Fast, easy, clean approaches to large-scale green chemistry and enzyme catalysis

Introduction:

I am currently a junior at the University of Connecticut, studying as a joint Chemistry and Physiology & Neurobiology major. I am interested in research at the cutting edge of chemistry that has a biological application. I would like to be a part of the University Scholar program in order to satiate my curiosities in the field of microwave chemistry. Specifically, I would like to integrate microwave chemistry with biology. In my first two years, (and summers) at the University of Connecticut, I have spent a considerable amount of time working with Dr. Leadbeater in his chemistry lab, doing microwave chemistry. These experiences have sparked my growing interest in the field of chemistry, as well as my desire to develop protocols to make complex molecules, in a solvent- free (or minimized) way, which are in demand in the pharmaceutical industry. I plan on taking graduate level chemistry classes, to increase my chemistry background, and to further develop my lab techniques. My senior thesis is the best way to finalize an in depth study, and share this work with the scientific community. My undergraduate research has sparked my desire to undertake an MD / PhD after graduation. Graduating with two degrees will allow me to integrate my chemical knowledge with my understanding of the human body and understand how the most basic and relevant parts of science and life work.

 

Experience:

Microwave-promoted synthesis (frequency around 2.45 GHz) is an area of increasing research interest as evidenced by the growing number of papers and recent reviews appearing in the literature.[1]^{,[2],[3],[4],[5]} As well as being a very efficient mechanism of transferring energy to molecules, microwaves can enhance the rate of reactions, and in many cases improve product yields. Working in Dr. Leadbeater’s lab has made me very proficient in the field of microwave chemistry, and I have worked with a wide range of reactions and I have perfected several laboratory protocols. In my first semester working in the group, I worked with a range of different phase transfer agents in order to investigate the scope of possibilities for accelerating reactions, with microwave irradiation, that conventionally are slow and give low yields. My work was successful and we came up with a fast (5 minute) reaction that could be run in water as solvent, by using microwave heating. Traditionally, the reaction takes 16 hours using an organic solvent and is heated conventionally. I was able to add many skills to my portfolio. I also learned how to run NMR spectra (Nuclear Magnetic Resonance), read, and interpret them. During the summer of 2004, I worked on a project which concerned reactions in the microwave using very low catalyst concentrations, but my main project was to work on a methodology called “heating while cooling.” This was sponsored by CEM Microwave Technology, the leading manufacturer of scientific microwave apparatus. This was an innovative technique, which helped to stabilize substrates in the microwave which decompose quickly, due to excessive amounts of heat when reacting. During the fall of 2004 and spring 2005, I worked on investigating different reactions using the heating while cooling technique. In the summer of 2005, I worked on developing methods for larger scale chemistry using microwave heating. I received a grant from CBIA/Pfizer which paid for my housing at the University of Connecticut for the summer. I have been a part of two publications thus far, one on heating while cooling, and another on ultra-low catalyst loading in a reaction called the Suzuki coupling.[6]^,[7] Currently, we are in the process of submitting a third paper for review on my work from the summer of 2005.[8]

 

Background Information:

Microwave chemistry offers the “next generation” of heating a chemical reaction, opening new avenues for research, as well as decreased reaction times from 50 hours (a little over 2 days) to 5 minutes, in some cases. Due to the advantages of microwave chemistry, it is very likely that the next generation of heating will be with microwave irradiation. Much of the work in this field to date has been conducted using modified domestic microwave ovens (similar to those in the kitchen at home) to promote reactions when working in conventional organic solvents.[9] There are problems associated with this, such as poor reproducibility of reactions and the fact that it is hard to control reactions precisely.[10] It is also dangerous to perform scientific reactions in a conventional microwave, because of lack of safety features. In recent years, with the advent of scientific focused microwave systems, many of these major problems have been overcome. Using scientific microwaves it is possible to control the temperature, pressure, microwave power and reaction times very easily and with a high degree of reproducibility. One must also consider that by decreasing reaction times drastically, this makes it possible to screen many more reactions with less cleanup, time, and also to accelerate the rate of scientific discovery.

