Advances in bioconversion of anthracycline antibiotics

Biotech. Adv. Vol. 7. pp. 215-239, 1989

0734-9750/89 $O.OO + .50 ~ 1989 Maxwell Pergamon Macmillan plc

Printed in Great Britain. All Rights Reserved

A D V A N C E S IN B I O C O N V E R S I O N OF ANTHRACYCLINE ANTIBIOTICS* U. GRAFE, K. DORNBERGER, C. WAGNER and K. ECKARDT C e n t r a l Institute

of Microbiology a n d Experimental Therapy, GDH Academy of Sciences, P.O. Box 73, lena 6900, G.D.R.

ABSTRACT During

the

tential

last decade

usefulness

in c a n c e r

new microbial

strains

les

cells

employing

bioconversion late

new anthracycline-type treatment

or e n z y m e s .

transformations

have been

a n d by b i o c o n v e r s i o n s

of a n t h r a c y c l i n e

structures

s u c h as are

supplied

of p r e c u r s o r

We h i g h l i g h t structures

w i t h po-

recent

b o t h by molecu-

advances

w i t h the m a i n

in

f o c u s on

c a r r i e d o u t by o x i d o r e d u c t a s e s .

KEYWORDS

Anthracycline oxidations lations,

antibiotics,

and reductions,

methylations,

bioconversions

by cells

glycosylations,

and enzymes,

reductive

deglycosy-

acylations. INTRODUCTION

The anthracyclines related tes.

form a still

antibiotics

mostly

Some representatives

position

in a n t i c a n c e r

unomycin

and adriamycin

modifications Both state

of

les h a v e

the art been

The continuing fies

leading

the b i o s y n t h e s i s

of t h i s

have

to m o r e

family occupy

been

potent

in b i o c o n v e r s i o n s

more

(1,2,3).

subjected and

of a n t h r a c y c l i n e s

development

f a m i l y of

from cultures

chemotherapy

excellently

highlighting

growing

derived

in the

an o u t s t a n d i n g

In p a r t i c u l a r , so e x t e n s i v e

less (4,5)

toxic

field

findings

derivatives.

215

reviewed

molecu(6,7,8,9).

of a n t h r a c y c l i n e s and evaluating

*To Prof. Dr. F. Bergter on the occasion of his 65th

da-

chemical

a n d the p r e s e n t

of a n t h r a c y c l i n e - t y p e

and comprehensively

recent

structurally of a c t i n o m y c e -

birthday

them

justiin

U. GRAFE etal.

216

comparison

with

Referring

cyclines.

deal

evidence.

pattern

with

Particular

role

cycline

foregoing

to the b a s i c

preferentially

their

the

of b i o s y n t h e s i s ,

late m i c r o b i a l

reference

in r e d u c t i v e

the

cyclines

first and have

tetracyclic

description

Bauer been

aglycone

revealed

with

one p r o p i o n a t e

15,16),

cinones,

acid,

known

The

that

formations Negative

the

one hand,

from

Thus, only

aglycone

and

has

IU-222

been

in the

This

case

the

late

lation

and

splitting

release

the

through

absence

to o t h e r of

supported

mutants

classes the

were

pathway off

of any

a series

5,11, and

of c o n v e r s i o n

of

on the other, precedes

musug-

trans(14).

by S t r e p t o m y c e s

view The

that • - r h o d o m y same

picture

has

~riseus

mu-

found

belonging grown

to be c o s y n t h e -

to d i f f e r e n t

in m i x e d

culture

~on

of a so far u n i d e n t i f i e d

for a s e r i e s catalyzing

the m e t h o x y c a r b o n y l a glycosylated

i;

Streptomyces

were

of d a u n o m y c i n s

(Fig.

daof

7-O-rhodosaminyl-E-rhodo-

(14).

presence

is r e s p o n s i b l e

of

B-pyrromy-

anthracyclines

the

with

i;

many

by b a u m y c i n - n e g a t i v e

MEI30-A4

glycosides

intermedi(Fig.

from e-rhodomycinone

c-rhodomycinone

studies

where

indicated

which

c- and

microorganisms

glycosylated

cosynthetic

during

comitant

of

as

of

H-isorhodomycinones,

and b a u m y c i n s

leading

daunomycinone

complex

but

early

from w h i c h

7-O-daunosaminylaklavinone

rhodomycinone-producing media.

and

coeruleorubidus

mutant

in fact,

drawn

e-

such

stu-

starting

via a s e r i e s

aklaviketone

are d e r i v e d

in the b i o c o n v e r s i o n

aklavin

coeruleorubidus

solid

aglycones

pathway

leads

aklavinone,

at the

Biosynthetic

detected

and

of d a u n o m y c i n

glycosylation

the

results

mycinone,

enzymic

recently

the p r o d u c i n g

to d a u n o m y c i n

at

units

anthra-

substituents

moieties.

ester

intermediate,

of S t r e p t o m y c e s

sized

three

its m e t h y l

within

in the

in 1 9 5 0 by

occurring

the o l i g o k e t i d e

acetate

adriamycinone

on the

daunomycinone

tants.

and

of a n t h r a -

published

150 n a t u r a l l y

sugar

that

nine

biosynthesis

aklavinone,

cinone,

in the

anthracycline

and

bioconversions 12,13).

than

e ,B,~, ~ - r h o d o m y c i n o n e s ,

unomycinone,

been

and

to a u n i q u e

other

and

including

aklanonic

gested

conversions

OF A N T H R A C Y C L I N E S

differing

unambiguously

transformations

tants

and

of a n t h r a c y c l i n e s

(iO) m o r e isolated,

dies

the

of a n t h r a -

to o x i d o r e d u c t a s e s

deglycosylation

will

trisaccharides.

