Microsoft word - res and schol grant 2009 v5.doc

Biocatalysis: Utilizing Engineered E. coli towards Pharmaceutical Precursors
Brent D. Feske - Department of Chemistry and Physics

Need and Significance - The synthesis of pharmaceutical-grade compounds is often a difficult and
complex task, which is a major contributing factor for the high cost of drugs. Many academic and industrial scientists are working to find a simpler, more affordable way to synthesize these complex molecules. Developing these molecules through biocatalysis (reactions with enzymes or living cells) is a potential solution to this problem. Recently, many industries (Merck, DuPont, Pfizer, etc.) have implemented large-scale syntheses of pharmaceutical-grade compounds using biocatalysis. This process is not only advantageous since it is often a direct route to these complex molecules, but these reactions take place in water thus eliminating the use of toxic solvents. This field of ‘green chemistry’ has become very popular with industry due to the high cost of disposing toxic waste and to build a better image with the surrounding communities. Using biocatalysis as a practical application to the pharmaceutical industry has proven to be difficult. However, with the advancement of molecular biology (cloning and the manipulation of DNA), this field of science has now been given the tools it needs to be successful. Recently, Stewart et. al from the University of Florida developed 20 different engineered Escherichia coli (E. coli) that have been found to do a variety of complex reactions. However, we have only scratched the surface on these organisms’ synthetic ability. Cultures of these 20 engineered E. coli have been obtained by Armstrong Atlantic State University, and these organisms are presently being screened for their reaction capabilities. Fortunately, we have been successful in finding external funding. An NSF-Research Undergraduate Institutions (RUI) grant was funded for three years and started spring 2009 ($195,421) and a NSF-Major Research Instrumentation (MRI) grant was recently funded ($277,326) which supplies a state of the art instrument called an LCMS- qqTOF, which should arrive January 2010. This external funding supports this research with equipment, supplies, travel, and even summer stipends for faculty and students. Unfortunately, what these grants do not support is a course reduction to allow the PI to focus more of his efforts in making sure that these grants are successfully executed. This is in particular importance for the spring 2010 semester. As mentioned above, the LCMS-qqTOF will arrive. This instrument (which retails over a half a million dollars without discounts) is a highly technical instrument that neither myself, nor anyone else at AASU, has ever been trained to use. As a result, it will take a significant time investment to learn how to run the instrument, to do routine maintenance, and to be sure that the other 6 PI’s on the grant are trained properly so we can successfully execute our proposed research using this instrument. Validity - Utilizing engineered E. coli for the synthesis of biologically active molecules has
become a great interest to pharmaceutical companies. Recently, DSM Pharmaceutical and Merck have purchased the aforementioned E. coli cultures with hopes to utilize them for their commercial synthesis of drugs. Not only are reactions using these E. coli of interest to the pharmaceutical industry, but they have proven to be of interest to the academic world. Over the past 3 years, several publications have reported the reactive diversity of these organisms and the enzymes they possess. We have shown that Taxol® (the highest selling cancer drug in the history of the pharmaceutical market) and Bestatin® (a known antibiotic and anticancer agent) can be synthesized from the biocatalytic products of these E. coli.1,2 In addition, recent work performed by Dr. Feske (PI) and 7 AASU undergraduate students resulted in the formal synthesis of Prozac® (and other serotonin reuptake inhibitors) and β-blockers using the engineered E. coli as the key Feasibility and Personnel Qualifications - Dr. Feske (PI) has assisted with the engineering of the
