Nonactin biosynthesis: Unexpected patterns of label incorporation from 4,6-dioxoheptanoate show evidence of a degradation pathway for levulinate through propionate in Streptomyces griseus

Article abstract of DOI:10.1021/np100421v

The polyketide nonactin, a polyketide possessing antitumor and antibacterial activity, is produced by an unusual biosynthesis pathway in Streptomyces griseus that uses both enantiomers of the nonactin precursor, nonactic acid. Despite many studies with labeled precursors, much of the biosynthesis pathway remains unconfirmed, particularly the identity of the last achiral intermediate in the pathway, which is believed to be 4,6-diketoheptanoyl-CoA. We set out to confirm the latter hypothesis with feeding studies employing [4,5-¹³C₂]-, [5,6- ¹³C₂]-, and [6,7-¹³C₂]-4,6- diketoheptanoate thioester derivatives. In each case the isotopic label was incorporated efficiently into nonactin; however, at positions inconsistent with the currently accepted biosynthesis pathway. To resolve the discrepancy, we conducted additional feeding studies with a [3,4-¹³C ₂]levulinate thioester derivative and again observed efficient label incorporation. The latter result was intriguing, as levulinate is not an obvious precursor to nonactin. Levulinate, however, is known to be efficiently degraded into propionate even though the pathway for the conversion is not known. On the basis of both our levulinate and diketoheptanoate isotope incorporation data we can now postulate a pathway from levulinate to propionate that can also account for the conversion of 4,6-diketoheptanoate into levulinate in S. griseus.

Full text of DOI:10.1021/np100421v

J. Nat. Prod. 2010, 73, 2009–2012
2009
Nonactin Biosynthesis: Unexpected Patterns of Label Incorporation from 4,6-Dioxoheptanoate
Show Evidence of a Degradation Pathway for Levulinate through Propionate in Streptomyces
griseus
Jian Rong, Micheal E. Nelson, Brian Kusche, and Nigel D. Priestley*
Department of Chemistry and Biochemistry, The UniVersity of Montana, 32 Campus DriVe, Missoula, Montana 59812, United States
ReceiVed June 25, 2010
The polyketide nonactin, a polyketide possessing antitumor and antibacterial activity, is produced by an unusual
biosynthesis pathway in Streptomyces griseus that uses both enantiomers of the nonactin precursor, nonactic acid. Despite
many studies with labeled precursors, much of the biosynthesis pathway remains unconfirmed, particularly the identity
of the last achiral intermediate in the pathway, which is believed to be 4,6-diketoheptanoyl-CoA. We set out to confirm
the latter hypothesis with feeding studies employing [4,5-13C2]-, [5,6-13C2]-, and [6,7-13C2]-4,6-diketoheptanoate thioester
derivatives. In each case the isotopic label was incorporated efficiently into nonactin; however, at positions inconsistent
with the currently accepted biosynthesis pathway. To resolve the discrepancy, we conducted additional feeding studies
with a [3,4-13C2]levulinate thioester derivative and again observed efficient label incorporation. The latter result was
intriguing, as levulinate is not an obvious precursor to nonactin. Levulinate, however, is known to be efficiently degraded
into propionate even though the pathway for the conversion is not known. On the basis of both our levulinate and
diketoheptanoate isotope incorporation data we can now postulate a pathway from levulinate to propionate that can also
account for the conversion of 4,6-diketoheptanoate into levulinate in S. griseus.
Streptomyces griseus subsp. griseus ETH A7796 (DSM40695)
makes a series of ionophore antibiotics known as the macrotetrolides
(Figure 1).1 The most prevalent homologue, nonactin, has been
shown to possess antitumor activity and to be an effective inhibitor
of the P170-glycoprotein-mediated drug resistance in cancer cells.2
The 32-membered ring of a macrotetrolide is composed of four
monomers, the latter being either nonactic acid or homononactic
acid. In the case of nonactin, two monomers of (+)-nonactic acid
and two monomers of (-)-nonactic acid are linked such that
nonactin has S4 symmetry and is achiral. Recently, we were able
to demonstrate that diastereoisomers of nonactin, derived from only
one enantiomer of nonactic acid, were inactive.3 The production
of both enantiomers of the precursor acids, and that such acids
originate from succinic acid,4 show that nonactin is an unusual
polyketide.
