Epimerization of an Ascaroside-Type Glycolipid Downstream of the Canonical β-Oxidation Cycle in the Nematode Caenorhabditis nigoni

Article abstract of DOI:10.1021/acs.orglett.9b03808

A species-specific ascaroside-type glycolipid was identified in the nematode Caenorhabditis nigoni using HPLC-ESI-(-)-MS/MS precursor ion scanning, HR-MS/MS, and NMR techniques. Its structure containing an l-3,6-dideoxy-lyxo-hexose unit was established by

Full text of DOI:10.1021/acs.orglett.9b03808

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Epimerization of an Ascaroside-Type Glycolipid Downstream of the
Canonical β‑Oxidation Cycle in the Nematode Caenorhabditis nigoni
†
Celia P. Bergame, Chuanfu Dong,‡,∥ Sylvain Sutour,§ and Stephan H. von Reuß*,†,‡,§
́
†
̂
Laboratory for Bioanalytical Chemistry, Institute of Chemistry, University of Neuchatel, Avenue de Bellevaux 51, CH-2000
Neuchatel, Switzerland
̂
‡
̈
Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Hans-Knoll Straße 8, D-07745 Jena, Germany
§
̂
̂
̂
Neuchatel Platform for Analytical Chemistry (NPAC), University of Neuchatel, Avenue de Bellevaux 51, CH-2000 Neuchatel,
Switzerland
S
* Supporting Information
ABSTRACT: A species-specific ascaroside-type glycolipid was
identified in the nematode Caenorhabditis nigoni using HPLC-ESI-
(−)-MS/MS precursor ion scanning, HR-MS/MS, and NMR
techniques. Its structure containing an L-3,6-dideoxy-lyxo-hexose
unit was established by total synthesis. The identification of this
novel 4-epi-ascaroside (caenorhabdoside) in C. nigoni along with
the previous identification of 2-epi-ascarosides (paratosides) in
Pristionchus pacificus indicate that nematodes can generate highly
specific signaling molecules by epimerization of the ascarylose
building block downstream of the canonical β-oxidation cycle.
3,6-Dideoxyaldohexoses constitute essential building blocks of
bacterial lipopolysaccharides1 and nematode glycolipids.2 Of
the eight theoretically possible isomers (Figure 1), six have
(4) is known as a terminal glycan unit of glycoproteins of the
parasitic nematode Trichinella spiralis.9 The enantiomeric L-
ascarylose (3) was first described from lipophilic glycolipids
located in the eggs of parasitic nematodes of the Ascaris
genus.10 Over the past decade, L-ascarylose (3) has been
recognized as the core building block of a large diversity of
nematode-derived glycolipids, commonly known as ascaro-
sides, which represent key signaling molecules in nematode
chemical ecology.2 Nematode ascarosides include a large
diversity of homologous fatty acid like aglycones derived from
the peroxisomal β-oxidation cycle and, in addition, incorporate
a variety of building blocks from primary metabolic pathways
to form a modular library of signaling molecules.11 Recently, a
sixth 3,6-dideoxysugar, the enantiomeric L-paratose (5, 3,6-
dideoxy-L-ribo-hexose), was identified as a rare building block
of 2-epi-ascaroside-type glycolipids such as 12 from the
nematode Pristionchus pacificus.12 Here we describe the
identification of a novel 4-epi-ascaroside-type glycolipid 11
from the nematode Caenorhabditis nigoni that represents the
first natural product carrying the L-3,6-dideoxy-lyxo-hexose
unit (7) that we like to call L-caenorhabdose. The exclusive
specificity of these rare glycolipids for certain aglycones
suggests that nematodes can generate species-specific 3,6-
dideoxyaldohexoses via 2- and 4-epimerization of the L-
ascarylose building block (3) downstream of the highly
conserved peroxisomal β-oxidation cycle.
Figure 1. Eight possible stereoisomers of 3,6-dideoxyaldohexose, of
which five were identified from microorganisms (1−4, 6) and four
from nematodes (3−5, and 7, this work).
