In Situ Electrochemical Monitoring of Caged Compound Photochemistry: An Internal Actinometer for Substrate Release.

Caged compounds are molecules that release a protective substrate to free a biologically active substrate upon treatment with light of sufficient energy and duration. A notable limitation of this approach is difficulty in determining the degree of photoactivation in tissues or opaque solutions because light reaching the desired location is obstructed. Here, we have addressed this issue by developing an in situ electrochemical method in which the amount of caged molecule photorelease is determined by fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes. Using p-hydroxyphenyl glutamate (pHP-Glu) as our model system, we generated a linear calibration curve for oxidation of 4-hydroxyphenylacetic acid (4HPAA), the group from which the glutamate molecule leaves, up to a concentration of 1000 µM. Moreover, we are able to correct for the presence of residual pHP-Glu in solution as well as the light artifact that is produced. A corrected calibration curve was constructed by photoactivation of pHP-Glu in a 3 µL photoreaction vessel and subsequent analysis by high-performance liquid chromatography. This approach has yielded a linear relationship between 4HPAA concentration and oxidation current, allowing the determination of released glutamate independent of the amount of light reaching the chromophore. Moreover, we have successfully validated the newly developed method by in situ measurement in a whole, intact zebrafish brain. This work demonstrates for the first time the in situ electrochemical monitoring of caged compound photochemistry in brain tissue with FSCV, thus facilitating analyses of neuronal function.

Competing pathways for photoremovable protecting groups: the effects of solvent, oxygen and encapsulation.

Extending the applications of Photoremovable Protecting Groups (PPGs) to "cage" phenols has generally met with unusually complex PPG byproducts. In this study, we demonstrate that the p-hydroxyphenacyl (pHP) cage for both simple and complex phenolics, including tyrosine, dispenses free phenols. With the simpler unsubstituted phenols, the reaction is governed by their Brønsted Leaving Group ability. On the other hand, the byproducts of the cage vary with these phenols. For the more acidic phenols the cage byproduct follows the Favorskii rearrangement to form p-hydroxyphenylacetic acid whereas for the weaker phenols other reactions such as reduction and hydrolysis begin to emerge. When the photolysis is conducted in octa acid (OA) containers, non-Favorskii, unrearranged fragments of the cage and other byproducts arise.

Supramolecular photochemistry of encapsulated caged ortho-nitrobenzyl triggers.

ortho-Nitrobenzyl (oNB) triggers have been extensively used to release various molecules of interest. However, the toxicity and reactivity of the spent chromophore, o-nitrosobenzaldehyde, remains an unaddressed difficulty. In this study we have applied the well-established supramolecular photochemical concepts to retain the spent trigger o-nitrosobenzaldehyde within the organic capsule after release of water-soluble acids and alcohols. The sequestering power of organic capsules for spent chromophores during photorelease from ortho-nitrobenzyl esters, ethers and alcohols is demonstrated with several examples.

Excitation by Axon Terminal GABA Spillover in a Sound Localization Circuit.

Synapses from neurons of the medial nucleus of the trapezoid body (MNTB) onto neurons of the lateral superior olive (LSO) in the auditory brainstem are glycinergic in maturity, but also GABAergic and glutamatergic in development. The role for this neurotransmitter cotransmission is poorly understood. Here we use electrophysiological recordings in brainstem slices from P3-P21 mice to demonstrate that GABA release evoked from MNTB axons can spill over to neighboring MNTB axons and cause excitation by activating GABAAR. This spillover excitation generates patterns of staggered neurotransmitter release from different MNTB axons resulting in characteristic "doublet" postsynaptic currents in LSO neurons. Postembedding immunogold labeling and electron microscopy provide evidence that GABAARs are localized at MNTB axon terminals. Photolytic uncaging of p-hydroxyphenacyl (pHP) GABA demonstrates backpropagation of GABAAR-mediated depolarizations from MNTB axon terminals to the soma, some hundreds of microns away. These somatic depolarizations enhanced somatic excitability by increasing the probability of action potential generation. GABA spillover excitation between MNTB axon terminals may entrain neighboring MNTB neurons, which may play a role in the developmental refinement of the MNTB-LSO pathway. Axonal spillover excitation persisted beyond the second postnatal week, suggesting that this mechanism may play a role in sound localization, by providing new avenues of communication between MNTB neurons via their distal axonal projections. Significance statement: In this study, a new mechanism of neuronal communication between auditory synapses in the mammalian sound localization pathway is described. Evidence is provided that the inhibitory neurotransmitter GABA can spill over between axon terminals to cause excitation of nearby synapses to further stimulate neurotransmitter release. Excitatory GABA spillover between inhibitory axon terminals may have important implications for the development and refinement of this auditory circuit and may play a role in the ability to precisely localize sound sources.

