The functionalization of C-H bonds is an essential reaction in biology and chemistry. Metalloenzymes that often exhibit this type of reactivity contain metal-oxido intermediates which are directly involved the initial cleavage of the C-H bonds. Regulation of the cleavage process is achieved, in part, by hydrogen bonds that are proximal to the metal-oxido units, yet our understanding of their exact role(s) is still emerging. To gain further information into the role of H-bonds on C-H bond activation, a hybrid set of urea-containing tripodal ligands has been developed in which a single H-bond can be adjusted through changes in the properties of one ureayl N-H bond. This modularity is achieved by appending a phenyl ring with different parasubstituents from one ureayl NH group. The ligands have been used to prepare a series of Mn IIIoxido complexes and a Hammett correlation was found between the pK a values of the complexes and the substituents on the phenyl ring that was explained within the context of changes to the Hbonds involving the Mn III-oxido unit. The complexes were tested for their reactivity toward 9,10dihydroanthracene (DHA) and a Hammett correlation was found between the second-order rate constants for the reactions and the pK a values. Studies to determine activation parameters and the kinetic isotope effects are consistent with a mechanism in which rate-limiting proton transfer is an important contributor. However, additional reactivity studies with xanthene found a significant increase in the rate constant compared to DHA, even though the substrates have the same pK a (C-H) values. These results suggest do not support a discrete proton-transfer/electron transfer process, but rather an asynchronous mechanism in which the proton and electron are transferred unequally at the transition state.

Abstract. Changes in the amounts of cellular eumelanin and pheomelanin have been associated with carcinogenesis. The goal of this work is to develop methods based on two-photon-excited-fluorescence (TPEF) for measuring relative concentrations of these compounds. We acquire TPEF emission spectra (λ ex ¼ 1000 nm) of melanin in vitro from melanoma cells, hair specimens, and in vivo from healthy volunteers. We find that the pheomelanin emission peaks at approximately 615 to 625 nm and eumelanin exhibits a broad maximum at 640 to 680 nm. Based on these data we define an optical melanin index (OMI) as the ratio of fluorescence intensities at 645 and 615 nm. The measured OMI for the MNT-1 melanoma cell line is 1.6 AE 0.22 while the Mc1R gene knockdown lines MNT-46 and MNT-62 show substantially greater pheomelanin production (OMI ¼ 0.5 AE 0.05 and 0.17 AE 0.03, respectively). The measured values are in good agreement with chemistry-based melanin extraction methods. In order to better separate melanin fluorescence from other intrinsic fluorophores, we perform fluorescence lifetime imaging microscopy of in vitro specimens. The relative concentrations of keratin, eumelanin, and pheomelanin components are resolved using a phasor approach for analyzing lifetime data. Our results suggest that a noninvasive TPEF index based on spectra and lifetime could potentially be used for rapid melanin ratio characterization both in vitro and in vivo.

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Background and Objective Achieving local control of gliomas with photodynamic therapy (PDT) requires the delivery of adequate light fluences to depths of 1–2 cm in the resection margin where the majority of local recurrences originate. This is clinically impractical with current single-shot, intraoperative PDT treatments due to the length of time required to deliver adequate fluences. Multiple or extended treatment protocols would therefore seem to be required. The response of human glioma spheroids to 5-aminolevulinic acid (ALA)-mediated PDT using single or, repetitive light delivery protocols was investigated at both low and ultra low fluence rates. Study Design/Materials and Methods Human glioma spheroids (400 μm diameter) were subjected to sub-threshold light fluence (1.5, 3, or 6 J cm−2) ALA–PDT consisting of four light delivery schemes: single treatment given over either 1 or 24 hours, repetitive treatment given either as four 1 hour light treatments separated by a 4 day interval, or 24 hours light delivery, consisting of four 24 hours treatments separated by a 3 day interval. Treatment efficacy was evaluated using a growth assay. In some cases, confocal microscopy was used to image cell viability. Results The repetitive and single light treatment protocols were most effective when delivered at ultra low (μW cm−2) fluence rates. In all cases, growth inhibition was light dose-dependent. The repetitive ultra low fluence rate treatment (1.5 J cm−2; irradiance = 17 μW cm−2) light delivery protocol was the most effective resulting in total growth inhibition during the 2-week observation period. Conclusion Ultra low light fluence rate ALA–PDT results in significant spheroid growth inhibition. Repeated administration of ALA was required during repetitive and/or protracted single PDT treatment protocols. The existence of a lower fluence rate limit, below which the efficacy of threshold light fluences diminish was not found in these studies.