diazaoctane Ring System of Prenylated Indole Alkaloids

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Fungal Origins of the Bicyclo[2.2.2]diazaoctane Ring System of Prenylated Indole

Alkaloids

Finefield, Jennifer M.; Frisvad, Jens Christian; Sherman, David H.; Williams, Robert M.

Pub lished in:
Journal of Natural Products

Link to article, DOI:
10.1021/np200954v

Pub lication date:
2012

Document Version
Early version, also known as pre-print
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Citation (APA):
Finefield, J. M., Frisvad, J. C., Sherman, D. H., & Williams, R. M. (2012). Fungal Origins of the
Bicyclo[2.2.2]diazaoctane Ring System of Prenylated Indole Alkaloids. Journal of Natural Products, 75(4), 812-
833. DOI: 10.1021/np200954v
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Review
pubs.acs.org/jnp
Fungal Origins of the Bicyclo[2.2.2]diazaoctane Ring System
of Prenylated Indole Alkaloids
Jennifer M. Finefield,† Jens C. Frisvad,‡ David H. Sherman,§ and Robert M. Williams*,†,⊥
†
Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523, United States
‡
Department of Systems Biology, Building 221, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark
§
Life Sciences Institute and Departments of Medicinal Chemistry, Microbiology & Immunology, and Chemistry,
University of Michigan, Ann Arbor, Michigan 48109, United States
⊥
University of Colorado Cancer Center, Aurora, Colorado 80045, United States
ABSTRACT: Overeightdifferentfamiliesofnaturalproducts
consisting of nearly 70 secondary metabolites that contain
the bicyclo[2.2.2]diazaoctane ring system have been isolated
from various Aspergillus, Penicillium, and Malbranchea species.
Since 1968, these secondary metabolites have been the focus
of numerous biogenetic, synthetic, taxonomic, and biological
studies and, as such, have made a lasting impact across
multiple scientific disciplines. Thisreview covers the isolation,
biosynthesis, and biological activity of these unique secondary
metabolites containing the bridging bicyclo[2.2.2]diazaoctane
ringsystem.Furthermore,thediversefungaloriginofthesenaturalproductsiscloselyexaminedand,inmanycases,updatedto
reflect the currently accepted fungal taxonomy.
■
INTRODUCTION known to produce metabolites containing the bridging bicycle.
SpecificincorporationofintactC units,combinedwiththe13C
Naturalproductsderivedfromfungalsourceshavebeenanarea 2
coupling patterns observed by 13CNMR spectroscopy, demon-
of intense research for decades due to the vast structural
strated that the isoprene units found in these metabolites arise
diversity and biological activity of these secondary metabolites.
from dimethylallyl pyrophosphate (DMAPP), a product of the
Numerous fungal-derived natural products serve as clinical
mevalonic acid pathway (Scheme 1).11,12
therapeutics for both human and animal diseases, and thus the
Of the natural products containing the unusual bridging
search for new classes of bioactive secondary metabolites from
bicycle, two distinct stereochemical configurations have been
terrestrial and marine fungal sources has garnered additional
interest.1 Natural products containing a unique bicyclo[2.2.2]- noted with respect to the relative configuration at the C-19
position (brevianamide numbering). The rare anti-configuration
diazaoctaneringsystemarepartofadiverseclassofbiologically
(1)andthemorecommonsyn-configuration(2)aredetermined
active prenylated indole alkaloids isolated mainly from various
Penicillium and Aspergillus species of fungi.2 The breviana- by the position of the C−H proton at the bridgehead of
mides,3 paraherquamides,4 stephacidins,5 notoamides,6 asper- the bicyclic core in relation to the bridging secondary lactam
paralines,7 marcfortines,8 chrysogenamides,9 and malbranchea- (Figure 2).13 The brevianamides are currently the only family
mides10 exhibit a range of interesting structural features, such of secondary metabolites to display the anti-configuration about
the bridging bicycle; however, it was recently discovered that
as a bicyclo[2.2.2]diazaoctane nucleus and a complex, oxidized
two novel metabolites chrysogenamide A and versicolamide B
amino acid skeleton (Figure 1). Structurally, these natural
alsodisplaytherareanti-stereochemistry.6f,9Contrarytothis,all
products are comprised of tryptophan, a cyclic amino acid
other known metabolites containing the core bridging bicycle
(proline, pipecolic acid, or derivatives of proline or pipecolic
display the syn-configuration.
