Total Synthesis of the Death Cap Toxin Phalloidin: Atropoisomer Selectivity Explained by Molecular-Dynamics Simulations

Article abstract of DOI:10.1002/chem.201901888

Phallotoxins and amatoxins are a group of prominent peptide toxins produced by the death cap mushroom Amanita phalloides. Phalloidin is a bicyclic cyclopeptide with an unusual tryptathionin thioether bridge. It is a potent stabilizer of filamentous actin and in a fluorescently labeled form widely used as a probe for actin binding. Herein, we report the enantioselective synthesis of the key amino acid (2S,4R)-4,5-dihydroxy-leucine as a basis for the first total synthesis of phalloidin, which was accomplished by two different synthesis strategies. Molecular-dynamics simulations provided insights into the conformational flexibility of peptide intermediates of different reaction strategies and showed that this flexibility is critical for the formation of atropoisomers. By simulating the intermediates, rather than the final product, molecular-dynamics simulations will become a decisive tool in orchestrating the sequence of ring formation reactions of complex cyclic peptides.

Full text of DOI:10.1002/chem.201901888

A Journal of
Accepted Article
Title: Total synthesis of the death cap toxin Phalloidin - atropoisomer-
selectivity explained by molecular-dynamics simulations
Authors: Roderich Süssmuth, Guiyang Yao, Jan-Oliver Joswig, and
Bettina Keller
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To be cited as: Chem. Eur. J. 10.1002/chem.201901888
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Total synthesis of the death cap toxin phalloidin - atropoisomer-
selectivity explained by molecular-dynamics simulations
Guiyang Yao,[a] Jan-Oliver Joswig,[b] Bettina G. Keller[b] and Roderich D. Süssmuth*[a]
Abstract: Phallotoxins and amatoxins are a group of prominent
peptide toxins produced by the death cap mushroom Amanita
phalloides. Phalloidin is a bicyclic cyclopeptide with an unusual
tryptathionin thioether bridge. It is a potent stabilizer of filamentous
actin and in a fluorescently labeled form widely used as a probe for
actin binding. Herein, we report on the enantioselective synthesis of
the key amino acid (2S,4R)-4,5-dihydroxy-leucine as a basis for the
first total synthesis of phalloidin, which was accomplished by two
different synthesis strategies. Molecular-dynamics simulations
provided insights into the conformational flexibility of peptide
intermediates of different reaction strategies and showed that this
flexibility is critical for the formation of atropoisomers. By simulating
the intermediates, rather than the final product, molecular-dynamics
contains tryptathionin, a thioether amino acid constituting the
bridge between the Cys sidechain and the 2-position of the
indole ring of Trp.
Contrary to amatoxins, phalloidin is orally non-toxic. Its main
toxicity (injected into the bloodstream) has been attributed to the
uptake by the Organic Anion Transporting Polypeptide
transporters OATP1P1[7] expressed on the sinusoidal side of
hepatocytes.[8] Once taken up by the hepatocytes, its tight and
selective binding to filamentous (F-)actin ultimately causes the
destruction of the liver cells.[9] Modified derivatives of phalloidin
containing fluorescent dyes are widely used to visualize F-actin
for biomedical microscopy purposes.[10] A previous synthetic
attempt toward [Ala]7-phalloidin[11] however yielded two
derivatives. It has been suggested that these derivatives are
simulations will become
a decisive tool in orchestrating the
sequence of ring formation reactions of complex cyclic peptides.
atropoisomers, i.e. isomers generated by the hindered rotation
11]
around the tryptathionin bridge,[5,
which places the indole
sidechain of Trp either on the upper or the lower side of the
plane of the macrolactam (Fig 2A and Fig 2B). However, further
experimental details remained elusive.
In the past years peptides have received increased attention as
potential drugs, since they could close a gap between small
molecules and proteins:[1] They are smaller in size than proteins,
but can address the inhibition of protein-protein interactions,
while being commonly synthetically accessible and cheaper to
manufacture. In this context, bioactive cyclic peptides constitute
an excellent basis for studies directed towards the
understanding of active and passive peptide transport and the
design of peptides aiming for the delivery through membranes.[2]
The cyclic peptide structure confers structural rigidity and thus
bioactivity, while the absence of charges may facilitate transport.
