Photoinduced signal amplification through controlled externally sensitized fragmentation in masked sensitizers

Article abstract of DOI:10.1021/ja066692u

Addition of lithiated dithianes to diaryl ketones, potential electron-transfer sensitizers, disrupts conjugation between the two aromatic moieties, effectively masking the sensitizer. A novel photoamplification strategy is developed based on photosensitiz

Full text of DOI:10.1021/ja066692u

Published on Web 11/02/2006
Photoinduced Signal Amplification through Controlled Externally Sensitized
Fragmentation in Masked Sensitizers
Rudresha Kottani, Janaki R. R. Majjigapu, Alexei Kurchan, Kavitha Majjigapu,
Tiffany P. Gustafson, and Andrei G. Kutateladze*
Department of Chemistry and Biochemistry, UniVersity of DenVer, DenVer, Colorado 80208-2436
Received September 19, 2006; E-mail: akutatel@du.edu
Detection of molecular recognition events has always been an
area of primary focus in bioanalytical sciences, with methods based
on fluorescence being of preference due to their sensitivity. How-
ever, high throughput fluorescence binding assays require the tested
ligands to be either spatially addressable or segregated on support
beads for mechanical sorting. Solution-phase libraries, either unsup-
Figure 1. Diarylketone sensitizing its own unmasking, as monitored by
ported or immobilized on submicrometer-sized carriers, present a
challenge, as there were no direct methods to assay them. Our recent
methodology for direct screening of solution-phase libraries, encod-
ed with photolabile tags, resolves this limitation.1 It uses dithiane-
based molecular systems, capable of photoinduced fragmentation,
but only in the presence of external sensitizers; i.e., the fragmenta-
tion in such systems is made contingent on a molecular recognition
event, which brings the sensitizer in the proximity of the photolabile
unit (Scheme 1).
UV absorption @ 360 nm (Ar ) p-MeOPh-).
Scheme 2
The next logical question is whether a molecular recognition
event can trigger not just one fragmentation, but rather set off a
progression of photochemical events, releasing multiple copies of
the encoding dithiane tags, thus enhancing the sensitivity of
detection. In this communication we prove that such photoampli-
fication can be achieved.
Non-PCR amplification of molecular recognition has been
implemented via enzymatic catalysis,2a polymerization,2b or massive
liquid crystal reorientation,2c all in a spatially addressable fashion.
Amplification of electrophoretic tags via sensitized generation of
singlet oxygen, with the effect localized due to limited diffusion
of 1O2, has also been proposed.3 Our amplification strategy is
fundamentally different. Addition of lithiated dithianes to diaryl
ketonesspotential sensitizerssdisrupts conjugation between the two
aromatic moieties, effectively masking the sensitizer. The photo-
sensitized fragmentation in such adducts releases more diaryl
ketone, capable of sensitization. In the experiment shown in Figure
1, 3 mol % of 4,4′-dimethoxybenzophenone was added to its
dithiane adduct in acetonitrile and irradiated, while UV absorption
of the solution was monitored at 360 nm. A typical autocatalytic
curve was obtained, indicating that photorelease of the masked
ketone accelerated this bimolecular fragmentation. While this
process resembles a chain reaction, with the released sensitizer
carrying the chain, it is controlled better than radical polymeriza-
tions, as the photoamplification chain can be stopped and/or
reinitiated at any time by removing or applying the UV source.
The amplification uses a source of UV photons to amplify the
amount of sensitizer, which often needs to be replenished in
photoinduced ET reactions to compensate for irreversible reduction.
Another outcome of the amplification is the mass release of
dithianes, triggered with a very small amount of the initiator.
In conjunction with spatial segregation on surfaces, this approach
can be utilized for bioanalytical purposes; Scheme 2 illustrates this
principal concept. The tested ligand is sparsely tethered to a surface,
which displays the dithiane-masked sensitizer as a bulk material.
Incubation with a receptor, modified with a tethered sensitizer,
brings the sensitizer into the vicinity of adducts (A, red arrow). As
irradiation commences, the initiator unmasks proximal sensitizers
on the surface which, in turn, can sensitize the unmasking of their
neighbors (B, red arrows) yielding more sensitizer. Ideally, the
process is continued in expanding concentric ripples, until all of
the sensitizer is unmasked. As a result, an amplified release of
dithiane tags into solution takes place, allowing for detection with
enhanced sensitivity by GC/MS. When the ligands are spatially
segregated from each other on discrete polymer beads or dendrim-
ers, as in the one-bead-one-compound libraries, they can be
encoded with 2-alkyl- substituted dithiane adducts for subsequent
mass-discriminating analysis of the released tags.
Scheme 1
Figure 2 shows how suitable the dithianes are for GC/MS detec-
tion: the first derivative single-ion monitoring (SIM) trace of six
alkyl dithianes encoding decimal 207, binary 0011001111, is ob-
tained with 0.5 pmoles of each dithiane per injection (S/N > 10).
N-Hydroxysuccinimide esters of the masked 3-carboxy-ben-
