Scaling Amatoxin Synthesis with an Improved Route to (2 S,3 R,4 R)-Dihydroxyisoleucine Exemplified by a Toxic, Clickable α-Amanitin Analogue

Article abstract of DOI:10.1021/acs.joc.0c03022

Here we report a scalable synthesis of the key amino acid residue, (2S,3R,4R)-4,5-dihydroxyisoleucine (DHIle) in α-amanitin, that in turn enables the scalable synthesis of an equipotent analogue, Asn(N-ethylazide)-S,6′-dideoxy-α-amanitin, suitable for CuAAC conjugation to empower studies on therapeutic antibody-drug conjugates.

Full text of DOI:10.1021/acs.joc.0c03022

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Note
Scaling Amatoxin Synthesis with an Improved Route to
(2S,3R,4R)‑Dihydroxyisoleucine Exemplified by a Toxic, Clickable
α‑Amanitin Analogue
Chido M. Hambira,# Kaveh Matinkhoo,# Alla Pryyma, Brian O. Patrick, and David M. Perrin*
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ABSTRACT: Here we report a scalable synthesis of the key amino
acid residue, (2S,3R,4R)-4,5-dihydroxyisoleucine (DHIle) in α-
amanitin, that in turn enables the scalable synthesis of an
equipotent analogue, Asn(N‑ethylazide)-S,6′-dideoxy-α-amanitin, suit-
able for CuAAC conjugation to empower studies on therapeutic
antibody-drug conjugates.
lpha-amanitin (Figure 1), isolated over 80 years ago, is
the deadliest member of the amatoxin family of peptides
applications,18 chemical synthesis enables access to new
derivatives and provides a roadmap to scalability. Defying
synthetic access for nearly seven decades, α-amanitin contains
several synthetically challenging components, notably,
(2S,3R,4R)-4,5-dihydroxyisoleucine (DHIle), and a 6-hy-
droxy-tryptathionine-(R)-sulfoxide staple, unique among all
natural products. Whereas the tryptathionine staple, which is
critical for activity, is easily accessed by a variety of
methods,19−23 neither the (R)-sulfoxide nor the synthetically
challenging 6′-hydroxy group contributes significantly to
toxicity and thus may be dispensable when entertaining
analogs for clinical applications.24−26 By contrast, DHIle is
required for toxicity as epimers thereof are considerably less
toxic.27 As such, this deceptively simple amino acid presents
synthetic challenges that have gravely limited scalability and
defied synthetic access for decades.
In 1966, DHIle was synthesized without regard for
stereoselectivity28 while later reports addressed diastereose-
lective syntheses that posed difficulty in chiral resolution.27,29
It has only been since 2018 that three total syntheses finally
addressed enantioselective access to DHIle by a variety of
classical approaches.30−32 Key steps in Perrin et al.30 involved:
(i) Brown crotylation to introduce the stereogenic carbons at
C-3 and C-4;33 (ii) Upjohn dihydroxylation reaction that
generates undesired osmium waste in addition to its high
A
Figure 1. Structure of α-amanitin and key synthetic challenges.
produced by the death-cap mushroom A. phalloides. Benefiting
from an extremely rich history,1−3 α-amanitin is an orally
available, rigid, bicyclic octapeptide and one of the most lethal
natural products known to humankind (LD50 = 50−100 μg/
kg).4,5 In contrast to numerous antimitotics that inhibit tubulin
depolymerization in growing cells, α-amanitin is a highly
selective allosteric inhibitor of RNA polymerase II (Pol II)
with a Ki of 1−10 nM6−12 that is thus capable of killing both
quiescent and growing cells. Owing to desirable characteristics
of stability, potency, and a unique mechanism of action, α-
amanitin has attracted considerable attention as a drug payload
in the development of antibody-drug conjugates (ADCs) for
use in cancer treatment.13−17 Indeed, α-amanitin, with its
fascinating pharmacology, holds extraordinary promise for
targeted therapeutics.
Received: December 24, 2020
Published: March 12, 2021
To date, most amanitin conjugates have been synthesized
using naturally sourced toxins. While fungal culture might
provide sufficient quantities of this toxin for various
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cost,34 and (iii) an asymmetric mismatched Strecker reaction
using KCN to furnish the amino nitrile intermediate with very
poor diastereoselectivity (1.1:1) (Figure 2A). Subsequent
a
Scheme 1. Synthesis of 7
a
Conditions: (a) ethylene glycol (10.6 equiv), TBDPSCl (1 equiv),
imidazole (0.95 equiv), THF, RT, 24 h, 75%; (b) oxalyl chloride (1.5
equiv), DMSO (3.1 equiv), Et3N (4.4 equiv), DCM, −78 °C (3 h) to
RT (1 h), 85%; (c) L-proline (0.2 equiv), propanal (5 equiv), 1,4-
dioxane, 4 °C, 48 h, d.r. 4:1; (d) TBDMSOTf (1.05 equiv), 2,6-
lutidine (1.25 equiv), DCM, −78 °C, 52% over two steps, one
diastereomer (ca. 99:1 anti:syn).
decomposition, and notably, previous reports do not report
the separation of the diastereomers.39−43 As in our hands, β-
hydroxyacetaldehyde 6 proved too fragile for isolation as single
diastereomer, we performed silylation immediately upon
extraction. To our delight, this afforded aldehyde 7 as a single
diastereomer (the other diastereomer was not detected) in
albeit slightly lower isolated yields (52%) compared to
previous reports. We surmise that silylation “locked in” the
desired diastereomer, presumably via an in-situ kinetic
resolution that exclusively favored the anti orientation due to
increased steric crowding of the silylether in the syn
conformation.
Since the next step involved a mismatched Strecker reaction
(according to Felkin−Ahn’s model), it is important to note
that, even with (S)-α-phenylethylamine ((S)-α-PEA) as a
chiral auxiliary, it is challenging to obtain good diastereose-
lectivity for the all-anti configuration. Several standard
conditions were investigated, none of which led to product
formation or improved the efficiency of the reaction (Table 1,
entries 1−4). We obtained our best results by using excess
titanium(IV) alkoxide (ethoxide or isoproproxide) as an
additive, which enabled reaction completion in as little as 4
h (entries 6−7; see Supporting Information (SI) for details).
