The utilization of secondary phosphine oxide synthons for the preparation of novel tertiary phosphine oxide modified DNA

Article abstract of DOI:10.1055/s-1998-1632

Novel tertiary phosphine oxides are described which act as masked secondary phosphine oxides. The protection of the reactive P-H bond allows generation of a carbanion alpha to phosphorus and subsequent alkylation with an oxetane. The free P-H can then be liberated with anhydrous acid. This functionality can then be further alkylated to generate novel tertiary phosphine oxide backbone modified DNA.

Full text of DOI:10.1055/s-1998-1632

March 1998
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283
The Utilization of Secondary Phosphine Oxide Synthons for the Preparation of Novel Tertiary
Phosphine Oxide Modified DNA
Stephen P Collingwood and Roger J Taylor
Novartis Pharmaceuticals Central Research, Hulley Road, Macclesfield, Cheshire, SK10 2NX, UK
Received 8 November 1997
Abstract: Novel tertiary phosphine oxides are described which act as
masked secondary phosphine oxides. The protection of the reactive P-H
bond allows generation of a carbanion alpha to phosphorus and
subsequent alkylation with an oxetane. The free P-H can then be
liberated with anhydrous acid. This functionality can then be further
alkylated to generate novel tertiary phosphine oxide backbone modified
DNA.
readily prepared by two alternative methods. Treatment of the ketal
protected hypophosphorous acid adduct 2 with methyl magnesium
4
chloride gave the protected methyl phosphine oxide 3 which was
readily methylated under basic conditions to give 1a in good yield.
Alternatively 1a could be prepared from the corresponding parent
5,7
phosphine oxide 4 by Lewis acid catalysed alkylation with triethyl
orthoacetate (Scheme 1). The latter route was also effective for the
preparation of the protected phosphine oxide 1b from phenyl methyl
8
phosphine oxide 5 which was prepared to demonstrate the flexibility of
our synthesis strategy with regard to the group R (Fig 2).
The utilisation of antisense oligonucleotides as therapeutics in man is
currently undergoing evaluation in several clinical trials, with particular
focus on cancer and viral targets. The oligomers under study are
1
mainly phosphorothioates, simple backbone modified DNA analogues,
which have been termed first generation antisense oligonucleotides.
Nevertheless the search for second generation antisense
oligonucleotides with increased nuclease stability, stronger binding to
complementary RNA, lower toxicity and higher bioavailability still
2
3
continues. In this letter we describe the replacment of three of the
phosphate oxygens in DNA with carbon, thus generating a phosphine
oxide. We hoped that such modified DNA might ameliorate stability to
nucleases, increase bioavailability and due to the known highly polar
nature of phosphine oxides generate an effective mimic of poly ionic
DNA.
Good general synthetic methods for the synthesis of highly
functionalised phosphine oxides are not available. We required a
methodology that would be relatively tolerant of functionality. Figure 1
shows our planned disconnection, utilizing two electrophilic nucleic
acid synthons and a novel masked secondary phosphine oxide reagent
such as the ketal protected derivative 1. We reasoned that, in analogy to
Scheme 1
4
related studies from this laboratory, a secondary phosphine oxide
reactive PH bond could be protected by alkylation with an orthoacetate
5
to produce a ketal. This group should be stable under neutral and basic
conditions but readily cleaved by acid to reveal the PH moiety ready for
subsequent reaction (Fig.2). This strategy has proved extremely
effective for the protection of PH functionality in phosphinate
6
derivatives.
Figure 2
A thymidine-thymidine dinucleotide was chosen as the initial target.
Related synthetic studies, in the area of phosphorus esters, from this
6
9
laboratory and in particular from Tanaka et al demonstrated the
effectiveness of thymidine oxetane 6 as the electrophilic component in
reactions with alpha phosphorus anions. We envisioned that this
chemistry might also be appropriate for phosphine oxides. Accordingly
o
reaction of both 1a and 1b with butyl lithium at -78 C in THF followed
by quenching with oxetane 6 under boron trifluoride catalysis provided
the required phosphine oxides 7a and 7b as essentially 1:1 mixtures of
diastereoisomers at phosphorus. The acid catalysed cleavage of the ketal
group to provide the free PH bond was then demonstrated by conversion
of 7b to the secondary phosphine oxide 8. This substrate readily
Figure 1
10
underwent base catalysed addition to the aldehyde 9
phenyl phosphine oxide dinucleotide 10 as
yielding the
mixture of 4
a
diastereoisomers. However for incorporation into oligonucleotides
inversion at C3’ of 7 was required. Mitsunobu reaction of 7a with
benzoic acid gave a moderate yield of the required product 11. The
We chose as our initial target the simplest possible trialkyl phosphine
oxide (R=Me, Fig 2) to minimise the steric difference to wild type
DNA. The ketal protected prochiral dimethyl phosphine oxide 1a was

