81- Linker synthesis: The linker is the most synthetically tunable component of a
bioreactive tether; thus, most of the creativity and chemistry of a bioreactive
tether entails the chemical synthesis of the linker or tether. Three classes of
linkers that have been synthesized to probe ion channel structure, function, and
modulation are: (i) libraries of different-length linkers to measure distances; (ii)
photoisomerizable linkers to instantly and reversibly change tether length; (iii)
cleavable linkers to deliver small molecule probes.
Libraries of different-length bioreactive tethers are used to determine distances
in ion channel complexes because the modification rates are dependent on linker
length. Because a panel of reagents must be synthesized, high-yielding chemical
reactions such as peptide bond formation are used to generate a systematic series
of bioreactive tethers with defined tether lengths. In fact, Blaustein and co-workers
simply used glycines as spacer residues to synthesize a panel of cysteine reactive
QAs with linkers of 21–45 Å (Blaustein et al. 2000 ). Polyglycine linkers longer than
Fig. 2 Syntheses of tethered blockers with quaternary ammoniums. a Synthesis of a carboxylic
acid triethylammonium precursor. b Synthesis of a calmodulin-based tethered blocker. ( To p)
Structure of the maleimido-quaternary ammonium linkers: n, number of glycines in the linker;
d, extended length of linkers rounded to the nearest ångström from the center of the quaternary
ammonium to the olefinic carbons on the maleimide. ( Bottom) Cartoon depiction of the calmodu-
lin protein labeling procedure using a calmodulin cysteine mutant. c Synthesis of a maleimido-
quaternary ammonium (MAL-AZO-QA) containing an azobenzene linker
Bioreactive Tethers