Frucooligosaccharides purification: Complexing simple sugars with phenylboronic acid

Article abstract of DOI:10.1016/j.foodchem.2019.01.130

Prebiotic fructooligosaccharides (FOS) are currently obtained by enzymatic reaction with fructosyltransferases (FTFs) using sucrose as both donor and acceptor. In these reactions glucose results as the most abundant by-product, arising from each fructosyl transfer event and, together with fructose, because of the inherent hydrolytic activity of the FTFs. As FOS are mainly used as prebiotic in nutraceutical foods, the reduction or total elimination of monosaccharides is required. In this work the selective elimination of monosaccharides from a synthetic FOS mixture was achieved through the selective complexation of glucose and fructose with phenyl boronic acid (PBAc) followed by ethyl-acetate extraction. The process was applied to a complex mixture of FOS obtained in an enzymatic synthesis reaction containing 40% glucose, 15.8% fructose and 35% of FOS, elimination of the sugars was achieved through 3:1 molar reactions, resulting in a levan-type FOS product with 97% purity.

Full text of DOI:10.1016/j.foodchem.2019.01.130

Accepted Manuscript
Frucooligosaccharides purification: complexing simple sugars with phenylbor-
onic acid
R. Porras-Domínguez Jaime, Rodríguez-Alegría María Elena, Miranda Alfonso,
Álvarez-Berber Laura Patricia, Castillo Edmundo, López Munguía Agustín
PII:
S0308-8146(19)30205-5
DOI:
https://doi.org/10.1016/j.foodchem.2019.01.130
FOCH 24225
Reference:
Food Chemistry
To appear in:
Received Date:
Accepted Date:
9 October 2018
23 January 2019
Please cite this article as: Jaime, R.P., Elena, R.M., Alfonso, M., Patricia, A.L., Edmundo, C., Agustín, L.M.,
Frucooligosaccharides purification: complexing simple sugars with phenylboronic acid, Food Chemistry (2019),
doi: https://doi.org/10.1016/j.foodchem.2019.01.130
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Frucooligosaccharides purification: complexing simple
sugars with phenylboronic acid.
Porras-Domínguez Jaime R.1, Rodríguez-Alegría María Elena1, Miranda Alfonso1,
Álvarez-Berber Laura Patricia2, Castillo Edmundo1 & López Munguía Agustín1 (*).
1Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad
Nacional Autónoma de México. Av. Universidad 2001, Col. Chamilpa, C. P. 62210,
Cuernavaca, Morelos, México.
2Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos. Av.
Universidad 2001, Col. Chamilpa, C.P. 62210, Cuernavaca, Morelos, México
* Corresponding author (agustin@ibt.unam.mx).
Key words: phenyboronic acid, fructooligosacarides, fructans, levans, prebiotics,
sugar purification.
Abstract
Prebiotic fructooligosaccharides (FOS) are currently obtained by enzymatic
reaction with fructosyltransferases (FTFs) using sucrose as both donor and
acceptor. In these reactions glucose results as the most abundant by-product,
arising from each fructosyl transfer event and, together with fructose, because of the
inherent hydrolytic activity of the FTFs. As FOS are mainly used as prebiotic in
nutraceutical foods, the reduction or total elimination of monosaccharides is
required. In this work the selective elimination of monosaccharides from a synthetic
mixture FOS was achieved through the selective complexation of glucose and
fructose with phenyl boronic acid (PBA) followed by ethyl-acetate extraction. The
process was applied to a complex mixture of FOS obtained in an enzymatic
synthesis reaction containing 40 % glucose, 15.8 % fructose and 35% of FOS,

elimination of the sugars was achieved through 3: 1 molar reactions, resulting in a
levan-type FOS product with 97% purity.
1. Introduction
The use of fructooligosaccharides (FOS) as nutraceuticals has become an
important trend in food science due to their prebiotic properties. Indeed, their
application in the direct regulation of the human gut microbiota, the treatment and
prevention of several diseases and the general well-being associated with their
regular consumption have been extensively demonstrated (Ritsema & Smeekens,
2003). Evidence of these properties have been established through different in vitro
and in vivo experimental approaches (M. Roberfroid, 2007), as well as the modern -
omic techniques: transcriptomics, proteomics and genomics applied under a wide
variety of experimental conditions (Candela, Maccaferri, Turroni, Carnevali, &
Brigidi, 2010). Consequently, different strategies have been developed for the
extraction and hydrolysis of plant inulins as well as for the direct synthesis of FOS of
varied structure and molecular weight, through biocatalytic strategies (ref). Actually,
the most popular industrial procedure adopted for FOS production is through
enzymatic hydrolysis of inulins extracted from vegetal sources, mainly chicory and
dahlia roots, using endo-inulinases (Sangeetha, Ramesh, & Prapulla, 2005).
The most abundant products of these reactions are FOS, also known as
oligofructosides or oligofructose. Due to the nature of the hydrolytic reaction and
considering the intrinsic inulin structure, FOS are characterized by a reducing
fructosyl end and the absence of a terminal glucose. Actually, inulins are mainly

