Meeting key synthetic challenges in amanitin synthesis with a new cytotoxic analog: 5′-hydroxy-6′-deoxy-amanitin

Article abstract of DOI:10.1039/d0sc04150e

Appreciating the need to access synthetic analogs of amanitin, here we report the synthesis of 5′-hydroxy-6′-deoxy-amanitin, a novel, rationally-designed bioactive analog and constitutional isomer of α-amanitin, that is anticipated to be used as a payload for antibody drug conjugates. In completing this synthesis, we meet the challenge of diastereoselective sulfoxidation by presenting two high-yielding and diastereoselective sulfoxidation approaches to afford the more toxic (R)-sulfoxide.

Full text of DOI:10.1039/d0sc04150e

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Meeting key synthetic challenges in amanitin
0
synthesis with a new cytotoxic analog: 5 -hydroxy-
Cite this: DOI: 10.1039/d0sc04150e
0
6
-deoxy-amanitin†
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Alla Pryyma,
Kaveh Matinkhoo,
Antonio A. W. L. Wong
and David M. Perrin
*
0
Appreciating the need to access synthetic analogs of amanitin, here we report the synthesis of 5 -hydroxy-
Received 29th July 2020
Accepted 2nd October 2020
0
6
-deoxy-amanitin, a novel, rationally-designed bioactive analog and constitutional isomer of a-amanitin,
that is anticipated to be used as a payload for antibody drug conjugates. In completing this synthesis, we
meet the challenge of diastereoselective sulfoxidation by presenting two high-yielding and
diastereoselective sulfoxidation approaches to afford the more toxic (R)-sulfoxide.
DOI: 10.1039/d0sc04150e
rsc.li/chemical-science
7
drug-tolerant cell subpopulations. Examples include ADCs for
Introduction
targeting human epidermal growth factor receptor 2 and pros-
8
9
a-Amanitin, the deadliest of the amatoxins produced by the tate specic membrane antigen. With but a few exceptions,
death-cap mushroom Amanita phalloides, is a potent, orally nearly all bioconjugates to a cytotoxic amanitin have emerged
1
available inhibitor of RNA polymerase II (pol II) (K ꢀ 10 nM),
from naturally-sourced a-amanitin. To date, conjugation
i
that has been validated as a payload for targeted cancer handles used for a-amanitin-based bioconjugates include the d-
therapy. First described in 1907 and isolated in 1941, a- hydroxyl of (2S,3R,4R)-4,5-dihydroxyisoleucine (DHIle), the
amanitin is a compact bicyclic octapeptide that has been asparagine side chain, and the 6 -hydroxyl of the tryptathio-
indispensable for probing RNA pol II-catalysed transcription in nine staple. Of these, increasingly, the latter has been favored
eukaryotes. While the toxin makes numerous contacts with RNA as a site for bioconjugation to the intact toxin.
pol II, the lack of synthetic access has limited the ability to
generate discrete analogs to probe these contacts, most notably synthesis will likely serve as the main means for accessing large
the role of the 6 -hydroxyl group on the indole. Of the few amounts of toxin needed to address structure–activity rela-
2
3
4
2a
10
0
11
12
As biosynthetic fermentation is currently low-yielding,
0
0
naturally occurring amatoxins wherein the 6 -OH is replaced by tionships and provide new handles for bioconjugation. This
H, amaninamide and amanin are only 25–50% as toxic as the heightened interest in a-amanitin has inspired three total
13
natural product (Fig. 1) indicating moderate importance of the syntheses that address synthetic challenges embedded in
5
OH group.
three key components: (i) an enantioselective synthesis of
0
Its putative contribution to affinity is understood from two DHIle, (ii) creation of an oxidatively challenged 6 -OH-
apparent interactions seen in a recent co-crystal structure of a- tryptathionine-sulfoxide that demands formal oxidation at
amanitin-RNA pol II: (i) the 6 -hydroxyl group forms an H-bond
0
with Ser-782 accounting for the difference in K 's between
i
mammalian and yeast RNA pol II; (ii) the hydroxyl group
increases the electron density of the indole which forms
6
a cation–p interaction with Arg-749. Besides contributing to
0
toxicity, the 6 -OH provides a preferred handle for chemical
functionalization for graing to antibodies.
