Molecular-shape selectivity by molecular gel-forming compounds: Bioactive and shape-constrained isomers through the integration and orientation of weak interaction sites

Article abstract of DOI:10.1039/c1cc13397g

A molecular gel system was assembled on carrier particles and the integrated effect of weak interaction sites enabled highly efficient separation of the bioactive and shape-constrained isomers of tocopherols, β-carotene, and polycyclic aromatic hydrocarbons (PAHs) by multiple interaction mechanisms.

Full text of DOI:10.1039/c1cc13397g

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COMMUNICATION
Molecular-shape selectivity by molecular gel-forming compounds:
bioactive and shape-constrained isomers through the integration and
orientation of weak interaction sitesw
Abul K. Mallik, Hongdeng Qiu, Tsuyoshi Sawada, Makoto Takafuji and Hirotaka Ihara*
Received 8th June 2011, Accepted 3rd August 2011
DOI: 10.1039/c1cc13397g
A molecular gel system was assembled on carrier particles and
the integrated effect of weak interaction sites enabled highly
efficient separation of the bioactive and shape-constrained
isomers of tocopherols, b-carotene, and polycyclic aromatic
hydrocarbons (PAHs) by multiple interaction mechanisms.
aggregates constructed of highly ordered assembly like aqueous
lipid bilayer membranes. A number of self-assembling systems
have been used as separation phases.5a–c
In this communication, we demonstrate that a molecular gel
system can be applied as a highly-ordered organic phase for
highly selective liquid chromatography. This idea is based on
the expectations that highly efficient integration of weak
interaction groups can be generated through molecular
gelation and consequently lead to highly efficient separation
of the bioactive and shape-constrained isomers,3a–e such that
SRM1647e,3a tocopherol, and b-carotene can be realized in
multiple interaction mechanisms. For this purpose, we focused
on L-glutamide-derived low-molecular organogelator as a base
unit. Our previous investigations showed that dialkyl
L-glutamide-derived amphiphilic lipids form nanotubes,6a
nanohelices,6b and nanofibers6c based on lipidic bilayer struc-
tures in water and on the fact that intermolecular hydrogen
bonding among the amide moieties contributes to self-
assembly. In addition, lipophilic derivatives of L-glutamide
showed self-organization even in organic solvents.6d,e
Although the bioactivities of biological molecules are often
different, they have various isomers of closely related species.
Therefore, the separation of these isomers is one of the most
challenging issues facing the analyst, and solving this challenge
is important in the disciplines of environmental, clinical and
food sciences. For example, the cis-isomers of b-carotene have
attracted attention because their bioavailability is totally
different from that of their corresponding trans-isomers1a
and they may have specific functions.1b Similarly, among the
isomers of tocopherol, a and g significantly decrease endo-
genous cholesterol synthesis and apo-Al-mediated cholesterol
secretion in Caco-2 cells.1c
To solve the separation challenges, we have designed and
synthesized poly(octadecyl acrylate)-grafted silica (Sil-ODAn)2a
as a lipid membrane-analogous organic phase. This polymer
cannot form a bilayer structure in water but instead forms
nanogels, which undergo a temperature-responsive phase
transition between ordered and disordered structures, similar
to aqueous lipid membrane systems. The organic phase also
showed remarkably enhanced selectivity for PAHs via multiple
carbonyl–p interactions at lower temperatures due to ordered
functional groups (carbonyl groups).2b–e However, the
separation of the bioactive and shape-constrained isomers of
large molecules such as tocopherol and b-carotene3a–e has
been difficult to resolve.
The molecule 1 was newly designed and synthesized for the
above-mentioned purpose (see Scheme S1, ESIw). Its functions
can be emphasized by being immobilizable with a terminal
carboxyl group and the fact that five amide bonds are
integrated per molecule (Schemes 1 and 2). Furthermore, its
self-assembling function can be detected by monitoring the
chiroptical property. Fig. 1 shows the temperature dependency
of the circular dichroism (CD) intensity as an indicator of
chirality. NK-77 as a cationic dye has no CD signal on its
absorption band because NK-77 is an achiral molecule
(see Fig. S1, ESIw). However, when NK-77 was mixed with
anionic assemblies from 1, an extremely large and positive CD
signal around the absorption band of NK-77 was detected,
especially at temperatures below 50 1C. The molecular
ellipticity [y]552 reached 1.8 Â 106 deg cm2 dmolÀ1. This large value
cannot be explained by simple electrostatic interaction of
NK-77 with chiral 1. Similar enhancement of the CD signal
has been recognized by induction of a chiral arrangement of
achiral dyes on chirally-ordered assemblies.6b,e Therefore, the
CD pattern indicates that NK-77 interacts with the chiral
molecular gel to produce R-chirally stacked dimers or
polymers, as shown in Fig. 1. In support of this estimation, the
CD intensity showed remarkable temperature dependency
On the other hand, self-assembling low-molecular-weight
gelators (LMWGs) have attracted interest because of their
unique properties and usefulness in a wide range of applications
in various fields.4a–f The gelation is induced by three-dimensional
network formation from nanosized one-dimensional fibrillar
Department of Applied Chemistry and Biochemistry,
Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, Japan.
E-mail: ihara@kumamoto-u.ac.jp; Fax: +81 96-342-3662;
Tel: +81 96-342-3662
w Electronic supplementary information (ESI) available: Experimental
details, characterizations, and additional results. See DOI: 10.1039/
c1cc13397g
c
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Chem. Commun., 2011, 47, 10341–10343 10341

