Stereoselective synthesis of highly functionalised P-stereogenic nucleosides via palladium-catalysed P-C cross-coupling reactions

Article abstract of DOI:10.1016/j.tetlet.2008.09.102

We have shown that the configuration at the P-stereogenic centre in a range of H-phosphonates can be assigned using a combination of chromatographic mobility, ¹H and ³¹P NMR data. We have also shown that the individual P-stereoisomers can be cross-coupled with vinyl and aryl halides, in the presence of a palladium(0) catalyst, to afford the corresponding vinyl- and arylphosphonates in a stereocontrolled manner.

Full text of DOI:10.1016/j.tetlet.2008.09.102

Tetrahedron Letters 49 (2008) 6984–6987
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Stereoselective synthesis of highly functionalised P-stereogenic nucleosides
via palladium-catalysed P–C cross-coupling reactions
*
Benjamin Whittaker, Manuel de Lera Ruiz, Christopher J. Hayes
School of Chemistry, University of Nottingham, University Park, Nottingham, NG7 2RD, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
We have shown that the configuration at the P-stereogenic centre in a range of H-phosphonates can be
assigned using a combination of chromatographic mobility, 1H and 31P NMR data. We have also shown
that the individual P-stereoisomers can be cross-coupled with vinyl and aryl halides, in the presence
of a palladium(0) catalyst, to afford the corresponding vinyl- and arylphosphonates in a stereocontrolled
manner.
Received 13 August 2008
Accepted 17 September 2008
Available online 20 September 2008
Ó 2008 Elsevier Ltd. All rights reserved.
The ability to modulate gene function in a sequence-specific
manner at either the RNA (antisense,1 RNAi2) or the DNA (anti-
gene)3 level has stimulated much interest in the synthesis and bio-
been observed previously for other modified oligomers that bear
P-stereogenic centres.11 We now wish to report our initial findings
on the development of such a method, which allows stereocon-
trolled access to P-stereogenic vinylphosphonates using palla-
dium-catalysed P–C cross-coupling as a key step.
It is well know that the stereogenic centre in P-chiral H-phos-
phonates is configurationally stable at room temperature,12
and that the P–H bond can be oxidised with overall retention of
logical evaluation of
a wide range of nucleic acid-derived
oligomers. Many modifications to the natural phosphodiester oli-
gonucleic acid backbone have been examined in order to overcome
problems associated with nuclease degradation, and significant
progress has been made towards increasing their useful lifetime.4
Our own studies in this area have focussed upon replacing the
internucleotide phosphodiester linkage with a nuclease-resistant
vinylphosphonate moiety 1,5 and we have used these materials
as probes with which to study helicase,6 nuclease7 and polymerase
activity7 (Scheme 1). The vinylphosphonate internucleotide modi-
fication was incorporated into oligomers by using suitably modi-
fied dinucleotide phosphoramidite reagents 3, which in turn
were synthesised from the corresponding H-phosphonate 4 and
vinyl bromide 5 building blocks via palladium(0)-catalysed P–C
cross-coupling8 (Scheme 2).
O
O
NH
O
NH
O
N
N
O
O
O
P
O
P
O
O
O
O
O
O
O
O
NH
O
NH
O
R
R3N
N
N
O
n
O
Neutral
Cationic
internucleotide
internucleotide
linkage
linkage
O
O
1
2
As a prelude to examining the potential therapeutic applica-
tions of these modified oligonucleotides, we wished to explore
the possibility of synthesising neutral 19 and cationic 210 versions
of the vinylphosphonate internucleotide linkage as these may be
better candidates for efficient transport across cellular membranes.
In principle, we could easily prepare neutral versions by using
base-stable alkyl groups on the H-phosphonate in place of the cya-
noethyl-protecting group used previously, and cationic versions
could be prepared in a similar way using pendant groups that
could be protonated under physiological conditions (i.e., amines
and guanidines).10 As the target phosphonate diesters possess a
P-stereogenic centre, which will be retained in the final oligomer,
we needed to develop a method for accessing single P-stereoiso-
mers of these compounds, as different biological activities had
O
O
NH
O
NH
O
N
O
DMTO
N
O
O
P
DMTO
O
O
O
RO
Pd(0), ligand
base
O
P
NH
O
RO
H
N
4
O
O
Br
NH
O
3
O
N
O
P
NiPr2
O
NC
RO
5
, R = TBDPS
6, R = H
* Corresponding author. Tel.: +44 0115 951 3045; fax: +44 0115 951 3564.
E-mail address: chris.hayes@nottingham.ac.uk (C. J. Hayes).
Scheme 1. Retrosynthesis of vinylphosphonate-linked oligonucleotides.
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.09.102

