Iron(III)-templated macrolactonization of trihydroxamate siderophores

Article abstract of DOI:10.1021/ol301869x

A method was developed to synthesize macrocyclic trihydroxamate siderophores using optimized Yamaguchi macrolactonization conditions. The natural ability of siderophores to bind iron(III) was exploited to template the reactions and allowed for rapid react

Full text of DOI:10.1021/ol301869x

ORGANIC
LETTERS
2012
Vol. 14, No. 17
4390–4393
Iron(III)-Templated Macrolactonization
of Trihydroxamate Siderophores
Timothy A. Wencewicz, Allen G. Oliver, and Marvin J. Miller*
Department of Chemistry and Biochemistry, University of Notre Dame,
251 Nieuwland Science Hall, Notre Dame, Indiana 46556, United States
mmiller1@nd.edu
Received July 7, 2012
ABSTRACT
A method was developed to synthesize macrocyclic trihydroxamate siderophores using optimized Yamaguchi macrolactonization conditions. The
natural ability of siderophores to bind iron(III) was exploited to template the reactions and allowed for rapid reaction rates, high product conversions,
and the formation of large macrolactone rings up to 35 atoms. An X-ray structure of a 33-membered macrolactone siderophoreÀFe(III) complex is
presented.
Siderophores are low molecular weight, multidentate
iron(III) chelators used by microorganisms for assimila-
tion of otherwise insoluble and biounavailable iron(III)
minerals.1 Siderophore structures are finely tuned to selec-
tively bind iron(III) with high affinity from a mixed metal
environment, which makes them ideal candidates for
chelation-based treatments of human iron overload dis-
eases such as β-thalassemia.2 The limited choice of drugs
and far from ideal treatment regimes for currently ap-
proved iron chelation therapies have driven the search for
more effective iron(III) chelating agents.3
macrocyclic siderophore scaffolds. A macrocyclic scaffold
preorganizes the ligands for metal chelation, increases
iron(III) binding constants, and stabilizes the resulting
siderophoreÀiron(III) complexes which gives the producing
organism a competitive advantage for sequestering the
essential nutrient.4 Herein we report a method to synthe-
size macrocyclic trihydroxamate siderophores using an iron-
(III)-templated Yamaguchi macrolactonization reaction.
Linear trihydroxamate siderophores from the ferrioxa-
mine family were some of the first siderophores isolated
and have been used extensively in the clinic for treating
iron-overload diseases in humans.1,2 Linear ferrioxamine
siderophores form 1:1 octahedral complexes with iron(III)
and have iron(III) binding constants of 1030, while a value
One approach adapted by microbes to improve a side-
rophore’s ability to chelate iron(III) is to biosynthesize
(1) (a) Handbook of Microbial Iron Chelates; Winkelmann, G., Ed.;
CRC Press: Boca Raton, FL, 1991. (b) Miethke, M.; Marahiel, M. A.
Microbiol. Mol. Biol. Rev. 2007, 71, 413–451.
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Inorg. Chem. 1989, 28, 2189–2195. 5. (b) Schwarzenbach, G.; Schwar-
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L’Eplattenier, F.; Schwarzenbach, G. Helv. Chim. Acta 1963, 46, 1409–
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(2) Nick, H. Curr. Opin. Chem. Biol. 2007, 11, 419–423.
(3) (a) Kontoghiorghes, G. J.; Eracleous, E.; Economides, C.;
Kolnagou, A. Curr. Med. Chem. 2005, 12, 2663–2681. (b) Liu, J.; Obando,
D.; Schipanski, L. G.; Groebler, L. K.; Witting, P. K.; Kalinowski, D. S.;
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r
10.1021/ol301869x
Published on Web 08/20/2012
2012 American Chemical Society

