Synthesis and Characterization of Mannose-Functionalized Polymeric Micelles

Lauren Abougi

 Self-assembling glycopolymers have garnered increased attention from the scientific community as an effective solution to the complications presented by current therapeutics. Using glycopolymers to encapsulate highly toxic drugs, targeted delivery can be achieved by exploiting the enhanced permeation and retention (EPR) effect and by functionalizing polymers with sugar moieties specific to receptors present on tumorassociated cell lines. With this combination of passive and active targeting in mind, amphiphilic diblock copolymer poly(2-(α-D-mannosyloxy)ethyl methacrylate)-copoly(tert-butyl acrylate), or pMannHEMA-co-ptBA, was synthesized in seven different molecular weight and diblock compositions using reversible addition fragmentation chain transfer (RAFT) polymerization.

These glycopolymers exhibited low critical micelle concentrations, ranging from 0.08 mg/mL to 0.93 mg/mL in ddH2O. By dynamic light scattering and transmission electron microscopy, the micelles were shown to be spherical in shape and between 70 and 100 nm in diameter. The binding behavior of the copolymer with concanavalin A (ConA) was also examined by measuring the turbidity change rate. Each of the copolymers displayed increased absorbance in the presence of ConA, indicating receptor recognition. Consequently, this work provides a general synthetic route to selfassembling glycopolymers. Due to this self-assembling nature and receptor recognition abilities, glycopolymers such as these are distinguished as potentially clinically relevant molecules in the future of drug delivery.

 Manipulation of the Electronic and Optical Properties of Phenylenevinylene-Based Oligomers Through the Incorporation of Polycyclic Aromatic Hydrocarbons

Christopher Corbett

 The Earth’s increasing energy demands combined with the diminishing reserves of fossil fuels motivates the exploration of renewable and cleaner energy sources. Solar energy has the potential to meet the Earth’s growing energy needs, but current solid state devices are costly, require time to produce, and have less than ideal efficiencies. Organic electronic devices, and specifically organic solar cells are of great interest in designing the next generation electronic devices to replace current solid state devices. Organic solar cells have the potential to be lighter, cheaper, easier to produce, and more efficient than current silicon-based devices. Work in the Park Lab, which previously focused on designing these devices, has focused on the active materials that are necessary for charge excitation and transport, and are ultimately responsible for the production of current. Our goal is to alter the structure and properties of the electron donor component of the active layer through the incorporation of polycyclic aromatic molecules into the parent oligomer. Several variations of the parent trimer were produced that incorporated these highly conjugated arenes. The increase in conjugation due to the addition of these compounds has significant effects on the absorption and emission properties of our oligomers.

Construction of Biological and Chemical Systems for Novel Antibiotic Drug Discovery

Bryn Falahee

 Antibiotics were arguably one of the most important discoveries of the 20th century. With the invention of antibiotics, the average lifespan of the human population substantially increased. However, many microbes have developed resistance to antibiotics currently used in the clinic. If new antibiotics are not developed, diseases and infections that were once considered easy to treat may become incurable. To develop novel antibiotics, we will target histidine kinase CckA. This kinase mediates an essential signaling pathway in Caulobacter crescentus. Additionally, histidine kinases are highly conserved in the bacterial genome, making CckA a good target.

Using disulfide exchange screening, we synthesized small drug fragments to use in fragment based drug discovery. Here, we provide a successful synthetic approach for building disulfide drug fragments. We also present the optimization of the protein system that will be used to test the drug fragments. An unnatural cysteine residue was designed and incorporated into CckA so that the protein can be used for disulfide exchange screening. Two cysteine mutants were expressed and purified, T498C and V488C. We also compared kinetics of the mutated CckA constructs to the wild type protein to verify that the mutations did not dramatically alter the active site of the protein. Preliminary inhibition data of AMP-PNP was obtained for the V488C mutant. Additionally, the T498 and wild type proteins were tested on LCMS to verify that the proteins could be visualized for tethering analysis. Moving forward, protein expression and purification should be optimized and kinetics data should be verified. The work accomplished thus far prepares the lab to move forward with initial disulfide exchange screening.

