Total synthesis of the actinoallolides and a designed photoaffinity probe for target identification

Article abstract of DOI:10.1039/d0ob01831g

The actinoallolides are a family of polyketide natural products isolated from the bacterium Actinoallomurus fulvus. They show potent biological activity against trypanosomes, the causative agents of the neglected tropical diseases human African trypanosomiasis (sleeping sickness) and Chagas disease, while exhibiting no cytotoxicity against human cell lines. Herein, we give a full account of our strategy evolution towards the synthesis of this structurally unique class of 12-membered macrolides, which culminated in the first total synthesis of (+)-actinoallolide A in 20 steps and 8% overall yield. Subsequent late-stage diversification then provided ready access to the congeneric (+)-actinoallolides B-E. Enabled by this flexible and efficient endgame sequence, we also describe the design and synthesis of a photoaffinity probe based on actinoallolide A to investigate its biological mode of action. This will allow ongoing labelling studies to identify their protein binding target(s). This journal is

Full text of DOI:10.1039/d0ob01831g

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Biomolecular Chemistry
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Total synthesis of the actinoallolides and a
designed photoaffinity probe for target
identification†
Cite this: DOI: 10.1039/d0ob01831g
Matthew J. Anketell,
Theodore M. Sharrock and Ian Paterson
*
The actinoallolides are
a family of polyketide natural products isolated from the bacterium
Actinoallomurus fulvus. They show potent biological activity against trypanosomes, the causative agents
of the neglected tropical diseases human African trypanosomiasis (sleeping sickness) and Chagas disease,
while exhibiting no cytotoxicity against human cell lines. Herein, we give a full account of our strategy
evolution towards the synthesis of this structurally unique class of 12-membered macrolides, which cul-
minated in the first total synthesis of (+)-actinoallolide A in 20 steps and 8% overall yield. Subsequent late-
stage diversification then provided ready access to the congeneric (+)-actinoallolides B–E. Enabled by this
flexible and efficient endgame sequence, we also describe the design and synthesis of a photoaffinity
probe based on actinoallolide A to investigate its biological mode of action. This will allow ongoing label-
ling studies to identify their protein binding target(s).
Received 4th September 2020,
Accepted 25th September 2020
DOI: 10.1039/d0ob01831g
rsc.li/obc
Fortunately, the natural world possesses a rich and structu-
rally diverse source of antiparasitic compounds.17 The acti-
Introduction
Among communicable illnesses, parasitic diseases are the noallolides (Fig. 1), a family of novel polyketides, are one such
fourth largest cause of mortality worldwide, being responsible example, isolated by Iwatsuki and co-workers from a cultured
for over 500 000 deaths in 2015.1,2 Two such conditions are the strain
(MK10-036)
of
the
actinomycete
bacterium
neglected tropical diseases human African trypanosomiasis Actinoallomurus fulvus, obtained from the roots of Capsicum
(sleeping sickness)3–5 and Chagas disease,6–10 both caused by
protozoan parasites of the genus Trypanosoma. These illnesses
are estimated to account for a loss of 455 000 disability-
adjusted life years annually.11 Trypanosomes are extremely
adept at evading the immune system, and these neglected tro-
pical diseases can only be cleared from the body with the help
of chemotherapeutic agents.12 However, the ongoing develop-
ment of effective pharmaceutical agents against the causative
agents of these diseases has been challenging due to two
major factors.13,14 First, the eukaryotic nature of protozoan
parasites means that trypanosomes are biologically more
closely related than bacteria to human cells. This renders the
development of selective anti-trypanosomal drugs more chal-
lenging.15 Secondly, as these diseases mainly affect less econ-
omically developed countries, a lack of financial incentive to
develop new drugs has led to an urgent need for better, more
effective and safer treatments for these neglected diseases.16
University Chemical Laboratory, University of Cambridge, Lensfield Road, CB2 1EW,
UK
†Electronic supplementary information (ESI) available: Full experimental details
and characterisation of new compounds, together with NMR comparisons of the
synthetic and natural actinoallolides. See DOI: 10.1039/D0OB01831G
Fig. 1 3D structures of the five actinoallolide congeners 1–5 and the
designed photoaffinity probe analogue 6.
