Synthesis of the C-Terminal Macrocycle of Asperipin-2a

Article abstract of DOI:10.1021/acs.orglett.9b00488

A synthetic approach to the C-terminal macrocycle of asperipin-2a is presented. Two epimers were prepared, possessing R- and S-configurations at the β-position of Tyr³. Comparison of NMR data of the natural product with these isomers and X-ray

Full text of DOI:10.1021/acs.orglett.9b00488

Letter
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Synthesis of the C‑Terminal Macrocycle of Asperipin-2a
Sadegh Shabani, Jonathan M. White, and Craig A. Hutton*
School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, Victoria
3
010, Australia
*
S Supporting Information
ABSTRACT: A synthetic approach to the C-terminal macrocycle of asperipin-2a is presented. Two epimers were prepared,
3
possessing R- and S-configurations at the β-position of Tyr . Comparison of NMR data of the natural product with these
3
isomers and X-ray crystallographic data for one macrocycle support assignment of the 2S,3S-configuration of Tyr . Key steps in
the synthesis include a stereoselective benzylic oxidation of the tyrosine residue and Lewis-acid-catalyzed ring opening of the
subsequently generated aziridine.
ibosomally synthesized and post-translationally modified
peptides (RiPPs) are a class of peptide natural products in
the genome of A. flavus as responsible for the biosynthesis of
9
−12
R
ustiloxin B 2.
The ustiloxin gene cluster encodes the UstYa/
which the core structures are encoded directly in precursor
UstYb proteins, which were identified as responsible for the
hydroxylation of the benzylic position of the tyrosine residue
and oxidative cyclization to generate the tertiary alkyl−aryl ether
1
−4
proteins. In the biosynthesis of RiPPs, a core peptide is fused
to leader and/or follower peptides that facilitate installation of
post-translational modifications (PTMs). Following modifica-
tion of the core peptide, the leader and/or follower regions are
removed to release the final product. PTMs dramatically
increase the chemical and functional space of mature peptides.
These modifications include hydroxylation, chlorination, and
side-chain oxidation and cross-linking, leading to, for example,
highly functionalized cyclic and bicyclic peptides such as the
ustiloxins, amanitins, and asperipin-2a (Figure 1).
12,13
linkage present in the ustiloxins.
Umemura and co-workers screened the genome sequences of
Aspergillus for the presence of ustYa/ustYb gene homologues in a
9
,11
search for novel RiPP natural products. From these studies, a
RiPP metabolite was isolated and designated as asperipin-2a 1
9
(
Figure 2).
The ustiloxins were originally isolated from the pathogenic
5
−8
fungus Ustilaginoidea virens
and subsequently from Aspergil-
9
,10
lus flavus. A biosynthetic gene cluster (ust) was identified in
Figure 2. Structure and numbering of asperipin-2a.
Asperipin-2a 1 is a bicyclic peptide that contains three
tyrosine residues, threonine and glycine residues, and a 2-
hydroxy-3-phenylpropanoic acid (Hpp). The gene cluster
encoding this bicyclic peptide includes an ustYa/Yb homologous
gene, with the UstY-like protein identified as the sole oxidative
enzyme.
Asperipin-2a was characterized by 2D NMR spectroscopy and
9
mass spectrometry, which indicated the presence of ether cross-
Received: February 5, 2019
Figure 1. RiPP natural products.
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b00488
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
links between the phenolic oxygen of Tyr and the β-position of
Letter
6
Scheme 2. Synthesis of Aziridine 14
3
3
Tyr and between the phenolic oxygen of Tyr and the β-
1
position of Hpp (Figure 2). More recently, improved
production yield of 1 allowed determination of the absolute
configuration by chemical degradation, chiral HPLC, and more
14
detailed NMR analysis.
The unusual structure of asperipin-2a, together with the lack
of information from the initial report regarding the stereo-
13
chemistry at positions C1, C2, and C16, prompted us to
investigate a route toward the total synthesis of asperipin-2a.
Here, we report the first synthesis of the C-terminal macrocycle
of asperipin-2a and confirmation of the stereochemical
3
configuration of the central β-functionalized Tyr residue.
