Profiling substrate specificity of two series of phenethylamine analogs at monoamine oxidase A and B

Article abstract of DOI:10.1039/c4ob01377h

The membrane bound enzyme monoamine oxidase exist in two splice variants designated A and B (MAO-A and MAO-B) and are key players in the oxidative metabolism of monoamines in mammalians. Despite their importance and being a prevalent target for the development of inhibitors as drugs, no systematic study of substrate specificity has been reported. In this study we present a systematic study of the MAO-A and MAO-B substrate specificity profile by probing two series of phenethylamine analogs. K_mand k_catvalues were determined for four N-alkyl analogs 2 -5 and four aryl halide analogs 6-9 at MAO-A and MAO-B. A following in silico study disclosed a new adjacent compartment to the MAO-B substrate pocket defined by amino acids Tyr188, Tyr435, Tyr398, Thr399, Cys172 and Gly434. This new insight is important for the understanding of the substrate specificity of the MAO-B enzyme and will be relevant for future drug design within the field of monoamines.

Full text of DOI:10.1039/c4ob01377h

Organic &
Biomolecular Chemistry
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PAPER
Profiling substrate specificity of two series
of phenethylamine analogs at monoamine
oxidase A and B†
Cite this: Org. Biomol. Chem., 2014,
12, 8689
Egon Heuson,a,b Morten Storgaard,c Tri H. V. Huynh,c Franck Charmantray,a,b
Thierry Gefflaut*a,b and Lennart Bunch*c
The membrane bound enzyme monoamine oxidase exist in two splice variants designated A and B
(MAO-A and MAO-B) and are key players in the oxidative metabolism of monoamines in mammalians.
Despite their importance and being a prevalent target for the development of inhibitors as drugs, no
systematic study of substrate specificity has been reported. In this study we present a systematic study
of the MAO-A and MAO-B substrate specificity profile by probing two series of phenethylamine analogs.
Km and kcat values were determined for four N-alkyl analogs 2–5 and four aryl halide analogs 6–9 at
MAO-A and MAO-B. A following in silico study disclosed a new adjacent compartment to the MAO-B
substrate pocket defined by amino acids Tyr188, Tyr435, Tyr398, Thr399, Cys172 and Gly434. This new
insight is important for the understanding of the substrate specificity of the MAO-B enzyme and will be
relevant for future drug design within the field of monoamines.
Received 2nd July 2014,
Accepted 11th September 2014
DOI: 10.1039/c4ob01377h
www.rsc.org/obc
isoforms.5 Because the MAO enzymes are key actors in the
metabolism of the monoaminergic neurotransmitters, a large
Introduction
Monoamine oxidase (MAO) is a flavin adenine dinucleotide number of inhibitors have been synthesized to discover novel
(FAD)-dependent enzyme bound to the outer mitochondrial drugs for the treatment of psychiatric and/or neurological dis-
membrane and is responsible for the oxidative deamination orders:6,7 MAO-A inhibitors have found use as antidepress-
of monoamines such as monoaminergic neurotransmitters, ant8,9 and anxiolytic drugs,10,11 whereas MAO-B inhibitors
dietary amines and hormones.1,2 The enzyme exists in two iso- are used for the treatment of Parkinson’s12,13 and Alzheimer’s
forms, MAO-A and MAO-B, which display 70% amino acid diseases.7
residue identity.3 While MAO-A is found in the brain, liver, the
In contrast, a limited number of MAO substrates have been
gastrointestinal tract and in placenta, MAO-B is only found in reported: Kynuramine14 (Fig. 1), a metabolite of melatonin,15 is
the brain and blood platelets.4 Aside from differences in tissue a substrate for both MAO-A and B and used in in vitro MAO
distribution, the A/B isoforms also differ in substrate speci- assays.16 The substrate 1-methyl-4-phenyl-1,2,3,6-tetrahydro-
ficity. MAO-A is the predominant isoform for deamination of pyridine (MPTP)17 is converted into a neurotoxin by MAO-B18
e.g. the neurotransmitters serotonin, adrenalin and noradrena- and has been explored as a scaffold for the synthesis of other
lin,4 whereas MAO-B preferentially deaminates lipophilic MAO-substrates including 1-methyl-3-phenyl-2,5-dihydro-1H-
monoamines e.g. benzylamine, phenethylamine (PEA, 1) pyrrole,19,20 2-methylisoindolines,19 4-(1-methyl-1H-pyrrol-2-yl)-
and octylamine. The primary amines, dopamine, tyramine, 1-(prop-2-yn-1-yl)piperidine,21 3,4-cyclopropyl analogs of MPTP22
kynuramine and tryptamine are common substrates for both as well as 1-methyl-3-phenylpyrrolidine and the 3,4-cyclopropyl
analogs hereof23 (Fig. 1). Finally the MAO A/B substrate specificity
of some neurological drugs have been investigated in order to
aClermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand,
get a more detailed understanding of the drug’s pharmaco-
BP 10448, F-63000 Clermont-Ferrand, France
kinetic properties (Sumatriptan – an anti-migraine drug24 and
bCNRS, UMR 6296, ICCF, 63177 Aubière, France.
