The relationship between the structure of the headgroup of sphingolipids and their ability to form complex high axial ratio microstructures

Article abstract of DOI:10.1016/S0009-3084(00)00204-8

Ceramides with chemically modified polar headgroups were prepared and examined for their ability to form complex high axial ratio microstructures (CHARMS), potential drug delivery vehicles. In general, if the modified ceramide had either a hydrogen bond donor or acceptor at C-1 and C-3, including hydrophobic or hydrophilic groups attached to C-1 microstructures formed. Tolerated groups include amides, esters, sulfonates, and ethers. If modification at C-3 added significant bulk (greater than four carbons regardless of hydrophilicity), then amorphous aggregates formed. Ceramides with C-1 and C-3 bridged through a cyclic structure also made microstructures. By using a sphingolipid with an amine headgroup, CHARMs may be modified covalently after formation.

Full text of DOI:10.1016/S0009-3084(00)00204-8

Chemistry and Physics of Lipids
109 (2001) 1–14
www.elsevier.com/locate/chemphyslip
The relationship between the structure of the headgroup of
sphingolipids and their ability to form complex high axial
ratio microstructures
Alex S. Goldstein a, Michael H. Gelb a, Paul Yager b,*
a Departments of Chemistry and Biochemistry, Uni6ersity of Washington, Box 351700, Seattle, WA 98195-1700, USA
b Molecular Bioengineering Program, Department of Bioengineering, Uni6ersity of Washington, Box 352255, Seattle,
WA 98195-2255, USA
Received 5 July 2000; received in revised form 1 September 2000; accepted 1 September 2000
Abstract
Ceramides with chemically modified polar headgroups were prepared and examined for their ability to form
complex high axial ratio microstructures (CHARMS), potential drug delivery vehicles. In general, if the modified
ceramide had either a hydrogen bond donor or acceptor at C-1 and C-3, including hydrophobic or hydrophilic groups
attached to C-1 microstructures formed. Tolerated groups include amides, esters, sulfonates, and ethers. If modifica-
tion at C-3 added significant bulk (greater than four carbons regardless of hydrophilicity), then amorphous aggregates
formed. Ceramides with C-1 and C-3 bridged through a cyclic structure also made microstructures. By using a
sphingolipid with an amine headgroup, CHARMs may be modified covalently after formation. © 2001 Published by
Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Sphingolipid; Complex high axial ratio microstructure; Ceramide
perfluorinated phospholipids (Krafft et al.,
1996a,b), alkylated sugars (Pinteric et al., 1973;
1. Introduction
Boettcher et al., 1996), and sphingolipids
(Archibald and Yager, 1992; Archibald and
Mann, 1994; Kulkarni and Brown, 1996; Kulka-
rni et al., 1995; Goldstein et al., 1997). In some
cases, these lipids microstructures have been in-
vestigated for use as components in drug delivery
systems. One requirement for such use could be
covalent attachment of therapeutic agents of the
surfactants. To date, sphingolipids have not been
used to deliver therapeutic agents.
A variety of bisalkyl lipids can form complex
high axial ratio microstructures (CHARMs), non-
liposomal supramolecular assemblies with defined
morphologies. These include diacetylenic phos-
pholipids (Yager and Schoen, 1984; Carlson et al.,
1997), dialkylglutamide (Yamada et al., 1984;
Shimizu and Hato, 1993; Lee et al., 1998, 1999),
* Corresponding author.
0009-3084/01/$ - see front matter © 2001 Published by Elsevier Science Ireland Ltd. All rights reserved.
PII: S0009-3084(00)00204-8

2
A.S. Goldstein et al. / Chemistry and Physics of Lipids 109 (2001) 1–14
A sphingolipid consists of two divergent re-
was stirred 75 min and then more triphenylphos-
phine (0.016 g, 64.8 mmol) was added. The now
clear heterogeneous solution was stirred 75 min,
whereupon 20 ml diisopropylazodicarboxylate was
added. The solution was stirred for 1.5 h, then
zinc azide bis pyridine complex (0.020 g, 64.8
mmol) was added and the solution stirred an
additional 1.5 h. The mixture was absorbed onto
silica gel and purified by flash chromatography
(8:1–1:1 hexane:EtOAc) to provide target (0.01 g,
7%) as a clear film, Rf (1:1 hexane:EtOAc) 0.36;
1H NMR (500 MHz), 5.76 (m, 1H, C-5), 5.47 (dd,
1H, C-4, J=3.4, 7.7 Hz), 5.35 (t, 2H, C-15%,
C-16%, J=7.7 Hz), 4.20 (m, 3H, C-1, C-3), 3.96
(m, 1H, C-2), 2.17–1.95 (m, 8H, C-6, C-2%, C-14%,
C-17%), 1.62 (m, 2H, C-3%), 0.88 (t, 6H, C-18,
C-24%, J=6.6 Hz); IR (neat) 3294, 2925, 2100,
gions, a hydrophilic headgroup that encompasses
a 1° alcohol, N-acylamine, and 2° allylic alcohol,
and a hydrophobic region that contains the hy-
drocarbon chains. The ability of sphingolipids
(ceramide and galactocerebroside) with varying
hydrocarbon lengths and degrees of unsaturation
to form CHARMs has been studied (Kulkarni
and Brown, 1996; Kulkarni et al., 1995, 1999;
Goldstein et al., 1997); however, studies examin-
ing chemical modification within the headgroup
region are very limited in scope and depth. This
paper takes a more detailed look at substitutions
within the headgroup region to see which linkages
may be used to tether model drug to lipid without
inhibiting the formation of CHARMs. Secondly,
since not all sphingolipids derivatives form mi-
crostructures, it will be shown that CHARMs
may be covalently modified after formation.
1751 cm−1
.
2.2. 1-O-triphenylmethyl-16:0-Cer (3)
2. Experimental
16:0-Cer (0.112 g, 208 mmol), triphenylmethyl
chloride (0.064 g, 229 mmol) and DMAP (0.051 g,
416 mmol) in 20 ml dry toluene was refluxed
overnight under N2. The solvent was removed by
rotary evaporation and the residue purified by
flash chromatography (9:1–2:1 hexane:EtOAc) to
provide the target as a white solid (0.089 g, 55%),
1H NMR spectra were obtained in CDCl3 using
a Bruker 500 MHz (500) or Bruker 300 MHz
(300) NMR spectrometer with tetramethylsilane
as an internal standard. Silica gel (EM Science
Silica Gel 60, 230–400 mesh) was used for all
flash chromatography. Infrared (IR) spectroscopy
was determined using a Perkin Elmer 1600 FTIR.
Thin layer chromatography (TLC) was performed
using plates coated with 250 mm Silica Gel 60 F254
(EM Science). All reagents were used as received.
Transmission electron micrographs (TEM) were
obtained using a Philips EM 410 electron micro-
scope operating at an acceleration potential of 80
kV. Samples were applied to Formvar-coated 150
mesh copper TEM sample grids. In some cases,
2% aqueous ammonium molybdate (pH 5.1) was
applied.
1
Rf (3:1 hexane:EtOAc) 0.32; H NMR (500 MHz)
7.41–7.25 (15H), 6.07 (d, 1H, NH, J=7.4 Hz),
5.62 (m, 1H, C-5), 5.27 (dd, 1H, C-4, J=6.2, 15.5
Hz) 4.17 (m, 1H, C-2), 4.06 (m, 1H, C-3), 3.39
(dd, 1H, C-1, J=3.7, 9.9 Hz), 3.5 (m, 2H, C-1),
2.20 (t, 2H, C-2%, J=6.8 Hz), 1.92 (m, 2H, C-6),
1.64 (m, 2H, C-3%), 0.88 (t, 6H, C-18, C-16%,
J=6.8 Hz).
2.3. 1-O-triphenylmethyl-8:0-Cer (4)
8:0-Cer (0.132 g, 310 mmol), triphenylmethyl
chloride (0.086 g, 310 mmol) and DMAP (0.038 g,
310 mmol) in 50 ml toluene was refluxed
overnight. The solvent was removed by rotary
evaporation and the residue purified by flash
chromatography (8:1–2:1 hexane:EtOAc) to the
target as a white solid (0.116 g, 56%), Rf (3:1
2.1. 1-Azido-24:1-Cer (1)
To 24:1-Cer (0.014 g, 21.6 mmol), triphenylphos-
phine (0.011 g, 43.2 mmol) in 9 ml dry CH2Cl2 was
added crushed zinc azide bis pyridine complex
(0.033 g, 108 mmol) and then diisopropylazodicar-
boxylate (8.5 ml, 43.2 mmol). The orange solution
1
hexane:EtOAc) 0.23; H NMR (500 MHz) 7.41–
7.25 (15H), 6.06 (d, 1H, NH, J=8.0 Hz), 5.62 (m,

