Angewandte
Communications
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cated that metabolite 1 has two ring systems. Although
considerable overlap was observed between the proton
1
signals in the upfield region of the H NMR spectrum of
metabolite 1 (d 1.23–1.99 ppm), extensive analysis of its 2D
H
NMR data, including COSY, TOCSYand HMQC-conjugated
TOCSY spectra successfully revealed the presence of two
spin systems, the first of which was H -1/H -2/H -3/H -4
2
2
2
2
(Figure 2). The downfield shift in the
chemical shift values of CH -1 (d
2
H
3
.06, 3.13 ppm; dC 41.2 ppm) sug-
gested that C-1 was attached to
a nitrogen atom. HMBC analysis
revealed correlations from H -1 and
2
H -4 to C-5, which suggested the
2
presence of a piperidine ring. We
subsequently focused on the second
spin system, H -6/H-7/H -8/H-9,
where C-7 and C-9 were determined
to be oxymethine carbons. HMBC
Figure 2. COSY and key
HMBC correlations of
the spirohemiaminal
moiety of streptoami-
2
2
analysis revealed correlations from nal-9n (1).
Figure 1. Chemical structures of streptoaminals and 5aTHQ-9i.
a) Structures of streptoaminals-9n (1), À9i (2), and À8n (3). b) Struc-
ture of 5aTHQ-9i. c) A molecular signature in the mass spectrum and
an LC-MS chromatogram of streptoaminals. Left: The mass spectrum
of a fraction containing streptoaminals showed a cluster of ion peaks
with 14 mass unit intervals. Right: LC-MS data. The total ion
H-7 and H -6 to C-5, as well as
2
a correlation from H -6 to C-4, which
2
suggested that C-4 and C-6 were
connected through C-5. The chemical shift of the quaternary
carbon C-5 (dC 86.9 ppm) implied the presence of a hemi-
aminal functionality, suggesting that C-9 was connected to C-5
though an oxygen atom to form 1-aza-9-oxaspiro-
chromatogram (TIC) and mass chromatograms of streptoaminals (m/
z 270, 284, 298, 312, and 326) are shown. The m/z values of
metabolites (see arrows): 1 298, 2 298, 3 284. A fraction containing
streptoaminals 1–3 was analyzed by LC-MS on a COSMOSIL-5C -ARII
[
5,5]undecan-7-ol. The remaining portion to be assigned was
1
8
C H , which was deduced to be a nonyl group consisting of
9
19
column by gradient elution with 70–100% MeOH and 0.1% formic
acid.
eight methylenes and one methyl group. COSY analysis
revealed that this alkyl chain was connected to C-9. Taken
together, these data suggested that metabolite 1 existed as the
planar structure shown in Figure 1.
by LC-MS analysis. In this way, we identified a mixture of
metabolites with mass ions in the range 270 to 326, which were
separated by 14 mass unit intervals, suggesting that these
metabolites contained an odd number of nitrogen atoms and
had alkyl chains of different lengths (Supporting Information,
Figure S1). The elution pattern of these congeners by LC-MS
analysis was similar to that of the 5aTHQ congeners, which
suggested that these new metabolites were chemically related
to 5aTHQs (Figures 1; Supporting Information, Figure S1). It
is noteworthy that these metabolites were produced in much
larger quantities in the combined-culture system than they
were in the presence of S. nigrescens alone (Supporting
Information, Figure S1). Fractions containing streptoaminals
were combined and further purified by column chromatog-
raphy over silica gel, ODS and amino silica gel to afford
a mixture of the metabolites containing different alkyl chains.
Despite the broad and overlapping peak shapes obtained over
the preparative HPLC columns, we successfully purified three
congeners (1–3) by including TFA in the mobile phase.
The molecular formula of metabolite 1 was determined to
be C H NO by HR-ESI-MS (m/z 298.2731, calcd for
The spirohemiaminal structure of metabolite 1 was shared
by metabolites 2 and 3, as evidenced by the similarities in
their NMR spectra (Supporting Information, Table S1).
Indeed, the only significant difference between the structures
of metabolites 1–3 was the nature of the alkyl chain at their C-
[10f]
9 position. In a similar manner to 5aTHQs,
metabolite
1 was designated as streptoaminal-9n, based on the molecular
formula of its alkyl chain being C H with a normal-type
9
19
terminal structure. Metabolite 2 had a C-9 alkyl chain with an
iso-type branched methyl group, and was therefore desig-
nated as streptoaminal-9i. Metabolite 3 was designated as
streptoaminal-8n because it had a normal-type octyl chain at
its C-9 position. Although we were only able to purify three
congeners in this study, LC-MS analysis of the crude extract
suggested that there were several other streptoaminals with
various alkyl chains, indicating the existence of a biosynthetic
relationship to 5aTHQs (Supporting Information, Figure S1).
The relative stereochemistry of streptoaminal-9n (1) was
deduced by the careful analysis of its NMR spectra and its
chemical conversion to the corresponding isopropylidene
derivative 4. The conformation of the tetrahydropyran ring
1
8
34
2
+
1
13
2
98.2741, C H NO [M + H] ). The H, C, DEPT, and
18 35 2
3
HMQC NMR data for metabolite 1 revealed the presence of
one CH , fourteen CH , and two CH (d 63.9 and 67.8 ppm)
was initially analyzed by NOESY correlations and JH/H
3
values (Figure 3a). A large coupling constant ( J
=
3
2
C
H-8a/H-9
groups, as well as one quaternary carbon (d 86.9 ppm), all of
which were determined to be sp -hybridized (Supporting
Information, Table S1). Two degrees of unsaturation indi-
11.8 Hz) revealed that H-8a and H-9 were configured with
an axial orientation. In contrast, the small coupling constants
between H-7 and all of the vicinal protons (2.2–5.9 Hz)
C
3
2
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Angew. Chem. Int. Ed. 2016, 55, 1 – 6
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