Biosynthetic pathways to isocyanides and isothiocyanates; precursor incorporation studies on terpene metabolites in the tropical marine sponges Amphimedon terpenensis and Axinyssa n.sp.

Article abstract of DOI:10.1039/b315894b

The biosynthetic origins of the isocyanide and isothiocyanate functional groups in the marine sponge metabolites diisocyanoadociane (1), 9-isocyanopupukeanane (10) and 9-isothiocyanatopupukeanane (11) are probed by the use of [(14)C]-labelled precursor experiments. Incubation of the sponge Amphimedon terpenensis with [(14)C]-labelled thiocyanate resulted in radioactive diisocyanoadociane (1) in which the radiolabel is specifically associated with the isocyanide carbons. As expected, cyanide and thiocyanate were confirmed as precursors to the pupukeananes 10 and 11 in the sponge Axinyssa n.sp.; additionally these precursors labelled 2-thiocyanatoneopupukeanane (12) in this sponge. To probe whether isocyanide-isothiocyanate interconversions take place at the secondary metabolite level, the advanced precursor bisisothiocyanate 17 was supplied to A. terpenensis, but did not result in significant labelling in the natural product isocyanide 1. In contrast, in the sponge Axinyssa n.sp., feeding of [(14)C]-9-isocyanopupukeanane (10) resulted in isolation of radiolabelled 9-isothiocyanatopupukeanane 11, while the feeding of [(14)C]-11 resulted in labelled isocyanide 10. These results show conclusively that isocyanides and isothiocyanates are interconverted in the sponge Axinyssa n.sp., and confirm the central role that thiocyanate occupies in the terpene metabolism of this sponge.

Full text of DOI:10.1039/b315894b

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Biosynthetic pathways to isocyanides and isothiocyanates;
precursor incorporation studies on terpene metabolites in the
tropical marine sponges Amphimedon terpenensis and
Axinyssa n.sp.†‡
Jamie S. Simpson§ and Mary J. Garson*
Department of Chemistry, School of Molecular and Microbial Sciences,
The University of Queensland, Brisbane QLD 4072, Australia.
E-mail: m.garson@mailbox.uq.edu.au
Received 9th December 2003, Accepted 30th January 2004
First published as an Advance Article on the web 26th February 2004
The biosynthetic origins of the isocyanide and isothiocyanate functional groups in the marine sponge metabolites
diisocyanoadociane (1), 9-isocyanopupukeanane (10) and 9-isothiocyanatopupukeanane (11) are probed by the
use of [14C]-labelled precursor experiments. Incubation of the sponge Amphimedon terpenensis with [14C]-labelled
thiocyanate resulted in radioactive diisocyanoadociane (1) in which the radiolabel is specifically associated with the
isocyanide carbons. As expected, cyanide and thiocyanate were confirmed as precursors to the pupukeananes 10 and
11 in the sponge Axinyssa n.sp.; additionally these precursors labelled 2-thiocyanatoneopupukeanane (12) in this
sponge. To probe whether isocyanide-isothiocyanate interconversions take place at the secondary metabolite level, the
advanced precursor bisisothiocyanate 17 was supplied to A. terpenensis, but did not result in significant labelling in
the natural product isocyanide 1. In contrast, in the sponge Axinyssa n.sp., feeding of [14C]-9-isocyanopupukeanane
(10) resulted in isolation of radiolabelled 9-isothiocyanatopupukeanane 11, while the feeding of [14C]-11 resulted in
labelled isocyanide 10. These results show conclusively that isocyanides and isothiocyanates are interconverted in
the sponge Axinyssa n.sp., and confirm the central role that thiocyanate occupies in the terpene metabolism of this
sponge.
Introduction
Marine invertebrates produce an abundance of unique and
bioactive compounds, notable among which are the terpene iso-
cyanides.1,2 Following the first reports of their isolation in the
mid-1970’s,3 interest in the biosynthesis of these metabolites,
particularly in the origin of the isocyanide group,3b,4 has led to a
number of experimental studies on them.5,6 Research from our
group was the first to show that the isocyanide groups of diiso-
cyanoadociane (1) are derived from inorganic cyanide through
incorporation of [14C]-cyanide into the sponge Amphimedon
terpenensis. The radioactivity in the resulting samples of the
diterpene 1 was proven to be specifically associated with the
isocyanide carbons through stepwise hydrolytic degradation to
the corresponding formamide 2, and then to amines 3 and 4.5
Subsequent studies by Karuso and Scheuer showed that [14C]-
cyanide is a precursor for 2-isocyanopupukeanane (5) in Cio-
calypta sp. and the diterpene kalihinol F (6) in Acanthella sp.
They also confirmed that the carbon and nitrogen atoms of
the isocyanide moiety in 9-isocyanoneopupukeanane (7) are
derived intact from [13C, 15N]-cyanide.6b
Many terpene isocyanides are isolated with the corresponding
organisms.9,11 Incorporation studies with Australian specimens
of Acanthella cavernosa established that thiocyanate ion was a
precursor to the isothiocyanate moiety in axisothiocyanate-3
isothiocyanate substituted metabolites, for example the spiro-
axane axisonitrile-3 (8) together with axisothiocyanate-3 (9)7
and the tricyclic 9-isocyanopupukeanane (10) with 9-iso-
(9), but more surprisingly, that this precursor also labelled the
thiocyanatopupukeanane (11).8–10 The related thiocyanates
isocyanide group of axisonitrile-3 (8).12 Furthermore, cyanide
was a precursor to both the isocyanide and isothiocyanate
(such as 12), have been isolated less frequently from marine
moieties in this sponge. These results suggested that marine
sponges might interconvert cyanide and thiocyanate at the
inorganic level (Scheme 1; reactions a and b). The conversion of
cyanide to thiocyanate by rhodanese is a well-studied irrevers-
ible reaction in microorganisms, plants and higher animals,13
while peroxidases are known to catalyse the oxidation of
thiocyanate to cyanide and sulfate.13b Alternatively, marine
isocyanides and isothiocyanates might be interconverted at
† Dedicated to the memory of Paul J. Scheuer (1915–2003) who was a
pioneer of marine isocyanide chemistry.
‡ Electronic supplementary information (ESI) available: 1H and 13C
NMR spectra for compounds 14–18. See http://www.rsc.org/suppdata/
ob/b3/b315894b/
§ Current address: Research School of Chemistry, Institute of
Advanced Studies, The Australian National University, Canberra ACT
0200, Australia.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 9 3 9 – 9 4 8
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Table 1 Incorporation of inorganic precursors into Amphimedon terpenensis and Axinyssa n.sp
Compound
Precursor, amount
Incorporation period (days)
Molar specific activity (µCi mmolϪ1
)
Incorporation (%)
1
1
SCNϪ, 25 µCi
SCNϪ, 50 µCi
12
19
0.48
0.92
0.049
0.14
10
10
10
10
CNϪ, 100 µCi
CNϪ, 100 µCi
SCNϪ, 12.5 µCi
SCNϪ, 25 µCi
16
27
3
0.006
0.001
0.002
0.003
0.0009
0.00005
0.0075
0.0015
16
11
11
11
CNϪ, 100 µCi
SCNϪ, 12.5 µCi
SCNϪ, 25 µCi
16
3
16
13.9
6.4
2.6
0.3
1.14
0.35
12
12
12
12
CNϪ, 100 µCi
CNϪ, 100 µCi
SCNϪ, 12.5 µCi
SCNϪ, 25 µCi
16
27
3
1.23
0.007
0.36
0.15
0.079a
0.00009
0.047
16
0.029a
a These values have been recalculated since our earlier publication.16
had indicated that while this sponge converts both cyanide and
thiocyanate into the isothiocyanate 11, the isocyanide 10 from
both thiocyanate and cyanide feedings appeared not to be
labelled.17 This unconvincing biosynthetic result was most
unexpected given the significant incorporations into the diter-
pene and sesquiterpene isocyanides described above. We there-
fore report additional precursor incorporation data that enable
us to properly describe isocyanide/isothiocyanate biosynthesis
in Axinyssa n.sp.
Results and discussion
Precursor incorporation studies were performed using our
standard protocols5,12 on healthy species of A. terpenensis or of
Axinyssa n.sp. collected at Heron Island or at Lizard Island
on the Great Barrier Reef. The specimens were equilibrated
in aquaria after collection, and then maintained at ambient
temperature and with aeration in seawater in small jars. After
addition of the precursor, the radioactivity content in the
seawater was monitored to confirm precursor uptake during
incubation. After ca. 12 h under these static conditions, the
sponges were either maintained in aquaria under flowing
fresh seawater conditions for an interval, typically 10–20 days
depending on the health of the specimens, or returned under-
water for longer incorporation periods. Sponges were then
frozen for subsequent workup and isolation of terpene metab-
olites. In all experiments the isolated products were rigorously
purified to constant radioactivity.
