Transformations of cyclic nonaketides by Aspergillus terreus mutants blocked for lovastatin biosynthesis at the lovA and lovC genes

Article abstract of DOI:10.1039/b207721c

Two mutants of Aspergillus terreus with either the lovC or lovA genes disrupted were examined for their ability to transform nonaketides into lovastatin 1, a cholesterol-lowering drug. The lovC disruptant was able to efficiently convert dihydromonacolin L 5 or monacolin J 9 into 1, and could also transform desmethylmonacolin J 15 into compactin 3. In contrast, the lovA mutant has an unexpectedly active β-oxidation system and gives only small amounts of 1 upon addition of the immediate precursor 9, with most of the added nonaketide being degraded to heptaketide 22. Similarly, the lovA mutant does not accumulate the polyketide synthase product 5 and rapidly degrades any 5 added as a precursor via two cycles of β-oxidation and hydroxylation at C-6 to give 20. The possible involvement of epoxides 21a and 21b in the biosynthesis of 1 was also examined, but their instability in fermentation media and fungal cells will require purified enzymes to establish their role.

Full text of DOI:10.1039/b207721c

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Transformations of cyclic nonaketides by Aspergillus terreus
mutants blocked for lovastatin biosynthesis at the lovA and lovC
genes
John L. Sorensen,a Karine Auclair,b Jonathan Kennedy,c C. Richard Hutchinsonc and
John C. Vederas*a
a Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
E-mail: john.vederas@ualberta.ca; Fax: 00 1 780 492 5475; Tel: 00 1 780 492 8231
b Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal,
Quebec H3A 2K6, Canada. E-mail: karine.auclair@mcgill.ca; Fax: 00 1 514 398 3797;
Tel: 00 1 514 398 2822
c Kosan Biosciences Inc., 3832 Bay Center Place, Hayward, California 94545, USA.
E-mail: hutchinson@kosan.com; Fax: 00 1 510 732 8401; Tel: 00 1 510 732 8400
Received 7th August 2002, Accepted 18th September 2002
First published as an Advance Article on the web 21st November 2002
Two mutants of Aspergillus terreus with either the lovC or lovA genes disrupted were examined for their ability to
transform nonaketides into lovastatin 1, a cholesterol-lowering drug. The lovC disruptant was able to efficiently
convert dihydromonacolin L 5 or monacolin J 9 into 1, and could also transform desmethylmonacolin J 15 into
compactin 3. In contrast, the lovA mutant has an unexpectedly active β-oxidation system and gives only small
amounts of 1 upon addition of the immediate precursor 9, with most of the added nonaketide being degraded to
heptaketide 22. Similarly, the lovA mutant does not accumulate the polyketide synthase product 5 and rapidly
degrades any 5 added as a precursor via two cycles of β-oxidation and hydroxylation at C-6 to give 20. The possible
involvement of epoxides 21a and 21b in the biosynthesis of 1 was also examined, but their instability in fermentation
media and fungal cells will require purified enzymes to establish their role.
Introduction
Lovastatin 1 (also known as mevinolin, monacolin K, and
Mevacor) is a potent cholesterol-lowering agent1 and a pre-
cursor for synthesis of the widely-prescribed drug, simvastatin
2 (Zocor).2 Its 6-desmethyl analogue, compactin 3 (also
designated as ML-236B or mevastatin) can be converted by
microbial oxidation to the 6β-hydroxy derivative, pravastatin
4 (Pravachol).3,2b These compounds and certain fully syn-
thetic mimics block (3S)-hydroxy-3-methylglutaryl-coenzyme
A reductase, the rate-limiting enzyme in the biosynthesis of
cholesterol.1–4
Fig. 1 Lovastatin biosynthesis genes.
add the C-8 hydroxyl to form monacolin J 9.7 The 2-methyl-
butyryl side chain is produced by a separate PKS encoded by
lovF, and further used by LovD to directly acylate 9 and yield
1.5 The function of LovA, a protein essential for the formation
of 1, is not fully understood, but it has sequence homology
to P450 enzymes and is probably involved in the oxidative
conversion of 5 to introduce the diene system present in 9. Bio-
synthetic studies on compactin 3 using the fungus Penicillium
aurantiogriseum indicate a labelling pattern analogous to that
seen in lovastatin 1,8a but the genetic machinery required for
the formation of 3 has not yet been reported.†
Biosynthetic studies on lovastatin 1 with fungi, such as
Aspergillus terreus, show that its assembly via a polyketide
pathway involves intial construction of dihydromonacolin L 5.
The cooperation of polyketide synthases (PKSs) encoded by
the lovB and lovC genes (Fig. 1) accomplish the ca. 35 steps
necessary to generate 5 (Scheme 1).5,6 In the absence of the
LovC protein, which imparts enoyl reductase activity to the
complex, the LovB iterative type I PKS enzyme generates trun-
cated pyranones 6 and 7.5 During normal biosynthesis, oxid-
ative transformations of the PKS product 5 introduce a second
double bond in the decalin (decahydronaphthalene) system and
Preliminary studies using an A. terreus mutant with the lovC
gene disrupted demonstrated that cyclic nonaketide precursors
such as dihydromonacolin L 5 and monacolin J 9 could be
transformed to lovastatin 1 by this organism.9 We now report
full details of this work as well as transformations of other
cyclic nonaketides by this mutant and by an A. terreus mutant
having a disrupted lovA gene. The results suggest that gene
† The gene cluster for compactin 3 has recently been characterised
(see ref. 8b).
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 5 0 – 5 9
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
50

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Scheme 1 Biosynthesis of Lovastatin.
disruption can alter the profile of secondary metabolites in
triketide stage. However, expression of both the lovC and lovB
ways that cannot be easily predicted based solely on their
function in a biosynthetic pathway.
genes in A. nidulans results in complete biosynthesis of dihydro-
monacolin L 5, thereby indicating that LovC is responsible
for providing the required enoyl reductase function to the
LovB PKS system. The structure of 5 produced by these
cultures has been confirmed by spectroscopic techniques and
X-ray crystallography (Fig. 2).
Results and discussion
In order to probe the role of the lovA and lovC genes in the
biosynthetic pathway, two A. terrus mutants were examined in
which one of these genes was disrupted, thereby blocking the
production of lovastatin 1. In the first instance we employed an
A. terreus mutant with its lovC gene disrupted.‡ As mentioned
above, the lovC gene and the LovC protein produced by it have
previously been demonstrated to be necessary for the bio-
synthesis of dihydromonacolin L 5.5,6 It has also been shown
that heterologous expression of the lovastatin biosynthesis gene
lovB in Aspergillus nidulans results in production of the trun-
cated polyketide metabolites 6 and 7.5 These appear to arise
after failure to complete the proper enoyl reductase step at the
Biotransformations by A. terreus lovC mutant
Careful HPLC analysis of fermentation extracts of the A. terreus
mutant deficient in the lovC gene confirmed that it did not
produce any 5 or 1. However the entire post-PKS machinery
required to transform 5 into 1 would still be expected to be
intact and functional. In order to test this, a series of biotrans-
formation experiments were conducted. Initially, formation of
the diketide side chain (under the control of lovF gene) and its
esterification onto monacolin J 9 (under the control of lovD
gene) to afford 1 were examined. Monacolin J 9 was added
(EtOH solution) to fermentation cultures of A. terreus lovC
mutant in mid-log phase. Ten hours later, the culture broth was
extracted and examined for the presence of 1. HPLC analysis
‡ For a detailed description of the construction of A. terreus dis-
ruptants and A. nidulans transformants see the supporting information
for ref. 5.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 5 0 – 5 9
51

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Fig. 2 Crystal structure of 5 (top) and 21a (bottom).
revealed that about 70% of 9 had been converted into 1. This
demonstrates that the lovastatin diketide synthase (LDKS) is
present and fully functional, and unlike the lovastatin non-
aketide synthase (LNKS i.e. LovB), does not require LovC for
proper assembly of the diketide side chain.
