Boronic acid recognition of non-interacting carbohydrates for biomedical applications: Increasing fluorescence signals of minimally interacting aldoses and sucralose

Article abstract of DOI:10.1039/c7ob01893b

To address carbohydrates that are commonly used in biomedical applications with low binding affinities for boronic acid based detection systems, two chemical modification methods were utilized to increase sensitivity. Modified carbohydrates were analyzed

Full text of DOI:10.1039/c7ob01893b

Organic &
Biomolecular Chemistry
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Boronic acid recognition of non-interacting
carbohydrates for biomedical applications:
increasing fluorescence signals of minimally
interacting aldoses and sucralose†
Cite this: DOI: 10.1039/c7ob01893b
a
b
a
b
Angel Resendez,
Md Abdul Halim,
Jasmeet Singh, Dominic-Luc Webb
a
and Bakthan Singaram
*
To address carbohydrates that are commonly used in biomedical applications with low binding affinities
for boronic acid based detection systems, two chemical modification methods were utilized to increase
sensitivity. Modified carbohydrates were analyzed using a two component fluorescent probe based on
boronic acid-appended viologen–HPTS (4,4’-o-BBV). Carbohydrates normally giving poor signals
(
4
fucose, L-rhamnose, xylose) were subjected to sodium borohydride (NaBH ) reduction in ambient con-
ditions for 1 h yielding the corresponding sugar alcohols from fucose, L-rhamnose and xylose in essen-
tially quantitative yields. Compared to original aldoses, apparent binding affinities were increased 4–25-
fold. The chlorinated sweetener and colon permeability marker sucralose (Splenda), otherwise undetect-
able by boronic acids, was dechlorinated to a detectable derivative by reactive oxygen and hydroxide
2 2
intermediates by the Fenton reaction or by H O and UV light. This method is specific to sucralose as
Received 30th July 2017,
Accepted 2nd November 2017
other common sugars, such as sucrose, do not contain any carbon–chlorine bonds. Significant
fluorescence response was obtained for chemically modified sucralose with the 4,4’-o-BBV–HPTS
probe system. This proof of principle can be applied to biomedical applications, such as gut permeability,
malabsorption, etc.
DOI: 10.1039/c7ob01893b
rsc.li/obc
hinder the use of this method in large scale studies that
require analysis of multiple samples per day. For permeability
1
. Introduction
7
Analysis of carbohydrates in solution continues to be an testing to advance to routine use, more rapid and cost-effective
important aspect of several areas of research for the environ- analyses of sugars and sugar alcohols in biological buffers,
1
2
3
4
mental, food, pharmaceutical, biomedical, and petrochem- urine and blood are desirable.
5
ical industries. Biomedical application include gastrointesti-
Methodologies developed over the past few years, for sugar
nal (GI) permeability and malabsorption. GI permeability analysis, can be divided into two main analytical categories:
assessment uses oral ingestion and subsequent analysis of enzymatic or non-enzymatic techniques. The former is usually
combinations of sugars and sugar analogs in urine. substrate specific and the latter method usually involves chro-
Performing GI permeability assessment provides an alternative matographic techniques (HPLC, GC or CE). Since carbohydrates
approach to screening for malnutrition and maladies associ- have no intrinsic chromophore, labeling them with a chromo-
6
8–10
ated with several gastrointestinal diseases. Existing analytical phore allows detection and characterization.
Although
methods require the use of expensive instruments, such as effective, there are a few drawbacks that come with using chrom-
HPLC coupled to mass spectrometry. Although sensitive, the atography. For biomedical facilities with limited resources to
labor-intensive nature of these methods and cost per analysis adapt assays such as permeability tests routinely, effective and
user-friendly analytical procedures are needed. From this, non-
enzymatic methods have been developed that take advantage of
a
Department of Chemistry and Biochemistry, University of California Santa Cruz,
Santa Cruz, CA 95064, USA. E-mail: singaram@ucsc.edu
other chemistries, such as supramolecular and boronic acid
chemistries; these techniques have developed into major
b
Department of Medical Sciences, Gastroenterology and Hepatology Unit,
11,12
research areas that focus on developing glucose sensors,
Uppsala University, 751 85 Uppsala, Sweden
13
and chemoreceptors for other carbohydrate targets.
