Total synthesis and stereochemical assignment of baringolin

Article abstract of DOI:10.1002/anie.201302372

The thiopeptide antibiotic baringolin has been synthesized, and its structure and stereochemistry have been confirmed. The use of a strategy based on palladium-catalyzed cross-couplings permitted a modular construction of this natural product. Copyright

Full text of DOI:10.1002/anie.201302372

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DOI: 10.1002/anie.201302372
Natural Products
Total Synthesis and Stereochemical Assignment of Baringolin**
Xavier Just-Baringo, Paolo Bruno, Lars K. Ottesen, Librada M. CaÇedo, Fernando Albericio,*
and Mercedes ꢀlvarez*
Thiopeptides are a family of naturally occurring, peptide-
derived molecules with high sulfur content formed by a central
nitrogen-containing six-membered heterocycle decorated
with many azoles in a macrocyclic array.[1,2] These natural
products have drawn the attention of many research groups
mainly owing to their interesting antibiotic activities[3] and
their challenging structures.[4] One member of this family,
thiostrepton, which is an ingredient of Panolog, has reached
the market.
Baringolin is a novel thiopeptide of the d series,[1] and thus
contains a central 2,3,6-trisubstituted pyridine (for structure
see Scheme 1). It was isolated by Biomar SA from fermenta-
tion of the marine-derived bacterium Kucuria sp MI-67-EC3-
038 strain of the Micrococcaceae family, found at the coast of
Alicante (southern Spain). Important antibacterial activity at
nanomolar concentrations was found in several strains, such
as Staphylococcus aureus, Micrococcus luteus, Propionibacte-
rium acnes, and Bacillus subtilis.[5] The structure of baringolin
was established by spectroscopic methods.[6] The macrocycle
in baringolin contains, in addition to three natural amino acids
(Tyr, Phe, and Asn), a pyridine, three thiazoles, a methylox-
azole ring, and also some motifs not present in other
thiopeptides at the same time, such as a thiazoline with an
a-chiral center and a pyrrolidine motif derived from a Pro
[*] X. Just-Baringo, Dr. P. Bruno, Dr. L. K. Ottesen, Prof. F. Albericio,
Prof. M. ꢀlvarez
Institute for Research in Biomedicine
Barcelona Science Park–University of Barcelona
Baldiri Reixac 10, 08028 Barcelona (Spain)
and
CIBER-BBN, Networking Centre on Bioengineering Biomaterials
and Nanomedicine, 08028 Barcelona (Spain)
E-mail: fernando.albericio@irbbarcelona.org
mercedes.alvarez@irbbarcelona.org
Homepage: http://www.pcb.ub.edu/fama/htm/home.htm
Prof. F. Albericio
Department of Organic Chemistry, University of Barcelona
08028 Barcelona (Spain)
Prof. M. ꢀlvarez
Laboratory of Organic Chemistry, Faculty of Pharmacy
University of Barcelona, 08028 Barcelona (Spain)
Scheme 1. Retrosynthesis of baringolin. Boc=tert-butyloxycarbonyl,
Alloc=allyloxycarbonyl, Fmoc=9-fluorenylmethoxycarbonyl.
Dr. L. M. CaÇedo
Instituto Biomar, S.A. Parque Tecnolꢁgico de Leon-M10.4
24009 Armunia, Leꢁn (Spain)
[**] We gratefully acknowledge support from the Spanish Science and
Innovation Ministry, CICYT (CTQ2012-30930) and the Generalitat
residue. A long peptidic tail is attached to the pyridine
de Catalunya (2009SGR 1024). X.J. thanks ISCIII for a PFIS grant. We
through a fourth thiazole. This tail is a pentapeptide contain-
ing three methylidenes resulting from dehydration of Ser.[7]
Surprisingly and to our knowledge, this is the longest tail
reported to date for this family of antibiotics. Baringolin
also thank Dr. Antonio Fernandez from Biomar SA for supplying
a sample of the natural product for comparison.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201302372.
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contains seven stereocenters, the configuration of which was
considered to be that of natural l-amino acids. A thorough
review of literature precedents shows that all thiopeptides of
which the stereochemistry has been confirmed to date are
made out of l-amino acids; this finding is consistent with their
ribosomal origin.[7]
The aim of this work was not only to synthesize this new
entity, but also to develop a synthetic strategy that would
fulfill our aspiration for an easy construction of closely related
new entities to evaluate the structure-activity relationships
(SAR) of this interesting family of antibiotics. Moreover, this
first synthesis should also serve as the ultimate confirmation
of the structure and stereochemical assignment of baringolin.