Figure 2. (A) Previous retrosynthetic analysis (top). (B) Current
route to DHIle (bottom).
reports by Sussmuth et al.31 and Muller et al.32 employed a
common strategy that ultimately relied on asymmetric
dihydroxylation35 of dehydro-Ile that was achieved in 2−
2.5:1 diastereoselectivity. For synthesizing dehydro-Ile, dia-
stereoselectivities were higher. Muller et al. obtained the
dehydro-Ile through a β-methylation of an extensively
protected (Nα-benzyl-Nα-phenylfluorenyl-CαOtBu-Cγ-OMe)
aspartate in 5:1 diastereoselectivity (see Wolf and Rapoport),36
followed by over-reduction and reoxidation to the semi-
aldehyde and then Wittig olefination; Sussmuth et al. arrived at
dehydro-Ile via a sophisticated reaction sequence that included
an enantioselective ruthenium-catalyzed alkylation (6:1 d.r.) of
a glycine Zn-enolate with a chiral allylcarbonate (see Bayer and
Kazmaier)34,37 procured from Zn(0)-mediated reductive ring
opening of an iodo-epoxide obtained by Sharpless asymmetric
epoxidation.38
Table 1. Strecker Reaction Optimization
Despite the use of enantioselective methods, these reports
highlight the inextricable challenges associated with stereo-
selective synthesis of DHIle. Given the importance of amanitin
to cancer biology, a scalable and efficient route to DHIle is
needed to empower applications in amanitin-targeted ther-
apeutics. Herein, we report a concise synthesis of DHIle
summarized in Figure 2B that we exploit to generate an
equipotent synthetic amatoxin with an azide handle that
permits bio-orthogonal conjugation. We suggest that this
report represents an essential advance toward chemical
translation of this venerated toxin and analogues thereof.
Starting with the cheap and abundant ethylene glycol, mono-
silylation, followed by a Swern oxidation, furnished the desired
α-silyloxy-aldehyde 5. Aldehyde 5 was then subjected to an L-
proline-catalyzed cross-aldol reaction with propanal (Scheme
1).
This aldol reaction is reported to afford a modest d.r. (ca.
5:1), yet the product is prone to dehydration and
a
(S)-α-PEA·HCl. All other reactions used (S)-α-PEA as a free base.
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Using the optimized conditions, (S)-α-phenylethylamine
was condensed with aldehyde 7 in the presence of excess
Ti(OEt)4 to give imine 10 in 1.5 h. TMSCN was then added
to furnish the desired aminonitrile 8 as a separable mixture of
two diastereomers in good yield and improved diastereomeric
ratio of 2:1 (Scheme 2).
resin, followed by coupling, to obtain a linear heptapeptide 15
which was then cyclized via the Savige−Fontana reaction that
provided monocyclic heptapeptide 16 concomitantly with
resin cleavage (Scheme 3). Following isolation, heptapeptide
16 was coupled to 12 to yield a monocyclic octapeptide in
yields of ∼75%.
Finally, macrocyclization formed amanitin 17, which was
found to be nearly as potent as α-amanitin (EC50 of 0.36 μM
compared to 0.2−0.5 μM for α-amanitin against CHO cells).
Although up to 0.5 g of the monocyclic octapeptide was
successfully scaled, out of an abunance of caution for safety,
only milligram quantities of the final toxin were synthesized per
batch. By contrast, this immediate seco-precursor to α-amanitin
was found to be nontoxic up to 100 μM (see SI) and was
therefore stockpiled for small-batch conversion to the toxic
end-product.
a
Scheme 2. Synthesis of Lactone 11
In summary, we disclose a route to DHIle 12 that obviates
late-transition metals or pyrophoric reagents with a reduction
in total steps over our first report. Key to this approach is a
simple L-proline-catalyzed cross aldol reaction between an α-
hydroxy-acetaldehyde and propanal to set the configurations of
the β- and γ-positions, the diastereoselectivity of which is
greatly enhanced by silylation to provide aldehyde 7 as a single
diastereomer. Prior synthesis of aldehyde 6, reportedly
obtained in >99% e.e., afforded a mixture of diastereomers at
∼5:1 that were not reportedly separated;40 in our own hands,
attempts to purify 6 from its syn stereoisomer led to
dehydration, decomposition, and polymerization. Hence,
silylation nicely afforded cpd 7 in extremely high purity for
use in the Strecker reaction.
Despite the challenges of a mismatched Strecker reaction,
the use of a readily available chiral auxiliary, (S)-α-phenyl-
ethylamine, along with Ti(OEt)4 ultimately affords gram
quantities of DHIle in respectable yield and higher
diastereoselectivity over our first report. While approaches to
catalytic asymmetric Strecker reactions are worthy of future
consideration, in the context of a mismatched Strecker that
normally favors the opposite diastereomer, this represents a
significant improvement in terms of yield, diastereoselectivity,
and reaction efficiency. We attribute enhanced efficiency to the
dehydrating activity and Lewis acidity afforded by Ti(OEt)4 for
activating both the aldehyde and imine. Yet explanations for
enhanced diastereoselectivity are tenuous: whereas a 1,3-Ti4+-
chelate would be an attractive explanation for the enhanced
a
Conditions: (a) (i) (S)-α-PEA (1.05 equiv), Ti(OEt)4 (4 equiv),
DCM, RT, 2 h; (b) TMSCN (2 equiv), 70%; (c) 6 M HCl, reflux, 3
h, 56%. For reaction conditions to convert lactone 11 to the fully
protected DHIle 12, see ref 30. Ellipsoid contour percent probability
level is 50%.
Standard workup followed formation of a titanium-edte
(N,N,N′,N′-tetrakis(2-hydroxyethyl)ethylenediamine) com-
plex,39 which was readily removed by washing with aqueous
NH4OH. Hydrolysis of the aminonitrile 8 in 6 M aq. HCl at
reflux yielded the lactone 11 (Scheme 2). The remaining steps
to convert 11 to the final solid-phase peptide synthesis-(SPPS)
compatible DHIle 12 followed our previously reported DHIle
synthesis and provided 1.6 g of the final material (a crystal was
obtained and submitted for diffraction).
With 12 in hand, we turned our attention to incorporating it
into the fully elaborated dideoxy-amanitin while adding an
azide conjugation handle for eventual use in ADC applications.
To this end, N-2-azidoethyl asparagine 13 was prepared by
coupling 2-azidoethylamine to aspartic acid, while fluoropyrro-
loindoline 14 was obtained quantitatively as a pair of equally
reactive diastereomers (syn-cis/anti-cis) through oxidative
fluorocyclization of tryptophan (Scheme 3).23 Fmoc-Hyp-
(tBu)-OH was loaded onto a 2-chlorotrityl chloride (CTC)
a
Scheme 3. Solid-Phase Synthesis of Asn(N‑ethylazide)-S,6′-dideoxy-α-amanitin, 17
a
Our previously reported SPPS method was used to synthesize 17. See the SI for synthesis of 13 and 14. Conditions: (a) TFA:CH2Cl2 (1:1, 6 mL),
RT, 1 h; (b) DHIle 12 (2.5 equiv), iPr2EtN (to pH ∼ 8.5), DMF, RT, 48 h; then Et2NH, 2 h; then Bu4NF, 1 h, 50% over 3 steps; (c) HATU (7
equiv), Pr2EtN (to pH 9), DMA, RT, 2 h.
i
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anti-Felkin diastereoselectivity,44 silylethers are weakly Lewis
basic,45,46 and prior reports on Ti4+-mediated enolate addition
reactions to 3-substituted aldehydes might challenge this
assertion.47
EDTA), 0.85% Trypan blue, and the antibiotic mixture Pen/Strep
(10K U/mL penicillin, 10K mg/mL streptomycin) were also
purchased from Gibco. All cell culture plastic ware was obtained
from Corning or Falcon. Cells were cultured at 37 °C in a humidified
chamber with 5% CO2. When used in cell culture, DMSO was
purified by filtration through a 0.2 mm filter. All experiments are
carried out in a laminar flow culture cabinet, unless otherwise noted.
