Monoamine oxidase B-catalyzed reactions of cis- and trans-5-aminomethyl- 3-(4-methoxyphenyl)dihydrofuran-2(3H)-ones. Evidence for a reversible redox reaction

Article abstract of DOI:10.1021/ja9811041

Monoamine oxidase B (MAO B) was previously shown to catalyze the decarboxylation of cis- (1) and trans-5-(aminomethyl)-3-(4- methoxyphenyl)dihydrofuran-2(3H)-one hydrochloride (2) (Silverman, R. B.; Zhou, J. J. P.; Ding, C. Z.; Lu, X. J. Am. Chem. Soc. 1995, 117, 12895- 12896). By [¹⁴C]-labeling of the aryl methoxyl group, it is now shown that the decarboxylated product is 4-(4-methoxyphenyl)-butanal (7), which is in the same oxidation state as the substrate. Two other products are produced, 4-carboxy-4-(4-methoxyphenyl)butanal (8), and 5-formyl-3-(4- methoxyphenyl)dihydrofuran-2(3H)-one (9). Only 9 is an oxidation product; 7 and 8 are in the same oxidation state as the substrate (1 or 2). No products are detected under strictly anaerobic conditions. All of these products can be rationalized as arising from the formation of an α-carbon radical, generated either by single-electron amine oxidation and loss of a proton or direct hydrogen atom abstraction to 10 (Scheme 5). This intermediate then can undergo second electron oxidation and hydrolysis of the iminium ion to give 9 (the normal oxidation product). However, it also can suffer either homolytic C-O bond cleavage, decarboxylation, and electron return from the active site to give 7 or heterolytic cleavage and electron return from the active site to give 8. 5-(4-Methoxyphenyl)tetrahydrofuran-2-ol (14), an oxidation product from the intermediate that leads to 7, is not detected. These results suggest that MAO B can catalyze reversible redox reactions.

Full text of DOI:10.1021/ja9811041

J. Am. Chem. Soc. 1998, 120, 10583-10587
10583
Monoamine Oxidase B-Catalyzed Reactions of cis- and
trans-5-Aminomethyl-3-(4-Methoxyphenyl)dihydrofuran-2(3H)-ones.
Evidence for a Reversible Redox Reaction
Xingliang Lu and Richard B. Silverman*
Contribution from Department of Chemistry and Department of Biochemistry, Molecular Biology, and
Cell Biology, Northwestern UniVersity, EVanston, Illinois 60208-3113
ReceiVed April 2, 1998. ReVised Manuscript ReceiVed July 30, 1998
Abstract: Monoamine oxidase B (MAO B) was previously shown to catalyze the decarboxylation of cis- (1)
and trans-5-(aminomethyl)-3-(4-methoxyphenyl)dihydrofuran-2(3H)-one hydrochloride (2) (Silverman, R. B.;
Zhou, J. J. P.; Ding, C. Z.; Lu, X. J. Am. Chem. Soc. 1995, 117, 12895-12896). By [14C]-labeling of the aryl
methoxyl group, it is now shown that the decarboxylated product is 4-(4-methoxyphenyl)butanal (7), which is
in the same oxidation state as the substrate. Two other products are produced, 4-carboxy-4-(4-methoxyphenyl)-
butanal (8), and 5-formyl-3-(4-methoxyphenyl)dihydrofuran-2(3H)-one (9). Only 9 is an oxidation product;
7 and 8 are in the same oxidation state as the substrate (1 or 2). No products are detected under strictly
anaerobic conditions. All of these products can be rationalized as arising from the formation of an R-carbon
radical, generated either by single-electron amine oxidation and loss of a proton or direct hydrogen atom
abstraction to 10 (Scheme 5). This intermediate then can undergo second electron oxidation and hydrolysis
of the iminium ion to give 9 (the normal oxidation product). However, it also can suffer either homolytic
C-O bond cleavage, decarboxylation, and electron return from the active site to give 7 or heterolytic cleavage
and electron return from the active site to give 8. 5-(4-Methoxyphenyl)tetrahydrofuran-2-ol (14), an oxidation
product from the intermediate that leads to 7, is not detected. These results suggest that MAO B can catalyze
reversible redox reactions.
