Fluorinated Boronic Acid-Appended Pyridinium Salts and 19F NMR Spectroscopy for Diol Sensing

Article abstract of DOI:10.1021/jacs.7b01167

The identification and discrimination of diols is of fundamental importance in medical diagnostics, such as measuring the contents of glucose in the urine of diabetes patients. Diol sensors are often based on fluorophore-appended boronic acids, but these

Full text of DOI:10.1021/jacs.7b01167

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Article
Fluorinated Boronic Acid-Appended Pyridinium
Salts and 19F NMR Spectroscopy for Diol Sensing
Jörg Axthelm, Sven H.C. Askes, Martin Elstner, Upendar Reddy
Gandra, Helmar Görls, Peter Bellstedt, and Alexander Schiller
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b01167 • Publication Date (Web): 18 Jul 2017
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Fluorinated Boronic Acid-Appended Pyridinium Salts and 19F NMR Spectros-
copy for Diol Sensing
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Jörg Axthelm,† Sven H. C. Askes,† Martin Elstner,† Upendar Reddy G.,† Helmar Görls,†
Peter Bellstedt,† Alexander Schiller†*
†Institute for Inorganic and Analytical Chemistry (IAAC), Friedrich Schiller University Jena, Humboldtstr. 8, Dꢀ07743 Jena, Germany
ABSTRACT: The identification and discrimination of diols is of fundamental importance in medical diagnostics, such as measurꢀ
ing the contents of glucose in the urine of diabetes patients. Diol sensors are often based on fluorophoreꢀappended boronic acids,
but these severely lack discrimination power and their response is oneꢀdimensional. As an alternative strategy, we present the use of
fluorinated boronic acidꢀappended pyridinium salts in combination with 19F NMR spectroscopy. A pool of 59 (bio)analytes was
screened, containing monosaccharides, phosphorylated and Nꢀacetylated sugars, polyols, carboxylic acids, nucleotides and amines.
The majority of analytes could be clearly detected and discriminated. In addition, glucose and fructose could be distinguished up to
1:9 molar ratio in mixtures. Crucially, the receptors feature high sensitivity and selectivity, are water soluble, and their 19FꢀNMR
analyte fingerprint is pHꢀrobust, thereby making them particularly wellꢀsuited for medical application. Finally, to demonstrate this
applicability, glucose could be detected in synthetic urine samples down to 1 mM using merely a 188 MHz NMR spectrometer.
Introduction
The identification and discrimination of diols is of fundamenꢀ
tal importance in medical diagnostics. For instance, the selecꢀ
tive detection of glucose in urine is of high interest for diagꢀ
nosing Diabetes mellitus,1, 6, 12 whereas the detection of cateꢀ
cholamines, such as dopamine or epinephrine, may be used for
the detection of Alzheimer’s disease, neuroblastoma, and
Figure 1. Current boronic acid receptor scaffolds with at-
pheochromocytoma.20, 27, 34 As a consequence, numerous sensꢀ
tached fluorine probes used for diol sensing via 19F NMR
ing approaches have been developed to detect these important
spectroscopy with workgroups and year of publication.3, 16, 21,
bioanalytes. To exploit the strong affinity of boron for diols
37
under aqueous conditions, most detection strategies featured
attracted considerable interest. In 1994, the group of Gabel
investigated biologically relevant diols with 4ꢀfluorophenyl
boronic acid.21 Surprisingly, only few approaches have been
published with fluorinated phenylboronic acids (Figure 1). The
group of James used a fluorinated formylphenylboronic acid
and a chiral auxiliary amine for the determination of the enanꢀ
tiopurity of diols via 19F NMR.37 Moreover, related systems
boronic acidꢀlabeled receptors connected directly to a chroꢀ
mophore with a luminescent or colorimetric response.8ꢀ9, 11, 15,
19, 25, 31
Although, fluorescenceꢀbased receptors demonstrate
high sensitivity for diols even in the nanoꢀmolar range, they
lack a characteristic analyte response because the fluorescence
emission is only modulated in one dimension, i.e. more or less
signal intensity. To accomplish true applicability of boronic
acid receptors in medical diagnostics, sensors have to respond
directly and specifically to the structural character and concenꢀ
tration of the analyte, even in a complex matrix with potentialꢀ
ly interfering substrates.