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fruitescens collected in Thailand.18 The 3D structure of acti-
noallolide A (1) was determined by spectroscopic analysis and
X-ray crystallography, which revealed the presence of 10 stereo-
centres, two trisubstituted alkenes and a 12-membered macro-
lactone incorporating
a
five-membered hemiacetal. In
addition, the structures of actinoallolides B–E (2–5) were con-
firmed by chemical correlation with actinoallolide A.
The actinoallolides were tested against three strains of
Trypanosoma and compared with several commonly used anti-
trypanosomal drugs. Actinoallolides A–E were also tested for
biological activity against Trypanosoma brucei brucei, the strain
responsible for animal trypanosomiasis (Nagana disease) and
compared with three of the most commonly used drug treat-
ments. They all compared favourably, with actinoallolide A
determined as the most potent by two orders of magnitude,
with an IC50 of 8.3 nM. Actinoallolide A (1) was also tested
against Trypanosoma brucei rhodesiense, a strain responsible
for human African trypanosomiasis and Trypanosoma cruzi,
the causative agent of Chagas disease, showing double the
potency of benznidazole. Actinoallolide A (1) showed no obser-
vable activity against MRC-5 human cells, giving it a high
selectivity index (>20 000).19 It was inactive against Gram-posi-
tive and negative bacteria and against yeast and fungi, indicat-
ing a highly specific mechanism of antitrypanosomal activity
that is yet to be determined.
The isolation group have also identified the actinoallolide
biosynthetic gene cluster, proposed a biosynthesis,20 and
reported preliminary synthetic studies.21 Herein, we give a full
account of the first total synthesis of the actinoallolides,22
which was enabled by a challenging ring-closing metathesis
reaction to form the macrocyclic trisubstituted alkene, the
most complex example of its kind.23 As the biological target
and mechanism of action of the actinoallolides are unknown,
we sought to leverage our total synthesis to shed light on these
unanswered questions. Based on a highly efficient endgame,
we now report the design and synthesis of the actinoallolide
A-based probe 6 for target identification using photoaffinity
labelling.24,25
Scheme 1 Initially proposed retrosynthesis of actinoallolide A with the
three key aldol reactions highlighted in green.
membered hemiacetal after deprotection and oxidation at C3
to give actinoallolide A.
Results and discussion
Retrosynthetic analysis of actinoallolide A
The side chain contains two distinct stereoclusters. The
C
11–C14 stereotetrad was envisaged to be constructed by a
Our proposed key bond disconnections (Scheme 1) were of the substrate-controlled allylation of 9 preceded by a lactate
C1 ester and the C8–C9 alkene to form two fragments of aldol reaction to reveal, after redox adjustment, ester 10. The
roughly equal size and complexity: the macrocycle precursor 7 isolated C14 stereocentre in 10 could then be installed by an
and the side chain 8. A major advantage of this approach is Ireland–Claisen rearrangement from the diester 11. The ‘all
that it allows a flexible fragment coupling strategy whereby the syn’ stereotetrad in 12 can then be formed by a titanium-
two coupling steps could be undertaken in either order; cross mediated aldol reaction/in situ reduction between methacro-
metathesis could be followed by macrolactonisation, or esteri- lein and ethyl ketone 13, itself synthesised in three steps
fication could be followed by ring-closing metathesis as from (R)-Roche ester (14). The decision to attach the term-
desired.
The C1–C8 fragment 7 would be forged from natural (S)- form the C21 ketone as late as possible to facilitate late-stage
lactic acid with key bonds formed via a Seebach alkylation and diversification.
more complex organometallic reagent
inal ethyl group at such a late stage was made in order to
A
a diastereoselective aldol reaction. Protection of the C3 and C6 could be used to add any modified tail in place of the ethyl
alcohols would allow late-stage unmasking of the delicate five- group.