†
Electronic supplementary information (ESI) available: Experimental pro-
Supramolecular chemistry takes advantage of a host–guest type
cedures, characterization of boronic acid receptor (4,4′-o-BBV), and additional
fluorescence data. See DOI: 10.1039/c7ob01893b
interaction through intermolecular forces upon interaction, the
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1
4,15
system produces a detectable change in a signal.
In con- corresponding reduced forms, such as sorbitol relative to
2
4
trast, boronic acid recognition of saccharides displays reversible glucose. Sugar alcohols can have multiple syn-1,2 and/or syn-
covalent bonding of diols in saccharides to form boronate 1,3-diols where boronate ester formation occurs preferen-
1
2,16
24
esters.
Boronic acid-based methodologies have utilized fluo- tially. It has been shown that certain acyclic sugar alcohols
1
7
18
rescent or electrochemical means for quantifying the reco- can have higher binding affinity towards boronic acids com-
gnition event. Advantages of using boronic acid based chemo- pared with the corresponding aldose or ketose forms.
receptors include ease of synthesis, ability to operate in a wide pH Moreover, boronic acids lack binding affinity to non-reducing,
range, and ability to be designed around a plethora of possible cyclic carbohydrates (aldoses and ketoses), such as sucrose
19
reporter–recognition systems. Based on the intrinsic affinity of and the artificial sweetener sucralose, as they lack the hemi
boronic acids to cis-1,2 or cis-1,3 diols, we devised a modular, acetal/ketal group essential for boronic acid binding.
2
5,26
In
two-component sensing system comprising the anionic fluo- these sugars and sugar derivatives, the C-1 hydroxyl (hemi
rescent dye, 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium acetal/ketal) group on the anomeric carbon is part of the glyco-
salt (HPTS), and a boronic acid-appended viologen (4,4′-o-BBV). sidic linkage and is unavailable for boronic acid binding.
This two-component BBV system acts dually as a quencher and Consequently, no fluorescent signals are generated in our two-
a receptor operating at neutral or physiological pH. In the component system for sucrose and sucralose, important
absence of saccharide, a ground-state complex is formed by cou- markers used in applications such as assessing gut per-
2
7
lombic attraction between the anionic dye and cationic meability. To develop boronic acid-recognition of saccharides
quencher, with a decrease of HPTS fluorescence intensity. Upon with low binding affinity that are commonly used as gut per-
saccharide binding, the boronic acid moiety forms a tetrahedral meability markers, such as rhamnose, xylose, and sucralose,
anionic boronate ester. The negatively charged boronate ester we needed to generate the signal through chemical modifi-
then neutralizes the cationic viologen, diminishing the quench- cation of these sugars. Signal enhancement with a fluo-
ing efficacy of BBV, liberating de-quenched (i.e., fluorescent) rescence-based boronic acid chemoreceptor has been reported
HPTS. The strength of the fluorescent signal generated upon where an amphiphilic, glucose-selective monoboronic acid
dissociation of the ground state complex is dependent on the exhibited an excimer emission enhancement upon aggrega-
2
8
saccharide concentration (Scheme 1). This system has been tion. To our knowledge, chemically induced signal amplifica-
extensively studied by our group for the recognition of various tion of the saccharide boronic acid binding event has not been
20–22
saccharides and sugar alcohols (glycitols).
quantitatively measured. It is known that acyclic sugar alcohols
The present analytes were chosen because they have bio- can have higher binding affinity toward boronic acids com-
6
22,29
medical applications, such as gut permeability, malabsorp- pared to the corresponding aldose or ketose forms.
