Isolation of Imaqobactin, an Amphiphilic Siderophore from the Arctic

Article Cite This: J. Nat. Prod. 2018, 81, 858−865

pubs.acs.org/jnp

Isolation of Imaqobactin, an Amphiphilic Siderophore from the Arctic Marine Bacterium Variovorax Species RKJM285 Andrew W. Robertson,†,‡ Nicholas G. McCarville,‡ Logan W. MacIntyre,§ Hebelin Correa,‡ Brad Haltli,†,‡,§ Douglas H. Marchbank,†,‡ and Russell G. Kerr*,†,‡,§ †

Department of Chemistry, University of Prince Edward Island, Charlottetown, PEI, Canada C1A 4P3 Nautilus Biosciences Canada Inc., Duffy Research Center, 550 University Avenue, Charlottetown, PEI, Canada C1A 4P3 § Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PEI, Canada C1A 4P3 ‡

S Supporting Information *

ABSTRACT: The amphiphilic siderophore imaqobactin was isolated from the Arctic bacterium Variovorax sp. RKJM285, a strain isolated from marine sediment collected from an inlet near Clyde River, Nunavut, Canada. The 2D structure of imaqobactin was determined by a combination of LC-HRMS, MS/MS, and NMR spectroscopic methods. The absolute configuration of the depsipeptide core was determined by Marfey’s analysis, and the relative configuration of the 4,7diamino-3-hydroxy-2-methylheptanoic acid moiety was determined by NOESY and selective NOE experiments. The photoreductive properties of imaqobactin were tested and are discussed. Initial tests for antimicrobial and cytotoxic activity of imaqobactin were also performed, identifying moderate antimicrobial activity.

I

adapted to this extreme climate and low nutrient availability by developing a unique chemical diversity. This has renewed interest in the Canadian Arctic as a source of unique chemistry.10,11 In an ongoing bioprospecting partnership with the Canadian territory Nunavut, our laboratory has accumulated an extensive library of Arctic microorganisms.12 The Gram-negative genus Variovorax has gained interest as a source of unique natural products, with recent genome-mining methodologies successfully identifying several new siderophores, including the variochelins (V. boronicumulans),13 the vacidobactins (V. paradoxus S110),14 and the variobactins (V. paradoxus P4B)14 (Figure 1). Herein we report the discovery and structural characterization of the amphiphilic lipopeptide siderophore imaqobactin (1), from the Arctic marine bacterium Variovorax sp. RKJM285 through an LC-HRMS-guided bioprospecting approach (Figure 1). Metal-binding and photoreductive properties of 1 are discussed. This represents the first siderophore characterized from a marine Arctic source and highlights the importance of continued efforts toward screening for chemical diversity within this extreme environment.

ron is an essential micronutrient used by all organisms for metabolic processes requiring iron-dependent heme enzymes.1,2 Despite the biological importance of iron and its high natural abundance on earth, the predominant oxidation state in aerobic environments, including terrestrial, aquatic, and marine, is Fe(III), often existing as insoluble ferric oxides.3 Due to this poor solubility, iron bioavailability within these environments is severely limited.4 To counter this, many microorganisms release high-affinity small-molecule Fe(III) chelators known as siderophores. These natural products are secreted into the surrounding environment to scavenge Fe(III), which is then actively transported back into the organism, where it can be further processed for use in cellular operations.5 Siderophore biosynthesis is postulated to be an important factor in environmental iron cycling and thus can have a direct impact on microbial community development.6 The ecological importance of these molecules has resulted in the continued interest in the discovery of new siderophores to understand their chemical and biological roles in shaping microbiomes and has led to over 250 unique siderophores identified to date.2,7 The Canadian Arctic represents a relatively unexploited microbiome for natural product discovery. The Arctic marine environment has been typically dismissed as a rich source of natural products due to the belief that low biodiversity within the area will yield low chemical diversity. Marine natural products from arctic environments represent approximately only 3% of marine natural products characterized to date.8,9 Conversely, recent literature suggests microorganisms have © 2018 American Chemical Society and American Society of Pharmacognosy

Received: November 8, 2017 Published: April 4, 2018 858

DOI: 10.1021/acs.jnatprod.7b00943 J. Nat. Prod. 2018, 81, 858−865

Journal of Natural Products

Article

Figure 1. Structures of siderophores variochelin A (V. boronicumulans),13 variobactin A (V. paradoxus P4B),14 vacidobactin A (V. paradoxus S110),14 and imaqobactin (Variovorax sp. RKJM285), isolated from Variovorax strains. Common key structural features are bolded, including an extended 4,7-diamino-3-hydroxy-2-methylheptanoic acid (blue), 3-hydroxybutanoic acid (red), dodecanoic acid (orange), and N-formylhydroxamate (green).

