Nitrogen-Containing Volatiles from Marine ... - ACS Publications

Article Cite This: J. Nat. Prod. 2017, 80, 3289−3295

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Nitrogen-Containing Volatiles from Marine Salinispora pacif ica and Roseobacter-Group Bacteria Tim Harig,† Christian Schlawis,† Lisa Ziesche,† Marion Pohlner,‡ Bert Engelen,‡ and Stefan Schulz*,† †

Institute of Organic Chemistry, Technische Universität Braunschweig, Hagenring 30, 38106 Braunschweig, Germany Institute for Chemistry and Biology of the Marine Environment (ICBM), Carl von Ossietzky Universität Oldenburg, Carl von Ossietzky Straße 9-11, 26129 Oldenburg, Germany

‡

S Supporting Information *

ABSTRACT: Bacteria can produce a wide variety of volatile compounds. Many of these volatiles carry oxygen, while nitrogen-containing volatiles are less frequently observed. We report here on the identification and synthesis of new nitrogen-containing volatiles from Salinispora pacif ica CNS863 and explore the occurrence in another bacterial lineage, exemplified by Roseobacter-group bacteria. Several compound classes not reported before from bacteria were identified, such as dialkyl ureas and oxalamides. Sulfinamides have not been reported before as natural products. The actinomycete S. pacif ica CNS863 produces, for example, sulfinamides N-isobutyl- and N-isopentylmethanesulfinamide (5, 6), urea N,N′-diisobutylurea (16), and oxalamide N,N′diisobutyloxalamide (17). In addition, new imines such as (E)-1-(furan-2-yl)-N-(2-methylbutyl)methanimine (8) and (E)-2((isobutylimino)methyl)phenol (13) were identified together with several other imines, acetamides, and formamides. Some of these compounds including the sulfinamides were also released by the Roseobacter-group bacteria Roseovarius pelophilus G5II, Pseudoruegeria sp. SK021, and Phaeobacter gallaeciensis BS107, although generally fewer compounds were detected. These nitrogen-containing volatiles seem to originate from biogenic amines derived from the amino acids valine, leucine, and isoleucine.

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volatiles, the obligate marine actinomycete genus Salinispora23 and members of the Roseobacter group24,25 that were also investigated by Dickschat et al.26,27 On first sight, the marine environment does not seem to be suited for mostly hydrophobic apolar volatile compounds because of the hydrophilic water phase. Nevertheless, apolar volatile compounds are used as signals by many marine organisms, e.g., brown algae that produce apolar hydrocarbons as pheromones28,29 or other algae.30 During our investigation of the volatiles released by Salinispora pacif ica CNS863 several unknown nitrogencontaining volatiles were detected, which in part also occurred in some Roseobacter-group bacteria of the genera Roseovarius, Pseudoruegeria, and Phaeobacter. Many of them showed unusual EI-mass spectra that eluded identification by simple database comparison procedures. The identified compounds are shown in Chart 1. We will report here on the identification and synthesis of these compounds, which are produced in submicrogram quantities by the bacteria.

olatile compounds are released by organisms in any habitat on earth. Such volatiles may be metabolites serving a certain function or are waste products of the emitter’s metabolism. With this odor bouquet information is transported that potentially can be used by other organisms, revealing information on the status of the emitter. The structures of volatiles from important producers such as plants1−3 or animals4 have been investigated for a long time, as has the function of such compounds. In contrast, much less is known on the structures of volatiles released by bacteria5−8 and their functions.9−11 Most of the volatiles known so far from bacteria are either hydrocarbons or their derivatives containing O or S.5,12 Nitrogen-containing compounds are less frequently found, with a few exceptions such as pyrazines.5,13 Other examples include the ubiquitous signaling compound indole,14,15 which is produced by many bacteria8 and is a characteristic odor compound of Escherichia coli.16 Another common bacterial compound is 2-aminoacetophenone, a signal of Pseudomonas aeruginosa.17 Besides very volatile ammonia and hydrogen cyanide, several compound classes occasionally found include amines,18−20,20 imines,18,19,21 amides,20,21 oximes,6 nitro compounds,6 or aromatic compounds such as pyridines.22 In an effort to understand the chemical ecology of marine bacteria, we have investigated two groups for the production of © 2017 American Chemical Society and American Society of Pharmacognosy

