A 2-O-Methylriboside Unknown Outside the RNA World Contains Arsenic

Article abstract of DOI:10.1002/anie.201706310

Lipid-soluble arsenic compounds, also called arsenolipids, are ubiquitous marine natural products of currently unknown origin and function. In our search for clues about the possible biological roles of these compounds, we investigated arsenic metabolism in the unicellular green alga Dunaliella tertiolecta, and discovered an arsenolipid fundamentally different from all those previously identified; namely, a phytyl 5-dimethylarsinoyl-2-O-methyl-ribofuranoside. The discovery is of particular interest because 2-O-methylribosides have, until now, only been found in RNA. We briefly discuss the significance of the new lipid in biosynthesis and arsenic biogeochemical cycling.

Full text of DOI:10.1002/anie.201706310

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Accepted Article
Title: A 2-O-methylriboside unknown outside the RNA world contains
arsenic
Authors: Ronald Alexander Glabonjat, Georg Raber, Kenneth Bendix
Jensen, Nikolaus Guttenberger, Klaus Zangger, and Kevin
Francesconi
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A 2-O-methylriboside unknown outside the RNA world contains
arsenic
Ronald A. Glabonjat, Georg Raber, Kenneth B. Jensen, Nikolaus Guttenberger, Klaus Zangger, and
Kevin A. Francesconi*
Abstract: Lipid-soluble arsenic compounds, also called arsenolipids,
are ubiquitous marine natural products of currently unknown origin
and function. In our search for clues about the possible biological
roles of these compounds, we investigated arsenic metabolism in
the unicellular green alga Dunaliella tertiolecta, and discovered an
arsenolipid fundamentally different from all those previously
identified; namely,
a
phytyl 5-dimethylarsinoyl-2-O-methyl-
ribofuranoside. The discovery is of particular interest because 2-O-
methylribosides have, until now, only been found in RNA. We briefly
discuss the significance of the new lipid in biosynthesis and arsenic
biogeochemical cycling.
Figure 1.
unicellular alga Dunaliella tertiolecta.
A phytyl 5-dimethylarsinoyl-2-O-methyl-ribofuranoside from the
High resolution electrospray mass spectrometry of an
ethanol/dichloromethane extract of the alga showed an ion with
m/z 547.3337 [M+H]+ corresponding to a molecular formula of
The biological chemistry of arsenic is highly topical, with re-
ported human health issues ranging from detrimental effects[1]
+
C28H56AsO5 (∆m = -0.26 ppm) (Figure 2). Tandem MS analysis
from arsenic exposure to arsenic’s beneficial use as
a
therapeutic agent.[2] Arsenic has even been reported to have an
essential role in extremophile biology, in that it could replace
phosphorus in DNA[3], a claim that has been vigorously refuted.[4]
We now report the discovery of an unusual arsenic-containing
lipid from a unicellular marine alga that incorporates a 2-O-
methylriboside bound to phytol. Methylation of the ribose ring in
the 2-O position has never been found in simple natural
carbohydrates, yet is found in all major classes of RNA,[5,6] and
has been hypothesized as the evolutionary link between RNA
and DNA.[5,7] The discovery of this new arsenylated 2-O-
of this molecular ion yielded an arsenic-containing product with
[M+H]+ of m/z 269.0366 resulting from a neutral loss of the lipid
moiety corresponding to a C20H38-unit (m/z 278.2972; see below).
The mass spectrum also revealed fragment ions at m/z 123 and
137, which are characteristic of the dimethylarsinoyl (Me2AsO)
group. Treatment of the extract of D. tertiolecta with H2S or
MeI/DTT produced, respectively, thioxo and trimethylated
derivatives of the natural product, which supported this
dimethylarsinoyl assignment (Figure S2 & Table S1). Acid
hydrolysis of the arsenolipid quantitatively produced a water-
soluble arsenical with m/z 269 [M+H]+, identical with the MS
fragmentation product, whereas base hydrolysis produced no
change suggesting that the lipid contained a glycosidic bond
(Figure S2 & Table S1).
methylriboside could again stimulate speculation about
biological role for arsenic in early life.
a
We first detected the new arsenolipid in extracts of the
marine unicellular alga Dunaliella tertiolecta and in extracts from
oceanic phytoplankton. HPLC/elemental mass spectral analysis
revealed that Dunaliella contained small amounts of several
arsenolipids including two known compounds,[8-10] and one major
arsenic-containing lipid with properties quite different from
previously reported compounds (Figure S1). By combining mass
spectral data with data from degradation and derivatization
experiments, partial chemical synthesis, and NMR spectroscopy
on a trace of isolated natural product, as detailed below, we
identified the new compound as phytyl 5-dimethylarsinoyl-2-O-
methyl-ribofuranoside (Figure 1).