Scheme 1 Proposed biosynthetic pathways for A. cavernosa.
the secondary metabolite level, as suggested for the spiroaxane
series of metabolites in Scheme 1 (reactions c and d). Using
novel experimental techniques that left the sponge specimens
under study in their natural habitat, the Scheuer group found
that 2-isocyanopupukeanane (5) was a precursor to the corre-
sponding isothiocyanate 13 in Hymeniacidon sp. (= Ciocalypta
sp.), but they could not detect the reverse isothiocyanate to
isocyanide conversion. These experiments used [13C]-labelled
precursors and were analysed by GC-MS, using the [M ϩ 1]ϩ
ion to measure the incorporation of [13C]-label.14
Inorganic precursor experiments with A. terpenensis and
Axinyssa n.sp
The precursor status of cyanide5 and of thiocyanate15 in diiso-
cyanoadociane (1) biosynthesis has previously been probed in
A. terpenensis. In our original experiment using thiocyanate, a
sponge sample from Lizard Island was treated with 50 µCi
[14C]-thiocyanate giving radiolabelled diisocyanoadociane
(6240 dpm mgϪ1; 0.14%) after 19 days incubation (Table 1).15
Subsequently, a sponge sample collected at Heron Island was
supplied with sodium [14C]-thiocyanate (25 µCi), and an
In this paper we provide full details of sensitive [14C]-labelling
experiments that compare isocyanide–isothiocyanate inter-
conversions in two marine sponges, firstly in A. terpenensis
which is characterised by isocyanides such as 1,15 and in which
isothiocyanates are minor metabolites, and then in Axinyssa
n.sp. which contains the 9-isocyanopupukeanane (10) and
9-isothiocyanatopupukeanane (11) pair of metabolites, together
with thiocyanate 12.9,16 As we show below, the two sponges
provide contrasting results in these advanced precursor
incorporations. Further, our earlier work with Axinyssa n.sp.
incorporation of 0.049% (3270 dpm mgϪ1; 0.48 µCi mmolϪ1
)
was obtained after 12 days incorporation. As with cyanide,5
incorporations levels into 1 appear to increase as the incubation
time is extended. The specificity of labelling in the diisocyano-
adociane sample isolated from the Lizard Island sponge
was investigated using the degradation sequence developed
earlier.5,15 Glacial acetic acid treatment gave bisformamide
2 and resulted in the retention of 95.1% of the radioactivity.
The removal of a single formyl group was accomplished using
2.5 M sodium hydroxide to give monoamide 3, which retained
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 9 3 9 – 9 4 8
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Table 2 Degradation of diisocyanoadociane (1) isolated from [14C]-thiocyanate incorporation experiments with Amphimedon terpenensis
Compound
Mol. spec. activity (µCi mmolϪ1
)
Relative activity (%)
1
4
0.918
0.025
100.0
2.4
1
2
3
4
0.143
0.136
0.071
0.006
100.0
95.1
49.7
4.2
Table 3 Conversion of labeled metabolites 10, 11, and 12 from [14C]-cyanide incorporation experiments with Axinyssa n.sp
Compound
Mol. spec. activity (µCi mmolϪ1
)
Relative activity (%)
10
14
0.0063
0.0063
100
100
11
15
13.9
14.8
100
106a
12
16
1.23
0.001
100.0
0.1
a The radiochemical yield shows that the degradation product was of slightly higher radiochemical purity than the starting material.
Table 4 Conversion of labeled metabolites 10, 11, and 12 from [14C]-thiocyanate incorporation experiments with Axinyssa n.sp
Compound
Mol. spec. activity (µCi mmolϪ1
)
Relative activity (%)
10
14
0.0032
0.0030
100
94
11
15
2.6
2.7
100
104a
12
16
0.150
0.0005
100.0
0.3
a See footnote to Table 3.
49.7% of the radioactivity. The second formyl group was
removed by refluxing with 6 M HCl to give bisamine 4, with
only 4.2% of the radioactivity remaining, while direct
hydrolysis of the isolated 1 to 4 with 6 M HCl also gave
unlabelled material (Table 2).
9-Isocyanopupukeanane (10) (19 dpm mgϪ1 above background;
0.0075%; 11.1 mg isolated), 9-isothiocyanatopupukeanane (11)
(53500 dpm mgϪ1; 1.14%; 5.9 mg), and 2-thiocyanatoneo-
pupukeanane (12) (3036 dpm mgϪ1; 0.047%; 4.3 mg) were
isolated, thus the low incorporation into 10 was not related to
the length of the incubation period. Comparing the various
incorporation experiments, over time the specific activity of the
isothiocyanate 11 and thiocyanate 12 metabolites decreases.
Conversion of the isolated samples of isocyanide 10 into the
corresponding formamide 14 occurred with significant reten-
tion of radioactivity (Tables 3 and 4) which supported genuine
labelling. Because of the low 14C content of the samples, further
degradation could not be undertaken. By analogy with the
results from A. terpenensis in Australia,5 and from Hawaiian
sponges,6b we infer that the label is associated with the iso-
cyanide carbon atom. The radioactive isothiocyanate products
11 from the cyanide and thiocyanate (16 day) incubations
were refluxed with diethylamine, yielding samples of N,N-
diethylthiourea 15 that retained the radioactivity content
(Tables 3 and 4). The isothiocyanate 11 and thiourea 15 resisted
hydrolytic degradation, preventing experimental confirmation
of the site of labelling; again we inferred by analogy with earlier
research that the [14C]-label was associated with the iso-
thiocyanate functionality. This assumption was subsequently
supported by advanced precursor experimentation as explained
below. The thiocyanate metabolite 12 was labelled in all
experiments; it was possible to reduce samples of thiocyanate
12 by brief treatment with LiAlH4 to give the thiol 16.11
Reaction of thiocyanate 12 from the [14C]-cyanide incubation
gave thiol 16 which was found to be unlabelled (Table 3).
Similarly, the thiocyanate 12 isolated from incorporation of
The roles of cyanide and thiocyanate in the biosynthesis of
the Axinyssa metabolites 10–12 next required investigation.
These experiments were conducted at Heron Island, and have
in part been the subject of a preliminary communication.17
Firstly, sponge material was incubated with [14C]-cyanide for
16 days, yielding 9-isocyanopupukeanane (10) with low radio-
activity (60 dpm mgϪ1 above background; 0.006 µCi mmolϪ1
;
0.0009% incorporation; 33.9 mg isolated), 9-isothiocyanato-
pupukeanane (11) with much higher 14C content (116270 dpm
mgϪ1; 13.9 µCi mmolϪ1; 0.3%; 5.7 mg), and 2-thiocyanato-
neopupukeanane (12) (10270 dpm mgϪ1; 1.23 µCi mmolϪ1
0.079%; 17.0 mg) (Table 1). A second incorporation experiment
of 27 days duration resulted in 9-isocyanopupukeanane (10) (10
dpm mgϪ1 above background; 5 × 10Ϫ5%; 10.0 mg isolated) and
2-thiocyanatoneopupukeanane (12) (58 dpm mgϪ1; 9 × 10Ϫ5%;
3.6 mg). Incorporation of [14C]-thiocyanate for 16 days yielded
9-isocyanopupukeanane (10), again with low radioactivity
(30 dpm mgϪ1 above background; 0.0015%; 27.9 mg isolated),
9-isothiocyanatopupukeanane (11) with much higher 14C
content (22000 dpm mgϪ1; 0.35%; 8.8 mg isolated), and
2-thiocyanatoneopupukeanane (12) (1255 dpm mgϪ1; 0.029%;
12.9 mg).
We speculated that the low incorporation of cyanide and
thiocyanate into the isocyanide was a result of the extended
incorporation period, and therefore tested this using thio-
cyanate in an incorporation experiment of 3 days duration.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 9 3 9 – 9 4 8
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[14C]-thiocyanate ion gave unlabelled thiol 16 on reduction
(Table 4).
17 and of the pupukeanane metabolites 10 and 11. We chose to
follow the synthetic approach of Hagadone et al.,14 who used
the amine derived from the natural isocyanide as starting
material for their labelled synthesis. Owing to the ease of
isolation of diisocyanoadociane (1), we first explored synthesis
of 17 (Scheme 3). Thus diisocyanoadociane (1) was converted
to the bisamine 4 by acid hydrolysis, then conversion to the
formamide 2 was explored. Standard methods for the form-
ation of amide bonds such as DCC coupling gave poor yields,
but use of formic pivalic anhydride gave acceptable yields of 2.
[14C]-Formic acid is available commercially as its sodium salt,
however trial reactions using unlabelled sodium formate gave
low yields of formamide 2, in contrast to reactions using formic
acid. Sodium [14C]-formate (400 µCi) was therefore equilibrated
with unlabelled formic acid before reaction with pivaloyl
chloride, then the amine 4 was added to this [14C]-labelled
mixed anhydride. The reaction was then forced to completion
by the addition of excess unlabelled mixed anhydride. Workup
of the reaction gave 2 in excellent chemical yield, but only in
30% radiochemical yield which likely reflects the effectiveness
of the formate equilibration step. The formamide product was
immediately dehydrated with toluenesulfonyl chloride in dry
pyridine to give [14C]2-diisocyanoadociane (1) in 57% radio-
chemical yield after purification by silica chromatography and
repeated recrystallisation from hexane. A portion of the [14C]2-
diisocyanoadociane (1) sample was degraded by acid hydrolysis
to the formamide 2 which retained 94.5% radioactivity, then
using 2.5 M NaOH to the monoamide 3 which retained 35.1%
of the [14C]-label. Thus both isocyanide groups of the diterpene
1 were labelled, but the equatorial, and more nucleophilic,
amino substituent of the parent bisamine 4 was preferentially
labelled over the axial amino group by our procedure. Treat-
ment of the remaining [14C]2-diisocyano-adociane (1) with
sulfur at 120 ЊC then gave [14C]2-bis-isothiocyanatoadociane
(17) in 40% yield (with 33% retention of radioactivity) after pre-
parative TLC. This material was used in incorporation studies
at Lizard Island, as reported in our preliminary communi-
cation.15 A second batch of [14C]2-bisisothiocyanatoadociane
was prepared subsequently for incorporation study at Heron
Island. Starting from amine 4 and sodium [14C]-formate (250
µCi), the formamide 2 was prepared in 12.1% radiochemical
yield, then dehydrated (TsCl/Pyr) to the diisocyanide 1 (59%
radiochemical yield) and treated with sulfur at 120 ЊC to give
[14C]2-bisisothiocyanatodociane 17 in 97% radiochemical yield
after preparative TLC.
