synthesis. Postsynthesis activation of the safety-catch by mild
acidic hydrolysis enables selective photorelease (Figure 1).
Synthesis of the SC-PLL (Scheme 1) began with 3′,5′-
dimethoxy-4′-hydroxyacetophenone 1 and ethylene glycol.
Ethylene glycol was monoprotected with TBS-Cl, and then
coupled to 1 via Mitsunobu reaction to yield 2 (87%).
Treatment of 2 with PhI(OAc)2 in basic MeOH gave 3
(84%).10 The free alcohol was protected with NPPoc-Cl11
and deprotected with TBAF to give 4 (89%). Finally,
treatment with 2-cyanoethyl diisopropylchlorophosphora-
midite gave phosphitlylated linker 5 (86%), ready for
attachment to functionalized surfaces.12
To investigate the conditions for activation and photore-
lease of SC-PLL 5, monohydroxysilane derivatized glass
slides3a were prepared and extended with two NPPoc-
hexaethylene glycol-phosphoramidite spacers (analogous to
previously reported MeNPoc-hexaethyleneglycol-phosphor-
amidites),11c followed by coupling of the SC-PLL 5. Inter-
mediate phosphites were oxidized to the phosphotriesters
with I2/H2O/pyridine solution. A series of square features
containing T20-oligomers were synthesized via standard
protocols,13 using iterative cycles of photodeprotection and
coupling/oxidation of NPPoc-protected nucleoside phos-
phoramidites (Proligo), followed by deprotection with a 1:1
solution of ethylenediamine and ethanol. Small squares inside
the larger square features were irradiated for varying times
either before or after hydrolysis of the dimethyl ketal with
acid. For acid activation of the linker, 3% trichloroacetic acid
(w/v) in 95:5 acetone/MeOH was used to minimize nonspe-
cific stripping from the glass slides and potential depurination
of oligonucleotides, while allowing rapid activation of the
linker. Thioxanthone or 1-chloro-4-propoxy-9H-thioxanthone
(CPT) was used to enable the efficient, long wavelength
(>360 nm) photolysis of the phenacyl group. The exact
mechanism of thioxanthone mediated photorelease remains
undetermined; however, it may function as a photoreductant
or as a triplet sensitizer.14 The slide was visualized by
hybridization to a complementary Cy3-labeled A29 probe
(Figure 2a). Selective and spatially controlled photorelease
of oligonucleotides was revealed by loss of fluorescence in
small inner squares only after acid activation of the safety-
catch. No photorelease of the oligomer was observed prior
to acid activation.
Figure 1. Use of a safety-catch photolabile linker for light-directed
synthesis and release of oligonucleotides.
could aid the development of a robust gene assembly
platform. Chemical methods of elution, such as the basic
hydrolytic cleavage of linkers,5 have the disadvantage of
releasing oligonucleotides from all exposed areas of the array
surface. Selective release of oligonucleotides from the array
surface could be accomplished through a microfluidic
approach, such that a cleavage reagent is delivered only to
specific regions of the array. Here we report a safety-catch
photolabile linker (SC-PLL) that allows for both light-
directed synthesis and spatially selective photorelease of
oligonucleotides in microarrays.
A review of photolabile protecting groups (PLPGs) and
photolabile linkers (PLLs)6 presented two potential strategies
for linker design: (1) wavelength selective photolysis or (2)
latent photocleavable linkers. The independent, orthogonal
deprotection of PLPGs has been achieved through the use
of compounds that respond selectively to light of differing
wavelengths.7 However, the reported selectivity is not
absolute and is limited by the broad absorption spectra of
these photolabile protecting groups. As the synthesis of an
oligonucleotide microarray requires many irradiation cycles
prior to release, wavelength selective release was not deemed
practical. SC-PLL’s based on the benzoin PLPG have
previously been developed to reduce light-sensitivity and
improve the chemical stability of PLPGs used in syntheses.8
A SC-PLL for oligonucleotide synthesis and release must
be compatible with the conditions of DNA synthesis, and
activation/photolysis conditions must be compatible with
DNA. We chose to develop our safety-catch based on the
phenacyl PLPG9 since masking the carbonyl as a dimethyl
ketal renders this group photoinert during oligonucleotide
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