Liposomal circular dichroism (L-Cd) of arenoyl derivatives of sphingolipids. Amplification of cotton effects in ordered lipid bilayers

Article abstract of DOI:10.3390/md15120352

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.

Full text of DOI:10.3390/md15120352

marine drugs
Article
Liposomal Circular Dichroism (L-CD) of Arenoyl
Derivatives of Sphingolipids. Amplification of
Cotton Effects in Ordered Lipid Bilayers
Tadeusz F. Molinski 1,2,*, Caroline D. Broaddus 1 and Brandon I. Morinaka 1,†
1
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive
MC0358, La Jolla, CA 92093, USA; broaddusc@yahoo.com (C.D.B.); phambi@nus.edu.sg (B.I.M.)
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 9500 Gilman
2
Drive MC0358, La Jolla, CA 92093, USA
*
†
Correspondence: tmolinski@ucsd.edu; Tel.: +1-858-534-7115
Present address, Department of Pharmacy, National University of Singapore, 18 Science Drive 4,
Singapore 117543, Singapore.
Received: 13 October 2016; Accepted: 6 December 2017; Published: 20 December 2017
Abstract: 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
0
0
0
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.
Keywords: marine natural product; circular dichroism; exciton coupling; absolute configuration;
liposome; sphingosine; sphingolipid
1
. Introduction
The assignment of relative configuration (RC) and absolute configuration (AC) of marine
natural products, in particular long-chain flexible polyketides and lipids, has no general solution;
stereochemical elucidation is approached on a case-by-case basis. Creative integrated approaches,
based on a number of methods (e.g., modified Mosher’s ester method for secondary alkanols [
degradation-synthesis, nOe and J-based NMR approaches [ ], circular dichroism [ ] (CD)—and
more recently, vibrational circular dichroism (VCD) [ ]—have often led to successful outcomes in
numerous instances [ ]. CD, with comparisons of synthetic model compounds of known configuration,
has been used to assign AC in long-chain lipids with native chromophores, albeit with weak Cotton
effects (CEs) [ ]. Interpretation of exciton coupled circular dichroism (ECCD) of perbenzoates of
acyclic polyols [ 11], and benzoate/benzamide pairs of vicinal aminoalkanols [12] give rise to
1,2],
3
4,5
6
7
8
9–
characteristic bisignate Cotton effects (CEs) that reflect their absolute configuration [13]. These methods,
largely developed by Nakanishi, Berova, and Gawronski, exploited pair-wise intramolecular exciton
coupling to develop “fingerprint” CD spectra of all possible diastereomeric combinations of polyols,
and 2-amino-3-alkanols and 2-amino-1,3-alkanediols—sphingolipids—and has been applied to the
assignment of absolute configuration of D-erythro-sphingosine (1a, Figure 1), sphinganine (1b) and
related acylic natural products [14–16].
Mar. Drugs 2017, 15, 352; doi:10.3390/md15120352
www.mdpi.com/journal/marinedrugs

Mar. Drugs 2017, 15, 352
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Figure 1. D-Sphingosine (1a), its dihydro-derivative, sphinganine (1b), and “two-headed” sphingolipid
natural products.
More complex C28 and C30 sphingolipids—the so-called antifungal “two-headed”
sphingolipids [17 20]—which comprise pseudo-dimeric -bis-aminoalkanols with four or
–
α,ω
more stereocenters of heterogeneous configurations—have also been addressed by empirical variants
of this method. We showed that deconvolution of complex superposed CEs (a corollary of van’t Hoff’s
principle of optical superposition) [21] enables assignment of absolute and relative configurations of
four stereocenters in two-headed sphingolipids by matching their measured spectra against calculated
“
hybrid-CD spectra” generated by linear combinations of simple threo and erythro benzoyl derivatives
of diastereomeric 2-amino-3-alkanol and 2-amino-1,3-alkanediol models [20,21].
The latter method has been applied to the complete stereo-assignments of calyxoside from the red
alga, Calyx sp. [18], and the sponge natural products rhizochalin (2) and rhizochalinins [20,21] from
Rhizochalina incrustata [22], oceanapiside ( ) from Oceanapia phillipensis [23
3
,24], leucettamol A (4) from
Leucetta microrhaphis [25], and the truncated-chain sphingolipid, (2S,3R)-2-aminodocecan-3-ol from
the tunicate, Clavelina oblonga [26]. In the latter examples, the CEs arise from pair-wise intramolecular
exciton coupling of benzoate/benzamide chromophores and their magnitudes are modest to weak, up
−
1
3
−1
to ∆ε~7.5–11 mol
·
dm cm for the perbenzoyl D-erythro-sphingosine (the threo isomer is weaker).
·
The corresponding 1,3-dinaphthoate/2-naphthamide pairs show larger dichroism, with ∆ε values up
−
1
3
−1
to 56–68.6 mol
·
dm cm , or 5–9-fold higher in magnitude for a comparable model compound of
the same configuration [27].
·
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
0
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.
2
. Results
For the purposes of this study, model compounds containing benzoyl or 6-methoxy-2-naphthoyl
chromophores (herein, abbreviated Np) were prepared (Scheme 1) for measurements of CD spectra
under two sets of conditions: isotropic media (MeOH) or liposomal formulations (L-CD, liposomes
18:0), as previously described [30], where the DPPC or DSPC [31] bilayers comprise an anisotropic
medium [32 33]. Natural D-sphingosine (1a) was converted (N-benzoylimidazole ( ), DBU, CH CN,
,
5
3

