usually reacted with the free amine functions of UAPol-NH 2 . Provided a suitably functionalized
version of the fluorophore to be grafted is commercially available, which is frequently the case, a
single step is required for the synthesis, purification remaining, usually, the most time-consuming task.
A large variety of FAPols has been synthesized and used for a wide variety of experiments, both
in vitro and in vivo (Table 4.5 and Chap. 8, Fig. 8.9).
In a number of cases, labeling has to be carried out in aqueous solution, after A8-35 or A8-75
particles have already formed (routes C and D in Fig. 4.30). Such is the case for an oligonucleotide
(ODN)-carrying version of A8-35 (OligAPol; Le Bon et al. 2014a). As compared to the syntheses
mentioned above, coupling an ODN to an A8-35 particle presents special challenges, given that the
reaction involves two large partners (~6.6 and ~40 kDa, respectively), both of them polyanions and,
therefore, repulsing each other. Carboxylate functions of A8-35 were reacted with an aminefunctionalized ODN in the presence of zero-length cross-linkers, ethyl-3-[3-dimethylaminopropyl]
carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), leading to the formation of an
amide function. The initial objective of 1 ODN per particle could not be reached, the grafting ratio
being limited to ~0.5 ODN per A8-35 particle. This level of grafting was nevertheless sufficient to
show that OligAPols assemble into well-behaved particles (see § 4.4.2) and to validate them as
efficient tools to trap, handle, and immobilize MPs (Le Bon et al. 2014a; see Chap. 13).
The same approach was used to synthesize SAPols. Derived with 40% isopropylamine, using
HMPAS, A8-75 yields A8-35 (Fig. 4.2). If isopropylamine is replaced with taurine, the end product is
a sulfonated APol (“SAPol”) (Fig. 4.1). However, SAPols cannot be efficiently synthesized by
HMPAS because of the poor solubility of taurine in NMP. Taurine is therefore grafted in aqueous
buffer, in the presence of EDC, onto the surface of already formed A8-75 particles (Dahmane et al.
2011) (route C). It can be assumed that taurine is grafted more or less randomly onto water-exposed
carboxylates lying at their surface. Therefore, whereas the distribution of the octyl chains along the
PAA backbone of SAPols is expected to be random (Magny et al. 1992), that of the taurine residues is
likely biased, because carboxylate-rich regions of the chains stand a higher probability of reacting than
octylamide-rich ones (cf. Fig. 4.18).
4.4.2
Solution Behavior of Labeled or Tagged Derivatives of A8-35 and A8-75
In general, labeling or tagging does not affect the solution properties of A8-35 or A8-75. Such is the
case, for instance, for all FAPols (see e.g. Zoonens et al. 2007; Giusti et al. 2012; Opačić et al. 2014b)
as well as for biotinylated A8-35 (unpublished data) or isotopically labeled APols, except, of course, as
regards DAPol and perDAPol, whenever the properties measured depend on the mass or scattering
length (cf. § 4.3.1.2.2).
Some tags however do affect the solution behavior of APol particles. Such is the case of the
hexahistidine tag of HistAPol or the imidazole moieties of ImidAPol. The batch of ImidAPol most
extensively investigated carried ~6 imidazole moieties per 100 PAA units, i.e. ~20 per 40-kDa particle.
In basic aqueous solutions, its behavior resembles very much that of underivatized A8-35; in acidic
solutions, however, ImidAPol precipitates quantitatively only when the pH is close to 3, due to the
additional hydrophilicity provided, at acidic pH, by protonated imidazoles (F. Giusti, unpublished
results). HistAPol was tagged to the level of 4–5 His 6 tags per particle, or 25–30 imidazole groups, i.e.
a total level slightly higher than that of ImidAPol, and with quite a different distribution, since
imidazoles, in HistAPol, come in groups of six. As is the case for ImidAPol, the solution properties
of HistAPol under acidic conditions differ from those of plain A8-35, a large fraction of the polymer
remaining in solution at pH < 5 (Giusti et al. 2015). At basic pH, HistAPol assembles into more
heterogeneous and slightly larger particles than A8-35 or ImidAPol. From a practical point of view,
4.4 Labeled and Functionalized Amphipols
203
version of the fluorophore to be grafted is commercially available, which is frequently the case, a
single step is required for the synthesis, purification remaining, usually, the most time-consuming task.