Figure 1. Structures of linear and macrocyclic trihydroxamate
siderophores from the ferrioxamine family.
Figure 2. Design for Fe(III)-templation to bring the hydroxyl
and carboxyl groups into closer proximity.
of 1032.5 was reported for the macrolactam ferrioxamine
siderophore desferrioxamine E, 2 (Figure 1).5 Desferriox-
amine G, 1, is a direct biosynthetic precursor for desfer-
rioxamine E, 2, in Streptomyces coelicolor, and the
macrolactonization event is facilitated by the action of
DesD in an ATP-dependent fashion via the mixed anhy-
dride with release of PPi.6 The existence of the hydroxy
acid siderophore desferridanoxamine, 3, suggests that a
macrolactone counterpart, 4, might exist in nature since an
increase in iron(III)-binding affinity would give the produ-
cing organism a competitive growth advantage to com-
pensate for extra energy spent on biosynthesis of the
macrolactone ring.
The total synthesis of desferrioxamine E was accomplished
by Bergeron and co-workers using a DPPA-mediated
macrolactamization of tri-O-benzyl-desferrioxamine G. The
macrolactamization took 4 days and gave a 54% yield of the
benzyl protected desferrioxamine E precursor.7 A similar
benzyl protecting group strategy was adopted by our group
to synthesize desferridanoxamine, 3.8 However, a macrocy-
clization approach to desferridanoxamine macrolactone, 4,
using a protected hydroxy acid precursor was not possible
since the terminal alcohol was also protected as a benzyl
ether.9 Thus, a different synthetic approach was envisioned
using an iron(III)-templated macrolactonization (Figure 2).
Precomplexation of desferridanoxamine, 3, with iron(III)
was anticipated to bring the alcohol and carboxylate in close
proximity in the resulting siderophoreÀiron(III) complex,
5a. The template-induced proximity decreases the effective
macrolactone ring size to 14, compared to the untemplated
33-membered ring, and was expected to promote a more
facile macrolactonization event.
While there are many reagents and methods for per-
forming macrolactonizations of large rings,10 we first
tested the iron(III)-templation model by screening the
well-known Keck11 (DCC, DMAP, DMAP-HCl) and
Yamaguchi12 (2,4,6-trichlorobenzoyl chloride, iPr2EtN,
DMAP) macrolactonization conditions using danoxa-
mine, 5a, as a substrate. Keck macrolactonization condi-
tions failed to give any detectable macrolactone product
according to LC-MS analyses (data not shown) while the
initial Yamaguchi conditions screened gave a 43% con-
version to the macrolactone, 5b (Table 1, entry 1). When
desferridanoxamine, 3, was used as the substrate under
Keck and Yamaguchi conditions, no macrolactone pro-
duct was detected by LC-MS (data not shown).
Inspired by these initial results the Yamaguchi condi-
tions were optimized using danoxamine, 5a, as a model
substrate (Table 1, entries 1À4). Reactions were monitored
by quenching aliquots of the reaction mixture with methanol
followed by analysis with LC-MS and analytical HPLC
using visible detection at a wavelength of 427 nm (λmax
for 1:1 trihydroxamate siderophore:Fe(III) complexes)1a,5
which allowed for accurate calculation of product ratios.
The LC-MS studies showed the presence of four side-
rophore products: macrolactone 5b, methyl ester 5c,
dimethyl amide 5d, and dimer 5e. Increasing the reac-
tion temperature to 50 °C enhanced the reaction rate
but did not increase conversion to the desired macrolactone
(Table 1, entry 2). Increasing the amount of Yamaguchi
(10) (a) Parenty, A.; Moreau, X.; Campagne, J.-M. Chem. Rev. 2006,
106, 911–939. 10. (b) Schweitzer, D.; Kane, J. J.; Strand, D.; McHenry,
P.; Tenniswood, M.; Helquist, P. Org. Lett. 2007, 9, 4619–4622.
(11) (a) Boden, E. P.; Keck, G. E. J. Org. Chem. 1985, 50, 2394–2395.
(b) Keck, G. E.; Sanchez, C.; Wager, C. A. Tetrahedron Lett. 2000, 41,
8673–8676.
(12) (a) Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi,
M. Bull. Chem. Soc. Jpn. 1979, 52, 1989–1993. (b) Dhimitruka, I.;
SantaLucia, J., Jr. Org. Lett. 2006, 8, 47–50.
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Nat. Chem. Biol. 2007, 3, 652–656.
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(8) Roosenberg, J. M., Jr.; Miller, M. J. J. Org. Chem. 2000, 65, 4833–
4838.
(9) See Scheme S1 of the Supporting Information.
Org. Lett., Vol. 14, No. 17, 2012
4391