B. Whittaker et al. / Tetrahedron Letters 49 (2008) 6984–6987
6985
pure H-phosphonates. Although we could separate the two
isomers, we did not know at this stage which was the RP and which
was the SP configured material, so our next task was to assign the
absolute configuration at the P-stereogenic centre.
O
P
R1O
R2O
L
L
Br
R3
Pd
R3
reductive
oxidative
insertion
elimination
As we knew of no direct method for assigning the stereochem-
istry at phosphorus, we decided to oxidise the H-phosphonates to
the corresponding phosphorothioates, 11 and 12, and then corre-
late these with the same materials produced via an independent,
stereocontrolled route. For this purpose, we used the P-chiral in-
dole-substituted phosphoramidite 13 of Just17 as our starting
material, as this had been used previously to prepare P-chiral
phosphorothioates of known stereochemistry. Thus, Just’s phos-
phoramidite 13 was prepared according to the known procedure17
in good yield, and nucleophilic substitution with the chosen
alcohol then gave the corresponding phosphites, which were
immediately oxidised with Beaucage’s reagent18 to give the
phosphorothioate diesters 14 with retention of stereochemistry.
Elimination of the chiral auxiliary with DBU finally gave the phos-
phorothioates 11 as single SP stereoisomers, whose 1H and 31P NMR
data were then compared to those obtained for the equivalent
compounds produced previously via oxidation of the H-phospho-
nates 9 and 10 (Scheme 3).
It was found that the data for phosphorothioates 11 (produced
from the ‘Fast’ H-phosphonates 9) were identical to those produced
for the same compounds prepared from Just’s phosphoramidite 13,
thus confirming them as the SP stereoisomers. As it is known that
oxidation proceeds with retention of stereochemistry, we could as-
sign the precursor ‘Fast’ H-phosphonates 9 as possessing the RP
absolute configuration,19 and also the remaining ‘Slow’ H-phos-
phonates 10 as having the SP configuration. It is striking that in
all cases the RP configured H-phosphonates have higher chromato-
graphic mobility (denoted ‘Fast’) than the SP configured materials
(denoted ‘Slow’).20 Inspection of the 1H and 31P NMR data for the
RP and SP H-phosphonates also revealed characteristic trends with-
in the two stereochemical series. For example, the chemical shift of
the P–H 1H resonance was always at higher field for the RP series
than the equivalent proton in the SP series. Also the 31P resonance
was always at higher field in the RP series than it was in the SP ser-
ies. It thus appears that a combination of chromatographic mobil-
ity, 1H and 31P NMR data can be used to assign the absolute
configuration of the P-stereogenic centre (Table 1).21
O
P
OR2
OR1
L
L
L
Br
Pd
L
Pd
R3
R3
O
P
OH
P
Base HBr
R1O
R1O
R2O
H
R2O
Scheme 2. Proposed catalytic cycle for P–C cross-coupling.
stereochemistry.13 Furthermore, it has been shown that P-chiral
phosphinates14 and phosphine–borane complexes15 undergo tran-
sition metal-mediated cross coupling reactions with overall reten-
tion of stereochemistry at phosphorus. Thus, as a starting point for
our own work, we decided to explore the use of single H-phospho-
nate P-stereoisomers in the palladium-catalysed cross-coupling
reaction, as this should afford single isomers of the desired vinyl-
phosphonate products (Scheme 2).
A range of H-phosphonates were synthesised by either hydroly-
sis of the corresponding phosphoramidite,16 or sequential substitu-
tion of PCl3 with 50-OTBS-thymidine 7, an appropriate alcohol and
then water (Scheme 3). A 50:50 mixture of (RP)-9 and (SP)-10 iso-
mers was obtained during this reaction, and these were separated
using flash column chromatography to afford the stereochemically
O
O
NH
O
NH
O
(i) PCl3, py
(ii) ROH, py
N
TBSO
RO
N
TBSO
O
O
(iii) H2O
(54-72%)
P
O
HO
7
8
HO
a, R = PhthN(CH2)5; b, R = 5-hexenyl
c, R = 5-hexynyl; d, R = o-nitrobenzyl
Having determined the stereochemistry at the P-stereogenic
centre of some H-phosphonates, we were ready to examine their
behaviour in palladium-catalysed P–C cross-coupling reactions.
Thus, each diastereoisomer of the phthalimide-containing H-phos-
phonates (RP)-9a and (SP)-10a was reacted with the thymidine-de-
rived vinyl bromide 55 using a catalyst system derived from
Pd(OAc)2, Ph3P and propylene oxide (Scheme 4).
Pleasingly, the desired vinylphosphonates (SP)-15 (67%) and
(RP)-16 (50%) were isolated as single diastereoisomers (Scheme
4). In order to demonstrate the potential utility of the phthaloyl-
protected amines, each was exposed to hydrazine hydrate in
O
O
NH
O
NH
TBSO
N
O
N
O
TBSO
O
P
O
+
O
O
O
P
RO
RO
X
X
(SP
)
(RP)
9, X = H
(Slow) 10, X = H
(Fast)
)
S8, pyridine
(RP)
(SP
12, X = SH
11, X = SH
Table 1
O
O
DBU
Entry
Compound
Mobilitya
1H d ppmb (P–H)
31P d ppmc (P–H)
RP/SP
NH
O
NH
1
2
3
4
5
6
7
8
9a
10a
9b
10b
9c
10c
9d
Fast
Slow
Fast
Slow
Fast
Slow
Fast
6.86
6.89
6.83
6.86
6.83
6.87
7.06
7.09
8.2
8.4
7.9
8.1
7.9
8.1
8.5
8.6
RP
SP
RP
SP
RP
SP
RP
SP
TBSO
N
TBSO
N
O
O
O
(i) DBU, ROH,
THF
NC
P
O
P
O
O
(R)
N
RO
S
NC
O
(ii) Beaucage's
reagent
13
NH
14
10d
Slow
(48-70%)
a
Relative chromatographic mobility on SiO2 gel.
500 MHz, CDCl3.
121.5 MHz, CDCl3.
b
c
Scheme 3. Synthesis and stereochemical assignment of H-phosphonates.

