Synthesis and hybridisation properties of phosphonamidate ester modified nucleic acid

Article abstract of DOI:10.1055/s-2001-12328

The replacement of the two backbone oxygen atoms of the phosphodiester linkage of DNA with carbon and nitrogen in the form of a phosphonamidate ester linkage, in both possible regioisomeric forms, is described. An alkylation reaction with the 3′-C-P-N-5′ methyl phosphonamidate regioisomer yielded the corresponding N-methylated analogues. For each example separation into individual phosphonamidate ester diastereoisomers was performed prior to incorporation into oligonucleotides. The effect upon duplex stability for DNA oligonucleotides containing each of these modifications with complimentary RNA is reported.

Full text of DOI:10.1055/s-2001-12328

LETTER
467
Synthesis and Hybridisation Properties of Phosphonamidate Ester Modified
Nucleic Acid
Robin A. Fairhurst,1* Stephen P. Collingwood,1 David Lambert, Roger J. Taylor1
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
as the ester generates a chiral environment at phosphorus
Abstract: The replacement of the two backbone oxygen atoms of
and hence diastereoisomers for each regioisomer as
the phosphodiester linkage of DNA with carbon and nitrogen in the
shown in Figure 1. The importance of phosphorus stere-
ochemistry in determining the stability of duplexes in
form of a phosphonamidate ester linkage, in both possible regioiso-
meric forms, is described. An alkylation reaction with the 3’-C-P-
N-5’ methyl phosphonamidate regioisomer yielded the correspond- which such chiral modified oligonucleotides are incorpo-
ing N-methylated analogues. For each example separation into indi-
rated has been described for a number of backbone modi-
fications.8 Therefore, a sequence was sought which would
vidual phosphonamidate ester diastereoisomers was performed
prior to incorporation into oligonucleotides. The effect upon duplex
allow access to both diastereoisomers of the modifications
stability for DNA oligonucleotides containing each of these modifi-
1 and 2. In common with many other workers, a thymi-
dine-thymidine dimer approach was selected for the initial
cations with complimentary RNA is reported.
Key words: antisense, modified oligonucleotides, nucleosides,
evaluation requiring the preparation of phosphonamidate
phosphorus protecting groups
linked dinucleosides for incorporation into standard phos-
phoramidite oligonucleotide synthesis.
The application of modified oligonucleotides for disease
control, based upon the antisense concept of translational
arrest, is currently an important area of medicinal chemis-
try research.2 First-generation antisense oligonucleotides
were based upon the phosphorothioate modification as a
replacement for the enzymatically labile phosphodiester
linkage of ‘wild-type’ DNA. Current attention is focused
upon developing antisense agents with even greater in
vivo stability, improved cell penetration and enhanced
binding properties with complimentary RNA.3 As part of
Figure 1
a project to identify such novel second-generation oligo-
nucleotide modifications, we have been interested in
Our initial approach involved phosphorus to nitrogen
bond construction through a Staudinger reaction with H-
phosphinate and azido substituted nucleoside precursors.9
A retrosynthetic analysis outlining this strategy for forma-
tion of the key phosphonamidate linkage is shown in Fig-
ure 2. This disconnection required the synthesis of the
suitably protected 3’- and 5’-homologated H-phosphin-
ates 3, 4, and 5, the syntheses for which are described be-
low and shown in Schemes 1 and 2. The azide coupling
partners 6 and 7 were prepared from thymidine following
literature procedures.10
phosphorus containing replacements for the phosphodi-
ester backbone linkage of ‘wild-type’ DNA.4,5 In particu-
lar, the possibility of C-P-N replacements attracted our
attention in light of the reported enhanced binding to com-
plimentary RNA for the phosphoramidate 3’-N-P(O-)-O-
5’ modification, in which nitrogen replaces the 3’-oxy-
gen.6 Replacement of the second oxygen atom with car-
bon was anticipated to be beneficial due to the increased
nuclease stability associated with C-P backbone linkag-
es.4 Additionally, our own work has demonstrated en-
hanced hybridisation properties with the 3’-methylene
phosphonate modification 3’-C-P(O-)-O-5’, in which car-
bon replaces the 3’-oxygen.5 Recently, the related 3’-N-
P(Me)-O-5’ methyl phosphonamidate modification has
been described, but to date no hybridisation data has been
disclosed for this linkage.7 In this letter, we present our
initial results from the incorporation of the phosphonam-
idate ester C-P(OR)-N backbone modification into DNA.
