Introduction

Click chemistry is widely used in biology and materials science because it is selective, fast, and reliable. Among different click reactions, strainpromoted azide–alkyne cycloaddition (SPAAC) is especially useful for biological systems, since it does not require copper catalysts, which can damage cells and sensitive biomolecules.

DBCO PEG (dibenzocyclooctyne–polyethylene glycol) is one of the most common reagents used for SPAAC. It combines a highly reactive strained alkyne (DBCO) with PEG, which improves water solubility, flexibility, and biocompatibility. This design overcomes many drawbacks of earlier click reagents, such as poor solubility, nonspecific interactions, and limited in vivo compatibility. As a result, DBCO PEG is now widely used in drug delivery, diagnostics, and biomaterials research.

This article gives a clear overview of how DBCO PEG works, why it is useful, where it is applied, and how it is typically used in the lab.

How DBCO PEG SPAAC Works

DBCO PEG enables SPAAC through two main components:

1. DBCO’s Strained Alkyne

Dibenzocyclooctyne contains an eightmembered ring that is under significant strain. This strain makes the alkyne highly reactive toward azide (–N₃) groups, allowing a fast cycloaddition reaction without any copper catalyst. The reaction forms a stable 1,2,3triazole linkage, which is well suited for longterm biological and chemical applications.

2. The Role of PEG

The PEG chain improves performance in biological systems:

• Increases water solubility of the DBCO group
• Reduces nonspecific protein adsorption
• Improves biocompatibility and circulation behavior

In most cases, PEG molecular weights of 2000–5000 Da provide a good balance between solubility, flexibility, and minimal steric hindrance during conjugation.

Why DBCO PEG Is Useful

Copperfree and biocompatible: Suitable for livecell labeling, in vivo imaging, and nucleic acid delivery

Highly selective: Reacts specifically with azides and does not interfere with amines, thiols, or carboxyl groups

Multiple formats available: Common derivatives include DBCOPEGNHS, DBCOPEGmaleimide, and DBCOPEGCOOH

Stable under mild conditions: Effective across pH 5.0–8.5; lyophilized products are typically stable for up to 18 months at −20 °C

Typical Applications

1. Bioconjugation and Drug Delivery

Antibody–Drug Conjugates (ADCs)
DBCO PEG is often used to link cytotoxic drugs to azidemodified antibodies. The PEG spacer improves solubility and circulation time, while the triazole linkage provides excellent stability and reduces the risk of premature drug release.

mRNA and LNP Functionalization
In lipid nanoparticle (LNP) systems, DBCO PEG allows targeting ligands (such as folate or RGD peptides) to be attached via azide groups. This improves cellular uptake without disrupting particle structure.

2. Materials Science and Diagnostics

Surface Functionalization
DBCO PEG is used to functionalize nanoparticles, hydrogels, biosensors, and medical implants. PEG helps reduce nonspecific protein binding, while SPAAC ensures stable and reproducible surface modification.

Fluorescent and Imaging Probes
Fluorophores conjugated through DBCO PEG show reduced aggregation and more stable signals, making them suitable for both in vitro assays and in vivo imaging.

Simple Lab Workflow (Protein / IgG Example)

Preparation

Exchange IgG (≈1 mg/mL) into 0.1 M sodium carbonate buffer, pH 8.5 to remove competing reagents

Prepare DBCOPEGNHS (≈2000 Da) at 10 mg/mL in anhydrous DMSO

Use a 10:1 molar ratio of DBCOPEGNHS to antibody (optimize if needed)

Reaction and Quenching

Add the DBCO PEG solution slowly to the antibody while gently mixing

Incubate for 1 h at room temperature (or overnight at 4 °C for sensitive proteins)

Quench unreacted NHS ester with 50 mM glycine for 15 min

Purification and Analysis

Remove excess reagent using 7 kDa MWCO desalting columns equilibrated with PBS (pH 7.4)

Confirm conjugation by SDSPAGE or UV–Vis spectroscopy

Troubleshooting

 Low conjugation efficiency: Check buffer pH, use freshly prepared DBCO PEG, or increase the molar ratio (up to 15:1)

 Protein aggregation: Keep DMSO ≤5 %, add reagent slowly, and avoid vigorous mixing

 Nonspecific interactions: Choose PEG with Mw ≥2000 Da for better steric shielding

FAQ

What PEG molecular weight is best?

Usually 2000–5000 Da, balancing solubility and minimal steric effects.

Is DBCO PEG suitable for in vivo use?

Yes. Its copperfree mechanism and PEG biocompatibility make it suitable for ADCs, LNPs, and imaging applications.

How stable are DBCO PEG solutions?

DMSO stocks are typically stable for 1–2 weeks at −20 °C; fresh solutions are recommended for critical experiments.

Can DBCO PEG be used with DNA or mRNA?

Yes. It is compatible with azide or thiol/aminemodified nucleic acids for delivery systems and molecular probes.

Conclusion

DBCO PEG is a practical and reliable reagent for SPAACbased bioconjugation. Its copperfree reactivity, PEGdriven biocompatibility, and broad applicability make it valuable for drug development, diagnostics, and biomaterials research. With appropriate PEG selection and reaction conditions, consistent and reproducible results can be achieved for both research and translational use.