The occurrence of ansamers in the synthesis of cyclic peptides

Abstract

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.

Full text

Article https://doi.org/10.1038/s41467-022-34125-8
The occurrence of ansamers in the synthesis
of cyclic peptides
Guiyang Yao
1,2,6
, Simone Kosol
1,6
,MariusT.Wenz
3,6
, Elisabeth Irran
1
,
Bettina G. Keller
3
,OliverTrapp
4,5
& Roderich D. Süssmuth
1
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 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.
The chemical synthesis of constrained peptide macrocycles of natural
origin or of designed artificial 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
Aanddefined non-canonical atropisomers, a family of shape-defined
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 classification5. Ultimately, the existence of
atropisomers has also been postulated for the peptide toxins phalloi-
dins and amanitins6–12.
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-
ified peptides (RiPPs) and display high toxicity with low lethal doses
in vivo animal experiments. α-Amanitin 1, a slow acting toxin (LD
50
=
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
Check for updates
1
Institut für Chemie, Technische Universität Berlin, Strasse des 17. Juni 124, 10623 Berlin, Germany.
2
Center for Innovative Drug Discovery, Greater Bay Area
Institute of Precision Medicine (Guangzhou), School of Life Sciences, Fudan University, Shanghai, PR China.
3
Department of Biology, Chemistry, Pharmacy,
Freie Universität Berlin, Arnimallee 22, 14195 Berlin, Germany.
4
Department of Chemistry and Pharmacy, Ludwig-Maximilians-University, Butenandtstr. 5-13,
81377 Munich, Germany.
5
Max-Planck-Institute for Astronomy, Königstuhl 17, 69117 Heidelberg, Germany.
6
These authors contributed equally: Guiyang Yao,
Simone Kosol, Marius T. Wenz. e-mail: suessmuth@chem.tu-berlin.de
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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 configuration of ste-
reoisomers of bridged cyclic systems, which can exist as
configurational 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 first total synthesis of
natural atropisomer
bridge above bridge below
α
11'
In/out isomerism
AA
4AA
3
AA
2
AA
1
AA
5AA
6
AA
7
Norbornapeptides
AA
4
AA
3AA
2
AA
1
AA
5
AA
6
AA
7
N
N
b)a) c) Non-canonical atropisomerism
d) Amanitin atropisomerism
e)
L-Glu
NH HN
O
D-Glu
HN
O
O
O
N
NH HN
O
D-Glu
HN
O
O
O
N
L-Glu
Asp Asp
Asp Asp
HH
HH
g) Ansamer configurations
Ala1
Trp2
Tyr 3
Tyr 6
Trp5
Ile4
Tyr 6
Trp5
Ile4
Tyr 3
Trp2
Ala1
N
H
O
NH
O
NH
O
N
H
O
N
H
SH
OH
H
N
O
HN
OH
OH
H
O
N
OH
N
O
H
2
N
O
HO O
Asn
1
Hyp
2
DHile
3
OHTrp
4
Gly
5
Ile
6
Gly
7
Cys
8
-amanitin (1)
non-natural atropisomer
COOH
a
b
c
d
a
b
c
d
S/M
f) Ansa-compounds
Pansa
Mansa
N→C
Directionality
of the main
ring:
11'
Pansa Mansa
α
α
L
L
P
Fig. 1 | Peptide isomerism types and suggested determination of the ansamer
configuration of cyclic amanitin-type systems. Several nomenclatures have
been proposed to describe the isomerism of peptides displaying ring systems
(a–c). 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
configuration for ansa-compounds24.gDetermination of ansamer-configurations:
(1) the main cycle and the directionality is identified (light blue): from N- to
C-terminus. (2) Bridge-up/bridge-down cases in view of the priority order in the
main cycle. Identification of the leading atom/group L of the bridge next to the
bridgehead atom αis assigned. (3) The descriptor P
ansa
or M
ansa
is assigned
according to the directionality (clockwise/counter-clockwise) from the position of
the leading atom/group L.
Article https://doi.org/10.1038/s41467-022-34125-8
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 simplified 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 (2a–2d and 3a–3d) were synthesized
following our previous work19 and the subsequent final 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
significant 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 3.1.8.2 and Supplementary Fig. 19), we
concluded that different bicyclization reagents and conditions do not
significantly 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
(2a–2d), repeatedlyresulted in two peaks (4a and 4b,R
t
= 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
90º
0º
270º
180º
90º
90º
270º
180º180º 0º
10%
10% 10%
5%
5%
5%
HATU/1:2.7 / 58%
HATU/1:0 / 34%
HATU/1:0 / 60%
HATU/1:0 / 61%
θA,C
θA,B
θC,B
Bicyclization
Bicyclization
θA,C
a) b)
c)
4a
4b
3b 270º
0º
3b
vs
EDC, HOAt/1:0.7 / 82%
810
Time (min)
7.67
8.13
6
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 defined planes (A-ring in gray, B-ring in blue and tryptathionine
bridgein red, as in a,bare shown as lines,their relativeorientation is defined by the
angles θas indicated (top left). For each angle (θ
A,C
,θ
C,B
,andθ
A,B
), 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.
Article https://doi.org/10.1038/s41467-022-34125-8
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,wedefined 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 Marfey’smethod
20 showed identical configuration 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 °C→90 °C→20°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
-10
0
10
20
190 210 230 250 270 290
MRE
HN
C-Ile-N
N-Trp-C
C-Asn-N
N-Hyp-C N-Gly-C
C-Gly-N
C-Cys-N
N-Ile-C
H
HH
H
H
H
H
O
OO
O
O
O
OO
S
O
1
2345
6
7
8
H2N
C-Ile-N
N-Trp-C
C-Asn-N
N-Hyp-C N-Gly-C
C-Gly-N
C-Cys-N
N-Ile-C
H
HH
H
H
H
H
O
OO
O
O
O
OO
S
O
1
2345
6
7
8
H2N
C-Ile-N
N-Trp-C
C-Asn-N
N-Hyp-C N-Gly-C
C-Gly-N
C-Cys-N
N-Ile-C
H
HH
H
H
H
H
O
OO
O
O
O
OO
S
O
1
2345
6
7
8
H2N
C-Ile-N
N-Trp-C
C-Asn-N
N-Hyp-C N-Gly-C
C-Gly-N
C-Cys-N
N-Ile-C
H
HH
H
H
H
H
O
OO
O
O
O
OO
S
O
1
2345
6
7
8
7.0
8.0
9.0
1.0
2.0
3.0
4.0
5.0
6.0
1H [ppm]
G7
W4
G5
I3
N1
I6
G
7
C8
I
6
I
3
N
1
1
1
1
1
C
8
8
W
4
G
G
G
G
G
G
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
2
Rel. abs.
4b
4a
0
50
100
wave length [nm]
043083024022062003
4a 4b
1H [ppm]
a)
b)
c)
d)
e)
4a 4b
-Il
T
-G
n
e
-G
T
y-
s-
ys
y-
e
n
-I
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*dmol−1]. 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).
Article https://doi.org/10.1038/s41467-022-34125-8
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
temperatures.
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
t
=5.88min; [M+H]
+= 825.4253Da) are structurally iden-
tical (Supplementary Fig. 7: LC-MS after desulfurization). This was
further confirmed by NMR spectroscopy of HPLC-purified 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
configuration of all amino acids in 4b is identical with 4a, corrobor-
ating our results from Marfey’s analysis that no epimers were formed
during macrocyclization. Remarkably, in the X-ray structure of 4b,the
trans-amide bond between Asn1and Hyp2is flipped 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 reflected 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 defined 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 flipping 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 flipping of the
bridged cyclic structures leads to a conformational change in the cycle.
As we find that 4a and 4b cannot readily interconvert even under
heating we would assign the isomers the same molecular formula and
same bond connectivities to configurational 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)identification of the main cycle27–29 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
ansa
or M
ansa
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
4a,totheP
ansa
and the non-natural isomer 4b to the M
ansa
isomer.
Hence, the biosynthesis of α-amanitin 1,isP
ansa
-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 flavoprotein 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
ansa
/M
ansa
nomenclature could
be also applied to previously described norbornapeptides by
Article https://doi.org/10.1038/s41467-022-34125-8
Nature Communications | (2022) 13:6488 5
Content courtesy of Springer Nature, terms of use apply. Rights reserved

