Synthesis of heterobactins A and B and Nocardia heterobactin

Article abstract of DOI:10.1016/j.tet.2011.03.003

The synthesis of the Rhodococcus erythropolis siderophores heterobactins A and B, and the structurally related Nocardia heterobactin, is described. Two approaches for the assembly of these asymmetric ligand donor chelators are explored. In the first approach, a scheme predicated on the biosynthesis of the Paracoccus denitrificans siderophore, parabactin, is employed. In this approach, the central donor synthon is added last. In the second scheme, the central donor and the terminal 2,3-dihydroxybenzoyl fragment are first fixed to the ligand's d-ornithine backbone. This is followed by condensation with the cyclic ornithine hydroxamate glycine segment. The schemes offer a flexible approach to other heterobactins. Job's plots suggest that heterobactin A and Nocardia heterobactin form 1:1 ligand/metal complexes, while heterobactin B forms a 3:2 ligand/metal complex.

Full text of DOI:10.1016/j.tet.2011.03.003

Tetrahedron 67 (2011) 3163e3169
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of heterobactins A and B and Nocardia heterobactin
*
Raymond J. Bergeron , Shailendra Singh, Neelam Bharti
Department of Medicinal Chemistry, University of Florida, Box 100485, Gainesville, FL 32610-0485, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 January 2011
Received in revised form 25 February 2011
Accepted 1 March 2011
Available online 8 March 2011
The synthesis of the Rhodococcus erythropolis siderophores heterobactins A and B, and the structurally
related Nocardia heterobactin, is described. Two approaches for the assembly of these asymmetric ligand
donor chelators are explored. In the first approach, a scheme predicated on the biosynthesis of the
Paracoccus denitrificans siderophore, parabactin, is employed. In this approach, the central donor synthon
is added last. In the second scheme, the central donor and the terminal 2,3-dihydroxybenzoyl fragment
are first fixed to the ligand’s D-ornithine backbone. This is followed by condensation with the cyclic
Keywords:
ornithine hydroxamate glycine segment. The schemes offer a flexible approach to other heterobactins.
Job’s plots suggest that heterobactin A and Nocardia heterobactin form 1:1 ligand/metal complexes,
while heterobactin B forms a 3:2 ligand/metal complex.
Natural products
Total synthesis
Heterobactin
Siderophore
Iron complex
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
candidates as tools for bioremediation.13 This potential application
adds value to the current work. The Rhodococcus siderophores are
Iron serves as a prosthetic for many different redox enzymes,1
which are essential for life itself. Although it comprises about 5%
of the earth’s crust, the metal is not easily accessible to biological
systems.
now synthetically accessible for analytical standards or biosynthetic
studies. This will help investigators to monitor and understand the
organism’s iron-regulated growth properties. This is key to opti-
mizing the use of Rhodococcus in oil remediation.
The concentration of ferric iron required to support the growth of
most microorganisms lies in the range of 5ꢀ10ꢁ8 to 1ꢀ10ꢁ6 mol/L.2
However, because of the extreme insolubility of ferric hydroxide at
physiological pH, about 10ꢁ18 mol/L, microorganisms had to develop
a means of sequestering and transporting this metal.3 They secrete
large quantities of siderophores, low molecular weight chelators,
which form complexes with iron(III), providing a transport vector.4
Most siderophores present with either hydroxamate donors, e.g.,
desferrioxamine5 and nocardamine,6 or catecholamide donors, e.g.,
petrobactin7 and vibriobactin8 (Fig. 1). These particular ligands form
1:1 complexes with Fe(III). Catecholamide chelators typically form
Interestingly, the siderophores isolated from both Rhodococcus and
Nocardia are in many ways similar to the ligands predicated on poly-
amine backbones, e.g., parabactin9 and agrobactin14 (Fig. 2). For exam-
ple, with parabactin, the bidentate fragments, 2,3-dihydroxybenzoyl
tighter Fe(III) complexes, e.g., parabactin9 (Kf¼1048
M
ꢁ1) (Fig. 2), than
their hydroxamate counterparts, e.g., desferrioxamine (1028 Mꢁ1
)
(Fig. 1).10
The current study focuses on the assembly of the heterobactins A
and B (Fig. 2), isolated from Rhodococcus erythropolis11 and a heter-
obactin analogue (Nocardia heterobactin or JBIR-16) derived from
a human pathogen Nocardia tenerifensis.12 R. erythropolis has drawn
considerable attention recently because of the ability of particular
strains to metabolize oil, making these organisms attractive
* Corresponding author. Fax: þ1 352 392 8406; e-mail address: rayb@ufl.edu
(R.J. Bergeron).
Fig. 1. Catecholamide and hydroxamate siderophores.
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.03.003

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R.J. Bergeron et al. / Tetrahedron 67 (2011) 3163e3169
Scheme 1. Retrosynthetic analysis of heterobactins A, B and Nocardia heterobactin.
Fig. 2. Derivation of heterobactin A and Nocardia heterobactin from heterobactin B by
analogy to parabactin and agrobactin.
key to the success of Scheme 1 is the effective use of N- and
O-protecting groups.
groups, are fixed to a linear polyamine backbone at the terminal nitro-
gens via amide linkages. The remainder of the hexacoordinate donor
array is fixed to the central nitrogen as the amide of 2-(2-hydroxy-
phenyl) -(4S,5R)-trans-5-methyl-4-oxazolinecarboxylic acid. The donor
ligands consist of the terminal 2,3-dihydroxybenzoyl hydroxyls, the
central oxazoline nitrogen, and the internal aromatic hydroxyl. Heter-
obactin A (1) and B (2), from Rhodococcus,11 and the heterobactin ana-
logue (3) from Nocardia12 also present with terminal bidentate
fragments, although unsymmetrical: on one end, a 2,3-dihydroxy-
An alternative retrosynthetic view of Nocardia heterobactin (3)
begins by bis-acylation of
D
-ornithine (10) with activated 2,3-
dihydroxybenzoic acid (5), generating bis-catecholamide 11
(Scheme 2). Peptide coupling of glycine derivative 6 with carboxylic
acid 11 will produce the natural product 3 by a more convergent
synthesis than in Scheme 1. As before, the route in Scheme 2 is
dependent on optimal fragment protecting groups.
benzoyl, on the other, a hydroxamate derived from cyclized N5eOHe
L-
ornithine. The central donor in heterobactin A (1), a 5-hydroxy-4-car-
boxyl-benzoxazole may employ the aromatic hydroxyl and the amide
function, as has been previously observed.11 A similar central donor
scenario, a substituted salicylamide array, can also be invoked with
Nocardia heterobactin (3) with its central 2,3-dihydroxybenzamide unit.
