Vibriobactin antibodies: A vaccine strategy

Article abstract of DOI:10.1021/jm900119q

A new target strategy in the development of bacterial vaccines, the induction of antibodies to microbial outer membrane ferrisiderophore complexes, is explored. A vibriobactin (VIB) analogue, with a thiol tether, 1-(2,3-dihydroxybenzoyl)-5,9-bis[[(4S,5R)-

Full text of DOI:10.1021/jm900119q

J. Med. Chem. 2009, 52, 3801–3813
3801
Vibriobactin Antibodies: A Vaccine Strategy
Raymond J. Bergeron,*,† Neelam Bharti,† Shailendra Singh,† James S. McManis,† Jan Wiegand,† and Linda G. Green‡
Department of Medicinal Chemistry and the Hybridoma Core Laboratory, Interdisciplinary Center for Biotechnology Research,
UniVersity of Florida, GainesVille, Florida 32610-0485
ReceiVed January 30, 2009
A new target strategy in the development of bacterial vaccines, the induction of antibodies to microbial
outer membrane ferrisiderophore complexes, is explored. A vibriobactin (VIB) analogue, with a thiol tether,
1-(2,3-dihydroxybenzoyl)-5,9-bis[[(4S,5R)-2-(2,3-dihydroxyphenyl)-4,5-dihydro-5-methyl-4-oxazolyl]car-
bonyl]-14-(3-mercaptopropanoyl)-1,5,9,14-tetraazatetradecane, was synthesized and linked to ovalbumin
(OVA) and bovine serum albumin (BSA). The antigenicity of the VIB microbial iron chelator conjugates
and their iron complexes was evaluated. When mice were immunized with the resulting OVA-VIB conjugate,
a selective and unequivocal antigenic response to the VIB hapten was observed; IgG monoclonal antibodies
specific to the vibriobactin fragment of the BSA and OVA conjugates were isolated. The results are consistent
with the idea that the isolated adducts of siderophores covalently linked to their bacterial outer membrane
receptors represent a credible target for vaccine development.
Introduction
Iron occurs in oxidation states from -2 to +6 depending on
both pH and the nature of the ligating groups surrounding the
metal.1 It is these dependencies that nature has exploited so
effectively in enlisting the metal as a central component in a
myriad of redox processes.2 In fact, life without iron is virtually
nonexistent.3 However, while the metal composes some 5% of
the earth’s crust, it is nevertheless difficult for living systems
to access. In the biosphere, iron exists largely as Fe(III), in a
variety of water insoluble forms, at pH 7. The concentration of
free Fe(III) under these conditions is ∼1.4 × 10-9 M,4 somewhat
lower than that required to support most life forms. In the
presence of phosphate ions in culture media and potential animal
hosts, the free Fe(III) concentration in solution drops even
Figure 1. Naturally occurring iron chelators (siderophores): hydrox-
amates (1) and catecholamides (2 and 3).
further, by a factor of 10.
Both prokaryotes and eukaryotes have overcome the problem
of iron accessibility by developing iron-binding ligands and
associated transport systems.5-12 Prokaryotes produce a group
of iron chelators, siderophores (generally low molecular weight,
iron-specific ligands), that they secrete into the environment.10
Iron acquisition becomes somewhat more problematic for
microorganisms in an in vivo situation (e.g., in humans).
Pathogens have additional iron acquisition hurdles to overcome
beyond low metal solubility. Animals, for example, have an
iron-withholding system: proteinaceous iron chelators that make
iron acquisition difficult for microorganisms. There is little of
the free metal available in animals. It is generally bound to
heme10 (iron-containing enzymes)10 by transferrin12 (an iron
shuttle protein) or stored in ferritin.11 In each instance, iron is
not easily accessible to microorganisms.
The opportunistic microorganism Vibrio Vulnificus nicely
illustrates how pathogens can overcome host iron-withholding.20,38
The siderophore produced by Vibrio Vulnificus,21 vulnibactin
(2) (Figure 1), cannot remove iron from transferrin, the ever-
present iron shuttle protein in plasma, in spite of the fact that 2
binds iron more tightly than transferrin. The chelator cannot
access transferrin iron, as it is bound within the protein. To
solve this problem, the microorganism secretes a protease, which
cleaves transferrin, thus releasing iron. The metal is then
sequestered by 2, and the ferrisiderophore is taken up via an
intermembrane receptor ViuA.39-41
These ligands often present very large formation constants (e.g.,
13
1048 M-1
)
and can effectively remove the metal from other
donor arrays. The resulting metal complex, a ferrisiderophore,
is then taken up by microorganisms,14-16 most often beginning
with binding to an outer membrane receptor.17 This is followed
by shuttling the iron complex through the periplasm and finally
to the cytoplasm, where the iron is freed up. These ferrisidero-
phore transporters are energy-dependent, often exploiting the
tonB system.18-20 In most instances, the desferrisiderophore is
released to further gather iron.
There have now been over 500 different siderophores
identified.21-25 While there are certainly exceptions, the two
main classes of natural product iron chelators are hydroxama-
tes,26-31 such as desferrioxamine (1) and catecholamides,13,32-36
including vulnibactin (2) and vibriobactin (3) (Figure 1). Some
microorganisms can, in fact, utilize more than one type and/or
class of siderophores.37
* To whom correspondence should be addressed. Phone: (352) 273-7725.
Fax: (352) 392-8406. E-mail: rayb@ufl.edu.
In fact, Vibrio Vulnificus mutants without the vulnibactin
transporter have reduced pathogenicity in mice.42 This uptake
apparatus has been shown to have significant homology with
the Vibrio cholerae receptor.20,38,39 However, while it seems
†
Department of Medicinal Chemistry.
Hybridoma Core Laboratory, Interdisciplinary Center for Biotechnology
‡
Research.
10.1021/jm900119q CCC: $40.75
2009 American Chemical Society
Published on Web 06/03/2009

3802 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Bergeron et al.
Figure 2. Retrosynthetic analysis of vibriobactin-OVA (4) and vibriobactin-BSA (5) conjugates from vibriobactin thiol (6).
clear from studies with genetically altered microorganisms that
shutting down the siderophore iron-uptake system can slow
growth and reduce pathogenicity, microorganisms can still
access iron via other mechanisms.43-45 For example, Vibrio
cholerae can utilize transferrin and heme as iron sources. The
issue then becomes how useful a target the siderophore transport
apparatus is in antimicrobial design strategies.
Miller has, in a series of classic studies, employed sidero-
phores and the corresponding transporters as vectors for the
delivery of antibiotics.46 Alternatively, Esteve-Gassent was able
to demonstrate that a vaccine developed to treat eels infected
with Vibrio Vulnificus serovar E. contained antigens to the
putative receptor for vulnibactin. Esteve-Gassent point out that
the antibody could be blocking siderophore uptake, could trigger
classical complement activation, or “mark bacteria for op-
sonophagocytosis”.47
There is now significant literature that supports the idea that
many microorganisms present with outer membrane receptors
for the binding and internalization of their ferrisiderophore
complexes. It is not unreasonable to assume that on binding to
the microbial receptors, the iron siderophore complex is at least
initially exposed. If sufficiently antigenic, this “ferrisiderophore
face” could represent a significant target in vaccine development.
The question then becomes what should the expectations be
regarding the antigenicity of a ferrisiderophore fixed to a large
carrier molecule? If indeed it were very antigenic, this would
merit the assembly of ferrisiderophores with functionality that
allow for covalent linkage to the transporter and isolation of
the adduct as a potential vaccine.
The antigenicity of a ferrisiderophore bound to a large carrier
molecule is the focus of this manuscript. The specific questions
addressed here are the following: Is it possible (1) to assemble
a carrier siderophore conjugate, i.e., a protein carrier conjugate,
(2) to raise antibodies to the conjugate in mice, and (3) to assess
the antigenicity of the protein siderophore and its iron complex?
utilization in Vibrio cholerae.48,49 This was attractive for two
reasons: Vibrio cholerae represents an important pathological
target,50-52 and we had established critical information about
vibriobactin chemistry in earlier studies.32-36 Accordingly, we
elected to investigate an ovalbumin (OVA)-vibriobactin protein
conjugate (4, OVA-VIB) as an antigen.
The fundamental issue would be appending a tether to
vibriobactin (3) (Figure 1), which would allow for fixing the
ligand to a carrier protein, in this case, both OVA and bovine
serum albumin (BSA). This demanded a synthetic approach very
different from the assembly of vibriobactin itself.35 The
OVA-VIB conjugate (4) would be used as an antigen to raise
antibodies in mice, and the BSA-VIB conjugate (5) (Figure
2) would be utilized in an enzyme-linked immunosorbent assay
(ELISA), first for the detection of serum polyclonal antibodies
and, finally, vibriobactin-specific IgG monoclonal antibodies.
Thus, choosing the appropriate activated tether for the vibrio-
bactin protein conjugate was the first hurdle. While a number
of different tethers were considered (e.g., acyl, halo, thiol),
previous experience with hypusine antibody generation53 en-
couraged pursuit of a thiol-containing tether. The final ligand
would be 1-(2,3-dihydroxybenzoyl)-5,9-bis[[(4S,5R)-2-(2,3-di-
hydroxyphenyl)-4,5-dihydro-5-methyl-4-oxazolyl]carbonyl]-14-
(3-mercaptopropanoyl)-1,5,9,14-tetraazatetradecane (6) or vibri-
obactin thiol (Figure 2).
The first approach to vibriobactin thiol (6) began with the
conversion of norspermidine (7) to polyamine reagent 12, that
is, thermospermine54 protected at the aminobutyl terminus
(Scheme 1). Specifically, norspermidine (7) was heated at reflux
with 4-chloro-1-butanol in 1-butanol in the presence of K2CO3
and KI, producing linear triamino alcohol 8. The amine groups
of 8 were masked as tert-butyl carbamates using di-tert-butyl
dicarbonate in THF to generate alcohol 9 in 42% yield for two
steps. In spite of the moderate yield, the conversion of 7 to 9 in
Scheme 1 is considerably shorter than our previous route to
N1-(4-hydroxybutyl)-N1,N4,N7-tris(tert-butoxycarbonyl)norsper-
midine (9).55 The hydroxyl of 9 was activated as its tosylate 10
in 92% yield by treatment with TsCl in CH2Cl2 and NEt3.55
Heating 10 with potassium phthalimide in DMF at 85 °C gave
the fully protected tetraamine 11 in 71% yield. The carbamates
of 11 were cleaved quantitatively with trifluoroacetic acid (TFA),
affording reagent 12. Activation of 2,3-dimethoxybenzoic acid
with 1,1′-carbonyldiimidazole (CDI) and acylation of the
primary amine of 12 in CH2Cl2 and NEt3 gave masked
catecholamide 13 in 70% yield. Next, the N-hydroxysuccinimide
ester of N-tert-butoxycarbonyl-L-threonine56 (3 equiv) was
coupled to the internal nitrogens of 13 in DMF to produce
Results and Discussion
Antigen Design Concept. The current study focuses on the
generation of antibodies against vibriobactin (3, VIBa), the
hexacoordinate iron chelator, a siderophore, responsible for iron
a Abbreviations: VIB, vibriobactin; BSA, bovine serum albumin; OVA,
ovalbumin; ELISA, enzyme-linked immunosorbent assay; TFA, trifluoro-
acetic acid; CDI, 1,1′-carbonyldiimidazole; OD, optical density; ICP-MS,
inductively coupled plasma mass spectroscopy; NMS, normal mouse sera;
sc, subcutaneously; EWB, ELISA wash buffer; PBS, phosphate buffered
saline; mAb, monoclonal antibody; DMSO, dimethyl sulfoxide; THF,
tetrahydrofuran; DMF, N,N-dimethylformamide.

