An efficient chemoenzymatic approach towards the synthesis of rugulactone

Article abstract of DOI:10.3390/molecules23030640

Rugulactone is a natural product isolated from the plant Cryptocarya rugulosa. It has shown very important biological activity as an inhibitor of the nuclear factor κB (NF-κB) activation pathway. A new chemoenzymatic approach towards the synthesis of rugu

Full text of DOI:10.3390/molecules23030640

molecules
Article
An Efficient Chemoenzymatic Approach towards the
Synthesis of Rugulactone
Theodore Tyrikos-Ergas, Vasileios Giannopoulos and Ioulia Smonou *
Department of Chemistry, University of Crete, Vasilika Vouton, 71003 Heraklion, Crete, Greece;
thoriaki@yahoo.gr (T.T.-E.); billgianno@hotmail.com (V.G.)
*
Correspondence: smonou@uoc.gr; Tel.: +30-281-054-5010
Received: 13 February 2018; Accepted: 7 March 2018; Published: 12 March 2018
Abstract: Rugulactone is a natural product isolated from the plant Cryptocarya rugulosa. It has
shown very important biological activity as an inhibitor of the nuclear factor κB (NF-κB) activation
pathway. A new chemoenzymatic approach towards the synthesis of rugulactone is presented here.
The chirality, induced to the key intermediate by a stereoselective enzymatic reduction utilizing
NADPH-dependent ketoreductase, is described in detail.
Keywords: chemoenzymatic; α-pyrone; ketoreductase; bioreduction; rugulactone; inhibitor
1
. Introduction
Rugulactone
Cryptocarya rugulosa [
exhibit up to five-fold induction of inhibitor of
g/mL [ ]. The transcription factor NF- B was discovered in 1986 as a nuclear factor regulating
the formation of the variety of the light chain (one of two subunits from which immunoglobin
is built) [ ]. Soon afterwards, it became clear that these proteins, which harbor this specific DNA
binding activity, are expressed in nearly all cell types. It is well known that NF- B affects most
aspects of cellular physiology—from immunity and inflammation to apoptosis, cell survival, growth,
and proliferation [ ]. Consistent with this role, incorrect regulation of NF- B has been linked to
cancer [ ] and inflammatory [10 11] and autoimmune [12] diseases. Thus, there is an intensive
1
is a secondary metabolite, isolated in 2009 from organic extracts of the plant
]. It has shown very important biological activity and has been reported to
B (I B) proteins of nuclear factor B (NF- B) at
1
κ
κ
κ
κ
25
µ
1
κ
κ
2
κ
3
–
7
κ
8,
9
,
necessity for the exploration of new biomimetic compounds that can inhibit and control NF-
κB
activity effectively.
Rugulactone is a rather new dihydro
α-pyrone that inhibits the NF–κB activation pathway.
This natural product shows an interesting structural pattern with two electrophilic groups of potential
Michael acceptors (highlighted in red, Figure 1), which covalently bind to target protein active
sites, an
inhibitory properties of rugulactone, it has been a challenging subject of research for synthetic organic
chemists. Recent studies have verified that many analogues [13 14] and derivatives [ ] of rugulactone
α,β-unsaturated γ-lactone together with an α,β-unsaturated ketone. Due to the remarkable
,
8
demonstrate similar or better antibacterial and antifungal activity as compared to the natural product,
even with the opposite configuration of the natural product. Several synthetic schemes have been
reported where chirality has been induced using various methods [13
–22], such as a chiral pool [20],
proline-catalyzed -aminooxylation of aldehydes [13], Jacobsen’s hydrolytic kinetic resolution of
α
epoxides [16], Keck’s asymmetric allylation [17], allylation of carbonyl compounds with chiral boronic
esters [19], or asymmetric catalytic Overman esterification [14]. Also, a chemoenzymatic synthesis
was reported by Fadnavis et al. [18], involving an enzymatic kinetic resolution of racemic homoallylic
alcohols employing Candida rugosa lipase. However, in this case, the maximum conversion to the
desired enantiomer could not exceed 50%. The majority of all of these synthetic routes include several
synthetic steps and utilize expensive starting materials and peculiar reagents.
