Nitrogen-Containing Volatiles from Marine Salinispora pacifica and Roseobacter-Group Bacteria

Article abstract of DOI:10.1021/acs.jnatprod.7b00789

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 pacifica 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. pacifica 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.

Full text of DOI:10.1021/acs.jnatprod.7b00789

Article
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Nitrogen-Containing Volatiles from Marine Salinispora pacifica and
Roseobacter-Group Bacteria
Tim Harig,† Christian Schlawis,† Lisa Ziesche,† Marion Pohlner,‡ Bert Engelen,‡ and Stefan Schulz*,†
†Institute of Organic Chemistry, Technische Universitat Braunschweig, Hagenring 30, 38106 Braunschweig, Germany
̈
‡Institute for Chemistry and Biology of the Marine Environment (ICBM), Carl von Ossietzky Universitat 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 pacifica
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. pacifica 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.
volatiles, the obligate marine actinomycete genus Salinispora23
olatile compounds are released by organisms in any
and members of the Roseobacter group24,25 that were also
Vhabitat on earth. Such volatiles may be metabolites serving
investigated by Dickschat et al.26,27 On first sight, the marine
a certain function or are waste products of the emitter’s
metabolism. With this odor bouquet information is transported
environment does not seem to be suited for mostly
that potentially can be used by other organisms, revealing
hydrophobic apolar volatile compounds because of the
information on the status of the emitter. The structures of
volatiles from important producers such as plants1−3 or
hydrophilic water phase. Nevertheless, apolar volatile com-
animals4 have been investigated for a long time, as has the
pounds are used as signals by many marine organisms, e.g.,
function of such compounds. In contrast, much less is known
on the structures of volatiles released by bacteria5−8 and their
functions.9−11
brown algae that produce apolar hydrocarbons as phero-
mones28,29 or other algae.30
During our investigation of the volatiles released by
Salinispora pacifica CNS863 several unknown nitrogen-
containing 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 sub-
microgram quantities by the bacteria.
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 com-
pounds,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
Received: September 15, 2017
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Chart 1
RESULTS AND DISCUSSION
■
Figure 1. Total ion chromatograms of Salinispora pacifica CNS 863
(A), Roseovarius pelophilus G5II (B), and Pseudoruegeria sp. SK021
(C).
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-isobutylmethane-
sulfinamide (5). Similarly, ions m/z 43, 64, 71, 92, and 134 led
to the identification of compound J as N-isopentylmethane-
sulfinamide (6), which coeluted with another unknown
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 Addition-
ally, L was identified as an isomer of 12, (E)-N-(2-
methylbutyl)-1-phenylmethanimine (11). The mass spectra of
imines are characterized by a very intense peak derived from α-
cleavage 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 2-
methylbutyl 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
B
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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) N-
isopentylmethanesulfinamide (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
Scheme 1. Synthesis of Nitrogen-Containing Volatiles
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′-
C
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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 pacifica CNS863, Roseovarius
pelophilus G5II, Pseudoruegeria sp. SK021, and Phaeobacter gallaeciensis
BS107. S. pacifica 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. pacifica 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.
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. pacifica
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-methyldecy-
lurea 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-2-
methylpropan-1-imine (18), known from the essential oil of
Perilla frutescens,41 and the amides N-isopentyl-4-methylpenta-
namide (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.
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.
The nitrogen-containing 1−20 volatiles seem to originate
from biogenic amines isobutyl-, isopentyl-, and 2-methylbutyl-
amine, 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.
EXPERIMENTAL SECTION
■
General Experimental Procedures. One- and two-dimensonal
1H NMR and 13C NMR analyses were performed using the following
instruments: Bruker AV-II 300 (1H 300 MHz, 13C 75.5 MHz), AV III-
400 (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
I = 1143; IR (solid) νmax 3184, 2957, 2926, 2871, 1733, 1712, 1523,
1
1470, 1415, 1386, 1367, 1301, 1252, 1176, 1052, 979, 949 cm−1; H
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,
1
1367, 1302, 1135, 1059, 991, 962, 881 cm−1; H NMR (CDCl3, 300
D
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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.
= 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;
1H 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).
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,
1
1641, 1560, 1467, 1368, 1260, 1220, 1161 cm−1; H 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.
(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;
1H 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).
N,N′-Diisobutylurea (15). 15 was synthesized from S,S′-dimethyl-
dithiocarbonate (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,
1
1434, 1387, 1368, 1336, 1272, 1164, 1127, 1057, 920, 818 cm−1; H
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
(E)-N-(2-Methylbutyl)-1-phenylmethanimine (11). 11 was synthe-
sized 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,
1
1034, 1005, 974, 919, 849, 768, 754 cm−1; H 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),
E
DOI: 10.1021/acs.jnatprod.7b00789
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
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).
ORCID
Stefan Schulz: 0000-0002-4810-324X
Notes
The authors declare no competing financial interest.
(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).
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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ASSOCIATED CONTENT
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* Supporting Information
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ChemBioChem 2005, 6, 2023−2033.
The Supporting Information is available free of charge on the
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Dobler, I.; Brinkhoff, T.; Schulz, S. ChemBioChem 2015, 16, 2094−
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Corresponding Author
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*E-mail: stefan.schulz@tu-bs.de. Phone: +495313917353. Fax:
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