Brockmann

ates

review

bioconversions

is g i v e n

BIOSYNTHESIS

After

this

of c o n v e r s i o n s both

groups,

intermediate

glycosy-

without (17).

con-

ch

co

r,o

(3 0

0

0

c~

(3

o o~ ~<

\

~X

~ OH 0

""

(or tautomeric forms)

o ,onon,c oc,0

~ via methyl ester 0 ~1~ COOMe

OH 0

/

decaketide

~" ~ L [ I /1 J " O H ~ ~ pyrrom,cmon~,~ X ~HT~OH~ "~ \ n alw COOHe a__driamycinone ~ daunfmycinone ~- rhodomycinone ~ ~ "'OH cklavincne .._carmiAnom),cinone. ~_isorhVodom),cinone~~h.l~H ~) 6H OH i3 jsorh'~'od0mycinone 10-demethoxycarbonyI-Erhodomycinone v • -~2- rhodomycinone _j3- rhodomycinone V ~- rhodomycinone

(3-Pyrrom~cinonex

a ,ov,ooo,

4"

1 propionyl-CoA 9 malonyl- CoA

COOCoA

N

N

Z

Z

> Z -]

Z ©

© Z

¢0

218

U. G R A F E et al.

Variations enzyme occur such

of

the scheme depicted

A i n s t e a d of p r o p i o n y l in t h e b i o s y n t h e s i s

as n o g a l a m y c i n s

Until

now,

sugars

tives

Possibly,

amine,

the starting

of a c l a c i n o m y c i n has been

tially

t i o n by a s p e c i f i c

to L - c i n e r u l o s e

to a c l a c i n o m y c i n s

containing

other

the pertinent

pyrromycinones) thracycline sugars

A and,

(Fig.

2;

the

deriva-

in turn,

19).

Similarly,

formed

Other

either of

out w i t h

ini-

2-deoxy-L-fu-

sugar

oxida(L-rhodi-

and L-cinerulose trisaccharides

by g l y c o s y l a t i o n

the a g l y c o n e

during

an-

or a c y l a t i o n s

the g l y c o s i d e s

(18),

information

of

(e.g. c -

late conversions

but d e t a i l e d

triyiel-

SI)

Subsequent

to L - a c u l o s e

s u c h as m e t h y l a t i o n s

free sugars,

(MA 144

could

In the

of a k l a v i n o n e

of t h e t e r m i n a l

a n d B.

are

m a y a l s o be c a r r i e d

than with

(12,18,19).

under-

L-daunos-

conversions.

disaccharide

or o x i d a t i o n

biosynthesis

like

(5) which,

formation

later,

A, Y,

aglycones

aglycones

nucleoside

sugars

of L - r h o d o s a m i n e ,

oxidoreductase

B led

with

been

to be a s t e p by s t e p p r o c e d u r e

composed

nose)

18 d i f f e r e n t

less w e l l

for a d d i t i o n a l

(MA 144 NI)

co-

block

antibiotics

so far k n o w n has

yield

(aklavin),

a trisaccharide

the

starting

biosynthesis

and L-rhodinose

acetyl

building

(7).

and L-rhodinose

shown

via monosaccharide

cose,

of

might

material

1 involving initial

anthracycline-type

aglycones

transformations

2-deoxy-L-fucose,

saccharides ding

pathway

to a n t h r a c y c l i n e

of g l u c o s e - 6 - p h o s p h a t e

provide case

of o t h e r

steffimycins

the b i o g e n e t i c

bound

stood.

and

in Fig.

C o A as the

of

rather

is not y e t

available. EARLY Much

w o r k has

quence netic The

of

depicted

or t h e r e a f t e r

tetracyclic

the

system,

reader

or s u g a r s ,

of a n t h r a c y c l i n e 3 summarizes

i;

15,16,88).

giving

are

and even

activity.

antibiotics

Transformations

details

cannot

to e a r l i e r

reactions

structural

be g r o u p e d

starting

diversity

be g i v e n

of

here,

(4,5,6,7).

of

reducing as

pro-

of the

to b i o l o g i c a l l y

changes

to d e c o m p o s i t i o n s

They could

parts

reviews

leading

i).

so far

the b i o s y n t h e t i c

at d i f f e r e n t

se-

bioge-

(Fig.

bioconversions

r i s e to the n a t u r a l

Further

to p a r t i c u l a r

the p r o p e r

a n d the g e n e r a l

throughout

oxidations

m a y be r e f e r r e d

transformations

biological

thus

structure.

anthracyclines, nes

(Fig.

BIOSYNTHESIS

to e x p l o r e

biosynthesis

occurring

involve

ANTHRACYCLINE

in the past,

in Fig.

at t h e a g l y c o n e

anthracycline

Late

spent,

in a g l y c o n e

interrelationship

with aklavinone

and

BIOCONVERSIONSIN

been

events

scheme

known cess

AND LATE

their

active aglyco-

or d e l e t i n g

follows:

BIOCONVERSION OF ANTHRACYCLINE ANTIBIOTICS

~./ Akn- Rhn

~///"

219

yHL-rhodosamine H (Rhn) NMe-2

k / ~ 2OH-L-deoxyf ucose H~)O~H (dFuc)

Akn-Rhn-dFuc

~

~H L_rhodinos e

H

(Rho) Akn-Rhn-dFuc-Rho (MAI44NI)

Akn-Rhn-dFuc- cinerulose A {aclacinornycinA)

!

Akn- Rhn-dFuc-L-aculose(aclacinomycin Y )

~k

~L formationof ether linkage Akn-Rhn-dFuc-cinerulose B (aclacinomycin B)

Fig.

2.

