The conversion of indole-3-acetic acid to 3-methylene-oxindole in the presence of peroxidase

BIOCHEMICAL

Vol.5,No.4,1961

AND BIOPHYSICAL

The Conversion of Indole-3-Acetic oxindole

RESEARCH COMMUNICATIONS

Acid to 3-Methylene-

in the Presence of Peroxidase.

Richard L. Hinman, CarolBauman,

and Joan Lsng

Union Carbide Research Institute 32 Depot Plaza White Plains,

N.Y.

Received May 29, 1961 The existence oxidative

degradation

has been recognized

and that

that

related

clusive

proof

IAA, catalyzed

that

the IA&oxidase

of the oxidation

of structure

peroxidases

products,

we report

of the principal

by crystalline

horseradish

have

(Ray, 1958). type (Ray, 1s58),

would probably yield here the first

product

con-

from the oxidation

peroxidase,

the

acid (IAA)

attempts

systems are of the peroxidase

oxidases and purified

if not identical

catalyze

growth hormone indole-3-acetic

the organic products

the natural

closely

of the plant

plants

for many years and numerous unsuccessful

been made to identify Recognizing

of enzyme systems in higher

of

in the absence of

added hydrogen peroxide. Experimental.

3-Bromo&indole-3-acetic

N-bromosuccinimide

acid,

and IAA by a procedure

prepared by the reaction

to be described

in a forthcoming

paper, melted at 150-151" dec. Anal. Calcd. for CIO%BrNO

c, 44.46;

H, 2.98;

H, 3.20;

N, 5.05;

out as described

previously

3'

N, 5.19;

Br, 29.59.

Found:

C, 44.59;

of

Br, 29.80. Enzymatic reactions (Hinman and Frost, peroxidase

1961).

(calculated

of M.W. WOO).

product,

Final

concentrations

by assuming that

were 10w4M IAA and 10m7M

the weighed protein

IAA, measured by the modified

Weber, 1951), was completely final

were carried

Salkowski test

consumed in about one hour.

observed spectrophotometrically, 250

was pure enzyme

continued

(Gordon and

Formation

of the

for upwards of

Vol. 5, No. 4,196l

BIOCHEMICAL

48 hours at 25’,

but was complete in lo-15

changes were observed at @I 3.5 2H 6.5

(0.067

AND BIOPHYSICAL

(0.12

RESEARCH COMMUNICATIONS

minutes at 100'.

citrate),

T$ 5.0

of 5% aqueous sodium bisulfite

were effected

to 2.5 ml. of reaction

shown in Fig.

1 are:

after

at 25O, or after

No. 2 + N&E03;

correction

_Macetate),

and

for blank;

(1) IAA (10m4@;

(4) Water 1aFr

followed

of No. 3 after

(5) Dec. product

buffer

(2) IAA (10-4$

~7 min. at 25'

by adding 0.05 - 0.10 ml. mixture

All spectra were measured in 0.2 _Macetate

(3)

spectral

_Mphosphate).

Reactions with sodium bisulfite

26 km.

(0.2

Similar

at 2H 5.0. at 2H 5.0.

+ peroxidase by llmin.

extraction

Those (10w7@

at 100"; with ether and

of 3-bromo~xindole-y-acetic

e-C’

acid

M I

Fig.

WAVELENGTH

t-H/U)

1

BIOCHEMICAL

Vol. 5, No. 4,1961

AND BIOPHYSICAL

(5 x l.0-5$;

(6) No. 5 + NeaS03;

Discussion.

We have found that

concentration oxindole

(7) 3-Methyloxindole the peroxidase-catalyzed

the first

A non-acidic

of the second product

product

indolic

of the overall

increases with increasing

and shove only indolic

oxidation

material

In this is j-methylene-

The proportion

IAA concentration;

The structure

is formed.

of IAA

is composed

reaction

component is also formed.

x l/3).

(ordinate

(Ray, 1956).

of which is enzyme-catalyzed

range the principal

(I).

(10w4$

From 10 -5 - 10V4M IAA the oxidation

is concentration-dependent. of two stages,

RESEARCH COMMUNICATIONS

at 10 -3

of this

product

is

under investigation. Structure

ultraviolet

spectrum (Fig.

an exocyclic

1958;

Julian

l),

a feature

1953).

and Printy,

was obtained

released together

(Wenhert, Bernstein,

An ultraviolet

bearing

and Udelhofen,

spectrum with peaks at the

when +bromokindole-j-acetic

acid (II)

of II to I is base-catalyzed

with one mole of carbon dioxide

a combined dehydrohalogenation the base-catalyzed

278 sh;

of oxindoles

was

in water. The conversion

(Fittig

characteristic

double bond at the 3-position

same wavelengths dissolved

by the two maxima near 250 mp in its

J was suggested

and Binder, caax

1879).