A.S. Goldstein et al. / Chemistry and Physics of Lipids 109 (2001) 1–14
3
1H, C-5), 5.26 (dd, 1H, C-4, J=6.2, 15.5 Hz),
4.17 (m, 1H, C-2), 4.07 (dd, 1H, C-3, J=3.7, 8.0
Hz), 3.39 (dd, 1H, C-1, J=3.7, 9.9 Hz), 3.30 (39
(dd, 1H, C-1, J=3.7, 9.9 Hz), 2.20 (t, 2H, C-2%,
J=7.5 Hz), 1.92 (m, 2H, C-6), 1.64 (m, 2H, C-3%),
0.88 (t, 6H, C-18, C-8%, J=6.8 Hz).
1H, C-1, J=3.7, 9.9 Hz), 3.57 (75 (dd, 1H, C-1,
J=3.7, 9.9 Hz) 3.41 (d, 1H, OH, J=8.0 Hz),
2.22 (t, 2H, C-2%, J=7.4 Hz), 2.00 (m, 6H, C-6,
C-14%, C-17%), 1.63 (m, 2H, C-3%), 0.88 (t, 6H,
C-18, C-24%, J=6.8 Hz).
2.5.2. 3-O-allyl-24:1-Cer (18)
2.4. 1-O-(4,4%-dimethoxytriphenylmethyl)-
24:1-Cer (5)
Rf (1:1 hexane:EtOAc) 0.42; 1H NMR (300
MHz) 6.16 (d, 1H, NH, J=7.3 Hz), 5.80 (m, 2H,
C-5, CHꢀCH2), 5.28 (t, 2H, C-15%, C-16%, J=4.5
Hz), 5.12 (t, 2H, CHꢀCH2), 3.90 (m, 4H, C-2,
C-1, OCH2), 3.72 (dd, 1H, C-1, J=5.9, 12.7 Hz),
3.52 (bd, 1H, C-3, J=9.8 Hz), 2.93 (bs, 1H, OH),
2.15 (t, 2H, C-2%, J=6.8 Hz), 2.00 (m, 6H, C-6,
C-14%, C-17%), 1.53 (m, 2H, C-3%), 0.81 (t, 6H,
C-18, C-24%, J=6.8 Hz).
To 24:1-Cer (0.031 g, 47.8 mmol), and DMAP
(0.006, 52.6 mmol) in 10 ml toluene was added
4,4%-dimethoxytriphenylmethyl chloride (0.018 g,
52.6 mmol). The reaction was refluxed for 21 h
and the solvent removed by rotary evaporation.
The yellow residue was purified by flash chro-
matography (8:1–2:1 hexane:EtOAc) to provide
the target as a white solid (0.010 g, 23%), Rf (3:1
2.6. 1-O-allyl-16:0-Cer (7) and
-1-O-allyl-16:0-Cer (19)
1
hexane:EtOAc) 0.14; H NMR (500 MHz) 7.39–
6.82 (m, 13H), 6.05 (d, 1H, NH), 5.62 (dt, 1H,
C-5), 5.35 (t, 2H, C-15%, C-16%), 5.29 (dd, 1H,
C-4), 4.18 (bt, 1H, C-3), 3.79 (s, 6H, OCH3), 3.42
(d, 1H, OH), 3.41 (dd, 1H, C-1) 3.31 (dd, 1H,
C-1), 2.19 (t, 2H, C-2%), 2.01 (m, 4H, C-14%, C-17%).
1.92 (m, 2H, C-6), 1.59 (m, 2H, C-3%), 0.88 (t, 6H,
C-18, C-24%).
To 16:0-Cer (0.057 g, 106 mmol) in 9 mL ben-
zene was added powdered sodium hydroxide
(0.051 g, 1.3 mmol), allyl bromide (183 mL, 2.1
mmol). The reaction was heated to reflux for 15 h.
After cooling to room temperature, the solvent
was removed under reduced pressure and the
residue purified by flash chromatography (8:1-0:1
hexane:EtOAc) to provide 3-O-allyl-16:0-Cer
(0.024 g, 39%) and 1-O-allyl-16:0-Cer (0.021 g,
34%) as white films.
2.5. 1-O-allyl-24:1-Cer (6) and
3-O-allyl-24:1-Cer (18)
3-O-allyl-16:0-Cer (7): Rf (1:1 hexane:EtOAc)
0.45; 1H NMR (300 MHz) 6.27 (d, 1H, NH,
J=7.3 Hz), 5.80 (m, 2H, C-5, CH=CH2), 5.38
(dd, 1H, C-4, J=7.8, 15.6 Hz), 5.12 (t, 2H,
CH=CH2), 4.0 (m, 4H, C-2 C-1, OCH2), 3.72
(dd, 1H, C-1, J=5.9, 12.7 Hz), 3.72 (bd, 1H, C-3,
J=13.7 Hz), 3.04 (bs, 1H, OH), 2.22 (t, 2H, C-2%,
J=7.3 Hz), 2.05 (t, 2H, C-6, J=6.8 Hz), 1.62 (m,
2H, C-3%), 0.88 (t, 6H, C-18, C-16%, J=6.8 Hz).
1-O-allyl-16:0-Cer (19): Rf (1:1 hexane:EtOAc)
0.62; 1H NMR (300 MHz) 6.18 (d, 1H, NH,
J=7.8 Hz), 5.86 (m, 2H, C-5, CH=CH2), 5.46
(dd, 1H, C-4, J=5.4, 15.6 Hz), 5.18 (dd, 2H,
CH=CH2), 4.17 (bs, 1H), 4.01 (bs, 1H), 3.93 (bs,
2H, OCH2), 3.72 (dd, 1H, C-1, J=3.4, 9.8 Hz),
3.52 (75 (dd, 1H, C-1, J=3.4, 9.8 Hz) 3.38 (d,
1H, OH, J=8.8 Hz), 2.19 (t, 2H, C-2%, J=7.3
Hz), 2.01 (m, 2H, C-6), 1.60 (m, 2H, C-3%), 0.85 (t,
6H, C-18, C-16%, J=6.8 Hz).
To 24:1-ceramide (0.029 g, 45 mmol) in 10 ml
benzene was added powdered sodium hydroxide
(0.065 g, 1.5 mmol) allyl bromide (90 ml, 1 mmol).
The reaction was heated to reflux for 18.5 h. After
cooling to room temperature, the solvent was
removed under reduced pressure and the residue
purified by flash chromatography (7:1–0:1 hex-
ane:EtOAc) to provide 3-O-allyl-24:1-Cer (0.010
g, 32%) and 1-O-allyl-24:1-Cer (0.009 g, 29%) as
white films.
2.5.1. 1-O-allyl-24:1-Cer (6)
Rf (1:1 hexane:EtOAc) 0.58; 1H NMR (500
MHz) 6.22 (d, 1H, NH, J=8.0 Hz), 5.86 (m, 1H,
CHꢀCH2), 5.76 (dt, 1H, C-5), 5.49 (dd, 1H, C-4
J=9.3, 15.5 Hz), 5.35 (t, 2H, C-15%, C-16%, J=4.3
Hz), 5.24 (dd, 2H, CHꢀCH2), 4.16 (bs, 1H), 4.05
(bs, 1H), 3.96 (t, 2H, OCH2, J=6.2 Hz), 3.75 (dd,