linear polydisperse polymers of fructose consisting of β(2–1) fructosyl–fructose
linkages with usually a glucose moiety linked to the end of the chain by an α1–2
bond as in sucrose (M. Roberfroid, 2007, Nollet, L. M., & Toldrá, F, 2012).
As these types of processes are carried out with inulin as substrate, depending on
the nature and specificity of the inulinase preparation, either endo- or, to a minor
extent, exo-inulinases, small quantities of fructose are obtained as by-product. This
is also the case of chemical hydrolysis of inulin using organic or mineral acids, strong
acid cation resins, zeolites or activated carbon as catalysts. Nevertheless, all these
processes inevitably result in a certain amount of fructose contaminating the
prebiotic FOS mixture (Sangeetha et al., 2005).
In contrast, synthesis of FOS may also be based on fungal FTFs synthesis, through
transfer reactions using sucrose as substrate (M. B. Roberfroid, 2000; Sangeetha et
al., 2005). The products of this kind of synthesis are mainly non-reducing-FOS with
a terminal glucose molecule in one end (GFn) and a (n-1) fructose engaged in a C2-
OH glycosidic linkage with the last fructose in the other (C1-OH or C6-OH linkage).
This is the case of 1- and 6-kestose, 1 and 6-nystose, 1 and 6 f-nystose), etc.,
corresponding to inulin or levan-type FOS respectively, among others. Nevertheless,
and important feature in this type of synthesis is the presence of monosaccharides
derived from sucrose hydrolysis, but also the important amount of glucose released
as an intrinsic consequence of the FTFs mechanism, each time a sucrose molecule
acts as a fructosyl donor. Inevitably, the final FOS mixture produced from FTFs
contains glucose and fructose as by-products, which, due to their unselective effect
on the hostage microbiota, require elimination from FOS when applied as prebiotics.

Methods to separate oligosaccharides from mixtures containing
monosaccharides based on ultrafiltration and nanofiltration membranes have been
reported. However, in this type of processes, are expensive and efficient separations
are achieved only when low concentrations of monosaccharides are present (e.g.
less than 5% w/v) otherwise, low productivity and/or large membrane surfaces are
required (Kamada, Nakajima, Nabetani, Saglam, & Iwamoto, 2002).
An alternative available in the food industry is ion-exchange chromatography,
commonly described in the production of high fructose corn syrups, to reduce the
glucose content of glucose-fructose mixtures, among other processes. The physical-
chemical basis of this separation is not exclusively ion-exchange, but rather the
formation of a complex between metallic cations and hydroxyl groups of the
carbohydrate molecule in a specific orientation, the stability of the complexes
depending on the hydroxyl group orientation and the nature of the cation. The resins
used to separate monosaccharides are charged with s-metal cations such as Na+
and Ca2+. The complexes formed require 3 hydroxyl groups oriented in cis position.
However, the stability constants are found in the 0.1-6.0 range, which are relatively
low for an efficient separation (Alekseev, Garnovskii, & Zhdanov, 2007).
Consequently, separation of glucose and fructose in a Ca2+ ions chromatography
require very large columns and several steps of elution.
Boron and boronic compounds can also form complexes with carbohydrates. In
fact, these compounds behave as Lewis acids due to their electron deficiency nature
at the central boron atom possessing a vacant -orbital, where the hydroxyl groups
of sugars may act as electron pair donors (Sivaev & Bregadze, 2014).

The specificity of this kind of compounds is derived from the fact that the distance
between the two hydroxyl groups in boron acids is equivalent to the distance
between two vicinal hydroxyl groups of the carbohydrate in cis- position. A
description of how sugars containing hydroxyl groups in cis- orientation may react
with boron acids to obtain stables complexes has been described in detail (Hall,
2006).The equilibrium constants between boron acids with glucose and fructose are
found in the range of 10-1 – 102 M-1 in water at pH 5 to 8.5, which are higher than
those reported for sugars and s-metal cations, suggesting a covalent character of
the interaction between these compounds (Alekseev et al., 2007). In fact, several
procedures to detect glucose involving colorimetric techniques and based on
reactions with boron acids have been developed (Wu et al., 2013).
Despite the numerous reports dealing with boron complex formation with
carbohydrates, the technical feasibility for FOS or saccharides purification from
complex sugars mixtures using the boron complex approach has not been studied.
In this paper, we propose a new method to purify FOS starting with a levan-type FOS
mixture produced through a combination of levansucrase and endolevanase
reactions from sucrose, as recently reported (Porras J et al, 2017), where the FOS
product (35% w/v), contains a high concentration of glucose (40% w/v) and fructose
(15% w/v). The process consists in the separation of fructose and glucose through
the selective formation of non-soluble phenyl boronic acid complexes, easily
extracted from the reaction bulk to render a FOS solution with high purity and low
monosaccharides content suitable for consumption as prebiotic.