Scheme 2 Syntheses of dihydromonacolin J 12 and dihydrolovastatin
14.
Compound 12 could be synthesised in 4 steps from 1 as
shown in Scheme 2. Basic hydrolysis of 1 gave 9, which could be
selectively protected with TBDMSCl in DMF to afford 10.
Selective hydrogenation of 10 using Ir(cod)py(Pcy3)PF6
catalyst11 in MeOH generated 11 in 86% yield. Removal of the
protecting group with buffered TBAF produced 12 in 34%
overall yield from 1. Intermediate 11 could also be acylated
with (S)-2-methylbutyric anhydride12 yielding 13 which, with
subsequent removal of the protecting group, affords the target
dihydrolovastatin 14 in 35% overall yield from 1.13 Compound
12 was added to cultures of the A. terreus lovC disruptant, but
formation of 14 could not be detected by extensive HPLC
analysis of the culture broth using synthetic 14 as a standard.
Dihydrolovastatin 14 has been previously isolated from the
fermentations of wild type A. terreus in 8% yield relative
to lovastatin 1, as determined by HPLC.14 This earlier study
in combination with the current results suggests that either
disruption of lovC unexpectedly alters the capability of the
organism to transform 12 to 14, or that this conversion does
occur, but with alteration of the metabolism which further
transforms 14 and brings its concentration below our detection
limit.
The specificity of the late stage enzymes was probed by exam-
ining whether 6-desmethylmonacolin J 15 could be transformed
into compactin 3 by the A. terreus lovC mutant. When 15
(obtained by the basic hydrolysis of 3) was added to fermen-
tation cultures of this mutant, 3 was isolated from the culture
broth in 45% yield after purification. This demonstrates that the
acyltransferase enzyme (LovD) can tolerate the lack of a
methyl group at the C-6 position of monacolin J 9. In a separate
experiment ML-236C, the 6-desmethyl analogue of monacolin
L 8, was added to cultures of the A. terreus lovC mutant. How-
ever, no compactin 3 could be detected by HPLC in the extract
of the culture broth. This result suggests that either the enzyme
responsible for hydroxylation at C-8 has a stringent require-
ment for the C-6 methyl group, or that 8 and ML-236C are not
intermediates on the biochemical route to 1 and 3, respectively.
The latter possibility may be more probable, as occurrence of 8
on the direct lovastatin biosynthetic pathway has been disputed
and is still uncertain.10
To help determine if the A. terreus lovC disruptant could be
used effectively to make dihydrolovastatin analogues, possible
biotransformation of dihydromonacolin J 12 was examined.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 5 0 – 5 9
52

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To verify that the post-PKS enzyme system required to oxid-
atively introduce the second double bond of the diene moiety
is intact in the A. terreus lovC mutant, dihydromonacolin L 5
(isolated from A. nidulans lovB ϩ lovC) was added to a fermen-
tation of this organism and the broth extract was examined by
HPLC for the presence of 1. Precursor 5 was transformed into 1
in 40% isolated yield, thereby demonstrating that all the neces-
sary post-PKS steps for lovastatin biosynthesis are present in
this mutant (Scheme 3).
Biotransformations by A. terreus lovA mutant
The transformation of 5 to 8 requires at least one oxidative
enzyme, and it seemed that the lovA gene product, whose
sequence is similar to cytochrome P450 enzymes, could be
involved in this process. In order to investigate this, the
biotransformations performed by an A. terreus mutant dis-
rupted at the lovA gene were examined. This organism would be
expected to accumulate dihydromonacolin L 5, as it has an
intact PKS system (i.e. LovB ϩ LovC enzymes) and it should
lack the ability to produce 1. HPLC analysis of cultures of
A. terreus lovA disruptant did not reveal the presence of any 1
(as expected), but 5 could not be detected either. This
unexpected result suggests that either the A. terreus lovA
mutant does not have the ability to produce 5, or that 5 is
somehow further transformed by the fungus.
In order to determine the fate of 5, 14C-dihydromonacolin L
(produced by addition of sodium 14C-acetate to A. nidulans
lovB ϩ lovC cultures), was fed to cultures of A. terreus lovA
mutant. The extract of the culture was fractioned by thin layer
chromatography, and the band containing most of the radio-
activity was isolated to yield a chromatographically homo-
geneous compound. Full structural assignment reveals that 20
is the major product of 5 in the A. terreus lovA disruptant. This
compound presumably arises as a result of two cycles of
β-oxidation,16 first yielding the octaketide 18 followed by the
heptaketide 19. This is then terminated by a P450 type hydroxy-
lation at C-6 to produce 20 (Scheme 5). This process for
Scheme 3 A. terreus lovC biotransformations.
The substrate specificity of the post-PKS machinery could be
further tested by attempted transformation of a dihydromon-
acolin L derivative 16 having an unsaturation in the upper ring
(Scheme 4). Elimination from the methanesulfonates of 5 or 1
Scheme 5 Proposed pathway to 20.
metabolising 5 does not appear to be unique to the A. terreus
lovA mutant, as 20 was also isolated from extracts of the cul-
tures of A. nidulans lovB ϩ lovC, along with a small amount of
the intermediate heptaketide 19. Careful examination of the
radioactive 5 obtained from this A. nidulans strain and used as
the precursor for the biotransformation confirmed that it was
not contaminated by 19 or 20. Since β-oxidation is ubiquitous
in fungi such as Aspergillus,16 and hydroxylation at C-6 is com-
mon in the metabolism of lovastatin 1,17 20 appears to arise
from a widespread metabolic pathway in Aspergillus which is
unrelated to lovastatin biosynthesis. Although this explains the
lack of dihydromonacolin L 5 in the A. terreus lovA mutant
cultures, it is perhaps surprising that blockage of a key oxid-
ative enzyme on the route to lovastatin 1 should shunt the
metabolism so completely.
Scheme 4 Synthesis of unsaturated analogues.
gave α,β-dehydrodihydromonacolin L 16 and α,β-dehydro-
lovastatin 17, respectively.15 Addition of 15 to cultures of the
A. terreus lovC disruptant followed after 10 hours by HPLC
analysis of the culture showed no detectable trace of 17,
suggesting that changes in the upper ring are not tolerated.
The results of successful biotransformation experiments with
A. terreus lovC mutant are summarised in Scheme 3.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 5 0 – 5 9
53

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Another well established mode of reaction of P450 enzymes
is the epoxidation of a double bond such as that present in 5.18
Hydroxylation with allylic rearrangement to directly form the
proposed intermediate, α-hydroxy-3,5-dihydromonacolin L19
(Scheme 1), would actually be an unexpected reaction for this
type of enzyme,20 which alternatively may be capable of
dehydrogenation of 5 directly to the diene system. In order to
determine if the LovA enzyme could form an epoxide during
biosynthesis of lovastatin, the two diastereomeric epoxides 21a
and 21b were prepared from 5 by treatment with MCPBA, fol-
lowed by separation of the stereoisomers by HPLC (Scheme 6).
To verify that the late stages of the post-PKS machinery are
still functional in the A. terreus lovA mutant, monacolin J 9
was added to cultures of this organism. Examination of the
extracts by HPLC allowed isolation of a trace amount (ca. 1%)
of lovastatin 1 along with an additional metabolite. The latter
was identified as 22. Similar to the fate of 5, compound 9 is
primarily metabolised by β-oxidation enzymes to a truncated
product. However in this case, the additional double bond in
the decalin system appears to hinder hydroxylation at C-6. The
biotransformations performed by A. terreus lovA mutant are
summarised in Scheme 7.
Scheme 7 Transformations by A. terreus lovA mutant.
