Stereopermutation on the putative structure of the marine natural product mucosin

Article abstract of DOI:10.3390/molecules22101720

A stereodivergent total synthesis has been executed based on the plausibly misassigned structure of the unusual marine hydrindane mucosin (1). The topological connectivity of the four contiguous all-carbon stereocenters has been examined by selective permutation on the highlighted core. Thus, capitalizing on an unprecedented stereofacial preference of the cis-fused bicycle[4.3.0]non-3-ene system when a Michael acceptor motif is incorporated, copper-mediated conjugate addition furnished a single diastereomer. Cued by the relative relationship reported for the appendices in the natural product, the resulting anti-adduct was elaborated into a probative target structure 1?.

Full text of DOI:10.3390/molecules22101720

molecules
Article
Stereopermutation on the Putative Structure of the
Marine Natural Product Mucosin
Simen G. Antonsen 1, Harrison Gallantree-Smith 1, Carl Henrik Görbitz 2
,
ID
Trond Vidar Hansen 1,3, Yngve H. Stenstrøm 1
and Jens M. J. Nolsøe 1, ,†
*
ID
1
Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003,
1433 Ås, Norway; simen.antonsen@nmbu.no (S.G.A.); lithium_87@hotmail.co.uk (H.G.-S.);
t.v.hansen@farmasi.uio.no (T.V.H.); yngve.stenstrom@nmbu.no (Y.H.S.)
Department of Chemistry, University of Oslo, P.O. Box 1033, 0315 Oslo, Norway; c.h.gorbitz@kjemi.uio.no
Department of Pharmaceutical Chemistry, University of Oslo, P.O. Box 1068, 0316 Oslo, Norway
Correspondence: jens.mj.nolsoe@nmbu.no; Tel.: +47-6723-2467
2
3
*
†
Dedication: Dedicated to Lars Skattebøl on the occasion of his 90th birthday.
Received: 19 September 2017; Accepted: 5 October 2017; Published: 13 October 2017
Abstract: A stereodivergent total synthesis has been executed based on the plausibly misassigned
structure of the unusual marine hydrindane mucosin (1). The topological connectivity of the
four contiguous all-carbon stereocenters has been examined by selective permutation on the
highlighted core. Thus, capitalizing on an unprecedented stereofacial preference of the cis-fused
bicycle[4.3.0]non-3-ene system when a Michael acceptor motif is incorporated, copper-mediated
conjugate addition furnished a single diastereomer. Cued by the relative relationship reported for the
appendices in the natural product, the resulting anti-adduct was elaborated into a probative target
structure 1*.
Keywords: marine hydrindane natural product; asymmetric synthesis; stereodivergent strategy;
structural elucidation; eicosanoid
1. Introduction
Certain metabolites derived from polyunsaturated fatty acids (PUFAs) play a key role in
mammalian physiology, where they orchestrate both inflammatory response as well as the return to
homeostasis [
a number of distinct eicosanoids and docosanoids have been identified, which are active in the cascade
elicited by noxious stimuli [ 23]. As a result, natural products with an underlying PUFA motif are of
1–4]. By combining total synthesis with chemical biology and molecular pharmacology,
5
–
great interest as potential immunomodulators.
Since antiquity, sea dwelling organisms have proven to be a particularly abundant source of new
chemical entities, set apart from those found in the terrestrial environment [24
Phoenicians were renowned for their trading with Tyrian purple from the Murex sea snail [26
Rising above mere prospecting, modern-day discovery, enabled by the advent of powerful analytical
instruments and methods, has found a wealth of bioactive compounds in the marine environment [28 34].
Ostensibly, mucosin ( ) is a natural product that was isolated from the Mediterranean sponge
Reniera mucosa as methyl ester 35]. Formally classified as an eicosanoid, it has been conjectured to
originate from arachidonic acid ( ), based on the C20-architechture (Figure 1). While sharing some
noticeable features with the prostane scaffold, the compound differs by having an unusual bicyclic core.
Clearly, in the structure proposed for mucosin ( ), the characteristic cyclopentane ring is integrated
,25]. Thus, the ancient
,27].
–
1
2
[
3
1
in a cis-fused bicyclo[4.3.0]non-3-ene system. However, turning to the elucidation, the assignment of
topology poses a challenge. Though only a small molecule, the structure is compact in terms of the
four contiguous stereocentres. In NMR experiments on the isolated methyl ester, pertaining to both 1D-
Molecules 2017, 22, 1720; doi:10.3390/molecules22101720
www.mdpi.com/journal/molecules

Molecules 2017, 22, 1720
2 of 18
and 2D-techniques, the distinguishing resonances/correlations are ensconced in a crowded aliphatic
region. Consequently, with the absence of any coupling pattern to corroborate the configuration of the
carbocycle, the assignment published by Casapullo et al. does not convince on its own [35].
6
6
5
H
8
8
1
5
1
11
12
9
11
12
9
CO2R
16
CO2H
14
14
15
H
20
20
16
3 Arachidonic Acid
1 R = H, Mucosin
2 R = Me
Figure 1. Suggested structure of mucosin and its relation to arachidonic acid.
Fascinated by the structure and the prostane-like motif, we devised a practical, divergent and
synthetically unambiguous strategy to establish the proposed stereochemistry. At the end of the
campaign, capitalizing on X-ray crystallography to pinpoint the relative arrangement, it was concluded
that mucosin (
1) does not represent the portrayed compound [36]. In a pursuit to identify the
natural product isolated from Reniera mucosa, our intent is to achieve the goal by manipulation
of the bicyclo[4.3.0]non-3-ene system. We herein detail synthesis of the mucosin diastereomer 1*
demonstrating aspects of the chosen route with regard to stereochemical control (Figure 2). From the
point of potential biological activity, the putative structure of mucosin shares some apparent structural
similarities with bicyclic prostaglandins. Thus, providing that sufficient amounts could be made
available, material could be screened in similar assays. Currently, it is the bearer of unknown properties.
,
H
CO2R
H
1* R = H, exo-Mucosin
2* R = Me
Figure 2. Stereopermutation on the cis-bicyclo[4.3.0]non-3-ene scaffold.
2. Results and Discussion
In 2012, Whitby and co-workers reported that they had completed the first total synthesis
of antipodal mucosin (ent-1) [37]. Using zirconium induced co-cyclisation as the pivotal feature,
the preliminary experimental work led them to conclude that thermodynamic control would favour the
relative stereochemistry assigned by Casapullo et al. [35]. Applied to the actual sequence, elaboration
of the key zirconacycle afforded a ~3:1 mixture of diastereomers [37]. While it was conjectured that
the major component could be processed to ent-1, the minor component would in turn yield ent-1*.
However, although Whitby and co-workers provided data that aligned with the natural product,
the authors of the present paper demonstrated irrefutably, that mucosin is not represented by the
relative topological connectivity featured in structure
1 [36]. This therefore raised the question as
to which diastereomer had been taken on by Whitby and co-workers, and whether mucosin in fact
corresponds to structure 1*. In order to resolve this pressing issue, we designed a strategy to access
structure 1*.
A central feature in our divergent strategy (Scheme 1) was to take advantage of the efficient
desymmetrization of meso-ketone
sequence to obtain meso-ketone
matter of introducing a functional pattern amenable for subsequent stereoiteration. Ideally, in order
4
[36]. (Supplementary Materials pp. S3, S4 provides the synthetic
4) After a chiral foothold had been established, it would then be a

Molecules 2017, 22, 1720
3 of 18
to uncover the topologically deviant point(s), the prerequisite cis-fused keto ester
5
should also be
interconvertible with the trans-fused system if need be. However, we first chose to examine the
configuration at the appended positions. Thus, along these lines and having previously established
a diastereochemical bias, addition of some suitable nucleophile to conjugate ester
7 was judged to
follow the precognised trend. By completing the sequence, a new compound 1* with the topology
inverted at C8 and C16 would result. Aptly, this could then be named exo-mucosin 1*, since the bulky
group added during the stereodifferentiating step was projected to occupy the exo face of the bicycle
(Figure 3). Once exo-mucosin 1* had been made, the physical data recorded could be compared against
those published for the natural product.
side-chain
elaboration
H
CO2R
H
1* R = H
2* R = Me
Desymmetrization
Alkyne iteration
CO2R
CO2R
Bu
conjugate
addition
H
H
H
H
elimination
CO2Me
H
H
H
Michael addition
8
7
Target Molecule
CO2R
LG
CO2R
O
asymmetric
acylation
H
H
reduction
H
H
6
5
H
O
H
4
Scheme 1. Key strategic points towards synthesis of exo-mucosin (1*).
O
OR
Exo
face
H
H
Bu
H
Attack from less
hindered face
CO2R
H
Endo
face
O
OR
X
H
H
H
Attack from more
hindered face
Bu
Figure 3. Projected diastereofacial bias in the key conjugate addition.
By the developed protocol, our synthesis commenced with desymmetrization of meso-ketone
using Mander’s reagent in combination with the lithium amide of (+)-bis[(R)-phenyethyl]amine (Scheme 2).
This chiral amide is sometimes also referred to as Simpkins’ base [38 40]. Then, with asymmetric keto
ester in hand, conjugated ester 10 was prepared by a three-step procedure, involving sequential
manipulation of the keto moiety. Accordingly, the ketone in was reduced, whereupon the corresponding
4 [36],
–
9
9
alcohol was turned into a mesylate. Finally, the intermediate mesylate was subjected to base-induced
elimination, whereby the Michael acceptor motif was produced.

