A new strategy for the design of monoamine oxidase inactivators. Exploratory studies with tertiary allylic and propargylic amino alcohols

Article abstract of DOI:10.1021/ja973978d

A new strategy for the design of monoamine oxidase (MAO) inhibitors is proposed. The strategy is based on the premise that tertiary-amine containing MAO-inactivators which operate by alkylation of active site nucleophiles are activated in situ by single e

Full text of DOI:10.1021/ja973978d

5864
J. Am. Chem. Soc. 1998, 120, 5864-5872
A New Strategy for the Design of Monoamine Oxidase Inactivators.
Exploratory Studies with Tertiary Allylic and Propargylic Amino
Alcohols
Kelly A. Van Houten,‡ Jong-Man Kim,‡ Michael A. Bogdan,‡ Dino C. Ferri,‡ and
Patrick S. Mariano*,†
Contribution from the Department of Chemistry and Biochemistry, UniVersity of Maryland,
College Park, Maryland 20742, and the Department of Chemistry, UniVersity of New Mexico,
Albuquerque, New Mexico 87131-1096
ReceiVed NoVember 20, 1997
Abstract: A new strategy for the design of monoamine oxidase (MAO) inhibitors is proposed. The strategy
is based on the premise that tertiary-amine containing MAO-inactivators which operate by alkylation of active
site nucleophiles are activated in situ by single electron transfer (SET) to the MAO-flavin cofactor to form
aminium cation radicals which undergo secondary fragmentation reactions to produce reactive electrophiles.
The purpose of the current work was to assess the feasibility and applicability of this proposal for the design
of new families of MAO-inactivators. Based on the documented retro-aldol type fragmentation reactivity of
â-amino-alcohol cation radicals, tertiary â-allylic and -propargylic â-amino-alcohols were expected to serve
as precursors of conjugated ketones in SET-promoted processes. Evidence supporting this hypothesis was
gained from studies of model SET-photoreactions of members of this amino-alcohol family with 3-methyl-
lumiflavin (3MLF). The efficient production of 4a- and 4a,5-flavin adducts in these excited-state reactions
demonstrates that aminium radicals, arising by SET-oxidation of tertiary â-allylic and -propargylic â-amino-
alcohols, fragment to generate R,â-unsaturated ketones which react rapidly with the simultaneously formed
3MLF-hydroflavin anion. The second feature of the MAO-inactivator design strategy pathway was tested by
examining reactions of the MAOs with substances which contain electrophilic, conjungated enone and ynone
moieties tethered to amine functions to ensure delivery to the enzyme active sites. The covalent modification
of active site cysteine thiol residues by the unsaturated ketone groups in these substances was confirmed by
demonstrating that they serve as active site-directed, time-dependent, nonredox based, inactivators of MAO-A
and MAO-B. In the key test of the feasibility of the new MAO-inactivator design strategy, it was shown that
selected tertiary â-allylic and -propargylic â-amino-alcohols undergo redox reactions in the MAO-A active
site which result in inactivation of the enzyme via covalent modification of a single cysteine residue. The
experimental results which support the conclusions stated above are presented and discussed in this paper.
Introduction
preferentially oxidizes serotonin is selectively and irreversibly
inhibited by the tertiary propargylic amine clorgyline (1), an
agent used in the treatment of depression.4 On the other hand,
MAO-B is preferentially inactivated by the inhibitor deprenyl
(2) which is used in conjunction with L-DOPA to treat
Parkinson’s disease.5
The monoamine oxidases MAO-A and MAO-B are two
structurally homologous, redox active enzymes found in the
mitochondrial membranes of most mammalian tissues.1 The
primary function of the MAOs is to regulate the concentrations
of primary and secondary amines by catalyzing their oxidative
deamination to produce amine and carbonyl products. Among
the more important substrates of these enzymes are the
neurologically active amines epinephrine, dopamine, and sero-
tonin. As a consequence, the MAOs play key roles in regulating
the level of these neurotransmitters in the human brain.2 MAO-
inhibitors cause an elevation in the levels of the neuroactive
amines and are thus of medicinal importance. MAO-A which
Both MAO-A and MAO-B contain a flavin (FAD) cofactor,
covalently bound through a cysteine thioether linkage to the
8-position of the isoalloxazine ring.3 The chemical steps of
(3) 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.
(4) For a general review, see: (a) Tripton, K. F.; Dostert, P.; Benedetti,
M. S. Monoamine Oxidase and Disease; Academic Press: New York, 1984.
(b) Dawson, J. H. J. Nerual. Transm. 1987, 23, 121.
(5) Youdim, M. B. H.; Finberg, J. P. AdVances in Neurology; Yahr, M.
D., Bergmann, J., Eds.; Raven Press: New York, 1986; Vol. 45, pp 127-
136. Knoll, J. In AdVances in Neurology; Yahr, M. D., Bergman, K. J.,
Eds.; Raven Press: New York, 1986; Vol. 45, pp 107-110.
† University of New Mexico.
‡ University of Maryland.
