Comparative Ascaroside Profiling of Caenorhabditis Exometabolomes Reveals Species-Specific (ω) and (ω - 2)-Hydroxylation Downstream of Peroxisomal β-Oxidation

Article abstract of DOI:10.1021/acs.joc.8b00094

Chemical communication in nematodes such as the model organism Caenorhabditis elegans is modulated by a variety of glycosides based on the dideoxysugar l-ascarylose. Comparative ascaroside profiling of nematode exometabolome extracts using a GC-EIMS scree

Full text of DOI:10.1021/acs.joc.8b00094

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Article
Comparative Ascaroside Profiling of Caenorhabditis
Exometabolomes Reveals Species-Specific (#) and (# – 2)-
Hydroxylation Downstream of Peroxisomal #-Oxidation.
Chuanfu Dong, Douglas K. Reilly, Célia Bergame, Franziska
Dolke, Jagan Srinivasan, and Stephan Heinrich von Reuss
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b00094 • Publication Date (Web): 26 Feb 2018
Downloaded from http://pubs.acs.org on February 27, 2018
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Comparative Ascaroside Profiling of Caenorhabditis Exometabolomes
Reveals SpeciesꢀSpecific (ω) and (ω – 2)ꢀHydroxylation Downstream of
Peroxisomal βꢀOxidation.
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Chuanfu Dong,1,2 Douglas K. Reilly,3 Célia Bergame,4 Franziska Dolke,1 Jagan Srinivasan,3
and Stephan H. von Reuss*,1,4
1: Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, HansꢀKnoell Strasse 8, Dꢀ
07745 Jena, Germany
2: Department for Integrative Evolutionary Biology, Max Planck Institute for Developmental Biology, Maxꢀ
PlanckꢀRing 9, Dꢀ72076 Tübingen, Germany
3: Department of Biology and Biotechnology, Worcester Polytechnic Institute, 60 Prescott Street, Worcester,
MA 01605, United States
4: Laboratory of Bioanalytical Chemistry, University of Neuchatel, Avenue de Bellevaux 51, CHꢀ2000
Neuchâtel, Switzerland
* Corresponding author: Stephan H. von Reuss
Phone: 0041 (0)32ꢀ718ꢀ2510
Fax: 0041 (0)32ꢀ718ꢀ2511
Eꢀmail: stephan.vonreuss@unine.ch
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ABSTRACT
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Chemical communication in nematodes, such as the model organism Caenorhabditis elegans,
is modulated by a variety of glycosides based on the dideoxysugar Lꢀascarylose. Comparative
ascaroside profiling of nematode exometabolome extracts using a GCꢀEIMS screen reveals
that several basic components including ascr#1 (ascꢀC7), ascr#2 (ascꢀC6ꢀMK), ascr#3 (ascꢀ
ꢁC9), ascr#5 (ascꢀωC3), and ascr#10 (ascꢀC9) are highly conserved among the
Caenorhabditis. Three novel, side chain hydroxylated ascaroside derivatives were exclusively
detected in the distantly related C. nigoni and C. afra. Molecular structures of these speciesꢀ
specific, putative signaling molecules were elucidated by NMR spectroscopy and confirmed
by total synthesis and chemical correlations. Biological activities were evaluated using
attraction assays. The identification of (ω)ꢀ and (ω – 2)ꢀhydroxyacyl ascarosides demonstrates
how GCꢀEIMSꢀbased ascaroside profiling facilitates the detection of novel ascaroside
components and exemplifies how speciesꢀspecific hydroxylation of ascaroside aglycones
downstream of peroxisomal βꢀoxidation increases the structural diversity of this highly
conserved class of nematode signaling molecules.
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INTRODUCTION
Research with the model organism Caenorhabditis elegans and the development of novel
analytical techniques promoted the characterization of ascarosides, a modular glycolipid
library based on the 3,6ꢀdideoxysugar Lꢀascarylose linked to fatty acid derived aglycones
(Scheme 1).1 In C. elegans, ascarosides modulate a large diversity of biological responses
including dauer formation,2ꢀ6 reproduction,6,7 stress resistance,7,8 lifespan,8,9 and behavior.10ꢀ14
The diversity of biological responses modulated by ascarosides is paralleled by their large
structural diversity. Even small changes in molecular structures,7,14,15 synergistic effects,10,11,16
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and variations in ascaroside compositions7,17,18 can dramatically alter their biological activity.
Homologous series originate from chain shortening of very long chain precursors upon
peroxisomal βꢀoxidation (Scheme 1A) to furnish ascarosides carrying acyl (1, ascꢀC#), enoyl
(2, ascꢀꢁC#), and (3R)ꢀhydroxyacyl (3, ascꢀβOHꢀC#) aglycones.19ꢀ23 2ꢀKetoalkyl (6, ascꢀC#ꢀ
MK) and 2ꢀhydroxyalkyl (7, ascꢀC#ꢀOH) aglycones are presumably produced via
decarboxylation and subsequent reduction of labile 3ꢀketoacyl intermediates (4). Furthermore,
(ω)ꢀascarosides such as ascꢀωC3 (8, ascr#5) demonstrate that (ω – 1) and (ω)ꢀlinked
components enter the peroxisomal βꢀoxidation cycle. Downstream of the peroxisomal βꢀ
oxidation cycle, the resulting basic ascaroside skeletons serve as scaffolds for the attachment
of additional structural units derived from primary metabolic pathways to furnish a modular
library of speciesꢀspecific signaling components such as the indole ascaroside ICꢀascꢀC5 (9,
icas#9) (Scheme 1B).5,12,16,22,24
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Scheme 1. Ascaroside diversity originating from: (A) side chain modification and shortening
via peroxisomal βꢀoxidation, (B) subsequent attachment of additional metabolic units from
primary metabolism, or (C) specific (ω), (ω – 2), or (ω – 3)ꢀhydroxylation downstream of
peroxisomal βꢀoxidation.
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Ascaroside signaling is highly conserved in intraspecies nematode communication,16,25ꢀ29 and
also involved in interꢀgenotypic competition30,31 and cross kingdom interactions,32ꢀ34
demonstrating that ascarosides represent key regulators of nematode chemical ecology.
Considering the small amounts of ascarosides present in nematode exometabolomes, the large
diversity of homologous structures, and the complexity of the background matrix, mass
spectrometric techniques are indispensable for comprehensive ascaroside analysis. Mass
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spectrometric screens that employ specific fragment ions as markers are capable to highlight
putative nematodeꢀderived ascarosides and their biosynthetic precursors and mask the
background matrix. We have previously developed a HPLCꢀESIꢀ(˗)ꢀMS/MS precursor ion
screen that employs an ascaryloseꢀderived fragment ion at m/z 73.1 [C3H5O2]˗ for the selective
detection of known as well as yet unidentified components.22 ESIꢀ(–)ꢀMS/MS precursor ion
screening has been employed in various studies22,25ꢀ27,29,33,34 but ultimately requires a triple
quadrupole instrument. Considering the importance of ascaroside signaling in nematode
chemical ecology, we recently developed a complementary GCꢀEIMS technique35 that
employs an ascaryloseꢀderived K1ꢀfragment ion at m/z 130.1 [C6H14OSi]+● along with A1 and
A2 fragment ions at m/z 275.1 [C12H27O3Si2]+ and m/z 185.1 [C9H17O2Si]+ as characteristic
markers to facilitate selective ascaroside profiling in trimethylsilyl (TMS) derivatized crude
nematode exometabolome extracts (Scheme 2). Furthermore, aglyconeꢀspecific fragment ions
for a rearranged oxonium ion (J1) at [M – 173]+ and a carbocation (J2) at [M – 291]+ facilitate
the identification of compound specific sidechains. Here we employ the GCꢀEIMS screen for
comparative ascaroside profiling in a variety of Caenorhabditis species to demonstrate its
potential for the discovery of novel ascaroside components in crude unfractionated nematode
exometabolome extracts.
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Scheme 2. EIꢀinduced fragmentation of TMSꢀderivatized ascarosides.
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RESULTS AND DISCUSSION
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Following a phylometabolomic approach we performed a GCꢀEIMSꢀbased comparative
analysis of exometabolome extracts from 13 Caenorhabditis species with a strong focus on
the Elegans group that harbors the model organism C. elegans.36,37 Liquid cultures were
established in Sꢀmedium and propagating nematodes were fed with concentrated E. coli OP50
for 7 days, after which cultures were starved for another 7 days.38 The media supernatant
representing the exometabolome was collected, lyophilized, and extracted with methanol.
