Stereochemical assignment and stereoselective synthesis of 3′-C-P-N-5′ Rp-ethyl phosphonamidate modified nucleic acid

Article abstract of DOI:10.1055/s-2001-12329

The configuration of the duplex stabilising 3′-C-P-N-5′ ethyl phosphonamidate nucleic acid backbone modification has been identified as R_p following the preparation of cyclic nucleoside analogues and NOE studies. Complimentary routes for formation of the key phosphonamidate nitrogen to phosphorus bond from stereochemically defined H-phosphinate intermediates, involving both a retention and inversion at the phosphorus centre, have been identified.

Full text of DOI:10.1055/s-2001-12329

LETTER
473
Stereochemical Assignment and Stereoselective Synthesis of 3’-C-P-N-5’
RP-Ethyl Phosphonamidate Modified Nucleic Acid
Robin A. Fairhurst,1* Stephen P. Collingwood,1 David Lambert1
Novartis Pharmaceuticals, Central Research Laboratories, Hulley Road, Macclesfield, Cheshire, SK10 2NX, UK
Fax +44 (0) 1403 323307; E-mail: robin.fairhurst@pharma.novartis.com
Received 25 January 2001
phosphonamidate dinucleoside. To this end, removal of
the ketal protecting group from either 3 or 4 led to signif-
icant epimerisation at the phosphorus centre under the
standard deprotection conditions of trimethylsilyl chlo-
Abstract: The configuration of the duplex stabilising 3’-C-P-N-5’
ethyl phosphonamidate nucleic acid backbone modification has
been identified as RP following the preparation of cyclic nucleoside
analogues and NOE studies. Complimentary routes for formation of
the key phosphonamidate nitrogen to phosphorus bond from ste- ride, ethanol, chloroform.5 Reducing the quantity of hy-
reochemically defined H-phosphinate intermediates, involving both
a retention and inversion at the phosphorus centre, have been iden-
tified.
drogen chloride liberated under the above deprotection
conditions and performing the reaction in dichlo-
romethane with a minimum amount of ethanol enabled the
extent of epimerisation to be maintained consistently be-
Key words: antisense, modified oligonucleotides, phosphorus pro-
tecting groups, stereoselective
low 5%. Following this modified procedure the individual
RP and SP H-phosphinate diastereoisomers 5 and 6 were
readily prepared in multigram quantities from 3 and 4 re-
spectively with greater than 95% stereochemical purity.6
The stereochemistry of modified nucleic acid in which the
phosphodiester is replaced with a chiral phosphorus con-
taining linkage has been shown to have a significant im-
pact upon the duplex stabilising properties of such
molecules.2 In the preceding paper3 a single phosphorus
diastereoisomer of 3’-C-P-N-5’ ethyl phosphonamidate
modified nucleic acid was identified as enhancing binding
with complimentary RNA to the extent of Tm+0.9°C per
modification.3 During this initial exploratory study, indi-
vidual phosphonamidate diastereoisomers were obtained,
after the separation of a 1:1 mixture, at the penultimate
stage of the synthesis. To further evaluate the potential of
this modification a more efficient route to prepare nucle-
oside dimers containing the above duplex stabilising
phosphonamidate diastereoisomer was desired. Addition-
ally, an assignment of the phosphorus stereochemistry
was sought in order to allow a rationalisation of the ori-
gins of the enhanced duplex stability.
Following the route employed in the initial investigation,
thymidine was homologated at the 3’-position to give the
3’-iodomethyl derivative 1. Alkylation of the potassium
anion of the differentially protected hypophosphorous
acid synthon 2 with the iodide 1 occurs cleanly to give a
1:1 mixture of the phosphinate diastereoisomers 3 and 4.
In the original preliminary study, these isomers were car-
ried through the synthesis without separation.3 However,
they are readily separable by normal phase medium pres-
sure chromatography, on a multigram scale, to provide the
individual diastereoisomer 3 and 4, assigned as SP and RP
respectively vide infra, Scheme 1.4 Our initial interest was
to establish if either of the individual H-phosphinates 3 or
4 could be carried through the original sequence, whilst
retaining the stereochemical integrity at the phosphorus
centre to prepare selectively a single diastereoisomeric
Scheme 1 i) 4 equiv. 2, 4 equiv. KHMDS, THF, -78 ºC to rt (85-
94%); ii) MPLC separation diastereoisomers; iii) 1.1 equiv. Me3SiCl,
1.25 equiv. EtOH, CH2Cl2, 0 ºC to rt (78-92%).