Prominent examples of that type of peptide from the group of
fungal peptides are the amatoxins and the phallotoxins. Of the
latter, phalloidin is the main representative.
Herein, we report on the total synthesis of phalloidin, which
we envision as a biologically interesting and synthetically
challenging molecule. Furthermore, our theoretical and
experimental study aims for a rational and optimized synthesis
strategy, as well as to explaining previously reported effects of
atropoisomery. A robust total synthesis could further provide
access to phalloidin derivatives for chemical biology studies, e.g.
for the design of molecular probes. Finally, it opens the way to
extended studies on structure-activity-relations, of transport
phenomena across membranes, as well as provide options for
further structural diversification.[12]
Phalloidin 1 (Figure 1) is a bicyclic heptapeptide and its structure
has been elucidated[3] and characterized by NMR spectroscopy
studies[4] and the X-ray crystallographic analysis of [Ala]7-
phalloidin.[5] Characteristic structural features are one D-Thr and
the unusual amino acids (2S,4R)-4,5-dihydroxy-L-leucine
(DHLeu) and (2S,4S)-4-hydroxy-proline (Hyp). The latter is the
less abundant cis-epimer, since commonly this post-translational
hydroxylation, e.g. occurring in collagen, exclusively renders
trans-4-hydroxyproline.[6] Finally, the phalloidin structure
[a]
Dr. G. Yao, Prof. Dr. R. D. Süssmuth
Institut für Chemie, Technische Universität Berlin
Strasse des 17. Juni 124, 10623 Berlin (Germany)
E-mail: suessmuth@chem.tu-berlin.de
Figure 1. Structure of phalloidin (1) and strategic macrolactamization sites
(strategy A and B) for the final ring closure; (2S,4R)-4,5-dihydroxy-L-leucine
(DHLeu), (2S,4S)-4-hydroxy-proline (Hyp).
[b] J.-O. Joswig, Prof. Dr. B. G. Keller
Department of Biology, Chemistry, Pharmacy, Freie
Universität Berlin, Takustr. 3, 14195 Berlin (Germany).
Supporting information for this article is given via a link at
the end of the document.
Key challenges of the synthesis were the access to a suitably
protected (2S,4R)-4,5-dihydroxy-leucine (DHLeu) and the
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establishment of the tryptathionin bridge, while orchestrating an
elegant sequence of ring formation reactions – if possible –
avoiding atropoisomers. Two distinctive mechanisms for the
formation of atropoisomers can be envisioned: (i) isomerization
of natural phalloidin, or (ii) synthesis-based, during the assembly
of the bicycle. We performed microsecond molecular-dynamics
(MD) simulations of both phalloidin atropoisomers 1 and 1’
(Figure 2A and B). Neither at 300 K, nor at an elevated
temperature of 500 K, spontaneous isomerization occurred.
Furthermore, the peptide cycle formed multiple highly-populated
intramolecular hydrogen bonds. Fully open ring structures were
hardly ever sampled and had mean lifetimes of less than 2 ps.
Especially, because the open ring structures are so short-lived, it
seems very unlikely that spontaneous isomerization can occur
even on timescales which are much larger than one
microsecond. These results were confirmed by NMR
spectroscopic investigations: HPLC-MS and NMR spectroscopic
control of natural phalloidin 1 heated to elevated temperatures
(90°C, DMSO) only led to signal shifts, but the compounds
stayed intact (see Figure S7). Accordingly, the synthesis-based
sequence of ring cyclization reactions must determine the
occurrence of atropoisomers.
For the synthesis of the bicyclic structures of the phalloidin-
type there exist two fundamentally different strategies: first the
formation of the macrocycle followed by the I2-mediated
formation of the tryptathionin bridge [Glu]7-phalloidin,[13] or the
thioether formation prior to macrolactamization [Ala]7-
phalloidin.[11] The latter route is based on thioether formation of
the Trp-Leu-Ala-D-Thr-Cys pentapeptide followed by attachment
of a Pro-Ala dipeptide and macrolactamization ([5+2] strategy).
