Boronic Acid: A Bio-Inspired Strategy to Increase the Sensitivity and Selectivity of Fluorescent NADH Probe

Article abstract of DOI:10.1021/jacs.6b05810

Fluorescent probes have emerged as an essential tool in the molecular recognition events in biological systems; however, due to the complex structures of certain biomolecules, it remains a challenge to design small-molecule fluorescent probes with high sensitivity and selectivity. Inspired by the enzyme-catalyzed reaction between biomolecule and probe, we present a novel combination-reaction two-step sensing strategy to improve sensitivity and selectivity. Based on this strategy, we successfully prepared a turn-on fluorescent reduced nicotinamide adenine dinucleotide (NADH) probe, in which boronic acid was introduced to bind with NADH and subsequently accelerate the sensing process. This probe shows remarkably improved sensitivity (detection limit: 0.084 μM) and selectivity to NADH in the absence of any enzymes. In order to improve the practicality, the boronic acid was further modified to change the measurement conditions from alkalescent (pH 9.5) to physiological environment (pH 7.4). Utilizing these probes, we not only accurately quantified the NADH weight in a health care product but also evaluated intracellular NADH levels in live cell imaging. Thus, these bio-inspired fluorescent probes offer excellent tools for elucidating the roles of NADH in biological systems as well as a practical strategy to develop future sensitive and selective probes for complicated biomolecules.

Full text of DOI:10.1021/jacs.6b05810

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Communication
Boronic Acid: A Bio-Inspired Strategy to Increase the
Sensitivity and Selectivity of Fluorescent NADH Probe
Lu Wang, Jingye Zhang, Beomsue Kim, Juanjuan Peng, Stuart N. Berry,
Yong Ni, Dongdong Su, Jungyeol Lee, Lin Yuan, and Young-Tae Chang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b05810 • Publication Date (Web): 08 Aug 2016
Downloaded from http://pubs.acs.org on August 8, 2016
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Boronic Acid: A Bio-Inspired Strategy to Increase the Sensitivity and
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Lu Wang,1 Jingye Zhang,2 Beomsue Kim,3 Juanjuan Peng,3 Stuart N. Berry,3 Yong Ni,3 Dongdong Su,3
Jungyeol Lee,1 Lin Yuan,4 YoungꢀTae Chang*1,3
1Department of Chemistry and Medicinal Chemistry Programme, National University of Singapore, Singapore 117543
2Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai 201203,
PR China
3Laboratory of Bioimaging Probe Development, Singapore Bioimaging Consortium, Singapore 117543
4State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan
University, Changsha 410082, PR China
Supporting Information Placeholder
Among the complex biomolecules, the representative reduced
nicotinamide adenine dinucleotide (NADH) consists of one adeꢀ
nine, one nicotinamide, two ribose rings and a pair of bridging
phosphate groups (Figure S1). The complicated structure makes it
difficult to design ideal probes to detect in biological systems.
However, NADH and its oxidized form, NAD+, are the most inꢀ
dispensable coenzymes found in all living cells and are required
in multiple biological processes.12ꢀ17 Thus, it is of considerable
interest to prepare probes for monitoring NADH levels in biologiꢀ
cal systems.
ABSTRACT: Fluorescent probes have emerged as an essential
tool in the molecular recognition events in biological systems;
however, due to the complex structure of certain biomolecules, it
remains a challenge to design smallꢀmolecule fluorescent probes
with high sensitivity and selectivity. Inspired by the enzymeꢀ
catalyzed reaction between biomolecule and probe, we present a
novel combinationꢀreaction twoꢀstep sensing strategy to improve
sensitivity and selectivity. Based on this strategy, we successfully
prepared a turnꢀon fluorescent NADH (reduced nicotinamide
adenine dinucleotide) probe, in which boronic acid was introꢀ
duced to bind with NADH and subsequently accelerate the sensꢀ
ing process. This probe shows remarkably improved sensitivity
(detection limit: 0.084 µM) and selectivity to NADH in the abꢀ
sence of any enzymes. In order to improve the practicality, the
boronic acid was further modified to change the measurement
conditions from alkalescent (pH 9.5) to physiological environꢀ
ment (pH 7.4). Utilizing these probes, not only did we accurately
quantify the NADH weight in a health care product, but also evalꢀ
uate intracellular NADH levels in live cell imaging. Thus, these
bioꢀinspired fluorescent probes offer excellent tools for elucidatꢀ
ing the roles of NADH in biological systems, as well as a practical
strategy to develop future sensitive and selective probes for comꢀ
plicated bioꢀmolecules.