The precursor experiments described above establish the
precursor status of inorganic cyanide and thiocyanate in the
biosynthesis of terpene isocyanides, isothiocyanates and thio-
cyanates in A. terpenensis and Axinyssa n.sp. The labeling
of thiocyanate 12 by cyanide supports the interconversion of
cyanide and thiocyanate as it is difficult to envisage the
incorporation of cyanide into a terpene, followed by insertion
of sulfur to give an organic thiocyanate. The alternative cyan-
ation of a thiol suggested by Pham et al.11 is less likely given the
incorporation of both cyanide and thiocyanate. Sulfur insertion
into an isocyanide to give an isothiocyanate (by an enzyme
functionally equivalent to rhodanese)13,18 followed by isomeriz-
ation remains a possibility, however, the isomerization equi-
librium usually favours an isothiocyanate over a thiocyanate.19
The high incorporation into the isothiocyanate metabolite 11
is in striking contrast to the low incorporation into the major
isocyanide metabolite 10. These contrasting incorporation
levels can be partly explained by the quantities of metabolite
isolated. 9-Isocyanopupukeanane (10) is the major metabolite
of Axinyssa n.sp., and the background levels of this metabolite
in the sponge at the start of an incorporation experiment dilute
the radioactivity content of the isolated product. However, in
our earlier work with A. cavernosa,12 we had observed higher
levels of incorporation from [14C]-cyanide and [14C]-thiocyanate
into the spiroaxane metabolite axisonitrile-3 (8) compared to
axisothiocyanate-3 (9), despite the status of axisonitrile-3 as a
major metabolite in this sponge. Precursor dilution during bio-
synthesis by unlabelled metabolite cannot adequately explain
the Axinyssa results. We therefore propose that in Axinyssa
n.sp., the major biosynthetic route (Scheme 2 reactions a, f and
d) is from cyanide to thiocyanate, followed by incorporation
into the thiocyanate 12 or isothiocyanate 11 and subsequent
conversion of the isothiocyanate into the isocyanide 10. If these
steps occur at a slow rate, then isocyanide 10 would be labelled
at a lower level than the isothiocyanate 11. The alternative
direct utilisation of cyanide for isocyanides (Scheme 2 reaction
e) then conversion to isothiocyanate 11 (reaction c), may occur,
but does not adequately account for the significant labelling
found in the thiocyanate 12, or the low level of labelling of the
isocyanide 10. We reasoned that incorporation of [14C]-labelled
samples of 10 and 11 into Axinyssa n.sp. would enable us to
evaluate isocyanide–isothiocyanate interconversions at the
secondary metabolite level, and hence to explore the status of
the individual biosynthetic steps in Scheme 2.
Scheme 3 Synthesis of [14C]2-bisisothiocyanate 17. (a) 6 M HCl,
reflux, 68%; (b) [14C]-formic pivalic anhydride, Et3N, CH2Cl2, 88%; (c)
p-TsCl in pyridine, 60%; (d) Sulfur, 120 ЊC, 56%.
Scheme 2 Interconversions in Axinyssa n.sp.
In synthesizing the samples of [14C]2-bisisothiocyanato-
adociane (17) from the diisocyano analog 1, we recognized that
there is a risk of contamination from unreacted [14C]2-diiso-
cyanoadociane (1), which would label the biosynthetic product
of the incorporation experiment, and possibly lead to a
misleading result. To avoid this, a cold carrier dilution and
separation protocol was used. The sample of [14C]2-17 (11.7 mg;
6.7 µCi) for use at Heron Island was mixed with unlabelled
diisocyanide 1 (11 mg), and the mixture separated by silica
chromatography resulting in the bisisothiocyanate 17 (9.5 mg;
containing 5.98 µCi) and diisocyanoadociane (1) (11.0 mg;
containing 0.01 µCi). Purification of the recovered isocyanide
sample by normal phase HPLC and recrystallisation from
hexane gave diisocyanoadociane (9.5 mg) with a very low level
The terpene chemistry of A. terpenensis is dominated by
isocyanides over isothiocyanates; bisisothiocyanate 17 has not
been isolated from this sponge, however two isomeric mono-
isothiocyanates have been isolated from the related sponge
Cymbastela hooperi.20 A precursor incorporation study with 17
was undertaken to complement the experiments in Axinyssa
n.sp. by enabling us to evaluate whether isothiocyanates are
precursors to isocyanides in A. terpenensis.
Synthesis of advanced precursors
To test these biosynthetic ideas, we required syntheses of [14C]-
labelled samples of the proposed cycloamphilectane precursor
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Table 5 Advanced precursor incorporations with Amphimedon terpenensis and degradation of diisocyanoadociane (1)
Compound
Precursor
Molar specific activity (µCi mmolϪ1
)
Incorporation (%)
Relative activity (%)
1
2
[14C]-17a
0.059
0.057
0.02
—
100.0
97b, c
1
4
[14C]-17a
0.011
0.001
0.002
—
100.0
9.1d
1
[14C]-17e
<0.0059
<0.0002
a Incorporation of 11.5 µCi. b Samples of 2 are hygroscopic which slightly lowers the measured specific activity. c Insufficient material for further
degradation. d The amine 4 is difficult to purify, resulting in residual activity, which is unlikely to be genuine labelling. e Incorporation of 5.3 µCi.
of radioactivity (<70 dpm mgϪ1; 0.0003 µCi). In a second cycle
of this purification, the radiolabelled 17 was again mixed with
unlabelled 1 (10.0 mg), and recovered by preparative TLC,
yielding 9.2 mg of 17 (5.3 µCi) and 9.4 mg of 1 (0.001 µCi). This
procedure removed any unreacted [14C]2-diisocyanide from the
synthetic bisisothiocyanate product and confirmed the radio-
chemical purity of the recovered bisisothiocyanate 17 for use in
incorporations.
Synthesis of the required radiolabelled pupukeanane metab-
olites was next undertaken. Amine 18, available by 6 M HCl
hydrolysis of the sponge-derived isocyanide 10, was used as
starting material (see Scheme 4). The [14C]-label was incorpor-
it had been prepared from radioactive 1. Any carry-through of
unreacted radiolabelled diisocyanide during purification of the
bisisothiocyanate precursor would interfere with the incorpor-
ation results. Subsequently, an incorporation experiment was
conducted on A. terpenensis at Heron Island using a sample
of bisisothiocyanate 17 that had been carefully treated with
unlabelled 1 as described above to remove any traces of residual
[14C]2-1. The diisocyanoadociane isolated from this biosynthetic
experiment had a radioactivity content of 4 dpm mgϪ1 above
background, and was essentially non radioactive. Thus there
was no incorporation of bisisothiocyanate 17 into diisocyano-
adociane 1 in this experiment. Although a negative biosynthetic
result does not necessarily disprove a biosynthetic conversion,
our data suggest that this precursor may not be part of the
biosynthetic pathway to 1 in this sponge. The bisisothiocyanate
has not been reported from A. terpenensis or the closely-related
C. hooperi,20 so it is possible that the lack of incorporation
occurs because the enzymes responsible for the pathway to
diisocyanide 1 do not recognize bisisothiocyanate 17 as a
substrate.
We next explored advanced precursor studies with Axinyssa
n.sp.16 Prior to these experiments, the radiochemical purity of
the precursors used was defined by the cold carrier dilution
method described above. [14C]-9-Isocyanopupukeanane (10)
was dissolved in methanol and incubated with a small piece of
Axinyssa n.sp. overnight, followed by a 21 day incorporation
period. Extraction of the sponge, followed by purification
to constant specific activity gave a sample of 9-isothiocyanato-
pupukeanane (11) which was significantly radioactive (Table 6).
Fractions containing 9-isocyanopupukeanane (10) were highly
radioactive, but were not purified further. Confirmation of
the radiochemical purity of the isolated sample of 9-iso-
thiocyanatopupukeanane (11) was obtained by preparation of
the thiourea 15 (Et2NH reflux), which retained the radioactivity
after purification. In a second experiment, [14C]-9-isothio-
cyanatopupukeanane (11) was dissolved in acetone and
supplied to a specimen of Axinyssa n.sp. in a similar incorpor-
ation. The sample of 9-isocyanopupukeanane (10) isolated from
this incorporation experiment was significantly radioactive (see
Table 6). Fractions containing 9-isothiocyanatopupukeanane
(11) were highly radioactive, but were not purified further.