1
using H, 11B, 13C NMR have been studied.10, 29, 32 Micouin et
al. used commercially available fluorinated boronic acid
chemosensors for monitoring boronic acidꢀdiol interaction in
aqueous solution.16 Recently, our group published an array of
fluorinated boronic acidꢀappended bipyridinium salts.3 Herein,
19F NMR spectra were processed into twoꢀdimensional QRꢀ
like barcodes. This enabled an easy approach for simple diol
discrimination.
We propose that this issue can be addressed by using fluoriꢀ
nated boronic acids as sensitive and selective probes to proꢀ
duce characteristic 19F Nuclear Magnetic Resonance (NMR)
fingerprints.5 19F NMR spectroscopy benefits from 100%
natural abundance of the 19Fꢀnucleus, high sensitivity towards
electronic environmental changes, a high gyromagnetic ratio,
the broad chemical shift range which prevents signal overlap
and usually absent background signals. Remarkable results
were achieved by Swager et al., who used 19F probes on tungꢀ
sten calixarene complexes for the identification of neutral
organic compounds,39ꢀ40 chiral amines,41 anions,13 and bioacꢀ
tive molecules.38 The new field of boronic acidꢀcontaining
receptors with 19F probes for sensing diolꢀcontaining analytes
via 19F NMR has
In the present study, a collection of fluorinated boronic acidꢀ
appended benzylpyridinium salts 1ꢀ3 (FꢀoꢀBBpy’s) and 4
(CF3ꢀoꢀBBpy) were synthesized. These one component diol
sensors contain one boronic acid function and a single fluoꢀ
rine/trifluoromethane moiety directly attached to the arene
core (Scheme 1). Optimally performing sensor 1 was used for
qualitative and quantitative 19FꢀNMR fingerprinting of 59
relevant bioanalytes, such as monosaccharides, phosphoryꢀ
lated and Nꢀacetylated sugars, polyols, carboxylic acids, nuꢀ
cleotides and amines.
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Subtle structural modifications in the probes influenced the
19Fꢀ
117.21, and –117.26 ppm (two overlapping signals, Scheme
2). New peaks appeared baseline separated and upfield shifted.
Scheme 2: Boronic Acid – Boronate Equilibrium and 19F
Diol Fingerprinting using Fluorinated Boronic Acid-
Appended Receptors under Physiological Aqueous Condi-
tionsa
1
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4
Scheme 1: Reaction Scheme of Pyridinium Salts 1-6 and
Molecule Structures of 1, 2 and 4a
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aMethylphenyl precursors have been photoꢀbrominated to corꢀ
responding benzylbromides followed by Nꢀalkylation with pyriꢀ
dine. Solvent molecules, bromide anions, and hydrogens of moleꢀ
cule structures are omitted for clarity; gray = C, green = F, blue =
N, red = O, pink = B.
NMR response to those bioanalytes. Furthermore, several
application studies were performed: discrimination of glucose
and fructose in mixtures, detection of enzymatically produced
Dꢀfructose, and the detection of glucose in synthetic urine as a
potential Diabetes test.
aThe 19F probe of 1 (10 mM) recognizes the binding of
Dꢀ
Results and Discussion
glucose (100 mM) at the boronic acid binding site leading to
highly characteristic 19F fingerprints produced by several possible
binding modes (three resulting shifts). Chemical shifts have been
predicted via DFT and support the trend of shift direction. Dashed
lines illustrate calculated shifts (ωB97XD/6ꢀmethod) of 1 (sp2 /
sp3 of boron in the equilibrium).
Compounds 1ꢀ6 were synthesized in two steps in high yields
and excellent purities (Scheme 1). All compounds have been
characterized by NMR spectroscopy, mass spectrometry,
elemental analysis, UVꢀVis absorption, infrared spectroscopy
and potentiometric pH titration (see ESI). The molecule strucꢀ
tures of 1, 2 and 4 support their characterization (Scheme 1
and ESI). Crucially, all compounds exhibit high solubility (up
to 77 mM for 1) and photo stability in aqueous solution due to
the high polarity of the ionic pyridinium moiety. Thus, diol
screening and discrimination under physiological conditions
within a broad concentration range is applicable.