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Finally, the decision to utilise two sets of orthogonal pro- Claisen rearrangement used in the total synthesis of ebelac-
tecting groups was informed by our planned endgame. The tone,32 we began investigating this key step. Initial attempts at
hydroxyl groups at C21 and C3 require oxidation, whereas those performing this reaction led to appreciable formation of the
at C6, C13 and C19 do not. Thus it was planned to use benzyl C14 α-silylated by-product. This TMS group could be removed
ether-based groups for the former and silyl ethers for the latter with K2CO3 in MeOH but, as would be expected, led to signifi-
hydroxyl groups.
cant epimerisation. The epimer was visible by NMR and pro-
vided evidence that the Ireland–Claisen product had been
obtained as a single diastereomer. This side reaction is pro-
posed to occur via enolisation of the formed silyl ester by
excess LDA and trapping of this enolate with TMSCl. As such,
it was important to precisely control the equivalents of LDA
used. In order to avoid quenching any LDA, it is necessary to
remove all traces of HCl from the large excess of TMSCl used.
In the ebelactone work, this was accomplished by premixing
TMSCl with Et3N and centrifuging the resulting suspension to
remove the precipitate. In this work, removal of the precipitate
was more conveniently accomplished via filtration through a
syringe tip filter (PVDF membrane, 0.45 μm pores). Optimising
the quantity of LDA used increased the yield to 73% with no
observable formation of the α-silylated by-product. For ease of
isolation, the carboxylic acid product was methylated without
purification to afford methyl ester 10.
Pleasingly, the DIBAL-H reaction of this ester 10 proceeded
well, reducing the methyl ester at C13 to the corresponding
aldehyde and liberating the free hydroxyl group at C19, setting
the stage for the second strategic aldol reaction. While sub-
strate control was used to construct all other stereocentres in
side chain fragment 8, the anticipated mismatch between the
Felkin–Anh selectivity of aldehyde 15 and the desired configur-
ation at the C13 hydroxyl stereocentre made it impossible in
this case. Using boron aldol methodology developed in the
group,33,34 we were able to couple aldehyde 15 with lactate-
derived ketone 16 to form anti adduct 17 as a single diastereo-
mer, overriding the inherent facial selectivity of aldehyde 15 to
install the C12 and C13 stereocentres. Protection of both
Synthesis of the C9–C21 side chain fragment (8)
The synthesis of side chain fragment 8 commenced with ethyl
ketone 13.26 In previous work, the ‘all-syn’ aldol selectivity
required for the construction of 12 had been achieved by use
of tin(II) triflate as a Lewis acid.27,28 While effective, the use of
tin(II) triflate was procedurally cumbersome, especially on
scale owing to its capricious preparation. We sought to
improve upon this process by combining two recent develop-
ments in aldol chemistry. Romea and Urpí have reported the
use of titanium Lewis acids for the highly selective aldol reac-
tions of protected β-hydroxy ketones29 and α-hydroxy ketones
with an in situ reduction to afford the syn-1,3-diol.30 It was
reasoned that combining these two processes would afford the
desired ‘all-syn’ stereotetrad in diol 12. In the event, appli-
cation of this in situ reduction with LiBH4 to the aldol addition
of ketone 13 and methacrolein provided diol 12 in 90% yield
as a single diastereomer (Scheme 2). Owing to the oxophilicity
of the titanium and boron complexes formed in the reaction,
an extended workup incorporating treatment with hydrogen
peroxide and multiple washings with Rochelle salt solution
was found to be necessary for release of 12.
After selective monoesterification of the allylic alcohol in
diol 12 was found to be problematic, it was decided to proceed
through the required Ireland–Claisen rearrangement31 with
diester 11. Only the O17 ester was correctly positioned to
undergo the [3,3]-sigmatropic rearrangement while the O19
ester would be removed during the later reduction of the C13
methyl ester to the aldehyde. Guided by a similar Ireland–
Scheme 2 Synthesis of the side chain fragment 8.