We
2
3
tion, and plant physiology. While many sugars, such as lactu- explored pre-treatment reactions for saccharides with low or
lose and mannitol, give meaningful signals in our fluorescent no binding affinity (i.e., fucose, rhamnose, or xylose) by a
probe based on boronic acid appended viologen (4,4′-o-BBV), simple reductive conversion to corresponding alditols. Aldoses
many others do not. In this study, fucose, xylose, L-rhamnose can be reduced by sodium borohydride (NaBH
4
) in aqueous
and sucralose, which give weak or no signal, were modified to solution to generate corresponding sugar alcohols in moder-
3
0
give increased binding (i.e., higher S/N). In general, boronic ate-to-good yields. Oligosaccharides can also be reduced by
acids lack specificity and selectivity for alcohol groups on NaBH to corresponding alditols. Usually, enzymatic methods
4
cyclic forms of carbohydrates, but can have high affinity to the are used to quantify product alditols, such as D-mannitol and
Scheme 1 Proposed mechanism of signal transduction for a two-component fluorescent probe based on boronic acid-appended viologens (left).
Aldoses and sucralose to be chemically modified (right).
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3
1,32
D-sorbitol.
Our recent work has involved developing a non- where F
0
is the fluorescence intensity in the absence of
enzymatic, multiwell fluorescent assay to quantify these sugar analyte; F is the fluorescence intensity after the addition of
markers which are routinely used to monitor gut per- analyte; Fmax is the fluorescence intensity at which no further
2
2,33
meability.
b
We were interested in quantifying xylose and signal is obtained with further analyte addition; K is the
L-rhamnose, as these aldoses have also been used to study apparent binding constant and [A] is analyte concentration. Kb
3
4
intestinal permeability. Sucralose, a non-reducing trichlori- was solved using Origin lab software (OriginLab Corp,
nated sucrose, is used as a marker for colon permeability as it Northampton, MA, USA, was used to perform the calculation)
3
5,36
is resistant to fermentation by colonic bacteria.
Carbon– following the Benesi–Hildebrand plot where it is assumed that
3
9,40
chloride bonds in sucralose were converted into carbon– a 1 : 1 binding stoichiometry is present.
Binding constants
hydroxyl bonds by the Fenton reaction. This chemical modifi- were calculated to compare before and after modification of
cation is a proof of principle that will enable use of non-inter- aldoses.
acting carbohydrates that are used for gut permeability
test (i.e. xylose, rhamnose, or sucralose). This modification
2
.4. NaBH reduction of aldoses
4
was anticipated to make the product amenable for recognition To a 10 mL round bottom flask, the aldose sugar (0.1 mmol)
by the boronic acid viologen (4,4′-o-BBV) in our two-com- and 0.05 M sodium phosphate-HEPES buffer (5 mL) was
ponent fluorescent system. In gut permeation studies, we have added and the aldose solution (20 mM) was stirred for
control on what kind of sugar is administered to the subjects. 5 minutes. Then, NaBH (0.011 g, 0.3 mmol) was added, and
4
Consequently, we do not have to address the sugar selectivity the reaction mixture stirred for 1 h at 25 °C. The resulting solu-
problem. We can control this by giving only specific non-inter- tion was serially diluted with 0.05 M sodium phosphate-
fering sugars, such as lactulose and/or sucralose. Herein we HEPES buffer to obtain the desired concentration points
report the quantification of mono- and disaccharides after (10 mM–0.1 mM) for fluorescence measurements.
chemical modifications to amplify the signal in our two-com-
2
.5. Dechlorination of sucralose by Fenton reaction
To a 25 mL round-bottomed flask, sucralose (0.0795 g,
.2 mmol), FeSO ·7H O (0.0278 g, 0.1 mmol), and deionized
O (9.979 mL) were added and stirred for 5 min. Then,
21 µL, 0.2 mmol) of a 30% w/w solution of H (9.79 M) was
ponent fluorescent probe system.
0
H
2
(
4
2
2
. Experimental
2 2
O
2
.1. Chemicals and reagents
added to the reaction mixture, producing a translucent bronze
color; the mixture was then stirred for 1 h at 25 °C. The reac-
tion mixture containing the dechlorinated sucralose was
diluted in 0.1 M sodium phosphate buffer, pH 7.4 to obtain
fluorescence intensity values for different concentrations
All reagents and saccharides were purchased from Sigma
Aldrich Co. (USA). Sucralose was purchased from TCI Chemicals
and used as received. Hydrogen peroxide (H O , 30% w/w) was
purchased from Fisher Scientific International. All solvents were
at least HPLC grade. Acetonitrile (MeCN) was obtained from a
solvent purification system, sent to an ampoule under argon
2
2
(
10 mM–0.1 mM). Samples were centrifuged at 2500 relative
centrifugal force (RCF) for 5 min. Supernatants were pipetted
onto a 96 well plate and analyzed for modified sucralose by
the 4,4′-o-BBV–HPTS assay.
