N3,5′-Cycloxanthosine, the first natural occurrence of a cyclonucleoside

Article abstract of DOI:10.1021/np0502692

An Eryus sp. of marine sponge from the Great Australian Bight has yielded the first reported natural occurrence of a cyclonucleoside, N ³,5'-cycloxanthosine. The structure of N³,5'- cycloxanthosine was confirmed by detailed spectroscopic analysis and total synthesis. 2005 American Chemical Society and American Society of Pharmacognosy.

Full text of DOI:10.1021/np0502692

J. Nat. Prod. 2005, 68, 1689-1691
1689
N3,5′-Cycloxanthosine, the First Natural Occurrence of a Cyclonucleoside
Robert J. Capon* and Nicholas S. Trotter
Centre for Molecular Biodiversity, Institute for Molecular Bioscience, The University of Queensland, St Lucia,
Queensland, 4072, Australia
Received July 26, 2005
An Eryus sp. of marine sponge from the Great Australian Bight has yielded the first reported natural
occurrence of a cyclonucleoside, N3,5′-cycloxanthosine. The structure of N3,5′-cycloxanthosine was
confirmed by detailed spectroscopic analysis and total synthesis.
During investigations into bioactive metabolites from
Australian marine sponges we had cause to examine an
Eryus sp. (class, Demospongia; order, Astrophorida; family,
Geodiidae) obtained in July 1995 during scientific trawling
operations in the Great Australian Bight. Although the
aqueous EtOH extract of this specimen did not demonstrate
significant biological activity in our target bioassays (an-
timicrobial and antiparasitic), we were intrigued by the
presence of unidentified “aromatic” compounds in the H2O-
soluble partition. In past years we have observed that
sponge extracts can yield novel H2O-soluble metabolites,
such as the unprecedented sulfur sugar 5-thio-D-mannose,1
the nematocidal amino acid analogues echinobetaines A2
and B3, and the acetylcholine mimic esmodil.4 In each of
these cases, the structural proofs were supported by
detailed spectroscopic analysis and total synthesis. Encour-
aged by these experiences and the prospect that the Eryus
sp. may also contain novel metabolites, we embarked on a
detailed chemical analysis.
for 2 (see Table 1) were deceptively simple, indicative of a
nuclear base bearing a single aromatic proton attached to
a sugar moiety, suggestive of a modified nucleoside. Lack
of a measurable 1H NMR coupling or 2D NMR COSY
correlation to the proposed anomeric proton H-1′ (δ 6.15)
was interpreted as evidence of a restricted conformation
featuring dihedral angles that disfavored J1′,2′ coupling.
This analysis was further tempered by the observation of
2D NMR gHMBC correlations from H-1′ to C-2′, C-3′, and
C-4′. Connection of the sugar moiety through N9 was
supported by 2D NMR gHMBC correlations from H-1′ to
C-4 and C-8. The UV and 2D NMR data for 2, while
supportive of a modified nucleoside, did not provide con-
clusive evidence from which to assign unambiguously a
molecular structure. To more fully explore this possibility
and provide an unambiguous assignment of molecular
structure, we embarked on a structure proof by total
synthesis.
An early and plausible candidate structure for 2 was that
of 8,5′-O-cycloinosine; however, although known as a
synthetic compound since 1976,10 the published spectro-
scopic data for 8,5′-O-cycloinosine proved inadequate for
modern comparison purposes. Employing literature pro-
cedures,10-13 we completed a total synthesis of 8,5′-O-
cycloinosine in five steps from adenosine, as outlined in
Scheme 1 (for experimental details see the Supporting
Information). To our disappointment, 8,5′-O-cycloinosine
did not coelute on HPLC with, and the 1H NMR (d6-DMSO
or d4-MeOH) data clearly differed from that of, the natural
product 2. Despite these differences, the data were suf-
ficiently similar for us to conclude that 2 is indeed a
cyclonucleoside, just not 8,5′-O-cycloinosine.
The freeze-dried H2O-soluble partition of an aqueous
EtOH extract of the Eryus sp. was fractionated by elution
through Sephadex G-10 (H2O) followed by C18 HPLC, to
yield the two pure “aromatic” metabolites. The first of these
metabolites was identified as C2-R-D-mannosylpyranosyl-
tryptophan (1). This unusual C-glycosylated tryptophan
was first reported in 1994 by Hofsteenge et al.5 during an
investigation into novel post-translational modification in
human RNases and was subsequently detected by the same
authors in a range of other human proteins including IL-
126 and the erythropoietin receptor.7 Although first recog-
nized as a rare post-translational C-glycosylation event in
human proteins, the free glycosylated amino acid 1 was
subsequently reported in 2000 by Riguera et al.8 from a
marine ascidian. Following this study, in 2001 C2-R-D-
mannosylpyranosyltryptophan (1) was acknowledged by
Yonemura et al.9 as a novel marker of renal function, being
an accurate measure of insulin clearance in patients with
chronic renal failure. Our isolation of C2-R-D-manno-
sylpyranosyltryptophan (1) from an Australian marine
sponge, Eryus sp., suggests that tryptophan C-glycosylation
as a biosynthetic event may be more widespread than
previously documented.
While the reisolation of C2-R-D-mannosylpyranosyltryp-
tophan was noteworthy, of greater interest to us was the
discovery of the co-metabolite, 2. On the basis of high-
resolution ESI(+)MS measurements (M + Na ∆mmu -0.2),
2 was attributed the molecular formula C10H10N4O5, re-
A reappraisal of the data for 2 suggested an alternative
cyclonucleoside structure, namely, N3,5′-cycloxanthosine.
First synthesized in 1963,14 and further characterized by
ORD studies,15 N3,5′-cycloxanthosine remained dormant in
the scientific literature until 2004, at which time it came
to our attention through the publication16 of a convenient
1
quiring eight double-bond equivalents. The H NMR data
1
one-step synthesis from xanthosine. Although the H and
* Corresponding author. Tel: +61-7-33462979. Fax: +61-7-33462101.
E-mail: r.capon@imb.uq.edu.au.
13C NMR (d6-DMSO) data reported for N3,5′-cycloxan-
10.1021/np0502692 CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/02/2005

