Volatiles from the Psychrotolerant Bacterium Chryseobacterium polytrichastri

Article abstract of DOI:10.1002/cbic.202000503

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.

Full text of DOI:10.1002/cbic.202000503

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Volatiles from the Psychrotolerant Bacterium
Chryseobacterium polytrichastri
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Lukas Lauterbach[a] and Jeroen S. Dickschat*[a]
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The flavobacterium Chryseobacterium polytrichastri was inves-
tigated 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 com-
pounds. Compound identification was based on a comparison
to library mass spectra for known compounds and on a
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synthesis of authentic standards for unknowns. Important
classes were phenylethyl amides and a series of corresponding
imines and pyrroles.
1. Introduction
2. Results and Discussion
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 phylogeneti-
cally 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 Chryseobac-
terium 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] Psychro-
tolerance has been reported within this genus before, the most
astonishing example probably being Chryseobacterium green-
landense which was isolated from a 120000-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.
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 head-
space 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).
For several well-known volatiles the identification by
comparison of mass spectra to library spectra and of retention
indices to previously published data was possible. Among
terpenoids this included the nor-carotenoids[13] 6-methylhept-5-
en-2-one (1), geranyl (2) and farnesyl acetone (3), nerolidol (4),
and the saturated derivative 6,10,14-trimethylpentadecan-2-one
(5). The widely distributed sulfur volatiles dimethyl disulfide (6)
and dimethyl trisulfide (7) and the less frequently observed
methanesulfonamide (8) were also identified (Figure 2).[14,15]
The most abundant compound class were nitrogen-contain-
ing volatiles, including pyrazine (9) and a series of alkylated
derivatives (10–19, Figure 3), altogether making up around 13%
of the headspace extract. Pyrazines with diverse substitution
patterns are known as headspace constituents from many fungi
[a] L. Lauterbach, Prof. Dr. J. S. Dickschat
Kekulé Institute for Organic Chemistry and Biochemistry
University of Bonn
Gerhard-Domagk-Straße 1, 53121 Bonn, Germany
E-mail: dickschat@uni-bonn.de
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cbic.202000503
This article is part of a Special Collection on Chemical Proteomics and
Metabolomics. To view the complete collection, visit our homepage
© 2020 The Authors. Published by Wiley-VCH GmbH. This is an open access
article under the terms of the Creative Commons Attribution License, which
permits use, distribution and reproduction in any medium, provided the
original work is properly cited.
Figure 1. Total ion chromatogram of the headspace extract obtained from
CLSA analysis of C. polytrichastri. Peaks marked with asterisks represent the
plasticiser dibutyl phthalate and major unidentified aromatic compounds.
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nitriles, benzonitrile (38) and phenylacetonitrile (36), were
observed as minor constituents (Figure 4).
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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-phenyl-
ethyl)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-
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Figure 2. Structures of terpenoids and sulfur volatiles observed in the
headspace extract of C. polytrichastri.
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Figure 3. Structures of heteroaromatic compounds found in the headspace
extract of C. polytrichastri.
and bacteria,[14À 16] and a biosynthetic pathway for alkylated
compounds via acetoin (20) has been discovered by
a
combined labelling and mutation study in Corynebacterium
glutamicum.[17] This biosynthetic origin is also plausible for C.
polytrichastri since butane-2,3-diol (25), potentially arising by
reduction of 20, is present in the headspace extract. Alter-
natively, pyrazines with branched groups such as 14 might arise
by dimerisation of amino acids or amino aldehydes.[18,19]
Figure 4. Structures of aromatic compounds contributing to the headspace
extract of C. polytrichastri.
Other heteroaromatic nitrogen compounds were found in
traces, represented by 2-acetylthiazole (21), 2-acetylpyrrole (22),
the pyridine derivatives 2,4,6-trimethylpyridine (23) and 2-
cyanopyridine (24), and the four bicyclic compounds benzothia-
zole (26), indole (27), 4-methylquinazoline (28) and 4-meth-
ylquinoline (29; Figure 3). Aromatic compounds include the
main compound 2-phenylethanol (30, 13.6%), well-known from
several bacteria,[15] and some of its derivatives such as phenyl-
acetaldehyde (34), the acetate (31) and benzoate esters (32).
Further compounds were benzyl alcohol (35), that has pre-
viously been observed in other Flavobacteriaceae,[4] benzalde-
hyde (37) and diphenylethanedione (39), acetophenone (40),
benzophenone (41) and 2-aminoacetophenone (42). The latter
might be a precursor for 29 that is a formal condensation
product of 42 with acetaldehyde. Intriguingly, the combination
of both compounds was previously observed in Myxococcus
xanthus and Streptomyces caviscabies.[20,21] Additionally, the two
amides benzamide (43), N-phenylacetamide (44), and two
Scheme 1. A) Synthesis of N-(2-phenylethyl)amides. B) McLafferty rearrange-
ment from 49 leading to m/z 163.
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phenylethyl)amides from formic to octanoic acid. A synthesis
starting from 42 and the acid chlorides (Scheme 1A) confirmed
this hypothesis.
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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-phenyl-
ethyl)-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
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Clauson-Kaas
reaction
from
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and
2,5-dimeth-
oxytetrahydrofuran (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-Phenyl-
ethyl)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 C3H6N+) and 42
(tentatively assigned to C2H4N+) 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-phenyl-
ethylamine for 62 was delineated. Another imine was tenta-
tively identified as N-(2-furylmethylidene)-2-phenylethylamine
Figure 5. EI-MS spectra of A) N-(2-phenylethyl)formamide (46), B) N-(2-
phenylethyl)butanamide (49), C) N-(2-phenylethyl)octanamide (52), D) N-(2-
phenylethyl)-3-methylbutanamide (53) and E) N-(2-phenylethyl)benzamide
(54). Red indicates ions arising from benzyl group cleavage, blue indicates
fragments originating from McLafferty rearrangement.