Mar. Drugs 2017, 15, 352
3 of 10
◦
7
0 C) to the corresponding perbenzoate (
6
) which was subsequently hydrogenated (H , 1 atm, 10%
2
◦
0
Pd-C, EtOAc, 23 C) to give N,O,O -tribenzoylsphinganine (
gave pernaphthoyl derivative,
7
) [16]. A parallel sequence of reactions
◦
8
, by acylation of 1a (N-Np-imidazole,
9
, DBU, CH CN, 60 C), which
3
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
1
2, prepared from the known long-chain primary alcohol 13 [7], were also secured.
Scheme 1. Synthesis of sphingolipid benzoyl and (6-methoxy-2-naphthoyl) derivatives. a, EDCI,
DMAP, 6-methoxy-2-naphthoic acid, CH Cl ; b, N-benzoylimidazole (5), DBU, CH Cl ; c, H (1 atm),
2 2 2
2
2
0 0
0% Pd-C, EtOAc; d, N-(6 -methoxy-2 -naphthoyl)imidazole (9), DBU, CH Cl .
2 2
1
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.
0
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 ( ~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 , formulated in DSPC [31] unilamellar liposomes (aqueous), shows
more complex behavior dominated by three strong CEs with greatly amplified magnitudes: a shorter
(
−
λ
,
6
wavelength negative band (∆ε −42 (
λ
= 210 nm), a significantly longer wavelength (∆ε +28.0 (241 nm))
255 nm, ∆ε −36 that tails off toward longer wavelengths, and
and a strong, negative broad band (
λ
stronger edge features at λ ~200 nm compared with 6 in MeOH.

Mar. Drugs 2017, 15, 352
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◦
0
Figure 2. Circular dichroism (CD) spectra (23 C) of (a) (red) N,O,O -tri-(benzoyl)-D-sphingosine
−
5
−4
(
6
) in MeOH (c = 6.17
×
10 M) and (b) (blue)
6
in DSPC liposomes (c = 1.63
×
10 M) (c) (red)
−
10 M) and (d) (blue) 7 in DSPC liposomes
4
tri-(benzoyl)-D-sphinganine (
7
) in MeOH (c = 2.2
×
c = 5.28 × 10− M).
5
(
Table 1. Isotropic CD (MeOH) and liposomal CD (L-CD). Summary of Parameters [∆ε (λ)].
Entry
Cmpd.
MeOH
+8.2 (224)
L-CD
1
2
3
4
5
6
6
7
8
10
11
12
−2.2 (240)
−
41.5 (210) +28.0 (241)
+17.6 (216)
44.5 (249)
−
−
−
36.0 (255)
13.0 (227) +33.3 (242)
31.5 (266)
-
-
+6.3 (220)
+71.2 (232)
−
−
−
10.1 (238)
43.7 (255)
45.2 (255)
−14 (204)
−
12.6 (253)
+114 (228)
+227 (230)
−
−
14.5 (303)
17.4 (301)
-
-
-
-
+52.2 (231)
−104 (249)
−80 (262)
−
1
1
1
1
-
-
-
-
1
1
1
1
1
1
−
23.8 (213) +34.6 (233)
(S)-14 2
7
-
-
-
2
1
1
2
1
1
(
±)-14
1
1
1
1
1
8
9
-
-
-
-
-
-
2
15 2
Baseline, only; 2 Reference [33].
Hydrogenation of the ∆4 double bond in
rise to a CD (MeOH) spectrum (Figure 2c, Table 1) that is qualitatively similar to the latter (Figure 2a)—a
simple bisignate CE, but stronger in magnitude (∆ε +6.3 (220 nm), 10.1 (238)). Reconstituting
in DSPC [31] liposomes gives rise to a dramatically different L-CD spectrum with complex features
13.0 (227), +33.3 (242), 12.6 (253)) and smaller
6 to give the tri-benzoylsphinganine derivative 7 gives
−
8
dominated by five CEs (∆ε −14 (204 nm), +18 (216),
−
−
edge effects. The latter two CEs occurred at longer wavelengths than those arising from chromophores
in isotropic media, and the last one tapered off toward longer wavelengths, as did 6 (Figure 2d).
Replacement of the Bz groups in
sphinganine 10—gave isotropic CD spectra in MeOH (Figure 3a,c, respectively, Table 1) qualitatively
similar to and (e.g., 10 ∆ε +52 (231 nm), 46 (255)), but with CEs of higher magnitudes. Under
L-CD conditions, the CD spectra of and 10 (Figure 3b,d, respectively)—to our surprise—were not
even similar to the L-CD spectra of tri-benzoyl derivatives and . In the L-CD spectra of 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
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
6 and 7 with Np groups–the triacyl sphingosine 8 and
6
7
:
−
8
6
7
8
(
6
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