Terminal

steps

of a c l a c i n o m y c i n

by S t r e p t o m y c e s - glycosylations

galilaeus

1

H2

biosynthesis

(17,18)

of a n t h r a c y c l i n o n e s

- oxidations

and

- reductions

of a n t h r a c y c l i n o n e s

- methylations - acylations - oxidations

and

reductions

GLYCOSYLATIONS Investigations

synthesized

confirm

the p r o p o s e d

through

mutant

directed

components

OF A N T H R A C Y C L I N O N E S

into glycosylation

mically

of b l o c k e d

of s u g a r

of n a t u r a l l y

anthracyclinones biosynthetic

strains)

have

pattern

or to i s o l a t e

biosynthesis,

BY I N T A C T C E L L S occurring

been performed (e.g.

either

to

by c o m p l e m e n t a t i o n

new unusual

mutasynthesis,

or c h e -

structures

and hybrid

biosyn-

220

U.GRAFE etal. -demethylation, decarboxylation, !/ oxidation -oxidation

1 ~ J~ C O ~ y Ja t i o n

-oxidation /

COOMe

- methylation 1 ~ '

-oxidation

~

' ~-

- g lucu ronida tion - glucosylation

-deglycosylation, reduction

--~r"~ _gl ycosylat ion s, - ~condory transformations of sugars 0 ~-reduction / II -glycosylation of 13-dihydro

~'i@

-daunomycinone

deri~tive

~

~idation

partial structure

6. Fig.

3.

Biosynthetic cyclinone

thesis

(20).

are only briefly

cells

of b l o c k e d

of

the tetracyclic

CIO,

been tes

of mono,

observed

the g l y c o s y l a t i o n s

strains

nucleus,

and C13 hydroxy

formation

of

mentioned

mutant

similar

have been

while disaccharides

so far r e p o r t e d ,

have been

converting

but

groups

here,

trisaccharides

in t h e b i o s y n t h e s i s

of the a n t h r a -

skeleton

The majority

which

modifications

carried

out by

the C T - h y d r o x y

glycosylations

established.

group

of C4,

In g e n e r a l ,

and even pentasaccharides have

been

shown

of t h e t r i s a c c h a r i d e s

as

(Fig.

has

intermedia2;

18}.

C7-O-glycosides Observed

monoglycosylation

nes b y a g l y c o n e - n e g a t i v e biosynthetic matic

and other cones

Feeding

strains

of a n t h r a c y c l i n e s by

Streptomyces

s u c h as

mycinone

idiotrophic spp.

have

E-rhodomycinone,

sugars

as s h o w n

strains

been

of

confirmed

of

reported

~-pyrromycinone,

fed a g l y c o the o v e r a l l

in Fig. S.

i. E n z y -

coeruleorubidus

for v a r i o u s

agly-

and e-isorhodo-

(20,21). of

synthetic lines

pathway

glycosylations

with different

mutants

aglycones pathway

characteristic

and

adriamycin,

the

fed a g l y c o n e

which

occupy

to b l o c k e d of

the p a r e n t a l

or m o d i f i e d (e.g.

an e a r l i e r

mutants

yielded strain

glycosides

due

isorhodomycinone,

position either

in the b i o -

anthracyc-

s u c h as d a u n o m y c i n to t r a n s f o r m a t i o n s

see b e l o w ) .

However

of

BIOCONVERSION OF ANTHRACYCLINE ANTIBIOTICS

there

may

be s t r a i n - s p e c i f i c

missing

glycosylation

mutants

of

S. c o e r u l e o r u b i d u s

as E - p y r r o m y c i n o n e genera, fied was

e.g.

were

noticed

procedure,

qoeruleorubidus

E-pyrromycinone

dihydrodaunomycin

from

may

glycosides,

recognize been

provided

by

that

daunosaminyl modified

have

employed

lonolide, biotic

(as an

specificity.

to y i e l d the

by d e s o s a m i n e ,

has

sp.

Further,

appear

yielded

naturally

peucetius In the grown

to p o s s e ~

et al.

(30)

vat.

car-

case

of

in the

biosynthesis) rather

fed e.g.

of

followed

as a p o s s i b l e

anti-

Streptomyces

protylonolide was

broad-

proty-

of the m a c r o l i d e

mutant

biosynthesis

protylonolide

in the

which

(24).

precursor

of h y b r i d

of the a d d e d

could

13-deoxycarminomycinone

23-hydroxy-desosaminyl

product

machinery

of p o l y k e t i d e

a blocked

me-

possibility

s_p_. K A - 4 6 4

inhibitor

from

glycosylation

(I).

formed

via C 2 3 -

by s u b s e q u e n t

intermediate

of d a u n o s -

biosynthesis.

Otherwise, and

(29). as

antibiotics

aglycone

(obtained

This

of S t r e p t o m y c e s

Sadakane

hydroxylation

amine

such

isolated

by c h e m i c a l

B-isorhodomycinone

Streptomyces

a 16-membered

Obviously,

to

to new a n t h r a c y c -

of a S t r e p t o m y c e s

derivatives

by m u t a n t s

aglycones

access

structure.

glycosyltransferase(s)

tylosin

fradiae),

or

new a n t i t u m o r

of c e r u l e n i n

substrate

mu-

to l - h y d r o x y - 1 3 -

the g l y c o s y l a t i n g

aglycones

the d a u n o m y c i n - p r o d u c i n g

range

employed

biosynthesis

or s y n t h e s i z e d

fermentation

but

the p e r t i n e n t

(21)

of u n u s u a l

for g e t t i n g

unusual

the p e r t i n e n t

to y i e l d

et al.

in d a u n o m y c i n

(23,24,25,26)

occurring

presence

and

of m o d i -

N-formyl-l-hydroxy-13-dihydrodaunomycin.

of ~ - c i t r o m y c i n o n e

been

such

species

formation

E-isorhodomycinone

be u s e f u l

demonstrated

minatus

aglycones

of o t h e r

MuWI,

Yoshimoto

blocked

and

the p e r t i n e n t

presence

when

by

through

l-hydroxy-ll-deoxy-carminomycin

glycosylations

mutants

(27,28)

line

and

enzymatic

blocked

thods

Otherwise,

to c u l t u r e s

as

instance,

(22).

of S,

In future,

such

for

~rhodomycinone

roseoviolacea

structures

In a m u t a s y n t h e t i c

convert

shown,

and

(20).

supplied

Actinomadura

glycoside

Cants

differences

of a k l a v i n o n e

221

mutations

aglycone

cosides

could

could

formation

alter

in s u c h

be o b t a i n e d .