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Figure 6. EI-MS spectra of A) N-(2-phenylethyl)pyrrole (56), B) 2-methyl-N-(2-
phenylethyl)pyrrole (58) and C) 2,5-dimethyl-N-(2-phenylethyl)pyrrole (60).
Red indicates a fragment ion arising from benzyl group cleavage.
Figure 7. EI-MS spectra of A) N-(3-methylbutylidene)-2-phenylethylamine
(62), B) N-(2-furylmethylidene)-2-phenylethylamine (64), C) N-benzylidene-2-
phenylethylamine (65) and D) 3-methyl-N-(2-phenylethylidene)-1-butan-
amine (67). Red indicates fragment ions arising from benzyl group cleavage,
blue indicates ions originating from McLafferty rearrangement.
spectra as N-benzylidene-2-phenylethylamine (65) and 3-meth-
yl-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
Scheme 2. Synthesis of N-(2-phenylethyl)pyrroles.
(64) by comparison of its mass spectrum (Figure 7B) to a
database spectrum. Compound 65, one of the major constitu-
ents in the headspace (10.9%), and 67 were identified from
their mass spectra (Figure 7C and D) by comparison to library
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Scheme 3. Synthesis of imine reference substances. Yields refer to condensa-
tion over molecular sieves.
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-butan-
amine (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.
One more group contributing to the headspace extract
consists of methyl ketones, similar to the volatiles emitted by
other Flavobacteriaceae (Figure 8).[8] Among these are the
unbranched methyl ketones 68 to 74, spanning chain lengths
from C6 to C17, and the ωÀ 1 methyl branched compounds 12-
methyl-2-tridecanone (75) and 13-methyl-2-tetradecanone (76),
which were identified by comparison to their known mass
spectra and retention indices.[8]
Three further compounds exhibited very similar mass
spectra (Figure 9A–C), but showed molecular ions increasing by
14 Da and simultaneously increasing retention indices by
100 units, suggesting a series of structurally related homolo-
gous compounds. The observed fragment ion pattern was in
agreement with 3-methyl-2-ketones for which the base peak at
m/z 72 is explained by McLafferty rearrangement, while the
strong fragment ion at m/z 43 can arise by α-cleavage.
Furthermore, the retention indices of 85 and 86 matched
recently published data for 3-methyl-2-undecanone and 3-
methyl-2-dodecanone (Table S1).[40]
Scheme 4. A) Compounds 33, 34 and 37 are produced by Phe degradation,
B) 61 and 66 arise from degradation of Leu. PAL: phenylalanine ammonia
lyase, DC: PLP-dependent decarboxylase, PPDC: phenyl pyruvate decarbox-
ylase, AADD: aryl aldehyde dehydrogenase, BCAT: branched-chain amino
transferase, KDCA: α-ketoacid decarboxylase, LDC: leucine decarboxylase.
Figure 8. Structures of methyl ketones from C. polytrichastri.
2-one (86) were synthesised by alkylation of ethyl 2-methyl-3-
oxobutanoate (77) with the suitable alkyl bromides (78–80),
yielding the β-keto esters 81–83, which were further converted
by
saponification
with
spontaneous
decarboxylation
(Scheme 5). The obtained compounds showed identical mass
spectra and retention indices to those of the natural products.
The ketones have all been reported as natural products before,
84 in the marking fluid of Panthera tigris, and 85 and 86 as
pheromones in Ptomascopus morio.[40,41]
To confirm their identity the three ketones 3-methyldecan-
2-one (84), 3-methylundecan-2-one (85) and 3-methyldodecan-
Scheme 5. Synthesis of the methyl ketones 77, 78 and 79.
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discovery of diterpene synthases yielding diterpenes of new
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skeletons.[9] However, the terpenes produced by the known
synthases from Chryseobacterium were not found in the head-
space 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.
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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.
Analytical methods: NMR spectroscopy was performed at 297 K
either on an Avance I (400 MHz), Avance I, Avance III HD Ascend
(both 500 MHz) or an Avance III HD Ascend (700 MHz) from Bruker
(Billerica, MA, USA). Spectra were referenced against solvent peaks
(1H NMR: CDCl3 δ=7.26 ppm, C6D6 δ=7.16 ppm; 13C NMR: CDCl3
δ=77.16 ppm, C6D6 δ=128.06 ppm).[42] Multiplicities are given by d
(doublet), t (triplet), q (quartet), sept (septet) and br (broad). IR
spectra were recorded on a Bruker α with a diamond ATR probe
head, intensities of bands are indicated by w (weak), m (medium)
and s (strong). GC/MS analysis was performed on a 7890B GC–
5977 A MD system (Agilent, Santa Clara, CA, USA) fitted with a HP5-
MS UI column (30 m, 0.25 mm i.d., 0.50 μm film). The GC was
operated with 1) an inlet pressure of 77.1 kPa at an He flow of
23.3 mLminÀ 1, 2) an injection volume of 1 μL (synthetic samples) or
2 μL (headspace extracts), 3) a temperature ramp starting with
Figure 9. EI mass spectra of A) 3-methyldecan-2-one (84), B) 3-meth-
ylundecan-2-one (85) and C) 3-methyldodecan-2-one (86). Blue indicates a
McLafferty rearrangement leading to the base peak at m/z 72.
À 1
À 1
°
°
°
5 min at 50 C, increasing at 10 C min (synthetic) or 5 C min
°
(headspace) to 320 C, 4) 60 s valve time and 5) He carrier gas flow
of 1.2 mLminÀ 1. The parameters of the MS were 1) source temper-
°
°
3. Conclusion
ature: 230 C, 2) transfer line temperature: 250 C, 3) quadrupole
temperature: 150 C and 4) electron energy: 70 eV. Retention
°
indices were determined in comparison to a homologous series of
n-alkanes (C7À C40). GC/MS-QTOF analyses were conducted on a
7890B GC–7200 accurate-mass Q-TOF detector system (Agilent).
