A BH3 masked H-phosphonate for coupling in oligonucleotide synthesis

Article abstract of DOI:10.1016/j.tetlet.2013.02.054

A method of masking 3′-H-phosphonate group for the solution-phase synthesis of ribonucleotide by H-phosphonate approach was described. The phosphonic acid group was masked by bis-(2-cyanoethyl) boranophosphate during the reactions. After a successive demasking treatment by triethylamine, trityl cation, and triethylamine, the triethylammonium 3′-H-phosphonate nucleotide can be obtained efficiently ready for the coupling cycle in synthesis of oligonucleotide.

Full text of DOI:10.1016/j.tetlet.2013.02.054

Tetrahedron Letters 54 (2013) 2183–2186
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A BH3 masked H-phosphonate for coupling in oligonucleotide synthesis
⇑
Jinyu Huang, Wei Lu, Zhen Xi
The State Key Laboratory of Elemento-Organic Chemistry and Department of Chemical Biology, Nankai University, Tianjin 300071, China
a r t i c l e i n f o
a b s t r a c t
A method of masking 30-H-phosphonate group for the solution-phase synthesis of ribonucleotide by
H-phosphonate approach was described. The phosphonic acid group was masked by bis-(2-cyanoethyl)
boranophosphate during the reactions. After a successive demasking treatment by triethylamine, trityl
cation, and triethylamine, the triethylammonium 30-H-phosphonate nucleotide can be obtained
efficiently ready for the coupling cycle in synthesis of oligonucleotide.
Article history:
Received 17 November 2012
Revised 30 January 2013
Accepted 19 February 2013
Available online 26 February 2013
Ó 2013 Published by Elsevier Ltd.
Keywords:
Protecting group
H-Phosphonate
Boranophosphate
Block coupling
Oligoribonucleotide synthesis
Solution-phase synthesis of oligonucleotide, compared to solid-
phase synthesis, has the advantages of easy characterization of
reaction intermediates and easy scale-up, which should be useful
in the synthesis of large scale of specific sequence oligonucleotide
for therapeutical application such as siRNA. In our previous work,1
we reported the synthesis of siRNA targeting on human superoxide
dismutase gene in solution by the phosphotriester approach,2
which has successfully yielded siRNA showing same efficiency of
the gene silencing effect as its solid phase phosphoamidite ap-
proach synthesized counterpart.3,4 The phosphotriester approach
generally gives quite efficient coupling in less than 14 mer oligori-
bonucleotides, while still needs to be optimized for longer than 20
mer synthesis.
BH3 group was frequently used as the protecting group for
phosphine derivatives,20 and boranophosphate nucleotide was
synthesized successfully.21–23 Wada group has done some excel-
lent work using BH3 to protect phosphonic acid.24,25 They have
synthesized a boranophosphate-linked disaccharide, and con-
verted it into H-phosphonate diester disaccharide. Inspired by their
results, herein we have tested whether boranophosphotriester
(compound 3 in Scheme 1) could be demasked to give H-phospho-
nate diester (compound 4 in Scheme 1), then further be demasked
to give H-phosphonate monoester (compound 1 in Scheme 1) for
coupling in oligonucletide synthesis. Several aromatic and alkyl
groups can be used as a leaving group in the protection of phos-
phate.26 Cyanoethyl group (CE), a frequently-used protecting group
in oligonucleotide synthesis, has been chosen here as the protect-
ing group for nucleotide 30-phosphate. After BH3 treatment, thus
bis-(2-cyanoethyl) boranophosphate nucleotide (compound 2 in
Scheme 1) was synthesized. Also, it is known that BH3 group can
be demasked by trityl cation in acidic solution,24,25,27 in order to
avoid the simultaneous deprotection on 50-DMTr protecting group,
the trityl group, which is much stable in acid than DMTr group, was
used as we have found that when the reaction was carried out in
0.2% dichloroacetic acid and 2.5 equiv of TMTrOH in CH2Cl2 in
10 min, the coordinated BH3 group could be demasked by TMTrOH
rapidly without observed deprotection on 50-trityl group. Fortu-
nately, the 2-cyanoethyl group and the BH3 group were success-
fully demasked successively to give 30-H-phosphonate monoester
nucleotide.
H-Phosphonate approach,5–18 which has high coupling effi-
ciency, is regarded as an alternative approach for oligonucleotide
synthesis in solution. As same as phosphotriester approach, the
H-phosphonate approach can adopt ‘block’ condensation reactions
in solution-phase synthesis.10 During synthesis of those building
block intermediates, a suitable protecting group for 30 end is cru-
cial. Levulinyl (Lev) ester group was used as 30-OH protecting
group in the synthesis of DNA by H-phosphonate approach in solu-
tion.10,12 However, when using the same protecting group for the
synthesis of RNA, demasking Lev group from 30-OH would induce
20?30 migration of 20-protecting group such as TBDMS.19 It would
be advantageous if we can develop an efficient masking–demask-
ing protocol for 30-H-phosphonate, in which 30-H-phosphonate
was masked during the coupling reaction, and demasked to give
30-H-phosphonate monoester for the next nucleotide coupling.
The nucleoside 5 in THF and 3-hydroxypropionitrile was added
successively into a THF solution of PCl3 and 2,6-dimethylpyridine
at ꢀ78 °C under argon atmosphere. Followed by adding 1 M
BH3ꢁTHF in THF solution at ꢀ20 °C. The resulting solution was
⇑
Corresponding author. Tel./fax: +86 22 23504782.
E-mail address: zhenxi@nankai.edu.cn (Z. Xi).
0040-4039/$ - see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.tetlet.2013.02.054

