Silent catalytic promiscuity in the high-fidelity terpene cyclase δ-cadinene synthase

Article abstract of DOI:10.1039/c8ob02821d

δ-Cadinene synthase (DCS) is a high-fidelity sesquiterpene synthase that generates δ-cadinene as the sole detectable organic product from its natural substrate (E,E)-FDP. Previous work with this enzyme using substrate analogues revealed the ability of DCS

Full text of DOI:10.1039/c8ob02821d

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Silent catalytic promiscuity in the high-fidelity
terpene cyclase δ-cadinene synthase†
Cite this: DOI: 10.1039/c8ob02821d
Marianna Loizzi, David J. Miller
and Rudolf K. Allemann
*
δ-Cadinene synthase (DCS) is a high-fidelity sesquiterpene synthase that generates δ-cadinene as the sole
detectable organic product from its natural substrate (E,E)-FDP. Previous work with this enzyme using
substrate analogues revealed the ability of DCS to catalyse both 1,10- and 1,6-cyclisations of substrate
analogues. To test whether this apparent promiscuity was an artefact of alternate substrate use or an
inherent property of the enzyme, aza analogues of the proposed α-bisabolyl cation intermediate were
prepared since this cation would be formed after an initial 1,6-cyclisation of FDP. In the presence of
250 μM inorganic disphosphate both (R)- and (S)-aza-bisaboyl cations were potent competitive inhibitors
of DCS (Ki = 2.5 0.5 mM and 3.44 1.43 μM, respectively). These compounds were also shown to be
potent inhibitors of the 1,6-cyclase amorpha-4,11-diene synthase but not of the 1,10-cyclase aristolo-
chene synthase from Penicillium roquefortii, demonstrating that the 1,6-cyclase activity of DCS is most
likely an inherent property of the enzyme even when the natural substrate is used and not an artefact of
the use of substrate analogues.
Received 13th November 2018,
Accepted 10th December 2018
DOI: 10.1039/c8ob02821d
rsc.li/obc
phobic active site shelters this high energy intermediate from
bulk solvent.2b,c The active site, lined with hydrophobic and
Introduction
Terpene synthases catalyse some of the most complex reac- aromatic amino acid residues then steers the initial carbo-
tions in the natural world. From a small pool of isoprenyl cation through a series of ring closures and rearrangements
diphosphates they generate a myriad of hydrocarbons and prior to quench of the final carbocation either by proton loss
alcohols that are often processed into thousands of terpenoids or nucleophilic attack by water.2b,c,6e Usually this is tightly con-
with diverse biological activities with many potential appli- trolled by the enzyme, with a single enantiomer dominating
cations for instance as agrochemicals or therapeutic agents.1
the product pool whereby several rings and stereocentres are
The details of terpene synthase chemistry2 have been inves- often generated in a single chemical step from an achiral pre-
tigated by site directed mutagenesis and with non-natural cursor. Control of this process is thought to arise from a
amino acids,3 analogues of substrates,4 and putative reaction product-like active site contour in combination with direction
intermediates,5 X-ray crystallography,2b,c,6 and computational of carbocation location in the intermediates through the nega-
modelling.2d,7 Together these investigations revealed a fasci- tive charge on the diphosphate anion and aromatic amino
nating, yet still incomplete picture. A series of X-ray crystal acid side chains that can stabilise carbocations at certain
structure of aristolochene synthase from Aspergillus terreus in locations through cation–π interaction.2b,c,4a A small subset of
both closed and open conformations along with complexes terpene synthases, on the other hand, exhibit significant
containing the complete substrate (or analogue), diphosphate promiscuity, presumably through having a less structured and/
anion and/or Mg2+ co-factors8 revealed the physical steps of or flexible active site that allows the intermediates to sample a
the catalytic cycle. Binding of a Mg2+-ion is followed by coordi- large number of reactive conformations prior to final carbo-
nation of the prenyl diphosphate substrate and a second Mg2+ cation quench. For example, δ-selinine synthase and
ion; coordination of a third Mg2+ ion triggers active site γ-humulene synthases from Abies grandis generate 34 and 52
closure to form the Michaelis complex.8 Diphosphate cleavage products from farnesyl diphosphate (1), respectively.9 Terpene
is then triggered to form an initial carbocation and the hydro- synthases have been postulated to evolve through such pro-
miscuous intermediates prior to further evolution into high-
fidelity synthases.3e The modern δ-cadinene synthase (DCS)
from Gossypium arboreum is a high-fidelity sesquiterpene
School of Chemistry, Cardiff University Main Building, Park Place, Cardiff,
CF10 3AT, UK. E-mail: allemannrk@cardiff.ac.uk
synthase that catalyses the formation of the bicyclic hydro-
†Electronic supplementary information (ESI) available: General experimental
carbon (+)-δ-cadinene (7),10 the first committed step in the bio-
procedures, enzyme preparation and purification, kinetics data, gas chromato-
synthesis of the phytoalexin gossypol.11 The catalytic domain
grams, mass spectra and NMR spectra. See DOI: 10.1039/c8ob02821d
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is situated in the C-terminal domain and adopts the α-helical cation, an intermediate common to both pathways. In previous
fold domain, typical of class 1 terpene synthases.2b,c,12 It con- work, using substrate analogues we were unable to definitively
tains the conserved aspartate rich motif D307DTYD311 on helix rule out pathway (b) and indeed when 6-fluorofarnesyl di-
