Self-assembling properties of 6-O-alkyltrehaloses under aqueous conditions

Article abstract of DOI:10.1016/j.carres.2012.05.014

In this study, we report the self-assembling properties of 6-O-alkyltrehaloses with different chain lengths, that is, octyl, decyl, dodecyl, tetradecyl, and hexadecyl, under aqueous conditions. The materials were synthesized from trehalose via five reacti

Full text of DOI:10.1016/j.carres.2012.05.014

Carbohydrate Research 357 (2012) 32–40
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Self-assembling properties of 6-O-alkyltrehaloses under aqueous conditions
⇑
Manami Kanemaru, Kazuya Yamamoto, Jun-ichi Kadokawa
Department of Chemistry, Biotechnology, and Chemical Engineering, Graduate School of Science and Engineering, Kagoshima University, 1-21-40 Korimoto,
Kagoshima 890-0065, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, we report the self-assembling properties of 6-O-alkyltrehaloses with different chain
lengths, that is, octyl, decyl, dodecyl, tetradecyl, and hexadecyl, under aqueous conditions. The materials
were synthesized from trehalose via five reaction steps. The scanning electron microscopy (SEM), trans-
mission electron microscopy (TEM), powder X-ray diffraction (XRD), and dynamic light scattering (DLS)
measurements indicated that the self-assembling property of 6-O-dodecyltrehalose was completely dif-
ferent from that of the other derivatives. The former primarily formed spherical micelles in water, which
further assembled into face-centered cubic-based aggregates when dried. The latter derivatives, on the
other hand, formed vesicle-type particles via the formation of lamellar-like planes, which further merged
with each other, most likely by the fusion of the planes, to construct larger aggregates.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 20 March 2012
Received in revised form 13 May 2012
Accepted 15 May 2012
Available online 23 May 2012
Keywords:
6-O-Alkyltrehalose
Self-assembly
Vesicle
Lamellar plane
Micelle
1. Introduction
ester-linked carbohydrate amphiphiles because of the simpler
structure of the ether linkage and the exclusion of the possibility
Amphiphilic molecules, that is, amphiphiles, possess hydro-
philic and hydrophobic moieties in the same molecule. Under
aqueous conditions, amphiphiles form self-assembling aggregates
with various controlled morphologies such as spherical micelles,
cylindrical micelles, spherical vesicles, planer bilayers, and tubes.1
Because amphiphiles containing carbohydrates such as glycolipids
as a hydrophilic part exhibit important in vivo functions in living
systems,2 synthetic carbohydrate-based amphiphiles have been
studied extensively and have been shown to exhibit a large variety
of self-assembling morphologies.1,3 For example, the self-assem-
bling properties and applications, as surfactants, of fatty acid esters
of disaccharides such as sucrose and trehalose have been stud-
ied.4,5 Moreover, sucrose fatty acid esters have been used as effec-
tive food additives in various industries in the form of emulsifiers.6
The self-assembling properties of sucrose fatty acid esters change,
depending on the lengths and types of the fatty acid chains and
have been investigated extensively.7 In comparison, fewer studies
have looked at the self-assembling properties of amphiphilic su-
crose ethers, in which a sucrose residue is attached to the alkyl
chains by ether linkages.8 Because ether linkages are more stable
than ester linkages in aqueous conditions, these new ether-linked
carbohydrate amphiphiles can be expected to find applications as
emulsifiers and additives in various food industries. Moreover,
we have considered that ether-linked carbohydrate amphiphiles
exhibit self-assembling properties that are different from those of
of hydrogen bonding by the carbonyl group. In a previous paper,
we reported the self-assembling properties of a mixture of 6-O-
and 60-O-hexadecylsucroses under aqueous conditions, which were
new sucrose-based amphiphiles having the structure of a sucrose
residue connected with a single hexadecyl chain by an ether link-
age at either the 6- or 60-position.9 On the basis of the results of our
analysis, we had proposed the following process for the self-
assembly of the mixture of sucrose ethers under aqueous condi-
tions. The mixture primarily formed spherical micelles with a
diameter of ca. 5–7 nm in water. The micelles organized in a regu-
lar manner and formed a face-centered cubic (FCC) structure dur-
ing the drying of the aqueous dispersion that led to nanoparticles
with a diameter of ca. 50 nm. Moreover, several numbers of
the nanoparticles further assembled together to form larger
aggregates.
