Inactivation of mitochondrial monoamine oxidase B by methylthio-substituted benzylamines

Article abstract of DOI:10.1016/S0968-0896(03)00486-3

Mitochondrial monoamine oxidase was inactivated by o-mercaptobenzylamine (1) and o- (2) and p-methylthiobenzylamine (5). Experiments were carried out to provide evidence for possible mechanisms of inactivation. The corresponding o- (3) and p-hydroxybenzylamine (4) are not inactivators. Four radiolabeled analogues of 2 and 5, having radioactivity at either the methyl or benzyl groups, were synthesized, and all were shown to incorporate multiple equivalents of radioactivity into the enzyme. Inactivation in the presence of an electrophile scavenger decreased the number of molecules incorporated, but still multiple molecules became incorporated; catalase did not further reduce the number of inactivator molecules bound. Two inactivation mechanisms are proposed, one involving a nucleophilic aromatic substitution (S_NAr) mechanism and the other a dealkylation mechanism. Evidence for both mechanisms is that inactivation leads to reduction of the flavin (oxidation of the inactivator), but upon denaturation the flavin is reoxidized, indicating that attachment is not at the flavin. A cysteine titration indicates the loss of four cysteines after inactivation and denaturation. Support for the S _NAr mechanism was obtained by showing that o- and p-chlorobenzylamine also inactivate MAO. Chemical model studies were carried out that also support both S_NAr and dealkylation mechanisms.

Full text of DOI:10.1016/S0968-0896(03)00486-3

Bioorganic & Medicinal Chemistry 11 (2003) 4423–4430
Inactivation of Mitochondrial Monoamine Oxidase B by
Methylthio-Substituted Benzylamines
Xingliang Lu, Marıa Rodrıguez,y Wenxin Gu and Richard B. Silverman*
Department of Chemistry, Department of Biochemistry, Molecular Biology, and Cell Biology, and the Drug Discovery Program,
Northwestern University, Evanston, IL 60208-3113, USA
Received 15 April 2003; accepted 30 June 2003
Abstract—Mitochondrial monoamine oxidase was inactivated by o-mercaptobenzylamine (1) and o- (2) and p-methylthiobenzyla-
mine (5). Experiments were carried out to provide evidence for possible mechanisms of inactivation. The corresponding o- (3) and
p-hydroxybenzylamine (4) are not inactivators. Four radiolabeled analogues of 2 and 5, having radioactivity at either the methyl or
benzyl groups, were synthesized, and all were shown to incorporate multiple equivalents of radioactivity into the enzyme. Inacti-
vation in the presence of an electrophile scavenger decreased the number of molecules incorporated, but still multiple molecules
became incorporated; catalase did not further reduce the number of inactivator molecules bound. Two inactivation mechanisms are
proposed, one involving a nucleophilic aromatic substitution (SNAr) mechanism and the other a dealkylation mechanism. Evidence
for both mechanisms is that inactivation leads to reduction of the flavin (oxidation of the inactivator), but upon denaturation the
flavin is reoxidized, indicating that attachment is not at the flavin. Acysteine titration indicates the loss of four cysteines after
inactivation and denaturation. Support for the SNAr mechanism was obtained by showing that o- and p-chlorobenzylamine also
inactivate MAO. Chemical model studies were carried out that also support both SNAr and dealkylation mechanisms.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
ephinephrine and serotonin, and MAO B selectively
oxidizes dopamine.
Mitochondrial monoamine oxidase (MAO; EC 1.4.3.4)
is one of the enzymes responsible for the catabolism of
various monoamine neurotransmitters, such as trypta-
mine, dopamine, norepinephrine, epinephrine, and
serotonin, and therefore plays an important role in the
regulation of the intracellular concentrations of these
compounds.1 These amines mediate arousal and emo-
tion and influence temperature regulation, eating beha-
vior, behavioral aspects of sexuality and aggression,
sleep and consciousness, and pituitary regulation.2 It
has been found that compounds that inhibit mono-
amine oxidase have many important pharmacological
properties, especially as antidepressant agents3 or anti-
parkinsonian agents.4 In 1968, it was found that MAO
exists in two different isozymic forms (MAO A and
MAO B) in humans;5 MAO A selectively oxidizes nor-
The primary sequences of human liver MAO A and
MAO B were deduced from the corresponding cDNA
clones.6 The Aand B forms of MAO are homodimers
with subunit molecular weights of 59,700 and 58,800,
respectively. The two isozymes are derived from sepa-
rate genes but have about 70% sequence homology.
