**1. Introduction**

The development of novel antimicrobial and insecticidal agents is urgently needed to improve public health worldwide. Indeed, drug-resistant pathogenic microorganisms such as methicillin-resistant *Staphylococcus aureus* (MRSA), vancomycin-resistant *S. aureus* (VRSA), and vancomycin-resistant *Enterococci* (VRE) have emerged [1]. Other Gram-negative pathogens are particularly worrisome because they are becoming resistant to nearly all of the antibiotic drugs currently available, including carbapenems [2]. These pathogens have become a major clinical problem, causing significant mortality in both healthy hosts and in those with underlying comorbidities [3]. Thus, it is essential to investigate new drugs to address the decreasing efficiency of the currently available antibiotics [2]. Another major public health problem concerns mosquitoes and their ability to spread pathogens such as malaria parasites, dengue, chikungunya, and more recently the Zika virus. The World Health Organization (WHO) estimated that more than 80% of the world's population is at risk of contracting vector-borne diseases,

and each year more than 700,000 people die as a result [4]. *Aedes* sp. mosquitoes are the primary vector for transmitting arboviruses worldwide [5]. Today, the two main approaches to control them involve genetic modification or the application of chemicals. Synthetic chemicals including pyrethroids or organophosphates such as temephos and malathion have been sprayed into the environment for decades. However, most of these synthetic chemicals have adverse effects, leading to the development of resistance, environmental pollution, and the introduction of toxic hazards to humans and other nontarget organisms [6]. Furthermore, the abuse and/or misuse of these compounds has resulted in the loss of vector control efficacy. Thus, specific safer pesticides are urgently needed [7].

Natural products have proven to be an immeasurable source of bioactive compounds [8]. Entomopathogenic microorganisms, the natural enemies of insects, are known to produce bioactive metabolites that have been implicated in complex defense and self-protection mechanisms. Indeed, to achieve ecological success, they produce several chemical entities including insecticides and/or antimicrobial metabolites [9].

As part of our investigation into the secondary metabolites produced by entomopathogenic microorganisms, a collection of 53 strains was extracted and screened against *Aedes aegypti* mosquito larvae and human pathogenic microorganisms (*Staphylococcus aureus*, MRSA, *Candida albicans*, and *Trichophyton rubrum*). The extract from the bacterium *Pantoea* sp. SNB-VECD14B exhibited a mortality rate of 97.2% against *Ae. aegypti*. larvae at a concentration of 100 ppm and a minimum inhibitory concentration (MIC) of 16 μg/mL against *T. rubrum*. In addition, the full strain collection was profiled by high-resolution mass spectrometry and the resulting fragmentation data were organized as a single molecular network. This molecular networking approach allowed us to organize untargeted tandem MS datasets according to their spectral similarity and thus to group analytes by structural similarity [10]. Similar approaches have served as powerful tools to navigate the chemical space of complex biological matrices and can be used to view the chemical constituents of a wide variety of extracts in a single map [11,12]. Appropriate taxonomical color mapping allowed us to highlight a structurally related series of compounds also produced by *Pantoea* sp. that was selected for further investigation.

#### **2. Results and Discussion**

#### *2.1. Isolation and Structure Elucidation*

The EtOAc extract of *Pantoea* sp. SNB-VECD14B was subjected to bioguided preparative high performance liquid chromatography (HPLC) using a C18 silica gel column to yield three pure compounds (**1**–**3**) (Figure 1).

**Figure 1.** Compounds **(1**–**3)** isolated from *Pantoea* sp.

The molecular formula of compound **1** was determined to be C23H37NO4 via HRESI-TOFMS analysis which gave a pseudomolecular ion at *m*/*z* 392.2769 [M+H]<sup>+</sup> (calculated for C23H38NO4 +, 392.2795), indicating six degrees of unsaturation. The structure of this compound was deduced from NMR spectral data (Table 1). The 1H and 13C NMR spectra of **1** suggested the presence of one methyl group (δ<sup>H</sup> 0.90) that appeared as a triplet in the 1H NMR spectrum, fatty acid methylenes integrating for 20 protons (δ<sup>C</sup> 30.9, δ<sup>H</sup> 1.29) that appeared as a characteristic broad signal in the 1H NMR spectrum, two other distinct methylenes at δ<sup>H</sup> 1.40 and 2.29/2.34, and six methines, one of which was hydroxylated (δ<sup>H</sup> 3.85) with the other five methines corresponding to aromatic protons with chemical shifts in the 1H NMR spectrum between 7.18 and 7.24 ppm. The sequence of 1H-1H COSY signals from H2 to H5 and from H12 to H14 allowed us to determine the presence of a hydroxylated fatty acid moiety. 1H-13C correlations observed in the HMBC spectrum between H2 /C1 , C3 and C4 confirmed that compound **1** has a tetradecanoate moiety that is hydroxylated at C3 (Figure 2). Another 1H-1H correlation was observed between H2 and H3 and 1H-13C correlations between H2/C1, C3 and H3/C1, C2, C4, and C5 along with the C1 chemical shift at δ 176.0 demonstrated the presence of a phenylalanine unit. Although no correlation was observed (COSY, HMBC, NOESY) between the phenylalanine and fatty acid moieties, the only possible linkage between these two units is via an amide bond. This is consistent with the chemical shifts at C1 (δ<sup>C</sup> 173.8), C2 (δ<sup>C</sup> 55.9), and H2 (δ<sup>H</sup> 4.60). Thus, compound **1** was identified as *N*-(3-hydroxytetradecanoyl)phenylalanine. At this stage, the asymmetric carbons configurations could not be determined. The stereochemistry of this molecule and the following compounds will be demonstrated by synthesis.

