Welcome to the research page of the Moyna Group. Here you will find information related to the research endeavors in which the group is currently involved, the tools that are used for such purposes, the group members behind the different research projects, as well as collaborations between the Moyna Group and other research groups and institutions.
Our main interests are directed towards the elucidation of the mode of action of biologically active molecules at the atomic level. For such purpose, we work on two different but complementary fields of chemistry: Part of the group works on computational chemistry methods, and is interested in the development of molecular modeling tools that can be employed in the study of the conformational behavior and dynamics of biomolecules. Other members of the group use synthetic organic chemistry in the design and preparation of new drugs with targets as varied as malaria and cancer. Please read on to learn more about these exciting projects.
Application of Chemical Shifts in Conformational Studies
Nuclear Magnetic Resonance (NMR) is one of the most important tools for the determination of three-dimensional (3D) structures, conformational preferences, and solution dynamics of biologically relevant molecules, including natural products, peptides, and polysaccharides. Normally, information on relative distances between protons in the molecule, obtained by nuclear Overhauser effects (NOEs), are employed to constraint the molecular conformations and, together with a plethora of molecular modeling methods, obtain plausible structures. Recently, researchers have started using non conventional NMR constraints, such as those derived from chemical shift data, as additional sources of structural information. One of the goals of the Moyna Group is to employ 1H and 13C chemical shifts to aid in the determination of 3D structures of polypeptides and polysaccharides. We have already shown that 1H chemical shifts can be important in cases were other NMR data is scarce.1,2,3 Currently, we are attempting to determine the variation of the 13C chemical shift of the anomeric carbons in model disaccharides versus the <f,y> angles of the glycosidic bond by ab initio methods, in order to use the resulting surfaces in molecular modeling simulations.4,5,6 The surface of computed shifts for the anomeric carbon (=) of the glycopeptide GlcNAc-b-Ser is shown below. These mathematical relationships can be used directly in structure refinement, or employed in 13C shift back-calculations. The long term objective of the project is to derive similar surfaces for other important glycosidic bonds, such as those found in glycolipids and glycoproteins, and saponins, and use them as a tool to study the structure of these biologically relevant molecules by NMR.
As part of this project we are also studying the applicability of Density Functional Theory (DFT) methods to the calculation of 13C chemical shifts. For this purpose, we are comparing the performance of different DFT correlation and exchange functionals, as well as theory levels (i.e., basis sets) to results from calculations done using Hartee-Fock methods.7 Our results will establish which of the common DFT methods commonly employed is better suited for the calculations of 13C chemical shifts in sugars, as well as provide us with a more economical route to the estimation of 13C chemical shift surfaces for these molecules.
Design and Synthesis of Simple and Novel Antimalarials
Due to its high morbidity and mortality, malaria is a disease of enormous importance in tropical countries. Over 2 billion people live in areas of high incidence, and it is estimated that there are 200 million infected humans. Its principal agent is Plasmodium falciparum, a parasite transmitted by the bite of Anopheles mosquitoes. P. falciparum is a particularly resistant parasite which is known to have high adaptability by mutation. This mutability makes quite likely the development of resistance to vaccines and chemotherapies currently being introduced. Even for drugs with novel modes of action, such as artemisinin and other natural and synthetic endoperoxides, there is a high likelihood that resistant Plasmodia strains will evolve. This would be especially problematic, because there is high probability that the mechanism of resistance will involve the inactivation of the endoperoxide moiety, and thus would render these strains resistant to all drugs with similar chemical structure. The trademark of artemisinin action is the interaction between its endoperoxide bond with active iron (II) species in the food vacuole of the parasite, which catalyses the generation of highly cytotoxic radicals. While the bond energy of the peroxo linkage (-O-O-) is 32 Kcal/mole, the corresponding bond energies of the -N-O- and -N-N- bonds are 36 and 38 Kcal/mole respectively. Thus, compounds bearing these moieties could follow a reaction pattern, and thus biological activity, related to that of endoperoxides. However, the differences in chemical structure would make these compounds unaffected by the potential Plasmodium variants capable of inactivating endoperoxide moieties which are likely to appear. Our group is therefore attempting to prepare simple compounds bearing nitroso and hydrazo moieties. They are based on simple tropolones and benzotropolones to which the -N-O- and -N-N- groups are attached by means of an hetero Diels-Alder reaction. Some of the compounds being considered are shown below:
In this figure, X represents either nitrogen or oxygen, and R1, R2, and R3 are subsituents which will be modified to modulate the activity and pharmacological properties of the derivatives once biological data for the different trial series are obtained. Currently, 24 compounds from the first series bearing -N-O- and -N-N- moieties have been prepared, and we are awaiting results from biological assays against P. Falciparium.9 Using these data, we can perform QSAR and CoMFA using a variety of molecular descriptors to determine which modifications to the variable groups are needed to improve activity and pharmacological properties. We are hopeful that our endeavors will result in the generation of malaria therapies active against resistant strains of Plasmodia which are likely to appear.