25,400;

The spectral 4200),

features

acid to stycene

of I ( X max 247, 253,

as prepared from II,

show the

to those of the known 3-ethylideneoxindole

244 sh, 247, 253, 287; c -25,200; 3-isopropylideneoxindole

( X -

29,600;

25,700;

258, 292;

252,

indicating

in a manner analagou~ to

of E-bromo-@henylpropionic

25,500;

expected relationship

per mole of II,

and decarboxylation,

conversion

and bromide ion is

<,,

(

A max

4550) and 26,m;

28,800;

6750). As an cr,p-unsaturated addition either

to the exocyclic the oxidation

8600;

double bond.

product

produced a new product,

Addition

of sodium bisulfite

of IAA or the decomposition

the spectrum of which ( h -

2500 for the product

known 3-methyloxindole

amide I would be expected to undergo facile

derived

from II)

( x max 247.5, 252

product

248.5,

resembled closely

275 sh;

f ,,

8360;

to of II

275 sh; that 1680)

< -

of the as would

Vol. 5, No. 4,196l

BIOCHEMICAL

AND BIOPHYSICAL

be expected. from the proposed structure:

RESEARCH COMMUNICATIONS

the sodium salt

of 3-(sulfomet~l)-

oxindole.

Br 1. 2. non-enzymatic

%J02" J 03

N '0 H

II

H CH$03Na

'Ilhe additional

product(s)

by maxima at 280 and 285 rnp product

(Fig.

mixture

by extraction

(Fig. that

formed in the enzymfktic reaction,

in the spectra of both I and its

material

features

more clearly.

The fact

remained in the aqueous phase is in agreement

with the proposed sulfonic

since 3-methyloxindole

acid structure,

is readily

into ether. E msx of I, calculated

Using the to I is quantitative, 22 hrs.

reaction

The spectrum of the aqueous phase

with ether.

1, curve 4), then showed the oxindolic

extracted

reduction

1, curves 2 and 3), was removed from the bisulfite

the oxindolic

detected

the conversion

of IAA to I was estimated

at 25O when the rate of reaction Solutions

reagent,

Ehrlich's

Although

I was fairly

above lo -51: -, the intensity at 10e2g a precipitate was probably

Elanan's reagent,

stable

the product

gave negative

(probably isolated

tests

and diazotized

at the lower concentrations

of its

of II

as 60% after

was very slow.

of-3-methyleneoxindole reagent,

by assuming the conversion

sulfa&lie

acid.

(10F5 - 10m4@,

spectrum diminished

rapidly

polymeric)

(Material

formed.

with Salkowski

with time, of this

and type

by Ray and !Chimann (1956) from the reaction 253

BIOCHEMICAL

Vol. 5, No. 4,196l

AND BIOPHYSICAL

Under chromatographic

of IAA with the oxidase from Omphalia). particularly

in the ammoniacal solvents

IAA and its

oxidation

instability

of I, which precludes

multiplicity

products,

of products The conversion

for the oxidation oxidation when light principal

its

reported

isolation,

of IAA.

The reported 1958)

the

indicate

(III)

converted

general pathway

spectra that

Moreover,

for the photolytic

I is a principal it is now clear

of IAA with acidified

1961) is compound I.

product that

the

hydrogen peroxide

t&;cr-Dimethylindole-3-acetic

to 3-isopropylideneoxindole

that

This

helps to explain

ultraviolet

is used.

from the reaction

with confidence

of

by other workers.

(Rinman and Frost,

predicted

used for chromatography

of IAA to I appears to be a fairly

of 302 and 313 rnp

is similarly

generally

conditions,

I underwent rapid decomposition.

of IAA (Ray and Curry,

product

RESEARCH COMMUNICATIONS

and it

acid can be

the last

compound is also formed in the non-

of III

in the presence of the oxidase from

enzymatic stage of the oxidation Omphalia (Ray and Thimann, 1956).

Acknowledgement.

The authors

express their

of the Union Carbide Research Institute New York Botanical

indebtedness

to Dr. Sanford Siegel

and to Dr. Richard Klein

Garden for many helpful

of the

discussions.

References R. Fittig and F. Binder, Ann., 2, 131 (1879). S. A. Gordon and R. P. Weber, Pl. Physiol., 26, 1% (1951). R. L. Hinman and P. Frost, in "Plant Growth Regulation," R. Klein, ea., Iowa State University Press, Ames, Iowa, 1961, p. 205. P. L. Julian and H. C. Printy, J. Am. Chem. Sot., 3, 5301 (1953). P. M. Ray, Arch. Biochem. Biophys., 64, 193 (1956). P. M. Ray, Ann. Rev. Plant Phys., $,-8l (1958). P. M. Ray and G. M. curry, Nature, 181, 895 (1958). P. M. Ray and K. V. Thimann, Arch. %&hem. Biophys., 64, 175 (1956). E. Wenkert, B. S. Bernstein, and J. H. Udelhofen, J. K Chem. Sot., 80, W9 (1958).

254