PESTICIDE

BIOCHEMISTRY

A Comparison
P. GADHER,*J

AND

PHYSIOLOGY

19, l- 10 (1983)

of the Potency of Some Fungicides

Sterol 14-Demethylation
E. I. MERCER,*T~

B. C. BALDWIN,?

as Inhibitors

of

AND T. E. WIGGINS?

*Department of Biochemistry and Agricultural Biochemistry, University College of Wales.

Aberystwyth, Dyfed SY23 300, and Imperial Chemical Industries (PLC),
Plant Protection Division, Jealotts Hill Research Station,
Bracknell, Berkshire RGl2 6EY, Great Britain
Received December 22, 1981; accepted July 27, 1982
An enzymatic assay system has been developed to measure the relative potency of fungicides
such as triadimefon, triarimol, triforine, and buthiobate as inhibitors of sterol ICdemethylation.
The enzyme preparation used is the 8000g supematant derived from a homogenate of an aerobically
adapted, anaerobically grown, high sterol strain of Saccharomyces cerevisiae. After incubation of
the enzyme with [2-%]mevalonic acid and the fungicide the ratio, radioactivity in 4,4-dimethyl
sterolsiradioactivity in 4-demethyl sterols is determined. The higher this ratio is, the more efficient
is the fungicide as an inhibitor of fungal sterol 14-demethylation, The ratio has been determined for
a number of commercial fungicides and two series of triazole compounds. A similar assay system
based on the 10,OOOgsupematant from a rat liver homogenate was also tested but gave an inaccurate assessment of the relative potency of fungicides as inhibitors of fungal sterol 14-demethylation.
INTRODUCTION

This paper describes the development of

an enzymatic assay which enables the relative efficacy of this type of fungicide to be
determined.

Several systemic fungicides introduced

during the past decade inhibit ergosterol
biosynthesis
in fungi. These include
triadimefon (I), triforine (2), buthiobate (3),
triarimol
(4), fenarimol
(S), nuarimol (5)
(Fig. 1), imazalil (1-[(2,4-dichlorophenyl)2-(2-propenyloxy)ethyl]-lH-imidazole)
(6),
fluotrimazole
(1-(3-trilluoromethyltriphenyl)1,2,4-triazole) (7), active on phytopathogens,
and clotrimazole (bisphenyl-(2-chlorophenyl)I-imidazolyl-methane)
(7), used against animal mycoses. Their main inhibitory site appears to be the sterol 1Cdemethylase complex, thus causing the accumulation in the
fungi of ergosterol precursors that retain the
14cr-methyl group. Such sterols are unable to
pack satisfactorily with the fatty acyl chains of
the phospholipids in the fungal membranes
(8) whose formation is therefore disrupted.
This markedly limits fungal growth.

MATERIALS

AND METHODS

Fungicides.
Triadimefon
(Bayer A.G.),
triforine
(Celamerk
GmbH & Co.), triarimol and fenarimol
(Eli Lilly & Co.),
buthiobate (Sumitomo Chemical Co.), and
prochloraz (Boots Co. Ltd.) (Fig. 1) were
gifts from the respective companies. Figure 2 shows the structures of two series
of triazole compounds
with fungicidal
properties discovered by the Plant Protection Division of Imperial Chemical Industries PLC which were used in this
study. Compound I is 1-tert-butyl-2-( 1,2,4triazol- I-yl)-3-(4-chlorophenyl)-1-propanone.
Compounds II-V are isomeric forms of ltert- butyl-2-( 1,2,4-triazol- I-yl)-3-(4-chlorophenyl)-propanl-01, the secondary alcoholic reduction product of I which has
chiral centers at positions 1 and 2 (Fig. 2). II
is a mixture of the two forms of this product, one of which has the R configuration

In receipt of a CASE studentship from the Science

Research Council; present address: Department of
Biochemistry, Medical School. University of Edinburgh.
* To whom all correspondence should be addressed.
1

004%3575/83/OlOOOl-10$03.00/O
Copyright
All rights

@ 19g3 by Academic Press. Inc.

of reproduction
in any form reserved.