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hydroxyl groups as TES ethers then afforded 18 which under- cedure. The first attempt to convert the dioxolanone to the
went a standard two-step auxiliary removal procedure to propyl ketone directly by addition of propylmagnesium
provide aldehyde 9.33 Addition of the allyl moiety to 9 to com- bromide, however, was unsuccessful, yielding either unreacted
plete the side chain fragment 8 required some optimisation starting material or the double addition product. Ring
with the use of allylmagnesium chloride or allyltrimethylsilane opening to the Weinreb amide 21 was trialled next and pro-
leading to low diastereoselectivity. Fortunately, it was found ceeded in excellent yield using the in situ generated anion of
that the utilisation of allyltributylstannane in a Hosomi– N,O-dimethylhydroxylamine. With this result, a one-pot con-
Sakurai reaction35,36 with the non-chelating Lewis acid version to the desired propyl ketone was examined. However,
BF3·OEt2 afforded 8 in high yield with 15 : 1 dr.
the conversion of 21 to the corresponding propyl ketone in situ
This completed the scalable and high-yielding synthesis of proved unsuccessful even with warming to room temperature.
the side chain fragment 8 in 13 linear steps from (R)-Roche Weinreb amide 21 was next silyl protected and converted to
ester (14) with an overall yield of 21%. This excellent yield (an propyl ketone 22, required for the planned C3–C4 bond-
average of 89% per step) and the scalability of the route forming lithium-mediated aldol reaction. As the protecting
allowed 2.3 g to be prepared, providing ample material for group strategy utilised a silyl group at C6 and a PMB group at
development of the endgame.
C3, the C1 protecting group was required to be orthogonal to
both.
Synthesis of the C1–C8 macrocycle precursor fragment
From propyl ketone 22, two routes to macrocycle precursor
fragment 7 were initially considered. The first plan was to
utilise an orthoester protecting group on aldehyde 23, as this
would allow access to C1 at the desired carboxylic acid oxi-
dation level. This lithium-mediated aldol reaction afforded syn
adduct 24, providing the desired diastereomer in good yield
and with useful diastereoselectivity. We had planned to protect
the C3 alcohol with a PMB group, however all attempted con-
ditions led to no reaction or to degradation, presumably owing
to steric congestion. Furthermore, removal of the orthoester
protecting group could not be achieved without degradation
due to the acid and base sensitivity of the aldol adduct.
With this approach proving to be unviable, C1 was next
introduced at the protected alcohol oxidation state. Noting
that PMB groups can be oxidatively transposed,40 this second
plan was to utilise a PMB group on aldehyde 25 to produce
The synthesis of the macrocycle precursor fragment began
with the construction of dioxolanone 19 via a two-step pro-
cedure (Scheme 3),37 allowing an enolate alkylation38 with
methallyl bromide to install the C6 stereocentre. This reaction
proved difficult to optimise, with a 53% yield of 20, albeit as a
single diastereomer. This was due to formation of a major by-
product due to self-reaction of the dioxolanone.39 However, the
modest yield was deemed sufficient and allowed the prepa-
ration of 20 on a multi-gram scale.
The removal of the pivaldehyde-derived auxiliary via ring
opening of dioxolanone 20 was the next task to be accom-
plished. In preliminary studies, the desired conversion to
propyl ketone 22 was achieved using a four-step sequence com-
mencing with ring opening to the methyl ester. We envisaged
abridging this sequence by using a different ring-opening pro-
Scheme 3 Synthesis of the key propyl ketone 22 and attempts at elaboration.
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aldol adduct 26. As before, the lithium-mediated aldol reaction
These frustrations en route to the macrocycle precursor frag-
afforded the syn adduct in good yield, in a 4 : 1 ratio of diaster- ment led us to consider more substantial modifications to the
eomers. The major diastereomer was then converted to PMP route. The efforts thus far had focused on installing a PMB
acetal 27 by treatment with DDQ under anhydrous conditions. protecting group at C3, either by a ‘PMB transposition’of the
Disappointingly, subsequent attempts at reductive PMP PMB group at C1, or by direct protection of the alcohol at C3.
opening to the primary alcohol were all unsuccessful, with the The former method had failed due to the selectivity problems
use of DIBAL-H40 or NaCNBH3/TMSCl41 causing side-reactions with opening the intermediate PMP acetal and the latter had
or reacting with the undesired regioselectivity to revert to sec- failed due to the steric hindrance of aldol adduct 26 and its
ondary alcohol 26. This unexpected selectivity may be due to instability under forcing conditions. To circumvent these pro-
preferential chelation of the Lewis acid between the C3 oxygen blems within the constraints of the overall protecting group
and the ketone rather than the less hindered C1 oxygen as strategy – that is to allow for the chemoselective oxidative
would normally be expected.
