Comparison of the supramolecular structures of two glyco lipids with chiral and nonchiral methyl-branched alkyl chains from natural sources

Article abstract of DOI:10.1021/jp0460238

Two alkyl glycosides with the same type of disaccharide headgroups (melibiose) and different methyl-branched alkyl chains, short chiral [(2R,4R,6R,8R)-2,4,6,8-tetramethyldecyl, extracted from an animal source] and long nonchiral (3,7,11,15-tetramethylhexadecyl, from a plant source), were synthesized. The supramolecular aggregate structure formed in dilute solutions was investigated by small-angle neutron scattering and surface tension measurements. The lyotropic phase diagram was studied by differential scanning calorimetry and water penetration scans. The thermotropic phase behavior was investigated by polarizing microscopy. The compounds showed unusual phase behavior: (i) The liquid-crystalline polymorphism is reduced to only form smectic A phases in the pure state; the formation of lyotropic phases such as hexagonal or lamellar phases was not observed, (ii) The compound with the longer nonchiral alkyl chain is more soluble in water than the one with the shorter chiral chain, most likely because of the different flexibilities of the chains, (iii) For the long-chain compound, the formation of micelles is observed, whereas the short-chain compound forms large disklike/bilayer aggregates. The method of methylation of the chain controls the self-assembly and can explain different biological functions for either plants (variable temperature) or animals (constant temperature).

Full text of DOI:10.1021/jp0460238

J. Phys. Chem. B 2005, 109, 1599-1608
1599
Comparison of the Supramolecular Structures of Two Glyco Lipids with Chiral and
Nonchiral Methyl-Branched Alkyl Chains from Natural Sources
Go1tz Milkereit,† Vasil M. Garamus,‡ Jun Yamashita,§ Masakatsu Hato,§ Michael Morr,| and
Volkmar Vill*,†
Institute of Organic Chemistry, UniVersity of Hamburg, Martin Luther King Platz 6, D-20146 Hamburg,
Germany, GKSS Research Centre, Max Planck Straâe 1, D-21502 Geesthacht, Germany, Nanotechnology
Research Institute, AIST, 1-1-1 Higashi, Tsukuba Central 5, Tsukuba, Ibaraki 305-8565, Japan, and
Gesellschaft fu¨r Biotechnologische Forschung mbH, Mascheroder Weg 1, D-38124 Braunschweig, Germany
ReceiVed: September 3, 2004; In Final Form: October 26, 2004
Two alkyl glycosides with the same type of disaccharide headgroups (melibiose) and different methyl-branched
alkyl chains, short chiral [(2R,4R,6R,8R)-2,4,6,8-tetramethyldecyl, extracted from an animal source] and long
nonchiral (3,7,11,15-tetramethylhexadecyl, from a plant source), were synthesized. The supramolecular
aggregate structure formed in dilute solutions was investigated by small-angle neutron scattering and surface
tension measurements. The lyotropic phase diagram was studied by differential scanning calorimetry and
water penetration scans. The thermotropic phase behavior was investigated by polarizing microscopy. The
compounds showed unusual phase behavior: (i) The liquid-crystalline polymorphism is reduced to only form
smectic A phases in the pure state; the formation of lyotropic phases such as hexagonal or lamellar phases
was not observed. (ii) The compound with the longer nonchiral alkyl chain is more soluble in water than the
one with the shorter chiral chain, most likely because of the different flexibilities of the chains. (iii) For the
long-chain compound, the formation of micelles is observed, whereas the short-chain compound forms large
disklike/bilayer aggregates. The method of methylation of the chain controls the self-assembly and can explain
different biological functions for either plants (variable temperature) or animals (constant temperature).
1. Introduction
The first observation of lyotropic liquid-crystalline phase
behavior was in 1884 when Koch observed unusual optical
textures upon the investigation of aqueous dispersions of extracts
from tuberculosis bacteria.1 The observation of thermotropic
liquid crystallinity is also related to alkylated carbohydrates.
Fischer and Helferich observed a double melting of hexadecyl-
â-D-glucopyranoside.2 These amphotropic molecules that form
both thermotropic and lyotropic mesophases gained much
interest during recent years.3-7 They are mainly nontoxic and
biodegradable and can be obtained from renewable sources.
The principal phase behavior of these compounds is shown
in Figure 1.8,9 A typical amphiphile is represented by a vertical
line and usually exhibits only one mesophase, but in the case
of an unusual geometry of the hydrophilic headgroup, poly-
morphism can occur. In this case, cubic phases may also be
observed,10 while simple amphiphiles normally exhibit only a
smectic A (SA) phase thermotropically.11
Figure 1. Principal phase behavior of amphiphilic compounds.8,9
addition of paraffin, it is possible to induce inverse phases (e.g.,
inverse micelles), with the polar headgroup curved away from
the solvent.
The discontinuous cubic phases are based upon various
packings of spherical or slightly anisotropic micelles, whereas
the bicontinuous cubic phases with interwoven fluid porous
structures are based upon underlying infinite periodic minimal
surfaces.12 This phase, in particular, is of great biological
interest.13-18 The lamellar (LR) phase with its multibilayer
structure has long been well-known to biologists, but there is
much evidence today that the bicontinuous cubic phase may
play an important role in processes such as membrane fusion.
A problem in lipid and surfactant investigations is solubility.
Surfactants that are soluble in water show no polymorphism
that would be interesting for biophysical investigations, whereas
compounds that show this polymorphism are often not soluble
Deviation from the previously mentioned vertical line may
also be seen upon the addition of solvents, the most common
of which is water, because of its biological relevance. Whereas
small amounts of water will not change the phase type and
transition temperatures, larger amounts will introduce new
phases, such as cubic and columnar phases (HI/II). Upon the
* Corresponding author. Phone: +49 40-42 838 4269. Fax: +49 40-42
838 4325. E-mail: vill@liqcryst.chemie.uni-hamburg.de.
† University of Hamburg.
‡ GKSS Research Centre.
§ Nanotechnology Research Institute, AIST.
| Gesellschaft fu¨r Biotechnologische Forschung mbH.
10.1021/jp0460238 CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/04/2005

1600 J. Phys. Chem. B, Vol. 109, No. 4, 2005
Milkereit et al.
Figure 2. Chemical structures of the investigated glycolipids 1-O-[(2′′R,4′′R,6′′R,8′′R)-2′′,4′′,6′′,8′′-tetramethyldecyl]-6-O-(R-D-galactopyranosyl)-
â-D-glucopyranoside (Mel-â-TMD) and 1-O-(3′′,7′′,11′′,15′′-tetramethylhexadecyl)-6-O-(R-D-galactopyranosyl)-â-D-glucopyranoside (Mel-â-Phy).
in water because of the length of the alkyl chain. With increasing
chain length, the melting temperature and the Krafft temperature
(or gel to liquid-crystalline phase transition; TK) rise. For
example, Tc of dodecyl-maltoside is below 25 °C and rises for
stearyl-maltoside to 40 °C.19
As alkyl chains, we chose the phytanyl chain with a chain length
of 16 methylene groups (4 additional methylene groups in the
branched part) and the (all-R) tetramethyldecyl chain with a
chain length of 10 methylene groups (4 additional methylene
groups in the branched part). The thermotropic properties were
studied using polarizing microscopy, the lyotropic phase
sequence was studied by the contact preparation method, and
differential scanning calorimetry was performed at defined water
concentrations. The critical micelle concentration (cmc) was
measured by the surface tension method, and additionally,
geometric properties were calculated from this. The micellar
aggregate structures in dilute solution were investigated using
small-angle neutron scattering (SANS).
