Nucleic acid containing 3′-C-P-N-5′ ethyl phosphonamidate ester and 2′-methoxy modifications in combination; synthesis and hybridisation properties

Article abstract of DOI:10.1055/s-2002-25332

The preparation of thymidine-thymidine and thymidine-5-methylcytidine dinucleosides containing a 3′-C-P-N-5′ ethyl phosphonamidate ester linkage, with defined phosphorus stereochemistry, in combination with a 2′-methoxy substituent in the lower sugar residue, is described. Incorporation of these dinucleosides into DNA oligonucleotides and the effect upon duplex stability with complimentary RNA is reported.

Full text of DOI:10.1055/s-2002-25332

LETTER
763
Nucleic Acid Containing 3 -C-P-N-5 Ethyl Phosphonamidate Ester and
2 -Methoxy Modifications in Combination; Synthesis and Hybridisation
Properties
Nucleic
A
cidCon
o
taining 3-
C
b
-P-N
-
5
E
thy
i
l
Phosp
n
honamidate
E
ste
r
A. Fairhurst,*1 Stephen P. Collingwood,1 David Lambert, Elke Wissler
Novartis Pharmaceuticals, Central Research Laboratories, Hulley Road, Macclesfield, Cheshire, SK10 2NX, UK
Fax +44(1403)323307; E-mail: robin.fairhurst@pharma.novartis.com
Received 29 January 2002
achieve this the incorporation of a 2 -electronegative sub-
stituent has been well described as an effective route to the
desired ribose conformation.7 In particular, 2 -methoxy
substituents have been demonstrated to be well suited
Abstract: The preparation of thymidine-thymidine and thymidine-
5-methylcytidine dinucleosides containing a 3 -C-P-N-5 ethyl
phosphonamidate ester linkage, with defined phosphorus stere-
ochemistry, in combination with a 2 -methoxy substituent in the
lower sugar residue, is described. Incorporation of these dinucleo- to this role, both individually,8 and as rationalised above,
sides into DNA oligonucleotides and the effect upon duplex stabil-
ity with complimentary RNA is reported.
in combination with both a 4 -C-aminomethyl sugar
modification and a carboxamide backbone linkage.9 In
Key words: antisense, modified oligonucleotides, nucleosides,
phosphorus, stereoselective
this letter we present our findings for combination of
the 3 -C-P-N-5 ethyl phosphonamidate ester linkage and
2 -methoxy modifications, and extend the scope of inves-
tigation beyond linking T-T residues (Figure).
Significant effort continues to be expended on the identi-
fication of modified oligonucleotides for antisense appli-
cations.2 The primary focus being upon: enhancing
nuclease resistance, increasing the affinity for comple-
mentary RNA and improving cellular uptake by reducing
the overall charge. Changes to all of the nucleic acid struc-
tural elements, base, sugar and phosphate linkage, have
been investigated individually, and in a smaller number of
instances, in combination.3 Through the appropriate amal-
gamation of stabilising modifications, oligonucleotides
have been produced with impressive affinities for their
RNA targets. Such modifications are particularly effec-
tive for the flanking sequences of gapmer oligonucle-
otides, where they complement a central unmodified, or
phosphorothioate, RNase H supporting region.4 In these
applications the flanking modifications are primarily re-
sponsible for the overall stability and affinity of the anti-
sense oligonucleotide. Previously we have described
DNA incorporating thymidine-thymidine (T-T) dimers
modified with the neutral 3 -C-P-N-5 (RP)-ethyl phos-
phonamidate ester linkage 1, which stabilise hybrid du-
plexes to the extent of Tm +0.9 °C per modification.5,6
The enhanced hybridisation properties are rationalised as
arising from a 3 -endo conformational preference in the
upper sugar of the modified dinucleoside promoting the
formation of a more stable A-type hybrid duplex. To en-
hance the affinity of oligonucleotide sequences contain-
ing dinucleosides with this linkage further, the incor-
poration of a complementary 3 -endo conformational bias
in the lower sugar residue would be anticipated to rein-
force the preference for an A-type duplex. As a way to
Figure
To evaluate the outcome of combining the two modifica-
tions fully our initial approach was to prepare both dia-
stereoisomeric 3 -C-P-N-5 ethyl phosphonamidate ester
T-T dinucleosides substituted with a 2 -methoxy moiety
in the lower sugar ring 2. A sequence involving a
Staudinger reaction, between nucleosides bearing H-
phosphinate and azide functionality, in the key dinucleo-
side forming step was selected, and is shown in Scheme 1.