Both isozymes have been expressed in mammalian cells
and are localized to the mitochondrial outer membrane.
The two subunits were shown to be identical,7 and each
subunit contains one flavin adenine dinucleotide (FAD)
coenzyme8 covalently bound via its 8a-methyl group to
an active-site cysteine residue.9 It is known that the fla-
vin coenzyme must be in its oxidized form for the reac-
tion to take place, and upon oxidation of the
monoamine, the flavin is converted to its reduced
form.10 As is true for all flavin oxidases, molecular
oxygen is then required to convert the reduced flavin
back into its oxidized form with production of hydrogen
peroxide (Scheme 1).
*Corresponding author at the Department of Chemistry: Tel.: +1-
847-491-5653; fax: +1-847-491-7713; e-mail: agman@chem.north
western.edu
yPermanent address: School of Chemistry, Faculty of Sciences, Uni-
versidad Central de Venezuela, Caracas, Venezuela.
Much experimental evidence supports a single-electron
transfer mechanism for MAO (Scheme 2).11 Because of
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00486-3

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X. Lu et al. / Bioorg. Med. Chem. 11 (2003) 4423–4430
the support for an electron-transfer mechanism and the
evidence for the formation of radical intermediates, it
was thought that a novel approach for the inactivation
of MAO would be by an intramolecular hydrogen atom
transfer mechanism as shown in Scheme 3 for o-mer-
captobenzylamine (1). Initially, it was thought that
attachment of the thiyl radical could occur either at the
flavin or at the previously-proposed active site cysteine
residue.12 However, the crystal structure of MAO B has
shown that there are no cysteine residues in the active
site.13 Compound 1 was, in fact, found to be an inacti-
vator, but so was the corresponding o-methylthio-
benzylamine (2). In this paper, we describe experiments
to elucidate the mechanism for the unexpected inacti-
vation of MAO B by 2 and related compounds.
to our surprise, 2 also was a concentration- and time-
dependent inactivator of MAO B with KI=25 mM and
kinact=0.47 minÀ1
. To test whether intramolecular
methyl radical transfer was responsible for inactivation,
the corresponding isomer, p-methylthiobenzylamine (5),
was used, and it too was found to cause inactivation
with a KI=27 mM and kinact=0.38 minÀ1. It is apparent
that the mechanism in Scheme 3 does not apply to
inactivation of MAO B by the methylthiobenzylamines.
Results and Discussion
o-Mercaptobenzylamine (1) was found to be a concen-
tration- and time-dependent inactivator of mitochon-
drial MAO B with inactivation kinetic constants
KI=350 mM and kinact=0.13 minÀ1. To gain support for
the proposed inactivation mechanism shown in Scheme 3,
the corresponding phenol 3 was prepared, which was
shown to exhibit no inactivation of MAO B. Abstrac-
tion of a hydrogen atom from a phenol is a much higher
energy process than abstraction from a mercaptan.
Likewise, p-hydroxybenzylamine (4) also was not an
inactivator. Further support for the mechanism in
Scheme 3 was sought using o-methylthiobenzylamine
(2). In this case, a methyl radical would have to be
transferred, which is a much less likely event. However,
To determine the fate of the methyl and benzyl groups
during inactivation by 2 and 5, four radioactively
labeled analogues were synthesized, [methyl-14C]-2,
[methyl-14C]-5 (Scheme 4), [benzyl methylene-3H]-2, and
[benzyl methylene-3H]-5 (Scheme 5). The results of the
inactivation experiments with these radiolabeled inacti-
vators are shown in Table 1. Multiple molecules of
inactivator are incorporated into the enzyme, even after
denaturation, suggesting that an electrophilic species is
generated which indiscriminantly attaches to enzyme
nucleophiles. This is reminiscent of the inactivation of
MAO B by allylamine,14 which is oxidized to acrolein
(propenal), a highly electrophilic product that continued
to react with the enzyme even after all activity was lost.
In order for the inactivator to accumulate in MAO B,
an electrophilic species must be released and either
attach to a nucleophile on the way out or re-enter after
release. When the inactivation was carried out in the
presence of a large excess of b-mercaptoethanol to trap
released electrophiles, there was a decrease in the number
Scheme 1.
Scheme 2.
Scheme 3.