To circumvent this problem, alkyl glycosides with unsaturated
chains were synthesized;19,20 the melting point and Tc could be
lowered by this to below 0 °C. The disadvantage of these
molecules is the lability of the double bond, which is unstable
in oxidative environments and often not stable during chemical
modifications. A different approach is the use of methyl-
branched alkyl chains, with a homogeneous alternating sequence
of branching. There are two main sources for this type of chain.
The isoprenoid-type alcohols can be obtained from plant sources,
as short-chain (gernaniol) and long-chain alcohols (phytanol).
Several lipids of these plant-based carbohydrates were synthe-
sized, and their lyotropic properties were thoroughly
investigated.21-26 A shorter-chained methyl-branched fatty
alcohol can be obtained from animal sources. The preen gland
of the domestic goose is a source for the chiral alcohol
(2R,4R,6R,8R)-tetramethyldecanol. Several glyco glycero lipids
and alkyl glycosides with this type of alcohol have been
synthesized.27-29 These compounds also showed liquid-crystal-
line phase behavior at ambient temperature.
For biological model systems, it is often necessary to also
test lipids with an uncommon carbohydrate headgroup. Whereas,
for technical applications, glucosides and maltosides are used,
uncommon or expensive carbohydrate headgroups are less
frequently used and investigated. Nevertheless, it is useful if
the results obtained for branched maltosides (TK is lowered)
can be applied to other disaccharide compounds. Additionally,
it would be interesting to see if these compounds would then
still show a phase behavior similar to that of the n-alkyl
analogues, which is often much more complex than that of
glucosides or maltosides.
2. Materials and Methods
2.1. Materials. R-D-Melibiose monohydrate was purchased
from Acros Organics. Solvents were distilled prior to use. 6-O-
(2′,3′,4′,6′-Tetra-O-acetyl-R-D-galactopyranosyl)-1,2,3,4-tetra-
O-acetyl-â-D-glucopyranoside (melibiose peracetate) was pre-
pared according to standard procedures. 3,7,11,15-Tetra-
methylhexadecan-1-ol was prepared by hydrogenation of
3,7,11,15-tetramethylhexadec-2-en-1-ol (Merck, Darmstadt) in
the presence of palladium on charcoal (10%; Fluka) as described
by Minamikawa et al.21,30 The chiral fatty alcohol (2R,4R,6R,8R)-
tetramethyldecan-1-ol was prepared using extracts from rump
glands of Anser a.f. domesticus.31,32
Thin-layer chromatography (TLC) was performed on silica
gel (Merck GF254), and detection was effected by spraying with
a solution of ethanol/sulfuric acid (9:1, v/v), followed by heating.
Column chromatography was performed using silica gel (Merck,
0.063-0.200 mm, 230-400 mesh). NMR spectra were recorded
on a Bruker AMX 400 or DRX5001 spectrometer (mc
)
centered multiplet, d ) doublet, t, ) triplet, dd ) double
doublet, and dt ) doublet triplet). J values are given in hertz.
2.2. Synthesis. 1-O-[(2′′R,4′′R,6′′R,8′′R)-2′′,4′′,6′′,8′′-Tet-
ramethyldecyl]-6-O-(2′,3′,4′,6′-tetra-O-acetyl-R-D-galactopyran-
osyl)-2,3,4-tri-O-acetyl-â-D-glucopyranoside (1). Totals of 3.39
g (5 mmol) of melibiose peracetate and 1.07 g (5 mmol) of
(2R,4R,6R,8R)-2,4,6,8-tetramethyldecan-1-ol were dissolved in
50 mL of dry dichloromethane under an atmosphere of dry
nitrogen. A total of 994 mg (880 µL; 7 mmol) of boron
trifluoride etherate was added, and the solution was stirred for
In this paper, we report about the synthesis of two different
alkyl glycosides with the same disaccharide headgroups and
different alkyl chains (Figure 2). As a carbohydrate headgroup,
we chose the R1f6-linked sugar melibiose, consisting of a
galactose moiety and a glucose moiety. These R1f6-linked
headgroups are of particular interest because they show a much
more complex polymorphism than the commonly used R1f4-
or â1f4-linked disaccharides (for example, maltose or lactose).

Comparison of the Structures of Two Glyco Lipids
J. Phys. Chem. B, Vol. 109, No. 4, 2005 1601
6 h at ambient temperature until TLC revealed the reaction to
be complete. The reaction was quenched with 50 mL of a
saturated solution of sodium hydrogen carbonate, the organic
layer was separated, and the aqueous layer was extracted twice
with dichloromethane. The combined organic phases were
washed twice with water, dried over magnesium sulfate, and
evaporated in vacuo. The residue was purified by column
chromatography [light petroleum (bp 50-70 °C)/ethyl acetate
for compound 1. Yield: 1.28 g (28%). C46H76O18 (944.3906).
1H NMR (400 MHz, CDCl3 + TMS): δ 5.42 (dd, 1H, H-4′),
5.32 (dd, 1H, H-3′), 5.18 (dd, 1H, H-3), 5.18 (dd, 1H, H-3),
5.13 (d, 1H, H-1′), 5.08 (dd, 1H, H-2′), 5.04 (dd, 1H, H-4),
4.90 (dd, 1H, H-2), 4.45 (d, 1H, H-1), 4.28 (ddd, 1H, H-5′),
4.08 (dd, 1H, H-6a′), 4.03 (dd, 1H, H-6b′), 3.80 (dt, 1H, H-Ra),
3.73 (dd, 1H, H-6a), 3.62 (ddd, 1H, H-5), 3.55 (dd, 1H, H-6b),
3.42 (dt, 1H, H-Rb), 2.10, 2.09, 2.01, 2.00, 1.97, 1.95 (each s,
3H, OAc), 1.45-1.64 (m, 4H, H-γ, H-η, H-λ, H-ï), 0.98-1.64
(m, 20H, CH2-â, CH2-δ, CH2-ꢀ, CH2-ú, CH2-υ, CH2-ι, CH2-ø,
CH2-µ, CH2-ν, CH2-ê), 0.87 (d, 3H, CH3-δ), 0.84 (d, 6H, CH3-
1
(2:1)]. Yield: 0.99 g (24%). C40H64O18 (860.2967). H NMR
(400 MHz, CDCl3 + TMS): δ 5.38 (dd, 1H, H-4′), 5.28 (dd,
1H, H-3′), 5.14 (t, 1H, H-3), 5.09 (d, 1H, H-1′), 5.04 (dd, 1H,
H-2′), 5.02 (t, 1H, H-4), 4.87 (dd, 1H, H-2), 4.40 (d, 1H, H-1),
4.17 (mc, 1H, H-5′), 3.99-4.08 (m, 3H, H-6a, H-6a′, H-6b′),
3.69 (dd, 1H, H-6b), 3.58 (ddd, 1H, H-5), 3.53 (mc, 1H, H-Ra),
3.23 (mc, 1H, H-Rb), 2.06, 1.99 (je 1, 3H, OAc), 1.98 (s, 6H,
OAc), 1.97, 1.94, 1.91 (each s, 3H, OAc), 1.78 (mc, 1H, H-â),
1.57 (mc, 1H, H-δ), 1.55 (mc, 1H, H-ú), 1.40 (mc, 1H, H-θ),
1.37 (mc, H-ιa), 1.32 (mc, 1H, H-γa), 1.22 (mc, H-ηa), 1.19 (mc,
1H, H-ꢀa), 1.03 (mc, 1H, H-ιb), 0.95 (d, 3H, CH3-â), 0.86-
0.92 (m, 2H, H-γb, H-ꢀb), 0.88 (d, 3H, CH3-δ), 0.86 (t, 3H,
alkyl-CH3), 0.81-0.85 (m, 1H, H-ηb), 0.84 (d, 3H, CH3-θ),
3
3
η, CH3-λ), 0.81 (d, 6H, CH3-ï, CH′3-ï); J1,2 ) 8.1, J2,3
)
)
3
3
3
3
3
9.7, J3,4 ) 9.7, J4,5 ) 9.7, J5,6a ) 5.1, J5,6b ) 2.5, J6a,b
11.2, 3J1′,2′ ) 3.6, 3J2′,3′ ) 10.7, 3J3′,4′ ) 3.1, 3J4′,5′ ) 1.0, 3J5′,6a′
3
3
3
3
) 5.6, J5′,6b′ ) 6.6, J6a′,b′ ) 11.2, JRa,â-CH ) 6.1, JRb,â-CH
2
2
3
3
) 6.6, JRa,b ) 9.7, JCH ,CH ) 6.6.