The GC was equipped with a HP5-MS fused silica capillary column
(30 m, 0.25 mm i. d., 0.50 μm film). GC parameters were 1) injection
volume: 1 μL, 2) split ratio: 10:1, 60 s valve time, 3) carrier gas: He
°
In conclusion, this study provides the first insights into the
secondary metabolism of a bacterium from the genus Chrys-
eobacterium 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 nitro-
gen-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)pentana-
mide (50), 2-methyl-N-(2-phenylethyl)-pyrrole (58), 2,5-dimeth-
yl-N-(2-phenylethyl)pyrrole (60), N-(3-methylbutylidene)-2-phe-
nylethylamine (62), N-(2-furylmethylidene)-2-phenylethylamine
(64) and N-benzylidene-2-phenylethylamine (65), thus demon-
strating that the genus Chryseobacterium is of high interest to
natural product chemists. This was also reflected in our recent
at 1 mLminÀ 1
,
and 4) temperature program: 5 min at 50 C
°
À 1
°
increasing at 10 C min to 320 C. MS parameters were 1) inlet
pressure: 83.2 kPa, He at 24.6 mLminÀ 1, 2) transfer line: 250 C, 3)
°
electron energy 70 eV.
Culture conditions and preparation of headspace extracts: C.
polytrichastri DSM 26899 was obtained from the DSMZ (Braunsch-
weig, Germany) and was cultivated on 123 TGY (5.0 g LÀ 1 tryptone,
5.0 g LÀ 1 yeast extract, 1.0 g LÀ 1 glucose, 1.0 g LÀ 1 K2HPO4, Àp1H 6.9,
°
autoclaved at 121 C for 20 min; for solid medium 20.0 g L 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
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°
culture grown for 3 days and were cultivated for 3 days at 28 C
before submitting them to the CLSA. The volatiles were collected
for 24 h and were extracted from the activated charcoal using small
amounts of dichloromethane (7×10 μL). The extracts were directly
injected to the GC/MS.
7.4 Hz), 1.62 (tq, 2H, JH,H =7.4 Hz, 7.2 Hz), 0.91 (t, 3H, JH,H =7.2 Hz)
ppm. 13C NMR (126 MHz, CDCl3, 298 K): δ=173.2 (Cq), 139.0 (Cq),
128.9 (2xCH), 128.7 (2xCH), 126.6 (CH), 40.7 (CH2), 38.8 (CH2), 35.8
(CH2), 19.3 (CH2), 13.9 (CH3) ppm.
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9
N-(2-Phenylethyl)pentanamide (50): Yield: 0.325 g (1.58 mmol,
Synthesis of phenethyl benzoate (32):[43] To a cooled (0 C) solution
78%). 1H NMR (500 MHz, CDCl3, 298 K): δ=7.31 (ddd, 2H, JH,H
=
3
°
5
3
4
of 2-phenylethanol (2.10 g, 17.3 mmol, 1.0 equiv) and pyridine
(1.67 mL, 20.7 mmol, 1.2 equiv) in dichloromethane (50 mL) was
slowly added benzoyl chloride (2.40 mL, 20.7 mmol, 1.2 equiv) and
the mixure was stirred for 18 h at room temperature. The mixture
7.3 Hz, 6.8 Hz, JH,H =1.5 Hz), 7.23 (tt, 1H, JH,H =7.4 Hz, JH,H =1.4 Hz),
3 4
7.19 (dd, 2H, JH,H =7.0 Hz, JH,H =1.4 Hz), 5.68 (brs, 1H), 3.52 (dt, 2H,
3JH,H =5.9 Hz, 6.9 Hz), 2.81 (t, 2H, 3JH,H =7.0 Hz), 2.14 (t, 2H, JH,H
=
3
3
3
7.6 Hz), 1.57 (tt, 2H, JH,H =7.4 Hz, 7.5 Hz), 1.31 (tq, 2H, JH,H =7.5 Hz,
7.6 Hz), 0.91 (t, 3H, 3JH,H =7.4 Hz) ppm. 13C NMR (126 MHz, CDCl3,
298 K): δ=173.4 (Cq), 139.0 (Cq), 128.9 (2xCH), 128.8 (2xCH), 126.6
(CH), 40.7 (CH2), 36.6 (CH2), 35.8 (CH2), 28.0 (CH2), 22.5 (CH2), 13.9
(CH3) ppm.
°
was cooled to 0 C and the reaction was terminated by addition of
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
water. The organic phase was washed subsequently with NaOH
(1 m, 2×50 mL), HCl (1 m, 50 mL) and brine (50 mL), dried with
MgSO4 and evaporated to dryness. The crude mixture was purified
by flash chromatography (cyclohexane/ethyl acetate 5:1) to yield
the title compound as colourless oil (2.80 g, 12.3 mmol, 72%). TLC
N-(2-Phenylethyl)hexanamide (51): Yield: 0.386 g (1.76 mmol,
87%). 1H NMR (500 MHz, CDCl3, 298 K): δ=7.31 (dd, 2H, JH,H
=
3
1
(cyclohexane/ethyl acetate 5:1): Rf =0.45. H NMR (500 MHz, CDCl3,
3
3
7.4 Hz, 7.6 Hz), 7.23 (t, 1H, JH,H =7.5 Hz), 7.19 (dd, 2H, JH,H =7.4 Hz,
298 K): δ=8.08 (dd, 2H, 3JH,H =7.7 Hz, 4JH,H =1.6 Hz), 7.57–7.62 (m,
4JH,H =1.2 Hz), 5.46 (brs, 1H), 3.52 (dt, 2H, JH,H =6.1 Hz, 6.8 Hz), 2.81
3
3
1H), 7.48 (dd, 2H, JH,H =6.0 Hz, 7.7 Hz), 7.32–7.40 (m, 4H), 7.26–7.32
(t, 2H, 3JH,H =7.0 Hz), 2.11 (t, 2H, 3JH,H =7.7 Hz), 1.58 (tt, 2H, JH,H
=
3
(m, 1H), 4.59 (t, 2H, 3JH,H =7.1 Hz), 3.13 (t, 2H, JH,H =7.0 Hz) ppm. 13C
3
7.2 Hz, 7.7 Hz), 1.22–1.35 (m, 4H), 0.88 (t, 3H, JH,H =7.0 Hz) ppm. 13C
NMR (126 MHz, CDCl3, 298 K): δ=173.2 (Cq), 139.1 (Cq), 128.9
(2xCH), 128.7 (2xCH), 126.6 (CH), 40.6 (CH2), 36.9 (CH2), 35.9 (CH2),
31.5 (CH2), 25.5 (CH2), 22.5 (CH2), 14.0 (CH3) ppm.
