Biosynthesis, Synthesis, and Activities of Barnesin A, a NRPS-PKS

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Biosynthesis, Synthesis and Activities of Barnesin A, a NRPSPKS Hybrid Produced by an Anaerobic Epsilonproteobacterium Maja Rischer, Luka Raguz, Huijuan Guo, Francois Keiff, Gabriele Diekert, Tobias Goris, and Christine Beemelmanns ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00445 • Publication Date (Web): 14 Jun 2018 Downloaded from http://pubs.acs.org on June 15, 2018

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ACS Chemical Biology

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Biosynthesis, Synthesis and Activities of Barnesin A, a NRPS-PKS Hybrid Produced by an Anaerobic Epsilonproteobacterium

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Maja Rischer,[a] Luka Raguž,[a] Huijuan Guo,[a] Francois Keiff,[a] Gabriele Diekert,[b] Tobias Goris,[b]* and

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Christine Beemelmanns[a]*

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[a] Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute,

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Beutenbergstraβe 11a, D‐07745 Jena, Germany

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[b] Department of Applied and Ecological Microbiology, Institute of Microbiology, Friedrich Schiller

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University, Philosophenweg 12, D‐07743 Jena, Germany

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* corresponding authors: tobias.goris@uni‐jena.de; Christine.Beemelmanns@hki‐jena.de

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Abstract

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Despite the wealth of physiological knowledge and plentiful genomes available, only few natural

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products of anaerobic bacteria have been identified until today and even less have been linked to

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their biosynthetic gene cluster. Here, we analyzed a unique NRPS‐PKS hybrid gene cluster from an

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anaerobic Epsilonproteobacterium (Sulfurospirillum barnesii). Phylogenetic analysis of key

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biosynthetic genes, gene expression studies and comparative metabolomics resulted in the

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identification of the first anoxically biosynthesized NRPS‐PKS hybrid metabolite, a lipo‐dipeptide with

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a vinylogous side chain, named barnesin A. The absolute structure was verified by a modular total

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synthesis and barnesin and derivatives were found to have antimicrobial activity as well as selective

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and nanomolar inhibitory activity against pharmacological important cysteine proteases, such as

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cathepsin B.

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Introduction

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Traditionally, the search for pharmaceutical important secondary metabolites was based on

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activity screening of the most prolific bacterial lineages, actinomycetes and myxobacteria.1,2

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However, high rediscovery rates are the major drawback of studying these well‐known

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natural product producers. Recent bioinformatic studies on thousands of gene sequences

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allowed to map the global abundance of biosynthetic gene clusters (BGCs) within all bacterial

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lineages.3‐5 Most notably, genomes of rarely investigated (facultative) anaerobic bacteria,

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previously believed to be unable to produce natural products,6,7 were found to carry genes

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encoding for polyketide synthases (PKS) and non‐ribosomal peptide synthetases (NRPS); both

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key to the production of complex and bioactive secondary metabolites.8,9 Despite their high

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abundance and industrial and medicinal importance of anaerobes,10 almost none of the ACS Paragon Plus Environment

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identified BGCs have yet been linked to the production of a specific metabolite. In particular

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complex culture conditions, the tight regulation of gene expression and the genetic

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inaccessibility have set back in depth metabolomic analysis.6,7 The few examples of natural

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products from anaerobes include closthioamides, the first antibiotics isolated from the

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anaerobic bacterium Clostridium cellulolyticum,11 clostrubin produced by the plant pathogen

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Clostridium puniceum (Fig. 1),12 a new antibiotic lactocillin13 and a group of dipeptide

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aldehydes and derivatives (e.g. phevalin) produced by a broad range of common gut

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microbes.14 However, other anaerobic bacterial lineages, in particular free‐living species, have

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so far been overlooked in the search of unique bioactive secondary metabolites and thus

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represent an unexplored area of chemical space that holds considerable promise for natural

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product discovery and potential new bioactivities.

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Fig. 1. Absolute structure of the first anoxically biosynthesized NRPS‐PKS hybrid molecule barnesin A (1); and structures of the PKS‐derived clostrubin and closthioamide isolated from anaerobic Clostridium spp.

