Dimerization of conserved ascaroside building blocks generates species-specific male attractants in Caenorhabditis nematodes.

Comparative ascaroside profiling of Caenorhabditis nematodes using HPLC-ESI-(-)-MS/MS precursor ion scanning revealed a class of highly species-specific ascaroside dimers. Their 2- and 4-isomeric, homo- and heterodimeric structures were identified using a combination of HPLC-ESI-(+)-HR-MS/MS spectrometry and high-resolution dqf-COSY NMR spectroscopy. Structure assignments were confirmed by total synthesis of representative examples. Functional characterization using holding assays indicated that males of Caenorhabditis remanei and Caenorhabditis nigoni are exclusively retained by their conspecific ascaroside dimers, demonstrating that dimerization of conserved monomeric building blocks represents a yet undescribed mechanism that generates species-specific signaling molecules in the Caenorhabditis genus.


Introduction
Chemical communication in nematodes is mediated by ascarosides, glycolipids of the 3,6-dideoxy-arabino-aldohexose L-ascarylose linked to a wide range of homologous aglycones derived from the peroxisomal β-oxidation cycle (Fig. 1). [1][2][3][4][5][6][7][8] Ascaroside signalling is widely conserved in nematodes 9 and modulates a large diversity of biological functions. 7,8 Over the past decade the development of sensitive ascaroside-selective screens based on characteristic fragment ions observed during electrospray ionization-tandem mass spectrometry (ESI-MS/MS) 10,11 or electron ionization-mass spectrometry (EI-MS) 12 along with comparative analysis of wild-type and peroxisomal β-oxidation mutant metabolomes [13][14][15] has facilitated the identification of several hundreds of ascaroside structures, most of which have not yet been characterized with respect to their potential biological functions. Systematic bioassays with ascarosides, while limited in number and scope, established that even minor changes in molecular structure dramatically impact their biological activities, 16 which demonstrates that ascaroside signalling represents a complex "chemical language". Comparative analysis of several closely related Caenorhabditis species revealed that most simple ascarosides are highly conserved. Short chain ascarosides carrying (ω-1)linked odd numbered C5, C7, C9, and C11 acyl chains as aglycones (1) are particularly abundant and widely distributed. 17 These common ascarosides serve as scaffolds for speciesspecific modifications including the hydroxylation of the aglycone 17,18 and epimerization of the L-ascarylose unit (e.g. 2), 19 as well as the attachment of additional building Fig. 1 General structure of common ascarosides (1) along with species-specific derivatives from C. nigoni (2 and 3) and C. remanei (4); IC-asc-C5 (3) 23 from C. nigoni or the fatty acid ascarosides dominated by asc-C4-cyC11 (4) from C. remanei. 24 A yet uncharacterized class of putative ascarosides was observed in small quantities in both C. nigoni and C. remanei (Fig. 2) as well as other Caenorhabditis species. Their universal molecular formula C (20+n) H (34+2n) O 11 (n = 0-10) was determined by high resolution mass spectrometry (HR-MS), which suggested a homologous series of ascaroside dimers (Table S1 †). Systematic analysis of the ESI-(-)-HR-MS/MS spectra (Fig. 3) revealed neutral loss of an ascarylose unit [M − C 6 H 13 O 4 ] to form ion I along with a monomeric ascaroside building block II from cleavage of the ester linkage, thus, facilitating the differentiation of isomeric homo and heterodimers. ESI-(+)-HR-MS/MS results in an oxonium ion III corresponding to the esterified ascaroside building block, as well as the loss of the terminal aglycone to yield fragment ion IV composed of both ascarylose units connected by their aglycone linker, which ultimately facilitates the assignment of both monomeric building blocks along with their order of attachment.

Conclusions
In conclusion, combination of HR-MS/MS and NMR techniques along with total synthesis facilitated the identification of 2-and 4-isomeric homologous series of ascaroside dimers in Caenorhabditis spp. The homodimeric 4′-(asc-C7)-asc-C7 (SMID: dasc#1) 30 has previously been shown to act as a modulator of mouth form dimorphism in the androdioecious nematode Pristionchus pacificus, diplogastridae, which also produces a variety of 2-linked ascaroside dimers that carry an additional 4-linked ureido-isobutyrate moiety. 31,32 Among the gonochoristic C. remanei and C. nigoni the males are exclusively retained by ecologically significant amounts of their conspecific ascaroside dimers, demonstrating that dimerization represents an effective mechanism to generate species-specific signalling molecules. Considering just the most prevalent monomeric ascaroside building blocks with (ω-1)-linked acyl aglycones ranging from 4 to 11 carbons, their dimerization via the 2-or 4-position results in 128 theoretically possible structures of which 27 were now characterized in Caenorhabditis species using HR-MS/MS techniques. Incorporation of unsaturated aglycones as well as the assembly of ascaroside trimers further expands the structural diversity of these components.
Remarkably, dimers carrying even numbered aglycones were particularly prevalent, although the corresponding monomeric building blocks such as asc-C4 (1, n = 1) and asc-C6 (1, n = 3) are rare and cannot be produced by canonical β-oxidation of the predominating homologous series of odd numbered ascarosides. Along with the hydroxylation of the ascaroside aglycones, epimerization of the ascarylose unit, and attachment of additional building blocks from primary metabolic pathways, the dimerization of monomeric units represents a highly effective mechanism to generate species-specific ascarosides, which form a complex chemical language in nematode communication. Species-specific retention of C. remanei and C. nigoni in response to 100 fmol of the ascaroside dimers shows male-specific retention to conspecific components (different letters indicate statistically significant differences between groups, P < 0.01, ANOVA with Dunett's post hoc test, n = 20, values represent means ± 1 SD).

