Chemistry Honors Papers

Facile access to cyclooctanoid systems via microwave-‐assisted sequential 6‐exo‐dig cyclization/Claisen rearrangement

Aaron Feldman — Wed, 22 May 2013 12:41:24 PDT

The practical preparation of eight-membered rings remains a challenging synthetic pursuit. Herein, we have successfully demonstrated the synthesis ofcyclooctanoid systems via a microwave-assisted tandem 6-exo-dig cyclization/Claisen rearrangement on appropriately substituted 5-hexyn-1-ol precursors. The initial base-catalyzed cyclization is facilitated by activation of the triple-bond with an electron-withdrawing substituent. Subsequent Claisen rearrangement of the resulting allyl vinyl ether gives the desired eight-membered ring.

[diagram omitted]

This thesis describes initial optimization and investigates the scope of this novel methodology through the synthesis of a variety of cyclooctanone analogs. Further investigations will include expansion of reaction scope, as well as application of this methodology to natural product synthesis.

Studies Toward the Total Synthesis of Frondosin D

Taryn Campbell — Fri, 18 May 2012 09:31:36 PDT

Nanostructured Manganese Oxide Catalysts

David Kriz — Tue, 31 May 2011 12:09:46 PDT

Study Towards the Synthesis of Guanacastepene A

Sarah Spiegel — Tue, 31 May 2011 12:09:44 PDT

Studies of Firefly Luciferase: Development of a Ratiometric BRET-FRET-based Factor Xa Protease Assay and Attempts to Chemically Trap Luciferase in the Second Partial Reaction Conformation

Kelsey Taylor — Tue, 31 May 2011 12:09:43 PDT

Improved Red-emitting Firefly Luciferase Mutant for Biotechnical Applications

Audrey L. Davis — Fri, 05 Jun 2009 11:34:16 PDT

Luciferase from the North American firefly Photinus Pyralis catalyzes a highly efficient bioluminescent reaction that produces yellow-green light (557 nm at pH 7.8). This bioluminescence system has been adapted for a variety of applications including gene reporter assays, detection of bacteria and toxins, whole-cell biosensor measurements, and in vivo imaging. A luciferase variant with a red-shifted bioluminescence spectra and high specific activity, two qualities that are challenging to achieve simultaneously, would be especially useful when paired with a green-emitting enzyme in a dual-color reporter assay, or used alone for improved imaging in living animals.

Our lab previously reported a red-emitting mutant named Ppy RE, which contained a single mutation at S284T that shifted the emission maximum from the 557 nm to 615 nm at pH 7.8. In a subsequent publication, Ppy RE was enhanced with five mutations to make Ppy RE-TS, which exhibited improved thermostability at 37°C, but at the expense of reducing the red-shift to 610 nm. In this report, we further improve upon Ppy RE-TS with four rational point mutations that red-shift the emission maximum to 617 nm, dramatically increase the enzyme thermostability, and improve the total light emission during 8-second assays. This new enzyme, named PRET7, is characterized and evaluated for performance in E. Coli and HEK293 mammalian cell lines. Compared to a commercially available enzyme called CBR, which has been codon-optimized for expression in mammalian cells, PRET7 produces a stronger signal over 8-second assays in which the least expensive reagents available are used. Aiming to improve the already impressive performance of PRET7, we are currently awaiting results from collaborators who are evaluating a human codon-optimized version of PRET7 in HEK293 cells.

Green Flourescent Protein: A Computational Study of Hula-Twisting, GFPuv Thermostability and the Role of Arginine96 in Chromophore Formation

Nana Yaa A. Baffour-Awuah — Mon, 04 May 2009 13:03:05 PDT

Green fluorescent protein (GFP) has revolutionized in vivo cellular and molecular labeling. Three features of GFP that could potentially affect the engineering of better GFPs have been investigated by computational methods.

1. Loss of fluorescence has been documented in GFP and this probably occurs through fast internal conversion. The volume conserving hula twisting HT which was thought to be the mechanism of fast internal conversion does not explain radiationless decay since the φ-one bond flip OBF requires the same volume as the concerted HT.

2. GFPuv is a thermostable mutant that is 45 times more fluorescent at ambient temperatures than the wild-type GFP. The reduced distance between the amide N of Gly67 and the carbonyl C of Ser65, as well as the reduced distance between Arg96 and the carbonyl O of Tyr66 are structural features possibly contributing to the improved thermostability of GFPuv.

3. Arg96 is conserved in almost all GFP mutants and is suspected to play a critical role in one or more of the chromophore forming steps. Our data shows Arg96 is

closer to the carbonyl O of Ser65 than to the corresponding carbonyl of Tyr66.