Absorbance measurements of the 96-well plates were obtained using a
Beckman-Coulter DTX 880 multimode detector, equipped with an
excitation filter of 595 nm. Immortalized CHO cells had been stored
in liquid nitrogen. To revive cells, a 1 mL tube of the frozen cells in a
medium containing 10% DMSO was warmed in a 30 °C water bath
and diluted with 9 mL of fresh media. Media contained 10% FBS and
100 U/mL penicillin and 100 mg/mL streptomycin, unless otherwise
indicated. The cells were incubated in a T-25 flask at 37 °C at 5%
CO2. After 24 h, the medium was aspirated and replaced with fresh
medium. When cells reached a level of 90−100% confluence, they
were sub-cultured. The medium was removed, and the cells were
treated with 0.25% trypsin containing 1.3 mM EDTA in the
incubator. Once the cells were detached from the tissue culture
flask, 3−5 mL of media was added to quench the trypsin and
transferred to a 10 mL centrifuge tube. The mixture was centrifuged
for 5 min at 8000 rpm, and the supernatant was discarded. The cells
were suspended in fresh medium, diluted as required, and transferred
to a new culture flask.
To assay cell viability, a nearly confluent tissue culture flask was
trypsinized, and the cells were counted following treatment with
Trypan blue, using a hemocytometer. The cells were then diluted to
the appropriate stock concentrations in fresh medium and transferred
in 100 μL to a 96-well plate using a multi-channel pipet. The number
of cells plated varied from experiment to experiment. These were
incubated at 37 °C and 5% CO2 for a 24 h period to allow for
adherence. The medium was aspirated, and fresh medium was added,
which contained the desired additives in DMSO. The cells were then
re-incubated for 72 h. At the completion of the experiment, a 100 μL
aliquot of 25 mg/mL MTT in PBS was added to each well. The plate
was incubated 3 h further to allow for the formation of the formazan
product in viable cells. The medium was carefully aspirated, and the
purple product was solubilized in DMSO. The absorbance of each
well was recorded at 595 nm. Data were processed in Microsoft Excel
and GraphPad Prism. Experiments were performed in triplicates
unless otherwise noted, and the error bars were calculated as the
standard error of the mean.
In conclusion, while, for safety reasons, we did not produce
gram quantities, we suggest that this method, particularly in
regard to the production of DHIle, can empower scalable and
practical syntheses of cytotoxic amanitins, the translation of
which into a viable antibody-drug conjugate is currently under
way. Although we chose the azide owing to its use in “click”
reactions, we anticipate that many other handles and linkers for
bioconjugation can be readily appended at this position, while
access to various hydroxylated tryptathionines as well as
defined sulfoxide diastereomers, is also readily anticipated.48
This report now sets the stage for accessing numerous
amanitin analogues and eventually enabling their synthesis
on scale.
EXPERIMENTAL SECTION
■
General Methods. All reactions were performed under an argon
atmosphere in flame-dried glassware and dried solvents at room
temperature, unless otherwise stated. Anhydrous solvents were
prepared by distillation under a nitrogen atmosphere or drying over
3 or 4 Å molecular sieves for at least 48 h. Ethers were distilled from
sodium in the presence of benzophenone as indicator. Triethylamine,
dichloromethane, and hexanes were distilled over calcium hydride.
Methanol was distilled from magnesium. DMSO and DMF were dried
over 4 Å molecular sieves under an argon atmosphere. All reagents
and solvents were purchased from Sigma-Aldrich, Alfa Aesar, Acros
Organics, Strem Chemicals, Matrix Scientifics, AK Scientific, Oak-
wood Chemicals, or TCI America, unless otherwise stated. Authentic
α-amanitin was purchased from Sigma-Aldrich. Proton (1H NMR)
and carbon (13C NMR), spectra were obtained using Bruker AV-300
(300 MHz), AV-400inv (400 MHz), AV600-CRP (600 MHz), and
Bruker Avance III (850 MHz equipped with xyz-gradient TCI
cryoprobe) spectrometers. Chemical shifts were standardized against
the deuterated solvent peak, and the acquired data were analyzed
using Topspin and MestReNova software. Optical rotation data were
acquired using a JASCO polarimeter in CH2Cl2 or MeOH. IR
absorption frequencies were measured using a PerkinElmer Frontier
FT-IR spectrometer. All analytical HPLC chromatograms were
generated on an Agilent 1100 system equipped with an auto injector,
a fraction collector, and a diode array detector. Final peptides were
purified by HPLC chromatography with a solvent gradient of 0.1%
formic acid in acetonitrile (Solvent A) and 0.1% formic acid in water
(Solvent B). Analytical HPLC purifications were performed using an
Agilent Eclipse XDB C-18 (4.6 × 250 mm) column with a flow rate of
2 mL/min. All preparatory HPLC chromatograms were generated on
an Agilent 260 Infinity II system equipped with an auto injector, a
fraction collector, and a diode array detector. An Agilent 5 Prep C-18
column (50 × 21.2 mm, L × ID) fitted with a column guard was
employed. Low-resolution mass spectra (LRMS) using electrospray
ionization (ESI) mode were obtained using a Waters ZQ mass
spectrometer equipped with an ESCI ion source and a Waters 2695
HPLC. High-resolution mass spectra (HRMS) in ESI mode were
obtained using a Micromass LCT-TOF mass spectrometer equipped
with an ESI ion source. A JASCO spectrometer was employed to
acquire the CD spectrum in methanol and 10% formate buffer at ∼20
°C. Quantification of peptides was done via UV−vis spectroscopy
using an extinction coefficient of 10 000 M−1 cm−1 at the wavelength
with the maximum absorbance (290−305 nm) for synthetic peptides,
except for α-amanitin that has an extinction coefficient of 13 500 M−1
cm−1. Sample preparation for cell toxicity assays: Following
quantification, solutions with toxic peptides were re-lyophilized and
re-suspended in a given volume of DMSO or H2O to provide a 1 mM
solution, which was then used in cell toxicity assays. Cell culture: Cells
were cultured in a-MEM or high-sucrose DMEM, purchased from
Gibco. Fetal bovine serum (FBS), 0.25% trypsin (with 1.3 mM
Trypsinized cells were diluted to a concentration of 3.3 × 104 cells/
mL for CHO cells. Each cell line was plated in a 96-well plate, with
100 μL of the stock solution per well (5000 cells per well) and
incubated for 24 h. Stocks of α-amanitin or analogues were prepared
at various concentrations, containing a maximum of 0.8% DMSO, and
added to various wells, according to the desired final concentration.
