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The biosynthesis of fulvic acid, a fungal metabolite of heptaketide origin
Tetrahedron Letters Vol. 27, pp 493 - 496 @Pergamon Press Ltd. 1981. Printed in Great Britain
0040-4039/f31/0201-0493$02.00/O
THE BIOSYNTHESIS OF FULVIC ACID, A FUNGAL METABOLITE OF HEPTAKETIDE ORIGIN Itsuo Kurobane and C. Richard Hutchinson* School of Pharmacy, University of Wisconsin, Madison, WI 53705, U.S.A. and Leo C. Vining Department of BioZogy, Ddhousie University, HaZifax, Nova Scotia, B3H 4J1, Canada Swmary: Analyses of the regiochemistryof [13C]acetate incorporation into fulvic acid (2) by PeniciZtiwn brefetdirmwn indicates that the metabolite is biosynthesized via a heptaketid; intermediate assembled as a single chain of seven C units rather than from two smaller polyketide chains. This information favours a route le0ding through comnon Cl4 intermediates to the three fungal metabolites, citromycetin, fulvic acid and fusarubin. Within the large class of fungal metabolites of polyketide origin, those formed from seven C2 units have been subdivided by Turner,' on the basis of structure and available biosynthetic information, into eight groups. Compounds in the first seven are logically derived from a comnon linear heptaketide intermediatewhich is made from one acetyl and six malonyl units. The different groups of end-products are postulated to arise from folding pattern variations during subsequent cyclizing reactions. Citromycetin (1) and fulvic acid (_2),the two compounds in Turner's eighth group, were distinguished from the heptaketide-basedmetabolites in that their structures could not be derived by simple linear chain folding. Birch and co-workers' showed that seven C2 units originating from [14C]acetatewere incorporated into 1 by &nicitliwn frequentans. Later, Gatenbeck and Mosbach3 found evidence for two starter units by chemical degradation of 1 labeled biosyntheticallyfrom diethyl [2-'4C]malonate and concluded that two polyketide chains contributed to the structure. A 13C NMR study of 1 labeled from [l-13C),[2-'3C]-and [1,2-13Clacetate showed a labelling pattern consistent with a two-chain origin4 but also did not exclude pathways in which a single chain is cyclized and then ring-opened to a branched intermediate such as has been postulated on several occasions5'6 to be the progenitor of 1, ,2and fusarubin (2). Two such pathways have been noted, one involving a palitantin-type'and the other a fusarubin-type'folding of the heptaketide. We are persuaded by evidence presented here for the biosynthesis heptaketide
that 1, ,2 and 3 arise from common
ketide folding
pattern
route to the 4a,lOa-dihydrofusarubins, idea has recently
been validated
Oxidative
of the indicated
fission
C,4 intermediates
(A) that gives B upon intramolecular
as shown in Fig. 1.
aldol cyclization
from which 3 is formed by non-enzymic
by labeling
experiments
with
which
a direct
oxidation.8
This
acetate.'
bond in B, followed by appropriate bond reconnections (shown
of all fungal metabolites
single linear heptaketide
The hepta-
provides
[13C]- and C2H1-labeled
later in Scheme 1) would give rise to 1 and ,2. The hypothesis for the biogenesis
of ,2 via a single-chain
is modified
containing
is attractive
because
seven C2 units as derivatives
by organism-specific
cyclizing
it accounts of a
and processing
reactions. PeniciZZizun brefeldianwn Raulin-Thom
medium.lO*ll
Dodge (NRRL 2083) produces ,2 and palitantin
Both products reach their maximum 493
yield
(_4) when grown
in
on day 5 of the fermentation.
494
citromycetin(!)
Figure
Probable
3.
Experiments precursor
with
biogenetic
fulvic acid(?I
fusarubin(3)
origin of citromycetin,
[l- 14C]acetate
fulvic acid and fusarubin.
showed that 14C enrichment
of ,2 was highest
(18-19%)
if the
was fed on days 2 and 3 whereas _4 was maximally enriched by precursor feeding on days 13 To achieve high C incorporation, therefore, we administered [l-13C]acetate and
3 and 4.
