Synthesis of deuterated isopentyl pyrophosphates for chemo-enzymatic labelling methods: GC-EI-MS based 1,2-hydride shift in epicedrol biosynthesis

Article abstract of DOI:10.1039/c9ra00163h

A sesquiterpene epicedrol cyclase mechanism was elucidated based on the gas chromatography coupled to electron impact mass spectrometry fragmentation data of deuterated (²H) epicedrol analogues. The chemo-enzymatic method was applied for the specific synthesis of 8-position labelled farnesyl pyrophosphate and epicedrol. EI-MS fragmentation ions compared with non-labelled and isotopic mass shift fragments suggest that the ²H of C6 migrates to the C7 position during the cyclization mechanism.

Full text of DOI:10.1039/c9ra00163h

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Synthesis of deuterated isopentyl pyrophosphates
for chemo-enzymatic labelling methods: GC-EI-MS
based 1,2-hydride shift in epicedrol biosynthesis†
Cite this: RSC Adv., 2019, 9, 28258
Madhukar S. Said,ab Govinda R. Navale,ab Jayant M. Gajbhiyeab
a
*
and Sandip S. Shinde
A sesquiterpene epicedrol cyclase mechanism was elucidated based on the gas chromatography coupled
to electron impact mass spectrometry fragmentation data of deuterated (2H) epicedrol analogues. The
chemo-enzymatic method was applied for the specific synthesis of 8-position labelled farnesyl
pyrophosphate and epicedrol. EI-MS fragmentation ions compared with non-labelled and isotopic mass
shift fragments suggest that the 2H of C6 migrates to the C7 position during the cyclization mechanism.
Received 8th January 2019
Accepted 19th August 2019
DOI: 10.1039/c9ra00163h
rsc.li/rsc-advances
Terpenoids are one of the most valuable sub-classes of natural spiro-albatene9 and 18-hydroxydo labella-3,7-diene10 have been
products, consisting of thousands of interesting structures with studied by gas chromatography coupled to electron impact
well functionalized scaffolds.1 Numerous terpene cyclase mass spectrometry (GC/EI-MS). The key advantage of GC-MS
enzymes exist, isolated from bacteria, fungi and plants, but few analysis is that it requires less product compared to NMR
have been investigated and characterized.2 The universal spectroscopy, and is also a highly sensitivity, rapid method of
precursor isopentenyl pyrophosphate (IPP) plays a key role in analysis.11
extending linear alkylpyrophosphates, such as geranylpyr-
The chemical synthesis of specic position labelled linear
ophosphates (GPP), farnesylpyrophosphates (FPP) and ger- prenyl pyrophosphate is time consuming, laborious, and
anylgeranyl pyrophosphates (GGPP), which when nally requires a multistep reaction process.12 To overcome these
triggered by terpene cyclase lead to the production of diverse problems, a chemo-enzymatic strategy can be considered as an
hydrocarbon skeletons.3 This terpene cyclase catalysed mecha- alternative protocol for the rapid synthesis of a specic
nism involves specic rearrangements such as multiple cascade precursor.13
cyclisation, hydride shis, and stereoselective reactions via
cationic intermediates to achieve unique cyclic hydrocarbon AF157059) has been isolated from Artemisia annua L.14 Epi-
skeletons.4
cedrol cyclase (ECS) converts linear FPP into the unique
A bioactive sesquiterpene epicedrol (EC, GenBank:
The biosynthesis mechanisms of enzymes have been inves- tricyclic complex alcohol epicedrol. Recently, we studied the
tigated by feeding isotope-labelled precursors to terpene GC-EI-MS fragmentation of epicedrol derived from enzyme
cyclase. These isotopes include deuterium, (2H), carbon (13C) assay in H218O, which revealed that the source of the oxygen
and tritium (3H), and have been commonly used for the char- atom in EC was derived from water not from the pyrophos-
acterization of some enzymes, including patchouli alcohol phate of FPP.15 In continuation of epicedrol biosynthesis
synthase, cadinene synthase, and geosmin synthase etc.5 The study, herein a chemo-enzymatic strategy is applied to syn-
conventional method to trace isotopically-labelled products thesise (2H)-labelled-EC and investigate the cyclisation
derived from enzyme catalysts is nuclear magnetic resonance mechanism of ECS by EI-MS fragmentation ions compared
(NMR) spectroscopy, which generally requires milligram with non-labelled species and isotopic mass shi fragments
amounts of product for complete analysis, thus to synthesis (Fig. 1). In this strategy, the chemically prepared synthetic
a sufficient amount of product requires high quantities of four (D)-IPPs 2–5 were used for the elongation to (2H)-FPP
labelled precursors and enzymes. Recently, the mechanisms of with dimethylallyl pyrophosphate (DMAPP) and GPP
complex cyclic terpenes such as epicubebol,6 iso-dauc-8-en-11- through FPP synthase from Santalum album L. and GPP syn-
ol,6 and pristinol synthase,7 diterpene synthase, phomopsene8 thase from Catharanthus roseus. The subsequent cyclisation
by ECS yielded specic 8-position labelled EC analogues,
which were analysed by GC-MS.
