New latent fluorophore for DT diaphorase

Article abstract of DOI:10.1021/ol052635e

This study describes the design and synthesis of a novel latent fluorophore 3 for DT diaphorase based on the trimethyl lock effect and characterization of its enzymatic kinetics. Fluorophore 3 is also a sensitive fluorimetric reagent for detecting glucose when coupled with DTD and glucose dehydrogenase.

Full text of DOI:10.1021/ol052635e

ORGANIC
LETTERS
2
006
Vol. 8, No. 2
65-268
New Latent Fluorophore for DT
Diaphorase
2
Sheng-Tung Huang*,† and Yuh-Ling Lin‡
College of Engineering, Department of Chemical Engineering and Biotechnology,
National Taipei UniVersity of Technology, Taipei 106, Taiwan, R.O.C., and
College of Medicine, Department of Medicine, Fu-Jen Catholic UniVersity,
Hsin-chuang, Taipei Hsien 24205, Taiwan, R.O.C.
ws75624@ntut.edu.tw
Received October 31, 2005
ABSTRACT
This study describes the design and synthesis of a novel latent fluorophore 3 for DT diaphorase based on the trimethyl lock effect and
characterization of its enzymatic kinetics. Fluorophore 3 is also a sensitive fluorimetric reagent for detecting glucose when coupled with DTD
and glucose dehydrogenase.
DT diaphorase (DTD) (EC 1.6.99.2) is a homodimeric,
cytosolic flavoprotein widely distributed and ubiquitously
present in all of the tissues of nearly all animal species. It
and normal tissue, this enzyme has attracted considerable
attention as a potential candidate for targeted anticancer
therapy. In addition, DTD and the DTD-targeted fluorescent
1
6
catalyzes the two-electron reduction of various quinones,
quinone epoxide, and aromatic nitrocompounds, using NADH
or NADPH as an electron donor. DTD is generally regarded
chromogen coupled with dehydrogenase have been broadly
used for inspecting and diagnosing morbid states, instead of
conventional chemical reagents.
2
7
as a protective enzyme. The reduction of electrophilic, redox-
active quinones by the enzyme is believed to result in
detoxification. Recent reports have indicated that DTD
Fluorescence has long been viewed as a powerful tool for
basic research in the biological sciences, the development
of new drugs, the assurance of food safety and environmental
3
7
activity maintains the reduced states of ubiquinones and
R-tocopherolquinone, thereby promoting their antioxidant
function in membranes.4 On the other hand, DTD is
overexpressed in many cancerous tissues compared to normal
tissue, especially nonsmall cell lung carcinoma, colorectal
quality, and the clinical diagnosis of diseases. Currently,
employing a redox reaction and a fluorimetric redox indicator
is a sensitive approach for glucose diagnostics. In this case,
an oxidizing or reducing with the presence of glucose results
in the reduction or oxidation of the fluorimetric redox
indicator or via a mediator, which allows for a qualitative
or quantitative determination. The known fluorimetric detec-
tion reagents for determining glucose have some drawbacks.
Oxygen-dependent fluorescent indicators for glucose are
5
carcinoma, liver cancers, and breast carcinomas. Because
of the difference in the DTD expression between a tumor
†
National Taipei University of Technology.
Fu-Jen Catholic University.
‡
(
1) Ernster L. Methods Enzymol. 1967, 10, 309-317.
2 2
determined by detecting H O generated by the glucose
oxidase. This reaction is catalytically supported by the
(
2) (a) Knox, R. J.; Friedlos, F.; Boland, M. P. Cancer Metastasis ReV.
1
1
993, 12, 195-212. (b) Hajo, A. K. D.; Winston, G. W. J. Biochem. Toxicol.
991, 6, 277-282. (c) Huang, M.-T.; Miwa, G. T.; Cronheim, N.; Lu, A.
Y. J. Biol. Chem. 1976, 254, 11223-11227.
(5) (a) Schlarger, J. J.; Powis, G. Int. J. Cancer 1990, 45, 403-409. (b)
Marin, A.; Lopez de Cerain, A.; Hamilton, E. Br. J. Cancer 1995, 72, 917-
921.
(6) Gutierrez, P. L. Free Rad. Biol. Med. 2000, 29, 263-275.
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Wiley-VCH: Weinhiem, Germany, 2002. (b) Iwata, K.; Kawahara, K.;
Nakajima, H. U.S. Patent 5,912,139, 1999.
(
3) (a) Hosoda, S.; Nakamura, W.; Hayashi, K. J. Biol. Chem. 1974,
49, 6416-6423. (b) Iyanagi, T. Chem. Scripta 1987, 27, 31-36.
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2
(
Burgess, J. R.; Navas, P.; Villalba, J. M. Biofactors 1999, 163-170. (b)
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1
0.1021/ol052635e CCC: $33.50
© 2006 American Chemical Society
Published on Web 12/16/2005

