Synthesis of mixed sequence borane phosphonate DNA

Article abstract of DOI:10.1021/ja061757e

A novel solid-phase phosphoramidite-based method has been developed for the synthesis of borane phosphonate DNA. Keys to this new approach are replacement of the common 5′-dimethoxytrityl blocking group with a 5′-silyl ether and the use of new protecting

Full text of DOI:10.1021/ja061757e

Published on Web 06/06/2006
Synthesis of Mixed Sequence Borane Phosphonate DNA
Heather Brummel McCuen,‡ Mary S. Noe´, Agnieszka B. Sierzchala, Adrian P. Higson,§ and
Marvin H. Caruthers*
Department of Chemistry and Biochemistry, UniVersity of Colorado, Boulder, Colorado 80309-0215
Received March 14, 2006; E-mail: marvin.caruthers@colorado.edu
For some time now,1 oligodeoxyribonucleotides (ODNs) bearing
internucleotide borane phosphonate linkages have been of consider-
able interest for applications in diagnostic and therapeutic areas
because they mimic natural DNA in various biological processes.1-5
The problem with this analogue is the lack of high yielding,
chemical methods for its synthesis. In this paper, we report a new
strategy that generates mixed sequence borane phosphonate DNA
of high yield and purity.
To date, the most successful synthesis approach has been
conversion of deoxyoligonucleotide H-phosphonates, via silylation
followed by boronation, to oligomers having exclusively borane
phosphonate internucleotide linkages.2-5 Results with unprotected
Figure 1. 5′-O-Silyl-2′-deoxynucleoside-3′-phosphoramidites and borane
bases suggest that 10mers can be prepared with maximum yields
of 20-30%.5 Recently, an alternative method,6 featuring mono-
nucleotide borane phosphonates and a phosphotriester strategy, has
been used to prepare borane phosphonate dinucleotides having all
four bases but with coupling yields from 72 to 92%. Unless
improved considerably, these results suggest that neither approach
is viable for the preparation of oligomers rapidly in high yields on
supports.
To overcome these challenges, we have developed a new strategy
for synthesizing borane phosphonate DNA. Previous research has
shown that the 5′-dimethoxytrityl group, which transiently protects
each synthon during natural DNA chemical synthesis, is incompat-
ible with the preparation of borane phosphonate DNA.2 This
observation forced us to explore alternative strategies. On the basis
of earlier research,7 we prepared 2′-deoxynucleosides having 5′-
O-[benzhydroxybis(trimethylsilyloxy)]silyl protection and discov-
ered that this silyl ether could be removed under conditions
compatible with the synthesis of borane phosphonate DNA. As a
result, this group has been substituted for dimethoxytrityl as a
transient 5′-protecting group (Figure 1).
Another serious challenge has been protection of the 2′-
deoxynucleoside bases. This is because borane reagents reduce the
commonly used amide protecting groups to N-alkyl or aryl exocyclic
amines or form stable, irreversible borane adducts with these bases.
This problem has been solved by using a relatively new protecting
group strategy.8 Thus for adenine and cytosine, the exocyclic amines
are protected with dimethoxytrityl and trimethoxytrityl, respectively.
Protection of guanine with N2-(9-fluorenylmethoxycarbonyl) is
based upon our observations that certain carbamates are resistant
to reduction with borane. Similarly with thymine, the use of anisoyl
on N3 eliminates possible reduction by borane reagents2 and also
protects from N-methylation of thymine via the methyl phosphate
internucleotide linkage.
phosphonate ODNs. Silyl ) benzhydroxybis(trimethoxysilyloxy)silyl. B′
) protected base. B ) cytosine, thymine, adenine, and guanine. X,Y )
combinations of phosphate and borane phosphonate linkages.
Table 1. Mass Spectrum Analysis of Oligodeoxynucleotides
molecular weight
no.
ODNa
calculated
observed
3
4
5
6
7
8
9
10
d[(TpTpTb)4TpT]
d[(TbTp)6TbT]
d[(GpTpGbTpGpTb)2GpT]
d[(GbTpGbTp)3GbT]
d(Tb)9T
d[(AbTb)4AbT]
d[(CbTb)4CbT]
d(TbCbTbTbAbCbTbGbAbT)
4185.7
4179.8
4360.8
4354.9
2960.8
3005.9
2885.8
2973.9
4183.4b
4177.5b
4360.7b
4355.2b
2954.5c
3000.7c
2881.5c
2967.5c
a p ) phosphate; b ) borane phosphonate. b Perseptive Biosystems
Voyager Biospectrometry Workstation using a previously published pro-
cedure.8 c HPLC-ESI-Q-TOF-MS Instrument System.
acetonitrile and tetrazole to generate a family of dimers having a
phosphite triester internucleotide linkage. These dimers are then
reacted with either THF‚BH3 or a peroxyanion solution.8 Removal
of 5′-silyl protection with triethylammonium hydrogen fluoride
generates a family of dinucleotides having any of the four bases
and either a P-IV phosphonium borane adduct or a phosphate
triester linkage. These dimers can then be extended using the same
