Department of Chemistry

Investigations carried out in our laboratory encompass a wide range of interests in synthetic organic chemistry including heterocyclic and medicinal chemistry.

Current efforts are in the following areas: (1) synthesis and chemistry of five-membered heterocycles and natural products containing such units; (2) synthesis and chemistry of fungal metabolites; (3) synthesis and chemistry of cyclopeptide alkaloids; (4) synthesis of biologically important depsipeptides; (5) synthesis of novel ninhydrins; (6) synthesis of anti- angiogenic agents.

1. Utilization of D-ribonolactone and other sugars as precursors in the synthesis of several structurally challenging molecules is currently underway in our laboratory.
2. The synthesis of naturally occurring fungal metabolites containing a common hexasubstituted aromatic ring but different side chains such as colletochlorin D, ascofuranone and ascochlorin are another area of interest. The biological activities of those natural products range from high hypolipidemic action to anticancer and antiprotozoan activity.
3. Cyclopeptide alkaloids are natural products found in many plant families. A broad program aimed at developing methodology for the synthesis of the most commonly found thirteen- and fourteen-membered ring cyclopeptide alkaloids is currently underway. Sanjoin, used in Chinese folk medicine is one of our targets. Other antitumor cyclic peptides provenient from plants, the astins, are also under investigation.
4. Didemnins are a new class of depsipeptides isolated from a Carribean tunicate of the family Didemnidae, a species of the genus Trididemnum. These cyclic peptides have shown highly active antiviral and antitumor agents. The synthetic studies carried out in our laboratory have produced synthetic and spectral evidence for the absolute configuration of the asymmetric centers of the hydroxyisovalerylpropionyl (HIP) unit of the macrocycle, thereby requiring a revision of the original stereochemistry. The stereocontrolled total synthesis of these natural products has already been accomplished. The synthesis of several beta-turn mimics and constrained analogs are under investigation. Because of a broad program to develop efficient synthetic routes to the didemnins, other cyclodepsipeptides have been chosen as the next targets. The choice of these compounds was not only based on their relationship to didemnins but also on previous synthetic studies of products originating from polyketide biosynthesis, and earlier investigations of carbohydrates.
5. Novel ninhydrins are being synthesized as reagents for the detection of amino acids.
6. We have found that sulfated beta-cyclodextrin mimicked heparin advantageously. This effective synthetic product is of utmost importance in the control of angiogenesis and has other important applications in medicine. This recent discovery uncovers a new class of anti-angiogenic agents, consisting of a hydrophilic carrier and a hydrophobic angiostat, and offers a unique opportunity for the development of chemical technologies which will have important applications in the bio- and medical sciences. We are therefore continuing these studies with several goals in mind. We are investigating new and more effective carriers, we are designing single species that contain both the angiostat and carrier, and we are looking for new and more effective angiostats.
7. 

Identification, characterization and optimization of chemical probes of biological systems; Identification of novel agents to treat neurodegenerative diseases such as Alzheimer's Disease; Design of novel chemical libraries to probe biological systems.

The focus of our research is to study how proteins fold from random or quasi-random coils to their biologically functional conformations. We are particularly interested in the kinetic aspects of the folding mechanisms. Novel laser spectroscopic methods are being used and developed to study the early folding events and folding intermediates.

Fast events in protein folding

Understanding how folding proceeds at early time is apparently essential to the elucidation of the entire folding mechanism. To access and characterize the early folding events requires a fast initiation method and a probe that has structural specificity. Our general approach is to use novel laser-induced temperature-jump and fast-mixing techniques to initiate refolding/unfolding on nanosecond or microsecond timescales, and use time-resolved infrared and fluorescence spectroscopies to probe the subsequent folding dynamics and structural ordering along the folding/unfolding pathways. This approach provides not only fast time resolution, but also the necessary structural sensitivity, since both infrared and fluorescence are well-established conformation probes. Recent works involve the study of the helix-coil transition, helix-helix interaction, and ß-sheet formation. 

Single-molecule study of protein conformation dynamics

Recently, a new view of the kinetics of protein folding has emerged based on the new conceptual framework of statistical mechanical models, replacing the pathway concept with the broader notion of rugged energy landscapes. The heterogeneity in folding kinetics therefore can be realized as a result of the motions of an ensemble of protein conformations on the rugged energy hypersurface that is biased towards the native state, analogous to parallel diffusion-like processes. Studying folding dynamics statistically using single-molecule techniques will provide unique information regarding a protein's folding energy landscape, which may not be obtained by conventional ensemble studies since the conventional measurements of molecular dynamics in the condensed phase represent only averages over large numbers of molecules and events. Currently, confocal fluorescence spectroscopy and microscopy are being used to study protein spontaneous fluctuation and folding dynamics at single-molecule level. 

