Total synthesis of pyoverdin D

Article abstract of DOI:10.1021/ol400490s

Pyoverdin D is an important siderophore that is used by the human pathogen Pseudomonas aeruginosa to import iron and gain a competitive advantage. This unique partially cyclic octapeptide bears four nonproteinogenic amino acids, including ^δN-formyl-^δN-hydroxy-l-ornithine, and a catechol containing chiral chromophore. Here, we report the first total synthesis of pyoverdin D.

Full text of DOI:10.1021/ol400490s

ORGANIC
LETTERS
2013
Vol. 15, No. 7
1702–1705
Total Synthesis of Pyoverdin D
Roi Mashiach and Michael M. Meijler*
Department of Chemistry and the National Institute for Biotechnology in the Negev,
Ben-Gurion University of the Negev, Be’er-Sheva 84105, Israel
meijler@bgu.ac.il
Received February 22, 2013
ABSTRACT
Pyoverdin D is an important siderophore that is used by the human pathogen Pseudomonas aeruginosa to import iron and gain a competitive
advantage. This unique partially cyclic octapeptide bears four nonproteinogenic amino acids, including δN-formyl-δN-hydroxy-L-ornithine, and a
catechol containing chiral chromophore. Here, we report the first total synthesis of pyoverdin D.
When subjected to iron starvation conditions, Pseudomonas
aeruginosa, an opportunistic human pathogen, secretes
vast amounts of its main siderophore, pyoverdin D, along
with several other iron chelators.1 Pyoverdin D is a unique,
partially cyclic octapeptide that presents a number of
interesting structural features, such as the side chain to
backbone macrocycle that forms between the lysine residue
and the C-terminal threonine carboxylate. Other important
features include the presence of four nonproteinogenic
amino acids found as part of the backbone of the molecule,
namely two δN-formyl-δN-hydroxy-L-ornithine residues
and two D-serine residues. In addition to these traits, the
major characteristic of this class of molecules is the presence
of an N-terminal dihydroxyquinoline-derived chromophore.2,3
To date, no total synthesis for this key natural product has
been reported.
stability constant of approximately 1032 À1, and once
M
recognized by a cognate outer membrane transporter, it is
immediately shuttled into the periplasm, where it dissoci-
ates and is subsequently recycled for further use. Presently,
the precise regulation of these processes is not fully under-
stood, although it is known that synchronization of
population-wide siderophore synthesis is regulated through
quorum sensing.4À6 Thus far, more than 100 different
pyoverdin molecules have been identified,5 with the highly
abundant pyoverdin D, in particular, having been shown
by Meyer et al. to be of paramount importance not only in
iron metabolism but also in terms of the producer’s ability
to acquire virulence.1
When our efforts to synthesize pyoverdin D began, the
structure of the molecule had already been elucidated, not
only by NMR2,3 but also from the crystallographic data
obtained on the molecule within its receptor.7 In addition,
the synthesis of the racemic protected chromophore had
been described previously in a seminal study by Miller and
The catechol-bearing chromophore, together with the
two hydroxamic acids, serve as bidentate ligands for iron-
(III) and together form a strong octahedral complex upon
chelation of this vital metal. The resulting complex has a
(4) Cornelis, P. Appl. Microbiol. Biotechnol. 2010, 86, 1637–1645.
(5) Greenwald, J.; Hoegy, F.; Nader, M.; Journet, L.; Mislin, G.;
Graumann, P.; Schalk, I. J. Biol. Chem. 2007, 282, 2987–2995.
(6) Schalk, I. J.; Abdallah, M. A.; Pattus, F. Biochemistry 2002, 41,
1663–1671.
(7) Cobessi, D.; Celia, H.; Folschweiller, N.; Schalk, I. J.; Abdallah,
M. A.; Pattus, F. J. Mol. Biol. 2005, 347, 121–134.
(1) Meyer, J. M.; Neely, A.; Stintzi, A.; Georges, C.; Holder, I. A.
Infect. Immun. 1996, 64, 518–523.
(2) Demange, P.; Wendenbaum, S.; Linget, C.; Mertz, C.; Cung,
M. T.; Dell, A.; Abdallah, M. A. BioMetals 1990, 3, 155–170.
(3) Briskot, G.; Taraz, K.; Budzikiewicz, H. Liebigs Ann. Chem. 1989,
375–384.
r
10.1021/ol400490s
Published on Web 03/26/2013
2013 American Chemical Society

