Jack F. Eichler

Tumor cytotoxicity of 5,6-dimethyl-1,10-phenanthroline and its corresponding gold(III) complex.

A gold(III) complex possessing 5,6-dimethyl-1,10-phenanthroline (5,6DMP) was synthesized and fully characterized using standard spectroscopic techniques, as well as X-ray crystallography and elemental analysis. The complex [(5,6DMP)AuCl(2)][BF(4)] (2) was found to possess a distorted square planar geometry about the gold(III) center, commonplace for d(8) Au(III) cations possessing sterically un-hindered polypyridyl ligands. Compound 2 was evaluated for its potential use as an anticancer therapeutic. It was determined that the complex is stable in phosphate buffer over a 24-hour period, thought it does undergo rapid reduction in the presence of equimolar amounts of reduced glutathione (GSH) and ascorbic acid. The DNA binding and in vitro tumor cytotoxicity of the title compound 2 were also determined. It was found to undergo weak and reversible binding to calf thymus DNA, and was more cytotoxic towards a panel of human cancer cell lines than the commonly used chemotherapy agent cisplatin. Cytotoxicity experiments with the free 5,6DMP ligand indicate that the ligand has IC(50) values that are slightly lower than those observed for the gold complex (2), and coupled with the fact that the ligand appears to be released from the gold(III) metal center in reducing environments, this suggests the ligand itself may play an important role in the antitumor activity of the parent gold complex.

Biosynthetic incorporation of fluorohistidine into proteins in E. coli: a new probe of macromolecular structure.

Histidine is important for carrying out a number of protein functions. For instance, it can act as a general acid/base in enzymatic catalysis, it plays a role in ligating metal cations in metalloproteins, and stabilizes protein structure by metal binding, hydrogen bonding, or electrostatic interactions. One noteworthy example of histidine’s potential role in protein stability is observed in the pathogenesis of the anthrax toxin, where protonation of His side chains in one of the proteins leads to a large structural perturbation and subsequent toxicity. Given the unique role of this amino acid in such processes, the development of methods that probe the structural and mechanistic features of His in proteins would be extremely valuable. The biosynthetic incorporation of unnatural amino acids into proteins provides the experimentalist with a variety of methods that can probe protein structure and function. In particular, fluorinated amino acids can be used to achieve a relatively isosteric change (by replacing a single hydrogen with fluorine) that results in quite different electronic properties. Additionally, F NMR can be used to monitor changes in protein conformation in response to changes in the environment that are sometimes not detectable by other techniques. Although incorporating fluorine-labeled amino acids is not a new idea, access to an expanding tool box of fluorinated protein building blocks has promoted a renaissance in this area of research. Over 30 years ago, a photochemical Schiemann reaction was developed for synthesizing 2-fluorohistidine (2-FHis) and 4-fluorohistidine (4-FHis). To our knowledge this still represents the only fluorination procedure available for accessing these imidazole derivatives. The pKa of the side chain of both 2-FHis and 4-FHis has been measured previously, and is decreased from approximately 6.0–6.5 to 1 and 3, respectively. Because of this, these analogues provide a means for verifying the role of native His in pH-dependent processes. To this end, 4-FHis has been incorporated into the S peptide of ribonuclease, and into full-length ribonuclease A with chemical synthetic methods. Early experiments demonstrated that tritium-labeled 2-FHis could be incorporated into bacterial protein, and these analogues (2-FHis more so than 4-FHis) had an inhibitory effect on E. coli growth. However, in order to achieve high levels of incorporation for structural studies, it is typical to employ the use of bacterial auxotrophs. While the use of auxotrophs for the biosynthetic incorporation of novel His analogues into E. coli has been reported, a similar protocol for the incorporation of fluorohistidine derivatives has yet to be described. Herein, we provide unequivocal evidence for the incorporation of both 4-FHis and 2-FHis into a mutant form of the chaperone PapD by using an E. coli strain that is auxotrophic for His. PapD is the prototype for a wide variety of highly homologous chaperones that utilize the chaperone-usher pathway for the assembly of P-pili, and has been previously labeled with fluorophenylalanine and fluorotryptophan in protein-folding studies. The wild-type (WT) protein does not contain any His residues. Thus, site-specific labeling can be accomplished by the introduction of a single His residue by site-directed mutagenesis, and biosynthetic labeling can be performed according to previously described protocols. In this work, we used site-directed mutagenesis to introduce a single His residue at Arg200 in PapD. Among the chaperones that are homologous to PapD, the most similar is PmFD from Proteus mirabilis (47% identity), which possesses a His residue at position 200. Therefore, an R200H substitution was not expected to alter the structure and stability of PapD. To confirm this, urea denaturation studies were performed on PapD (R200H) and it was found to have a similar stability as that previously reported for WT PapD; PapD(WT): DG8=8.95 kcal [a] Dr. J. F. Eichler, Prof. Dr. J. G. Bann Department of Chemistry, Wichita State University Wichita, KS 67226 (USA) Fax: (+1)316-978-7373 E-mail : jim.bann@wichita.edu [b] Dr. J. C. Cramer, Dr. K. L. Kirk Laboratory of Bioorganic Chemistry National Institute of Diabetes & Digestive & Kidney Diseases National Institutes of Health Bethesda, MD 20892 (USA) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