4-Hydroxyphenacyl Ammonium Salts: A Photoremovable Protecting Group for Amines in Aqueous Solutions.

Irradiation of N-protected p-hydroxyphenacyl (pHP) ammonium caged derivatives at 313 nm releases primary and secondary amines or ammonia in nearly quantitative yields via the photo-Favorskii reaction when conducted in acidic or neutral aqueous buffered media. The reaction efficiencies are strongly dependent on the pH with the most efficient and highest yields obtained when the pH of the media maintains the ammonium and p-hydroxyl groups as their conjugate acids. For example, the overall quantum yields of simple secondary amines release are 0.5 at acidic pH from 3.9 to 6.6 dropping to 0.1 at neutral pH 7.0 and 0.01 at pH 8.4. Speciation studies provide an acid-base profile that helps define the scope and limitations of the reaction. When the pKa of the ammonium group is lower than that of the phenolic hydroxyl group, as is the case for the α-amino-protected amino acids, the more acidic ammonium ion deprotonates as the media pH is changed from acidic toward neutral or basic, thus diminishing the leaving group ability of the amino group. This, in turn, lowers the propensity for the photo-Favorskii rearrangement reaction to occur and opens the reaction pathway to alternative competing photoreduction process.

2-Diazo-1-(4-hydroxyphenyl)ethanone: a versatile photochemical and synthetic reagent.

α-Diazo arylketones are well-known substrates for Wolff rearrangement to phenylacetic acids through a ketene intermediate by either thermal or photochemical activation. Likewise, α-substituted p-hydroxyphenacyl (pHP) esters are substrates for photo-Favorskii rearrangements to phenylacetic acids by a different pathway that purportedly involves a cyclopropanone intermediate. In this paper, we show that the photolysis of a series of α-diazo-p-hydroxyacetophenones and p-hydroxyphenacyl (pHP) α-esters both generate the identical rearranged phenylacetates as major products. Since α-diazo-p-hydroxyacetophenone (1a, pHP N2) contains all the necessary functionalities for either Wolff or Favorskii rearrangement, we were prompted to probe this intriguing mechanistic dichotomy under conditions favorable to the photo-Favorskii rearrangement, i.e., photolysis in hydroxylic media. An investigation of the mechanism for conversion of 1a to p-hydroxyphenyl acetic acid (4a) using time-resolved infrared (TRIR) spectroscopy clearly demonstrates the formation of a ketene intermediate that is subsequently trapped by solvent or nucleophiles. The photoreaction of 1a is quenched by oxygen and sensitized by triplet sensitizers and the quantum yields for 1a-c range from 0.19 to a robust 0.25. The lifetime of the triplet, determined by Stern-Volmer quenching, is 31 ns with a rate for appearance of 4a of k = 7.1 × 10(6) s(-1) in aq. acetonitrile (1 : 1 v : v). These studies establish that the primary rearrangement pathway for 1a involves ketene formation in accordance with the photo-Wolff rearrangement. Furthermore we have also demonstrated the synthetic utility of 1a as an esterification and etherification reagent with a variety of substituted α-diazo-p-hydroxyacetophenones, using them as synthons for efficiently coupling it to acids and phenols to produce pHP protect substrates.

Photorelease of phosphates: Mild methods for protecting phosphate derivatives.