acid), and one or two isoprene units. In addition to their
Since the first isolation of these bicyclo[2.2.2]diazaoctane-
structuraldiversity,avarietyofbioactivitiesarealsopresentfor
containing metabolites, substantial effort has focused on both
these compounds, including but not limited to insecticidal,
the synthetic and the biosynthetic aspects of this unique
cytotoxic, anthelmintic, and antibacterial properties.
ring system.14,15 However, although relatively little is known
Initial characterization of these types of natural products led
about the microbial assembly and modification of metabolites
to the conclusion that two isoprene units are present in these
containing this core bridging bicycle, new genomic and bio-
alkaloid families, in which one of the two isoprene units is
chemical approaches are beginning to clarify important aspects
involved in the formation of the bicyclo[2.2.2]diazaoctane
ring system.11 In order to determine the biosynthetic pathway
fromwhichtheisopreneunitsoriginate,[13C ]-acetatehasbeen Received: December 8,2011
2
utilized in precursor incorporation studies with fungal cultures Published: April15, 2012
©2012AmericanChemicalSocietyand
AmericanSocietyofPharmacognosy 812 dx.doi.org/10.1021/np200954v|J.Nat.Prod.2012,75,812−833
Journal of Natural Products Review
Figure 1. Structures ofseveral secondary metabolites that contain the core bicyclo[2.2.2]diazaoctane ringsystem.
Scheme 1. Classical Mevalonic Acid Pathway Showing the Labeling Pattern via 1,2-Doubly Labeled Acetatea
aNote the change in carbon numbering.
Scheme 2. Proposed Biosynthetic Diels−Alder Reaction16
Figure 2. Anti- and syn-relationship of the bicyclo[2.2.2]diazaoctane
core.
biomimetic syntheses of several advanced metabolites.15−17
of these pathways. Originally proposed by Porter and Sammes Concurrently, substantial effort has been put forth to isolate
in 1970, the biosynthetic formation of the bicyclo[2.2.2]- and characterize the putative Diels−Alderase enzyme respon-
diazaoctane ring system is believed to arise via an intra- sible for this biosynthetic transformation, but, to date, no such
molecular hetero Diels−Alder reaction (IMDA) of a 5-hydro- enzyme has been isolated.
xypyrazine-2(1H)-one (Scheme 2).16 Extensive synthetic ThePenicilliumandAspergillusfungalproducersofsecondary
research carried out by Williams and co-workers has aimed at metabolites containing the bicyclo[2.2.2]diazaoctane ring have
supporting a biosynthetic Diels−Alder reaction via the beendefinedtaxonomicallybasedonearlyphenotypicmethods
813 dx.doi.org/10.1021/np200954v|J.Nat.Prod.2012,75,812−833
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Table 1. Culture Collection Strains Producing Secondary Metabolites with a Bicyclo[2.2.2]diazaoctane Unit and Related
Compounds (new data: Frisvad, unpublished)
secondarymetabolite fungalspecies producers:isolatesavailableininternationalculturecollections
malbrancheamides Malbranchea (RRC1813)b(UCSC086937A)
aurantiacaa
M.graminicola
chrysogenamide P.chrysogenum (No.005)b
brevianamides P.viridicatum IMI039755ii,cIMI351305,IMI351306,NRRL869,NRRL870,NRRL959,NRRL962,NRRL3586,
NRRL3600,NRRLA-15402,NRRLA-18563,NRRLA-26909
P.brevicompactum CBS257.29,cCBS256.31,CBS316.59,CBS317.59,CBS480.84,CBS100068,CBS110069,NRRL867,
NRRL32579
A.carlsbadensis CBS123894c
deoxybrevianamides A.pseudoustus NRRL5856,cMRC096
P.italicum ATCC48952,CBS339.48,CBS278.58,CBS349.75,CBS489.84,
P.ulaiense CBS136.41,CBS210.92,CBS722.92,CBS262.94
paraherquamides P.brasilianum CBS253.55,cCBS338.59
P.cluniae CBS276.89c
P.glaucoroseum NRRL908c
P.cf.canescens ATCC20841,IMI332995
paraherquamideE A.aculeatinus CBS204480
A.fijiensis CBS313.89,cIMI337664a
asperparalines A.aculeatinus ATCC204480=JV-23,CBS101.43,CBS186.67,CBS610.78,CBS621.78,CBS116.80,CBS121060,
CBS121875
A.fijiensis CBS313.89,cCBS119.49,IMI337664a
notoamides(including A.bridgeri CBS350.81c
(−)-versicolamide) A.melleus CBS546.65,cNRRL394