2. Results and discussion
2.1. Synthetic strategy
From a biosynthetic perspective, it seems reasonable that both
heterobactin A (1) and Nocardia heterobactin (3) derived from
heterobactin B (2) (Fig. 2). However, although heterobactin B (2)
was isolated from R. erythropolis,11 it remains to be identified in
Nocardia sp. It is interesting that the same biosynthetic sequence
was shown with both parabactin9 and agrobactin.14 Both were
derived from the tetracoordinate precursor, N1,N8-bis(2,3-dihy-
droxybenzoyl)spermidine.9 These observations suggested a bio-
mimetic, retrograde synthetic scheme for the heterobactins
described in this work. Initially, the key intermediate would indeed
be a protected form of ligand 2 (Fig. 2).
Scheme 2. Alternative retrosynthesis of Nocardia heterobactin.
An approach to heterobactins 1e3 derives from the following
retrosynthetic analysis, which requires the regiospecific formation
of three amide bonds (Scheme 1). In the final step the amino group
of heterobactin B (2) could be acylated with activated 5-hydroxy-4-
benzoxazolecarboxylic acid (4) to provide heterobactin A (1) or 2,3-
dihydroxybenzoic acid (5) to give Nocardia heterobactin (3). The
inner amide bond of 2 could be formed by coupling dipeptide 6
2.2. Total synthesis
The biomimetic approach is defined by four segments, (1) as-
sembly of the hydroxamate, cyclic N5-hydroxy- -ornithine-N2-gly-
L
cyl fragment (15, Scheme 3), (2) construction of the N5-(2,3-
dibenzyloxy)- -ornithine segment (18, Scheme 4), (3) coupling of
D
with monoacylated
hydroxy-2-piperidinone (8), a cyclic
glycine (9) would furnish 6; condensation of
5-amino group with 2,3-dihydroxybenzoic acid (5) would give 7. A
D
-ornithine 7. N-Acylation of (S)-3-amino-1-
-ornithine hydroxamate, with
-ornithine (10) at the
the above fragments and deprotection of the central nitrogen to
liberate the key intermediate 20 (Scheme 5), and (4) N-acylation of
tri-O-protected heterobactin B (20) to fully protected heterobactin
A (21) or Nocardia heterobactin (22, Scheme 6).
L
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R.J. Bergeron et al. / Tetrahedron 67 (2011) 3163e3169
3165
Scheme 3. Synthesis of protected cyclic ornithine hydroxamate peptide 15.
Scheme 4. Synthesis of protected catecholamide fragment 18.
Scheme 6. Synthesis of heterobactins A (1), B (2), and Nocardia heterobactin (3).
TEA in CH2Cl2 to generate the intermediate 19 in 80% yield. The tert-
butoxycarbonyl protecting group of 19 was removed by treatment
with TFA and triethylsilane in CH2Cl2, providing 20 in 95% yield.
When tripeptide 20 was debenzylated under a hydrogen at-
mosphere over Pd/C in CH3OH and a catalytic amount of 1 N HCl,
heterobactin B (2) was obtained in 96% yield (Scheme 6). N-Acyl-
ation of intermediate 20 with 5-hydroxy-4-benzoxazolecarboxylic
acid (4) as its NHS ester provided peptide 21 (45% yield). Treatment
of 21 with H2 over Pd/C, led to heterobactin A (1) quantitatively.
5-Hydroxy-4-benzoxazolecarboxylic acid (4) was generated
from methyl 2-amino-3,6-dihydroxybenzoate (23)17 as in Scheme 7.
Methyl ester 23 was converted into oxazole 24 in 75% yield with
triethyl orthoformate in refluxing EtOH. While saponification of the
methyl ester of 24 with LiOH failed, treatment of 24 with LiI in
refluxing THF for 18 h provided carboxylic acid 4 in 76% yield.
Scheme 5. Synthesis of the key intermediate, 20.
Synthesis of the cyclic hydroxamate of -ornithine/glycine (15)
L
(Scheme 3)fragmentwasaccomplishedbya1,10-carbonyldiimidazole
(CDI) mediated couplingof (S)-3-amino-1-benzyloxy-2-piperidinone
hydrobromide (12)15 with N-(tert-butoxycarbonyl)glycine (13) in
triethylamine (TEA) and CH2Cl2, providing peptide 14 in 68% yield.
The peptide was subjected to a trifluoroacetic acid (TFA) removal of
the Boc-protecting group in the presence of triethylsilane to provide
O-benzylated hydroxamate 15 as its TFA salt in quantitative yield.
In order to construct the terminal catecholamide of hetero-
bactins 1e3, 2,3-bis(benzyloxy)benzoic acid (17)16 was activated as
its N-hydroxysuccinimide (NHS) ester using N,N0-dicyclohex-
Scheme 7. Synthesis of 5-hydroxy-4-benzoxazolecarboxylic acid (4).
Alternatively, acylation of the amino group of 20 with 2,3-diben-
zyloxybenzoic acid (17) as its NHS ester provided penta-O-protected
peptide 22 (30% yield). Hydrogenolysis of 22 over Pd/C led to the
Nocardia heterobactin (3) quantitatively.
The moderate yields (30e45%) associated with the formation of
intermediates 21 or 22 by the methodology of Scheme 6 prompted
us to consider another route to both heterobactin A (1) and
Nocardia heterobactin (3), shown in Scheme 8. The alternative
synthesis of the latter siderophore began with the condensation of
ylcarbodiimide (DCC) and coupled with
-N2-Boc ornithine (16),
D
producing N2-Boc-N5-(2,3-dibenzyloxybenzoyl)-
-ornithine (18) in
D
70% yield (Scheme 4).
The key intermediate (20, Scheme 5) required for the assembly
of heterobactin A (1), heterobactin B (2), and Nocardia heterobactin
(3) (Scheme 6) was accessed by first coupling 15 with 18 using CDI/

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R.J. Bergeron et al. / Tetrahedron 67 (2011) 3163e3169
Scheme 8. Alternate synthesis of 22.