Received: September 15, 2017 Published: December 1, 2017 3289

DOI: 10.1021/acs.jnatprod.7b00789 J. Nat. Prod. 2017, 80, 3289−3295

Journal of Natural Products

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Chart 1

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RESULTS AND DISCUSSION Bacterial cultures on agar or in the liquid phase often produce a range of various volatiles in low quantities.5 The GC/MS analysis of the volatiles of liquid cultures of S. pacifica CNS863 by closed-loop stripping analysis (CLSA)31 revealed the presence of 16 nitrogen-containing compounds, A−Q (Figure 1A), indicated by their often odd-numbered molecular ions. Compounds B−D and F proved to be the known amides 1−4. N-Isobutyl- (2)32 and N-isopentylacetamides (4)21,32 are known from other bacteria, as is N-isopentylformamide (3),21 while N-isobutylformamide (1) has not been reported from bacteria before. Compound G showed a mass spectrum not easy to interpret (Figure 2G). HR-GC/MS revealed its molecular composition to be C5H13NOS (HRMS found 135.07374, calcd 135.07178). The CLSA extract contained only low amounts of material in the nanogram range. To obtain additional IR data of G, we used a GC/IR instrument, which condenses the GC effluent on a rotating cooled ZnSe disk. By repeated injection enough material in the nanogram range was collected to obtain an IR spectrum that revealed the presence of a strong absorption at 1051 cm−1, characteristic for a sulfinamide group in the solid state (IR spectrum in the Supporting Information).33,34 The strong N−H stretch band at 3180 cm−1 indicated a secondary or primary sulfinamide.33,34 Ions m/z 57 and 43 in the mass spectrum point to an isobutyl group, leaving a methyl group as the second hydrocarbon fragment of G. The composition of ion m/z 64, CH4OS, placed the methyl group next to the sulfur. Compound G was therefore identified as N-isobutylmethanesulfinamide (5). Similarly, ions m/z 43, 64, 71, 92, and 134 led to the identification of compound J as N-isopentylmethanesulfinamide (6), which coeluted with another unknown

Figure 1. Total ion chromatograms of Salinispora pacif ica CNS 863 (A), Roseovarius pelophilus G5II (B), and Pseudoruegeria sp. SK021 (C).

compound. The structures of the sulfinamides were proven by synthesis according to Scheme 1. Methylsulfinyl chloride was prepared starting from dimethyl disulfide,35 followed by the addition of isobutylamine to furnish 5. Because of the tedious workup, homologue 6 was prepared directly from dimethyl disulfide and isopentylamine under copper catalysis. 36 Sulfinamides have not been reported from nature before. Various imines (A, E, I, K−N, Q) were present as well. Compounds K and M were identified as (E)-N-isobutyl- (10) and (E)-N-isopentyl-1-phenylmethanimine (12), while A was N-isobutylidenisobutylamine (18). These volatiles are known from Bacillus popillae18 and Stigmatella aurantiaca.21 Additionally, L was identified as an isomer of 12, (E)-N-(2methylbutyl)-1-phenylmethanimine (11). The mass spectra of imines are characterized by a very intense peak derived from αcleavage next to N and formal cleavage of the CN bond including H-transfer, leading to ions m/z 118 and 91 in the case of 11 (Figure 2L). Compounds E and I showed such ions at m/z 81 and 108, indicating likely a furan ring. Therefore, furyl imines 7 and 9 were synthesized from furfural and proved to be identical to E and I. Both compounds represent new natural products. Slightly earlier than I compound H with a similar mass spectrum occurred (Figure 2H), exhibiting different intensities in some ions such as m/z 122 and 71 due to a 2methylbutyl side chain. This compound was tentatively assigned to be (E)-1-(furan-2-yl)-N-(2-methylbutyl)methanimine (8). Finally, a similar approach led to the 3290



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Figure 2. Mass spectra and gas chromatographic retention index I on an apolar HP-5 phase. (G) N-isobutylmethanesulfinamide (5), (J) Nisopentylmethanesulfinamide (6), (L) (E)-N-(2-methylbutyl)-1-phenylmethanimine (11), (E) (E)-1-(furan-2-yl)-N-isobutylmethanimine (7), (H) (E)-1-(furan-2-yl)-N-(2-methylbutyl)methanimine (8), (I) (E)-1-(furan-2-yl)-N-isopentylmethanimine (9), (N) (E)-2-((isobutylimino)methyl)phenol (13), (Q) (E)-2-((isopentylimino)methyl)phenol (14), (O) N,N′-diisobutylurea (15), (P) N,N′-diisobutyloxalamide (17).

identification of N and Q as hydroxyphenylimines. The ions m/z 107 and 134 are shifted by 16 amu compared to the respective ions in 10 and 12, indicating an additional O in these compounds. The synthesis of such imines starting from o-, m-, or p-hydroxybenzaldehyde revealed the natural compounds to be derived from salicyl aldehyde. Comparison of the synthetic materials with the natural compounds by GC/MS revealed N to be (E)-2-((isobutylimino)methyl)phenol (13) and Q to be (E)-2-((isopentylimino)methyl)phenol (14). Imines 8, 9, 13, and 14 have not been reported before from nature, while 7 is known from Yunnan truffle.37 The mass spectrum of compound O showed some similarity to that of 2 in the lower mass range. The molecular ion was 71 amu heavier, suggesting an additional C4H9N unit. Therefore, we proposed this compound to be N,N′-diisobutylurea (15), which was synthesized according to Scheme 1, confirming our assignment. Compound P, with a molecular ion at m/z 200, was initially thought to be the corresponding N,N′-