The Specific Role of Side Chain Functional Groups in Taxol
Taxol is one of the most effective chemotherapeutic agents against several cancers. Taxol binds and stabilizes microtubules, interfering with cell division and ensuing the death of rapidly proliferating cancerous cells. Despite that extensive SAR and structural studies have been carried out, there are still questions regarding the specific interactions between functional groups crucial for activity in the taxol molecule and the binding site of the drug in microtubules. One such group is the hydroxyl group at position C2' of the side chain. Deletion or blockage of this group with a protective group such as methoxy or acetyl results in diminished activity. One of the hypothesis is that this hydroxyl functionality constraints the very flexible side chain of the molecule into a single conformation which corresponds to the bioactive conformation by formation of an intermolecular hydrogen bond to the carbonyl at position C1' of the side chain. Therefore, this presents the molecule to the tubulin binding site always in the right conformation, maximizing its binding affinity. However, if that is the case, inactive analogs in which the 2'-OH group are masked should not adopt the same conformations than the parent drug. This is not the case, as our previous studies have shown that taxol analogs with a masked or absent 2'-OH group (2'-methoxytaxol, 2'-acetyltaxol, and 2'-deoxytaxol) adopt very similar conformation to that of the parent drug in a variety of solvents and conditions.10,11 Therefore, our hypothesis is that the 2'-OH group does not hydrogen bonds intramolecularly stabilizing a preferred conformation, but with a specific group in the tubulin binding site. In order to add support to this hypothesis, we are in the process of preparing analogs bearing hydrogen bond donors at the C2' position of the side chain. Two of these analogs, 2'-aminotaxol and 2'-thiotaxol, are shown below:
Our approach to the synthesis of these analogs is based on the preparation of suitably protected side chain synthons and subsequent coupling to the diterpene core of taxol, Baccatin III. We have already designed a route to suitable side chains which relies on the preparation of b-lactam precursors.12 We are currently in the final stages of the preparation of the first analog of the series, 2'-aminotaxol (X = NH2). If active, this analog would not only help us to prove our hypothesis, but the possibility of preparing different salt derivatives could also help with the solubility problems of the parent drug, and would open the door to a new generation of taxol pro-drugs.
Relevant Publications and Presentations
1) Moyna, G.; Zauhar, R. J.; Williams, H. J.; Nachman, R. J.; Scott, A. I. Comparison of Ring Current Methods for use in Molecular Modeling Refinement of NMR Derived Three Dimenssional Structures. J. Chem. Inf. Comp. Sci. 1998, 38, 702-709.
2) Moyna, G.; Williams, H. J.; Nachamn, R. J.; Scott, A. I. Detection of Nascent Polyproline II Helices in Solution by NMR in Synthetic Insect Kinin Neuropeptide Mimics Containing the X-Pro-Pro-X Motif. J. Peptide Res. 1999, 53, 294-301.
3) Moyna, G. Use of NMR and Molecular Modeling in Conformational Studies of Insect Kinin Neuropeptides Analogs. University of the Sciences in Philadelphia, August, 1998 (PDF copy).
4) Moyna, G.; Zauhar, R. J. Derivation of 13C Chemical Shift Surfaces for the Anomeric Carbons of Polysaccharides Using ab initio Methodology. 219th American Chemical Society National Meeting, New Orleans, Louisiana, August 22-26, 1999 (PDF copy).
5) DeGrazia, M.; Swalina, C.; Moyna, G. 13C Chemical Shift Surfaces as tools for Conformational Studies of Oligosaccharides. Applications to Molecular Dynamic Simulation Trajectories of the Disaccharide a-Manp-(1->3)-b-Glcp-OMe. American Chemical Society Mid Atlantic Regional Meeting, University of Delaware, Wilmington, Delaware, May 15-17, 2000 (PDF copy).
6) Swalina, C. W.; Zauhar, R. J.; DeGrazia, M. J.; Moyna, G. Derivation of 13C Chemical Shift Surfaces for the Anomeric Carbons of Oligosaccharides and Glycopeptides Using Ab Initio Methodology. J. Biomolec. NMR 2001, 21, 49-61.
7) Swalina, C.; DeGrazia, M.; Moyna, G. Computation of Oligosaccharide 13C Chemical Shifts. A Comparative Study of Hartree-Fock and DFT Ab InitioMethods at Different Theory Levels. American Chemical Society Mid Atlantic Regional Meeting, University of Delaware, Wilmington, Delaware, May 15-17, 2000 (PDF copy).