3
2
1-O-(3′′,7′′,11′′,15′′-Tetramethylhexadecyl)-6-O-(R-D-galac-
topyranosyl)-â-D-glucopyranoside (Mel-â-Phy). A total of 1.20
g (1.31 mmol) of 2 was deprotected as described for compound
Mel-â-TMD. Yield: 741 mg (91%). C32H62O11 (622.4292).
[R]20 +48 (c 1.0, MeOH). HIRESFAB-MS (m/z): 645.0382
D
[M + Na]+. 1H NMR (400 MHz, methanol-d4): δ 4.89 (d, 1H,
H-1′), 4.31 (d, 1H, H-1), 4.01 (dd, 1H, H-6a), 3.90-3.95 (m,
2H, H-4′, H-5′), 3.52-3.86 (m, 6H, H-2′, H-3′, H-6a′, H-6b,
H-6b′, H-Ra, H-Rb), 3.50 (ddd, 1H, H-5), 3.43 (dd, 1H, H-4),
3.38 (dd, 1H, H-3), 3.21 (dd, 1H, H-2), 1.65-1.75 (m, 4H, H-γ,
H-η, H-λ, H-ï), 1.01-1.64 (m, 20H, CH2-â, CH2-δ, CH2-ꢀ,
CH2-ú, CH2-υ, CH2-ι, CH2-ø, CH2-µ, CH2-ν, CH2-ê), 0.94 (d,
3H, CH3-δ), 0.84 (d, 6H, CH3-η, CH3-λ), 0.90 (d, 6H, CH3-ï,
0.82 (d, 3H, CH3-ú); 3J1,2 ) 8.1, 3J2,3 ) 9.7, 3J3,4 ) 9.7, 3J4,5
)
3
3
3
3
3
9.7, J5,6a ) 4.8, J5,6b ) 2.6, J6a,b ) 11.2, J1′,2′ ) 3.6, J2′,3′
3
3
3
3
) 10.7, J3′,4′ ) 3.5, J4′,5′ ) 1.3, JCH -â,â ) 6.6, JCH -δ,δ
)
3
3
6.6, 3JCH -ú,ú ) 6.6, 3JCH -θ,θ ) 6.6, 3Jalkyl-CH ,ι ) 6.6. 13C NMR
3
3
3
(400 MHz, CDCl3 + TMS): δ 170.52, 170.30, 170.18, 169.84,
169.32, 169.28 (CdO, OAc), 101.42 (C-1), 96.75 (C-1′), 75.08
(C-R), 72.94 (C-3), 72.61 (C-5), 71.32 (C-2), 69.10 (C-4), 68.05,
68.03 (C-4′, C-2′), 67.42 (C-3′), 66.47 (C-5′), 66.39 (C-6), 61.60
(C-6′), 45.36 (C-ꢀ), 44.58 (C-η), 41.03 (C-γ), 31.52 (C-θ), 30.35
(C-â), 28.81 (C-ι), 27.43 (C-ú), 27.31 (C-δ), 21.11 (CH3-δ),
20.98 (CH3-ú), 20.77, 20.70, 20.67, 20.64 (-CH3, OAc), 20.08
(CH3-θ), 17.78 (CH3-â), 11.24 (alkyl-CH3).
3
3
3
3
3
CH′3-ï); J1,2 ) 8.1, J2,3 ) 9.2, J3,4 ) 9.2, J4,5 ) 9.2, J5,6a
3
2
3
) 4.1, J5,6b ) 2.0, J6a,b ) 11.2, J1′,2′ ) 4.1.
2.3. Surface Tension. Surface tension was measured on a
Kru¨ss K6 tensiometer (Kru¨ss), using the de Nou¨y ring method.
Measurements were carried out using bidistilled water with a
surface tension of σ ) 72-73 mN/m. All values were corrected
for the temperature. Corrections for the ring geometry and the
hydrostatic lifted volume of the liquid were made using the
method described by Harkins and Jordan33 or by Zuidema and
Waters.34
2.4. SANS. SANS experiments were performed with the
SANS-1 instrument at the FRG1 research reactor at the GKSS
Research Centre.35 Four sample-to-detector distances (from 0.7
to 9.7 m) were employed to cover the range of scattering vectors
q from 0.005 to 0.25 Å-1. The neutron wavelength λ was 8.1
Å with a wavelength resolution of 10% (full-width at full-
maximum).
The solutions were prepared in D2O (Deutero GmbH; purity
99.98%). The samples were kept in quartz cells (Hellma) that
had a path length of 5 mm. The samples were placed in a
thermostated holder for isothermal conditions T ) 25.0 ( 0.5
and 50.0 ( 0.5 °C. The raw spectra were corrected for the
background from the solvent, sample cell, and other sources
by conventional procedures.36 The two-dimensional isotropic
scattering spectra were azimuthally averaged, converted to an
absolute scale, and corrected for detector efficiency by dividing
them by the incoherent scattering spectra of pure water,36 which
was measured with a 1 mm pathlength quartz cell (Hellma).
The average excess scattering-length density per unit mass
(∆Fm) of the surfactant in deuterated water was determined from
the known chemical composition, and it was equal to -4.92 ×
1010 cm/g for compound Mel-â-Phy and -4.51 × 1010 cm/g
for compound Mel-â-TMD.
1-O-[(2′′R,4′′R,6′′R,8′′R)-2′′,4′′,6′′,8′′-Tetramethyldecyl]-6-O-
(R-D-galactopyranosyl)-â-D-glucopyranoside (Mel-â-TMD). A
total of 0.97 g (1.16 mmol) of 1 was dissolved in 50 mL of
anhydrous methanol, and sodium methoxide was added (pH
8-9). The solution was stirred at ambient temperature until TLC
revealed the reaction to be complete. It was neutralized using
Dowex 50WX ion-exchange resin (protonated form), filtered,
and evaporated in vacuo. The product was recrystallized from
propan-2-ol. Yield: 588 mg (94%). C26H50O11 (538.3353).