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and disordered structures. The differential scanning calori-
metry (DSC) thermogram is direct evidence indicating that the
bending point is closely related to the peak-top temperature in
the phase transition (Fig. 1). These results indicate that 1 has a
self-assembling ability with chirally ordered structures. In
addition, 1 forms a gel containing nanometre-scale organized
assemblies in organic solvent. Fig. S3 (ESIw) shows a TEM
image of fibrous aggregates of 1 prepared from benzene. A
typical example of the 1 benzene gel is shown in Fig. S4 (ESIw).
The driving force for the aggregation is mainly intermolecular
hydrogen bonding among the amide bonds. Interestingly, 1
can form gel even after grafting on silica. As a proof, FT-IR
and diffuse reflectance infrared Fourier transform (DRIFT)
spectra have characteristic peaks for 1 and Sil-FIP carbonyl
groups at 1647 and 1642 cmÀ1 in THF (sol state). The peaks
shift to 1634 and 1637 cmÀ1 in benzene (gel state), respectively
(Fig. S5, ESIw), which is attributed to the hydrogen bond
formation of carbonyl groups.
Scheme 1 Schematic illustration of the concept of immobilization of
molecular gel. The functional groups can be integrated with ordered
structure on silica.
The molecular gel-forming 1 was then immobilized onto
3-aminopropyltrimethoxysilane (APS)-grafted silica by covalent
linkages to obtain a functional group-integrated organic phase
on silica (Sil-FIP) (Scheme 2). The Sil-FIP phase was
characterized by elemental analysis, thermogravimetric analysis
(TGA), DRIFT, solid-state 13C cross-polarization magic angle
spinning (CP/MAS), suspended-state 1H NMR and 29Si
CP/MAS NMR spectroscopy.
Detailed characterization results are given in Table S1 and
Fig. S6–S9 in ESI.w
The molecular-shape selectivity of Sil-FIP was evaluated by
liquid chromatographic retention of a packed column with
SRM 869b and SRM1647e as shown in Fig. S10 and S11
(ESIw).3a As shown in Table 1, Sil-FIP had considerably
higher selectivity (a) than monomeric, polymeric C18
(C18-mono and C18-poly), and C30 columns. For example,
the highest molecular planarity (ap-terphenyl/m-terphenyl = 12.6)
and linearity selectivity (anaphthacene/benzo[a]anthracene = 5.50) was
observed with Sil-FIP compared with other phases (Table 1).
From the above findings of the very high molecular-shape
selectivity of Sil-FIP, we attempted to separate shape-
constrained biologically important isomers of vitamin E
(tocopherol) and b-carotene.
Scheme 2 Immobilization of the molecular gel onto APS-grafted
silica by covalent linkage.
Comparative chromatograms for the separation of b- and
g-isomers of tocopherol with C18-mono, C18-poly, C30, and Sil-
FIP are shown in Fig. 2. It is clear that Sil-FIP can separate b- and
g-isomers, while other phases cannot separate the isomers at all.
The other isomers (a and d) are excluded here because their
separation is not difficult due to the hydrophobicity differences.
Similarly, the difficulty in the separation of b-carotene
isomers is that they differ only in the characteristics of their
Fig. 1 CD spectra of 1 at variable temperature in the presence of
cyanine dye in benzene. [1] = 1.0 mmol; [NK-77] = 0.002 mmol.
Temperature dependency curve indicates phase transition temperature.
The dashed line indicates DSC thermogram obtained in the heating
process in benzene (50 mmol) (left). Binding behavior of NK-77 in the
chiral assemblies (right).
Table 1 Separation factors (a) of PAHs and their isomers for various
stationary phases
Organic Triphenylene/ p-/m-
Naphthacene/
phases o-terphenyl Terphenyls benzo[a]anthracene (BaP/TBN)
TBN/BaP
C18
-
1.95
1.01
1.13
1.97 (0.50)
mono
C18-poly 3.87
C30 3.85
Sil-FIP 11.9
with a bending point around 55 1C and the CD intensity reduced
to 1/100 at temperatures above the bending point. This pheno-
