Preparation of thymine dinucleotide methylphosphonate analogs via thymine methylpnosphonofluoridate

Full text of DOI:10.1021/jo951395l

1540
J . Org. Chem. 1996, 61, 1540-1542
P r ep a r a tion of Th ym in e Din u cleotid e
Meth ylp h osp h on a te An a logs via Th ym in e
Meth ylp h osp h on oflu or id a te
Michael C. Pirrung*,1 and N. Chidambaram
Department of Chemistry, P. M. Gross Chemical Laboratory,
Duke University, Durham, North Carolina 27708-0346
Received J uly 28, 1995
Analogs of the charged and enzymatically-labile phos-
phodiester bonds in oligonucleotides have been used in
the antisense approach to drug development which
targets nucleic acids.2 Methylphosphonates,3 originally
prepared by Miller and Ts’o using phosphonodiester
chemistry,4 can be prepared using either tricoordinate
or tetracoordinate phosphorus chemistry.5 Methods that
have been developed recently for the synthesis of meth-
ylphosphonate nucleotides have focused on phosphorus-
(V) derivatives in which active esters of 3′-methylphos-
phonates are coupled to 5′-alcohols. Stec and co-workers
have reported the synthesis of oligomers up to pentamers
using DBU/LiCl to couple diastereomerically-pure nu-
cleoside selenomethyl methylphosphonates with the 5′-
alcohols of protected nucleosides.6 The use of hexaflu-
oroisopropyl7 and p-nitrophenyl8 leaving groups with
t-BuMgCl as base is also a known coupling protocol.
We were interested in developing a method that would
allow near “reagent-less” couplings to be conducted in
solution to facilitate the preparation of methylphospho-
nate dinucleotides on a significant scale. We focused on
the condensation of 5′-O-(trimethylsilyl)nucleosides with
a 3′-O-methylphosphonofluoridate to produce the di-
nucleotide and the volatile trimethylsilyl fluoride (eq 1).
Methods for the preparation of phosphonofluoridates
from phosphonothioates were known,9 but an efficient
method for the preparation of phosphonothioates was
needed.
ester derivatives of both alkyl phosphates and meth-
ylphosphonate.10 Compound 1 can be stored as a stock
solution and coupled with 5′-O-(dimethoxytrityl)thymi-
dine. However, unlike van Boom’s example with the
PdO compounds, this coupling requires addition of the
nucleophilic catalyst DMAP. Hydroxypropionitrile is
coupled to the resulting monobenzotriazolyl phosphono-
thioate in the presence of N-methylimidazole to afford 2
as a mixture of diastereoisomers (eq 2). Treatment of 2
The bis(benzotriazolyl) phosphonothioate (1) is pre-
pared by treating methylphosphonothioic dichloride with
hydroxybenzotriazole by analogy to the results of van
Boom, who has prepared O,O-bis(benzotriazolyl) active
(1) Fellow of the J ohn Simon Guggenheim Memorial Foundation,
1994-1995.
(2) Zon, G. Ann. N.Y. Acad. Sci. 1990, 616, 161-72. Miller, P. S.
Bio/ Technology 1991, 9, 358-62. Antisense Nucleic Acids Proteins;
Mol, J . N. M., Van der Krol, A. R., Eds.; Dekker: New York, 1991.
Uhlmann, E.; Peyman, A. Chem. Rev. 1990, 90, 543-84.
(3) For recent reviews, see: Miller, P. S.; Cushman, C. D.; Levis, J .
T. In Oligonucleotides and Analogues; Eckstein, F., Ed.; Oxford
University Press: Oxford, 1991; pp 137-159. Miller, P. S.; Ts’o, P. O.
P.; Hogrefe, R. I.; Reynolds, M. A.; Arnold, L. J ., J r. In Antisense
Research and Applications; Crooke, S. T., Lebleu, B., Eds.; CRC Press,
Inc.: Boca Raton, FL, 1993; pp 189-203.
with freshly-condensed ammonia in ethanol (∼1:3) for 1
h at room temperature yields salt 3, which can be
fluorinated in a convenient procedure using the com-
mercially-available 4-fluoro-3,5-dinitrobenzotrifluoride
(4) to give 5. This material exhibits the expected large
one-bond P-F coupling (J ) 1049 Hz). The stoichiometry
of fluorinating agent and the solvent affect this fluorina-
tion reaction, which could be monitored by 31P NMR.