Scheme 1. Total Synthesis of Pyoverdin D
co-workers.8 Nevertheless, most of the chemical research
regarding pyoverdin D had focused on derivatization of
pyoverdins obtained through fermentation.9À12 Indeed,
the lack of a synthetic pathway for pyoverdin D has thus
far narrowed the scope of derivatization sites available on the
molecule, thereby limiting the questions that can be answered
by using such molecular tools. With this in mind, we started
the total synthesis of pyoverdin D. We chose to employ a
solid-phase peptide synthesis (SPPS) approach,13,14 where
the introduction of different or even nonproteinogenic amino
acids is possible during backbone build-up.15
either a linear or a partially cyclic peptide bearing two
metal-chelating amino acids and possessing a chelating
chromophore at the N terminus.8,9,16
With these conserved features in mind, and based on
the elegant work of Miller and co-workers, we developed
a relatively simple strategy that can serve as a general
methodology for the syntheses of those pyoverdin mole-
cules that carry a similar chromophore. In order to enable
an SPPS approach to synthesize the peptide moiety,
representing a facile combinatorial method for analog
Here, we present a concise synthetic route for pyoverdin D
that can serve as the basis for further efforts aimed at the
synthesis of a variety of related pyoverdins and analogs. Such
syntheses will further extend our ability to study the important
and still enigmatic relationship between iron signaling and
P. aeruginosa behavior at the single and multicellular levels.
The convergent synthetic route leading to pyoverdin D
generation employed in this study is depicted in Scheme 1.
The family of known pyoverdin molecules is derived from
Scheme 2. Synthesis of δN-Formyl-δN-benzyloxy-L-ornithine
(8) Kolasa, T.; Miller, M. J. J. Org. Chem. 1990, 55, 4246–4255.
(9) Budzikiewicz, H. Curr. Top.Med. Chem. 2001, 1, 73–82.
(10) Schons, V.; Atkinson, R. A.; Dugave, C.; Graff, R.; Mislin,
G. L. A.; Rochet, L.; Hennard, C.; Kieffer, B.; Abdallah, M. A.; Schalk,
I. J. Biochemistry 2005, 44, 14069–14079.
(11) Kinzel, O.; Budzikiewicz, H. J. Pept. Res. 1999, 53, 618–625.
(12) Hennard, C.; Truong, Q. C.; Desnottes, J.-F.; Paris, J.-M.;
Moreau, N. J.; Abdallah, M. A. J. Med. Chem. 2001, 44, 2139–2151.
(13) Chan, W. C.; White, P. D. Fmoc Solid Phase Peptide Synthesis-A
Practical Approach; Oxford University Press: Oxford, 2000.
(14) Lloyd-Williams, P.; Albericio, F.; Giralt, E. In Chemical Ap-
proaches to the Synthesis of Peptides and Proteins; CRC Press: Boca Raton,
FL, 1997.
(15) Humphrey, J. M.; Chamberlin, A. R. Chem. Rev. 1997, 97, 2243–
2266.
(16) Drechsel, H.; Jung, G. J. Pept. Sci. 1998, 4, 147–181.
Org. Lett., Vol. 15, No. 7, 2013
1703