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we investigated growth kinetics at 15, 22, and 30 °C (Figure S2). A similar maximal cell density was attained at 15 and 22 °C; however growth was approximately 2-fold faster at 22 °C. Growth was lowest at 30 °C, reaching only 60% of the maximal cell density attained at the lower temperatures (Figure S2). These results suggest that siderophore production by the pyschrotolerant RKJM285 is favored at lower temperatures. Inhibition of siderophore production by elevated temperatures has been reported in several bacteria, including those of marine origin.19−21 Generally, siderophore production is not observed at maximal growth temperatures; however, the mechanism for this temperature-based regulation of siderophore production is not known. To obtain sufficient material to support structural characterization, a 3 L fermentation was performed, and the growth media was extracted with Diaion HP-20 resin and eluted stepwise with an increasing concentration of MeOH in H2O. LC-HRMS analysis detected the unknown metabolite in the MeOH eluate. Material was further purified by LH-20 sizeexclusion chromatography. LC-HRMS analysis of the purified metabolite identified the deferrated ion of 1 with an m/z of 934.5038 [M + H]+, which suggested a molecular formula of C41H71N7O17. The m/z (987.4124 [M − 2H + Fe(III)]+) of the Fe(III)-1 was consistent with the expected molecular formula of C41H68FeN7O17, which confirmed 1 as a ferric iron chelator. LH-20 fractions containing the two ions (m/z 987 and 934) of interest were pooled. For NMR characterization, complete iron removal was necessary. Material was loaded and eluted through a Chelex resin column with 100% MeOH. This was followed by treatment with 8-hydroxyquinoline according to literature protocols, to yield metal-free 1.22 Final purification of 1 was accomplished by semipreparative HPLC using a reversed-phase pentafluorophenyl(2) (PFP) column, yielding 11.6 mg of an off-white solid. Unfortunately, NMR analysis of 1 was hindered by peak broadness and poor resolution in several different solvents tested, likely owing to conformational changes or

RESULTS AND DISCUSSION Variovorax sp. RKJM285 was isolated on kerosene-supplemented marine Bushnell-Haas15 medium from a marine sediment sample collected near Clyde River, Nunavut (70°28′03.4″ N, 68°36′05.0″ W). RKJM285 was identified as a strain of Variovorax via analysis of its partial 16S rRNA gene sequence (GenBank accession number MF671834), which was obtained as described previously.16 Analysis of the partial sequence using the EZBioCloud server17 indicated the closest relatives to RKJM285 were V. boronicumulans BAM-48T (99.28% similarity) and V. paradoxus NBRC 15149T (99.17% similarity). Further taxonomic analysis would be required to accurately assign RKJM285 to a species. Small-scale (10 mL) media screening was performed to identify new metabolites in EtOAc extracts by liquid chromatography high-resolution mass spectrometry (LCHRMS). Analysis of extracts identified two coeluting ions with m/z of 934.5038 [M + H]+ and 987.4168 [M + H]+ in ISP2 medium supplemented with marine salts (ISP2m). The mass difference between these ions corresponded to 53 mass units relative to the m/z 987 ion. This mass loss is consistent with the loss of Fe(III), typical of iron-binding siderophores.18 Interestingly, no production was observed in ISP2 medium prepared without the addition of marine salts, suggesting that salt is required for production of the observed metabolite. As RKJM285 was isolated from a cold environment, we probed the effect of temperature on the production of the unknown metabolite. The bacterium was cultivated at 4, 15, 22, and 30 °C. The bacteria grew at all temperatures tested; however growth was slow at 4 °C and was not pursued further. LCHRMS analysis of extracts revealed prodigious production of the metabolite by cultures incubated at 15 and 22 °C, but only low-level production at 30 °C. Interestingly, the highest production was observed at 15 °C, suggesting temperaturedependent production of the metabolite (Figure S1). To determine if metabolite production was correlated with growth, 859