When it is performed with 1 equiv of 4 in acetone, the
thioester 6, presumably an intermediate in the overall
(4) Miller, P. S.; Yano, J .; Yano, E.; Carroll, C.; J ayaraman, K.; Ts’o,
P. O. P. Biochemistry 1979, 18, 5134.
(5) Vyazovkina, E. V.; Komarova, N. I.; Lebedev, A. V. Bioorg. Khim.
1993, 19, 86 and references cited therein.
(6) (a) Wozniak, L. A.; Pyzowski, J .; Wieczorek, M.; Stec, W. J . J .
Org. Chem. 1994, 59, 5843. (b) Lesnikowski, Z. J .; Zabawska, D.;
J aworska-Maslanka, M. M.; Schinazi, R. F.; Stec, W. J . New J . Chem.
1994, 18, 1197.
(7) Lesnikowski, Z. J .; Wolkanin, P. J .; Stec, W. J . In Proceedings
of the Second International Symposium on Phosphorus Chemistry
Directed Towards Biology; Bruzik, K. S., Stec, W. J ., Eds.; Elsevier
Science Publishers B. V.: Amsterdam, 1987; pp 189-194. Lesnikowski,
Z. J .; Wolkanin, P. J .; Stec, W. J . Tetrahedron Lett. 1987, 28, 5535.
Lesnikowski, Z. J .; J aworska-Maslanka, M. M.; Stec, W. J . Nucleosides
Nucleotides 1991, 10, 733.
(8) Cormier, J . F.; Pannunzio, T. Tetrahedron Lett. 1991, 32, 7161.
(9) Boter, H. L.; Ooms, A. J . J .; Van Der Berg, G. R.; Van Dijk, C.
Recl. Trav. Chim. Pays-Bas 1966, 85, 147. Benschop, H. P.; De J ong,
L. P. A. Acc. Chem. Res. 1988, 21, 368.
(10) Marugg, J . E.; de Vroom, E.; Dreef, C. E.; Tromp, M.; van der
Marel, G. A.; van Boom, J . H. Nucl. Acids Res. 1986, 14, 2171.
(11) Attempts to form the diastereomerically-pure phosphonofluo-
ridate from chromatographically-separated diastereomers of 2 by
analogy to earlier results in the preparation of optically-active Sarin
(isopropyl methylfluorophosphonate)9 (using picryl fluoride) failed. It
is unclear if this is related to the relatively subtle change to fluorinating
agent 4 or to the difference between isopropoxy and the nucleoside 3′
oxygen. We were unable to reproduce literature preparations of picryl
fluoride.
0022-3263/96/1961-1540$12.00/0 © 1996 American Chemical Society

Notes
J . Org. Chem., Vol. 61, No. 4, 1996 1541
toluene) and pyridine (1.5 mL) in anhydrous dioxane (50 mL)
at room temperature. The reaction mixture was stirred for 2 h,
and the pyridinium hydrochloride salts were removed by filtra-
tion under anhydrous conditions to give a stock solution of 1
(0.11M). 31P NMR (dioxane): δ 121.31.
fluorination process, also results. In pyridine, it gives 5
and a compound postulated to be Meisenheimer complex
7 [J P-S-C-F ) 47 Hz).11
5′-O-(Dim et h oxyt r it yl)t h ym id in e 3′-O-(2′′-cya n oet h yl
m eth ylp h osp h on oth ioa te) (2).14 5′-O-(Dimethoxytrityl)thy-
midine (1.09 g, 2 mmol) was dissolved in anhydrous pyridine (5
mL) and evaporated to dryness. This material was dissolved in
CH3CN (15 mL) and transferred through a cannula to a flask
containing a solution of 1 (22 mL, 1.21 mmol) and DMAP (0.27
g, 2.2 mmol, dried in a drying pistol over refluxing toluene). After
0.5 h, 3-hydroxypropionitrile (0.35 g, 5 mmol, 0.34 mL) was
added followed by N-methylimidazole (0.8 mL, 10 mmol). Stir-
ring was continued for 2 h. Solvents were removed in vacuo,
and the residue was dissolved in CH2Cl2 (40 mL), washed with
0.1 M triethylammonium bicarbonate (15 mL), and water (15
mL), dried over MgSO4, and concentrated. The crude compound
was purified by flash chromatography (CH2Cl2:EtOH 98:2) to
afford 2 (1.12g, 81%) as a mixture of diastereoisomers. Rf: 0.49,
0.53 (EtOH:CHCl3 6:94). 1H NMR (CDCl3): δ 8.48 (s, 1H), 8.44
(s, 1H), 7.60 (s, 1H), 6.46 (m, 1H), 6.43 (m, 1H), 5.45 (m, 1H),
4.21 (d, J ) 2.2 Hz, 1H), 3.81 (s, 6H), 3.46 (m, 2H), 2.77 (t, J )
6.2 Hz, 2H), 2.56 (m, 1H), 2.46 (m, 1H), 1.91 (d, J ) 14.1 Hz,
3H), 1.86 (d, J )14.4 Hz, 3H), 1.48 (s, 3H), 1.46 (s, 3H). 31P
NMR (CDCl3): δ 98.30, 98.77 (identical to literature).
