Discrimination of redox-responsible biomolecules by a single molecular sensor

Article abstract of DOI:10.1021/ol303403x

A new application of a fluorescent sensor (PyDPA) for the discrimination of redox-responsible molecules is reported. Nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NAD ⁺/NADP⁺) and flavin mononucleotid

Full text of DOI:10.1021/ol303403x

ORGANIC
LETTERS
2013
Vol. 15, No. 6
1210–1213
Discrimination of Redox-Responsible
Biomolecules by a Single Molecular
Sensor
Jinrok Oh and Jong-In Hong*
Department of Chemistry, College of Natural Sciences, Seoul National University,
Seoul 151-747, Korea
jihong@snu.ac.kr
Received January 15, 2013
ABSTRACT
A new application of a fluorescent sensor (PyDPA) for the discrimination of redox-responsible molecules is reported. Nicotinamide adenine dinucleotide/
nicotinamide adenine dinucleotide phosphate (NADþ/NADPþ) and flavin mononucleotide/flavin adenine dinucleotide (FMN/FAD) were differentiated by
means of ratiometric fluorescence change from excimerꢀmonomer equilibrium and time-dependent fluorescence change, respectively.
Redox reactions are ubiquitous in biological systems
because they are involved in protein regulation, oxidative
phosphorylation, and metabolic reactions.1 Redox-respon-
sible functional groups, such as N-alkyl-β-nicotinamide and
the isoalloxazine moiety, are involved in biological redox
reactions.
Nicotinamide adenine dinucleotide (NADþ) and nico-
tinamide adenine dinucleotide phosphate (NADPþ) con-
tain β-nicotinamide, which is responsible for hydride-
transfer reactions. In particular, NADþ and NADPþ are
known to be extensively involved in oxidative reactions
and reductive reactions, respectively.1 Flavin mononucleo-
tide (FMN), flavin adenine dinucleotide (FAD), and ribo-
flavin have the isoalloxazine moiety in common, and they
serve as either electron acceptors in the oxidized form or as
electron donors in the reduced form. As a prosthetic group
in flavoproteins, they are involved in metabolic reactions
and the redox reactions of electron transport chains.2 The
most intriguing feature about these molecules is the
relationship between their structures (Figure 1) and their
roles. For example, NADPþ has the same structure as that
of NADþ except for an additional phosphate group at the 20
position of the ribose ring connected to the adenine moiety.
The slight structural difference between NADþ and NADPþ
leads to a significant difference in their roles: NADþ for
oxidative and NADPþ for reductive reactions.
Capillary electrophoresis (CE) techniques have been de-
veloped for the detection of nicotinamide derivatives3 and
flavins.4 However, CE cannot be used for the real-time or in
vivo detection of those redox-responsible biomolecules. It is
known that the adenine unit of FAD significantly decreases
the fluorescence of isoalloxazine by an intramolecular photo-
induced electron transfer (PeT) quenching mechanism,5
which has attracted considerable attention in the detection
and discrimination of flavins using fluorescence methods.
Recently, we successfully demonstrated that sensors deri-
ved from the bis(Zn(II)-DPA) complex (DPA, dipicolylamine)
could detect phosphate-containing molecules (Figure 2):
(3) Soga, T.; Igarashi, K.; Ito, C.; Mizobuchi, K.; Zimmermann,
H. P.; Tomita, M. Anal. Chem. 2009, 81, 6165.
(4) Britz-McKibbin, P.; Markuszewski, M. J.; Iyanagi, T.; Matsuda,
K.; Nishioka, T.; Terabe, S. Anal. Biochem. 2003, 313, 89.
(5) Sarma, R. H.; Dannies, P.; Kaplan, N. O. Biochemistry 1968, 7,
4359.
(1) Berg, J. M.; Tymoczko, J. L.; Stryer, L. Biochemistry; W. H.
Freeman and Company: New York, 2006.
(2) (a) Walsh, C. Acc. Chem. Res. 1980, 13, 148. (b) Bruice, T. C. Acc.
Chem. Res. 1980, 13, 256. (c) Massey, V. Biochem. Soc. Trans. 2000, 28,
283.