X. Lu et al. / Bioorg. Med. Chem. 11 (2003) 4423–4430
4425
Scheme 4.
particular mechanism of oxidation to the iminium ion,
it is not specified in these schemes). Evidence for the
mechanisms in Schemes 6 and 7 was obtained by
demonstrating that inactivation of MAO B with p-
methylthiobenzylamine leads to reduction of the flavin,
but denaturation of the inactivated enzyme results in
reoxidation of the flavin (data not shown), despite the
fact that multiple inactivator molecules are still bound
(see Table 1). This indicates that the inactivator
becomes oxidized during inactivation and attachment of
the inactivator does not occur at the flavin. Atitration
of cysteine residues with DTNB indicated that after
inactivation and dialysis only three cysteine residues
remained (the control without inactivator contained 6.9
cysteine residues), yet all of the enzyme activity
returned. This supports a random modification of
enzyme nucleophiles, including cysteine residues, which
have little effect on the active site.
Scheme 5.
of inactivator molecules attached to the enzyme (Table
1), but still five molecules of the benzyl part of the
compound remained bound to the dimeric enzyme and
14 or 15 molecules derived from the labeled methyl
group remained bound to the dimer. Although there are
seven free cysteine residues in each subunit (14 for the
dimer), nucleophiles other than cysteine may be
involved in the modification event, again similar to what
was observed with acrolein as an inactivator.14 There
was little or no change when catalase was added to the
inactivation mixture, indicating that hydrogen peroxide
generated from normal turnover of amines is not
responsible for the electrophilic species.
The mechanism in Scheme 6 is nucleophilic aromatic
substitution. If this is relevant, then it suggests that the
methylthio group is simply acting as a leaving group,
and other leaving groups could be substituted. Conse-
quently, a series of ortho- and para-substituted halo-
benzyl amines was tested for inactivation. In fact, both
ortho- and para-chlorobenzylamine were time-depen-
dent irreversible inhibitors of MAO B with KI=65 mM
and kinact=0.09 minÀ1 for the ortho-isomer and
The mechanisms shown in Schemes 6 and 7 are con-
sistent with the observation that radioactivity from both
the benzyl group as well as from the methyl group,
respectively, is incorporated into the enzyme (note that
because these experiments provide no support for any
Scheme 6.
Scheme 7.

4426
X. Lu et al. / Bioorg. Med. Chem. 11 (2003) 4423–4430
KI=23 mM and kinact=0.09 minÀ1 for the para-isomer.
However, when either the ortho- or para-positions were
substituted with fluorine or bromine, no time-dependent
inactivation occurred. It is not clear why these com-
pounds do not inactivate the enzyme, although all of the
halo-substituted benzylamines are substrates for MAO
B (Table 2), and possibly the inactivation pathway for
the chloro-substituted analogues is somewhat competi-
tive with turnover, but not in the case of the other two.
30% yield) and para-ethylthiobenzoic acid (8, 22%
yield), indicating that a SNAr reaction took place. The
conversion of the aldehyde to an approximately equal
amount of both the alcohol and carboxylic acid is
reminiscent of a Cannizzaro reaction, which generally is
carried out with hydroxide as the nucleophile, but has
been reported also to occur with thiols.15 However,
substitution of the methylthiol group by ethylthiolate
does not indicate whether the reaction was a direct
SNAr with ethanethiolate or if it proceeded via initial
demethylation to the para-mercapto analogue (as in
Scheme 8, pathway B) followed by SNAr reaction of
that product with ethanethiolate (with loss of HSÀ) or
both. To determine if demethylation had occurred, GC–
MS of the reaction products was carried out (Fig. 1).
The results are summarized in Scheme 9. Direct deme-
thylation of 9 by ethylthiolate gives ethyl methyl sulfide
Chemical model reactions (Scheme 8) were run to test
for the feasibility of the reactions depicted in Schemes 6
and 7. As a model for the unstable para-methylthio-
benzylimine, para-methylthiobenzaldehyde (6) and ethyl
para-methylthiobenzoate (9) were used. As a mimic for
an enzyme cysteine residue, sodium ethylthiolate was
used as the nucleophile. Although these reactions are
run under non-biological conditions, the enzyme has
entropic and enthalpic advantages that can allow these
reactions to proceed under more mild conditions. The
reaction with 6 gave para-ethylthiobenzyl alcohol (7,
Table 2. Kinetic constants for ortho- and para-substituted benzyla-
mines
Table 1. Equivalents of radioactivity bound to MAO B upon inacti-
vation by radiolabeled methylthio-substituted benzylamines
X
Km (mM)
kcat (minÀ1
)
KI (mM)
kinact (minÀ1
)
Compd
Conditions Aa Conditions Bb Conditions Cc
2-MeS
4-MeS
2-Cl
4-Cl
2-F
4-F
2-Br
4-Br
0.56
6.6
0.98
0.18
0.88
39
38
11.5
3.1
19
25
27
65
23
a
0.50
0.38
0.09
0.09
a
[methyl-14C]-5
27
49
8
15
14
5
—
13
—
—
[methyl-14C]-2
[benzylmethylene-3H]-5
[benzylmethylene-3H]-2
8
5
a
a
a
a
a
a
50
b
51
b
aCompound (40 mM) was incubated with MAO B (17.5 mM) at pH 7.3
until inactive, then dialyzed.