5′-O-(Dim eth oxytr ityl)th ym id in e 3′-O-Meth ylp h osp h o-
n oth ioic Acid Am m on iu m Sa lt (3). Freshly-condensed NH3
in EtOH (∼1:3, 3 mL) was added to 2 (0.054 g, 0.078 mmol),
and the solution was stirred for 1 h at room temperature to
afford 3. 31P NMR (EtOH): δ 77.38, 78.19. This material was
used without further purification.
5′-O-(Dim eth oxytr ityl)th ym id in e 3′-O-Meth ylp h osp h o-
n oflu or id a te (5). The salt obtained from the above reaction
(0.078 mmol) was dried over anhydrous pyridine and dissolved
in acetone (2 mL). Compound 4 (0.04 g, 0.16 mmol, Marshallton
Research Labs) was added, and the reaction mixture was stirred
at room temperature for 1-1.5 h. Progress of the reaction was
monitored by phosphorus NMR. 31P NMR (acetone): δ 31.84
(d, J ) 1048.42 Hz), 31.90 (d, J ) 1049.46 Hz). Evidence for
the elemental composition of this compound could not be
obtained because of its instability.15
To serve as coupling partners for 5, 3′-O-benzoyl-dT
and the 3′-O-benzoyl-protected nucleosides dCBz and dABz
were silylated using hexamethyldisilazane and chloro-
trimethylsilane in pyridine at room temperature for 0.25
h12 to afford 5′-O-(trimethylsilyl)deoxynucleosides 8-10
in near-quantitative yields. 3′-O-Benzoyl(N2-i-Bu)dG was
silylated with neat TMSCN to give a mixture of 11 and
starting material in a 2:1 ratio.
Thymine dinucleoside methylphosphonate analogs can
be prepared by coupling 5 with trimethylsilyl ethers
8-11 in the presence of KF13 and DMAP (useful in
preventing detritylation) in THF at 60 ˚C for 24-48 h
(eq 3). Control experiments show that both additives are
required, though an explicit role in the reaction has been
ascribed to neither. Other fluoride sources (CsF and Bu4-
NF) and milder conditions (with 18-crown-6) do not lead
to cleaner products. The isolated yields of the dinucle-
otides are good (68-79%), and the products are readily
obtained in pure form by filtration and chromatography,
removing salts and any unreacted starting material. No
aqueous workup is required.
5′-O-(Dim eth oxytr ityl)th ym id in e 3′-O-((S-(2′′,6′′-Din itr o-
4′′-(tr iflu or om eth yl)p h en yl)m eth ylp h osp h on oth ioa te) (6).
31P NMR (acetone): δ 90.85, 91.36. Evidence for the elemental
composition of this compound could not be obtained because of
its instability.
Gen er a l P r oced u r e for t h e P r ep a r a t ion of 5′-O-(Tr i-
m eth ylsilyl)-3′-O-ben zoyl d eoxyn u cleosid es 8-10. Hexa-
methyldisilazane (1.2 mL, 0.92 g, 5.69 mmol) and chlorotri-
methylsilane (0.6 mL, 0.51 g, 4.72 mmol) were added to 3′-O-
benzoylthymidine14 (0.4 g, 1.16 mmol) in pyridine (4 mL) and
stirred at room temperature for 0.25 h. The pyridine was
removed in vacuo, and its final traces were removed by coevapo-
rating with toluene (10 mL). Diethyl ether (75 mL) or CH2Cl2
(40 mL) was added to precipitate the products.