Crude exometabolome extracts were converted into their TMS derivatives using NꢀMethylꢀNꢀ
(trimethylsilyl) trifluoroacetamide (MSTFA) and subsequently analyzed by GCꢀEIMS to
show a large diversity of primary and secondary metabolites, some of which could be
tentatively identified using the NIST 14 mass spectral library (Figure 1A). Putative
ascarosides were detected by inspection of the extracted ion chromatograms for the highly
characteristic ascaryloseꢀderived K1 fragment ion at m/z 130.1 [C6H14OSi]+● (Figures 1B and
S1). Individual ascaroside structures were identified based on their aglyconeꢀspecific J1 [M –
173]+ and J2 [M – 291]+ fragment ion signals and Kovats retention indices by comparison
with a collection of more than 200 components that carry (ω – 1)ꢀ or (ω)ꢀlinked acyl (1),
enoyl (2), (3R)ꢀhydroxyacyl (3), 2ꢀketoalkyl (6), or 2ꢀhydroxyalkyl (7) sidechains previously
characterized in C. elegans wildꢀtype and peroxisomal βꢀoxidation mutants.35
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Figure 1: Ascaroside profiling of the C. nigoni exometabolome. (A) Total ion
chromatogram (TIC) of the TMSꢀderivatized C. nigoni crude exometabolome; (B) Extracted
ion chromatogram (EIC) for the K1 fragment at m/z 130.1 [C6H14OSi]+●; (C) HPLCꢀESIꢀ
MS/MS precursor ion screen for m/z 73.1 [C3H5O2]–.
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These analyses demonstrate that basic ascarosides with sidechains ranging from 3 to 11
carbons are highly conserved in all Caenorhabditis wildꢀtype isolates tested, although their
relative compositions vary significantly between the different species (Figure 2). A set of five
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compounds, ascꢀC7 (
1
, n = 2, X = OH; ascr#1, daumone#1),2 ascꢀC9 (
, n = 4, X = OH; ascr#3, daumone#3),3 ascꢀC6ꢀMK (
, asc#5)4 consistently represent the dominating components,
1, n = 4, X = OH;
ascr#10),12 ascꢀꢁC9 (
2
6
, n = 2; ascr#2,
daumone#2),3 and ascꢀωC3 (
8
and, taken together, account for more than 60% of the total ascarosides identified. An
exception is the C. portoensis metabolome that predominantly contains ascꢀC11 ( , n = 6, X =
OH; ascr#18). Furthermore, ascꢀC6ꢀMK ( , n = 2; ascr#2) is absent in C. nigoni
C. afra, and C. portoensis, whereas ascꢀωC3 ( , asc#5) is absent in C. nigoni
and C. portoensis. AscꢀC6ꢀOH (
MK ( , n = 2; ascr#2) supporting the assumption of a common biosynthetic origin (Scheme
1
6
,
C. japonica
,
,
8
, C. japonica
7
, n = 2; ascr#6) was exclusively detected alongside ascꢀC6ꢀ
6
1). While these basic ascarosides are common in most Caenorhabditis species tested, GCꢀ
EIMS based ascaroside profiling also revealed some highly speciesꢀspecific components
including a yet unidentified component with a J2 fragment at m/z 186.1 from C. elegans, ascꢀ
βOHꢀC13 (
3, n = 8, X = OH; bhas#22) from C. japonica, and ascꢀC5ꢀEA (1, n = 0, X =
NHCH2CH2OH; easc#9) from C. portoensis 35
.
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Figure 2: Comparative GCꢀEIMS ascaroside profiling of 13 Caenorhabditis
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exometabolomes.
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In addition, we observed three putative ascarosides that accounted for around 10% of the total
ascarosides detected in the exometabolome of C. nigoni (Figures 2 and 1B) but did not match
any of the more than 200 basic ascaroside structures previously identified in C. elegans wildꢀ
type and mutant metabolomes.35 Inspection of their 70 eV EIMS spectra (Figure 3) revealed
dominating K1ꢀfragment ion signals at m/z 130.1 [C6H14OSi]+●, along with A1ꢀfragments at
m/z 275.2 [C12H27O3Si2]+, and A2ꢀfragments at m/z 185.1 [C9H17O2Si]+ that are characteristic
for the ascarylose unit (Scheme 2). Several aglyconeꢀspecific signals for J1 fragment ions at
m/z 433.2 [C19H41O5Si3]+, J2 fragment ions at m/z 315.2 [C15H31O3Si2]+, and (J2 – TMSOH)
fragment ions at m/z 225.1 [C12H21O2Si]+ indicated nine carbon side chains with one
additional unit of unsaturation and one trimethylsilyloxy moiety for compounds 10 and 12.
The third compound 11 displayed diagnostic signals for a J1 fragment at m/z 435.3
[C19H43O5Si3]+, a J2 fragment at m/z 317.2 [C15H33O3Si2]+, and a (J2 – TMSOH) fragment at
m/z 227.1 [C12H23O2Si]+ indicative for a trimethylsilyloxyꢀsubstituted nine carbon side chain.
Comparison with the known (3R)ꢀhydroxylated ascꢀβOHꢀC9 (3, n = 4, X = OH; bhas#10),
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previously characterized in the exometabolome of Panagrellus redivivus,26,35 demonstrated
that both compounds are different (Figure S2) and excluded a βꢀoxidationꢀderived 3ꢀ
hydroxyacyl aglycone due to the lack of the characteristic fragment ion at m/z 233.1
[C9H21O3Si2]+ derived from αꢀcleavage. However, the identification of a homologous
fragment ion signal at m/z 289.2 [C13H29O3Si2]+ suggested a 7ꢀhydroxyacyl structure for ascꢀ
7OHꢀC9 (11) from C. nigoni, demonstrating how EIMS fragmentation can aid in structure
assignment.
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Figure 3: GCꢀEIMS spectra of (ω) and (ω – 2) hydroxyacyl ascarosides from C. nigoni.
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In conclusion, comparative GCꢀEIMSꢀbased ascaroside screening revealed three speciesꢀ
specific, side chain hydroxylated compounds in the C. nigoni exometabolome. The same
components with molecular ion signals at m/z 317.2 [M – H]˗ for 10 and 12 and m/z 319.2 [M
– H]˗ for 11 were also detected using the HPLCꢀESIꢀ(–)ꢀMS/MS precursor ion screen for m/z
73.1 [C3H5O2]– (Figure 1C), demonstrating that the GCꢀEIMS and HPLCꢀMS/MS methods
complement each other. However, in contrast to GCꢀEIMS, the MS/MS precursor ion screen
also revealed additional derivatives including large amounts of indole ascarosides such as ICꢀ
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ascꢀC5 (9, icas#9), a male attractant in C. nigoni,16 demonstrating that GCꢀEIMS is restricted
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to the most basic ascaroside compounds.
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To identify the molecular structures of the speciesꢀspecific ascarosides (10ꢀ12), the
exometabolome extract of 1.6 L C. nigoni liquid culture supernatant was fractionated by solid
phase extraction (RPꢀC18ꢀSPE) using a 10%ꢀstepwise gradient of aqueous methanol as eluent.
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1
Fractions were screened for ascarosides by GCꢀEIMS (Figure S3) and H NMR spectroscopy
(Figure S4) using the K1 fragment ion at m/z 130.1 [C6H14OSi]+● and the anomeric proton at
approximately δH 4.65 ppm (s, 1H) as characteristic markers, respectively. These analyses
confirmed the assignment of several known ascarosides and traced the target compounds to a
fraction eluted with 40% methanol (Figure S3) that contained predominantly ascꢀC7 (1, n = 2
, X = OH; ascr#1) along with a diversity of additional metabolites such as indole acetic acid
(IAA, auxine) and anthranilic acid (Figure S5). Subsequent separation by semipreparative
HPLC using a C18 column furnished fractions of sufficient purity to facilitate structure
assignment.