3
NMR (126 MHz, CDCl3, 298 K): δ=166.6 (Cq), 138.0 (Cq), 133.0 (CH),
130.4 (Cq), 129.7 (2xCH), 129.1 (2xCH), 128.7 (2xCH), 128.5 (2xCH),
126.7 (CH), 65.6 (CH2), 35.4 (CH2) ppm.
Synthesis of N-(2-phenylethyl)formamide (46):[22] A mixture of 2-
phenylethylamine (2.26 mL, 18.7 mmol, 1.0 equiv) and ethyl for-
mate (30 mL, 27.5 mmol, 1.5 equiv) was stirred under reflux over-
night. Excess of ethyl formate was removed under high vacuum to
yield the pure amide as viscous oil (2.57 g, 17.2 mmol, 92%; mixture
of E and Z diastereomers). 1H NMR (500 MHz, CDCl3, 298 K): δ=8.13
N-(2-Phenylethyl)octanamide (52): Yield: 0.443 g (1.80 mmol,
89%). 1H NMR (500 MHz, CDCl3, 298 K): δ=7.31 (ddd, 2H, JH,H
=
3
5
3
4
7.4 Hz, 7.4 Hz, JH,H =1.5 Hz), 7.23 (tt, 1H, JH,H =7.4 Hz, JH,H =1.4 Hz),
3
4
7.19 (dd, 2H, JH,H =7.4 Hz, JH,H =1.3 Hz), 5.63 (brs, 1H), 3.52 (dt, 2H,
3JH,H =6.2 Hz, 6.9 Hz), 2.82 (t, 2H, 3JH,H =7.0 Hz), 2.13 (t, 2H, JH,H
=
3
3
7.4 Hz), 1.58 (tt, 2H, JH,H =7.2 Hz, 7.4 Hz), 1.22–1.31 (m, 8H), 0.87 (t,
3
(s, 1H, Z), 7.89 (d, 1H, JH,H =10.9 Hz, E), 7.28–7.34 (m, 2H), 7.15–7.26
3H, JH,H =6.9 Hz) ppm. 13C NMR (126 MHz, CDCl3, 298 K): δ=173.5
3
3
(m, 3H), 5.83 (brs, 1 H), 3.57 (t, 2H, JH,H =6.4 Hz, Z), 3.47 (t, 1 H,
(Cq), 139.0 (Cq), 128.9 (2xCH), 128.8 (2xCH), 126.7 (CH), 40.7 (CH2),
36.9 (CH2), 35.8 (CH2), 31.8 (CH2), 29.4 (CH2), 29.1 (CH2), 25.9 (CH2),
22.7 (CH2), 14.2 (CH3) ppm.
3JH,H =7.0 Hz, E), 2.85 (t, 2H, JH,H =6.5 Hz) ppm. 13C NMR (126 MHz,
3
CDCl3, 298 K): δ=164.6 (CH, E), 161.4 (CH, Z), 138.5 (Cq, Z), 129.0
(2xCH, E), 128.9 (2xCH, Z), 128.8 (4xCH, 2xE, 2xZ), 127.1 (CH, E), 126.8
(CH, Z), 43.3 (CH2, E), 39.4 (CH2, Z), 37.8 (CH2, E), 35.6 (CH2, Z) ppm.
N-(2-Phenylethyl)-3-methylbutanamide (53): Yield: 0.378 g
(1.84 mmol, 92%). 1H NMR (500 MHz, CDCl3, 298 K): δ=7.31 (dd, 2H,
General procedure for the synthesis of N-(2-phenylethyl)
amides:[25] To a solution of 2-phenylethylamine (1.65 equiv) in
dichloromethane (0.25 m) was added a solution of acid chloride
(1.0 equiv) in dichloromethane (0.4 m). The mixture was stirred for
1 h at room temperature and was washed subsequently with
NaHCO3 solution (5%), HCl (1 m) and NaCl solution (1 m). The
organic phase was dried with MgSO4 and evaporated to dryness to
yield the amides in >95% purity.
3JH,H =7.2 Hz, 7.4 Hz), 7.22 (t, 1H, 3JH,H =7.2 Hz), 7.19 (d, 2H, JH,H
=
3
3
7.2 Hz), 5.83 (brs, 1H), 3.54 (dt, 2H, JH,H =6.2 Hz, 6.6 Hz), 2.83 (t, 2H,
3JH,H =7.0 Hz), 2.08 (tsept, 1H, 3JH,H =7.0 Hz, 6.4 Hz), 2.02 (d, 2H,
3JH,H =7.0 Hz), 0.91 (d, 6H, 3JH,H =6.4 Hz) ppm. 13C NMR (126 MHz,
CDCl3, 298 K): δ=173.0 (Cq), 139.0 (Cq), 128.9 (2xCH), 128.8 (2xCH),
126.7 (CH), 46.0 (CH2), 40.8 (CH2), 35.8 (CH2), 26.3 (CH), 22.5 (2xCH3)
ppm.