Synthesis of the 5’-homologated H-phosphinate 3 was
achieved as follows. The 5’-homologated phosphinate 8
was made available from a boron trifluoride catalysed ox-
etane ring opening with a phosphinate stabilised carban-
ion, following the procedure previously reported by this
group.4 Subsequent hydrolysis of the 3’-benzoate fol-
lowed by silylation yielded 9. Selective removal of the
mixed orthoester protecting group from 9 upon treatment
with trimethylsilylchloride, ethanol and chloroform at
room temperature liberated the ethyl H-phosphinate 10.11
Hydrolysis with triethylamine, water and ethanol pro-
Direct replacement of the phosphodiester moiety of DNA
with the unsymmetrical phosphonamidate C-P-N linkage
leads to the two regioisomeric possibilities 1 and 2, Figure
1. Additionally, incorporation of the phosphorus moiety
Synlett 2001 No. 4, 467–472 ISSN 0936-5214 © Thieme Stuttgart · New York

468
R. A. Fairhurst et al.
LETTER
logue that could be readily converted to the iodide 12
upon treatment with methyltriphenoxyphosphonium io-
dide in DMF. The iodide 12 was then employed in an
alkylation reaction with the potassium anion of the differ-
entially protected hypophosphorous acid synthon 13 to
give the ethyl phosphinate 14.5,11 Deprotection of 14 in an
analogous manner to that described above for 10, using in
situ generated hydrogen chloride, yielded the ethyl H-
phosphinate 4. Subsequent hydrolysis followed by re-es-
terification gave the methyl H-phosphinates 5, Scheme 2.
Figure 2
duced the corresponding H-phosphinic acid, which was
re-esterified with allyl alcohol, using DCC as the con-
densing agent, to produce the allyl H-phosphinate 3,
Scheme 1.
Scheme
2
i) NaBH4, EtOH, rt (95-97%); ii) 1.1 equiv.
MeP+(OPh)3I-, 2 equiv. 2,6-lutidine, DMF, rt (68-76%); iii) 4 equiv.
13, 4 equiv. KHMDS, THF, -78ºC to rt (85-94%); iv) Me3SiCl, EtOH,
CHCl3, rt (70-87%); v) Et3N, EtOH, H2O, rt (87-92%); vi) MeOH,
DCC, 2 mol% DMAP, THF, rt (75-86%).
Following the above sequences, all the mononucleoside
H-phosphinates 3, 4 and 5 were obtained as essentially 1:1
mixtures of phosphorus diastereoisomers as adjudged by
31P NMR.
With the required intermediates in hand, dimer formation
was achieved upon bis(trimethylsilyl)trifluoroacetamide
catalysed reaction of the H-phosphinates 3, 4 and 5 with
the appropriate azidonucleoside derivative in pyridine at
room temperature, as shown in Scheme 3.9 In each case
after completion of the reaction, removal of volatiles pro-
duced mixtures of the desired products and trimethylsil-
ylated intermediates that were converted completely
through to the desired products upon refluxing in a mix-
ture of chloroform and methanol.13 From these reactions,
the three phosphonamidate dinucleosides 15, 16 and 17
were obtained in good yields, each as 1:1 mixtures of dia-
stereoisomers epimeric at the phosphorus centre. In the
case of 15, separation into the individual phosphorus dia-
stereoisomers, via flash column chromatography, was
readily achieved to provide the first eluting diastereoiso-
Scheme 1 i) NaOMe, MeOH, rt (85%); ii) TBDPS-Cl, imidazole,
DMF, rt (91%); iii) Me3SiCl, EtOH, CHCl3, rt (71%); iv) Et3N, EtOH,
H2O, rt (95-98%); v) allyl alcohol, DCC, 2 mol% DMAP, THF, rt (69-
73%).
