Biological activity of trisporoids and trisporoid analogues in Mucor mucedo (-)

Article abstract of DOI:10.1016/j.phytochem.2005.04.022

In the course of their sexual interactions, zygomycete fungi communicate via an elaborate series of carotene-derived compounds, namely trisporic acid and its biosynthetic progenitors. A novel building-block strategy allowed the systematic generation of st

Full text of DOI:10.1016/j.phytochem.2005.04.022

PHYTOCHEMISTRY
Phytochemistry 66 (2005) 1358–1365
www.elsevier.com/locate/phytochem
Biological activity of trisporoids and trisporoid analogues in
Mucor mucedo (ꢀ)
a
b,
b
Doreen Schachtschabel , Christine Schimek *, Johannes Wo¨stemeyer ,
a
Wilhelm Boland
a
¨
Abteilung Bioorganische Chemie, Max-Planck-Institut fu¨r chemische Okologie, Hans-Kno¨ll-Straße 8, D-07745 Jena, Germany
b
Lehrstuhl fu¨r Allgemeine Mikrobiologie und Mikrobengenetik, Institut fu¨r Mikrobiologie, Friedrich-Schiller-Universita¨t Jena,
Neugasse 24, D-07743 Jena, Germany
Received 17 February 2005; received in revised form 1 April 2005
Available online 23 May 2005
Abstract
In the course of their sexual interactions, zygomycete fungi communicate via an elaborate series of carotene-derived compounds,
namely trisporic acid and its biosynthetic progenitors. A novel building-block strategy allowed the systematic generation of struc-
turally modified trisporoids along with putative early biosynthetic precursors for physiological tests. The impact of discrete struc-
tural elements was documented by the ability of individual compounds to induce sexually committed hyphae in Mucor mucedo. The
activity screening contributed to establish general structure–function relationships for trisporoid action. Most crucial for activity
were the dimension of the longer side chain, the polarity of functional groups at C(4) and C(13), and the number of conjugated
double bonds in the side chain. The presence of an oxygen substituent at the cyclohexene ring is not essential for function. The over-
all biological activity apparently results from the combination of the various structural elements.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Mucor mucedo; Zygomycetes; Sex hormone; Trisporic acid; Precursors; Chemosynthesis; Bioactivity
1. Introduction
trisporols and trisporins, are defined by their substitu-
ents at C(1) and C(4) (Figs. 1 and 3).
The C18 b-carotene derivate trisporic acid and its pre-
cursors are pheromones involved in the regulation of
mating reactions in Zygomycetes (Zygomycota) (Goo-
day, 1978; Schimek et al., 2003). As the same basic sig-
nal system is used throughout such a wide systematic
range, interspecific reactions occur frequently and abun-
dantly. Nevertheless, species-specificity of the mating
reactions is maintained, ascertaining the formation of
mature zygospores only in compatible mating pairs of
the same species. The various trisporoid types produced
during biosynthesis, i.e., trisporates, methyl trisporates,
Trisporic acids are the products of a complex, coop-
erative pathway involving both the (+) and the (ꢀ) mat-
ing type. In a simplified overview the early biosynthetic
steps towards, for example, trisporic acid B take place in
both mating partners, beginning with the cleavage of b-
carotene until the production of 4-dihydrotrisporin (2).
Compound 2 is the last common intermediate and is
converted to trisporin (4) in the (ꢀ) mating type, and
to 4-dihydromethyltrisporate (3) in the (+) mating type
(BuꢀLock et al., 1976). The compounds act as phero-
mones towards putative partner strains (Fig. 1).
As each mating type is unable to convert its own
intermediate into trisporic acid, production of the latter
depends on exchange of the mating type-specific
precursors between the complementary mycelia. The
*
Corresponding author. Tel.: +49 3641 949 327; fax: +49 3641 949
312.
E-mail address: b9sccr@uni-jena.de (C. Schimek).
0031-9422/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2005.04.022

