DOI: 10.1039/C4CC10395E
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ene and
the two
-terpineol reflect the absence of destabilizing effects in
-terpinyl carbocations. From C1 and C2, the cyclization
cascade proceeded to carbocation D which was subsequently
rearranged to the tertiary carbocations E1 and E2. Because the
positive charge fully surrounded by deuterium atoms is less effi-
ciently stabilized as by C-H hyperconjugation, strong deuterium
isotope effects on the formation of sabinene, sabinene hydrate
and -thujene were observed. Obviously, E1 and E2 preferentially
stabilize by reacting with water as a nucleophile rather than los-
ing a positively charged hydrogen isotope.
Conclusions
Multiproduct terpene synthases TPS4 and TPS5 from Zea mays
show isotopically sensitive branching in the reaction cascade of
prenyl diphosphates en route to mono- and sesquiterpene volat-
iles via a common carbocationic intermediate along a branched
reaction sequence. The primary kinetic isotope effects of deuter-
ium atoms on terminating deprotonations and effects resulting
from lower stabilization of the reactive intermediates by hyper-
conjugation direct the reaction to an enhanced formation of alco-
hols instead of olefinic products. Accordingly, the extensive
deuterium labeling of intermediary cations is a valuable tool to
identify branching points in complex cyclization sequences of
multiproduct terpenoid synthases.
Figure 4. Proposed reaction mechanism for the formation of
monoterpenes by TPS4 and TPS5 from (E)-[2H6]-GDP [7b].
From [2H6]-FDP 5d, after the initial ionization-isomerization-
ionization sequence, the cyclization cascade is initiated by the
formation of (S)- and (R)-bisabolyl cations (A and B). These first
carbocations can be directly deprotonated to produce (S)--
bisabolene without noticeable KIEs (the positive charge being
located far from the deuterated center). Tertiary carbocations A1
and B1 are almost as stable as A and B because the positive
charge is distant from the deuterium labeled carbon center and,
hence, can be stabilized by C-H hyperconjugation. Secondary
carbocations A3 and B3, which are energetically less favorable
than tertiary ones (e.g. A4 and B4), are relatively stable due to
surrounding hydrogen atoms. Carbocations A4, A5, B4 and B5 are
the least stable ones, since the positive charge is fully surrounded
by deuterium atoms and hence, cannot be delocalized and stabi-
lized by C-D hyperconjugation. The stability of terminal carbo-
cations results in the production of the corresponding cyclic
terpene. Taken together, these observations are consistent with
the product distribution obtained by isotopically sensitive bran-
ching experiments. Deuterium isotope effects are less pronounced
in the monodeuterated analogues (Table 1). Nevertheless, strong
KIEs for the formation of sesquithujene, 7-epi-sesquithujene or
sesquisabinenes A and B were observed after incubation of both
enzymes with hexadeuterated substrates (Fig. 4). In case of the
bisabolyl carbocations A and B, (Fig. 3), minor KIEs were ob-
Notes and references
a
Max Planck Institute for Chemical Ecology, Hans-Knöll-Strasse 8, D-
b
Institute for Pharmacy, University of Halle, Hoher Weg 8, D-06120
Halle, Germany
Electronic Supplementary Information (ESI) available:
See DOI: 10.1039/c000000x/
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occur spontaneously and are not affected by kinetic isotope ef-
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substrates. These minor KIEs observed for limonene, -terpinol-
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