GADHER

Monochlorophmyl

t+o+-=&
N

ET AL.
swims

Dichlorophenyl

series

F=O

Triadimefon
H

n
*-w-L,
aNA .-+H

Cl

G-

n=tA~Ag;

of lR.ZR L 15.25

YU=tAA,t

m=f$aix;

of lR.lS&lS.ZR

~=Mx&x~::

FIG. 2. Structures
paper.

c,A+

IP.lR.2Riwm.r
P= 15.25 isomer

,S(W),C%

.--YAW
9-.;

of lR.ZR6

c,
/
15.25

of lR.2S 6 lS.2R

11~ IR.ZR isomer

X- IS.25 isomw

assayed

in this

according to Bucher and McGarrahan

as outlined by Popjak (11).

(10)

of

ICI compounds

N==C
t-8u

Buthiobate

Preparation
of the yeast S8 enzyme system. The yeast was grown semi-anaerobiProchloroz

OHChIN,
HC w

Nn~-c~NH.CHo
-

c(a),

Triforine

FIG. 1. Structures
sayed in this paper.

of commercial

fungicides

as-

(9) at positions 1 and 2 while the other has

the S configuration
(9) at these positions.
III is a mixture of the lR,2,S and lS,2R isomers while IV and V are the lR,2R and
lS,Z isomers, respectively. Compound VI
is the dichlorophenyl
analog of I and
VII-X
are the dichlorophenyl
analogs of
II-V, respectively.
Organisms.
Male rats of the SpragueDawley strainweighing
about 3O@g were
the source of the liver used to prepare the
SIO enzyme system. Saccharomyces
cerevisiae (NCYC 739, high sterol strain) was
used in the preparation of the yeast SB enzyme system; it was maintained on Sabourand-Dextrose agar (Oxoid).
Preparation
of the rat liver S,, enzyme
system. This enzyme system was prepared

cally for 48 hr at 30C in 2-liter conical

flasks filled to the neck with a medium containing per liter, (NHJ,S04,
2 g; KH2POI,
2 g; Na,HPO,,
0.5 g; MgS04.7H,0,
0.25 g;
MnSO,.4H,O,
0.025 g; D-glucose, 20 g; and
yeast extract (Difco), 1 g. The cells were harvested by centrifugation, washed twice with
0.1 M phosphate buffer pH 6.2, and then resuspended in the same buffer to which 10%
(w/v) glucose had been added and shaken
(160 rpm in an orbital incubator shaker at
25C) for 150 min according to the method
of Klein (12). The resulting aerobically
adapted cells were harvested by centrifugation and washed twice with cold 0.1 M
phosphate buffer, pH 7.4, containing 30
mM nicotinamide,
5 mM MgC12, and 5 mM
N-acetylcysteine.
After resuspension
in
this medium (0.75 ml/g wet wt of cells) the
resulting paste was passed once through a
precooled French pressure cell operating at
0.1379GPa (20,000 psi). The homogenate of
disrupted cells was centrifuged at 1OOOgfor
10 min at 4C. The resulting supernatant
was filtered through glass wool to remove
the floating lipid layer and centrifuged at
SOOOg for 20 min at 4C. The supernatant
was filtered through glass wool and used
immediately
as the S8 enzyme system.