23.1
81
bSame as Conditions A, except with the addition of 0.2 mM 2-mer-
captoethanol, followed by urea denaturation.
aNot an inactivator.
bNot a substrate.
cSame as Conditions B, except with the addition of 500 mg of catalase.
Scheme 8.
Scheme 9.

X. Lu et al. / Bioorg. Med. Chem. 11 (2003) 4423–4430
4427
Figure 1.
Scheme 10.
Scheme 11.
(10). Dimethyl sulfide (11) is the product of initial SNAr
displacement of methylthiolate by ethylthiolate followed
by methylthiolate demethylation of 9. Diethyl sulfide
(12) could arise from initial SNAr displacement of
methylthiolate (13) to give ethyl 4-ethylthiobenzoate
followed by ethylthiolate deethylation. The disulfide
products (14 and 15) are oxidation products of methyl-
thiolate and ethylthiolate, respectively (diethyldisulfide
also was produced, but that would derive from direct
oxidation of ethylthiolate).
nitrogen, which activates the 4-alkyl group for homolytic
cleavage by the active site heme radical. The corre-
sponding mechanism for MAO B is shown in Scheme 11.
The problem with this mechanism stems from the eluci-
dation of the active site as shown in the crystal struc-
ture,13 namely, there are no cysteine residues in the
active site, the most likely protein source of radicals.
.
Also, if X in Scheme 11 is the flavin semiquinone, then
alkylation of the flavin would result; however, dena-
turation of inactivated enzyme leads to reoxidation of
the flavin. Other inactivators that we have reported to
attach to the flavin produce stable flavin adducts that
remain reduced even upon denaturation.17
Both the radioactive inactivator studies with MAO B
and the model studies support the proposed mechan-
isms shown in Schemes 6 and 7 as reasonable possibi-
lities. An alternative to Scheme 7 that should be
mentioned is a one-electron mechanism, related to the
proposed mechanism of inactivation of cytochrome
P450 by dihydropyridines (Scheme 10).16 This mechan-
ism involves one-electron oxidation of the pyridine
These enzyme and chemical model results support the
mechanisms shown in Schemes 6 and 7. Although the
compounds are not very potent inactivators, their
mechanisms of inactivation appear to be unique for
monoamine oxidase.

4428
X. Lu et al. / Bioorg. Med. Chem. 11 (2003) 4423–4430
Experimental
3H); HRMS calcd for C8H11SN 153.0612, Found
153.0607. Anal. calcd for C8 H12 ClNS: C, 50.65; H,
6.33; N, 7.39. Found: C, 50.45; H, 6.25; N, 7.28.
Chemicals and analytical methods
NMR spectra were recorded on either a Varian 300-
MHz or Varian Unity Plus 400-MHz spectrometer.
Chemical shifts are reported as d values in parts per
million down field from Me4Si as the internal standard
in CDCl3. Thin-layer chromatography was performed
on EM/UV silica gel plates with a UV indicator. Mass
spectra were obtained on a VG instruments VG70-
250SE high-resolution spectrometer. GC–MS spectra
were recorded on a HP 6890 GC system with mass
selective detector. UV spectra were recorded on a Per-
kin-Elmer Lambda 10UV/Vis spectrometer. Radio-
activity was measured on a TRI-CARB 2100 TR Liquid
Scintillation Analyzer. Column chromatography was
performed with Merck silica gel (230–240 mesh). [14C]-
Dimethyl sulfate and [3H] sodium borohydride were
purchased from American Radiolabeled Chemicals (St.
Louis, MO, USA). Other chemicals were purchased
from Aldrich Chemical Co. Biochemicals and enzymes
were purchased from Sigma Chemical Co.