Mar. Drugs 2017, 15, 352
5 of 10
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.
◦
0
0
0
Figure 3. CD spectra (23 C) of (a) (red) N,O,O -tri-(6 -methoxy-2 -naphthoyl)-D-sphingosine (8)
−
5
−5
in MeOH (c = 1.4
×
10 M) and (b) (blue)
8
in DSPC liposomes (c = 5.87
×
10 M); (c) (red)
10 M) and (d) (blue) 10 in
0
0
−5
tris-(6 -methoxy-2 -naphthoyl)-D-sphinganine (10) in MeOH (c = 1.4
DSPC liposomes (c = 1.4 × 10− M).
×
5
3
. Discussion
CD spectra of perbenzoyl sphingolipids
bisignate split-Cotton effects. The new derivatives
6
and
7
in MeOH solution (Table 1) show the expected
8
and 10, bearing the Np chromophore bearing
donor-acceptor substituents, confer higher oscillator strengths for the intramolecular charge transfer
1
0
transition (the B band) than most p-substituted benzoates or 2 -naphthoates. Consequently, the
b
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
Entries 3 and 4) are 114 and 97.4, respectively, compared to only 10.4 and 16.4 for and , (Entries
and 2), respectively. Given the ~10-fold greater magnitude of CEs, and comparable ease of Np
8 and 10
(
6
7
1
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.

Mar. Drugs 2017, 15, 352
6 of 10
(
a)
(b)
Figure 4.
Cartoon of hypothetical intermolecular interactions between paired
0
N,O,O -tri-acylsphingosine molecules within the liposomal bilayer. Red spheres = polar head
groups of DSPC, Np = 6-methoxy-2-naphthoyl,
band). For clarity, only two Np groups are shown, and intramolecular interactions are not depicted.
a) Close packed chromophores (b) lipid chain-separated chromophores.
ε = electronic transition dipole moment (charge transfer
(
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 = 0.2 mL). Under L-CD conditions the limit of detection of
f
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].
4
. Materials and Methods
General methods can be found elsewhere [36]. All CD spectra were recorded on a Jasco J-810
◦
1
spectropolarimeter at 23 C. NMR spectra were recorded on a Varian Mercury 400 ( H NMR, 400 MHz;
13
1
13
C NMR, 100 MHz) with a dual-tuned H- C 5 mm room temperature probe, or a Jeol ECA 500 with
a 5 mm H{ C} inverse detect probe ( H NMR, 500 MHz; C NMR, 125 MHz). Chemical shifts are
referenced to internal solvent peaks (residual CHCl , δ 7.24 ppm; CDCl , δ 77.0 ppm).
1
13
1
13
3
H
3
C

