Typically, there are three biological functional groups on the peptide for the further conjugation: amino ( –NH2 ), carboxyl ( –COOH), and thiol ( –SH). The most effective way is to utilize the free thiol groups from cysteine. The reaction of maleimides with thiols is widely used for bioconjugation and labeling of biomolecules.
Click Chemistry to Conjugate Peptides With Other Biomolecules
The click chemistry is another efficient method to conjugate the peptide with other biomolecules. The peptide can be modified with azide groups (–N3). The novel Copper-free Click Chemistry is based on the reaction of a diarylcyclooctyne moiety (DBCO) with an azide-peptide reaction partner. This click reaction is very fast at room temperature and does not require a cytotoxic Cu(I) catalyst, resulting in almost quantitative yields of stable triazoles. The DBCO allows Copper-free Click Chemistry to be done with live cells, whole organisms, and non-living samples. Within physiological temperature and pH ranges, the DBCO group does not react with amines or hydroxyls, which are naturally present in many biomolecules. The reaction of the DBCO group with the azide group is significantly faster than with the sulfhydryl group (–SH, thiol).
One example of the peptide drug conjugations is the antibody-biomolecule conjugate.
click chemistry: DBCO-azide
A simple protocol: Click chemistry of antibody-DNA conjugationPre-conjugation considerations
Remove all additives from antibody solutions using dialysis or desalting.
Remove BSA and gelatin from antibody solutions.
Concentrate the antibody after dialysis or purification.
Activation of antibodies with DBCO-NHS ester
Mix antibody with 20-30 fold molar excess over antibody of DBCO-NHS ester dissolved in DMSO.
Incubates at room temperature for 30 min or 2 hours on ice.
Quenching activation reaction
Add Tis-Hcl (50-100mM, pH 8) to the reaction.
Incubate at RT for 5 min or 15 minutes on ice.
Equilibration and removal of non-reactive DBCO-NHS ester by Zeba column (Follow the manufacturer’s instruction)Copper-Free click reaction
Mix DBCO-NHS ester labeled antibody with 2-4 times molar excess of azide-modified Oligos.
Incubated overnight (around 10-12 hours) at 4°C or 3-4 hours at room temperature.
Validation of conjugation and purification by HPLCSelected References:
Simon et al. (2012) Facile Double-Functionalization of Designed Ankyrin Repeat Proteins using Click and Thiol Chemistries. Bioconjugate Chem. 23(2):279.
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.