The

the

intensely

(~-L-Daunosaminyl)-B-rhodomycinone) from

cultures

myces

strain

Barminomycins cinone

of m u t a n t D788 (32)

obtained

specifity

a manner

OXA-1874

(II)

that

potent was

of g l y c o s y l a t i o n

new u n u s u a l

monogly-

oxaunomycin

recently

(7-0-

isolated

of a b a u m y c i n - p r o d u c i n g

Strepto-

(31). and

unusual

by c u l t i v a t i o n

glycoside of

derivatives

S. p e u c e t i u s

of d a u n o m y -

in p r e s e n c e

of

222

U. GRAFEet al. NHe 2

~

:

HO ---0 ~ 0

---

CH20 H thio-

and

newly

discovered

I

fluorobarbituric

acid

anthracyclines

0

(33)

are a d d i t i o n a l

from m i c r o b i a l

OH

examples

of

sources.

OH

"'OH it

OH 7-O-Triqlycosides Aklavinone

and e - p y r r o m y c i n o n e

trisaccharides

mycins

cinerubins

formed

and

cosides by Oki

though et al.

C34)

a step (18)

by s t e p

several

by a p i g m e n t - n e g a t i v e aclacinomycins he,

(34)~

with chain

growth

different

mutant

of

Aglycones

~-isorhodomycinone,

by m u t a n t

are

e- and

MAI44-MI-KE303

from of

the

sugar

as ~ -,

glycosylation of e x p e r i m e n t s galilaeus,

Surprisingly,

were

the p e r t i n e n t

feeding

at C I O p o s i t i o n with

blocked

the h y b r i d

(35)

antibiotics

of

CGIO

As s h o w n

were

formed

transformed

A as sugar led

In a n o t h e r

S. c o e r u l e o r u b i d u s and

of

triglycosides

of y - r h o d o m y c i n o n e

(see below).

mutants

chain.

B- and e - r h o d o m y c i n o -

L-rhodosaminyl-2-deoxy-L-fucosyl-L-cinerulose (35,36).

monogly-

as a p r o d u c e r

B-pyrromycinone

to y i e l d

as a c l a c i n o -

pertinent

trisaccharides

S. g a l i l a e u s such

such

the

CGII

to series

and 2.

7-O-(L-rhodos-

aminyl-2-deoxyiL-fucosyl-L-cinerosyl. A)-B-isorhodomycinone

and

BIOCONVERSION OF ANTHRACYCLINE ANTIBIOTICS

-~2-rhodomycinone ever,

feeding

cinone,

and

obtained

and

strain,

KE303,

4;

mutant

not

yield

trisarubicinol

aglycones

qalilaeus

such

added

carsame

was

triglycosides

obtained

(a p i g m e n t - l e s s

strain)

able

was

auramycinone,

to c o n v e r t

sulfurmyci-

and

e-pyrro-

ll-hydroxysulfurmycinone

containing

sugar

chains

either

of L - r h o d o s a m i n e - 2 - d e o x y - L - f u c o s e - L - c i n e r u l o s e

L-rhodosamino-2-deoxy-L-fucose-L-cinerulose

B (Fig.

Feeding

mutant

laeus

When

to the

l-hydroxysulfurmycinone,

ll-hydroxy-auramycinone,

to the p e r t i n e n t composed

adriamy-

glycoside.

were

of

2-hydroxyaklavinone

MAI44-MI

yielded,

to a b l o c k e d

quite

How-

OBB-III-838

as a k l a v i n o n e ,

l-hydroxy-auramycinone,

mycinone,

any

A)-13-dihy~rocarminomycinone)

Streptomyces

223

(37).

(7-O-(L-rhodosaminyl-2-deoxy-

of an a c l a c i n o m y c i n - p r o d u c i n g

several ~one,

38).

et al.

13-deoxodaunomycinone,

did

13-dihydrocarminomycinone

L-fucosyl-L-cinerosyl (Fig.

by Y o s h i m o t o

of d a u n o m y c i n o n e ,

steffimycinone

minomycinone mutant

were

similar,

A or of

4;

28/39).

of ~.

gali-

2-hydroxyaclacinomycin

A

(40).

If c h e m i c a l l y occurred IOR

synthesized

solely

with

configuration.

specificity 111-848, tion:

type

Despite

and

the

were

not

cinerubins

production

strains.

For

by S t r e p t o m y c e s

aklavinone

possessing

the a p p a r e n t

chemically

Ro-22-8507)

gesting

racemic isomer

of g l y c o s y l a t i n g

several

cinomycins

the

enzyme(s)

linones

instance,

the k e s a r i r h o d i n s DSM

2658

cer

cell

lines

been

(L1210,

P388

A and

etc.),

excep-

to a c l a sug-

by w i l d -

B produced

of

13-deoxy-

containing

other

( $-rhodomycinone-7-O-(rho-

described

in v i t r o

OBB-

7-O-L-rhodosaminyl-2-

a trisaccharide

trisaccharide

promising

so.

information

A or B d e r i v a t i v e s

Further,

new

are

9R,

substrate

In a d d i t i o n

trisaccharides

previously

possess

(28).

