# Development and Applications of Bioorthogonal Chemistry

> **NIH NIH R35** · STATE UNIVERSITY OF NEW YORK AT BUFFALO · 2021 · $396,169

## Abstract

Development and Applications of Bioorthogonal Chemistry
ABSTRACT
This MIRA application combines two research efforts in our lab in tackling the critical barriers in the study of
class B GPCR biophysics and signaling in live cells, i.e., the lack of suitable tools for constructing functional
GPCR biosensors as well as capturing the transient and highly dynamic GPCR-interacting proteins involved in
biased agonism. We have a long-standing interest in developing the reactivity-based chemical tools to address
significant biological problems that are difficult to solve using conventional molecular biology techniques. In the
past five years we continued to make progress in both tool development and the applications of these tools to
address important biological problems. For instance, we optimized several bioorthogonal reactions, including
the photoinduced tetrazolealkene cycloaddition reaction (‘photoclick’ chemistry), the spiroalkenetetrazine
ligation reaction, the palladium-mediated cross-coupling reactions, and the sequence-specific 2-cyanobenzo-
thiazolecysteine ligation reaction. Together with the genetic code expansion involving a spiroalkene amino
acid, two of these reactions (photoclick chemistry and tetrazine ligation) were harnessed for site-specific
introduction of organic fluorophore (fluorescein, Cy3 and Cy5) at the extracellular loop 3 of GLP-1R and GCGR,
two members of the class B GPCRs implicated in diabetes and obesity, for an ongoing single-cell FRET study
of the domain movement during ligand-induced receptor activation in live cells. In addition, we made a
serendipitous discovery that 2-aryl-5-carboxytetrazole (ACT) offers a new proximity-dependent photo-cross-
linker, which was then used in the design of the photo-affinity labels that enabled in situ capture and
subsequent identification of the drug targets as well as a genetically encoded amino acid for site-specific
incorporation and subsequent capture of the transient EGFRGrb2 interaction complex in mammalian cells.
Built upon these results, in this application we plan to continue our studies of orthogonal chemical reactivity at
the chemistry-biology interface and pursue the following two related projects. In Project 1, we will construct the
FRET-based biosensors of GLP-1R and GCGR via bioorthogonal labeling to probe the conformational
dynamics involved in the receptor activation and signaling in live cells. A new set of fluorescence ‘turn-on’
reagents will be designed for bioorthogonal, fluorescent labeling of the intracellular loop 3 (ICL3) of GLP-1R
and GCGR to allow single-cell intra- and intermolecular FRET analysis of receptor conformations in live cells.
In Project 2, we will develop a genetically encoded ACT photo-cross-linker containing an alkyne group and
apply this photo-cross-linker to map the time-dependent GLP-1R and -arrestins interactomes by mass
spectrometry in response to ligand stimulation. We expect that these studies will not only validate new
chemical t...

## Key facts

- **NIH application ID:** 10071168
- **Project number:** 5R35GM130307-03
- **Recipient organization:** STATE UNIVERSITY OF NEW YORK AT BUFFALO
- **Principal Investigator:** Qing Lin
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $396,169
- **Award type:** 5
- **Project period:** 2019-01-01 → 2023-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10071168

## Citation

> US National Institutes of Health, RePORTER application 10071168, Development and Applications of Bioorthogonal Chemistry (5R35GM130307-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10071168. Licensed CC0.

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