Cyanide and thiocyanate-based biosynthesis in tropical marine sponges

9 Pages·1999·3.2 MB·English

by  J S Simpson

CYANIDE AND THIOCYANATE-BASED BIOSYNTHESIS IN TROPICAL MARINE
SPONGES
JAMIE S. SIMPSON AND MARY J. GARSON
Simpson, J.S. & Garson, M.J. 1999 06 30: Cyanide and thiocyanate-based biosynthesis in
tropical marinesponees. MemoirsoftheQueenslandMuseum44: 559-567. Brisbane. ISSN
0079-8835.
The sponge Axinyssa n.sp. incorporates both sodium l4C] cyanide and sodium 14C]
[ [
thiocyanateinto2-thiocyanatoneopupukeananeaswellas into9-isothiocyanatopupukeanane,
however these 2 precursors were not incorporated into 9-isocyanopupukeanane. The
specificity of incorporation into the thiocyanate carbon was confirmed by chemical
degradation. Stylotella aurantium incorporates sodium 14C] cyanide and sodium l4C]
thiocyanate intothedichloroiminefunctionalityofthestyl|otellanesAand B,aswell as[ into
farnesy isothiocyanate.Thespecificityofincorporationintothedichloroiminecarbonatom
I
was confirmed by chemical degradation. These experiments represent the first detailed
studyofthebiosyntheticoriginoforganicthiocyanatesanddichtoroimines, andextendthe
range of functionality known to be biosynthesised from cyanide and thiocyanate. Our
results raise the interesting question of the interconversion of inorganic cyanide and
thiocyanate and/or the interconversion of the resulting organic metabolites in marine
sponges. An isothiocyanate-isocyanide conversion was demonstrated in Amphimedon
terpenensis by incorporation of a l4C-labelled sample of diisothiocyanatoadociane into
diisocyanoadociane.OPorifera, Amphimedonterpenensis,Axinyssa, Stylotellaaurantium,
biosynthesis, cyanide, dichloroimines. isocyanides, isothiocyanates,secondarymetabolites,
terpenes, thiocyanates.
JamieS. Simpson& MatyJ. Garson(email:[email protected]). Departmentof
Chemistry, UniversityofQueensland, StLucia4072, Australia; 30November 1998.
Marine sponges of the order Axinellida, Furthermore inorganic thiocyanate was shown
Halichondrida and Haplosclerida often contain also to be a precursorto both the isocyanide and
bioactive terpenes with isocyanide, isothio- the isothiocyanate metabolites in this sponge.
cyanate and formamide functionality; the rarer From these experimental results a biosynthetic
isocyanate and thiocyanate substituents are also link was inferred between the two inorganic
known (Scheuer, 1992; Chang & Scheuer, 1993; precursors or between the two metabolite types
Garson et al., 1998). These unique metabolites (Dumdei et al., 1997).
have been novel targets for study with ,4C- and
The origin ofthe thiocyanato group has been
l3C-labelled precursors to determine the bio- the subject of much biosynthetic speculation
saytnotmhe(tiGcarsoorni,gin19o8f9;thCehannogn-&terSpcehneouiedr,ca1r9b9o0n; (Garson, 1993). Pham et ai. (1991)suggested the
cyanation of a thiol, which appears to be a
Garson, 1993; Garson et al., 1999). Workby our reasonable pathway to the amino acid-derived
research group on the sponge Amphimedon psammaplin thiocyanate (Jimenez & Crews,
terpenensis has shown that marine isocyanides 1991). In contrast, in those sponges in which
suchasdiisocyanoadociane(Fig. 1A)arederived thiocyanates co-occur with isocyanides or with
by functionalisation ofa terpene precursor with isothiocyanates,theinvolvementoftheambidenl
i1n9o8r8g)a.niKcarcuysaonid&e S(cGahresuoenr,(11998869;)Fsouobkseesqueetntall.y, t1h9i9o2c;vaWnaaltkeerio&nhBaustlbeere,n1i9n96v)o.keTdhe(Hdeicehtlaol.r,o1i9m8i9n;e
showed that both diterpene (eg. Fig. IB) and (= carbonimidic dichloride) moiety represents a
sesquiterpene (eg. Fig. 1C) isocyanides are rare example of a functional group containing
cyanide-derived, and further demonstrated the both nitrogen and carbon which has previously
intact incorporation oftheNpCi unit. Ourrecent been found in terpene metabolites ofthe Indo-
work with Acanthella cavernosa has shown the Pacific sponge Pseudaxinvssa pitvs (Wratten &
utilisationofcyanide forthebiosynthesisofboth Faulkner, 1977; 1978a; 1978b). Thecoocurrence
a sesquiterpene isocyanide (Fig. ID) and an ofan isothiocyanatetogetherwith dichloroimines
isothiocyanate (Fig. 1E) inthis axinellid sponge. in P. pitys suggested to us the involvement of
.