5′-O-(Tr im eth ylsilyl)-3′-O-ben zoylth ym id in e (8) (0.46 g,
95%). Mp: 169.9-171.9 ˚C. Rf: 0.56 (EtOH:CHCl3 (6:94)). 1H
NMR (CDCl3): δ 9.55 (s, 1H), 8.05 (d, J ) 7.3 Hz, 2H), 7.73 (s,
1H), 7.60 (br t, J ) 7.5, 7.3 Hz, 1H), 7.47 (br t, J ) 7.7, 7.5 Hz,
2H), 6.51 (dd, J ) 9.0, 5.5 Hz, 1H), 5.48 (d, J ) 5.7 Hz, 1H),
4.27 (s, 1H), 3.93 (d, J ) 1.9 Hz, 2H), 2.26, 2.55 (m, 2H), 1.94 (s,
3H), 0.20 (s, 9H). HRMS calcd for C20H27N2O6Si [M + 1]:
419.526, found 419.1638.
5′-O-(Tr im e t h ylsilyl)-N 4,3′-O-d ib e n zoyl-2′-d e oxycyt i-
d in e (9) (0.21 g, 95%), colorless solid, mp dec ∼180 °C. Rf: 0.52
(EtOH:CHCl3 6:94). 1H NMR (CDCl3): δ 8.47 (d, J ) 7.5 Hz,
1H), 8.06 (d, J ) 7.2 Hz, 1H), 7.92 (d, J ) 7.5 Hz, 1H), 7.64-
7.46 (m, 7H), 6.53 (dd, J ) 8, 6 Hz, 1H), 5.52 (d, J ) 6.1 Hz,
1H), 4.40 (d, J ) 1.8 Hz, 1H), 3.98 (d, J ) 1.7 Hz, 2H), 2.29,
Exp er im en ta l Section
Bis-O-(1-Ben zotr ia zolyl) Meth ylp h osp h on oth ioa te (1).
A solution of methylphosphonothioic dichloride (0.79 mL, 1.12
g, 7.5 mmol, J ohnson Matthey) in anhydrous dioxane (15 mL)
was added dropwise to a stirring solution of 1-hydroxybenzo-
triazole (2.05 g, 5.2 mmol, dried in a drying pistol over refluxing
(14) Niewiarowski, W.; Lesnikowski, Z. J .; Wilk, A.; Guga, P.;
Okruszek, A.; Uznanski, B.; Stec, W. J . Acta Biochim. Pol. 1987, 34,
217. Lebedev, A. V.; Rife, J . P.; Wickstron, E. Tetrahedron Lett. 1990,
31, 855.
(15) Misiura, K.; Pietrasiak, D.; Stec, W. J . J . Chem. Soc., Chem.
Commun. 1995, 613-614.
(12) Sweeley, C. C.; Bentley, R.; Makita, M.; Wells, W. W. J . Am.
Chem. Soc. 1963, 85, 2497.
(13) Phosphorofluoridates have been coupled with silanes using KF
promotion: Semchenko, F. M.; Eremin, O. G.; Martynov, B. I. Zh.
Obshch. Khim. 1992, 62, 473.

1542 J . Org. Chem., Vol. 61, No. 4, 1996
Notes
2.91 (m, 2H), 0.19 (s, 9H). HRMS calcd for C26H30N3O6Si [M +
1]: 508.636, found 508.1898.
transfer all of the material. KF (0.022 g, 0.38 mmol) and DMAP
(0.27 g, 0.22 mmol) were added. The vial was sealed tightly and
stirred on a heating block at 60 °C, with reaction monitoring by
31P NMR. After the reaction was complete, the mixture was
filtered through a small pad of Celite which was then washed
with CH2Cl2:EtOH (90:10). The filtrate was concentrated to a
crude material that was purified by flash chromatography using
EtOH:CHCl3 (2-6%) to obtain the pure dinucleotide.
T-T (12)6a (0.057 g, 79%). Rf: 0.36 (EtOH:CHCl3 6:94).
Faster: 1H NMR (CDCl3) δ 1.54 (d, J ) 17.7 Hz, 3H); 31P NMR
(CDCl3) δ 32.78. Slower: 1H NMR (CDCl3) δ 1.59 (d, J ) 17.4
Hz, 3H); 31P NMR (CDCl3) δ 32.20.
5′-O-(Tr im eth ylsilyl)-N6,3′-O-d iben zoyl-2′-d eoxya d en os-
in e (10) (0.25 g, 97%), pale yellow solid, mp 143.5-144.5 °C.
