*3.6. Synthesis of Compound 3*

To a mixture of the palmitoleic acid (50 mg), L-phenylalanine methyl ester (80 mg) and HATU (143 mg) was added DMF (5 mL) followed by DIEA (97 mg) under a N2 atmosphere. The reaction mixture was stirred at RT overnight. A saturated solution of ammonium chloride (NH4Cl) was added and the reaction mixture was extracted with *tert*-butylmethylether (3 times). The combined organic phases were dried with Na2SO4 and concentrated in vacuo. The crude mixture was purified by flash chromatography using C18 4g silica gel to give 52.5 mg of **3-OMe**.

To a solution of **3-OMe** (15 mg) in MeOH (5ml) and water (0.5 mL) was added LiOH.H2O (5 equiv.). The reaction mixture was stirred at RT for 4 h and aqueous HCl (1M) was added to adjust the pH to 2–3. The resulting mixture was extracted twice with ethyl acetate, dried with Na2SO4 and concentrated in vacuo to provide 14 mg of **3**. [*α*] 20 *<sup>D</sup>* +49 (*c* 0.5, CHCl3); HRESITOFMS *m/z* 402.2976 [M+H]+ (calculated for C25H40NO3 +, 402.3003); 1H and 13C NMR spectroscopic data, see S76-S77.

The same synthesis was done with D-phenylalanine methyl ester to give *ent-***3-OMe** (65.6 mg) and *ent***-3** (13.5 mg) with [*α*] 20 *<sup>D</sup>* -49 (*c* 0.5, CHCl3); HRESITOFMS *m/z* 402.2977 [M+H]+ (calculated for C25H40NO3 +, 402.3003); 1H and 13C NMR spectroscopic data, see S100-S101.

#### *3.7. Molecular Networking*

#### 3.7.1. Mass Spectrometry Analysis Parameters

Chromatographic separation was performed on a Waters ACQUITY UPLC system interfaced to a Q-Exactive Focus mass spectrometer (Thermo Scientific, Bremen, Germany), with a heated electrospray ionization (HESI-II) source. Thermo Scientific Xcalibur 3.1 software was used for instrument control. The LC (Liquid Chromatography) conditions were as follows: Column, Waters BEH C18 50 × 2.1 mm, 1.7 μm; mobile phase, (A) water with 0.1% formic acid, (B) acetonitrile with 0.1% formic acid; flow rate, 600 <sup>μ</sup>L·min−1; injection volume, 1 <sup>μ</sup>L; gradient, linear gradient of 2−100% B over 6 min and then isocratic at 100% B for 0.6 min. An ACQUITY UPLC photodiode array detector was used to acquire PDA spectra, which were collected from 210 to 450 nm. In positive ion mode, the diisooctyl phthalate C24H38O4 [M+H]+ ion (*m/z* 391.28429) was used as the internal lock mass. The optimized HESI-II parameters were as follows: Source voltage, 4.0 kV (pos); sheath gas flow

rate (N2), 55 units; auxiliary gas flow rate, 15 units; spare gas flow rate, 3.0; capillary temperature, 275.00 ◦C (pos), S-lens RF level, 45. The mass analyzer was calibrated using a mixture of caffeine, methionine−arginine−phenylalanine−alanine−acetate (MRFA), sodium dodecyl sulfate, sodium taurocholate, and Ultramark 1621 in an acetonitrile/methanol/water solution containing 1% formic acid by direct injection. The data-dependent MS/MS events were performed on the three most intense ions detected in the full scan MS (Top3 experiment). The MS/MS isolation window width was 1 Da, and the stepped normalized collision energy (NCE) was set to 15, 30, and 45 units. In data-dependent MS/MS experiments, full scans were acquired at a resolution of 35,000 FWHM (at *m/z* 200) and MS/MS scans at 17,500 FWHM both with an automatically determined maximum injection time. After being acquired in a MS/MS scan, parent ions were placed in a dynamic exclusion list for 2.0 s.

#### 3.7.2. MS Data Pretreatment

The MS data were converted from the .RAW (Thermo) data format to the .mzXML format using MSConvert software, which is part of the ProteoWizard package [15]. The converted files were treated using the MzMine software suite [17].