560 MEMOIRS OF THE QUEENSLAND MUSEUM
Island (23°27\S, 151°55'E) or at North Point
(12-16m depth). Lizard Island (H^'S,
145°27'E) on the Great Barrier Reef, Australia
Of
under permit numbers G96/050, G97/097,
G98/037andG98/227 issuedjointly bytheGreat
Barrier Reef Marine Park Authority and the
QueenslandNationalParksand WildlifeService.
Sponge samples used in biosynthetic exper-
iments were maintained in running seawatcr at
ambienttemperatureandlightconditionspriorto
use. Voucher specimens ofthe spongesAxittyssa
n. sp., (accession number QMG312575), Stylo-
tteelrlpaeanennrsainst,i(uAmM(ZQ4M9G7380;7Q1M33G)3an1d42A2m8)p,hiarmcehdeolnd
Q R=NC at the Queensland Museum (QM), Brisbane or
£ R NCS the Australian Museum (AM), Sydney.
Isolationofmetabolites. I)Axinyssan. sp. Anor-
FIG. 1. Structures of isocyanide and isothiocyanatc ganic extract was prepared from frozen sponge
metabolites investigated in biosynihetic experi- (49.6g wet wt) and further purified by normal
ments. A, diisocyanoadociane. B, kalihinol F. C,
2-isocyanopupukeanane. D, axisonitrile-3. E, phase flash chromatography (gradient elution
with hexanes/EtOAc) and normal phase HPLC
axisothiocyanate-3.
using 0.25% EtOAc in hexanes to give
cyanide/thiocyanate in the biosynthesis of the (-)-9-isocyanopupukeanane (Fig. 2A; 107.6mg),
dichloroimine group. (-)-9-isothiocyanatopupukeanane (Fig. 2B;
In this paper we present the results of bio- 3.5mg), and (-)-2-thiocyanatoneopupukeanane
synthetic experiments with the sponge Axinyssa (Fig. 2C; 31.4mg)togetherwith smalleramounts
n. sp. which provide evidence for a cyanide/ of other isocyanides and isothiocyanates as
thiocyanate origin of the thiocyanato function- describedbySimpsonetal. (1997b).2)Stylotella
ality. We test the possibility using Stylotella aurantium. Anorganicextractwaspreparedfrom
auremtium that dichloroimine metabolites are frozensponge(204gwet wt) and furtherpurified
biosynthesisedfromfarnesylpyrophosphateusing by normalphase flashchromatography (gradient
cyanide or thiocyanate to supply the Ni-Cj elution with hexanes/EtOAc) and by normal
moiety. The role ofinorganic thiocyanate and of phase HPLC using 0.2% EtOAc in hexanes to
an organic isothiocyanatc in diisocyanoadociane givestylotellaneA(Fig.2E;9mg), stylotellaneB
biosynthesis are also explored. (Fig. 2F; 75.6mg), and farnesyl isothiocyanate
(Fig. 2G; 2mg) as described by Simpson et al.