(1) For comprehensive reviews of MAO-biochemistry, see: (a) Singer,
T. P. J. Neur. Trans. 1987, Suppl. 23, 1. (b) Singer, T. P. In Chemistry and
Biochemistry of FlaVoenzymes; Muller, F., Ed.; CRC Press: Boca Raton,
FL, 1991; Vol. 3, pp 437-470. (c) Weyler, W.; Hsu, Y. P. P.; Breakfield,
X. O. Pharmac. Ther. 1990, 47, 391.
(2) Schildkraut, J. T.; Kety, S. S. Science 1967, 156, 21.
S0002-7863(97)03978-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/05/1998

Design of Monoamine Oxidase InactiVators
J. Am. Chem. Soc., Vol. 120, No. 24, 1998 5865
Scheme 1
Scheme 3
Scheme 4
Scheme 2
cofactor to generate aminium radical intermediates.9 However,
less is known about the events which lead to enzyme covalent
adduct formation and, specifically whether this involves radi-
cal12,13 or nucleophile-electrophile8,14 coupling processes. We
believe that a reasonable argument can be made for operation
of a general route for MAO inactivation by tertiary amines
which proceeds via amine cation radical fragmentation to form
electrophilic intermediates which then alkylate a nucleohilic
residue in the enzyme active site region (Scheme 3). The
reasoning embodied in this scheme leads to the general
prediction that tertiary amines whose cation radicals haVe a
high propensity to fragment to produce electrophilic intermedi-
ates should serVe as effectiVe MAO-inactiVators.
We have initiated studies to test this approach to the rational
design of new tertiary amine containing MAO-inactivators.
Based on the documented retro-aldol type fragmentation reactiv-
ity of â-hydroxy-aminium radicals,15 we anticipated that tertiary
â-allylic and â-propargylic â-amino alcohols of general structure
3 would serve as precursors of conjugated ketones in SET-
promoted processes (Scheme 4) and, consequently, as MAO-
inactivators.
MAO catalysis, represented in Scheme 1, involve simultaneous
oxidation of the amine substrate and reduction of the oxidized
flavin cofactor to generate imine and 1,5-dihydroflavin products.
Hydrolysis of the imine and flavin oxidation by molecular
oxygen then complete the catalytic cycle.
The first partial reaction, oxidation of the amine substrate,
has been the focus of much mechanistic speculation. While
some investigators favor a single electron transfer (SET)/radical
mechanism6 for the oxidation of primary and secondary amines,
the polar addition-elimination mechanism originally proposed
by Hamilton7 is equally feasible8 (Scheme 2). Tertiary amines,
on the other hand, because of their reduced nucleophilicity and
increased electron-donor ability, are perhaps better candidates
for the SET initiated mechanistic pathway.9
Much of what is known about the chemical mechanism of
the amine oxidation partial reaction catalyzed by MAO has
derived from the results of studies of the mechanism(s) for
inactivation of the enzyme by mechanism based inhibitors.10,11
There seems to be agreement that the respective catalytic and
inactivation processes involving the MAOs and tertiary amine
substrates and inactivators are initiated by SET to the flavin
A combination of photochemical and biological studies with
selected members of this family of compounds have provided
preliminary results which demonstrate that (1) SET-induced
photochemical reactions of these substances with the model
flavin, 3-methyllumiflavin, results in production of conjugated
ketone intermediates, (2) conjugated ketones which are tethered
to tertiary amine functions to ensure active site binding inactivate
the MAOs by covalent modification of a single cysteine thiol
residue, and (3) selected tertiary â-allylic and â-propargylic
â-hydroxyamines inactivate MAO-A by a pathway that involves
redox participation by the flavin cofactor and that results in
alkylation of an active site cysteine thiol grouping.
(6) Silverman, R. B. In AdVances in Electron-Transfer Chemistry;
Mariano, P. S., Ed.; JAI-Press: Greenwich, CT, 1992; pp 177-211.
(7) Hamilton, G. A. Prog. Bioorg. Chem. 1971, 1, 83.
(8) Kim. J.-M.; Hogey, S. E.; Mariano, P. S. J. Am. Chem. Soc. 1994,
116, 1000.
(9) Richter, D. Biochem. J. 1937, 31, 2022. Kalgutkar, A. S.; Castignoli,
K.; Hall, A.; Castignoli, N. J. Med. Chem. 1994, 37, 944 and references
therein.
(12) Lewis, F. D.; Simpson, J. T.; Krantz, A.; Kokel, B. J. Am. Chem.
Soc. 1982, 104, 7155. Lewis, F. D.; Simpson, J. T. J. Am. Chem. Soc. 1980,
102, 7593. Krantz, A.; Lipkowitz, G. S. J. Am. Chem. Soc. 1977, 99, 4156.
(13) Silverman, R. B. Acc. Chem. Res. 1995, 28, 235.
(10) Singer, T. P.; Husain, M. In FlaVins and FlaVoproteins; Massey,
V., Williams, C. H., Eds.; Elsevier: New York, 1982; pp 389-401. Singer,
T. In Monoamine Oxidase Structure, Function and Altered Function; Singer,
T. P., Von Korff, R. W., Murphy, D. L., Eds.; Academic Press: New York,
1979; pp 7-37.
(14) Kim, J.-M.; Bogdan, M. A.; Mariano, P. S. J. Am. Chem. Soc. 1991,
113, 925.