8) Dejoux, A.; Cieplak, P.; Hannick,, N.; Moyna, G.; Dupradeau, F.-Y. AmberFFC, A Flexible Program to Convert AMBER and GLYCAM Force Fields for use with Commercial Molecular Mechanics Packages. J. Mol. Model. 2001 (accepted for publication). Visit the AMBERFFC web-site.
9) Ren, H.; Grady, S.; Gamenara, D.; Heinzen, H.; Moyna, P.; Croft, S. L.; Kendrick, H.; Yardley, V.; Moyna, G. Design, Synthesis, and Biological Evaluation of a Series of Simple and Novel Potential Antimalarial Compounds. Bioorg. Med. Chem. Lett. 2001, 11, 1851-1854.
10) Moyna, G.; Williams, H. J.; Scott, A. I.; Ringel, I.; Gorodetsky, R.; Swindel, C. S. Conformational studies of taxol analogs modified at the C-2' position in hydrophobic and hydrophilic solvent systems. J. Med. Chem. 1997, 40, 3305- 3311.
11) Williams, H. J.; Moyna, G.; Scott, A. I.; Swindell, C. S.; Chirlian, J. M.; Heerding, J. M.; Williams, D. K. NMR and molecular modeling study of the conformation of taxol 2'-acetate in chloroform and aqueous dimethylsulfoxide solutions. J. Med. Chem. 1996, 39, 1555-1559.
12) Moyna, G.; Williams, H. J.; Scott, A. I. Preparation of aminated taxol side chain precursors. A simple approach to 2,3-diaminoacids using the b-lactam synthon method. Syn. Comm. 1997, 27, 1561-1567.
See the complete publication list
Instrumentation and Facilities
Laboratories
The Moyna group has a considerable ammount of laboratory space. There is one laboratory for exclusive use of the group which has ample hood and bench space for two people, as well as space shared with Dr. Joel Kauffman in a larger laboratory with a total of four hoods and one walk-in hood. Both laboratories are well equipped with all the necessary glassware, roatary evaportators, UV lamps, and other small instrumentation needed during routine synthetic work.
Members of the Moyna group have access to a newly upgraded Anasazi EFT-90 90 MHz NMR spectrometer, with 1H, 13C, mutipulse, and 2D capabilities for use in routine structure determination and characterization of reaction products and intermediates. The Deparment has also secured funds from NSF for the purchase of a state-of-the-art 400 MHz NMR spectrometer with inverse detection, pulse field gradients, and the capability of performing the latests multinuclear pulse sequences. This instrument will be installed on campus by the Summer of 2001. Dr. Moyna is responsible for the maintenance of the EFT-90 and he will also be in charge of the 400.
The Moyna Group also maintains an NMR software page, in which you can find EFT-90 pulse programs, C++ code for-post processing of EFT-90 spectra, and Perl scripts for the generation and analysis of shaped selective pulses.
Computer Resources
Most of the molecular modeling simulations carried out by members of the group are performed in the newly established West Center for Computer-Aided Drug Discovery. This facility has several Silicon Graphics workstations, dual processor P-III workstations, and Macintosh desktop computers which can be used as X-Windows terminals or as standalone machines. The most impressive computer in the West Ceter is chaingang, a Beowulf supercomputer cluster with 16 compute nodes and a server, capable of performing extremely demanding calculations.
In addition to state-of-the-art hardware, all workstations in the West Center house a number of packages used in molecular modeling simulations. Among the extensive list of software are the ab initio packages Gaussian98 and GAMESS-US, the well known molecular mechanics and dynamics suite AMBER95, as well as Spartan 5.0.1 and Sybyl 6.6, which have powerful molecular graphics tools.
As part of a grant from the National Computational Science Alliance (CH99xxx), the Moyna group has allocations on the ALIANCE HP N-4000 12-node/96-processor cluster (High Performance Computing Group - University of Kentucky), and an SGI Origin 2000 (Boston University - Scientific Computing and Visualizationn) supercomputers. These supercomputers were used for most of the 13C chemical shift calculations before the Beowulf supercomputer cluster was installed in the West Center, and are currently used when the load on the local supercomputer and workstations is high.
Our group has a number of on-going collaborations with several research groups, both at USP and throughout the world.
One of the most important joint research efforts is with the group of Dr. Randy J. Zauhar here at USP. Both groups have been working in the development of ALMS (Automated Ligand binding with Multiple Substitutions), a system that runs under the SYBYL modelling package from Tripos, Inc. ALMS uses a combinatorial builder to generate large numbers of candidate ligands for a receptor site, and a genetic algorithm to optimize their orientations. The development of the algorithm was entirely done by the Zauhar group, while members of the Moyna group added functionality to the program and developed a graphical user interface (GUI) for ease of use. Additionally, both groups are the main users and mainteiners of the computer facilities at West Center at USP.