and stored no more than 4 weeks before use. Ultra-pure water
1
(
>14 MΩ cm− ) obtained from a Millipore water system was
used for buffers and other solutions. The synthesis of 4,4′-o-
2
.5.1 Dechlorination of sucralose by photooxidation. Serial
diluted preparations of sucralose were prepared in 1 : 1 30%
w/w) H solution and deionized water. Sucralose was found
21,22
BBV was performed as previously reported.
(
2 2
O
2
.2. Instrumentation
to have an absorbance peak near 265 nm with no absorbance
above 300 nm, whereas H O had a narrow peak at 357 nm. A
For multiwell fluorescence measurements, solutions were
pipetted onto a 96-well plate (#3694, Corning, USA) and a fluo-
rescence plate reader (Envision® 2103 Multi-label,
PerkinElmer) was used (excitation filter, 405 nm; emission
filter, 535 nm, emission aperture, normal, measurement
height from the bottom, 6.5 mm; number of flashes, 10). A
monochromator based plate reader was used to scan for
optimal wavelengths for HPTS fluorescence and sucralose
absorbance (Spark 10 M, Tecan, Austria).
2
2
Spectroline TVC 312R/F transilluminator equipped with an
Watt lamp with a peak at 360 nm (negligible below 300 nm)
was used to selectively photo-oxidize H O of different sucra-
8
2
2
lose dilutions, which were incubated for 2 h. Others were kept
−1
under ambient conditions. After UV treatment, 1 µg µL cata-
lase was added to the tubes and incubated for 5 min to seques-
ter remaining H O . All the samples were vortexed and centri-
2 2
fuged at 2500 RCF for 10 min. Supernatants were pipetted
onto a 96 well plate and analyzed for modified sucralose by
the 4,4′-o-BBV–HPTS assay.
2
.3. Data analysis
Apparent binding constants were determined by non-linear
3
7,38
curve fitting using the following equation.
2.6. Fluorescence recovery measurements by BBV–HPTS
ꢀ
ꢁ
probe of chemically modified aldoses and sucralose
Fmax
F0
1
þ
Kb½Aꢀ
F
Ready-made plates were prepared by adding 10 µL of the 4,4′-o-
BBV–HPTS probe solution as 4-fold concentrate (1.6 mM 4,4′-o-
¼
ð1Þ
F0
1 þ Kb½Aꢀ
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BBV and 16 µM HPTS) in 0.1 M sodium phosphate HEPES
buffer pH 7.4 containing 0.04% Triton X-100 (4× buffer). At the
time of running the assay, each well received 30 µL of sample
in quadruplicate. Blank wells were given 10 µL 4× buffer with
neither HPTS nor 4,4′-o-BBV. Baseline fluorescence wells con-
tained 30 µL of buffer and 10 µL of probe solution.
Fluorescence recovery of HPTS was measured on the plate
reader. After blank subtraction, fluorescence intensity (F) for
each analyte concentration relative to initial quenched (F
0
)
HPTS (F/F ) ratio was calculated. Under these conditions, F is
0
0
a non-zero value after background subtraction with about 20%
of maximum fluorescence intensity of HPTS.
3
. Results and discussion
3
.1. Reduction of low affinity aldoses increases fluorescence
signal in the BBV–HPTS system
The present analytes were chosen because they have bio-
6
medical applications, such as gut permeability and malab-
sorption. While many sugars, such as lactulose and mannitol,
give meaningful signals in our fluorescent probe based on
boronic acid appended viologen (4,4′-o-BBV), many others do
not. In this study, fucose, xylose, L-rhamnose, which give weak
or no signal, were modified by sodium borohydride to give
increased binding. Sodium borohydride is used widely in
organic synthesis, using aprotic polar and aqueous solvent
systems. To increase the aldose-boronic acid binding affinity,
the aldoses fucose, L-rhamnose and xylose were reduced with
Fig. 1 Normalized HPTS (4 µM final concentration) fluorescence recov-
ery (F
0
= initial baseline fluorescence, F = recovered fluorescence) with
NaBH
4
in neutral buffer at ambient conditions. Resulting aldi-
4,4’-o-BBV as the boronic acid receptor of each aldose before and after
tols were quantified using 4,4′-o-BBV–HPTS probe (Scheme 2).