Citalopram – an antidepressive drug,25 Fig. 1).
E-mail: thierry.gefflaut@univ-bpclermont.fr; Fax: +33 473407717;
Tel: +33 473407866
Systematic studies of MAO-A and MAO-B substrate specifici-
ties find applications in diverse research areas such as drug
cDepartment of Drug Design and Pharmacology, Faculty of Health and Medical
Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen Ø,
discovery, development of enzyme activity assays, and new
Denmark. E-mail: lebu@sund.ku.dk; Tel: +45 35336244
†Electronic supplementary information (ESI) available: 1H NMR of compounds
3 and 4. See DOI: 10.1039/c4ob01377h
enzyme-based synthetic methodology. In contrast to the
diverse and chemically complex MAO substrates depicted in
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Fig. 1 Examples of MAO substrates include endogenous metabolites, synthetic compounds and drugs.
Fig. 1, phenethyl amine (PEA, 1) is a simple arylalkyl amine and bulkiness, while PEA analogs 6–9 (Fig. 2) were included in
which comprises the chemical features commonly found in the study to explore the influence on substrate specificity of a
MAO-A and MAO-B substrates. Therefore it attracted our atten- aryl halide substituent.
tion to investigate the substrate specificity of two series of PEA
Synthesis of N-alkyl PEA (1) analogs
analogs (N-alkyl analogs 2–5 and aryl halide analogs 6–9,
Fig. 2) at the MAO-A and MAO-B enzymes, and follow-up these
results with an in silico study.
Whereas PEA (1), N-methyl analog 2, N,N-dimethyl analog 5,
and aryl halide analogs 6–9 were purchased from commercial
suppliers, the N-ethyl analog 3 and N-propyl analog 4 were syn-
thesized, following a standard protocol of reductive amination
of PEA (1) and the respective aldehyde, using NaBH3CN as the
reducing agent (Scheme 1).27 The desired secondary amines 3
and 4 were obtained in modest yields with the corresponding
tertiary amines as the major side products (not shown).
Results and discussion
It has previously been shown that PEA (1, Fig. 2) is a preferred
substrate for MAO-B over MAO-A.26 We designed two distinct
series of PEA (1) analogs to probe the substrate specificity of
the MAO-A and -B enzymes: PEA analogs 2–5 (Fig. 2) are
simple aliphatic N-alkyl analogs increasing the chain length
MAO-A and B substrate profiling
The four N-alkyl PEA analogs 2–5 were then investigated as
potential substrates of both MAO-A and MAO-B. Kinetic con-
stants for the MAO-catalyzed oxidation of substrates 2–9 with
O2 were estimated using a continuous fluorescence assay
based on hydrogen peroxide (H2O2) co-product titration.
Horseradish peroxidase was used to catalyse a non-rate-limit-
ing coupled reaction between H2O2 and 10-acetyl-3,7-dihydroxy-
phenoxazine (Amplex® Red) producing the fluorescent
resorufin. This assay was previously used for the evaluation of
MAO substrates or inhibitors.28 PEA (1) and kynuramine were
Fig. 2 Chemical structure of N-alkyl PEA (1) analogs 2–5 and aryl
halide analogs 6–9.