The main challenge in the total synthesis of asperipin-2a is the
formation of the benzyl−aryl ether linkages in the highly
functionalized bicyclic system. A number of methods for the
synthesis of benzyl−aryl ether linkages have been reported,
nucleophile in combination with metal triflate catalysts for the
23,28−30
preparation of tryptophan derivatives.
Accordingly,
combinations of aziridine 14 with tyrosine 15 in the presence
of a range of Lewis acids were screened for their efficiency in
generating ether adduct 16 (Table 1). Of the catalysts screened,
1
5
16,17
including epoxide and aziridine
ring openings and
18,19
Mitsunobu reactions.
We postulated that an aziridine/
epoxide ring opening approach would be suitable for the
synthesis of both of the ether linkages in asperipin-2a. The
devised retrosynthetic analysis is shown in Scheme 1.
Table 1. Optimization of Aziridine Ring Opening
Scheme 1. Retrosynthesis of Asperipin-2a 1
entry
catalyst
solvent
equiv
yield (%)
1
2
3
4
5
6
7
8
9
Cs CO3
TBD
Cu(OTf)2
Sc(OTf)3
Yb(OTf)3
Bi(OTf)3
DMF
1.1
1.1
1.1
1.1
1.1
1.1
0.2
0.5
1.1
0
0
10
5
0
0
25
50
75
2
toluene
toluene
toluene
toluene
toluene
DCM
BF ·OEt2
3
BF ·OEt2
DCM
DCM
3
BF ·OEt2
3
it was found that only BF ·OEt was effective for activation of the
3
2
aziridine. Under optimized conditions, treatment of aziridine 14
with 15 in the presence of 1.1 equiv of BF ·OEt generated the
3
2
ether adduct 16 in 75% yield and 65:35 dr. No evidence of C-
1
6
alkylation was observed. The poor diastereoselectivity
presumably indicates the aziridine ring opening proceeds via
Generation of the N-terminal macrocycle to complete 1 could
be approached through intramolecular substitution of a
an S 1 process with the benzylic carbocation stabilized by the p-
N
3
cinnamate-derived epoxide 4 with the phenolic group of Tyr .
OMe group. Though the dr is poor, when these studies were
initiated, no information regarding the stereochemistry at C16
of asperipin-2a was available, and as such, access to both
diastereomers was deemed a favorable pathway.
The C-terminal macrocycle 6 could be approached through an
analogous ring opening of aziridine 7 with the phenolic group of
6
Tyr . The required aziridine would be accessed via the
corresponding β-hydroxytyrosine 9.
With the conditions optimized for aziridine ring opening in
the model study, application to a fully functionalized peptide was
investigated. Accordingly, synthesis of aziridine-containing
tripeptide 21 was undertaken. An N-nosyl-protected aziridine
was chosen to facilitate subsequent sulfonamide deprotection.
Accordingly, the oxazolidinone 11 was treated with nosyl
chloride followed by hydrolysis of the oxazolidinone and methyl
ester functionalities to give β-hydroxytyrosine 17 (Scheme 3).
Boc-Thr(Bn)-OMe 18 was coupled with Gly-OtBu, followed by
selective deprotection of the Boc group in the presence of the
tert-butyl ester, to give dipeptide 19. Coupling of the dipeptide
19 to β-hydroxytyrosine 17 in the presence of EDC gave
tripeptide 20 in good yield. The β-hydroxytyrosine moiety in 20
was then converted to the aziridine 21 through a Mitsunobu-
The required aziridine component was synthesized through
C−H activation of Boc-Tyr(Me)-OMe 10 as described by
2
0
Shimamoto et al. to give the oxazolidinone 11. Oxazolidinone
1
1 was N-tosylated and subsequently hydrolyzed to give the β-
hydroxytyrosine 13 in good yield. The β-hydroxytyrosine 13 was
then converted to the aziridine 14 in quantitative yield through a
Mitsunobu-type process (Scheme 2).