The Moyna group is also working in a project with the group of Dr. Francois Yves-Dupreadeau at the Faculte de Pharmacie, Universite de Picardie Jules Verne, Amiens, France. The goal of the project is the determination of AMBER force field parameters by ab initiomethods for the -SO3- and -S-SO3- functional groups. These functional groups are present in certain analogs of insulin, and the force field parameters are required for accurate simulations of the molecules in solution, as well as in the study of their structure by NMR. As part of the project, the team has developed the AMBERFFC program, which allows for the easy incorporation of different versions of the AMBER parameter files into commercial molecular modeling packages.
We have very close ties with the laboratory of Dr. Horacio Heinzen at the Catedra de Farmacognosia y Productos Naturales, Facultad de Quimica, UDELAR, Montevideo, Uruguay. The two groups are working together in the development of novel and simple malaria therapeutics, some of which are described above. While we are concentrating on a limited series of starting materials, the group at Montevideo is employing a larger variety of frameworks, including diene-containing Natural Products such as euacarvone, as well as dienes obtained from the biotransformation of halogenated aromatic compounds by Pseudomona putida.
As part of the malaria project we also work in collaboration with Dr. Simon L. Croft at the Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK. All the in vitro and in vivo assays against P. falciparium for the potential antimalarials prepared by the group are being tested in the laboratories of Dr. Croft.
The Fearless Leader
Guillermo Moyna, Ph.
D.
Dr. Moyna is a native of Uruguay, were he received him Bs. in Chemistry at the UDELAR. He moved to College Station, TX, in August of 1993, were he worked on his Ph. D. at the Department of Chemistry, Texas A&M Univeristy, under the direction of Prof. A. I. Scott. He completed his doctoral studies in October of 1998, and moved to Philadelphia in December, where he worked as a post-doctoral student for Dr. Zauhar at USP. In August of 1999 Dr. Moyna took his current possition as Assistant Professor of Chemistry in the Department of Chemistry & Biochemistry.
In this picture we can see him next to his magic concoction, mate tea, which gives him strength, wisdom, a clear mind, a valid reason to visit the restroom many times a day, and a wonderful conversation topic. Learn more about mate tea, a traditional drink from the south of South America, following this link.
Graduate Students
Hongyu Ren (MSc)
Ren was the first graduate in the group, and she joined in the Summer of 2000. Ren received a MSc. degree in Pharmacy from Shandong Medical Unversity, PRC, and then moved to the United States. She is leading the efforts in the preparation of potential antimalarias based on tropolones and benzotropolones dervatives. Together with Shannon Grady, Ren has been able to prepare 24 new compounds in a very short time which are currently being tested against P. falciparium.
Kristen Barlett (MSc)
Kris, a USP graduate from Whitehall, PA, is currently performing a rotation with the group. She is working together with Mike DeGrazia in the preparation of 2'-amino and 2'-thiol derivatives of the antineoplastic drug taxol described above. We are hoping that Kris will stay around to complete and expand the project after she is done with her rotation.
Undergraduate Students
Mike DeGrazia ('01
- AKA "Paranoia")
Mike was the first member of the Moyna group, and joined in the Fall of 1999. Mike, currently a Senior in the Department, joined USP after obtaining an Asoc. degree in Biology from Middlesex County College, NJ. Mike, seen here next to his beloved hood, is working in the preparation of 2'-amino and 2'-thiol derivatives of taxol. He also participated in several of the 13C chemical shift computational studies together with Chet Swalina.
Chet Swalina ('01)
Chet was the second member of the Moyna group, and he joined right after Mike in the Fall of 1999. Chet is from Morristown, NJ, and he trasferred to USP from County College of Morris, NJ, after obtaining a double Asoc. degree in Science and Social Sciences. Chet is working in the estimation of ab initio 13C chemical shift surfaces for oligosaccharides. As part of the project, he is also comparing the accuracy and precission of Hartree-Fock and DFT methods in the estimation of 13C chemical shifts in sugars.
Shannon Grady ('01)
Shannon joined the group in the Summer of 2000. Originally from Harrsiburgh, PA, she is currently in her senior year here at USP. Shannon, seen here distilling a precursor in the synthesis of tropolone, is working on the synthesis of potential antimalarial compounds, and has helped Ren in the preparation of several of our first potential antimalarials. Although she says she wants to own and tend a bar after College, we know she loves working in the laboratory!
Matthew Banghart ('02)
Matt started working in the lab in the Fall of 2000. He is from West Chester, PA, and he is currently a Junior in the Department. He is working together with Ren and Shannon in the preparation of benzotropolone derivates for use in the preparation of potential antimalarials. Matt has very diverse interests, and last year he carried out a very original reasearch project with Dr. Catherine Bentzley.
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