Total synthesis of biselide A

Article abstract of DOI:10.1039/d0sc06223e

A total synthesis of the marine macrolide biselide A is described that relies on an enantiomerically enriched α-chloroaldehyde as the sole chiral building block. Several strategies to construct the macrocycle are presented including a macrocyclic Reformatsky reaction that ultimately provides access to the natural product in a longest linear sequence of 18 steps. Biological testing of synthetic biselide A suggests this macrolide disrupts cell division through a mechanism related to the regulation of microtubule cytoskeleton organization. Overall, this concise synthesis and insight gained into the mechanism of action should inspire medicinal chemistry efforts directed at structurally related anticancer marine macrolides.

Full text of DOI:10.1039/d0sc06223e

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Total synthesis of biselide A†
Venugopal Rao Challa,‡ Daniel Kwon,‡ Matthew Taron, Hope Fan, Baldip Kang,
Darryl Wilson, F. P. Jake Haeckl, Sandra Keerthisinghe, Roger G. Linington
Cite this: Chem. Sci., 2021, 12, 5534
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
*
and Robert Britton
A total synthesis of the marine macrolide biselide A is described that relies on an enantiomerically enriched
a-chloroaldehyde as the sole chiral building block. Several strategies to construct the macrocycle are
presented including a macrocyclic Reformatsky reaction that ultimately provides access to the natural
product in a longest linear sequence of 18 steps. Biological testing of synthetic biselide A suggests this
Received 12th November 2020
Accepted 15th February 2021
macrolide disrupts cell division through
a mechanism related to the regulation of microtubule
cytoskeleton organization. Overall, this concise synthesis and insight gained into the mechanism of
action should inspire medicinal chemistry efforts directed at structurally related anticancer marine
macrolides.
DOI: 10.1039/d0sc06223e
rsc.li/chemical-science
(MIC ¼ 0.03 mg mLꢀ1).4 Biselide A (7) was found to be ꢁ5 to
Introduction
10 fold less active than haterumalide NA against a panel of
10 human cancer cell lines. However, unlike the hater-
umalides, biselide A showed no toxicity at concentrations as
high as 50 mg mLꢀ1 in a brine shrimp assay. This later result
led to speculation that C20 oxidation in the biselides makes
these compounds generally less toxic and thus better drug
leads.9 Several simplied synthetic analogues of hater-
umalide NA have also been reported, and these studies
found that both the macrolide and side chain are critical for
biological activity.12
The haterumalides and biselides are structurally related
macrocyclic polyketides isolated from several different
sources.1 In 1999, the rst member of the haterumalide
family, haterumalide B (1), was reported from extracts of an
Okinawan ascidian Lissoclinum sp. by Ueda, and its structure
and partial stereochemistry were assigned using NMR
spectroscopic methods.2 At the same time, Uemura reported
the haterumalides NA (2), NB (3), NC (5) and ND (6) from
extracts of the Okinawan sponge Ircinia sp. and determined
the absolute stereochemistry of 2 using a modied Mosher's
method.3 Soon thereaer, Strobel reported the isolation of
haterumalide NA (2) from a strain of Serratia marcescens4 and
additional haterumalides have since been reported from the
soil bacteria Serratia plymuthica5,6 and Serratia liquefaciens.7
8
In 2004 and 2005,9 Kigoshi reported the closely related
natural products biselide A (7) and B (8), respectively, which
primarily differ from the haterumalides by oxygenation at
C20. Notably, the polyketide synthase gene cluster that
encodes for the biosynthesis of the haterumalides was
identied by Salmond,10 and Piel has disclosed an unusual
oxygen insertion reaction critical to production of the
terminal carboxylic acid function.11
Broad interest in both the haterumalides and biselides
has been stimulated by their potentially useful biological
activity.4 For example, haterumalide NA (2) is cytotoxic to
P388 cells (IC50 ¼ 0.32 mg mLꢀ1)3 and a potent antimycotic
Fig. 1 Representative examples of haterumalide (1–3, 5 and 6) and
biselide (6 and 7) natural products.
Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, V5A
1S6, Canada. E-mail: rbritton@sfu.ca
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0sc06223e
‡ Contributed equally.