Vibriobactin Antibodies
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3803
Scheme 2. Synthesis of 20a
Scheme 1. Synthesis of 17a
a Reagents: (a) 60% NaH, 4-methoxybenzyl bromide (3.5 equiv), DMF,
62%; (b) 2 N NaOH, dioxane, 90%.
generating primary amine 15, 2 equiv of which were connected
using 3,3′-dithiodipropionic acid (CDI in CH2Cl2), furnishing
disulfide 16 in 40% yield. Threonylamine and catecholamide
deprotection and also disulfide cleavage occurred in the presence
of BBr3 in CH2Cl2, giving dihydrobromide salt 17 in 73% yield.
Unfortunately, any attempt at cyclization of the threonyl
fragments of 17 with ethyl 2,3-dihydroxybenzimidate34 resulted
in complex mixtures, including thioesters, that were virtually
impossible to separate, thus dooming the route of Scheme 1 to
failure in the last step.
A catechol protecting group other than methyl, that is, one
that could be removed concurrently with the BOC functionality
while leaving the disulfide intact, was required. Thus, 2,3-
dihydroxybenzoic acid (18) was converted to its trianion with
NaH in DMF and treated with excess 4-methoxybenzyl bromide
to make ester 19 in 62% yield. Hydrolysis of 19 with NaOH
(aqueous) in dioxane produced 2,3-bis(4-methoxybenzyloxy)-
benzoic acid (20) in 90% recrystallized yield (Scheme 2).
Activation of an equivalent of carboxylic acid 20 with CDI and
stirring with N12-(phthaloyl)thermospermine (12) in CH2Cl2 and
NEt3 afforded diamine 21 in 57% yield (Scheme 3).
The secondary amines of 21 were acylated with the active
ester of N-(BOC)-L-threonine as before to generate tetraamine
derivative 22 in 60% yield. The phthalimide functionality of
22 was cleaved in 90% yield with hydrazine hydrate in EtOH
at room temperature, yielding primary amine 23, 2 equiv of
which were joined utilizing 3,3′-dithiodipropionic acid (CDI in
CH2Cl2), resulting in disulfide 24 in 60% yield. The threonyl
carbamates and the 4-methoxybenzyl ethers57 of 24 were
simultaneously cleaved, using TFA in anisole and CH2Cl2;
Sephadex LH-20 purification resulted in a 60% yield of disulfide
25, a tetrakis(TFA) salt. The threonyl moieties of 25 were next
condensed with excess ethyl 2,3-dihydroxybenzimidate34 in
refluxing EtOH to produce tetrakis(oxazoline) disulfide 26 in
20% yield. Finally, the disulfide bond of iron chelator 26 was
reduced to the free thiol 6 in 60% yield, utilizing H2 (3 atm)
over Pd black in CH3OH under iron-free conditions (Scheme
3).
Functionalization of Vibriobactin Thiol (6) with OVA
and BSA Protein Carriers. Vibriobactin thiol (6), freshly
generated from disulfide 26 (Scheme 3), was incubated with a
maleimide-activated OVA (27) or BSA (28) protein carrier for
8 h. Unreacted maleimide was capped by conjugating it further
with cysteine for 8 h, resulting in Michael adduct OVA-VIB
(4) or BSA-VIB (5), respectively (Scheme 4). Both 4 and 5
were purified on a dextran desalting column. Positive fractions,
as determined by the optical density (OD) at 280 nm, were
analyzed for protein concentration using a Coomassie assay.58
a Reagents: (a) 4-chloro-1-butanol, K2CO3, KI, 1-butanol, 125 °C, 1 d;
(b) di-tert-butyl dicarbonate, THF, 42%; (c) 92%;55 (d) potassium phthal-
imide, DMF, 90 °C, 48 h, 71%; (e) TFA, CH2Cl2, 0 °C f room temp, 2 h,
quantitative; (f) 2,3- dimethoxybenzoic acid, CDI, CH2Cl2, NEt3, 70%; (g)
N-(BOC)-L-threonine N-hydroxysuccinimide ester (3 equiv), DMF, 55%;
(h) hydrazine hydrate, EtOH, 65%; (i) 3,3′-dithiopropionic acid (0.4 equiv),
CDI, CH2Cl2, 40%; (j) BBr3, CH2Cl2, 73%; (k) ethyl 2,3-dihydroxybenz-
imidate, failed.
triamide 14 in 55% yield. The phthalimide protecting group of
14 was removed in 65% yield with hydrazine hydrate in EtOH,

3804 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Scheme 3. Synthesis of Vibriobactin Thiol 6a
Bergeron et al.
Scheme 4. Coupling of Vibriobactin Thiol 6 to Carrier Proteinsa
a Reagents: (a) 50% DMSO, 0.1 M phosphate buffer (pH 7.4), 8 h; (b)
cysteine.
excess ferric nitrilotriacetate in phosphate buffer for 2 h. At
this point, desferrioxamine (1) (Figure 1) was added to complex
excess iron. The mixture was then purified on a G-25 Sepharose
column. The same ferration procedure, including purification,
was carried out on the maleimide-activated OVA (27), producing
ferric protein complex 32. The iron content of the purified
ferrivibriobactin protein adduct (31), the iron-treated maleimide
protein (32), and the elution buffer were determined by
inductively coupled plasma mass spectroscopy (ICP-MS). The
background iron, elution buffer iron, and maleimide protein iron
were subtracted from the iron content associated with the
ferrivibriobactin OVA complex (31). From this measurement,
and assuming that Fe(III) and vibriobactin form a 1:1 complex,
the coupling efficiency of OVA maleimide (27) to 6 was 30%.
Vibriobactin (3) and vibriobactin disulfide (26) iron(III)
complexes, 33 and 34, respectively, which were to be evaluated
as potential antigens, were prepared as previously described.59
The resulting suspensions were separated on a small C-18
column, eluting with EtOH (aqueous). Colored fractions were
pooled and lyophilized.
Development of Murine Monoclonal Antibodies against
Vibriobactin. The procedures were similar to those of Kao and
Klein60 and Simrell, et al.61 Briefly, the OVA-VIB conjugate
(4) was used as an antigen to raise antibodies in mice. Antigenic
response was determined via an ELISA. Once an adequate IgG
response was observed, an immunized mouse was given a final,
prefusion booster of the antigen without adjuvant. The mouse
was euthanized 4 days later, and antibody-forming cells from
the animal’s spleen were fused to tumor cells grown in culture.
The resulting hybridomas were screened for antibody produc-
tion; antibody-producing hybridomas were cloned. Monoclonal
antibodies were then produced and purified.
Polyclonal Antibody Response. Two mice (M1 and M2)
were immunized with 4. The immune response (serum titer) of
these animals and nonimmunized mice (normal mouse sera,
NMS) was determined via ELISA for polyclonal antibodies
against the following potential antigens: (a) BSA-VIB conju-
gate (5), (b) BSA-cysteine conjugate (30), (c) vibriobactin thiol
(6), (d) vibriobactin disulfide (26), (e) vibriobactin disulfide-iron
complex (34), (f) vibriobactin (3), and (g) vibriobactin-iron
complex (33). Twenty-three days after the second immunization,
a Reagents: (a) 20, CDI, CH2Cl2, NEt3, 57%; (b) N-(BOC)-L-threonine
N-hydroxysuccinimide ester (2.5 equiv), DMF, 40 °C, 60%; (c) hydrazine
hydrate, EtOH, 90%; (d) 3,3′-dithiopropionic acid, CDI, CH2Cl2, NEt3, 60%;
(e) TFA, anisole, CH2Cl2, 0 °C f room temp, 2 h, 60%; (f) ethyl 2,3-
dihydroxybenzimidate, EtOH, reflux, 36 h, 20%; (g) H2, 3 atm, Pd black,
EtOH, 1 d, 60%.
Functionalities 29 and 30 were also generated (Scheme 4); 30
was used as a negative control in evaluating the capacity of 6
as an antigenic determinant when bound to BSA.
Iron Complexes of 4, 27, 3, and 26. Protein desferrivibrio-
bactin-OVA complex 4 was converted to the corresponding
ferric siderophore complex 31 by mixing the conjugate with