Both approaches have limitations in applicability and the
occurrence of by-products, e.g. unwanted dimerization and
atropoisomers.
Which of the two atropoisomers is formed is determined by
whether the ring formation occurs from above or from below the
ring plane of the intermediate. In our MD simulations, we
measured the orientation of the tryptathionin bridge with respect
to the ring plane using the torsion angles indicated in Fig. 2C
and 2D and compared to the corresponding distributions in
phalloidin 1 and its atropoisomer 1’ (Fig. 2E). If the torsion angle
is zero the tryptathionin bridge lies in the ring plane. The two
atropoisomers are characterized by distributions on either side
of the zero, corresponding to orientations of the tryptathionin
bridge above and below the ring plane. Intermediate a contains
the smaller and therefore less flexible β turn type I. Its torsion
angle distribution coincides with this of natural phalloidin 1 and
does not cross the zero line. The formation of 1’ is thus sterically
impossible. By contrast, intermediate b contains the larger and
therefore more conformationally flexible ring. Its torsion angle
distribution crosses the zero line and shows overlap with the
torsion angle distributions of both atropoisomers. Thus, ring
formation from above and below the ring plane is possible for b.
The synthesis strategy of [Glu]7-phalloidin also yields selectively
the natural atropoisomer, which is in line with our MD simulation
of the intermediate of this strategy (c). Although the
tryptathionine bridge is not yet formed, the side chains of Cys3
and Trp6 stayed on the side of the macrocycle which leads to the
natural atropoisomer (see Supporting Information).
Figure 2: 3D structures of phalloidin 1 (A), its atropoisomer 1’ (B), ring
structure of intermediate a or a’ (C), ring structure of intermediate b (D).
Peptide rings to be closed in subsequent synthesis steps are shown
transparently in (C) and (D). The torsion angle of atom 6C2 with the ring plane
is shown as a dotted line in (C) and (D). (E): Distribution of these two torsion
angles in intermediate a/a’ (blue) and intermediate b (orange) compared to the
torsion angle distribution in 1 and 1’ (measured by MD simulations, see SI).
Based on these predictions from MD simulations, in our
retrosynthetic analysis (Scheme 1), two strategies with final
macrolactamization sites were chosen: between Cys3 and D-
Thr2 or Trp6 and DHLeu7, respectively. In the first approach
([4+3]-strategy), we envisioned the assembly of the linear
tetrapeptide cyclized by I2-mediated tryptathionin formation to
thioether 5 (β turn type I), which was then followed by coupling
of a tripeptide 3 and macrolactamization to phalloidin (1a). In the
alternative route (heptapeptide strategy), linear heptapeptide 19
(see Scheme 4) was synthesized on solid support, followed by
the I2-mediated tryptathionin formation, cleavage from the resin
(monocyclic peptide 7) and final macrolactamization to yield
phalloidin 1b.
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group of DHLeu, others employed a 4,5-didehydro-L-leucyl-L-
alanine dipeptide. However, the subsequent Sharpless
asymmetric dihydroxylation (ADH)[17] rendered inseparable
mixtures of diastereomers. Our initial strategy with a 4,5-
didehydro-L-leucine (Ddl)[18]-containing tripeptide (Fmoc-Ddl-Ala-
Thr(Bzl)-OMe) 10, (see Scheme 2A and Supporting Information)
and subsequent Sharpless ADH rendered the desired peptide
11 with a diastereoselectivity (dr 4.8:1), which was transformed
into tripeptide 3 for the subsequent attachment to monocyclic
tetrapeptide 5.