previously been described, which represent three pairs of
enantiomers (1−6). In many Gram-negative bacteria, 3,6-
dideoxyhexoses located at the terminal ends of lipopolysac-
charide chains constitute major epitopes that determinate
antigen activity3 such as L-colitose (1, 3,6-dideoxy-L-xylo-
hexose) from E. coli O111,4 D-abequose (2, 3,6-dideoxy-D-xylo-
hexose) from Salmonella abortus equi,5 L-ascarylose (3, 3,6-
dideoxy-L-arabino-hexose) from Yersinia pseudotuberculosis,6 D-
tyvelose (4, 3,6-dideoxy-D-arabino-hexose) from Salmonella
typhi,5,7 and D-paratose (6, 3,6-dideoxy-D-ribo-hexose) from
Salmonella paratyphi.8 Within the animal kingdom, D-tyvelose
Received: October 25, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b03808
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Comprehensive ascaroside profiling of the Caenorhabditis
nigoni exometabolome extract using two complementary
techniques, a HPLC-ESI-(−)-MS/MS precursor ion scan13
for the ascaroside specific fragment ion at m/z 73.0 [C3H5O2]−
(Figure S1) and GC-EIMS analysis of TMS-derivatized crude
metabolome extracts14 using extracted ion chromatograms for
the K1 fragment ion at m/z 130.1 [C6H14OSi]•+, resulted in
the identification of several common simple ascarosides (9, n =
0−7), along with some species-specific derivatives such as the
hydroxyacyl ascaroside asc-7OH-ΔC9 (10) (Figure 2).15
Table 1. 400 MHz NMR Data of the Hydroxyacyl
Ascaroside-Type Glycolipids asc-7OH-ΔC9 (10)15 and cae-
7OH-ΔC9 (11) from C. nigoni (in CD3OD)
asc-7OH-ΔC9 (10)
cae-7OH-ΔC9 (11)
1H
13C
1H
13C
1
2
4.65 s
3.75 s.br
97.8 4.78 s
69.9 3.57 s.br
98.8
68.2
32.3
3ax 1.80 ddd, 13.1, 11.0, 3.0
3eq 1.95 dt, 13.1, 3.8
35.9 2.06 dt, 14.3, 3.1
1.93 ddt, 14.3, 1.0, 3.4
68.3 3.57 s.br
71.4 3.99 dq, 1.0, 6.6
18.1 1.18 d, 6.6
4
3.52 ddd, 11.3, 9.3, 4.3
3.64 dq, 9.3, 6.2
1.22 d, 6.2
69.1
68.5
17.2
170.8
123.7
150.0
33.0
25.8
33.0
75.0
75.7
14.8
5
6
1′
2′
3′
4′
5′
6′
7′
8′
9′
-
170.6
-
5.82 d, 15.6
6.95 dt, 15.6, 7.0
2.27 m
1.54−1.68 m
1.48−1.62 m
3.53 m
123.2 5.82 d, 15.6
150.5 6.91 dt, 15.6, 7.0
33.1 2.26 m
25.9 1.53−1.70 m
33.0 1.52−1.59 m
74.9 3.52 m
3.74 dq, 3.9, 6.2
1.14 d, 6.2
75.3 3.75 dq, 3.9, 6.3
14.7 1.15 d, 6.3
Figure 2. Core structure of nematode ascarosides (9) along with the
species-specific hydroxyacyl ascaroside asc-7OH-ΔC9 (10) and 4-
epimeric hydroxyacyl caenorhabdoside cae-7OH-ΔC9 (11) from
Caenorhabditis nigoni, as well as the 2-epimeric L-paratoside par-C5
(12) from Pristionchus pacificus.
and, thus, suggested the cae-7OH-ΔC9 structure for
caenorhabdoside 11 (SMID:16 caen#1).
The structure assignment of cae-7OH-ΔC9 (11) was
unambiguously established by total synthesis as shown in
Scheme 1. The threo-configured aglycone (19) was prepared in
9.5% yield over six steps as previously described (Scheme
1a).15 (+)-Methyl D-lactate (13) was converted to the para-
methoxybenzyl (PMB) ether (14), reduced to the aldehyde 15,
and reacted with 4-pentenyl magnesium bromide to furnish the
threo-configured 2-O-PMB-3-hydroxy-oct-7-ene (16) with a
One additional putative ascaroside-type component (11)
was enriched from the C. nigoni exometabolome extract by
reverse-phase C18 solid-phase extraction (SPE) and finally
isolated as a 1:1 mixture along with coeluting asc-C7 (SMID:16
ascr#1, 9, n = 3) using semipreparative RP-C18-HPLC (Figure
S2). The molecular formula of C15H26O7 was established by
HRMS (found m/z 317.1603 [M − H]−, Δ 0.9 ppm), whereas
HR-MS/MS analysis suggested a hydroxynonenoic acid
1
diastereoisomeric excess of de > 92% as determined by H
NMR. Benzoylation of 16 gave 17 with de > 99% after column
chromatography. Cross metathesis of 17 with ethyl acrylate
using Grubbs second-generation catalyst furnished the non-
enoate ester 18, which was converted to the aglycone unit 19
upon selective cleavage of the PMB group. The 2,4-di-O-
benzoyl protected L-caenorhabdose building block (28) was
prepared in 1% yield over eight steps as shown in Scheme 1b.