Confirmation of the radiochemical purity of the isolated 9-
isocyanopupukeanane (10) was achieved by hydration (g.
AcOH) to give formamide 14 which retained the radioactivity
after purification. In these two incorporation experiments,
the specificity of incorporation was not tested, and is not in
question as the precursors supplied were specifically labelled at
the isocyanide or isothiocyanate carbons. The high incorpor-
ation levels obtained do not suggest that metabolic degradation
and re-utilization of labelled breakdown products via general
metabolism had occurred.
Scheme 4 Synthesis of [14C]-isocyanide 10 and [14C]-isothiocyanate 11.
(a) 6 M HCl, reflux, 66%; (b) [14C]-formic pivalic anhydride, Et3N,
CH2Cl2, 59%; (c) p-TsCl in pyridine, 84%; (d) sulfur, 120 ЊC, 33%.
ated by formylation of amine 18 with 750 µCi of [14C]-formic
pivalic anhydride to give the [14C]-formamide 14 with the reten-
tion of 19% of the radioactivity. Dehydration of the formamide
with toluenesulfonyl chloride in dry pyridine gave [14C]-9-
isocyanopupukeanane (10) in 93% radiochemical yield. A
portion of this isocyanide was converted to the isothiocyanate
11 by treatment with sulfur at 120 ЊC in 45% radiochemical
yield. The sample of [14C]-11 was mixed with unlabelled 10, the
mixture separated by preparative TLC and the two individual
compounds purified. Two cycles of this purification protocol
resulted in unlabelled 10 (<0.0001 µCi), confirming the radio-
chemical purity of the recovered [14C]-9-isothiocyanato-
pupukeanane (11) for use in incorporations.
Advanced precursor incorporation
Precursor incorporation experiments were performed in the
field according to our usual protocol.5,12 First, at Lizard Island,
[14C]2-bisisothiocyanate 17 was dissolved in acetone and pro-
vided equally to two small pieces of A. terpenensis in aerated
seawater. After 19 days incorporation, the diisocyanoadociane
isolated from each specimen was found to contain a low level of
radioactivity (Table 5). One sample of 1 was degraded to the
formamide 2, which retained 97% of the radioactivity, but lack
of material prevented further degradation to amine products,
while the other sample was hydrolysed directly to the bisamine
4 that contained 9.1% of the activity of the diisocyanide. The
higher than expected 14C content of this sample of 4 was
attributed to side products of the hydrolysis reaction, and the
difficulty of purification. At first sight, these results were con-
sistent with a low conversion of precursor 17 to diisocyanide 1
by the sponge.15 On reflection, interpretation of the data from
these initial advanced precursor studies was complicated by
concerns about the purity of the precursor supplied, given that
These results show the conversion of isocyanides to iso-
thiocyanates and also of isothiocyanates to isocyanides in
Axinyssa n.sp. Although the incorporation percentages are
similar for the isocyanide to isothiocyanate conversion (0.16%)
and the reverse isothiocyanate to isocyanide conversion
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Table 6 Advanced precursor incorporations with Axinyssa n.sp
Compound
Precursor
Molar specific activity (µCi mmolϪ1
)
Incorporation (%)
Relative activity (%)
11
15
[14C]-10
4.6
4.1
0.16a
—
100
89b
10
14
[14C]-11
0.14
0.14
0.12a
—
100
100
a These values were inadvertently transposed in our earlier publication.20 b The small amount and difficulty in purification meant the specific activity
was lower than desired, however it is unlikely that this level of labeling would be retained after purification of an unlabelled compound.
(0.12%), the endogenous levels of these metabolites (and the
consequent dilution) mean that the rate of transfer of iso-
cyanide (major metabolite) to isothiocyanate (minor metabol-
ite) is significantly faster than the reverse conversion. The
possibility that these conversions occur without the action of
enzymes was considered. The procedures used for the synthesis
of the radiolabelled metabolites provide limited evidence in
support of the enzyme-mediated nature of these conversions. In
the cold carrier dilution and separation experiments, labelled
isothiocyanate is mixed with unlabelled isocyanide, and the
isolated isocyanide was found to be non radioactive. Any
chemical conversion would result in labelling of the isolated
isocyanide, unless the conversion was extremely slow at room
temperature. Additionally the lack of utilization of bisiso-
thiocyanate 17 by A. terpenensis, suggests that isothiocyanates
are not converted chemically to isocyanides under incorpor-
ation conditions within sponge tissue. Although this evidence is
not conclusive, it suggests that the non-enzymatic conversion of
isothiocyanates to isocyanides either does not occur, or occurs
only at a very slow rate under normal conditions.
conversions detected in Axinyssa n.sp. represent an equilibrium
within the sponge that allows adjustment of the concentration
of the metabolites to suit the environmental needs of the
sponge.2,21 Additional biochemical roles for cyanide, thio-
cyanate and their organic counterparts are under investigation
in our laboratory.22
Experimental
General experimental
Thin layer chromatography (TLC) was carried out using
Kieselgel 60 F254 silica containing a UV fluorescence indicator
coated on aluminium plates and visualised under UV light (254
nm). Further visualisation was achieved with vanillin spray (50
mg vanillin, in 25% sulfuric acid in ethanol (50 mL)) followed
by heating. Flash chromatography was carried out using
Kieselgel 60 230–400 mesh silica. Silica PTLC was carried out
on Merck 05717 2 mm glass backed plates with a UV fluor-
escence indicator (254 nm) and visualised under UV light.
Bands were scraped off and the silica placed in a column, then
the compounds were eluted with CH2Cl2 and EtOAc. High
performance liquid chromatography (HPLC) was carried
out on Waters µPorasil (10 µm) columns (300 mm × 7.8 mm)
with isocratic elution using hexane/ethyl acetate mixtures;
compounds were detected by refractive index detection.
Nuclear magnetic resonance (NMR) spectra were recorded
on a Bruker DRX500 instrument at 500 MHz in deuterio-
chloroform and referenced to residual solvent (δH CHCl3 = 7.25
ppm, δC CDCl3 = 77 ppm). Electron impact (EI) mass spectra
were recorded on a Kratos MS25RFA spectrometer at 70 eV.
Electrospray ionisation (ESI) mass spectra were recorded on a
P E SCIEX API III triple quadrupole mass spectrometer for
solutions in methanol.
Summary
The issue of whether the isocyanide and isothiocyanate second-
ary metabolites can be interconverted by marine sponges
was first addressed by Scheuer et al. using [13C]-labelled
precursor incorporation.14 Some mass spectrometric evidence
was obtained for the conversion of the tricyclic metabolite
2-isocyanopupukeanane into 2-isothiocyanatopupukeanane by
the Hawaiian sponge Ciocalypta sp., however the reverse trans-
formation (isothiocyanate isocyanide) was not detected using
this methodology. Our data, using sensitive [14C]-labelling
experiments, do not provide convincing evidence for an iso-
thiocyanate to isocyanide conversion in the Great Barrier Reef
sponge A. terpenensis. The incorporation values obtained are
low and variable, possibly because the isothiocyanate precursor
supplied is not a natural metabolite of this sponge. In con-
trast, we have obtained definitive evidence for isocyanide–
isothiocyanate interconversions by advanced precursor experi-
ments with Axinyssa n.sp. In this sponge, the efficiency of
incorporation is similar in either direction. Incorporation of the
inorganic precursors cyanide or thiocyanate into the Axinyssa
metabolites 10 and 11 gives contrasting data for the two
metabolites. High incorporations of these two precursors were
achieved with 11, whereas the isocyanide 10 was poorly
labelled. These combined experimental results suggest to us
that the major pathway operating in Axinyssa n.sp. is
cyanide thiocyanate isothiocyanate 11 isocyanide 10,
which implies that there may be a major biosynthetic role for
thiocyanate ion in this sponge. The labelling of thiocyanate
metabolite 12 by cyanide ion via thiocyanate ion (Scheme 2
reactions a and g) in this sponge is an additional piece of
biosynthetic information which adds weight to the proposed
pathway as it also confirms a key role for thiocyanate ion.17 The
biochemical origin of the thiocyanate or cyanide ion used for
marine terpene biosynthesis in these sponges remains elusive.
Finally we note that it is possible that the organic inter-
The specific activities of the [14C]-radiolabelled precursors
were sodium cyanide 40–60 µCi mmolϪ1, sodium thiocyanate
5–25 µCi mmolϪ1 and sodium formate 40–60 mCi mmolϪ1, and
were purchased from ICN.
