Solid-Phase-Based Total Synthesis and Stereochemical Assignment of the Cryptic Natural Product Aurantizolicin

Article abstract of DOI:10.1021/acs.orglett.8b03940

The total synthesis and stereochemical assignment of the polyazole cyclopeptide aurantizolicin was achieved by connecting the solution synthesis of building blocks with solid-phase peptide synthesis. Macrothiolactonization and an aza-Wittig reaction provi

Full text of DOI:10.1021/acs.orglett.8b03940

Letter
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Solid-Phase-Based Total Synthesis and Stereochemical Assignment
of the Cryptic Natural Product Aurantizolicin
†
‡
‡
,†
Ansgar Oberheide, Sebastian Pflanze, Pierre Stallforth, and Hans-Dieter Arndt*
†
Friedrich-Schiller-Universitat
̈
, Institut fu
Leibniz Institute for Natural Product Research and Infection Biology, Hans Kno
Communication, Beutenbergstr. 11A, D-07745 Jena, Germany
S Supporting Information
̈
r Organische und Makromolekulare Chemie, Humboldtstr. 10, D-07743 Jena, Germany
‡
̈
ll Institute (HKI), Chemistry of Microbial
*
ABSTRACT: The total synthesis and stereochemical assign-
ment of the polyazole cyclopeptide aurantizolicin was achieved
by connecting the solution synthesis of building blocks with
solid-phase peptide synthesis. Macrothiolactonization and an
aza-Wittig reaction provided the natural product macrocycle
in high yield as well as key stereoisomers. NMR comparison as
well as isolation of the natural product from the producer
organism Streptomyces aurantiacus confirmed the presence and
sequence of one L-Ile and one D-allo-Ile residue in
aurantizolicin.
arine organisms produce a broad panel of structurally
M
diverse metabolites that often display unique chemical
1
properties as well as distinct biological activities. Especially
natural products containing five-membered heterocycles such
as imidazoles, oxazoles, or thiazoles receive interest with
respect to their isolation, biosynthesis, bioactivity, and
2
synthetic accessibility. Beyond the large class of linear
3
azol(in)e-containing peptides (LAPs), several macrocyclic
4
5
6,7
8
9,10
peptides 1, 2, 3, 4, and 5
have been isolated and
structurally characterized that feature five catenated azoles
bridged by a lipophilic tripeptide unit (Figure 1).
While being produced by different marine actinobacteria, all
5
the octapeptide-derived macrocycles curacozole (2), uruktha-
6
,7
8
pelstatin A (3), mechercharstatin (4), and YM-216391
9
,10
(5)
inhibit the cell growth of different human cancer cell
lines with high potency. Neither the molecular mechanism of
action of these natural products nor the environmental cues
triggering their production have been clarified to date. Closely
1
1
related to compounds 1−5 is the smaller marthiapeptide A
(
6) that shows antibacterial activity against Gram-positive
bacteria but is considerably less cytotoxic.
Figure 1. Polyazole cyclopeptides 1−5 of the YM-216391 family and
the related but smaller marthiapeptide A (6).
The biosynthesis of YM-216391 (5) was shown to feature
posttranslational modifications of a ribosomally synthesized
1
2
octaprepeptide. Based on the analysis of bacterial genomes
for the rapid modification of all α-amino acid residues (Scheme
1). Ideally, the central thiazole ring could be simplified via an
4
and biosynthesis genes, the structures of aurantizolicin (1)
1
4
5
intramolecular aza-Wittig reaction to the less strained
and curacozole (2) were predicted. These natural products
azidothiolactone 7 that should be easily available from the
ω-mercapto carboxylic acid 8. This highly modified octapep-
tide might be assembled by three consecutive amide couplings
to identify the biazoles 9 and 11 as well as two Ile derivatives
were isolated in small amounts and structurally assigned.