epimerizable, they are common to both enantiocomplementary
pathways that generate nonactic acid, and the divergence in
stereochemistry must occur later in the pathway. The divergence
presumably arises through the stereoselectivity of dehydrogenase
enzymes acting on such substrates. In this case 10 would not be an
intermediate in nonactin biosynthesis. Consequently, we would have
strong evidence that our notion of a thioester translocation was valid
if we were able to show that 10 was indeed a biosynthesis
intermediate, a hypothesis testable using a traditional feeding
experiment.
Results and Discussion
Our first approach to testing our hypotheses was to attempt the
obvious feeding experiment with a 13C-labeled derivative of 10.
We chose a dual-13C labeling pattern for our probe 10a (Scheme
2, compound 15), as the dual label offers greater sensitivity and
allows us to determine if degradation of the intermediate occurs
prior to incorporation into nonactin. Benzyl acetate was prepared
The broad outlines of the nonactin biosynthesis pathway were
originally established by stable isotope feeding experiments with
primary metabolites5-9 and more advanced precursors.4,10 The latter
data showed that after an initial condensation of malonyl-CoA and
succinyl-CoA (Scheme 1) an adipate derivative 8 was generated.
Acylation of 8 with acetyl-CoA led to the generation of both
enantiomers of nonactic acid; acylation with propionyl-CoA led to
both enantiomers of homononactic acid. The conversion of 9a/b
into 10a/b can be accomplished by transposition of the CoASH
thioester followed by spontaneous decarboxylation. Such a trans-
thioesterification reaction is supported by data from the sequencing
and annotation of the nonactin biosynthesis gene cluster.11,12 The
deduced product of orf8 has high sequence identity to E-glutaconate:
acetyl-CoA CoASH transferase13-15 as well as a number of other
enzymes that catalyze the transfer of CoASH.16-19 The E-
glutaconate transferase acts in a ping-pong mechanism to first
transfer CoASH from the donor thioester to a glutamate residue.
In a second step, the CoASH moiety is transferred from the
glutamate to the acceptor carboxylate.15 The thioester transposition
proposed in nonactin biosynthesis (9 to 10) is the intramolecular
analogue of the succinyl-CoA:3-ketoacid CoASH transferase-
catalyzed reaction. As intermediates 9 and 10 are achiral or readily
Figure 1. Structures of the naturally occurring macrotetrolides and
their precursors.
* Corresponding author. Tel: +1 44 (406) 243-6251. Fax: +1 44 (406)
243 4227. E-mail: nigel.priestley@umontana.edu.
10.1021/np100421v  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/07/2010

2010 Journal of Natural Products, 2010, Vol. 73, No. 12
Scheme 1. Early Steps of the Nonactin Biosynthesis Pathway
Rong et al.
Figure 2. Summary of feeding results obtained with [4,5-13C2]-
(green), [5,6-13C2]- (blue), and [6,7-13C2]-4,6-diketoheptanoate (red)
derivatives. The 13C label is efficiently incorporated into nonactin,
via nonactic acid, at positions derived from propionate.
Scheme 3. Synthesis of a Thioester Derivative of
[3,4-13C2]Levulinate
Scheme 2. Synthesis of Labeled 4,6-Diketoheptanoate
Derivatives
remained intact, although the incorporation of the label at the
C1-C2 (Figure 2, blue highlight) positions rather than the expected
C7-C8 positions was a surprise. In repetitions of the experiment,
label incorporations of 1% to 10% were obtained, with higher
incorporations being observed at lower overall levels of mac-
rotetrolide synthesis. The levels of 13C incorporation were compa-
rable to those observed in feeding experiments with labeled
propionate. No significant enrichments by single 13C labels were
observed. Furthermore, the intact 13C-13C label was found at
C1-C2 and C8-C9 in methyl homononactate. Analysis of dias-
tereoisomeric derivatives made from both methyl nonactate and
methyl homononactate showed that there was no stereoselectivity
associated with incorporation. Both enantiomers of each acid were
labeled to the same extent and at the same positions. The observed
labeling pattern is inconsistent with the proposed biosynthesis
pathway but does suggest that 15 is degraded to propionate, as the
observed labeling patterns match those observed from feeding [2,3-
13C2]propionate to S. griseus.