Molecules 2018, 23, 640; doi:10.3390/molecules23030640
www.mdpi.com/journal/molecules

Molecules 2018, 23, 640
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Figure 1. Rugulactone.
Biocatalysis has long been known as an alternative technology, capable of delivering highly
stereo-, chemo-, and regioselective transformations [23 24]. Recent contributions demonstrate the broad
,
diversity of impressive opportunities for chemoenzymatic processes, which underline their potential
as valuable solutions for current synthetic challenges in the synthesis of valuable chiral building blocks
towards the production of pharmaceuticals and biologically active natural products [23–26].
As a part of our interest in applications of bioreductions in the synthesis of biologically interesting
compounds, we report here a new chemoenzymatic approach towards the synthesis of rugulactone 1.
2
. Results and Discussion
Our synthetic approach consisted of eight simple synthetic steps utilizing inexpensive starting
materials. The chirality of the natural product was achieved by a stereoselective enzymatic reduction
utilizing NADPH-dependent ketoreductase. Initially, the racemic rugulactone was synthesized
according to the retrosynthetic approach shown in Scheme 1.
Scheme 1. Retrosynthetic analysis of rugulactone.
Rugulactone (
±
)-
1
can be derived from a Grubbs’ cross metathesis between the two terminal
can be synthesized from the dihydroxy ester , which can be derived from
. On the other hand, enone can be obtained easily
alkenes and . Lactone
2
3
2
5
the commercially available methyl-2-cyanoacetate
6
3
from the commercially available aldehyde 4.
Initially, the synthesis of intermediate
5
was achieved through keto ester
7 (Scheme 2).
The synthesis of compound was reported many years ago [27
7
,28]. Unfortunately, in our case,
the classic C–C coupling involving Claisen or Grignard, as well as Reformatsky and aldol, reactions
led to unexpected byproducts. Therefore, these synthetic methods were not useful. Next, we chose an
alternative and simpler approach for the synthesis of
7, utilizing an organometallic Barbier reaction [29].
The carbonyl moiety was then reduced by NaBH in MeOH to furnish the corresponding hydroxy
4
ester
commercially available tert-butyl acetate, followed by a carbonyl reduction. Furthermore, a single-step
cyclization of dihydroxy compound with simultaneous dehydration, catalyzed by p-toluenesulfonic
acid, led to γ-lactone 2 in good yield (Scheme 2).
Furthermore, enone was synthesized from commercially available 3-phenylpropanal
steps involving alkylation of the aldehyde through Grignard vinylation followed by 2-iodoxybenzoic
acid (IBX) oxidation of the resulting allylic alcohol (Scheme 2). Finally, the cross-metathesis of the two
8. In addition, compound 5 was obtained from a Claisen reaction between the hydroxy ester 8 and
5
3
4 in two
9

Molecules 2018, 23, 640
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terminal alkenes—the lactone
2
and the enone
3
—catalyzed by Grubbs (II) catalyst [30], led successfully
to the formation of ( )-rugulactone 1 in a good reaction yield of 75%, whereas the overall yield of the
±
synthesis was found to be 21% (Scheme 3). The spectroscopic data are identical to those reported in
the literature [1].
◦
Scheme 2.
(
A
) Reagents and conditions: (
a
) allyl bromide, Zn powder, tetrahydrofuran (THF), 0 C–r.t.,
◦
5
−
2%; (
b
) NaBH , MeOH, 0 C, 95%; (
c
) (i) lithium diisopropylamide (LDA), tert-butyl acetate, THF,
4
◦
◦
78 C (ii) NaBH , MeOH, 0 C, 79% over two steps; (
d
) p-toluenesulfonic acid (PTSA), toluene, reflux,
4
◦
9
7%; (
B
) (e
) vinyl magnesium chloride, THF, 0 C–r.t., 86%; (
f
) 2-iodoxybenzoic acid (IBX), dimethyl
sulfoxide (DMSO), r.t., 88%.
