A new efficient photocaging system with a fluorescence reporting function has been developed. The photolabile latch is based on adducts of C-nucleophiles with aromatic ketones, such as thioxanthones and xanthones. The system is designed to quantify the release of biological effectors and to monitor their spatial distribution and localization by single- and two-photon fluorescence microscopy. In the armed state the ketone's conjugation is disrupted by nucleophilic addition, resulting in a blue shift of the absorption maxima and a dramatic decrease in fluorescence intensity. The mechanism of the photoinduced uncaging involves homolytic C-C bond fragmentation followed by radical disproportionation, regenerating the carbonyl moiety and restoring fluorescence. The uncaging can be initiated via either a one- or two-photon process, offering a new powerful tool for molecular life sciences. The synthesis and uncaging of dendrimer- and polymeric bead-based model systems are described.

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. However, 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 unsupported or immobilized on sub-micron carriers, present a challenge, as there were no direct methods to assay them. Our recent methodology for direct screening of solution phase libraries, encoded 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 fragmentation in such systems is made contingent on a molecular recognition event, which brings the sensitizer in the proximity of the photolabile unit (Scheme 1).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, and thus enhancing the sensitivity of detection. In this Communication we prove that such photoamplification 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 1 O 2 , has also been proposed. 3 Our amplification strategy is fundamentally different. Addition of lithiated dithianes to diaryl ketones -potential sensitizers -disrupts conjugation between the two aromatic moieties, effectively masking the sensitizer. The photosensitized 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 polymerizations, as the photoamplification chain can be stopped and/or re-initiated 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.akutatel@du.edu. Supporting Information AvailableExperimental procedures and spectra. This material is available free of charge via the Internet at http://p...

Externally sensitized photoinduced fragmentation in dithiane adducts of carbonyl compounds [1] requires the presence of an electron-transfer sensitizer, and therefore can be made contingent on a molecular recognition event. This event "arms" the binary photoactive system by bringing the adduct and the sensitizer into the immediate proximity of each other.[2] Such conditional photorelease of dithianes, which are readily detectable at subpicomolar levels, presents new opportunities for useful bioanalytical applications.We have recently developed this concept into a fundamentally novel methodology for direct screening of solutionphase combinatorial libraries, in which various 2-alkylsubstituted dithianes are used as encoding digits.[3] By immobilizing dithiane-masked benzophenone units on polymeric beads or dendrimers, we have further shown that one molecular recognition event can trigger the release of multiple copies of the encoding dithiane tags, which amounts to amplification on the surface.[4] Such photoamplification is possible because each externally sensitized fragmentation of the benzophenone-dithiane adducts unmasks more sensitizer which, in turn, unmasks its neighbors carrying the amplification chain and therefore boosting the sensitivity.The next logical question is whether this photoamplification methodology can be implemented in a linear array of masked sensitizers, and-if such amplification is possiblewhether the propagation of the effect and the release of dithiane tags occurs sequentially (that is, not unlike the onedimensional propagation in the Bickford fuse), randomly, or in some other unusual order. Herein, we report on photoamplification in linear polypeptide scaffolds.The synthesis of masked benzophenone sensitizers tethered to Fmoc-protected lysine is outlined in Scheme 1. We chose four 2-substituted dithianes, ethyl (a), propyl (b), pentyl (c), and octyl (d), to positionally encode the lysine residues in the polypeptide chain. The lithiated dithianes were reacted with 3-benzoylbenzoic acid sodium salt (1), thus furnishing adducts 2 a-d, which were converted into N-hydroxysuccinimide (NHS) esters 3 a-d and tethered to Fmoc-protected lysine. The modified lysines 4 a-d, in the form of their NHS esters 5 a-d, were utilized in solid-state peptide synthesis on TentaGel beads (0.48 mmol g À1 ). The peptides were capped with lysine-tethered benzophenone as the photoinitiator. As linear amplification in a single strand was the focus of our study, we chose the low-loading beads as a scaffold mimicking infinite dilution in solvent (that is, preventing interstrand sensitization; see Supporting Information).The following tetra-, hepta-, and decapeptides, which contain photoactive lysine residues and spacers, were synthesized:Lys BP -Lys DT1 -Lys DT2 -Lys DT3 -BEAD (6 a) Lys BP -Gly-Lys DT1 -Gly-Lys DT2 -Gly-Lys DT3 -BEAD (6 b) Lys BP -Met-Lys DT1 -Met-Lys DT2 -Met-Lys DT3 -BEAD (6 c) Lys BP -Gly-Gly-Lys DT1 -Gly-Gly-Lys DT2 -Gly-Gly-Lys DT3 -BEAD (6 d) where Lys DTX represents a lysine residue outfitted ...

