NHS esters in Reactive groups

Primary or secondary amino groups are ubiquitous in biomolecules. They are present in proteins and peptides as ε-amino groups of lysines and N-terminal amino groups. Many natural and synthetic small molecules are amines, and DNAs with amino groups can be prepared by solid phase synthesis, and enzymatically.

Amino groups can be modified by acylation with various activated carbonyl compounds. Among these compounds, activated N-hydroxysuccinimide esters are most suitable for biolabeling and bioconjugation applications.

Compared to other activated esters, NHS derivatives possess good stability in aqueous environment. Optimal pH for the coupling of amines with NHS esters in aqueous buffers is 8.3-8.5. Labeling is also possible in organic solvents. Polar aprotic solents, such as DMF or DMSO, are preferred. One equivalent of tertiary amine base, such as triethylamine, or diisopropylethylamine (DIPEA) is necessary to achieve efficient amine acylation in non-aqueous solution.