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Structurally, the haterumalides and biselides possess a 14- macrolactonization as a key step (29 steps in LLS). Both of these
membered macrolide that incorporates a trans-substituted syntheses relied on an aluminum hydride reduction of a prop-
tetrahydrofuran (THF) ring in addition to an unusual (Z,Z)-1,4- argylic alcohol to construct a Z-congured C7–C8 olen and
chlorodiene fragment (C4–C8). In 2003, Kigoshi reported the a Nozaki–Hiyama–Kishi (NHK)14–16 reaction to append the side
rst total synthesis of ent-haterumalide NA methyl ester (4) and chain to the fully functionalized macrolide core.
reassignment of the stereochemical relationship between the
The rst total synthesis of the natural product haterumalide
C14 and C15 centers as erythro (Fig. 2).13 Since the absolute NA (2) was reported by Hoye in 2005 (20 steps in LLS),17 who
stereochemical assignment of 2 was based on modied demonstrated the 14-membered macrolide could be formed
Mosher's ester analysis at the C15 alcohol,3 this synthetic work through a Pd-catalyzed chloroallylation that also introduced the
also resulted in a reassignment of the absolute stereochemistry Z-vinyl chloride function. In 2008 Roulland disclosed
of the haterumalides and biselides. Key to the success of this a synthesis of haterumalide NA via a process involving a Suzuki–
rst total synthesis (26 steps in longest linear sequence (LLS)) Miyaura cross-coupling of a 1,1-dichloroalkene to form the C8–
was the development of an intramolecular Reformatsky reac- C9 bond, followed by macrolactonization (19 steps in LLS).18
tion to construct both the macrocycle and the C2–C3 bond.13 In Kigoshi also reported a second generation synthesis of hater-
the same year, Snider reported the second total synthesis of ent- umalide NA in 2008 that involved a similar coupling strategy to
haterumalide NA methyl ester that involved a Yamaguchi construct the C8–C9 bond but exploited a macrolactonization
reaction (33 steps in LLS).12,19 In the same year Borhan reported
the rst synthesis of haterumalide NC (5) (18 steps in LLS) using
a chlorovinylidene chromium carbenoid to construct the C8–C9
bond.20 More recently, the rst synthesis of biselide A (7) was
reported by Hayakawa and Kigoshi in 2017 (34 steps in LLS).21
Here, again, a Suzuki–Miyaura coupling was used to construct
the C8–C9 bond and a macrolactonization reaction was
employed. Notably, the C3 stereogenic center was introduced
using an auxiliary controlled asymmetric aldol reaction.
Based on the intriguing structure and biological activity of
biselide A (7), and our longstanding interest in the synthesis of
THF-containing marine natural products,22–31 we became intent
on developing a total synthesis of 7 that would also support
additional biological testing. We have previously reported22
straightforward synthetic routes to hydroxytetrahydrofurans
that exploit diastereoselective aldol reactions between lithium
enolates and enantiomerically enriched a-chloroaldehydes32
(Fig. 2, 9). These later materials can be prepared in excellent
enantiomeric excess via the organocatalytic a-chlorination of
aldehydes using processes developed by Jørgensen,33 MacMil-
lan34,35 or Christmann.36,37 As depicted in Fig. 2, we planned to
exploit this strategy using the chloroketone 17 38 to rapidly
access the THF 13. From here, a sequence involving a metath-
esis reaction,17 esterication,21 or Reformatsky13 reaction would
expectedly provide the 14-membered ring. Our efforts to explore
each of these individual reactions as macrocyclization strategies
and the ultimate realization of a total synthesis of biselide A (7)
and biological testing of our synthetic material is described
below.
Results and discussion
Enantioselective synthesis of a-chloroaldehyde 24
We have previously reported the aldol reaction of 17 and (ꢂ)-18
(Fig. 2, PG ¼ TBS) provides a route the tetrahydrofuran 13 in
racemic form.38 To support an enantioselective synthesis of
biselide A (7), we examined the enantioselective a-chlorination
of protected b-hydroxyaldehydes 19 and 20. Unfortunately, in
both cases a-chlorination resulted predominantly in elimina-
Fig. 2 Previous syntheses of haterumalide and biselide natural prod-
tion, affording acrolein as the major product (Table 1, entries 1
and 2). Using D-proline catalysis, the TBS-protected alcohol 21
ucts and a chlorohydrin-based strategy for the synthesis of biselide A
(7). LLS ¼ longest linear sequence.