Thus, indication is given that glucose binds to 1, and that the
equilibrium between free 1 and complexed 1 is slow on the
NMR timescale. More importantly, this binding event had a
significant effect on the electronic environment of the aroꢀ
matic system and resulted in characteristic chemical shifts of
the 19F probe, even over five bond lengths. The presence of
three peaks reflects three distinct binding modes of glucose in
the esterified sensorꢀglucose complex, and represents a unique
19F NMR “fingerprint”. The shift to negative ppm value of the
three peaks with respect to the parent compound was caused
by a change in hybridization from sp2 to sp3, which was conꢀ
firmed by predicted 19F NMRꢀshifts from DFT calculations
(Scheme 2 and ESI). Interestingly, the chemical shift of the
fingerprint did not vary within a tested pH range of 6.6 to 8.2
(ESI), demonstrating good pH robustness within the physioꢀ
logical pH range. This is in strong contrast to most fluorescent
based boronic acids that are sensitive for changes in pH.31
Overall, these initial results with sensor 1 established that the
synthesized fluorinated boronic acids have an excellent potenꢀ
tial as sensing and discrimination platform for diols without
the need of complicated chemometric techniques.7, 28
Potentiometric pH titration experiments were performed to
investigate the influence of the fluorine probe(s) upon the
boronic acid’s equilibrium between its free (sp2 boron, Scheme
2) and the boronate form (sp3 boron, see ESI). The nonꢀ
fluorinated receptor 6 exhibits the highest pKa value (8.50).
Decreased pKa values were observed for all fluorinated recepꢀ
tors: 2 (8.39) > 1 (8.11) > 3 (7.46) > 4 (7.14). Overall, the
acidity raised with increasing content of fluorine (6 < 1 < 4)
due to an increase in electron withdrawing (H < F < CF3),
while the positioning of fluorine resulted in an order in acidity
of 2 < 1 < 3. The high pKa of 2 seems to have its origin in
possible fluorineꢀhydrogen bonding. Lowered pKa values were
found for 1 in presence of
Dꢀglucose, Dꢀfructose and Dꢀ
mannitol with pKa values of 7.2, 5.4 and 6.0, respectively. It is
a result of preferred OH– abstraction due to a lowered angle
strain of the boronate ester (rehybridization sp2ꢀsp3).18, 22 As a
matter of fact, the drop in pKa is desirable for the applicability
of the sensor under physiological conditions.
As an initial 19F NMR spectroscopy experiment, 100 mM
Dꢀ
glucose was probed by 10 mM of compound 1 in aqueous
Next, the 19F NMR sensing behavior was considered of the
monoꢀfluorinated receptors 4ꢀFꢀoꢀBBpy (2) and 3ꢀFꢀoꢀBBpy
(3), which are isomers of 1. We were interested in how esteriꢀ
fication affected their chemical shifts with respect to the direcꢀ
tion, complexity and shift change. Our results showed, that the
shift is strongly dependent on the position of the fluorine atom
at the arene moiety. In detail, 19F signals of the pure receptors
HEPES buffer solution (100 mM, pH 7.4, 10% D2O). In abꢀ
sence of glucose, a single 19F NMR peak was observed at δF
–
111.75 ppm for unbound 1, whereas three additional peaks
were observed in presence of ꢀglucose at δF –115.97, –
are located at
δF –111.75 (1), –113.74 (2) and –104.84/–112.48
D
ppm (3) with fluorine in para, meta and ortho position to
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boron, respectively (Figure 2a). Although only one single
1
2
3
signal was expected for all three receptors alone, 3 is the only
compound which shows a minor peak (δF –112.48 ppm) beꢀ
side the strongly
4
5
6
7
downfield shifted main peak (δF –104.84 ppm). Addition of
diols, such as
D
ꢀglucose,
Dꢀfructose or catechol, lead to strongꢀ
ly upfield shifted (ꢀ
δ
F up to 5 ppm) 19F signals for 1.
