Design of Monoamine Oxidase InactiVators
J. Am. Chem. Soc., Vol. 120, No. 24, 1998 5871
dissolved in ether. Filtration gave 17 mg (24%) of 3-MLF as the
precipitate. Concentration of the filtrate in vacuo gave a residue which
was subjected to preparative TLC on silica gel (ether) to provide 55
mg (68%) of the 4a-adduct 8.14
Triton X-100 at 25 °C containing either amphetamine, amphetamine
and the inactivator, or only inactivator were incubated at 25 °C.
Aliquots were removed at various time intervals, and enzyme activity
was assayed by use of the kynuramine assay procedure.
Irradiation of 3MLF and 4-(N,N-Dimethylamino)-3-phenylbut-
1-yn-3-ol (7). A solution of 50 mg (0.19 mmol) of 3MLF and 105
mg (0.56 mmol) of the amino alcohol 7 in 150 mL of MeOH was
irradiated for 2 h. The photolyzate was concentrated in vacuo giving
a residue which was mixed with ether, followed by filtration to give
26 mg of 3-MLF as the precipitate. The filtrate was concentrated in
vacuo giving a residue which was subject to preparative TLC on silica
gel (ether) to yield 27 mg of recovered amino alcohol 7, 18 mg (50%)
of the adduct 10E (mp 229-231 °C), and 16 mg (45%) of the adduct
10Z (mp 211-213 °C).
Sulfhydryl Titration of MAOs. Solutions of MAO A with and
without added inactivator in 100 mM sodium phosphate buffer (pH
7.2) containing 10% glycerol and 0.2% Triton X-100 were incubated
at 25 °C. When the enzyme activity was <5% of the control activity,
the solutions were dialyzed for 4 h against three changes (500 mL) of
100 mM sodium phosphate buffer (pH 7.2), containing 10% glycerol
and 0.2% Triton X-100. The dialyzed solutions were assayed for
enzyme activity and protein content.
Thiol titrations with 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB) were
performed according to a modification of the literature procedure.25
A
10E: 1H NMR: 2.19 (s, 6H, C-7 and C-8), 3.31 (s, 3H, N-10), 3.67
(s, 3H, N-3), 5.08 (s, 1H, NH), 6.65 (d, 1H, J ) 15.5, H-1′), 6.69 (s,
1H, C-6), 6.84 (s, 1H, C-9),6.92 (d, 1H, J ) 15.5, H-2′), 7.37, 7.55,
7.66 (m, 5H, aromatic); 13C NMR 19.4 (C-7 and C-8 CH3), 28.4 (N-
10 CH3), 32.4 (N-3 CH3), 65.8 (C-4a), 117.0 (C-1′), 117.7 (C-2′), 125.7
(C-8), 128.6, 128.7, 133.6, 136.6 (aromatic), 129.0 (C-7), 129.4 (C-
9a), 130.2 (C-5a), 134.9 (C-6), 134.9 (C-9), 155.6 (C-10a), 160.0 (C-
2), 167.0 (C-4), 188.3 (C-3′); IR 3294, 1713, 1678, 1654, 1560, 1063;
EIMS 402 (M, 15), 345 (30), 240 (41), 105 (100), 149 (52), 77 (33),
51 (45); HRMS (EI) m/z 402.1702 (C23H22N4O3 requires 402.1692).
10Z: 1H NMR: 1.76 (s, 3H, C-8), 2.05 (s, 3H, C-7), 3.29 (s, 3H,
N-10), 3.46 (s, 3H, N-3), 5.34 (s, 1H, NH), 5.75 (d, 1H, J ) 12.7,
H-1′), 6.26 (s, 1H, C-6), 6.63 (d, 1H, J ) 12.7, H-2′), 6.66 (s, 1H,
C-9), 7.37, 7.55, 7.60 (m, 5H, aromatic); 13C NMR 18.9 and 19.2 (C-7
and C-8 CH3), 28.5 (N-10 CH3), 32.3 (N-3 CH3), 59.0 (C-4a), 117.2
(C-1′), 118.0 (C-2′), 125.6 (C-8), 128.4, 128.7, 134.1, 136.4 (aromatic),
128.6 (C-7), 130.0 (C-9a), 133.5 (C-6), 134.4 (C-5a), 134.8 (C-9), 155.8
(C-10a), 160.0 (C-2), 166.4 (C-4), 191.3 (C-3′); IR 3346, 2943, 1725,
1667, 1557, 1283, 1146; EIMS 402 (M, 19), 345 (100), 303 (22), 240
(60), 105 (38), 77 (27), 51 (30); HRMS (EI) m/z 402.1698 (C23H22N4O3
requires 402.1692).
200 µL aliquot of either the control or inactivated MAO A solution
was added to a solution of 380 µL of deionized water, 200 µL of 100
mM sodium phosphate buffer (pH 8.0), and 100 µL of 20% sodium
dodesyl sulfate (NaDodSO4) with 1 mg/mL EDTA. The absorbance
of each solution at 412 nm was recorded as an enzyme absorbance
background reading. The blank absorbance was zeroed by using a
solution of 380 µL deionized water, 200 µL of 100 mM sodium
phosphate buffer (pH 8.0), 100 µL of 20% NaDodSO4 with 1 mg/mL
EDTA, and 200 µL of 10% glycerol in 100 mM sodium phosphate
buffer (pH 7.2), containing 0.2% Triton X-100. A 20 µL aliquot of 4
mg/mL of DTNB in 100 mM sodium phosphate buffer (pH 8.0) was
added to each enzyme solution, and the absorbance at 412 nm was
measured over a 30 min period. A 20 µL aliquot of the DTNB solution
was added to the solution used to prezero the spectrometer, and the
absorbance was measured and used as a DTNB absorbance background.
