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Dibenzylcyclooctyne-PEG4-NHS ester

Cat. No. Amount Price (EUR) Buy / Note
CLK-A134-10 10 mg 168,50 Add to Basket/Quote Add to Notepad
CLK-A134-100 10 x 10 mg 877,90 Add to Basket/Quote Add to Notepad
CLK-A134-500 500 mg 2.726,00 Add to Basket/Quote Add to Notepad
Structural formula of DBCO-PEG4-NHS ester (Dibenzylcyclooctyne-PEG4-NHS ester)
Structural formula of DBCO-PEG4-NHS ester

For research use only!

Shipping: shipped at ambient temperature

Storage Conditions: store at -20 °C
store undissolved, for use prepare a fresh solution

Shelf Life: 12 months after date of delivery (undissolved)

Molecular Formula: C34H39N3O10

Molecular Weight: 649.69 g/mol

CAS#: 1427004-19-0

Purity: > 95 % (HPLC)

Form: oil

Color: slightly orange to slightly brown

Solubility: DCM, DMF, DMSO, THF

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

NHS ester for introduction of a dibenzylcyclooctyne to an available amine functionality. The PEG5 spacer is very hydrophilic. It reduces non-specific binding and enhances solubility in water as well as in commonly used organic solvents of moderate polarity. The 30 Å (25 atoms) long spacer gives ready accessibility to the dibenzylcyclooctyne for the azide binding. Moreover, it enhances accessibility of the amine sites, which may be buried.

Important Product Information

  • NHS esters are moisture-sensitive. To avoid moisture condensation onto the product always let the vial come to room temperature before opening, be careful to limit exposure to moisture and restore under an inert atmosphere. The NHS-ester moiety readily hydrolyzes and becomes non-reactive, therefore, prepare stock solutions immediately before use. Stock solutions in anhydrous solvents can be kept for several days (freeze when not in use).
  • Hydrolysis of the NHS ester is a competing reaction. Conjugation with primary amines of proteins/peptides (i.e. acylation) is favored at near neutral pH (6 - 9) and with concentrated protein solutions. For conjugation, use non-amine-containing buffers at pH 7 - 9 such as PBS (20 mM sodium phosphate, 150 mM sodium chloride, pH 7.4), 20 mM HEPES, 100 mM carbonate/biocarbonate, or 50 mM borate buffer.
  • Do not use buffers that contain primary amines (e.g. Tris, Glycine).
  • Avoid buffers that contain azides, which can react with DBCO.
  • Dissolve DBCO-PEG4-NHS ester in a dry water-miscible organic solvent such as DMSO or DMF before diluting in final reaction buffer. DBCO-PEG4-NHS ester is soluble in aqueous buffers up to 5.5 mM.
  • Reactions with DBCO and azides are more efficient at high concentrations and temperatures (i.e. 4 - 37 °C). Typical reaction times are less than 4 hours, however, incubating for longer can improve efficiency.

Additional Materials Required

  • Water-miscible organic solvent such as dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF)
  • Reaction buffer: Phosphate-buffered saline (PBS) or other buffer at pH 5 - 9
  • Quenching buffer: 1 M Tris-HCl, pH 8.0
  • Spin Desalting Columns or Slide-A-Lyzer Dialysis Cassettes

Protein Derivatization

  • Prepare proteins in PBS.
  • Immediately before use, prepare 10 mM of the DBCO-PEG5 - NHS reagent in DMSO or DMF.
  • Add the NHS reagent to the protein sample at a final concentration of 0.5 - 2 mM. If the protein concentration is ≥5 mg/ml, use a 10 - fold molar excess of the reagent. For samples < 5 mg/ml, use a 20 - to 50 - fold molar excess.
  • Incubate the reaction at room temperature for 30 minutes or on ice for 2 hours.
  • Stop the reaction by adding Quenching Buffer to a final concentration of 50 - 100 mM Tris.
  • Incubate the reaction at room temperature for 5 minutes or on ice for 15 minutes.
  • Remove non-reactive reagent by dialysis or desalting

Long-term aqueous Stability of DBCO-labeled Samples

DBCO modified goat IgG (DOL 7) losses about 3 - 5 % of its reactivity toward azides over 4 weeks at 4 °C or -20 °C. For long time storage azide- and thiol-containing buffers should be avoided.

Direct Measurement of DBCO Incorporation

The degree of DBCO incorporation (i.e. the number of DBCO per protein molecule) can be determined from the absorbance scan of the purified conjugate (235 - 400 nm).

Copper-free Click Reaction

  • Prepare the azide-containing sample in reaction buffer.
  • Add DBCO-protein conjugate to azide-containing sample.
  • Recommendation: Add 1 mol equivalent of limiting protein to 1.5 - 3.0 mol equivalents of highest abundance reagent.
  • Incubate the reaction at room temperature for 1 - 4 hours. Incubation at 4 °C requires 4 - 12 hours.
  • The reaction is now ready for purification by size exclusion chromatography if required.


Problem: No conjugation of DBCO with azide

  • Possible reason: One or more sample is not labeled
    - Confirm molecules were labeled or repeat activation process
  • Possible reason: NHS-ester hydrolyzed
    - Allow product to equilibrate to room temperature before opening
    - Prepare new solutions in the indicated dry solvents
    - Avoid buffers that contain primary amines such as Tris and glycine
  • Possible reason: Excess reagent not quenched or removed
    - Remove non-reacted reagent by dialysis or desalting

Problem: Low conjugation of DBCO and azide

  • Possible reason: Suboptimal reaction conditions
    - Increase incubation time
    - Optimize conjugation conditions by altering molar excess
    - Perform conjugation reactions at 37 °C

Product Citations:
Please click the black arrow on the right to expand the citation list. Click publication title for the full text.

Selected References:
Simon et al. (2012) Facile Double-Functionalization of Designed Ankyrin Repeat Proteins using Click and Thiol Chemistries. Bioconjugate Chem. 23 (2):279.
Zeng et al. (2012). 64Cu Core-Labeled Nanoparticles with High Specific Activity via Metal-Free Click Chemistry. ACS Nano. 6 (6):5209.
Arumugam et al. (2011). [18F]Azadibenzocyclooctyne ([18F]ADIBO): A biocompatible radioactive labeling synthon for peptides using catalyst free [3+2] cycloaddition. Bioorg. Med. Chem. Lett. 21:6987.
Campbell-Verduyn et al. (2011). Strain-Promoted Copper-Free Click Chemistry for 18F Radiolabeling of Bombesin. Angew. Chem. Int. Ed. 50:11117.
Debets et al. (2010) Aza-dibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3+2) cycloaddition. Chem. Commun. 46:97.