Synthesis of dinucleotides containing nitrone, hydroxylamine and amidoxime linkages

Article abstract of DOI:10.1016/S0040-4039(03)01355-8

Three new thymidine dinucleotides with nitrone, hydroxylamine and amidoxime backbone linkages, suitable for incorporation into oligonucleotide chains, were easily synthesized, by coupling of readily available thymidine monomers and applying short, simple

Full text of DOI:10.1016/S0040-4039(03)01355-8

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 5679–5681
Synthesis of dinucleotides containing nitrone, hydroxylamine and
amidoxime linkages
John K. Gallos* and Constantinos C. Dellios
Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki 541 24, Greece
Received 27 March 2003; revised 21 May 2003; accepted 30 May 2003
Abstract—Three new thymidine dinucleotides with nitrone, hydroxylamine and amidoxime backbone linkages, suitable for
incorporation into oligonucleotide chains, were easily synthesized, by coupling of readily available thymidine monomers and
applying short, simple and efficient procedures.
© 2003 Elsevier Ltd. All rights reserved.
The synthesis of oligonucleotides with modified back-
bone linkages remains a very active area of research.1
Current efforts are focused on the complete replace-
ment of the phosphorus atom in order to achieve higher
affinity for the RNA target, enhanced nuclease resis-
tance and improved permeability and cellular uptake.
towards nucleases.2 Oligomers with positively charged
guanidine3 and methylthiourea4 linkers 2 and 3 are also
promising leads in the development of new antisense
therapeutics, since the attractive forces between them
and the negatively charged DNA or RNA contribute
significantly to the stability of heteroduplex and triplex
structures formed between these species. In addition,
they are stable to enzymatic hydrolysis due to the lack
of a phosphodiester linkage. Interestingly, oligonucle-
otides containing the hydroxamate functionality5 4 dis-
play a binding affinity similar to the natural analogues
and substantial resistance towards nucleases. Moreover,
the hydroxamate functionality can effectively chelate
with a ferric ion, which can undergo Fenton chemistry
to generate hydroxy radicals. Thus, hydroxamate
nucleic acids might cleave the target RNA through
radical reactions.
From the plethora of modifications reported hitherto,
oligonucleotides with the amide linkage 1 (Fig. 1) have
shown very promising antisense properties by display-
ing higher binding affinity and very good resistance
We now report the synthesis of three new dinucleotides
containing nitrone 5, hydroxylamine 6 and amidoxime
7 functionalities as internucleotide backbone linkages
(Fig. 2). In all these cases the existing 3%-CH2 sub-
stituent might favor the C3%-endo conformation, which
is beneficial for an RNA binding behavior.1,6 The
nitrone and amidoxime functionalities in 5 and 7,
respectively, have a structural preorganization similar
to that of the amide functionality in 1 and the hydroxa-
mate in 4. Due to their neutral character at physiologi-
cal pH, all these linkages will contribute to the overall
charge reduction and therefore might potentially be
favorable for penetration through cellular membranes.
Finally, the hydrophilicity of the nitrone, hydroxyl-
amine and amidoxime backbones will increase the solu-
bility of these molecules in biological fluids.
Figure 1.
Keywords: antisense oligonucleotides; thymidine; nitrone; hydroxyl-
amine; amidoxime.
* Corresponding author. Tel.: +30-2310-997714; fax: +30-2310-
997679; e-mail: igallos@chem.auth.gr
0040-4039/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)01355-8

