4-(N-methylhydrazino)-7-nitro-2,1,3-benzooxadiazole (MNBDH): A novel fluorogenic peroxidase substrate

Article abstract of DOI:10.1002/(SICI)1521-3773(20000417)39:8<1453::AID-ANIE1453>3.0.CO;2-7

A novel peroxide substrate with a benzooxadiazole backbone: 4-(N- methylhydrazino)-7-nitro-2,13-benzooxadiazole (MNBDH; 1) is introduced as a prototype for a new class of fluorogenic peroxide substrates. The advantages of 1 lie in its lower detection and quantification limits, a favorable red- shifted fluorescence signal, and a greater signal, and a greater signal stability after the successful reaction. The new substrate was validated by the determination of α-D-glucose in beverages.

Full text of DOI:10.1002/(SICI)1521-3773(20000417)39:8<1453::AID-ANIE1453>3.0.CO;2-7

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4
-(N-Methylhydrazino)-7-nitro-2,1,3-
6
benzooxadiazole (MNBDH): A Novel
Fluorogenic Peroxidase Substrate**
2
Jörg Meyer, Andrea Büldt, Martin Vogel, and
Uwe Karst*
Enzymes are characterized by a range of attractive features
for applications in analytical chemistry. Low analyte detection
limits, even in complex matrices, are realized by their high
catalytic activity and substrate selectivity. In this context,
peroxidases are of special interest because of the possibility of
coupling H O detection withreactions yielding H O . In
addition to the type and activity of the peroxidase, the lower
detection limit of the enzymatic reaction is influenced by the
chromogenic or fluorogenic substrate used, as the properties
of the detected reaction product play a crucial role. A variety
of organic compounds are used as chromogenic substrates, for
example, aromatic amines like o-phenylenediamine (OPD),
3,3'-5,5'-tetramethylbenzidine (TMB),[ and also 2,2'-azino-
bis(3-ethylbenzothiazolin)-6-sulfonate as the diammonium
salt (ABTS) . In case of the more sensitive fluorogenic
methods the p-hydroxyphenylcarboxylic acids, especially p-
hydroxyphenylacetic acid (pHPA), have found widespread
application.[
The main disadvantage of the p-hydroxyphenylcarboxylic
acids and other fluorogenic substrates is the difference
between the optimum pH for the enzymatic reaction, which
lies in a moderately acidic range,[ and for fluorescence
detection of the products, where alkaline media are re-
[
[
2
2
2
2
[
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[6]
fluorophore and, in some cases, not even the exact structure
of the fluorescent compounds obtained could be elucidated.
Hydrazine reagents are the most popular group of deriva-
tising reagents for carbonyl compounds.[ In this work, we
describe the use of a hydrazine reagent as a fluorogenic
peroxidase substrate. Surprisingly, the nonfluorescent 4-(N-
methylhydrazino)-7-nitro-2,1,3-benzooxadiazole (MNBDH),
which was recently introduced as reagent for the determi-
[
[
7]
3
6, 722 ± 724.
[
15] K. Barlos, D. Gatos, J. Kallitsis, G. Papaphotiu, P. Sotiriu, Y.
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Kuhlmann, Nature 2000, 403, 223 ± 226.
nation of carbonyl groups[ and nitrite ions, is oxidized by
H O in presence of peroxidase (POD) to the intensively
8]
[9]
[
2
2
fluorescing
4-(N-methylamino)-7-nitro-2,1,3-benzooxa-
diazole (MNBDA) [Eq. (1)]. The identity of the reaction
product was verified by NMR, UV/Vis, and fluorescence
spectroscopy, mass spectrometry, and HPLC. The enzymatic
reaction and fluorescence detection are carried out in a mildly
[
*] Priv.-Doz. Dr. U. Karst, Dipl.-Chem. J. Meyer, Dipl.-Chem. M. Vogel
Anorganisch-chemisches Institut
Westfälische Wilhelms-Universität Münster
Wilhelm-Klemm-Strasse 8, 48149 Münster (Germany)
Fax : (‡49)251-83-33169
E-mail: uwe.karst@uni-muenster.de
Dr. A. Büldt
BASF AG, Ludwigshafen (Germany)
[
**] The authors would like to thank the Fonds der Chemischen Industrie
Frankfurt/Main) for financial support.
(
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acidic medium (pH 5.8). In case of MNBDH, similar pH
optima for conversion of the substrate and detection of the
product are observed. The HPLC experiments, with UV/Vis
detection of the reaction mixture, show only one additional
peak in the chromatogram; its retention time and UV/Vis
spectrum matcht ha t of pure MNBDA. Withfluorescence
detection (excitation wavelength: 470 nm, emission wave-
length: 547 nm) only the MNBDA peak appears in the
chromatogram. No other reaction products could be observed
by liquid chromatography.
Figure 1. Fluorescence spectrum of MNBDA.