4
A.S. Goldstein et al. / Chemistry and Physics of Lipids 109 (2001) 1–14
2.7. 1-O-t-butyldiphenylsilyl-24:1-Cer (9)
7.57 (m, 2H), 6.04 (d, 1H, NH), 5.79 (dt, 1H,
C-5), 5.55 (dd, 1H, C-4), 5.35 (t, 2H, C-15%,
C-16%), 4.63 (dd, 1H, C-1), 4.46 (m, 2H, C-1, C-3),
4.31 (bs, 1H, C-2), 2.97 (bs, 1H, OH), 2.19 (t, 2H,
C-2%), 2.01 (m, 6H, C-6, C-14%, C-17%), 1.59 (m,
2H, C-3%), 0.88 (t, 6H, C-18, C-24%).
To 3-O-tBDPS-24:1-Cer (0.032 g, 36 mmol) in 3
ml dry THF was added NaH (0.001 g, 40 mmol).
After stirring for 10 min, 2-bromoethanol 3.1 ml,
43 mmol) was added and the reaction stirred for 3
h. The solvent was removed by rotary evapora-
tion and the residue purified by flash chromatog-
raphy (10:1–0:1 hexane:EtOAc) to provide in
addition to starting material some target as a
clear film (0.007 g, 21%) Rf (3:1 hexane:EtOAc)
2.9. 1-Biotinamido-24:1-Cer (11)
To 1-phthalimido-3-O-tBDPS-24:1-Cer (inter-
mediate in synthesis of compound 15, 0.046 g,
45.3 mmol) in 12 ml 95% EtOH was added 0.23 ml
hydrazine hydrate. The solution was heated to
reflux for 2 h, then the solvent removed by rotary
evaporation, and the residue purified by flash
chromatography (1:0–9:1 EtOAc:MeOH) to give
1-amino-3-O-tBDPS-24:1-Cer (0.036 g, 90%) as a
1
0.31; H NMR (500 MHz) 7.70–7.32 (m, 10H),
6.14 (d, 1H, NH, J=7.4 Hz), 5.83 (dt, 1H, C-5),
5.54 (dd, 1H, C-4), 5,41 (t, 2H, C-15%, C-16%), 4.02
(m, 2H, C-1, C-3), 3.81 (bd, 1H, C-1), 3.58 (d, 1H,
OH), 2.25 (t, 2H, C-2%), 2.07 (m, 6H, C-6, C-14%,
C-17%), 1.66 (m, 2H, C-3%), 1.14 (s, 9H), 0.95 (t,
6H, C-18, C-24%, J=6.2 Hz).
1
clear film, Rf (MeOH) 0.48; H NMR (500 MHz)
7.77–7.34, (m, 10H) 6.02 (d, 1H, amide), 5.34 (m,
4H, C-4, C-5, C-15%, C-16%), 4.25 (m, 1H, C-3),
3.99 (m, 1H, C-2), 3.02 (bs, 2H, C-1), 2.55 (bs,
2H, NH2) 2.02–1.83 (m, 8H, C-6, C-2%, C-14%,
C-17%), 1.05 (s, 9H, t-Bu), 0.88 (t, 6H, C-18, C-24%
J=7.2 Hz).
2.8. 1-O-(2-napthoic acid)-24:1-Cer (10)
To 3-O-tBDPS-24:1-Cer (0.063 g, 71.1 mmol),
2-napthoic acid (0.013 g, 78.2 mmol) and DMAP
(0.010 g, 78.2 mmol) in 10 ml dry 1:1
CH3CN:CH2Cl2 was added dicyclohexylcarbodi-
imide (0.016 g, 78.2 mmol). The reaction was
stirred for 22 h and the white precipitate removed
by filtration. The solvent was evaporated and the
residue purified by flash chromatography (8:1–2:1
hexane:EtOAc) to provide 1-O-(2-napthoyl)-3-O-
tBDPS-24:1-Cer (0.052 g, 70%, as a white residue:
To 1-amino-3-O-tBDPS-24:1-Cer (0.021 g, 23.7
mmol) in 12 ml dry THF was added 18 ml of 1.0 M
(in THF) tetrabutylammonium fluoride. The reac-
tion stirred 3 h, the solvent removed by rotary
evaporation, and the residue purified by flash
chromatography (1:0–0:1 EtOAc:MeOH). Frac-
tions containing 1-amino-24:1-Cer were com-
bined, diluted with H2O, extracted with 5×Et2O,
and the extracts concentrated to provide the de-
sired material as a clear film (0.013 g, 87%), Rf
1
Rf (3:1 hexane:EtOAc) 0.57; H NMR (500 MHz)
7.98–7.29 (m, 17H), 5.57 (d, 1H, NH), 5.50 (dd,
1H, C-4), 5.47 (dt, 1H, C-5), 5.35 (t, 2H, C-15%,
C-16%), 4.66 (dd, 1H, C-1), 4.50 (dd, 1H, C-1),
4.46 (m, 1H, C-2), 4.38 (bt, 1H, C-3), 2.03–1.83
(m, 8H, C-6, C-2%, C-14%, C-17%), 1,41 (m, 2H,
C-3%). 0.88 (t, 6H, C-18, C-24%).
1
(1:1 hexane:EtOAc) 0.36; H NMR (500 MHz),
5.76 (m, 1H, C-5), 5.47 (dd, 1H, C-4, J=3.4, 7.7
Hz), 5.35 (t, 2H, C-15%, C-16%, J=7.7 Hz), 4.20
(m, 3H, C-1, C-3), 3.96 (m, 1H, C-2), 2.17–1.95
(m, 8H, C-6, C-2%, C-14%, C-17%), 1.62 (m, 2H,
C-3%), 0.88 (t, 6H, C-18, C-24% J=6.6 Hz); IR
To 1-O-(2-napthoyl)-3-O-tBDPS-24:1-Cer
(0.051 g, 49 mmol) in 15 ml dry THF was added
1.0 M tetrabutylammonium fluoride (14 ml). The
reaction was stirred 3 h, whereupon the solvent
was removed by rotary evaporation and the
residue purified by flash chromatography (6:1–2:1
hexane:EtOAc) to provide the title compound
(0.026 g, 67%) as a clear residue, Rf (1:1 hex-
(neat) 3294, 2925, 2100, 1751 cm−1
.
To 1-amino-24:1-Cer (0.005 g, 7 mmol) in 5 ml
dry DMF was added biotin-tetrafluorophenyl es-
ter (0.006 g, 16 mmol). After stirring overnight,
the solvent evaporated in vacuo. Flash chro-
matography of the residue (9:1 CHCl3:MeOH)
provided the target as a clear oil (0.001 g, 14%),
1
ane:EtOAc) 0.63; H NMR (500 MHz) 8.59 (bs,
1
1H), 8.03 (dd, 1H), 7.95 (d, 1H), 7.87 (d, 2H),
Rf (5:1 CHCl3:MeOH) 0.79; H NMR (500 MHz)