4-Methylthiobenzyl[methylene-3H]amine. This com-
pound was synthesized by a literature procedure.19 4-
Methylthiobenzonitrile (149 mg, 1 mmol) and cobalt
chloride hexahydrate (476 mg, 2 mmol) were dissolved
in 99% methanol (4 mL), and a solution of [3H]sodium
borohydride (19.5 mCi, 170 mCi/mmol) and sodium
borohydride (380 mg) in 4 mL methanol was added in
portions with stirring at 20 ꢀC. Evolution of hydrogen
gas was observed. When the addition was completed,
stirring was continued for 1 h at 20 ꢀC. 3 N Hydrochloric
acid (2 mL) was poured into the reaction mixture and stir-
red until the black precipitate dissolved. After removal of
methanol by evaporation, the unreacted 4-methylthio-
benzonitrile was extracted with ether. The aqueous layer
was made alkaline with ammonium hydroxide solution
and then was extracted with ether (3 Â 10 mL). The com-
bined extracts were washed with saturated sodium chloride
solution, and the organic phase was extracted with 1 N
hydrochloric acid (3 Â 3 mL) and evaporated. The residue
was recrystallized from ethanol-ether to give the product
as a white solid (114 mg, 60%); specific radioactivity: 2.8 Â
109 dpm/mmol; radiopurity: 96% (by TLC).
2-Methylthiobenzylamine hydrochloride (2).18 To a sus-
pension of 2-methylthiobenzaldehyde (3.04 g, 20 mmol)
in 95% ethanol (120 mL) and sodium carbonate (2.44 g,
23 mmol) in water (24 mL) was dropwise added hydroxyl-
amine hydrochloride (2.1 g, 30 mmol) in water (8 mL)
over a period of 0.5 h. After addition was complete the
reaction mixture was stirred for 24 h followed by fil-
tration to remove the inorganic salts and evaporation of
ethanol. To the residue was added water to precipitate
the oxime which was filtered, washed with water and
dried overnight under vacuum, yielding 3 g (90%) of the
2-Methylthiobenzyl[methylene-3H]amine. This com-
pound was synthesized by the same procedure used to
make 4-methylthiobenzyl[methylene-3H]amine described
above, but the yield was 88 mg (50%); specific radio-
activity: 2.0 Â 109 dpm/mmol; radiopurity: 95%.
4-[methyl-14C]Methylthiobenzylamine.
4-Mercapto-
1
oxime; H NMR (CDCl3) d 8.17 (s, 1H), 7.52 (d, 2H),
7.26 (d, 2H), 2.44 (s, 3H).
benzonitrile was synthesized by a literature procedure.20
4-Methylthiobenzonitrile (745 mg, 5 mmol) in chloro-
form (15 mL) was oxidized with meta-chloroperoxy-
benzoic acid at 0 ꢀC for 2 h. Then the mixture was
stirred with Ca(OH)2 (555 mg, 7.5 mmol) at 20 ꢀC for
20 min followed by filtration and evaporation to give
the essentially pure sulfoxide, which was heated in tri-
fluoroacetic anhydride (10 mL) at reflux for 30 min. The
volatile components were removed by evaporation, the
residue was dissolved in methanol–triethylamine (1:1,
100 mL) and evaporated to dryness. The residue was
dissolved in chloroform, washed with NH4Cl (satu-
rated), dried over sodium sulfate, and evaporated to
dryness. The residue was purified by silica gel chromato-
graphy to give mercaptobenzonitrile (219 mg, 32%).
To a solution of the oxime (3 g, 18 mmol) in 95% etha-
nol (150 mL) was added zinc powder (13 g, 200 mmol)
with vigorous stirring. The mixture was brought to
reflux and 10% hydrochloric acid (70 mL) was added
over 2 h. The mixture was refluxed overnight followed
by filtration of excess zinc and removal of ethanol from
the filtrate. To the resulting white emulsion was added
20% sodium hydroxide (30 mL) followed by filtration of
the resulting zinc hydroxide. The filtrate was saturated
with sodium chloride, and the filter cake was triturated
with chloroform. The chloroform triturate was used to
extract the filtrate saturated with sodium chloride. The
combined chloroform extracts were washed with brine.
