Volatiles from the Psychrotolerant Bacterium Chryseobacterium polytrichastri

Abstract The flavobacterium Chryseobacterium polytrichastri was investigated for its volatile profile by use of a closed‐loop stripping apparatus (CLSA) and subsequent GC‐MS analysis. The analyses revealed a rich headspace extract with 71 identified compounds. Compound identification was based on a comparison to library mass spectra for known compounds and on a synthesis of authentic standards for unknowns. Important classes were phenylethyl amides and a series of corresponding imines and pyrroles.


Introduction
The genus Chryseobacterium is the second largest within the family Flavobacteriaceae with more than 100 described members. [1] The number of identified species has increased quickly over the last decades, from only seven species being known in 2002. [2] Chryseobacterium spp. are present in various habitats of different environmental conditions including soil, [3] Antarctic sea water, [4] diseased fish [5] and human tissue samples like for the pathogen Chryseobacterium gleum. [6] Although the ecology and pathogenicity of chryseobacteria and phylogenetically closely related bacteria has been well investigated, their secondary metabolism so far remained disregarded. First studies include the isolation of sulfobacins A and B from Chryseobacterium sp. NR 2993, [7] a report on volatile methyl ketones from bacteria of distinctly related genera isolated from arctic sea water, [8] and the identification of two diterpene synthases from Chryseobacterium polytrichastri and Chryseobacterium wanjuense producing diterpenes with novel skeletons. [9] C. polytrichastri DSM 26899 investigated in this study was isolated from the moss Polytrichastrum formosum which was collected from the Gawalong glacier in Tibet, China. [10] Psychrotolerance has been reported within this genus before, the most astonishing example probably being Chryseobacterium greenlandense which was isolated from a 120 000-year-old layer of ice. [11] Here we report on the volatile organic compounds (VOCs) emitted by C. polytrichastri and provide one of the first studies on the secondary metabolism within the genus Chryseobacterium.

Results and Discussion
An agar-plate culture of C. polytrichastri was subjected to a closed-loop stripping apparatus (CLSA) [12] and the volatiles were collected on charcoal filter traps for 24 hours. The charcoal filter was extracted with dichloromethane and the obtained headspace extract was analysed directly by GC-MS. The total ion chromatogram (TIC) showed a rich bouquet consisting of 71 compounds identified by this study, originating from various compound classes (Figure 1, Figure S1 and Table S1 in the Supporting Information).
2-Phenylethylamine (33) was also found, while several of its derivatives could not be identified simply from their mass spectra, since no good hits to library spectra were conceived. Therefore, structural suggestions were made based on the fragment ions observed in the EI mass spectra. One additional compound included in our MS library was N-(2-phenylethyl) formamide (46), while no literature retention index for this compound was available. A synthesis from ethyl formate (45) and 33 (Scheme 1) [22] confirmed the identity of the synthetic compound with the natural product. With this as a starting point a homologous series of 2-phenylethyl amides (47-52), was suspected from their mass spectra, all showing a similar fragmentation pattern to the pattern reported for N-(2-phenylethyl)amides before, [23] with a characteristic base peak at m/z 104 and molecular ions from m/z 149 for 46 increasing stepwise by 14 Da to m/z 247 for 52 ( Figures 5 and S2). A second important fragment ion indicative of the chain length arising by cleavage of the benzyl group was observed from m/z 58 increasing to m/z 156 for 52. Taken together, these data suggested the compounds 46-52 to represent a series of N-(2-   phenylethyl)amides from formic to octanoic acid. A synthesis starting from 42 and the acid chlorides (Scheme 1A) confirmed this hypothesis.
According to the molecular ion 53 was an isomer of 50, but eluted earlier from the GC, in agreement with a branched acid portion. The non-branched amides with longer alkyl chains exhibited diagnostic fragment ions arising by McLafferty rearrangement with cleavage of the acid side chain (Scheme 1B, highlighted in blue in Figures 5 and S2). [24] The corresponding McLafferty ion of 53 was observed at m/z 163, indicating an isovalerate portion, while the 2-methylbutyrate derivative would require m/z 177. The structural proposal of N-(2-phenylethyl)-3-methylbutanamide for 53 was confirmed by synthesis (Scheme 1A). [25] Furthermore, the structure of N-(2-phenylethyl) benzamide for 54 was identified from its mass spectrum ( Figure 5E), whose molecular ion indicated an acid side chain with four additional degrees of unsaturation. Also the enhanced fragment ions at m/z 77 and 51 pointed to a phenyl group as in the benzoate derivative. The structure of N-(2-phenylethyl) benzamide for 54 was confirmed by synthesis of reference material. Compound 48 was previously isolated from the limnic bacterium Bacillus sp. GW90a, [26] while 51-54 were reported before from Xenorhabdus doucetiae. [25] Compound 54 is known as a moderate inhibitor of N-acylhomoserine lactone sensors in Escherichia coli MT102 and Pseudomonas putida F117. [27] The odd molecular ions for 56, 58 and 60 with mass spectra not included in our libraries also pointed to nitrogen-containing compounds ( Figure 6). All three compounds showed fragment ions at m/z 104 and 91 and further typical fragment ions of aromatic compounds (m/z 77, 65 and 39), suggesting they might likewise contain N-phenylethyl groups. The base peak at m/z 80 for 56, arising by loss of a benzyl group, indicated a nitrogen-containing portion with three degrees of unsaturation as in pyrrole, and is also typical for its N-alkylated derivatives. For 58 and 60 the base peak was increased by 14 Da (m/z 94) and 28 Da (m/z 108), respectively. Methylations of the pyrrole at C2 for 58 and at C2 and C5 for 60 were considered most likely. A synthesis of all three reference compounds, of 56 by a Clauson-Kaas reaction from 33 and 2,5-dimethoxytetrahydrofuran (55), [28] and of 58 and 60 in a solvent-free Paal-Knorr reaction from 33 and the dicarbonyl compounds 57 and 59, [29] confirmed the suggested structures in all three cases (Scheme 2). Detailed proposed fragmentation pathways for all three compounds are presented in Schemes S1-S3. N-(2-Phenylethyl)pyrrole (56) has been reported before from Abelmoschus esculentes [30] and Saccharomyces cerevisiae, [31] whereas 58 and 60 represent new natural products.
Compound 62 showed a molecular ion of m/z 189 suggesting the presence of nitrogen, and fragment ions at m/z 105, 91 and 77 indicating a phenethyl group ( Figure 7A). The fragment ions at m/z 56 (tentatively assigned to C 3 H 6 N + ) and 42 (tentatively assigned to C 2 H 4 N + ) are typical for imines, and together with the α-fragmentation leading to m/z 132 and the neutral loss of propene through McLafferty rearrangement [24] the structural suggestion of N-(3-methylbutylidene)-2-phenylethylamine for 62 was delineated. Another imine was tentatively identified as N-(2-furylmethylidene)-2-phenylethylamine (64) by comparison of its mass spectrum ( Figure 7B) to a database spectrum. Compound 65, one of the major constituents in the headspace (10.9 %), and 67 were identified from their mass spectra ( Figure 7C and D) by comparison to library spectra as N-benzylidene-2-phenylethylamine (65) and 3-methyl-N-(2-phenylethylidene)-1-butanamine (67), respectively. Full hypothetical fragmentation pathways for 62, 64, 65 and 67 are shown in Schemes S4-S7. To verify the tentatively assigned structures all four imines were prepared by condensation of the corresponding amines and aldehydes over molecular sieves (Scheme 3) [32] and proved to be identical to the volatiles from C. polytrichastri by MS and retention index. Because of its  instability compound 67 could not be isolated in pure form, but was obtained in a synthetic mixture containing one component that showed the same mass spectrum as the natural product from C. polytrichastri. 3-Methyl-N-(2-phenylethylidene)-1-butanamine (67) has been observed as a volatile from Tuber melanosporum before, [33] whereas the imines 62, 64 and 65 represent new natural products.
The biosynthesis of the imines could proceed analogously to their synthesis by condensation of an aldehyde and an amine. This hypothesis is supported by the presence of several of the needed precursors in the headspace extracts, including the aldehydes 34 and 37 and the amine 33. These building blocks can be formed by degradation of phenylalanine, [34,35] while 61 and 66 that are not observed in the headspace can derive from leucine (Scheme 4). [36,37] Furfural (63) is a sugar degradation product associated to spoilage or fermentation processes, [38,39] but no biosynthetic pathway in bacteria was reported so far.