Scheme 1 Synthesis of N-alkyl analogs 3 and 4 via a one-pot reductive
amination of PEA (1) with appropriate aldehydes.
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Table 1 MAO-A and -B substrate activities of kynuramine, PEA (1), N-alkyl analogs 2–5, and aryl halide analogs 6–9. Km in [µM], kcat in [min−1] and
kcat/Km in [min−1 μM−1 × 1000]
MAO-A
MAO-B
Cmpd no.
R1
R2
Km
kcat
kcat/Km
Km
kcat
kcat/Km
Kynuramine
PEA, 1
—
H
Me
Et
Pr
Me, Me
H
H
H
H
—
H
H
H
H
H
o-Cl
m-Cl
p-Cl
p-Br
378 17.3
4550 549
402 9.9
n.s.
n.s.
n.s.
70.9 2.5
24.3 1.4
152 8.1
178 14.0
7.90 0.22
9.63 1.09
5.84 0.10
n.s.
n.s.
n.s.
16.8 0.27
5.21 0.09
1.87 0.06
1.46 0.07
20.9 1.1
2.1 0.4
14.5 0.4
n.s.
n.s.
n.s.
237 9.0
214 13.1
12.3 0.8
8.2 0.7
37.3 1.4
7.0 0.4
5.4 0.4
343 22.6
515 61.9
112 15.1
2.9 0.2
3.2 0.3
2.4 0.2
2.3 0.2
1.36 0.02
3.12 0.10
2.25 0.07
2.20 0.10
1.26 0.12
0.96 0.03
1.85 0.07
1.97 0.14
3.41 0.12
3.84 0.15
36.4 1.5
444 27.6
419 32.2
6.4 0.5
2.4 0.4
8.5 1.2
638 45.9
623 68.1
1430 117
1690 147
2
3
4
5
6
7
8
9
n.s.: not substrate.
included as reference substrates, and results summarized in in affinity (Km), while the turnover number was roughly the
Table 1. As N-alkyl PEA analogues 2–5 release simple N-alkyl- same (kcat) in comparison to 1. In summary, a chloride in
amines upon MAO-catalyzed oxidation and hydrolysis, the MAO either the o- or m-position gave little change in activity,
activity with N-methyl-, N-ethyl-, N-propyl- and N,N-dimethyl- whereas a halide in the p-position improved MAO-B activity by
amine was needed to be addressed. For both MAO-A and 4-fold.
MAO-B isoforms, no activity was detected with any of these
four N-alkyl amines.
The comparison of the activities of the two isoforms MAO-A
and B towards this substrate series confirms that MAO-B
In summary for MAO-A activity, the N-Me analog 2 displays shows a much stronger affinity for PEA, and analogs thereof,
a 10-fold increase in affinity (Km) compared to PEA (1). On the than MAO-A. A Km ratio of 650 was obtained for PEA, whereas,
other hand, the turnover number (kcat) for 2 is roughly 50% the different analogues 2 and 6–9 showed a drop in selective
lower. Elongation of the N-alkyl chain (N-ethyl analog 3 and affinity with Km ratio ranging from 7.6 to 77. Conversely,
N-propyl analog 4) resulted in complete loss of MAO-A activity. higher kcat values were measured with MAO-A for several com-
Furthermore, the more bulky N,N-dimethyl analog 5 was not a pounds. N-Methyl analog 2, m-chloro derivative 7 and PEA
substrate for the MAO-A enzyme. Exploration of an aryl halide itself displayed a 2 to 3 fold higher value, whereas the o-chloro
substituent, analogs 6–9, resulted in a 25–200-fold increase in PEA derivative 6 showed a nine fold higher kcat value for
affinity for MAO-A. The effect on kcat spanned from a 2-fold MAO-A compared to MAO-B. However, the kcat values which are
increase (o-Cl, compound 6), over a 2-fold decrease (m-Cl, com- calculated from protein content independently for each
pound 7) to a 6-fold decrease (p-Cl and p-Br, compound 8 enzyme should be compared with some carefulness, even
and 9) (Table 1). Overall, compared to PEA, 6 and 7 were though commercial MAO-A and MAO-B batches are reported to
100-fold better substrates for MAO-A, while 8 and 9 were only be of high purity and to display the same level of activity in the
4–6 fold better.
