The oxidation of organic anions by flavins

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 41, No. 5, 1970

THE OXIDATION

OF ORGANIC ANIONS BY FLAVINS

James A. Rynd* and Morton

J.

Gibian

Department of Chemistry University of California Riverside, California 92502 Received September 14, 1970 The reduction of flavins by a-hydroxyketone enolates leads to dihydroflavins and diketones. Isoalloxazines related to flavins (good analogs for their redox chemistry) are reduced by 9,10-dihydroanthracene and succinonitrile carbanions and by propiophenone enolate (in the latter case to give the isoalloxazine Alkali metal alcoholates, with an appropriate radical anion). isoalloxazine, produce 50% yields of isoalloxazine radical ion, presumably via a substitution and electron transfer process. The ease of?&-banion (and complete lack of conjugate acid) oxidation suggests a general mode of flavin redox function. Recent

spectroscopic

the characterization plexes tion

of both states.

of many of the

free

1

However,

free

With

flavin

generally

only

a few notable

with

various

non-reactive

substrates

flavin

might

indications

protein

portions Flavins

ganic

of the

react

flavin

isoalloxazine. redox

seem relevant *National

states

7

oxida-

been direc-

substrates

have not

under

transfer

which

electrons

of the possible

been other-

to or from

functions

of the

enzymes.

either While

with

a wide variety

photoreduction contributing

and structure,

to a chemical

Institutes

and corn-'

in various

the conditions

photochemically

compounds to give

of the of

give

exceptions

would

forms

in

redox chemistry of 2-6 , attempts to react

physiological

wise

have resulted

has attention

organic

Finding

successful.

flavins

recently

the basic

studies

amphoteric

and protein-bound

ted to understanding flavins.

and chemical

of Health

Predoctoral 1097

or photoalkylation to the understanding

these

elucidation

of or-

processes

of the normal Fellow,

do not dark

1968-70.

re-

BIOCHEMICAL

Vol. 41, No. 5,197O

actions

of flavoproteins.

tronically

excited

Photochemical

states.

generated

by complexing

enzymatic

or not,

(dark)

flavin

suggest

redox

such energetic

involve

states

elec-

may be

in any thermal

reaction,

related

state

to be shown. new closely

systems function

involving

organic

of some of the

ground substrates

which

apoenzymes.

and Methods

Reactions

were run anaerobically

gon in Schlenk fit

processes

or association

several

a possible

Materials

That

has yet

We now report

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

tubes

equipped

by first

with

bubbling

1 cm square

Pyrex

with

bottoms

arto

Flavins were 14 or Beckman DU spectrophotometer. 8 All reagents were obby published procedures.

in a Cary

synthesized tained

from Aldrich

Doering.

'

Chemical

Dimethylformamide

and +-BuOK was prepared

(DMF) and dimethylsulfoxide

from CaH2 and stored

were distilled Epr spectra was calibrated

were taken using

the

nitriledithiolene)-nickel tration

Co.,

was determined

over

on a Varian

molecular

after (DMSO)

sieves. The instrument

V-4502.

tetrabutylammonium salt of bis-(maleo10 anion or DPPH in DMF. Spin concen-

from the

integrated

spectra

or estimated

from peak heights.

Results

and Discussion

We have found duce oxidized

that

flavins

in polar

was judged

by the

reoxidized

by molecular

When the mixed tion

a variety

enolate

anaerobically of flavin

ability

of

carbanions

aprotic

solvents.

of the

flavin

True

re-

reduction

to be rapidly

oxygen. of benzoin

with

occurs,

reduced

and enolates

(I,R=C6H5,

3-benzyllumiflavin Quantitative

1098

yields

see Scheme I) in DMF rapid of dihydroflavin

is reducand

BIOCHEMICAL

Vol. 41, No. 5,197O

benzil*

(III)

an a-amino rapidly

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

are obtained. ketone

The reaction

(C,H,COCH(NH2)C6H5)

oxidizes

acetoin

(I,R=CH3)

is equally

and base.

facile

Flavin

with

also

and 2-hydroxycyclohexanone

to

the a-diketones. 0 OH

00 II II RC-CR)

O-OH

Ri-&HR + OH--+

R-&=;R

--

I

IV

RC=CR + HFR* IV

III One-half equivalent produces

an equivalent

of the

semidione half-reduced

(IV)11

(II)

An additional

or full

flavin.

equivalent

Since

upon mixing,

all

these

two one-electron all

these

(Scheme I). of flavin

or one of flavin

A slight

(FR.-),

excess

identified

of by

(470nm, 400nm, sh, 370nm) and epr

half-equivalent

of hydroxyketone

of semidione reactions

experiments steps

(I)

added to one equivalent

flavin

the radical-anion lb visible characteristic

spectra.

Scheme I

of the a-hydroxyketone

base produces its

III

(IV) take

cannot

produces place

enolate

fully

reduced

instantaneously

be used to differentiate

from one two-electron

step

for

the

over-

reaction. In order

to study

hydroxyketones, void

cf

tain

flavin

it

anions

of compounds less

was desirable

to utilize

acidic

than

isoalloxazines

the de-

the C-8 and C-10 methyl groups that cause the basic side lb reactions of flavins. Thus, lo-phenylisoalloxazines (V) re-

erably

redox

more stable

*Identified by tic dry box under N20

and spectral in basic

using

properties

but

should

be consid-

solution.

three

different

1099

solvent

systems

in a

BIOCHEMICAL

Vol. 41, No. 5, 1970

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a)R=H b) R = CH2C6H5

Va and Vb behaved scribed tered

below.

