A novel palladium-catalysed coupling strategy for the rapid synthesis of nucleic acid analogues bearing modified backbones

Article abstract of DOI:10.1055/s-1999-2767

The synthesis of a 5'-deoxy-5'-methylidene phosphonate-containing thymidine dimer 14 has been achieved using a palladium-catalysed cross coupling reaction as a key step. The E-vinyl bromide 9 was synthesised from the corresponding 1,1-dibro-moolefin 7 via

Full text of DOI:10.1055/s-1999-2767

1124
LETTER
A Novel Palladium-Catalysed Coupling Strategy for the Rapid Synthesis of
Nucleic Acid Analogues Bearing Modified Backbones
Sahar Abbas, Christopher J. Hayes*
The School of Chemistry, The University of Nottingham University Park, Nottingham, NG7 2RD, UK
Fax +44 (115) 951 3564; E-mail: Chris.Hayes@nottingham.ac.uk
Received 26 April 1999
nate 3 and a vinyl bromide 4 to form the crucial C6’-P
bond in the modified backbone (Figure 1).
Abstract: The synthesis of
a 5’-deoxy-5’-methylidene phos-
phonate-containing thymidine dimer 14 has been achieved using
a palladium-catalysed cross coupling reaction as a key step. The E-
vinyl bromide 9 was synthesised from the corresponding 1,1-dibro-
moolefin 7 via selective reduction using dimethylphosphite and
triethylamine.
N-Base
O
O
O
O
P
Key words: antisense, palladium coupling, vinyl bromide, oligonu-
cleotide, modified backbone
N-Base
O
O
O
O
O
R
O
O
R
P
O
N-Base
O
5'
N-Base
The ability of small nucleic acid fragments to regulate the
production of gene products (i.e. RNA and proteins) has
stimulated a considerable amount of research into their
possible use in the treatment of a variety of diseases (e.g.
HIV and cancer).1 The antisense approach relies upon the
binding of a short (antisense) oligonucleotide drug to a
(sense) RNA target to form a stable supramolecular com-
plex.2 As a result of forming this complex, the process of
translation is disrupted and protein production from the
target RNA transcript is inhibited. At first glance, natural-
ly occurring oligonucleotides appear to be ideal drug can-
didates, especially as it has been known for sometime that
bacteria are capable of regulating gene expression by us-
ing antisense RNA molecules.3 Unfortunately, the pres-
ence of the internucleoside phosphodiester group 1
(Figure 1) renders naturally occurring nucleic acids sus-
ceptible to degradation by a number of nuclease enzymes.
In order to increase their nuclease resistance, a number of
modifications to the phosphodiester backbone have been
made. These modifications have ranged from replacing
one or both of the non-bridging oxygens with sulfur, giv-
ing phosphorothioates and phosphorodithioates respec-
tively, all the way to replacing the sugar-phosphate
backbone completely in oligomers such as the protein nu-
cleic acids (PNAs).4
O
6'
R=H (DNA), OH (RNA)
O
2
1
Palladium (0)
catalysed
coupling
N-Base
RO
O
Br
N-Base
O
+
O
O
P
RO
R'O
H
3
4
Figure 1
The E-vinyl bromide 9 was synthesised in good overall
yield from commercially available thymidine in 6 steps
(Scheme 1). Selective protection of the primary alcohol of
thymidine gave the corresponding 5’-TBDMS silyl ether 5
in high yield.7 TBDPS protection of the resulting second-
ary alcohol, followed by treatment with HF (48% aq) in
acetonitrile next provided the primary alcohol 6. Oxida-
tion of 6 with Dess-Martin periodinane8 then afforded the
aldehyde 8. A number of alternative oxidising agents
(Swern, SO3◊pyridine:DMSO, PDC) were examined in or-
der to effect this transformation, but the results obtained
by using the periodinane reagent were found to be superi-
or. Homologation of 8 to the 1,1-dibromo olefin 7 was
next achieved using the procedure of Ramirez et. al.9 Ste-
reoselective reduction of 7 using dimethylphosphite in the
presence of triethylamine10 yielded the desired E-vinyl
bromide 9 and the Z-vinyl bromide 10 as a separable 3:1
mixture in 84% yield.
In this letter, we wish to describe our own studies in this
area which have been directed towards replacing the hy-
drolytically labile phosphodiester linkage with a 5’-
deoxy-5’-methylidene phosphonate group 2 (Figure 1).
The synthesis of a thymidine dimer containing this modi-
fication has been reported recently by Caruthers and
Zhao,5 and they have shown that oligomers containing
this modification are significantly stabilised towards en-
zymatic degradation by snake venom phosphodiesterase.
In contrast to the route developed by Caruthers and Zhao,
we planned to utilise a palladium-catalysed coupling
reaction6 between a suitably functionalised H-phospho-
The H-phosphonate coupling partner 12 was synthesised
in two steps from the TBDMS-protected thymidine 5. Re-
Synlett 1999, No. 07, 1124–1126 ISSN 0936-5214 © Thieme Stuttgart · New York