To further understand the unusual incorporation of 15, we
prepared the isotopomers [4,5-13C2]- and [6,7-13C2]-4,6-diketohep-
tanoate through analogous synthetic chemistry. Incorporation of the
latter labels was again evident in the macrotetrolides but at positions,
and in patterns, consistent with the degradation of 4,6-diketohep-
tanoate into propionyl-CoA. The efficient incorporation of 13C label
shows that the probe compounds are capable of entering the cell
and participating in metabolism. The lack of incorporation of label
at the expected positions is suggestive that 15 is not an intermediate
in nonactin biosynthesis. However, to discount 15 as an intermedi-
ate, we need to understand how 15 is efficiently converted into
propionate. Such a metabolic pathway is not immediately obvious,
as the 6-keto group of 15 must become a methylene and the
5-position of 15 must become the carboxyl group of propionate
without scrambling or breaking of the C5-C6 and C6-C7 bonds
of 15.
from [2-13C1]acetate and then used to generate an enolate that, at
low temperature, was quenched with an imidazolide generated from
[1-13C1]acetic acid. The benzyl ester was chosen so as to add
molecular weight to the synthetic intermediates and as a useful
chromophore to aid in purification. Acylation of 13 using Mg2+
/
pyridine20 to generate the required enolate worked well. Hydro-
genolysis of 14 occurred with spontaneous decarboxylation. The
latter process is analogous to the conversion of 9 to 10 in nonactin
biosynthesis, where we postulate that the trans-thioesterification
reaction also occurs with concomitant, spontaneous decarboxylation.
Straightforward replacement of the methyl ester with N-caprylcys-
teamine under standard conditions21 gave our first required, labeled
probe. The probe was administered to a fermentative culture of S.
griseus at the onset of nonactin production, and after 48 h
macrotetrolides were recovered.4 A preliminary 13C NMR survey
showed significant incorporation of 13C label into the macrotetrolide
mixture. To simplify the analysis, the macrotetrolides were subjected
to acid methanolysis to afford methyl nonactate (6-OMe) and methyl
homononactate (7-OMe), which were separated by chromatography.
Reduction of methyl nonactate with LiAlH4 gave the expected,
known diol10 as a mixture of enantiomers. Derivatization of the
diol with Mosher’s chloride was followed by 13C NMR analysis. It
was evident that the contiguous 13C-13C labeling of the precursor
At this point we decided to regroup and evaluate some less likely
pathways to nonactic acid. One such pathway involved a key
reaction in which levulinate could be acylated to generate 4,6-
diketoheptanoate. We prepared, therefore, a thioester derivative of
[3,4-13C2]levulinate as
a probe (Scheme 3). Benzyl [2,3-
13C2]acetoacetate was generated from benzyl [2-13C1]acetate and
[1-13C1]acetic acid as described above for 13. Deprotonation of 16
with NaH, followed by reaction with ethyl 2-bromoacetate, led to

Nonactin Biosynthesis
Journal of Natural Products, 2010, Vol. 73, No. 12 2011
Scheme 4. Primary Metabolic Pathways That Account for the Conversion of 4,6-Diketoheptanoyl-CoA into Levulinoyl-CoA and
Levulinoyl-CoA into Propionyl-CoAa
a The pathways account for the labeling patterns observed in the feeding experiments and for the use of levulinate as a propionate precursor in fermentation.
17, which, upon saponification, underwent spontaneous decarboxy-
lation to afford [3,4-13C2]levulinic acid. The latter compound was
converted to activated N-caprylcysteamine thioester 18 using
standard procedures.21
and allow us to propose a pathway for the latter conversion that
accounts for all our feeding results.