However, no YmG epimerase homolog had been identified,
leaving amino acid stereochemistry unclear. Heterologous
12
overexpression of 1 succeeded recently by using promoter
1
3
exchange. Marfey’s analysis suggested the presence of one D-
allo-Ile residue in the product such obtained. We have
therefore designed a synthesis of aurantizolicin (1) that allows
Received: December 10, 2018
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b03940
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Scheme 1. Synthesis Planning for Aurantizolicin (1)
oxazole 17 in a two-step procedure including oxazoline
16
formation by using diethylaminosulfur trifluoride (DAST)
17
and oxidation with DBU/BrCCl3. Methyl ester 17 was
cleanly deprotected with lithium hydroxide to give carboxylic
acid 18. In order to prepare the building block for the
macrocyclization chemistry, the N-Boc-thiazolidine was
deprotected using TFA and the free thiol was reprotected
with TrCl in TFA. Then, a ZnSO -catalyzed diazo transfer to
4
the free amine gave access to the advanced azide building block
9
in 66% yield over three steps. Crucial for the success of the
18
diazo transfer was the use of ZnSO instead of ZnCl . This
4
2
sequence features a late-stage incorporation of the reactive
azide functionality which was found to be labile during oxazole
formation as well as to be prone to racemization and β-
14
elimination under basic conditions. Furthermore, the use of
toxic tin reagents for the saponification of the methyl ester is
19
avoided.
For the synthesis of bioxazole 11, suitably protected glycine
9 and serine 20 were coupled to dipeptide 21 (90% yield,
1
Scheme 3) and cyclodehydrated with DAST. Oxidation with
a
Scheme 3. Synthesis of Bioxazole 11
1
0 as essential building blocks. By using solid-phase peptide
synthesis (SPPS) for chain assembly, rapid and high yielding
construction of the target molecule should be achieved.
The synthesis of biazole 9 began with transforming L-
15
cysteine into the fully protected thiocarboxamide 13, which
was then combined with methyl bromopyruvate in a Hantzsch-
type synthesis to afford thiazole 14 in 86% yield (Scheme 2).
Saponifying methyl ester 14 using LiOH provided carboxylic
acid 15 (97% yield). Subsequent amide coupling with racemic
β-hydroxy phenylalanine methylester hydrochloride yielded
thiazolopeptide 16 as a diastereomeric mixture in 84% yield.
The β-hydroxy amide 16 was then smoothly transferred into
a
Scheme 2. Synthesis of Biazole 9
a
EDCl = 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydro-
chloride, HOBt = 1-hydroxybenzotriazole.
DBU/BrCCl afforded oxazole 22 with 69% yield over both
3
steps. The methyl ester was cleaved (LiOH), and the
corresponding acid 23 was coupled with serine methyl ester
hydrochloride to give β-hydroxy amide 24 in 79% yield.
Bioxazole 25 was obtained in 65% yield by using DAST and
20
DBU/BrCCl3. The synthesis of building block 11 was
completed by saponification of the methyl ester (LiOH) and
exchange of the Boc for an Fmoc protecting group for SPPS.
Carboxylic acid 11 was obtained in 65% yield over three steps.
With both biazole building blocks 9 and 11 in hand, the
pseudo-octapeptide 8 was assembled on solid support (Scheme
4
). 2-Chlorotrityl resin was selected to allow retaining the STr
group upon peptide release (HFIP). Bioxazole 11 was attached
to the resin at 0.60 mmol/g which resulted in a loading of
0.37 mmol/g. Excess bioxazole 11 from the resin loading was
partially recovered (72% of theoretical yield). Cleavage of the
terminal Fmoc groups was performed with 20% piperidine in
DMF. The coupling of the two α-amino acids (Fmoc-Ile and
a
DAST = diethylaminosulfur trifluoride, DBU = 1,8-diazabicyclo-
[
5.4.0]undec-7-ene, HBTU = O-(benzotriazole-1-yl)-1,1,3,3-tetra-
methyluronium, pyr = pyridine, TFAA = trifluoroacetic anhydride,
Tr = trityl.
B
DOI: 10.1021/acs.orglett.8b03940
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
a
Scheme 4. Assembly of the Cyclization Precursor 8 by SPPS and Completion of the Total Synthesis of Aurantizolicin (1)
a
The diastereomeric cyclopeptides 26 and 27 were analogously synthesized. HFIP = 1,1,1,3,3,3-hexafluoro-2-propanol, 2,6-lutid. = 2,6-
dimethylpyridine, PyBOP = (benzotriazol-1-yl-oxy)tripyrrolidinophosphonium hexafluorophosphate, TFA = trifluoroacetic acid.