[1,2-'3C2]acetate the two-day
(each containing
periods
of maximum
90 atom % l3 C) to give 5 rnfjbroth concentrations
metabolite
The labeled
production.
products were
twice during
isolated
on
day 5. The 6c relative were assigned pyridine
to internal
comparison
on the basis of:
and their respective
in the 13C NMR spectrum
Me4Si of all signals
trimethyl
of 6c val;;s
for ,2 and anhydrofulvic ,5 and _7,inchloroform;
derivatives,
of fulvic acid acid (5)" the $H
in
coupling
relationships in these four compounds observed by gated proton irradiation (NOE) experiments; 1 13 C NMR spectra of 13C-enriched ,5 and ,7. THE NOE &c coupling data obtained from the
and
spectrum
of 2 had two triplet signals
a high-field
quartet
and a mid-field
in the upfield doublet
region;
appeared
that of _6 had only one.
in the NOE spectrum
this evidence
we could assign the 6c of C-2, C-7, C-9 and C-11 securely.
ments enabled
us to use the unique
in the spectrum
of l3 C-enriched
l*c
The 6c of C-3a and C-4 were distinguished coupling
for C-3a but the absence
could assign constants upfield
with
C-3a and with C-4.
of long-range
also labeled
of [1,2-13C2]acetate
and
per site.
Its (+)
From these data we carbon
coupling
by its characteristic
in 2 vs. 5 and 6 vs. 7.
spectral
regiochemistry
into fulvic acid by analyzing
of 2.6+0.2%
for C-4.
of a 3&H
of C-5 and of C-6 was based on the relative
6c values
The pertinent
the complete
assign-
intact C2 units
and by the presence
the value of their respective
data areshown
([1-13C]Acetate
in Figure 2.
of the incorporation of [1-13C]acetate and 13 C NMR spectra of 13C enriched 2, 7; $, the
Fulvic acid was labeled only at the (0) carbons
enrichment
for all seven
The 6c for C-12 is also defined
patterns
of 2 and ,5. From The resulting
shifts of C-2a, C-7a, C-8a and C-10.
of any long range C,H coupling
Finally assignment
C-5 and not C-6.)
We then determined
and 7.
+H
visible
by the value of 'J -C4C12
the 6c of C-3 and C-12 by noting
shift on methylation.
magnitudes
relationships
,7 to assign the chemical
Similarly,
(Fig. 2) by [l-13C]acetate
carbon pairs were labeled
with a
by [l,E-13C]acetate
C
495
Figure 2.
13C NMR data for fulvic acid and its derivatives. J values shown for 5 and ,7are +1.2 Hz and all spectra were determined at 22.5 MHz-!?t27°C.
(Fig. 2) with a l3C enrichment of 1.&0.2% per site. Since we could clearly see the small quartet resonances due to '%C
coupling between carbons in neighboring 13C2 sub-sets for at
least one carbon of each C2 unit in the 13C NMR spectrum of 5, we could calculate that the 13C enrichment of the individual acetyl/malonyl units was 45+2% at the time of polyketide chain 9 assembly. The manner in which fulvic acid is labelled by [13C]acetate is compatible with its formation from a single heptaketide chain folded as in A (Fig. 1), which is then cyclized and ringopened without C2-unit bond fission as in the biosynthesis of citromycetin. The nearly equal enrichment by
["Clacetate of all one- and two-carbon subsets and the consistent high 13C
enrichment of all seven C2 subsets at the time of chain assembly are more likely for a singlechain origin than for one in which separately formed chains come together. In particular, the intermolecularenrichment of two polyketide chains would probably have fallen below the observed 45% due to dilution with unlabeled material before fusion occurred; we should not then have observed coupling between the neighboring carbons of C2 units joined in the fusion process. Finally, we can discount palitantin as a precursor of fulvic acid in P. brefeZdianm 12 because ,2was enriched by acetate significantly earlier than 4. The weight of evidence from this study and that on fusarubin' thus favours the conclusion 13 Scheme 1 shows a reasonable sequence that 1, 2 and ,3have a common biosynthetic parentage. of biochemical transformationsby which these metabolites could be formed from intermediateB 14 of Fig. 1.
496
Scheme 1.
Biogenetic hypothesis for the formation of citromycetin fusarubin (2) from a comnon intermediate @).
(I), fulvic acid (_2) and
References and Notes
(1) (2)
Turner,
W.B.,
Birch, A.J., 1958, 4576. WI__
(3) Gatenbeck,
"Fungal Metabolites",
Academic
Press,
Fitton, P., Pride, E., Ryan, A.J.,
S. and Mosbach,
(4) Evans, G.E. and Staunton,
K., Biochem. J., J.C.S.
Biophys.
London,
H.B., J. Chem. Sot.,
Res. Cornnun., ."__... 1963, 11, 166.
Chem. Comm., _..,__ 1976, 760.