From a knowledge of the interactions among molecules, it is possible in principle to predict the structure and the thermodynamic properties of materials as well as the dynamics of molecular processes. The overall objective of our research program is twofold: to evaluate the potential energies of intermolecular interactions for various systems as accurately as possible and to study by means of statistical mechanics the influence of these potentials of intermolecular force on the structure and properties of macroscopic systems.

Our group is interested to study the effect of nano-confinement on structure, dynamics and other properties of materials. Materials behave differently on surfaces, interfaces or small length scales compared to their bulk properties.  Understanding such differences are crucial in many technological applications where materials are constrained in nanometer size spaces, such as organic electronics, polymer applications and drug delivery. One can take advantage of such difference to produce novel materials, such asexceptionally stable glasses or harvest light for various applications. In biological systems, most of the dynamics happens in nanometer size proximity of surfaces and interfaces, and understanding the properties in confinement is a key in predicting function. We focus our efforts on understanding the origins of such modified properties on a fundamental level as well as possible application of such phenomena in producing novel materials or experimental tools.

Enhanced Mobility at the Surface of Polymeric and Organic Glasses: 

We study the properties of glasses at the air/glass interface. Our studies show that below the glass transition temperature, where the bulk of the material is in an out of equilibrium state, the interfacial dynamics are many orders of magnitude faster that the bulk dynamics. As a result a layer close to the interface maintains equilibrium properties. We study the dynamics of this layer, its thickness, and its effect on the properties of the underlying glass. The interfacial layer can strongly modify properties of amorphous materials in nanometer length scales. They also allow one to produce near-equilibrium structures at temperatures well below bulk glass transition temperature, through physical vapor deposition.

Exceptionally Stable Glasses: 

The enhanced mobility of the interfacial layer allows us to produce near-equilibrium glasses at temperatures well below the bulk glass transition temperature, Tg, by means of physical vapor deposition (PVD). Exceptionally stable glasses are formed when the substrate temperature during PVD is maintained just below the glass transition temperature. We study the morphology and the kinetics of PVD films during formation and their relationship to the final properties of the stable glass. These studies provide information on mechanisms of rapid aging below Tg and stable glass formation. We also investigate exceptional material properties of these glasses and the role of the chemical structure in these properties such as the optical birefringence and electronic properties.

Novel Emergent Optical Properties in Disordered Nanoparticle Clusters: 

Using simple synthetic routs we can produce dielectric core-gold shell nanoparticles decorated with randomly packed nanoparticles of various shapes and sizes. Spiky nanoparticles are a good example of such nanoparticles. Broadband and tunable structure of spiky gold nanoshells makes them ideal for various applications such as enhanced Raman scattering, temperature and index sensing and sensors for biological and light harvesting applications. Exceptional properties, such as higher order quadrupoloar scattering and magnetic dipole plasmons in these nanoparticles are due to inherent disorder in their structure and random packing arrangements. We explore optical properties of these nanoshells, using various theoretical and experimental tools. We also develop new techniques that allow us to study properties of meta materials formed from these types of particles.

Surface Mediated Self-assembly of Amyloid Aggregrates: 

Surface self-assembly provides an alternative pathway for amyloid aggregation that is not available in bulk solutions. We us high-resolution atomic force microscopy and other imaging techniques to study the adhesion and diffusion of peptides on various surfaces and their role in facilitating amyloid fibril formation through self-assembly routs. We also use our exceptional capabilities in high-resolution imaging to study the conformation of amyloids formed under various conditions in aqueous conditions.

Our lab is developing chemical and biophysical tools to study and manipulate complex biological systems. Projects span many areas of synthetic organic, inorganic, and biophysical chemistry; molecular, cell, and developmental biology; and bioengineering. We are particularly interested in developing new technologies for biomolecular imaging and the fabrication of functional bio-nanomaterials.

Hyperpolarized 129Xe Biosensors for Early Cancer Detection

Molecular imaging technologies hold great promise for early cancer diagnosis and intervention. Our goal is to develop new reagents that extend the capabilities of magnetic resonance imaging (MRI) for monitoring multiple cancer markers simultaneously in vivo. 129Xe has found increasing use for biological imaging applications, due to its biological compatibility (xenon is an anesthetic at high concentrations), hyperpolarizability (this enhances signals 1,000-fold), and high affinity for organic cages such as cryptophanes. The chemical shift of 129Xe varies by a remarkable 200 ppm, depending on its molecular environment: Thus, a 129Xe atom encapsulated inside a cryptophane is a sensitive reporter of perturbations outside the cage. Based on this principle, our lab is generating new biosensors that will identify biomarkers associated with cancers of the breast, lungs, brain, and pancreas. The long-range goal of this project is to use MRI to detect aberrant proteins that cause cancer in humans, years before the formation of a tumor.