A.ochraceus CBS108.08,cCBS589.68,CBS624.78,NRRL6319,(CL41582)
A.ostianus CBS103.07,cCBS101.23,CBS311.80,IMI177964b,IMI348937,NRRL422,NRRL5225,NRRLA-15156,
(01F313)
A.persii CBS112795c
A.petrakii CBS105.57c
A.sclerotiorum CBS549.65,cATCC18413,NRRL5167,(CNC358)
A.westerdijkiae CBS112803c=NRRL3174,CBS112791,CBS112804,NRRL5175,NRRL5221,NRRL5228,
notoamides(including A.protuberus CBS602.74,cCBS601.74
(+)-versicolamide) A.versicolor CBS118.50,CBS106.57,CBS599.65,IMI096225,NRRL35600
A.elongatus CBS387.75c
austamide A.pseudoustus NRRL5856,cMRC096
citrinadins P.citrinum CBS139.45,c(N-059)
PF1270citrinadins P.westlingii (PF1270)
talathermophilins Talaromyces CBS116.72,(YM1−3)
thermophilus
aPossiblemisidentification. bStrain unavailable. cType strain.
of Raper and Thom18 for Penicillium and Raper and Fennell19 inacaveinMexico(Table2).10Shortlyfollowingtheisolation
for Aspergillus. However, several of these species have since of malbrancheamide and malbrancheamide B, Mata and co-
been reclassified based on modern molecular methods. workersreportedthepresenceofthenonhalogenatedprecursor
Extensive taxonomical research carried out by Frisvad and malbrancheamide C, which was later named premalbranchea-
co-workershasprovidednewphylogeneticinformationforeach mide.10c This unique family of halogenated secondary
fungal culture known to produce the bicyclo[2.2.2]diazaoctane metabolites has expanded recently with the isolation of two
ring system described in this review, and both the original and newchlorinatedmetabolites,(−)-spiromalbramideand(+)-iso-
updated fungal strain nomenclature will be mentioned when malbrancheamideB,fromMalbrancheagraminicola,asreported
applicable. Furthermore, numerous strains of these fungal by Crews and co-workers.10d Furthermore, two brominated
cultures have been determined to produce the same sets of analogues, (+)-malbrancheamide C (hereafter referred to as
secondary metabolites (Table 1). Taxonomic revisions of (+)-malbrancheamide “C”) and (+)-isomalbrancheamide C,
species and reidentifications of the strains are based on a were obtained from M. graminicola when the growth medium
polyphasic approach, including morphological, physiological, was enriched with bromine salts.
chemical, and DNA sequence data. Mostly sequences from This family of natural products represents the only bicyclo-
β-tubulinandcalmodulingeneshavebeenvaluableinassigning
[2.2.2]diazaoctane-containing metabolites to be reported from
s■trains to new or known species. a fungal source outside the genera Penicillium and Aspergillus.
Taxonomically, the strain of M. aurantiaca (RRC1813) may
MALBRANCHEAMIDES
either be that species or represent the species M. graminicola,
First identified in 2006 by Mata and co-workers, malbranchea- since three strains of M. aurantiaca from the CBS collection
mide and malbrancheamide B were isolated from Malbranchea (CBS 127.77, Utah, ex culture contaminant, type strain; CBS
aurantiaca RRC1813, a fungus obtained from bat guano found 759.74 and CBS 309.76, both ex soil, India) did not produce
814 dx.doi.org/10.1021/np200954v|J.Nat.Prod.2012,75,812−833
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Table 2. Fungal Origin of the Malbrancheamides
Scheme 3. Proposed Biosynthesis of the Malbrancheamides
themalbrancheamidesorpenicillicacid.10eHowever,theisola- Following the isolation of the malbrancheamides, interest
tion of (+)-malbrancheamide, (+)-malbrancheamide B, wassparkedinelucidatingthebiosynthetictimingoftheindole
(+)-premalbrancheamide, and (−)-spiromalbramide from chlorination. As shown in Scheme 3, the initially proposed
M.graminicolaindicatesthatthesecompoundsarecommon biogenesis of the malbrancheamides begins with the con-
in the genus Malbranchea. densation of tryptophan, proline, and dimethylallyl pyrophos-
Structurally, the malbrancheamides are the first members of phatetoaffordtheknownnaturalproductdeoxybrevianamideE.