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presence of a larger spin system was evident, consisting of a methyl (doublet, δH 0.93, δC 12.3), three methines, a chain of three methylene units, a hydroxy group, and an amine moiety. The spin system was identified as a 4,7-diamino-3-hydroxy-2methylheptanoic acid. This moiety is identical to those identified in the variochelins,13 the vacidobactins,14 and the variobactins14 (Figure 1), likely arising from a Claisen condensation of ornithine/arginine and methylmalonate, followed by ketoreduction as proposed by Nett and coworkers.13 Further analysis showed a singlet corresponding to H-20 (δH 8.02), which correlated to C-20 (δC 152.2) by HSQC. HMBC correlations from H-20 to C-19 (δC 51.2) identified the position of this spin system at the terminal nitrogen of the 4,7-diamino-3-hydroxy-2-methylheptanoic acid residue. These chemical shifts were consistent with the presence of an N-formylhydroxamate group, another feature observed in metal-binding siderophores (Figure 1).28 The final spin system was identified as a 3-hydroxybutanoic acid (Hbu) group capping the Nδ-position of the ornithine residue (Figure 2A). HMBC correlations from the amide protons to adjacent carbonyl carbons identified the linear peptide sequence as SerOrn(Hbu)-Thr-Hya (Figure 2A). Cyclization of the peptide core was confirmed by an HMBC correlation from H-40 of the Hbu moiety to the α-carbonyl (C-34) of Hya. This confirmed the presence of a cyclic depsipeptide linked between Orn and Hya residues via Hbu. The Ser residue was then located adjacent to the 4,7-diamino-3-hydroxy-2-methylheptanoic acid moiety by HMBC correlation from NH-23 (δH 8.40) to C-13 (δC 174.9). Finally, an HMBC correlation between NH-16 (δH 7.94) and C-1 (δC 171.6) of the dodecanoic acid moiety placed it on the NH-16-position coupled via an amide linkage. A loss of 182 mass units from several ions in the MS/MS experiments was also observed, confirming the identity of the dodecanoic acid (Figure 2B). Siderophores typically possess three chelating groups for Fe(III) binding, including but not limited to hydroxamate and α-hydroxycarboxylic acid groups.28 The chemical formula, determined by HRMS analysis of 1, and its metal adducts suggested the presence of an additional hydroxy group unaccounted for by NMR analysis. Because all spin systems had been thoroughly characterized, the only possible location left to accommodate this group was the Nδ-positions of the Orn residue, consistent with similar hydroxamate-containing siderophores (Figure 2A).25−27 To confirm the location of the hydroxamates HR-MS/MS analysis of 1 was performed. Fragmentation identified a sequential loss of 18 (H2O) from initial cleavage of the depsipeptide,29,30 followed by loss of 131 (Hya), 101 (Thr), and finally 198 corresponding to the Nhydroxybutylornithine group (Figure S43). This fragmentation pattern corroborated the cyclic depsipeptide sequence determined by NMR and strongly suggests the presence of a hydroxamate at the N-29-position (Figure 2B and Figure S43). Additionally, an MS/MS fragment ion at m/z 486 was observed, corresponding to the N-hydroxyformyl-4,7-diamino3-hydroxy-2-methylheptanoic acid core coupled with serine and dodecanoic acid (Figure 2B and Figure S43). This confirmed the presence of the second hydroxamate at the N-19 position, consistent with other N-hydroxyformyl-containing siderophores (Figure 1). This analysis gave the complete 2D-structure of 1. A complete list of chemical shifts and correlations can be found in Table 1. The compound was named imaqobactin, in reference to the Inuktitut word for sea or ocean (imaq) and in

incomplete iron removal (Figures S30−S33). In acetic acid-d4, peak sharpness was obtained, but hydrolysis of 1 occurred quickly, as observed by LC-HRMS, through the emergence of an m/z of 952.5118 [M + H]+. Characterization of other siderophores has been documented using gallium adducts for NMR analysis.22,23 The Ga(III)-1 salt was prepared and purified as described in the Experimental Section, yielding 10.3 mg of an off-white solid (observed m/z 1000.4042 [M − 2H + Ga(III)] + , calculated m/z = 1000.4007 for C41H68GaN7O17). All NMR analysis was performed on the gallium adduct. 2D-structural characterization of Ga(III)-1 was accomplished using a combination of 1H, 13C, COSY, HSQC, HMBC, TOCSY, and NOESY NMR spectroscopy experiments, as well as MS/MS fragmentation. Initial NMR analysis of the COSY and TOCSY spectra revealed the presence of a lipopeptide containing eight spin systems (Figure 2). This included the four amino acids serine (Ser), threonine (Thr), ornithine (Orn), and β-hydroxyaspartic acid (Hya). Hya is a common feature in other siderophores,24−26 typically involved in metal chelation and ligand-to-metal charge transfer during photoreduction of iron.27 A dodecanoic acid moiety was identified by 1H NMR integration and later confirmed by MS/MS analysis. The

Figure 2. 2D-structural characterization of 1, illustrating (A) important COSY (bold lines) and HMBC (solid arrows) correlations and proposed ligands for Ga(III) binding (dash lines) for the structural characterization of imaqobactin (1); (B) characteristic fragments of 1 in tandem MS experiments. Dashed lines through amide linkages illustrate “y” and “b” fragments obtained. aA loss of 182 mass units was observed from several fragments in tandem MS experiments indicating the presence of a dodecanoic acid moiety. b Represents the “b” fragments observed corresponding to loss of single amino acids after initial depsipeptide ester cleavage (wavy line). (C) Structure of 1 with absolute and relative (*) configuration assigned. 860



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Table 1. NMR Spectroscopic Data for Imaqobactin in DMSO-d6 (1H 600 MHz, 13C 151 MHz) pos. dodecanoic 1 2 3 4 5 6 7 8 9 10 11 12

δC, type

δH (J in Hz)

acida 171.6, C 35.5, CH2 25.2, CH2 28.9, CH2 28.7, CH2 28.8, CH2 29.0, CH2 29.0, CH2 29.1, CH2 31.3, CH2 22.1, CH2 14.0, CH3

1.99, m 1.45, m 1.17−1.25, obs. 1.17−1.25, obs. 1.17−1.25, obs. 1.17−1.25, obs. 1.17−1.25, obs. 1.17−1.25, obs. 1.24, obs. 1.27, obs. 0.85, t (7.0)

