The two currently accepted mechanisms for MAO-catalyzed oxidations

TANKO RESEARCH GROUPWhat do Designer Drugs, Parkinson’s Disease, and the Persistent Radical Effect have in Common?Nathan Price (presenting)

Parkinson’s Disease/Parkinsonism

Clinical syndrome characterized by tremor, bradykinesia, rigidity, and postural instability

• Lewy bodies (abnormal aggregates of protein that develop inside nerve cells)

• Destruction of dopaminergic neurons in the substantia nigra

• Generally a disease that affects the elderly

N

O

CH3

CH2CH3

O Δ

NCH3

- HOAc

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

(MPTP)

• MPTP is lipophilic and can cross blood-brain barrier• Undergoes oxidation catalyzed by monoamine oxidase B to produce a potent

neurotoxin• Kills primarily dopamine-producing neurons in a part of the brain called the pars

compacta of the substantia nigra• MPTP used to simulate Parkinson’s disease to better understand etiology, develop

treatment strategies, etc.

What is a designer drug? A structural or functional analog of a controlled substance that has been designed to mimic the pharmacological effects of the original drug, while avoiding classification as illegal and/or detection in standard drug tests

N

Ph

CH3

Fl Fl

N

Ph

CH3

- H

N

Ph

CH3H

HH

N

Ph Fl Fl

N

Ph

N

Ph

HH

H

- H

N

Ph

HH

fast

SET Hypothesis for MAO oxidation of amines (Current Status)

• Paramagnetic intermediates derived from the flavin moiety have never been detected

• Chemical model studies have shown that rearranged products (and flavin-adducts) can arise via a polar, nucleophilic pathway

• Numerous experimental & computational studies strongly support other mechanisms

Polar mechanism:

Hydride transfer mechanism:

N

N

N

N O

OR

R

N

N

N

N O

OR

R

H2NCHPh

H

NH

N

N

HN O

OR

R

+ PhCH=NHPhCH2NH2

NH

N

N

N O

OR

R

+ PhCH=NH2

NH

N

N

HN O

OR

R

+ PhCH=NH

N

N

N

N O

OR

R

CH

PhH

NH2

A glaring omission from recent studies – none have considered the reaction of MAO with tertiary amines

Tertiary amines are not MAO substrates, with the exception of MPTP and itsstructural analogs are the only reported tertiary amines with MAO-B substrateproperties

This work: To ascertain the mechanism of reaction of chemical model compounds(biomimetics) with an MPTP derivative

MMTP: 1-methyl-4-(1-methyl-1H-pyrrol-2-yl)-1,2,3,6-tetrahydropyridine

N CH3NCH3

MMTP

N

N

N

N

O

O

N

N

N

N

O

O

CH3CH2

ClO4

3MLF Fl+

Reaction of MMTP with Fl+ gave rise to two principal products, in nearly quantitative yield: 1) the expected oxidation product MMP+ and 2) MMTPH+, the protonated form of the starting material MMTP.

Two small molecules, molecular oxygen (O2) and water (H2O) play critical roles in this chemistry

Water (in CH3CN) introduces a ”sideshow” equilibrium. This reaction needs to be studied under rigorously dry conditions and in a glovebox.

N CH3 N CH3

MMTP MMP+

NCH3

HN N NCH3CH3CH3

Fl +

MMTPH+

N

N

N

N

O

O

CH3CH2

Fl+ + MMTP + H2O

HOMMTPH+

N

C6H6N

H CH3

+

Headline News: It works!

5.56.06.57.07.58.08.59.0f1(ppm)

1

2

3

4

5

6

15min

30min

60min

90min

120min

180min

N CH3 N CH3

MMTP MMP+

NCH3

HN N NCH3CH3CH3

Fl +

MMTPH+

Reaction conducted in the presence of O2, monitored by 1HNMR (d 5.5 – 9.0)

MMTPH+

MMP+

N

N

6.06.57.07.58.08.5f1(ppm)

1

2

3

4

12days

17days

23days

afteradditionofair

Reaction conducted under anaerobic conditions: “Silent” NMR – only MMTPH+ is detected

The final two pieces of the puzzle: The UV/Vis spectrum of the reaction of MMTP and Fl+ (anaerobic conditions) ...and a heartbeat! The EPR spectrum of the reaction of MMTP and Fl+ (anaerobic conditions)

0. 0

0. 5

1. 0

1. 5

2. 0

300 340 380 420 460 500 540 580 620 660 700 740 780

Abs

orba

nce

Wavelength (nm)

B

N

N

N

N

O

O

CH3CH2N

N

N

N

CH3CH2 O

O

Fl+ Fl

e-

- 600

- 400

- 200

0

200

400

600

• SET is back on the menu as a viable mechanism for the MAO catalyzed oxidation of tertiary amines such as MPTP. Failure to detect this pathway in previous studies is because other mechanisms operate for primary and secondary amines.

• The ability to form a stabilized radical such as MMTP•, which drives the SET process, is unique to allylic tertiary amines such as MPTP and derivatives.

Conclusions and implications

Fl +

+ Fl

N

Ar

HH

N

Ar

H

N

Ar

HN

Ar

H

O2

O2

N

Ar

+ H+

O2H2O2

N

Ar

H

+ FlO2 H2O22 H+ +

• Some mechanism-based MAO-B inhibitors such as the propargylic aminespargyline and selegiline (deprenyl) possess similar functionality, and thus, mayalso operate via a single electron transfer process.

• While the role of O2 in completing the catalytic cycle for MAO is known, apossible role of O2 in the early steps of the mechanism has not been considered.Future studies of MAO catalysis under anaerobic conditions are needed.

N

pargyline

N

deprenyl

Mechanistic studies with 5-ethyl-3-methyllumiflavinium (Fl+) perchlorate, a biomimetic model for flavoenzyme monoamine oxidase B (MAO-B) catalysis, and the tertiary, allyl amine 1-methyl-4-(1-methyl-1H-pyrrol-2-yl)-1,2,3,6-tetrahydropyridine (MMTP) reveal that proton coupled electron transfer (PCET) may be an important pathway for MAO catalysis. The first step involves single electron transfer (SET) leading to the free radicals Fl• and MMTP•, the latter produced by deprotonation of the initially formed and highly acidic MMTP•+. Molecular oxygen (O2) is found to play a hitherto unrecognized role in the early steps of the oxidation. MMTP and several structurally similar tertiary amines are the only tertiary amines oxidized by MAO, and their structural/electronic properties provide the key to understanding this behavior. A general hypothesis about the role of SET in MAO catalysis, and the recognition that PCET occurs with appropriately substituted substrates is presented.

+

+

MMTPH+