Chemical Protein Synthesis
We seek to expand the capabilities of chemical protein synthesis through development of new chemical methods for protein synthesis and ligation.
Solid Phase Peptide Synthesis enables the routine synthesis of 50-amino-acid peptide fragments through coupling and deprotection cycles, with no intermediate purifications. Modern methodologies in development expand these capabilities up toward 100 or sometimes even 150 amino acids, but for chemical synthesis of large proteins it is still necessary to develop methods for the ligation of smaller peptide fragments into a complete protein product. Native chemical ligation (NCL) has been the premier methodology for combining unprotected peptide fragments to intact proteins since I developed it as a graduate student in the lab of Steve Kent (Science 1994). Since then my group has been at the forefront of making NCL accessible to more researchers through development in increasingly synthetically simple methods. This has ranged from development of among the earliest and most widely used methods for accessing thioesters through Fmoc chemistry (Angewandte 2008, JACS 2015), to our most recent work on an extremely mild in-situ activation that enables multiple sequential ligations in one pot without intermediate purification (Angewandte 2018).
Currently students in my lab are working to expand these methodologies so as to:
- Enable fragment amide coupling from peptide-pyrazole precursors
- Develop methodologies for performing native chemical ligation on the solid phase
- Expand the capabilities of native chemical ligation to oxygen nucleophiles (Serine and Threonine)
Bioconjugation
We seek to expand the scope and versatility of bioconjugation methodologies through development of classic organic chemistry for protein applications in-vitro and in-vivo.
Bioconjugation methodologies seek to enable site-selective incorporation of various useful chemical moieties onto biological molecules, ranging from small peptides all the way to cell-surface proteins and chromosomal DNA. Our work in this area has sought to leverage our knowledge of classic synthetic organic methods and physical chemistry to develop reactions that are fast, operationally simple, and broadly applicable. This is perhaps best emphasized by our work on nucleophilic catalysis of oxime and hydrazone ligations (Angewandte 2006, Bioconj. Chem. 2008, Bioconj. Chem. 2013), which applied simple physical chemistry insights to greatly increase reaction rates. This has allowed use to use oxime and hydrazone ligations in many diverse applications, from display of peptides and glycans on quantum dots (ACS Nano 2010, JACS 2010, Bioconj. Chem. 2018), to assembly of multifunctional viral nanoparticles (Nano Lett. 2010), and even to labeling of the surface of live cells (Nat. Methods 2009). In recent years we have developed Glaser’s 19th century copper chemistry into a modern methodology for protein conjugation and peptide stapling with a rigid diyne linker (Angewandte 2017, Chembiochem 2018). Additionally, we have utilized selenomethionine, a naturally occurring and easily expressible amino acid, for selective reversible or irreversible bioconjugation to the interior and N- and C-termini of proteins independently (Manuscript in preparation).
Currently students in my lab are further developing and utilizing these methods to:
- Develop potent antimicrobial peptides efficacious against drug resistant bacteria
- Design protease inhibitors by using rigid peptide staples to enforce strict backbone conformation
- Perform selctive selenomethionine conjugations to expressed proteins
Reversible Adsorption to Solid Support (RASS)
We seek to leverage our expertise on the physical chemical properties of the interactions between biomolecules and macro/mesoscale surfaces facilitate and enable complex synthetic reactions on biopolymers.
Purification of synthetic peptides has long relied on HPLC/LC-MS technologies, in which a non-polar solid phase (C18 Silica) is used to bind the peptide, while a mobile phase of water/acetonitrile mixtures elute the peptide. What is less often discussed are the underlying physical properties, that rely on the multivalent binding kinetics of biomacromolecules, allow for robust separation of highly similar peptide mixtures. Peptides, as well as DNA and RNA, are biopolymers with a backbone of repeating structure, which allows them to interact with a solid support at multiple positions along their length. This means that for a peptide to move down the column most or all the contacts must be broken in favor of solvation, causing peptides to be almost completely static in solvent mixtures well below the elution condition. Small molecules, by contrast, will slowly travel down a column under isocratic conditions. Moving out of the HPLC setting, this means we can bind a peptide to a sorbent, such as C18 silica, perform reactions under various conditions, and wash the small molecules away while leaving the peptide bound. In our lab we call this method RASS, for Reversible Adsorption to Solid Support, and we initially developed it for synthesis of click-chemistry-based peptide libraries (ACS Comb. Sci. 2016). This application was based on performing sequential aqueous reactions without intermediate purification, but we have since gone on to develop the method so as to allow for organic-phase reactions, which would be difficult or intractable under standard handling conditions (Angewandte 2018). This technology has also opened up new fields of study for our group, enabling us to develop revolutionary new methods for the creation of DNA-Encoded Libraries with unprecedented scope of accessible reactions and scaffolds (JACS 2019).
Currently students in my lab are using this technology to:
- Develop an ever-expanding scope of reactions for use in DNA-Encoded Libraries
- Enable site-specific chemical modification of native DNA
- Perform a diverse array of chemical modifications on peptides and proteins
Understanding biomolecules through chemistry. 