D, but instead of the usual characteristic NSE/DTE Mg2+ phosphate (6F-FDP) was used as a substrate analogue it proved
binding motif, DCS has
a second aspartate rich motif to be a potent inhibitor (Ki = 2.4 μM), giving no detectable
D451DVAE455 on helix H.6e Despite only generating a single pentane-extractable products when incubated with DCS. This
detectable hydrocarbon product, extensive mechanistic ana- result is consistent with an initial 1,6-cyclisation pathway since
lysis of the DCS-catalysed reaction pathway has not unambigu- it would be expected to undergo 1,10-ring closure and give an
ously defined the chemical steps of its catalytic cycle. abortive product rather than inhibit the enzyme in the latter
Moreover, conversion of fluorinated and stereochemically scenario. On the other hand, 2-fluorofarnesyl diphosphates
altered FDP analogues with DCS revealed an underlying (2F-FDP) and 10-fluorofarnesyl diphosphate (10F-FDP) gave
mechanistic promiscuity with products arising from an initial products arising from 1,10- and 1,11 ring-closures, respect-
1,10-, 1,6- or 1,11-ring closure depending upon the substrate ively, consistent with an initial 1,10-ring closure mechanism.12
analogue used (vide infra).12 Two chemical mechanisms
Hence examination of the catalytic mechanism of DCS
remain plausible for the formation of δ-cadinene from FDP using FDP analogues has led to inconclusive, yet intriguing
(Scheme 1). Both pathways involve initial formation of (3R)- results, showing that this enzyme has the potential to use
nerolidyl diphosphate ((3R)-NDP, (2)) as an enzyme-bound alternative reaction pathways. Yet the question arises, is this
intermediate. In pathway (a), a 1,10-macrocyclisation occurs to simply an artefact of the substrate used or is this an inherent
generate cis-germacradienyl cation (4). A subsequent [1,3]- property of the enzyme? The work described here provides
hydride shift is followed by a 1,6-electrophilic ring closure to alternative mechanistic data for the DCS-catalysed transform-
cadinenyl cation (6), from which δ-cadinene (7) is formed after ation of FDP to δ-cadinene using aza-analogues of putative car-
proton loss from C6. In pathway (b), a 1,6 ring-closure of 2 is bocation intermediates. Although the highly unstable carbo-
followed by a [1,3]-hydride shift from C1 to C7; subsequently a cationic intermediates formed during terpene synthase cataly-
second ring closure and a [1,5]-hydride shift lead to cadinenyl sis, cannot be isolated, it is possible to replace the sp2 hybri-
dised carbocationic carbon of a given intermediate with an sp3
hybridised nitrogen in an amine analogue or with a sp2 hybri-
dised nitrogen in an iminium ion. Although the tetrahedral
tertiary ammonium ions inherently are imperfect geometric
analogues of the planar carbocations, these aza-terpenoids are
thought to mimic the topological and electrostatic properties
of carbocations generated by these enzymes.5 However, since
they cannot be processed by the enzyme, they often act as
tightly bound competitive inhibitors of terpene synthases.5c,13
Hence, the use of strategically designed aza-analogues may
enable the disentanglement of the possible reaction mecha-
nisms catalysed by DCS. Here we report the stereoselective syn-
thesis of the two enantiomers of aza-bisabolyl cation and their
kinetic evaluation as inhibitors of DCS. Comparison of their
effect upon catalysis by AS and amporpha-4,11-diene synthase
(ADS), enzymes that follow 1,10- and 1,6-ring-closure mecha-
nisms, validate the result that DCS has inherent 1,6- as well as
1,10 ring closure activity.
Results and discussion
If α-bisabolyl cation 8 is a reaction intermediate on the
pathway to δ-cadinene (7), one or both of enantiomeric aza-
analogues of 11 (Fig. 1) should act as competitive inhibitors of
DCS.
Both enantiomers of 11 have previously been prepared.14a
Here we report an alternative synthesis that is more concise
and avoids the use of harsh reaction conditions. Key to the
synthesis of both enantiomers is an enantioselective synthesis
of the two enantiomers of carboxylic acid 18 (Scheme 2). This
was achieved through asymmetric Diels–Alder reaction of an
Scheme 1 Possible chemical steps for DCS catalysed production of
δ-cadinene from FDP (1).
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was in fact optimal for both enantiomers but due to the high
cost of L-pantolactone not used for bulk preparation for the
R-enantiomer of 18. Optical purity of all subsequent com-
pounds was checked using chiral GC, HPLC and/or polarime-
try and in all cases no loss of optical purity was detected in
later synthetic steps.
Both syntheses now proceeded in identical manner and
Scheme 2 only illustrates the synthesis of the S-enantiomer of
11. Hydrolysis of 14 using LiOH in an equivolume mixture of
THF, water and methanol for 1 h at 50 °C gave carboxylic acid
18 in near quantitative yield. 18 was converted to p-methoxy-
benzyl urethane derivative 19 by treatment with diphenylpho-
sphorylazide (DPPA) followed by a Curtius rearrangement in
the presence of p-methoxybenzyl alcohol, which proceeded
with strict retention of stereochemistry.15b The overall yield of
the urethane product 19 was 60% over the two steps. Final con-
version to (S)-11 was achieved first through reduction with
LiAlH4 in anhydrous Et2O (50%) then HBTU mediated coup-
ling to 4-methypent-3-enoic acid (70%) followed by a second
reduction with LiAlH4 in Et2O. To prevent air oxidation upon
storage the product was converted to its hydrochloride salt
with HCl in ether, yielding (S)-11·HCl in 55% yield over the
final two steps. The optical purity of (S)-11 was estimated to be
≥98% by comparison with previously reported data.14a Similar
results were obtained for the synthesis of (R)-11.
