Solid-phase synthesis of oligonucleotides containing a bipyridine ligand at the 3′-3′ inversion of polarity site

Article abstract of DOI:10.1016/S0960-894X(00)00673-9

The preparation of a solid support useful for the synthesis of oligonucleotides with a 3′-3′ inversion of polarity, via a linker containing a chelating molecule, namely 2,2′-bipyridine, is described.

Full text of DOI:10.1016/S0960-894X(00)00673-9

Bioorganic & Medicinal Chemistry Letters 11 (2001) 383±386
Solid-Phase Synthesis of Oligonucleotides Containing a Bipyridine
Ligand at the 30-30 Inversion of Polarity Site
Aldo Galeone, Luciano Mayol,* Giorgia Oliviero, Daniela Rigano and Michela Varra
Dip. di Chimica delle Sostanze Naturali, Univ. di Napoli ``Federico II'', Via D. Montesano, 49 I-80131 Napoli, Italy
Received 28 July 2000; revised 20 November 2000; accepted 23 November 2000
AbstractÐThe preparation of a solid support useful for the synthesis of oligonucleotides with a 30-30 inversion of polarity, via a
linker containing a chelating molecule, namely 2,20-bipyridine, is described. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
In this frame, we have designed and prepared a solid-
phase support useful for the synthesis of oligonucleo-
tides with 30-30 inversion containing a chelating agent,
namely a bipyridine moiety. Transition metal com-
plexesÐcontaining oligonucleotides (ODNs)10,11Ð
represent a topic in constant growth. Such conjugates
are involved in the study of electron transfer
processes12 14 as well as in the development of arti®cial
nucleases characterized by a high sequence-speci®city
and eciency.15,16 In fact, a metal centre tethered to a
particular sequence could enable the complex to oxida-
tively modify or cleave a nucleic acid target.
There is a growing interest in design and synthesis of
newly modi®ed oligonucleotides (ODNs) as potential
drugs in anticancer or antiviral therapy.1,2 Such mole-
cules, indeed, may be very ecient tools in the selective
inhibition of gene expression, both in the antisense
approach, where the target is an mRNA, and in the
antigene strategy, through the formation of a triple helix
complex (triplex) with a selected double-stranded DNA
sequence.2 6 In the latter strategy, one of the major
restrictions is that a stable triplex, via Hoogsteen triads
formation, can be envisaged under physiological condi-
tions only for relatively long (15±17 bases) homopurine
tracts within the same strand of a double helical DNA
fragment and such a requirement is rarely met in biolo-
gically important regions of DNA. To recognize a wider
number of DNA sequences, a possible solution is the
use of ODNs containing a 30-30 inversion of polarity,
able to target (purine)m(pyrimidine)n sequences by
hybridization of the adjacent purine blocks on alternate
strands and by switching strand at the junction between
the oligopurine and the oligopyrimidine domains.7 9
From a chemical point of view, the 30-30 inversion can
be ful®lled by a suitable linker capable of crossing the
major groove and whose properties can be, in addition,
exploited to supply the oligonucleotide with useful
characteristics. For example, the 30-30 linker may incor-
porate an intercalating agent or a major groove ligand
in order to improve the hybridization between the probe
ODN and the target duplex.
Chemistry
The synthetic route (see Scheme 1) used for the pre-
paration of oligomers with a 30-30 inversion containing a
bipyridine moiety is based on a three-functionalized
molecule (2-amino-1,3-propandiol) that allows: (i)
anchorage to the polymeric support (Tentagel-NH2); (ii)
linkage to the metal complexing unit (2,20-dipyridine);
(iii) oligonucleotide chain assembly.
2-Amino-1,3-propandiol (1) is protected at the amino
function and at one of the two hydroxyls by 9-¯uor-
enylmethoxy-carbonyl (Fmoc) and 4,40-dimethoxytrityl
protecting groups, respectively (derivatives 2 and 3).17,18
Subsequently, the Fmoc group is removed by piperidine
thus giving derivative 419 which, in turn, is reacted with the
penta¯uorophenolic ester of 2,20-bipyridine-4,40-dicar-
boxylic acid (6)20 a€ording derivative 7.21 Compound 6
represents the activated chelating molecule and is obtained
by reaction of commercially available 2,20-bipyridine-4,40-
dicarboxylic acid (5) with the penta¯uorophenolic ester of
*Corresponding author. Fax: +39-81-678552; e-mail: mayoll@unina.it
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00673-9