N-(2-Phenylethyl)benzamide (54): Yield: 0.800 g (1.32 mmol, 65%).
1H NMR (500 MHz, CDCl3, 298 K): δ=7.70 (ddd, 2H, 3JH,H =8.4 Hz,
N-(2-Phenylethyl)acetamide (47): Yield: 0.216 g (1.32 mmol, 65%).
1H NMR (500 MHz, CDCl3, 298 K): δ=7.30 (dd, 2H, 3JH,H =6.5 Hz,
5
3
4
4JH,H =1.5 Hz, JH,H =3.1 Hz), 7.48 (tt, 1 H, JH,H =6.5 Hz, JH,H =1.4 Hz),
3
4
3
7.3 Hz), 7.22 (tt, 1H, JH,H =7.5 Hz, JH,H =1.5 Hz), 7.19 (d, 2H, JH,H
=
7.40 (ddd, 2H, 3JH,H =8.3 Hz, 7.7 Hz, 4JH,H =1.5 Hz), 7.33 (ddd, 2H,
3
6.9 Hz), 5.79 (brs, 1H), 3.51 (dt, 2H, JH,H =6.8 Hz, 7.0 Hz), 2.82 (t, 2H,
3JH,H =7.0 Hz), 1.95 (s, 3H) ppm. 13C NMR (126 MHz, CDCl3, 298 K):
δ=170.4 (Cq), 138.9 (Cq), 128.9 (2xCH), 128.8 (2xCH), 126.7 (CH),
40.9 (CH2), 35.7 (CH2), 23.3 (CH3) ppm.
4
3JH,H =6.5 Hz, 6.5 Hz, JH,H =1.5 Hz), 7.23–7.27 (m, 3H), 6.20 (brs, 1H),
3.72 (dt, 2H, 3JH,H =5.9 Hz, 6.9 Hz), 2.94 (t, 2H, 3JH,H =6.9 Hz) ppm. 13
C
NMR (126 MHz, CDCl3, 298 K): δ=167.6 (Cq), 139.0 (Cq), 134.8 (Cq),
131.5 (CH), 128.9 (2xCH), 128.8 (2xCH), 128.7 (2xCH), 126.9 (2xCH),
126.7 (CH), 41.3 (CH2), 35.8 (CH2) ppm.
N-(2-Phenylethyl)propanamide (48): Yield: 0.326 g (1.83 mmol,
3
91%). 1H NMR (500 MHz, CDCl3, 298 K): δ=7.30 (ddd, 2H, JH,H
=
Clauson-Kaas reaction to 56:[29] To a solution of 2,5-dimeth-
oxytetrahydrofuran (3.93 mL, 29.2 mmol, 1.0 equiv) in glacial acetic
acid (15 mL) was added 2-phenylethylamine (3.68 mL, 29.2 mmol,
1.0 equiv). After stirring the reaction mixture under reflux for 1 h,
acetic acid was removed at the rotary evaporator. The residue was
redissolved in ethyl acetate (150 mL). The organic layer was washed
subsequently with HCl (1 m), K2CO3 solution (10%) and brine
(150 mL each), dried with MgSO4 and evaporated to dryness. The
residue was subjected to column chromatography (cyclohexane/
ethyl acetate 15:1) to yield 56 as colourless oil (3.81 g, 22.2 mmol,
66%). TLC (cyclohexane/ethyl acetate 20:1) Rf =0.46. 1H NMR
5
3
4
7.2 Hz, 7.3 Hz, JH,H =1.5 Hz), 7.23 (tt, 1H, JH,H =7.2 Hz, JH,H =2.1 Hz),
3
4
7.19 (dd, 2H, JH,H =7.2 Hz, JH,H =1.5 Hz), 5.68 (brs, 1H), 3.52 (dt, 2H,
3
3JH,H =6.4 Hz, 7.0 Hz), 2.82 (t, 2H, 3JH,H =7.0 Hz), 2.17 (q, 2H, JH,H
=
7.4 Hz), 1.12 (t, 3H, 3JH,H =7.5 Hz) ppm. 13C NMR (126 MHz, CDCl3,
298 K): δ=174.1 (Cq), 139.0 (Cq), 128.9 (2xCH), 128.8 (2xCH), 126.6
(CH), 40.7 (CH2), 35.8 (CH2), 29.8 (CH2), 10.0 (CH3) ppm.
N-(2-Phenylethyl)butanamide (49): Yield: 0.386 g (2.0 mmol,
1
3
100%). H NMR (500 MHz, CDCl3, 298 K): δ=7.30 (ddd, 2H, JH,H
=
5
3
4
7.4 Hz, 7.8 Hz, JH,H =1.4 Hz), 7.23 (tt, 1H, JH,H =7.6 Hz, JH,H =1.3 Hz),
3
4
7.19 (dd, 2H, JH,H =7.8 Hz, JH,H =1.4 Hz), 5.59 (brs, 1H), 3.52 (dt, 2H,
3
3
3JH,H =5.5 Hz, 7.2 Hz), 2.82 (t, 2H, 3JH,H =7.0 Hz), 2.11 (t, 2H, JH,H
=
(500 MHz, CDCl3, 298 K): δ=7.30 (dd, 2H, JH,H =6.5 Hz, 7.4 Hz), 7.11
ChemBioChem 2020, 21, 1–11
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(d, 2H, 3JH,H =7.3 Hz), 7.22–7.27 (m, 1H), 6.61 (s, 2H), 6.14 (s, 2H), 4.12
(t, 2H, 3JH,H =7.5 Hz), 3.06 (t, 2H, 3JH,H =7.6 Hz) ppm. 13C NMR
(126 MHz, CDCl3, 298 K): δ=138.6 (Cq), 128.8 (2xCH), 128.7 (2xCH),
126.7 (CH), 120.6 (2xCH), 108.1 (2xCH), 51.3 (CH2), 38.5 (CH2) ppm.