The total amount of free 5-mercapto-2-nitrobenzoate produced was
calculated from the absorbance at 412 nm of the DTNB treated MAO-A
solution minus the two background readings. The assay was performed
in triplicate.
Changes in the Flavin UV-Visible Spectrum upon Inactivation
of MAO-A. Solutions of MAO-A (25 µM) in 50 mM potassium
phosphate buffer, at pH 7.2 containing 0.2% Triton X-100, were placed
in individual cuvettes that were then sealed with rubber septa. These
cuvettes were repeatedly, sequentially purged with argon and evacuated.
A solution of each inactivator was then added by using an airtight
syringe, and the UV-visible spectra were recorded periodically over
27 min time period. These experiments were performed in duplicate.
Enzyme Assays. MAO-A was assayed by the method of Weiss-
bach.19 MAO-A in 50 mM sodium phosphate buffer (pH 7.2)
containing 0.2% Triton X-100 at 25 °C was mixed with 1 mM
kynuramine as the substrate. The rate of the kynuramine reaction to
form 4-hyroxyquinoline was measured by monitoring the increase in
absorbance at 314 nm. The enzyme activity of MAO-B was measured
by use of a modified procedure of Tabor.36b MAO-B in 50 mM sodium
phosphate buffer (pH 7.2) containing 0.2% Triton X-100 at 25 °C was
mixed with benzylamine as the substrate. The rate of reaction to
produce benzaldehyde was measured by monitoring the increase in
absorbance at 250 nm. One unit of enzyme activity equals that amount
needed to form 1 µmol of product per minute.
Inhibition of MAO-A Catalysis of Kynuramine Oxidation. Initial
velocities for MAO-A catalyzed oxidation of kynuramine19 were
determined by monitoring the changes in absorbance at 314 nm.
Reactions were initiated by adding MAO-A (final concentrations in
the range of 5.95-10.4 µM) to solutions containing varying concentra-
tions of kynuramine and the selected inhibitors in 50 mM sodium
phosphate buffer at pH 7.2 containing 0.2% Triton X-100 at 25 °C.
The Ki value for each inhibitor was determined by use of a Lineweaver-
Burk20b plot of 1/ [kynuramine] vs 1/initial velocity and the Cleland
enzyme kinetic computer analysis.21 All Ki determinations were made
in duplicate.
Dimethylamine Production in the Reaction of MAO-A with
â-Hydroxyamine 5. MAO-A (100 µM) was inactivated with â-hy-
droxyamne 5 (25 mM) in 50 mM sodium phosphate buffer, at pH 7.2,
containing 5% glycerol and 0.2% Triton X-100. The mixture was then
made basic by addition of saturated NaHCO3 and mixed with 1 mL of
a 2 mg/mL solution of dabsyl chloride in acetone.30 The solution was
kept at 25 °C for 1 h and extracted with chloroform. The extracts
were dried, and a 10 µL portion was removed for HPLC analysis
(Beckman Ultrasphere ODS C-18 column (0.46 cm × 25 cm), eluted
with hexane:2-propanol (80:20) at a flow rate of 1 mL/min). TLC
(ether) was performed with dabsyl chloride (Rf ) 0.76), â-amino alcohol
5 (Rf ) 0.52), and N,N-dimethyldabsylamide (21) (Rf ) 0.64).30 The
remaining solution was subjected to preparative TLC (ether). The band
that comigrated with N,N-dimethyldabsylamide (21) was collected and
1
subjected to H NMR analysis. A control containing 25 mM 5 in 50
mM sodium phosphate buffer, at pH 7.2, containing 5% glycerol and
0.2% Triton X-100 was also treated with dabsyl chloride in the same
manner.
Time Dependent Inactivation of MAO-A. Aliquots of an MAO-A
stock solution were added to solutions containing varying concentrations
of the inactivators in 50 mM sodium phosphate buffer (pH 7.2),
containing 0.2% Triton X-100. After mixing, the samples were
incubated at 25 °C and periodically assayed for enzyme activity by
use of the kynuramine assay procedure.19 All inactivations were
performed either in duplicate or triplicate. Plots of the natural log of
the MAO-A activity remaining versus time in each case gave apparent
inactivation rate constants from which the kinetic constants Kinact and
kinact were determined by the use of Kitz and Wilson plots.20a
Inhibition of MAO-B Catalyzed Oxidation of Benzylamine.
Initial velocities for MAO-B catalyzed oxidation of benzylamine36b were
determined by monitoring the changes in absorbance at 250 nm.
Reactions were initiated by adding aliquots of MAO-B to solutions
containing varying concentrations of benzylamine and the inhibitors
in 50 mM sodium phosphate buffer at pH 7.2 containing 0.2% Triton
X-100 at 25 °C. The Ki value for each inactivator was determined by
use of the Lineweaver-Burk20b plot and Cleland enzyme kinetic
computer analysis21 methodologies. All Ki determinations were made
in duplicate.
Effect of Amphetamine on the Inactivation of MAO-A. Solutions
of MAO-A in 50 mM sodium phosphate buffer at pH 7.2 with 0.2%