[R]20 +52 (c 0.8, MeOH). HIRESFAB-MS (m/z): 561.6758
D
[M + Na]+. 1H NMR (500 MHz, methanol-d4): δ 4.89 (d, 1H,
H-1′), 4.31 (d, 1H, H-1), 4.01 (dd, 1H, H-6a), 3.90-3.95 (m,
2H, H-4′, H-5′), 3.88 (dt, 1H, H-Ra), 3.69-3.80 (m, 4H, H-2′,
H-3′, H-6a′, H-6b, H-6b′), 3.68 (dd, 1H, H-Ra), 3.58 (dt, 1H,
H-Rb), 3.50 (ddd, 1H, H-5), 3.48 (dd, 1H, H-Rb), 3.43 (dd,
1H, H-4), 3.38 (dd, 1H, H-3), 3.21 (dd, 1H, H-2), 1.93 (mc,
1H, H-â), 1.61-1.65 (m, 2H, H-δ, H-ú), 1.49 (mc, 1H, H-θ),
1.44 (mc, H-ιa), 1.42 (mc, 1H, H-γa), 1.32 (mc, H-ηa), 1.27 (mc,
1H, H-ꢀa), 1.10 (mc, 1H, H-ιb), 0.99 (d, 3H, CH3-â), 0.96 (mc,
1H, H-γb), 0.95 (mc, 1H, H-ꢀb), 0.94 (d, 3H, CH3-δ), 0.93 (t,
3H, alkyl-CH3), 0.92 (d, 3H, CH3-θ), 0.90 (d, 3H, CH3-ú), 0.88
(mc, 1H, H-ηb); 3J1,2 ) 8.1, 3J2,3 ) 9.2, 3J3,4 ) 9.2, 3J4,5 ) 9.2,
3
2
3
3
3J5,6a ) 4.1, J5,6b ) 2.0, J6a,b ) 11.2, J1′,2′ ) 4.1, JH-Ra,â
)
)
7.6, 3JH-Rb,â ) 5.1, 3JH-Ra,b ) 9.2, 3JCH -â,â ) 6.6, 3JCH -δ,δ
6.6, JCH -ú,ú ) 6.6, JCH -θ,θ ) 6.1, Jalkyl-CH ,ι ) 6.6.
3
3
3
3
3
3
3
3
1-O-(3′′,7′′,11′′,15′′-Tetramethylhexadecyl)-6-O-(2′,3′,4′,6′-tetra-
O-acetyl-R-D-galactopyranosyl)-2,3,4-tri-O-acetyl-â-D-glucopy-
ranoside (2). Totals of 3.39 g (5 mmol) of melibiose peracetate,
1.49 g (5 mmol) of 3,7,11,15-tetramethylhexadecan-1-ol, and
994 mg (880 µL; 7 mmol) of boron trifluoride etherate in 50
mL of anhydrous dichloromethane were reacted as described
2.5. Polarizing Microscopy and Contact Preparation. An
Olympus BH optical polarizing microscope equipped with a

1602 J. Phys. Chem. B, Vol. 109, No. 4, 2005
Milkereit et al.
Figure 3. Synthetic procedure for the preparation of Mel-â-TMD and Mel-â-Phy.
Mettler FP 82 hot stage and a Mettler FP 80 central processor
was used to identify thermal transitions and characterize
anisotropic textures.
in different ways. Phythanol (3,7,11,15-tetramethylhexadecan-
1-ol), for the preparation of Mel-â-Phy, was prepared by a
simple catalytic hydrogenation of phythol (3,7,11,15-tetra-
methylhexadec-2-en-1-ol), as described by Minamikawa et
al.21,30
The chiral methyl-branched fatty alcohol (2R,4R,6R,8R)-
2,4,6,8-tetramethyldecanol was prepared from natural sources
as described by Morr et al.31,32 Using extracts from the preen
glands of the domestic goose (Anser a.f. domesticus), the alcohol
is prepared in a multistep synthesis by transesterification,
Spaltrohr distillation, and reduction of the fatty acid methyl ester.
The alcohol was obtained with a diastereomeric excess of more
than 99%.
For the contact preparation, a small amount of sample was
placed on a microscope slide and covered with a cover glass
before heating it to the liquid-crystalline phase (SA or Col).
Afterward, a small amount of solvent was placed on the slide
at the edge of the cover glass. As soon as the solvent had moved
under the cover glass and the sample was completely surrounded
by the solvent, it was again placed, for a few seconds, on the
hot stage at a temperature of 100-120 °C, and the phase
behavior was investigated immediately afterward by polarizing
microscopy.
The synthesis of compounds Mel-â-TMD and Mel-â-Phy was
carried out using standard procedures.6 Protection of the sugar
in acetic acid anhydride/sulfuric acid afforded the â-peracetate
in a good yield. The glycosylation reaction using boron
trifluoride etherate as a Lewis acid gave the desired â-glycosides.
Deprotection using the standard Zemple´n procedure finally led
to the desired products.
3.2. Thermotropic Liquid-Crystalline Properties. The
thermotropic properties were investigated using polarizing
microscopy. The following liquid-crystalline phases and transi-
tion temperatures were found (Cr ) crystalline; g ) glass; I )
isotropic):
2.6. Differential Scanning Calorimetry (DSC). The thermal
phase behavior of the investigated compounds was investigated
using a Seiko SSC/560U differential scanning calorimeter. The
lipid/water mixtures were sealed in silver DSC capsules. To
fully develop crystals of Mel-â-Phy, the samples (49 wt % lipid)
were incubated at -20 °C for 3 h before a heating run was
initiated at a rate of 0.2 °C/min. For Mel-â-TMD, the samples
(54 wt % lipid) were incubated at -40 °C for 3 h before
initiating a heating run at a rate of 0.4 °C/min. A cooling run
was not performed because of a great degree of supercooling
of the samples.
3. Results and Discussion
Mel-â-TMD
Mel-â-Phy
Cr
g
<20.0
SA
SA
156.9
178.6
I
I
?
3.1. Compounds and Synthesis. Figure 3 shows the synthetic
route for the preparation of the investigated compounds.
Although the preparations of both glycosides follow the same
route, the alcohols needed for the preparations were prepared
Compound Mel-â-TMD, with the short alkyl chain, is already
a liquid crystal at ambient temperature. The compound forms a

Comparison of the Structures of Two Glyco Lipids
J. Phys. Chem. B, Vol. 109, No. 4, 2005 1603
Figure 4. Water-penetration scan for Mel-â-Phy at 23 °C (×40). There
was no sign of water penetration, and crystals remained, even after 24
h of contact with water. When the temperature exceeded 40 °C, the
crystals melted and quickly turned into a transparent isotropic solution
(see text).
Figure 5. Heating curve of a 49 wt % Mel-â-Phy/water system (heating
rate: 0.2 °C/min).
ally observed in water-penetration experiments. Upon cooling
of the transparent aqueous solution, the solid again started to
appear below 20 °C. This observation is consistent with the
DSC measurements. Figure 5 shows a typical example of the
DSC thermogram of the Mel-â-Phy/water system (49 wt %),
in which an endothermic peak (∆H ) 38 kJ/mol) associated
with the glass T LR phase transition of Mel-â-Phy was observed
between 33 and 43 °C. The endothermic peak that starts at about
-15 °C and ends at about 1 °C belongs to the melting of frozen
water in the Mel-â-Phy/water system. Compared to normal ice
melting, the water started to melt at significantly lower
temperatures. This suggests that the melting of ice in the Mel-
â-Phy/water system, occurring throughout a wide temperature
range, is a result of different water organization.
Mel-â-TMD/Water System. Dry Mel-â-TMD exhibited bire-
fringent textures at 23 °C. Upon addition of water, an isotropic
region started to grow from the Mel-â-TMD/water interface,
with a concomitant decrease in the birefringent Mel-â-TMD
region (see Figure 6). The birefringent Mel-â-TMD region
eventually disappeared, resulting in a two-phase-coexistence
system of the isotropic phase (I) and the aqueous system (W).
