Precursor-Directed Biosynthesis: Stereospecificity for Branched-Chain Diketides of the β-Ketoacyl-ACP Synthase Domain 2 of 6-Deoxyerythronolide B Synthase

Article abstract of DOI:10.1002/hlca.200390327

Modular polyketide synthases such as 6-deoxyerythronolide B synthase (DEBS) catalyze the biosynthesis of structurally complex natural products. Streptomyces coelicolor CH999/pJRJ2 harbors a plasmid encoding DEBS(KS1 ⁰), a mutant form of 6-deoxyerythronolide B synthase that is blocked in the formation of 6-deoxyerythronolide B (1, 6-dEB) due to a mutation in the active site of the ketosynthase (KS1) domain that normally catalyzes the first polyketide chain-elongation step of 6-dEB biosynthesis. Administration of (2S,3R,4S)- and (2S,3R,4R)-3-hydroxy-2,4-dimethylhexanoic acid N-acetylcysteamine (SNAC) thioesters (= S-[2-(acetylamino)ethyl] (2S,3R,4S)- and (2S,3R,4R)-3-hydroxy-2,4-dimethylhexanethioates) 3 and 4 in separate experiments to cultures of Streptomyces coelicolor CH999/pJRJ2 led to production of the corresponding (14S)- and (14R)-14-methyl analogues of 6-dEB, 10 and 11, respectively. Unexpectedly, when a 3:2 mixture of 4 and 3 was fed under the same conditions, exclusively branched-chain macrolactone 11 was isolated. In similar experiments, feeding of 3 and 4 to S. coelicolor CH999/pCK16, an engineered strain harboring DEBS1 + TE(KS1⁰), resulted in formation of the branched-chain triketide lactones 13 and 14, while feeding of the 3:2 mixture of 4 and 3 gave exclusively 14. The biochemical basis for this stereochemical discrimination was established by using purified DEBS module 2 + TE to determine the steady-state kinetic parameters for 3 and 4, with the k_cat/K_M for 4 shown to be sevenfold greater than that of 3.

Full text of DOI:10.1002/hlca.200390327

Helvetica Chimica Acta Vol. 86 (2003)
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Precursor-Directed Biosynthesis: Stereospecificity for Branched-Chain
Diketides of the b-Ketoacyl-ACP Synthase Domain 2 of
6-Deoxyerythronolide B Synthase
by Kenji Kinoshitaa), Chaitan Khoslab), and David E. Cane*a)
a) Department of Chemistry, Box H, Brown University, Providence, Rhode Island 02912-9108, USA
(e-mail: David Cane@brown.edu)
b) Departments of Chemical Engineering, Chemistry, and Biochemistry, Stanford University, Stanford,
California 94305-5025, USA
Dedicated with thanks and admiration to Professor Duilio Arigoni, teacher and friend, on the occasion of his
75th birthday
Modular polyketide synthases such as 6-deoxyerythronolide B synthase (DEBS) catalyze the biosynthesis
of structurally complex natural products. Streptomyces coelicolor CH999/pJRJ2harbors a plasmid encoding
DEBS(KS10), a mutant form of 6-deoxyerythronolide B synthase that is blocked in the formation of 6-
deoxyerythronolide B (1, 6-dEB) due to a mutation in the active site of the ketosynthase (KS1) domain that
normally catalyzes the first polyketide chain-elongation step of 6-dEB biosynthesis. Administration of
(2S,3R,4S)- and (2S,3R,4R)-3-hydroxy-2,4-dimethylhexanoic acid N-acetylcysteamine (SNAC) thioesters (ˆ S-
[2-(acetylamino)ethyl] (2S,3R,4S)- and (2S,3R,4R)-3-hydroxy-2,4-dimethylhexanethioates) 3 and 4 in separate
experiments to cultures of Streptomyces coelicolor CH999/pJRJ2led to production of the corresponding (14 S)-
and (14R)-14-methyl analogues of 6-dEB, 10 and 11, respectively. Unexpectedly, when a 3 :2mixture of 4 and 3
was fed under the same conditions, exclusively branched-chain macrolactone 11 was isolated. In similar
experiments, feeding of 3 and 4 to S. coelicolor CH999/pCK16, an engineered strain harboring DEBS1 ‡
TE(KS10), resulted in formation of the branched-chain triketide lactones 13 and 14, while feeding of the 3 :2
mixture of 4 and 3 gave exclusively 14. The biochemical basis for this stereochemical discrimination was
established by using purified DEBS module 2 ‡ TE to determine the steady-state kinetic parameters for 3 and 4,
with the kcat/KM for 4 shown to be sevenfold greater than that of 3.
Introduction.
Modular polyketide synthases (PKSs) are large (Mr 100
1,000 kDa) multifunctional enzyme systems that are responsible for the biosynthesis
of structurally complex natural products from simple building blocks such as malonyl-,
methylmalonyl-, ethylmalonyl-, and methoxymalonyl-CoA [1]. Polyketide natural
products possess a wide variety of pharmacologically important activities, including
antibiotic, antifungal, antiparasitic, antitumor, and immunosuppressive properties [2].
Modular polyketide synthases are organized into coordinated groups of active sites,
known as modules, in which each module is responsible for one complete cycle of
polyketide chain extension and functional-group modification [3]. The individual
modules are, in turn, made up of a set of catalytic domains of 100 400 amino acids,
each of which is similar in both function and amino acid sequence to the analogous
enzymes of fatty acid biosynthesis. The polyketide chain-building step is a decarbox-
ylative condensation reaction catalyzed by a b-ketoacyl-ACP synthase (b-ketosynthase,
KS) domain [4]. Almost all PKS modules possess a core set of three domains: a b-
ketosynthase (KS), an acyl transferase (AT), and an acyl carrier protein (ACP)

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domain. (Interestingly, Shen and co-workers have recently reported that the individual
modules of the gene cluster encoding leinamycin biosynthesis lack their own AT
domains [5].) The degree of functional-group modification taking place during each
chain-elongation cycle is determined by the specific combination of additional b-keto
reductase (KR), dehydrase (DH), and enoyl reductase (ER) domains found in each
module. Further structural diversity arises from the use of different starter and chain-
elongation units as well as the generation of new stereogenic centers. Modular PKSs are
responsible for the biosynthesis of the polyketide precursors of a wide variety of
metabolites including erythromycin [6], methymycin [7], tylosin [8] epothilone [9],
rapamycin [10] and rifamycin [11].
The best-understood modular PKS is 6-deoxyerythronolide B synthase (DEBS)
from Saccharopolyspora erythraea, which catalyzes the formation of 6-deoxyerythro-
nolide B (6-dEB, 1), the parent macrolide aglycone of the broad-spectrum antibiotic
erythromycin A [3] [6] (Fig. 1,a). The formation of 6-dEB involves six condensation
steps beginning with a propionyl-CoA starter unit and six methylmalonyl-CoA
extender units. The six modules of DEBS are organized into three bimodular,
homodimeric proteins, DEBS1, DEBS2, and DEBS3, each of subunit Mr > 330 kDa
[12]. At the N-terminus of module 1 in DEBS1 is a specialized set of AT and ACP
domains that, together, are responsible for loading of the propionyl-CoA primer unit.
The C-terminus of DEBS3 carries a thioesterase (TE) domain that releases the
heptaketide product from ACP6 by cyclization to the 14-membered macrolactone
6-dEB (1).
Fig. 1. Modular organization of DEBS. a) Each of the six modules contains
a ketosynthase (KS),
acyltransferase (AT), and acyl carrier protein (ACP), as well as variable numbers of supplementary domains,
such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER). The loading didomain primes
module 1 and a thioesterase (TE) domain cyclizes the heptaketide and generates 6-dEB (1). b) Formation of 6-
dEB by DEBS(KS10) is blocked but can be restored by addition of the diketide-SNAC 2.

