An improved procedure for the synthesis of vinylphosphonate-linked nucleic acids

Article abstract of DOI:10.1016/S0040-4039(00)00653-5

The synthesis of a range of 5'-deoxy-5'-methylidene phosphonate- containing thymidine dimers has been achieved using a palladium-catalysed cross coupling reaction as a key step. The optimised reaction conditions comprising of palladium acetate, 1,1'-bis(diphenylphosphino)ferrocene and propylene oxide in THF were found to couple a range of H-phosphonates and vinyl bromide 3 in fair to excellent yield. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00653-5

Tetrahedron Letters 41 (2000) 4513±4517
An improved procedure for the synthesis of
vinylphosphonate-linked nucleic acids
Sahar Abbas and Christopher J. Hayes*
The School of Chemistry, The University of Nottingham, University Park, Nottingham NG7 2RD, UK
Received 24 March 2000; accepted 20 April 2000
Abstract
The synthesis of a range of 50-deoxy-50-methylidene phosphonate-containing thymidine dimers has
been achieved using a palladium-catalysed cross coupling reaction as a key step. The optimised reaction
conditions comprising of palladium acetate, 1,10-bis(diphenylphosphino)ferrocene and propylene oxide in
THF were found to couple a range of H-phosphonates and vinyl bromide 3 in fair to excellent yield.
# 2000 Elsevier Science Ltd. All rights reserved.
Keywords: antisense; palladium coupling; vinyl bromide; oligonucleotide; modi®ed backbone.
The potential to inhibit protein synthesis in vivo by binding a short `antisense oligonucleotide'
to an appropriate `sense' messenger RNA (mRNA) has stimulated considerable interest in the
potential use of these materials as therapeutic agents.1 Due to a number of well-documented
limitations, naturally occurring phosphodiester-linked nucleic acids (DNA and RNA 1, Fig. 1)
are not suitable drug candidate molecules. One of their main limitations is their instability
towards a number of nuclease enzymes. Many attempts have been made to increase nuclease
resistance by chemical modi®cation of the oligonucleotide backbone,1,2 and we have recently
reported our own work in this area.3 In particular we have been interested in the synthesis of
nucleotides possessing a vinylphosphonate internucleotide linkage4 and we were able to show that
a thymidine dimer possessing this backbone modi®cation could be synthesised using a palladium
catalysed carbon±phosphorous coupling as a key step (Fig. 1).3,5 Using tetrakis(triphenylphosphine)-
palladium(0) as catalyst, triethylamine as base and DMF as the solvent, we were able to obtain
the modi®ed dimer 4 in a pleasing 42% yield. In this letter we now wish to describe further studies
in this area which have led to the development of a new coupling protocol and the synthesis of a
new range of interesting modi®ed nucleotide dimers.
Although we were pleased at the initial 42% yield for this coupling, we realised that it needed
to be improved upon if we were going to have a feasible route to modi®ed oligonucleic acids.
* Corresponding author. Tel: (0115) 951 3045; fax: (0115) 951 3564; e-mail: chris.hayes@nottingham.ac.uk
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00653-5

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Figure 1.
Upon examining the composition of the mass balance of the reaction it was obvious that the
major by-product formed was the secondary alcohol 5. The formation of 5 can easily be
accounted for by the hydrolysis of the H-phosphonate coupling partner 2. Control reactions
quickly con®rmed this hypothesis, as 2 was hydolysed to 5 in quantitative yield by treatment with
triethylamine in DMF at room temperature in less than one hour. This result was slightly con-
cerning as it appeared that decomposition of the H-phosphonate coupling partner was a rather
facile process under the coupling reaction conditions. In order to address this potentially serious
problem we needed to either stabilise the H-phosphonate towards hydrolysis or improve the rate
of the coupling reaction. Ideally we hoped to ®nd conditions to achieve both of these goals, but
we ®rst decided to address the problem of increasing the rate of coupling step. Over the last few years
there have been massive improvements in the area of transition metal-catalysed amine arylation
reactions.6 It has been found that sterically demanding and/or chelating ligands are especially
good in this type of transformation. Due to the obvious similarities between this and our carbon±
phosphorous coupling reaction, we wondered whether analogous changes to our catalyst would
result in a useful rate enhancement.
We therefore decided to examine the use of the catalyst generated in situ from commercially
available 1,10-bis(diphenylphosphino)ferrocene (dppf) and palladium acetate in re¯uxing THF.
Rather gratifyingly, we found that this combination of catalyst and solvent resulted in the formation
of the coupled product 4 in a much improved 78% yield. Changing the ligand to dppf and solvent
to THF also resulted in an unexpected simpli®cation of the isolation and puri®cation procedure.
After the reaction was judged complete by TLC analysis, the volatiles were removed in vacuo and
the crude residue was subjected to puri®cation by ¯ash column chromatography. When PPh3
was used as ligand, the puri®cation of 4 was rather laborious, as it was often contaminated with
the almost co-polar triphenylphosphine oxide. The use of dppf as ligand completely removed this
inconvenience, and 4 could be isolated in pure form from the initial isolation and puri®cation
procedure. With this success in hand we now wanted to test the scope and limitations of this
catalyst system. In order to do this we ®rst needed to synthesise a range of additional H-phosphonate
coupling partners. The H-phosphonates 8a±c were synthesised from the alcohol 5, via the phosphor-
amidites 7a±c, using standard procedures (Scheme 1).7
We were now in a position to examine the palladium-catalysed couplings of 8a±c with the vinyl
bromide 3. Firstly, 8a and 8b were coupled using the newly developed dppf/THF catalyst system
(the cyanoethanol-protected H-phosphonate 11 was not subjected to these reaction conditions
due to its sensitivity to amine bases). In both cases we were able to isolate the desired coupled
products 9 and 10, albeit in rather modest yields (21% and 24%, respectively, Fig. 2). We were