With this premise in mind, the total synthesis of baringolin
was designed using only commercially available l-amino
acids as the sole source of chirality to confirm if the previous
hypothesis was correct.
The retrosynthetic analysis started with the disconnection
of the peptidic tail (Scheme 1) to give two synthetic frag-
ments, macrocycle 1 and pentapeptide 2. In turn, macrocycle
1 could be obtained from trisubstituted pyridine 3 and
building blocks 4–6. The concourse of orthogonal protecting
groups was key to the success of the synthesis of these
complex molecules. This was clearly evidenced in the
structure of 3.
First of all, the synthesis of the central polyheterocyclic
core 3 was attempted. A cross-coupling-based strategy[2,8] was
chosen, since it would offer a modular approach to the target
structure (Scheme 2). The synthetic approach was based on
the chemoselective derivatization of commercial 2,6-dichloro-
nicotinic acid (7a), which can be easily converted into 2-
Scheme 3. Synthesis of pyridine building block 3. Reagents and
conditions: a) tBuOK, MeOH, 658C, 4 days, 85%; b) 8, PyBOP, DIPEA,
THF, 08C, 3 h, 89%; c) Dess–Martin periodinane, CH2Cl2, RT, 6 h,
95%; d) PPh3, I2, NEt3, CH2Cl2, 08C to RT, 15 h, 78%; e) 10, [Pd-
(PPh3)4], 1,4-dioxane, 808C, 48 h, 88%; f) HBr, AcOH, RT, 28 h, 73%;
g) (Boc)2O, NEt3, CH2Cl2, 08C, 4 h, 94%; h) Tf2O, 2,6-lutidine, DMAP,
CH2Cl2, 08C to RT, 3 h, 88%; i) 9, [Pd(PPh3)4], DMA, 458C, 1 h, quant.
PyBOP=(1H-benzotriazol-1-yloxy)tris(pyrrolidino)phosphonium hexa-
fluorophosphate, DIPEA=diisopropylethylamine, DMAP=4-(dimethyl-
amino)pyridine, DMA=dimethylacetamide.
tion with Thr 8, followed by Dess–Martin oxidation of the side
chain into the corresponding methyl ketone and subsequent
cyclization to yield the desired biaryl 11.[12] Stille cross-
coupling between chloropyridine 11 and enantiopure tri-
methyltin derivative 10 rendered methoxypyridine 12, which
could be converted into triflate 13 after acidolysis of the
methoxy group. Lastly, 13 was subjected to Negishi cross-
coupling conditions with thiazole zinc bromide 9 to render
quantitatively the desired central polyheterocyclic core 3,
which was suitably functionalized for subsequent orthogonal
deprotections.
Construction of the pentapeptide tail 2 was carried out by
solid-phase peptide synthesis (SPPS) using Fmoc chemistry
and Rinkamide ChemMatrix resin,[13] using l-alanine and l-
proline, as well as Fmoc-l-phenylselenocysteine[14] as precur-
sor of dehydroalanine residues (Scheme 4). Condensation of
the different Fmoc-protected amino acids (Fmoc-AA-OH)
was carried out with N,N’-diisopropylcarbodiimide and
Oxyma Pure[15] as coupling agents. Deprotection before the
introduction of a new Fmoc-AA-OH was achieved with
piperidine. The final cleavage with trifluoroacetic acid (TFA)
afforded pentapeptide 2 with the free amine and a C-terminal
amide ready for condensation with the carboxylic acid of the
macrocycle in the last steps of the synthetic process.
Scheme 2. Retrosynthesis of pyridine building block 3.
chloro-6-methoxynicotinic acid (7b),[9] which contains two
differentiated a-positions along with a carboxylic acid that
serves as a precursor of the methyloxazole motif.
The other building blocks for the construction of 3 were
benzyl-protected Thr 8, zinc thiazole 9,[10] and bithiazole
pyrrolidine 10. The synthesis of the later has been recently
reported by us,[11] and it was prepared as a suitable building
block for a cross-coupling-based strategy.
Transformation of pyridine carboxylic acid 7b into
pyridine oxazole 11 (Scheme 3) was performed by condensa-
Thiazole 4 was prepared by protecting-group manipula-
tion of a previously described synthon (see the Supporting
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Information).[16] The last building block was the Phe-derived
thiazoline 5, the synthesis of which was addressed by
cyclization of the corresponding dipeptide 14 (Scheme 5).[17]
Both the condensation and the cyclization steps yielded
products 14 and 15 in excellent yields. Further saponification
of the methyl ester with trimethyltin hydroxide[18] afforded
acid 5 in an excellent diastereomeric ratio (d.r. 96:4).
