Altering the Sex Pheromone Cyclo(l-Pro-l-Pro) of the Diatom Seminavis robusta towards a Chemical Probe

As a major group of algae, diatoms are responsible for a substantial part of the primary production on the planet. Pennate diatoms have a predominantly benthic lifestyle and are the most species-rich diatom group, with members of the raphid clades being motile and generally having heterothallic sexual reproduction. It was recently shown that the model species Seminavis robusta uses multiple sexual cues during mating, including cyclo(l-Pro-l-Pro) as an attraction pheromone. Elaboration of the pheromone-detection system is a key aspect in elucidating pennate diatom life-cycle regulation that could yield novel fundamental insights into diatom speciation. This study reports the synthesis and bio-evaluation of seven novel pheromone analogs containing small structural alterations to the cyclo(l-Pro-l-Pro) pheromone. Toxicity, attraction, and interference assays were applied to assess their potential activity as a pheromone. Most of our analogs show a moderate-to-good bioactivity and low-to-no phytotoxicity. The pheromone activity of azide- and diazirine-containing analogs was unaffected and induced a similar mating behavior as the natural pheromone. These results demonstrate that the introduction of confined structural modifications can be used to develop a chemical probe based on the diazirine- and/or azide-containing analogs to study the pheromone-detection system of S. robusta.


Contents
Walz Mess-und Regeltechnik, Effeltrich, Germany) and microscopic observations. The cultures were filtered when they reached the late-exponential phase, yielding 3 L of sterile spent medium. The medium was aliquoted in 50 mL falcons and stored at -20 °C.

Potency evaluation
The potency of the filtered medium was assessed using the interference assay described above.
Cultures with 1 mL of ASW were conditioned with different dilutions of the filtered 85B medium: 1 mL aliquots of two dilutions (1:10 and 1:100) and an aliquot of undiluted spent medium were added to a 1 mL culture, resulting in three dilution factors (DF): 2, 20 and 200.
To some wells, cyclo(L-Pro-L-Pro) 1 was added in a concentration of 100 nM. Assessing the attractiveness of the beads was carried out manually (threshold = 1) and the results are shown in Figure S2. As the effect of dilution was not significant, the filtered medium was used as a 1/100 dilution for every assay in this study. of the dilutions for the three treatments is not significant (p > 0.05). If the differences between the mutual treatments (positive control -100 nM, 100 nM -negative control and positive -negative control) are considered, the dilution effect on these differences are also found to be not significant. The graph represents the actual attractiveness, with the mean ± 95% confidence interval (n = 4). DF = dilution factor. Figure S3. The optimal parameters for the interference assay are a density of 7 · 10 7 cells m -2 and a threshold of 1 cell bead -1 . Each bar represents the difference in relative attractivity towards pheromone-coated beads (2 nmol mg -1 ) between the positive control and cultures treated with 10 µM cyclo(L-Pro-L-Pro) prior to bead addition (n = 3), with a given density and threshold. As can be seen on the graph, the threshold optimum is different for every density: the higher the density, the higher the optimal threshold is. The relative difference is maximal using a low density and a low threshold. The density of the two least dense cultures was determined after the medium renewal and before the dark-adaptation of the cultures by manual counting. The density of the remaining cultures was estimated by extrapolating the manual counts of the least dense cultures. The represented values are based on the difference between the estimated average attraction presented in Figure S4. The 95% confidence intervals of the differences are presented in Table S1. Table S1 | Threshold optimization: calculation of the 95% confidence intervals of the difference in relative fraction of attractive beads. The differences of the estimated average fraction of attractive beads between the positive control and 1 at 10 µM was calculated for every threshold and density. The error values of these differences were used to calculate the 95% c.i. of the estimated differences presented in Figure S3. D = density, T = threshold, Epos = estimated average of the positive control, EDKP1 = estimated average of 1 at 10 µM.      Table S8 | Calculation of the 95% confidence interval of the interference values. The differences of the estimated average fraction of attractive beads between the synthetic analogs and 1 were calculated. The error values of these differences were used to calculate the 95% c.i. of the estimated fraction attractive beads (ln(E)). The estimated average interference values and confidence intervals were calculated relative to the difference of 1 at 100 nM and the positive control (see section S5.2).