Recently, several offline methods, including a conventional enꢀ
zymatic cycling assay,18 high performance liquid chromatogꢀ
raphy,19 and capillary electrophoresis,20 have been developed to
quantify NADH in vitro. Further, the intrinsic fluorescence of
NADH has been used in the quantification and imaging of biologꢀ
ical samples,21,22 however, its short excitation/emission waveꢀ
length can be easily interfered with by other biomolecules, such as
proteins. To the best of our knowledge, except macromolecular
fluorescent probes based on genetically encoded proteins and
nanoparticles,23ꢀ26 several smallꢀmolecule fluorescent NADH
probes have been reported but only two were applied in live cell
imaging.27ꢀ31 The current design strategy is mainly based on the
redox reaction between NADH and probe, along with alteration of
the probes’ fluorescence intensity or wavelength. However, due to
lack of recognition groups in the above probes, the selectivity of
these probes to NADH is still not sufficient. Furthermore, other
deficiencies, such as turnꢀoff fluorescent response and low sensiꢀ
tivity also limited their application in cell imaging. Thus, it is
urgent to prepare a fluorescent probe that selectively and sensiꢀ
tively detects NADH in live cells.
Optical imaging as a nonꢀinvasive imaging technique has been
widely used in exploration of biological processes, disease diagꢀ
nosis, and treatment.1ꢀ3 Fluorescent probes sensing the target bioꢀ
molecules or biological microenvironment are an indispensable
part in the imaging process.4ꢀ9 Ideal fluorescent probes should
contain several basic requirements, such as suitable sensitivity,
high selectivity, good stability, short response time, and dynamic
changes of fluorescent properties.10 Among the various of characꢀ
ters of the fluorescent probes, sensitivity and selectivity are parꢀ
ticularly important to detect specific biomolecules, especially in
complicated biological systems, such as live cells.11 However, due
to the complex structures of certain biomolecules, it remains a
challenge to reasonably design fluorescent probe with satisfied
sensitivity and selectivity simultaneously.
Since first reported in the 1960s, the enzymatic cycling assay
has been used to quantify NADH concentrations in vitro and
evaluate NADHꢀrelated enzyme activities.32 As shown in Figure
1A, in the presence of enzymes, probe and NADH can be fixed
into the binding sites of the enzyme and localized in close proximꢀ
ity in specific sites, which promotes hydride transfer and accelerꢀ
ates the redox reaction. However, the high price and strict measꢀ
urement condition of this method limit the application in bioꢀ
samples, especially in live cells. Inspired by the enzymeꢀcatalyzed
reaction for NADH detection and ligandꢀdirected protein
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Figure 1. A) Scheme of the enzymeꢀcatalyzed NADH sensing
process. B, C) The design and structures of NADH probes.
labeling,33 we proposed a novel combinationꢀreaction twoꢀstep
sensing strategy to design smallꢀmolecule fluorescent probes.
Specifically, a recognition group was introduced in the probe to
combine with NADH, which subsequently accelerates the sensing
process in the absence of any enzymes (Figure 1B). As shown in
Figure S1, NADH can be divided into two parts: reaction site –
nicotinamide and recognized sites – ribose or pyrophosphate linkꢀ
er. In this work, resazurin was chosen as the fluorophore due to its
excellent performance for evaluating reductase activity.34 In the
case of binding group, boronic acid was selected because it has
been widely applied in the recognition of diolꢀcontaining biomolꢀ
ecules, such as carbohydrates35 and nucleoside polyphosphates
(NPPs)36. It was hypothesized that the combination of boronic
acid of the probe and ribose of NADH would shorten the spatial
distance and facilitate hydride transfer, thereby accelerating the
reduction of weakly fluorescent resazurin derivatives to strongly
fluorescent products. Thus, 3ꢀaminophenylboronic acid was conꢀ
nected to resazurin via an alkane linker (Figure 1C and Scheme
S1). The probes were prepared and characterized by spectroscopy,
1H NMR, 13C NMR, and high resolution mass spectrometry (supꢀ
porting information).