Vibriobactin Antibodies
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3805
Table 1. ELISA Determination of Reactivity of Polyclonal Antibodies
(Serum Titer) against 5 and 30
optical density at 405 nma
M1
M2
NMS
5
30
5
30
5
30
serum
dilution 23 d 56 d 56 d 23 d 56 d 56 d 23 d 56 d 56 d
1:200
1:400
1:800
3.192 3.675 3.464 3.381 3.647 3.496 0.111 0.111 0.087
3.295 3.749 3.340 3.370 3.692 3.496 0.106 0.095 0.077
2.986 3.708 2.787 3.148 3.664 3.359 0.113 0.107 0.074
1:1600 2.230 3.586 1.676 2.867 3.637 2.975 0.087 0.087 0.072
1:3200 1.370 3.535 0.883 2.188 3.486 2.025 0.109 0.096 0.076
1:6400 0.905 2.919 0.447 1.255 2.724 1.270 0.179 0.100 0.072
1:12800 0.476 1.756 0.254 0.809 1.718 0.736 0.106 0.095 0.080
1:25600 0.296 0.830 0.167 0.431 1.012 0.379 0.100 0.091 0.094
a The optical density of the p-nitrophenol product was determined 1 h
after the addition of the p-nitrophenyl phosphate substrate. M1 was
immunized with 4 twice sc at a dose of 50 µg/immunization. M2 was
immunized with the conjugate twice sc at 100 µg/immunization. Sera were
obtained 23 and 56 d after the second immunization. Normal mouse sera
(NMS) served as a negative control.
the serum from the mouse immunized with a higher dose of 4
(M2) seemed more active against antigen 5 than M1, with a
higher p-nitrophenol OD at 405 nm at all dilutions (Table 1).
However, by day 56, there was little difference in the reactivity
of serum from M1 vs M2. At this time, even after a 25600-fold
dilution, the serum titers of the immunized mice against 5 were
over 9 times greater than that of the NMS (Table 1).
The mouse sera also contained polyclonal antibodies against
30 that were nearly as active as antibodies against 5 (Table 1).
Since cysteine was used to cap unreacted maleimide sites in
the synthesis of 4, this was expected. As will be discussed below,
this was not an issue for the purified monoclonal antibodies;
we were able to select for antibodies specific against 4. The
mouse sera did not react against any of the other antigens, c-g
(data not shown). This could have been attributed to a simple
lack of activity in the case of antigens c-g or the nature of the
ELISA itself. Antigens c-g are relatively low molecular weight,
moderately water-soluble ligands. These compounds may not
have adhered to the ELISA wells, or they may have been
removed during the washing steps. In order to settle this issue,
a series of competitive binding ELISAs were performed.
Competitive Binding ELISA. In the competitive binding
ELISA, sera from immunized mice or nonimmunized mice were
first incubated with potential antigens f and g, or with 4, at
antigen concentrations ranging from 0 to 250 µg/mL. If an
antigen is an effective competitor, it will bind to the antibody
during this initial incubation, leaving less antibody available to
bind to a second antigen coated on the ELISA plate. This
“competition” will be reflected in lower p-nitrophenol optical
density values.
Figure 3. Competitive binding ELISA of diluted polyclonal sera
(1:10000) from mice immunized with the OVA-VIB conjugate (4).
Animal M1 (A) was immunized with 4 twice sc at 50 µg/injection,
while mouse M2 (B) was immunized with 4 twice sc at 100
µg/injection. The sera were incubated with varying concentrations of
antigen 4, prior to being tested via ELISA against antigen 5. The dotted
line indicates the amount of 4 (∼0.05 µg/mL) needed to reduce the
optical density of the p-nitrophenol product to 50% of that of the sera
not incubated with any 4. The closed circles indicate the optical density
of unconjugated OVA (27) (10 µg/mL).
Table 2. Competitive Binding ELISA of Serum from Immunized Mice
and Nonimmunized Mice
antigen 5: optical density at 405 nma
antigen 4
conc (µg/mL)
M1
M2
NMS
0
1.147
0.486
0.250
0.135
0.114
0.106
0.108
0.048
1.152
1.800
0.926
0.581
0.243
0.137
0.113
0.109
0.044
1.655
0.113
0.109
0.108
0.105
0.108
0.108
0.111
0.045
0.113
0.08
0.4
2
10
50
250
Medium
27
Antigen 4 was found to be an effective competitor at all
concentrations tested (Figure 3, Table 2). The smaller antigens
f and g were not effective competitors (data not shown),
verifying the necessity for a large carrier molecule in order for
the antibody to recognize vibriobactin (3). Unconjugated OVA
(27) was not an effective competitor. The p-nitrophenol optical
density of serum incubated with 27 against antigen 5 was 92%
greater than serum first incubated with 4 at the same concentra-
tion (Figure 3). It is clear that the antibody “recognizes” the
siderophore on the protein carrier.
Cell Fusion-Hybridoma Formation. Four months after the
second immunization, mouse M2 was given a prefusion booster
of 4. Fusion was done following the method of Simrell et al.61
except that the myeloma cell line used was Sp2/0. In short,
spleen cells were fused with Sp2/0 cells such that the ratio of
a The optical density of the p-nitrophenol product was determined 1 h
after the addition of the p-nitrophenyl phosphate substrate. Sera from
immunized mice (M1 and M2) and normal mice (NMS), diluted 1:10000,
were incubated with antigen 4 for 2 h at room temperature. A portion of
this mixture was then transferred to an ELISA plate that had been coated
with antigen 5, 10 µg/mL. Medium was used as a negative control;
unconjugated OVA (27) (10 µg/mL) served as a positive control.
spleen cells to Sp2/0 cells was 7:1. After incubation for 11 days,
the supernatants from the resulting hybridomas were tested via
ELISA against antigen 5. The hybridomas that gave the strongest
ELISA signal were further cultured for 7 days. Their superna-
tants were tested again via ELISA against 5, BSA-VIB-iron
(35), and 30. The class of antibody (IgG or IgM) was also
determined. The 12 most active hybridomas that were 5 and 35
positive and 30 negative are shown in Table 3. One hybridoma,

3806 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Bergeron et al.
Table 4. Recloning of 5A6 Pooled Multiple Colony Wells: ELISA
Table 3. Reactivity of Antibody-Containing Hybridoma Supernatants
against 5 and Other Antigens as Measured by an ELISA
Screening of Supernatants from Resulting Single Colony Wells against
5, 35, and 30
optical density at 405 nma
optical density at 405 nma
hybridoma
5
35
30
IgG (γ-chain) IgM (µ-chain)
5
1H8
2C11
2D6
3.662 3.040 0.100
3.564 3.427 0.246
2.481 2.340 0.157
3.173 2.832 0.180
2.830 2.903 0.161
3.331 3.449 0.217
2.824 3.663 0.486
1.979 2.022 0.196
3.154 2.962 0.555
2.399 2.130 0.170
3.359 3.488 0.390
4.000 4.000 0.548
0.093 0.101 0.107
3.350 3.443 1.002
0.608
3.652
0.825
3.530
2.331
3.537
3.808
2.491
3.532
3.500
2.889
4.000
0.103
3.438
0.091
0.432
3.083
0.120
0.085
0.133
0.389
0.084
0.094
0.097
0.090
0.120
0.106
0.386
clone
1G2
1G8
2D5
2F4
2G4
3B9
3E9
3G8
4B5
4B7
Medium
5A6 (PC)
primary
secondary
35
30
4.000
4.000
3.408
3.382
3.436
3.395
3.395
3.457
3.421
3.419
0.235
3.359
3.472
3.558
3.575
3.522
3.548
3.473
3.420
3.479
3.503
3.503
0.125
3.488
3.492
3.444
3.579
3.586
3.587
3.418
3.386
3.549
3.534
3.563
0.127
3.488
0.721
0.807
0.689
0.831
1.075
1.130
0.604
0.929
0.636
0.862
0.099
0.667
2F5
2F10
3F11
3H9
4A5
4A7
4G3
5A6
5D8
Medium
M2 (PC)
a The optical density of the p-nitrophenol product was determined 1 h
after the addition of the p-nitrophenyl phosphate substrate. Medium served
as a negative control; immune mouse serum (M2) in a 1:1000 dilution was
used as a positive control (PC). Hybridoma culture supernatants were
undiluted and were screened for antigenic activity 18 days postfusion. Rabbit
antimouse IgG (whole molecule), at a dilution of 1:1000, was used to assess
the reactivity of the BSA-containing antigens. The class of antibody (IgG
or IgM) was determined by the use of goat antimouse IgG (γ-chain specific)
or goat antimouse IgM (µ-chain specific) at a dilution of 1:4000.
a The optical density of the p-nitrophenol product was determined 1 h
after the addition of the p-nitrophenyl phosphate substrate. Medium was
used as a negative control; supernatant from the pooled multiple colony
wells from 5A6 served as a positive control (PC). Hybridoma clone culture
supernatants were undiluted and were assessed for activity against 5 10
days after recloning (primary). The hybridomas were incubated for an
additional 5 days, and the supernatants were screened again (secondary).
Activity against 35 and 30 were determined 15 days after recloning.
Table 5. Reactivity of 5A6 and 2F10 Clone Supernatant against 5 and
Other Antigens As Measured by an ELISA
2D6, was IgM-positive; the remaining 11 hybridomas were IgG-
positive (Table 3). Two of the most promising IgG-positive
hybridomas, 5A6 and 2F10, were cloned.
optical density at 405 nma
clone
5
35
30
IgG (γ-chain) IgM (µ-chain)
Cloning of Antibody-Producing Hybridomas. Cells from
the 5A6 and 2F10 hybridomas were diluted to a concentration
of one or two cells per well. After incubation for 4 days, the
plates were scanned microscopically and were scored for single
colony and multiple colony wells. Ten days after seeding,
supernatant from the wells that contained cells underwent
screening via ELISA against 5. Supernatants from the single
colony wells of 5A6 were not very active against 5 (data not
shown). Because of this, the four most 5 positive multiple colony
wells of 5A6 were pooled, diluted, and replated as single cells.
After 10 days, supernatants from the resulting clones were tested
by an ELISA against 5. The 10 single colony wells with the
strongest ELISA positives (primary) against 5 are shown in
Table 4. After incubation for an additional 5 days, the
supernatants were tested again (secondary) via ELISA against
5, 35, and 30. All 10 clones were highly active against 5 and
35 and showed little activity toward 30 (Table 4). A positive 5
and 35 response and a negligible 30 response were a clear
indication that the antigenic determinants of the antibody were
associated with the vibriobactin (3) segment of 5 and not with
BSA (28) itself.
Recloning of the 2F10 hybridoma was unnecessary: 2F10-1A9
and 2F10-2A3 were highly active against 5 and 35 and poorly
responsive to 30 (Table 5). These two clones, as well as two
clones from 5A6 (5A6-2D5 and 5A6-1G8), were selected for
further evaluation. The clone supernatants were assayed for the
class of antibody, IgG or IgM, and were shown to be IgG-
positive (Table 5). Multiple stocks from each cell line were
frozen. 5A6-1G8 and 2F10-2A3 were tested for mycoplasma
contamination and were found to be negative. Additional
monoclonal antibodies (mAb) derived from 5A6-2D5 and
2F10-1A9 were produced and purified.
5A6-1G8
5A6-2D5
5A6 (PC)
2F10-1A9 3.410 3.335 0.413
2F10-2A3 2.675 2.656 0.300
3.613 3.565 0.663
3.576 3.443 0.792
3.359 3.488 0.667
3.610
2.825
3.659
3.522
2.825
2.729
0.192
0.220
0.460
0.250
0.413
0.460
0.301
0.315
2F10 (PC)
Medium
2.347 2.185 0.362
0.172 0.155 0.166
a The optical density of the p-nitrophenol product was determined 1 h
after the addition of the p-nitrophenyl phosphate substrate. Hybridoma clone
culture supernatants were undiluted. Medium served as a negative control;
supernatant from the pooled multiple colony wells from 5A6, and
supernatant from the 2F10 uncloned parent hybridoma, served as positive
controls (PC). Rabbit antimouse IgG (whole molecule), at a dilution of
1:1000, was used to assess the reactivity of the BSA-containing antigens.
The class of antibody (IgG or IgM) was determined by the use of goat
antimouse IgG (γ-chain specific) or goat antimouse IgM (µ-chain specific)
at a dilution of 1:4000.
transferred to an ELISA plate that had been coated with antigen
5. Antigen 4 was found to be an effective competitor at all
concentrations tested (Figure 4, Table 6); 27 was not an effective
competitor. The p-nitrophenol optical density of the mAb
initially incubated with 27 against antigen 5 was approximately
90% greater than those of the mAb first incubated with 4 at the
same concentration (Table 6). It is clear that the antibody
“recognizes” the siderophore on the protein carrier.
Antigenic Determinants. One of the observations that stands
out with the data from the antibody-containing hybridoma
supernatants is the lack of difference in reactivity between 5
and its iron complex (35) (Tables 3 and 5). There are profound
differences in structure between vibriobactin (3) and its 1:1 iron
complex (33). Complex 33 would have all of the donor groups
(e.g., aromatic hydroxyls and oxazoline nitrogen) folded into
the metal.48 Catecholamides such as 3 bind iron very tightly,
Competitive Binding ELISA Studies: Determination of
the Sensitivity of the Purified Monoclonal Antibodies.
Competitive binding ELISA studies using the purified mAb were
conducted. The mAb were first incubated with varying con-
centrations of antigen 4, from 0 to 250 µg/mL, prior to being
with formation constants of nearly 1048 M-1 13 This means that
.
because of the ubiquitous nature of iron, the iron complexes 31
and 35 were probably formed in vitro. In fact, it is likely that
antibodies in animals are being formed against the OVA-VIB
iron complex (31).