2.- MATERIALS AND METHODS
2.1 Expression and purification of fusion enzyme LevB1SacB. The Escherichia
coli Rosetta 2 strain was transformed with the plasmid pet22b+levb1sacb containing
a fusion gene coding for the fusion enzyme LevB1SacB, this containing both
levansucrase and endolevanase activities designed for the synthesis of levan type
FOS (Porras et al 2017). 50 ml of Luria Bertani medium containing triptone 10g/L
(Bacto ™) , yeast extract (5 g/L Bacto ™,), NaCl 10g/L (J.T. BAKER® A.C.S) and
ampicillin 200 μg/mL (Sigma Aldrich,), were inoculated with a single transformed
colony and incubated for 18 h at 37°C and 200 rpm. The 50 ml served as inoculum
of 2 L Luria Bertani medium with 200 μg/mL ampicillin distributed in two 2.5 L Fern
Bach flasks. The cultures were incubated at 37°C and 200 rpm until an OD of 0.5
was reached (8h approximately) then the culture was induced with 0.4 mM IPTG
(Gold Biotechnology, USA) and incubated for additional 18 h at 18°C and 200 rpm.
Cells were then harvested by centrifugation at 8000 rpm for 20 min and washed
twice with 50 mM, pH 6.0 potassium phosphate buffer (J.T. BAKER® A.C.S.
Reagent), sonicated in an Ultrasonic Processor (Cole-Parmer,) with 3 pulses of 15
s with an amplitude of 70% and finally centrifuged at 9000 rpm for 30 min. The
enzyme was recovered in the supernatant and purified by affinity chromatography in
His-Trap columns (His-Trap 5 mL,) charged with nickel sulfate (J.T. BAKER®
A.C.S.,). The enzyme was eluted in an ÄKTA Protein Purification Systems, GE
Healthcare Life Science, using an imidazole (J.T. BAKER® A.C.S.) pH 6.5 buffer.

The pure enzyme fraction was dialyzed in dialysis bags with a 10 kDa molecular
weight cut off against a 50 mM, pH 6.0 acetate sodium buffer, prepared with acetic
acid (J.T. BAKER® A.C.S.,) and sodium acetate (J.T. BAKER® A.C.S.,) with a 1 mM
of CaCl2 (J.T. BAKER® A.C.S.). Finally, the levansucrase activity of the fusion
enzyme was measured following the release of reducing sugars from sucrose
through the DNS method as previously reported (Porras-Domínguez, Rodríguez-
Alegría, Ávila-Fernández, Montiel-Salgado, & López-Munguía, 2017).
2.2 Synthesis of levan-type FOS. The standard conditions for the FOS synthesis
were carried out in 50 ml volume reactors containing 7 U/ml LevB1SacB activity and
60% w/v sucrose as substrate (Sigma Aldrich,) in pH 6.0 acetate sodium buffer
containing 1 mM CaCl2 (J.T. BAKER® A.C.S). The reaction medium was incubated
at 37°C and 300 rpm and monitoring for glucose, fructose and sucrose every hour
by HPLC (6h or 90% conversion). The reaction was stopped by thermal inactivation,
boiling the samples in a water bath for 10min.
2.3 Measurement of sugar concentration. The concentration of glucose, fructose
and sucrose was measured by HPLC (Waters ™) in an aminated column (Prevail,
Grace®, USA), with acetonitrile (J.T. Baker® HPLC-9012) / water 75:25 v/v, as mobile
phase at a flow of 1.0 ml/min at 30°C, equipped with a refraction index detector.
Glucose, fructose and sucrose (Sigma Aldrich,) were used as standards. Levan type
FOS were measured as fructose equivalents after complete hydrolysis. For this
purpose, 6 g/ml of total FOS samples in 50 mM pH 6.0 acetate buffer at 50°C were
treated with 60 U/ml of Fructozyme (Novozyme). The reactions were stopped by

incubation in a boiling water bath for 10 minutes and fructose measured as
described. FOS concentration is reported as fructose equivalents, considering that
the molar mass of fructose incorporated in oligosaccharides correspond to 162
g/mol.
2.4 Levan type FOS purification.
In order to reduce the simple sugars content of the FOS solution, residual glucose
and fructose were complexed with phenylboronic acid (PBAc). Reaction conditions
were explored in order to obtain an efficient separation.
2.4.1 Effect of phenylboronic acid (PBAc) equivalents. A volume of 5 ml of
ethanol was added to 5 ml of the FOS samples with a given glucose and/or fructose
content as specified in the next section. 1, 2 or 3 PBAc molar equivalents (Sigma
Aldrich,) per sugar molar were added to the FOS mixture. PBAc added with
continuous stirring until complete dissolution.
The pH was then adjusted to 7.0 with sodium hydroxide, and the solution evaporated
in a rotatory evaporator first at 60°C under a 400 mmHg vacuum to evaporate
ethanol, followed by an additional treatment at 70°C under 80 mmHg until complete
dryness. The white powder obtained was washed with 10 volumes of ethyl acetate
in a Büchner flask to eliminate the phenylboronic-monosaccharides complexes.
Finally, the purified FOS obtained as powder were stored at room temperature.
2.4.2 Solvent effect. Alternative solvents were explored for the complex extraction:
the procedure described in section 2.4.1 was repeated with acetonitrile or methanol

