Nonenzymatic oxidative steps accompanying action of the cytochrome P450 enzymes StaP and RebP in the biosynthesis of staurosporine and rebeccamycin

Article abstract of DOI:10.1021/ja0743801

In the biosynthesis of the indolocarbazole natural products staurosporine and rebeccamycin, a complex set of oxidative transformations results in dimerization and oxidative cross-linking of a pair of l-tryptophan monomers. Most intriguing among the oxidative enzymes involved in this pathway is StaP (or its homologue, RebP), which appears to catalyze the four- to eight-electron oxidation of chromopyrrolic acid to give a set of three aglycones, differing in oxidation state in the pyrrole-derived ring. In this work, we sought to clarify the catalytic role of StaP in this process and gain insight into the apparent lack of specificity in its product profile. Through the preparation of a putative intermediate, we show that all steps downstream of aryl-aryl coupling can occur nonenzymatically in solution, pinpointing the function of StaP as a two-electron aryl-aryl coupling catalyst that acts on chromopyrrolic acid. StaP thus joins a small but growing family of aryl-aryl coupling enzymes that use P450-based chemistry to facilitate this oxidative transformation. Furthermore, using ring-deuterated substrate, we show that this aryl-aryl coupling process is not a rate-limiting step in the overall formation of the aglycones. This work expands on our previous studies on the biosynthetic enzymes involved in the staurosporine and rebeccamycin pathways and acts to illuminate the specific roles of StaP and Rebp in the context of these biosyntheses. Copyright