Vibriobactin Antibodies
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3807
efficiency of 3 in the rats, i.e., the actual amount of iron cleared
by the ligand vs the theoretical iron clearance, was only 4.3 (
1.1%. This implied that up to 95% of the free ligand remains
or is cleared uncomplexed. In the bile duct-cannulated rats this,
of course, all unfolds in a matter of hours. However, in the
mouse immunization experiments, 4 had weeks to become
saturated with iron; the most important issue is the iron to ligand
ratio. Assuming that 3.23 µg atoms of iron/kg is available for
chelation, in a 25 g mouse 1.45 × 10-9 mol of iron is available.
The mice were effectively given 0.33 × 10-9 (M1) or 0.66 ×
10-9 (M2) mol of 4. In view of the protracted exposure of the
ligand to iron, it is difficult to imagine that all of the 3 bound
to OVA would also not be iron-bound.
Conclusion
The current study focused on methodologies for assembling
antigens that would allow for the assessment of the antigenic
properties of vibriobactin fixed to large carrier molecules, e.g.,
OVA (27) and BSA (28). Choosing the appropriate activated
tether for the vibriobactin protein conjugate was the first hurdle.
While a number of different tethers were considered, a thiol
analogue was chosen, 1-(2,3-dihydroxybenzoyl)-5,9-bis[[(4S,5R)-
2-(2,3-dihydroxyphenyl)-4,5-dihydro-5-methyl-4-oxazolyl]car-
bonyl]-14-(3-mercaptopropanoyl)-1,5,9,14-tetraazatetradecane
(6) or vibriobactin thiol (Figure 2).
The first attempt at synthesizing 6 (Scheme 1) unfortunately
failed in the last step. Cyclocondensation of ethyl 2,3-dihy-
droxybenzimidate34 with the threonyl units of thiol 17 led to
intractable mixtures, including thioesters. It became clear that
the free thiol could not be released prior to this cycloconden-
sation. In a second approach (Scheme 2), a different catechol
protecting group was employed in 2,3-bis(4-methoxybenz-
yloxy)benzoic acid (20). This could be removed concurrently
with the BOC functionality of the key intermediate 24 to
produce 25 while leaving the requisite disulfide intact (Scheme
3). The threonyl moieties of 25 were next condensed with excess
ethyl 2,3-dihydroxybenzimidate.34 Finally, disulfide iron chelator
26 was cleaved to the vibriobactin thiol (6), utilizing H2 (3 atm)
over Pd black in CH3OH under iron-free conditions (Scheme
3). The thiol (6) was then incubated with a maleimide-activated
OVA (27) or BSA (28) protein carrier, resulting in Michael
adduct OVA-VIB (4) or BSA-VIB (5), respectively (Scheme
4). Conjugate 4 was mixed with an adjuvant and was success-
fully used as an antigen to raise antibodies in mice, and
conjugate 5 was used in an ELISA, first for the detection of
serum polyclonal antibodies and ultimately vibriobactin-specific
IgG monoclonal antibodies.
Figure 4. Competitive binding ELISA of purified mAb from 5A6-2D5
(A) and 2F10-1A9 (B). The mAb (0.11 µg of protein/mL) were
incubated with varying concentrations of antigen 4, prior to being tested
via ELISA against antigen 5. The dotted line indicates the amount of
4 (∼0.05-0.06 µg/mL) needed to reduce the optical density of the
p-nitrophenol product to 50% of that of the mAb not incubated with
any 4. The closed circles indicate the optical density of 27 (10 µg/
mL).
Table 6. Competitive Binding ELISA of Purified Monoclonal
Antibodies Derived from Clones of 5A6-2D5 and 2F10-1A9
antigen 5: optical density at 405 nma
antigen 4
conc (µg/mL)
5A6-2D5
(purified mAb)
2F10-1A9
(purified mAb)
0
2.132
0.669
0.483
0.189
0.145
0.113
0.093
0.092
2.190
3.572
0.889
0.879
0.497
0.137
0.109
0.095
0.090
3.214
0.08
0.4
2
10
50
250
medium
27
a The optical density of the p-nitrophenol product was determined 1 h
after the addition of the p-nitrophenyl phosphate substrate. Purified
monoclonal antibodies (mAb) were incubated with antigen 4 for 2 h at
room temperature. A portion of this mixture was then transferred to an
ELISA plate that had been coated with antigen 5, 10 µg/mL. Medium was
used as a negative control; 27 (10 µg/mL) served as a positive control.
It is clear that further characterization of the antibody-antigen
binding is required, e.g., stoichiometries, formation constants,
etc. The results to date are consistent with the idea that
vibriobactin (3) presents strong antigenic determinants when
fixed to a large carrier molecule. The fact that 4 given to rodents
produced an antibody so active against 5 begs the question
regarding the antibodies’ potential in controlling the course of
a vibrio infection in an animal model. We believe the data are
further in keeping with the idea that covalently linking sidero-
phore analogues to siderophore bacterial outer membrane
receptors38 or to defined siderophore outer membrane receptor
c-terminal peptide fragments represents a credible target for
vaccine development. Although there are many potential pitfalls,
To support this idea, bile duct-cannulated rats were given 3
subcutaneously (sc) at a dose of 75 µmol/kg. The rodents’ bile
and urine were collected for 48 h to determine if 3 sequestered
and promoted the excretion of iron. The iron content of the
bile and urine was determined using atomic absorption spec-
troscopy. We have used this model for many years to evaluate
the efficiency with which iron chelators promote the excretion
of iron from animals.62,63 Vibriobactin (3) was indeed found to
sequester iron in vivo. Recall that 3 forms a tight 1:1 iron
complex with Fe(III). Rats given 75 µmol/kg of 3 would be
expected to clear 75 µg atoms/kg of iron if the binding and
clearance were 100% efficient. However, the iron clearing

