Preparation and evaluation of a molecular recognition bionic solid phase extraction column for separation of glucosides

Article abstract of DOI:10.1002/jccs.201000185

Amolecular recognition bionic solid phase extraction (SPE) column for separation of glucosides has been prepared using a positively charged β-glucosylamidine as the ligand inwhich a glyconmoiety is connected via an N-glycoside linkage. β-Glucosylamidine, highly potent and selective inhibitors of β-glycosidase, is immobilized through a one-step synthesize procedure involving the addition of β-glucosylamine and 2-iminothiolane. HCl simultaneously to a matrix modified with maleimido groups via an appropriate spacer to give a molecular recognition absorbent for β-glucosides. N-octyl-β-D-glucopyranoside and β-D-galactopyranoside or α-D-mannopyranoside was directly chromatographed through the bionic chromatographic column, resulting in a much stronger retention of β-D-glucopyranoside than β-D-galactopyranoside and α-D-mannopyranoside. The retained glucopyranoside could only be eluted by glucose solution. This indicates that the binding of the glucoside was of specific nature that corresponds to the glycon substrate specificity of the glucoside. The ease of preparation and the selective nature of the molecular recognition bionic chromatography should promise a large-scale preparation of the molecular recognition adsorbent for the purification and removal of glucosides according to their glycon substrate specificity.

Full text of DOI:10.1002/jccs.201000185

1248
Journal of the Chinese Chemical Society, 2010, 57, 1248-1256
Communication
Preparation and Evaluation of a Molecular Recognition Bionic Solid Phase
Extraction Column for Separation of Glucosides
Ping-Ping Tangb (
Yun Gaob (
), Ji-Bao Caia,b* (
),
)
) and Qing-De Sub,** (
aCenter of R&D, China Tobacco Jiangxi Industrial Co. Ltd., Nanchang 330096, P. R. China
bDepartment of Chemistry, University of Science and Technology of China, Hefei, 230026, P. R. China
A molecular recognition bionic solid phase extraction (SPE) column for separation of glucosides has
been prepared using a positively charged b-glucosylamidine as the ligand in which a glycon moiety is con-
nected via an N-glycoside linkage. b-Glucosylamidine, highly potent and selective inhibitors of b-glyco-
sidase, is immobilized through a one-step synthesize procedure involving the addition of b-glucosylamine
and 2-iminothiolane. HCl simultaneously to a matrix modified with maleimido groups via an appropriate
spacer to give a molecular recognition absorbent for b-glucosides. N-octyl-b-D-glucopyranoside and
b-D-galactopyranoside or a-D-mannopyranoside was directly chromatographed through the bionic chro-
matographic column, resulting in a much stronger retention of b-D-glucopyranoside than b-D-galactopy-
ranoside and a-D-mannopyranoside. The retained glucopyranoside could only be eluted by glucose solu-
tion. This indicates that the binding of the glucoside was of specific nature that corresponds to the glycon
substrate specificity of the glucoside. The ease of preparation and the selective nature of the molecular
recognition bionic chromatography should promise a large-scale preparation of the molecular recognition
adsorbent for the purification and removal of glucosides according to their glycon substrate specificity.
Keywords: Molecular recognition; Solid phase extraction; Glucosides; Separation.
INTRODUCTION
combined with biology science and separation technology.4
The field of biochromatography employs separation tech-
niques to characterize biomolecules, such as proteins, pep-
tides, nucleic acids and carbohydrates. As an accurate, sen-
sitive, and reproducible alternative to electrophoretic
methods, biochromatography has the ability to observe bio-
molecules while retaining their biological activity (non-de-
naturation). But as it uses biomolecules as its ligand, the re-
taining of its bioactivity is a big problem during separation
process.
Chemical derivatives of O-linked glucose at C-1 can
produce glucosides. Glucoside is one of a large series of
amorphous crystalline substrates, occurring very distrib-
uted in plants, rarely in animals, and regarded as influential
agents in the formation and disposition of sugars. By the ac-
tion of ferments, or dilute acids, they break down into some
characteristic substrates (such as acid, aldehyde, alcohol,
phenole, or alkaloid) and glucose.1
Glucosides are tightly related to the chemical and
physical properties of the organisms. For example, the glu-
cosides in tea can be hydrolyzed to release volatile aroma
compounds that are important sources of tea scent.2 A re-
view by Hechman pointed out that glucosides were among
the most important flavor precursors for tobacco and
smoke.3 But until now there is no effective way to separate
glucosides from other carbohydrates.