STEROL

I4-DEMETHYLATION-INHIBITING

CAPACITY

OF

FUNGICIDES

Incubation
with the rat liver S,O enzyme
Extraction and purification
of the unsasystem. The incubation mixtures contained
ponifiable lipid. The reaction mixtures were
the following components in a final volume
heated for 1 hr at 70C in an atmosphere of
of 5 ml: 4.5 ml S,, enzyme system (-40 mg oxygen-free
nitrogen; their volume was
protein rnr l); 2 PCi DL-[2- i4C]mevalonic
maintained by the addition of ethanol as reacid; 15 pmol ATP; 5 ,umol NADPH;
15 quired. They were cooled, diluted with 4
pmol D-glucose-6-phosphate; 5 pmol MnCl,;
vol water, and the unsaponifiable lipid was
11.6 nkat (0.7 IU) glucose-6-phosphate
de- extracted with peroxide-free
diethylether
hydrogenase (EC 1.1.1.49); and 25 ~1 meth(hereafter
referred to as ether) in the
anolic solution of the fungicide to give a usual way.
final concentration
in the incubation mixThe unsaponifiable
lipid, dissolved in 2
ture of usually 50 or 125 m.
No-fungiml ether, was added to a 2-g column of
cide control incubations contained 25 ~1 acid-washed, Brockmann grade III alumimethanol.
na. A further 20 ml ether was passed
The incubation mixtures were shaken for through the column and all the eluent col1 hr at 30C. The reaction was terminated
lected. The eluent was then evaporated to
by the addition of 7 ml ethanol and 2 g dryness in an atmosphere of oxygen-free
KOH. This was immediately
followed by nitrogen to yield the purified unsaponifiable
the addition of 1 mg each of squalene,
lipid. This procedure removed an unknown,
lanosterol, and cholesterol dissolved in 1 ml non-sterol, polar component which would
ethanol; these acted as nonradioactive
car- otherwise mar the subsequent thin-layer
riers. The flasks were then filled with
chromatography.
An aliquot of the purified
oxygen-free nitrogen, tightly corked, and unsaponifiable
lipid was removed
fol
stored overnight at 2C.
radioassay.
Thin-layer chromatography.
The purified
Incubations
with the yeast S8 enzyme
system. The incubation mixtures contained
unsaponifiable
lipid from each incubation
the following components in a final volume
mixture was chromatographed
as a 5-cmof 5 ml: 4.5 ml S8 enzyme system (-30 mg tong streak on 20 x 20-cm Whatman LK6
protein rnk); 2.5 &i DL-[2-14C]mevalonic
silica gel TLC plates using a 9: 1 (v/v) mixacid; 15 pmol ATP; 5 pmol NADPH;
15 ture of benzene and ether for development.
pmol D-glucose-6-phosphate;
15 pmol
Two streaks were placed on each plate
NAD+; 10 pmol L-methionine;
15 pmol re- along with markers of lanosterol and choduced glutathione; 25 ,umol MgCl,; 5 pmol
lesterol or ergosterol as appropriate.
MnCl,;
11.6 nkat (0.7 iU) glucose-6After development
the plates were subphosphate dehydrogenase; and 25 ~1 meth- jected to radioautography
using Kodak
anolic solution of the fungicide. The final
No-screen X-ray film. After 4 days the films
concentration of fungicide in the incubation
were developed with Kodak DX-80 developer and fixed with Kodak FX-40 fixer.
mixture was usually in the range I-50
control incubations
Using the radioautogram
to accurately
PM- No fungicide
delineate
their
positions,
the
radioactive
contained 25 ~1 methanol.
The incubation mixtures were shaken for zones corresponding
to the 4,Qdimethyl
3 hr at 30C. The reaction was terminated
sterols (R, - 0.4), 4a-methyl
sterols (&
by the addition of 7 ml ethanol and 2 g -0.36), and 4-demethyl sterols (R, -0.29)
KOH. This was immediately
followed by were scraped off the plate into glass scintillation vials.
the addition of 0.75 mg each of squalene,
Radioassay.
Nuclear Enterprises
Ltd.
lanosterol, and ergosterol dissolved in 1 ml
NE
216
liquid
scintillator
(10
ml)
was
added
ethanol. The flasks were then filled with
vial containing
the
oxygen-free nitrogen, tightly corked, and to each scintillation
stored overnight at 2C.
silica gel scraped from a TLC sterol zone.

GADHER

The vial was shaken to elute the sterol into

the liquid scintillator
and then, after the
powder had settled, assayed in an Intertechnique SL-33 scintillation
counter programmed to correct for quenching. Recovery of radioactivity
applied to the plates
was 85%.
Aliquots of purified unsaponifiable
lipid
were dissolved in 10 ml of NE 216 liquid
scintillator
and radioassayed in a similar
manner.
Protein determination. The protein concentration of the S8 and S,, enzyme systems
was determined by the method of Lowry et
al. (13) using bovine serum albumin as the
standard.
Difference spectra. Rat liver microsomes
(100,000-g pellet from S,, preparation) were
resuspended to give a protein concentration
of about 0.5 mg mll. The difference spectra
were recorded
in l-cm cuvettes in an
Aminco DW 2 spectrophotometer
between
350 and 500 nm after the addition of 20~1 of
ethanolic solutions of VII, IX, and X to the
sample, and 20 ~1 ethanol to the reference
cuvette.

ET AL.