7S,

(with one

recent

purpurascens

these

Streptomyces

is m o r e

with e-rhodomycinone

of

of

natural

sugars

some

range

there

(41).

Though

conversion

of o t h e r

carminomycinone

d i n o s e ) 3) has

fed,

natural

aglycones

glycosylated

deoxy-b-fucosyl-L-cinerulosyl

combined

broad of

synthesized

was

the

(42). derivatives

activities so

of a n t h r a c y c -

against

far n o n e h a v e

some

been

can-

employ-

ed t h e r a p e u t i c a l l y . CiO-O-glycosides An

interesting

CiO-O-monoglycoside

aminyl)-y-rhodomycinone) specific

recombinant

iremyceticus

(III)

because

strain (43,44).

is i r e m y c i n of

its

of S t r e p t o m y c e s Other

rare

(iO-O-(L-rhodos-

isolation

cases

from

violaceus in w h i c h

an

inter-

subsD. only

C10-

224

U. GRAFE et al,

%

.0

I

~I0

i

11

0

R1

Rg

)H

R4

R7

R9

RIO

RII

RI2

aklavinone

H

OH

OH

Et

COOMe

H

H

B-rhodomycinone

H

OH

OH

Et

OH

OH

H H

e-rhodomycinone

H

OH

OH

Et

COOMe

OH

e-isorhodomycinone

OH

OH

OH

~%

COOMe

OH

H

c-pyrromycinone

OH

OH

OH

Et

COOMe

H

H H

~-pyrromycinone

OH

OH

OH

Et

OH

H

B-isorhodomycinone

OH

OH

OH

Et

OH

OH

H

~2-rhodomycinone

OH

OH

OH

Et

OH

OH

H

carminomycinone

H

OH

OH

COMe

H

H

H OH

2-OH-aklavinone

H

OH

OH

Et

COOMe

H

auramycinone

H

OH

OH

Me

COOMe

H

H

sulfurmycinone

H

OH

OH

CH2COMe

COOMe

H

H

l-OH-auramycinone

OH

OH

OH

Me

COOMe

H

H

l-OH-sulfurmycinone

OH

OH

OH

CH2COMe

COOMe

H

H

ll-OH-auramycinone

H

OH

OH

Me

COOMe

OH

H

ll-OH-sulfurmycinone

H

OH

OH

CH2COMe

OGOMe

OH

H

Fig.

4.

7-O-Triglycosylation mutants

of 2.

of a g l y c o n s

qalilaeus

and 2.

by b l o c k e d

coeruleorubidus

225

BIOCONVERSION OF ANTHRACYCLINE ANTIBIOTICS

OH

0

glycosides

have

been

OH

formed

0

are CG8

compound

(iO-O-(L-rhodos-

aminyl-2-deoxy-L-fucosyl-L-cinerosyl

A)-Y-rhodomycinone)

tained

to a b l o c k e d

by

feeding

galilaeus

(18)

of y - r h o d o m y c i n o n e

and

the

naturally

occurring

ob-

mutant

cosmomycins

of 2.

A

(iO-O-

(L-rhodosaminyl-L-rhodinosyl-L-rhodinosyl)-y-rhodomycinone)

and

B (iO-O-(L-rhodosaminyl-2-deoxy-L-fucosyl-L-rhodinosyl)-Y-rhodomycinone)

(45,46).

(For

the

occurrence

of y - r h o d o m y c i n o n e

see M a t s u z a w a

Serirubicins

ditrisarubicins

MG344-hF49, different

and

several

from

in

(47)

moieties

chains

A447

both

aglycones.

as a g l y c o n e s

~ I~

while

cyaneus

cyaneus

SD.

representing

see

C and

IV) D

(48)

(49)

the

from ~.

are

complexes

containing

either

at 7-0 and

iO-O positions

Thus,

contain ~ -citromycinone

Streptomyces

(i) c.f.)

from ~.

formula

hexasaccharides

anthracycline

respectively A447

of s u g a r

trisaccharide

droxyserirubicin

cited

(general

D S M and a n t i b i o t i c s

of d i f f e r e n t

or m o d i f i e d of

cytorhodins

combinations

purpurascens composed

(47),

of a i O - O - p e n t a g ~ y c o s i d e

serirubicin

identical

and

l-hy-

and ~ 2 - r h o d o m y c i n o n e

cytorhodins,

ditrisarubicin,

are d e r i v a t i v e s

of

and

B-rhodomycinone

'"OH

R1.R2=sugarl-sugar2-sug or3 $ugarl"Rhn s u g a r 2 : d F u c or Rho sugar3" Rho or CinAor Acu or Cin B

228

U. GRAFE et al.

(49).

The

cosmocarcins

biosynthetic cells

studies

or e n z y m e s

possesssimilar

on e n z y m a t i c

have

not

been

structures

(50).

Up to now,

ClO-O-glycosylations

by e n t i r e

reported.

C4-O-glycosides Recently,

a new

class

of b i o s y n t h e t i c

of e - r h o d o m y c i n o n e

and

et al.

of

as p r o d u c t s

Earlier, lacea

by way

MuWI

succeeded

none

(V).

e-glycosides, compounds. too,

the

of

that

transformation

same

routes

(glucuronides

been

by C a s s i n e l l i

described

peucetius

employing

vat.

castaneus

Actinomadura

e-rhodomycinone,

known

or by

compounds

appears

anthracyclines

(51).

roseovio-

Nakagawa

et al.

4-O-(B-D-glucopyranosyl)-e-rhodomyci-

previously

new

It thus

the

cyclines

the

fermentations

microbial

ciple,

Streptomyces

in i s o l a t i n g

Unlike from

has

of g l u c o s y l a t i o n

for b i o c o n v e r s i o n

(52)

isolated

aklavinone

anthracyclinone

feeding

were

found

with

respect

which

are

to be B - g l y c o s i d i c to 4 - O - g l y c o s y l a t i o n ,

of a n t h r a c y c l i n e s

as d e s c r i b e d

glycosides

experiments,

follows,

for t r a n s f o r m a t i o n

in p r i n -

of a n t h r a -

in m a m m a l s .

