blocked mutant of A. pretiosum. In fact, aminobenzoic
acid 620 was not loaded onto the PKS or processed by the
polyketide synthase in A. pretiosum and thus formation of
the desired ansamitocin derivative 7 was not encountered.
Instead, when supplementing a culture of this AHBA
blocked mutant with 6, 13 new metabolites 8À20 were
isolated as pure compounds after extensive preparative
HPLC purification. Except for metabolite 12 for which
only HRMS data were available all metabolites were
characterized by NMR spectroscopy and HRMS. Re-
markably, none of these metabolites contained an azido
group. In all cases, the reduced anilines were formed and in
several cases the amino group was further modified by
acetylation (see 9,10,13,14,19,and20), methylation (see 11),
or amidination to yield the corresponding urea derivative
15 (Figure 1).
The ability of A. pretiosum to reduce aryl azide 6 is
unusual as was demonstrated by two additional experi-
ments. First, feeding of aryl azide 6to the AHBA(À) blocked
mutant of the geldanamycin producer S. hygroscopicus
yielded azido-geldanamcin derivative 21 (Scheme 2).
Scheme 3. Feeding of 3-Azido-5-hydroxybenzoic Acid 22 and
3,5-Diaminobenzoic acid 23 to the AHBA(À) Blocked Mutant
of A. pretiosum
Further tailoring steps (epoxidation, chlorination, and
N-methylation) did occur in all cases. Ansamitocin deri-
vatives 16À18 bearing a chlorine substituent at C19 are
rare examples of incorporation of chlorine into an un-
chlorinated substrate.
Second, we fed 2-azido-3-hydroxybenzoic acid 22 to A.
pretiosum and found that AP3 (3b) was formed in good
yield (Scheme 3). Again, the azido group must have been
reduced during bioprocessing. However, when this experi-
ment was repeated with the AHBA blocked mutant of
S. hygroscopicus, geldanamycin was not formed. This further
confirms the differences between A. pretiosum and
S. hygroscopicus.
Scheme 2. Feeding of Azido Benzoic Acid 6 to the AHBA(À)
Blocked Mutant of S. hygroscopicus
At this point, it remains unclear at which stage of the
biosynthesis the reduction in A. pretiosum takes place.
Either aryl azides 6 or 22 are loaded onto the PKS which
means that, somewhere en route to the final PKS module,
reduction of the azido moiety occurs so that the amide
synthase can perform macrolactamization of the seco-acid.
Alternatively, and more likely, reductions of 6 and 22
occur prior toloading onto the PKS starter module, sothat
the whole process would proceed in the “natural” manner.
The fact that mutasynthon 23 (Scheme 3) was not
further processed, though it is the reduction product of
azide 6, may be ascribed to its high polarity that could
reduce its ability to penetrate the bacterial membrane.
Alternatively, in vivo activation to the CoA ester may be
hampered compared to 6.
In order to rule out chemical reduction of the azide group
by natural thiols we treated azide 22 with ethanethiol,
thiophenol, and glutathione in degassed pH 7 buffer for 3
days. MS-analysis revealed no conversion of 22 by thiophe-
nol, and only traces (less than 0.5%) of the reduction product
was found in the presence of glutathione and ethanethiol.
Furthermore, we fed benzyl azides 24À26 in order to
probe the selectivity of A. pretiosum. None of these three
arenes were processed by A. pretiosum. In contrast, benzyl
azide 26 was transformed to seco-proreblastatinamide
derivative 27 by the (À)AHBA mutant strain of S. hygro-
scopicus (Figure 2). In this case, almost complete process-
ing took place; however, amide formation took place at
Commonly, the halogenase requires the phenolic group
at C20 and even when the phenol at C20 is O-methylated
chlorination is suppressed. Obviously, also a free amino
group at C20 can act as a directing group, probably
through H-bonding in the active site of the halogenase. It
is noteworthy that feeding of 3,5-diaminobenzoic acid 23
to the mutant strain of A. pretiosum did not provide any of
these anilino ansamitocins or other advanced metabolites.
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