Scheme 1. Synthesis of Nitrogen-Containing Volatiles

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Scientific Inc. modified with workbooks provided by Dani Instruments. Gas chromatographic retention indices I were determined from a homologous series of n-alkanes. Commercially available starting materials and solvents were purchased from Sigma-Aldrich or Fisher Scientific and used without further purification. Technical solvents were distilled before use. All reactions involving water-sensitive chemicals were performed in heat-dried glassware with magnetic stirring under a nitrogen atmosphere. TLC was performed on Polygram SIL G/UV254 plates (Macherey-Nagel) with detection by UV (254 nm). Flash chromatography was performed on silica gel M60 (0.04−0.063 mm, 230−400 mesh ASTM, Macherey-Nagel) under pressure. Strains, Culture Conditions, and Extraction. Bacterial strains used in this study were Salinispora pacif ica CNS863, Roseovarius pelophilus G5II, Pseudoruegeria sp. SK021, and Phaeobacter gallaeciensis BS107. S. pacif ica was collected in 2006 from the Fiji islands. This strain has been sequenced with the GenBank accession number LGVV00000000. While R. pelophilus G5II was isolated from tidal-flat sediment on the German North Sea coast, Pseudoruegeria sp. SK021 was isolated in 2011 from surface sediments located between Denmark and Norway. The strains have been sequenced with the GenBank accession numbers AJ968650 and HG423263, respectively. P. gallaeciensis BS107, accession number NR_027609, was isolated from rearing of the scallop Pecten maximus. S. pacif ica was incubated at 28 °C in A1 medium (10 g/L soluble starch, 4 g/L yeast extract, 2 g/L peptone, 30 g/L instant ocean), while Roseobacter-group strains were grown at 20 °C in marine broth medium (MB, Carl Roth). Precultures were routinely grown in Erlenmeyer flasks at 20/28 °C on a rotary shaker at 150 rpm. Erlenmeyer flasks (500 mL) containing 100 mL of A1/MB were inoculated with 2% preculture. After growth of the culture (3−6 days) headspace extracts were obtained by CLSA as described earlier.42 The extracts were prepared with 30 μL of CH2Cl2 and analyzed by GC/MS. The detected volatile compounds were identified by comparison of their mass spectra, fragmentation patterns, and gas chromatographic retention indices with those of reference compounds, synthesized as described. N-Isobutylmethanesulfinamide (5). A solution of dimethyl disulfide (0.38 mL, 4.25 mmol) and acetic acid (0.49 mL, 8.49 mmol) was cooled to −20 °C. Sulfuryl chloride (0.27 mL, 3.40 mmol) was added dropwise to the stirred almost frozen mixture. After the addition, the reaction mixture was stirred for 30 min at −20 °C, then slowly warmed to room temperature (rt) and stirred for a further 2 h. The solution was concentrated under reduced pressure to give crude yellow sulfinyl chloride,35 which was diluted with 42 mL of CH2Cl2. Isobutylamine (1.19 mL, 12.02 mmol) was added dropwise, and the mixture was stirred for 18 h at rt. The reaction solution was washed once with saturated NaHCO3 solution, then with H2O and brine, and dried with MgSO4. The solvent was removed under reduced pressure. Chromatographic purification on silica gel (EtOAc) gave 5 (39 mg, 7%) as a colorless liquid. I = 1143; IR (solid) νmax 3184, 2957, 2926, 2871, 1733, 1712, 1523, 1470, 1415, 1386, 1367, 1301, 1252, 1176, 1052, 979, 949 cm−1; 1H NMR (CD3OD, 300 MHz) δ 2.99−2.72 (m, 2H), 2.63 (s, 3H), 1.75 (hept, J = 6.7 Hz, 1H), 0.94 (dd, J = 6.7, 1.6 Hz, 6H); 13C NMR (CD3OD, 76 MHz) δ 51.07, 41.00, 30.55, 20.47, 20.42; EIMS m/z 135 (22), 120 (99), 92 (62), 77 (6), 76 (9), 64 (36), 63 (24), 57 (100), 43 (20), 41 (55); HREIMS m/z 135.07375 (calcd for C5H13NOS, 135.07178). N-Isopentylmethanesulfinamide (6). CuI (20 mg, 0.11 mmol) and 2,2′-bipyridine (17 mg, 0.11 mmol) were added to a mixture of dimethyl disulfide (100 mg, 1.06 mmol), isopentylamine (204 mg, 2.34 mmol), and ammonium hexafluorophosphate (173 mg, 1.06 mmol) in DMSO (2.1 mL) and H2O (0.7 mL). The mixture was stirred at 80 °C for 18 h in the presence of air, provided by a balloon. The residue was dissolved in diethyl ether, washed once with H2O and brine, and dried with anhydrous Mg2SO4.36 Chromatographic purification on silica (diethyl ether/pentane, 1:1) gave 6 (27 mg, 8%) as a colorless liquid. I = 1251; IR (solid) νmax 3173, 2957, 2930, 2871, 1470, 1413, 1385, 1367, 1302, 1135, 1059, 991, 962, 881 cm−1; 1H NMR (CDCl3, 300