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Expression of the entire set of DEBS structural genes in heterologous hosts such as
Streptomyces coelicolor CH999 or engineered strains of Escherichia coli, which carry
auxiliary genes for post-translational pantotheinylation of the individual ACP domains
of the PKS and for biosynthesis of the necessary propionyl-CoA and methylmalonyl-
CoA building blocks, results in accumulation of substantial titers of 6-dEB [13][14].
Inactivation by site-directed mutagenesis of the KS1 domain that normally catalyzes
the first condensation step of 6-dEB biosynthesis gives a mutant construct,
DEBS(KS10), that no longer produces 6-dEB. Formation of 6-dEB (1) can be
restored, however, when (2S,3R)-3-hydroxy-2-methylpentanoic acid-SNAC thioester
(2), an N-acetylcysteamine (ˆ2-(acetylamino)ethanethiol, HSNAC) thioester deriv-
ative of the natural diketide chain elongation intermediate, is administered to cultures
of S. coelicolor CH999 harboring plasmid pJRJ2encoding DEBS(KS1 0) [15a]
(Fig. 1,b). Moreover, by utilizing surrogate diketide and triketide analogues, it is
possible to exploit the apparently broad substrate specificity of the DEBS KS2domain
to generate a wide range of 6-dEB derivatives [15]. We have recently extended the
versatility of this precursor-directed biosynthesis technology by the development of an
E. coli strain harboring a DEBS mutant from which the entire loading didomain and
DEBS module 1 have been deleted [15e].
To further explore the utility of this methodology and to better define the substrate
specificity of the DEBS KS2domain, we have now extended these studies to an
investigation of the utilization of the diastereoisomeric branched-chain diketide
substrates 3 and 4. Both 3 and 4 share a common substitution pattern and configuration
at C(2) and C(3) with the natural diketide 2. Diketides 3 and 4 can be thought of as
diastereoisomeric branched-chain analogues of 2 or, alternatively, as 5-deoxy-6-
demethyl analogues of a triketide intermediate. The results of our studies are detailed
below.
Results. Preparation of the Branched-Chain Diketide Analogues 3 and 4. The
branched-chain diketide substrate (2S,3R,4S)-3-hydroxy-2,4-dimethylhexanoic acid N-
acetylcysteamine (SNAC) thioester 3 was readily prepared from the known (S)-2-
methylbutanal ((S)-5) by using the chiral oxazolidinone method of Evans et al. [16] to
prepare the acid 7 followed by conversion to the corresponding thioester 3 (Scheme 1).
The diastereoisomeric (2S,3R,4R)-thioester 4 was in turn synthesized from racemic
(Æ)-5, by using silica-gel chromatography to separate the resulting mixture of
diastereoisomeric acyloxazolidinones 6 and 8. The (4R)-epimer 8 was then converted
to the corresponding (2S,3R,4R)-3-hydroxy-2,4-dimethylhexanoic acid SNAC thioester
4. In like manner, the mixture of 8 and 6 was also used to prepare a 3 :2mixture of
(2S,3R,4R)-4 and (2S,3R,4S)-3.
Feeding of the Branched-Chain Diketides to S. coelicolor CH999/pJRJ2. According
to previously developed protocols [15d], the diastereoisomeric branched-chain
diketide substrates 3 and 4 were individually fed to 3-day-old agar cultures of S.
0
coelicolor CH999/pJRJ2harboring DEBS(KS1 ). After an additional 4 d incubation,
the combined mycelia and media from 20 plates (25 ml each) were homogenized and
extracted with AcOEt, and the resulting polyketide products were purified from the
concentrated extract by SiO2 column chromatography and preparative TLC. The
isolated macrolactones were analyzed by a combination of 1D- and 2D-NMR

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Scheme 1. Synthesis of Branched-Chain Diketide-SNAC Esters. A) Preparation of (4S)-3; B) preparation of
(4R)-4; C) preparation of 3 :2mixture of 4 and 3.
a) Bu2BOTf, (i-Pr)2NEt, (4S)-4-benzyl-3-propanyl-oxazolidinone. b) LiOH, H2O2. c) i. (PhO)2PN3, Et3N; ii.
HSNAC. d) SiO2 Column chromatography.
spectroscopy and HR-FAB-MS, as well as by comparison to the spectra of 6-dEB (1).
Both branched-chain diketide substrates 3 and 4 were recognized by DEBS module 2
and processed by the downstream modules to give the expected macrolactone products,
the (14S)-13-(s-butyl) 6-dEB analogue 10 (6% yield, based on diketide 3) and the
(14R)-13-(s-butyl) 6-dEB analogue 11 (25% yield, based on diketide 4; Scheme 2).
Unexpectedly, feeding of the 3 :2(4 R/4S) mixture of 4 and 3 under the same conditions
gave a single macrolide product (40% yield, based on diketide 4), identical in all
respects to the sample of 11 that had been obtained from the feeding of the (4R)-
branched-chain diketide thioester 4 alone.
Feeding of the Branched-Chain Diketides to S. coelicolor CH999/pCK16. S.
coelicolor CH999/pCK16 is an engineered strain harboring DEBS1 ‡ TE(KS10),
corresponding to the loading didomain and the first two modules of DEBS, but with an
inactive KS1 domain [17]. Feeding of the natural diketide 2 to S. coelicolor CH999/
pCK16, which can bypass the biochemical block in module 1, has been shown to result
in production of the well-known triketide lactone 12, the product of TE-catalyzed
cyclization of the acyclic triketide acyl-ACP intermediate normally generated by DEBS
module 2( Scheme 3). Administration of the individual branched-chain diketides 3 and
4 to agar cultures of S. coelicolor CH999/pCK16 gave the predicted triketide lactones 13
and 14, respectively, isolated in 10 and 50% yield based on administered branched-
chain diketide diastereomer (Scheme 3). Similar to the results obtained with the
complete DEBS(KS10) system, feeding of the 3 :2mixture of 4 and 3 to S. coelicolor
CH999/pCK16 yielded only triketide lactone 14 as a single component (60% yield

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Scheme 2. Conversion of Branched-Chain Diketide-SNAC Esters to 14-Methyl-6dEb Analogues by S. coelicolor
CH999/JRJR Harboring DEBS(KS10)
based on 4), identical to that obtained from the feeding the (4R)-branched-chain
diketide thioester 4.
Synthesis of Triketide Lactones. To confirm the structure and the configurational
assignments of the triketide lactones 13 and 14 that were produced by precursor-
directed biosynthesis from branched-chain diketides with S. coelicolor CH999/pCK16,
authentic samples of both 13 and 14 were each prepared directly from the individual
acyloxazolidinone intermediates 6 and 8, respectively (Scheme 4). The b-OH groups of
each diastereoisomer were protected with (t-Bu)Me2SiOTf (TBDMSOTf), affording
the TBDMS ethers 15 and 16, which were converted to the aldehydes 19 and 20,
respectively, by straightforward LiAlH4 reduction followed by PDC oxidation. These
aldehydes each served as substrates for a second asymmetric aldol reaction, yielding
compounds 21 and 22, respectively, which were each cleaved from the chiral auxiliary
with LiOH/H2O2. Treatment of the resulting carboxylic acids 23 and 24 with HF
1
effected silyl ether cleavage and subsequent lactonization to form 13 and 14. The H-
and 13C-NMR spectra of the individual synthetic triketide lactones 13 and 14 were

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Scheme 3. Conversion of Diketide-SNAC Esters to Triketide Lactones by S. coelicolor CH999/pCK16
Harboring DEBS1 ‡ TE(KS10)
Scheme 4
a) TBDMSOTf, (i-Pr)2EtN. b) LiAlH4. c) PDC. d) Bu2BOTf, (i-Pr)2NEt, (4R)-4-benzyl-propanoyloxazol-
idinone. e) LiOH, H2O2. f) HF.

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identical to those of the compounds obtained from the feeding of the branched-chain
diketides 3 and 4 to S. coelicolor CH999/pCK16.
Enzymatic Conversion of Branched-Chain Diketides to Triketide Lactones. To
probe further the biochemical basis for the apparent stereoselectivity for the (4R)-
methyl branched-chain diketide 4 compared to its diastereoisomer, (4S)-3, we carried
out individual incubations of both 3 and 4 with isolated DEBS module 2 ‡ TE [18] in
the presence of [2-14C]methylmalonyl-CoA and NADPH to give the expected triketide
lactones 13 and 14, as determined by radio-TLC/phosphoimaging (Scheme 5). We then
Scheme 5. Enzymatic Conversion of Diketide-SNAC Esters to Triketide Lactones Catalyzed by DEBS Module
2 ‡ TE
carried out a series of incubations with varying concentrations of each substrate
(Fig. 2), and fit the resulting data to the MichaelisÀMenten equation to calculate the
characteristic kcat, KM, and kcat/KM values for each branched-chain diketide (Table 1).
For comparison, we also measured the steady-state kinetic parameters for the natural
diketide 2 by using the same enzyme preparations of DEBS module 2 ‡ TE. Notably, at
concentrations above 1 mm (ca. 2times KM), (4R)-methyl diketide 4 showed
pronounced substrate inhibition (Fig. 2,c). The relevant steady-state parameters for
4 were, therefore, calculated from the data obtained at concentrations of 4 ꢀ 1 mm.
The specificity constant kcat/KM for each substrate could also be calculated directly from
the slope of the V vs. [S] curve at low substrate concentrations (0.1 1 mm).
Comparison of the relative kcat/KM values revealed that the specificity for (4S)-methyl
3 was similar to that of the natural diketide substrate 2, while the (4R)-methyl
diastereoisomer 4 showed a sevenfold increase in kcat/KM relative to 3 (5.5-fold relative
to 2).
Competition experiments were also carried out with DEBS module 2 ‡ TE in which
variable concentrations of natural diketide 2 were incubated with [2-14C]methylmal-