Conclusion
In conclusion, this study provides the first insights into the secondary metabolism of a bacterium from the genus Chryseobacterium by identification of the emitted volatiles. Although some similarities to distinctly related Flavobacteriaceae can be observed with respect to the production of methyl ketones, the most pronounced class of volatiles was represented by nitrogen-containing compounds including pyrazines and other aromatic heterocycles, besides amides, pyrroles and imines mostly deriving from 2-phenylethylamine. Several new natural products were identified, including N-(2-phenylethyl)pentanamide (50), 2-methyl-N-(2-phenylethyl)-pyrrole (58), 2,5-dimethyl-N-(2-phenylethyl)pyrrole (60), N-(3-methylbutylidene)-2-phenylethylamine (62), N-(2-furylmethylidene)-2-phenylethylamine (64) and N-benzylidene-2-phenylethylamine (65), thus demonstrating that the genus Chryseobacterium is of high interest to natural product chemists. This was also reflected in our recent discovery of diterpene synthases yielding diterpenes of new skeletons. [9] However, the terpenes produced by the known synthases from Chryseobacterium were not found in the headspace extract, suggesting that the corresponding genes are not expressed under laboratory culture conditions. Some of the simple compounds reported here such as pyrazine (9) might (partially) originate from the medium, which is also possible for benzaldehyde (37) and furfural (63) used as building blocks in the formation of imines. Clarification of a biosynthetic origin could be obtained by feeding of isotopically labelled potential biosynthetic precursors. Future studies in our laboratories will include investigations on the volatiles released by bacteria of other untapped genera.

Experimental Section
General experimental details: Reactions were performed in dried flasks under an Ar atmosphere and in dried solvents. Chemicals were used as purchased from the supplier. Cooling was maintained using ice/water (0°C) or liquid nitrogen/acetone (À 78°C) for time periods up to 1 h. For longer periods of time a cooling unit was used. Column chromatography was performed on silica gel (0.04-0.06 nm, Acros Organics, Geel, Belgium) with previously distilled solvents.
Culture conditions and preparation of headspace extracts: C. polytrichastri DSM 26899 was obtained from the DSMZ (Braunschweig, Germany) and was cultivated on 123 TGY (5.0 g L À 1 tryptone, 5.0 g L À 1 yeast extract, 1.0 g L À 1 glucose, 1.0 g L À 1 K 2 HPO 4 , pH 6.9, autoclaved at 121°C for 20 min; for solid medium 20.0 g L À 1 agar was added before autoclaving). Liquid cultures were inoculated from glycerol stocks and were cultivated at 28°C with shaking at 160 rpm. Plates were inoculated using 400-600 μL of a liquid