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The occurrence of ansamers in the synthesis of cyclic peptides


Abstract and Figures

Amanitin is a bicyclic octapeptide composed of a macrolactam with a tryptathionine cross-link forming a handle. Previously, the occurrence of iso-mers of amanitin, termed atropisomers has been postulated. Although the total synthesis of α-amanitin has been accomplished this aspect still remains unsolved. We perform the synthesis of amanitin analogs, accompanied by in-depth spectroscopic, crystallographic and molecular dynamics studies. The data unambiguously confirms the synthesis of two amatoxin-type isomers, for which we propose the term ansamers. The natural structure of the P-ansamer can be ansa-selectively synthesized using an optimized synthetic strategy. We believe that the here described terminology does also have implications for many other peptide structures, e.g. norbornapeptides, lasso peptides, tryp-torubins and others, and helps to unambiguously describe conformational isomerism of cyclic peptides.
Physicochemical differences of amaninamide isomers UV-absorbance spectra a show different maxima at λ = 289 nm and 293 nm for 4a (purple) and 4b (orange). b CD spectra of 4a (purple) and 4b (orange) show opposite Cotton effects at 225 nm. MRE = mean residue ellipticity in 10³x [deg*cm²*dmol⁻¹]. c Differences in chemical shifts of 4a (purple) and 4b (orange) in the amide region of the ¹H-¹H-TOCSY NMR spectra. d Crystal structures of 4a¹⁵ and 4b. The A-ring and B-ring are colored gray and blue, respectively. The tryptathionine bridge is shown in red. e H-bonds found in the crystal structures of 4a (left) and 4b (right) are indicated by dashed green lines (top schematics). Amides with small chemical shift changes in VT-NMR measurements (ΔδHN/ΔT > −3.0 ppbK-1) indicative of H bonding are highlighted in green, solvent-exposed amides with large shift changes (ΔδHN/ΔT < −4.6 ppbK-1) are highlighted in red and amides with weak shielding/H-bonding are shaded in orange (top schematics). H-bonds observed in MD simulations are shown as blue dashed lines with the line width indicating the average population over 20 µs simulation time (bottom schematics; only H-bonds that occurred in >10% of the populations are shown, see Supplementary Fig. 12). In the MD simulations, amide groups highlighted with green ellipses showed small calculated solvent accessible surface areas (SASA < 0.02 nm²), amides with large SASA ( > 0.04 nm²) are circled in red and amides with intermediate accessibility are circled in orange (exact values are listed in Supplementary Table 4).
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The occurrence of ansamers in the synthesis
of cyclic peptides
Guiyang Yao
, Simone Kosol
, Elisabeth Irran
Bettina G. Keller
& Roderich D. Süssmuth
α-Amanitin is a bicyclic octapeptide composed of a macrolactam with a
tryptathionine cross-link forming a handle. Previously, the occurrence of iso-
mers of amanitin, termed atropisomers has been postulated. Although the
total synthesis of α-amanitin has been accomplished this aspect still remains
unsolved. We perform the synthesis of amanitin analogs, accompanied by in-
depth spectroscopic, crystallographic and molecular dynamics studies. The
data unambiguously conrms the synthesis of two amatoxin-type isomers, for
which we propose the term ansamers. The natural structure of the P-ansamer
can be ansa-selectively synthesized using an optimized synthetic strategy. We
believe that the here described terminology does also have implications for
many other peptide structures, e.g. norbornapeptides, lasso peptides, tryp-
torubins and others, and helps to unambiguously describe conformational
isomerism of cyclic peptides.
The chemical synthesis of constrained peptide macrocycles of natural
origin or of designed articial peptides sometimes leads to the
occurrence of isomers, which have been designated with various
terms. There exist various literature reports: Wareham et al. describe
the homeomorphic isomerism of macrobicyclic peptidic compounds
which involved a passage of the bridge chain through the macrolactam
(Fig. 1a)1. Bartoloni et al. investigated the diastereomeric norborna-
peptides as potential drug scaffolds which showed bridge-up/down
orientations according to the NMR solution structure (Fig. 1b)2.
The Yudin group reported an unusual tunable atropisomeric peptidyl
macrocycle which is made possible by controlling the conformational
interconversion3. More recently, Baran and co-workers accomplished
the reported total synthesis of the peptidic indole alkaloid tryptorubin
Aanddened non-canonical atropisomers, a family of shape-dened
molecules that are distinguished by bridge below/bridge above
arrangements (Fig. 1c)4. However, the vernacular nomenclature incited
some controversy and Crossley and co-workers suggested a composite
phenomenon using polytope formalism which is the fundamental
of akamptisomerism classication5. Ultimately, the existence of
atropisomers has also been postulated for the peptide toxins phalloi-
dins and amanitins612.
Phallotoxinsand amatoxins are two bicyclic peptide toxin families
isolated from the death cap mushroom Amanita phalloides. They both
belong to the ribosomally synthesized and post-translationally mod-
ied peptides (RiPPs) and display high toxicity with low lethal doses
in vivo animal experiments. α-Amanitin 1, a slow acting toxin (LD
50 100 μg/kg), has been reported to be a selective inhibitor of RNA
polymerase II13,14. Its bicyclic octapeptide structure contains a 6-
hydroxy-tryptathionine-(R)-sulfoxide cross-link (Fig. 1d). With the
macrolactam ring as an imaginary plane, in a typical presentation, the
tryptathionine bridge is located as a handle above the macrolactam, as
it can be derived from a previously published X-ray structure (Fig. 1e)15.