(2xCH), 126.4 (1 C,CH), 112.1 (CH), 111.7 (CH), 63.6 (CH2), 37.9 (CH2)
ppm.
1
2
3
4
5
6
7
8
9
N-Benzylidene-2-phenylethylamine (65): Yield: 8.81 g (42.1 mmol,
1
4
84%). H NMR (400 MHz, C6D6, 298 K): δ=7.88 (t, 1H, JH,H =1.4 Hz),
General procedure for Paal-Knorr synthesis:[30] A mixture of 2-
phenylethylamine (1.0 equiv) and a 1,4-dicarbonyl compound (57,
59; 1.0 equiv) was stirred at room temperature for 1 h. The water
formed during the reaction was removed under high vacuum. The
products were purified by column chromatography (cyclohexane/
ethyl acetate 30:1).
7.71 (dd, 2H, JH,H =7.8 Hz, JH,H =1.6 Hz), 7.07–7.21 (m, 8H), 3.74 (td,
3 4 3
3
4
2H, JH,H =7.3 Hz, JH,H =1.3 Hz), 2.98 (t, 2H, JH,H =7.3 Hz) ppm. 13C
NMR (100 MHz, C6D6, 298 K): δ=160.8 (CH), 140.6 (Cq), 137.1 (Cq),
130.5 (CH), 129.4 (2xCH), 128.7 (2xCH), 128.6 (2xCH), 128.5 (2xCH),
126.4 (CH), 63.4 (CH2), 38.0 (CH2) ppm.
3-Methyl-N-(2-phenylethylidene)butanamine (67): EI-MS (70 eV)
m/z (%): 175 (7), 174 (20), 160 (9), 146 (11), 132 (33), 118 (100), 104
(24), 98 (23), 91 (55), 77 (9), 65 (4), 55 (3), 43 (6), 41 (5; Figure 7,
Scheme S7).
2-Methyl-N-(2-phenylethyl)pyrrole (58): Yield: 0.880 g (4.75 mmol,
95%). TLC (cyclohexane/ethyl acetate 30:1) Rf =0.28. 1H NMR
(500 MHz, CDCl3, 298 K): δ=7.33 (ddd, 2H, 3JH,H =7.1 Hz, 8.1 Hz,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
3
4
5JH,H =1.3 Hz), 7.27 (tt, 1H, JH,H =8.1 Hz, JH,H =2.1 Hz), 7.13 (dd, 2H,
General procedure for the synthesis of ethyl 2-acetyl-2-meth-
ylalkanoates (81–83): To a suspension of potassium carbonate
(2.0 equiv) in acetone (0.25 m), ethyl 2-methylacetoacetate
(3.0 equiv) and alkyl bromide (78–80; 1.0 equiv) were added. The
reaction mixture was refluxed overnight and filtered after cooling
to room temperature. The filter was washed with acetone and the
filtrate was dried with MgSO4 and evaporated to dryness. The
residue was subjected to column chromatography (cyclohexane/
ethyl acetate 10:1) to yield the pure ethyl 2-acetyl-2-meth-
ylalkanoates (81–83) as colourless oils.
3JH,H =7.2 Hz, 5JH,H =1.3 Hz), 6.56 (brs, 1H), 6.08 (s, 1H), 5.89 (brs,
3
3
1H), 4.04 (t, 2H, JH,H =7.5 Hz), 3.02 (t, 2H, JH,H =7.5 Hz), 2.14 (s, 3H)
ppm. 13C NMR (126 MHz, CDCl3, 298 K): δ=138.6 (Cq), 128.9 (2xCH),
128.7 (2xCH), 128.4 (Cq), 126.7 (CH), 119.7 (CH), 106.9 (CH), 106.6
(CH), 48.3 (CH2), 38.3 (CH2), 11.9 (CH3) ppm, (Figures S3–S5). HRMS
(EI): m/z: 185.1202 (calc. for [C13H15N]+ 185.1199). EI-MS (70 eV): m/z
(%): 185 (34), 104 (20), 94 (100), 78 (11), 65 (7), 53 (6), 51 (4), 41 (6),
39 (5; Figure S3, Scheme S1). IR (diamond ATR): ν˜=3027 (w), 2929
(w), 2861 (w), 1682 (m), 1603 (w), 1553 (w), 1493 (m), 1453 (m),
1420 (m), 1360 (w), 1295 (m), 1236 (w), 1156 (w), 1140 (w), 1074 (w),
1029 (w), 974 (w), 887 (w), 751 (m), 732 (m), 696 (s), 614 (m), 563
Ethyl 2-acetyl-2-methylnonanoate (81): Yield: 0.377 g (1.65 mmol,
33%). TLC (cyclohexane/ethyl acetate 10:1) Rf =0.33. 1H NMR
(m), 495 (m)cmÀ 1
.
3
(700 MHz, CDCl3, 298 K): δ=4.18 (q, 2H, JH,H =7.1 Hz), 2.13 (s, 3H),
2,5-Dimethyl-N-(2-phenylethyl)pyrrole
(60):
Yield:
1.658 g
1.83–1.90 (m, 1H), 1.69–1.77 (m, 1H), 1.31 (s, 3H), 1.20 À 1.30 (m,
(8.33 mmol, 83%). TLC (cyclohexane/ethyl acetate 30:1) Rf =0.31.
