4.4
Labeled and Functionalized Amphipols
One of the attractive characteristics of APols is that it is relatively easy to label them, isotopically or
otherwise. Because, if they are not displaced by another surfactant, APols bind tightly to the TM
surface of MPs (Chap. 5), they can therefore be used to label or tag non-covalently but essentially
irreversibly any MP without having to modify it (Chap. 13). As previously observed for longer
polymers (Ringsdorf et al. 1991; Morishima et al. 1995), a broad range of chemical modifications
can be brought to the basic structure of APols without, as a rule, compromising the solubility of the
molecules, their ability to self-associate and to adsorb onto MP TM surfaces, nor the solution
properties of the resulting MP/APol complexes. Furthermore, because (i) each MP binds several
APol molecules – BR, for instance, a small MP (27 kDa), binds ~54 kDa of A8-35 (Gohon et al.
2008) (see Chap. 5), i.e. ~12 molecules – and (ii) APols mix freely both in particles and at the surface
of MPs (Zoonens et al. 2007), complexes carrying two or more functions can be formed by the simple
device of trapping the protein with a mixture of APols (see e.g. Della Pia et al. 2014a, b; Le Bon et al.
2014a, and Chap. 13).
Labeled or tagged APols can be put to a multitude of uses, among which:
• Studying the solution properties of APols (this chapter) and MP/APol complexes (Chaps. 5
and 9); the miscibility of APols (Chap. 5, § 5.7); their biodistribution upon injection into
living organisms (Chap. 15); their association with MPs and the composition, structure, and
dynamics of MP/APol complexes (Chap. 5, §§ 5.3, 5.4, 5.5, and 5.6); the exchange of
surfactants at the surface of MPs (Chap. 5, § 5.7); or the distribution of APols and MP/APol
complexes in fractionation experiments (Chap. 5, § 5.2.1);
• Immobilizing MPs onto solid supports (Chap. 13);
• Modulating the contrast between APols and solvent or MPs in SANS or AUC experiments
(Chap. 9) or improving NMR spectra (Chap. 10);
• Associating an adjuvant to a MP used as an immunogen (Chap. 15);
as well as many other applications still to be validated, some of which are tentatively indicated in
Table 4.5.
Labeling or functionalizing APols can take various courses, each of which has its specific
constraints and advantages regarding both the synthesis and the purification of the derivative. Once
a basic APol structure has been validated, labeling it or endowing it with functional groups is, in
general albeit not always, relatively straightforward (reviewed in Le Bon et al. 2014b). An overview of
functionalized APols that have been validated for practical use is schematically presented in Fig. 4.29
and a list of labeled or functionalized variants of A8-35 and A8-75 in Table 4.5, along with some
indications and references about the uses they have been or could be put to. As regards other types of
APols, a biotinylated derivative of PC-APols has been described by Basit et al. (2012), a biotinylated
NAPol by Ferrandez et al. (2014), and a thiolated SMA by Lindhoud et al. (2016).
4.4.1
Synthesis of Labeled or Tagged Derivatives of A8-35 and A8-75
The various routes toward labeled or functionalized derivatives of A8-35 and A8-75 have been
recently reviewed and their respective fields of application, advantages, and drawbacks discussed by
Le Bon et al. (2014b). Only a simplified overview is provided here. Further details on the chemistry of
the syntheses are provided in Annex 4.6 (§ 4.6.6).
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4 Chemical Structure, Synthesis, and Physical-Chemical Properties of Amphipols
Labeled and Functionalized Amphipols
One of the attractive characteristics of APols is that it is relatively easy to label them, isotopically or
otherwise. Because, if they are not displaced by another surfactant, APols bind tightly to the TM
surface of MPs (Chap. 5), they can therefore be used to label or tag non-covalently but essentially
irreversibly any MP without having to modify it (Chap. 13). As previously observed for longer
polymers (Ringsdorf et al. 1991; Morishima et al. 1995), a broad range of chemical modifications
can be brought to the basic structure of APols without, as a rule, compromising the solubility of the
molecules, their ability to self-associate and to adsorb onto MP TM surfaces, nor the solution
properties of the resulting MP/APol complexes. Furthermore, because (i) each MP binds several
APol molecules – BR, for instance, a small MP (27 kDa), binds ~54 kDa of A8-35 (Gohon et al.
2008) (see Chap. 5), i.e. ~12 molecules – and (ii) APols mix freely both in particles and at the surface
of MPs (Zoonens et al. 2007), complexes carrying two or more functions can be formed by the simple
device of trapping the protein with a mixture of APols (see e.g. Della Pia et al. 2014a, b; Le Bon et al.
2014a, and Chap. 13).
Labeled or tagged APols can be put to a multitude of uses, among which:
• Studying the solution properties of APols (this chapter) and MP/APol complexes (Chaps. 5
and 9); the miscibility of APols (Chap. 5, § 5.7); their biodistribution upon injection into
living organisms (Chap. 15); their association with MPs and the composition, structure, and
dynamics of MP/APol complexes (Chap. 5, §§ 5.3, 5.4, 5.5, and 5.6); the exchange of
surfactants at the surface of MPs (Chap. 5, § 5.7); or the distribution of APols and MP/APol
complexes in fractionation experiments (Chap. 5, § 5.2.1);
• Immobilizing MPs onto solid supports (Chap. 13);
• Modulating the contrast between APols and solvent or MPs in SANS or AUC experiments
(Chap. 9) or improving NMR spectra (Chap. 10);
• Associating an adjuvant to a MP used as an immunogen (Chap. 15);
as well as many other applications still to be validated, some of which are tentatively indicated in
Table 4.5.
Labeling or functionalizing APols can take various courses, each of which has its specific
constraints and advantages regarding both the synthesis and the purification of the derivative. Once
a basic APol structure has been validated, labeling it or endowing it with functional groups is, in
general albeit not always, relatively straightforward (reviewed in Le Bon et al. 2014b). An overview of
functionalized APols that have been validated for practical use is schematically presented in Fig. 4.29
and a list of labeled or functionalized variants of A8-35 and A8-75 in Table 4.5, along with some
indications and references about the uses they have been or could be put to. As regards other types of
APols, a biotinylated derivative of PC-APols has been described by Basit et al. (2012), a biotinylated
NAPol by Ferrandez et al. (2014), and a thiolated SMA by Lindhoud et al. (2016).
4.4.1
Synthesis of Labeled or Tagged Derivatives of A8-35 and A8-75
The various routes toward labeled or functionalized derivatives of A8-35 and A8-75 have been
recently reviewed and their respective fields of application, advantages, and drawbacks discussed by
Le Bon et al. (2014b). Only a simplified overview is provided here. Further details on the chemistry of
the syntheses are provided in Annex 4.6 (§ 4.6.6).
198
4 Chemical Structure, Synthesis, and Physical-Chemical Properties of Amphipols