For the determination of H O , based on the procedure
2
2
À8
described below, the limit of detection (LOD) is 2.2 Â 10
m
À8
and the limit of quantification (LOQ) is 7.5 Â 10 m. These
values are superior by a factor of three compared to those
obtained for pHPA. In addition, the selectivity is improved
using MNBDH because of the red-shifted excitation and
emission maxima.
The MNBDH method was validated by comparison with
Figure 2. Time-based development for the reaction of MNBDH and
pHPA withperoxidase and H O .
2 2
two established peroxidase substrates, ABTS[ and pHPA,
10]
[11]
through the determination of glucose in four different
beverages. Glucose is oxidized by aerial oxygen in the
presence of glucose oxidase, and forms hydrogen peroxide.
The latter is detected in a peroxidase-catalyzed reaction based
on one of the substrates mentioned above.
The analytical figures of merit of the three methods are
listed in Table 1. The photometric method had the smallest
relative standard deviation but it is an order of magnitude less
sensitive than the fluorimetric techniques. The LOD and
LOQ withMNBDH are lower by a factor of t hr ee compared
to pHPA. Further advantages of MNBDH are the red-shifted
excitation and emission maxima (see also the fluorescence
spectrum depicted in Figure 1) and an improved signal
stability (Figure 2). Advantages for pHPA are the better
water solubility and a higher reaction rate.
Table 2. Results for the determination of glucose in soft drinks. All values
refer to the content of a-d-glucose.[
a]
À1
Method
Apple juice
Orange juice
Cola [gL
]
Instant tea
À1
À1
À1
À1
[
gL
]
[gL
]
[gL
]
[%ww
]
ABTS405
ABTS649
ABTS732
MNBDH
pHPA
20.6 Æ 0.4
20.9 Æ 0.6
21.0 Æ 0.5
22.0 Æ 0.4
20.8 Æ 2.1
25.4 Æ 1.1
26.0 Æ 1.4
26.0 Æ 1.0
26.4 Æ 0.5
26.1 Æ 1.4
41.1 Æ 0.6
42.4 Æ 0.8
42.7 Æ 0.6
44.2 Æ 2.1
50.0 Æ 1.7
20.6 Æ 1.0
20.6 Æ 1.3
20.3 Æ 1.0
20.0 Æ 1.2
15.3 Æ 1.4
[
a] Deviations are calculated as the standard deviation of the mean value of
eight separate determinations.
directed optimization should yield substrates with increased
potential.
The results of the determination of a-d-glucose in soft
drinks are presented in Table 2. Apart from the data for cola
and lemon-flavored instant tea obtained by using pHPA, all
results correspond well.
The MNBDH reagent may therefore be seen as prototype
of a new and powerful class of peroxidase substrates for which
Experimental Section
Commercial samples investigated in this study were apple juice, orange
juice, cola, and lemon-flavored instant tea. Liquid samples were diluted
directly (by a factor of 10000 for detection withABTS or pHPA, and by a
Table 1. Parameters for the determination of glucose using the peroxidase substrates ABTS, MNBDH, and pHPA.
Method
Limit of
detection [m][
Limit of
quantification [m]
Averaged
standard deviation [%]
Smallest/largest
standard deviation [%][d]
Linear
range [m]
a]
[b]
[c]
ABTS405[e]
ABTS649
ABTS732
MNBDH
pHPA
6 Â 10
À7
2 Â 10
À6
2.7
2.8
1.9
3.6
5.3
5.7/0.3
7.3/0.4
4.2/0.4
5.6/2.7
8.8/2.7
2 Â 10 ± 2 Â 10
À6
À4
À4
À4
À5
À5
À6
À6
À6
1 Â 10
3 Â 10
3 Â 10 ± 2 Â 10
À7
À6
À6
5 Â 10
2 Â 10
2 Â 10 ± 2 Â 10
À8
À7
À7
5 Â 10
2 Â 10
2 Â 10 ± 2 Â 10
À7
À7
À7
2 Â 10
5 Â 10
5 Â 10 ± 2 Â 10
[
a] Calculated as the triple standard deviation of the blank. [b] Calculated as the tenfold standard deviation of the blank. [c] Relative standard deviation of
eight separate determinations, averaged for the linear range. [d] Relative standard deviation of eight separate determinations, smallest and largest value of
the linear range. [e] Subscripts correspond to the wavelengths (in nanometers) used for photometric measurements.
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factor of 50000 for MNBDH). The instant tea was weighed and dissolved in
bidistilled water (0.5 g in 10 mL water) and diluted in the same way.
s-Homoconjugation in Cyclically Preoriented
N4-(Radical) CationsÐN ´´´ N Bond Lengths
All measurements were carried out using microplate readers.