aOrganic Chemistry Division, CSIR-National Chemical Laboratory (CSIR-NCL), Dr
Homi Bhabha Road, Pune-411008, India. E-mail: ss.shinde@ncl.res.in
bAcademy of Scientic and Innovative Research (AcSIR), Ghaziabad, 201 001, India
† Electronic supplementary information (ESI) available: NMR, GCMS, and HRMS
data of pure compound. See DOI: 10.1039/c9ra00163h
To explore the structure-based mechanistic pathway of epice-
drol biosynthesis, initially all four (2H)-IPPs 2–5 were synthesized
from the starting material methyl acetoacetate (1). The syntheses
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Scheme 2 Synthesis of 4 and 5. (k) PPh3CD3Br (98% atom of D), n-
BuLi THF, 0 ꢁC, 3 h; (l) Br2, aq. NaOH, dioxane, 5 h; (m) CDI, CH3-
NHOCH3Cl, DCM 12 h; (n) CD3MgI (98% atom of D), THF, ꢀ78 ꢁC, 3 h.
transformation to 16 involves the same protocols of TBDPS
deprotection and tosylation of 9 (Scheme 1). Then, pyro-
phosphorylation of 16 gives C4-labelled 4. In the synthesis of
IPP 5, compound 14 is subjected to a haloform reaction in the
presence of Br2 in NaOH to afford 17 as a carboxylic acid.18
Acid 17 can be converted into an amide in the presence of
CD3I and N,O-dimethyl hydroxylamine hydrochloride, then
Grignard reaction with CD3MgI at ꢀ10 ꢁC leads to the
formation of product 18. Further synthesis of 20 is achieved
via a similar reaction pathway as that of tosylated 9. Pyro-
phosphorylation of 20 gives C5-labelled 5.
Fig. 1 Strategy to specifically synthesise deuterium-labelled epicedrol
using a chemo-enzymatic method.
of labelled (1-2H2)-IPP (2) and (2-2H2)-IPP (3) were achieved by
following the general synthesis steps shown in Scheme 1. Initially,
the ketone protecting group of 1 reacts with ethylene glycol in the
presence of PTSA (p-toluenesulfonic acid) under azeotropic
conditions to afford the desired ester 6.
Further, the ester group is reduced with 2H followed by the use
of Bouveault–Blanc conditions16 to obtain the corresponding
alcohol, which when treated with TBDPSCl (tert-butyl-diphenyl-
chlorosilane) gives protected-OH 7. The selective deprotection of
the ketone of 7 is achieved in the presence of PPTS (pyridinium p-
toluenesulfonate) in aqueous acetone. Furthermore, the free
ketone is subjected to carbon Wittig olenation using methyl tri-
phenyl phosphonium bromide, to afford 8 in quantitative yield.