384
A. Galeone et al. / Bioorg. Med. Chem. Lett. 11 (2001) 383±386
the tri¯uoroacetic acid. Derivative 7 is turned into
compound 10, the building block to be linked to the
solid support, by treatment, in sequence, with: (i) t-
butyl-dimethyl-silyl-chloride22 in order to protect one of
the two hydroxyl-groups; (ii) succinic anhydride23 to
obtain a carboxylic function useful for the anchorage to
the solid support; (iii) the penta¯uorophenolic ester of the
tri¯uoroacetic acid for the activation of the carboxylic
function.24 Finally, 10 is anchored to the resin (Tentagel-
NH2),25 to give the functionalized support (11) whose
loading (88 mmol/g on average) is estimated by spectro-
scopic measurement of 4,40-dimethoxytriphenylmethyl
cation released by acidic treatment on a weighed sample
of the support.
The so obtained Tentagel resin 11 can be used for the
assembly of 30-30 inverted symmetric sequence by stan-
dard phosphoramidite chemistry.
Results and Discussion
The functionalized support 11 was subjected to one
coupling cycle in an automated DNA synthesizer, using
Scheme 1. (a) Fmoc-N-hydroxysuccinimide ester in dry DMF; (b) 4,40-dimethoxytrityl chloride, 4-(dimethylamino)pyridine in dry Py; (c) piperidine
in dry DMF; (d) penta¯uorophenolic ester of tri¯uoroacetic acid (CF3COOC6F5) in dry DMF; (e) dry DMF; (f) t-butyldimethylsilylchloride
(TBDMSiCl), imidazole, in dry DMF; (g) succinic anhydride, 4-(dimethylamino)pyridine, in dry Py; (h) penta¯uorophenolic ester of tri¯uoroacetic
acid (CF3COOC6F5) in dry DMF; (i) tentagel-NH2 in dry DMF; (l) coupling cycle and trityl groups removal; (m) 25% aqueous NH3.

