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Abstracts of Oak Ridge Posters |
1 Affymetrix Research Laboratories, Affymetrix, Inc., 3380 Central Expressway, Santa Clara, CA 95051
aauthor for correspondence: fax 408-731-5408, e-mail andrea_cuppoletti{at}affymetrix.com
High-density DNA probe arrays are finding widespread use in biomedical research and diagnostics because of their ability to simultaneously address large numbers of genes. GeneChip® probe arrays are manufactured by use of photolithography techniques (1)(2)(3)(4). At the base of the current Affymetrix process of synthesis of oligo-DNA probes is the use of nucleoside monomers protected with photo-removable groups (Fig. 1A
). Irradiation of the partially built oligomer with near-ultraviolet wavelengths (mainly 365 nm) deprotects the terminal hydroxyl group, and the use of appropriate masks allows for control of the sequence of the probe and the size of the features. The sensitivity of DNA to ultraviolet radiation limits the range of wavelengths usable for the photolitographic step to mainly the Hg atomic emission at 365 nm. Therefore, the choice of photo-removable hydroxyl protecting groups depends on the absorbance at this wavelength (4).
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Our ongoing investigations concerning the fabrication of DNA microarrays have several goals, including increasing the rate and the efficiency of photo-deprotection. The rate of deprotection, or photospeed (PS), is a function of the molar absorptivity of the chromophore at the wavelength of irradiation and the quantum yield of the desired photochemical product formation process. Increasing the molar absorptivity at 365 nm and the quantum efficiency of the process will generally lead to an increase in PS. In general, the rate and yield of photo-deprotection can be controlled by tuning each of these variables.
Several coumarins and variants of nitro aromatic systems, such as nitrocoumarins, nitroanilines, and nitroindolines, were synthesized and studied for their photoreactivity (Table 1
). To screen this series of compounds, we synthesized the corresponding acetate derivatives and tested them in solution for their rate of photocleavage. The more interesting ones (e.g., those with a large PS or a large molar absorptivity) were synthesized as nucleoside-3'-phosphoramidites and tested for their rate of deprotection and the efficiency of DNA synthesis on a solid support.
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For the solution photolysis study, we dissolved the acetate in methanol to reach an absorbance of 
0.1 at 365 nm. The solution was then irradiated with a 500 mW collimated mercury light source. The progress of the photocleavage was monitored by both ultraviolet absorbance and HPLC analysis. Half-lives (t1/2) were calculated by linear regression analysis (4) of plots of ln(area of the species) vs time (t1/2 = 0.693/slope). PS was then calculated as the inverse of the half-life multiplied by the intensity of the light source expressed in Joules [PS = 1/(t1/2 x intensity)]. A procedure for the measurement of the deprotection yield has been already described (4).
Coumarins were chosen for their generally high molar absorptivity at the wavelength of irradiation. To take advantage of the fast and efficient photo-induced homolysis/heterolysis (5)(6) of benzylic ethers, we have functionalized several coumarins for this purpose. As anticipated, substitution on the aromatic ring with an electron-donating group (Et2N- or MeO-) enhanced the molar absorptivity at 365 nm as well as the rate of deprotection of the acetates. The deprotection/coupling efficiency for generation of T-oligomers using 5'-coumarin carbonyl-protected dT-3'-phosphoramidites as monomers was >90%.
Encouraged by the results achieved with the coumarin series, we expanded the study to include another series of photogroups, nitrocoumarins. This series was chosen for their anticipated large molar absorptivities at 365 nm. Contrary to our expectations, however, nitro group substitution in either position 3 or 5 of the coumarin moiety decreased the molar absorptivity of the acetate derivatives up to 10-fold. This decrease was paralleled by a similar decrease in their rate of photocleavage. In line with these findings, during the photochemical study, we observed an absence of fluorescence for all of the nitro-substituted coumarin compounds. It is likely that substitution with a nitro group generates a low-lying excited triplet state that reduces the reactivity of these compounds.
Contrary to the series of compounds discussed above, the mechanism of photocleavage of substituted nitroanilines and indolines has been more extensively investigated (7)(8)(9). The mechanistic pathway is dependent on the presence of water or other nucleophiles. Photo-induced generation of the excited triplet state triggers an intramolecular rearrangement and elimination of the acyl group (Fig. 1B
). We have studied a series of nitroaniline, nitroindoline, and nitroquinoline carbamates as possible 5'-OH photoprotecting groups.
In the case of the anilines, the molar absorptivity at 365 nm is dependent on the substitution on the aromatic ring. In fact, electron-donating groups increased the molar absorptivity and the PS. The deprotection/coupling efficiency, however, did not follow the same trend. The monosubstituted 4-methoxy analog had the best coupling efficiency (97%), followed by the unsubstituted aniline analog (93%) and finally the methylenedioxy analog, with a coupling efficiency of only 88%. Nitroindolines were faster than the anilines within the same pattern of substitution on the aromatic ring. Of this series the 4-methoxy analog was the fastest to react on irradiation (PS = 5.0) and the one with the largest coupling efficiency (93%). Finally, in the case of the quinoline compound, the rate of photo-deprotection was 10-fold slower than that for the corresponding unsubstituted indoline. This result seems to be suggestive of a steric demand for the efficiency of the photocleavage. Unfortunately, the preparation of carbonate derivatives of nitroquinoline was complex, and we do not have data on the coupling efficiency.
In conclusion, several classes of compounds have been studied and shown to give fast and efficient photo-deprotection of 5'-hydroxyl groups for in situ DNA microarray synthesis. These same compounds might find application in other areas of synthetic chemistry as well. By appropriate substitution it is possible to increase the molar absorptivity of the substrate and influence the rate of deprotection. The deprotection/coupling efficiency can also be altered, but the outcome is more difficult to predict. Our efforts in designing, synthesizing, and testing still more photogroups are ongoing.
References
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