Fig. 1 Chemical structures of the α-bisabolyl cation (8) and corres-
ponding aza analogues (R)-11 and (S)-11.
To validate any results obtained for these compounds as
inhibitors of DCS, they were tested as inhibitors of aristolo-
chene synthase from Penicillium roqueforti (AS) and amorpha-
4,11-diene synthase (ADS). These two enzymes are known to
proceed via 1,10- and 1,6-cyclisations of the initial carbocation
during their catalytic cycle (Scheme 3).1g Hence aza-bisabolyl
cations 11 should act as poor inhibitors of AS and potent
inhibitors of ADS, as they closely resemble a reaction inter-
mediate in the latter case only.
Scheme 2 Synthesis of aza-analogues (R)- and (S)-11. Reagents and
conditions: (i) acryloyl chloride, BuLi, THF, 35%. (ii) Et2AlCl, CH2Cl2,
−100 °C, 54%. (iii) Acryloyl chloride, NEt3, CH2Cl2, 60%. (iv) TiCl4,
CH2Cl2, −10 °C, 84%. (v) LiOH, THF, H2O, MeOH, 50 °C, 99%. (vi) DPPA,
NEt3. (vii) p-Methoxybenzyl alcohol, toluene, 60% over two steps. (viii)
LiAlH4, Et2O, 50%, (ix) 4-methylpent-3-enoic acid, EtNPri2, HBTU, DMF,
70%. (x) LiAlH4, Et2O. (xi) HCl in Et2O, 55% two steps.
Recombinant AS and ADS were prepared and purified
according to previously published procedures17,18 and both
acrylate derivatised with a chiral auxiliary with a butadiene.15
Oxazolidin2-one 12 was alkylated with acryloyl chloride after
deprotonation with n-butyl lithium with 35% yield. The result-
ing ester 13 was then subjected to an asymmetric Diels–Alder
reaction with 2-methylbutadiene.15 The enantioselectivity and
yield were optimal at −100 °C in CH2Cl2 (52%, ee >95%, de
>95% (see ESI† for details). This produced the key compound
to generate the S enantiomer of the aza-analogue 11. The equi-
valent R configured ester was generated using D-pantolactone
(15) as a chiral auxiliary.15 After alkylation with acryloyl chlor-
ide, using NEt3 as the base in CH2Cl2, diester 16 was isolated
in 60% yield. Again, an asymmetric Diels–Alder reaction with
2-methylbutadiene was carried out, this time at −10 °C in
CH2Cl2 using TiCl4 as a Lewis acid catalyst yielding the R ester
Scheme 3 Initial catalytic chemical steps leading to (c) (+)-aristolo-
chene (22) and (d) amorpha-4,11-diene (23) follow 1,10- and 1,6-cyclisa-
in 84% yield (ee = 92% and de = 97%).16 The latter procedure tion of FDP, respectively.
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(R)-11 and (S)-11 were tested as inhibitors using a standard by both enantiomers, improving the Ki approximately 20-fold
radiolabelled assay involving conversion of tritium labelled (Ki = 1.5 and 3.7 μM for the S and R enantiomers respectively)
FDP by each enzyme and scintillation counting of the pentane demonstrating that the active site of ADS prefers a cation–
extractable products.5b Terpene synthases are known to anion pair in its active site.5d,13
efficiently bind cation-PPi pairs and inhibition was assessed
Recombinant DCS was generated with a C-terminal hexahis-
both in the presence and absence of 250 μM diphosphate tidine tag (DCS-His6) as previously described.19 Inhibition
(Table 1). Synergistic inhibition of aza-analogues 11 with assays were carried out using the same protocol used for AS
diphosphate has been observed previously for a variety of and ADS. Both aza analogues were found to be competitive
other terpene synthases.5d,13c,14
inhibitors of DCS-His6 in the presence of PPi but only poor
Kinetic data were fitted by non-linear regression to the inhibitors in its absence (Table 1). DCS clearly requires a
Michaelis–Menten equation (v0 = kcat[E][S]/(KM + [S]). The cation–anion pair in its active site for effective inhibition by
mode of inhibition was determined by inspection of double aza-analogues. Our results provide strong evidence for 1,6-
reciprocal plots and observed to be competitive in all cases cyclase activity for DCS.
where inhibition was significant at low concentrations of 11.
KI was determined from a plot of inhibitor concentration
versus K′M/(kcat[E]) where K′M = KM(1 + [I]/KI).