Figure 2. (A) UVꢀVis absorption and (B) fluorescence spectra of
BA-Resa with titration of NADH as function of concentration (0ꢀ
50 µM). DMSO/PBS (1/99, pH 9.5). C,D) Plot of the UV absorbꢀ
ance ratio (A1/A0) and fluorescence intensity ratio (F/F0) of probe
BA-Resa, Con-Resa and Resazurin in the NADH titration (0ꢀ
100 µM). BA-Resa: A480/A575, Resazurin: A575/A610. DMSO/PBS
(1/1, pH 9.5). E) Fluorescence response of BA-Resa to various
biomolecules. Analytes (2ꢀ7, 1 mM; 8ꢀ19, 50 µM): 1) buffer, 2)
Ca2+, 3) K+, 4) Na+, 5) Fe3+, 6) Zn2+, 7) Mg2+, 8) Tyrosine, 9)
Cysteine, 10) Methionine, 11) GSH, 12) Glucose, 13) Ribose, 14)
Fructose,15) ATP, 16) ADP, 17) NAD+, 18) NADH, 19) NADPH.
F/F0 represents the fluorescence intensity ratio at 575 nm and F0 is
the fluorescence intensity of BA-Resa without NADH. Measureꢀ
ment condition: [BA-Resa] = 10 µM, 10 mM phosphate buffer
saline (pH 9.5) solution, λex = 480 nm. 37 ˚C, and incubation
time: 20 min. Results are expressed as mean ± standard deviation
of three independent experiments.
Initially, the spectroscopic properties of BA-Resa (10 µM)
responding to NADH were examined in the buffer solution (pH
9.5; DMSO/water = 1/99 v/v, 10 mM PBS) (Table S1). As disꢀ
played in Figure 2A and S2, the decreased absorption band at 535
nm and the increased band at 485 nm were observed simultaneꢀ
ously upon the addition of NADH (0 – 50 µM). At the same time,
the maximum fluorescence wavelength was shifted from 620 nm
to 575 nm in accompaniment with a 38.6ꢀfold emission turnꢀon
response (Figure 2B and S3). This is consistent with the reduction
of the weakly fluorescent resazurin to strongly fluorescent resoruꢀ
fin.37 Notably, the response of BA-Resa to NADH at low concenꢀ
trations (0 – 1 µM) also showed excellent linearity with detection
limit as low as 0.087 µM (Figure S4). Furthermore, in control
experiments with Con-Resa and Resazurin, there were few specꢀ
tral changes, thus demonstrating the remarkably enhanced sensiꢀ
tivity of this twoꢀstep sensing strategy (Figure 2C, 2D and Figure
S5). It was also shown that this sensing process could be achieved
within 25 minutes (Figure S6). pH titration experiments indicated
that higher pH benefits the fluorescent response of BA-Resa to
NADH (Figure S7), which is probably due to the favorable comꢀ
bination between probe and NADH in alkalescent environment.
of NAD+ even at high concentration and long incubation time
(Figure S8). Meanwhile, we further evaluated the selectivity of
BA-Resa towards NADH by comparing with common biomoleꢀ
cules. As shown in Figure 2C, negligible fluorescence enhanceꢀ
ment was observed after incubation with metal ions (2ꢀ7), amino
acids (9ꢀ11), carbohydrates (12ꢀ14) and NPPs (15ꢀ16), demonꢀ
strating the potential of this NADH probe in live cell imaging.
Interestingly, even though it shares the same reducing functional
group with NADH, reduced nicotinamide adenine dinucleotide
phosphate (NADPH, 19) generated a much lower fluorescence
signal (Figure 2C and S9). This could be because the partially
phosphorylated ribose in NADPH reduces the possibility of comꢀ
bination with boronic acid of the probe, thereby slowing the reacꢀ
tion rate.
To confirm the function of boronic acid in the proposed sensing
process, control probe Con-Resa without boronic acid was preꢀ
pared (Scheme S1). As shown in Figure S5, after incubation with
NADH (0 ꢀ 100 µM), few spectral changes of control probe Con-
Resa were observed. However, the mixture of NADH and boronic
acidꢀcontaining probe BA-Resa displayed blueꢀshifted waveꢀ
length and increased fluorescence intensity. These spectral changꢀ
es indicate the production of reduced fluorophore (BA-Resa-P),
Oxidized nicotinamide adenine dinucleotide (NAD+), the oxiꢀ
dized form of NADH, is a persistent source of interference in
traditional NADH detection methods. However, it was shown that
the fluorescence signal of BA-Resa remains silent in the presence
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Figure 3. The proposed mechanism of probe BA-Resa respondꢀ
ing toward NADH. The binding with boronic acid could be to
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either of two riboses in NADH (blue).
which is consistent with the calculated results (Figure S10).