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In our quest to explore so far undiscovered bacterial physiological capabilities and unique

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biosynthetic pathways and based on our recent work on the genetic basis of organohalide

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respiration in Epsilonproteobacteria,15‐17 we performed a detailed activity and genome‐wide

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comparison of several members of this genus. Here, we report that Sulfurospirillum barnesii,18

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a free‐living Epsilonproteobacterium found in anoxic sediments contaminated with arsenate

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or selenate, harbors a unique and amongst anaerobic bacteria very rare NRPS‐PKS hybrid

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biosynthetic gene cluster (BGC designated as brn), whereas all other Sulfurospirillum spp.

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lacked any signs of NRPS or PKS related gene clusters (Fig. S2).19 The phylogenetic analysis of

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key biosynthetic enzymes revealed the substrate specificity of single domains, and also

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showed that they are evolutionary distant to from currently reported domains. Based on this

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gene‐to‐molecule approach, we proved our structure proposal using an ‘omics’‐guided

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isolation approach and total synthesis resulting in the identification of the first anoxically

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biosynthesized NRPS‐PKS derived natural product, named barnesin A.

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ACS Paragon Plus Environment

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Results and Discussion

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First, we performed a comparative gene cluster analysis. The brn gene cluster (approximately

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20 kbp in size) encodes nine proteins, annotated by us as BrnA–BrnI (Fig. 2A, Table S1),

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including a NRPS (BrnE, SMUL_748), a PKS (BrnF, SMUL_749), a PPTase (BrnI, SMUL_750), and

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a cyclic peptide transporter (Sulba_0745‐747, BrnBCD). Sequence repeat search via RePuter

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led to the identification of two conserved direct repeats (24 and 12 bp) flanking the brn

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cluster,20 which are indicative for a possible horizontal gene transfer. A blastp query of the

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complete cluster resulted in the identification of a highly similar non‐characterized gene

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cluster within the genome of Geovibrio sp. L21‐Ace‐BES (RefSeq, Acc. No.

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NZ_ATWF00000000.1) which belongs to the order Deferribacterales and was assembled from

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a metagenome obtained from a hypersaline mat.21 No repeats were found up‐ and

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downstream of the gvb hybrid gene cluster and no other evidence for horizontal gene

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transfer could be detected within the Geovibrio genome. We then analyzed the specificity of

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biosynthetic domains of both clusters (brn and gvb) to predict the possible core structure of

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the encoded metabolites.22‐24 The first C‐domain (BrnE C1) was assigned as starter C‐domain

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and suspected to catalyze a fatty acid transfer to form lipopetides.25 The second C‐domain

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(BrnE C2) was found to promote the condensation of two L‐amino acids (Table S2, S3).

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Subsequently, we analyzed both A‐domains; the first A domain (BrnE, A1) was predicted to

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accept aromatic amino acids (Fig. 2A, darker shaded color code) and belongs to an own,

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previously not identified, subgroup within this particular group of A‐domains. The second

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A‐domain (BrnE, A2) analysis was ambiguous, but protein modeling using Swiss Model clearly

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indicated a binding pocket fitting and stabilizing arginine or lysine residues (Fig. S3).26 The

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trans‐AT PKS (BrnF) includes a ketosynthase (KS), ketoreductase (KR), dehydratase (DH) and

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thioesterase (Fig. 2B, darker shaded color code BnrF KS1, Table S4).27 We also identified all AT

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domains encoded within the genome of S. barnesii and Geovibrio sp. L21‐Ace‐BES and found

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candidate genes (Sulba_0581 and WP_022851336.1) having the highest similarity to other

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discrete AT domains that form a loose but distinctive clade distant from cognate ATs (Table

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S4, S5).28 Overall, the phylogenetic BGC analysis revealed that brn and gvb encode enzymes

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responsible for the production of a modified lipo‐dipeptide containing at least one tyrosine

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moiety and one basic amino acid.