(5) Birch, A.J., Fortschr. Chem. Org. Naturstoffe, 1957, 14, 1_9_5_8, 131; Thomas, R., Biochem. J., MY, 7& 748. (6) Dean, F.M., Eade, R.A., Moubasher,
1971, pp. 140-156.
Smith, H. and Whalley,
R.A. and Robertson,
186; Whalley,
A., ) Nature
W.B.,
1957 ____'
-’179
Chem. and Ind. 366.
(7) Whalley, W.B., in 'Recent Developments in the Chemistry of Natural Phenolic Compounds' ed. W.D. Ollis, Pergamon Press, Oxford, 1961, p. 49; Money, T., Nature, 1_9_6_3, 199, 592; Stickings, C.E. and Mahmoudian, A., Chem. and Ind., -WI-_ 1962, 1718. (8) Kurobane,
I., Vining,
L.C., McInnes,
A.G. and Smith,
(9) Kurobane,
I., Vining,
L.C., McInnes,
A.G. and Walter,
(10) Dean, F.M., Eade, R.A., Moubasher, (11) F
Czapek-Dox
medium
the fungus
D.G., Can. J. Chem., _I__ 1978, 56, 1593.
R.A. and Robertson,
is variously
reported
J.A.,
Can. J. Chem.,
1980, 3, ..,_-_
1380.
A., J. Chem. Sot., _I__ 1957, 3497. to produce only brefeldin
A (Harri,
1963, 46, 1235) ., Loeffler, W., Sigg, H.P., Stahelin, H. and Tamn, Ch., Helv. Chim. Acta, _I-._
or chiefly
griseofulvin
(ref. 6 above).
(12) We determined the regiochemistry of 4 labeled from [13C]acetate and found it to be as previously reported for [14C]acetate-'(Birch, A.J. and Cocor, M., J. Chem. Sot., __-I 1960, 866; Chaplen, P. and Thomas, R., Biochem. J., __I_ 1960, 77, 91). (13) Although the results cited in ref. 3 do not support this conclusion we do not wish to cormnent until the biosynthesis of 1 is reexamined with appropriate 13C-labeled precursors. (14) This research was supported in part by a grant (GM 25799) from the National Institutes of Health, by a Research Career Award to C.R.H. (CA 00257) for 1976-81 and by an operating grant from the Natural Sciences and Engineering Research Council of Canada. (Received
in USA 14 October
1980)
0040-4039/f31/0201-0493$02.00/O
THE BIOSYNTHESIS OF FULVIC ACID, A FUNGAL METABOLITE OF HEPTAKETIDE ORIGIN Itsuo Kurobane and C. Richard Hutchinson* School of Pharmacy, University of Wisconsin, Madison, WI 53705, U.S.A. and Leo C. Vining Department of BioZogy, Ddhousie University, HaZifax, Nova Scotia, B3H 4J1, Canada Swmary: Analyses of the regiochemistryof [13C]acetate incorporation into fulvic acid (2) by PeniciZtiwn brefetdirmwn indicates that the metabolite is biosynthesized via a heptaketid; intermediate assembled as a single chain of seven C units rather than from two smaller polyketide chains. This information favours a route le0ding through comnon Cl4 intermediates to the three fungal metabolites, citromycetin, fulvic acid and fusarubin. Within the large class of fungal metabolites of polyketide origin, those formed from seven C2 units have been subdivided by Turner,' on the basis of structure and available biosynthetic information, into eight groups. Compounds in the first seven are logically derived from a comnon linear heptaketide intermediatewhich is made from one acetyl and six malonyl units. The different groups of end-products are postulated to arise from folding pattern variations during subsequent cyclizing reactions. Citromycetin (1) and fulvic acid (_2),the two compounds in Turner's eighth group, were distinguished from the heptaketide-basedmetabolites in that their structures could not be derived by simple linear chain folding. Birch and co-workers' showed that seven C2 units originating from [14C]acetatewere incorporated into 1 by &nicitliwn frequentans. Later, Gatenbeck and Mosbach3 found evidence for two starter units by chemical degradation of 1 labeled biosyntheticallyfrom diethyl [2-'4C]malonate and concluded that two polyketide chains contributed to the structure. A 13C NMR study of 1 labeled from [l-13C),[2-'3C]-and [1,2-13Clacetate showed a labelling pattern consistent with a two-chain origin4 but also did not exclude pathways in which a single chain is cyclized and then ring-opened to a branched intermediate such as has been postulated on several occasions5'6 to be the progenitor of 1, ,2and fusarubin (2). Two such pathways have been noted, one involving a palitantin-type'and the other a fusarubin-type'folding of the heptaketide. We are persuaded by evidence presented here for the biosynthesis heptaketide
that 1, ,2 and 3 arise from common
ketide folding
pattern
route to the 4a,lOa-dihydrofusarubins, idea has recently
been validated
Oxidative
of the indicated
fission
C,4 intermediates
(A) that gives B upon intramolecular
as shown in Fig. 1.