3808 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Bergeron et al.
as above, and p-nitrophenyl phosphate at a concentration of 1.0
mg/mL, 100 µL/well, was added. The plates were read using an
ELISA plate reader at 405 nm, tracking the absorbance (OD) of
the yellow water-soluble product, p-nitrophenol. A positive response
was considered a test well with a p-nitrophenol OD value 3 times
greater than that of the negative control.
nevertheless, because of the above data, we feel compelled to
look at this approach. This will be the subject of a future
manuscript.
Experimental Section
The class of antibody (IgG or IgM) of the hybridoma supernatant
(Table 3) or the clone supernatant (Table 5) was determined by an
ELISA, replacing the rabbit antimouse IgG (whole molecule) with
goat antimouse IgG (γ-chain specific) or goat antimouse IgM (µ-
chain specific) at a dilution of 1:4000 (50 µL/well).
Competitive Binding ELISA Procedure. Competitive binding
ELISAs were performed on (1) serum from immunized mice that
contained polyclonal antibodies and (2) purified mAb derived from
the cloning of 5A6-2D5 and 2F10-1A9. Medium was used as a
negative control, while 27 (10 µg/mL, 50 µL/well) served as a
positive control for the competitive binding ELISA of the polyclonal
serum (Table 2) and purified mAb (Table 6). Two types of plates
were utilized: a conical bottom polypropylene plate was used for
the incubation of the antibody-antigen mixture, whereas a 96-well
MaxiSorp plate was used for the ELISA.
Biological Materials and Methods. The animal-related protocols
were approved by the University of Florida Institutional Animal
Care and Use Committee. Three female Balb/c ByJ mice (6-7
weeks old) were purchased from The Jackson Laboratory (Bar
Harbor, ME). TiterMax Adjuvant was obtained from Sigma (St.
Louis, MO). ELISA plates (MaxiSorp) were purchased from Nalge
Nunc International Co. (Naperville, IL). An automatic microplate
washer (model EL404, Bio-Tek Instruments, Inc., Winooski, VT)
and microplate reader (Spectromax Plus 384, Molecular Devices,
Union City, CA) were utilized. The rabbit antimouse IgG (whole
molecule), goat antimouse IgG (γ-chain specific), and goat anti-
mouse IgM (µ-chain specific) antibodies were purchased from
Sigma (St. Louis, MO). Dulbecco’s modified Eagle’s medium
(HyClone) was obtained from Thermo Fisher Scientific (Waltham,
MA). Hybridoma plates were incubated in a Forma Scientific
Incubator, model 3154 (Marietta, GA). A MycoAlert Kit (Lonza,
Allendale, NJ) was used to assess potential mycoplasma contamina-
tion. Monoclonal antibodies were produced using hybridoma
production media, BD Cell Mab Medium, Quantum Yield (BD
Biosciences, San Jose, CA), supplemented with 10% low IgG fetal
bovine serum (HyClone, Thermo Fisher Scientific, Waltham, MA)
in CELLine CL 350 flasks (Sartorius Stedim, New York, NY).
Amicon Ultra-15 centrifugal filters with a 30 kDa cutoff were
obtained from Millipore (Billerica, MA). Male Sprague-Dawley
rats (400-450 g) were procured from Harlan Sprague-Dawley
(Indianapolis, IN). Cremophor RH-40 was provided by BASF
(Parsippany, NJ). An atomic absorption spectrometer, Perkin-Elmer
model 5100 PC (Norwalk, CT), was used to determine the iron
content of the rat bile and urine samples.
Monoclonal Antibody Production: Immunization. To produce
antibodies to small molecules in animals, conjugation to larger
carrier proteins (e.g., 27 or 28) is generally required. Compound 6
was conjugated with 27 using a linker to prepare 4, which was
mixed with an adjuvant and used as an antigen to immunize two
mice. One mouse (M1) received 50 µg of 4 per sc injection, and
the other mouse (M2) received 100 µg of 4 per sc injection. The
mice were given a second immunization of 4 at the same doses
four weeks later.
Noncompetitive Binding ELISA Procedure. The method
followed the approach of Kao and Klein.60 Briefly, the assay
involved coating a potential antigen (50 µL/well) on the ELISA
plates, utilizing solutions of antigens ranging in concentration from
1 to 40 µg/mL. The antigens (a) BSA-VIB conjugate (5), (b)
BSA-cysteine conjugate (30), (c) vibriobactin thiol (6), (d)
vibriobactin disulfide (26), (e) vibriobactin disulfide-iron complex
(34), (f) vibriobactin (3), and (g) vibriobactin iron complex (33)
were diluted in blocking buffer (1% BSA in PBS with 0.02% azide).
Normal mouse sera (NMS) or medium served as negative controls.
Polyclonal serum from an immunized mouse (M2) in a 1:1000
dilution or hybridoma supernatant (Tables 4 and 5) were used as
positive controls.
In brief, the polypropylene incubation plate was blocked with
300 µL of blocking buffer (1% BSA in PBS with 0.02% azide)
and was allowed to incubate overnight at 4 °C. The following day,
the blocking buffer was removed (flicked) from the plate, and the
plate was blotted on a paper towel. A 96-well ELISA plate was
coated with antigen 5 (10 µg/mL, 50 µL/well) that had been diluted
in PBS with 0.02% azide. After incubation overnight at 4 °C, the
plate was washed 4 times (300 µL/wash) as described above,
blocked with 300 µL of blocking buffer for 1 h, and washed again.
Antigens d-g were diluted in blocking buffer in microcentrifuge
tubes at antigen concentrations ranging from 0 to 250 µg/mL. The
diluted antigens (50 µL/well) were transferred from the microcen-
trifuge tubes to the polypropylene incubation plate that had been
blocked and washed. A 50 µL aliquot of the diluted polyclonal
mouse serum (1:10000 dilution) or mAb (0.11 µg protein/mL) was
added to the incubation plate wells containing the diluted antigens.
The polypropylene plate was then incubated on a rocking platform
for 2 h at room temperature, allowing time for the antigen-antibody
complexes to form.
A portion of the antigen/antibody mixture (50 µL) was transferred
from the polypropylene incubation plate to the 5-coated ELISA
plate that had been blocked and washed. The ELISA plate was
incubated with gentle agitation for 1 h at room temperature and
was washed 4 times with the EWB. Rabbit antimouse IgG
antibodies conjugated to alkaline phosphatase were added (50 µL/
well); the IgG antibody was diluted (1:1000) in 1% BSA-blocking
buffer. After 1 h, the ELISA plate was washed 4 times with the
EWB to remove any unreacted IgG antibodies. p-Nitrophenyl
phosphate substrate (1.0 mg/mL, 100 µL/well) was added. The
ELISA plate was allowed to incubate with gentle agitation for 1 h
at room temperature and was read at 405 nm, tracking the OD of
the yellow water-soluble product, p-nitrophenol. Reduction in the
OD value of a test well compared to the positive control reflects
the effectiveness of the competitor binding to the antibody.
Cell Fusion-Hybridoma Formation. Four months after the
second immunization, 4 days before fusion, a mouse (M2) was
given a prefusion booster of 100 µg of 4 without adjuvant. This
final immunization was given intraperitoneally (ip). On the day of
fusion, the mouse was anesthetized, exsanguinated, and euthanized.
The spleen was removed and washed with Dulbecco’s modified
Eagle’s medium to remove the antibody-forming cells. The medium
was supplemented with 10% equine serum and 1× antibiotic-
antimycotic (per mL: 100 IU of penicillin, 0.10 mg of streptomycin,
0.25 µg of amphotericin B, and 50 µg of gentamycin). The fusion
was performed by the procedures described in Simrell et al.61 except
that the myeloma cell line used was Sp2/0 (a murine myeloma
aminopterin-resistant cell line with a defect in purine metabolism).
Spleen cells were mixed with myeloma cells at a 7:1 ratio. The
fusion was performed with 50% polyethylene glycol 1500 followed
by a controlled dilution with media. A centrifugation step
The plates were allowed to incubate overnight at 4 °C. An ELISA
wash buffer (EWB) containing PBS with 0.02% azide and 0.5%
Tween-20 was used to wash the plates. The plates were washed 4
times (300 µL/wash) using an automatic microplate washer. The
wells were then blocked with 1% BSA in PBS with 0.02% azide
for 1 h at room temperature and washed again. A 50 µL aliquot of
diluted polyclonal mouse serum (1:200-1:25600), undiluted hy-
bridoma supernatant, or purified mAb (0.11 µg protein/mL) was
added to each well. The plates for this and each subsequent step of
the ELISA were incubated with gentle agitation for 1 h at room
temperature. The plates were then washed 4 times as above, and
rabbit antimouse IgG (whole molecule), conjugated to alkaline
phosphatase, was added (50 µL/well); the IgG antibody was diluted
(1:1000) in BSA-blocking buffer. The plates were washed 4 times

Vibriobactin Antibodies
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3809
(1500-1800 rpm for 8 min) was performed to pellet the fused cells.
