Biomimetic Synthesis of Urukthapelstatin A by Aza-Wittig Ring Contraction

Article abstract of DOI:10.1002/ejoc.201600994

Marine bacteria produce highly cytotoxic polyheterocyclic cyclopeptide natural products by ribosomal peptide biosynthesis. Among them, urukthapelstatin A features a chain of five 2,4′-connected azole rings within a cyclo-octapeptide framework. We report a novel synthesis design that uses only α-amino acids as starting materials that leads to an efficient and stereoselective total synthesis of urukthapelstatin A. Kinetically favored macro-thiolactonization and high-yielding aza-Wittig heterocyclization to contract the macrocycle were crucial for success. These investigations additionally uncovered the unsuspected configurational lability of the embedded enamide substructure in solution.

Full text of DOI:10.1002/ejoc.201600994

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Accepted Article
Title: Biomimetic synthesis of urukthapelstatin A by Aza-Wittig ring contraction
Authors: Sebastian Schwenk; Cyril Ronco; Ansgar Oberheide; Hans-Dieter Arndt
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201600994
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European Journal of Organic Chemistry
10.1002/ejoc.201600994
Biomimetic synthesis of urukthapelstatin A by Aza-Wittig ring contraction**
Sebastian Schwenk, Cyril Ronco, Ansgar Oberheide, and Hans-Dieter Arndt*
[*] M. Sc. S. Schwenk, Dr. C. Ronco+, B. Sc. A. Oberheide, Prof. Dr. H.-D. Arndt
Friedrich-Schiller-Universität, Institut für Organische Chemie und Makromolekulare Chemie
Humboldtstr. 10, 07743 Jena (Germany)
E-mail: hd.arndt@uni-jena.de
http://www.arndtgroup.uni-jena.de/
+New address:
Prof. Dr. C. Ronco, Université de Nice Sophia-Antipolis, Institut de Chimie, 28 Avenue
Valrose, 06108 Nice Cedex 2 (France)
[**]This research was supported in part by the Alexander-von-Humboldt foundation (fellowship to
C.R.) and the TMWWDG (grant No. 43-5572-321-12040-12). We thank Ms. Heidi Zdon for lab
support and the NMR platform Jena (Dr. Peter Bellstedt) for excellent service.
Abstract
Marine bacteria produce highly cytotoxic polyheterocyclic cyclopeptide natural products by
ribosomal peptide biosynthesis. Among them, urukthapelstatin A features a chain of five 2,4’-
connected azole rings within an cyclooctapeptide framework. We report on a novel synthesis design
that uses only -amino acids as starting materials and has led to an efficient and stereoselective total
synthesis of urukthapelstatin A. A kinetically favored macrothiolactonizations and a high-yielding
Aza-Wittig heterocyclization for contracting the macrocyle were crucial for success. These investi-
gations have additionally uncovered the unsuspected configurational lability of the embedded
enamide substructure in solution.

