Angewandte
Chemie
equilibrium shift and catalytic activity of OxyB upon compar-
ing these substrates.
interesting observation is that feeding 3-fluoro-b-hydroxytyr-
osine to a bhp deletion mutant (Bhp is required for Bht
biosynthesis) allowed isolation of a balhimycin derivative
fluorinated in residues 2 and 6 (fluorobalhimycin), showing
that OxyB can turn over in vivo, at least slowly, a fluorinated
linear peptide precursor.[13] This result also illustrates the
potential of the mutabiosynthesis approach for generating
novel glycopeptide antibiotic analogues. Unfortunately, there
have been no reports so far of in vitro activity with the
halogenase, so its substrate preferences are unknown. Pres-
ently, this makes it difficult to define the preferred order of
the chlorination and cross-linking steps. We anticipate,
however, that the synthetic methods described herein may
facilitate in vitro studies on this key halogenase.
To test how chlorination of a heptapeptide-S-PCP sub-
strate influences turnover by OxyB, a heptapeptide-S-PCP7
substrate 18 and the analogue 19 (Scheme 2) containing
(2S,3R)-b-hydroxy-m-chlorotyrosine (Cht) at position 6 were
synthesized (see the Supporting Information). The PCP7
thioester conjugates (18 and 19) were assayed with OxyB
under standard conditions. As reported earlier,[4a] model
heptapeptide 18 was converted (60–70%) into the C-O-D
macrocyclic product 20. However, similar assays with 19
repeatedly failed to reveal the formation of any C-O-D ring
product; only the corresponding linear peptide hydrazide was
recovered (ca. 90%; Supporting Information, Figure S13).
This dramatic effect observed in vitro upon introducing the
chlorine substituent suggests that a linear heptapeptide-S-
PCP chlorinated in residue 6 is not a viable intermediate in
vancomycin assembly, as it would not be a substrate for OxyB.
Two key observations reported herein provide important
new insights into vancomycin/balhimycin biosynthesis. First,
the conclusion that a chlorinated linear heptapeptide-S-PCP
is not a viable intermediate in vancomycin assembly rests on
the proviso that in vitro studies with the unnatural substrates
reveal properties of the biosynthetic reactions as they occur
naturally within the cell. Although difficult to prove unam-
biguously, this seems a reasonable conclusion, as OxyB
catalyzes efficient cross-linking in vitro on hexapeptide-S-
PCP substrates. On the other hand, fully chlorinated linear
(that is, non-cross-linked) hexa- and heptapeptides have been
isolated from different null mutants of the balhimycin
producer.[2a,10] It is possible, however, that these chlorinated
linear peptides might be shunt products produced in minor
amounts over long fermentation times. One very interesting
shunt metabolite isolated from one of the mutants (a dpgA
mutant) was a cross-linked heptapeptide containing Hpg7 in
place of Dpg7.[10]
The second point concerns the timing of the halogenation
steps. The halogenase is unusual in that it must recognize two
rather different sites to chlorinate residues 2 and 6 in the
peptide backbone. Free Cht is not a naturally occurring
precursor and is not used for glycopeptide biosynthesis,[11] so
chlorination most likely occurs on an NRPS-bound inter-
mediate. Chlorination might take place directly after incor-
poration of each Bht residue on the NRPS. The in vitro
experiments described above, however, show that a chlorine
substituent in residue 6 of the hexapeptide-S-PCP slows
dramatically the cross-linking reaction catalyzed by OxyB.
This raises the question as to whether, during the biosynthesis,
cross-linking by OxyB might occur before chlorination.
Although earlier molecular genetic studies revealed chlori-
nated hexa- and heptapeptides from balhimycin null muta-
nts,[2a,10] later in-frame deletions of the oxygenase genes,
designed to avoid polar effects on downstream genes
(including the halogenase), gave oxyA or oxyC mutants that
produced only singly or doubly cross-linked but fully chlori-
nated products, whereas an oxyB mutant gave only non-
chlorinated linear hexa- and heptapeptide products, suggest-
ing that the action of the halogenase might depend in some
way on the presence of OxyB as well as the NRPS.[12] A final
Received: June 8, 2012
Revised: August 1, 2012
Published online: October 16, 2012
Keywords: cytochrome P450 · glycopeptide antibiotics ·
.
halogenase · phenol coupling · vancomycin
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