MATERIALS AND METHODS ( 1 997a). 3) Amphimedon terpenensis
Diisocyanoadociane (Fig. 1A; 16mg) was
Abbreviations.GC-MS,gaschromatography-mass isolated from frozen sponge (25g wet wt) as
spectrometry; TLC, thin layer chromatography; describedby Fookes et al. (1988).
NMR, nuclear magnetic resonance; HPLC, high
Biosynthetic experiments. 1) Pieces ofAxinyssa
performance liquid chromatography.
n. sp. (approx. 80g wet wt) were placed in an
Chemicals and biochemicals. Solvents used in aquarium containing 200ml aerated seawater at
the extraction of compounds from sponge ambient temperature (20-23°C Sodium 14C]
samples were glass distilled. All radioactive cyanide (100|iCi) or sodium |)4.C] thiocya[nate
[
precursorswerepurchasedfromSiijmaChemical (25uCi) was added and the sponge allowed to
Co. (St Louis, MO). assimilateradioactivityfor 12hr. Thespongewas
Biologicalmaterials. Samples ofAxinyssa n. sp. keptinrunningseawaterina 1 litre aquarium at
(Halichondrida: Halichondriidae), Stylotella ambient temperaUire for 16 days, then frozen for
entrantium (Halichondrida: Halichondriidae), subsequent radiochemical analysis. Metabolites
Kelly-Borges & Bergquist, 1988, and Amphi- were purified according to the above protocol.
medon terpenensis (Haplosclerida: Niphatidae) The radioactivity content was monitored at each
Fromont, 1993, were collected using SCUBA at stage ofthe purification sequence, and terpenes
Coral Gardens, Experimental Gardens or Coral weresubjectedtorepeatedHPLCuntilthespecific
Spawning dive sites (12-16m depth), Heron activitywasconstant.2)Stylotellaaurantium(24g
d
I
BIOSYNTHESIS IN TROPICAL MARINE SPONGES
wet wt) was placed in an
aquarium containing 200ml
aerated seawater at ambient
temperature(20-2S°C Sodium
)
I ' C] cyanide (50|lCi ) was F
addedand thespongeallowedto
assimilate radioactivity lor I2hr
overnight. Thi was kept
i
ill running seawater in ;i 10L
aquarium at ambient temp- R Hi I I
erature for 9 days, then frozen m tJ t |i r r mi
for subsequent radiochemical
bis. Metabolites were pur-
ified according to the above
protocol.The radioactivity con-
tentwasmonitoredat each
oi the punlie. aience. A
um *C] thiocyanate
(UpCii; C)[ d' ays incorporation) PIG, i olated i ..,
experiment, used a 1Zg piece of 'Mm. lucts. -\,
sponge. 3) Ampkimedon ter- thiol from 2-thioayanatunei opupuk!eauatii lo Hani
penetfuis (26g wet wt) was ilellane B. 0, famesyl isothi liiyf cai w«ni ran
placed in an aquarium nylotellane B. I, amine fVom stylot
tontaining 4 ml., acrate
seawateratambienttemperature 121)-_'3°CJ | ' 11- innci tissue contained high populai
Diisothiocyanatoadocianei, 1 1 uCi)wasadded and diverse bacterial cell types ill addition t1
t1h2ehrsspoonvegrenaiglhlto.weTdhetosapsosnimgielawtaesrakedpitoaienfrnuintning mceelmlbsranAen-bAorucnhdaneuac-lleoiikde symbianl tIiers!
