Liposomal Circular Dichroism (L-CD) of Arenoyl Derivatives of Sphingolipids. Amplification of Cotton Effects in Ordered Lipid Bilayers

Liposomal circular dichroism (L-CD) of acyclic amino alcohols exhibit amplification of Cotton effects when measured in highly uniform, unilamellar liposomes. The effect is likely due to intermolecular associations—H-aggregates—that self-assemble spontaneously within the lipid bilayer, and persists over long time scales. L-CD spectra of N,O,O′-tri-(6′methoxy-2′-naphthoyl)-d-erythro-sphingosine, or the corresponding dihydro-derivative (sphinganine), shows ~10-fold amplification of magnitudes of Cotton effects over conventional CD spectra recorded in isotropic solution.

Although the above CD methods for acyclic systems are sensitive (the sample requirement is of the order of µg) the critical drawback is still the limit of detection. Free bond rotation and other degrees of freedom can average out CEs that arise from weak perturbations of chromophores within an asymmetric sphere, or even the stronger pair-wise exciton couplings, although-as we have shown above-this can be partially compensated for by use of chromophores with stronger electronic transition dipole moments [12]. Here, we describe a CD method that produced significant gains in the magnitudes of the Cotton effects of N,O,O′-triacyl sphingolipids when their CD spectra were measured in liposomal formulations. When arrayed in uniform, unilamellar liposomes, prepared by membrane extrusion methods, the membrane-bound lipids, bearing arylcarboxylate chromophores, assemble into ordered arrays that exhibit intermolecular interactions through delocalized excitons-the so-called J-and H-aggregates [28,29]. The corresponding liposomal circular dichroism (L-CD) spectra exhibit more complex features including longer wavelength CEs with dramatic amplifications of magnitude.
Although the above CD methods for acyclic systems are sensitive (the sample requirement is of the order of µg) the critical drawback is still the limit of detection. Free bond rotation and other degrees of freedom can average out CEs that arise from weak perturbations of chromophores within an asymmetric sphere, or even the stronger pair-wise exciton couplings, although-as we have shown above-this can be partially compensated for by use of chromophores with stronger electronic transition dipole moments [12]. Here, we describe a CD method that produced significant gains in the magnitudes of the Cotton effects of N,O,O -triacyl sphingolipids when their CD spectra were measured in liposomal formulations. When arrayed in uniform, unilamellar liposomes, prepared by membrane extrusion methods, the membrane-bound lipids, bearing arylcarboxylate chromophores, assemble into ordered arrays that exhibit intermolecular interactions through delocalized excitons-the so-called J-and H-aggregates [28,29]. The corresponding liposomal circular dichroism (L-CD) spectra exhibit more complex features including longer wavelength CEs with dramatic amplifications of magnitude.
(N-benzoylimidazole (5), DBU, CH3CN, 70 °C) to the corresponding perbenzoate (6) which was subsequently hydrogenated (H2, 1 atm, 10% Pd-C, EtOAc, 23 °C) to give N,O,O′-tribenzoylsphinganine (7) [16]. A parallel sequence of reactions gave pernaphthoyl derivative, 8, by acylation of 1a (N-Np-imidazole, 9, DBU, CH3CN, 60 °C), which was subsequently hydrogenated under the above conditions to give the pernaphthoyl sphinganine 10. For purposes of comparison, the long-chain 2-vinylnaphthalene 11 and the simple primary Np ester 12, prepared from the known long-chain primary alcohol 13 [7], were also secured. The CD spectra of the 6-8, 10-12 were measured under two sets of conditions: isotropic (MeOH solution) and anisotropic conditions obtained by formulation into membrane-bound unilamellar liposomes. Each of the latter samples was prepared from crude liposomes (sonication) by repeated extrusion through a porous membrane (100 Å pore size), which, as shown previously [30,32,33], gave uniform spherical liposomes of approximately 25-30 nm in diameter. [30] Measurement of chiroptical properties of these uniform nanoparticles can be made with little or no light scattering, and reveal complex features (absent from CD spectra recorded under isotropic conditions) that we attribute to intermolecular excitons.