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Fig. 2. Plot of V vs. [S] for incubation of branched-chain diketide-SNAC substrates with DEBS module 2 ‡ TE.
a) (4S)-Methyl diketide 3; b) (4R)-methyl diketide 4, 0.15 1.0 mm; c) (4R)-methyl diketide 4, 0.15 10.0 mm,
showing substrate inhibition
onyl-CoA and NADPH in the presence of 0, 0.2, and 0.5 mm 4, with the formation of
triketide lactone 12 assayed by TLC/phosphoimaging. As is evident from the double
reciprocal LineweaverÀBurk plot for these reactions (Fig. 3), 4 behaved as a typical
noncompetitive inhibitor, based on the observed decrease in Vmax but absence of

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Table 1. Steady-State Kinetic Parameters for Incubation of Branched-Chain Diketides with DEBS Module 2‡ TE
Substrate
KM
[mm]
kcat
[minÀ1
kcat/KM
[mmolÀ1minÀ1
kcat/KM
[rel]
]
]
2a)
(4R)-3
(4S)-4
4.37 Æ 0.61
2.90 Æ 0.13
0.49 Æ 0.04
1.56 Æ 0.120.36
80.82 Æ 0.020.2
0.96 Æ 0.04
100
78
1.98
550
a) The steady-state parameters previously determined for 2 for DEBS module 2 ‡ TE were KM > 3.2m m, kcat
4.6 minÀ1, kcat/KM 0.75 mmolÀ1 minÀ1 [18].
>
change in the apparent Vmax/KM for 2. Direct fitting of these limited data to the
appropriate version of the MichaelisÀMenten equation gave a calculated Ki of ca.
0.3 mm for 6 vs. natural diketide 2, close to the independently measured KM for 4 of
0.5 mm.
Fig. 3. LineweaverÀBurk plot showing noncompetitive inhibition by branched-chain diketide 4 for processing of
natural diketide 2 by DEBS module 2 ‡ TE
Discussion. The use of modular polyketide synthases blocked in specific reactions
in combination with surrogate substrates has provided a powerful tool for the
engineered biosynthesis of novel analogues of medicinally important natural products
[15]. A major requirement for the successful implementation of this promising tool is a
better understanding of the structural and configurational features that influence the
processing of substrate analogues by individual polyketide synthase modules.
DEBS(KS10) is a mutant of the parent DEBS PKS in which the b-ketoacyl-ACP
synthase domain of module 1 (KS1) has been inactivated by replacement of the active-
site cysteine by alanine [15a]. A variety of analogues of the natural (2S,3R)-3-hydroxy-
2-methylpentanoyl-SACP diketide, the normal product of module 1, have been fed as
the corresponding SNAC thioesters to cultures of S. coelicolor harboring plasmid
0
pJRJ2that encodes DEBS(KS1 ), resulting in the formation of the corresponding
structural variants of 6-deoxyerythronolide B (1) [15].
Although feeding analogues of natural biosynthetic intermediates to strategically
blocked PKS mutants harbored within intact microbial hosts has substantial utility for

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the controlled generation of novel bioactive polyketides, only qualitative information
about relative substrate specificity can be gleaned from comparison of the relative
product yields, due to lack of information about numerous experimental variables,
including differences in precursor stability, uptake, competing metabolism, and end-
product stability, as well as variations in in vivo enzyme expression and activity.
Fortunately, the availability of substantial quantities of recombinant PKS modules,
expressed and purified from E. coli, has made possible quantitative evaluation of
substrate specificity under well-defined experimental conditions. In comparing the
behaviors of different substrate analogues with a single module, the most-informative
kinetic parameter is the specificity constant, kcat/KM. Since kcat/KM for any enzyme is a
function of only individual rate constants up to and including the first irreversible
reaction and since the KS-catalyzed decarboxylative condensation is effectively
irreversible, it is evident that the observed kcat/KM values for diketides 2, 3, and 4
reflect exclusively the properties of the KS domain of DEBS module 2.
Significantly, both diastereoisomeric branched-chain diketides, 3 and 4, when fed
individually to S. coelicolor CH999 harboring either DEBS(KS10) or DEBS1 ‡
TE(KS10), were converted efficiently to the corresponding branched-chain analogues
of 6-dEB, 10 and 11, or to the triketide lactones 13 and 14, respectively (Schemes 2 and
3). Surprisingly, however, when 3 :2mixtures of 4 and 3 were fed to the same strains of
S. coelicolor, the only isolated lactone products were the single diastereomers 11 and
14, respectively, corresponding effectively to exclusive mobilization of the (4R)-4-
methyl diketide 4.
The biochemical basis for this apparent kinetic resolution of the diastereoisomeric
mixture of branched-chain diketides is evident by comparison of the relative kcat/KM
values for processing of the individual substrates by isolated DEBS module 2 ‡ TE,
with 4 being favored over 3 by a ratio of > 7:1, reflecting an intrinsic preference of
DEBS KS2for the (4 R)-methyl substrate 4. At a diastereoisomeric ratio of 3 :2, this
difference in relative kcat/KM should translate into a competitive kinetic advantage for 4
of > 10 :1. That the preferred branched-chain diketide substrate 4 can act as a
noncompetitive inhibitor of the diketide 2 at concentrations < 1 mm requires that 4
bind to both free protein as well the enzyme-substrate complex of DEBS module 2 ‡
TE and 2 with equal affinity. Although the detailed molecular mechanism for this non-
competitive inhibition is not known, it is conceivable that binding of branched-chain
diketide 4 to one KS2domain of the DEBS module 2 ‡ TE homodimer could influence
turnover of a diketide 2 by the paired KS2domain. Such cross-talk between the paired
KS2domains could also account for the marked substrate self-inhibition displayed by 4
(Fig. 2,c). Alternatively, the observed noncompetitive inhibition of chain elongation of
2 by 4 could also be accounted for by a relatively rapid rate of acylation but slower rate
of release of 4 by the TE domain as compared to 2. The DEBS TE domain is known to
recognize and hydrolyze substituted acyl thioesters, and shows a markedly higher
specificity for longer chain substrates such as 4 relative to shorter-chain substrates such
as 2 [19]. Since the TE must recognize the distal end of the substrate chain, it is,
therefore, possible that the branched-chain diketide might remain more tightly bound
to the TE than would diketide 2. Whatever the mechanism of non-competitive
inhibition of turnover of natural diketide 2 by branched-chain diketide 4, it is likely that
4 might also act as a noncompetitive inhibitor of the turnover of branched-chain

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diketide diastereoisomer 3, whose steady-state kinetic parameters are similar to those
of natural diketide 2. (Direct measurement of such inhibition using TLC/phosphoi-
maging was complicated by the very similar TLC Rf values of branched-chain triketide
lactones 13 and 14.) Such noncompetitive inhibition would further enhance the
intrinsic kinetic advantage for 4 over 3 in processing of the 3 :2mixture by
DEBS(KS10) or DEBS1 ‡ TE(KS10). In any event, formation of minor amounts
(< 5% relative yield) of the diastereoisomeric lactones 10 and 13 might well have gone
undetected by the isolation and analytical procedures used in the precursor-directed
biosynthesis experiments described.
It is interesting to compare the sevenfold relative preference in kcat/KM for (4R)-4-
methyl-4 over (4S)-4-methyl-3 exhibited by DEBS KS2with the striking diastereoiso-
meric differentiation shown by DEBS KS2towards the unsaturated triketides 25 28
(Scheme 6) [20]. We have previously reported that both 25a and 26 are efficiently
processed to the corresponding tetraketides 29a and 30 by DEBS module 2 ‡ TE, with
relative kcat/KM values compared to natural diketide 2 (kcat/KM ˆ 100) of 120 and 23,
respectively. Significantly, both 25a and 26 have the same d-methyl configuration at
C(4) as the preferred branched-chain diketide 4. Similarly, the 6-demethyl analogue
25b, which has the same d-methyl configuration at C(4), is also a good substrate for
DEBS module 2 ‡ TE, with a kcat/KM(rel) of 71, while the 4-demethyl analogue 25c has
a kcat/KM(rel) of 83. By contrast, neither unsaturated triketide 27 nor 28, with l-methyl
substituents at C(4), is a substrate for DEBS module 2 ‡ TE.
Scheme 6. Processing of Unsaturated Triketide Analogues by DEBS Module 2 ‡ TE