microsomal expression system. Besides, regarding the kcat/Km
The activity profile observed for MAO-B with the substrate values, compounds 6 and 7 clearly appear as good substrates
series 2–9 was highly different. The activity measured with the for both enzymes, much better than kynuramine which is
N-methyl analog 2 was comparable with that of 1 both with commonly used as a non-selective substrate in MAO assays. In
respect to Km and kcat. Extending the N-alkyl chain to an addition, the p-bromo derivative 9, displays a high selectivity
N-ethyl group, compound 3, gave a significant (50-fold) drop in for MAO-B with kcat/Km 200 times higher compared to MAO-A.
affinity (Km) for MAO-B. With a further extension to N-propyl, Although this selectivity ratio is similar to that observed with
compound 4, a 70-fold drop in Km was observed in comparison PEA, compound 9 shows a 4 times higher activity and consti-
with 1. On the other hand, N,N-dimethyl analog 5 displayed tute therefore a new valuable substrate for MAO-B selective
only a 15-fold drop in affinity (Km = 112 μM) as compared with assays.
1. For all four analogs 2–5, the turnover number, was deter-
In regard to already published studies on the role of MAO-A
mined to be within the same range (kcat = 0.96–2.25 min−1) – and MAO-B enzymes in drug metabolism, the results of N-alkyl
slightly lower than for 1. The effect of an arylic chloride or PEA analogs 2–5 are intriguing. It has been shown, that
bromide, analogs 6–9, was manifested in a 2 to 3-fold increase the anti-migraine drug Sumatriptan, which comprises an
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N,N-dimethyl amine moiety, is metabolized primarily by interactions are of stabilizing nature in contrast to repulsive
MAO-A.24 Furthermore, it has been shown that the anti- π-electron–free-amine interactions, the amine substrates likely
depressant drug Citalopram, which also comprises an N,N- remain protonated at the stage of entrance and are directed to
dimethyl amine moiety, is metabolized by both the MAO-A the substrate pocket by exchanging π–cation interactions with
and MAO-B enzymes.2
the aromatic residues in the entrance and substrate pockets.
Modeling. The MAO-B enzyme has previously been crystal- Approaching the FAD, the ammonium group protonates N(5)
lized with covalently bound FAD (PDB code: 2VRL).29 In the preceding its nucleophilic attack on C(4a) to form key inter-
proximity of the covalently bound FAD molecule the substrate mediate A (Scheme 2). This covalently-bound FAD-substrate
binding pocket has been identified and is of lipophilic nature complex was selected as the starting point for a computational
(Fig. 3). In connection hereto, a second pocket has been searching for binding mode of the active substrates reported
defined and following named the entrance pocket. The substrate herein.
and entrance pockets are narrowed in at the site of their
joining by the side chains of amino acid residues Leu171, bound substrate is not possible due to its transient nature, the
Tyr326 and Ile199, although the latter is in its open form.29
crystal structure of covalently-bound inhibitor, benzylhydra-
While X-ray crystal structure determination of an enzyme-
Two distinct mechanisms have been suggested for the cata- zine with MAO-B (PDB code: 2VRL), has been reported29 and
lytic action of the MAO-A and MAO-B enzymes; a radical reac- was selected as a suitable starting template for the modeling
tion and a polar nucleophilic mechanism.2 While the latter is study. Firstly, structural changes were made to the FAD-ligand
the most commonly accepted one, details on nitrogen protona- complex as to represent key intermediate A (Scheme 2) com-
tion states are disputed.2 Earlier it has been proposed that the prising the appropriate N-substituent. For analogs 2–4 both
substrate is at its free amine form when it is directed to the geometries of the positively charged nitrogen were built and
substrate pocket. However, based on the fact that π–cation modeled independently, while the symmetry of dimethyl
analog 5 allowed for building of only one. Each structural
complex was submitted to a LowModeMD simulation and the
sample of conformers was examined. Overall, analogs 2–5 were
well-accommodated by the MAO-B enzyme.