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Table 1 Enantioselective a-chlorination of aldehyde 21
provided the chloroester in >97% ee. Protection of the alcohol
as a TBS ether and reduction then gave the chlorohydrin 30,
which could be routinely prepared on >10 g scale via this 4-step
process. Oxidation of 30 using PCC gave the a-chloroaldehyde
24 in excellent yield (95%), though again we noted an erosion in
enantiomeric purity. As such, we examined other oxidation
protocols and found that using Dess–Martin periodinane40 with
the addition of solid NaHCO3, the a-chloroaldehyde 24 could be
prepared on up to 15 g scale in good yield and enantiomeric
purity (>92% ee). While this sequence is longer (5 steps vs. 3
steps) than that involving a direct enantioselective a-chlorina-
tion (Table 1), it provided a reliable alternative when large
amounts of a-chloroaldehyde 24 were required.
Entry
Aldehyde
Methoda
Time (h)
Yield (%)
ee (%)
1
2
3
4
5
6
19
20
21
21
21
21
A
A
A
B
C
Cc
4
4
4
18
16
16
<10b
<10b
90
na
na
15
80
94
90
30d
35d
40d
Ring closing metathesis approach to biselide A
Prior studies on the haterumalides by Hoye and co-workers17
involved use of a relay ring closing metathesis (RRCM)41
approach to form both the C4–C5 alkene and the 14-membered
macrocycle. From these studies it was found that substrates
with an intact THF ring failed to undergo RRCM, likely due to
strain in the resulting bridged bicycle. However, in “relaxed”
model systems that lacked the THF ring, RRCM reactions were
successful.41 Inspired by this work, we rst explored a strategy in
which the THF would be assembled aer macrocyclization, and
the macrocycle itself would be produced through a process
involving a “relaxed” RRCM reaction (e.g., 31 / 32, Scheme 2).
The synthesis of the RRCM precursor followed a straightforward
sequence of reactions that initiated with the known ester 33,42
prepared in 97% ee via kinetic resolution using a Sharpless
asymmetric epoxidation reaction.43 Protection of the alcohol as
a TBS ether, oxidative cleavage of the alkene function and
subsequent Wittig reaction and hydrolysis gave the Z-alkene 36
in good overall yield. While this material lacks oxygenation at
the position corresponding to C20 in biselide A, it was viewed as
a good model substrate for exploring the RRCM reaction and
would yield access to the haterumalide family of marine mac-
rolides (e.g., 2, Fig. 1). Synthesis of the required chloroalkene 38
a
A: 25 (10 mol%), NCS (1 equiv.), CH2Cl2, rt; B: 26 (10 mol%), NCS (1
equiv.), CH2Cl2; C: 27 (15 mol%), LiCl (1.5 equiv.), Cu(TFA)2 (0.5
b
equiv.), NasS2O8c(1 equiv.), H2O (2.1 equiv.), rt. The major product
d
was acrolein.
Reaction at 10 ꢃC.
Product accompanied by
formation of acrolein.
was cleanly converted into the a-chloroaldehyde 24 in good
yield albeit expectedly33 low ee. We next examined the use of D-
prolinamide and found that the a-chloroaldehyde 24 could be
prepared in much improved enantioselectivity (entry 4), but was
again accompanied by predominant formation of acrolein.
Finally, we explored the a-chlorination process reported by
MacMillan35 (entries 5 and 6). Here, the a-chloroaldehyde 24
was produced in up to 94% ee and modest yield (40%). Efforts
focused on further optimizing these conditions by reducing the
reaction temperature (e.g., entry 6), adding pH 8 buffer, proton
scavengers (e.g., 2,6-di-tert-butyl-4-methylpyridine) or varying
the amount of water, failed to improve the result summarized in
entry 5.