Trehalose is a nonreducing disaccharide, in which two glucose
units are linked by an a,a
-1,10-glycosidic linkage.10 Because treha-
lose has been produced industrially from starch using enzymes and
has become available at low costs, it can be regarded as a new
renewable resource that is comparable to sucrose. Naturally occur-
ring glycolipids containing a trehalose residue (trehalolipids) are
found in nature. An example is trehalose 6,60-dimycolate, the most
reported trehalolipid, which is an
a-branched-chain mycolic acid
in which the 6-position of each glucose moiety is esterified.11
Therefore, studying the synthesis and properties of new amphi-
philes composed of a trehalose residue as the hydrophilic part
could contribute to the development of emulsifiers and novel food
additives. Although the preparation and surfactant properties of
⇑
Corresponding author. Fax: +81 99 285 3253.
E-mail address: kadokawa@eng.kagoshima-u.ac.jp (J. Kadokawa).
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.05.014

M. Kanemaru et al. / Carbohydrate Research 357 (2012) 32–40
33
synthetic amphiphilic trehalose esters have been previously re-
ported,5 to the best of our knowledge, amphiphilic trehalose ethers
have hardly been investigated.
intermediate, that is, 6,60-di-O-trityltrehalose (1). The compound
1 was synthesized by modifying a previously published proce-
dure.12 Since we could not directly obtain purified 1 from the crude
products containing unreacted trehalose by a simple silica gel
chromatographic method, the products were once acetylated using
acetic anhydride. Then, 6,60-di-O-trityltrehalose hexaacetate (10)
was isolated from the acetylated products by silica gel chromatog-
raphy, followed by deacetylation using sodium methoxide, to yield
1. Then, procedures including benzylation, detritylation, mono-
etherification using alkyl bromides with different chain lengths,
and debenzylation were performed, in the same manner as re-
ported in our previous study,9 to obtain the five 6-O-alkyltreha-
loses (mono-C8, C10, C12, C14, and C16).
With the above-mentioned points in mind, in this paper, we re-
port the synthesis and self-assembly, under aqueous conditions, of
a series of new trehalose-based amphiphiles, which are 6-O-aklylt-
rehaloses having different chain lengths, that is, octyl, decyl, dode-
cyl, tetradecyl, and hexadecyl (mono-C8, C10, C12, C14, and C16,
respectively). Consequently, we found that mono-C8, C10, C14,
and C16 showed hierarchically self-assembling processes based
on the primary formation of lamellar-like planes, leading to vesi-
cle-type particles and their further aggregates, whereas self-
assembling property of mono-C12 was completely different from
that of the others but similar to that of the previously reported
mixture of 6-O- and 60-O-hexadecylsucrose, which was, based on
the FCC-based organization of the primary formed spherical
micelles.
The structures of all the products were confirmed by 1H NMR
and MALDI-TOF MS measurements. Figure 1a shows the 1H NMR
spectrum of mono-C8 in CD3OD. All the signals were assignable
to the structure of mono-C8 as follows; 1H NMR (CD3OD) d 0.80
(t, 3H, CH3, J = 6.8 Hz), 1.14–1.32 (m, 10H, –(CH2)5–CH3), 1.44–
1.49 (m, 2H, –O–CH2–CH2–), 3.35–3.85 (m, 14H, H-
2,20,3,30,4,40,5,50,6,60, –O–CH2–CH2–), 4.996, 5.004 (2d, 2H, H-1,
J = 2.8, 3.6 Hz). The mono-etherified structure was confirmed by
the appearance of two doublet sets of anomeric signals (d 4.996
and 5.004) and the integrated ratio of the signal due to the CH3
moiety of the octyl group to the signal due to an H-1 proton of
the glucose residue, which was 3:1. The MALDI-TOF MS spectrum
showed a significant peak corresponding to the molecular mass of
[mono-C8] Na+ (found: m/z = 477.4337; calcd: m/z = 477.2312). To
further confirm the structure, the product was acetylated using
acetic anhydride and pyridine. The 1H NMR spectrum (Fig. 1b) of
the acetylated derivative in CDCl3 showed that the chemical shift
of the signals assignable to the H-6 protons was not changed from
that before the acetylation (Fig. 1a), whereas the signals due to the
2. Results and discussion
The synthesis of amphiphilic 6-O-alkyltrehaloses (mono-C8,
C10, C12, C14, and C16) from trehalose was carried out by the five
reaction steps according to Scheme 1, which were the successive
ditritylation at the 6- and 60positions, benzylation of the other free
hydroxy groups, detritylation, monoetherification at the 6-positi-
ton, and debenzylation. Although another synthetic approach, via
monotritylation instead of ditritylation was also considered, we se-
lected the aforementioned route via the ditritylation. Because we
are investigating the self-assembly of other trehalose derivatives,
namely, 6,60-di-O-alkyltrehaloses (the results will be reported else-
where), as well, and these derivatives can be synthesized simulta-
neously with the present 6-O-alkyltrehaloses from the same
OTr
OTr
O
OH
O
O
BnO
RO
RO
HO
HO
TrCl
OBn
BnO
OR
O
OH
O
NaH
DMF
BnBr, TBAB
OTr
OR
OH
OH
OTr
OBn
OBn
pyridine
O
O
O
O
HO
RO
OR
BnO
OH
1; R = H
trehalose
2
acetic anhydride /
pyridine
CH3ONa
1'; R = Ac
OH
O(CH2)nCH3
O
O
OBn
BnO
BnO
BnO
BnO
conc. HCl
acetic acid / ClCH2CH2Cl / water
OBn
O
NaH
DMF
CH3 (CH2)n
Br
, TBAB
OH
O
OH
OBn
OBn
OBn
O
O
OBn
BnO
BnO
4-C8; n = 7
3
4-C10; n = 9
4-C12; n = 11
4-C14; n = 13
4-C16; n = 15
O(CH2)nCH3
O
HO
HO
OH
O
H2, Pd-C
OH
OH
Bu
Ph
butanol / methanol / water
TBAB = Bu N+ Bu
Bn =
CH2
Tr =
C
Ph
O
Bu
Br
Ph
HO
OH
mono-C8; n = 7
mono-C10; n = 9
mono-C12; n = 11
mono-C14; n = 13
mono-C16; n = 15
Scheme 1. Synthesis of 6-O-alkyltrehaloses from trehalose.

