Characterisation of the broadly-specific O-methyl-transferase jerf from the late stages of jerangolid biosynthesis

Article abstract of DOI:10.3390/molecules21111443

We describe the characterisation of the O-methyltransferase JerF from the late stages of jerangolid biosynthesis. JerF is the first known example of an enzyme that catalyses the formation of a non-aromatic, cyclic methylenolether. The enzyme was overexpressed in E. coli and the cell-free extracts were used in bioconversion experiments. Chemical synthesis gave access to a series of substrate surrogates that covered a broad structural space. Enzymatic assays revealed a broad substrate tolerance and high regioselectivity of JerF, which makes it an attractive candidate for an application in chemoenzymatic synthesis with particular usefulness for late stage application on 4-methoxy-5,6-dihydro-2H-pyran-2-one-containing natural products.

Full text of DOI:10.3390/molecules21111443

molecules
Communication
Characterisation of the Broadly-Specific
O-Methyl-transferase JerF from the Late Stages of
Jerangolid Biosynthesis
Steffen Friedrich 1, Franziska Hemmerling 1,2, Frederick Lindner 2, Anna Warnke 1,
Johannes Wunderlich 2, Gesche Berkhan 1,2 and Frank Hahn 1,2,
*
1
Zentrum für Biomolekulare Wirkstoffe, Leibniz-Universität Hannover, Schneiderberg 38, 30167 Hannover,
Germany; steffen.friedrich@oci.uni-hannover.de (S.F.); franziska.hemmerling@oci.uni-hannover.de (F.H.);
anna.warnke@oci.uni-hannover.de (A.W.); gesche.berkhan@oci.uni-hannover.de (G.B.)
Professur für Organische Chemie (Lebensmittelchemie), Fakultät für Biologie, Chemie und
Geowissenschaften, Universitätsstraße 30, 95447 Bayreuth, Germany;
2
frederick.lindner@uni-bayreuth.de (F.L.); johannes.wunderlich1@uni-bayreuth.de (J.W.)
Correspondence: frank.hahn@uni-bayreuth.de; Tel.: +49-921-55-553660; Fax: +49-921-55-555365
*
Academic Editor: Tobias A. M. Gulder
Received: 4 October 2016; Accepted: 21 October 2016; Published: 29 October 2016
Abstract: We describe the characterisation of the O-methyltransferase JerF from the late stages of
jerangolid biosynthesis. JerF is the first known example of an enzyme that catalyses the formation of
a non-aromatic, cyclic methylenolether. The enzyme was overexpressed in E. coli and the cell-free
extracts were used in bioconversion experiments. Chemical synthesis gave access to a series of
substrate surrogates that covered a broad structural space. Enzymatic assays revealed a broad
substrate tolerance and high regioselectivity of JerF, which makes it an attractive candidate for an
application in chemoenzymatic synthesis with particular usefulness for late stage application on
4-methoxy-5,6-dihydro-2H-pyran-2-one-containing natural products.
Keywords: methyltransferases; enzymes; chemoenzymatic synthesis; natural products;
chemoselectivity; methylenolethers
1. Introduction
α
-Pyrones and
examples for biologically active
extensively used as fragrances, anticoagulants or rodenticides [
have also been identified as signalling molecules in bacterial communication [2].
γ
-pyrones are abundant structures in microbial natural products. Prominent
-pyrones are coumarin and isocoumarin derivatives, which are
]. More recently, 4-hydroxyl- -pyrones
α
1
α
O-Methylated pyrones occur in polyketide natural products with highly interesting biological
activity such as enterocin (2) and (+)-(R)-aureothin (6), which are produced by Streptomyces thioluteus
and Streptomyces maritimus, respectively (see Scheme 1). For both compounds, methyl transfer
takes place during the final tailoring steps of their biosynthesis and is catalyzed by class I
S-(50-adenosyl)-L-methionine (SAM)-dependent O-methyltransferases (O-MTs; EncK for
for 6) ]. Although EncK and AurI possess very high sequence similarity, they react with different
2, AurI
[
3
chemoselectivity on the same structural element [4,5].
Molecules 2016, 21, 1443; doi:10.3390/molecules21111443
www.mdpi.com/journal/molecules

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Scheme 1. Enterocin (
active bacterial polyketides harbouring O-methylated pyrone structures. (
O-methyltransferases AurI and EncK are known to methylate 4-hydroxyl-pyrones
(b) JerF is supposed to methylate the enol tautomer 7 in the course of jerangolid biosynthesis [6–9].
2
), (+)-(R)-aureothin (
6
) and jerangolid D (10) are examples for biologically
) The SAM-dependent
and , respectively;
a
1
3
Extensive studies by Hertweck and Moore showed that AurI methylates the enol in
-pyrone , whereas EncK methylates the 5-hydroxyl group of desmethyl-5-deoxyenterocin (
form the -pyrone 10 12]. EncK was able to complement O-methylation activity in a aurH mutant
of the aureothin producer. Interestingly, this supplemented strain produced the aureothin derivative
, showing that EnkC keeps its natural 4-O-selectivity for methylation while at the same time being
3
to form
the
γ
4
1
) to
γ
2
[
–
∆
5
highly substrate tolerant. Such O-methyltransferases are highly interesting candidates for application
in combinatorial biosynthesis [5].
Another promising approach for accessing novel natural product derivatives is the application
of biosynthesis enzymes for chemoenzymatic synthesis [3,13]. Methyltransferases are particularly
attractive candidates for this purpose. Not only do they introduce specific methylation patterns on
complex structures and thus enable streamlining a synthetic route by exploiting their high chemo-
and regioselectivity. Furthermore, it has been shown on several examples that the tolerance of MTs
regarding the transferred group can be used to bring in alternative residues. For example, propargyl
groups can be introduced onto specific positions and later addressed for biorthogonal coupling
reactions like click chemistry [14
–17]. Apart from natural product synthesis, this strategy has also been
used for site specific labelling of biomolecules like DNA, RNA or proteins [18–21].

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The jerangolids (jerangolid D (10) is shown in Scheme 1) are myxobacterial, reduced polyketide
natural products with unusual structural elements, resulting from extensive tailoring of the primary
polyketide synthase (PKS) product. They contain a 3-(hydroxymethyl)-4-methoxy-5,6-dihydro-2H-
pyran-2-one, which is formed via action of the PKS-thioesterase (TE) domain followed by the O-MT
JerF, the Rieske-FeS-cluster protein JerL and the monooxygenase JerO [
formation of a methylenolether starting from a ketone, thus leading to a cyclic, non-phenolic methyl
ether. Apart from the two above-mentioned examples from enterocin ( ) and aureothin ( ) biosynthesis,
6]. JerF is responsible for the
2
6
no O-MTs that form methylated pyrones have been characterised yet.
2. Results
2.1. Synthesis of Substrate Surrogates
To confirm the postulated biosynthetic role of JerF and to evaluate its potential for
chemoenzymatic synthesis, we set out to investigate the catalytic activity of JerF in vitro. Synthetic
precursor surrogates of varying complexity (rac
structural space (Scheme 2; Figures S1–S26). Two series of compounds were obtained, containing either
the 3-methyl-6-vinyldihydro-2H-pyran-2,4(3H)-dione (rac 14d ) that is present in the biosynthetic
precursor or 3-desmethyl analogs (rac 14a ). All substrates were readily available by vinylogous
aldol reaction of -ketoester enolates 11a or 11b, respectively, followed by lactonization under basic
conditions (Scheme 2a) [22 25]. The relative orientation of the substituents on ring positions 3 and
6 in rac 14d
was shown to be predominantly syn, according to 1H-NMR spectroscopy and NOE
correlation spectroscopy (see NMR spectra and Figure S26 in the Supplementary Materials). The
methylenolethers rac 15a were synthesised from rac 14a by O-methylation using MeI and K2CO3
or NaH, respectively.
-14a–h) were synthesised that covered a broad
-
–h
-
–c
β
–
-
–h
-
–g
-
–g
Scheme 2. (a) General synthetic route to JerF precursor surrogates rac-14a–h. Product surrogates were
obtained by chemical methylation using MeI and base. For exact reaction conditions, see the materials
and methods section; (b) Substrate surrogates that were applied in the assays.

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2.2. Cloning, Expression and Establishment of Assay Conditions
A codon-optimised gene of jerF (GenBank accession number: ABK32292.1) was cloned into
pET28a(+), pET20b(+) and pCOLDI plasmids for overexpression in E. coli BL21 (DE3). Only for
the C-terminally His6-tagged fusion protein (derived from expression of jerF-pET20b(+)), minor
amounts of purified but catalytically inactive protein were obtained after Ni-affinity chromatography.
We therefore decided to conduct bioconversion experiments with the cell-free extracts.
Catalytically active cell-free extracts were obtained from jerF-pCOLDI expression in E. coli
◦
BL21 (DE3) at 16 C for 16 h in the presence of 0.1 mM IPTG. Cell disruption was achieved by
sonication on ice in pH 8.8 reaction buffer (40 mM Tris HCl, 100 mM NaCl). Initial bioconversion
experiments were carried out with the racemic mixture of substrate surrogates rac
5 mM MgCl2 and 0.97 mM SAM tosylate for 16 h at 28 C (Figure 1, for unprocessed spectra see
-
14d (0.25 mM),
◦
Figures S48–S53) [26,27]. HPLC-MS analysis showed that rac-14d was almost completely converted
into the respective O-methylated product mixture rac-15d (Figure 1f).
Figure 1. HPLC-MS analysis of in vitro conversions catalyzed by JerF. Chromatograms are shown
for m/z = 191 (blue trace) and m/z = 205 (red trace). (
a) synthetic rac-14d and synthetic rac
-15d
;
(
b) cell-free extract containing JerF with 0.94 mM SAM tosylate in reaction buffer (40 mM Tris
HCl, 100 mM NaCl, 5 mM MgCl2, pH 8.8); (c) reaction buffer with SAM tosylate and rac
-14d
;
(d) heat-inactivated cell-free extract containing JerF in reaction buffer with SAM tosylate and rac-14d;
(
(
e
) cell-free extract from expression of pCOLD-I (devoid of jerF) with SAM tosylate and rac
-14d;
f
) cell-free extract from expression of jerF_pCOLD-I with SAM tosylate and rac 14d. Conditions of the
-
conversion assay in (f): 0.25 mM rac-14d, 5 mM MgCl2, 1.0 mM SAM-tosylate, 0.2 mL cell-free extract
◦
(2.7 mg/mL), 28 C, 16 h; rac-14d: [M + Na]+ = 191.0684, rac-15d: [M + Na]+ = 205.0841.
To unambiguously assign the methylation activity to the jerF gene product, we conducted a series
of control experiments. Methylation occurred only in the presence of MgCl2, SAM tosylate and
the lysate from the jerF-pColdI expression (Figure 1f). If any of these components was left out, no
formation of rac-15d was obtained (Figure 1b,c). The same was true if the lysate was denatured by
heat treatment prior assaying, which attributes the activity to a component of the cell lysate (Figure 1d).
The lysate of a pColdI vector expression (devoid of jerF) in E. coli BL21 did also not cause the formation
of rac-15d, clearly highlighting that the expression product of jerF is responsible for the observed
activity (Figure 1e).