◦
Scheme 3. Reagents and conditions: Grubbs(II) catalyst (5 mol %), dry CH Cl , 45 C, 75%.
2
2
After the successful completion of this attractive synthetic procedure of racemic rugulactone,
we applied this methodology for the synthesis of the optically active rugulactone by the intervention
1
of a key enzymatic step. Our research group has lengthy experience in biocatalytic reductions of various
carbonyl compounds and their applications for the chemoenzymatic synthesis of natural products
with biological activity [31
enzymes, like ketoreductases, are valuable and powerful catalysts in the synthesis of optically active
intermediates and precursors for many pharmaceuticals [33 40]. In the present work, the asymmetric
reduction of the keto ester using NADPH-dependent ketoreductases was the key step introducing
the chirality of the natural product . Before conducting the screening for active enzymes, we tested
under the usual enzymatic reaction conditions (phosphate buffer 200 mM, pH 6.9,
,32]. Moreover, we and other research groups have shown that reductive
–
7
1
the stability of
and 37 C) [33
7
,
◦
34]. Unfortunately, under those conditions, substrate
7
was decomposed, as it was
1
observed by the H-NMR spectroscopy. After the clarification of the optimum conditions (phosphate
◦
buffer 200 mM, pH 6.9, and temperature 5–8 C), the stereoselective reduction of
7 was carried out
with several ketoreductases to screen and identify the most suitable biocatalyst for the desirable
transformation. The enzymes chosen for the screening have displayed high activity in structurally
similar carbonyl substrates [32,36,37]. The well-established system for the NADPH recycling was
applied in all enzymatic reactions (Scheme 4) [33]. Many enzymes demonstrated activity towards the
reduction of 7, and the best results are shown in Table 1.
Ketoreductases (Kreds) A1C, A1D, B1F, 101, and 119 showed excellent activity (>99% conv.)
towards the reduction of keto ester
7 (conv > 99%). Kreds A1C, A1D, and 119 displayed

Molecules 2018, 23, 640
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excellent enantioselectivity (ee > 99%) (Scheme 4), whereas Kreds B1F and 101 showed reasonable
enantioselectivity. All positive enzymes catalyzed efficiently the reduction to form the optically active
hydroxy ester
stereochemistry of the hydroxy group by the use of methoxy phenyl acetic esters (MPA esters) [41] and
was found to be the (S)- enantiomer. Compound is the key chiral synthon for the chemoenzymatic
synthesis of the (S)-epimer of natural product according to our proposed synthesis (Scheme 2).
8. The absolute configuration of the hydroxy ester was determined by assigning the
8
8
1
The synthesis of this epimer is very useful since it has demonstrated high biological activity like many
analogues [15] and derivatives [8].
Table 1. Enzymatic reduction of methyl 3-oxohex-5-enoate 7.
Substrate Kred a
Conv. b (time)
Yield c
ee d
Product
A1C
A1D
119
>99%
>99%
>99%
>99%
>99%
(12 h)
(12 h)
(12 h)
(12 h)
(12 h)
62%
64%
79%
62%
83%
>99%
98%
>99%
58.6%
72%
(S)-8
(S)-8
(S)-8
(S)-8
(S)-8
7
B1F
1
01
a
Ketoreductase; b Conversions were derived from H-NMR spectra; c Isolated; d Enantiomeric excess was derived
1
from chiral column GC analysis.
Scheme 4. Asymmetric enzymatic reduction of 7 using Kred 119.
It is worth noting that under the enzymatic reaction conditions, substrate
bond isomerization to form a small amount (6%) of compound 7a, as it was observed by H-NMR
7
showed a double
1
spectroscopy (Scheme 5).
Scheme 5. Isomerization of keto ester 7.
