Synthesis of a novel nucleic acid mimic: P-boranomethylphosphonate

Article abstract of DOI:10.1081/NCN-100002547

A new type of non-ionic nucleotide analogue with a doubly modified internucleotide linkage, P-boranomethylphosphonate, has been successfully synthesized and characterized. Dithymidine boranomethylphosphonate 5 is the first example of a P-boranomethylphosphonate compound; it is a highly lipophilic phosphodiester analog, which is almost totally resistant to both snake venom phosphodiesterase (SVPDE) and bovine spleen phosphodiesterase (BSPDE). P-boranomethylphosphonates are expected to be promising candidates for mechanistic, diagnostic and therapeutic applications.

Full text of DOI:10.1081/NCN-100002547

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SYNTHESIS OF A NOVEL NUCLEIC ACID MIMIC: P-
BORANOMETHYLPHOSPHONATE
Jinlai Lin a & Barbara Ramsay Shaw b
a Paul M. Gross Chemical Laboratory, Department of Chemistry , Duke University ,
Durham, North Carolina, 27708-0346, U.S.A.
b Paul M. Gross Chemical Laboratory, Department of Chemistry , Duke University ,
Durham, North Carolina, 27708-0346, U.S.A.
Published online: 07 Feb 2007.
To cite this article: Jinlai Lin & Barbara Ramsay Shaw (2001) SYNTHESIS OF A NOVEL NUCLEIC ACID MIMIC: P-
BORANOMETHYLPHOSPHONATE, Nucleosides, Nucleotides and Nucleic Acids, 20:4-7, 1325-1328, DOI: 10.1081/
NCN-100002547
To link to this article: http://dx.doi.org/10.1081/NCN-100002547
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 1325–1328 (2001)
SYNTHESIS OF A NOVEL NUCLEIC ACID MIMIC:
P-BORANOMETHYLPHOSPHONATE
Jinlai Lin and Barbara Ramsay Shaw∗
Paul M. Gross Chemical Laboratory, Department of Chemistry,
Duke University, Durham, North Carolina 27708-0346
ABSTRACT
A new type of non-ionic nucleotide analogue with a doubly modified internu-
cleotide linkage, P-boranomethylphosphonate, has been successfully synthe-
sized and characterized. Dithymidine boranomethylphosphonate 5 is the first
example of a P-boranomethylphosphonate compound; it is a highly lipophilic
phosphodiester analog, which is almost totally resistant to both snake venom
phosphodiesterase (SVPDE) and bovine spleen phosphodiesterase (BSPDE).
P-boranomethylphosphonates are expected to be promising candidates for
mechanistic, diagnostic and therapeutic applications.
INTRODUCTION
Non-ionic nucleotide and oligonucleotide analogs (1) have long attracted at-
tention. Improved uptake by cells and extended biological half-life make them
attractive candidates for use as antiviral and antisense agents in both cell culture
and animals. These analogues are interesting in their own right because studies on
their conformation and interactions with proteins and nucleic acids can give new
insights into factors affecting nucleic acid structure and function. Of the uncharged
modified oligonucleotides, the nucleoside methylphosphonates, wherein the neg-
atively charged phosphate oxygen is replaced by a methyl group, are among the
most promising because they are highly resistant to nucleases (2).
∗Corresponding author. E-mail: brshaw@chem.duke.edu
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Copyright 2001 by Marcel Dekker, Inc.
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LIN AND SHAW
Figure 1. Internucleotide linkages: (a) methylphosphonate, (b) boranophosphate, (c) boranometh-
ylphosphonate.
By structurally combining the methylphosphonate (2) and boranophosphate
(3) backbones, we have created a new phosphodiester linkage, the boranomethyl-
phosphonate [CH3–P–BH3] (Fig. 1), wherein the two nonbridging oxygen atoms
of a phosphodiester group are replaced by methyl and borane groups. Since these
groups impart lipophilicity to oligonucleotides, it was expected that the resulting
Scheme 1.

