During oligonucleotide synthesis, convenient amino group protection methodology is important not only for exocyclic amines but also for side chain amino groups ("aminolinkers" or "aminotethers"). These amino group-containing tethers can be conveniently deprotected and used to attach various functionalities to modify the biological or chemical properties of oligonucleotides (e.g., to conjugate groups which can improve uptake of antisense oligonucleotides by living cells); to attach chemical nucleases targeting the pathogenic genes; and to attach reporter groups (such as fluorescein or biotin) which are extensively used in DNA based diagnostics in following cellular trafficking of antisense oligonucleotides (Manoharan, M., in Antisense Research and Applications, S. T. Crooke and B. Lebleu (eds.), CRC Press, Boca Raton, Fla., 1993, 303-349). In spite of their widespread use, the conventional protecting groups used in oligonucleotide chemistry for aminotethers are either too labile during the monomer synthesis (e.g., CF.sub.3 CO--, Fmoc) or somewhat inert, thereby requiring harsh conditions during, for example, oligonucleotide deprotection. The phthalimido group, for example, requires CH.sub.3 NH.sub.2 in addition to the standard ammonium hydroxide conditions. The acid labile MMT (monomethoxytrityl) group is sometimes used, but generally to protect an aminolinker only at the 5'-end of the oligonucleotide.
To overcome these problems the alloc (allyloxycarbonyl) group (Kunz. H. Angew. Chem. 96, 426, 1984; Hayakawa, Y.; Wakabayashi, S.; Kato, H.; Noyori, R. J. Am. Chem. Soc. 112, 1691, 1990) has been adopted as a protecting group for aminolinkers, as it can be removed using zerovalent palladium (Pd (0)) either in solution phase or in solid phase (Barber-Peoch, I; Manoharan, M.; Cook, P. D. Nucleosides & Nucleotides 16, 1407-1410, 1997; Nelson, P. S.; Muthini, S.; Kent, M. A; Smith T. H.; Nucleosides & Nucleotides 16, 1951-1959, 1997). The chloroformate Cl--(C.dbd.O)--O--CH2-CH2-CN also has been used to protect nucleobase amines. Chloroformates, however, are unstable and difficult to use. It would therefore be desirable to provide alternative reagents for protecting amine and other groups during synthesis.
USPTO Backgrounds
USPTO Backgrounds