For this reason, the final product of the enzymatic reduction in every case was a mixture of the
desired optically active hydroxy ester
well as the carbonyl moiety reduction of the isomer 7a. This observation was in accordance with our
previous unpublished results with several ketoreductases, which can also reduce -unsaturated keto
8 and a side product derived from the double bond reduction as
α,β
moieties together with the double bond reduction. This can most likely be attributed to contaminations
of these commercially available enzymes.
3
. Materials and Methods
3
.1. General
Unless otherwise noted, all solvents and reagents were purchased from Sigma-Aldrich (Munich,
Germany) in the highest purity and were used without any further purification. Dry tetrahydrofuran
THF) was used after distillation into a Soxhlet in the presence of metallic Na and benzophenone. Kreds,
glucose dehydrogenase, and NADPH were commercially available (Codexis, Redwood City, CA, USA).
(

Molecules 2018, 23, 640
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NMR spectra were generally recorded at room temperature on Bruker Avance series 500 and
00 spectrometers (Billerica, MA, USA). Chemical shifts ( ) are reported in ppm relative to the residual
solvent peak (CDCl3, : 77.0), and the multiplicity of each signal is designated
: 7.26, 13CDCl3,
by the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad.
3
δ
δ
δ
1
13
Coupling constants (J) are quoted in Hz. H- and C-NMR spectra of compounds 1–3, 5, and 7–9
,
1
and H-NMR of (R)-MPA and (S)-MPA esters of (S)-methyl 3-hydroxyhex-5-enoate can be found
in Supplementary Materials. Thermo LTQ-Orbitap XL with an electron transfer dissociation (ETD)
ion trap mass spectrometer (Waltham, MA, USA) was used for the high resolution mass spectra
(HRMS). Column chromatographic separations were carried out of a flash chromatography system
using silica gel and hexane/ethyl acetate or petroleum ether/ethyl acetate solvent mixtures. For thin layer
chromatography (TLC), Merck silica gel (grade 60 F245, Merck & Co., Kenilworth, NJ, USA) was used.
The progress of the enzymatic reactions and the selectivities were determined by gas chromatography
using SHIMADZU GC-2014 gas chromatograph (Shimadzu, Japan) equipped with an flame ionization
detector (FID) detector (HP GC column: 30 m
permethylated, Cyclodextrin-B, Part No. 19091G-B233).
×
0.25 mm
×
0.25 µm chiral capillary column, 20%
3
.2. Synthetic Procedures
3
.2.1. Methyl 3-Oxohex-5-enoate (7)
Aluminum trichloride (160 mg, 1.2 mmol) was added all at once to a solution of zinc powder
780 mg, 12 mmol), methyl 2-cyanoacetate (267 L, 3 mmol), and crotyl bromide (390 L, 4.5 mmol)
(
µ
µ
◦
in anhydrous THF (12 mL) at 0 C (ice-water bath). The reaction mixture was warmed to room
temperature and then stirred at room temperature. After the reaction was completed (monitored by
TLC), aqueous HCl (2 M, 5 mL) was added to the reaction mixture and was stirred at room temperature
for 5 min. The reaction mixture was passed through a short silica gel column and the organic solvent
was removed directly under reduced pressure. Further purification was achieved by flash column
chromatography (hexane/EtOAc, v/v, 30/1) to give the corresponding ester
7 with 52% isolated yield
1
(
222 mg). H-NMR (500 MHz; CDCl ; Me Si):
δ
5.87–5.95 (m, 1H), 5.22–5.25 (m,1H), 5.16–5.20 (m, 1H),
3
4
13
3.74 (s, 3H), 3.50 (s, 2H), 3.305 (d, J = 7.0 Hz, 2H). C-NMR (500 MHz, CDCl3): δ 200.5, 167.45, 129.5,
1
19.8, 52.4, 48.3, 47.7.