aldol cyclization
from which 3 is formed by non-enzymic
by labeling
experiments
with
which
a direct
oxidation.8
This
acetate.'
bond in B, followed by appropriate bond reconnections (shown
of all fungal metabolites
single linear heptaketide
The hepta-
provides
[13C]- and C2H1-labeled
later in Scheme 1) would give rise to 1 and ,2. The hypothesis for the biogenesis
of ,2 via a single-chain
is modified
containing
is attractive
because
seven C2 units as derivatives
by organism-specific
cyclizing
it accounts of a
and processing
reactions. PeniciZZizun brefeldianwn Raulin-Thom
medium.lO*ll
Dodge (NRRL 2083) produces ,2 and palitantin
Both products reach their maximum 493
yield
(_4) when grown
in
on day 5 of the fermentation.
494
citromycetin(!)
Figure
Probable
3.
Experiments precursor
with
biogenetic
fulvic acid(?I
fusarubin(3)
origin of citromycetin,
[l- 14C]acetate
fulvic acid and fusarubin.
showed that 14C enrichment
of ,2 was highest
(18-19%)
if the
was fed on days 2 and 3 whereas _4 was maximally enriched by precursor feeding on days 13 To achieve high C incorporation, therefore, we administered [l-13C]acetate and
3 and 4.
[1,2-'3C2]acetate the two-day
(each containing
periods
of maximum
90 atom % l3 C) to give 5 rnfjbroth concentrations
metabolite
The labeled
production.
products were
twice during
isolated
on
day 5. The 6c relative were assigned pyridine
to internal
comparison
on the basis of:
and their respective
in the 13C NMR spectrum
Me4Si of all signals
trimethyl
of 6c val;;s
for ,2 and anhydrofulvic ,5 and _7,inchloroform;
derivatives,
of fulvic acid acid (5)" the $H
in
coupling
relationships in these four compounds observed by gated proton irradiation (NOE) experiments; 1 13 C NMR spectra of 13C-enriched ,5 and ,7. THE NOE &c coupling data obtained from the
and
spectrum
of 2 had two triplet signals
a high-field
quartet
and a mid-field
in the upfield doublet
region;
appeared
that of _6 had only one.
in the NOE spectrum
this evidence
we could assign the 6c of C-2, C-7, C-9 and C-11 securely.
ments enabled
us to use the unique
in the spectrum
of l3 C-enriched
l*c
The 6c of C-3a and C-4 were distinguished coupling
for C-3a but the absence
could assign constants upfield
with
C-3a and with C-4.
of long-range
also labeled
of [1,2-13C2]acetate
and
per site.
Its (+)
From these data we carbon
coupling
by its characteristic
in 2 vs. 5 and 6 vs. 7.
spectral
regiochemistry
into fulvic acid by analyzing
of 2.6+0.2%
for C-4.
of a 3&H
of C-5 and of C-6 was based on the relative
6c values
The pertinent
the complete
assign-
intact C2 units
and by the presence
the value of their respective
data areshown
([1-13C]Acetate
in Figure 2.
of the incorporation of [1-13C]acetate and 13 C NMR spectra of 13C enriched 2, 7; $, the
Fulvic acid was labeled only at the (0) carbons
enrichment
for all seven
The 6c for C-12 is also defined
patterns
of 2 and ,5. From The resulting
shifts of C-2a, C-7a, C-8a and C-10.
of any long range C,H coupling
Finally assignment
C-5 and not C-6.)
We then determined
and 7.
+H
visible
by the value of 'J -C4C12
the 6c of C-3 and C-12 by noting
shift on methylation.
magnitudes
relationships
,7 to assign the chemical
Similarly,
(Fig. 2) by [l-13C]acetate
carbon pairs were labeled
with a
by [l,E-13C]acetate
C
495
Figure 2.
13C NMR data for fulvic acid and its derivatives. J values shown for 5 and ,7are +1.2 Hz and all spectra were determined at 22.5 MHz-!?t27°C.