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Fig. 2. A) Comparative gene maps of brn (S. barnesii sp. SES‐3) and gvb (Geovibrio sp. L21‐Ace‐BES); each arrow represents a gene (ORF). Marked with an asterisk is the gene of BrnH (putative membrane protein not annotated as an ORF). Amino acid identities of the orthologues are given above the gvb genes. B) Phylogenetic analysis of adenylation domains with predicted substrate specificity. C) Phylogenetic analysis of trans‐AT associated ketosynthase domains. Predictions of the NRPS Prediction Blast Server were used for labeling predicted amino acids of the phylogenetic tree. Peptide sequences of adenylation domains were aligned using ClustalW The best distance matrix was calculated and the tree was constructed using Mega 6. Probability values >50 are shown at the nodes based on 1000 bootstraps. Darker shaded fields represent the S. barnesii and Geovibrio sp. subgroup.

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To address whether the NRPS‐PKS hybrid cluster is transcribed under laboratory growth

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conditions, we performed time‐dependent gene expression studies and found a robust level

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of transcription (gene brnE) in the used cultivation conditions (Fig. 3 and S4). This prompted

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us to conduct a comparative NMR‐ and UHPLC‐MS‐based metabolomic analysis of S. barnesii

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and two closely related species (S. deleyianum and S. multivorans) lacking the NRPS‐PKS

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cluster. Metabolites of each culture supernatant were enriched using C‐18 based solid‐phase

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extraction and subsequent HPLC/LC‐HRMS analysis revealed one distinct UV‐absorption peak

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(λmax 214 nm), which correlated to a unique protonated molecular ion peak at 488.2864

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[M+H]+ not reported by AntiBase29 and SciFinder. For full structural analysis, we pursued

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preparative scale fermentation (70 L) and MS‐ and NMR‐guided purification of culture

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extracts led to the isolation of 1.0 mg pure compound, named barnesin A (1) (Fig. 1). The


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molecullar formula was assign ned as C25H 37O5N5 bassed on ESI‐HRMS anal ysis (m/z 488.2864 4

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[M+H]+,, calculated d 488.2867, Δ = ‒0.69 9 ppm). Detailed analy ysis of the obtained 2D 2 NMR

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spectra (DMSO‐d6) and HR‐MS S/MS fragm mentation id dentified the e predicted metabolite e to be a

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eptide conssisting of aa tyrosine, a vinylogous argininee and an attached a modified lipo‐dipe

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ey analysis verified tyyrosine to be b L‐configuured, which h was in unsaturrated fatty acid. Marfe

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agreement with previous phylogenetic an nalysis.

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Fig. 3 A A) brnE Gen ne expression at differeent cultivation time points using cconventional reverse transcrip ptase PCR (R RT‐control (w without reve rse transcrip ptase enzyme), gDNA (D NA of S. barrnesii), C‐ (control without DN NA or RNA template). t B B) HPLC‐base ed analysis of o culture exxtracts of S. barnesii obtained d from differrent cultivatiion time poi nts (represe entative UV ttrace at 214 nm). C) Com mparative HPLC‐baased analysiss of culture extracts from e m three diffe erent Sulfuro ospirillum sttrains (representative UV tracee at 214 nm).

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Fig. 4. P Putative biossynthetic asssembly of baarnesin A (1 1) formation; abbreviatioon (ACP: acyl carrier protein; PCP: peptiidyl carrier protein; C: condensation; A: aden nylation; KSS: ketosynthase; DH: dehydratase; KR: kettoreductase;; TE: thioesteerase).

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Scheme 1. Total synthesis of barnesin A and derivatives: a) HBTU, DIPEA, DCM, r.t., o.n., 78%; b) 10%aq KOH, MeOH, r.t., 3 h, quant.; c) 1. CuCO3∙Cu(OH)2, H2O, r.t., 15 min; 2. bis‐Boc‐ pyrazolocarboxamidine, DIPEA, formamide, dioxane, r.t., o.n., 3. EDTA∙2Na∙2H2O, NaHCO3, H2O then Fmoc‐OSu, acetone, r.t., o.n. 70% (three steps); d) N,O‐dimethylhydroxylamine hydrochloride, HBTU, DIPEA, DCM, r.t., o.n., 71%; e) 1. LiAlH4, THF, 0 °C, 30 min, 2. triethyl phosphonoacetate, NaH, THF, 0 °C, 45 min, 32% (two steps); f) 20% piperidine in DMF, r.t., 4 h, 86%; g) COMU, DIPEA, DMF, 0 °C, 6 h, 27%; h) TFA, DCM, 0 °C to r.t., o.n.; i) porcine liver esterase, DMSO, H2O, Tris/HCl buffer, 37 °C, 5 d, 47% (two steps).

