Synthesis of novel 3'-methylene H-phosphonate thymidines

Article abstract of DOI:10.1016/S0040-4039(00)01358-7

The synthesis of novel 5'-DMT- and 5'-MMT-protected 3'-methylene H-phosphonate thymidines 1-6, having methoxy, fluoro, hydrogen, and methoxyethoxy substituents at the 2'-position is reported. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01358-7

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 7813–7816
Synthesis of novel 3%-methylene H-phosphonate thymidines
Haoyun An,* Tingmin Wang and P. Dan Cook
Isis Pharmaceuticals, Inc., 2292 Faraday Avenue, Carlsbad, CA 92008, USA
Received 13 July 2000; accepted 1 August 2000
Abstract
The synthesis of novel 5%-DMT- and 5%-MMT-protected 3%-methylene H-phosphonate thymidines 1–6,
having methoxy, fluoro, hydrogen, and methoxyethoxy substituents at the 2%-position is reported. © 2000
Elsevier Science Ltd. All rights reserved.
Keywords: 3%-methylene; H-phosphonate; H-phosphonic acid; nucleotide; thymidine; Arbuzov reaction.
Utilization of antisense oligonucleotides as therapeutics in humans is undergoing evaluation in
a number of clinical trials, and the first antisense drug has been approved.1 These oligonucleotides
are mainly phosphorothioates, simple backbone-modified DNA analogues with improved nuclease
resistance but limited target-binding affinity and oral bioavailability. Recently, methylphospho-
nate,2 3%,5%-methylene phosphine oxide,3 phosphoramidate,4 amide,5 and other6 backbone-modified
oligonucleotides have received attention for their potential usefulness as antisense drugs.
3%-Methylene modified oligonucleotides are expected to increase stability against nucleases and
target-binding affinity (Tm) to complimentary RNA based on the initial X-ray analysis of an
octamer having a single 3%-methylene phosphonate (MP) linkage.7 In addition, 3%-methylene
modified oligonucleotides may improve the bioavailability and cellular uptake, as well as alter
the distribution because of their higher lipophilicity and isoelectronic/isosteric structures to natural
DNA. However, 3%-methylene oligonucleotide has received little attention because of the synthetic
difficulty encountered in the preparation of appropriate monomers and oligonucleotides. Synthesis
of 3%-methylene phosphates (PV) has been explored.8 The 3%-methylene phosphonate dimer9 and
trimer10 have been prepared, and the dimer has been incorporated into an octamer by a
solution-phase approach.7 However, the 3%-methylene phosphate or phosphonate monomer has
not been utilized in oligonucleotide synthesis by a solid-phase approach.
In this communication, we describe a new effective strategy for the synthesis of novel 5%-DMT-
and MMT-protected 3%-methylene H-phosphonate thymidines 1–6 with 2%-O-methyl, fluoro,
hydrogen, and O-(2-methoxy)ethyl substituents (Schemes 1 and 2). 3%-Methylene H-phosphonates
1–6 thus obtained serve as effective monomers for the synthesis of 3%-methylene modified
oligonucleotides by a solid-phase approach.11
* Corresponding author. Tel: 760-603-3834; fax: 760-929-0036; e-mail: han@isisph.com
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01358-7

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Scheme 1.
Scheme 2.