diisopentylurea (16), but the mass spectrum did not match. The loss of fragments with 43 and 57 amu together with the molecular mass hinted again at two isobutyl groups in the compound, and the ion at m/z 100 suggested a symmetric compound. Therefore, N,N′-diisobutyloxalamide (17) was proposed as structure P, and synthesis (Scheme 1) confirmed this assignment. Although we could not detect 16 in S. pacif ica CNS863, it was present in Roseovarius pelophilus G5II. Urea derivatives and oxalamides have not been reported before as bacterial volatiles. Bacteria of the Staphylococcus intermedius group secrete small nonvolatile N-monosubstituted urea compounds such as N-(2-phenethyl)urea that act as quorum quenching compounds.38 N-Methyl-N′-9-methyldecylurea has been identified from Photorhabdus luminescens.39 No natural volatile oxalamides are known, although bis(agamatine)oxalamide has been identifed from spider venom.40 We were also interested in the distribution of these compounds in other bacteria. Therefore, we investigated several Roseobacter-group bacteria, an important marine bacterial lineage. We could identify several of the discussed N-containing volatiles in Roseobacter, although the number of compounds in single strains was lower compared to Salinispora. As examples, results from Roseovarius peolphilus G5II, Pseudoruegeria sp. SK021, and Phaeobacter gallaeciensis BS107 are shown (Figure 1, Table S1 in the Supporting Information). In addition to the compounds 1−3, 5, 6, and 14−17, Roseovarius pelophilus G5II contained imine (E)-N-isobutyl-2methylpropan-1-imine (18), known from the essential oil of Perilla f rutescens,41 and the amides N-isopentyl-4-methylpentanamide (20) and N-isobutyl-3-methylbutanamide (19), not previously reported as natural products. Amides 19 and 20 were identified along the lines discussed for the other compounds and synthesized as described in Scheme 1. The nitrogen-containing 1−20 volatiles seem to originate from biogenic amines isobutyl-, isopentyl-, and 2-methylbutylamine, derived from the amino acids valine, leucine, and isoleucine. They are condensed with small activated aldehyde or acyl metabolites of the bacteria to form these compounds. Their presence in widely different bacterial lineages hints at the widespread occurrence of these bacterial volatiles.

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EXPERIMENTAL SECTION

General Experimental Procedures. One- and two-dimensonal 1 H NMR and 13C NMR analyses were performed using the following instruments: Bruker AV-II 300 (1H 300 MHz, 13C 75.5 MHz), AV III400 (1H 400 MHz, 13C 100 MHz), or AV II-600 (1H 600 MHz, 13C 151 MHz). Chemical shifts are reported in ppm relative to tetramethylsilane as an internal standard (δ = 0 ppm). High-resolution MS data were obtained with a Agilent 6890 gas chromatograph coupled to a JMS-T100GC (GCAccuTOF, JEOL) equipped with a ZB5-MS (Phenomenex, 30 m × 0.25 mm i.d. × 0.25 μm) column. GC/MS was performed on an HP 6890 gas chromatograph coupled to an MSD 5973 (EI 70 eV, Hewlett-Packard) and on a GC 7890A coupled to an MSD 5975C (Agilent Technologies). All gas chromatographic separations were performed on an HP5-MS (Agilent Technologies, 30 m × 0.25 mm i.d. × 0.25 μm) fused-silica capillary column. GC/IR analysis was performed using a GC 7890B (Agilent Technologies) gas chromatograph coupled to a DiscovIR solid phase instrument (Dani Instruments). The samples eluting from the GC column were deposited on a cooled ZnSe disc at −40 °C using a disc speed of 4 mm/min. The gas chromatograph was equipped with an Agilent HP-5 column (30 m × 0.25 mm i.d. × 0.25 μm) with helium as the carrier gas. The resulting infrared spectra had a resolution of 4 wavenumbers, ranging from 700 to 4000 cm−1, and were normalized and processed using GRAMS/AI 9.2 software by Thermo Fisher 3292