4-amino-7-N-hydroxyformyl-3-hydroxy-2-methylheptanoic 13 174.9, C 14 42.7, CH 2.63, m 15 77.3, CH 3.38, obs. 16 48.9, CH 3.88, m 17 21.5, CH2 a: 2.58, m b: 1.56, m 18 27.5, CH2 a: 1.78, m b: 1.26, obs. 19 51.2, CH2 a: 3.76, obs. b: 3.26, obs. 20 152.2, CH 8.02, s 21 12.3, CH3 0.93, d (6.2) 16-NH 7.94, bd (9.3) 15-OH 4.50, d (8.1) serine (Ser) 22 171.2, C 23 56.5, CH 24 61.6, CH2 23-NH 24-OH

a: b:

4.16, m 3.77, obs. 3.73, obs. 8.40 (bd, 7.6) 4.89 (bt, 5.5)

HMBC

pos.

1, 3 2, 4 obs. obs. obs. obs. obs. obs. obs. 10, 12 10, 11 acid 13, 15, 21 13, 14, 16, 17, 21 1, 14, 17, 18 16, 19 16, 19 17 17 17 17 18, 19 13, 14, 15 1, 16 14,15

22, 22, 22, 13, 23,

δC, type

ornithine (Orn) 25 171.5, C 26 54.1, CH 27 23.1, CH2

δH (J in Hz)

HMBC

obs. m obs. m obs m obs. bd (5.2)

25, 26 26 26, 26, 27 27 22,

27, 28

26-NH

4.10, 2.08, 1.23, 2.27, 1.52, 3.95, 3.26, 8.29,

threonine (Thr) 30 168.4, C 31 57.7, CH 32 65.3, CH 33 20.0, CH3 31-NH 32-OH

4.09, 4.27, 0.92, 7.11, 5.13,

obs. m d (5.7) bd (9.2) bd (4.4)

25, 30, 31, 25, 31,

30, 32, 33 33 32 31, 32 32, 33

28

27.2, CH2

29

50.5, CH2

a: b: a: b: a: b:

27 27

26, 28

β-hydroxyaspartic acid (Hya) 34 168.7, C 35 55.9, CH 36 75.5, CH 37 175.5, C 35-NH

4.44, dd (9.1, 3.7) 3.75, d (3.7)

34, 36 34, 35, 37

7.20, bd (9.1)

30, 35, 36

3-hydroxybutanoic acid (Hbu) 38 162.4, C 39 35.4, CH2 a: b: 40 67.4, CH 41 19.9, CH3

2.73, 2.53, 5.24, 1.12,

38, 38, 34, 38,

dd (15.2, 11.4) obs. m d (6.4)

40, 40, 38, 39,

41 41 39, 41 40

24 23 23 23 24

a13

C NMR chemical shifts of carbons C-5 through C-9 were arbitrarily assigned and are interchangeable.

retention time of the second peak, confirming the configuration as L-erythro-β-hydroxyaspartic acid. Efforts to assign the configuration of the Hbu moiety involving hydrolysis of 1 followed by derivatization of the released Hbu with chiralresolving agents and comparison to pure standards were unsuccessful.25 Despite attempting several hydrolysis and derivatization conditions, we were unable to unambiguously assign the configuration at this position. The relative configuration of the 4,7-diamino-3-hydroxy-2methylheptanoic acid core was determined by NOESY and 1D NOE NMR experiments. Position-16 was arbitrarily assigned as S to correspond with the reported structure of variochelin A.13 Irradiation of the H-21 methyl group resulted in an NOE to H14 and H-16, but not with H-15. In addition, H-14 showed an NOE with H-15 but not with H-16. The relative configuration was assigned as (14S*, 15S*, 16S*). This assignment is in agreement with that of Nett and co-workers, who observed identical results for the variochelins.13 The relative stereochemical assignment at these three positions is open to interpretation due to possible free rotation. Further experimental evidence is required to corroborate these results. All

acknowledgment of the partnership between the Kerr laboratory and Nunavut Tunngavik Inc. and the Nunavut Research Institute (NRI). Amino acid configurations of 1 were determined by Marfey’s analysis. Compound 1 was hydrolyzed with 6 M HCl and then treated with 1-fluoro-2−4-dinitrophenyl-5-L-alanine amide (LFDAA). The hydrolysate was compared to L-FDAA-derivatized amino acid standards of known configuration. Analysis established the configurations to be L-serine, D-ornithine, and L-threonine. Standards of DL-threo-β-hydroxyaspartic acid and DL-erythro-β-hydroxyaspartic acid were prepared from transepoxysuccinic acid and cis-epoxysuccinic acid, respectively, according to literature protocols.31 The mixtures were derivatized with L-FDAA and compared to the LC-HRMS profiles of the L-FDAA-treated hydrolysate. The hydrolysate contained one peak with the correct mass and the same retention time (14.9 min) as one of the erythro-βhydroxyaspartic acid diastereomers (Figure S15). Previously it has been established that the elution order with C18 reversedphase conditions for L-FDAA-erythro-β-hydroxyaspartic acid diastereomers is D → L.32 The hydrolysate peak matched the 861