3
.2.2. Methyl 3-Hydroxyhex-5-enoate (8)
Sodium borohydride (0.26 mmol, 10 mg) was added in dry ethanol (10 mL) under nitrogen,
◦
and the mixture was cooled to 0 C. After stirring for 5 min a solution of dry methanol (5 mL)
containing methyl 3-oxohex-5-enoate
for 4 h at 0 C, the reaction was quenched with saturated ammonium chloride, and methanol was
concentrated by rotor evaporator. Then, water (20 mL) was added and extracted twice with EtOAc
7 (111 mg, 0.78 mmol) was added dropwise. After stirring
◦
(
2
×
20 mL). The organic layer was dried over MgSO and the solvents were evaporated to dryness.
4
The residue was purified by flash column chromatography (hexane/EtOAc, v/v, 20/1) to afford
1
the methyl 3-hydroxyhex-5-enoate
ppm):
dd, J1 = 16.6 Hz, J = 3.5 Hz, 1H), 2.44 (dd, J = 16.6 Hz, J = 9 Hz, 1H), 2.23–2.33 (m, 2H). C-NMR
8
with 95% yield (107 mg). H-NMR (500 MHz; CDCl ; Me Si;
3
4
δ
δ 5.78–5.86 (m, 1H), 5.13–5.16 (m, 1H), 5.11–5.12 (m, 1H), 4.06–4.12 (m, 1H), 3.71 (s, 3H), 2.53
13
(
(
2
1
2
500 MHz, CDCl ): δ 173.2, 133.9, 118.2, 67.3, 51.8, 40.9, 40.4.
3
3
.2.3. tert-Butyl 3,5-dihydroxyoct-7-enoate (5)
Under nitrogen atmosphere, dry diisopropyl amine (621
µ
L, 4.72 mmol) was dissolved in dry
◦
THF (5 mL). The mixture was cooled to 0 C and BuLi 1.6 M in hexane (3.0 mL, 4.4 mmol) was added
◦
◦
dropwise. After stirring for 20 min at 0 C the mixture was cooled to
−
78 C and a solution of tert-butyl
◦
acetate (439 mg, 2 mmol) in dry THF (2 mL) was added and the mixture was stirred at
−
78 C for
◦
2
0 min. Then, the mixture was cooled to
−
78 C and a solution of methyl 3-hydroxyhex-5-enoate

Molecules 2018, 23, 640
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8
(340 mg, 2.36 mmol) in dry THF (3 mL) was added and the reaction mixture was stirred until the
completion of the reaction, which was observed by TLC analysis of reaction aliquots. The reaction
mixture was quenched with saturated NH Cl (20 mL) and was extracted with EtOAc (2 15 mL).
×
4
The combined organic layers were dried over MgSO and evaporated to dryness. Then, the crude
4
product was dissolved in dry methanol (5 mL) and the methanol mixture was added dropwise to a
stirring solution of sodium borohydride (45 mg, 0.87 mmol) in dry methanol (8 mL) under nitrogen
◦
at 0 C. The reaction mixture was stirred until the completion of the reaction monitored by TLC.
The reaction was quenched with saturated ammonium chloride, and the methanol was concentrated
by rotor evaporator. Then, water (20 mL) was added and the aqueous layer was extracted twice with
EtOAc (2
×
20 mL). The organic layer was dried over MgSO and the solvents were evaporated to
4
dryness. The residue was purified using silica gel chromatography (hexane/EtOAc, v/v, 15/1) to
1
afford the tert-butyl 3,5-dihydroxyoct-7-enoate
CDCl ; Me Si):
5
with 79% overall yield (363 mg). H-NMR (500 MHz;
δ
5.78–5.86 (m, 1H), 5.07–5.14 (m, 2H), 4.20–4.42 (m, 1H), 3.90–4.15 (m, 1H), 2.36–2.63
3
4
13
(m, 2H), 2.20–2.36 (m, 2H), 1.52–1.70 (m, 2H), 1.46 (s, 9H). C-NMR (500 MHz, CDCl3):
δ 172.4, 172.08,
1
34.6, 134.5, 118, 117.8, 81.5, 71.1, 69.0, 67.5, 65.7, 43.2, 42.5, 42.1, 42.0, 41.5, 41.4, 28.1. HRMS (TOF-ESI)
+
m/z: [M + H] Calcd. for C H O +H, 231.1552; Found 231.1592.
