Microbiological synthesis of 16α, 18-dihydroxydeoxycorticosterone

697 MICROBIOLOGICAL lQ, R. Section

School

Received:

lb,

S.

Boston,

was established

appeared

product.

of Medicine,

Boston

by microbiological

Uni-

Yield

roseochromogenus studies,

of the product

about 60%

of the substrate

method of synthesis

in human and experimental

of 16a,

13400).

lb,

18-dihy-

formation

and

of substrate

and,

18-OH-DOC isolated

found

of the compound

(ATCC

derivative

of fermentation

was con-

in lower

in the acidic

yield

conditions

18-dihydroxy-DOC

for further

specific

of 18-OH-

produced

was about 38%

18-dihydroxy-DOC

way to make large quantities

(1) and 1, of high

hydroxylation

to human adrenal

A second product This

18-dihydroxy-DOC)

18-dihydroxy-DOC)

by chromatographic

to be a dimer of 11&,

of the fermentation. role

MELBY

02118.

(lb,

product

for losses in recovery, to this

C.

Department

by Streptomyces

chromatography.

allowing

J.

Massachusetts

in good yield

of the fermentation

droxy-DOC

AND

(1,2-3H-l&,

and 1, 2-3H-18-OH-DOC

gas-liquid

OF

ABSTRACT

were obtained

identity

tical

DALE

and Metabolism,

of Medicine,

18-dihydroxy-DOC

verted

L.

18-Dihydroxydeoxycorticosterone

activity DOC

BRAHAM,

9/5/75

2-3H-lQ,

The

L.

of Endocrinology

versity

SYNTHESIS

18-DlHYDROXYDEOXYCORTlCOSTERONE

is a prac-

study of its possible

hypertension. INTRODUCTION

Recent

studies

steroidogenesis isolation

from this

in “low

renin”

and identification

as a product

Normal

adrenal

cer were shown metabolite, of single Further effect

adrenal

greater glands

Vohne

26, Number 6

5) investigating

hypertension metabolite

a mean of 15%

taken

of 18-OH-DOC: This slices

S

l&I,

metabolite

with

with

“low Kagawa

was initially

to further

-X?LIEOXDI

study

the role

in incubations

the sodium

by the presence 16a,

can-

to the 1Q

hypertension

assay,

was enhanced

the

18-dihy-

for breast

was found

renin”

with

1,2-3H-18-0H-DOC.

adrenalectomy

to the 16a metabolite

in the modified

I n order

adrenal

of added 1, 2-3H-18-OH-DOC

from patients

that

altered

have been concerned

from women undergoing

doses of aldosterone (5).

4,

of human adrenal

conversion

have shown

of subthreshold

18-dihydroxy-DOC

taken

3,

18-dihydroxy-DOC).

of incubation

to convert

while

studies

(lQ,

glands

(2,

essential

of a polar

droxydeoxycorticosterone found

laboratory

(5). retaining of lb,

18-dihydroxy-DOC

December, 2975

may play tities

of the compound

supply

Since

the first

streptomycetes

perimental nus (S. -

tissue

roseochromogenus Recent

roseochromogenus)

16~1 hydroxylate Marsheck

Steroids

keto reduced. the salt OH-DOC

with

properties

has weaker

decreases

with

have shown

larger

quan-

the limited

that

function,

ability

activity

a property

have and ex-

roseochromoge-

steroids,

and only

than

for industrial

as noted

function

of

many workers

Streptomyces

most C-21

but will

in a review

not

by

these steroids

are 20-

thus far has been to reduce

and synthetic

retaining

7) of the ability

nucleus,

use of 16a hydroxylation of natural

(6,

streptomycetes

16o hydroxylate

retaining

12),

the steroid

a 17-oxygen

sodium

the salt (11,

and coworkers

a 17-oxygen

The principal

retaining

hydrocortisone

with

to synthesize

from incubations

and other

studies

will

steroids

(8).

by Perlman

to 16a hydroxylate

purposes.

necessary

available.

description

used Streptomyces

tion

than could be obtained

of human adrenal

certain

it was found

in human hypertension,

steroids DOC

(9-12).

