# Development and Applications of Bioorthogonal Chemistry

> **NIH NIH R35** · STATE UNIVERSITY OF NEW YORK AT BUFFALO · 2024 · $439,679

## Abstract

Development and Applications of Bioorthogonal Chemistry
ABSTRACT
This MIRA renewal application combines three ongoing research projects in the PI’s lab to further develop
bioorthogonal covalent chemistry to tackle significant biophysical, biochemical, and in vivo imaging problems in
biomedical research. We have a long-standing interest in developing reactivity-based chemical tools to
address biomedical problems that are difficult to solve using conventional molecular biology techniques alone.
In the last five years, we continued our progress in tool development and applications of these tools to study
class B G protein-coupled receptors (GPCRs) in live cells and small domain antibodies such as monobodies.
Specifically, we optimized the tetrazole-based photoclick chemistry by designing new genetically encoded
hydrophilic azaspiroalkene reporters, the sterically shielded tetrazoles for fast bioorthogonal ligation reactions
with strained alkenes and alkyne on live cell surface, the tetrazole-based fluorescent reporters of hydrogen
peroxide in mammalian cells, a new class of reagents called hydrazonyl sultones (HS) that release NI through
tautomerization and display a balanced stability and cycloaddition reactivity, and biocompatible genetically
encoded crosslinkers based on 2-arboxy-4-aryltriazoles and -lactam for efficient inter- and intramolecular
crosslinking. In conjunction with genetic code expansion, we showed that these chemical tools can be used to
install an organic fluorophore into class B GPCRs for FRET studies as well as endowing cell penetration to
small domain antibodies through orthogonal crosslinking. In this application, we plan to leverage our most
powerful tools developed recently, namely, hydrazonyl sultones (HS) and -lactam-lysine (BeLaK), and
prepare modified membrane receptors, enzymes, and nanobodies with new capabilities using bioorthogonal
chemistry. Specifically, in Project 1, we will design hydrazonyl sultone (HS)-based turn-on fluorophores for
constructing FRET-based biosensors to probe class B GPCR activation and signal transduction dynamics in
live cells. The organic fluorophore will be installed at the intracellular loop 3 of class B GPCRs via
bioorthogonal HS−BCN ligation reaction to permit FRET-based single-cell kinetic analysis of receptor activation
and recruitment of G protein and -arrestin. In Project 2, we will design β-lactam-encoded enzyme traps for
covalent capture and subsequent identification of protein lysine methyltransferase substrates in living cells. In
Project 3, we will develop covalent nanobody-based immunoPET radiotracers with enhanced performance.
Both BeLaK-mediated, proximity-driven crosslinking chemistry and HS-mediated, environment-dependent
bioorthogonal ligation reactions will be exploited to enhance tumor uptake of the nanobody-based PET tracers
for greater sensitivity without unwanted nephrotoxicity. We expect these studies will offer new capabilities of
covalent chemistry to biomedica...

## Key facts

- **NIH application ID:** 10841908
- **Project number:** 2R35GM130307-06
- **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:** 2024
- **Award amount:** $439,679
- **Award type:** 2
- **Project period:** 2019-01-01 → 2029-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10841908

## Citation

> US National Institutes of Health, RePORTER application 10841908, Development and Applications of Bioorthogonal Chemistry (2R35GM130307-06). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10841908. Licensed CC0.

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