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2.3. Comparative Assaying of Synthetic Substrates and Assay Upscaling
Under the conditions established for rac
-
14d, compound rac
-
14h was completely methylated,
highlighting the broad substrate tolerance of JerF (Figure 2). The higher conversion compared to
rac-14d reflects the closer structural similarity of rac-14h to the proposed biosynthetic precursor 9.
Figure 2. HPLC-MS analysis of in vitro conversions of rac-14h catalyzed by JerF. Chromatograms
are shown for m/z = 259 (blue trace) and m/z = 273 (red trace). (
incubation in buffer; ( ) incubation of rac-14h with cell-free extract containing JerF; (
of rac-14h with cell-free extract containing JerF and SAM tosylate in reaction buffer. The minimal
conversion is probably caused by residual amounts of SAM in the cell-free extract. ( ) HR-MS analysis
of the assay product of ( ); ( ) HR-MS analysis of the assay product of ( ); Conditions of the conversion
a) synthetic rac-14h after overnight
b
c) incubation
d
b
e
c
ass◦ay: 0.25 mM rac-14h, 5 mM MgCl2, 1.0 mM SAM-tosylate, 0.2 mL cell-free extract (3.0 mg/mL),
28 C, 16 h, pH 8.8.
Insertion of the other synthetic substrate surrogates rac-14b, rac-14c and rac-14e-g under similar
conditions showed partial conversion of all substrates containing a 3-Me group (rac-14e-g) and full
conversion of the non-branched substrates rac-14b and rac-14c (Figures S54–S58). In all cases, an
exclusive methylation on the 4-O and no reaction on 2-O or C-3 occurred, showing that JerF acts
highly chemo- and regioselectively. However, for the non-branched substrates rac-14b and rac-14c
,
the absolute amount of formed product was unexpectedly low.
Overnight incubation of compounds rac-14b, rac-14c and rac-14e–g in the absence of JerF
revealed that all substrates undergo slow, spontaneous degradation at pH 8.8. This trend is more
pronounced for the non-branched lactones rac-14b and rac-14c and seems to be accelerated by
uncharacterised components of the lysate. Accordingly, attempts to conduct the reaction with rac-14d
on the semi-preparative scale (up to 7 mg starting material) led to hardly reproducible results and
yields below 10%.
A markedly increased stability of the lactones as well as the corresponding methylenolethers
rac
with compound mixture rac
and compound stability were observed at pH 7.5 (Figure S59). The experiments were thus repeated
-
15b
,
rac
-
15c and rac
-
15e
–g was observed at near-neutral pH. In comparative enzymatic assays
-14e and JerF at different pH values, the best results in terms of conversion

Molecules 2016, 21, 1443
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at this pH value with substrates rac
-
-
14b
14c
Figures S60–S64). The compound mixture rac
presence of small amounts of starting material was still visible. The 3-desmethyl substrates rac
and rac 14c were fully methylated according to HPLC-MS analysis. However, complete degradation of
rac 14b and rac 14c without any conversion into rac 15b and rac 15c was observed in some repetitions
,
,
rac
rac
-
-
14c and rac
14e and rac
14g was also methylated to a large extend, however the
14b
-
14e
–
g
. Full conversion was reproducibly
obtained for compounds rac
-14b
,
rac
-14f (Figure 3, for unprocessed spectra see
-
-
-
-
-
-
-
of the experiment, indicating that destructive side reactions caused by the lysate could be partially
responsible for this positive result.
Figure 3. HPLC-MS analysis of in vitro conversions catalyzed by JerF. (
for m/z of substrates rac 14b rac 14c and rac 14e (blue traces) and the respective O-methylated
products rac 15b rac 15c and rac 15e (red traces). General conditions of the conversion assays:
a–j) Chromatograms are shown
-
,
-
-
–g
-
,
-
-
–g
cell-free extract of the JerF expres◦sion (7.8 mg/mL) with 4.18 mM SAM tosylate, 5 mM MgCl2 and
0.25 mM substrate incubated at 28 C for 20 h in reaction buffer (25 mM HEPES, 100 mM NaCl, pH 7.5).
rac
rac
-
-
14b: [M + Na]+ = 205.0820; rac
14c: [M + Na]+ = 239.0663; rac
-
-
14e/rac
-
15b: [M + Na]+ = 219.0976; rac 15e: [M + Na]+ = 233.1133;
-
15c: [M + Na]+ = 253.0820; rac 15f: [M + Na]+ = 267.0976;
-
14f/rac
-
rac-14g: [M + Na]+ = 289.1759; rac-15g: [M + Na]+ = 303.1915.
Reaction upscaling was also more successful at pH 7.5 and gave reproducible results (Table 1,
Figures S43–S47). Approximately 4 mg of compounds rac 14c rac 14e and rac 14f were individually
incubated with the cell-free extract from a jerF expression in a total assay volume of 10 mL. Conversions
of 27%–42% into the respective methylenolethers rac 15c rac 15e and rac 15f were obtained. The crude
products of rac 15e and rac 15f were partially purified by column chromatography on silica gel.
In both cases, an aliphatic impurity was co-purified, which could not be removed.
-
,
-
-
-
,
-
-
-
-

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Table 1. Conversions, yields and scale of the semi-preparative conversions with JerF.
Conversion a
Crude yield
Scale
b
Compound
e.e.
rac-14c
rac-14e
rac-14f
42%
27%
35%
n.d.
38%c
13%c
84%
42%
<10%
4.6 mg
4.6 mg
4.3 mg
a
The conversion was determined by integration of the signals of the protons at position 6 of the dione in the
1H-NMR spectra; b The e.e. was calculated from the peak areas of the chiral HPLC chromatograms. Crude
yield is given after column chromatography on silica gel. A non-removable, aliphatic impurity was co-purified
(see Figures S45 and S47).
c
The crude products of the semi-preparative conversions as well as the samples from column
chromatography were analysed by chiral HPLC. In the cases of rac 14c and rac 14e, the racemic
starting material was converted into product enriched in one stereoisomer with an enantiomeric
ratio of 92:8 and 71:29, respectively (Table 1). For rac 14f, an only negligible enantiomeric excess
-
-
-
was observed. JerF thus discriminates between the inserted stereoisomers, however, with a strong
dependence on the substrate structure.
3. Discussion
We were able to characterise the O-methyltransferase JerF from jerangolid biosynthesis by assaying
of the enzyme in bioconversion experiments with synthetic substrate surrogates. JerF is the first
characterised case of an O-methyltransferase that forms a cyclic, non-aromatic methylenolether. The
enzyme shows promising substrate tolerance. It accepts aliphatic and aromatic residues (R in Scheme 3)
of varying size as well as substrates that lack the methyl group on C-3, which is present on the natural
precursor. Furthermore, the enzyme is fully selective for methylation on 4-O and is not reactive towards
the other two potential methylation sites at 2-O and C-3. During chemical synthesis of substrates
rac-15a–h, only C-3-methylated side products were obtained, suggesting that an inherent selectivity for
4-O-methylation exists under the conditions applied. In this context, it would be interesting to evaluate
if the O-MT AurI keeps its confirmed 2-O-selectivity in the reaction with this kind of substrates.
Scheme 3.
JerF shows broad substrate tolerance in the reaction with diverse
6-vinyldihydro-2H-pyran-2,4(3H)-diones rac-14a–h.
Reactions with the enzyme could be conveniently performed using the cell-free extract from a
jerF expression. Reactions on the analytical scale with substrates rac-14a–h proceeded with high to
complete conversion. Problems arising from slow spontaneous degradation during the reaction could
be reduced by performing the reaction at pH 7.5.
Upscaling of the enzymatic reactions with substrate mixtures rac-14c, rac-14e and rac-14f was
successful at pH 7.5, giving conversions of 27%–42%. A further optimisation of the reaction conditions
will probably improve this result. It is furthermore known that SAM-dependent MTs are often inhibited
by S-adenosylhomocysteine (SAH) that is formed during the reaction. Addition of a SAH-hydrolase
or SAH nucleosidase could thus also be helpful. Analysis of the semi-preparative scale conversions

Molecules 2016, 21, 1443
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by chiral HPLC revealed a discrimination of JerF between the inserted stereoisomers. The degree of
selectivity strongly depended on the constitution of the inserted substrates.
The insights gained about relevant features of JerF like substrate tolerance and chemoselectivity
suggest further investigations on its applicability in chemoenzymatic synthesis. Future studies will
concentrate on a thorough optimisation of the enzyme overexpression and the reaction conditions on
the analytical and the (semi)preparative scale. Further studies on its substrate tolerance will help to
evaluate the scope of the enzyme. Finally, the enzyme will be applied in the chemoenzymatic total
synthesis of 4-methoxy-5,6-dihydro-2H-pyran-2-one-containing natural products.
4. Materials and Methods
4.1. General Information
4.1.1. Chemistry Methods and Materials
All reactions were performed in oven dried glassware under an atmosphere of nitrogen
gas unless otherwise stated. Dry solvents were purchased from Sigma-Aldrich Chemie GmbH
(Steinheim, Germany) and Acros (Geel, Belgium) or taken out of a solvent system from M. Braun
(Garchingen, Germany). Dry reagents were ordered from Sigma-Aldrich, Arcos, abcr GmbH (Karlsruhe,
Germany) and Roth (Karlsruhe, Germany). NMR spectra were recorded with DRX-500, DPX-400
and AVANCE-400 instruments (Bruker, Billerica, MA, USA) with the residual solvent signal as
internal standard (CHCl3 = 7.26 ppm). multiplicities are described using the following abbreviations:
s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, b = broad. 13C-NMR spectra are reported
as values in ppm relative to residual solvent signal (CHCl3 = 77.06 ppm) as internal standards. The
multiplicities are elucidated using the distortionless◦enhancement by polarisation transfer (DEPT)
◦
spectral editing technique, with secondary pulses at 90 and 135 . Multiplicities are reported using the
following abbreviations: q (quarternary carbon), t (tertiary carbon = methine), s (secondary carbon
= methylene), p = (primary carbon = methyl). High resolution mass spectra are obtained with a
Micromass LCT via loop-mode injection from an Alliance 2695 HPLC system (Waters, Milford, MA,
USA). Alternatively, a Micromass Q-TOF in combination with a Waters Acquity Ultra performance LC
system is employed. Ionisation is achieved by ESI or APCI. Modes of ionisation, calculated and found
mass are given. Reversed phase-HPLC-applications were performed with membrane-filtrated and
double distilled water as well as commercial available HPLC-grade solvents (methanol or acetonitrile).
Semi-preparative HPLC was performed with a Merck Hitachi HPLC system (Darmstadt, Germany;
Pump L-7150, Interface D-7000, Diode Array Detector L-7450) under use of C18-SP stationary phase.
Solvents, columns, operating procedures and retention times (tR) are given with the corresponding
experimental and analytical data. (Abbreviations: PE = petroleum ether; EtOAc = ethyl acetate).
Chiral HPLC-applications were performed with a Waters Alliance HPLC (Waters 2695 Separation
Module, Waters 2487 Dual
λ Absorbance Detector). In all cases the flow was 0.8 mL/min and the
detection wavelength was 215 nm. Gradient conditions were: 1: OD-3, n-hexane:i-PrOH = 93:7; 2:
AD-H, n-hexane:i-PrOH = 90:10; 3: AD-H, n-hexane:i-PrOH = 95:5; 4: OD-3, n-hexane:i-PrOH = 80:20;
5: OD-3, n-hexane:i-PrOH = 85:15; 6: AD-H, n-hexane:i-PrOH = 90:10. The retention times (tR) are
given with the corresponding experimental and analytical data (abbreviations for columns: OD-3:
Daicel Chiralcel® OD-3; AD-H: Daicel Chiralpak® AD-H). Samples were applied in form of 10
µL of
membrane-filtrated solution in a concentration of approximately 1 mg/mL in n-hexane:iPrOH = 85:15.
4.1.2. Biochemistry Methods and Materials
All chemicals and antibiotics were purchased from Sigma-Aldrich and Roth. Cell disruption
was conducted by sonication (Sonoplus Typ UW3100) from Bandelin (Berlin, Germany). His-bind
nickel chelate chromatography resin was purchased from Novagen. Millipore Amicon® ultra