3
3
8H), 1.25 (t, 3H, JH,H =7.1 Hz), 1.11–1.17 (m, 2H), 0.87 (t, 3H, JH,H
=
1H NMR (500 MHz, CDCl3, 298 K): δ=7.33 (ddd, 2H, 3JH,H =7.1 Hz,
7.1 Hz) ppm. 13C NMR (176 MHz, CDCl3, 298 K): δ=206.0 (Cq), 173.3
(Cq), 61.3 (CH2), 59.9 (Cq), 34.9 (CH2), 31.9 (CH2), 30.1 (CH2), 29.2
(CH2), 26.3 (CH3), 24.3 (CH2), 22.7 (CH2), 18.9 (CH3), 14.2 (2xCH3) ppm
(Figures S9–S11). HRMS (EI): m/z: 200.1775 (calculated for the
McLaffety fragment ion [C14H26O3–C2H2O]+ 200.1771, the molecular
ion was not observed). EI-MS (70 eV) m/z (%): 200 (48), 171 (12), 157
(15), 144 (36), 129 (7), 115 (100), 98 (18), 87 (57), 69 (16), 55 (15), 43
(62), 41 (19; Figure S12). IR (diamond ATR): ν˜=2955 (m), 2927 (m),
2857 (w), 1739 (m), 1712 (s), 1463 (w), 1377 (w), 1356 (w), 1298 (w),
1240 (s), 1179 (m), 1144 (s), 1123 (m), 1096 (m), 1024 (m), 970 (w),
5
8.1 Hz, JH,H =1.3 Hz), 7.26–7.30 (m, 1H), 5.80 (brs, 2H), 7.15 (d, 2H,
3JH,H =7.5 Hz), 3.98 (t, 2H, 3JH,H =7.5 Hz), 2.93 (t, 2H, 3JH,H =7.5 Hz),
2.19 (s, 6H) ppm. 13C NMR (126 MHz, CDCl3, 298 K): δ=138.7 (Cq),
129.0 (2xCH), 128.7 (2xCH), 127.5 (2xCq), 126.8 (CH), 105.3 (2xCH),
45.4 (CH2), 37.7 (CH2), 12.5 (2xCH3) ppm.
General procedure for the synthesis of imines:[31] To a stirred
suspension of activated molecular sieves (4 Å, 0.5 g molÀ 1) in diethyl
ether (3 mL mmolÀ 1) the aldehyde (34, 37, 61, 63; 1.0 equiv) and
the amine (33, 66; 1.0 equiv) were added. The mixture was stirred
overnight and the molecular sieves were filtered off and washed
with diethyl ether. The filtrate was evaporated to dryness to yield
the imines in high purity (>95%).
860 (w), 807 (w), 769 (w), 723 (w), 602 (w), 539 (w) cmÀ 1
.
Ethyl 2-acetyl-2-methyldecanoate (82): Yield: 0.464 g (1.81 mmol,
36%). TLC (cyclohexane/ethyl acetate 10:1) Rf =0.36. 1H NMR
3
N-(3-Methylbutylidene)-2-phenylethylamine (62): Yield: 8.14 g
(43 mmol, 86%). 1H NMR (500 MHz, CDCl3, 298 K): δ=7.53 (t, 1H,
3JH,H =5.7 Hz), 7.26–7.33 (m, 2H), 7.16–7.22 (m, 3H), 3.65 (t, 2H,
3JH,H =7.4 Hz), 2.94 (t, 2H, 3JH,H =7.4 Hz), 2.08–2.15 (m, 2H), 1.85
(sept, 1H, 3JH,H =6.6 Hz), 0.90 (d, 6H, 3JH,H =6.6 Hz) ppm. 13C NMR
(126 MHz, CDCl3, 298 K): δ=165.4 (CH), 140.0 (Cq), 129.1 (2xCH),
128.4 (2xCH), 126.1 (CH), 63.0 (CH2), 44.8 (CH2), 37.5 (CH2), 26.4 (CH),
22.6 (2xCH3) ppm (Figures S6–S8). HRMS (EI): m/z: 189.1512 (calc. for
[C13H19N]+ 189.1512). EI-MS (70 eV) m/z (%): 189 (1), 188 (1), 174 (3),
147 (47), 132 (4), 105 (28), 104 (19), 98 (27), 91 (37), 77(24), 65 (15),
56 (60), 42 (100), 39 (13; Figure 7, Scheme S4). IR (diamond ATR):
ν˜=3062 (w), 3027 (w), 2953 (s), 2867 (m), 1653 (m), 1603 (w), 1495
(w), 1454 (m), 1363 (m), 1254 (w), 1166 (m), 1089 (w), 1030 (w),
(500 MHz, CDCl3, 298 K): δ=4.18 (q, 2H, JH,H =7.2 Hz), 2.13 (s, 3H),
1.31 (s, 3H), 1.83–1.90 (m, 1H), 1.69–1.77 (m, 1H), 1.22–1.30 (m,
3
3
10H), 1.25 (t, 3H, JH,H =7.2 Hz), 1.10–1.18 (m, 2H), 0.87 (t, 3H, JH,H
=
7.2 Hz) ppm. 13C NMR (126 MHz, CDCl3, 298 K): δ =206.0 (Cq), 173.3
(Cq), 61.3 (CH2), 59.8 (Cq), 34.9 (CH2), 32.0 (CH2), 30.1 (CH2), 29.5
(CH2), 29.3 (CH2), 26.3 (CH3), 24.3 (CH2), 22.8 (CH2), 18.9 (CH3), 14.2
(2xCH3) ppm (Figures S13–S15). HRMS (EI): m/z: 214.1928 (calculated
for the McLaffety fragment ion [C15H28O3 À C2H2O]+ 214.1927, the
molecular ion was not observed). EI-MS (70 eV) m/z (%): 214 (35),
171 (15), 157 (20), 144 (36), 129 (7), 115 (100), 98 (19), 87 (60), 69
(20), 55 (21), 43 (84), 41 (32; Figure S12). IR (diamond ATR): ν˜=
2924 (s), 2854 (m), 1740 (m), 1712 (s), 1463 (w), 1377 (w), 1356 (w),
1243 (s), 1229 (m), 1175 (m), 1144 (s), 1125 (m), 1099 (m), 1022 (m),
1002 (w), 922 (w), 868 (w), 744 (s), 697 (s), 571 (w), 497 (m) cmÀ 1
.