Introduction
Scheme 1
Monoamine oxidase (EC 1.4.3.4; MAO) is a flavoenzyme
that is important in the degradation of a variety of biogenic
amines. Considerable evidence has been presented that is
consistent with an electron-transfer mechanism for this enzyme
(Scheme 1, pathway a),1 although others have suggested a direct
hydrogen atom abstraction mechanism (Scheme 1, pathway b)2
and a nucleophilic mechanism.3 Recently, it has been proposed
that MAO contains a redox-active disulfide at the active site
and that electrons from the substrate are transferred initially to
the disulfide and from the disulfide to the flavin.4 This
intriguing result may explain why a flavin semiquinone inter-
mediate has not yet been detected during enzymatic turnover.2
The first electron transfer to the flavin depicted in pathway a is
generally written as a reversible reaction to rationalize the
isotope effect on C-H bond cleavage that is often observed,
although there is no evidence that this step is reversible. To
provide chemical evidence for the intermediacy of an R-radical
during MAO-catalyzed amine oxidation (by either pathway a
or b), two different approaches were taken. In the first approach,
it was shown that MAO B-catalyzed oxidation of (aminometh-
yl)cubane led to destruction of the cubane structure, supporting
the generation of a cubylcarbinyl radical.5 Secondly, cis- (1)
and trans-5-(aminomethyl)-3-(4-methoxyphenyl)dihydrofuran-
2(3H)-one hydrochloride (2) were synthesized with [14C] labels
at the carbonyl carbon atoms, and it was shown that MAO B
catalyzed the decarboxylation of both analogues; the mechanism
proposed for this decarboxylation reaction proceeds via R-radical
3 (drawn via an amine radical cation intermediate) as shown in
Scheme 2.6 The fate of radical 4, however, was not clear. In
* Address correspondence to the Department of Chemistry. Phone: (847)
491-5653. Fax: (847) 491-7713. E-mail: Agman@chem.nwu.edu.
(1) (a) Silverman, R. B. Acc. Chem. Res. 1995, 28, 335-342. (b)
Silverman, R. B. In AdVances in Electron-Transfer Chemistry; Mariano,
P. S., Ed.; JAI Press: Greenwich, CT, 1992; Vol. 2, pp 177-213.
(2) (a) Walker, M. C.; Edmondson, D. E. Biochemistry 1994, 33, 7088-
7098. (b) Miller, J. R.; Edmondson, D. E.; Grissom, C. B. J. Am. Chem.
Soc. 1995, 117, 7830-7831. (c) Jonsson, T.; Edmondson, D. E.; Klinman,
J. P. Biochemistry 1994, 33, 14871-14878.
(3) (a) Kim, J.-M.; Bogdan, M. A.; Mariano, P. S. J. Am. Chem. Soc.
1993, 115, 10591-10595. (b) Kim, J.-M.; Hoegy, S. E.; Mariano, P. S. J.
Am. Chem. Soc. 1995, 117, 100-105.
(5) Silverman, R. B.; Zhou, J. P.; Eaton, P. E. J. Am. Chem. Soc. 1993,
115, 5, 8841-8842.
(6) Silverman, R. B.; Zhou, J. J. P.; Ding, C. Z.; Lu, X. J. Am. Chem.
Soc. 1995, 117, 12895-12896.
(4) Sablin, S. O.; Ramsay, R. R. J. Biol. Chem. 1998, 273, 14074-14076.
10.1021/ja9811041 CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/30/1998

10584 J. Am. Chem. Soc., Vol. 120, No. 41, 1998
Lu and SilVerman
this paper, we report our investigation of the products formed
by the MAO B-catalyzed reaction of 1 and 2, which, unexpect-
edly, provides evidence for an apparent reversible redox reaction
of MAO B and possible reversible electron transfer.