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P-BORANOMETHYLPHOSPHONATE
1327
hybrid nucleoside P-boranomethylphonates should be more lipophilic and more
resistant to nucleases than either methylphosphonates or boranophosphates.
Here, we report the first example of a boranomethylphosphonate compound,
specifically the dithymidine P-boranomethylphosphonate (TBPMeT, 5), its synthe-
sis, characterization, and some biochemical properties.
The synthesis of dinucleoside P-boranomethylphosphonate 5 is outlined in
Scheme 1. 5ꢁ-O-DMT-thymidine was phosphitylated with (i-Pr2N)P(Cl)(CH3)
catalyzed by triethylamine to give 1. Phosphite 1 was treated in situ with 3ꢁ-O-
acetylthymidine and tetrazole in DMF to give compound 2 (31P NMR signals at
189.1 and 188.1 ppm), which was then treated with excess borane-methyl sul-
fide complex to afford phosphite-borane 3 with a 31P NMR signal at 152.5 ppm
(br). The low boiling-point solvents were removed under reduced pressure; and
the syrup was treated with DMT removal reagent (2.5% dichloroacetic acid in
dichloromethane) to yield 4 which was converted to 5 with NH4OH/CH3OH at
room temperature. The crude mixture was purified by column chromatography on
silica gel to give 5 which was further purified by reverse-phase HPLC by isocratic
elution with 84% 50 mM triethylammonium acetate (TEAA) and 16% CH3CN to
obtain pure 5, Rt = 12.1 min. The crude 5 could also be purified on a silica gel
HPLC colunm, eluting with 9% methanol and 91% ethyl acetate, Rt = 16.3 min.
The overall yield of dithymidine P-boranomethylphosphonate 5 (TBPMeT) was
about 42%. The chemical structure of 5 was established via UV λmax = 267nm;
31P NMR (DMSO-d6) δ (ppm) 147.6 (br); 1H NMR (not shown); MS (FAB+): m/z
for (M+H)+ 543.2; HRMS (FAB−) calcd for C21H31O10N4PB (M–H)− 541.1869,
found 541.1856. Successful separation of two diastereomers (Rp and Sp) of 5 was
achieved by reverse-phase HPLC with 18% CH3CN and 82% 50 mM triethylam-
monium acetate; for the first eluted diastereomer 5a, Rt (5a) = 14.7 min; for the
second eluted diastereomer 5b, Rt (5b) = 16.2 min. The method described above
should be applicable to the synthesis of other deoxyribo- or ribo-oligonucleoside
boranomethylphosphonates.
RESULTS AND DISCUSSION
Replacementofanonbridgingoxygenatominthenaturalphosphodiesterlink-
−
age by S−, CH3 or BH3 generally preserves the ability of oligodeoxynucleotides
(ODN) to form complementary duplexes, although with somewhat lower stability
(4–5). The phosphorothioate internucleotide analog has emerged as the linkage of
choice for the development of antisense oligonucleotides as therapeutic agents. Al-
though oligonucleoside phosphorothioates (5) have many virtues, new analogs are
still of interest due to the poor cellular permeation properties of phosphorothioate
ODNs. Numerous backbone modifications have been made to increase the cellular
permeability of ODNs but few have resulted in an improvement. Thus, we decided
to introduce methyl and borane into the nucleotide phosphodiester linkage. As
expected, double replacement of two oxygen atoms in the natural phosphodiester

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LIN AND SHAW
linkage greatly enhances the lipophilicity of the oligonucleotide and imparts almost
total resistance to the 3ꢁ and 5ꢁ endonucleases.
Our studies indicated that the P-boranomethylphosphonate internucleotide
linkage is: (1) very stable toward neutral and acidic hydrolysis; (2) extremely resis-
tant toward cleavage by both snake venom phosphodiesterase (SVPDE) and bovine
spleen phosphodiesterase (BSPDE), and (3) highly lipophilic. The TBPMeT dimer
was 6800- and 370-fold more lipophilic than normal TpT (dithymidine phosphate)
and TpBT (dithymidime boranophosphate) accordingly.
Thus, the non-ionic [CH3–P–BH3]-ODN constitutes an entirely new and
intriguing class of modified nucleic acids. High lipophilicity and resistance to
enzymatic cleavage, in conjuction with possible utility as carriers of 10B in boron
neutron capture therapy (BNCT) (6) for the treatment of cancer, make the bora-
nomethylphosphonate a promising candidate for further mechanistic, diagnostic
and therapeutic investigation.
ACKNOWLEDGMENT
We thank Dr. Vladimir Rait for his suggestions and encouragement. This work
was supported by grants 1R01-GM57693-01 from NIH and DE-FG05-97ER62376
from DOE to B.R.S.
REFERENCES
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1981, 20, 1874–1880 and references cited therein. (b) Uhlmann, E.; Peyman, A.
Chem. Rev., 1990, 90, 543–584. (c) Zhang, J.; Matteucci, M. D. Bioorg. & Med.
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(2) (a) Smith, C. A.; Aurelian, L.; Reddy, M. P.; Miller, P. S.; Ts’o, P. O. P. Proc. Natl.
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(3) (a) Sood, A.; Shaw, B. R.; Spielvogel, B. F. J. Am. Chem. Soc., 1990, 112, 9000– 9001.
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