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10.1021/ol303403x
Published on Web 03/05/2013
2013 American Chemical Society

PTZ-DPA [PTZ-DPA consists of bis(Zn(II)-DPA) and
phenothiazine (PTZ)] was utilized to discriminate between
FAD and FMN. FAD was detected by the fluorescence
increase through the catalytic self-lysis of FAD into cyclic
FMN (cFMN) and adenosine monophosphate (AMP),
while the fluorescence of FMN was significantly decreased
by PeT from the PTZ in PTZ-DPA to the isoalloxazine
group.6 PyDPA shows selective excimer-based recognition of
ppGpp which contains two pyrophosphate groups.7 These
results inspired us to study further the ability of PyDPA to
discriminate between redox-responsible molecules.
NADþ and NADP in 1 mM HEPES buffer solution using
PyDPA. Because NADPþ has an additional phosphate
group, we expected that only NADPþ could induce ex-
cimer formation. As depicted in Figure 3, the addition of
either NADþ or NADPþ resulted in a decrease in mono-
meric fluorescence of pyrene, and concomitantly, only
NADPþ showed increased excimer emission centered at
470 nm and the shape of excimer emission was the same as
reported previously.7,8 This result indicates that it is pos-
sible to discriminate NADPþ from NADþ by the fluoro-
metric method.
What would cause the decrease in the monomeric pyrene
emission upon the addition of either NADþ or NADPþ?
In a previously reported screening test, binding of adeno-
sine derivatives to PyDPA resulted in enhancement of the
monomeric pyrene emission;7 thus, the nicotinamide moi-
ety seems to be responsible for emission quenching of
pyrene. The underlying quenching mechanism was deter-
mined by performing electrochemical studies using 5
(Scheme S1) and BnN (Scheme S2) as model compounds
of pyrene and nicotinamide, respectively. Reduction poten-
tials of 5 and BnN were estimated to be ꢀ3.52 and ꢀ3.75 eV
from the vacuum level (Table S1). This slightly higher lowest
unoccupied molecular orbital (LUMO) level of pyrene
(∼0.2 eV) suggests that oxidative PeT quenching from the
excited pyrene to NADþ is possible. On the other hand, the
Figure 1. Chemical structures of NAD(P)þ (left) and FMN(FAD)
(right).
Figure 2. Chemical structures of PyDPA and PTZ-DPA.
First, we developed a new strategy for the synthesis of
PyDPA, as shown in Scheme S1. The previously reported
synthetic route employs several low-yielding steps such as
metalꢀhalogen exchange followed by transition metal-free
aliphatic halide substitution, and nucleophilic phenol sub-
stitution. The new synthesis replaced these two low-yielding
steps by a malonic acid Knoevenagel condensation-catalytic
hydrogenation-reduction sequence, and a Mitsunobu re-
action, respectively. Although the number of steps is in-
creased, overall yield is greatly improved. Notably, single
chromatographic separation at the final stage is sufficient
to produce pure PyDPA (for a detailed procedure and
characterization, see Supporting Information).
We conducted fluorescence titration experiments to
investigate the possibility of discrimination between
Figure 3. Fluorescence spectral changes upon the addition of
either (a) NADþ or (b) NADPþ in the presence of 20 μM
PyDPA in 1 mM HEPES buffer solution (0.1% DMSO,
pH = 7.40), λex = 344 nm.
(6) Rhee, H.-W.; Choi, S. J.; Yoo, S. H.; Jang, Y. O.; Park, H. H.;
Pinto, R. M.; Cameselle, J. C.; Sandoval, F. J.; Roje, S.; Han, K.; Chung,
D. S.; Suh, J.; Hong, J.-I. J. Am. Chem. Soc. 2009, 131, 10107.
(7) Rhee, H.-W.; Lee, C.-R.; Cho, S.-H.; Song, M.-R.; Cashel, M.;
Choy, H. E.; Seok, Y. J.; Hong, J.-I. J. Am. Chem. Soc. 2008, 130, 784.
Org. Lett., Vol. 15, No. 6, 2013
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highest occupied molecular orbitals (HOMOs) of 5 and
BnN were estimated to be ꢀ6.87 and ꢀ8.17 eV, respectively.
However, the calculated HOMO level of NADþ is not
expected to be in the nicotinamide core9 and the large energy
level difference (1.3 eV) is undesirable for electron transfer.10
Therefore, the D-type-PeT (electron transfer from the ex-
cited pyrene to nicotinamide species) mechanism seems
most plausible for quenching.