instead of ethanol, in extractions using 2.5 equivalents of PBAc per reducing sugar
equivalent.
2.4.3 Monosaccharides extraction kinetics. The monosaccharides extraction
kinetics was studied repeating the procedure described in section 2.4.1 using 2.5
equivalents of PBAc with incubation at room temperature, measuring the extraction
reached after 0, 30, 60, 90 and 120 min of reaction after PBAc addition and pH
adjustment. After each complexation time, the levan-type FOS purification procedure
described above was applied.
2.4.4 Synthesis of glucose- and fructose- PBAc complexes. The procedure
described in section 2.4.1 was applied to a 5ml standard solutions containing 60%
w/v glucose or fructose in 50mM pH 6.0 acetate buffer to synthesize the phenyl
boronic-glucose and phenyl boronic-fructose complexes 3 PBAc equivalents were
added to monosaccharides solutions. The PBA-monosaccharides complexes were
extracted with 10 volumes of ethyl-acetate- The extraction solution was evaporated
in a rotatory evaporator under 400 mmHg vacuum at 50°C to concentrate the
monosaccharide complexes and residual PBAc. The concentrated solution was kept
in ice to crystalize the residual PBAc and removed later by filtration. The filtrate was
further evaporated until complete dryness in the rotatory evaporator under 400
mmHg and 50°C. A sample of 100mg of each complex was used for RMN 1H & 13C
spectroscopy analysis.
2.4.5 Thin Layer chromatography. Complexation reaction products were also
analyzed by Thin Layer Chromatography in silica gel Plates 60 (MERCK) as the
stationary phase and an ethanol:butanol:water (15:9:6) v/v mixture as the mobile

phase. Plates were eluted twice in the mobile phase and revealed with α-naftol
solution and heat treatment.
2.4.6 Binding analysis of glucose and fructose with phenyl boronic acid via
NMR Spectrometry.
NMR spectra were acquired on a Varian Unity NMR Spectrometers operating at 500
1
13
MHz for H and 175 MHz for C nuclei, respectively. Chemical shifts are listed in
parts per million (ppm), referenced to CHCl3 and were made based on 1H–1H COSY,
HSQC, and HMBC spectral analyses as required. All coupling constants are given
as numerical values.
Analytical:1H-NMR (500 MHz, CDCl3): δ (ppm) 8.31 – 7.20 (aromatic protons of
complexed PBAC), 5.14 (dd, 1H, J=2.5 Hz, J=9.1 Hz, H-4), 4.88 (d, 1H, J=2.5 Hz,
H-3), 4.72 (dd, 1H, J=1.75 Hz, J=8.25 Hz, H-5), 4.0 (d, 1H, J=13.5 Hz, H-1a), 3.88
(d, 1H, J=12.0 Hz, H-1b), 3.80 (dd, 1H, J=2.0 Hz, J=14.0 Hz, H-6a), 3.71(d, 1H,
J=12.0 Hz, H-6b). 13CNMR (175 MHz, CDCl3): δ (ppm) 140-125 (aromatic carbons
of complexed PBAC), C-2 104.55, C-4 86.47, C-5 75.04, C-3 70.86, C-1 64.15, C-6
61.91.
1D- and 2D-NMR spectroscopic measurements were performed to analyze the
binding of monosaccharides D-glucose and D-fructose to PBAC. We used CDCl3 as
solvent for experiments under neutral non-aqueous conditions. Depending on the
saccharide under investigation different binding complexes between the
phenylboronic acid and monosaccharides were proposed. The structures were

deduced by comparing the chemical shifts and coupling constants of the boronic
acid-glucose or fructose complexes with those described.
By mixing the ligand and monosaccharides in a 3:1 stoichiometric ratio, the 1H NMR
spectra of the solution showed signals from complexed sugars ( 3.7 -  4.5 for D-
glucose and  3.7-  5.5 ppm for D-fructose). Furthermore, different peaks could be
detected which were shifted downfield. Signals corresponding to free fructose or
glucose did not appear.

3.- Results
3.1 FOS synthesis and by-products
As in all FOS production processes where FTFs are involved using
sucrose as substrate, glucose accumulates as the main by-product besides fructose
and residual sucrose. Although there are no toxic consequences derived from these
sugars, their presence in the product is not convenient considering that their final
destination is the intestinal microbiota, where only soluble fiber and FOS favor the
establishment of a beneficial microbiota and the overall gastric health. Considering
that sucrose is used both as fructosyl donor and acceptor, one free glucose molecule
will be obtained for each fructosyl unit incorporated to FOS. We have previously
described the simultaneous levan synthesis/hydrolysis reaction involving the use of
a levansucrase and a endolevanase for the production of a new levan-type FOS
series from sucrose, where an inconvenient amount of monosaccharides are
obtained as by products (Porras-Domínguez et al., 2017). In order to design a
process for FOS purification, reaction conditions were stablished to ensure a
systematic content of glucose, fructose and residual sucrose in the product reaction
batches (M & M section 2.2). In this context, 1.7L reactors were employed for the
synthesis of levan-type FOS, with a composition consisting of 35 % (w/w) of levan-
type FOS with potential prebiotic properties, but also a simple sugars content
including 40 %(w/w) glucose, 15 % (w/w) fructose and 10% (w/w) residual sucrose,
as summarized in Table 1. This composition results from a compromise between the
amount of residual sugars (glucose and fructose), sucrose conversion, and FOS
diversity in the final product distribution. The larger the reaction time, the larger the