Recently, a-amanitin has been showcased as a highly effec-
2a
tive payload in antibody drug conjugates (ADCs). Unlike other
ADC payloads, a-amanitin targets both dividing and quiescent
cells and shows potential for preventing cancer relapse from
Fig. 1 Chemical structure of a/b-amanitin and structure–activity
relationships of natural and synthetic amatoxins with indole modifi-
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver,
B.C., V6T 1Z1, Canada. E-mail: dperrin@chem.ubc.ca
cations; a 2–4 fold-increase in K
i
as a result of indole modification is
0
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/d0sc04150e
relative to the 6 -OH containing counterparts; commonly used
conjugation sites are highlighted.
1
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positions 2 and 6 of the indole, and (iii) stereoselective oxida-
tion of the thioether to give (R)-sulfoxide, which is approxi-
13a
mately 8-fold more toxic than the (S)-sulfoxide.
In 2018, Perrin et al. opted for a 6-boronated-L-tryptophan as
a masked phenol equivalent that underwent oxidative uo-
rocyclization followed by Savige–Fontana reaction to form the
14
13a
tryptathionine staple with the pendent boronate. Oxidative
deborylation, peptide elongation, and macrocyclization gave
the S-deoxy-amanitin that was readily sulfoxidized with
moderate diastereoselectivity (ca. 2 : 1) favoring the (R)-
sulfoxide.
In 2020, S u¨ ssmuth et al. described a convergent “5 + 1 + 2”
strategy whereby the tryptathionine staple was synthesized rst
by reacting protected cysteine-sulfenyl chloride with orthogo-
nally protected 6-OBn-L-tryptophan. The tryptathionine was
then elaborated to a pentapeptide followed by addition of DHIle
13b
and Pro–Asn and macrolactamization.
A third report
advanced a scalable synthesis of 6-OAc-Trp, and utilized Savige–
1
4
0
Fontana reaction to introduce the 6 -OH-tryptathionine sta-
ple.
13c
0
Fig. 2 Docking pose of 50-OH-60-deoxy-amanitin 2 showing favor-
While all three reports provide access to 6 -OH-
0
0
able interactions with RNA-pol II; 5 -OH-6 -deoxy-amanitin 2 –
orange, a-amanitin 1 – magenta, green dashed line – distance
between Ser-782 and indole alcohol.
tryptathionine, they noted the severe challenges of selective
oxidative thiolation of the electron-rich indole.
0
In contrast to the immediate interest in accessing the 6 -
hydroxyl group, interest in generating the (R)-sulfoxide remains
academic. Nevertheless, because Nature exclusively produces
the (R)-sulfoxide, logic suggests that the (R)-sulfoxide may
provide privileged pharmacological/toxicological properties. As
such, diastereoselective access of the (R)-sulfoxide continues to
pose a signicant synthetic challenge that was met with but
moderate selectivity in the rst total synthesis and remained
unprogressed in the two subsequent ones.
also engage in an H-bonding interaction with Ser-782. A dock-
ing study validated this hypothesis: the 5 -OH-6 -deoxy-amanitin
0
0
(
2) was docked into the RNA pol II using Autodock Vina
15
molecular docking program.
0
0
The most energetically favorable pose of 5 -OH-6 -deoxy-
amanitin was compared to the crystal structure pose of a-
amanitin (Fig. 2). Further assessment using PyMOL Molecular
Graphics System conrmed that the aforementioned key inter-
actions between the enzyme and the indole ring are still
achievable and the modied indole does not alter the overall
Despite these synthetic efforts, there remains a demand for
biologically active and more synthetically accessible a-amanitin
analogs that: (i) capture the putative p–cation interaction for
enhanced affinity and (ii) provide an indole-situated conjuga-
tion handle for ADC development. Towards these ends, herein
we report the synthesis of a constitutional isomer of near-native
16
conrmation of the t. Importantly, the docking pose sug-
gested that the 5 -hydroxyl group should still form the antici-
0
pated H-bond with Ser-782 (Fig. 2, green line). Based on these
observations, we reasoned that the incorporation of 5-OH-
tryptophan should maintain the toxicity of the new amanitin
analog.
0
toxicity that introduces a 5 -OH-tryptathionine, which is readily
achieved from cheap, commercially available 5-OH-L-trypto-
phan (5-OH-Trp).
