Photolabile thymidine cleavable with a 532 nanometer laser

Article abstract of DOI:10.1016/j.tetlet.2011.07.052

The synthesis and characterization of a novel 3′ p-hydroxyphenacyl- caged thymidine bearing a 5′ N,N-diisopropylcyanoethylphosphoramidite is presented representing a new methodology for the photoregulation of PCR and gene expression. Solid phase oligonucleotide synthesis affords a primer blocked at the 3′ position, which could function as a phototrigger for polymerase activity. The caging group exhibits quantitative photolysis in 15 s using a 532 nm green hand laser.

Full text of DOI:10.1016/j.tetlet.2011.07.052

Tetrahedron Letters 52 (2011) 4989–4991
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Photolabile thymidine cleavable with a 532 nanometer laser
⇑
Chad J. Pickens, Kyle R. Gee
Molecular Probes-Life Technologies, 29851 Willow Creek Road, Eugene, OR 97402, United States
a r t i c l e i n f o
a b s t r a c t
The synthesis and characterization of a novel 30 p-hydroxyphenacyl-caged thymidine bearing a 50 N,N-
diisopropylcyanoethylphosphoramidite is presented representing a new methodology for the photoregu-
lation of PCR and gene expression. Solid phase oligonucleotide synthesis affords a primer blocked at the 30
position, which could function as a phototrigger for polymerase activity. The caging group exhibits
quantitative photolysis in 15 s using a 532 nm green hand laser.
Article history:
Received 31 March 2011
Revised 8 July 2011
Accepted 12 July 2011
Available online 28 July 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Caged thymidine
p-Hydroxyphenacyl
Phosphoramidite
Photolysis
PCR
Spatiotemporal regulation through light irradiation presents an
attractive method to trigger biochemical events, as it enables high-
resolution control for the release of active molecular species, often
for the elucidation of complex biological processes. Enabling the
site-specific control of a cellular process in its native environment
is intriguing, and many recent developments have been made.1–3
A
multitude of caged compounds and macromolecules have been
reported with a variety of applications, including proteins,4–6 pep-
tides,7 neurotransmitters,8,9 and nucleotides.10,11 Photoactivatable
oligonucleotides have been shown to regulate genetic events, such
as transcription,12,13 translation,14 DNAzyme reactions,15,16 RNA
interference,17 aptamer binding,18 and PCR,19,20 but the need for
an improved caging methodology is apparent, especially for nucleic
acids.
Two main approaches to caging DNA have been utilized. The
first involves alteration of the DNA backbone, either a statistical
but nonspecific caging of the phosphodiester bonds21,22 or the
insertion of a photocleavable linker in the backbone.23 While this
approach has proven effective, it is difficult to achieve a completely
binary (on/off) system capable of fully restoring function. The
second approach is to cage the nucleobase, disrupting the
Watson–Crick base pairing. This method is effective in disrupting
DNA hybridization and is more capable of achieving binary behav-
ior. Here we report a novel alternative by caging the 30 position to
inhibit primer extension while still allowing hybridization (Fig. 1).
This provides facile access to 30 caged primers via reverse phase
oligonucleotide synthesis.
Figure 1. Caging methodologies to regulate oligo synthesis.
Of the common ortho-nitrobenzyl, coumarin, and benzoin
caging groups, the para-hydroxyphenacyl (pHP) group is a promis-
ing alternative given its rapid photolysis, aqueous solubility, and
biocompatibility.24,25 A variety of pHP caged bioactive compounds
have been reported (including ATP, GABA, and bradykinin), with
⇑
Corresponding author. Tel.: +1 541 335 0366 fax: +1 541 335 0206.
E-mail address: kyle.gee@lifetech.com (K.R. Gee).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.07.052

