Sensitized two-photon photochemical deprotection

Article abstract of DOI:10.1039/c0cc00782j

The photoremovable protecting group NPPoc has little sensitivity to two-photon excitation, limiting its use in applications requiring high spatial control of its photochemistry. In the presence of a triplet sensitizer with a large two-photon absorption cross-section, however, the two-photon uncaging action cross-section is improved to levels useful in a variety of applications.

Full text of DOI:10.1039/c0cc00782j

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Sensitized two-photon photochemical deprotection
Michael C. Pirrung,*a Timothy M. Dore,*b Yue Zhub and Vipul S. Ranaa
Received 6th April 2010, Accepted 1st June 2010
First published as an Advance Article on the web 16th June 2010
DOI: 10.1039/c0cc00782j
The photoremovable protecting group NPPoc has little sensitivity
to two-photon excitation, limiting its use in applications requiring
high spatial control of its photochemistry. In the presence of a
triplet sensitizer with a large two-photon absorption cross-section,
however, the two-photon uncaging action cross-section is improved
to levels useful in a variety of applications.
length of the sequences that can be prepared. Superior
performance in DNA synthesis is exhibited by photoremovable
groups of the NPPoc family (eqn (1)), which give near-
quantitative yields.8
ð1Þ
Two-photon photochemistry provides some advantages over
conventional one-photon processes, particularly in the bio-
logical sciences. The use of two IR photons in lieu of a near-UV
photon obviates the risk of damage to UV-sensitive biomolecules
and provides high spatial control at the focal point of an IR
laser, where high photon flux meets the requirements for
two-photon absorption (TPA). This principle is used in two-
photon fluorescence microscopy1 (where TPA creates an
excited state that fluoresces) and two-photon uncaging (where
the excited state undergoes a chemical reaction).1,2 The latter
process relies on a photoremovable protecting group that is
responsive to two-photon excitation, an area of ongoing
scientific interest and challenge.
One characteristic of essentially all photoremovable groups
is that they asymptotically approach completion (expected
for any first-order process), which as shown in our earlier
kinetic modeling9 affects the spatial definition of photochemical
deprotection in microarray fabrication. One exception to this
behavior has been reported, however: when the deprotection
of a NPPoc group at 420 nm on a microarray is performed
with sensitization by thioxanthone, the reaction proceeds
essentially linearly to completion.10 While unexpected and
uncommon, Steiner et al. have performed experiments and
provided theory that explains this behavior.11 Thioxanthone’s
significantly higher 365 nm absorption (e 4918 cmꢀ1 Mꢀ1
compared to 225 cmꢀ1 Mꢀ1 for NPPoc) followed by energy
transfer to the NPPoc group also provides a 2–3-fold enhance-
ment in deprotection rate on the microarray.
A number of protecting groups that are removable by TPA
have been reported, such as the Bhc and BHQ groups.3 A key
characteristic of any photoremovable protecting group is its
two-photon uncaging action cross-section, or du, which is
measured in units of Goppert-Mayer (GM), or 10ꢀ50 cm4
s
Our initial efforts to improve upon the performance of
photoremovable groups for light-directed synthesis relied on
sequential one-photon processes to accomplish the equivalent
of two-photon deprotection.9 Aiming to develop a more
robust technology that offers the attractive cycle yields and
kinetics of sensitized NPPoc deprotection and adds the high
spatial definition of TPA, we found examples in stereolitho-
graphy where thioxanthone has been used as a two-photon
photo-initiator of polymerization.12 We postulated that TPA
of thioxanthone (TPA cross-section, da = 5 GM)13 with high
peak power laser pulses would create excited states in a
localized area that could then transfer their energy only to
nearby NPPoc groups for photochemical deprotection.
¨
photonꢀ1. A protecting group should have du > 0.5 GM to be
useful in two-photon photodeprotection. The du’s of Bhc and
BHQ are around 0.8 GM at 740 nm,3 whereas some newer
groups (such as BNSF) have a du of 5–10 GM.4
DNA microarray fabrication relies on photochemical
deprotection using a modified photolithographic method,
light-directed parallel chemical synthesis.5 Methods for semi-
conductor photolithography have advanced at a rapid pace,
particularly concerning the feature sizes that can be practically
created. Current capabilities enable features as small as 65 nm
to be written, reflecting Moore’s law (the doubling every two
years of the number of transistors on an integrated circuit).6
The same progress has not been seen with DNA microarrays.
Feature size in DNA photolithography began at 500 mm and
has reached only as low as 4 mm.7
In the fabrication of DNA microarrays, a parameter as
important as feature size is the cycle yield (reflecting both a
photodeprotection step and a phosphoramidite coupling step)
in the addition of successive nucleotide subunits. It controls