12
22
4
3
.2.4. Allyl-5,6-dihydro-2H-pyran-2-one (2)
Under nitrogen atmosphere, tert-butyl 3,5-dihydroxyoct-7-enoate
5 (100 mg, 0.46 mmol) was
dissolved in dry toluene (8 mL) and p-toluenesulfonic acid monohydrate (45 mg, 0.26 mmol) was
added. After stirring the solution at room temperature for 5 min, the reaction mixture was refluxed
for 5 h. After completion of the reaction, the reaction mixture was washed twice with sodium
carbonate (2
×
8 mL). The organic layer was dried over MgSO , and the solvent was evaporated to
4
dryness. Pure 6-allyl-5,6-dihydro-2H-pyran-2-one
2
was obtained after silica gel chromatography
1
(hexane/EtOAc v/v, 10/1) with 97% yield (62 mg). H-NMR (500 MHz; CDCl ; Me Si;
δ
ppm):
3
4
δ
6.86–6.90 (m, 1H), 6.02 (d, J = 9.7 Hz, 1H), 5.79–5.88 (m, 1H), 5.15–5.19 (m, 2H), 4.46–4.51 (m, 1H),
2
.53–2.59 (m, 1H). 2.44–2.50 (m, 1H), 2.33–2.36 (m, 2H) 13C-NMR (500 MHz, CDCl3):
δ
164.3, 145.0,
1
32.2, 121.4, 118.9, 77.0, 39.0, 28.6.
3
.2.5. 5-Phenylpent-1-en-3-ol (9)
To an ice-cold solution of 3-phenylpropanal
8 (700 µL, 5.14 mmol) and dry THF (8 mL),
vinyl magnesium chloride 1 M (1.5 mL, 1.4 mmol) was added dropwise and the reaction was
stirred at room temperature for 3 h under nitrogen dry atmosphere. When the reaction was
complete (TLC), it was quenched with saturated aqueous NH Cl, and the mixture was extracted
4
with Et O (3 × 50 mL). The combined organic layers were dried (MgSO ) and concentrated under
2
4
reduced pressure. The residue was purified by column chromatography (hexane/EtOAc v/v, 20/1)
to produce the corresponding homoallylic alcohol as a clear liquid with 86% isolated yield (195 mg).
1
H-NMR (500 MHz; CDCl ; Me Si):
δ 7.27–7.30 (m, 2H), 7.17–7.216 (m, 3H), 5.9 (ddd, J = 17.2 Hz,
1
3
4
J = 10.4 Hz, J = 6.2 Hz, 1H), 5.23–5.27 (m, 1H), 5.12–5.15 (m, 1H), 4.11–4.14 (m, 1H), 2.66–2.78 (m, 2H),
2
3
13
1.80–1.91 (m, 2H). C-NMR (300 MHz, CDCl ): δ 141.8, 141.0, 128.5, 128.40, 125.9, 115.0, 72.5, 38.5, 31.6.
3
3
.2.6. 5-Phenylpent-1-en-3-one (3)
The homoallylic alcohol obtained above (0.5 mL, 4.92 mmol) was added to a stirred solution of
IBX (1.4 g, 5.14 mmol) in dry DMSO (10 mL) at room temperature, and the mixture was stirred for 6 h.