We have developed a new photoremovable protecting group for caging phosphates in the near UV. Diethyl 2-(4-hydroxy-1-naphthyl)-2-oxoethyl phosphate (14a) quantitatively releases diethyl phosphate upon irradiation in aq MeOH or aq MeCN at 350 nm, with quantum efficiencies ranging from 0.021 to 0.067 depending on the solvent composition. The deprotection reactions originate from the triplet excited state, are robust under ambient conditions and can be carried on to 100% conversion. Similar results were found with diethyl 2-(4-methoxy-1-naphthyl)-2-oxoethyl phosphate (14b), although it was significantly less efficient compared with 14a. A key step in the deprotection reaction in aq MeOH is considered to be a Favorskii rearrangement of the naphthyl ketone motif of 14a,b to naphthylacetate esters 25 and 26. Disruption of the ketone-naphthyl ring conjugation significantly shifts the photoproduct absorption away from the effective incident wavelength for decaging of 14, driving the reaction to completion. The Favorskii rearrangement does not occur in aqueous acetonitrile although diethyl phosphate is released. Other substitution patterns on the naphthyl or quinolin-5-yl core, such as the 2,6-naphthyl 10 or 8-benzyloxyquinolin-5-yl 24 platforms, also do not rearrange by aryl migration upon photolysis and, therefore, do not proceed to completion. The 2,6-naphthyl ketone platform instead remains intact whereas the quinolin-5-yl ketone fragments to a much more complex, highly absorbing reaction mixture that competes for the incident light.

The pivotal role of oxyallyl diradicals in photo-Favorskii rearrangements: transient spectroscopic and computational studies.

The photochemistry of the hydroxybenzocycloalkanonyl derivatives 6b-e provides the triplet oxyallyl diradicals (3)9 that decay via intersystem crossing to their more stable singlet isomers (1)9. Vibrationally resolved transient spectra of (3)9 were recorded by pump-probe spectroscopy and laser flash photolysis. It was found that the ring strain dependent rate of intersystem crossing is the rate-limiting step in the formation of photo-Favorskii or solvolysis reaction products in water. The reactivities of open-shell singlet oxyallyls (1)9a-e determine the product ratios due to their relative abilities to form the corresponding cyclopropanones 10. The smallest five-membered derivative, (1)9b, represents the first example of an oxyallyl diradicaloid that cannot form cyclopropanone 10b or the isomeric allene oxide 13b; instead, it is eventually trapped by water to form the sole solvolysis product 12b. Our observations provide a comprehensive overview of the role of oxyallyl diradicals in reaction mechanisms and offer a new strategy to stabilize open-shell singlet diradicals.

Stereochemically probing the photo-Favorskii rearrangement: a mechanistic investigation.

Using model (R)-2-acetyl-2-phenyl acetate esters of (S)- or (R)-α-substituted-p-hydroxybutyrophenones (S,R)-12a and (R,R)-12b, we have shown that a highly efficient photo-Favorskii rearrangement proceeds through a series of intermediates to form racemic rearrangement products. The stereogenic methine on the photoproduct, rac-2-(p-hydroxyphenyl)propanoic acid (rac-9), is formed by closure of a phenoxy-allyloxy intermediate 17 collapsing to a cyclopropanone, the "Favorskii" intermediate 18. These results quantify the intermediacy of a racemized triplet biradical (3)16 on the major rearrangement pathway elusively to the intermediate 18. Thus, intersystem crossing from the triplet biradical surface to the ground state generates a planar zwitterion prior to formation of a Favorskii cyclopropanone that retains no memory of its stereochemical origin. These results parallel the mechanism of Dewar and Bordwell for the ground state formation of cyclopropanone 3 that proceeds through an oxyallyl zwitterionic intermediate. The results are not consistent with the stereospecific S(N)2 ground state Favorskii mechanism observed by Stork, House, and Bernetti. Interconversion of the diastereomeric starting esters of (S,R)-12a and (R,R)-12b during photolysis did not occur, thus ruling out leaving group return prior to rearrangement.

A photo-Favorskii ring contraction reaction: the effect of ring size.