(15) (a) Davidson, R. S.; Orton, S. P. J. Chem. Soc., Chem. Commun.
1974, 209. (b) Gaillard, E. R.; Whitten, D. G. Acc. Chem. Res. 1996, 29,
292. (c) Burton, R. D.; Bartberger, M. D.; Zhang, Y.; Eyler, J. R.; Schanze,
K. S. J. Am. Chem. Soc. 1996, 118, 5655. (d) Su, Z.; Falvey, D. E.; Yoon,
U. C.; Mariano, P. S. J. Am. Chem. Soc. 1997, 119, 0000.
(11) Silverman, R. B.; Cesarone, J. M.; Lu, X. J. Am. Chem. Soc. 1993,
115, 4955 and references therein.

5866 J. Am. Chem. Soc., Vol. 120, No. 24, 1998
Van Houten et al.
Table 1. Data for Competitive Inhibition and Inactivation of
MAO-A by Aminoenones 11-14, Aminoynones 15 and 16, Related
Substances 17-19, and â-Hydroxyamines 5-7
Results
SET-Photochemical Studies. It is well-recognized that the
triplet excited state of 3-methyllumiflavin (3MLF), produced
by excitation and intersystem crossing, is an excellent one-
electron oxidizing agent owing to its large excited-state reduction
potential (ca.+1.3 V).12,16 As such, photoreactions of this flavin
serve as useful models to explore SET-induced reactions of
electron donors. To determine if the aminium radicals derived
from tertiary â-allylic and â-propargylic â-hydroxyamines 3 do
indeed undergo efficient retro-aldol type fragmentation reactions
to generate electrophilic conjugated ketones, we have examined
the SET-promoted photoreactions of the â-hydroxyamines 4-7
with 3-methyllumiflavin.
compd
11
12
13
14
15
16
17
18
Ki (mM)
Kinact (mM)
kinact × 102 (min-1
)
4.8 ( 0.5
4.1 ( 0.5
1.5 ( 0.1
4.0 ( 0.6
2.8 ( 0.2
0.4 ( 0.3
1.0 ( 0.1
0.8 ( 0.2
1.9 ( 0.2
2.1 ( 0.2
1.4 ( 0.1
2.3 ( 0.4
0.4 ( 0.03
2.0 ( 0.3
13.8 ( 0.3
7.6 ( 0.5
3.7 ( 0.1
6.0 ( 0.2
7.3 ( 0.2
3.7 ( 0.5
a
9.5 ( 0.1
13.0 ( 0.3
1.2 ( 0.4
1.4 ( 0.4
1.9 ( 0.2
1.2 ( 0.3
a
a
a
1.7
1.3
a
a
19
5
13.9 ( 0.5
11.8 ( 0.4
2.5 ( 0.5
a
(+)-(R)-5
(-)-(S)-5
1.4 ( 0.1
a
0.7 ( 0.08
6
7
2.6 ( 0.2
a Not determined.
17 and 18 and tertiary R-silylamine 19 are recorded in Table 1.
The synthetic sequences used to prepare 4-7 are provided
in Supporting Information. Photoreactions were performed by
irradiating N2-purged MeOH solutions containing 3MLF and
the allylic and propargylic amino alcohols with Uranium glass
filtered-light (λ>320 nm, ensuring that 3MLF is the primary
light absorbing species). The progress of each photoreaction
was monitored by UV-visible spectroscopy, and irradiation was
terminated when the absorbance at 450 nm associated with
3MLF reached ca. 20% of its original value. Product separation
was accomplished by either silica gel TLC or precipitation (in
the case of flavin 4a-adducts). The results in terms of products
and yields are summarized in Schemes 5. The efficient
formation of flavin adducts in these photoreactions is consistent
with the proposed retro-aldol type reactivity of intermediate
tertiary â-allylic and â-propargylic â-hydroxyaminium radicals
which results in generation of conjugated ketone intermediates.
The ability of the amino-enones and -ynones to serve as
inactivators of MAO-A was examined next. MAO-A (50 mM)
in Na2HPO4 buffer (pH 7.2) containing 0.2% Triton-X at 25
°C was independently reacted with 11-16. Catalytic activity
was monitored periodically by using the kynuramine assay.19
As exemplified in Figure 1 for the case of amino-enone 11,
MAO-A inactivation occurs in a time and concentration
dependent manner. Kitz-Wilson replots20a of the reciprocals
of the apparent rate constants for the inactivation processes vs
the reciprocals of the inactivator concentrations provide the rate
constants for inactivation, kinact, and the inactivation dissociation
constants, Kinact, listed in Table 1. Significantly, the amino-
ketones 17 and 18 and the tertiary R-silylamine 19 do not serve
as efficient inactivators of MAO-A over the time periods in
which their R,â-unsaturated carbonyl analogues bring about
complete inactivation of the enzyme, suggesting that the
reactions occurring between MAO-A and 11-16 must involve
the respective enone or ynone functionalities.