removal of the C3 and C21 protecting groups in one operation,
Given that this selectivity problem was likely due to the pro- it was proposed to switch the C3 protecting group from PMB to
pensity of the C5 ketone to coordinate with the adjacent C3 PMBM (para-methoxybenzyloxymethyl).44 Like the PMB group,
oxygen, it was proposed that reducing the C5 ketone and tran- it can be oxidatively removed orthogonally to silyl groups but
siently protecting the resulting alcohol as a TMS ether might can be installed under far milder conditions. However, as the
circumvent this impasse. Ketone 27 was thus reduced using PMBM group was liable to cleavage under oxidative conditions,
LiBH4 to form secondary alcohol 28 in 70% yield and 2 : 1 dr. this alteration necessitated a revision of the C1 protecting
The diastereomers were inseparable by chromatography so the group such that it could be removed orthogonally to the
alcohols were then silylated to form 29. This mixture of dia- PMBM group. Given that the aromatic ring in the DMB (3,4-
stereomeric PMP acetals then underwent reductive opening dimethoxybenzyl)45 group is more electron rich than in the
with DIBAL-H. Surprisingly, the two epimeric acetals reacted PMBM group, it was anticipated that selective oxidative clea-
with opposing regioselectivities, with the major diastereomer vage of the DMB group would be possible.46
forming the undesired secondary alcohol 30 and the minor
diastereomer forming the desired primary alcohol 31.
The corresponding DMB-protected aldehyde 32 required for
the aldol coupling was prepared via a three-step procedure.47
In light of this mixed result, it was reasoned that if we This aldehyde then underwent an analogous lithium-mediated
could override the inherent substrate selectivity of ketone 27 to aldol reaction with propyl ketone 22, providing syn adduct 33
reduction, and obtain the 1,3-anti diastereomer selectively, (Scheme 4) in similar yield and dr to that obtained from the
this might provide a viable route to the macrocycle fragment. PMB-protected variant. This was subsequently PMBM pro-
Testing a range of simple reducing agents was fruitless, with tected using the mild base DIPEA in refluxing acetonitrile over-
each one either giving the undesired selectivity or no reaction. night to afford PMBM ether 34 in excellent yield. With triply-
It was next decided to attempt the reduction of the C5 ketone protected aldol adduct 34 in hand, selective DMB cleavage was
prior to PMP acetal formation, using the C3 alcohol in 26 to next attempted. Fortunately, one equivalent of DDQ with
direct
a
1,3-anti Evans–Tishchenko42 or Evans–Saksena careful monitoring afforded primary alcohol 35 in excellent
reduction.43 With prolonged reaction time and an excess of yield, with complete retention of the PMBM group. This then
the samarium catalyst,42 it was possible to achieve a low yield underwent a double oxidation sequence; first was a Swern oxi-
of 10% with 2 : 1 dr for the former reaction, but no product dation to give aldehyde 36, then a Pinnick oxidation provided
was obtained under the latter conditions. This was attributed the complete macrocycle precursor fragment 37.
to the steric hindrance of the C5 ketone with its quaternary α
stereocentre incorporating a bulky OTMS group.
Overall, macrocycle precursor fragment 37 was synthesised
in 10 linear steps from (S)-lactic acid with an overall yield of
Scheme 4 Completion of the synthesis of macrocycle precursor fragment 37.
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Table 1 Screening of ring-closing metathesis conditions for the for-
mation of macrocycle 39 from precursor 38 (G-II = Grubbs second-
17%. This represents an average of 84% per step, and the scal-
ability of the route allowed 600 mg to be prepared, providing
ample material for development of the endgame.
generation catalyst, HG-II
catalyst)
= Hoveyda–Grubbs second-generation
Fragment union and endgame
Entry
Cat. (mol%)
Solvent (mM)
Temp.