As seen in Figure 6c, the isotropic (most probably Mel-â-TMD-
rich) phase dispersed as spherical liquid droplets in an aqueous
solution phase. Thus, the isotropic phase observed in the water-
penetration scan (Figure 6a,b) was not a cubic phase. The turbid
two-phase state remained unaltered at least up to 70 °C. When
the two-phase solution was incubated at 4 °C, the liquid droplets
transformed into birefringent solid particles.
The observation is consistent with the DSC thermogram of
the Mel-â-TMD/water system (54 wt %). The thermogram
consists of two endothermic peaks. The lower endothermic peak
is most presumably the one associated with the melting of frozen
water in the Mel-â-TMD/water system. The second peak,
overlapping with the first endothermic peak, is associated with
the phase transition of Mel-â-TMD. The endotherm completed
at about 18 °C (indicated by an arrow in Figure 7).
To summarize the results, the Mel-â-Phy/water system
displayed a TK of about 33 °C, above which the system formed
an isotropic transparent solution with moderate foaming ability.
This indicates that Mel-â-Phy is soluble in water by forming
normal micelles at temperatures above its TK. Compound Mel-
â-TMD, on the other hand, was not soluble in water, even at
temperatures above its TK. The Mel-â-TMD/water system forms
a two-phase solution that consists of a lipid-rich phase and an
aqueous solution (most probably built up by isotropic disklike
aggregates/bilayer aggregates; see section 3.5). As shown in
section 3.5, the concentration of Mel-â-TMD in the aqueous
solution is about 10-3 g/mL (∼18 times the cmc). Thus, Mel-
â-Phy, with its phytanyl chain (a total of 20 carbon atoms), is
SA phase over the whole temperature range. Compound Mel-
â-Phy also exclusively forms a LR phase. The clearing temper-
ature is 21.7 K higher compared to that of Mel-â-TMD. Both
compounds cannot be obtained in a crystalline form, like most
of the known branched-chain glycolipids. The glass temperature
(Tg) of Mel-â-TMD is below room temperature, whereas Mel-
â-Phy has a Tg above room temperature. It forms a rigid glass
structure with cracks resulting from shrinking during cooling
of the glassy state.
Unfortunately, no reliable data on the thermotropic and
lyotropic phase sequence and transition temperatures for the
corresponding n-decyl-, n-hexadecyl-, or n-eicosanyl-melibio-
sides can be found. Despite this, the data for the n-dodecyl-
and n-octadecyl-melibiosides19 provide sufficient information
for a comparison. n-Dodecyl-melibioside forms a cubic phase
above the melting point (155 °C) upon heating, whereas upon
cooling from the isotropic phase, a SA phase is formed.
However, n-octadecyl-melibioside has a melting point of 152
°C, and above that, a SA phase is formed throughout the whole
temperature range up to the clearing temperature (262 °C).
The melting point is lowered in both cases. In the case of
the tetramethyldecyl chain, the effect is even much stronger than
that for the tetramethylhexadecyl chain. This means that the
distance between the methyl groups in the branching has to be
taken into account. With decreasing distances between the
methyl groups, the disturbance of the packing density in the
chains rises. Another effect is the change in the molecular shape.
The molecular geometry of n-dodecyl-melibioside lies between
a rodlike and a wedge-shaped structure, which can be seen from
the phases formed. The tetramethyldecyl chain then leads to a
stable rodlike structure; thus, a stable thermotropic SA phase is
formed. Mel-â-Phy also forms a SA phase, but compared to
n-octadecyl-melibioside, the temperature range of this phase is
small, indicating that the shape is still rodlike. However, upon
heating, a change of shape is favored.
3.3. Thermal and Lyotropic Phase Behavior of the Lipid/
Water Systems. Mel-â-Phy/Water System. Water-penetration
(contact-penetration) experiments of Mel-â-Phy displayed rather
unusual phase behavior. Dry Mel-â-Phy was in the glass state
at ambient temperature (23 °C) and did not show any sign of
water penetration, even after 2 days of contact with water (see
Figure 4). With increasing temperature (above 35 °C), the glass
disappeared rapidly, forming a clear transparent solution. This
may be attributable to the dissolving of Mel-â-Phy in water.
Solutions of Mel-â-Phy then showed a moderate foaming ability.
Because of the rapid change upon heating, it was not possible
to observe any stable water-penetration textures that are gener-

1604 J. Phys. Chem. B, Vol. 109, No. 4, 2005
Milkereit et al.
stretching vibration band in Fourier transform infrared spec-
troscopy, which is found upon the investigation of glycosyl
diacyl glycerols with tetramethyldecyl chains,29 that is a sensitive
marker of the alkyl and acyl chain order.38 This can result in a
rigid structure of compound Mel-â-TMD, even above TK. The
hydration and self-assembly that occurs upon the addition of
water will be energetically more unfavorable than it will for
the more flexible Mel-â-Phy.
Second, the good aqueous solubility of Mel-â-Phy appears
to be in marked contrast to the phase behavior of another
phytanyl-chained glycolipid, 1-O-phythanyl-â-D-maltoside,
â-Mal2(Phyt). Having a common phytanyl chain, their molecular
structures differ only in their headgroup part, for example,
melibiose for Mel-â-Phy and maltose for â-Mal2(Phyt). It is
interesting to note that the â-Mal2(Phyt)/water system forms a
water (dilute aqueous lipid solution) and a LR two-phase region
followed by a one-phase region of the LR phase as the lipid
concentration increases.39 This again adds an intriguing example
in which the saccharide headgroup has a significant effect on
the physical behavior of glycolipid/water systems.40
Lowering TK by using branched alkyl chains instead of n-alkyl
chains is a common method employed in surfactant chemistry.41
Unfortunately, no literature data are available on the TK values
of n-tetradecyl- or n-eicosanyl-melibiosides. Only data for
n-dodecyl-melibioside (TK < 0 °C) and n-octadecyl-melibioside
(TK ) 67.5 °C) can be found.19 From these data, the values for
n-tetradecyl-glycoside (20-30 °C) and n-eicosanyl-glycoside
(80-100 °C) can be estimated. When these values are compared
with the DSC results, the difference between the branched chains
is again visible. Whereas TK of Mel-â-Phy (33 °C) is signifi-
cantly lower than even that for octadecyl-melibioside, TK of
Mel-â-TMD (18 °C) seems to be in the same range as those
for the corresponding n-alkyl analogues.
3.4. Surface Tension and Geometric Properties. Table 1
shows the geometrical properties of the investigated compounds.
The carbon-chain volume and the extended chain length were
calculated according to the method described by Tanford.42 For
the calculations, we assumed that the volume of the carbon
chains totaled 14 and 20 carbon atoms for Mel-â-TMD and Mel-
â-Phy, respectively. For the chain length, we assumed that the
branching of the chain does not affect the chain length; therefore,
the chain length will be, in the case of Mel-â-TMD, 10 carbon
atoms and, in the case of Mel-â-Phy, 16 carbon atoms.
In Figure 8, the adsorption isotherms of the investigated
compounds at the air solution interface are shown. The
interfacial absorption behavior was investigated at 40 °C. Both
compounds formed homogeneous solutions in the measured
concentration range; no phase separation occurred, and no
multiphase systems formed. No phase separation occurred above
the cmc for compound Mel-â-Phy. For Mel-â-TMD at a
concentration of 1 × 10-3 g/mL, the solution became cloudy,
indicating the formation of bigger aggregates/phases or a
solubility limit.
Figure 6. Water-penetration scan of Mel-â-TMD at 23 °C (×40). W:
water region. I: isotropic region. L: lipid region. Microscopic pictures
at (a) 2 min of water penetration and (b) 5 min of water penetration.