5680
J. K. Gallos, C. C. Dellios / Tetrahedron Letters 44 (2003) 5679–5681
amines 10 and 11 were accessible from thymidine,
applying the method developed by Miller,8 namely
replacement of the 5%-OH by BocNHOCbz under Mit-
sunobu conditions, followed by deprotection (Scheme
1).
Thymidine hydroxylamines 10 and 11 were readily con-
densed with the thymidine aldehyde 8 to give the
desired unprotected and protected dimers 17 and 18,9
respectively, isolated as white foams in high yield
(Scheme 2). It is interesting that the 3%-OH of the
hydroxylamine moiety needs no protection prior to the
condensation. Although the existing data did not allow
the unequivocal assignment of the nitrone geometry, it
is most probable that this group adopts the thermo-
dynamically more stable Z-form, as happens in all
heavily substituted nitrones.10
Reduction of the nitrone group in 17 could apparently
result in a dinucleotide with the hydroxylamine back-
bone 6 (Fig. 2). It was considered, however, that the
presence of the free N-OH group could not be dis-
cerned from 3%-OH in the phosphoramidite formation
reaction. For this reason, we decided to protect the
N-OH group as N-OAc, as it could be regenerated after
formation of the oligonucleotide chain. Dinucleotide 18
was reduced by NaBH4 and the N-OH group thus
formed was acetylated, in high yield (Scheme 3). Hav-
ing protected the internucleotide hydroxylamine group,
the 3%-OH was deprotected by treatment with TBAF to
give the desired dimer 21,9 unfortunately accompanied
by the doubly unprotected one 22.
Figure 2.
The amidoxime backbone linker in 7 (Fig. 2) could be
prepared according to the most general method of
making such molecules,11 namely addition of the
known primary amine 12 to the nitrile oxide generated
from the oxime 9. To this end, the latter was converted
to chloroxime 23 (Scheme 4) by addition of pyridine to
a solution of NCS in dry chloroform followed by
careful addition of 9 to the resulting mixture. This
procedure was necessary in order to avoid hydrolysis of
the dimethoxytrityl ether. The formation of compound
23 was detected by TLC and without isolation, its
Scheme 1. Reagents and conditions: (i) BocNHOCbz, PPh3,
DEAD, THF, 0“20°C, 12 h, 47% for 15 and 80% for 16. (ii)
Pd/C, H2, MeOH, 20°C, 2 h; then 10% TFA, CH2Cl2, 20°C,
2 h, 94% for 10 and 81% for 11.
Scheme 2. Reagents and conditions: (i) Na2CO3, EtOH, 20°C,
15 min for 5 (95%) and 12 h for 17 (84%).
Thymidine dinucleotides 5, 6 and 7 (B=thymine) were
easily prepared by condensation of aldehyde 8 and its
oxime 9 with the hydroxylamines 10 and 11 or the
amine 12. Compounds 8, 9, and 12 were prepared
according to the literature methods,7 while hydroxyl-
Scheme 3. Reagents and conditions: (i) NaBH4, EtOH, 20°C,
12 h, 92%. (ii) Ac2O, pyridine, 0“20°C, 12 h, 92%. (iii)
TBAF, THF, 20°C, 10 min, 50% of 21 and 46% of 22.