Meanwhile, the redox reaction in the sensing process was also
demonstrated by liquid chromatographyꢀmass spectrometry (LCꢀ
MS) (Figure S11). Therefore, a twoꢀstep sensing mechanism of
the boronic acidꢀcontaining probe responding to NADH was proꢀ
posed and displayed in Figure 3. Firstly, the boronic acid of BA-
Resa and either of diols in NADH initially form the boronic ester
of intermediate 1. Secondly, the shorter distance in intermediate 1
facilitates the hydride transfer from NADH to probe BA-Resa,
thereby accelerating the reduction of the weakly fluorescent BA-
Resa to the strongly fluorescent BA-Resa-P (More discussion in
Figure S11 legend). Notably, the assumptive boronic esterꢀ
containing intermediate 1 was confirmed by MALDIꢀTOF mass
spectrometry (Figure S12).
Figure 4. A) The structures of NADH probe BA-Resa and RA-
Resa. The emission spectra of BA-Resa (B) and RA-Resa (C)
responding to NADH at physiological pH (7.4). D) Fluorescence
response of RA-Resa to various biomolecules. Analytes (2ꢀ7, 1
mM; 8ꢀ19, 100 µM): 1) buffer, 2) K+, 3) Na+, 4) Ca2+, 5) Zn2+, 6)
Fe3+, 7) Mg2+, 8) Tyrosine, 9) Cysteine, 10) Methionine, 11) GSH,
12) Glucose, 13) Ribose, 14) Fructose,15) ATP, 16) ADP, 17)
NAD+, 18) NADH, 19) NADPH. F/F0 represents the fluorescence
intensity ratio and F0 is the fluorescence intensity at 575 nm of
RA-Resa without NADH. Measurement condition: [Probes] = 10
µM, 10 mM phosphate buffer saline (pH 7.4) solution, λex = 480
nm, 37 ˚C, and incubation time: 20 min. Results are expressed as
mean ± standard deviation of three independent experiments.
Even though BA-Resa was successfully applied to detect
NADH in buffer solution, the alkalescent measurement conditions
limit the applications in biological systems. To address this probꢀ
lem, the phenylboronic acid of BA-Resa was replaced by 2ꢀ
(hydroxymethyl)phenylboronic acid, which forms boronic esters
with diols of ribose at physiological pH (7.4).38 As shown in Figꢀ
ure 4 and S13, in pH 7.4 buffer solution, only slightly enhanced
fluorescence intensity of BA-Resa was obtained in the presence
of NADH; however, probe RA-Resa emitted a remarkably inꢀ
creased fluorescence with detection limit as low as 0.41 µM under
the same conditions. This is mainly because 2ꢀ
(hydroxymethyl)phenylboronic acid of RA-Resa is favorable for
binding with NADH at pH 7.4, accelerating the redox reaction
(Figure S14). The pH effect on the spectral changes of RA-Resa
responding to NADH was examined and listed in Figure S15.
Interestingly, in pH 7.4 buffer solution, probe RA-Resa not only
showed significantly enhanced sensitivity to NADH, but also
displayed improved selectivity in the presence of biomolecules
(Figure 4D, S16ꢀ18). With the advantages of RA-Resa demonꢀ
strated, we next tested the feasibility of RA-Resa to detect NADH
produced in an enzyme system. As shown in Figure S19, only in
the presence of alcohol dehydrogenase, NAD+ and ethanol, was
increased fluorescence intensity observed, indicating the potential
of RA-Resa to study the NAD+ꢀdependent enzyme activities. In
addition, NADH is contained in lots of dietary supplements, but
its strong reduction ability makes it easily oxidized to NAD+. In
this work, probe RA-Resa was also successfully used to accurateꢀ
ly measure the NADH content of dietary supplement in a fluoresꢀ
cence assay (Figure S20).