6986
B. Whittaker et al. / Tetrahedron Letters 49 (2008) 6984–6987
O
O
NH
NH
O
N
O
TBSO
O
P
N
O
Pd(OAc)2, Ph3P,
propylene oxide
TBSO
O
O
P
O
O
O
5
, THF
O
O
O
N
NH
O
O
N
H
N
O
O
O
10a, (SP)
9a, (RP)
15, (SP) 67%
TBDPSO
16
, (RP) 50%
H2N-NH2,
MeOH
H2N-NH2,
MeOH
O
O
NH
O
NH
O
N
N
O
TBSO
TBSO
O
P
O
O
O
O
O
O
P
NH
O
NH
O
O
O
H2N
H2N
N
N
O
O
18
17
TBDPSO
TBDPSO
Fmoc-Arg(Pbf)-OH
EDCI, HOBt
Fmoc-Arg(Pbf)-OH
EDCI, HOBt
O
O
NH
N
NH
NH
NH
N
O
TBSO
TBSO
O
O
O
P
O
HN
NHPbf
HN
N
NHPbf
O
O
O
O
O
O
O
P
NH
O
NH
O
O
O
N
H
Fmoc
N
H
Fmoc
N
O
N
N
H
O
H
20
19
TBDPSO
TBDPSO
50%, 2 steps
66%, 2 steps
Scheme 4. Stereospecific palladium-catalysed cross-coupling reactions.
methanol to facilitate deprotection to afford the corresponding pri-
mary amines (SP)-17 and (RP)-18 (Scheme 4). As the primary
amines were quite difficult to handle due to their polarity, we
decided to show that they could be further derivatised via pep-
tide-bond formation. Thus, conjugation of 17 and 18 with Fmoc-
Arg(Pbf)-OH22 gave the corresponding amides 19 and 20 in good
yields for the two-step procedure.
Stimulated by this success, and with the obvious potential to
use this chemistry to prepare nucleic acid-peptide conjugates, we
next explored the direct cross-coupling of nucleotide and amino
acid-derived coupling partners. Thus, the hexenyl-substituted H-
phosphonate (RP)-9b was cross-coupled with the protected p-iod-
ophenylalanine derivative 21 to afford the nucleic acid-amino acid
hybrid 22 (73%) as a single stereoisomer (Scheme 5).
We concluded this study by performing a cross-coupling reac-
tion between the phthalimide-containing H-phosphonate (RP)-9a
and the thymidine-derived vinylbromide 6, which contains an
unprotected 30-OH group. As expected, the desired vinylphos-
phonate 23 was isolated in good yield (80%) as a single isomer,
and this material was then converted into its corresponding
O
O
NH
NH
N
O
TBSO
N
O
TBSO
O
P
O
P
O
O
Pd(OAc)2, Ph3P,
propylene oxide
O
O
THF, 73%
O
O
H
I
9b
O
22
OMe
O
N
OMe
H
Boc
21
N
H
Boc
Scheme 5. Synthesis of nucleic acid-amino acid hybrid.

