TCO-PEG₄-NHS ester

For research use only!

Shipping: shipped at ambient temperature

Storage Conditions: store at -20 °C

Shelf Life: 12 months after date of delivery

Molecular Formula: C₂₄H₃₈N₂O₁₀

Molecular Weight: 514.57 g/mol

CAS#: 1621096-79-4

Purity: > 90 % (HPLC)

Form: oil

Solubility: Chloroform, DCM, DMF, DMSO

Applications:
Protein-peptide conjugates
Protein-antibody conjugates
Peptide-small molecule conjugates
¹⁸F radiolabelling
Protein-oligonucleotide conjugates
Surface modification

Description:
The inverse-electron demand Diels-Alder cycloaddition reaction of trans-Cyclooctenes (TCO) with tetrazines is a bioorthogonal reaction that possesses exceptional kinetics (k > 800 M^-1s^-1) and selectivity. Such excellent reaction rate constants are unparalleled by any other bioorthogonal reaction pair described to date.
The extremely fast kinetics and selectivity enables the conjugation of two low abundance biopolymers in an aqueous and otherwise complex chemical environment through the formation of a stable dihydropyridazine. This bioorthogonal reaction possesses extreme selectivity and biocompatibility, such that the complimentary reagents can form covalent bonds within richly functionalized biological systems, in some cases, living organisms. The TCO-tetrazine click reaction is a very powerful tool in catalyst-free protein-protein bioconjugation.

Advanced Features

• By far, the fastest kinetics among any other bioorthogonal reaction pairs
• Reactions complete in 30-60 minutes at low protein concentrations (5-10 μM)
• TCO functional group remains stable in aqueous buffered media (weeks at 4°C, pH 7.5)
• Conjugation efficiency > 99 % without requiring a toxic catalyst (e.g. Cu(I))

Important Product Information

• NHS esters are moisture-sensitive and readily hydrolyze. Avoid moisture condensation by allowing product to come to room temperature before opening. Prepare a working stock solution immediately before use and discard unused portion.
• Hydrolysis is a competing reaction with primary amines of proteins/peptides.Acylation is favored using concentrated protein solutions (1 - 5 mg/ml) at pH 7 - 9. For NHS ester reactions, use an amine-free buffer such as 100 mM sodium phosphate, 150 mM sodium chloride pH 7.5. Do not use buffers containing primary amines (e.g. Tris, Glycine). Prior to use, dissolve the reagent in a dry water-miscible organic solvent such as DMSO or DMF.
• Reactions between tetrazine and TCO are complete in 30-60 minutes at 5-10 μM.

Additional Material Required

• Water-miscible organic solvent such as dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF)
• Reaction buffer: Phosphate-buffer (100 mM sodium phosphate, 150 mM NaCl, pH 7.5) or other suitable amine-free buffer at pH 7 - 9
• Quenching buffer: 1 M Tris-HCl pH 8.0
• Spin Desalting Colum (e.g. ThermoScientific Zeba™)

Procedure for Labeling Proteins

• Buffer exchange proteins into phosphate reaction buffer at 1 - 5 mg/ml using a desalt spin column.
• Immediately before use prepare 10 mM TCO-PEG₄-NHS reagent in DMSO or DMF.
• Add a 20-fold molar excess NHS reagent to the protein sample and incubate for 1 hour at room temperature.
• Stop the reaction by adding Quenching buffer to a final concentration of 50-100 mM, incubate for 5 minutes.
• Remove excess reagent by desalting the labeled protein through a desalt spin column or by dialysis.

Long-term Aqueous Stability of TCO-labeled Samples

TCO-PEG₄-modified goat IgG (17.2 DOL) losses about 10.5 % of its reactivity toward tetrazines when stored at 4°C for 4 weeks and 7 % when stored at -20°C in 100 mM sodium phosphate, 150 mM sodium chloride solution at pH 7.5. For long-term storage azide and thiol-containing buffers should be avoided.

Protein-Protein Tetrazine/TCO Conjugation

• Calculate volume tetrazine-labeled protein (1 - 5 mg/ml) equivalent to a 2 - 5 fold molar excess over desired volume TCO-labeled protein (1 - 5 mg/ml).
• Mix calculated volume tetrazine -labeled protein with desired volume of TCO-labeled protein.
• Allow reaction to proceed for 60 minutes at room temperature.
• Store conjugate at 4°C until ready for purification or use.

Troubleshooting

Problem: No or poor labeling of protein with TCO

• Possible reason: NHS-ester hydrolyzed
  - Allow product to equilibrate to room temperature before opening. Use only high quality, anhydrous water-miscible solvents such as DMSO or DMF
• Possible reason: Amine-contaminants in protein labeling reaction buffer (e.g. Glycine, Tris)
  - Buffer exchange proteins into an amine-free buffer before labeling (e.g. 100 mM sodium phosphate, 150 mM sodium chloride, pH 7.5)
• Possible reason: Sub-optimal reaction conditions
  - Optimize labeling conditions by altering molar excess

Product Citations:
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Selected References:
Cobley et al. (2020) Reversible Thiol Oxidation Inhibits the Mitochondrial ATP Synthase in Xenopus Laevis Oocytes Antioxidants 9 (3):215.
Devaraj et al. (2009) Fast and Sensitive Pre-Targeted Labeling of Cancer Cells through a Tetrazine/trans-Cyclooctene Cycloaddition. Angew. Chem. Int. Ed. 48:7013.
Haun et al. (2009) Probing Intracellular Biomarkers and Mediators of Cell Activation Using Nanosensor and Bioorthogonal Chemistry. ACS Nano. 5:3204.
Blackman et al. (2008) Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity. J. Am. Chem. Soc. 130:13518.
Devaraj et al. (2008) Tetrazine-Based Cycloadditions: Application to Pretargeted Live Cell Imaging. Bioconjugate Chem. 19:2297.