The parameters were adjusted as follows: The centroid mass detector was used for mass detection with the noise level set to 1.0 × <sup>10</sup><sup>6</sup> when the MS level was set to 1, and to 0 when the MS level was set to 2. The ADAP chromatogram builder was used and set to a minimum group size of 5 scans, minimum group intensity threshold of 1.0 × 105, minimum highest intensity of 1.0 × 105, and *m/z* tolerance of 8.0 ppm. For chromatogram deconvolution, the wavelets (ADAP) algorithm was used. The intensity window S/N was used as the S/N estimator with a signal-to-noise ratio set at 25, a minimum feature height at 10,000, a coefficient area threshold at 100, a peak duration range from 0.02 to 0.9 min and the RT wavelet range from 0.02 to 0.05 min. Isotopes were detected using the isotope peak grouper with a m/*z* tolerance of 5.0 ppm, an RT tolerance of 0.02 min (absolute), and the maximum charge set at 2. The representative isotope used was the isotope that was the most intense. An adduct (Na+, K+, NH4 +, ACN+, CH3OH+, Isoprop+) search was performed with the RT tolerance set at 0.1 min and the maximum relative peak height at 500 %. A complex search was also performed using [M+H]+ in ESI positive mode, with the RT tolerance set at 0.1 min and the maximum relative peak height at 500%. Finally, a custom database search was performed using the Dictionary of Natural Products 2018 (v. 26.2) database (http://dnp.chemnetbase.com) where the search was restricted to fungal or bacterial metabolites. Peak alignment was performed using the join aligner method where the *m*/*z* tolerance was set at 8 ppm, absolute RT tolerance at 0.065 min, weight for *m/z* at 10 and weight for RT at 10. The peak list was gap-filled with the same RT and *m/z* range gap filler (*m*/*z* tolerance at 8 ppm). Eventually the resulting aligned peak list was filtered using the peak-list rows filter option to keep only those features associated with MS2 scans. Full parameters are available as a .xml file in the Supplementary Materials (Entomopathogens\_MzMine\_parameters.mzmine).

#### 3.7.3. Molecular Networks Generation

To maintain the retention time and exact mass information and to allow the separation of isomers, the molecular networks were created using the .mgf file resulting from the MzMine pretreatment step detailed above: https://bix-lab.ucsd.edu/display/Public/Feature+Based+Molecular+Networking. Spectral data were uploaded on the Global Natural Products Social molecular networking platform [10]. A network was then created where edges were filtered to have a cosine score above 0.65 and more than 6 matched peaks. Further edges between two nodes were kept in the network if and only if each of the nodes appeared in each other's respective top 10 most similar nodes. The spectra in the network were then searched against GNPS spectral libraries. The library spectra were filtered in the same manner as the input data. All matches kept between network spectra and library spectra were required to have a score above 0.7 and at least 6 matched peaks. The output was visualized using Cytoscape 3.6 software [18]. The full MS data set was uploaded and is accessible on the GNPS servers as Massive Data sets N◦ MSV000082940. The molecular network analysis and clustered

spectra are accessible at the following link: https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task= b036dd9fcb964ca49fe8dc4345944b86.

#### *3.8. Evaluation of Larvicidal Activity*

#### 3.8.1. Insect Collection and Rearing

The *Aedes aegypti* strain was used for extract and compound Testing. The laboratory strain Paea, came from French Polynesia and had been maintained at the Institut Pasteur de la Guyane in French Guiana for over a decade. This strain is susceptible to all insecticides. *Ae. aegypti* eggs were conserved on dried paper strips. Hatching was induced by dropping these strips in water and placing them under vacuum pressure for at least 1 h. The larvae were fed with yeast pellets. Larval rearing was performed under natural conditions at 28 ◦C ± 2 ◦C, 80 % ± 20 % RH and with a photoperiod of 14 h dark and 10 h light. Late third or early fourth-instar larvae were used in the tests. All crude extracts and fractions were investigated using the WHO procedure for testing mosquito larvicides [19,20]. The larvicidal activity of pure compounds was evaluated using a tube assay.