Commercially available L-rhamnose (20) was converted to the
1-O-benzyl rhamnoside (21) and the 2,3-positions protected as
isoproylidene ketal (22). After formation of the 4-trifluor-
omethylsulfonate (triflate) ester (23) and hydrolysis of the
ketal, the resulting 24 was cyclized to the oxirane (25) under
slightly alkaline conditions.17 Regioselective oxirane ring
opening using lithium triethylborohydride furnished the L-1-
O-benzyl caenorhabdoside (26).18
After benzoylation to the ester 27, palladium-catalyzed
hydrogenation furnished the 2,4-di-O-benzoyl-caenorhabdose
building block (28) as a 7:3 mixture of the α- and β-anomers.
Glycoside formation between the aglycone (19) and the
caenorhabdose (28) unit was accomplished using the
trichloroacetimidate route19 to furnish 29 as a 3.4:1 mixture
of the α- and β-isomers. Subsequent deprotection of isolated
29 under alkaline conditions afforded cae-7OH-ΔC9 (11),
which displayed identical NMR (Figure S7) and LC-MS
(Figure S8) data as the natural product isolated from C. nigoni.
Potential behavioral activity was evaluated using a holding
assay that quantifies nematode retention in cae-7OH-ΔC9
(11) conditioned scoring regions as well as a two-spot
chemotaxis assay that quantifies nematode preference for
1
aglycone (Figure S3). Differential analysis of the H NMR
spectrum with those of a preceding fraction rich in asc-C7 (9, n
= 3) highlighted signals corresponding to the target compound
11 (Figure S4). Comparative analysis of the high-resolution
dqf-COSY spectrum of 11 with those of asc-7OH-ΔC9 (10),
previously identified from C. nigoni,15 suggested the presence
of a similar (ω − 1)-linked 7OH-ΔC9 aglycone, whereas the
7,8-threo-configuration was derived from the vicinal coupling
3
constant of J = 3.9 Hz by comparison with the threo- and
erythro-configured synthetic standards prepared as previously
described (Figure S5).15 Because the target compound 11 was
different from threo-asc-7OH-ΔC9 (10), the stereochemistry
of its 3,6-dideoxyhexose moiety was reevaluated based on the
chemical shifts and 1H,1H-coupling constants (Table 1, Figure
S6). Structure assignment was complicated by the fact that the
chemical shifts for H-2 and H-4 were almost indistinguishable,
but both methines were found to exhibit small coupling
constants with both anisochoric H-3 methylene protons at δH
1.93 ppm (dt, 2J = 14.2 Hz, 3J = 3.5 Hz) and 2.06 ppm (dt, 2J =
14.2 Hz, 3J = 3.1 Hz). Furthermore, the methine proton H-5 at
3
δH 3.99 ppm appeared as a broad quartet with J5,6 = 6.6 Hz,
3
3
instead of the dq-signal with J5,4 = 9.3 Hz and J5,6 = 6.2 Hz
commonly observed in ascarosides, indicating that the vicinal
3
coupling constant J5,4 is small. Along with the very small
(unresolved) coupling constant for the anomeric proton H-1,
these results indicated the α-configured 4-epimer of ascarylose
for the sugar moiety that we like to call caenorhabdose (7)
B
DOI: 10.1021/acs.orglett.9b03808
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
a
Scheme 1. Total Synthesis of Caenorhabdoside cae-7OH-ΔC9 (11) from C. nigoni
a
PMB-TCA: para-methoxybenzyl trichloroacetimidate, CSA: camphor-10-sulfonic acid, EA: ethyl acrylate. Grubbs cat. M2a: Grubbs second-
generation catalyst, 2,2-DMP: 2,2-dimethoxypropane, LiTEBH: lithium triethylborohydride, TCA: trichloroacetonitrile, DBU: 1,8-diazabicyclo-
undec-7-ene.
feeding in cae-7OH-ΔC9 (11) conditioned E. coli spots in
comparison with solvent-treated control. We found that cae-
7OH-ΔC9 (11), individually as well as in combination with
asc-7OH-ΔC9 (10), does not elicit any significant response in
adult C. nigoni males or females. Additional experiments will be
required to unravel the biological activity and ecological
function of these highly species-specific potential signaling
compounds.