D. Schachtschabel et al. / Phytochemistry 66 (2005) 1358–1365
1359
-carotene
retinal
O
-C18-ketone (1)
O
OH
4-dihydrotrisporin (2)
(+)-mating type
(-)-mating type
COOCH3
O
O
diffusion
OH
O
4-dihydromethyltrisporate (3)
trisporin (4)
CH2OH
COOCH3
O
O
O
O
trisporol (5)
methyltrisporate (6)
COOH
O
O
trisporic acid B (7)
Fig. 1. Postulated biosynthesis of trisporic acid B (van den Ende, 1976; Sutter et al., 1989; modified). Analogous pathways are expected to generate
the trisporic acids A, C, D, and E.
precursors are taken up by the mating partner, and are
converted in sequence of mating type-specific
enzymatic steps into trisporic acid (reviewed by Gooday,
1978, 1983).
et al., 1974; Sutter and Whitaker, 1981a) as is exempli-
fied for the different series of trisporic acids (7) shown
in Fig. 2. This divergence may be the result of biosyn-
thetic ramifications during speciation and the overall
evolution of this fungal group. The compound pattern
within each species is supposed to be the basis for spe-
cies-specificity of the sexual response (Sutter et al.,
1989). Complete reactions, culminating in the formation
of mature zygospores, are restricted to crossings be-
tween compatible partners of the same species. On the
other hand, the absolute amount of individual com-
pounds of the blend may also influence the response of
the mating partner. These conceptional uncertainties re-
main to be resolved. Also, the actual physiological func-
tions of the biosynthetic intermediates (Fig. 1) are not
yet known in detail; the same applies to the sequence
a
Trisporoids exert a number of responses in suscepti-
ble mycelium. They induce directed growth and thus
facilitate partner finding and contact between the puta-
tive partner hyphae. They also directly induce the for-
mation of sexually committed hyphae, the zygophores,
in Mucor mucedo and a number of other species. They
further elicit a range of more general responses, among
them up-regulation of b-carotene synthesis and, e.g., in
Phycomyces, arrest of vegetative growth as preliminary
step to sexual morphogenesis (Drinkard et al., 1982).
Previous studies established the presence of a mixture
of derivates in each species (van den Ende, 1968; Sutter

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D. Schachtschabel et al. / Phytochemistry 66 (2005) 1358–1365
COOH
O
trisporic acid A
O
OH
COOH
COOH
O
O
trisporic acid B
trisporic acid C
O
OH
COOH
COOH
HO
HO
trisporic acid D
O
trisporic acid E
O
Fig. 2. The trisporic acids are shown as the end products of the pathways.
of biosynthetic step(s) in the different species. Especially,
the sequence of the oxidative functionalization of the
early intermediates and the physiological importance
of the resulting products remain to be established. This
lack of knowledge prompted us to start a systematic and
comprehensive study of structure–function relationships
in trisporoid action and biosynthesis. As the existing
methods do not allow the isolation and purification of
natural trisporoids at a preparative scale, the approach
depends on the synthesis of potential metabolites. Ow-
ing to their interesting biological activities, the trispor-
oids have been repeatedly targets for synthesis. Methyl
trisporates have been synthesized by six groups so far
(Edwards et al., 1971; Isoe et al., 1971; Secrist et al.,
1977; Trost and Ornstein, 1983; Takabe and White,
1983; White et al., 1985; Takahashi et al., 1988). The
free trisporic acids have been produced by different
strategies by White et al. (1985), and by Reeder and
Meyers (1999). Several of the proposed early precursors
were synthesized by the groups around BuꢀLock et al.
(1973, 1976) and Sutter (1986), and trisporol was synthe-
sized also by White and coworkers (Prisbylla et al.,
1979). However, most previous routes are lengthy and
suffer from low overall yields. Since the current project
focuses only on the early intermediates of trisporoid bio-
synthesis, the major obstacle of previous syntheses,
namely the presence of the carboxylate group in the
molecule, could be ignored. The most important struc-
tural features to be analyzed are exemplified in Fig. 3.
As shown, the analysis of structure–functional rela-
tionships of the early trisporoids addresses in particular
the functional and structural principles linked to the oxi-
dative functionalization at C(4) and C(13). This covers
the presence of keto- or alcohol functions at both car-
bon atoms as well as combinations of ketones and alco-
hols. In the case of alcohols both centres become chiral.
Moreover, the overall chain length, the stereochemistry,
the number of double bonds, and the relative distance
between the double bond and the position of the oxygen
functions are of principal interest. Here we report on the
biological activity of compounds obtained along a novel
and highly flexible synthesis towards early trisporoids.
First data on their physiological effects (growth arrest
and zygophore induction) of the compounds are
presented.
2. Results and discussion
2.1. Synthetic concept
The novel concept towards trisporoid-type com-
pounds addresses only compounds that lack the car-
boxyl- or ester group of the late trisporates. To
minimize the synthetic effort towards the different test
compounds (Figs. 3 and 4), we developed a building
block strategy which allows the generation of all of the
different trisporoids from a single intermediate.
The strategy is outlined in Fig. 4. Commercial b-io-
none (8) is first converted into the central (E,Z)-vinyl
bromide 9. The bromide can be easily functionalized
at C(4) by oxidation with CrO3 Æ pyridine (Sarett et al.,
1952) to ketone (10), or to an acetate by allylic oxidation
with Pd(CF3COO)2 (McMurry and Kocovsky, 1984).
The latter can be readily converted to 10. The second
strategic element of the novel synthesis is based on the
facile alkylation of the vinylic bromides 9 and 10 with
organozinc reagents in the presence of Pd(0)-catalysts
(Knochel et al., 1998). The organozincates can be read-
ily obtained from functionalized halides without the

