Systematic Evaluation of Bioorthogonal Reactions in Live Cells with Clickable HaloTag Ligands: Implications for Intracellular Imaging

  • J Am Chem Soc. 2015 Sep 9;137(35):11461-75. doi: 10.1021/jacs.5b06847.
Heather E Murrey  1 ,  Joshua C Judkins  1 ,  Christopher W Am Ende  1 ,  T Eric Ballard  1  2 ,  Yinzhi Fang  3 ,  Keith Riccardi  2 ,  Li Di  2 ,  Edward R Guilmette  4 ,  Joel W Schwartz  4 ,  Joseph M Fox  3 ,  Douglas S Johnson  1
Affiliations
  • 1. Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development , Cambridge, Massachusetts 02139, United States.
  • 2. Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research and Development , Groton, Connecticut 06340, United States.
  • 3. Brown Laboratories, Department of Chemistry and Biochemistry, University of Delaware , Newark, Delaware 19716, United States.
  • 4. Neuroscience and Pain Research Unit, Pfizer Worldwide Research and Development , Cambridge, Massachusetts 02139, United States.
PMID: 26270632 DOI: 10.1021/jacs.5b06847
Abstract

Bioorthogonal reactions, including the strain-promoted azide-alkyne cycloaddition (SPAAC) and inverse electron demand Diels-Alder (iEDDA) reactions, have become increasingly popular for live-cell imaging applications. However, the stability and reactivity of reagents has never been systematically explored in the context of a living cell. Here we report a universal, organelle-targetable system based on HaloTag protein technology for directly comparing bioorthogonal reagent reactivity, specificity, and stability using clickable HaloTag ligands in various subcellular compartments. This system enabled a detailed comparison of the bioorthogonal reactions in live cells and informed the selection of optimal reagents and conditions for live-cell imaging studies. We found that the reaction of sTCO with monosubstituted tetrazines is the fastest reaction in cells; however, both reagents have stability issues. To address this, we introduced a new variant of sTCO, Ag-sTCO, which has much improved stability and can be used directly in cells for rapid bioorthogonal reactions with tetrazines. Utilization of Ag complexes of conformationally strained trans-cyclooctenes should greatly expand their usefulness especially when paired with less reactive, more stable tetrazines.

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