Table 1. NMR data for (ω) and (ω – 2)ꢀhydroxyacyl ascarosides (10ꢀ12) isolated from C.
nigoni (400 MHz, CD3OD).
threoꢀascꢀ7OHꢀꢁC9 (10) threoꢀascꢀ7OHꢀC9 (11)
ascꢀ9OHꢀꢁC9 (12)
a
b
a
b
a
b
Position
δC
δH , mult δC
δH , mult
δC
δH , mult
(J, Hz)
(J, Hz)
(J, Hz)
ꢀ
1
2
nd
C
ꢀ
nd
C
ꢀ
nd
C
124.8 CH
5.83 d
(15.5)
6.86 dt
(15.5, 7.0)
37.5 CH2 2.21 t (7.6) 122.9 CH
5.81 d
(15.6)
6.95 dt
(15.6, 6.9)
3
148.1 CH
26.9 CH2 1.63 m 150.7 CH
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32.7 CH2
25.6 CH2 1.54ꢀ1.68
m
32.8 CH2 1.48ꢀ1.62
m
2.25 m
30.4 CH2
27.0 CH2
1.40 m
1.40 m
33.1 CH2
29.3 CH2
2.25 m
1.52 m
6
32.9 CH2 1.48ꢀ1.53
m
26.3 CH2
1.50 m
9
7
8
74.8 CH
75.5 CH
3.53 m
3.74 dq
(3.9, 6.3)
75.2 CH
75.6 CH
3.52 m
3.73 dq
(3.8, 6.2)
1.14 d
32.8 CH2
78.6 CH
1.59 m
3.69 m
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9
14.5 CH3 1.14 d (6.3) 14.6 CH3
64.6 CH2
3.50 dd
(11.7, 5.6)
3.60 dd
(11.7, 4.2)
4.75 s
3.84 s.br
1.95 dt
(13.0, 3.8)
1.78 ddd
(13.0, 11.2,
3.1)
(6.2)
1’
2’
3’ax
3’eq
97.9 CH
69.7 CH
35.8 CH2
4.65 s
3.76 s.br
1.95 dt
(13.0, 3.8)
1.80 ddd
(13.0, 11.4,
3.0)
3.52 ddd
(11.3, 9.3,
4.3)
3.64 dq
(9.3, 6.3)
97.8 CH
69.8 CH
35.8 CH2
4.65 s
3.76 s.br
1.95 dt
(13.0, 3.8)
1.81 ddd
(13.0, 11.4,
3.0)
3.51 ddd
(11.4, 9.5,
4.4)
3.66 dq
(9.5, 6.3)
1.22 d
99.8 CH
69.6 CH
35.9 CH2
4’
68.2 CH
71.3 CH
68.4 CH
71.4 CH
68.4 CH
71.4 CH
3.53 m
5’
6’
3.67 m
17.8 CH3 1.22 d (6.2) 17.8 CH3
18.1 CH3 1.22 d (6.1)
(6.2)
a from HSQC spectrum. b from 1H NMR and dqfꢀCOSY spectra.
The molecular formula of C15H26O7 for compound 10 (~275 ꢂg) was established by HRꢀ
EIMS. Inspection of oneꢀ and twoꢀdimensional NMR spectra (1H NMR, dqfꢀCOSY, HSQC)
confirmed an αꢀconfigured ascarylose moiety along with an (ω – 1)ꢀlinked α,βꢀunsaturated C9
side chain (Table 1). Furthermore, the (ω – 2)ꢀposition of the additional hydroxy group was
deduced based on dqfꢀCOSY correlations from the terminal (ω)ꢀmethyl group at δH 1.14 ppm
(d, J = 6.3 Hz, 3H), δC 14.5 ppm to the (ω – 1)ꢀoxymethine group at δH 3.74 ppm (dq, J = 3.9
Hz, J = 6.3 Hz, 1H), δC 75.5 ppm and further on to the (ω – 2)ꢀposition at δH 3.53 ppm (m,
1H), δC 74.8 ppm. While all (ω – 1)ꢀlinked homologous ascarosides that have been identified
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so far share the same (R)ꢀconfiguration at the penultimate carbon,1 the stereochemistry of the
(ω – 2)ꢀhydroxymethine group of ascꢀ7OHꢀꢁC9 (10) could not be unambiguously assigned
based solely on the vicinal H,Hꢀcoupling constant of 3.9 Hz.39
9
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Compound 11 (~110 ꢂg) with a molecular formula of C15H28O7 according to ESIꢀHRMS
exhibits almost identical NMR data for the ascarylose unit and the (ω)ꢀpart of the sidechain,
but displays a triplet signal at 2.21 (t, J = 7.6 Hz, 2H) instead of the signals for an α,βꢀ
unsaturated enoyl moiety (Table 1), thus, suggesting the corresponding dihydroꢀderivative
structure ascꢀ7OHꢀC9 (11). Compound 12 (~130 ꢂg) with a molecular formula of C15H26O7
according to ESIꢀHRMS was obtained as a 1:1 mixture with ascꢀ7OHꢀC9 (11). Comparative
analysis of their dqfꢀCOSY spectra indicated an ascarylose unit with considerably different
chemical shifts (Table 1). Furthermore, an α,βꢀunsaturated side chain was identified due to δH
5.81 (d, JE = 15.6 Hz, 1H) and 6.95 (dt, JE = 15.6 Hz, J = 6.9 Hz, 1H), along with a
2
3
2
hydroxymethylene group at δH 3.50 (dd, J = 11.7 Hz, J = 5.6 Hz, 1H), 3.60 (dd, J = 11.7
Hz, 3J = 4.2 Hz, 1H), and δC 64.6 ppm, that displayed vicinal H,Hꢀcoupling correlation to the
(ω – 1)ꢀoxymethine proton at δH 3.69 (m, 1H) and δC 78.6 ppm, thus indicating the terminal
(ω)ꢀposition for the hydroxylation of the aglycone in ascꢀ9OHꢀꢁC9 (12). In conclusion,
analysis of one and twoꢀdimensional NMR spectra revealed three novel side chain modified
ascarosides that carry additional hydroxy functions at the 7ꢀposition (ω – 2) and the 9ꢀposition
(ω), the structures of which were finally established by total synthesis and chemical
correlations.
Both diastereomeric (ω – 2)ꢀhydroxy ascarosides, (7R,8R)ꢀthreoꢀ10a and (7S,8R)ꢀerythroꢀ10b
were synthesized as shown in Scheme 3. (R)ꢀMethyl lactate (14) was converted to the paraꢀ
methoxybenzyl (PMB) ether 15 and reduced to the aldehyde 16 using DIBALꢀH. Addition of
4ꢀpentenylmagnesium bromide afforded (6R,7R)ꢀthreoꢀ6ꢀhydroxyꢀ7ꢀPMBꢀOꢀ1ꢀoctene (17)
with a diastereomeric excess of de 92% due to asymmetric induction via a Cram chelate
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complex.40 Esterification of 17 with benzoyl chloride and pyridine or benzoic acid under
Mitsunobu conditions (PPh3, DIAD) afforded the diastereomeric benzoates (6R,7R)ꢀthreoꢀ18a
or (6S,7R)ꢀerythroꢀ18b, respectively, in excellent diastereomeric purities of de >99% after
column chromatography (Figure S6). Cross metathesis with ethyl acrylate using Grubbs 2nd
generation catalyst41 furnished the corresponding ethyl 7ꢀBzꢀOꢀ8ꢀPMBꢀOꢀ(2E)ꢀnonenoates
(7R,8R)ꢀthreoꢀ19a or (7S,8R)ꢀerythroꢀ19b that were subsequently deprotected using 2,3ꢀ
dichloroꢀ5,6ꢀdicyanoꢀ1,4ꢀbenzoquinone (DDQ)42 to afford (7R,8R)ꢀthreoꢀ20a or (7S,8R)ꢀ
erythroꢀ20b. Coupling with 2,4ꢀdiꢀOꢀbenzoyl protected ascarylose via the
trichloroacetimidate route43 furnished the diastereomeric ascarosides (22a or 22b) that were
finally deprotected using alkaline hydrolysis to give the desired (ωꢀ2)ꢀhydroxy ascarosides
(7R,8R)ꢀthreoꢀ10a or (7S,8R)ꢀerythroꢀ10b. In addition, an undesired intramolecular
cyclization product (23a or 23b) was obtained. Comparison of the NMR (Figure S7) and GCꢀ
EIMS (Figure S8) data of diastereomeric (7R,8R)ꢀthreoꢀ10a and (7S,8R)ꢀerythroꢀ10b with
those of the natural product (10) isolated from the C. nigoni exometabolome confirmed its
(7R,8R)ꢀthreo configuration. Furthermore, the (7R,8R)ꢀthreoꢀconfiguration of the dihydroꢀ
derivative ascꢀ7OHꢀC9 (11) from C. nigoni was unambiguously established by comparison of
the mass spectra and GC retention times with those of the hydrogenation products of (7R,8R)ꢀ
threoꢀ10a and (7S,8R)ꢀerythroꢀ10b (Figure S9).