_

R2

H H '~O

~

0

0

%

~00Me

OH

OH

RI:HOrOH OH

R2= COOH or CH20H

vE Cl3-O-glycosides Recently, ted

to

4-demethoxydaunomycinone

of S.

peucetius

A~uga

reptans

were

found

cinone

way

The cell

(M-99-FCE)

L (labiateae)

(53).

and

Daucus

latter

example

cultures

above

Similarly, carota

reduce

were

conver-

by a m u t a n t

L.

plant

strain

cells

of

(Umbr@lliferae)

and g l y c o s y l a t e

daunomy-

13(S)-dihydro-O(B-D-glycopyranosyl)-daunomycinone

of b i o c o n v e r s i o n

scribed

(VI)

to s t e r e o s p e c i f i c a l l y

to y i e l d

(VII). plant

and d a u n o m y c i n o n e

13-(S)-dihydro-13-O-B-D-glucopyranosides

illustrates

for the p r o d u c t i o n (54).

In a n a l o g y

the C l 3 - O - g l y c o s i d e s

are

nicely

the p o t e n t i a l

of

of new a n t h r a c y c l i n e s to the C 4 - O - d e r i v a t i v e s B-glycosides.

by de-

BIOCONVERSION OF ANTHRACYCLINE ANTIBIOTICS

OH

0

227

0

""OH

Ivie~

0

O, R

o.

R=h.leorH

6 'JT'

~

o"

"OH

ONe 0

OH

OH

VI_/I Other The

glycosylations

newly

discovered

cilorubicin) galamine

moiety

7-O-bonded enzymatic

An e a r l y

of

methylation

cosides

similar

common

structures bound

aglycone,

type

sugar

to the

occurrence

of an

(55).

anthracyclinones ester

is a late

instance,

and

adriamycin

step

during (Fig.

in d a u n o conversion i;

56). of

or a d r i a m y c i n

4-O-demethyl-ll-desoxyadriamycin

is the

(15,16,17).

or by a d d i t i o n

to d a u n o m y c i n

4-O-demethyldaunomycinone

no-

in a d d i t i o n

the

for

in m u t a n t s

de-

moieties

to m o s t

group

(e.g.

to b i c y c l i c

of g l y c o s y l a t i o n

to its m e t h y l

to d a u n o m y c i n blocked

chain

suggesting

this and

acid

or s u l p h a n i l a m i d e

strains, of

out

occurring,

was

the

chain

the C 4 - h y d r o x y

carminomycin

ducing

step

of a k l a n o n i c

biosynthesis

ethionine

C2 of

aglycone

methylation

Methylation

of

carrying

at the

and

a trisaccharide

at C1 and

system

conversion

When

arugomycin

tetrasaccharide

Methylations

mycin

contain

(57)

(carminomycinone)

pro-

and g l y -

228

U. GRAFE et 01.

(58)

were

vine

was

produced.

As a p r o o f

synthesized

via

of N - m e t h y l a t i o n

N-methylation

of sugars,

of d a u n o s a m i n y l

akla-

aklavinone

(59). Acylations

of

sugar

N-acylations (14,60) lation

have

Oxidation part

of

been may

at C12

volved

have

In some

prior

to the

cases,

so far

group

e.g.

in Fig. been

have

steps

as

to c a r b o n y l

and

subsequent

oxidation

to y i e l d

adriamycin

(doxorubicin)

a single

step

e£ al.

(1988)

adriamycin

by m u t a n t

Reductions

of

As

the

rule,

inactive

of

latter

showing

aromatic and

pathway

The

two o t h e r

(5,16)

of C l 4 - m e t h y l

published

the c o n v e r s i o n

aglycone

constituents

of a n t h r a c y c l i n e s

at the

aglycone

reactions

lead

for

recent-

of d a u n o m y c i n

(63).

to m u c h

less

as

to C14

proof

S. p e u c e t i u s

The m a i n

such

group

Final

was

the in-

of C l 3 - m e t h y l e n e

function

process

of

oxidases

In a d d i t i o n ,

(56).

strain-

C I O and CII

to

or e v e ~

are

13S-dihydroderivatives

of d a u n o m y c i n ,

carminomycin,

adriamycin

- analogous cyclines

These

reduction

agents

iO-keto

deglycosylation

transformations

mammalian

of

group

of

steffimycin-type

anthra-

(64)

- reductive

organisms,

are

of C 7 - O - b o u n d e d commonplace

involving

sugars

to m a n y m i c r o b i a l

nucleotide

coenzymes

but

also

as r e d u c i n g

(65,6,8).

Reduction

of the

cycline-producing as by o t h e r

side

chain

organisms

microbial

by c o n c o m i t a n t

have

of p a r t i c u l a r

been

of c a r b o n y l

carbonyl under

species

companied

city

(succiny-

76 of

reductions

products.

formation and

the

in the

C8,

oxidation

hydroxyl

reaction

the

biosynthetic

reported:

in £ - r h o d o m y c i n o n e

CI,

i (5,6,7,8,62).

characterized,

been

of

subsequent

at p o s i t i o n s

of the g e n e r a l

not

of o x i d a t i o n s

as well

of o x y g e n

parts

types

ly by M e r l i

sugar

O-acylations

cyclisation

nucleus

as s h o w n

in d a u n o m y c i n

of d a u n o s a m i n

aglycone

introduction

constitutive

formylation)

(61).

tetracyclic

anthracyclines

and

reported. occur

at the

the

specific are

(acetylation

e.g.)