When the reaction was complete, the mixture was filtered, diluted with H O (10 mL), and extracted
9
2
with CH Cl (2
×
20 mL). The combined organic layers were washed with brine (20 mL), dried (MgSO4),
2
2
and concentrated in vacuo. Purification by column chromatography (petroleum ether/EtOAc v/v,
1
2
7
5/1) furnished compound
.28–7.30 (m, 2H), 7.19–7.21 (m, 3H), 6.36 (dd, J = 17.8 Hz, J = 10.6 Hz, 1H)), 6.22 (d, J = 17.8 Hz, 1H),
3
with 88% isolated yield (693 mg). H-NMR (500 MHz; CDCl ; Me Si):
δ
3
4
1
2

Molecules 2018, 23, 640
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5
1
.83 (d, J = 10.6 Hz, 1H), 2.90–2.98 (m, 4H). 13C-NMR (500MHz, CDCl3):
28.33, 128.27, 126.1, 41.2, 29.8.
δ
199.8, 141.0, 136.5, 128.5,
3
.2.7. (E)-6-(4-Oxo-6-phenylhex-2-enyl)-5,6-dihydro-2H-pyran-2-one (1)
Under nitrogen atmosphere, Grubbs’ (II) catalyst (15 mg, 5 mol %) was added to a stirred
solution of 6-allyl-5,6-dihydro-2H-pyran-2-one
2
(50 mg, 0.36 mmol) and 5-phenylpent-1-en-3-one
◦
3
(174 mg, 1.1 mmol) in dry CH Cl (10 mL), and the reaction mixture was stirred at 45 C for 12 h.
2
2
After completion of the reaction, the solvent was evaporated to dryness and the residue was purified
using silica gel chromatography (hexane/EtOAc, v/v, 4/1) to afford
1 with 75% isolated yield (73 mg).
1
H-NMR (500 MHz; CDCl ; Me Si):
δ 7.26–7.29 (m, 2H), 7.17–7.20 (m, 3H), 6.86–6.90 (m, 1H), 6.77–6.83
3
4
(
2
m, 1H), 6.19 (d, J = 15.9 Hz), 6.04 (dt, J = 9.8 Hz, J = 1.8 Hz, 1H), 4.52–4.57 (m, 1H),2.88–2.96 (m, 4H),
1 2
.58–2.70 (m, 2H), 2.32–2.34 (m, 2H). C-NMR (500 MHz, CDCl3): δ 199.0, 163.7, 144.6, 141.0, 140.0,
13
1
33.5, 128.5, 128.4, 126.1, 121.5, 76.0, 41.7, 37.5, 29.9, 28.9.
3
.3. General Procedure for Enzymatic Reductions
The reductions were performed as follows: In a phosphate buffered solution (1 mL, 200 mM,
pH 6.9), the substrate (5 mg, 0.035 mmol), the corresponding Kred (2 mg), glucose (21 mg), glucose
dehydrogenase (2 mg), and NADPH (2 mg, 2.5 mM) were added. The reactions were incubated at
◦
3
–8 C. After completion of the reactions, the products were isolated by extracting the crude reaction
mixture with EtOAc (3
to dryness.
×
1.5 mL). The combined organic layers were dried over MgSO and evaporated
4
(
S)-Methyl 3-hydroxyhex-5-enoate, (S)-8
The enzymatic reduction of methyl 3-oxohex-5-enoate
7
catalyzed by Kred-119 was completed
after 3 h and the optically active alcohol
data are identical to those of compound 8. H-NMR (500 MHz; CDCl ; Me Si):
8
was produced with 79% yield and > 99% ee. The spectroscopic
1
δ 5.78–5.86
3
4
(
m, 1H), 5.13–5.16 (m, 1H), 5.11–5.12 (m, 1H), 4.06–4.12 (m, 1H), 3.71 (s, 3H), 2.53 (dd, J = 16.6 Hz,
1
13
J = 3.5 Hz, 1H), 2.44 (dd, J = 16.6 Hz, J = 9 Hz, 1H), 2.23–2.33 (m, 2H). C-NMR (500 MHz, CDCl3):
2
1
2
δ
173.2, 133.9, 118.2, 67.3, 51.8, 40.9, 40.4. GC data: (column: 30 m
×
0.25 mm
×
0.25 µm chiral capillary
◦
◦
column, Cyclodextrin-B 150 C for 10 min, rate: 0 C/min; carrier gas: He, press 90 kPa). t = 2.61 min.
