Oligomeric building block approach to the synthesis of diastereomerically pure pentathymidine 3′,5′-methanephosphonates

Article abstract of DOI:10.1021/ol991376o

formula presented A method for a large-scale synthesis of stereodefined oligo(nucleoside 3′,5′-methanephosphonates) has been developed, based on transient 3′-O protection, which allows for the conversion of the protecting chirally defined methanephosphonanilidate group, located at the 3′ end of a stereoregular oligomer, into diastereomerically pure oligomeric building blocks for stereospecific coupling with the 5′-OH group of another oligonucleotide.

Full text of DOI:10.1021/ol991376o

ORGANIC
LETTERS
2000
Vol. 2, No. 6
771-773
Oligomeric Building Block Approach to
the Synthesis of Diastereomerically Pure
Pentathymidine
3′,5′-Methanephosphonates
Jaroslaw Pyzowski, Lucyna A. Wozniak, and Wojciech J. Stec*
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences,
90 363 Ło´dz, Poland
wjstec@bio.cbmm.lodz.pl
Received December 21, 1999
ABSTRACT
A method for a large-scale synthesis of stereodefined oligo(nucleoside 3′,5′-methanephosphonates) has been developed, based on transient
3′-O protection, which allows for the conversion of the protecting chirally defined methanephosphonanilidate group, located at the 3′ end of
a stereoregular oligomer, into diastereomerically pure “oligomeric building blocks” for stereospecific coupling with the 5′-OH group of another
oligonucleotide.
Oligo(nucleoside 3′,5′-methanephosphonates) (oligo-PMe)
have been considered as potentially useful candidates for
drugs in antisense and antigene strategy.1 They are capable
of sequence-specific recognition and binding to their target
molecules (mRNA or DNA),2 exhibit resistance toward
cellular nucleases,3 and are able to gain access to the
intracellular environment.4
The methanephosphonate internucleotide phosphorus atom
constitutes a chiral center; therefore, in the case of oligo-
PMe obtained via elongation of the oligomer within n - 1
nonstereospecific coupling reactions, the final product con-
sists of a mixture of 2n-1 P diastereomers. The strong
influence of the P diastereomerism of oligo-PMe on their
physicochemical properties5 was first recognized for chimeric
oligomers with alternating methanephosphonate/phosphodi-
ester bonds6 and further for stereoregular octathymidine
methanephosphonates7 obtained by the first stereospecific
method.8
(5) Miller, P.; Agris, C.; Aurelian, L.; Blake, K.; Lin, S-.B.; Murakami,
A.; Reddy, M.; Smith, C.; Ts’o, P. O. P. In Interrelationship Among Aging,
Cancer and Differentiation, Pullman, B., Ed.: Reidel: Dordrecht, The
Netherlands, and Boston, 1985; pp 207-219.
(1) Ts’o, P. O. P.; Aurelian, L.; Chang, E.; Miller, P. S. Ann. N.Y. Acad.
Sci. 1992, 660, 159-177.
(2) Miller, P. S. Prog. Nucleic Acid Res. Mol. Biol. 1996, 52, 261-291.
(3) Smith, C. C.; Aurelian, L.; Reddy, M. P.; Miller, P. S.; Ts’o, P. O.
P. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 2887-2893.
(4) Agrawal, S.; Temsamani, J.; Galbraight, W.; Tang, J. Clin. Phar-
macokinet. 1995, 28, 7-16.
(6) Miller, P. S.; Dreon, N.; Pulford, S. M.; McParland, K. B. J. Biol.
Chem. 1980, 255, 9659-9665.
(7) Lesnikowski, Z. J.; Jaworska, M.; Stec, W. J. Nucleic Acids Res.
1990, 18, 2109-2115.
(8) Lesnikowski, Z.; Jaworska, M.; Stec, W. J. Nucleic Acids Res. 1988,
16, 11675.
10.1021/ol991376o CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/24/2000