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Marsden et al. have recently used a chimeric form of DEBS1 ‡ TE (−DEBS1-TE× in
their terminology) in which the natural DEBS loading didomain, ATL-ACPL, has been
replaced by the corresponding loading didomain of the avermectin PKS (AVR_lm) in
order to generate polyketides derived from branched-chain starters [21]. The
avermectins are tridecaketide macrolides produced by S. avermitilis, and are
themselves derived from either a (2S)-2-methylbutyrate (avermectin B1a (31)) or an
isobutyrate (avermectin B1b (32)) starter unit, the (2S)-2-methylbutyryl-CoA
originating from (2S,3S)-isoleucine (Scheme 7) [22]. In fact, the avermectin loading
domain is known to tolerate a broad range of carboxylic acid starter units, and this
natural promiscuity had previously been exploited to generate a variety of avermectin
analogues derived from novel starter units [23]. Consistent with this permissiveness
toward starter-unit structure, strains of S. coelicolor CH999 harboring the hybrid
AVR_lm ‡ DEBS1 ‡ TE produced a 8 :2:2:5 mixture of four triketide lactones 12, 33,
34, and 35, in which 35 carried an s-Bu substituent of unspecified configuration
(Scheme 8) [21]. The latter branched-chain triketide lactone was reported to be
identical to a synthetically prepared lactone, of known structure but unassigned
configuration at C(6) [21][24]. In fact, comparison of the 1H-NMR spectrum reported
by Marsden et al. [21] for 35 with the spectra of each of the authentic triketide lactones
13 and 14 has now established the identity of 35 and 14, thereby allowing unambiguous
assignment of the (6R)-configuration to the s-Bu side chain of 35 (Table 2).
Scheme 7. Biosynthesis of Avermectins B1b (31) and B1a (32) by S. avermitilis from Isobutyryl-CoA and (2S)-
Methylbutyryl-CoA Derived from Valine and Isoleucine, Respectively
1
Table 2. Comparison of H-NMR Data for Branched-Chain Triketide Lactones
H-Atom
d
13
35a)
14
HÀC(5)
HÀC(6)
HaÀC(7)
HbÀC(7)
MeÀC(6)
3.85
1.73
1.921.48
1.24
0.89
3.83
1.74
3.86
1.75
1.51
1.09
1.08
1.05
1.10
a) Supplemental Material: [21].

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Scheme 8. Formation of Triketide Lactones by AVR_lm ‡ DEBS1 ‡ TE
Interestingly, 35 (ˆ14) can only be derived from a (2R)-2-methylbutyryl-CoA starter,
rather than the (2S)-2-methylbutyryl-CoA originating from isoleucine. Thus, although
the chimeric AVR_lm ‡ DEBS1 ‡ TE produces a mixture of four triketide lactones,
derived more or less indiscriminately from acetate, propionate, isobutyrate, and
2-methylbutyrate starters, it is only the (2R)-3-methylbutyrate enantiomer that is
utilized by the chimeric AVR_lm ‡ DEBS PKS. It has already been shown that the
avermectin loading domain can utilize either enantiomer of the 2-methylbutyrate
starter [23b]. The observed chiral discrimination leading to exclusive formation of
(6R)-methyl triketide lactone 35 (ˆ14) by the hybrid AVR_lm ‡ DEBS1 ‡ TE cannot,
therefore, be due to AVR_lm, nor can it be attributed to DEBS KS2, since DEBS1
would be already committed irreversibly to synthesizing polyketide by the time that the
(4S)-4-methyl branched-chain diketide intermediate is generated and presented to
DEBS KS2. The apparently strict discrimination against the (2S)-2-methylbutyrate
starter must, therefore, be due to the intrinsic stereoselectivity of the downstream
DEBS KS1 domain.
Tolerance of unnatural intermediates has been widely observed in genetically
engineered PKSs. [25]. The results with branched-chain diketides demonstrate the
utility of precursor-directed biosynthesis as a method for the controlled generation of
novel polyketides having a stereogenic chiral center at C(14) of erythromycin A.
Experimental Part
General. Q Sepharose fast-flow resin was purchased from Pharmacia. TLC was carried out on Whatman
silica-gel plates (AL SIL G/UV). [2-14C]Methylmalonyl-coenzyme A (54 mCi/mmol) was purchased from
American Radiolabeled Chemicals, Inc. All other commerical reagents, buffer components, and enzymes were
purchased from Sigma-Aldrich Chemical Co. and were of the highest purity available. Culture medium
components were purchased from Difco. NMR Spectra were recorded on a Bruker Avance 300 spectrometer.
High-resolution mass spectra HR-MS were measured on a Kratos MS80RFA mass spectrometer. Quantification
of radio-TLC components for kinetic assays utilized a BioRad GS-363 Molecular Imager and Molecular Analyst
software calibrated with standards of known radioactivity. Protein concentration was determined by the method
of Bradford [26] with the kit from BioRad. Kinetic data were analyzed by direct fitting to the relevant version of
the MichaelisÀMenten equation with the KaleidaGraph data analysis software (Synergy Software).
(4S)-4-Benzyl-3-[(2S,3R,4RS)-3-hydroxy-2,4-dimethylhexanoyl]oxazolidin-2-one (3 :2mixture 8/6). To a
soln. of 1.0 g (4.26 mmol, 1.0 equiv.) of (4S)-4-benzyl-3-propanoyloxazolidin-2-one in 20 ml of CH2Cl2 at 08
under N2 was added 4.7 ml of Bu2BOTf (1.0m in CH2Cl2, 1.1 equiv.), followed by the addition of 0.9 ml EtN(i-
Pr)2 (5.12mmol, 1.2equiv.). The soln. was stirred at 0 8 for 30 min, then cooled to À788 (dry ice/acetone), stirred
for 30 min, and 690 ml of freshly distilled (Æ)-2-methylbutanal (6.39 mmol, 1.5 equiv.) was added rapidly via
syringe. The resulting soln. was stirred for 2h at À788, then for 1.5 h at 08 before quenching with 20 ml of MeOH