seawater in a 20L aquarium at ambient er ai., this volume; Kierst ct al., L991
stuebmspeeqruaetnutreradii\o*rche1m9icdaalysa,naltyshiesn. MFrcotzaebn- Poi ipCi iSmoedniuoml A| 'x[iCn]ystxhaiocn.ysapn.atmeaiwnatsaisnuepdpliineda
Twhereerapduiroiafciteidviatycccoorndtienngttwoasthemoanbiotvoeredpraottoecaoclh. . 98i)m.1 1AHlntenrdeiISetdaaLy,s19a9q7u;arSiaumps
i of the purification sequence. A sodium bation. the sponge sample was fro/en and
\]lC\ thiocyanate (50pCi; 19 days incorporation) : Mianc (Fig. 2CJ
experiment used a 45g piece ofsponge. N ited and rigor: ou;sly purified by MPLC to
Procedures used in the synthesis oi I CJ- constant specific radioactivity. The thu>e>
edlisieswohtehrieoc(ySainmaptsooando&ciGaanresowni.llin bperepdatration). (iFniTga.b2lCe :I consistneinfftcawntiltnytrhaediuosaectoifvet,hiaoscsyianate
for the biosynthesis ofthe thioeyanato group as
RESULTS
shown in Pig 3, To test the specificity oi n
corporation, 2-thiocyana
sIe)sqIuv/i't/ne.rvspYe/nne.smpetacbololleictteesdbayt HGeCr-oMnSI,. cTonLtCaiannedd . 2C) was degraded lo die thiol (J
N\1K; the hc\anc-soluh|es were processed as using L1AIH4. The thiol product was not radio-
tJ-purrpiubkeedainnanSeimpissoocnyaentiadle. 1is1o9t9h7ibo)cytaonaglievepdaiier eacxtcilvuesi(vTealbyleas2s)octihaetreefdorweitthhe t|he'V| label nato
carbon, Incorporation of sodium C]
(lag. 2A,H) and 2-thiocyanatoneopupukeanane f
(Fig. 2Cy The GC-MS profile of the sesqui- into a second piece of sponge also gave radio-
terpene fraction showed a number ofother peaks active 2-Lhiocvanaloncop" Lnfi (Tab
including isoeyamdcs and isothiocyanates. Light Degradation resulted in unlabclled thiol product
able 2) indicating the label was again
and electron microscopic inspection ofAxinysso t I
n.sp. revealed the presence of mieiohial syni- elusivelyassociated wiih Lb anatomoiety
^
bionts. The outer layers sponge tissue were Our experiments i1 monitor
rich in cyanobactei ia ofa or] ically isocyanide/isothiocyanate biosynthesis in thia
similar to Aphanocapsa feldmanm while the sponge When die isoc)anide/teothiocyanaie pair
562 MEMOIRS OF THE QUEENSLAND MUSEUM
TABLE 1. Molarspecific activities ofAxinyssa n. sp. TABLE 2. Molarspecific activities ofAxinyssa n. sp.
metabolites, a, published incorporation values were degradationproducts.
notpercentage values (Simpson& Garson, 1998); b,
incorporation of25uCi; c, <10~2%; d, incorporation Compound Molarspecific Radioactivity
oflOO^Ci. (Fig.no.) Precursor (mCaic/timvMitoyle) (%)
Compound Molarspecific Incorporation^ 2C Na[14qSCN 0.150 100.0
(Fig.no.) Precursor (mCaic/timvMitoyle) (%) 2D Na[l4C]SCN O.001 0.3
2C Na[,4C]CN 1.230 100.0
2A Na[l4C]SCNb 0.004 <=
2B Na[l4C]SCNb 2.630 0.08 2D Na[I4C]CN 0.001 0.1
2C Na[l4C]SCNb 0.150 0.02
shown in Table 3, consistent with the use of
2A Na[l4C]CNd 0.014 c
cyanideforthebiosynthesisofthedichloroimine
2B Na[l4ClCNd 13.900 0.3 group(Fig.4,routenotation 'a'). Thepercentage
2( Na[l4C]CNd 1.230 0.2 incorporation levels measured were low as a