Mar. Drugs 2017, 15, 352
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(S)(+)-12-(tert-Butyldimethylsilyloxy)-2-methyldodecyl 6-methoxy-2-naphthoate (12). A solution of alcohol
1
3
(6 mg, 0.0181 mmol), 4-dimethylaminopyridine (0.54 mg, 0.44
µ
mol) and 6-methoxy-2-naphthoic
◦
acid (5 mg, 0.0247 mmol) in CH Cl (0.50 mL) was cooled to 0 C and treated with EDCI
2
2
(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, 2.7 mg, 1.74 µmol) and the mixture was stirred at
room temperature overnight. The solvent was removed under a stream of N , and the residue was
2
separated using preparative thin layer chromatography (1:9 EtOAc/hexanes) to give ester 12 (1.12 mg,
1
1
1
1
1
3%). [α
] +1.29 (c 1.63, CHCl ). H NMR (CDCl , 500 MHz)
δ
8.52 (s, 1H), 8.04 (dd, J = 8.6, 1.8 Hz,
D
3
3
H), 7.85 (d, J = 8.6 Hz, 1H), 7.76 (d, J = 9.2 Hz, 1H), 7.20 (dd, J = 9.2, 2.6 Hz, 1H), 7.16 (d, J = 2.6 Hz,
H), 4.26 (dd, J = 10.3, 5.8 Hz, 1H), 4.15 (dd, J = 10.3, 6.9 Hz, 1H), 3.59 (t, J = 6.9 Hz, 2H), 1.63 (m, 1H),
.50 (m, 1H), 1.31–1.25 (m, 20 H), 1.05 (d, J = 6.8 Hz, 3H), 0.89 (s, 6H), 0.04 (s, 9H).
N-Benzoylimidazole (5). The method of Nakanishi and coworkers was adapted, for the preparation of
5
[
37]. A solution of benzoyl chloride (2 mL) in benzene (20 mL) was added dropwise to a solution of
◦
imidazole (2.32 g, 34.1 mmol) and benzene (200 mL). The resulting mixture was cooled to 8 C, then
allowed to warm to room temperature and stirred overnight. After removal of solvent, pure
obtained as a clear oil. The H NMR data were consistent with literature values [27].
5 was
1
0
D-(E)-(–)-erythro-N,O,O -(Tribenzoyl)sphingosine (
6
) [16]. A solution of D-erythro-sphingosine (1a, 10 mg,
0
.03 mmol) and N-benzoylimidazole (82 mg, 0.476 mmol) in acetonitrile (5 mL) was treated with DBU
◦
(49
µL, 0.33 mmol) and the mixture stirred at 70 C for 24 h, then at room temperature for 24 h. After
removal of solvent, the mixture was separated using flash chromatography (1:9 EtOAc: hexane), which
yielded perbenzoylated D-erythro-sphingosine (
6
[
16], 11.9 mg, 58% yield). [
α
]D
−
5.90 (c 2.06, CHCl3).
1
H NMR (CDCl , 400 MHz)
δ 8.02 (m, 4H), 7.74 (m, 2H), 7.58–7.39 (m, 9H), 6.73 (d, J = 8.8 Hz, 1H), 5.96
3
(dt, J = 15.5, 7.8 Hz, 1H), 5.80 (t, J = 6.5 Hz, 1H), 5.66 (dd, J = 15.4, 7.0 Hz, 1H), 4.94 (m, 1H), 4.72 (dd,
J = 11.7, 6.4 Hz, 1H), 4.60 (dd, J = 11.7, 4.3 Hz, 1H), 2.06 (q, J = 7.0 Hz, 2H), 1.35–1.21 (m, 18H), 0.88 (t,
J = 7.0 Hz, 3H).
0
D-erythro-N,O,O -(Tribenzoyl)sphinganine (
7). A mixture of
6
(5.9 mg, mmol) and 10% Pd-C (1 mg)
in ethyl acetate (0.6 mL) was stirred under an atmosphere of H at room temperature for 48 h then
2
1
filtered (syringe filter, 0.45-µm) to give
7
. The H NMR and CD spectroscopic data were consistent
with literature values [23].
0
0
0
D-(E)-erythro-N,O,O -Tri-(6 -methoxy-2 -naphthoyl)sphingosine (
(
8
). A mixture of D-erythro-sphingosine
L, 0.33 mmol), and N-(6-methoxy-2-naphthoyl)imidazole (
08 mg, 0.43 mmol) and dry acetonitrile (5 mL) was stirred overnight at room temperature, then stirred
1a, 11.6 mg, 0.039 mmol), DBU (49
µ
9,
1
◦
at 60 C for an additional 24 h. The solution was then concentrated under a stream of Ar and solvent
traces were removed under high vacuum for 30 min to give a crude product which was separated
by flash chromatography (3:7 EtOAc-hexane) followed by HPLC (silica, 10
×
25 mm column, 3:7
8.51 (s, 1H), 8.48 (s, 1H), 8.22 (s,
H), 8.02 (d, J = 8.6 Hz, 1H), 7.98 (d, J = 8.6 Hz, 1H), 7.82–7.66 (m, 6H), 7.18–7.08 (m, 6H), 6.97 (m, 2H),
.03 (dt, J = 15.6, 7.8 Hz, 1H), 5.93 (t, J = 5.3 Hz, 1H), 5.76 (dd, J = 15.4, 7.1 Hz, 1H), 5.05 (m, 1H), 4.86
−
EtOAc-hexane, 4 mL min ) to give 8. H NMR (CDCl , 400 MHz) δ
3
1
1
·
1
6
(
(
dd, J = 11.7, 5.8 Hz, 1H), 4.75 (dd, J = 11.7, 4.6 Hz, 1H), 2.09 (q, J = 7.0 Hz, 2H), 1.38–1.17 (m, 18H), 0.88
t, J = 7.0 Hz, 3H).
0
0
N-(6 -Methoxy-2 -naphthoyl)imidazole (
9). The method of Nakanishi and coworkers was adapted for
the preparation of 38]. A solution of 6-methoxy-2-naphthoyl chloride (147 mg, 0.668 mmol) in
9
[
toluene (1.5 mL) was added to a suspension of imidazole (91.1 mg, 1.34 mmol) in toluene (5 mL)
and the mixture stirred at room temperature overnight. The mixture was filtered through Celite,
and the filter bed washed with additional toluene. After removal of solvent from the combined
filtrates under reduced pressure, a clear glass of N-(6-methoxy-2-naphthoyl)imidazole (
108.4 mg, 64%).
9) was obtained
(
0
0
0
D-erythro-N,O,O -Tri-(6 -methoxy-2 -naphthoyl)sphinganine (10). Naphthoyl derivative
8 (1.7 mg, mmol)
was added to a vial containing a suspension of 10% Pd on carbon (0.3 mg) in EtOAc (0.5 mL). The