Scheme 2. Postulated Biosynthesis of Highly Species-
Specific Signaling Molecules in the Nematodes C. nigoni and
P. pacificus Based on Epimerization of the L-Ascarylose
Building Block Downstream of the β-Oxidation Cycle
In conclusion, chemical analysis of the C. nigoni metabolome
resulted in the identification of a novel 4-epi-ascaroside-type
glycolipid (11), the first natural product carrying the 3,6-
dideoxy-lyxo-hexose unit (7). While the biogenesis of various
3,6-dideoxyhexoses in bacteria (1−4, 6) is well understood,20
the de novo pathway to L-ascarylose (3) as the core building
block of nematode ascarosides remains enigmatic. Functional
characterization of C. elegans genes with homology to those
implicated in the biogenesis of bacterial 3,6-dideoxyhexoses
demonstrated that they are involved in L-rhamnose (6-
deoxymannose) biosynthesis.21
Comprehensive ascaroside profiling of the C. nigoni
metabolome demonstrated that formation of the 4-epimeric
caenorhabdoside 11 is highly specific for the 7-OH-ΔC9
aglycone (Scheme 2). While its biosynthetic precursor asc-
ΔC9 (SMID: ascr#3, 30) is highly abundant, the correspond-
ing 4-epimeric caenorhabdoside with a ΔC9 aglycone could
not be detected (Figure S9), indicating that 4-epimerization
occurs downstream from the β-oxidation cycle and side-chain
hydroxylation. Similarly, analysis of P. pacificus daf-22, a
mutant defective in peroxisomal β-oxidation, did not reveal any
long-chain paratoside precursors,22 which suggests that par-C5
(SMID: part#9, 12) is derived from the corresponding asc-C5
(SMID: ascr#9, 31) via 2-epimerization downstream of the β-
oxidation cycle. Enzymes capable of catalyzing epimerization of
nonactivated hydroxymethine groups are well-known from
C
DOI: 10.1021/acs.orglett.9b03808
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
carbohydrate metabolism.23 Taken together, these results
demonstrate how epimerization of the L-ascarylose building
block acts along with peroxisomal β-oxidation, side chain
hydroxylation, and modular assembly to generate highly
species-specific glycolipids that form the chemical language
of nematodes.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.9b03808.
■
S
Detailed experimental procedures, supporting tables and
figures as described in the mail text, and NMR spectra of
isolated and synthetic compounds (PDF)
(10) Bartley, J. P.; Bennett, E. A.; Darben, P. A. J. Nat. Prod. 1996,
59, 921−926.
(11) Srinivasan, J.; von Reuss, S. H.; Bose, N.; Zaslaver, A.; Mahanti,
P.; Ho, M. C.; O’Doherty, O. G.; Edison, A. S.; Sternberg, P. W.;
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: stephan.vonreuss@unine.ch.
ORCID
(13) von Reuss, S. H.; Bose, N.; Srinivasan, J.; Yim, J. J.; Judkins, J.
C.; Sternberg, P. W.; Schroeder, F. C. J. Am. Chem. Soc. 2012, 134,
1817−1824.
́
Celia P. Bergame: 0000-0001-9075-505X
Chuanfu Dong: 0000-0003-3043-7257
Sylvain Sutour: 0000-0001-9676-7123
Stephan H. von Reuß: 0000-0003-4325-5495
Present Address
∥Department of Integrative Evolutionary Biology, Max Planck
Institute for Developmental Biology, Max-Planck-Ring 9, D-
(14) von Reuss, S. H.; Dolke, F.; Dong, C. Anal. Chem. 2017, 89,
10570−10577.
(15) Dong, C.; Reilly, D. K.; Bergame, C.; Dolke, F.; Srinivasan, J.;
von Reuss, S. H. J. Org. Chem. 2018, 83, 7109−7120.
(16) The nomenclature of ascarosides utilizes structure-based
abbreviations according to: (head group-)asc-(ω)(Δ)C#(terminal
group) as well as Small Molecule IDentifiers (SMIDs) that facilitate
database searching for small molecule metabolites (see: www.smid-db.
org).
̈
72076 Tubingen, Germany.
Notes
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347. (b) Cuccarese, M. F.; Wang, H.-Y. L.; O’Doherty, G. A. Eur. J.
Org. Chem. 2013, 2013, 3067−3075.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Financial support by the University of Neuchatel (UniNE), the
■
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731−732.
Max Planck Society (MPG), and the Swiss National Science
Foundation (SNSF) is gratefully acknowledged. All nematode
strains were provided by the Caenorhabditis Genetics Center,
which is funded by the NIH Office of Research Infrastructure
Programs (P40 OD010440).
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DOI: 10.1021/acs.orglett.9b03808
Org. Lett. XXXX, XXX, XXX−XXX