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.

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.

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.

Spectral Properties of Fluorogenic Thiophene-derived Triarylmethane Dyes.

Spectral properties and fluorogenic behaviors of five novel thiophene variants of malachite green (MG), termed MGTs, were determined. Appreciable changes as a function of homologation and substitution pattern, including absorption band positions and intensities and fluorescence quantum yields were observed. In particular, the shorter wavelength y-band absorption was found to shift over a nearly 200 nm range based on aryl group variation, allowing fine-tuning of the excitation wavelength for these dyes. In addition, the fluorescence intensity of some MGTs increased significantly (up to 4600-fold) when the dye was bound to a cognate protein partner, which is potentially useful for cell imaging studies.

p-Hydroxyphenacyl photoremovable protecting groups - Robust photochemistry despite substituent diversity.

A broadly based investigation of the effects of a diverse array of substituents on the photochemical rearrangement of p-hydroxyphenacyl esters has demonstrated that common substituents such as F, MeO, CN, CO2R, CONH2, and CH3 have little effect on the rate and quantum efficiencies for the photo-Favorskii rearrangement and the release of the acid leaving group or on the lifetimes of the reactive triplet state. A decrease in the quantum yields across all substituents was observed for the release and rearrangement when the photolyses were carried out in buffered aqueous media at pHs that exceeded the ground-state pKa of the chromophore where the conjugate base is the predominant form. Otherwise, substituents have only a very modest effect on the photoreaction of these robust chromophores.

Fluorinated photoremovable protecting groups: the influence of fluoro substituents on the photo-Favorskii rearrangement.

To further explore the nature of the photo-Favorskii rearrangement and its commitment to substrate photorelease from p-hydroxyphenacyl (pHP), an array of ten new fluorinated pHP gamma-aminobutyric acid (GABA) derivatives was synthesized and photolyzed. The effects of fluorine substitution on the chromophore and the photophysical and photochemical properties of these new chromophores were shown to be derived primarily from the changes in the ground state pK(a) of the phenolic groups. The quantum yields and rate constants for release are clustered around Phi(dis) = 0.20 +/- 0.05 and k(r) = 8 +/- 7 x 10(7) s(-1) (H2O), respectively. The triplet lifetimes of the pHP GABA derivatives were concentrated in the range of 0.4-6.0 ns (H2O). The corresponding deprotonated conjugate bases displayed reduced efficiencies by 50% or more (one exception) and exhibited a weak fluorescence in pH 8.2 buffer. Pump-probe spectroscopy studies have further defined the rates of intersystem crossing and the lifetimes of the reactive triplet state of the fluoro pHP chromophore.

Deoxycholate interacts with IpaD of Shigella flexneri in inducing the recruitment of IpaB to the type III secretion apparatus needle tip.

Type III secretion (TTS) is an essential virulence function for Shigella flexneri that delivers effector proteins that are responsible for bacterial invasion of intestinal epithelial cells. The Shigella TTS apparatus (TTSA) consists of a basal body that spans the bacterial inner and outer membranes and a needle exposed at the pathogen surface. At the distal end of the needle is a "tip complex" composed of invasion plasmid antigen D (IpaD). IpaD not only regulates TTS, but is required for the recruitment and stable association of the translocator protein IpaB at the TTSA needle tip in the presence of deoxycholate or other bile salts. This phenomenon is not accompanied by induction of TTS or the recruitment of IpaC to the Shigella surface. We now show that IpaD specifically binds fluorescein-labeled deoxycholate and, based on energy transfer measurements and docking simulations, this interaction appears to occur where the N-terminal domain of IpaD meets its central coiled-coil, a region that may also be involved in needle-tip interactions. TTS is initiated as a series of distinct steps and that small molecules present in the bacterial milieu are capable of inducing the first step of TSS through interactions with the needle tip protein IpaD. Furthermore, the amino acids proposed to be important for deoxycholate binding by IpaD appear to have significant roles in regulating tip complex composition and pathogen entry into host cells.