Cyclization and Derivatization of the Potasium Channel Antagonist Tertiapin

Abstract

The potent potassium channel antagonist tertiapin-Q was used in order to expand the application of data previously published by Clark et al. and also to further examine the usefulness of a newly published peptide backbone cyclization technique. This peptide was also used in order to investigate the possibility of creating a cyclic, fluorescent peptide probe for the ROMK1 channel, which builds upon the thesis work of previous students in the Bingham laboratory. The data published by Clark et al. describes a correlation between N-to-C termini distance and required linker length for a cyclic peptide. To date, this relationship has not been used to cyclize a peptide toxin that did not originate from Conus spp., therefore using it to cyclize the peptide toxin tertiapin-Q represents a unique challenge to these data. After using solid phase peptide synthesis techniques to synthesize 3 length variants of tertiapin-Q, only the variant with the predicted optimal linker length was able to cyclize successfully, thereby indicating that the previously published data does apply to non-Conus toxins and can therefore be reliably used to cyclize toxins from other organisms, which could lead to new and exciting drug leads. The newly published Fmoc-based cyclization technique represents a safer and more accessible alternative to cyclizing peptides than traditional native chemical ligation since it does not require the use of highly corrosive HF or TFMSA, the former of which necessitates the use of specialty Teflon™ coated lab apparatus. Using this technique to successful cyclize tertiapin-Q during this project represented the first non-Conus toxin to be cyclized in this manner, thereby expanding the application of this relatively untested protocol. With this knowledge, future researchers can utilize this technique, in conjunction with the data published by Clark et al., in order to cyclize any given peptide sequence. Lastly, building upon the work of previous students, conjugating a fluorophore to a cyclic derivative of tertiapin-Q would allow it to be used a fluorescent probe which could be used to trace the distribution and investigate the mechanisms of the molecular targets of this peptide, namely ROMK1 and GIRK1/4. Using the Staudinger reaction to conjugate a DyLight 650 fluorophore to the peptide resulted in poor yields (approximately 3%) of the desired product. This could indicate that the reactive azide moiety is sterically hindered by the surrounding charged/bulky amino acid side-chains, which suggests that the inclusion of an extra carbon chain would negate this effect, allowing the reaction to proceed to completion. If this is coupled with an increase in the ratio of peptide to fluorophore, then the reaction would be almost guaranteed to proceed with little to no trouble.