
pyridine
into pentane.
in anhydrous
The reaction
mixture
Of considerable
of 8 and 10 could be sepa-
react
of N-methylimidazole
(159 pl, 2 mmol)
showed one peak at 87.1 ppm having When this phosphonothioating
result
pyridine
used to synthesize
The first step was
(5 ml) were allowed
twice with water
with
1 (242 mg, 0.5 mmol), to react for 17 hr.
(2 ml) was added and the reaction
was extracted
11 was
(2 ml) and 20 ml CH2C12.
9.
(223 mg. 1.5 mmol).
in dry CH2CL2
reagent
that the symmetri-
(987 mg, 4 mmol) was reacted
in anhydrous
for
85% of
(12 ml) for 48 hr, only compound
led to the unexpected
dichloride
This reagent was syn-
(247 Pl, 2 mmol) with methylphosphono-
when thymidine
pyridine
inter-
using either procedure.16-18
components.
was therefore
(162 ~1, 2 mmol)
of 6 molar equivalents
of 7, 8, and 10 via flash column chroma-
in pyridine
(149 mg, 1 mmol)
Methylphosphonothioic
(986 mg, 4 mmol) (3 hr).
mixture
of this observation
cal 12 was formed in quantitative methylphosphonothioic
(1 or 2, and anhy-
to form the 3'-O-bis-
Addition
bulky model compound.
in the presence
(986 mg, 4 mmol)
sub-
this was shown using O-o.u-dimethy-E-cyano-
a sterically
value of all 31P containing
deoxynucleoside
that the alkoxymethylchloridothioates
Initially
Inves-
(2 ml) gave (4hr) 7, 8 or 10 in 70-76%
3,3-dimethyl-3-hydroxypropionitrile
was reacted with thymidine
dichloride.
that the chlorine
(224 mg, 1.5 mmol)
that the diastereomers
was the observation
31P NHR of the reaction
two hours at 35°C.
pyridine
of 7 were not resolved
(223 mg, 1.5 mmol)
Protected
Dimerization
and precipitation
with deoxynucleosides.
by reacting
oxidato the
for 17 hr in order to obtain quantita-
of the diastereomers
phosphonochloridothioate,
thioic dichloride
due presumably
indicating
was not observed.
in anhydrous
It was observed
tic.
the diastereomers
regioselectively ethylmethyl thesized
separability
(5 m1)15
derivative
yield after flash column chromatography
Of particular
tic
and subsequent
products
dichloride
tively either
or preparative
Formation
on an analytical
using methylphosphonothioic
by earlier
(161 pl, 2 mmol) in dry CH2CL2
rated whereas
Dowex-50WX-4
into pentane.l3
with cyanoethanol,
to decomposition
drouspyridine
tography
of 1 g anhydrous
was not observed
0.5 mmol) was first treated with methylphosphonothioic
est was the relative
intermedi-
heat and transesterification.l0,14
with this reagent
of the appropriate
of
of 1 mmol of 1 with bis
and precipitation
condensation
of 7-10 were completed
tigations
(thymidilyl)
could not be
via phosphonamidite
by condensation
v/v/v)
sulfur in CS2 leads mainly
syntheses
The compounds
were
of
7-10 were based upon modifications
methylphosphonite
of 3 with tetraxole,lO
of 4 to moisture,
Successful
instability
The yield of 3 was 68% after flash chromatography
65:30:5,
3'-3' dinucleoside
Activation
work-up.
methylphosphonates
11 (1.5 mmol)
and acetonitrile
tion with elemental
compounds
(3) was prepared
(toluene:ethylacetate:triethylamine; of a symmetrical
distillative
of nucleoside
(N,N-diisopropylamino)methylphosphine form)l*
side-products
to be the thermal
reactions.
The methylphosphonamidite
(pyridinium
appeared
allowed
to
to remove 12. The organic
3207
NC CH, C(CH,),-OCH3
HC)
HO
II
HO \
’
6H
12 layer was concentrated ated from side-products The diastereomers overall
isolated
having
deoxynucleosides
and starting
material
of 9 were then separated
a mixture
by reversed
of diastereomers,
phase tic (acetone:
by reverse phase hplc (CH3CN:H20;
was fraction-
H20; 3:7, v/v). 3:2, v/v) with an
yield of 56% from 1, and characterized.2l
These results thioates
to a dry foam and the product,
outline
a pathway
for synthesizing
a 3'-5' internucleotide followed
linkage.
by their incorporation
thymidine
Extension
dinucleotide
of this synthetic
nethylphosphonoroute to the other
into DNA should now be possible.
Acknowledgements We wish to thank P. Sadecky and R. Barkley for the FAB mass spectral analysis and M. Ashley for recording the COSY NMR spectra. This work was supported by NIH (GM25680). This is paper XX Pauer XIX is H. H. Caruthers. R. Kierzek and J.-Y Tann in Phosphorus on Nucleotide Chemistrv. Chemistry Directed To&ds B>ology (W. J. Stec, Ed.) Elsevier, Amsterdam, in prgss. References 1. 2. 3.
4. 5. 6.
7. 8. 9. 10. 11.
R. L. Hilderbrand in The Role of Phosphonates in Living Systems, C.R.C. Press, Inc., Boca Raton, FL (1982). P. S. Miller, M. P. Reddy. A. Murakami, K.R. Blake, S-B Lin, and C.H. Agric, Biochemistry . . . . 25, 5092-5097 (1986) and references cited therein. H.-J. Fritz, W.-B. Frommer, W. Kramer, and W. Werr in Cheml'cal and Enzymatic Synthesis of Gene Fragments (eds. H. G. Gassen and A. Lang) p. 43, Verlag Chemie, Weinheim, Germany (1982). F. W. Adamiak, N. 2. Barciszewska, E. Biala, K. Grzeskowiak, R. Kierzek, A. Kraszewski. W. T. Markiewicz and W. Wiewiorowski, Nucleic Acids Res. 3, 3397-4015 (1976). F. Hoffmann, D. Wadsworth and H. Weiss, J. Am. Chem. Sot. 80. 3945-3948 (1958). 3lP NMR and 1~ NMR for all compounds were recorded in CDC13 unless otherwise specified. For 31P NMR, 85% H3P04 was the external reference. UV spectra were recorded in dichloromethane. Rf values refer to separation on silica gel tic plates unless otherwise specified. All isomer fractionation6 on hplc were performed using a 250 mm X 10 mm Econosil C-18 column from Altech Applied Science. 0-B-Cyanoeth lmethylphosphonochloridothioate. Mass Spectrum, 183 CM+), 148 (N-Cl)+; 31P NMR 6 97.7; YH NMR 6 4.4 (M, a-CH2), 2.9 ( 8-CH2), 1.9-25 (ABX, 3, P-CH3). ?*H NMR:6 3.95 (d, o-CH2 of n-octyl), 1.8-1.1 0-FOctylmethylphosphonochloridothioate. (m, CH2 of n-octyl, P-CH3), 0.88 (CH3 of n-octyl). M. A. Dorman, S. A. Noble, L. J. McBride and M. H. Caruthers. Tetrahedron 40, 95-102 (1984). A. Jiiger and J. Engels. Tetrahedron Lett 25, 1437-1440 (1984). Bis (N,N-diisopropylamino)methylphosphine. 31P NMR 6 131.6; 1H NMR 6 4.5 (He, CH(CH3)2), 3.3 (d JHP * 18H2, p-CH3). 1.4 (d, CH3)2).