 Role of Proteases and a Protease Inhibitor in Streptomyces coelicolor Development

Emily Gao

Streptomyces coelicolor is the model organism for the Streptomyces genus of gram-positive, soil dwelling bacteria with a complex multicellular life cycle intimately coordinated with its production of secondary metabolites such as antibiotics. The NY415 bald mutant is a developmental mutant of the species incapable of forming an aerial mycelium and thus provides a prime opportunity to study the molecular pathways and interactions that give rise to the organism’s complex life cycle. The NY415 bald mutant has a higher extracellular protease activity and produces much less Streptomyces trypsin-like inhibitor (STI) than the wild type strain. Previously, it was found that exogenous addition of wild type culture supernatant to NY415 bald colonies was capable of recovering normal development. This suggests that some species secreted by the wild type is crucial and missing in NY415 culture supernantant and provides further incentive to monitor the protein differences between the mutant and wild type. Overexpression of SCO0732 was found to delay and possibly reduce sporulation, suggesting that lower levels of SCO0732 expression are needed for normal sporulation. Mutation of SCO0762, the gene for Streptomyces trypsin-like inhibitor (STI), also led to delays in and disruption of the sporulation process. Analysis of the protease activity of the SCO0762 mutant and the SCO0732 overexpression strain suggested that both strains have increased protease activity in comparison to the wild type. An assay was developed to quantify protease inhibitor activity, and tested using the protease inhibitor leupeptin. Finally, it was found that exogenous addition of concentrated wild type culture supernatant to NY415 cells could not consistently cause a recovery of normal development in this bald mutant, suggesting that the species responsible for this recovery is only effective at the proper concentration.

 Manipulation of the Electronic and Optical Properties of Polycyclic Aromatic Hydrocarbons Through the

Incorporation of Electron-Withdrawing Functionality

Alejandro Gimenez

As we envision newer, more modern, and greener devices to address our current energy concerns, there are limitations with relying on current solid state materials, as they are costly, heavy, and exhibit less than ideal efficiencies. Organic electronic devices, which have the potential to be cheaper, lighter, and more efficient, may be better suited for the applications of the future. These devices are fundamentally based on polycyclic aromatic hydrocarbons, which exhibit interesting optical and electronic properties that can be fine-tuned. In the Park Lab, we have explored various conjugated systems, which could serve as potential electron donor or acceptor materials in the active layer of solar cells. In particular, we introduced a set of electron-withdrawing substituents to various arenes in the hope of tuning their electronic structure. We probed the effects of various substituents on the absorption and emission properties of anthracene, pyrene, and perylene, and found that the electronic structure of these various arenes can be tuned systematically.

 Copper(I) and Copper(II) Complexes with Tetradentate Pyridine-amine Ligands as Atom Transfer Radical Polymerization Catalysts

Sarah Guillot

Atom transfer radical polymerization (ATRP) is a versatile, metal-mediated method of producing polymers with controlled composition and molecular weights. The redox couple of the metal tunes control by modulating the equilibrium between the growing, active radical state and the capped, dormant state of polymer chains. Ligand structure has been shown previously to strongly impact the catalysts’ ability to control the polymerization. Polydentate ligands of amine and pyridine donor moieties have produced some of the most active ATRP catalysts. In this work, copper bromide complexes of neutral tetradentate pyridine-amine ligands with additional amine, ether or thioether donor atoms were synthesized and utilized in the ATRP of styrene. We explored how differences in ligand design affected the efficiency of the ATRP of styrene. All complexes were active ATRP catalysts with fast polymerization rates and good molecular weight control. Redox potentials ranged from -0.1 to -0.3 V (vs. SCE). As expected, the variation in donor atoms impacted the degree of control and rates of polymerizations. Diverse structural motifs in the solid state and NMR evidence of ligand fluxionality in solution relate to differences in polymerization rates and control, and affirm ligand design as an effective tool to tune the rate and degree of control in ATRP.

 Characterization of a Novel Carrier Protein Phosphodiesterase, SCO6672, in Streptomyces coelicolor A3(2)

Sora Kim

 The Streptomyces are a genus of filamentous, Gram-positive soil bacteria that are known to produce a wide array of secondary metabolites, many of which have therapeutic significance as antibiotics. Many of these antibiotic molecules are nonribosomal peptide or polyketide compounds. Essential to biosynthesis of these antibiotic molecules is the post-translational attachment of the 4’-phosphopanthetheine (Ppant) group onto the carrier protein(s) catalyzed by phosphopantetheinyl transferases (PPTases) to generate holo-carrier protein(s). The SCO6673 PPTase is one of three PPTases encoded in the S. coelicolor A3(2) genome, the model organism for the genus, and this enzyme has been shown to be required for calcium-dependent antibiotic (CDA) biosynthesis.