To demonstrate that the MAO inactivation occurs while the
tethered enones/ynones are bound to the MAO-A active site,
inhibition of the inactivation processes by the potent MAO
competitive inhibitor, (+)-(S)-amphetamine (Ki ) 34 µM for
MAO-A)22 was tested. Solutions of MAO-A containing the
inactivators 11 and 15 (6 mM) and (S)-amphetamine (1.6 mM)
were incubated for ca. 2.5 h time periods with periodic
monitoring of enzyme activity. MAO-A inactivation is com-
pletely prevented in the presence of (S)-amphetamine. Thus,
Determination of Enzymatic Inhibition and Inactivation
Constants for Tethered Amino-Enones and Ynones. The
known silylmethylamino-enone 1117 and -ynone 1518 and their
homologues and non-silicon containing analogues 12, 14, and
16 were prepared (Supporting Information) in order to determine
if, and how, active site-bound, conjugated ketones inactivate
the MAOs. Accordingly, these substances were first tested as
inhibitors of the MAOA-catalyzed oxidation reaction of
kynuramine.19 Double recipricol plots20b of the initial velocities
versus kynuramine concentration at various inhibitor concentra-
tions showed that 11-16 are all reversible, competitive inhibi-
tors of this enzyme. The inhibition constants, Ki, determined21
for these substances as well as for the saturated amino-ketones
(16) Novak, M.; Miller, A.; Bruice, T. C. J. Am. Chem. Soc. 1980, 102,
1465. Eweg, J. K.; Muller, F.; Visser, A. J.; Veeger, C.; Bebelaar, D.; Van
Voorst, J. D. Photochem. Photobiol. 1979, 30, 463. Franke, J.; Ott, U.;
Kramer, H. E. A.; Traber, R. H. Photochem. Photobiol. 1989, 49, 131. See
also ref 13.
(17) Jeon, Y. T.; Lee, C. P.; Mariano, P. S. J. Am. Chem. Soc. 1991,
113, 8847.
(18) Khim, S. K.; Cederstrom, E.; Ferri, D. C.; Mariano, P. S.
Tetrahedron 1996, 52, 5222.
(19) Weissbach, H.; Smith, T. E.; Daley J. D.; Witkop, B.; Udenfriend,
S. J. Biol. Chem. 1960, 235, 1160.
(20) (a) Kitz, W.; Wilson, B. J. Biol. Chem. 1962, 237, 3245. (b)
Lineweaver H.; Burke, D. J. Am. Chem. Soc. 1934, 56, 658.
(21) Cleland, W. W. In The Enzymes, 3rd ed.; Boyer, P. D., Ed.;
Academic Press: New York, 1970; Vol. 2, pp 1-65.
(22) Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70.

Design of Monoamine Oxidase InactiVators
J. Am. Chem. Soc., Vol. 120, No. 24, 1998 5867
Table 2. Free-Thiol Determinations on MAO-A and Its
Derivatives Arising by Inactivations with Aminoenones 11 and 12,
Aminoynone 15, and Hydroxyamines 5 and 7
Scheme 5
enzyme
MAO-A
MAO-A + 11
MAO-A + 12
MAO-A + 15
MAO-A + 5
MAO-A + 7
no. of thiolsa
6.8 ( 0.1
5.8 ( 0.1
5.9 ( 0.1
1.3 ( 0.1
5.7 ( 0.2
5.6 ( 0.1
a Determined by use of the DTNB titration method (ref 25) and three
independent experiments each.
flavine moiety. UV-visible spectroscopic methods were used
to distinguish between the two mechanistic pathways for
MAO-A inactivation. Accordingly, three deoxygenated, sealed
curvettes containing MAO-A alone, MAO-A and the amino-
enone 11, and MAO-A and kynuramine were incubated for a 2
h time period leading to ca. 97% inactivation of the enzyme.
The solution containing MAO-A and kynuramine, as expected,
experiences a rapid decrease in the flavin absorbance in the
350-550 nm region as a result of reduction to the 1,5-dihydro
form.24 In contrast, the oxidized flavin spectrum of the solution
containing MAO-A and 11 remains unchanged during this time
period. Thus, the flavin cofactor in MAO-A is not redox active
in the reaction with 11.
Scheme 6
To demonstrate that amino-enone inactivation is a conse-
quence of alkylation of an MAO cysteine thiol function, the
number of free thiols within the enzyme before and following
inactivation was determined by using the 5,5′-dithio-bis-(2-
nitrobenzoic acid) (DTNB) titration method.22 Accordingly,
native MAO-A was found to contain seven free thiol functions
in agreement with the seven known (i.e., predicted from the
gene sequence)25 free cysteine residues of this enzyme (Table
2). In contrast, DTNB titrations of the enzymes, inactivated
by the amino-enones 11 and 12, showed that both contain ca.
6-titratable thiol moieties (Table 2). However, the amino-ynone
15 serves as a much less selective alkylating agent for MAO-
A, indicated by the data in Table 2; complete inactivation of
MAO-A by 15 results in a modified enzyme that contains ca.
1-free thiol only. The combined results demonstrate conclu-
sively that the major, if not exclusive, process involved in MAO-
inactivation by the amino-enones (and perhaps -ynones) is active
site thiol alkylation.
