Aggregation behavior and dynamics of synthetic amphiphiles that self-assemble to anion transporters

The amphiphilic heptapeptides-referred to as synthetic anion transporters (SATs)-mediate chloride transport in planar lipid bilayer membranes, synthetic liposomes, and mammalian cells. The SATs described have the general formula R1(2)NCOCH2OCH2CO-(Gly)3-Pro-(Gly)3-OR2. Substitution at R1 and R2 with various aliphatic or aromatic groups alters the ability of SATs to transport chloride through a phospholipid bilayer membrane. Despite extensive structure-activity relationship studies concerning Cl(-)-mediated transport by SATs, relatively little was known about the mechanism of insertion and pore-formation in the membrane. In the current study, the mechanistic behavior of SATs was investigated in aqueous solution and at the air-water interface. In the latter case, Langmuir trough studies and Brewster angle microscopy (BAM) revealed the extent of monolayer stability and organization for SATs. Dynamic light scattering and transmission electron microscopy (TEM) confirmed these results and defined the aggregation behavior of SATs in solution. SAT derivatives that showed low chloride transport activity organized into stable monolayers at the air-water interface, while more active SATs formed less stable monolayers. The relationship between intermolecular organization of SATs and pore-formation in the membrane is discussed along with its implications for chloride transport.     

Air–water interfacial behavior of amphiphilic peptide analogs of synthetic chloride ion transporters

A family of heptapeptide-based chloride transporters (called synthetic anion transporters, SATs) were designed to insert into phospholipid membrane bilayers and form pores. Many of these compounds have proved to be chloride selective transporters. The transporters were designed to incorporate hydrophilic heptapeptides that could serves as headgroups and hydrocarbon tails that could serve as hydrophobic membrane anchors. Insertion of the SAT molecules into a bilayer requires approach to and insertion at the aqueous-membrane surface. The studies reported here were conducted to model and understand this process by studying SAT behavior at the air–water interface. A Langmuir trough was used to obtain surface pressure–area isotherm data. These data for amphiphilic SATs were augmented by Brewster angle microscopy (BAM), molecular modeling, and calculations of the hydrophobicity parameter log P. The analyses showed that the heptapeptide (hydrophilic) module of the SAT molecule rested on the water surface while the dialkyl (hydrophobic) tails oriented themselves in the air, perpendicular to the water surface. Brewster angle microscopy visually confirmed a high order of molecular organization. Results from these studies are consistent with the previously proposed mechanism of SAT membrane insertion and pore formation.     

Anion transport properties of amine and amide-sidechained peptides are affected by charge and phospholipid composition

Four synthetic anion transporters (SATs) having the general formula (n-C(18)H(37))(2)N-COCH(2)OCH(2)CO-(Gly)(3)Pro-Lys(epsilon-N-R)-(Gly)(2)-O-n-C(7)H(15) were prepared and studied. The group R was Cbz, H (TFA salt), t-Boc, and dansyl in peptides 1, 2, 3, and 4 respectively. The glutamine analog (GGGPQAG sequence) was also included. A dansyl-substituted fluorescent SAT was used to probe peptide insertion; the dansyl sidechain resides in an environment near the bilayer's midpolar regime. When the lysine sidechain was free or protected amine, little effect was noted on final Cl(-) transport rate in DOPC : DOPA (7 : 3) liposomes. This stands in contrast to the significant retardation of transport previously observed when a negative glutamate residue was present in the peptide sequence. It was also found that Cl(-) release from liposomes depended on the phospholipid composition of the vesicles. Chloride transport diminished significantly for the free lysine containing SAT, 2, when the lipid was altered from DOPC : DOPA to pure DOPC. Amide-sidechained SATs 1 and 5 showed a relatively small decrease in Cl(-) transport. The effect of lipid composition on Cl(-) transport was explained by differences in electrostatic interaction between amino acid sidechain and lipid headgroup, which was modeled by computation.     