Molecules 2017, 22, 1720
4 of 18
Having carried out the delineated transformation, the key stereoiterative concept could be
tested in the elaboration of conjugated ester 10. While addition to the less hindered exo-face seemed
inevitable, the resulting stereochemistry at the ester-appended chiral centre was somewhat uncertain
a priori. Simplistically, depending on whether the supervening ester enolate is intercepted by H+
at the equatorial or axial position of C8, the protonated species will correspond to the kinetic and
the thermodynamic product, respectively. Reflecting the ambivalent stereochemical nature of the
C8-carbanion, and based on our previous experience [36], conjugate addition to 10 could consequently
lead to a mixture of epimers.
1) NaBH4 in MeOH, rt
2) MsCl, Et3N, CH2Cl2
1) BuMgCl, TMSCl
CuI (10 mol%)
2) NH4Cl (aq)
CO2Me
O
CO2Me
H
0 oC to rt
H
H
H
(+)-Simpkin’s base
methyl cyanoformate
3) DBU, toluene, rt
O
56% (3 steps)
THF
THF
-78 oC
69% (ee: 99%)
-35 o
C
H
H
H
81%
4
9
10
1) MsCl, Et3N, CH2Cl2
0 oC to rt
2) KCN, DMSO, 70 o
3) DIBAL-H, hexane
-78 oC
C
OH
Bu
CHO
Bu
CO2Me
Bu
H
H
H
O-B reagent, K2CO3
DIBAL-H
hexane
0
MeOH
0 oC to rt
78%
60% (3 steps)
oC to rt
H
H
H
92% (ee: > 99%)
12
11
13
1) Cp2ZrCl2, DIBAL-H
THF, 0 o
C
H
H
H
2) I2, THF, 0 oC to rt
CO2R
Bu
3) EtO2C(CH2)3ZnBr
(Ph3P)4Pd (10 mol%)
THF, rt
H
14
86% (3 steps, one-pot)
15 R = Et
1* R = H: LiOH, THF/MeOH/H2O, rt, 95%
2* R = Me: TMSCHN2, toluene/MeOH, rt, 96%
Scheme 2. The total synthesis of exo-mucosin (1*) and its methyl ester 2*.
In reality, with Cu(I)-catalysed conjugate addition, using BuMgCl as nucleophile in the presence
of TMSCl, the reaction gave ester 11 as the sole compound (Scheme 3). Presumably, the Lewis
acid takes on dual roles: Not only does TMSCl lower the LUMO of the Michael acceptor, but also
stabilizes the ester enolate [41–46]. Hence, ester 11 ought to be the conjectured thermodynamic product.
Subsequent reduction provided the corresponding carbinol 12, which could also be readily derivatized for
the purpose of X-ray analysis. By obtaining suitable crystals of the dinitrobenzoate 12-DNB, the relative
configuration of the four contiguous stereocentres could be established (Figure 4). This also confirmed
the exo-facial and thermodynamic preference in the reaction of 10, using the specified conditions.
(Supplementary Figure S-74 provides a side perspective of the single crystal X-ray structure 12-DNB).
With the intended topological pattern confirmed, carbinol 12 was taken through a course of four steps
to install an alkyne handle by the Ohira-Bestmann protocol [47–50]. For the last step, it may be noted that
Taber et al. have provided an interesting alternative to the rather pricy reagent [50]. Although 1H-NMR
of the natural product clearly indicates the presence of an E-alkene [35], the en route aldehyde 13
could also serve as a relay point for Z-selective olefination. However, with the cited observation in
mind, alkyne 14 was transformed accordingly to provide the featured E-configured alkenyl ester motif.
This was achieved by performing three consecutive reactions in one-pot. Thus, by means of stereospecific
hydrometallation [51–55] and halodemetallation [56], alkyne 14 rendered the corresponding E-vinyl
halide as substrate for Pd-catalysed cross-coupling with a commercial zinc reagent [57
,58]. The target
molecule, exo-mucosin 1*, was then obtained after hydrolysis of ester 15. Finally, re-esterification gave
methyl ester 2*, to be compared with the data published by Casapullo et al. [35].
The cis-fused bicyclo[4.3.0]non-3-ene system is not often encountered in nature. Adhering to
the supposition that arachidonic acid (3) is the biogenetic origin of mucosin [59–61], the geometry
proposed for the core structure invokes a formal disrotatory ring-closure [62]. At a more profound level,

Molecules 2017, 22, 1720
5 of 18
the machinery leading to the natural product may traverse any number of pericyclic pathways [36].
Of particular interest, though, is the ongoing discussion regarding whether or not enzyme-catalysed
Diels-Alder reactions are implicated in biological systems [63]. The preceding biosynthetic transformation
of
species [59
transformative impetus [78–85], the authors of this paper have found no example of cis-fusion.
3, into a suitable conjugated precursor for cycloaddition, is known to take place in several marine
–
61 64 77]. However, in all the cases where a Diels-Alderase could be claimed to provide the
, –
Scheme 3. Observed divergent diastereoselectivity in the conjugate addition to Michael acceptor 10.
Figure 4. Single crystal X-ray structure obtained from the 3,5-dinitrobenzoate of the advanced
intermediate 12 at 298 K. The structure is deposited at Cambridge Crystallographic Data Centre
as CCDC 1535632.
Cycloaddition via a non-enzymatic pathway is also possible. Thus, Gerwick has proposed allylic
carbocations as conceptual intermediates in the biogenesis of marine carbocyclic oxylipins, such as

Molecules 2017, 22, 1720
6 of 18
prostaglandin A2 (PGA2) [86
–
88]. In this sense, arachidonic acid (3) provides a link between mucosin
and the prostanoid scaffold, pointing towards a possible mechanism. Yet, for the majority of examples
found, the annulation produces a trans-1,2-disubstituted cyclopentane ring [89–94].
Although being an uncommon structural feature, it would be premature to conclude that the
cis-fused bicyclo[4.3.0]non-3-ene system was incongruous. Nevertheless, when recordings were
made on methyl ester 2*, the data did not match those reported for the compound isolated from
Reniera mucosa. This was most convincingly demonstrated by comparing the 13C-NMR spectra (Table 1):
Out of the 20 resonances that are observable for the carbon framework, excluding the methoxy group,
16 display deviating shifts (see also Supplementary Figure S-38). Furthermore, the optical rotation
of 2* did not only differ in magnitude, but also in sign: While the naturally occurring material and its
purported structure
2
have values of [α]2D6 = −35.5◦ and
−
9.8◦ (c = 0.8, hexane), respectively [35
,36]
the diastereomer 2* had an [α]2D6 = +64.0◦ (c = 0.8, hexane). Whitby and co-workers have reported
[α]2D6 = +38.2◦ (c = 0.8, hexane) for the material obtained via zirconium induced co-cyclisation [37].
†,‡
Table 1. Comparative 13C-NMR of methyl ester 2* (δ-values).
Entry
Casapullo et al. [35]
Whitby et al. [37]
Previous Work [36]
This Work
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
174.2
130.0
129.8
127.0
127.0
52.1
51.4
47.1
42.1
39.9
36.7
36.5
36.4
33.2
32.0
31.7
31.5
30.7 §
24.5
22.6
13.8
174.2
130.3
129.8
127.3
127.1
52.2
51.4
47.2
42.3
40.1
37.0
36.74
36.68
33.4
32.4
31.9
31.6
30.7
24.7
22.9
14.1
174.2
130.4
129.9
126.3
126.1
51.4
51.0
44.0
40.3
38.1
37.7
37.1
34.9
33.4
31.9
31.0
27.8
27.7
24.8
22.9
14.1
‡
174.2
131.2
129.0
125.3
125.1
51.6
51.4
41.3
37.2
36.2
35.5
35.4
33.4
33.0
31.9
31.0
26.9
24.7
23.0
21.7
14.1
†
The italic bold numbers indicate deviating
able to procure the original 13C-spectrum from the authors quoted in Ref. [35] for comparison. According to
communication rendered in the supporting information accompanying Ref. [37], the resonance at 30.7 had been
36.3 was observed. The data were subsequently revised and this
δ-values compared to Ref. [35]. Upon request, we have not been
§
δ
omitted in Ref. [35], while an additional signal at
fact is not touched upon in the main paper.
δ
By achieving a rational synthesis of exo-mucosin 2*, the target selection has been narrowed down.
Yet, in terms of the cis-fused bicycle, there are permutants still unaccounted for. However, given the
obvious sterical encumbrance of the two remaining syn-diastereomers, they seemed unlikely
candidates considering the biogenesis of marine carbocyclic oxylipins [86–88]. Additionally, it is worth
noticing that the anti-relationship of the appended groups seems to rest on a sounder foundation:
Diagnostic correlations between the C7-methylene group and the C16-proton have been observed by
NOESY and ROESY [35]. Furthermore, DFT calculations by Whitby and co-workers on the relative
stability of zirconacycles [37], indicate that the anti-geometries are favoured over the syn-geometries.
Hence, based on our synthetic endeavours, it was inferred that the natural product named mucosin
has a trans-fused bicyclo[4.3.0]non-3-ene ring system and the featured substituents are anti-related.
Albeit that the outlined synthesis did not yield the ultimate target, the sequence provided
an answer to a central question: namely, the question about the geometry of the fused bicycle.

Molecules 2017, 22, 1720
7 of 18
Moreover, taken together with what we have detailed before [36], the current findings have demonstrated
a fascinating chemical aspect of the cis-fused bicyclo[4.3.0]non-3-ene scaffold, that unfolds when a Michael
acceptor motif is incorporated. Thus, swapping the functional group at the β-position of the Michael
acceptor with the functional group of the Michael donor, a complete inversion of diastereoselectivity
was observed. The transformation proved to be doubly orthogonal, as even the stereochemistry at the
α-position was inverted in the process. Bearing in mind the chosen protocol, it must be assumed that
the favoured epimer is not obtained via spontaneous equilibration of the incipient ester enolate anion.
Rather, the reactive constellation between BuMgCl and TMSCl intercepts the Michael adduct as a silyl
ketene acetal; it is therefore in the succeeding protonation of the trapped ester enolate anion that the
observed epimeric configuration is established (Scheme 4). The stereoselective protonation of enolates is
a concept, which has received a great deal of attention and in its purest form constitutes a biomimetic
approach to establish
α-chirality [95–98]. In our example, the pre-existing topology works in consonance
to dictate which face is being protonated.
Scheme 4. Silyl ketene acetal as the source of face-selective protonation.
In summary, the conjugate system shown in Scheme 3 (vide supra) displays a remarkable
diastereotopic preference, enabling excellent control over the reactive manifold.
3. Experimental Section
3.1. General Information
All commercially available reagents and solvents were used in the form they were supplied without
any further purification. (+)-Bis[(R)-1-phenylethyl]amine hydrochloride (optical purity
≥
99% ee by GLC)
was purchased from Sigma-Aldrich (St. Louis, MO, USA). The stated yields are based on isolated material.
The melting points are uncorrected. Thin layer chromatography was performed on silica gel 60 F254
aluminum-backed plates fabricated by Merck (Kenilworth, NJ, USA). Flash column chromatography was
performed on silica gel 60 (40–63 µm) fabricated by Merck. NMR spectra were recorded on a Bruker
AscendTM 400 (Bruker, Billerica, MA, USA) at 400 MHz for 1H-NMR and at 100 MHz for 13C-NMR.
Coupling constants (J) are reported in hertz (Hz) and chemical shifts are reported in parts per million
1
(
δ
) relative to the central residual protium solvent resonance in H-NMR (CDCl3 =
δ
7.27) and the
central carbon solvent resonance in 13C-NMR (CDCl3 =
δ 77.00 ppm). The following abbreviation,
appt, has been used to designate an apparent triplet. Mass spectra were recorded at 70 eV on Waters
Prospec Q spectrometer (Waters Corporation, Milford, MA, USA) using EI as the method of ionization.
IR spectra (4000–600 cm−1) were recorded on a Perkin-Elmer Spectrum BX series FT-IR spectrophotometer
(Waltham, MA, USA) using a reflectance cell (HATR). Optical rotations were measured using a 1 mL cell
with a 1.0 dm path length on a Perkin Elmer 341 polarimeter using the stated solvents. Determination
of enantiomeric excess was performed by GLC on an Agilent Technologies 7820A GC instrument
(Agilent Technologies, Santa Clara, CA, USA) with split (1:30) injection, FID detector and equipped with a
chiral stationary phase (Agilent J&W GC columns, CP-Chirasil-DEX CB, 25 m, 0.25 mm, 0.25
µm) applying
the conditions stated. X-ray crystallography was performed on a Bruker D8 Venture diffractometer