Scheme 2
Results
Synthesis of cis- (5) and trans-5-Aminomethyl-3-[meth-
oxyl-14C]-(4-methoxyphenyl)dihydrofuran-2(3H)-one Hy-
drochloride (6). The cis-(5) and trans-5-aminomethyl-3-
[methoxyl-14C]-(4-methoxyphenyl)dihydrofuran-2(3H)-one hy-
drochlorides (6) were synthesized by the same route previously
reported for the syntheses of cis- and trans-[2-14C]-5-amino-
methyl-3-(4-methoxyphenyl)dihydrofuran-2(3H)-one, which
started from 4-methoxyphenylacetonitrile.6,7 For the synthesis
of 5 and 6, 4-[14C]methoxyphenylacetonitrile was first synthe-
sized from 4-hydroxyphenylacetonitrile and [14C]dimethyl sul-
fate.
Compound 7 was synthesized by pyridinium chlorochromate
oxidation of 4-(4-methoxyphenyl)-1-butanol (Aldrich Chemical
Co.). Compound 8 was prepared by the route shown in Scheme
3. Esterification of 4-(methoxyphenyl)acetic acid (Aldrich
Chemical Co.) produced 16, which was converted to the
malonate ester (17) with diethyl oxalate and sodium hydride.
Michael addition of the malonate ester anion to acrolein gave
18. Following acetal protection of the aldehyde with ethylene
glycol and saponification/decarboxylation, 20 was deprotected
with HCl to give 8. Aldehyde 9 was unstable under the
conditions of the enzyme experiment, so the corresponding 2,4-
dinitrophenylhydrazone was prepared. The cis and trans alde-
hydes (9) were synthesized from the corresponding iodides6,7
by a DMSO oxidation.8 Ceric ammonium nitrate oxidation of
4-(4-methoxyphenyl)butyric acid (Aldrich Chemical Co.) gave
lactone 15, which was reduced with DIBAL to the lactol 14
(Scheme 4).
compounds 8, 7, and 9, respectively. On the basis of the amount
of enzyme used, 119 equiv of 7 (0.83 µmol of 7/0.007 µmol of
MAO B) and 9 equiv of 8 (0.065 µmol of 8/0.007 µmol of
MAO B) were produced. Because of the instability of 9, it
could not be quantified. No 14 was detected; 0.1 equiv of 14
could have been detected had it been formed. The addition of
catalase to decompose any hydrogen peroxide generated during
the oxidation reaction had no effect on the product formation
or on their relative concentrations. When the experiment was
repeated with 1 under strictly anaerobic conditions, no metabo-
lites were detected.
Discussion
MAO catalyzes the oxidation of a variety of primary,
secondary, and tertiary amines. However, upon reaction of
MAO B with either 5 or 6 three enzyme-derived products are
formed, two of which, 7 and 8, are in the same oxidation state
as 5 and 6. The formation of these metabolites can be accounted
for by an elaboration of the mechanism shown in Scheme 1 for
MAO B-catalyzed decarboxylation of 5 and 6 (Scheme 5).
Following either one-electron transfer and proton transfer or
direct hydrogen atom abstraction (not specifically shown in
Scheme 5) intermediate 10 could decompose by a homolytic
(pathway a) or a heterolytic (pathway b) cleavage to 11 or 13,
respectively. Homolytic C-C bond cleavage of 11 leads to
decarboxylation and formation of intermediate 12, as previously
reported.6 To get to 7 from 12 a one-electron reduction is
required, which could occur by electron transfer from the flavin
semiquinone to the benzylic radical (followed by proton transfer
and hydrolysis). This returns the oxidation state to that of the
substrate. The corresponding oxidation of 12, i.e., electron
transfer from 12 to the flavin semiquinone, apparently, is not
favorable, since that would lead to 14, which was not detected
(a fraction of an equivalent could have been detected). Het-
erolytic cleavage of 10 (pathway b) would give 13; to get to 8
from 13, again, a one-electron reduction by electron transfer
from the flavin semiquinone is required. Compound 8, but not
the major product, compound 7, also could arise from a
nucleophilic mechanism. The expected normal oxidation
product 9 can arise from 10 by second electron transfer (pathway
c) and hydrolysis of the resultant iminium ion.
