Biosynthesis of the ethyl side chain of stigmasterol derivatives by the slime mould Dictyostelium discoideum

Tetrahedron Letters

No.18,

pp. 1049-1052,

$iosynthesis derivatives Maryse

1967.

Perguwn Prere Ltd. Printed in Great Britain.

of the ethyl side chain of stigmasterol

by the slime

LENFANT,

Elise

mould Dictvostelium ZISSMANN

Inetitut de Chimie des Substances (Essonne),

discoideum

and Edgar LEDERER

Naturelles, France.

C. N. R. S. , Gif-sur-Yvette

‘(Received 12 January 1967) The ethyl and ethylidene synthesized

side chains at G-24,

in vivo by two successive

the methyl group of two molecules tle et.

C-methylation

of methionine

, 1 ; Bader et al. ,2 ; Villanueva et. leading to ergosterol

the methyl of methionine atoms of methionine

(1) in 1963.

atoms derived

(Cas-

et.

,4) that during the C-

to study the fate of the hydrogen reaction

mentioned

in a scheme

proposed

step can lead to a methylene step

above. by Castle

compound (I) which

; this can either lead to an ethyli-

or to an ethyl derivative

(IV) containing -five hydrogen

frommethionine.

In previous rol,

interesting

for the second C-methylation

(III) containing four,

precursor

acid , one of the hydrogen atoms of

have already been considered

The first’c-methylation

could be the substrate dene derivative

it seemed

are

the carbon atoms of

of a phytosterol

(Jaureguiberry

during the double C-methylation

The two alternatives et.

steps transferring

onto C-24

and to tuberculostearic

is lost,

of the phytosterols,

,3).

Since it has been shown in our laboratory methylation

characteristic

have generally

biogenetic

schemes

been considered

logy with the methyl derivatives tion of an intermediate

(5,6,7,8)

ethylidene

to be precursors

derivatives

of the ethyl derivatives

(V) which have been shown recently

methylene

derivative

(III) such as fucoste(IV),

to originate

in ana-

by satura-

(I) (9,10,11).

Mercer

sterol

(7) and Goad et al. (8) have recently studied the biosynthesis of the phyto3 side chain using (14C. H-methyl) -methionine ; they conclude that the ethyl side chain

(of eitosterol methionine,

of maize

and larch leaves)

thus indicating the sequence

As the conclusions tope effect of tritium, CD3.

drawn from

In the course of the slime

(I)-+(II)+

such experiments

was to choose a biological

from

(III) --+ (IV). can be endangered

we thought that the same problem

The only difficulty

poration of the labeled

contains only four hydrogen atoms derived

by the strong iso-

should be studied using methionine-

system

permitting

a high enough incor-

methionine.

of a discussion

mould Dictvostelium

of this problem, discoideum,

Professor

the sterols

1049

D.Arigoni

suggested

the use

of which had been studied exten-

1050 No.12

sively

by Heftmann

Dr.

M. I. Krichevsky

less

mutant

u.‘(lZ)

and Johnson

made

of E. coli

the excellent

this

slime

that we should

and then feed

these

mould first

cells

the

formulae

above

it is not yet We

show

,. mass

without

spectrometry

an ethylidene

proceeds

either

on E. coli,

feeds

grow

a methionine-

to the slime

mould.

from

is also

intense

peak

of -five

and indicating

ion,

m/e

414)

into the ethyl that

or by elimination

reaction of Ht

was

3

(12)

side

obshows

chain,

(II)-

(IV)

(not via III) lea-

can then be reduced, p-acetate

spectrum

peak

at m/e

A peak isopropyl

deuterated

(“)

gal,

incorporated

1

(VI)

showed

gave

the molecular

an intense

peak

ion peak

at m/e

at m/e

456

344

corresponding

due to the loss

of the e,thyl

sample

group (VI)

in the molecular deuterium

427

observed

atoms

(M-29)

apparently

at m/e

by an allylic from

413

is considered

cleavage.