Paper
Organic & Biomolecular Chemistry otroph) were obtained from the Caenorhabditis elegans Genetics Center (CGC).

Preparation of exometabolome extracts
Fourteen wild-type Caenorhabditis species were cultivated at 23°C on Nematode Growth Medium (NGM) agar 33 seeded with E. coli OP50. Mixed stage nematodes from five 10 cm plates were collected in M9 phosphate buffer 33 to serve as inoculums for liquid cultures grown in 100 ml S-medium 33 at 23°C and 150 rpm. Concentrated E. coli OP50 bacteria pellet (from an overnight culture in lysogeny broth (LB) medium at 37°C and 170 rpm and collected by centrifugation at 5000g for 10 min) 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 5000g). The filtered supernatant representing the exometabolome was frozen at −80°C, lyophilized, and extracted with 3 × 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 analysed by HPLC

Liquid chromatography-electrospray ionization-high resolution-tandem mass spectrometry (HPLC-ESI-HR-MS/MS)
HPLC-ESI-HRMS analysis of crude nematode exometabolome extracts and exometabolome fractions was performed using a Dionex UltiMate 3000 HPLC instrument coupled to a Bruker Maxis ultrahigh resolution (UHR) qTOF mass spectrometer equipped with an electrospray ionization (ESI) unit operated in positive or negative mode. Chromatographic separations were achieved using an Agilent ZORBAX Eclipse XDB-C18 column (250 × 3 mm, 5 µm particle diameter) with a flow rate of 400 µl min −1 and gradient elution starting at 3% acetonitrile in 0.5% aqueous acetic acid (v/v) for 5 minutes followed by a linear increase to 100% acetonitrile with 0.5% acetic acid (v/v) within 35 minutes. Data were analysed with the Compass DataAnalysis 4.3 software (Bruker).

Enrichment of dimeric ascarosides from Caenorhabditis exometabolome extracts
Dimeric ascarosides were enriched from exometabolome extracts obtained from 1.6 L of a Caenorhabditis nigoni JU1422 liquid culture supernatant 17 and 1.5 L of a Caenorhabditis remanei PB4641 liquid culture supernatant 24 obtained as previously described. The filtered supernatant was frozen at −80°C, lyophilized, and the residue extracted with 3 × 100 ml methanol for 12 h each. The filtered extracts were concentrated to dryness under reduced pressure and the resulting exometabolome extract were adsorbed onto 2 g of Celite and fractionated by solid phase extraction (SPE) on 5 g reverse phase C18 cartridges (Chromabond, Macherey-Nagel) using a stepwise gradient of methanol in water as eluent (0 to 100% in 10% steps, v/v) to afford 10 fractions (20 ml each). Fractions were concentrated to dryness under reduced pressure and analyzed by HPLC-ESI-HR-MS/MS. Fraction eluting with 40-70% methanol that were found to be rich in dimeric ascarosides were either fractionated again by solid phase extraction (SPE) on 1 g reverse phase C18-endcapped cartridges (Chromabond, Macherey-Nagel) using a stepwise gradient of methanol in water as eluent (0 to 100% in 10% steps, v/v) to afford 10 fractions (5 ml each) and/or fractions containing the target components according to HPLC-ESI-(−)-HR-MS were subsequently submitted to semi-preparative HPLC using an Agilent HP-1100 HPLC instrument equipped with a Grom-Sil 120 ODS-4 HE column (250 × 8 mm, 5 µm) coupled to a Gilson 206 Abimed fraction collector. A flow rate of 2 ml min −1 with gradient elution was used starting at 3% acetonitrile in 0.5% aqueous acetic acid (v/v) for 3 minutes, followed by a linear increase to 100% acetonitrile with 0.5% acetic acid (v/v) within 30 minutes. Aliquots of 10 µl were analysed by HPLC-ESI-(−)-HR-MS as described before. Fractions containing the target compounds were concentrated to dryness under reduced pressure, dried in high vacuum, and the residues were dissolved in 550 µl CD 3 OD and analysed by one-and two-dimensional NMR spectroscopy.  (Table S1 †).

Holding assay to evaluate nematode behavioural response
Nematode preference for environments conditioned with 100 fmol of ascaroside dimers was measured using a modified holding assay. On a 6 cm Petri dish filled with 6 ml peptonefree nematode growth medium (NGM) agar, circular scoring regions of 9 mm diameter were marked. Next, 1 μl of 10% aqueous methanol (v/v, as solvent control) or 100 nM solutions of ascaroside dimers such as natural 4′-(asc-C4)-asc-C5 (5) isolated from C. remanei PB4641, synthetic 2′-(asc-C6)-asc-C5 (7) and 2′-(asc-C6)-asc-C6 (8) identical to the natural products isolated from C. nigoni JU1422, or their synthetic 4-isomers 4′-(asc-C6)-asc-C5 (13a) and 4′-(asc-C6)-asc-C6 (13b) in 10% aqueous methanol (v/v) were placed in the centre of the scoring areas onto the agar and left to dry for 5 minutes. Young adult nematodes from non-starved and non-crowded 6 cm NGM agar plates seeded with E. coli OP50 were sorted by sex and transferred to peptone-free unseeded NGM agar plates for approximately 30 min before being used for the assay to minimize the amount of concomitant bacteria. Individual worms (up to 3) were placed into the centre of the conditioned scoring region and the time required for the nematodes to leave the scoring region was recorded. Nematodes were defined to have left the scoring area when no part of the nematode was still within the circular boundary. A total number of 20 worms per condition were analysed and experiments were repeated on two separate days with comparable results. A oneway ANOVA with Dunett's post-test was performed using the SPSS statistics software version 26 (IBM) to evaluate the effect of ascaroside dimers versus solvent control on mean times nematode spent in scoring regions.