this class of metabolites to display halogenation of the indole From here, two distinct biosynthetic pathways are possible to
moiety. While malbrancheamide was determined to contain form the known metabolite premalbrancheamide. One
chlorinationattheC-5andC-6positionoftheindole,theexact potential pathway involves oxidation of deoxybrevianamide E
structure of malbrancheamide B was unclear. By synthesizing to yield the Diels−Alder precursor 5-hydroxypyrazin-2(1H)-
both the C-5-chlorinated and C-6-chlorinated potential one (4a), which would undergo [4+2] cycloaddition to afford
compounds,itwasestablishedthatmalbrancheamideBcontains keto-premalbrancheamide. Subsequent carbonyl reduction of
C-6monochlorination.21Inconjunctionwiththeuniqueindole the tertiary amide would provide premalbrancheamide. On
chlorination, the malbrancheamides are also characterized by the other hand, carbonyl reduction could occur prior to the
the syn-configuration about the bridging bicycle, as well as the IMDA reaction, in which 4b would serve as the key achiral
presence of a monoketopiperazine moiety in the core bicycle. intermediate and direct intermediate to premalbrancheamide.
Biologically, the malbrancheamides were determined to display Two individual and concurrent halogenases would then lead
calmodulin inhibitory activity of CaM-dependent phosphodies- from premalbrancheamide to malbrancheamide B and then to
terase (PDE1).10c,f malbrancheamide.
815 dx.doi.org/10.1021/np200954v|J.Nat.Prod.2012,75,812−833
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To test whether premalbrancheamide and/or keto-premal- and pyran rings. Chrysogenamide A is the first and only
brancheamide serve as biosynthetic precursors to the metabolite of these families of natural products to display
halogenated metabolites, doubly 13C-labeled premalbranchea- ■neurocyteprotectionagainstoxidativestress-inducedcelldeath.9
mide and keto-premalbrancheamide were synthesized by
Williams et al. and provided to M. aurantiaca in separate BREVIANAMIDES
precursor incorporation studies (Scheme 4).10f Results from Between 1969 and 1972, Birch and co-workers isolated six
novel secondary metabolites, brevianamides A−F (Figure 3),
Scheme 4. Precursor Incorporation Studies with
M. aurantiaca.
thesefeedingstudiesshowedsignificantincorporationof[13C]-
2
premalbrancheamide into malbrancheamide B (5.5% incorpo-
ration), but surprisingly, [13C ]-keto-premalbrancheamide did
2
not incorporate. The latter result suggests that the timing
for the reduction of the tryptophan-derived amide carbonyl
residue must occur prior to the [4+2] cycloaddition; thus the
biosynthetic formation of premalbrancheamide most likely
arises from the proposed intermediate 4b. Future genetic and
biochemical analysis of the malbrancheamide biosynthetic Figure 3. Structures of the brevianamides and related metabolites.