Rf: 0.45 (EtOH:CHCl3 6:94). 1H NMR (CDCl3): δ 9.07 (br s,
1H), 8.84 (s, 1H), 8.52 (s, 1H), 6.76 (dd, J ) 7.9, 6.4 Hz, 1H),
5.69 (dd, J ) 3.0, 1.6 Hz, 1H), 4.45 (d, J ) 1.6 Hz, 1H), 3.97 (d,
J ) 2.4 Hz, 2H), 2.86 (m, 2H), 0.21 (s, 9H). HRMS calcd for
C
27H30N5O5Si [M + 1]: 532.686, found 532.2021.
5′-O-(Tr im et h ylsilyl)-N2-isob u t yr oyl-3′-O-b en zoyld eox-
ygu a n osin e (11). Cyanotrimethylsilane (0.13 mL, 0.094 g, 0.95
mmol) was added to 3′-O-benzoyldeoxyguanosine16 (0.42 g, 0.95
mmol), and the solution was stirred at room temperature under
nitrogen for 0.5 h. The gas outlet was passed through sodium
hydroxide to trap the released hydrocyanic acid. TLC showed
starting material, and 1H NMR showed a starting material to
silyl ether ratio of 1:2. Rf: 0.45 (EtOH:CHCl3 6:94). 1H NMR
(CDCl3): δ 8.36 (s, 1H), 6.35 (m, 1H), 5.67 (d, J ) 5.73 Hz, 1H),
4.38 (d, J ) 1.96 Hz, 1H), 3.88 (m, 2H), 2.83 (m, 1H), 2.67 (m,
2H), 1.28 (m, 6H), 0.15 (s, 9H). HRMS calcd for C24H32N5O6Si
[M + 1]: 514.626, found 514.2114. Attempts to purify this
compound by column chromatography or preparative TLC were
not successful, and it was used as obtained.
Gen er a l P r oced u r e for th e Syn th esis of Din u cleosid e
Meth ylp h osp h on a tes. Acetone was evaporated from the 5′-
silyl ether (0.075 mmol) under vacuum, and the resulting
material was handled in an inert atmosphere box. THF (2 mL)
was added, and the dissolved material was added to 5 (0.063 g,
0.15 mmol) in a V-Vial, with 2 × 1 mL washings of THF to
T-C (13)17 (0.057 g, 70%). Rf: 0.52, 0.56 (EtOH:CHCl3 10:
90). Faster: 1H NMR (CDCl3) δ 1.52 (d, J ) 17.4 Hz, 3H); 31P
NMR (CDCl3) δ 32.58. Slower: 1H NMR (CDCl3) δ 1.60 (d, J )
17.4 Hz, 3H); 31P NMR (CDCl3) δ 33.10.
T-A (14)18 (0.054 g, 68%). Rf: 0.54, 0.58 (EtOH:CHCl3 10:
90). Faster: 1H NMR (CDCl3) δ 1.48 (d, J ) 17.4 Hz, 3H); 31P
NMR (CDCl3) δ 32.45. Slower: 1H NMR (CDCl3) δ 1.54 (d, J )
17.4 Hz, 3H); 31P NMR (CDCl3) δ 32.91.
T-G (15)6a (0.075 g, 72%). Rf: 0.55, 0.59 (EtOH:CHCl3 12:
88). Faster: 1H NMR (CDCl3) δ 1.46 (d, J ) 17.4 Hz, 3H); 31P
NMR (CDCl3) δ 33.71. Slower: 1H NMR (CDCl3) δ 1.55 (d, J )
17.4 Hz, 3H); 31P NMR (CDCl3) δ 32.13.
Ack n ow led gm en t. Financial support was provided
by Genta, Inc.
Su p p or tin g In for m a tion Ava ila ble: NMR spectra of
compounds 8-11 (4 pages). This material is contained in
libraries on microfiche, immediately follows this article in the
microfilm version of the journal, and can be ordered from the
ACS; see any current masthead page for ordering information.
(16) de Rooji, J . F. M.; Wille-Hazelger, G.; van Deursen, P. H.;
Serdin, J .; van Boom, J . H. Recl. Trav. Chim. Pays.-Bas 1979, 98, 537.
(17) Vyazovkina, E. V.; Engels, J . W.; Lebedev, A. V. Bioorg. Khim.
1993, 19, 197.
(18) Kan, L. S.; Cheng, D. M.; Miller, P. S.; Yano, J .; Ts’o, P. O. P.
Biochemistry 1980, 19, 2122.
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