NADH in live oral squamous cell carcinoma (OSCC) cells. Iniꢀ
tially, we performed the MTT assay by incubating with RA-Resa
at different concentrations for 6 h and 24 h respectively. The reꢀ
sults indicate the low cytotoxicity of the probe RA-Resa in live
cells (Figure S21). Then, the subcellular distribution of RA-Resa
was examined in live OSCC cells. The colocalization of signal
from probe and organelle markers indicated that the probe could
distribute across the whole cell, mainly in the cytoplasm rather
than in specific organelles (Figure S22). To demonstrate the capaꢀ
bility of RA-Resato detect intracellular NADH, live cells were
incubated with glucose at different concentrations prior to treatꢀ
ment with probe. This is because intracellular NADH levels greatꢀ
ly depend on the glycolysis process, which is affected by the gluꢀ
cose concentration. As expected, when treated with 1 mM gluꢀ
cose, RA-Resa stained cells showed slightly stronger fluoresꢀ
cence signal in yellow than in the absence of glucose (Figure 5
and S23). Meanwhile, further enhanced fluorescence was obꢀ
served after incubation with a higher concentration of glucose (20
mM). This result is therefore consistent with previously reported
work.24 Meanwhile, negligible fluorescent signal was observed in
live OCSS cells after incubation with control probe Con-Resa
(Figure S24). In addition, it was reported that the interconversion
between lactate and pyruvate affects the balance of intracellular
NADH and NAD+ species.26 As shown in Figure 5E and 5F,
strong fluorescence signal of the cells was observed after incubaꢀ
tion with 10 mM lactate, while the cells treated with pyruvate and
lactate simultaneously displayed much weaker fluorescence, indiꢀ
cating the roles of lactate and pyruvate in changing intracellular
NADH levels. Additionally, The similar phenomena were also
observed in human cervical cancer cell (HeLa) line and Chinese
Inspired by the excellent performance of this NADH probe in
vitro, we evaluated the application of RA-Resa for detecting
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Figure 5. Microscopic images of probe RA-Resa (5 µM, 15 min)ꢀ
stained live OSCC cells after incubation in following conditions.
A,D) no glucose for 30 min; B) 1 mM glucose for 30 min; C) 20
mM glucose for 30 min; E) 10 mM lactate for 10 min; F) 10 mM
lactate and 5 mM pyruvate for 10 min. Scale bar: 50 µm.
hamster ovary cell (CHO) lines (Figure S25 and S26). Furtherꢀ
more, the NADH levels under the above conditions were quantiꢀ
fied by NADH assay kit, which are consistent with microscopic
images (Figure S27).
In conclusion, inspired by the enzymeꢀcatalyzed sensing proꢀ
cess, we present a novel combinationꢀreaction twoꢀstep sensing
strategy to develop fluorescent probes. Based on this bioꢀinspired
strategy, the representative complex biomolecule, NADH, was
successfully detected by the boronic acidꢀcontaining fluorescent
probe, BA-Resa. This probe showed excellent sensitivity (detecꢀ
tion limit: 0.084 µM) and selectivity to NADH in alkalescent
conditions in the absence of any enzymes. Subsequently, this
NADH probe was further modified to give RA-Resa, which seꢀ
lectively responds to NADH at intracellular concentrations at
physiological pH (7.4). Significantly, the probe RA-Resa not only
provides a simple way to detect NADH in vitro, but also displays
the feasibility to evaluate NADH levels in live cell imaging. Altꢀ
hough there are still some drawbacks to this probe, such as photoꢀ
instability and ease of washout, this work provides a novel and
practical approach to increase sensitivity and selectivity when
designing fluorescent probes for sensing complicated biomoleꢀ
cules.
ASSOCIATED CONTENT
Supporting Information
Synthetic procedures and compound characterization data, quanꢀ
tum efficiency determination details, and computational data. This
material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
chmcyt@nus.edu.sg
Notes
The authors declare no competing financial interests.
(36) Wang, L.; Yuan, L.; Zeng, X.; Peng, J.; Ni, Y.; Er, J. C.; Xu, W.;
Agrawalla, B. K.; Su, D.; Kim, B.; Chang, Y. T. Angew. Chem. Int. Ed.
2016, 55, 1773.
(37) Guilbault, G. G.; Kramer, D. N.; Goldberg, P. J. Phys. Chem.
1965, 69, 3696.
ACKNOWLEDGMENT
This research was supported by an intramural funding from the
A*STAR Biomedical Research Council and Singapore Ministry
of Education Academic Research Fund Tier 1 (Rꢀ143ꢀ000ꢀ602ꢀ
112). We thank SBICꢀNikon Imaging Centre for confocal microsꢀ
copy facilities.
(38) Iannazzo, L.; Benedetti, E.; Catala, M.; EtheveꢀQuelquejeu, M.;
Tisne, C.; Micouin, L. Org. Biomol. Chem. 2015, 13, 8817.
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