conversion of sucrose, but also the larger the reduction of FOS average molecular
weight due to endolevanase hydrolysis.
This preparation was the basis to develop a specific FOS purification procedure
eliminating glucose and fructose, through a phenyl boronic acid complexation
extraction.
3.2 Extraction stoichiometry.
The first step in the design of the extraction process was to define the required molar
rate of PBAc for the removal of both sugars. To address this question a set of
experiments was designed using molar ratios from 1:1 to 3:1 PBA: glucose &
fructose in the reaction medium (Figure 1). The 3:1 molar ratio is equivalent to 6 M
PBAc for the 2 M glucose & fructose present in the FOS solution (1333 mM and
526 mM of glucose & fructose respectively). The experiments were carried out
following the procedure described in section 2.2 in Materials and Methods. In Figure
1 the distribution and relative amount of the main components of the FOS solution
are shown before and after treatment with the different PBAc: sugar ratios. A control
experiment is included, for an extraction in the absence of PBAc. In all the
extractions no significant change in FOS concentration was observed, suggesting
that PBAc does not interact with FOS. In all experiments, glucose content (1333 mM)
was substantially reduced, particularly at high molar PBAc ratios. Although with a

different performance, it is important to point out that fructose concentration is also
reduced. When looking at the results in detail, it is clear that equimolar amounts of
PBAc and sugars (1 PBAc eq per reducing sugar equivalent) was not enough for an
efficient removal of all monosaccharides. If a total removal is the objective in a
reasonable time, then 3 PBAc equivalents are the best option. Moreover, it seems
that it exists a particular selectivity for the different sugars or FOS present in the
mixtures. Actually, from Figure 1, glucose was preferentially removed at all
PBAc:sugars ratios evaluated, followed by fructose, which is removed when PBAc:
sugars ratio is higher than 2:1;finally sucrose was removed only at the highest
PBAc:sugars ratio. Therefore, all three sugars were almost completely removed at
the 3:1 PBAc:sugars ratio.
This behavior may be explained by the set of different factors that influence the
removal of sugars in solution: the PBAc-sugar equilibrium constant together with the
individual concentration of each sugar and the equilibria of the different isomeric
forms of the sugars in solution.
The multiple equilibrium PBAc-sugar forms present in solution have already been
reported (Springsteen & Wang, 2002). These authors demonstrate the higher affinity
of PBAc to form complexes with fructose than with glucose. In fact, the PBAc-
fructose equilibrium formation constant is forty times higher (Keq = 160 M-1) than the
PBAc- glucose constant (Keq = 4.6 M-1). Therefore, and in contrast to the results we
have just described, complexes should be preferentially formed with fructose.
However, glucose concentration in the FOS mixture is almost 4 times higher than

fructose, resulting in a preferential PBAc-glucose complex formation, even at low
PBAc:sugars equivalents, particularly in ethanol/water 50:50 pH 7.0, where reduced
differences between the equilibrium constants of PBAc with the two sugars have
been reported. In fact, in Figure 1 it may be observed that fructose is removed once
glucose concentration has been substantially reduced, at PBA/sugar equivalent
ratios higher than 1.5.
In terms of the equilibria of the different structural isomeric forms of sugars in
solution,
The formation of PBAc complexes with glucose, fructose and sucrose is mainly
determined by the presence of cis-vicinal hydroxyl groups (Figure 2). Hence, there
is an interaction of PBAc with the two vicinal hydroxyl groups of C1 and C2 positioned
in cis orientation in the α-D glucopyranose configuration of glucose. Nevertheless,
several alternative configurations of PBAc:glucose complexes have been proposed.
The α-glucofuranose is a glucose conformer whose equilibrium proportion in water
solution is less than 1%. Nevertheless, a recent report describes the formation of
complexes, with 2 PBAc molecules in the hydroxyl groups in C5 - C6, and C1 - C2
(Wu et al., 2013), explaining the PBAc molar excess requirement for total glucose
removal.
The PBAc selectivity for glucose and fructose in the FOS mixture is explained by the
fact that in the structure of levan type FOS, oligofructosides composed mainly of β-
fructofuranose conformers, the most abundant oligosaccharides, have no hydroxyl
groups in cis configuration, with the C2 compromised in the glycosidic bond with the
C6 hydroxyl group of the next fructose molecule. In the case of free fructose, an