4cY-methyl sterols (& - 0.36), and (iii) those

having no methyl groups at C-4, designated
the 4-demethyl sterols (&--0.29). The three
sterol classes match closely the normal sequence of demethylation
in animal and fungal sterol biosynthesis,
which is 4,4,14trimethyl sterols + 4,4-dimethyl
sterols -+
4a-methyl
sterols -+ 4-demethyl
sterols.
Thus the first two members of the demethylation sequence fall into the TLC class
designated as 4,4-dimethyl sterols. Since fungicides inhibiting the removal of the 14cymethyl group block the demethylation
sequence at the first step it would be expected
that, in incubations
of [2-14C]mevalonic
acid with the S,, and S8 enzyme systems,
they would cause the accumulation
of
radioactivity in the 4,4-dimethyl sterols and
a depletion in the 4-demethyl sterols. This
is what was found. A similar result was obTABLE 1
Effect of Fungicides on the Ratio of Radioactivity in
the 4,4-Dimethyl Sterols to That in the I-Demethyl
Sterols Formed from [2-Y]Mevalonic
Acid by the
Rat Liver SIO Enzyme System
Dpm ratio [Mean k SEM
(No. of Expts)]

RESULTS

Enzyme Characterization

Fungicide

50 ELM

125 /LlW

It was found that none of the fungicides

Triadimefon
59
a 8 (9)
70
k 13 (5)
reduced the incorporation
of radioactivity
Triarimol
3.4 2 0.9 (2)
8.8, k 0.7 (5)
from [2-14C]mevalonic acid into the purified
Fenarimol
4.3 -c 0.3 (2) 20
k 3.8 (3)
Buthiobate
unsaponifiable material. Under the incuba51
-r- 13 (3)
Prochloraz
44
tion conditions used the rat liver S,, enTriforine
1.1 +- 0.4 (2)
5.3 -r- 1.0 (3)
zyme system incorporated about 40% of the
26
I
-c 5 (5)
47
2 8.5 (2)
biosynthetically
active mevalonic acid isoII
1.28
k
0.2
(3)
6.0
mer into unsaponifiable
lipid regardless of
III
27
_ 7 (3)
72
the presence of fungicide while the yeast S8
IV
2.1 2 0.1 (2)
enzyme system incorporated
about 7%;
V
1.8 2 0.7 (2)
there was, however, some variation in inVI
5.6 -e 1.1 (5) 23
corporation from one enzyme preparation
VII
3.5 k 0.7 (2)
7.7
to another.
VIII
1.0 -e 0.2 (2)
8.4
IX
4.1
The TLC system used to analyze the unX
1.6
saponifiable lipid separated squalene (R,
-0.75), squalene-2,3-oxide
(R, -0.62), and
Control
0.50
0.50
three classes of sterols: (i) those having two
a dpm ratio = (disintegrations min in 4,4-dimethyl
methyl groups at C-4, designated the 4,4- sterols)/(disintegrations min- in 4-demethyl sterols)
dimethyl sterols (R,-0.4); (ii) those having
adjusted to a control ratio of 0.5 (the control ratio
one methyl group at C-4, designated the was always within the range 0.24- 1.66).

STEROL

14-DEMETHYLATION-INHIBITING

CAPACITY

TABLE
Effect

of Fungicides

on the Ratio of Radioactivity

Formed from [2-14CjMevalonic

OF

FUNGICIDES

in the 4,4-Dimethyl
Sterols to That in the I-Demethyl
Acid by the Yeast S8 Enzyme
System

Dpm ratioa [Mean k SEM (No. of Expts)]

Fungicide

50 pM

Triadimefon
Triarimol
Fenarimol
Buthiobate
Prochloraz
Triforine

122
53
6.2
116
3.7

I
II
III
IV
V

113

56
69

51
81
74

VI
VII
VIII
IX
X

33
36

11
2.6
39
2.1

17
2.1

52

138
57
84
22

57

33

166
21
-

2.00

544
17
4
4

186
53
-

Control

10 /LM

Sterols

--.-1 PM

3 (4)

3.9
-

1.9

17
179
37
79
9.5

16
136
13
163

2.8

2.00

2.00

9.2
5.1
-

1.3 (2)

2 34 (3)
+ 0.4 (2)
t 29 (4)

3.2
2.8

3.1
43
2.5

k 4.9 (4)

2.00

a dpm ratio = (disintegrations min- in 4,4-dimethyl sterols)/(disintegrations min- in 4-demethyl sterols.

adjusted to a control ratio of 2.0 (the control ratio was always within the range 1.78-3.62).