Flipped classroom modules for large enrollment general chemistry courses: a low barrier approach to increase active learning and improve student grades

In the face of mounting evidence revealing active learning approaches result in improved student learning outcomes compared to traditional passive lecturing, there is a growing need to change the way instructors teach large introductory science courses. However, a large proportion of STEM faculty continues to use traditional instructor-centered lectures in their classrooms. In an effort to create a low barrier approach for the implementation of active learning pedagogies in introductory science courses, flipped classroom modules for large enrollment general chemistry course sequence have been created. Herein is described how student response systems (clickers) and problem-based case studies have been used to increase student engagement, and how flipped classroom modules have integrated these case studies as collaborative group problem solving activities in 250–500 seat lecture halls. Preliminary evaluation efforts found the flipped classroom modules provided convenient access to learning materials that increased the use of active learning in lecture and resulted in a significant improvement in the course grade point average (GPA) compared to a non-flipped class. These results suggest this approach to implementing a flipped classroom can act as a model for integrating active learning into large enrollment introductory chemistry courses that yields successful outcomes.

Monitoring anthrax toxin receptor dissociation from the protective antigen by NMR

The binding of the Bacillus anthracis protective antigen (PA) to the host cell receptor is the first step toward the formation of the anthrax toxin, a tripartite set of proteins that include the enzymatic moieties edema factor (EF), and lethal factor (LF). PA is cleaved by a furin‐like protease on the cell surface followed by the formation of a donut‐shaped heptameric prepore. The prepore undergoes a major structural transition at acidic pH that results in the formation of a membrane spanning pore, an event which is dictated by interactions with the receptor and necessary for entry of EF and LF into the cell. We provide direct evidence using 1‐dimensional 13C‐edited 1H NMR that low pH induces dissociation of the Von‐Willebrand factor A domain of the receptor capillary morphogenesis protein 2 (CMG2) from the prepore, but not the monomeric full length PA. Receptor dissociation is also observed using a carbon‐13 labeled, 2‐fluorohistidine labeled CMG2, consistent with studies showing that protonation of His‐121 in CMG2 is not a mechanism for receptor release. Dissociation is likely caused by the structural transition upon formation of a pore from the prepore state rather than protonation of residues at the receptor PA or prepore interface.

The design and synthesis of heterometallic alkoxide-amides and their application in the MOCVD of zirconium-tin-titanate (ZTT)

The novel heterometallic alkoxide-amide species, [Ti{OPri}3{N(SnMe3)2}] (1), [Ti{OBut}3{N(SnMe3)2}] (2), and [Ti{OPri}3{N(SnMe3)(SiMe3)}] (3) have been synthesized and fully characterized. Compounds 1–3 were used in combination with tetrakis(tert-butoxide)zirconium(IV) to obtain zirconium-tin-titanate (ZTT) films via metal organic chemical vapor deposition (MOCVD). A low-pressure vapor draw (pressure ∼ 5 Torr) was used to deliver the precursor to an oxygen-rich, hot-walled tube furnace (temperature = 430 °C) where metal oxide thin films were deposited on silicon wafers approximately 1.5 cm × 1.5 cm in size. The surface morphology of the as-deposited films has been characterized by SEM, and preliminary XPS measurements indicate that the metals of interest are transported to the substrate surface in the deposition process. The ZTT film grown from a separate vapor draw of 2 and Zr(OBut)4 was determined to have an approximate composition of Zr0.97Sn0.12Ti0.05O3.33.

A GENERAL ROUTE TO TIN-NITROGEN HETEROCUBANES

The reaction of the lithiated bis-stannylamine, ((CH3)3)Sn2NLi·THF (1), with (C5H5)2ZrCl2 yields the imido-bridged zirconium dimer, [(C5H5)2ZrN(Sn(CH3)3)]2 (3), which was characterized by single crystal x-ray diffractometry. The heteroleptic stannylamine, [{(CH3)3Sn}{(CH3)3Si}]NLi·Et2O(2), prepared using(1)as a synthetic precursor, reacts with SnCl2 to yield the tin-nitrogen heterocubane, [Sn(μ3-NSi(CH3)3)]4 (4), which is fully characterized by single crystal x-ray diffractometry and standard spectroscopic techniques. The use of this class of stannylamine ligands allows access to heterometallic amido complexes and provides a general route to obtaining tin-nitrogen heterocubanes.

