Total Synthesis of the Putative Structure of Asperipin-2a and Stereochemical Reassignment

Article abstract of DOI:10.1021/acs.orglett.0c02884

The total synthesis of the putative structure of asperipin-2a is described. The synthesis features ether cross-links between the phenolic oxygen of Tyr6 and the β position of Tyr3 and the phenolic oxygen of Tyr3 and the β position of Hpp1 in the unique 17- and 14-membered bicyclic structure of asperipin-2a, respectively. The synthesized putative structure does not match the natural product, and a stereochemical reassignment is postulated.

Full text of DOI:10.1021/acs.orglett.0c02884

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Letter
Total Synthesis of the Putative Structure of Asperipin-2a and
Stereochemical Reassignment
Sadegh Shabani, Jonathan M. White, and Craig A. Hutton*
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ABSTRACT: The total synthesis of the putative structure of asperipin-2a is described. The synthesis features ether cross-links
between the phenolic oxygen of Tyr6 and the β position of Tyr3 and the phenolic oxygen of Tyr3 and the β position of Hpp1 in the
unique 17- and 14-membered bicyclic structure of asperipin-2a, respectively. The synthesized putative structure does not match the
natural product, and a stereochemical reassignment is postulated.
The main challenge in the total synthesis of asperipin-2a is
speripin-2a is a ribosomally synthesized and post-
Atranslationally modified peptide (RiPP) isolated from the formation of the two alkyl−aryl ether linkages in the highly
Aspergillus flavus (Figure 1).1
functionalized bicyclic system. A number of methods including
epoxide ring opening12 and Mitsunobu reactions13,14 have
been reported for the synthesis of benzyl−aryl ethers. While
we initially postulated an aziridine/epoxide ring-opening
approach toward both ether linkages in asperipin-2a,11 after
much experimentation, we decided to pursue a Mitsunobu
approach to the ether linkage in the N-terminal ring.
Accordingly, (R)-glyceraldehyde acetonide 2 was treated with
phenylmagnesium bromide in the presence of CuI to give the
alcohol 3 with 99:1 d.r. (Scheme 1).15,16 Analysis of 3 by X-ray
crystallography confirmed the (R,R) configuration (inset). The
Figure 1. Proposed structure of asperipin-2a 1.
Mitsunobu reaction of alcohol 3 with tyrosine derivative 5
under sonication and at high concentration (3 M) successfully
generated ether adduct 6. Compound 6 was nosylated; then,
Asperipin-2a is a bicyclic hexapeptide derived from the
sequence FYYTGY. The 2D structure of asperipin-2a was
reported in 2016,1 with the biosynthetic pathway and absolute
configuration unveiled in 2019.2−7 The unique structure of
asperipin-2a, together with the lack of information regarding its
biological role or activity and our interest in cross-linked
peptide natural products,8−10 prompted us to investigate a
total synthesis of this compound. We previously reported a
route to the C-terminal macrocycle of asperipin-2a.11 Herein
we report the first total synthesis of the putative structure of
asperipin-2a and postulate a stereochemical reassignment of
the 2-hydroxy-3-phenylpropanoic acid (Hpp) residue on the
N-terminal ring.
the oxazolidinone and ester groups of 7 were hydrolyzed to
afford the β-hydroxytyrosine derivative 8 (Scheme 1).
The β-hydroxytyrosine 8 was coupled to Thr(Bn)-Gly-OtBu
9 to give the peptide 10 in 80% yield (Scheme 2). The
formation of the Tyr-3−Tyr-6 ether cross-link was then
Received: September 3, 2020
https://dx.doi.org/10.1021/acs.orglett.0c02884
© XXXX American Chemical Society
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Macrocyclization of peptide 13 using HATU/HOAt
afforded a diastereomeric mixture of 14 in 55% yield. The
isomers were separated by HPLC, and the minor diastereomer
14b was successfully crystallized and analyzed by X-ray
crystallography (Figure 2), which confirmed the 1R,2S,16R
Scheme 1. Synthesis of β-Hydroxytyrosine 8 (Inset: ORTEP
of 3)
Figure 2. ORTEP diagram of 14b (ellipsoids at the 30% probability
level).
Scheme 2. Synthesis of Macrocycle 14
stereochemistry numbering according to that of asperipin-2a).
The configuration of the central β-hydroxytyrosine moiety was
in accordance with our previous study.11 Thus the major
isomer 14a possesses 1R,2S,16S stereochemistry. In this stage
of our synthesis, the absolute configuration of asperipin-2a was
reported by Ye et al. as 1R,2S,16S,7 suggesting that the 2S and
16S configurations of 14a match those of the natural product.
The configuration at the C1 secondary alcohol of 14a is the
opposite of that predicted by Ye et al., requiring an inversion
step later in the synthesis (Note: the R/S notation at C1 is
switched due to a difference in priorities between the primary
alcohol present in 14a and the amide group in asperipin-2a 1).