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Helvetica Chimica Acta Vol. 86 (2003)
and 50 mm phosphate buffer (pH 7.2). After 5 min, 10 ml of 30% aq. H2O2 was added dropwise. After being
stirred an additional 20 min at 08, the mixture was extracted with 3 Â 2 0 ml CHCl2, dried (Na2SO4), and
2
concentrated. Purification by flash column chromatography (CC; SiO2; 20% AcOEt/hexanes) afforded 950 mg
of a 3 :2mixture 8/6 as a colorless oil (70% yield). Rf (20% AcOEt/hexanes) 0.28. [a]D ˆ ‡31.2( c ˆ 1.0,
CHCl3); The IR, and 1H- and 13C-NMR data for the mixture corresponded to the sum of the spectra of the
individual components 8 and 6, whose spectroscopic data are given below. HR-FAB-MS (NBA/NaI): 342.1693
‡
([M ‡ Na] , (C18H25NO4)Na ‡ ; calc. 342.1681).
(2S,3R,4RS)-3-Hydroxy-2,4-dimethylhexanoic Acid (3 :2mixture 9/7). To a stirred soln. of 350 mg
(1.10 mmol, 1.0 equiv.) of the 3 :2mixture 8/6 in 12ml of THFand 4 ml of H 2O at 08 was added under N2 1.06 ml
(8.8 mmol, 8 equiv.) of 30% aq. H2O2, followed by 92 mg (2.20 mmol, 2 equiv.) of LiOH ¥ H2O in one portion.
The resulting mixture was warmed to r.t. overnight before quenching with 1.25 g of Na2SO3 in 5 ml of H2O. THF
was evaporated, and the aq. soln. was extracted with CH2Cl2 (2 Â 20 ml). The aq. layer was adjusted to pH 1.0
with 1n HCl and extracted with AcOEt (3 Â 20 ml). The org. soln. was dried (Na2SO4) and concentrated in
vacuo to yield 135 mg of a mixture 9/7 as a yellow oil, which was used immediately in the next step [16b].
S-[2-(Acetylamino)ethyl] (2S,3R,4RS)-3-Hydroxy-2,4-hexanethioate (3 :2mixture
4/3). Crude
(2S,3R,4RS)-3-hydroxy-2,4-dimethylhexanoic acid (135 mg, 0.84 mmol, 1.0 equiv.) in 5 ml of DMF under N2
was treated with diphenyl phosphorylazide (0.28 ml, 1.26 mmol, 1.5 equiv.) and Et3N (0.24 ml, 1.68 mmol,
2equiv.) at 0 8. After 2h, 210 mg (1.68 mmol, 2equiv.) of N-acetylcysteamine (ˆ2-(acetylamino)ethanethiol)
was added. The mixture was allowed to stir at r.t. for 2h, followed by addition of 5 ml of H 2O and extraction
with AcOEt (3 Â 25 ml). The org. extract was dried (Na2SO4) and concentrated in vacuo. Purification by CC
(SiO2; 60% AcOEt/hexanes) afforded 178 mg (80%) of a 3 :2mixture of (4 R)-4/(4S)-3 as a colorless oil. Rf
(AcOEt) 0.40. The IR and 1H- and 13C-NMR data for the mixture corresponded to the sum of the spectra of the
individual components 4 and 3, whose spectroscopic data are given below. HR-FAB-MS (NBA/NaI): 284.1291
‡
‡
([M ‡ Na] , (C12H23NO3S)Na ; calc. 284.1296).
(4S)-4-Benzyl-3-[(2S,3R,4S)-3-hydroxy-2,4-dimethylhexanoyl]oxazolidin-2-one (6). To a suspension of
3.8 g (10.0 mmol) of pyridinium dichromate in CH2Cl2 was added dropwise 440 mg (5.0 mmol) of (2S)-2-
methylbutan-1-ol at r.t. After 2h, an additional 3.8 g (10.0 mmol) of pyridinium dichromate was added to the
mixture. The mixture was allowed to stir at r.t. overnight and then passed through a short column of Florisil (30
mesh, washed with CH2Cl2). The filtrate was concentrated under reduced pressure on ice water to afford (2S)-2-
methylbutanal (5), which was used immediately in the preparation of 6 by the method described above for the
diastereoisomeric mixture 8/6.
Data of 6: Rf (10% Et2O/CH2Cl2) 0.59. [a]D ˆ ‡41.0 (c ˆ 1.0, CHCl3). IR (film): 3534, 2966, 1778, 1692.
1H-NMR (300 MHz, CDCl3): 0.91 (t, J ˆ 7.3, Me(6')); 0.99 (d, J ˆ 6.6, MeÀC(4')), 1.00 1.20 (m, HbÀC(5'));
1.27 (d, J ˆ 7.0, MeÀC(2')); 1.40 1.58 (m, HÀC(4'), HaÀC(5')); 2.79 (dd, J ˆ 3.4, 9.5, 1 H, PhCH2); 3.26 (dd,
J ˆ 13.5, 3.3, 1 H, PhCH2); 3.69 (dd, J ˆ 7.1, 3.7, HÀC(3')); 3.97 (m, HÀC(2')); 4.21 (m, CH2(5)); 4.71 (m,
HÀC(4)); 7.20 7.38 (m, arom. H); 13C-NMR (75.5 MHz, CDCl3): 177.6; 153.0; 135.2; 129.5; 129.0; 127.5; 75.1;
‡
‡
66.2; 55.2; 40.0; 37.8; 37.3; 25.7; 14.6; 11.4. HR-FAB-MS (NBA/NaI): 342.1692. ([M ‡ Na] (C18H35NO4)Na ;
calc. 342.1681).
S-[2-(Acetylamino)ethyl] (2S,3R,4S)-3-Hydroxy-2,4-dimethylhexanethioate (3). Compound 3 was prepared
from 6 by the same procedure used to prepare the diastereoisomeric mixture 4/3. Rf (AcOEt). 0.40 [a]D ˆ ‡11.7
(c ˆ 1.0, CHCl3). IR (film): 3293, 3079, 2965, 2933, 2876, 1657, 1554. 1H-NMR (300 MHz, CDCl3): 0.92( t, J ˆ 7.4,
3 H); 0.96 (d, J ˆ 6.8, MeÀC(4)); 1.10 1.25 (m, HbÀC(5)); 1.27 (d, J ˆ 7.1, Me ÀC(2)); 1.35 1.55 (m, HaÀC(5),
HÀC(4)); 1.98 (s, COMe); 2.90 (m, HÀC(2)); 3.05 (m, CH2S); 3.46 (m, CH2N); 3.73 (t, J ˆ 5.7, HÀC(3)); 5.84
(br., NH). 13C-NMR (100 MHz, CDCl3): 204.0; 170.5; 75.4; 51.2; 39.5; 37.1; 28.4; 26.0; 23.2; 13.9; 12.4; 10.9. HR-
‡
‡
FAB-MS (NBA/NaI): 284.1290 ([M ‡ Na] , (C12H23NO3S)Na ; calc. 284.1296).
(4S)-4-Benzyl-3-[(2S,3R,4R)-3-hydroxy-2,4-dimethylhexanoyl)oxazolidin-2-one (8). Compound 8 was
separated by CC (SiO2; CH2Cl2) from the 3 :2mixture 8/6. Rf (10% Et2O/CH2Cl2) [a]D ˆ ‡42.5 (c ˆ 1.0,
CHCl3) . IR (film): 3525, 2967, 2878, 1781, 1697. 1H-NMR (300 MHz, CDCl3): 0.87 (d, J ˆ 6.8, MeÀC(4')); 0.93
(t, J ˆ 7.5, Me(6')); 1.10 1.25 (m, HbÀC(5')); 1.26 (d, J ˆ 7.0, MeÀC(2')); 1.45 1.60 (m, HaÀC(5')); 1.75 1.90
(m, HÀC(4')); 2.81 (dd, J ˆ 13.4, 9.5, 1 H, PhCH2); 3.28 (dd, J ˆ 13.5, 3.2, 1 H, PhCH2); 3.64 (dd, J ˆ 9.1, 2.2
HÀC(3')); 3.97 (m, HÀC(2')); 4.22 (m, CH2(5)); 4.73 (m, HÀC(4)); 7.20 7.38 (m, 5 arom. H). 13C-NMR
(75.5 MHz, CDCl3): 177.9; 152.8; 135.0; 129.3; 128.8; 127.3; 74.7; 66.0; 55.0; 38.4; 37.6; 36.9; 25.0; 14.6; 10.8. HR-
‡
‡
FAB-MS (NBA/NaI): 342.1692. ([M ‡ Na] ), (C18H35NO4)Na ; calc. 342.1681.
S-[2-(Acetylamino)ethyl] (2S,3R,4R)-3-Hydroxy-2,4-dimethylhexanethioate (4). Compound 4 was pre-
pared according to the same procedure used to prepare 3. Rf (AcOEt) 0.40: [a]D ˆ ‡16.3 (c ˆ 1.0, CHCl3). IR
(film): 3288, 3081, 2965, 2934, 2877, 1659, 1551. 1H-NMR (300 MHz, CDCl3): 0.86 (d, J ˆ 6.9, MeÀC(4)); 0.93 (t,