Antitumor Activity of a Polypyridyl Chelating Ligand: In Vitro and In Vivo Inhibition of Glioma:

Glioblastoma multiforme is an extremely aggressive and invasive form of central nervous system tumor commonly treated with the chemotherapeutic drug Temozolomide. Unfortunately, even with treatment, the median survival time is less than 12 months. 2,9-Di-sec-butyl-1,10-phenanthroline (SBP), a phenanthroline-based ligand originally developed to deliver gold-based anticancer drugs, has recently been shown to have significant antitumor activity in its own right. SBP is hypothesized to initiate tumor cell death via interaction with non-DNA targets, and considering most glioblastoma drugs kill tumors through DNA damage processes, SBP was tested as a potential novel drug candidate against glial-based tumors. In vitro studies demonstrated that SBP significantly inhibited the growth of rodent GL-26 and C6 glioma cells, as well as human U-87, and SW1088 glioblastomas/astrocytomas. Furthermore, using a syngeneic glioma model in mice, in vivo administration of SBP significantly reduced tumor volume and increased survival time. There was no significant toxicity toward nontumorigenic primary murine and human astrocytes in vitro, and limited toxicity was observed in ex vivo tissues obtained from noncancerous mice. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining and recovery assays suggest that SBP induces apoptosis in gliomas. This exploratory study suggests SBP is effective in slowing the growth of tumorigenic cells in the brain while exhibiting limited toxicity to normal cells and tissues and should therefore be further investigated for its potential in glioblastoma treatment.

Synthesis, characterization, and stability of iron (III) complex ions possessing phenanthroline-based ligands

It has previously been demonstrated that phenanthroline-based ligands used to make gold metallotherapuetics have the ability to exhibit cytotoxicity when not coordinated to the metal center. In an effort to help assess the mechanism by which these ligands may cause tumor cell death, iron binding and removal experiments have been considered. The close linkage between cell proliferation and intracellular iron concentrations suggest that iron deprivation strategies may be a mechanism involved in inhibiting tumor cell growth. With the creation of iron (III) phen complexes, the iron binding abilities of three polypyridal ligands [1,10-phenanthroline (phen), 2,9-dimethyl-1, 10-phenanthroline (methylphen), and 2,9-di-sec-butyl-1, 10-phenanthroline ( sec-butylphen)] can be tested via a competition reaction with a known iron chelator. Therefore, iron (III) complexes possessing all three ligands were synthesized. Initial mass spectrometric and infrared absorption data indicate that iron (III) tetrachloride complex ions with protonated phen ligands (RphenH+) were formed: [phenH][FeCl4], [methylphenH][FeCl4], [ sec-butylphenH][FeCl4]. UV-Vis spectroscopy was used to monitor the stability of the complex ions, and it was found that the sec-butylpheniron complex was more stable than the phen and methylphen analogues. This was based on the observation that free ligand was observed immediately upon the addition of EDTA to the [phenH][FeCl4] and [methylphenH] [FeCl4] complex ions.

Antitumor Activity of 2,9-Di-Sec-Butyl-1,10-Phenanthroline.

The anti-tumor effect of a chelating phen-based ligand 2,9-di-sec-butyl-1,10-phenanthroline (dsBPT) and its combination with cisplatin were examined in both lung and head and neck cancer cell lines and xenograft animal models in this study. The effects of this agent on cell cycle and apoptosis were investigated. Protein markers relevant to these mechanisms were also assessed. We found that the inhibitory effect of dsBPT on lung and head and neck cancer cell growth (IC50 ranged between 0.1–0.2 μM) was 10 times greater than that on normal epithelial cells. dsBPT alone induced autophagy, G1 cell cycle arrest, and apoptosis. Our in vivo studies indicated that dsBPT inhibited tumor growth in a dose-dependent manner in a head and neck cancer xenograft mouse model. The combination of dsBPT with cisplatin synergistically inhibited cancer cell growth with a combination index of 0.3. Moreover, the combination significantly reduced tumor volume as compared with the untreated control (p = 0.0017) in a head and neck cancer xenograft model. No organ related toxicities were observed in treated animals. Our data suggest that dsBPT is a novel and potent antitumor drug that warrants further preclinical and clinical development either as a single agent or in combination with known chemotherapy drugs such as cisplatin.