Carboxylate anion diminishes chloride transport through a synthetic, self-assembled transmembrane pore

Six amphiphilic heptapeptides with the structure (C18H37)2NCOCH2OCH2CO-(Gly)3-Pro-(Gly)n-(Glx)-(Gly)m-O(CH2)6CH3, in which Glx represents glutamic acid or its benzyl ester and n+m=2, have been studied. In addition, the glutamate residue in the GGGPGGE sequence was esterified by fluorescent 1-pyrenemethanol. These compounds insert into phospholipid bilayers and form anion-conducting pores. Hill plots based on carboxyfluorescein release indicate that the pores are at least dimeric. Studies that involved ion-selective electrode techniques showed that transport of chloride varied with the position of glutamate within the peptide chain and whether glutamic acid was present as the free acid or its benzyl ester. Chloride transport activity was significantly higher for the glutamate esters than for free carboxylates irrespective of the glutamate position. Activity was highest when the glutamate residue in approximately (Gly)3-Pro-(Xxx)3 approximately was closest to the C terminus of the peptide. A fluorescent pyrene residue was introduced to probe the aggregation state of the amphiphile. The selectivity of the pore for Cl(-) over K+ was maintained even when the carboxylate anion was present within it. Complexation of Cl(-) by the ionophoric peptides was confirmed by negative ion mass spectrometry. Planar bilayer voltage clamp experiments confirmed that pores with more than one conductance state may form in these dynamic, self-assembled pores.     

Aggregation and supramolecular membrane interactions that influence anion transport in tryptophan-containing synthetic peptides

Self-assembly is a desired property in supramolecular chemistry, but extensive aggregation may be counterproductive. Rigid systems typically have better organization, but are inherently less dynamic. This work shows that ion transport by amphiphilic heptapeptides (synthetic anion transporters or SATs) is affected by aggregation of the monomers in the bulk aqueous phase to which they are added and within the bilayer. Ion transport was assessed for all compounds by assay of Cl(-) release from liposomes. The mechanism of ion transport was confirmed by planar bilayer conductance studies for two compounds at opposite ends of the efficacy scale. Dynamic light scattering, the Langmuir trough, transmission electron microscopy, ion release from liposomes, and planar bilayer conductance studies were used to assess the importance of self-assembly versus aggregation in ion transport. Generally, greater aggregation was has an adverse effect on the transport, although at least dimerization is required for amphiphilic heptapeptides to readily transport Cl(-). Anion transport in these systems was found to be sensitive to changes in the C-terminal portion of the (Gly)(3)Pro(Gly)(3) sequence. Moreover, a significant difference in transport efficacy was apparent when L-Trp was replaced by D-Trp in the same position.     

Aggregation and Supramolecular Membrane Interactions that Influence Anion Transport in Tryptophan‐Containing Synthetic Peptides

Self‐assembly is a desired property in supramolecular chemistry, but extensive aggregation may be counterproductive. Rigid systems typically have better organization, but are inherently less dynamic. This work shows that ion transport by amphiphilic heptapeptides (synthetic anion transporters or SATs) is affected by aggregation of the monomers in the bulk aqueous phase to which they are added and within the bilayer. Ion transport was assessed for all compounds by assay of Cl^? release from liposomes. The mechanism of ion transport was confirmed by planar bilayer conductance studies for two compounds at opposite ends of the efficacy scale. Dynamic light scattering, the Langmuir trough, transmission electron microscopy, ion release from liposomes, and planar bilayer conductance studies were used to assess the importance of self‐assembly versus aggregation in ion transport. Generally, greater aggregation was has an adverse effect on the transport, although at least dimerization is required for amphiphilic heptapeptides to readily transport Cl^?. Anion transport in these systems was found to be sensitive to changes in the C‐terminal portion of the (Gly)₃Pro(Gly)₃ sequence. Moreover, a significant difference in transport efficacy was apparent when L ‐Trp was replaced by D ‐Trp in the same position.     

Transport of chloride and carboxyfluorescein through phospholipid vesicle membranes by heptapeptide amphiphiles