Scheme 1
peroxidase and is prone to interference by electron donors,
such as urea or bilirubin. In contrast, redox indicators that
were inspired by the trimethyl lock effect as a means to reveal
the cloaked fluorescence, which had been demonstrated by
Raines’s group. The rapid formation of 4,4,5,7-tetrameth-
8
12c
directly accept an electron from an oxidizing enzyme instead
9
of oxygen are preferred. The resazurin, transition metal Os,
ylhydrocoumarin from the corresponding o-hydroxycinnamic
10
13
and Ru complexes are the oxygen-independent fluorescent
indicators for glucose. In the case of transition metals, their
fluorescence efficiency varies with the oxygen content of
the sample. On the other hand, the nonfluorescent resazurin
is converted into fluorescence-developing resorufin by DTD,
acid is a result of the trimethyl lock effect. The unfavorable
steric interactions between three methyl groups enhance the
nucleophilicity of the phenolic oxygen and lead to a rapid
lactone formation, and this is an example of the use of strain
to enhance the reactivity. Our strategy for the optical
detection of the DTD activity also relies on the trimethyl
lock effect (Scheme 1). The chemistry of the trimethyl lock
effect of quinone as a masked phenol has been thoroughly
+
â-nicotinamide adenine dinucleotide (NAD ), and glucoses
9
dehydrogenase (GDH) in the presence of glucose. How-
ever, the resorufin can be further reduced by DTD to yield
a nonfluorescent product,11 and the emission bands of
resorufin formed by the redox reaction strongly overlap the
absorption bands of the nonreacted resazurin, which sig-
nificantly reduces the sensitivity of the glucose determina-
tion.
14
15
studied and utilized in the pro-drug strategies; however,
there have been no reports of this chemistry for designing
latent fluorophores. We envisioned that the trimethyl lock
effect would be suitable for the design of a novel latent
fluorophore for DTD. We selected rhodamine 110 (2) as the
fluorophore to be masked because of its high quantum yield,
long emission, excitation wavelength, and popularity for
Latent fluorophores are stable probes that unmask their
intense fluorescence only by a user-designated chemical
reaction, and they are especially useful tools for basic
research in the biological sciences. Recently, there are great
interests in developing latent fluorophores due to their unique
selectivity and minimal interference from the probe concen-
16
basic research in the biological sciences. We reasoned that
the installation of quinone acid 1 at the 3′ and 6′ positions
of a xanthenone scaffold would allow this platform to adopt
a closed and nonfluorescent lactone form. Upon selective
reduction of the quinone moiety in 3, the highly reactive
phenol 4 was generated followed by rapid lactone formation
with the concomitant release of the open and fluorescent
rhodamine 110 (2).
1
2
tration, excitation intensity, and emission sensitivity. We
(
8) Haughland, R. Handbook of Fluorescent Probes and Research
Chemicals, 6th ed.; Molecular Probes, USA 1996.
9) Matsu-Ura, S.; Yamauchi, Y.; Ohmori, H.; Maeda, H. Bunseki Kagaku
002, 51, 111-115.
10) (a) Ryabov, A. D.; Firsova, Y. N.; Ershov, A. Y.; Dementiev, I. A.
(
2
(
J. Biol. Inorg. Chem. 1999, 4, 175-182. (b) Woltman, S. J.; Even, W. R.;
(13) (a) Milstien, S.; Cohen, L. A. J. Am. Chem. Soc. 1972, 94, 9158-
9165. (b) Karle, J. M.; Karle, I. L. J. Am. Chem. Soc. 1972, 94, 9182-
9189.
Webber, S. G. Anal. Chem. 1999, 71, 1504-1512.
(11) Nims, R. W.; Prough, R. A.; Lubet, R. A. Arch. Biochem. Biophys.
1
984, 229, 459-465.
12) (a) Zlokarnik, G.; Negulescu, P. A.; Knapp, T. E.; Mere, L.; Burres,
N.; Feng, L.; Whitney, M.; Roemer, K.; Tsien, R. Y. Science 1998, 279,
(14) Borchardt, R.; Cohen, L. A. J. Am. Chem. Soc. 1972, 94, 9175-
(
9182.
(15) Gharat, L.; Taneja, R.; Weerapreeyakul, N.; Rege, B.; Polli, J.;
Chikhale, P. J. Int. J. Pharm. 2001, 219, 1-10.
(16) Leytus, S. P.; Melhado, L. L.; Mangel, W. F. Biochem. J. 1983,
209, 299-307.
8
4-88. (b) Chang, M. C. Y.; Pralle, A.; Isacoff, E. Y.; Chang, C. J. J. Am.
Chem. Soc. 2004, 126, 15392-15393. (c) Chandran, S.; Dickson, K. A.;
Raines, R. T. J. Am. Chem. Soc. 2005, 127, 1652-1653.
266
Org. Lett., Vol. 8, No. 2, 2006