R
3
.4. Preparation of MPA-Esters
3
.4.1. General Method for the Synthesis of MPA Esters of Secondary Alcohols
To a solution of the corresponding secondary alcohol (0.1 mmol) in dry CH Cl were added
2
2
1
.1 equivalent of N,N’-dicyclohexylcarbodiimide (DCC) (0.11 mmol, 23 mg) and 1.1 equivalent of the
◦
corresponding (R) or (S) MPA (0.11 mmol, 18 mg) and the reaction mixture was stirred at 0 C for
4–6 h. After completion of the reaction, the produced urea was filtered and the filtrate was evaporated
and then purified by column chromatography with 5/1 Hex/EtOAc. The produced corresponding
MPA-ester was isolated with 90% isolated yield.
(
R)-MPA Ester of (S)-Methyl 3-hydroxyhex-5-enoate
1
H NMR (500 MHz; CDCl ; Me Si):
δ 7.40–7.42 (m, 2H), 7.317–7.36 (m, 3H), 5.41–5.49 (m, 1H),
3
4
5
.30–5.35 (m, 1H), 4.84–4.90 (m, 2H), 4.72 (s, 1H), 3.60 (s, 3H), 3.41 (s, 3H), 2.52–2.62 (m, 2H), 2.218–2.223
(m, 2H).
(
S)-MPA Ester of (S)-Methyl 3-hydroxyhex-5-enoate
1
H NMR (500 MHz; CDCl ; Me Si):
δ 7.40–7.42 (m, 2H), 7.31–7.36 (m, 3H), 5.65–5.74 (m, 1H),
3
4
5.26–5.35 (m, 1H), 5.05–5.08 (m, 2H), 4.71 (s, 1H), 3.39 (s, 3H), 3.37 (s, 3H), 2.44–2.50 (m, 2H), 2.37–2.41
(m, 2H).

Molecules 2018, 23, 640
8 of 10
4
. Conclusions
In summary, a total synthesis of (
±
)-rugulactone has been achieved in a highly efficient and
concise way—in eight steps with an overall yield of 21%. This flexible synthetic pathway can be
applied to the synthesis of a variety of –unsaturated -pyrones, which bear two potential Michael
α
,β
α
acceptors. Concerning the chemoenzymatic synthetic approach of the optically active (S) epimer of the
natural product 1, we identified through a screening procedure three suitable highly stereoselective
ketoreductases, which catalyzed efficiently the formation of the chiral key intermediate
8 as the (S)
enantiomer in high yield and excellent ee, using ketoreductases for the first time. Further studies
focused on screening other anti-Prelog reductive enzymes for the synthesis of the (R)-8 enantiomer are
currently underway.
Supplementary Materials: Supplementary materials are available online at http://www.mdpi.com/1420-3049/
13
1
13
1
13
2
3/3/640/s1. 1H- and C-NMR of compound
7
2
1
: S2, H- and C-NMR compound
8
9
: S3, H- and C-NMR
1
13
1
13
1
13
compound
compound
3
5
: S4, H- and C-NMR compound
: S7, H- and C-NMR compound
-hydroxyhex-5-enoate: S9.
: S5, H- and C-NMR compound
: S6, H- and C-NMR
1
13
1
3
: S8, H-NMR of (R)-MPA and (S)-MPA esters of (S)-methyl
Acknowledgments: This work was supported by the Special Account for Research Funds of University of Crete
(
KA 4453 and KA 4065, SARF UoC). We thank C. Markou for some preliminary experiments. We are also thankful
to the PROFI (Proteomics Facility at IMBB-FORTH) for performing the HRMS analysis.
Author Contributions: I.S., T.T.-E. and V.G. conceived and designed the experiments; T.T.-E. and V.G. performed
the experiments and analyzed the data; I.S. contributed reagents/materials/analysis tools; I.S., T.T.-E. and V.G.
wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
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