Helvetica Chimica Acta Vol. 86 (2003)
3903
J ˆ 7.4, 3 H); 1.10 1.25 (m, HbÀC(5)); 1.21 (d, J ˆ 7.0, MeÀC(2)); 1.35 1.55 (m, HbÀC(5)); 1.70 1.85 (m,
HÀC(4)); 1.98 (s, COMe); 2.89 (m, HÀC(2)); 3.05 (m, CH2S); 3.46 (m, CH2N); 3.69 (dd, J ˆ 8.5, 3.2, HÀC(3));
5.83 (br., NH). 13C-NMR (100 MHz, CDCl3): 204.1; 170.6; 75.6; 50.6; 39.1; 37.1; 28.3; 24.5; 22.8; 15.0; 10.8; 10.1.
‡
‡
HR-FAB-MS (NBA/NaI): 284.1292 ([M ‡ Na] , (C12H23NO3S)Na ; calc. 284.1296).
(4S)-4-Benzyl-3-{(2S,3R,4R)-3-[(tert-butyl)dimethylsiloxy]-2,4-dimethylhexanoyl}oxazolidin-2-one (16).
Compound 8 (230 mg, 0.72 mmol, 1.0 equiv.) was dissolved in 5 ml of CH2Cl2 and cooled to 08. EtN(i-Pr)2
(216 ml, 1.18 mmol, 1.7 equiv.) and TBDMSOTf (208 ml, 1.10 mmol, 1.6 equiv.) were added, and the mixture was
allowed to warm slowly to r.t., then stirred for an additional 2h. The reaction was quenched with 1 ml of brine,
the mixture was extracted with 4 Â 5 ml CH2Cl2, dried (Na2SO4), and concentrated. Purification by CC (SiO2;
5% AcOEt/hexanes) afforded 290 mg (93%) of 16. Colorless oil. 16: Rf (10% AcOEt/hexanes) 0.55. [a]D
ˆ
‡52.8 (c ˆ 1.0, CHCl3). IR (film): 2959, 2858, 1783, 1699, 1460, 1382, 1210, 1117, 1103, 1016, 969, 868, 837, 774,
700 cm. 1H-NMR (300 MHz, CDCl3): 0.06 (s, MeSi); 0.08 (s, MeSi); 0.82 0.99 ( m, Me3C, Me(6'), MeÀC(4'));
1.13 1.32( m, HbÀC(5')); 1.16 (d, J ˆ 6.4, MeÀC(2'); 1.38 1.59 (m, HÀC(4'), HaÀC(5')); 2.78 (dd, J ˆ 13.3, 9.6,
1 H, PhCH2); 3.28 (dd, J ˆ 13.3, 3.1 1 H, PhCH2); 3.88 4.04 (m, HÀC(2'), HÀC(3')); 4.18 (m, CH2(5)); 4.65
(m, HÀC(4)); 7.15 7.38 (m, 5 arom. H). 13C-NMR (75.5 MHz, CDCl3): 176.0; 152.7; 135.2; 129.3; 129.0; 127.2;
76.1; 65.8; 55.6; 40.8; 40.7; 26.0; 24.8; 15.2; 13.8; 12.3; À2.9; À3.4. HR-FAB-MS (NBA/NaI): 456.2539 ([M ‡
‡
‡
Na] , (C24H39NO4Si)Na ; calc. 456.2546.
(2S,3R,4R)-3-[(tert-Butyl)dimethylsiloxy]-2,4-dimethylhexanal (20). Compound 16 (290 mg, 0.67 mmol,
1.0 equiv.) was dissolved in 10 ml of freshly distilled Et2O and cooled to À788. LiAlH4 (1.0 ml of a 1.0m soln. in
hexanes, 1.0 mmol, 1.5 equiv.) was added by syringe, and the mixture was stirred for an additional 30 min and
then warmed to 08. The reaction was quenched successively with 50 ml of H2O, 60 ml of 15% NaOH, and 160 ml of
H2O at 08. After 10 min, the mixture was filtered through a silica-gel layer with Et2O. The filtrate was dried
(Na2SO4), and concentrated in vacuo. The residue, which consisted of a mixture of 18 and 20, was used
immediately for the next reaction (18: Rf (10% AcOEt/hexanes) 0.31. To a stirred suspension of 1.0 g
(2.64 mmol) of pyridinium dichromate in 10 ml of CH2Cl2 was added dropwise a mixture 18/20 in 5 ml of
CH2Cl2. After 16 h, the mixture was passed through a short column of Florisil (30 mesh, 20 g) with 5% AcOEt/
hexanes, and the filtrate was concentrated in vacuo to afford 36 mg (58% based on 16) of 20. Rf (10% AcOEt/
hexanes) 0.72. IR (film): 2960, 2860, 1709. 1H-NMR (300 MHz, CDCl3): À0.06 (s, MeSi); 0.00 (s, MeSi); 0.79
0.87 (m, Me3C, Me(6), MeÀC(4)); 1.04 (d, J ˆ 6.9, MeÀC(2)); 0.97 1.12 (m, HbÀC(5)); 1.40 1.65 (m,
HÀC(4), HaÀC(5)); 2.32 2.45 (m, HÀC(2)); 3.96 4.01 (m, HÀC(3)); 9.75 (br., CHO). 13C-NMR (75.5 MHz,
CDCl3): 205.3; 74.9; 50.0; 39.7; 25.9; 25.2; 14.1; 8.6; À4.2.
(4R)-4-Benzyl-3-{(2R,3S,4R,5R,6S)-5-[(tert-butyl)dimethylsiloxy]-3-hydroxy-2,4,6-trimethyloctanoyl}ox-
azolidin-2-one (22). Bu2BOTf (110 ml of a 1.0m soln. in CH2Cl2) was added by syringe at 08 to 23 mg of (4R)-4-
benzyl-3-propanoyloxazolidin-2-one (0.1 mmol, 1.0 equiv.) in 0.2 ml of dry CH2Cl2, followed by dropwise
addition of 21 ml (0.12mmol, 1.2equiv.) of EtN(i-Pr) 2. The mixture was cooled to À788, and 20 mg of 20 was
added. The mixture was stirred at À788 for 30 min, then warmed to r.t., and stirred for an additional 16 h. A
mixture of 0.2ml of phosphate buffer (1.0 m Na3PO4, pH 7.4) and 0.4 ml of MeOH was added, followed by
dropwise addition of 0.2ml of 30% H 2O2. After 30 min, the mixture was extracted with 2 Â 10 ml of CH2Cl2.
The org. layers were combined, dried (Na2SO4), and concentrated in vacuo. FC (5 g of SiO2; 0 10% AcOEt/
hexanes) afforded 10 mg (29%) of 22: Rf (10% AcOEt/hexanes) 0.25. [a]D ˆ À22.1 (c ˆ 1.0, CHCl3). IR (film):
2957, 1785, 1681. 1H-NMR (300 MHz, CDCl3): 0.00 (s, MeSi); 0.02( s, MeSi); 0.75 0.85 (m, Me3C, Me(8'),
MeÀC(4'), MeÀC(6')), 0.85 1.02( m, HbÀC(7')); 0.97 1.12( m, HbÀC(7')); 1.13 (d, J ˆ 7.0, MeÀC(2'); 1.43
1.58 (m, HÀC(6'), HaÀC(7')); 1.27 145 (m, HaÀC(7')); 1.48 1.65 (m, HÀC(6')); 1.70 1.88 (m, HÀC(4'));
2.69 (dd, J ˆ 9.6, 13.3, 1 H, PhCH2); 3.22 (dd, J ˆ 3.2, 13.3, 1 H, PhCH2); 3.74 3.90 (m, HÀC(2'), HÀC(3'),
HÀC(5')); 4.05 4.20 (m, CH2(5)); 4.60 4.68 (m, HÀC(4)); 7.10 7.30 (m, 5 arom. H). 13C-NMR (75.5 MHz,
CDCl3): 177.5; 152.9; 135.2; 129.4; 128.9; 127.4; 72.2; 66.2; 55.4; 39.8; 39.1; 38.5; 37.8; 31.6; 26.0; 22.6; 18.3; 16.2;
‡
‡
14.1; 11.9; 11.2; 8.8; À4.1; À4.3. HR-FAB-MS (NBA/NaI): 514.2985 ([M ‡ Na] , (C27H45NO5Si)Na ; calc.
514.2965.
(2R,3S,4R,5R,6R)-3-Hydroxy-2,4,6-trimethyloctano-5-lactone (14). Imide 22 (10 mg, 0.02mmol) was
dissolved in 2ml of THF. H 2O (0.5 ml) was added, and the soln. was cooled to 08 with vigorous stirring,
while 20 ml of 30% H2O2 was added, followed by 2mg (0.05 mmol) of LiOH. The mixture was allowed to warm
slowly to r.t. After 24 h, the reaction was quenched with 35 mg of Na2SO3. After stirring for 10 min, the mixture
was concentrated in vacuo to remove THF and then acidified to pH 1 by dropwise addition of conc. HCl. The
resulting cloudy mixture was extracted with 3 Â 10 ml of CH2Cl2. The combined org. layers were dried (Na2SO4)
and concentrated in vacuo to give (2R,3S,4R,5R,6S)-5-[tert-butyl)dimethylsiloxy]-3-hydroxy-2,4,6-trimethyloc-
1
tanoic acid (24). Rf (40% AcOEt/hexanes) 0.38; H-NMR (300 MHz, CDCl3): 0.01 (s, MeSi); 0.03 (s, MeSi);