Molecules 2016, 21, 1443
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centrifugal filters (10,000 and 30,000 MW Cut-off) and PD-10 desalting columns from GE Healthcare
(Buckinghamshire, UK) were used for protein concentration and buffer exchange respectively.
4.2. Synthesis of Substrate Surrogates
4.2.1. General Procedures
Aldol Reaction
A solution of LDA was freshly prepared by adding n-BuLi (2.5 M in hexane, 2.5 equiv.)
to diisopropylethylamine (0.7 M, 2.5 equiv.) in THF at
−
78 ◦C. The solution was stirred at
room temperature for 30 min and after cooling to
−
78 ◦C, DMPU (1.0 equiv.) was added.
Methyl-2-methyl-3-oxobutanoate or methyl-3-oxobutanoate (1 M, 1.0 equiv.), respectively, in THF was
added and the solution was stirred for further 50 min. The aldehyde (1.1 equiv.) was added and the
solution stirred for 2 h. The reaction was quenched by addition of 2 M HCl. After separation of the
layers, the aqueous layer was extracted by Et2O and the combined organic layers were dried over
MgSO4. The solvent was removed in vacuo.
Lactonization
The product of the aldol reaction was dissolved in 1 M KOH and stirred at room temperature for
5 h. After cooling to 0 ◦C, 2 M HCl was added until a pH value of 0 was reached. The resulting solid
was filtrated, washed with H2O and the desired product purified by column chromatography.
O-Methylation of the Dihydropyran-2,4-diones
After dissolving the lactone (0.2 M, 1.0 equiv.) in DMFabs, the solution was cooled to 0 ◦C and
MeI (1.0 equiv.) and K2CO3 (1.5 equiv.) were added. After 1 h, the solution was warmed to room
temperature and stirred overnight. The layers were separated and the aqueous layer was extracted
with EtOAc. The combined organic layers were washed with brine, dried over MgSO4 and the solvent
was removed in vacuo. The crude product was purified by column chromatography.
O-Methylation of the 3-Methyldihydropyran-2,4-diones
After dissolving the lactone (0.2 M, 1.0 equiv.) in THFabs, the solution was cooled to 0 ◦C and
NaH (60% in mineral oil, 1.3 equiv.) was added. After 1 h, MeI (1.2 equiv.) was added and the solution
was warmed to room temperature. After stirring overnight, the reaction was quenched by the addition
of H2O. After separation of the layers, the aqueous layer was extracted with EtOAc, the combined
organic layers were washed with brine and dried over MgSO4. The solvent was removed in vacuo and
the crude product was purified by column chromatography.
4.2.2. Substrate Synthesis
Compounds rac-14a
–
g
and rac-15b
–
h
were synthesised according to the route
shown in Schemes 4–6.
Scheme 4. Synthesis of compound rac-14a [28–30].

Molecules 2016, 21, 1443
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Scheme 5. General synthetic route to substrates rac-14b–g and reference compounds rac-15b–g.
Scheme 6. Synthesis of dione rac-14h from TBS-protected Roche ester I.
(E)-6-(Prop-1-en-1-yl)dihydro-2H-pyran-2,4(3H)-dione (rac-14a)
Under nitrogen atmosphere, sodium hydride (60% suspension, 900 mg, 22.5 mmol, 1.2 equiv.)
was suspended in 40 mL dry THF and cooled to 0 ◦C. To this reaction mixture, methyl acetoacetate
(2.0 mL, 18.7 mmol, 1.0 equiv.) was added dropwise. After gas evolution had ceased, the reaction
was cooled to
−
78 ◦C followed by dropwise addition of n-BuLi (8.3 mL, 20.6 mmol, 1.1 equiv., 2.5 M
78 ◦C.
in hexanes). The reaction was allowed to warm to 0 ◦C for 30 min and then cooled again to
−
(E)-crotonaldehyde (1.7 mL, 20.6 mmol, 1.1 equiv.) was added dropwise over 5 min and the reaction
was stirred for 30 min at r.t. The reaction was quenched by addition of 20 mL saturated NH4Cl/H2O
(1:1) at 0 ◦C. The aqueous solution was washed twice with 50 mL EtOAc and then acidified to pH 1
using concentrated hydrochloric acid. The resulting precipitate was dissolved in 20 mL EtOAc and the
aqueous phase was extracted twice with 20 mL EtOAc. The combined organic phases were dried over
Na2SO4 and filtered. The solvents were removed under reduced pressure. Compound rac-14a was
obtained as a pale-yellow solid (20%, 588 mg, 4.50 mmol).
Methyl-(E)-5-hydroxy-2-methyl-3-oxooct-6-enoate (rac-13d)
Under argon atmosphere, diisopropylamine (1.9 mL, 13.5 mmol, 2.5 equiv.) was dissolved in
20 mL dry THF and cooled to
−
78 ◦C. To this solution, n-BuLi (5.3 mL, 13.5 mmol, 2.5 M in hexanes,
2.5 equiv.) was added dropwise. The reaction was allowed to warm to 0 ◦C for 30 min. At −78 ◦C,
DMPU (670 µL, 5.40 mmol, 1.0 equiv.) was added dropwise and the resulting mixture was stirred

Molecules 2016, 21, 1443
11 of 23
for 30 min. Methyl-3-oxobutanoate (702
µL, 5.40 mmol, 1.0 equiv.) was dissolved in 5 mL dry
THF and this solution was added to the reaction mixture followed by stirring for 50 min at −78 ◦C.
(E)-crotonaldehyde (490 µL, 5.90 mmol, 1.1 equiv.) was then added followed by stirring for 2 h
at −78 ◦C. The reaction was quenched by addition of 30 mL 2 M hydrochloric acid at −78 ◦C and
allowed to warm to r.t. The aqueous phase was extracted three times with 50 mL Et2O. The combined
organic phases were dried over Na2SO4 and filtered. The solvents were removed under reduced
pressure. 1.06 g of crude rac-13d were obtained and used without further purification.
(E)-3-Methyl-6-(prop-1-en-1-yl)dihydro-2H-pyran-2,4(3H)-dione (rac-14d)
Crude methyl ester rac
-
13d (414 mg, 2.10 mmol, 1.0 equiv.) was dissolved in 20 mL 1 M potassium
◦
hydroxide solution and was stirred at r.t. for 7 h. The solution was cooled to 0 C followed by addition
of 6 M hydrochloric acid to pH 1.0. The resulting precipitate was filtered using a glass frit and the
obtained crystals were dried under reduced pressure to give compound rac-14d as yellow crystals
(263 mg, 1.50 mmol, 70%, syn:anti = 5:1).
(E)-4-Methoxy-3-methyl-6-(prop-1-en-1-yl)-5,6-dihydro-2H-pyran-2-one (rac-15d)
Under nitrogen atmosphere, (E)-3-Methyl-6-(prop-1-en-1-yl)dihydro-2H-pyran-2,4(3H)-dione
(
25a, 50.0 mg, 300
µ
mol, 1.0 equiv.) was dissolved in 3.0 mL dry THF and the resulting solution was
mol, 1.2 equiv.) was added and the reaction
L, 360 mol, 1.2 equiv.). The reaction
◦
cooled to 0 C. NaH (60% suspension, 13.9 mg, 350
mixture was stirred for 30 min followed by addition of MeI (22
µ
µ
µ
mixture was stirred for 1 h at r.t. and then quenched by the addition of 3 mL H2O. The aqueous phase
was extracted three times with 10 mL EtOAc. The combined organic phases were washed twice with
brine and then dried over Na2SO4. After filtration, the solvents were removed under reduced pressure.
Purification by flash column chromatography (petroleum ether (PE/EtOAc = 4:1 to 2:1) yielded the
product (rac-15d, 30 mg, 165 µmol, 55%) as a pale-yellow solid.
(E)-6-(3-Methylbut-1-en-1-yl)dihydro-2H-pyran-2,4(3H)-dione (rac-14b)
Following the general procedures 1 and 2, dione rac-14b was prepared from LDA (816 mg,
7.65 mmol, 2.5 equiv.), DMPU (333 mL, 2.78 mmol, 1.0 equiv.), methyl-3-oxobutanoat (299 mL,
2.78 mmol, 1.0 equiv.) and (E)-4-methylpent-2-enal (300 mg, 3.06 mmol, 1.1 equiv.). After column
chromatography on silica gel (PE/EtOAc = 5:1
622 µmol, 20%) was obtained as a yellow solid.
→
2:1), the mixture of the diones rac-14b (113 mg,
(E)-4-Methoxy-6-(3-methylbut-1-en-1-yl)-5,6-dihydro-2H-pyran-2-one (rac-15b)
Following the general procedure 3, methoxy-pyran-2-one rac 15b was prepared from dione
14b (50.0 mg, 270 mol, 1.0 equiv.), MeI (17.0 L, 270 mol, 1.0 equiv.) and K2CO3 (56.0 mg,
400 µmol, 1.5 equiv.). After column chromatography on silica gel (PE/EtOAc = 4:1 2:1), the mixture
-
rac
-
µ
µ
µ
→
of the methoxy-pyran-2-ones rac-15b (9.40 mg, 48.0 µmol, 18%) was obtained as a yellow solid.
(E)-3-Methyl-6-(3-methylbut-1-en-1-yl)dihydro-2H-pyran-2,4(3H)-dione (rac-14e)
Following the general procedures 1 and 2, dione rac
5.10 mmol, 2.5 equiv.), DMPU (244 mL, 2.04 mmol, 1.0 equiv.), methyl-2-methyl-3-oxobutanoate
265 mg, 2.04 mmol, 1.0 equiv.) und (E)-4-methylpent-2-enal (200 mg, 2.04 mmol, 1.0 equiv.). After
-14e was prepared from LDA (546 mg,
(
column chromatography on silica gel (PE/EtOAc = 2:1), the mixture of the diones rac
1.13 mmol, 55%, d.r. = 10:1) was obtained as a yellow solid.
-14e (222 mg,
(E)-4-Methoxy-3-methyl-6-(3-methylbut-1-en-1-yl)-5,6-dihydro-2H-pyran- 2-one (rac-15e)
Following the general procedure 4, methoxy-pyran-2-one rac 15e was prepared from dione
14e (E)-3-methyl-6-(3-methylbut-1-en-1-yl)dihydro-2H-pyran-2,4(3H)-dion (50.0 mg, 260 mol,
-
rac
-
µ