Molecular recognition is based on selectivity and dif-
ferential integrating between biomolecules. It means re-
searching chemical and biologic combinations on molecu-
lar level that widely exist in chemical and biologic sys-
tems.5 Lai et al deeply studied the molecular recognition
theory and developed a series of analysis methods of speci-
ficity and highly selectivity that can be applied to chemical
and biological analysis of complex biological samples.6
Biochromatography was developed in 1980s. It is
* Corresponding author. Tel: +86-551-360-6642; Fax: +86-551-360-6642; E-mail: jbcai@ustc.edu.cn
** Co-corresponding author. E-mail: qdsu@ustc.edu.cn

Molecular Recognition SPE for Separation of Glucosides
J. Chin. Chem. Soc., Vol. 57, No. 6, 2010 1249
Be enlightened by this, we wonder if we can integrate
biochromatography and molecular recognition to get mo-
lecular recognition bionic chromatography. That means we
can biomimetic the molecular structure of the biomolecules
and apply them to the ligands of biochromatography. As
they are not real biomolecules, this will help us solve the
problem of bioactivity loss during separation.
tals were separated, washed with methanol, and air-dried.
The crude product was recrystallized from an equal weight
of a 1:10 mixture of concentrated ammonium hydroxide
and water, by the successive addition of 2 volumes of meth-
anol and 2 volumes of ethanol. After 48 h, the crystals were
separated, washed with methanol, air-dried and stored in
refrigerator until use.
In this paper, we report the preparation and character-
ization of a molecular recognition bionic chromatography
adsorbent for b-D-glucoside. In preparing the adsorbent, a
one-pot procedure for in-situ formation and immobiliza-
tion of b-glucosylamidine has been developed so as to tai-
lor the ligand conveniently to the glycon substrate specific-
ity of glucoside. This new method was successfully used
for the separation of glucopyranoside and the selective na-
ture of the ligand-glucoside interaction was proved.
1H NMR (300 MHz, D2O): d 4.05-4.12 (d, 1H, H-1);
d 3.80-3.90 (d, 2H, H-6, H-7); d 3.60-3.72 (dd, 1H, H-5); d
3.45-3.50 (m, 1H, H-4); d 3.32-3.40 (m, 1H, H-3); d 3.10-
3.18 (m, 1H, H-2). 13C NMR (50 MHz, D2O): d 85.21 (C-
1). Chemical shifts were uncertain to ± 0.05 ppm. All the
data was consistent with the literature data of b-glucosyl-
amine8,9,13 and a-anomer couldn’t be detected.
The calculation data for C6H13NO5 is: C, 40.2%; H,
7.31%; N, 7.82%. In our experiment, the data we found
was: C, 39.93%; H, 7.35%; N, 7.30%. Considering the ex-
periment error and a little glucose impurity that can’t be
eliminated, we can assure that the found data is consistent
with the theoretical data.
EXPERIMENTAL SECTION
Reagents and apparatus
Dimethyl sulfoxide (DMSO) and pyridine were dis-
tilled from CaH2 to remove water and stored under nitro-
gen. Other solvents were used as received with no further
treat. 2-Iminothiolane.HCl and N-(g-maleimidobutyry-
loxy) succinimide ester (GMBS) was purchased from
Pierce Chemical Co. TSKgel AF-Amino Toyopearl 650 M
was purchased from Tosoh Co., Japan. Other chemicals
were obtained from commercial sources and were of the
highest purity available. 1H NMR spectra were recorded on
a Varian VXR-300 (300 MHz) spectrometer using tetra-
methylsilane (for D2O) as an internal standard. Chemical
shifts were recorded in ppm with the internal standard at dH
= 0.00. Elemental analyses were performed on an Elemen-
tar Vario EL-III. Mass spectra were obtained on a JEOL
JMS700 spectrometer. A Shimadzu UV-1600 UV-Vis spec-
trophotometer was used for UV-Vis analysis.