The TBDPS group of 8 is deprotected in the presence of TBAF
(tetra-n-butylammonium uoride), then subsequent tosylation
gives 9. The pyrophosphorylation of precursor 9 can be success-
fully performed according to the procedure by Davisson et al.,17 to
achieve (1-2H2)-2. Proton exchange with a deuterium atom using
D2O can then be carried out on the active methylene group of 1,
followed by ketone protection to give 10. The reduction of 10 with
LiAlH4, and subsequent protection with TBDPSCl affords 11.
Further, 11–13 can be achieved by following the same protocol
used for the synthesis of 7–9, where pyrophosphorylation of 13 and
ion exchange give C2-deuterated 3.
Feeding enzyme assay of (2H)-IPPs 2–5 with GPP synthase
and FPP in the presence of DMAPP and GPP afforded labelled
FPPs. The unit-1 (U1) in the FPP labelling was achieved by FPP
synthase assay between GPP and 2–5, to give (1-2H2), (2-2H),
(4-2H2) and (15-C2H3)-FPP. Subsequent cyclisation by ECS yiel-
ded products to be analysed by GC/EI-MS. The obtained frag-
mentation ion data were compared with non-labeled epicedrol,
as shown in Fig. 2.
The other four positions of the U2 of FPP were labeled as
(5-2H2), (6-2H), (8-2H2) and (14-C2H3)-FPP by similar chemo-
Scheme 2 shows the synthesis of (4-2H2)-IPP 4 and (5-
C2H3)-IPP 5. Building block 14 is produced by sequential
reduction of ester 6, then TBDPS protection, and deketala-
tion under similar reaction conditions to those used in
Scheme 1. The Witting reaction of 14 using (2H3)-methyl-
triphenylphosphonium iodide affords compound 15. Further
Scheme 1 Synthesis of 2 and 3. (a) Ethylene glycol, PTSA, toluene,
reflux; (b) Na, MeOD, hexane 15 min; (c) TBDPSCl, TEA, DCM, 0 ꢁC, 6 h;
(d) PPTS, H2O/acetone 5 : 1, RT, 12 h; (e) PPh3CH3Br, n-BuLi, THF, 0 ꢁC,
3 h; (f) TBAF, THF, RT, 2 h; (g) TsCl, DMAP, DCM, 0 ꢁC, 10 h; (h) Fig. 2 Mass spectra of epicedrol derived from (1), FPP, (2) C1–2H2 FPP,
(Bu4N)3PO7H, ACN, RT; (i) D2O, RT, 12 h; (j) LiAlH4, THF, 2 h, 0 ꢁC.
(3) C2–2H FPP, (4) C3–2H2 FPP, and (5) C15–2H3 FPP.
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enzymatic methods using DMAPP, then subsequently exposed isopropyl group. An alternative possibility for the formation of
to FPP synthase, and ECS yielded deuterated EC analogues (as EC161 is via the cleavage of a C6–C10 bond to produce H+c, which
shown in ESI†). The products were analysed by GC/EI-MS and occurs during ring opening with the elimination of an isobutene
the comparative fragmentation patterns of unlabelled and (2H)- unit, followed by an ethylene unit, with concomitant hydrogen
epicedrols were studied.
Their plausible structures were drawn aer comparing the and the formation of ion m/z ¼ 119. The mass fragment ion m/z ¼
increased isotope mass shi fragments, as shown in Fig. 3. 150 clearly indicates its formation from epicedrol by the a-
rearrangement and multiple hydrogen eliminations to give N+
The selected major fragments of epicedrol for analysis are the cleavage of the C2–C3 bond followed by inductive cleavage with
molecular ion [M]+ peak at m/z ¼ 222, m/z ¼ 95 (EC95), m/z ¼ 204 the neutral loss of iso-butyraldehyde. This cation intermediate
(EC204), m/z ¼ 161 (EC161), m/z ¼ 150 (EC 150) and m/z 119 (EC119). stabilizes via a tertiary carbocation and radical. Aer the initia-
The origin of fragment EC204 reveals a position-specic mass tion of A+c fragmentation, the formation of the structurally stable
shi in the formation of m/z ¼ 204 by the elimination of water base ion fragment m/z ¼ 95 is achieved aer proton transfer to
molecules with the loss of hydrogen. The fragment ion was oxygen to yield P+c and subsequent hydrogen rearrangements via
observed for all 8 position-labelled ECs with an increased mass of Q+ and R+ followed by inductive loss of acetone via a ring opening
+1, as observed for m/z ¼ 205 in the experiment with (2-2H)-FPP. reaction. Furthermore, the B and C rings formed in epicedrol
Mass shis of +2 and +3 were observed at m/z ¼ 206 and m/z ¼ originate from isoprene U2 and U3-FPP.