The CD spectrum of N,O,O′-tribenzoyl sphingosine 6 in MeOH ( Figure 2a, Table 1) has relatively simple features: bisignate peaks due to a simple ECCD effect of relatively weak magnitude (∆ε +8.2 (224 nm), −2.2 (240 nm)), and a short-wavelength edge (λ ~ 205 nm) of a CE masked by end-absorption of the MeOH solvent: essentially identical to that reported from earlier studies [16,23] In sharp contrast, the L-CD spectrum of the 6, formulated in DSPC [31] unilamellar liposomes (aqueous), shows more complex behavior dominated by three strong CEs with greatly amplified magnitudes: a shorter wavelength negative band (∆ε −42 (λ = 210 nm), a significantly longer wavelength (∆ε +28.0 (241 nm)) and a strong, negative broad band (λ 255 nm, ∆ε −36 that tails off toward longer wavelengths, and stronger edge features at λ ~ 200 nm compared with 6 in MeOH. The CD spectra of the 6-8, 10-12 were measured under two sets of conditions: isotropic (MeOH solution) and anisotropic conditions obtained by formulation into membrane-bound unilamellar liposomes. Each of the latter samples was prepared from crude liposomes (sonication) by repeated extrusion through a porous membrane (100 Å pore size), which, as shown previously [30,32,33], gave uniform spherical liposomes of approximately 25-30 nm in diameter. Measurement of chiroptical properties of these uniform nanoparticles can be made with little or no light scattering, and reveal complex features (absent from CD spectra recorded under isotropic conditions) that we attribute to intermolecular excitons.
Replacement of the Bz groups in 6 and 7 with Np groups-the triacyl sphingosine 8 and sphinganine 10-gave isotropic CD spectra in MeOH (Figure 3a,c, respectively, Table 1) qualitatively similar to 6 and 7 (e.g., 10: ∆ε +52 (231 nm), −46 (255)), but with CEs of higher magnitudes. Under L-CD conditions, the CD spectra of 8 and 10 (Figure 3b,d, respectively)-to our surprise-were not even similar to the L-CD spectra of tri-benzoyl derivatives 6 and 7. In the L-CD spectra of 8 and 10, the long-wavelength features were diminished and, in both compounds, the CEs collapsed into a single dominant, more intense broad band; for example, in 10 (∆ε +227 (230 nm)), close to the positive CE of 6 and 7, along with two less intense negative CEs (∆ε −104 (249), −80 (262)).
Replacement of the Bz groups in 6 and 7 with Np groups-the triacyl sphingosine 8 and sphinganine 10-gave isotropic CD spectra in MeOH (Figure 3a,c, respectively, Table 1) qualitatively similar to 6 and 7 (e.g., 10: ∆ε +52 (231 nm), −46 (255)), but with CEs of higher magnitudes. Under L-CD conditions, the CD spectra of 8 and 10 (Figure 3b,d, respectively)-to our surprise-were not even similar to the L-CD spectra of tri-benzoyl derivatives 6 and 7. In the L-CD spectra of 8 and 10, the long-wavelength features were diminished and, in both compounds, the CEs collapsed into a single dominant, more intense broad band; for example, in 10 (∆ε +227 (230 nm)), close to the positive CE of 6 and 7, along with two less intense negative CEs (∆ε −104 (249), −80 (262)).
Finally, measurement of the CD spectra of 11 and 12 (Table 1), either in MeOH or under L-CD conditions (DSPC), showed no CEs, only baseline. Reformulation of 11 into liposomes, prepared from fatty acyl phosphatidylglycerols of differing chain lengths (DLPC, DMPC, DPPC, DSPC) [31], did not change the spectra. On occasion, we have observed that the ordered self-assembly of long-chains appended with arylcarboxylate chromophores in liposomes [32] is kinetically limited and requires annealing over time or temperature, however, in the case of 11 and 12, CEs did not appear in the respective L-CD spectra, even after 24 h at 23 • C.
Mar. Drugs 2017, 15, 352 5 of 10 and requires annealing over time or temperature, however, in the case of 11 and 12, CEs did not appear in the respective L-CD spectra, even after 24 h at 23 °C.

Discussion
CD spectra of perbenzoyl sphingolipids 6 and 7 in MeOH solution (Table 1) show the expected bisignate split-Cotton effects. The new derivatives 8 and 10, bearing the Np chromophore bearing donor-acceptor substituents, confer higher oscillator strengths for the intramolecular charge transfer transition (the 1 Bb band) than most p-substituted benzoates or 2′-naphthoates. Consequently, the interactions between Np ester-ester and ester-amide pairs show superposed CEs of higher absolute magnitudes than the corresponding Bz derivatives with more pronounced, symmetric bisignate split CEs. For example, the absolute magnitude of peak-to-trough ∆ε values (the A parameter) for 8 and 10 (Entries 3 and 4) are 114 and 97.4, respectively, compared to only 10.4 and 16.4 for 6 and 7, (Entries 1 and 2), respectively. Given the ~10-fold greater magnitude of CEs, and comparable ease of Np derivatization and per-benzoylation, we recommend Np as a chromophore over Bz or 2-naphthoyl for AC assignments of aminoalkanols using the canonical "dibenzoate method" [13].