Fmoc-D-allo-Ile) succeeded under regular conditions for
2
1
SPPS.
A slight excess of the advanced biazole 9 (1.5 equiv) was
found to be optimal for the final coupling. HFIP-induced
release from resin delivered octapeptide 8 in 70% yield.
Benefits for increasing amounts of 9 were minute, whereas
reduced stoichiometry compromised yields (1.1 equiv: 56%).
Acid-mediated detritylation of peptide 8 afforded the ω-
mercapto carboxylic acid as the cyclization precursor. The
14
crucial macrocyclization proceeded smoothly. Slow addition
of the cyclization precursor to PyBOP in moderate dilution
delivered macrothiolactone 7 in 80% yield. The PPh -mediated
3
aza-Wittig reaction of azidothiolactone 7 formed the
corresponding thiazoline, which was directly oxidized with
DBU/BrCCl to provide aurantizolicin (1, 48% yield).
3
1
The comparison of the reported H chemical shifts of the
natural product to our synthetic sample revealed a very high
similarity (average Δδ = 13.3 ppb). Compared to linear
peptides for which extensive NMR studies have been
Figure 2. Deviations of 1H chemical shifts (ppm) between the
synthetic cyclopeptides 1, 26, and 27 compared to reported values in
DMSO-d6 (500 and 600 MHz, respectively). Positions 1−16
represent the tripeptide section, and positions 17−37, the polyazole
backbone.
22
performed to distinguish L-Ile and D-allo-Ile, changes of the
configuration of amino acids embedded in a macrocycle or
protein can lead to strongly altered conformations and thus
23
individual key signatures in NMR spectra. To confirm the
stereochemical assignment and further test our synthesis
design, we synthesized the diastereomeric cyclopeptides 26
and 27 within 7 days in 22% and 17% yield respectively,
starting from loaded resin (see Supporting Information (SI)
for details). For these isomers either both isoleucine residues
feature the L-configuration (cyclopeptide 26) or the order of L-
whereas the data of synthetic cyclopeptide 1 perfectly match
1
3
those reported (see SI). Furthermore, C NMR comparison
led to similar conclusions. The ample amount of material
available from synthesis also allowed detecting and assigning all
carbon resonances (see SI for details). Taken together, our
data confirm the tripeptide sequence of the naturally occurring
aurantizolicin (1) is composed of Gly-L-Ile-D-allo-Ile.
2
4
Ile and D-allo-Ile is inverted (cyclopeptide 27).
For the structurally very similar curacozole (2), analogous
results were obtained. The spectra of synthetic cyclopeptide 1
and of curacozole (2) coincide strikingly. The crucial CHα
signal chemical shifts as well as coupling constants match up
very well (see SI). Based on these data, we conclude that the
configuration of the recently reported curacozole (2) is
identical to that of aurantizolicin (1) and other known
members of this class of natural products.
1
Analysis of the H NMR spectra of the three synthetic
stereoisomers (Figure 2) revealed large deviations in the H
1
chemical shifts of stereoisomers 26 and 27 when compared to
reported values (average Δδ = 108 and 47.5 ppb, respectively).
As expected, the configuration of the tripeptide does not
1
strongly influence the H chemical shifts of the polyazole
backbone. However, marked differences between the stereo-
isomers 26 and 27 and the naturally occurring aurantizolicin
In order to unequivocally confirm that synthetic aurantizo-
licin is identical to the natural product, we isolated it from the
were observed for the CH and NH signals of the tripeptide,
α
C
DOI: 10.1021/acs.orglett.8b03940
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
4
,13
original producer S. aurantiacus.
Extensive screening efforts
(2) (a) Jin, Z. Nat. Prod. Rep. 2013, 30, 869−915. (b) Jin, Z. Nat.