Figure 2. HR-ESMS of the phytyl 5-dimethylarsinoyl-2-O-methyl-
ribofuranoside from the unicellular alga Dunaliella tertiolecta.
R. A. Glabonjat, Dr. G. Raber, Dr. K. B. Jensen,
Dr. N. Guttenberger, Dr. K. Zangger, Dr. K. A. Francesconi
Institute of Chemistry, NAWI Graz
University of Graz
Universitaetsplatz 1/I, 8010 Graz (Austria)
E-mail: kevin.francesconi@uni-graz.at
Further tandem mass spectral analysis of the m/z 269 ion
from the original arsenolipid revealed a fragmentation pattern
similar to that shown by dimethylarsinoylribosides[11] except that
some characteristic fragment-ions had an additional 14 Da. This
pattern was consistent with a methoxy substitution of one of the
sugar ring hydroxyl groups (Figure S9); O-methylation of the
intact lipid with HBF4/TMSCHN2 gave a product (m/z 561.3494)
with an additional 14 Da, which supported the presence of a
Supporting information for this article is given via a link at the end of
the document.
1
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10.1002/anie.201706310
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single methoxy in the original lipid, and its location at the 2-O-
position was derived from tandem MS data before and after 3-O-
methylation (Figure S2 & Table S1).
tertiolecta in a medium enriched with arsenate with a view to
obtaining sufficient material for NMR analysis (Figure S7). In this
way, we produced about 2 g of dry cells containing ca 20 µg As
as the new arsenolipid, which was purified (Figure S8) by
solvent partitioning, silica column chromatography, and
Encouraged by the preliminary results showing similarities
between our compound and arsenoribosides, while remaining
sceptical about the 2-O-methoxy motif, we then synthesized the
model methyl 5-dimethylarsinoyl-2-O-methyl-β-D-ribofuranoside
(7) for comparison with the natural product (Scheme 1). On
electrospray ionization in the tandem mass spectrometer, the
model compound obligingly produced the 5-dimethylarsinoyl-2-
O-methyl ribose, which then fragmented in a manner identical
with that of the natural product (Figures 3, S3 & S4). In contrast,
two previously synthesized non-O-methylated arsenic-containing
cyclic ethers, with exact masses identical with that of 5-
dimethylarsinoyl-2-O-methyl ribose, did not match this
fragmentation pattern.[12]
preparative reversed-phase HPLC to provide
a material
weighing ca 0.1 mg and containing 15 µg As. Full spectra
1
assignment of all H and 13C signals (Table 1) corroborated the
1
structure shown in Figure 1. H NMR clearly showed the signals
corresponding to the proposed dimethylarsinoylriboside
structure, including the 2-O-methyl group. Equally informative
was the hydrophobic portion of the molecule, which revealed a
branched hydrocarbon chain consistent with phytol (3,7,11,15-
tetramethylhexadec-2-en-1-ol), an algal natural product
commonly incorporated into lipids including chlorophyll-a and
vitamin K1.
Table 1. 1H and 13C NMR results for phytyl 5-dimethylarsinoyl-2-O-methyl-
ribofuranoside extracted from Dunaliella tertiolecta.