In addition, we identify new methodologies to create the (R)-
sulfoxide with high diastereoselectivity (>15 : 1) along with the
0
Encouraged by this result, we undertook a synthesis of 5 -
0
OH-6 -deoxy-amanitin (2) that would rely on two key compo-
(S)-sulfoxide diastereomer and sulfone for comparison. Antici-
nents: (i) introduction of the fully protected DHIle-NHS ester as
previously reported, and (ii) synthesis and characterization of
the 3a-uoro-pyrroloindoline produced from 5-OH-tryptophan
that could be subjected to late-stage Savige–Fontana reaction
pating success with chemoselective conjugation akin to afore-
0
mentioned conjugates to the 6 -OH-indole, this work now
expands the toolbox of available synthetic amanitin analogs for
consideration as payloads for use in translational applications.
0
to give the 5 -OH-tryptathionine (Scheme 1). In addition to this
synthetic feat, we addressed mid- and late-stage sulfoxidation
with high diastereoselectivity to afford the desired (R)-sulfoxide.
Following the approach used in the rst total synthesis of a-
Results
In approaching the synthetic challenge of creating a hydroxy-
tryptathionine staple, we appreciated the high natural abun-
dance of 5-OH-tryptophan in a number of biosynthetic path-
ways along with its commercial availability as a single
enantiomer. Hence, we hypothesized that 5-OH-L-tryptophan
could be introduced in lieu of the synthetically-demanding 6-
13a
amanitin and supported by more recent work on the oxidative
uorocyclization of tryptophan to give uoropyrroloindoline

17
(
FPI) for Savige–Fontana tryptathionylation, we started with
commercially available 5-OH-tryptophan. Following Fmoc-
protection, oxidative uorocyclization with N-uoro-2,4,6-
trimethylpyridinium triate (FP-T300) gave a diastereomeric
0
OH-L-tryptophan. We also reasoned that the 5 -hydroxyl could
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0
Scheme 1 Retrosynthesis of 5 -OH-6 -deoxy-amanitin 2, showing major disconnection sites and key intermediates. DHIle-NHS ester 3 is
0
coupled to the monocyclic 5 -OH-heptapeptide 4 obtained from a 5-OH-tryptophan-derived FPI precursor 6a/b and solid-phase accessible
hexapeptide 5.
pair of uoropyrroloindolines (syn-cis/anti-cis). Unfortunately, deprotection decreased steric encumbrance to facilitate mac-
these decomposed upon isolation, presumably due to the rocyclization that successfully effected 11, the penultimate and
electron-rich indole ring (data not shown). To mitigate this rst appreciably toxic species that would await sulfoxidation to
0
0
problem, we employed a facile protection of the hydroxyl group give the target (R)-5 -OH-6 -deoxy-amanitin (Scheme 3).
as a tert-butyldimethylsilyl ether that greatly improved the With a robust synthetic approach to a new thioether analog
stability of the diastereomeric pair of FPI's 6a/b. Thus, TBS- with equal toxicity compared to S-deoxy-amanitin (vide infra),
0
0
protected 8 was treated with FP-T300 in DCM to yield 6a/b as the nal obstacle towards 5 -OH-6 -deoxy-amanitin was the
a diastereomeric mixture in 87% yield (Scheme 2, 6a/b syn-cis/ diastereoselective sulfoxidation of the tryptathionine bridge to
anti-cis, 1.8 : 1).
the (R)-congured sulfoxide. Of note, in the a-amanitin series,
With uorocyclization proceeding in high yield, we directly the (R)-sulfoxide is the most potent of the toxins, followed by the
coupled the diastereomeric mixture 6a/b to hexapeptide 5 that equipotent sulfone and thioether and then the unnatural (S)-
18
had been prepared on the solid-phase using standard Fmoc sulfoxide that shows 8-fold reduced potency. The relationship
solid-phase peptide synthesis conditions (Scheme 3). It is easily between the stereochemical conguration of the sulfoxide and
appreciated that the 1.8 : 1 diastereomeric distribution of the the biological activity has fascinated scientists for decades as
FPI precursor is immaterial since the stereochemistry is lost numerous efforts fail to provide a structural rationalization for
during the Savige–Fontana reaction. Fmoc-deprotection, fol- this intriguing difference in toxicity (vide infra,
5
b,18,19
lowed by TFA/DCM (5 : 1) treatment resulted in global depro- discussion).
tection, resin cleavage, and thioetherication (Savige–Fontana
reaction) to afford the desired 5 -OH-tryptathionine-containing unmet synthetic challenge in this context. Previously, it was
Achieving diastereoselective sulfoxidation still poses an
0
monocycle 4. The monocyclic heptapeptide 4 was coupled shown that oxidation of the thioether with H
2
O
2
in acetic acid
13a
with the fully protected NHS-ester of DHIle (3). Subsequent produces sulfoxides in a 1 : 2 ratio favoring the (S)-diaster-
5b,20
Fmoc-deprotection revealed
a
free amine, while TBS eomer.