Conclusions
Biotransformations of nonaketides accomplished by mutants
of A. terreus deficient in two lovastatin biosynthesis genes (lovA
or lovC) have been examined, and the structures of the resulting
metabolites were determined. The mutant disrupted at the lovC
gene lacks the ability to produce dihydromonacolin L 5 or lova-
statin 1, but still contains a highly functional set of post-PKS
enzymes. This is established by the ability of the A. terreus lovC
mutant to effectively transform either 5 or monacolin J 9 into 1.
In addition, this organism can transform 15 into 3, thereby
demonstrating that it can accept a slightly modified substrate.
This work suggests that it may be possible to use the A. terreus
lovC mutant to convert a variety of precursors into lovastatin
analogues. Studies to accomplish this are in progress. As differ-
ent substrate analogues may exhibit varying transport proper-
ties and diverse efficiency of transformation, cell-free studies
with purified enzymes will be critical for detailed understanding
of the biochemical conversions.
The mutant of A. terreus disrupted at the lovA gene displays
quite different characteristics. In contrast to the lovC mutant,
its β-oxidation system is highly active, with the consequence
that there is rapid degradation of exogenously-added non-
aketides which are known to be intermediates on the bio-
synthetic route to lovastatin 1. For example, only a small
amount of monacolin J 9, the immediate precursor of 1, is
converted into the target drug, with most of the added com-
pound being oxidatively degraded to 22. This lovA mutant also
does not accumulate dihydromonacolin L 5 as expected, but
rather metabolises it rapidly to 20. The LovA protein, although
resembling a P450 enzyme and clearly required for produc-
tion of lovastatin 1, may also be involved in generating com-
pounds which exercise control over expression or operation of
the β-oxidation system in A. terreus.
Scheme 6 Synthesis of 21a and 21b.
The stereochemical assignment of the two epoxides was con-
firmed by X-ray crystallography of the α-isomer (Fig. 2). In a
separate experiment, 14C labelled 5 was used in the synthesis
so that the transformation of 21a and 21b could be followed
by tracer analysis. Each 14C-labelled diastereomer was fed
to separate cultures of A. terreus lovA mutant, which were
subsequently analysed by thin layer chromatography with
radioisotopic scanning (radio-TLC). If an epoxide were the
product of the LovA enzyme and therefore a true intermediate
on the pathway to lovastatin, then either 21a or 21b should
presumably be converted through the rest of the post-PKS
pathway to yield 1. However, no radioactive 1 could be
detected by radio-TLC analysis. Analogous experiments
wherein 21a and 21b were fed separately to cultures of A.
terreus lovC mutant, yielded similar results: no radioactive 1
could be detected. In all cases, none of the original epoxide
could be detected or recovered. Control experiments show
that the epoxides are quite sensitive to the culture media, and
decompose to complex mixtures of polar compounds that are
not easily extracted into organic solvents. Treatment of 21a
with the methyl ester of glycine results in rapid nucleophilic
opening of the epoxide ring by the amino group (data not
shown). Hence, it is likely that these epoxides can react with
nucleophiles either in the media or in the fungal cells prior to
exposure to post-PKS enzymes in the lovastatin pathway.
Further experiments to determine the intermediacy of 21a or
21b will require purified enzymes.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 5 0 – 5 9
54

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flask. All biological media solutions and equipment were
autoclaved at 121 ЊC and 15 psi of steam pressure for 15 min.
Experimental
General Experimental
4a,5-Dihydromonacolin L 5
All chemicals were purchased from Aldrich Chemical company
(Madison, WI) or Sigma Chemicals (St. Louis, MO). All sol-
vents, unless otherwise indicated, were HPLC grade and used as
such. Dry CH2Cl2 was prepared by distilling under Ar from
CaH2. Diazomethane was prepared as a diethyl ether solution
from DIAZALD (N-methyl-N-nitrosotoluene-p-sulfonamide)
according to Aldrich technical bulletin AL-113. Evaporation
refers to removal of solvent under reduced pressure on a
rotary evaporator. Preparative TLC was done on glass plates
(20 × 20 cm) pre-coated (0.25, 0.5, 1.0 mm) with silica gel
(EM Science, Kieselgel 60 F254). Analytical TLC was done on
glass plates (5 × 1.5 cm) pre-coated (0.25 mm) with silica gel
(EM Science, Kieselgel 60 F254). Compounds were visualized
by exposure to UV light and by dipping the plates in a 1%
Ce(SO4)2ؒ4H2O 2.5% (NH4)Mo7O24ؒ4H2O in 10% H2SO4 fol-
lowed by heating on a hot plate. Flash column chromatography
was performed with Silicycle silica gel (60 Å, 230–400 mesh).
Preparative HPLC was performed on a RANIN Dynamax
high performance liquid chromatograph employing a manual
injector, fraction collector and single wavelength detector.
Reversed phase HPLC was conducted utilizing a Waters radial
compression module with a Bondapak C18 25 × 100 mm
column (15–20 µm particle size) with a guard column of
similar packing. The mobile phase for RP-HPLC typically con-
sisted of 100% H2O to 70% CH3CN–30% H2O over 25 min,
linear gradient. Normal phase HPLC was done on the same
system using a Waters radial compression Porasil silica gel
column (125 Å, 25 × 100 mm). The NP-HPLC mobile phase
employed was 30% EtOAc–70% hexane to 70% EtOAc–30%
hexane over 27 min.
NMR spectra were recorded on a Varian Inova 600, Inova
300 or Unity 500 spectrometer. For 1H (300, 500 or 600 MHz)
δ values are referenced to CHCl3 (7.24 ppm) and for 13C (125.8
or 150.9 MHz) referenced to CDCl3 (77.0 ppm).
Melting points are uncorrected and were determined on a
Kauffman Block microscope or a Thomas-Hoover apparatus
using open capillary tubes. Optical rotations were measured on
a Perkin-Elmer 241 polarimeter with a micro cell (100 mm
path length, 1 mL). IR spectra were recorded on a Nicolet
Magna-IR 750 with a Nic-Plan microscope FT-IR spectro-
meter. Mass spectra were recorded on a Krator AEI MS–50
(HREIMS) and a ZabSpec Isomass VG (HRESMS).
Radioactivity was determined using standard liquid scintil-
lation procedures in plastic 10 ml scintillation vials with a
Beckman Ready-gel scintillation cocktail. The scintillation
counter used was a Beckman LS 5000TD with automatic
quench control to directly determine decompositions per min-
ute (dpm) in the labelled samples against a quench curve pre-
pared from Beckman 14C quenched standards. Radiolabelled
compounds were also detected by a TLC assay, using a
Berthold LB2760 2D-TLC scanner.
4a,5-Dihydromonacolin L 5 was isolated from a strain of
Aspergillus nidulans expressing the lovastatin lovB and lovC
biosynthesis genes following the cloning procedure described
elsewhere.5 The typical isolation procedure is described below.
10 µL of a concentrated spore suspension was used to inoculate
1 L of A. nidulans growth media and the culture was incubated
at 30 ЊC and 200 rpm for 4 days. The mycelia were harvested by
filtering through sterile miracloth (Calbiochem) and were thor-
oughly rinsed with 2 L of 1% lactose solution. The wet mycelia
were divided into 4 equal portions and used to inoculate 4 × 1 L
of A. nidulans production media. Cultures were incubated at 30
ЊC and 200 rpm for 2 days, after which point the growth was
supplemented with 1 g LϪ1 sodium acetate added each day for
an additional 5 days. The cultures were harvested by removing
the mycelia by vacuum filtration and the combined broth (4 L)
was acidified with 2 M HCl (pH < 2) and extracted with CH2Cl2
(2 × 1.5 L). The combined organic layers were dried (Na2SO4),
filtered and evaporated to provide ca. 500 mg of crude extract
which was fractionated by flash column chromatography to
provide 300 mg of pure 4a,5-dihydromonacolin L 5 (typical
yields, 50–70 mg LϪ1 of production media).