aforementioned E. coli and has spent the last 9 years optimizing and applying these organisms towards the synthesis of pharmaceuticals. Presently, four undergraduate students (Chem 2900/3900 – total 4 credit hours) have joined this project and are actively researching here at AASU. The Chemistry and Physics department and NSF has monetarily invested in this project by purchasing important analytical tools and basic equipment that has allowed us to adequately sustain an active research project. As this research progresses and expands the PI’s needs begin to expand also, thus the Research and Scholarship grant can play a crucial role in his success by funding a course reduction. The PI’s research has been funded by two Research and Scholarship Grants, which has resulted in the following outcomes: 1. Two peer reviewed journal articles with a total of 7 student coauthors (one still in progress) 2. Preliminary results that led to 2 – NSF research grants totaling ~$473,000 (as the PI) 3. Several presentations (faculty and student) at regional and national meetings 4. Helped support nine students in independent research courses totaling 14 credit hours Assessment and Implementation Plan - Popular biocatalytic approaches to pharmaceutical-grade
molecules utilize wild-type (non-engineered) organisms that are found in nature; the most popular of organisms being bakers’ yeast because of its availability. Bakers’ yeast, along with many other organisms (E. coli, yeasts, fungi), has been screened for a variety of ketone reductions (Scheme 1).
Unfortunately, these wild type organisms rarely afford a product in the pure form needed for pharmaceutical-grade compounds, instead forming products with a mixture of ‘chiral’ centers. Scheme 1 – General example of a reaction with wild-type organisms
Recent studies on our engineered E. coli found that they can produce high purity products (Scheme 2). In addition, due to the genetic makeup of these cells, they can be used on a large-
scale more efficiently than wild-type organisms. Scheme 2 – General reaction using our engineered E. coli
Presently, β-keto esters and α-chloro-β-keto esters (Scheme 2) have been the only class of
substrates fully investigated with these E. coli. Our latest work here at AASU, screened and characterized the reaction of β-keto nitriles with the engineered E. coli. The next step of this process is too utilize these pharmaceutical grade compounds as precursors for the synthesis of lactones, which are compounds known for their strong biological activity (Scheme 3).
submitted to Th e Jou r nal of Bi oor ga nic and Med icin al Chemistr y L etter s Scheme 3 – Overall scheme towards chiral lactones.
Upon completion of the lactone synthesis, this work will be assessed by publishing a manuscript presenting the results in a peer-reviewed journal. In addition, some of this research has already been presented at National and Regional Meetings of the American Chemical Society (ACS), at the AASU undergraduate research symposiums, and we will continue to present at appropriate meetings. Lastly, assessment of student success will also be conducted by documenting student achievement after graduation, such as many of the PI’s research students have moved on to Dental, Medical, Pharmacy, and Graduate school. Budget - The proposal asks for $2,500 for a 3 credit hour course reduction for PI – Brent Feske.
This reduction would afford more time to be spent in the laboratory conducting research with undergraduate students, which will increase student and faculty productivity that will result in an increased scholarly product that can be reported in peer reviewed manuscripts and in the mandatory annual NSF reports. In addition, this will give me more time to meet with the instrument technicians during their training sessions for the new LCMS-qqTOF and to be sure that all new users are quickly trained on this instrument so we can promptly utilize it in our research
References (undergraduate coauthors underlined)
1. Feske B. D.; Kaluzna I. A.; Stewart J. D. “Enantiodivergent, Biocatalytic Routes to Both Taxol Side Chain Antipodes.” J. Org. Chem. 2005, 70(23), 9654-9657.
2. Feske B.D.; Stewart, J.D. “Chemoenzymatic Formal Total Synthesis of (-)-Bestatin.” Tetrahedron: Asymmetry 2005, 16(18), 3124-3127.
3. Nowill, R.; Patel, T.; Beasley, D.; Alvarez, J.; Anuskiewitz, R.; Hizer, T.; Ghiviriga, I.; Mateer, S. C.; Feske, B. D. Biocatalytic Strategy towards Asymmetric β-Hydroxy
Nitriles and γ-Amino Alcohols. Bioorganic and Medicinal Chemistry letters – 2009 -
submitted.
4. Hammond, R. J.; Poston, B. W.; Ghiviriga, I.; Feske, B. D. “Biocatalytic Synthesis Towards Both Antipodes of 3-hydroxy-3-phenylpropanitrile a precursor to
Fluoxetine, Atomoxetine and Nisoxetine.” Tetrahedron Lett. 2007, 48, 1217-1219.

Source: http://www.armstrong.edu/images/faculty_development/Biocatalysis.pdf