A.S. Goldstein et al. / Chemistry and Physics of Lipids 109 (2001) 1–14
5
7.01 (m, 1H, NH), 6.38 (s, 1H, NH) 6.25 (s, 1H,
NH), 5.77 (m, 1H, C-5), 5.49 (dd, 1H, C-4), 5.35
(m, 2H, C-15%, C-16%), 4.51 (s, 1H, biotin bridge-
head), 4.34 (s, 1H, biotin bridgehead), 4.11 (m, 1H,
C-3), 3.37 (m, 1H, C-2), 3.29 (m, 3H, C-1, biotin
methine), 2.91 (m, 1H, biotin methylene), 2.74 (m,
1H, biotin methylene), 2.33 (m, 2H, a-biotin
amide), 1.25 (m, 4H, biotin), 1.47 (m, 2H, d-biotin
amide), 0.88 (t, 6H, C-18, C-24% J=6.8 Hz).
C-16%, J=4.3 Hz), 4.24 (1H, C-3), 3.99 (s, 2H,
HOCH2C(O)), 3.95 (m, 1H, C-2), 3.50 (m, 2H,
C-1), 2.65 (bt, 1H, HOAc), 2.02–1.84 (m, 8H, C-6,
C-2%, C-14%, C-17%), 1.44 (m, 2H, C-3%), 1.08 (s, 9H,
t-Bu), 0.88 (t, 6H, C-18, C-24% J=6.2 Hz); FAB-
MS 944 (M+, 3%), 886 (M-t-Bu, 15%) 866 (M-Ph,
6%), 687 (M-tBDPSiOH, 47%).
2.12. 1-Hydroxyacetamido-24:1-Cer (14)
To
1-bromoacetamido-3-O-tBDPS-24:1-Cer
2.10. 1-Bromoacetamido-24:-1-Cer (12)
(0.010 g, 10 mmol) in 2 ml N-methyl-2-pyrrolidone
was added water (0.3 ml, 17 mmol) and NaHCO3
(0.003 g, 30 mmol). The reaction was incubated
under dry N2 (g) at 105°C for 23.5 h. After cooling
to room temperature, 10 ml H2O was added and
the solution was extracted twice with 10 ml Et2O.
The organic layers were washed with 10 ml H2O,
10 ml saturated NaCl (aq), and 10 ml H2O. The
solvent was removed by rotary evaporation and the
residue purified by flash chromatography (3:1–0:1
hexane:EtOAc) to provide the desired material as
To 1-bromoacetamido-3-O-tBDPS-24:1-Cer
(0.003 g, 3 mmol) in 3 ml THF was added 25 ml of
3% HCl/MeOH (v:v). The solution was stirred for
22 h whereupon 10 ml Et2O was added. The
mixture was washed with 3 ml 5% NaHCO3 (aq)
and 5 ml H2O. The solvent was removed under
vacuum and the residue purified by flash chro-
matography (1:1–0:1 hexane:EtOAc) to provide
the desired material as a clear film (0.001 g, 50%),
1
Rf (1:1 hexane:EtOAc) 0.19; H NMR (500 MHz)
1
a clear film (0.002 g, 20%), Rf (EtOAc) 0.07; H
6.06 (d, 1H, NH, J=6.8 Hz), 5.77 (dt, 1H, C-5),
5.49 (dd, 1H, C-4), 5.35 (t, 2H, C-15%, C-16%, J=4.9
Hz), 4.18 (1H, C-3), 4.06 (1H, C-2), 3.65 (1H), 3.50
(m, 2H), 3.32 (m, 1H), 2.75 (m, 1H), 2.20–1.98 (m,
8H, C-6, C-2%, C-14%, C-17%), 0.88 (t, 6H, C-18,
C-24%); ES-MS 769 (M+, 72%).
NMR (500 MHz) 7.05 (s, 1H, AcNH), 6.28 (d, 1H,
NH, J=7.4 Hz), 5.81 (m, 1H, C-5), 5.55 (dd, 1H,
C-4), 5.40 (t, 2H, C-15%, C-16%, J=4.3 Hz), 4.15 (m,
4H, C-2, C-3, HOCH2), 3.67 (m, 1H, C-1), 3.50 (m,
1H, C-1), 3.16 (bs, 1H, OH), 2.80 (bs, 1H, OH)
2.24–1.84 (m, 8H, C-6, C-2%, C-14%, C-17%), 0.88 (t,
6H, C-18, C-24% J=6.2 Hz); FAB-MS 706 (M+,
6%), 688 (M-H2O, 12%).
2.11. 1-Hydroxyacetamido-3-O-t-butyldiphenyl-
silyl-24:1-Cer (13)
2.13. 1-Phthalimido-24:1-Cer (15)
To
3-O-tBDPS-1-bromoacetamido-24:1-Cer
(0.109 g, 108 mmol) in 20 ml N-methyl-2-
pyrrolidone was added water (0.3 ml, 17 mmol) and
NaHCO3 (0.009 g, 108 mmol). The reaction was
incubated under dry N2 (g) at 93°C for 18.5 h. After
cooling to room temperature, 20 ml H2O was added
and the solution was extracted twice with 20 ml
Et2O. The organic layers were washed with 15 ml
H2O, 15 ml saturated NaCl (aq), and 15 ml H2O.
The solvent was removed by rotary evaporation
and the residue purified by flash chromatography
(3:1–0:1 hexane:EtOAc to provide the desired
material as a clear film (0.073 g, 72%), Rf (EtOAc)
0.54; 1H NMR (500 MHz) 7.68–7.37 (m, 10H), 6.81
(s, 1H, AcNH), 5.74 (d, 1H, NH, J=8.7 Hz),
5.47–5.40 (m, 2H, C-4, C-5), 5.35 (t, 2H, C-15%,
To-3-O-tBDPS-24:1-Cer (0.111 g, 125 mmol),
triphenylphosphine (0.164 g, 626 mmol), and ph-
thalimide (0.020 g, 138 mmol) in 20 ml dry THF was
added diisopropylazodicarboxylate (27 ml, 138
mmol). The initially orange solution was stirred for
3 h. The solvent was evaporated and the residue
purified by flash chromatography (15:1–5:1 hex-
ane:EtOAc) to provide 1-phthalimido-3-O-tBDPS-
24:1-Cer as a white solid (0.108 g, 85%), Rf (3:1
1
hexane:EtOAc) 0.65; H NMR (500 MHz) 7.81–
7.34 (m, 14H), 5.62 (d, 1H, NH), 5.52 (m, 2H, C-4,
C-5), 5.35 (t, 2H, C-15%, C-16%), 4.29 (m, 2H, C-2,
C-3), 3.96 (dd, 2H, C-1), 1.99 (m, 8H, C-6, C-2%,
C-14%, C-17%), 1.60 (m, 2H, C-3%), 1.11 (s, 9H,
t-BuSi), 0.88 (t, 6H, C-18, C-24%).

