Biotin-PEG4-Azide

Product Information

A biotin labeling reagent for biomolecules using azide alkyne cycloaddition (Click Chemistry)

What is biotin

Biotin, also known as vitamin B7, is a sulfur and nitrogen containing bicyclic heterocycle that functions as an essential coenzyme in cellular metabolism. It is required for carboxylation reactions catalyzed by enzymes such as acetyl CoA carboxylase and pyruvate carboxylase.

Beyond its biological role, biotin is renowned for forming one of the strongest known non covalent interactions with the proteins avidin and streptavidin. This exceptionally stable interaction is widely exploited in modern biochemical research, particularly in peptide, protein, and nucleic acid applications.

Applications of biotin

Peptide/protein research:

The biotin–streptavidin (or avidin) interaction underpins numerous techniques in peptide and protein research. Biotinylation of peptides and proteins is widely used for affinity purification, as biotin labeled targets can be captured on streptavidin or avidin coated magnetic beads, resins, or columns, enabling rapid one step purification.

In addition, biotin labeling allows sensitive detection of proteins through streptavidin conjugated to reporter molecules, most commonly fluorophores or enzymes such as horseradish peroxidase (HRP). Consequently, biotinylation is frequently employed in immunoassays including ELISA, Western blotting, and immunohistochemistry.

Biotin tagging also supports the enrichment of specific protein populations, such as newly synthesized proteins, for downstream mass spectrometry analysis. Furthermore, immobilization of biotinylated proteins on streptavidin coated surfaces enables binding assays to study protein–protein interactions.

Nucleic acid research:

In nucleic acid research, biotin labeling is primarily used for purification and capture applications. The strong interaction between biotin and streptavidin or avidin allows biotinylated DNA or RNA to be immobilized on beads, columns, or resins, facilitating efficient separation from impurities.

Biotin containing probes are commonly applied in in situ hybridization (ISH), Southern blotting, and Northern blotting to target specific DNA or RNA sequences. After hybridization, these probes can be visualized and localized using streptavidin  or avidin based detection reagents, typically conjugated to fluorophores or enzymes.

Biotin Labeling via Click Chemistry

All of the above applications require reliable production of biotin labeled biomolecules. Click chemistry offers a simple, cost effective, and highly specific approach to achieve this. For click based labeling, the target biomolecule must carry a terminal alkyne or a strained alkyne functionality.

Strained alkynes enable catalyst free reactions and are especially suitable for sensitive biological applications. Common examples include DBCO and BCN groups. Biomolecules containing these modifications are widely available. baseclick offers biotin labeled oligonucleotides and mRNA as well as custom synthesis of alkyne or DBCO modified nucleic acids for flexible downstream labeling. For other targets, support is available via support@baseclick.eu.

Chemical Properties of Biotin-PEG4-Azide

Biotin-PEG4-Azide is a versatile reagent for biotin labeling under mild conditions. Covalent conjugation to target molecules can be achieved via two click chemistry-based reactions:

• Strain promoted azide–alkyne cycloaddition (SPAAC): A copper free reaction requiring neither heat nor catalyst, resulting in a stable triazole linkage with strained alkynes.

• Copper catalyzed azide–alkyne cycloaddition (CuAAC): A selective reaction forming a 1,4 substituted triazole linkage between biotin and the target molecule.

Both reactions are considered bioorthogonal, meaning they proceed without interfering with native biological functionalities. The hydrophilic PEG4 spacer reduces steric hindrance between the biotin and the labeled biomolecule while significantly increasing water solubility.

These properties make Biotin-PEG4-Azide an ideal choice for biotin labeling of a wide range of alkyne modified biomolecules, including peptides, proteins, and nucleic acids. The option to use copper free SPAAC click chemistry further enables in vivo labeling applications.

Because click chemistry relies on non-naturally occurring functional groups, the site of biotin attachment can be precisely controlled, in contrast to traditional labeling approaches such as NHS ester based biotinylation.

LITERATURE

Discovery of an NAD+ analogue with enhanced specificity for PARP1, X. N. Zhang et al., 2022, Chemical Science, Vol. 13(7), p. 1982-1991.

https://doi.org/10.1039/D1SC06256E

Epitopes displayed in a cyclic peptide scaffold bind SARS‐CoV‐2 antibodies, C. Eriksson et al., 2023, ChemBioChem, Vol. 24(15), e202300103.

https://doi.org/10.1002/cbic.202300103

Functionalized scout fragments for site-specific covalent ligand discovery and optimization, V. M. Crowley et al., 2021, ACS Central Science, Vol. 7(4), p. 613-623.

https://doi.org/10.1021/acscentsci.0c01336