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Helvetica Chimica Acta Vol. 86 (2003)
0.75 (d, J ˆ 7.1, Me ÀC(6)); 0.78 0.92( m, Me3C, Me(8), MeÀC(4)); 0.93 1.08 (m, HbÀC(7)); 1.12( d, J ˆ 7.2,
MeÀC(2)); 1.45 1.65 (m, HÀC(6), HaÀC(7)); 1.66 1.80 (m, HÀC(4)); 2.52 2.62 (m, HÀC(3)); 3.72 3.75
(m, HÀC(2)); 3.97 4.03 (m, HÀC(5)). The sample of 24 was dissolved in 2ml of MeCN and 0.5 ml of H 2O. To
the cloudy mixture, HF (20 ml of a 48% aq. soln.) was added dropwise. The mixture gradually cleared to a
colorless soln. and was stirred at r.t. for 4 h before being quenched with sat. aq. NaHCO3 to a pH of 7.5. The
resulting mixture was extracted with 3 Â 10 ml of CH2Cl2. The combined org. extract was dried (Na2SO4) and
concentrated in vacuo. The residue was purified by chromatography (1 g of silica gel, 20% AcOEt/hexanes) to
afford 14 (3 mg, 69%) whose physical and spectroscopic properties were identical to those of the biosyntheti-
cally-generated lactone 14 described below.
(4S)-4-Benzyl-3-{(2S,3R,4S)-3-[(tert-butyl)dimethylsiloxy]-2,4-dimethylhexanoyl}oxazolidin-2-one (15).
Compound 6 (40 mg, 0.44 mmol, 1.0 equiv.) was converted to 15 as described above for 16 to afford, after
purification, 190 mg (100%) of 15. Colorless oil. Rf (10% AcOEt/hexanes) 0.55. [a]D ˆ ‡43.3 (c ˆ 1.0, CHCl3).
1
IR (film): 2958, 1783, 1699. H-NMR (300 MHz, CDCl3): 0.07 (s, MeSi); 0.08 (s, MeSi); 0.82 0.99 ( m, Me3C,
Me(6'), MeÀC(4')); 1.11 1.22 (m, HbÀC(5')); 1.25 (d, J ˆ 6.8, MeÀC(2')); 1.27 1.43 (m, HaÀC(5')); 1.47 1.59
(m, HÀC(4')); 2.76 (dd, J ˆ 13.3, 9.6, 1 H, PhCH2); 3.27 (dd, J ˆ 13.4, 3.1, 1 H, PhCH2); 3.87 4.02( m, HÀC(2'),
HÀC(3')); 4.19 (m, CH2(5)); 4.64 (m, HÀC(4)); 7.19 7.37 (m, 5 arom. H). 13C-NMR (75.5 MHz, CDCl3):
176.3; 152.9; 135.3; 129.4; 128.9; 127.3; 76.0; 65.9; 55.5; 41.6; 41.0; 40.6; 37.6; 26.4; 26.1; 25.6; 18.4; 18.0; 14.9;
‡
‡
14.0; 12.3; À3.6; À3.7; À4.0. HR-FAB-MS (NBA/NaI): 456.2545 ([M ‡ Na] ; (C24H39NO4Si)Na ; calc.
456.2546.
(2S,3R,4S)-3-[(tert-Butyl)dimethylsiloxy]-2,4-dimethylhexan-1-ol (17). Compound 15 (190 mg, 0.44 mmol,
1.0 equiv.) was reduced with LiAlH4 as described above to give 49 mg (43%) of 17 as a colorless oil after
purification by FC (5 g of SiO2; 0 5% AcOEt/hexanes). Rf 0.31 (10% AcOEt/hexanes). IR (film): 3362.
1H-NMR (300 MHz, CDCl3): 0.00 (s, MeSi); 0.02( s, MeSi); 0.75 0.85 (m, Me3C, Me(6), MeÀC(2),
MeÀC(4)); 0.96 1.20 (m, HbÀC(5)); 1.35 1.50 (m, HÀC(4), HaÀC(5)); 1.84 1.92( m, HÀC(2)); 3.42 (dd,
J ˆ 5.9, 10.5, HbÀC(1)); 3.62 3.70 ( m, HaÀC(1), HÀC(3)). 13C-NMR (75.5 MHz, CDCl3): 66.2; 39.3; 37.7; 27.1;
25.8; 18.1; 15.1; 12.3; À3.8; À3.9.
(2S,3R,4S)-3-[(tert-Butyl)dimethylsiloxy]-2,4-dimethylhexanal (19). Compound 17 was oxidized with
pyridinium dichromate to the corresponding aldehyde 19 (36 mg, 73%) as described for 20. Rf 0.72(10%
1
AcOEt/hexanes). IR (film): 2959, 1728. H-NMR (300 MHz, CDCl3): À0.03 (s, MeSi); 0.01 (s, MeSi); 0.75
0.95 (m, Me3C, Me(6), MeÀC(4)); 1.02( d, J ˆ 6.9, MeÀC(2)); 1.00 1.18 (m, HbÀC(5)); 1.20 1.57 (m,
HÀC(4), HaÀC(5)); 2.40 2.56 (m, HÀC(2)); 3.90 4.01 (m, HÀC(3)); 9.75 (br. s, CHO); 13C-NMR
(75.5 MHz, CDCl3): 205.4; 75.3; 50.8; 39.1; 30.4; 26.5; 25.9; 23.7; 14.1; 9.3; À4.1; À4.3.
(4R)-4-Benzyl-3-{(2R,3S,5R,6S)-5-[(tert-burtyl)dimethylsiloxy]-3-hydroxy-2-,4-,6-trimethyloctanoyl}ox-
azolidin-2-one (21). Compound 21 was prepared from 19 as described for 22 to afford, after FC (5 g of SiO2; 0
10% AcOEt/hexanes) 40 mg (58%) of 21. Rf 0.25 (10% AcOEt/hexanes). [a]D ˆ À13.8 (c ˆ 1.0, CHCl3). IR
(film): 2958, 1784, 1682. 1H-NMR (300 MHz, CDCl3): 0.07 (s, MeSi); 0.08 (s, MeSi); 0.79 1.02( m, Me3C,
Me(8'), MeÀC(4'), MeÀC(6')); 1.20 (d, J ˆ 7.0, MeÀC(2')); 1.08 1.22 (m, HbÀC(7')); 1.27 1.45 (m,
HaÀC(7')); 1.48 1.65 (m, HÀC(6')); 1.70 1.88 (m, HÀC(4')); 2.68 (dd, J ˆ 9.6, 13.4, 1 H, PhCH2); 3.25 (dd,
J ˆ 3.3, 13.3, 1 H, PhCH2); 3.78 4.03 (m, HÀC(2'), HÀC(3'), HÀC(5')); 4.19 4.23 (m, CH2(5)); 4.65 4.72
(m, HÀC(4)); 7.19 7.37 (m, 5 arom. H). 13C-NMR (75.5 MHz, CDCl3): 176.8; 153.1; 135.3; 129.4; 128.9; 127.3;
72.7; 66.2; 55.6; 40.2; 39.4; 37.7; 37.5; 27.7; 26.0; 18.3; 12.0; 8.5; À4.1; À4.3. HR-FAB-MS (NBA/NaI): 514.2975
‡
([M ‡ Na] , (C27H45NO5Si)Na ‡ ; calc. 514.2965).
(2R,3S,4S,5R,6S)-3-Hydroxy-2,4,6-trimethyloctano-5-lactone (13). Imide 21 (40 mg, 0.08 mmol) was
hydrolyzed, lactonized, and deprotected as described for 14 to give, after chromatography (1 g of SiO2, 2 0%
AcOEt/hexanes), 11 mg (69%) of 13, whose physical properties were identical to those of the biosynthetically-
derived product 13.
0
General Precursor Feeding and Extraction Conditions. S. coelicolor CH999/pJRJ2encoding DEBS(KS1
)
[15a] and S. coelicolor CH999/pCK16 encoding DEBS1 ‡ TE (KS10) [17] were each cultivated at 308 on 25-ml
R2YE agar [27] plates for 3 d. Each plate was then overlaid with 1 ml of diketide soln. in 5% DMSO/H2O. After
an additional 4 d, the combined mycelia and agar (20 plates, 500 ml total) were homogenized and extracted with
AcOEt (3 Â 500 ml) at ca. 408. The combined org. extracts were dried (MgSO4), concentrated to ca. 5 ml in
vacuo, then washed with sat. NaHCO3 and sat. NaCl, dried (Na2SO4), and concentrated in vacuo.
Purification and Structure Determination. The crude mixture obtained from each precursor feeding was
subjected to initial purification by SiO2 CC (ca. 1 g SiO2 in a Pasteur pipette) with 20% AcOEt/hexanes. The
relevant fraction thus obtained was further purified by prep. TLC (6 cm  10 cm) with 40% AcOEt/hexanes.
The product band was excised and eluted with AcOEt, and the resultant product was characterized by 1H-