Scientific aims:

In applying for the University Scholar program, I am interested in undertaking a project which has two parts, one which adresses needs in the chemistry community, and a second which fufills academic curiosity. Pfizer has expressed interest in this project due to the environmentally friendly chemistry component (fast, clean, efficient heating), as well as the exploration of the effects of scaleup in the microwave. Although I will not be obtaining financial support from Pfizer, one of the lab heads has written a letter on my behalf supporting the project. Outside funding will come from CEM in the form of scientific equipment, as well as providing a site to do much larger scaleup for which the Leadbeater group does not have the equipment. I will be be receiving a dual Bachelor of Science in December of 2007, for reasons which will be described later, and for the spring of 2008, I am interested to continue my work with Dr. Leadbeater and CEM, so that it will be possible to devote all of my time to the lab. Research which has a use outside of academia is of growing importance in the scientific world today, because it would more rapidly be available to benefit society as a whole. Using the Leadbeater group’s state of the art scientific microwaves, I am interested in the use of water as a solvent, because it is environmentally friendly, as well as being a good solvent which readily absorbs microwave energy.[11]

The first component of this project focuses on scale-up in the microwave, to make kilograms of material rather than just a few milligrams. Traditionally, microwave reactions are built for small scale synthesis, and reactions take place in 1- 5 milliliter reaction vessels, under pressure. There is a real demand in the chemical industry to be able to use microwave heating for large scale synthesis, as well as for, as is already done, preliminary optimization and screening of reactions. Working in collaboration with CEM Microwave Technology, I am interested in investigating and overcoming problems concerning scale-up of reactions, and working towards a solution to a widespread “fear” of using microwaves for scale up in the pharmaceutical industry by more “conventional” chemists. Previous work has been undertaken by the Leadbeater group in this field as well, in order to investigate several different closed- vessel alternatives (flow-through, batch and parallel microwave irradiation synthesis).[12] This past summer, working in a project funded by Pfizer and the Connecticut Business and Industry Association, I investigated scaling up one of our microwave reactions run in water with very low catalyst concentrations. This work was successful and forms the foundation of my future research in this area. The second component of this project focuses on enzymatic reactions (esterases and lipases) in the microwave. Traditionally, enzymatic reactions on the industrial scale can take very long times to reach completion, but a few reports have suggested that microwave irradiation can accelerate this process.^[13]

When irradiating a sample with microwaves, it is important to be sure to use the right microwave apparatus. Using a domestic microwave is difficult, because the energy that they put into the reaction is not consistent, and the microwave field may also not be homogeneous. This can cause enzymes to lose their structural integrities more readily, as well as making it difficult to repeat results.^[14],[15] In order for an enzyme to maintain its function, it must maintain its structure. Recently there have been many publications in scientific journals focusing on enzymatic reactions and “non-thermal” microwave effects.^{[16],[17],[18]} “Non-thermal” microwave effects are envoked when scientists cannot explain the acceleration of reactions by simple chemical heating principles. Many of these findings are suspect because they are based on reactions which have been run in conventional microwaves, rather than in scientific microwaves. While investigating these effects, it is important to disconnect the relationship between temperature and power. In previous papers, power was increased, but temperature also increased as an effect of this.^[19] With the extensive research that I have undertaken on “heating while cooling” I now know how to use this feature in order to separate effects due to temperature and power in a microwave reaction. This is possible because with our apparatus we can maintain a reaction at a set temperature, while feeding a pre-determined power into it, since as the reaction temperature rises, cooled air is blown over the reaction vessel walls. This allows for a reaction to continue with a set amount of power being put into it, without increasing the temperature beyond a point where the enzymes being used are thermally stable. This will allow for continuous microwave irradiation to be used without a significant rise in temperature of the reaction mixture. Enzyme catalysis is a mature field and reactions can be very chemo- and stereo-selective.[20] In addition, there are a large number of enzymes that are readily available that can be used for a range of synthetic transformations. One of the major drawbacks with the use of enzymes is that reaction times can take days, however with the application of scientific microwaves; it may be possible to decrease these reaction times to minutes.[21]

 

Methods:

If accepted as a University Scholar, the first part of my project will be focused on scaling up chemical reactions in the microwave. Due to the academic and scientific foundations that I have developed, I would be able to provide valuable insights into this topic. My past work focused on the Suzuki reaction (a key reaction in pharmaceutical synthesis). I would be interested to further explore this reaction, and scale it up farther, using a batch reactor. The second reaction to investigate would be a Diels-Alder reaction, because it is solvent free (environmentally friendly) and it would also allow us to do direct comparisons between batch and flow-through reactors. A third reaction would be a multi-component reaction, (possibly the Hantzsch dihydropyridine synthesis) since this is commonly used in medicinal chemistry projects. This is a multi-component reaction, because more than one thing is happening at once. I would like to see if this reaction is passed through a flow through microwave, it will work, or if the three substrates used need to be “premixed” in a batch reactor. It would be possible to adapt this reaction to a flow-through or batch apparatus. It is viable to perform reactions without the need for solvent by adsorbing the reagents on to a solid support. I would like to study one of these reactions, using large scale microwave heating. This method would only be adaptable to a batch reactor since it is not possible to pump solids in a flow-through apparatus. Finally, a reaction involving a somewhat hard-to-handle reagent such as sodium azide would add another interesting dimension to this project. Using a substrate which a chemist would not normally consider for scale up would be useful, because I could dilute the reaction mixture, and apply it to flow- through. Clearly, due to potential explosions, this scale up method would not be suitable for a batch reactor.