Scheme 2: Synthesis of phalloidin 1a ([4+3]-strategy). A) Synthesis of
tripeptide 3, a) H-L-Ala-OtBu, HATU, DIPEA, DMF; b) TFA/DCM (1:1), 1h; c)
H-D-Thr(OBn)-OMe HATU, DIPEA, DMF; d) AD-mix-α, tBuOH/H2O, 0°C, 12 h,
58% for the correct diastereomer after silica column; e) Et2NH/MeCN (1:1), 15
min; f) Cbz-Cl, DIPEA, DMF, 5 h; g) 0.5 N LiOH/MeOH/THF (1:1:1), 2 h, then
acidified by AcOH; h) H2 (1 atm), Pd-C 10%, MeOH. B) Application of the
[4+3]-strategy. a) piperdine/DMF (1:4); b) Fmoc-L-Ala-OH, TBTU, DIPEA,
DMF; c) Fmoc-cis-4-L-Hyp(OTBDMS)-OH, TBTU, DIPEA, DMF; d) Fmoc-L-
Cys(Trt)-OH, TBTU, DIPEA, DMF; e) 2 mg/ml I2 in DMF, 2.5 h; f) TFA/TIS/H2O
(95:2.5:2.5); g) DCC, N-hydroxysuccinimide (NHS), DMF, 12 h; h) tripeptide 3,
DIPEA, DMF, 6 h; i) Et2NH/MeCN, 15 min; j) EDCI (5 eq), HOAt (5 eq), DIPEA
(20 eq), DCM/DMF (peptide concentration, 0.05 mM).
However, in order to enable a greater flexibility of the overall
synthesis, we pursued the synthesis of an appropriately
protected amino acid building block: The synthesis of protected
enantiomerically pure (2S,4R)-Fmoc-4,5-DHLeu(TMDMS)2-OH 8
(Scheme 3) was ultimately accomplished starting from
methacryloyl chloride 14, which was transformed into a Weinreb
amide. After Sharpless ADH and silylation of the diol group with
TBDMS, compound 15 was obtained in 65% yield with 90% ee.
Subsequent to reduction with LiAlH4, aldehyde[19] 16 was used in
a Horner-Wadsworth olefination to form 2,3-didehydroamino
acid ester 17.[20] The protected dihydroxy-Leu derivative 17 was
stereoselectively hydrogenated using with Ru(COD)2BF4 and
(R)-MonoPhos as a ligand.[21] The hydrolysis of the methyl ester,
hydrogenolytic removal of the Cbz-group and protection with
Fmoc, rendered compound 8 in gram scale in 25% total yield.
The stereochemistry of 8 was determined as (2S,4R) by acid-
Scheme 1. Retrosynthetic analysis of phalloidin. A) [4+3]-strategy via
monocyclic heptapeptide 2; B) heptapeptide strategy from monocyclic peptide
7, [Asymmetric Sharpless dihydroxylation (ADH)].
From the unusual amino acid building blocks of phalloidin, we
had to consider cis-4-hydroxy-L-proline (4-Hyp) and (2S,4R)-4,5-
DHLeu: Previously, 4-Hyp was reported to lactonize (4-OH
group with the carboxy group) during peptide coupling.[14]
Therefore, we prepared the TBDMS-protected cis-4-hydroxy-L-
proline according to published protocols (see Supporting
Information, Scheme S1).[15] A significant experimental challenge
was
a suitable synthetic approach to (2S,4R)-4,5-DHLeu:
previous attempts as well as a recent chemoenzymatic route
according to Renata et al.[16] yielded massive amounts of
lactonized product, rendering these approaches futile. In order to
prevent lactonization from esters or activation of the carboxy
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mediated lactonization to S6 and compared to NMR spectra
resin of monocyclic thioether-peptide 7 was performed with
(see Figure S6).
TFE/HOAc/DCM (1:1:8) and preparative HPLC purification
rendered monocyclic peptide
7 in 30% yield. The final
macrolactamization to phalloidin was most favorable for
HATU/collidine in DCM/DMF (5:1) as a coupling reagent. After
removal of the silyl protecting groups from the hydroxy functions
of DHLeu with TBAF/THF, the crude peptide was purified by
reversed-phase HPLC. Synthetic phalloidin 1b was obtained in
an overall yield of 10%.