pathway will undoubtedly resolve the mechanism of mono-
k■etopiperizine assembly by M. aurantiaca.1 fromPenicilliumbrevicompactum.3Ofthesesixmetabolites,four
compounds were found to contain the unique bicyclo[2.2.2]-
CHRYSOGENAMIDE diazaoctane core. Additional research revealed that breviana-
midesAandBarediastereoisomers,whilebrevianamidesCand
In 2008, Zhu and co-workers were interested in identifying
Dareartifactsofisolationduetolightirradiation.3cStructurally,
newneuroprotectivecompoundsfromendophyticfungionthe
the brevianamides were found to display the anti-configuration
parasitic plant Cistanche deserticola collected in northwest
mainland China (Table 3).9 From the roots of C. deserticola, aboutthebicycliccore,aswellasthespiro-indoxylmoiety,and
an unsubstituted indole ring. Furthermore, the brevianamides
threeactivestrainswerescreenedforneuroprotectiveeffectson
are a class of natural products that display modest biological
SH-SY5Y cells, and the fungal culture Penicillium chrysogenum
activities,inwhichbrevianamidesAandDhavebeenshownto
No.005demonstratedsignificantactivity.FromP.chrysogenum,
possess antifeedant and insecticidal effects.25
the novel metabolite chrysogenamide A was isolated. From
a taxonomic perspective, even though the original culture was As shown in Table 4, the brevianamides have been isolated
not available for examination, other secondary metabolites predominately from two Penicillium species. The two main
characteristic of P. chrysogenum were also isolated,9 supporting producers of brevianamide A are P. brevicompactum3,22,23 and
the identity.22−24 P. viridicatum,3e,22,23 of which both are consistent producers of
From a structural point of view, this novel metabolite is brevianamide A.26 The isolate of P. ochraceum reported to
closely related to the marcfortines (discussed below) in that produce brevianamide A3d was determined to be a brown-
chrysogenamide A was found to contain a bicyclo[2.2.2]- spored mutant of P. viridicatum.26 Deoxybrevianamide E is
diazaoctane ring system, a spiro-oxindole moiety, and a producedbyP.italicum,27d,eP.ulaiense,22andafungusidentified
pipecolic acid unit with C-17 methylation. Additionally, asAspergillusustusNRRL5856.27a−cThelatterisolatehasbeen
chrysogenamide A displays the rare anti-configuration about the basis for the description of a new species, Aspergillus
the bridging bicycle and an isoprene unit at the C-7 indole pseudoustus;28however,A.pseudoustusistheonlyspeciesknown
position,asopposedtothemorecommonlyobserveddioxepin to produce austamide (discussed below). The first biosynthetic
Table 3. Fungal Origin and Bioactivity of Chrysogenamide A
816 dx.doi.org/10.1021/np200954v|J.Nat.Prod.2012,75,812−833
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Table 4. Fungal Origin of the Brevianamides system. Thus, it was proposed that deoxybrevianamide E is first
convertedto3R-hydroxyindolenine(6)viaanR-selectiveindole
compound fungalspecies bioactivity reference
oxidase,whichcouldthenproceedalongoneofthreepathways.
brevianamideA P.brevicompactum, antifeedant 3a−c,e, Following an irreversible nucleophilic addition, 6 could be
P.viridicatum, insecticidal d,25
P.ochraceuma readily converted to brevianamide E, or 6 could undergo a
brevianamideB P.brevicompactum 3a−c pinacol-typerearrangementtoindoxyl7.Asecondoxidation−
brevianamideC P.brevicompactum 3a−c enolizationsequencewouldthenyieldthechiralandoptically
brevianamideD P.brevicompactum insecticidal 3a−c,25 pure azadiene (3), followed by an IMDA reaction to directly
brevianamideFb A.versicolor, antibacterial 6f,3c,30 give brevianamides A and B. It should be noted that efforts to
P.brevicompactum substantiatethisalternativebiosyntheticpathwayremainelusive
brevianamideE P.brevicompactum 3a−c duetocomplicationsregardingthesynthesisof7.Despiteexten-
deoxybrevianamideE Aspergillussp.MF297-2,c 6a,22, sive synthetic efforts, 7 appears to be unstable and spontane-
A.ustus,dP.ulaiense, 27 ously suffers a retro-Michael reaction to give the dehydroalanine
P.italicum
piperazinedione and the reverse-prenylated indoxyl.
aMisidentified fungal culture; correct identification = Penicillium
Alternatively, reordering these steps would involve the
viridicatum. bWidely distributed biosynthetic precursor; thus only
relevantfungaloriginsarelisted.cUndergoingreidentification(possibly 3R-hydroxyindolenine (6) undergoing two-electron oxidation
Aspergillus sclerotiorum). dReclassified asAspergillus pseudoustus. and enolization to the corresponding azadiene followed by
intramolecularDiels−Aldercycloadditionandfinalpinacol-type
precursor is the common dioxopiperazine brevianamide F rearrangement of the Diels−Alder cycloadducts, each of which
(cyclo-L-Trp-L-Pro).Thisalkaloidhasbeenshowntoserveasthe ■was constituted by a 3R-hydroxyindolenine.