The chloroform solution was extracted with 3% hydro-
chloric acid followed by evaporation under the vacuum,
and the residue was recrystallized from ethanol-ether to
To
a solution of 4-mercaptobenzonitrile (203 mg,
1.5 mmol) in dry THF (5 mL) was added sodium
hydride (43 mg, 1.8 mmol) and stirred for 3 h at room
temperature. To this solution was added [14C] dimethyl
sulfate (0.3 mCi, 0.4 mCi/mmol) in THF (2 mL) fol-
lowed by dimethyl sulfate (150 mL, 1.6 mmol). The
solution was stirred overnight at room temperature,
acidified with 3 N hydrochloride acid, and extracted
with ether (3 Â 15 mL). The combined extracts
were dried over MgSO4 and evaporated. The resi-
due was reduced with sodium borohydride and
cobaltous chloride hexahydrate, as was described
above for 4-methylthiobenzyl[methylene-3H]amine. The
1
give 2 as a white solid (1.07 g, 40%); H NMR (D2O) d
7.10–7.28 (m, 4H); 4.10 (s, 2H); 2.34 (s, 3H). Anal. calcd
for C8 H12 ClNS: C, 50.65; H, 6.33; N, 7.39. Found: C,
50.75; H, 6.41; N, 7.33.
4-Methylthiobenzylamine hydrochloride (5). 4-Methyl-
thiobenzylamine hydrochloride (5) was prepared by the
same procedure as 2-methylthiobenzylamine hydro-
chloride described above. The yield was 510 mg (19%);
1H NMR (D2O) d 7.35 (q, 4H), 4.37 (s, 2H), 2.46 (s,

X. Lu et al. / Bioorg. Med. Chem. 11 (2003) 4423–4430
4429
reduced product was recrystallized from ethanol–ether
to give 4-[14C]-methyl]thiobenzylamine as a white solid
(80 mg, 30%); specific radioactivity: 6.9 Â 107 dpm/
mmol; radiopurity 95%.
(0.5 M, pH 4.0). Dilute MAO B (20 mM) was prepared
by mixing stock MAO B (110 mM) with the sodium
phosphate buffer (pH 7.0 containing 10 mM NaN3). All
solutions and the spectrometer were kept at 25ꢀC with the
use of a temperature controlled water bath/recirculator
and a block heater. The substrate solutions and control
without substrate (90 mL) were incubated with diluted
MAO (10mL) for 1, 2, 3, 4, or 5 min (depending on activ-
ity), then a 10-mL aliquot was mixed with the assay solu-
tion (490 mL) and the absorbance was measured at 414 nm
versus a blank of assay solution (500 mL), dilute MAO
(10 mL), and buffer (0.1 M sodium phosphate, pH 7.0 buf-
fer with 10 mM sodium azide). The reported extinction
coefficient of 24,600 molÀ1 cmÀ1 for the ABTS dye was
used to convert the absorbance measurements into con-
centration units. The concentration of hydrogen peroxide
was calculated from the calibration curve, and the data
were then plotted in a double reciprocal plot (Lineweaver–
Burke) for approximate kcat and Km determination.24
2-[methyl-14C]Methyl]thiobenzylamine. This compound
was synthesized by the same method as for 4-
[methyl-14C]methylthiobenzylamine, but in a yield of
8% (22 mg); specific radioactivity: 6.9 Â 107 dpm/mmol;
radiopurity 99%.
The reaction of 4-methylthiobenzaldehyde and ethyl 4-
methylthiobenzoate with sodium ethanethiolate
Asolution of 4-methylthiobenzaldehyde (304 mg,
2 mmol) and sodium ethanethiolate (840 mg, 10 mmol)
in 1-methyl-2-pyrrolidinone (NMP) was heated at
100 ꢀC for 14 h. The solution was acidified with 2 N HCl
(10 mL) to pH 2 and extracted with ether (3 Â 10 mL).
The combined extracts were washed with 10% sodium
carbonate solution. The ether solution was then dried
and evaporated. The residue was purified by silica gel
chromatography. The product was identified by 1H
NMR and GC–MS as 4-ethylthiobenzyl alcohol
Inactivation of MAO B by compounds 3, 4, 5 and halo-
genated benzylamine
Asolution of the benzylamine analogues (10–50 mM) in
sodium phosphate buffer (0.2 M, pH 7.2, 95 mL) and
MAO B (130 mM, 5 mL) in sodium phosphate (50 mM,
pH 7.2) was incubated at room temperature. Aliquots of
the incubation solution (10 mL) were assayed periodi-
cally with a cinnamylamine solution (1.01 mM, 490 mL)
in Tris buffer (20 mM, pH 9.0). The rate of oxidation
was determined at 290 nm, the wavelength correspond-
ing to the formation of cinnamaldehyde. Acontrol
without inactivator also was assayed. The inactivation
rate corresponding to each concentration of the inacti-
vitor was obtained, and the KI and kinact values were
determined by the method of Kitz and Wilson.25
1
(100 mg, 30%); H NMR (CDCl3): d 7.25 (m, 4H); 4.60
(s, 2H); 2.91 (q, 2H); 1.27 (t, 3H).