34
M. Kanemaru et al. / Carbohydrate Research 357 (2012) 32–40
Figure 1. 1H NMR spectra of (a) mono-C8 (CD3OD) and (b) its acetylated derivative (CDCl3).
other positions (besides anomeric signals) shifted to a lower mag-
netic field. This data indicated that only the 6-position was etheri-
fied in the aforementioned product, and hence, was not acetylated
by acetic anhydride. All the analytical and acetylation results men-
tioned above support the structure of mono-C8. The structures of
the other derivatives were also confirmed in the same manner.
Then, the products were subjected to the following investigations
of their self-assembling properties under aqueous conditions.
In the SEM images of the samples on aluminum plates (Fig. 2),
which were prepared by drying dispersions of all the derivatives
in water (1.0 ꢀ 10ꢁ5 mol/L), particle-like nanoaggregates were
seen. The average diameters of the aggregates of mono-C8, C10,
C14, and C16 decreased with an increase in the alkyl chain lengths
and were 136.4, 114.1, 97.4, and 78.1 nm, respectively (the values
of the standard deviations are shown in Table 1). However, the
average diameter (58.3 nm) of the aggregates of mono-C12 was
smaller than that of the other aggregates, regardless of the alkyl
chain length. To further confirm the hierarchical structures of the
nanoaggregates, TEM measurements of the samples were per-
formed. Dispersions of mono-C8–C16 in water (1.0 ꢀ 10ꢁ5 mol/L)
were placed on carbon film-coated grids. After the negative-stain-
ing technique was carried out, the TEM samples were prepared by
drying the preparative materials. The TEM images of the mono-C8,
C10, C14, and C16 samples (Fig. 3a, b, d, and e) exhibited that sev-
eral vesicle-type particles with diameters of approximately 30–
60 nm assembled to form larger aggregates with sizes of approxi-
mately 80–100 nm. Furthermore, a magnified TEM image of the
sample mono-C10 (Fig. 3b0) showed that the vesicles were con-
structed from lamellar-like planes probably by the self-organiza-
tion of the alternating hydrophilic trehalose and hydrophobic
decyl layers. Interestingly, the TEM image also showed that the
planes at the interfacial area between the two vesicles were fused,

M. Kanemaru et al. / Carbohydrate Research 357 (2012) 32–40
35
Figure 2. SEM images of the samples prepared from dispersions of (a) mono-C8, (b) C10, (c) C12, (d) C14, and (e) C16 in water (1.0 ꢀ 10ꢁ5 mol/L).
Table 1
SEM and DLS results of 6-O-alkyltrehaloses
Sample
SEM results
DLS results in water (1.0 ꢀ 10ꢁ5 mol/L)
Average diameter (nm)
136.4
114.1
58.3
97.4
78.1
Standard deviation
Average diameter (nm)
Polydispersity index
Mono-C8
30.6
22.4
12.5
17.5
18.2
123.6
123.3
12.9
104.4
84.4
0.247
0.258
0.584
0.730
0.590
Mono-C10
Mono-C12
Mono-C14
Mono-C16
Figure 3. TEM images of the samples prepared from dispersions of (a) mono-C8, (b) C10, (c) C12, (d) C14, and (e) C16 in water (1.0 ꢀ 10ꢁ5 mol/L) and (b0) magnified image of
(b).
which probably contributed to the stabilization of the larger aggre-
gates in water (vide section on the dynamic light scattering (DLS)
measurements). On the other hand, in the TEM image of the
mono-C12 sample, some particles with diameters of approxi-
mately 15–17 nm were found to be forming aggregates with sizes
of approximately 60 nm, but each particle did not exhibit a vesicle-
like morphology. The SEM and TEM results suggested that the self-
assembling process of mono-C12 under aqueous conditions was
different from that of the other materials under the same
conditions.