precursor for several related secondary metabolites, those
MARCFORTINES
lacking the bridging bicycle, such as the fumitremorgins and
spirotryprostatins in Aspergillus fumigatus.29 In 1980, Polonsky and co-workers reported the isolation of
Following the isolation of these unique natural products in threenewalkaloidsfromPenicilliumroqueforti(laterreclassified
1969, which were the first metabolites to be isolated to display as P. paneum), an essential fungus used in the production of
theunusualbicyclo[2.2.2]diazaoctaneringsystem,Birchandco- many varieties of blue cheese that contain mold.8a,b From the
workerscarriedoutseveralradiolabeledprecursorincorporation strainB26ofP.paneum,marcfortinesA,B,andCwereisolated
studies in order to elucidate early stages of the biosynthetic (Figure 4). From a structural point of view, these metabolites
pathway of the brevianamides.3a−c,31 Birch demonstrated that contain the bicyclo[2.2.2] ring system with the syn-config-
the brevianamides are derived biogenetically from tryptophan, urationandamonoketopiperazinering.Structurallyresembling
proline, and mevalonic acid and later showed that radiolabeled chrysogenamide A, the marcfortines contain a spiro-oxindole
cyclo-L-Trp-L-Pro (brevianamide F) is also a precursor to moiety and an unsubstituted pipecolic acid unit. Furthermore,
brevianamide A in cultures of P. brevicompactum. On the basis all three marcfortine natural products display substitution on
oftheseresults,BirchproposedthatbrevianamideFarisesfrom the indole ring at the C-6 and C-7 positions; marcfortines A
the condensation of tryptophan and proline, which is followed andBbothcontainadioxepinring,whereasmacfortineChasa
by C-2 reverse prenylation to afford deoxybrevianamide E. pyran moiety.
Oxidation and cyclization of deoxybrevianamide E would give As shown in Table 5, the marcfortines were originally
rise to brevianamides A and B (Scheme 5). isolatedfromastrainidentifiedasP.roqueforti;8a,bhowever,this
Williams later demonstrated through tracer incorporation species was subsequently subdivided into three distinct species,
studies with P. brevicompactum that radiolabeled deoxybrevia- P.roqueforti,P.carneum,andP.paneum,33anditwasdetermined
namide E incorporates into brevianamides A, B, and E in that only the latter can produce marcfortines.8d,e,22,23 Addition-
significant radiochemical yields.32 At the same time, Williams ally, Aspergillus carneus has been reported to produce
showed that neither tritium-labeled brevianamide E nor the marcfortine A;4c however, the isolate producing marcfortine A
proposed racemic, 13C-labeled cycloadduct 5 incorporates into and acyl aszonalenin differed from the culture ex type of
brevianamide A or B when provided to P. brevicompactum in A. carneus, which produced citrinin. Therefore, the identity of
isotopically labeled precursor incorporation studies (Scheme 6). the marcfortine-producing Aspergillus is questionable.
Theseresultsledtothecurrentlyproposedbiosyntheticpathway The biosynthesis of marcfortine A has been investigated by
of brevianamides A and B, as shown in Scheme 6. Since Kuoetal.,and,asshowninScheme7,thismetaboliteisderived
cycloadduct 5 does not incorporate into the brevianamides, it from L-tryptophan (oxindole moiety), L-methionine (via SAM
was suggested that stereoselective indole oxidation must occur methylationofthemonoketopiperazineN),L-lysine(pipecolate
prior to the formation of the bicyclo[2.2.2]diazaoctane ring residue), and acetate (isoprene units).34 On the basis of these
Scheme 5. Birch’s Proposed Biosynthesis of the Brevianamides
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Scheme 6. Alternative Biosynthetic Pathway for the Brevianamides
Scheme 7. Biosynthetic Derivatives of Marcfortine A14
Scheme 8. Two Biogenetic Routes from L-Lysine to
Pipecolic Acid34b
Figure 4. Themarcfortine family ofnatural products.