The combined sodium carbonate solutions were acid-
ified with 2 N HCl to pH 2, then it was extracted with
ether (3 Â 10 mL). The extracts were dried and evapo-
rated to dryness. The residue was identified as 4-ethyl-
1
thiobenzoic acid ( 81 mg, 22%); H NMR (CDCl3): d
7.95 (dd, 2H); 7.28 (dd, 2H); 3.00 (q, 2H), 1.35 (t, 3H).
As a control reaction, benzaldehyde was treated with
sodium ethanethiolate under the same conditions as the
reaction above with 4-methylthiobenzaldehyde. The
products, benzyl alcohol and benzoic acid, were identi-
Incorporation of radioactivity into MAO B by [3H]- or
[14C]-labeled methylthiobenzylamines
1
fied by H NMR and GC–MS.
Asolution of ethyl 4-methylthiobenzoate and sodium
ethanethiolate in the NMP was heated under the same
conditions as above. After workup, the product was
identified by 1H NMR and GC–MS as 4-ethylthio-
benzoic acid; H NMR (CDCl3): d 7.99 (dd, 2H); 7.31
(dd, 2H); 3.03 (q, 2H); 1.38 (t, 3H).
MAO B (112 mM, 50 mL) was incubated with [3H]- or
[14C]-labeled compounds (50 mM) in 200 mM sodium
phosphate buffer (500 mL, containing 30% DMSO), pH
7.3 at 25 ꢀC. Acontrol without inactivator was run
simultaneously at one-fifth the scale. MAO B treated
with inactivator was devoid of activity when checked
after 2 days. To the inactivated enzyme solution was
added 480 mg of urea, and the solution was heated at
50 ꢀC overnight to denature the enzyme. This solution
was dialyzed at 4 ꢀC versus 8 M urea in 50 mM sodium
phosphate buffer (500 mL). The dialysis buffer was
changed four times over 24 h, during which time the
radioactivity of the denatured enzyme was monitored.
The denatured enzyme solution was assayed for radio-
activity and protein concentration, from which the
number of equivalents of radioactivity attached per
MAO B molecule was calculated.
1
Enzyme and assay
Bovine liver MAO B was isolated and assayed as pre-
viously reported.21
Determination of Km and kcat
The method of Szutowicz et al.22 as previously mod-
ified23 was used to assay formation of hydrogen perox-
ide. Solutions of the benzylamine analogues were
prepared (0.1, 0.2, 0.4, 0.7, 1.0, 1.5 and 2.0 mM in 0.1 M
sodium phosphate, pH 7.0 buffer with 10 mM sodium
azide). An assay solution was prepared containing
horseradish peroxidase (0.05 mg/mL) and 2,20-azino-
bis(3-ethylbenzothiaxoline-6-sulfonic acid) diammo-
nium salt (ABTS, 18 mM) in phosphate–citrate buffer
Cysteine titration of MAO B before and after inactiva-
tion with 2-methylthiobenzylamine
The procedure of Silverman and Zieske26 was followed.
The number of enzyme sulfhydryl groups was determined

4430
X. Lu et al. / Bioorg. Med. Chem. 11 (2003) 4423–4430
by the 5,50-dithiobis(2-nitrobenzoic acid) (DTNB) assay
of Fernandez Diez et al.27 MAO B (140 mM, 20 mL) in
sodium phosphate buffer (50 mM, pH 7.2) was added to
2-methylthiobenzylamine (33 mM, 880 mL), and the
solution was incubated for two days. The inactivated
enzyme solution was dialyzed against sodium phosphate
buffer (66 mM, pH 7.0, 4 Â 500 mL) to remove the
excess inactivator. The MAO B activity returned to the
same activity of the control without inactivator. To the
enzyme solution was added a solution of 300 mL of 20%
NaDodSO4 in water containing EDTA(1 mg/mL) and
DTNB (40 mg/mL, 20 mL). Two controls, one without
MAO B and one without DTNB, were carried out as
well. The absorbance at 412 nm was recorded for the
three solutions from which the number of sulfhydryl
groups could be determined.
6. Bach, A. W. J.; Lan, N. C.; Johnson, D. L.; Abell, C. W.;
Bembenek, M. E.; Kwan, S.-W.; Seeburg, P. H.; Shih, J. C.
Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 4934.
7. Lan, N. C.; Chen, C.; Shih, J. C. J. Neurochem. 1989, 52,
1652.