Considering these challenges, we investigated the conver-
sion of L-serine into 24 using a process reported by De Kimpe for
the amino acids Ile, Phe and Val (Scheme 1).39 For our purpose,
L-Ser (28) was rst converted into the chloroester 29 via double
Waldon inversion39 followed by esterication. It was critical that
this chlorination reaction was executed at temperatures below
exploited
a palladium catalysed chloroallylation reaction
developed by Kaneda.44,45 Alkylation of ethylacetoacetate fol-
lowed by decarboxylation then gave the methyl ketone 39 as
a 1 : 1 mixture of E- and Z-isomers as indicated.
ꢃ
ꢃ
ꢀ15 C to avoid racemization. For example, at 0 C the chlor-
With the required fragments in hand, a lithium aldol reac-
tion between the enolate derived from methyl ketone 39 and the
a-chloroaldehyde 24 gave the ketochlorohydrin 40 22,31,38 in
modest yield (53%, d.r. ¼ 4 : 1). Esterication of the chlorohy-
drin using the carboxylic acid 36 then gave the ketoester 41. A
subsequent RRCM reaction using conditions reported by Hoye41
gave truncation products (ꢁ40% yield) along with the E,Z-
macrocycle 42 (45% yield). The E-conguration of the C4–C5
alkene function in 42 was assigned by nOe analysis. In an effort
to access the desired Z,Z-isomer, several reaction parameters
were evaluated including temperature (40 ꢃC to 110 ꢃC), solvent
(toluene, CH2Cl2, hexanes), catalyst (Grubbs II,46 Hoveyda–
Grubbs II47), concentration, addition rate and use of various
additives known to prevent isomerization48 (e.g., benzoqui-
none). In no instance was the desired Z,Z-macrocycle formed.
oester was produced in 60% ee, while reaction at ꢀ20 ꢃC reliably
Scheme 1 Multigram synthesis of a-chloroaldehyde 24.
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Macrolactonization approach to biselide A
Based on these results, we revised our approach and instead
pursued macrolactonization strategy to form the 14-
a
membered ring (Scheme 3, 45 / 46).49 Toward this goal, the
methyl ketone 48 was prepared using Kaneda's procedure44 in
three steps from propargyl bromide (37). A subsequent lithium
aldol reaction22,38 with the enantiomerically enriched a-chlor-
oaldehyde 24 provided the ketochlorohydrin 49 in excellent
yield as a mixture of diastereomers. DIBAl reduction of the
carbonyl function in 49 then gave the chlorodiol 50 as a 4 : 1
mixture of diastereomers that were not separated. Heating this
mixture in methanol (120 ꢃC)23 in a sealed vessel in a microwave
effected the removal of the TBS protecting group followed by
cyclization to afford the THF 51, which was isolated as a single
diastereomer in excellent yield. Attempts to avoid the removal of
the TBS protecting group by addition of buffers (e.g., pH 7
phosphate) or base (e.g., 2,6-di-tertbutyl-4-methylpyridine) were
not successful. Notwithstanding, the diol could be readily pro-
tected as the corresponding bis-TBS ether 52 or acetonide 53 in
good yield.
At this point, we examined diastereoselective cross metath-
esis reactions to incorporate both the C3–C4 fragment of bise-
lide A along with the C20 hydroxymethyl group using various
Scheme 2 A relay ring closing metathesis strategy for biselide A.
Considering Hoye had reported that functional groups adjacent
to the site of RRCM (e.g., alkene, alcohol, ketone) play a signif-
icant role in determining the ultimate conguration of the
alkene,17 we also prepared the diastereomeric ester 43 to assess
the impact of C3 conguration on the RRCM reaction. Thus,
ester ent-33 was synthesized via a Sharpless asymmetric epoxi-
dation using (ꢀ)-DIPT and progressed to the C3 epimer 43 in
the same manner outlined for 41. Unfortunately, RRCM reac-
tion of 43 again gave the E,Z-isomer 44 as the only macro-
cyclization product.
Scheme 3 Synthesis of the tetrahydrofurans 52 and 53.
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Table 2 Cross metathesis reactions of 2-methylene-1,3-propanediol
and derivatives
could be selectively unmasked in a mixture of THF–H2O–HOAc
(2 : 1 : 0.1), affording seco-acid 63.