D-ornithine (10) itself with 2 mol of 2,3-dibenzyloxybenzoic acid
(17) activated as NHS ester to produce diamide acid 25 in 77% yield.
The carboxylate group of 25 was then coupled with the amine of 15
to produce the benzyl-protected precursor (22) to the Nocardia
siderophore in 55% yield. As the debenzylation of 22 was quanti-
Fig. 3. Job’s plots of the Fe(III) complex of heterobactins A (1), B (2), and Nocardia
heterobactin (3). Solutions containing different ligand/Fe(III) ratios were prepared such
that [ligand]þ[Fe(III)]¼0.9 mM at pH 7.4.
tative, the overall yield of Nocardia heterobactin (3) from D-orni-
thine (10) via Scheme 8 was 37%, while the previous route from
D-
N2-Boc ornithine (16) provided 3 in only a 16% overall yield.
A similar approach was also invoked for the assembly of het-
erobactin A (1) but failed. The N-Boc protecting group of amide 18
(Scheme 4) was quantitatively removed with TFA. Unfortunately,
we were unable to condense oxazolecarboxylic acid 4 with the
resulting primary amine using either DCC or CDI.
the donor fragments are already in place. The latter scheme worked
well for Nocardia heterobactin (3) but was not viable for hetero-
bactin A (1). The former approach (Scheme 1) provided all of the
heterobactins and would also permit the generation of synthetic
heterobactin analogues for biological testing, especially in vivo
metal decorporation. Specifically, amine 20 (Scheme 6) could be
reacted with a wide range of activated carboxylic acids followed by
removal of the O-benzyl protecting groups.
2.3. Stoichiometry
In the original papers describing the structures of heterobactins A
(1) and B (2)11 and Nocardia heterobactin (3),12 theauthorsoffer some
preliminaryevidence regardingthestoichiometryof thesiderophore/
ironcomplexes. The evidence, largely mass spectraldata, isinkeeping
withtheidea thatheterobactins A andB and Nocardiaheterobactinall
form 1:1 ligand/metal complexes. Winkelmann,11 further speculates
that the tetracoordinate heterobactin B may also form a 3:2 ligand/
metal complex. While the mass spectral data is certainly consistent
with the 1:1 ligand/metal complexes, more complete support for the
solution chemistry seemed necessary.
In order to further substantiate the nature of the metal com-
plexes, Job’s plots were run for all three ligands at pH 7.4. The total
siderophore plus iron concentrations were kept constant while the
mole fraction was varied. The absorbance max was read at 520 nm
for the heterobactin A and Nocardia heterobactin iron complexes
and at 508 nm for the heterobactin B iron complex (Fig. 3). The Job’s
plots for the heterobactin A and Nocardia heterobactin iron com-
plexes complement the mass spectral data and are consistent with
1:1 ligand/metal complexes. However, the Job’s plot for the hetero-
bactin B iron complex suggests that a 3:2 ligand/metal chelator
exists at pH 7.4.
With the ligands available in sufficient quantity, we were able to
evaluate the iron complex stoichiometries utilizing Job’s plots. Both
heterobactin A (1) and Nocardia heterobactin (3) formed 1:1 li-
gand/metal complexes. Heterobactin B (2) was shown to form a 3:2
ligand/metal complex. These findings support previous structural
studies predicated on mass spectral data.11 The included work will
be valuable for investigators interested in the potential oil spill
bioremediation properties of Rhodococcus. They now have facile
access to heterobactins A (1) and B (2), analytical standards, and
biosynthetic intermediates that will help define the role of iron in
microbial growth.
4. Experimental
4.1. General procedures
Reagents were purchased from Aldrich Chemical Co. (Milwau-
kee, WI). Fisher Optima-grade solvents were routinely used, and
THF was distilled from sodium/benzophenone. Reactions were run
under a nitrogen atmosphere, and the organic extracts were dried
with sodium sulfate and then filtered. Silica gel 70e230 from Fisher
Scientific (Pittsburgh, PA) was utilized for column chromatography,
and silica gel 40e63 from SiliCycle, Inc. (Quebec City, Quebec,
Canada) was used for flash column chromatography. Compounds
1e3 were chromatographed using Sephadex LH-20, which was
obtained from Amersham Bioscience (Piscataway, NJ). Fractions
were spotted on a silica gel TLC plate and sprayed with 1% ferric
chloride in ethanol. Distilled solvents and glassware that had been
presoaked in 3 N HCl for 15 min, washed with distilled water and
distilled ethanol, and oven dried were used in the isolation of 1e3.
Optical rotations were run at 589 nm (sodium D line) at 20 ꢂC
utilizing a PerkineElmer 341 polarimeter, with c being concentra-
tion in grams of compound per 100 mL of solution (CHCl3 not in-
dicated). The iron content of the Fe(III)-NTA solution was verified
3. Conclusion
Siderophores from R. erythropolis, heterobactins A (1) and B (2),
and from N. tenerifensis, Nocardia heterobactin (3), are now syn-
thetically accessible. Two approaches were investigated. One
scheme is based on the likely biosynthesis of heterobactin A and
Nocardia heterobactin, a biomimetic design (Scheme 1). With this
methodology, the central donor fragments for both heterobactin A
and the Nocardia heterobactin are added to the heterobactin B
backbone last.
In the second design, the cyclic
the ligand is coupled to the N2,N5-bis(2,3-dihydroxybenzoyl)-
ornithine ‘end’ of the siderophore (Scheme 2). In this instance, all of
L-ornithine hydroxamate ‘end’ of
D
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R.J. Bergeron et al. / Tetrahedron 67 (2011) 3163e3169
3167
using a PerkineElmer 5100 PC Atomic Absorption Spectropho-
tometer (AAS). Data for the Job’s plots were recorded on a UV-2550
UVevis spectrophotometer. High resolution mass spectra were
obtained on an Agilent 6210 TOF mass spectrometer using elec-
trospray ionization (ESI). The base peaks are reported for the high
resolution mass spectra. NMR spectra were obtained at 400 MHz
(1H) or 100 MHz (13C) on a Varian Mercury 400 BB instrument.