14C-4a,5-Dihydromonacolin L 5 was produced from 1 L of A.
nidulans production culture fermented as described above,
except that 250 µCi of sodium [1-14C]acetate (fed as a 5 mL
aqueous solution 4 times per day) was added in addition to the
unlabelled sodium acetate. The culture was harvested and
extracted as above and the crude extract (97 mg) was fraction-
ated by preparative TLC to give 22.5 mg of pure 14C-4a,5-
dihydromonacolin L 5 with a specific activity of 0.185 µCi
mgϪ1. The 1H NMR of the 14C-4a,5-dihydromonacolin L 5 was
identical to that of the unlabelled compound.
For unlabelled 5: mp 162–163ЊC, lit. mp 163–164 ЊC;21 [α]2D5
=
ϩ 115 (c 0.22, methanol), lit. [α]2D5 = ϩ 123.9 (c 0.5, methanol);
UV (MeCN solution) λmax 228 nm (br 210–234); IR (micro-
scope) 3391 (br m), 3018 (w), 2910 (s), 1713 (s), 1254 (s), 1062
(s), 1047 (s) cmϪ1; HREIMS 306.21902 (306.21948 calcd. for
C19H30O3) (Mϩ, 27%), 288.20876 (M Ϫ H2O, 18%), 161.13299
(M Ϫ C7H13O3, 100%), 105.07044 (C8H9ϩ, 99%). 1H NMR (600
MHz, CDCl3) δ 5.57 (ddd, 1H, J = 9.8, 4.9, 2.7 Hz, H-3), 5.28
(d, 1H, J = 9.8 Hz, H-4), 4.67 (m, 1H, H-11), 4.38 (m, 1H,
H-13), 2.72 (dd, 1H, J = 17.6, 5.13 Hz, H-14), 2.60 (ddd, 1H,
J = 17.6, 3.7, 1.6 Hz, H-14), 2.21 (m, 1H, H-2), 2.00 (m, 1H,
H-6), 1.95 (m, 1H, H-12), 1.89 (m, 1H, H-4a), 1.81–1.73 (m,
2H, H-10 and H-12), 1.62 (m, 2H, H-9), 1.58–1.42 (m, 5H, H-5,
H-7, H-8a and H-10), 1.07 (dq, 1H, J = 11.9, 3.7 Hz, H-8), 0.97
(dq, 1H, J = 11.9, 3.0 Hz, H-8), 0.96 (d, 3H, J = 7.3 Hz, 6-CH3),
0.82 (d, 3H, J = 6.9 Hz, 2-CH3); 13C NMR (125 MHz, CDCl3)
δ 170.4 (C-15), 132.6 (C-3), 131.6 (C-4), 76.1 (C-11), 62.8 (C-
13), 41.4 (C-8a), 40.0 (C-8), 38.9 (C-5), 38.7 (C-14), 37.3 (C-4a),
36.1 (C-12), 33.2 (C-10), 32.3 (C-7), 32.0 (C-2), 27.5 (C-6), 23.7
(C-1), 23.6 (C-9), 18.2 (6-CH3), 14.9 (2-CH3).
Aspergillus nidulans growth media was prepared by dissolv-
ing 20 g of glucose, 20 g of yeast extract, 1 g of peptone and 1
mL of a 1 mg per mL solution of p-aminobenzoic acid in 1 L of
distilled deionized water. Aspergillus nidulans production media
was prepared by dissolving in 1 L of distilled deionized water
1 mL trace element solution (1.0 g FeSO4ؒ7H2O, 8.8 g
ZnSO4ؒ7H2O, 0.4 g CuSO4ؒ5H2O, 0.15 g MnSO4ؒ4H2O, 0.1 g
Na2B7O7ؒ10H2O, 0.05 g (NH4)6Mo7O24ؒ4H2O, 0.5 mL conc.
HCl dissolved in 1 L H2O), 100 mL of 10 × AMM (Aspergillus
Minimal Media) salts (60 g NaNO3, 5.2 g KCl, 15.2 g KH2PO4
dissolved in 1 L of H2O and adjusted to pH 6.5), 1 mL of a
1 mg per mL p-aminobenzoic acid solution, and 0.9 mL of
cyclopentanone and autoclaving. After cooling to room tem-
perature 2.5 mL of a sterile solution of 20% MgSO4ؒ7H2O and
25 mL of a sterile 40% lactose solution was added to the above
Crystal data for 4a,5-dihydromonacolin L 5
Data were acquired on a Bruker P4/RA/SMART 1000 CCD
diffractometer. All intensity measurements were performed
using graphite monochromated Mo-Kα radiation (λ = 0.71073
Å). 4a,5-Dihydromonacolin L 5 (C19H30O3) was obtained as
white crystals, space group P212121 (No. 19), a = 5.5947 (9),
b = 9.6504 (17), c = 31.930 (6) Å, V = 1723.9 (5) Å3, Z = 4,
T = Ϫ80 ЊC, ρcalcd = 1.181 g cmϪ3, µ = 0.078 mmϪ1. 10870 reflec-
tions were measured, 3287 unique which were used in all least
2
squares calculations, R1(F) = 0.0620 (for 899 reflections with Fo
2
≥ 2σ(Fo )), wR2(F 2) = 0.2159 for all unique reflections. The abso-
lute stereochemistry cannot be directly determined from the
X-ray data, but it is correct as shown based on transformation
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 5 0 – 5 9
55

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of 5 to 1. CCDC reference number 191453. See http://
www.rsc.org/suppdata/ob/b2/b207721c/ for crystallographic
files in CIF or other electronic format.
according to the procedure of Stork and Kahne.22 The selective
reduction of tert-butyldimethylsilyloxymonacolin J 10 was
achieved by modification of the procedure of DeCamp et al.11
To a solution of tert-butyldimethylsilyloxymonacolin J 10
(0.103 g, 0.24 mmol) in CH2Cl2 (1.7 mL) was added MeOH
(0.1 mL) and the Ir complex (2 mg, 2.5 µmol). The mixture was
hydrogenated for 10 min. Analytical TLC displayed the pres-
ence of starting material, therefore more Ir complex (2 mg,
2.5 µmol) was added and the mixture hydrogenated for an addi-
tional 10 min. This process was repeated until no starting
material remained by TLC (a total of 8 mg Ir complex was
added). The mixture was passed through a pad of Fluorosil to
remove the catalyst and the solvent was evaporated under
reducedpressuretogive4a,5-dihydro(tert-butyldimethylsilyloxy)-
monacolin J 11 (105 mg, quant.) as a mixture (86 : 14) of the
desired olefin and over-reduced tetrahydro material. The tetra-
hydro product was removed by flash column chromatography
(50% EtOAc in hexanes) to give pure 4a,5-dihydro(tert-butyl-
dimethylsilyloxymonacolin J 11.
Monacolin J 9
Monacolin J 9 was produced by the basic hydrolysis of
lovastatin as follows. 200 mg of lovastatin 1 was placed in a
25 mL round bottomed flask and suspended in 10 mL of aque-
ous 1 M LiOH and heated at reflux overnight. The reaction was
cooled to room temperature, acidified with 2 M HCl (pH < 2)
and extracted with CH2Cl2 (3 × 10 mL). The combined organic
extracts were dried (Na2SO4), filtered and evaporated to give
243 mg crude residue which was dissolved in 25 mL toluene and
lactonized by heating at reflux with a CaH2 Soxhlet apparatus
for one hour. After cooling to room temperature the solvent
was evaporated and the residue (208 mg) was fractionated by
flash column chromatography (40% EtOAc in hexanes) to give
140 mg (89% yield) pure monacolin J 9.