Table 1. Optimization and Scope of the Yamaguchi Macrolactonization for Trihydroxamate SiderophoreÀIron(III) Complexes
5aÀ8a
relative % composition after 72 hc
ring
size
TBCb
iPr2EtN
temp
% acid
% lactone
% Me ester
% NMe2 amide
% dimer
entrya
substrate
n
(equiv)
(equiv)
(°C)
(5aÀ8a)
(5bÀ8b)
(5cÀ8c)
(5dÀ8d)
(5eÀ8e)
1
2
3
4
5
6
7
8
5a
5a
5a
5a
6a
5a
7a
8a
2
2
2
2
1
2
3
4
33
33
33
33
32
33
34
35
1.0
1.0
3.0
3.0
3.0
3.0
3.0
3.0
2.0
2.0
6.0
6.0
6.0
6.0
6.0
6.0
22
16
26
0
43
12
8
24
20
10
7
5
7
50
39
22
73
9
8
50
0
74
8
12
23
16
11
4
50À22d
0
77e
47
0
0
22
0
28
0
9
50À22d
50
0
89e
96f
0
0
0
0
a Reactions were performed in anhydrous DMF using 5 mg of danoxamine 5a (entries 1À4) or 1 mg of siderophoreÀFe(III) hydroxy acids 5aÀ8a
(entries 5À8) at a concentration of 0.005 M and 0.5 equiv of DMAP. b TBC: 2,4,6-trichlorobenzoyl chloride. c Relative percent compositions were
determined after 72 h by quenching the reaction mixture with MeOH and integrating peak areas from HPLC chromatograms monitored at 427 nm.
d Reaction mixture was heated to 50 °C to solublize the siderophoreÀFe(III) complex then cooled to rt for the remaining reaction time. e Percent
composition was reached after 1 h and was stable throughout the course of the reaction. f Percent composition was reached after 6 h and was stable
throughout the course of the reaction.
reagent (2,4,6-trichlorobenzoyl chloride) and Hunig’s
base noticeably increased conversion to the desired
macrolactone 5b and suppressed formation of bypro-
ducts 5d and 5e (Table 1, entry 3). Increasing the reaction
temperature, in combination with excess Yamaguchi
reagent and Hunig’s base, enhanced the reaction rate
but did not increase conversion to the macrolactone
(Table 1, entry 4). The presence of methyl ester
(formed during the MeOH quench) after 72 h indicated
that the activated ester is long-lived under the reaction
conditions.
To evaluate the scope of the optimized Fe(III)-tem-
plated Yamaguchi macrolactonization, additional sidero-
phore substrates, 6aÀ8a, with carboxyl termini of variable
chain length (n = 1À4; ring sizes =32À35) were screened
using the same HPLC assay described previously. All of
the siderophore substrates, 6aÀ8a, successfully gave the
macrolactone (Table 1, entries 5À8). Surprisingly, the
percent conversion to macrolactone followed the trend 8b
(96%, n = 4) > 7b (89%, n = 3) > 6b (77%, n = 1) > 5b
(47%, n = 2). The reason for this trend is unknown, but we
hypothesize that the carboxylic acid terminus could interact
with the Lewis acidic Fe(III) center and participate in
chelation which might hinder formation of the Yamaguchi
activated ester (Figure 2).
To probe for this Lewis acid effect we studied the relative
rates of hydrolysis of purified siderophoreÀFe(III) com-
plex methyl esters 5cÀ8c (Table 1). Reaction half-lives
were calculated using first-order rate plots generated by
integrating siderophore methyl ester and carboxylic acid
HPLC peak areas at 427 nm. At pH 10, all the methyl ester
siderophoreÀFe(III) complexes were stable, as indicated
by UV/vis analysis of the reaction mixtures, and gave clean
hydrolysis reactions. The half-lives of ester hydrolysis fol-
lowed the order 6c (1.6 h, n = 1) > 5c (8.1 h, n = 2) > 7c
(13.0 h, n = 3) > 8c (19.9 h, n = 4), showing a clear trend
that increasing the distance of the ester from the Fe(III)
center increased the ester’s half-life.13 Since all the esters
4392
Org. Lett., Vol. 14, No. 17, 2012