not only the quality of the ultimate DNA probes but also the
Such a sensitization strategy enables a modular approach
for maximizing the spatial definition and yield in photo-
deprotection. That is, a protecting group with a high photo-
chemical deprotection yield for a particular functional group
can be paired with a sensitizer with a high two-photon cross-
section, so long as the energy transfer is favorable. This
approach is far more attractive than seeking novel photo-
removable protecting groups that provide both quantitative
deprotection yields and high TPA. Neither property is under-
stood well enough that it can be predicted a priori, so empirical
approaches based on synthesis and examination of many novel
groups would be required. Other work has used similar
‘‘antenna’’ strategies for the generation of singlet oxygen14
a Department of Chemistry, University of California, Riverside, CA,
92521, USA. Fax: +1 951 827 2749; Tel: +1 951 827 2722
b Department of Chemistry, University of Georgia, Athens, GA, 30602,
USA. Fax: +1 706 542 9454; Tel: +1 706 583 0423
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Fig. 2 Time course of sensitized deprotection of 30-NPPoc-T at
766 nm. The initial concentration was 100 mM, and the sample size
was 20 mL. The line is a least-squares fit of a single exponential decay.
Error bars represent the standard deviation of the measurement.
Fig. 1 Time course of sensitized deprotection of 30-NPPoc-T at
365 nm in the presence and absence of the triplet sensitizer isopropyl
thioxanthone (THX). The lines are a least-squares fit of a single
exponential decay.
methods;3 it is 0.86 GM. The distinction between one-photon
and two-photon sensitized deprotection is apparent, where at
the 20ꢂ concentration of thioxanthone, the latter is greatest
and the former is poorest.
and for the release of biological effectors (but only in an
intramolecular sense).15
To establish a method to measure the rate of a sensitized
two-photon deprotection, sensitized one-photon deprotection of
30-NPPoc-T (eqn (2)) was first studied. 30-NPPoc-T was irradiated
in non-degassed acetonitrile using a Spectroline SB-100P Hg lamp
(365 nm). The sensitizer was isopropyl thioxanthone (THX),
which is obtained commercially as an (immaterial) mixture of
positional isomers. The concentrations of starting material and the
product thymine (T) were determined by HPLC using DMT-T as
an internal standard and were normalized to the % conversion.
Representative data are shown in Fig. 1. Interestingly, increasing
concentrations of sensitizer slowed deprotection. The fastest rate
was observed with 10 mol% of thioxanthone, and the slowest was
observed with 20 equiv. (vide infra). Steiner made similar observa-
tions in his solution experiments and attributed them to oxygen
quenching of the THX triplet.16
Sensitized deprotection of 30-NPPoc-T using TPA was
studied at 766 nmw using a fs-pulsed, mode-locked, Ti:sapphire
laser as described in past work.3 The average laser power was
390 mW, the pulsewidth (dt) was 137 fs, and the repetition
rate was 76 MHz. Study of the dependence of deprotection
efficiency on thioxanthone concentration showed that higher
concentrations were best. The fastest deprotection was
observed at the highest concentration examined, a 20ꢂ excess
of sensitizer (eqn (2)). The normalized conversion determined
by HPLC (average of 3 runs) is shown in Fig. 2.
The reported results establish a new method for two-photon
sensitized photochemical removal of a protecting group. They
suggest that a broad range of photoremovable groups may be
adapted to TPA via sensitization, and that this principle
should apply to many sensitizer/photoremovable group pairs,
provided they have downhill energy transfer. The method
provides the ability to modify the protecting group and
sensitizer at will to fit the requirements of particular molecules
to be protected/deprotected. The specific combination
examined here provides a two-photon uncaging cross-section
du that is comparable to many existing two-photon photo-
removable groups used in caging studies. This result is
superior to one reported method for two-photon uncaging
based on the NPPoc group.17 The high concentration of THX
could be addressed by using a NPPoc group that carries its
own thioxanthone sensitizer,16 essentially a variant on the
intramolecular ‘‘antenna’’ strategy. It also seems likely that
improvements can be made on thioxanthone as a two-photon
sensitizer, since two-photon photoinitiators with da > 100 GM
are known, such as 7-(benzo[d]thiazol-2-yl)-9,9-didecyl-N,N-
diphenyl-9H-fluoren-2-amine (DPABz).13 This method may
find its greatest application in the de-caging of biological
effectors, but could also be used in direct-write methods in
molecular photolithography.18
This work was supported in part by an NSF CAREER
award to TMD (CHE-0349059).
Notes and references
ð2Þ
w This wavelength was chosen because it is twice the lmax of the
thioxanthone sensitizer (383 nm) and probably close to the optimum
wavelength for TPA.
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