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Figure 3. Photoreaction of Fe(III)-1 illustrating (A) control reaction shielded from light showing no degradation of Fe(III)-1; (B) reaction exposed to light illustrating loss of Fe(III)-1 with the emergence of photolysis products at m/z 945, 913, and 873, including the structures of two proposed photolysis products. Complexed ferric iron in the photolysis products is likely from excess iron in the system or spontaneous oxidation of ferrous iron to Fe(III). Sites of iron binding in photolysis products are unknown and thus not depicted.

3 h appeared red after treatment. Samples shielded from light remained colorless (Figure S11). The UV-B-treated samples showed an increase in absorption at 535 nm from 0.004 ± 0.002, prior to UV-B exposure, to 0.035 ± 0.004 after light exposure for 3 h. Reactions not exposed to light (control) showed no significant difference over the same time frame, illustrated by an absorbance of 0.004 ± 0.002 at the start of incubation and an absorption of 0.006 ± 0.001 after 3 h in the dark. These data confirm imaqobactin (1) as a photoreactive siderophore implicated in the reduction of ferric iron to ferrous iron. Metal Binding. To test the metal-binding capabilities of the siderophore, aliquots of 1 were treated with various metal salts, including Al(III)Cl3, V(III)Cl3, and Au(III)Cl3, and then analyzed by LC-HRMS. In addition to the aforementioned stable Fe(III) and Ga(III) adducts, compound 1 was also capable of forming a stable adduct with Al(III), as observed by the complete conversion from an m/z of 934.5038 to an m/z of 958.4585 [M − 2H + Al(III)]+, which was consistent with the expected molecular formula (C41H68AlN7O17) of the aluminum salt (Figure S7). Upon treatment of 1 with VCl3, the emergence of two new ions was observed with m/z values of 918.5072 and 902.5150 [M + H]+, corresponding to the loss of one and two oxygens, respectively. Vanadium(III) has been shown to reduce hydroxamates to the corresponding amide derivatives.33 These masses likely represent the mono- and diamide derivatives of 1. Treatment of 1 with AuCl3 resulted in complete loss of the m/z of 934, with the emergence of a series of unidentified ions. Siderophore−gold reactivity has been observed previously with mixed hydroxamate/β-hydroxyaspartic acid-containing siderophores involved in the reduction

other examples of siderophores containing a similar moiety have yet to have the absolute configuration assigned by a chemical approach.13,14,26 Photoreactivity of Imaqobactin. Mixed hydroxamate/βhydroxyaspartic acid-containing siderophores have been implicated in the photoreduction of ferric iron to ferrous iron. Reduction occurs via a ligand-to-metal charge transfer resulting in the cleavage of the siderophore at or around the chelating Hya residue.13,18,25,27 To test the photoreductive properties of imaqobactin, samples of Fe(III)-1 were exposed to UV-B radiation for a period of 3 h; a second control sample was protected from light for the same period. Samples were analyzed by LC-HRMS for the presence of cleavage products. LC-HRMS profiles identified the presence of several photolysis products in the UV-B-treated sample with complete loss of Fe(III)-1, while the sample protected from light showed no degradation (Figure 3A). Of the photolysis products, three compounds with m/z values of 945.4015, 913.4137, and 873.4182 were the most prominent (Figure 3B). These masses corresponded to losses of C2H2O, C2H2O3, and C4H2O4, respectively. Proposed photolysis products are illustrated in Figure 3B. Products were identified as complexed with Fe(III). The ability of siderophore photolysis fragments to retain Fe(III) affinity has been previously demonstrated in the structurally similar aquachelins27 and marinobactins.18 The presence of the corresponding iron-free ions (m/z = 892.4902, 860.5014, and 820.5064 [M + H]+) was also identified by single-ion monitoring. To confirm this reaction as a true photoreductive process, Fe(II) was then trapped through a bathophenanthrolinedisulfonic acid (BPDS) assay according to literature protocols.13,25 Samples of Fe(III)-1 treated with UV-B light for 862



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of Au(III).26 The siderophore 1 appears to be reactive with gold, although further studies are required to determine if this is a reductive process similar to that described by Magarvey and co-workers.26 Bioactivity. Compounds 1, Fe(III)-1, and Ga(III)-1 were screened for their antimicrobial and cytotoxic activities. None of the compounds showed appreciable cytotoxicity against any of the cell lines tested, while Fe(III)-1 and Ga(III)-1 salts also showed no antimicrobial activity. Compound 1 showed moderate activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), Staphylococcus warneri, and Proteus vulgaris, exhibiting IC50 values of 35, 11, 28, and 14 μM, respectively. This activity is likely due to the ability of 1 to bind Fe(III), inhibiting microbial proliferation via cellular iron depletion.