Determination of MAO Binding Constants for the â-Al-
lylic- and â-Propargylic-â-hydroxyamines. The â-hydroxy-
amines 5-7, although lacking phenolic functionality, are
structural analogues of the aryl-ethanolamine containing MAO
inhibitors/substrates, amphetamine, epinephrine, and norepi-
nephrine. Thus, modest binding affinities to the MAO active
sites were expected. Indeed, the steady-state initial velocity data
listed in Table 1 show that 5-7 are reversible competitive
inhibitors of MAO-A, with dissociation constants which are of
comparable magnitude to those of other tertiary amines (e.g.,
Ki ) 7.1 mM for MAO-B by N,N-dimethyl-N-(2-phenylethyl)-
amine)26 and sterically crowded primary amines (e.g, Ki ) 0.2
mM for MAO-B by 1-phenylcyclopropylamine).27
MAO-A inactivations by the amino-enones and -ynones depends
on these compunds binding in the active site of the enzyme.
The amino-enones and -ynones also serve as affinity labeling
reagents of MAO-B. For example, the amino-ynone 15, is a
reversible competitive inhibitor of MAO-B (Ki ) 1.3 mM) as
well as a time and concentration dependent inactivator (Kinact
) 7.3 mM and kinact ) 1.9 × 10-2 min-1). The inactivation of
MAO-B by 15 is also prevented by (S)-amphetamine.
Determination of the Alkylation Site in MAO Inactivation
by Tethered Amino-Enones and -Ynones. As shown in
Scheme 6, two pathways are possible for the inactivation
reactions of the tethered R,â-unsaturated ketoamines. One
involves simple nucleophilic Michael-addition of the cysteine
thiol group which has been previously characterized as being
located in the MAO active site region.23 The other route
involves redox participation by the flavin cofactor and results
in the generation of a covalent adduct containing a reduced
To determine if the individual enantiomers of these â-amino
alcohols have different MAO-A binding affinities, resolution
of 5 was carried out. Separation of the antipodes was performed
(24) Miller, J. R.; Edmondson, D. E.; Grissom, C. B. J. Am. Chem. Soc.
1995, 117, 7830 and references therein.
(25) Fernandez-Diez, M. J.; Osuga, D. T.; Feeney, R. E. Arch. Biochem.
Biophys. 1964, 107, 449.
(23) (a) Erwin, V. G.; Hellerman, L. J. Biol. Chem. 1967, 242, 4230.
(b) Weyler, W.; Salach, J. I, J. Biol. Chem. 1985, 260, 13199.
(26) Silverman, R. B.; Ding, C. Z. J. Med. Chem. 1993, 36, 3606.
(27) Silverman, R. B.; Zieske, P. A. Biochemistry 1985, 24, 2128.

5868 J. Am. Chem. Soc., Vol. 120, No. 24, 1998
Van Houten et al.
Figure 2. Plot of the natural log of the percent activity remaining vs
time for inactivation of MAO-A by 5. MAO-A (10 mM) was incubated
with 5 (0, square; 1, diamond; 2, circle; 4, triangle; 8, filled square; 10
mM, filled diamond) in 50 mM Na2HPO4 buffer (pH 7.2) at 25 °C.
MAO-A activity was monitored by use of the kynuramine assay.
Figure 1. A plot of the natural log of the percent activity remaining
vs time for inactivation of MAO-A by the amino-enone 11. MAO-A
(8.5 µm) was incubated with 11 (0, square; 1, diamond; 2.5, circle;
5.0, triangle; 7.5, filled square; 10.0, filled diamond mM) in 50 mM
Na2HPO4 (pH 7.2) solutions containing 0.2% Triton-X at 25 °C.
Aliquots (50 µL) were removed periodically and assayed for MAO-
activity by use of the kynuramine procedure.
by HPLC through the use of a Chiralcel OJ column or by
crystallization of and regeneration from the (+)-D-dibenzoyl-
tartrate salt. Absolute stereochemical assignments to (+)-5 and
(-)-5 as (R) and (S), respectively, were made based on X-ray
crystallographic analysis of the tartrate salt 20 of (-)-5
(Supporting Information). As indicated by the similar magni-
tudes of the inhibition constants exhibited by the (R)-enantiomer
and (S)-enantiomer of 5 (Table 1), the enzyme displays little
stereoselectivity in binding the allylic â-amino alcohol antipodes.
This is not an unexpected result based on the observation28 that
MAO-A displays only a small binding preference for the (R)-
(Ki ) 0.1 mM) vs the (S)- (Ki ) 0.11 mM) enantiomers of
N-methylamphetamine.
Figure 3. UV-visible spectra of the flavin-cofactor absorbance region
of MAO-A during the course of its inactivation by the propargylic-â-
amino alcohol 7. MAO-A (25 µM) was incubated with 7 (23 mM) in
50 mM Na2HPO4 (pH 7.2) containing 0.2% Triton-X under anaerobic
conditions. Spectra were recorded before addition of 7, 27 min after
addition of 7 under anaerobic conditions, and after admission of air to
the reaction mixture.
active site protection experiments were carried out. Specifically,
the inactivation reactions were carried out with 16 mM of each
inactivator and in the presence or absence of saturating (+)-
(S)-amphetamine (1.6 mM). As expected for mechanism-based
inactivation, (+)-(S)-amphetamine completely blocks the reac-
tions of 5 and 7 (16 mM) with this enzyme. In addition,
application of the DTNB titration method shows that MAO-A
inactivated by both the allylic- and propargylic-tertiary amino
alcohols, 5 and 7, contains one less cysteine-thiol function than
the native enzyme (see Table 2).
