Jad Mills and Dr. Sandra Burnett, Molecular Biology and Microbiology
The synthetic dimerizer AP20187 initiates targeted dimerization of mutant FK506 binding proteins (FKBP) to study cell functions, cell signaling pathways, and protein functions. In Macrophage Fas-Induced Apoptosis (Mafia) transgenic mice, AP20187 is used to initiate Macrophage apoptosis . To determine the mechanism behind some depletion variability in Mafia mice, AP20187 was characterized and degradation was monitored using hydrogen and carbon nuclear magnetic resonance (1H- and 13C-NMR), high performance liquid chromatography (HPLC), electro spray mass spectrometry (ESMS), polarimetry, and spectrophotometry.
The variety of cell signalling pathways and protein interactions elucidated by the AP20187 CID model and described in the Literature Review of this thesis confirm that the model is important to the future of microbiology and biochemistry research. The chemical analyses conducted on AP20187 in this thesis have increased our understanding of some of the physical characteristics of the compound, and enhanced our ability to use it.
The absorbance studies showed that AP20187 concentration can be tracked using spectrophotometry by following the λmax of 294 nm and using the standardization equations (Eq. 1 and Eq. 2) to convert absorbance to dimerizer concentration. Future research may study whether AP20187 concentration changes during depletion studies may impact depletion results. The Polarimetry study obtained an optical rotation of 22.4°. This confirmed that the AP20187 dimerizer is chiral and not a racemic mixture, and provides a basis for enantiomeric separation studies should enantiomers be determined to be the cause of depletion variability.
NMR studies confirm that the AP20187 comprises a structure consistent with the expected structure. Although one structural change was registered after one week, other studies failed to register structural changes after even six months of storage. Because of this lack of repeatability and the large dimerizer concentrations required for NMR analysis, NMR proved to be valuable for evaluating the structure of the compound but ineffective for monitoring structural degradation over time.
ESMS analysis showed that AP20187 is liable to spontaneous degradation when stored in the dark at -20° C. as it is during depletion experiments. Whereas fresh AP20187 showed only one peak at the expected 1482.76 AMU, after six months impurities were detected in the sample, and the intensity of the peak at 1482.8 AMU had decreased from 7.37% of the material being eluted to 0.03%.
The degradation effects of various amounts of HCl, NaOH, heat, and UV light were effectively monitored using ESMS. One treatment caused a 92.83% decrease in the proportion of AP20187 per total counts over the course of 35 days. This treatment was repeated for a larger sample of AP20187, resulting in an 88.11% decrease in proportion of AP20187 per total counts over the course of 8 days. This sample of degraded AP20187 achieved depletion results similar to mock treatment, providing an activity confirmation of the ESMS analysis that the AP20187 was degraded.
With the combination of academic and experimental research contained in this thesis I have determine that AP20187 is liable to spontaneous structural degradation in standard storage conditions over a period of six months, that this degradation results in an inability of AP20187 to initiate dimerization, and that this degradation may be effectively monitored using ESMS. I have further characterized AP20187 by providing 1H- and 13C- NMR spectra, a λmax and standardization equations, and an optical rotation. This has led to a better understanding of the AP20187 dimerizer, and provided an effective means for characterizing it and similar compounds. This thesis explored the stability of the compound by subjecting AP20187 to extremes in temperature and pH and thereby inducing degradation.
Future studies may explore the effects of dimerizer concentration changes during depletion experiments or may attempt to isolate impurities in AP20187 and determine whether or not some of them represent AP20187 enantiomers or other isomers. Because depletion experiments generally take place within one week of solubilization of the dimerizer, structural degradation is most likely not the cause of observed depletion variable. Future studies may with to focus attention on whether the variability may be caused by inconsistent transgene expression in the Mafia mice.
Because of the support of the ORCA grant, I have been able to complete this research in time to submit it as an BYU undergraduate Honors Thesis. I have been studying AP20187 since Spring term 2006, and this Thesis is a culmination of 1.5 years of research. The ORCA grant allowed me to have the time and means to complete this ambitious project. With the grant I was able to pay for the lab expenses for the depletion experiment, and spend the necessary time to finish the sometimes laborious process of conducting the necessary experiments, interpreting the data, and writing-up the results. The Thesis has already been submitted to my advisor for review and will be defended in February 2008. After the defense the final Thesis will be published by the BYU Honors Department with copies placed with the Chemistry Department and with Harold B. Lee Library.