Development and Applications of Photoinducible Bioorthogonal Chemistry ABSTRACT Bioorthogonal chemistry has emerged as a powerful tool in probing biomolecular structure and function in living systems. Combining with recent developments in introducing novel chemical reactivity into biomolecules site- selectively in vivo, bioorthogonal chemistry offers an unprecedented opportunity to monitor and expand biomolecular function in living systems. Our long term goal is to develop a toolbox of photoinducible bioorthogonal reactions and apply them to study protein function in living systems. The bioorthogonal reactions we are developing build from our chemical insights into unusual heterocycles which are thermodynamically stable, and yet undergo rapid photoinduced ring openings to generate the highly reactive intermediates. These intermediates then react selectively with their cognate, externally introduced partners in living systems. In the Preliminary Studies, we show the first photoinducible bioorthogonal reaction between diaryltetrazoles and alkenes, and its application in the site-specific modification of proteins both in biological buffer and in living E. coli cells. In this project, we propose to significantly expand the scope and the utility of this reaction toolbox by: 1) identifying tetrazoles with enhanced reactivity toward unactivated alkenes;2) developing a photoinducible diarylazirine-based bioorthogonal reaction;3) developing a general strategy for functionalizing newly synthesized proteins in living cells;and 4) probing protein posttranslational modifications such as lipidation and phosphorylation in living cells. We hope these new developments will enable functional study of proteins in vivo with exquisite specificity at the molecular level and operational simplicity at the system level.
Our specific aims are the follows: (1) To optimize the reactivity of tetrazoles and develop a diarylazirine- based photoinducible bioorthogonal reaction. Substituent effect based on a """"""""push-pull"""""""" hypothesis will be explored to achieve the selective and enhanced reactivity toward unactivated alkenes. (2) To develop a general strategy for labeling newly synthesized proteins in mammalian cells through co-translational alkene incorporation followed by selective functionalization with the tetrazole-based chemistry. Experiments are proposed to examine the co-translational activities of several activated alkene amino acids and their subsequent functionalization by the tetrazole compounds. (3) To apply the tetrazole-based bioorthogonal chemistry to model Ras lipidation in living cells and probe the role of lipid structures on Ras membrane targeting dynamics, specificity, and function. Both the intein-mediated chemical ligation and the amber codon suppression methods will be employed in constructing the tetrazole-encoded N-Ras mutant for this study. (4) To apply the tetrazole-based bioorthogonal chemistry to mimic STAT-1 tyrosine phosphorylation by incorporating a tetrazole amino acid at the tyrosine phosphorylation site (Tyr-701) using both native chemical ligation and amber codon suppression techniques. We will examine the effect of chemical phosphorylation on the engineered STAT-1 dimerization, nuclear transport, and transcriptional activation in living cells.

Public Health Relevance

The development of chemical tools for the study of complex and dynamic biological problems represents a central challenge in chemical biology. As a new class of chemical tools, the bioorthogonal reactions have significantly advanced our understanding of biomolecular structure, function, and dynamics in living systems, however, various limitations of currently available bioorthogonal reactions prevent their wider applications in the biomedical research. This proposal addresses the development of a class of photoinducible bioorthogonal reactions with many desirable reaction attributes, and their applications in functionalizing newly synthesized proteins as well as the study of the dynamics of protein posttranslational modifications such as lipidation and phosphorylation in living cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM085092-05
Application #
8460102
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Fabian, Miles
Project Start
2009-04-01
Project End
2015-03-31
Budget Start
2013-04-01
Budget End
2015-03-31
Support Year
5
Fiscal Year
2013
Total Cost
$283,879
Indirect Cost
$104,178
Name
State University of New York at Buffalo
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
038633251
City
Buffalo
State
NY
Country
United States
Zip Code
14260
An, Peng; Lin, Qing (2018) Sterically shielded tetrazoles for a fluorogenic photoclick reaction: tuning cycloaddition rate and product fluorescence. Org Biomol Chem 16:5241-5244
Tian, Yulin; Lin, Qing (2018) Genetic encoding of 2-aryl-5-carboxytetrazole-based protein photo-cross-linkers. Chem Commun (Camb) 54:4449-4452
An, Peng; Lewandowski, Tracey M; Erbay, Tu?çe G et al. (2018) Sterically Shielded, Stabilized Nitrile Imine for Rapid Bioorthogonal Protein Labeling in Live Cells. J Am Chem Soc 140:4860-4868
An, Peng; Wu, Hsuan-Yi; Lewandowski, Tracey M et al. (2018) Hydrophilic azaspiroalkenes as robust bioorthogonal reporters. Chem Commun (Camb) 54:14005-14008
An, Peng; Lewandowski, Tracey M; Lin, Qing (2018) Design and Synthesis of a BODIPY-Tetrazole Based ""Off-On"" in-Cell Fluorescence Reporter of Hydrogen Peroxide. Chembiochem 19:1326-1333
Antoine John, Alford; Lin, Qing (2017) Synthesis of Azobenzenes Using N-Chlorosuccinimide and 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU). J Org Chem 82:9873-9876
Tian, Yulin; Jacinto, Marco Paolo; Zeng, Yu et al. (2017) Genetically Encoded 2-Aryl-5-carboxytetrazoles for Site-Selective Protein Photo-Cross-Linking. J Am Chem Soc 139:6078-6081
Ramil, Carlo P; Dong, Maoqing; An, Peng et al. (2017) Spirohexene-Tetrazine Ligation Enables Bioorthogonal Labeling of Class B G Protein-Coupled Receptors in Live Cells. J Am Chem Soc 139:13376-13386
Herner, András; Marjanovic, Jasmina; Lewandowski, Tracey M et al. (2016) 2-Aryl-5-carboxytetrazole as a New Photoaffinity Label for Drug Target Identification. J Am Chem Soc 138:14609-14615
Ramil, Carlo P; An, Peng; Yu, Zhipeng et al. (2016) Sequence-Specific 2-Cyanobenzothiazole Ligation. J Am Chem Soc 138:5499-502

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