(c) Microscopic picture of a 3 wt % Mel-â-TMD/water system at 23
°C (×40). Spherical droplets of the water-swollen lipid phase (disklike/
bilayer aggregates) are dispersed in the aqueous solution phase.
Figure 7. Heating curve of a 54 wt % Mel-â-TMD/water system
(heating rate: 0.4 °C/min).
more soluble in water than Mel-â-TMD, with its smaller
hydrophobic chain (a total of 14 carbon atoms in the hydro-
phobic chain). A definite explanation will need a more detailed
phase diagram, but a reason for this unexpected behavior might
be found in the difference between the branched alkyl chains.
In comparison to that of n-alkyl chains, the flexibility of the
alkyl chain should be reduced, in the cases of both the phytanyl
and tetramethyldecyl chains.37 Nevertheless, the phytanyl chain
should show some flexibility with its alternating -(CH2)3-
CHCH3- sequence. However, the tetramethyldecyl chain, with
the alternating -CH2-CHCH3- sequence, should show less
flexibility. This is also indicated by the absence of the symmetric
The data obtained from the surface tension measurements are
presented in Table 1.
The cmc value of Mel-â-TMD is higher than that of
compound Mel-â-Phy, which has a longer alkyl chain. The
decrease of the cmc from Mel-â-TMD to Mel-â-Phy is in
accordance with the theory predicting a decrease in cmc values
with an increase in the alkyl chain length.43
From the pre-cmc slope, the headgroup area per molecule at
the air-water interface was calculated. From this, the critical
packing parameters (CPPs) were calculated.41 As shown in Table
1, CPP predicts the formation of a bilayer structure for the

Comparison of the Structures of Two Glyco Lipids
J. Phys. Chem. B, Vol. 109, No. 4, 2005 1605
TABLE 1: Adsorption of Mel-â-TMD and Mel-â-Phy at the Air-Aqueous-Solution Interface and Data Calculated from the
Pre-cmc Slope, T ) 40 °C
carbon chain extended chain
critical packing
γcmc
γmin
volumea
lengthb
headgroup areac
parameterd
cmc [mM L-1
]
cmc [g/mL]
[mN/m] [mN/m]
υc [Å3]
lc [Å]
a0 [Å2]
υc/lca0
Mel-â-TMD 0.1 ( 2 × 10-2
5.4 × 10-5
35.6 35.2
∼36 ∼36
404
565.4
14.15
21.74
23.1 ( 4
37 ( 15
∼1
Mel-â-Phy
0.03 ( 1 × 10-2 1.87 × 10-5
0.7
a The values were calculated using υc ) 27.4 + 26.9nc. b Calculated with lc ) 1.5 + 1.265nc.42 c Calculated from the pre-cmc slope. d Calculated
from the carbon chain volume, the extended chain length, and the headgroup area per molecule at the air-water interface obtained from the
pre-cmc slope.
Figure 9. Scattering curves and model fits for solutions of Mel-â-
TMD (c ) 1.0 × 10-3 g/mL; empty symbols) and Mel-â-Phy (c ) 5.0
Figure 8. Surface tension plots for Mel-â-TMD and Mel-â-Phy at 40
× 10-4 g/mL; filled symbols) in D2O, T ) 50 °C. The curves are
°C.
normalized to the concentration of surfactant in aggregates (c - cmc).
The solid lines represent fits or approximations. The arrow points to
the position of the maximum in the scattering data of Mel-â-TMD.
shorter-chained lipid Mel-â-TMD. For compound Mel-â-Phy,
the prediction of the micellar shape is more difficult. The error
in the determination of a0 from the pre-cmc slope is very high
because it is difficult to estimate the correct part in the slope of
the curve (the value of CPP predicts a cylindrical structure of
the micelles).
3.5. Analysis of SANS Data. SANS data were collected for
solutions of Mel-â-TMD (c ) 1.0 × 10-3 g/mL) and Mel-â-
Phy (c ) 5.0 × 10-4 g/mL) in D2O at a temperature of 50 °C
for Mel-â-Phy and 20, 50, and 70 °C for Mel-â-TMD. These
concentrations are approximately 20-25 times the cmc value
for both compounds. A detailed description of the analysis of
the SANS data is given in the appendix.
diameter, or thickness, respectively. The IFT routine for rodlike
aggregates was applied to the large q part (q > 0.02 Å-1). The
large q part is chosen to ensure the validity of the approximation
for rodlike aggregates (infinite long cylinder) q > 1/L, where L
is the length of the cylinder.
We obtained the following values for the parameters: RCS,g
) 13.4 ( 0.2 Å (the corresponding radius of the homogeneous
circular cross section of Mel-â-Phy aggregates is 19.0 ( 0.4
Å) and ML ) (1.4 ( 0.1) × 10-13 g/cm (this gives ∼13 Mel-
â-Phy molecules per 10 Å of aggregate length and ∼90 Å2 per
one Mel-â-Phy molecule at the surface of the cylinder). The
radius of the cylindrical cross section, calculated from the radius
of gyration, points to an extended conformation of the alkyl
chain in the rodlike aggregates (Table 1).
The analysis was continued with the model fitting. A
cylindrical model was chosen and applied to the experimental
data. In Figure 9, the experimental data and the applied model
are shown. The applied model and the experimental scattering
curve fit together throughout the whole interval of the scattering
vector q. Using the data, we calculated the radius (R ) 19.2 (
0.4 Å) and the length of the cylinder (L ) 290 ( 10 Å). The
radii of the cylinder obtained by the two different methods (IFT
analysis and model fitting) are in accordance. Both of the
methods gave consistent results, which can be interpreted as
the formation of short cylindrical aggregates in dilute solution.
The results are summarized in Table 2.
The obtained scattering curves are very different for the two
glycosides (shown in Figure 9). Compound Mel-â-TMD shows
a high scattering at the lowest interval of the scattering vector
(q < 0.01 Å-1). At an intermediate q range (0.01-0.1 Å-1),
the scattering curve changes and shows a disklike behavior
according to dΣ(q)/dΩ ∼ q-2. For large values of q, a diffraction
maximum (q ≈ 0.15 Å-1) is observed.
In contrast, the scattering curve of the lipid Mel-â-Phy shows
a rodlike behavior at an intermediate range of the scattering
vector (dΣ(q)/dΩ ∼ q-1).
It can be assumed that the two lipids form different aggregate
structures in an aqueous solution: (1) Mel-â-TMD forms large
aggregates with partial-plane or disk-shaped substructures. (2)
Mel-â-Phy forms small rodlike aggregates.
Mel-â-Phy Solutions. We started the analysis of the scattering
data for compound Mel-â-Phy by applying the indirect Fourier
transformation (IFT) method developed by Glatter,44 using the
version reported by Pedersen.45 This model-independent ap-
proach needs only minor additional (model) information on the
possible aggregate structure: dimensions (spherelike, rodlike,
or disklike) and a maximal value of the diameter, cross-sectional
The observations of the water-penetration scans of Mel-â-
Phy are consistent with the results from SANS. Above TK, Mel-
â-Phy forms cylindrical micelles at a concentration that is 27
times above the cmc. The formation of micelles is similar to
that of the n-alkyl counterparts. For example, for cis-9-

1606 J. Phys. Chem. B, Vol. 109, No. 4, 2005
Milkereit et al.