36
M. Kanemaru et al. / Carbohydrate Research 357 (2012) 32–40
To study the self-assembling processes of mono-C8–C16 in
diameter of mono-C12 (12.9 nm) in the DLS profile (Fig. 5c) was
much smaller than that observed in the SEM image (58.3 nm).
The former probably corresponded to either a spherical micelle
or the assembly of several micelles in the solution state, whereas
the latter represented further aggregates from the micelles accord-
ing to the FCC organization in the solid (dried) state.
even greater detail, powder X-ray diffraction (XRD) and DLS mea-
surements were conducted. Figure 4 shows the XRD profiles of
the five samples, which were prepared by drying aqueous disper-
sions of mono-C8–C16 (1.0 ꢀ 10ꢁ3 mol/L). In the XRD profiles of
the mono-C8, C10, C14, and C16 samples (Fig. 4a, b, d, and e),
the diffraction peaks due to (001) and further peaks assignable
to the (002), (003), and/or (004) Bragg reflections of the lamellar
patterns were observed.13 From the XRD patterns, it was found
that the widths of each layer increased with an increase in the alkyl
chain lengths and were calculated to be 3.1 nm for mono-C8, 3.3
nm for mono-C10, 3.8 nm for mono-C14, and 3.9 nm for mono-
C16. On the other hand, the XRD profile of the mono-C12 sample
(Fig. 4c) exhibited diffraction peaks ascribable to the (111),
(200), and (311/222) Bragg reflections of the FCC structure.14
Using the XRD patterns, the diameter of a sphere, which took part
in the FCC structure, was calculated according to a previously de-
scribed method15 and found to be 5.1 nm. The molecular size
(length) of mono-C12 was calculated by the MM2 method using
the CS Chem3D software program and found to be approximately
3 nm. Accordingly, when mono-C12 molecules formed a spherical
micelle having hydrophilic trehalose residues in the outer layer
and hydrophobic dodecyl chains in the inner layer, the diameter
of the sphere could be estimated to be approximately 6.0 nm,
which was in good agreement with that of the sphere calculated
from the XRD results. The DLS results of dispersions of all the mate-
rials in water (1.0 ꢀ 10ꢁ5 mol/L) (Fig. 5) showed monomodal pro-
files with relatively small polydispersity indices (Table 1). The
average particle diameters of the mono-C8, C10, C14, and C16
samples decreased in this order and were found to be 123.6,
123.3, 104.4, and 84.4 nm, respectively, (Fig. 5a, b, d, and e). These
values were in relatively good agreement with the average diame-
ters of the nanoaggregates observed in the SEM images. The DLS
profiles and SEM/TEM images of mono-C8, C10, C14, and C16 sug-
gested that the stable nanoaggregates with diameters of around
100 nm were constructed from several vesicles by the fusion of
the lamellar-like planes. On the other hand, the average particle
On the basis of the above-mentioned analytical results, the
following self assembling processes of the present materials are
proposed (Fig. 6). The derivatives mono-C8, C10, C14, and C16
form vesicle-type particles that are constructed from lamellar-like
planes. The particles are further merged, most likely because of the
fusion of the planes at the interfacial area between the vesicles
(Fig. 6a). Therefore, the DLS profile of the aqueous dispersions of
these materials showed average diameters corresponding to the
merged vesicles, but to each vesicle. On the other hand, the self-
assembling process of mono-C12 under aqueous conditions is
completely different from that of the other four derivatives. The
self-assembly of mono-C12 does not induce the formation of ves-
icle-type particles, but leads to the formation of spherical micelles
in water, with a diameter of approximately 5–13 nm. These mi-
celles are present in individual form, or only a few of which assem-
bles in the aqueous dispersion. On the drying of the dispersion, the
micelles assembled according to the FCC organization to construct
the nanoaggregates with a size of approximately 60 nm (Fig. 6b).
The reason for the difference in the self-assembling processes of
mono-C12 and the other derivatives is not yet clear, but it is obvi-
ously suggested that the chain lengths in 6-O-alkyltrehaloses
strongly affect their self-assembling properties under aqueous con-
ditions. It is likely that the balance between the hydrophilic and
hydrophobic parts in mono-C12 is more favorable for the forma-
tion of spherical micelles as compared to those in the other deriv-
atives under aqueous conditions. More detailed studies using
methods based on calculations that consider factors such as critical
packing parameters are now in progress to further investigate the
reasons for the difference in the self-assembling processes of
6-O-alkyltrehaloses, as well as to compare these reasons with the
results of our previous study.9
Figure 4. XRD profiles of the samples prepared from dispersions of (a) mono-C8, (b) C10, (c) C12, (d) C14, and (e) C16 in water (1.0 ꢀ 10ꢁ3 mol/L).