10
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Scheme 3: Synthesis of (ω – 2)ꢀhydroxyacyl ascarosides threoꢀascꢀ7OHꢀꢁC9 (10a) and
erythroꢀascꢀ7OHꢀꢁC9 (10b).
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Reagents and conditions: (i) 4ꢀmethoxybenzyl trichloroacetimidate, trimethylsilyl triflate,
CH2Cl2, 0 °C, 3 h, 62%; (ii) DIBALꢀH, CH2Cl2, ꢀ78 °C, 0.5 h, 100%; (iii): 4ꢀ
pentenylmagnesium bromide, Et2O, 0 °C, 1 h, 58%; (iv) benzoyl chloride, pyridine, CH2Cl2, 0
°C, 12 h, 87% (18a); (v) benzoic acid, PPh3, DIAD, THF, 4 h, 55% (18b); (vi): ethyl acrylate,
Grubbs 2nd generation catalyst, CH2Cl2, 40 °C, 9 h, 76% (19a) and 81% (19b); (vii): DDQ,
CH2Cl2/H2O, 1 h, 70% (20a) and 72% (20b); (viii): 2,4ꢀdiꢀOꢀbenzoylꢀascarosylꢀ1ꢀ(2,2,2ꢀ
trichloroacetimidate) (21), trimethylsilyl triflate, CH2Cl2, 0 °C, 3 h, 68% (22a) and 64%
(22b); (ix): lithium hydroxide, water, MeOH, 12 h, 36% (10a), 46% (10b), 39% (23a), and
42% (23b).
The (ω)ꢀhydroxylated ascaroside ascꢀ9OHꢀꢁC9 (12) was synthesized as shown in Scheme 4.
Copper(I)ꢀcatalyzed addition of pentenyl magnesium bromide to tertꢀbutyldimethylsilyl (S)ꢀ
glycidyl ether (24) afforded (S)ꢀ8ꢀtertꢀbutyldimethylsilyloxyꢀ7ꢀhydroxyꢀ1ꢀoctene (25). Cross
metathesis with ethyl acrylate using Grubbs 2nd generation catalyst41 gave the corresponding
(8S,2E)ꢀethyl 8ꢀhydroxyꢀ9ꢀtertꢀbutyldimethylsilyloxyꢀ2ꢀnonenoate (26). Coupling of 26 to
2,4ꢀdiꢀOꢀbenzoylꢀascarosyl trichloroacetimidate (21) furnished 27, which was subsequently
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deprotected using tetrabutylammonium fluoride (TBAF) to give the alcohol 28, followed by
alkaline hydrolysis to give ascꢀ9OHꢀꢁC9 (12) identical to the natural product from C. nigoni
as shown by comparison of the GCꢀEIMS and NMR data (Figures S8 and S10). In addition,
an undesired intramolecular cyclization product 29 with an oxacyclooctan ring was obtained.
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Scheme 4. Synthesis of the (ω)ꢀhydroxyacyl ascaroside ascꢀ9OHꢀꢁC9 (12).
Reagents and conditions: (i) 4ꢀpentenylmagnesium bromide, copper(I)iodide, THF, 0 °C, 3 h,
94%; (ii) ethyl acrylate, Grubb’s 2nd generation catalyst, CH2Cl2, 40 °C, 9 h, 81%; (iii) 2,4ꢀdiꢀ
Oꢀbenzoylascarosylꢀ1ꢀ(2,2,2ꢀtrichloroacetimidate) (21), trimethylsilyl triflate, CH2Cl2, 0 °C, 3
h, 53%; (iv) tetrabutylammonium fluoride, THF, 3 h, 78%; (v) lithium hydroxide, water,
MeOH, 12 h, 25% (12) and 69% (29).
Using the synthetic compounds as reference standards, threoꢀascꢀ7OHꢀꢁC9 (10a), threoꢀascꢀ
7OHꢀC9 (11a), and ascꢀ9OHꢀꢁC9 (12) could also be identified in the exometabolome extract
of Caenorhabditis afra (sp.7) strain JU1286 from Ghana (Figures S11ꢀ12), a member of the
distantly related Japonica group (Figure 2)36,37 suggesting that (ω) and (ω – 2)ꢀhydroxylation
in C. nigoni and C. afra has a polyphyletic origin. Targeted screening for homologous
compounds using GCꢀEIMS and HPLCꢀMS demonstrated that (ω) and (ω – 2)ꢀhydroxylation
in C. nigoni and C. afra is tightly controlled and exclusively affects C9 and ꢁC9 aglycones,
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indicating that this speciesꢀspecific modification of ascaroside aglycones occurs downstream
of peroxisomal βꢀoxidation.
6
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9
Aiming to characterize the biological functions of the novel ascarosides, behavioral response
of C. nigoni males or females to 1 µM ascꢀ(7R)ꢀOHꢀ∆C9 (10a), ascꢀ(7S)ꢀOHꢀ∆C9 (10b), ascꢀ
9OHꢀ∆C9 (12), as well as ascꢀ∆C9 (2, n = 4, X = OH; ascr#3) was evaluated using a spot
attraction assay. While these analyses demonstrate that the common ascꢀ∆C9 (2, n = 4, X =
OH; ascr#3) acts as a potent male attractant in C. nigoni, reminiscent of the attraction of C.
elegans males,10 the speciesꢀspecific (ω – 2) and (ω)ꢀhydroxylated derivatives (10a, 10b, and
12) are not active (Figure S13). Additional experiments will be required to clarify the
biological functions of these compounds and unravel the ecological significance of speciesꢀ
specific (ω) and (ω – 2)ꢀhydroxylation in C. nigoni and C. afra.
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CONCLUSION
Our results demonstrate that comparative GCꢀEIMSꢀbased ascaroside profiling represents a
powerful technique to characterize ascaroside diversity and detect novel speciesꢀspecific
components in TMSꢀderivatized crude nematode exometabolomes. We have isolated three
new (ω) and (ω – 2)ꢀhydroxylated ascarosides from C. nigoni and determined their structures
using NMR spectroscopy. Structure assignments were unambiguously established by total
synthesis and chemical correlations. Comparative analysis of 13 Caenorhabditis species
demonstrates that within the Elegans group both (ω)ꢀ and (ω – 2)ꢀhydroxylated ascarosides
(10ꢀ12) are highly specific for C. nigoni (sp.9), but the same compounds were also detected in
the rather distantly related C. afra (sp.7), a member of the Japonica group, thus, suggesting a
polyphyletic origin for the hydroxylation steps. In addition, we found that (ω)ꢀ and (ω – 2)ꢀ
hydroxylation in C. nigoni and C. afra is highly specific for ascarosides carrying C9 and ꢁC9
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sidechains, strongly suggesting that the hydroxylation step occurs downstream of peroxisomal
βꢀoxidation. While traces of (3R)ꢀhydroxyacyl ascarosides, intermediates of the peroxisomal
βꢀoxidation cycle, are widespread in nematode exometabolomes, sidechain hydroxylation
downstream of βꢀoxidation such as (ω)ꢀ and (ω – 2)ꢀhydroxylation in C. nigoni and C. afra
has so far only been described as maleꢀspecific (ω – 3)ꢀhydroxylation in Panagrellus
redivivus (Scheme 1C).26 However, while the dihydroxylated dhas#18 (13) represents a maleꢀ
produced femaleꢀattractant in Panagrellus redivivus,26 the hydroxy ascarosides (10ꢀ12) did
not attract C. nigoni males or females. Additional research will be required to elucidate the
biological functions of these speciesꢀspecific components and decipher the ecological
significance of (ω)ꢀ and (ω – 2)ꢀhydroxylation of ascaroside aglycones as a means to further
increase structural diversity of this highly conserved class of nematode signaling molecules.