Thus,

l&-

(9) and 16a hydroxyla-

of 9a-fluoroprednisolone

utilized

in the synthesis

(8) details

the many factors

and 9a-fluoro-

of triamcinolone

(9a-

fluoro-lQ-hydroxyprednisolone). An excellent sults

recent

in the microbiological

roseochromogenus portance

by -S. (Cu*,

transformation

that

were carried

high ferrous out in iron

roseochromogenus. Al-,

Co*,

Addition Ni*,

addition

or ferric tanks,

Cr*,

of steroids. medium

and 16a hydroxylation,

but the method of substrate

it was found tions

review

composition

ion concentrations, caused isomerization

Zntt,

Specifically

may be so (7,

of equivalent Mnff,

affecting

and re-

in terms of S.

is not of critical 13).

im-

In one experiment

obtained

when fermenta-

of triamcinolone

concentrations Sn++)

yields

of other

did not induce

formed cations isomeriza-

S tion

699

TBstOXDI

(14). As the iQI

is necessary amounts

hydroxylase

to induce

it by certain

of radioactive

progesterone

steroids

to induce

but present

is constitutive, C-21

steroids

of high specific

the 16~1 hydroxylase

in only

in order

small

to l&I

activity.

it

hydroxylate

Janoski

for synthesis

amounts,

trace

et al (15) used

of 16a-hydroxypregneno-

fone- 3H. The

subject

enus of l&x,

of this

report

is the microbiological

18-dihydroxy-DOC

18-dihydroxy-DOC

from

from

synthesis

l8-OH-DOC

by 2,

and synthesis

roseochromog-

of 1,2-‘H-l&I,

1,2-3H-18-OH-DOC. EXPERIMENTAL

Gas-liquid

chromatography

chromatograph. scanning

thin

A Packard layer

3H-18-0H-DOC DOC

A.

Malt

culture

13400)

Malt

yeast extract

of

yeast broth (Difco)

A lyophilized

Excellent

colonies

from the plate

of malt yeast broth. strokes

per minute

fermentation. fresh

malt

taining

at 28’

yeast broth

substrate

culture

18-OH-DOC

and Co.

gas

was used for

The

and 1,2-

1,2-3H-18-0H-

of S.

broth C.

cultures The

96 hours,

after

and computation

roseochromoge-

malt extract broth

agar”

(Difco)

with

72 hours.

were placed of this flask.

was mixed

These

remained culture This

colonies

flasks

on a rotary

11 cc culture

in 0.5

shaker 4.5

were

containing

50 cc

at 100

to 5.0

during

the

was added to 10 cc of

culture

was allowed

was added to a flask

had been deposited

cc

onto malt yeast agar

250 cc Erlemeyer

pH of the cultures

on the walls

to con-

of the flask

used as a solvent.

of large quantities

was used as substrate

“Bacto-yeast malt extract

inoculated

a 1 cc aliquot

which

(S.

Cdlection.

roseachromagenus

in a 125 cc Erlemeyer

of the ethanol

Culture

of 5 grams/liter.

mixturewere

were observed

18-OH-DOC,

For preparation isolation

from dried

hours and then the entire

upon evaporation

OH-DOC

model 7401

Scanner

roseochromogenus

from dehydrated

and used to inoculate All

After

grow for 48-72

The

Searle

added at a concentration

and loops of this

lifted

D.

from Americziype

was prepared

Fermentations: water

G,

Streptomyces

was purchased

of sterile plates.

from

yeast agar was prepared

(Difco).

on a Packard

Radiochromatogram

of 51 Ci/‘mm.

lyophilized

nus) (ATCC -

was performed

7201

and paper chromatograms.

were purchased

hod a S. The

(TLC)

(GLC)

Model

of 16a,

in each flask.

of yields,

18-dihydroxy-DOC, In initial

experiments,

1 pCi of 1,2-3H-18-0H-DOC

2 mg of 18to facilitate was added with

the 18-OH-DOC.

The fermentation

For preparation specific One

activity,

of small broth

to another

the induction

flask

Isolation

of 19,

as substrate

twice,

washed with

GF-254

(Brinkman

l/10

on its walls.

to proceed

volume

Instruments)

of water

of the two product

steroids

given

two products

1.