the methionine-CD3

ion region

at m/e

in the molecule

to be due to the eli-

461

feeding (456

experiments

t 5) corresponding

showed to an

(*+)*

(*)

In the culture

(*“)

the intensity of the Mt t5 peak is approximately three times that of the Mt peak. There are also, as expected, peaks at m/e 458 and m/e 459 corresponding probably molecules of (VI) having two and three deuterium atoms, respectively, in the side -

obtained stigmast-22-en-3 pal is accompanied ion peak Th e molecular bably stigmastanp-01 (m/e 416). shifted by five mass units in the experiment with deuterated

chain.

;

of the side-chain.

a small

of the terminal

incorporation

which

the mass

the side-chain.

been

as intermediate

of (II) by a hydride

of methionine-CD

(stigmast-2Z-en-3

have

because

(14).

incorporation

sterol,

(N)

is not shown

it is introduced

a good

atoms

stigmast-22-en-3

of a part

A purified the most

derivative

fragments

to the elimination

There

deuterium

compounds

Non deuterated

in fact,

(III) unsaturation

at what stage

that,

by reduction

ding to unsaturated

the C-22

of the isolated

that five

excluding

other

clear

in the following

any doubt

mination

w

(1) ln

group

As

b*H;q9H;H3

PO

among

(13).

suggestion

on methionine-CD3

QH+qH2

tained

et.

by a saturated sterol. proof this compound is also methionine.

In both

sterols to

1051

No.12

The

peak

rated

sterol,

peak

at m/e

that

413

group

is apparently

deuterated ethyl

valid

does

418

in the spectrum deuterium

in the spectrum

not contain

group

of the corresponding

atoms

are

with the presence

The

unshifted

of all five

peak

at m/e

in the spectrum

deuterium

The

; this shows

compound

deuterium.

remains

deute-

in the side -chain.

of the deuterated

the incorporated

of the ethyl

in agreement

results,

will

of Goad

427

of the

atoms

in the

transfer

be possible

presence

of p ntact

cases

the

the intact

meter)

methyl

of the side-chain,

that analogous

so as to compare

group

double

bonds

medium

are

experiments

the results

of course

using

obtained

methio-

with those

at 23’

extracted

by CH

2

- D. discoideum of Banner are

2

are

-en-3

Acknowledgments

for

We thank

Dr.

G.Gerisch

Finally, the mass would

for the strain

we wish

not have

15) proving

It thus

until

Prof.

seems

the

that in

now the transfer and other

of

strong-

HCl

-22 -en-3

E. coli. buffer

(160

(12)

mm

dia-

At the end of the M/60,

pH 6 (19)

D (methionine-less) and agitated

(200

grown

with

in the

r. p. m. ) culture

at the end of the vegetative

;

the neutral

of silicic

801 is thus

Si02:2,

dishes

fraction

acid.

obtained.

(191

50 mg of a 1:4 After

hexane_benzene,5:1).

mg)

acetylation

33 mg of stig-

obtained.

for his

advice

Dr.

and Dr.

as well of isotopic

M. I. Krichevsky

on the culture

E.Heftmann

for

conditions

a culture

as the Commissariat

for their

most

of the slime

of the same a 1’Energie

help-

mould

organism, Atomique,

compounds.

to express

our appreciation E. I. instrument)

possible.

304

on a column

D.Arigoni

of E. coli,

in 20 Petri

are harvested

(on a MS9-A. been

(see

of aromatic

of living

(3 l/min)

by dilute

(AgN03:l, are

for the purchase

spectra

of E. coli

aerated

and stigmast

of D. discoideum,

a grant

case

bond.

,

in 10 1 of a Sarensen

chromatographed

149-151”

is grown

in presence

and the cells

by TLC

:

and for the strain G. Cohen

m. p.

double

as a whole

C-methylations

vlZ/Ml

suspended

hydrolyzed

stanol

separated

801,

ful suggestions,

are

and then

of the saturated

the acetates

for

(18),

The

in a fermentor

Cl

only

of 20 1 of a culture

cells

aliphatic

is transferred

shown

of 3 g of methionine-CD3.