Table 5. Fungal Origin of the Marcfortines
compound fungalspecies bioactivity references
marcfortineA A.carneus,P.paneum, anthelmintic 8c−e,a,b,f
P.roquefortia
marcfortineB P.paneum,P.roquefortia 8d,e,a,b
marcfortineC P.paneum,P.roquefortia 8d,e,a,b
metabolizes L-lysine to the pipecolic acid unit, Kuo and co-
aReclassified asPenicillium paneum. workers carried out separate feeding studies with [α-15N]- and
[ε-15N]-L-lysine. NMR and MS analysis revealed that the
results, it is clear that the isoprene residues originate from [α-15N]-L-lysinewasincorporatedattheα,β,andγnitrogenof
mevalonic acid; however, there are two possible biosynthetic marcfortine A, with a rate of 1.8%, 13%, and 9%, respectively.
routes for the formation of pipecolic acid from L-lysine, as Contrary to this result, [ε-15N]-L-lysine showed an incorpo-
shown in Scheme 8.34b To determine which pathway ration rate of 54%, 2.7%, and 3.9% respectively, for the three
818 dx.doi.org/10.1021/np200954v|J.Nat.Prod.2012,75,812−833
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nitrogens, thus leading to the conclusion that the [ε-15N]-L- canescens, and P. cluniae, which are all soil fungi and the main
lysine gave a more specific and higher incorporation at the producers of paraherquamides (Table 6).
pipecolate nitrogen. On the basis of these data, pathway A is Structurally, all of the paraherquamides contain the
the preferred method of L-lysine metabolism to the pipecolic bicyclo[2.2.2]diazaoctane core with the syn-configuration;
acid moiety.34b however, the paraherquamide family displays a plethora of
■
skeletal diversity. For example, the paraherquamides exhibit
PARAHERQUAMIDES varying substitution and oxygenation on not only the proline
ring but also the spiro-oxindole moiety, in that several of the
One of the largest groups of metabolites containing the
paraherquamidescontainanunusualdioxepinring,whileothers
bicyclo[2.2.2]diazaoctane ring system is the paraherquamides,
contain a pyran moiety. Within this family of secondary
which were first isolated from Penicillium paraherquei,4a a
metabolites, VM55599 and pre-paraherquamide are the only
synonym of P. brasilianum,35 and later from an isolate ATCC two compounds that do not contain either a spiro-oxindole
20841,originallyidentifiedasP.charlesii.4hHowever,P.charlesii functionality or an oxygenated indole ring system.
was stated incorrectly36 to be a synonym of P. fellutanum by The paraherquamides have attracted considerable interest
Pitt,37 andATCC 20841 has therefore been cited asbelonging due to their potent anthelmintic activity. Parasitic nematodes
tooneofthesespeciesinlaterpublications.Consequently,this causeseverehealthproblemsinhumansanddomesticanimals,
isolate and another similar isolate producing paraherquamides and the severity of this problem is increasing due to the
(IMI 332995)4e represent a new species closely related to development of drug-resistant strains of parasites.38 Initial
P. canescens and will be called hereafter P. cf. canescens. Other anthelmintic activity was detected in Trichostrongylus colubri-
producers of paraherquamides include P. cluniae4d and formis,39 and subsequent investigations showed that para-
Aspergillus aculeatinus.4j herquamide A is effective against strains of parasites that are
Paraherquamide A (Figure 5) was the first member of this resistant to broad-spectrum anthelmintics.40 However, the
family to be isolated by Yamazaki et al. from P. paraherquei toxicity of paraherquamide A was determined to be too great
(=Penicilliumbrasilianum).4aAdecadelater,paraherquamideA for use as a broad-spectrum anthelmintic.41 To decrease the
was isolated from a fungus identified as P. charlesii (= P. toxicity, but still maintain potency, several paraherquamide
fellutanumsensu Pitt37)along withsix structurally relatednovel derivatives have been synthesized and tested for anthelmintic
analogues, paraherquamides B−G.4b,c The original isolate activity.38 Of these derivatives, 2-deoxyparaherquamide A
(ATCC20841)andanewisolate(IMI332995)bothrepresent (Figure6),laternamedderquantel(Pfizer),displayedexcellent
a new species, provisionally called P. cf. canescens. In the activity in models of Hemonchus contortus, T. colubriformis, and
followingyears,theparaherquamidefamilyofprenylatedindole Ostertagia ostertagi, while maintaining a safe level of toxicity.