LETTER
A Novel Palladium-Catalysed Coupling Strategy
1125
O
O
O
O
NH
NH
O
NH
O
NH
O
N
N
N
N
TBSO
TBSO
TBSO
HO
O
O
O
O
O
i
i, ii
HO
O
HO
TBDPSO
P
NiPr2
5
6
MeO
5
11
iii
ii
O
O
O
O
NH
NH
Br
Br
NH
O
NH
N
iv
O
N
O
N
N
O
O
TBSO
TBSO
O
O
P
O
O
TBDPSO
TBDPSO
O
MeO
O
O
7
8
P
OH
MeO
H
v
12
13
O
O
Reagents: i, iPr2NP(OMe)Cl, Et3N, DMAP, CH2Cl2 (87%); ii, tetrazole,
H2O, MeCN (88%)
NH
O
NH
O
Br
Scheme 2
N
N
+
O
O
Br
coupling reaction as well as developing procedures for the
incorporation of these modified nucleotides into oligode-
oxyribonucleotide sequences, which will be suitable for
biological evaluation.
TBDPSO
TBDPSO
9
10
Reagents: i, TBDPS-Cl, imidazole, DMF (90%); ii, HF (48% aq), MeCN
(88%); iii, Dess-Martin periodinane, CH2Cl2 (80%); iv, CBr4, Ph3P,
CH2Cl2 (63%); v, (MeO)2P(O)H, Et3N, DMF (3:1, trans:cis 84%)
Scheme 1
O
NH
action of 5 with N,N-diisopropylmethylphosphonamidic
chloride11 produced a 1:1 diastereomeric mixture of the
phosphoramidite 11. Tetrazole-mediated hydrolysis of 11
then yielded the desired H-phosphonate 12,12 again as a
1:1 mixture of diastereoisomers at phosphorus. Unfortu-
nately, the diastereoisomers could not be separated at this
stage and the 1:1 mixture was used in the subsequent cou-
pling reaction.
N
TBSO
O
P
O
Pd(OAc)2, Ph3P,
O
+
9
12
O
O
Et3N, DMF, 70°C
(42%)
NH
O
MeO
N
O
TBDPSO
14
With the two coupling partners in hand we were in a posi-
tion to examine the key palladium-catalysed coupling re-
action. To our delight, when one equivalent of the vinyl
bromide 9 was reacted with two equivalents of the H-
phosphonate 12 in the presence of Pd(PPh3)4 (generated in
situ from Pd(OAc)2 and PPh3) the desired coupled product
14 was obtained in a pleasing 42% isolated yield (1:1 mix-
ture of separable diastereoisomers at phosphorus).13 The
presence of characteristic 1H-31P coupling constants in the
1H NMR spectrum of 14 clearly showed that the C6’-P
bond had been formed during the coupling reaction and
1H-1H coupling constants confirmed that the E-olefin ge-
ometry had been retained in the coupled product. High
resolution FAB mass spectrometry showed a molecular
ion of 909, thus confirming the identity of 14. We are cur-
rently examining methods to improve the yield of this
Scheme 3
In summary, we have demonstrated that the synthesis of
5’-deoxy-5’-methylidene phosphonate backbone-modi-
fied dinucleotides can be achieved using a novel palladi-
um-catalysed carbon-phosphorus coupling reaction as a
key stratagem. We have also shown that the coupling part-
ners (viz. 9 and 12) are readily available in good overall
yield in only a few steps from commercially available
starting materials. The convergent nature and mild condi-
tions employed for the synthesis of 14 should allow access
to a range of backbone-modified nucleotides and not just
Synlett 1999, No. 07, 1124–1126 ISSN 0936-5214 © Thieme Stuttgart · New York