The design and synthesis of the bioreductive quinone-
based cytotoxic analogue is an ongoing research effort in
our group.17 We are interested in developing a latent
fluorophore which is based on the trimethyl lock effect, with
the quinone as a masked phenol for detecting the activity of
DTD. In addition, glucose diagnostics could still benefit from
a latent fluorimetric reagent because the DTD and DTD-
targeted fluorescent chromogen coupled with GDH are ideal
oxygen-independent fluorimetric reagents for glucose diag-
nostics. Hence an objective of the present study was to
provide an alternative redox-active latent fluorophore as the
detection reagent for the fluorimetric determination of
glucose, which can at least partially eliminate the disadvan-
tage of the prior art. This study involves the synthesis and
characterization of the novel DTD latent fluorophore 3. In
addition, we established the latent fluorophore 3 as a sensitive
fluorimetric reagent for detecting glucose.
ylhydrocoumarin based on fluorescence intensity of rhodamine
110 generated. Employing the fluorescence calibration curve
of rhodamine 110, this enzymatic reaction synthesized the
4,4,5,7-tetramethylhydrocoumarin in 32% yield based on the
fluorescence intensity of rhodamine 110. The solution
containing 3 with DTD and NADH in PBS became fluo-
rescent within 1 min (Figure 2A). In contrast, the incubation
The synthesis of 3 was accomplished in one step by
15
coupling of the known acid 1 with rhodamine 110 (2) with
moderate yield (Scheme 1). The excitation and emission
spectra of 3 exhibited a near baseline even with co-incubation
with DTD alone (Figure 1), 1 mM NADH alone (data not
Figure 2. (A) Time course of the generation of fluorescence (λex
)
3
492 nm, λem ) 520 nm) by rhodamine (dashed line, 5 µM) and
(solid line, 5 µM) in PBS at 25 °C. Data are normalized at the
maximum emission. (B) Double-reciprocal plot of reduction of 3
per molecule per second (V) versus substrate concentration ([3]).
of rhodamine 110 with DTD (1 unit) and NADH (1 mM)
exhibited no reaction, thus the rhodamine 110 (2) is not the
substrate of DTD (Figure 2A). The apparent kinetic param-
eters of 3 with DTD have also been determined. The value
Figure 1. Excitation and emission spectra of 3 (10 µM) incubated
for 4 h at 37 °C in PBS with or without NADH.
5
-1 -1
of kcat/K
m
is 2.35 × 10 M s , and the K
m
value is 26.8
1
9
µM (Figure 2B). The fluorescence signal results from two
steps, quinone reduction followed by the intramolecular
lactonization. The rate-determining step for the fluorescence
signal seems to be the quinone reduction step because the
intramolecular lactonization induced by the trimethyl lock
effect has an effective rate of 10 M s . The fluores-
cence signal that resulted from quinone reductions designed
herein is 120-fold faster than the hydrolysis counterpart,
shown), or after months of storage in phosphate buffer (PBS)
at pH 7.3 (data not shown). The introduction of one unit of
human recombinant DTD and 1 mM NADH to the solution
resulted in a large increase in the fluorescence characteristic
of rhodamine 110 (2) after 4 h (Figure 1). The presence of
mild reduction agent dithiothreitol (1 mM) alone with 3 (10
µM) in PBS also resulted in an increase in the fluorescence
characteristic of rhodamine 110 after 4 h incubation at 37
6
-1 -1 13b
1
2
which was designed by Raines’s group. The affinity of the
quinone moiety in 3 toward DTD seems to be lower when
compared to that of other similar DTD target quinone-based
°
C. The quinone moiety in 3 is commonly found in various
18
1
8a
anticancer therapeutic agents; this might be due to the steric
interference of the bulky xanthenone scaffold in 3. This novel
DTD-targeted fluorogenic substrate 3 is a useful latent
fluorophore, a stable molecule with an intense fluorescence
that is unmasked by a user-designated chemical reaction.
We next assessed the ability of 3 as a fluorimetric reagent
for determining glucose. NADH is the necessary cofactor
DTD substrates, and one molecular rhodamine 110 in 3
requires two 4,4,5,7-tetramethylhydrocoumarins to be formed;
therefore, we estimated the yield of the 4,4,5,7-tetrameth-
(17) (a) Huang, S. T.; Kuo, H. S.; Hsiao, C. L.; Lin, L. Y. Bioorg. Med.
Chem. 2002, 10, 1947-1952. (b) Huang, S. T.; Tsai, H. D.; Kuo, H. S.;
Yang, Y. P.; Peng, Y. C.; Lin, Y. L. ChemBioChem 2004, 5, 797-803.
(18) Many known quinone base anticancer agents usually exhibit a low
micromolar affinity toward DTD. (a) Hargreaves, H. J.; Mayalarp, S. P.;
Butler, J.; McAdam, S. R.; O’Hare, C. C.; Hartley, J. A. J. Med. Chem.
(19) The kinetic data were obtained in PBS containing one unit of DTD
and 1 mM NADH. Unmasking of the second amino group leads to the
manifestation of a ca. 90% fluorescence.
1
997, 40, 357-361. (b) Phillips, R. M. Biochem. Pharm. 1996, 52, 1711-
1
718.
Org. Lett., Vol. 8, No. 2, 2006
267