Molecules 2017, 22, 1720
8 of 18
with InCoatec ImuS Microfocus radiation source and Photon 100 CMOS detector. Data collection with
Apex2 [99], data integration and cell refinement with SAINT,1 absorption correction by SADABS [99],
structure solution with SHELXT [100], structure refinement with SHELXL [101]. Molecular graphics from
Mercury [102].
3.2. Synthesis of Keto Ester 9
(+)-Bis[(R)-1-phenylethyl]amine hydrochloride (2.5 g, 9.60 mmol, 1.58 equiv.) was added in one
portion to dry THF (10 mL) at ambient temperature and stirred for 5 min. The stirring suspension
was then cooled to
−
78 ◦C and BuLi (2.5M in hexane) (7.67 mL, 19.18 mmol, 3.16 equiv.) was added
◦
dropwise. The suspension changed colour from cloudy white to pale orange. After stirring at
15 min the suspension was warmed to ambient temperature whereby a transparent yellow solution was
formed. This recooled to (0.826 g, 6.07 mmol,
1.0 equiv.) was added dropwise over 10 min in dry THF (10 mL). This mixture was then stirred for 45 min
whereby a purple colour evolved. Methyl cyanoformate (0.96 mL, 12.14 mmol, 2.0 equiv.) was then
added dropwise over 5 min. and the mixture immediately turned bright yellow in colour. This mixture
−
78 C for
−
78 ◦C and meso-(1S,6R)-bicyclo[4.3.0]non-3-ene-8-one
4
was left stirring for 2.5 h and then quenched by addition of H2O (2 mL) at
then warmed to r.t. and extracted with EtOAc (2 50 mL). The resulting organic layer was then washed
with H2O (2 100 mL), 0.5 M HCl (1
−
78 ◦C. The mixture was
×
×
× 100 mL) and brine (1 × 100 mL). The organic layer was then
dried over MgSO4, filtered and concentrated in vacuo. The resulting crude keto ester was purified by
column chromatography (hexane/EtOAc 5:1) to form a colourless oil. This oil was then recrystallised
◦
from hexane at 0 C, filtered and air dried to obtain the compound
9
as white crystals. All spectroscopic
and physical data were in full agreement with those reported in the literature [103]. Yield: 0.812 g
(69%); [α]2D6 = −161◦ (c = 0.1, CHCl3); 1H-NMR (400 MHz, CDCl3):
δ 5.73–5.66 (m, 2H), 3.76 (s, 3H),
3.04 (d, J = 11.1 Hz, 1H), 2.88–2.83 (m, 1H), 2.52–2.38 (m, 3H), 2.33–2.21 (m, 2H), 2.04 (dd, J = 1.9, 18.2 Hz,
1H), 1.67–1.61 (m, 1H); 13C-NMR (100 MHz, CDCl3):
δ 211.6, 169.7, 124.9, 123.9, 57.7, 52.4, 46.6, 37.3,
29.7, 26.8, 25.3; IR (neat, cm−1) 3034 (w), 2945 (m), 2908 (m), 2837 (w), 1751 (s), 1718 (s) 1656 (w) 1433◦(s)
1404 (m); HRMS (EI+): Exact mass calculated for C11H14O3 [M]+: 194.0943 found 194.0933; m.p.: 59–61 C;
TLC (hexane/EtOAc 4:1, KMnO4 stain): Rf = 0.42.
3.3. Synthesis of Michael Acceptor 10
3.3.1. Synthesis of α-Hydroxy Ester pre-10a
To stirring solution of 9
(1.40 g, 7.21 mmol, 1.0 equiv.) in MeOH (50 mL) at 0 ◦C was added NaBH4
(0,410 g, 10.8 mmol, 1.5 equiv.). The reaction was monitored by TLC and was deemed complete after
◦
1 h. Then, dilute aq. HCl (10 mL, 1 M) was added drowise at 0 C. The quenched reaction was concentrated
in vacuo to afford a crude mixture. This was poured over Et2O (50 mL), whereupon water (50 mL) was
added. The organic layer was separated and the aqueous layer was extracted with Et2O (2 × 50 mL).
The organic layers were combined, washed with brine (1 × 100 mL), dried over MgSO4, filtered and
concentrated in vacuo to afford a colourless, oily, residue. The crude was purified by column
chromatography on silica (hexane/EtOAc 7:3) to afford the C8-epimeric compound pre-10a as colourless
oil. Yield: 1.07 g (76%); 1H-NMR (400 MHz, CDCl3):
2.65–2.50 (m, 1H), 2.38–2.10 (m, 6H), 2.10–1.84 (m, 2H), 1.52–1.42 (m, 1H)
175.5 (major), 174.8 (minor), 127.5 (minor), 126.7 (minor), 126.6 (major), 125.6 (major), 75.3 (major),
δ
5.81–5.71 (m, 2H), 4.57–4.39 (m, 1H), 3.73 (s, 3H),
13C-NMR (100 MHz, CDCl3)
;
:
δ
73.0 (minor), 57.3 (major), 53.9 (minor), 51.8 (major), 51.7 (minor), 42.1 (minor), 40.9 (major), 39.0 (major),
−1
38.0 (minor), 34.0 (minor), 33.6 (major), 27.9 (minor), 27.7 (major), 26.3 (major), 26.2 (minor); IR (neat, cm
)
3439 (br), 3026 (w), 2914 (w), 2841 (w), 1712 (s), 1438 (m); HRMS (EI+): Exact mass calculated for C11H16O3
[M]+: 196.1099, found 196.1087; TLC (hexane/EtOAc 4:1, KMnO4 stain): Rf = 0.30.

Molecules 2017, 22, 1720
9 of 18
3.3.2. Synthesis of Mesylate pre-10b
To a stirring solution of C8-epimer pre
-10a (1.07 g, 5.45 mmol, 1.0 equiv.) in dry CH2Cl2 (50 mL)
at ambient temperature was added Et3N (1.14 mL, 8.18 mmol, 1◦.5 equiv.) in a dropwise manner.
The resulting mixture was left stirring for 5 min. and then cooled to 0 C. Subsequently, methanesulfonyl
chloride (0.51 mL, 6.59 mmol, 1.2 equiv.) was added in a dropwise manner and the reaction mixture was
left stirring for 10 min. with continued cooling. Then, the cooling was discontinued and the reaction
mixture was allowed to attain ambient temperature overnight. At this point the reaction mixture had
turned from colourless to yellow. Brine (20 mL) was added in a dropwise manner and the volatiles were
removed in vacuo. The resulting yellow liquid was poured over EtOAc (50 mL) and satd. aq. NaHCO3
(50 mL) was added. The organic layer was separated and the aqueous layer was extracted with EtOAc
(2
×
50 mL). The organic layers were combined and washed with brine (1
×
100 mL), dried over
MgSO4, filtered and concentrated in vacuo to afford a yellow, oily, residue. The crude was purified by
column chromatography on silica (hexane/EtOAc 4:1) to afford the C8-epimeric compound pre-10b as
a yellow oil. Yield: 1.16 g (77%); 1H-NMR (400 MHz, CDCl3)
δ 5.81–5.77 (m, 0.3H, minor), (m, 1.7H,
major), 5.37–5.31 (m, 1H), 3.74 (s, 2.6H, major), 3.72 (s, 0.4H, minor), 3.01 (s, 2.6H, major), 2.96 (s, 0.4H,
minor), 2.88–2.82 (m, 1H), 2.4.06 (m, 6H), 2.02–1.87 (m, 1H), 1.86–1.78 (m, 1H); 13C-NMR (100 MHz,
CDCl3)
δ 173.9, 127.3 (minor), 126.8 (minor), 125.6 (major), 124.4 (major), 83.9 (major), 83.8 (minor),
54.6 (major), 53.6 (minor), 52.2 (major), 51.9 (minor), 40.9 (minor), 39.8 (major), 39.1 (major), 38.3 (minor),
37.9 (major), 36.4 (minor), 33.9 (major), 33.5 (minor), 27.6 (minor), 26.6 (major), 26.0 (minor), 25.4 (major);
IR (neat, cm−1
) 3031 (w), 2942 (w), 2847 (w), 1734 (s), 1438 (w), 1354 (s); HRMS (EI+): Exact mass
calculated for C12H18O5S [M]+: 274.0875, found 274.0865; TLC (hexane/EtOAc 4:1, KMnO4 stain):
Rf = 0.45.
3.3.3. Synthesis of Michael Acceptor 10
To a stirring solution of C8-epimer pre-10b (1.40 g, 5.10 mmol, 1.0 equiv.) in dry toluene (30 mL)
at ambient temperature was added DBU (1.73 mL, 11.6 mmol, 2.3 equiv.) in a dropwise manner
over 5 min. The reaction mixture was stirred overnight at the stated conditions. Having deemed the
reaction complete by TLC, water (10 mL) and dilute aq. HCl (10 mL, 0.5 M) was added. The resulting
mixture was poured over Et2O (20 mL) and the organic layer was separated. The aqueous layer was
extracted with Et2O (2
×
30 mL). The organic layers were combined, washed in succession with
50 mL), dried over MgSO4, filtered and concentrated in vacuo to
water (1 × 50 mL) and brine (1
×
obtain a oily residue. The crude was purified by column chromatography on silica (hexane/EtOAc
9:1) to afford the compound 10 as colourless oil. Yield: 0.866 g, (95%); [α]2D6 = +180◦ (c = 0.8, CHCl3);
1H-NMR (400 MHz, CDCl3)
δ
6.80–6.69 (m, 1H), 5.95–5.84 (m, 1H), 5.84–5.74 (m, 1H), 3.74 (s, 3H),
3.04–3.92 (m, 1H), 2.67–2.51 (m, 2H), 2.50–2.36 (m, 1H), 2.34–2.12 (m, 2H), 2.00–1.82 (m, 2H); 13C-NMR
(100 MHz, CDCl3)
165.6, 143.5, 140.8, 128.3, 127.0, 51.2, 41.2, 39.4, 36.0, 27.8, 26.6; IR (neat, cm−1
δ
)
3031 (w), 2931 (w), 2841 (w), 1712 (s), 1628 (w), 1438 (m); HRMS (EI+): Exact mass calculated for
C11H14O2 [M]+: 178.0994, found 178.1000; TLC (hexane/EtOAc 4:1, KMnO4 stain): Rf = 0.60.
3.4. Synthesis of Michael Adduct 11
To a solution of Michael acceptor 10 (0.421 g, 2.36 mmol, 1.0 equiv.) in dry THF (20 mL) at
−
35 ◦C was added in succession CuI (0.045 g, 0.24 mmol, 0.1 equiv.) and TMSCl (0.641 g, 0.75 mL,
5.90 mmol, 2.5 equiv.). The resulting slightly heterogenous mixture caused by suspended CuI was
stirred for 5 min, whereupon BuMgCl (2.0 M in THF) (2.36 mL, 4.72 mmol, 2.0 equiv.) was added
◦
in a dropwise manner during the course of 2 h, maintaining the temperature between at
−
35 C.
Initial colours cycled between clear and yellow, but gradually took a transient purple hue while
reverting to clear. Upon completing the addition, the purple colour persisted (cloudy amethyst).
At this point, TLC revealed that the starting material had been consumed. The reaction was treated
with aq. satd. NH4Cl (5 mL) and diluted with Et2O/water (30 mL, 2:1). The phases were separated and

