Tieing Up Bad DNA:

Formation and Reaction of Ene-Diyne Molecules

Dr. Thomas M. Mitzel

Department of Chemistry

The discovery that natural products containing ene-diyne reactive centers inhibit growth of mutated DNA strands, retarding the development of cancer cells and tumors, has prompted much excitement in the scientific community.. Although this research appears promising, it is presently slowed by difficult synthetic routes and low yields of target ene-diyne structures.

This proposal addresses the use of novel synthetic routes which will alleviate this impediment. Use of water as a solvent in the formation of ene-diynes will introduce an environmentally benign methodology allowing for rapid advances in a very stimulating, important, yet untapped area of chemistry.

1.0 Introduction

Imagine being able to determine the full blueprint of an entire building based on information contained within one brick located in the foundation. As incredible as this may seem, nature does just that within biological systems. Deoxyribonucleic Acid (DNA), discovered in cell nuclei in 1868 by Fritz Meischer a Swiss Physicist, and given its modern structure in 1953 by Watson and Crick (who were subsequently awarded the Nobel prize in 1962), is involved in storage and transmission of genetic information.1 Everything about us including our hair color, body type, musical talent (or lack thereof), etc. is contained within this one molecule. Our "blueprint" or genetic code, is built into us at birth, and carried in our DNA.

DNA replicates at each cell division. As such, every cell contains information about the entire organism. The process for DNA replication is shown in Scheme 1. DNA is a two stranded molecule in a

Scheme 1: DNA Replication Within Our Cells

helix shape made up of millions of base fragments. In solution (our bodies are 75% water), DNA "unwinds" to give single strands, which bond with free bases in solution, to form complimentary strands, thereby reproducing another DNA molecule. A single DNA chain may be up to 12 centimeters in length and contain 250 million base pairs.1 Despite the enormous size of these molecules, the replication sequence is carried out in relative harmony with errors occurring only about once in every 10-100 billion base bonding interactions.2

Although errors do not occur often in a numerical sense, any error may interrupt the genetic code. An incorrect transmission of genetic information by our DNA in replication of these 250 million or so bases may lead to a mutation. Many mutations are harmless, and our "blueprint" is not affected by them, however, some mutations may lead to uncontrolled replication.2 In most DNA sequences, our body knows when replication should be controlled, but when mutations occur, the body is overridden, and replication does not stop. Uncontrolled replication of DNA, and cells, leads to growth of malignant tumors, and cancer.

2.0 Ene-Diyne Molecules

In an effort to fight this uncontrolled growth, chemists have been trying to find molecules to arrest formation of the malignant tumors. These molecules are known as antitumor antibiotics.3 A type of antibiotics known as ene-diynes have shown to arrest formation of malignant tumors by "tieing" the two strands of DNA together.3,4 By tieing the DNA strands together, ene-diynes prevent them from unraveling, arresting the replication process.3 One of the ene-diynes, Dynemicin A (Figure 1), has been particularly effective. As shown in Figure 1, the ene-diyne portion of this molecule closes to form a cyclic structure. This cyclic structure has two radical two radical centers (Figure 1). Radical centers have only 1/2 the number of electrons they should, and are extremely reactive due to this phenomenon. These radical

Figure 1: Structure of Dynemicin with Ene-Diyne Portion of Molecule Shown

centers interact with DNA , forming labile centers, which then form a covalent bond (ties itself together) across the two strands, rendering it unable to unravel or replicate (Scheme 2). Mutated, or cancerous cells replicate faster than normal cells, so ene-diynes will have more of an affect on the mutated DNA,

Scheme 2: Ene-Diyne Method of Tieing DNA Strands Together

helping to prevent the spread of tumorous tissue. The major problem, however, is that Dynemicin A, and other ene-diynes, also react with healthy DNA, stopping all replication processes. As a result, these potential antibiotics are presently too toxic for widespread use in cancer therapy.

3.0 Current Work

Many drugs are made more potent, or more safe, by altering their structure in such a manner that they are attracted only to certain portions of the biological system. In reference to Dynemicin A, the reactive ene-diyne portion of the molecule is quite small in relation to the overall size of the compound. It should be possible to alter the skeletal structure of ene-diyne moieties, tailoring them to be attracted more toward mutated DNA structures than to healthy DNA molecules. Research toward this goal has been noted in several groups.5 In these studies, authors have designed an ene-diyne with differing skeletal structures constructed around the reactive portion of the molecule. Although some of these studies have shown nominal success, research is limited by lengthy syntheses of these ene-diyne molecules, and harsh conditions during the construction of the skeletal structures. Altered ene-diynes did show more propensity to interact with mutated DNA than with healthy DNA, but yields of these molecules were low and widespread testing was unable to be undertaken. What is needed is a more simple, environmentally benign method leading to construction of these ene-diyne moieties, allowing more thorough research into honing the structures for targeting mutated DNA and cancerous cells.