with H2O (25 mL) and saturated NaCl (25 mL) and concentrated
under reduced pressure. Column chromatography with 10%
CH3OH/CHCl3 (Rf 0.55) provided 0.39 g (70%) of 18 as a white solid,
mp 65e66 ꢂC: [
a
] ꢁ9.0 (c 0.2). 1H NMR
d
1.36e1.95 (mþs, 13H),
3.12e3.20 (m, 1H), 3.32e3.44 (m, 1H), 4.34 (q, 1H, J¼4.4), 5.09 (s,
2H), 5.15 (s, 2H), 7.15e7.18 (m, 2H), 7.30e7.50 (m, 10H), 7.68e7.73
(m, 1H), 8.17 (br s, 1H). 13C NMR
d 25.0, 25.6, 28.5, 30.1, 33.8, 39.1,
Chemical shifts (
d
) for 1H spectra are given in parts per million
52,8, 71.5, 76.7, 80.1, 117.4, 123.5, 124.6, 126.7, 127.8, 128.4, 128.8,
128.9, 129.0, 129.0, 136.4, 136.5, 147.1, 151.8, 155.9, 166.1, 174.9.
HRMS m/z calcd for C31H36N2O7 549.2595 [MþH]þ, 571.2415
[MþNa]þ, 1119.4937 [2MþNa]þ; found 549.2584 [MþH]þ, 571.2410
[MþNa]þ, 1119.4888 [2MþNa]þ.
downfield from tetramethylsilane for organic solvents (CDCl3 not
indicated) or sodium 3-(trimethylsilyl)proponate-2,2,3,3-d4 for
D2O. Chemical shifts (
million referenced to 1,4-dioxane (
solvent resonance in CDCl3 77.16) (not indicated) or CD3OD (d
d
) for 13C NMR spectra are given in parts per
d
67.19) in D2O or to the residual
(d
49.00). Coupling constants (J) are in hertz. Rf values were calculated
from silica gel TLC plates run on the column chromatography sol-
vent unless otherwise specified. Melting points were recorded on
a Fisher-Johns melting point apparatus and are uncorrected.
4.2.4. tert-Butyl [(R)-1-[[2-[[(S)-1-(benzyloxy)-2-oxopiperidin-3-yl]
amino]-2-oxoethyl]amino]-5-[2,3-bis(benzyloxy)benzamido]-1-ox-
opentan-2-yl]carbamate (19). CDI (0.109 g, 0.67 mmol) was added to
a solution of 18 (0.370 g, 0.67 mmol) in CH2Cl2 (2 mL) and stirred for
1 h at room temperature. The resulting solution was cooled to 0 ꢂC
and was added to a suspension of 15 (0.262 g, 0.67 mmol) and TEA
(0.14 mL,1.34 mmol) at 0 ꢂC. The solutionwas stirred for 15 h at room
temperature, followed by the addition of CH2Cl2 (20 mL). The re-
action mixture was washed with 8% NaHCO3 (25 mL), 0.5 M citric
acid (25 mL), saturated NaCl (25 mL). The organic phase was con-
centrated by rotary evaporation. Flash chromatography eluting with
5% CH3OH/CH2Cl2 (Rf 0.4) afforded 0.43 g (80%) of 19 as a colorless
4.2. Procedures
4.2.1. tert-Butyl (S)-[2-[[1-(benzyloxy)-2-oxopiperidin-3-yl]-amino]-
2-oxoethyl]carbamate (14). CDI (0.162 g, 1.0 mmol) was added to
a solution of 13 (0.175 g, 1.0 mmol) in CH2Cl2 (5 mL). After stirring
for 1 h, a solution of 12 (0.301 g,1.0 mmol) in CH2Cl2 (2 mL) and TEA
(0.202 g, 2.0 mmol) was added. The solution was stirred for 24 h at
room temperature and diluted with CH2Cl2 (25 mL). The organic
layer was washed with 8% NaHCO3 (25 mL), 0.5 M citric acid
(25 mL), and saturated NaCl (25 mL) and was concentrated by ro-
tary evaporation. Flash chromatography using 5% CH3OH/CH2Cl2 (Rf
solid, mp 72e73 ꢂC: [
a
] þ44.35 (c 0.115). 1H NMR
d 1.43 (s, 9H),
1.48e1.58 (m, 2H), 1.72e1.84 (m, 6H), 2.21e2.25 (m, 1H), 3.03e3.16
(m, 1H), 3.24e3.30 (m, 1H), 3.33e3.42 (m, 1H), 3.49e3.58 (m, 1H),
3.90 (dd,1H, J¼5.6,16.8), 4.04 (dd,1H, J¼5.6,16.8), 4.18e4.27 (m,1H),
4.28e4.35 (m, 1H), 4.82 (2d, 2H, J¼10.4, 10.4), 5.08 (s, 2H), 5.14 (s,
2H), 5.30 (br s, 1H), 7.01 (br s, 1H), 7.14 (d, 2H, J¼5.2), 7.27e7.48 (m,
0.5) generated 0.256 g (68%) of 14 as a viscous oil: [
a
] þ98.0 (c 0.10).
1H NMR
d
1.46 (s, 9H), 1.82e1.89 (m, 2H), 2.34e2.42 (m, 1H),
3.31e3.38 (m, 1H), 3.39e3.48 (m, 1H), 3.81 (dd, 1H, J¼16.8, 4.8),
3.94 (dd, 1H, J¼17.2, 4.8), 4.40 (quintet, 1H, J¼6.0), 4.92 (2d, 2H,
J¼10.8, 10.8), 5.26 (br s, 1H), 6.98 (d, 1H, J¼6.0), 7.36e7.43 (m, 5H).
15H), 7.67 (t,1H, J¼4.8), 8.10 (br s,1H).13C NMR
d 21.1, 26.1, 27.6, 28.5,
30.1, 38.6, 43.2, 51.3, 53.9, 71.4, 76.0, 76.6, 77.6, 80.3, 117.1, 123.3,
124.6, 125.4, 127.4, 127.8, 128.4, 128.4, 128.6, 128.8, 128.9, 129.0,
129.0,129.2,129.7,135.3,136.5,146.9,151.8,165.9,167.5,169.3,173.0.
HRMS m/z calcd for C45H53N5O9 808.3916 [MþH]þ, 830.3736
[MþNa]þ; found 808.3886 [MþH]þ, 830.3706 [MþNa]þ.