D. Schachtschabel et al. / Phytochemistry 66 (2005) 1358–1365
1361
R2=R3=H
R1= CH3;
CH2OH;
R2=R3=O
R2 R3
13
R1
E,Z
R2=H R3=OH
COOCH3;
COOH
1
7
configuration
4
distance
OH
chiral center,oxidation
(
alcohol or ketone)
Fig. 3. Relevant structural elements of trisporoids for biological activity.
1
need for protecting groups (Huo, 2003). This minimizes
the number of transformations on the generally acid- or
base sensitive trisporoid backbone. For example, trispo-
rin (4) is synthesized in a single operation by a Pd(0)-
catalyzed alkylation of 10 with an organozinc reagent
directly obtained from 4-iodo-butan-2-one. The same
strategy can be followed to generate a derivative with
a secondary alcohol 14 or to produce aliphatic sides
chains of variable chain length, e.g., 11 and 12. Heck-
type alkylation of 9 with buten-2-one (Naora et al.,
1988) leads to 1 and biosynthetic precursors of trisporin
(4). Aromatic derivatives representing analogues with
restricted conformational freedom or fluorescent probes
can be obtained along a similar protocol. The approach
also allows the preparation of deuterium labelled com-
pounds via H/2H-exchange on the level of b-ionone.
Full experimental details for the novel route will be pub-
lished elsewhere.
2.2. Biological activity tests
The biological activities of the test compounds have
been monitored in M. mucedo (ꢀ) as the most sensitively
reacting species (Sutter and Whitaker, 1981a). Also with
respect to the previously postulated intermediate prefer-
ences (Fig. 1), the (ꢀ) strain of M. mucedo is particularly
well suited. Despite its preference for complementary
precursors, M. mucedo (ꢀ) also responds, albeit less pro-
nounced, to (ꢀ)-mating type-specific compounds from
other species (Sutter and Whitaker, 1981a; Schimek
O
8
O
R1
R2
R1
R2
13 R1=R2=H 77%
4 R1=R2=O 71%
11 R1=R2=H 85%
12 R1=R2=O 81%
Br
R1
R2
9 R1=R2=H 82%
10 R1=R2=O 70%
O
OH
R1
R2
R1
R2
1 R1,R2=H 84%
14 R1=R2=H 69%
OCH3
15 R1=R2=H 88%
16 R1=R2=O 73%
R1
R2
Fig. 4. Unified synthetic route to early intermediates of trisporic acids.