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allowed us to identify culture conditions that led to the
expression of the aurantizolicin biosynthesis genes in sufficient
amounts for HPLC- and LC-MS-based comparison. Mass
selected LC-MS experiments as well as comparison of HPLC
profiles showed that synthetic 1 and natural aurantizolicin
match perfectly, whereas the diastereoisomers 26 and 27
displayed different retention times (see SI). Beyond con-
firmation of structural identity, our experiments show that
expression of the aurantizolicin biosynthesis genes is tightly
regulated. This finding prompts the question for cues necessary
to trigger the production of this cytotoxic metabolite of S.
aurantiacus in Nature.
In summary, we completed the first total synthesis of
aurantizolicin (1) and of two stereoisomers 26 and 27 by
designing a rapid assembly of the cyclization precursor
peptides on solid support, followed by an optimized, high-
yielding macrocyclization procedure. NMR data and the
comparison with isolated materials from the producer strain
S. aurantiacus confirm that synthetic cyclopeptide 1 is identical
to the naturally occurring aurantizolicin. This efficient new
synthesis format provides sufficient material for in-depth
biological studies and will equally facilitate investigations of
non-natural analogs. The stable synthetic L-Ile epimer suggests
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25
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ASSOCIATED CONTENT
(
■
*
S
Supporting Information
(
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.8b03940.
(
Chem. 2013, 2013, 3290−3315.
General methods; synthetic procedures and physical
data; NMR spectra (PDF)
(19) Nicolaou, K. C.; Estrada, A. A.; Zak, M.; Lee, S. H.; Safina, B. S.
Angew. Chem., Int. Ed. 2005, 44, 1378−1382.
(20) Double cyclodehydration reaction of the tripeptide NBoc-Gly-
Ser-Ser-OMe by using DAST smoothly delivered the corresponding
bioxazoline. However, the subsequent oxidation reaction turned out
to be problematic, giving the desired bioxazole 26 in low yield only
AUTHOR INFORMATION
Corresponding Author
■
(
20%).
21) (a) El-Faham, A.; Albericio, F. Chem. Rev. 2011, 111, 6557−
602. (b) Kates, S. A.; Albericio, F. Solid-phase synthesis: A practical
guide; CRC Press: New York, 2000.
22) Anderson, Z. J.; Hobson, C.; Needley, R.; Song, L.; Perryman,
*
E-mail: hd.arndt@uni-jena.de.
ORCID
(
6
Hans-Dieter Arndt: 0000-0002-0792-1422
Notes
(
M. S.; Kerby, P.; Fox, D. J. Org. Biomol. Chem. 2017, 15, 9372−9378.
(23) (a) Kikuchi, H.; Hoshikawa, T.; Fujimura, S.; Sakata, N.;
Kurata, S.; Katou, Y.; Oshima, Y. J. Nat. Prod. 2015, 78, 1949−1956.
(b) Dang, B.; Shen, R.; Kubota, T.; Mandal, K.; Bezanilla, F.; Roux,
B.; Kent, S. B. H. Angew. Chem., Int. Ed. 2017, 56, 3324−3328.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
A.O. and S.P. gratefully acknowledge PhD fellowships from the
Carl-Zeiss-Stiftung and the IRLS for Microbial and Bio-
molecular Interactions, respectively. P.S. and H.D.A. were
supported by the DFG (SFB1127 ChemBioSys). P.S. is
thankful to the Leibniz Association for funding. The authors
thank Dr. Johannes Arp (HKI) for initial culturing experiments
and the NMR platform Jena for excellent support. This work
benefitted in part from equipment grants of the TMWWDG
(24) The presence of D-Ile or two D-allo-Ile residues was found
unlikely considering the biosynthesis logic of these natural products.
This analysis was substantiated in parallel to our work by Pei et al.; see
ref 13.
(25) Spontaneous changes of configuration have been observed for
ribosomally expressed azole-containing cyclopeptides; e.g., see ref 14
and: Schoof, S.; Arndt, H.-D. Chem. Commun. 2009, 7113.
(
43-5572-321-12040-12) and the DFG (INST 275/331-1
FUGG).
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DOI: 10.1021/acs.orglett.8b03940
Org. Lett. XXXX, XXX, XXX−XXX