Next, selective oxidation of the primary alcohol of 14a, in
the presence of the secondary alcohol, was pursued. The
treatment of 14a with TEMPO and NaOCl was effective,
generating carboxylic acid 15 in 70% yield (Scheme 3).24
Coupling of 15 with Tyr(Bn)-OtBu 16 afforded peptide 17 in
80% yield; then, deprotection of the nosyl group afforded 18 in
75% yield. Deprotection of the tert-butyl ester, followed by
macrocyclization using HATU and HOAt, provided the
Scheme 3. Synthesis of Protected Asperipin 20
pursued using our previously developed aziridine strategy. The
β-hydroxytyrosine moiety of peptide 10 was converted to the
aziridine 11 in 90% yield as a single diastereomer. The
treatment of aziridine 11 with protected tyrosine Boc-Tyr-OAll
12 in the presence of BF3·OEt2, followed by the deprotection
of acetonide, tBu, and Boc groups, yielded the ether-linked
adduct 13 in 72% yield with 2.4:1 d.r. (Scheme 2). While the
diastereoselectivity of the aziridine ring opening is moderate, it
was an improvement over our model studies.11 Ring-opening
reactions of 3-arylaziridine-2-carboxylates to generate β-
functionalized arylalanine derivatives have been reported
using a variety of acid catalysts and with a range of
stereochemical outcomes.17−22 The major isomer of 13 is
formed with the retention of stereochemistry (vide infra),
suggesting that aziridine 11 undergoes a Lewis-acid-catalyzed
SN1 process with substrate-directed selectivity for the attack of
the tyrosine phenol at the intermediate benzylic carbocation,23
generating the (15S,16S) isomer of 13 as the major product.
B
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bicyclic compound 19 in 15% yield. The corresponding
cyclodimer was isolated as the major product in 25% yield.
Despite the low yielding macrocyclization, with the bicyclic
framework of asperipin-2a successfully constructed, conversion
to asperipin-2a was pursued. Inversion of the secondary
alcohol at C1 was required to generate the absolute
configuration of asperipin deduced by Ye et al. Accordingly,
a Lattrell-Dax reaction25,26 was performed on 19 to provide
protected asperipin-2a 20 in 70% yield. Unfortunately, at this
stage all attempts to cleave the benzyl groups from 20 resulted
in decomposition. We postulate that the ether linkages in 20
are not stable to hydrogenolysis conditions.
To access a total synthesis of asperipin-2a, a modified
protecting group strategy was required. Accordingly, we
employed a global allyl-protecting group strategy. Furthermore,
given the issue of cyclodimerization during the formation of
the second macrocycle, an alternative macrocyclization
position was pursued. Allyl-protected threonine derivative 21
was first coupled to β-hydroxytyrosine 8, and the resultant
peptide was converted to the aziridine 22 in 90% yield
(Scheme 4). Subsequent aziridine ring opening of 22 with Boc-
Tyr-OAll 12 afforded the ether-linked adduct 23 with an
improved 3:1 dr. Following separation of the diastereomers,
the major isomer 23a was treated with TFA to effect
deprotection of the acetonide, tBu, and Boc groups.
Subsequent macrocyclization using HATU/HOAt afforded
macrocycle 24 in 60% yield. Selective oxidation of the primary
alcohol in the presence of the secondary alcohol was again
effected by treatment with TEMPO and NaOCl to generate
the corresponding carboxylic acid in 85% yield. Deprotection
of the nosyl group afforded hydroxy acid 25 in 85% yield
(Scheme 4).
To investigate an improved route to the N-terminal ring, we
decided to perform the macrocyclization at the Hpp-1−Tyr-2
amide bond rather than the Tyr-2−Tyr-3 attempted
previously. Accordingly, Boc-Tyr(allyl)-OH 26 was activated
with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),
then coupled to the amine of 25 to afford peptide 27 in 80%
yield. The Boc group was removed by treatment with TFA,
and subsequent macrocyclization using HATU and HOAt
successfully provided the bicyclic peptide 28 in good yield
(64%) with no evidence of cyclodimerization, validating the
revised route to the N-terminal ring. The Lattrell-Dax
inversion of the secondary alcohol at C1 of 28 was effected
with tetrabutyl ammonium nitrite, which proceeded in better
yield than using KNO2, to afford the protected asperipin-2a 29.
Global deprotection of the three allyl groups was effected by
treatment with Pd(PPh3)4/PhSiH3 to provide putative
asperipin-2a 1 in 48% yield (Scheme 4).
1
A comparison of the H NMR data of the natural product
and the synthesized compound revealed significant differences,
most notably for the signals corresponding to the hydrogen at
C2. A sample of asperipin-2a natural product was kindly
1
donated by Prof. Oikawa. The H2 signal in the H NMR
Scheme 4. Synthesis of the Proposed Structure of Asperipin-
2a 1
spectrum of natural asperipin-2a is observed as a broad singlet
peak at δ 5.96, with the small coupling constant to H1
indicative of a dihedral angle H1−C1−C2−H2 of close to 90°.