result of loss ofvolatile metabolites during the
(Fig. 2A,B) were isolated, the isothiocyanate purificationprocess combinedwiththe chemical
samples were radioactive (>150,000dpm/mg), instabilityofthedichloroiminegroup.Totestthe
whereas the isocyanide samples from both specificity of incorporation, stylotellane B was
thiocyanate and cyanide feedings were not degraded to the methyl carbamate (Fig. 2H) and
significantly labelled (<100dpm/mg). The theamine(Fig. 21) using0.INphosphoricacidin
specificity of labelling of 9-isothiocyanato- 95% methanol. The carbamate product was
pupukeanane is currently under investigation. radioactive, whereas the amine was devoid of
2) Extracts of the sponge Stylotella aurantium wraadsioaecxtcivliutsyiv(eTlaybleas4s),octihaetreedforweitthhe t[h14eC]imlaibneel
weakly inhibited the growth of a P388 mouse carbon. Incorporation of sodium ,4C] thio-
TleLuCkaaenmdiaNcMeRll.lTinheeaDnCdM-csonotlauibnleed cteormppeonneesnbtys cyanate into a secondpiece ofsponge[ also gave
radioactivemetabolites (Table 3), howeverthere
oaSfnidmtphseBone(xFettirgaac.l.t(2w1Ee9,9rF7e)a,)prttooocggeiesvtseheetdrheawssitytdlehostcefrlailrbaneneedssyiAln awtaison.inIsnufefiaccihenetxpmearteirmieanlt,fotrhecihseomliactaeld fdaergnreasdy-l
isothiocyanate (Fig. 2G) was also radioactive.
isothiocyanate (Fig. 2G). Light microscopic in-
spectionofspongetissuerevealedthe absenceof 3) 50uGi Sodium 14C] thiocyanate was then
[
microbial symbionts otherthan bacteria. supplied to a specimen of A. terpenensis
50uGi Sodium I4C] cyanidewas suppliedto a accordingtoourestablishedprotocols(Fookeset
[
specimen of S. aurantium according to our al., 1988; Dumdei et al., 1997). After 19 days
established protocols (Dumdei et al., 1997; aquarium incubation, the sponge sample was
Simpson et al, 1997a). After 9 days aquarium frozen and diisocyanoadociane isolated and
incubation, the sponge sample was frozen and rigorouslypurifiedby HPLC, thenrecrystallised
stylotellanes A and B were isolated and to constant specific radioactivity. The sample
rigorouslypurifiedbyHPLC toconstantspecific was significantly radioactive consistent with the
radioactivity. The samples ofstylotellanesAand use of thiocyanate for the biosynthesis of the
B (Fig. 2E,F) were significantly radioactive, as isocyanide group (Fig. 5). Degradative exper-
iments are in progress to
confirm the specific
$ labelling. When a sample of
sex.
^5 • diisothiocyanatoadociane,
l4C-labelled in both isothio-
CN- SCN- cyanate groups, was
f provided to A. terpenensis,
the diisocyanoadociane
isolated was found to be
radioactive. The specificity
FIG 3. Incorporation into isothiocyanate and thiocyanate metabolites of °* labelling is under
Axinyssan. sp investigation.
BIOSYNTHESIS IN TROPICAL MARINE SPONGES 563
TABLE 3. Molar specific activities ofS. aurantium TABLE 4. Molar specific activities ofS. aurantium
metabolites, a, incorporation of 50uCi; b, degradation products, a, after dilution with
incorporation of13uCi. unlabelled metabolite.