Further study on the binding between PyDPA and
NAD(P)þ was performed. In the case of NADþ, the
fluorescence spectral changes at 398 nm fitted well with a
one-to-one binding equilibrium, and its binding constant
(Ka) was estimated to be 4.4 ꢁ 104 Mꢀ1, which is similar to
the binding affinity toward monohydrogen phosphate.11
NADPþ showed more efficient quenching of the emission
of monomeric pyrene than did NADþ, and it does not
follow a one-to-one binding equilibrium. Interestingly, the
emission changes of PyDPA when a 2-fold concentration
of NADPþ (R2 = 0.983) is used indicates that PyDPA
interacts withNADPþ witha 2:1 binding stoichiometry via
coordination with diphosphate and phosphate moieties;
the two corresponding binding constants were assumed to
be very similar. Job’s plot analysis also supports a 2:1
binding stoichiometry. The apparent Ka of NADPþ was
estimated tobe 3.7ꢁ 105 Mꢀ1, whichis about 8timeslarger
than that of NADþ. One intriguing observation is that the
pyrene dimer formed in the 2:1 complex was not destroyed
upon addition of even large amounts of NADPþ during
the titration experiment. This is completely different from
the binding of PyDPA to ppGpp, in which the excimer
emission decreased at a high level of ppGpp.7,12 This
saturation behavior and ratiometric property toward
NADPþ would be advantageous in the selective sensing
of NADPþ, while ppGpp shows concentration-dependent
pyrene excimer emission, showing the highest excimer
emission at optimal guest concentration. The fact that
PyDPA-NADPþ complexation exhibits an increased Ka
compared to that of PyDPA binding with NADþ and
maintains the pyrene dimeric structure even at high con-
centrations of NADPþ can be understood in terms of the
cooperative effect of the aromatic groups involved in the
termolecular complexation.13
Figure 4. (a) Fluorescence spectral change upon addition of
FMN in the presence of 20 μM PyDPA. (b) Fluorescence
intensity change at 526 nm with time in the presence of 15 μM
FAD and either 1 μM (red circle) or 10 μM (black square) of
PyDPA. In both cases, experiments were performed in 1 mM
HEPES buffer solution (pH = 7.40), excitation = 344 nm.
enhanced, which may indicate that fluorescence resonance
energy transfer (FRET) between the two fluorophores
(pyrene and isoalloxazine) occurs (Figure 4a). However,
the normalized excitation spectra of FMN did not differ,
regardless of PyDPA presence, which implies that no
efficient FRET between PyDPA and FMN takes place
(Figure S8). Also, addition of pyrophosphate14 resulted in
a great enhancement in the emission intensity of both
PyDPA and FMN (Figure S9). These results suggest that
there is extensive mutual quenching between PyDPA and
FMN. Interestingly, however, we were able to detect green
fluorescence from isoalloxazine in a 1:1 mixture of PyDPA
and FMN under 365 nm UV light.
Next, we wondered if PyDPA can discriminate flavin
species by taking advantage of isoalloxazine’s fluores-
cence. A fluorescence titration experiment for FMN re-
vealed that the monomeric emission of PyDPA was greatly
decreased, while FMN emission centered at 526 nm was
The quenching mechanism of PyDPA by FMN (and
vice versa) was also studied by the electrochemical method.
Reduction potentials of both pyrene and FMN were
measured to be ꢀ3.52 eV, as shown in Table S1, indicating
that reversible electron transfer between the two LUMOs
of the fluorophores is possible. However, the estimated
HOMOs of pyrene and FMN are at ꢀ6.87 and ꢀ5.92 eV,
respectively, from the vacuum level. These data suggest
that efficient A-type PeT [electron transfer from the
(8) Schazmann, B.; Alhashimy, N.; Diamond, D. J. Am. Chem. Soc.
2006, 128, 8607.
(9) The HOMO of the nicotinium core is estimated to be ꢀ8.17 eV.
The value is estimated from its reduction potential and optical band gap,
4.4 eV, corresponding to the absorption onset point, 280 nm.
(10) Siders, P.; Marcus, R. A. J. Am. Chem. Soc. 1981, 103, 748.
(11) Han, M. S.; Kim, D. H. Angew. Chem., Int. Ed. 2002, 41, 3809.
