David B. Smithrud

Real-time sensing and discrimination of single chemicals using the channel of phi29 DNA packaging nanomotor.

A highly sensitive and reliable method to sense and identify a single chemical at extremely low concentrations and high contamination is important for environmental surveillance, homeland security, athlete drug monitoring, toxin/drug screening, and earlier disease diagnosis. This article reports a method for precise detection of single chemicals. The hub of the bacteriophage phi29 DNA packaging motor is a connector consisting of 12 protein subunits encircled into a 3.6 nm channel as a path for dsDNA to enter during packaging and to exit during infection. The connector has previously been inserted into a lipid bilayer to serve as a membrane-embedded channel. Herein we report the modification of the phi29 channel to develop a class of sensors to detect single chemicals. The lysine-234 of each protein subunit was mutated to cysteine, generating 12-SH ring lining the channel wall. Chemicals passing through this robust channel and interactions with the SH group generated extremely reliable, precise, and sensitive current signatures as revealed by single channel conductance assays. Ethane (57 Da), thymine (167 Da), and benzene (105 Da) with reactive thioester moieties were clearly discriminated upon interaction with the available set of cysteine residues. The covalent attachment of each analyte induced discrete stepwise blockage in current signature with a corresponding decrease in conductance due to the physical blocking of the channel. Transient binding of the chemicals also produced characteristic fingerprints that were deduced from the unique blockage amplitude and pattern of the signals. This study shows that the phi29 connector can be used to sense chemicals with reactive thioesters or maleimide using single channel conduction assays based on their distinct fingerprints. The results demonstrated that this channel system could be further developed into very sensitive sensing devices.

Synthesis and investigation of host-[2]rotaxanes that bind metal cations.

Materials that bind metal cations are highly sought after for new devices. In this report, we show that rotaxanes can transfer metal cations with picrate, perchlorate, or chloride counterions from an aqueous solution into chloroform. The rotaxanes contain a dibenzyl-24-crown-8 ether as the wheel with either a benzyl-18-crown-6 ether (CEBG-R1-3) or a 3,5-dimethylbenzyl moiety (ArBG-R) as one blocking group. Alkali and alkaline picrate salts were efficiently extracted from an aqueous solution, presented in the millimolar range, into chloroform. Large association constants were derived for the complexes in chloroform, especially for the divalent cation Mg(2+). Switching the counterion to chloride greatly diminished the amount of salt extracted. To explore the transfer mechanism of the rotaxanes, a comparison was made in the amount of NaClO(4), KClO(4), NaCl, and KCl extracted by CEBG-R1, ArBG-R, benzyl-18-crown-6 ether (B18C6), and two model compounds, which were used to represent the crown-ether blocking group and the axle of a rotaxane. Two-dimensional NMR analysis was performed on the rotaxane-cation complexes in CDCl(3). We found that the host rotaxanes transfer the perchlorate salts poorly when compared to B18C6, but they transfer chloride salts from 1 M salt solutions, whereas B18C6 does not. The transfer of chloride salts appears to rely on an allosteric type relationship between the binding of the chloride ion and metal cation to a rotaxane. Accordingly, when chloride binds to the dialkylammonium ion of the axle, the wheel moves along the axle and forms a binding site for a metal cation. In this report we demonstrate that host rotaxanes can bind metal cations, change their geometries upon cation and anion association, and operate through allosteric mechanisms, making them promising candidates for molecular devices.

The state of antibody catalysis

One of the fascinations of catalytic antibodies is the possibility of harnessing the mechanisms available to enzymes for chemical transformation and applying them to the broad realm of chemistry encountered in organic synthesis. Recently, the catalytic repertoire of antibodies has been extended to include mechanistically more complex bimolecular reactions and the immunological response to the hapten can be more thoroughly examined as a result of the advent of new screening technology using bacterial phages or auxotrophic cell lines.

Host-rotaxanes with oligomeric axles are intracellular transport agents

Polymeric macromolecules are promising drug delivery devices with endocytotic properties that need to be resolved. Host-rotaxanes (HRs) also deliver materials into cells but require improved in vivo targeting capacity. Combining the targeting properties of nanoparticles with the transport function of HRs may improve drug efficacy. Our prototype HR (HR 1) has a short axle and is an efficient transporter. Here, we have constructed HRs that contain an oligo(ethylene glycol) (HR 2) or an oligoalkyl (HR 3) axle with the future goal of combining them with nanoparticles. HR 2 more efficiently delivers Fl-peptides into ovarian cancer cells than HR 3 and, in most cases, than HR 1. HR 2 appears to possess the appropriate balance between water solubility and lipophilicity to be an efficient transporter along with a suitable structure for incorporation into a larger nanoparticle.

Condensation reactions of calix[4]arenes with unprotected hydroxyamines, and their resulting water solubilities

Abstract In this report, we describe a convenient method to create neutrally charged, water soluble calix[4]arenes that contain hydroxyamides attached to their lower rims. Selective amidation reactions of a diacid calix[4]arene with several unprotected hydroxyamines was achieved using 2-ethoxy-1,2-dihydroquinoline as the coupling agent. The solubilities of the derivatized calix[4]arenes depended on the structure of the hydroxyamide, as well as, the number of hydroxyl groups. Molecular simulations of the derivatized compounds in water revealed that intramolecular H-bond formation is an important component of solubility. Calix[4]arenes containing 10 hydroxyl groups were as soluble in water as a calix[4]arene that contained two carboxylates.