Molecules 2017, 22, 1720
10 of 18
the aq. phase was extracted with Et2O (3
×
20 mL). The combined org. phases were washed with brine
(15 mL), dried over MgSO4, filtered and the solvent was evaporated in vacuo. The residue was purified
by column chromatography on silica (hexane/EtOAc 90:10) to afford compound 11 as a colourless oil.
Yield: 0.496 g (81%); [α]2D6 = +63◦ (c = 0.8, CHCl3); 1H-NMR (400 MHz, CDCl3)
δ
5.66–5.56 (m, 2H),
3.67 (s, 3H), 2.58–2.46 (m, 2H), 2.36–2.16 (m, 3H), 1,95–1.68 (m, 4H), 1.45–1.35 (m, 2H), 1.35–1.15 (m, 5H)
,
0.87 (t, J = 7.0 Hz, 3H); 13C-NMR (100 MHz, CDCl3)
δ 174.8, 124.7, 124.6, 56.0, 51.3, 38.9, 37.4, 36.8, 36.0,
35.3, 30.4, 26.5, 22.8, 22.6, 14.1; IR (neat, cm−1) 3020 (w), 2925 (m), 1734 (s); HRMS (EI+): Exact mass
calculated for C15H24O2 [M]+: 236.1776, found 236.1763; TLC (hexanes/EtOAc 80:20, KMnO4 stain):
Rf = 0.70.
3.5. Synthesis of Carbinol 12
Michael adduct 11 (0.496 g, 2.10 mmol, 1.0 equiv.) was dissolved in hexane (10 mL) at ambient
temperature and stirred for 5 min. The solution was then cooled to 0 ◦C and DIBAL-H (1M in hexane)
(4.2 mL, 4.20 mmol, 2.0 equiv.) was added dropwise over 5 min. The reaction was then left to warm
◦
to r.t. After 1 h the reaction was cooled back to 0 C and quenched with sat. aq. NH4Cl (5 mL).
The reaction mixture was allowed to warm to ambient temperature whereby a cloudy suspension occurred.
This suspension was poured over sat. aq. NH4Cl (20 mL) and the organic layer separated. The aqueous
layer was extracted with EtOAc (2
1 × 100 mL), brine (1 100 mL), dried over MgSO4, filtered and concentrated in vacuo to give a crude
cloudy oil. This was then purified by column chromatography on silica (hexane/EtOAc 95:5) to afford
×
50 mL) and the organic layers combined, washed with H2O
(
×
compound 12 as a colourless oil. Yield: 0.400 g, (92%); [α]2D6 = +104 (c = 0.8, CHCl3); 1H-NMR (400 MHz,
CDCl3) 5.77–5.51 (m, 2H), 3.77–3.58 (m, 2H), 2.38–2.22 (m, 1H) 2.22–2.05 (m, 2H), 2.05–1.71 (m, 4H)
1.71–1.52 (m, 2H), 1.52–1.35 (2H) 1.35–1.14 (m, 6H), 0.88 (t, J = 7.1 Hz, 3H); 13C-NMR (100 MHz, CDCl3)
125.3, 124.9, 63.3, 53.7, 38.1, 36.8, 36.3, 35.5, 35.4, 30.8, 26.6, 22.9, 21.6, 14.1; IR (neat, cm−1) 3328 (br.),
◦
,
δ
3020 (w), 2925 (s); HRMS (EI+): Exact mass calculated for C14H24O [M]+: 208.1827, found 208.1832;
TLC (hexane/EtOAc 4:1, KMnO4 stain): Rf = 0.40. The enantiomeric excess was determined by chiral
GLC analysis (CP-Chirasil-DEX CB, using the following program: 60 ◦C (45 min)—1 degrees/min to
160 ◦C—160 ◦C (5 min)): tr(e1, major) = 65.08 min and tr(e2, minor) = 65.67 min; ee: > 99% [104].
3.6. Synthesis of 3,5-Dinitrobenzoate 12-DNB
To a stirring solution of carbinol 12 (0.129 g, 0.546 mmol, 1.0 equiv.) in dry DCM (20 mL) was
◦
added Et3N (0.23 mL, 1.64 mmol, 3.0 equiv.) dropwise. The solution was then cooled to 0 C and
3,5-dinitrobenzoyl chloride (0.215 g, 0.933 mmol, 1.7 equiv.) was added in one portion. The reaction was
slowly warmed to ambient temperature and monitored by TLC until completion. After 2 h, the reaction
mixture was poured over H2O (10 mL) and the organic layer separated. The aqueous layer was then
extracted with DCM (2
×
10 mL) and the organic layers combined. The organic layers were then
30 mL), dried with MgSO4, filtered and concentrated in vacuo
washed with H2O (1 30 mL), brine (1
×
×
to form a crude orange oil. This was purified by column chromatography on silica (hexane/EtOAc,
95:5) to afford the compound 12-DNB as a white powder. Yield: 0.193 g (88%), [α]2D6 = +42◦
(
c = 0.8, CHCl3); 1H-NMR (400 MHz, CDCl3)
δ
9.23 (t, J = 2.2 Hz, 1H), 9.14 (d, J = 2.2 Hz
4.49 (s, 1H), 4.47 (d, J = 1.9 Hz, 1H), 2.37–2.10 (m, 4H), 2.02–1.88 (m, 2H),
1.58–1.43 (m, 2H), 1.40–1.23 (m, 5H), 0.89 (t, J = 6.7 Hz, 3H); 13C-NMR (100 MHz,
162.5, 148.7, 134.1, 129.3, 125.4, 124.3, 122.3, 67.7, 49.5, 38.6, 36.9, 36.7, 35.5, 35.4, 30.8, 26.5,
,
2H), 5.70–5.61 (m, 2H)
1.88–1.68 (m, 3H)
CDCl3)
,
,
δ
22.9, 21.8, 14.1; IR (neat, cm−1) 3098 (w), 3020 (w), 2931 (m), 1723 (s), 1538 (s); HRMS (EI+): Exact mass
calculated for C21H26N2O6 [M]+: 402.1791, found 402.1797; m.p.: 117 ◦C; TLC (hexane/EtOAc 4:1,
KMnO4 stain): Rf = 0.75. [105]