Phosphonamidate formation with either of the separated
H-phosphinate diastereoisomers 5 or 6 and 5’-azidothy-
midine 7, upon activation with bis(trimethylsilyl)trifluo-
roacetamide, in an analogous manor to that previously
described with the mixed diastereoisomers,3 led to the for-
mation of the corresponding monosilylated dimers 8 and
9 respectively. No significant loss of stereochemical in-
tegrity at the phosphorus centre was detected in these re-
actions.7 Transformations of this type involving the
formation of O-silylated P(III) intermediates and their
subsequent addition to electrophiles are precedented to
occur with retention of configuration at the phosphorus
Synlett 2001 No. 4, 473–476 ISSN 0936-5214 © Thieme Stuttgart · New York

474
R. A. Fairhurst et al.
LETTER
centre.8 Subsequent removal of the 5’-silyl group from the inversion of phosphorus stereochemistry,11 thus the over-
dimers 8 and 9 followed by selective mono-tritylation at all result being an inversion of the phophorus configura-
the 5’-position provided intermediates which could be tion. Applying such conditions with the H-phosphinate 5
correlated with the original synthesis. This allowed the H- and the 5’-aminothymidine derivative 1212 as the nucleo-
phosphinate 6 to be identified as the required diastereo- philic component produced the desired bis-silylated phos-
isomer for synthesis, by this route, of the desired DNA- phonamidate dimer 13 without significant loss of chiral
RNA stabilising modification. Further confirmation of integrity at the phosphorus centre.13 Stereochemical cor-
this stereochemical assignment was obtained following relation with an intermediate of established configuration
phosphitylation of the required stereoisomer 10, derived from the original sequence was made possible after desil-
from 6, to provide the phosphoramidite 11 as a 1:1 mix- ylation of 13 followed by selective 5’-monotritylation to
ture of P(III) epimers for oligonucleotide synthesis, again give the RP phosphonamidate 10, Scheme 3. Thus,
Scheme 2.9
establishing that the anticipated opposite configurational
outcome to the azide sequence, of inversion, had occurred
at phosphorus.
Scheme 3 i) CCl4, Et3N, CH2Cl2, pyridine, 3 molecular sieves,
0 ºC to rt (75%); ii) 3 equiv. Bu4N+F-, THF, 0 ºC (80%); iii) 1.2 equiv.
DMTrCl, pyidine, rt (78%).
Methodology has hence been established to improve the
overall efficiency by enabling both diastereoisomeric
alkylation products 3 and 4 to be employed in the prepa-
ration of the required RP phosphonamidate dinucleoside
linkage, via stereochemically complimentary reactions.
Scheme 2 i) 6 equiv. Me3SiNC(OSiMe3)CF3, pyridine, 0 ºC to rt
(64-79%); ii) 2 equiv. Bu4N+F-, THF, 0 ºC (94%); iii) 1.2 equiv. DM-
TrCl, pyidine, rt (71%); iv) 3 equiv. (iPr2N)2POCH2CH2CN, 5 equiv.
Diisopropylammonium tetrazolide, CH2Cl2, rt (72%).