The cells were incubated for 72 h, at which point viability was
assessed as described.
2-((tert-Butyldiphenylsilyl)oxy)ethan-1-ol (S1). To ethylene
glycol (215.6 mL, 3.86 mol) in dry THF (1.9 L) was added imidazole
(23.5 g, 345.6 mmol). Then TBDPSCl (100 g, 363.8 mmol) was
added to the mixture under Argon. The resulting mixture was stirred
for 24 h at RT (19−21 °C), quenched with H2O (500 mL), and
extracted with Et2O (3 × 500 mL). The combined organic layers were
dried over MgSO4, concentrated, and purified by flash column
chromatography using silica gel (EtOAc/hex 1:4) to afford S1 as a
colorless oil/white solid (81.9 g, 75%). Note: alternatively, the crude
product of this reaction can be used in the next step without further
1
purification. TLC (EtOAc:hex 10:90 v/v): Rf = 0.18; H NMR (400
MHz, Methylene Chloride-d2) δ 7.72−7.64 (m, 4H), 7.49−7.34 (m,
6H), 3.76 (t, J = 4.8 Hz, 2H), 3.69−3.62 (m, 2H), 2.06−1.99 (m,
1H), 1.06 (s, 9H); 13C{1H} NMR (75 MHz, Methylene Chloride-d2)
δ 136.1, 134.0, 130.3, 128.3, 65.8, 64.2, 27.2, 19.6; HRMS (ESI-TOF,
m/z) calculated for C18H24NaO2Si [M + Na]+, 323.1443; found
323.1441.
2-((tert-Butyldiphenylsilyl)oxy)acetaldehyde (5). To a sol-
ution of (COCl)2 (8.56 mL, 99.8 mmol) in dry DCM (70 mL) at
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−78 °C was added dropwise a solution of DMSO (14.7 mL, 206.3
mmol) in DCM (70 mL). The reaction mixture was stirred for 30 min
at −78 °C, and a solution of S1 (20 g, 66.6 mmol) in DCM (140 mL)
was added slowly. The resulting mixture was stirred for 30 min at −78
°C. After a solution of Et3N (40.8 mL, 293 mmol) in DCM (140 mL)
was added to the mixture at −78 °C, the reaction was warmed up to
RT and stirring was continued for 1 h. The contents of the reaction
were partitioned between H2O (300 mL) and DCM, and the aqueous
layer was extracted with DCM (3 × 150 mL). The combined organic
layers were washed with sat’d aq. KH2PO4 (2 × 200 mL) and brine,
dried over Na2SO4, and concentrated to yield the crude aldehyde,
which was purified by flash column chromatography using silica gel
(EA/hex 1:99 to 5:95, gradient) to afford 5 as a colorless oil (16.9 g,
85%). TLC (EtOAc:hex 10:90 v/v): Rf = 0.64 (visualized with DNP);
1H NMR (300 MHz, Methylene Chloride-d2) δ 9.69 (s, 1H), 7.74−
7.61 (m, 4H), 7.50−7.34 (m, 6H), 4.24 (s, 2H), 1.11−1.07 (m, 9H);
13C{1H} NMR (75 MHz, Methylene Chloride-d2) δ 201.7, 136.1,
was added and the reaction mixture was heated at 55 °C for 15 min
using an oil bath. After cooling to room temperature, 25% NH4OH
(100 mL) was added and the mixture was transferred to a separatory
funnel for a standard extractive workup. The aqueous layer was
extracted with DCM (3 × 75 mL). The combined organic layers were
washed with brine, dried over anhydrous Na2SO4, filtered, and
concentrated. The crude residue was purified by flash column
chromatography using silica gel (EA/hex/Et3N, 1:99:0.1) to afford 8
as a clear oil and a single diastereomer (d.r. 2:1 in favor of the desired
diastereomer) (7.4 g, 70%). TLC (EtOAc:hex 40:60 v/v): Rf = 0.63
(visualized with Ninhydrin); 1H NMR (400 MHz, Methylene
Chloride-d2) δ 7.66−7.62 (m, 4H), 7.47−7.36 (m, 6H), 7.35−7.20
(m, 5H), 4.06 (q, J = 6.4 Hz, 1H), 3.67 (dd, J = 10.7, 5.1 Hz, 1H),
3.62−3.52 (m, 3H), 2.24−2.15 (m, 1H), 1.36 (d, J = 6.5 Hz, 3H),
1.05 (s, 9H), 1.02 (d, J = 6.9 Hz, 3H), 0.63 (s, 9H), −0.13 (s, 3H),
−0.19 (s, 3H); 13C{1H} NMR (101 MHz, Methylene Chloride-d2) δ
144.2, 136.2, 133.9, 130.3, 130.3, 129.3, 128.3, 128.2, 128.0, 127.4,
120.0, 75.4, 66.4, 57.2, 50.6, 40.0, 27.3, 26.0, 25.5, 19.7, 18.2, 13.2,
−4.0, −5.1; FT-IR (film) cm−1: 3331, 2958, 2929, 2857, 1728, 1472,
133.2, 130.6, 128.4, 70.68, 27.02, 19.67; LRMS-ESI (m/z) calculated
for C18H22NaO2Si [M + Na]+, 321.13; found 321.20.
(2S,3R)-4-((tert-Butyldiphenylsilyl)oxy)-3-hydroxy-2-meth-
ylbutanal (6). Into a suspension of 5 (5 g, 16.8 mmol) and L-proline
(390 mg, 3.35 mmol) in dioxane (18 mL) at 4 °C was added
dropwise a solution of propanal (6.0 mL, 83.9 mmol) in dioxane (16
mL) over 24 h via a syringe pump. The reaction mixture was stirred
for 24 h at 4 °C, at which point it was diluted with Et2O/hex (1:1) (2
× reaction volume) and vigorously washed with H2O (20 × 20 mL)
and one portion of brine (20 mL). The organic layer was dried over
Na2SO4 and concentrated at 4 °C to afford 6 as a light-yellow oil. The
crude oil was immediately used in the next step following
concentration but without further purification (d.r. 4:1 from the
crude 1H NMR). Note: the product of this reaction 6 is unstable and
degrades at RT (19−21 °C) within a few hours. Concentration under
reduced pressure should be performed with flask immersed in ice
water until a crude oil is obtained, whereupon it must be immediately
used in the next reaction. TLC (EtOAc:hex 40:60 v/v): Rf = 0.47
(visualized with p-anisaldehyde); LRMS-ESI (m/z) calculated for
C21H28O3SiNa [M + Na]+, 379.17; found 379.26.