equilibrium of 4 different conformers exists in solution, where β-fructopyranose, is
the preferential isomer form (70% in water solutions) wherein pairs of hydroxyl
groups (C4- C5, as well as C2- C3) may complex with PBA; an additional isomer, β-
fructofuranose (25% in water solutions) may also form a complex with PBAc with its
C2-C3 hydroxyl groups. In the case of the less abundant conformers, while α or 
fructopyranose may react with their C4-C5 hydroxyls, α–fructofuranose does not
form complexes, but its concentration may be reduced as a consequence of the
overall isomers equilibrium during the complexation reaction. In spite of the 4
possible interactions between fructose and PBAc, only the β-fructopyranose linked
through the C4- C5 hydroxyl groups has been reported in reactions between fructose
and boronic acids (Kim et al., 2008).
In conclusion, the multiple equilibrium sugar isomers and sugar isomers-PBAc forms
in the reaction between PBAc and sugars substantially explain why a large molar
excess of PBAc is required to remove all monosaccharides. A Thin layer
chromatography plate comparing the FOS distribution before and after the extraction
with 3 PBAc equivalents and the recovered sugars demonstrates the efficiency of
the process (Figure 3).
3.3 Extraction optimization.
The initial experiments were performed assuming an immediate interaction between
PBAc and sugars to reach equilibrium. We therefore explored the evolution of the
extraction profile with extraction time as described in M&M section 2.2.2. It was

verified that the complexation is instantaneous as no differences were found in the
amount of glucose and fructose removed after mixing or after 120 min of interaction.
We may conclude that the reaction equilibrium is reached almost immediately with
a concomitant decay in pH as previously reported for reactions carried out in
aqueous systems (Springsteen & Wang, 2002). We also explored alternative
solvents for the reaction. Two additional solvents were selected: acetonitrile and
methanol. For each alternative solvent the extent of monosaccharides removed was
quantified. Although ethanol has a hydroxyl group, it is not able to react with PBAc
due to the absence of 2 hydroxyl groups in cis position, separated by a C-C bond
distance, a requirement already discussed (Hall, 2006). This explains the similar
results found for the extractions carried out with both methanol and ethanol
(supplementary material). Finally, no differences in monosaccharides efficiency
removal were found neither in acetonitrile, a solvent with a similar polarity as ethanol,
where PBAc is soluble (supplementary material). In conclusion, all three solvents
are suitable for the extraction process.
3.4 RMN analysis and structures.
In order to clearly define the major complexes structure between glucose, fructose
and PBAc among all possible structures described, fructose, glucose and PBAc
complexes were synthetized and extracted as described in M&M, section 2.4.4, for
RMN 1H and 13C analysis.
The binding of fructose to boronic acids has been reported both in neutral non-
aqueous and alkaline aqueous solutions. Extensive NMR experiments have shown
that furanose binds with boronic acids by the β-D-fructofuranose and β-D-

fructopyranose forms. In alkaline aqueous solution seven different complexes were
observed; while, under non-aqueous conditions, signals emerged from 13 different
compounds. The number of isomers obtained and the anomers involved depend on
the ratio between fructose and the boronic acid concentration (Norrild & Eggert,
1996). In our case, only one complex fructose-phenylboronic acid was observed
1
according with the following evidence: H NMR spectrum of PBAC-fructose showed
seven signals in the saccharide region, three of them shifted downfield. The signals
in the aromatic region of proton spectrum indicated also the presence of complexed
PBAc. (Figure 4a). The J-coupling were particularly useful to distinguish the
conformation of the D-fructose moiety. By comparing the proposed binding
complexes with the different anomers present during the mutarotation of fructose,
1
signals corresponding to only one complex appeared in the H NMR and 13C NMR
spectra. The data obtained provides evidence that the phenylboronic acid was
complexed to the β-D-pyranose form of fructose, which exists in aqueous solution at
around 75% (Norrild & Eggert, 1996). We deduce an 1,2:4,5-α-D-fructopyranose
complex with PBAC on the basis of a contemporary NMR study and reports data
(Figure 4b) (Nicholls & Paul,2004.; Wood, 1974 ). 13C chemical shifts for the boronic
acid part of the complexes are given in the Experimental Section. The chemical shift
assignments were in agreement with information from 2D NMR spectra obtained for
the complex studied. β-D-fructopyranose binds favorably to boronic acids due to the
preponderant presence of syn-periplanar hydroxyl groups in this anomer.
As far as glucose interactions with PBAc, the situation is more complex. The 1H NMR
and 13C NMR spectra of PBAC and D-glucose showed chemical shifts for the sugar