In details, the in silico study concluded that the N-alkyl moi-
eties reach into an adjacent hydrophobic compartment
defined by the side chains of amino acids Tyr188, Tyr435,
Tyr398 and Thr399, and backbone carbonyl groups of Cys172
and Gly434 (Fig. 3B). It is noteworthy that a water molecule is
located in this compartment and it engages in a hydrogen
bonding interaction with the carbonyl group of Cys172. Thus,
for N-alkyl group larger than methyl this water molecule must
be displaced. Moreover, the N-phenethyl groups are well-fitted
in the substrate binding pocket. For analogs 2–4 the major
difference between their two protonated states is the orien-
tation of the N-proton, which engages in π–cation interactions
with residues Tyr398 or Tyr435. We propose that the proto-
nated state, which forms π–cation interaction with Tyr435
(Fig. 3B and 4) is favorable due to the spatial orientation of the
α-hydrogen atom, as this is believed to undergo N(5)-base cata-
lyzed elimination (Scheme 2, step c).
Positively charged quaternary amine 5 revealed similar
binding modes as described above for analogs 2–4, but orient-
ing one N-methyl group towards Tyr435 or Tyr398. The spatial
orientation of the phenethyl moiety is similar but the confor-
mation of the methyl groups suggests that the conformer in
which the N-methyl group points towards Tyr435 is favored
due to the N(5)-base catalyzed elimination of α-hydrogen atom
Fig. 3 Amino acid residues forming the entrance pocket, the substrate
(Scheme 2, step c). The 20-fold loss in affinity (compound 2 vs.
binding pocket and the newly discovered adjacent compartment of the
MAO-A/B enzymes. Filled circles designate residue backbone as pocket compound 5) could be due to a possible steric clash between
wall and unfilled circles designate residue side chain as pocket wall. Red
circles designate residue of differentiation between MAO-A and MAO-B.
(A) MAO-A amino acid residue alignment onto X-ray structure of MAO-B
(2VRL). (B) X-Ray structure of MAO-B (2VRL) with covalently-bound FAD
the one N-methyl group of 5 and Tyr435.
To investigate the observed differences in binding affinity
of 2–5 at MAO-A and MAO-B, the amino acid sequence of
human MAO-A was aligned with the X-ray structure of MAO-B
used in the in silico study (2VRL) (Fig. 3A). Only the Cys172
in green and covalently bound protonated substrate N-methyl-phe-
nethylamine (2) in blue.
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Scheme 2 Suggested polar nucleophilic mechanism for MAO-B-catalyzed oxidation of amines.
chloride increases affinity in both cases. However, both para
aryl chloride and bromide are disfavored for MAO-A, but well-
accepted for MAO-B. These difference are likely to be explained
by the distinct amino acid residues defining the doorstep
between the substrate pocket and the entrance pocket of
MAO-A and MAO-B (Fig. 3, MAO-B → MAO-A: Leu171 → Ile180,
Ile199 → Phe208 and Tyr326 → Ile335).
Conclusion
In conclusion, the MAO-A and MAO-B substrate specificity
profile was studied for two series of PEA (1) analogs: for the
N-alkyl PEA series, analogs 2–5, an N-methyl group, compound
2, was tolerated by both isoforms. On increasing the alkyl
chain length or bulkiness complete loss of enzymatic activity
at MAO-A was observed while the affinity for MAO-B was
lowered by 20–70 fold. The series comprising analogs with an
aryl halide substituent, compounds 6–9, showed that the posi-
tion of substitution has a strong influence on isoform prefer-
ence, with the para position favoring substrate specificity
towards the MAO-B isoform. An in silico study suggested that
the N-alkyl substituents occupy an adjacent compartment to
the substrate pocket defined by amino acids Tyr188, Tyr435,
Tyr398, Thr399, Cys172 and Gly434 (Fig. 3B and 4). This new
insight is relevant to the understanding of MAO-B substrate
specificity and will be of importance to future drug design
within the field of monoamines.
Fig. 4 Amino acids occluding the adjacent compartment in MAO-B.