UV-visible spectroscopic monitoring of the reaction of
MAO-A with â-amino alcohol 7 has provided information about
the redox-participation of the flavin cofactor in the process. As
shown by the spectra reproduced in Figure 3, anaerobic reaction
of MAO-A with 7 is associated with a change from the typical
enzyme oxidized flavin spectrum with a maximum at ca. 450
nm to one that contains a maximum at ca. 410 nm. This change,
which is reversed by admission of oxygen into the reaction
solution, is not consistent with complete reduction of the flavin
residue in the enzyme to produce either the 1,5-dihydro form
Determination of the Thermodynamic and Kinetic Con-
stants for MAO Inactivation with the â-Allylic- and â-Pro-
pargylic-â-Hydroxyamines. The â-allylic and â-propargylic
â-hydroxyamines 5-7 were tested as irreversible inhibitors of
MAO-A by use of time dependent reactions in which catalytic
activity was assayed by use of the kynuramine method.19 The
apparent rate constants for inactivation, derived from the slopes
of plots of the natural log of activity remaining vs time (Figure
2 for 5), were evaluated. From the Y-intercepts of double
recipricol plots20b of apparent kinact value vs inactivator con-
centrations, the kinact value were derived, and from the X-
intercepts, the dissociation constants, Kinact, for MAO-inactivatior
were determined. The inactivation constants determined in this
manner for 5 and 7 are recorded in Table 1. Interestingly, the
primary â-amino alcohol 6 was found not to be an MAO-A
inactivator.
To determine if MAO-A inactivation by the â-amino alcohols
5 and 7 is associated with covalent modification of the enzyme,
(28) Robinson, J. B. Biochem. Pharmacol. 1985, 34, 4105.

Design of Monoamine Oxidase InactiVators
J. Am. Chem. Soc., Vol. 120, No. 24, 1998 5869
Scheme 7
Scheme 8
tation route dominates alternative decay reactions of the
aminium radicals involving (e.g., RCH-deprotonation)31 espe-
cially in the cases of the R-aryl analogues.15 R-Amino radicals
are known to be exceptionally powerful reducing agents.32
As a result, SET from these intermediates to the hydroflavin
radical is facile and results in formation of the nucleophilic
hydroflavin anion. Michael addition of the hydroflavin anion
to the conjugated carbonyl products then produces the 4a-
adducts.
or a 4a- or N5-adduct.24 In fact, previous efforts29 have shown
that the 410 nm absorption band is associated with an anionic
flavin-semiquinone radical. Thus, this species might be a meta-
stable product in the â-amino alcohol inactivation pathway (see
below) suggesting that the flavin cofactor of MAO-A is involved
in the mechanism for inactivation by 5 and 7.
As shown in Scheme 4, fragmentation of aminium radicals
derived from the allylic and propargylic â-amino alcohols 5
and 7 forms a dimethylaminomethyl radical which would be
further transformed by oxidation and subsequent hydrolysis to
N,N-dimethylamine. Dabsyl-chloride30 treatment of the crude
mixture obtained from reaction of MAO-A with amino-alcohol
5 leads to isolation of the dimethylamine derived sulfonamide
Observations made in our investigations with the tethered
amino-enones and -ynones lend credence to suggested mech-
anisms for MAO-inactivation involving the generation and
addition reactions of electrophilic intermediates. These sub-
stances serve as affinity labeling agents owing to the presence
of both the amine functions which lead to active site binding
and the R,â-unsaturated ketone groups which provide Michael
addition reactivity. The active nucleophile in these inactivation
processess is a cysteine thiol which earlier chemical studies23
have identified as being required for catalytic activity.
1
2130 characterized by TLC, HPLC, and H NMR analyses.
The results point to the operation of mechanism based
pathways for MAO-A inactivation by the â-hydroxyamines 5
and 7. In a manner consistent with our proposal, the enzymatic
sequence (Scheme 8) is most likely initiated by SET within the
enzyme-inactivator complex which is then followed by retro-
aldol fragmentation of the intermediate tertiary aminium radical.
This step might be facilitated by the same basic residue that
participates as a general base in the MAO-catalytic reaction.
The R,â-unsaturated carbonyl product formed in this manner
serves as a Michael acceptor in reaction with a cysteine thiol
to produce the inactivated enzyme. In the presence of oxygen,
both the anionic flavin semiquinone, possibly responsible for
the 410 nm absorption band under anaerobic conditions, and
the dimethylaminomethyl radical are converted to the respective
oxidized flavin and the iminium cation precursor of dimethy-
lamine.
Finally, MAO-inactivation by the allylic- and propargylic-
amino alcohols 5 and 7 appears to be selective for MAO-A.
For example, although 5 is a modest competitive inhibitor of
MAO-B (Ki ) 2.7 mM), neither it nor its acetylene analogue 7
are inactivators of the B-enzyme.
