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Angewandte
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recording growth curves over 72 h. The growth curves for
mutants grown in the presence of 1 and 2 and mutants grown
in medium containing PABA (10 mm) were similar, with only
the rescued DpabA mutant reaching a lower optical density
with 1 and 2 than with directly added PABA (Figure 2C). We
delineated the requirements for successful auxotroph rescue
with a series of control reactions, in which we varied the
components of the defined minimal medium and measured
the final OD600 values after 48 h at 378C (Table S3 in the
Supporting Information). We found that the presence of both
substrate 1 and catalyst 2 is essential. Thiol addition had
a minimal impact on growth, perhaps indicating that the
improved conversion observed with these additives does not
affect the efficiency of rescue. This result could also suggest
that thiols generated by E. coli are involved in the depro-
tection in vivo.
An alternate explanation for auxotroph rescue could be
the accumulation of mutations during the course of the
experiment that either alleviate PABA auxotrophy or enable
the strains to generate PABA from substrate 1. We inves-
tigated these possibilities by re-inoculating rescued cultures
back into minimal medium lacking PABA (Figure S3 and
Table S4 in the Supporting Information). None of the rescued
cultures grew under these conditions, which confirms that
they were still PABA auxotrophs. The rescued strains also
remained unable to grow in PABA-free medium containing
substrate 1 (10 mm), a result that indicates that they had not
acquired the ability to deprotect Alloc-PABA. All of the
rescued cultures retained the ability to grow in medium
containing PABA and could be rescued a second time in
PABA-free medium containing substrate 1 and catalyst 2.
Finally, the fact that three auxotrophic strains, each contain-
ing a distinct mutation, could be rescued using this depro-
tection reaction adds to the weight of evidence against genetic
changes leading to bacterial growth in these experiments.
A final piece of data supporting the link between Alloc
removal and growth was the detection of PABA in rescued
cultures. We determined the concentration of PABA neces-
sary to achieve rescue by inoculating DpabA and DaroC
mutants into M9 glycerol minimal media containing varying
amounts of PABA (10 mm–10 pm). We found that at least
10 nm of PABA was required to rescue either mutant strain
(Table S5 in the Supporting Information). We then used liquid
chromatography–mass spectrometry (LC–MS) to quantify
the PABA produced during a rescue experiment (Table S6 in
the Supporting Information). Analysis of the culture medium
after rescue of the DpabA mutant revealed a PABA concen-
tration of 60 nm; therefore, the concentration of PABA
generated from the Ru-catalyzed deprotection of 1 is suffi-
cient to allow growth of these auxotrophs. Although we
detected PABA in the medium, we do not know to what
extent deprotection occurs intracellularly versus extracellu-
larly; gaining a deeper understanding of where biocompatible
reactions take place is a future area of interest.
transformation that had not previously been utilized in the
presence of living organisms. The hydroxylation of aromatic
substrates in water using low-valent transition metals and
molecular oxygen has been extensively investigated, because
these systems mimic the reactivity of monooxygenase
enzymes.[13] In particular, oxygenation using iron(II) com-
plexes, molecular oxygen, and reducing agents (ascorbic and
citric acids) has been previously carried out in aqueous
solvent at ambient temperatures, conditions that could
potentially be compatible with living organisms.[14] These
hydroxylations are thought to proceed through formation of
reactive oxygen species, including hydroxyl radicals, making it
unclear whether this type of transformation could be bio-
compatible.[15] However, this reaction has biological rele-
vance, because it resembles chemistry that may have occurred
during the oxygenation of the Earthꢀs atmosphere, as
molecular oxygen reacted with iron present in the environ-
ment.[16]
We envisioned rescuing the DaroC mutant, a p-hydroxy-
benzoic acid (PHBA) auxotroph, through an iron-catalyzed
para hydroxylation of benzoic acid (3, Figure 3A). We first
confirmed that the DaroC mutant could not grow in PHBA-
free M9 glycerol minimal medium, and that medium contain-
ing benzoic acid (100 mm) did not rescue growth (Figure 3B).
In a manner analagous to that of the earlier rescue of PABA
auxotrophy, the media contained all nutrients required for
growth except PHBA. We then inoculated the DaroC mutant
into PHBA-free minimal media containing 3 (10, 100, or
200 mm), FeSO4 (10, 100 or 200 mm), and citric acid (0.2, 2, or
4 mm).[14e] The cultures containing higher concentrations of
substrates and catalysts were rescued. Control experiments
revealed that all reaction components were required for
growth (Table S7 in the Supporting Information). Compar-
ison of the growth curves for cultures grown in the presence of
PHBA with those for rescued cultures revealed a more
pronounced delay of entry into the exponential growth phase
during rescue than was observed for the PABA auxotrophs
(Figure 3C). We hypothesize that this difference may be due
to cell death arising from reactive oxygen species generated
during the hydroxylation reaction, an effect that would reduce
the inoculum. Similarly to the rescued PABA auxotrophs, the
rescued PHBA auxotrophs remained unable to grow in the
presence of 3, thus indicating a key role for catalysis in the
restoration of growth (Table S8 and Figure S4 in the Support-
ing Information). Culturing the DaroC mutant in media
containing varying amounts of PHBA revealed that
10–100 nm PHBA is sufficient for growth (Table S9 in the
Supporting Information). We confirmed that PHBA was
produced in rescued cultures by using LC–MS, and detected
70 nm of PHBA in the medium after rescue (Table S10 in the
Supporting Information).
Unlike most enzymatic transformations that functionalize
aromatic scaffolds, the iron-catalyzed hydroxylation used to
generate PHBA is not expected to be regioselective. To
establish the selectivity of this reaction, we performed the
hydroxylation in culture medium using 3 (1 mm). At this
higher substrate concentration, we were able to separate and
quantify all three monohydroxylation products by using
HPLC (Figure S5 and Table S11 in the Supporting Informa-
This initial demonstration of auxotroph rescue relied
upon a reaction that had previously been applied in living
cells. We decided to explore the generality of this phenom-
enon by rescuing a different type of auxotrophy using
iron(II)-catalyzed hydroxylation, a chemically challenging
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Angew. Chem. Int. Ed. 2013, 52, 11800 –11803