With the seco-acids 62 and 63 in hand, macrolactonization
attempts using basic (e.g., Yamaguchi51 and Boden–Keck52),
acidic (e.g., Trost53 and Yamamoto54) and near neutral (e.g.,
modied Mukaiyama55 and Corey–Nicolaou56) conditions were
explored. Unfortunately, none of these reactions gave the
desired macrocycles 64 or 65, and instead resulted primarily in
decomposition. Hayakawa and Kigoshi have reported that21
macrolactonization of a less labile seco-acid, in which the
alcohol functions at C3/C20 are protected as silyl ethers and not
acetates, was successful using Yamamoto conditions. Consid-
ering as well that the choice of protecting group at C15 also
plays a key role in related macrolactonizations,13 advancing
seco-acids 62 or 63 to the desired macrocycles 64 or 65 would
require several tedious protecting group manipulations
including replacement of the acetate functions at C3 and C20
that are required for biselide A with orthogonal protecting
groups to that at C15, and thus add signicantly to the overall
length of the process.
Entrya
Alkene
R1
R2
THF
Product
Yield (%)
1
2
3
54a
54b
54c
54d
54d
54e
54e
54e
TES
TBS
H
TBS
H
H
H
TBS
TBS
53
53
53
53
53
53
53
52
55ab
55bc
55c
55d
55d
55e
55e
56e
30
33
17
4
16
5d
6
TBS
<10e
60
C(CH3)2
C(CH3)2
C(CH3)2
7f
8
77
46
a
Conditions: 52 or 53 (1 equiv.) was dissolved in toluene (0.05 M), 54a–e
was added (5 equiv.), reaction mixture was sparged with N2, heated to
60 ꢃC and Hoveyda–Grubbs catalyst (2nd generation, 5 mol%) was
Curiously, during these studies we noticed that protons
assigned to the C1–C13 region of the molecule were distinct for
b
added. The resulting mixture was heated at reux for 2 h. E : Z ratio
c
d
¼ 1 : 1. E : Z ratio ¼ 1 : 1.5. Stewart–Grubbs catalyst was used
e
f
(5 mol%). The major product was the dimer of 53 (ꢁ50% yield). 10
equiv. of 54e was added.
derivatives of 2-methylene-1,3-propanediol. As summarized in
Table 2, cross metathesis of 52 with mono-TES or mono-TBS
protected diols 54a and 54b gave little E/Z selectivity. In fact,
the metathesis reaction with 54b favoured the undesired Z-
isomer (entries 1 and 2). We also investigated cross metathesis
reactions involving diol 54c (entry 3) or the protected diols 54d
and 54e (entries 4 to 6). From these studies the acetonide 54e
proved to be the best metathesis partner and gave the diene 56e
in 77% yield (entry 7). A similar cross metathesis reaction using
the bis-TBS ether 52 gave the acetonide 56e (entry 8).
While regioselective deprotection of the bis-acetonide 55e
(Table 2, entry 8) was unsuccessful, the acetonide protecting
group in bis-silyl ether 56e was readily removed to afford the
diol 57 (Scheme 4). From here, following a procedure reported
by Imai,50 treatment of 57 with vinyl acetate and porcine
pancreatic lipase (PPL) in 1,4-dioxane gave the mono-acetate 58
in 56% yield. The E-conguration of the alkene function in 58
was conrmed by NOE analysis. Oxidation of the allylic alcohol
using Dess–Martin periodinane (DMP)40 afforded the unstable
aldehyde 59 that was prone to isomerization. As such, this
material was reacted directly with the lithium enolate derived
from ethyl acetate to afford a near equal mixture of hydroxy
esters (not shown). Unfortunately, we were unable to hydrolyze
the ethyl ester function without signicant degradation and, as
such, a small collection of related esters were prepared
including OtBu, OPMB, OPh and the StBu thioester. Of these,
only the p-methoxybenzyl ester17 60 was hydrolyzed without
effecting signicant degradation. Thus, acylation of the C3–OH
function, removal of the TBS protecting groups and ester
hydrolysis gave the seco-acid 62. Following reprotection of both
alcohol functions in 62 as TBS ethers, the secondary alcohol
Scheme 4 Synthesis of the seco acids 62 and 63.