Conclusions
The inhibition data for AS and ADS validate both of these
compounds as valuable mechanistic probes for the present The aza-bisabolyl cations 11 were potent competitive inhibi-
investigation since they are poor inhibitors of AS and potent tors of ADS, a 1,6-cyclase yet were much poorer inhibitors of
inhibitors of ADS. PPi had little effect on the ability to inhibit PR-AS, a known 1,10-cyclase. When DCS was challenged with
AS (KI > 200 μM in both the presence and absence of PPi for these aza-analogues in the presence of diphosphate anion they
AS). Both enantiomers of 11 acted as competitive inhibitor of were potent inhibitors of the conversion of FDP to
ADS, showing that they are able to compete effectively with the (+)-δ-cadinene (7), which would only be expected if DCS had a
natural substrate FDP at the active site. As these aza-com- 1,6-cyclase activity. The use of a variety of substrate analogues
pounds cannot be turned over by ADS, these result support the possessing different stereochemistry and heteroatoms did not
intermediacy of an α-bisabolyl cation in the biosynthesis of lead to clear results regarding whether DCS follow a 1,6 or 1,10
amorpha-4,11-diene, in agreement with the findings of Picaud pathway.12 If the proposed initial isomerism of the substrate to
et al.18b who used deuterated farnesyl diphosphate and deuter- nerolidyl diphosphate (2) was suppressed using a fluorine
ium exchange experiments to suggest that the R-enantiomer of atom at C2 then a 1,10 cyclisation was observed (Fig. 2).
the α-bisabolyl cation is the sole intermediate formed in the 2-Fluorogemacrene A (25) was the DCS catalysed product from
biosynthesis of amorpha-4,11-diene. Therefore, only the R the transoid (2Z,6E)-2-fluorofarnesyl diphosphate (24) while
enantiomer of 11 would be expected inhibit ADS; however, if the cisoid substrate analogue 26 gave the cisoid product 2F-
the S-enantiomer was a slightly more potent inhibitor (Ki = helminthogermacrene A (27).12 However, in nearly every other
50 μM for (R)-11 versus 25 μM for (S)-11) Table 1. This is con- case involving the use of substrate analogues with DCS, 1,6-
sistent with a flexible model for sesquiterpene active sites, cyclisation was observed at least in-part (Fig. 2).12 These
according to which an active site can accommodate a variety of results may simply reflect the use of different substrates rather
intermediates of different shape and charge distribution than an inherent ability of DCS to catalyse the conversion of
without being rigidly complementary to a single intermediate FDP to 7 along two distinct reaction paths.20 The observation
or transition state species. For example, work by Cane et al. that 11 acts as a competitive inhibitor of the DCS catalysed
showed that both enantiomers of the aza-analogue 11 were conversion of FDP provides strong evidence that DCS can
equally effective inhibitors of trichodiene synthase.14a It is also efficiently use a 1,6-cyclisation pathway.
notable that the presence of PPi enhanced inhibition of ADS
The fact that inorganic diphosphate led to a more tightly
bound active site carbocation/diphosphate ion pair is consist-
ent with previous work where often the active site recognises a
cation-PPi pair more effectively than the cation alone.5b,c–7a,14
The fact that (R)-11 acts as a weak inhibitor in the absence of
PPi is more difficult to explain. It was previously suggested
that, in the DCS active site pocket, the alkenyl chain of (3R)-
nerolidyl diphosphate (2) is ideally positioned to ensure the
formation of the α-bisabolyl cation with an R configuration at
C6 (Scheme 1).12 The C1–C7 hydride shift from 8 to 9 then
occurs to the same Si face of C7 in cation 8, therefore a (7R)-9
formation is expected (Scheme 1). Hence the (R)-11 should
mimic better the α-bisabolyl cation generated by this enzyme,
and therefore act as competitive inhibitor with higher binding
affinity when compared with the S-enantiomer. The evidence
Table 1 Kinetic data for inhibition of ADS, AS and DCS by (R)-(11) and
(S)-11. Uninhibited kinetic data for each enzyme: ADS KM = 2 0.15 μM
k
cat = 1.19 × 10−2 52 × 10−5 s−1. AS KM = 2.42 0.11 μM, kcat = 1 × 10−2
2 × 10−5 s−1. DCS-His6 KM = 0.58 11 μM kcat = 1.26 × 10−3 5 ×
10−6 s−1
Enzyme
ADS
Aza-analogue
Ki (μM) (+250 μM PPi)
Ki (μM)
25
(S)-11
(R)-11
(S)-11
(R)-11
(S)-11
(R)-11
1.5 0.5
3.7 1.9
255 23
489 62
3.44 1.43
2.5 0.5
5
50 17
AS
295 23
472 48
273 77
1700 300
DCS
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their redesign to produce nature-like compounds that are not
found in the biosphere.23,24
Experimental
General experimental procedures, enzyme preparation and
purification are described in ESI† along with kinetics data, gas
chromatograms, mass spectra and NMR spectra.
(R)-4,4-Dimethyl-2-oxotetrahydrofuran-3-yl acrylate (16)
Freshly distilled propenoyl chloride (0.41 mL, 5 mmol) was
added over 1 h to a stirred solution of (R)-pantolactone
(500 mg, 3.84 mmol) and Et3N (583 mg, 5.76 mmol) in an-
hydrous CH2Cl2 (10 mL) at −24 °C. The resulting mixture was
stirred for 5 h at −24 °C, and subsequently washed with
aqueous 1 M HCl (10 mL). The aqueous phase was then
extracted with CH2Cl2 (3 × 20 mL). The combined organic
phases were washed with saturated NaHCO3 solution (3 ×
20 mL), water (3 × 20 mL) and brine (3 × 20 mL). The organic
phase was dried over MgSO4, concentrated under reduced
pressure and the residue was purified by flash chromatography
on silica (EtOAc : hexane 4 : 6) to yield the pure compound as a
yellow oil (375 mg, 53%). δH (300 MHz, CDCl3) 6.48 (1 H, d, J =
17.5, CHHvC), 6.18 (1H, dd, J = 17.5, 10.5 Hz, CHvCH2), 5.93
(1H, d, J = 10.5 Hz, CHHvC), 5.40 (1H, s, CvOCH–O), 4.03 (2
H, s, CH2–OCvO), 1.17 (3 H, s, CH3), 1.08 (s, 3 H, CH3);
δC (63 MHz, CDCl3) 174.7 (OCvOCHO), 172.5 (OCvOCvH2),
134.0
(H2CvCHCvO),
118.7
(H2CvCHCvO),
76.1
(OCvOCHO), 74.6 (OCH2CH), 40.2 (C–(CH3)2), 23.4 (CH3),
23.0 (CH3). αD +10° (CH2Cl2, c = 17). Data are in agreement
with previous work.15a
(R)-4.4-Dimethyl-2-oxotetrahydrofuran-3-yl-(S)-4-
Fig. 2 Summary of 1,10 and 1,6 cyclisation products generated through
DCS catalysis.