Products Generated upon Incubation of 5 and 6 with
MAO B. Incubation of 5 or 6 with purified beef liver MAO
B9 produced three enzyme-derived products (7-9), as detected
by HPLC (Figure 1). Six different radioactive products were
detected. The peaks labeled A and B are diastereomers of a
nonenzymatic rearrangement product of 5 and 6, namely, 21;10
Since 7 and 8 return the flavin to the oxidized form, if
cleavage of the C-O bond of 10 (pathways a and b) is faster
than electron transfer to the iminium ion (pathway c), then even
under anaerobic conditions, products 7 and 8 should be detected.
When the reaction was carried out anaerobically, no products
were detected, suggesting that formation of 9, which gives
reduced flavin and inactivates the enzyme, is a much faster
process than cleavage of the C-O bond. This same conclusion
was arrived at previously6 when 1 and 2 were [14C]-labeled at
the lactone carbonyl carbon; only 0.5 and 6 equiv of 14CO2 was
D is the starting material (5 in this experiment), C, E, and F are
(7) Ding, Z.; Silverman, R. B. J. Med. Chem. 1992, 35, 885-889.
(8) Johnson, A. P.; Pelter, A. J. Chem. Soc. 1964, 520.
(9) Weyler, W.; Salach, J. I. Arch. Biochem. Biophys. 1981, 212, 147-
153.
(10) Lu, X.; Silverman, R. B. Bioorg. Med. Chem. in press.

Monoamine Oxidase B-Catalyzed Reactions
J. Am. Chem. Soc., Vol. 120, No. 41, 1998 10585
Scheme 3
Scheme 4
resolution spectrometer. UV spectra were recorded on a Perkin-Elmer
Lambda 10 UV/vis spectrometer. Radioactivity was measured on a
TRI-CARB 2100TR liquid scintillation analyzer. Column chromatog-
raphy was peiformed with Merck silica gel (230-400 mesh). [14C]-
Dimethyl sulfate was purchased from American Radiolabeled Chemicals
Inc. (St. Louis, MO) Other chemicals were purchased from Aldrich
Chemical Co. Biochemicals and enzymes were purchased from Sigma
Chemical Co.
4-(4-Methoxyphenyl)butanal (7). To a stirred suspension of
pyridinium chlorochromate (3.22 g, 15 mmol) and sodium acetate (0.24
g, 3 mmol) in anhydrous methylene chloride (28 mL) was added 4-(4-
methoxyphenyl)-butanol (1.80 g, 10 mmol) in methylene chloride (5
mL). After 2 h of stirring, anhydrous ether (20 mL) was added, and
the suspension was filtered through a pad of Celite. The filtrate was
then concentrated in vacuo, and the residue was purified by flash
chromatography (hexane/ethyl acetate, 4:1) to give 1.4 g (79%) of 7
as an oil. 1H NMR (CDCl3): δ 9.75 (s, 1 H), 7.11 (AB, 2 H), 6.85
(AB, 2 H), 3.79 (s, 3 H), 2.60 (t, 2 H), 2.47 (t, 2 H), 1.93 (m, 2 H).
HRMS calcd for C11H14O2 178.0994, found 178.0993.
Figure 1. HPLC of metabolites from incubation of MAO B with either
5 or 6. Peaks A and B are diastereomers of a nonenzymatic rearrange-
ment product10, peak C is 8, peak D is starting material (5), peak E is
7, and peak F is 9.