for DTD to reduce the quinone moiety in 3, and NADH is
phore 3 coupled with DTD and GDH is a sensitive system
to detect glucose in the low micromolar range, and this
sensitivity is comparable to that of other fluorimetric reagent
systems, such as resazurin coupled with glucose oxidase.20
The fluorescence efficiency of the redox reaction and a
fluorimetric redox indicator developed herein is not influ-
enced by the oxygen content of the sample. Since the
fluorophore rhodamine 110 (2) is not the substrate for DTD
also the product of many dehydrogenases.
The GDH oxidizes glucose in the presence of NAD to
+
yield glucono-δ-lactone and NADH. The NADH generated
from the oxidation reaction coupled with DTD selectively
reduced the quinone moiety in 3, and the highly reactive
phenol 4 was generated followed by rapid lactone formation
with the concomitant release of the open and fluorescent
rhodamine 110 (2). Thus, 3 can be a useful fluorimetric
reagent for monitoring the GDH activities and glucose
concentration. As expected, the mixture of 3 (5 µM) with
(Figure 2A), the fluorescence intensity of rhodamine 110 is
not affected, and the sensitivity of the fluorimetric redox
indicator for the glucose determination is not reduced. The
study herein demonstrates that the DTD-targeted latent
fluorophore 3 is a fluorescent indicator for glucose upon
coupling with the NADH generating GDH.
In conclusion, this report describes the design of a new
class of fluorogenic substrates of DTD and the characteriza-
tion of its enzymatic kinetics, and it demonstrates its
applicability as a fluorimetric reagent for detecting glucose.
The new fluorogenic substrate is easy to make, simple to
use for detecting DTD activity, and has a high sensitivity
for determining the glucose concentration. Use of this latent
fluorophore to identify a high-affinity DTD substrate is
underway in our laboratory.
+
DTD (1 unit), GDH (1 unit), and NAD (40 µM) was nearly
nonfluorescent even after 1 h of incubation at 25 °C. The
addition of glucose with a 1 h incubation triggers a prompt
fluorescence increase that is characteristic of rhodamine 110
(2). A plot of the fluorescence intensity and the concentration
of glucose reveals a linear relationship at a glucose concen-
tration between 1 and 6 µM (Figure 3). The latent fluoro-
Acknowledgment. This work was supported by grants
from NSC (NSC 94-2113-M-027-004).
Supporting Information Available: Procedures for the
syntheses and analyses reported herein (PDF). This material
is available free of charge via the Internet http://pubs.acs.org.
OL052635E
Figure 3. Relationship of glucose concentration with fluorescence
intensity observed after a 1 h incubation of a mixture of 3 (5 µM),
+
glucose, DTD (1 unit), GDH (1 unit), and NAD (40 µM).
(20) Maeda, H.; Matsu-Yra, S.; Yamauchi, Y.; Ohmori, H. Chem. Pharm.
Bull. 2001, 49, 622-625.
268
Org. Lett., Vol. 8, No. 2, 2006