207 in the spectra of (1-2H2), (4-2H2) and (15-C2H3)-FPP, as shown
The mechanism of hydride migration from C6 to C7 was
in Fig. 2. The electron impact ionization data of the ECs suggest studied by conversion of (6-2H)-FPP to its corresponding epi-
the loss of an electron from the oxygen lone pairs to form B+c, cedrol. The position-specic mass shi analysis of m/z ¼ 95
followed by hydrogen elimination with the loss of H2O to generate shows an increase in the mass by +1 and even a loss of the C6
the fragment m/z ¼ 204. This fragment ion structurally supports portion in the case of the fragment m/z ¼ 161 (see the ESI†). The
our previous H218O assay of epicedrol biosynthesis, where the extracted information from all the GC-EI-MS spectra reveals that
tertiary cation intermediate was quenched with water.15
an exceptional labelling fragmentation pattern was observed in
The structural fragment EC189 suggests that the generation the MS analysis of the C7-containing fragment. These results
of the fragment ion m/z ¼ 189 can be traced through the methyl suggest that the hydrogen migrated in a 1,2-hydride shi
groups of FPP. Thus, in the fragmentation generated from the manner to quench bisabolyl cation D.
experiments on (14-C2H3)-FPP and (15-C2H3)-FPP, we compared
Based on the comparative fragmentation analysis of the GC/EI-
the MS fragment ions to reveal that the labelled CD3 group MS data of non-labeled and labelled epicedrol, we proposed
disappears from (15-C2H3)-FPP at C15, but this is not observed a possible mechanism for epicedrol biosynthesis, as mapped out in
in (14-C2H3)-FPP. This fragment ion indicates that the fragment Scheme 3. The isomerization of the OPP (O-pyrophosphate) group
originates from precursor m/z ¼ 204 via the loss of a methyl of FPP forms the stable isomer nerolidyl diphosphate intermediate
radical.
B. The ring cyclisation of the C1 and C6 carbons generates inter-
The fragment m/z ¼ 161 indicates the generation of the ion by mediate C, then the axial hydride in the C6 position was transferred
the loss of a neutral iso-propyl group from fragment EC189. A to C7 to generate bisabolyl carbocation D. The cyclisation driving
plausible mechanism for this involves the sequential elimination force of the C10 double bond results in subsequent ring closure to
of water then a-fragments from H+c followed by the loss of an form a new bond from C6–C10 and spiro-intermediate E followed
by the formation of a third ring through the new bond between the
C2 double bond and C11 carbocation via C-ring closure. Finally,
carbocation G was quenched by an external water source to produce
a stable epicedrol.
Fig. 3 Possible fragmentation of epicedrol. The red lines indicate
deuterium a-cleavage, rH: hydrogen rearrangement.
Scheme 3 Proposed mechanism of epicedrol biosynthesis.
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In summary, four synthetic (2H)-IPP isomers 2–5 were
prepared from the common starting material methyl acetoace-
tate and successfully used for (2H)-FPP synthesis through
enzyme catalysts. The GC-EI-MS fragmentation based mecha-
nism of epicedrol demonstrates that its biosynthesis proceeds
via 1,2-hydride migration and cyclisation reactions. This
RSC Advances
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There are no conicts to declare.
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