Receptor 3 shifted downfield (ꢀδF up to –2 ppm) whereas 2
showed minor upfield shifted (ꢀδF up to 0.6 ppm) fluorine
signals. This trend supports the enhanced ability of paraꢀ and
orthoꢀpositioned fluorine probes, compared to the meta posiꢀ
tion, to strongly respond to changes of boron rehybridization
upon diol binding via the conjugated system. The reversed
direction in shifting of the ortho isomer 3 could be explained
by the fluorine’s proximity directly to the boron binding site
associated with BꢀOꢀHꢀꢀFꢀ hydrogen bonding.14, 23 Overall, the
number of newly appearing receptorꢀdiol signals is identical
for all three isomers. This verifies that each 19F probe (paraꢀ,
metaꢀ and orthoꢀ) recognizes the same number of formed
receptorꢀanalyte complexes. 19F NMR spectra of the monoꢀ
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fluorinated receptors and in presence of
Dꢀglucose (b), Dꢀ
fructose (c), and catechol (d) are shown in Figure 2. Going
beyond, 1ꢀ3 can be used as an arrayꢀlike sensor ensemble with
a significantly enhanced diol discrimination power as we have
recently reported using a set of different set of fluorinated
boronic acid probes.3
Figure 2. Influence of the fluorine position on the 19F NMR
sensing behavior of the receptors 1-3 (4 mM) and in presence
of selected diols (40 mM, HEPES 100 mM, pH 7.4, 10% D2O,
188 MHz, 256 scans).
Overall, receptor 1 was found to be the best performing diol
receptor with receptorꢀanalyte spectra of excellent signalꢀtoꢀ
noise ratio (S/N), baselineꢀseparated signal shifting and strong
photo stability (ESI). To exclude 19F’s response to other propꢀ
erty changes than esterification the negative control 3ꢀFꢀBpy
(5) was introduced. In detail, the 19F NMR spectrum of the
nonꢀboronic acid compound 5 (Figure S56) shows no new 19F
NMR signals beside the original peak at δF –112.46 ppm in
boronic acid / boronate equilibrium seen in Scheme 2. Thus,
fluorine exclusively responds to diols bound at the boronic
acid moiety.
Receptor 5ꢀCF3ꢀoꢀBBpy (4) with a trifluoromethyl group was
primarily introduced to increase the sensitivity in 19F NMR. A
higher fluorine content should increase the S/N. To our delight
the S/N of 4 was improved compared with 1 (~ten times, 10
mM, 188 MHz, 256 scans). However, no significant shifts in
presence of catechol (b) or
Dꢀfructose (c). Furthermore, the
signals of 5 are very sharp without distinct lineꢀbroadening
from a possible
19F NMR of 4 upon esterification with catechol and
D
ꢀfructose
(Figure S56c, d) could be recorded. Only a minor downfield
shift of the unbound receptor at F –62.66 to –62.43 ppm could
be observed in the case of ꢀglucose (Figure S57). Thus, unꢀ
δ
D
fortunately compound 4 provided only very poor sensing
potential due to interrupted conjugation between the boronic
acid and the fluorines in the CF3 moiety and the lack of charꢀ
acteristic 19F fingerprints. These results signify that connecting
the fluorine directly to the arene boronic acid is a crucial reꢀ
quirement for detecting diol binding events at boron. As a
result, only receptors 1ꢀ3 fulfill this requirement with special
preference of 1.
Qualitative Screening of Bioanalytes using Sensor 1
Consequently, compound 1 was selected as best performing
sensor and used to qualitatively screen a wide variety of anaꢀ
lyte classes to check (i) crucial structural features of target
analytes for binding, (ii) the discrimination power of the sysꢀ
tem, and (iii) to investigate 19F patterns and their correlation of
structurally related target molecules. The main focus was laid
on monosaccharides (aldoses and ketoses), glyceraldehyde,
dihydroxyacetone, hexoses (e.g. fructose, glucose, and galacꢀ
tose), and phosphorylated or Nꢀacetylated sugars. The pool of
59 tested analytes also included disaccharides, carboxylic
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