1690 Journal of Natural Products, 2005, Vol. 68, No. 11
Notes
Table 1. NMR (d6-DMSO, 600 MHz) Data for N3,5′-Cycloxanthosine (2) and 8,5′-O-Cycloinosine
2
8,5′-O-cycloinosine
position
δC
δH (m, J (Hz)) (m,J)
COSY
gHMBCa
δC
δH [m, J (Hz)]
1
7.00 (br s)
8.04 (s)
2
151.0
H-5′a
145.8
3
4
140.7
117.8
157.4
H-8, H-1′, H-5′a
H-8
H-8
145.8
119.3
155.9
5
6
7
8
134.5
7.81 (s)
H-1′
152.5
9
1′
92.5
75.8
70.1
83.7
51.3
51.3
6.15 (s)
OH-2′, H-4′, H-5′a
H-1′, OH-2′, OH-3′
H-1′, OH-3′, H-4′, H-5′a,b
H-1′, H-2′, OH-3′, H-5′b
H-3′, H-4′
89.1
77.1
71.0
88.2
74.5
74.5
5.93 (s)
2′
3.87 (dd, 5.2, 5.2)
4.20 (br m)
4.56 (m)
OH-2′, H-3′
H-2′, OH-3′, H-4′
H-3′, H-5′a,b
H-4′, H-5′b
H-4′, H-5′a
H-2′
4.27 (d, 6.0)
4.45 (d, 6.1)
4.56 (s)
3′
4′
5′a
5′b
OH-2′
OH-3′
4.56 (m)
4.56 (d, 13.1)
4.05 (d, 13.1)
3.71 (dd, 15.2, 3.2)
5.50 (d, 5.0)
5.44 (d, 6.6)
H-3′, H-4′
H-3′
a Protons correlated to carbon resonances in the δC column.
Scheme 1. Synthesis of 8,5′-O-Cycloinosinea
8,5′-O-cycloinosine or N3,5′-cycloxanthosine. By contrast,
8,3′-O-cycloadenosine has been described as an inhibitor
of rhodopsin kinase,21 and the pyrimidine analogues,
exemplified by 2,2′-O-cyclocytidine and 2,5′-O-cyclocytidine,
have been acknowledged as antitumor22 and antiviral
agents,23 respectively. Carbocyclic cyclonucleoside ana-
logues such as 8,5′-cycloadenosine are known as products
of oxidative damage to DNA.24 Most recently, in 2004, the
value of cyclonucleosides has been revisited, with N3,5′-
cycloxanthosine (2) being patented25 as a member of a
family of “synthetic” nucleosides with antiviral properties.
a Reagents: (i) Br2, NaOAc buffer; (ii) p-TsOH, acetone; (iii) NaH, 1,4-
dioxane; (iv) 1 M H2SO4; (v) NaNO2, acetic acid.
Experimental Section
Scheme 2. Synthesis of N3,5′-Cycloxanthosine (2)a
General Experimental Procedures. See Supporting In-
formation.
Biological Material. The marine sponge Eryus sp. (Mu-
seum of Victoria Registry Number MVF83533) was collected
by beam trawl in July 1995 from the Great Australian Bight
at a depth of 50 m at position 33°35′ S, 114°46′ E. The sponge
was diced, steeped in EtOH, and kept at -20 °C prior to
extraction.
Extraction and Isolation. The EtOH extract was decanted
and concentrated in vacuo to yield a crude extract (4.68 g).
Trituration with DCM returned insoluble material (4.62 g,
99%), which was partitioned between n-BuOH (2.32 g, 50%)
and H2O (2.25 g, 48%). The freeze-dried H2O-soluble material
was fractionated by Sephadex G-10 (H2O) and C18 HPLC (2.5
mL min-1 5% isocratic CH3CN/H2O through a 10 µm Phenom-
enex Spherex 5 C18 250 × 10 mm column) chromatography to
return the previously reported C2-R-D-mannosylpyranosyltryp-
tophan8,26,27 (1) (10.0 mg, 0.21%) and (-)-N3,5′-cyclo-
xanthosine14-16 (2) (9.5 mg, 0.20%). In subsequent isolations
of natural products 1 and 2, the C18 HPLC column of choice
proved to be a 5 µm Agilent Zorbax SB-Aq 150 × 9.4 mm
column (4.2 mL min-1 H2O elution). All yields for purified