1109, 824; HRMS (ESI-TOF, m/z) calculated for C36H53N2O2Si2 [M
23.5
+ H]+, 601.3646; found 601.3644. [α]D
CH2Cl2).
= −38.1° (c = 1.0,
(3S,4R,5R)-5-(Hydroxymethyl)-4-methyl-3-(((S)-1-phenyl-
ethyl)amino)dihydrofuran-2(3H)-one (11). A 6 M aqueous
solution of HCl (32 mL) was added to 11 (3 g, 5 mmol, 1 equiv)
in a round-bottom flask equipped with a condenser. The resulting
heterogeneous mixture was heated to reflux using an oil bath, at which
point the mixture turned into a dark-brown homogeneous solution.
Following 3 h of reflux, the reaction mixture was cooled down and
washed with EA/hex (1:1, 2 × 20 mL). The pH of the aqueous layer
was adjusted to 6 by addition of saturated aq. Na2CO3. At this point,
two-thirds of the water present in the aqueous solution was removed
in vacuo. The pH of the remaining aqueous layer was further adjusted
to 11−12 by addition of more saturated aq. Na2CO3. The resulting
solution was extracted with EtOAc (6 × 20 mL), and the combined
organic layers were washed with brine, dried over anhydrous Na2SO4,
filtered, and concentrated. The crude residue was purified by flash
column chromatography using silica gel (EA/hex 20:80 to 30:70,
gradient) to afford 11 as a colorless oil (700 mg, 56%). TLC
(EtOAc:hexanes 60:40 v/v): Rf = 0.27; 1H NMR(300 MHz,
CD2Cl2): δ 7.40−7.26 (m, 5H), 4.17−4.10 (m, 2H), 3.75 (dd, J =
12.4, 3.2 Hz, 1H), 3.58 (dd, J = 12.4, 5.1 Hz, 1H), 3.47 (d, J = 7.8 Hz,
1H), 2.20 (m, 1H), 1.36 (d, J = 6.7 Hz, 3H), 1.02 (d, J = 7.2 Hz, 3H);
13C{1H} NMR(75 MHz, CD2Cl2): δ 177.6, 145.1, 128.5, 127.2,
(2S,3R)-3-((tert-Butyldimethylsilyl)oxy)-4-((tert-butyl-
diphenylsilyl)oxy)-2-methyl Butanal (7). TBDMSOTf (8.08 mL,
35.16 mmol) was added dropwise to a solution of 6 (crude, 11.94 g,
33.5 mmol) and 2,6-lutidine (4.85 mL, 41.9 mmol) in DCM (230
mL) at −78 °C. After stirring for 1 h at this temperature, the reaction
mixture was warmed up to RT over 1 h and its contents were poured
into saturated aq. NaHCO3 (100 mL). Then the resulting layers were
separated. The aqueous layer was extracted with DCM (3 × 100 mL),
and the combined organic layers were dried over Na2SO4 and
concentrated. The crude product was purified by flash column
chromatography using silica gel (EA/hex 2:98) to afford 7 as a light-
yellow oil (7.67 g, 52% over two steps, anti:syn = 99:1). TLC
(EtOAc:hex 40:60 v/v): Rf = 0.65 (visualized with p-anisaldehyde and
127.1, 85.2, 63.2, 57.6, 56.9, 35.5, 24.3, 13.0; FT-IR (film) cm−1:
3437, 3324, 2966, 2930, 2874, 1765, 1452, 1169, 1117, 1004, 764,
703; HRMS (ESI-TOF, m/z) calculated for C14H19NO3Na [M +
Na]+, 272.1265; found 272.1266. [α]D21.3 = −41.1° (c = 1.0, CH2Cl2).
2,5-Dioxopyrrolidin-1-yl (2S,3R,4R)-2-((((9H-fluoren-9-yl)-
methoxy)carbonyl)amino)-4,5-bis((tert-butyldimethylsilyl)-
oxy)-3-methylpentanoate (12). The remaining steps of this
synthesis were performed according to our previously reported
synthesis of DHIle and the first total synthesis of α-amanitin.27
2-Azidoethan-1-amine (S2). This was prepared following
standard procedures as previously reported.49 Upon completion, the
reaction was cooled down to room temperature and then placed in an
ice bath. Potassium hydroxide (1.45 g, 259 mmol) was added slowly
while stirring gently. The reaction mixture was extracted with diethyl
ether (3 × 15 mL), washed with brine (30 mL), dried over anhydrous
Na2SO4, filtered, and concentrated by vacuum distillation at 0 °C to
afford the product S2 as a colorless oil (1.74 g, 20.2 mmol, 78%).
Note: The starting material was completely consumed; however, due
to the volatility of the product and associated safety considerations,
complete removal of diethyl ether was not achieved. Therefore,
diethyl ether is observed in the NMR spectra. This material was
carried forward to the next reaction without further purification. TLC
1
DNP); H NMR (400 MHz, Methylene Chloride-d2) δ 9.80 (d, J =
1.7 Hz, 1H), 7.66 (tt, J = 6.2, 1.7 Hz, 4H), 7.47−7.37 (m, 6H), 4.09−
3.99 (m, 1H), 3.69−3.57 (m, 2H), 2.77−2.65 (m, 1H), 1.11 (d, J =
7.0 Hz, 3H), 1.03 (s, 9H), 0.82 (s, 9H), 0.01 (s, 3H), −0.10 (s, 3H);
13C{1H} NMR (100 MHz, Methylene Chloride-d2) δ 204.0, 136.2,
136.1, 133.7, 133.6, 130.4, 128.3, 128.3, 74.5, 65.9, 50.2, 27.1, 26.0,
19.5, 18.4, 10.2, −4.3, −4.9; FT-IR (film) cm−1: 3073, 2956, 2931,
2886, 2858, 1727, 1109, 837; HRMS (ESI-TOF, m/z) calculated for
21.4
C27H42NaO3Si2 [M + Na]+, 493.2570; found 493.2568. [α]D
+27.7° (c = 1.0, CH2Cl2).