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Polyheterocyclic, macrocyclic natural products enjoy continued interest both in biosynthesis
research as well as unique hit compounds for drug design.[1] Among them, purely amino acid
derived polyazole cyclopeptides have been identified that are produced by Actinomycetes from
marine environments. Three compounds, urukthapelstatin A (1, Figure 1),[2] mechercharstatin (2,
also known as mechercharmycin or IB-01211),[3] and YM-216391 (3)[4] are derived from eight
amino acids and share very similar structures with five contiguously catenated azoles linked via their
2 and 4’-positions, all closed by a lipophilic tripeptide section. They are produced by different
species but show all high cytotoxicity in cancer cell line growth inhibition in the 10 nM range,
potentially indicating a common ecological purpose or similar molecular target that remains unclear
to date. The related but smaller marthiapeptide (4) contains four catenated thiazol(in)es and displays
reduced cytotoxicity (sub-M).[5] In contrast, telomestatin (5) features eight cyclocatenated azoles
and is considerably less toxic (M).[6] It however displays interesting selectivity that is attributed to
high affinity of the almost planar polyazole 5 to G-quadruplex structures in telomeres and its
resulting interference with telomere signaling in sensitive cell lines.[7]
The biosynthesis of polyazole cyclopeptides can be assumed to proceed by ribosomal peptide
biosynthesis and posttranslational modification (RiPPs), as it was clearly shown in the case of YM-
216391.[8] This pathway is shared by the structurally related bacteriocins, a large class of metabolites
from marine cyanobacteria that is rich in cyclopeptide congeners containing azole rings.[9]
Bacteriocins with directly connected azole rings have not (yet) been described, however.
Due to their remarkable bioactivity which seems to be linked to the polyazole fragments embedded
in a ring structure, total syntheses have been pursued for urukthapelstatin A (1),[10] mechercharstatin
(2),[11] YM-216391 (3),[12] telomestatin (4),[13] and marthiapeptide (5).[14] These reports differ
conceptually in the methods employed for closing the macrocycle and for azole formation in late
stage intermediates. However, most of these works report either a problematic macrocycle formation
event or difficulties when creating the ultimate azole ring in the macrocycle.
These issues likely relate to the molecular structure of oxazole and thiazole-containing scaffolds
(Figure 2). Gas phase measurements report opening angles of 146° for oxazole[15] and 134° for
thiazole[16] for substituents attached on a 2,4-disubstituted azole 11. While more elaborate analyses
are certainly possible, simple geometry suggests that a ring of such azoles should suffer from angle
strain when it is composed from less than ten oxazoles or less than eight thiazoles (12, average
corner angle  for a decagon: 144°; octagon: 135°). Likewise, enthalpic and/or entropic strain may
increase when an azole ring is constructed within a macrocycle already formed. Either a change in

European Journal of Organic Chemistry
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angle occurs when side-chain functionalized peptides are employed (path A) or the chain length is
shortened when the chain is formed via the heteroatom (path B). However, path B features a larger
ring size than A, and hence could be more easily accessed by initial macrocyclization. In terms of
this analysis an Aza-Wittig reaction should be suited very well for azole formation as it’s precursor
topology conforms to path B and azole formation will be induced under neutral conditions in an
intramolecular fashion via an iminophosphorane intermediate.[17]
In order to probe this hypothesis we designed a novel synthesis of urukthapelstatin A based on the
considerations above (Figure 1). As Aza-Wittig reactions perform especially well for thiazole
formation, retrosynthetic dehydration (a) and Aza-Wittig disconnection (b) led us to the azido-
macrothiolactone 6, that should be easily prepared by thiolactonization (c) from -mercapto ester 7.
Precursor 7 features a turn-inducing DLL-configured tripeptide in its center procting two semi-rigid
appendages that should facilitate ring closure.[18] It disconnects favorably into bis-azole fragment 8,
regular tripeptide 9, and aminoalcohol 10 by peptide coupling (d) and condensing oxazole formation
(e). Bis-azole 8 should be similarly accessed from a mono-thioazole acid (f). Overall, all building
blocks can such be assembled from natural amino acid, leading to a biomimetic synthesis design.[1c]
The synthesis began with the construction of the azole building blocks (Scheme 1). Two charges of
serine (13) were divergently protected as allyl ester 14 (85%) or by installing TBS- and Boc-
protecting groups (→ 15, 95%)[26] and then united to dipeptide 16 by using EDC (76% yield).
Oxazole formation mediated by DAST[19a] followed by BrCCl3/DBU oxidation[20] performed
reproducibly to deliver oxazole 17 in 68% yield. Deprotection to amino alcohol 10 was then
quantitatively achieved with aqueous TFA.
For the N-terminus cysteine derivative 18 was converted to azide 20 by azide transfer[17] and used to
construct thiazole 21 by high yielding one-pot Tr-deprotection - thioesterification - Aza-Wittig -
oxidation sequence employing acid 19 (4 steps, 87% yield). Its NHBoc group was swiftly exchanged
for an azide group (→ 22, 95%).[17] Attempts to hydrolyze methyl ester 22 were characterized by
side reactions, notably STr-elimination and degradation. After considerable experimentation it was
found that NaOH buffered with TFE created conditions that would cleanly deliver acid 23 (99%
yield) and could circumvent the usage of toxic tin reagents.[21] Coupling to H--OH-Phe-Me
(mixture of stereoisomers) then proceeded smoothly and gave dipeptide 24 in 85% yield. For
oxazoline formation DOF was then favorably used,[19b] but the following oxidation had to be
performed with limiting reagent and at low temperature to avoid overoxidation (36% over two
steps). Methyl ester deprotection by using Me3SnOH[22] then cleanly delivered acid 8.