The pellet was resuspended in Dulbecco’s modified Eagle’s
medium-high glucose, supplemented with 20% equine serum, 25%
Sp2/0 myeloma conditioned medium and 1× hypoxanthine, ami-
nopterin, thymidine (HAT) and was seeded in five 96-well plates
at 2.8 × 105 cells per well. The plates were incubated at 37 °C,
with a CO2 concentration of 7%.
Eleven days postfusion, supernatants from the resulting HAT-
resistant hybridomas were subjected to a primary ELISA screening
to detect if the cells were secreting antibodies against 5. Cells from
51 wells that were positive in the primary screening against 5 were
transferred to three 24-well plates and further incubated. The
supernatant was assessed again 1 week later in a secondary ELISA
screening against 5, 35, and 30 (Table 3). The hybridomas that
were 5 and 35 positive and 30 negative were further tested as
described above to determine the class of antibody, IgG or IgM
(Table 3).
Cloning of Antibody-Producing Hybridomas. Two of the most
promising hybridomas, 5A6 and 2F10, were cloned. Cells from
the hybridomas were diluted to a concentration of one or two cells
per well and were seeded in eight 96-well plates (four plates each
for 5A6 and 2F10) over a feeder layer of irradiated 3T3 mouse
fibroblasts. After incubation for 4 days, the plates were scanned
microscopically and were scored for single colony and multiple
colony wells. For the 5A6 cell line, 96 single colony wells and 60
multiple colony wells were found, whereas the 2F10 cell line
presented 120 single colony wells and 47 multiple colony wells.
Ten days after seeding, the supernatants from all 323 wells that
contained cells underwent screening via ELISA against 5.
Supernatants from the single colony wells of 5A6 were not very
active against 5 (data not shown). Because of this, the four most 5
positive multiple colony wells of 5A6 were pooled, diluted, and
replated as single cells in four 96-well plates. Ten days after this
recloning, the 10 single colony wells with the strongest ELISA
positives (primary) against 5 were chosen (Table 4) and cultured
in a 24-well plate. After incubation for an additional 5 days, the
supernatant was tested again (secondary) via ELISA against 5, 35,
and 30. All 10 clones were highly active against 5 and 35 and
showed little activity toward 30 (Table 4).
Recloning of the 2F10 hybridoma was unnecessary. The 10 single
colony wells with the strongest ELISA response against 5 from
the four 96-well plates were transferred to a 24-well plate. After
incubation for an additional 5 days, the supernatants were tested
against 5, 35, and 30. Two clones from 2F10 (2F10-1A9 and
2F10-2A3), as well as two clones from 5A6 (5A6-2D5 and
5A6-1G8), were chosen for further evaluation. Cell lines 5A6-
1G8, 5A6-2D5, 2F10-2A3, and 2F10-1A9 were tested for myco-
plasma contamination and were found negative (Table 5). Multiple
stocks from each of the four cell lines were frozen.
ficient of 14. For an IgG antibody with a molecular weight of
approximately 150000, this corresponds to a molar extinction
coefficient (ε) of 210000 M-1 cm-1
.
Iron Clearance in Non-Iron-Overloaded, Bile-Duct Cannu-
lated Rats. Four rats were subjected to bile duct-cannulation as
previously described.62,63 The rats were given 3 sc at a dose of 75
µmol/kg. The drug was solubilized in 40% Cremophor RH-40/
water. Bile samples were collected from the rats at 3 h intervals
for 48 h. The urine samples were taken at 24 h intervals. Sample
collection and handling are as previously described.62,63 The iron
content of the bile and urine were assessed by atomic absorption
spectroscopy. Iron clearing efficiency was calculated as set forth
elsewhere.65 The theoretical iron output of the chelator was
generated on the basis of a 1:1 vibriobactin-iron complex.48
Synthetic Materials and Methods. Reagents were purchased
from Aldrich Chemical Co. (Milwaukee, WI). Fisher Optima-grade
solvents were routinely used, and DMF was distilled. Organic
extracts were dried with sodium sulfate and then filtered. Distilled
solvents and glassware that had been presoaked in 3 N HCl for 15
min were employed in reactions involving chelators. Silica gel 70-
230 from Fisher Scientific was utilized for column chromatography,
and silica gel 40-63 from SiliCycle, Inc. (Quebec City, Quebec,
Canada) was used for flash column chromatography. Sephadex LH-
20 was obtained from Amersham Biosciences (Piscataway, NJ).
An ICP-MS X 0675, manufactured by Thermo Electron Corporation
(England), was used for the determination of the iron content of
selected solutions/compounds. Derivatized OVA or BSA protein
containing maleimide moieties 27 and 28, respectively, were
obtained from Pierce (Rockford, IL).66 NMR spectra were obtained
at 400 MHz (1H) or 100 MHz (13C) on a Varian Mercury-400BB.
1
Chemical shifts (δ) for H spectra are given in parts per million
downfield from tetramethylsilane for organic solvents (CDCl3 not
indicated) or sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 for D2O.
Chemical shifts (δ) for 13C spectra are given in parts per million
referenced to 1,4-dioxane (δ 67.19) in D2O or to the residual solvent
resonance in CDCl3 (δ 77.16). Coupling constants (J) are in hertz.
The base peaks are reported for the high resolution mass spectra.
Elemental analyses were performed by Atlantic Microlabs (Nor-
cross, GA) and were within (0.4% of the calculated values.
OVA-VIB (4) and BSA-VIB (5). Derivatized proteins 2766
or 2866 (2 mg each) were dissolved in the conjugation buffer (100
µL) and incubated with a freshly prepared solution of 6 (2 mg in
200 µL of 50% aqueous DMSO) for 8 h. Unreacted maleimide
was capped by conjugating it further with cysteine (2 mg in 50 µL
degassed water) for 8 h. After incubation, 4 and 5 respectively were
purified on a dextran desalting column (5 mL), eluting with 30%
DMSO/purification buffer. Fractions (0.5 mL) were collected, and
the OD was measured at 280 nm. Positive fractions of each
conjugate were pooled, and the protein concentration of each
conjugate was estimated by a Coomassie assay.58
Production and Purification of Monoclonal Antibodies.
5A6-2D5 or 2F10-1A9 cloned hybridoma cells were grown in
hybridoma production media supplemented with 10% low IgG fetal
bovine serum in CELLine CL 350 flasks. The flasks were incubated
at 37 °C with a CO2 concentration of 7%. One harvest per week
was taken for 3 weeks. The cells were centrifuged at 2000 rpm for
15 min, and the resulting supernatant was collected and purified
by circulating through a 5 mL protein G Sepharose 4B column.
The supernatant recirculated through the column for 90 min (∼23
passes). The column was then rinsed with PBS (70-150 mL). The
antibodies were eluted using 0.1 M glycine, pH 2.8. Ten fractions
of 3 mL each were collected. The eluted fractions were neutralized
with 2.0 M Tris, pH 9.0. Absorbance was measured at 280 nm.
Fractions with the highest absorbance readings were pooled,
desalted, and concentrated using Amicon Ultra-15 centrifugal filters
with a 30 kDa cutoff. The concentrated, purified monoclonal
antibodies were recovered in a final volume of 0.6 mL in PBS.
The protein concentration of the purified mAb were measured
spectrophotometrically at 280 nm. Most mammalian antibodies (i.e.,
immunoglobulins) have protein extinction coefficients (εpercent) in
the range of 12-15.64 The final protein concentration of the purified
IgG antibody was estimated assuming a protein extinction coef-
1-(2,3-Dihydroxybenzoyl)-5,9-bis[[(4S,5R)-2-(2,3-dihydroxy-
phenyl)-4,5-dihydro-5-methyl-4-oxazolyl]carbonyl]-14-(3-mer-
captopropanoyl)-1,5,9,14-tetraazatetradecane (6). Palladium black
(0.127 g) was added to a solution of 26 (0.231 g, 0.133 mmol) in
anhydrous degassed EtOH (5 mL), and the mixture was stirred under
H2 at 45 psi for 24 h. After filtration through Celite and washing
the solids with degassed EtOH (2 × 5 mL), the filtrate was
concentrated in vacuo to afford 0.137 g (60%) of 6 as a brown
solid: 1H NMR δ 1.35-1.75 (m, 10 H), 1.8-2.2 (m, 4 H),
2.47-2.59 (m, 2 H), 2.82-2.91 (m, 2 H), 3.02-3.91 (m, 12 H),
4.72-5.0 (m, 2 H), 5.21-5.36 (m, 2 H), 6.58-6.77 (m, 3 H),
6.84-7.2 (m, 3 H), 7.24-7.8 (m, 3 H); HRMS m/z calcd for
C42H53N6O12S, 865.3358 (M + H); found, 865.3466.
N1-(4-Hydroxybutyl)-N1,N4,N7-tris(tert-butoxycarbonyl)nor-
spermidine (9). 4-Chloro-1-butanol (16.5 g, 0.150 mol) was
introduced to a mixture of 7 (39.4 g, 0.300 mol), KI (2.475 g, 15.0
mmol), and K2CO3 (10.5 g, 75.0 mmol) in anhydrous 1-butanol
(375 mL). The mixture was stirred at 125 °C for 24 h, slowly cooled
to room temperature, filtered, and concentrated under vacuum to
afford 8 as a viscous oil, which was dissolved in 50% aqueous
THF (500 mL). Di-tert-butyl dicarbonate (130.8 g, 0.600 mol) in

