Organic Letters
Letter
Compounds 1 and 9c−e were then heated in methanol to
provide the corresponding isoxazolidinone derivatives (2, 9h−
j) via rearrangement reactions.
CAS assays were performed with these analogues in hand
(Figure 3A). First, the role of the hydroxamate group of 1 in
the iron-binding propensity of any of these analogues,
suggesting that the ethylene linker is flexible and thus well
capable of positioning either amine atom toward the metal
center.
A series of growth promotion assays were conducted with A.
baumannii ΔbasD to correlate the iron-binding abilities of the
acinetobactin analogues to their iron delivery capabilities
(Figure 3B).18 Strikingly, hydroxamate-modified analogues 9a
and 9b failed to exhibit growth promotion activities despite
their Fe(III) binding capabilities. This observation implicates
that although the hydroxamate bridge of 1 may not function as
an iron chelator, it is a critical element for the recognition of
acinetobactin (1) by the relevant cellular uptake machinery. At
this point, it is uncertain which component (e.g., BauA, BauD,
or BauF) would directly interact with the hydroxamate moiety;
this will be investigated in due course.
The growth promotion activities of the prepseudomonine
analogues (9e−g) well correlated with their iron-binding
affinities; only prepseudomonine (9e) could act as a side-
rophore, and its activity was analogous to that of 1 at
concentrations below 6.25 μM. Interestingly, pseudomonine
(9j) could promote bacterial growth despite its weak Fe(III)
binding, but this activity was BauA-independent, similar to 2
(Figure S5B). The imidazole-modified analogues exhibited
somewhat complex results. Among the oxazoline analogues
(9c,d), N1-methylated 9d showed an activity similar to that of
1, while the activity of 9c was much lower. This indicates that
the conformation of the 9d−Fe(III) complex is more
compatible with the acinetobactin uptake system, and it also
suggests that the N1 position of 1 would be a suitable site for
conjugating an antibiotic molecule. Among the isoxazolidinone
analogues (9h,i), N1-methylated 9i was unable to promote
growth of A. baumannii, while 9h exhibited BauA-independent
Figure 3. Functional characterization of 10 acinetobactin analogues:
(A) CAS assay results; (B) growth promotion assay results using A.
baumannii ΔbasD.
Collectively, this study discloses the intriguing structure−
function relationships of various acinetobactin analogues, and
several crucial implications for the successful design of effective
acinetobactin−antibiotic conjugates were derived. First, oxazo-
line 1 is likely the physiologically relevant siderophore structure
for A. baumannii based on the growth-promotion assay results
using mutant strains. This conclusion suggests that 1 would
serve better as the antibiotic delivery vehicle targeting A.
baumannii, as it more appropriately exploits the acinetobactin
uptake machinery than 2, the key requirement for conferring
the anticipated potency and selectivity to the antibiotic
conjugates based on acinetobactin.2 As for the conjugate
design, the hydroxamate bridge seems to be critical for cellular
uptake because its modification (9a and 9b) completely
abolished iron delivery. In addition, the N1 position of the
pendant imidazole appears to be a promising drug conjugation
site because modification at this site (9d) did not abrogate the
iron delivery function. Notably, modification of N1 can be easily
introduced by applying the van Leusen reaction, as
demonstrated herein.
iron binding was investigated using 9a and 9b. Although these
analogues possess modifications that disrupt the metal binding
of the hydroxamate (9a: O-methylation, 9b: a simple amide
linkage), both could elicit color changes in the CAS assay,
indicating that they were capable of binding Fe(III). This
observation was consistent with the proposed iron-binding
mode of acinetobactin 1 involving the catechol dihydroxyl and
imidazole groups, not the hydroxamate (vide supra).
Four phenolic derivatives (9e,f,g,j) were prepared and tested
to investigate the role of the catechol moiety. Notably, these
compounds comprise pseudomonine (9j), another siderophore
produced by some Pseudomonas strains, and its analogues.16
Prepseudomonine (9e) exhibited a strong CAS signal even at a
low concentration (50 μM), indicating that the 3″-hydroxyl
group of 1 is not critical for iron binding. This result is not
unexpected because the phenolic oxazoline moiety is often
found in other siderophores, such as mycobactin.17 Mod-
ifications of the hydroxamate bridge in 9f and 9g appeared to
considerably weaken the iron binding, contrary to that for the
catechol siblings (9a,b). Surprisingly, pseudomonine (9j) failed
to elicit a noticeable color change in the CAS assay, even at 1.25
mM, unlike 2. Nevertheless, the fluorescence quenching of 9j
by Fe(III) was observed (Figure S3), which indicates that 9j is
still capable of binding Fe(III), albeit not tightly.
The siderophore-based antibiotic delivery approach provides
an alternative and powerful approach to overcome the
threatening problems of drug resistance of various pathogens.
The insights provided in this study will serve as an
indispensable foundation for the success of ensuing efforts in
this direction and may lead to the discovery of potent
acinetobactin−antibiotic conjugates that will be effective in
treating drug-resistant A. baumannii.
Analogues 9c/9h and 9d/9i were designed to identify which
amine atom, N1 or N3, of the pendant imidazole ring is
responsible for iron binding. Interestingly, the CAS assay
results reveal that N-methylation did not significantly decrease
C
Org. Lett. XXXX, XXX, XXX−XXX