Longstanding questions are whether so-called atropisomers
indeed existed and if so, under which circumstances they would occur,
and what type of isomerism this would be? Previous studies of surro-
gate molecules of phalloidin and amanitin reported NMR spectro-
scopic data accompanied by CD spectroscopic analysis8and molecular
dynamics (MD) simulations7. However, epimerization of sidechain
Received: 20 May 2022
Accepted: 14 October 2022
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Institut für Chemie, Technische Universität Berlin, Strasse des 17. Juni 124, 10623 Berlin, Germany.
Center for Innovative Drug Discovery, Greater Bay Area
Institute of Precision Medicine (Guangzhou), School of Life Sciences, Fudan University, Shanghai, PR China.
Department of Biology, Chemistry, Pharmacy,
Freie Universität Berlin, Arnimallee 22, 14195 Berlin, Germany.
Department of Chemistry and Pharmacy, Ludwig-Maximilians-University, Butenandtstr. 5-13,
81377 Munich, Germany.
Max-Planck-Institute for Astronomy, Königstuhl 17, 69117 Heidelberg, Germany.
These authors contributed equally: Guiyang Yao,
Simone Kosol, Marius T. Wenz. e-mail:
Nature Communications | (2022) 13:6488 1
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stereocenters as an alternative explanation could not be unambigu-
ously ruled out6.
Here, in a systematic approach combining various analytical
methods (Marfey analytics, X-ray crystallography, NMR, and CD
spectroscopy) with MD simulations, we determine the structure and
dynamics of this sought isomer. Furthermore, we investigate different
macrolactamization sites and show that an optimized strategy can
ensure atroposelective synthesis. Finally, we propose the term ansa-
mer to describe and unambiguously assign the conguration of ste-
reoisomers of bridged cyclic systems, which can exist as
congurational stereoisomers, depending on the position of the
bridge, above or below the main ring. We suggest applying this
terminology also to other conformationally restricted cyclic peptides,
such as norbonapeptides or lasso peptides.
Results and discussions
Site-dependent macrolactamization and cycloisomer formation
Apart from semi-synthetic attempts, four total syntheses of α-amanitin
have been reported to date. These contain three basic synthetic
approaches to install the characteristic tryptathionine: An initial route
employed the Savige-Fontana reaction via an Hpi (3a-hydroxy-pyr-
rolo[2,3-b] indole)16 intermediate by the team from Heidelberg Pharma
GmbH. This reaction was also used in a more sophisticated fashion by
Perrin and co-workers to accomplish the rst total synthesis of
natural atropisomer
bridge above bridge below
In/out isomerism
b)a) c) Non-canonical atropisomerism
d) Amanitin atropisomerism
Asp Asp
Asp Asp
g) Ansamer configurations
Tyr 3
Tyr 6
Tyr 6
Tyr 3
-amanitin (1)
non-natural atropisomer
f) Ansa-compounds
of the main
Pansa Mansa
Fig. 1 | Peptide isomerism types and suggested determination of the ansamer
conguration of cyclic amanitin-type systems. Several nomenclatures have
been proposed to describe the isomerism of peptides displaying ring systems
(ac). dStructure of α-amanitin with the tryptathionine bridge highlighted in red.
DHIle (2 S,3 R,4R-dihydroxyisoleucine), Hyp (4-trans-L-hydroxyproline), OHTrp (6-
OH-tryptophan). eProposed conformational isomers of α-amanitin. The A and
B-rings are colored in gray and blue, respectively. fDetermination of the
conguration for ansa-compounds24.gDetermination of ansamer-congurations:
(1) the main cycle and the directionality is identied (light blue): from N- to
C-terminus. (2) Bridge-up/bridge-down cases in view of the priority order in the
main cycle. Identication of the leading atom/group L of the bridge next to the
bridgehead atom αis assigned. (3) The descriptor P
or M
is assigned
according to the directionality (clockwise/counter-clockwise) from the position of
the leading atom/group L.
Nature Communications | (2022) 13:6488 2
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amanitin17. One approach from our lab was using a preformed tryp-
tathionine from the reaction of indoles with sulfenyl chlorides, in a
convergent [5+1+2] synthesis strategy
18. Recently, we developed a
robust and versatile iodine-mediated tryptathionine formation proto-
col that enabled us to synthesize various amanitin analogs for detailed
SAR studies19. When we used the protocol to sample different macro-
lactamization sites (Fig. 2a), we noticed in somecases the formation of
a by-product with an identical molecular mass as the desired amanitin
analog. This could be interpreted as the formation of a diastereomer. A
preliminary Marfey analysis20 could however not prove epimerization,
which led us to assume another isomer effect.