1126
S. Abbas, C. J. Hayes
LETTER
(34 mg, 0.13 mmol) was added to a stirring solution of
palladium (II) acetate (7.2mg, 0.032 mmol) in DMF (1mL)
and the resulting mixture was stirred for 20 minutes.
Triethylamine (100mL, 0.73 mmol), a solution of the vinyl
bromide 9 (90 mg, 0.16 mmol) in DMF (1 mL) and a solution
of the H-phosphonate 12 (141 mg, 0.32 mmol) in DMF (1 mL)
were then added and the resulting yellow solution was heated
at 70 °C for 20 hrs. After cooling to room temperature, ethyl
acetate (20 mL) and water (20 mL) were added and the
separated aqueous phase was washed with ethyl acetate
(3 x 20 mL). The combined organic layers were washed with
brine (20 mL) and dried (MgSO4). Removal of the solvent in
vacuo and purification of the residue by column
thymidine dimers. The results of our studies in this area
and the incorporation of these materials into oligonucle-
otides will be published in due course.
Acknowledgement
We would like to thank the Nuffield Foundation (NUF-
NAL) and the School of Chemistry, University of Not-
tingham for financial support and The University of Not-
tingham for the provision of a Ph.D. studentship (SA).
chromatography (SiO2, ethyl acetate) provided the desired
product 14 as a 1:1 mixture of separable diastereoisomers (62
mg, 42%): IR (CHCl3 soln) 3392, 2956, 2991, 2859, 1694,
1591, 1464, 1438, 1364, 1322, 1276, 1120, 1049, 1000, 976,
908, 939, 648 cm-1; least polar isomer: Rf 0.30 (ethyl acetate);
1H NMR(CDCl3, 500 MHz) d 8.84 (s, 1H), 8.74 (s, 1H), 7.70-
7.38 (m, Ar, 11H), 6.99 (s, 1H), 6.55 (ddd, J 23, 17, 5 Hz, 1H),
6.33 (dd, J 9, 5 Hz, 1H), 6.22 (dd, J 8, 6 Hz, 1H), 5.73 (ddd, J
20, 17, 2 Hz, 1H), 5.01 (app t, J 6 Hz, 1H), 4.48 (m, 1H), 4.35
(m, 1H), 4.20, (m, 1H), 3.90 (dd, J 11, 3 Hz, 1H), 3.84 (dd, J
11, 3 Hz, 1H), 3.65 (d, J 11 Hz, 3H), 2.48 (dd, J 14, 5 Hz, 1H),
2.24 (ddd J 14, 6, 3 Hz, 1H), 2.20 (m, 1H), 2.01 (ddd, J 14, 9,
5 Hz, 1H), 1.92 (s, 3H), 1.89 (s, 3H), 1.10 (s, 9H), 0.93 (s, 9H),
0.12 (s, 6H); most polar isomer: Rf 0.22 (ethyl acetate) 1H
NMR(CDCl3, 500 MHz) d 8.33 (s, 1H), 8.28 (s, 1H), 7.70-
7.41 (m, Ar, 11H), 6.93 (s, 1H), 6.52 (ddd, J 23, 17, 5 Hz, 1H),
6.41 (dd, J 8, 6 Hz, 1H), 6.37 (dd, J 9, 5 Hz, 1H), 5.70 (ddd, J
20, 17, 2 Hz, 1H), 5.01 (app t, J 7 Hz, 1H), 4.48 (m, 1H), 4.28
(m, 1H), 4.16, (m, 1H), 3.87 (dd, J 12, 2 Hz, 1H), 3.81 (dd, J
12, 2 Hz, 1H), 3.68 (d, J 11 Hz, 3H), 2.50 (dd, J 14, 5 Hz, 1H),
2.30 (ddd J 14, 6, 3 Hz, 1H), 2.11 (m, 1H), 1.98-1.90
References and Notes
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(obscured m, 1H), 1.93 (s, 3H), 1.88 (s, 3H), 1.10 (s, 9H), 0.93
(s, 9H), 0.12 (s, 3H), 0.12 (s, 3H); HRMS (FAB positive ion)
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Article Identifier:
1437-2096,E;1999,0,07,1124,1126,ftx,en;L06399ST.pdf
Synlett 1999, No. 07, 1124–1126 ISSN 0936-5214 © Thieme Stuttgart · New York