A fluorescent probe for the detection of NAD(P)H

Article abstract of DOI:10.1039/a904971a

NAD(P)H may be monitored by using reduction to release the fluorophore umbelliferone from a precursor conjugate with a quinoxalinium salt.

Full text of DOI:10.1039/a904971a

A fluorescent probe for the detection of NAD(P)H
Carl A. Roeschlaub, Nicola L. Maidwell, M. Reza Rezai and Peter G. Sammes*
Molecular Probes Unit, Department of Chemistry, School of Physical Sciences, UNIS, Guildford, Surrey,
UK GU2 5XH. E-mail: p.sammes@surrey.ac.uk
Received (in Cambridge, UK) 22nd June 1999, Accepted 16th July 1999
NAD(P)H may be monitored by using reduction to release
the fluorophore umbelliferone from a precursor conjugate
with a quinoxalinium salt.
to the 1,2-dihydro isomer 9, isolated in high yield ( > 90%). In
contrast to the dihydrophenazine 4, the dihydroquinoxaline 9 is
relatively stable, although, upon exposure to air it is oxidised to
give a variety of highly coloured products. It is to be noted that
both 4 and 8 are both formally anti-aromatic and this
undoubtedly contributes to their chemical reactivity. Reduction
NADH and its phosphate ester NADPH are ubiquitous reducing
agents in nature. In rapidly proliferating cervical cancer cells the
turnover of NAD(P)H is greater than in normal cells and thus it
4
of the salt 7 can also be effected with NaBH , although with an
1
can be used as a general biological marker for cancer. A
excess of this reagent, over-reduction to give the tetra-
hydroquinoxaline 10 was observed. None of 10 was observed
upon reduction of 7 with NADH.
potential fluorescent probe for the detection of these aberrant
2
cells has been described that uses the reduction of the weakly-
fluorescent dye resazurin 1 to the fluorescent derivative
Because of its bimolecular nature, the rate of reduction of the
quinoxalinium salt 7 by NADH is concentration dependent. At
concentrations in the micromolar range the reduction takes
several hours to complete but at millimolar concentrations
reaction is complete within seconds.
In order to generate a useful fluorescent probe, 2-methylqui-
noxaline 5 was brominated at the methyl group, using NBS, and
the bromomethyl compound 6 then conjugated with umbellifer-
one 16 to produce the derivative 11. Methylation of this base
with MeOTf afforded the triflate salt 12. Neither of the
compounds 11 nor 12 showed any significant fluorescence.
Reduction of the salt 12 with NADH was effected by adding
a solution of the salt to a stirred solution of NADH at pH 7.5 and
immediately re-measuring the emission spectrum. A signal
corresponding to that of free umbelliferone 16 formed almost
immediately. That this was due to liberated umbelliferone 16
was also confirmed by TLC and direct isolation; in a
quantitative experiment > 85% 16 could be extracted from the
solution. In the absence of NADH no formation of umbellifer-
one 16 was observed, confirming that the liberation was a direct
consequence of reduction by NADH.
resorufin 2. A disadvantage of this system is that direct
reduction of resazurin to resorurufin does not occur and the
reaction has to be ‘catalysed’ by an electron carrier such as
The fate of the quinoxalinium moiety has also been explored.
Reduction is assumed to give, initially, the 1,4-dihydro
intermediate 13. Rather than tautomerising, in the manner
observed for the conversion of the unsubstituted quinoxalinium
salt (7 to 8 to 9), to give 14, which has not been observed,
elimination of umbelliferone occurs to give the quinoxalinium
3
phenazinium methosulfate 3 or an enzyme such as diaphorase.
2
The mechanism of this reduction has been investigated and, for
the catalyst phenazinium methosulfate, shown to proceed via
initial formation of the dihydrophenazine derivative 4, which is
extremely unstable and rapidly undergoes further redox reac-
tions.
NAD(P)H is also co-produced by a number of enzyme-based
redox systems, such as glucose-6-phosphate dehydrogenase4
and is used in a number of enzyme linked immunosorbent
5
assays (ELISA). In these assays a catalyst has also to be used,
often another enzyme such as diaphorase, to act as the electron
carrier in conjunction with either resazurin, reduced to the
3
fluorescent resorufin, or of a leuco-dye, such as a tetrazolium
salt, which is reduced to the corresponding, highly coloured
formazan.6
In order to avoid the need for these two-component systems
involving an electron carrier, conjugation of a masked fluor-
ophore to a reducible heteroaromatic system was considered, in
which the reduction would unmask the fluorophore. After
studying several systems we focused on use of derivatives of the
quinoxalinium system. 2-Methylquinoxaline 5 was found to
react regioselectively with methylating agents, such as MeI and
MeOTf, to produce the corresponding 1,3-dimethylquinox-
alinium salt 7.† Reduction of these salts with NADH at pH 7.5
rapidly produces a dihydro compound. Whilst mechanistic
studies with NADH predict that this will be the 1,4-dihydro
7
isomer 8, this was not obtained since it efficiently tautomerises
Scheme 1 Reagents and conditions: i, NADH, pH 7.5.
Chem. Commun., 1999, 1637–1638
1637