TABLE 2: Results of SANS Data Analysis by a
Model-Independent Approach (IFT), Model Fitting, and
Calculated Parameter of Aggregates for Mel-â-Phy (c ) 5.0
× 10-4 g/mL, T ) 50 °C)
excluded. For example, n-tetradecyl-melibioside forms spherical
micelles.38
The CPP values calculated from the surface tension data
(section 3.4) are in accordance with the results from the SANS
analysis. Mel-â-Phy forms cylindrical micelles (CPP ∼ 0.7),
whereas Mel-â-TMD forms bilayer aggregates (CPP ∼ 1).
micellar shape
cylindrical
19.0 ( 0.4
290 ( 10
(1.4 ( 0.1) × 10-13
∼13
radius of cylindrical cross section [Å]
length [Å]
mass per unit length [g/cm]
number of molecules per 1 nm of length
surface area per molecule [Å2]
4. Conclusions
90
Two new glycolipids with methyl-branched alkyl chains of
different lengths and chiralities were synthesized. The combina-
tion of different analytical standard methods (polarizing mi-
croscopy, contact preparation, DSC, surface tension measure-
ments, and SANS) gave a comprehensive characterization of
the biophysical properties in the pure state and in an aqueous
solution. The effect of the branching is obvious. The compounds
show liquid-crystalline phase behavior near or at ambient
temperature, although the phase behavior is changed. n-Alkyl-
melibiosides exhibit a more complex polymorphism leading to
the formation of lamellar, cubic, and columnar mesophases,
because of the wedge-shaped geometry of the molecules, than
was found for the investigated compounds. The use of methyl-
branched alkyl chains instead of n-alkyl chains led to a general
change of the physical properties. It is interesting to note that
the solubility of Mel-â-TMD is low even above TK. This is
similar to the observations made for the tetramethyldecyl wax
esters of the preen glands, which are responsible for the good
water protection of the feathers of the domestic goose.46 The
method of methylation of the chain controls the self-assembly
and can explain different biological functions for plants (variable
temperature) and animals (constant temperature). The different
influences of the branched chains can be used for the applica-
tions. Mel-â-Phy shows normal surfactant behavior starting with
micelle formation and can be used as a sugar surfactant,
exhibiting a galactose residue at the end of the disaccharide
head. Conversely, Mel-â-TMD will form stable bilayers and
can be used for building model membranes with an unusual
carbohydrate head or more simple stabilized membranes.
The main observations that were made can be summarized
as follows.
(1) Similar properties of Mel-â-Phy and Mel-â-TMD:
(a) The liquid-crystalline polymorphism is reduced to only
form SA phases in the pure state; the formation of lyotropic
phases, such as hexagonal or lamellar phases, was not observed.
(b) No cubic or columnar phases were formed, because of
the branched alkyl chains, therefore changing the molecular
shape from wedge-shaped to a rodlike shape.
(c) The stability of the liquid-crystalline phase is increased.
(d) The melting points and TK’s are lowered.
(2) Differences between Mel-â-Phy and Mel-â-TMD:
(a) Mel-â-Phy forms cylindrical micelles.
(b) The less flexible TMD chain disables the formation of a
large micellar solution region; compound Mel-â-TMD forms
disklike aggregates/bilayers of small or medium size instead
because no birefringent textures were observed for this phase
region (Figure 6).
(c) Mel-â-TMD is not very soluble in water, even above TK.
(d) Mel-â-Phy has a greater hydrophobic moiety than Mel-
â-TMD, but it is more soluble in water, as a result of the more
flexible phytanyl chain.
TABLE 3: Results of SANS Data Analysis by
Model-Independent Approach (IFT), Model Fitting, and
Calculated Parameter of Aggregates for Mel-â-TMD (c )
1.0 × 10-3 g/mL, T ) 25, 50, and 70 °C)
micellar shape
bilayer/large aggregates
∼4000 Å (large aggregates)
39 ( 2 (bilayer)
radius [Å]
thickness of layer for [Å]
repeat distance between layers [Å]
mass per surface unit [g/cm2]
surface area per molecule [Å2]
40-50 (bilayer)
(8.3 ( 0.5) × 10-10 (bilayer)
100 (bilayer)
octadecenyl-melibioside, the formation of long cylindrical
micelles was observed.38
For compound Mel-â-Phy, the area per headgroup in the
micelles obtained from the SANS data is about 2-3 times higher
than that at the liquid-air interface (Table 2). This shows that,
in the micelle, a wedge-shaped packing of the molecules with
a dense hydration of the carbohydrate headgroup can be
assumed, whereas at the liquid-air interface, a nearly rodlike
packing of the monolayers occurs.
Mel-â-TMD Solution. The scattering data of Mel-â-TMD
show the so-called Porod behavior at the lowest interval of q
(q < 0.01 Å-1). It points to a smooth and sharp interface
between large aggregates and the surrounding solvent. The
following parameters of smaller disklike/bilayer aggregates were
obtained from the analysis of the intermediate part of the
scattering curve: a radius gyration of thickness of RT,g ) 11.4
( 0.4 Å (the corresponding thickness of bilayers of Mel-â-
TMD is 39 ( 2 Å) and a mass per surface unit of MS ) (8.3 (
0.5) × 10-10 g/cm2 (100 Å2 per one Mel-â-TMD molecule in
the surface of the bilayer).
We can summarize the results of the structural analysis as
follows: Large aggregates (R ∼ 4000 Å) are formed. The
surface is smooth/sharp. Substructural units are disklike particles
with a thickness of approximately 40 Å, and the formation of
ordered bilayers with a repeat distance between the layers of d
∼ 40-50 Å is observed. It is probable that we observed a phase
transition at this concentration. The results are summarized in
Table 3.
For compound Mel-â-TMD, the headgroup area in the bilayer
obtained from SANS is about 4 times higher than that at the
liquid-air interface (Table 3). This is due to the formation of
different aggregates in solution that makes the calculation of
the exact concentration in the single aggregates impossible. From
the theoretical point of view, it is expected that the headgroup
area in the bilayer should be in a range similar to that at the
air-water interface.41
Contrary to the results obtained for solutions of Mel-â-Phy,
compound Mel-â-TMD formed no micelles at a concentration
of 1 × 10-3 g/mL (18 times above the cmc). In this case, the
formation of large aggregates/lipid bilayers was observed. This
can again be an indication that the alkyl chain of Mel-â-TMD
is very rigid, which makes the formation of micelles, even at
low concentrations, unfavorable. Nevertheless, concentrations
below this were not investigated; thus, the formation of micelles
in a very narrow concentration range near the cmc cannot be
Acknowledgment. Financial support by the Deutsche For-
schungsgemeinschaft (Graduiertenkolleg 464, SFB 470) is
gratefully acknowledged.

Comparison of the Structures of Two Glyco Lipids
J. Phys. Chem. B, Vol. 109, No. 4, 2005 1607
Appendix: Analysis of SANS Data
Pcyl(q) is the form factor of a cylinder of length L and radius
R:48
Mel-â-Phy Solutions. The scattering intensities for rodlike
aggregates are written via the cross-sectional pair distance
distribution function pCS(r):
2
sin[qL/(2 cos â)]2J1[qR sin â]
[qL/(2 cos â)][qR sin â]
π/2
P (q) ) 4
sin â dâ
∫
cyl
{
}
0
(6)
dΣ(q)/dΩ ) 2π ∞p (r) J (qr) dr
(1)
π
q
∫
CS
0
( )
0
where J1 is the first-order Bessel function.
P
rod(q,L) is the form factor of an infinitely thin rod of length
where J0 is the zeroth-order Bessel function.
The pCS(r) function is given by44
L:
P
rod(q) ) 2Si(qL)/(qL) - 4 sin2(qL/2)/(q2L2)
(7)
c - cmc
2πML
pCS(r) )
r∆F(r′) ∆F(r + r′) dr′
(2)
∫
where
where ∆F(r) is the contrast [difference between the scattering-
length density of aggregates at the point r, F(r), and the averaged
scattering-length density of the solvent, Fs; ∆F(r) ) F(r) - Fs];
the vectors r and r′ are lying in the cross-sectional plane.