Molecules 2016, 21, 1443
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1.0 equiv.), NaH (13.2 mg, 330
µmol, 1.3 equiv.) and MeI (20.0
µL, 312
µmol, 1.2 equiv.). After column
chromatography on silica gel (PE/EtOAc = 5:1
rac-15e (5.20 mg, 25.0 µmol, 10%) was obtained as a yellow solid.
→
2:1), the mixture of the methoxy-pyran-2-ones
Methyl-(E)-5-hydroxy-3-oxo-7-phenylhept-6-enoate (rac-13c)
NaH (60% in mineral oil, 440 mg, 11.0 mmol, 1.1 equiv.) was solved in 25 mL THFabs at 0 ◦C and
methyl-3-oxobutanoate (1.08 mL, 10.0 mmol, 1.0 equiv.) was added. After 20 min, the solution was
◦
cooled to
−
78 C, n-BuLi (2.5 M in hexane, 4.40 mL, 11.0 mmol, 1.1 equiv.) was added and it was stirred
for 1 h at this temperature. After adding cinnamic aldehyde (1.39 mL, 11.0 mmol, 1.1 equiv.) to the
reaction mixture, the solution was stirred for 3 h at
◦
−
78 C. The reaction was quenched by the addition
of 2 M HCl. After separation of the layers, the aqueous layer was extracted with Et2O (3 × 50 mL),
the combined organic layers were dried over MgSO4 and the solvent was removed in vacuo. After
purification by column chromatography on silica gel (PE/EtOAc = 4:1), the desired alcohol rac-13c
(632 mg, 2.72 mmol, 27% yield) was obtained as an orange solution.
(E)-6-Styryldihydro-2H-pyran-2,4(3H)-dione (rac-14c)
Methyl-(E)-5-hydroxy-3-oxo-7-phenylhept-6-enoate (rac
solved in 2 mL MeOHabs and K2CO3 (89.3 mg, 650 mol, 1.5 equiv.) was added at room temperature.
The reaction mixture was stirred for 3 h at r.t., the resulting solid was filtered and washed with H2O.
-13c, 100 mg, 430 µmol, 1.0 equiv.) was
µ
After purification by column chromatography on silica gel (PE/EtOAc = 4:1
rac-13c (58.8 mg, 272 µmol, 63% yield) was obtained as a colourless solid.
→
2:1), the desired lactone
(E)-4-Methoxy-6-styryl-5,6-dihydro-2H-pyran-2-one (rac-15c)
Following the general procedure 3, methoxy-pyran-2-one rac
14c (50.0 mg, 230 mol, 1.0 equiv.), MeI (14.5 L, 230 mol, 1.0 equiv.) and K2CO3 (47.7 mg,
350 µmol, 1.5 equiv.). After column chromatography on silica gel (PE/EtOAc = 4:1 1:1), the mixture
-15c was prepared from dione
rac
-
µ
µ
µ
→
of the methoxypyran-2-ones rac-15c (17.0 mg, 130 µmol, 56%) was obtained as a yellow solid.
(E)-3-Methyl-6-styryldihydro-2H-pyran-2,4-(3H)-dione (rac-14f)
Following the general procedures 1 and 2, dione rac
1.92 mmol, 2.5 equiv.), DMPU (118 mL, 799 mol, 1.0 equiv.), methyl-2-methyl-3-oxobutanoate
100 mg, 0.77 mmol, 1.0 equiv.) and cinnamic aldehyde (101 L, 799 mol, 1.1 equiv.). After column
-14f was prepared from LDA (205 mg,
µ
(
µ
µ
chromatography on silica gel (PE/EtOAc = 2:1), the mixture of the diones rac-14f (67.3 mg, 292 µmol,
37%) was obtained as a white solid.
(E)-4-Methoxy-3-methyl-6-styryl-5,6-dihydro-2H-pyran-2-one (rac-15f)
Following the general procedure 4, methoxy-pyran-2-one rac
14f (25.0 mg, 109 mol, 1.0 equiv.), NaH (6.00 mg, 131 mol, 1.2 equiv.) and MeI (8.00
1.2 equiv.). After column chromatography on silica gel (PE/EtOAc = 4:1 2:1), the mixture of the
-
15f was prepared from dione
rac
-
µ
µ
µL, 131 µmol,
→
methoxypyran-2-ones rac-15f (3.50 mg, 14.3 µmol, 13%) was obtained as a white solid.
(E)-6-(Dec-1-en-1-yl)-3-methyldihydro-2H-pyran-2,4(3H)-dione (rac-14g)
Following the general procedures 1 and 2, dione rac
3.75 mmol, 2.5 equiv.), DMPU (180 mL, 1.50 mmol, 1.0 equiv.), methyl-2-methyl-3-oxobutanoat
195 mg, 1.5 mmol, 1.0 equiv.) und (E)-undec-2-enal (278 mg, 1.70 mmol, 1.1 equiv.). After column
chromatography on silica gel (PE/EtOAc = 10:1 1:1), the mixture of the diones rac 14g (279 mg,
1.05 mmol, 62%, d.r. = 12.5:1) was obtained as a yellow solid.
-14g was prepared from LDA (402 mg,
(
→
-

Molecules 2016, 21, 1443
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(E)-6-(Dec-1-en-1-yl)-4-methoxy-3-methyl-5,6-dihydro-2H-pyran-2-on (rac-15g)
Following the general procedure 4, methoxy-pyran-2-one rac
14f (50.0 mg, 190 mol, 1.0 equiv.), NaH (69.80 mg, 240 mol, 1.3 equiv.) and MeI (14.0
230 µmol, 1.2 equiv.). After column chromatography on silica gel (PE/EtOAc = 5:1 2:1), the mixture
-15g was prepared from dione
rac
-
µ
µ
µL,
→
of the methoxypyran-2-ones rac-15g (50 mg, 30.3 µmol, 16%) was obtained as a white solid.
Methyl-(R)-3-((tert-Butyldimethylsilyl)oxy)-2-methylpropanoate (I)
(R)-Methyl-3-hydroxy-2-methylpropionate (10.0 mL, 90.0 mmol, 1.0 equiv.), DMAP (110 mg,
0.90 mmol, 0.01 equiv.) and imidazole (9.9 g, 144 mmol, 1.6 equiv.) were dissolved in 100 mL ◦CH2Cl2.
TBSCl was dissolved in 20 mL CH2Cl2 and added dropwise to the reaction mixture at 0 C. The
resulting suspension was stirred at r.t. for 1 h. The reaction was quenched by the addition of 150 mL
H2O. The aqueous phase was extracted three times with 50 mL CH2Cl2. The combined organic phases
were washed once with H2O and dried over MgSO4. After filtration, the solvent was removed under
reduced pressure. The compound
further purification.
I was obtained as a pale-yellow oil (20.1 g) and was used without
(R)-3-((tert-butyldimethylsilyl)oxy)-N-methoxy-N,2-dimethylpropanamide (II)
TBS-protected Roche ester (5.00 g, 21.51 mmol, 1 equiv.) was solved in 40 mL THF.
I
N,O-Dimethylhydroxylamine hydrochloride (3.27 g, 33.6 mmol, 1.56 equiv.) was added and the
◦
resulting solution cooled to
added dropwise and stirred at
−
20 C. Isopropylmagnesium chloride (32 mL, 64.7 mmol, 3.01 equiv.) was
−
10 ◦C for 1 h. The reaction was quenched by the addition of 15 mL
saturated NH4Cl solution and three times extracted with Et2O. The combined organic layers were
dried over MgSO4 and the solvent removed in vacuo. After column chromatography on silica gel (15%
Et2O in hexane), the amide II (5.62 g, 21.5 mmol, 99% yield) was obtained as a yellow oil.
(R)-3-((tert-Butyldimethylsilyl)oxy)-2-methylpropanal (III)
◦
Amide II (2.51 g, 9.60 mmol, 1 equiv.) was solved in 10 mL THF and cooled to
−
78 C. DIBAL-H
solution (19.2 mL, 19.20 mmol, 2 equiv., 1 M in hexane) was added dropwise. After 1 h, the reaction
was quenched by pouring it into 20 mL of a saturated solution of K-Na-tartrate and stirring for 1 h. The
aqueous layer was three times extracted with Et2O and the combined organic layers were dried over
MgSO4. After removal of the solvents under reduced pressure and column chromatography on silica
gel, (PE/Et2O = 10:1) the aldehyde III (1.94g, 9.60 mmol, 99% yield) was obtained as a pale-yellow oil.
(S)-tert-Butyl-((2,4-dimethylpent-3-en-1-yl)oxy)dimethylsilane (IV)
To a suspension of bromo(isopropyl)triphenyl-
15 mL THF was slowly added n-BuLi (2.60 mL, 6.50 mmol, 1.7 equiv., 2.5 M in hexane) at
stirred at room temperature for 30 min until a dark-red colour appeared. It was cooled to
aldehyde III (774 mg, 3.83 mmol, 1 equiv.) in a small amount of THF was added dropwise. After 16 h,
water was added to the white suspension. The aqueous layer was three times extracted with CH2Cl2.
The combined organic layers were dried over MgSO4 and the solvent removed in vacuo. After column
chromatography on silica gel (PE), compound IV (600 mg, 2.63 mmol, 69% yield) was obtained.
λ
5-phosphane (2.95 g, 7.65 mmol, 2 equiv.) in
◦
−
78 C. It was
−
78 ◦C and
(S)-2,4-Dimethylpent-3-en-1-ol (V)
Alkene IV (570 mg, 2.50 mmol, 1 equiv.) was solved in 10 mL MeOH and PPTS (3.76 g, 14.97 mmol,
◦
6 equiv.) was added. After stirring for 24 h at 50 C, the solvent was removed under reduced pressure.
The residual solid was washed with 3 mL water and the aqueous layer was three times extracted with
CH2Cl2. The combined organic layers were washed with saturated NaHCO3 solution and saturated
NaCl solution and then dried over MgSO4. After removal of the solvent under reduced pressure and

