Rokita Laboratory Research Summary
Our research program is united by a common interest in describing the structure and activity of biological macromolecules through their essential chemical reactivity. The methods of organic synthesis, physical organic chemistry, protein and nucleic acid chemistry, biochemistry and genetic manipulation are applied to questions of enzyme catalysis and nucleic acid modification. Current projects include: enzymatic dehalogenation; DNA photochemistry and electron transfer; quinone methide generation and reactivity; target promoted alkylation of nucleic acids; and metal-mediated recognition and oxidation of nucleic acids.

Rokita Curriculum Vitae

Current Laboratory Members

Previous Laboratory Members

Recent Undergraduate and Graduate Courses
CHEM 648 Fall 2004
BCHM 463 Spring 2004
BCHM 463 Spring 2005
CHEM 231 Fall 2005
BCHM 462 Spring 2006
CHEM 231 Fall 2007
BCHM 462 Spring 2008

Enzymatic dehalogenation. The catalytic properties of the enzyme iodotyrosine deiodinase are currently under investigation to identify the source of its unique reactivity. This enzyme is necessary for conserving iodide for biosynthesis of thyroxine and is one of only two types of mammalian enzymes known to effect reductive dehalogenation. Iodotyrosine deiodinase is reported to contain an essential flavin although the role of flavin in the cleavage of the carbon-iodine bond is unprecedented and not yet understood.

Our description of catalysis to date has relied on 1) reversible enzyme inhibition to define both substrate recognition patterns and catalytic transition-state (reactive intermediate) properties, 2) mechanism-based inactivation to characterize substrate activation and 3) protein expression and mutagenesis to identify amino acid residues responsible for its key catalytic and structural properties. Evidence derived from a series of transition-state analogues suggests a catalytic mechanism involving substrate tautomerization and transient loss of aromatic stability.

DNA photochemistry and electron transfer. Genetic changes caused by exposure to sunlight occur along sequences of DNA at widely variable rates. Often pre-mutagenic lesions are localized at sites referred to as hotspots. The molecular determinants of this phenomenon are under investigation by examining the photochemistry of defined oligonucleotide and polynucleotide models. In a complementary approach, we are also characterizing the nature of excess electron transfer through DNA using aromatic amines as photoactivated electron donors and bromodeoxyuridine as an electron acceptor. Reductive electron transfer in general has yet to be described to the extent known for the complementary process of oxidative transfer. Both types of transfer affect the distribution of lesions in DNA formed by environmental stress.

Quinone methide generation and reactivity. Quinone methides are highly electrophilic and transient intermediates generated through bioactivation. These intermediates cause DNA alkylation by drugs such as mitomycin and tamoxifen, food additives such as butylated hydroxytoluene (BHT), and some natural products. Our laboratory has developed a series of simple precursors that form ortho quinone methide intermediates upon addition of the chemical trigger fluoride. This approach allows for easy control of reaction under biomimetic conditions. Functional groups that are both strongly nucleophilic and reasonably acidic (nuc1) react with quinone methides quickly but transiently to reform this intermediate. In contrast, weaker nucleophiles (nuc2) that are not good leaving groups react more slowly but form stable adducts .

Target promoted alkylation of nucleic acids. The reversibility of quinone methide alkylation has provided a strategy to create stable derivatives that unfurl a latent quinone methide only after they bind to their target sequence of nucleotides. (1) Intramolecular and reversible formation of a self adduct generated between a quinone methide and its conjugated oligonucleotide remains dominant in the presence of non-complementary DNA and even strong nucleophiles such as 2-mercaptoethanol. (2) Intermolecular reaction only becomes competitive when the thermodynamics of base pairing drives a conformational change that allows for efficient transfer of the quinone methide to its intended target. Our efforts now focus on developing related conjugates for applications in vivo and ultimately, the general principle of target promoted alkylation may be applied to a wide variety of biological macromolecules and reactive intermediates. Recent publication of this work in Proc. Natl. Acad. Sci. USA was highlighted in Chemical and Engineering News.

Metal-mediated recognition and oxidation of nucleic acids. Transition metal complexes have received great attention in the field of nucleic acids because of their role in carcinogenesis, chemotherapy and structural recognition. A number of functions vital to our health are dependent on nickel and copper and yet many detrimental activities are also associated with these metals. Our laboratory focuses on protein and nucleic acid oxidation caused by various complexes of nickel and copper. Attention is directed most specifically to the relationship between the oxidative activity of biomimetic complexes and their nuclearity and coordination chemistry in order to discovery new chemotherapeutic agents. Most recently in collaboration with Professor Kenneth Karlin (Department of Chemistry, Johns Hopkins University), we are examining the mechanism of selective recognition and oxidation of DNA by multinuclear copper complexes as illustrated below.