Scheme 3. Synthesis of the Protected Chromophore
preparation, we first developed a succinct method for the
synthesis of 9-fluorenylmethyloxy carbonyl (Fmoc)-
protected17 δN-formyl-δN-hydroxy-L-ornithine.
bulkiness was proven to be crucial for the synthesis of the
chromophore.8 The synthetic pathway is depicted in
Scheme 3, starting from commercially available L-DOPA
(9), which was treated with thionylchloride in methanol
to furnish 10. The R-amine was then treated with Boc
anhydride and Et3N in methanol to generate 11.
The Fmoc SPPS approach developed also required the
enantiopure preparation of a chromophore with suitable
protecting groups. Following synthesis of the peptide, our
design proceeded with the incorporation of the chromo-
phore, cleavage of the linear-protected pyoverdin from
the resin, formation of the macrolactam, and global de-
protection. We commenced with the synthesis of properly
protected formylhydroxyornithine (Scheme 2).
Our strategy was based on a similar synthetic route
designed by Fennell et al.18 where a protected formylhy-
droxylysine was synthesized. We started from commer-
cially available Boc-L-Orn-OtBu (3) which was oxidized
by treatment with dibenzoyl peroxide to furnish 4. For-
mylation was achieved via coupling with formic acid,
using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)
as the coupling reagent, to obtain the δN-formylated prod-
uct that was further reacted to give 6 through one-pot
solvolysis and benzylation reactions. Deprotection of the
tert-butyloxycarbonyl (Boc) and tert-butyl ester moieties by
exposure to trifluoroacetic acid (TFA) provided the free
amine and carboxyl moieties (7). Subsequent exposure to
Fmoc-OSu gave 8 with an overall yield of 23%. With the
hydroxamate-containing amino acid in hand, we next set
out to synthesize the chromophore. Our synthesis of the
protected dihydroxyquinoline was inspired by the synthetic
route published by Miller and co-workers. The Boc protect-
ing group was incorporated early in the synthesis, since its
The dibenzyl-protected catechol was synthesized by
treatment of 11 with benzyl bromide in the presence of
K2CO3 and NaI in acetone. The nitration of 12 was carried
out under relatively mild acidic conditions (1.5 equiv of
nitric acid in an acetic acid/acetic anhydride mixture at
15 °C) to prevent cleavage of the acid-labile Boc group,
affording 13. The next step, one-pot reduction and cycliza-
tion to form the quinoline moiety, was achieved by treating
13 with zinc dust and NH4Cl in a THF/methanol mixture.
The resulting amine and 14 were then extracted from
the reaction mixture and heated in methanol at reflux
temperature to give the Boc-protected quinoline 14.19
Thionation was performed by reacting 14 with
Lawesson’s reagent20 to give the thioquinoline 15. The
thioquinoline was reacted with freshly prepared 4-amino-
2-hydroxybutanoate (20) in the presence of mercuric ace-
tate, which not only led to ligation of the two fragments
but also to the expected oxidation of the C7ÀC8 bond,
generating the fluorescent scaffold as a mixture of E and Z
isomers (21). After mesylation of the alcohol, we observed
that both of the isomers had collapsed to the same prod-
uct upon the concomitant one-pot cyclization, yielding
(19) The extraction was necessary since one of the benzyl groups was
shown to be partially cleaved in the presence of zinc under reflux
conditions
(20) Clausen, K.; Thorsen, M.; Lawesson, S. O. Tetrahedron 1981, 37,
3635–3639.
(17) Carpino, L. A.; Han, G. Y. J. Am. Chem. Soc. 1970, 92, 5748–
5749.
(18) Fennell, K. A.; Miller, M. J. Org. Lett. 2007, 9, 1683–1685.
1704
Org. Lett., Vol. 15, No. 7, 2013