284
LETTERS
SYNLETT
Scheme 2
reduced yield here was due to competitive 1,2-elimination. Cleavage of
the diethyl ketal with anhydrous acid gave the secondary phosphine
oxide 12 in good yield. Subsequent base catalysed addition to the
aldehyde 9, again readily allowed construction of the dinucleotide
skeleton, giving 13 as a mixture of four diastereoisomers. The earlier
Mitsunobu reaction provided the necessary differentiation of the
secondary alcohols. Thus selective Barton deoxygenation alpha to
phosphorus gave dinucleotide 14, both activation and reduction having
occurred in moderate unoptimised yield. One pure isomer of 14 was
readily obtained by flash silica chromatography at this stage. This
isomer and the remainder (as a 2:1 mixture favouring the other isomer)
were separately converted through established deprotection, protection
and activation protocols to give two samples of phosphoramidites 16
ready for oligonucleotide synthesis.
In conclusion we have developed a relatively direct synthesis of
phosphine oxide modified DNA. The strategy described herein should
be generally applicable to the synthesis of phosphine oxide modified
DNAs where the group R is varied (Figure 2). In addition the simple
protected phosphine oxide synthons which we have developed may
prove generally useful for the synthesis of functional phosphine oxides
for diverse applications.
Both samples of phosphoramidites 16 were separately incorporated into
11
two oligonucleotide sequences, whose identities were confirmed by
12
MALDI-TOF mass spectral analysis. The hybridisation of these
sequences to complementary RNA and DNA was assessed and the
13
results are presented in the Table. Both the pure and mixed isomers
bound to RNA and DNA significantly less strongly than wild type
DNA. Clearly the destabilisation is greater for binding to DNA than to
RNA. A large difference in binding affinity between isomers is also
suggested by this data which interestingly has also been observed for
Typical experimental procedures
The synthesis and synthetic application of the novel protected dialkyl
phosphine oxides are described here for reference.
14
methylphosphonates.

