Nucleotides part LXXX: Synthesis of 3'-O fluorescence labeled thymidine derivatives and their 5'-O-triphosphates.

A new labeling technique attaching a fluorescent pteridine derivative (3, 5) via a linker onto the 3'-OH group of 5'-O-dimethoxytritylthymidine (7) was developed to lead to the conjugates 8 and 11. After detritylation to give 9 and 12, the final conversion into the corresponding 5'-triphosphates (13, 14), which were isolated as sodium salts, was performed by known methods.

Intramolecular sensitization of photocleavage of the photolabile 2-(2-nitrophenyl)propoxycarbonyl (NPPOC) protecting group: photoproducts and photokinetics of the release of nucleosides.

Novel photolabile protecting groups based on the 2-(2-nitrophenyl)propoxycarbonyl (NPPOC) group with a covalently linked thioxanthone as an intramolecular triplet sensitizer exhibit significantly enhanced light sensitivity under continuous illumination. Herein we present a detailed study of the photokinetics and photoproducts of nucleosides caged with these new protecting groups. Relative to the parent NPPOC group, the light sensitivity of the new photolabile protecting groups is enhanced by up to a factor of 21 at 366 nm and is still quite high at 405 nm, the wavelength at which the sensitivity of the parent compound is practically zero. A new pathway for deprotection of the NPPOC group proceeding through a nitroso benzylalcohol intermediate has been discovered to complement the main mechanism, which involves beta elimination. Under standard conditions of lithographic DNA-chip synthesis, some of the new compounds, while maintaining the same chip quality, react ten times faster than the unmodified NPPOC-protected nucleosides.

Recent highlights on photolithic oligonucleotide array in situ synthesis.

Light directed synthesis of high-density oligonucleotide microarrays is currently performed using either ortho-nitro-benzyl-type [MeNPOC] (Pease, A.C.; Solas, D.; Sullivan, E.J.; Cronin, T.M.; Holmes, C.P.; Fodor, S.P.A. Proc. Natl. Acad Sci U.SA. 1994, 91, 6333.) or ortho-nitrophenylethyl-type [NPPOC] (Hasan, A.; Stengele, K.P.; Giegrich, H.; Cornwell, P.; Isham, K.R.; Sachleben, R.A.; Pfleiderer, W.; Foote, R.S. Tetrahedron 1997, 53, 424Z) protecting groups as the 5'-O-carbonate ester of the phosphoramidite building block. The synthesis cycle uses a combinatorial approach attaching one specific base per cycle, thus as many as 100 cycles need to be run to make an array of 25-mers. Time needed for deprotection/activation of the growing oligo chain determines overall manufacturing time and consequently also cost. In this report we demonstrate the development of photoprotected posphoramidite monomers for light directed array synthesis with increasing sensitivity to the UV light used. If combined with maskless array synthesis, this technology allows for synthesis of arrays with >780,000 different 25-mer oligonucleotides in about one hour and allows for high flexibility in array design and reiterative redesign. The arrays synthesized show high quality and reproducibility in our standard hybridization based assay.