13C NMR
d 21.0, 27.8, 28.5, 44.3, 51.4, 51.4, 76.2, 80.4, 128.7, 129.0,
129.7, 135.2, 156.1, 167.9, 169.9. HRMS m/z calcd for C19H27N3O5
378.2023 [MþH]þ, 400.1843 [MþNa]þ, 777.3794 [2MþNa]þ; found
378.2021 [MþH]þ, 400.1838 [MþNa]þ, 777.3794 [2MþNa]þ.
4.2.5. N-[(R)-4-Amino-5-[[2-[[(S)-1-(benzyloxy)-2-oxopiperidin-3-yl]
amino]-2-oxoethyl]amino]-5-oxopentyl]-2,3-bis(benzyloxy) benzamide
trifluoroacetate (20). TFA (0.45 mL, 6.0 mmol) and triethylsilane
(0.16 mL, 1.0 mmol) were successively added to 19 (0.320 g,
0.4 mmol) in CH2Cl2 (2 mL) with ice bath cooling, and the solution
was stirred at 0 ꢂC for 1 h and at room temperature for 3 h. After the
removal of volatiles by rotary evaporation, the residue was dried
under high vacuum to give 0.306 g (95%) of 20 as a white solid, mp
4.2.2. (S)-2-Amino-N-[1-(benzyloxy)-2-oxopiperidin-3-yl]-acet-
amide trifluoroacetate (15). TFA (1.96 mL, 26.4 mmol) was added to
14 (0.66 g, 1.75 mmol) in CH2Cl2 (2 mL) with ice bath cooling fol-
lowed by triethylsilane (0.70 mL, 4.4 mmol), and the solution was
stirred for 1 h at 0 ꢂC and 2 h at room temperature. Volatiles were
removed under reduced pressure. The procedure was repeated with
toluene, and the residue was dried under high vacuum to give 0.65 g
(quantitative) of 15 as a white solid, mp 91e92 ꢂC: [
H2O). 1H NMR (D2O)
1.72e2.10 (m, 4H), 3.54e3.62 (m, 2H), 3.84 (s,
2H), 4.46e4.52 (m, 1H), 4.97 (s, 2H), 7.40e7.54 (m, 5H). 13C NMR
(D2O)
a] þ5.2 (c 0.115,
94e95 ꢂC: [
a
] ꢁ2.5 (c 0.12, CH3OH). 1H NMR
d 1.54e1.98 (m, 8H),
d
2.94e3.05 (m, 1H), 3.16e3.24 (m, 1H), 3.32e3.41 (m, 2H), 3.72e3.80
(m, 1H), 4.11e4.17 (m, 1H), 4.24e4.30 (m, 1H), 4.43e4.52 (m, 1H),
4.79 (2d, 2H, J¼10.4,10.4), 5.04 (s, 2H), 5.12 (s, 2H), 7.06e7.12 (m, 2H),
7.27e7.33 (m, 13H), 7.36 (m, 5H), 7.56 (dd, 1H, J¼2.0, 7.6), 8.10 (d, 1H,
d
23.2, 29.4, 43.4, 52.9, 53.6, 78.6, 119.3 (q, J¼290.2), 131.7,
132.3,133.0,137.2,166.0 (q, J¼35.2),169.8,170.7. HRMS m/z calcd for
C14H19N3O3 (free amine) 278.1499 [MþH]þ, 300.1319 [MþNa]þ,
555.2926 [2MþH]þ, 577.2725 [2MþNa]þ; found 278.1486 [MþH]þ,
300.1305 [MþNa]þ, 555.2905 [2MþH]þ, 577.2724 [2MþNa]þ.
J¼7.6), 8.19 (t, 1H, J¼6.4), 8.67 (br s, 1H). 13C NMR
d 21.0, 25.2, 27.2,
28.4, 29.9, 37.8, 43.3, 50.7, 52.7, 71.4, 75.7, 76.7, 116.4 (q, J¼290.6),
117.4, 122.9, 124.6, 126.8, 127.5, 127.8, 128.0, 128.5, 128.6, 128.7, 128.8,
128.9, 129.0, 129.0, 129.6, 129.7, 134.9, 136.3, 136.4, 147.0, 151.9, 161.6
(q, J¼35.9), 166.5, 167.7, 169.7, 171.0. HRMS m/z calcd for C40H46N5O7
(free amine) 708.3353 [MþH]þ, 730.3217 [MþNa]þ; found 708.3390
[MþH]þ, 730.3220 [MþNa]þ.
4.2.3. (R)-5-[2,3-Bis(benzyloxy)benzamido]-2-[(tert-butoxy-car-
bonyl)amino]pentanoic acid (18). A solution of DCC (0.206 g,
1.0 mmol) in THF (1 mL) was added dropwise to a mixture of 17
(0.334 g, 1.0 mmol) and NHS (0.115 g, 1.0 mmol) in THF (2 mL) at
0 ꢂC. The mixture was stirred for 6 h at room temperature and was
filtered. The filtrate was added to a solution of 16 (0.232 g,
1.0 mmol) and KHCO3 (0.20 g, 2.0 mmol) in 50% aqueous THF
(20 mL) at pH w8. After the reaction mixture was stirred at room
temperature for 24 h, THF was removed by rotary evaporation. The
residue was treated with 0.5 M citric acid (20 mL) and extracted
with EtOAc (2ꢀ25 mL). The combined organic portion was washed
4.2.6. N-[(R)-1-[[2-[[(S)-1-(Benzyloxy)-2-oxopiperidin-3-yl]-amino]-
2-oxoethyl]amino]-5-[2,3-bis(benzyloxy)benzamido]-1-oxopentan-2-
yl]-5-hydroxy-4-benzoxazolecarboxamide (21). A solution of DCC
(0.166 g, 0.81 mmol) in THF (1 mL) was added dropwise to a mix-
ture of 4 (0.143 g, 0.81 mmol) and NHS (0.092 g, 0.81 mmol) in THF
(2 mL) at 0 ꢂC. The reaction mixture was stirred overnight at room
temperature and was filtered. The filtrate was concentrated to

3168
R.J. Bergeron et al. / Tetrahedron 67 (2011) 3163e3169
dryness under reduced pressure. The residue was added to a solu-
tion of 20 (0.64 g, 0.80 mmol) and TEA (0.160 g, 1.6 mmol) in 5%
aqueous CH3CN (20 mL) followed by stirring at room temperature
for 48 h. Volatiles were removed under reduced pressure, and the
residue was treated with 0.25 M citric acid (20 mL) and extracted
with EtOAc (2ꢀ25 mL). The combined organic extracts were
washed with H2O (25 mL) and saturated NaCl (25 mL), and con-
centrated. Column chromatography with 8% CH3OH/CH2Cl2 (Rf 0.6)
filtered through a bed of Celite and silica gel, and the filtrate was
concentrated under reduced pressure. Column chromatography
eluting with 5% CH3OH/CH2Cl2 (Rf 0.65 in 10% CH3OH/CH2Cl2)
provided 0.265 g (75%) of 24 as a yellow solid, mp 134e135 ꢂC: 1H
NMR
d
4.13 (s, 3H), 7.07 (d, 1H, J¼8.8), 7.7 (d, 1H, J¼8.8), 8.19 (s, 1H),
11.30 (s, 1H). 13C NMR
d
53.1, 103.9, 116.3, 118.0, 139.1, 143.9, 154.4,
161.0, 170.5. HRMS m/z calcd for C9H7NO4 216.0267 [MþNa]þ,
238.0087 [MꢁHþ2Na]þ, 409.0642 [2MþNa]þ; found 216.0267
[MþNa]þ, 238.0080 [MꢁHþ2Na]þ, 409.0629 [2MþNa]þ.