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ISP2m) were incubated at rt with shaking at 200 rpm for 48 h. Fermentations were conducted in 10 mL of ISP2m broth (see agar recipe above but with agar omitted), inoculated with 3% (v/v) of the seed culture, and incubated at 15, 22, and 30 °C with shaking at 200 rpm for 6 days. All temperatures were tested in triplicate. Growth was observed in all fermentations, and the media blanks remained sterile. Following the fermentation, MeOH-activated Diaion HP-20 resin (5% w/v) was added to each tube, and the tubes were then shaken for 30 min at 200 rpm. Cells and resin were collected by centrifugation at 10000g for 10 min, and then the pellets were washed twice with 15 mL of Milli-Q H2O. The washed resin was freeze-dried and then extracted with 10 mL of MeOH. Extracts were dried and then prepared at 0.5 mg mL−1 in MeOH for LC-HRMS analysis. Peak areas for ions of interest (m/z = 987.4168) were measured and compared. Large-Scale Fermentation, Extraction, and Isolation. RKJM285 was maintained on ISP2m agar plates for a period of 5−7 days before inoculation into seed cultures. Cells were inoculated into 250 mL of ISP2m liquid media in a 1 L Erlenmeyer flask and grown at rt for 48 h (200 rpm). Seed growth (3% v/v) was inoculated into 3 L (3 × 1 L in 2.8 L Fernbach flasks) of ISP2m liquid media and incubated at rt (200 rpm) for 6 days. Fermentations were pooled, and MeOHactivated HP-20 resin (20% w/v) was added and stirred at rt for 2 h. Aqueous media was removed via vacuum filtration through coffee filters. HP-20 resin was resuspended in 3 L of H2O and stirred at rt for 1 h, followed by solvent removal as previously described. This process was repeated three times (9 L of H2O total), and the H2O fractions were discarded. The HP-20 was resuspended in a 1:1 MeOH/H2O mixture (1 L), mixed for 30 min at rt with stirring, and filtered. This process was repeated one additional time (2 L 1:1 MeOH/H2O total volume). The HP-20 was finally extracted with MeOH by resuspending in 1 L and stirring for 30 min at rt followed by filtration. This process was repeated once (2 L MeOH total volume), and the MeOH fractions and MeOH/H2O (1:1) fractions were pooled separately. Extracts were dried in vacuo, yielding 636 mg (MeOH extract) and 2.214 g (MeOH/H2O) of extract. The ion of interest (m/ z 987.4168) was located in the 100% MeOH extract by LCMS analysis. The mixture was dissolved in MeOH, filtered through glass wool to remove any residual insoluble material, and dried in vacuo to yield 551 mg of a yellow solid. The material was further purified via LH-20 size exclusion chromatography (column dimensions 15 × 2 cm) using MeOH as the eluent. Fractions were collected (2 mL volume) and analyzed for the presence of m/z 987.4168 and m/z 934.5023 by LC-HRMS. Fractions containing the ions of interest were pooled and dried in vacuo, yielding 108 mg of a yellow mixture. Ferric iron was removed according to literature protocols.28 For the following procedures, all work was carried out in iron-free glassware.28 The mixture was dissolved in 40 mL of deionized H2O, added to 25 mL of a 8-hydroxyquinoline solution in MeOH (145 mg, 1 mM), and stirred at rt for 24 h. The MeOH was removed in vacuo, and the remaining aqueous solution was extracted with CH2Cl2 (5 × 20 mL). The aqueous layer was dried in vacuo, yielding 50 mg of crude 1. Material was dissolved in 1 mL of phosphate-buffered saline (PBS) buffer (pH 7.6), and the sample was treated with excess Ga2(SO4)3 (100 mg) and allowed to mix for 1 h at rt. The sample was loaded onto a MeOH-conditioned Hypersep C18 (Thermo Scientific) flash cartridge (50 mg), washed with H2O (20 mL) to remove excess PBS and gallium, eluted with MeOH (10 mL), and dried in vacuo. Complete conversion to the gallium adduct was observed by LCHRMS analysis, showing the presence of an m/z 1000.4042 [M − 2H + Ga(III)]+ corresponding to a molecular formula of C41H68GaN7O17. Final purification of Ga(III)-1 was accomplished via semipreparative HPLC using a reversed-phase Phenomenex Luna 5 μm PFP 100 Å column (250 × 10.00 mm). Purification was accomplished by injecting 50 μL aliquots of a 10 mg mL−1 solution of Ga(III)-1 in MeOH. Elution was achieved with an isocratic gradient of 100% MeOH over 10 min, with a flow rate of 3 mL min−1, monitoring by ELSD. The retention time of Ga(III)-1 was found to be 4.50 min. All fractions containing purified Ga(III)-1 were pooled, dried in vacuo, and lyophilized for 7 days, yielding 10.2 mg of the title compound as an