4990
C. J. Pickens, K. R. Gee / Tetrahedron Letters 52 (2011) 4989–4991
O
ODMT
O
O
OH
O
N
P
O
Br
N
O
O
O
P
Cl
N
N
HN
O
N
HN
O
O
O
ODMT
O
HO
N
O
O
HN
O
O
O
5
N
40% AcOH
in MeOH
2
HN
O
O
LiHMDS, THF
DIEA, CH2Cl2
O
OH
N
O
1
HO
HO
3
4
HO
6
λ = 532 nm
100%
O
15 sec
OH
O
N
HN
O
OH
7
Scheme 1. Synthesis of pHP caged thymidine phosphoramidite 6. DMT = dimethoxytrityl.
diisopropylamide fails to produce 3 under the same conditions
(Scheme 1).
DMT deprotection with acetic acid followed by phosphitylation
of the free 50-OH gives the desired phosphoramidite 6. A minor
reaction product (10%) with substitution at the p-OH was isolated,
and the structure was confirmed by gCOSY and NOESY experi-
ments (see Supplementary data). Given flash chromatography fol-
lowing each step (three overall), the total isolated yield is 19.1%.
Importantly, pHP caging of the substrate does not introduce a chi-
ral center (as opposed to some other caging groups), alleviating the
need to separate diastereomers.
Figure 2. RP-HPLC photolysis studies of compound 4 at 532 nm (5–30% MeCN in
H2O, observed at 280 nm).
Absorbance spectra of 4 indicate a maximum at 273 nm (see
Supplementary data). An optimal two-photon irradiation wave-
length will be twice this value, or 546 nm. HPLC photolysis studies
confirm quantitative conversion to thymidine 7 (10 lM solution in
fast photolysis rates and favorable photolysis quantum yields
1:1 MeOH:H2O) in 15 s using a 532 nm green hand laser (Fig. 2).
This photolysis wavelength and source are substantially cheaper
to use than the conventional one- and two-photon uncaging
sources.28 Also, the photolysis efficiency appears substantially
higher than that typically achieved with ortho-nitrobenzyl caging
groups.32 Compound 4 is stable, neat or in solution for at least
one week when exposed to ambient light, while the phosphorami-
dite 6 undergoes partial photolysis (ꢀ50%) after four days in
DMSO-d6 at room temperature exposed to ambient light. Com-
pound 6 is not stable under aqueous conditions following the ini-
tial flash chromatography over deactivated silica.
(U
).26,27 The byproduct, p-hydroxyphenylacetic acid, is non-toxic
and its absorbance is blue-shifted so as to not interfere with the
photolysis reaction. Two-photon removable groups are promising
for biological applications by replacing UV excitation with simulta-
neous absorption of two IR photons equal in total energy.28 This
approach eliminates potential thymine dimerization, which is
known to occur through a reversible [2+2] photocycloaddition un-
der UVA exposure.29,30 Application of a two-photon group allows
the selected irradiation source to be well outside the UVA region,
and better tissue penetration with less phototoxicity is observed
with longer wavelength sources. Two-photon excitation also en-
ables three-dimensional control over substrate release. However,
it remains difficult to predict two-photon uncaging efficiency from
caging group structure;31 ortho-nitrobenzyl groups have proven
particularly resistant to efficient two-photon uncaging.32
Future endeavors will include feasibility studies of the oligonu-
cleotide primers synthesized using 6, as photo-initiators of the PCR
reaction.
Acknowledgments
Alkylation of the 30 position of 50-DMT-thymidine with
p-hydroxyphenacyl bromide was initially attempted with NaH
(in varying stoichiometric ratios). Unsuccessful efforts were origi-
nally attributed to competing substitution between the p-OH and
the 30-OH. Yet, attempts with p-OMe substituted phenacyl bromide
(data not shown) also did not yield the desired product. Subse-
quent experiments revealed that any significant excess of NaH is
We wish to thank Dr. Vladimir Martin and Dr. Upinder Singh for
their helpful discussions on synthetic strategy.
Supplementary data
Supplementary data (experimental procedures and associated
characterization data) associated with this article can be found,
in the online version, at doi:10.1016/j.tetlet.2011.07.052.
sufficient to deprotonate the a-position of 2 and form phenacyl di-
mers prior to nucleophilic attack by the alkoxide. But, without a
large excess of hydride, primarily N-alkylation of the nucleobase
is observed (as with K2CO3 and other bases). 1,8-Diazabicy-
clo[5.4.0]undec-7-ene forms a mixture of N- and O-alkylated prod-
ucts on the thymine ring, but only lithium hexamethyldisilazide
gives the desired 30 alkylation product 3 (70% yield); lithium
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