The 3’-homologated H-phosphinates 4 and 5 were made
available from the previously described 3’-formylated
thymidine derivative 11.12 Reduction of 11 with sodium
borohydride in ethanol furnished the hydroxymethyl ana-
Synlett 2001, No. 4, 467–472 ISSN 0936-5214 © Thieme Stuttgart · New York

LETTER
Synthesis of Phosphonamidate Ester Modified Nucleic Acid
469
mer 15A and second eluting isomer 15B.14 These individ-
ual isomers were readily desilylated with
tetrabutylammonium fluoride (TBAF) in THF to yield the
corresponding 3’-alcohols, which were phosphitylated to
give the phosphoramidates 18A and 18B ready for oligo-
nucleotide synthesis. For the 3’-C-P-N-5’ analogues sep-
aration into the individual diastereoisomers immediately
after the coupling reaction proved to be tedious. However,
following desilylation of 16 with TBAF in THF and sub-
sequent selective 5’-tritylation allowed the readily separa-
ble 3’-alcohol diastereoisomers 19A and 19B to be
obtained.14 Phosphitylation of these individual diastereo-
isomers produced the dinucleoside phosphoramidites 20A
and 20B. In the methyl phophonamidate example 17 only
minimal differences in Rf between diastereoisomers were
observed throughout the analogues sequence and the
mixed isomers were investigated without separation.
Thus, the mixed isomer methyl phosphonamidate 5’-tri-
tylated dinucleoside 21 was phosphitylated to give 22 as a
1:1:1:1 mixture of diastereoisomers epimeric at both the
P(V) and P(III) centres.
N-Alkylation of the phosphonamidate linkage provides a
site for tethering additional functionality and reducing the
overall polarity, with the potential to improve cell perme-
ability and chemical stability.15 We selected N-methyl-
ation to establish the effect upon duplex stability for the
3’-C-P-N-5’ phosphonamidate system. Utilising the reac-
tion between H-phosphinate and azide to prepare the
phosphonamidate linkage precluded the introduction of
an N-alkyl substituent until after that step in the synthesis.
As a consequence, competitive alkylation at the N-3 posi-
tion of the thymine residues was anticipated to be prob-
lematic. This was overcome by selective introduction of
benzyloxymethyl protecting groups at these sites in the
bis-silylated intermediate 17, upon treatment with benz-
yloxymethyl chloride and diazabicycloundecane (DBU)
in acetonitrile at room temperature. No evidence of com-
petitive phosphonamidate alkylation was observed during
the protection of the two thymine residues. The product
from this reaction could then be N-methylated at the de-
sired position with an excess of methyl iodide, following
deprotonation with sodium hydride. At this point in the
synthesis, in contrast to the N-H precursors, separation of
the phosphonamidate diastereoisomers via flash column
chromatography was now readily achieved to give 23A
and 23B.14 Subsequent hydrogenolysis removed the benz-
yloxymethyl protecting groups to give the N-methylated
analogues of dinucleoside 17. These were then trans-
formed into the corresponding 5’-dimethoxytrityl 3’-
phosphoramidites 24A and 24B in an analogue manner to
that described above for the N-H derivatives, as shown in
Scheme 4.
Scheme 3 i) 5 equiv. Me3SiNC(OSiMe3)CF3, pyridine, 0 ºC to rt
(81-87%); ii) flash column chromatographic separation diastereoiso-
mers, silica gel, eluent; 5% methanol in ethyl acetate; iii) 1.5 equiv.
Bu4N+F-, THF, 0 ºC (83-92%); iv) 3 equiv. (iPr2N)2POCH2CH2CN, 5
equiv. diisopropylammonium tetrazolide, CH2Cl2, rt (69-78%); v)
3 equiv. Bu4N+F-, THF, 0 ºC (96%); vi) 1.2 equiv. DMTrCl, pyidine,
rt (68-88%); vii) flash column chromatography, silica gel, eluent;
10% ethanol in chloroform.
Oligonucleotides were prepared incorporating each of the
above seven modified dinucleosides following standard
phosphoramidite coupling protocols.16 Using this ap-
Synlett 2001, No. 4, 467–472 ISSN 0936-5214 © Thieme Stuttgart · New York

470
R. A. Fairhurst et al.
LETTER
is not anticipated to account for the majority of the differ-
ence in duplex stability between the two regioisomeric
modifications. Phosphonamidate N-methylation with
modification 2 resulted in oligonucleotides exhibiting
similar affinities to their unsubstituted counterparts, but
again direct comparison with individual diastereoisomers
is complicated by different ester residues. However, com-
paring the mean Tm value for N-methylated methyl ester
diastereoisomers 24A and 24B with the corresponding
mixed N-H diastereoisomers 22, in the singularly modi-
fied sequence (-1.8 ºC vs. -1.8 ºC), indicates no substantial
difference in affinity.