Seven synthetic anion transporters (SAT) of the general form R(2)N-COCH(2)OCH(2)CO-(Gly)(3)-Pro-(Gly)(3)-OR' were prepared. Three pairs of compounds each contained twin n-hexyl, n-decyl, and n-octadecyl (R) groups at the N-terminus and one contained twin n-tetradecyl groups. Three of the compounds were C-terminated by benzyl and three by heptyl (R') residues. The ability of these compounds to mediate ion release from phospholipid vesicles was assessed. Chloride release was measured by ion selective electrode measurements and by chloride quenching of the fluorescent dye lucigenin. Transport of the anion carboxyfluorescein (CF) was measured by fluorescence dequenching. Differences in both the C- (R') and N-terminal (R) residues within the ionophores affected anion transport. The chloride release data acquired by ion selective electrode and fluorescence methods were similar but not identical. A possible carrier mechanism for Cl(-) transport was discredited. Both Cl(-) and CF anions were released from vesicles by these compounds. The results of CF and Cl(-) transport showed good consistency when the ionophore's N-terminal chains were either decyl or octadecyl but not when they were hexyl. The transport of CF and Cl(-) appears to be fundamentally different when R is C(6) compared to C(10) or C(18). Differences between the behavior of SATs with Cl(-) and CF were also reflected in negative ion mass spectrometric studies.     

Effects of amphiphile topology on the aggregation of oligocholates in lipid membranes: macrocyclic versus linear amphiphiles

A macrocyclic and a linear trimer of a facially amphiphilic cholate building block were labeled with a fluorescent dansyl group. The environmentally sensitive fluorophore enabled the aggregation of the two oligocholates in lipid membranes to be studied by fluorescence spectroscopy. Concentration-dependent emission wavelength and intensity revealed a higher concentration of water for the cyclic compound. Both compounds were shown by the red-edge excitation shift (REES) to be located near the membrane/water interface at low concentrations, but the cyclic trimer was better able to migrate into the hydrophobic core of the membrane than the linear trimer. Fluorescent quenching by a water-soluble (NaI) and a lipid-soluble (TEMPO) quencher indicated that the cyclic trimer penetrated into the hydrophobic region of the membrane more readily than the linear trimer, which preferred to stay close to the membrane surface. The fluorescent data corroborated with the previous leakage assays that suggested the stacking of the macrocyclic cholate trimer into transmembrane nanopores, driven by the strong associative interactions of water molecules inside the macrocycles in a nonpolar environment.     

Fluorescence quenching of anthracene by indole derivatives in phospholipid bilayers

The quenching of anthracene fluorescence by indole, 1,2-dimethylindole (DMI), tryptophan (Trp) and indole 3-acetic acid (IAA) in palmitoyloleoylphosphatidylcholine (POPC) lipid bilayers was investigated. A very efficient quenching of the anthracene fluorescence in the lipid membrane is observed. Stern-Volmer plots are linear for DMI but present a downward curvature for the other quenchers. This was interpreted as an indication of the presence of an inaccessible fraction of anthracene molecules. By a modified Stern-Volmer analysis the fraction accessible to the quenchers and the quenching constant were determined. The changes in the fluorescence emission spectrum of indole and DMI have been used to calculate the partition constants of these probes into the membranes, and bimolecular quenching rate constants were determined in terms of the local concentration of quencher in the lipid bilayer. The rate constants are lower than those in homogeneous solvents, which may be ascribed to a higher viscosity of the bilayer. No changes in the emission spectra of Trp and IAA are observed in the presence of vesicles, indicating that these probes locate preferentially in the aqueous phase, or in close proximity to the vesicular external interface in a medium resembling pure water. In these cases quenching rate constants were determined in terms of the analytical concentration. In the quenching by DMI a new, red shifted, emission band appears; it is similar to that observed in non-polar solvents and it is ascribable to an exciplex emission. The exciplex band is absent in the quenching by IAA and Trp and only very weakly present when the quencher is indole. From the position of the maximum of the exciplex emission, a relatively high local polarity could be estimated for the region of the bilayer where the quenching reaction takes place.     

Transmission of binding information across lipid bilayers

A synthetic transmembrane receptor that is capable of transmitting binding information across a lipid bilayer membrane is reported. The binding event is based on aggregation of the receptor triggered by copper(II) complexation to ethylenediamine functionalities. By labelling the receptor with fluorescent dansyl groups, the copper(II) binding event could be monitored by measuring the extent of fluorescence quenching. Comparing the receptor with a control receptor lacking the transmembrane linkage revealed that the transmembrane receptor binds copper(II) ions more tightly than the non-spanning control receptor at low copper(II) concentrations. Since the intrinsic binding to copper(II) is the same for both receptors, this effect was attributed to synergy between the connected interior and exterior binding sides of the transmembrane receptor. Thus, this is the first reported artificial signalling event in which binding of a messenger on one side of the membrane leads to a cooperative binding event on the opposite side of the membrane, resembling biological signalling systems and helping us to get a better understanding of the requirements for more effective artificial signalling systems.     