Reymond et al.2and in an extended version, it may prove helpful to
describe the conformation of in/out-isomers1,33,thelassopeptides
34,35
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 reflects 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 scientific
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 fixed in differently bridged con-
formations, have been thoroughly characterized by spectroscopic and
crystallographicmethodsaswellasbyMDsimulations.Forsteric
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.
Methods
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 first 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
2
-mediated thioether formation. After removal of
the Fmoc-group and followed cleavage from the resin, the monocyclic
peptide was obtained following subsequent HPLC purification. The
synthesis and characterization data of all compounds have been
reported in the supplementary information.
General procedure for the bicyclic peptide synthesis
Monocyclic octapeptide 2a–2d and 3a–3d (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 purified 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
macrolactam
To obtain resonance assignments for NOE assignment and structure
calculations 4a,4b, and desulfurized macrolactam 5were dissolved
in deuterated DMSO-d
6
(~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 file 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
dimethylformamidewerecarriedoutinGROMACS
39–41.Theforcefield
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 findings 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 files 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 file 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.
python.org/downloads/), Jupyter (IPython 8.5.0, notebook version
6.4.12, https://jupyter.org); Visualization: VMD for MACOSXX86_64,
version 1.9.4a57 (April 27, 2022, https://www.ks.uiuc.edu/Research/
vmd/). MD data isstored together with custom code according to DFG
regulations, and they are available from B.G.K. (bettina.keller@fu-
berlin.de) upon request. Other data is available from the correspond-
ing author. Source data are provided with this paper.
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Acknowledgements
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
Article https://doi.org/10.1038/s41467-022-34125-8
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
suggestions.
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
manuscript.
Funding
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
https://doi.org/10.1038/s41467-022-34125-8.
Correspondence and requests for materials should be addressed to
Roderich D. Süssmuth.
Peer review information Nature Communications thanks the anon-
ymous reviewer(s) for their contribution to the peer review of this
work. Peer reviewer reports are available.
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