>
2 Š**
Glucose oxidase (GOD) reaction (I): 50 mL of a solution of GOD (15 mg)
in acetate buffer (10 mL; pH ˆ 5.5; 0.01m) were added to 100 mL of a
glucose solution. After mixing thoroughly, the solution was incubated for
Kai Exner,* Birgit Groûmann, Georg Gescheidt,
Jürgen Heinze, Manfred Keller, Thomas Bally,
Pavel Bednarek, and Horst Prinzbach*
1
5 min at 378C.
GOD reaction (II): as I, but 20 mL GOD solution and 40 mL glucose
solution were used.
(
pp)s-Conjugation, the electron delocalization between
GOD reaction (III): as I, but the GOD solution was prepared with
collinear p-orbitals, is a borderline case for the theory of
chemical bonding.[ It occurs efficiently only in compounds
difficult to synthesize and with specific steric/stereoelectronic
prerequisites. Making use of cage-fixed cyclobutanes A (X ˆ
C, (iso)pagodanes), of preoriented dienes B (X ˆ C, (iso)-
pagodadienes/(seco)dodecahedradienes), and bisdiazenes/
bisdiazenetetroxides B (X ˆ N, NO), the effective s-homo-
conjugation or s-bishomoaromaticity in 3C/3(2)e cations D
phosphate buffer (pH 5.8; 0.01m).
1]
Glucose determination using POD and ABTS: 50 mL of a solution
containing POD (0.5 mg) and ABTS (5.5 mg) in acetate buffer (10 mL;
pH 5.5; 0.01m) were pipetted to the mixture of reaction I. After the sample
had been mixed thoroughly and incubated for 10 min at room temperature,
the absorbance of the samples was measured at 405, 649, and 732 nm.
Glucose determination using POD and pHPA: 50 mL of a solution
containing POD (2.5 mg) and pHPA (7.6 mg) in ammonium buffer
(
10 mL; pH 9.5; 0.01m) were pipetted to the mixture of reaction II. After
[2]
[3, 4]
(
X ˆ C), in 4C(N)/3(2)e cations C (X ˆ C, NO),
and in
the sample had been mixed thoroughly and incubated for 15 min at room
temperature, the fluorescence of the samples was measured at excitation
and emission wavelengths of 320 and 405 nm, respectively.
[5]
4
N/5(6)e anions C (X ˆ N) have been observed. Here, we
Glucose determination using POD and MNBDH: 1 mg MNBDH was
dissolved in 10 mL acetonitrile. Of this solution, 1.4 mL are added to a
solution containing POD (2.5 mg) in phosphate buffer (10 mL; pH 5.8;
0
.01m). From this mixture, 40 mL were pipetted into the mixture of
reaction III. After the sample had been mixed thoroughly and incubated
for 10 min at room temperature, the fluorescence of the samples was
measured at excitation and emission wavelengths of 470 and 545 nm,
respectively.
Received: December 6, 1999 [Z14358]
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[
[
[
[
[
*] Dr. K. Exner, Prof. Dr. H. Prinzbach, Dr. M. Keller
Institut für Organische Chemie und Biochemie der Universität
Freiburg
[
7] R. K. Beasley, C. E. Hoffmann, M. L. Rueppel, J. W. Worley, Anal.
Chem. 1980, 52, 1110 ± 1114.
[
8] A. Büldt, U. Karst, Anal. Chem. 1999, 71, 1893 ± 1898.
Albertstrasse 21, 79104 Freiburg (Germany)
Fax : (‡49)761-203-5987
[
9] A. Büldt, U. Karst, Anal. Chem. 1999, 71, 3003 ± 3007.
[
[
10] ABTS is oxidized to an intensely green radical cation and shows UV/
Vis absorption maxima at 405, 415, 649, 732, and 815 nm.
11] The phenol derivative pHPA is dimerized in the peroxidase-catalyzed
reaction to form the corresponding biphenol, which fluoresces in
alkaline media (pH 9.5) withexcitation and emission maxima of 323
and 403 nm, respectively.
E-mail: horst.prinzbach@organik.chemie.uni-freiburg.de
Dr. B. Groûmann, Prof. Dr. J. Heinze
Institut für Physikalische Chemie der Universität Freiburg
Albertstrasse 21, 79104 Freiburg (Germany)
Priv.-Doz. Dr. G. Gescheidt
Institut für Physikalische Chemie der Universität Basel
Klingelbergstrasse 80, 4056 Basel (Switzerland)
Prof. Dr. T. Bally, Dipl.-Chem. P. Bednarek
Institut für Physikalische Chemie, Universität Fribourg
Perolles, 1700 Fribourg (Switzerland)
[
**] This project has been supported by the Deutsche Forschungsgemein-
schaft, the Fonds der Chemischen Industrie and BASF AG. We thank
Dipl.-Chem. V. Peron and B. Geiser for technical assistance.
Supporting information for this article is available on the WWW under
http://www.wiley-vch.de/home/angewandte/ or from the author.
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