part of the complex. In general, the results show that protons within the saccharide
are more deshelled after binding to PBAc. Nevertheless, for D-glucose a readily
interpretation of the spectra was not possible since the measurement of coupling
constants, which are necessary for a further analysis, cannot be performed due to
peak broadening.
However, the 1H NMR spectra showed signals corresponding to the presence of five
α anomer protons (=6.26, 6.28, 6.29, 5.77 and 6.55 ppm), corresponding to five
complexes (ratio 25:25:42:1.5:6.5). β anomers were not detected (Figure 5a)
The results from NMR spectrum analysis, results suggest that, under our conditions,
the PBAc probably bound one and two pairs of hydroxyl groups. The positions at C-
1 and C-2 were critical for binding, while the remaining hydroxyl groups played a
secondary role at C-3,5, C-5,6 and C-4,5,6 respectively. As the boronate template
was synthesized in organic solvent under azeotropic distillation, neutral trivalent
boronate esters instead of negatively charged tetravalent structures are expected as
reported in the literature (Norrild & Eggert, 1995; Shiomi & Saisho, 1993).
In conclusion NMR data indicated that four complexes in the furanose form and one
in the pyranose form were formed by glucose and PBAc. As we mentioned before
glucose could interact with two boronic acid units in its furanose form, and we
suggest the formation of α-D-glucofuranose-1,2:3,5-bis(borate), α-D-glucofuranose-
1,2:5,6-bis(borate), and α-D-glucofuranose-1,2:3,5,6-bis(borate), and with one

boronic acid unit to yield α-D-glucofuranose-1,2 mono(borate) and α-D-
glucopyranose-1,2 mono (borate) complexes (Figure 5b).
There are reports demonstrating the formation of boronic acid-D-glucose complexes
by using 1H and 13C NMR spectroscopy under both neutral non-aqueous and alkaline
aqueous conditions. From these studies, it was concluded that the preferred binding
form is the α–D-glucofuranose bound to two boronic acid groups. In both neutral
non-aqueous and alkaline aqueous conditions, the first binding site was position
(1,2) of the furanose form of D-glucose. The binding site for the second boronic acid
group is (3,5) and (3,5,6). Other papers mention that there is an intramolecular
arrangement of glucose between furanose and pyranose forms during complexes
formation, and that is a prerequisite for strong boronate complexation (Norrild &
Eggert, 1995; Shiomi & Saisho, 1993).
4.- Conclusions
The purification of FOS obtained by different enzymatic synthesis strategies, may
be efficiently performed through complexation of glucose, fructose and residual
sucrose in an extraction process with PBAc. The process has a high selectivity
towards glucose, fructose, and eventually sucrose, which are eliminated from the
solution without interaction with the FOS product. When a 3:1 molar PBAc : sugars
ratio is used in the extraction, a purity of around of 97% is obtained. The efficiency
in terms of individual sugars separation is affected by the PBAc:sugars ratio and is

strongly affected by the large excess of glucose present in the FOS solution. We
demonstrate through RMN analysis, that an excess of PBAc is required for an almost
complete elimination of sugars as multiple sugar isomer forms coexist in equilibrium,
particularly in the case of glucose. This simple and strait forward methodology can
be used obtain high purity FOS obtained from FTFs reactions with sucrose to avoid
hindrance of the FOS prebiotic effect by the simple sugars.
5.-Aknowledgments.
Jaime Porras acknowledges the National Council of Science and Technology
(CONACyT) support during his PhD thesis. This work was supported by CONACyT-
BMBF-2015-267620 and the National Autonomous University of Mexico grants
UNAM-PAPIIT IT200316 and IN209016. We particularly thank Fernando Gonzalez
for analytical support, as well as Martín Patiño and Mario Trejo for their advice in
technology transfer procedures.

4.-References
Alekseev, Y. E., Garnovskii, A. D., & Zhdanov, Y. a. (2007). Complexes of natural
carbohydrates with metal cations. Russian Chemical Reviews, 67(8), 649–
669. https://doi.org/10.1070/RC1998v067n08ABEH000343
Candela, M., Maccaferri, S., Turroni, S., Carnevali, P., & Brigidi, P. (2010).
Functional intestinal microbiome, new frontiers in prebiotic design.
International Journal of Food Microbiology, 140(2–3), 93–101.
https://doi.org/10.1016/j.ijfoodmicro.2010.04.017
Hall, D. G. (2006). Structure, Properties, and Preparation of Boronic Acid
Derivatives. Overview of Their Reactions and Applications. Boronic Acids:
Preparation and Applications in Organic Synthesis and Medicine.
https://doi.org/10.1002/3527606548.ch1
Kamada, T., Nakajima, M., Nabetani, H., Saglam, N., & Iwamoto, S. (2002).
Availability of membrane technology for purifying and concentrating
oligosaccharides. European Food Research and Technology, 214(5), 435–
440. https://doi.org/10.1007/s00217-001-0486-6
Kim, N. H., Kim, H. J., Kang, D. Il, Jeong, K. W., Lee, J. K., Kim, Y., & Oh, D. K.
(2008). Conversion shift of D-fructose to D-psicose for enzyme-catalyzed
epimerization by addition of borate. Applied and Environmental Microbiology,
74(10), 3008–3013. https://doi.org/10.1128/AEM.00249-08
Nicholls, M. P., & Paul, P. K. C. (n.d.). Structures of carbohydrate-boronic acid
complexes determined by NMR and molecular modelling in aqueous alkaline