tained by Marriot (14) using a cell-free homogenate from Candida albicans. This redistribution
of radioactivity
between the
4,4-dimethyland the 4-demethyl sterols
can be expressed numerically as the ratio,
disintegrations
per minute in the 4,4dimethyl sterols/disintegrations
per minute
in the 4-demethyl sterols (hereafter called
the dpm ratio). The greater the inhibition
of sterol 14-demethylation
exerted by a
fungicide the higher will be the dpm ratio
relative to that of the no-fungicide control.
Tables 1 and 2 show the ratios obtained
with the fungicides shown in Figs. 1 and 2
when added at various concentrations
to
the S,, and S, enzyme systems, respectively. Figure 3 shows plots of dpm ratio
versus concentration for the fungicides triadimefon, IV, and IX obtained with both
the S,, and SB enzyme systems.
Activity
With

of Fungicides
the rat liver

S,, enzyme

system

FIG. 3. Curves relating dpm ratio to concentration

offzzngzczde
in incubations
with the yeast and rat liver
enzyme systems
(dpm ratio=
disintegrations
min- in
4,4-dimethyl
sterolsldisintegrations
min-
in 4demethyl
sterols;
l , compound
IX with the yeast enzyme system;
0, compound
IX with the rat liver enzyme system;
A, compound
IV with the yeast enzyme
system:
A, compound
IV with the rat liver enzyme
system:
H, triadimefon
with the yeast enzyme system;
0, triadimefon
with the rat liver enzyme system).

GADHER

triadimefon, buthiobate, and prochloraz are

the most effective sterol ICdemethylase
inhibitors of the named fungicides, while
triforine is the least effective. Of the ICI
compounds the dichlorophenyl
series (VIX) was less effective than the monochlorophenyl series (I-V).
Within the latter
the ketone (I) is almost as effective as
the best of its secondary alcoholic reduction products, namely III. This product, the
lR,2S + lS,2R isomeric mixture, was much
more effective than II, the lR,2R+
lS,2S
isomeric mixture, a fact borne out by ineffectiveness of IV and V, the resolved isomers of II.
With the yeast S, enzyme system a
somewhat different picture emerged. Of the
named fungicides triadimefon,
triarimol,
fenarimol, and prochloraz were the most
effective with triforine and buthiobate the
least effective. Of the ICI compounds the
ketones of both mono- and dichlorophenyl
series (I and VI, respectively) were almost
equally effective but nothing like so effective as the best of their secondary alcoholic
reduction products. Of these the lR, 2R +
lS,2S isomeric mixtures (II and VII) were
far more effective than the lR,2S + lS,2R
mixtures (III and VIII); this is the reverse
of the rat liver S,, finding. Of the resolved
isomers of II, namely IV (lR,2R) and V
(lS,2S), and of VII, namely IX (lR,2R) and
X (lS,2S), the lR,2R isomers were extremely effective while the lS,2S isomers
were almost inactive. In fact IX, the lR,2R
isomer in the dichlorophenyl
series, was by
far the most active as an inhibitor of sterol
14-demethylation
of all the compounds
tested.
The dpm ratio versus fungicide concentration curves shown in Fig. 3 are sigmoidal and eventually plateau out, showing
that complete inhibition
of the sterol 14demethylase
is eventually achieved. The
concentration at which this occurs is 25- 3.5
fl for triadimefon with both the liver S,,
and the yeast S8 enzyme systems. However
in the case of IV and IX there is a marked
difference between the concentrations
at

ET AL.

which it occurs in the two enzyme systems.

With the yeast Ss enzyme system complete
inhibition is achieved by IV at 5-- 1Ofl and
by IX at about 2 fl whereas neither compound shows any effective inhibition with
the liver S10enzyme system even at 50 m.
Cytochrome

P-450 Binding

Difference spectra obtained when VII,

IX, and X were added to rat liver microsomal preparations
indicated
that each
compound gave a Type II difference spectrum (15). Figure 4 shows the spectrum for
VII, with a maximum at 427 and minimum
at 389 nm, which indicates the interaction
of a nitrogen atom (probably N4 of the
triazole ring) with the iron of the haem.
DISCUSSION

The assay procedure described in this

paper using the rat liver S,, and yeast S,
enzyme systems can be used to compare
the efficacy of compounds such as the fungicides shown in Figs. 1 and 2, as inhibitors
of sterol 1Cdemethylation.
It may be used
in two different ways, (i) by measuring the
dpm ratio for each of the fungicides to be
compared at a series of different concentrations so as to produce dpm ratio versus
fungicide concentration
curves like those
shown in Fig. 3; the more potent the fungicide is in inhibiting
sterol 14-demethylation the lower will be the concentration at which the curve reaches the plateau, or (ii) by comparing the dpm ratio
for each fungicide at the same concentra-

FIG. 4. Difference
spectrum
of rat liver microsomes
with compound
VII (2 x IO+ M) against untreated
microsomes.