Experimental Section
General Experimental Procedures. NMR spectra were acquired
at 400 MHz (1H) and 100 MHz (13C), were referenced to the residual
solvent, and are reported as chemical shift (δ/ppm), intensity, splitting
pattern, and coupling constant (J/Hz). Solvents were obtained from
Fisher Scientific. All other reagents, unless noted otherwise, were
obtained from Aldrich. The solvents CH2Cl2 (over CaH2) and THF (over
Na/K-benzophonone) were dried and distilled prior to use. Solutions
were concentrated by evaporation in Vacuo. All synthesis procedures,
unless noted otherwise, were carried out under a slight positive pressure
of dry argon gas.
We used both levulinic acid and 18 in feeding experiments in
fermentative cultures of S. griseus. The macrotetrolides recovered
from these experiments were converted into methyl nonactate and
methyl homononactate and analyzed by 13C NMR. The dual label
was incorporated intact at C1-C2 of nonactate and both C1-C2
and C8-C9 of homononactate, a pattern identical to that observed
in the feeding experiment of the [5,6-13C2]-4,6-diketoheptanoate
thioester. Resolution of the enantiomers of nonactic acid was
achieved this time using a Rhodococcus-mediated biotransformation,
a stereoselective oxidation of methyl (-)-nonactate to methyl (-)-
8-ketononactate.22 Dual 13C-labeling was observed in both methyl
(+)-nonactate and methyl (-)-8-ketononactate, confirming our
expectation that no stereoselection is observed in the conversion
of levulinate.
Microorganism. Streptomyces griseus subsp. griseus ETH A7796
was obtained from the Deutsche Sammlung von Mikroorganismen and
Zellkulturen GmbH (DSM 40695). Fermentation of S. griseus and
feeding studies were performed according to our standard procedures.4
Benzyl [2-13C1]Acetate (12). Benzyl bromide (2.40 mL, 20.0 mmol)
was added to a solution of sodium [2-13C1]acetate (0.836 g, 10.0 mmol)
in anhydrous DMF (20 mL). The solution was heated at 96 °C for
three days. Water (80 mL) and Et2O (120 mL) were added to the cooled
mixture, which was then stirred. The organic layer was recovered, and
the aqueous layer then extracted with Et2O (2 × 120 mL). The Et2O
fractions were combined, dried over MgSO4, filtered, and concentrated.
Chromatography on silica gel, eluting with EtOAc-hexanes (15:85),
The conversion of levulinate to propionyl-CoA, such that
levulinate is a precursor of nonactin, is, at best, unlikely. The
levulinate feeding experiment, however, was of significance, as it
drew our attention to pathways for levulinate degradation and
provided the final clue needed to make a rational hypothesis for
the unusual degradation of 4,6-diketoheptanoate into propionate that
was observed in our original set of feeding experiments. It is known
that levulinate can act as a source of propionate and is often used
as such in fermentation media, although the details of the conversion
are not clear.23 Our initial feeding experiments can be rationalized
on the basis of the degradation of 4,6-diketoheptanoyl-CoA into
levulinoyl-CoA (Scheme 4).
1
gave the product as a liquid (1.36 g, 89.8%): H NMR (400 MHz,
1
13
CDCl3) δ 7.30 (5H, m), 5.10 (2H, s), and 2.08 (3H, d, J H- C ) 134.5
13
13C-13
C
Hz); C NMR (100 MHz, CDCl3) δ 170.7 (d, J
) 59.4 Hz),
135.8, 128.1, 66.1, and 20.9.
Benzyl [2,3-13C2]-3-Oxobutanoate (13). Diisopropylamine (1.3 mL,
9.2 mmol) was added to dry THF (10 mL) in a 50 mL three-neck round-
bottom flask, cooled in a dry ice-acetone bath at -60 °C. n-BuLi (5.1
mmol) in hexanes (17.4 mL) was added via syringe. Compound 12
(1.16 g, 7.7 mmol) was dissolved in dry THF (5 mL), and the solution
added dropwise via syringe. The temperature was maintained below
-60 °C for 45 min. [1-13C1]Acetic acid (0.27 mL, 4.6 mmol) was added
via syringe to a stirred suspension of recrystallized 1,1′-carbonyldi-
imidazole (0.845 g, 5.20 mmol) in dry THF (5 mL) in a 10 mL round-
bottom flask. Stirring was maintained for 1 h at rt. The acetylimidazole
solution was then transferred to the enolate solution via syringe over 5
min, maintaining the internal temperature below -20 °C. The reaction
was stirred for 45 min, quenched by addition of water (25 mL), acidified
to pH 4 with 2 M HCl, and then extracted with CHCl3 (3 × 50 mL).