Oxidations

moieties

is c a r r i e d

oxygen

and g e n e r a

N-acetylation assistance

reduction

(66),

(67).

to c o n f i r m

of d a u n o m y c i n

out

limitation

by a n t h r a (20)

as well

occasionally

ac-

NMR e x p e r i m e n t s the

stereospecifi-

by S. w i l l m o r i i

to y i e l d

BIOCONVERSION OF ANTHRACYCLINE ANTIBIOTICS

the

pertinent

Side the

chain

13(S)-hydroxy

carbonyl

excellent

derivative

reductions

surveys

have

of M a r s h a l l

229

(67,68).

been

reviewed

et al.

and

in d e t a i l

Fujiwara

in

and H o s h i n o

(6,7,8). Reductive

deglycosylation

aglycones

is a n o t h e r

and

tissues,

plant

of any chain

a given has

rases type

(69),

and w i t h

under

oxygen

monas

hydrophila,

capable

with

diaphorase, plant

of t r a n s f o r m a t i o n

A number

E. coli

pendent

that

from

the

the

the

feature

capacity

as well.

microbes

of a g i v e n

The

such

as Aero-

glycosides

deglycosylation

microbe

same strains

is l i k e w i s e

anthracycline

of r e d u c t i v e

c-reduc-

streptomyces

freundii

side

s u c h as

cytochrome

reductase

of o t h e r

added

7-O-sugar

flavoproteins

producing

and C i t r o b a c t e r

of d e g l y c o s y l a t i n g

suggesting

within

in m a m m a l i a n

Decomposition

containing

oxidase,

ferredoxin

7-deoxy-

both

(5,6,7,8).

mammalian

xanthin

occurs

limitation.

cells

antibiotic

demonstrated

P450,

the

of a n a e r o b i o s i s

and m i c r o b i a l

anthracycline

been

cytochrome

to g i v e p r e f e r e n t i a l l y

consequence

to form

is inde-

anthracyc-

lines. Studies

onto

the d e c o m p o s i t i o n

preparations as

intermediates

posed has

radical

been

within under

The

end of

completeness,

Redox

a specific enzymatic

full

but

also the

to the

activity

such

recovery

reference

should

the

usually

to o x i d a t i o n

as

beer.

decomposition spec.

of

the

be g i v e n

AM 33352

respiratory

prevented

to form

formed

and

ef-

7-deoxy-akla-

and d e t e r g e n t s not of

Thus,

frequently

sugar

pro-

2,4-dinitrophenol

only with the

on

regard

recovery

of

decomposition

to be a v o i d e d

of the p r o d u c e d

in or-

antibiotic.

to a k r o b o m y c i n

(73)

For as

(VIII).

moieties as m i x t u r e

of a c l a c i n o m y c i n

oxidoreductase conversion

radicals the

9,10-anhydro-13-deoxo-carminomycin

within are

with

2.

detergents

improvement

needs

maximum

microsomal free

7,7"-bis(7-deoxyaklavinone)

interesting

fermentation

of

accord

2,4-dinitrophenol seems

liver

Obviously,

of a c l a c i n o m y c i n s of

fermentation

reactions

B due

requires

effect

occurring

Aclacinomycins and

of

reaction.

of ATP p r o d u c t i o n

from

to a c h i e v e

a naturally

In full

of a c l a c i n o m y c i n - p r o d u c i n g

deglycosylation

its m e c h a n i s m

by rat

involvement

or m e m b r a n e - d i s o r i e n t i n g

anthracyclines at the

the

formation

deglycosylation

(72).

mechanism

with

70,6,7,8).

as a side

limitation

mg/l)

reductive

5;

mechanism,

Uncouplers

fectively

der

(Fig.

observed

oxygen

(60-200

vinone

compatible

the m y c e l i u m

chain.

to

were

subsequent

to a c l a c i n o m y c i n

of c o m p o n e n t s

A to a c l a c i n o m y c i n enzymatic

B (Fig.

or e v e n

2; 4 , 1 8 , 3 4 ) .

A, Y, Y by nonAs a

U. GR,~FE et al.

230

~I

C O O M e •"OH

~

u

c

2e.[e H+

-

ACN-A Cn iA

2,4-dinit rophenol

~ .....

0 0

OOMe

COOMe

"'"OH

552 COOOMeH

7- DAK N

BDAKN

Fig.

5. Reductive cleavage of 7-O-glycosides of various anthracyclinones ACM-A:

aclacinomycin

7DAKN:

7-deoxyaklavinone;

A;

BDAKN:

7.7"-bis(7-deoxyaklavinone)

BIOCONVERS1ON OF ANTHRACYCLINE ANTIBIOTICS

0

side

effect,

tion

of the

rubin

Y

specific

activity

was

oxidoreductase

A to L - a c u l o s e at

later

mogeneity

(m.w.

MAI44-MI

72000)

molecular

aclacinomycin, et al.

isolated

B. The

causes

forma-

corresponding

recently

(77)

shown

and

from

cine-

streptomyces

tail

possible

that

medium

from

cytoplasm.

strain, iOOOO

the

the

shown

been

culture

highly

lacinomycin

A has

In fact,

effective been

(76).

(48)

which

Recently,

it

to the

occurrence (78).

in the

mycelia

the

(76)

be s e c r e t e d

AM 3 3 3 5 2

localized

Aretz

the C I O ~ t r i g l y -

intracellular spec.

producing

specifically

while

could

conversion

carried

out

of a c l a c i n o m y c i n - p r o d u c i n g AM 33352

was

has

In this

supernatant higher

cultures, fractions

(80). was

The

S.

activity,

associated

were

with

inactive.

of a c l a c i n o m y c i n

by w a s h e d

qalilaeus

the

than

in

converting

aclacinomycin

versa,

localized

compartments

such

This

are

as the

finding

intra

and

supported

OBB-III

the

cells,

extracellular

and

in y o u n g

while

B and,

different

speculations

(79)

activities

A to Y and

within

B to ac-

or c o m p l e t e

a maximum

entire

Apparently,

spectively

vice

mycelia

attaining

oxidoreductases

membrane.