The incorporation of a less electronegative 3’-substituent
in modifications 1 and 2 is anticipated to favour a 3’-endo
sugar conformation. This primary structural bias has
formed the basis for rationalising the origin of the en-
hanced hybridisation properties for a number of such
backbone modifications.18 These enhanced affinities are
considered to arise from the above furanose conforma-
tional preference promoting the formation of a more sta-
ble A-type hybrid duplex. Therefore, the difference
between 1 and 2 may be attributed to the facility with
which the internucleotide linkage can be incorporated
whilst adopting a conformation required for the formation
of such an A-type duplex. Comparison of 1 with the du-
plex stabilising 3’-N-P-O-5’ phosphoramidates,6 and 2
with the duplex destabilising 3’-C-P-C-5’ phosphinates,4
indicates that in both instances the interchange of a 5’-het-
eroatom bonded to phosphorus for a carbon atom produc-
es a marked decrease in Tm. Inclusion of such a 5’-carbon
substituent results in the incorporation of two adjacent
methylenes into the backbone linkage. The detrimental ef-
fect upon free energy of duplex formation arising from
this structural feature most probably has key contributions
due to excess conformational flexibility19 and from per-
turbation of hydration due to hydrophobicity.20
Scheme 4 i) PhCH2 OCH2Cl, DBU, CH3CN, 0 ºC (83-85%); ii) 100
equiv. MeI, NaH, toluene, 40 ºC, sealed bomb (91-95%); iii) flash co-
lumn chromatographic separation diastereoisomers, silica gel, eluent;
ethyl acetate; iv) 5% Pd on carbon, 1 atmosphere H2, EtOH, rt (84-
90%); v) 3 equiv. Bu4N+F-, THF, 0 ºC (89-94%); vi) 1.2 equiv. DM-
TrCl, pyidine, rt (65-71%); vii) 3 equiv. (iPr2N)2POCH2CH2CN, 5
equiv. diisopropylammonium tetrazolide, CH2Cl2, rt (63-74%).
proach 15mer and 17mer sequences, containing either one
or five of the phosphonamidate modified linkages respec-
tively, were synthesised and the melting data obtained
with complimentary RNA are shown in the Table.17
From the melting data it is apparent that only the longer
retained diastereoisomer 20B of the 3’-C-P-N-5’ phos-
phonamidate modification 2 was found to enhance duplex
stability over ‘wild-type’ DNA. Comparison of the Tm
values of the methyl ester 22 with the mean value for the
ethyl esters 20A and 20B, in the singularly modified
15mer sequence (-1.8 ºC vs. -0.23 ºC), suggests a possible
beneficial effect by increasing the size of the ester residue
from methyl to ethyl for the modification 2. In contrast,
modification 1, in which carbon and nitrogen are
switched, resulted in a significant reduction in hybridisa-
tion energy for both diastereoisomers. Direct comparison
of the two modifications is complicated by the presence of
different phosphonamidate ester residues. However, this
In summary, examples of all four possible isomeric phos-
phonamidate ester C-P-N replacements for the phospho-
diester linkage of DNA have been investigated. From
these studies one diastereoisomer of the 3’-C-P-N-5’ iso-
mer was identified as enhancing the stability of duplexes
with complimentary RNA relative to ‘wild-type’ DNA.
Table
Tm for TTTTTCTCTCTCTCT ‘wild-type’ DNA with complimentary RNA 52.7 ºC.
Tm for GCGTTTTTTTTTTTGCG ‘wild-type’ DNA with complimentary RNA 50.2 ºC.
‡tt indicates modified dimer with phosphonamidate linkage.
Synlett 2001, No. 4, 467–472 ISSN 0936-5214 © Thieme Stuttgart · New York

LETTER
Synthesis of Phosphonamidate Ester Modified Nucleic Acid
to warm room temperature and stirred for 18 h before
471
The introduction of an N-methyl residue into this modifi-
cation was discovered to have no pronounced effect upon
duplex stability. Further investigations with this potential-
ly interesting phosphonamidate modification 20B, includ-
ing determination of the phosphorus stereochemistry, are
reported in the following letter.21
quenching by the addition of methanol (10 ml). Removal of
volatiles followed by further addition of methanol (10 ml) and
evaporation yielded a crude mixture of trimethylsilylated
phosphonamidate dimers. Cleavage of the trimethylsilyl
residues was achieved after refluxing the crude product for 5
h in chloroform (15 ml) and methanol (5 ml). Removal of
volatiles followed by flash column chromatography on silica
gel, eluting with a gradient from 2% to 10% ethanol in
chloroform, yielded the phosphonamidate dimer 16 as a
viscous clear colourless oil (686 mg, 87%): 31P NMR (CDCl3,
162 MHz) 32.3, 31.6 ppm.
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.