C(oFimgp.onuon.)d Precursor M(omlCaaicr/timsvpMieotcylifei)c Incor<p%or)ation C(oFmigp.onuon.)d Precursor M(omlCaaicrt/imsvpMietocylifei)c Radioactivity
2E Na[l4C]CNa 1.136 0.004 2F Na['V]CN 0.332a 100.0
2F Na[,4C]CNu 1.472 0.033 2H Na[uC]CN 0.326 98.2
2E Na[l4qSCNb 0.354 0.00034 21 Na[l4C]CN 0.004 1.2
2F Na['4C]SCNb 0.224 0.00056
utilisation ofthis ambident precursor. Likewise
cyanide is utilised for both thiocyanate and iso-
DISCUSSION thiocyanate biosynthesis. Our proposal is that
cyanide is converted in Axinyssa n. sp. to thio-
OurbiosynthelicexperimentswithAxinyssan. cyanate by the action of an enzyme similar to
sp. and with S. aurantium, together with the rhodanese (Scheivelbein et al., 1969; Westley,
earlierwork on A. terpenensis andA. cavernosa 1973) and then incorporated into either
(Garson, l986;FookesetaL, 1988;DumdeietaL, 9-isothiocyanatopupukeanane or 2-thiocyanato-
1997), reveal that cyanide and thiocyanate are neopupukeanane. The alternative possibility that
precursors involved in the biosynthesis of four cyanide is converted first to the isocyanide then
NpCi functional groups found in marine ter- by sulphur insertion to an isothiocyanate is less
penes, namely isocyanides, isothiocyanates, likely since cyanide was not utilised for the bio-
thiocyanates and dichloroimines. synthesis of9-isocyanopupukeanane inthe same
A number ofdifferent biosynthetic pathways specimen ofAxinyssa n. sp.
can be invoked to explain the origin ofthe thio- Stylotella aurantium uses both cyanide and
cyanate group. Pham et al. (1991) suggested the thiocyanate as precursors for the biosynthesis of
cyanation of a terpene thiol, however this the dichloroimine and isothiocyanate groups.
proposal does not adequately explain the co- Fig.4showstwoplausiblebiosynthetic routesto
occurrence of thiocyanates and isothiocyanates the stylotellanes A and B, one route (a)using an
inthe samesponge. The insertionofsulphurinto isonilrile intermediate andthe other(b) invoking
an organic cyanide or isocyanide to give a an isothiocyanate intermediate. The isolation of
thiocyanate is mechanistically unprecedented. farnesyl isothiocyanate, but not of farnesyl
Sulphur insertion into an isocyanide to give an isocyanide (Fig. 4A), from this sponge is con-
isothiocyanate (Hagadone et al., 1984), perhaps sistent with the operation of path (b). The
using an enzyme functionally equivalent to dichloroimine metabolites are among the most
rhodanese (Westley, 1973), followed by iso- unusual ofthe cyanide and thiocyanate-derived
merisation of the isothiocyanate to the terpenes. In the laboratory, isocyanide dihalides
thiocyanate is a plausible biosynthetic pathway. can be synthesised by addition of chlorine to
In the laboratory however, the isothiocyanate- isocyanidesorbychlorinationofisothiocyanates
thiocyanate equilibrium usually favours an (Ktihleetal., 1967).Thebiosyntheticmechanisms
isothiocyanate over a thiocyanate (Hughes, proposed invoke the use of a chloroperoxidase
o1f975a)n.aAmbfiindaelntbiotshyinotchyeatniacteposasniiboilnittyoisattthaeckusea eWanlzkyemre, 1t9o93c;hlWoarilnkaetre&inBtuetrlmeerd,ia1t9e9s6).(Butler &
terpene carbenium ion or its functional equiv- In A. terpenensis, our results are consistent
&
alent (He et al., 1989; 1992; Walker Butler, with the use ofboth thiocyanate and ofcyanide
1996). Thiocyanate either reacts through the for isocyanide biosynthesis. Thiocyanate may
nitrogen centre generating an isothiocyanate perhaps be converted to cyanide by use of a
derivative or through the sulphur generating a peroxidase enzyme, as hasbeen demonstrated in
thiocyanate. some bacteria (Ohkawa et al., 1971; Pollock &
Results on the biosynthesis ofthe thiocyanate Goff, 1992;Westley, 1981),whichisthenutilised
moiety are particularly informative. The incorp- for isocyanide biosynthesis (Fig. 5).
oration of inorganic thiocyanate into the We havepreviously suggestedthatA. cavernosa
thiocyanate and isothiocyanatemetabolites (Fig. is able to interconvert inorganic cyanide and
2B,C) ofAxinyssa n. sp. is consistentwith direct thiocyanate (Dumdei et al., 1997). Our current
564 MEMOIRS OF THE QUEENSLAND MUSEUM
sponge contains isothio-
cyanates as minor met-
abolites (unpublished
results). When radiolabel-
ed diisothiocyanatoadociane
was supplied to samples of
A. terpenensis, the
diisocyanoadociane
isolated was shown to be
radioactive. Chemical
degradation is currently in
progress to confirm the
specificity of labelling in
this advanced precursor
experiment.