To establish a simplied model system to further investigate this
observation, we decided to use readily available amino acids. There-
fore, in the pursuit of the synthetic amatoxin we replaced DHIle3with
Ile3to obtain Ile3-S-deoxo-amaninamide (Fig. 2). Thus, eight over-
lapping monocyclic peptides (2a2d and 3a3d) were synthesized
following our previous work19 and the subsequent nal macrocycliza-
tion was performed by using either HATU or EDC/HOAt as coupling
agent, rendering eight corresponding bicyclic peptides. For the bicy-
clization of 2b, the coupling reagent HATU was initially tested but
signicant amounts of the guanidination product were detected. To
suppress the formation of the guanidination product, EDC/HOAt was
selected as coupling reagent. Since the ratio of 4a and 4b is not
changed much under different coupling conditions (see Supplemen-
tary information section and Supplementary Fig. 19), we
concluded that different bicyclization reagents and conditions do not
signicantly change the ansa-selectivity of the reaction. The crude
peptides were carefully analyzed by HPLC-MS (see Supplementary
Fig. 1). The synthesis route via monocyclic peptides containing ring A
(2a2d), repeatedlyresulted in two peaks (4a and 4b,R
= 7.67 min and
8.13 min, respectively; Supplementary Fig. 1). Both peaks had the
identical molecular mass of bicyclic Ile3-S-deoxo-amaninamide
([M + H]+= 855.3851 Da), albeit occurring at different ratios (1:0 to
1:0.7, see Fig. 2a). In contrast, for precursor peptides with monocycle B
as intermediate (3a-3d) only one peak was observed, with the excep-
tion of 3c which favored the formation of 4b (ratio 1:2.7, Fig. 2a). When
screening different macrolactamization sites, the ratio of isomer yields
did not follow a clear trend. Precursors with preformed A-ring (2a-2d)
tended to result more often in isomeric product mixtures, while pre-
cursors with preformed B-rings (3a-3d) mostly yielded the natural
isomer. The NMR spectra of the two isomers formed by different
macrolactamization strategies correspond to each other which also
excluded an epimerization during bicyclization (see Supplementary
Figs. 14 and 15).
In our established iodine-mediated tryptathionine formation
protocol, we employed precursor 3b which exclusively yields 4a
(Fig. 2a). To investigate if 3b exhibits conformational pre-organization
favoring 4a, we performed classical MD simulations of the precursor as
well as of 4a and 4b. Indeed, in the simulations, 3b forms a stable
hydrogen bond network (Fig. 2b, Supplementary Fig. 2b), which allows
HATU/1:0.2 / 70%
HATU/1:0.1 / 65%
HATU/1:0.3 / 68%
θC,B θA,B
10% 10%
HATU/1:2.7 / 58%
HATU/1:0 / 34%
HATU/1:0 / 60%
HATU/1:0 / 61%
a) b)
3b 270º
EDC, HOAt/1:0.7 / 82%
Time (min)
Fig. 2 | Peptide precursor choice impacts isomer yields. a Bicyclization condi-
tions (HATU or EDC/HOAt) and different ring closure sites with preformed A-ring
2a-d (gray) or B-ring 3a-d (blue) lead to different isomer ratios (4a:4b)andyields
(4a+4b). bMD-simulated structure of precursor 3b with highest probability.
Hydrogen bonds are shown as green dashed lines. The structure is colored
according to the scheme in Fig. 1e. cIllustration of the model system to assess the
relative orientation of the A-ring, B-ring, and tryptathionine bridge in the amanitin
scaffold. The three dened planes (A-ring in gray, B-ring in blue and tryptathionine
bridgein red, as in a,bare shown as lines,their relativeorientation is dened by the
angles θas indicated (top left). For each angle (θ
), the average
distribution over 20 µs simulation data is presented in a circular plot. The dis-
tributions of angles observed in simulations of 4a (violet), 4b (orange), and 3b
(cyan) are shown as lines.
Nature Communications | (2022) 13:6488 3
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the molecule to adopt a conformation in which the C- and N-termini
are spatially close and the tryptathionine bridge is located above the
B-ring (Fig. 2b, Supplementary Fig. 3d). To compare the structural
organization of 3b with 4a and 4b,wedened three planes and the
angles (θ) between them: one plane for ring A and B, respectively, and a
third plane for the tryptathionine (Fig. 2a, c). We measured the angles
in the MD simulations of 3b,4a and 4b to compare the orientation of
the bridge relative to the macrocycle in the three molecules. Satisfy-
ingly, in 3b, the tryptathionine and the ring planes are positioned as in
4a with angles between 90° and 180° (Fig. 2c, Supplementary Fig. 3d).
This likely explains the selective reaction of monocyclic 3b to bicyc-
lic 4a.
Analytical characterization of bicyclic amaninamide isomers
Since UV absorption of tryptathionines is highly distinctive, it has been
previously employed to characterize tryptathionines21. Inte restingly, the
UV maximum absorption of isobaric 4a and 4b is slightly different with
λ=289nm and λ= 293 nm, respectively (Fig. 3a). To clearly exclude
epimerization during bicyclization, enantiomer analysis of the amino
acids by Marfeysmethod
20 showed identical conguration for every
amino acid in both isomers 4a and 4b (see Supplementary Fig. 4).
Further analysis revealed that the CD and NMRspectroscopic data
of peptide 4a are fully consistent with Ile3-S-deoxo-amaninamide as
previously characterized15. In contrast, the CD and NMR spectra of 4b
are noticeably different (Fig. 3b, Supplementary Fig. 13) and suggest a
different 3D structure. As reported previously for amanitin analogs8,
this could be consistent with the formation of conformational isomers.
For compound 4a, a positive Cotton effect was observed between
λ= 210 nm and 230 nm (in accordance with reported CD data of the
natural conformer). In contrast, the potential non-natural conformer
4b shows a negative Cotton effect at these wavelengths (Fig. 3b).