methylcyclohex-3-ene-1-carboxylate (17)
To a solution of 16 (302 mg, 1.67 mmol) in anhydrous CH2Cl2
(5 mL) at −10 °C, TiCl4 (0.82 mL, 0.82 mmol, 1.0 M solution in
CH2Cl2) was added, and the resulting solution was stirred
that both enantiomers of 11 are equally as effective in the pres- under argon at −10 °C for 1 h. 2-Methylbutadiene (0.23 mL,
ence of PPi is consistent with a permissive model of the active 2.3 mmol) was then added over 5 min and the mixture was left
site structure, according to which an active site should accom- stirring for 3 h at −10 °C. The reaction was quenched by
modate a variety of rearranged intermediates of different addition of 10% Na2CO3 in water (5 mL). The aqueous phase
shape and charge distribution without being rigidly comp- was then extracted with CH2Cl2 (3 × 20 mL). The organic layers
lementary to a single intermediate. On the other hand, their were combined, washed with H2O (3 × 10 mL), brine (3 ×
lack of inhibitory effects on the 1,10-cyclase PR-AS shows that 10 mL), dried over anhydrous MgSO4, filtered and concen-
a major difference in the connectivity of the aza-analogue com- trated under reduced pressure. The residue was purified by
pared to the carbocation intermediate (i.e. bisabolyl cation flash chromatography on silica (EtOAc : hexane 2 : 8) to yield
rather than the 10-membered ring containing germacrenyl pure 17 as a colourless oil (353 mg, 85% yield). δH (300 MHz,
cation) renders them ineffective as inhibitors; hence the 1,6- CDCl3) 5.32 (1 H, s, OCHCvO,) and (1 H, br CHv), 3.98 (2 H,
cyclase activity of DCS postulated previously12 is intrinsic to s, CHCH2O), 2.69–2.51 (1 H, m, CH–CvO), 2.19 (2 H, m broad,
the enzyme.
CHO–CH2Cv), 1.98 (3 H, m, CHH–CH2), 1.73 (1H, m, –CHH–
Terpene synthases can generate great structural and stereo- CH2), 1.58 (s, 3H, CH3Cv), 1.13 (3H, s, CH3), 1.04 (3 H, s,
chemical complexity in one synthetic step and have therefore CH3). δC (75 MHz, CDCl3) 174.7 (OCvOCHO), 172.5
potential as powerful synthetic biocatalysts for the generation (OCvOCvH2), 134.0 (HCvCCH3), 118.7 (HCvCCH3), 76.1
of many bioactive compounds.4f,18a,21,22 A clear understanding (OCvOCHO), 74.6 (OCH2C), 40.2 (C–(CH3)2), 38.9 (CH2CH–
of the catalytic strategies employed by these enzymes can aid CvO),
28.9
(HCvCCH2),
27.7
(CvCHCH2),
25.2
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(HCvCCH2CH2), 23.4 (CCH3), 23.0 (CCH3), 19.8 (CH3CvCH). solved in H2O (10 mL) and extracted with CH2Cl2 (3 × 5 mL).