As for the mass spectrum experiment, we found the
molecular ion peak at 179.0794 m/e, which is consistent
with the molecular weight.
The melting point was found as 127-128 °C which is
also consistent with the literature reported data.16
The new, crystalline b-D-glucosylamine could be kept
for short period in refrigerator. But after about a month,
some of it had been hydrolyzed. So the glucosylamine was
used freshly prepared.
Soluble form of b-D-glucosylamidine
b-D-glucosylamine (1) (179 mg, 1.0 mmol) was sus-
pended in dry pyridine (5 mL) at 0 °C. 2-iminothiolane.HCl
(138 mg, 1.0 mmol) was added to the suspension and the
mixture was stirred for 4.5 h at 0 °C under a nitrogen atmo-
sphere. The mixture gave a clear solution as the reaction
proceeded. The Ellman’ reagent (DTNB solution) was pre-
pared by dissolving 39.6 mg of 5,5¢-dithiobis (2-nitroben-
soic acid) (DTNB) in 10 mL phosphate buffer (pH = 7.0).
After the reaction had been completed, the reaction mixture
was immediately dilute with 10 mL phosphate buffer (pH =
8.0) and 1 mL of DTNB solution was added. The color de-
veloped rapidly (about 2 min). Then the absorbance at 412
nm was determined by UV-Vis spectroscopy.
b-D-Glucosylamine (1)
b-D-glucosylamine was synthesized according to the
literature.8 To 50 mL of anhydrous methanol were added 40
g of D-glucose and 0.5 g of ammonium chloride. And the
mixture was treated with ammonia gas at 0 °C until the
sugar had all dissolved. The solution was stored in refriger-
ator until satisfactory crystallization had occurred. The
first crystallization was slow, and required storage of the
solution for about a month. Subsequent crystallizations
with the aid of seed crystals were more rapid, but maximum
yield required storage for a week or two longer. The crys-
Solid support modified by GMBS (2)
TSKgel Amino TOYOPEARL 650 M (dry weight, 40
mg; wet volume, 0.2 mL; total titrable NH2 group, 20 mmol)

1250 J. Chin. Chem. Soc., Vol. 57, No. 6, 2010
Tang et al.
was suspended in dry DMSO (0.5 mL). GMBS (44.8 mg,
0.16 mmol, 8 equiv of total NH2 group) was added. The
mixture was shaken at room temperature for 10 h under ni-
trogen atmosphere. The resulting suspension was filtered,
and the gel was washed successively with water, EtOH and
hexane, and air-dried.
matography column. Firstly, we balanced the column with
citrate buffer (pH = 6.0). Secondly, we passed 2 mL of com-
plex solution of p-nitrophenyl-b-D-galacto-pyranoside
(0.1 mol/L) and n-octyl-b-D-gluco-pyranoside (0.1 mol/L)
in citrate buffer (pH = 6.0) and collected the outflow (out-
flow 1). Thirdly, we used 5 mL citrate buffer (pH = 6.0) to
wash the column and also collected the outflow (outflow
2). At last, we eluted the column with 5 mL 10% glucose-
citrate solution and collected the eluent.
The moles of the ligand were calculated from the
moles of the amino groups of the gel before and after the
modification with GMBS. The gel (dry weight, 40 mg; wet
volume, 0.2 mL) was suspended in DMSO (0.5 mL), and
p-nitropheyl acetate (0.120 g, 0.67 mmol) was added. The
mixture was shaken at room temperature for 2.5 h. Then the
reaction mixture was diluted with 50 mM Tris-HCl buffer
(pH = 8.0). The concentration of pNP was determined by
measuring the absorbance at 400 nm. As reference, the re-
actant of gel and GMBS was dealt with the same procedure.