Molecules 2016, 21, 1443
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column chromatography on silica gel (PE/Et2O = 5:1), alcohol
obtained as a colourless oil.
V
(262 mg, 2.30 mmol, 92% yield) was
(R,E)-Ethyl-4,6-dimethylhepta-2,5-dienoate (VII)
◦
Alkohol
V
(560 mg, 4.90 mmol, 1 equiv.) was solved in 20 mL CH2Cl2 at 0 C and NaHCO3
(1.24 g, 14.7 mmol, 3 equiv.) and DMP (3.83 g, 9.03 mmol, 1.8 equiv.) added. After 5 h, a cooled
◦
solution (0 C) of ethyl-2-(triphenylphosphoranylidene)acetate (7.28 g, 20.89 mmol, 4.3 equiv.) in
5 mL CH2Cl2 was added and stirred for further 4 h. The solution was diluted with 20 mL CH2Cl2
and 10 mL of a saturated NaHCO3 solution was slowly added. The aqueous layer was three times
extracted with CH2Cl2. The combined organic layers were washed with saturated Na2S2O3 solution,
dried over MgSO4 and the solvent was removed under reduced pressure. After purification by column
chromatography on silica gel (PE/Et2O = 20:1), ethyl ester VII (500 mg, 2.74 mmol, 56% yield over
two steps) was obtained as a colourless oil.
(R,E)-4,6-Dimethylhepta-2,5-dien-1-ol (VIII)
Ester VII (65 mg, 0.36 mmol, 1 equiv.) was solved in 6 mL THF and cooled to
of DIBAL-H (1.10 mL, 1.10 mmol, 3.08 equiv., 1 M in hexane) that was cooled to 0 C was added
−
78◦◦C. A solution
◦
dropwise. It was stirred for 2 h at
−
78 C, for 1 h at room temperature, 3 mL of a solution of saturated
K-Na-tartrate solution was added together with 1 mL Et2O. It was stirred for further 23 h. The aqueous
layer was three times extracted with Et2O. The combined organic layers were washed with saturated
NaCl solution, dried over MgSO4 and the solvent was removed under reduced pressure. After column
chromatography on silica gel (PE/Et2O = 5:1), alcohol VIII (47 mg, 0.34 mmol, 94% yield) was isolated.
(R,E)-4,6-Dimethylhepta-2,5-dienal (IX)
Alkohol VIII (20.5 mg, 150 µmol, 1 equiv.) was solved in 3 mL CH2Cl2 and MnO2 (240 mg,
2.76 mmol, 19 equiv.) added. After 24 h, the reaction mixture was filtrated over diatomaceous earth,
the residual solid was washed with CH2Cl2 and the solvent was removed under reduced pressure.
Aldehyde IX (20 mg, 0.15 mmol, 99% crude yield) was obtained without further purification.
((R,E)-3,5-Dimethylhexa-1,4-dien-1-yl)-3-methyldihydro-2H-pyran-2,4(3H)-dione (rac-14h)
Under argon atmosphere, diisopropylamine (52
0.5 mL dry THF and the resulting solution was cooled to
0.37 mmol, 2.5 M in hexanes, 2.5 equiv.) was added dropwise. The reaction was allowed to warm
to 0 ◦C for 30 min. At 78 ◦C, DMPU (19
L, 0.15 mmol, 1.0 equiv.) was added dropwise and the
resulting mixture was stirred for 30 min. Methyl-3-oxobutanoate (19.4 mg, 0.15 mmol, 1.0 equiv.)
µ
L, 0.37 mmol, 2.5 equiv.) was dissolved in
−
78 ◦C. To this solution, n-BuLi (150
µL,
−
µ
was dissolved in 250
stirring for 1 h at
was then added followed by stirring for 2 h at
1 mL 3 M hydrochloric acid at
three times with 10 mL Et2O. The combined organic phases were dried over Na2SO4 and filtered.
The solvents were removed under reduced pressure. The crude product was taken up in 4 mL 1 M
potassium hydroxide solution and the resulting solution was stirred for 7 h at r.t. At 0 C, the solution
µL dry THF and this solution was added to the reaction mixture followed by
−
78 ◦C. Aldehyde IX (21 mg, 0.15 mmol, 1.0 equiv.), dissolved in 250
µL dry THF,
◦
−
78 C. The reaction was quenched by addition of
−
78 ◦C and allowed to warm to r.t. The aqueous phase was extracted
◦
was acidified to pH 1.0 by addition of 6 M hydrochloric acid. The aqueous phase was extracted
three times with 10 mL EtOAc. The combined organic phases were dried over Na2SO4 and filtered.
The solvents were removed under reduced pressure. Purification by flash column chromatography
(EtOAc/PE = 1:10) yielded 40% of compound rac-14h as a pale-yellow oil (16 mg, 0.15 mmol).

Molecules 2016, 21, 1443
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4.2.3. Analytical Data
1
(E)-6-(Prop-1-en-1-yl)dihydro-2H-pyran-2,4(3H)-dione (rac-14a): Rf (EtOAc/PE = 1:4): 0.1; H-NMR
400 MHz, CDCl3): δH = 5.95–5.86 (m, 1H, 7-CH), 5.61–5.55 (m, 1H, 6-CH), 5.13–5.08 (m, 1H, 5-CH),
(
3.54 (d, 1H, 2J = 19.2 Hz, 2-CH2), 3.44 (d, 1H, 2J = 19.1 Hz, 2-CH2), 2,76 (dd, 1H, 2J = 18.2 Hz, 3J = 3.6 Hz,
4-CH2), 2,62 (dd, 1H, 2J = 18.1 Hz, 3J = 9.5 Hz, 4-CH2), 1.77 (m, 3H, 8-CH3); 13C-NMR (100 MHz, CDCl3):
δC = 17.92 (8-CH3), 43.57 (4-CH2), 47.12 (2-CH2), 75.64 (5-CH), 126.67 (6-CH), 132.03 (7-CH), 167.14 (1-C),
199.96 (3-C); HRMS (ESI): m/z: calc. for C8H10NaO3 [M + Na]+ 177.0528, found 177.0529 [M + Na]+.
1
Methyl-(E)-5-hydroxy-2-methyl-3-oxooct-6-enoate (rac-13d): H-NMR (400 MHz, CDCl3): δH = 5.77–5.68
(m, 1H, 8-CH), 5.52–5.45 (m, 1H, 7-CH), 4.57–4.50 (m, 1H, 6-CH), 3.73 (s, 3H, 10-CH3), 3.55 (q, 1H,
3J = 7.1 Hz, 2-CH), 2.83–2.72 (m, 2H, 5-CH2), 2.70–2.68 (m, 1H, OH), 1.70–1.68 (m, 3H, 9-CH3), 1.35 (dd,
3H, 3J = 7.1 Hz, 5J = 2.4 Hz, 3-CH3); 13C-NMR (100 MHz, CDCl3): δC = 206.2, 206.1 (4-C), 170.8, 170.7
(1-C), 131.9, 131.9 (8-CH), 127.6, 127.6 (7-CH), 68.7, 68.6 (6-CH), 53.5, 53.4 (10-CH3), 52.7, 52.7 (2-CH),
48.3, 48.1 (5-CH2), 17.8 (9-CH3), 12.7, 12.7 (3-CH3). HRMS (ESI): m/z: calc. for C10H16O4Na [M + Na]+
223.0945, found 223.0946 [M + Na]+.
1
(E)-3-Methyl-6-(prop-1-en-1-yl)dihydro-2H-pyran-2,4(3H)-dione (rac-14d): H-NMR (400 MHz, CDCl3):
δH = 5.98–5.89 (m, 1H, 7-CH), 5.60–5.54 (m, 1H, 6-CH), 5.16–5.11 (m, 1H, 5-CH), 3.58 (q, 1H, 3J = 6.6 Hz
,
2
3
2
3
2-CH), 2.76 (dd, 1H, J = 19.0 Hz, J = 3 Hz, 4-CH), 2.55 (dd, 1H, J = 19.0 Hz, J = 11.6 Hz,
3
4
3
4-CH), 1.78 (dd, 3H, J = 6.5 Hz, J = 1.6 Hz, 8-CH3), 1.37 (d, 3H, J = 6.5 Hz, 9-CH3); 13C-NMR
(
100 MHz, CDCl3): δC = 201.4 (3-C), 169.7 (1-C), 132.2 (7-CH), 126.6 (6-CH), 74.8 (5-CH), 51.9 (2-CH),
43.5 (4-CH2), 17.9 (8-CH3), 8.0 (9-CH3); HRMS (ESI): m/z: calc. for C9H12NaO3 [M + Na]+ 191.0684,
found 191.0685 [M + Na]+.
(E)-4-Methoxy-3-methyl-6-(prop-1-en-1-yl)-5,6-dihydro-2H-pyran-2-one (rac-15d): 1H-NMR (200 MHz,
CDCl3): δH = 5.96–5.71 (m, 1H, 8-CH), 5.68–5.54 (m, 1H, 7-CH), 4.80–4.68 (m, 1H, 6-CH), 3.78 (s, 3H,
10-CH3), 2.62–2.53 (m, 2H, 5-CH2), 1.79–1.77 (m, 3H, 3-CH3), 1.76–1.72 (m, 3H, 9-CH3).