STEROL

I4-DEMETHYLATION-INHIBITING

CAPACITY

OF

FUNGICIDES

tion. Method (i) has the advantage that it ylation?

This is exemplified
by the fact
does not rely upon the absolute accuracy of that (i) triarimol
is just as good an inthe dpm ratios, potency being expressed as hibitor of the yeast enzyme system as
the fungicide concentration required to ef- triadimefon but is markedly less good with
fect complete inhibition. However it has the that from rat liver, (ii) buthiobate is just as
disadvantage of being more time consuming
good an inhibitor of the rat liver enzyme
and expensive than method (ii). The latter is system as triadimefon but is markedly less
probably the method of choice for routine
good with that from yeast, (iii) compounds
work because it is quicker and cheaper.
II and VII (lR,2R + lS,2s isomeric mixHowever, because it relies upon the accu- tures) are poor inhibitors of the rat liver enracy of dpm ratios a number of precautions
zyme but excellent inhibitors of that from
must be taken. First, all the fungicides to be yeast, and (iv) compounds
III and VIII
compared should be incubated with the (lR, 2s + lS, 2R isomeric mixtures) are exsame preparation of the So or S8 enzyme
cellent inhibitors of the rat liver enzyme
system. If there are too many to do this system but poor inhibitors
of that from
there should be an adequate overlap of fun- yeast. This raises two questions;
first,
gicides from preparation
to preparation.
which of the two enzyme systems is betMoreover it is advisable to select a fun- ter suited to assessing the relative efficacies
gicide, e.g., triadimefon,
to act as a stan- of these fungicides as inhibitors of sterol
dard and to assay it, along with the fun- i4-demethylation
in fungal phytopathogicides under investigation,
with each en- gens? and second, why should there be
zyme preparation.
These precautions
this difference between the two enzyme
minimize the slight variations between en- systems?
zyme preparations.
Second, care must be
The answer to the first question was obtaken over the choice of the concentration
tained by comparing the relative efftcacies
at which the fungicides are to be assayed. If of II versus III and VII versus VIII, with
the concentration
is too high sterol 14- their ability to inhibit the growth of the
demethylation
may be almost completely
phytopathogenic
fungus, Ustilago maydis
inhibited with the result that the radioactiv(maize loose smut). The ED,, values (conity in the 4,4-demethyl
sterols will be too centration required produce a 50% reduclow to produce a well-delineated
zone on tion in growth) were found to be: II, 1.0
radioautography
of the thin-layer
chro- @4; III, 21 M and VII, 0.2 pM; VIII, 83
matogram. This causes imprecision in the pM (B.C. Baldwin and T. E.Wiggins, unscraping of the zone from the plate and thus published data). These compare favorably
in its subsequent radioassay. When the dpm with the yeast dpm ratios obtained, for inratio is high, as will be the case in this in- stance, at 5 pM fungicide concentration,
stance, a small error in the determination
of i.e., II, 179; III, 37 and VII, 136; VIII, 13
the disintegrations
per minute in the 4- and unfavorably
with the rat liver dpm
demethyl sterols causes a disproportionately
ratios, obtained, for instance, at 125 PM,
large error in the dpm ratio. This can be i.e., II, 6.0; III, 72 and VII, 7.7; VIII, 8.4.
avoided by using a fungicide concentration
Clearly the yeast enzyme is the system of
which causes only partial inhibition of sterol
choice to assess the relative efficacies of
Id-demethylation,
thus ensuring a relatively
fungicides as inhibitors of fungal sterol 14low dpm ratio and a well-defined
4- demethylation.
demethyl sterol TLC zone.
Although
the yeast assay system deIt is clear that the two enzyme systems
scribed in this paper may be useful in opgive a different answer to the question
timizing
the structure
of sterol 14-dewhat are the relative efficiencies of the methylation
inhibitors,
the resulting comfungicides in inhibiting
sterol 16demethpounds will not necessarily be good control