To assign the phosphorus stereochemistry in the above re-
action sequences cyclic nucleoside analogues originating
from 3 and 4 were anticipated to provide derivatives from
which the original phosphorus stereochemistry could be
deduced by NMR. To this end, intramolecular phosphon-
amidate bond formation with a 5’-azido nucleoside deriv-
ative was investigated, as outlined in Scheme 4.14 Thus,
functional group manipulation at the 5’-position with ei-
ther 3 or 4, following a sequence of desilylation, iodin-
ation and nucleophilic displacement, produced the 5’-
azides 17 and 18 respectively. Subsequent removal of the
ortho ester protecting groups, under the improved non-
epimerising conditions described above, provided the H-
phosphinate azides 19 and 20.15 Subsequent cyclisation of
19 or 20 upon activation with bis(trimethylsilyl)trifluoro-
acetamide produced trimethylsilylated cyclic phosphon-
amidates which could be readily desilylated with tetrabu-
tylammonium flouride to produce 21 and 22 respective-
This new sequence described above for the preparation of
the desired dinucleoside building block 11 represents an
improvement in efficiency over the original route, as an
earlier diastereoisomeric separation is made. However,
still half the material at the alkylation step remains redun-
dant in the form of the H-phosphinate 5. If conditions
could be identified for phosphonamidate formation with 5
that involved an inversion of phosphorus stereochemistry
then this material could also be utilised. The Atherton-
Todd reaction represents an alternative strategy for P-N
bond formation from an amine and suitably activated P-H
species.10 Stereochemically these reactions involve the
formation of an electrophilic P-Cl intermediate which
would be anticipated to occur with retention of configu-
ration followed by a nucleophilic displacement of chlo-
ride ion, which in the majority of examples proceeds with
Synlett 2001, No. 4, 473–476 ISSN 0936-5214 © Thieme Stuttgart · New York

LETTER
Stereochemical Assignment and Stereoselective Synthesis
475
Scheme 4 i) 1.1 equiv. Bu4N+F-, THF, 0 ºC (88-87%); ii) 1.2 equiv. MeP+(OPh)3I-, 2 equiv. 2,6-lutidine, DMF, rt (53-65%); iii) 5 equiv.
NaN3, DMF, 80 ºC (89-95%); iv) 1.1 equiv. Me3SiCl, 1.25 equiv. EtOH, CH2Cl2, 0 ºC to rt (63-67%); v) 5 equiv. Me3SiNC(OSiMe3)CF3,
pyridine, 0 ºC to rt (60-66%); vi) 1 equiv. Bu4N+F-, THF, 0 ºC (>95%).
ly.16 Throughout the above sequences the stereochemical In summary, two complimentary routes for the synthesis
integrity at phosphorus was shown to be retained to the of the duplex stabilising 3’-C-P-N-5’ RP-ethyl phosphon-
extent of >95% by 31P NMR. The stereochemistry at the amidate modification have been developed which utilise
phosphonamidate centre was then readily assigned in the both diastereoisomeric H-phosphinate intermediates. The
cyclic nucleoside derivatives following NOE experi- phosphorus stereochemistry has been assigned as RP in the
ments, 21 as SP and 22 as RP.17
modification of interest following NMR experiments after
conversion of individual diastereoisomers of the key H-
phosphinate intermediates to the corresponding cyclic nu-
cleoside analogues. Further studies with this interesting
Assuming the same stereochemical outcome for the inter- duplex stabilising 3’-C-P-N-5’ RP-ethyl phosphonamidate
molecular and intramolecular reactions between the H- modification will investigate the incorporation into a
phosphinate and azido functionality’s enables the assign- broader range of sequences beyond those containing adja-
ment of RP to be made to the duplex stabilising dinucleo- cent thymidines.
side modification 11 of interest. Extrapolating further
allows an assignments of RP and SP to be applied to the
starting H-phosphinates 5 and 6 respectively. This is
Acknowledgement
We would like to thank Dr. B. Everett for performing NMR studies.
based upon transformations having occurred with inver-
sion and retention of phosphorus configuration to produce
the phosphonamidate dinucleoside intermediates 13 and 9
respectively. Additionally, deprotection of the mixed
ortho ester protecting group, to liberate the H-phosphin-
ates 5 and 6, is anticipated to occur with retention of con-
figuration, based upon analogous dealkylation reactions at
phophorus.18 Thus, enabling the assignments of SP and RP
to be made to the diastereoisomeric alkylation products 3
and 4 respectively.
References and Notes
(1) New address: Novartis Horsham Research Centre,
Wimblehurst Road, Horsham, West Sussex, RH12 5AB, UK.
(2) Lebedev, A. V.; Wickstrom, E. Perspectives in Drug
Discovery and Design 1996, 4, 17. Endo, M.; Komiyama, M.