the protected chromophore 22. Treatment of 22 with
p-toluenesulfonic acid (TsOH) in acetonitrile,21 provided
the free amine 23,22 which was treated with succinic
anhydride in chloroform at 50 °C to provide 24. The tert-
butyl ester was then cleaved by exposure to 50% TFA,
forming 25, which was further treated with sodium acetate
in acetic anhydride at 60 °C to facilitate the formation of
the succinimide (26) from the open succinate at a 33%
overall yield. Finally, the synthesis of pyoverdin D
(Scheme 1) was carried out on super acid-labile Rink acid
resin23 using Fmoc chemistry. Double loading of the resin
by treatment with 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-
triazole (MSNT) and 1-methylimidazole (MeIM) in
CH2Cl2 had been reported to minimize racemization.24
The peptide was elongated using (benzotriazol-1-yloxy)-
tripyrrolidinophosphoniumhexafluorophosphate
(PyAOP)25 as the coupling reagent.26 The chromophore
was then coupled to the peptide, followed by treatment
with 4% TFA, yielding the linear pyoverdin D skeleton
(1) as a mixture of a succinimide and a methyl succinate
(probably due to washings with MeOH after cleavage).
We performed the cyclization reaction in solution phase,
as possible formation of cross-links on the solid support
was probable due to the steric hindrance promoted by
the bulky protecting groups. Cyclization was achieved
by treating the semiprotected linear peptide with 10 equiv
of HATU27 and N,N-diisopropylethylamine (DIEA) in
DMF,28 affording the partially cyclic octapeptide 2.
Further deprotection by catalytic hydrogenation fol-
lowed by basic hydrolysis furnished pyoverdin D. HPLC
analysis (Supporting Information) of synthetic and
natural pyoverdin D (extracted and purified from wild-
type P. aeruginosa strain PAO1) showed the synthetic
compound and the extracted pyoverdin to be identical
while detailed NMR (1H NMR, TOCSY and ROESY)
and CD analyses showed that the extracted and synthetic
samples perfectly matched each other (Figures 1À3, Sup-
porting Information).
under iron-limited conditions, effected through addition
of human transferrin, a high-affinity iron chelator that
is present in human plasma, unless pyoverdin is added
to the medium. The concentration-dependent responses in
growth stimulation were nearly identical for synthetic and
natural pyoverdin D.
In summary, we report the first total synthesis of pyo-
verdin D, with its identity and configuration confirmed by
comparison with extracted and purified samples from wild-
type P. aeruginosa. Our versatile and modular synthetic
strategy can be applied to generate a large number of
pyoverdin analogs, related siderophores and drug conju-
gates, enabling the pursuit of questions that carry major
significance for understanding bacterial survival and com-
petition for resources, as well as potential crosstalk be-
tween population-wide signaling of cell density and iron
availability.
Figure 1. Effects of synthetic (S) and extracted (E) pyoverdin D
on growth of the pyoverdin- and pyochelin-deficient P. aeruginosa
strain PAO1ΔpvdDΔpchEF (in CAA medium incubated at
37 °C for 8 h), in the presence and absence of 4 μM human apo-
transferrin (Tf). The transferrin effectively depletes the medium
of available iron and prevents growth of the double mutant in the
absence of pyoverdin D. Other siderophores are able to chelate
iron under iron-rich conditions, enabling growth of pyoverdin-
deficient P. aeruginosa strains.
We also tested the biological activities of the synthetic
and natural samples using a P. aeruginosa strain lacking
the pyoverdin synthetase (PAO6606) and thus handi-
capped in its ability to take up iron through the pyoverdin
pathway. Figure 1 shows that this strain does not grow
Acknowledgment. We thank Professors E. P. Greenberg
(University of Washington) and Ehud Banin (Bar-Ilan
University) for generously providing bacterial strains,
Dr. Tali Scherf (Weizmann Institute of Science) for help
with pyoverdin 1D and 2D NMR, and Dr. Josep Rayo and
Dr. Yohai Dayagi for technical assistance.This research
was supported by the EuropeanResearch Council (Starting
Grant No. 240356, M.M.M.), the Israel Science Founda-
tion (Grant No. 749/09), and The Edmond J. Safra Center.
(21) Pozdnev, V. F. Zh. Obshch. Khim. 1988, 56, 5.
(22) Because of the observed instability of the chromophore under
strongly acidic conditions, we decided to modify our SPPS strategy using
super-acid-sensitive resin and side-chain protecting groups.
(23) Rink, H. Tetrahedron Lett. 1987, 28, 3787–3790.
(24) Blankemeyer-Menge, B.; Nimtz, M.; Frank, R. Tetrahedron
Lett. 1990, 31, 1701–1704.
(25) Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio, F. Chem.
Commun. 1994, 201–203.
Supporting Information Available. Detailed experimen-
tal procedures, characterizations, and copies of 1H and 13
C
(26) Arginine was coupled using PyBOP, since it was observed that
PyAOP promotes the formation of δ lactam, which hampers the
coupling reaction and gives rise to the formation of a deletion sequence.
(27) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397–4398.
(28) He, L.; Yang, L.; Castle, S. L. Org. Lett. 2006, 8, 1165–1168.
NMR spectraofnew compounds.Thismaterialisavailable
free of charge via the Internet at http://pubs.acs.org.
The authors declare no competing financial interest.
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