The effect of ring size on the photo-Favorskii induced ring-contraction reaction of the hydroxybenzocycloalkanonyl acetate and mesylate esters (7a-d, 8a-c) has provided new insight into the mechanism of the rearrangement. By monotonically decreasing the ring size in these cyclic derivatives, the increasing ring strain imposed on the formation of the elusive bicyclic spirocyclopropanone 20 results in a divergence away from rearrangement and toward solvolysis. Cycloalkanones of seven or eight carbons undergo a highly efficient photo-Favorskii rearrangement with ring contraction paralleling the photochemistry of p-hydroxyphenacyl esters. In contrast, the five-carbon ring does not rearrange but is diverted to the photosolvolysis channel avoiding the increased strain energy that would accompany the formation of the spirobicyclic ketone, the "Favorskii intermediate 20". The six-carbon analogue demonstrates the bifurcation in reaction channels, yielding a solvent-sensitive mixture of both. Employing a combination of time-resolved absorption measurements, quantum yield determinations, isotopic labeling, and solvent variation studies coupled with theoretical treatment, a more comprehensive mechanistic description of the rearrangement has emerged.

Applications of p-hydroxyphenacyl (pHP) and coumarin-4-ylmethyl photoremovable protecting groups.

Most applications of photoremovable protecting groups have used o-nitrobenzyl compounds and their (often commercially available) derivatives that, however, have several disadvantages. The focus of this review is on applications of the more recently developed title compounds, which are especially well suited for time-resolved biochemical and physiological investigations, because they release the caged substrates in high yield within a few nanoseconds or less. Together, these two chromophores cover the action spectrum for photorelease from >700 nm to 250 nm.

2-Hydroxyphenacyl ester: a new photoremovable protecting group.

A 2-hydroxyphenacyl moiety absorbing below 370 nm is proposed as a new photoremovable protecting group for carboxylates and sulfonates. Laser flash photolysis and steady-state sensitization studies show that the leaving group is released from a short-lived triplet state. In addition, DFT-based quantum chemical calculations were performed to determine the key reaction steps. We found that triplet excited state intramolecular proton transfer represents a major deactivation channel. Minor productive pathways involving the triplet anion and quinoid triplet enol intermediates have also been identified.

Gas-phase fragmentation of deprotonated p-hydroxyphenacyl derivatives.

Electrospray ionization of methanolic solutions of p-hydroxyphenacyl derivatives HO-C(6)H(4)-C(O)-CH(2)-X (X = leaving group) provides abundant signals for the deprotonated species which are assigned to the corresponding phenolate anions (-)O-C(6)H(4)-C(O)-CH(2)-X. Upon collisional activation in the gas phase, these anions inter alia undergo loss of a neutral "C(8)H(6)O(2)" species concomitant with formation of the corresponding anions X(-). The energies required for the loss of the neutral roughly correlate with the gas phase acidities of the conjugate acids (HX). Extensive theoretical studies performed for X = CF(3)COO in order to reveal the energetically most favorable pathway for the formation of neutral "C(8)H(6)O(2)" suggest three different routes of similar energy demands, involving a spirocyclopropanone, epoxide formation, and a diradical, respectively.

The Discovery, Development and Demonstration of Three Caged Compounds.

An overview of the history, mechanistic aspects and applications is provided for p-hydroxyphenacyl (pHP) and benzoin photoremovable protecting groups, which release biologically important leaving groups upon photolysis with UV light. Also discussed is (7-diethylaminocoumarin-4-yl)methyl (DEACM), a photoremovable protecting group that absorbs visible light. These are followed by the α-keto amides and naphtho- and benzothiophene-2-carboxanilides as caging groups, which eliminate leaving groups via photochemically produced zwitterionic intermediates. Also covered are amino-1,4-benzoquinones, which upon exposure to green and red wavelengths of light photorearrange to an unstable photoproduct that subsequently eliminates leaving groups in aqueous media. Selected examples are given that use these photoremovable protecting (caging) groups for the light-activated release of biologically important substrates under physiological conditions in cells and tissue as practical applications in biology, biochemistry and physiology. These caging groups have found significant applications because their photochemistry is efficient and a single coproduct is formed in addition to the photoreleased substrate.

Visible photorelease of liquid biopsy markers following microfluidic affinity-enrichment.