(14) Flash column chromatography was performed using Merck
Silica Gel 60 (0.040-0.063mm). First eluting diastereoisomer
assigned A, second eluting diastereoisomer assigned B. NMR
spectra were recorded with a Bruker AC400 or Bruker
DRX500 instrument. 31P NMR shifts are given as ppm values
relative to phosphoric acid.
References and Notes
(1) New address: Novartis Horsham Research Centre,
Wimblehurst Road, Horsham, West Sussex, RH12 5AB, UK.
(2) Wahlestedt, C.; Good, L. Curr. Opin. in Drug Discov. Dev.
1999, 2, 142. Bennett, C. F. Exp. Opin. Invest. Drugs 1999, 8,
237. Ma, D. D. F.; Rede, T.; Naqvi, N. A.; Cook, P. D.
Biotechnol. Annu. Rev. 2000, 5, 155.
(3) Uhlmann, E.; Peyman, A. Chem Rev. 1990, 90, 544.Crooke, S.
T.; Lebleu, B.; Antisense Research and Applications; CRC
Press: Boca Raton, 1993. Matteucci, M. D.; Martin, J. C. J.
Med. Chem. 1993, 36, 1923. Crooke, S. T. Med. Res. Rev.
1996, 16, 319. Freier, S. M.; Altmann, K-H. Nucleic Acids
Res. 1997, 25, 4429. Uhlmann, E. Curr. Opin. in Drug Discov.
Dev. 2000, 3, 203.
(4) Collingwood, S. P.; Baxter, A. D. Synlett 1995, 703.
Collingwood, S. P.; Taylor, R. J. Synlett 1998, 283.
(5) Collingwood, S. P.; Douglas, M. E.; Natt, F.; Pieles, U.
Phosphorus, Sulphur and Silicon 1999, 144-146, 645.
(6) Gryaznov, S.; Chen, J. K. J. Am. Chem. Soc. 1994, 116, 3143.
Chen, J-K.; Schultz, R. G.; Lloyd, D. H.; Gryaznov, S. M.
Nucleic Acid Research 1995, 23, 2661. Gryaznov, S. M.;
Lloyd, D. H.; Chen, J-K.; Schultz, R. G.; DeDionisio, L. A.:
Ratmeyer, L.; Wilson, W. D. Proc. Natl. Acad. Sci. USA 1995,
92, 5798. Gryaznov, S.; Skorski, T.; Cucco, C.; Nieborowska-
Skorska, M.; Chiu, C. Y.; Lloyd, D.; Chen, J-K.;
Koziolkiewicz, M.; Calabretta, B. Nucleic Acids Research
1996, 24, 1508. McCurdy, S. N.; Nelson, S. N.; Hirschbein, B.
L.; Fearon, K. L. Tetrahedron Lett. 1997, 38, 207. Tereshkov,
S.; Gryaznov, S.; Egli, M. J. Am. Chem. Soc. 1998, 120, 269.
(7) Kers, I.; Bollmark, M.; Kraszewski, A.; Stawinski, J.
Phosphorus, Sulfur and Silicon 1999, 144-146, 637.
(8) Lebedev, A. V.; Wickstrom, E. Perspectives in Drug
Discovery and Design 1996, 4, 17.
15A; 31P NMR (CDCl3, 202 MHz) 35.44 ppm; 1H (CDCl3,
400 MHz) 10.16 (s, 1H), 8.96 (s, 1H), 7.65-7.58 (m, 4H),
7.52 (s, 1H), 7.44-7.18 (m, 15H), 6.94 (s, 1H), 6.81 (d, 4H,
J = 9 Hz), 6.54 (t, 1H, J = 7 Hz), 6.22 (t, 1H, J = 7 Hz), 5.78-
5.67 (m, 1H), 5.18-5.03 (m, 2H), 4.80-4.69 (m, 1H), 4.40-4.34
(m, 1H), 4.25-4.14 (m, 1H), 4.10-4.05 (m, 1H), 3.96-3.90 (m,
1H), 3.87-3.70 (m, 2H), 3.75 (s, 6H), 3.44 (dd, 1H, J = 2 and
11 Hz), 3.24 (dd, 1H, J = 2 and 11 Hz), 2.38-2.21 (m, 2H),
2.18-2.10 (m, 1H), 2.02-1.89 (m, 1H), 1.81 (s, 3H), 1.74-1.39
(m, 3H), 1.49 (s, 3H), 1.05 (s, 9H).