1362
D. Schachtschabel et al. / Phytochemistry 66 (2005) 1358–1365
et al., 2003). Altogether this is the most reliable strain
to monitor a broad response. The typical responses,
observed after exposure of growing cultures of (ꢀ)
M. mucedo are surveyed in Table 1.
The induction of zygophores can be considered as a
reliable proof for sexual stimulation and can be quanti-
fied, if certain limitations are taken into consideration.
That special type of aerial hypha is easily discernible
from the sporangiophores, the vegetative reproductive
structures. The zygophores are small, not exceeding
about 500 lm in height and 15 lm in width in the (ꢀ)
mating type (data not shown). In contrast, young spo-
rangiophores have a diameter of at least 50 lm, and la-
ter their tips swell characteristically. Differentiation can
be induced at the nanogram level of trisporoids (Sutter
and Whitaker, 1981a), however at very low concentra-
tions zygophore production is no longer quantifiable
and occurs as an all-or-nothing reaction. At high con-
centrations, quantification is hampered by the high den-
sity of zygophores that can be no longer reliably
counted.
2.3. Biological activity of individual compounds
As safeguard in case of weak responses, rather high
amounts (10–100 lg) of the compounds were applied.
The strongest reaction, determined as the ability to in-
duce zygophores, was detected for compound 13 with
50 zygophores at the most. Zygophore induction was
also observed for compounds 1, 11 and 12. Their effects
were constantly weaker, yielding on average 4–20 zygo-
phores for each of the three compounds.
Compound 12 induced typical zygophores only at
high concentration (ca. 100 lg). At lower concentrations
(25 lg) formation of atypical, branched aerial hyphae
(designated as antlers) was observed. The antlers have
about the same dimensions as the non-branched zygo-
phores and have been observed with natural trisporoids,
too (Schimek, unpublished observations). Antlers are
probably the result of weakly positive reactions, where
the stimulus is not strong enough to override all other
morphogenetic signals.
For natural metabolites upstream of 4-dihydrotrispo-
rin (2) only very few data on physiological activities are
available. As yet, these compounds have not been iso-
lated from natural sources. With the synthetic racemate
of 4-dihydrotrisporin B (2) (custom synthesized by ARC
Laboratories, Minden, The Netherlands) we could dem-
onstrate that 4-dihydrotrisporin (2) does not elicit zygo-
phore induction in M. mucedo (ꢀ) or (+) strains,
although the compound already exhibits all necessary
structural features. Since biological activity was postu-
lated only for compounds downstream of 4-dihydrotri-
sporin (2), its putative biosynthetic progenitor 1, the
b-C18-ketone, should also be inactive in M. mucedo
(ꢀ) (BuꢀLock et al., 1976). However, synthetic com-
pound 1 was found to be active in our assays. Com-
pound 13, which differs from 1 only by reduction of
the a,b-conjugated double bond in the carbonyl com-
pound, was the most active analogue of all. This con-
firms the data presented by Sutter et al. (1996) who
observed zygophore production in carotene deficient
mated mutant strains of Phycomyces blakesleeanus when
stimulated with synthetic compound 13. Compound 13
differs from inactive 4-dihydrotrisporin (2) by lacking
the polar hydroxyl group at C(4). The other two active
analogues, namely 11 and 12, correspond to the series
Table 1
Physiological effects of synthetic trisporoid analogues in Mucor mucedo
(ꢀ)
zygophore
induction
growth
arrest
compound
mixture of natural
trisporoids extracted
from mated cultures of
Blakeslea trispora
yes
no
no
yes
no
O
8
yes
yes
11
no
no
12
O
OH
OH
O
no
no
17
14
13
1
no
no
yes
O
yes
no
yes
no
O
2
OH
OCH3
OCH3
no
yes
no
15
n.d.a
16
O
a not determined