Matrix with b-glucosylamidine as the ligand (3)
TSKgel Amino TOYOPEARL 650 M (dry weight,
400 mg; wet volume, 2 mL; total titrable NH2 group, 200
mmol) was suspended in dry DMSO (5 mL). GMBS (448
mg, 16 mmol, 8 equiv of total NH group) was added, and
the mixture was shaken at room temperature for 10 h under
nitrogen atmosphere. The resulting suspension was fil-
tered, and the gel was washed successively with water,
EtOH and hexane, and air-dried. Unreacted amino groups
were blocked by shaking the gel in a mixture of acetic an-
hydride (2 mL) and pyridine (2 mL) for 3 h. The resulting
gel (2) was filtered and washed successively with water,
EtOH and hexane. To a suspension of the resulting gel in
dry pyridine (5 mL) were added b-glucosylamine (1) (180
mg, 1 mmol) and 2-iminothiolane.HCl (138 mg, 1 mmol) at
room temperature. The mixture was shaken at room tem-
perature for 24 h under nitrogen atmosphere and filtered.
The resulting gel was washed successively with water,
EtOH and hexane. The unreacted maleimido group was
blocked by shaking the gel with 2-mercaptoethanol (0.34
mL, 5 mmol) in dry pyridine (5 mL) at room temperature
for 4 h. The resulting gel was filtered and washed succes-
sively with water, EtOH and hexane, and air-dried. This
final gel was stored in citrate buffer (pH = 6.0) until use.
Separation function of the synthesized molecular rec-
ognition bionic chromatography
Then the two outflows and the eluent were hydro-
lyzed in disodium hydrogen phosphate-citric acid buffer
(pH = 2.6) at 80 °C for 3 h respectively. Each of the hydro-
lyzed solution was equally divided into two parts. One part
was extracted with dichloromethane and then analyzed by
GC-MS. The other part was basified with 10% NaOH solu-
tion to pH 8.0 and then analyzed by UV-Vis spectrometer to
determine the absorption at 400 nm of pNP.
The same procedure was applied to the separation of
n-octyl-b-D-glucopyranoside and metyl-a-D-mannopy-
ranoside except that methanol was detected by GC-MS.
RESULTS AND DISCUSSION
Synthesis of b-glucosylamine
Ammonia can directly react with sugar to yield glu-
cosylamine (Fig. 1).
In a typical synthesis procedure, a sugar is dissolved
in an alkanol medium at a temperature of 0 °C-10 °C and
ammonia gas is introduced into the medium. The ammoni-
ated solution is maintained under cooling conditions for a
period of a month or more. Usually the glucosylamine
product crystallizes out of the alkanol medium during the
reaction period.9
Condensation of the sugar and ammonia can presum-
ably take place either through the neutral, carbonyl modifi-
cation of the sugar or through the acyclic cation
formed by acid catalysis, but the acyclic cation is far more
reactive (Fig. 2). This hypothesis accounts for the effec-
OH
H
OH
H
H
NH3
O
H
O
HO
HO
HO
NH2
OH
HO
H
OH
H
OH
We packed the white powder synthesized in the above
procedure into a stainless glass column (2 mm i.d, 5 cm
length) to get a desired molecular recognition bionic chro-
H
H
H
H
Fig. 1. Reaction of D-glucose and ammonia.

Molecular Recognition SPE for Separation of Glucosides
J. Chin. Chem. Soc., Vol. 57, No. 6, 2010 1251
Fig. 2. Mechanism of formation of glucosylamine.
peaks are totally the same. The peak at 1620-1625 cm-1 is
the characteristic peak of N-H bond bending vibration
(dN-H). The peak at 1200-1030 cm-1 is the characteristic
peak of C-N bond stretching vibration (nC-N). The stretch-
ing vibration peak of N-H (nN-H) is at about 3400 cm-1
which is overlapped by the broad peak of O-H stretching
vibration (nO-H).
tiveness of general acid catalysts (NH4Cl) in promoting
formation of glucosylamine. Condensation of the acyclic
cation with ammonia does not take place in strongly acidic
solution, because under these conditions, the nucleophilic
character of the ammonia is satisfied by combination with
hydrogen ion (ammonium salt formation). The high ammo-
nia concentration can hasten crystallization.
In the final reaction solution, the D-glucosylamine
establishes equilibrium mixtures consisting almost entirely
of the b-pyranose anomer (as the optical ratio showed). The
stability of the b-pyranose forms of D-glucosylamine pre-
sumably arises from its assuming normal chair conforma-
tion, in which all of the groups attached to the ring, except
the hydrogen atoms, lie in equatorial positions.11-12
Crude glucosylamine (contaminated with little sugar)
was used without further purification because glucose did-
n’t interfere with next synthesis steps.14
Synthesis of soluble form of b-glucosylamidine
Before immobilizing the b-glucosylamidine as the
molecular recognition ligand, a soluble form of the ligand
was synthesized to examine the immobilization method.