NaBH
4
reduction. A. Non-reduced B. reduced aldoses fucose,
Using NaBH in an aqueous system can create an alkaline L-rhamnose, and xylose in 0.05 M sodium phosphate-HEPES buffer
4
solution, pH 7.4. Error bars represent standard deviation of quadrupli-
cate responses.
solution. The boronic acids in our two-component fluorescent
probe are sensitive to pH changes of the medium and can
4
1
generate false positive signals in pH > 8. This system has
been extensively studied and we have determined that the two-
component fluorescent probe is unaffected by borate salts or
high salt concentration. To further prevent this false positive
Similarly, the binding constants for each aldose versus
signal produced by increasing pH of the medium, optimiz- reduced aldose provided a significant difference. Apparent
ation of NaBH
equivalences and buffer system was conducted. binding constants for reduced aldoses provided 4, 7, and 25
4
It was determined that three equivalences of NaBH4 to the times higher for reduced fucose, L-rhamnose, and xylose
aldose sugar in 0.05 M sodium phosphate HEPES buffer was respectively (Table S-1 & Fig. S-2†). We observed the following
optimal to achieve an alditol specific fluorescent signal under order of apparent binding affinities: L-rhamnose > xylose >
the reaction conditions (Fig. S-1†). Finally, the strong buffering fucose. This demonstrates that the reduced forms of each
capacity of the 4× buffer in the wells clamps the pH at 7.4 aldose has a higher binding affinity for the boronic acid recep-
during the fluorescence measurements. Compared to tor 4,4′-o-BBV, reconfirming that sugar alcohols have higher
unreacted aldose, reduced aldoses provided significantly binding affinities compared to their aldose form when com-
enhanced fluorescent signals with at least a 4-fold increase for paring the diastereomers fucose and L-rhamnose, and xylose
fucose and up to 2-fold difference for reduced L-rhamnose at (Fig. S-3†). In addition, sensitivity for each sugar improved
1
0 mM concentration (Fig. 1A & B).
upon reduction, as indicated by lower detection limits (signal
Scheme 2 Reaction scheme for the reduction of aldoses and quantification by the 4,4’-o-BBV–HPTS probe.
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to noise ratio greater than 3 standard deviations of baseline particular, for colon permeability due to gut diseases, such as
values) (Table S-2†). IBD, IBS, Crohn’s disease, colitis, and colon cancer. In gut per-
Because reduction is carried out under ambient tempera- meation studies, we have control on what kind of sugar is
ture and near neutral pH, we suspected that these aldoses administered to the subjects. Consequently, we do not have to
were reduced without any epimerization. Since xylitol is com- address the sugar selectivity problem. We can control this by
mercially available, the fluorescent signal for commercial giving only specific non-interfering sugars, such as lactulose
xylitol and reduced xylose were compared to determine the and/or sucralose. We envisaged that development of rapid,
extent of epimerization, if any, during the reduction step. The cost-effective, and reliable analytical methods for the in-field
reduction of xylose was performed under the same conditions analysis of sucralose will continue to grow in importance. We
as previously described, and commercially available xylitol was were interested in quantifying sucralose by boronic acid based
used to compare the fluorescence recovery for both standard detection methods as it can provide a platform for low-cost,
sugar alcohol solutions using 4,4′-o-BBV as the boronic acid practical, and rapid gut permeation analysis compared to con-
receptor (Fig. 2).
