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(3) In addition to its specificity, for the explicit analysis, 13C2-
DA is designed as an activatable probe, which can produce a 1H
signal in an off-to-on manner (left to right in Figure 1). Under
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the multiple-resonance pulse scheme for H next to successive
aliphalic-13C (13CA) and carbonyl-13C (13CO) sequence, the
DA metabolite 13C2-DOPAC can be specifically detected
because of the presence of the 1H−13CA−13CO sequence
(red in Figure 1). Again, we expected that the designed probe
would specifically and explicitly “sense” DA metabolism in
complex biological systems.
13C2-DA was synthesized in a three-step process using
dimethylformamide-carbonyl-13C and 13C-nitromethane as the
13C sources (Supporting Information).
NMR measurements showed that the designed 13C2-DA
actually worked as a probe in the analysis of DA metabolism.
13C2-DA was incubated in the presence or absence of the
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purified enzymes MAO-A and ALDH, and subjected to H−
{13CA−13CO} NMR analysis (Figure 2a). 13C2-DA itself
produced no detectable signal (upper left in Figure 2a). In
marked contrast, after 13C2-DA was incubated with the enzymes
for 4 h, a clear singlet signal appeared around 3.3 ppm (upper
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right in Figure 2a), which was confirmed to be H of 13C2-
DOPAC by comparison with an authentic sample. The addition
of a known MAO-A-selective inhibitor, clorgyline, completely
suppressed the signal (bottom right in Figure 2a). A time
course analysis showed an incubation-time-dependent increase
of the 13C2-DOPAC signal (left in Figure 2b), which was
compatible with that observed in an HPLC−UV analysis
(Figure 2b, right). The consumption of 13C2-DA and
production of 13C2-DOPAC were also confirmed by 1D-
HSQC analysis (Figure S1). These results support our idea that
the signal derived from 13C2-DOPAC, which was produced
enzymatically by MAO-A and ALDH.
Naturally, but importantly, the time course for the
production of 13C2-DOPAC from 13C2-DA was the same as
that for the production of DOPAC from nonisotope-enriched
DA (Figure S2). These data suggest that 13C2-DA works well as
a probe, reflecting the chemical and biological properties of
naturally occurring DA.
Because of the high selectivity of the multiple resonance
NMR technique, the probe can be used to detect DA
metabolites in real tissue samples. Figure 2c shows the NMR
spectrum for 13C2-DOPAC (100 μM) in a mouse liver
Figure 2. (a) 1H−{13CA−13CO} NMR spectrum of 13C2-DA (1 mM)
in reaction buffer (100 mM potassium phosphate, pH 7.4) in the
presence or absence of MAO-A (12 units/mL), with or without the
MAO-A-selective inhibitor, clorgyline (200 μM). Each reaction buffer
contains NAD+ (1 mM), ascorbic acid (1 mM), EDTA (0.7 mM), and
ALDH (5000 units/mL). After incubation for 4 h at 37 °C, the
samples were subjected to NMR analysis. (b) Time course change in
the 1H−{13CA−13CO} NMR spectrum (left) and reversed-phase
HPLC profile (right) of 13C2-DA (1 mM) in the reaction buffer
containing MAO-A (12 units/mL) and ALDH (5000 units/mL). (c)
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homogenate. In conventional H NMR measurements, the H
signal of 13C2-DOPAC overlaps the extensive background
signals derived from endogenous molecules (Figure 2c, left). In
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Conventional H NMR (left) and H−{13CA−13CO} NMR (right)
spectra of 13C2-DOPAC (100 μM) in mouse liver homogenate.
contrast, under the H−{13CA−13CO} experiments, a clear
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singlet 1H signal was observed around 3.3 ppm, with no
background signal (Figure 2c, right). This clear contrast
indicates the validity of our probe for the analysis of DA
metabolism in complex biological samples.
A.8 The affinity of clorgyline for MAO is known to vary with
substitutions at the aromatic moiety.8 Therefore, we anticipated
that clorgyline derivatives, modified at the aromatic moiety,
would show different inhibitory efficacy against DA metabo-
lism.
With a promising 13C2-DA probe in hand, we proceeded on
to the in situ screening and evaluation of inhibitors of DA
metabolism in complex biological samples. The experimental
scheme is shown in Figure 3a. Candidate inhibitors were
incubated in tissue homogenate (15 min). 13C2-DA was then
added and incubated for 5 h, and its metabolism was monitored
Clorgyline derivatives a−g (Figure 3b) were designed and
synthesized from the corresponding phenol derivative in a one-
step procedure (Supporting Information) and their inhibitory
efficacies were evaluated with 1H−{13CA−13CO} NMR
directly with H−{13CA−13CO} NMR measurements.
analysis. As shown in Figure 3c, the H signal from 13C2-
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We chose the MAO-A selective inhibitor, clorgyline, as a lead
compound (Figure 3b). Clorgyline consists of a propargylamine
moiety, which reacts with cofactor FAD to inhibit MAO-A
activity irreversibly, and an aromatic moiety, which acts as a
binding unit to a hydrophobic pocket at the active site of MAO-
DOPAC was observed around 3.3 ppm after 13C2-DA was
incubated in a mouse liver sample (middle in the upper line).
The addition of clorgyline strongly, but not completely,
suppressed the production of 13C2-DOPAC (left in the upper
line). This incomplete suppression is reasonable if we consider
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dx.doi.org/10.1021/ja305051u | J. Am. Chem. Soc. 2012, 134, 12398−12401
Ueki, Ryosuke