Full text of DOI:10.1021/ja0743801

Published on Web 08/17/2007
Nonenzymatic Oxidative Steps Accompanying Action of the Cytochrome P450
Enzymes StaP and RebP in the Biosynthesis of Staurosporine and
Rebeccamycin
Annaleise R. Howard-Jones and Christopher T. Walsh*
Department of Biological Chemistry & Molecular Pharmacology, HarVard Medical School, 240 Longwood AVenue,
Boston, Massachusetts 02115
Received June 15, 2007; E-mail: christopher_walsh@hms.harvard.edu
Scheme 1. Overall Reaction Catalyzed by StaP/RebP, StaC, and
RebC
Biosynthesis of the antitumor indolocarbazoles rebeccamycin and
staurosporine involves a complex series of oxidative coupling
reactions, catalyzed by heme and flavin-based enzymes, starting
from the common amino acid L-tryptophan.1-3 Most intriguing
among these is the four- to eight-electron oxidation of chromopy-
rrolic acid (CPA) 1 to the three aglycones, K252c 3, 7-hydroxy-
K252c 4, and arcyriaflavin A 5, which can be catalyzed by the
single cytochrome P450 enzyme, StaP (or its homologue from the
rebeccamycin pathway, RebP) (Scheme 1).4 The presence of StaC
enhances production of K252c 3 over other oxidation states of the
aglycone, while RebC directs the preferential formation of arcyri-
aflavin A 5. This activity is dependent on the presence of dioxygen.4
Our previous work had suggested that aryl-aryl coupling preceded
decarboxylation and oxidation of the pyrrole ring.4 Hence, we
postulated that the role of StaP/RebP may be simply to facilitate
aryl-aryl coupling (a net two-electron oxidation), leading to the
formation of 5,7-dicarboxyaglycone 2 (5,7-dicarboxy-12,13-dihy-
dro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole) (Scheme 2), with
subsequent steps occurring spontaneously in solution.4 In order to
verify this assertion, here we explore a synthetic route toward the
putative intermediate 5,7-dicarboxyaglycone 2 to assess its stability
and its ability to convert to the aglycones 3, 4, and 5. The presence
of a spontaneous, nonenzymatic route from the aryl-aryl-coupled
intermediate 2 to the aglycones would provide strong evidence that
the singular role of StaP/RebP is to perform a two-electron aryl-
aryl coupling reaction on CPA 1. Although such aryl-aryl coupling
catalysts are not uncommon in biosynthetic clusters, few have been
studied in any detail.5-8 We also probe the deuterium kinetic isotope
effect of the StaP-mediated reaction to determine which of the aryl-
aryl coupling or subsequent oxidative steps are rate-limiting.
The dimethyl ester of 5,7-dicarboxyaglycone was prepared by
palladium-catalyzed aryl-aryl coupling9 of the dimethyl ester of
CPA (Supporting Information Scheme 1). This compound was
easily isolable, and its identity was confirmed by NMR spectro-
scopic analysis. Upon deprotection in the presence of lithium
hydroxide (in 1:1 methanol/water at room temperature), a suite of
products were obtained, none of which corresponded to 5,7-
dicarboxyaglycone 2. The main two isolated products from this
reaction mixture were 7-carboxy-K252c 7 and 7-carboxy-7-hy-
droxy-K252c 8, as confirmed by NMR and mass spectroscopy
(Scheme 2, Supporting Information), which were formed in a ratio
of approximately 3:1. These data suggest that compounds 7 and 8
are the product of spontaneous reactivity of 5,7-dicarboxyaglycone
2 under mild aerobic conditions, and hence we suggest that they
are representative of true intermediates formed in solution on
reaction of StaP with CPA 1.
to determine if they were in fact en route to the rebeccamycin and
staurosporine aglycones, 3, 4, and 5.
7-Carboxy-K252c 7 was highly unstable and converted spontane-
ously to two peaks by HPLC, the major peak corresponding to
7-hydroxy-K252c 4 and the minor one to arcyriaflavin A 5 (data
not shown), which formed in a 2:1 ratio. This result was confirmed
by mass spectroscopy: a clear peak at m/z 356, corresponding to
the intact 7-carboxy-K252c 7, was seen in the originally isolated
sample; after 30 min in DMSO, this was replaced by the mass of
7-hydroxy-K252c 4 (m/z 328) (Scheme 2). Thus it seems that
7-carboxy-K252c 7 is directly en route to the two more oxidized
aglycones (7-hydroxy-K252c 4 and arcyriaflavin A 5), previously
observed to form by StaP-mediated oxidation of CPA 1. Interest-
ingly, K252c 3 is not observed to form in the initial hydrolysis
reaction nor in the reaction of 7-carboxy-K252c 7, suggesting that
spontaneous formation of this aglycone follows a divergent route.
The second product, 7-carboxy-7-hydroxy-K252c 8, was found
to be relatively stable under standard conditions at room temperature
(Scheme 2). Furthermore, it was not a substrate for any combination
of RebC, StaC, or StaP, as determined by HPLC analysis (data not
shown). This more highly oxidized form of compound 7 therefore
appears to be off-pathway with respect to aglycone formation in
the rebeccamycin/staurosporine manifolds. That this apparently
“dead-end” compound has not been isolated from StaP enzymatic
assays suggests that it may not be formed in the StaP-catalyzed
reaction.
These data suggest that aryl-aryl bond formation is indeed the
only necessary enzyme-catalyzed step in the aglycone-forming
reaction, with the remainder of the pathway occurring spontane-
ously. 7-Hydroxy-K252c 4 and arcyriaflavin A 5 form via the
intermediacy of 7-carboxy-K252c 7, whereas K252c 3 is likely
formed via an alternative, less oxidized intermediate (Scheme 2).
Therefore, we propose that K252c 3 derives from an intermediate
We next sought to determine the reactivity of compounds 7 and
8, either spontaneously or in the presence of StaP, StaC, or RebC,
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10.1021/ja0743801 CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Scheme 2. Proposed StaP-Mediated and Spontaneous Steps in
the Biosynthesis of Aglycones 3, 4, and 5, Showing the
Intermediacy of 7-Carboxy-K252c 7 and Divergence to Oxidized
Off-Pathway Species, 7-Carboxy-7-hydroxy-K252c 8
Table 1. Rates of Aglycone Production by StaP Using Protio-CPA
(kH) and (Indole-d10)-CPA (kD) as Substrates
aglycone
1
1
produced
k
H (min-
)
k
D (min-
)
kH/kD
K252c 3
arcyriaflavin A 5
0.046 ( 0.001
0.031 ( 0.002
0.048 ( 0.005
0.033 ( 0.004
0.96 ( 0.11
0.94 ( 0.14
formation of aglycones 3, 4, and 5. Rather, subsequent decarboxy-
lation and oxidation of the coupled scaffold, which we have
determined to be spontaneous processes, are the rate-limiting stages
of this transformation.
The indolocarbazole scaffolds of the rebeccamycin and stauro-
sporine natural products, and perhaps also the rearranged scaffolds
of the violaceins11,12 and chromoviridins,13 arise by a set of enzyme-
mediated oxidations using flavin and heme cofactors, admixed with
autoxidative processes that reflect the instability of electron-rich
pyrrole frameworks in aerobic environments. The results described
in this report substantially simplify the role of StaP (and its
homologue RebP) in mediating formation of a suite of aglycones
by four- to eight-electron oxidation of CPA 1. That is, StaP/RebP
is simply responsible for the two-electron intramolecular aryl-aryl
coupling of CPA 1 to give the six-ring intermediate 2, with all
subsequent steps occurring nonenzymatically in solution.
In the two enzyme StaP/StaC or StaP/RebC systems,4 the partner
flavoenzymes StaC and RebC likely act to intercept intermediate
2 or subsequent intermediates such as 6 or 7 formed en route to
the aglycones (Scheme 2). Recent crystallographic work has
identified density attributable to a tautomer of 7 in the active site
of RebC.14 Our current study on the nonenzymatic steps following
StaP-mediated aryl-aryl bond formation corroborates the proposed
role of RebC (and by extension StaC) in intercepting and redirecting
intermediates en route to the aglycones, 3, 4, and 5.
Acknowledgment. This work was supported by NIH grant
GM020011. We thank Catherine L. Drennan and Katherine S. Ryan
for helpful discussions.
such as 6, though this precursor has not been isolated. Nonetheless,
it is apparent that 7-carboxy-7-hydroxy-K252c 8 is not an inter-
mediate in the formation of aglycones 3, 4, or 5. These data suggest
a cascade of nonenzymatic decarboxylations and oxidations, totaling
two to six electron oxidations, following the two-electron oxidative
aryl-aryl coupling effected by the cytochrome P450 enzyme, StaP,
or RebP (Scheme 2).
Supporting Information Available: Experimental procedures and
complete ref 6. This material is available free of charge via the Internet
at http://pubs.acs.org.
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