GADHER

agents for plant pathogenic

fungi. The
structure has not only to be tailored to
inhibit the fungal sterol 1Cdemethylase
but also to allow for (i) uptake into, transport, and metabolism
within the infected
plant, (ii) uptake into the fungal mycelium
and access to the sterol lCdemethylase,
(iii) lack of access to or inability to inhibit
the plant sterol lQdemethylase,
and (iv) no
adverse effect on plant growth. The testing
of such diverse requirements
in a compound can still only be satisfactorily
accomplished with a plant infected with the
fungus.
Similarly the specificity of action must be
examined in vivo; the fact that liver sterol
demethylase is inhibited by the fungicides
does not reflect upon the situation if fungicides sprayed on crops are ingested by
animals, because uptake, metabolism,
and
excretion have all to be considered. With
compound IX we have observed a marked
selectivity with respect to the yeast and
liver sterol 14-demethylase
complexes.
Maximum
inhibition
of the former is
achieved at a concentration of about 2 /..GU
while virtually no inhibition of the latter is
caused by 25 times that concentration.
In answer to the second question, we
have clearly demonstrated by our results
that different compounds,
and different
isomers and enantiomers of the same compounds, can inhibit the yeast and liver 14demethylase systems to a different amount.
All evidence to date indicates that the two
enzymes follow the same multistep reaction
sequence. Although the detailed mechanisms of several of its individual reactions
have yet to be elucidated the outline of the
reaction sequence (Fig. 5) is clear. Reactions 1, 2, and 3 are catalyzed by mixed
function oxygenases, one of which, that
catalyzing reaction 1, utilizes cytochrome
P-450 (16). Since it is well known that the
nitrogen atom of heterocyclic rings binds to
the protohaem iron of cytochrome P-450,
thereby competing with the natural substrate oxygen and producing the Type II
difference sprectrum (15), we consider it

ET

AL.

FIG. 5. Outline
tion sequence.

of the

sterol ll-demethylation

reac-

likely that the sterol 14-demethylationinhibiting fungicides, all of which have nitrogen heterocycles, block reaction I. This is
supported by the fact that these fungicides
(i) bind powerfully to cytochrome P-450,
causing typical Type II difference spectra
(Fig. 4) and (ii) they cause the accumulation
of 14cY-methyl sterols rather than 14ahydroxymethyl
or 14a-formyl
sterols as
would occur if they inhibited reactions 2 or
3. We therefore postulate that these fungicides block the cytochrome P-450 component of the mixed function oxygenase
catalyzing reaction 1. The blockage is probably primarily
due to the binding of the
heterocyclic nitrogen atom of the fungicide
to the protohaem iron atom thus excluding
oxygen, the natural sixth ligand of the iron.
However, since not all nitrogen heterocycles are equally good inhibitors of sterol
lCdemethylation,
it is clear that the rest of
the molecule has an important bearing on
the binding of the fungicide to the cytochrome P-450. We suggest therefore that
the non-N-heterocyclic
part of the molecule
binds to the lipophilic binding site of cytochrome P-450 which is normally occupied
by the 14a-methyl sterol (Fig. 6). Differences in the nature of the lipophilic binding

STEROL

14-DEMETHYLATION-INHIBITING

cyt

P-450

structures
P-450-lanosterol
Il-demethylation
complex
(a) and the cytochrome
plex (b).

OF FUNGICIDES

triadimenol can be relieved by the application of gibberellins and that plants treated
with these fungicides contained substantially lower levels of gibberellin than normal
(17). These findings suggest that triadimefon and triadimenol
inhibit
gibberellin
biosynthesis, and the suggestion is strengthened by the observation that ancymidol,
a pyrimidine
related to the fungicides triarimol and fenarimol,
inhibits the mixed
function oxygenase-catalyzed
steps in gibberellin biosynthesis,
namely ent-kaurene
--+ enr-kaurenol -+ ent-kaurenal, which are
cytochrome P-450 mediated (18, 19).
We have found the assay described here
very useful in comparing the intrinsic activity of candidate fungicides as inhibitors
of the sterol 14-demethylase system.