All L-CD spectra recorded for sphingosine-sphinganine pairs, 6, 7 and 8, 10, showed dramatically amplified CEs with new bands appearing at longer wavelengths compared to those observed in MeOH. Two factors contribute to these phenomena: restricted rotation of the chromophore-appended head-groups of sphingolipids that impedes conformational averaging, and intermolecular assembly of membrane-bound chromophores into H-aggregates. This assembly of triacyl-sphingolipids, largely driven by dipole-dipole and π-π stacking interactions (Figure 4), leads to delocalized chromophores with longer wavelength absorptions. These assemblies, normally weak in isotropic dilute solution where solvent-solute interactions dominate, are consolidated and strengthened by hydrophobic packing forces (and possibly augmented by hydrogen bonding) that organize the long-chain tails within the interior of the liposomal lipid bilayer. Inherent amplification of chirality through self-organizing polymers or oligomers has been reported before [34], but the use of liposomes in the present context achieves a similar effect and adds another dimension to CD as a tool to augment stereoassignment.

Discussion
CD spectra of perbenzoyl sphingolipids 6 and 7 in MeOH solution (Table 1) show the expected bisignate split-Cotton effects. The new derivatives 8 and 10, bearing the Np chromophore bearing donor-acceptor substituents, confer higher oscillator strengths for the intramolecular charge transfer transition (the 1 B b band) than most p-substituted benzoates or 2 -naphthoates. Consequently, the interactions between Np ester-ester and ester-amide pairs show superposed CEs of higher absolute magnitudes than the corresponding Bz derivatives with more pronounced, symmetric bisignate split CEs. For example, the absolute magnitude of peak-to-trough ∆ε values (the A parameter) for 8 and 10 (Entries 3 and 4) are 114 and 97.4, respectively, compared to only 10.4 and 16.4 for 6 and 7, (Entries 1 and 2), respectively. Given the~10-fold greater magnitude of CEs, and comparable ease of Np derivatization and per-benzoylation, we recommend Np as a chromophore over Bz or 2-naphthoyl for AC assignments of aminoalkanols using the canonical "dibenzoate method" [13].
All L-CD spectra recorded for sphingosine-sphinganine pairs, 6, 7 and 8, 10, showed dramatically amplified CEs with new bands appearing at longer wavelengths compared to those observed in MeOH. Two factors contribute to these phenomena: restricted rotation of the chromophore-appended head-groups of sphingolipids that impedes conformational averaging, and intermolecular assembly of membrane-bound chromophores into H-aggregates. This assembly of triacyl-sphingolipids, largely driven by dipole-dipole and π-π stacking interactions (Figure 4), leads to delocalized chromophores with longer wavelength absorptions. These assemblies, normally weak in isotropic dilute solution where solvent-solute interactions dominate, are consolidated and strengthened by hydrophobic packing forces (and possibly augmented by hydrogen bonding) that organize the long-chain tails within the interior of the liposomal lipid bilayer. Inherent amplification of chirality through self-organizing polymers or oligomers has been reported before [34], but the use of liposomes in the present context achieves a similar effect and adds another dimension to CD as a tool to augment stereoassignment.  The orientations of the electronic transition dipole moments ε of the arenoyl groups (Figure 4; only the primary O-Np groups are shown) within a monomer and between monomers are not known; indeed, this is a complex problem. Nevertheless, first order ensembles (considering only pairs) are likely offset in a manner reminiscent of conjugated monomeric molecules in other H-aggregates [35]. Although the bilayer is considered a "fluid mosaic", closeness of spacing of paired monomeric chromophores (e.g., close packed form, Figure 4a) should influence the degree of delocalization of excitons. Further refinement of this model, beyond that implied in a simplified pair-wise interaction depicted in Figure 4, is wanting, but beyond the scope of this paper.
Earlier, we showed that appearance of the CEs in L-CD of substituted naphthamide 14 [33] was dependent only upon the configuration at C-2 (R and S enantiomers showed opposite CD spectra of equal magnitude), and exhibited a reversible temperature dependence that followed the expected gel phase transition temperature of DSPC that comprise the liposomes. It is likely that similar non-bonded interactions are also operative in the liposomal formulations of 6, 7, 8 and 10. It is important to note again that racemic (±)-14 or achiral naphthamide 15 show no CEs (baseline only) in MeOH or in DSPC liposomes, proving that the asymmetry of the bilayer itself is not the origin of induced CEs in L-CD [33].
