The cytochrome P450 enzymes represent an important class of heme containing enzymes. There is considerable interest in immobilizing these enzymes on a surface so that interactions between a single enzyme and other species can be studied with respect to electron transfer, homodimer or heterodimer interactions, or for construction of biological based chips for standardizing cytochrome P450 metabolism or for high throughput screening of pharmaceutical agents. Previous studies have generally immobilize P450 enzymes in a matrix or on a surface. Here, we have attached CYP2C9 to gold substrates such that the resulting construct maintains the ability to bind and metabolize substrates in the presence of NADPH and cytochrome P450 reductase. The activity of these chips is directly dependent upon the linkers used to attach CYP2C9 and to the presence of key molecules in the active site during enzyme attachment. A novel method to detect substrate-enzyme binding, namely superconducting quantum interference device (SQUID) magnetometry, was used to monitor the binding of substrates. Most significantly, conditions that allow measurable CYP2C9 metabolism to occur have been developed. The cytochrome P450 enzymes represent an important class of heme containing enzymes. They are responsible for a significant portion of xenobiotic metabolism and have been extensively studied for mechanistic and practical reasons. More recently there has been interest in immobilizing these enzymes on a surface to study the electron transfer in a single enzyme, 1 homodimer or heterodimer interactions, 2 or for construction of biological based chips for high throughput screening of pharmaceutical agents.Previous efforts to immobilize P450 enzymes have been made 3 including CYP2E1 on gold electrodes 1 bacterial P450 BM3 on graphite, 4 P450cam immobilized in sol-gel films, 5 pgannett@hsc.wvu.edu. CYP1A2 and CYP3A4 in polyion films, 6 and CYP119 in dimethyldidodecylammonium poly (p-styrene sulfonate). 5 In these studies the detection of substrate binding is often made electrochemically by observing shifts in the redox potential upon substrate binding. 7 Enzymelike metabolic reactions have also been observed by the application of an electrochemical current. 1 However, to be amenable to study and manipulation at the single molecule level it may be desirable to minimize the surrounding matrix. Also, it is highly desirable for the enzyme to metabolize substrates utilizing endogenous enzymes and co-factors to more closely model the corresponding biological system. To our knowledge, this has not been achieved with cytochrome P450 enzymes. NIH Public AccessIn this work we have attached CYP2C9 to gold substrates such that the resulting construct maintains the ability to bind and metabolize substrates in the presence of NADPH and cytochrome P450 reductase. The activity of these chips is dependent upon the linkers used to attach CYP2C9 and to the presence of key substrates during the attachment. A novel method to detect substrate binding, namely superconducti...

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C8-Aryl purines, their nucleosides, and phosphoramidites has been synthetic targets for more than 60 years. Interest in these compounds stems from their utility as fluorescent markers, they have therapeutic uses, are biomarkers, biomolecular probes, supramolecular building blocks, and for conformational studies. Until recently, the selective arylation of the C8-position of purines has been a challenging task. Several approaches have been explored including building them up from a pyrimidine or selective C8-modification of an unsubstituted purine. Neither of these approaches has proven to have broad scope. The discovery that C8-aryl purine nucleosides can be made via the Suzuki cross-coupling reaction has allowed a diverse array of analogues to be prepared and, in turn, the corresponding phosphoramidites. The latter is particularly significant as C8-aryl purine adducts are a major mutation observed from aromatic carcinogens and ready access to C8-aryl phosphoramidites will facilitate the synthesis and study of C8-aryl purine biomarkers and modified oligonucleotides.

DNA has many different conformations however only Z‐DNA adopts a left‐handed helix. The zigzag phosphate backbone of Z‐DNA brings the negatively charged phosphate residues closer together rendering Z‐DNA a higher energy form of DNA. Z‐DNA has been implicated in carcinogenesis from its role in gene expression and mutagenesis because Z‐DNA formation can stimulate large scale gene deletions, translocations, and rearrangements in vivo. The inability to generate a stable Z‐DNA structure under physiological conditions has made investigation of B‐ to Z‐DNA interconversion difficult since Z‐DNA requires external stimuli to stabilize its formation like chemical modifications or specific proteins known to stabilize Z‐DNA, Z‐DNA binding proteins (ZBP). We have demonstrated that C8‐arylguanine DNA adducts, derived from carcinogenic arylhydrazines, drive Z‐DNA formation and the addition of MgCl2 stabilizes the Z form under physiological conditions. The conformational effects of the C8‐arylguanine mutation were determined by nuclear magnetic resonance (NMR) and circular dichroism (CD). In addition, preliminary results using in silico molecular dynamics and CD have demonstrated that ZBPs prefer to bind to the C8‐arylguanine modified DNA. C8‐Arylguanine can be used as a tool to clarify the mechanism of ZBP mediated B‐ to Z‐DNA transition. The current view fails to explain the ZBP selectivity for the Z form and leads to a contradictory conclusion that ZBPs cannot bind Z‐DNA. C8‐Arylguanine modified oligonucleotides can be used to fully elucidate the binding of ZBPs to DNA, as both the B and Z forms can be generated under physiological conditions, using surface plasmon resonance (SPR). This may reveal the underlying factors that control ZBP‐DNA interactions which could become a potential therapeutic target. (Supported by a fellowship to BCT (HEPC.dsr.09013) and (NSF EPS‐1003907)