971 (w), 859 (w), 770 (w), 722 (w), 603 (w), 540 (w) cmÀ 1
.
N-(2-Furylmethylidene)-2-phenylethylamine (64): Yield: 9.27 g
(46.5 mmol, 93%). 1H NMR (500 MHz, C6D6, 298 K): δ=7.71 (brs, 1H),
Ethyl 2-acetyl-2-methylundecanoate (83): Yield: 0.648 g
(2.40 mmol, 48%). TLC (cyclohexane/ethyl acetate 10:1) Rf =0.41.
3
3
5.97–5.99 (m, 1H), 7.12 (dd, 2H, JH,H =7.2 Hz, 7.5 Hz), 7.09 (d, 2H,
1H NMR (500 MHz, CDCl3, 298 K): δ=4.18 (q, 2H, JH,H =7.1 Hz), 2.13
3
4
3JH,H =7.4 Hz), 7.04 (tt, 1H, JH,H =7.2 Hz, JH,H =1.6 Hz), 7.00 (brs, 1H),
(s, 3H), 1.83–1.90 (m, 1H), 1.69–1.77 (m, 1H), 1.31 (s, 3H), 1.22–1.30
3
3
4
3
6.59 (d, 1H, JH,H =3.3 Hz), 3.61 (td, 2H, JH,H =7.3 Hz, JH,H =1.2 Hz),
(m, 12H), 1.26 (t, 3H, JH,H =7.2 Hz), 1.11–1.19 (m, 2H), 0.87 (t, 3H,
2.90 (t, 2H, JH,H =7.3 Hz) ppm. 13C NMR (126 MHz, C6D6, 298 K): δ=
3JH,H =7.0 Hz) ppm. 13C NMR (176 MHz, CDCl3, 298 K): δ=206.0 (Cq),
173.3 (Cq), 61.3 (CH2), 59.9 (Cq), 34.9 (CH2), 32.0 (CH2), 30.1 (CH2), 29.6
(CH2), 29.5 (CH2), 29.4 (CH2), 26.3 (CH3), 24.3 (CH2), 22.8 (CH2), 18.9
3
153.0 (Cq), 150.0 (CH), 144.2 (CH), 140.4 (Cq), 129.4 (2xCH), 128.6
ChemBioChem 2020, 21, 1–11
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(CH3), 14.2 (2xCH3) ppm (Figures S16–S18). HRMS (EI): m/z: 228.2083
(calculated for the McLaffety fragment ion [C16H30O3 À C2H2O]+
228.2084, the molecular ion was not observed). EI-MS (70 eV) m/z
(%): 228 (93), 199 (3), 171 (36), 157 (14), 144 (47), 129 (8), 115 (100),
98 (16), 87 (48), 69 (17), 55 (18), 43 (93), 41 (30; Figure S12). IR
(diamond ATR): ν˜=2925 (s), 2855 (w), 1740 (m), 1713 (s), 1621 (w),
1463 (w), 1377 (w), 1356 (w), 1234 (s), 1176 (m), 1144 (s), 1124 (m),
1099 (s), 1022 (m), 971 (w), 859 (w), 808 (w), 768 (w), 723 (w), 603
Acknowledgements
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We thank Neran M. Reuber for assistance in the synthesis of
reference compounds. Open access funding enabled and organ-
ized by Projekt DEAL.
(w), 539 (w) cmÀ 1
.
Conflict of Interest
General procedure for the synthesis of 3-methylalkan-2-ones
(84–86): To a solution of the ethyl 2-acetyl-2-methylalkanoates (81–
83; 1.0 equiv) in ethanol (0.2 m) was added potassium hydroxide
solution (3 m, 2.0 equiv) and the mixture was refluxed for 3 h. After
cooling to room temperature HCl (3 m) was added and the mixture
was diluted with ethyl acetate The separated organic phase was
washed with water, dried with MgSO4 and evaporated to dryness.
The residue was purified by column chromatography (cyclohexane/
ethyl acetate 20:1) to give the 3-methylalkan-2-ones (84–86) as
colourless oils.
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The authors declare no conflict of interest.
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Keywords: fragmentation mechanisms
products · structure elucidation · volatile organic compounds
· GC-MS · natural
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298 K): δ=2.49 (ddq, 1H, JH,H =6.9 Hz, 6.9 Hz, 6.9 Hz), 2.12 (s, 3H),
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298 K): δ=2.49 (ddq, 1H, JH,H =6.9 Hz, 6.9 Hz, 6.9 Hz), 2.12 (s, 3H),
1.58–1.67 (m, 1H), 1.20–1.37 (m, 13H), 1.06 (d, 3H, 3JH,H =7.0 Hz),
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72 (100), 57 (19), 55 (7), 43 (52), 41 (12; Figure 9). IR (diamond ATR):
ν˜=2958 (m), 2924 (s), 2855 (m), 1712 (s), 1461 (m), 1356 (m), 1232
(w), 1166 (w), 1137 (w), 1106 (w), 952 (w), 804 (w), 721 (w), 602 (w),
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0.87 (t, 3H, 3JH,H =6.9 Hz) ppm. 13C NMR (176 MHz, CDCl3, 298 K): δ=
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Manuscript received: July 22, 2020
Revised manuscript received: August 11, 2020
Accepted manuscript online: August 12, 2020
Version of record online: ■■■, ■■■■
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Over 70 volatile organic compounds
have been identified in a headspace
extract from C. polytrichastri. The
extract was particularly rich in
nitrogen-containing natural
products, many of which were struc-
turally derived from 2-phenylethyl-
amine. Several compounds are new
natural products, their suggested
structures are based on the observed
mass spectral fragmentation,
L. Lauterbach, Prof. Dr. J. S. Dickschat*
1 – 11
Volatiles from the Psychrotolerant
Bacterium Chryseobacterium polytri-
chastri
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confirmed by synthesis of reference
substances.