Stapled peptides are an application of olefin metathesis first demonstrated by Grubbs and Blackwell1 using o-allyl amino acids, and later improved upon by Verdine and coworkers2 using alpha-methyl substituted olefinic amino acids. The olefinic amino acids are installed at the i, i+4, or i, i+7 positions and a subsequent ring closing metathesis reaction tethers the two positions. We’ve covered this topic in the past, and there have been interesting developments in the field since then.
An appropriately designed stapled peptide can show better cell permeability and better resistance to proteolysis than their untethered counterparts, but deciding where to install the hydrocarbon staple is still relegated to trial and error. A recent paper by Walensky and Neuberg3 tackles the question of cellular uptake in a rigorous and quantitative manner. In order to systematically screen various parameters, such as staple position or point mutations in the peptide chain, a high-content fluorescence imaging and analysis method was utilized. The fluorescently tagged stapled peptides were visualized and quantified for their cellular uptake, and the data subjected to statistical analysis to determine the most important variables. They found that a middling level of helicity, 61-86%, and moderate hydrophobicity, as measured by HPLC retention time, are the key parameters in determining cellular uptake. Just like in the story of Goldilocks, the best peptides have neither not too much, nor too little of helicity and hydrophobicity.
A very interesting point to me is what is not included in this publication. The synthetic methods for preparing the peptides and the subsequent ring closing metathesis are not covered in great detail! As this is a blog on olefin metathesis, I would be remiss not to point readers to details of how this chemistry is performed here.4 It is a testament to the robust nature of the ring closing reaction that preparation of these peptides is routine and allows the authors to focus on biological aspects of this field.
[1] Blackwell, H. E.; Grubbs, R. H. Angew. Chem., Int. Ed. 1998, 37, 3281.
[2] Schafmeister, C. E.; Po, J.; Verdine, G. L. J. Am. Chem. Soc. 2000, 122, 5891.
[3] Bird, G. H.; Mazzola, E.; Opoku-Nsiah, K.; Lammert, M. A.; Godes, M.; Neuberg, D. S.; Walensky, L. D. Nat. Chem. Biol. 2016, 12, 845.
[4] Kim, Y-W.; Grossmann, T. N.; Verdine, G. L. Nature Protocols. 2011, 6, 761.
Leave a Reply