Since b-glucosylamidine was synthesized readily from the
corresponding b-glucosylamine and thioimidate without
protecting the hydroxyl groups,7 a one-pot procedure for in
situ formation of the b-glucosylamidine was developed by
using a cyclic thioimidate (2-iminothiolane.HCl). It is
shown in Fig. 4.
We studied the IR spectrum of the product (Fig. 3).
As we can see from Fig. 3, when using 0.5 g catalyst
and 1.0 g catalyst, the position and the intensity of the
2-Iminothiolane.HCl, also called Traut’s reagent, is a
useful reagent for chemical modification of proteins to
yield amidinated derivatives which contain sulfhydryl
groups that can be used in the next steps, for example, re-
acting with maleimido groups. It reacts with primary amines
(-NH2) to introduce sulfhydryl (-SH) groups while main-
taining charge properties similar to the original amino
Fig. 3. The IR spectrum of glucosylamine.
Fig. 4. Formation of soluble form of b-glucosylamidine.

1252 J. Chin. Chem. Soc., Vol. 57, No. 6, 2010
Tang et al.
groups.6
neutral pH 6.5-7.5 to form stable thioether bonds.15
The reaction was clear after a period of reaction. When
the reaction finished, the reactant was immediately treated
with Ellman’s reagent (DTNB) to determine the existence
of free sulfhydryl groups by UV-Vis absorbance at 412 nm
(Fig. 5).14
As for the solid support, we choose a gel with amino
groups (2) (TSKgel AF-Amino Toyopearl 650 M). Toyopearl
gel remains dimensionally stable within wide extremes of
pH and ionic strength. It can be used to couple ligands us-
ing their carboxylate groups (peptide bond formation) or
aldehyde groups (reductive amination). The gel can be
treated with GMBS to introduce maleimido groups via a
spacer of four carbon atoms in length (Fig. 6).
From the intensive absorbance at 412 nm after treated
with Ellman’s reagent, we can see that the product con-
tained sulfhydryl groups. But because the sulfhydryl groups
can be oxidated easily in air, the test should be conducted
immediately.
To calculate the modification efficiency of GMBS,
we reacted the gel before and after modification with pNPA
(p-nitrophenyl acetate) respectively, and measured the
absorbance at 400 nm of the result pNP (p-nitrophenol) to
determine the moles of amine groups on the amine gel (Fig.
7).
Modification of Amine Matrix with GMBS
The soluble form should be attached to a solid sup-
port when performing separation analysis. A “spacer arm”
which can conjugate molecules together by a covalent
bound should be used in the attachment process. GMBS is a
heterobifunctional cross-linker that contains N-hydroxy-
succinimide (NHS) ester and maleimide groups that allow
covalent conjugation of amine- and sulfhydryl-containing
molecules. NHS esters react with primary amines (in fact
on its deprotonated form) at pH 7-9 to form amide bonds,
while the maleimide reacts very specifically with sulfhy-
dryl groups (nucleophilic attack of the thiolate anion) at
From the result of UV-Vis spectrum, we can calculate
that 71.5% of the amines on the amine gel have been modi-
fied. The hydrolysis of GMBS that increases with pH and
the high dilutions of the molecule can account for the effi-
ciency of the reaction (Fig. 8).
Also, we compare the IR spectrum of GMBS and that
of the gel after modification. The wide peak at 3400-3500
cm-1 becomes stronger and wider in modified gel, which
shows that the amine group has been attached to GMBS.
Fig. 7. The reaction of amine and pNPA.
Fig. 5. Determination of sulfhydryl groups.
O
O
N
GMBS
N
H
NH2
DMSO
3
2
O
Fig. 8. The UV-Vis spectrum before and after modifi-
cation.
Fig. 6. The reaction of amine gel and GMBS.

