New methods based on photolithography and surface fluorescence were used to determine photodeprotection rates and stepwise yields for light-directed oligonucleotide synthesis using photolabile 5‘-(((α-methyl-2-nitropiperonyl)oxy)carbonyl)(MeNPOC)-2‘-deoxynucleoside phosphoramidites on planar glass substrates. Under near-UV illumination (primarily 365 nm) from a mercury light source, the rate of photoremoval of the MeNPOC protecting group was found to be independent of both the nucleotide and length of the growing oligomer (t 1/2 = 12 s at 27.5 mW/cm2). A moderate dependence on solvent polarity was observed, with photolysis proceeding most rapidly in the presence of nonpolar solvents or in the absence of solvent (e.g., t 1/2 = 10−13 s at 27.5 mW/cm2). In solution, the photolysis rate was linearly dependent on light intensity over the range 5−50 mW/cm2. Average stepwise yields for the synthesis of dodecamer oligonucleotides were in the range of 92−94%, using monomers based on N 6-(phenoxyacetyl)-2‘-deoxyadenosine, N 2-isobutyryl-2‘-deoxyguanosine, N 4-isobutyryl-2‘-deoxycytidine, and thymidine. By comparison, an efficiency of 98%/step was obtained using a conventional 5‘-dimethoxytrityl monomer with acid deprotection on the same support. The lower yields associated with the photochemical process appears to be due to incomplete recovery of free 5‘-hydroxyl groups after photolysis on the support, although high yields of 5‘-OH nucleosides (≥96%) are consistently observed when 5‘-MeNPOC monomers are photolyzed in solution.

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Deoxynucleoside phosphoramidites can be prepared in good yield from deoxynucleosides, bis-dialkylaminophosphines, and the corresponding dialkylamine hydrotetrazolide or tetrazole as catalysts. These phosphoramidites generated in situ lead to the direct synthesis of deoxyoligonucleotides on polymer supports. INTRODUCTIONThe current phosphite triester methodology for deoxyoligonucleotide synthesis requires the condensation of deoxynucleoside phosphoramidites 7a-d or 8a-d, activated by tetrazole, with the 5'-hydroxyl group of a deoxynucleoside or deoxyoligonucleotide attached covalently to a polymer support (1-4). Although these phosphoramidites can be prepared by existing methods (5,6) from the appropriately protected deoxynucleosides la-d, the chlorophosphines 2 and 3 used in forming 7a-d and 8a-d, respectively, are difficult to prepare and easily react with trace amounts of water. Moreover, the high reactivity of 2 and 3 and the concomitant production of insoluble amine hydrochloride salts preclude their use for any strategy involving the in situ generation of deoxynucleoside phosphoramidites for deoxyoligonucleotide synthesis on solid supports (7). Because of our interest in the latter approach, we were prompted to investigate the relative stability and reactivity of the aminophosphines 4, 5, and 6 towards phosphodiester formation. We wish to report that the phosphoramidites 7a-d and 8a-d can be prepared in good yields by the reaction of suitably protected deoxynucleosides la-d and bis-dialkylaminophosphines 4 or 6 using amine salts 9 and 10, respectively, as catalysts. This method was applied to a

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High-density DNA probe arrays provide a massively parallel approach to nucleic acid sequence analysis that is transforming gene-based biomedical research and diagnostics. Light-directed combinatorial oligonucleotide synthesis has enabled the large-scale production of GeneChip probe arrays which contain several hundred of thousand oligonucleotide sequences on glass "chips" about one cm2 in size. Due to their very high information content, GeneChip probe arrays are finding widespread use in the hybridization-based detection and analysis of mutations and polymorphisms ("genotyping"), and in a wide range of gene expression studies. The manufacturing process integrates solid-phase photochemical oligonucleotide synthesis with lithographic techniques adapted from the microelectronics industry. The present-generation methodology employs MeNPOC photo-activatable nucleoside monomers with proximity photolithography, and is currently capable of printing individual 10 microns 2 probe features at a density of 10(6) probes/cm2.