EXSY NMR analysis indicates that 4a and 4b are not readily
exchangeable conformers. Interconversion does not occur at elevated
temperatures in DMSO (150 °C, 10 h; Supplementary Fig. 17), or water
(CD spectroscopy: cycle 20 °C90 °C20°C) which is consistent with
VT-NMR measurements (see Supplementary Figs. 5 and 6). Inter-
conversion of the two isomers would require the indole sidechain to
pass through the macrolactam ring which appears sterically
190 210 230 250 270 290
N-Hyp-C N-Gly-C
N-Hyp-C N-Gly-C
N-Hyp-C N-Gly-C
N-Hyp-C N-Gly-C
1H [ppm]
Rel. abs.
wave length [nm]
4a 4b
1H [ppm]
4a 4b
Fig. 3 | Physicochemical differences of amaninamide isomers. UV-absorbance
spectra ashow different maxima at λ= 289 nm and 293 nm for 4a (purple) and 4b
(orange). bCD spectraof 4a (purple) and4b (orange) show opposite Cotton effects
at 225 nm. MRE = mean residue ellipticity in 103x [deg*cm2*dmol1]. cDifferences in
chemical shifts of 4a (purple) and 4b (orange) in the amide region of the 1H-
1H-TOCSY NMR spectra. dCrystal structures of 4a15 and 4b. The A-ring and B-ring
are colored gray and blue, respectively. The tryptathionine bridge is shown in red.
eH-bondsfound in the crystalstructures of 4a (left) and 4b (right) are indicated by
dashed green lines (top schematics). Amides with small chemical shift changes in
VT-NMR measurements (ΔδHN/ΔT>3.0 ppbK-1) indicative of H bonding are
highlighted in green, solvent-exposed amides with large shift changes (ΔδHN/
ΔT<4.6 ppbK-1)are highlightedin red and amides withweak shielding/H-bonding
are shaded in orange (top schematics). H-bonds observed in MD simulations are
shown as blue dashed lines with the line width indicating the average population
over 20 µs simulation time (bottom schematics; only H-bonds that occurred in
>10% of the populations are shown, see Supplementary Fig. 12). In the MD simu-
lations, amide groups highlighted with green ellipses showed small calculated
solvent accessible surface areas (SASA < 0.02nm2), amides with large SASA
( > 0.04 nm2) are circled in red and amides with intermediate accessibility are cir-
cled in orange (exact values are listed in Supplementary Table 4).
Nature Communications | (2022) 13:6488 4
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impossible without the breakage and reformation of covalent bonds
(Fig. 3).This is supported by our MD simulations where we also do not
observe a transition between 4a and 4b (Fig. 2c), even at elevated
To further characterize the relationship between isomers 4a and
4b both compounds were desulphurized with Raney Nickel at 80 °C to
give the corresponding macrolactams (see Supporting Information).
LC-MS analysis of the paralleled reactions indicated that the mono-
cycles 5(R
=5.88min; [M+H]
+= 825.4253Da) are structurally iden-
tical (Supplementary Fig. 7: LC-MS after desulfurization). This was
further conrmed by NMR spectroscopy of HPLC-puried 5(Supple-
mentary Fig. 8: calculated structure), suggesting that the tryptathio-
nine bridge is the key factor of isomer formation.
Final proof for the isomeric nature of 4a15 (CCDC deposition
number: 1128063) and 4b was obtained from X-ray crystallography.
Crystals of compound 4b were grown in 10% EtOH aqueous solution
and the structure of the peptide was obtained (CCDC: 2153904, Fig. 3d
and Supplementary Fig. 9). In the crystal of 4b, the tryptathionine
bridge is clearly located below the plane of the macrolactam. The
conguration of all amino acids in 4b is identical with 4a, corrobor-
ating our results from Marfeys analysis that no epimers were formed
during macrocyclization. Remarkably, in the X-ray structure of 4b,the
trans-amide bond between Asn1and Hyp2is ipped to a cis-amide
conformation with the carbonyl-group of Asn1facing towards the
outside of the macrolactam instead of the inside (the trans character
of the hydroxy-group of Hyp2is maintained). Overall, the backbone
geometry and H-bonding pattern are drastically different from 4a,
giving the molecule a more compact appearance (Fig. 3e, Supple-
mentary Fig. 16). This promotes the formation of a hydrophobic patch
by Ile3and Ile6in 4b, whereas in 4a these are oriented in opposite
directions. These conformational differences also substantially alter
the physicochemical properties of the bicyclic peptides: hence the
isomer 4b is insoluble in water at 2 mM (Supplementary Fig. 10).
Besides the differences between crystal structures of 4a and 4b,
which are also reected in the angle populations shown in the Rama-
chandran plots (Supplementary Fig. 2a), the MD simulations also
reveal differences in the dynamic behavior of the two isomers. We
calculated the atomic RMSF for the simulated structures of 4a and 4b
compared to their respective crystal structure (Fig. 3d, Supplementary
Fig. 3).The RMSF values suggest that 4b is generally more rigid tha n 4a
which is in line with the backbone angle distributions of 4a being
broader than of 4b (Supplementary Figs. 2a and 3). Interestingly,
quantum mechanical calculations (TDDFT level) on 100 optimized
structures out of each MDdata set showed that 4b is also energetically
favorable compared to 4a (difference ~30 kJ/mol).
The MD simulations and the crystal structures are in very good
agreement, as the all-atom RMSD is <0.4 nm and the backbone RMSD
<0.2 nm (Supplementary Fig. 21). However, we noted some differences
between the hydrogen bond patterns observed in the crystal struc-
tures and the simulated structures (Fig. 3e). For 4a,inthemajorityof
trajectories, the hydrogen bond pattern matches the crystal structure
(Supplementary Fig. 11). Interestingly, in the MD simulations, we
observed a subset of structures with a different set of hydrogen bonds
compared to the crystal structure. Correlation analysis of the H-bonds
in this subset and in the crystal structure (Supplementary Fig. 2b)
shows that they are mutually exclusive, suggesting that a different
minor conformation could exist which we did not observe in our
experiments.Overall, the MD simulationssuggest that the structure 4a
adopted in the crystal is likely the most stable conformation of this
isomer (Supplementary Fig. 11, Supplementary Table 3), which is sup-
ported by the very good agreement in the NOE distances between the
NMR and MD ensembles (Supplementary Fig. 13c).