Ferritin Templates for Nanoparticle Synthesis and Assembly

The goal of this project is to use ferritin proteins as templates for synthesizing and assembling inorganic nanoparticles with nanometer precision. Ferritins contain 24 four-helix bundle subunits that self-assemble to create a large central cavity. We have made water-stable, 10-12-nm gold and silver nanoparticles inside ferritin (gray sphere). Particles are fully characterized using facilities at the UPenn Laboratory for Research on the Structure of Matter (LRSM). We are functionalizing the surface of these ferritin-metal nanoparticles for sensing and nano/biomaterials applications. We are also performing computational protein design, in collaboration with the Saven lab, to mutate residues inside the ferritin cavity to enhance their metal-binding properties. Methods for organizing ferritin metal nanoparticles in 2- and 3-dimensions are being developed, in order to build very small conducting circuits. 

Laser-Activated Chemical Biology: Controlling Genes with Light

The goal of this project is to develop methods for turning genes "on" and "off" with light inside neurons and developing zebrafish embryos with high spatial and temporal control. As a first step, we have developed methods for incorporating a photoactive blocking group in the middle of a DNA or RNA oligonucleotide. In one application, we modulated primer extension by DNA polymerase (KF) using UV light. Photoactivation was monitored using a fluorescent reporter. We are now developing methods to control protein translation by the ribosome using similarly caged RNA. Blocking groups mask the messenger RNA start codon, and are designed to prevent translation until photocleavage. We will control complex gradients of proteins involved in cell signaling during zebrafish development and wound healing, using a state-of-the-art UV confocal microscope in the lab.

For many years the Dailey group has been involved in the areas of reactive intermediates, strained-ring chemistry, computational chemistry, matrix isolation, and organo-fluorine chemistry.

More recently we have turned our attention to the study of the mechanism of anesthesia. Many of the currently used inhalation anesthetics are small fluorinated molecules. One of the currently most widely used intravenous anesthetics is Propofol, a drug which allegedly contributed to the demise of Michael Jackson. Anesthetics are some of the most dangerous drugs currently used today, and their mechanism of action (both good and bad) remains largely unknown. New photoaffinity anesthetic compounds which mimic anesthetics but which can be photoactivated so that they can bind to potential molecular targets are being developed in our group. Using these compounds we are investigating the mechanism of anesthesia in collaboration with Dr. Roderick Eckenhoff's group at the School of Medicine at Penn. This collaboration was recently highlighted in C&E News.

Structures of several currently used anesthetics and the corresponding photoaffinity labeling analogs prepared in our laboratory.

Our overall thrust is to study the linkage between biological structure and function, using a broad array of chemical, physical, and biological tools. Our major efforts fall in three principal areas.

Ribosomes 

We are interested in the structure and function of the bacterial ribosome, which is the site of protein biosynthesis in the cell. Most recently we have been focusing on the application of kinetic and spectroscopic approaches, including the use of single molecule and single-turnover studies in conjunction with fluorescence resonance energy transfer (FRET), to elucidate mechanisms for ribosomal catalysis of protein synthesis. We are particularly interested in the functions of G-proteins on the ribosome, and how these functions are altered by antibiotics and by mutations of tRNAs and ribosomal RNA. We are also pursuing studies on how the rate of protein synthesis is modulated by specific mRNA and oligopeptide sequences.

Ribonucleotide reductase (RR) 

RR catalyzes the reduction of nucleoside diphosphates to deoxynucleoside diphosphates and is the key enzyme controlling the rate of DNA synthesis. As such it is highly regulated and is a target enzyme for cancer chemotherapy. Our studies focus on the RRs derived from mammalian cells. We are developing novel and specific inhibitors of this enzyme, using both a rational design approach and combinatorial methods, and based in part on our detailed studies of the allosteric regulation of this enzyme. This work utilizes synthetic organic chemistry, biochemistry, molecular modeling and X-ray crystallography approaches.