Radiochemical measurements
Scintillation counting was carried out on a Wallac 1410 liquid
Scintillation counter using Wallac Optiphase Hisafe aqueous
organic scintillation liquid. Each chemical sample was counted
in triplicate. For diisocyanoadociane samples of low activity,
aliquots of recrystallised disocyanoadociane were weighed to
the nearest 0.01 mg and transferred directly into scintillation
vials, then dissolved with 0.1 mL benzene and diluted with
3.9 mL of scintillant. Other organic samples were counted
in triplicate in either benzene or methanol, using 1% of the
available material in each scintillation vial. Samples were dis-
solved in 10 mL solvent and 0.1 mL aliquots transferred for
counting in 3.9 mL scintillant. For samples of 10 mg or less,
samples were dissolved in 1 mL solvent, and 0.01 mL aliquots
transferred for counting in 4.0 mL scintillant. Samples of
low activity were then recounted using 30% of the available
material in each scintillation vial. All samples were counted
until the number of counts recorded reached a 2σ confidence
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 9 3 9 – 9 4 8
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limit. Sample radioactivity values were corrected for back-
ground radiation by subtraction of an averaged value obtained
by counting in triplicate blank samples containing the same
volume of the same solvent.
dissolved in ethanol (0.75 mL) and 2.5 M sodium hydroxide
(0.75 mL) was added. The solution was heated to reflux
for three hours, then cooled to room temperature overnight.
The reaction mixture was then applied to a reverse phase
chromatography cartridge (eluted with H2O, 30% MeOH/H2O,
MeOH, then CH2Cl2) to give monoamine 3 (7.9 mg, 99%; 0.078
µCi mmolϪ1, 57%). Further reverse phase cartridge chromato-
graphy (eluting with H2O, 30% MeOH/H2O, MeOH, then
Animal material
Specimens of A. terpenensis were collected by hand using
SCUBA (Ϫ10–15 m) at dive sites on the southern side of Heron
Island or at North Point, Lizard Island, Great Barrier Reef,
Australia. A voucher sample (registry number QM G307133)
was identified as Amphimedon terpenensis (Order: Haplo-
sclerida, Family: Niphatidae), and is lodged at The Queensland
Museum, Brisbane.
Specimens of Axinyssa n.sp were collected by hand using
SCUBA (Ϫ10–15 m) at Coral Spawning dive site at Heron
Island on the Great Barrier Reef, Australia. A voucher sample
(registry number QM G312575) was identified as Axinyssa n.sp.
(1939) (Order: Halichondrida, Family: Halichondriidae), and is
lodged at The Queensland Museum, Brisbane.
CH2Cl2) gave monoamine 3 (6.5 mg, 81%; 0.071 µCi mmolϪ1
,
49.7%).5
Hydrolysis of [14C]-monoamine 3 to bisamine 4. Monoamine
3 (3.4 mg, 0.071 µCi mmolϪ1) was dissolved in acetone (2 mL)
and added to hydrochloric acid (1.5 mL, 6 M) and heated to
reflux for 3 h, then cooled to room temperature and stirred
overnight. The reaction mixture was washed with EtOAc
(2 mL), the EtOAc layer extracted with hydrochloric acid (1.5
mL, 6 M) and the combined acid layers made basic with sodium
hydroxide. The aqueous solution was applied to a reverse phase
chromatography cartridge and eluted with H2O, 20% MeOH/
H2O, MeOH, then CH2Cl2 to give bisamine 4 (1.5 mg, 49%;
0.006 µCi mmolϪ1, 4.2%), identical with previously reported
material.5
Incorporation experiments with inorganic precursors
[14C]-Thiocyanate and A. terpenensis. A piece of A. terpenen-
sis (30.5 g wet wt.) was placed in a beaker containing unfiltered,
continuously aerated seawater (200 mL) and maintained at
ambient temperature by a flowing seawater bath surrounding
the beaker. Precursor (NaS14CN, 25 µCi) was then added, and
the sponge allowed to assimilate the precursor for a 12 h period
overnight. Water samples were taken at regular intervals which
revealed that 33% of the precursor was taken up during this
period. The sponge was then transferred to a large flowing
seawater aquarium for a 12 day incorporation period, then
frozen for subsequent workup. The sponge was sliced finely and
extracted with CH2Cl2 : MeOH (1 : 1, 4 × 100 mL). The extracts
were evaporated to an aqueous suspension, which was extracted
with CH2Cl2 (3 × 60 mL). The CH2Cl2 layers were dried and
evaporated to give a green residue (300 mg, 0.98% chemical
yield; 6140 dpm mgϪ1, 3.3% radiochemical incorporation)
which was chromatographed on silica eluting stepwise with
hexane to ethyl acetate. Fractions were combined on the basis
of radioactivity and TLC comparison with an authentic sample
of diisocyanoadociane (1). The fraction containing diisocyano-
Hydrolysis of diisocyanoadociane (1) to bisamine 4. Undiluted
diisocyanoadociane (1) (8.9 mg, 0.918 µCi mmolϪ1) was added
to hydrochloric acid (6 M, 3 mL) and refluxed for 3 h, then
allowed to cool to room temperature overnight. The reaction
solution was made basic with sodium hydroxide, then applied
to a reverse phase chromatography cartridge (eluted with
H2O, 20% MeOH/H2O, MeOH, then CH2Cl2) to give bisamine
4 (2.6 mg, 31%; 0.025 µCi mmolϪ1, 2.4%).
[14C]-Cyanide and Axinyssa n.sp. The incorporation of
Na14CN (100 µCi) was performed in the same way using a piece
of Axinyssa n.sp. (88 g wet wt.) in 200 mL seawater. After a
16 day incorporation, the sponge was extracted with CH2Cl2 :
MeOH (1 : 1) (4 × 140 mL). The extract was then evaporated
to an aqueous residue which was extracted with hexane (3 ×
80 mL). The hexane layers were dried and evaporated to give a
green residue (906 mg). This residue was fractionated on a silica
flash column by stepwise gradient elution using hexane/ethyl
acetate mixtures,9 and fractions combined on the basis of TLC,
NMR and radioactivity. Purification by repeated normal
phase HPLC (0.25% EtOAc/hexane) gave 9-isocyanopupuke-
anane (10) (33.9 mg, 0.038%; 60 dpm mgϪ1, 0.0009%) and
2-thiocyanatoneopupukeanane (12) (17.0 mg, 0.019%; 10270
dpm mgϪ1, 0.079%).9 Repeated normal phase HPLC (hexane)
of another fraction gave 9-isothiocyanatopupukeanane (11)
(5.7 mg, 0.0065%; 116270 dpm mgϪ1, 0.3%).9 A second piece of
sponge (70 g) was treated identically with Na14CN (100 µCi).
The sponge was then transferred to an underwater grid
at Ϫ14 m for 27 days. The sponge was extracted as above to give
adociane (1) (31.7 mg, 0.1% chemical yield; 5220 dpm mgϪ1
,
0.3% radiochemical incorporation) was further purified by
repeated normal phase HPLC (5% EtOAc in hexane), followed
by two recrystallisations from hexane to give diisocyano-
adociane (1) (8.2 mg, 0.027%; 3270 dpm mgϪ1, 0.049%), identi-
cal in all respects with authentic material and literature data.5,23
A second piece of sponge (45 g wet wt.) was treated with
precursor (NaS14CN, 50 µCi) for an incorporation period of 19
days, followed by workup as above, resulting in diisocyano-
adociane (1) (24.8 mg, 0.055%; 6240 dpm mgϪ1, 0.14%).
9-isocyanopupukeanane (10) (10.1 mg, 0.014%; 10 dpm mgϪ1
,
Hydration of [14C]2-diisocyanoadociane (1) to [14C]2-
bisformamide 2. A small sample of [14C]-diisocyanoadociane (1)
was diluted with unlabelled material, then recrystallised before
use in degradation studies. [14C]2-Diisocyanoadociane (1) (26.2
mg, 0.143 µCi mmolϪ1) was dissolved in glacial acetic acid (1
mL) and allowed to stand at room temperature overnight. The
reaction was diluted with water (5 mL), and saturated sodium
bicarbonate solution (1 mL), then extracted with CH2Cl2
(4 × 10 mL). The CH2Cl2 layers were washed with saturated
sodium bicarbonate solution (3 × 10 mL) until effervescence
ceased. The CH2Cl2 layers were dried and evaporated to give a
colourless oil (22.2 mg). This residue was purified by silica
chromatography (eluting with CH2Cl2, then EtOAc stepwise to
MeOH) to give [14C]2-bisformamide 2 (19.0 mg, 65% chemical
yield; 0.136 µCi mmolϪ1, 95.1% radiochemical yield), identical
with previously reported material.5
0.00005%) and 2-thiocyanatoneopupukeanane (12) (3.6 mg,
0.0051%; 58 dpm mgϪ1, 0.00009%). The small amount of 11
isolated in this experiment was not able to be purified to
constant specific activity.
Hydration of 9-isocyanopupukeanane (10) to formamide 14.
9-Isocyanopupukeanane (10) (14.2 mg, 0.006 µCi mmolϪ1) was
dissolved in glacial acetic acid (2 mL) and left to stand
overnight. The resulting solution was poured onto saturated
sodium bicarbonate solution (20 mL), extracted with CH2Cl2
(3 × 20 mL), dried and evaporated to give a colourless oil.