The

was eluted

Identification

of 16a,

chromatographed (2

with

: 1 : 2) with 68-OH-E

with

ethanol I&

renal DOC

separately

i,2-

on silica

reference

standard.

18-OH-DOC

as as-

scanning

with

gel

: ace-

peaks are

ethanol

and the

extraction. The most polar

of the products

$1 paper in the system benzene

reference

and

of dichloromethone

The

Standard.

single

was

: acetone : water

band found

was eluted

and dried.

18-Dihydroxy-DOC

slices

hydroxy-

18-OH-DOC

18-OH-DOC

were eluted

18-dihydroxy-DOC:

on Whatman

using

5 volumes

3H ro d’roactive

dichloromethane

for

H-18-OH-DOC,

1,2-

and the unchanged

bands and corresponding

in Table

was left

was then added

system of dichloromethane

with

sessed by UV absorbing 18-OH-DOC

culture

and then chromatographed

in the thin-layer

and al-

72 hours.

Fermentations with

broth

solutiog

25 pCi of

another

were extracted

: water (70 : 25 : 5 : 0.7)

Mobilities

This This

had been deposited

was allowed

t8-dihydroxy-DOC:

3H-18-OH-DOC

: methanol

deposited

of high

was then added to a flask

of the 16a-hydroxylase.

on whose walls

and the fermentation

11 CC culture

hours.

et al (15) was performed.

was added to 10 cc of ma-yeast

The entire

2 mg of progesterone

96 hrs to allow

tone

culture

for 72-96

18-dihydroxy-DOC

of the method of Janoski

to grow over 48 hours.

containing

to proceed

of 1,2-‘H-l&l,

amounts

a modification

cc of growing

lowed

was then allowed

was made by incubating

by methods previously

DOC

described

(A) was compared

(3).

18-OH-DOC

This

to the fermentation

adrenal

product

with

product

human ad16a,

18-di-

18-dihydroxy-

lb,

(F): Both compounds

acetone eluates

were chromatographed

from both products

(16) and chromatographed in benzene

: ethanol

(9

paper in a benzene Finally, (TMSE)

on Whatman

: water (2 : 1 : 2) system with a +-OH-E were

: 1).

The

: cyclohexane

the two oxidized

and submitted

supelcoport

column

then oxidized

on silica

6 feet

with

standard. with

The

periodic

18-OH-DOC

:

single acid

lactone

standard

on Whatman

#1

: water (5 : 3 : 5 : 1) system.

were converted

chromatography

X 2 mm with

paper in a chloroform

were chromatographed

: methanol

products

to gas-liquid

to the lactone

gel GF-254 lactones

#l

reference

column

to the trimethylsilyl

on OV

temperature

101 on 100/l 255’

C,

esters 20 mesh

nitrogen

flow

of 42 ml/min. identification

of Second

Product

tation

was seen on the initial

dized

with

ethanol

(9

periodic

TLC

of Fermentation: system,

acid and chrom~tographed

: 1) with reference

standards

A second product

In order

to characterize

on silica

of III-OH-DOC

gel GF-254 Iactone

of the fermenthis,

it was oxi-

in benzene

and I6a,

:

18-dihy-

S droxy-

DOC

lactone

Radioactivity tion

(New

efficiency

Nuclear)

of the counter

was 40%.

To assess the percent

conversion

Carb Model

3330

Counting

diluted

to one liter

of 18-OH-DOC

liquid

solution

in aliquots

was used to compute

with

of

toluene.

to each of the two products,

of the unchanged

recovery

scintilla-

consisted

was added to 2 mg of 18-OH-DOC

of the radioactivity

the two products

Tri

radioactivity.

England

1 uCi of l,2-3H-18-OH-DOC Counting

A Packard

Studies:

was used for assaying

42 ml of Iiquifluor Tritium

701

(A).

and Yield

counter

WBEOXDI

in each flask.

18-OH-DOC

and in

and yields.

RESULTS Three

separate

UV absorbing

TLC

system.

is given

with

a Rl8_OH_DOC

16a,

1.

18-dihydroxy-DOC,

be a dimer

of 16a,

previously

described

unchanged ing.