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The synthesis of 3%-methylene H-phosphonate monomers 1–6 having ꢀOCH3, ꢀF, ꢀH, and
ꢀOCH2CH2OCH3 substituents at the 2%-position starting from the corresponding 3%-iodomethyl
nucleosides 8–1112 is depicted in Schemes 1 and 2. 2%-O-Methylthymidine 8 was utilized to
develop the synthetic strategy. Ammonium phosphinate and 1,1,1,3,3,3-hexamethyldisilazane
were heated at 110°C for 2 h to generate the intermediate bis(trimethylsilyloxy)phosphine
(BTSP) (7).13 BTSP has been utilized for the synthesis of other small molecule H-phospho-
nates,14 but it has not been used in nucleotide chemistry. We have utilized BTSP as the
phosphorus source to form a PꢀC bond of 3%-methylene H-phosphonates in one step from the
corresponding iodomethyl nucleosides. The resulting novel H-phosphonates can be used for
oligonucleotide synthesis by employing a typical solid-phase H-phosphonate strategy instead of
historical solution-phase phosphodiester or phosphotriester approaches.
3%-Iodomethylthymidine 8 was reacted in CH2Cl2 with BTSP 7, prepared in situ, by an
Arbuzov-type reaction. Hydrolysis of the resulting silyl intermediate with methanol provided the
desired product 16 in 31% yield as the H-phosphinic acid after chromatographic purification. A
reduced product, 3%-methyl compound 17, was obtained in 59% yield as a by-product. The
5%-TBDPS protecting group of 16 was removed by treating it with triethylamine trifluoride
(Et3N·3HF) providing H-phosphinic acid 18 in 97% yield. Compound 18 was then reacted with
DMT-Cl to generate the desired final H-phosphonate monomer 1 in 65% yield as its Et3N salt.
Treatment of 8–11 with Et3N·3HF provided the corresponding unprotected nucleosides 12–15 in
greater than 90% yields (Scheme 1). 2%-O-Methyl-3%-iodomethyl compound 12 was reacted with
BTSP under the same conditions as described above to give H-phosphinic acid 18 in 24% yield
and the corresponding reduced product 19 in 53% yield.
Compound 12 was reacted with DMT-Cl to provide 5%-DMT-protected compound 20 in 99%
yield (Scheme 2). Arbuzov reaction between 20 and BTSP did not provide the desired compound
1; instead, deprotected products 18 and 19 were obtained because the DMT-protecting group
was removed by H-phosphinic acid formed in the reaction. However, by adding diisopropylethyl-
amine (Hu¨nig’s base), the Arbuzov reaction between 20 and BTSP generated the desired product
1 in 37% yield and the corresponding reduced product 22 in 54% yield after chromatographic
purification. By the same manner as 2%-O-methyl derivatives, compound 13 was protected with
DMT to provide 2%-fluoro-3%-iodomethyl nucleoside 21 in an excellent yield. Arbuzov reaction
between 21 and BTSP under the same conditions in the presence of Hu¨nig’s base finally gave
2%-fluoro-3%-methylene H-phosphonate thymidine 2 and the reduced product 23.
5%-DMT-3%-methylene H-phosphonate thymidines 1 and 2 have been successfully used for the
synthesis of oligonucleotides by solid-phase H-phosphonate chemistry.11 However, the DMT-
protecting group in the H-phosphonate nucleotide system is not an ideal protecting group. In
order to utilize 3%-methylene H-phosphonate monomers for oligonucleotide synthesis more fully,
we have considered the less acid labile MMT-protecting group. The reaction of deprotected
compounds 12–15 with 3 equivalents of MMT-Cl provided the corresponding MMT-protected
compounds 24–27 in 84–99% yields. Arbuzov reaction between 24–27 and BTSP under the same
conditions as described above finally gave 5%-MMT-protected 3%-methylene H-phosphonate
thymidines 3–6 and the corresponding reduced products 28–31. MMT-protected products 3–6
are more stable than 1 and 2, and subsequently more easily handled. Monomers 3–5 have been
effectively utilized in solid-phase oligonucleotide synthesis.11 New compounds 12–31 and novel
3%-methylene H-phosphonate thymidines 1–6 were characterized by NMR and high-resolution
mass spectroscopic analyses. The synthesis of 3%-methylene H-phosphonate thymidines 1–6 from
the corresponding 3%-iodomethyl nucleosides 8–11 was straightforward and efficient. This

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strategy can potentially be used for the synthesis of other nucleobases with various 2%-
sustituents.
In conclusion, we have developed a useful strategy for the synthesis of various 2-substituted
3%-methylene H-phosphonate nucleotides. New 5%-DMT- and 5%-MMT-3%-methylene H-phospho-
nate thymidines 1–6 with four different substituents at the 2%-position were synthesized success-
fully by this strategy. Two types of protecting groups at the 5%-position provided more flexibility
and the corresponding monomers have been utilized successfully in solid-phase oligonucleotide
synthesis. Syntheses of 3%-methylene H-phosphonates using other nucleobases and 3%-methylene
modified oligonucleotides, as well as their antisense property studies, are in progress and will be
reported in due course.
Acknowledgements
The authors thank Drs. Mano Manoharan, Martin Maier, Bruce Ross, Ramesh Bharadwaj,
Bal Bhat, and Andrei Guzaev for their helpful discussions.
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