Purification by silica chromatography gave formamide 14 (14.5
mg, 95%; 0.006 µCi mmolϪ1, 100%). δH (2 rotamers) 0.74 (3H,
s), 0.83 (3H, d, J 6.3), 0.84 (3H, d, J 6.3), 0.98 (3H, s), 1.01 (1H,
m), 1.14 (1H, m), 1.25 and 1.35 (1H, m), 1.26 (1H, m), 1.30 (2H,
m), 1.36 (1H, m), 1.46 (1H, m), 1.84 (1H, m), 2.04 (1H, m), 2.20
(1H, m), 3.11 and 3.86 (1H, m), 5.65 and 5.95 (1H, br m), 7.98
and 8.18 (1H, m). δC (2 rotamers) 21.5, 21.7, 24.3, 26.8, 26.9,
28.4, 28.5, 30.0, 31.5, 31.7, 33.9, 38.3, 38.8, 43.9, 44.0, 46.8,
Hydrolysis of [14C]2-bisformamide 2 to [14C]-monoamine 3.
Bisformamide 2 (8.7 mg, 0.024 mmol; 0.136 µCi mmolϪ1) was
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47.2, 48.5, 49.4, 49.5, 55.4, 162.2, 167.5. m/z (EI) = 249 (Mϩ ),
Synthesis of 14C-labelled advanced precursors
ؒ
ؒ
204 (100%), 161, 133, 106, 41. m/z (EI) 249.2089 [Mϩ
]
Synthesis of [14C]2-bisisothiocyanatoadociane (17). Hydrolysis
of diisocyanoadociane (1) to bisamine 4. Diisocyanoadociane
(1) (500 mg, 1.54 mmol) was hydrolysed with hydrochloric acid
(6 M, 3 mL) as described above to give bisamine 4 (319 mg,
68%).
C16H27NO requires 249.2092.
Conversion of 9-isothiocyanatopupukeanane (11) to diethyl-
thiourea 15. 9-Isothiocyanatopupukeanane (11) (2.8 mg, 13.9
µCi mmolϪ1) was refluxed in diethylamine (2.5 mL) overnight,
then evaporated. Silica chromatography gave a residue (2.7 mg)
which was purified using normal phase HPLC (5% EtOAc in
Formylation of bisamine 4 to give [14C]2-bisformamide 2.
Sodium formate (400 µCi) was equilibrated with formic acid
(17.7 µL, 0.46 mmol) and added to pivaloyl chloride (69.4 µL,
0.56 mmol) in dry THF (3 mL) and triethylamine (92 µL, 0.66
mmol) and cooled to Ϫ80 ЊC. Bisamine 4 (80.4 mg) in dry THF
(3 mL) was then added, and the reaction mixture allowed to
warm to room temperature. To ensure complete reaction a fur-
ther amount of pre-prepared pivalic formic anhydride (formic
acid (31.9 µL, 0.84 mmol) and pivaloyl chloride (123 µL, 1.0
mmol)) was added at Ϫ80 ЊC, and the reaction again allowed to
warm to room temperature. The reaction mixture was diluted
with CH2Cl2 (10 mL) then washed with sodium hydroxide solu-
tion (10 mL, 2.5 M) followed by sulfuric acid (10 mL, 25%),
then dried and evaporated. Purification by silica chromato-
graphy (eluting with CH2Cl2, then EtOAc stepwise to MeOH)
gave [14C]-labelled bisformamide 2 (105.3 mg, 110%; 120 µCi,
30%). The >100% recovery presumably results from the hygro-
scopic nature of 2; further efforts to purify this material were
not worthwhile at this point.
hexane) giving thiourea 15 (1.8 mg, 50.6%; 14.8 µCi mmolϪ1
,
106%; see footnote to Table 3). δH 0.80 (3H, s), 0.83 (3H, d,
J 7.8), 0.84 (3H, d, J 7.9), 0.98 (3H, s), 1.09 (1H, m), 1.14 (1H,
m), 1.23 (6H, t, J 7.0), 1.24 (1H, m), 1.26 (1H, m), 1.28 (1H, m),
1.30 (1H, m), 1.37 (1H, m), 1.45 (1H, m), 1.48 (1H, m), 1.84
(1H, dd, J 13 and 11), 2.07 (1H, m), 2.47 (1H, m), 3.65 (4H, q,
J 7), 4.34 (1H, m), 5.40 (1H, br d, J 8). δC 12.9, 21.6, 21.8, 24.7,
27.1, 29.2, 30.0, 31.7, 34.2, 38.3, 38.7, 44.1, 45.0, 48.5, 48.7,
49.3, 57.2, 179.9. m/z (ESI) 337.3 (M ϩ Hϩ, 100%). m/z (EI)
337.2684 C20H37N2S requires 337.2677.
Reduction of 2-thiocyanatoneopupukeanane (12) to thiol 16.
2-Thiocyanatoneopupukeanane (12) (6.8 mg, 0.025 mmol, 1.23
µCi mmolϪ1) in dry THF (1.2 mL) was added to LiAlH4 (4.9
mg, 0.13 mmol) and stirred at room temperature for 8 minutes,
then poured into saturated aqueous ammonium chloride
(5 mL) and extracted with EtOAc (3 × 10 mL), dried over
anhydrous magnesium sulfate and evaporated to give 6.6 mg of
a residue. Purification by silica chromatography followed by
normal phase HPLC (0.05% EtOAc in hexane) gave thiol 16
(2.4 mg, 39%; 0.001 µCi mmolϪ1, 0.1%).17 δH 0.78 (3H, d, J 6.5),
0.86 (2H, m), 0.89 (3H, d, J 6.5), 0.92 (1H, m), 0.95 (1H, m),
1.00 (3H, s), 1.05 (3H, s), 1.20 (1H, m), 1.25 (1H, m), 1.34 (1H,
m), 1.38 (1H, m), 1.42 (1H, m), 1.50 (1H, m), 1.78 (1H, ddd,
J 3.5, 11.0 and 14.5), 2.10 (1H, ddd, J 5.5, 9.5 and 14.5), 2.80
(1H br d, J 7). δC 20.9, 21.3, 22.1, 26.3, 26.7, 32.2, 32.2, 32.7,
Dehydration of [14C]2-bisformamide
2
to give [14C]2-
diisocyanoadociane (1). The labelled bisformamide 2 (105.3
mg, 0.29 mmol, 120 µCi) was dissolved in dry pyridine (2 mL)
and added to toluenesulfonyl chloride (332 mg, 1.75 mmol) and
refluxed for 3 h during which time the initially formed purple
colour faded to a light brown. The reaction mixture was poured
into hexane (10 mL), washed with sodium hydroxide solution
(10 mL, 2.5 M) to remove unreacted toluenesulfonyl chloride,
followed by sulfuric acid (2 × 10 mL, 25%) to remove pyridine,
then dried and evaporated. Purification by silica chromato-
graphy (eluting with hexane to CH2Cl2 stepwise then EtOAc)
followed by recrystallisation from hexane gave diisocyanide 1
(36.0 mg, 38%; 68.6 µCi, 57%).
37.9, 39.5, 40.9, 45.7, 44.6, 50.5, 53.3. m/z (EI) 238 (Mϩ ), 223,
ؒ
205 (100%), 149, 107, 95, 41.
[14C]-Thiocyanate and Axinyssa n.sp. The incorporation of
NaS14CN (25 µCi) was performed in the same way using
Axinyssa n.sp. (77 g wet wt.) in 200 mL of seawater. After a
16 day incorporation, the sponge was extracted to give
Reaction of [14C]2-diisocyanoadociane (1) with sulfur to give
[14C]2-bisisothiocyanatoadociane (17). The labelled diiso-
cyanide 1 (36.0 mg, 0.11 mmol, 68.6 µCi) was added to sulfur
(25 mg, 0.78 mmol) in a reaction vial and heated at 120 ЊC
overnight, after which time TLC analysis indicated no remain-
ing starting material. The reaction mixture was chromato-
graphed on a silica PTLC plate to give [14C]2-bisisothiocyanate
17 (17.2 mg, 40%; 23 µCi, 33 %). δH 0.65 (1H, ddd, J 10, 10 and
10), 0.74 (1H, m), 0.82 (1H, m), 0.84 (1H, m), 0.85 (3H, d,
J 6.5), 0.91 (1H m), 0.99 (3H, d, J 6.6), 1.01 (1H, m), 1.08 (2H,
m), 1.09 (1H, m), 1.10 (1H, m), 1.23 (1H, m), 1.26 (3H, s), 1.36
(3H, s), 1.38 (1H, m), 1.42 (1H, m), 1.50 (1H, m), 1.53 (1H, m),
1.77 (1H, ddd, J 13.4, 13.4 and 3.8), 1.87 (1H, dddd, J 13.6, 3.6,
3.6 and 3.6), 1.94 (1H, m), 1.98 (1H, m), 2.05 (1H, m). δC 16.4,
19.6, 20.4, 23.7, 25.6, 26.1, 26.2, 36.4, 38.2, 40.6, 40.7, 41.6,
42.2, 45.9, 46.6, 47.7, 49.0, 50.2, 64.4, 67.9, 130.0, 130.5. m/z
9-isocyanopupukeanane (10) (27.9 mg, 0.036%; 30 dpm mgϪ1
,
0.0015%), 2-thiocyanatoneopupukeanane (12) (12.9 mg,
0.017%; 1255 dpm mgϪ1, 0.029%) and 9-isothiocyanato-
pupukeanane (11) (8.8 mg, 0.011%; 22000 dpm mgϪ1, 0.35%).