(1H)
(13C)
(1H)
(13C)
Position
Position
[ppm]
[ppm]
[ppm]
[ppm]
1
5.03
103.7
84.2
75.7
77.9
37.2
16.2
58.7
64.4
119.3
141.9
40.0
25.2
36.8
15
1.41
32.7
37.3
24.5
37.4
32.8
37.4
24.8
39.4
28.0
22.6/22.7
16.4
19.7
19.7
2
3.73
16
1.09/1.29
1.22/1.33
1.09/1.29
1.41
3
4.23
17
Scheme 1. Synthesis of methyl 5-dimethylarsinoyl-2-O-methyl-β-D-
ribofuranoside. Reagents and conditions: (a) cat. H2SO4, MeOH, 0 °C  rt,
overnight; b) 1.1 eq 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane, anhydrous
4
4.23
18
5
2.45
19
pyridine,
0 °C  rt, 60 min; (c) 3.1 eq NaH, MeI/1,3-dimethyl-2-
imidazolidinone, rt, 40 min; (d) 3.0 eq TBAF.3H2O, THF, rt, 30 min; (e) PPh3,
CCl4, anhydrous pyridine, 50-55 °C, 30 min; (f) Me2AsI/Na, THF, rt overnight,
then H2O2 (for full details see Supporting Information).
6, 7
8
1.83
20
1.09/1.29
1.30
3.56
21
9
4.05/4.20
5.31
22
1.17
10
11
12
13
14
23
1.56
---
24, 28
25
0.90
2.03
1.68
1.44
26
0.89
1.13/1.31
27
0.89
The presence of phytol in the arsenolipid was also consistent
with the previous tandem MS observation of a neutral loss of a
C20H38-unit (m/z 278.2972; ∆m = -0.40 ppm). We then acid-
hydrolyzed the purified lipid and quantified the two products; the
data showed excellent agreement with a 1:1 molar ratio of
arsenosugar/phytol (Figure S5). On the basis of the experiments
reported here, the new arsenolipid was identified as ((3-hydroxy-
2-methoxy-1-((3,7,11,15-tetramethylhexadec-2-en-1-
yl)oxy)tetrahydrofuran-4-yl)methyl)dimethylarsine oxide (Figure
1).
Figure 3. Tandem mass spectral comparison between synthetic methyl 5-
dimethylarsinoyl-2-O-methyl-β-D-ribofuranoside (7), and the phytyl 5-
dimethylarsinoyl-2-O-methyl-ribofuranoside from the unicellular alga Dunaliella
tertiolecta.
Algae are responsible for about 50 % of the total global
primary production of carbon, and most of the algal productivity
is attributed to unicellular algae (phytoplankton).[13] It is likely that
the new arsenolipid will prove to be widespread among
unicellular algae, and thus its biosynthesis and degradation will
play a pivotal role in the biogeochemical cycling of arsenic.
Macroalgae, however, do not appear to synthesize this
Concurrent with our attempts to arrive at a viable structure by
mass spectrometry and synthesis, we cultured more D.
2
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10.1002/anie.201706310
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arsenolipid, but rather they produce lipids incorporating an
arsenoriboside.[8,14] The transformation of arsenate by
methylation is a common process in biology, possibly invoked as
a detoxification step. This process takes place through the
universal methyl donor S-adenosylmethioneine, which can also
be responsible for 2’-O-methylation of RNA,[5] and in the case of
macroalgae, is further thought to serve as the source of the
ribose ring itself.[15] Whether the 2-O-methyl analogue of S-
adenosylmethioneine can act in a similar manner is unknown.
The fundamental difference in the arsenolipids produced by
unicellular compared with macroalgae might reflect an early
evolutionary change in their ability to handle or use arsenic in an
evolving world still exploring possible elemental substrates.
While there are no data that clearly demonstrate a biological
role for arsenic, it is increasingly being reported in molecules
that play an important role in biology, for example bound into
phosphatidylcholines as constituents of membrane lipids.[10] The
hydrophobic phytol part of the arsenolipid described here might
also be relevant to arsenic’s role in lipid chemistry by anchoring
the molecule in the membrane of the chloroplast, a role similar
to phytol in chlorophyll-a. We hope the report of this unusual
new natural product will stimulate research into arsenic’s
biological chemistry.
Keywords: arsenic • arsenolipid • phytol • arsenosugar • 2-O-
methyl-riboside
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We thank Josef Ehgartner, Elliott G. Duncan and William A.
Maher for providing algae samples cultured in the Ecochemistry
Laboratory at the University of Canberra. This research was
supported by the Austrian Science Fund (FWF) project numbers
23761-N17 and I2412-B21. We also thank NAWI Graz for
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