generated 0.316 g (45%) of 21 as a viscous oil: [
NMR
a
] ꢁ5.0 (c 0.11). 1H
d
1.50e1.70 (m, 3H), 1.71e2.10 (m, 4H), 2.24e2.31 (m, 1H),
3.12e3.21 (m, 1H), 3.24e3.32 (m, 1H), 3.36e3.43 (m, 1H), 3.61e3.71
(m, 1H), 3.91 (dd, 1H, J¼16.8, 5.6), 4.11 (dd, 1H, J¼16.8, 6.0), 4.31
(quintet, 1H, J¼5.6), 4.83 (2d, 2H, J¼10.0, 10.0), 5.07 (s, 2H), 5.14 (s,
2H), 6.98 (d, 1H, J¼9.2), 7.05 (d, 1H, J¼6.8), 7.12e7.15 (m, 2H),
7.27e7.47 (m, 15H), 7.50 (t, 1H, J¼6.0), 7.55 (d, 1H, J¼9.2), 7.66 (t, 1H,
J¼5.6), 8.09 (s, 1H), 8.12 (t, 1H, J¼5.6), 9.62 (d, 1H, J¼7.2), 12.61 (s,
4.2.9. 5-Hydroxy-4-benzoxazolecarboxylic acid (4). Anhydrous LiI
(0.47 g, 11.9 mmol) was added to a solution of 24 (0.23 g, 1.19 mmol)
inTHF (20 mL), and the mixturewas refluxed for 18 h in the dark. The
solvent was removed by rotary evaporation. The residue was dis-
solved in H2O (10 mL), and the pH was adjusted to w2 with 1 N HCl.
The mixture was extracted with EtOAc (2ꢀ20 mL), and the organic
phase was concentrated under reduced pressure. Column chroma-
tography eluting with 10% CH3OH/CH2Cl2 (Rf 0.28) afforded 0.162 g
(76%) of 4 as light yellow solid, mp 171e172 ꢂC: 1H NMR (CD3OD):
1H). 13C NMR
d 21.0, 26.1, 27.6, 30.2, 38.5, 43.3, 51.3, 51.4, 52.7, 71.4,
76.0, 76.6, 104.3, 116.3, 116.8, 117.0, 123.2, 124.6, 127.5, 127.8, 128.4,
128.6, 128.8, 128.8, 128.9, 129.0, 129.7, 135.3, 136.4, 136.5, 137.8,
143.0, 146.9, 151.8, 153.7, 160.5, 165.9, 167.5, 169.2, 169.2, 172.0.
HRMS m/z calcd for C48H48N6O10 891.3324 [MþH]þ, 913.3144
[MþNa]þ; found 891.3317 [MþH]þ, 913.3129 [MþNa]þ.
d
7.05 (d,1H, J¼9.2), 7.81 (d,1H, J¼8.8), 8.56 (s,1H).13C NMR (CD3OD)
¼104.6,117.1,118.8,139.9,145.0,156.8,162.3,172.3. HRMS m/z calcd
d
for C8H5NO4 223.9930 [MꢁHþ2Na], 245.9750 [Mꢁ2Hþ3Na]þ;
found 223.9932 [MꢁHþ2Na], 245.9744 [Mꢁ2Hþ3Na]þ.
4.2.7. N,N0-[(R)-5-[[2-[[(S)-1-(Benzyloxy)-2-oxopiperidin-3-yl]-
amino]-2-oxoethyl]amino]-5-oxopentane-1,4-diyl]bis[2,3-bis-(benzy-
loxy)benzamide] (22). A solution of DCC (0.103 g, 0.5 mmol) in THF
(1 mL) was added dropwise to a mixture of 17 (0.167 g, 0.5 mmol)
and NHS (0.058 g, 0.5 mmol) in THF (2 mL) at 0 ꢂC, and the reaction
mixture was stirred at room temperature for 16 h, filtered, and
concentrated to dryness under reduced pressure. The residue was
added to a solution of 20 (0.402 g, 0.5 mmol) and TEA (0.101 g,
1.0 mmol) in 10% aqueous CH3CN (20 mL). After the mixture was
stirred at room temperature for 40 h, volatiles were removed by
rotary evaporation. The residue was treated with 0.5 M citric acid
(20 mL) and extracted with EtOAc (2ꢀ25 mL). Organic extracts
were washed with H2O (25 mL) and saturated NaCl (25 mL), and
concentrated in vacuo. Column chromatography with 10% CH3OH/
CHCl3 generated 0.153 g (30%) of 22 as a glassy solid.