The synthesis of stereodefined oligo-PMe can be ap-
proached via nonstereospecific condensation reactions fol-
lowed by separation of diastereomers9 or via stereospecific
coupling reactions of diastereomerically pure monomers, but
both are limited to relatively short oligomers.10
Scheme 2
As demonstrated earlier, condensation of a pure diastere-
omer of 5′-O-DMT-nucleoside 3′-O-(S-methyl methane-
phosphonothioate) (2; prepared from diastereomerically pure
5′-O-DMT-nucleoside-3′-O-methanephosphonanilidate (1))
with 3′-O-acetylthymidine (3) provides 5′-O-DMT-3′-O-
acetyl dinucleoside 3′,5′-methanephosphonate (4).11 This
condensation performed in the presence 1,8-diazabicyclo-
[5.4.0]undec-7-ene (DBU)/LiCl is stereospecific and occurs
with inversion of configuration.12 Removal of the 5′-O
protective group and further condensation of the resulting
3′-O-acetyl dinucleoside 3′,5′-methanephosphonate with an
appropriate S-alkyl methanephosphonothioate 2 allow for
elongation of dinucleotide into 3′-O-Ac-protected tris-
(nucleoside methanephosphonate) (5; Scheme 1). Performed
appropriate dithymidine methanephosphonate with a pendant
3′-O-S-methyl methanephosphonothioate (9). Compound 9
can be used as a dimeric “building block” for condensation
5′-OH
with the trimer
T
TPMeT3′-Oac (10), giving pentakis-
PMe
(thymidine methanephosphonate) (11) of predetermined
configuration at the phosphorus atom of each internucleotide
methanephosphonate linkage.
Scheme 1
The key assumption of the present work was that the
presence of a 3′-O-methanephosphonanilidate pendant group
of mono- or oligonucleotide will not interfere with the
process of nucleophilic substitution at the phosphorus atom
of the S-Me methanephosphonothioate function attached at
the 3′-O position of another mono- or oligonucleotide. Since
the coupling process requires the presence of a strong organic
base, namely DBU/LiCl, one may speculate that an excess
of DBU may generate an anionic site at the pendant
methanephosphonanilidate which competes with 5′-OH of
the same molecule in the process of nucleophilic substitution
at phosphorus. That speculation was not unmerited, since
DBU can be used as an alternative to NaH base for PN f
PX conversion.15 Therefore, diastereomerically pure (RP)-1
in the same manner, further stepwise elongation of the
oligonucleotide chain allowed us to obtain diastereomerically
pure pentakis(nucleoside methanephosphonate) with prede-
termined absolute configuration at phosphorus of each
internucleotide methanephosphonate linkage.13
In this communication we present evidence that the 5′-O
protective group of substrate 1 (B ) Thy) can be selectively
removed, and the resulting thymidine 3′-O-methanephos-
phonanilidate (6) can be coupled with S-methyl methane-
phosphonothioate 2 (B ) Thy), providing dithymidine 3′,5′-
methanephosphonate with a 3′-O pendant methanephosphon-
anilidate function (7; Scheme 2). The 3′-O-methanephos-
phonanilidate group of 7 can be converted into the corre-
sponding methanephosphonothioate of 8 in a chemoselective
manner (PN f PS),14 providing, after alkylation, the
1
(B ) Thy; 31P NMR δ 30.22 ppm; H NMR (CDCl3) δ
(slow) 1.90 ppm (d, 3H, JP-H ) 16.90 Hz, P-Me)) was
treated with 3% trichloroacetic acid in dichloromethane (15
min), providing (RP)-5′-OH-thymidine 3′-O-methanephos-
phonanilidate (6: 86% yield; 31P NMR δ 30.25 ppm;
C17H22O6N3P, MS FAB [M - H] 395, calcd 395.3).
Condensation of (RP)-6 with (SP)-2 (B ) Thy) (in a 3:1
molar ratio) in the presence of DBU (9 molar equiv) and
LiCl (9 equiv) yielded dinucleotide 7 with an internucleotide
methanephosphonate of SP configuration possessing a 3′-O
pendant methanephosphonanilidate group of RP configura-
tion. Compound 7 was purified by means of silica gel column
chromatography (silica gel 60, 230-400 mesh, eluted with
an increasing gradient of ethanol (0-4%) in chloroform; Rf
) 0.5 in chloroform-methanol (9:1)), and the yield of pure
compound was 53% (31P NMR δ 32.54 ppm, 30.01 ppm;
C49H55O14N5P2, MS FAB [M - H] 998.9, calcd 998.94).
Treatment of a DMF solution of 7 with NaH (2 equiv, 50%
suspension in mineral oil) and CS2 (10 equiv), followed by
alkylation of the terminal sodium 3′-O-methanephosphonate
(9) Vyazovkina, E. V.; Savchenko, E. V.; Lokhov, S. G.; Engels, J. M.;
Wickstrom, E.; Lebedev, A. V. Nucleic Acids Res. 1994, 22, 2404-2409.
(10) Wozniak, L. A. In ReViews on Heteroatom Chemistry; Oae, S., Ed.;
MYU: Tokyo, 1999; Vol. 19, pp 173-203.
(11) Stec, W. J.; Wozniak, L. A.; Pyzowski, J.; Niewiarowski, W.
Antisense Nucleic Acids Drug DeV. 1997, 7, 383-397.
(12) Wozniak, L. A.; Pyzowski, J.; Wieczorek, M.; Majzner, W.; Stec,
W. J. J. Org. Chem. 1998, 63, 5395-5402.
(13) Wozniak, L. A.; Pyzowski, J.; Wieczorek, M.; Stec, W. J. Org.
Chem. 1994, 59, 5843-5846.
(14) Stec, W. J. Acc. Chem. Res. 1983, 16, 411-417.
(15) Baraniak, J.; Wozniak, L. A. Unpublished results.
772
Org. Lett., Vol. 2, No. 6, 2000