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Rudolph Autopol III polarimeter using a 50 mm microcell (1.2 mL). Absorbance was measured on a NanoDrop (ND1000) spectrophotometer. Infrared (IR) spectra were recorded using attenuated total reflectance on a Thermo Nicolet 6700 FT-IR spectrometer. NMR spectra were collected using a Bruker Avance III NMR spectrometer (1H: 600 MHz, 13C: 151 MHz) equipped with a 5 mm cryoprobe. All Hya 1H and 13C NMR spectra were acquired on a 400 MHz Bruker AVANCE III NMR spectrometer operating at 400 and 100 MHz, respectively, and equipped with a broadband SmartProbe and AVANCE III HD NanoBay console. All chemical shifts are referenced to residual solvent peaks [1H (DMSO-d6): 2.50 ppm; (H2O): 4.80; 13C (DMSO-d6): 39.51 ppm]. Direct infusion (MS/MS analysis) high-resolution mass spectrometry analysis was performed on an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific) using an ESI ion source operating in positive mode with a resolution of 30 000, monitoring a mass range from 150 to 2000 atomic mass units (amu). The mass spectrometer was operated under the following conditions: spray voltage, 3.0 kV; capillary temperature, 300 °C; S-lens RF voltage, 60.0%; maximum injection time (ms), 10; microscans, 1. The system was controlled by XCalibur software modules (Thermo Scientific). LC-HRMS analysis of compounds and extracts was carried out with an ESI-HRMS Exactive (Thermo Scientific) operating in positive mode with a resolution of 30 000, monitoring a mass range from 190 to 2000 amu. Chromatography was accomplished using a Core-Shell 100 Å C18 column (Phenomenex, Kinetex, 1.7 μm 50 × 2.1 mm). A linear solvent gradient from 95% H2O/0.1% formic acid (solvent A) and 5% CH3CN/0.1% formic acid (solvent B) to 100% solvent B over 4.8 min followed by a hold for 3.2 min with a flow rate of 500 μL min−1 and 10 μL injection volume was used. Eluent was detected by ESI-MS, ELSD, and UV 200−600 nm. UV-B photoreduction reactions were performed in quartz cuvettes in a Spectroline UV Cross-linkiner Select Series instrument, and UV−vis absorbance spectra were recorded using a Nanodrop ND-1000 spectrophotometer. All reagents were purchased from commercial sources and used without further purification. All solvents used for compound purification were HPLC grade or better. Small-Scale Media Screening Fermentations. Variovorax sp. RKJM285 was maintained on ISP2m agar (0.4% dextrose, 0.4% yeast extract, 1% malt extract, 1.8% Instant Ocean, 1.5% Bacto agar) at room temperature (rt). Seed cultures were conducted in 10 mL of ISP2m medium and incubated at rt for 48 h (200 rpm). Small-scale fermentations (10 mL) were inoculated with 3% v/v seed inoculum and allowed to proceed for 6 days at rt (200 rpm). Fermentations were extracted with EtOAc (2 × 10 mL), and organic layers were pooled and dried in vacuo. Extracts were dissolved in MeOH (100 μg mL−1) and screened by LC-HRMS. Temperature Effect on Production. Variovorax sp. RKJM285 was maintained on ISP2m agar plates at rt. Seed cultures (5 mL of 863



Article

aeruginosa ATCC 14210, Proteus vulgaris ATCC 12454, and Candida albicans ATCC 14035. Optical density was recorded using a Thermo Scientific Varioskan Flash plate reader at 600 nm, recording at time zero and then again after incubation for 22 h (37 °C) to determine percent growth inhibition. Bioactivity: Cytotoxicity. Compounds 1, Fe(III)-1, and Ga(III)-1 were tested for cytotoxicity against the Vero kidney cell line from African green monkey, adult human epidermal keratinocytes, MCF7 human breast adenocarcinoma (ATCC HTB-22), and human breast adenocarcinoma cells (ATCC HTB-26). All assays were carried out as described previously.36 Fluorescence was monitored using a Thermo Scientific Varioskan Flash plate reader at 560/12 excitation, 590 nm emission both at time zero and 4 h after Alamar blue addition to calculate IC50 values.