FIG. 6. Postulated

CAPACITY

of

the cytochrome
transition
state
P-150-fkgicide
com-

site and/or its position relative to the protohaem would then account for the different
responses of the yeast and rat liver enzyme
systems to the fungicides.
The concept of fungicide-cytochrome
P-450 binding described above can account for the relatively
poor sterol 14demethylation-inhibiting
ability of triforine
with both yeast and rat liver enzyme systems. Triforine differs from all the other
fungicides tested in having its heterocyclic
nitrogen atoms substituted.
These bulky
substituents would prevent the heterocyclic
nitrogens from binding efficiently to the
protohaem iron. It is possible that the limited inhibition demonstrated is due to the
binding of one of the formamido nitrogens
to the protohaem iron.
The concept of fungicide-cytochrome
P-450 binding can also account for the
stunting of plant growth which is caused by
some of these fungicides. It has been shown
that the stunting effect of triadimefon and
its secondary alcoholic reduction product

REFERENCES
1. H. Buchenauer, Mode of action of triadimefon in
Ustilago

avenae.

Pestic.

Biochem.

Physiol.

7,

309 (1977).
2. J. L. Sherald and H. D. Sisler, Antifungal mode of
action of triforine. Pestic. B&hem.
Physiol.
5,
477 (1975).
3. T. Kato, S. Tanaka, M. Ueda, and Y. Kawase,
Inhibition of sterol biosynthesis in Monilinia
fructigena
by the fungicide S-1358, Agr. Biol.
Chem. 39, 169 (197.5).
4. N. N. Ragsdale and H. D. Sisler, Inhibition of ergosterol synthesis in Ustilago maydis by the
fungicide triarimol, Biochem.
Biophys.
Res
Commun.

46, 2048 (1972).

5. H. Buchenauer, Biochemical effects of the systemic fungicides fenarimol and nuarimol in Ustilago

avenae,

Z.

Pfanzenkr.

Pjlanzenschut:

84, 286 (19771.

6. H. Buchenauer. Mechanism of action of the fungicide imazalil in Ustilago
avenae,
J. PJlanzenkr. Pflanzenschutz
84, 440 (1977).
7. H. Buchenauer, Analogy in the mode of action of
fluotrimazole and clotrimazole in Ustilago avenae,

Pestic.

Biochem.

Physiol.

8, 15 (1978).

8. K. Bloch, Speculations on the evolution of sterol

structure and function,
CRC
Crit.
Rev.
Biochem.

9, l(1979).

9. R. S. Cahn. C. K. Ingold, and V. Prelog, SpeciIication of molecular chirality, Angew.

Chem.
Int. Ed. Engl. 5, 385 (1966).
10. N. L. R. Bucher and K. McGarrahan,
The
biosynthesis of cholesterol from acetate-l-C
by cellular fractions of rat liver, J. Biol. Chem.
222, 1 (1956).

10

GADHER

Il.

G. Popjak, Enzymes of sterol biosynthesis in liver

and intermediates of sterol biosynthesis, In
Methods in Enzymology
(R. B. Clayton,
Ed.), Vol. 15, p. 393, Academic Press, New
York, 1969.
12. H. P. Klein, Synthesis of lipids in resting cells of
Saccharomyces

cerevisiae,

J. Bacterial.

69,620

(1955).
13. 0. H. Lowry, N. Rosenbrough, A. L. Farr, and
R. T. Randall, Protein measurement with the
Folin phenol reagent, J. Biol. Chem. 193, 265
(1951).
14. M. S. Marriot, Inhibition of sterol biosynthesis in
Candida
albicans
by imidazole-containing
antifungals, J. Gen. Microbial.
117, 253 (1980).
15. J. B. Schenkman, H. Remmer, and R. W. Estabrook, Spectral studies of drug interaction with
hepatic microsomal cytochrome (rat), Mol.
Pharmacol.
3, 113 (1967).

ET

AL.

16. G. F. Gibbons, C. R. Pullinger, and K. A. Mitropoulos, Studies on the mechanism of lanosterol 14o-demethylation; a requirement for two
distinct types of mixed function oxidase,
Biochem.
J. 183, 309 (1979).
17. H. Buchenauer
and E. Rohner, Effect of
triadimefon and triadimenol on growth of various plant species as well as on gibberelfin content and sterol metabolism in shoots of barley
seedlings, Pestic. Biochem.
Physiol.
15, 58,
(1980).
18. R. C. Coolbaugh, S. S. Hirano, and C. A. West,
Specificity of action of ancymidol, a plant
growth inhibitor, Plant Physiol.
Suppl.
59,
77 (1976).
19. R. C. Coolbaugh and R. Hamilton, Inhibition of
ent-kaurene oxidation and growth by a-cyclopropyl-cu-(p-methoxyphenyl)-5-pyrimidine
methyl
alcohol, Plant Physiol. 57, 245 (1976).