27 g of moist

is the second

(15,16,17).

the spores residue

(VI)

to a non activated

had been

on the solid stage,

in the sterol

of methionine

part

is incubated

Saclay,

by degradation

It is hoped

plants,

group

group

group

the centrifugation presence

of a CD3

Experimental

sporulation

mast-22

be confirmed studied.

in higher

methyl

-CH3

ly nucleophilic

mixture

will

mould

et al. (8). The

stage.

which

for the slime

nine-CD3

work

unshifted that all five

to m/e

due to loss

compound,

Our

med

remained

group.

only

Dr.

344

demonstrating

is shifted

the isopropyl

which

all

at m/e

clearly

and thanks

;

without

to Dr. B. C. Das his

interest

who perfor-

and help

this

is

No.12

REFERENCES

1)

M. Castle,

2)

S. Bader,

3)

V. Il. Villanueva. M. Barbier and E. Lederer. Bull. Sot. Chim. Fr. ,1423 (1964) trage sur Biochemie und Physiologie van Naturstoffen”, K. Mothes Festschrift, Gustav Fischer Verlag, Jena, 509 (1965).

4)

5)

G. Blondin and W.R. L. Guglielmetti

Nes,

J.Amer.

and D. Arigoni,

Chem.Soc.,

@. 3306 (1963).

Proc. Chem. Sot.

, 16 (1964).

G. Jaureguiberry, 3587 (1964).

J. H. Law,

J.A. McCloskey

and E. Lederer,

C. R.Acad.

G. Jau&guiberry, (1965).

J.H. Law,

J.A.McCloskey

and E. Lederer,

Biochemistry,

M. F.Hiigel,

W. Vetter,

H.Audier,

M. Barbier

and E. Lederer,

; “BeiVEB

Sci.,

258, 4.

Phytochemistry,

347, 3,7,

(1964). 6)

E. Lederer, Biochem. J. , 93, 449 (1964) cal Sciences, 1, 1129 (1965).

7)

E.I.Mercer,

8)

L. J. Goad,

A. S.A. Hammam,

9)

M.Akhtar,

M.A.

Biochem.

J.,

Parvez

A. Dennis and T. W.Goodwin,

and P. F. Hunt, Biochem.

D. H. R. Barton,

11)

G. Jaurhguiberry, 855 (1966).

M. Lenfant,

12)

E. Heftmann,

Wright

13)

D. F. Johnson,

B.E.

14)

D. F. Johnson,

E. Heftmann and G.V.

15)

G. Jaurdguiberry, 27 (1966).

16)

B.E.

17)

G. Jaurkguiberry, 259, 3108 (1964).

18)

J. T. Banner,

19)

G. Gerisch,

Tropp,

20,

473 (1964)

; Israel J.Medi-

96, 17P (1965).

10)

D. M.Harrison

B.E.

; Experientia,

and G. P. Moss, R. Toubiana,

and C. U. Liddel,

B.C.

, 12,

Arch. Entwicklungs

Bioph.,

97, 232 (1962).

Tetrahedron,

, 104, 102 (1966). suppl. S, part I,

_3, 1837 (1964). and E. Lederer,

1 (1947). 152,

Chem.Comm.

91, 266 (1960).

Arch. Bioch. Bioph.

H.Audier

1322 (1966).

595 (1966).

and E. Lederer,

Arch.Bioch.

Biochemistry,

Mechanik,

@,

(1966).

Arch. Bioch. Bioph.,

C. Houghland,

G.Farrugia-Fougerouse,

J. Exptl. 2001.

Chem. Comm.,

Das and E. Lederer,

J. H. Law and J.M.Hayes,

Nature -’

, 100, 3%

R.Azerad

Wright and E. Heftmann,

M. Lenfant,

J.

632 (1960).

C. R.Acad.Sci.,