For 4b, a bifurcated backbone hydrogen bond is present in the
crystal between the carbonyl oxygen of Ile3and amide hydrogens on
the opposite site of the peptide ring (Ile6and Cys8). MD simulations
suggest a trifurcated H-bond instead, also involving the amide of Gly7
(Fig. 3e, Supplementary Fig. 12). Unfortunately, it was not possible to
determine if the amide proton of Gly7is shielded by a H-bond in
solution as it was not resolved in the VT-NMR experiments. However,
the calculated solvent-accessible surface areas of the amide groups in
the crystal and MD structures agree well with each other (Fig. 3e,
Supplementary Fig. 12d, Supplementary Table 4). As in 4a,theNMR
and the MD ensembles are in very good agreement with each other
(Supplementary Fig. 13c).
Ansamers a concept for assigning conformational isomers of
cyclic peptides
Our structural and physicochemical characterization of the two iso-
mers shows that the isomers 4a/4b are not enantiomers, but diaster-
eomers,lastly not only because of the pronounced differences in bond
geometries, but also due to the chirality of amino acids. Previously,
isomers arising from differing bond geometries at the bridgehead in
bicyclic peptide systems have been categorized as atropisomers22,
non-canonical atropisomers4or akamptisomers5. This has led to some
controversy and at this time there is no accurate and simple term to
describe such a pair of isomers23. Atropisomers are clearly dened as
stereoisomers, which are interconverted by rotation of a single bond
between connecting moieties, which are typically sterically hindered.
The herein described bridged cyclic peptide structures 4a and 4b are
different from such atropisomers, because the interconversion of
these stereoisomers is not just attributed to the rotation around a
hindered single bond, but a ipping of thebridged cyclic structure. For
that matter, the hindered bond-angle inversion that leads to akamp-
tisomerization appears to be a better descriptor of the observed iso-
merism. But in case of 4a and 4b it is planes instead of bonds that
undergo an angle inversion (Fig. 2c) and application of heat will not
convert one diastereomer to the other. In addition, the ipping of the
bridged cyclic structures leads to a conformational change in the cycle.
As we nd that 4a and 4b cannot readily interconvert even under
heating we would assign the isomers the same molecular formula and
same bond connectivities to congurational rather than conforma-
tional isomers. We therefore propose the term ansamer to stereo-
chemically describe the two bridged isomers 4a and 4b.Comparedto
ansa-compounds, which consist of bridged planar chiral phenylene
systems24 (Fig. 1f), in the here presented ansamers, the bridged main
cycle is structurally strained (ring strain or a clamp bridge), which
increases the interconversion barrier between the two isomers. In
contrast to atropisomers, ansa-compounds (lat. ansa= handle), e.g.
cyclophanes, are interconverted by rotation of the handle around the
planar phenylene. Similar to ansa-compounds23 the assignment of the
stereochemical descriptor can be made as follows in agreement with
the CIP rules25,26:(1)identication of the main cycle2729 with the pre-
ferred directionality (Fig. 1g, from N to C terminus), (2) assignment of
the leading atom/group of the bridge (Fig. 1g, leading atom/ group L)
next to the bridgehead atom (α), followed by (3) assignment of the
priority from the position of L: clockwise/counter-clockwise sense of
the main cycle (Fig. 1g). (4) The descriptors P
or M
can be
assigned accordingly (Fig. 1g). These assignment rules are unambig-
uous and correctly describe existing enantiomers, epimers, and dia-
stereomers. This procedure would attribute conformational isomer
and the non-natural isomer 4b to the M
Hence, the biosynthesis of α-amanitin 1,isP
-selective in establish-
ing the tryptathionine bridge, as only the isomer with the indole above
the ring has been found in peptides from the Amanita mushroom
family. A member of the avoprotein monooxygenase (FMO) family
has been suggested to catalyze C-S bond formation30, however,
little is known about this step while the cleavage of the leader peptide
and cyclization by a prolyl oligopeptidase (POPB) are well
characterized31,32. Theherein proposed P
nomenclature could
be also applied to previously described norbornapeptides by
Nature Communications | (2022) 13:6488 5
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Reymond et al.2and in an extended version, it may prove helpful to
describe the conformation of in/out-isomers1,33,thelassopeptides
and even tryptorubin4(see Supplementary Fig. 18).
Inspired by the terminology used for ansa-compounds, the term
ansa (handle) illustrates the cause of the isomerism and at the same
time reects the planar aspect shared with benzene ansa-compounds.
In this sense the nomenclature is consistent. We hope the inten-
ded terminological similarity between ansa compound for aromates
and ansamer will raise attention and spur discussions in the scientic
community about the underlying problem to accurately categorize
this type of isomerism.
In summary, we have proven the occurrence of isomers in the
synthesis of bicyclic amanitin analogs, which have been previously
postulated and termed as atropisomers. The crucial step is the mac-
rolactamization of monocyclic tryptathionine-containing peptides.