=
(2S,3R,4R)-4-((tert-Butyldimethylsilyl)oxy)-5-((tert-butyldi-
phenylsilyl)oxy)-3-methyl-2-(((S)-1-phenylethyl)amino)-
pentanenitrile (8). To a solution of 7 (8.3 g, 17.6 mmol) in dry
DCM (160 mL) were added (S)-α-phenylethylamine (4.72 mL, 37.07
mmol) and Ti(OEt)4 (26.1 mL, 123 mmol). The resulting solution
was stirred at RT (19−21 °C) for 2 h, until the formation of the imine
intermediate 10 was complete (1H NMR). Then, TMSCN (9.83 mL,
69.8 mmol) and additional dry DCM (160 mL) were added, and the
reaction mixture was stirred for 2 h at RT (19−21 °C). Upon
completion, edte (30.5 g, 129 mmol, 1.05 equiv relative to Ti(OiPr)4)
1
(MeOH:CH2Cl2 98:2, 0.1% Et3N v/v): Rf = 0.32; H NMR (400
MHz, (CD3)2SO): δ 3.24 (t, J = 6.0 Hz, 2H), 2.70 (t, J = 5.9 Hz, 2H);
13C{1H} NMR (100 MHz, (CD3)2SO): δ 53.8, 41.2; HRMS (EI-
5366
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Note
TOF, m/z) calculated for C2H7N4 [M + H]+, 87.0671; found
87.0667.
resin was filtered, and a fresh batch of capping solution was added for
an additional 10 min. The resin was filtered and washed with DMF (8
mL × 3), then CH2Cl2 (8 mL × 3), and dried on high vacuum over
P2O5 to remove residual solvent. Resin loading was determined by
measuring the UV absorbance of the liberated dibenzofulvene
following treatment with 2% DBU at 304 nm, with an absorption
coefficient of ε304 = 7624 M−1 cm−1.
tert-Butyl N2-(((9H-Fluoren-9-yl)methoxy)carbonyl)-N4-(2-
azidoethyl)-L-asparaginate (S3). To a flame-dried round-bottom
flask containing a solution of Fmoc-Asp-OtBu (4.82 g, 11.7 mmol),
EDC·HCl (2.58 g, 13.5 mmol), and HOBt·H2O (1.82 g, 13.5 mmol)
in anhydrous CH2Cl2 (44 mL) was added S2 (1.16 g, 13.5 mmol),
followed by slow addition of DIPEA (3.78 g, 29.3 mmol). The
reaction mixture was left to stir at RT overnight under Argon. Upon
completion, water (20 mL) was slowly added and the reaction
mixture was stirred for an additional 5 min. Following phase
separation, the aqueous layer was extracted with CH2Cl2 (3 × 15
mL). Combined organic layers were washed with brine (50 mL),
dried over anhydrous Na2SO4, filtered, and concentrated. The crude
material was purified by flash column chromatography using silica gel
(EA/hex 40:60) to afford S3 as a white foamy solid (2.93 g, 6.1 mmol,
The resin (1 g, 0.79 mmol/g) was transferred to a Zeba spin
column, and DMF was added and left to shake for 30 min (6 mL × 3,
10 min each with draining of DMF after each swelling period). The
solvent was drained and 6 mL of 20% piperidine in DMF (6 mL)
solution was added. The reaction was left to shake at room
temperature for 10 min, and then the solvent was drained. A fresh
batch of 20% piperidine in DMF (6 mL) was added, and the reaction
was left for an additional 5 min. The solvent was drained, and the
resin was washed with DMF (6 mL × 3), CH3CN (6 mL × 3), EtOAc
(6 mL × 3), and DMF (6 mL × 3). A solution of 13 (90 mg, 0.213
1
52%). TLC (EtOAc:hexanes 45:55 v/v): Rf = 0.35; H NMR (400
i
MHz, CD3OD): δ 7.80 (d, J = 7.5 Hz, 2H), 7.66 (d, J = 7.5 Hz, 2H),
7.39 (t, J = 7.4 Hz, 2H), 7.31 (tt, J = 7.5, 1.1 Hz, 2H), 4.45 (dd, J =
7.0, 5.5 Hz, 1H), 4.40−4.29 (m, 2H), 4.23 (t, J = 6.9 Hz, 1H), 3.41−
3.33 (m, 4H), 2.69 (qd, J = 15.3, 6.3 Hz, 2H), 1.45 (s, 9H); 13C{1H}
NMR (100 MHz, CD3OD): δ 172.5, 171.9, 158.3, 145.2, 142.6,
128.8, 128.2, 126.2, 120.9, 83.1, 68.1, 53.0, 51.5, 39.9, 38.6, 28.2;
HRMS (ESI-TOF, m/z) calculated for C25H29N5NaO5 [M + Na]+,
502.2066; found 502.2072.
mmol), PyBOP (110.8 mg, 0.213 mmol), and Pr2EtN (33.0 mg,
0.255 mmol) in 6 mL of DMF was added to the resin. The reaction
was left to shake for 4 h. The solution was drained, and the resin was
washed with DMF (6 mL × 3), CH3CN (6 mL × 3), EtOAc (6 mL ×
3), and DMF (6 mL × 3). Coupling with a solution of 13 was
repeated for 4 h. The resin was washed with DMF (6 mL × 3),
CH3CN (6 mL × 3), and EtOAc (6 mL × 3), and then capped with a
solution of Ac2O:Collidine:EtOAc (1:2:2, 6 mL) for 15 min. The
solution was drained, and the resin was washed with DMF (6 mL ×
3), CH3CN (6 mL × 3), EtOAc (6 mL × 3), and CH2Cl2 (6 mL ×
3).
N2-(((9H-Fluoren-9-yl)methoxy)carbonyl)-N4-(2-azidoethyl)-
L-asparagine (13). To a round-bottom flask containing a solution of
S3 (2.89 g, 6.0 mmol) in CH2Cl2 (13 mL) was added TFA (19.4 g,
169.8 mmol) dropwise. The reaction mixture was left to stir at RT
under Argon for 4 h. Upon completion, the solvent was removed
under reduced pressure. CH2Cl2(15 mL) was added to the crude
material, followed by a brine wash (10 mL × 2). The organic phase
was concentrated, and the crude material was purified by flash column
chromatography using silica gel (EA/hex/HOAc 45:55:0.1) to afford
13 as a white amorphous solid (2.31 g, 5.5 mmol, 90%). TLC
Following the coupling of Asn(N‑ethylazide) (13), 5 equiv of Nα-Fmoc-
amino acids (Cys(STr), Gly, Ile, Gly), HBTU (5 equiv), and DIPEA
(10 equiv) in DMF (6 mL) were sequentially added to the N-
terminus of the growing peptide. The general Fmoc coupling protocol
was followed: Resin was pre-swollen in DMF for 30 min (6 mL × 3,
10 min each with draining of DMF after each swelling period). After
draining DMF, unreacted sites on the resin were capped with
CH2Cl2:CH3OH:iPr2EtN (8 mL of a 80:15:5 mixture, 10 min) and
then washed with CH2Cl2 (8 mL × 3), DMF (6 mL × 3), CH3CN (6
mL × 3), EtOAc (6 mL × 3), and DMF (6 mL × 3). The N-terminal
Fmoc protecting group was removed by washing with 20% piperidine
in DMF (8 mL for 5 min, 8 mL for 10 min). Following deprotection,
the resin was washed with DMF (6 mL × 3), CH3CN (6 mL × 3),
EtOAc (6 mL × 3), and DMF (6 mL × 3). The next amino acid was
coupled to the resin and left on the shaker for 2 h. For a typical
coupling reaction, (Fmoc-Xaa(R)-OH, 5 equiv), coupling agent
HBTU (5 equiv), and DIPEA (10 equiv) in DMF (8 mL) were used.