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MHz) δ 3.68 (t, J = 5.9 Hz, 1H), 3.22−3.05 (m, 2H), 2.61 (s, 3H), 1.65 (hept, J = 6.7 Hz, 1H), 1.53−1.35 (m, 2H), 0.92 (d, J = 6.6 Hz, 6H); 13C NMR (CDCl3, 76 MHz) δ 41.99, 40.73, 39.57, 25.75, 22.40; EIMS m/z 136 (4), 135 (6), 134 (83), 132 (42), 92 (29), 78 (6), 77 (6), 72 (5), 71 (84), 64 (12), 63 (18), 55 (10), 47 (7), 43 (100), 41 (28). General Synthesis of Amides. Acetic acid chloride (1 equiv) was dissolved in CH2Cl2 (0.1 M). The amine (2.83 equiv) was added dropwise to the solution and stirred for 2 h at rt. The reaction mixture was then washed once with H2O, NaHCO3 solution, and brine and dried over MgSO4. Removing the solvent under reduced pressure yielded the desired product without further purification. N-Isobutyl-3-methylbutanamide (19). 19 was synthesized with the use of isovaleryl chloride (150 mg, 1.24 mmol) and isobutylamine (257 mg, 3.52 mmol) as described above in 90% yield (157 mg) as a colorless oil: I 1226; IR (ATR) νmax 3300, 3094, 2957, 2930, 2872, 1641, 1560, 1467, 1368, 1260, 1220, 1161 cm−1; 1H NMR (CDCl3, 300 MHz) δ 5.48 (s, 1H, NH), 3.02 (dd, J = 6.9, 6.0 Hz, 2H CH2), 2.22−1.91 (m, 3H, CH, CH2), 1.71 (hept, J = 6.8 Hz, 1H), 0.89 (d, J = 6.4 Hz, 6H), 0.84 (d, J = 6.7 Hz, 6H); 13C NMR (CDCl3, 76 MHz,) δ 172.85 (C), 47.17 (CH2), 46.76 (CH2), 28.92 (CH), 26.59 (CH), 22.87 (2× CH3), 20.50 (2× CH3); EIMS m/z 158 (4), 157 (20), 156 (2), 142 (12), 115 (76), 114 (27), 102 (50), 100 (8), 86 (4), 85 (67), 73 (10), 72 (22), 69 (8), 68 (2), 60 (39), 59 (8), 58 (27), 57 (100), 56 (23), 55 (5), 53 (4), 44 (46), 43 (43), 42 (19), 41 (81), 40 (10), 39 (39). N-Isopentyl-4-methylpentanamide (20). 20 was synthesized from 4-methylvaleryl chloride (167 mg, 1.24 mmol) and isopentylamine (307 mg, 3.52 mmol) as described above in 89% yield (204 mg) as a colorless oil: I 1448; IR (solid) νmax 3271, 3091, 2957, 2934, 2871, 1640, 1564, 1469, 1385, 1367, 1279, 1231, 1201, 1171, 1117 cm−1; 1H NMR (CDCl3, 300 MHz) δ 5.41 (s, 1H, NH), 3.26−3.14 (m, 2H, CH2), 2.14−2.04 (m, 2H, CH2), 1.63−1.40 (m, 4H, CH2 and 2× CH), 1.38−1.27 (m, 2H, CH2), 0.85 (d, J = 6.6, 6H, 2× CH3), 0.83 (d, J = 6.4 Hz, 6H, 2× CH3); 13C NMR (76 MHz, CDCl3) δ 173.16 (C), 38.54 (CH2), 37.77 (CH2), 34.87(CH2), 34.64 (CH2), 27.80 (CH), 25.85 (CH), 22.43 (2× CH3), 22.29 (2× CH3); EIMS m/z 186 (1), 185 (5), 184 (2), 171 (5), 170 (40), 156 (7), 143 (4), 142 (40), 130 (9), 129 (100), 128 (14), 116 (19), 115 (3), 114 (31), 100 (9), 99 (34), 98 (4), 87 (13), 86 (24), 84 (5), 82 (4), 81 (37), 74 (7), 73 (84), 72 (24), 71 (32), 70 (11), 69 (8), 68 (2), 60 (7), 59 (3), 58 (5), 57 (10), 56 (15), 55 (36), 54 (5), 53 (7), 45 (5), 44 (46), 43 (88), 42 (15), 41 (61), 40 (5), 39 (23). General Synthesis of Ureas. S,S’-Dimethyldithiocarbonate (1 equiv) was heated to 60 °C; then a 40% aqueous solution of an amine (3 equiv) was added. The solution was stirred for 2 h at 60 °C, whereby a white solid precipitated, followed by stirring for 16 h at rt. H2O was removed under reduced pressure. Toluene was added and the azeotrope was removed under reduced pressure. The crude product was purified by recrystallization from toluene. N,N′-Diisobutylurea (15). 15 was synthesized from S,S′-dimethyldithiocarbonate (150 mg, 1.23 mmol) and isobutylamine (0.37 mL, 3.68 mmol) as described above as a white solid in 96% yield (203 mg): I 1460; IR (solid) νmax 3325, 2958, 2929, 2872, 1631, 1579, 1469, 1434, 1387, 1368, 1336, 1272, 1164, 1127, 1057, 920, 818 cm−1; 1H NMR (CDCl3, 300 MHz) δ 5.13 (t, J = 5.7 Hz, 2H, 2× NH), 2.90 (dd, J = 6.8, 5.9 Hz, 4H, 2× CH2), 1.65 (hept, J = 6.8 Hz, 2H, 2× CH), 0.83 (d, J = 6.7 Hz, 12H, 4× CH3); 13C NMR (CDCl3, 76 MHz) δ 159.08 (C), 47.85 (2× CH2), 29.02 (2× CH), 20.10 (4× CH3); EIMS m/z 173 (7), 172 (63), 158 (3), 157 (23), 130 (4), 129 (19), 117 (5), 116 (2), 115 (5), 101 (89), 100 (3), 87 (4), 86 (8), 85 (8), 74 (8), 73 (7), 72 (16), 58 (48), 57 (40), 56 (35), 55 (20), 54 (5), 44 (8), 43 (49), 42 (15), 41 (100), 40 (8), 39 (41). N,N′-Diisopentylurea (16). 16 was synthesized from S,S′dimethyldithiocarbonate (150 mg, 1.23 mmol) and isopentylamine (0.43 mL, 3.68 mmol) as described above as a white solid in 94% yield (231 mg): I = 1678; IR (solid) νmax 3316, 2957, 2930, 2871, 1627, 1579, 1469, 1384, 1367, 1280, 1253, 1230, 1171 cm−1; 1H NMR (CDCl3, 300 MHz) δ 4.51 (t, J = 5.1 Hz, 2H, NH), 3.10 (td, J = 7.5, 5.6 Hz, 4H, 2× CH2), 1.54 (hept, J = 6.8 Hz, 2H, 2× CH), 1.32 (dt, J