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EXPERIMENTAL SECTION
Preparation of exometabolome extracts. Wildꢀtype isolates of thirteen Caenorhabditis
species were cultivated at 23 °C on NGM agar seeded with E. coli OP50: C. elegans N2
(Bristol), C. nigoni (sp.9) JU1422, C. briggsae AF16, C. sinica (sp.5) JU727, C. remanei
PB4641, C. tropicalis (sp.11) JU1373, C. wallacei (sp.16) JU1904, C. doughertyi (sp.10)
JU1771, C. brenneri (sp.4) PB2801, C. japonica DF5081, C. afra (sp.7) JU1286, C.
portoensis (sp.6) EG4788, and C. n. sp. 8 (sp.8) QX1182. Mixed stage nematodes from five
10 cm plates collected in M9 buffer served as inoculums for liquid cultures grown in 100 ml
Sꢀmedium at 23 °C and 150 rpm. Concentrated E. coli OP50 bacteria pellet from an overnight
culture in LB medium at 37 °C and 170 rpm was provided as food from day 1 to day 7, after
which the cultures were starved for 7 days. After 14 days, nematodes were separated by
centrifugation (5 min at 5000 g). The filtered supernatant representing the exometabolome
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was frozen at ꢀ80 °C, lyophilized, and extracted with 3 x 100 ml methanol for 12 h each. The
combined extract was filtered, concentrated to dryness at 40 °C under reduced pressure,
reconstituted in 1 ml methanol, and aliquots were analyzed by HPLCꢀHRMS, HPLCꢀMS/MS
precursor ion screening for m/z 73.1, as well as GCꢀEIMS. All experiments were performed in
triplicate.
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Preparation of Trimethylsilyl (TMS) Derivatives for GCꢀEIMS Analysis. Aliquots of
crude nematode exometabolome extracts, C. nigoni exometabolome fractions, and synthetic
ascaroside standards were concentrated to dryness. The residues were treated with 10 µl Nꢀ
methylꢀNꢀ(trimethylsilyl) trifluoroacetamide (MSTFA) at 60 °C for 40 minutes, diluted with
10 µl DCM, and 1 µl of the solution analyzed by GCꢀEIMS.
Gas ChromatographyꢀElectron ImpactꢀMass Spectrometry (GCꢀEIMS). Separation of
volatile TMS derivatives and acquisition of their 70 eV electron impact mass spectra was
performed using a Trace GC 2000 series (Thermo Scientific) equipped with a Zebron ZBꢀ5
Guardian column (15 m, 0.25 mm ID, 0.25 ꢂm film thickness; with 10 m guardian end)
coupled to a single quadrupole ThermoQuest Trace MS (Finnigan). Helium was used as the
carrier gas at a flow rate of 1 ml/min. A temperature program starting at 130 °C for 5 min,
followed by a linear gradient of +10 °C/min to 350 °C was applied. A total volume of 1 µl
was injected using a 10:1 split ratio and an injector temperature of 250 °C. Electron ionization
(EI, 70 eV) mass spectra were acquired from m/z 35–650 amu. Data were analyzed with the
Xcalibur 3.1 software (Thermo Fisher Scientific).
Liquid ChromatographyꢀElectrospray IonizationꢀHigh ResolutionꢀMass Spectrometry
(HPLCꢀESIꢀHRMS). HPLCꢀESIꢀHRMS analysis of crude nematode exometabolome
extracts and C. nigoni exometabolome fractions was performed using a Dionex UltiMate 3000
HPLC instrument coupled to a Bruker Maxis ultrahigh resolution (UHR) qTOF mass
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spectrometer equipped with an electrospray ionization (ESI) unit operated in positive or
negative mode. Chromatographic separations were achieved using an Agilent ZORBAX
Eclipse XDBꢀC18 column (250 x 3 mm, 5 µm particle diameter) with a flow rate of 400
µl/min and gradient elution starting at 3% acetonitrile in 0.5% aqueous acetic acid for 5
minutes followed by a linear increase to 100% acetonitrile with 0.5% acetic acid within 35
minutes. Data were analyzed with the Compass DataAnalysis 4.3 software (Bruker).
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Liquid Chromatography Electrospray Ionization Precursor Ion Screening. HPLCꢀ
MS/MS precursor ion screening for m/z 73.1 was performed using an Agilent 1260 HPLC
instrument (Agilent Technologies) coupled to an API5000 Triple Quadrupole LC/MS/MS
mass spectrometer (AB Sciex, Darmstadt) equipped with an electrospray ionization (ESI) unit
operated in negative mode. A CID energy of ꢀ34 was applied. Chromatographic separations
were achieved using an Agilent ZORBAX Eclipse XDBꢀC18 column (50 x 4.6 mm, 1.8 ꢂm
particle diameter) (Agilent Technologies) with a flow rate of 1.1 ml/min and gradient elution
starting at 5% acetonitrile in 0.05% aqueous formic acid followed by a linear inꢀcrease to 95%
acetonitrile with 0.05% formic acid within 10 minutes. Data were analyzed with the Analyst
1.6 software (AB Sciex).
1
NMR spectroscopy. NMR spectra were recorded in CD3OD or CDCl3 at 400 MHz for H
and 100 MHz for 13C using a Bruker AMX400 instrument. Residual solvent signals were used
as internal standard with 1H at 3.31 ppm and 13C at 49.05 ppm for CD3OD or 1H at 7.26 ppm
and 13C at 77.16 ppm for CDCl3. Twoꢀdimensional homonuclear double quantum filtered
(dqf)ꢀCOSY spectra were recorded using phase cycling for coherence selection. For the
isolated compounds a total of 32 scans were acquired using a time domain of 8k in F2
(acquisition time of 1.2 s) and 512 increments in F1. For two dimensional heteronuclear
HSQC spectra 96 scans were acquired using a time domain of 1k in F2 and 256 increments in
F1. Spectra were zeroꢀfilled to 8k x 4k (COSY) or 4k x 2k (HSQC) prior to Fourier
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transformation, phased manually, and baseline corrected using the Topspin 3.2 (Bruker) and
MNova 9.0 (Mestrelab Research) software.
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Spot Retention Assay. Assays were performed as described previously.10,44 50ꢀ60 larvalꢀ
stage 4 (L4) worms were segregated by sex and stored at 20 °C for 5 hours to overnight to be
assayed as young adults. 0.6 µL of vehicle control or ascaroside solution was placed in each
scoring region. As the working stock of ascaroside was made in MilliQꢀpurified ultrapure
H2O, this was used as the vehicle control. Five animals were placed on each “X” of the assay
plate, which was then transferred to a microscope containing a camera and recorded for 20
minutes. Each sex and compound was assayed over three plates per day on at least three
different days.
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Isolation of hydroxy ascarosides from the C. nigoni exometabolome. Hydroxyacyl
ascarosides of C. nigoni were isolated from 1.6 L of the liquid culture supernatant. The
filtered supernatant was frozen at ꢀ80 °C, lyophilized, and the residue extracted with 3 x 100
ml methanol for 12 h each. The filtered extract was concentrated to dryness under reduced
pressure and the resulting C. nigoni exometabolome extract was adsorbed onto 2 g of celite
and fractionated by reverse phase chromatography on 5 g RPꢀC18ꢀSPE cartridges
(Chromabond, MachereyꢀNagel) using increasing concentrations of methanol in water as
eluent to afford 10 fractions (20 ml each). Aliquots of 10 µl were concentrated to dryness
under reduced pressure, treated with 10 µl MSTFA at 60 °C for 30 min, diluted with 10 µl
DCM, and analyzed by GCꢀEIMS (Figure S3). Fractions were concentrated to dryness under
1
reduced pressure and analyzed by H NMR spectroscopy (Figure S4). The 40% methanol
fraction containing the target components according to GCꢀEIMS was subsequently submitted
to semiꢀpreparative HPLC using an Agilent HPꢀ1100 HPLC instrument equipped with a
GromꢀSil 120 ODSꢀ4 HE column (250 x 8 mm, 5 µm) coupled to a Gilson 206 Abimed
fraction collector. A flow rate of 2 ml/min with gradient elution was used starting at 3%
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acetonitrile in 0.5% aqueous acetic acid for 3 minutes, followed by a linear increase to 100%
acetonitrile with 0.5% acetic acid within 30 minutes. Aliquots of 10 µl were analyzed by GCꢀ
EIMS and HPLCꢀESIꢀ(ꢀ)ꢀHRꢀMS as described before. Fractions containing the target
compounds were concentrated to dryness, dissolved in 650 µl CD3OD, and analyzed by oneꢀ
and twoꢀdimensional NMR spectroscopy.
