Chemistry
Applications for consideration for Fall undergraduate research funding
1. Abstract
In an effort to produces a novel peptide containing cyclobutane molecule cyclized from combretastatin A-4, methods of peptide attachment to carboxylic acid residues are explored to provide an effective approach for synthesis of the target molecule. Several approaches to forming an amide linkage from a carboxylic acid residue and the primary amine of the peptide are explored including techniques of amide formation between carboxylic acids and amines using SOCl2 and triethylamine (TEA), and commonly used peptide coupling reactions using DCC/DMAP and HBTU are assessed to determine an efficient method of peptide attachment necessary for the formation of our target molecule, a novel combretastatin A-4 analog which will be biologically evaluated for it's ability to bind to the cholchicine site of tubulin , it's water solubility, and cytotoxicity. Current results, including several potentially successful amide formations, are discussed as well as an outline of reactions currently underway and those to come in determining an effective method of peptide attachment on the combretastatin derived cyclobutane.
2. Description of Research.
Combretastatins are naturally occurring molecules originally derived from the African Willow tree (Combretum caffrum), and have demonstrated the ability to cause vascular disruption in tumors. The vascular disruption keeps the cancer tumor from being supplied blood from the body, and it dies. Combretastatin A-4 is the most effective form of the molecule that has been found so far, thus making it a good model for a new potential ant-cancer drug for cancers that involve tumor formation1. There are several critical drawbacks to using combretastatin A-4 for use in human treatment of cancer. These include its chemical stability, toxicity toward health cells, and its insolubility in blood. Structural modifications often attempt to create new drugs that are based on the reactivity of combretastatin A-4, but that attempt to correct it's shortcomings for therapeutic use.
The proposed purpose of my research focuses a new combretastatin A-4 analog that will contain peptide attachments intended to both improve the water solubility of the molecule, and to direct the drug specifically to the cancer tumor while avoiding healthy cells. The initial body of the research will focus on performing different well established peptide coupling reactions to find a sufficient method of attachment needed to create the new molecule. After an appropriate method of attachment is determined the focus of the work will shift on developing the new combretastatin A-4 analogs, to which these peptides will be attached. A variety of molecules will be developed that differ in the peptides that they have attached. The new molecules will be evaluated on their effect on cultures of cancer cells versus cultures of healthy cells, as well as their ability to dissolve in water, since blood is a water based environment.
2.1 Technical details
Peptide coupling is usually carried out in the laboratory by first activating the carboxyl group of an N-protected amino acid, which increases the rate of reaction, and then introducing it to an unprotected amino acid to which coupling is desired2. The two classes of activating reagents commonly used are carbodiimides and triazolols. The most common carbodiimides are dicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIC). DCC is a waxy solid at room temperature and can be difficult to work with, so DIC was developed as an easier to work with alternative. A lesser used carbodiimide that is water soluble is ethyl-(N’,N’-dimethylamino)propylcarbodiimide hydrochloride (EDC), which is useful for reactions that modify proteins4. A common problem encountered with using carbodiimides is that they are so reactive that racemization of the product is often a problem. Triazols were developed as a less reactive alternative to carbodiimides, and allow much better control over stereochemistry. The most common triazoles currently used are 1-hydroxy-benzotriazole (HOBt) and 1-hydroxy-7-aza-benzotriazole (HOAt)4. These substances form an active ester which is much less reactive and greatly reduces racemization, allowing much better control over the final structure of the synthesized product. The above mentioned procedures will be performed and compared on the basis of their ability to form the desired product. The more successful of the procedures will be carried on to attach peptides to the cyclobutane containing combretastatin A-4 analog.
1. Keira Gaukroger, John A. Hadfield, Lucy A. Hepworth, Nicholas J. Lawrence, and Alan T. McGown, Novel Syntheses of Cis and Trans Isomers of Combretastatin A-4,” Journal of Organic Chemistry 66 (November 2001): 8135-8138
2. Bogdan Falkiewicz, “Comparison of the efficiency of various coupling systems in the acylation of model secondary amines with thymin-1-ylacetic acid,” Nucleic Acids Symposium Series 42 (1999): 153-154
3. Rosa Ortuno, Albertina Moglioni, and Graciela Moltrasio, “Cyclobutane biomolecules: Synthetic approaches to amino acids, peptides and nucleosides” Current Organic Chemistry 9 (2005): 237
4. Toni Brown, Hilary Machay, Mark Turlington, Arden Sutterfield, and Traci Smith, ”Modifying the N-terminus of poyamides: PyImPyIm has improved sequence specificity over f-ImPyIm,” Bioorganic & Medicinal Chemistry 16 (2008): 5266
3. Time Period. The work will take place from June 2010 to May 2011
4. Budget with Justification.
Funding for the project will be used specifically for chemical reagents needed to perform coupling reactions, as well as peptides that will be used for attachment in the reactions.
DCC (Dicyclohexylcarbodiimide) $34.53
DMAP (4-Dimethylaminopyridine) $30.00
HBTU (2-(1H-Benzotriazole-1-yl)-1 1 3 3-tetramethyluronium) $33.00
DIPEA (N,N-Diisopropylethylamine): $27.80
EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) $81.27
HoBT $168.00
L-Aspartic acid dimethyl ester hydrochloride (peptide) $208.00
FMOC-N-trityl-L-Asparagine (Peptide) $66.00
L(+)-Glutamic Acid Monosodium Salt Monohydrate 99% $98.00
L-Tyrosine (peptide) $75.00
L-Methionine $67.00
5. Are you seeking additional funding from other sources? Not at this time
6. Publication outlet.
The results will be published in the University of North Carolina at Asheville Journal of undergraduate research, and will be presented to the community at both the Fall and Spring undergraduate research symposium. The most recent updates on the work will be presented at symposium.
7. Human Subject Form. No human subjects will be used for this research.