Serine proteinase inhibitors ("serpins") 

Serpins are known to be of great importance for inflammation process in mammals. We seek to understand the structural basis for the specificity of interaction of these serpins with a variety of serine proteases, using a combination of chemical modification, single turnover FRET kinetics, FT-IR spectroscopy and genetic engineering approaches to elucidate the basic mechanisms underlying such specificity. A second area of interest is serpin polymerization, which underlies several diseases associated with serpin malfunction. These studies are being carried out using single molecule confocal microscopy.

Dynamics of Ribosomal Conformational Change

We are interested in structural aspects of the mechanisms of hydrolytic metalloenzymes in the arginase-deacetylase family. To date, we have determined the crystal structures of rat arginase I, human arginase I, human arginase II, and arginases from Plasmodium falciparum, Leishmania mexicana, and Schistosoma mansoni. Structural and enzymological data suggest a mechanism for arginine hydrolysis in which both manganese ions activate a bridging hydroxide ion for nucleophilic attack at the guanidinium group of arginine in the first step of catalysis. Based on our structural and mechanistic analyses, we designed and synthesized boronic acid analogues of arginine such as 2-amino-6-boronohexanoic acid (ABH, Kd = 5 nM) [Baggio et al. (1997) J. Am. Chem. Soc. 119, 8107]. The boronic acid moiety of ABH similarly undergoes nucleophilic attack by the metal-bridging hydroxide ion to yield a metal-bound boronate anion that mimics the tetrahedral intermediate and its flanking transition states in catalysis (Figure 1), as shown in X-ray crystallographic studies of rat arginase I [Cox et al. (1999) Nature Struct. Biol. 6, 1043], human arginase I [Di Costanzo et al. (2005) Proc. Natl. Acad. Sci. USA, 102, 13058], P. falciparum arginase [Dowling et al. (2010) Biochemistry 49 5600], and L. mexicana arginase [D' Antonio et al. (2013) Arch. Biochem. Biophys. 535, 163]

Figure 1: Human arginase I-ABH complex. (a) Omit electron density map of ABH bound in the enzyme active site at 1.29 Å resolution. Water molecules appears as red spheres and Mn(II) ions appears as larger pink spheres. (b) Summary of arginase-ABH interactions; manganese coordination interactions are designated by green dashed lines, and hydrogen bonds are indicated by black dashed lines. (c) Stabilization of the tetrahedral intermediate (and flanking transition states) in the arginase mechanism based on the binding mode of ABH.

We have also used ABH as a chemical tool for probing the role of arginase in regulating arginine bioavailability for nitric oxide (NO) biosynthesis in tissues and in live animals. We discovered that arginase inhibition by ABH enhances smooth muscle relaxation in ex vivo organ bath studies. Since smooth muscle relaxation in the corpus cavernosum of the penis is necessary for erection, we concluded that human penile arginase is a potential target for the development of new therapies in the treatment of erectile dysfunction [Cox et al. (1999) Nature Struct. Biol. 6, 1043]. Our subsequent in vivo studies demonstrated that arginase inhibition by ABH enhances erectile function and vasocongestion in the male and female genitalia, so we concluded that both male erectile dysfunction and female sexual arousal disorder are potentially treatable by ABH [Cama et al. (2003) Biochemistry 42, 8445; Christianson (2005) Acc. Chem. Res. 38, 191]. More recent studies show that ABH may also be useful in the treatment of certain cardiovascular disorders such as atherosclerosis [Santhanam et al. (2007) Circulation Res. 101, 692; Ryoo et al. (2008) Circulation Res. 102, 923]. The biopharmaceutical company Arginetix was founded in 2008 based on our arginase inhibitor technology.

Our work with metal-dependant histone deacetylases recently yielded the first crystal structure of a histone deacetylase complexed with a macrocyclic depsipeptide inhibitor (Figure 2) [Cole et al. (2011) J. Am. Chem. Soc. 133, 12474]. Additionally, we recently showed that mutations in histone deacetylase 8 identified in patients diagnosed with Cornelia de Lange Syndrome compromise catalytic activity by causing structural changes in the active site that perturb substrate binding and catalysis [Deardorff et al. (2012) Nature 489, 313; Decroos et al. (2014) ACS Chem. Biol., in press.]. In addition to our work with arginase, we are studying other metalloenzymes that adopt the arginase fold, such as polyamine deacetylase [Lombardi et al. (2011) Biochemistry 50, 1808].