Molecules 2016, 21, 1443
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(E)-6-(3-Methylbut-1-en-1-yl)dihydro-2H-pyran-2,4(3H)-dione (rac-14b): Rf (PE/EtOAc = 2:1): 0.20;
1H-NMR (400 MHz, CDCl3):
= 5.86 (ddd, J = 15.6, 6.6, 1.2 Hz, 1H, CHCHCH(CH3)2), 5.50 (ddd,
δ
J = 15.6, 6.2, 1.4 Hz, 1H, CHCHCH(CH3)2), 5.11 (ddd, J = 9.7, 6.2, 3.6 Hz, 1H, CH2CH), 3.49 (q,
J = 19.1 Hz, 2H, COCHCO), 2.77 (dd, J = 18.2, 3.6 Hz, 1H, CH2), 2.63 (dd, J = 18.2, 9.5 Hz, 1H, CH2),
2.42–2.29 (m, 1H, CH(CH3)2), 1.02 (dd, J = 6.8, 1.4 Hz, 6H, CH(CH3)2); 13C-NMR (100 MHz, CDCl3):
δ = 199.9 (q, CHCH2CO), 167.2 (q, OCO), 143.7 (t, CHCHCH(CH3)2), 122.6 (t, CHCHCH(CH3)2), 75.8
(t, CH2CH), 47.1 (s, COCH2CO), 43.7 (s, CHCH2), 31.0 (t, CH(CH3)2), 22.0 (p, CH3), 21.9 (p, CH3);
HRMS (ESI): m/z for C10H15O3 [M + Na]+: calculated 183.1024, observed: 183.1021.
(E)-4-Methoxy-6-(3-methylbut-1-en-1-yl)-5,6-dihydro-2H-pyran-2-one (rac-15b): Rf (PE/EtOAc = 2:1): 0.32;
1H-NMR (400 MHz, CDCl3):
δ = 5.81 (ddd, J = 15.5, 6.5, 1.0 Hz, 1H, CHCHCH(CH3)2), 5.51 (ddd,
J = 15.5, 6.7, 1.3 Hz, 1H, CHCHCH(CH3)2), 5.15 (d, J = 1.4 Hz, 1H, CHCOCH3), 4.81 (ddd, J = 11.0, 6.7,
4.2 Hz, 1H, CH2CHO), 3.74 (s, 3H, OCH3), 2.61–2.50 (m, 1H, CH2), 2.40 (dd, J = 17.1, 4.2 Hz, 1H, CH2),
2.37–2.25 (m, 1H, CH(CH3)2), 1.01 (d, J = 1.3 Hz, 3H, CH(CH3)2), 0.99 (d, J = 1.3 Hz, 3H, CH(CH3)2);
13C-NMR (100 MHz, CDCl3):
δ = 172.7 (q, COCH3), 167.2 (q, OCO), 142.6 (t, CHCHCH(CH3)2),
123.8 (t, CHCHCH(CH3)2), 90.6 (t, CHCOCH3), 76.5 (t, CH2CHO), 56.2 (p, OCH3), 33.5 (s, CH2), 30.9
(t, CH(CH3)2), 22.0 (p, CH(CH3)2), 22.0 (p, CH(CH3)2); HRMS (ESI): m/z for C11H16O3 [M + Na]+:
calculated 219.0997, observed: 219.0997.
(E)-3-Methyl-6-(3-methylbut-1-en-1-yl)dihydro-2H-pyran-2,4(3H)-dione (rac-14e): Rf (PE/EtOAc = 2:1):
1
0.24; H-NMR (400 MHz, CDCl3):
δ = 5.97 (ddd, J = 15.5, 6.5, 0.9 Hz, 1H, CHCHCH(CH3)2), 5.58
(ddd, J = 15.5, 6.8, 1.4 Hz, 1H, CHCHCH(CH3)2), 5.31–5.14 (m, 1H, CH2CH), 3.65 (q, J = 6.7 Hz, 1H,
COCHCH3), 2.85 (dd, J = 19.0, 3.1 Hz, 1H, CH2), 2.64 (dd, J = 19.0, 11.6 Hz, 1H, CH2), 2.52–2.35 (m, 1H,
CH(CH3)2), 1.45 (d, J = 6.6 Hz, 3H, COCHCH3), 1.11 (d, J = 1.6 Hz, 3H, CH(CH3)2), 1.10 (d, J = 1.5 Hz
,
3H, CH(CH3)2); 13C-NMR (100 MHz, CDCl3):
δ
= 201.3 (q, CH2COCH), 169.8 (q, OCO), 143.8 (t,
CHCHCH(CH3)2), 122.5 (t, CHCHCH(CH3)2), 74.9 (t, CH2CH), 51.9 (t, COCHCH3), 43.6 (s, CH2), 30.9
(p, COCHCH3), 22.0 (p, CH(CH3)2), 21.9 (p, CH(CH3)2), 8.0 (p, COCHCH3); HRMS (ESI): m/z for
C11H16O3 [M + Na]+: calculated 219.0999, observed: 219.0997.
(E)-4-Methoxy-3-methyl-6-(3-methylbut-1-en-1-yl)-5,6-dihydro-2H-pyran-2-one (rac-15e): Rf (PE/EtOAc
= 2:1): 0.78; 1H-NMR (400 MHz, CDCl3):
δ = 5.82 (ddd, J = 15.5, 6.5, 1.0 Hz, 1H, CHCHCH(CH3)2),
5.54 (ddd, J = 15.5, 6.9, 1.4 Hz, 1H, CHCHCH(CH3)2), 4.84–4.67 (m, 1H, CH2CH), 3.79 (s, 3H, OCH3),
2.75–2.47 (m, 2H, CH2), 2.45–2.23 (m, 1H, CH(CH3)2), 1.78 (dd, J = 1.8, 1.2 Hz, 3H, CCH3), 1.02 (d,
J = 0.7 Hz, 3H, CH(CH3)2), 1.00 (d, J = 0.7 Hz, 3H, CH(CH3)2); 13C-NMR (100 MHz, CDCl3):
δ = 168.5
(q, COCH3), 165.2 (q, OCO), 142.6 (t, CHCHCH(CH3)2), 124.1 (t, CHCHCH(CH3)2), 103.8 (q, CCH3),
75.4 (t, OCH), 55.6 (p, OCH3), 30.9 (s, CH2), 29.9 (t, CH(CH3)2), 22.1 (p, CH(CH3)2), 22.1 (p, CH(CH3)2),
9.0 (p, CCH3); HRMS (ESI): m/z for C12H19O3 [M + Na]+: calculated 211.1330, observed: 211.1334.

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1
Methyl-(E)-5-hydroxy-3-oxo-7-phenylhept-6-enoate (rac-13c): H-NMR (400 MHz, CDCl3):
δ
= 7.44–7.21
(m, 5H, Haromat.), 6.68 (dd, J = 15.9, 1.0 Hz, 1H, Holefin.), 6.23 (dd, J = 15.9, 6.2 Hz, 1H, Holefin.), 4.87–4.74
(m, 1H, CHOH), 3.77 (s, 3H, OCH3), 3.55 (s, 2H, COCH2CO), 2.90 (d, J = 6.0 Hz, 2H, CHOHCH2CO).
The analytical data was consistent with the literature [30].
1
(E)-6-Styryldihydro-2H-pyran-2,4(3H)-dione (rac-14c): H-NMR (400 MHz, CDCl3):
δ = 7.47–7.29 (m, 5H,
H
aromat.), 6.77 (dd, J = 16.0, 1.1 Hz, 1H, CCHCHCHO), 6.24 (dd, J = 16.0, 6.0 Hz, 1H, CCHCHCHO),
5.40–5.29 (m, 1H, CHCH2), 3.56 (q, J = 19.2 Hz, 2H, CH2COO), 2.89 (dd, J = 18.2, 3.6 Hz, 1H, CHCH2CO),
2.75 (dd, J = 18.2, 9.6 Hz, 1H, CHCH2CO). The analytical data was consistent with the literature [30].
(E)-4-Methoxy-6-styryl-5,6-dihydro-2H-pyran-2-one (rac-15c): Rf (PE/EtOAc = 2:1): 0.21; 1H-NMR (200
MHz, CDCl3):
6.2 Hz, 1H, PhCHCH), 5.24 (d, J = 1.1 Hz, 1H, OCCH), 5.11 (ddd, J = 11.2, 5.5, 1.2 Hz, 1H, CH2CH),
3.81 (s, 3H, OCH3), 2.87–2.47 (m, 2H, CH2); 13C-NMR (100 MHz, CDCl3):
= 172.4 (q, COCH3), 166.9
δ = 7.51–7.24 (m, 5H, Haromat.), 6.78 (dd, J = 16.0, 1.0 Hz, 1H, PhCHCH), 6.30 (dd, J = 16.0,
δ
(q, OCO), 135.9 (t, Caromat.), 133.3 (t, Caromat.), 128.8 (t, Caromat.), 128.5 (t, PhCHCH), 126.884 (t, Caromat.),
125.6 (t, PhCHCH), 90.7 (t, OCCH), 76.0 (t, OCH), 56.3 (p, OCH3), 33.5 (s, CH2); HRMS (ESI): m/z for
C14H15O3 [M + Na]+: calculated 231.1021, observed: 231.1018.
1
(E)-3-Methyl-6-styryldihydro-2H-pyran-2,4-(3H)-dione (rac-14f): Rf (PE/EtOAc = 1:1): 0.44; H-NMR
(400 MHz, CDCl3)
6.5 Hz, 1H, CCHCH), 5.49–5.20 (m, 1H, CH2CH), 3.65 (q, J = 6.6 Hz, 1H, COCH), 3.09–2.82 (m, 1H,
CH2), 2.79–2.52 (m, 1H, CH2), 1.41 (d, J = 6.6 Hz, 3H, CH3); 13C-NMR (100 MHz, CDCl3):
= 200.9
(q, COCH2), 269.5 (q, OCO), 135.4 (1C, PhCH), 134.4 (1C, PhCHCH), 129.0 (1C, Caromat.), 128.9 (2C,
aromat.), 127.0 (2C, Caromat.), 123.8 (1C, Caromat.), 74.6 (1C, CH2CH), 52.0 (1C, CH2COCHCO), 43.6 (1C,
CH2), 8.1 (1C, CH3); HRMS (ESI): m/z for C14H14O3 [M + Na]+: calculated 253.0840, observed: 253.0841.
δ = 7.58–7.28 (m, 5H, Haromat.), 6.80 (d, J = 15.9 Hz, 1H, PhCH), 6.23 (dd, J = 15.9,
δ
C