5-(4-Methoxyphenyl)dihydrofuran-2(3H)-ol (14). Lactone 15 was
prepared by a a known procedure.11 A mixture of 4-methoxyphen-
ylbutanoic acid (0.97 g, 5 mmol), ceric ammonium nitrate (5.48 g, 10
mmol), 0.5 N nitric acid (10 mL), and acetonitrile (20 mL) was refluxed
for 8 h. The resulting solution was cooled to room temperature and
extracted with methylene chloride. The organic layer was washed
successively with 10% aq sodium bicarbonate and water, dried over
Na2SO4, and filtered. The solution was concentrated in vacuo, and
the crude product was purified by flash column chromatography
(hexane/ethyl acetate ) 2:1) to give 0.5 g (52%) of 15. mp 52-53
°C; 1H NMR (CDCl3): δ 7.25-7.27 (AB, 2 H), 6.89-6.91 (AB, 2 H),
5.42-5.49 (m, 1 H), 3.79 (s, 3 H), 2.57-2.67 (m, 3 H), 2.18-2.22
(m, 1 H). HRMS calcd for C11H12O3 192.079, found 192.078.
A solution of 15 (0.5 g, 2.6 mmol) in toluene (4 mL) was cooled to
-78 °C under a nitrogen atmosphere. Then, a 1.5 M solution of DIBAL
in toluene (4 mL, 6 mmol) was added dropwise over 45 min. The
resulting mixture was stirred at -78 °C for 3.5 h, and the excess DIBAL
was destroyed by the addition of a solution of isopropyl alcohol (2 M)
in toluene (3 mL). The solution was then allowed to warm to 0 °C,
and water (0.1 mL), THF (10 mL), and a mixture of silica gel (1.5 g)
and magnesium sulfate (3 g) were added successively. Between
trapped (presumably leading to the formation of 7) upon
inactivation of MAO B by the cis- and trans compounds,
respectively, but 146 and 281 equiv of nonamines (the sum of
7-9), respectively, were produced. Presumably, the reason that
not even 1 equiv of 9 was detected was because of its instability.
Both the conversion of 12 to 7 and 13 to 8 support a reversible
redox reaction that may involve an electron-transfer mechanism
from the substrate to the flavin or from the substrate to a redox-
active disulfide.4 This provides strong evidence that a reversible
initial electron transfer from the amine substrate to the flavin is
plausible and reasonable and suggests that the MAO-catalyzed
oxidation of amines may be completely reversible.
Experimental Section
General Methods. NMR spectra were recorded on either a Varian
300-MHz or a Varian Unity Plus 400-MHz spectrometer. Chemical
shifts are reported as δ values in parts per million downfield 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-
(11) Nagarajan, A.; Porchezhiyan, V.; Srinivasaramanujam; Balasubra-
manian, T. R. Ind. J. Chem. 1985, 24B, 1202-1203.

10586 J. Am. Chem. Soc., Vol. 120, No. 41, 1998
Lu and SilVerman
Scheme 5
each addition the solution was stirred for 30 min. After filtration, the
solid was washed with THF (2 × 10 mL) and methylene chloride (10
mL).The filtrate was dried over magnesium sulfate, and the solvent
was evaporated under reduced pressure to give 0.4 g of 14 as an oil
(80%) containing a mixture of diastereomers. 1H NMR (CDCl3): δ
7.38 (d, 1 H), 7.25 (AB, 2 H), 7.85 (AB, 2 H), 5.71 (t, 0.5 H), 5.59 (t,
0.5 H), 5.20 (t, 0.5 H), 4.96 (t, 0.5 H), 3.78 (s, 3 H), 2.95 (d, 0.5 H),
2.85 (d, 0.5 H), 2.41 (m, 0.5 H), 2.22 (m, 0.5 H), 2.05 (m, 1 H), 1.98
(m, 0.5 H), 1.89 (m, 0.5 H). Anal. Calcd for C11H14O3: C, 68.04%;
H, 7.22%. Found: C, 67.78%; H, 7.41%. HRMS calcd for C11H14O3
194.094, found 194.094.
Ethyl 4-Methoxyphenylacetate (16). To a solution of 4-methox-
yphenylacetic acid (5 g, 30 mmol) in benzene (30 mL) was added
p-toluenesulfonic acid (60 mg) and ethanol (10 mL). The reaction was
refluxed using a Dean-Stark trap to azeotropically remove the water.