zophenone, both for direct coupling (i.e., 3) and tethered via GABA
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10.1021/ja066692u CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. SIM trace of 2-Me-, Et-, Pr-, Bu-, octyl- and nonyl-dithianes
encoding decimal 207.
Scheme 3
Figure 3. Release of methyldithiane due to photoamplification of minute
amounts of free sensitizer planted with the bulk of the masked benzophen-
one.
polymeric matrix and (ii) the fact that the tethered freshly unmasked
sensitizer can only unmask a few of its immediate neighbors, as
the amplification front moves on. In solution, due to free diffusion,
any sensitizer can carry the chain as long as it manages to escape
the irreversible reduction. Thus, xanthones are preferred, as the
reduction potential of the triplet xanthone is higher than that of
benzophenone.5 Yet, xanthones are less prone to hydrogen abstrac-
tion and survive longer in reducing matrices.6
The same amplification experiments with small amounts of
planted sensitizer were carried out using PAMAM-NH2 G5 den-
drimers. In several experiments, according to sulfur elemental
analysis, 86 to 99 of 128 primary amino groups on dendrimer’s
surface were coupled with the methyldithiane adduct of xanthone-
3-carboxylate. After this, 5 to 6 molecules of free sensitizer were
tethered to the dendrimer surface and irradiated. In all cases
amplified amounts of released dithianes were detected in solution,
i.e. exceeding the amounts of the planted sensitizer. The dendrimers
were also solubilized in micellar solutions of SDS to prove the
amplification concept in an aqueous environment. The size of a
particle in 30 mM SDS was estimated to be 8.8 nm by pulse field
gradient NMR. The micelle-free dendrimer in DMSO-d6 has an
estimated size of 6.4 nm, which implies that with aqueous detergent
one micelle contains one dendrimer molecule. Irradiation, followed
by extraction with hexane and GC/MS analysis, again revealed mass
release of dithiane tags.
(4) or 11-aminoundecanoic acid (5), were synthesized as shown in
Scheme 3. Masked xanthones 6,7 were synthesized similarly.
To emulate a small amount of the initiator, brought in by a
molecular recognition event, we planted 5% of free sensitizer on
polystyrene high loading beads (PS-NH2 1.86 mmol/g, 200 µm).
The rest of the surface material was the masked sensitizer. Actual
loading was determined by sulfur elemental analysis to be 1.17
mmol/g. The beads were suspended in MeCN, purged with argon,
and irradiated with a 320 nm long pass filter, and dithiane release
was monitored by GC/MS. Figure 3 shows a typical release
profile: dithiane concentration increases, reaching a maximum,
followed by a slow decrease due to secondary photodegradation.
Control experiments were performed to rule out interbead sensi-
tization and self-cleavage. For this, two additional types of beads
were prepared, containing (A) 100% immobilized adducts and (B)
100% immobilized sensitizer. Irradiation of a 50-50 mixture of
beads A and B did not release dithianes, suggesting no interbead
sensitization. Second, the masked sensitizer was tested for direct
fragmentation under the same irradiation conditions, because such
nonsensitized cleavage undermines amplification, producing false
positives not triggered by molecular recognition. Ultimately, at a
shorter wavelength, any adduct is capable of self-cleavage. Am-
plification is feasible if, at a given wavelength, the sensitized
fragmentation is overwhelmingly more efficient than self-cleavage,
i.e., ꢀketφsens . ꢀaddφself, where ꢀket and ꢀadd are the extinction
coefficients of the free sensitizer and its adduct, φsens and φself are
the quantum yields of the sensitized process and self-cleavage.
Addition of dithianes to aromatic ketones disrupts conjugation,
causing large blue-shifts. Yet, some adducts retain significant
absorptionsin thioxanthones it is due to the surviving diphenyl-
sulfide moietysand quantum yields of self-cleavage remain rather
high.4 The adducts of benzophenone-3- and xanthone-2-carboxa-
mides (beads A) showed no detectable self-cleavage when irradiated
for 5 h above 320 nm at 9.5 × 1019 photons/h (the sensitized
reaction in solution is fully completed within 1.5 h at this flux).
Unlike amplification in solution (Figure 1), dithiane release from
the beads lacks the S-shaped autocatalytic curve. This is due to (i)
unwanted reduction or cross-linking of the sensitizer in the
To conclude, using micro- and nanosized polymeric supports we
proved the concept of photoamplification on surfaces, whereby a
sensitizer autocatalyzes its own release, concomitant with the release
of encoding tags. Such amplified release can be made contingent
on a molecular recognition event, offering a promising methodology
for high throughput bioanalytical applications.
Acknowledgment. This research is supported by NIH
(GM067655).
Supporting Information Available: Experimental procedures and
spectra. This material is available free of charge via the Internet at
http://pubs.acs.org.
References
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(3) Singh, S.; Chan-Hui, P.-Y. U.S. Patent 6,949,347, 2005.
(4) Nonsensitized self-cleavage in the adducts of fluorescent ketones is utilized
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