repetitive cycle to generate an ODN of the appropriate length.
Protecting groups are removed sequentially. Initially and with
the ODN attached to the support, 80% acetic acid is used to
eliminate trityl groups from adenine and cytosine8 (the P-IV borane
adduct is compatible with acetic acid). Next the oligomer is treated
with a dithiolate10 to remove internucleotide methyl protection.
Finally, ammonium hydroxide eliminates carbamate and anisoyl
groups from guanine and thymine, respectively, and generates 2.
Purification is by reverse phase HPLC. A typical result for a 10mer
(compound 10, Table 1) having all four bases and borane phos-
phonate internucleotide linkages is shown in Figure 2 (total reaction
mixture). The major peak (excluding the first, anisic acid peak) is
the product (99% coupling yield, isolated yield 88%). As expected
Using these appropriately protected 2′-deoxynucleoside phos-
phoramidites (Figure 1), a new, high yielding synthesis cycle has
been developed.9 Starting with a 2′-deoxynucleoside attached to
polystyrene, the first step is condensation with 1a-d in anhydrous
‡ Current address: Agilent Laboratories, Santa Clara, CA 95051.
§ Current address: Ultrafine, Manchester M15 65Y, U.K.
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J. AM. CHEM. SOC. 2006, 128, 8138-8139
10.1021/ja061757e CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. Reverse phase HPLC analysis of the total, crude reaction mixture from the synthesis of compound 10. Inset A: Gel electrophoresis results from
total, crude reaction mixtures. Lanes 1-5, d(TpTp)4TpT, 7, 8, 9, and 10, respectively. Inset B: Phosphorus NMR of compound 10.
Acknowledgment. Supported by the University of Colorado.
The technical assistance of Richard Shoemaker in the NMR
Laboratory is gratefully acknowledged.
from many P-chiral centers and the resulting large number of
stereoisomers, this peak is broad.
Numerous oligomers have been prepared using this synthesis
Supporting Information Available: Procedures for the synthesis
strategy with isolated yields of 70-89%. Table 1 lists mass data
of 1a-d and a table outlining the steps for one synthesis cycle. Also
for examples having combinations of 2′-deoxynucleoside bases and
included are several HPLC profiles from the synthesis of various ODNs,
internucleotide phosphate and borane phosphonate linkages. The
a table summarizing both the total yields and coupling yields for various
observed masses for all ODNs correspond to those as calculated.
Similarly, phosphorus NMR analyses display a broad signal at 96
ppm (borane phosphonate) and a sharp peak at -2 ppm when
phosphate is part of the backbone. By phosphorus NMR, when all
internucleotide linkages are borane phosphonate (Table 1, com-
pound 10), phosphate cannot be detected (inset B, Figure 2). 11B
NMR spectra for all oligomers consist of a broad signal at -40
ppm (characteristic of the borane phosphonate linkage). Neither
the mass spectral nor boron NMR data suggest the presence of
boronated bases or sugars. When total, unpurified reaction mixtures
are analyzed by gel electrophoresis, only one major band which
corresponds to the product is observed (inset A, Figure 2). As
expected from previous research,1-6 these borane phosphonate
ODNs are resistant to degradation by exonucleases and DNase I.
Now that a chemistry has been developed for synthesizing borane
phosphonate DNA in high yield and having any combination of
the four common bases as well as both phosphate and borane
phosphonate internucleotide linkages, many biological and bio-
chemical studies are possible. These include the use of borane
phosphonate ODNs for various diagnostic and therapeutic applica-
tions as well as the further development of borane phosphonate
oligomers having, among others, phosphorothioate, phospho-
rodithioate, and methyl phosphonate internucleotide linkages.
ODNs, and gel electrophoresis analysis of borane phosphonate ODNs
following degradation with SVP and DNase I. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
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(9) For deoxyoligonucleotide synthesis, a modified Applied Biosystems Model
392 instrument is used. Each synthesis cycle consists of the following
reactions with appropriate washes: 65 s coupling with phosphoramidite
(0.05 M) and tetrazole (0.22 M) in acetonitrile, conversion of the phosphite
triester to the borane adduct using 25 mM BH3‚THF in THF or to the
phosphate triester with an aqueous peroxyanion buffer8 and 70
s
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ethanolamine in N,N-dimethylformamide (pH 9). Syntheses are completed
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