LRMS (EI+) m/z: 252.13 (50%), 122.07 (20), 94.08 (100), 79.05 The resulting aqueous phase was acidified to pH = 2 at 0 °C
(30), 67.05 (10). αD −51.3 (CHCl3, c = 1). Data are in agreement with 15% HCl, extracted with a mixture of n-pentane : CH2Cl2
with previous work.16
(98 : 2 3 × 10 mL), dried over anhydrous Na2SO4, and concen-
trated under reduced pressure to give 18 as a white powder
(35 mg, 80%). δH (CDCl3, 300 MHz) δ 5.32 (1H, s, CHv),
(S)-3-Acryloyl-4-benzyloxazolidin-2-one (13)
To
5.60 mmol) in anhydrous THF (12 mL) at −78 °C, n-BuLi (3 H, m, CHH–CH2), 1.69 (1H, m, CHH–CH2), 1.59 (3H, s,
(2.1 M in THF, 3.14 mL, 6.59 mmol) was added dropwise over CH3). δC (100 MHz, CDCl3) 182.3 (HOCvO), 133.8
a
solution of (S)-4-benzyloxazolidin-2-one (1.00 g, 2.54–2.39 (1 H, m, CH–COOH), 2.17 (2H, m, –CH2CHvC), 1.93
δ
30 minutes and the mixture stirred for a further 3 h at −78 °C. (HCvCCH3), 119.0 (CH2–HCvCCH3), 39.0 (HC–COOH), 29.13
Freshly distilled acryloyl chloride (557 mg, 6.16 mmol) was (CH2CvCH3), 27.3 (CH2CHvCCH3), 25.5 (CH2CH2CvCH),
added dropwise over 20 minutes and the reaction stirred for 23.5 (CH3). (S)-18: αD −80.6 (CHCl3, c = 0.5); −106.4 (95%
2 h at −78 °C. The reaction was then allowed to warm to room EtOH, c = 4). (R)-18: αD +93 (CHCl3, c = 0.5); +105.5 (95% EtOH,
temperature overnight. The reaction was quenched with sat. c = 4). M.p. 82–92 °C. LRMS (EI+) m/z 140.06 (100%, M+),
NH4Cl (20 mL) and extracted with diethyl ether (3 × 30 mL). 136.06 (15), 125.05 (40), 122.06 (100), 95.87 (100), 94.06 (100),
The organic layer was washed with water (3 × 40 mL), saturated 93.07 (80), 79.04 (100), 77.03 (100), 68.06 (90), 67.04 (100) Data
aqueous NaHCO3 (3 × 40 mL), dried (MgSO4), filtered, and are in agreement with previous work.15,16
concentrated under reduced pressure. Flash chromatography
on silica gel (hexane : ethyl acetate 6 : 4) afforded 13 as a color-
less solid (452 mg, 35%). δH (CDCl3, 300 MHz) 7.45 (dd, 1H,
(R)- and (S)-4-Methoxybenzyl [(4-methylcyclohex-3-en-1-yl)
methyl]-carbamate (19)
J = 6.0, 18.0, CHvCH2), 7.23 (m, 5H, aromatic Hs), 6.54 (dd, To a solution of 18 (1.3 g, 9.3 mmol) in anhydrous toluene
1H, JH, H = 18.0, 18.0, CHHvCH2), 5.87 (dd, 1H, J = 9.0, 9.0, (20 mL) at
0 °C, diphenylphosphoryl azide (2.2 mL,
CHHvC), 4.68 (m, 1H, CHN), 4.14 (m, 2H, CH2O), 3.29 (dd, 10.2 mmol) and Et3N (3.9 mL, 27.8 mmol) were added. The
1H, J = 9.0, 9.0, CvCHHPh), 2.74 (dd, 1H, J = 12.0, 12.0, resulting mixture was left stirring for 3 h at 100 °C before
CHHPh) αD −86° (CH2Cl2, c = 0.65). Data are in agreement 4-methoxybenzyl alcohol (1.27 mL, 10.2 mmol) was added,
with previous work.14a
and the reaction was left to stir for 16 h at 100 °C. The reaction
was then allowed to cool to room temperature and the solution
was concentrated under reduced pressure. The residue was the
purified by flash chromatography on silica gel (EtOAc : n-
(R)-4-Benzyl-3-((S)-4-methylcyclohex-3-enecarbonyl)oxazolidin-
2-one (14)
To a stirred solution of 13 (200 mg, 0.86 mmol) at −100 °C, hexane 1 : 9) to yield 19 as a yellow crystalline solid (1.28 g,
were added 2-methylbutadiene (1.72 mL, 17.2 mmol) in anhy- 80%). δH (300 MHz, CDCl3) δ 7.23 (2 H, dt, J = 2.9 and 5.3 CH
drous CH2Cl2 (5.0 mL) and Et2AlCl (1.2 mL, 1.5 eq.). The reac- ArCH), 6.81 (2 H, dt, J = 2.9 and 5.3, ArCH), 5.21 (1 H, br,
tion was stirred at −100 °C for 30 min then the mixture was HCvC), 4.95 (2 H, s, –OCH2Ph), 4.67–460 (1 H, m, CHN), 3.74
poured into ice cold aqueous hydrochloric acid (1 M, 20 mL). (3 H, s, –OCH3), 2.29–2.20 (2 H, m, CH2–CH2CHN), 1.93 (2 H,
The mixture was extracted with CH2Cl2 (2 × 10 mL). The com- m, CH2–CH2CHN), 2.01–1.86 (2 H, m, CH–CH2CHN), 1.55
bined organic layers were dried over anhydrous MgSO4, (3 H, s, CH3CvCH). δC (75 MHz, CDCl3) δ 159.5 (vCO–CH3),
filtered, and concentrated under reduced pressure. The 155.8 (NHCvO), 134.1 (CvCCH3), 130.0 (CvC aromatic),
product was purified by flash chromatography on silica 128.7 (CCH2O), 118.3 (CvCCH3), 113.9 (CvC aromatic), 66.3
(EtOAc : hexane : Et3N 92 : 7 : 1) to yield the Diels Alder adduct (CCH2O), 62.8 (CHN), 55.3 (OCH3), 31.9 (CH2CHN), 28.4
(14) as a white crystalline solid (139 mg, 54%). δH (300 MHz, (CH2CvC), 28.0 (CH2CvC), 23.4 (vCCH3). νmax (thin film,
CDCl3) 7.34–7.10 (5 H, m, ArCH), 5.36 (1 H, br., CHvC), 4.63 cm−1) 3300 (N–H stretch), 2900–2700 (C–H stretch), 1650
(1 H, dt, J = 16.6, 6.9, CHN), 4.23–3.95 (2 H, m, CH2O), 3.60 (CvO ester stretch), 1250 (C–N stretch), 830 (aromatic CH
(1 H, t, J = 8.8, CHCvO), 3.20 (1 H, dd, J = 13.2, 3.3, CHHPh), bending); (S)-19: αD −9.3, (c = 0.6, CHCl3) (R)-19: αD +12 (c =
2.70 (1 H, dd, J = 13.3, 9.5, CHHPh), 2.20–1.6 (6 H, m, 0.6, CHCl3) m.p. 69–71 °C LRMS (EI+) m/z: 275.15 (100% M+),
CH2CH2CHCH2CvC), 1.64 (3H, s, CH3) αD +79 (CH2Cl2, c = 276.15 (20), 259.12 (18), 258.12 (60), 231.12 (25), 228.1128(12),
1.4). LR-MS (EI+) m/z: 299.15 (100% M+), 300.16 (15), 269.06 214.16 (100). HRMS (EI+) 275.1522; C16H21NO3 requires
(18), 267.07 (50), 232.10 (20), 178.08 (100), 146.07 (30), 140.03 275.1521.