J. K. Gallos, C. C. Dellios / Tetrahedron Letters 44 (2003) 5679–5681
5681
Y., Ed.; Wiley-VCH: Weinheim, 1998; pp. 523–536; (e)
Herdewijn, P. In Carbohydrate Mimics: Concepts and
Methods; Chapleur, Y., Ed.; Wiley-VCH: Weinheim,
1998; pp. 553–579; (f) Temsamani, J.; Agrawal, S. In
Advances in Antiviral Drug Design; De Clercq, E., Ed.;
JAI Press: London, 1996; Vol. 2, pp. 1–39; (g) Varna, R.
S. Synlett 1993, 621–637; (h) Englisch, U.; Gauss, D. H.
Angew. Chem., Int. Ed. Engl. 1991, 30, 613–629; (i)
Herdewijn, P. Liebigs Ann. 1996, 1337–1348; (j) Egli, M.
Angew. Chem., Int. Ed. Engl. 1996, 35, 1894–1909; (k)
Wengel, J. Acc. Chem. Res. 1999, 32, 301–310; (l) Freier,
S. M.; Altmann, K.-H. Nucleic Acids Res. 1997, 25,
4429–4443.
2. (a) De Mesmaeker, A.; Ha¨ner, R.; Martin, P.; Moser, H.
E. Acc. Chem. Res. 1995, 28, 366–374; (b) Jung, P. M. J.;
Beaudegnies, R.; De Mesmaeker, A.; Wendeborn, S. Tet-
rahedron Lett. 2003, 44, 293–297 and references cited
therein.
3. (a) Blasko´, A.; Dempcy, R. O.; Minyat, E. E.; Bruice, T.
C. J. Am. Chem. Soc. 1996, 118, 7892–7899; (b) Blasko´,
A.; Dempcy, R. O.; Minyat, E. E.; Bruice, T. C. Bio-
chemistry 1997, 36, 7821–7832; (c) Luo, J.; Bruice, T. C.
J. Am. Chem. Soc. 1998, 120, 1115–1123; (d) Linkletter,
B. A.; Szabo, I. E.; Bruice, T. C. J. Am. Chem. Soc. 1999,
121, 3888–3896; (e) Kojima, N.; Bruice, T. C. Org. Lett.
2000, 2, 81–84; (f) Kojima, N.; Szabo, I. E.; Bruice, T. C.
Tetrahedron 2002, 58, 867–879.
4. (a) Arya, D. P.; Bruice, T. C. J. Am. Chem. Soc. 1998,
120, 6619–6620; (b) Arya, D. P.; Bruice, T. C. J. Am.
Chem. Soc. 1998, 120, 12419–12427; (c) Arya, D. P.;
Bruice, T. C. J. Am. Chem. Soc. 1999, 121, 10680–10684.
5. (a) Ramasamy, K. S.; He, L.; Stisavljevic, V.; Harpham,
B.; Seifert, W. Tetrahedron Lett. 2000, 41, 4317–4321; (b)
Li, H.; Miller, M. J. Tetrahedron Lett. 2000, 41, 4323–
4327.
Scheme 4. Reagents and conditions: (i) NCS, pyridine, CHCl3,
20°C, 30 min. (ii) 12, Et3N, CHCl3, 20°C, 12 h, 57% overall.
chloroform solution was treated with the amine 12 and
triethylamine to afford the desired dimer 24,9 in satis-
factory overall yield, apparently with intermediate for-
mation of the respective nitrile oxide.
In conclusion, the nitrone, hydroxylamine and amid-
oxime dithymidines reported here represent three new
types of modification in the internucleotide backbone
bridges with potential antisense properties. These
dimers were easily prepared by coupling of known and
readily available thymidine monomers, applying short,
simple and efficient procedures. Our efforts directed
towards the exploration of the compatibility of other
bases with these procedures as well as in the incorpora-
tion of the prepared dimers into oligonucleotide chains
and investigation of their biophysical properties are
now in progress and results will be reported in due
course.
6. Mohan, V.; Griffey, R. H.; Davis, D. R. Tetrahedron
1995, 51, 6855–6868.
Acknowledgements
7. (a) Stork, G.; Zhang, C.; Gryaznov, S.; Schultz, R.
Tetrahedron Lett. 1995, 36, 6387–6390; (b) Luo, P.;
Leitzel, J. C.; Zhan, Z.-Y. J.; Lynn, D. G. J. Am. Chem.
Soc. 1998, 120, 3019–3031; (c) Tronchet, J. M. J.; Grivet,
C.; Grand, E.; Seman, M.; Dilda, P. Carbohydr. Lett.
2000, 4, 5–12.
We are grateful to The Hellenic General Secretariat for
Research and Technology for financial support of this
work and to the ‘Leonidas Zervas’ Foundation for a
fellowship to C.C.D.
8. Li, H.; Miller, M. J. J. Org. Chem. 1999, 64, 9289–9293.
9. All new compounds were characterised from their spec-
troscopic (1H and 13C NMR, MS) and analytical (C,H,N
microanalyses and/or HRMS) data, which are consistent
with the proposed structures. These data, detailed experi-
mental procedures and the results from the oligonucle-
otide formation attempts will be given in a full paper.
10. Torsell, K. B. G. Nitrile Oxides, Nitrones, and Nitronates
in Organic Synthesis; VCH: Weinheim, 1989.
11. Nicolaides, D. N.; Varella, E. A. In The Chemistry of
Functional Groups; Supplement B: The Chemistry of Acid
Derivatives; Patai, S., Ed.; John Wiley & Sons: New
York, 1992; Vol. 2, pp. 875–966.
References
1. (a) Uhlmann, E.; Peyman, A. Chem. Rev. 1990, 90,
543–584; (b) Sanghvi, Y. S.; Cook, P. D. In Nucleosides
and Nucleotides as Antitumor and Antiviral Agents; Chu,
C. K.; Baker, D. C., Eds.; Plenum Press: New York,
1993; pp. 311–324; (c) Uhlmann, E. In Antibiotics and
Antiviral Compounds: Chemical Synthesis and Modifica-
tion; Krohn, K.; Kirst, H. A.; Maag, H., Eds.; VCH:
Weinheim, 1993; pp. 463–470; (d) Sanghvi, Y. S. In
Carbohydrate Mimics: Concepts and Methods; Chapleur,