Molecules 2017, 22, 1720
11 of 18
3.7. Synthesis of Aldehyde 13
3.7.1. Synthesis of Mesylate pre-13a
To a stirring solution of carbinol 12 (0.400 g, 1.92 mmol, 1.0 equiv.) in dry CH2Cl2 (5 mL) at ambient
temperature, was added Et3N (0.54 mL, 3.84 mmol, 2.0 equiv.) dropwise. This solution was left stirring
◦
for 5 min then cooled to 0 C. Then methanesulfonyl chloride (0.45 mL, 5.76 mmol, 3.0 equiv.) was added
dropwise and the reaction was left at 0 ◦C for 10 min then warmed to ambient temperature and left
overnight. The reaction mixture turned colourless to yellow. Then, brine (10 mL) was added dropwise
and the volatiles concentrated in vacuo to afford a yellow liquid. This was poured over EtOAc (50 mL)
and sat. aq. NaHCO3 (50 mL) was added. The organic layer was separated and the aqueous layer
extracted with EtOAc (2
× 50 mL). The organic layers were combined and washed with brine (1 × 50 mL),
dried over MgSO4, filtered and concentrated in vacuo to afford a crude yellow oil. This was then purified
by column chromatography on silica (hexane/EtOAc 95:5) to afford the compound pre-13a as a colourless
oil. Yield: 0.497 g, (90%); [α]2D6 = +79◦ (c = 0.8, CHCl3); 1H-NMR (400 MHz, CDCl3) δ 5.60–5.50 (m, 2H)
,
4.21–4.12 (m, 2H)
1.71–1.55 (m, 3H)
CDCl3)
,
,
2.94 (s, 3H)
1.40–1.30 (m, 2H)
,
2.25–2.11 (m, 1H), 2.11–1.99 (m, 2H), 1.98–1.83 (m, 2H)
, 1.83–1.76 (m, 1H),
,
1.28–1.10 (m, 5H)
,
0.82 (t, J = 7.0 Hz, 3H); 13C-NMR (100 MHz,
δ
125.3, 124.4, 70.3, 50.0, 38.1, 37.4, 36.6, 36.4, 35.4, 35.3, 30.7, 26.4, 22.8, 21.4, 14.1; IR (neat, cm−1
)
3020 (w), 2925 (m), 1354 (s); HRMS (EI+): Exact mass calculated for C15H26O3S2 [M]+: 286.1603,
found 286.1627; TLC (hexane/EtOAc 4:1, KMnO4 stain): Rf = 0.50.
3.7.2. Synthesis of Nitrile pre-13b
To a stirring solution of mesylate pre-13a (0.497 g, 1.74 mmol, 1.0 equiv.) in dry DMSO (30 mL) was
added solid KCN (0.675 g, 10.4 mmol, 6.0 equiv.) in one portion. The reaction mixture was then heated
◦
to 70 C for 2 h. The reaction mixture changed from colourless to yellow. Then, the reaction was cooled
to r.t. and H2O (5 mL) was added dropwise. The reaction mixture turned from yellow to colourless.
This was then poured over EtOAc (20 mL) and the organic layer separated. The aqueous layer was
extracted with EtOAc (2
×
20 mL) and the organic layers combined. They were then washed with brine
(1 50 mL), dried over MgSO4, filtered and concentrated in vacuo to afford a crude brown oil. This was
×
then purified by column chromatography on silica (hexane/EtOAc 98:2) to give the compound
pre-13b as a colourless oil. Yield: 0.325, (86%); [α]2D6 = +111◦ (c = 0.8, CHCl3); 1H-NMR (400 MHz,
CDCl3)
δ
5.82–5.47 (m, 2H), 2.42–2.28 (m, 2H), 2.18–2.14 (m, 2H), 2.04–1.85 (m, 3H), 1.74–1.65 (m, 3H),
125.4,
3026 (w),
1.51–1.35 (m, 2H), 1.33–1.19 (m, 5H), 0.89 (t, J = 7.0 Hz, 3H) 13C-NMR (100 MHz, CDCl3)
δ
124.2, 119.6, 47.3, 41.4, 37.9, 35.8, 35.7, 35.1, 30.6, 26.5, 22.8, 21.5, 17.7, 14.1; IR (neat, cm−1
2919 (s), 2248 (w), 1465 (w) 1436 (w); HRMS (EI+): Exact mass calculated for C15H23N [M]+: 217.1830,
)
found 217.1845; TLC (hexane/EtOAc 4:1, KMnO4 stain): Rf = 0.80.
3.7.3. Synthesis of Aldehyde 13
A stirring solution of nitrile pre-13b (0.322 g, 1.48 mmol, 1.0 equiv.) in hexane (10 mL) was cooled to
◦
−
78 C. Then DIBAL-H (1M in hexane) (2.20 mL, 2.22 mmol, 1.5 equiv.) was added dropwise over 5 min
and the reaction left to stir for 20 min. Then sat. aq. Rochelle salt (5 mL) was added dropwise to the reaction
mixture and then left to warm to ambient temperature. The resulting cloudy suspension was poured
over EtOAc (20 mL) and sat. aq. Rochelle salt (20 mL). The organic layer was separated and the aqueous
phase extracted with EtOAc (2
1 × 50 mL), dried over MgSO4, filtered and concentrated in vacuo to afford a crude cloudy oil. This was
then purified by column chromatography on silica (hexane/EtOAc, 95:5) to afford the compound 13 as
a colourless oil. Yield: 0.253 mg, (78%); [α]2D6 = +101◦ c = 0.8, CHCl3); 1H-NMR (400 MHz, CDCl3)
9.79 (t, J = 2.3 Hz, 1H), 5.68–5.51 (m, 2H), 2.49–2.44 (m, 2H) 2.35–2.23 (m, 1H), 2.22–2.12 (m, 1H), 2.10–1.98
(m, 2H), 1.92–1.78 (m, 2H), 1.74–1.60 (m, 3H) 1.47–1.35 (m, 2H), 1.35–1.15 (m, 5H), 0.88 (t, J = 7.0 Hz, 3H);
13C-NMR (100 MHz, CDCl3)
202.9, 125.3, 124.7, 45.2, 44.7, 41.4, 37.5, 35.7, 35.6, 34.9, 30.8, 26.7, 22.9,
×
20 mL). The organic phases were combined and washed with brine
(
(
δ
,
,
δ

Molecules 2017, 22, 1720
12 of 18
22.1, 14.1; IR (neat, cm−1
)
3020 (w), 2919 (m), 2712 (w), 1723 (s); HRMS (EI+): Exact mass calculated for
C15H24O [M]+: 220.1827, found 220.1824; TLC (hexane/EtOAc 4:1, KMnO4 stain): Rf = 0.80.
3.8. Synthesis of Alkyne 14
To a stirring solution of aldehyde 13 (0.253 g, 1.15 mmol, 1.0 equiv.) in dry MeOH (6 mL) at 0 ◦C
was added solid K2CO3 (0.381 mg, 2.76 mmol, 2.4 equiv.) in one portion and Ohira-Bestmann reagent
(10% w/w in MeCN) (3.9 mL, 3.32 g, 1.73 mmol, 1.5 equiv.). The suspension was then warmed to
ambient temperature and left stirring 1 h. After analysis by TLC the mixture was treated with sat.
aq. NaHCO3 (20 mL), and the resulting mixture poured over CH2Cl2 (20 mL). The organic phase
was separated and the aqueous phase was washed with CH2Cl2 (3
×
10 mL). The organic phases
were then combined, dried over Na2SO4, filtered and concentrated in vacuo to afford a crude oil.
This was purified by column chromatography on silica (hexane/EtOAc, 95:5) to afford the compound
14 as a colourless oil. Yield: 0.193 g, (78%); [α]2D6 = +119◦ (c = 0.8, CHCl3); H-NMR (400 MHz,
1
CDCl3)
δ 5.72–5.58 (m, 2H), 2.35–2.24 (m, 2H), 2.18–2.09 (m, 3H), 2.07–1.98 (m, 1H), 1.91 (t, J = 2.7 Hz,
1H), 1.89–1.83 (m, 1H), 1.83–1.75 (m, 1H), 1.75–1.55 (m, 3H), 1.55–1.45 (m, 1H), 1.45–1.36 (m, 1H),
1.36–1.13 (m, 5H), 0.89 (t, J = 7.1 Hz, 3H); 13C-NMR (100 MHz, CDCl3)
δ; 125.2, 125.0, 84.4, 67.9,
50.2, 41.2, 37.7, 36.1, 35.9, 35.2, 30.8, 26.8, 22.9, 21.4, 18.9, 14.1; IR (neat, cm−1) 3311 (m), 3020 (w),
2919 (s), 1432 (m); HRMS (EI+): Exact mass calculated for C16H24 [M]+: 216.1878, found 216.1867;
TLC (hexane, KMnO4 stain and anisaldehyde dip): Rf = 0.25.
3.9. Synthesis of exo-Mucosin 1*
3.9.1. Synthesis of Ethyl Ester 15
To a stirring solution of Cp2ZrCl2 (0.400 g, 1.37 mmol, 2.0 equiv.) in dry THF (8 mL) at 0 ◦C was
added DIBAL-H (1M in hexane) (1.37 mL, 1.37 mmol, 2.0 equiv.) via dropwise addition. The resulting
◦
homogenous mixture was then protected from light and stirred at 0 C for 1 h after which time a
colourless heterogeneous mixture formed. Then alkyne 14 (0.148 g, 0.684 mmol, 1.0 equiv.) dissolved in
dry THF (4 mL) was added dropwise to the reaction mixture at 0 ◦C. After 1 h at 0 ◦C iodine (0.260 g,
1.02 mmol, 1.5 equiv.) was added in one portion to the homogeneous yellow reaction mixture. The reaction
mixture was then warmed to ambient temperature and stirred for 1 h. To the preformed vinyl iodide
was successively added 4-ethoxy-4-oxobutylzinc bromide solution (0.5M in THF) (2.7 mL, 1.37 mmol,
2.0 equiv.) dropwise and (Ph3P)4Pd (0.079 g, 0.068 mmol, 0.1 equiv.) in one portion. The resulting light
brown mixture was stirred at ambient temperature for 1 h and monitored by TLC. Once the reaction
had gone to completion 1 M HCl (10 mL) was added dropwise and the reaction poured over Et2O
(15 mL). The aqueous phase was extracted with Et2O (3
×
50 mL) and the organic phases combined,
dried over MgSO4, filtered and concentrated in vacuo to form a crude brown oily mixture. This oily
mixture was purified by column chromatography on silica (hexane/EtOAc, 95:5) to afford the compound
15 as a colourless oil. Yield: 0.196 g, (86%); [α]2D6 = +67◦ (c = 0.8, CHCl3); 1H-NMR (400 MHz,
CDCl3)
3H), 2.16–1.82 (m, 8H)
13C-NMR (100 MHz, CDCl3)
δ
5.69–5.56 (m, 2H), 5.48–5.33 (m, 2H), 4.12 (q, J = 7.1 Hz, 2 H), 2.31–2.21 (appt, J = 7.5 Hz
1.75–1.50 (m, 6H), 1.48–1.41 (m, 1H), 1.39–1.22 (m, 9H), 0.88 (t, J = 7.1 Hz, 3H);
173.8, 131.2, 129.1, 125.3, 125.1, 60.2, 51.6, 41.3, 37.3, 36.2, 35.6, 35.5,
,
,
δ
33.7, 33.0, 31.9 31.0, 26.9, 24.8, 23.0, 21.7, 14.2, 14.1; IR (neat, cm−1) 3020 (w), 2925 (m), 1734 (s);
HRMS (EI+): Exact mass calculated for C22H36O2 [M]+: 332.2715, found 332.2709; TLC (hexane/EtOAc
95:5, KMnO4 stain): Rf = 0.65.
3.9.2. Synthesis of exo-Mucosin 1*
To a stirring solution of ethyl ester 15 (0.177 g, 0.533 mmol, 1.0 equiv.) in THF/MeOH/H2O (2:2:1)
(5 mL) at ambient temperature was added lithium hydroxide monohydrate (0.783 mg, 18.7 mmol,
35.0 equiv.) in one portion. The reaction mixture was left stirring and monitored by TLC. Left overnight,
the reaction had gone to completion and was acidified to pH 2 by 1M HCl (5 mL). The reaction mixture

