DBCO-PEG4-Maleimide

Dibenzylcyclooctyne-PEG4-Maleimide

Cat. No. Amount Applications: Protein-peptide conjugates CLK-A108P-10 10 mg

CLK-A108P-100 100 mg Peptide-small molecule conjugates

CLK-A108P-500 500 mg 18F radiolabelling

Protein-oligonucleotide conjugates

Surface modification N O O O O O O N O O N N H H Description: Sulfhydryl reactive reagent with enhanced solubility in water as O well as in commonly used organic solvents of moderate polarity. Structural formula of DBCO-PEG4-Maleimide The PEG4 hydrophilic spacer will reduce or eliminate aggregation or precipitation problems when labeling antibodies and other biolo- For research use only! gical molecules. Due to its length, the 26.3 Å (22 atoms) long spacer enhances accessibility of the azide-reactive dibenzylcyclooctyne and of reactive thiols, which may be buried. Shipping: shipped at ambient temperature Important Product Information Storage Conditions: store at -20 °C • Molecules, which shall react with maleimide compounds, must Additional Storage Conditions: store undissolved, for use prepare a have free (reduced) sulfhydryls. Reduce peptide disulfide fresh solution bonds with disulfide reducing reagents such as Immobilized TCEP Disulfide Reducing Gel (Pierce Biotechnology). Reduce di- Shelf Life: 12 months after date of delivery (undissolved) sulfide bonds in high molecular weight proteins using 5 mM

Molecular Formula: C36H42N4O9 TCEP (1:100 dilution) for 30 minutes at room temperature, fol- lowed by TCEP removal using a desalting column. Proteins (e.g. Molecular Weight: 674.75 g/mol antibodies) can be inactivated by complete reduction of their CAS#: 1480516-75-3 disulfide bonds. Selective reduction of hinge-region disulfide bonds in IgG can be accomplished with 2-Mercaptoethylamine- Purity: > 90 % (HPLC) HCl (2-MEA). Sulfhydryls can be added to molecules using N- succinimidyl S-acetylthioacetate (SATA) or 2-iminothiolane-HCl Form: oil (Traut’s Reagent), which modify primary amines. Color: slightly yellow • Do not use buffers that contain sulfhydryl-containing compon- ents (e.g. DTT). Solubility: DCM, DMF, DMSO, THF • Avoid buffers that contain azides, which can react with DBCO. • The maleimide group reacts predominantly with free sulfhy- dryls at pH 6.5 - 7.5, forming stable thioether bonds. At pH val- ues > 7.5, reactivity toward primary amines and hydrolysis of the maleimide groups can occur. At pH 7, the maleimide group is 1,000 times more reactive toward a free sulfhydryl than to an amine. • Do not use DTT, TCEP or β-mercaptoethanol, because they will reduce the azide.

Additional Materials Required: • Water-miscible organic solvent such as dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF) • Reducing reagents such as Immobilized TCEP Disulfide Redu- cing Gel (Pierce Biotechnology) • Reaction buffer: Phosphate-buffered saline (PBS) or other

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Dibenzylcyclooctyne-PEG4-Maleimide

sulfhydryl-free buffer at pH 6.5 - 7.5. Include 5 - 10 mM EDTA The degree of DBCO incorporation (i.e. the number of DBCO to help prevent the reoxidation of disulfides by trace divalent per protein molecule) can be determined from the absorbance scan metals. of the purified conjugate (235 - 400 nm). • (Optional): Quenching buffer: concentrated (0.5 - 1 M) cycteine, DDT or other thiol containing reducing agents Troubleshooting • (Optional): Spin Desalting Columns or Slide-A-Lyzer Dialysis Cassettes Problem: No conjugation of DBCO with azide • Possible reason: One or more sample is not labeled - Confirm molecules were labeled or repeat activation process Protein Derivatization • Possible reason: DBCO-Maleimide decomposed • Prepare sulfhydryl-containing protein, prepared as described - Allow product to equilibrate to room temperature before in the Important Product Information section. opening • Immediately before use, weigh a small quantity of DBCO- - Prepare new solutions in the indicated dry solvents Maleimide and dissolve it in dimethylformamide (DMF) or di- - Avoid buffers that contain sulfhydryl methylsulfoxide (DMSO) at a 5 - 20 mM concentration. • Possible reason: Excess reagent not quenched or removed • Dissolve protein(s) in Conjugation Buffer at 0.1 mM (e.g. 5 mg in - Remove non-reacted reagent by dialysis or desalting 1 ml for a 50 kDa protein). • Add DBCO-Maleimide solution to the dissolved protein(s) at 0.4 mM final concentration (ca. four-fold molar excess for 0.1 mM Problem: Low conjugation of DBCO and azide protein solution). • Possible reason: Suboptimal reaction conditions • Note: The reaction solution may appear cloudy as a result of the - Increase incubation time low aqueous solubility of DBCO-Maleimide, usually, such solu- - Optimize conjugation conditions by altering molar excess tions become clearer as the reaction proceeds. Many proteins - Perform conjugation reactions at 37 °C will precipitate when the DMF or DMSO concentration exceeds 10 - 15 % of the final reaction volume, if protein solubility is not an issue, there is no limit to the DMF or DMSO concentration that may be used. Selected References: • Incubate reaction mixture for 1 hour at room temperature or for Simon et al. (2012) Facile Double-Functionalization of Designed Ankyrin Repeat 2 hours at 4 °C. Proteins using Click and Thiol Chemistries. Bioconjugate Chem. 23 (2):279. • Quench reaction by adding Quenching Solution at 10 - 50 mM fi- Zeng et al. (2012). 64Cu Core-Labeled Nanoparticles with High Specific Activity nal and incubating for 15 minutes at room temperature. Altern- via Metal-Free Click Chemistry. ACS Nano. 6 (6):5209. atively (or in addition) remove the excess nonreacted reagent by desalting or dialysis. 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. Copper-free Click Reaction • Prepare the azide-containing sample in reaction buffer. Campbell-Verduyn et al. (2011). Strain-Promoted Copper-Free Click Chemistry 18 • Add DBCO-protein conjugate to azide-containing sample. for F Radiolabeling of Bombesin. Angew. Chem. Int. Ed. 50:11117. • Recommendation: Add 1 mole equivalent of limiting protein to Debets et al. (2010) Aza-dibenzocyclooctynes for fast and efficient enzyme 1.5 - 3.0 mol equivalents of highest abundance protein. PEGylation via copper-free (3+2) cycloaddition. Chem. Commun. 46:97. • Incubate the reaction at room temperature for 2 - 4 hours. In- cubation at 4 °C requires 4 - 12 hours. • The reaction is now ready for purification.

Long-term aqueous Stability of DBCO-labeled Samples

DBCO modified goat IgG (DOL 7) losses about 3 - 5 % of its re- activity 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

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