De Novo Design of Proteins - Peptide and Protein Design for Biopharmaceutical Applications - page 224

potential of using fluorous interactions in the design of proteins [104].
The van der Waals radius of fluorine is 1.35 ˚ ,only0.15 ˚ larger than
hydrogen; however, the C-F bond is significantly longer ( 1.4 ˚ )than
the C-H bond ( 1.0 ˚ ) and fluorine is better considered an isostere of
oxygen. Obviously, a C-F bond has the opposite polarity of a corre-
sponding C-H bond and introduction of fluorine induces polarization
of C-H bonds in the proximity and has an effect on the pK a several
bonds away.
Fluorocarbon molecules appear much more hydrophobic than the
analogous hydrocarbon molecules. Interestingly, although fluorocarbons
are hydrophobic, perfluorinated molecules tend also to be poorly soluble
in hydrocarbon solvents. Hexane, water and perfluorohexane are
mutually immiscible and separate into three layers after mixing.
Perfluorocarbons are better described as fluorophilic, rather than lipo-
philic or hydrophobic. However, the cohesive dispersion forces between
hydrocarbon molecules are larger than those between fluorocarbon
molecules, as hydrocarbons are more polarizable, which excludes fluor-
ocarbons from hydrocarbons, and the 'fluorous effect' is thus at least to
some extent caused by this exclusion from hydrocarbons. This fluorous
effect is responsible for the nonstick property of Teflon, as it interacts
with neither hydrophilic nor lipophilic compounds. The use of fluorous
interactions in the de novo design of proteins has focused on the interior
of proteins, especially fluorinated substitutes for Leu and Val. Ideally,
fluorous-fluorous interactions in proteins would be orthogonal to other
interactions.
Tirrel and coworkers have prepared a fluorinated coiled-coil ensemble
[105]. In this Leu zipper, Leu in the d positions of the heptad was
substituted by trifluoroleucine (Tfl, racemic mixture). The sequence
was expressed in vivo , but not with 100% incorporation. It formed a
stable dimer, but at high-mM concentrations it associated to higher-order
species. It proved highly resistant to denaturation.
Kumar and coworkers [106-108] designed a 30-residue coiled-coil
sequence with Lys at e positions and Glu at g positions, which due to
unfavourable interhelical electrostatic in the antiparallel arrangements
favoured the parallel alignment. A single Asn was placed in the core such
that it could only hydrogen bond in the parallel arrangement, and the
sequence was N-terminally extended with Cys-Gly-Gly to allow inter-
strand disulfide formation. In addition to H b with an all-Leu core, Kumar
and coworkers also prepared F b with hexafluoro-leucine (hFLeu) in the
core. In a key experiment, the disulfide-linked heterodimer H b F b was
allowed to 'self-sort' under redox conditions. Homodimerization into