The resulting isomers which appear xed in differently bridged con-
formations, have been thoroughly characterized by spectroscopic and
reasons, the indole sidechain of the tryptathionine cannot thread
through the macrolactam ring. In a stereochemical description of
these stereoisomers, we devise the term ansamers which can unam-
biguously describe these isomers and which can be applied also to
various other cyclic peptides.
General procedure for the monocyclic peptide synthesis
2-CTC resin (1 g, 0.98 mmol/g) was pre-swollen for 20 min in DCM in a
manual solid phase peptide synthesis vessel (10 mL). After the solvent
was drained, the rst amino acid Fmoc-AA1-OH (0.3 mmol) and DIPEA
(0.26mL, 1.5 mmol) in DCM (5 mL) were added to the resin. The mix-
ture was agitated for 2 h and before the solvent was drained. The resin
was rinsed with DMF (4 ×3 mL). Then a mixture of MeOH/DIPEA/DCM
(1:1:8) was added to cap the remaining 2-chlorotrityl chloride on the
resin. The mixture was agitated for 0.5 h. Then the solvent was drained
and the resin was washed with DMF (4 × 3 mL). The resin loading was
determined to be 0.30 mmol/g. The Fmoc-group was removed with
20% piperidine in DMF solution. Fmoc-AA2-OH (4 eq) was coupled to
the deprotected resin according to TBTU mediated coupling. The
Fmoc-group of the resulting resin was removed employing 20%
piperidine in DMF. The following six amino acids were coupled to the
deprotected. The tryptathionine formation was carried out on the
solid support using I
-mediated thioether formation. After removal of
the Fmoc-group and followed cleavage from the resin, the monocyclic
peptide was obtained following subsequent HPLC purication. The
synthesis and characterization data of all compounds have been
reported in the supplementary information.
General procedure for the bicyclic peptide synthesis
Monocyclic octapeptide 2a2d and 3a3d (1.0 eq) was dissolved in DMF
(1 mM). Then, DIPEA (2.2 eq) and HATU(2.0eq)wereaddedat0°C.The
reaction mixture was allowed to warm to r.t. for 12 h and concentrated
under reduced pressure. The crude product was puried using pre-
parative HPLC to afford bicyclic octapeptide as a white powder. Since
large amounts of guanidinylation product were detected during mac-
rolactamization of 2b, the alternative coupling condition EDC (2 eq) and
HOAt (2 eq) was employed to cyclize the monocyclic peptide 2b.All
results of LC-MS runs are shown in Supplementary Fig. 1. In addition, the
detritylation was performed after macrolactamization of 3c.Theyield
and ratio of 4a and 4b isshowninFig.2.
NMR assignment and structure calculation of desulfurized
To obtain resonance assignments for NOE assignment and structure
calculations 4a,4b, and desulfurized macrolactam 5were dissolved
in deuterated DMSO-d
(~10 mM). TOCSY, COSY, NOESY, and
1H-13C-HSQC spectra were recorded on a Bruker Avance III 700 MHz
spectrometer with a TXI 5 mm probe. Standard Bruker pulse programs
were used and all spectra were acquired at 298 K. Residual solvent
methyl peaks (DMSO-d7 δ=2.502for1Handδ=39.0ppmfor13C) were
used for chemical shift referencing. 2D homonuclear spectra were
measured with acquisition times of 70 and 18ms for the direct and
indirect dimensions, respectively. TOCSY and NOESY spectra were
accumulated with 16 or 32 (in case of 4b) scans and COSY spectra with
eight scans. The TOCSY and NOESY mixing times were set to 100 and
300 ms, respectively. Natural abundance 1H-13C-HSQC spectra were
measured with 140 scans and acquisitiontimes of 14 and 120 msfor the
direct and indirect dimensions. The spectra were processed and ana-
lyzed using TopSpin 3.5 (Bruker) and CcpNmr 2.3.136. After shift
assignment (Supplementary Table 1), the NOE correlations of 4a and
4b were manually assigned and residue interaction matrices of 4a and
4b were generated using CcpNmr. For structure determination, the
manually assigned chemical shifts of the desulfurized macrolactam
and NOESY peak lists were supplied to CYANA for automated NOE
assignment and structure calculation (Supplementary Table 2). The
program CYLIB37 was used to generate a CYANA library le for
4-hydroxyproline. A set of 1000 structures was calculated and the 100
best were visually inspected with UCSF Chimera38.
Molecular dynamics simulations
All-atom classical MD simulations of peptides 4a,4b,and3b in explicit
parameters for the peptides were obtained with ACPYPE42. The simu-
lations were conducted at the NpT ensemble with p=1bar and
T=300KorT= 400 K. The simulation time was 20 μs for each system
at T= 300 K, and 0.1 μs for each system at T=400K. See SI for a
detailed protocol.
Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this article.
Data availability
All processed data that support the ndings of this study are available
within the article and its Supplementary Information (experimental
details; synthetic procedures; X-ray diffraction, NMR, UV/Vis, VT-NMR,
MD simulations). All information to redo the MD simulations including
topology, starting, and structure les are stored on Zenodo [DOI:
10.5281/zenodo.6974777] together with the highest-probability struc-
tures or simulated crystal structures of 4a,4b,3b,and3c as well as
downsampled trajectories with a visualization state for the open-
source program VMD. The highest-probability structures for 4a,4b,
3b,and3c are provided as source data le alongside the manuscript.