M. Kanemaru et al. / Carbohydrate Research 357 (2012) 32–40
37
Figure 5. DLS profiles of (a) mono-C8, (b) C10, (c) C12, (d) C14, and (e) C16 in water (1.0 ꢀ 10ꢁ5 mol/L).
Figure 6. Plausible self-assembling processes of (a) mono-C8, C10, C14, and C16 and (b) mono-C12 under aqueous conditions.
(mono-C8, C10, C12, C14, and C16) with different chain lengths
under aqueous conditions. The materials were synthesized from
trehalose by five reaction steps. The structures of the products
were established by 1H NMR and MALDI-TOF MS measurements
3. Conclusion
This study investigated the characteristic processes responsible
for the self-assembling properties of five 6-O-alkyltrehaloses

38
M. Kanemaru et al. / Carbohydrate Research 357 (2012) 32–40
as well as by an acetylation experiment. The self-assembling prop-
erties of the materials under aqueous conditions were evaluated
using SEM, TEM, XRD, and DLS measurements. Consequently, the
four materials mono-C8, C10, C14, and C16 were found to form
vesicle-type particles via the formation of lamellar-like planes in
water. These particles merged further probably because of fusion
of the planes at the interfacial area between the vesicles, to form
nanoaggregates with average diameters of ca. 80–100 nm. On the
other hand, mono-C12 formed spherical micelles with a diameter
of ca. 5–13 nm in water, which then assembled according to the
FCC organization during the drying process from the aqueous dis-
persion to construct nanoaggregates with a size of ca. 60 nm. The
above findings indicated that the chain lengths of the materials
strongly affected their self-assembling properties under aqueous
conditions. A more detailed study of the effect of the chain lengths
on the self-assembling properties of the present materials is now
in progress, and its results will be published in a future paper.
(v/v)) to give 2 (2.51 g, 1.83 mmol) quantitatively. 1H NMR (CDCl3)
d 3.04 - 4.24 (m, 12H, H-2,20,3,30,4,40,5,50,6,60), 4.64–4.95 (m, 12H,
CH2-Ph), 5.46 (d, 2H, H-1,10, J = 3.6 Hz), 6.76–7.57 (m, 60H,
aromatics).
A mixture of 2 (2.37 g, 1.73 mmol) in a mixed solvent of acetic
acid (5.0 mL) and 1,2-dichloroethane (5.0 mL) was refluxed for dis-
solution. Water (1.50 mL), 1,2-dichloroethane (5.0 mL), and con-
centrated hydrochloric acid (approximately 15 drops) were
added, in this order, to the mixture at that temperature and the
mixture was kept at that temperature for another 10 min. After
the reaction mixture was cooled to room temperature and diluted
with 1,2-dichloroethane, it was neutralized by the addition of sat-
urated aqueous NaHCO3 with stirring, diluted with water, and ex-
tracted with chloroform. The organic layer was dried over
anhydrous Na2SO4, filtered, and evaporated. The residue was sub-
jected to column chromatography using silica gel (eluent; hex-
ane/ethyl acetate = 5:1?3:1 (v/v)) to give 3 (0.99 g, 1.12 mmol)
in 64.9% yield. 1H NMR (CDCl3)
d 3.49–3.59 (m, 8H, H-
2,20,4,40,6,60), 4.04 - 4.09 (m, 4H, H-3,30,5,50), 4.63–5.01 (m, 12H,
CH2-Ph), 5.12 (d, 2H, H-1,10, J = 3.6 Hz), 7.26–7.37 (m, 30H,
aromatics).
4. Experimental
4.1. Materials
Under argon, to a dispersion of NaH (0.11 g, 1.70 mmol) in DMF
(15.0 mL) was added a solution of 3 (0.51 g, 0.57 mmol) in DMF
(5.0 mL) at room temperature and the mixture was stirred at that
temperature for 30 min. Then, 1-bromooctane (0.33 mL,
1.70 mmol) and tetrabutylammonium bromide (small amount)
were added to the mixture and the reaction was allowed to pro-
ceed at 80 °C for 24 h. After the reaction mixture was diluted with
water and extracted with chloroform, the organic layer was dried
over anhydrous Na2SO4, filtered, and evaporated. The residue was
subjected to column chromatography on silica gel (eluent; hex-
ane/ethyl acetate = 40:1?10:1?5:1 ? 3:1 (v/v)) to give 4-C8
(0.18 g, 0.16 mmol) in 28.9% yield. 1H NMR (CDCl3) d 0.85 (t, 3H,
CH3, J = 6.8 Hz), 1.22–1.28 (m, 10H, –(CH2)5–CH3), 1.42–1.55 (m,
2H, –O–CH2–CH2–), 3.25–3.70 (m, 10H, H-2,20,4,40,6,60, –O–CH2–
CH2–), 4.01–4.11 (m, 4H, H-3,30,5,50) 4.58–5.01 (m, 12H, CH2-Ph),
5.16, 5.18 (2d, 2H, H-1,10, J = 3.2, 4.0 Hz), 7.24–7.36 (m, 30H,
aromatics).