#### 3.8.2. Cup Assays

A total of 100 larvae were exposed in each bioassay. Four replicates of 25 larvae were prepared in cups containing 99 mL of distillated water. A 1 mL aliquot of the extract/fraction in absolute ethanol was added to each cup. The crude extract and the fractions were all tested at a concentration of 100 ppm. Absolute ethanol (1 mL) served as a negative control. Larval mortality was recorded 24 h after exposure.

#### 3.8.3. Tube Assays

Fifty larvae were tested at each concentration with ten replicates per concentration (10 test tubes × 5 larvae). Each compound was tested at a concentration of 10 μg/mL. A stock solution was prepared at 1 mg/mL in absolute ethanol, and then, 30 μL of this solution was added to each test tube (75 × 12 × 0.8 − 1.0 soda rimLess, catalog #212-0013, VWR, International) containing 2.97 mL of distilled water. Larval mortality was recorded 24 h after exposure.

#### 3.8.4. Statistical Analysis

Abbott's formula for mortality was applied if the mortality rate in the control was between 5 and 20%. The test was invalidated if the mortality rate in the control was greater than 20% [21].

#### *3.9. Antimicrobial Assays*

The ATCC strains were purchased from the Pasteur Institute and clinical isolates were provided by Phillipe Loiseau (University Paris Sud, Châtenay-Malabry, France). The strains used in this study were: *Candida albicans* ATCC10231, *Staphylococcus aureus* ATCC29213, methicillin-resistant *S. aureus* ATCC33591, and *Trichophyton rubrum* SNB-TR1. Extracts, fractions, and pure compounds were tested according to the reference protocol of the European Committee on Antimicrobial Susceptibility Testing (EUCAST, http://www.eucast.org, 11 April 2016). The standard microdilution test as described by the Clinical and Laboratory Standards Institute guidelines (M27-A2, M7-A8 and M38-A) was used to determine minimal inhibition concentrations (MIC) against dermatophyte fungi, bacteria, and yeasts [22–24]. Crude extracts and pure compounds were tested at concentrations ranging from 256 to 0.5 μg/mL. The microplates were incubated at 32 ◦C, and MIC values were calculated after 24 h for bacteria, 48 h for yeast and 5 days for *T. rubrum*. The MIC values reported in Table 2 refer to the lowest concentration preventing visible growth in the wells. All assays were conducted in triplicate.

#### **4. Conclusions**

In conclusion, our study highlights that *Pantoea* sp. SNB-VECD14B can biosynthesize diverse lipoamino acids with antimicrobial and/or insecticidal activities. Three hydroxyacyl-phenylalanine derivatives were isolated and characterized from EtOAc extracts, and different analogs were synthesized to demonstrate structure-activity relationships. In short, we observed that the free carboxylic acid group was important for biological activity. Compounds with a long carbon chain (16 to 18 carbons) and a *Z* double bond, exhibited the highest larvicidal activity, while compounds with a saturated C12 or C14 chain demonstrated the best antimicrobial activity. The studied lipoamino acids appear to have similar biological and structural properties compared to free fatty acids [25,26]. Their amphipathic nature provides a wide range of activities. Given the observed biological activities, these molecules could provide an alternative to common antimicrobial agents for application in agriculture, food preservation, or cosmetics.

Finally, the molecular networking-based approach revealed several close analogues of lipoamino acids in the *Pantoea* sp. extract. The global molecular network established over a wide collection of entomopathogenic strains indicated that this structural family was indeed typical of the *Pantoea* genus. These lipoamino acids have diverse biological activities, and it can be hypothesized that the strain has acquired the ability to protect itself from various environmental aggressions (fungi, bacteria, and insects) by producing a truly diverse mix of lipoamino acids that exhibit complementary activities.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/5/ 1083/s1.

**Author Contributions:** Conceptualization, D.S. and V.E.; formal analysis, S.T., L.P., P.-M.A. and I.D.; funding acquisition, D.S. and V.E.; investigation, S.T.; methodology, S.D., D.S. and V.E.; project administration, V.E.; resources, I.D.; supervision, J.-L.W., I.D., D.S., and V.E.; validation, P.-M.A. and I.D.; writing—original draft, S.T. and D.S.; writing—review and editing, V.E.

**Funding:** This work has benefited from an "Investissement d'Avenir" grant managed by Agence Nationale de la Recherche (CEBA, ref. ANR-10-LABX-0025).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