(55), 122.07 (20), 91.00 (65), 63.00 (30). Data are in agreement
(R)- & (S)-N-Methyl-1-(4-methylcyclohex-3-en-1-yl)methanamine
(20)
with previous work.14a
(R)- and (S)-4-Methylcyclohex-3-ene-1-carboxylic acid (18)
To a stirred solution of carbamate 19 (100 mg, 0.4 mmol) in
To a solution of 14 or 17 (0.32 mmol) in THF : MeOH : H2O anhydrous diethyl ether (7 mL) at 0 °C, was added LiAlH4
(1 : 1 : 1, 1.5 mL), LiOH (67 mg, 1.6 mmol) was added, and the (50 mg, 1.28 mmol). The mixture was then heated to reflux for
resulting mixture was vigorously stirred for 1 h at 50 °C. The 5 h. The reaction was cooled to 0 °C before it was quenched by
reaction was then cooled to room temperature and concen- the addition of water (6 mL) and an excess of 15% NaOH solu-
trated under reduced pressure. The resulting slurry was dis- tion (6 mL). The resulting mixture was left to stir at 0 °C for
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1 h and the precipitate was removed by filtration through organic layers were dried over anhydrous Na2SO4, concentrated
a Celite pad. The organic phase was extracted with water under reduced pressure, and the residue was purified by flash
(2 × 10 mL) and the pooled organic layers were then washed chromatography on silica (Et2O : MeOH 1 : 9) to yield the
with 10% HCl (2 × 10 mL) and the organic fraction was dis- amine as a yellow oil (32 mg, 65% yield). δH (300 MHz, CDCl3)
carded. The combined aqueous layers were adjusted to pH 12 5.27 (1 H, d, J = 2.4, H3CCvCH), 5.03 (1H, t, J = 5.6,
by dropwise addition of 10% NaOH (15 mL). The product was (CH3)2CvCH), 2.64–2.44 (1 H, m, CHN), 2.38 (2 H, ddd, J = 7.6,
extracted with diethyl ether (4 × 15 mL), then dried over anhy- 5.8 and 2.6, CH2N), 2.23 (3 H, s, CH3N), 2.08 (2 H, dd, J = 15.5
drous MgSO4, and filtered. The product was then concentrated and 7.2, CH2CH2N), 2.04–1.85 (4 H, m, CH2CH2CHN),
carefully under reduced pressure to give 20 as a volatile color- 1.85–1.67 (2 H, m, CvCHCH2CHN), 1.62 (3 H, s, CH3CvCH),
less oil (20 mg, 40%). δH (300 MHz, CDCl3) 5.24 (1 H, m br, 1.55 and 1.57 (2 × 3 H, 2 × s, (CH3)2CvCH). δC (75 MHz,
CHvC), 2.55 (1 H, dt, J = 16.6 and 8.0, CHNH2), 2.37 (3 H, s, CDCl3)
δ 133.9 (HCvCCH3), 132.6 (CHvCCH3), 122.1
HNCH3), 2.25–2.10 (1 H, m, NH), 1.99–1.87 (2 H, m, (CHvCCH3), 120.0 (NCH2CH2CHvCCH3), 58.9 (CHN), 53.5
CH2CHvC), 1.87–1.66 (2 H, m, CH2CH2CvCH), 1.61 (1 H, (CH2N), 37.9 (CH2CH2CHN), 30.8 (vCHCH2N), 27.2
broad m, CH2CH2CvCH), 1.60 (3 H, s, H2CvCCH3), 1.45–1.26 (CH2CCH3), 26.5 (CH3N), 25.7(CH2CH2N), 25.6 (CH3CCH3),
(1 H, m, CH2CH2CvCH). δC (75 MHz, CDCl3) 134.0 23.2 (CH3CCH3), 17.8 (CH3Cv). HRMS (EI+) 207.1990;
(CvCCH3), 119.1 (CvCCH3), 54.8 (CHNH), 32.7 (CH2CHNH), C14H25N requires 207.1987. (S)-11: αD −61 (CHCl3, c = 1) (R)-11
32.1 (CvCHCH2CH), 29.7 (CH3N), 29.0 (CH2Cv), 23.4 αD +63 (CHCl3, c = 1). Data are in agreement with previous
(CH3Cv) (S)-20: αD −79 (c 1.00, CHCl3) (R)-20: αD +84 (c 1.00, work.14a The amine was then dissolved in Et2O (1 mL) and HCl
CHCl3) Data in agreement with previous work.14a
(1 M in anhydrous Et2O) was added slowly. A light yellow pre-
cipitate was formed. The ether was concentrated under
reduced pressure and the salt stored in 1.2 mL of deionised
water. δH (300 MHz, MeOD) 5.38 (1 H, br, H3CCvCH), 5.14
(R)- and (S)-N,4-Dimethyl-N-(4-methylcyclohex-3-en-1-yl)pent-3-
enamide (21)