However, the corresponding signal of H2 in the synthesized
compound appeared as a doublet at δ 5.58 with coupling
constant of 9.0 Hz to H1 (Figure 3). Furthermore, HPLC
analysis of the natural and synthesized compounds indicated
that they are not identical (Figure 4).
Figure 3. NMR comparison of natural and synthetic asperipin.
With the synthesized asperipin not matching the natural
product, we sought to determine the point of discrepancy. The
configuration at all α-C atoms was unambiguously established
by Oikawa and coworkers through hydrogenolysis−hydrolysis
of the natural product and analysis of the constituent amino
acids through L-FDLA (1-fluoro-2,4-dinitrophenyl-5-L-leucina-
mide) derivatization and LC−MS.7 Chiral HPLC analysis of
the underivatized hydrolysate confirmed the presence of (R)-3-
phenyllactic acid, indicating the configuration at C1 (the α-C
C
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near-perpendicular orientation. Further evidence is provided
by NMR analysis of precursor 28, in which the H1 signal is
observed as a doublet at δ 5.96 ppm with a small coupling
constant of 3.2 Hz (Figure S88), similar to that observed in the
natural product. This similarity suggests that 28 and natural
asperipin-2a possess the same relative stereochemistry at C1−
C2. Because 28 possesses 1S,2S configuration, this supports
the 1R,2R assignment for natural asperipin-2a (Figure 5C,D).
In conclusion, we have completed the first total synthesis of
the putative structure 1 of the bicyclic peptide natural product
asperipin-2a. The synthesized compound was not identical to
the natural product, prompting reanalysis of the stereo-
chemistry of asperipin-2a. We conclude that natural asper-
ipin-2a possesses (1R,2R) stereochemistry.
Figure 4. HPLC traces of coinjected natural and synthetic asperipin.
Synthetic product retention time: 12.16 min. Natural product
retention time: 12.61 min. Wavelength: 214 nm.
of Hpp-1) to be R. The configuration of the β-position of Tyr-
3 (C16) was assigned by Oikawa and coworkers by NOESY
NMR analysis and confirmed by our X-ray crystallographic
analysis (of 14b and ref 11).
Thus only the configuration at C2 remains ambiguous.
Given that the synthetic material is the 1R,2S isomer and is not
the natural product, it follows that natural asperipin-2a is the
1R,2R isomer.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c02884.
Oikawa and coworkers proposed that the small coupling
between H1 and H2 is indicative of a Gauche or near-
perpendicular orientation, as shown in Figure 5A, thereby
Experimental procedures and characterization (NMR
and MS spectra, HLPC traces, X-ray crystallography
data) (PDF)
Accession Codes
CCDC 2019840−2019841 contain the supplementary crys-
tallographic 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.
AUTHOR INFORMATION
Figure 5. Newman projections of C1−C2 isomers of asperipin. S =
■
small predicted JH1−H2, L = large predicted JH1−H2
.
Corresponding Author
Craig A. Hutton − School of Chemistry and Bio21 Molecular
assigning the 2S-configuration at C2. However, the stereo-
chemistry of the synthetic compound is established as 2S by
the following analysis: X-ray crystallographic analysis of 14b
reveals the 1R,2S (erythro) absolute stereochemistry, and thus
14a, and by analogy 24, is also 1R,2S-configured. Compound
24 is transformed to the 1S,2S (erythro) precursor 28, due to
the change in Cahn−Ingold−Prelog (CIP) priorities upon
oxidation of the primary alcohol to the acid. Latrell-Dax
inversion at C1 of 28 must therefore generate the 1R,2S-
(threo-) configured 29, which after deprotection yields the
1R,2S product (Figures 5B and 6). Thus, with the synthetic
and natural compounds being nonidentical and C2 of the
natural product being the only ambiguous stereocenter, natural
asperipin-2a must be the 1R,2R isomer.
Science and Biotechnology Institute, The University of
Melbourne, Melbourne, Victoria 3010, Australia; orcid.org/
0000-0002-2353-9258; Email: chutton@unimelb.edu.au
Authors
Sadegh Shabani − School of Chemistry and Bio21 Molecular
Science and Biotechnology Institute, The University of
Melbourne, Melbourne, Victoria 3010, Australia; orcid.org/
0000-0003-4542-9858
Jonathan M. White − School of Chemistry and Bio21 Molecular
Science and Biotechnology Institute, The University of
Melbourne, Melbourne, Victoria 3010, Australia; orcid.org/
0000-0002-0707-6257
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.0c02884
The small JH1−H2 value observed for the natural product is
consistent with the 1R,2R configuration adopting the
conformation shown in Figure 5D, with H1 and H2 in a
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This project was funded by the Australian Research Council
(DP180101804). S.S. thanks the University of Melbourne for a
Melbourne Research Scholarship (MRS) and an Award from
the Albert Shimmins Fund. We thank Prof. Oikawa (Hokkaido
University) for the provision of a sample of natural asperipin-
2a.
Figure 6. Reassigned stereochemical assignment of asperipin-2a.
D
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Hydroxy-α-Amino Acids via α-Hydroxy-β-Amino Esters. Tetrahedron
2013, 69, 8885−8898.
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