To a solution of 4-C8 (0.033 g, 0.030 mmol) in a mixed solvent
of butanol (4.0 mL), methanol (0.40 mL), and water (0.13 mL) was
added 10% palladium on carbon (0.0155 g) and the mixture was
stirred at 60 °C for 3 d under hydrogen atmosphere. The reaction
mixture was then filtered, and the filtrate was evaporated and
dried under reduced pressure to give mono-C8 (0.015 g,
0.031 mmol) quantitatively. 1H NMR (CD3OD) d 0.80 (t, 3H, CH3,
J = 6.8 Hz), 1.14–1.32 (m, 10H, –(CH2)5–CH3), 1.44–1.49 (m, 2H, –
O–CH2–CH2–), 3.35–3.85 (m, 14H, H-2,20,3,30,4,40,5,50,6,60, –O–
CH2–CH2–), 4.996, 5.004 (2d, 2H, H-1,10, J = 2.8, 3.6 Hz); MALDI-
TOF MS: Calcd [C20H38O11]Na+: m/z 477.2312. Found: m/z
477.4337.
All reagents were obtained commercially and used without fur-
ther purification. N,N-Dimethylformamide (DMF) was purified by
distillation over calcium hydride. Other solvents were used as
received.
4.2. Synthesis of 6-O-octyltrehalose (mono-C8)
A solution of triphenylmethyl chloride (14.0 g; 50.0 mmol) in
pyridine (25.0 mL) was added to a dispersion of trehalose dihy-
drate (3.78 g, 10.0 mmol) in pyridine (15.0 mL) at room tempera-
ture under argon and the mixture was stirred at 90 °C for 7 h.
After the reaction mixture was cooled to room temperature, acetic
anhydride (18.9 mL, 200 mmol) was added and the solution was
stirred for 24 h at that temperature. After the mixture was coevap-
orated with toluene three times for concentration and diluted with
chloroform, the solution was successively washed with water,
dried over anhydrous Na2SO4, filtered, and evaporated. The residue
was subjected to column chromatography using silica gel (eluent;
hexane/ethyl acetate = 4:1 (v/v)) to give the 10 (6.96 g, 6.19 mmol)
in 61.9% yield. 1H NMR (CDCl3) d 1.75, 1.89, 1.99 (3s, 18H, CH3),
3.10 (d, 4H, H-6,60, J = 3.7 Hz), 4.08–4.13 (m, 2H, H-5,50), 5.14,
5.19 (t, dd, 4H, H-2,20,4,40, J = 10.8 Hz, J = 10.8, 4.4 Hz), 5.45 (t, 2H,
H-3,30, J = 9.8 Hz) 5.46 (d, 2H, H-1,10, J = 3.6 Hz), 7.15–7.41 (m,
30H, aromatics).
Under argon, a mixture of 10 (6.96 g, 6.19 mmol) and sodium
methoxide (0.065 g, 1.02 mmol) in a mixed solvent of methanol
(35.0 mL) and THF (35.0 mL) was refluxed for 5 d. After the reac-
tion mixture was cooled to room temperature, it was treated with
cation-exchange resin (Amberlite IR-120 H+ form) for 30 min. The
resin was filtered off and the filtrate was concentrated to give 1
(3.67 g, 6.45 mmol) quantitatively. 1H NMR (DMSO-d6 + D2O) d
3.00–4.06 (m, 12H, H-2,20,3,30,4,40,5,50,6,60), 5.15 (d, 2H, H-1,10,
J = 3.2 Hz), 7.10–7.44 (m, 30H, aromatics).
Under argon, to a dispersion of NaH (1.63 g, 27.1 mmol) in DMF
(20.0 mL) was added a solution of 1 (1.50 g, 1.81 mmol) in DMF
(12.0 mL) at room temperature and the mixture was stirred at that
temperature for 30 min. After benzyl bromide (1.95 mL,
16.3 mmol) and tetrabutylammonium bromide (small amount)
were added to the mixture, the reaction was conducted at room
temperature for 6 d. After the resulting mixture was concentrated
and diluted with ethyl acetate, the solution was washed with
water, dried over anhydrous Na2SO4, filtered, and evaporated.
