6
isolated from P. sclerotiorum in 1940. Since then, 1 has
been found to inhibit multiple therapeutic targets.
The native promoter of afoA, the gene that encodes for the
pathway-specific transcription activator of the asperfura-
none pathway, was replaced with the inducible alcohol
dehydrogenase promoter, alcA. The afoD gene, which
codes for the hydroxylase in the asperfuranone pathway,
was deleted to enable the accumulation of intermediate,
compound 5. It should be noted that the afo cluster is
silent under normal laboratory growing conditions. The
wildtypestrain, thus, didnot producedetectablequantities
of compound 5 and asperfuranone (4) by LC/MS analysis.
The mutant strain was initially cultured in a liquid lactose
minimal medium under inducing conditions (refer to
Supporting Information) at 37 °C for 3 days to produce
nearly 200 mg/L of 5 without need for further purification
since 5 is poorly dissolved in aqueous media.
7
Sclerotiorin belongs to an important class of natural
products called azaphilones. Azaphilones are structurally
diverse polyketides that share a highly oxygenated bicyclic
core and chiral quaternary center. These polyketides are
known for their 4H-pyran motif, which reacts with amines
1
1
8
to produce the corresponding vinylogous γ-pyridones.
Early synthetic studies by Whalley and co-workers re-
ported the total synthesis of several azaphilones, which
9
included compound 8 prepared in 14 steps. Recent syn-
thetic efforts by Porco and co-workers have shown assem-
bly of the azaphilone core through a copper-mediated
enantioselective dearomatization route. The application
of their asymmetric methodology was demonstrated on
(
ꢀ)-S-15183a (2), (ꢀ)-mitrorubin (3), and more recently
We altered cultureconditions inseveral waystooptimize
the titer of compound 5. First, culture time prior to the
induction of alcA was investigated. Cultures were incu-
bated from 12 to 36 h before the chemical inducer cyclo-
pentanone, necessary to induce the alcA promoter, was
introduced. Thereafter the culture remained under induc-
ing conditions for an additional 72 h. The experiment
revealed the production of 5 was enhanced by growth
10
with 1 (Figure 1).
30ꢀ36 h before induction (Figure S1). We next examined
a second parameter, the culture time postinduction. The
A. nidulans strain was cultured for 7 days after induction,
and samples were collected at 1-day intervals from day
three through day seven (Figure S2). An increase and
then decline was observed over the period, with the
accumulation of 5 peaking on day five. Under optimized
expression conditions our engineered strain produced the
polyketide (5) abundantly, providing a titer of 900 mg/L.
The elevated production of this advanced metabolite
allowed us to employ it for the preparation of a small
library of azaphilones.
Figure 1. Azaphilone natural products.
We focused on applying our semisynthetic route to
prepare (þ)-sclerotiorin by treating 5 with p-toluenesulfo-
nic acid (Scheme 2) to form the 2-benzopyrilium salt (6),
which is then oxidized by lead tetraacetate to generate the
Although many azaphilones have been isolated and
identified, their biosynthetic pathways remained unknown
until our recent identification of the asperfuranone (4)
biosynthetic pathway in Aspergillus nidulans. A mutant
strain from the previous study provided aldehyde 5 as a
stable intermediate, which has been isolated from other
azaphilone-producing organisms. Our work aims to en-
hance the production of the putative azaphilone intermedi-
ate (5) and use synthetic chemistry to structurally diversify
9
1
1
nonhalogenated azaphilone (7). Although the acetoxyla-
tion at C-7 would be nonstereospecific, the diastereomers
1
13
were indistinguishable (t.l.c., H and C NMR) and could
not separated by HPLC.
Electrophilic chlorination of azaphilone 7 introduces a
chlorine atom at C-5 by using a slight excess of N-chloro-
succinimide to provide the natural product (þ)-sclero-
tiorin and 7-epi-sclerotiorin (8) in 61% yield. Despite the
recalcitrant purification of 8, the diastereomers were sepa-
rated by analytical chiral HPLC to reveal close to a 1:1
ratio of (þ)-sclerotiorin and 7-epi-sclerotiorin.
5
into natural and non-natural azaphilones.
In this study, the fungal strain used to overproduce
compound 5 contains two genetic alterations (Scheme 1).
(
(
6) Curtin, T. P.; Reilly, J. Biochem. J. 1940, 34, 1419.
7) Osmanova, N.; Schultze, W.; Ayoub, N. Phytochem. Rev. 2010, 9,
Additionally, several azaphilone analogs were also pre-
pared from 5 (Scheme 3). To create a more efficient
synthetic route, we were interested in hypervalent-iodine-
mediated phenol oxidative dearomatization with o-iodoxy-
benzoic acid (IBX), a method developed by Pettus and
315.
(
8) Wei, W.-G.; Yao, Z.-J. J. Org. Chem. 2005, 70, 4585.
9) Chong, R.; King, R. R.; Whalley, W. B. J. Chem. Soc. (C) 1971,
(
3
566.
10) (a) Zhu, J.; Grigoriadis, N. P.; Lee, J. P.; Porco, J. A. J. Am.
Chem. Soc. 2005, 127, 9342. (b) Zhu, J.; Porco, J. A. Org. Lett. 2006, 8,
169. (c) Germain, A. R.; Bruggemeyer, D. M.; Zhu, J.; Genet, C.;
O’Brien, P.; Porco, J. A. J. Org. Chem. 2011, 76, 2577.
11) Chiang, Y.-M.; Szewczyk, E.; Oakley, B. R.; Wang, C. C. C.
(
1
2
co-workers. The reaction proceeds with the formation
5
(
(12) Marsini, M. A.; Gowin, K. M.; Pettus, T. R. R. Org. Lett. 2006,
8, 3481.
J. Am. Chem. Soc. 2009, 131, 2965.
Org. Lett., Vol. 14, No. 4, 2012
973