3810 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Bergeron et al.
THF (100 mL) was added with continuous stirring. After 16 h,
volatiles were removed, and the residue was dissolved in H2O (300
mL) and extracted with EtOAc (200 mL, 3 × 100 mL). The
combined organic layers were washed with 0.5 M citric acid (100
mL), H2O (100 mL), and saturated NaCl (100 mL) and were
concentrated under reduced pressure. Column chromatography using
49:49:2 hexanes/EtOAc/CH3OH provided 31.73 g (42%) of 9 as a
furnished 2.17 g (55%) of 14 as a white foam: 1H NMR δ
1.14-1.22 (m, 6 H), 1.38-1.46 (m, 18 H), 1.54-2.02 (m, 8 H),
2.90-3.61 (m, 10 H), 3.68-3.76 (m, 2 H), 3.89 (s, 3 H), 3.91 (s,
3 H), 3.98-4.14 (m, 2 H), 5.44-5.68 (m, 2 H), 7.04 (d, 1 H, J )
8.4), 7.14 (d, 1 H, J ) 8.0), 7.62-7.67 (m, 1 H), 7.70-7.72 (m, 2
H), 7.84-7.86 (m, 2 H); HRMS m/z calcd for C45H67N6O13,
899.4767 (M + H); found, 899.4783. Anal. (C45H66N6O13) C,
H, N.
1
viscous colorless oil. H NMR δ 1.44, 1.45, and 1.46 (3 s, 27 H),
N4,N8-Bis[(N-tert-butoxycarbonyl-L-threonyl]-N1-(2,3-dimetho-
xybenzoyl)thermospermine (15). Hydrazine hydrate (5 mL) was
added to a solution of 14 (0.350 g, 0.389 mmol) in EtOH (10 mL),
and the reaction mixture was stirred at room temperature for 16 h.
Solid was filtered, and the filtrate was concentrated in vacuo. The
residue was taken up in 1 N NaOH (10 mL) and extracted with
CHCl3 (3 × 10 mL), and organic extracts were concentrated. Flash
chromatography, eluting with 1% concentrated NH4OH/MeOH,
gave 195 mg (65%) of 15 as a viscous solid: 1H NMR δ 1.14-1.22
(m, 6 H), 1.38-1.46 (m, 18 H), 1.54-2.02 (m, 8 H), 2.68-2.76
(m, 2 H), 2.90-3.61 (m, 10 H), 3.89 (s, 3 H), 3.91-3.94 (m, 3 H),
3.98-4.14 (m, 2 H), 4.44-4.62 (m, 2 H), 5.46-5.64 (m, 2 H),
7.04 (d, 1 H, J ) 8.4), 7.14 (t, 1 H, J ) 8.0), 7.62-7.67 (m, 1 H);
HRMS m/z calcd for C37H65N6O11, 769.4713 (M + H); found,
769.4709. Anal. (C37H64N6O11) C, H, N.
1.5-1.8 (m, 9 H), 3.02-3.34 (m, 10 H), 3.67 (t, 2 H, J ) 5.9),
4.78 and 5.27 (2 br s, 1 H); 13C NMR δ 25.20, 27.84, 28.57, 28.60,
28.61, 29.82, 37.72, 44.10, 45.01, 47.01, 62.57, 79.57, 155.75,
156.16, 174.84; HRMS m/z calcd for C25H50N3O7, 504.3648 (M +
H); found, 504.3640. Anal. (C25H49N3O7) C, H, N.
N1-[4-(Tosyloxy)butyl]-N1,N4,N7-tris(tert-butoxycarbonyl)nor-
spermidine (10). 10 was prepared from 9 by our previous reaction
conditions.55 HRMS m/z calcd for C32H56N3O9S, 657.3659 (M +
H); found, 657.3672. Anal. (C32H55N3O9S) C, H, N.
N1-[4-(Phthalimido)butyl]-N1,N4,N7-tris(tert-butoxycarbonyl)-
norspermidine (11). Potassium phthalimide (1.138 g, 6.15 mmol)
was added to 10 (2.70 g, 4.10 mmol) in DMF (50 mL), and the
reaction mixture was heated at 90 °C for 48 h. Solvent was removed
in vacuo, and the residue was treated with H2O (50 mL) and
extracted with CHCl3 (3 × 50 mL). The combined organic phase
was washed with saturated NaCl and concentrated under reduced
pressure. Column chromatography with 20% EtOAc/CHCl3 gener-
ated 1.84 g (71%) of 11: 1H NMR δ 1.43-1.45 (3 s, 27 H),
1.52-1.76 (m, 8 H), 3.06-3.29 (m, 10 H), 3.71 (t, 2 H, J ) 6.8),
7.70-7.73 (m, 2 H), 7.83-7.87 (m, 2 H); 13C NMR δ 25.98, 27.46,
28.48, 29.49, 37.40, 37.60, 43.71, 44.87, 46.52, 78.88, 79.43, 79.65,
123.25, 123.41, 132.10, 133.99, 134.11, 155.45, 156.03, 168.41.
HRMS m/z calcd for C33H53N4O8, 633.3858 (M + H); found,
633.3920. Anal. (C33H52N4O8) C, H, N.
1,36-Bis(2,3-dimethoxybenzoyl)-15,22-dioxo-18,19-dithia-
1,5,9,14,23,28,32,36-octaaza-5,9,28,32-tetrakis[(N-tert-butoxycar-
bonyl-L-threonyl]hexatriacontane (16). CDI (0.10 g, 0.65 mmol)
was added to a solution of 3,3′-dithiopropionic acid (0.06 g, 0.32
mmol) in CH2Cl2 (2 mL). After the mixture was stirred for 2 h, a
solution of 15 (0.60 g, 0.78 mmol) in CH2Cl2 (5 mL) was added to
the reaction mixture. The solution was stirred for 15 h at room
temperature and was diluted with CH2Cl2 (25 mL). The organic
layer was washed with 1 N NaOH (15 mL) and saturated NaCl
(15 mL) and was concentrated in vacuo. Flash chromatography
using 15% EtOH/CHCl3 generated 0.218 g (40%) of 16 as a pale-
N1-[4-(Phthalimido)butyl]norspermidine Tris(trifluoroace-
tate) (12). TFA (25 mL) was added to 11 (3.46 g, 5.47 mmol) in
CH2Cl2 (25 mL) with ice bath cooling, and the solution was stirred
for 1 h at 0 °C and 1 h at room temperature. After removal of
volatiles in vacuo, the residue was treated with toluene and dried
by high vacuum to give 3.69 g (quantitative) of 12 as a white solid:
1H NMR (D2O) δ 1.72-1.75 (m, 4 H), 2.05-2.15 (m, 4 H),
3.08-3.19 (m, 10 H), 3.71 (t, 2 H, J ) 6.4), 7.81-7.87 (m, 4 H);
13C NMR δ (D2O) 23.24, 23.56, 24.37, 25.47, 37.10, 37.53, 44.90,
45.20, 45.29, 47.79, 123.91, 131.71, 135.37, 171.17; HRMS m/z
calcd for C18H29N4O2 333.2285 (M + H, free amine); found,
333.2324. Anal. (C24H31F9N4O8 ·1.5H2O) C, H, N.
1
brown solid: H NMR δ 1.10-1.25 (m, 12 H), 1.29-1.50 (m, 36
H), 1.52-2.05 (m, 16 H), 2.50-2.65 (m, 4 H), 2.90-3.08 (m, 4
H), 3.09 -3.60 (m, 24 H), 3.85-3.98 (m, 12 H), 3.99-4.60 (m, 8
H), 5.40-5.65 (m, 4 H), 6.98-7.15 (m, 2 H), 7.18-7.20 (m, 2 H),
7.60-7.69 (m, 2 H); HRMS m/z calcd for C80H135N12O24S2,
1711.9155 (M + H); found, 1711.9170.
1-(2,3-Dihydroxybenzoyl)-5,9-bis[(N-tert-butoxycarbonyl-L-
threonyl]-14-(3-mercaptopropanoyl)-1,5,9,14-tetraazatetrade-
cane Dihydrobromide (17). Boron tribromide in CH2Cl2 (1 M,
7.56 mL, 7.56 mmol) was added to a mixture of 16 (340 mg, 0.199
mmol) in CH2Cl2 (20 mL) at -78 °C, and after being stirred for
1 h, the reaction mixture was warmed to room temperature and
was stirred for 15 h. The reaction mixture was quenched cautiously
at 0 °C with H2O (10 mL) and was stirred for 2 h. The aqueous
layer was extracted with CH2Cl2 (20 mL) and was concentrated in
vacuo, and the residue was subjected to a Sephadex LH-20 column,
eluting with 40% EtOH/toluene to give 115 mg (73%) of 17 as a
white solid: 1H NMR (D2O) δ 1.15-1.25 (m, 6 H), 1.46-2.01 (m,
8 H), 2.56-2.63 (m, 2 H), 2.94-3.00 (m, 2 H), 3.03-3.68 (m, 12
H), 4.04-4.20 (m, 2 H), 4.24-4.39 (m, 2 H), 6.83 (t, 1 H, J )
8.4), 7.15 (d, 1 H, J ) 8.0), 7.30 (d, 1 H, J ) 7.6); HRMS m/z
calcd for C28H49N6O8S, 629.3333 (M + H, free amine); found,
629.3329. Anal. (C28H50Br2N6O8S·H2O) C, H, N.
4-Methoxybenzyl 2,3-Bis(4-methoxybenzyloxy)benzoate (19).
Sodium hydride (60%, 4.69 g, 0.117 mol) was added in portions
to 18 (5.47 g, 35.5 mmol) in DMF (150 mL) with ice bath cooling,
and the mixture was stirred at 0 °C for 45 min and at room
temperature for 1 h. A solution of 4-methoxybenzyl bromide (25.0
g, 0.124 mol) in DMF (50 mL) was added to the reaction mixture
over 30 min. After the mixture was stirred for 20 h, quenching
with H2O (30 mL) at 0 °C was performed, and solvents were
removed under high vacuum. The concentrate was dissolved in
EtOAc (200 mL), which was washed with H2O (100 mL) and
saturated NaCl (100 mL); solvent was removed in vacuo. Flash
chromatography, eluting with 5:1 hexanes/EtOAc, gave 11.33 g
(62%) of 19 as a white solid, mp 108 °C: 1H NMR δ 3.77 (s, 3 H),
3.79 (s, 3 H), 3.81 (s, 3 H), 4.95 (s, 2 H), 5.03 (s, 2 H), 5.25 (s, 2
N1-(2,3-Dimethoxybenzoyl)-N7-[4-(phthalimido)butyl]norsper-
midine (13). CDI (0.563 g, 3.48 mmol) was added to a solution of
2,3-dimethoxybenzoic acid (0.633 g, 3.48 mmol) in CH2Cl2 (5 mL).
After being stirred for 1 h, the solution was cooled to 0 °C and
was added to a suspension of 12 (2.82 g, 4.18 mmol) and NEt3
(2.46 g, 24.4 mmol) in CH2Cl2 (30 mL). The reaction mixture was
stirred for 15 h at room temperature, diluted with CH2Cl2 (100 mL),
and washed with 8% NaHCO3 (50 mL). The organic phase was
concentrated in vacuo, and the residue was subjected to flash
chromatography, eluting with 5% concentrated NH4OH/MeOH to
afford 1.20 g (70%) of 13 as a colorless oil: 1H NMR δ 1.48-1.57
(m, 2 H), 1.62-1.85 (m, 6 H), 2.61-2.73 (m, 8 H), 3.53 (q, 2 H,
J ) 6.0), 3.70 (t, 2 H, J ) 7.6), 3.88 (s, 3 H), 3.89 (s, 3 H), 7.03
(dd, 1 H, J ) 8.0, 1.6), 7.14 (t, 1 H, J ) 8.0), 7.64 (dd, 1 H, J )
8.0, 1.6), 7.69-7.71 (m, 2 H), 7.82-7.84 (m, 2 H), 8.15 (br, 1 H,
J ) 7.2); HRMS m/z calcd for C27H37N4O5, 497.2765 (M + H);
found, 497.2671. Anal. (C27H36N4O5) C, H, N.
N4,N7-Bis[(N-tert-butoxycarbonyl-L-threonyl]-N1-(2,3-dimetho-
xybenzoyl)-N7-[4-(phthalimido)butyl]norspermidine (14). A solu-
tion of freshly prepared N-tert-butoxycarbonyl-L-threonine N-hy-
droxysuccinimide ester56 (4.17 g, 13.2 mmol) in DMF (20 mL)
was added to a solution of 13 (2.2 g, 4.4 mmol) in DMF (20 mL).
After the mixture was stirred for 72 h, the solvent was removed
under vacuum and the residue was taken up in CHCl3 (50 mL).
The organic layer was washed with 5% NaHCO3 (3 × 50 mL),
H2O (50 mL), and saturated NaCl (50 mL) and was concentrated
in vacuo. Flash chromatography, eluting with 10% EtOH/EtOAc,