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(7
R,8R,2E)ꢀthreoꢀ8ꢀ[(3’,6’ꢀdideoxyꢀαꢀLꢀarabinoꢀhexopyranosyl)oxy]ꢀ7ꢀhydroxyꢀ2ꢀ
nonenoic acid (threoꢀascꢀ7OHꢀꢁC9, 10) isolated from the C. nigoni exometabolome (275 ꢂg,
c = ~720 nmol/L), for H and C NMR data see table 1; HRMS (ESIꢀTOF) m/z (M – H)ꢀ
1
13
Calcd for C15H25O7 317.1606, found 317.1619.
(7
R,8R)ꢀthreoꢀ8ꢀ[(3’,6’ꢀdideoxyꢀαꢀLꢀarabinoꢀhexopyranosyl)oxy]ꢀ7ꢀhydroxynonanoic
acid (threoꢀascꢀ7OHꢀC9, 11) isolated from the C. nigoni exometabolome (110 ꢂg, ~285
nmol/L), for H and C NMR data see table 1; HRMS (ESIꢀTOF) m/z (M – H)ꢀ Calcd for
1
13
C15H27O7 319.1762, found 319.1771.
(2
E
,8
S)ꢀ8ꢀ[(3,6ꢀdideoxyꢀαꢀLꢀarabinoꢀhexopyranosyl)oxy]ꢀ9ꢀhydroxyꢀ2ꢀnonenoic
acid
(ascꢀ9OHꢀꢁC9, 12) isolated from the C. nigoni exometabolome (130 ꢂg, ~340 nmol/L), for
1H and 13C NMR data see table 1; HRMS (ESIꢀTOF) m/z (M – H)ꢀ Calcd for
C15H25O7 317.1606, found 317.1617.
(R
)ꢀMethyl 2ꢀ(4ꢀmethoxybenzyloxy)propanoate (15).45 Under argon atmosphere a solution
of (R)ꢀ(+)ꢀmethyl 2ꢀhydroxypropanoate (14) (1.04 g, 10 mmol) and 4ꢀmethoxybenzyl 2,2,2ꢀ
trichloroacetimidate (2.8 g, 10 mmol) in dry DCM (15 ml) at 0 °C was treated with
trimethylsilyl triflate (10 µl). After stirring at 0 °C for 3 h the reaction was quenched by
addition of saturated NaHCO3 solution (1 ml), the mixture was diluted with DCM (15 ml),
and washed with saturated NaHCO3 solution (2 x 10 ml), dried over Na2SO4, and
concentrated under reduced pressure. The product was isolated by column chromatography
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(silica gel, 9:1 v/v hexane/ethyl acetate elution, Rf = 0.22) to afford 15 (1.38 g, 6.2 mmol,
62%) as a yellowish oil. 1H NMR (400 MHz, CDCl3) δ 7.28 (d, J = 8.7 Hz, 2H), 6.87 (d, J =
8.7 Hz, 2H), 4.61 (d, J = 11.3 Hz, 1H), 4.38 (d, J = 11.3 Hz, 1H), 4.05 (q, J = 6.8 Hz, 1H),
13
3.79 (s, 3H), 3.75 (s, 3H), δ 1.41 (d, J = 6.8 Hz, 3H); C NMR (100 MHz, CDCl3) δ 173.9,
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159.5, 129.72, 129.70, 113.9, 73.7, 71.8, 55.4, 52.0, 18.8.45
(R
)ꢀ2ꢀ(4ꢀMethoxybenzyloxy)propanal (16).45 Under argon atmosphere a solution of 15 (672
mg, 3 mmol) in dry DCM (10 ml) at ꢀ78 °C was treated dropwise with a 1 M DIBALꢀH
solution (3.3 ml, 3.3 mmol) in toluene. After stirring at ꢀ78 °C for 30 minutes the reaction was
quenched with methanol (0.5 ml) and saturated sodium potassium tartrate solution (10 ml)
and stirred for 1 h and extracted with DCM (2 x 20 ml). The combined organic phase was
dried over Na2SO4 and concentrated under reduced pressure. Column chromatography (silica
gel, DCM elution) afforded 16 (580 mg, 3 mmol, 100% yield) as a colorless oil that was
1
directly used for the next step. H NMR (400 MHz, CDCl3) δ 9.63 (d, J = 1.8 Hz, 1H), 7.29
(d, J = 8.7 Hz, 2H), 6.89 (d, J = 8.7 Hz, 2H), 4.57 (d, J =11.4 Hz, 1H), 4.54 (d, J = 11.4 Hz,
1H), 3.87 (dq, J = 1.8 Hz, J = 7.0 Hz, 1H), 3.80 (s, 3H), 1.31 (d, J = 7.0 Hz, 3H); 13C NMR
(100 MHz, CDCl3) δ 203.7, 159.7, 129.8, 129.5, 114.1, 79.3, 71.9, 55.4, 15.4.45
(2R,3R)ꢀthreoꢀ3ꢀHydroxyꢀ2ꢀ(4ꢀmethoxybenzyloxy)ꢀ7ꢀoctene (17). Under argon atmosphere
a solution of 4ꢀpentenylmagnesium bromide (6 mmol) in diethyl ether (10 ml), prepared from
5ꢀbromoꢀ1ꢀpentene (900 mg, 6 mmol) and magnesium (150 mg, 6.2 mmol), was cooled to 0
°C and treated dropwise with 16 (580 mg, 3 mmol) in Et2O (2 ml) over the course of 5 min.
The resulting mixture was stirred at 0 °C for 1 h, quenched with saturated aqueous NH4Cl
solution (10 ml) and the aqueous layer extracted with ethyl acetate (2 x 10 ml). The combined
organic phases were dried over Na2SO4 and concentrated under reduced pressure. The product
was isolated by column chromatography (silica gel, 4:1 v/v hexane/ethyl acetate elution, Rf =
0.42) to afford 17 (460 mg, 1.74 mmol, 58% yield) with a diastereoisomeric excess of de =
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92% as determined by 1H NMR spectroscopy. 1H NMR (400 MHz, CDCl3) δ 7.25 (d, J = 8.6
Hz, 2H), 6.88 (d, J = 8.6 Hz, 2H), 5.80 (ddt, J = 17.2 Hz, J = 10.2 Hz, J = 6.7 Hz, 1H), 5.00
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2
2
(dbr, J = 17.2 Hz, 1H), 4.94 (dbr, J = 10.3 Hz, 1H), 4.60 (d, J = 11.1 Hz, 1H), 4.36 (d, J =
9
11.1 Hz, 1H), 3.80 (s, 3H), 3.40 (m, 1H), 3.34 (dq, J = 6.0, J = 6.3 Hz, 1H), 2.09 (m, 2H),
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1.62 (m, 1H), 1.47 (m, 1H), 1.44 (m, 2H), 1.17 (d, J = 6.0 Hz, 3H); C NMR (100 MHz,
CDCl3) δ 159.4, 138.9, 130.6, 129.5, 114.6, 114.0, 78.2, 74.9, 70.8, 55.3, 33.8, 32.4, 24.9,
15.7.
(2R,3R)ꢀthreoꢀ3ꢀBenzoyloxyꢀ2ꢀ(4ꢀmethoxybenzyloxy)ꢀ7ꢀoctene (18a). A solution of 17
(316.5 mg, 1.2 mmol) and dry pyridine (290 µl, 3.6 mmol) in dry DCM (2 ml) at 0 °C was
treated with benzoylchloride (280 µl, 2.4 mmol) in dry DCM (1 ml). After stirring at RT for
12 h the mixture was diluted with DCM (10 ml), washed with 1 M HCl (10 ml), saturated
aqueous NaHCO3 solution (10 ml), dried over Na2SO4, and concentrated under reduced
pressure. Column chromatography of the residue (silica gel, 9:1 v/v hexane/ethyl acetate
elution, Rf = 0.37) afforded (2R,3R)ꢀthreoꢀ18a (386 mg, 1.05 mmol, 87% yield) as a colorless
oil. 1H NMR (400 MHz, CDCl3) δ 8.09 (d, J = 7.6 Hz, 2H), 7.59 (m, 1H), 7.47 (m, 2H), 7.26
(d, J = 8.5 Hz, 2H), 6.86 (d, J = 8.5 Hz, 2H), 5.79 (ddt, J = 17.0 Hz, J = 10.3 Hz, J = 6.7 Hz,
1H), 5.25 (m, 1H), 5.01 (d, J = 17.1 Hz, 1H), 4.96 (d, J = 10.3 Hz, 1H), 4.62 (d, J = 11.5 Hz,
1H), 4.49 (d, J =11.5 Hz, 1H), 3.81 (s, 3H), 3.74 (dq, J = 4.9 Hz, J = 6.3 Hz, 1H), 2.10 (m,
13
2H), 1.77 (m, 2H), 1.46 (m, 2H), 1.23 (d, J = 6.4 Hz, 3H); C NMR (100 MHz, CDCl3) δ
166.4, 159.3, 138.5, 133.0, 130.7, 129.8, 129.4, 129.0, 128.5, 114.9, 113.9, 76.1, 74.6, 70.9,
55.4, 33.7, 29.0, 25.0, 15.5.
