80 K. Mruk and W. R. Kobertz
2 Approach
Bioreactive tethers have three components: two targeting moieties linked together
via a tether. The methodological challenge is connecting these three pieces together
to obtain the desired reagent. For this reason, simple peptide or thiol-specific chem-
istries (maleimides) are primarily used for bond formation. Access to standard or-
ganic synthesis equipment, including a NMR and mass spectrometer is required. In
addition, most of the bioreactive tethers are water soluble and require C18 reverse-
phase HPLC purification. Because every synthesis and chemical derivatization is
unique, the following approach section provides basic synthetic strategies with ex-
emplars for creating and employing bioreactive tethers.
- Chemical Derivatization of Ion Channel Targeting Moieties: The revers-
ible ion channel modifier is the primary source of specificity and thus must be
modified such that its binding is minimally perturbed. For small molecules, the
compound is typically derivatized to perform peptide bond chemistry. This strat-
egy is exemplified by the workhorse of the K+ channel community, Quaternary
Amines (QAs). QAs are routinely used because one of the alkyl groups can
be substituted with a different pendant arm without affecting binding. Fig. 2a
shows the synthesis of a triethylammonnium derivative (Blaustein et al. 2000 )
that enables attachment of a QA via standard peptide chemistry. In general, this
classic SN2-displacement reaction works well with trimethyl- and triethylamine,
but longer chain QA’s (up to butyl) are achievable with extended reaction times
(Lu et al. 2011 ). In addition, maleimide QAs have also been synthesized (Lu
et al. 2011 ). Carboxylic acid versions are purified by standard organic chemistry
approaches (crystallization and trituration); the maleimide QAs by C18 reverse-
phase HPLC, eluting with 0.1 % trifluoroacetic acid to prevent base hydrolysis
of the maleimide.
In addition to small molecules, peptides and small proteins can also be used as a
reversible targeting moiety. These “biologics” often provide higher binding affini-
ties than small molecules, can be readily expressed in E. coli as wild type or fusion
proteins, and purified by standard biochemical methods. To date, peptides and pro-
teins containing an exogenous cysteine residue have been used as the attachment
point for the tether (Shimony et al. 1994 ). However, the recent emergence of genetic
approaches to incorporate unnatural amino acids into proteins (Chin 2014 ) enables
alternative chemoselective and bioorthogonal chemistries to be utilized (e.g. azides,
alkynes, ketones, and aldehydes). Figure 2c shows the derivatization of a reduced
calmodulin (CaM) cysteine mutant with excess maleimide (~ 1–10 mM) to ensure
rapid and complete modification (Mruk et al. 2012 ). After derivatization, the pep-
tide or protein is purified by reverse-phase HPLC and the presence of the modifying
group confirmed by mass spectrometry. For modifications that utilize excess bis-
maleimide to yield a cysteine-reactive protein or peptide (Hua et al. 2011 ; Morin
and Kobertz 2007 , 2008a), it is essential that 0.1 % trifluoroacetic acid is included
in the chromatography eluents to prevent maleimide hydrolysis.