A novel and convenient synthetic strategy has been designed for the preparation of achiral nonnucleosidic phosphoramidites for automated solid-phase oligonu-cleotide synthesis. It is based on DMTr-protected 4-(2-hydroxyethyl)-morpholine-2,3-dione as the key component and a family of building blocks obtained by its ring-opening by primary aliphatic amine functional blocks. A series of nonnucleosidic phosphoramidites was synthe-sized containing various side-chain functionalities, and corresponding oligodeoxyribonucleotides were prepared incorporating modified units in single or multiple posi-tions along the chain.
Recently we have developed a novel strategy for the synthesis of nonnucleosidic phosphoramidites for nucleic acid functionalization, which is a merger of two approaches: 1) ring-opening of lactones by aliphatic amines, and 2) the use of activated oxalate esters for oligonucleotide derivatization. We have applied our new building block strategy for the preparation of achiral phosphoramidites bearing various side-chains. [1,2] The approach requires two separately functionalized building blocks. One is a key block that introduces the two hydroxyls needed for incorporation into the DNA chain. Another is a functional block tagged with side-chain groups to convey specific properties to oligonucleotides. The key block is a common reagent for the preparation of a family of synthons with different side-chain tags. In each case oligonucleotide modification is accomplished by employing an appropriately functionalized monomer or a combination of monomers during standard solid phase synthesis.
We have selected diethanolamine as an inexpensive starting material that provides both hydroxyl groups required for the incorporation into the oligonucleotide chain, and a secondary amino group for the attachment of a side chain. Previously, the problem of selective protection of only one of the two identical hydroxyls had significantly lowered yields of the products. We have solved the problem by simultaneous protection-activation of only one of the two hydroxyls as a cyclic oxalamide ester. The reaction of diethanolamine with inexpensive diethyl oxalate gives lactone 1 in high yield and purity. The lactone could be easily O-dimethoxytritylated in good yield affording DMTr-lactone as our key block, which was used in all subsequent reactions with functional blocks. Those are primary aliphatic mono-amines, which are either commercially available or easily synthesized. The structure of the side chain of a functional block defines a specific property that is conveyed to a modified oligonucleotide such as a bioconjugation site e.g. an alkynyl group, a hydrophobic aromatic or aliphatic moiety, a positively charged residue, a fluorescent label or an intercalator. Phosphitylation of alcohols 4 yields a family of nonnucleosidic phosphoramidites 5 tagged with different side chains. The monomers were successfully employed in solid-phase synthesis of functionalized oligonucleotides according to standard phosphoramidite protocol.
ACKNOWLEDGEMENT
This work was partly carried out with the aid of the Russian Government support for research projects implemented under supervision of world leading scientists (agreement No. 14.B25.31.0028 with Sidney Altman as the leading scientist), RFBR grant No. 13-04-01176, and interdisciplinary grant from SB RAS.
REFERENCES
1. Kupryushkin, M. S.; Pyshnyi, D. V. Russ. J. Bioorg. Chem. 2012, 38, 662.
2. Kupryushkin, M. S.; Konevetz, D. A.; Vasilyeva, S. V.; Kuznetsova, A. S.; Stetsenko, D. A.; Pyshnyi, D. V.Nucleosides, Nucleotides Nucleic Acids 2013, 32, 306.