The steroid

Of

steroids

the initial

experiments

have indicated

as 1,2-“H-l

In order

agreed

in high Q,

58%

with

specific

1 .OO was assumed to be

were recovered

to be 50%.

dimer and 17%

by weighing

these results. activity,

18-dihydroxy-DOC,

to check the above assumptions

remained

In the synthesis

21%

of the initial

that all

the nature

three

to lQI,

unchanged.

substrate

and prod-

of l,2-3H-16a, number

18-

of cpm was re-

and 15% as the presumed about

18-di-

18-OH-DOC.

was converted

unlabelled

for count-

as 16a,

Assuming

of the 18-OH-DOC

ratio

to

to the dimer of 18-OH-DOC

dimer and 9% as unchanged recovery

to be

was presumed

of each band were taken 25%

steroids

was presumed

of 0.76

Rl8_OH_DOC

added,

to the presumed

and conversion

closely

dihydroxy-DOC

of counts

equally,

with

of eluates

as the presumed

25%

of yields

steroid

Aliquots

were recovered

uct steroids

covered

The

number

13%

18-dihydroxy-DOC, Analysis

(17).

analogous

scan-

of the three

of 0.41

a Rl8_OH_DOC

18-dihydroxy-DOC

18-OH-DOC.

hydroxy-DOC, Previous

the one with

H radioactive

The mobilities

ning peaks were seen in the first in Table

3

bands and corresponding

dimer.

of the two product

702 steroids

several

presumed

additional

16a,

3

scanning

18-dihydroxy-DOC

product,

16a,

(3).

changed

compound and lactone

lactones

were

mer of 16a, mine this, lactone. lactone

of the

in a paper

: acetone : water (2 : 1 : 2).

(F),

vs 16a,

are given

Only

one UV

mobilities

in GLC.

of the fermentation

18-dihydroxy-DOC

in Table

2.

Identical

mobility

(A),

Finally,

TMSE’S

retention

as unof the two

times of 7.2

min-

for both products. with

a R18_0H_DOC

18-dihydroxy-DOC the compound

The mobility is given

and chromatographed

Ch romatographic

run for comparison

The steroid

The eluate

peak was seen and it had the characteristic

18-dihydroxy-DOC

utes were found

were performed.

was dried

system of benzene

H radioactive

of l&,

chromatographies

18-dihydroxy-DOC

chromatographic and

TEEOXDl

s

in Table

or a dimer of the compound.

was oxidized

of the oxidized

in Table

1 was assumed to be either

with

periodic

acid

compound vs 16a,

In order

in an attempt

an iso-

to deterto make a

18-dihydroxy-DOC

(A)

3. DISCUSSION

In order

to continue

research

into

the role of 16a,

human and experimental

hypertension,

quantities

than were possible

renals,

of the steroid the only

other

16a,

18-dihydroxy-DOC

DOC

of high specific

enus to hydroxylate Two

products

method

yet reported.

and microcurie activity

through

quantities

of fermentation

available were

18-dihydroxy-DOC

necessary incubation

to synthesize with

Mil I igram quantities

were synthesized

commercially

was shown to be 16a,

it was found

18-dihydroxy-DOC

18-OH-DOC

isolated. by TLC

larger

human ad-

of unlabel led

of 1,2-3H-16a,

successfully

in

using 5.

18-dihydroxyroseochromog-

and 1 ,2-3H-18-0H-DOC.

One was made in high yield and paper chromatography,

and deriv-

S ative

formation The

ative that

and

Several

fermentation

(8,

Dominguez periodic

can perform 15).

16a,

(18) and called

acid,

it formed

the only

on steroids

a dimer

lactone.

formed

Mobilities

and yields

roseochromogenus. water

(70

that

lacking

by Goodman

with As this

After

that

with

extracted

oxidation

compound

“L”

by

with to

of the

is a dimer

of

of the fermentation.

after

dichloromethane

of

mobility

to the behavior

the acid conditions

of steroids

form

chromatographic

this

is

(14) or a com-

designated

is analogous

we feel

under

TLC

identical

function

the second product

and Smith

(17).

by deriv-

transformation

a 17-oxygen

assumed that

by Birmingham

of the dimer of 18-OH-DOC,

1.

have found

form of 18-OH-DOC

a compound

18-dihydroxy-DOC

Table

was investigated

It was therefore

to the less polar

18-dihydroxy-DOC

lactone

of fermentation

was an isomer as described

pound analogous

160,

investigators

roseochromogenus

lQ-hydroxylation

703

.