Triazole‐Tailored Guanosine Dinucleosides as Biomimetic Ion Channels to Modulate Transmembrane Potential

A “click” ion channel platform has been established by employing a clickable guanosine azide or alkyne with covalent spacers. The resulting guanosine derivatives modulated the traffic of ions across the phospholipid bilayer, exhibiting a variation in conductance spanning three orders of magnitude (pS to nS). Förster resonance energy transfer studies of the dansyl fluorophore with the membrane binding fluorophore Nile red revealed that the dansyl fluorophore is deeply embedded in the phospholipid bilayer. Complementary cytosine can inhibit the conductance of the supramolecular guanosine channels in the phospholipid bilayers.     

Photoinduced electron transfer in thin layers composed of fullerene-cyclic peptide conjugate and pyrene derivative

A bilayer structure was constructed on gold by Langmuir-Blodgett deposition of a fullerene (C 60)-cyclic peptide-poly(ethylene glycol) (PEG) conjugate and thereafter a pyrene derivative from the air/water interface. The cyclic peptide moiety acts as a scaffold to prevent the fullerenes from self-aggregation and accordingly makes the monolayer homogeneous and stable. In addition to this gold/C 60-cyclic peptide-PEG/pyrene bilayer, a pyrene monolayer, a gold/C 60-PEG conjugate/pyrene bilayer (lacking the peptide scaffold), and a gold/pyrene/C 60-cyclic peptide-PEG bilayer (with the opposite order of layers) were also prepared, and their anodic photocurrent generation were studied in an aqueous solution containing a sacrifice electron donor. The most efficient photocurrent generation was observed in the gold/C 60-cyclic peptide-PEG/pyrene bilayer. It is considered that the C 60 unit acts not only as sensitizer but also as an electron acceptor facilitating the electron transfer from the excited pyrene unit to gold, and that the fullerene layer suppresses quenching of the excited pyrene unit by energy transfer to gold. Furthermore, the cyclic peptide scaffold helps the fullerenes disperse without aggregation in the membrane and seems to protect their redox properties or inhibit self-quenching of their excited state. It is thus concluded that a bilayer structure with desired orientation of functional units is important for efficient photoinduced electron transfer and that a cyclic peptide scaffold is useful to locate hydrophobic functional groups properly in a thin layer.     

Electrostatic contributions to indole-lipid interactions

The role of electrostatic forces in indole-lipid interactions was studied by (1)H and (2)H NMR in ether- and ester-linked phospholipid bilayers with incorporated indole. Indole-ring-current-induced (1)H NMR chemical shifts of lipid resonances in bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, and 1,2-di-O-octadecenyl-sn-glycero-3-phosphomethanol show a bimodal indole distribution, with indole residing at the upper hydrocarbon chain/glycerol region of the lipid and near the choline group, when present. (2)H NMR of indole-d(7)-incorporated lipid bilayers reveals that the former site is occupied by about two-thirds of the indole, which adopts a distinct preferred orientation with respect to the bilayer normal. The results suggest that the upper hydrocarbon chain/glycerol location is dictated by many factors, including interactions with the electric charges and dipoles, van der Waals interactions, entropic contributions, and hydrogen bonding. Indole diffusion rates are higher in lipids with ester bonds and lower in choline-containing lipids, suggesting that interactions between indole and carbonyl groups are of minor importance for lipid-indole association and that cation-pi interactions with choline drive the second indole location. Nuclear Overhauser effect spectroscopy cross-relaxation rates suggest a 30-ns lifetime for indole-lipid associations. These results may have important implications for sidedness and structural transitions in tryptophan-rich membrane proteins.     