Time
39 : 40
With key fragments 8 and 37 in hand, the stage was set to
explore the pivotal fragment union sequence. The retrosyn-
thetic plan (Scheme 1) had left the ordering of these steps flex-
ible. However, preliminary investigations had indicated that an
efficient and stereoselective cross metathesis to give the trisub-
stituted 8E alkene was unlikely to succeed, so we proceeded
with exploring the esterification/RCM sequence. The frag-
ments 37 and 8 were coupled together under standard
Yamaguchi conditions48 to afford ester 38 in an excellent yield
of 99%, enabling investigation of the “do-or-die” macrocycle-
forming RCM step (Scheme 5).
1
2
3
4
5
6
7
8
9
10
G-II (20)
G-II (50)
G-II (20)
G-II (20)
HG-II (20)
HG-II (40)
HG-II (40)
HG-II (40)
HG-II (40)
HG-II (40)
CH2Cl2 (5)
40 °C
40 °C
80 °C
110 °C
110 °C
110 °C
110 °C
110 °C
145 °C
110 °C
18 h
18 h
18 h
18 h
18 h
72 h
72 h
72 h
72 h
7 d
0 : 1
0 : 1
0 : 1
0 : 1
CH2Cl2 (10)
Benzene (1)
Toluene (1)
Toluene (1)
Toluene (1)
Toluene (10)
Toluene (0.25)
Xylene (0.25)
Toluene (1)
1 : 1
1.5 : 1
1.3 : 1
2 : 1
1.2 : 1
3 : 1
The first attempt at cyclisation of 38 using Grubbs second- sibly return to the dimer. Much more slowly, but irreversibly,
generation catalyst (G-II)49,50 in refluxing degassed CH2Cl2 pro- this ruthenium carbene can undergo an intramolecular reac-
vided only the undesired dimer 40 in quantitative yield tion with the 1,1-disubstituted alkene to afford desired macro-
(Table 1, entry 1). Increasing the catalyst loading to 50 mol% cycle 39. To help encourage the slow, irreversible RCM step,
(entry 2) or the use of higher-boiling solvents (entries 3 and 4) the duration of the reaction was increased to seven days,
failed to provide any macrocyclic product, in each case return- increasing the product : dimer ratio to 3 : 1 and providing the
ing only dimer 40. As an additional measure, a solution of desired product with an improved yield of 75% (entry 10). To
catalyst was added portionwise over the initial few hours of the our knowledge, this challenging and remarkable transform-
reaction to reduce catalyst decomposition, although the colour ation is the most complex example of an RCM reaction to form
change from purple to brown was observed within 30 min of a trisubstituted alkene in a medium-sized ring.23
each addition, indicating rapid catalyst degradation. In light of
Having prepared an abundant supply of advanced inter-
these failures, we switched to the more reactive Hoveyda– mediate 39, the removal of both PMB-containing protecting
Grubbs second-generation catalyst (HG-II).51,52 Gratifyingly, an groups was accomplished by treatment with DDQ, affording
initial RCM reaction of 38, overnight in refluxing toluene, diol 41 in 94% yield (Scheme 6). Chemoselective oxidation of
afforded an inseparable 1 : 1 mixture of dimer 40 and desired the primary alcohol at C21 in the presence of the C3 secondary
macrocycle 39 (entry 5). Increasing the catalyst loading to alcohol was achieved using a TEMPO/BAIB oxidation53,54
40 mol% and the reaction time to 72 h increased the product : which afforded aldehyde 42 in 90% yield. Subsequently,
dimer ratio to 1.5 : 1 (entry 6). In an attempt to increase the chemoselective addition of ethylmagnesium bromide to the
reaction rate and yield, the solvent was changed to o-xylene to newly formed aldehyde in the presence of the C5 ketone pro-
allow reflux at a higher temperature. Surprisingly, this led to a ceeded smoothly, affording an inseparable epimeric mixture of
reduction in the product : dimer ratio to 1.2 : 1 (entry 9), indi- 43 in 91% yield, with no observable attack at the ketone.
cating that there was a ‘sweet spot’ temperature at around
110–140 °C, below which the reaction is prohibitively slow and lolides, our attention now turned to the final two steps of oxi-
above which catalyst decomposition occurs.
dation and global deprotection. Oxidation of both secondary
Having completed the full carbon backbone of the actinoal-
By this point, a mechanistic hypothesis was proposed alcohols at C3 and C21 was initially attempted under Swern
whereby the terminal alkene present in 38 rapidly dimerises. conditions, however, this was found to be unreliable.