6
A.S. Goldstein et al. / Chemistry and Physics of Lipids 109 (2001) 1–14
To 1-phthalimido-3-O-tBDPS-24:1-Cer (0.037 g,
7.08 (m, 1H, C-5), 6.70 (1H, NH), 6.26 (d, 1H, C-4,
J=17.3 Hz), 4.88 (1H, C-2), 3.94 (1H, C-1), 3.79
(1H, C-1), 3.27 (1H, OH), 2.25 (m, 4H, C-6, C-2%),
0.87 (t, 6H, C-16%, C-18).
36.4 mmol) in 12 ml dry THF was added 1.0 M (in
THF) tetrabutylammonium fluoride (11.6 ml, 40.1
mmol). The solution was stirred for 4 h. The solvent
was evaporated and the residue purified
by flash chromatography (6:1–1:1 hexane:EtOAc)
to provide the title material as a white solid (0.007
2.16. 1-Phthalimido-3-keto-24:1-Cer (18)
1
g, 25%), Rf (3:1 hexane:EtOAc) 0.09; H NMR
To 3-keto-24:1-Cer (0.005 g, 7.7 mmol),
triphenylphosphine (0.010 g, 38.7 mmol), and ph-
thalimide (0.001 g, 8.5 mmol) in 1 ml dry THF was
added diisopropylazodicarboxylate (0.002 g, 1.7
ml). The reaction was stirred overnight. The solvent
was removed by rotary evaporation and the residue
was purified by flash chromatography (6:1–3:1
hexane:EtOAc) to give the product (0.002 g, 33%)
(500 MHz) 7.88–7.71 (m, 4H), 6.15 (d, 1H, NH),
5.75 (dt, 1H, C-5), 5.50 (dd, 1H, C-4), 535 (t, 2H,
C-15%, C-16%), 4.35 (m, 1H, C-2), 4.23 (bs, 1H, C-3),
3.87 (dd, 2H, C-1), 3.07 (bs, 1H, OH), 2.11 (m, 2H,
C-2%), 2.02 (m, 6H, C-6, C-14%, C-15%), 1.50 (m, 2H,
C-3%), 0.88 (t, 6H, C-18, C-24%).
1
2.14. 3-Keto-24:1-Cer (16)
as a white film, Rf (3:1 hexane:EtOAc) 0.26; H
NMR (500 MHz) 7.90–7.76 (m, 4H), 6.41 (s, 1H,
NH), 5.71 (dt, 1H, C-5), 5.54 (d, 1H, C-4), 5.35 (t,
2H, C-15%, C-16%), 5.11 (d, 2H, C-1), 4.97 (t, 1H,
C-2), 2.39 (t, 2H, C-2%), 2.02 (m, 6H, C-6, C-14%,
C-17%), 1.66 (m, 1H, C-3%), 0.88 (t, 6H, C-18, C-24%).
To 24:1-Cer (0.012 g, 18.5 mmol) in 20 ml dry
benzene was refluxed with manganese(IV) oxide
(0.012 g, 122.4 mmol) for 5 h and then stirred an
additional 12 h at room temperature. The solvent
was removed by rotary evaporation and the residue
was purified by flash chromatography (5:1–2:1
hexane:EtOAc) to provide desired material (0.005
g, 42%) as a white film, Rf (1:1 hexane:EtOAc) 0.45;
1H NMR (500 MHz) 7.10 (dt, 1H, C-5), 6.77 (bs,
1H, amide), 6.26 (d, 1H, C-4), 5.35 (t, 2H, C-15%,
C-16%), 4.90 (m, 1H, C-2), 3.89 dd, 2H, C-1), 3.31
(bs, 1H, OH), 2.27 (m, 4H, C-2%, C-6), 2.01 (m, 4H,
C-14%, C-17%), 1.65 (m, 1H, C-3%), 1.46 (m, 1H, C-3%),
0.88 (t, 6H, C-18, C-24%).
2.17. 1-O-triphenylmethyl-3-O-methoxymethyl-
24:1-Cer (21)
To 1-O-triphenylmethyl-24:1-Cer (0.040 g, 45
mmol) and diisopropylethylamine (1 ml) in 25 ml
dry THF was added bromomethylmethyl, ether (16
ml, 195 mmol). The reaction was stirred overnight
and the solvent removed by rotary evaporation.
The residue was purified by flash chromatography
(8:1–0:1 hexane:EtOAc) to provide starting mate-
rial and pure target as a clear film (0.016 g, 38%),
2.15. 3-Keto-24:1-Cer (17)
1
Rf (3:1 hexane:EtOAc) 0.58; H NMR (500 MHz)
16:0-Cer (0.070 g, 0.13 mmol) and DDQ (0.295
g, 1.30 mmol) were refluxed for 5 h in dry benzene
and then stirred at 20°C for 4 days. The solvent was
removed by rotary evaporation and the residue
purified by flash chromatography (6:1–2:1 hex-
ane:EtOAc) to provide the desired material as a red
solid. This red solid was dissolved in 10 ml Et2O
and washed with 8×5 ml H2O, 5 ml brine, 5 ml
saturated NaHCO3 (aq) and 3×5 ml H2O. The
gold colored ether layer was filtered through celite,
evaporated and the residue purified by flash chro-
matography (3:1–1:1 hexane:EtOAc) to provide
the title compound as a white solid (0.010 g, 14%),
7.44–7.22 (m, 15H), 5.67 (m, 2H, NH, C-5), 5.35
(t, 2H, C-15%, C-16%, J=4.3 Hz), 5.23 (dd, 1H, C-4,
J=8.0, 15.5 Hz), 4.62 (d, 1H, OCH2O, J=6.8 Hz),
4.46 (d, 1H, OCH2O, J=6.8 Hz), 4.25 (m, 1H,
C-2), 4.19 (t, 1H, C-3, J=6.8 Hz), 3.38 (dd, 1H,
C-1, J=5.0, 9.9 Hz), 3.25 (dd, 1H, C-1, J=5.0, 9.9
Hz), 3.21 (s, 3H, OCH3), 2.10 (t, 2H, C-2%, J=6.8),
2.00 (m, 6H, C-6, C-14%, C-17%), 1.67 (m, 2H, C-3%),
0.88 (t, 6H, C-18, C-24%, J=6.2 Hz).
2.18. 3-O-methoxymethyl-24:1-Cer (22)
To 1-O-triphenylmethyl-3-O-methoxymethyl-
24:1-Cer (0.016 g, 17 mmol) in 10 ml 1:1
1
Rf (1:1 hexane:EtOAc) 0.26; H NMR (500 MHz)