In view ofthe previous
successful experiments
with both diterpene
isocyanides (Garson,
1986;FookesetaL, 1988;
FIG. 4. Biosynthesis of dichloroimines in Stylotella aurantium. A, farnesyl Karuso & Scheuer, 1989)
isocyanide. B, farnesyl isothiocyanate. C, stylotellaneA. D, stylotellane B. and sesquiterpene
isonitriles (Karuso &
results suggestAxinyssan. sp., & aurantium and Scheuer, 1989; Dumdei et al, 1997), it is quite
A. terpenensis are able to interconvert these 2 extraordinarythat we have not demonstratedthe
inorganic precursors. We have also speculated incorporation of cyanide into the major iso-
that enzymic transformations which parallel the cyanide component (Fig. 6) ofAxinyssa n. sp.
cyanide-thiocyanate interconversion may Likewise thiocyanate appears not to be used for
transform organic isocyanides into isothio- isocyanide biosynthesis in this sponge, in
cyanates, or the reverse, in marine sponges contrast toA. cavernosa in which thiocyanate is
(Dumdei et al, 1997). Figure 6 illustrates these used for isocyanide biosynthesis (Dumdei et al.,
suggestedbiosyntheticrelationshipsforAxinyssa 1997) and also in contrast toA. terpenensis (this
n.sp. Thiocyanate is used to make 9-isothio- paper). The lack ofincorporation ofthiocyanate
cyanatopupukeanane which then undergoes into 9-isocyanopupukeanane (Fig. 6) suggests
desulphurisation togive9-isocyanopupukeanane; that eitherthethiocyanate to cyanide conversion
alternatively, cyanide is used forisocyanide bio- isinefficientinthisspongeorthattheconversion
synthesis, then the isocyanide is converted into ofisothiocyanate into isocyanide does not occur.
the isothiocyanate by an enzyme functionally We are currently isolating some of the other
equivalent to rhodanese. minor isocyanide metabolites from Axinyssa
In pioneering biosynthetic experiments, samples labelled by thiocyanate or cyanide in
Hagadone et al. (1984) inferred the precursor order to investigate the role of cyanide and
status of an isocyanide terpene metabolite in thiocyanate in isocyanide biosynthesis in this
isothiocyanate formation in Ciocalypta sp. sponge. A clearer picture ofthe complex meta-
They explored the in vivo conversion of 2-iso- bolic interrelationships in Axinyssa n. sp. will
cyanpupukeanane into the corresponding emerge when we test the utilisation of
formamide and isothiocyanate metabolites. The
natural product status offormamide metabolites CN
has however been questioned by Tada et al.
(1988). A second concern with the work of
Hagadone et al. (1984) is their use of the
relatively insensitive l C label in conjunction
with mass spectrometric detection.
SCN-
Our preliminary results with A. terpenensis
suggest that an isothiocyanate to isocyanide FIG. 5. Biosynthesis of diisocyanoadociane in A.
transformationmayoccurinthis sponge.The terpenensis.
BIOSYNTHESIS IN TROPICAL MARINE SPONGES 565
now exploring Nj-Cj biosynthesis provide us
with additional candidates to study the cellular
localisationofterpenemetabolitesandtoexplore
the role ofsymbionts in biosynthesis.