(2R,3S)ꢀerythroꢀ3ꢀBenzoyloxyꢀ2ꢀ(4ꢀmethoxybenzyloxy)ꢀ7ꢀoctene (18b). Under argon
atmosphere a solution of 17 (294.5 mg, 1.11 mmol), triphenylphosphine (668.8 mg, 2.55
mmol), and benzoic acid (300.4 mg, 2.46 µmol) in dry THF (6 ml) at 0 °C was treated with
440 µl diisopropyl azodicarboxylate (DIAD, 440 µl, 2.23 mmol). After stirring at RT for 4 h
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the solvent was removed under reduced pressure. Column chromatography of the residue
(silica gel, 9:1 v/v hexane/ethyl acetate elution, Rf = 0.37) afforded (2R,3S)ꢀerythroꢀ18b
(223.5 mg, 606.6 µmol, 55% yield) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 8.09 (d, J
= 7.6 Hz, 2H), 7.59 (m, 1H), 7.47 (m, 2H), 7.27 (d, J = 8.5 Hz, 2H), 6.85 (d, J = 8.5 Hz, 2H),
5.80 (ddt, J = 17.0 Hz, J = 10.2 Hz, J = 6.7 Hz, 1H), 5.28 (m, 1H), 5.03 (d, J = 17.1 Hz, 1H),
4.98 (d, J = 10.3 Hz, 1H), 4.59 (d, J = 11.5 Hz, 1H), 4.54 (d, J =11.5 Hz, 1H), 3.79 (s, 3H),
3.74 (dq, J = 3.8 Hz, J = 6.3 Hz, 1H,), 2.11 (m, 2H), 1.80 (m, 2H), 1.52 (m, 2H), 1.28 (d, J =
6.4 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 166.3, 159.2, 138.5, 132.9, 130.68, 130.74,
129.8, 129.4, 128.5, 114.9, 113.9, 76.2, 75.4, 70.8, 55.3, 33.7, 29.3, 25.0, 16.0.
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(7R,8R,2E)ꢀthreo-Ethyl 7ꢀbenzoyloxyꢀ8ꢀ(4ꢀmethoxybenzyloxy)ꢀ2ꢀnonenoate (19a). Under
argon atmosphere a solution of 18a (368.5 mg, 1 mmol) and ethyl acrylate (545 µl, 5 mmol)
in DCM (30 ml) was treated with GrubbsꢀII catalyst (50 mg, 59 µmol) and stirred at 40 °C for
9 h. The solution was concentrated under reduced pressure and the residue purified by
chromatography (silica gel, 4:1 v/v hexane/ethyl acetate elution, Rf = 0.40) to afford 19a
1
(335.7 mg, 762 µmol, 76% yield) as a green oil. H NMR (400 MHz, CDCl3) δ 8.07 (d, J =
7.2 Hz, 2H), 7.57 (m, 1H), 7.47 (m, 2H), 7.25 (d, J = 8.6 Hz, 2H), 6.93 (dt, J = 15.6, J = 7.0
Hz, 1H), 6.86 (d, J = 8.6 Hz, 2H), 5.82 (d, J = 15.6 Hz, 1H), 5.24 (m, 1H), 4.62 (d, J = 11.5
Hz, 1H), 4.48 (d, J = 11.5 Hz, 1H), 4.19 (q, J = 7.0 Hz, 2H), 3.81 (s, 3H), 3.73 (dq, J = 4.6, J
= 6.4 Hz, 1H), 2.23 (m, 2H), 1.78 (m, 2H), 1.52 (m, 2H), 1.30 (t, J = 6.9 Hz, 3H), 1.23 (d, J =
13
6.4 Hz, 3H); C NMR (100 MHz, CDCl3) δ 166.7, 166.4, 159.3, 148.6, 133.1, 130.7, 130.4,
129.8, 129.5, 128.5, 121.8, 113.9, 75.8, 74.4, 70.9, 60.3, 55.4, 32.1, 29.1, 24.2, 15.4, 14.4.
(7
S,8
R
,2E
)ꢀerythroꢀEthyl
7ꢀbenzoyloxyꢀ8ꢀ(4ꢀmethoxybenzyloxy)ꢀ2ꢀnonenoate
(19b).
Under argon atmosphere a solution of 18b (223.5 mg, 607 µmol) and ethyl acrylate (330 µl, 3
mmol) in DCM (20 ml) was treated with GrubbsꢀII catalyst (30 mg, 35 µmol) and stirred at 40
°C for 9 h. The solution was concentrated under reduced pressure and the residue purified by
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chromatography (silica gel, 4:1 v/v hexane/ethyl acetate elution, Rf = 0.40) to afford 19b
(218.2 mg, 495 µmol, 81% yield) as a brownish oil. 1H NMR (400 MHz, CDCl3) δ 8.07 (d, J
= 7.2 Hz, 2H), 7.59 (m, 1H), 7.47 (m, 2H), 7.26 (d, J = 8.5 Hz, 2H), 6.94 (dt, J = 15.6, J = 6.9
Hz, 1H), 6.84 (d, J = 8.5 Hz, 2H), 5.82 (d, J = 15.6 Hz, 1H), 5.24 (dt, J = 9.1 Hz, J = 3.9 Hz,
1H), 4.55 (s, 2H), 4.19 (q, J = 7.1 Hz, 2H), 3.79 (s, 3H), 3.72 (dq, J = 3.9 Hz, J = 6.4 Hz, 1H),
2.25 (m, 2H), 1.79 (m, 2H), 1.56 (m, 2H), 1.29 (t, J = 7.0 Hz, 3H), 1.26 (d, J = 6.4 Hz, 3H);
13C NMR (100 MHz, CDCl3) δ 166.7, 166.2, 159.2, 148.5, 133.0, 130.6, 130.5, 129.7, 129.4,
128.5, 121.8, 113.8, 76.0, 75.3, 70.8, 60.2, 55.3, 32.0, 29.3, 24.2, 16.0, 14.4.
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(7R,8R,2E)ꢀthreoꢀEthyl 7ꢀbenzoyloxyꢀ8ꢀhydroxyꢀ2ꢀnonenoate (20a). A solution of 19a
(330 mg, 750 µmol) in DCM (5 ml) was treated with water (260 µl) and 2,3ꢀdichloroꢀ5,6ꢀ
dicyanoꢀ1,4ꢀbenzoquinone (187 mg, 825 µmol). After stirring at RT for 1 h the mixture was
quenched with water (1 ml) and the aqueous phase extracted with DCM (5 x 1 ml). The
combined organic phase was dried over Na2SO4 and concentrated under reduced pressure.
Column chromatography (silica gel, 2:1 v/v hexane/ethyl acetate elution, Rf = 0.42) afforded
20a (168.7 mg, 527 µmol, 70% yield) as a yellowish oil.
1H NMR (400 MHz, CDCl3) δ 8.05 (m, 2H); 7.57 (m, 1H), 7.45 (m, 2H), 6.91 (dt, J = 15.7, J
= 7.0 Hz, 1H), 5.80 (d, J = 15.7 Hz, 1H), 5.05 (dt, J = 7.8 Hz, J = 5.0 Hz, 1H), 4.16 (q, J = 7.2
Hz, 2H), 3.94 (dq, J = 4.8, J = 6.4 Hz, 1H), 2.23 (m, 2H), 1.77 (m, 2H), 1.55 (m, 2H), 1.26 (t,
J = 7.0 Hz, 3H), 1.23 (d, J = 6.5 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 166.7, 166.6, 148.4,
133.3, 130.1, 129.8, 128.6, 121.9, 77.9, 69.0, 60.3, 32.0, 30.2, 24.1, 19.6, 14.4; HRMS (ESIꢀ
TOF) m/z (M + NH4)+ Calcd for C18H28NO5 338.1962, found 338.1979.