B. Whittaker et al. / Tetrahedron Letters 49 (2008) 6984–6987
6987
O
NH
N
O
TBSO
O
P
O
O
Pd(OAc)2, Ph3P,
propylene oxide
O
NH
O
O
N
O
TBSO
O
P
O
N
THF, 80%
H
9a
O
O
O
O
O
NH
O
O
N
Br
NH
O
N
O
N
O
O
23
HO
HO
O
6
1H-tetrazole, 24
NH
N
CH2Cl2, 80%
TBSO
O
O
NiPr2
P
O
O
NC
NiPr2
O
O
O
P
NH
O
24
O
N
N
O
P
O
25
O
NiPr2
O
NC
Scheme 6. Preparation of a vinylphosphonate-containing phosphoramidite reagent for oligonucleotide synthesis.
7. Doddridge, Z. A.; Bertram, R. D.; Hayes, C. J.; Soultanas, P. Biochemistry 2003, 42,
3239.
8. Hirao, T.; Masunaga, T.; Yamada, N.; Ohshiro, Y.; Agawa, T. Bull. Chem. Soc. Jpn.
1982, 55, 909.
phosphoramidite 25 using the standard procedure (Scheme 6). We
are now studying the incorporation of these modified nucleotides
into oligomers, and we will report our findings on this work in
due course.
In conclusion, we have shown that the configuration at the P-
stereogenic centre in a range of H-phosphonates can be assigned
using a combination of chromatographic mobility, 1H and 31P
NMR data. We have also shown that the individual P-stereoisomers
can be cross-coupled with vinyl- and aryl halides, in the presence
of a palladium(0) catalyst, to afford the corresponding vinyl- and
arylphosphonates in a stereocontrolled manner.
9. (a) Micklefield, J.; Fettes, K. J. Tetrahedron 1998, 54, 2129; (b) Fettes, K. J.;
Howard, N.; Hickman, D. T.; Adah, S. A.; Player, M. R.; Torrence, P. F.;
Micklefield, J. Chem. Commun. 2000, 765; (c) Blättler, M. O.; Wenz, C.; Pingoud,
A.; Benner, S. A. J. Am. Chem. Soc. 1998, 120, 2674; (d) Richert, C.; Roughton, A.
L.; Benner, S. A. J. Am. Chem. Soc. 1996, 118, 4518; (e) Roughton, A. L.; Portmann,
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Acknowledgements
11. (a) Thiviyanathan, V.; Vyazovkina, K. V.; Gozansky, E. K.; Bichenchova, E.;
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12. Johansson, T.; Stawinski, J. Bioorg. Med. Chem. Lett. 2001, 9, 2315.
13. Seela, F.; Kretschmer, U. J. Org. Chem. 1991, 56, 3861.
We thank the EPSRC (EP/D50368X/1) and The University of
Nottingham for financial support of this work.
14. (a) Xu, Y.; Wei, H.; Zhang, J.; Huang, G. Tetrahedron Lett. 1989, 30, 949; (b)
Zhang, J.; Xu, Y.; Huang, G.; Guo, H. Tetrahedron Lett. 1988, 29, 1955; (c) Xu, Y.;
Zhang, J. J. Chem. Soc., Chem. Commun. 1986, 1606.
15. (a) Al-Masum, M.; Kumaraswamy, G.; Livinghouse, T. J. Org. Chem. 2000, 65,
4776; (b) Oshiki, T.; Imamoto, T. J. Am. Chem. Soc. 1992, 114, 3975.
16. For the synthesis of H-phosphonates, including the o-nitrobenzyl-substituted
derivative 8d, using this method see Ref. 5b.
17. Wang, J.-C.; Just, G. J. Org. Chem. 1999, 64, 8090.
18. Iyer, R. P.; Egan, W.; Regan, J. B.; Beaucage, S. L. J. Am. Chem. Soc. 1990, 112,
1253.
19. It is important to note that no inversion occurs during this process, and the
apparent change in absolute configuration is simply due to the order of the
group priorities used in the CIP rules.
20. Similar chromatographic mobility trends had been observed previously for P-
stereogenic H-phosphonates. See Ref. 13.
21. NMR data previously had been used in a similar manner to assign configuration
at phosphorus: (a) Farnham, W. B.; Murray, R. K., Jr.; Mislow, K. J. Am. Chem.
Soc. 1970, 92, 5809; (b) Lewis, R. A.; Korpiun, O.; Mislow, K. J. Am. Chem. Soc.
1968, 90, 4847.
22. Fmoc-Arg(Pbf)-OH is commercially available; pbf = 2,2,4,6,7-pentamethyldi-
hydrobenzofuran-5-sulfonyl.
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