Another piece of sponge (24 g) was treated identically with
NaS14CN (12.5 µCi). After a 3 day incorporation period, the
sponge was worked up to give 9-isocyanopupukeanane (10)
(11.1 mg, 0.046%; 19 dpm mgϪ1, 0.0075%), 2-thiocyanato-
neopupukeanane (12) (4.3 mg, 0.0018%; 3036 dpm mgϪ1
,
0.047%) and 9-isothiocyanato-pupukeanane (11) (5.9 mg,
0.024%; 53500 dpm mgϪ1, 1.14%).
Hydration of 9-isocyanopupukeanane (10) to formamide 14.
9-Isocyanopupukeanane (10) (7.4 mg, 0.0032 µCi mmolϪ1) was
treated with acetic acid (2 mL) as before and gave formamide 14
(6.9 mg, 87%; 0.0030 µCi mmolϪ1, 94%).
Conversion of 9-isothiocyanatopupukeanane (13) to diethyl-
thiourea 15. 9-Isothiocyanatopupukeanane (11) (4.0 mg, 2.6
µCi mmolϪ1) was refluxed in diethylamine as before to give
thiourea 15 (2.4 mg, 47%; 2.7 µCi mmolϪ1, 104%; see footnote
to Table 3).
(EI) 388 (Mϩ ), 345, 330, 271 (100%), 201, 55, 41. m/z (EI)
ؒ
388.2001 [Mϩ ] C22H32N2S2 requires 388.2006. This sample was
ؒ
split and used in two incorporation experiments at Lizard
Island.
Reduction of 2-thiocyanatoneopupukeanane (12) to thiol 16.
2-Thiocyanatoneopupukeanane (12) (4.1 mg, 0.015 mmol,
0.15 µCi mmolϪ1) in dry THF (1.2 mL) was added to LiAlH4
(2.9 mg, 0.076 mmol) and stirred at room temperature for
8 minutes, then poured into saturated aqueous ammonium
chloride (5 mL) and extracted with EtOAc (3 × 10 mL), dried
and evaporated to give 4.8 mg of a residue. Purification by silica
chromatography followed by normal phase HPLC (0.05%
EtOAc in hexane) gave thiol 16 (1.6 mg, 44%; 0.0005 µCi
mmolϪ1, 0.3%).
Degradation of synthetic [14C]2-diisocyanoadociane (1).
Hydration of diisocyanoadociane (1) to bisformamide 2. A small
sample (<0.1 mg) of synthetic [14C]-diisocyanoadociane (1) was
diluted with unlabelled material, then recrystallised before use
in degradation studies. The resulting diisocyanoadociane (1)
(7.1 mg, 0.022 mmol, 0.065 µCi mmolϪ1) was treated with
glacial acetic acid as before and gave bisformamide 2 (6.9 mg,
88%; 0.061 µCi mmolϪ1, 94.5%).
Hydrolysis of [14C]2-bisformamide 2 to [14C]-monoamine 3.
Bisformamide 2 (6.7 mg, 0.019 mmol; 0.061 µCi mmolϪ1) was
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 9 3 9 – 9 4 8
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hydrolysed as before and gave monoamine 3 (4.4 mg, 70%;
purified by silica chromatography to give [14C]-9-isocyano-
0.022 µCi mmolϪ1, 35.1%).
pupukeanane (10) (16.3 mg, 40.6 µCi) for biosynthetic use.
Reaction of [14C]-9-isocyanopupukeanane (10) with sulfur to
give [14C]-9-isothiocyanatopupukeanane (11). [14C]-9-Isocyano-
pupukeanane (10) (28.4 mg, 0.123 mmol, 61.5 µCi) was placed
in a reaction vial and sulfur (25.3 mg, 0.79 mmol) was then
added together with toluene (0.5 mL) and the mixture then
heated for 24 h at 120 ЊC after which time TLC analysis showed
some remaining starting material. Further sulfur (20 mg, 0.63
mmol) was then added and the reaction heated at 120 ЊC for a
further 24 h. The reaction mixture was chromatographed on a
silica PTLC plate to give 9-isothiocyanatopupukeanane (11)
(18.7 mg, 58%; 27.9 µCi, 45%). Removal of traces of [14C]-9-
isocyanopupukeanane (10) was accomplished by mixing of
[14C]-9-isothiocyanatopupukeanane (11) (18.7 mg, 27.9 µCi)
with 9-isocyanopupukeanane (10) (14 mg) and then separation
(silica chromatography) to give 9-isocyanopupukeanane (10)
which was further purified by repeated normal phase HPLC
(0.25% EtOAC in hexane) to give 10 (5.2 mg, 0.0001 µCi), and
[14C]-9-isothiocyanatopupukeanane (11) (11.1 mg, 27.9 µCi).
This process was repeated with a second batch of unlabelled 10
(10.0 mg) which gave 9-isocyanopupukeanane (10) (4.4 mg,
<0.0001 µCi) after normal phase HPLC, and [14C]-9-
isothiocyanatopupukeanane (11) (10.7 mg, 25.4 µCi, 622.3 µCi
mmolϪ1) for biosynthetic use.
Repeat synthesis of [14C]2-bisisothiocyanatoadociane (17).
Sodium [14C]-formate (250 µCi) was used in reaction with
bisamine 4 (62.7 mg, 0.21 mmol) as before to give [14C]-labelled
bisformamide 2 (65.4 mg, 88%; 30 µCi, 12.1%). Dehydration
with toluenesulfonyl chloride gave diisocyanide 1 (35.0 mg,
60%; 17.7 µCi, 59%). Reaction of the diisocyanide 1 (16.4 mg,
0.506 mmol, 6.9 µCi) with sulfur gave bisisothiocyanate 17 (11.7
mg, 71%; 6.7 µCi, 97%). To remove any traces of unreacted
starting material, [14C]-bisisothiocyanate 17, (11.7 mg, 6.7 µCi)
was mixed with unlabelled 1 (11 mg), and the mixture separated
by silica chromatography (eluting with hexane to CH2Cl2 step-
wise) to give [14C]2-bisisothiocyanate 17, (9.5 mg, 5.98 µCi),
and diisocyanoadociane (1) (11.0 mg, 0.01 µCi). Further
purification of isocyanide 1 by normal phase HPLC (5% EtOAc
in hexane) resulted in unlabelled diisocyanoadociane 1, (9.5 mg,
0.0003 µCi). A repeat of this process used unlabelled isocyanide
1 (10.0 mg) and gave [14C]2-bisisothiocyanate 17 (9.2 mg, 5.3
µCi; 224.7 µCi mmolϪ1) and diisocyanoadociane (1), (9.4 mg,
0.001 µCi). Purification by normal phase HPLC (5% EtOAc in
hexane) resulted in unlabelled diisocyanoadociane (1), (8.2 mg,
0.00005 µCi).
Synthesis of [14C]-9-isocyanopupukeanane (10) and [14C]-9-
isothiocyanatopupukeanane (11). Hydrolysis of 9-isocyano-
pupukeanane (10) to amine 18. 9-Isocyanopupukeanane (10)
(143 mg, 0.62 mmol) was dissolved in methanol (3 × 1 mL) and
added to HCl (6 M, 20 mL) and the solution refluxed for 3 h
and then left to stir overnight at room temperature. The result-
ing solution was then made basic with sodium hydroxide
and extracted with ethyl acetate (3 × 40 mL), dried and
evaporated to give the amine 18 (89.8 mg, 66%). δH 0.81 (3H,
d, J 6.3), 0.83 (3H d, J 6.3), 0.96 (3H, s), 1.00 (1H, m), 1.07 (3H,
s), 1.10 (1H, dd, J 8.2 and 13.4), 1.28 (1H, m), 1.30 (1H, m),
1.40 (1H, m), 1.45 (1H, m), 1.65 (1H, m), 1.84 (1H, dd, J 10.3
and 13.5), 1.90 (1H br d, J 12), 2.02 (1H, m), 2.28 (1H, br t,
J 10.5), 3.05 (1H, br s), 8.34 (1H, br s). δC 21.6, 21.7, 24.4,
25.5, 26.3, 29.9, 30.8, 34.4, 38.0, 39.0, 45.7, 48.4, 49.6, 43.5,
Incorporation experiments with advanced precursors
Incorporation of [14C]2-bisisothiocyanatoadociane (17) into A.
terpenensis (Lizard Island). A specimen of A. terpenensis (26 g
wet wt.) in 400 mL seawater, was incubated with advanced pre-
cursor [14C]2-bisisothiocyanatoadociane (17) (8.6 mg, 11.5 µCi)
in acetone (0.3 mL) for a 12 h period overnight. Water samples
taken at regular intervals revealed that 95% of the precursor
was taken up during this period. The sponge was then trans-
ferred back to the large aquarium for a 19 day incorporation
period. Extraction as before gave diisocyanoadociane (1) (11.8
mg, 0.045%; 398 dpm mgϪ1, 0.02%). The fraction containing
[14C]-bisisothiocyanate 17 was highly radioactive (6.1 mg,
0.026%; 37420 dpm mgϪ1, 0.9%). In a parallel incorporation
experiment, the sponge (24 g wet wt.) was treated identically
with advanced precursor [14C]2-bisisothiocyanatoadociane (17)
to give, after purification, diisocyanoadociane (1) (6.6 mg,
0.028%; 76 dpm mgϪ1, 0.002%). The fraction containing [14C]-
bisisothiocyanate 17 was highly radioactive (3.1 mg, 0.013%;
56910 dpm mgϪ1, 0.69%).