media. https://doi.org/10.1039/b312760e
Nollet, L. M., & Toldrá, F. (Eds.). (2012). Handbook of analysis of active
compounds in functional foods. CRC Press, Boca Raton, FL. USA.
Norrild, J. C., & Eggert, H. (1995). Evidence for Mono- and Bisdentate Boronate
Complexes of Glucose in the Furanose Form. Application of1JC-CCoupling
Constants as a Structural Probe. Journal of the American Chemical Society,
117(5), 1479–1484. https://doi.org/10.1021/ja00110a003
Norrild, J. C., & Eggert, H. (1996). Boronic acids as fructose sensors. Structure
determination of the complexes involved using 1 J CC coupling constants.
Journal of the Chemical Society, Perkin Transactions 2, 0(12), 2583.
https://doi.org/10.1039/p29960002583
Porras-Domínguez, J. R., Rodríguez-Alegría, M. E., Ávila-Fernández, Á., Montiel-
Salgado, S., & López-Munguía, A. (2017). Levan-type fructooligosaccharides
synthesis by a levansucrase-endolevanase fusion enzyme (LevB1SacB).
Carbohydrate Polymers, 177(May), 40–48.
https://doi.org/10.1016/j.carbpol.2017.08.040
Ritsema, T., & Smeekens, S. (2003). Fructans: Beneficial for plants and humans.
Current Opinion in Plant Biology, 6(3), 223–230.
https://doi.org/10.1016/S1369-5266(03)00034-7
Roberfroid, M. (2007). Prebiotics: the concept revisited. The Journal of Nutrition,
137(3 Suppl 2), 830S–7S. Retrieved from
http://www.ncbi.nlm.nih.gov/pubmed/17311983

Roberfroid, M. B. (2000). Prebiotics and probiotics: are they functional foods? The
American Journal of Clinical Nutrition, 71(6 Suppl), 1682S–7S; discussion
1688S–90S. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/12495454
Sangeetha, P. T., Ramesh, M. N., & Prapulla, S. G. (2005). Recent trends in the
microbial production, analysis and application of Fructooligosaccharides.
Trends in Food Science & Technology, 16(10), 442–457.
https://doi.org/10.1016/j.tifs.2005.05.003
Shiomi, Y., & Saisho, M. (1993). Specific complexation of glucose with a
diphenylmethane-3, 3′-diboronic acid derivative: correlation between the
absolute configuration of mono-and di-saccharides. J. Chem. Soc., Perkin ….
Retrieved from
http://pubs.rsc.org/en/content/articlehtml/1993/p1/p19930002111
Sivaev, I. B., & Bregadze, V. I. (2014). Lewis acidity of boron compounds.
Coordination Chemistry Reviews, 270–271(1), 75–88.
https://doi.org/10.1016/j.ccr.2013.10.017
Springsteen, G., & Wang, B. (2002). A detailed examination of boronic acid–diol
complexation. Tetrahedron, 58(26), 5291–5300.
https://doi.org/10.1016/S0040-4020(02)00489-1
Wood, P. J., & S~diqui, I. R. (1974). AND MASS SPE&RA OF SOME
BORONIC ESTERS OF CARBOHYDRATES*. Carbohydrate Research (Vol.
36). Retrieved from https://ac.els-cdn.com/S0008621500830451/1-s2.0-
S0008621500830451-main.pdf?_tid=ab8b4252-91ee-4c5d-8576-

3a0bb9e5e648&acdnat=1536867419_c968f3b5074e31956ba8b3ecfdb52aa1
Wu, X., Li, Z., Chen, X.-X., Fossey, J. S., James, T. D., & Jiang, Y.-B. (2013).
Selective sensing of saccharides using simple boronic acids and their
aggregates. Chemical Society Reviews, 42(20), 8032.
https://doi.org/10.1039/c3cs60148j

Figure Legends.
Table 1. Composition of the final enzymatic reaction after levan-type FOS synthesis with LevB1SacB
(*)
Figure 1. Effect of PBAc equivalents per sugars equivalent in the purification process to remove
glucose and fructose (1M) in a solution containing FOS after an enzymatic synthesis from sucrose.
Sucrose (diagonal lines), glucose (dots), fructose (light grey), and FOS (dark grey).
Figure 2. -Glucose and -fructose isomer forms in solution, highlighting the cis-vicinal hydroxyl
groups required for interaction with phenylboronic acid. Complexes are not formed with
fructooligosaccharides (levantetraose is included as an example).
Figure 3. Thin layer chromatography of A) Initial mixture of FOS reaction, B) FOS mixture purified
using 3 equivalents of PBAc, and C) monosaccharides complexes removed from the mixture.
Figure 4. (a) 1H NMR spectroscopic data (500 MHz) for the interaction between PBAC and D-
fructose in CDCl3. Displayed are here the aromatic region A. (b) Proposed structure of D-fructose
complex formed with phenylboronic acid.
Figure 5. (a) 1H NMR spectrum (500 MHz) for the interaction between PBAC and D-glucose
in CDCl3. (b) Proposed structures of D-glucose complexes formed with phenylboronic acid.