To initiate the sequence, the previously described 3 -ho-
mologated H-phosphinate 3 was employed as a 1:1
mixture of diastereo-isomers epimeric at phosphorus.5
The remaining coupling partner 5 -azido-5 -deoxy-2 -
methoxythymidine 4 was prepared from 2 -methoxythy-
midine following reported procedures.5,10 Thus, activation
of the H-phosphinate 3 with bis(trimethylsilyl)trifluoroac-
etamide (BSTFA) facilitated reaction with azide 4 to pro-
duce the dinucleoside diastereoisomers 5 and 6 in good
yield, after removal of trimethylsilyl residues. At this
stage separation via flash column chromatography al-
lowed the first eluting (SP)-diastereoisomer 5 and second
eluting (RP)-diastereoisomer 6 to be isolated, vide infra.11
These individual isomers were separately desilylated
upon treatment with tetrabutylammonium fluoride in THF
to give the corresponding diols. Subsequent selective 5 -
Synlett 2002, No. 5, 03 05 2002. Article Identifier:
1437-2096,E;2002,0,05,0763,0766,ftx,en;D01702ST.pdf.
© Georg Thieme Verlag Stuttgart · New York
ISSN 0936-5214

764
R. A. Fairhurst et al.
LETTER
tritylation followed by phosphitylation of the 3 -alcohol
under standard conditions, yielded the phosphoramid-
ites 7 and 8 ready for oligonucleotide synthesis.
Scheme 2 i) 6 equiv Me3SiNC(OSiMe3)CF3, pyridine, 0 ºC to r.t.,
then CHCl3, CH3OH, 4 h, reflux (73%).
To explore the potential utility of the 3 -C-P-N-5 (RP)-
ethyl phosphonamidate ester modification further for an-
tisense applications, a study into the stabilising effect
upon sequences in which it is located beyond the linking
of adjacent thymidines is required. One well studied oli-
gonucleotide sequence incorporates seven consecutively
linked dinucleosides, including modifications between
thymidine and cytidine residues.3a To investigate this pos-
sibility a 5 -azido-5 -deoxy-2 -methoxy-5-methylcytidine
derivative was prepared from the thymidine derived azide
4, as shown in Scheme 3. Silylation of 4 yielded the 3 -
protected alcohol 10, in a suitable form for subsequent
manipulation of the 4-keto group of the pyrimidine moi-
ety. This was achieved utilising a standard sequence, of
activation as the 4-(1,2,4-triazoyl) intermediate followed
by displacement with ammonia to produce the 5-methyl
cytidine derivative 11.12 Protection of 11 with a slight ex-
cess of benzoyl chloride delivered 12, the required precur-
sor for the Staudinger coupling reaction.
Scheme 1 i) 6 equiv Me3SiNC(OSiMe3)CF3, pyridine, 0 ºC to r.t.,
then CHCl3, CH3OH, 4 h, reflux (77%); ii) flash column chromato-
graphic separation of diastereoisomers, silica gel, eluant; 10% ethanol
in chloroform; iii) 1.3 equiv Bu4N+F–, THF, 0 ºC (67–80%); iv) 1.4
equiv DMTr-Cl, pyridine, r.t. (62–87%); v)
3 equiv (i-
Pr2N)2POCH2CH2CN, 5 equiv diisopropylammonium tetrazolide,
CH2Cl2, r.t. (60–70%).
Scheme 3 i) 1.1 equiv TBDPS-Cl, imidazole, DMF, r.t. (>95%); ii)
10 equiv 1,2,4-triazole, 5 equiv POCl3, pyridine, 50 °C (>95%); iii)
0.88 equiv NH3, 1,4-dioxan, r.t. (>95%); iv) 1.2 equiv PhCOCl, 3
equiv Et3N, Et2O, r.t. (92%).