After 10 h, the benzene solution was cooled to room temperature,
diluted with an equal volume of ether, washed with saturated NaHCO3
(2 × 20 mL), and then dried over MgSO4 and filtered. The oil
remaining after removal of the solvent under vacuum was 16 (5 g,
87%). 1H NMR (CDCl3): δ 7.21 (AB, 2 H), 6.87 (AB, 2 H), 4.15 (q,
2 H), 3.79 (s, 3 H), 3.56 (s, 2 H), 1.25 (t, 3 H). HRMS calcd for
C11H14O3 194.094, found 194.094.
sodium salt of diethyl (4-methoxyphenyl)malonate precipitated as a
voluminous colorless solid, which was stirred with anhydrous ether
(10 mL), and the solid was collected by filtration. The solid was washed
with ether (3 × 10 mL), dried in air, and stirred into a mixture of 20
mL of saturated NaCl and 5 mL of glacial acetic acid. The solution
was extracted with ether (4 × 10 mL), and the combined extracts were
washed with water (2 × 10 mL) and dried over Na2SO4. The solvent
was evaporated to give 4.5 g (85%) of crude 17, which was used for
the next step without further purification. 1H NMR (CDCl3): δ 12.8
(s, 0.3 H), 7.24 (AB, 1.3 H), 7.08 (d, 0.7 H), 6.84-6.91 (AB, 2 H),
6.30 (s, 0.7 H), 4.20-4.31 (m, 3.3 H), 4.13 (t, 0.7 H), 3.80 (s, 3 H),
1.23-1.32 (m, 5 H), 1.02 (t, 1 H). HRMS calcd for C14H18O5 266.115,
found 266.115.
4,4-Dicarbethoxy-4-(4-methoxyphenyl)butanal (18). This com-
pound was prepared by the procedure of Warner and Moe.13 Compound
17 (2.66 g, 10 mmol) was added to a solution of sodium (6 mg) in
absolute ethanol (20 mL). The solution was cooled to 0 °C, and acrolein
(1.12 g, 20 mmol) was added dropwise. The reaction temperature was
maintained at approximately 5 °C during the reaction. After the reaction
was complete (2 h), the reaction mixture was neutralized with glacial
acetic acid. The solvent was removed under vacuum, and the residue
was dissolved in benzene (15 mL) and then washed with water (3 ×
15 mL). The organic solution was dried over anhydrous sodium sulfate.
Removal of the solvent gave 18 as an oil (2.1 g, 65%). 1H NMR
(CDCl3) δ 9.65 (s, 1 H), 7.39 (AB, 2 H), 6.86 (AB 2 H), 4.08-4.30
(m, 4 H), 3.77 (s, 3 H), 2.20-2.70 (m, 4 H), 1.20-1.28 (m, 6 H).
HRMS calcd for C17H2206 322.142, found 322.142.
Diethyl 4-Methoxyphenylmalonate (17). This compound was
prepared by a known procedure.12 A solution of diethyl oxalate (2.92
g, 20 mmol) and 16 (3.88 g, 20 mmol) in 5 mL of anhydrous
cyclohexane was added dropwise to a stirred suspension of sodium
hydride (60% in oil: 800 mg, 20 mmol) in 20 mL of cyclohexane at
60 °C. Within several minutes, vigorous gas evolution and refluxing
occurred. The mixture was heated for about 1 h until gas evolution
ceased and was allowed to stand for 15 h at room temperature. The
3,3-Dicarbethoxy-2-[4-(4-methoxyphenyl)butyl]-1,3-dioxolane (19).
A mixture of compound 18 (2 g, 6.2 mmol), p-toluenesulfonic acid
(60 mg), and ethylene glycol (5 g, 80 mmol) was suspended in benzene
(40 mL) and refluxed using a Dean-Stark trap. After 5 h of reflux,
(12) Tietze, L. F.; Eicher, Th. Reaction and Syntheses in the Organic
Chemistry Laboratory; University Science Books: Mill Valley, CA, 1989;
p 182.
(13) Warner, D. T.; Moe, O. A. J. Am. Chem. Soc. 1948, 70, 3470-
3472.