Aziridine 14 was employed in model studies with tyrosine
derivative 15 to investigate ring opening reactions to furnish the
required ether-linked adduct. The ring opening of aziridine-2-
carboxylates with various nucleophiles has been employed in the
synthesis of a range of β-substituted amino acid deriva-
1
6,21−27
tives.
We and others have employed indole as the
B
DOI: 10.1021/acs.orglett.9b00488
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Scheme 3. Synthesis of Aziridine Tripeptide
type reaction (Scheme 3). The aziridine-containing tripeptide
2
1 was isolated as a single stereoisomer.
Treatment of the aziridine-containing tripeptide 21 with
protected tyrosine 22 under the conditions developed in the
optimized model study (1.1 equiv of BF ·OEt , −78 °C) yielded
3
2
the ether adduct 23 in 75% yield with 60:40 dr (Scheme 4).
Scheme 4. Synthesis of the C-Terminal Macrocycle
1
Figure 3. (A) H NMR analysis of epimers of C-terminal macrocycle,
3
2
4a and 24b, showing signal from β-H of Tyr . (B) ORTEP diagram of
macrocycle 24b.
In conclusion, the C-terminal macrocycle of asperipin-2a has
been synthesized employing an aziridine ring opening reaction
3
6
to generate the Tyr −Tyr alkyl−aryl ether linkage. We have
3
confirmed that the configuration at the β-position of Tyr in
asperipin-2a is “S”. Synthetic efforts toward the total synthesis of
asperipin-2a are continuing.
ASSOCIATED CONTENT
Supporting Information
■
*
S
Subsequent deprotection of the Boc and tBu groups with TFA
was followed by macrocyclization in the presence of HATU to
provide the macrocycle 24 in 50% yield as a mixture of epimers
at the tyrosine β-position.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.9b00488.
Experimental procedures, 1H and 13C NMR spectra of
1
0−14 and 16−24, HPLC traces of 16 and 24 (PDF)
The epimeric macrocycles 24a and 24b were separated by
HPLC and analyzed by NMR spectroscopy, and their spectra
were compared with that of asperipin-2a. A distinctive difference
in the NMR spectra of isomers 24a and 24b was observed for the
Accession Codes
CCDC 1886340 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge via
www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_
request@ccdc.cam.ac.uk, or by contacting The Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge
CB2 1EZ, UK; fax: +44 1223 336033.
3
signal from the β-H of Tyr (corresponding to H16 in asperipin-
2
a). For isomer 24a, the signal for the Tyr β-H occurs as a
doublet at δ 5.12 ppm with a coupling constant of 9.9 Hz, which
is a reasonably close match to that for the equivalent β-H in
12,13
asperipin-2a (δ 5.23 ppm, d, J = 8.4 Hz) (Figure 3A).
contrast, the signal for the Tyr β-H of isomer 24b occurs at δ
.51 ppm with a much smaller coupling constant of 2.7 Hz.
In
AUTHOR INFORMATION
Corresponding Author
■
5
These data suggests that the configuration at position C16 of
asperipin-2a matches that of isomer 24a.
*
E-mail: chutton@unimelb.edu.au.
Further, isomer 24b was successfully crystallized and analyzed
by X-ray crystallography (Figure 3B), which showed this
compound possesses the R-configuration at the β-position of
ORCID
Jonathan M. White: 0000-0002-0707-6257
Craig A. Hutton: 0000-0002-2353-9258
Notes
3
Tyr . Thus, the combination of NMR and X-ray analysis suggest
3
that the Tyr in asperipin-2a is 2S,3S-configured, which
14
corroborates the recent NMR analysis of Ye et al.
The authors declare no competing financial interest.
C
DOI: 10.1021/acs.orglett.9b00488
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
(
13) Nagano, N.; Umemura, M.; Izumikawa, M.; Kawano, J.; Ishii, T.;
ACKNOWLEDGMENTS
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This work was supported by the Australian Research Council
DP180101804) and The University of Melbourne (MRS to
S.S.). The authors acknowledge the support of the Bio21
Institute Mass Spectrometry and Proteomics Facility.
(
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Org. Lett. XXXX, XXX, XXX−XXX