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the C3 epimers in both 62 and 63 when spectra were recorded in
CDCl3 but not in CD3OD. Considering that 8 bonds separate the
C3 stereocenter from the THF core, the distinct resonances for
each diastereomer suggested that these compounds adopt
conformations dominated by hydrogen bonding between the
carboxylic acid and the THF function. In this way, the relative
congurations of C3 impacts the overall conformation and
chemical shi values for protons throughout the molecule in
CDCl3. In CD3OD, where hydrogen bonding would be disrupted,
C3 epimers had nearly identical 1H NMR spectra. Further, when
the seco-acid 62 was converted into the corresponding methyl
ester (TMSCHN2) the C3 epimers had identical 1H NMR spectra
in CDCl3. To probe the role of hydrogen bonding in these seco-
acids a molecular dynamics conformational search of both C3
epimers of 62 was carried out using the Tinker molecular
modelling package.57 Conformers within 4 kcal molꢀ1 of their
respective minima were then further rened by geometry opti-
mization in Gaussian 09 58 at the PCM59 (solvent: CHCl3)–PM6 60
level of theory (see ESI† for details). Using the PM6 Gibbs free
energies we then visually examined conformations within
2 kcal molꢀ1 of their respective minima to interrogate the
different modes of intramolecular hydrogen bonding within the
seco-acid. For both C3 epimers, the lowest energy conforma-
tions included ones where hydrogen bonding between the
carboxylic acid and the C2-, C3-hydroxy functions and/or tetra-
hydrofuran oxygen constrain the molecule in a pseudo-
macrocyclic conformation (e.g., Scheme 4, inset). Unfortu-
nately, attempts to exploit the biased pseudo-macrocyclic
conformation of the seco-acid through hydrogen bond-
templated macrolactonization promoted by various acid cata-
lysts in aprotic solvents (e.g., CHCl3) also failed to provide the
desired macrocycle.
Scheme 5 Synthesis of the bromoacetate 77.
Macrocyclic Reformatsky approach to biselide A
above for the related aldehyde 59 (Scheme 4). With freshly
prepared aldehyde 77 in hand, the key ring closure reaction was
investigated using rhodium catalysis as reported by Honda.61
Disappointingly, using these standard conditions as well as
variants where concentration, temperature and catalyst loading
were examined gave only mixtures of degradation products.
Considering the noted inuence of the C15 protecting group on
productivity of macrocyclizations within this family of mole-
cules,13,62 and the failure of a related C15 TBDPS–ether to
undergo ring closure via macrolactonization,13 we reluctantly
returned to the TBS ether 68 and exhaustively examined
methods to advance this material to the corresponding bro-
moacetate 74 in improved yield.
Following the cross-metathesis reaction between the
acetonide-protected 2-methylene-1,3-propanediol 54e and TBS
ether 68, we suspected that incomplete removal of ruthenium
catalyst contributed to the partial degradation and low yields of
diol 74. Grubbs63 and others64–67 have noted that highly coloured
residual ruthenium catalysts can promote isomerization and
decomposition of metathesis products. Several strategies for
removing the ruthenium catalysts have been reported and were
evaluated separately by us, including stirring the crude
metathesis reaction mixture with (i) DMSO or Ph3PO prior to
Having failed to assemble the macrocyclic core of biselide A via
RRCM or macrolactonization, we nally focused on a macrocy-
clic Reformatsky reaction that had proven successful in
Kigoshi's rst synthesis of ent-haterumalide methyl ester
(Fig. 2).13 As summarized in Scheme 5, we expected that the
bromoacetate 77 could be accessed directly from intermediates
generated in our earlier studies. Towards this goal, monop-
rotection of the chlorodiol 51 gave the TBS ether 68, which was
converted into the bromoacetate 70 in excellent overall yield.