= 7.4, 6.9 Hz, 4H, 2× CH2), 0.84 (d, J = 6.6 Hz, 12H, 4× CH3); 13C NMR (CDCl3, 76 MHz) δ 158.08 (C), 38.74 (2× CH2), 38.34 (2× CH2), 25.34 (2× CH), 22.06 (4× CH3); EIMS m/z 201 (6), 200 (42), 186 (3), 185 (28), 158 (2), 157 (17), 145 (3), 144 (39), 132 (2), 131 (17), 115 (10), 114 (11), 102 (5), 101 (44), 89 (2), 88 (25), 87 (13), 86 (7), 85 (5), 84 (1), 75 (2), 74 (19), 73 (8), 72 (11), 71 (14), 70 (19), 69 (5), 58 (3), 57 (8), 56 (21), 55 (24), 54 (3), 53 (6), 45 (6), 44 (100), 43 (84), 42 (19), 41 (76) 40 (6), 39 (27). General Synthesis of Imines. To a stirred 0.1 M solution of the amine in toluene with 3 Å molecular sieves was added the respective aldehyde (1 equiv), and the mixture was heated to reflux for 26 h. The reaction mixture was filtered over Celite and washed with CH2Cl2. The solvent was removed under reduced pressure to give the desired product with no further purification needed. (E)-1-(Furan-2-yl)-N-isobutylmethanimine (7). 7 was synthesized with the use of isobutylamine (0.16 mL, 1.64 mmol) and furfural (0.14 mL, 1.64 mmol) as described above to yield 7 (121 mg, 49%) as a colorless oil: I = 1115; UV (CH2Cl2) λmax (log ε) 265 (4.22); IR (solid) νmax 3105, 2957, 2929, 2898, 2871, 2823, 1727, 1651, 1486, 1469, 1384, 1366, 1340, 1275, 1243, 1202, 1157, 1076, 1031, 1016, 934, 884, 828, 777, 751 cm−1; 1H NMR (CDCl3, 400 MHz) δ 7.96 (t, J = 1.4 Hz, 1H), 7.42 (dd, J = 2.4, 0.7 Hz, 1H), 6.64 (dd, J = 3.5, 0.8 Hz, 1H), 6.39 (dd, J = 3.4, 1.8 Hz, 1H), 3.32 (dd, J = 6.7, 1.3 Hz, 2H), 1.97 (hept, J = 6.7 Hz, 1H), 0.87 (d, J = 6.8 Hz, 6H); 13C NMR (CDCl3, 101 MHz) δ 151.62 (C), 149.50 (CH), 144.46 (CH), 113.47 (CH), 111.45 (CH), 70.05 (CH2), 29.42 (CH), 20.63 (2× CH3); EIMS m/z 151 (6), 136 (4), 123 (5), 108 (100), 94 (2), 81 (63), 53 (11), 52 (7), 41 (7), 39 (9). (E)-1-(Furan-2-yl)-N-(2-methylbutyl)methanimine (8). 8 was synthesized with the use of 2-methylbutylamine (0.18 mL, 1.56 mmol) and furfural (0.13 mL, 1.56 mmol) as described above to yield 8 (237 mg, 92%) as a yellow oil: I = 1230; UV (CH2Cl2) λmax (log ε) 264 (4.21); IR (solid) νmax 3109, 2961, 2876, 2826, 1651, 1582, 1566, 1486, 1463, 1396, 1378, 1275, 1156, 1080, 1017, 937, 884, 749 cm−1; 1 H NMR (CDCl3, 400 MHz) δ 8.07−8.02 (m, 1H), 