[α]2D5 = ϩ17.3 (c 0.14, CH3OH); IR (microscope) 3236 (br s),
2927 (s), 2879 (s), 1707 (m), 1645 (m), 1451 (s), 1318 (s), 1093
[α]2D5 = ϩ76 (c 1.20, CH2Cl2); FTIR (cast) 3463 (br), 1733 (s),
1253 (s) cmϪ1; HREIMS [M]ϩ 436.30047 (436.30090 calcd. for
C25H44O4Si) 436 (1%), 418 (6%), 361 (28%), 343 (8%) and 75
(100%); 1H NMR (300 MHz, CDCl3) δ 5.61 (ddd, 1H, J = 9.7,
4.9, 2.6 Hz), 5.36 (d, 1H, J = 9.8 Hz), 4.66 (m, 1H,), 4.27 (m,
1H), 4.16 (m, 1H), 2.59 (dd, 1H, J = 17.5, 4.3 Hz), 2.56 (ddd,
1H, J = 17.5, 3.5, 1.4 Hz), 2.52–1.01 (m, 20H), 1.19 (d, 1H,
J = 7.4 Hz), 0.87 (m, 9H), 0.81 (d, 3H, J = 7.1 Hz), 0.06 (3 H, s)
and 0.05 (3 H, s); 13C NMR (125 MHz, CDCl3) δ 174.0, 170.3,
132.6, 131.0, 76.3, 69.8, 63.6, 41.8, 41.7, 39.3, 38.6, 37.6, 36.8,
35.6, 33.2, 31.3, 30.9, 26.7, 26.7, 25.7, 23.1, 21.0, 16.5, 14.9,
11.7, Ϫ4.8, Ϫ4.9.
(s), 1075 (s), 1060 (s), 1049 (s), 1026 (s), 973 (s), 858 (s) cmϪ1
;
HREIMS [M]ϩ 320.19827 (320.19876 calcd. for C19H28O4)
320.2 (2%), 302.2 (9%), 198.1 (58%), 157.1 (100%), 105.1 (41%);
1H NMR (500 MHz, CD3OD) δ 5.93 (d, 1H, J = 10.0 Hz, H-4),
5.75 (dd, 1H, J = 10.0, 6.2 Hz, H-3), 5.45 (br s, 1H, H-5), 4.23
(br dd, 1H, J = 6.5, 2.8 Hz, H-8), 9.92 (m, 1H, H-11), 3.79 (m,
1H, H-11), 3.79 (m, 1H, H-11), 3.79 (m, 1H, H-13), 3.68 (br t,
2H, J = 6.5 Hz, H-14), 2.38 (m, 2H, H-2, H-6), 2.13 (br dd, 1H,
J = 12.1, 2.8 Hz, H-13), 1.92–1.68 (m, 4H, H-7, Ha-9, Ha-12),
1.67–1.50 (m, 3H, H-1, Ha-10, Hb-12), 1.41 (m, 1H, Hb-10), 1.31
(m, 1H, H-9), 1.18 (d, 3H, J = 7.4 Hz, 6-Me), 0.89 (d, 3H,
J = 6.9 Hz, 2-Me); 13C NMR (125 MHz, CD3OD) δ 190.0
(C-15), 134.1 (C-4), 133.2 (C-4a), 130.6 (C-3) 130.0 (C-5), 71.7
(C-8), 69.2 (C-11), 65. 9 (C-13), 60.1 (C-14), 45.4 (C-7), 41.0
(C-10), 39.8 (C-8a), 37.6 (C-1), 37.1 (C-12), 35.6 (C-9), 32.1,
29.1 (C-2, C-6), 23.65 (6-Me), 14.3 (2-Me).
4a,5-Dihydromonacolin J 12
A sample of 30.0 mg (0.071 mmol) 4a,5-dihydro(tert-butyldi-
methylsiloxy)monacolin J 11 was dissolved in 4 mL THF in a 10
mL round bottomed flask and cooled to 0 ЊC. 20 µL (0.355
mmol, 5 eq.) glacial acetic acid was added, followed 10 min later
by 100 µL of a 1 M solution of tetrabutylammonium flouride
(TBAF) in THF (0.100 mmol, 1.5 eq.). The reaction was stirred
for 2 h at 0 ЊC, then warmed to room temp. and stirred for an
additional 2 h. Analytical TLC displayed mostly starting
material so an additional 100 µL of 1 M TBAF in THF was
added and the reaction stirred at room temp. overnight. The
solvent was evaporated and the residue was dissolved in 20 mL
CH2Cl2 and extracted with 0.4 M CaCl2 (1 × 10 mL), washed
with H2O (3 × 10 mL), dried (Na2SO4), filtered and evaporated
to give 20.0 mg crude product. 4a,5-Dihydromonacolin J 12 was
purified by flash column chromatography over silica gel (1–5%
i-PrOH in CH2Cl2) to yield 13.6 mg (60% yield) pure product.
[α]2D5 = ϩ137.4 (c 0.38, MeOH); FTIR (cast), 3361 (br), 2906
(s), 1703 (s), 1259 (s), 1039 (s) cmϪ1; HREIMS [M]ϩ 322.21394
(322.2144 calcd. for C19H30O4) 322.2 (1%), 286.2 (10%), 200.1
(18%), 159.1 (79%), 105.1 (100%); 1H NMR (600 MHz, CDCl3)
δ 5.60 (ddd, 1H, J = 9.7, 4.8, 2.6 Hz), 5.35 (d, 1H, J = 10.1 Hz),
4.68 (m, 1H), 4.36 (quintet, 1H, J = 3.7 Hz), 4.14 (d, 1H, J = 2.8
Hz), 2.71 (dd, 1H, J = 18.0, 5.1 Hz), 2.60 (ddd, 1H, J = 18.0, 3.8,
1.8 Hz), 2.01–1.90 (m, 3H), 1.82–1.20 (m, 16 H), 1.18 (d, 3H,
J = 7.5 Hz), 0.83 (d, 3H, J = 7.0 Hz); 13C NMR (150 MHz,
CDCl3) δ 170.4, 132.3, 131.5, 76.1, 67.1, 62.8, 42.7, 39.3, 39.0,
38.6, 37.0, 36.3, 32.8, 31.4, 29.7, 27.0, 23.0, 21.6, 15.0.
tert-Butyldimethylsilyloxymonacolin J 10
tert-Butyldimethylsilyloxymonacolin
J
10 was prepared
according to the procedure of Willard and Smith.12 100 mg
(0.312 mmol) monacolin J and 106.2 mg (1.56 mmol, 5 eq.)
imidazole were placed in a 10 mL round bottomed flask and
dissolved in 4 mL dimethylformamide (DMF). To this was
added 117 mg (0.78 mmol, 2.5 eq.) tert-butyldimethylsilyl chlor-
ide as a solution in 1mL DMF. The reaction was stirred at room
temperature for 18 hours, then diluted with 30 mL CH2Cl2 and
extracted with 0.2 M HCl (1 × 10 mL). The organic layer was
washed with H2O (3 × 10 mL), dried (Na2SO4), filtered and
evaporated to give 197.5 mg crude residue. The product
was purified by flash column chromatography (20% EtOAc in
hexanes) to give 100 mg (74% yield) tert-butyldimethylsilyl-
oxymonacolin J 10.