March 1998
SYNLETT
285
Secondary phosphine oxide synthon 1a
Acknowledgements
4
To a solution of methyl(1,1-diethoxyethyl)phosphine oxide 4 (89.3g,
We thank Dr C Schmit and Dr U Pieles (Novartis Basle) for the
synthesis of the phosphoramidites and their incorporation into nucleic
acid together with Dr D Hüsken (Novartis Basle) and Dr S M Freier
(ISIS Pharmaceuticals) for performing hybridisation experiments. We
also thank Dr A D Baxter for helpful discussions.
o
0.5mol) in THF (900ml) at -78 C under argon was added a solution of
KHMDS (688ml, 0.52mol) in toluene over 15min. After stirring for 1h
methyl iodide (34ml, 0.55mol) was added over 5min. After a further 1h
o
at -78 C the reaction was quenched by the addition of a 1% solution of
NaH PO (450ml) and the mixture allowed to slowly warm to ambient
2
4
temperature. The resulting mixture was partially concentrated,
dichloromethane was added (600ml) and the organic phase separated
References and Notes
(1) Szymkowski, D.E.; Drug Development Tech., 1996, 1, 415.
and washed with a 1% solution of NaH PO (500ml) followed by water
2
4
(2) Altmann, K.-H., Fabbro, D.; Dean, N. M.; Geiger, T.; Monia, B.
(500ml). Drying over MgSO , concentration and purification by silica
4
P.; Müller, M.; Nicklin, P. Biochem. Soc. Trans.,1996, 24, 630.
flash column chromatography (chloroform-ethanol 40:1) followed by
fractional distillation gave dimethyl (1,1-diethoxyethyl)phosphine oxide
(3) Baxter, A. D.; Baylis, E. K.; Collingwood, S. P.; Taylor, R. J.;
o
Weetman, J. EP 629633, 1994.
1a (41.7g, 43%) b.p 100 C at 0.05mmHg. Found C 49.1, H 10.2, P
31
1
16.1% C H O P requires C 49.5; H 9.85; N P 15.95%. P nmr
H
(4) Hall, R. G.; Riebli, P. EP 501702, 1991.
8
19 3
1
decoupled (CDCl , 36MHz) δ 48.3 ppm. H nmr (CDCl , 400MHz) δ
3
3
(5) Baylis, E. K.; Tetrahedron Lett., 1995, 36, 9385.
(6) Collingwood, S. P.; Baxter, A. D. Synlett, 1995, 703.
(7) Hays, H. R. J. Org. Chem., 1968, 33, 3690.
0.98 (3H, t, 7Hz), 1.28 (6H, d, 11Hz), 1.33 (3H, d, 10Hz), 3.48 (3H, q,
7Hz).
(8) Compound 5 was prepared by a directly analogous route to that
described by Xu, Y.; Wei, H.; Xia, J. Liebigs Ann. Chem., 1988,
1139.
Protected 5’-methylene secondary phosphine oxide 7a
To a solution of phosphine oxide 1a (11g, 56.6mmol) in dry THF
o
(100ml) at -78 C under argon was added n-butyl lithium (35ml, 1.6M in
(9) Tanaka, H.; Fukui, M.; Haraguchi, K.; Maskai, M.; Miyasaka, T.
hexanes) dropwise over 10min. After 2h boron trifluoride etherate (7ml,
57mmol) was added, immediately followed by the dropwise addition of
a solution of 1-(3,5-anhydro-β-D-threo-pentafuranosyl) thymine 6
Tetrahedron Lett., 1989, 30, 2567.
(10) Sanghvi, Y. S.; Ross, B.; Bharadwaj, R.; Vasseur, J-J.
Tetrahedron Lett., 1994, 35, 4697; Sanghvi, Y. S.; Bharadwaj, R.;
Debart, F.; De Mesmaeker, A. Synthesis, 1994, 1163.
o
(2.51g, 11.2mmol) in dry THF over 45min. After 2h at -78 C a
suspension of NaHCO (5g) in water (10ml) was added. The resulting
3
(11) Each oligonucleotide was prepared on an ABI 390 DNA
synthesiser using standard phosphoramidite chemistry according
to : Gait, M. J.; Oligonucleotide Synthesis: A Practical Aproach,
IRL Press, Oxford, 1984.
mixture was allowed to slowly warm to ambient temperature and was
then concentrated. Dichloromethane (200ml) and magnesium sulphate
were added the solid was separated by filtration and washed with
additional dichloromethane (4x100ml). Concentration of the combined
dichloromethane filtrate and washings followed by flash silica column
chromatography (gradient elution: ethyl acetate-ethanol 20:1-8:1) gave
compound 7a (2.1g, 45%) as a white foam, isolated as a 1:1 mixture of
(12) Pieles, U.; Zürcher, W.; Schär, M.; Moser, H. Nucleic Acids Res.,
1993, 21, 3191.
(13) The thermal denaturation of DNA/RNA duplexes was monitored
at 260 nm using a Gilford Response II spectrophotometer.
Absorbance vs temperature profiles were measured at 0.004mM
of each strand in 10mM phosphate pH 7.0 (Na Salts), 100mM
total (Na+) 0.1mm EDTA. Tm’s were obtained from fits of
absorbance vs. temperature curves to a two state model with linear
slope baselines (Freier, S. M.; Albrego.; T. D.; Turner, D. H.
Biopolymers, 1992, 22, 1107.) All values are averages of at least
1
31
diastereoisomers as judged from H and P nmr. Found C 49.4; H 7.8;
N 6.3; P 6.8%. C N O P.H O requires C 49.5; H 7.6; N 6.4; P
H
18 31
2
7
2
31
1
7.1%. P nmr H decoupled (CDCl , 162MHz) δ 54.6 and 53.2 ppm.
H nmr (CDCl , 400MHz) δ 1.19-1.27 (m, 6H), 1.49-1.60 (m, 6H),
3
1
3
1.83-1.90 (m, 1H), 1.91 (d, 3H, J 0.9Hz, isomer A), 1.93 (d, 3H, J
0.9Hz), 2.05 (dd, 1H, J 2.5, 15.0 Hz), 2.09 (dd, 1H, J 2.4, 15.1 Hz,
isomer A), 2.12-2.37 (m, 3H), 2.58-2.66 (m, 1H), 3.64-3.77 (m, 7H),
4.29-4.34 (m, 1H), 6.19 (dd, 1H, J 2.3, 8.8Hz, isomer A), 6.24 (dd, 1H, J
2.4, 8.8Hz), 7.85 (d, 1H, J 1.1Hz, isomer A), 7.90 (d, 1H., J 1.2Hz), 8.82
(s, 1H), 8.87 (s, 1H, isomer A).
o
three experiments. The absolute error of the Tm values is +-0.5 C.
(14) Lesnikowski, Z. L.; Jaworska, M.; Stec, W. J. Nucl. Acids Res.,
1990, 18, 2109; Milligan, J. F.; Matteucci, M. D.; Martin, J. C. J.
Med. Chem., 1993, 36, 1923.