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(E)-4-Methoxy-3-methyl-6-styryl-5,6-dihydro-2H-pyran-2-one (rac-15f) Rf (PE/EtOAc = 2:1): 0.44; 1H-NMR
(400 MHz, CDCl3): = 7.45–7.28 (m, 5H, Haromat.), 6.75 (d, J = 16.0 Hz, 1H, PhCHCH), 6.29 (dd, J = 16.0
6.4 Hz, 1H, PhCHCH), 5.04–4.91 (m, 1H), 3.81 (s, 3H, OCH3), 2.81–2.58 (m, 2H, CH2), 1.82 (dd,
J = 1.9, 1.2 Hz, 3H, CCH3); 13C-NMR (100 MHz, CDCl3):
= 165.0 (q, CH2CO), 135.9 (q, OCO), 133.3
δ
,
δ
(t, PhCHCH), 128.9 (t, Caromat.), 128.5 (t, PhCHCH), 127.2 (t, OCHCHCHCH), 126.8 (t, Caromat.), 125.9
(t, Caromat.), 104.0 (p, CCH3), 74.9 (t, OCH), 55.7 (p, OCH3), 29.9 (s, CH2), 9.1 (p, CCH3); HRMS (ESI):
m/z for C15H16O3 [M + Na]+: calculated 267.0996, observed: 267.0997.
(E)-6-(Dec-1-en-1-yl)-3-methyldihydro-2H-pyran-2,4(3H)-dion (rac-14g): Rf (PE/EtOAc = 20:1): 0.11;
1H-NMR (400 MHz, CDCl3):
δ
= 6.04–5.83 (m, 1H, CH2CHCHCHO), 5.54 (ddt, J = 15.4, 6.9, 1.4 Hz
1H, CH2CHCHCHO), 5.31–5.04 (m, 1H, CH2CHO), 3.57 (q, J = 6.6 Hz, 1H, CH3CH), 2.77 (dd, J = 19.0,
3.0 Hz, 1H, CH2), 2.56 (dd, J = 19.0, 11.6 Hz, 1H, CH2), 2.13–2.01 (m, 2H, CHCHCH2), 1.37 (d, J = 6.6 Hz
3H, CHCH3), 1.25 (t, J = 11.0 Hz, 12H, CH2CH2CH2CH2CH2CH2), 0.88 (t, J = 6.8 Hz, 3H, CH2CH3);
13C-NMR (100 MHz, CDCl3):
= 201.4 (q, CH3CHCO), 169.7 (q, OCO), 137.3 (t, CH2CHCHCHO),
,
,
δ
125.2 (t, CH2CHCHCHO), 74.8 (t, CHCHO), 51.9 (t, CH3CH), 43.6 (s, COCH2), 32.3 (s, CHCHCH2), 32.0
(s, CHCH2CH2), 29.5 (s, CH2), 29.4 (s, CH2), 29.3 (s, CH2), 28.8 (s, CH2), 22.8 (s, CH2), 14.3 (p, CHCH3),
8.0 (p, CH2CH3); HRMS (ESI): m/z for C16H26O3 [M + Na]+: calculated 289.1781, observed: 289.1780.
(E)-6-(Dec-1-en-1-yl)-4-methoxy-3-methyl-5,6-dihydro-2H-pyran-2-on (rac-15g): Rf (PE/EtOAc = 2:1): 0.53;
1H-NMR (400 MHz, CDCl3):
δ = 5.84 (ddd, J = 7.6, 7.2, 3.8 Hz, 1H, CH2CHCHCHO), 5.58 (ddt, J = 15.4,
6.8, 1.4 Hz, 1H, CH2CHCHCHO), 4.80–4.64 (m, 1H, CCH2CHO), 3.78 (s, 3H, OCH3), 2.68–2.48 (m,
2H, CH2COCH3), 2.06 (dd, J = 13.9, 7.0 Hz, 2H, CHCHCHCH2), 1.78 (d, J = 0.6 Hz, 3H, CCH3),
1.33–1.18 (m, 12H, CH2CH2CH2CH2CH2CH2), 0.88 (t, J = 6.8 Hz, 3H, CH2CH3); 13C-NMR (100 MHz,
CDCl3): δ = 168.5 (q, COCH3), 165.1 (q, OCO), 136.0 (t, CH2CHCHCHO), 126.9 (t, CH2CHCHCHO),
103.8 (q, CCH3), 75.3 (t, CHCHO), 55.6 (p, OCH3), 32.3 (s, CHOCH2), 32.0 (s, CHCHCH2), 29.9
(s, CH2CH2CH2CH2CH2), 29.6 (s, CH2CH2CH2CH2CH2), 29.4 (s, CH2CH2CH2CH2CH2), 29.3 (s,
CH2CH2CH2CH2CH2), 29.0 (s, CH2CH2CH2CH2CH2), 22.8 (s, CH2CH2CH2CH2CH2), 14.3 (p, CCH3),
9.0 (p, CH2CH3); HRMS (ESI): m/z for C17H28O3 [M + Na]+: calculated 303.1936, observed: 303.1936.
Methyl-(R)-3-((tert-Butyldimethylsilyl)oxy)-2-methylpropanoate (
I
): Rf = 0.9 (petroleum ether/Et2O 10:1);
[α]2D3
=
−
0.1 (c = 0.86, CH2Cl2); 1H-NMR (400 MHz, CDCl3): δH = 3.78 (dd, 1H, J = 9.7 Hz,
2
2
3
3J = 6.9 Hz, 3-CH), 3.67 (s, 3H, 5-CH3), 3.64 (dd, 1H, J = 9.8 Hz, J = 6.0 Hz, 3-CH), 2.65 (sext, 1H,
3J = 6.8 Hz, 2-CH), 1.13 (d, 3H, 3J = 6.8 Hz, 4-CH3), 0.87 (s, 9H, 7-CH3), 0.03 (d, 6H, 3J = 1.4 Hz, 6-CH3);
13C-NMR (100 MHz, CDCl3): δC = 175.6 (1-C), 65.4 (3-CH2), 51.6 (5-CH3), 42.7 (2-CH), 25.9 (7-CH3),
18.3 (8-C), 13.6 (4-CH3), −5.4 (6-CH3).

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(R)-3-((tert-Butyldimethylsilyl)oxy)-N-methoxy-N,2-dimethylpropanamide (II): Rf (PE/EE 3.5:1): 0.50; [α]D20
15.8 (c = 1.1, CH2Cl2); 1H-NMR (400 MHz, CDCl3):
= 3.83 (d, J = 9.6, 8.2 Hz, 1H, CH2-OTBS), 3.71
:
−
δ
(s, 3H, OMe), 3.53 (d, J = 9.6, 6.2 Hz, 1H, CH2-OTBS), 3.19 (m, 4H, CH-CH3, N-Me), 1.07 (d, J = 6.8 Hz,
3H, CH3-CH2), 0.87 (m, 9H, OTBS, 0.04 (s, 3H, OTBS), 0.03 (s, 3H, OTBS) ppm; 13C-NMR (100 MHz,
CDCl3):
δ = 171.3 (q, CO-N), 65.7 (s, CH2-OTBS), 61.5 (p, OMe), 38.0 (t, CH-CH3), 31.6 (p, NMe), 25.9
(p, OTBS), 18.3 (q, OTBS), 13.8 (p, CH3-CH),
[M + Na]+: ber. 284.1658 gef. 284.1659.
−
5.5 (p, OTBS) ppm; HRMS [ESI] m/z for C12H27NO3SiNa
(R)-3-((tert-Butyldimethylsilyl)oxy)-2-methylpropanal (III): [α]D23
(
=
−
0.1 (c = 0.68, CH2Cl2); H-NMR
1
200 MHz, CDCl3): δH = 9.74 (d, 1H, 3J = 1.6 Hz, 1-CH), 3.83 (m, 2H, 3-CH2), 2.61–2.44 (m, 1H, 2-CH),
1.09 (d, 3H, 3J = 7.0 Hz, 4-CH3), 0.87 (s, 9H, 6-CH3), 0.05 (s, 6H, 5-CH3); 13C-NMR (100 MHz, CDCl3):
δC = 204.9 (1-CH), 65.0 (3-CH2), 42.0 (2-CH), 25.9 (6-CH3), 18.3 (7-C), 13.2 (4-CH3), −5.4 (5-CH3).
(S)-tert-Butyl-((2,4-dimethylpent-3-en-1-yl)oxy)dimethylsilane (IV): Rf = 0.5 (petroleum ether); [α]2D3 = +0.1
(c = 0.8, CH2Cl2); 1H-NMR (400 MHz, CDCl3): δH = 4.88–4.85 (m, 1H, 3-CH), 3.43 (dd, 1H, 2J = 9.7 Hz,
3J = 6.0 Hz, 1-CH2), 3.31 (dd, 1H, 2J = 9.8 Hz, 3J = 7.6 Hz, 1-CH2), 2.57–2.46 (m, 1H, 2-CH), 1.68 (d,
3H, 4J = 1.2 Hz, 5-CH3), 1.63 (d, 3H, 4J = 1.2 Hz, 5-CH3), 0.92 (d, 3H, 3J = 6.6 Hz, 6-CH3), 0.89 (s, 9H,
9-CH3), 0.03 (s, 6H, 7-CH3); 13C-NMR (100 MHz, CDCl3): δC = 132.0 (4-C), 127.8 (3-CH), 68.3 (1-CH2),
35.7 (2-CH), 26.1 (9-CH3), 18.5 (8-C), 18.2 (5-CH3), 17.6 (6-CH3), −5.1 (7-CH3).
(S)-2,4-Dimethylpent-3-en-1-ol (V δ = 4.88 (dquin,
): Rf (PE:EE 5:1): 0.26; 1H-NMR (400 MHz, CDCl3):
J = 9.5, 1.4 Hz, 1H, CH=C), 3.51–3.43 (m, 1H, CH2-CH), 3.34–3.28 (m, 1H, CH2-CH), 2.66–2.54 (m,
1H, CH-CH3), 1.73 (d, J = 1.3 Hz, 3H, Me-C), 1.67 (d, J = 1.0 Hz, 3H, Me-C), 1.38 (bs, 1H, OH), 0.92
(d, J = 6.5 Hz, 3H, CH3-CH) ppm; 13C-NMR (100 MHz, CDCl3):
δ = 134.3 (q, C=CH), 127.1 (t, CH=C),
67.9 (s, CH2-OH), 35.5 (t, CH-CH3), 25.9 (p, Me-C), 18.2 (p, CH3-CH), 17.0 (p, Me-C) ppm; [α]D21
(c = 1.1, CH2Cl2).
:
−
39.4
1
(R,E)-Ethyl-4,6-dimethylhepta-2,5-dienoate (VII): Rf (PE/EE 5:1): 0.65; H-NMR (400 MHz, CDCl3):
δ = 6.87 (dd, J = 15.7, 6.5 Hz, 1H, CH=CH), 5.75 (dd, J = 15.7, 1.4 Hz, 1H, CH=CH), 4.94 (dquin, J = 8.7,
1.4 Hz, 1H, CH=C), 4.18 (q, J = 7.1 Hz, 2H, OEt), 3.19 (dddd, J = 15.3, 13.7, 6.8, 1.5 Hz, 1H, CH-CH3)
1.70 (d, J = 1.4 Hz, 3H, Me-C), 1.62 (d, J = 1.4 Hz, 3H, Me-C), 1.28 (t, J = 7.2 Hz, 3H, OEt), 1.10 (d, J = 7.2
Hz, 3H, CH3-CH) ppm; 13C-NMR (100 MHz, CDCl3):
δ = 167.1 (q, CO2Et), 153.1 (t, CH=CH), 133.0
(q, C=CH), 126.4 (t, CH=C), 128.9 (t, CH=CH), 60.2 (s, OEt), 35.4 (CH-CH3), 25.8 (p, Me-C), 20.1 (p,
CH3-CH), 18.0 (p, Me-C), 14.3 (p, OEt) ppm; HRMS [ESI] m/z for C11H18O2Na [M + Na]+: ber. 205.1204
gef. 205.1206; [α]22: +124.0 (c = 1.3, CH2Cl2).
D