Vibriobactin Antibodies
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3811
H), 6.76 (d, 2 H, J ) 8.8), 6.86 (d, 2 H, J ) 8.8), 6.89 (d, 2 H, J
) 8.4), 7.04 (t, 1 H, J ) 7.8), 7.11 (dd, 1 H, J ) 8.0, 2.0), 7.18 (d,
2 H, J ) 8.8), 7.33-7.36 (m, 5 H); 13C NMR δ 55.32, 55.37, 55.40,
66.81, 71.20, 75.31, 112.97, 113.61, 114.02, 118.16, 123.93, 127.08,
128.19, 128.75, 129.51, 129.73, 130.31, 130.41, 148.43, 152.95,
159.43, 159.62, 159.70, 166.44; HRMS m/z calcd for C31H30NaO7,
537.1889 (M + Na); found, 537.1884.
7.22-7.26 (m, 3 H), 7.4 (d, 2 H, J ) 8.4); HRMS m/z calcd for
C51H77N6O13, 981.5543 (M + H); found, 981.5589. Anal.
(C51H76N6O13) C, H, N.
1,36-Bis[2,3-bis(4-methoxybenzyloxy)benzoyl]-15,22-dioxo-
18,19-dithia-1,5,9,14,23,28,32,36-octaaza-5,9,28,32-tetrakis[(N-
tert-butoxycarbonyl-L-threonyl]hexatriacontane (24). CDI (0.063
g, 0.372 mmol) was added to a solution of 3,3′-dithiopropionic acid
(0.039 g, 0.186 mmol) in CH2Cl2 (2 mL). After the mixture was
stirred for 2 h, a solution of 23 (0.550 g, 0.56 mmol) in CH2Cl2 (5
mL) was added followed by NEt3 (0.025 g, 0.25 mmol). The
solution was stirred for 15 h at room temperature and was diluted
with CH2Cl2 (25 mL). The organic layer was washed with 8%
NaHCO3 (20 mL) and saturated NaCl (20 mL) and concentrated
in vacuo. Flash chromatography, using 8% CH3OH/CHCl3, gener-
ated 0.238 g (60%) of 24 as a pale-brown solid: 1H NMR δ
1.10-1.23 (m, 12 H), 1.25-1.47 (m, 36 H), 1.48-2.00 (m, 16 H),
2.52-2.70 (m, 4 H), 2.80-3.01 (m, 4 H), 3.07-3.55 (m, 24 H),
3.80-3.81(m, 6 H), 3.83 (s, 6 H), 3.95-4.17 (m, 4 H), 4.27-4.62
(m, 4 H), 5.01-5.04 (m, 4 H), 5.07 (2 s, 4 H), 5.42-5.65 (m, 4
H), 6.82-6.86 (m, 4 H), 6.93 (2 d, 4 H, J ) 8.4), 7.12-7.18 (m,
4 H), 7.22-7.27 (m, 6 H), 7.4 (d, 4 H, J ) 8.4); HRMS m/z calcd
for C108H159N12O28S2 2137.0859 (M + H), found 2137.0867. Anal.
(C108H158N12O28S2) C, H, N.
2,3-Bis(4-methoxybenzyloxy)benzoic Acid (20). A solution of
19 (8.60 g, 16.71 mmol) in dioxane (84 mL) and 2 N NaOH (42
mL) was stirred for 24 h at room temperature. The reaction mixture
was concentrated in vacuo. The residue was stirred with H2O (100
mL) and then acidified to pH 2 with 1 N HCl. The white solid was
filtered, washed with hexane, and recrystallized from EtOAc/
hexanes to generate 5.93 g (90%) of 20 as a white crystalline solid,
1
mp 129 °C: H NMR δ 3.8 (s, 3 H), 3.85 (s, 3 H), 5.12 (s, 2 H),
5.20 (s, 2 H), 6.83 (d, 2 H, J ) 8.8), 6.96 (d, 2 H, J ) 9.2), 7.18
(t, 2 H, J ) 8.0), 7.22-7.27 (m, 2 H), 7.41 (d, 2 H, J ) 8.4), 7.73
(dd, 1 H, J ) 1.2, 8.0); 13C NMR δ 55.36, 55.37, 55.44, 55.46,
71.42, 114.26, 119.12, 123.04, 124.37, 125.00, 126.87, 128.02,
129.73, 131.21, 147.17, 151.45, 159.94, 160.44, 165.45; HRMS
m/z calcd for C23H22NaO6, 417.1332 (M + Na); found, 417.1332.
N1-[2,3-Bis(4-methoxybenzyloxy)benzoyl]-N7-[4-(phthalimi-
do)butyl]norspermidine (21). CDI (0.239 g, 1.48 mmol) was added
to a solution of 20 (0.583 g, 1.48 mmol) in CH2Cl2 (2 mL). After
being stirred for 1 h, the solution was cooled to 0 °C and added to
a suspension of 12 (1.0 g, 1.48 mmol) and NEt3 (1.05 g, 10.4 mmol)
in CH2Cl2 (2 mL) at 0 °C. The solution was stirred for 15 h at
room temperature and was diluted with CH2Cl2 (50 mL). The
reaction mixture was washed with 8% NaHCO3 (25 mL) and was
concentrated. Flash chromatography, eluting with 5% concentrated
NH4OH/MeOH, afforded 0.597 g (57%) of 21 as a colorless oil:
1H NMR δ 1.48-1.72 (m, 8 H), 2.52-2.63 (m, 8 H), 3.36 (q, 2 H,
J ) 6.0), 3.69 (t, 2 H, J ) 7.2), 3.80 (s, 3 H), 3.84 (s, 3 H), 4.98
(s, 2 H), 5.07 (s, 2 H), 6.83 (d, 2 H, J ) 8.8), 6.93 (d, 2 H, J )
8.4), 7.13 (d, 2 H, J ) 4.8), 7.23 (m, 3 H), 7.4 (d, 2 H, J ) 8.4),
7.68-7.7 (m, 2 H), 7.81-7.83 (m, 2 H), 8.12 (t, 1 H, J ) 7.2);
HRMS m/z calcd for C41H49N4O7, 709.3596 (M + H); found,
709.3611. Anal. (C41H48N4O7) C, H, N.
1,36-Bis(2,3-dihydroxybenzoyl)-15,22-dioxo-18,19-dithia-
1,5,9,14,23,28,32,36-octaaza-5,9,28,32-tetrakis(L-threonyl)hexatri-
acontane Tetrakis(trifluoroacetate) (25). TFA (25 mL) was added
to a mixture of 24 (1.16 g, 0.547 mmol) and anisole (5 mL) in
CH2Cl2 (25 mL) with ice bath cooling, and the solution was stirred
for 1 h at 0 °C and 1 h at room temperature. After removal of
volatiles in vacuo, the concentrate was subjected to a Sephadex
LH-20 column, eluting with 40% EtOH/toluene to give 0.54 g of
1
25 (60%) as a white solid: H NMR (D2O) δ 1.24-1.30 (m, 12
H), 1.34-1.60 (m, 8 H), 1.74-2.21 (m, 8 H), 2.57-2.66 (m, 4 H),
2.84-2.93 (m, 4 H), 3.0-3.64 (m, 24 H), 4.04-4.20 (m, 4 H),
4.24-4.38 (m, 4 H), 6.80-6.86 (m, 2 H), 7.03-7.07 (m, 2 H),
7.18-7.21 (m, 2 H); HRMS m/z calcd for C56H95N12O16S2,
1255.6425 (M + H, free amine); found, 1255.6417. Anal.
(C64H98F12N12O24S2 ·2.5H2O) C, H, N.
N4,N7-Bis[(N-tert-butoxycarbonyl-L-threonyl]-N1-[2,3-bis(4-
methoxybenzyloxy)benzoyl]-N7-[4-(phthalimido)butyl]norsper-
midine (22). A solution of freshly prepared N-tert-butoxycarbonyl-
L-threonine N-hydroxysuccinimide ester56 (1.12 g, 3.54 mmol) in
DMF (10 mL) was added to a solution of 21 (1.0 g, 1.41 mmol) in
DMF (10 mL). After the mixture was stirred for 72 h at 40 °C, the
solvent was removed under vacuum, and the residue was taken up
in CHCl3 (50 mL). The organic layer was washed with aqueous
5% NaHCO3 (3 × 50 mL), H2O (50 mL), saturated NaCl (50 mL)
and was concentrated in vacuo. Flash chromatography, eluting with
10% EtOH/EtOAc, afforded 0.945 g (60%) of 22 as a white foam:
1H NMR δ 1.14-1.21 (m, 6 H), 1.37-1.49 (m, 18 H), 1.52-1.8
(m, 8 H), 3.10-3.60 (m, 10 H), 3.68-3.74 (m, 2 H), 3.79-3.81
(m, 3 H), 3.84 (s, 3 H), 3.97-4.11 (m, 2 H), 4.32-4.60 (m, 2 H),
5.02 (s, 2 H), 5.07 (s, 2 H), 5.44-5.62 (m, 2 H) 6.82-6.86 (m, 2
H), 6.93 (d, 2 H, J ) 8.4), 7.11-7.13 (m, 2 H), 7.22-7.26 (m, 3
H), 7.39 (d, 2 H, J ) 8.4), 7.69-7.72 (m, 2 H), 7.81-7.85 (m, 2
H), 8.02 (br s, 1 H); HRMS m/z calcd for C59H79N6O15, 1111.5598
(M + H); found, 1111.5650. Anal. (C59H78N6O15) C, H, N.
N4,N8-Bis[(N-tert-butoxycarbonyl-L-threonyl]-N1-[2,3-bis(4-
methoxybenzyloxy)benzoyl]thermospermine (23). Hydrazine hy-
drate (5 mL) was added to a solution of 22 (250 mg, 0.225 mmol)
in EtOH (10 mL), and the reaction mixture was stirred at room
temperature for 16 h. Solid was filtered, and the filtrate was
concentrated under high vacuum. The residue was taken up in 1 N
NaOH (10 mL) and extracted with CHCl3 (3 × 10 mL), and organic
extracts were concentrated. Flash chromatography, eluting with 1%
concentrated NH4OH/MeOH, afforded 198 mg (90%) of 23 as a
1,36-Bis(2,3-dihydroxybenzoyl)-15,22-dioxo-18,19-dithia-
1,5,9,14,23,28,32,36-octaaza-5,9,28,32-tetrakis[[(4S,5R)-2-(2,3-di-
hydroxyphenyl)-4,5-dihydro-5-methyl-4-oxazolyl]carbonyl]hexatri-
acontane (26). Ethyl 2,3-dihydroxybenzimidate34 (0.231 g, 1.27
mmol) was added to a solution of 25 (0.35 g, 0.21 mmol) in
anhydrous EtOH (15 mL). The mixture was heated at reflux under
N2 for 36 h and was concentrated in vacuo. Column chromatography
on Sephadex LH-20, eluting with 15% EtOH/toluene, afforded
0.072 g (20%) of 26 as a gray solid: 1H NMR δ 1.35-1.75 (m, 20
H), 1.8-2.2 (m, 8 H), 2.47-2.59 (m, 4 H), 2.82-2.91 (m, 4 H),
3.02-3.91 (m, 24 H), 4.72-5.0 (m, 4 H), 5.21-5.36 (m, 4 H),
6.58-6.77 (m, 6 H), 6.84-7.02 (m, 6 H), 7.08-7.24 (m, 6 H);
HRMS m/z calcd for C84H103N12O24S2, 1728.6669 (M + H); found,
1728.7344. Anal. (C84H102N12O24S2) C, H, N.
BSA-Cysteine Conjugate (30). Maleimide-activated protein
2866 (2 mg in 100 µL buffer) and cysteine (2 mg in 200 µL of
degassed water) were incubated for 8 h at room temperature. After
incubation, 30 was purified on a dextran desalting column, eluting
with a purification buffer. Fractions (0.5 mL) were collected, and
the OD was measured at 280 nm. Positive fractions were pooled,
and protein concentration was estimated by Coomassie assay.58
Iron Complex of OVA-VIB Protein Conjugate (31). A 1 mL
solution of 4 (1.685 mg/mL) was incubated at pH 7.4 with 1 mM
FeNTA (500 µL) at room temperature. After the mixture was
incubated for 2 h, excess 1 was added and the mixture again
incubated for 2 h. The mixture was subjected to a G-25 Sepharose
column, using 30% DMSO/phosphate buffer as an eluting solvent.
Fractions (0.5 mL) were collected, and the first colored band
(fractions 3-6) was eluted; the OD was checked at 280 nm. Protein-
containing fractions were pooled (4 mL) and subjected to ICP-MS
for estimation of iron content.
1
white solid: H NMR δ 1.11-1.22 (m, 6 H), 1.37-1.43 (m, 18
H), 1.58-2.02 (m, 8 H), 2.71 (q, 2 H, J ) 6.0), 2.90-3.60 (m, 10
H), 3.80-3.81 (m, 3 H), 3.84 (s, 3 H), 3.98-4.14 (m, 2 H),
4.30-4.60 (m, 2 H), 5.00-5.08 (m, 4 H), 5.42-5.65 (m, 2 H),
6.82-6.86 (m, 2 H), 6.93 (d, 2 H, J ) 8.4), 7.13-7.19 (m, 2 H),

3812 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
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Acknowledgment. This paper is dedicated to Dr. William
R. Weimar in recognition of his many contributions to our
research program over the years. The project described was
supported by Grant Numbers R37DK049108 (National
Institute of Diabetes and Digestive and Kidney Diseases) and
R01AI074068 (National Institute of Allergy and Infectious
Diseases). The content is solely the responsibility of the
authors and does not necessarily represent the official views
of the National Institute of Diabetes and Digestive and Kidney
Diseases, the National Institute of Allergy and Infectious
Diseases, or the National Institutes of Health. We thank
Elizabeth M. Nelson for her technical assistance and Miranda
E. Coger for her editorial and organizational support. We
acknowledge the spectroscopy services in the Chemistry
Department, University of Florida, for the mass spectrometry
analyses and Diane G. Duke of the Hybridoma Core
Laboratory, Interdisciplinary Center for Biotechnology Re-
search, University of Florida, for the ELISA assessments of
antibodies against the OVA-VIB conjugate.
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Supporting Information Available: Elemental analytical data
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