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(
Scheme S1). The elongations were performed using the
backside of the projection plane (Scheme 1) and is thus
perfectly oriented to take up the proton in the final step.
A BLAST search using the SvS sequence as a probe was
performed to identify closely related enzymes in other
bacteria. A phylogenetic tree constructed from SvS and the
65 closest hits is shown in Figure S17. Organisms that were
available from the DSMZ strain collection and that encode
the most closely related enzymes to the SvS from S. violens
were investigated for their production of 1, which could be
shown for the streptomycetes S. ochraceiscleroticus, S. sclero-
tialus, and S. ambofaciens as well as the actinomycete
Allokutzneria albata (Figure S18). Closely related enzymes
include the cyclases for (+)-(1(10)E,4E,6S,7R)-germacra-
GGPP synthase (GGPPS) from Streptomyces cyaneofuscatus.
Only (2- C)GGPP and (20- C)GGPP were directly synthe-
sized (Scheme S2). Small-scale reactions (1 mg) of the 20
isotopomers of ( C )GGPP with SvS followed by extraction
with C D6 and direct C NMR analysis without product
purification showed the C label in the expected positions in
all cases (Figure S11). For the geminal methyl groups C16 and
C17 of GGPP SvS showed a strictly controlled stereochemical
course (Figures S11R + S), as observed for most terpene
1
3
13
1
3
1
1
3
6
1
3
[5d,e,14]
cyclases.
14)-diene synthase from Fusarium fujikuroi, which
reveals a distribution of the label between C12 and C13 of
One of the few exceptions is (À)-guaia-6,10-
(
[
15]
[13]
FPP.
dien-6-ol (8, Scheme 2), tsukubadiene (9), and cyclooctat-
Stereoselectively deuterated OPPs can be used for the
determination of the absolute configurations of terpenes and
of the intermediate a-terpinyl cation en route to the achiral
[
5f,13d,16,17]
monoterpene 1,8-cineol.
The enzymatic conversion
of these probes results in products with the deuterium label in
diastereotopic positions, which can be distinguished by NMR
spectroscopy. To increase the sensitivity of the probes in
1
3
2
HSQC spectroscopy both enantiomers of (1- C,1- H)GPP
1
3
2
and (1- C,1- H)FPP were synthesized with an additional
Scheme 2. Terpenes produced by enzymes with a close relationship to
SvS (8–10) and structure of cyclooctat-9-en-7-ol (11).
1
3
C label (Scheme S3) and with high enantiomeric purity
Figure S12). Incubation of SvS and GGPPS with (R)- and
(
(
1
3
2
S)-(1- C,1- H)FPP and IPP followed by product analysis by
[
6b]
HSQC and comparison to the unlabeled compound allowed
assignment of one of the two stereochemically different
7(8),10(14)-diene (10). Although their enzymes are phylo-
genetically distant, the structural similarity of the products 10
and cyclooctat-9-en-7-ol (11) suggests that the cyclization
mechanism for 10 may be similar to the mechanism for 11,
which proceeds through a surprising rearrangement of a cyclo-
1
3
hydrogen atoms at the C-labeled carbon atom to the site of
deuterium incorporation and the other one to the site of
protium incorporation, with a known absolute configuration
of the stereocenter at this carbon atom (Figure S13). The
absolute configuration of the other stereocenters in 1 was
deduced by assignment of their relative configuration with
respect to the stereocenter at the labeled carbon atom, thus
resulting in the structure of (3R,6R,10S,11R,14R)-1. Consis-
tent findings were obtained using the same strategy for the
conversion of (R)- and (S)-(1- C,1- H)GPP (Figure S14).
These experiments make use of the known inversion of
configuration in chain elongations of OPPs with IPP by OPP
synthases.
The 1,3-hydride shift from 6 to 7 was investigated by
conversion of (3- C,2- H)GGPP, which was prepared by
using a reported method for deuterium introduction at C2
Scheme S4).
resulted in a direct bond between deuterium and the C-
labeled carbon atom in 1, as indicated by a triplet in the
C NMR spectrum of the obtained product as a result of C-
H spin coupling (Figure S15). The stereochemical course of
the final deprotonation step was addressed using (R)- and (S)-
1- H)GGPP (Scheme S3). GC/MS analysis of the enzyme
products revealed the specific loss of deuterium from (S)-(1-
[
6a]
propane intermediate.
This prompted us to investigate
whether 9 is also formed by a related mechanism.
For this purpose, the gene for the tsukubadiene synthase
(TdS) from Streptomyces tsukubaensis NRRL 18488 was
cloned and expressed in E. coli (Figure S2). Consistent with
the reported findings by heterologous expression in Strepto-
1
3
2
[6b]
myces avermitilis,
the purified protein converted GGPP
into a single product (Figure S19), while GPP and FPP were
not accepted. Since only the planar structure of 9 had been
reported, the diterpene product was isolated and its structure
fully elucidated by extensive NMR spectroscopy (Table S4,
Figures S20–S26). The relative configuration determined by
NOESY experiments, as shown in Scheme 2, was in line with
the suggested structure based on a comparison of the
recorded and calculated NMR data for all stereoisomers of
[18]
1
3
2
[
19]
(
With this probe, the hydride migration
1
3
[
21]
9.
1
3
13
The proposed cyclization mechanism of TdS starts with
2
the cyclization of GGPP to 12 (Scheme 3). The further
reactions, including a ring expansion and ring contraction by
WMR, a 1,2-hydride migration, as well as a 7,9- and a 2,6-
cyclization to form 13, may be a concerted process with
retention of configuration at C14 to avoid secondary cation
intermediates. Two subsequent 1,2-hydride migrations via 14
to generate 15 may be followed by a rearrangement of the
cyclopropane ring, similar to a key step in the biosynthesis of
2
(
2
H)GGPP but not from the R enantiomer (Figure S16).
These experiments demonstrate that the proton syn to the
migrating hydride in the reaction from 6 to 7 is abstracted, if
inversion of configuration at C1 is assumed for the intitial
[
6a]
1
ses.
,11-cyclization, as described for several terpene cycla-
11. A ring opening to produce 17, 1,2-hydride migration to
form 18, and deprotonation yield 9. This suggested mecha-
nism was tested by conversion of all 20 isotopomers of
[
13b,20]
A candidate base for the deprotonation of 7 is the
diphosphate anion that leaves C1 of GGPP towards the
2
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Angew. Chem. Int. Ed. 2017, 56, 1 – 5
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