In other metalloenzyme work, we have determined the crystal structure of A. aeolicus LpxC, a zinc-requiring enzyme that catalyzes the first step of lipid A biosynthesis in Gram-negative bacteria [Whittington et al. (2003) Proc. Natl. Acad. Sci. USA 100, 8146] (Figure 3). Subsequent structural studies have allowed us to pinpoint regions of the active site that interact with the fatty acid and diphosphate moieties of the substrate [Gennadios et al. (2006) Biochemistry 45, 7940; 15216], and these studies have guided the first steps in the structure-based design of new LpxC inhibitors that may ultimately be useful in the treatment of Gram-negative bacterial infections [Shin et al. (2007) Bioorg. Med. Chem. 15, 2617]. To date, we have broadened these structural studies to include LpxC enzymes from Gram-negative pathogens Y. pestis (bubonic plague) and F. tularensis (tularemia) [Cole et al. (2011) Biochemistry 50, 258.]

Figure 3: Structure and biological function of LpxC. This zinc enzyme catalyzes the first committed step of lipid A biosynthesis; lipid A is the hydrophobic anchor of lipopolysaccharide, which comprises the outer leaflet of the outer membrane of Gram-negative bacteria. The crystal structure of LpxC reveals a hydrophobic tunnel in the active site that accommodates the fatty acid moiety of the substrate, and this binding interaction is required for the active site to adopt a catalytically-active conformation.

Structural Basis of Terpenoid Biosynthesis

The family of terpenoid natural products currently numbers more than 70,000 members found in all forms of life. Terpenoids, are involved in diverse biological functions such as the mediation of plant-parasite interactions or the modulation of membrane fluidity. Since times of antiquity, terpenoid natural products have also been essential components of the pharmacopeia as analgesics, antibiotics, and anti-cancer compounds (e.g., Taxol). We are interested in the enzymes that catalyze the biosynthesis of different cyclic terpenoids [Christianson (2006) Chem. Rev. 106, 3412; Christianson (2008) Curr. Opin. Chem. Biol. 12, 141]. We have determined the three-dimensional crystal structures of terpenoid cyclases from various bacterial, fungal, and plant sources, such as epi-isozizaene synthase from S. colicolor [Aaron et al. (2010) Biochemistry 49, 1787], bornyl diphosphate synthase from culinary sage [Whittington et al. (2002), Proc. Natl. Acad. Sci. USA 99, 15375], aristolochene synthase from A. terreus [Shishova et al. (2007) Biochemistry 46, 1941], trichodiene synthase from F. sporotrichioides [Rynkiewicz et al. (2001) Proc. Natl. Acad. Sci. USA 98, 13543], δ-cadinene synthase from cotton [Gennadios et al. (2009) Biochemistry 48, 6175] and taxadiene synthase from the Pacific yew (which catalyzes the first committed step in the biosynthesis of Taxol, a potent cancer chemotherapeutic compound), [Köksal et al. (2011) Nature 469, 116]. To illustrate, structures of bornyl diphosphate synthase and taxadiene synthase are shown in Figures 4 and 5, respectively. These structures guide the study of site-specific mutants and alternative substrates as we explore the structural basis of diversity in terpenoid biosynthesis [e.g., see: Vedula et al. (2005) Biochemistry 44, 12719; Vedula et al. (2008) Arch. Biochem. Biophys. 469, 184; Christianson (2007) Science 316, 60], Köksal et al. (2012) Biochemistry 51, 3003, 301.

Figure 4: Reaction catalyzed by bornyl diphosphate synthase. Aza analogues of carbocation intermediates are shown in boxes; crystal structures of their complexes with the synthase reveal structural inferences on catalysis. The enzyme undergoes significant conformational changes upon the binding of 3 Mg2+ ions and pyrophosphate (or a substrate diphosphate group). These conformational changes sequester the active site from bulk solvent and trigger substrate ionization to initiate catalysis [Whittington et al. (2002) Proc. Natl. Acad. Sci. USA 99, 15375].

Figure 5: Structural relationships among terpenoid cyclases.The class I terpenoid cyclase fold of pentalenene synthase (blue) contains metal-binding motifs DDXXD and (N,D)DXX(S,T)XXXE (red and orange, respectively); in 5-epi-aristolochene synthase, this domain is linked to a smaller, vestigial domain (green). A related domain is found in the class II terpenoid cyclase fold of squalene-hopene cyclase, where it contains the general acid motif DXDD (brown) and a second domain (yellow) inserted between the first and second helices; a hydrophobic plateau flanking helix 8 (gray stripes) enables membrane insertion. Taxadiene synthase contains both class I and class II terpenoid cyclase folds, but only the class I domain is catalytically active. The role of N-termini (purple) in class I plant cyclases is to "cap" the active site, as shown for 5-epi-aristolochene synthase.