Steven E. Rokita

Macrocyclic nickel complexes in DNA recognition and oxidation

Nickel(I1) macrocyclic complexes have been found to promote site- and conformation-specific oxidation of DNA. The systematic study of ligand effects has demonstrated that the crucial features in regulating DNA reactivity are in-plane donor strength and flexibility of the macrocyclic ligand. Based upon investigations of conformation specificity, macrocyclic ligand effects, and oxidant requirements, a mechanism has been proposed. In this mechanism, a nickel(II1) complex containing a ligated guanine and oxidant molecule is postulated as a key intermediate. The oxidation of DNA has also been extended to macrocyclic complexes containing cobalt(III), rhodium(II1) and chromium(II1).

DNA MODIFICATION PROMOTED BY WATER-SOLUBLE NICKEL(II) SALEN COMPLEXES : A SWITCH TO DNA ALKYLATION

Reaction of a 17-base hairpin-forming oligonucleotide with [N,N-bis(salicylaldehyde)-meso-1,2-bis(4- trimethylaminophenyl)ethylenediimino]nickel(II) perchlorate, 2, and KHSO5 produced two types of high molecular weight products, an alkaline-labile species and a nonalkaline-labile species, which co-migrated on gel electrophoresis. Upon treatment with piperidine, the base-labile derivative led to strand scission products only at accessible guanine residues that were not part of a Watson-Crick duplex. The formation of higher molecular weight species is proposed to occur via a highly reactive ligand-centered radical acting as a DNA alkylating agent.

Flavoprotein iodotyrosine deiodinase functions without cysteine residues.

Flavoproteins are able to catalyze a variety of metabolic processes due to the wide range of reactions promoted by flavin. Appreciation for the catalytic power of flavin continues to increase as still more flavin-dependent processes are discovered. Typically, once a new activity is detected in one organism, related reactions are often identified soon thereafter in numerous other organisms, as recently illustrated by the growing class of flavin-dependent halogenases. In contrast, only one highly conserved flavoprotein has yet been directly associated with reductive dehalogenation. This enzyme, iodotyrosine deiodinase (IYD), is critical in the thyroid for recycling iodide from the by-products of thyroxine (T4, 4-(4-hydroxy-3,5diiodophenoxy)-3,5-diiodophenylalanine) biosynthesis (Scheme 1). Reductive dehalogenation is unusual in aerobic life and particularly rare in higher organisms. Humans express only two representatives, the flavoprotein mentioned above that acts on iodinated tyrosine and a selenoprotein iodothyronine deiodinase (ID) that acts on T4 and its derivatives. Similar mechanisms have been proposed for these enzymes based on their reported dependence on a cysteine and selenocysteine residue, respectively. ID is thought to stabilize a nonaromatic tautomer of T4 and then release an equivalent of I that oxidizes its selenocysteine residue (Scheme 2). An analogous process of stabilizing a substrate tautomer and reacting with an active site cysteine has also been proposed for a reductive dechlorination catalyzed by the bacterial enzyme tetrachlorohydroquinone dehalogenase (TD) (Scheme 2). Similarly, IYD appears to stabilize a nonaromatic tautomer of diiodotyrosine because a pyridone derivative that mimics the intermediate tautomer binds to the enzyme with very high affinity. Subsequent evidence was then expected to implicate involvement of one or more cysteine residues in IYD turnover as well. The results described herein reveal instead that all aspects of turnover are independent of cysteine, and neither ID nor TD provides precedence for IYD. The major structural domain of IYD belongs to the NADH oxidase/flavin reductase superfamily, and prior modeling suggested that two of the three cysteines (C217 and C239, Figure 1) were located in the proposed active site adjacent to the bound flavin mononucleotide (FMN). This proximity is reminiscent of the many flavoproteins that contain redoxactive cysteines, and hence the flavin of IYD was originally expected only to mediate electron transfer to an active site cysteine. Although NADPH is thought to act as the physiological source of reducing equivalents, its effect is lost after detergent is used to disrupt the membrane surrounding IYD. Under these conditions, dithionite is required as an alternative electron donor. Thiols such as GSH and DTT do not support turnover by IYD in contrast to their ability to promote deiodination by ID and dechlorination by TD. Still, thiols are chemically Scheme 1. Iodide homeostasis is maintained in part by recycling iodide from monoand diiodotyrosine.

Synthesis and reactivity of 6-(fluoromethyl)indole and 6-(difluoromethyl)indole

Abstract The N-1 BOC protected precursors of 6-(fluoromethyl)indole and 6-(difluoromethyl)indole were prepared and deprotected via flash vacuum thermolysis. The stability of these newly prepared, unprotected indole derivatives has been characterized and compared to that of a previously known compound, 6-(trifluoromethyl)indole.

Chemical Reagents for Investigating the Major Groove of DNA

Chemical modification provides an inexpensive and rapid method for characterizing the structure of DNA and its association with drugs and proteins. Numerous conformation‐specific probes are available, but most investigations rely on only the most common and readily available of these. The major groove of DNA is typically characterized by reaction with dimethyl sulfate, diethyl pyrocarbonate, potassium permanganate, osmium tetroxide, and, quite recently, bromide with monoperoxysulfate. This commentary discusses the specificity of these reagents and their applications in protection, interference, and missing contact experiments.

DNA Cleavage vs. Cross-Linking Using Nickel Peptides

Rarely do the late transition metal ions Ni2+ and Cu2+ exist as aquated ions in vivo; rather they find protein residues as effective chelating agents. In serum, albumin is one of the principal scavengers of these ions, in part via the N-terminus which in the human protein begins with the sequence Asp-Ala-His (DAH) (Harford and Sarkar, 1997). Since the characterization of this site, the N-terminal XXH motif has been identified in a number of other peptides and proteins, including human spermine protamine HP2a, histatin 3, and the neuromedins C and K (Bal et al., 1997). Nickel has long been known as a carcinogenic metal, especially in the form of nickel ore particulates which more readily enter cells by phagocytosis (Costa et al., 1994). Once inside the cell, complexation to nuclear proteins, especially histones, could be one important mechanism for toxicity in which redox active nickel species are capable of inflicting oxidative damage on proteins and DNA (Kasprzak, 1996; Kasprzak, 1995). Both DNA strand breaks and DNA-protein cross-links have been identified as lesions resulting from exposure to excess nickel (Ciccarelli et al., 1981; Ciccarelli and Wetterhahn, 1982). Meanwhile, bioconjugates containing the N-terminal XXH motif have been designed for the express purpose of protein or DNA modification. In order to understand the molecular mechanisms underlying nickel toxicity as well as to provide a foundation for the design of DNA-targeting agents, we have carried out a series of mechanistic investigations pertaining to nickel peptide mediated DNA oxidation. Here we review our current understanding of these processes.