The ruthenium catalyst can then reinsert into the new di- Alternatively, the oxidation was performed using the milder
substituted alkene in this dimer and can then undergo one of Dess–Martin periodinane.55 Double oxidation under these con-
two processes. In most cases, this undergoes an inter- ditions was found to be clean and reliable, providing triketone
molecular reaction with another molecule of dimer 40 to rever- 44 in 99% yield.
Scheme 5 Fragment union and RCM-mediated formation of the 12-membered macrocycle 39.
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Scheme 6 Endgame and completion of the total synthesis of actinoallolide A (1).
The only obstacle now remaining was the final de-
protection. The conditions should ensure complete cleavage of
the C6 TMS ether and the C13 and C19 TES ethers, while also
forming the transannular hemiacetal. The conditions were
also required to be sufficiently mild to avoid any possible dele-
terious side reactions. Preliminary experience with the lability
of the hemiacetal motif led to the hypothesis that actinoallo-
lides C–E may be artefacts of the elaborate chromatographic
isolation process. Consequently, we were particularly cautious
to avoid elimination across the C3–C4 bond and transesterifica-
tion of the macrolactone in 44. Initial attempts using TBAF
were unsuccessful, leading either to incomplete deprotection
or degradation. Pleasingly, acidic fluorous deprotection con-
ditions proved successful, with treatment of 44 with
a
1 : 3 mixture of HF·py/py with warming to 40 °C effecting quan-
titative conversion to (+)-actinoallolide A (1). The resulting syn-
thetic actinoallolide A possessed comparable specific rotation
and identical NMR spectra to those of the natural product (see
the ESI†).
The total synthesis of (+)-actinoallolide A (1) was thus
achieved in 20 linear steps from (R)-Roche ester (14) with an
overall yield of 8%, representing an average yield of 88% per
step. Importantly, this first total synthesis gave an orthogonal
validation of the full 3D structure and absolute configuration
of actinoallolide A and will enable further biological investi-
gations of this highly potent family of natural products.
Scheme 7 Conversion of actinoallolide A (1) to actinoallolides B–E
(2–5).
lolide C (3) and actinoallolide E (5). This unexpected result
indicated the instability of the cyclic hemiacetal moiety to
basic conditions, perhaps explaining why global deprotection
was unsuccessful under basic fluorous conditions. This spon-
taneous conversion of actinoallolide A (1) to actinoallolides C
(3) and E (5) on alumina also provides evidence that these
Conversion of actinoallolide A to actinoallolides B–E
We next sought to access the other four congeners, actinoallo- compounds are isolation artefacts rather than genuine natural
lides B–E (2–5), by replication of the conversion protocols in products. This serendipitous result completed our synthesis of
the isolation paper (Scheme 7).18 Actinoallolide A (1) under- all five members of the actinoallolides.
went a 1,3-syn hydroxyl-directed reduction using triethyl–
Based on the assumption that actinoallolides C and E are
borane followed by sodium borohydride to afford actinoallo- isolation artefacts, it is likely that actinoallolide D is also an
lide B (2) in 93% yield and as a single diastereomer. artefact, arising from dehydration of actinoallolide B. This
Subsequent treatment of actinoallolide B (2) with trifluoroace- would leave actinoallolides A and B as the only true natural
tic acid caused dehydration of the cyclic hemiacetal to provide products, differing only in the oxidation level at C21. In exam-
actinoallolide D (4) in 99% yield.
ining the proposed biosynthesis of actinoallolide A,20 the
During the initial purification of actinoallolide A using authors had identified an inactive ketoreductase domain (KR1)
silica chromatography, some minor by-product formation was in the polyketide synthase responsible for the biosynthesis of
observed. Using alumina instead, attempted purification of actinoallolide A. They commented that “although KR1 has
actinoallolide A led to conversion to a 1 : 1 mixture of actinoal- catalytic amino acids and the NADPH binding motif, it seems
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to be inactive, as predicted from the structure of actinoallolide actinoallolide B, containing the C21 alcohol, is produced, and
A that has a ketone at C21”. If, rather than being inactive, the when transfer to module 2 occurs before the reduction can
KR1 domain were instead partially active, this identified poly- take place, the product is instead actinoallolide A, with the
ketide synthase would be able to generate both actinoallolides unreduced C21 ketone. On inspection of the amino acid
A and B. When the KR1 domain performs the C21 reduction, sequence of the KR1 domain, it possesses both sets of charac-
teristic amino acids which determine whether reduction
affords the L- or D-configuration, potentially explaining its
partial inactivity.