A.S. Goldstein et al. / Chemistry and Physics of Lipids 109 (2001) 1–14
7
CH2Cl2:MeOH was added p-toluenesulfonic acid
monohydrate (0.008 mg, 42 mmol). The reaction
was stirred overnight and then reduced to 25% of
the initial volume by rotary evaporation. The
solution was diluted with 25 ml Et2O and washed
with 20 ml saturated NaHCO3 (aq) and 20 ml H2O.
The organic layer was evaporated and the residue
purified by flash chromatography (5:1–0:1 hex-
ane:EtOAc) to provide the target as a white film
1,2-dibromoethane (0.5 ml). The reaction was incu-
bated at 97°C for 17 h under inert atmosphere.
After cooling to room temperature, 30 ml H2O was
added and the solution extracted with 2×20 ml
Et2O. The ethereal solution was washed with 20 ml
H2O, 10 ml saturated NaCl (aq) and 10 ml H2O,
and then concentrated. The residue was purified by
flash chromatography (8:1–0:1 hexane:EtOAc) to
give the target as a white film (0.005 g, 15%), Rf (1:1
hexane:EtOAc) 0.52; 1H NMR (500 MHz) 5.80 (dt,
1H, C-5), 5.55 (m, 2H, C-4, NH), 5.38 (t, 2H, C-15%,
C-16%), 5.05 (d, 1H, OCH2O) 4.65 (d, 1H, OCH2O),
4.54 (d, 1H, OCH2O, J=6.8 Hz), 4.18 (dd, 1H,
C-3), 3.96 (m, 2H, C-1), 3.45 (m, 1H, C-2), 2.16 (m,
2H, C-2%), 2.05 (m, 6H, C-6, C-14%, C-17%), 1.62 (m,
2H, C-3%), 0.88 (t, 6H, C-18, C-24).
1
(0.010 g, 83%), Rf (1:1 hexane:EtOAc) 0.23; H
NMR (500 MHz) 6.23 (d, 1H, NH, J=7.4 Hz),
5.75 (m, 1H, C-5), 5.35 (m, 3H, C-4, C-15%, C-16%),
4.60 (d, 1H, OCH2O, J=6.8 Hz), 4.54 (d, 1H,
OCH2O, J=6.8 Hz), 4.21 (m, 1H, C-3), 3.96 (m,
2H, C-1), 3.66 (m, 1H, C-2), 3.38 (s, 3H, OCH3),
2.21 (m, 2H, C-2%), 2.05 (m, 6H, C-6, C-14%, C-17%),
1.62 (m, 2H, C-3%), 0.88 (t, 6H, C-18, C-24%, J=6.8
Hz).
2.21. (1,3-O-hexyl acetal)-24:1-Cer (25)
2.19. 3-O-methoxyethoxymethyl-24:1-Cer (23)
To 24:1-Cer (0.005 g, 8 mmol) in 15 ml CH2Cl2
was added hexanal (2 ml, 15 mmol) and p-toluene-
sulfonic acid monohydrate (0.0003 g, 1 mmol). The
reaction was heated to reflux overnight under inert
atmosphere. After cooling to room temperature,
the solvent was removed under reduced pressure
and the residue purified by flash chromatography
(8:1–3:1 hexane:EtOAc) to provide target as a
white film (0.002 g, 33%), Rf (3:1 hexane:EtOAc)
0.13; 1H NMR (500 MHz) 5.77 (dt, 1H, C-5), 5.45
(dd, 1H, C-4), 5.35 (t, 2H, C-15%, C-16%), 4.91 (d,
1H, NH), 4.52 (t, 1H, acetal), 4.22 (dd, 1H, C-3),
3.87 (m, 1H, C-2), 3.78 (t, 1H, C-1), 3.35 (t, 1H,
C-1), 2.16 (m, 2H, C-2%), 2.05 (m, 6H, C-6, C-14%,
C-17%), 1.64 (m, 2H, C-3%), 0.87 (t, 6H, C-18, C-24%).
To 1-O-tBDPS-24:1-Cer (0.025 g, 28 mmol) in 15
ml dry CH2Cl2 was added diisopropylethylamine
(9.8 ml g, 56 mmol) and 2-methoxyethoxymethyl
chloride (3.5 ml, 31 mmol). The reaction was stirred
for 4 h after which additional DIPEA (0.50 ml) and
MEMCI (0.25 ml) was added to the reaction stirred
overnight. The solution was washed with 30 ml
H2O and 30 ml saturated NaCl (aq). The solvent
was removed by rotary evaporation. The residue
was dissolved in 5 ml THF and stirred with 0.25 ml
1.0 M tBAF for 2 h. The solvent was removed by
rotary evaporation and the residue purified by flash
chromatography (3:1–0:1 hexane:EtOAc) to give
target as a clear film (0.016 g, 76%): (EtOAc) 0.41;
1H NMR (500 MHz) 6.06 (d, 1H, NH, J=8.0 Hz),
5.64 (dt, 1H, C-5), 5.27 (m, 3H, C-4, C-15%, C-16%),
4.66 (d, 1H, OCH2O, J=6.8 Hz), 4.51 (d, 1H,
OCH2O, J=6.8 Hz), 4.07 (t, 1H, C-3, J=7.4 Hz),
3.92 (m, 2H, C-1), 3.79 (bt, 1H, C-2, J=8.7), 3.55
(m, 4H, OCH2 CH2O), 3.35 (s, 3H, OCH3), 2.08 (t,
2H, C-2%), 1.95 (m, 6H, C-6, C-14%, C-17%), 1.54 (m,
2H, C-3%), 0.81 (t, 6H, C-18, C-24%, J=6.2 Hz).
2.22. (1,3-O-(3%hydroxy)-propyl acetal)-24:1-Cer
(26)
To 24:1-Cer (0.042 g, 65 mmol) in 10 ml CH2Cl2
was added 3-O-triphenylmethyl-propanal (0.019
mg, 71 mmol) and p-toluenesulfonic acid monohy-
drate (0.012 mg, 65 mmol). The reaction was heated
to reflux for 16 h. After cooling to room tempera-
ture, the reaction was washed with 10 ml saturated
NaHCO3 (aq) and 10 ml H2O. The solvent was
removed under reduced pressure and the residue
purified by flash chromatography (4:1–0:1 hexane:
EtOAc) to provide (1,3-O-(3%-hydroxy)-propyl ace-
2.20. (1,3-O-formyl acetal)-24:1-Cer (24)
To 3-O-methoxymethyl-24:1-Cer (0.036 g, 52
mmol) in 10 ml N-methyl-2-pyrrolidone was added

8
A.S. Goldstein et al. / Chemistry and Physics of Lipids 109 (2001) 1–14
tal)-24:1-Cer as a white film (0.014 g, 30%), Rf
(EtOAc) 0.57; H NMR (500 MHz) 5.76 (dt, 1H,
prepared, albeit in poor yield, using a zinc azide
bispyridine complex (Viaud and Rollen, 1990).
1-O-triphenylmethyl-24:1-Cer (2), (Goldstein et al.,
1997), 1-O-triphenylmethyl-16:0-Cer (3), and 1-O-
triphenylmethyl-8:0-Cer (4) were formed by treat-
ing the appropriate ceramide with triphenyl-
methylchloride. The 1-O-dimethoxytriphenyl-
methyl-24:1-Cer (5) was prepared using dimethoxy-
triphenylmethylchloride. 1-O-allyl-24:1-Cer (6)
and 1-O-allyl-16:0-Cer (7) were generated in
moderate yields when the 1° alcohol of the appro-
priate ceramide was reacted with sodium hy-
droxide and allylbromide. The 1-O-p-toluene-
sulfonyl-24:1-Cer (8) was formed, albeit in very
1
C-5), 5.44 (dd, 1H, C-4), 5.35 (t, 2H, C-15%,
C-16%), 4.92 (d, 1H, NH), 4.78 (t, 1H, acetal), 4.26
(dd, 1H, C-3), 3.92 (m, 1H, C-2), 3.78 (bd, 2H,
C-1), 3.37 (t, 1H, OH), 2.10 (m, 2H, C-2%), 2.01
(m, 4H, C-14%, C-17%), 1.93 (M, 2H, C-6), 0.88 (t,
6H, C-18, C-24%, J=6.8 Hz).
3. Conjugation to preformed CHARMs
To 6.5 mg of NFA-GalCer CHARMs (nonhy-
droxy fatty acid containing portion of galacto-
cerebroside) in 200 ml 0.15 M NaHCO3 (aq) was
stirred for 96 h in the dark with 1.0 mg BODIPY-
FL-NSSE (Molecular Probes). The assemblies
were pelleted by centrifugation (10 min, 10 000×
g) and the supernatant removed. The pellet was
washed with 4×1 ml H2O and 2×1 ml 1 M
NaCl (aq) with centrifugation (10 min, 10 000×
g) between washes.
3.1. 1:3 1-amino-Cer:NFA-GalCer
As above except the lipid assembly (4.9 mg) of
tubules was stirred with 0.85 mg BODIPY-FL-
NSSE.
4. Synthesis
A variety of headgroups were created through
covalent modification of the sphingosine
molecule. These headgroups include azide, ethers,
silylethers, sulfonyl esters, esters and amides. The
synthesis of the analogs began with 24:1-Cer,
16:0-Cer, 8:0-Cer, 3-silyl-24:1-Cer, 1-trityl-24:1-
Cer or 1-amino-24:1-Cer as shown in Figs. 1 and
2. These materials are commercially available,
described within this paper (1-amino-24:1-Cer), or
previously published. (Goldstein et al., 1997).
4.1. C-1 modification of 24:1-Cer
The following materials were generated by re-
acting the 1° alcohol of 24:1-Cer with a variety of
reagents. The 1-azido-24:1-Cer (1) material was
Fig. 1.