Assignment of the phosphorus stereochemistry in the sep-
arated dimers 5 and 6, was made following coupling of 9,
the (SP)-H-phosphinate diastereoisomer of 3, with the
azide 4, as shown in Scheme 2. Reaction under the BST-
FA promoted conditions employed for the preparation of
5 and 6 have previously been shown to proceed with re-
tention of phosphorus configuration.6 Hence, the product
derived from 9 is the (RP)-phosphonamidate, which could
be retrospectively correlated with the second eluting iso-
mer 6, obtained after separation in the sequence shown
above in Scheme 1.
Azide 12 underwent a BSTFA mediated coupling with the
(SP)-H-phosphinate 9 to yield the anticipated dinucleoside
13 with >95% retention of stereochemical integrity at the
phosphorus centre, as shown in Scheme 4.13 Repeating the
same sequence of alcohol group manipulation, as em-
ployed in the preparation of 7 and 8, involving desilyla-
Synlett 2002, No. 5, 763–766 ISSN 0936-5214 © Thieme Stuttgart · New York

LETTER
Nucleic Acid Containing 3 -C-P-N-5 Ethyl Phosphonamidate Ester
765
The SP increasing by 2.6 °C per modification and the RP
increasing by 1.4 °C per modification, in comparative T-
T containing sequences. This increase for the (RP)-diaste-
reoisomer being similar to the incorporation of a single 2 -
methoxy-modification into the above 15mer DNA se-
quence, of Tm +1.2 °C.16 The reason for the relatively
large increase in Tm for the (SP)-ethyl phosphonamidate
linkage is unclear. However, the anticipated preference
for the (RP)-diastereoisomer to provide the most stable
modification is conserved, and is rationalised as arising
from a more favourable orientation of the ethyl residue
away from the backbone axis in the hybrid duplex.17
Incorporation of seven T-T and T-MeC dimers to give the
15mer sequence with alternating (RP)-phosphonamidate/
phosphodiester linkages produced a Tm value of +3.1 °C
per modification. Correcting this value for the established
stabilising effect for the introduction of a 5-methyl residue
into cytidine, of +0.5 °C per modification,18 normalises to
a slightly greater Tm value when compared to that ob-
served with the exclusively T-T containing sequences, of
+2.7 °C per modification. Hence, extending the scope of
the (RP)-phosphonamidate linkage to T-MeC containing
sequences resulted in no loss of stabilisation, and suggests
that the modification may be suitable for general applica-
tion. Additionally, the presence of consecutively modified
(RP)-phosphonamidate dinucleosides produced the se-
quence with the highest Tm value. Indicating no loss of
the desired stabilising properties upon incorporation into
a highly modified oligonucleotide, again suggesting the
possibility for application across a broad range of se-
quences.
In summary, T-T and T-MeC dinucleosides combining
both diastereoisomeric forms of the ethyl phosphonami-
date linkage and a 2 -methoxy substituent in the lower
sugar residue have been synthesised and incorporated into
DNA oligonucleotides. Hybridisation data with comple-
mentary RNA has validated the proposed combination of
these structural elements to produce a highly stabilising
modification, and in the case of the (RP)-diastereoisomer,
displaying many attributes required for antisense applica-
tions.
Scheme 4 i) 5 equiv Me3SiNC(OSiMe3)CF3, pyridine, 0 ºC to r.t.,
then CHCl3, CH3OH, 4 h, reflux (89%); ii) 3.0 equiv Bu4N+F–, THF,
0 ºC (74%); iii) 1.3 equiv DMTr-Cl, pyridine, r.t. (87%); iv) 3 equiv
(i-Pr2N)2POCH2CH2CN, 5 equiv diisopropylammonium tetrazolide,
CH2Cl2, r.t. (77%).
tion, selective 5 -tritylation followed by 3 -phos-
phitylation, gave the thymidine-5-methylcytidine (T-MeC)
phosphoramidite 14 ready for oligonucleotide synthesis.
Oligonucleotide sequences were prepared incorporating
the modified dinucleosides 7, 8 and 14 utilising standard
phosphoramidite coupling protocols.14 Following this
strategy a 17mer and a 15mer sequence, containing five
and one or seven modified linkages respectively, were
synthesised and the melting data with complimentary
RNA determined.15 The results are shown in the Table and
compared with data for the singularly modified 3 -C-P-N-
5 ethyl phosphonamidate ester linkage 1.5
Comparison of the melting data indicates that incorpora-
tion of the 2 -methoxy-substituent into the lower sugar
residue to give 2 produced the anticipated increase in hy-
brid duplex stability for both diastereoisomeric forms of
the phosphonamidate linkage, when compared with 1.