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The absolute structure of 1 matched the proposed biosynthetic assembly line as depicted in

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Fig. 4. In short, octenoic acid, presumably originating from fatty acid biosynthesis, is coupled

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via a specific condensation starter domain (C1) to L‐tyrosine. The resulting lipo‐peptide is

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then coupled to arginine (A1) via the second condensation domain (C2). A switch to the trans‐

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AT PKS module allows the elongation via decarboxylative addition of an ACP‐bound malonyl

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extender unit and the combined action of the KR and DH domains yields the vinylogous

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arginine moiety. Due to the high homologies between both gene clusters, brn and gvb, we

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currently hypothesize that a highly similar compound is produced by the designated

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Geovibrio sp. L21‐Ace‐BES. Here, it is interesting to note that the biosynthetic enzymes (Brn)

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appear highly substrate specific as LC‐MS/MS analysis revealed barnesin A (1) to be the only

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derivative present within the culture extracts.

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Barnesin A belongs to the few natural products containing a vinylogous amino acids,30 and the

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only other natural product known to contain a vinylogous arginine moiety is the pentapeptide

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miraziridine A (Scheme 1), isolated from the marine sponge Theonella aff. Mirabilis.31 Other

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structurally related natural products containing vinylogous amino acid are cyclotheonamides

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derived from the marine sponge Theonella swinhoei,32 syringolin A isolated from the

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bacterium Pseudomonas syringae,33‐35 and the related glidobactin A, produced by the ACS Paragon Plus Environment

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bacterium Polyangium brachysporum sp.36,37 Here it is interesting to note that syringolin A

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was identified as a virulence factor, inhibiting the 20S proteasome via a 1,4‐Michael addition

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reaction of the proteasome to the electrophilic unsaturated amide moiety within the

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macrolactam scaffold.35

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To gain access to larger quantities of compound 1, to proof our structural hypothesis and to

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perform bioactivity studies, we performed a short and modular synthesis (Scheme 1). First,

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coupling of trans‐2‐octenoic acid 2 with L‐tyrosine methyl ester hydrochloride 3, followed by

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saponification yielded acid 4 in 78% over two steps. Next, the vinylogous arginine was

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synthesized using two different strategies.38,39 Then three‐fold Boc‐protected arginine 6 was

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converted to the respective aldehyde, and the latter was subjected to a Horner‐Wadsworth‐

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Emmons transformation. As we faced several synthetic unreliabilities with both, synthetic and

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commercial protected arginine 6,40 we decided to use Fmoc‐protected derivative 7, which

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allowed us to selectively deprotect the ‒position and pursue the coupling reaction with 4 to

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give protected precursor 9. Final Boc‐deprotection using TFA and biocatalytic saponification

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with porcine liver esterase resulted in the desired compound 1. The obtained spectral data of

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the synthetic derivative 1 was in full agreement with analytical data obtained from the

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natural isolate proving our structural assumption (Table S7). Moreover, change of fatty acid

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precursor and reduction of the vinylogous double bond let to barnesin derivative 11‐13,

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respectively.

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We then evaluated the biological activities of barnesin A (1) and derivatives 10‐13, and

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identified barnesin derivative 10, 11 and 13 to have moderate to good inhibitory activity

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against Mycobacterium vaccae (10670), human‐pathogenic Staphyloccocus aureus (SG511)

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and Pseudomonas aeruginosa (K799/61 B9) (Table S11). We also confirmed that all tested

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compounds showed no significant antiproliferative or cytotoxic effects (Table S12). As small

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peptides and vinylogous systems have a history in being highly potent protease inhibitors,41‐43

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we analyzed the activity of all barnesin derivatives (1, 10‐13) and found derivatives (1, 10 and

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11) to inhibit cysteine proteases, including Cathepsin B,44 in the nanomolar range, whereas

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derivatives 12 and 13 were inactive (Table 1). These results suggest that barnesin derivatives

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(1, 10‐13) act as a Michael acceptor responsible for irreversible covalent binding to active site

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of cysteine protease.