Monoamine Oxidase B-Catalyzed Reactions
J. Am. Chem. Soc., Vol. 120, No. 41, 1998 10587
the suspension was washed with saturated sodium carbonate and then
dried over MgSO4 and filtered. Removal of the solvent gave 19 as an
oil (2 g, 90%). 1H NMR (CDCl3): δ 7.43 (AB, 2 H), 6.86 (AB, 2 H),
4.85 (t, 1 H), 4.18-4.28 (m, 4 H), 3.92 (t, 2 H), 3.82 (t, 2 H), 3.77 (s,
3 H), 2.30-2.52 (m, 2 H), 1.55-1.70 (m, 1 H), 1.35-1.50 (m, 1 H),
1.20-1.37 (m, 6 H). HRMS calcd for C19H26O7 366.168, found
366.130.
3-Carboxy-2-[4-(4-methoxyphenyl)butyl]-1,3-dioxolane (20). A
mixture of compound 19 (400 mg, 1.1 mmol), potassium hydroxide
(560 mg, 10 mmol), water (3 mL), and ethanol (4 mL) was refluxed
for 2 h. After a clear solution was formed, 8 mL of water was added
and the solution was washed with ether (3 × 7 mL). Then, the solution
was acidified with 10% aqueous HCI to pH 2-3. The acidic solution
was extracted with ether (3 × 10 mL), and the combined extracts were
dried over MgSO4, filtered, and concentrated under vacuum, giving
20 as an oil (200 mg, 75%). 1H NMR (CDCl3): δ 7.21 (AB, 2 H),
6.81 (AB, 2 H), 4.85 (t, 1 H), 3.95 (t, 2 H), 3.82 (t, 2 H), 3.77 (s, 2 H),
3.55 (t, 1 H), 2.10-2.23 (m, 1 H), 1.82-1.97 (m, 1 H), 1.54-1.72 (m,
2 H). HRMS calcd for C14H18O5 266.115, found 266.115.
phosphate buffer, pH 7.2 (2200 µL, containing 10% of DMSO) at 25
°C until the remaining activity of MAO B was less than 10% compared
to the control without inactivator, which was run simultaneously at
one-fifth the scale. The incubation solution was extracted with
methylene chloride (4 × 5 mL). The combined extracts were washed
with water (5 mL) and evaporated. Compounds 7-9 were added as
standards, and the residue was taken up in acetonitrile/water (6:4) and
analyzed by analytical reversed-phase HPLC using a C18 column,
eluting with a gradient elution between water (containing 5% acetonitrile
and 0.06% trifluoroacetic acid) and acetonitrile (containing 5% water
and 0.06% trifluoroacetic acid) and monitoring at 215 nm; aliquots
from 0.5 min fractions were removed for scintillation counting. The
experiment was repeated with nonradiolabeled 5 and 6, and the
metabolites were analyzed directly by LC/electrospray mass spectrom-
etry. The masses corresponded to those obtained with the synthetic
compounds for 7-9, respectively. MS/MS fragmentation patterns of
7-9 also corresponded to those with the corresponding synthetic
compounds.
Oxygen Dependence of the Inactivation of MAO-B by cis-5-
Aminomethyl-3-(4- methoxyphenyl)dihydrofuran-2(3H)-one (5). A
preincubation solution containing cis-5-aminomethyl-3-(4-methoxy-
phenyl)-dihydrofuran-2(3H)-one (17.5 mM, 1100 µL) in sodium
phosphate buffer (100 mM, pH 7.2, containing 10% DMSO) and
protocatechuate 3,4-dioxygenase (1 unit) in a 5 mL Eppendorf tube
sealed with a rubber septum was frozen in liquid nitrogen, and then
the system was pumped under vacuum through a needle. After 5 min,
the tube was thawed at 4 °C and the system was filled with argon. The
freeze-pump-thaw-argon cycle was repeated three times. Another
preincubation solution in an Eppendorf tube sealed with a rubber
septum, which contained MAO B (50 µL, 117 mM) and protocatechuic
acid (20 µL, 12.5 mM), was deaerated by the same freeze-pump-
thaw-argon process. Then the deaerated MAO B mixture was
transferred to the deaerated inactivator solution with a gastight syringe.