The SCO6672 gene is adjacent to and upstream of SCO6673 and encodes a protein with a putative metallophosphoesterase domain that shares homology to calcineurin-like phosphoesterases. SCO6672 was previously shown to catalyze hydrolysis of the Ppant group from a PCP domain of the CDA synthetase in vitro, and this is hypothesized to serve as a post-translational mechanism of control of antibiotic biosynthesis by regulating the availability of holo-carrier proteins. These preliminary observations of SCO6672 phosphodiesterase activity were confirmed with a general phosphodiesterase substrate, bis-pNPP, and two more PCP domains of the CDA synthetase as assayed through MALDI-TOF mass spectrometry and conformationally-sensitive polyacrylamide gel electrophoresis.

The physiological function of SCO6672 in vivo was investigated by studying the effect of complementation on antibiotic production and morphological differentiation of a DSCO6672-6673::apr mutant. We found that undecylprodigiosin (RED) production in complemented strains was restored to levels comparable to the wild-type strain, and that overexpression of SCO6672 led to underproduction of RED; effects of complementation and overexpression on CDA production or morphological differentiation were inconclusive. These results suggest that the physiological role of SCO6672 in S. coelicolor A3(2) is complex, and that it may influence multiple aspects of antibiotic biosynthesis.

 Amino Acid Based Thermoresponsive Micelles

Alexander Lou

 Micelles assembled from amphiphilic copolymers have garnered interest for their potential application to targeted drug delivery. We have synthesized several polymers of the form poly(N-acryloyl-(amino acid)-N-isopropylamine)-block-poly(acrylic acid) using methionine, alanine, valine, isoleucine, and phenylalanine. Benzyl containing monomers, whether the benzyl moiety was an end group or in the side chain, were the least polymerizable. Their self-assembly and thermoresponsive behaviors were probed by dynamic light scattering (DLS) and circular dichroism (CD). The copolymers exhibited stable CD spectra, independent of assembly state or temperature. DLS revealed methionine-based micelles of 15 and 33 nm in radius, and phenylalanine-based micelles having a critical micelle temperature (CMT) of 45 – 50˚C. Self-assembly and potential for tunable thermoresponsive behavior makes these polymers promising as drug delivery systems.

 Targeting LexA Cleavage to Prevent the Development of Antibiotic Resistance: In vivo Characterization of LexA Cleavage Inhibitors

Lovemore Makusha

 The phenomenon of antibiotic resistance has resulted in the need for new strategies to combat bacterial infections. Bacteria quickly develop resistance, rendering current antibiotics ineffective soon after discovery. In this context, the SOS system, an inducible DNA repair mechanism whose activation has been implicated in the development of drug resistance, has emerged as an intriguing drug target. Herein, we report of the systematic discovery of compounds that inhibit LexA cleavage, a key step in the activation of the mutagenic SOS response. Through a high-throughput assay previously developed in our lab, together with in vitro protein assays, we discovered ten compounds that inhibit Escherichia coli LexA cleavage. We developed an in vivo reporter assay using a Bacillus subtilis strain containing the lacZ gene under the transcriptional control of an SOS promoter and showed that all five of the compounds we tested mitigated the induction of the SOS response, presumably through inhibition of LexA cleavage. Moreover, the concentration dependence of in vivo inhibition correlated well with in vitro results in the distantly related E. coli species. Together, our results indicate the feasibility of targeting LexA cleavage to abrogate the production of resistance-conferring mutations in the bacterial genome.

 Deterministic and Stochastic Analysis of a 2-gene Minimal Oscillator

Steve Mendoza

We analyze a 2 gene minimal oscillator with differential and stochastic techniques. We find conditions that maximize the amount of oscillations. In particular, we find that oscillations in the stochastic regime are optimized by larger particle numbers and higher cooperativity constants. We also try to generalize our results to a circadian rhythms model in Drosophila. Doing this, we find that our results are qualitatively similar to those found in the paper. We argue that our model could serve as a template for other oscillatory genetic circuits in nature.

 Advancements Toward the Total Synthesis of Jerangolid D

Mika Nakashige

 Efforts towards the total asymmetric synthesis of jerangolid D are elucidated in this document. Jerangolid D is a polyketide natural product isolated from the Sorangium cellulosum bacterium and is known to exhibit antifungal properties. Jerangolid D contains two rings: a cis–dihydropyran and an α,β-unsaturated δ-lactone. These two rings are joined by a doubly allylic remote stereogenic center. Ambruticin, another antifungal secondary metabolite, shares several structural components. The synthesis of the cis–dihydropyran highlights the reductive carbon-Ferrier rearrangement reaction that was optimized during the course of this research. From here, we strategically intercepted Jacobsen’s synthesis of ambruticin and take advantage an asymmetric hydroformylation to set the remote stereogenic center. The remaining steps were adapted from methods for kavalactone syntheses developed in this lab to create the α,β-unsaturated δ–lactone and ultimately yield jerangolid D.