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(R,E)-4,6-Dimethylhepta-2,5-dien-1-ol (VIII): Rf (PE/EE 5:1): 0.21; 1H-NMR (400 MHz, CDCl3):
δ
= 5.60
(ddt, J = 5.0, 3.8, 9.7 Hz, 2H, CH=CH), 4.95 (dquin, J = 9.0, 1.4 Hz, 1H, CH=C), 4.09 (m, 2H, CH2-OH),
3.11–3.01 (m, 1H, CH-CH3), 1.69 (d, J = 1.2 Hz, 3H, Me-C), 1.62 (d, J = 1.0 Hz, 3H, Me-C), 1.25 (bs, 1H,
OH), 1.03 (d, J = 6.8 Hz, 3H, CH3-CH) ppm; 13C-NMR (100 MHz, CDCl3):
δ = 137.7 (t, CH-CH2OH),
131.1 (q, C=CH), 128.4 (t, CH=CHCH2), 126.5 (t, CH=C), 63.9 (s, CH2-OH), 35.1 (CH-CH3), 25.7 (p,
Me-C), 20.9 (p, CH3-CH), 17.9 (p, Me-C) ppm; [α]22: +74.7 (c = 1.0, CH2Cl2).
D
(R,E)-4,6-Dimethylhepta-2,5-dienal (IX): Rf (PE/EE 5:1): 0.58; 1H-NMR (400 MHz, CDCl3):
δ = 9.51 (d,
J = 7.8 Hz, 1H, H-CO), 6.73 (dd, J = 15.5, 6.3 Hz, 1H, CH=CHCO), 6.06 (ddd, J = 15.6, 7.9, 1.4 Hz, 1H,
CH=CHCH), 4.96 (dquin., J = 8.8, 1.4 Hz, 1H, CH=C), 3.33 (dqd, J = 15.2, 6.8, 1.3 Hz, 1H, CH-CH3),
1.72 (d, J = 1.4 Hz, 3H, Me-C), 1.63 (d, J = 1.4 Hz, 3H, Me-C), 1.15 (d, J = 6.8 Hz, 3H, CH3-CH) ppm;
13C-NMR (100 MHz, CDCl3):
δ = 194.5 (t, CO), 162.5 (t, CH=CHCO), 133.9 (t, CH-CO), 130.5 (q, C=CH),
125.6 (t, CH=C), 35.9 (CH-CH3), 25.7 (p, Me-C), 19.8 (p, CH3-CH), 18.0 (p, Me-C) ppm.
((R,E)-3,5-Dimethylhexa-1,4-dien-1-yl)-3-methyldihydro-2H-pyran-2,4(3H)-dione (rac-14h): Rf (EtOAc/PE
1
3
4
= 1:10): 0.3, H-NMR (500 MHz, CDCl3): δH = 5.83 (ddd, 1H, 3J = 9.3 Hz, J = 6.2 Hz, J = 0.9 Hz,
8-CH), 5.49 (ddt, 1H, 3J = 15.5 Hz, 3J = 6.8 Hz, 4J = 1.6 Hz, 7-CH), 5.17–5.12 (m, 1H, 6-CH), 4.93 (d, 1H,
3J = 9.0 Hz, 11-CH), 3.57 (q, 1H, 3J = 6.6 Hz, 2-CH), 3.11 (ddt, 1H, 3J = 7.0 Hz, 3J = 1.7 Hz, 9-CH), 2.77
(dd, 1H, 2J = 19.0 Hz, 3J = 2.7 Hz, 5-CH2), 2.56 (dd, 1H, 2J = 19.0 Hz, 3J = 11.7 Hz, 5-CH2), 1.70 (d, 3H,
3J = 1.1 Hz, 13-CH3), 1.62 (s, 3H, 13-CH3), 1.37 (d, 3H, 3J = 6.7 Hz, 3-CH3), 1.06 (dd, 3H, 3J = 6.9 Hz,
4J = 1.3 Hz, 10-CH3); 13C-NMR (125 MHz, CDCl3): δC = 201.3 (4-C), 169.8 (1-C), 141.3 (8-CH), 132.4
(12-C), 127.3 (11-CH), 122.9 (7-CH), 74.9 (6-CH), 51.9 (2-CH), 43.6 (5-CH2), 35.3 (9-CH), 25.9 (13-CH3),
20.7 (10-CH3), 18.9 (13-CH3), 8.0 (3-CH3). HRMS (ESI): m/z: calc. for C14H20NaO3 [M + Na]+ 259.1310,
found 259.1312 [M + Na]+.
4.3. Cloning and Enzyme Expression
4.3.1. Molecular Cloning
A codon-optimised gene for jerF was obtained as a plasmid jerF_pMK-T (Invitrogen, Waltham,
MA, USA) provided with the NdeI (50-CAT ATG-30) and EcoRI (50-GAA TTC-30) restriction sites used
for standard restriction cloning into pET-28a(+) (Novagen, Billerica, MA, USA) and pCOLD-I (Takara
Bio USA Inc., Mountain View, CA, USA). For this purpose, jerF_pMK-T was double digested by NdeI
(New England Biolabs, Ipswich, MA, USA) and EcoRI (New England Biolabs). The gel-purified insert
was ligated into NdeI/EcoRI-treated pET-28a(+) and pCOLD-I respectively using T4 DNA Ligase
(Thermo Fisher Scientific, Waltham, MA, USA) to generate recombinant plasmids jerF_pET-28a(+) and
jerF pCOLD-I for expression of N-terminal His6-tagged fusion proteins.
For cloning into pET-20b(+) (Novagen), jerF was amplified by PCR under standard conditions
using jerF_pMK-T as a template with primers as follows: forward, 50-AGG CTC GAG TGC CGG
ACT TTC GGT GC-30; reverse, 50-TGA GAT CTC ATA TGC GTA CCA GTG ATG C-30. Gel-purified
amplicons were double digested using XhoI (New England Biolabs) and NdeI, followed by the ligation
into XhoI/NdeI-treated pET-20b(+) vector using T4 DNA Ligase to generate recombinant plasmid
jerF_pET-20b(+) for expression of C-terminal His6-tagged fusion protein. All recombinant plasmids
were transformed into E. coli TOP10 chemically competent cells for plasmid propagation.

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4.3.2. Protein Sequence
>JerF
MRTSDAVWAGAAGYTRARLQVYDFFIYGFNSPVAWKCPGEELLENYNRHVSGNHLDVGVGTG
YLLDRCRFPTAKPRVFLMDLNPDALQVTAQRLHRFQPQTLRRNVLDPIRFDGEPFDSIGMNYLMHCV
PGSIPEKAVMFDHLSALLKPGGVIFGSTVLSEGVDKGIVARAIMDRFNKKGIFSNTRDAASDLTRALEE
RFDDVSVRVVGCVGLFSARKRTCAGTESPA
4.3.3. Enzyme Expression
Recombinant plasmids jerF_pET-28a(+), jerF_pET-20b(+) and jerF_pCOLD-I were used to
transform E. coli BL21 (DE3) chemically competent cells. The resulting transformants were used to
generate overnight cultures in LB broth under appropriate antibiotic selection (50
µg
mL−1 kanamycin
·
for jerF_pET-28a(+), 50
µ
g mL−1 carbenicillin for the others). The next day, these cultures were used
to inoculate 50 mL LB broth in 250-mL-flasks containing appropriate antibiotics to an initial OD600
of 0.05. The inoculated cultures were grown at 37 ◦C, 180 rpm for 2–3 h until OD600 reached 0.5–1.0.
Isopropyl-β-d-thiogalactopyranoside (IPTG) was then added to a final concentration of 0.1 mM to
induce gene expression. For jerF_pCOLD-I, the cells were cultured at 16 ◦C overnight. The cells were
harvested at 10,000 g for 45 min at 4 ◦C. The cell pellet was resuspended in 10 mL/g reaction buffer
(40 mM Tris HCl, 100 mM NaCl, pH 8.8) and applied to sonication. Cell debris was removed by
◦
centrifugation at 10,000 g for 40 min at 4 C to obtain cell-free extract which were immediately used in
enzyme assays.
4.4. Enzyme Assays
4.4.1. Establishment of Assay Conditions
Initial enzyme assays with substrate rac
Tris HCl, 100 mM NaCl, 5 mM MgCl2, pH 8.8) containing JerF (2.7 mg/mL total protein), 0.25 mM
rac
14d and 0.97 mM SAM tosylate as methyl donor, incubated at 28 ◦C for 16 h. The reaction was
quenched by the addition of 100 L brine followed by extraction with EtOAc (2 400 L). The
-14d were carried out in 200 µL of reaction buffer (40 mM
-
µ
×
µ
resulting organic extracts were dried and redissolved in 1 mL MeCN. LC-MS analysis were carried out
with a Q-ToF Premier (Waters) in combination with a Waters Acquity Ultra performance LC system
(H2O/MeCN = 95:5 + 0.1% FA → 5:95 + 0.1% FA, 0.4 mL/min, 8 min).
4.4.2. Comparative Assaying of Synthetic Substrate Surrogates
Enzymatic substrate conversions were carried out in 200 µL of reaction buffer (25 mM HEPES,
100 mM NaCl, 5 mM MgCl2, pH 7.5) containing JerF (8.4 mg/mL total protein), 0.25 mM substrate
◦
surrogates and 4.18 mM SAM tosylate as methyl donor, incubated at 28 C for 20 h. The reaction
was quenched by the addition of 100
resulting organic extracts were dried and redissolved in 200
µ
L brine followed by extraction with EtOAc (2
×
200 µL). The
µL MeCN. LC-MS analysis were carried
out with a micromass LCT via loop-mode injection from a Waters Alliance 2695 HPLC system.
4.4.3. Semi-preparative Scale Conversions
Semi-preparative scale JerF assays were carried out in a total volume of 10 mL, containing
4.3–4.6 mg of substrate and 3.6–4.0 mg/mL of total protein in reaction buffer (25 mM HEPES, 100 mM
◦
NaCl, 5 mM MgCl2, pH 7.5) and 1.67 mM SAM tosylate as methyl donor, incubated at 28 C for
16 h. The reaction was quenched by the addition of 5 mL brine followed by extraction with EtOAc
(
3 mL × 15 mL). The resulting organic extracts were dried, redissolved in deuterated chloroform and
subjected to 1H-NMR analysis and HPLC-MS analysis.

Molecules 2016, 21, 1443
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Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
11/1443/s1.
Acknowledgments: Funding from the Emmy Noether program of the DFG (HA 5841/2-1), the Marie Curie
program of the European Union (project number 293430) and the Graduate School MINAS (doctoral stipend to
G.B.) are gratefully acknowledged. We thank the NMR facility and the mass facility of the OCI as well as Breuning
and his group for access to chiral HPLC.
Author Contributions: S.F., F. Hemmerling and F. Hahn conceived and designed the experiments;
S.F., F. Hemmerling, A.W., G.B., F.L. and J.W. performed the experiments; S.F., F. Hemmerling, A.W., G.B., F.L.,
J.W. and F. Hahn analyzed the data; S.F. and F. Hahn wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).