In the 2018 report, m-chloroperbenzoic acid
(mCPBA)-mediated sulfoxidation successfully yielded the (R)-
sulfoxide in modest ꢀ2 : 1 excess over (S)-sulfoxide. Inexpli-
cably, selectivity could be tuned by varying the reaction solvent;
iPrOH/EtOH and tBuOH/EtOH co-mixtures were effective at
providing moderate selectivity for the desired (R)-sulfoxide
3 2
while polar solvents (e.g. DMF, HOAc, CF CH OH) greatly
13a,c
favored the (S)-sulfoxide.
In order to verify that 11 would undergo similar sulfox-
idation as S-deoxy-amanitin, we commenced our investigation
by subjecting it to mCPBA treatment in iPrOH/EtOH (2 : 1)
(Fig. 3). The HPLC and mass spectrometry analysis of the
reaction revealed full consumption of 11 aer one hour and
formation of three distinct products in a ratio of 4.4 : 4.6 : 1,
which were identied by mass spectrometry as two sulfoxides
a
Scheme 2 Synthesis of N -Fmoc-FPI(5-OTBS)-OH (6a/b) as a pair of
diastereomers; *NMR yield.
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Scheme 3 Synthesis of 5 -OH-tryptathionine and incorporation of DHIle to give bicyclic precursor 11.
and a sulfone (see Fig. S2 in ESI†). This transformation was using MTS colorimetric cell viability and RNA pol II transcrip-
accompanied by a predictable change in the UV-Vis absorbance tion assays (Fig. 3). To our delight, MTS assay on Chinese
signifying a modication of the chromophore. The rst of the Hamster Ovary (CHO) cells revealed that 2 and 11–13 showed
two sulfoxides eluting from the HPLC was tentatively assigned toxicities in keeping with the chromatographic elution prole
as the desired (R)-sulfoxide based on the shorter HPLC reten- observed for the (R)-, (S)-sulfoxides and the sulfone of a-
tion time observed for a-amanitin with respect to its (S)-sulf- amanitin (Fig. 3) and leading us to assign a tentative (R)-
oxide diastereomer. It is well-known that the (S)-sulfoxides in designation to the sulfoxide born on 2.
a series of amanitin analogs are signicantly less potent
Whereas 2 was nearly as cytotoxic as a-amanitin itself,
inhibitors of RNA pol II in vitro, compared to the corresponding strikingly, the putative (S)-sulfoxide 12 showed signicantly
5
c
(
R)-sulfoxides (although previous reports did not provide reduced cytotoxicity even compared to (S)-sulfoxide of a-ama-
13a,18,21
unambiguous structural elucidation and relied only on corre- nitin.
While many synthetic amanitin analogs reported in
lations with chromatographic retention times and toxicity to the recent studies have relied solely on IC values from cyto-
5
0
9,22
assign stereochemistry).
toxicity assays as a measure of activity, such analysis assumes
Applying the correlation between chromatographic retention equal cellular uptake. To obtain a direct measure of activity
times of these compounds and authentic a-amanitin to make against the intracellular target RNA pol II, we performed an in
a tentative stereochemical assignment, we then assessed the vitro transcription assay using HeLa-Scribe nuclear extract
3
2
biological activity of the new amanitin analogs 2 and 11–13 transcription system and [ P]-a-rGTP. Interestingly, the K
i
Fig. 3 MTS and RNA pol II assay results of oxidized products; *yield in sulfoxide; IC50: CHO cells, MTS, 72 h, n ¼ 3; K
i
: RNA pol II transcription
assay, n ¼ 3.