alkaloids continued to expand with the isolation of para- Manufacturing of 2-deoxyparaherquamide relies on a semi-
herquamides H and I and several related compounds being synthetic procedure wherein paraherquamide is isolated from
reported, including VM55595, VM55596, VM55597, large-scale fermentation of P. simplicissimum, purified, and
VM55599,SB200437,andSB203105.4Morerecently,Sherman, chemically converted to the 2-deoxyparaherquamide ana-
Williams, and co-workers reported the occurrence of pre- logue.38 This new spiroindole was released in July 2010 as a
paraherquamideinculturesoftheparaherquamideA-producing combination treatment with abamectin and is currently
P. cf. canescens, as well as in the asperparaline A-producing marketed under the brand name Startect for use as an
fungus Aspergillus japonicus (later identified as A. aculeatinus).4j anthelmintic in sheep.42
Furthermore,itwasdeterminedthatparaherquamidesAandB Elucidating thebiosynthesisofparaherquamideAhasbeenan
are also produced by A. aculeatinus. P. brasilianum, P. cf. areaofintenseresearchforseveralyears,largelybyWilliamsetal.
Figure 5. Structures ofthe paraherquamides and relatedcompounds.
819 dx.doi.org/10.1021/np200954v|J.Nat.Prod.2012,75,812−833
Journal of Natural Products Review
Table 6. Fungal Origin of the Paraherquamides
compound fungalspecies bioactivity references
paraherquamideA P.charlesii,aP.cluniae,P.brasilianum(=P.paraherquei),Penicilliumsp.IMI332995,a anthelmintic 4b−d,a,e,g,
Penicilliumspecies antinematodal 38−42
paraherquamideB P.charlesii,aP.cluniae antinematodal 4b−d
paraherquamideC P.charlesiia antinematodal 4b,c,h
paraherquamideD P.charlesiia antinematodal 4b,c,h
paraherquamideE Aspergillussp.IMI337664,A.aculeatus,P.charlesii,aP.cluniae, antinematodal 4f,b,c,h,k,d,e,g
Penicilliumsp.IMI332995,aPenicilliumspecies
paraherquamideF P.charlesii,aPenicilliumsp.IMI332995,Penicilliumspecies antinematodal 4b,c,h,e,g
paraherquamideG P.charlesii,aPenicilliumsp.IMI332995,aPenicilliumspecies antinematodal 4b,c,h,e,g
paraherquamideH P.cluniae 4d
paraherquamideI P.cluniae 4d
VM55595 Penicilliumsp.IMI332995a 4e
VM55596 P.cluniae insecticidal 4d
VM55597 P.cluniae,Penicilliumsp.IMI332995a insecticidal 4d,e
VM55599 P.paneum,Penicilliumsp.IMI332995,aA.japonicus,bP.charlesiia 4i,k,e,j
SB200437 Aspergillussp.IMI337664c anthelmintic 4f
SB203105 Aspergillussp.IMI337664c anthelmintic 4f
pre-paraherquamide A.japonicus,bP.charlesiia 4j
aReclassified asa newspecies relatedto Penicillium canescens.bReclassified as Aspergillus aculeatinus.cReclassified as Aspergillus fijiensis.
that the structurally similar paraherquamides follow a com-
parable biosynthetic pathway. To determine which amino acid
residues are incorporated into paraherquamide A, feeding
experimentswereperformedwithP.cf.canescensusing[1-13C]-
L-tryptophan, [methyl-13C]-L-methionine, [1-13C]-L-proline,
and [1-13C]-L-isoleucine (Scheme 9).43 Through 13CNMR
spectroscopy and electrospray mass spectrometry, the position
Figure 6. Structure of 2-deoxyparaherquamide A (derquantel).
and percentage of 13C incorporation were analyzed, and as
expected, [1-13C]-L-tryptophan incorporated at the C-12
On the basis of the results obtained from the precursor incor- (paraherquamide numbering) position of the oxindole ring
poration studies of the brevianamides, this group postulated with 2.5% incorporation. Moreover, similar to results obtained
Scheme 9. (A) 13C-Labeled Amino Acid Incorporation into Paraherquamide A in Penicillium cf. canescens; (B) Proposed
Biosynthetic Pathway for the Conversion of L-Isoleucine to 3(S)-Methyl-L-proline43
820 dx.doi.org/10.1021/np200954v|J.Nat.Prod.2012,75,812−833