J. Org. Chem. 1996, 61, 1994. Mag, M.; Jahn, K.;
Kretzschmar, G.; Peyman, A.; Uhlmann, E. Tetrahedron
1996, 52, 10011. Mujeeb, A.; Reynolds, M. A.; James, T. L.
Biochemistry 1997, 36, 2371. Laurent, A.; Naval, M.; Debart,
F.; Vasseur, J-J.; Rayner, B. Nucleic Acids Res. 1999, 27,
4151.
The above assignment of the duplex stabilising diastereo-
isomer as RP is consistent with previously reported exam-
ples in which data for stereochemically defined chiral
phosphorus containing linkages has been obtained,2 with
the orientation of the ethyl phosphonamidate ester residue
within a helical duplex being dependant on the phospho-
rus chirality. Incorporation of the SP phosphonamidate
diasteroisomer into a helical duplex would be anticipated
to induce a pseudoaxial configuration in which the ethyl
residue is oriented into the major groove. In contrast, the
RP diastereoisomer is anticipated to adopt a pseudoequito-
rial conformation orienting the ethyl residue away from
the backbone axis and into the surrounding solvent. Ratio-
nalisations implicating both steric and entropic factors to
account for the greater stability observed with the pseu-
doequitorial conformation, equating to the RP configura-
tion in this example, have been proposed.19
(3) Fairhurst, R. A.; Collingwood, S. P.; Lambert, D.; Taylor, R.
J. Synlett 2001, 467.
(4) Stationary phase Kiesegel 60 (80-230 mesh); eluent 5%
ethanol in ethyl acetate. First eluting diastereoisomer assigned
3, second eluting diastereoisomer assigned 4. NMR spectra
were recorded with a Bruker AC400 or Bruker DRX500
instrument. 31P NMR shifts are given as ppm values relative to
phosphoric acid.
3; White foam; 31P NMR (MeOH-d4, 202 MHz) 49.58 ppm;
1H (MeOH-d4, 500 MHz) 7.66-7.58 (m, 4H, m-Ph), 7.45 (s,
1H, H6), 7.38-7.27 (m, 6H, o,p-Ph), 6.06-6.02 (m, 1H, H1’),
4.17-4.06 (m, 2H, POCH2CH3), 4.04-3.98 (m, 1H, H5’), 3.87-
3.83 (m, 1H, H5’), 3.78-3.73 (m, 1H, H4’), 3.67-3.54 (m, 4H,
COCH2CH3), 2.83-2.72 (m, 1H, H3’), 2.39-2.32 (m, 1H, H2’),
2.28-2.20 (m, 1H, H2’), 2.13-2.04 (m, 1H, H6’), 1.76-1.66 (m,
1H, H6’), 1.40 (d, 3H, J = 12 Hz, PCCH3), 1.37 (s, 3H, 5-Me),
1.24 (t, 3H, J = 7 Hz, POCH2CH3), 1.13-1.04 (m, 6H,
COCH2CH3), 0.99 (s, 9H, SiCMe3).
4; White foam; 31P NMR (MeOH-d4, 202 MHz) 49.99 ppm;
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476
R. A. Fairhurst et al.
LETTER
1H (MeOH-d4, 500 MHz) 7.65-7.58 (m, 4H, m-Ph), 7.47 (s,
1H, H6), 7.38-7.26 (m, 6H, o,p-Ph), 6.06-6.02 (m, 1H, H1’),
4.16-4.08 (m, 2H, POCH2CH3), 4.04-3.98 (m, 1H, H5’), 3.85-
3.80 (m, 1H, H5’), 3.76-3.73 (m, 1H, H4’), 3.66-3.54 (m, 4H,
COCH2CH3), 2.84-2.73 (m, 1H, H3’), 2.47-2.39 (m, 1H, H2’),
2.28-2.19 (m, 1H, H2’), 2.03-1.95 (m, 1H, H6’), 1.84-1.75 (m,
1H, H6’), 1.42 (d, 3H, J = 12 Hz, PCCH3), 1.38 (s, 3H, 5-Me),
1.20 (t, 3H, J = 7 Hz, POCH2CH3), 1.14-1.05 (m, 6H,
COCH2CH3), 1.00 (s, 9H, SiCMe3).