The X-raycrystallographic data for 4b was deposited at the Cambridge
Crystallographic Data Centre (CCDC) under deposition number CCDC
2153904 [DOI: 10.5517/ccdc.csd.cc2b99sr]. The open-source software
used in this study is available under: MD simulations and analysis:
GROMACS 2019.4 and GROMACS 2020.6 (https://manual.gromacs.
org/documentation/), Custom code: Python 3.9.2 (https://www., Jupyter (IPython 8.5.0, notebook version
6.4.12,; Visualization: VMD for MACOSXX86_64,
version 1.9.4a57 (April 27, 2022,
vmd/). MD data isstored together with custom code according to DFG
regulations, and they are available from B.G.K. (bettina.keller@fu- upon request. Other data is available from the correspond-
ing author. Source data are provided with this paper.
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Dr. Guiyang Yao thanks the Alexander von Humboldt Foundation for a
postdoctoral fellowship and the National Natural Science Foundation
of China (82204189). The work was funded by the Deutsche For-
schungsgemeinschaft (DFG, German Research Foundation, RTG 2473
Nature Communications | (2022) 13:6488 7
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Bioactive Peptides to R.D.S., M.T.W., and B.G.K.). M.T.W. and B.G.K. thank
the Paderborn Center for Parallel Computing PC2 and ZEDAT of FU Berlin
for computing time. Molecular graphics and analyses were performed
with UCSF Chimera, developed by the Resource for Biocomputing,
Visualization, and Informatics at the University of California, San Fran-
cisco, with support from NIH P41-GM103311. The authors thank Manuel
Gemander and Hengshan Wang for helpful discussions and critical
Author contributions
G.Y, S.K., and R.D.S. designed the experiments. G.Y. performed the
synthesis of all shown compounds. S.K. and G.Y. designed and super-
vised the CD, NMR, and X-ray studies. E.I. recorded the crystallographic
data and solved the structure. O.T. made contributions to the isomery
interpretation and to the ansamer nomenclature. S.K., M.T.W., and B.G.K.
performed the theoretical calculations of the precursor, ansamers, and
macrolactam. All authors wrote, read, discussed, and approved the
Open Access funding enabled and organized by Projekt DEAL.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary information The online version contains
supplementary material available at
Correspondence and requests for materials should be addressed to
Roderich D. Süssmuth.
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ymous reviewer(s) for their contribution to the peer review of this
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... 26-28 However, recently we were able to synthesise, isolate and characterise both isomers of the amatoxin amanullin. 29 We also proposed the term 'ansamers' for this particular isomerism, where P ansa is the natural isomer with the bridge above the macrolactam ring, and M ansa is the newly synthesized non-natural isomer with the bridge below the macrolactam ring. Formally, the two ansamers can be interconverted by rotating the tryptathionine bridge around the (imaginary) axis between the two bridgeheads, but this rotation is hindered by the macrolactam ring. ...
... Spectroscopic data indicated that amanullin only forms a single dominant backbone conformation in each of the ansamers of amanullin (with two conformations of the Ile3-ethyl group in the crystal structure of the M -ansamer). 29 The conformation of the P -ansamer is stabilized by a strong hydrogen bond from the amide of Gly5 to the carbonyl oxygen of Asn1. This hydrogen bond is known to contribute to the overall rigidity of the amatoxin scaffold. ...
... This hydrogen bond is known to contribute to the overall rigidity of the amatoxin scaffold. 29,40,41 Here, we investigate the amatoxin Gly5Sar-amanullin, in which the amide of Gly5 has been methylated (sarcosine), thereby prohibiting the stabilizing hydrogen bond. N-methylation is a well-established strategy to modify the structure and dynamics of monocyclic peptides. ...
Full-text available
Amatoxins are strong inhibitors of RNA polymerase II, and cause cell death. Because of their cytotoxicity, they are candidates for anti-cancer drugs, and understanding their structure-activity relationship is crucial. Amatoxins have a rigid bicyclic scaffold which consists of a cyclic octapeptide bridged by cysteine and tryptophan side chain forming a tryptathionine bridge. Here, we show the influence of the N-methylation on the amatoxin scaffold by studying Gly5Sar-amanullin with MD simulations and NMR experiments. Since we have shown recently that the amatoxin scaffold allows for two isomeric forms (ansamers), we studied both isomers of Gly5Sar-amanullin. We found that both isomers of Gly5Sar-amanullin form two long-living conformations, which is unusual for amatoxins, and that they are differently affected by the N-methylation. The natural Gly5Sar-amanullin forfeits the hydrogen bonds to Gly5 due to the N-methylation, which is expected from existing crystal structures for alpha-amanitin. Our results however indicate that this does not cause more flexibility due to a shift in the hydrogen bond pattern. In the unnatural isomer, we observe an interesting cis-trans-isomerisation of the backbone angles in Trp4 and Gly7, which is enabled by the N-methylation. We expect that our perspective on the effect of N-methylation in amatoxins could be a starting point for further SAR-studies which are urgently needed for the design of better anti-cancer agents.
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Mechanically interlocked molecules (MIMs), such as rotaxanes and catenanes, have captured the attention of chemists both from a synthetic perspective and because of their role as simple prototypes of molecular machines. Although examples exist in nature, most synthetic MIMs are made from artificial building blocks and assembled in organic solvents. The synthesis of MIMs from natural biomolecules remains highly challenging. Here, we report on a synthesis strategy for interlocked molecules solely made from peptides, that is, mechanically interlocked peptides (MIPs). Fully peptidic, cysteine-decorated building blocks were self-assembled in water to generate disulfide-bonded dynamic combinatorial libraries consisting of multiple different rotaxanes, catenanes and daisy chains as well as more exotic structures. Detailed NMR spectroscopy and mass spectrometry characterization of a [2]catenane comprising two peptide macrocycles revealed that this structure has rich conformational dynamics reminiscent of protein folding. Thus, MIPs can serve as a bridge between fully synthetic MIMs and those found in nature.