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In order to assemble the backbone structure, manual peptide synthesis on Tr-resin was employed
after loading with protected Thr (0.7 mmol/g, Scheme 2). Regular Fmoc/tBu chemistry and
terminally Boc-protected D-allo-Ile delivered tripeptide acid 9 in 96% isolated yield overall.
Coupling of acid 9 to amino alcohol 10 by using HBTU succeeded in > 90% yield. However,
significant epimerization of the Thr residue in amide 26 occurred under these conditions (*d.r. 2:1).
Careful optimization (PyAOP, EtN(iPr)2, -3 °C → 20 °C) reduced diastereomer formation (*d.r.
> 9:1), at the expense of yield (50%). Anyhow, the minor R-isomer(*) was easily separated by
chromatography and its solubility in EtOAc.1 Hydroxymethyl peptide 26 was then transformed into
bis-oxazole 27 by using DAST,[19] followed by oxidation with DBU and excess CCl4[13] in a solvent
mixture containing pyridine, which was crucial to minimize side product formation. After acid-
mediated tBu- and Boc-deprotection of bis-oxazole 27 bis-azole acid 8 was attached to give
octapeptide precursor 7 (82% yield). Deallylation proceeded cleanly with morpholine as allyl
scavenger,[23] and acid-mediated detritylation furnished cyclization precursor 28 (97% yield).
The crucial macrocyclization performed excellently by using PyBOP (88% yield, Scheme 3) when
slow addition of the precursor to the reagent was employed (syringe pump).[24] Interestingly, macro-
thiolactone 6 was stable to isolation and chromatography, suggesting low residual ring strain.
Furthermore, ring contraction of azidothioester 6 by Aza-Wittig reaction in 2,6-lutidine as solvent[17]
followed by oxidation delivered the penta-azole macrocycle 29 in gratifying 79% yield. E2-type anti
elimination then proceeded cleanly via the stable mesylate that was eliminated smoothly by
employing DBU to give Z-configured urukthapelstatin A (1, 61%). Analytical data (1H, 13C, HRMS,
TLC) of the final product were identical to data reported for the natural product.[2,10] As a control,
syn elimination conditions (CuCl/EDC)[25] gave an isomer distinct of 1 to which E-configuration
was assigned by NOESY-NMR. The stereoisomeric purity was high in both cases (d.r. >95:5).
While performing these studies and in contrast to an earlier report,[10] we found the Z- and E-isomers
of 1 to be distinct and separable by TLC and HPLC (Figure 3 and supporting info). To our surprise,
apparent isomer “contaminations” appeared even after stringent separation by prep. HPLC. Closer
inspection and monitoring revealed that isomers of 1 slowly interconvert between Z and E forms
when dissolved in aqueous acetonitrile (pH 3) or SDS buffer (pH 7.4) (d.r. 95:5 → 75:25 during
96 h at 20°C, Figure 3). Hence, although the compound seems to be configurationally stable in the
solid state and can be crystallized,[2] it is not fully stable in solution. This observation will be
important for future biological testing and compound design.
1 The minor isomer R-26 was converted to the final product 1 as well by using the synthesis described and an
interchange of final elimination conditions, thereby confirming independently the stereochemical assignments.

