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to the opposite enantiomer as in the plant ([a]D20 = À14.8),
establishing the terpene from Streptomyces pratensis as
(+)-(1(10)E,4E,6S,7R)-germacradien-6-ol. The absolute con-
figuration of the plant metabolite 1 was recently confirmed by
total synthesis.[9] Unlike the other terpene synthase products,
1 is not found in S. pratensis laboratory cultures (not shown).
The complex NMR spectra prevented a full assignment of
1
the H and 13C NMR signals to 1a and 1b, as in a previous
report.[8a] To overcome this problem, all fifteen (13C1)FPP
isotopomers were synthesized (Figures S3–6)[10] and con-
verted with germacradienol synthase. Each product was
extracted with (2H8)toluene and analyzed by 13C NMR
spectroscopy, resulting in two strong signals for the labeled
carbons of conformers 1a and 1b (Figure S7). These data
unambiguously established which 13C NMR signals of
1 belonged to which carbon center, but it was not possible
to assign which of the two 13C NMR signals observed in each
single experiment belonged to which conformer. Therefore,
completely labeled (13C15)FPP was synthesized and incubated
with germacradienol synthase and the product was analyzed
by 13C, 13C COSY NMR experiments.[11] This experiment
revealed two distinct sets of cross-peaks (Figure 2; for an
enlarged version see Figure S8) that allowed for an unambig-
uous assignment of all 30 carbon signals to each of the 15
carbon atoms of 1a and 1b (Table 1). The assignment of most
Figure 2. 13C,13C COSY spectrum of (13C15)-1 obtained by enzymatic
conversion of (13C15)FPP. The two sets of cross-peaks for the con-
formers are shown in yellow (for 1a) and red (for 1b).
identical to 2a and 2b in terms of their mass spectra and GC
retention times. Their structures were determined by one- and
two-dimensional NMR spectroscopy (Table 1), resulting in
their identification as shyobunol (2a) and 5,10-di-epi-shyo-
bunol (2b).[12] The relative configurations were determined
from key NOESY correlations (Figure 4A). The assignment
of NMR data was confirmed by Cope rearrangement of
(13C15)-1, obtained by enzymatic conversion of (13C15)FPP, and
subsequent analysis of the product by 13C, 13C COSY NMR
experiments (Figure S10). From these experiments, two
distinct sets of cross-peaks were detected for 2a and 2b that
gave direct insights into the carbon–carbon connectivities.
The absolute configurations of 2a and 2b can be deduced
from the stereocenters at C-6 and C-7 of 1 that are not
affected by the Cope rearrangement, as is known for various
1
1H NMR resonance signals was possible from H,1H COSY,
HSQC, and HMBC correlations (Figure 3) of the unlabeled
compound. For a few cases, the HSQC spectra of the relevant
(13C1)-1 isotopomers were very useful (Figure S9).
For structure elucidation of the two Cope rearrangement
products observed during GC–MS analysis, 1 was subjected to
a microwave reaction in toluene at 2258C. The products were
separable by column chromatography and proved to be
Table 1: NMR data of the conformers 1a and 1b of (1(10)E,4E,6S,7R)-germacradien-6-ol in (2H8)toluene recorded at À508C, and of 2a/2b in
(2H6)benzene at 258C.[a]
1+2
1a (DD)
1b (UD)
2a
2b
C[a]
1H
13C
1H
13C
1H
13C
1H
13C
1
2
CH 4.80 (d, J=11.6, 1H) 129.0 4.87 (t, J=7.5, 1H) 121.5 5.78 (dd, J=17.5, 10.8, 1H) 150.3 5.85 (dd, J=17.6, 10.8, 1H) 150.2
CH2 2.17 (m, 1H)
1.95 (m, 1H)
CH2 2.04 (m, 1H)
2.00 (m, 1H)
24.8 2.24 (m, 1H)
1.89 (m, 1H)
39.2 2.09 (m, 1H)
1.94 (m, 1H)
24.6 4.98 (dd, J=17.5, 1.2, 1H, E) 110.1 4.90 (dd, J=17.6, 0.8, 1H, E) 110.4
4.94 (dd, J=10.8, 1.2, 1H, Z) 4.86 (dd, J=10.8, 0.8, 1H, Z)
37.5 5.02 (br s, 1H, Z) 113.3 4.90 (br s, 1H)
3
113.9
4.91 (br s, 1H, E)
–
4.70 (br s, 1H)
–
4
5
6
7
8
Cq
–
131.8
–
132.0
146.8
145.5
57.4
71.9
44.6
22.3
CH 5.06 (d, J=7.0, 1H) 135.1 5.04 (d, J=8.5, 1H) 133.0 1.69 (d, J=1.7, 1H)
CH 4.54 (d, J=6.0, 1H) 68.5 4.55 (d, J=6.0, 1H) 68.4 3.81 (br s, 1H)
CH 0.75 (d, J=9.0, 1H) 49.7 0.66 (d, J=9.5, 1H) 52.5 0.74 (m, 1H)
56.6 2.32 (d, J=6.8, 1H)
70.2 3.94 (m, 1H)
49.8 1.54 (m, 1H)
21.1 1.56 (m, 2H)
CH2 1.95 (m, 2H)
1.39 (d, J=13.8, 1H)
30.7 1.80 (m, 1H)
1.30 (m, 1H)
25.6 1.63 (m, 1H)
1.49 (m, 1H)
9
CH2 2.44 (d, J=13.1, 1H) 36.1 2.14 (m, 1H)
41.8 1.48 (m, 1H)
1.29 (m, 1H)
41.1 1.53 (m, 1H)
1.25 (m, 1H)
33.7
1.62 (t, J=13.5, 1H)
–
1.79 (m, 1H)
–
10 Cq
11 CH 1.78 (m, 1H)
12 CH3 1.00 (d, J=6.0, 3H) 21.4 1.09 (d, J=6.3, 3H) 21.2 0.92 (d, J=6.8, 3H)
13 CH3 1.05 (d, J=6.5, 3H) 21.6 1.00 (d, J=6.0, 3H) 21.3 0.94 (d, J=6.9, 3H)
14 CH3 1.55 (s, 3H)
15 CH3 1.32 (s, 3H)
135.3
138.5
–
40.2
–
30.3
27.4
21.9
23.1
23.1
26.8
32.0 1.73 (m, 1H)
32.6 1.68 (m, 1H)
29.4 1.88 (oct, J=6.8, 1H)
20.8 0.94 (d, J=6.8, 3H)
21.3 1.20 (d, J=6.6, 3H)
20.3 0.93 (s, 3H)
22.0 1.48 (s, 3H)
16.3 1.30 (s, 3H)
16.9 1.46 (s, 3H)
16.2 1.72 (s, 3H)
27.8 1.68 (s, 3H)
[a] Carbon numbering as shown in Figure 1. Chemical shifts d in ppm, multiplicity m (s=singlet, d=doublet, t=triplet, oct=octet, m=multiplet,
br=broad), coupling constants J are given in Hertz. Carbon assignments for 1 were deduced from incubation experiments with 13C-labeled FPP
isotopomers (see the main text).
Angew. Chem. Int. Ed. 2015, 54, 13448 –13451
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim