Substrate recognition and channeling of monomodules from the pikromycin polyketide synthase

Article abstract of DOI:10.1021/ja034841s

The unique ability of the pikromycin (Pik) polyketide synthase to generate 12- and 14-membered ring macrolactones presents an opportunity to explore the fundamental processes underlying polyketide synthesis, specifically the mechanistic details of the cha

Full text of DOI:10.1021/ja034841s

Published on Web 09/23/2003
Substrate Recognition and Channeling of Monomodules from
the Pikromycin Polyketide Synthase
Brian J. Beck,†,| Courtney C. Aldrich,†,| Robert A. Fecik,‡ Kevin A. Reynolds,§ and
David H. Sherman*,†,|
Contribution from the Department of Microbiology and Biotechnology Institute,
UniVersity of Minnesota, Minneapolis, Minnesota 55455; Department of Medicinal Chemistry,
UniVersity of Minnesota, Minneapolis, Minnesota 55455; and Department of Medicinal
Chemistry and Institute for Structural Biology and Drug DiscoVery, Virginia Commonwealth
UniVersity, Richmond, Virginia 23219
Received February 24, 2003; E-mail: davidhs@umich.edu
Abstract: The unique ability of the pikromycin (Pik) polyketide synthase to generate 12- and 14-membered
ring macrolactones presents an opportunity to explore the fundamental processes underlying polyketide
synthesis, specifically the mechanistic details of the chain extension process. We have overexpressed
and purified PikAIII (module 5) and PikAIV (module 6) and assessed the ability of these proteins to generate
tri- and tetraketide lactone products using N-acetylcysteamine-activated diketides and 14C-methylmalonyl-
CoA as substrates. Comparison of the stereochemical specificities for PikAIII and PikAIV and the reported
values for the DEBS modules reveals significant differences between these systems.
Introduction
the four diastereomeric NAC thioesters of 2-methyl-3-hydroxy-
pentanoic acid have been investigated with native bimodular
Polyketides are a diverse class of natural products that possess
antibiotic, anticancer, and immunosuppressive bioactivities as
well as others.1 Their biosynthesis shares a common mechanism
where the repetitive condensation of simple malonic acid
derivatives are catalyzed by modular, multifunctional proteins,
the polyketide synthases (PKSs). A prototype type I PKS
enzyme is organized into modules each of which catalyzes one
cycle of chain elongation. A module minimally consists of three
domains: a ketosynthase (KS), an acyltransferase (AT), and
an acyl carrier protein (ACP). Additionally, a module may
include optional reductive domains (ketoreductase (KR), dehy-
dratase (DH), and enoylreductase (ER)) that are responsible for
adjusting the oxidation state of the â-position. The terminal
module of the PKS assembly line contains a thioesterase (TE)
domain which releases the fully elongated polyketide chain
either as a macrolactone or as a carboxylic acid.1
DEBS proteins, as well as DEBS proteins that have been
reengineeredandpurifiedasnon-nativemonomodularenzymes.2-6
The DEBS modules tolerate these short diketide chain lengths
and have a universal specificity for the (2S, 3R) stereochemistry.
“Docking” of the domains at the NH2 and COOH ends of these
bimodular proteins (DEBS 1, 2, and 3) has been proposed to
promote the sequential interaction of PKS modules and the
channeling of polyketide chain elongation intermediates.4-6
Generalization and utilization of these observations require
validation by a comparative analysis with other PKS systems.
Streptomyces Venezuelae ATCC 15439 produces the 12- and
14-membered ring aglycones, 10-deoxymethynolide (10-Dml,
1), and narbonolide (Nbl, 2), respectively, through the activity
of the pikromycin (Pik) PKS (Figure 1).7 Initiation of polyketide
biosynthesis by PikAI and successive elongation through PikAII
and PikAIII provide a hexaketide leading to 10-Dml, whereas
Nbl production requires an additional extension of the hexa-
ketide chain intermediate with methylmalonyl-CoA catalyzed
by PikAIV. In vivo expression studies have led to alternative
models for the partitioning of the hexaketide chain produced
by PikAIII. Initially, an N-terminally truncated form of PikAIV
lacking its docking domain was proposed to exclusively catalyze
Much of our current knowledge of PKS systems is largely
based from the study of the 6-deoxyerythronolide B polyketide
synthase (DEBS). DEBS is organized into three large bimodular
proteins (DEBS1, 2, and 3) that direct the biosynthesis of the
clinically important drug Erythromycin. The chain elongation
and processing functions of DEBS modules have been studied
in-vitro using simple diketide N-acetylcysteamine (NAC) thioesters
to mimic the natural polyketide chain intermediates. Specifically,
(2) Weissman, K. J.; Bycroft, M.; Cutter, A. L.; Hanefeld, U.; Frost, E. J.;
Timoney, M. C.; Harris, R.; Handa, S.; Roddis, M.; Staunton, J.; Leadlay,
P. F. Chem. Biol. 1998, 5, 743-754.
† Department of Microbiology and Biotechnology Institute, University
of Minnesota.
(3) Chuck, J. A.; McPherson, M.; Huang, H.; Jacobsen, J. R.; Khosla, C.; Cane,
D. E. Chem. Biol. 1997, 4, 757-766.
(4) Tsuji, S. Y.; Cane, D. E.; Khosla, C. Biochemistry 2001, 40, 2326-2331.
(5) Wu, N.; Tsuji, S. Y.; Cane, D. E.; Khosla, C. J. Am. Chem. Soc. 2001,
123, 6465-6474.
‡ Department of Medicinal Chemistry, University of Minnesota.
§ Virginia Commonwealth University.
| Current address: Department of Medicinal Chemistry, Department of
Chemistry, and Department of Microbiology and Immunology, University
of Michigan, Ann Arbor, MI 48109-1065.
(6) Wu, N.; Kudo, F.; Cane, D. E.; Khosla, C. J. Am. Chem. Soc. 2000, 122,
4847-4852.
(7) Xue, Y.; Zhao, L.; Liu, H.-W.; Sherman, D. H. Proc. Natl. Acad. Sci. U.S.A.
1998, 95, 12111-12116.
(1) Katz, L. Chem. ReV. 1997, 97, 2557-2575.
9
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J. AM. CHEM. SOC. 2003, 125, 12551-12557
12551

A R T I C L E S
Beck et al.
Figure 1. Biosynthesis of 10-deoxymethynolide and narbonolide by the Pikromycin (Pik) PKS in S. Venezuelae.
Figure 2. Synthesis of multiple lactones by PikAIII and PikAIV using N-acetylcysteamine (NAC)-activated thioesters 3-6. (A) Synthesis of 7-10 and
11-14 requires the extension of the diketide by PikAIII and PikAIV, respectively, whereas synthesis of 15-18 requires elongation of the diketide through
PikAIII and PikAIV. (B) Radio-TLC identification of reaction products 7, 11, and 15 by incubation with NAC diketide 3.
the synthesis of 10-Dml.8 Subsequently, site-directed mutants
Results
of PikAIV indicated that the hexaketide chain may “skip”
Protein Purification and Assays for Substrate Incorpora-
tion. PikAIII and PikAIV holoenzymes were overexpressed and
through the domains of PikAIV leading to macrolactonization
by the thioesterase.9 To understand the mechanistic details of
purified as previously described.12 Purified PikAIII and PikAIV
polyketide chain extension in this system, we sought to identify
were individually reacted with 2-[14C]-methylmalonyl-CoA and
each of the diketide NAC thioesters 3-6.13 The tri- and tetra-
the substrate specificity and the interaction between these
monomodular proteins using diketide substrates. We reasoned
that acceptance of a diketide by either PikAIII or PikAIV would
quantified by radio-TLC (Figure 2B). A time-course analysis
produce a triketide that could be cyclized to generate triketide
lactones (TKLs) 7-10 and 11-14, respectively. Accordingly,
PikAIV extension of the triketide chain produced by PikAIII
would yield tetraketides 15-1810,11 (Figure 2A).
ketide products were identified using synthetic standards and
of both PikAIII and PikAIV demonstrated that enzyme activity
remained constant during the first 120 min of reaction (data
not shown). The initial rates, V, at a given [S] were determined
by single time-point stopped-time incubations at 90 min. All
reactions were run in triplicate employing a fresh enzyme
preparation. Apparent steady-state kinetic values (kcat and Km)14
were determined for enzyme-substrate pairings that yielded a
detectable product by fitting the normalized V versus [S] plots
to the Michaelis-Menten equation. (The normalized V versus
(8) Xue, Y.; Sherman, D. H. Nature 2000, 403, 571-574.
(9) Beck, B. J.; Yoon, Y. J.; Reynolds, K. A.; Sherman, D. H. Chem. Biol.
2002, 9, 575-583.
(10) TKL 8-10, 13, and 14 and tetraketides 16-18 were not observed by radio-
TLC.
(11) The C-2 stereocenters of compounds 11, 12, 15, and 16 are configurationally
labile and, for simplicity, are represented as the enol tautomers. In aqueous
and methanolic solutions, â-keto-δ-lactones 11-12 and 15-16 exist
exclusively as the enol tautomers. In chloroform, TKL 11 and 12 exist as
a ratio of keto:enol tautomers which varied from 10:1 to 2:1. Tetraketide
15 is only sparingly soluble in chloroform but exists as a 5.5:1 mixture of
keto:enol tautomers. The insolubility of tetraketide 16 in chloroform
prevented evaluation of its tautomeric ratio.
(12) Beck, B. J.; Aldrich, C. C.; Fecik, R. A.; Reynolds, K. A.; Sherman, D. H.
J. Am. Chem. Soc. 2003, 125, 4682-4683.
(13) Harris, R. C.; Cutter, A. L.; Weissman, K. J.; Hanefeld, U.; Timoney, M.
C.; Staunton, J. J. Chem. Res. (S) 1998, 283.
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Chiral Recognition by the Pikromycin Polyketide Synthase
A R T I C L E S
Table 1. Steady-State Kinetic Parameters for the Formation of
Triketide Lactones by PikAIII and PikAIV
k
cat/Km
module
substrate
k
cat (min-1
0.0045 ( 0.0003
naa
)
Km (mM)
(min-1 mM-1
)
PikAIII
(2S,3R)-3
(2R,3S)-4
(2S,3S)-5
(2R,3R)-6
3.1 ( 0.8
na
0.0015
na
na
na
na
na
na
na
PikAIV (2S,3R)-3
(2R,3S)-4
0.013 ( 0.002
0.0076 ( 0.0007
na
na
10.5 ( 3.1
5.7 ( 1.6
na
0.0012
0.0013
na
na
(2S,3S)-5
(2R,3R)-6
na
Figure 3. Production of TKL 7 and tetraketide 15 by PikAIII and PikAIV
interaction. (2) TKL 7; (9) tetraketide 15; (b) total production.
a na, no activity detected.
Scheme 1 a
[S] plots of these reactions are available in the Supporting
Information.)
Verification of Reaction Products. Reaction products were
identified by comigration with authentic standards, and GC-
MS or LC-MS analysis was employed to rigorously confirm
these assignments. PikAIII only reacted with syn-diketide (2S,
3R)-3 while PikAIV only accepted syn-diketides (2S,3R)-3 and
(2R,3S)-4. To provide sufficient product for detection, PikAIII
and PikAIV were incubated for 16 h with the appropriate dike-
tide NAC esters. Incubation of PikAIII with (2S,3R)-3 produced
TKL 7 (retention time (tret), 7.73 min) as the only diastereomeric
product as determined by GC-MS analysis detecting at m/z
171 (M-H)+ in single-ion mode employing chemical ionization
(see Supporting Information for GC-MS traces and conditions).
Incubation of PikAIV with (2S,3R)-3 and (2R,3S)-4 afforded
the enantiomeric keto-lactones 11 and 12 (tret, 21.00 min) as
confirmed by LC-MS analysis detecting at m/z 169 (M-H)-
in negative ion mode using electrospray ionization (ESI). Tetra-
ketide 15 (tret, 21.90 min) was similarly analyzed by LC-MS
detecting at m/z 227 (M-H)- in negative ion mode (ESI). (See
Supporting Information for LC-MS traces and HPLC condi-
tions.)
a Key: (a) 1 N aq HCl:THF (4:1), 60 °C, 40 h; (b) (COCl)2, DMSO,
Et3N, CH2Cl2, -78 °C f -20 °C; (c) Zn dust, AcOH, CH2Cl2, 0 °C.
provided a KD of 95 ( 32 nM.16
V
max[P4]
V )
(1)
KD + [P4]
Synthesis of Tri- and Tetraketide Standards. A flexible
synthetic strategy was desired that could efficiently provide all
diastereomeric tri- and tetraketide products employing a common
intermediate. This was most effectively accomplished utilizing
Evans diketide synthon 19.18 Aldol reaction of â-ketoimide 19
with simple aldehydes provides a rapid entry to triketides
wherein the choice of Lewis acid (TiCl4, Sn(OTf)2, or (c-
hex)2BCl) allows access to three of the four possible diastere-
omeric triketide products.18,19 Additionally, the choice of
reducing agent allows the stereochemistry of the â-keto function
to be adjusted as desired. Triketide 2017 was prepared according
to Evans; subsequent δ-lactonization proceeded in quantitative
yield under aqueous acidic conditions to afford lactone 7
(Scheme 1).20 Ketolactone standard 1121 is available directly
by oxidation of 7; however initial attempts employing the Dess-
Determination of the Dissociation Constant Between
PikAIII and PikAIV. The apparent dissociation constant, KD,
of the PikAIII and PikAIV interaction was determined using
the analysis reported by Tsuji et al.4 When PikAIII and PikAIV
were reacted together with (2S,3R)-3, a product mixture of TKLs
7 and 11 and tetraketide 15 was observed by radio-TLC (Figure
2B). In the present system, PikAIV partitions the incoming
triketide into two products, TKL 7 and tetraketide 15; therefore,
the formation of both products is a measure of the interaction
of PikAIII with PikAIV. However, as 7 is also produced by
PikAIII, albeit at a reduced rate, this contribution was subtracted
from the overall observed production of 7. Variation of PikAIV
(P4) at a constant concentration of PikAIII (P3) under saturating
concentrations of all substrates (MMCoA, NADPH, and (2S,3R)-
3) provided the saturation curve in Figure 315 which is described
by eq 1, where V is the sum of the velocities of both tetraketide
15 and the corrected velocity of triketide 7. The maximal
velocity, Vmax, is equal to kcat[P3]0, where [P3]0 is the total
concentration of PikAIII. Nonlinear regression analysis of eq 1
(16) The apparent KD can be taken as an upper limit; thus, the binding interaction
may actually be stronger than calculated employing this analysis. The
dissociation constant KD is equal to [P3]L[P4]/[P3L‚P4]. Also, [P3]0 ) [P3]L
+ [P3]F, where P3L is the concentration of PikAIII ACP5-loaded triketide
and P3F is equal to concentration of free-PikAIII without a loaded triketide.
If interaction between P3L and P4 results in channeling of the triketide to
P4 resulting in the formation of 7 and 15, then we can write V ) kcat[P3L‚
P4]. This is alternatively expressed by eq 1 in terms of the known
parameters, [P4] and [P3]0. However, this analysis neglects the effect of
[P3]F on the overall process. In fact, [P3]F will act as a competitive inhibitor
of P4. The net effect is to underestimate KD by a factor of (1 + [P3]F/Ki).
(17) Evans, D. A.; Kim, A. S.; Metternich, R.; Novack, V. J. J. Am. Chem.
Soc. 1998, 120, 5921-5942.
(18) Evans, D. A.; Clark, J. S.; Metternich, R.; Novack, V. J.; Sheppard, G. S.
J. Am. Chem. Soc. 1990, 112, 866-868.
(14) Since these are complex multifunctional systems reacting with non-natural
substrates, the rate-determining step remains to be determined. Thus, the
presented kcat values represent a composite of individual rate constants for
the loading of diketide substrates, the Claisen condensation performed by
the KS domain, reductive processing of the keto function, and lactonization.
(15) TKL 7 production shown in Figure 3 results from PikAIV stimulation only.
These data were obtained by subtracting the contribution due to spontaneous
lactonization (kcat ) 0.0045 min-1) from the observed production of 7.
(19) Evans, D. A.; Ng, H. P.; Clark, S.; Rieger, D. L. Tetrahedron Lett. 1992,
48, 2127-2142.
(20) For an alternative synthesis of TKL 7, see: Wilkinson, A. L.; Hanefeld,
U.; Wilkinson, B.; Leadlay, P. F.; Staunton, J. Tetrahedron Lett. 1998, 39,
9827-9830.
(21) Luo, G.; Pieper, R.; Rosa, A.; Khosla, C.; Cane, D. E. Bioorg. Med. Chem.
1996, 4, 995-999.
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A R T I C L E S
Beck et al.
Scheme 2 a
with expulsion of the oxazolidinone auxiliary to furnish tetra-
ketide 23 with no competing R,â-elimination of propionic acid.34
Deprotection of tetraketide 23 with Dowex cationic exchange
resin yielded the desired tetraketide 15 in quantitative yield and
analytical purity after filtration from the resin. Synthetic
tetraketide 15 was identical to a biosynthetically derived sample
1
by comparison of H NMR, 13C NMR, and HRMS spectral
data.35 This short six step sequence starting from 19 allowed a
very efficient synthesis of the tetraketide target, and an
analogous series of reactions provided tetraketide 16.30
Discussion
a Key: (a) TBSOTf, 2,6-lutidene, CH2Cl2, 0 °C, 5 min; (b) (EtCO)2O,
DMAP, CH2Cl2, 50 °C, 7 h; (c) KHMDS (4.0 equiv), THF, -78 °C, 2 h;
(d) Dowex 50WX2-H+, THF:H2O (1:1), 50 °C, 20 h.
Comparison of the specificity constants (kcat/Km) reveals that
PikAIV does not distinguish between syn-diketides (2S,3R)-3
and (2R,3S)-4. In contrast, DEBS 3 (module 6) was reported to
accept both syn-diketide substrates with a 20-fold preference
for (2S,3R)-3.6 The specificity constants for PikAIV with
(2S,3R)-3 and (2R,3S)-4 are, respectively, 890-fold and 45-fold
less than those reported for DEBS 3 (module 6) with the same
substrates. Thus, PikAIV displays extraordinary specificity and
processes the synthetic non-natural diketides at levels 2 to 3
orders of magnitude lower than the corresponding DEBS system.
Additionally, PikAIV does not accept anti-diketides (2S,3S)-5
and (2R,3R)-6 which is consistent with the behavior of DEBS
modules.6,36
Martin periodinane22 or PCC resulted in decomposition to
unidentifiable products. It was found that Swern conditions23,24
successfully oxidized the alcohol of TKL 7 to the desired keto
function but additionally resulted in chlorination to afford
R-chloroketone 21.25 The decomposition pathway observed with
other oxidation reagents is likely due to overoxidation of the
initially formed keto-lactone.26 The success of the Swern
oxidation thus lies in the protection afforded by chlorination at
the C-2 position which serves to prevent further oxidation at
this position.27 Dechlorination was achieved by reduction with
zinc and acetic acid to provide the desired ketolactone 11.11,28,29
TKL standards 8 and 12 were prepared in an analogous fashion
from â-ketoimide 19.30
Triketide 20 was rapidly elaborated to the desired tetraketide
target 15 by taking advantage of a novel Claisen-like cyclization
developed by Branda¨nge and Leijonmarck.31-33 First, regiose-
lective protection of triketide 20,17 followed by acylation of the
remaining secondary alcohol with propionic anhydride, provided
22 in nearly quantitative yield (Scheme 2). Treatment of 22
with potassium bis(trimethylsilyl)amide in THF at -78 °C
resulted in regioselective deprotonation of the side chain
propionate group and intramolecular Claisen-like cyclization
We have also characterized native PikAIII, which lacks a
C-terminal TE domain. This represents the first example where
a monomodule has been evaluated without such a domain.
Previously, unnatural DEBS monomodules were investigated
with a TE domain fused to the C-terminal end of the protein.
We found that PikAIII produces a TKL product in the absence
of a TE domain. In this case, lactonization is not enzymatically
mediated but is due to spontaneous cyclization. PikAIII
discriminates between the syn diastereomers elongating only
the (2S,3R)-3 diketide substrate, while no TKL product was
observed with (2R,3S)-4. Since PikAIII has been observed to
extend (2R,3S)-4 generated through an intramolecular priming
reaction,12 the KS5 domain likely discriminates against diffusive
loading of this enantiomer. This stereochemical selectivity
distinguishes PikAIII from the reported activities of individual
DEBS modules that accept both syn configurations albeit with
a preference for (2S,3R)-3.6 Without enzymatic release, the
observed kcat value cannot be directly compared to DEBS values;
however, the relative kcat observed for PikAIII is still insightful.
Cane and co-workers have recently reported the activity of a
PikAIII-TE hybrid and observed nearly identical results with a
(22) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277-7287.
(23) Omura, K.; Swern, D. Tetrahedron 1978, 34, 1651-1660.
(24) Oxidation to â-keto-δ-lactones is reported to be problematic; however,
modified Swern conditions employing TFAA/DMSO have been successfully
employed. See: Sakai, N.; Ohfune, Y. J. Am. Chem. Soc. 1992, 114, 998-
1010.
(25) A single diastereomer was observed; however, the stereochemistry depicted
for R-chloroketone 21 has not been rigorously confirmed. An NOE
enhancement was not observed between the C-2 methyl group and H-4
which suggests that it should reside in the equatorial position indicated.
(26) When 1 equiv of oxalyl chloride was employed in the Swern oxidation,
the reaction went to approximately 50% conversion to R-chloroketone 21,
indicating that chlorination of the initially formed keto-product proceeds
faster than oxidation of the starting alcohol.
k
cat 4-fold greater than our value.37 Last, PikAIII did not accept
(27) Interestingly, the reported oxidation by Cane and co-workers21 of triketide
lactone 1 under Swern conditions afforded a pyrone product (30%), along
with recovered starting material (40%) and unidentified material (30%).
We found that R-chloroketones 21 and 23 were unstable to silica gel
chromatography which may explain the discrepancy in the reported results.
(28) For an alternative synthesis of â-keto-δ-lactones 11 and 12, see refs 12
and 21.
anti-diketides (2S,3S)-5 and (2R,3R)-6 as similarly reported for
the DEBS modules.6,36
To extend this work further, the channeling of a non-natural
substrate and protein-protein interactions between PikAIII and
(29) Interestingly, in chloroform, the keto-tautomer of 11 exists as a 16:1 mixture
of 2R:2S epimers. Gas phase calculations at the AM1 level indicate that
both epimeric keto-lactones exist in a twist-boat conformation and are
approximately equienergetic. GC-MS analysis in fact reveals two peaks
at m/z) 168 (M-2H)+ in a ratio of 58:42 in support of the semiempirical
calculations; thus, the epimeric ratio in chloroform reflects a difference in
solvation.
(34) Sorenson and co-workers32 have demonstrated the importance of the
potassium counterion in this reaction as lithium and sodium enolates
reportedly31 afforded an 11-membered ring due to a competing endo-attack
on the oxazolidinone carbonyl.
(35) Kao, C. M.; Luo, G.; Katz, L.; Cane, D. E.; Khosla, C. J. Am. Chem. Soc.
1996, 118, 9184-9185.
(36) During the review of this manuscript, a complementary report by Cane
and co-workers37 reported a modest preference for the (2S,3R)-3 NAC
diketide by PikAIV and the same diastereoselectivity with PikAIII modified
with a thioesterase from the pikromycin cluster.
(30) See Supporting Information.
(31) Branda¨nge, S.; Leijonmarck, H. Tetrahedron Lett. 1992, 33, 3025-3028.
(32) Vanderwal, C. D.; Vosburg, D. A.; Weiler, S.; Sorenson, E. J. Org. Lett.
1999, 1, 645-648.
(33) Hinterding, K.; Singhanat, S.; Oberer, L. Tetrahedron Lett. 2001, 42, 8643-
8645.
(37) Yin, Y.; Lu, H.; Khosla, C.; Cane, D. E. J. Am. Chem. Soc. 2003, 125,
5671-5676.
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Chiral Recognition by the Pikromycin Polyketide Synthase
A R T I C L E S
Experimental Section
PikAIV were investigated using the (2S,3R)-3 diketide substrate.
When PikAIII and PikAIV were reacted together with (2S,3R)-
3, a product mixture of TKL 7 and 11 and tetraketide 15 was
observed by radio-TLC (Figure 2B). Previous studies involving
channeling of triketide chain intermediates between DEBS
monomodules with natural docking domains reportedly gave
exclusive formation of tetraketide 15.4 Introduction of a linker
mismatch into the DEBS modules resulted in premature release
of the chain intermediate and formation of TKL 7. Interestingly,
the rate of TKL 7 synthesis by PikAIII was accelerated
approximately 4-fold (kcat ) 0.017 min-1) with the addition of
PikAIV. Indeed, PikAIII with a covalently attached TE domain
was recently reported to form TKL 7 at a comparable rate.37
We note that the PikAIV dependent release of the triketide chain
from PikAIII resembles the natural mechanism of 10-Dml
formation; however, this consequence is an artifact of the non-
natural substrate and the apparent substrate specificity of these
modules. Significantly, we observed that PikAIV containing an
inactivated (C207A) KS6 domain did not show this rate
enhancement, while PikAIV containing an inactivated (S1196A)
TE domain retained activity for TKL 7 cyclization, indicating
a role for KS6 in lactonization (see Supporting Information for
radio-TLC). In contrast, the TE domain is required for macro-
lactonization of the hexaketide leading to 10-Dml.8 These results
indicate that the KS6 domain can apparently function as a
thioesterase when provided with a triketide chain intermediate.
Presumably, the oxy anion hole38 that mediates the Claisen
condensation also stabilizes the tetrahedral intermediate formed
during “spontaneous” lactonization. Thus, despite the natural
interaction of PikAIII and PikAIV, PikAIV does not permit
complete elongation of the triketide chain channeled by PikAIII,
suggesting a reduced tolerance for the triketide intermediate that
is released as TKL 7. This reduced tolerance and the apparent
lactonization activity of KS6 likely explain the synthesis of TKL
7 (but not tetraketide 15) with the in vivo pairing of DEBS1
and PikAIV,39 whereas the combination of DEBS1 and DEBS
Module3+TE produced a greater amount of tetraketide 15
relative to TKL 7.40,41 The calculated dissociation constant for
the PikAIII-PikAIV interaction of 100 nM is approximately
10-fold lower than the KD reported for the interaction between
DEBS modules indicating a more specific interaction inherent
to Pik module 5 and module 6.4
Protein Overexpression and Purification. The construction of
expression plasmids for PikAIII and PikAIV has been previously
reported.12 Proteins were individually expressed in Escherichia coli
BL21 (DE3) using plasmids pET28b and pET24b, respectively
(Novagen). The PikAIII protein was engineered with an NH2-6×HIS
tag, and PikAIV was modified to contain a COOH-6×HIS tag. Both
proteins were coexpressed with the Bacillus subtilis sfp gene encoding
a phosphopantetheine transferase for post-translational modification of
the ACP domains.42 Overexpression cultures were grown at 37 °C to
an A600 ) 0.5-0.6, equilibrated to 22-25 °C for 20 min, then induced
using 0.1 mM of isopropyl-1-thio-D-galactopyranoside (IPTG), and
incubated overnight. Cells were harvested by centrifugation at 3000 ×
g and suspended in lysis buffer (50 mM NaH2PO4, pH 8.0, 150 mM
NaCl, 10 mM imidazole, 1 mM DTT, 10% glycerol). After disruption
of the cells using a French press, the clarified lysate was loaded onto
Ni-NTA resin (Qiagen) previously equilibrated with lysis buffer. Both
PikAIII and PikAIV were eluted from the Ni-NTA resin in lysis buffer
containing 250 mM imidazole. Fractions containing the eluted protein
were pooled, and a PD-10 column (Pharmacia) was used to exchange
to storage buffer (100 mM NaH2PO4, pH 7.2, 1 mM EDTA, 1 mM
DTT, 20% glycerol). The purity of each preparation was determined
to be >90% by SDS-PAGE.
In Vitro Polyketide Production. The reactivity of PikAIII and
PikAIV with NAC diketides 3-6 was determined by measuring the
incorporation of 2-[14C]-methylmalonyl-CoA into the resulting tri- and
tetraketide lactone products. 2-[14C]-Methylmalonyl-CoA (55 mCi/
mmol) was purchased from American Radiolabeled Chemicals, Inc.,
and all other chemicals were purchased from Sigma. Enzymatic
reactions were run in 400 mM NaH2PO4 buffer (pH 7.2) containing 5
mM NaCl, 1 mM EDTA, 1 mM DTT, 800 µM NADPH (for reactions
with PikAIII), 20% glycerol, and 5% DMSO in a final volume of 100
µL. In reactions designed to determine the individual reactivity of each
protein with each diketide diastereomer, protein was added to a
concentration of 2 µM, and diketide concentrations ranged from 1 to
25 mM. For experiments investigating the interaction between PikAIII
and PikAIV, assays and product detection were performed as described
above with 2 µM PikAIII, 0.06-2 µM PikAIV, and 12 mM (2S,3R)-3.
In all reactions, enzyme was equilibrated in buffer for 5 min at 30 °C
and the reaction was initiated with the addition of 2-[14C]-methylmal-
onyl-CoA (diluted for a specific activity ) 1.35 mCi/mmol) for a final
saturating concentration of 435 µM. The reaction was maintained at
30 °C for 1.5 h and then quenched, and the products were extracted
with EtOAc (2 × 450 µL). The extracts were dried under a stream of
N2, and the products were resolved on silica gel TLC plates using 60%
EtOAc/hexanes as the solvent; polyketide products were identified using
authentic reference compounds. Separation of reaction extracts by TLC
allowed visualization of radiolabeled products by autoradiography.
Product formation was quantified using ImageQuant (Molecular
Dynamics) using a standard curve generated with 2-[14C]-methylma-
lonyl-CoA, and apparent kinetic values were determined using Graph-
Pad Prism software.
Significance. The observation that Pik modules 5 and 6 show
greater specificity with di- and triketide substrates compared
to DEBS modules highlights important distinctions between
these systems. Comparative analysis using authentic substrates
with other PKS systems will determine whether the apparent
stringency of the Pik PKS or the flexibility of DEBS is more
typical of these multifunctional enzymes. Thus, the mechanisms
that modular PKSs have evolved to control processing of
unnatural chain intermediates represents a key consideration for
full development of combinatorial biosynthesis for generating
novel natural products.
Synthesis of Substrates and Reference Compounds. The diketide
NAC esters 3-6 were synthesized essentially as described except for
two minor modifications43 and determined to have a de > 99% by
HPLC.13 See Supporting Information for general procedures.
(3R,4S,5S,6R)-6-Ethyl-4-hydroxy-3,5-dimethyl-tetrahydro-pyran-
2-one (7). A solution of triketide 2017 (299 mg, 0.857 mmol) in THF:1
(42) Lambalot, R. H.; Gehring, A. M.; Flugel, R. S.; Zuber, P.; LaCelle, M.;
Marahiel, M. A.; Reid, R.; Khosla, C.; Walsh, C. T. Chem. Biol. 1996, 3,
923-936.
(43) The original procedure of Evans was employed for the LiOOH-mediated
hydrolysis of the oxazolidinone auxiliary. See: Evans, D. A.; Britton, T.
C.; Ellman, J. A. Tetrahedron Lett. 1987, 28, 6141-6144. In the subsequent
coupling reaction with N-acetylcysteamine, DCC was replaced by EDC
and the urea byproduct was removed with an aqueous 1 N HCl wash. Also,
the reported DCC coupling reaction was allowed to stir for 24-72 h;
however, the reaction was complete in less than 1 h at 0 °C.
(38) Huang, W.; Jia, J.; Edwards, P.; Dehesh, K.; Schneider, G.; Lindqvist, Y.
EMBO J 1998, 17, 1183-1191.
(39) Kim, B. S.; Cropp, T. A.; Florova, G.; Lindsay, Y.; Sherman, D. H.;
Reynolds, K. A. Biochemistry 2002, 41, 10827-10833.
(40) Pieper, R.; Gokhale, R. S.; Luo, G.; Cane, D. E.; Khosla, C. Biochemistry
1997, 36, 1846-1851.
(41) Gokhale, R. S.; Hunziker, D.; Cane, D. E.; Khosla, C. Chem. Biol. 1999,
6, 117-125.
9
J. AM. CHEM. SOC. VOL. 125, NO. 41, 2003 12555

A R T I C L E S
Beck et al.
N aq HCl (4:1, 50 mL) was heated at 60 °C for 40 h. The reaction was
cooled to room temperature, and the THF was removed under reduced
pressure. The resulting mixture was partitioned between EtOAc and
H2O. The organic layer was separated, and the aqueous layer was
extracted with EtOAc (2×). The combined organic extracts were
washed with saturated aq NaCl, dried (Na2SO4), filtered, and concen-
trated under reduced pressure to an oil. Chromatography on silica gel
(20 g) with 30% EtOAc/hexanes (300 mL) to 50% EtOAc/hexanes
(150 mL) afforded 147 mg (0.857 mmol, 100% yield) of a colorless
oil. Additionally, 150 mg (99%) of the oxazolidinone auxiliary was
recovered. Lastly, 34 mg of 4-chlorobutanol was isolated as a result of
decomposition of THF under the reaction conditions. mp 37-41 °C;
The product was further purified by recrystallization by dissolving the
product in a minimum of hot CH2Cl2, followed by careful addition of
a layer of pentane (approximately 5 times the volume of CH2Cl2) on
top of the CH2Cl2 layer. The product recrystallized from the resulting
two-layer solution at -10 °C overnight to afford 16 mg (0.0941 mmol,
60% yield) of fine white needles that had a pleasant brown sugar-
molasses odor. mp 110-113 °C; TLC Rf ) 0.36 (50% EtOAc/hexanes);
[R]25D -57.0 (c ) 3.05, MeOH); 1H NMR (CDCl3) 25:1:8 mixture of
9R-keto:9S-keto:enol isomers δ 0.98 (t, 1H, J ) 7.5 Hz, enol CH3),
1.07 (t, 3H, J ) 7.8 Hz, keto CH3), 1.11 (d, 3H, J ) 7 Hz, keto CH3),
1.35 (d, 3H, J ) 6.9 Hz, keto CH3), 1.58-1.72 (m, 1.33H, keto +
enol CH2), 1.78 (s, 1H, enol HOsCdCsCH3), 1.78-1.94 (m, 1.33H,
keto + enol CH2), 2.38-2.48 (m, 0.3H, enol CdCsH), 2.62 (qd, 1H,
J ) 7.5, 2.7 Hz, keto CH), 4.22 (ddd, 0.3H, J ) 9.6, 6, 3 Hz, enol
TLC Rf ) 0.45 (40% EtOAc/hexanes); [R]25 +122 (c ) 1.29, CH2-
D
Cl2); 1H NMR (CDCl3) δ 0.95 (ovlp d, 3H, J ) 6.9 Hz), 0.99 (ovlp t,
3H, J ) 7.5 Hz), 1.39 (d, 3H, J ) 7.5 Hz), 1.57 (dp, 1H, J ) 13.2, 7.8
Hz), 1.82 (dp, 1H, J ) 13.2, 7.8 Hz), 2.0 (br s, 1H), 2.12-2.20 (m,
1H), 2.46 (dq, 1H, J ) 10.5, 7.2 Hz), 3.81 (dd, 1H, J ) 10.2, 3.9 Hz),
4.12 (ddd, 1H, J ) 8.7, 6.6, 2.7 Hz); 13C NMR (CDCl3) δ 4.5, 10.0,
14.4, 25.3, 36.7, 39.8, 73.9, 81.3, 173.5; IR (film) 3440 (w, br), 2972
(m), 2941 (w), 2883 (w), 1711 (s), 1458 (m), 1360 (m), 1217 (m),
1111 (m), 980 (m) cm-1; HRMS (EI) m/z calcd for C9H16O3 (M+),
172.1099; found, 172.1094.
CHOCdO), 4.65 (ddd, 1H, J ) 8.1, 5.1, 3.0 Hz, keto CHOCdO); 13
C
NMR (CDCl3) δ 8.3, 9.9, 10.0, 24.1, 44.4, 50.4, 78.5, 169.9, 205.2; 1H
NMR (CD3OD) δ 1.01 (t, 3H, J ) 7.5 Hz), 1.08 (d, 3H, J ) 6.9 Hz),
1.52-1.64 (m, 1H), 1.70 (s, 3H), 1.68-1.81 (m, 1H), 2.38 (qd, 1H, J
) 6.9, 3.3 Hz), 4.23 (ddd, 1H, J ) 8.7, 5.7, 3.0 Hz); 13C NMR (CD3-
OD) δ 8.9, 10.2, 10.9, 25.3, 37.5, 80.7, 98.0, 172.3, 173.1; IR (film)
3116 (m, br), 2975 (m), 2941 (m), 2883 (m), 2689 (w, br), 1652 (s),
1539 (w), 1456 (m), 1393 (s), 1369 (s), 1354 (s), 1128 (s), 987 (m),
768 (m) cm-1; HRMS (EI) m/z calcd for C9H14O3 (M+), 170.0943;
found, 170.0952.
(3R,5S,6R)-3-Chloro-6-ethyl-3,5-dimethyl-dihydro-pyran-2,4-di-
one (21). To a solution of oxalyl chloride (102 µL, 1.19 mmol, 5.0
equiv) in CH2Cl2 (2 mL) at -78 °C was added dropwise a solution of
DMSO (127 µL, 1.79 mmol, 7.5 equiv) in CH2Cl2 (0.5 mL), and the
resulting solution was stirred for 15 min. A solution of alcohol 7 (40.7
mg, 0.238 mmol, 1.0 equiv) in CH2Cl2 (1 mL + 1 mL wash) was added
dropwise, and the resulting solution was stirred for 30 min. Et3N (332
µL, 2.38 mmol, 10.0 equiv) was added to the cloudy solution to afford
a clear solution which was stirred for 1 h at -78 °C and warmed to 0
°C over another hour to afford a milky white heterogeneous mixture.
The reaction was partitioned between Et2O and 0.1 N aq HCl. The
organic layer was separated and washed successively with H2O and
saturated aq NaCl, dried (Na2SO4), filtered, and concentrated under
reduced pressure to 46 mg (0.225 mmol, 95% yield) of a colorless oil.
The crude 1H NMR spectrum indicates a purity greater than 80%. The
product decomposes on silica gel; however, rapid flash chromatography
on silica gel (2 g) with 50% EtOAc/hexanes afforded 12 mg (0.0588
mmol, 25% yield) of a colorless oil. TLC Rf ) 0.54 (20% EtOAc/
hexanes); [R]25D +46.0 (c ) 0.135, CH2Cl2); 1H NMR (CDCl3) δ 1.08
(t, 3H, J ) 7.8 Hz), 1.15 (d, 3H, J ) 6.9 Hz), 1.60-1.80 (m, 2H),
1.85 (s, 3H), 3.01 (qd, 1H, J ) 7.2, 3.3 Hz), 5.01 (ddd, 1H, J ) 8.4,
5.1, 3.0 Hz); 13C NMR (CDCl3) δ 9.9, 10.0, 21.6, 24.5, 43.1, 59.0,
78.4 167.1, 199.9; IR (film) 2975 (w), 2929 (w), 2882 (w), 2854 (w),
1763 (s), 1732 (s), 1457 (w), 1377 (w), 1278 (m), 1126 (m), 1107 (m),
991 (w), 971 (w) cm-1; HRMS (EI) m/z calcd for C9H1335ClO3 (M+),
204.0553; found, 204.0555.
4-(R)-Benzyl-3-[(2R,3S,4S,5R)-5-(tert-butyldimethylsilanyloxy)-3-
propionyloxy-2,4-dimethylheptanoyl]-oxazolidin-2-one (22). To a
solution of triketide 20 (206 mg, 0.59 mmol, 1.0 equiv) and 2,6-lutidene
(77 µL, 0.66 mmol, 1.1 equiv) in CH2Cl2 (5 mL) at 0 °C was added
TBSOTf (152 µL, 0.66 mmol, 1.1 equiv), and the reaction was stirred
for 5 min. The reaction mixture was diluted with CH2Cl2 and washed
successively with 1 N aq HCl, H2O, saturated aq NaHCO3, and saturated
aq NaCl, dried (Na2SO4), filtered, and concentrated under reduced
pressure to afford 278 mg (102% yield) of a yellow oil.17 To a solution
of the yellow oil in CH2Cl2 (5 mL) was added propionic anhydride
(304 mL, 2.36 mmol, 4.0 equiv), Et3N (249 mL, 2.36 mmol, 4.0 equiv),
and 4-DMAP (15 mg, 0.12 mmol, 0.2 equiv), and the solution was
refluxed for 7 h until complete by TLC. The reaction was cooled to
room temperature, diluted with EtOAc, and washed consecutively with
1 N aq HCl, H2O, saturated aq NaHCO3, and saturated aq NaCl, dried
(Na2SO4), filtered, and concentrated under reduced pressure to an oil.
Chromatography on silica gel (25 g) with 10% EtOAc/hexanes afforded
300 mg (0.58 mmol, 97% yield) of a colorless oil. TLC Rf ) 0.60
1
(20% EtOAc/hexanes); [R]25 -75.2 (c ) 1.10, CH2Cl2); H NMR
D
(CDCl3) δ 0.00 (s, 3H), 0.04 (s, 3H), 0.88 (t, 3H, J ) 7.5 Hz), 0.92 (s,
9H), 0.98 (d, 3H, J ) 6.9 Hz), 1.19 (t, 3H, J ) 7.5 Hz), 1.21 (d, 3H,
J ) 7.2 Hz), 1.57 (p, 2H, J ) 7.5 Hz), 1.85-1.90 (m, 1H), 2.37 (ddd,
2H, J ) 15.3, 7.5, 2.7 Hz), 2.82 (dd, 1H, J ) 13.2, 10.2 Hz), 3.33 (dd,
1H, J ) 13.2, 3.3 Hz), 3.57 (td, 1H, J ) 7.8, 1.2 Hz), 4.16 (dd, 1H, J
) 8.4, 1.8 Hz), 4.24 (ddd, 1H, J ) 13.8, 7.2, 1.8 Hz), 4.32 (t, 1H, J )
7.8 Hz), 4.48 (dddd, 1H, J ) 10.2, 7.8, 3.3, 1.8 Hz), 5.21 (dd, 1H, J
) 10.5, 2.1 Hz), 7.25-7.40 (m, 5H); 13C NMR (CDCl3) δ -5.2, -3.5,
7.9, 8.2, 9.5, 10.4, 18.2, 26.0, 27.7, 28.2, 37.4, 38.0, 39.1, 56.5, 66.4,
72.4, 74.1, 127.0, 128.7, 129.3, 135.6, 153.6, 173.9, 174.7; IR (neat)
2946 (m), 2883 (w), 2857 (w), 1783 (s), 1734 (m), 1701 (m), 1463
(5S,6R)-6-Ethyl-4-hydroxy-3,5-dimethyl-5,6-dihydro-pyran-2-
one (11). To a solution of oxalyl chloride (66 µL, 0.77 mmol, 5.0 equiv)
in CH2Cl2 (1 mL) at -78 °C was added dropwise a solution of DMSO
(82 µL, 1.16 mmol, 7.5 equiv) in CH2Cl2 (0.5 mL), and the resulting
solution was stirred for 15 min. A solution of alcohol 7 (26.5 mg, 0.154
mmol, 1.0 equiv) in CH2Cl2 (0.5 mL + 0.5 mL wash) was added
dropwise, and the resulting solution was stirred for 30 min. Et3N (215
µL, 1.54 mmol, 10.0 equiv) was added, and the reaction was stirred
for 15 min at -78 °C and then slowly warmed to -20 °C over another
hour to afford a milky white heterogeneous mixture. Glacial AcOH
(176 µL, 3.08 mmol, 20 equiv) and Zn dust (206 mg, 3.08 mmol, 20
equiv) were added to the heterogeneous reaction mixture, and the
mixture warmed to 0 °C over 10 min. The reaction was partitioned
between CH2Cl2 and 0.1 N aq HCl. The organic layer was separated
and washed successively with H2O, saturated aq NaHCO3, and saturated
aq NaCl, dried (Na2SO4), filtered, and concentrated under reduced
pressure to an oil. Chromatography on silica gel (2 g) with 50% Et2O/
pentane afforded 30.7 mg (0.180 mmol, 117% yield) of a white solid.
(w), 1381 (m), 1245 (m), 1211 (m), 1104 (m), 1045 (m), 836 (m) cm-1
;
HRMS (EI) m/z calcd for C28H45NO6Si (M+), 519.3016; found,
519.2998.
(5R,6S)-6-[(1S,2R)-2-(tert-Butyldimethylsilanyloxy)-1-methyl-bu-
tyl]-4-hydroxy-3,5-dimethyl-5,6-dihydro-pyran-2-one (23). A solu-
tion of propionate 22 in THF (5 mL) at -78 °C was treated with a
freshly prepared solution of KHMDS (3.5 mL, 0.35 M in THF, 1.23
mmol, 4.0 equiv) and stirred for 2 h. The reaction was quenched with
a saturated aq NH4Cl/MeOH/H2O (1:1:1, 30 mL) mixture at -78 °C
and warmed to 0 °C. The reaction mixture was partitioned between
EtOAc and H2O. The aqueous layer was separated and acidified to pH
2 with 1 N aq HCl and extracted with EtOAc (3×). The combined
9
12556 J. AM. CHEM. SOC. VOL. 125, NO. 41, 2003

Chiral Recognition by the Pikromycin Polyketide Synthase
A R T I C L E S
organic layers were washed with saturated aq NaCl, dried (Na2SO4),
filtered, and concentrated under reduced pressure to a white solid.
Chromatography on silica gel (15 g) with 10% EtOAc/hexanes (100
mL) to 15% EtOAc/hexanes (200 mL) afforded 68 mg of a white solid.
The product was further purified by recrystallization from MeOH to
10.3, 6.9, 1.8), 2.66 (qd, 1H, J ) 7.5, 2.7 Hz), 3.70 (q, 1H, J ) 6.6
Hz), 4.07 (ddd, 1H, J ) 6.6, 4.8, 1.8 Hz), 4.84 (dd, 1H, J ) 10.2, 2.1
Hz); Identifiable peaks of the enol tautomer δ 1.80 (s, 3H, H-12), 2.38-
2.46 (m, 1H, H-4), 4.34 (dd, 1H, J ) 10.5, 3.3 Hz, H-5), 4.62 (ddd,
1H, J ) 9.6, 6.0, 3.0 Hz, H-7); 1H NMR (CD3OD) δ 0.82 (d, 3H, J )
6.9 Hz), 0.98 (t, 3H, J ) 7.3 Hz), 1.09 (d, 3H, J ) 7.2 Hz); 1.34-1.48
(m, 1H), 1.50-1.64 (m, 1H), 1.70 (s, 3H), 1.80 (dqd, 1H, J ) 10.5,
6.9, 1.8 Hz), 2.42 (qd, 1H, J ) 6.9, 3.0 Hz), 3.98 (ddd, 1H, J ) 8.7,
4.8, 1.8 Hz), 4.26 (dd, 1H, J ) 10.8, 3.0 Hz); 13C NMR (CD3OD) δ
7.8, 8.7, 10.5, 11.4, 28.6, 35.9, 39.0, 71.1, 79.4, 98.0, 172.1, 174.1; IR
(film) 3178 (w, br), 2973 (m), 2938 (m), 2882 (w), 2690 (w, br), 1654
(s, br), 1456 (w), 1390 (m), 1368 (m), 1312 (w), 1123 (m), 993 (m),
966 (w), 766 (w) cm-1; HRMS (EI) m/z calcd for C12H20O4 (M+),
228.1362; found, 228.1355.
afford 61 mg (0.17 mmol, 58% yield) of fine white needles. TLC Rf )
1
0.25 (20% EtOAc/hexanes); [R]25 -14.8 (c ) 0.894, CH2Cl2); H
D
NMR (CD3OD) δ 0.0 (s, 3H), 0.09 (s, 3H), 0.83 (d, 3H, J ) 7.2 Hz),
0.87 (s, 9H), 0.88 (t, 3H, J ) 7.5 Hz), 1.10 (d, 3H, J ) 6.9 Hz), 1.50-
1.63 (m, 2H), 1.70 (s, 3H), 1.88 (dqd, 1H, J ) 10.5, 6.9, 1.5 Hz), 2.44
(qd, 1H, J ) 6.9, 3.0 Hz), 4.14 (ovlp ddd, 1H, J ) 8.7, 6.3, 1.8 Hz),
4.18 (dd, 1H, J ) 10.8, 2.7 Hz); 13C NMR (CD3OD) δ -4.4, -3.9,
7.3, 8.7, 10.5, 10.6, 19.0, 26.5, 29.0, 36.0, 37.2, 67.9, 79.1, 98.0, 171.8,
174.2; IR (film) 2957 (s), 2930 (s), 2884 (m), 2857 (m), 1783 (m),
1733 (s), 1653 (m), 1461 (m), 1388 (s), 1257 (s), 1128 (s), 1027 (m),
835 (s), 775 (s) cm-1; HRMS (EI) m/z calcd for C18H34O4Si (M+),
342.2226; found, 342.2221.
Acknowledgment. This research was supported by grants
from NIH (GM48562) and NSF (NSF/BES-0118926) to D.H.S.
(5R,6S)-4-Hydroxy-6-[(1S,2R)-2-hydroxy-1-methyl-butyl]-3,5-di-
methyl-5,6-dihydro-pyran-2-one (15). Protected tetraketide 23 (23.5
mg, 0.069 mmol) was stirred with Dowex 50WX2-H+ resin (100 mg)
in THF:H2O (1:1, 2 mL) at 50 °C for 20 h. The Dowex 50WX2 resin
was prepared as follows: the resin was treated with 6 N aq HCl, washed
with H2O until the filtrate was pH 5.0, and then air-dried for 10 min.
The reaction mixture was filtered through a plug of cotton and
concentrated under reduced pressure to 15.7 mg (0.069 mmol, 100%
yield) of a white solid. The product is sparingly soluble in CDCl3 and
moderately soluble in MeOH. TLC Rf ) 0.23 (40% EtOAc/hexanes);
[R]25D 68 (c ) 0.66, MeOH); 1H NMR (CDCl3) exists as a 5.5:1 mixture
of keto:enol tautomers. Data for keto tautomer δ 0.86 (d, 3H, J ) 6.9
Hz), 1.00, (t, 3H, J ) 7.5 Hz), 1.13 (d, 3H, J ) 7.2 Hz), 1.34 (d, 3H,
J ) 6.6 Hz), 1.39-1.68 (m, 3H, C-8 CH2 + OH), 1.91 (ddq, 1H, J )
Supporting Information Available: Normalized V vesus [S]
plots for the reactions of (2S,3R)-diketide 3 with PikAIII and
PikAIV as well as (2R,3S)-diketide 4 with PikAIV. GC-MS
trace and conditions for compound 7 isolated from in vitro
reaction of (2S,3R)-diketide 3 with PikAIII. LC-MS traces and
HPLC conditions of compounds 11 and 15 isolated from in vitro
1
reactions with PikAIII + PikAIV. H NMR and 13C NMR
spectra of all compounds reported in Schemes 1 and 2. Full
experimental details and characterization for 8, 12, 16, and
intermediates required for their synthesis. This material is
available free of charge via the Internet at http://pubs.acs.org.
JA034841S
9
J. AM. CHEM. SOC. VOL. 125, NO. 41, 2003 12557