N-Acylated amino acid methyl esters from marine Roseobacter group bacteria

N-Acylated amino acid methyl esters from marine Roseobacter group bacteria Hilke Bruns1,§, Lisa Ziesche1,§, Nargis Khakin Taniwal1, Laura Wolter2, Thorsten Brinkhoff2, Jennifer Herrmann3, Rolf Müller3 and Stefan Schulz*1 Full Research Paper Address: 1Institute of Organic Chemistry, Technische Universität Braunschweig, Hagenring 30, 38106 Braunschweig, Germany, 2Institute for Chemistry and Biology of the Marine Environment, University of Oldenburg, Carl-von-Ossietzky-Straße 9–11, 26111 Oldenburg, Germany and 3Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research (HZI), Saarland University, Campus E8.1, 66123 Saarbrücken, Germany Email: Stefan Schulz* - Open Access Beilstein J. Org. Chem. 2018, 14, 2964–2973. doi:10.3762/bjoc.14.276 Received: 13 September 2018 Accepted: 15 November 2018 Published: 03 December 2018 Associate Editor: J. S. Dickschat © 2018 Bruns et al.; licensee Beilstein-Institut. License and terms: see end of document. * Corresponding author § Both authors added equally to this work and are therefore both first authors. Keywords: amino acid derivatives; 2-aminobutyric acid; homoserine lactones; natural products; quorum sensing Abstract Bacteria of the Roseobacter group (Rhodobacteraceae) are important members of many marine ecosystems. Similar to other Gramnegative bacteria many roseobacters produce N-acylhomoserine lactones (AHLs) for communication by quorum sensing systems. AHLs regulate different traits like cell differentiation or antibiotic production. Related N-acylalanine methyl esters (NAMEs) have been reported as well, but so far only from Roseovarius tolerans EL-164. While screening various roseobacters isolated from macroalgae we encountered four strains, Roseovarius sp. D12_1.68, Loktanella sp. F13, F14 and D3 that produced new derivatives and analogs of NAMEs, namely N-acyl-2-aminobutyric acid methyl esters (NABME), N-acylglycine methyl esters (NAGME), N-acylvaline methyl esters (NAVME), as well as for the first time a methyl-branched NAME, N-(13-methyltetradecanoyl)alanine methyl ester. These compounds were detected by GC–MS analysis, and structural proposals were derived from the mass spectra and by derivatization. Verification of compound structures was performed by synthesis. NABMEs, NAVMEs and NAGMEs are produced in low amounts only, making mass spectrometry the method of choice for their detection. The analysis of both EI and ESI mass spectra revealed fragmentation patterns helpful for the detection of similar compounds derived from other amino acids. Some of these compounds showed antimicrobial activity. The structural similarity of N-acylated amino acid methyl esters and similar lipophilicity to AHLs might indicate a yet unknown function as signalling compounds in the ecology of these bacteria, although their singular occurrence is in strong contrast to the common occurrence of AHLs. Obviously the structural motif is not restricted to Roseovarius spp. and occurs also in other genera. 2964 Beilstein J. Org. Chem. 2018, 14, 2964–2973. Introduction The identification and structural elucidation of naturally occurring compounds traditionally requires isolation and NMR investigation as key method to detect novel compounds and new structural classes. Although the advent of NMR spectrometers with high frequencies and cryoprobes with small diameters enables experiments to be performed in the μg scale with pure compound, the isolation of the pure material from complex samples as well as the access to the expensive equipment still pose a considerable challenge to find new compounds. The ongoing quest for new structures also increasingly addresses minor components, requiring larger amounts of the producing organism, not always readily accessible, to isolate a targeted compound [1-3]. though NAMEs are structurally similar to AHLs by an acyl chain linked to an amino acid derivative via an amide bond, they do not activate AHL receptors in roseobacters [21]. Instead, they show moderate antialgal activity [21]. In contrast to AHLs, the acyl chain can also be terminally oxidized [24]. During our analyses of different Roseobacter isolates, we encountered several compounds, which mass spectra show similarities to known NAMEs. These compounds proved to be either new NAMEs or constitute new classes of acylated amino acid methyl esters, derived from valine (NAVME), glycine (NAGME), or 2-aminobutyric acid (NABME). The identification of these compounds will be discussed based on the outlined approach including GC/MS analysis, interpretation of mass spectra, and verification by synthesis. An alternative methodology can avoid the laborious isolation procedure. Direct analysis by mass-spectrometric methods of natural materials, e.g., extracts, may give enough information to infer the structure of an unknown compound that is finally proven by synthesis and comparison with natural material. The use of GC/EI-MS is especially advantageous because such mass spectra often reveal key structural features. Furthermore, the availability of large cross-platform databases useful for dereplication allows focussing on new compounds. We are interested in natural compounds from Roseobacter group bacteria, an abundant class of marine bacteria occurring in diverse habitats with a broad metabolic potential [4-7]. Especially attached-living roseobacters produce diverse secondary metabolites, e.g., N-acylhomoserine lactones (AHLs) that the bacteria use for communication by quorum sensing [8-10]. AHLs are extensively investigated because of the broad knowledge on their biosynthesis, the underlying gene organization, as well as their function in many bacteria [11-13]. In the Roseobacter group, AHLs are involved, e.g., in antibiotic production [9] or cell differentiation [10]. Although many other bacterial signalling compounds must exist, only few of them have been characterized so far [14-18]. Such signalling compounds as well as many other unknown metabolites often occur in small amounts, which renders trace detecting methods like GC/MS a suitable approach for their detection and structure elucidation, provided their polarity falls into the analytical window of the method. A wide variety of AHLs, e.g., widespread (Z)-N-(tetradec-7enoyl)homoserine lactone (1, Z7-C-14:1-AHL, Figure 1), have been identified in roseobacters by these methods [19-22]. A related group of compounds occurring in Roseovarius only, are N-acylalanine methyl esters (NAMEs), e.g., (Z)-N-(hexadec-9enoyl)alanine methyl ester (2, Z9-C16:1-NAME), the major NAME produced by Roseovarius tolerans EL 164 [23]. Al- Figure 1: N-Acylhomoserine lactones 1 (Z7-C14:1-AHL) and N-acylalanine methyl esters 2 (Z9-C16:1-NAME) occurring in Roseobacter group bacteria. Results and Discussion The secondary metabolites released by liquid cultures of various roseobacters were collected by extraction via Amberlite XAD16 resin and analysed by GC/MS. Four of these strains, Ros (...truncated)