A. Galeone et al. / Bioorg. Med. Chem. Lett. 11 (2001) 383±386
385
thymidine phosphoramidite as a building block, to ver-
ify its resistance to the reagents and conditions usually
employed in DNA solid-phase synthesis. After treat-
ment with dichloroacetic acid to remove the trityl
groups, the resin underwent a mild treatment26 with
ammonia to detach the nucleotidic material and to
deprotect the phosphate groups, as well. Crude material
was puri®ed by reversed phase HPLC thus a€ording
two main products, which were shown to be, by ESI-
MS analyses, the completely deprotected dimer 50-thy-
mine-30-dipyridine-30-thymine-50 (13a) and the same
molecule still carrying the t-butyldimethylsilyl group
(13b). As expected, the latter compound could be turned
into the former one by treatment with tetra-
butylammonium ¯uoride.27 The same functionalized
support has been exploited for the preparation of a
number of conjugates of the type 50-ODN-30-dipyridine-
30-ODN-50, whose hybridization properties towards
speci®c double-stranded DNA targets are under inves-
tigation in order to verify whether the linker allows the
molecule to eciently cross the major groove without
causing severe distortions of the triple helix. Particu-
larly, we prepared successfully oligonucleotides 50-T8-30-
DiPy-30-T8-50 (I) and 50-(CT)4-30-DiPy-30-(TC)4-50 (II).28
10. Bigey, P.; Pratviel, G.; Meunier, B. Nucleic Acids Res.
1995, 23, 3894 and references cited therein.
11. Wiederholt, K.; McLaughlin, L. W. Nucleic Acids Res.
1999, 27, 2487 and references cited therein.
12. Kelley, S. O.; Holmlin, R. E.; Stemp, E. D. A.; Barton,
J. K. J. Am. Chem. Soc. 1997, 119, 9861.
13. Lewis, F. D.; Wu, T.; Zhang, Y.; Letsinger, R. I. Science
1997, 227, 673.
14. Breslin, D.; Coury, J.; Anderson, J.; McFail-Isom, L.;
Kan, Y.; Williams, L. D.; Bottomley, L.; Scuster, G. B. J. Am.
Chem. Soc. 1997, 119, 5043.
15. Sigman, D. S.; Mazumder, A.; Perrin, D. M. Chem. Rev.
1993, 93, 2295.
16. Pyle, A. M.; Barton, J. K. Prog. Inorg. Chem. 1990, 38,
413.
17. Procedure for the synthesis of 2: 1 g (10.9 mmol) of 2-
amino-1,3-propandiol (1) and 4.85 g (14.4 mmol) of 9-¯uor-
enylmethyl-N-succinimidylcarbonate (Fmoc-OSu) are dis-
solved in dry N,N-dimethylformamide (25 mL) and stirred for
24 h. The reaction is monitored by TLC (CHCl3/CH3OH
95:5). Crude material is puri®ed by extraction with H2O
(2%MeOH)/n-hexane. The aqueous phase is evaporated to
dryness recovering 3.1 g of product (90% yield). 1H NMR
(500 MHz, CD3OD): d 7.82 (2H, d, J=7.5 Hz), 7.70 (2H, d,
J=7.5 Hz), 7.42 (2H, t, J=7.5 Hz), 7.34 (2H, t, J=7.5 Hz),
4.39 (2H, d, J=6.8 Hz), 4.24 (1H, t, J=6.8 Hz), 4.25±3.55
(5H, m).
18. Procedure for the synthesis of 3: 3 g (9.6 mmol) of 2-[(9-
¯uorenylmethoxy-carbonyl)amino]-1,3-propandiol are dried
by coevaporations (Â3) with dry pyridine and then dissolved
in the same solvent (20 mL). 2.27 g (6.7 mmol) of 4,40-dime-
thoxytrityl chloride and a catalytic amount of dimethylamino
pyridine (50 mg, 0.4 mmol) are added to the solution. After
stirring for 2 h, the reaction is quenched with methanol and
evaporated to dryness. The product is puri®ed on a silica gel
column eluted with n-hexane/ethyl acetate 1:1. Yield: 3.5 g
The solid-phase support we have prepared allows the
synthesis of conjugates containing a ligand at the 30-30
inversion of polarity site which, to the best of our
knowledge, represents the ®rst example of such modi®ed
oligonucleotides. The metal-complexing molecule,
namely 2,20-bipyridine, was chosen on the basis of its
well-known coordination properties.29 The syntheses of
similar supports based on other linkers characterized by
di€erent ¯exibility, are currently in progress.
(59%). 1H NMR (500 MHz, CDCl3):
d 7.78 (2H, d,
J=7.5 Hz), 7.61 (2H, t, J=7.5 Hz), 7.42±7.20 (13H, m), 6.80
(4H, d, J=7.2 Hz), 4.42 (2H, d, J=6.8 Hz), 4.19 (1H, t,
J=6.8 Hz), 3.90±3.61 (11H, m).
Acknowledgements
19. Procedure for the synthesis of 4: 3.5 g (5.7 mmol) of 1-
(2,20-dimethoxytriphenylmethoxy)-2-[(9-¯uorenylmethoxy-car-
bonyl)amino]-propan-3-ol are treated with a freshly prepared
solution of 10% piperidine in dry N,N-dimethylformamide
(15 mL). After stirring for 3 h, the reaction mixture is evapo-
rated to dryness and puri®ed on a silica gel column eluted with
increasing amounts of CH3OH in CHCl3 (1% Py). Yield: 2.2 g
(98%). 1H NMR (500 MHz, CDCl3): d 7.51±7.20 (9H, m), 6.96
(4H, d, J=7.2 Hz), 3.80 (6H, s) 3.65 (1H, m), 3.50 (1H, m),
3.19 (1H, m), 3.10 (1H, m), 3.65 (1H, m).
20. Procedure for the synthesis of 6: 400 mg (1.6 mmol) of
2,20-bipyridine-4,40-dicarboxylic acid (5) are dried in vacuum
and dissolved in dry N,N-dimethylformamide (15 mL) to
which a few drops of pyridine have been added. Then, the
penta¯uorophenolic ester of tri¯uoroacetic acid (780 mL,
3.8 mmol) is added and the solution kept overnight under
stirring at room temperature. The reaction is monitored by
TLC (CHCl3/CH3OH 8:2). The mixture is diluted with ethyl
acetate and rinsed with HCl 0.1 N (Â3) and, successively, with
5% sodium bicarbonate (Â3). The organic phase is evaporated
to dryness to give 6 in 96% yield.
This work is supported by Italian M.U.R.S.T. and
C.N.R. The authors are grateful to ``Centro Ricerche
Interdipartimentale
C.R.I.A.S., for supplying NMR facilities.
di
Analisi
Strumentale'',
References and Notes
1. Cohen, J. S. Oligonucleotides, Antisense Inhibitors of Gene
Expression; Macmillan Press: London, 1989.
2. Wickstrom, E. Prospect for Antisense Nucleic Acids Ther-
apy of Cancer and AIDS; Wiley-Liss: New York, 1991.
3. Moser, H. E.; Dervan, P. B. Science 1987, 238, 645.
4. Uhlmann, E.; Peyman, A. Chem. Rev. 1990, 90, 543.
5. Englisch, U.; Gauss, D. H. Angew. Chem., Int. Engl. Ed.
1991, 30, 613.
6. Thuong, N. T.; Helene, C. Angew. Chem., Int. Engl. Ed.
1993, 32, 666.
7. Asseline, U.; Thuong, N. T. Tetrahedron Lett. 1994, 35,
5221.
21. Procedure for the synthesis of 7: 631 mg (1.09 mmol) of
the penta¯uorophenolic ester of 2,20-bipyridine-4,40-dicar-
boxylic acid (6) are dissolved in dry N,N-dimethylformamide
8. Zhou, B. W.; Marchand, C.; Asseline, U.; Thuong, N. T.;
Sun, J. S.; Garestier, T.; Helene, C. Bioconjugate Chem. 1995,
6, 516.
9. De Napoli, L.; Messere, A.; Montesarchio, D.; Pepe, A.;
Piccialli, G.; Varra, M. J. Org. Chem. 1997, 26, 9025.
(10 mL) containing a few drops of dry pyridine. 2.2 g
(5.6 mmol, 8 equiv) of derivative 4 are added under stirring
and the solution is kept at room temperature overnight. The
reaction is monitored by TLC (CHCl3/CH3OH 92:8). The
solvent is removed under reduced pressure and the product