Discussion
The results presented above demonstrate the validity of the
proposed strategy for MAO-inactivator design. It is clear that
SET-photochemical processes can serve as a predictive method
for the identification of tertiary amines which are capable of
reacting with the MAOs to generate electrophilic products in
the enzyme active site region. The observations made in the
current photochemical study confirm that tertiary aminium
radicals arising from â-allylic- and â-propargylic-â-hydroxyamines
undergo facile retro-aldol like fragmentation15 to produce
conjugated enone or ynone products. The operation of this
process is revealed by the formation of 4a-adducts (e.g., 8) as
the major if not exclusive substances produced in these
photoreactions. Accordingly, SET from the amine function in
the â-amino alcohols to the triplet excited state of 3MLF
generates an ion radical pair in which aminium radical retro-
aldol-like fragmentation occurs with simultaneous proton trans-
fer to the anion radical of 3MLF (Scheme 7). This fragmen-
A few additional observations made in this study are worthy
of comment. The first concerns the fact that the primary amine
6, although serving as a comparable reversible competitive
inhibitor of MAO-A, is not an inactivator of this enzyme. While
there are a number of possible reasons for the lack of reactivity
of this substance, one of these might be related to the fact that
(31) Pienta, N. J. In Photoinducd Electron Tranfer; Fox, M. A.. Channon,
M., Eds.; Elsevier: New York, 1988; Part C, pp 421-486. Lewis, F. D.
Acc. Chem. Res. 1986, 19, 401.
(32) Wayner, D. D. M.; McPhee, D. J.; Griller, D. J. Am. Chem. Soc.
1988, 110, 132.
(29) Yue, K. T.; Bhattacharyya, A. K.; Zhelyaskov, V. R.; Edmondson,
D. E. Arch. Biochem. Biophys. 1993, 300, 178.
(30) Lin, J. K.; Wang, C. H. Anal. Chem. 1980, 52, 630.

5870 J. Am. Chem. Soc., Vol. 120, No. 24, 1998
Van Houten et al.
Scheme 9
Experimental Section
General. All reactions were run under N2 or Ar atmospheres unless
otherwise noted and magnesium sulfate was used as a drying agent.
All new compounds were obtained as oils in >90% purity (by 1H and
13C NMR) unless otherwise noted. 1H NMR (200, 400, 500 MHz)
and 13C NMR (50 MHz) spectra were recorded on CDCl3 solutions
unless otherwise noted and chemical shifts are reported in parts per
million relative to CHCl3 (7.24 for 1H NMR and 77.00 for 13C NMR)
as an internal standard. Coupling constants are given in Hertz (Hz).
13C NMR resonance assignments were aided by use of the DEPT
technique to determine number of attached hydrogens. Mass spectro-
metric data was obtained by use of either electron impact (EI) or
chemical ionization (CI) techniques and fragments are recorded as m/z
(relative intensity). Infrared (IR) spectroscopic data are recorded in
units of cm-1. Optical rotations were recorded by using the sodium
D-line (589 nm). Thin-layer chromatography (TLC) was performed
on 0.25 mm silica coated glass or plastic plates. Preparative TLC was
performed on 20 × 20 cm plates coated with silica gel. Column
chromatography was performed with the use of 230-400 mesh silica
gel, 100-200 mesh fluorisil, or 80-200 mesh alumina. Unless
otherwise noted, materials obtained from commercial sources were used
without further purification. All distillations were performed under
dry N2 or Ar atmospheres unless otherwise noted. All reaction solvents
were dried and distilled prior to use. The solvent used for photore-
actions (MeOH) was of spectrograde quality.
Scheme 10
Preparative photochemical reactions were conducted by using an
apparatus consisting of a 450W Hanovia medium pressure, mercury
lamp surrounded by a Uranium glass filter (λ > 330 nm) within a quartz,
water cooled well immersed in the reaction solution (ca. 15 °C reaction
temperature). In each case, the photolysis solution was purged with
deoxygenated N2 before and during irradiation.
MAO-A (KM ) 0.12 mM, kcat ) 35 min-1) was purified35 from the
yeast Saccharomyces cereVisiae which was overexpresseed with human
liver MAO-A (gift from Walter Weyler). MAO-B was obtained from
beef liver, purchased fresh from a local slaughter house, and was
purified by a minor modification of the procedure of Salach.36a
Irradiation of 3MLF and the Amino Alcohol 4. A solution (170
mL) of 77 mg (0.26 mmol) of 3MLF and 120 mg (0.45 mmol) of the
amino alcohol 4 in MeOH was irradiated for 1 h. The photolyzate
was concentrated in vacuo to give a residue which was dissolved in
CHCl3 and diluted with n-hexane. The resulting precipitate was
collected by filtration to give 101 mg (96%) of the adduct 8.14
Concentration of the filtrate gave 5 mg (4%) of 3-(N-benzyl-N-methyl)-
aminopropiophenone (9).