15B; 31P NMR (CDCl3, 202 MHz) 35.89 ppm; 1H (CDCl3,
400 MHz) 10.23 (s, 1H), 8.99 (s, 1H), 7.63-7.57 (m, 4H),
7.47 (s, 1H), 7.44-7.17 (m, 15H), 6.83 (s, 1H), 6.80 (d, 4H,
J = 9 Hz), 6.49 (t, 1H, J = 7 Hz), 6.14 (t, 1H, J = 7 Hz), 5.92-
5.80 (m, 1H), 5.31-5.14 (m, 2H), 4.80-4.71 (m, 1H), 4.44-4.34
(m, 1H), 4.31-4.22 (m, 1H), 4.07-4.00 (m, 1H), 3.91-3.82 (m,
2H), 3.76 (s, 6H), 3.74-3.66 (s, 1H), 3.38 (d, 1H, J = 10 Hz),
3.22 (d, 1H, J = 10 Hz), 2.35-2.25 (m, 1H), 2.23-2.10 (m, 2H),
1.99-1.89 (m, 1H), 1.77 (s, 3H), 1.71-1.42 (m, 3H), 1.49 (s,
3H), 1.06 (s, 9H).
19A; 31P NMR (CDCl3, 162 MHz) 33.33 ppm; 1H (CDCl3,
400 MHz) 7.64 (s, 1H), 7.40 (d, 2H, J = 7 Hz), 7.32-7.24 (m,
9H), 7.23-7.16 (m, 1H), 7.09 (s, 1H), 6.84-6.77 (m, 4H), 6.04-
5.97 (m, 2H), 4.44-4.37 (m, 1H), 4.10-3.72 (m, 3H), 3.76 (s,
6H), 3.52 (d, 1H, J = 12 Hz), 3.30-3.07 (m, 3H), 2.79-2.53 (m,
3H), 2.37-2.19 (m, 3H), 1.92-1.77 (m, 1H), 1.84 (s, 3H), 1.68-
1.54 (m, 1H), 1.45 (s, 3H), 1.26-1.18 (m, 3H).
19B; 31P NMR (CDCl3, 162 MHz) 33.26 ppm; 1H (CDCl3,
500 MHz) 7.56 (s, 1H), 7.35 (d, 2H, J = 7 Hz), 7.28-7.08 (m,
11H), 6.75 (dd, 4H, J = 1 and 7 Hz), 5.98-5.86 (m, 2H), 4.46-
4.30 (m, 1H), 3.99-3.80 (m, 3H), 3.72 (s, 6H), 3.40 (d, 1H,
J = 12 Hz), 3.27-3.09 (m, 3H), 2.89-2.80 (m, 1H), 2.77-2.66
(m, 1H), 2.61-2.54 (m, 1H), 2.36-2.15 (m, 3H), 1.87-1.75 (m,
4H), 1.63-1.51 (m, 1H), 1.42 (s, 3H), 1.16 (t, 3H, J = 7 Hz).
23A; 31P NMR (CDCl3, 162 MHz) 34.18 ppm; 1H (CDCl3,
400 MHz) 7.86 (s, 1H), 7.68-7.55 (m, 8H), 7.44-7.18 (m,
23H), 6.55-6.49 (m, 1H), 6.16 (t, 1H, J = 7Hz), 5.51-5.42 (m,
4H), 4.71-4.64 (m, 4H), 4.07-3.91 (m, 3H), 3.83-3.78 (m, 1H),
3.75-3.70 (m, 1H), 3.39 (d, 3H, J = 17 Hz), 3.19-3.09 (m, 1H),
2.68-2.55 (m, 1H), 2.40-2.27 (m, 1H), 2.30 (d, 3H, J = 14 Hz),
2.25-2.16 (m, 1H), 2.12-2.02 (m, 1H), 1.89 (s, 3H), 1.76-1.57
(m, 4H), 1.61 (s, 3H), 1.08 (s, 9H), 1.05 (s, 9H).