Covalently-bound FAD in green with covalently-bound protonated sub-
strate N-methyl-phenethylamine (2) in blue. Key water molecule in type
code.
residue of MAO-B is altered to an Asn181 in MAO-A. Indeed
Asn181 comprises a more bulky side chain, but since it is the
backbone carbonyl group which defines the adjacent compart-
ment, the chemical nature of the side chain must have little
direct involvement. However, the water molecule found in the
X-ray of MAO-B is believed also to be present in MAO-A. Herein
it could be more tightly bound due to the before described
difference in amino acid residue. Displacement for larger
N-alkyl groups than methyl would then be disfavored, rendering
such N-alkyl analogs inactive as substrates for MAO-A.
Experimental section
Chemistry
The ortho and meta aryl positions direct substituents into Chemicals and reagents purchased from commercial suppliers
conserved space between MAO-A and MAO-B (Fig. 3) and a were used without further purification. Solvents were of HPLC
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quality and used as received. Evaporation in vacuo was per- was purchased from Sigma-Aldrich. The batches (0.5 mL,
formed on a rotary evaporator at approx. 40 °C (pressure: 2.5 mg) were thawed rapidly in a 37 °C water bath upon arrival,
20 mbar). TLC was performed using silica gel 60 F254 aluminium aliquoted and quickly refrozen to −80 °C until further use.
sheets. The plates were visualized in under UV light (254 nm) Before use, MAOs were thawed and then diluted to obtain a
followed by staining with KMnO4 or 0.5% ninhydrin in EtOH. 0.16 mg mL−1 solution in 50 mM phosphate buffer pH = 7.4. A
1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were 2 mM solution of Amplex red was prepared in 50 mM phos-
recorded on a Varian Mercury Plus and on a Varian Gemini phate buffer pH = 7.4, 10% v/v DMSO. A 4 µg mL−1 solution of
2000 instrument, respectively, using deuterated solvents as HRP was prepared in 50 mM phosphate buffer pH = 7.4. A
internal references. Chemical shifts (δ) are given in ppm and mixed solution of 0.5 mM Amplex red®, 1 µg mL−1 HRP in
coupling constants (J) in Hertz. LC-MS was recorded on an 50 mM phosphate buffer pH = 7.4, 2.5% v/v DMSO, was pre-
Agilent 1200 series solvent delivery system equipped with an pared just before use from the two previous solutions. 2 mM
Agilent 6400 series triple quadrupole mass spectrometer (ESI) solutions of the various amino substrates were prepared in
using a 10→90% MeCN gradient in H2O containing 0.05% 50 mM phosphate buffer pH = 7.4, 4% v/v DMSO. 1 µM to
HCOOH.
2 mM amino substrates solutions were then prepared by
N-Ethyl-2-phenethylamine (3). Acetaldehyde (0.20 mL, dilution in the same buffer.
3.5 mmol, 1.00 equiv.) was added to a stirred solution of PEA
MAO activity fluorescence assays was automated on a mono-
(1) (1.34 mL, 1.29 g, 10.6 mmol, 3.03 equiv.) in dry MeOH chromator-based fluorescence microplate reader (Safire™,
(30 mL) at 0 °C under a N2 atmosphere. The reaction mixture Tecan) using 96-well fluorescence microplates, incubated at
was stirred for 30 min at 0 °C and for 1.5 hours at rt. 37 °C in the dark. The reaction mixture was composed of
NaBH3CN (245 mg, 3.9 mmol, 1.11 equiv.) was added and 20 µL of MAO solution, 80 µL of the mixed solution containing
the reaction mixture was stirred at rt for 28 hours. After HRP and Amplex red®, and 100 µL of substrate solution. Enzy-
concentration in vacuo, the crude product was purified matic reactions were initiated with the substrate and moni-
by flash chromatography using isocratic EtOAc–MeOH–Et3N tored over 30 min by fluorimetry using excitation at λ = 563
(10 : 1 : 0.1). This afforded the titled compound (287 mg, 54%) 10 nm and detection at λ = 587 10 nm.
as a colorless oil. Rf 0.28 (EtOAc–MeOH–Et3N 10 : 1 : 0.1).