menon can be explained by a phase transition between ordered
1.40
1.74
12.6
1.48
1.75
5.50
0.86 (1.16)
0.59 (1.69)
0.16 (6.25)
c
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hydrophobic effects and hydrogen bonding are also possible,
depending on the structure of solutes.
In conclusion, we have described the separation of some
biologically important shape-constrained isomers on Sil-FIP in
a reversed-phase mode and compared them with commercially
available hydrophobized silicas such as monomeric and poly-
meric C18, and C30 phases. The newly developed molecular gel-
based material showed very high molecular shape selectivity by a
collection of interactions, depending on the structure of the
solute molecules. Application of a functional group-rich self-
assembled molecular gel in the science of molecular separation
will open the way to the solution of many of its challenges.
Dr Abul K. Mallik (ID# P10343) highly acknowledges the
Japan Society for the Promotion of Science (JSPS) for providing
financial support to carry out this research. This work was also
supported in part by a Grant-in-Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science, and
Technology of Japan.
Fig. 2 Separation of tocopherols on (a) C18-mono, (b) C18-poly, (c) C30,
and (d) Sil-FIP phases. Mobile phase: methanol–water (9 : 1 for (a) to
(c) and 8 : 2 for (d)), column temperature 50 1C, flow rate:
1.0 ml minÀ1. UV detection: 285 nm.
Notes and references
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molecular shape. Isomers of b-carotene were obtained by
photo-irradiation based on a method in the literature.7 The
chromatograms of all-E b-carotene with C18-mono, C18-poly,
C30, and Sil-FIP after photo-irradiation show the separation
ability of Sil-FIP (see Fig. S13 and S14, ESIw). Enlarged
chromatograms of Sil-FIP clearly show that before irradiation
one peak of all-E was recorded; however, after irradiation
several isomers were separated completely within 25 min. The
peak of all-E was identified by comparing the retention time of
injected samples before and after isomerization. Other peak
assignments are based on the work of Wegmann et al.8
In order to understand the difference between Sil-FIP and
simply-hydrophobized silica, we propose the direct interaction
with the integrated carbonyl groups from the molecular gel.
We have reported that carbonyl groups in Sil-ODAn are
polarized to d+ (carbon) and dÀ (oxygen) and a carbonyl–p
interaction has been discussed.2d This interaction in a model
complex of HCHO–benzene is much stronger (1.87 kcal molÀ1
)
than a CH4–benzene interaction (0.53 kcal molÀ1) and a plane-
to-plane interaction between two benzenes (0.49 kcal molÀ1).2e
Enhancement of selectivity through a carbonyl–p interaction
has been also discussed with Sil-ODAn by the retention time
of PAHs, whose resultant selectivity was higher, especially
at lower temperatures or a crystalline state of ODAn than for
C18 columns.2c This is attributed to the fact that the aligned
carbonyl groups in Sil-ODAn are effective for enhancing
higher selectivity toward PAHs through multiple carbonyl–p
interactions. On the other hand, we have clarified the fact
that five amide bonds are integrated per molecule of 1 and
the functional groups in Sil-FIP form a two-dimensionally
condensed layer by forming hydrogen bonding among the
glutamide moieties, which makes the carbonyl groups into an
ordered form favorable for multiple carbonyl–p interactions
with the guest molecules. 13C CP/MAS NMR spectral results
showed that alkyl chains are slightly ordered at lower
temperature in Sil-FIP, which synergistically increase the
degree of ordering of carbonyl groups. Since Sil-FIP contains
long alkyl chains and NH groups, other interactions like
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c
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Chem. Commun., 2011, 47, 10341–10343 10343