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.
High-density arrays of oligonucleotide probes are proving to be powerful new tools for large-scale DNA and RNA sequence analysis. A method for constructing these arrays, using light-directed DNA synthesis with photoactivatable monomers, can currently achieve densities on the order of 10 6 sequences͞cm 2 . One of the challenges facing this technology is to further increase the volume, complexity, and density of sequence information encoded in these arrays. Here we demonstrate a new approach for synthesizing DNA probe arrays that combines standard solid-phase oligonucleotide synthesis with polymeric photoresist films serving as the photoimageable component. This opens the way to exploiting high-resolution imaging materials and processes from the microelectronics industry for the fabrication of DNA probe arrays with substantially higher densities than are currently available.
The endo alcohol 8P-CN and its exo epimer (85:15, respectively) were also obtained (quantitatively) by heating tosylate 6P-CN in 80% dioxane/20% water at 160 °C for 3 weeks. Oxidation of the alcohol mixture with pyridinium chlorochromate (methylene chloride, 25 °C, 3.5 h), followed by the usual workup,37 led to 4-cyano-3(sj,n -8-diphenylbicyclo[3.2.1 ]oct-3-en-2-one (9P-CN), 65%, mp 154-156 °C (from benzene-petroleum ether); IR yCN 2250, i-co 1680 cm"1 2;
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