ventional methods, such as HPLC and MS. Sucralose is a non-
The fluorescence signal for commercial and synthetic reducing sugar; based on our earlier studies we have shown
reduced xylose) xylitol was up to 2-fold higher than xylose at that the hemiacetal (or hemiketal) in reducing sugars is criti-
(
1
0 mM. Additionally, there was no difference in apparent cal for boronic acid recognition. Consequently, sucralose could
−1
−1
33
binding constants (66 ± 8.6 M versus 52.3 ± 12.2 M ) for not be detected by our two-component HPTS–BBV system
commercial xylitol and synthetic xylitol. It was concluded with and warranted chemical modification induced signal amplifi-
high confidence that these reductions occur without any epi- cation of sucralose. Therefore, sucralose was subjected to
merization and afford mainly the corresponding alditols. It is chemical modification by reactive oxygen intermediates gener-
2 2
also clear that the glycitol product can be readily quantified ated in situ by the Fenton reaction using ferrous iron and H O
4
3,44
using boronic acid-based detection systems such as our two- as the oxidant.
component fluorescent probe (4,4′-o-BBV–HPTS).
Additionally, these reactive intermediates
2
2
can be generated using photolysis of H O by UV light, typi-
2
2
4
5
cally around 360 nm. Sucralose, which has real world appli-
cations, does not give a signal in this assay. The oxidation
product, however, interacts with BBV’s to give a measurable
enhanced signal. Our intent in this work, in which mM con-
centrations were convenient, was to demonstrate a chemical
process. Currently, a mixture of sugars, such as sucralose, lac-
tulose, mannitol, is orally administered. Among these, only
sucralose reaches the colon and escapes microbial fermenta-
tion. Consequently, we focused only on these “limited subset”
of saccharides that are important for gut permeation studies.
Sucralose was dechlorinated using ferrous sulfate hepta-
hydrate and 30% w/w H O in water under ambient conditions
3
.3. Fenton reaction and UV photooxidation of sucralose
yields detectable analyte in BBV–HPTS system
Sucralose has become one of the most widely used artificial
sweeteners in the food industry due to its poor metabolism
and 600 times higher sweetness index than sucrose. When
humans consume it, sucralose is excreted intact into the
environment, being resistant to metabolic degradation; it even
4
2
survives the wastewater treatment process. Consequently,
sucralose has become an excellent gut permeability marker, in
2
2
for over an hour to obtain the hydroxylated product. The
initially displaced chloride ions were confirmed by a qualitat-
ive AgNO3 test after one hour by observing precipitation of
4 2 2
AgCl. Optimal amounts of FeSO and H O were determined
to eliminate false positives and to achieve optimal measure-
ments of modified sucralose. The fluorescence signal for
dechlorinated sucralose product was compared to unreacted
sucralose and the control reaction (absence of sucralose),
which ruled out false positive signals (Fig. 3).
For the dechlorinated sucralose, there was up to a 13-fold
increase in fluorescence signal at 10 mM sucralose conversion
−
1
product with an apparent binding constant of 52.5 ± 4.3 M
HPTS spectral change Fig. S-4†). Untreated sucralose provided
(
no fluorescence signal and hence its binding affinity was not
determined. The reagents FeSO4 and H O alone, in the
2
2
absence of sucralose, gave small fluorescence signal above
mM concentration. Residual peroxide or hydroxyl radical can
oxidize C–B bond and release free HPTS. However, this side
reaction happens only at concentrations above mM.
Fig. 2 Normalized HPTS (4 µM final concentration) fluorescence recov-
4
0
ery (F = initial baseline fluorescence, F = recovered fluorescence) with
4,4’-o-BBV as the boronic acid receptor comparing commercially avail-
2
able xylitol (black) to reduced xylose (red) in 0.05 M sodium phosphate-
HEPES buffer, pH 7.4. Error bars represent standard error mean of qua-
druplicate responses.
Similarly, we sought to measure sucralose photooxidation of
H O by ultraviolet light to dechlorinate sucralose. This alle-
2
2
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ary alkyl chloride carbon bond. This gave m/z of 379 and 381
respectively. We anticipated, by analogy, that our chemical
modification of sucralose would lead to more than one
product as well. Given the significant fluorescence recovery, it
is plausible that a sugar alcohol derivative was generated.