[α]2D5 = Ϫ10.1 (c 1.35, CH2Cl2); FTIR (cast) 3446 (br), 2954
(s), 1734 (s), 1254 (s), 1082 (s), 735(s) cmϪ1; HREIMS [M]ϩ
434.28466 (434.28525 calcd. for C25H42O4Si), 434.3 (7%), 359.2
1
(17%), 284.2 (24%), 159.1 (100%), 101.0 (59%); H NMR (600
MHz, CDCl3) δ 5.95 (d, 1H, J = 9.5 Hz), 5.77 (dd, 1H, J = 6, 9.5
Hz), 5.52 (s, 1H), 4.65 (m, 1H), 4.27 (q, 1H, J = 3.5 Hz), 4.21
(s, 1H), 2.58 (dd, 1H, J = 17.5, 4 Hz), 2.53 (ddd, 1H, J = 17.5, 3,
1.5 Hz), 2.42 (m, 1H), 2.35 (s, 1H, J = 6.5), 2.14 (dd, 1H, J = 12,
2.5 Hz), 1.91–1.72 (m, 6H), 1.69 (ddd, 1H, J = 15, 3 Hz), 1.45
(m, 2H), 1.17 (d, 3H, J = 7.5 Hz), 0.88 (d, 3H, J = 7 Hz), 0.86 (s,
9H), 0.06 (s, 3H), 0.05 (s, 3H); 13C NMR (150 MHz, CDCl3) δ
170.5, 133.7, 131.3, 130.0, 128.4, 76.5, 65.3, 63.5, 39.3, 38.8,
36.9, 36.5, 35.8, 33.0, 30.8, 27.4, 25.7, 24.3, 23.8, 18.0, 14.0,
Ϫ4.8, Ϫ4.9.
4a,5-Dihydro(tert-butyldimethylsilyloxy)lovastatin 13
A sample of 14.0 mg (0.032 mmol) 4a,5-dihydro(tert-butyl-
dimethylsilyloxy)monacolin J 11 and 5 mg (0.041 mmol)
4-dimethylaminopyridine were placed in a 1 mL React-vial and
dissolved in 0.5 mL pyridine. To this mixture was added 100 µL
(93.4 mg, 0.50 mmol) (S)-2-methylbutyric anhydride and the
reaction was stirred at room temperature for 72 hours. The
reaction was diluted with 30 mL CH2Cl2 and washed with 1 M
4a,5-Dihydro(tert-butyldimethylsilyloxy)monacolin J 11
The iridium complex Ir(cod)py(Pcy3)PF6 was synthesized
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 5 0 – 5 9
56

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HCl (1 × 10 mL). The organic layer was washed with H2O
(3 × 10 mL), dried (Na2SO4), filtered and evaporated to give
ca. 50 mg of crude residue which was purified by flash column
chromatography to give 11.9 mg (72% yield) of pure 4a,5-di-
hydro(tert-butyldimethylsilyloxy)lovastatin 13.
EtOAc, 1 : 1) to give 10 mg (60%) pure α,β-dehydro-4a,5-di-
hydromonacolin L 16.
[α]2D5 = ϩ8.6 (c 1.00, CH2Cl2); FTIR (cast) 2915 (s), 1717 (s),
1389 (s), 1248 (s), 818 (s) cmϪ1; HREIMS [M]ϩ 288.20866
(288.20892 calcd. for C19H28O2) (19%), 273.2 (17%), 228.2
(22%), 176.1 (85%), 161.1 (71%), 105.1 (100%); 1H NMR
(600 MHz, CDCl3) δ 6.85 (ddd, 1H, J = 9.5, 9.5, 4.4 Hz), 6.00
(d, 1H, J = 9.8 Hz), 5.57 (ddd, 1H, J = 9.7, 4.7, 2.8 Hz), 5.28 (d,
1H, J = 9.8 Hz), 4.41 (dddd, 1H, J = 12.3, 12.3, 7.6, 4.7 Hz), 2.33
(m, 2H), 2.21 (m, 1H), 2.11 (m, 1H), 1.91–1.87 (m, 2H), 1.62–
1.41 (m, 8H), 1.34–1.22 (m, 2H), 1.08 (m, 1H), 0.99 (dd, 1H,
J = 10.3, 2.6 Hz), 0.96 (d, 3H, J = 7.3 Hz), 0.83 (d, 3H, J = 7.0);
13C NMR (150 MHz, CDCl3) δ 164.3, 144.8, 132.5, 131.6,
121.3, 78.0, 41.1, 39.6, 38.3, 38.2, 37.0, 32.0, 31.8, 31.5, 29.0,
27.1, 23.2, 17.7, 14.6.
[α]2D5 = ϩ73 (c 0.20, CHCl3); FTIR (cast) 2295 (s), 1727 (s),
1
1253 (s), 1079 (s) cmϪ1; H NMR (600 MHz, CDCl3) δ 5.62
(ddd, 1H, J = 9.5, 4.5, 2.5 Hz), 5.36 (d, 1H, J = 9.5 Hz), 5.15 (d,
1H, J = 2.5 Hz), 4.55 (m, 1H), 4.25 (quintet, 1H, J = 3.5 Hz),
2.57 (dd, 1H, J = 17, 4.5 Hz), 2.52 (dd, 1H, J = 17, 1 Hz), 2.45
(m, 1H), 2.41 (m, 1H), 2.32 (q, 1H, J = 7 Hz), 2.27 (m, 1H), 2.02
(m, 1H), 1.89 (d, 1H, J = 15 Hz), 1.83–1.80 (m, 2H), 1.72–1.44
(m, 5H), 1.32–1.22 (m, 2H), 1.17 (d, 2H, J = 7 Hz), 1.10 (d, 3H,
J = 7 Hz), 1.07 (d, 3H, J = 7.5 Hz), 0.93 (t, 3H, J = 7.5 Hz), 0.86
(s, 9H), 0.05 (s, 3H), 0.04 (s, 3H); 13C NMR (125 MHz, CDCl3)
δ 175.0, 170.1, 132.6, 131.0, 76.3, 69.8, 63.6, 41.8, 41.7, 39.3,
38.6, 37.6, 36.8, 35.6, 33.2, 31.3, 30.9, 26.8, 26.7, 26.6, 25.6,
23.1, 21.0, 17.9, 16.5, 16.4, 14.9, 11.7, Ϫ4.8, Ϫ4.9.
ꢀ,ꢁ-Dehydrolovastatin 17
33.0 mg (0.082 mmol) lovastatin 1 was placed in a 25 mL round
bottomed flask and dissolved in 3 mL CH2Cl2. To this solution
was added 34.7 µL (25 mg, 0.249 mmol, 3 eq.) triethylamine,
followed by 9.6 µL (14 mg, 0.123 mmol, 1.5 eq.) methane-
sulfonylchlorideandthereactionwasstirredatroomtemp.forone
hour. The reaction was diluted with CH2Cl2 and extracted with
0.1 M HCl (1 × 10 mL) and satd. NaHCO3 (1 × 10mL), washed
with H2O (3 × 10 mL), dried (Na2SO4) and filtered to give 40.4
mg of crude residue which was fractionated by flash column
chromatography to give 33.7 mg pure α,β-dehydrolovastatin 17.
[α]2D5 = ϩ136.4 (c 3.30, CH2Cl2); FTIR (cast) 2963 (s), 1723 (s),
1383 (s), 1247 (s), 818 (s) cmϪ1; HREIMS [M]ϩ 386.24530
(386.24570 calcd. for C24H34O4) 386.2 (2%), 284.2 (17%), 198.1
(64%), 159.1 (100%); 1H NMR (600 MHz, CDCl3) δ 6.83 (ddd,
1H, J = 9.8, 6.1, 2.4 Hz), 5.98 (ddd, 1H, J = 9.7, 2.6, 0.9 Hz),
5.97 (d, 1H, J = 10.5 Hz), 5.76 (dd, 1H, J = 9.5, 6.1 Hz), 5.50 (m,
1H), 5.36 (d, 1H, J = 3.2 Hz), 4.31 (dddd, 1H, J = 11.5, 11.5, 5.3,
3.5 Hz), 2.42 (m, 1H), 2.37–2.30 (m, 3H), 2.25–2.19 (m, 2H),
1.96–1.87 (m, 3H), 1.69–1.59 (m, 3H), 1.51–1.28 (m, 4H), 1.08
(d, 3H, J = 7.0 Hz), 1.05 (d, 3H, J = 7.4 Hz), 0.87 (d, 3H, J = 7.0
Hz), 0.85 (t, 3H, J = 7.5 Hz); 13C NMR (125 MHz, CDCl3)
δ 176.6, 164.2, 144.7, 133.0, 131.6, 129.7, 128.3, 121.5, 78.5,
67.7, 41.4, 37.3, 36.6, 32.7, 32.4, 30.7, 29.5, 27.4, 26.8, 24.2,
22.8, 16.2, 13.8, 11.7.