H-phosphinate 5 (285 mg, 500 mol) in anhydrous pyridine
(0.5 ml) and anhydrous dichloromethane (1 ml) at 0 ºC. The
reaction mixture was allowed to warm to room temperature,
stirred for 18 h at room temperature, filtered and evaporated to
give the crude product which was purified by flash column
chromatography, eluent 5% methanol in dichloromethane, to
give the phosphonamidate dimer 13 as an off-white foam (395
mg, 75%); 31P NMR (CDCl3, 162 MHz) 32.32 ppm.
Analysis of the crude reaction mixture indicates <5%
epimerisation having occurred resulting in formation of the
diastereoisomeric phosphonamidate dimer; 31P NMR (CDCl3,
162 MHz) 31.70 ppm.
(5) Baylis, E. K. Tetrahedron Lett. 1995, 36, 9385.
(6) Typical procedure; Trimethylsilyl chloride (1.07 ml, 8.39
mmol) was added dropwise to a solution of the phosphinate 3
(5.24 g, 7.63 mmol) in anhydrous ethanol (0.56 ml, 9.54
mmol) and anhydrous dichloromethane (38 ml) at 0 ºC. The
reaction mixture was allowed to warm to room temperature,
stirred at that temperature for 36 h at which point it was judged
to be complete by 31P NMR (aliquot of reaction mixture with
D2O insert shows consumption of the starting material at
46.59 ppm and formation of a predominant new resonance at
34.61 ppm) and drown-out into 50% saturated NaHCO3(aq)
(100 ml). Extraction with dichloromethane (3 X 50 ml),
drying of the organic layers over MgSO4 and removal of
volatiles gave the crude product which was purified by silica
gel flash column chromatography, eluting with 5% methanol
in dichloromethane, to give the H-phosphinate 5 as an off-
white foam (4.02 g, 92%); 31P NMR (CDCl3, 162 MHz)
34.20 ppm. Similarly, following the above procedure, the
(14) Hata, T.; Yamamoto, I.; Sekine, M. Chem. Lett. 1976, 601.
(15) Removal of the mixed orthoester protecting group from 17
and 18, in an analogous manner to that described above,6
produced the H-phosphinates 19 and 20 respectively with
<5% epimerisation as determined by 31P NMR analysis. 19;
Colourless glass; 31P NMR (CDCl3, 162 MHz) 33.14 ppm.
20; Colourless glass; 31P NMR (CDCl3, 162 MHz) 33.81
ppm.
(16) The H-phosphinate-azides 19 and 20 were cyclised with
bis(trimethylsilyl)trifluoroacetamide in pyridine to yield the
corresponding N-trimethylsilylated cyclic phosphonamidates
in an analogous fashion to that described in the previous
communication.3 In contrast to the dimer synthesis, removal
of the trimethylsilyl residues was best achieved upon
treatment with tetrabutylammonium fluoride (1.1 equiv) in
tetrahydrofuran at room temperature for 3 h. Evaporation of
volatiles and trituration with ether yielded the respective
cyclic phosphonamidate 21 or 22.
phosphinate 4 could be cleaved to the H-phosphinate 6; 31
P
NMR (CDCl3, 162 MHz) 34.42 ppm. Removal of the mixed
orthoester protecting group in both the above examples results
in 2-4% epimerisation at the phosphorus centre as judged by
31P NMR analysis of the crude reaction mixtures.
(7) Individual H-phosphinates 5 and 6 were reacted with 5’-
azido-5’-deoxythymidine 7 following similar reaction
conditions to those described in the preceding
21; White foam; 31P NMR (CDCl3, 162 MHz) 28.60 ppm; 1H
NMR (CDCl3, 400 MHz) 9.42 (s, br, 1H, HN3), 7.09 (s, 1H,
H6), 6.18 (d, 1H, J = 5 Hz, H1'), 4.16-4.04 (m, 2H, CH2CH3),
3.63-3.58 (m, 1H, H4'), 3.58-3.45 (m, 1H, H5' ), 3.39-3.26
(m, 1H, H5' ), 3.02-2.97 (m, br, PNH, 1H), 2.51-2.39 (m, 1H,
H3'), 2.37-2.24 (m, 1H, H6' ), 2.23-2.05 (m, 2H, H2'), 1.91 (s,
3H, 5-Me), 1.74-1.60 (m, 1H, H6' ), 1.31 (t, 3H, J = 6 Hz,
CH2CH3) ppm.