4.0 Proposed Work

Previous work in this lab has focused on formation of carbon-carbon bonds in an aqueous solvent using indium metal (Scheme 3).6 The advantage of this work over previous literature is that these reactions can be controlled with regard to spatial orientation of skeletal structures, and the reaction conditions are

Scheme 3: Coupling Reactions Using Indium Metal

very mild and environmentally friendly, using water as a solvent in lieu of harsh organic solvents. The success of our early work revealed that this new chemistry has the possibility of being tailored to allow formation of ene-diyne moieties as well. I propose to utilize this new, exciting, environmentally benign chemistry from our lab to form ene-diyne structures in order to elucidate a good skeletal structure for use in targeting mutated DNA to halt the replication of cancerous tumors.

Scheme 4: Simple System to Test Chemistry

Early work on this project would focus on the simple system shown in Scheme 4. This system would form a very simple "ene-diyne" structure as shown and is important for a number of reasons: 1) It would be the first ene-diyne formed under aqueous conditions. Since ene-diynes must work inside the biological system, which is 75% water, the formation of these molecules under similar conditions would give some insight into their biological stability. 2) The system is simple enough to allow conditions to be changed and perfected quickly for more elaborate tests to come. Many of the previous systems attempted by other authors have not been successful due to the long syntheses required to form the ene-diyne target molecule. The overall amount of material isolated in these previous studies has been low with little material isolated, reducing the number of tests that can be investigated.4,5 The system shown in Scheme 4 is only two steps in length. It will be quick to set up, and easy to institute any needed changes quickly, allowing us to gain access to larger amounts of material. 3) This molecule allows more than one method in formation of the ene-diyne. Nature is quite complex. It may be necessary to have more than one precursor that will allow cyclization of an ene-diyne. As Scheme 5 reveals, this system is flexible enough to allow access to several cyclization precursors. The chemistry needed to form each of the diradical molecules is simple and straight forward. This will allow the chemistry to be fine-tuned allowing for maximum yield of products. After this

Scheme 5: System Allows for Synthesis of Several Possible Cyclization Precursors

first portion of the project has been completed, and the best possible system of all those tested has been found, the work can be expanded to include construction of a better skeletal system surrounding the ene-diyne moiety. As Scheme 6 reveals, the ene-diyne may be "tethered" and the skeletal structure perfected.

Scheme 6: Building the Skeletal Structure for Fine Tuning of this System

By customizing the skeletal structure it will be possible to find an ene-diyne with a skeletal structure containing the correct shape allowing for targeting of mutated DNA molecules while bypassing healthy DNA structures.

5.0. Impact of Proposed Work and Future Goals

The ramifications of this research should lead to a breakthrough in how chemists view the formation of ene-diynes and how they interact with DNA. The use of water as a solvent could revolutionize industrial techniques from the use of traditional organic solvents to a more productive water solvent.

Work begun on this project with Trinity funds will help lay the groundwork needed to solicit outside funds (The National Science Foundation, National Institutes of Health, and others) that will allow the project to expand beyond the goals stated into larger and more complex systems allowing for specific targeting of DNA chains with very high accuracy. Future work would also include the use of bismuth metal which has been shown in our labs to mimic the reactivity of indium metal for ease of formation of carbon-carbon bonds, and thus, ene-diyne complexes.

All of the proposed research will incorporate the use of Trinity Undergraduate Students. The project would begin with one student during the summer of 2000 with more students being added to the project during the fall of 2000 once the preliminary results have been completed. The experience garnered in this type of atmosphere is irreplaceable and will prepare these students for their chemical and/or biochemical careers after they graduate from Trinity College.

6.0 References

1) Fox, M. A.; Whitesell, J. K.; Organic Chemistry, 2nd Edition, Jones and Bartlett Publishers, Boston, 1997, Chapter 19.

2) Frank-Kamenetskii, M. D. Unraveling DNA, VCH Publishers, 1993.

3) For a nice review of this material: Nicolaou, K. C.: Dai, W.-M. Angew. Chem. Int. Ed. Engl. 1991, 30 1387.

4) a) Myers, A. G.; Dragovich, P. S.; Kuo, E. Y. J. Am. Chem. Soc., 1992, 114, 9369. b) Lee, M. D.; Dunne, T. S.; Chang, C. C.; Ellestad, G. A. Siegel, M. M.; Morton, G. O.; McGahren, W. J.; Borders, D. B. J. Am. Chem. Soc. 1987, 109, 3466. c) Golik, J.; Dubay, G.; Groenewold, G.; Dawaguchi, H.; Konishi, M.; Krishnan, B.; Ohkuma, H.; Saitoh, K.; Doyle, T. W. J. Am. Chem. Soc., 1987, 109, 3462. d) Konishi, M.; Ohkuma, H.; Tsuno, T.; Oki, T.; VanDuyne, G.D.; Clardy, J. J. Am. Chem. Soc. 1900, 112 3715.

5) a) Nantz, M.H.; Spence, J.D.; Mass, D.K. J. Org. Chem. 1999, 64 4339. b) Dai, W.M.; Wu, J.; Fong, K.C.; Lee, M.Y.H.; Lau, C.W. J. Org. Chem. 1999, 64, 5062. c) Saalfrank, J. W. J. Org. Chem. 1999, 64, 6166.

6) Mitzel, T.M.; Engstrom, G.; Morelli, M.; Palomo, C. Tetrahedron Lett., 1999, 40, 5967.