4. Conclusions
Some aldoses have low binding affinities to our two-com-
ponent fluorescent probe based on boronic acid appended vio-
logen (4,4′-o-BBV–HPTS). These sugars are converted to their
4
glycitol form by NaBH reduction to increase HPTS fluo-
rescence response. This modification provided good to excel-
lent yields of the corresponding glycitols. Using the 4,4′-o-
BBV–HPTS probe, glycitols were measured by HPTS fluo-
rescence recovery. Compared to unreacted fucose, L-rhamnose,
Fig. 3 Normalized 4,4’-o-BBV–HPTS probe (4 µM HPTS final concen-
0
tration) fluorescence recovery (F = initial quenched fluorescence, F =
recovered fluorescence) for dechlorinated sucralose obtained by the
Fenton reaction (black), sucralose (red), and control reaction (green) in
0
.05 M sodium phosphate-HEPES pH 7.4 buffer. Inset is the fluo- and xylose, reduced aldoses yielded at least 4-fold difference in
rescence recovery in the lower range. Error bars represent standard binding affinity. Based on earlier findings, certain sugar alco-
error mean of quadruplicate responses.
hols have higher binding affinities for the boronic acid violo-
gen receptor (4,4′-o-BBV) compared to the corresponding
2
2,29
aldose forms.
using reactive oxygen and hydroxide intermediates generated
by the Fenton reaction or by photooxidation of H by ultra-
Further, in situ modification of sucralose
viated the need to use other reagents besides H
be considered to be a milder approach (Fig. 4).
2 2
O and might
2 2
O
Using this method, similar recoveries and detection limits
were obtained, with limit of detection and quantification of
violet light is demonstrated. Compared to native sucralose, sig-
nificantly increased fluorescence recovery was obtained by the
dechlorination reaction. When utilizing boronic acid based
detection methods for measuring aldose or sugar derivatives
for gut permeation analysis, such as sucralose, xylose or
L-rhamnose, simple chemically induced signal amplification
can be achieved to circumvent the need for using conventional
chromatographic methods for quantification of these sugars.
This proof of principle method to increase the fluorescence
signal for minimally binding or non-binding sugar gut
markers that are not metabolized is a step closure for adapting
gut permeability studies in low resource laboratories through
the use of simple and effective boronic acid based fluorescent
systems.
1
70 µM and 280 µM respectively. Having catalase to sequester
unreacted was critical for obtaining fluorescence
measurements. H can oxidize the boronic acid moiety,
2 2
H O
2 2
O
which would affect the quenched ground state complex and
subsequently generate a false positive signal. Elucidation of
the modified sucralose has been previously demonstrated
using mass spectrometry by photooxidation of H O in the
2
2
4
6
presence of UV light. Two dominant products were detected:
isomers from dechlorination of the C-1 primary alkyl chloride
carbon bond and those from dechlorination of the C-4 second-
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
The authors thank Mary N. Wessling and Ritchie A. Wessling
for proofreading and editing. A. R. also acknowledges the
graduate research fellowship support from the Initiative for
Maximizing Student Diversity (IMSD, grant 5R25GM058903).
D.-L. W gratefully acknowledges support from Bengt Ihre’s
Fig. 4 Fluorescence recovery of 4,4’-o-BBV–HPTS probe of sucralose
Foundation
(SLS-411011,
SLS-503061,
SLS-591271
&
recognition in the presence of H
and catalase (blue); in the absence of H
purple and cyan respectively). Error bars represent standard error mean
2
O
2
2 2
and UV light (green); H O , UV light
SLS-594831), Swedish Medical Association (SLS-411921 &
SLS-503131), RFR, Uppsala/Örebro region (RFR-476131),
Gastroenterology Research Foundation Sweden, (SLS-504191),
2 2
O
with and without UV light
(
for quadruplicate measurements.
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ALF funds 2015, Björklund’s Foundation (SLS-589741) and OE 23 U. Hanania, T. Ariel, Y. Tekoah, L. Fux, M. Sheva,
and Edla Johansson’s Science Foundation 2017.
Y. Gubbay, M. Weiss, D. Oz, Y. Azulay, A. Turbovski,
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4 W. J. Evans, E. J. McCourtney and W. B. Carney, Anal.
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