Molecules 2017, 22, 1720
13 of 18
was then poured over EtOAc (5 mL) and the aqueous phase extracted with EtOAc (3
×
5 mL).
The organic phases were combined and washed with brine (1
×
20 mL), dried over MgSO4, filtered
and concentrated in vacuo to provide a colourless oil. This was purified by column chromatography
on silica (hexane/EtOAc, 3:2) to afford the compound 1* as a colourless oil. Yield: 0.154 g, (95%);
[α]2D6 = +77◦ (c = 0.8, hexane); H-NMR (400 MHz, CDCl3)
δ 11.63 (br, 1H), 5.67–5.56 (m, 2H),
1
5.50–5.34 (m, 2H), 2.34 (t, J = 7.5 Hz, 2H), 2.33–2.22 (m, 1H), 2.15–2.12 (m, 8H), 1.77–1.49 (m, 6H),
1.48–1.40 (m,1H), 1.40–1.08 (m, 6H), 0.88 (t, J = 6.7 Hz, 3H); 13C-NMR (100 MHz, CDCl3)
δ
180.3,
131.4, 128.9, 125.3, 125.1, 51.6, 41.3, 37.3, 36.2, 35.6, 35.5, 33.4, 33.0, 31.8, 31.0, 26.9, 24.5, 23.0, 21.7, 14.2;
IR (neat, cm−1) 3020 (w), 2925 (m), 1712 (s); HRMS (EI+): Exact mass calculated for C20H32O2 [M]+:
304.2402, found 304.2400; TLC (hexane/EtOAc 3:2, KMnO4 stain): Rf = 0.40.
3.9.3. Synthesis of Methyl Ester 2*
To a stirring solution of exo-mucosin 1* (0.023 g, 0.076 mmol, 1.0 equiv.) in toluene/MeOH (3:2)
(5 mL) at ambient temperature was added TMS diazomethane solution (2M in hexane) (0.06 mL,
0.113 mmol, 1.5 equiv.) dropwise over 2 min. The reaction mixture bubbled and turned transparent
yellow. The reaction was monitored by TLC and after 1 h had gone to completion. The reaction
mixture was then concentrated in vacuo and directly purified by column chromatography on silica
(hexane/EtOAc, 95:5) to afford the compound 2* as a colourless oil. Yield: 23 mg, (96%); [α]2D6 = +64◦
1
(c = 0.8, hexane); H-NMR (400 MHz, CDCl3)
δ 5.69–5.56 (m, 2H), 5.49–5.32 (m, 2H), 3.66 (s, 3H),
2.29 (t, J = 7.5 Hz, 2H), 2.26–2.21 (m, 1H), 2.13–1.82 (m, 8H), 1.75–1.63 (m, 3H), 1.63–1.50 (m, 3H)
,
1.40–1.08 (m, 7H), 0.88 (t, J = 7.1 Hz, 3H); 13C-NMR (100 MHz, CDCl3)
δ; 174.2, 131.2, 129.0,
125.3, 125.1, 51.6, 51.4, 41.3, 37.2, 36.2, 35.5, 35.4, 33.4, 33.0, 31.9, 31.0, 26.9, 24.7, 23.0, 21.7, 14.1;
IR (neat, cm−1) 3020 (w), 2925 (s), 1745 (s); HRMS (EI+): Exact mass calculated for C21H34O2 [M]+:
318.2559, found 318.2552; TLC (hexane/EtOAc 95:5, KMnO4 stain): Rf = 0.65.
4. Conclusions
To recapitulate our findings, we have investigated the anti-diastereomer 2* of the proposed
structure 2. Through the execution of 13 discrete linear steps, the target molecule was obtained
in an overall yield of 11% and in multi-milligram quantities. By incorporating a Michael acceptor
motif, we have revealed an innate topological bias of the cis-fused bicyclo[4.3.0]non-3-ene system.
Combined with our previously developed three step one-pot alkyne iteration, we have shown
that exo-mucosin 1* is not identical to the natural product. In terms of the synthetic sequence
detailed by Whitby and co-workers [37], our findings must necessarily have some mechanistic
implications. Thus, we conclude that zirconium induced co-cyclisation did not deliver any of the
two anti-diastereomers,
1 and 1* respectively, by the published procedure. Achieving a synthesis that
establishes the true structure of mucosin will therefore also provide mechanistic insight into this matter.
Based on the present work and what has previously been published [36,37], the fusion geometry of the
hydrindane core embedded within mucosin is most likely trans.
Supplementary Materials: Electronic supplementary information is available online with full experimental
procedures and characterisation data for all new compounds; crystal data and refinement details are included to
give evidence of the relative stereochemistry of the late stage intermediate 12.
Acknowledgments: The authors are much indebted to Dag Ekeberg for the performance of mass spectrometric
analysis. Scholarships for S.G.A. and H.G.-S. from the Department of Chemistry, the Norwegian University of
Life Sciences, as well as funding from the Research Council of Norway for a research scholarship to J.M.J.N. and
grants to Y.H.S. (NFR 209335 and NFR 244351) are gratefully acknowledged.
Author Contributions: S.G.A. and H.G.-S. contributed equally to the practical work. C.H.G. performed the
crystallographic acquisition and analysis. T.V.H. and Y.H.S. supervised and oversaw the project. Y.H.S. prepared
the crystals for X-ray analysis. J.M.J.N. executed the diastereoselective addition and the alkyne iteration,
developed the key stereopermutation concept and wrote the manuscript. All authors read and approved the
final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2017, 22, 1720
14 of 18
References and Notes
1.
2.
3.
4.
Samuelsson, B. Role of basic science in the development of new medicines: Examples from the eicosanoid
field. J. Biol. Chem. 2012, 287, 10070–10080. [CrossRef] [PubMed]
Flower, R.J. Prostaglandins, bioassay and inflammation. Br. J. Pharmacol. 2006, 147, S182–S192. [CrossRef]
[PubMed]
Serhan, C.N. Pro-resolving lipid mediators are leads for resolution physiology. Nature 2014, 510, 92–101.
[CrossRef] [PubMed]
Basil, M.C.; Levy, B.D. Specialized pro-resolving mediators: Endogenous regulators of infection and
inflammation. Nat. Rev. Immunol. 2016, 16, 51–67. [CrossRef] [PubMed]
5.
6.
Goldblatt, M.W. A Depressor substance in seminal fluid. Chem. Ind. 1933, 52, 1056–1057.
Von Euler, U.S. Über die spezifische blutdrucksenkende substanz des menschlichen prostata- und
samenblasensekretes. Klin. Wochenschr. 1935, 14, 1182–1183. [CrossRef]
7.
8.
Bergström, S.; Sjövall, J. The isolation of prostaglandin. Acta Chem. Scand. 1957, 11, 1086. [CrossRef]
Bergström, S.; Ryhage, R.; Samuelsson, B.; Sjövall, J. Prostaglandins and related factors: 15. The structures of
prostaglandin E1, F1β and F1β. J. Biol. Chem. 1963, 238, 3555–3564.
9.
Hamberg, M.; Samuelsson, B. On the mechanism of the biosynthesis of prostaglandins E1 and F1β.
J. Biol. Chem. 1967, 242, 5336–5343. [PubMed]
10. Corey, E.J.; Weinshenker, N.M.; Schaaf, T.K.; Huber, W. Stereo-controlled synthesis of prostaglandins F2β
and E2 (dl). J. Am. Chem. Soc. 1969, 91, 5675–5677. [CrossRef] [PubMed]
11. Smith, W.L.; Lands, W.E.M. Stimulation and blockade of prostaglandin biosynthesis. J. Biol. Chem. 1971, 246,
6700–6702. [PubMed]
12. Corey, E.J.; Ensley, H.E. Preparation of an optically active prostaglandin intermediate via asymmetric
induction. J. Am. Chem. Soc. 1975, 97, 6908–6909. [CrossRef] [PubMed]
13. Dewitt, D.L.; El-Harith, E.A.; Kraemer, S.A.; Andrews, M.J.; Yao, E.F.; Armstrong, R.L.; Smith, W.L.
The aspirin and heme-binding sites of ovine and murine prostaglandin endoperoxide synthases. J. Biol. Chem.
1990, 265, 5192–5198. [PubMed]
14. Noverr, M.C.; Erb-Downward, J.R.; Huffnagle, G.B. Production of eicosanoids and other oxylipins by
pathogenic eukaryotic microbes. Clin. Microbiol. Rev. 2003, 16, 517–533. [CrossRef] [PubMed]
15. Ricciotti, E.; FitzGerald, G.A. Prostaglandins and inflammation. Arterioscler. Thromb. Vasc. Biol. 2011, 31,
986–1000. [CrossRef] [PubMed]
16. Serhan, C.N. The resolution of inflammation: The devil in the flask and in the details. FASEB J. 2011, 25,
1441–1448. [CrossRef] [PubMed]
17. Wagner, K.; Vito, S.; Inceoglu, B.; Hammock, B.D. The role of long chain fatty acids and their epoxide
metabolites in nociceptive signaling. Prostaglandins Other Lipid Mediat. 2014, 113–115, 2–12. [CrossRef]
[PubMed]
18. Aursnes, M.; Tungen, J.E.; Vik, A.; Colas, R.; Cheng, C.-Y.C.; Dalli, J.; Serhan, C.N.; Hansen, T.V. Total synthesis
of the lipid mediator PD1n-3 DPA: Configurational assignments and anti-inflammatory and pro-resolving
actions. J. Nat. Prod. 2014, 77, 910–916. [CrossRef] [PubMed]
19. Tungen, J.E.; Aursnes, M.; Vik, A.; Ramon, S.; Colas, R.A.; Dalli, J.; Serhan, C.N.; Hansen, T.V. Synthesis and
anti-inflammatory and pro-resolving activities of 22-OH-PD1, a monohydroxylated metabolite of protectin
D1. J. Nat. Prod. 2014, 77, 2241–2247. [CrossRef] [PubMed]
20. Serhan, C.N.; Chiang, N.; Dalli, J. The resolution code of acute inflammation: Novel pro-resolving lipid
mediators in resolution. Semin. Immunol. 2015, 27, 200–215. [CrossRef] [PubMed]
21. Primdahl, K.G.; Aursnes, M.; Walker, M.E.; Colas, R.A.; Serhan, C.N.; Dalli, J.; Hansen, T.V.; Vik, A.
Synthesis of 13(R)-hydroxy-7Z,10Z,13R,14E,16Z,19Z docosapentaenoic acid (13R-HDPA) and its biosynthetic
conversion to the 13-Series Resolvins. J. Nat. Prod. 2016, 79, 2693–2702. [CrossRef] [PubMed]
22. Ramon, S.; Dalli, J.; Sanger, J.M.; Winkler, J.W.; Aursnes, M.; Tungen, J.E.; Hansen, T.V.; Serhan, C.N.
The protectin PCTR1 is produced by human M2 macrophages and enhances resolution of infectious
inflammation. Am. J. Pathol. 2016, 186, 962–973. [CrossRef] [PubMed]
23. Serhan, C.N. Treating inflammation and infection in the 21st century: New hints from decoding resolution
mediators and mechanisms. FASEB J. 2017, 31, 1273–1288. [CrossRef] [PubMed]