Scheme 3: Synthesis of protected DHLeu derivative 8. a) N,O-
dimethylhydroxylamine hydrochloride, Et3N, DCM,
0 °C; b) AD-mix-β,
tBuOH/H2O, 0°C; c) TBDMSCl, imidazole, DMF, 35 °C, 12 h; d) LiAlH4, THF, -
78°C, 1h, then aq 2M NaOH; e) Schmidt´s phosphonoglycinate, DBU, DCM, -
20°C to rt, 16 h; f) H2 (20 bar), 4mol % (R)-MonoPhos, 2mol% [Ru(COD)2BF4],
DCM, 4 h, rt; g) 0.5N LiOH/MeOH/THF (1:1:1), 2 h; h) H2 (1 atm), Pd-C 10%,
MeOH; i) Fmoc-OSu, NaHCO3, 1,4-dioxane/H2O, 12 h.
Thus equipped with the required building blocks, we focussed on
peptide assembly and the establishment of the tryptathionine
bridge (Scheme 1). Although the Savige-Fontana reaction has
been wildly used for formation of the thioether peptide, including
the first total synthesis of α-amanitin, amatoxins as well as
phallotoxins,[22] the pre-oxidation of Trp yielding 3a-
hydroxypyrrolo [2,3-b]indoline (Hpi) and the subsequent reaction
with Cys involves a considerable number of steps. We decided
to use direct thionation of a non-protected Trp sidechain and
trityl-protected Cys by iodine oxidation, a mild an efficient
reaction for thioether coupling.[13, 23]
Scheme 4: Synthesis of phalloidin 1b from a linear heptapeptide precursor. a)
piperdine/DMF (1:4); b) Fmoc-L-Ala-OH, TBTU, DIPEA, DMF; c) Fmoc-cis-4-L-
Hyp(OTBDMS)-OH, TBTU, DIPEA, DMF; d) Fmoc-L-Cys(Trt)-OH, TBTU,
DIPEA, DMF; e) Fmoc-D-Thr-OH, HATU, HOAt, DIPEA, DMF; f) Fmoc-L-Ala-
OH, TBTU, DIPEA, DMF; g) 8, TBTU, DIPEA, DMF; h) 2 mg/ml I2 in DMF, 2.5
h; i) TFE (trifluoroethanol)/HOAc/DCM, (1:1:8); j) HATU (5 eq), collidine (20
eq), DCM/DMF (peptide concentration, 0.05 mM), 12 h; k) 1M TBAF in THF,
30 min.
According to our [4+3] strategy via the thioether tetrapeptide 5,
we performed the assembly of linear precursor peptide 12 on 2-
chlorotrityl (2-CTC) resin by Fmoc-solid phase peptide synthesis
(SPPS) starting with the immobilization of unprotected Fmoc-
Trp-OH (see Supporting Information). A resin loading of 0.30
mmol/g was preferred over higher loadings, which could
promote unwanted intramolecular tryptathionine formation.
Tetrapeptide 5 (Scheme 2) was synthesized according to a
sequence of amino acid couplings and Fmoc-deprotection
cycles: Fmoc-Ala-OH, Fmoc-cis-4-Hyp(OTBDMS)-OH and
Fmoc-Cys(Trt)-OH, followed by I2-mediated cyclization. Upon
cleavage from the resin with TFA/TIS/H2O (95:2.5:2.5), and
subsequent preparative HPLC purification, monocyclic
tetrapeptide 5 was obtained in an overall yield of 47%. Then, we
Notably, with our coupling strategies no atropoisomers were
observed unlike previously reported for the [5+2]-strategy.[10]
The circular dichroism (CD) spectra of synthetic phalloidins
1
1a/1b (Figure 3) and the H NMR spectra were consistent with
those of natural phalloidin 1 (see Figure S1). Likewise, the
calculated CD spectra of 1 are in very good agreement with the
experimental spectra. By contrast, the calculated CD spectrum
for atropoisomer 1’ differs clearly from the experimental and
calculated spectra of 1, in particular in the critical region of λ =
240 to 260 nm, in which the two spectra have different signs
(see SI).
turned to coupling of
5
and tripeptide
3
using
hydroxysuccinimide (NHS) ester-mediated coupling conditions
(DCC), which proceeded smoothly to yield heptapeptide 2. After
removal of the Fmoc protecting group, macrocyclization was
performed with EDCI/HOAt/DIPEA. HPLC purification yielded
synthetic phalloidin (1a) which was NMR spectroscopically
characterized and found to be identical with natural phalloidin 1
(see Supporting Information).
