Thermo Scientific Pierce Crosslinking Technical Handbook

Crosslinker Reactivities
Amine-reactive chemistries
Imidoesters
Imidoester crosslinkers react with primary amines to form amidine
bonds. The resulting amidine is protonated and, therefore, has a
positive charge at physiological pH (Figure 6). Imidoester homobifunctional
crosslinkers have been used to study protein structure
and molecular associations in membranes and to immobilize
proteins onto solid-phase supports. They also have been examined
as a substitute for glutaraldehyde for tissue fixation. Imidoesters
can penetrate cell membrane and crosslink proteins within
the membrane to study membrane composition, structure and
protein:protein and protein:lipid interactions. These crosslinkers
have also been used to determine or confirm the number and location
of subunits within multi-subunit proteins. In these experiments,
large molar excesses of crosslinker (100- to 1,000-fold) and low
concentrations of protein (1 mg/ml) are used to favor intramolecular
over intermolecular crosslinking.
Amine-containing
molecule
Imidoester
compound
Figure 6. Imidoester reaction scheme.
Amidine linkage
Imidoester crosslinkers react rapidly with amines at alkaline pH,
but have short half-lives. As the pH becomes more alkaline, the
half-life and reactivity with amines increases; therefore, crosslinking
is more efficient when performed at pH 10 than at pH 8.
Reaction conditions below pH 10 may result in side reactions,
although amidine formation is favored between pH 8-10. Studies
using monofunctional alkyl imidates reveal that at pH