Molecular Recognition SPE for Separation of Glucosides
J. Chin. Chem. Soc., Vol. 57, No. 6, 2010 1253
Fig. 9. The IR spectrum of GMBS and the modified gel.
And the peak at 1818 cm-1 becomes much weaker in modi-
fied gel, which shows that the NHS group of GMBS has
been eliminated by the reaction. Larger alkyl peaks of the
modified gel are due to the polymeric alkyl of the amine gel
(Fig. 9).
The resulting gel 3 was suspended in dry pyridine and
was treated with an excess amount of b-glucosylamine (1)
and 2-iminothiolane.HCl simultaneously to immobilize the
b-glucosylamidine as a ligand to yield the molecular recog-
nition adsorbent 4 (Fig. 10).
In conclusion, we can affirm that GMBS has been at-
tached to the amine gel through peptide bond formation re-
action.
Aspacer arm between the ligand and the solid support
is highly important in reducing the unfavorable steric inter-
action between the glucoside and the matrix.
The synthesized glucosylamidine is stable in acidic
media.7 The stability of the glucosylamidine probably co-
mes from the fact that the highly basic amidine is com-
pletely protonated under acidic conditions, thereby pre-
Synthesis of molecular recognition bionic chromatog-
raphy with b-glucosylamidine as the ligand
If the modified gel, 2-iminothiolane.HCl and b-glu-
cosylamine were all mixed together at once, the separation
functional groups could be easily attached to the solid sup-
port in one pot.
The block of the unreacted amino groups of the
TSKgel was very important for the one-pot procedure. Be-
cause 2-iminothiolane.HCl could also react with the amino
groups on the gel. So before add of 2-iminothiolane.HCl,
the unreacted amino groups of the matrix were blocked
with excessive acetic anhydride.
Fig. 11. The FTIR spectrum of the molecular recogni-
tion adsorbent.
Fig. 10. One-pot synthesis procedure of molecular rec-
ognition biochromatography.

1254 J. Chin. Chem. Soc., Vol. 57, No. 6, 2010
Tang et al.
venting the acid-catalyzed opening of the pyranose ring as
would easily occur with the parent glucosylamine. The mo-
lecular recognition adsorbent was washed with 0.1 M ci-
trate buffer (pH = 6.0) and suspended and stored in the
same buffer.
trate solution. The surplus 88% n-octyl-b-glucopyranoside
was eluted (GC-MS data not show).
Through separation analysis, we could see that most
of the p-nitrophenyl-b-D-galactopyranoside passed through
the column without retention. But most of the n-octyl-b-
D-glucopyranoside was retained by the column and could-
n’t be washed down by the citrate buffer. The retained b-
D-glucopyranoside could just be eluted by stronger condi-
tions such as glucose solution.
We analyzed the white powder of molecular recogni-
tion adsorbent with FTIR spectroscopy (Fig. 11).
As compared to the IR spectrometry of gel 3 (modi-
fied gel), we can clearly see that the three characteristic
peaks of maleimido ring have disappeared. It means that
the maleimido ring has disappeared by reacting with sulf-
hydryl group.
We also applied the molecular recognition bionic
chromatography column to the separation of n-octyl-b-D-
glucopyranoside and metyl-a-D-mannopyranoside. With
the same procedure, we could get almost the same result.
95% of the metyl-a-D-mannopyranoside passed through
the column without retention and just 8% of the n-octyl-b-
D-glucopyranoside passed through the column without
retention.
Separation function of the molecular recognition
bionic chromatography
To determine the separation function of the molecular
recognition adsorbent, first of all, we passed a mixture so-
lution of n-octanol and n-octyl-b-glucopyranoside in ci-
trate buffer (pH = 6.0) through the biochromatography col-
umn. As the GC-MS result showed, all the n-octanol passed
through the column without retention and most of the n-
octyl-b-glucopyranoside was retained by the adsorbent
(data not show).
These results suggest that the b-glucosylamidine
served as a substrate analog, where the glycon moiety was
primarily recognized as substrates by the glucoside. These
To determine the substrate recognition of the adsor-
bent, then, we applied it to the separation of n-octyl-b-glu-
copyranoside and p-nitrophenyl-b-D-galactopyranoside
(Fig. 12).
