In physiological compartments, provided with suitable reducing agents, transition metals are able to catalyze the activation of O 2 to various reactive oxygen species (ROS) 1 which readily attack biomolecules. The metal-catalyzed oxidation (MCO) of proteins plays an important role during oxidative stress and aging where modified proteins may accumulate in tissue (1-9). In addition, MCO also presents a stability problem for the biotechnological manufacturing of proteins (10, 11).A unique feature often observed during MCO of proteins is that only a few amino acid residues within a certain domain of the given protein are modified (2, 12). Such site specificity is attributed to the generation of ROS at specific metal-binding sites, where the highly reactive ROS attack labile functional groups nearby rather than diffuse into the bulk medium. It has been shown that most of the enzymes labile to MCO require metals for activity (13). The oxidation of these enzymes is less sensitive to the inhibition by exogenous ROS scavengers than would be expected on the basis of known rate constants for the reactions of specific ROS with these scavengers. However, one problem associated with all these studies is that neither the oxidizing species nor the intermediary protein-transition metal complexes formed during MCO of proteins have been well characterized. This is most pertinent to copper-catalyzed oxidation (i.e. to the frequently used ascorbate/Cu(II)/O 2 system) involving the Cu(II)/Cu(I) redox couple (2). Here, Cu(II) will most probably require a different geometry and different ligands within the protein ligand sphere than Cu(I). An initial reduction of Cu(II), bound to a metal-binding site, to Cu(I) may change the geometry of the protein-metal complex, and possibly even release Cu(I) from the metal-binding site. On the other hand, the metal may be retained when (i) the protein is flexible enough to adapt to the new geometrical requirements of the reduced metal, or (ii) re-oxidation of the metal by molecular oxygen or reduction products of molecular oxygen such as superoxide or hydrogen peroxide occurs faster than the release of the reduced metal from the metal-binding site.In a recent example for electron transfer coupled ligand dynamics in the model complex Cu(I/II)(TTCN) 2 , we could show that oxidation of tetrahedral [Cu(I)(TTCN) 2 ] ϩ potentially led to the loss of a ligand to yield [Cu(II)(TTCN)(H 2 O) 3 ] 2ϩ before geometrical reorganization around the Cu(II) atom allowed the addition of TTCN to give the final product, octahedral [Cu(II)(TTCN) 2 ] 2ϩ . 2 Analogous mechanisms may lead to kinetic complexities during protein oxidation which may be reflected in oxidation yields and kinetics as well as the nature of the oxidation products.One amino acid particularly affected by MCO is histidine (His) (15-17). There is as yet no detailed mechanism available for His oxidation in proteins, except a crystallographic study which showed the feasibility of metal-catalyzed processes within the metal-binding domain of glutamine syn...

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The 5,6-dihydroxytryptamine (5,6-DHT) derivatives 4-fluoro- and 7-fluoro-5,6-DHTs (26a,b) and 4,7-difluoro-5,6-DHT (26c) were synthesized from 3-fluoroanisole (1) and 1,4-difluoro-2,3-dimethoxybenzene (13), respectively. Efficient methods were developed for the conversion of 1 to 4-fluoro- and 7-fluoro-5,6-bis(benzyloxy)indoles (12a,b, respectively), and 13 to 4,7-difluoro-5,6-[( diphenylmethylene)dioxy]indole (19) via reductive cyclization of 2-nitro-beta-(dialkylamino)styrenes prepared in situ from 2-nitrotoluenes. Indoles 12a,b and 19 were then converted to 26a-c via the corresponding indole-3-acetonitriles. The fluorine-substituted 5,6-DHTs displayed increased phenol acidities, determined spectrophotometrically, and decreased inherent potential to undergo oxidation as determined by cyclic voltammetry. Fluorine substitution did not have a significant adverse effect on the cytotoxic potential as judged from the IC50 values of 117, 125, 135, and 92 microM for 26a,c and 5,6-DHT, respectively, for the inhibition of incorporation of [3H]thymidine into the DNA of neuroblastoma clone N-2a cells in culture. Surprisingly, 26a-c exhibited 32-, 23-, and 13-fold higher affinities, respectively, compared to 5,6-DHT for the serotonergic uptake system of N-2a cells as measured by the ability of 26a-c and 5,6-DHT to antagonize the uptake of [3H]5-HT into the N-2a cells. These desirable chemical and biological properties of 26a-c should make them useful tools for the study of the molecular mechanism of neurodegenerative action of 5,6-DHT.

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