Category: Seminars

Durga Pokharel

Advisor: Dr. Shiyue Fang

Doctoral candidate, Department of Chemistry

PhD Defense

Friday December 12, 2014   9:30am     Fisher 130

NON-CHROMATOGRAPHIC PURIFICATION OF SYNTHETIC BIOOLIGOMERS

Abstract

Synthetic oligonucleotides and peptides have found wide applications in industry and academic research labs. There are ~60 peptide drugs on the market and over 500 under development. The global annual sale of peptide drugs in 2010 was estimated to be $13 billion. There are three oligonucleotide-based drugs on market; among them, the FDA newly approved Kynamro was predicted to have a $100 million annual sale. The annual sale of oligonucleotides to academic labs was estimated to be $700 million. Both bio-oligomers are mostly synthesized on automated synthesizers using solid phase synthesis technology, in which nucleoside or amino acid monomers are added sequentially until the desired full-length sequence is reached. The additions cannot be complete, which generates truncated undesired failure sequences. For almost all applications, these impurities must be removed. The most widely used method is HPLC. However, the method is slow, expensive, labor-intensive, not amendable for automation, difficult to scale up, and unsuitable for high throughput purification. It needs large capital investment, and consumes large volumes of harmful solvents. The purification costs are estimated to be more than 50% of total production costs. Other methods for bio-oligomer purification also have drawbacks, and are less favored than HPLC for most applications.

To overcome the problems of known biopolymer purification technologies, we have developed two non-chromatographic purification methods. They are (1) catching failure sequences by polymerization, and (2) catching full-length sequences by polymerization. In the first method, a polymerizable group is attached to the failure sequences of the bio-oligomers during automated synthesis; purification is achieved by simply polymerizing the failure sequences into an insoluble gel and extracting full-length sequences. In the second method, a polymerizable group is attached to the full-length sequences, which are then incorporated into a polymer; impurities are removed by washing, and pure product is cleaved from polymer. These methods do not need chromatography, and all drawbacks of HPLC no longer exist. Using them, purification is achieved by simple manipulations such as shaking and extraction. Therefore, they are suitable for large scale purification of oligonucleotide and peptide drugs, and also ideal for high throughput purification, which currently has a high demand for research projects involving total gene synthesis. The savings with the new techniques compared with HPLC are estimated to be 70% to 90% depending on purification scale and throughput. We expect these new oligonucleotide and peptide purification technologies to be widely used in academic research labs, biotechnology companies, and pharmaceutical companies in the near future.

Mr. Robert K Brown

Advisor: Dr. Tarun K Dam

Master’s Candidate Department of Chemistry

Michigan Technological University

“Purification and Carbohydrate Binding Properties of Two New Plant Proteins”

Friday, December 12, 2014

10:00 – 11:00 AM 

Room 404 ~ Administration Building
Purification and Carbohydrate Binding Properties of Two New Plant Proteins

Abstract:

Protein glycosylation is an important post-translational modification for many biological processes such as cell recognition, intercellular communication and cell death. Proteins that are able to bind to glycosylated proteins via carbohydrates are called lectins. Hemolytic lectins are proteins or glycoproteins that undergo specific interactions with cell surface carbohydrates and subsequently induce cellular lysis. They are termed “hemo” lytic because of their ability to lyse erythrocytes. We have isolated a novel hemolytic lectin named HelyX from the bulbs of a monocot plant, as well as a mannose-binding lectin named DIL from a separate monocot species. HelyX is a uniquely robust hemolytic lectin. It shows concentration dependent reversible hemolytic/agglutinating properties against both human and rabbit erythrocytes. The activity was found to be carbohydrate dependent. HelyX was isolated using ammonium sulfate precipitation, size exclusion chromatography, and analyzed by gel electrophoresis. DIL was purified using a modified version of a newly developed protocol. DIL interacts with the plant enzyme invertase with high affinity. This high affinity interaction suggests that the binding site of DIL is complimentary to glycoproteins containing larger high mannose glycans. Invertase is central to plant metabolism and defense. Therefore DIL might play a modulatory role in plant metabolism and defense through its interaction with invertase. HelyX and DIL did not show lytic activity on free living amoeba, Acanthamoebae. Instead the lectins promoted cyst formation of amoeabae trophozoites indicating a lectin-mediated rearrangement of membrane architecture. This result indicates that the lytic activity of HelyX or DIL depends on the macromolecular landscape of the cell membrane.

MS Defense:  DM Ashraf Ul Habib
Chemistry Department

Lynn Mazzoleni, Advisor
Thursday, December 4  1pm,  Chem Sci 101
Evolution of Selected Isoprene Oxidation Products in Dark Aqueous Ammonium Sulfate

The climate of the world is changing but our understanding of atmospheric processes is limited. Atmospheric aerosol is a trace but very influential medium of the atmosphere. Especially little is known about the organic aerosol components and their aqueous phase chemical evolution. To address this, the aqueous phase processing of glyoxylic acid, py­ruvic acid, oxalic acid and methylglyoxal was studied simulating dark and radical free atmospheric aqueous aerosol. A novel observation of the cleavage of a carbon-carbon bond in pyruvic acid and glyoxylic acid leading to their decarboxylation was made in the presence of ammonium salts but decarboxylation was not observed from oxalic acid. The empirical rate constants for decarboxylation were determined and are competitive with nighttime OH radical reactions. The structure of the acid, ionic environment of the solu­tions and concentration of species were all found to affect the rate of decarboxylation. A tentative set of reaction mechanisms is proposed involving nucleophilic attack by ammo­nia on the carbonyl carbon leading to fragmentation of the carbon-carbon bond between the carbonyl and carboxyl carbons. Under similar conditions in atmospheric aerosol, the aqueous phase processing may markedly impact the physicochemical properties of aerosol.

Suntara (Boat) Fueangfung

Advisor: Dr. Shiyue Fang

Doctoral candidate, Department of Chemistry

Michigan Technological University

Friday, December 5, 2014, 9:00 am

Admin Building, Room 404

PhD Defense

Synthetic Oligodeoxynucleotide Purification via Catching by Polymerization

Abstract

Large quantities of pure synthetic oligodeoxynucleotides (ODNs) are important for preclinical research, drug development, and biological studies. These ODNs are synthesized on an automated synthesizer. It is inevitable that the crude ODN product contains failure sequences which are not easily removed because they have the same properties as the full length ODNs. Current ODN purification methods such as polyacrylamide gel electrophoresis (PAGE), reversed-phase high performance liquid chromatography (RP HPLC), anion exchange HPLC, and affinity purification can remove those impurities. However, they are not suitable for large scale purification due to the expensive aspects associated with instrumentation, solvent demand, and high labor costs.

To solve these problems, two non-chromatographic ODN purification methods have been developed. In the first method, the full-length ODN was tagged with the phosphoramidite containing a methacrylamide group and a cleavable linker while the failure sequences were not. The full-length ODN was incorporated into a polymer through radical acrylamide polymerization whereas failure sequences and other impurities were removed by washing. Pure full-length ODN was obtained by cleaving it from the polymer. In the second method, the failure sequences were capped by a methacrylated phosphoramidite in each synthetic cycle. During purification, the failure sequences were separated from the full-length ODN by radical acrylamide polymerization. The full-length ODN was obtained via water extraction. For both methods, excellent purification yields were achieved and the purity of ODNs was very satisfactory. Thus, this new technology is expected to be beneficial for large scale ODN purification.