4.2.2
Other Ionic Amphipols
As will be described in § 4.3, one of the limitations of A8-35 and its congeners is that they owe their
solubility to their carboxylate groups. They are therefore sensitive to factors that affect the solubility of
the latter, such as a low pH or the presence of Ca
2+ ions. Protonation of some of the carboxylates lowers
the solubility of A8-35 (Gohon et al. 2006), as does complexation by Ca
2+ and, to a much smaller extent,
Mg
2+ ions (Picard et al. 2006; see § 4.3.2). Under such conditions, free A8-35 particles tend to aggregate
and may precipitate, and so do MP/A8-35 complexes. The pH sensitivity is particularly annoying in
solution NMR experiments, where the observation of water-exposed amide protons is easiest at a slightly
acidic pH, which slows down their exchange with those of water. This led, quite rapidly, to attempts at
diversifying APol structures and, in particular, at developing APols that would be pH- and Ca
2+
-
insensitive. Several routes have been successfully explored (Table 4.1). One of them is to replace the
carboxylates with non-ionic moieties. The resulting non-ionic APols will be described in § 4.2.3. Another
is to endow ionic APols with additional or alternative ionic groups that will keep them soluble even at
low pH or in the presence of calcium ions, as described in the present section.
4.2.2.1 Sulfonated Amphipols
Sulfonated APols (SAPols) are variants of A8-35 in which the isopropylamine groups are replaced
with taurine ones (Fig. 4.1d). In terms of charge density along the chain, they are therefore similar to
A8-75. The development of SAPols was undertaken shortly after A8-35, and its congeners had been
validated for use in NMR studies (Zoonens et al. 2005), and a first series of comparative biochemical
experiments was published in 2006 (Picard et al. 2006). However, a full description of their synthesis
and properties and the validation of their use for solution NMR studies had to wait until 2011
(Dahmane et al. 2011). The synthesis of SAPols will be described below in § 4.4.1, along with that
of functionalized APols.
The purification of SAPols is more laborious than that of A8-35 (Dahmane et al. 2011). The
purification of A8-35 and its congeners takes advantage of the fact that they are insoluble at acidic pH,
so that several cycles of precipitation at pH < 2 followed by redissolution at pH > 8 make it straightforward to get rid of both hydrosoluble and poorly soluble contaminants, even on large batches (see § 4.5
Protocol 4.1). SAPols however cannot be precipitated in acidic solutions. They have to be separated from
contaminants by large-scale SEC, which is much more time-consuming and has hampered their marketing.
At low pH, or in the presence of Ca
2+ ions, some or all of the charges carried by the carboxylate
moieties of SAPols will be neutralized. Nevertheless, in keeping with the behavior of A8-35, which
comprises only 35% of ungrafted carboxylates, the 40% of monomers grafted with taurine are sufficient
to keep SAPols water-soluble down to pH À1 (Dahmane et al. 2011). Similarly, SAPol particles and
MP/SAPol complexes are insensitive to the presence of divalent cations (Picard et al. 2006).
4.2.2.2 Phosphorylcholine-Based Amphipols
The structures of phosphorylcholine-based zwitterionic APols (PC-APols; Fig. 4.1C) are similar to
those of A8-35 and A8-75, except that the free carboxylates are replaced with a phosphorylcholinebearing zwitterionic group. Their synthesis follows quite a different route, as it does not rely on
grafting a pre-existing PAA, but on (i) synthesizing a terpolymer precursor by reversible additionfragmentation chain transfer (RAFT; see Chiefari et al. 1998 and Annex 4.6, § 4.6.4.2) and (ii) grafting
phosphorylcholine groups onto it by reductive amination of phosphorylcholine glyceraldehyde (Diab
et al. 2007b). PC-APols are not currently commercial.
