highly stable,5 after the maleimide deprotection step. The
small amounts of [protected maleimido]-oligonucleotide
that may still be present in the crude do not react and can
be easily separated from the conjugate in the subsequent
purification step. Conjugates (Scheme 4) were obtained by
reacting maleimido-oligonucleotides 15aꢀ15e with 5ꢀ10
equiv of thiol at a slightly basic pH (7.7), overnight at room
temperature, in 0.5 M triethylammonium acetate (3:2
THF/triethylammonium acetate mixtures were used in
reactions with thiocholesterol). Conjugation reaction crudes
were analyzed by either HPLC or PAGE (denaturing con-
ditions), using C4 rather than a C18 stationary phase in the
case of cholesterol-oligonucleotide conjugates (Figures S7ꢀ
S10).12 Mass spectrometric characterization confirmed the
identity of all conjugates.
resin assembly of maleimido-oligonucleotides. [Protected
maleimido]-oligonucleotides were obtained after removal
of oligonucleotide protecting groups under standard condi-
tions. Subsequent retro-DielsꢀAlder reaction deprotected
the maleimide moiety in high yield, affording oligonucleo-
tides with fully reactive appending maleimide groups.
Both reaction of maleimido-oligonucleotides with thiols13
and DielsꢀAlder cycloadditions14 may be useful to pre-
pare oligonucleotide conjugates with improved pharma-
cokinetic properties. Maleimido-oligonucleotides may also
find application for immobilization on different surfaces,
as well as in the preparation of microarrays and nano-
sensors.15
Acknowledgment. This work was supported by funds
ꢀ
In summary, ammonia treatment (room temperature)
of the mixture formed upon reaction of maleimide-con-
taining compounds with 2,5-dimethylfuran allowed the
stable exo adducts to be easily isolated. Dimethylfuran-
protected maleimide derivatives provided with the required
functional group were used for the first time for the on-
from the Ministerio de Ciencia e Innovacion (Grants
CTQ2007-68014-C02-01 and CTQ2010-21567-C02-01, and
the projectRNAREG, Grant CSD2009-00080, fundedun-
der the programme CONSOLIDER INGENIO 2010) and
the Generalitat de Catalunya (2009SGR-208). A.S. was a
recipient fellow of the Generalitat de Catalunya.
(11) Richards, F. M.; Vithayathil, P. J. J. Biol. Chem. 1959, 234,
1459–1465.
(12) Bijsterbosch, M. K.; Rump, E. T.; De Vrueh, R. L. A.; Dorland,
R.; van Veghel, R.; Tivel, K. L.; Biessen, E. A. L.; van Berkel, T. J. C.;
Manoharan, M. Nucleic Acids Res. 2000, 28, 2717–2725.
Supporting Information Available. Experimental pro-
cedures, compound characterization data and spectra,
and HPLC profiles. This material is available free of
(13) (a) Verez-Bencomo, V.; Fernandez-Santana, V.; Hardy, E.;
Toledo, M. E.; Rodriguez, M. C.; Heynngnezz, L.; Rodriguez, A.; Baly,
A.; Herrera, L.; Izquierdo, M.; Villar, A.; Valdes, Y.; Cosme, K.; Deler,
M. L.; Montane, M.; Garcia, E.; Ramos, A.; Aguilar, A.; Medina, E.;
Torano, G.; Sosa, I.; Hernandez, I.; Martinez, R.; Muzachio, A.;
Carmenates, A.; Costa, L.; Cardoso, F.; Campa, C.; Diaz, M.; Roy,
R. Science 2004, 305, 522–525. (b) Sampathkumar, S.-G.; Li, A. V.;
Jones, M. B.; Sun, Z.; Yarema, K. J. Nat. Chem. Biol. 2006, 2, 149–152.
(c) Xu, H.; Baidoo, K.; Gunn, A. J.; Boswell, C. A.; Milenic, D. E.;
Choyke, P. L.; Brechbiel, M. W. J. Med. Chem. 2007, 4759–4765.
(d) Hakwere, H.; Perrier, S. J. Am. Chem. Soc. 2009, 131, 1889–1895.
(e) Huang, Y.-F.; Shangguan, D.; Liu, H.; Phillips, J. A.; Zhang, X.;
Chen, Y.; Tan, W. ChemBioChem 2009, 10, 862–868. (f) Ducry, K.;
Stump, B. Bioconjugate Chem. 2010, 21, 5–13. (g) Chan, J. M.; Zhang,
L.; Tong, R.; Ghosh, D.; Gao, W.; Liao, G.; Yuet, K. P.; Gray, D.; Rhee,
J.-W.; Cheng, J.; Golomb, G.; Libby, P.; Langer, R.; Farokhzad, O. C.
Proc. Natl. Acad. Sci. U.S.A. 2010, 107 (2213), 2218.
(14) (a) Tarasow, T. M.; Tarasow, S. L.; Eaton, B. E. Nature 1997,
389, 54–57. (b) Hill, K. W.; Taunton-Rigby, J.; Carter, J. D.; Kropp, E.;
Vagle, K.; Pieken, W.; McGee, D. P. C.; Husar, G. M.; Leuck, M.;
Anziano, D. J.; Sebesta, D. P. J. Org. Chem. 2001, 66, 5352–5358.
(c) Fruk, L.; Grondin, A.; Smith, W. E.; Graham, D. Chem. Commun.
€
2002, 2100–2101. (d) Tona, R.; Haner, R. Bioconjugate Chem. 2005, 16,
ꢀ
837–842. (e) Marchan, V.; Ortega, S.; Pulido, D.; Pedroso, E.; Grandas,
A. Nucleic Acids Res. 2006, 34, e24. (f) Steven, V.; Graham, D. Org.
Biomol. Chem. 2008, 6, 3781–3787. (g) Borsenerger, V.; Howorka, S.
Nucleic Acids Res. 2009, 37, 1477–1485.
(15) (a) Sassolas, A.; Leca-Bouvier, B. D.; Blum, L. J. Chem. Rev.
2008, 108, 109–139. (b) Williams, B. A. R.; Diehnelt, C. W.; Belcher, P.;
Greving, M.; Woodbury, N. W.; Johnston, S. A.; Chaput, J. C. J. Am.
Chem. Soc. 2009, 131, 17233–17241. (c) Niemeyer, C. M. Angew. Chem.,
Int. Ed. 2010, 49, 1200–1216.
Org. Lett., Vol. 13, No. 16, 2011
4367