**Figure 2.** Key 1H-1H COSY (bold lines) and HMBC (dashed arrows) correlations.

The molecular formula of compound **2** was determined to be C25H39NO4 based on the ion observed at *m/z* 418.2926 [M+H]<sup>+</sup> in the HRESI-TOFMS experiment (calculated for C25H40NO4 + , 418.2952), which corresponded to seven degrees of unsaturation. The 1H and 13C NMR spectra revealed several similarities to **1**. Only minor differences were observed, including the presence of a broad methylene signal integrating for just fourteen protons, along with two de-shielded protons at δ<sup>H</sup> 5.35 and two methylenes at δ<sup>H</sup> 2.04 corresponding to the methines and adjacent methylenes of a double bond. The COSY and HMBC data of **2** allowed us to determine that the fatty acid moiety is 3-hydroxyhexadec-9-enoate and that it is hydroxylated at C3. The (*Z*) configuration of the double bond was determined by comparison with the literature data based on the distinctive splitting of the olefinic protons [13]. The smaller coupling constant in the (*Z*) double bond impacts on the spacing of the

olefinic protons multiplet peaks and the overall aspect of the multiplet in 1H NMR. The double bond configuration was confirmed by comparison with analytical data of synthetic compounds described below. Thus, compound **2** was finally identified as (*Z*)-*N*-(3-hydroxyhexadec-9-enoyl)phenylalanine.


**Table 1.** Nuclear magnetic resonance (NMR) Spectroscopic Data (CD3OD) for compounds **1**, **2**, and **3.**

*<sup>a</sup>* Data recorded at 125 MHz. *<sup>b</sup>* Data recorded at 500 MHz. *<sup>c</sup>* Data recorded at 600 MHz. *<sup>d</sup>* Data recorded at 200 MHz.

The HRESI-TOFMS experiment for compound **3** indicated a molecular formula of C25H39NO3 with a *m*/*z* of 402.3004 [M+H]<sup>+</sup> (calculated for C25H40NO3 +, 402.3003), which corresponded to seven degrees of unsaturation. After examining the 1H and 13C NMR spectra, compound **3** was obviously similar to **2**. The only differences observed include signals pertaining to the hydroxy group that are absent in compound **3** as well as differences in the signals of the neighboring protons and carbons (Table 1). Ultimately, the fatty acid moiety was determined to be hexadec-9-enoate. The COSY and HMBC spectra showed the same correlations as in **2**, along with a correlation between H2/C1 that further confirmed the amide linkage between the two moieties. Compound **3** was therefore identified as (*Z*)-*N*-hexadec-9-enoylphenylalanine (Figure 2). The configuration of the amino acid subunit was determined to be L (*S*) after synthesis of **3** and *ent***-3** (described below) and comparison of their optical rotations. All synthetic (2*S*)-lipoamino acids had a positive optical rotation, as did compounds **1***–***3**. Altogether, the analytical data along with the obvious biosynthetic resemblance between **1**, **2**, and **3** led to the conclusion that the absolute stereochemistry of the phenylalanine moiety was L in **1** and **2** as well. The configuration of C-3 could not be determined. These three compounds were isolated for the first time from natural sources and the complete NMR data for molecules **1***–***3** is described for the first time in the literature [14,15].

#### *2.2. Synthesis of Lipoaminoacid Analogues*

Our structure activity relationship (SAR) investigation began with the synthesis of various acylated phenylalanine analogues of compound **3**. We modified the carbon chain length of the fatty acid, the configuration of the amino acid (L vs. D), and the number of double bonds in the fatty acid alkyl chain (Scheme 1). The synthetic method used classical conditions for peptides coupling [16]. Amide coupling between L- or D- amino acid methyl esters and fatty acids in the presence of HATU and Hunig's base, followed by saponification of the ester moiety with lithium hydroxide gave a series of 32 acylated phenylalanine derivatives. Thus, starting from the methyl ester of either L- or

D-phenylalanine, 16 ester derivatives (compounds **4** to **19**) and 16 free acids (compounds **20** to **35**) were prepared with varying lengths of the fatty acid chain (from 12 to 20 carbons) and different degrees of unsaturation. Compounds **3**, **3-OMe**, *ent***-3**, *ent***-3-OMe**, **8**, **15**, **24**, and **31** were *Z*-alkenes, whereas compounds **9**, **16**, **25**, and **32** had an *E*-alkene configuration. Four additional lipoamino acids (**18**, **19**, **34**, **35**) were prepared from palmitoleic acid and the methyl ester of either L-alanine or L-tyrosine following the same synthetic procedures. The general synthetic process is shown in Scheme 1. The structures of the newly synthesized compounds were confirmed by NMR and HRESI-TOFMS analysis.


**Scheme 1.** Synthetic compounds **3, 3-OMe**, *ent***-3**, *ent***-3-OMe**, and **4**–**35**.