Next, cross metathesis with acetonide-protected 2-methylene-
1,3-propanediol 54e (Table 2) gave the orthogonally protected
tetrol 72. Unfortunately, attempts to remove the acetonide
protecting group from this intermediate resulted in low and
variable yields of the desired diol 74 (15–20%), which was
accompanied by triol 76 as the major product. Thus, we
returned to diol 51 and protected the primary alcohol function
at C13 as a TBDPS ether. We were pleased to nd that with this
choice of protecting group, the diol 75 was accessible in excel-
lent overall yield following an identical sequence of reactions.
Enzymatic acylation50 of the E-allyl alcohol function followed by
oxidation gave the Reformatsky macrocyclization substrate 77,
which was prone to isomerization and degradation as noted
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purication,64 (ii) activated carbon followed by ltration TBS protected C13 alcohol, we were pleased to nd that the
through silica gel or neutral alumina,65 (iii) Pb(OAc)4 prior to macrocycle was formed in 54% yield as a 3.5 : 1 mixture of
ltration through silica gel or neutral alumina,66 or (iv) an iso- separable C3-epimers 79 and 80, further highlighting the crit-
cyanide (CNCH2CO2K) prior to ltration through silica gel or ical importance of the C15 protecting group to macro-
neutral alumina. Unfortunately, none of these procedures cyclizations in this family of natural products. While
completely removed the brown colour from the product, which a Mitsunobu reaction68 of the (3S)-epimer 79 gave a mixture of
proved to be a reasonable predictor for stability. As a last resort, elimination, stereochemical inversion and stereochemical
we explored removal of the ruthenium by-products by size retention products, a sequence of oxidation and reduction (see
exclusion chromatography (Sephadex LH-20 resin, MeOH). inset) converted 79 cleanly into the desired (3R)-epimer 80.
Here, we found that the rst few column fractions contained all Macrocyclic stereocontrol in a related reduction was also noted
of the coloured ruthenium by products and none of the aceto- by Hoye.17 In an attempt to improve the yield and diaster-
nide 72, and that the isolated acetonide was a clear colourless eoselectivity of the Reformatsky reaction, we examined several
ꢃ
oil.
With ruthenium-free metathesis product 72, removal of the
solvents (CH2Cl2, THF, MTBE), reaction temperatures (ꢀ20 C,
0
ꢃC, rt), addition rates of aldehyde 78, and reaction concen-
acetonide protecting group proceeded cleanly to provide the trations but failed to improve signicantly on this outcome.
diol 74 in now reproducibly excellent yield (75%) over two steps Finally, acetylation and removal of the TBS protecting group
(Scheme 6). From here, a straightforward sequence of acetyla- gave the alcohol 81, a compound previously reported by Hay-
tion and oxidation gave the aldehyde 78. At this point we akawa and Kigoshi in their synthesis of biselide A.21 Following
examined the macrocyclic Reformatsky reaction using the the same sequence of reactions used by these researchers (i.e.,
conditions described by Honda61 and Kigoshi.13 Now, with the NHK reaction14–16 and deprotection), the total synthesis of
biselide A (7) was completed. At this point, we were pleased to
nd that the spectroscopic data recorded on our synthetic
material (1H-, 13C- NMR, HRMS) was identical in all regards to
that reported by Hayakawa and Kigoshi21 for their synthetic
material as well as to that of the natural product.9 Thus, we were
able to access biselide A in ꢁ2% overall yield via a synthetic
route that required only 18 steps in the longest linear sequence
starting from propane diol (Table 1), or 20 steps from L-serine
(Scheme 1).
Biological testing of synthetic biselide A
To examine the effect of biselide A on mammalian cell devel-
opment, the synthetic natural product was subjected to image-
based phenotypic screening using the Cell Painting protocol.69
Here, hundreds of features, including uorescence intensity,
cell number, and texture, are extracted from images and used to
generate a biological activity prole (or ngerprint) of each
treatment condition. This ngerprint can then be compared to
those of reference compounds to identify similar phenotypes
associated with disruption of cell development.70 A dilution
series of biselide A (9.5 mM to 0.3 nM, 16 ꢄ two-fold dilutions)
was applied to cultures of U2OS human osteosarcoma cells,
incubated overnight, treated with a set of ve uorescent stains
that stain structural features and sub-cellular organelles, and
imaged using an automated high-content uorescence micro-
scope (Molecular Devices ImageXpress). Hierarchical clustering
of ngerprints from biselide A-treated cells with ngerprints
from the TargetMol reference library (Fig. 3) identied two
concentrations that clustered in a region enriched in reference
compounds that impact microtubule organization and
dynamics.