Scheme 1. Scaled up Macrolactonization of Danoxamine, 5a, and Deferration To Give Desferridanoxamine Macrolactone 4
have similar steric hindrance, the observed trend is consistent
with the Fe(III) coordination center acting as a Lewis acid to
polarize the ester carbonyl. This supports the possibility of
the terminal carboxylate chelating the siderophore’s Fe(III)
metal center, as discussed previously (Figure 2).
With reliable macrolactonization conditions in hand, the
reaction was scaled up using danoxamine, 5a (Scheme 1).
The scaled up reaction (75 mg of 5a) gave the macrolactone
5b in 53% yield (39 mg) after purification by Prep-HPLC. A
single crystal of macrolactone siderophoreÀFe(III) com-
plex 5b was obtained from wet MeOH/Et2O, and the crystal
structure was determined by X-ray diffraction (Figure 3).
This is one of only four ferrioxamine siderophore structures
that has been determined.14 Further structural characteriza-
tion of the macrolactone was obtained by removing the
Fe(III) using excess EDTA (Scheme 1) which allowed for
Figure 3. X-ray structure of the danoxamine macrolactoneÀ
NMR analysis of desferridanoxamine macrolactone 4.
The Fe(III)-templated Yamaguchi macrolactonizations
Fe(III) complex, 5b, displayed with 50% probability ellipsoids.
of siderophore-Fe(III) complex hydroxy acids 5aÀ8a were
Na,15a K,15b Sn,15c and Cu.15d To the best of our knowl-
fast and efficient. The use of an Fe(III) template brought
the terminal alcohol and carboxylates of the siderophore
edge, this is the first example using an Fe(III)-template for
substrates in closer proximity which enhanced the reaction
macrolactonization of a siderophore. We are currently
rate and percent conversion to the desired macrolactones
5bÀ8b. Iron also conveniently served as a chromophore
exploring the use of catalytic metals (Fe(III), Ga(III),
Al(II), etc.) in the macrolactonization reactions and eval-
uating the bioactivity and Fe(III)-affinity constants of the
macrocyclic siderophores.
(λmax = 427 nm) for monitoring reaction progress and a
protecting group for the nucleophilic hydroxamates. The
Fe(III) was removed using EDTA providing access to
desferri-siderophore macrolactones which are necessary
for treating iron-overload diseases.
Other metal-templated macrolactonizations are cited in
Acknowledgment. T.A.W. thanks the UND Chemistry-
Biochemistry-Biology Interface program (NIH T32GM075762)
for fellowship support. Crystallographic data were col-
the literature that use oxophilic ionophores complexed to
lected at Beamline 11.3.1 at the Advanced Light Source
(ALS), Lawrence Berkeley National Laboratory sup-
(13) See Supporting Information for exact experimental details,
HPLC chromatograms, and first-order rate plots.
ported by the U.S. Dept. of Energy, Office of Energy
Sciences, under contract DE-AC02-05CH11231.
(14) (a) van der Helm, D.; Poling, M. J. Am. Chem. Soc. 1976, 98, 82–
86. (b) Bilayet Hossain, M.; Jalal, M. A. F.; van der Helm, D. Acta
Crystallogr. 1986, C42, 1305–1310. (c) Bilayet Hossain, M.; Jalal,
M. A. F.; van der Helm, D.; Shimizu, K.; Akiyama, M. J. Chem.
Crystallogr. 1998, 28, 53–56. (d) Dhungana, S.; White, P. S.; Crumbliss,
A. L. J. Biol. Inorg. Chem. 2001, 6, 810–81813.
Supporting Information Available. General experimen-
tal details and characterization data (X-ray, NMR, MS,
HPLC) for synthesis of siderophores 4 and 5a,b,cÀ8a,b,c.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(15) (a) Carrillo, R.; Martin, V. S.; Martin, T. Tetrahedron Lett. 2004,
€
45, 5215–5219. (b) Furstner, A. Eur. J. Org. Chem. 2004, 943–958. (c)
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ꢀ ~
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Garcı
The authors declare no competing financial interest.
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Org. Lett., Vol. 14, No. 17, 2012
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