Transient Intermediates by Photodissociation and UV-Vis Action Spectroscopy

With support from the Chemical Mechanism, Function and Properties Program, Professor Frantisek Turecek at University of Washington will develop new reactions for chemical crosslinking in biomolecular ions representing components of proteins and nucleic acids, establish product structures, and study reaction mechanisms. Crosslinking is a process that has been used in industry to modify the properties of various commodity polymer materials such as tire rubber, polystyrene, and epoxy adhesives.

Its implementation in biomolecular structure studies relies on highly reactive intermediates that are produced by laser radiation and react rapidly by forming chemical bonds with other parts of the molecule or another molecule in a complex. The Frantisek research group has been developing chemical reactions to be used in crosslinking in peptide conjugates that are charged and isolated in the gas phase.

Under these conditions, reaction products and mechanisms can be studied free of interferences from the surrounding environment. Exploration of new types of organic reactions is important as it may have a broader impact in biochemistry and biology to provide new tools for molecular structure determination and advance scientific knowledge to foster science literacy.

The features of the proposed research, combining advanced experimental methods with quantum chemistry calculations, will contribute to the education of undergraduate and graduate students and a post-doctoral associate, including those from underrepresented groups, while also providing outreach to high-school students for research internships in the PI’s laboratories. Development of a diverse and globally competitive STEM workforce is a major goal.

In addition to the standard research training, the students and postdoctoral associates will continue having frequent opportunities to present their results at scientific conferences nationally and internationally. International collaborations and short exchange stays will foster culture immersion and professional growth of the participating students and trainees.

The research to be developed concerns four aims which are focused on the generation and properties of peptide and oligonucleotide conjugates of nitrile imines, exploring newly discovered crosslinking reactions of the involved functional groups. In the first aim, the PI and his team will explore methods for the synthesis of conjugates in which peptides and nucleotides are tagged with 2,5-diaryltetrazoles as nitrile imine precursors.

This research will provide access to stable precursors of reactive intermediates for nitrile-imine based covalent crosslinking. In the second aim, the PI and his team will carry out conformational analysis of the photo-tagged peptide ions and photodissociation products. The PI and collaborators will use cyclic ion mobility mass spectrometry to separate precursor ion conformers and product isomers and correlate their collision cross sections with those of low Gibbs energy isomers generated by Born-Oppenheimer molecular dynamics (BOMD) and density functional theory (DFT) calculations.

Progress in this project will provide the PI with a comprehensive view and understanding of the precursor conjugate stereochemistry. The third aim is focused on the combination of ultraviolet-visible (UV-VIS) action spectroscopy, time-dependent-DFT calculations, cyclic ion mobility mass spectrometry (c-IMS), and calculations of theoretical collision cross sections to aid identification of the photoproducts.

The results will help answer the critical question of transferring the efficient gas-phase crosslinking to the condensed phase for explorations of biomolecular structures with applications in structural biology. Finally, conjugates will be constructed in which 2,5-diaryltetrazoles and peptides are mounted on cyclic scaffolds to control steric access of the reactive nitrile-imine intermediate to the target moiety.

These synthetic peptide scaffolds will provide fine steric control of nitrile-imine-amide and nitrile-imine-nucleobase interactions leading to covalent bond formation and crosslinking. The study is aimed at elucidating the electronic and structural factors that play a role in nitrile-imine crosslinking to complex biomolecules.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.