under 24 min. Control fluorescence measurements on pure
umbelliferone, in the presence of 1 equiv. of the imine 7,
indicate that release of the umbelliferone from the salt 12 is
quantitative.
We thank the EPSRC for support of this work and the EPSRC
National Mass Spectrometry Service Centre, Swansea, Wales,
for accurate mass measurements.
Notes and references
†
All new compounds gave satisfactory microanalyses and/or accurate mass
measurements.
1
S. K. Jonas, C. Benedetto, A. Flatman, L. Micheletti, C. Riley, P. A.
Riley, D. Spargo, M. Zonca and T. F. Slater, Br. J. Cancer, 1992, 66,
185.
Fig. 1 Increase in emission with time; scans at 2 min. intervals; maximum
reached in < 24 min. See text for conditions.
2 L. P. Candeis, D. P. S. MacFarlane, S. L. W. McWhinnie, N. L. Maidwell,
C. A. Roeschlaub, P. G. Sammes and R. Whittlesey, J. Chem. Soc.,
Perkin Trans. 2, 1998, 2333.
tautomer 15, which then reforms the quinoxalinium species 7,
followed by a second NADH reduction step to produce the
imine 9, which has also been isolated and characterised (see
Scheme 1).
3
D. B. Cook and C. H. Self, Clin. Chem., 1993, 39, 965.
4 H. Shah, A. M. Saranko, M. Harkonen and H. Adlercreutz, Clin. Chem.,
1984, 30, 185.
5 For a review see A. Voller and D. E. Bidwell, Enzyme Immunoassays, in
Alternative Immunoassays, ed. W. P. Collins, Wiley, Chichester, 1985,
pp. 77–86.
Fig. 1 shows the results of a typical reaction. The increase of
umbelliferone 16 fluorescence is obtained by mixing a solution
6
E. Seidler, The Tetrazolium-Formazan System: Design and Histochem-
istry, in Progress in Histochemistry and Cytochemistry, Gustav Fischer
Verlag, Stuttgart, 1991, vol. 24, pp. 1–86.
G. Blankenhorn, in Pyridine Nucleotide Dependent Dehydrogenases, ed.
H. Sund, W. de Gruyter & Co., Berlin, 1997, pp. 185–205.
2
4
23
23
of the salt 12 at 1 3 10 mol dm with NADH at 1 3 10
2
3
mol dm in TRIS buffer at pH 7.5, taking aliquots at 2 min
intervals and diluting 100-fold in buffer (to give a sample at 1 3
7
2
6
23
1
(
l
0
mol dm ) before measuring the fluorescence spectrum,
l
ex 350 nm), monitoring the umbelliferone emission peak at
em 450 nm. Under these conditions reduction is observed in
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Chem. Commun., 1999, 1637–1638