communication3, but employing 6 equivalents of
bis(trimethylsilyl)trifluoroacetamide to give the
phosphonamidate dimers 8 and 9 respectively (64-79%). 8;
Clear colourless glass; 31P NMR (CDCl3, 162 MHz) 33.91
ppm. 9; Clear colourless glass; 31P NMR (CDCl3, 162 MHz)
34.38 ppm.
Analysis of the crude reaction mixture indicates <5%
epimerisation having occurred in preparation of either
diastereoisomer, as judged by 31P NMR.
22; White foam; 31P NMR (CDCl3, 162 MHz) 26.28 ppm; 1H
nmr (CDCl3, 400 MHz) 8.44 (s, br, 1H, HN3), 7.09 (s, 1H,
H6), 6.10 (d, 1H, J = 5 Hz, H1'), 4.18-4.03 (m, 2H, CH2CH3),
3.60-3.42 (m, 2H, H5'), 3.21-3.12 (m, br, 1H, PNH), 3.12-3.02
(m, 1H, H4'), 2.32-2.14 (m, 4H, H2', H3', H6' ), 1.94 (s, 3H,
5-Me), 1.72-1.59 (m, 1H, H6' ), 1.37 (t, 3H, J = 6 Hz,
CH2CH3) ppm.
(8) Van den Berg, G. R.; Platenburg, D. H. J. M.; Benschop, H. P.
Chem. Commun. 1971, 606. Dimukhametov, M. N.;
Nuredtinov, I. A. Izv. Akad. Nauk SSSR, Ser. Khim. 1983,
1214 (CAN 99:88285).
(9) Oligonucleotides prepared from 11 exhibited the same
hybridisation properties compared to analogue
(17) Stereochemical assignments were based upon NOE
experiments. The SP diastereoisomer 21 exhibited interactions
between the ethyl phosphonamidate methylene and the 5' ,
6' and 6' protons, suggesting multiple contributing
conformations. In contrast, the RP diastereoisomer 22
exhibited a strong interaction between the ethyl
oligonucleotides prepared as described in the previous
communication.3
phosphonamidate methylene and the 5' and 6' proton,
consistent with a single preferred conformation.
(10) Atherton, F. R.; Openshaw, H. T.; Todd, A. R. J. Chem. Soc.,
Chem.Commun. 1960, 660.
(11) Reiff, L. P.; Aaron, H. S. J. Am. Chem. Soc., Chem. Commun.
1970, 92, 5275.
(12) 5’-amino-3’-tert-butyldiphenylsilyl-5’-deoxythymidine 12
was prepared by catalytic hydrogenation of a methanol
solution of the corresponding azide,3 under 1 atmosphere of
hydrogen, over a palladium on carbon catalyst (10% by
weight), at room temperature for 18 h (>95%).
(18) Luckenbach, R. Phosphorus 1973, 3, 77. Omela czuk, J.;
Mikolajczyk, M. J. Am. Chem. Soc. 1979, 101, 7292.
(19) Pless, R. C.; Ts’o, P. O. P. Biochemistry 1977, 16, 1239.
Summers, M. F.; Powell, C.; Egan, W.; Byrd, R. A.; Wilson,
W. D.; Zon, G. Nucleic Acids Res. 1986, 14, 7421. Ferguson,
D. M.; Kollman, P. A. Antisense Research and Development
1991, 1, 243.
(13) Typical procedure; Anhydrous carbon tetrachloride (1 ml)
followed by anhydrous triethylamine (0.40 ml) was added
dropwise to a suspension of activated 3Å molecular sieves
(0.24 g) in a solution of the amine 12 (317 mg, 660 mol) and
Article Identifier:
1437-2096,E;2001,0,04,0473,0476,ftx,en;L21800ST.pdf
Synlett 2001, No. 4, 473–476 ISSN 0936-5214 © Thieme Stuttgart · New York