7.53−7.48 (m, 1H), 6.74−6.69 (m, 1H), 6.50−6.44 (m, 1H), 3.55 (ddt, J = 11.4, 5.9, 1.5 Hz, 1H), 3.33 (ddt, J = 11.4, 7.2, 1.4 Hz, 1H), 1.90−1.77 (m, 1H), 1.52−1.40 (m, 1H), 1.25−1.12 (m, 1H), 0.96−0.86 (m, 6H); 13C NMR (CDCl3, 101 MHz) δ 151.63 (C), 149.54 (CH), 144.46 (CH), 113.45 (CH), 111.45 (CH), 68.28 (CH2), 35.82 (CH), 27.44 (CH2), 17.65 (CH3), 11.35 (CH3); EIMS m/z 165 (6), 164 (9), 150 (3), 148 (3), 137 (18), 136 (25), 123 (3), 122 (14), 110 (4), 109 (53), 108 (100), 96 (4), 95 (11), 94 (12), 82 (5), 81 (64), 80 (11), 79 (4), 53 (15), 52 (10), 51 (7), 41 (12), 39 (12). (E)-1-(Furan-2-yl)-N-isopentylmethanimine (9). 9 was synthesized with the use of isopentylamine (0.20 mL, 1.72 mmol) and furfural (0.14 mL, 1.72 mmol) as described above to yield 9 (228 mg, 80%) as a colorless oil: I = 1234; UV (CH2Cl2) λmax (log ε) 264 (4.17); IR (solid) νmax 3102, 2957, 2927, 2870, 2844, 1649, 1580, 1566, 1485, 1469, 1384, 1367, 1275, 1156, 1079, 1018, 977, 931, 884, 750 cm−1; 1 H NMR (400 MHz, CDCl3) δ 8.09 (t, J = 1.1 Hz, 1H), 7.50 (dd, J = 1.9, 0.7 Hz, 1H), 6.71 (dd, J = 3.4, 0.8 Hz, 1H), 6.46 (dd, J = 3.4, 1.8 Hz, 1H), 3.59 (td, J = 7.3, 1.4 Hz, 2H), 1.67 (hept, J = 6.5 Hz, 1H), 1.60 (dt, J = 7.3, 7.2 Hz, 2H), 0.93 (d, J = 6.4 Hz, 6H); 13C NMR (CDCl3, 101 MHz) δ 151.62 (C), 149.24 (CH), 144.43 (CH), 113.37 (CH), 111.42 (CH), 59.86 (CH2), 39.79 (CH2), 25.79 (CH), 22.46 (2× CH3); EIMS m/z 165 (7), 164 (17), 150 (6), 137 (25), 136 (41), 122 (57), 109 (100), 108 (78), 95 (53), 94 (36), 81 (92), 67 (11), 53 (26). (E)-N-(2-Methylbutyl)-1-phenylmethanimine (11). 11 was synthesized with the use of 2-methylbutylamine (0.14 mL, 1.15 mmol) and benzaldehyde (0.12 mL, 1.15 mmol) as described above to yield 11 (181 mg, 90%) as a yellow oil: I = 1371; UV (CH2Cl2) λmax (log ε) 246 (4.23); IR (solid) νmax 3063, 3028, 2961, 2926, 2875, 2851, 1647, 1582, 1496, 1452, 1379, 1353, 1311, 1295, 1220, 1169, 1139, 1075, 1034, 1005, 974, 919, 849, 768, 754 cm−1; 1H NMR (CDCl3, 400 MHz) δ 8.17 (t, J = 1.4 Hz, 1H), 7.71−7.61 (m, 2H), 7.39−7.27 (m, 3H), 3.55−3.24 (m, 2H), 1.80−1.67 (m, 1H), 1.48−1.33 (m, 1H), 1.21−1.06 (m, 1H), 0.92−0.79 (m, 6H); 13C NMR (CDCl3, 101 MHz) δ 160.85 (C), 136.36 (CH), 130.38 (CH), 128.52 (2× CH), 3293