(Fig. 2) with a l3C enrichment of 1.&0.2% per site. Since we could clearly see the small quartet resonances due to '%C
coupling between carbons in neighboring 13C2 sub-sets for at
least one carbon of each C2 unit in the 13C NMR spectrum of 5, we could calculate that the 13C enrichment of the individual acetyl/malonyl units was 45+2% at the time of polyketide chain 9 assembly. The manner in which fulvic acid is labelled by [13C]acetate is compatible with its formation from a single heptaketide chain folded as in A (Fig. 1), which is then cyclized and ringopened without C2-unit bond fission as in the biosynthesis of citromycetin. The nearly equal enrichment by
["Clacetate of all one- and two-carbon subsets and the consistent high 13C
enrichment of all seven C2 subsets at the time of chain assembly are more likely for a singlechain origin than for one in which separately formed chains come together. In particular, the intermolecularenrichment of two polyketide chains would probably have fallen below the observed 45% due to dilution with unlabeled material before fusion occurred; we should not then have observed coupling between the neighboring carbons of C2 units joined in the fusion process. Finally, we can discount palitantin as a precursor of fulvic acid in P. brefeZdianm 12 because ,2was enriched by acetate significantly earlier than 4. The weight of evidence from this study and that on fusarubin' thus favours the conclusion 13 Scheme 1 shows a reasonable sequence that 1, 2 and ,3have a common biosynthetic parentage. of biochemical transformationsby which these metabolites could be formed from intermediateB 14 of Fig. 1.
496
Scheme 1.
Biogenetic hypothesis for the formation of citromycetin fusarubin (2) from a comnon intermediate @).
(I), fulvic acid (_2) and
References and Notes
(1) (2)
Turner,
W.B.,
Birch, A.J., 1958, 4576. WI__
(3) Gatenbeck,
"Fungal Metabolites",
Academic
Press,
Fitton, P., Pride, E., Ryan, A.J.,
S. and Mosbach,
(4) Evans, G.E. and Staunton,
K., Biochem. J., J.C.S.
Biophys.
London,
H.B., J. Chem. Sot.,
Res. Cornnun., ."__... 1963, 11, 166.
Chem. Comm., _..,__ 1976, 760.
(5) Birch, A.J., Fortschr. Chem. Org. Naturstoffe, 1957, 14, 1_9_5_8, 131; Thomas, R., Biochem. J., MY, 7& 748. (6) Dean, F.M., Eade, R.A., Moubasher,
1971, pp. 140-156.
Smith, H. and Whalley,
R.A. and Robertson,
186; Whalley,
A., ) Nature
W.B.,
1957 ____'
-’179
Chem. and Ind. 366.
(7) Whalley, W.B., in 'Recent Developments in the Chemistry of Natural Phenolic Compounds' ed. W.D. Ollis, Pergamon Press, Oxford, 1961, p. 49; Money, T., Nature, 1_9_6_3, 199, 592; Stickings, C.E. and Mahmoudian, A., Chem. and Ind., -WI-_ 1962, 1718. (8) Kurobane,
I., Vining,
L.C., McInnes,
A.G. and Smith,
(9) Kurobane,
I., Vining,
L.C., McInnes,
A.G. and Walter,
(10) Dean, F.M., Eade, R.A., Moubasher, (11) F
Czapek-Dox
medium
the fungus
D.G., Can. J. Chem., _I__ 1978, 56, 1593.
R.A. and Robertson,
is variously
reported
J.A.,
Can. J. Chem.,
1980, 3, ..,_-_
1380.
A., J. Chem. Sot., _I__ 1957, 3497. to produce only brefeldin
A (Harri,
1963, 46, 1235) ., Loeffler, W., Sigg, H.P., Stahelin, H. and Tamn, Ch., Helv. Chim. Acta, _I-._
or chiefly
griseofulvin
(ref. 6 above).
(12) We determined the regiochemistry of 4 labeled from [13C]acetate and found it to be as previously reported for [14C]acetate-'(Birch, A.J. and Cocor, M., J. Chem. Sot., __-I 1960, 866; Chaplen, P. and Thomas, R., Biochem. J., __I_ 1960, 77, 91). (13) Although the results cited in ref. 3 do not support this conclusion we do not wish to cormnent until the biosynthesis of 1 is reexamined with appropriate 13C-labeled precursors. (14) This research was supported in part by a grant (GM 25799) from the National Institutes of Health, by a Research Career Award to C.R.H. (CA 00257) for 1976-81 and by an operating grant from the Natural Sciences and Engineering Research Council of Canada. (Received
in USA 14 October
1980)