The N-3H isoalloxazine

on mixing

with

absorption.

significant

of

immediately

isoalloxazine

amounts was demonstrated (too

almost

100% original

sults

(Va)

reactions

de-

is

al-

&-BuOK,

The presence

slowly

anaerobic

in the

NaH, or even C H ONa to a species 65 a band at 400-410nm (~10,000), but no longer wave-

displaying length

somewhat differently

slowly

for

HCl also

were obtained

the carbanions

of

normal

reduced

isoalloxazine produced

by epr.

the

Shaking

oxidized

9,10-dihydroanthracene,

oxygen

restores

A slight

spectrum.

of a ten-fold

in

with

isoalloxazine)

spectrum.

upon addition

radical

excess Similar

re-

molar

excess

succinonitrile,

or

1.4

I,2

1.0

\

0.0 WAVELENGTH

Figure

1:

(nm)

Va (1x10-'M) plus sodium salt of 9,10-dihydroanthracene5x10-4) and excess 9,10-dihydroanthra-20 minutes: cene (1x10- 4E$ in DMSO, anaerobic. -.-. 6 hours: - - - previous sample after admission of air (1 minute). 1100

of

of

Vol. 4 1, No. 5, 1970

BIOCHEMICAL

propiophenone

(each produced

and slightly

less

than

for

the

shows spectra of oxidized

spectrum

upon oxygen

of unsubstituted

reduced

vb,

the

cut,

an isoalloxazine yield

showed close

to a very

addition

rapid

2:

mission

It philic produce transfers

is

indicative

results

of t-BuOK

spectrum spectrum

500

or C6H50Na

spectral

change to

in approximately is

left.

based on original

400

were obtained

50%

(Figure

2).

Epr

isoalloxazine,

600

Ad-

700

(nm)

--Vb(1.5x10W3M) in DMF; -.-. 1 minute after addition of asaerobic NaOC6H5; - - - previous sample after admission of air (1 minute? same after 6 hours or aerobic photolysis or acidification with HCl).

of air

alloxazine

of N-5 or

return

(seconds)

WAVELENGTH

Figure

rapid

1

Slow return

is typical

Addition

anion

50% spins

300

Figure

system.

surprising,

compound.

radical

to

of NaH in DMF).

while

also

c370) o No oxidized

by

compound

isoalloxazines.

but

3-benzyl

in DMSO or DMF led

from parent

9,10-dihydroanthracene

isoalloxazines,

with

anaerobically

an equivalent

C-4a substituted

More clear

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

led to the

spectrum seems likely

manner to the an alkylated

half that

immediate as intense &-BuO-

to FRox to generate

of oxidized

as in the original

iso-

spectrum,

or C6H50- adds in a nucleo-

isoalloxazine flavin

appearance

(possibly

anion the ‘101

which radical

at N-5 or C-4a)

subsequently anion

electron

(Scheme II).

to

Vol. 41,No.

5, 1970

BIOCHEMICAL

Such a process

12,13

has precedent.

+ C6H50e_,

FROX

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FROX

C6H5C-F'R

3 C6H5q-fiR + TR*-

\I

dimers?

L

Scheme II On the above,

FROX

other

and also

electron

with

It

It

oxidases,

acyl

inter P

ted)

a,

S to

that

the proteins

a-proton

fer

net

is

alia P,

flavin

4

to protein-bound

these

reactions

with

1,2-dihydro-

flavin

reduction

and succinate

is

oxidized

or

group,

acting

electron

dehydro-

This

resulting

amino

(dehydrogena-

as bases to

the

by either

as the

suggests

remove the

carbanion)

or hydride

(Scheme III).

Acknowledqments

(Grant

GM 15100)

support

of this

to

and the

the National Research

work. 1102

Institutes

Corporation

of Health for

and

trans-

Scheme III

We are grateful

by

in the work of Russell. 11,15

stabilizing

flavin

of Vb spec-

has occurred

That

stabilizing

may be in part

followed

of

in such flavoenzymes

each substrate

also

and base,complete

such case with

was suggested that

mentioned

are in progress.

reduction

recently.

a somewhat anion

then

studies

CoA dehydrogenases,

(possibly

this

14

products)

Another

be noted

genase,

that

that

is possible should

carbanions

and 100% return

Detailed

has been reported

carbanions

place

the oxidation

clear

three

7-cyanocycloheptatriene

and carbanions.

phthalate

the

upon aeration.

is

enolates

with

to Vb takes

(and especially

acid

hand,

transfer

trum occurs

O2

financial

Vol. 41,No.

5, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

References 1.

a) A. Ehrenberg and P. Hemmerich, in "Biological Oxidations," T. P. Singer, Ed., Interscience Publishers, New York, N. y., 1968. p. 239; b) A. Ehrenberg and P. Hemmerich, Europ. J_. Biochem., 2, 286 (1967): c) F. Muller, P. Hemmerich, A. Ehrenberg, G. Palmer, and V. Massey, i&i&., 14, 185 (1970).

2.

J.

3.

a) I. M. Gascoigne and G. K. Radda, Biochem. Biophys. 131, 448 (1967); b) M. J. Gibian and D. V. Winkelman, Letters, m. 44, 3901 (1969).

4.

G. 0. Weatherby

5.

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6.

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9.

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Biophys.

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A. Davison, N. Edelstein, 82, 2029 (1963).

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G. A. Rl1ssel1, 1807 (1964).

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14.

Observations

15.

G. A. Russell, R. Konaka, K-Y. Chang, and G. Kaupp,

and V. Massey,

J.

of D. L. Elliott

Europ.

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R. H.

E. G. Janzen,

Fed.

and A. H. Maki,

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T.

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E. T. Strom, W. C. Danen, ibid., 90, 4646 (1968).

1103

ibid.,

s.,