For each substrate, kinetic parameters were determined
1H NMR (D2O) δ 7.40 − 7.25 (m, 5H), 3.25 (t, J = 7 Hz, 2H), 3.05 using at least 7 different concentrations close to the estimated
(q, J = 7 Hz, 2H), 2.95 (t, J = 7 Hz, 2H), 1.18 (t, J = 7 Hz, 3H). Km value. Each experiment was reproduced three times using
13C NMR (CDCl3) δ 140.5, 129.0, 128.8, 126.5, 51.5, 44.4, 36.9, substrate mother solutions prepared independently. Maximal
15.7. MS (m/z) calcd for C10H15N [M + H]+ 150.1, found: 150.2.
slope were measured within the first 15 min (RFU min−1) and
N-Propyl-2-phenethylamine (4).27 Propionaldehyde (0.3 mL, converted to initial reaction velocities (µM H2O2 min−1), using
5.2 mmol, 1.00 equiv.) was added to a stirred solution of PEA a standard curve obtained with titrated H2O2 solutions under
(1) (2.0 mL, 1.92 g, 15.5 mmol, 2.98 equiv.) in dry MeOH the same assay conditions. Kinetic parameters and standard
(45 mL) at rt under a N2 atmosphere. The reaction mixture was errors were calculated from the Hanes–Woolf plot according to
stirred for 1 hour at rt. NaBH3CN (357 mg, 5.7 mmol, 1.10 the least-squares method and Gauss’s error propagation law.
equiv.) was added and the reaction mixture was stirred at rt for
5 hours. The reaction mixture was concentrated in vacuo. H2O
(25 mL) was added and the solution was extracted with
Modeling
DCM (3 × 30 mL). The combined organic phases were washed The comprehensive molecular modeling suite MOE (version
with H2O (30 mL) and brine (30 mL). The organic phase 2011.10, Chemical Computing Group) running on a Windows
was dried over anhydrous Na2SO4. After concentration 7 Professional 32 bit PC platform was used. The force field
in vacuo, the crude product was purified by flash chromato- PFROSST was used with solvation set to distance. For each
graphy using isocratic EtOAc–MeOH–Et3N (10 : 1 : 0.1) and the analog 2–5, PDB file 2VRL was imported and altered chemi-
product was further distilled under reduced pressure (b.p. 168 cally to represent key intermediate A with the appropriate
− 170 °C/250 mmHg) to afford the titled compound (232 mg, N-substituent. In cases where two protonation states were
34%) as a colorless oil. Rf 0.14 (EtOAc–MeOH–Et3N 10 : 1 : 0.1). possible, both were built and considered independently. Each
1H NMR (CDCl3) δ 7.30 − 7.16 (m, 5H), 2.91 − 2.85 (m, 2H), generated structural complex was first submitted to the LigX
2.83 − 2.77 (m, 2H), 2.58 (t, J = 7.5 Hz, 2H), 1.48 (sextet, J = command to restrain atoms further away than 8 Å from the
7.5 Hz, 2H), 0.89 (t, J = 7.5 Hz, 3H). 13C NMR (CDCl3) δ 140.2, ligand (standard setup). Subsequently a conformational search
128.8, 128.5, 126.3, 51.9, 51.4, 36.6, 23.3, 11.9. MS (m/z) calcd by LowModeMD (a short molecular dynamics simulation
for C11H17N [M + H]+ 164.1, found: 164.2.
using velocities with little kinetic energy on the high-frequency
vibrational modes30) was carried out using standard setup
with the following changes: RMS gradient = 0.1 and Energy
window = 1000.
MAO-A and B substrate assays
Human monoamine oxidase A and B (MAO-A and MAO-B)
Alignment of human MAO-A amino acid sequence onto
supersomes were purchased from BD Biosciences. Amplex red® MAO-B (2VRL) was done by standard setup of alignment func-
was purchased from Invitrogen. Horseradish peroxidase (HRP) tion in MOE (hMAO-A: 527).
8694 | Org. Biomol. Chem., 2014, 12, 8689–8695
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Organic & Biomolecular Chemistry
Paper
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Abbreviations
FAD
Flavin adenine dinucleotide
MAO Monoamine oxidase
MPTP 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine
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PAA
PEA
2-Phenylacetaldehyde
Phenethylamine
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