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values for compounds 2 and 11–13 were spread over a far
greater range compared to the IC50 values (Fig. 3). Thioether 11
and sulfone 13 showed similar affinity (79 ꢁ 10 and 100 ꢁ
2.2 nM, respectively), while (R)-sulfoxide 2 was approximately

ve-fold more active (18 ꢁ 1.5 nM) and the (S)-sulfoxide 12
showed greatly reduced inhibitory activity (710 ꢁ 150 nM)
compared to (R)-sulfoxide 2.
The near-native toxicity of 2 would suggest signicant
potential for use as an ADC payload and prompted us to develop
diastereoselective sulfoxidation methods in anticipation of
future translational needs. Hence, we turned our attention to
catalytic sulfoxidation as it represents one of the dominant
methods for enantioselective sulfoxidation. One of the earliest
and most convenient methods for asymmetric sulfoxidation
Fig. 4 HPLC chromatogram of the sulfoxidation reaction run in the
presence of Ti(OiPr) and L-DET showing formation of a-amanitin 1 as
4
the major diastereomer along with unreacted thioether 14.
nmol scale, Table 1, entry 5). To illustrate the utility of this
protocol in production of a-amanitin, we subjected S-deoxy-
amanitin 14 to these optimized conditions. Gratifyingly, the
employs titanium tetra-isopropoxide [Ti(OiPr)
4
] and (+)-diethyl-
L-tartrate (L-DET) as a ligand. The initial sulfoxidation
screening in the presence of Ti(OiPr) and L-DET was carried out
with two different oxidizing agents, cumene hydroperoxide
23
4
(R)-sulfoxide was obtained in 84% yield and with high diaster-
eoselectivity (Table 1, entry 6 and Fig. 4). The product identity
was conrmed by HPLC co-injection with an authentic sample
of a-amanitin, illustrating the rst case of a highly diaster-
eoselective access to a-amanitin (see Fig. S7, ESI†).
(
4
CHP) and urea hydroperoxide (UHP), in MeOH : DCM (1 : 1) at
C (Table 1, entries 1–3). Use of CHP as an oxidant yielded (S)-
ꢂ
sulfoxide in excess (Table 1, entry 1) while UHP gave the (R)-
sulfoxide in 2.6-fold excess when used in the presence of L-
While these results represent the rst-ever successful
approach to a highly diastereoselective installation of the (R)-
sulfoxide in any amanitin analog, we wondered if there could be
yet other means of accessing the desired (R)-sulfoxide without
resorting to chiral catalysis. Appreciating that the monocyclic
heptapeptide intermediate 4 likely adopts a different geometry
compared to the bicycle 11, we hypothesized that the sulfox-
idation of this intermediate may offer a unique opportunity for
inducing the desired (R)-conguration at an earlier stage in the
synthesis. We started our initial screening of the sulfoxidation
with mCPBA in iPrOH/EtOH (2 : 1); two products eluted from
the HPLC one minute apart. The rst product, designated as
compound 15, appearing to be a single diastereomer, was found
to be a sulfoxide by MS but of unknown stereochemistry, while
DET : Ti(OiPr)
DET : Ti(OiPr)
4
4
(Table 1, entry 2). In the absence of L-
, UHP gave the (S)-sulfoxide in 2.8-fold excess
4
(Table 1, entry 3). When L-DET : Ti(OiPr) were used in 4 : 1
ratio, the diastereoselectivity was improved to 95 : 5 (R : S) when
applied to 100-nmol (Table 1, entry 4) (lower selectivity was
observed on a 5-nmol scale). Use of excess L-DET ligand with
respect to Ti(OiPr)
ꢂ
4
(4 : 1 ratio) in 1 : 1 MeOH/DCM at 4 C over
2
7
0 hours (Table 1, entry 4), yielded the desired (R)-sulfoxide in
1% yield and without over-oxidation to the sulfone. Never-
theless, approximately 25% of thioether 11 remained uncon-
sumed aer 20 hours. Increasing the amount of UHP in the
presence of L-DET : Ti(OiPr) (8 : 1) and using DCM as the major
4
solvent improved both the yield and the diastereoselectivity (5-
a
Table 1 Oxidation of thioether 11 and 14 to (R)- and (S)-sulfoxides in the presence of Ti(OiPr)
4
and L-DET
11
Entry
Conditions
Substrate
(R : S)
or 12 (%)
Yield (%)
b
1
2
3
4
5
6
CHP/Ti(OiPr)
UHP/Ti(OiPr)
4
/L-DET (1 : 1 : 2), DCM/MeOH (1 : 1), 17 h
/L-DET (1 : 1 : 2), DCM/MeOH (1 : 1), 17 h
UHP, DCM/MeOH (1 : 1), 17 h
11
11
11
11
11
14
1 : 1.1
2.6 : 1
1 : 2.8
19 : 1
6.4 : 1
>50 : 1
39
44
20
25
4
61
b
4
66
b
80
71
c
UHP/Ti(OiPr)
UHP/Ti(OiPr)
UHP/Ti(OiPr)
4
/L-DET (1.2 : 0.8 : 3.2), DCM/MeOH (1 : 1), 20 h
b
4
/L-DET (2.5 : 1 : 8), DCM/MeOH (3 : 1), 17 h
96
b
4
/L-DET (2.5 : 1 : 8), DCM/MeOH (3 : 1), 17 h
16
84
a
b
c
(
R : S) distribution was determined by HPLC. HPLC sulfoxide yield. Isolated yield.