The pair distance distribution function is expressed as a sum
of N b splines evenly distributed in the interval [0, Dmax]. The
values of the coefficients are calculated numerically by a least-
squares fitting of the IFT model curve to the experimental data.
In the present study, the values of Dmax were carefully chosen
x
Si(x) ) t-1 sin t dt
∫
0
The function c(q) is related to the Fourier transform of the
“direct” correlation function, which we approximated by the
Fourier transform of the correlation hole around each site:45,47
3[sin(qD′) - qD′ cos(qD′)]
c(q) )
(8)
(qD′)3
to give both good fits to the experimental data and smooth pCS
(r) functions.
-
where the diameter of the correlation hole is chosen as D′ )
2R.
At the large q range (q > 0.02 Å-1), the experimental data
and the fitted curves coincide very well (data not shown).
The Gaussian shape of the pair distribution function is
characteristic of almost-homogeneous cylindrical aggregates
formed by compound Mel-â-Phy. Here, we got a first estimation
of the cross-sectional diameter of approximately 40 Å, which
is obtained from the maximum distance of pCS(r).
In the final fit, four parameters were fitted: the length of the
micelle, the cross-sectional radius, a correction factor for the
absolute scale, and a residual background. The correction factor
for the absolute scale takes small errors of the concentration
into account, and it is expected to be close to unity. The
correction factor for the absolute scale variation is within 5%
of unity, which reflects the accuracy of the absolute calibration
of SANS data and of the surfactant concentration. The experi-
mental data and the applied model are shown in Figure 9.
Mel-â-TMD Solution. The scattering data of Mel-â-TMD
show the so-called Porod behavior at the lowest interval of q
(q < 0.01 Å-1):48
After determination of the pair distance distribution function,
the mass per unit length of the aggregate ML and the radius of
gyration of the cross section of the micelle RCS,g will be
calculated. The radius of gyration can be written as
1/2
∞r2p (r) dr
∫
CS
0
RCS,g
)
(3)
dΣ(q)/dΩ ∼ Aq-4
(9)
∞
[
]
2
p (r) dr
∫
CS
0
This points to a smooth and sharp interface between aggregates
formed by Mel-â-TMD and the surrounding solvent. The
parameter A is connected with the interfacial area S by
The analysis was continued with the model fitting. A cylindrical
model was chosen and applied to the experimental data. The
model scattering cross section used for the fitting is based on
the integral equation theory for polymers and the polymer
reference interaction site model. For the isotropic solution of
rigid cylinders, the scattering cross section was taken as47
A ) 2π∆F2S
(10)
First, the parameter A was obtained from a fitting procedure at
the lowest q region. With knowledge of the volume fraction of
the surfactant and with the assumption that the formed ag-
gregates are spherical, the size of the aggregates was estimated
to be R ∼ 4000 Å. This means that, even at this very low
concentration, compound Mel-â-TMD forms not only simple
micelles but large aggregates as well. Most probably, these are
some sheets of lipid bilayers, which, with a further increase of
concentration, will form a lamellar phase.
Pcyl(q)
2
dΣ(q)/dΩ/(c - cmc) ) M∆Fm
1 + âc(q) Prod(q,L - 2 R )
(4)
where â ) [1 - S(0)]/S(0), with S(0) being the structure factor
calculated by the rigid cylinder model; that is, for a solution of
isotropic rigid cylinders, S(0) is calculated from the expression
for the osmotic compressibility as determined in the scaled
particle approximation:
The position of the diffraction maximum in the scattering
curve is connected to the distance between bilayers:
d ) 2π/qmax
(11)
(1 - B - C)4
S(0) )
(5)
[1 + 2(B + C)]2 + 2D[1 + B + (5/4)C]
From the quite-broad maximum in the SANS curve, d can be
estimated to be 40-50 Å.
In this equation, B ) πR2Ln, C ) 4/3πR3n, D ) 1/2πRL2n, L
is the length of the cylinders, R is the radius of the cylinders,
and n is the number concentration of the cylinders.
Next, the interval of q from 0.01 to 0.1 Å-1 was analyzed by
the IFT method. This interval shows the scattering behavior (dΣ-
(q)/dΩ ∼ q-2) of disklike (bilayer) aggregates.

1608 J. Phys. Chem. B, Vol. 109, No. 4, 2005
Milkereit et al.
(15) Siegel, D. P. Biophys. J. 1986, 49, 1155-1170.
The scattering intensities for disklike aggregates are written
in the form of the thickness pair distance distribution function
pT(r):
(16) Siegel, D. P. Biophys. J. 1986, 49, 1171-11783.
(17) Siegel, D. P. Chem. Phys. Lipids 1986, 42, 279-301.
(18) Siegel, D. P.; Banschbach, J.; Alford, J.; Ellens, H.; Lis, L. J.; Quinn,
P. J.; Yeagle, P. L.; Bentz, J. Biochemistry 1989, 28, 3703-3709.
(19) Minden, v. H. M.; Brandenburg, K.; Seydel, U.; Koch, M. H. J.;
Garamus, V.; Willumeit, R.; Vill, V. Chem. Phys. Lipids 2000, 106, 157-
179.
2π
dΣ(q)/dΩ )
π
∞p (r) cos(qr) dr
(12)
∫
T
2
( )
0
q
(20) Vill, V.; Minden, v. H. M.; Koch, M. H. J.; Seydel, U.; Branden-
burg, K. Chem. Phys. Lipids 2000, 104, 75-91.
The pT(r) function is given by44
(21) Minamikawa, H.; Hato, M. Langmuir 1997, 13, 2564-2571.
(22) Minamikawa, H.; Hato, M. Langmuir 1998, 14, 4503-4509.
(23) Korchowiec, B. M.; Baba, T.; Minamikawa, H.; Hato, M. Langmuir
1998, 17, 1853-1859.
c - cmc
2πMS
pT(r) )
∆F(r′) ∆F(r + r′) dr′
(13)
∫
(24) Hato, M. Curr. Opin. Colloid Interface Sci. 2001, 6, 268-276.
(25) Hato, M.; Minamikawa, H.; Salkar, R. A.; Matsutani, S. Langmuir
2002, 18, 3425-3429.
with r corresponding to the coordinate in the x direction and
MS corresponding to the mass per surface units of disklike
aggregates.
Now we are able to describe the intermediate q interval for
disklike aggregates by IFT (Figure 9). Similar to the method
used for rodlike aggregates, the radius of gyration (thickness
of RT,g) and the mass per surface unit MS can be calculated
from pT(r).
(26) Salkar, R. A.; Minamikawa, H.; Hato, M. Chem. Phys. Lipids 2004,
127, 65-75.
(27) Minden, v. H. M.; Morr, M.; Milkereit, G.; Heinz, E.; Vill, V. Chem.
Phys. Lipids 2002, 114, 55-80.
(28) Milkereit, G.; Morr, M.; Thiem, J.; Vill, V. Chem. Phys. Lipids
2004, 127, 47-63.
(29) Milkereit, G.; Gerber, S.; Brandenburg, K.; Koch, M. H. J.; Morr,
M.; Seydel, U.; Vill, V. To be published.
(30) Minamikawa, H.; Murakami, T.; Hato, M. Chem. Phys. Lipids 1994,
72, 111-118.
(31) Morr, M.; Wray, V.; Fortkamp, J.; Schmid, R. D. Liebigs Ann.
Chem. 1992, 5, 433-440.
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