8. Weyler, W. Biochem. J. 1989, 260, 725.
9. (a) Kearney, E. B.; Salach, J. I.; Walker, W. H.; Seng,
R. L.; Singer, T. P. Biochem. Biophys. Res. Commun. 1971,
42, 490. (b) Kearney, E. B.; Salach, J. I.; Walker, W. H.; Seng,
R. L.; Kenney, W.; Zeszotek, E.; Singer, T. P. Eur. J. Biochem.
1971, 24, 321.
10. Hellerman, L.; Chuang, H. Y. K.; DeLuca, D. C. Adv.
Biochem. Psychopharmacol. 1972, 5, 327.
11. (a) Silverman, R. B. Acc. Chem. Res. 1995, 28, 335. (b) Lu,
X.; Rodriguez, M.; Ji, H.; Silverman, R. B.; Vintem, A. P. B.;
Ramsay, R. R. In Flavins and Flavoproteins 2002: Proceedings
of the 14th International Symposium; Chapman, S. K., Perham,
R. N., Scrutton, N. S., Eds.; Rudolf Weber: Berlin, Germany,
2002; p 817.
12. Zhong, B.; Silverman, R. B. J. Am. Chem. Soc. 1997, 119,
6690.
13. Binda, C.; Newton-Vinson, P.; Hubalek, F.; Edmondson,
D. E.; Mattevi, A. Nat. Struct. Biol. 2002, 9, 22.
14. Silverman, R. B.; Hiebert, C. K.; Vazquez, M. L. J. Biol.
Chem. 1985, 260, 14648.
Flavin difference spectra of 4-methylthiobenzylamine-
inactivated MAO B
The procedure of Silverman and Banik was followed.28
Asolution of 4-methylthiobenzylamine (10 mM) in Tris
buffer (0.1 M, pH 7.0, 500 mL) and MAO B (110 mM) in
sodium phosphate buffer (50 mM, pH 7.0, 20 mL) was
incubated at room temperature for 5 h. Areaction
without inactivator also was run as a control. The
enzyme activity was assayed periodically. The enzyme
with inactivator was found to be devoid of activity. A
flavin difference spectrum was recorded, and then the
enzymes were denatured by the addition of sodium
dodecyl sulfate (20%, 400 mL), and the flavin difference
spectrum was again recorded.
15. Franzen, V. Chem. Ber. 1955, 88, 1361. (b) Franzen, V.
Chem. Ber. 1957, 90, 623.
16. (a) Augusto, O.; Beilan, H. S.; Ortiz de Montellano, P. R.
J. Biol. Chem. 1982, 257, 11288. (b) Ortiz de Montellano, P. R.;
Beilan, H. S.; Kunze, K. L. J. Biol. Chem. 1981, 256, 6708. (c)
DeMatteis, F.; Hollands, C.; Gibbs, A. H.; deSa, N.; Rizzar-
dini, M. FEBS Lett. 1982, 145, 87.
17. (a) Silverman, R. B.; Yamasaki, R. B. Biochemistry 1984,
23, 1322. (b) Silverman, R. B. Biochemistry 1984, 23, 5206. (c)
Silverman, R. B.; Zieske, P. A. Biochemistry 1985, 24, 2128.
18. Euerby, M. R.; Waigh, R. D. J. Chem. Res. Miniprint
1982, 9, 2417.
19. Satoh, T.; Suzuki, S.; Suzuki, Y.; Miyaji, Y.; Imai, Z.
Tetrahedron Lett. 1969, 4555.
Acknowledgements
20. Young, R. N.; Gauthier, J. Y.; Coombs, W. Tetrahedron
Lett. 1984, 25, 1753.
21. Weyler, W.; Salach, J. I. Arch. Biochem. Biophys. 1981,
212, 147.
We are grateful to the National Institutes of Health
(GM32634) for financial support of this research.
22. Szutowicz, A.; Kobes, R. D.; Orsulak, P. H. Anal. Bio-
chem. 1984, 138, 86.
References and Notes
23. Silverman, R. B.; Hawe, W. P. J. Enz. Inhib. 1995, 9, 203.
24. Lineweaver, H.; Burk, D. J. Am. Chem. Soc. 1934, 56,
658.
25. Kitz, R.; Wilson, I. B. J. Biol. Chem. 1962, 237, 3245.
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27. Fernandez Diez, M. J.; Osuga, D. T.; Feeney, R. E. Arch.
Biochem. Biophys. 1964, 107, 449.
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