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In summary, we report the first stereoselective synthesis of urukthapelstatin A (1) that proceeds
reproducibly in satisfactory yield. Critical steps were optimized by judicious synthesis design, most
notably the macrocyclization via a novel macrothiolactone and the azole formation via Aza-Wittig
ring contraction. In extension, the chemistry developed here should allow for a permutation of
individual building blocks, in order to prepare derivatives with improved stability for structural and
biological investigations. Such studies are currently underway in our laboratory.
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Figure 1. Polyazole cyclopeptide natural products originating from RiPPs and synthesis planning
for urukthapelstatin A with crucial azole rings highlighted. Abbreviations: All = Allyl; Alloc =
Allyloxycarbonyl; Boc = tert-Butyloxycarbonyl; DBU = 1,8-Diazabicyclo[5.4.0]undec-7-en; DAST
= Diethylaminosulfur trifluoride; DMAP = 4-Dimethylaminopyridine; DOF
methoxyethyl)aminosulfur trifluoride (“Deoxo-Fluor”); HBTU = O-(Benzotriazol-1’-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate; HOBt 1-Hydroxybenzotriazole; PyBOP
=
Bis(2-
=
=
(Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate; Ms = Methylsulfonyl; TBS
= tert-Butyldimethylsilyl; TFE = 2,2,2-Trifluoroethanol; Tr = Triphenylmethyl.

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Figure 2. Considerations for azole-forming reactions within macrocycles.

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Scheme 1. Synthesis of azole building blocks. Reagents and conditions: a) SOCl2 (2.2 equiv.), allyl
alcohol, 0 °C → 60 °C, 20 h; b) Boc2O (1.2 equiv., 2.5 M in 1.4-dioxane), Na2CO3 (1 equiv.), sat.
NaHCO3/H2O (1:1), 0 °C → 20 °C, 24 h; c) TBSCl (1.3 equiv.), imidazole (3 equiv.), DMF, 0 °C →
20 °C, 14 h; d) 13 (1.2 equiv.), EDC (1.1 equiv.), HOBt (1.1 equiv.), NEt3 (2 equiv.), CH2Cl2/DMF
(50:1), 20 °C, 19 h; e) DAST (1.1 equiv.), K2CO3 (2.1 equiv.), CH2Cl2, -78 °C → 20 °C, 3.5 h; f)
BrCCl3 (1.4 equiv.), DBU (2.4 equiv.), CH2Cl2, -30 °C → 20 °C, 5 h; g) TFA/H2O (17:2), 20 °C,
14 h; h) TFN3 (3 equiv.), ZnSO4 (0.05 equiv.), CH2Cl2/MeOH/H2O (2:5:1), 0 °C → 20 °C, 2 h; i)
TFA (5vol%), Et3SiH (5vol%), CH2Cl2, 20 °C, 1 h; j) 19 (1.2 equiv.), EDC x HCl (1.2 equiv.),
HOBt (1.1 equiv.), EtN(iPr)2 (1.3 equiv.), CH2Cl2/DMF (9:1), -10 °C → 20 °C, 15 min; k) PPh3
(1.35 equiv.), CH2Cl2/DMF (10:1), -10 °C → 20 °C, 15 h; l) 4 M HCl in 1,4-dioxane, 20 °C , 20 h;
m) 5 M NaOH/CH2Cl2/MeOH/TFE (0.1:2:1:1), 20 °C , 3 d; n) Ph-(CHOH)-(CHNH2)-COOMe
(1.2 equiv.), HBTU (1.2 equiv.), EtN(iPr)2 (2 equiv.), CH2Cl2/DMF (1:1), 0 °C → 20 °C, 3.5 h; o)
DOF (1.2 equiv.), pyridine (2 equiv.), THF, -65 °C → 20 °C, 4 h; p) BrCCl3 (0.9 equiv.), DBU
(1 equiv.), CH2Cl2, -50 °C → -25 °C, 30 h; q) Me3SnOH (2 equiv.), 1,2-dichloroethane, 80 °C, 22 h.