Scheme 4. Synthesis of pentapeptide 2. Reagents and conditions: a) 1.
Fmoc-AA-OH, N,N’-diisopropylcarbodiimide, Oxyma Pure, DMF, RT,
1.5 h; 2. 20% piperidine in DMF, RT (4 treatments); b) 95% TFA in
CH2Cl2, RT (4 treatments), 89%.
Next, it was taken into consideration that the thiazoline
moiety is prone to epimerization under both basic and acidic
conditions, and its manipulation should be limited to as few
steps as possible. Deprotection of the carboxylic acid of 3 by
hydrogenolysis of the Bn ester was performed with excellent
conversion by using Pd black (Scheme 6).[4j,19] Acid 16 was
condensed with Asn-derived thiazole 4 to yield 17 by using
EDC and HOAt as coupling agents, which would become the
reagents of choice for further amide formations. Fmoc-Tyr-
OH 6 was introduced next after elimination of the Boc group
at the pyrrolidine ring in 17. Fmoc removal under standard
conditions and subsequent condensation with thiazoline 5
rendered 19, the protected open form of the macrocycle. All
allyl-based protecting groups of 19 were simultaneously
removed by using catalytic [Pd(PPh3)4] and the crude was
subjected to macrocyclization conditions in the absence of
base, yielding the desired product 1. Ethyl ester hydrolysis of
1 was carried out by using trimethyltin hydroxide to avoid
epimerization of the thiazoline moiety under more common
Scheme 5. Synthesis of thiazoline 5. Reagents and conditions:
a) HBTU, DIPEA, CH2Cl2, RT, 1 h, 94%; b) Ph3PO, Tf2O, CH2Cl2,
À208C, 2 h, 86%; c) Me3SnOH, CH2Cl2, 608C, 4 h. HBTU=O-(1H-
benzotriazoyl-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophos-
phate, Trt=trityl.
Scheme 6. Total synthesis of baringolin. Reagents and conditions: a) H2 (1 atm), Pd black, CH2Cl2/EtOH (1:1), RT, 4 h, quant.; b) 4, EDC, HOAt,
DIPEA, DMF, 08C, 18 h, 82%; c) HCl, dioxane, RT, 7 h; d) 6, EDC, HOAt, DIPEA, DMF, 08C, 7 h, 71% (2 steps); e) piperidine, CH2Cl2, RT, 3 h,
87%; f) 5, EDC, HOAt, DIPEA, DMF, 08C, 3 h, 68% (2 steps); g) [Pd(PPh3)4], PhSiH3, CH2Cl2, RT, 7 h; h) EDC, HOAt, DMF (1 mm), 08C to RT,
21 h, 30% (2 steps); i) Me3SnOH, ClCH2CH2Cl, 608C, 19 h; j) 2, EDC, HOAt, DIPEA, DMF, 08C, 3 h, 81% (2 steps); k) tBuOOH, CH2Cl2, RT,
12 h, 66%. EDC=N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride, HOAt=1-hydroxy-7-azabenzotriazole.
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and drastic basic aqueous conditions.[18] Condensation of the
resulting acid with pentapeptide 2 yielded 20 in excellent
yield. Finally, oxidation with tert-butyl hydroperoxide and
in situ elimination of the resulting phenylselenide oxide
groups at room temperature rendered baringolin.
Coelution with a natural product sample of baringolin and
comparison of their NMR spectra showed that both com-
pounds are identical (see the Supporting Information).
Biological assessment against different strains of methicillin-
resistant S. aureus (MRSA) showed a minimum inhibitory
concentration (MIC) in the nanomolar range for both the
natural and synthetic compounds. These results confirm that
the structure of baringolin is based only on l-amino acids, as
other precedent thiopeptides are.
In conclusion, the total synthesis of baringolin was
achieved by a convergent strategy with a good overall yield.
The developed convergent synthetic procedure is especially
suitable for the preparation of baringolin analogues for SAR-
studies, which are currently ongoing. Furthermore, it could
also be applied to the preparation of other complex peptides
of the same family.
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Published online: June 18, 2013
Keywords: antibiotics · cross-couplings · heterocycles ·
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natural products · thiopeptides
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[19] Attempts of hydrogenolysis over palladium on charcoal under
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