We detail a heterobifunctional, 7-aminocoumarin photocleavable (PC) linker with unique properties to covalently attach Abs to surfaces and subsequently release them with visible light (400-450 nm). The PC linker allowed rapid (2 min) and efficient (>90%) release of CTCs and EVs without damaging their molecular cargo.

Competing pathways in the photo-Favorskii rearrangement and release of esters: studies on fluorinated p-hydroxyphenacyl-caged GABA and glutamate phototriggers.

Three new trifluoromethylated p-hydroxyphenacyl (pHP)-caged gamma-aminobutyric acid (GABA) and glutamate (Glu) derivatives have been examined for their efficacy as photoremovable protecting groups in aqueous solution. Through the replacement of hydrogen with fluorine, e.g., a m-trifluoromethyl or a m-trifluoromethoxy versus m-methoxy substituents on the pHP chromophore, modest increases in the quantum yields for the release of amino acids GABA and glutamate as well as improved lipophilicity were realized. The pHP triplet undergoes a photo-Favorskii rearrangement with concomitant release of the amino acid substrate. Deprotonation competes with the rearrangement from the triplet excited state and yields the pHP conjugate base that, upon reprotonation, regenerates the starting ketoester, a chemically unproductive or "energy-wasting" process. When picosecond pump-probe spectroscopy is employed, GABA derivatives 2-5 are characterized by short triplet lifetimes, a manifestation of their rapid release of GABA. The bioavailability of released GABA at the GABA(A) receptor improved when the release took place from m-OCF3 (2) but decreased for m-CF3 (3) when compared with the parent pHP derivative. These studies demonstrate that pKa and lipophilicity exert significant but sometimes opposing influences on the photochemistry and biological activity of pHP phototriggers.

Collagen cross linking agents: design and development of a multifunctional cross linker.

A new cross linking reagent based on the first-generation polyamidoamine dendrimer (G.1 PAMAM) has been synthesized by reaction of the PAMAM with eight equivalents of p-nitrophenyl diazopyruvate. The resulting water-soluble octadiazopyruvoyl PAMAM (8G.1 DAP, 1.3) was shown to undergo Wolff rearrangements upon photolysis in methanol at lambda > 300 nm to yield the methyl esters of the ketenes formed from the loss of nitrogen. 8G.1 DAP also forms strong bonds with dehydrated collagen with glass as high as 36 N cm(-2). Collagen to collagen bonds with tensile strengths as high as 92 N cm(-2) were observed with fully dehydrated tissues. The bonding decreased rapidly with increasingly hydrated tissue possibly due to the increased distance between the collagen fibrils and the competition of H2O for the free ketene functions.

Caged substrates applied to high content screening: an introduction with an eye to the future.

The use of photoremovable protecting groups in biology affords the end user high temporal, spatial, and concentration control of reagents and substrates. High content screening and other large-scale biology applications would benefit greatly from these advantages. Herein, we report progress in this field by highlighting the recent development of controllable siRNA (csiRNA), which is a dormant siRNA that can be activated using 365 nm light. Two different experimental designs are described to highlight the temporal and concentration variables that can be controlled. First, the RNAi process is activated at two timepoints, 24- and 48-h post-transfection, to demonstrate that the action of csiRNA does not begin until activated. Second, increasing light dosage exposure to cells transfected with csiRNA that controls the concentration of active siRNA molecules. All experiments are conducted in a 96-well format with light delivered through the UCOM device.

Melding Caged Compounds with Supramolecular Containers: Photogeneration and Miscreant Behavior of the Coumarylmethyl Carbocation.

By merging well-established concepts of supramolecular chemistry, protecting group strategy, and photochemistry, we have solubilized in water hydrophobic organic molecules consisting of a photoactive protecting group and masked carboxylic acids, released the desired acid, and confined a reactive carbocation intermediate within a capsule. Confinement of the photogenerated carbocation brought out the latent radical-like behavior. This observation is consistent with the recent theoretical prediction of the 7-(diethylamino)coumarinyl-4-methyl carbocation having a triplet diradical ground-state electronic contribution.