After hydrolyzing outflow 1 and outflow 2 and com-
paring the absorption at 400 nm, we could see that 83% of
the p-nitrophenyl-b-D-galactopyranoside had passed through
the column without retention (Fig. 13). By GC-MS analysis
of n-octanol, we can see that only 12% of the n-octyl-b-D-
glucopyranoside passed through the column. By experi-
menting GC-MS analysis of the hydrolyte of outflow 2, we
can see that no n-octyl-b-glucopyranoside is washed down
by citrate buffer (pH = 6.0).
Fig. 13. UV-Vis spectra of outflow 1 and outflow 2.
At last, we eluted the column with 10% glucose-ci-
Fig. 12. n-Octyl-b-glucopyranoside and p-nitrophenyl-b-D-galactopyranoside.

Molecular Recognition SPE for Separation of Glucosides
J. Chin. Chem. Soc., Vol. 57, No. 6, 2010 1255
characteristics are suitable for a molecular recognition
ligand for separating the glucosides according to their
glycon substrate specificities.
the glucoside. This adsorptive characteristic reflected the
fact that the b-glucosylamidine serves as an substrate ana-
log, and the analog strongly interacting with the glucoside
by the presence of a positive charge near the anomeric car-
bon to mimic the oxocarbenium ion intermediate of en-
zyme reaction.7 Hence the b-glucosylamidine ligand suc-
cessfully recruited the binding energy of the glucoside to-
wards the ligand to achieve selective binding to the corre-
sponding glucosides. This property, along with the ease of
synthesis, should promise useful applications not only for
purifying a target glucoside, but also for remove an unde-
sirable contaminating glucoside from the sample, based on
the glycon substrate specificities.
A question may arise here whether the glucoside was
separated by ion-exchange effects of the adsorbent, be-
cause the ligand is permanently and positively charged at
the pH used for the chromatography. But most of the galac-
topyranoside and mannopyranoside were not retained by
the chromatography. This adsorptive characteristic clearly
indicates that the glucopyranoside is bond to the ligand
with specific interaction between the glycon moiety of the
ligand and the glucopyranoside. A little amount of galacto-
pyranoside and mannopyranoside were also retained by the
adsorbent. It may be because of the weak interaction result-
ing from the similarity of the glucose pyranose and galac-
tose pyranose or mannose pyranose. This can also explain
the fact that the a-mannopyranoside was retained less than
b-galactopyranoside by the chromatography.
Many other molecular recognition bionic chromatog-
raphy adsorbents can be readily synthesized by choosing
appropriate carbohydrates added at the last stage of the
synthesis. Since the b-glycosylamidine was formed di-
rectly from the corresponding b-glucosylamine, this method
is applicable, in principle, to any glycosylamidine ligand
including those derived from oligosaccharides, provided
the precursor glycosylamines are available.16 Neither the
isolation of the intermediate b-glycosylamidine nor the
protection of the hydroxyl and the amido groups was nec-
essary for the immobilization.
The adsorptive characteristics of the molecular rec-
ognition bionic chromatography are affected by several
factors such as the steric interaction between the glucoside
and the matrix, the accessible surface area, the particle size
and the ability of the solute to diffuse in and out of the
microporous environment.16-17 We suppose that the bond
between b-glucosylamidine and b-glucoside may be hy-
drogen bond, static electronic bond, hydrophobic reaction,
van der Waals force or some other kind of weak bond. The
mechanism is still under research.
ACKNOWLEDGEMENT
This work was completed in National Synchrotron
Radiation Laboratory of China and China Tobacco Jiangxi
Industrial Co. Ltd. The authors thank to National Natural
Science Foundation of China (No.20405013) for the finan-
cial support.
But in our experiment, the column could just be used
for one time of separation experiment if it was eluted by
glucose solution. After it was eluted by glucose solution,
the separation function of the molecular recognition bionic
chromatography column decreased dramatically. We sup-
pose the reason is that after the column was washed with
glucose solution, the function spots of the molecular recog-
nition bionic chromatography were blocked by glucoses.
And until now, we haven’t found an efficient way to wash
these glucoses down and reproduce the separation function
of the column. As the expense for the column is large, this
is a big disadvantage of this method.
Received July 28, 2009.
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