4515
Scheme 1.
somewhat disappointed with these results, especially since we were using the `improved' catalyst
system. Once again we isolated signi®cant amounts of the hydrolysis product 5 from the reaction
mixtures, and it seemed that the rate of coupling was slower than the rate of decomposition for
the more hindered H-phosphonates 8a and 8b. To overcome this problem we next examined the use
of an alternative HBr scavenger to replace the triethylamine. The use of propylene oxide for this
purpose has been reported previously in the synthesis of arylphosphonates and phosphinates,8
and this looked ideal for our purposes. We therefore repeated the coupling reactions of 8a and 8b
with 3, using 1.2 equivalents of propylene oxide in place of the 4.5 equivalents of triethylamine.
To our pleasure this resulted in increased yields of the desired coupled products 9 and 10 (53%
and 65%, respectively). Having eliminated the need for amine base in the reaction conditions, we
could now examine the coupling of 8c without fearing removal of the cyanoethyl protecting
group. Thus, 8c was coupled with 3 using the new coupling conditions (Pd(OAc)2, dppf, THF,
propylene oxide (1.2 equiv.), re¯ux) to yield the desired dimer 11 in excellent yield (72%).
Figure 2. Conditions: (a) Pd(OAc)2, dppf, THF, Á, 20 h, Et3N (4.5 equiv.); (b) Pd(OAc)2, dppf, THF, Á, 20 h, propylene
oxide (1.2 equiv.)
These results indicate that the dppf/THF/propylene oxide coupling conditions are well suited
to a fairly wide range of H-phosphonate coupling partners. Although we were very pleased with
these ®ndings, we were acutely aware that if this methodology was to ®nd any use in synthesising

4516
biologically important modi®ed nucleic acid sequences we would have to be able to access materials
containing purine as well as pyrimidine bases. As a preliminary study in this area we synthesised
the N-benzoyl-protected H-phosphonate 12 to act as a purine-containing coupling partner.9
We were delighted to ®nd that coupling of 12 with 3, using the latest evolution of our coupling
conditions (vide supra), gave the desired N-protected AT dimer 13 in an excellent 92% yield
(Scheme 2).
Scheme 2.
In summary, we have developed a convenient coupling strategy to allow rapid access to a range
of backbone modi®ed nucleic acids. The conditions outlined above seem to have some degree of
generality and are tolerant of a number of potentially sensitive functional groups. We have also
demonstrated for the ®rst time that a mixed purine±pyrimidine sequence can be synthesised in
high yield. We are currently developing this methodology further, and examining the chemistry
necessary to incorporate these modi®ed dimers into oligonucleotide sequences. The results of
these studies will be reported in due course.
Acknowledgements
We would like to thank the Nueld Foundation (NUF-NAL) and the School of Chemistry,
University of Nottingham for ®nancial support and The University of Nottingham for the provision
of a Ph.D. studentship (SA).
References
1. (a) Uhlmann, E.; Peyman, A. Chem. Rev. 1990, 90, 543; (b) Szymkowski, D. E. Drug Discovery Today 1996, 1,
415; (c) Englisch, U.; Gauss, D. H. Angew. Chem., Int. Ed. Engl. 1991, 30, 613.
2. (a) Of the more recent modi®cations, the protein nucleic acids (PNAs) are worthy of special note. For a
comprehensive review, see: Uhlmann, E.; Peyman, A.; Breipohl, G.; Will, D. W. Angew. Chem., Int. Ed. Engl.
TM
1998, 37, 2797; (b) The ®rst `antisense oligonucleotide' therapeutic (Vitravene ), based upon a phosphorothioate
internucleotide linkage, was approved by the FDA in August 1998 for treatment of CMV retinitis (for more details
please see the Isis pharmaceuticals web site at www.isip.com).

4517
3. Abbas, S.; Hayes, C. J. Synlett 1999, 1124.
4. See also: Zhao, Z.; Caruthers, M. H. Tetrahedron Lett. 1996, 37, 6239.
5. Hirao, T.; Masunaga, T.; Yamada, N.; Ohshiro, Y.; Agawa, T. Bull. Chem. Soc. Jpn. 1982, 55, 909.
6. (a) Hammann, B. C.; Hartwig, J. F. J. Am. Chem. Soc. 1998, 120, 7369; (b) Harris, M. C.; Geis, O.; Buchwald,
S. L. J. Org. Chem. 1999, 64, 6019 and references cited therein.
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Tamatsukuri, S.; Ikehara, M. Tetrahedron Lett. 1986, 27, 199; (c) Marugg, J. E.; Burik, A.; Tromp, M.; van der
Marel, G. A.; van Boom, J. H. Tetrahedron Lett. 1986, 27, 2271 and references cited therein.
8. Lei, H.; Stoakes, M. S.; Schwabacher, A. W. Synthesis 1992, 1255.
9. The H-phosphonate 12 was synthesised in a similar manner to the H-phosphonate 2, which we have reported
previously. The synthesis involves selective TBS-50-protection of commercially available N-benzoyl-20-deoxyadenosine,
coupling with N,N-diisopropylmethylphosphonamidic chloride and then tetrazole-mediated hydrolysis in methylene
chloride.