Table Melting data for DNA oligonucleotides containing modifications 1 and 2 with complementary RNA
Modified dimer incorporated
Tm (ºC) per modificationa
Tm (ºC) per modificationa
Tm (ºC) per modificationa Average Tm (ºC)
tttttctctctctcT
TTTttCTCTCTCTCTc
GCGttttttttttTGCGd
per modificationb
(SP)-diastereoisomer 1
(RP)-diastereoisomer 1
(SP)-diastereoisomer 2
(RP)-diastereoisomer 2
–1.9
+1.6
+0.8
+2.5
–1.5
+0.1
+0.9
+2.0
–
–1.7
+0.9
+0.9
+2.3
–
–
+3.1
a Lower case indicates modified dimer with phosphonamidate linkage.
b Average for sequences containing exclusively T-T modified dimers.
c Tm for TTTTTCTCTCTCTCT ‘wild-type’ DNA with complementary RNA 52.7 °C.
d Tm for GCGTTTTTTTTTTTGCG ‘wild-type’ DNA with complementary RNA 50.2 °C.
Synlett 2002, No. 5, 763–766 ISSN 0936-5214 © Thieme Stuttgart · New York

766
R. A. Fairhurst et al.
LETTER
5: White amorphous foam; 1.07 g; 31P NMR (CDCl3, 162
MHz): = 32.52 ppm; 1H NMR (CDCl3, 400 MHz): = 9.67
(br s, 1 H), 9.52 (s, br, 1 H), 7.69–7.61 (m, 4 H), 7.45–7.34
(m, 7 H), 7.13 (s, 1 H), 6.10–6.02 (m, 1 H, H1 upper sugar),
5.50 (d, J = 2 Hz, 1 H, H1 lower sugar), 4.26–3.64 (m, 8 H),
3.50 (s, 3 H), 3.36–3.17 (m, 3 H), 2.74–2.61 (m, 1 H), 2.51–
2.42 (m, 1 H), 2.32–2.20 (m, 1 H), 2.04–1.82 (m, 2 H), 1.84
(s, 3 H), 1.74–1.59 (m, 1 H), 1.58 (s, 3 H), 1.27 (t, J = 7 Hz,
3 H), 1.04 (s, 9 H). MS (ES+): m/z (%) = 840(27) [M + H],
862(100) [M + Na].
Acknowledgement
We would like to acknowledge and thank Dr. U. Pieles and Dr. F.
Natt for oligonucleotide synthesis as well as Dr. S. M. Freier (Isis
Pharmaceuticals) for performing hybridisation experiments.
References
(1) New address: Novartis Horsham Research Centre,
Wimblehurst Road, Horsham, West Sussex, RH12 5AB,
UK.
6: White amorphous foam; 0.94 g; 31P NMR (CDCl3, 162
MHz): = 32.81 ppm; 1H NMR (CDCl3, 400 MHz): = 9.38
(br s, 1 H), 9.17 (s, br, 1 H), 7.70–7.62 (m, 4 H), 7.44–7.34
(m, 7 H), 7.13 (s, 1 H), 6.14–6.06 (m, 1 H, H1 upper sugar),
5.55 (d, J = 2 Hz, 1 H, H1 lower sugar), 4.24–3.64 (m, 8 H),
3.51 (s, 3 H), 3.50–3.18 (m, 3 H), 2.80–2.68 (m, 1 H), 2.50–
2.39 (m, 1 H), 2.34–2.23 (m, 1 H), 2.01–1.84 (m, 1 H), 1.84
(s, 3 H), 1.77–1.58 (m, 2 H), 1.61 (s, 3 H), 1.20–1.10 (m, 3
H), 1.04 (s, 9 H). MS (ES+): m/z (%) = 840(8) [M + H],
862(100) [M + Na].
(2) (a) Wahlestedt, C.; Good, L. Curr. Opin. Drug Discov. Dev.