Given their chemical structure, PC-APols are expected to carry no net charge at neutral and basic
pH. At acidic pH, however, the secondary amine they carry will protonate and they will become
162
4 Chemical Structure, Synthesis, and Physical-Chemical Properties of Amphipols
Other Ionic Amphipols
As will be described in § 4.3, one of the limitations of A8-35 and its congeners is that they owe their
solubility to their carboxylate groups. They are therefore sensitive to factors that affect the solubility of
the latter, such as a low pH or the presence of Ca
2+ ions. Protonation of some of the carboxylates lowers
the solubility of A8-35 (Gohon et al. 2006), as does complexation by Ca
2+ and, to a much smaller extent,
Mg
2+ ions (Picard et al. 2006; see § 4.3.2). Under such conditions, free A8-35 particles tend to aggregate
and may precipitate, and so do MP/A8-35 complexes. The pH sensitivity is particularly annoying in
solution NMR experiments, where the observation of water-exposed amide protons is easiest at a slightly
acidic pH, which slows down their exchange with those of water. This led, quite rapidly, to attempts at
diversifying APol structures and, in particular, at developing APols that would be pH- and Ca
2+
-
insensitive. Several routes have been successfully explored (Table 4.1). One of them is to replace the
carboxylates with non-ionic moieties. The resulting non-ionic APols will be described in § 4.2.3. Another
is to endow ionic APols with additional or alternative ionic groups that will keep them soluble even at
low pH or in the presence of calcium ions, as described in the present section.
4.2.2.1 Sulfonated Amphipols
Sulfonated APols (SAPols) are variants of A8-35 in which the isopropylamine groups are replaced
with taurine ones (Fig. 4.1d). In terms of charge density along the chain, they are therefore similar to
A8-75. The development of SAPols was undertaken shortly after A8-35, and its congeners had been
validated for use in NMR studies (Zoonens et al. 2005), and a first series of comparative biochemical
experiments was published in 2006 (Picard et al. 2006). However, a full description of their synthesis
and properties and the validation of their use for solution NMR studies had to wait until 2011
(Dahmane et al. 2011). The synthesis of SAPols will be described below in § 4.4.1, along with that
of functionalized APols.
The purification of SAPols is more laborious than that of A8-35 (Dahmane et al. 2011). The
purification of A8-35 and its congeners takes advantage of the fact that they are insoluble at acidic pH,
so that several cycles of precipitation at pH < 2 followed by redissolution at pH > 8 make it straightforward to get rid of both hydrosoluble and poorly soluble contaminants, even on large batches (see § 4.5
Protocol 4.1). SAPols however cannot be precipitated in acidic solutions. They have to be separated from
contaminants by large-scale SEC, which is much more time-consuming and has hampered their marketing.
At low pH, or in the presence of Ca
2+ ions, some or all of the charges carried by the carboxylate
moieties of SAPols will be neutralized. Nevertheless, in keeping with the behavior of A8-35, which
comprises only 35% of ungrafted carboxylates, the 40% of monomers grafted with taurine are sufficient
to keep SAPols water-soluble down to pH À1 (Dahmane et al. 2011). Similarly, SAPol particles and
MP/SAPol complexes are insensitive to the presence of divalent cations (Picard et al. 2006).
4.2.2.2 Phosphorylcholine-Based Amphipols
The structures of phosphorylcholine-based zwitterionic APols (PC-APols; Fig. 4.1C) are similar to
those of A8-35 and A8-75, except that the free carboxylates are replaced with a phosphorylcholinebearing zwitterionic group. Their synthesis follows quite a different route, as it does not rely on
grafting a pre-existing PAA, but on (i) synthesizing a terpolymer precursor by reversible additionfragmentation chain transfer (RAFT; see Chiefari et al. 1998 and Annex 4.6, § 4.6.4.2) and (ii) grafting
phosphorylcholine groups onto it by reductive amination of phosphorylcholine glyceraldehyde (Diab
et al. 2007b). PC-APols are not currently commercial.
Given their chemical structure, PC-APols are expected to carry no net charge at neutral and basic
pH. At acidic pH, however, the secondary amine they carry will protonate and they will become
162
4 Chemical Structure, Synthesis, and Physical-Chemical Properties of Amphipols