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The toxic bicyclic octapeptide α-amanitin is mostly found in different species of the mushroom genus Amanita, with the death cap (Amanita phalloides) as one of the most prominent members. Due to its high selective inhibition of RNA polymerase II which is directly linked to its high toxicity, particularly to hepatocytes, α-amanitin received an increased attention as a toxin-component of antibody-drug conjugates (ADC) in cancer research. Furthermore, the isolation of α-amanitin from mushrooms as the sole source severely restricts compound supply as well as further investigations, as structure-activity relationship (SAR) studies. Based on a straightforward access to the non-proteinogenic amino acid dihydroxyisoleucine we herein present a robust total synthesis of α-amanitin providing options for production at larger scale as well as future structural diversifications.
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Isomerism is a fundamental chemical concept, reflecting the fact that the arrangement of atoms in a molecular entity has a profound influence on its chemical and physical properties. Here we describe a previously unclassified fundamental form of conformational isomerism through four resolved stereoisomers of a transoid (BF)O(BF)-quinoxalinoporphyrin. These comprise two pairs of enantiomers that manifest structural relationships not describable within existing IUPAC nomenclature and terminology. They undergo thermal diastereomeric interconversion over a barrier of 104 ± 2 kJ mol-1, which we term 'akamptisomerization'. Feasible interconversion processes between conceivable synthesis products and reaction intermediates were mapped out by density functional theory calculations, identifying bond-angle inversion (BAI) at a singly bonded atom as the reaction mechanism. We also introduce the necessary BAI stereodescriptors parvo and amplo. Based on an extended polytope formalism of molecular structure and stereoisomerization, BAI-driven akamptisomerization is shown to be the final fundamental type of conformational isomerization.
Alpha-Amanitin and related amatoxins are studied for more than 6 decades mostly by isolation from death cap mushrooms. The total synthesis however remained challenging due to unique structural features such as posttranslational modified Isoleucine (dihydroxyisoleucine) and a tryptathion bridge dividing the macrocycle into two rings. Alpha-amanitin is a potent inhibitor of RNA polymerase II. Interrupting basic transcription processes of eukaryotes leads to apoptosis of the cell. This unique mechanism makes the toxin an ideal payload for antibody drug conjugates (ADC). Only microgram quantities of toxins, when delivered selectively to tumor sites by conjugating to antibodies are sufficient to eliminate malignant tumor cells of almost every origin. By solving the stereoselective access to dihydroxyisoleucine, a photochemical synthesis of the tryptathion precursor, solid phase peptide synthesis and macrolactamization we obtained a scalable synthetic route towards synthetic a-amanitin. This makes a-amanitin and derivatives now accessible for the development of new ADCs.
A twisted small-molecule synthesis Some molecules are easy to draw on paper, whereas others contain rings and contortions that require one to think in three dimensions. Reisberg et al. set out to synthesize the bicyclic small molecule tryptorubin A but found that their initial attempt produced a molecule with the right bonds but the wrong molecular shape, a form of noncanonical atropisomerism. The authors then devised a synthesis where they locked in the correct isomer before forming the second ring, which produced a product indistinguishable from the authentic natural product. Such structural isomers may be lurking when working with complex small molecules with constrained rotation. Science , this issue p. 458
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 simulations will become a decisive tool in orchestrating the sequence of ring formation reactions of complex cyclic peptides.
The synthesis of an inherently chiral all-carbon C2-symmetric [12](1,6)pyrenophane 1 is reported. The cyclophyne 1 was obtained via a ring-closing alkyne metathesis (RCAM) reaction using Mortreux’s catalyst molybdenum hexacarbonyl and 2-fluorophenol as phenol additive. The M and P enantiomers of the all-carbon pyrenophane 1 demonstrated to be very stable in their enantiopure form even upon prolonged heating at 200 °C. [12](1,6)pyrenophane-6-yne 1 was fully characterized by high resolution mass spectrometry (HRMS), NMR, UV-Vis and by measured and calculated electronic circular dichroism (ECD) spectroscopy.
Poisonous mushrooms have fascinated scientists and laypersons alike for thousands of years. Almost all mushroom fatalities are due to the genus Amanita, whose poetic common names (death cap, destroying angel) attest to their lethality. In his classic 1986 book, Theodor Wieland covered the state of our knowledge about the chemistry and biochemistry of the toxins of Amanita mushrooms up until that time, with a particular focus on the decades of chemical research by him and the Wieland dynasty (including his father, brother, brother-in-law, and cousin). Wieland’s book is now mainly of historical interest, with its exhaustive overview of the early chemical studies done without benefit of methods taken for granted by modern chemists. This book is a complete top-to-bottom revision of Wieland’s 1986 book. The material covers history, chemistry, and biology with equal thoroughness. It should be of interest to natural products chemists and biologists, professional and amateur mycologists, and toxicologists. The three scientific fields that are most relevant to the book are natural products chemistry, mycology, and fungal molecular genetics. Dr. Walton is an expert in all three. To maximize the broad utility and appeal of the book, care has been taken to define all technical terms specific to a particular discipline, so that, for example, mycologists will be able to understand the relevant chemistry, and chemists will be able to understand the relevant fungal biology.