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Table 1. Representative protease inhibition assays of barnesin A (1) and its derivatives in comparison to reported inhibitors. Compound

Papaina

Ficina

Cathepsin Bb

Trypsinc

Barnesin A (1)

15.96 µM (± 5.8)

3.43 µM (± 0.15)

91.72 nM (± 3.29)

>2000 µM

Derivative (10)

2.89 µM (± 0.13)

3.43 µM (± 0.38)

23.99 nM (± 5.3)

>2000 µM

Derivative (11)

4.78 µM (± 2.9)

1.16 µM (± 0.07)

87.56 nM (± 1.8)

>2000 µM

Derivative (12)

>2000 µM

> 2000 µM

> 2000 µM

n.t.

Derivative (13)

>2000 µM

> 2000 µM

> 2000 µM

n.t.

Leupeptin

0.86 µM

n.r.

21.5 nM

2.2 µM

Miraziridin A38,39

n.r.

n.r.

2.05 µM

60 µM

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Control reactions: inhibition: a) iodacetamide (2 mM): 98.8% inhibition of papain; 97.4% inhibition of ficin; b) leupeptin (1.5 mM): 99.9% inhibition of cathepsin B; c) PMF (2 mM): 100% inhibition of trypsin; n.r. = not reported in literature; n.t. = not tested.

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Conclusions

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Taken together, our experimental findings presented here reveal the first NRPS‐PKS based

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hybrid natural product from an anoxically cultivated free‐living bacterium (S. barnesii,

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Epsilonproteobacterium). Phylogenetic analysis of key genes of both biosynthetic clusters

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allowed for a NMR‐ and LC‐MS‐guided isolation approach of the first PKS‐NRPS hybrid

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metabolite (barnesin A (1)) produced from this bacterial lineage, and from anoxically

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cultivated bacteria in general. The absolute structure was proven by a modular total

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synthesis, which allowed accesses to non‐natural derivatives. A first pharmacological

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evaluation of this compound class revealed nanomolar inhibitory activity against cysteine

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proteases presumably via a 1,4‐Michael type addition mechanism. The ecological role and

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function of barnesin, however, remains to be elucidated. As the gene cluster analysis suggest

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a horizontal transfer between different phyla, similar compounds likely play a biological role

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in more than one free‐living species. Furthermore, the constitutive expression of the brn

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cluster and the production of such a reactive secondary metabolite impart a selective

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advantage to the producing organism by acting, e.g., as a defensive metabolite against other

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bacteria or protistan bacterivory.45

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In general, our findings demonstrate that comparative genome analysis of so far neglected

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natural product producers followed by a detailed phylogenetic biosynthetic pathways analysis

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allows for a targeted and highly efficient isolation approach. Given the large number of

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biosynthetic gene clusters of unknown function within many (facultative) anaerobes, such an


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approach holds great potential for discovering new chemical scaffolds and the identification

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of small‐molecule based microbial mediators in the anaerobic world.

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Conflicts of interest

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The authors declare a financial conflict of interest. A patent application has been filed

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(registration number: EP 18 160 274.9: Barnesin A, derivatives and uses thereof) and is

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currently under revision at the European patent office.

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Acknowledgements

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M. Rischer, L. Raguž and C. Beemelmanns are supported by the Leibniz Association, the Jena

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School for Microbial Communication and the German Research Council (DFG, grant

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BE4799/2‐1 EXSPHINGO and CRC ChemBioSys 1127), T. Goris by the DFG as part of the

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research group FOR1530. The authors thank B. Schenz, P. Brand‐Schön, S. Kruse (FSU Jena)

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and M. Küfner (HKI) for assisting in the cultivation of Sulfurospirillum barnesii. We thank A.

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Perner, H. Heinecke, H.‐M. Dahse and C. Weigel (HKI) for their help with MS and NMR

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measurements and activity assays, and H. Kries (HKI) for help in the modeling of the

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adenylation binding pocket.

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Notes and References

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† Electronic Supplementary Informa on (ESI) available: Fermenta on procedures; isola on

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procedures, ESI‐HRMS, 1H NMR, 13C NMR, and 2D NMR spectra as well as chemical modifications.

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TOC: graphical abstract 136x68mm (300 x 300 DPI)


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Biosynthesis, Synthesis, and Activities of Barnesin A, a NRPS-PKS

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