The strongest amplification of CEs under L-CD conditions was observed in 10. We estimated the limit of detection (LOD) from the peak-to-trough value of ∆ε for the major bisignate components in the L-CD spectrum of 10 ( Figure 2c, A = 334), the average noise level from λ = 300-400 nm and the nominal cuvette fill volume (l = 2 mm, Vf = 0.2 mL). Under L-CD conditions the limit of detection of 10 is estimated to be ~56 pmole, or almost 4-fold lower than 10 in MeOH. It is conceivable that the LOD may be further improved by appropriate replacement of the Np group with chromophores of higher oscillator strength, e.g., 2-anthracenoyl, 5-acetyl-7-dimethylamino-2H-chromen-2-one, p-methoxycinnamoyl [15].

Materials and Methods
General methods can be found elsewhere [36]. All CD spectra were recorded on a Jasco J-810 spectropolarimeter at 23 °C. NMR spectra were recorded on a Varian Mercury 400 ( 1 H NMR, 400 MHz; 13 C NMR, 100 MHz) with a dual-tuned 1 H-13 C 5 mm room temperature probe, or a Jeol ECA 500 with a 5 mm 1 H{ 13 C} inverse detect probe ( 1 H NMR, 500 MHz; 13 C NMR, 125 MHz). Chemical shifts are referenced to internal solvent peaks (residual CHCl3, δH 7.24 ppm; CDCl3, δC 77.0 ppm). The orientations of the electronic transition dipole moments ε of the arenoyl groups (Figure 4; only the primary O-Np groups are shown) within a monomer and between monomers are not known; indeed, this is a complex problem. Nevertheless, first order ensembles (considering only pairs) are likely offset in a manner reminiscent of conjugated monomeric molecules in other H-aggregates [35]. Although the bilayer is considered a "fluid mosaic", closeness of spacing of paired monomeric chromophores (e.g., close packed form, Figure 4a) should influence the degree of delocalization of excitons. Further refinement of this model, beyond that implied in a simplified pair-wise interaction depicted in Figure 4, is wanting, but beyond the scope of this paper.
Earlier, we showed that appearance of the CEs in L-CD of substituted naphthamide 14 [33] was dependent only upon the configuration at C-2 (R and S enantiomers showed opposite CD spectra of equal magnitude), and exhibited a reversible temperature dependence that followed the expected gel phase transition temperature of DSPC that comprise the liposomes. It is likely that similar non-bonded interactions are also operative in the liposomal formulations of 6, 7, 8 and 10. It is important to note again that racemic (±)-14 or achiral naphthamide 15 show no CEs (baseline only) in MeOH or in DSPC liposomes, proving that the asymmetry of the bilayer itself is not the origin of induced CEs in L-CD [33].
The strongest amplification of CEs under L-CD conditions was observed in 10. We estimated the limit of detection (LOD) from the peak-to-trough value of ∆ε for the major bisignate components in the L-CD spectrum of 10 ( Figure 2c, A = 334), the average noise level from λ = 300-400 nm and the nominal cuvette fill volume (l = 2 mm, V f = 0.2 mL). Under L-CD conditions the limit of detection of 10 is estimated to be~56 pmole, or almost 4-fold lower than 10 in MeOH. It is conceivable that the LOD may be further improved by appropriate replacement of the Np group with chromophores of higher oscillator strength, e.g., 2-anthracenoyl, 5-acetyl-7-dimethylamino-2H-chromen-2-one, p-methoxycinnamoyl [15].

Conclusions
Measurements of circular dichroism of 2 -naphthoyl and benzoyl derivatives of sphingosine in unilamellar DSPC liposomal preparations gave rise to large increases in the magnitudes of the Cotton effects and longer wavelength absorptions, compared to isotropic solution (MeOH). The dramatic effects are consistent with delocalized excitons: intermolecular associations in H-aggregates that self-assemble spontaneously within the lipid bilayer and persist over long time scales. The effect was not observed in the L-CD spectra of two counter examples measured: a long-chain vinyl naphthalene and a 2-naphthoate ester of a long-chain, methyl branched lipid. The liposomal CD method allows extension of detection limits down to sub-nanomole levels for critical chiroptical stereochemical assignments in sphingolipid natural products.