D. Schachtschabel et al. / Phytochemistry 66 (2005) 1358–1365
1363
of trisporate A. Published data on the trisporic acid A
series are inconsistent; trisporic acid A is described as
zygophore inducing by BuꢀLock et al. (1972), and as
inactive by White et al. (1985). In the latter study, trisp-
orol A, on the other hand, was found active towards M.
mucedo (+). As the trisporins are the biosynthetic pre-
cursors of trisporols (Gooday, 1983), which are func-
tional pheromones in some species, the activity of 12
does not surprise, and supports the statement of physio-
logical effects of intermediates of the trisporic acid A
series.
(3) An oxygen substituent at C(4) is not essential.
Analogues 11 and 13 are active without having
an oxygen atom at C(4). Compounds with a non-
functionalized cyclohexene ring may represent
early intermediates from asymmetric cleavage of
b-carotene. Formally, these compounds could be
considered as new trisporoid F derivates.
(4) With some reservation, the saturation of the C11–12
bond with the resulting enhanced rotational free-
dom may be important to adopt a conformation,
which is essential to fit to the macromolecular tar-
gets (receptors).
The lack of zygophore induction for 14 and 17 was
unexpected, since the compounds possess an oxygen
function at C(13), thus resembling intermediates of the
trisporic acid C series. On the other hand, a terminal hy-
droxyl group is also present in the inactive and chain-
shortened apotrisporol (van den Ende, 1968; Sutter
and Whitaker, 1981b), representing a degradation prod-
uct of trisporol. Formally, analogue 14 can be consid-
ered as an intermediate within the C or E-series of
trisporic acids which are generally physiologically ac-
tive. From the E-series, only methyl trisporate E has
been analyzed so far and was found to exhibit biological
activities similar to the corresponding methyl trisporate
C (Miller and Sutter, 1984). Trisporoids C have been de-
scribed as less active (BuꢀLock et al., 1972) by a factor of
10–20 (Sutter and Whitaker, 1981a) when compared to
those of the B series.
Steric, electronic and solubility factors may account
for the inactivity of the two aromatic compounds 15
and 16. Inactive are also compounds with a short side
chain, such as b-ionone (8) and a diketone, derived from
8 by oxidation at C(4). This is consistent with the inac-
tivity of apotrisporins and apotrisporols, which are con-
sidered as products of an inactivation process (Sutter
and Whitaker, 1981a; Sutter and Zawodny, 1984; Sutter,
1986). In summary, from the data in Table 1 several
functional correlations can be made:
(5) None of the above structural features alone seems
to be correlated with a specific response pattern.
Instead, the biological activity is conveyed by the
combination of the various elements. For example,
in the physiologically active C series the negative
effects of the hydroxyl group may be overcome
by the presence of other substituents at the ring
carbon atoms.
Another phenomenon often encountered as reaction
to chemical stimuli is a localized growth arrest. No
mycelium grows directly up to and beyond the filter disc,
which is then left in a more or less circular bay of uncov-
ered substrate. Growth arrest is a prerequisite to sexual
reactions in P. blakesleeanus (Drinkard et al., 1982), but
is generally not observed in M. mucedo. This behav-
ioural difference reflects the difference in zygophore ac-
tion in the two species. In M. mucedo, the aerial
zygophores with their limited length can only get into
contact with a partner zygophore, if the distance be-
tween them does not exceed ca. 1 mm. As zygophores
are not formed from very young mycelium directly at
the front of the growing colony, the partner mycelia
are forced to intermingle to allow for sexual reactions.
In Phycomyces, zygophores are the much shorter, lobed,
and bloated hyphal branches formed at the substrate
surface from the foremost hyphae of the growing col-
ony. As they cannot move freely in the air, their range
of action is very limited, and contact with a partner hy-
pha relies on the possibility of a direct encounter. It can-
not be excluded, however, that in Mucor, vegetative
growth might be reduced, too, in the course of switching
commitments. Another explanation for growth inhibi-
tion is, of course, a systemic toxic effect exerted by the
compound applied. Such toxic reactions can be triggered
by b-ionone (8), as was confirmed in this study for Mu-
cor (Table 1) and also for Phycomyces (data not shown).
Growth arrest was furthermore observed as reaction to
compounds 1 and 15. In Phycomyces, growth inhibition
was triggered also by 11. Carotene induction as a mar-
ker for sexual stimulation was occasionally observed,
but is not included in the table as no basis for evaluation
exists. A quantifiable assay for b-carotene production is
currently developed. The physiological reactions
(1) The length of the side chain appears to be critical
for activity. Chain-shortened compounds such as
b-ionone (8), abscisic acid, or apotrisporins, with
a C10- or C11-backbone, are inactive throughout.
A side chain with the correct length immediately
leads to active compounds, even if the carbonyl
group at C(13) is missing (cf. compounds 11 and
12). Earlier attempts with C15-backbone retinoids,
retinol, retinal, and retinoic acid demonstrated the
lack of biological activity for such compounds
(BuꢀLock et al., 1972).
(2) The polarity of functional groups at the longer side
chain is critical. While a hydrocarbon segment or
keto group at C(13) lead to the active compounds
11 and 13, a hydroxy group at C(13) 14 or an ester
moiety in the same position result in inactive
products.

1364
D. Schachtschabel et al. / Phytochemistry 66 (2005) 1358–1365
monitored in this study are not absolutely quantifiable
at the present stage. Nevertheless, the data obtained
provide a reasonable basis for unravelling of common
principles. With a synthetic concept allowing systematic
variation of individual structural features, a number of
very early trisporoid analogues have been generated
and their analysis helped to unravel basic correlations
determining the bioactivity of trisporoids, especially of
the C and E series (Fig. 2). Systematic evaluation of
the bioactivity of the trisporoids from the different series
with other members of the group of zygomycetes will
show, whether or not and to which extent discrete struc-
tural features have been developed to individualize a
species- and mating-type specific interaction. The large
number of currently known trisporoids may be indica-
tive for the development of a complex metabolic net-
work serving as a pool from which different bioactive
compounds may have been recruited during evolution
to generate functional and highly selective communica-
tion systems for sexual reproduction of the zygomycete
fungi.
are not included in the table. In the control reactions,
more than 200 zygophores were counted in all plates.
Absorbance spectra were recorded on a V-560 UV/
VIS Spectrophotometer (Jasco, Tokyo, Japan). Approx-
imative measurements of trisporoid concentrations were
calculated using the specific extinction coefficients for
trisporic acid ðE
1% cm
325 nm
¼ 572Þ and 4-dihydromethyl tris-
1% cm
282 nm
porate ðE
¼ 547Þ as derived from the work of Nie-
uwenhuis and van den Ende (1975).
Acknowledgement
This work was in part supported by the Deutsche
Forschungsgemeinschaft Priority Programme 1152,
‘‘Evolution of Metabolic Diversity’’.
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