9: 1H NMR 2.20 (s, 3H, N-CH3), 2.83 (t, J ) 7.0 Hz, 2H, H-2),
3.12 (t, J ) 7.0, 2H, H-3), 3.49 (s, 2H, benzylic), 7.25, 7.38, 7.50, and
7.90 (m, 10H, aromatic); 13C NMR 36.8 (C-2), 42.1 (N-CH3), 52.4
(C-3), 62.3 (benzylic), 126.9, 128.0, 128.1, 128.5, 128.9, 132.8, 136.9,
and 138.7 (aromatic Ph), 199.3, (CdO); IR 1682; EIMS 253 (M, 2),
176 (2), 162 (41), 134 (100), 105 (42), 91 (85), 77 (20); HRMS(EI)
m/e 253.1478 (C17H19NO requires 253.1467).
Irradiation of 3MLF and 4-(N,N-Dimethylamino)-3-phenylbut-
1-en-3-o1 (5). A solution of 71 mg (0.26 mmol) of 3MLF and 86 mg
(0.45 mmol) of the amino alcohol 5 in MeOH (150 mL) was irradiated
for 1 h. The photolyzate was concentrated in vacuo to give a residue
which was dissolved in 1 mL of CHCl3. To this solution was added
30 mL of n-hexane, and the resulting precipitate was collected by
filtration to give 98 mg of the 4a-adduct (91%) 8.14
Irradiation of 3MLF and 4-Amino-1-buten-3-phenyl-3-ol (6). A
solution of 71 mg (0.26 mmol) of 3MLF and 74 mg (0.46 mmol) of
the amino alcohol 6 in MeOH (150 mL) was irradiated for 1 h. The
photolyzate was concentrated in vacuo to give a residue which was
primary amines often have higher oxidation potentials than their
tertiary analogues.31,33 Indeed, the current photochemical studies
suggest that the aminium radical derived from 6 undergoes ready
retro-aldo like fragmentation. Thus, the inability of 6 to serve
as an MAO-A inactivator may be a consequence of the fact
that it, like other primary and secondary amine substrates and
inactivators, does not serve as an effective SET-donor to the
flavin group in the enzyme.
The possibility exists that several other well-known MAO
inactivation reactions, previously discussed in terms of radical
coupling mechanisms, may also follow routes involving genera-
tion of and alkylation by electrophilic intermediates. For
example, Silverman11 has shown that trans-2-phenylcyclopro-
pylamine 22 reacts with MAO-B to yield a single covalent
adduct via alkylation of the key cycteine residue. Bond
formation in this process may well be a result of the formation11
and Michael addition of cinnamaldehyde (or its imine) (see
Scheme 9).
In a similar way, MAO-B inactivation by the tertiary
propargylic amine, pargyline 2334 may be promoted by an initial
SET step,12 but the known flavocyanine adduct 25 might arise
by Michael addition to the R,â-unsaturated iminium cation
intermediate 38 (Scheme 10) rather than by a radical coupling
mechanism.
In summary, a novel mechanism-based design strategy has
been used to discover a new class of MAO-inactivators. Further
efforts are necessary, however, to fully elucidate the inter-
relationships that may or may not exist between MAO-
inactivation and catalytic reaction pathways and to refine the
design of â-amino alcohol inactivators to yield candidates with
higher binding affinities and inactivation rates.
(35) Weyler, W.; Titlow, C. C.; Salach, J. I. Biochem. Biolphys, Res.
Commun. 1990, 173, 1205 and ref 23b.
(36) (a) Salach, J. I.; Weyler, W. In Methods in Enzymology; Academic
Press: New York, 1987; Vol. 142, pp 627-637. Salach, J. I.; Weyler, W.
Arch. Biochem. Biophys. 1979, 192, 128. (b) Tabor, C. W.; Tabor, H. J.
Biol. Chem. 1954, 208, 645.
(33) Yoon, U. C.; Mariano, P. S. Acc. Chem. Res. 1992, 25, 233.
(34) Maycock, A. L.; Abeles, R. H.; Salach, J. I.; Singer, T. P.
Biochemistry 1976, 15, 114.
(37) Kim, J. M.; Cho, J. S.; Mariano, P. S. J. Org. Chem. 1991, 56,
4943.

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%

5872 J. Am. Chem. Soc., Vol. 120, No. 24, 1998
Van Houten et al.
Time Dependent Inactivation of MAO-B. Aliquots of an MAO-B
stock solution were added to solutions containing varying concentrations
of inactivators in 50 mM sodium phosphate buffer (pH 7.2), containing
0.2% Triton X-100. The samples were incubated at 25 °C and
periodically assayed for enzyme activity by use of the benzylamine
assay procedure.36b All assays were performed in duplicate.
MAO-A expression system and Professor Debra Dunaway-
Mariano for her helpful suggestions throughout the course of
this work.
Supporting Information Available: Synthetic procedures
for the preparation of 4-7, 12-14, and 16-18 and a Chem
3D-plot and supporting data of the crystallographically deter-
mined structure of 20 (40 pages, print/PDF). See any current
masthead page for ordering information and Web access
instructions.
Acknowledgment. Support for this effort by the National
Institutes of Health (GM-53822, for the biochemical studies)
and the National Science Foundation (CHE-9420889, for the
photochemical studies) is greatly appreciated. We thank Dr.
Walter Weyler for his generous gift of the yeast containing the
JA973978D