More efficient photolithographic synthesis of DNA-chips by photosensitization

Full text of DOI:10.1081/NCN-120022994

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 1395–1398, 2003
More Efficient Photolithographic Synthesis of DNA-Chips
by Photosensitization
1
D. Woll, S. Walbert, K.-P. Stengele, R. Green, T. Albert,
1
2
3
3
¨
W. Pfleiderer,1 and U. E. Steiner1,
*
1University of Konstanz, Konstanz, Germany
2Chemogenix GmbH, Pleiskirchen, Germany
3Nimblegen Systems Inc., Madison, Wisconsin, USA
Light-induced release of photolabile protecting groups is a key step in the
photolithographic in-situ synthesis of oligonucleotides in the production of
high-density DNA-chips.[1,2] Protecting groups of the o-nitrophenyl type that
are preferentially used for this purpose have rather low absorption coefficients
at 366 nm, the wavelength of the most conveniently used mercury line. The weak
absorptivity of these compounds leads to principal limitations of their light sen-
sitivity. Since they have photoreactive triplet states, however, this problem can be
overcome by introducing a triplet sensitizer that efficiently absorbs the light and
transfers the electronic energy to the reactive protecting group.
The sensitized photodeprotection was studied in detail for the o-nitrophenyl-
2-propoxycarbonyl (NPPOC) group[3] where the photo cleavage occurs via b-elim-
ination of the corresponding nitrostyrene derivative as shown in the scheme. The
first step of this reaction is photochemically initiated intramolecular H-atom
transfer from the a-position to the nitro group whereby an aci-nitro compound
is formed[3]. Protolytic dissociation of the aci-nitro compound is followed by
the b-elimination from the aci-nitro anion. As an example of the time-resolved
*Correspondence: U. E. Steiner, Universita¨t Konstanz, Fachbereich Chemie, Universita¨ts-
strasse 10, 78457 Konstanz, Germany; Fax: þ49 753 188 3138; E-mail: ulrich.steiner@uni-
konstanz.de.
1395
DOI: 10.1081/NCN-120022994
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

¨
Woll et al.
1396
observation of triplet sensitized formation of the intermediate aci-nitro com-
pound, laser flash spectroscopic signals at 420 nm, the absorption maximum of
the aci-nitro form, are shown in Fig. 1. Here the rise time of the aci-nitro form
related transient absorption is seen to decrease with increasing concentration of
the photolabile NPPOC-protected thymidine (NPPOC-T) functioning as the tri-
plet energy acceptor in this case.
Various potential sensitizers such as acridone and thioxanthone were inves-
tigated by using absorption, fluorescence and laser flash spectroscopy.
Furthermore, continuous irradiations followed by HPLC separation and charac-
terization of the peaks by absorption spectroscopy were used to determine the
kinetics of reactants and products and to assess the quantum yields of the photo-
sensitized reactions. Thioxanthone turned out to be the most suitable sensitizer
for the o-nitrophenyl-2-propoxycarbonyl (NPPOC) group. In homogenous
solutions (cf. Fig. 2) the light sensitivity could be enhanced by as much as a
factor of 10, while in terms of overall deprotection yield and avoidance of
side reactions the photosensitized reaction was as efficient as the direct photo-
reaction.
Figure 1. Transient absorption signals observed at 420 nm upon laser flash photolysis at
355 nm of an acetonitrile solution of 2-ethylthioxanthone containing increasing concentrations
of NPPOC-T (curves from below: 0.135, 0.247, 0.358, 0.480 M). The transient absorption sig-
nals are corrected for the overlapping triplet-triplet absorption of 2-ethylthioxanthone and
absorbance contributions resulting from direct photolysis of NPPOC-T.

Photolithographic Synthesis of DNA-Chips
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Figure 2. Kinetics of direct and thioxanthone sensitized photocleavage of NPPOC-T in aceto-
nitrile. The concentrations of NPPOC-T and thioxanthone were 0.091 mM and 0.113 mM,
respectively. The reaction progress was followed by HPLC analysis and monitoring the UV
absorbance of the peak of uncleaved NPPOC-T.
Photosensitization has also been tested for high-density DNA-chip synth-
esis and has been found to significantly reduce the cycle time of the synth-
esis without significant reduction in the quality of the chip.[4] Characteristic
kinetic traces are shown in Fig. 3. It is particularly worth of note that the
Figure 3. Kinetics of direct and sensitized photodeprotection of thymidine on a DNA chip.
A single layer of protected thymidin (NPPOC-T or MeNPOC-T) was bound to a silanized
microscope slide. After illumination with the given dose, the fluorescence label Cy3 amidite
was coupled to the array. The observed fluorescence intensity provides a relative measure of
free hydroxyl groups.

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Woll et al.
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reaction order of the sensitized reaction on the chips is close to zero,
which is particularly favorable because it shortens the necessary illumination
time.
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