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128.01 (2× CH), 67.96 (CH2), 35.96 (CH), 27.50 (CH2), 17.71 (CH3), 11.42 (CH3); EIMS m/z 175 (5), 174 (12), 160 (5), 147 (3), 146 (15), 132 (7), 119 (26), 118 (86), 117 (14), 106 (3), 105 (5), 104 (14), 103 (2), 98 (12), 97 (12), 92 (9), 91 (100), 90 (13), 89 (15), 78 (4), 77 (14), 76 (3), 65 (11), 64 (3), 63 (7), 62 (2), 52 (2), 51 (10), 50 (4), 43 (3), 42 (2), 41 (18), 40 (2), 39 (12). (E)-2-((Isobutylimino)methyl)phenol (13). 13 was synthesized with the use of isobutyl amine (0.20 mL, 2.05 mmol) and salicylaldehyde (0.21 mL, 2.05 mmol) as described above to yield 13 (352 mg, 97%) as a yellow oil: I = 1443; UV (CH2Cl2) λmax (log ε) 229 (4.24), 255 (4.20), 315 (3.76); IR (solid) νmax 3274, 3069, 2959, 2929, 2871, 2839, 1635, 1613, 1583, 1502, 1464, 1421, 1387, 1280, 1214, 1152, 1114, 1047, 1027, 758 cm−1; 1H NMR (CDCl3, 400 MHz) δ 13.73 (s, 1H), 8.30 (t, J = 1.3 Hz, 1H), 7.37−7.20 (m, 2H), 7.00−6.93 (m, 1H), 6.86 (td, J = 7.5, 1.1 Hz, 1H), 3.42 (dd, J = 6.5, 1.3 Hz, 2H), 1.97 (hept, J = 6.7 Hz, 1H), 0.98 (d, J = 6.7 Hz, 6H); 13C NMR (CDCl3, 101 MHz) δ 164.63 (CH), 161.43 (C), 132.02 (CH), 131.07 (CH), 118.76 (C), 118.33 (CH), 117.01 (CH), 67.43 (CH2), 29.56 (CH), 20.43 (2× CH3); EIMS m/z 178 (12), 177 (74), 135 (15), 162 (12), 145 (4), 135 (24), 134 (100), 133 (10), 121 (8), 120 (13), 119 (4), 108 (11), 107 (99), 106 (11), 93 (8), 91 (8), 79 (12), 78 (17), 77 (39), 66 (8), 65 (13), 64 (5), 52 (7), 51 (20), 41 (20), 39 (23). (E)-2-((Isopentylimino)methyl)phenol (14). 14 was synthesized with the use of isopentylamine (0.24 mL, 2.07 mmol) and salicylaldehyde (0.22 mL, 2.07 mmol) as described above to yield 14 (352 mg, 97%) as a yellow oil: I = 1563; UV (CH2Cl2) λmax (log ε) 228 (4.21), 254 (4.12), 314 (3.68); IR (solid) νmax 3061, 2957, 2930, 2870, 1634, 1614, 1583, 1501, 1462, 1419, 1385, 1367, 1245, 1281, 1222, 1209, 1152, 1054, 1037, 1024, 989, 967, 893, 859, 757, 738 cm−1; 1H NMR (CDCl3, 400 MHz) δ 13.61 (s, 1H), 8.26 (t, J = 1.2 Hz, 1H), 7.29−7.11 (m, 2H), 6.94−6.84 (m, 1H), 6.78 (td, J = 7.4, 1.1 Hz, 1H), 3.53 (td, J = 7.1, 1.3 Hz, 2H), 1.63 (hept, J = 6.9 Hz, 1H), 1.50 (dt, J = 7.3, 6.8 Hz, 2H), 0.87 (d, J = 6.6 Hz, 6H); 13C NMR (CDCl3, 101 MHz) δ 164.35 (CH), 161.35 (C), 131.97 (CH), 130.99 (CH), 118.82 (C), 118.33 (CH), 117.00 (CH), 57.59 (CH2), 39.81 (CH2), 25.76 (CH), 22.44 (2× CH3); EIMS m/z 192 (6), 191 (69), 190 (11), 176 (5), 162 (8), 149 (9), 147 (10), 146 (6), 134 (100), 133 (7), 132 (8), 131 (14), 121 (23), 120 (31), 118 (19), 108 (8), 107 (77), 106 (6), 93 (10), 91 (7), 77 (29), 66 (4), 65 (11), 51 (10), 41 (15), 39 (12). N,N′-Diisobutyloxalamide (17). To a solution of isobutylamine (210 mg, 2.87 mmol) in toluene (11.5 mL) was added diethyl oxalate (200 mg, 1.37 mmol), and the mixture was heated to reflux for 5 h. The reaction was then stirred at rt for 16 h, cooled to 0 °C, and diluted with 12 mL of pentane. The precipitating white solid was filtered of, washed with pentane, and dried under reduced pressure to afford 17 (148 mg, 54%) as a white solid: I = 1512; IR (solid) νmax 3297, 2961, 1656, 1526, 1472, 1434, 1389, 1361, 1268, 1229, 1159 cm−1; 1H NMR (CDCl3, 600 MHz) δ 7.59 (br s, 2H), 3.15 (dd, J = 6.9, 6.4 Hz, 4H), 1.84 (hept, J = 6.8 Hz, 2H), 0.95 (d, J = 6.8 Hz, 12H); 13C NMR (CDCl3, 151 MHz) δ 159.94, 46.99, 28.40, 20.02; EIMS m/z 201 (2), 200 (14), 185 (2), 157 (13), 145 (54), 143 (17), 102 (5), 101 (12), 100 (17), 72 (12), 59 (21), 58 (21), 57 (100), 46 (14), 41 (35).

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Stefan Schulz: 0000-0002-4810-324X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Deutsche Forschungsgemeinschaft for a grant through the Transregional Collaborative Research Center Roseobacter, SFB/TRR 51. We thank P. Jensen, Scripps Institution of Oceanography, UC San Diego, La Jolla, CA, USA, for the generous supply of the Salinispora strains.

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00789. 1

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REFERENCES

H NMR and 13C NMR of synthesized compounds, IR spectrum of 5, results of volatile analysis (PDF)

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