Systematic Evaluation of Bioorthogonal Reactions in Live Cells with Clickable HaloTag Ligands: Implications for Intracellular Imaging.
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.
Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development , Cambridge, Massachusetts 02139, United States.
Heather E
HE
Murrey
Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development , Cambridge, Massachusetts 02139, United States.
Joshua C
JC
Judkins
Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development , Cambridge, Massachusetts 02139, United States.
Christopher W
CW
Am Ende
Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development , Cambridge, Massachusetts 02139, United States.|Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research and Development , Groton, Connecticut 06340, United States.
T Eric
TE
Ballard
Brown Laboratories, Department of Chemistry and Biochemistry, University of Delaware , Newark, Delaware 19716, United States.
Yinzhi
Y
Fang
Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research and Development , Groton, Connecticut 06340, United States.
Keith
K
Riccardi
Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research and Development , Groton, Connecticut 06340, United States.
Li
L
Di
Neuroscience and Pain Research Unit, Pfizer Worldwide Research and Development , Cambridge, Massachusetts 02139, United States.
Edward R
ER
Guilmette
Neuroscience and Pain Research Unit, Pfizer Worldwide Research and Development , Cambridge, Massachusetts 02139, United States.
Joel W
JW
Schwartz
Brown Laboratories, Department of Chemistry and Biochemistry, University of Delaware , Newark, Delaware 19716, United States.
Joseph M
JM
Fox
Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development , Cambridge, Massachusetts 02139, United States.
Douglas S
DS
Johnson
D000475:Alkenes / Q000737:chemistry; D000480:Alkynes / Q000737:chemistry; D001386:Azides / Q000737:chemistry; D001896:Boron Compounds / Q000737:chemistry; D002470:Cell Survival; D061565:Cycloaddition Reaction; D034242:Cyclooctanes / Q000737:chemistry; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D042541:Intracellular Space / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D057054:Molecular Imaging; D015335:Molecular Probes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D000475:Alkenes; D000480:Alkynes; D001386:Azides; D001896:Boron Compounds; D034242:Cyclooctanes; D005456:Fluorescent Dyes; D008024:Ligands; D015335:Molecular Probes; D006867:Hydrolases; C046137:haloalkane dehalogenase; D019793:Fluorescein