metabolites were calculated against the crude ethanolic extract
(4.68 g).
a Reagents: (i) PPh3, diisopropylazodicarboxylate, DMF.
thosine in 2004 was a close match to the natural product
2, small discrepancies and the absence of an optical rotation
required that we repeat the synthesis and carry out an
independent comparison. On repeating the synthesis of
N3,5′-cycloxanthosine (Scheme 2) (for experimental details
see the Supporting Information), we observed that the
natural product 2 was in fact identical in all respects (1H
and 13C NMR, UV/vis, [R]D, and coelution on HPLC) to
N3,5′-cycloxanthosine.
The discovery of novel nucleosides from marine sponges
is not without precedence. Indeed, the discovery of spon-
gothymidine and spongouridine from a Caribbean sponge
in the 1950s by Bergmann et al.17-19 has been credited20
with inspiring development of the commercial antileukemic
agent Ara-C and the antiviral agent Ara-A. It has even
been suggested20 that knowledge of these sponge nucleo-
sides inspired development of the anti-AIDS drug AZT.
With respect to cyclonucleosides, although a target for
synthetic chemists as early as the 1960s, the scientific
literature is sparse on their application against biological
targets, with no primary literature on the biological screen-
ing or biological properties of the purine cyclonucleosides
C2-r-D-Mannosylpyranosyltryptophan8,26,27 (1): white
solid. Natural product 1 was identified by spectroscopic
analysis [1H and 13C NMR, ESI(()MS], optical rotation ([R]D),
and comparison with literature data.
(-)-N3,5′-Cycloxanthosine14-16 (2): white solid; [R]D -18°
(c 0.021, DMSO); UV (H2O) λmax (log ꢀ) 236 (3.91), 266 (3.99)
1
nm; H NMR (d6-DMSO, 600 MHz), see Table 1; (d4-MeOH,
600 MHz) δ 7.80 (s, H-8), 6.15 (s, H-1′), 4.76 (ddd, J ) 14.4,
2.3, 0.7 Hz, H-5′a), 4.68 (dt, J ) 3.6, 2.6, 2.6 Hz, H-4′), 4.34
(dd, J ) 5.5, 3.8 Hz, H-3′), 4.04 (d, J ) 5.6 Hz, H-2′), 3.87 (dd,
J ) 14.4, 2.7 Hz, H-5′b); 13C NMR (d6-DMSO, 150 MHz), see
Table 1; 13C NMR (d4-MeOH, 100 MHz) δ 159.7 (C, C-6), 152.9
(C, C-2), 142.5 br (C, C-4), 136.3 br (CH, C-8), 119.4 br (C,

Notes
Journal of Natural Products, 2005, Vol. 68, No. 11 1691
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531 [2M - H]-, 265 [M - H]-; HRESI(+)MS m/z 289.0547 ([M
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Acknowledgment. We acknowledge the CSIRO Division
of Oceanography, and the crew and scientific support staff
aboard the RV Franklin, for access and assistance in sample
collection. We also thank L. Goudie for taxonomic classifica-
tion, D. Howse for data management, and J. Ford and L.
Dempster for contributions toward chromatographic fraction-
ation.
Supporting Information Available: General experimental pro-
cedures, synthetic schemes, experimental procedures, and references
and notes for the synthesis of compounds 2 and 10. This material is
available free of charge via the Internet at http://pubs.acs.org.
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NP0502692