Mar. Drugs 2017, 15, 352
8 of 10
mixture was stirred under H (1 atm) at room temperature for 48 h, passed through a 0.45-micron
2
syringe filter, and the solvent removed from the clear solution to obtain 10. [
α
]D
−
1.5 (c 0.39, CHCl3).
1
H NMR (CDCl , 400 MHz) δ 8.49 (s, 1H), 8.40 (s, 1H), 8.28 (s, 1H), 8.02 (d, J = 8.6 Hz, 1H), 7.91–7.58
3
(
4
m, 8H), 7.39–7.04 (m, 6H), 5.51 (m, H), 4.98 (m, 1H), 4.83 (dd, J = 11.7, 5.8 Hz, 1H), 4.72 (dd, J = 11.7,
.6 Hz, 1H), 2.07 (m, 1H), 1.96 (m, 1H), 1.38–1.17 (m, 18H), 0.88 (t, J = 7.0 Hz, 3H).
Lipsomal CD Measurements. Liposomal formulations of 10 11 and 13 were prepared
according our published procedure [30 32 33]. Briefly, crude liposomes–prepared by ‘shell evaporation’
of a CHCl solution containing compound and DSPC or DPPC [31] (Avanti Polar Lipids, Alabaster,
6,
7,
8,
,
,
,
3
AL, USA) in a round bottom flask, followed by addition of distilled H O and sonication, followed by
2
◦
◦
annealing (heated to 60 C, cooled to 23 C; repeated twice)—were repeatedly extruded ( 25) through
×
polycarbonate membranes (100 nm pore) using gas-tight syringes (Liposofast, Avestin, Toronto, ON,
Canada). Final lipid concentration, c = 1 mg/mL. Isotropic CD spectra were measured on solutions
in MeOH at close to the same nominal concentration as L-CD spectra. All CD measurements were
carried out on prepared samples contained in a quartz cuvette (l = 2 mm).
5
. Conclusions
Measurements of circular dichroism of 2 -naphthoyl and benzoyl derivatives of sphingosine in
0
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.
Acknowledgments: This work was funded by the NIH (AI100776, CA122256). We are grateful to Doralyn Dalisay
for earlier contributions to the concept and execution of L-CD.
Author Contributions: Tadeusz F. Molinski conceived of and designed the experiments; Caroline D. Broaddus
and Brandon I. Morinaka performed the experiments; Caroline D. Broaddus, Tadeusz F. Molinski and
Brandon I. Morinaka analyzed the data; Tadeusz F. Molinski and Caroline D. Broaddus wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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(