(7S,8R,2E)ꢀerythroꢀEthyl 7ꢀbenzoyloxyꢀ8ꢀhydroxyꢀ2ꢀnonenoate (20b). A solution of 19b
(218.2 mg, 495 µmol) in DCM (5 ml) was treated with water (260 µl) and 2,3ꢀdichloroꢀ5,6ꢀ
dicyanoꢀ1,4ꢀbenzoquinone (124 mg, 545 µmol). After stirring at RT for 1 h the mixture was
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quenched with 1 ml water and the aqueous phase extracted with DCM (5 x 1 ml). The
combined organic phase was dried over Na2SO4 and concentrated under reduced pressure.
Column chromatography (silica gel, 2:1 v/v hexane/ethyl acetate elution, Rf = 0.42) afforded
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20b (113.5 mg, 354 µmol, 72% yield) as a yellowish oil. H NMR (400 MHz, CDCl3) δ 8.05
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(m, 2H), 7.57 (m, 1H), 7.45 (m, 2H), 6.91 (dt, J = 15.7, J = 6.9 Hz, 1H), 5.80 (d, J = 15.7 Hz,
1H), 5.11 (dt, J = 9.3 Hz, J = 3.7 Hz, 1H), 4.15 (q, J = 7.2 Hz, 2H), 4.00 (dq, J = 3.8, J = 6.4
Hz, 1H), 2.24 (m, 2H), 1.76 (m, 2H), 1.57 (m, 2H), 1.26 (t, J = 7.1 Hz, 3H), 1.23 (d, J = 6.3
Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 166.7, 166.7, 148.4, 133.2, 130.1, 129.7, 128.5,
121.9, 78.2, 69.3, 60.2, 31.9, 29.2, 24.2, 18.3, 14.3; HRMS (ESIꢀTOF) m/z (M + NH4)+ Calcd
for C18H28NO5 338.1962, found 338.1977.
Oꢀ(2,4ꢀdiꢀOꢀBenzoylꢀ3,6ꢀdideoxyꢀαꢀLꢀarabinoꢀhexopyranosyl) trichloroacetimidate (21).
A solution of 2,4ꢀdiꢀOꢀbenzoylꢀascarylose (53.5 mg, 150 µmol) in DCM (1 ml) was treated
with trichloroacetonitrile (32 µl, 320 µmol) and 1,8ꢀdiazabicyclo[5.4.0]undecꢀ7ꢀene (5 µl,
33.4 µmol). After stirring for 30 min the yellowish solution was concentrated under reduced
pressure. Column chromatography of the residue (silica gel, 4:1 v/v hexane/ethyl acetate
elution) afforded 20 (61.2 mg, 122.4 µmol, 81% yield) as a colorless oil that was directly used
for the next steps.
(7
R
,8
R,2E
)ꢀthreoꢀEthyl
7ꢀbenzoyloxyꢀ8ꢀ[(2,4ꢀdiꢀOꢀbenzoylꢀ3,6ꢀdideoxyꢀαꢀLꢀarabinoꢀ
hexopyranosyl)oxy]ꢀ2ꢀnonenoate (22a). A solution of 20a (14.7 mg, 45.9 µmol) and 21
(15.3 mg, 30.6 µmol) in dry DCM (1 ml) at 0 °C was treated with trimethylsilyl triflate (5 µl)
and stirred for 3 h. The reaction was quenched with sat. NaHCO3 solution (100 µl), dried over
Na2SO4, and concentrated under reduced pressure. Column chromatography (silica gel, 4:1
v/v hexane/ethyl acetate elution, Rf = 0.24) afforded 22a (13.8 mg, 20.9 µmol, 68% yield) as a
1
colorless oil. H NMR (400 MHz, CDCl3) δ 8.12 (m, 2H), 8.10 (m, 2H), 7.80 (d, J = 7.6 Hz,
2H), 7.58 (m, 3H), 7.47 (m, 2H), 7.46 (m, 2H), 7.42 (m, 2H), 6.94 (dt, J = 15.7, J = 7.0 Hz,
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1H), 5.83 (d, J = 15.6 Hz, 1H), 5.31 (m, 1H), 5.15 (s.br, 1H), 5.10 (ddd, J = 11.0, J = 9.8 Hz,
J = 4.7 Hz, 1H), 4.98 (s, 1H), 4.16 (q, J = 7.1 Hz, 2H), 4.08 (m, 1H), 4.01 (dq, J = 9.7 Hz, J =
6.3 Hz, 1H), 2.41 (dt, J = 13.7 Hz, J = 3.8 Hz, 1H), 2.28 (m, 2H), 2.19 (m, 1H), 1.80 (m, 2H),
1.61 (m, 2H), 1.29 (d, J = 6.5 Hz, 3H), 1.26 (t, J = 7.0 Hz, 3H), 1.03 (d, J = 6.2 Hz, 3H); 13C
NMR (100 MHz, CDCl3) δ 166.6, 166.1, 165.9, 165.7, 148.3, 133.4, 133.2, 133.2, 130.4,
130.3, 130.0, 129.91, 129.88, 129.7, 128.6, 128.5, 122.0, 93.3, 75.9, 72.5, 71.1, 70.4, 67.3,
60.3, 32.0, 29.9, 29.8, 24.0, 17.8, 15.0, 14.4; HRMS (ESIꢀTOF) m/z (M + NH4)+ Calcd for
C38H46NO10 676.3116, found 676.3133.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(7
S,8
R
,2E
)ꢀerythroꢀEthyl
7ꢀbenzoyloxyꢀ8ꢀ[(2,4ꢀdiꢀOꢀbenzoylꢀ3,6ꢀdideoxyꢀαꢀLꢀarabinoꢀ
hexopyranosyl)oxy]ꢀ2ꢀnonenoate (22b). A solution of 20b (14.7 mg, 45.9 µmol) and 21
(15.3 mg, 30.6 µmol) in dry DCM (1 ml) at 0 °C was treated with trimethylsilyl triflate (5 µl)
and stirred for 3 h. The reaction was quenched with sat. NaHCO3 solution (100 µl), dried over
Na2SO4 and concentrated under reduced pressure. The residue was chromatographed (silica
gel, 4:1 v/v hexane/ethyl acetate elution, Rf = 0.26) to afford 22b (12.9 mg, 19.5 µmol, 64%
yield) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 8.12 (m, 2H), 8.09 (m, 2H), 7.99 (d, J
= 7.8 Hz, 2H), 7.58 (m, 3H), 7.46 (m, 6H), 6.96 (dt, J = 15.8, J = 7.2 Hz, 1H), 5.85 (d, J =
15.5 Hz, 1H), 5.22 (dt, J = 15.2 Hz, J = 4.0 Hz, 1H), 5.15 (s.br, 1H), 5.12 (m, 1H), 4.94 (s,
1H), 4.16 (q, J = 7.1 Hz, 2H), 4.16 (dq, J = 3.8 Hz, J = 6.4 Hz, 1H), 4.13 (m, 1H), 2.41 (dt, J
= 13.2 Hz, J = 3.8 Hz, 1H), 2.30 (m, 2H), 2.19 (ddd, J =13.0 Hz, J = 11.7 Hz, J = 2.8 Hz, 1H),
1.93 (m, 1H), 1.82 (m, 1H), 1.64 (m, 2H), 1.29 (d, J = 6.5 Hz, 3H), 1.26 (t, J = 7.1 Hz, 3H),
13
1.03 (d, J = 6.2 Hz, 3H); C NMR (100 MHz, CDCl3) δ 166.7, 166.2, 165.9, 165.7, 148.4,
133.4, 133.3, 133.2, 130.4, 130.1, 130.0, 129.8, 128.6, 128.5, 122.0, 93.6, 76.6, 72.5, 71.0,
70.6, 67.2, 60.3, 32.1, 29.8, 28.6, 24.4, 17.7, 14.8, 14.4; HRMS (ESIꢀTOF) m/z (M + NH4)+
Calcd for C38H46NO10 676.3116, found 676.3129.
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