GLC

of the second product

identity

formation. S. -

~EEOTDI

fermentotion

with

S.

: acetone : methanoT :

: 25 : 5 : 0.7).

Steroid

R18-OH-DOC

16a,

18-dihydroxy-

DOC

lb,

18-dihydroxy-DOC

% Conversion

_Yield

0.41

29%

58

0.76

13%

25

9%

17

dimer 18-OH-DOC Table

2.

1 .oo Comparison di hydroxy-

A.

Whatman

#l

of mobilities DOC

(A)

of l&I,

18-dihydroxy-DOC

(F) and 16o,

D

paper in chloroform

Steroid

: acetone : water (2 : 1 : 2) R6fj-OH-E

6p-OH-E

1 .oo

I&,

18-dihydroxy-DOC

(F)

0.99

lb,

18-dihydroxy-DOC

(A)

0.98

18-

s

704 TLC

6.

benzene

: ethanol

(9

TmEOXDI

: 1)

Steroid

R1 8-OH-

lc&,

18-dihydroxy-DOC

lactone

(F)

0.75

16a,

18-dihydroxy-DOC

lactone

(A)

0.75

18-OH-DOC Whatman

C.

lactone #1 paper in benzene

: cyclohexane

RF11

Dye

16a,

18-dihydroxy-DOC

lactone

(F)

0.38

18-dihydroxy-DOC

lactone

(A)

0.38

dye

1 .oo

Gas-liquid

chromatography

Steroid

Retention

18-dihydroxy-

IQ, 16a, Table

: water (5 : 3 : 5 : 1)

: methanol

16a, Fll

DOC

18-dihydroxy-DOC Comparison

3.

TLC

in benzene

TMSE

(F)

7.2

lactone

TMSE

(A)

7.2

of lactones

of 16a,

18-dihydroxy-DOC

: ethanol

(9

during

(A)

fermentation.

: 1). R18-0H-DOC

18-dihydroxy-DOC

(min).

18-dihydroxy-DOC

found

Steroid

Oxidized

time

lactone

of mobilities

and dimer of lQ,

16at

lactone

1 .oo

Compound

D.

DOC

lactone

0.76

lactone

0.75

dimer ACKNOWLEDGEMENTS

This

work

was supported

07002-01,

PO 2-AM-08657-11

of Health,

Bethesda,

in part by Grants-in-Aid and ROl-HL15732-03

AM-l

2027-07,

TO

from the National

1 -AMInstitutes

Maryland. REFERENCES

1.

Steroid

Nomenclature:

18-hydroxydeoxycorticosterone 18,

lb,

18-dihydroxy-DOC

l&c-hydroxy-DOC

21 dihydroxypreg-4-ene-3,

= 16a,

(16a-OH-DOC)

18,

(18-OH-

21-trihydroxypreg-4-ene-3, = lc&,

21-dihydroxy-preg-4-ene-3,

20-dione deoxycorticosterone

(DOC)

= 21-hydroxypreg-4-ene-3,

DOC)

=

20-dione

20-dione

20-dione

s 9a-fluoroprednisolone

70.5

TPEOXD-

= 9a-fluoro-118, 3,

9a-fiuoro-hydrocortisone

17a,

= 9a-fluoro-1 3,

,Cpregnadiene-

l&hydroxypregnenolone

lB,

17a,

21-trihydroxypregn-4-ene-

20-dione

9a~fiuoro-lo-hydroxyprednisolone

= 3@,

= 9a-fluoro-llB,

16a,

hydroxy-1

, 4-pregnadiene-3,

17a,

21-tetra20-dione

l&dihydroxypregn-y-ene-2O-ane 17a, 21-trihydroxy-pregn-4-ene-3,

@-OH-E)

6@-hydroxycortisone

21-trihydroxy-I

20-dione

c 6@,

H,

20-trione 18-hydroxydeoxycorticosterone

lactone

(18-OH-DOC

androsten-178, 16a,

18-dihydroxydeoxycorticosterone

lactone

(lb,

18-Di-OH-DOC

lactone

iactone)

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