The reaction solution was incubated under argon at room temperature.
After 2 days, trichloroacetic acid (1000 µL, 5 mM) was added to the
incubation solution to denature the MAO B and quench the reaction.
This solution was extracted with chloroform, and the extracts were
analyzed by HPLC to identify the metabolites. A control experiment
without protocatechuic acid and protocatechuate 3,4-dioxygenase was
carried out also. In this case, the metabolites could be detected,
indicating that without the enzymatic scrubbers, oxygen is present in
sufficient concentration to drive the reaction. Another control reaction
was carried out using benzylamine as substrate. When the anaerobic
experiment was repeated, including the enzymatic oxygen scrubbers,
no benzaldehyde was found by GC with flame ionization detector
detection.
4-Carboxy-4-(4-methoxyphenyl)butanal (8). Compound 20 (100
mg, 0.38 mmol) was deprotected to the aldehyde with 1 N aqueous
HCl in THF at room temperature for 20 h. After evaporation in vacuo,
the remaining oil contained a small amount of starting material and an
impurity. Treatment of the crude 8 with 2,4-dinitrophenylhydrazine
reagent gave the 2,4-dinitrophenylhydrazone (8a) (8) 1H NMR
(CDCl3): δ 9.71 (s 1 H), 7.21 (AB, 2 H), 6.88 (AB, 2 H), 3.77 (s, 3
H), 3.56 (t, 1 H), 2.41 (m, 1 H), 2.33 (m, 0.5 H), 2.10 (m, 1 H), 1.89
(m, 0.5 H), 1.61 (m, 1 H). HRMS calcd for C12H14O4 222.089, found
222.089. (8a) 1H NMR (CDCl3): δ 11.0 (s, 1 H), 9.10 (s, 1 H), 8.25
(d, 1 H), 7.85 (d, 1 H), 7.42 (t, 1 H), 7.21 (AB, 2 H), 6.85 (AB, 2 H),
4.12 (m, 0.5 H), 3.80 (s, 3 H), 3.61 (m, 0.5 H), 2.41 (m, 2 H), 2.10 (m,
1 H), 1.21 (m, 1 H). Anal. Calcd for C18H18O7N4: C, 53.73%; H,
4.48%; N, 13.93%. Found: C, 53.44%; H, 4.45%; N, 13.84%.
2,4-Dinitrophenyl Hydrazone of 5-Formyl-3-(4-methoxylphenyl)-
dihydrofuran-2(3H)-one (9). A literature procedure8 was used to make
the aldehyde from the corresponding iodide.6,7 To a solution of 500
mg of sodium bicarbonate in DMSO (4 mL) was added the iodide
compound (1 mmol) at 150 °C for 4 min. The mixture was then poured
into water (12 mL), the solution was extracted with ether (4 × 10 mL),
and the combined extracts were washed with water and dried over
MgSO4. After concentration, 2,4-dinitrophenylhydrazine reagent was
added to the residue of 9 at room temperature and was stirred for 48
h. The mixture was extracted with chloroform followed by column
chromatography, giving the 2,4-dinitrophenylhydrazine derivative of
9. 1H NMR (CDCl3) δ 11.1 (s, 1 H), 9.16 (s, 1 H), 8.78 (s, 1 H), 8.38
(dd, 1 H), 7.90 (dd, I H), 7.21 (AB, 2 H), 6.88 (AB, 2 H), 4.70 (m, 1
H), 4.20 (m, 1 H), 4.01 (t, 1 H), 3.80 (s, 3 H), 3.48 (dd, 1 H), 3.35 (dd,
1 H).
Acknowledgment. We are grateful to the National Institutes
of Health (GM32634) for financial support of this research.
Reaction of MAO B with 5 and 6. MAO B (140 µM, 100 µL)
was incubated with 17.5 mM cis- (5) or trans-5-aminomethyl-3-(4-[
14C] methoxyphenyl)-dihydrofuran-2(3H)-one (6) in 100 mM sodium
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