A.S. Goldstein et al. / Chemistry and Physics of Lipids 109 (2001) 1–14
9
yacetamido-3-silyl-24:1-Cer
(13)
and
the
1-hydroxyacetamido-3-hydroxy-24:1-Cer
(14)
upon treatment with mild aqueous base in N-
methylpyrolidine (Hutchins and Taffer, 1983).
Treatment of 3-silyl-24:1-Cer with phthalimide
under Mitsunobu conditions followed by fluoride
desilylation formed 1-phthalimido-24:1-Cer (15).
Ceramides with functionality at the 2° allylic
alcohol (C-3) were also generated. The ketone,
allyl ether, methoxyethoxymethyl ether, and
methoxymethyl ether were prepared. Treatment of
24:1-Cer or 16:0-Cer with manganese oxide gener-
ated the respective 3-keto materials (16) and (17).
Using a Mitsunobu reaction, 3-keto-24:1-Cer (16)
was converted to 1-phthalimido-3-keto-24:1-Cer
(18). Treatment of 24:1-Cer or 16:0-Cer with allyl-
bromide and sodium hydroxide also formed the
3-O-allyl-24:1-Cer (19) and 3-O-allyl-16:0-Cer
(20). 1-O-triphenylmethyl-24:1-Cer was converted
to the 1-O-triphenylmethyl-3-O-methoxyethoxy
methyl material (21) which was also detritylated
under mild acid conditions to provide 3-O-
methoxyethoxymethyl-24:1-Cer (22). Similarly, 1-
O-tBDPS-24:1-Cer (9) was converted to the
3-O-methoxymethyl-24:1-Cer (23) by treatment
with methoxymethyl chloride followed by fluoride
mediated desilylation.
Ceramides with bridging functionality at C-1
and C-3 were also prepared. Originally, attempts
to O-alkylate C-1 in the presence of a 3-O-
methoxymethyl ether (22) under basic conditions
resulted in the formation of the 1,3-formyl acetal
(24). This unexpected result led to the further
investigations involving cyclic acetals. The 1,3-
hexyl acetal (25) was prepared by treating 24:1-
Cer with hexanal and a mild acid. Lastly,
treatment of 24:1-Cer with 3-O-triphenylmethyl-
propanal and p-toluenesulfonic acid generated the
3%-hydroxy-(1,3)-propyl acetal (26).
Fig. 2.
poor yield, using the corresponding sulfonyl chlo-
ride. A failed Williamson etherification reaction
caused migration of the 3-O-t-butyldiphenylsilyl
group to form the 1-O-tBDPS-24:1-Cer (9). The
1-O-napthoyl-24:1-Cer (10) was prepared using a
carbodiimide mediated coupling of napthoic acid
with a 3-silyl protected ceramide (Goldstein et al.,
1997) followed by acid catalyzed desilylation.
The following synthesized lipids have nitrogen
replacing the C-1 oxygen. The 3-silyl protected
ceramide (Goldstein et al., 1997) was converted to
the 1-phthalimdo-24:1-Cer using Mitsunobu
chemistry. The amine was revealed upon treat-
ment with hydrazine and silyl group could be
removed using fluoride. 1-Biotin-24:1-Cer (11)
material was formed by treatment of 1-amino —
24:1-Cer with biotin-tetrafluorophenyl ester
(Wilbur et al., 1997). 1-N-bromoacetamido-24:1-
Cer (12) was prepared by reaction of the bro-
moacetic anhydride (Robey and Fields, 1989) with
a 1-amino-3-silyl-24:1-Cer followed by fluoride
mediated desilylation. The 1-bromoacetamido-3-
silyl material was also converted to the 1-hydrox-
5. Results and discussion
In all cases, sphingolipids were precipitated
from a 1.0 mM DMF solution by the addition of
water (35% by volume) at room temperature. This
method has been found to be the most likely one
to form CHARMs of those tried to date. The

10
A.S. Goldstein et al. / Chemistry and Physics of Lipids 109 (2001) 1–14
fore, chemical modifications to this region may be
expected to affect CHARM topology (Pascher,
1976; Pascher and Sundell, 1977; Pascher et al.,
1992; Hamilton et al., 1993; Nandi and Bagchi,
1996; Selinger et al., 1996; Moore and Rerek,
1997). Furthermore, a sphingolipid’s hydrocarbon
packing energy stabilization may also play a fac-
tor in CHARM formation. Since we have no data
that bears on sphingolipid intermolecular interac-
tions, it is difficult to determine the exact reason
why lipids may or may not form CHARMs;
however, some trends appear to be present. In
general, sphingolipids lacking a H-bond donor/ac-
ceptor attached to C-1 (excluding the C-1 oxygen
if present) did not form CHARMs. The 1-azido-
24:1-Cer (1), 1-Tr-8:0-Cer (4), 1-DmTr-24:1-Cer
(5), and 1-tBDPS-24:1-Cer (9) formed amorphous
aggregates. Some of these results are surprising in
light of the fact that similar compounds do make
supramolecular assemblies. For example, 1-Tr-
resultant cloudy suspensions were allowed to
stand overnight; an aliquot was then removed for
examination by TEM. The observed morpholo-
gies are summarized in Table 1 and representative
example shown in Fig. 3. In 9 of 26 cases tried, no
type of recognizable CHARM formed; these pre-
cipitates are denoted as ‘amorphous’. When
CHARMs did form, their morphologies were ei-
ther ribbons or cylindrical structures. In many
cases, the ribbons tended to twist at irregular
intervals or fold over onto themselves. In most
cases, there existed up to a 2-fold variation in the
measured diameter. Since CHARMs tend to form
a tangled mesh (Archibald and Mann, 1994;
Kulkarni et al., 1995), determining the average
length of the supramolecular assemblies was not
usually feasible.
A sphingolipid’s CHARM morphology may be
attributed to specific patterns of inter- and intra-
molecular H-bonding of the headgroups; there-
Table 1
Compound c
Headgroup descriptor
Morphology
Dimensions
1
2
3
1-Azido-24:1
1-TrO-24:1
1-TrO-16:0
Amorphous
CHARM
Sheet
–
1.3 mm
–
4
5
6
7
1-TrO-8:0
1-DmTrO-24:1
1-O-allyl-24:1
1-O-allyl-16:0
Amorphous
Amorphous
Amorphous
Ribbon
–
–
–
0.1 mm
–
8
1-TsO-24:1
Ribbon
9
1-O-tBDPSi-24:1
1-Napthoyl-24:1
Amorphous
Hollow tube
Ribbon
CHARM
Amorphous
CHARM
Ribbon
Ribbon
Ribbon
Amorphous
Ribbon
Amorphous
CHARM
CHARM
Amorphous
Ribbon
–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
0.3 mm
0.2 mm
0.8 mm
–
1-Biotinamido-24:
1-Bromoacetamido-24:1
1-Hydroxyacetamido-3-O-tBDPSi-24:1
1-Hydroxyacetamdo-24:1
1-Phathalimido-24:1
3-Keto-24:1
3-Keto-16:0
1-Phthalimido-3-keto-24:1
3-O-allyl-24:1
–
0.2 mm
1.3 mm
50 nm
–
1.7 mm
–
0.3 mm
0.8 mm
–
3-O-allyl-16:0
1-TrO-3-OMOM-24:1
3-OMOM-24:1
3-OMEM-24:1
1,3-O-formylacetal-24:1
1,3-O-hexylacetal-24:1
1,3-O-(3%-hydroxy)-propylacetal-24:1
85 nm
CHARM
Ribbon
10×200 nm

A.S. Goldstein et al. / Chemistry and Physics of Lipids 109 (2001) 1–14
11
Fig. 3. TEM of (A) bromoacetamido-24:1-Cer (12). Scale bar is 10 mm; (B) 1-biotinamido-24:1-Cer (11). Scale bar is 2 mm; (C)
3-keto-24:1-Cer (16). Scale bar is 10 mm.