Note: when a noncommercially available amino acid was used, fewer
equivalents and longer coupling times were employed. Coupling was
performed twice when the free N-terminus was derived from
hydroxyproline residue or asparagine. A Kaiser test was employed
to qualitatively determine coupling efficiency. In the event that the
Kaiser test was inconclusive, a small amount of resin was extracted.
The peptide chain was cleaved from the resin with 25%
hexafluoroisopropanol (HFIP) in CH2Cl2 and analyzed by LRMS-
ESI. When the reaction was complete, the coupling mixture was
drained, and the resin was washed as described above. Finally, the
unreacted amine sites were capped with EtOAc/collidine/Ac2O
(2:2:1, 8 mL) for 20 min before washing the resin with CH2Cl2 (3 ×
8 mL), then DMF (3 × 8 mL), and then CH2Cl2 (3 × 8 mL) again.
Nα-Boc-FPI-OH (14) (764.0 mg, 2.37 mmol) was coupled to the N-
terminus of the hexapeptide as described above. Upon completion,
the resin was washed with DMF (6 mL × 3), MeCN (6 mL × 3),
EtOAc (6 mL × 3), and CH2Cl2 (6 mL × 3) and carried on to the
next reaction without further purification.
1
(EtOAc:hexanes:HOAc 30:70:0.1 v/v/v): Rf = 0.27; H NMR (400
MHz, CD3OD): δ 7.78 (d, J = 7.5 Hz, 2H), 7.67−7.63 (m, 2H),
7.41−7.35 (m, 2H), 7.30 (tt, J = 7.4, 1.1 Hz, 2H), 4.61−4.53 (m,
1H), 4.39−4.27 (m, 2H), 4.22 (t, J = 7.1 Hz, 1H), 3.40−3.33 (m,
4H), 2.85−2.65 (m, 2H); 13C{1H} NMR (100 MHz, CD3OD): δ
172.7, 145.3, 145.2, 142.6, 128.8, 128.2, 126.3, 120.9, 68.2, 51.4, 39.9,
38.6; FT-IR (film) cm−1:3311, 3061, 2955, 2105, 1704, 1656, 1533,
1449, 1259, 1051, 737; HRMS (ESI-TOF, m/z) calculated for
C21H22N5O5 [M + H]+, 424.1621; found 424.1614. [α]D23.5 = −16.4°
(c = 1. 0, MeOH).
(2S)-1-(tert-Butoxycarbonyl)-3a-fluoro-1,2,3,3a,8,8a-hexa-
hydropyrrolo[2,3-b]indole-2-carboxylic Acid (14). This was
prepared according to our previous reports.23,27 Briefly L-Boc-Trp-
OH (803.5 mg, 2.64 mmol) dissolved in 33 mL of anhydrous THF
was treated with 1-fluoro-2,4,6-trimethylpyridinium triflate (1.53 g,
5.27 mmol). Upon complete consumption of starting material, the
solvent was removed. The organic layer was washed with brine, dried
over Na2SO4, filtered, and concentrated under reduced pressure to
give both diastereomers of Boc-Fpi-OH 14 in the form of a light
brown solid that was immediately used in the next reaction without
further purification. TLC (EtOAc:CH2Cl2:HOAc 10:90:1 v/v/v): Rf
= 0.33.
Linear Heptapeptide (15). Standard Fmoc-SPPS procedures
were employed for the synthesis of the heptapeptide 15 on CTC
resin. In general, 5 equiv of Nα-Fmoc amino acids and 5 equiv of
coupling agent HBTU/iPr2EtN in DMF to make a coupling solution
were added sequentially to the growing N-terminus. To a flame-dried
round-bottom flask containing chlorotrityl resin (1 g, 100−200 mesh,
0.9 mmol/g) in anhydrous CH2Cl2, (8.2 mL) were added Fmoc-
Monocyclic Heptapeptide (16). The resin containing linear
peptide 15 was transferred to a falcon tube and stirred in
TFA:CH2Cl2 (1:1, 6 mL). After stirring at room temperature for 1
i
Hyp(OtBu)-OH (995 mg, 2.43 mmol) and Pr2EtN (791.0 mg, 6.1
mmol). The reaction was left to stir at room temperature overnight,
and then transferred to a spin column. The resin was washed three
times with DMF (8 mL × 3) and CH2Cl2 (8 mL × 3). Unreacted
sites of the resin were capped by adding a solution of
CH2Cl2:CH3OH:iPr2EtN (8 mL of a 80:15:5 mixture, 10 min). The
i
h, 2% H2O and 2% Pr3SiH (v/v) were added, and the reaction
mixture left to stir for an additional hour. The resin was filtered over
cotton wool and washed with CH2Cl2 (10 mL × 2), and then
concentrated. The residue was dissolved in a minimal amount of
5367
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Note
methanol and triturated with cold Et2O (4 mL × 2) and dried under
reduced pressure. The crude material was purified by a Sep-Pak C18
packed column eluting with 30−40% MeCN in H2O (0.1% Formic
acid). Combined fractions were lyophilized to afford 16 as a white
powder (165 mg, 0.20 mmol, 25% based on Hyp-OH loading to CTC
resin). 1H NMR (400 MHz, Methanol-d4) δ 7.62 (d, J = 8.1 Hz, 1H),
7.36−7.30 (m, 1H), 7.19−7.14 (m, 1H), 7.11−7.05 (m, 1H), 5.02
(dd, J = 8.5, 4.1 Hz, 1H), 4.51−4.39 (m, 4H), 4.22−4.17 (m, 1H),
4.13−4.02 (m, 3H), 3.87−3.79 (m, 3H), 3.76−3.70 (m, 2H), 3.57
(dd, J = 14.6, 8.1 Hz, 1H), 3.41−3.37 (m, 4H), 3.25−3.14 (m, 2H),
2.86−2.90 (m, 1H), 2.78−2.58 (m, 4H), 2.27−2.22 (m, 1H), 2.00−
2.01 (m, 2H), 1.89−1.86 (m, 1H), 1.64−1.57 (m, 1H), 1.25−1.18
(m, 1H), 0.96−0.91 (m, 7H); 13C{1H} NMR (101 MHz, DMSO) δ
173.2, 171.1, 169.6, 169.2, 169.0, 168.6, 163.5, 137.2, 126.9, 126.0,
122.3, 118.9, 118.7, 111.1, 68.7, 57.9, 54.4, 49.7, 47.9, 43.1, 42.4, 41.7,
38.2, 37.1, 35.3, 24.3, 15.6, 15.3, 11.1; HRMS (ESI-TOF, m/z)
calculated for C35H49N12O10S [M + H]+, 829.3415; found 829.3404.