Photophysics of anthracene-indole systems in unilamellar vesicles of DMPC and POPC: Exciplex formation and temperature effects

The quenching of anthracene fluorescence by indole (IN), 1,2-dimethylindole (DMI), tryptophan (Trp) and indole 3-acetic acid (IAA) in dimiristoylphophatidylcholine (DMPC) and palmitoyloleoylphosphatidylcholine (POPC) lipid bilayers was investigated. The studies were carried out at 25 degrees C in POPC vesicles and below (15 degrees C) and above (35 degrees C) the phase transition temperature (24 degrees C) of DMPC. A very efficient quenching of the anthracene fluorescence by IN and DMI in the lipid membrane is observed in all cases. It is less efficient in the case of Trp and IAA. Stern-Volmer plots are linear for DMI but present a downward curvature for the other quenchers. This was interpreted as an indication of the presence of an inaccessible fraction of anthracene molecules. By a modified Stern-Volmer analysis the fraction accessible to the quenchers and the quenching constant were determined. Partition constants of the quenchers were obtained from the changes in the fluorescence emission of the indole moiety caused by the presence of the phospholipid. Using the partition constants bimolecular quenching rate constants were determined in terms of the local concentration of quencher in the lipid bilayer. These corrected rate constants are lower than those in homogeneous solvents. In the case of DMPC values the gel phase are higher than in the liquid-crystalline phase. In the quenching by IN and DMI a new, red shifted, emission band appears which could be assigned to an exciplex emission. The exciplex band is absent in the quenching by IAA and Trp.     

Self-assembly of flexible β-strands into immobile amyloid-like β-sheets in membranes as revealed by solid-state 19F NMR

The cationic peptide [KIGAKI](3) was designed as an amphiphilic β-strand and serves as a model for β-sheet aggregation in membranes. Here, we have characterized its molecular conformation, membrane alignment, and dynamic behavior using solid-state (19)F NMR. A detailed structure analysis of selectively (19)F-labeled peptides was carried out in oriented DMPC bilayers. It showed a concentration-dependent transition from monomeric β-strands to oligomeric β-sheets. In both states, the rigid (19)F-labeled side chains project straight into the lipid bilayer but they experience very different mobilities. At low peptide-to-lipid ratios ≤1:400, monomeric [KIGAKI](3) swims around freely on the membrane surface and undergoes considerable motional averaging, with essentially uncoupled φ/ψ torsion angles. The flexibility of the peptide backbone in this 2D plane is reminiscent of intrinsically unstructured proteins in 3D. At high concentrations, [KIGAKI](3) self-assembles into immobilized β-sheets, which are untwisted and lie flat on the membrane surface as amyloid-like fibrils. This is the first time the transition of monomeric β-strands into oligomeric β-sheets has been characterized by solid-state NMR in lipid bilayers. It promises to be a valuable approach for studying membrane-induced amyloid formation of many other, clinically relevant peptide systems.     

The effect of cholesterol on the adsorption of phenyltin compounds onto phosphatidylcholine and sphingomyelin liposome membranes

Organometallic compounds are widely spread in the human environment sometimes, causing a substantial health risk. Their amphiphilic character enables them to intercalate and penetrate cell membranes, potentially affecting various vital cell functions. Compound adsorption onto the membrane depends on the compound properties, as well as on the membrane composition and state. When adsorbing onto the lipidic surface, phenyltins localize at areas where lipid bilayer organization is compatible with compound spatial requirements. The lipid bilayer is a dynamic and laterally nonuniform structure with complex local and global architecture correlated with a variety of cell functions. The selective binding of a toxic compound to selected membrane areas may, therefore, interfere with some types of cellular process. We present experimental results concerning phenyltin adsorption onto the lipid bilayer surface measured with the fluorescent probe fluorescein‐PE. Model lipid bilayers were formed from lipid mixtures mimicking various plasma membrane regions. The adsorption of Ph₃SnCl and P₂SnCl₂ onto the phosphatidylcholine–cholesterol bilayer was qualitatively different from sphingomyelin–cholesterol. The results presented indicate that phenyltins are likely to accumulate in areas containing phosphatidylcholine, outside of lipid rafts. Copyright © 2004 John Wiley & Sons, Ltd.     