 Identification and Characterization of Bacterial SOS Response Inhibitors

Asvelt J. Nduwumwami

 The development of bacterial resistance to current frontline antibiotics is becoming increasingly problematic in the treatment of bacterial infections. Recent evidence suggests that the bacterial SOS response plays an active role in tolerance to current therapeutic agents and is involved in pathways that lead to full-fledged antibiotic resistance. The SOS response is widespread among bacteria and its regulation is conserved. This SOS system is therefore an attractive target for development of antimicrobials. To validate the SOS response as a novel target in combating antibiotic resistance, we have screened 3520 small bioactive molecules for their ability to inhibit LexA cleavage, the prerequisite for the induction of the SOS response. We identified a total of eight compounds that inhibited LexA cleavage in addition to two previously found in the Lovett Laboratory. Seven of those compounds inhibited only RecA-mediated LexA cleavage in Escherichia coli and three of them inhibited both RecA–mediated cleavage and RecA-independent LexA autodigestion at pH 10.

Synthesis and Characterization of Substituted Acenes For Use as Electron Donors and Acceptors in Organic Solar Cells

Erica Wu

 Electronic devices fashioned out of organic compounds may offer numerous benefits compared to their solid-state, silicon-based counterparts; they have the potential to be cheaper, lighter, more flexible, and more efficient. In particular, organic solar cells are increasingly being researched in response to growing global energy consumption. The Park Lab is interested in exploring various conjugated systems for use in the active layer of organic solar cells, and this year, we worked on developing a synthetic library of various substituted acenes, linearly fused polycyclic aromatic hydrocarbons. Pentacene-based reaction chemistry was explored, as pentacene’s small bandgap and high charge mobility confers interesting optical and electronic properties for use in organic electronic devices. Pentacene-based compounds were synthesized and characterized in this project, and the addition of electron-withdrawing substituents was found to significantly impact the absorption and emission properties of pentacene, as well as lead to increased rates of photooxidation.

 Synthesis of Disulfide Fragments for Site-directed Ligand Discovery with CckA

Peter Young

 One promising method for discovering new, effective drugs is to find new cellular machinery to target. Histidine kinases (HKs), which mediate two-component signaling pathways (TCSs) in bacteria, may represent a viable new target for pharmacological inhibition. CckA, an essential HK in Caulobacter crescentus, is an excellent platform of discovery for TCS inhibitors. With site-directed ligand discovery (or “tethering”)—a fragment-based technique—small, weakly binding disulfide drug compounds can be covalently trapped in the enzyme active site by cysteine-capture, where they can be detected by whole-protein mass spectrometry. This technique allows the energy landscape of the active site to be probed and characterized so that a powerful CckA inhibitor can be constructed from scratch.

With this in mind, a CckA mutant with a cysteine within 5 Å of the ATP binding site, His6-CckA190-562-V488C, was engineered as a covalent handle for site-directed ligand discovery with CckA and identified by MALDI time of flight whole-protein mass spectrometry. It was found that the two-step, one-pot synthesis of disulfide fragments for screening with cys-CckA is best conducted with (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate instead of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) due to an unidentified impurity. A disulfide fragment library of nine compounds was synthesized and identified by 1H-NMR and LC/MS. Using cysteine-capture, these nine fragments can be screened for even weak binding activity in the CckA active site. Eventually, this technique could lead to the discovery of a novel, effective drug that functions by inhibition of histidine kinase mediated TCSs.

Towards the Total Synthesis of Enigmazole A: Model Studies Toward a C1-C12 Fragment

Menghan Zhao

 Enigmazole is a phosphorylated 18-membered macrolide natural product isolated from the sponge Cinachyrella enigmatica. The molecule exhibits potent cytotoxic activity, although its mechanism of action is unclear. Enigmazole A also possesses unique architectural elements, with eight stereogenic centers and a 2,4-disubstituted oxazole moiety, and has thus become an alluring synthetic target. The original synthetic plan for the molecule envisioned the use of Evans β-ketoimide aldol chemistry to rapidly and efficiently construct the C1–C4 dipropionate unit, while also selectively installing three of the eight stereogenic centers. However, the method furnishes a superfluous hydroxyl at the C3 position, and removal of this hydroxyl group has proven to be a formidable challenge. Herein we report on two distinct synthetic strategies: 1) continued efforts toward removal of the C3 hydroxyl through radical deoxygenation and 2) efforts toward construction of the C1–C12 fragment without the presence of a C3 hydroxyl, either through a rapid lactone synthesis developed in our lab or a Myers diastereoselective alkylation.