1999, 2, 142. (b) Bennett, C. F. Exp. Opin. Invest. Drugs
1999, 8, 237. (c) Ma, D. D. F.; Rede, T.; Naqvi, N. A.; Cook,
P. D. Biotechnol. Annu. Rev. 2000, 5, 155. (d) Bennett, C.
F.; Butler, M.; Cook, P. D.; Geary, R. S.; Levin, A. A.;
Mehta, R.; Teng, C.-L.; Deshmukh, H.; Tillman, L.; Hardee,
G. Gene Ther.; Marcel Dekker Inc.: New York, 2000, 305.
(e) Uhlmann, E. Curr. Opin. Drug Discovery Dev. 2000, 3,
203. (f) Galderisi, U.; Cipollaro, M.; Cascino, A. Emerging
Drugs 2001, 6, 69.
(3) (a) Freier, S. M.; Altmann, K.-H. Nucleic Acids Res. 1997,
25, 4429. (b) Altmann, K.-H.; Cuenoud, B.; Von Matt, P. In
Applied Antisense Oligonucleotide Technology; Krieg, A.
M.; Stein, C. A., Eds.; Wiley-Liss Inc.: New York, 1998.
(c) Egli, M.; Gryaznov, S. M. Cell. Mol. Life Sci. 2000, 57,
1440. (d) Engels, J. W.; Uhlmann, E. Pharm. Aspects
Oligonucleotides 2000, 35. (e) An, H.; Wang, T.; Maier, M.
A.; Manoharan, M.; Ross, B. S.; Cook, P. D. J. Org. Chem.
2001, 66, 2789.
(4) (a) Crooke, S. T.; Lemonidis, K. M.; Neilson, L.; Griffey, R.;
Lesnik, E. A.; Monia, B. P. Biochem. 1995, 312, 599.
(b) McKay, R. A.; Miraglia, L. J.; Cummins, L. L.; Owens,
S. R.; Sasmor, H.; Dean, N. M. J. Biol. Chem. 1999, 274,
1715. (c) Cramer, H.; Pfleiderer, W. Nuleosides,
Nucleotides, Nucleic Acids 2000, 19, 1765. (d) Malchere,
C.; Verheijien, J.; Van Der Laan, S.; Bastide, L.; Van Boom,
J.; Lebleu, B.; Robbins, I. Antisense Nucleic Acid Drug Dev.
2000, 10, 463. (e) Kværnø, L.; Wengel, J. Chem. Commun.
2001, 1419.
(12) (a) Kim, C. H.; Marquez, V. E.; Broder, S.; Mitsuya, H.;
Driscoll, J. S. J. Med. Chem. 1987, 30, 862. (b) Ross, B. S.;
Springer, R. H.; Vasquez, G.; Andrews, R. S.; Cook, P. D.;
Acevedo, O. L. J. Heterocycl. Chem. 1994, 31, 765.
(13) Reaction conditions were as described in ref.5 A reaction
carried out on a 1.46 mmol scale gave;
13: White amorphous foam; 1.54 g; 31P NMR (CDCl3, 162
MHz): = 32.70 ppm; 1H NMR (CDCl3, 400 MHz): = 8.62
(s, 1 H), 8.15 (d, 2 H, J = 7 Hz), 7.58–7.44 (m, 8 H), 7.35–
7.31 (m, 1 H), 7.30–7.17 (m, 16 H), 7.08 (s, 1 H), 5.96–5.90
(m, 1 H, H1 upper sugar), 5.42 (d, 1 H, J = 2 Hz, H1 lower
sugar), 3.97–3.89 (m, 1 H), 3.86–3.77 (m, 2 H), 3.69–3.53
(m, 4 H), 3.10 (s, 3 H), 2.98–2.81 (m, 2 H), 2.76–2.63 (m, 1
H), 2.59–2.48 (m, 1 H), 2.29–2.20 (m, 1 H), 2.03–1.94 (m, 1
H), 1.91 (s, 3 H), 1.76–1.60 (m, 1 H), 1.44 (s, 3 H), 1.38–1.14
(m, 1 H), 1.07–0.99 (m, 3 H), 0.94 (s, 9 H), 0.91 (s, 9 H).