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Novel photolabile amphiphiles containing thioxanthone-based fluorogenic caging groups are developed. Photoinduced fragmentation in dithiane-thioxanthone adducts was demonstrated to occur with 100% quantum efficiency at λ ~ 320 nm and more than 50% at λ ~ 360 nm. A plausible mechanism involves homolytic fission of a carbon-carbon single bond in the excited thioxanthone followed by disproportionation via hydrogen transfer. The critical feature of the system is that fluorescence of a substituted thioxanthone is recovered as a result of photofragmentation, making dithiane-thioxanthone adducts efficient fluorogenic caging groups. Photolabile amphiphiles containing these fluorogens are synthesized and their photoinduced disassembly is probed while following the fluorescence recovery. This methodology allows for destabilizing supramolecular assemblies of amphiphiles and at the same time offers a feedback mechanism for monitoring the process by fluorescence.

Externally sensitized photoinduced fragmentation in dithiane adducts of carbonyl compounds [1] requires the presence of an electron-transfer sensitizer, and therefore can be made contingent on a molecular recognition event. This event "arms" the binary photoactive system by bringing the adduct and the sensitizer into the immediate proximity of each other.[2] Such conditional photorelease of dithianes, which are readily detectable at subpicomolar levels, presents new opportunities for useful bioanalytical applications.We have recently developed this concept into a fundamentally novel methodology for direct screening of solutionphase combinatorial libraries, in which various 2-alkylsubstituted dithianes are used as encoding digits.[3] By immobilizing dithiane-masked benzophenone units on polymeric beads or dendrimers, we have further shown that one molecular recognition event can trigger the release of multiple copies of the encoding dithiane tags, which amounts to amplification on the surface.[4] Such photoamplification is possible because each externally sensitized fragmentation of the benzophenone-dithiane adducts unmasks more sensitizer which, in turn, unmasks its neighbors carrying the amplification chain and therefore boosting the sensitivity.The next logical question is whether this photoamplification methodology can be implemented in a linear array of masked sensitizers, and-if such amplification is possiblewhether the propagation of the effect and the release of dithiane tags occurs sequentially (that is, not unlike the onedimensional propagation in the Bickford fuse), randomly, or in some other unusual order. Herein, we report on photoamplification in linear polypeptide scaffolds.The synthesis of masked benzophenone sensitizers tethered to Fmoc-protected lysine is outlined in Scheme 1. We chose four 2-substituted dithianes, ethyl (a), propyl (b), pentyl (c), and octyl (d), to positionally encode the lysine residues in the polypeptide chain. The lithiated dithianes were reacted with 3-benzoylbenzoic acid sodium salt (1), thus furnishing adducts 2 a-d, which were converted into N-hydroxysuccinimide (NHS) esters 3 a-d and tethered to Fmoc-protected lysine. The modified lysines 4 a-d, in the form of their NHS esters 5 a-d, were utilized in solid-state peptide synthesis on TentaGel beads (0.48 mmol g À1 ). The peptides were capped with lysine-tethered benzophenone as the photoinitiator. As linear amplification in a single strand was the focus of our study, we chose the low-loading beads as a scaffold mimicking infinite dilution in solvent (that is, preventing interstrand sensitization; see Supporting Information).The following tetra-, hepta-, and decapeptides, which contain photoactive lysine residues and spacers, were synthesized:Lys BP -Lys DT1 -Lys DT2 -Lys DT3 -BEAD (6 a) Lys BP -Gly-Lys DT1 -Gly-Lys DT2 -Gly-Lys DT3 -BEAD (6 b) Lys BP -Met-Lys DT1 -Met-Lys DT2 -Met-Lys DT3 -BEAD (6 c) Lys BP -Gly-Gly-Lys DT1 -Gly-Gly-Lys DT2 -Gly-Gly-Lys DT3 -BEAD (6 d) where Lys DTX represents a lysine residue outfitted ...