The De-activation of Ammonium Sulfate Aerosol Particles as a function of Organic Acid Deposition onto the Particle Surface: Implications for Understanding Cloud Condensation Nuclei (CCN) Activity of Processed Ambient Aerosol Particles

Erica F. Gagne — Mon, 10 Sep 2007 10:09:37 PDT

The cloud condensation nuclei activity of organic coated ammonium sulfate particles was studied using a CCN counter in conjunction with the Aerodyne Aerosol Mass Spectrometer. The organic coatings studied were adipic acid, decanedioic acid (DEHS) and stearic acid. It was found, that the CCN activity of ammonium sulfate particles decreased as a function of insoluble (stearic, DEHS) and slightly soluble (adipic) organic coating on the surface of the aerosol particles. Due to the liquid state of DEHS, water molecules diffused through the coating and caused a slight increase in the critical supersaturation. Water was unable to diffuse through the thick and insoluble coating of stearic acid and caused the CCN activation of the ammonium sulfate core to be suppressed within the parameters of this experiment.

The Effect of the Protein Matrix on Fluorescence and an Analysis of the Effect of Fusing Residues 62 and 63 in Kindling Proteins

Scott L. Maddalo — Wed, 15 Aug 2007 11:12:35 PDT

In the ground state of the highly conjugated green fluorescent protein (GFP), the chromophore should be planar. However, numerous crystal structures of GFP and GFP-like proteins have been reported with slightly twisted chromophores. We have previously shown that the protein cavity surrounding the chromophore in wild-type GFP is not complementary with a planar chromophore. This study shows that the crystal structure of wild-type GFP is not an anomaly: most of the GFP and GFP-like proteins in the protein databank have a protein matrix that is not complementary with a planar chromophore. When the π-conjugation across the ethylenic bridge of the chromophore is removed the protein matrix will significantly twist the freely rotating chromophore from the relatively planar structures found in the crystal structures. The possible consequences of this non-planar deformation on the photophysics of GFP are discussed. In addition to GFP there are GFP-like proteins that can be reversibly photoswitched between a fluorescent and a nonfluorescent state. Conformational searching and molecular dynamics are used to examine the various kindling proteins with a cleaved chromophore. MD simulations supported that the chromophore does indeed start to move away from its initial trans configuration, and actually completes a full trans/cis isomerization. The nearby His197 residue does not provide a large barrier to the chromophore rotation when it is cleaved from the protein backbone. This implies that the rest of the protein backbone did not provide an obstacle to the isomerization. Fusion of the chromophore back to the protein backbone was also done and conformational analyses showed that the proteins with fused and cleaved chromophores did now show a great deal of change the φ and τ dihedral angles most cases.

Cloning, Sequencing, and Characterization of Luciola italica Luciferase

Jennifer P. DeAngelis — Tue, 15 Aug 2006 12:16:33 PDT

The characteristic yellow-green light of a firefly is the result of a multi-step reaction catalyzed by the luciferase enzyme. This enzyme has many applications in the biomedical field and ongoing work is being done to alter its properties to better fit these applications. The purpose of this project was to clone the Luciola italica luciferase cDNA and to express, purify and fully characterize the corresponding bioluminescence-catalyzing enzyme in hopes of obtaining novel bioluminescent materials. Fireflies were collected in the countryside of Bologna, Italy, flash frozen in liquid nitrogen and total RNA was extracted from the firefly lanterns. The L. italica luciferase cDNA was successfully cloned by RT-PCR using a gene-specific primer set based on the DNA sequence of the Eastern European Luciola mingrelica luciferase gene. The L. italica cDNA was determined to be 1647 base pairs in length with an open reading frame of 548 amino acids. Initial characterization of the enzyme showed that the L. italica protein exhibits bioluminescent activity similar in intensity to the common North American Photinus pyralis luciferase; however it produces light that is slightly red-shifted (having maximum emission at 564 nm). By steady state kinetics analysis, the L. italica Km for LH2 was found to be 0.095 mM, and that of P. pyralis is 0.015 mM. On the converse, both enzymes had similar Km values for Mg-ATP (0.160 mM for P. pyralis and 0.180 mM for L. italica). The L. italica enzyme was found to sustain its light in the visible region for a longer period of time than the P. pyralis enzyme. Phylogenetic analysis showed that the L. italica luciferase gene has 95.8% and 95.6% amino acid sequence identity to the Hotaria unmunsana (Korea) and Hotaria parvula (Japan) luciferase proteins, respectively. The processes that were used to clone the Luciola italica luciferase gene, characterize the protein, and optimize protein growth conditions are