TAXONOMIC NOTE. The sponge which we
have identified as 'Amphimedon'' terpenensis in
this paper has a chequered taxonomic history. It
was firstnamed in the literature as anAdocia sp.
bytheRochegroup(Bakeretal., 1976). Fromont
(1993)placed the sponge withinAmphimedon in
hertaxonomicstudiesonhaploscleridspongesof
the Great Barrier Reefand proposed the species
Q-isocyanopupukeanane 9-isothiocyanatopupukeanunc name for the large proportion of terpene
FIG. 6. Possible biosynthetic interconversions in metabolites. Van Soest et al. (1996) considered
Axinyssa n. sp. Solid lines indicate incorporation the skeletal characteristics were too irregular to
results or possible conversions, dotted lines indicate be compatible with Amphimedon. Based on
non-incorporation. structural characteristics and spicule analysis,
they proposed the combination Cymbastela
l4C-labelled isocyanide and isothiocyanate terpenensis, but acknowledged howeverthat the
precursors by this sponge. skeletalmorphology,growthformandtexturefor
The origin of the cyanide or the thiocyanate tdheescsrpiboendgebyweHroeopnoetrt&ypiBcearlgqoufisCtym(b1a9s9t2e)l.a,Thaes
used by marine sponges remains a tantalising
documented secondary metabolite chemistry of
mystery. Plants generate hydrogen cyanide by-
Cymbastela spp. consists ofpyrrole metabolites
hydrolysis of cyanogenic glycosides (Seigler,
1975). Some bacteria are known to produce from a New Caledonian species (Ahond et al.,
hydrogen cyanide (Knowles, 1976) orto convert 1988). SamplesofCymbastelasp.collected from
the amino acid cysteine to thiocyanate (Voet & HmeetraobnolIi.teanpdrofLiilzeabrydNI.MdRo naontdhGaCv-eMSa,sebcuotnhdaavrey
Voet, 1995),whilemethioninehasbeenimplicat-
ed in the formation ofcyanide as a byproduct of been shown to contain 24-isopropyl-5:' sterols
ethylene biosynthesis (Pirrung, 1985). To date (Stoilov et al., 1986), whereas A. terpenensis
experiments to determine an amino acid origin contains8 ' -sterols(Garsonetal., 1988).A more
for the isocyano group in diisocyanoadociane thorough taxonomic assessment of 'A/ ter-
have been unsuccesful (Fookes et al., 1988). penensis (and the related C. hooperi), including
consideration of live specimen characteristics,
Two sponges used in our biosynthetic ex- growth form, texture and spongin content and
periments have interesting symbiotic profiles. skeletal structure is required. It is possible that a
Amphimedon terpenensis has previously been new genus is required for these species but this
shown to contain high bacterial populations of requires substantially more corroborative
eubacteria together with a cyanobacterial evidence than is presently available (e.g. genetic
symbiont which morphologically resembles analyses).Forthepresentweretainthetaxon 'A.'
Aphanocapsafeldmcmni (Garson et al., 1992). terpenensis, but acknowledge it does not belong
Axinyssa n.sp. contains a cyanobacterial sym- with typical members ofAmphimedon (Haplo-
biont together with numerous bacteria, in sclerida; Niphatidae).
particular an archaeal-like bacteria which con-
tainsahighlyunusualmembrane-boundnucleoid ACKNOWLEDGEMENTS
(Fuerst,thisvolume;Fuerstetal., 1998).Wehave
previously demonstrated that A. terpenensis We thank John Hooper, Queensland Museum,
isocyanides are localised in sponge cells, for taxonomy, the Australian Research Council
primarily archaeocytes and choanocytes, and forfundingandtheAustralianGovernmentforan
infer that this is the site of synthesis of the APRA scholarship. The assistance ofthe staffof
metabolites (Garson et al., 1992). Terpene HeronIslandandLizardIslandResearchStations
metabolites in 2 other sponges have been shown in performing field work is gratefully acknow-
to be localised in sponge cells rather than sym- ledged.Thisresearchwasperformedunderpermits
biont cells (Uriz et al, 1996; Flowers et al., G96/050, G97/097, G98/037 andG98/227 issued
1998). The range ofsponges with which we are jointly by GBRMPA and QNPWS.
566 MEMOIRS OF THE QUEENSLAND MUSEUM
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