S. S. Shinde et al. / Tetrahedron Letters 57 (2016) 1161–1164
1163
2 2 2
Figure 2. (a) Marker and purified epicedrol synthase (lane 1). (b) GC–MS analysis (TIC) of epicedrol synthase assay of FPP and H O. (c) MS of product from FPP and H O
18O/H
1
8
assay. (d) MS of product from FPP and H
2
O assay.
+
and cedrol gave detectable molecular ion (M ) at m/z 222 along
with other peaks including m/z 204 formed from loss of water
molecule, respectively (Fig. 2). Notably, when epicedrol synthase
Table 1
Mass spectrometric analysis of molecular ion peaks of Epicedrol
EI mass (m/z)
222
96.58
80.13
224
3.42
19.86
Peak% (H1
6
1
was incubated with FPP in Tris–HCl buffer, pH 8.5, containing
MgCl in H O (21.6 at %), the product ratios were slightly chan-
2 2
2
O)
O)
8
18
Peak% (H
2
ged (Table 1). Interestingly, the enrichment of molecular ion at
m/z 224 for both epicedrol and cedrol was observed. This shift in
along with small traces cedrene and cedrol (Supporting informa-
tion, SI) which is in line with the earlier observation. This divalent
metal dependent sesquiterpene synthase could initiate the reac-
tion by the ionization of FPP to delocalized farnesyl/nerolidol car-
bocation followed by series of electrophilic cyclization including
a hydride shift from C-6 to C-7 to the generation of bisabolyl cation
1
8
the molecular ion clearly indicates the insertion of O to epicedrol
and cedrol molecules. Furthermore, there was no shift observed for
the peak at m/z 204 due to the loss of water molecule (Fig. 2d).
These results demonstrate that epicedrol and cedrol are formed
through the quenching of cedryl carbocation 3 by water molecule
present in the active site either from Si- or Re-face, respectively.
However, Si-face seems to be more favorable as the epicedrol is
(Scheme 1). Then si-face attack of the C-10 olefin initiated two sub-
sequent cyclizations to form exo-cyclic acoradane skeleton and
tricyclic cedrane skeleton by C-10 to C-6 and C-2 to C-11 ring
closure respectively. Generated tricyclic intermediate carbocation
1
8
the major enzymatic product. Furthermore, insertion of
derived from H
O
1
8
2
O implies that both epicedrol and cedrol might
not have formed through recapture of pyrophosphate anion pre-
sent in the active site followed by the hydrolysis of corresponding
diphosphate. The formation of corresponding sesquiterpene hydro-
carbons a-cedrene and b-cedrene might be formed through depro-
tonation at 4-H and 15-H of cedryl carbocation (3), respectively.
(
3, Scheme 1) may be quenched by following three pathways: (a)
deprotonation of either 15-H or 4-H which produces two
sesquiterpene hydrocarbons -cedrene and b-cedrene, respectively
or (b) stereo selective addition of water to form cedrol/epicedrol, or
c) capture pyrophosphate anion present in the active site followed
a
(
by hydrolysis to generate cedrol/epicedrol.
To address the ambiguous source of the oxygen atom in epice-
drol biosynthesis, epicedrol synthase was incubated with FPP in
both isotopically labeled and unlabeled conditions, then the
obtained extracted products were characterized by GC and GC-
MS analysis (SI). GC and GC-MS analysis of the control assay
extract obtained by incubating FPP with epicedrol synthase in nor-
mal buffer system (ESI) indicated the formation of epicedrol as a
Conclusions
In conclusion, we have demonstrated that the oxygen atom of
the hydroxyl functionality of epicedrol is derived from a water
molecule, not from the oxygen of diphosphate anion of farnesyl
diphosphate. The attack of water to obtain the isotopically labeled
epicedrol is preferred due to the steric blocking effects of the prox-
imal geminal dimethyl groups in cedryl carbocation. The detection
of traces of isotopically labeled cedrol implies that attack on the
unbound carbocation is at least partially responsible for the
observed product mixture.
major product along with traces of cedrol (1.2%),
a-cedrene
(
2.1%), and b-cedrene (3.2%) (SI, Table S1). The formation of these
compounds was confirmed by comparing retention time and GC
co-injection studies. EI MS of sesquiterpene alcohols, epicedrol,