HPLC purity 97%; λmax 288 nm.
data_request@ccdc.cam.ac.uk, or by contacting The Cam-
bridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
AUTHOR INFORMATION
■
Corresponding Author
David M. Perrin − Chemistry Department, The University of
British Columbia, Vancouver, BC V6T1Z1, Canada;
orcid.org/0000-0001-8342-6346; Email: dperrin@
chem.ubc.ca
Authors
Chido M. Hambira − Chemistry Department, The University
of British Columbia, Vancouver, BC V6T1Z1, Canada;
orcid.org/0000-0002-8119-9538
Kaveh Matinkhoo − Chemistry Department, The University of
British Columbia, Vancouver, BC V6T1Z1, Canada
Alla Pryyma − Chemistry Department, The University of
British Columbia, Vancouver, BC V6T1Z1, Canada;
orcid.org/0000-0003-2124-2853
Bicyclic Octapeptide (17). Monocyclic heptapeptide 16 (20 mg,
0.025 mmol) and (2S,3R,4R)-Oγ,Oδ-bis-TBS-Nα-Fmoc-dihydroxyiso-
leucine-OSu 12 (44.4 mg, 0.0625 mmol) were dissolved in DMF (400
μL). To the resulting solution was added iPr2EtN (8.5 μL, final pH 9),
and the reaction mixture was shaken on a vortexer for 72 h. Then,
Et2NH (20 μL) was added to the reaction, and shaking was continued
for another 2 h, followed by TBAF (150 μL) and further shaking of
the reaction mixture for 2 h. The reaction contents were diluted with
20% MeCN in H2O (0.1% formic acid, 4 mL) and directly purified by
preparatory HPLC to afford the monocyclic octapeptide as a fluffy
white powder (12 mg, 49% over 3 steps). HRMS-ESI [M + H]+
974.4142; λmax 286 nm. Monocyclic octapeptide (12 mg, 0.012
mmol) and HATU (380.2 mg, 0.111 mmol) were dissolved in DMA
Brian O. Patrick − Chemistry Department, The University of
British Columbia, Vancouver, BC V6T1Z1, Canada;
orcid.org/0000-0002-2921-8439
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.0c03022
Author Contributions
#C.M.H. and K.M. contributed equally to this work.
i
(0.006 M). To the resulting mixture was added Pr2EtN (14.6 mg,
final pH 9), and the solution was shaken for 2 h. The reaction
contents were diluted with 20% MeCN in H2O, and 0.1% formic acid
(4 mL), and directly purified by preparatory HPLC to afford the final
bicyclic octapeptide 17 as a fluffy white powder (2 mg, 0.0021 mmol,
Author Contributions
The manuscript was written through contributions of all
authors. Dr. Hambira performed the synthesis, optimization,
and characterization of all intermediates and final products; Dr.
Matinkhoo investigated the dihydroxyisoleucine synthesis; Dr.
Patrick acquired the X-ray diffraction data; Ms. Pryyma grew
the crystal of DHIle; and Dr. Perrin wrote and edited all drafts
of the manuscript and conceived of the work along with
contributions from all authors who have given approval to the
final version of the manuscript.
1
1
18% isolated yield). H NMR (400 MHz, DMSO) δ H NMR (850
MHz, DMSO-d6) δ 11.27 (s, 1H), 8.86 (dt, J = 21.9, 6.2 Hz, 1H),
8.37 (d, J = 3.6 Hz, 1H), 8.14 (d, J = 8.6 Hz, 1H), 7.95 (d, J = 10.0
Hz, 1H), 7.84 (t, J = 9.4 Hz, 1H), 7.57 (d, J = 8.2 Hz, 1H), 7.25−7.24
(m, 1H), 7.10−7.12 (t, J = 7.7 Hz, 1H), 7.01 (t, J = 7.5 Hz, 1H),
5.09−5.05 (m, 1H), 4.47−4.71 (m, 1H), 4.56 (ddd, J = 12.7, 10.0, 3.6
Hz, 1H), 4.52 (dd, J = 8.9, 4.9 Hz, 1H), 4.39−4.38 (m, 1H), 4.29 (dd,
J = 11.7, 7.0 Hz, 1H), 4.16 (dd, J = 19.0, 8.5 Hz, 1H), 3.90 (dd, J =
17.6, 7.6 Hz, 2H), 3.82−3.74 (m, 2H), 3.72 (dd, J = 8.7, 4.6 Hz, 1H),
3.56 (bs, 1H), 3.09−3.05 (m, 2H), 2.96 (dd, J = 16.4, 5.0 Hz, 1H),
2.90 (dd, J = 14.7, 6.6 Hz, 1H), 2.79 (dd, J = 11.3, 3.6 Hz, 1H), 2.20−
2.16 (m, 2H), 2.02−2.01 (m, 1H), 1.90−1.88 (m, 1H), 1.59−1.52
(m, 3H), 1.47−1.44 (m, 2H), 1.17 (t, J = 7.1 Hz, 1H), 1.11−1.08 (m,
2H), 0.86−0.84 (m, 3H), 0.82 (t, J = 7.4 Hz, 3H), 0.78 (d, J = 6.8 Hz,
3H) (Note: chemical shifts that are not reported were obscured by
the H2O/solvent peak; please see SI for HSQC data.); 13C{1H} NMR
(214 MHz, DMSO) δ 175.1, 174.2, 173.7, 173.4, 173.1, 173.0, 172.8,
171.2, 170.6, 169.0, 139.6, 130.1, 127.7, 125.3, 123.3, 121.7, 118.7,
114.2, 75.8, 71.8, 66.6, 65.1, 62.8, 62.0, 58.9, 57.8, 56.3, 55.3, 53.4,
52.6, 51.7, 45.4, 44.5, 41.6, 41.4, 40.8, 37.5, 37.4, 33.4, 23.8, 13.7;
HRMS (ESI-TOF, m/z): calculated for C41H57N13NaO12S [M + H]+,
978.3868; found 978.3858; λmax 287 nm.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge grants from the Canadian Institutes of Health
Research #220656 and the Canadian Cancer Sociey Research
Initiative #703374 and thank Dr. Maria Ezhova, Dr. Mark
Okon, and Dr. Lawrence McIntosh for help with NMR
acquisition and Dr. Elena Polishchuk and Ms. Jessie Chen for
help with cytotoxicity assays. C.M.H. was supported by a
postdoctoral fellowship competitively awarded by the Michael
Smith Foundation for Health Research.
REFERENCES
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.joc.0c03022.
■
■
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(3) Wienland, T.; Faulstich, H. Fifty Years of Amanitin. Experientia
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sı
NMR data (PDF)
Accession Codes
CCDC 2047129 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
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