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J. Huang et al. / Tetrahedron Letters 54 (2013) 2183–2186
Scheme 1. BH3 masked H-phosphonate for coupling cycle in oligonucleotide synthesis.
stirred at 0 °C for additional 30 min to give fully protected interme-
diate 7 in 86% yield (Scheme 2).
The triethylammonium H-phosphonate monoester 11 was
obtained by three sequential reactions: firstly, one of the two 2-
cyanoethyl groups was demasked by the treatment with
Et3N/CH3CN to give compound 9 in 91% yield; secondly, the coor-
dinated BH3 group in compound 9 was demasked to give H-phos-
phonate diester 10 in 78% yield by the treatment of 0.2%
dichloroacetic acid and 2.5 equiv of TMTrOH in CH2Cl2 in 10 min;
finally, the remaining 2-cyanoethyl group in compound 10 was
All other building blocks for H-phosphonate coupling reaction
such as compounds 8 and 11 were obtained from the fully pro-
tected mononucleotide 7 by different demasking reaction. 50-Trityl
group was cleaved by 3% TFA in the presence of 50 mM BH3ꢁPy and
50% Et3SiH as a trityl cation scavenger to avoid the demasking on
BH3 group.24,27 50-OH nucleotide 8 was thus obtained in 85% yield.
Scheme 2. Reagents and conditions: (i) PCl3, 2,6-dimethylpyridine, 3-hydroxypropionitrile, THF, ꢀ78 °C; (ii) BH3ꢁTHF/THF, ꢀ20 to 0 °C, 30 min; (iii) 3% TFA, BH3ꢁPy (50 mM),
CH2Cl2/Et3SiH (v:v = 1:1), 0 °C, 15 min; (iv) Et3N/CH3CN (v:v = 3:1), rt, 10 min; (v) 0.2% DCA, TMTrOH (12.5 mM), CH2Cl2, 0 °C, 10 min; (vi) Et3N/CH3CN (v:v = 4:1), rt, 5 min.

J. Huang et al. / Tetrahedron Letters 54 (2013) 2183–2186
2185
Figure 1. 31P NMR of compound 7, 9–11.
Scheme 3. Reagents and conditions: (i) pivaloyl chloride, BH3ꢁPy (0.1 M), C5H5N, 0 °C, 20 min. (ii) N-(phenylsulfanyl)-succinimide, C5H5N, 0 °C, 20 min; (iii) 3% TFA, BH3ꢁPy
(50 mM), CH2Cl2/Et3SiH (v:v = 1:1), 0 °C, 15 min; (iv) Et3N/CH3CN (v:v = 3:1), rt, 10 min; (v) 0.2% DCA, TMTrOH (12.5 mM), CH2Cl2, 0 °C, 10 min; (vi) Et3NH/CH3CN.

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J. Huang et al. / Tetrahedron Letters 54 (2013) 2183–2186
demasked by Et3N/CH3CN to give compound 11 in 51% yield. The
31P NMR (Fig. 1) clearly showed the transformation reaction from
compound 7 to 11.
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.tetlet.2013.02.054.
By coupling triethylammonium 30-H-phosphonate monoester
11 and 50-OH nucleotide 8, the dinucleotide 12 was synthesized.
The coupling reaction was activated by pivaloyl chloride in the
presence of 0.1 M BH3ꢁPy at 0 °C, then the thus formed H-phospo-
nate dinucleotide was reacted in situ with N-(phenylsulfanyl)-suc-
cinimide to give phosphorothioate triester 12 in 84% yield
(Scheme 3). The presence of 0.1 M BH3ꢁPy in the coupling reaction
can greatly reduce the partial demasking of P-BH3.
References and notes
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In conclusion, a BH3 masked H-phosphonate approach for cou-
pling cycle in oligonucleotide solution synthesis was established
herein successfully. This approach can not only take the advantage
of high coupling efficiency by H-phosphonate in solution, but also
avoid 20 ? 30 migration by 20-protecting group. This masking–
demasking protocol for 30-H-phosphonate, in which 30-H-phospho-
nate was masked by BH3 and CE during the coupling reaction, and
demasked to give 30-H-phosphonate monoester for next coupling,
may find new applications in the oligo-phosphate synthesis. The
further optimization of this BH3 masked H-phosphonate approach
for oligomer synthesis is under active investigation in our lab and
will be reported in due course.
Acknowledgments
This work was financially supported by Ministry of Science
and Technology of China (2010CB126102, 2009ZX09503-022,
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(20932005, 20872067).
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Supplementary data
Supplementary data (experimental procedures, characteriza-
tion data and 1H, 13C NMR, 31P NMR spectra and mass data)