4.2.10. (R)-2,5-Bis[2,3-bis(benzyloxy)benzamido]pentanoic acid (25). A
solution of DCC (0.206 g,1.0 mmol) in THF (3 mL) was added dropwise
to a mixture of 17 (0.334 g, 1.0 mmol) and NHS (0.115 g, 1.0 mmol) in
THF (3 mL) at 0 ꢂC. The solution was stirred for 16 h at room tem-
perature and was filtered. The filtrate was added to a solution of 10
(0.116 g, 0.5 mmol) and TEA (0.202 g, 2.0 mmol) in 10% aqueous THF
(20 mL). After the mixture was stirred at room temperature for 48 h,
volatiles were removed under reduced pressure. The residue was
dissolved in 0.5 M citric acid (15 mL) and extracted with EtOAc
(2ꢀ20 mL). The combined organic portion was washed with H2O
(20 mL) and saturated NaCl (25 mL) and then concentrated. Column
chromatography with 12% CH3OH/CHCl3 (Rf 0.33) provided 0.294 g
(77%) of 25 as a colorless solid, mp 45e46 ꢂC: [
CH3OH). 1H NMR
1.20e1.46 (m, 2H), 1.50e1.94 (m, 2H), 3.08
a] þ15.65 (c 0.65,
d
Alternate Method: CDI (0.041 g, 0.25 mmol) was added to a so-
lution of 25 (0.191 g, 0.25 mmol) in CH2Cl2 (1 mL) and stirred for 1 h
at room temperature. The resulting solution was added to a sus-
pension of 15 (0.94 g, 0.25 mmol) and TEA (0.07 mL, 0.67 mmol) at
0 ꢂC. The reaction mixture was stirred for 24 h at room temperature,
diluted with CH2Cl2 (10 mL), and washed with 8% NaHCO3 (10 mL),
0.5 M citric acid (10 mL), and saturated NaCl (10 mL). Concentration
of the organic phase under reduced pressure and flash chroma-
tography, eluting with 10% CH3OH/CHCl3 (Rf 0.6) afforded 0.141 g
(quintet, 1H, J¼6.4), 3.19 (quintet, 1H, J¼6.4), 4.66 (d, 1H, J¼4.8), 5.01
(s, 2H), 5.06e5.17 (mþs, 6H), 7.08e7.46 (m, 26H), 7.68e7.77 (m, 2H),
7.96 (br s, 1H), 8.55 (d, 1H, J¼7.6). 13C NMR
d 25.7, 29.4, 39.2, 52.5, 71.3,
71.4, 76.3, 76.5,117.2,117.5,123.4,123.4, 124.5,126.5,127.0,127.7,127.8,
127.9, 128.3, 128.6, 128.6, 128.7, 128.8, 128.8, 128.9, 129.0, 129.0, 136.2,
136.3, 136.4, 136.5, 147.0, 147.1, 151.7, 151.8, 165.6, 168.6, 174.4. HRMS
m/z calcd for C47H44N2O8 765.3170 [MþH]þ, 787.2990 [MþNa]þ,
809.2809 [MꢁHþ2Na]þ; found 765.3154 [MþH]þ, 787.2987
[MþNa]þ, 809.2797 [MꢁHþ2Na]þ.
(55%) of 22 as a glassy solid, mp 67e68 ꢂC: [
NMR
a
] þ30.67 (c 0.15). 1H
d
1.50e1.70 (m, 5H), 1.71e1.94 (m, 2H), 2.19e2.24 (m, 1H),
4.2.11. Heterobactin B (2). PdeC (10%, 0.75 g) and a catalytic
amount of 1 N HCl were added to a solution of 20 (0.141 g,
0.2 mmol) in CH3OH (5 mL), and the mixture was stirred under H2
at atmospheric pressure for 6 h. The mixture was filtered through
Celite, and the solids were washed with CH3OH (3ꢀ5 mL). The
combined filtrate was concentrated by rotary evaporation. The
residue was dissolved in CH3OH (10 mL), and Sephadex LH-20
(0.60 g) was introduced. After 4 h, the solvent was removed under
reduced pressure. The solid was loaded onto a preswelled LH-20
(3.0 g) column and was eluted with 1:14:85 H2O/EtOH/toluene. The
iron active fractions were combined and concentrated to afford
2.96e3.08 (m, 1H), 3.24e3.44 (m, 3H), 3.83 (dd, 1H, J¼16.8, 5.6),
4.04 (dd, 1H, J¼16.8, 6.0), 4.31e4.42 (m, 1H), 4.83 (2d, 2H, J¼10.0,
10.0), 5.05 (s, 2H), 5.11 (s, 2H), 5.15 (s, 2H), 5.13e5.20 (m, 2H), 6.89
(t, 1H, J¼6.0), 7.10e7.16 (m, 5H), 7.27e7.46 (m, 23H), 7.64e7.72 (m,
2H), 7.93 (t, 1H, J¼5.2), 8.39 (d, 1H, J¼5.6). 13C NMR
d 21.2, 26.1, 27.7,
28.9, 30.0, 38.6, 38.8, 43.3, 51.3, 51.3, 54.3, 71.5, 71.6, 76.0, 76.7,117.2,
117.8,123.5,124.6,124.7,126.9,127.5,127.9,127.9,128.5,128.7,128.9,
129.0, 129.0, 129.3, 129.8, 135.6, 136.5, 136.6, 136.6, 147.0, 147.4,
151.9, 151.9, 165.6, 166.5, 167.4, 169.5, 172.2. HRMS m/z calcd for
C61H61N5O10 1046.4311 [MþNa]þ, 2070.8761 [2MþNa]þ; found
1046.4356 [MþNa]þ, 2070.8798 [2MþNa]þ.
0.091 g (96%) of 2 as a white solid, mp 147e148 ꢂC: [
0.11, CH3OH). 1H NMR (CD3OD)
1.71e1.82 (m, 3H), 1.89 (m, 5H),
a
] ꢁ33.64 (c
d
4.2.8. Methyl 5-hydroxy-4-benzoxazolecarboxylate (24). Triethyl
orthoformate (1.21 mL, 7.29 mmol) was added to a solution of 23
(0.4 g, 1.82 mmol) in EtOH (2 mL). The reaction mixture was
refluxed for 24 h, cooled to room temperature and diluted with
acetone (20 mL). After treatment with charcoal, the mixture was
3.45 (t, 2H, J¼6.4), 3.56e3.64 (m, 2H), 3.94 (t, 1H, J¼6.4), 3.97 (s,
2H), 4.45e4.49 (m, 1H), 6.72 (t, 1H, J¼7.6), 6.92 (d, 1H, J¼8.0), 7.15
(d, 1H, J¼7.2). 13C NMR (CD3OD)
d 21.7, 26.0, 28.7, 29.9, 39.6, 43.2,
51.5, 52.6, 54.3, 116.7, 118.7, 119.6, 147.4, 150.2, 167.2, 170.6, 170.9,
171.7. HRMS m/z calcd for C19H27N5O7 (free amine) 438.1983

R.J. Bergeron et al. / Tetrahedron 67 (2011) 3163e3169
3169
[MþH]þ, 460.1803 [MþNa]þ, 875.3894 [2MþH]þ; found 438.1978
[MþH]þ, 460.1793 [MþNa]þ, 875.3859 [2MþH]þ.