Minor (SP)-diastereoisomer; 31P NMR (CDCl3, 162 MHz):
= 31.71 ppm; 1H NMR (CDCl3, 400 MHz): key
distinguishing resonances = 5.90–5.83 (m, 1 H, H1 upper
sugar), 5.29 (d, 1 H, J = 2 Hz, H1 lower sugar).
(5) Fairhurst, R. A.; Collingwood, S. P.; Lambert, D.; Taylor, R.
J. Synlett 2001, 467.
(6) Fairhurst, R. A.; Collingwood, S. P.; Lambert, D. Synlett
2001, 473.
(14) (a) Oligonucleotides were prepared using an ABI 390 DNA
synthesiser following standard phosphoramidite chemistry,
according to: Gait:, M. J. Oligonucleotide Synthesis: A
Practical Approach; IRL Press: Oxford, 1984. (b) For the
steps involving incorporation of modified dimers, double
couplings with double reaction times were employed.
(15) (a) The thermal denaturation of DNA/RNA hybrids was
performed at 260 nm using a Gifford Response II
(7) (a) Griffey, R. H.; Lesnik, E.; Freier, S.; Sanghvi, Y. S.;
Teng, K.; Kawasaki, A.; Guinosso, C.; Wheeler, P.; Mohan,
V.; Cook, P. D. ACS Symp. Ser. 1994, 580, 212.
(b) Manoharan, M. Biochim. Biophys. Acta 1999, 1489, 117.
(8) (a) Inoue, H.; Hayase, Y.; Imura, A.; Iwai, S.; Miura, K.;
Ohtsuka, E. Nucleic Acids Res. 1987, 15, 6131.
(b) Lamond, A. I.; Sproat, B. S. FEBS Lett. 1993, 325, 123.
(9) (a) De Mesmaeker, A.; Lesueur, C.; Bévièrre, M.-O.;
Waldner, A.; Fritsch, V.; Wolf, V. F. Angew. Chem., Int. Ed.
Engl. 1996, 35, 2790. (b) De Mesmaeker, A.; Lebreton, J.;
Jouanno, C.; Fritsch, V.; Wolf, R. M.; Wendeborn, S. Synlett
1997, 1287. (c) Pfundheller, H. M.; Wengel, J. Bioorg. Med.
Chem. Lett. 1999, 9, 2667.
spectrophotometer (Ciba-Corning Diagnostics Corp.,
Oberlin, OH) absorbance vs. temperature profiles were
measured at 4 m of each strand in 10 mM phosphate pH 7.0
(Na salts), 100 mM total (Na+) 0.1 mM EDTA. Tm’s were
obtained from fits of absorbance vs. temperature curves to a
two-state model with linear slope baselines: Freier, S. M.;
Albergo, T. D.; Turner, D. H. Biopolymers 1982, 22, 1107.
(b) All values are averages of at least three experiments. The
absolute error of the Tm values is 0.5 °C.
(10) Huang, J.; McElroy, E. B.; Widlanski, T. S. J. Org. Chem.
1994, 59, 3520.
(16) Martin, P. Helv. Chim. Acta 1995, 78, 486.
(17) (a) Pless, R. C.; Ts’o, P. O. P. Biochemistry 1977, 16, 1239.
(b) Summers, M. F.; Powell, C.; Egan, W.; Byrd, R. A.;
Wilson, W. D.; Zon, G. Nucleic Acids Res. 1986, 14, 7421.
(c) Ferguson, D. M.; Kollman, P. A. Antisense Research and
Development 1991, 1, 243.
(18) Sanghvi, Y. S.; Hoke, G. D.; Freier, S. M.; Zounes, M. C.;
Gonzalez, C.; Cummins, L.; Sasmor, H.; Cook, P. D. Nucleic
Acids Res. 1993, 21, 3197.
(11) Reaction conditions were as described in ref.5 Flash column
chromatography was performed using Merck Silica Gel 60
(0.040–0.063 mm). NMR spectra were recorded with a
Brucker AC400 instrument. Key distinguishing 1H
resonances for each diastereoisomer are assigned. 31P NMR
shifts are given as ppm values relative to phosphoric acid.
Mass spectroscopy was carried out using a Fisons
Instruments VG Platform II spectrometer. A reaction carried
out on a 3.11 mmol scale gave;
Synlett 2002, No. 5, 763–766 ISSN 0936-5214 © Thieme Stuttgart · New York