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the second proved to be an inseparable mixture of the second
sulfoxide and sulfone 16 (Fig. 5). To reduce the amount of
sulfoxide/sulfone by-products, solvent and temperature
screening was performed (Table S4,† entries 1–16). The lowest
amount of the by-products was obtained with dimethylforma-
mide (DMF) as the solvent (Table S4,† entry 11). The sulfox-
idation was further optimized by varying the reaction
temperature (Table S4,† entries 14–16). Temperatures below
0 0
4 Synthesis of the (R)-5 -OH-6 -deoxy-amanitin 2 to
Scheme
ꢂ
0
C were found to be optimal for suppressing the production of confirm the stereochemistry of the heptapeptide sulfoxidation,
ꢂ
incorporation of dihydroxyisoleucine and macrolactamization (A).
sulfoxide/sulfone. Running the reaction in DMF at ꢃ20 C on
a 1-mmol scale yielded the rst sulfoxide in 5-fold excess over the
second sulfoxide and inseparable sulfone (Fig. 5). This protocol
resulted in complete consumption of the thioether 4 in less
than 5 minutes.
0
0
HPLC chromatogram of the sulfoxide containing 5 -OH-6 -deoxy-
0
amanitin obtained through sulfoxide 15 and co-injection with (R)-5 -
0
OH-6 -deoxy-amanitin 2 produced via a reported mCPBA oxidation
route confirming (R)-sulfoxide formation during heptapeptide sul-
foxidation (B).
To conrm the stereochemical conguration of the mono-
0
0
cyclic sulfoxide, 15 was converted to the nal bicyclic 5 -OH-6 -
deoxy-amanitin 2 by the following approach (Scheme 4): the to the building block employed in the “5 + 1 + 2” strategy
heptapeptide sulfoxide 15 was coupled to a suitably protected developed by S u¨ ssmuth and coworkers. Somewhat surprisingly,
dihydroxyisoleucine to give the octapeptide, which was then this led to formation of two sulfoxides in 1 : 2 ratio. Similarly,
0
0
Fmoc- and TBS-deprotected and nally macrolactamized to when the monocyclic octapeptide (seco-5 -OH-6 -deoxy-
produce the bicyclic amanitin analog 2 (Scheme 4). The nal octapeptide S3) was subjected to mCPBA in DMF, two sulfox-
product was analysed by HPLC, including co-injection with an ides were formed with little diastereoselectivity (1 : 1.7) sug-
0
0
“
authentic sample” of (R)-5 -OH-6 -deoxy-amanitin (2) (Fig. 2), gesting that neither the pentapeptide nor the octapeptide
that was obtained through an mCPBA oxidation of the bicyclic adopts a conformation that is preordered to confer highly dia-
precursor 11 following a previously reported procedure for a- stereoselective attack by the oxidant.
13a
amanitin synthesis (Scheme 4A).