Design, synthesis and characterisation of a fluorescently labelled CyPLOS ionophore

A novel fluorescently labelled synthetic ionophore, based on a cyclic phosphate-linked disaccharide (CyPLOS) backbone and decorated with four tetraethylene glycol tails carrying dansyl units, has been synthesised in 12 steps in 26% overall yield. The key intermediate in the synthetic strategy is a novel glucoside building block, serving through its 2- and 3-hydroxy groups as the anchor point for flexible tetraethylene glycol tentacles with reactive azido moieties at their ends. To test the versatility of this glucoside scaffold, it was preliminarily functionalised with a set of diverse probes--as fluorescent, redox-active or hydrophobic tags--either by reduction of the azides followed by condensation with activated carboxylic acid derivatives, or by a direct coupling with a terminal alkyne in a Cu(I)-promoted 1,3-dipolar cycloaddition. Tagging of the monomeric building block with dansyl residues allowed us to prepare a fluorescent, amphiphilic macrocycle, which was investigated for its propensity to self-aggregate in CDCl(3)--studied by means of concentration-dependent (31)P NMR spectroscopy experiments--and in aqueous solution, in which combined dynamic light scattering (DLS) and small-angle neutron scattering (SANS) measurements provided a detailed physico-chemical analysis of the self-assembled systems, mainly organised in the form of large vesicles. Its ion-transport properties through phospholipid bilayers, determined by HPTS fluorescence assays, showed this compound to be more active than the previously synthesised CyPLOS congeners. Solvent-dependent fluorescence changes for the labelled ionophore in liposome suspension established that the dansyl moieties are dispersed in environments with polarity intermediate between those of CH(2)Cl(2) and propan-2-ol, suggesting that the CyPLOS tentacles infiltrate the mid-polar region of the membranes.     

Pyrene-sensitized electron transport across vesicle bilayers: Dependence of transport efficiency on pyrene substituents

Endoergic electron transport across vesicle bilayers from ascorbate (Asc-) in the inner waterpool to methylviologen (MV2+) in the outer aqueous solution was driven by the irradiation of pyrene derivatives embedded in the vesicle bilayers. The initial rate of MV2+ reduction is dependent on the substituent group of the pyrenyl ring; a hydrophilic functional group linked with the pyrenyl ring by a short methylene chain acts as a sensitizer for the electron transport. Mechanistic studies using (1-pyrenyl)alkanoic acids (1a-c) as sensitizers suggest that the electron transport is mainly initiated by the reductive quenching of the singlet excited state of the pyrene by Asc- and proceeds by a mechanism involving electron exchange between the pyrenes located at the inner and outer interface across the vesicle bilayer. We designed and synthesized novel unsymmetrically substituted pyrenes having both a hydrophilic group linked by a short methylene chain and a hydrophobic long alkyl group (5a-c), which acted as excellent sensitizers for the electron transport across vesicle bilayers.     

Voltage-dependent formation of anion channels by synthetic rigid-rod push-pull beta-barrels

Ion channels formed by p-octiphenyls equipped with amphiphilic, cationic tripeptide strands and either with (5) or without (6) axial dipole moment are described (preliminary communication: N. Sakai, S. Matile, J. Am. Chem. Soc. 2002, 124, 1184-1185). Fluorescence kinetics with variably polarized neutral or anionic vesicles, together with planar bilayer conductance measurements, reveal voltage dependence with weakly lyotropic anion selectivity, and deactivation by competing surface potentials of the ion channels formed by asymmetric 5. In planar bilayers, 5 forms short-lived, poorly organized channels--similar to those produced by alpha-helical natural antibiotics--capable of transforming into stable, ohmic p-octiphenyl "beta-barrel" ion channels similar to those of the >99 % homologous but symmetric 6. Fluorescence depth quenching and circular dichroism studies confirm the effect of membrane potentials in promotion of the partitioning of 5 (but not 6) into the bilayers, identifying partitioning as the voltage-dependent step.     

The unusual micelle micropolarity of partially fluorinated gemini surfactants sensed by pyrene fluorescence

New gemini surfactants having two fluorocarbon chains were prepared by refluxing partially fluorinated alkyl bromide with N,N,N',N'-tetramethyl-1,6-diaminohexane in acetonitrile. The partially fluorinated gemini surfactants containing a six-methylene spacer chain are easily soluble in water. The critical micelle concentrations (cmc's) were determined by various fluorescent probe methods. The hydrophobicity of a CF2 group was estimated to be 1.5 times that of a CH2 group according to the cmc values. The micelle micropolarity of a fluorocarbon gemini sensed by pyrene fluorescence was unusually high, suggesting an apparent iceberg-like environment in the location of pyrene. The significantly small micelle aggregation numbers of fluorinated gemini surfactants were ascertained by the pyrene fluorescence quenching method. The micelle ionization degree estimated by fluorescence quenching of 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) gave tendencies similar to those of the corresponding hydrocarbon geminis.