Molecules 2017, 22, 1720
15 of 18
24. Dias, D.A.; Urban, S.; Roessner, U. A historical overview of natural products in drug discovery. Metabolites
2012, 2, 303–306. [CrossRef] [PubMed]
25. Gerwick, W.H.; Moore, B.S. Lessons from the past and charting the future of marine natural products drug
discovery and chemical biology. Chem. Biol. 2012, 19, 85–98. [CrossRef] [PubMed]
26. McGovern, P.E.; Michel, R.H. Royal purple dye: Tracing the chemical origins of the industry. Anal. Chem.
1985, 57, 1514A–1522A.
27. Cooksey, C.J. Tyrian purple: 6,60-Dibromoindigo and related compounds. Molecules 2001, 6, 736–769.
[CrossRef]
28. Leal, M.C.; Puga, J.; Serôdio, J.; Gomes, N.C.M.; Calado, R. Trends in the discovery of new marine natural
products from invertebrates over the last two decades—Where and what are we bioprospecting? PLoS ONE
2012, 7, e30580. [CrossRef] [PubMed]
29. Montaser, R.; Luesch, H. Marine natural products: A new wave of drugs? Future Med. Chem. 2011, 3,
1475–1489. [CrossRef] [PubMed]
30. Blunt, J.W.; Copp, B.R.; Keyzers, R.A.; Munro, M.H.G.; Prinsep, M.R. Marine natural products. Nat. Prod. Rep.
2016, 33, 382–431. [CrossRef] [PubMed]
31. Blunt, J.W.; Copp, B.R.; Keyzers, R.A.; Munro, M.H.G.; Prinsep, M.R. Marine natural products. Nat. Prod. Rep.
2015, 32, 116–211. [CrossRef] [PubMed]
32. Blunt, J.W.; Copp, B.R.; Keyzers, R.A.; Munro, M.H.G.; Prinsep, M.R. Marine natural products. Nat. Prod. Rep.
2014, 31, 160–258. [CrossRef] [PubMed]
33. Blunt, J.W.; Copp, B.R.; Munro, M.H.G.; Northcote, P.T.; Prinsep, M.R. Marine natural products.
Nat. Prod. Rep. 2005, 22, 15–61. [CrossRef] [PubMed]
34. Faulkner, D.J. Marine natural products. Nat. Prod. Rep. 2001, 18, 1–49. [CrossRef] [PubMed]
35. Casapullo, A.; Scognamiglio, G.; Cimino, G. Mucosin: A new bicyclic eicosanoid from the Mediterranean
sponge Reniera mucosa. Tetrahedron Lett. 1997, 38, 3643–3646. [CrossRef]
36. Gallantree-Smith, H.C.; Antonsen, S.G.; Görbitz, C.H.; Hansen, T.V.; Nolsøe, J.M.J.; Stenstrøm, Y.H.
Total synthesis based on the originally claimed structure of mucosin. Org. Biomol. Chem. 2016, 14, 8433–8437.
[CrossRef] [PubMed]
37. Henderson, A.R.; Stec, J.; Owen, D.R.; Whitby, R.J. The first total synthesis of (+)-mucosin. Chem. Commun.
2012, 48, 3409–3411. [CrossRef] [PubMed]
38. Bunn, B.J.; Simpkins, N.S.; Spavold, Z.; Crimmin, M.J. The effect of added salts on enantioselective
transformations of cyclic ketones by chiral lithium amide bases. J. Chem. Soc. Perkin Trans. 1 1993, 24,
3113–3116. [CrossRef]
39. Bunn, B.J.; Cox, P.J.; Simpkins, N.S. Enantioselective deprotonation of 8-oxabicyclo[3.2.1]octan-3-one systems
using homochiral lithium amide bases. Tetrahedron 1993, 49, 207–218. [CrossRef]
40. Rodeschini, V.; Simpkins, N.S.; Wilson, C. Kinetic resolution in a bridgehead lithiation mediated by a chiral
Bis-lithium amide: Assignment of the absolute configuration of clusianone. J. Org. Chem. 2007, 72, 4265–4267.
[CrossRef] [PubMed]
41. Normant, J.F. Organocopper(I) compounds and organocuprates in synthesis. Synthesis 1972, 2, 63–80.
[CrossRef]
42. Posner, G.H. Conjugate addition reactions of organocopper reagents. Org. Reac. 1972, 19, 1–113.
43. Lipshutz, B.H. Applications of higher-order mixed organocuprates to organic synthesis. Synthesis 1987, 4,
325–341. [CrossRef]
44. Dambrin, V.; Villiéras, M.; Lebreton, J.; Toupet, L.; Amri, H.; Villiéras, J. Copper (I) mediated highly
diastereoselective conjugate addition of Grignard reagents to functionalised 6-membered ring cycloalkenols.
Synthesis of cyclohexanes derivatives substituted on three contiguous atoms. Tetrahedron Lett. 1999, 40,
871–874. [CrossRef]
45. Dambrin, V.; Villiéras, M.; Janvier, P.; Toupet, L.; Amri, H.; Lebreton, J.; Villiéras, J. Copper(I) mediated
highly diastereoselective conjugate addition of Grignard reagents to functionalised cycloalkenols: A general
and efficient route for the stereoselective synthesis of 5- and 6-membered ring trisubstituted cycloalkanols.
Tetrahedron 2001, 57, 2155–2170. [CrossRef]
46. Harutyunyan, S.R.; López, F.; Browne, W.R.; Correa, A.; Peña, D.; Badorrey, R.; Meetsma, A.; Minnaard, A.J.;
Feringa, B.L. On the mechanism of the copper-catalyzed enantioselective 1,4-addition of Grignard reagents
to α,β-unsaturated carbonyl compounds. J. Am. Chem. Soc. 2006, 128, 9103–9118. [CrossRef] [PubMed]

Molecules 2017, 22, 1720
16 of 18
47. Ohira, S. Methanolysis of dimethyl (1-diazo-2-oxopropyl) phosphonate: Generation of dimethyl
(diazomethyl) phosphonate and reaction with carbonyl compounds. Synth. Commun. 1989, 19, 561–564.
[CrossRef]
48. Müller, S.; Liepold, B.; Roth, G.J.; Bestmann, H.J. An improved one-pot procedure for the synthesis of alkynes
from aldehydes. Synlett 1996, 6, 521–522. [CrossRef]
49. Habrant, D.; Rauhala, V.; Koskinen, A.M.P. Conversion of carbonyl compounds to alkynes: General overview
and recent developments. Chem. Soc. Rev. 2010, 39, 2007–2017. [CrossRef] [PubMed]
50. Taber, D.F.; Bai, S.; Guo, P.-F. A convenient reagent for aldehyde to alkyne homologation. Tetrahedron Lett.
2008, 49, 6904–6906. [CrossRef] [PubMed]
51. Wilke, G.; Müller, H. Dialkylaluminiumhydride als sterisch spezifische reduktionsmittel für acetylene.
Chem. Ber. 1956, 89, 444–447. [CrossRef]
52. Hart, D.W.; Blackburn, T.F.; Schwartz, J. Hydrozirconation. III. Stereospecific and regioselective
functionalization of alkylacetylenes via vinylzirconium(IV) intermediates. J. Am. Chem. Soc. 1975, 97,
679–680. [CrossRef]
53. Brown, H.C.; Gupta, S.K. Hydroboration. XXXIX. 1,3,2-Benzodioxaborole (catecholborane) as a new
hydroboration reagent for alkenes and alkynes. General synthesis of alkane- and alkeneboronic acids and
esters via hydroboration. Directive effects in the hydroboration of alkenes and alkynes with catecholborane.
J. Am. Chem. Soc. 1975, 97, 5249–5255.
54. Zhao, Y.; Snieckus, V. A Practical in situ generation of the Schwartz reagent. Reduction of Tertiary amides to
aldehydes and hydrozirconation. Org. Lett. 2014, 16, 390–393. [CrossRef] [PubMed]
55. Yang, Z.; Zhong, M.; Ma, X.; Nijesh, K.; De, S.; Parameswaran, P.; Roesky, H.W. An aluminum dihydride
working as a catalyst in hydroboration and dehydrocoupling. J. Am. Chem. Soc. 2016, 138, 2548–2551.
[CrossRef] [PubMed]
56. Palei, B.A.; Gavrilenko, V.V.; Zakharkin, L.I. Comparative reactivity of alkyl, vinyl, and acetylene derivatives
of aluminum. Russ. Chem. Bull. 1969, 18, 2590–2595. [CrossRef]
57. Diederich, F.; Stang, P. (Eds.) Metal-Catalyzed Cross Coupling Reactions; J. Wiley-VCH: Weinheim, Germany, 1998.
58. Huo, S. Highly efficient, general procedure for the preparation of alkylzinc reagents from unactivated alkyl
bromides and chlorides. Org. Lett. 2003, 5, 423–425. [CrossRef] [PubMed]
59. Pohnert, G.; Boland, W. The oxylipin chemistry of attraction and defense in brown algae and diatoms.
Nat. Prod. Rep. 2002, 19, 108–122. [PubMed]
60. Andreou, A.; Brodhun, F.; Feussner, I. Biosynthesis of oxylipins in non-mammals. Prog. Lipid Res. 2009, 48,
148–170. [CrossRef] [PubMed]
61. Barbosa, M.; Valentao, P.; Andrade, P.A. Biologically active oxylipins from enzymatic and nonenzymatic
routes in macroalgae. Mar. Drugs 2016, 14, 23. [CrossRef] [PubMed]
62. Hoffman, R.; Woodward, R.B. The conservation of orbital symmetry. Acc. Chem. Res. 1968, 1, 17–22.
[CrossRef]
63. Klas, K.; Tsukamoto, S.; Sherman, D.H.; Williams, R.M. Natural Diels–Alderases: Elusive and irresistible.
J. Org. Chem. 2015, 80, 11672–11685. [CrossRef] [PubMed]
64. Jiang, Z.D.; Ketchum, S.O.; Gerwick, W.H. 5-Lipoxygenase-derived oxylipins from the red alga
Rhodymenia pertusa. Phytochemistry 2000, 53, 129–133. [CrossRef]
65. Bernart, M.; Gerwick, W.H. Isolation of 12-(S)-hepe from the red marine alga Murrayella periclados and
revision of structure of an acyclic icosanoid from Laurencia hybrida. Implications to the biosynthesis of the
marine prostanoid hybridalactone. Tetrahedron Lett. 1988, 29, 2015–2018. [CrossRef]
66. Jiang, Z.D.; Gerwick, W.H. Novel oxylipins from the temperate red alga Polyneura latissima: Evidence for an
arachidonate 9(S)-lipoxygenase. Lipids 1997, 32, 231–235. [CrossRef] [PubMed]
67. Lopez, A.; Gerwick, W.H. Two new icosapentaenoic acids from the temperate red seaweed Ptilota filicina J.
Agardh. Lipids 1987, 22, 190–194. [CrossRef] [PubMed]
68. Solem, M.L.; Jiang, Z.D.; Gerwick, W.H. Three new and bioactive icosanoids from the temperate red marine
alga Farlowia mollis. Lipids 1989, 24, 256–260. [CrossRef] [PubMed]
69. Hamberg, M.; Gerwick, W.H. Biosynthesis of vicinal dihydroxy fatty acids in the red alga
Gracilariopsis lemaneiformis: Identification of a sodium-dependent 12-lipoxygenase and a hydroperoxide
isomerase. Arch. Biochem. Biophys. 1993, 305, 115–122. [CrossRef] [PubMed]