I am interested in undertaking a pilot project to explore developing methods for microwave promoted enzyme-catalyzed reactions, with the aim of enhancing reaction rates and selectivity, and investigating the viability of a “non-thermal” microwave effect. I am in an excellent position to investigate this because of the versatility of the microwave system in Dr Leadbeater’s lab. An important factor when considering the use of microwave-promotion of enzyme-catalyzed reactions is the control of temperature. Conventionally, microwaves have been used for heating reaction mixtures to high temperatures very rapidly. This is not the optimal condition for enzyme catalysis due to denaturing of the enzymes, and it is therefore important to control the temperature of the reaction mixture. For this reason, I am interested in working with a broad scope of enzymes, which are thermally stable at different temperatures. I will start this project by using simple lipase and esterase reactions. I will compare and contrast the two methods of microwave promotion outlined above, this in itself being a valuable research objective. Screening will take place over a range of different powers, temperatures, solvents, and substrates in order to examine substrate compatibility as well as optimize reaction conditions. I will strive to use water or aqueous/ organic solvent mixtures due to a growing desire for green (environmentally friendly) chemistry in industry. The choice of enzyme will also be important. I plan to use commercially available enzymes, and some of the ones used will be specifically chosen because of their stability at moderate temperatures.

Along with Dr. Leadbeater (chemistry), I will work with Dr. Joseph Crivello (enzymatic analysis), who is one of my advisors for this project from UCONN, as well as Dr. Richard Vachet (from UMASS) who has recent expertise in biological chemistry applied to microwave irradiation.

 

Plan of Study:

For my final 4 semesters at the University of Connecticut, I will be spending time in the lab equivalent to at least taking three credits of independent study per semester, in order to carry out my University Scholar Project. I have already received independent study credits towards graduation, so the times blocked out for this really just signify credits which I could have been eligible for, had I not already had so many research credits on my transcript already. You may notice, that I am a semester behind as a Chemistry major. This is due to the fact that I came into the University of Connecticut declared a PNB major, and starting at here as a freshman, I was told to take Math 112, 113 and 114 (the slower calculus sequence). This set me back a semester because in order to take Math 210 (which I am currently taking) it is necessary to first take Math 116. I completed Math 116 over the summer of 2004. I am also off the classical track for a Chemistry major because I am taking physics in my junior year, instead of my sophomore year, due to priority being given to students who needed to take this class their sophomore year. As a PNB major, it is easy to push this class a year back and take it in the junior year. With an extra semester at UCONN, I will be able to graduate with a dual major in Chemistry and Physiology and Neurobiology, and I can space my classes out a little bit more, fit in some graduate classes which would be useful and interesting to me as well as having more time to devote to the lab. I will take a group 5 General Education over winter break of 2005, and the second group 4 General Education requirement over the winter break of 2006. After spring semester of 2007, I will have completed my General Education requirements. I would list the specific classes, however due to many conflicts that I have experienced in the past with the laboratory sciences, I will take what I am interested in, that happens to fit into my schedule at the time. As a diversion from the sciences, in the past I have enjoyed playing the clarinet and bass clarinet. I have always been interested in music, and my freshman year I had the opportunity to be a part of the Symphonic, and concert bands at the University of Connecticut, and I might be interested in playing in an ensemble in the future.

Classes which I am adding to my schedule, which deviate from the “normal” undergraduate classes that one would take having a chemistry and biology major are Chem 343: Adv. Organic Chem (3) or Chem 341: Organic Reactions (4), Chem 345: Determination of Organic Structure (3),and Chem 270 W: Undergraduate Thesis. I will decide between Chem 343 and 341 based on when these classes are offered, and how scheduling works out with other classes that I will need to take in the fall of 2006. These graduate level classes would further develop my chemistry skills, and general understanding of organic reactions, as well as further solidify what I already know based on previous lab experience. I will write my thesis based on what I have done in the lab, and since I am taking fewer classes during my final semester, I should be able to add additional lab time to repeat things if necessary to finish my thesis. It would be possible to take more graduate level classes, but my main goal is to be able to spend more time in lab, and to be successful in classes which are required for graduation from the University of Connecticut.