Then we assessed the alternative SPPS-based route from a
linear heptapeptide: Fmoc-based synthesis proceeded smoothly
employing TBTU as the coupling reagent in the sequence
(Scheme 4, see also Supporting Information): Fmoc-L-Ala-OH,
Fmoc-cis-L-Hyp(OTBDMS)-OH and Fmoc-L-Cys(Trt)-OH. Fmoc-
D-Thr-OH with free hydroxy group was coupled using
HATU/HOAt, followed by coupling of Fmoc-L-Ala-OH and Fmoc-
DHLeu(TMDMS)2-OH 8 using TBTU as a coupling reagent.
According to the protocol mentioned above, resin-bound peptide
19 was treated with I2 in DMF for 2.5 h.[13] Cleavage from the
Figure 3: Circular dichroism spectra of synthetic phalloidin (1a and 1b),
natural phalloidin (1) as well as simulation spectra of phalloidin
atropoisomer 1’.
1 and
In summary, we herein present the first total synthesis of the
multicyclic peptide toxin phalloidin, also giving access to an
enantioselective synthesis of (2S,4R)-4,5-dihydroxy-L-leucine
(DHLeu). The synthetic strategy to phalloidin is based on MD
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COMMUNICATION
[16]
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calculations which guided the way for the identification of suited
macrocyclization sites in an exclusive favor of the correct natural
atropoisomer. Furthermore, our study excludes the natural
occurrence of atropoisomery by a ring flip, and rather gives an
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[19]
[20]
[21]
[22]
The study shows that, for devising synthesis strategies of
complex and (multi)cyclic molecules, it is essential to have
precise knowledge of the conformational flexibility of all
intermediates - preferably already at the stage of a retrosynthetic
analysis. Molecular dynamics simulations provide this
information and have evolved into a tool, which can be used on
a routine basis. Thus far, molecular dynamics studies have
focused on the final peptides. We believe that by simulating the
intermediates rather than the final product, molecular dynamics
simulations will become a decisive step in the prior assessment
of synthetic methods. The synthesis will further facilitate the
synthesis of new phalloidin derivatives and structure activity-
relationship studies.
[23]
Acknowledgements
Dr. Guiyang Yao thanks the Alexander von Humboldt
Foundation for a postdoctoral fellowship. The work was funded
by the Deutsche Forschungsgemeinschaft (DFG, German
Research Foundation, RTG 2473 to B.G.K. and the cluster of
Excellence under Germany´s Excellence Strategy – EXC 2008/1
(UniSysCat) – 390540038" to R.D.S. and J.-O.J.).
Keywords: Phalloidin • Amanitia phalloides • cyclopeptide •
tryptathionine • thioether • molecular dynamics simulations
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10.1002/chem.201901888
Chemistry - A European Journal
COMMUNICATION
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COMMUNICATION
Guiyang Yao, Jan-Oliver Joswig, Bettina
G. Keller and Roderich D. Süssmuth*
Page No. – Page No.
Total synthesis of the death cap toxin
phalloidin - atropoisomer-selectivity
explained by molecular-dynamics
simulations
Text for Table of Contents
Molecular-dynamics simulations guide ring formation: The first total synthesis of phalloidin was achieved through the
enantioselective synthesis of (2S, 4R)-4,5-dihydroxy-leucine, which was accomplished by two different synthesis strategies.
Molecular-dynamics simulations showed that this flexibility of intermediates is critical for the formation of atropoisomers and will
become a decisive tool in orchestrating the sequence of ring formation reactions of complex cyclic peptides.
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