In an attempt to rationalize the outstanding selectivity in
Based on the HPLC co-injection data, we therefore conrmed oxidizing the heptapeptide to obtain the (R)-sulfoxide, we
that the sulfoxide 15 was indeed of the (R)-conguration examined the differences in geometries between the bicyclic
(Scheme 4B). This method thus represents a highly diaster- octapeptide 11 and monocyclic heptapeptide 4. Based on the
eoselective, mild, facile, and cost-effective approach for intro- geometry-optimized structure of the heptapeptide monocycle 4,
duction of (R)-sulfoxide at an earlier stage in the synthesis and, we hypothesize that the extensive H-bonding interactions
0
importantly, before the incorporation of the costly DHIle or observed between Asn-1, Hyp-2, 5 -OH-Trp-4 (Fig. 6A) and the
other analogs that may be of interest.
backbone may cause an increase in steric bulk on the si-face of
To examine whether this approach is generally applicable to the indole, disfavoring oxidant attack on the pro-S lone pair on
other monocyclic precursors, we attempted this mCPBA sul- the sulfur atom while leaving the pro-R lone pair more solvent-
0
foxidation on the monocyclic 5 -OH-pentapeptide S4, analogous accessible.
This can be observed by measuring distances between sulfur
and the neighboring atoms in the heptapeptide monocycle 4
and comparing these to bicycle 11 (Fig. 6B). Based on this
analysis, ve out of six neighboring atoms are in closer prox-
imity to sulfur in the heptapeptide. The Asn-1 side chain is
noteworthy since it shows the largest degree of displacement. In
contrast, the conformation of amanitin 11 shows that both lone
pairs on the sulfur atom share a similarly congested environ-
ment thus explaining the generally poor diastereoselectivity.
Finally, to complete this study, we evaluated certain key
products by circular dichroism (CD) to prove for key structural
elements such as a-helices, b-sheets, and b-turns that are
consistently found in non-random portions of proteins. Ama-
toxins are characterized by a type-II b-turn in ring II (formed by
Trp4–Gly5–Ile6–Gly7–Cys8), which is recognized to be crucial
for binding, and numerous natural toxins in this class as well as
synthetic derivatives have been characterized by CD. Notably,
Fig. 5 Oxidation of heptapeptide 4 to (R)-sulfoxide and (S)-sulfoxide/
(R)-sulfoxide 2 strongly matched the conformation of a-
ꢂ
sulfone in 5 : 1 ratio with mCPBA as an oxidant in DMF at ꢃ20 C; HPLC
amanitin; however, the nearly-equipotent thioether 11 gave
chromatogram of mCPBA oxidation; in reporting this, we show 15 to
have undefined stereochemistry at sulphur, which was determined a spectrum that differed signicantly from both 2 and a-
subsequently.
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added recognition, and (iii) preserve a conjugation handle on
the indole while bypassing the need to install the synthetically
0
challenging 6 -OH-tryptathionine staple. To direct our efforts,
we rst examined the interactions between the indole core and
the enzyme in the crystal structure along with the biological
activity scope of the known indole-modied analogs that have
been prepared from the natural product.
A review of the vast literature on derivatives of naturally
sourced amatoxins suggests that the indole core is relatively
tolerant to modications. Small modications generally result
in negligible reductions in RNA pol II affinity (Fig. 1). For
0
example, 6 -ether derivatives have been synthesized successfully
24
in the past by treating a-amanitin with alkyl halides. Similarly,
a two-fold reduction in affinity was observed when a-amanitin
0
was methylated or ethylated at position-6 to give 6 -OR-a-
amanitin (R ¼ Me, Et); methyl and ethyl ethers were two-fold
less active in an RNA pol II inhibition assay. However, longer
chain lipophilic derivatives exhibited a much-reduced affinity
towards RNA pol II even though their cell toxicity was increased
24
0
likely due to improved cell penetration capacity. Other 7 -
modied derivatives of a-amanitin have been synthesized, and
Fig. 6 Modelling of bicyclic precursor 11 (in green, left) and mono- this position was generally shown to be quite tolerant to
cyclic heptapeptide precursor 4 (in blue, right) shows greater exposure modications. Amine and arylazo substitutions were intro-
of the pro-R lone pair compared to the pro-S lone pair; H-bonding
interactions highlighted in yellow (A); distances from neighboring
atoms to S-atom (B).
0
25
duced at the 7 -position of tryptophan on a-amanitin. In 1982,
Morris et al. evaluated a series of six secondary-amine modi-
0
0
cations at the 7 -position and seven 7 -arylazo-amatoxins for
affinity towards RNA pol II and III. All of the tested analogs were
active towards RNA pol II, with K values up to 8 times that of a-
i
26
amanitin. These reports further underscored the plasticity
that the indole core offers and encouraged us to develop a new
tryptophan-modied analog while addressing the remaining
challenge of diastereoselective sulfoxidation.