off-white solid. All NMR spectra of 1 were recorded as the Ga(III) adduct. Imaqobactin (1): off-white solid; [α]25D −165 (c 0.075, MeOH); IR νmax 3362, 2933, 1720, 1659, 1589, 1521, 1456, 1383, 1338, 1126 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 934.4957 [M + H]+ (calcd for C41H71N7O17, 934.4979); m/z 987.4111 [M − 2H + Fe(III)]+ (calcd for C41H68FeN7O17, 987.4094); m/z 1000.4042 [M − 2H + Ga(III)]+ (calcd for C41H68GaN7O17, 1000.4007); m/z 958.4585 [M − 2H + Al(III)]+ (calcd for C41H68AlN7O17, 958.4560). Amino Acid Configuration by Marfey’s Analysis. To a microconical vial, 20 μL of Fe(III)-1 (10 mg mL−1 in MeOH) was added and then dried. A solution of 6 M HCl (250 μL) was added and heated to 80 °C for 60 min. The reaction was allowed to cool to rt, and 1 mL of 1 M NaHCO3 was added followed by 20 μL of L-FDAA in acetone (1% w/v). The reactions were heated at 37 °C for 1 h before quenching with 100 μL of 6 M HCl. The reaction mixture was dried in vacuo and dissolved in MeOH/H2O (1:1) at a concentration of 500 μg mL−1 for LC-HRMS analysis. Retention times were compared to those of authentic derivatized standards to determine the amino acid configurations. β-Hydroxyaspartic Acid Diastereomer Synthesis. Diastereomeric mixtures of DL-threo-β-hydroxyaspartic acid and DL-erythro-βhydroxyaspartic acid were prepared by treatment of cis-epoxysuccinic acid (1 g) and trans-epoxysuccinic acid (1 g) with 25 mL of a concentrated (28%) aqueous solution of NH4OH.31 Reactions were allowed to proceed for 24 h at 50 °C. Reaction mixtures were dried in vacuo, yielding thick syrups. The mixtures were dissolved in 10 mL of H2O and acidified to pH 3 with concentrated HCl. The DL-threo-βhydroxyaspartic acid mixture was maintained at 4 °C over a 72 h period. The white precipitate was filtered and washed with cold H2O (100 mL) and dried in vacuo. The material was then recrystallized from H2O and subsequently dried, yielding 785 mg (69.5%) as thin needles. This material was used without further purification. NMR spectra were in agreement with literature data.34 The DL-erythro-βhydroxyaspartic acid mixture was allowed to crystallize from H2O for 1 week at rt. The resultant crystals were filtered, washed with cold H2O (100 mL), and dried, yielding 622 mg (55.1%) as thin needles. This material was used without further purification. NMR spectra were in agreement with literature data.35 Photoreactivity Experiments: Degradation Products. A 1 mg sample of Fe(III)-1 was dissolved in 1 mL of PBS buffer (pH 7.5) and exposed to UV-B radiation (λ = 280−320 nm, 3.45 mW cm−2) for 3 h. An identical sample was shielded from UV radiation as a control. Samples were dried in vacuo, dissolved in MeOH (500 μg mL−1), and analyzed by LC-HRMS. Photoreactivity Experiments: BPDS Assay. Reduction of ferric iron to ferrous iron was carried out according to literature protocols using BPDS.13,25 Reactions contained 100 μM imaqobactin (1), 10 μM FeCl3, and 40 μM BPDS in PBS buffer (pH 7.5). Reactions were exposed to UV-B radiation (λ = 280−320 nm, 3.45 mW cm−2) for a period of 3 h. Control reactions receiving no UV-light exposure were completed in parallel. Reactions were performed in triplicate. UV absorption at 535 nm was measured for both UV-B-exposed and control samples before and after light exposure. Metal Binding. Compound 1 was fractioned into 500 μg aliquots, and each sample was dissolved in 1 mL of PBS buffer (pH 7.5). An excess (15 mg) of the corresponding metal chloride (AlCl3, AuCl3, VCl3) was added to the solutions and stirred at room temperature for 1 h. Samples were loaded onto MeOH-conditioned Hypersep C18 (Thermo Scientific) solid-phase extraction (SPE) cartridges (50 mg) and washed with 10 mL of H2O. The samples were eluted with 100% MeOH (5 mL) and dried in vacuo. Samples were dissolved in MeOH (500 μg mL−1) and analyzed by LC-HRMS. Antimicrobial Activity. Compounds 1, Fe(III)-1, and Ga(III)-1 were tested for antimicrobial activity. All testing was carried out in triplicate according to the Clinical Laboratory Standards Institute testing standards in a 96-well plate microbroth dilution assay as previously described.36 Compounds were tested against methicillinresistant Staphylococcus aureus ATCC 33591, S. warneri ATCC 17917, vancomycin-resistant Enterococcus faecium EF379, Pseudomonas

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00943. Experimental procedures, material, supporting figures, NMR spectra, LCHRMS profiles, and additional data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +1 902 566 0565. Fax: +1 902 566 7445. E-mail: rkerr@ upei.ca. ORCID

Andrew W. Robertson: 0000-0002-4617-7288 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank NSERC, the Canada Research Chair Program, the University of Prince Edward Island, the Atlantic Innovation Fund, the J.-Louis Lévesque Foundation, and Nautilus Biosciences Canada Inc. for funding this research. We would like to thank M. Fischer and Dr. C. Kirby of Agriculture and Agri-Food Canada (AAFC) for the NMR services provided on the 600 MHz instrument. The authors gratefully acknowledge funding from Atlantic Canada Opportunities Agency (ACOA) for the purchase of the 400 MHz NMR. We also acknowledge Nunavut Tunngavik Inc. for permission to collect sediment samples and J. Shirley (Nunavut Research Institute) for assistance with field work.

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REFERENCES

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Isolation of Imaqobactin, an Amphiphilic Siderophore from the Arctic

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