The residue was recrystallized from methanol/1-propanol (1:0.54
4.3. Synthesis of 6-O-decyltrehalose (mono-C10)
In the same way as 4-C8, 4-C10 was prepared from 3 in 31.4%
yield. 1H NMR (CDCl3) d 0.87 (t, 3H, CH3, J = 7.0 Hz), 1.22–1.26
(m, 14H, –(CH2)7–CH3), 1.42–1.52 (m, 2H, –O–CH2–CH2–), 3.27–
3.70 (m, 10H, H-2,20,4,40,6,60, –O–CH2–CH2–), 4.01–4.11 (m, 4H,
H-3,30,5,50) 4.58–5.01 (m, 12H, CH2-Ph), 5.16, 5.18 (2d, 2H, H-1,10,
J = 3.6, 4.0 Hz), 7.25–7.36 (m, 30H, aromatics).
Then, synthesis of mono-C10 was carried out in the same way
as mono-C8 from 4-C10 in 87.3% yield. 1H NMR (CD3OD) d 0.80 (t,
3H, CH3, J = 7.2 Hz), 1.32–1.40 (m, 14H, –(CH2)7–CH3), 1.43–1.51
(m, 2H, –O–CH2–CH2–), 3.35–3.85 (m, 14H, H-2,20,3,30,4,40,5,50,6,60,
–O–CH2–CH2–), 4.996, 5.004 (2d, 2H, H-1,10, J = 3.2, 3.2 Hz); MAL-
DI-TOF MS: Calcd [C22H42O11]Na+: m/z 505.2625. Found: m/z
505.7879.

M. Kanemaru et al. / Carbohydrate Research 357 (2012) 32–40
39
4.4. Synthesis of 6-O-dodecyltrehalose (mono-C12)
4.8. Preparation of samples for SEM measurements
Similarly to 4-C8, 4-C12 was prepared from 3 in 18.0% yield. 1H
NMR (CDCl3) d 0.87 (t, 3H, CH3, J = 6.8 Hz), 1.22–1.26 (m, 18H, –
(CH2)9–CH3), 1.47–1.58 (m, 2H, –O–CH2–CH2–), 3.23–3.70 (m,
10H, H-2,20,4,40,6,60, –O–CH2–CH2–), 4.01–4.11 (m, 4H, H-3,30,5,50)
4.58–5.01 (m, 12H, CH2-Ph), 5.16, 5.18 (2d, 2H, H-1,10, J = 3.6,
3.6 Hz), 7.24–7.36 (m, 30H, aromatics).
A
dispersion of 6-O-alkyltrehalose (1.0 ꢀ 10ꢁ5 mol/L) was
placed on an aluminum plate and it was left standing under ambi-
ent atmosphere until water was evaporated. Then, the resulting so-
lid sample was subjected to the measurements.
4.9. Preparation of samples for TEM measurements
The synthesis of mono-C12 was also carried out in the same
way as that of mono-C8 from 4-C12 in 88.2% yield. 1H NMR
(CD3OD) d 0.80 (t, 3H, CH3, J = 6.8 Hz), 1.14–1.31 (m, 18H, –
(CH2)9–CH3), 1.43–1.51 (m, 2H, –O–CH2–CH2–), 3.35–3.85 (m,
14H, H-2,20,3,30,4,40,5,50,6,60, –O–CH2–CH2-), 4.997, 5.004 (2d, 2H,
H-1,10, J = 2.8, 3.2 Hz); MALDI-TOF MS: Calcd [C24H46O11]Na+: m/z
533.2938. Found: m/z 533.4905.
A
dispersion of 6-O-alkyltrehalose (1.0 ꢀ 10ꢁ5 mol/L) was
placed on carbon film-coated copper grids. The negative-staining
technique was used for TEM sample preparation. Then, the pre-
parative material was left standing under ambient atmosphere un-
til water was evaporated. Then, the resulting solid sample was
subjected to the measurements.
4.10. Preparation of samples for XRD measurements
4.5. Synthesis of 6-O-tetradecyltrehalose (mono-C14)
A
dispersion of 6-O-alkyltrehalose (1.0 ꢀ 10ꢁ3 mol/L) was
In the same way as 4-C8, 4-C14 was prepared from 3 in 24.7%
yield. 1H NMR (CDCl3) d 0.88 (t, 3H, CH3, J = 6.6 Hz), 1.22–1.25
(m, 22H, –(CH2)11–CH3), 1.42–1.60 (m, 2H, –O–CH2–CH2–), 3.23–
3.70 (m, 10H, H-2,20,4,40,6,60, –O–CH2–CH2–), 4.01–4.14 (m, 4H,
H-3,30,5,50) 4.58–5.01 (m, 12H, CH2-Ph), 5.16, 5.18 (2d, 2H, H-1,10,
J = 3.6, 3.6 Hz), 7.24–7.36 (m, 30H, aromatics).
placed on a sample plate and it was left standing under ambient
atmosphere until water was evaporated. Then, the resulting solid
sample was subjected to the measurements.