Pt-rotaxanes as cytotoxic agents

Cytotoxic agents that specifically target cancer cells are in high demand. Modifying drugs with targeting groups however, can produce deleterious effects on drug pharmokinetics. In this study, platinum (Pt) was linked with host-rotaxanes to discover the effect on the cytotoxicity of Pt when carried by a highly modified rotaxane as a ligand. One host-rotaxane (Pt-BocRot) contains the basic components of a rotaxane: wheel (with a Boc protecting group), axle, and blocking group. A second rotaxane (Pt-ArgRot) contains arginine moieties on its wheel instead to potentially improve association with the phosphate groups on cell membranes or DNA backbone. The cytotoxicities of the rotaxanes and various model compounds were determined using ovarian cancer SKOV-3 cell line, which is resistant to cisplatin. We found Pt-ArgRot was slightly more cytotoxic than Pt-BocRot. Both were clearly more cytotoxic than rotaxanes without Pt and the model compounds. As importantly, they killed cells through an apoptotic mechanism. These results suggest that targeting agents for a particular cell type can be incorporated with Pt-complexes using the rotaxane architecture to improve drug specificity.

Synthesis of two bi-functional ligands for the QUEST three-hybrid system

Abstract The synthesis is described for two bi-functional ligands ( 1 ) and ( 2 ) that have been used as chemical inducers of dimerization for QUEST, 1 a three-hybrid system designed to detect novel enzyme activity in vivo.

Gankyrin Promotes Tumor-Suppressor Protein Degradation to Drive Hepatocyte Proliferation

Background & Aims Uncontrolled liver proliferation is a key characteristic of liver cancer; however, the mechanisms by which this occurs are not well understood. Elucidation of these mechanisms is necessary for the development of better therapy. The oncogene Gankyrin (Gank) is overexpressed in both hepatocellular carcinoma and hepatoblastoma. The aim of this work was to determine the role of Gank in liver proliferation and elucidate the mechanism by which Gank promotes liver proliferation. Methods We generated Gank liver-specific knock-out (GLKO) mice and examined liver biology and proliferation after surgical resection and liver injury. Results Global profiling of gene expression in GLKO mice showed significant changes in pathways involved in liver cancer and proliferation. Investigations of liver proliferation after partial hepatectomy and CCl4 treatment showed that GLKO mice have dramatically inhibited proliferation of hepatocytes at early stages after surgery and injury. In control LoxP mice, liver proliferation was characterized by Gank-mediated reduction of tumor-suppressor proteins (TSPs). The failure of GLKO hepatocytes to proliferate is associated with a lack of down-regulation of these proteins. Surprisingly, we found that hepatic progenitor cells of GLKO mice start proliferation at later stages and restore the original size of the liver at 14 days after partial hepatectomy. To examine the proliferative activities of Gank in cancer cells, we used a small molecule, cjoc42, to inhibit interactions of Gank with the 26S proteasome. These studies showed that Gank triggers degradation of TSPs and that cjoc42-mediated inhibition of Gank increases levels of TSPs and inhibits proliferation of cancer cells. Conclusions These studies show that Gank promotes hepatocyte proliferation by elimination of TSPs. This work provides background for the development of Gank-mediated therapy for the treatment of liver cancer. RNA sequencing data can be accessed in the NCBI Gene Expression Omnibus: GSE104395.

Ca2+ Selective Host Rotaxane Is Highly Toxic Against Prostate Cancer Cells

New therapies are needed to eradicate androgen resistant, prostate cancer. Prostate cancer usually metastasizes to bone where the concentration of calcium is high, making Ca2+ a promising toxin. Ionophores can deliver metal cations into cells, but are currently too toxic for human use. We synthesized a new rotaxane (CEHR2) that contains a benzyl 15-crown-5 ether as a blocking group to efficiently bind Ca2+. CEHR2 transfers Ca2+ from an aqueous solution into CHCl3 to greater extent than alkali metal cations and Mg2+. It also transfers Ca2+ to a greater extent than CEHR1, which is a rotaxane with an 18-crown-6 ether as a blocking group. CEHR2 was more toxic against the prostate cancer cell lines PC-3, 22Rv1, and C4-2 than CEHR1. This project demonstrates that crown ether rotaxanes can be designed to bind a targeted metal cation, and this selective cation association can result in enhanced toxicity.

A Versatile Axle for the Construction of Disassemblage Rotaxanes

Rotaxanes are unique mechanical devices that hold great promise as sensors. We report on two new rotaxanes that contain an acid or base sensitive trigger and readily disassemble in a wide range of environments. Disassemblage was observed under TLC and 1H-NMR analysis. The axle is highly charged, which enhances solubility in aqueous environments, and can be readily derivatized with sensor components. The trigger was swapped in a one-pot method, which is promising for the rapid production of a series of sensors.