were monitored at the visible lmax of the Fe(III) complexes (508 nm
for 2 and 520 nm for 1 and 3). A 25 mM MOPS buffer with 50%
CH3OH (v/v) was used to maintain the pH at 7.4. Solutions
containing different ligand/Fe(III) ratios were prepared by mixing
appropriate volumes of 0.9 mM ligand solution and 0.9 mM Fe(III)-
nitriloacetate (NTA) in MOPS-MeOH solution. The 0.9 mM Fe(III)-
NTA solution was prepared immediately prior to use by dilution of
a 45 mM Fe(III)-NTA stock solution with the MOPS/CH3OH mixture.
The Fe(III)-NTA stock solution was prepared by mixing equal vol-
umes of 90 mM FeCl3 and 180 mM trisodium NTA. The iron content
of the Fe(III)-NTA solution was verified by AAS.
4.2.12. Heterobactin A (1). PdeC (10%, 0.60 g) was added to a so-
lution of 21 (0.130 g 0.15 mmol) in CH3OH (5 mL), and the mixture
was stirred under H2 at atmospheric pressure for 4 h. The mixture
was filtered through Celite, and the solids were washed with
CH3OH (3ꢀ5 mL). The combined filtrate was concentrated by rotary
evaporation. The residue was dissolved in CH3OH (10 mL), and LH-
20 (0.5 g) was added. After 4 h, the solvent was removed under
reduced pressure. The solid was loaded onto a preswelled LH-20
(2.5 g) column, which was eluted with 2e50% EtOH in toluene. The
iron active fractions were combined and concentrated to afford
Acknowledgements
0.089 g (quantitative) of 1 as a white solid, mp 140e141 ꢂC: [
(c 0.24, CH3OH). 1H NMR (CD3OD)
1.69e2.13 (m, 8H), 3.46e3.63
a
] ꢁ7.5
d
Support of this work by grant number R37DK049108 from the
National Institutes of Health is most gratefully acknowledged. We
thank Dr. James S. McManis and Miranda E. Coger for their editorial
and organizational support. We acknowledge the spectroscopy
services in the Chemistry Department, University of Florida, for the
mass spectrometry analyses.
(m, 4H), 3.90 (m, 2H,), 4.38e4.46 (m,1H), 4.63e4.67 (m,1H), 6.34 (t,
1H, J¼7.6), 6.74 (d, 1H, J¼7.6), 6.93 (d, 1H, J¼8.8), 7.29 (d, 1H, J¼8.0),
7.62 (d, 1H, J¼9.2), 8.35 (s, 1H). 13C NMR (CD3OD)
d 21.7, 27.3, 28.7,
30.9, 39.4, 43.9, 51.5, 53.1, 54.9, 113.7, 115.0, 116.8, 118.3, 118.9, 118.9,
120.6, 139.6, 139.6, 143.7, 150.4, 155.8, 158.2, 164.8, 170.5, 171.6,
171.8, 175.0. HRMS m/z calcd for C26H31N5O10 597.1963 [MꢁH]ꢁ,
599.2096 [MþH]þ; found 597.1967 [MꢁH]ꢁ, 599.2028 [MþH]þ.
References and notes
4.2.13. Nocardia heterobactin (3). PdeC (10%, 0.20 g) was added to
a solution of 22 (0.283 g 0.4 mmol) in CH3OH (10 mL), and the
mixture was stirred under H2 at atmospheric pressure for 6 h. The
mixture was filtered through Celite, and the residue was washed
with CH3OH (3ꢀ5 mL). The combined filtrate was concentrated by
rotary evaporation. The residue was dissolved in CH3OH (10 mL),
and LH-20 (1.0 g) was added. After 4 h, the solvent was removed
under reduced pressure. The solid was loaded onto a preswelled
LH-20 (5.0 g) column, which was eluted with 1:10:89 H2O/EtOH/
toluene. The iron active fractions were combined and concentrated
to afford 0.229 g (quantitative) of 3 as a fluffy pale solid, mp
1. (a) Sahlin, M.; Petersson, L.; Graslund, A.; Ehrenberg, A.; Sjoberg, B.-M.; The-
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135e136 ꢂC:
[
a]
ꢁ12.0 (c 0.15, CH3OH). 1H NMR (CD3OD)
d
1.70e1.84 (m, 2H), 1.85e2.10 (m, 6H), 3.38e3.52 (m, 2H),
3.52e3.68 (m, 2H), 3.82e4.02 (m, 2H), 4.42e4.54 (m, 1H),
4.54e4.66 (m, 1H), 6.68e6.77 (m, 2H), 6.87e6.96 (m, 2H), 7.20 (d,
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1H, J¼7.2)), 7.35 (d, 1H, J¼7.2). 13C NMR (CD3OD)
d 21.9, 27.2, 28.9,
10. Model, B.; Berdoukas, V. The Clinical Approach to Thalassaemia; Grune & Strat-
30.3, 40.2, 43.8, 51.6, 52.7, 55.5, 116.9, 117.3, 118.8, 119.6, 119.7, 119.8,
120.0, 120.1, 147.4, 147.5, 149.8, 150.5, 167.3, 171.5, 171.6, 174.5, 174.9.
HRMS m/z calcd for C26H31N5O10 574.2144 [MþH]þ, 596.1963
[MþNa]þ; found 574.2143 [MþH]þ, 596.1966 [MþNa]þ.
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4.3. Job’s plots for heterobactins A (1), B (2) and Nocardia
14. Bergeron, R. J.; Xin, M. G.; Weimar, W. R.; Smith, R. E.; Wiegand, J. J. Med. Chem.
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heterobactin (3)
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The stoichiometries of the ligand-Fe(III) complexes of 1e3 were
determined spectrophotometrically using Job’s plots. Solutions