(12) From the energetic point of view, these phenomena might
originate from the similar interaction strengths of bisZn(DPA)-phos-
phate and pyrene-pyrene in water. This would result in stabilization of
the equilibrium state. However, in the ppGpp case, the binding strength
of bisZn(DPA)-pyrophosphate overwhelms that of pyrene-pyrene, and
thus, equilibrium would be shifted toward destroying the dimeric form
of pyrenes at high ppGpp concentration.
(14) Because pyrophosphate has a greater binding affinity toward the
1,3-bis(Zn-DPA) moiety than FMN, addition of pyrophosphate elim-
inates the weaker interaction between PyDPA and FMN.
(13) Kool, E. T. Chem. Rev. 1997, 97, 1473.
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Org. Lett., Vol. 15, No. 6, 2013

HOMO of FMN to the lower singly occupied molecular
orbital (SOMO) of excited PyDPA] quenching occurred.
From the relative energy level distribution, it is expected
that PyDPA is extensively quenched by FMN, while
FMN itself is partially quenched, which is exactly what
is observed.
catalysis (Figure 4b).6 This fluorescence change also agrees
well with pseudo-first-order kinetics, with an apparent rate
constant of 70 sꢀ1. These results indicate that PyDPA can
discriminate between riboflavin, FMN, and FAD with
respect to (time-course) fluorescence spectral change:
FMN is significantly quenched by the addition of a large
amount of PyDPA; FAD is detected by a gradual increase in
the fluorescence intensity of isoalloxazine with time upon
addition of PyDPA; and finally, the fluorescence of ribo-
flavin is not altered by PyDPA.
In conclusion, we developed a new synthetic strategy for
the production of PyDPA and investigated its further use
in the discrimination of redox-related biomolecules.
NADþ and NADPþ were distinguished by the presence
or absence of the excimer emission of PyDPA. FMN and
FAD were differentiated by means of the quenching of
FMN emission and the time-dependent fluorescence en-
hancement, respectively. Interesting features of sensing,
such as the cooperative effect of the aromatic groups
involved in the PyDPA-NADPþ complexation for main-
taining the pyrene dimeric structure or absorption sharing
of PyDPA and FMN in the detection of FMN, may give
insight indesigning sensorsand the analysisof complicated
emission profiles.
Our first attempt to rationalize the fluorescence changes
of PyDPA with respect to FMN concentration failed
under the assumption of a 1:1 binding mode. There are
two possible explanations for this: (1) more than one
quenching pathway is involved and (2) multiple binding
modes are involved. A SternꢀVolmer plot of PyDPA
(Figure S6), in the presence of FMN as a quencher, showed
two different proportional coefficients. This implies that
there are two major quenching pathways: PeT quenching
between isoalloxazine and pyrene, which is supported by
the electrochemical data; and absorption sharing of PyDPA
and FMN. Job’s plot reveals a 2:1 binding stoichiometry
between PyDPA and FMN (Figure S7), presumably
through coordination with phosphate and imide moieties.14
On the basis of these results and an assumption,15 the
PyDPA spectral change was successfully rationalized with
respect to FMN concentration. The apparent association
constant was estimated to be 1.93 ꢁ 105 Mꢀ1, and the
quenching efficiency of FMN to PyDPA was estimated to
be 1.0 (Figure S5), which is consistent with the results of
other experiments, such as the addition of pyrophosphate to
the PyDPA-FMN combination (Figure S9) and the excita-
tion spectra of FMN (Figure S8).
Acknowledgment. This workwas supported byan NRF
grant funded by the MEST (Grant No. 2012-0000159). J.O.
was supported by the Hi Seoul Science/Humanities Fellow-
ship from the Seoul Scholarship Foundation.
Because the catalytic group for the self-lysis of FAD
[bisZn(II)DPA] is exactly the same in PyDPA as in PTZ-
DPA, PyDPA is also expected to catalyze the lysis of FAD
into cFMN and AMP and show a similar fluorescence
enhancing behavior at 526 nm (which is exactly the emission
of isoalloxazine ring) with time as in the case of PTZ-DPA
Supporting Information Available. Synthesis details,
experimental details, calculation basis, fitting results,
additional spectroscopic data, and electrochemical data.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(15) As in the case of binding between PyDPA and NADPþ, the two
binding constants between PyDPA and FMN are assumed to be similar.
The authors declare no competing financial interest.
Org. Lett., Vol. 15, No. 6, 2013
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