4.11. Measurements
Then, synthesis of mono-C14 was carried out in the same way
as that of mono-C8 from 4-C14 in 83.5% yield. 1H NMR (CD3OD) d
0.80 (t, 3H, CH3, J = 7.2 Hz), 1.06–1.31 (m, 22H, –(CH2)11–CH3),
1.46–1.50 (m, 2H, –O–CH2–CH2–), 3.36–3.84 (m, 14H, H-
2,20,3,30,4,40,5,50,6,60, –O–CH2–CH2–), 5.018, 5.024 (2d, 2H, H-1,10,
J = 3.6, 3.6 Hz); MALDI-TOF MS: Calcd [C26H50O11]Na+: m/z
561.3251. Found: m/z 561.5306.
The 1H NMR spectra were recorded using a JEOL ECX400 spec-
trometer. The MALDI-TOF MS measurements were performed out
using a SHIMADZU Voyager Biospectrometry Workstation System
Ver.5.1 with 2.5-dihydroxybenzoic acid as the matrix containing
0.05% trifluoroacetic acid under the positive ion mode. The SEM
images were obtained using a Hitachi S-4100H electron micro-
scope. TEM measurements were performed using a Jeol JEM-
3010 at 200 kV. The XRD measurements were conducted using a
4.6. Synthesis of 6-O-hexadecyltrehalose (mono-C16)
PANalytical X’Pert Pro MPD system with Ni-filtered Cu K
a radia-
tion (k = 0.15418 nm). The DLS measurement was performed on a
Zetasizer Nano ZS (Malvern Instruments).
In the same way as 4-C8, 4-C16 was prepared from 3 in 25.2%
yield. 1H NMR (CDCl3) d 0.85 (t, 3H, CH3, J = 6.6 Hz), 1.21–1.25
(m, 26H, –(CH2)13–CH3), 1.43–1.55 (m, 2H, –O–CH2–CH2–), 3.24–
3.70 (m, 10H, H-2,20,4,40,6,60, –O–CH2–CH2–), 4.00–4.14 (m, 4H,
H-3,30,5,50) 4.58–5.01 (m, 12H, CH2-Ph), 5.16, 5.18 (2d, 2H, H-1,10,
J = 3.2, 3.6 Hz), 7.25–7.36 (m, 30H, aromatics).
Then, synthesis of mono-C16 was carried out in the same way
as that of mono-C8 from 4-C16 in quantitative yield. 1H NMR
(CD3OD) d 0.80 (t, 3H, CH3, J = 7.2 Hz), 1.14–1.27 (m, 26H, –
(CH2)13–CH3), 1.46–1.50 (m, 2H, –O–CH2–CH2–), 3.37–3.84 (m,
14H, H-2,20,3,30,4,40,5,50,6,60, –O–CH2–CH2–), 5.020, 5.026 (2d, 2H,
H-1,10, J = 4.2, 3.6 Hz); MALDI-TOF MS: Calcd [C28H54O11]Na+: m/z
589.3564. Found: m/z 589.9111.
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A typical example was as follows. Under argon, a mixture of
mono-C8 (0.0112 g; 0.025 mmol), acetic anhydride (0.25 mL),
and pyridine (0.25 mL) was stirred at room temperature for 24 h.
The reaction mixture was poured into a large amount of water to
precipitate the product. The precipitate was dried under reduced
pressure to give the acetylated product (0.0127 g, 0.017 mmol) in
67.8% yield. 1H NMR (CDCl3) d 0.88 (t, 3H, –CH2–CH3, J = 7.0 Hz),
1.19–1.37 (m, 10H, –(CH2)5–CH3), 1.62–1.65 (m, 2H, –O–CH2–
CH2–), 2.04–2.10 (7s, 21H, O@C–CH3), 3.33–3.48 (m, 4H, H-6, –
O–CH2–CH2–), 3.97–4.08 (m, 3H, H-5,50,60a), 4.26 (dd, 1H, H-60b,
J = 12.4, 5.2 Hz), 5.00–5.10 (m, 4H, H-2,20,4,40), 5.28 (d, 2H, H-1,10,
J = 4.4 Hz), 5.49, 5.49 (2t, 2H, H-3,30, J = 9.8, 10.0 Hz). Acetylations
of mono-C10, C12, C16, and C18 were carried out in the same
way as mentioned above.

40
M. Kanemaru et al. / Carbohydrate Research 357 (2012) 32–40
12. Kurita, K.; Masuda, N.; Aibe, S.; Murakami, K.; Ishii, S.; Nishimura, S.
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1997, 13, 3706–3714.
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Wiley: New York, 2004. Chapter 8.