Hydration of diisocyanoadociane (1) to bisformamide 2.
Diisocyanoadociane (1) (7.1 mg, 0.022 mmol, 0.059 µCi
mmolϪ1) was treated with glacial acetic acid as before to give
bisformamide 2 (6.92 mg, 88%; 0.057 µCi mmolϪ1, 97%).
Hydrolysis of diisocyanoadociane (1) to bisamine 4. Diiso-
cyanoadociane (1) (2.5 mg, 0.008 mmol, 0.011 µCi mmolϪ1) was
treated with 6 M hydrochloric acid as before to give bisamine 4
(0.9 mg, 40%; 0.001 µCi mmolϪ1, 9.1%).
55.4. m/z (EI) 221 (Mϩ ), 204, 178, 161, 133, 119, 107, 91, 56,
ؒ
43 (100%); m/z (EI) 221.2139 (Mϩ ), C15H27N requires
ؒ
221.2144.
[14C]-Formylation of amine 18 to give [14C]-formamide 14.
Sodium [14C]-formate (750 µCi) was dissolved in formic acid (28
µL, 0.58 mmol) and stirred for 5 minutes. Pivaloyl chloride (68
µL, 0.55 mmol) was then added and stirred for 25 minutes.
Amine 18 (116 mg, 0.53 mmol) in CH2Cl2 (1 mL) was then
added, followed by triethylamine (363 µL, 2.64 mmol), and the
resulting solution stirred for 20 minutes. A further equivalent of
pivalic formic anhydride (20 µL formic acid in 65 µL pivaloyl
chloride) was then added and stirred for a further 20 minutes.
The whole reaction mixture was then poured into saturated
aqueous sodium bicarbonate (5 mL) and then extracted with
CH2Cl2 (3 × 2 mL), dried and evaporated to give a residue
(150.3 mg), that was purified by silica chromatography to give
[14C]-formamide (14) (78.0 mg, 59.3%, 142.6 µCi, 19%).
Dehydration of [14C]-formamide 14 to give [14C]-9-isocyano-
pupukeanane (10). [14C]-Formamide 14 (78 mg, 0.31 mmol,
142.6 µCi) was dissolved in pyridine (6 mL) and added to tolue-
nesulfonyl chloride (239 mg, 1.25 mmol) and the solution
refluxed for 3 h and then stirred overnight at room temperature.
The resulting brown solution was poured into hexane (50 mL)
and washed with sodium hydroxide (2.5 M, 2 × 40 mL) then
sulfuric acid (25%, 2 × 40 mL), then dried and evaporated to
give a brown residue (74.0 mg). The residue was purified by
silica chromatography to give [14C]-9-isocyanopupukeanane
(10) (60.8 mg, 84%, 132 µCi, 93%). A portion of this sample
(28.4 mg, 61.5 µCi) was set aside for further reaction. The
remaining [14C]-9-isocyanopupukeanane (10) was then further
Incorporation of [14C]2-bisisothiocyanatoadociane (17) into A.
terpenensis (Heron Island). The sponge (16.8 g wet wt.) was
incubated with advanced precursor [14C]2-bisisothiocyanato-
adociane (17) (9.2 mg, 5.3 µCi) to give diisocyanoadociane (1)
(5.4 mg, 0.032%; 3.4 dpm mgϪ1, <0.0002%). The fraction
containing [14C]-bisisothiocyanate 17 was highly radioactive
(4.1 mg, 0.024%; 37790 dpm mgϪ1, 1.3%).
Incorporation of [14C]-9-isocyanopupukeanane (10) into
Axinyssa n.sp. A piece of sponge (19.2 g wet wt.) in 400 mL
seawater was incubated with [14C]-9-isocyanopupukeanane (10)
(8.1 mg, 20.3 µCi) in methanol (2 × 0.5 mL). After a 21 day
incorporation, the sponge was extracted as before to give
9-isothiocyanatopupukeanane (11) (2.4 mg, 0.012%; 29440
dpm mgϪ1, 0.16%).
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biosynthetic study among the marine natural products”; (b) C. W. J.
Chang and P. J. Scheuer, Comp. Biochem. Physiol., B: Biochem. Mol.
Biol, 1990, 97, 227–233 these authors wrote that “the biogenesis of
the isocyano group in secondary metabolism has been of great
interest. Today, . . ., we still only have partial answers”.
5 (a) M. J. Garson, J. Chem. Soc., Chem. Commun., 1986, 35–36; (b)
C. J. R. Fookes, M. J. Garson, J. K. MacLeod, B. W. Skelton and
A. H. White, J. Chem. Soc., Perkin Trans. 1, 1988, 1003–1011.
6 (a) P. Karuso, A. Poiner and P. J. Scheuer, J. Org. Chem., 1989, 54,
2095–2097; (b) P. Karuso and P. J. Scheuer, J. Org. Chem., 1989, 54,
2092–2095.
7 B. Di Blasio, E. Fattorusso, S. Magno, L. Mayol, C. Pedone,
C. Santacroce and D. Sica, Tetrahedron, 1976, 32, 473–478.
8 (a) B. J. Burreson, P. J. Scheuer, J. Finer and J. Clardy, J. Am. Chem.
Soc., 1975, 97, 4763–4768; (b) M. R. Hagadone, B. J. Burreson,
P. J. Scheuer, J. S. Finer and J. Clardy, Helv. Chim. Acta, 1979, 62,
2484–2494.
Conversion of 9-isothiocyanatopupukeanane (11) to diethyl-
thiourea 15. 9-Isothiocyanatopupukeanane (11) (0.8 mg, 0.003
mmol, 4.6 µCi mmolϪ1) was treated with diethylamine as before
to give thiourea (15) (0.4 mg, 39%; 4.1 µCi mmolϪ1, 89%).
Incorporation of [14C]-9-isothiocyanatopupukeanane (11) into
Axinyssa n.sp. A piece of sponge (18.1 g wet wt.) in 400 mL
seawater was incubated with [14C]-9-isothiocyanatopupuke-
anane (11) (6.2 mg, 14.6 µCi) in acetone (2 × 0.5 mL). After a 21
day incorporation, the sponge was extracted as above to give
9-isocyanopupukeanane (10) (29.8 mg, 0.165%; 1320 dpm
mgϪ1, 0.12%).
Hydration of 9-isocyanopupukeanane (10) to formamide 14.
9-Isocyanopupukeanane (10) (13.5 mg, 0.058 mmol, 0.14 µCi
mmolϪ1) was treated with glacial acetic acid to give formamide
14 (5.5 mg, 38%; 0.14 µCi mmolϪ1, 100%).
9 J. S. Simpson, M. J. Garson, J. N. A. Hooper, E. I. Cline and
C. K. Angerhofer, Aust. J. Chem., 1997, 50, 1123–1127.
10 Reference 8a illustrates the close links between field-based
observation and natural products isolation that were a hallmark of
Paul Scheuer’s research over many years.
Acknowledgements
We thank the Australian Research Council and The University
of Queensland for funding. The assistance of the late
Dr Andrew Flowers, who first collected Axinyssa n.sp. for us,
and of Richard Clark and the staff of Heron and Lizard Island
Research Stations in performing fieldwork are gratefully
acknowledged. Professor Raffaele Riccio provided us with
valuable assistance in the course of one field trip. We thank
Dr John Hooper, The Queensland Museum, for taxonomic
identification of samples. This research was performed under
permits G97/097 and G98/227 issued jointly by GBRMPA and
QNPWS.
11 A. T. Pham, T. Ichiba, W. Y. Yoshida, P. J. Scheuer, T. Uchida,
J. Tanaka and T. Higa, Tetrahedron Lett., 1991, 32, 4843–4846.
12 E. J. Dumdei, A. E. Flowers, M. J. Garson and C. J. Moore, Comp.
Biochem. Physiol., A: Physiol., 1997, 118, 1385–1392.
13 (a) H. Schiewelbein, R Baumeister and R. Vogel, Naturwissen-
schaften, 1969, 56, 416–417; (b) J. Westley, in Cyanide in Biology, eds.
B. Vennesland, E. E. Conn, C. J. Knowles, J. Westley and F. Wissing,
Academic Press, London, 1981; vol 1, pp 61–76.
14 M. R. Hagadone, P. J. Scheuer and A. Holm, J. Am. Chem. Soc.,
1984, 106, 2447–2448.
15 J. S. Simpson and M. J. Garson, Tetrahedron Lett., 1999, 40,
3909–3912.
16 J. S. Simpson and M. J. Garson, Tetrahedron Lett., 2001, 42,
4267–4269.
17 J. S. Simpson and M. J. Garson, Tetrahedron Lett., 1998, 39,
5819–5822.
18 J. Westley, Adv. Enzymol., 1973, 39, 327–368.
19 M. N. Hughes, in Chemistry and Biochemistry of Thiocyanic Acid
and its Derivatives, ed. A. A. Newman, Academic Press, London,
1975, pp 2–67.
20 A. D. Wright, G. M. König and C. K. Angerhofer, J. Org. Chem.,
1996, 61, 3259–3267.
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