4a,5-Dihydrolovastatin 14
7.7 mg (0.0148 mmol) of 13 was dissolved in 1 mL of 10% HCl
in MeOH, and stirred at room temp. for 1 hour. The reaction
was diluted with 30 mL CH2Cl2 and extracted with NaHCO3
(3 × 10 mL). The organic layer was washed with H2O
(3 × 10 mL), dried and evaporated to give 5.0 mg of 14.
1H NMR (300 MHz, CDCl3) δ 5.62 (m, 1H), 5.35 (d, 1H,
J = 9.6 Hz), 5.17 (m, 1H), 4.55 (m, 1H), 4.35 (m, 1H), 2.74 (d,
1H, J = 5.0 Hz), 2.68 (d, 1H, J = 5.0 Hz), 2.61 (dd, 1H, J = 3.8,
1.5 Hz), 2.52 (dd, 1H, J = 4.2, 1.5 Hz), 2.43 (d, 1H, J = 6.0 Hz),
2.4–2.2 (m, 2H), 2.05–1.75 (m, 6H), 1.70–1.40 (m, 10H), 1.15
(d, 3H, J = 7.0 Hz), 1.08 (d, 3H, J = 7.4 Hz), 0.88 (t, 3H, J = 8
Hz), 0.82 (d, 3H, J = 7.0 Hz).
6-Desmethylmonacolin J 15
A solution of compactin 3 (55 mg, 0.13 mmol) and LiOH
(60 mg, 1.3 mmol) in H2O (15 mL) was heated at reflux for one
day. The reaction mixture was cooled in an ice bath, acidified
with 2 M HCl (pH < 1) and extracted with EtOAc (5 × 30 mL)
and CHCl3 (4 × 20 mL). The combined organic extracts were
dried (Na2SO4), filtered and evaporated. The resulting resi-
due was taken up in toluene (60 mL) and heated at reflux
in a Sohxlet containing CaH2 for one hour. After cooling the
toluene was evaporated and the residue fractionated by flash
column chromatography (EtOAc, 100%) to yield 30 mg of 15 as
a colorless oil (75%).
Heptaketide 19
Heptaketide 19 was isolated from cultures of A. nidulans lovb ϩ
lovC as follows. 4 L of A. nidulans lovB ϩ lovC production
cultures, fermented as described above for the isolation of 5,
were extracted with CH2Cl2 (2 × 1.5 L) WITHOUT acidifi-
cationoftheculturebroth. Theorganiclayerwasdried(Na2SO4),
filtered and evaporated to give ca. 50 mg of crude residue. Puri-
fication by flash column chromatography (EtOAc 100%) gave
6.5 mg of pure 19.
[α]2D5 = ϩ78 (c 0.15, CHCl3); FTIR (cast) 3407 (br s), 2928 (s),
1710 (s), 1256 (s), 1075 (s), 754 (s); HREIMS [M]ϩ 306.18245
1
(306.18311 calcd. for C18H26O4); H NMR (500 MHz, CDCl3)
δ 5.92 (d, 1H, J = 9.6 Hz), 5.71 (dd, 1H, J = 9.6, 6.0 Hz), 5.52 (br
d, 1H, J = 2.1 Hz), 4.69 (m, 1H), 4.52 (m, 1H), 4.21 (br s, 1H),
2.67 (dd, 1H, J = 17.7, 5.0 Hz), 2.59 (ddd, 1H, J = 17.7,
3.7, 1.7 Hz), 2.31 (m, 2H), 2.14 (m, 2H), 1.96 (m, 2H), 1.82–
1.62 (m, 5H), 1.53–1.39 (m, 2H), 0.88 (d, 3H, J = 7.0 Hz); 13C
NMR (75 MHZ, CDCl3) δ 170.8, 133.3, 133.0, 128.3, 123.6,
76.2, 64.4, 62.6, 38.8, 38.5, 36.4, 36.1, 32.6, 30.8, 29.1, 23.8,
20.3, 13.9.
[α]2D0 ϩ120.3 (c 0.10, CHCl3); FTIR (cast) 3020, 2952, 2915,
2860, 1696, 1445, 1411, 1377, 1310, 1297, 1267, 1238, 1211, 720
cmϪ1; HREIMS [M]ϩ 236.1777 (236.1776 calcd. for C15H24O2);
1H NMR (500 MHz, CDCl3) δ 5.57 (ddd, 1H, J = 10.0, 5.0, 3.0
Hz), 5.28 (d, 1H, J = 10.0 Hz), 2.42 (ddd, 1H, J = 15.5, 10.0, 5.0
Hz), 2.26–2.19 (m, 2H), 2.00 (m, 1H), 1.96–1.97 (m, 2H), 1.60–
1.44 (m, 5H), 1.36 (m, 1H), 1.25 (dt, 1H, J = 13.0, 4.5 Hz), 1.10
(dq, 1H, J = 12.0, 4.0 Hz), 1.02–0.94 (m, 1H), 0.96 (d, 3H,
J = 7.5 Hz), 0.82 (d, 3H, J = 7.0 Hz); 13C NMR (125 MHz,
CDCl3) δ 179.9, 132.3, 131.5, 41.0, 39.9, 38.9, 37.3, 32.3, 32.0,
31.9, 27.5, 23.8, 23.6, 18.2, 15.0.
ꢀ,ꢁ-Dehydro-4a,5-dihydromonacolin L 16
A sample of 18.4 mg (0.060 mmol) 4a,5-dihydromonacolin L 5
was dissolved in 0.5 mL CH2Cl2. To this solution was added
7.1 µL (10.3 mg, 0.90 mmol, 1.5 eq.), methanesulfonyl chloride
and 2.5 µL (18.2 mg, 0.18 mmol, 3 eq.) triethylamine and
the reaction was stirred at room temp. 1 hour. The reaction
mixture was diluted with 30 mL CH2Cl2 and extracted with 0.2
M HCl (1 × 10 mL) and satd. NaHCO3 (1 × 10 mL). The
organic layer was washed with H2O (3 × 10 mL), dried
(Na2SO4), filtered and evaporated to give 21.0 mg crude prod-
uct. The product was fractionated by preparative TLC (hexane–
ꢁ-Oxidation product 20
β-Oxidation product 20, was isolated from a biotransformation
experiment as follows. 1 L of A. nidulans growth media was
inoculated with 10 mL of a concentrated suspension of
Aspergillus terreus lovA spores and incubated at 30 ЊC and 200
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 5 0 – 5 9
57

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rpm for 2 days. To the growing culture was added a solution of
unlabelled 4a,5-dihydromonaoclin L (30 mg) and 52.5 nCi
(ca. 0.5 mg) of 14C-4a,5-dihydromoancolin L in 6 mL EtOH.
0.5 mL of this dihydromonacolin L solution was added to the
cultures three times a day until completely fed. After seven days
total growth the mycelia were removed by vacuum filtration, the
broth acidified with 2 M HCl (pH < 2) and extracted with
CH2Cl2 (1 × 1 L, 1 × 0.5 L). The combined organic layers were
dried (Na2SO4), filtered and evaporated to give 207.2 mg crude
extract. The crude extract was fractionated by preparative TLC
to give a fraction (26.3 mg) containing the majority of the
radioactivity. A portion of this radioactive fraction was treated
with CH2N2 (Et2O solution), and fractionated by preparative
TLC to isolate the β-oxidation product as the major radioactive
component (1.7 mg, 3.4 nCi).