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Scheme 2. Peptide chain assembly and azole appendage. Reagents and conditions: a) trityl chloride
resin, Fmoc-L-Thr(tBu)-OH (1 equiv.), EtN(iPr)2 (3 equiv.), CH2Cl2/DMF (1:1), 20 °C, 5.5 h; resin
loading 0.7 mmol/g; b) piperidine/DMF (1:4), 20 °C , 22 min; c) Fmoc-L-Ala-OH (4equiv.), HOBt
(4 equiv.), HBTU (4equiv.), EtN(iPr)2 (8 equiv.), DMF, 20 °C, 7 h; d) Boc-D-allo-Ile-OH (1.5
equiv.), HOBt (1.5 equiv.), HBTU (1.5 equiv.), EtN(iPr)2 (2.5 equiv.), DMF, 20 °C, 6 h; e)
HFIP/CH2Cl2 (3:7), 20 °C, 30 min; f) 10 (0.9 equiv.), PyAOP (1.1 equiv.), EtN(iPr)2 (1.8 equiv.),
CH2Cl2/DMF (1:1), -5 °C → 20 °C, 22 h; g) DAST (1.1 equiv.), K2CO3 (2 equiv.), CH2Cl2, -78 °C
→ 20 °C, 5.5 h; h) DBU (10 equiv.), CCl4/pyridine/MeCN (2:3:3), -60 °C → 20 °C, 3 d; i) Anisole
(2.3 equiv.), TFA/CH2Cl2 (1:1), 0 °C → 20 °C, 3 h; j) 8 (1.2 equiv.), HBTU (1.6 equiv.), EtN(iPr)2
(3 equiv.), CH2Cl2/DMF (1:1), 0 °C → 20 °C, 2 d; k) Pd(dba)2 (0.2 equiv.), morpholine (20 equiv.),
CH2Cl2, 20 °C, 4 h; l) Et3SiH (5 vol%), TFA (3 vol%), CH2Cl2, 20 °C, 1 h.

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Scheme 3. Macrocyclization and synthesis of urukthapelstatin A (1) and its E-isomer. Reagents and
conditions: a) PyBOP (1.2 equiv.), EtN(iPr)2 (2 equiv.), CH2Cl2/DMF (6:1), 20 °C, 24 h; b) PPh3
(1.5 equiv.), 2,6-lutidine, 20 °C → 60 °C, 7 h; c) BrCCl3 (1.2 equiv.), DBU (1.5 equiv.), CH2Cl2,
-50 °C → 20 °C, 8 h; d) MsCl (2 equiv.), NEt3 (3 equiv.), CH2Cl2¸ 20 °C, 2.5 h; e) DBU (3 equiv.),
CH2Cl2, 20 °C, 1.5 h; f) CuCl (12.5 equiv.), EDC x HCl (3 equiv.), CH2Cl2/DMF (96:4), 20 °C,
30 h.

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Figure 3. E/Z-Isomerization of the enamide 1 monitored by HPLC. Z-Isomer after isolation (black,
Z/E = 95:5) and after 96 h (grey, Z/E = 72:28, normalized; see supp. info for details).

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Graphical abstract for the table of contents
Text suggestion for the table of contents:
Constraining well dosed. The first stereoselective total synthesis of urukthapelstatin A is reported
that employs a novel synthesis design. A kinetically favored macrothiolactonizations and a high-
yielding Aza-Wittig heterocyclization for contracting the macrocyle were crucial for success. The
embedded enamide substructure of the target molecules was found to slowly isomerize in solution.
Key topic:
Total synthesis