As part of our characterization, we provide CD spectra to
demonstrate what appears to be conformational differences
between the thioether, (R)- and (S)-sulfoxides along with the
sulfone. Since the sulfoxides show dramatic differences in the
CD spectra, it suggests that they adopt signicantly different
conformations. Previously, however, full structural elucidation
by NMR spectroscopy unexpectedly showed that the (R)- and (S)-
Fig. 7 CD spectra of amanitins 2, 11–13 (A) and heptapeptides 4, 15, 16
(
B).
18
sulfoxides had nearly overlapping structures, and while in the
amanitin in the 210–240 nm range (Fig. 7). The (S)-sulfoxide 12
and sulfone 13 both differed signicantly from 2 and a-
amanitin at nearly all wavelengths, and while both showed
more similar CD-spectra, these showed vastly different cyto-
crystalline state, structures of the (S)-sulfoxide and sulfone were
19a
nearly identical. To date, molecular dynamics calculations
have provided little additional insight into an enduring mystery
as to why the (S)-sulfoxide is uniquely less active than the
toxicities and K values in vitro. A similar trend is observed for
i
27
sulfone, the (R)-sulfoxide and the thioether. Hence, in terms of
the heptapeptides. The implications of these nding are dis-
cussed below.
this work, a structural rationalization for the differences in CD
spectra or cytotoxicity/K values, which correlate to those seen
i
with other amanitins, is not readily intuited.
While the advantages of using an (R)-sulfoxide over either
the thioether or sulfone as an ADC payload remain to be
Discussion
Herein we describe the synthesis of a new amanitin analog that assessed in preclinical animal models and human clinical
is a constitutional isomer of the natural product. In light of the studies, it is reasonable to hypothesize that Nature may have
0
importance of the 6 -OH for both enhancing toxicity and chosen to install the (R)-sulfoxide deliberately to enhance
providing a conjugation handle, and given the synthetic chal- systemic toxicity. Here, we have identied a means of achieving
lenges in accessing the 6-OH-tryptophan or its equivalents, we highly diastereoselective sulfoxidation on the precursor hepta-
aimed to design an analog that would: (i) impart greater elec- peptide, which now enables production of a congurationally
tron density into the indole system, (ii) provide an H-bond for stable precursor that awaits conjugation with DHIle, the most
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synthetically challenging monomer used in the synthesis. In
addition, the use of a chiral catalyst to introduce the (R)-sulf-
Acknowledgements
oxide late-stage now provides access to late-stage diaster- The authors acknowledge nancial support from the Canadian
eoselective sulfoxidation on bicyclic octapeptides.
Institutes for Health Research #220656 and the Canadian
The use of chiral sulfoxidation catalysts on S-deoxy-amanitin Cancer Society Research Initiative Grant #703374 and thank Dr
recapitulated similar diastereoselectivity that we report here for Maria Ezhova for help with NMR acquisition, Dr Elena Polish-
0
0
the 5 -hydrox-6 -deoxy analog demonstrating utility for resolving chuk and Ms Jessie Chen for help with cytotoxicity assays.
this question in the context of providing the natural product as
well. Although we did not evaluate this on the heptapeptide
precursor of amanitin, we believe that this method should
Notes and references
provide similar results and thus likely augurs for success in
accessing the natural product in high diastereomeric excess
without resoting to chiral catalysts. Finally, we posit that one
might use this approach to accomplish sulfoxidation on
amanitin analogs that are produced from circularly-permuted
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5
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8
9
11a,e
The (R)-sulfoxide 2 is signicantly more toxic than the (S)-
sulfoxide 12 in the context of this new analog. To access this
diastereomer, we developed a late-stage sulfoxidation approach
IV
utilizing Ti /L-DET-based catalytic system that is conveniently
2015, 16, 1420–1425.
applied to the bicyclic toxin as the ultimate synthetic step. We
suggest that this methodology has a high potential of being
translatable to a broad range of bicyclic amanitin analogs due to
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addition, a mild, facile, and cost-effective early stage sulfox-
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diastereoselectivity over the previously reported mCPBA-
assisted sulfoxidation. Finally, we believe this highly selective
method for (R)-sulfoxide formation may be broadly applicable
to use on other monocyclic amanitin precursors and presents
a signicant advancement for future large scale amanitin
syntheses.
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