Helvetica Chimica Acta Vol. 86 (2003)
3905
(300 MHz) and 13C-NMR (75 MHz) spectroscopy, HR-MS, and FT-IR. The 1H- and 13C-NMR spectra of each
product was fully assigned by a combination of 1H, 1H-COSY, DEPT and 1H, 13C-HETCOSY spectroscopy.
Conversion of 3 to (14S)-6-Deoxy-14-methylerythronolide B (10) by DEBS(KS10). Feeding of 20 ml of
2 0 mm 3 to S. coelicolor CH999/pJRJ2gave 10 mg of 10 (6%). White powder. Rf (40% AcOEt/hexanes) 0.49.
[a]D ˆ À50.2( c ˆ 0.963, CHCl3). IR (film): 3485, 2972, 2938, 1706. 1H-NMR (300 MHz, CDCl3): 0.88 (d, J ˆ 6.8
MeÀC(12)); 0.91 (d, J ˆ 6.7, MeÀC(14)); 0.94 (t, J ˆ 7.4, Me(16)); 1.00 1.10 (m, MeÀC(4), MeÀC(6),
MeÀC(8), MeÀC(10)); 1.05 1.20 (m, HbÀC(15)); 1.10 1.30 (m, HbÀC(7)); 1.30 (d, J ˆ 6.9, MeÀC(2)); 1.40
1.55 (m, HaÀC(15)); 1.66 (m, HaÀC(7)); 1.75 1.95 (m, HÀC(4), HÀC(12), HÀC(14)); 1.95 2.10 (m,
HÀC(6)); 2.50 2.70 (m, HÀC(8)); 2.70 2.85 (m, HÀC(10)); 2.81 (dq, J ˆ 10.4, 6.7, HÀC(2)); 3.67 (br. d, J ˆ
9.2, HÀC(11)); 3.92( d, J ˆ 10.5, HÀC(3)); 4.01 (dd, J ˆ 5.2, 2.0, HÀC(5)); 4.88 (dd, J ˆ 10.3, 1.3, HÀC(13)).
13C-NMR (75 MHz, CDCl3): 6.27 (MeÀC(8)); 6.86 (MeÀC(4)); 9.14 (MeÀC(12)); 10.41 (C(16)); 13.18
(MeÀC(10)); 14.69 (MeÀC(2)); 15.71 (MeÀC(14)); 16.52(Me ÀC(6)); 24.76 (C(15)); 35.00 (C(14)); 35.48
(C(6)); 37.32(C(7)); 37.43 (C(4)); 37.72(C(12)); 39.19 (C(8)); 43.62(C(2)); 44.01 (C(10)); 70.80 (C(11)); 76.44
(C(5)); 79.50 (C(13)); 79.5 (C(3)); 178.28 (C(1)); 213.48 (C(9)). HR-FAB-MS (NBA/NaI): 437.2877 ([M ‡
‡
‡
Na] , (C23H42O6)Na ; calc. 437.2879.
Conversion of 4 to (14R)-6-Deoxy-14-methylerythronolide B (11) by DEBS(KS10). Feeding of 20 ml of
5 mm 4 to S. coelicolor CH999/pJRJ2gave 10 mg (25%) of 11. Colorless crystals. M.p. 139 1408. Rf. (40%
AcOEt/hexanes) 0.49. [a]D ˆ À46.9 (c ˆ 0.625, CHCl3). IR (film): 3487, 2972, 2939, 1704. 1H-NMR (300 MHz,
CDCl3): 0.86 (d, J ˆ 6.9, MeÀC(12)); 0.88 (d, J ˆ 6.7, MeÀC(14)); 0.89 (t, J ˆ 7.4, MeÀC(16)); 1.00 1.10 (m,
MeÀC(4), MeÀC(6), MeÀC(8), MeÀC(10)); 1.05 1.20 (m, HbÀC(15)); 1.10 1.30 (m, HbÀC(7)); 1.29 (d,
J ˆ 6.8); 1.40 1.55 (m, HaÀC(15)); 1.66 (m, HaÀC(7)); 1.75 1.95 (m, HÀC(4), HÀC(12), HÀC(14)); 1.95
2.10 (m, HÀC(6)); 2.55 2.75 (m, HÀC(8)), 2.70 2.85 (m, HÀC(10)); 2.79 (dq, J ˆ 10.4, 6.8, HÀC(2)); 3.67
(br. d, J ˆ 9.3, HÀC(11)); 3.92( d, J ˆ 10.5, HÀC(3)); 4.01 (dd, J ˆ 5.2, 2.1, HÀC(5)); 4.88 (dd, J ˆ 10.3, 1.3,
HÀC(13)). 13C-NMR (75 MHz, CDCl3): 6.27 (MeÀC(8)); 6.84 (MeÀC(4)); 8.93 (MeÀC(12)); 10.39 (C(16));
13.18 (MeÀC(10)); 14.69 (MeÀC(2)); 14.72 (Me ÀC(14)); 16.51 (MeÀC(6)); 25.75 (C(15)); 35.13 (C(14));
35.45 (C(6)); 37.38 (C(7)); 37.58 (C(4)); 37.78 (C(12)); 39.09 (C(8)); 43.62 (C(2)); 44.00 (C(10)); 70.80 (C(11));
76.41 (C(5)); 78.34 (C(13)); 79.55 (C(3)); 178.29 (C(1)); 213.40 (C(9)). HR-FAB-MS (NBA/NaI): 437.2882
‡
‡
([M ‡ Na] , (C23H42O6)Na ; calc. 437.2879.
Conversion of the 3 :2 Mixture 4/3 to 11 by DEBS(KS10). Feeding of 20 ml of a 5 mm soln. of the 3 :2
mixture 4/3 to S. coelicolor CH999/pJRJ2gave 10 mg of 11 (40% yield, based on 4) as colorless crystals, whose
1H- and 13C-NMR spectra were identical with those of 11 obtained from the feeding of 4.
Conversion of 3 to 13 by DEBS1 ‡ TE(KS10). Feeding of 20 ml of 20 mm 3 to S. coelicolor CH999/pCK16
(DEBS1 ‡ TE KS10) gave 4 mg (10%) of 13. White powder. Rf (40% AcOEt/hexanes) 0.36. [a]D ˆ ‡82.8 (c ˆ
1.0, CHCl3). IR (film): 3440, 1716. 1H-NMR (300 MHz, CDCl3): 0.89 (d, J ˆ 6.9, MeÀC(6)); 0.93 (t, J ˆ 7.2,
Me(8)); 0.97 (d, J ˆ 7.0, MeÀC(4)), 1.24 (m, HbÀC(7)); 1.43 (d, J ˆ 7.1, Me ÀC(2)); 1.73 (m, HÀC(6)); 1.92( m,
HaÀC(7)); 2.28 (m, HÀC(4)); 2.48 (dq, J ˆ 10.4, 7.1, HÀC(2)); 3.80 (dd, J ˆ 10.3, 4.1, HÀC(3)); 3.85 (dd, J ˆ
10.2, 2.1, HÀC(5)). 13C-NMR: 173.7; 84.2; 74.2; 39.9; 35.4; 35.3; 24.1; 15.5; 14.4; 10.5; 4.4. HR-FAB-MS (NBA/
‡
‡
NaI): 223.1305 ([M ‡ Na] , (C11H20O3)Na ; calc. 223.1310.
Conversion of 4 to 14 by DEBS1 ‡ TE(KS10). Feeding of 20 ml of 5 mm 4 to S. coelicolor CH999/pCK16
(DEBS1 ‡ TE KS10) gave 5 mg (50%) of 14. White powder. Rf (40% AcOEt/hexanes) 0.36. [a]D ˆ ‡118 (c ˆ
0.35, CHCl3). IR (film): 3440, 1717. 1H-NMR (300 MHz, CDCl3): 0.94 (t, J ˆ 7.3, Me(8)); 0.97 (d, J ˆ 7.0,
MeÀC(4)); 1.05 (m, HbÀC(7)); 1.10 (d, J ˆ 6.5, MeÀC(6)); 1.42( d, J ˆ 7.1, Me ÀC(2)); 1.51 (m, HaÀC(7)); 1.75
(m, HÀC(6)); 2.31 (m, HÀC(4)); 2.48 (dq, J ˆ 10.3, 7.1, HÀC(2)); 3.80 (dd, J ˆ 10.4, 4.2, HÀC(3)); 3.86 (dd,
J ˆ 10.0, 2.1, HÀC(5)). 13C-NMR: 173.7; 83.4; 74.4; 40.0; 35.6; 35.4; 25.3; 14.4; 13.9; 10.6; 4.2. HR-FAB-MS
‡
‡
(NBA/NaI): 223.1307 ([M ‡ Na] , (C11H20O3)Na ; calc. 223.1310.
Conversion of the 3 :2 Mixture 4/3 to 14 by DEBS1 ‡ TE(KS10). Feeding of 20 ml of a 5 mm soln. of the 3 :2
mixture 4/3 to S. coelicolor CH999/pCK16 (DEBS1 ‡ TE KS10) gave 4 mg (60% yield based on 4) of 14 as a
white powder, whose 1H- and 13C-NMR spectra were identical with those of 14 obtained from the feeding of 4.
DEBS Module 2 ‡ TE. DEBS Module 2 ‡ TE was expressed in E. coli BL21(DE3) harboring plasmid
pRSG64 [18][28] and cotransformed with the sfp plasmid to ensure phosphopantetheinylation of the ACP
domain of the ACP domain [29]. The cells were grown in LB medium supplemented with ampicillin, kanamycin,
and chloramphenicol as previously described at 378 until OD600 ˆ 0.6. The cultures were then induced with 1 mm
final concentration of IPTG (isopropyl-b-d-thiogalactopyranoside) and grown at r.t. overnight. After
harvesting, the cell pellet was washed with buffer A (100 mm sodium phosphate (pH 7.2), 2 mm EDTA,
2.5 mm DTT, and 20% glycerol). The washed cells were then resuspended in disruption buffer (200 mm Na3PO4
(pH 7.2), 200 mm NaCl, 2.5 mm EDTA, 2.5 mm DTT, 30% glycerol, 1.5 mm benzamidine, 2mg/l pepstatin, and

3906
Helvetica Chimica Acta Vol. 86 (2003)
2mg/l leupeptin) and lysed with a French Press at 1000 psi. All steps were carried out at 48. After centrifugation,
the supernatant was treated with the final concentration of 50 mg/ml DNase and 10 mm MgCl2 for 20 min at 48,
and then subjected to a 0.15% PEI precipitation for 20 min. The proteins were purified on a 30-ml anion-
exchange chromatography column (Q Sepharose: Fast Flow resin from Pharmacia), which was washed with
100 mm Na3PO4 (pH 7.2) containing 20% glycerol and 200 mm NaCl, and then eluted with a gradient from 200 to
500 mm NaCl. The appropriate fractions containing DEBS module 2( > 70% of protein) eluted at ca. 250 mm
NaCl were pooled and concentrated on Centriprep 30 membranes (Amicon) to a protein concentration of ca.
110 mg/ml. The purity of this protein was determined to be > 60% by SDS-PAGE. The partially purified DEBS
module 2 ‡ TE protein soln. was placed in 500-ml Eppendorf tubes and stored at À808 before being used for the
kinetic studies.
Kinetic Assays. The kinetic assays were carried out with 0.31 mm DEBS module 2 ‡ TE, with concentrations
over a range of 125 mm 10 mm, plus 200 mm [2-14C]methylmalonyl-CoA (1 mCi/mmol), 1 mm NADPH, and
buffer A in 100-ml total volume at 308 for 1 h. The reactions were quenched by the addition of 100 ml of AcOEt
and subsequent vortexing for 1 min, and then separated by a micro-centrifuge (14000 rpm for 1 min). The
AcOEt extracts were loaded onto TLC plates, which were developed with 70% AcOEt/hexanes. Product yields
were quantitated by TLC-phosphoimaging.
This work was supported by grants from the NIH to D.E.C. (GM22172) and to C.K. (CA66736).
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Received July 31, 2003