4.2.3
Non-ionic Amphipols
There are several kinds of problems associated with the use of ionic polymers for handling MPs:
(i) Weak acids or bases, e.g. carboxylate groups, protonate or deprotonate depending on the
pH of the solution, which changes the global charge of the polymer and can either severely
diminish its solubility in water or turn a zwitterionic, globally neutral polymer into one with
a net charge. To take two examples, the global charge of A8-35 or SMA diminishes as the
pH drops below ~7 and some carboxylates protonate, making them more hydrophobic,
whereas PC-APols, which are globally neutral at pH 7, become cationic at acidic pH.
(ii) A net charge will affect the behavior of the MP/polymer complex in such experiments as
ion exchange chromatography or isoelectrofocusing.
(iii) Polymers with a higher charge density seem to stabilize MPs less efficiently (see Chap. 5),
which is reminiscent of the case of charged detergents discussed in Chap. 2. This can easily
be rationalized if one considers that disaggregation of a MP oligomer or expansion of a
denaturing MP monomer will be favored by electrostatic repulsion between the layers of
protein-bound surfactant.
(iv) No crystal structure of a MP has ever been solved using a globally charged detergent and
only very few using zwitterionic, globally neutral detergents (see e.g. Moraes et al. 2014),
making it dubious whether charged APols can be efficiently used for such a task (see
Chap. 11).
These considerations have provided a strong impetus to the search for non-ionic APols, which
was undertaken and carried out in collaboration with the organic chemist Bernard Pucci and his
colleagues. This, however, proved to be quite a difficult and lengthy endeavor, because it is not
straightforward to design, synthesize, and purify in a way that is reasonably economical and easy to
scale up a polymer that features (i) a high density of hydrophobic moieties and (ii) enough non-ionic
hydrophilic groups to endow it with the high solubility that is essential to its use in biochemistry and
biophysics. Two types of non-ionic polar moieties are commonly found in the detergents used for
biochemistry, the ether bonds of polyoxyethylene, as in Triton X-100 or C 12 E 8 , and the hydroxyl
groups carried by sugar moieties, as in octylglucoside or dodecylmaltoside (see Chap. 2, Fig. 2.1).
At this point, hydroxyl groups only have been tested with APols (Fig. 4.5).
4.2.3.1 THAM-Based Non-ionic Amphipols
Most of the non-ionic APols validated to date are derived from tris(hydroxymethyl)acrylamidomethane (THAM; Fig. 4.6), an approach pioneered by Bernard Pucci and his collaborators. The
three hydroxyl groups carried by THAM can be either left free, providing a moderately hydrophilic
monomer, or one or more of them can be grafted with sugar moieties, increasing the hydrophilicity,
and/or with an alkyl chain, creating an amphiphilic monomer. The synthesis of the first non-ionic
APols ever to be tested (Fig. 4.5A) relied on the co-telomerization of (i) an amphipathic monomer
carrying one n-heptyl or n-undecyl alkyl chain and (ii) a hydrophilic monomer carrying either three
free hydroxyls or two free hydroxyls plus one grafted with D-galactose (Fig. 4.7). A range of telomers
were synthesized, whose length varied from ~10 to ~130 monomers (hM n i ranging from ~3 to
~28 kDa), using various combinations of the two types of amphipathic monomers (grafted with either
a C 7 or a C 11 alkyl chain) and hydrophilic ones (ungrafted or carrying one D-galactosyl moiety). Their
solubility was usually ~30–50 gÁL
À1 , which is significantly lower than that of A8-35 (>320 gÁL
À1 ), but
sufficient for carrying out test biochemical experiments. Those were run on two model MPs, BR and
4.2 Amphipol Chemical Structure and Synthesis
165
Non-ionic Amphipols
There are several kinds of problems associated with the use of ionic polymers for handling MPs:
(i) Weak acids or bases, e.g. carboxylate groups, protonate or deprotonate depending on the
pH of the solution, which changes the global charge of the polymer and can either severely
diminish its solubility in water or turn a zwitterionic, globally neutral polymer into one with
a net charge. To take two examples, the global charge of A8-35 or SMA diminishes as the
pH drops below ~7 and some carboxylates protonate, making them more hydrophobic,
whereas PC-APols, which are globally neutral at pH 7, become cationic at acidic pH.
(ii) A net charge will affect the behavior of the MP/polymer complex in such experiments as
ion exchange chromatography or isoelectrofocusing.
(iii) Polymers with a higher charge density seem to stabilize MPs less efficiently (see Chap. 5),
which is reminiscent of the case of charged detergents discussed in Chap. 2. This can easily
be rationalized if one considers that disaggregation of a MP oligomer or expansion of a
denaturing MP monomer will be favored by electrostatic repulsion between the layers of
protein-bound surfactant.
(iv) No crystal structure of a MP has ever been solved using a globally charged detergent and
only very few using zwitterionic, globally neutral detergents (see e.g. Moraes et al. 2014),
making it dubious whether charged APols can be efficiently used for such a task (see
Chap. 11).
These considerations have provided a strong impetus to the search for non-ionic APols, which
was undertaken and carried out in collaboration with the organic chemist Bernard Pucci and his
colleagues. This, however, proved to be quite a difficult and lengthy endeavor, because it is not
straightforward to design, synthesize, and purify in a way that is reasonably economical and easy to
scale up a polymer that features (i) a high density of hydrophobic moieties and (ii) enough non-ionic
hydrophilic groups to endow it with the high solubility that is essential to its use in biochemistry and
biophysics. Two types of non-ionic polar moieties are commonly found in the detergents used for
biochemistry, the ether bonds of polyoxyethylene, as in Triton X-100 or C 12 E 8 , and the hydroxyl
groups carried by sugar moieties, as in octylglucoside or dodecylmaltoside (see Chap. 2, Fig. 2.1).
At this point, hydroxyl groups only have been tested with APols (Fig. 4.5).
4.2.3.1 THAM-Based Non-ionic Amphipols
Most of the non-ionic APols validated to date are derived from tris(hydroxymethyl)acrylamidomethane (THAM; Fig. 4.6), an approach pioneered by Bernard Pucci and his collaborators. The
three hydroxyl groups carried by THAM can be either left free, providing a moderately hydrophilic
monomer, or one or more of them can be grafted with sugar moieties, increasing the hydrophilicity,
and/or with an alkyl chain, creating an amphiphilic monomer. The synthesis of the first non-ionic
APols ever to be tested (Fig. 4.5A) relied on the co-telomerization of (i) an amphipathic monomer
carrying one n-heptyl or n-undecyl alkyl chain and (ii) a hydrophilic monomer carrying either three
free hydroxyls or two free hydroxyls plus one grafted with D-galactose (Fig. 4.7). A range of telomers
were synthesized, whose length varied from ~10 to ~130 monomers (hM n i ranging from ~3 to
~28 kDa), using various combinations of the two types of amphipathic monomers (grafted with either
a C 7 or a C 11 alkyl chain) and hydrophilic ones (ungrafted or carrying one D-galactosyl moiety). Their
solubility was usually ~30–50 gÁL
À1 , which is significantly lower than that of A8-35 (>320 gÁL
À1 ), but
sufficient for carrying out test biochemical experiments. Those were run on two model MPs, BR and
4.2 Amphipol Chemical Structure and Synthesis
165