Microtubules (MTs) are hollow cylindrical laments
comprised of tubulin dimers. MTs form a variety of highly
ordered structures (arrays) that perform key functions within
cells, including intracellular transport, cell migration, regula-
Scheme 6 Completion of the synthesis of biselide A (7).
5540 | Chem. Sci., 2021, 12, 5534–5543
tion of cell morphology, and cell division. Cell division is
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Fig. 3 Cell Painting results for biselide A (7). (A) Cell Painting fingerprints for dilution series of biselide A. Yellow ¼ positive deviation from control
values, blue ¼ negative deviation from control values. Comparison of Cell Painting fingerprints for active biselide A concentrations and
representative related reference compounds. (B) Original Cell Painting images including control and biselide A (top row), representative Tar-
getMol compounds with related MOAs (middle row) and TargetMol compounds with other, unrelated MOAs (bottom row). Images are composite
images from all fluorescence channels.
dependent on the mitotic spindle, a structure consisting of both Ultimately, an enantioselective a-chloroaldehyde-based
MTs and associated proteins, such as polo-like kinase 1 71,72 and approach was used to assemble the tetrahydrofuran core and
Aurora kinase,73,74 which are involved in spindle MT assembly the 14-membered macrocycle was constructed using a macro-
and organization.
Example Cell Painting proles and images are presented in molecule are installed using substrate-based stereocontrol that
Fig. 3A and B, respectively. Biselide A clustered with known originates from single a-chloroaldehyde. This concise
cyclic Reformatsky reaction. Notably, all 5 stereocenters in the
a
compounds that can be categorized into two classes based on approach compares well to contemporary syntheses of
their impact on MT arrays. Class I compounds (which includes members of the haterumalide and biselide family of natural
combretastatin A,75 TH588, TH287 hydrochloride,76 vinorelbine products. We also provide the rst biological testing data on any
tartrate,77 and epothilone B78) directly bind to tubulin dimers in synthetic member of this family and show that biselide A
all MT arrays, including the spindle, within the cell. Class II disrupts cell division through a mechanism related to regula-
compounds (which include INH6,79 HMN-176,80 dimethylenas- tion of microtubule cytoskeleton organization. This work
tron,81 MK8745,82 MLN905,83 and NMS-P937 84 target non MT- should support medicinal chemistry efforts directed at anti-
components of the mitotic spindle. Though these compounds cancer macrolides of this general structure class.
have different targets, they result in similar cellular phenotypes
due to their impact on MT arrays. Compounds in class I will
impact MT dynamics in all MT arrays and cause MT spindle
Author contributions
damage in a dose dependent manner,75 whereas class II
compounds largely affect MT array organization and MT
dynamics within the spindle.
V. R. C. and D. K. completed the total synthesis, M. T., H. F. and B.
K. developed methods for THF synthesis, metathesis and explored
macrocyclization reactions, D. W. carried out molecular modeling
studies, J. H. and S. K. were responsible for biological testing, R. G.
L. planned and supervised the biological testing and assisted with
manuscript preparation, R. B. planned and supervised the
synthetic work and managed manuscript preparation.
Clustering of biselide A in a region enriched in compounds
known to target MTs and MT dependent structures suggest that
biselide A may perturb MT associated processes, such as cell
division, through mechanisms related to regulation of MT
cytoskeleton organization.
Conclusion
Conflicts of interest
We report several generations of synthetic strategies aimed
towards the total synthesis of the marine macrolide biselide A. There are no conicts to declare.
© 2021 The Author(s). Published by the Royal Society of Chemistry
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25 M. T. Holmes and R. Britton, Chem.–Eur. J., 2013, 19, 12649–
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This work was supported by NSERC Discovery Grants to R. B.
and R. L., a MSFHR Career Investigator Award to R. B., and
a NSERC PGS for D. K., M. T., H. F., B. K. and D. W.
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