To a stirred solution of 20 (172 mg, 1.5 mmol) and DIPEA (1 H, t, J = 5.0, (CH3)2CvCH), 3.58–3.42 (1 H, m, CHN), 3.32
(775 mg, 6.0 mmol) in anhydrous DMF (6 mL), HBTU (1.15 g, (1 H, dd, J = 4.9, 1.6, CHHCH2N), 3.25–2.97 (1 H, m,
3.0 mmol) was added and the resulting mixture was stirred at CHHCH2N), 2.85 (3 H, s, CH3N), 2.59–2.39 (2 H, m,
room temperature for 20 min before 20 (358 mg, 1.5 mmol) CH2CH2CHN), 2.39–2.26 (2 H, m, CH2CH2CHN), 2.26–2.05
was added. The reaction was then stirred for 24 h at room (2 H, m, CvCHCH2CH2N), 1.84 (2 H, dd, J = 12.2, 10.4 Hz,
temperature. The mixture was concentrated under reduced CH3CvCHCH2), 1.76 (3 H, s, CH3CvCH), 1.72 (6 H, 2 × s,
pressure and the residue was dissolved in diethyl ether (CH3)2CvCH). δC (75 MHz, MeOD) δ 136.1 (HCvCCH3), 134.3
(20 mL). The solution was washed with water (2 × 25 mL), (CHvC(CH3)2)
117.6
(CHvCCH3)
116.5
(NCH2CH2
10% NaHCO3 (2 × 25 mL), 10% HCl (2 × 10 mL), and brine CHvCCH3), 61.8, 61.5 (CHN), 53.0, 52.5 (CH2N), 35.9, 35.2
(25 mL) before it was dried over anhydrous MgSO4 and con- (CH3N) 29.1, 29.0 (CH2CH2CHN), 25.7 (vCHCH2N), 24.5
centrated under reduced pressure. The residue was purified (CH2CCH3), 24.2, 24.0 (NCH2CH2), 23.25, 22.64 (CH3Cv),
by flash chromatography on silica gel (EtOAc : hexane 4 : 6) 21.64 (CH3CCH3), 16.65 (CH3CCH3). νmax (neat, cm−1) 2972
yielding 21 as a colorless oil (166 mg, 50%). δH (400 MHz, (broad, N–H stretch), 1379 (C–N stretch), 1161, 1051 and 1022
CDCl3) 5.24 (2 H, dd, J = 14.6 and 8.0, NCHCH2CHv and (C–N stretch), 950, 879, 815; HRMS (APCI+) 208.2057, C14H26N
vCHCH2CvO), 4.70–4.54 (0.5 H, m, CHN), 3.75 (0.5 H, dd, requires 208.2065; m.p. 131–133 °C, (S)-11 αD −62.2 (MeOH,
J = 12.6 and 9.7, CHN), 3.01 (2 H, dd, J = 12.6, 6.8 Hz, c = 0.09), (R)-11 αD +57.1 (MeOH, c = 0.09). Data are in agree-
CH2CvO), 2.75 (1.5 H, s, CH3N), 2.72 (1.5 H, s, CH3N), ment with previous work.14a
2.20–1.86 (5 H, m, CH2CH2CHCH2), 1.71–1.88 (1 H, m,
CH2CH2CHCH2), 1.75 (s, 3 H, CNCH2vCHCH3), 1.68 (3 H, s,
vCCH3), 1.66 (3 H, s, vCCH3). (S)-21: αD −9.5, (CHCl3, c =
Conflicts of interest
0.9) (R)-21: αD +10, (CHCl3, c = 0.9) HRMS (EI+) 221.1779;
C
14H23NO requires 221.1780. Data are in agreement with pre- There are on conflicts of interest to declare.
vious work.14a
(R)- and (S)-N,4-Dimethyl-N-(4-methylpent-3-en-3-yl)cyclohex-3-
en-1 ammonium chloride (11)
Acknowledgements
To a stirred solution of 21 (41 mg, 0.19 mmol) in anhydrous We thank Dr Robert Jenkins and Simon Waller for assistance
diethyl ether at 0 °C, was added LiAlH4 (33 mg, 0.87 mmol). with GC-MS experiments and Dr Juan A. Faraldos (all Cardiff
The mixture was heated to reflux for 6 h then allowed to cool University) for valuable scientific discussions during this
to room temperature and stirred for a further 12 h. The reac- work. This work was supported by the UK’s Biotechnology and
tion was quenched by the addition of water (6 mL) and 15% Biological Sciences Research Council (BBSRC) through grants
NaOH (6 mL) at 0 °C and stirred for 1 h at 0 °C. The white pre- (BB/H01683X/1, BB/M022463/1, BB/N012526/1). We thank
cipitate was removed by filtration on Celite and the filtrate was Cardiff University for the provision of a postgraduate student-
extracted with diethyl ether (2 × 25 mL). The combined ship to M. L.
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