S7 Syntheses
Solvents and commercially available reagents were obtained from Sigma-Aldrich (Missouri, USA), ChemPur (Karlsruhe, Germany), Acros (Geel, Belgium), Alfa Aesar (Ward Hill MA, USA), TCI Chemicals (Tokyo, Japan) and Air Liquide (Paris, France). All solvents were used without further purification. DMF was dried over activated molecular sieves for at least 48 h, THF was dried using an MBRAUN SPS-800 solvent purification system and dry methanol on molecular sieves was purchased from Acros. Boc anhydride was heated to its melting temperature prior to application. All reported temperatures were measured externally.
Reversed phase chromatography was carried out using a Grace Reveleris TM Flash All 1 H and 13 C NMR spectra were recorded at 400 and 100.6 MHz respectively, on a Bruker Avance III, equipped with 1 H/BB z-gradient probe (BBO, 5 mm). All spectra were processed using Topspin 3.2. 1 H, 13 C, COSY, HSQC, HMBC and APT spectra were acquired through the standard sequences available in the Bruker pulse program library. In all reported spectra CDCl3 and TMS were used as solvent and internal standard, respectively. The spectra were analyzed using Topspin 3.5.
The reaction mixture was extracted with petroleum ether (2 x 15 mL) and the combined organic fractions were extracted twice with 5 mL of a saturated NaHCO3 solution. The combined aqueous phases were acidified until pH 1 to 1. Spectral data were in accordance with literature. 3

S7.3 (S)-5-(tert-butoxycarbonyl)-1,2,5-triazaspiro[2.4]hept-1-ene-6carboxylic acid 12
The synthesis of diazirine 12 was adopted from Van der Meijden et al. 4 About 100 mL ammonia was condensed in a three-necked round-bottom flask containing oxoproline 11 (2.72 g, 12 mmol) and the solution was refluxed for 5 h. A suspension of 1.475 g hydroxylamine-O-sulfonic acid (13 mmol, 1.1 equiv.) in 7 mL of dry methanol at 12 °C (dry ice/dioxane bath) was added dropwise to the reaction mixture. The reaction mixture was refluxed for an extra 1.5 h and 15 mL dry methanol was added afterwards. The condenser was replaced by a cotton plug and the mixture was stirred for 16 h, allowing the ammonia to evaporate. The resulting slurry was filtered over a glass frit filter and the filter cake was washed with methanol (2 x 35 mL). The combined methanol phases are treated with 1.65 mL Et3N (11.9 mmol, 1 equiv.) and the resulting solution was concentrated to less than 15 mL. The

S7.11 Methyl (2S,4R)-4-azidopyrrolidine-2-carboxylate hydrochloride 24
A round-bottom flask containing 100 mL of dry MeOH was cooled to 0 °C. Acetyl chloride (1.47 mL, 20.6 mmol, 4 equiv.) was added dropwise and the mixture was allowed to react for 20 minutes, after which azidoproline 18 (1.47 g, 5.4 mmol) was added. The solution was cooled to 0 °C and 1.55 mL acetyl chloride (2.18 mmol, 4 equiv.) was added. The reaction mixture was stirred for 1 h, after which the solvent and excess acid was removed evaporatively. The product was obtained as a light brown oil in a quantitative yield, used without further purification. Spectral data were in accordance with literature. 9

S7.12 Methyl (2S,4S)-4-hydroxypyrrolidine-2-carboxylate hydrochloride 25
A round-bottom flask containing 100 mL dry MeOH was cooled to 0 °C. Acetyl chloride (1.47 mL, 20.6 mmol, 4 equiv.) was added dropwise and the mixture was allowed to react for 20 minutes, after which 1.26 g hydroxyproline 17 was added (5.14 mmol). The reaction mixture was stirred for 16 h, after which the solvent and excess acid was removed evaporatively. The product was obtained as a white solid in a quantitative yield, used without further purification.
Spectral data in accordance with literature. 10 Purification with reversed phase chromatography (water/ACN 100/0-20/80) yielded 0.90 g diketopiperazine (33%) as a white solid. 11 This procedure was used to synthesize diketopiperazines 3-9, Scheme S1 gives an overview of the proline analogs that were combined.