Synthesis of an actinoallolide A photoaffinity probe analogue
Having achieved a practical, convergent and high-yielding total
synthesis of the actinoallolides, we moved onto the design and
synthesis of a photoaffinity probe analogue (Scheme 8) to
identify their biological target. In order to minimally perturb
the structure of the native natural product, the photoaffinity
probe 6 was chosen to extend from the C23 terminus.56 The
photoreactive moiety is a diazirine, decomposing to a highly-
reactive carbene upon irradiation at 365 nm, facilitating cross-
linking to the bound target protein. The functional handle is a
terminal alkyne, allowing the facile conjugation of any desired
reporter tag using
cycloaddition.25
a
copper-catalysed azide–alkyne
The synthesis of probe
6
began by intercepting the
advanced intermediate 42. In the total synthesis, addition of
an ethyl group was required to complete the actinoallolide
carbon skeleton. Instead, the addition of the Grignard reagent
derived from halide 45 afforded diol 46 in 69% yield as an
inconsequential
1 : 1
mixture
of
diastereomers.
Chemoselective removal of the alkynyl TMS group was
achieved by treatment with AgNO3 and gave 47 in 65% yield.
Next, a copper-catalysed azide–alkyne cycloaddition between
alkyne 47 and linker fragment 48 (prepared from 1,4-cylohexa-
nedione in eight steps)57 proceeded to afford triazole 49 (99%).
The EDCI/DMAP-mediated amidation of 49 with propargyla-
mine proceeded smoothly, affording amide 50 in 99% yield.
This amide then underwent a double DMP-mediated oxidation
to afford triketone 51 (93%). Finally, global deprotection under
acidic fluorous conditions proceeded with an excellent yield of
99%, cleaving all three silyl ethers and inducing formation of
the transannular five-membered hemiacetal to form photo-
affinity probe 6. To date, this route has provided 26 mg of 6,
sufficient material for the required photoaffinity experiments.
In preliminary studies, the biological activities of synthetic
actinoallolide A (1) and photoaffinity probe 6 have been con-
firmed and photoaffinity studies are currently ongoing.
Conclusions
This account details the first total synthesis of the actinoallo-
lides, a family of polyketide natural products prized for their
promising anti-trypanosomal properties.58 This feat has been
accomplished in a scalable, high-yielding and stereoselective
fashion, giving actinoallolide A (1) in 20 linear steps from (R)-
Roche ester (14) with an overall yield of 8%. This was achieved
in a highly convergent manner, utilising two fragments 8 and
Scheme 8 Diversion of advanced intermediate 42 towards the syn-
thesis of photoaffinity probe analogue 6 of actinoallolide A.
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37 of similar complexity, which were obtained in 13 and 10
steps respectively. The stubbornness of C3 manipulation to
standard ‘PMB transposition’ conditions was frustrating and
necessitated modifications to the original strategy. Fortunately,
the judicious choice of alternative protecting groups allowed
us to overcome this obstacle.
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its kind. A high-yielding endgame finally afforded actinoallo-
lide A. Actinoallolide A was then converted to actinoallolides
B–E, completing the total synthesis of the entire natural
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Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We thank the Herchel Smith Fund (studentship to M. J. A.),
Dr Rachel J. Porter and Dr Simon Williams for assistance, and
Thomas Hayhow (AstraZeneca) for his interest. We are grateful
to Professor Terry Smith and Dr Gordon Florence at the
University of St Andrews for performing the preliminary bioac-
tivity assays. We also acknowledge the National Mass
Spectrometry Facility at Swansea University.
21 J. Oshita, Y. Noguchi, A. Watanabe, G. Sennari, S. Sato,
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22 For a preliminary communication, see: M. J. Anketell,
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