A large variety of FAPols has been synthesized and used for a wide variety of experiments, both
in vitro and in vivo (Table 4.5 and Chap. 8, Fig. 8.9).
In a number of cases, labeling has to be carried out in aqueous solution, after A8-35 or A8-75
particles have already formed (routes C and D in Fig. 4.30). Such is the case for an oligonucleotide
(ODN)-carrying version of A8-35 (OligAPol; Le Bon et al. 2014a). As compared to the syntheses
mentioned above, coupling an ODN to an A8-35 particle presents special challenges, given that the
reaction involves two large partners (~6.6 and ~40 kDa, respectively), both of them polyanions and,
therefore, repulsing each other. Carboxylate functions of A8-35 were reacted with an aminefunctionalized ODN in the presence of zero-length cross-linkers, ethyl-3-[3-dimethylaminopropyl]
carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), leading to the formation of an
amide function. The initial objective of 1 ODN per particle could not be reached, the grafting ratio
being limited to ~0.5 ODN per A8-35 particle. This level of grafting was nevertheless sufficient to
show that OligAPols assemble into well-behaved particles (see § 4.4.2) and to validate them as
efficient tools to trap, handle, and immobilize MPs (Le Bon et al. 2014a; see Chap. 13).
The same approach was used to synthesize SAPols. Derived with 40% isopropylamine, using
HMPAS, A8-75 yields A8-35 (Fig. 4.2). If isopropylamine is replaced with taurine, the end product is
a sulfonated APol (“SAPol”) (Fig. 4.1). However, SAPols cannot be efficiently synthesized by
HMPAS because of the poor solubility of taurine in NMP. Taurine is therefore grafted in aqueous
buffer, in the presence of EDC, onto the surface of already formed A8-75 particles (Dahmane et al.
2011) (route C). It can be assumed that taurine is grafted more or less randomly onto water-exposed
carboxylates lying at their surface. Therefore, whereas the distribution of the octyl chains along the
PAA backbone of SAPols is expected to be random (Magny et al. 1992), that of the taurine residues is
likely biased, because carboxylate-rich regions of the chains stand a higher probability of reacting than
octylamide-rich ones (cf. Fig. 4.18).
4.4.2
Solution Behavior of Labeled or Tagged Derivatives of A8-35 and A8-75
In general, labeling or tagging does not affect the solution properties of A8-35 or A8-75. Such is the
case, for instance, for all FAPols (see e.g. Zoonens et al. 2007; Giusti et al. 2012; Opačić et al. 2014b)
as well as for biotinylated A8-35 (unpublished data) or isotopically labeled APols, except, of course, as
regards DAPol and perDAPol, whenever the properties measured depend on the mass or scattering
length (cf. § 4.3.1.2.2).
Some tags however do affect the solution behavior of APol particles. Such is the case of the
hexahistidine tag of HistAPol or the imidazole moieties of ImidAPol. The batch of ImidAPol most
extensively investigated carried ~6 imidazole moieties per 100 PAA units, i.e. ~20 per 40-kDa particle.
In basic aqueous solutions, its behavior resembles very much that of underivatized A8-35; in acidic
solutions, however, ImidAPol precipitates quantitatively only when the pH is close to 3, due to the
additional hydrophilicity provided, at acidic pH, by protonated imidazoles (F. Giusti, unpublished
results). HistAPol was tagged to the level of 4–5 His 6 tags per particle, or 25–30 imidazole groups, i.e.
a total level slightly higher than that of ImidAPol, and with quite a different distribution, since
imidazoles, in HistAPol, come in groups of six. As is the case for ImidAPol, the solution properties
of HistAPol under acidic conditions differ from those of plain A8-35, a large fraction of the polymer
remaining in solution at pH < 5 (Giusti et al. 2015). At basic pH, HistAPol assembles into more
heterogeneous and slightly larger particles than A8-35 or ImidAPol. From a practical point of view,
4.4 Labeled and Functionalized Amphipols
203