Molecules 2017, 22, 1720
17 of 18
70. Gerwick, W.H.; Åsen, P.A.; Hamberg, M. Biosynthesis of 13R-hydroxyarachidonic acid, an unusual oxylipin
from the red alga lithothamnion corallioides. Phytochemistry 1993, 34, 1029–1033. [CrossRef]
71. Burgess, J.R.; De la Rosa, R.I.; Jacobs, R.S.; Butler, A. A new eicosapentaenoic acid formed from arachidonic
acid in the coralline red algae Bossiella orbigniana. Lipids 1991, 26, 162–165. [CrossRef]
72. Murakami, N.; Shirahashi, H.; Nagatsu, A.; Sakakibara, J. Two unsaturated 9R-hydroxy fatty acids from the
cyanobacterium Anabaena flos-aquae f. flos-aquae. Lipids 1992, 27, 776–778. [CrossRef]
73. Moghaddam, M.F.; Gerwick, W.H.; Ballantine, D.L. Discovery of 12-(S)-hydroxy-5,8,10,14-icosatetraenoic
acid [12-(S)-HETE] in the tropical red alga Platysiphonia miniata. Prostaglandines 1989, 37, 303–308. [CrossRef]
74. Weinberger, F.; Lion, U.; Delage, L.; Kloareg, B.; Potin, P.; Beltran, J.; Flores, V.; Faugeron, S.; Correa, J.;
Pohnert, G. Up-regulation of lipoxygenase, phospholipase, and oxylipin-production in the induced chemical
defense of the red alga Gracilaria chilensis against epiphytes. J. Chem. Ecol. 2011, 37, 677–686. [CrossRef]
[PubMed]
75. Kuo, J.-M.; Hwang, A.; Yeh, D. Purification, substrate specificity, and products of a Ca2+-stimulating
lipoxygenase from sea algae (Ulva lactuca). J. Agric. Food Chem. 1997, 45, 2055–2060. [CrossRef]
76. Mohamed, Y.M.A.; Hansen, T.V. Synthesis of methyl (5Z,8Z,10E,12E,14Z)-eicosapentaenoate. Tetrahedron Lett.
2011, 52, 1057–1059. [CrossRef]
77. Wise, M.L.; Hamberg, M.; Gerwick, W. Biosynthesis of conjugated triene-containing fatty acids by a novel
isomerase from the red marine alga Ptilota filicina. Biochemistry 1994, 33, 15223–15232. [CrossRef] [PubMed]
78. Kennedy, J.; Auclair, K.; Kendrew, S.G.; Park, C.; Vederas, J.C.; Hutchinson, C.R. Modulation of polyketide
synthase activity by accessory proteins during lovastatin biosynthesis. Science 1999, 284, 1368–1372.
[CrossRef] [PubMed]
79. Auclair, K.; Sutherland, A.; Kennedy, J.; Witter, D.J.; Van den Heever, J.P.; Hutchinson, C.R.; Vederas, J.C.
Lovastatin nonaketide synthase catalyzes an intramolecular Diels Alder reaction of a substrate analogue.
−
J. Am. Chem. Soc. 2000, 122, 11519–11520. [CrossRef]
80. Alt, S.; Wilkinson, B. Biosynthesis of the novel macrolide antibiotic anthracimycin. Chem. Biol. 2015, 10,
2468–2479. [CrossRef] [PubMed]
81. Oikawa, K.; Katayama, K.; Suzuki, Y.; Ichihara, A. Enzymatic activity catalysing exo-selective Diels–Alder
reaction in solanapyrone biosynthesis. J. Chem. Soc. Chem. Commun. 1995, 13, 1321–1322. [CrossRef]
82. Kasahara, K.; Miyamaoto, T.; Fujimoto, T.; Oguri, H.; Tokiwano, T.; Oikawa, H.; Ebizuka, Y.; Fujii, I.
Solanapyrone synthase, a possible Diels-Alderase and iterative Type I polyketide synthase encoded in a
biosynthetic gene cluster from Alternaria solani. ChemBioChem 2010, 11, 1245–1252. [CrossRef] [PubMed]
83. Kim, H.J.; Ruszczycky, M.W.; Choi, S.-H.; Liu, Y.-N.; Liu, H.-W. Enzyme-catalysed [4+2] cycloaddition is a
key step in the biosynthesis of spinosyn A. Nature 2011, 473, 109–112. [CrossRef] [PubMed]
84. Oikawa, H.; Yagi, K.; Watanabe, K.; Honma, M.; Ichihara, A. Biosynthesis of macrophomic acid:
Plausible involvement of intermolecular Diels–Alder reaction. Chem. Commun. 1997, 1, 97–98. [CrossRef]
85. Ose, T.; Watanabe, K.; Mie, T.; Honma, M.; Watanabe, H.; Yao, M.; Oikawa, H.; Tanaka, I. Insight into
a natural Diels–Alder reaction from the structure of macrophomate synthase. Nature 2003, 422, 185–189.
[CrossRef] [PubMed]
86. Gerwick, W.H. Carbocyclic oxylipins of marine origin. Chem. Rev. 1993, 93, 1807–1823. [CrossRef]
87. Gerwick, W.H. Epoxy allylic carbocations as conceptual intermediates in the biogenesis of diverse marine
oxylipins. Lipids 1996, 31, 1215–1231. [CrossRef] [PubMed]
88. Gerwick, W.H.; Singh, I.P. Structural diversity of marine oxylipins. In Lipid Biotechnology; Kuo, T.M.,
Gardner, H.W., Eds.; Marcel Dekker: New York, NY, USA, 2002; pp. 249–275.
89. Li, L.; Yu, P.; Tang, M.-C.; Zou, Y.; Gao, S.-S.; Hung, Y.-S.; Zhao, M.; Watanabe, K.; Houk, K.N.; Tang, Y.
The catalytic mechanism of a natural Diels–Alderase revealed in molecular detail. J. Am. Chem. Soc. 2016
138, 15837–15840. [CrossRef] [PubMed]
,
90. Kanoh, N.; Itoh, S.; Fujita, K.; Sakanishi, K.; Sugiyama, R.; Terajima, Y.; Iwabuchi, Y.; Nishimura, S.; Kakeya, H.
Asymmetric total synthesis of heronamides A–C: Stereochemical Confirmation and impact of long-range
stereochemical communication on the biological activity. Chem. Eur. J. 2016, 22, 8586–8595. [CrossRef]
[PubMed]
91. Booth, T.J.; Alt, S.; Capon, R.J.; Wilkinson, B. Synchronous intramolecular cycloadditions of the polyene
macrolactam polyketide heronamide C. Chem. Commun. 2016, 52, 6383–6386. [CrossRef] [PubMed]

Molecules 2017, 22, 1720
18 of 18
92. Yu, P.; Patel, A.; Houk, K.N. Transannular [6+4] and ambimodal cycloaddition in the biosynthesis of
heronamide A. J. Am. Chem. Soc. 2015, 137, 13518–13523. [CrossRef] [PubMed]
93. Lin, C.-I.; McCarty, R.M.; Liu, H.-W. The enzymology of organic transformations: A survey of name reactions
in biological systems. Angew. Chem. Int. Ed. 2017, 56, 3446–3489. [CrossRef] [PubMed]
94. Jeon, B.-s.; Ruszczycky, M.W.; Russell, W.K.; Lin, G.-M.; Kim, N.; Choi, S.-h.; Wang, S.-A.; Liu, Y.-N.;
Patrick, J.W.; Russell, D.H.; et al. Investigation of the mechanism of the SpnF-catalyzed [4+2]-cycloaddition
reaction in the biosynthesis of spinosyn A. Proc. Natl. Acad. Natl. USA 2017, 114, 10408–10413. [CrossRef]
[PubMed]
95. Cavelier, F.; Gomez, S.; Jacquier, R.; Verducci, J. Deracemization of silyl enol ethers. Tetrahedron Asymmetry
1993, 4, 2501–2505. [CrossRef]
96. Cavelier, F.; Gomez, S.; Jacquier, R.; Verducci, J. A first approach to asymmetric protonation via a polymer
supported chiral proton donor. Tetrahedron Lett. 1994, 35, 2891–2894. [CrossRef]
97. Diba, A.K.; Noll, C.; Richter, M.; Gieseler, M.T.; Kalesse, M. Intramolecular stereoselective protonation of
aldehyde-derived enolates. Angew. Chem. 2010, 122, 8545–8547. [CrossRef]
98. Mohr, J.T.; Hong, A.Y.; Stoltz, B.M. Enantioselective protonation. Nat. Chem. 2009, 1, 359–369. [CrossRef]
[PubMed]
99. Bruker. Bruker APEX3, SAINT+ and SADABS; Bruker AXS Inc.: Madison, WI, USA, 2016.
100. Sheldrick, G. SHELXT—Integrated space-group and crystal-structure determination. Acta Crystallogr. 2015
A71, 3–8. [CrossRef] [PubMed]
,
101. Sheldrick, G. Crystal structure refinement with SHELXL. Acta Crystallogr. 2015, C71, 3–8.
102. Macrae, C.F.; Bruno, I.J.; Chisholm, J.A.; Edgington, P.R.; McCabe, P.; Pidcock, E.; Rodriguez-Monge, L.;
Taylor, R.; van de Streek, J.; Wood, P.A. Mercury CSD 2.0—New features for the visualization and
investigation of crystal structures. J. Appl. Crystallogr. 2008, 41, 466–470. [CrossRef]
103. Nagao, Y.; Hagiwara, Y.; Tohjo, T.; Hasegawa, Y.; Ochiai, M.; Shiro, M. Highly enantioselective Claisen-type
acylation and Dieckmann annulation. J. Org. Chem. 1988, 53, 5983–5986. [CrossRef]
104. Preparation of the racemic reference compound rac-12 was performed according to the general procedures
outlined in Scheme 2, replacing (+)-Simpkin’s base with LDA to obtain rac-9 for the synthetic sequence.
105. The publCIF document accompanying compound 12-DNB is uploaded as separate file and the structure is
deposited at Cambridge Crystallographic Data Centre and identified as CCDC 1535632.
Sample Availability: Samples of exo-mucosin (1*) and methyl ester 2* are available from the authors.
©
2017 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/).