386
A. Galeone et al. / Bioorg. Med. Chem. Lett. 11 (2001) 383±386
puri®ed by a silica gel column eluted with increasing amounts
of CH3OH in CHCl3 (1% Py). Yield: 0.92 g (85%). ESI-MS:
993.4 m/z [M H] .
The reaction is stirred at room temperature overnight and
monitored by TLC (n-hexane/ethyl acetate 1:1). Product is
used in the next step without any puri®cation.
22. Procedure for the synthesis of 8: 1.1 g (1.1 mmol) of deri-
vative 7 are dried by coevaporations (Â3) with dry pyridine
and then dissolved in dry N,N-dimethylformamide (10 mL)
containing a few drops of pyridine. 100 mg (1.5 mmol) of imi-
dazole and 100 mg (0.7 mmol) of t-butyldimethylsilyl chloride
are added and the solution is kept overnight at room tem-
perature under stirring. The reaction is monitored by TLC
(CHCl3/CH3OH 95:5). The product is puri®ed on a silica gel
column eluted with increasing amounts of CH3OH in CHCl3
(1% Py). Yield: 700 mg (58%).
25. Attachment of derivative 10 to the resin: 65 mg of Tenta-
gel-NH2 are washed with CHCl3 (Â3), MeOH (Â3), acetone
(Â3) and dried under vacuum. The resin is then washed with
dry CH2Cl2 and allowed to swell at 50 ꢀC for 1 h. Finally it is
washed with dry Py and dried under vacuum, once again, for
3 h. Derivative 10 (210 mg) dissolved in dry DMF (1% dry Py)
(1 mL) is added to the resin and the suspension is kept 24 h in
an oscillating vessel. The resulting polymer is washed with dry
DMF (Â2) and CHCl3 (1% dry Py) (Â2) and dried under
vacuum.
23. Procedure for the synthesis of 9: A mixture of 700 mg
(0.6 mmol) of derivative 8 and 80 mg of dimethylamino pyri-
dine (0.6 mmol) is dried by coevaporations (Â3) with dry pyr-
idine and then dissolved in this solvent (8 mL). After addition
of 240 mg (2.4 mmol) of succinic anhydride the reaction is kept
overnight at room temperature under stirring. The reaction is
monitored by TLC (CHCl3/acetone 6:4). The product is pur-
i®ed on a silica gel column eluted with increasing amounts of
CH3OH in CHCl3 (1% Py), to give 704 mg of product (97%
yield). ESI-MS: 1231.7 m/z [M+Na]+.
26. Mild conditions (25% aqueous NH3, 1.5 h, room tem-
perature) are required in order to prevent hydrolysis of the
amide linkage between the bipyridine unit and 1,3-dihydroxy-
2-amino moiety. In the case of the synthesis of C-containing
sequences the more labile t-butylphenoxyacetyl (tBPA) pro-
tecting group for the exocyclic amino group of cytosine has
been used.
27. Gait, M. J.; Pritchard, C.; Slim, G. Oligonucleotides and
Analogues. A Practical Approach; IRL Press: Oxford, 1991; pp
36±37.
24. Procedure for the synthesis of 10: 740 mg (0.6 mmol) of
derivative 9 are dissolved in dry N,N-dimethylformamide
(5 mL). 0.5 mL (0.85 mmol) of the penta¯uorophenolic ester of
tri¯uoroacetic acid and 150 mL of dry pyridine were added.
28. ESI-MS analysis in the negative mode: measured mass
5190.1, expected mass 5182.8 (I); measured mass 5069.6,
expected mass 5062.8 (II).
29. Kalyanasundaram, K. Coord. Chem. Rev. 1982, 46, 159.