This same product could be isolated from the CH2Cl2 extract
of A. nidulans lovB ϩ lovC production cultures grown as
described above. A portion (23.2 mg) of the crude CH2Cl2
extract was treated with CH2N2 (Et2O solution) to give 26.1 mg
crude methylated product which was fractionated by HPLC to
give 2.2 mg pure β-oxidation product 20.
CDCl3) δ 170.2, 75.8, 62.8, 58.2, 57.0, 41.5, 38.6, 37.6, 35.9,
35.6, 34.6, 33.2, 31.7, 28.9, 26.9, 23.2, 22.7, 18.0, 9.5.
Crystal data for 4a,5-dihydromonacolin L ꢀ-epoxide 21a
Data were acquired on a Bruker P4/RA/SMART 1000 CCD
diffractometer. All intensity measurements were performed
using graphite monochromated Mo-Kα radiation (λ = 0.71073
Å). 4a,5-Dihydromonacolin L α-epoxide 20a (C19H30O4) was
obtained as white crystals, space group P212121 (No. 19), a =
5.5318 (11), b = 9.7123 (19), c = 32.163 (7) Å, V = 1728.0 (6) Å3,
Z = 4, T = Ϫ80 ЊC, ρcalcd = 1.239 g cmϪ3, µ = 0.085 mmϪ1. 9765
reflections measured, 3482 unique which were used in all least
squares calculations, R1(F) = 0.0538 (for 2478 reflections with
2
2
Fo ≥ 2σ(Fo )), wR2(F 2) = 0.1044 for all unique reflections. The
absolute stereochemistry of 21a cannot be directly determined
from the data, but it is correct as shown based on conversion of
5 to 1 and to 21a. CCDC reference number 191454. See http://
www.rsc.org/suppdata/ob/b2/b207721c/ for crystallographic
files in CIF or other electronic format.
ꢁ-Oxidation product 22
FTIR (cast) 3297 (br), 3008 (s), 1737 (s), 1437 (m), 1223 (s),
1170 (s) cmϪ1; HREIMS 248.17729 (248.1763 calcd. for
C16H24O2) [M Ϫ H2O]ϩ, 217.1 (18%), 192.1 (14%), 161.1
(100%), 119.1 (52%), 105.1 (78%); 1H NMR (600 MHz, CDCl3)
δ 5.58 (ddd, 1H, J = 9.5, 4.5, 2.5 Hz, H-3), 5.30 (d, 1H, J = 9.5
Hz, H-4), 3.65, (s, 3H, OCH3), 2.37 (ddd, 1H, J = 15, 10, 5 Hz,
H-10a/b), 2.24 (m, 1H, H-2), 2.18 (ddd, 1H, J = 15, 9.5, 6.5 Hz,
H-10a/b), 1.89 (m, 2H, H-9), 1.80, 1.78 (d, 1H, J = 2.5, H-4a),
1.69, 1.67, 1.47, 1.23 (s, 3H, 6-CH3), 1.02, 0.83 (d, 3H, J = 7 Hz,
2-CH3); 13C NMR (125 MHz, CDCl3) δ 171.3, 132.9, 130.3,
71.2, 51.6, 47.1, 41.1, 41.0, 40.8, 39.2, 32.0, 31.9, 27.0, 26.1,
24.3, 14.8.
β-Oxidation metabolite 22 was isolated as follows. A. terreus
lovA spores were used to inoculate flasks (2 × 100 mL) of
A. nidulans growth media after 4 days the mycelia were har-
vested by filtration and transferred to A. nidulans production
media (2 × 100 mL). After 48 hours, a solution of monacolin J
in EtOH (30 mg in 3.0 ml EtOH) was administered in intervals
(0.5 mL of the above solution every 12 hours until completely
fed). After seven days total growth in the production media, the
culture broth was harvested by vacuum filtration. The com-
bined broth was acidified and extracted with EtOAc (2 × 100
ml). The combined organic extracts were dried, filtered and
evaporated to give 42.0 mg crude extract. The crude extract was
fractionated by preparative TLC (EtOAc–hexane 4 : 1) to give a
crude sample of 22. This was further purified after treatment
with CH2N2 (Et2O solution) to give 0.1 mg of the methyl ester
of 22.
4a-5-Dihydromonacolin L epoxide 21a, 21b
A sample of 50.0 mg (0.163 mmol) dihydromonacolin L 5
was placed in a 10 ml round bottomed flask and dissolved in 4
mL of CHCl3 under Ar(g). To this solution was added dropwise
50.1 mg (0.244 mmol, 1.5 eq.) of an 85% preparation of
m-chloroperbenzoic acid in benzoic acid dissolved in 1 mL of
CHCl3. The reaction was stirred under Ar(g) at room temper-
ature for 90 min. The reaction was diluted with 25 mL of
CH2Cl2 and extracted with 5% NaHSO3 (1 × 10 mL) and satd.
NaHCO3 (1 × 10 mL), and washed with H2O (3 × 10 mL). The
organic layer was dried (Na2SO4), filtered and evaporated to
give 54.1 mg crude product which was fractionated by flash
column chromatography (40% EtOAc in hexanes) to yield 11.3
mg of 21a and 11.2 mg of 21b (44% total yield of pure 21a and
21b).
1H NMR (600 MHz, CDCl3) δ 5.95 (d, 1H, J = 10.0 Hz), 5.75
(dd, 1H, J = 9.1, 6.2 Hz), 5.55 (m, 1H), 4.28 (m, 1H), 3.68
(s, 3H), 2.50–2.20 (m, 6H), 1.80–1.20 (m, 10H), 1.16 (d, 3H,
J = 7.4 Hz), 0.88 (d, 3H, J = 7.0 Hz).
Acknowledgements
The authors thank Dr Robert McDonald (University of
Alberta) for crystallographic analyses, Dr Joanna Harris
(University of Alberta) for performing the selective reduction
of 10 to 11 and Karr-Hong Lee (University of Alberta) for
isolating 19. This work was supported by the Natural Sciences
and Engineering Research Council of Canada, the Canada
Research Chair in Bioorganic and Medicinal Chemistry, the
Canada Foundation for Innovation, and the Alberta Science
and Research Authority.
For 4a,5-dihydromonacolin L α-epoxide 21a: [α]2D5 = ϩ90
(c 0.95, CHCl3); FTIR (cast) 3392 (br), 2915 (s), 2876 (s), 1709
(s), 1257 (s), 1044 (s), 842 (s); HREIMS [M]ϩ 322.21451
(322.21442 calcd. for C19H30O4) 322 (5%), 307 (5%), 191 (48%),
1
179 (100%), 95 (65%); H NMR (500 MHz, CDCl3) δ 4.63
(dddd, 1H, J = 11.0, 11.0, 7.6, 3.8 Hz), 4.36 (m, 1H), 2.98
(d, 1H, J = 2.4 Hz), 2.72 (d, 1H, J = 3.7 Hz), 2.69 (d, 1H,
J = 5.0 Hz), 2.61 (dd, 1H, J = 3.6, 1.7 Hz), 2.56 (dd, 1H, J = 3.7,
1.7 Hz), 2.30 (m, 1H), 2.05 (m, 2H), 1.93 (m, 2H), 1.8–1.6 (m,
2H), 1.6–1.3 (m, 10H), 0.95 (d, 3H, J = 7.1 Hz), 0.89 (d, 3H,
J = 7.2 Hz); 13C NMR (125 MHz, CDCl3) δ 170.4, 76.0, 62.8,
58.8, 57.5, 38.6, 38.8, 37.4, 36.3, 36.2, 36.1, 33.0, 32.0, 30.3,
27.6, 23.9, 23.7, 18.1, 10.2.
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