Streptomyces

enzyme,

oxidizes

of d i s r u p t e d

cultures

cellular

ho-

From Yoshimoto's data

for S t r e p t o m y c e s

S.

growing

of

to near

medium.

Conversely,

spec.

of

A of a c l a -

maximum

to s t r e p t o m y c e t e s

enzyme

activity

purified

the

rhodirubins

the

affected.

intramycelial

was

filtrate

the o x i d o r e d u c t a s e

g centrifugation

L-cinerulose Y attaining

of

of c y t o r h o d i n s

seemed

been

not

culture

occurrence restricted

that

coside

recently

has

the

The was

chain

the

fermentation

from

(76).

cinerubins,

have

converting

of

oxygen,

7-O-trisaccharide

the

fermentation

A and

in a c l a c i n o m y c i n

stages

galilaeus

of

during

(74,75).

cinomycin

needs

(71)

cinerubins

(pyrraculomycin)

strains The

OH

Cl-oxidation pertinent

231

subof

re-

cellular

spaces

concerning

and

the

a role

232

U. GRAFE et al.

of o x i d o r e d u c t a s e s

in the

transport

of a k l a v i n o n e

glycosides

(80). In the too,

cytorhodin-producing

Aretz

et al.

oxidoreductase lose

in b o t h

occurrence observed (see The

(48,77)

oxidizing

diversity

chains,

enzyme

was

DSM

2658,

of a p a r t i c u l a t e A and L-acu-

to L - c i n e r u l o s e

at C7 a n d CIO p o s i t i o n s .

apparently

of n a t u r a l l y

DurDl]rasc~ns

the p r e s e n c e

L-rhodinose

trisaccharide

of this

StreDtomvces showed

the m a i n

occurring

cause

cytorhodin

The

of the

structures

IV). same

authors

to s p e c i f i c a l l y

used

oxidoreductase

oxidize

f r o m S_. g a l i l a e u s

the C 7 - s u g a r

chain

ATCC

for p r o d u c t i o n

+) torhodins

U (ClO-O-rhn-rod-rod;

C7-O-rhn-dfuc-cin

B)

31133

of cy-

and V

(ClO-

+) O-rhn-rod-rod, as e.g.

C7-O-rhn-rod-acu)

cytorhodin

B

from other

cytorhodins

(ClO-O-rhn-rod-rod; C 7 - O - r h n - d f u c - c i n

fermentation broth:

such A) +).

mixture of

aclocinomycins A , Y a n d B

I chromcltography ~. aclacinomycin A

aclocinom,,cin B

conversion within the / mycelium by dich

enzymic conver sion by oxidoreductas

/silica

~

.

enzymicconversion

/ Qclacinom'

gel

ohron'~-tography

fcin

by oxidoreductase

Y

conversion by mycelium Fig.

6.

Mutual

conversions

chemical

+)

footnote:

and

rhn:

procedures

L-rhodosamine

dfuc: cinA,

of a c l a c i n o m y c i n s

enzymatic

2-deoxy-L-fucose cinB:

L-cineruloses

rod:

L-rhodinose

acu:

L-aculose

A and

B

by

BIOCONVERSION OF ANTHRACYCLINE ANTIBIOTICS

233

By use of procedures

involving

similar

enzymatic

and non-enzyma-

tic conversions,

formation

of a c l a c i n o m y c i n s

A, Y, and B has

been

achieved

(Fig.

the

on the basis

of one fermentation

(80)

6). FUTURE

PERSPECTIVES

IN B I O C O N V E R S I O N

AND RELATED The advances sight

of modern

genetics

into the structures

k e t ~ e synthetases, well.

Genetic

by s w i t c h i n g - o n triguing

picture

tics

involving

gress

in this

efficient

structures

systems

streptomycetes

will profit

and polyketide

terest

pool

the t h e r a p e u t i c

value

(84,85)

of any given

the b i o s y n t h e t i c

alternate

chemical

for anthra-

future

could

steps

by the finding

However

of

inexhaus-

the practical

will mainly

product,

without

such as tetra-

from the seemingly

procedure

Proof

and actinorhodin

In addition,

of a n t h r a c y c l i n e s

advantage

antibio(81).

future work,

antibiotics

of the m i c r o o r g a n i s m s ( 8 7 , 8 8 ) .

or

the in-

work on biosynthesis

be stimulated

antibiotics

in b i o c o n v e r s i o n

The

genetic

related

fusion

by the elaboration

(82,83).

(77,86).

could

new hybrid

has been drawn

glaucescens

coelicolor

biotransformations

gene

and t r a n s f e c t i o n

as

of creating

At the horizon,

created

from extended

from Streptomyces

anthracyclines

by h e t e r o l o g o u s

structures

in-

such as poly-

the advantage

(43,44,63).

of t r a n s f o r m a t i o n

new a n t h r a c y c l i n e - t y p e tible

either

us to gain deeper enzymes,

and modify

field has been d e m o n s t r a t e d

Streptomyces

towards

enable

of b i o s y n t h e t i c a l l y

of a n t h r a c y c l i n e cenomycin

will

will offer

genes

anthracycline

cycline-producing any doubt,

ANTIBIOTICS

and to overproduce

"silent"

OF A N T H R A C Y C L I N E S

of b i o c o n v e r t i n g

recombinations

new a n t h r a c y c l i n e

from

procedure

depend

in-

on

and on the particular offer

in relation

to

routes. REFERENCES

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'

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