in 8 with methyl iodide (10 equiv), gave the compound
(SPRP)-9, which was isolated by means of silica gel flash
column chromatography, using a gradient of EtOH (1-3%)
in CHCl3 as the eluting system. The yield of (SPRP)-9 was
75% (31P NMR δ 32.50 ppm, 57.16 ppm; 1H NMR (CDCl3)
δ 2.31 (d, 13.3, 3H, P-SMe), 1.57 (d, 17.59, 3H, P-Me),
1.84 (d, 15.63, 3H, Me-P-SMe); C44H52O14N4P2S, MS FAB
[M - H] 954.3, calcd 954.9).
Scheme 3
It has to be pointed out that the 31P NMR spectrum of the
reaction mixture between S-methyl methanephosphonothioate
2 and methanephosphonanilidate 6 did not contain any
resonance signals indicating the formation of a P-N(Ph)-P
bond. Moreover, the conversion 6 f 7 was stereospecific
(no epimerization at the phosphorus of the phosphonanilidate
function in 7), since for the intermediary dithymidine
methanephosphonate with pendant 3′-O-sodium methane-
phosphonothioate (8) only two resonance lines at 32.21 ppm
(internucleotide methanephosphonate) and at 76.38 ppm (3′-
O-sodium methanephosphonothioate) were observed. Con-
sequently, alkylation of 8 with methyl iodide gave the single
compound 9. Overall, 7 f 9 conversion occurred with
retention of configuration.14
The second component for the in-solution block condensa-
tion, trimer 10, was prepared as follows: 3′-O-acetyl
thymidine 3 was condensed with (SP)-5′-O-DMT-thymidine
3′-O-(S-methyl methanephosphonothioate) (2), providing
(SP)-5′-O-DMT-3′-O-Ac-dithymidine 3′,5′-methanephospho-
nate (4; 31P NMR δ 32.23 ppm; MS FAB- [M - H] 887.3,
calcd 887.23). After the standard purification and the removal
of the 5′-O-DMT group (SP)-3′-O-acetyl dithymidine 3′,5′-
methanephosphonate (31P NMR δ 33.12 ppm) was condensed
with (SP)-2 (B ) T), and that condensation afforded (SPSP)-
5′-O-DMT-3′-O-acetyl tris(thymidine methanephosphonate)
(5; 31P NMR δ 31.29, 31.32 ppm; C55H64O20N6P2, MS FAB-
[M - H] 1188.7, calcd 1191.1). Treatment of fully protected
trimer 5 (B ) Thy) with 3% trichloroacetic acid in dichloro-
methane for 5 min provided (SPSP)-3′-O-acetyl tris(thymidine
methanephosphonate) (10; 31P NMR δ 31.16, 31.25 ppm in
CDCl3). The total yield of the partially deprotected trimer
10 was 64%.
using as eluent a gradient of EtOH (5-10%) in chloroform.
Compound 11 was isolated in 18% yield (6.3 mg; 31P NMR
δ 33.08, 32.81, 32.50 ppm in the ratio 1:2:1, respectively;
C54H74O29N10P4, MALDI TOF (after deprotection) 1450,
calcd 1450.4; HP TLC (10% MeOH in CHCl3) Rf 0. 31;
HPLC Econosphere C18 column, 0.1 M triethylammonium
bicarbonate (A, TEAB; pH 7.5), 80% MeCN in TEAB (B),
gradient 30-100% B, 2% B/min, Rt ) 16.4 min).
The corollary to this work was the proof that condensation
of 2 + 6 occurs without active participation of the pendant
3′-O-methanephosphonanilidate group of 6 and that this
group can be converted to 3′-O-S-methyl methanephospho-
nothioate 9. The overall process of preparation of 9 is
stereospecific. Because of the presence of the good S-Me
leaving group at the terminal phosphonate in 9, condensation
with the 5′-OH trinucleotide 10 was feasible. The preparation
of pentamer 11, although in modest yield (18%) of the final
coupling, provided a new example of stereospecific chemical
ligation of two oligonucleotide components. Since the scale-
up of the demonstrated process is achievable, this demon-
strates the possibility of the in-solution preparation of longer
oligo(nucleoside methanephosphonates) via the block-
condensation approach.
The advantage of the method presented here is the
relatively large scale of the synthesis and separation of
diastereomeric species only at the stage of monomeric
precursors, while the drawback of the presented strategy is
low yield (not optimized yet). Further studies involving the
application of this method to the preparation of hetero-
nucleoside sequences, with nucleobases other than thymidine,
are in progress.
The crucial step of condensation of the dimeric building
block 9 (2.7 equiv) and 5′-O deprotected trimer 10 (21 mg,
1 equiv) was performed in 0.2 M pyridine solution in the
presence of LiCl (10 equiv) and DBU (20 equiv) at room
temperature (1 h) (Scheme 3). The excess DBU was first
removed via precipitation of the concentrated (to one-fourth
of the total volume) reaction mixture with hexane, followed
by extraction of the reaction mixture, dissolved in chloro-
form, with a 0.05 M solution of citric acid. The final product
was purified by means of silica gel column chromatography,
Acknowledgment. Financial support from the State
Committee for Scientific Research (KBN, Grant No. 3TO9A
061 17 to W.J.S.) and Genta Inc., Lexington, MA, is
acknowledged.
OL991376O
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