12
A.S. Goldstein et al. / Chemistry and Physics of Lipids 109 (2001) 1–14
24:1-Cer (2) and 1-Tr-16:0-Cer (3) formed
CHARMs. On initial examination, one would
expect the structurally similar shorter acyl trityl
compound (4) and the dimethoxytrityl (5) to ag-
gregate into well defined morphologies. In the first
case, it appears that the bulky hydrophobic trityl
headgroup may disrupt the tight headgroup pack-
ing required for CHARM formation. However,
longer alkyl chain sphingolipids (2) and (3) have
stronger hydrocarbon interactions that may coun-
teract the disruption by the trityl group. In the
dimethoxytrityl case, either the added methoxy
groups could exceed the cross-sectional diameter
of a sphingolipid, thus preventing favorable head-
group–headgroup packing interactions (large
headgroup with small alkyl chains tends to favor
HII), or the increased electron density of the
methoxyphenyl group may perturb potential en-
ergy stabilizing dipole–dipole interactions (p-
stacking) between headgroups. When a phenyl
group is replaced with a t-butyl group, as in the
case of 1-tBDPS-24:1-Cer (9), microstructures did
not form, perhaps because of the absence of possi-
ble p-stacking in the headgroups, supporting the
above premise.
A majority of the modified headgroups are
similar in size but differ electronically. For exam-
ple, phthalimide (15), tosyl (8), and napthoyl (10)
substituents are ester linked, relatively nonpolar,
have significant p-clouds, and formed CHARMs.
On the contrary, biotin (11), bromoacetamide (12)
and hydroxyacetamide (14) lipids are amide
linked, relatively polar but lack significant p-
clouds, they also formed CHARMs. Perhaps ad-
ditional H-bond donor/acceptors in the amide
linked lipids (as compared with potential p-stack-
ing in the aromatic headgroups) are sufficient to
permit CHARM formation, or the hydrocarbon
interactions give sufficient stabilization.
Cer (6), 1-O-allyl-16:0-Cer (7), 3-O-allyl-24:1-Cer
(19), and 3-O-allyl-16:0-Cer (20).
The allyl group is hydrophobic but relatively
small, so it may try to ‘dive’ into the hydrophobic
region causing significant rearrangement of the
headgroup. Since 1-O-allyl-24:1-Cer (6) already
has a perturbed hydrocarbon region, further rear-
rangement of the H-bonding network of the head-
group and hydrocarbon packing may not favor
CHARM formation, resulting in amorphous ag-
gregates. On the other hand, 1-O-allyl-16:0-Cer
(7), existing in an all trans configuration, may be
able to better reorganize its hydrocarbon chains
to accommodate the small headgroup, thus allow-
ing CHARM formation.
When the allyl group was moved to the C-3
position, the 3-O-allyl-24:1-Cer (19) formed
CHARMs, whereas 3-O-allyl-16:0-Cer (20) did
not form CHARMs. In these cases, if the allyl
group dives into the hydrophobic region the head-
group does not have to significantly rearrange.
For the longer chain amphiphile (19) the greater
hydrocarbon length allows for greater flexibility in
accommodating packing perturbances than the
shorter hydrocarbon chains of amphiphile (20).
Other chemical modifications at C-3 of the
hydrophilic regions can be tolerated. For exam-
ple, the C-3 keto compounds (16) and (171
formed ribbonlike CHARMs. The size of the
attached hydrophilic group is important. The
smaller methoxymethyl material (22) did form
CHARMs whereas, the methoxyethoxymethyl
group (23), which contains one additional methyl-
ene, did not form microstructures.
It also appears that dual modification within
the hydrophilic region can be tolerated. 1-Trityl-3-
MOM-24:1-Cer (21) formed CHARMs. Acetals,
which include the C-1 and C-3 oxygens in a six
membered ring can form microstructures. The
acetals can be hydrophilic such as 1,3-formylac-
etal (24) or 1,3-prophylhydroxyacetal (26) or hy-
drophobic such as 1,3-hexylacetal (25).
In almost all cases, two different N-acyl groups,
16:0 (palmitoyl) and 24:1 (nervonoyl), were em-
ployed. Palmitoyl, being full saturated, can exist
in an all-trans configuration, the lowest hydrocar-
bon energy state. Nervonoyl contains a cis double
bond so the hydrocarbon tail has a ‘kink’ that
perturbs the hydrocarbon packing. The hydrocar-
bon packing differences may be responsible for
differences in the morphologies of 1-O-allyl-24:1-
6. Conjugation to preformed CHARMs
Since not all headgroup-modified sphingolipids
can independently form CHARMs, it is advanta-

A.S. Goldstein et al. / Chemistry and Physics of Lipids 109 (2001) 1–14
13
did fluoresce. In fact, CHARMs with amine
present at only 0.5 mol% were successfully conju-
gated (not shown) and all the amine was reagent
accessible when treated with 5-fold excess of acti-
vated fluorophore. When the fluorescent-
CHARMs are dissolved in solvent and
re-precipitated, the BODIPY-amine-sphingolipid
is excluded from the CHARMs.
7. Conclusions
Sphingolipid-based CHARMs tolerate a wide
variety of chemical moieties within the headgroup
region. Headgroups may include hydrophobic and
hydrophilic moieties and may be cyclic or acyclic
in nature. Based on the few compounds that
failed to form CHARMs used in this study, it
appears that as long as the H-bonding network of
the hydrophilic headgroup is not significantly per-
turbed, regular microstructures of some sort can
form. A sphingolipid with an amine-containing
headgroup may be chemically modified after
CHARM formation. These results suggest that a
wide range of therapeutic moieties could be incor-
porated into CHARMs for potential use as single
or multiple (co)drug delivery systems. The insolu-
bility of the long chain sphingolipids may immo-
bilize the CHARM at the desired site of action,
thus serving as a local drug depot and increasing
the residence time of hydrophilic drugs. This is
under exploration in our group at this time.
Fig. 4. Optical micrographs of (A) BODIPY treated NFA-
GalCer:1-amino-24:1-Cer CHARMs viewed under white light;
(B)
BODIPY
treated
NFA-GalCer:1-amino-24:1-Cer
CHARMs viewed under fluorescent light; (C) BODIPY
treated NFA-GalCer CHARMs viewed under white light; (D)
BODIPY treated NFA-GalCer CHARMs viewed under
fluorescent light.
geous to have a method with which to covalently
attach the drug to preformed CHARMs.
CHARMs composed of the nonhyxdroxy fatty
acid portion of galactocerebroside (NFA-GalCer)
or the 1:3 mixture of 1-amino-24:1-Cer and NFA-
GalCer were used as models for such a template.
CHARMs were treated with an activated fluores-
cent carboxylic acid (BODIPY); only the amine
should react with the fluorophore for conjugation.
After incubation, the supernatants were removed,
and the nanostructures washed with brine to re-
move any noncovalently associated BODIPY. As
shown in Fig. 4, optical micrographs of the
‘ropes’ of NFA-GalCer assemblies did not have
any significant fluorescence associated with the
microstructures whereas the mixed lipid system
Acknowledgements
This work was supported by a grant from the
Whitaker Foundation. The authors thank Dr
Paul Carlson and Dr Anatoly Lukyanov for per-
forming TEM.
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