(9) Letsinger, R. L.; Heavner, G. A. Tetrahedron Lett. 1975, 147.
Hata, T.; Yamamoto, I; Sekine, M. Chem. Lett. 1976, 601.
(10) 5’-O-dimethoxytrityl-3’-azido-3’-deoxythymidine 6 Mag,
M.; Schmidt, R.; Engels, J. W. Tetrahedron Lett. 1992, 48,
7319. 3’-O-tert.butyldiphenylsilyl-5’-azido-5’-
deoxythymidine 7 Prepared from 3’-O-
tert.butyldiphenylsilyl-5'-iodo-5'-deoxythymidine (Haung, J.;
McElroy, E. B.; Widlanski, T. S. J. Org. Chem. 1994, 59,
3520.) following treatment with sodium azide (5 equivalents)
in DMF at 85 °C for 5 h (95%).
(11) Baylis, E. K. Tetrahedron Lett. 1995, 36, 9385.
(12) 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.
(13) Typical procedure; Bis(trimethylsilyl)trifluoroacetamide
(1.00 ml, 3.76 mmol) was added dropwise to a solution of the
H-phosphinate 4 (430 mg, 753 mol) and azide 7 (404 mg,
791 mol) in anhydrous pyridine (7.5 ml) at 0 ºC. Upon
completion of the addition the reaction mixture was allowed
23B; 31P NMR (CDCl3, 162 MHz) 35.50 ppm; 1H (CDCl3,
400 MHz) 7.68-7.56 (m, 9H), 7.46-7.17 (m, 23H), 6.52-6.45
(m, 1H), 6.04-5.99 (m, 1H), 5.50-5.39 (m, 4H), 4.72-4.65 (m,
4H), 4.07-3.92 (m, 3H), 3.74-3.63 (m, 2H), 3.40 (d, 3H, J = 17
Hz), 3.35-3.24 (m, 1H), 2.69-2.57 (m, 1H), 2.40-2.25 (m, 2H),
2.29 (d, 3H, J = 14 Hz), 2.04-1.54 (m, 4H), 1.93 (s, 3H), 1.56
(s, 3H), 1.48-1.37 (m, 1H), 1.07 (s, 9H), 1.05 (s, 9H).
Synlett 2001, No. 4, 467–472 ISSN 0936-5214 © Thieme Stuttgart · New York

472
R. A. Fairhurst et al.
LETTER
(15) De Mesmaeker, A.; Häner, R.; Martin, P.; Moser, H. E. Acc.
Chem. Res. 1995, 28, 366.
Turner, D. H. Biopolymers 1982, 22, 1107.). All values are
averages of at least three experiments. The absolute error of
the Tm values is ±0.5 °C.
(16) 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. For the steps
involving incorporation of modified dimers, double couplings
with double reaction times were employed.
(18) Fraser, A.; Wheeler, P.; Cook, P. D.; Sanghvi, Y. S. J.
Heterocyclic Chem. 1993, 30, 1277. Gryaznov, S.; Schultz, R.
Tetrahedron Lett. 1994, 35, 2489. Mohan, V.; Griffey, R. H.;
Davis, D. R. Tetrahedron 1995, 51, 6855.
(19) De Mesmaeker, A.; Waldner, A.; Sanghvi, Y. S.; Lebreton, J.
Bioorg. Med. Chem. Lett. 1994, 4, 395.
(20) Varma, R. S. Synlett 1993, 621. Egli, M. Angew. Chem., Int.
Ed. Engl. 1996, 35, 1895.
(21) Fairhurst, R.A.; Collingwood, S.P.; Lambert, D. Synlett 2001,
473.
(17) The thermal dematuration of DNA/RNA hybrids was
performed at 260 nm using a Gifford Response II
spectrophotometer (Ciba-Corning Diagnostics Corp., Oberlin,
OH) absorbance vs temperature profiles were measured at
4 m 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.; Albergo, T. D.;
Article Identifier:
1437-2096,E;2001,0,04,0467,0472,ftx,en;L21700ST.pdf
Synlett 2001, No. 4, 467–472 ISSN 0936-5214 © Thieme Stuttgart · New York