The organic layers were combined and dried over MgSO4, filtered, and
concentrated. The product was purified by chromatography on silica
gel, eluting with EtOAc-hexanes (15:85), as an oil (0.46 g, 51.5%):
1H NMR (400 MHz, CDCl3) δ 7.35 (5H, m), 5.16 (2H, s), 3.48 (2H,
The conversion of 4,6-diketoheptanoyl-CoA into levulinoyl-CoA
would presumably occur via reduction of the ketone at C-4 of the
probe, followed by elimination of the intermediate hydroxy group
to give 19. Migration of the olefin into conjugation with the thioester
would afford intermediate 20, which can undergo one round of
standard ꢀ-oxidation to be converted into a levulinoyl-CoA.
Degradation of levulinoyl-CoA to propionyl-CoA would use the
same sequence of reduction-elimination (gives 21), olefin migration
(gives 22), and ꢀ-oxidation that then accounts fully for both the
conversion of 4,6-diketoheptanoate into levulinate and levulinate
into propionate and that agrees with the disposition of the 13C-
labeling patterns observed in our feeding experiments.
Our original plan to confirm that 4,6-diketoheptanoate was an
intermediate in nonactin biosynthesis through the use of labeling
experiments was foiled, as there is likely a very active pathway
for the degradation of levulinoyl-CoA to propionyl-CoA operating
in S. griseus. The latter pathway would account for the conversion
of 4,6-diketoheptanoyl-CoA into levulinoyl-CoA and the overall
conversion of our labeled probes into propionyl-CoA. The overall
result is that our initial feeding experiments can neither confirm
nor rule out 4,6-diketoheptanoyl-CoA as a nonactin biosynthesis
intermediate. Our labeling studies do shed light, however, on the
known but little understood conversion of levulinate into propionate
1
13
13H-13
dd, J H- C ) 130.4 Hz, 6.59 Hz), and 2.23 (3H, d, J
C ) 6.59 Hz);
13C NMR (100 MHz, CDCl3) δ 200.3 (keto, enriched, d, J
36.6 Hz), 175.8 (enol, enriched, d, J
128.4, 128.3, 89.5 (enol, enriched, d, J
(keto, enriched, d, J
)
13C-13
C
13C-13
13C-13
C
) 71.7 Hz), 135.1, 128.5,
C
) 73.2 Hz), 67.0, 49.9
13C-13
C
) 38.1 Hz), and 30.0.
1-Benzyl 6-Methyl [2-13C]-([1-13C]-2-Acetyl)-3-Oxohexanedioate
(14). 13 (1.31 g, 6.8 mmol) was added to a suspension of anhydrous
MgCl2 (0.65 g, 6.9 mmol) in dry CH2Cl2 (7 mL), which was then cooled
to 0 °C. Dry pyridine (1.1 mL, 13.7 mmol) was added via syringe to
the stirred suspension. After 15 min at 0 °C, carbomethoxypropionyl
chloride (0.85 mL, 6.9 mmol) was added via syringe. After a further

2012 Journal of Natural Products, 2010, Vol. 73, No. 12
Rong et al.
15 min at 0 °C the suspension was allowed to warm to rt, and stirring
continued for 2 h. Aqueous HCl solution (6 M, 5 mL) was added
carefully, and the mixture extracted with Et2O (3 × 25 mL). The organic
extracts were combined, dried, and concentrated. Chromatography on
silica gel, eluting with EtOAc-hexanes (2:3), gave the product as a
1H NMR (400 MHz, CDCl3) δ 10.9 (br), 2.88 (1H, q), 2.58 (4H, m),
and 2.16 (3H, dd, J H- ) 5.86 Hz, J H- ) 1.47 Hz); 13C NMR
1
1
13C
13C
13C-13
C
(100 MHz, CDCl3) δ 206.7 (d, J
) 38.15 Hz), 178.7, 37.5 (d,
13C-13
C
13C-13
) 38.1 Hz). 1,1-
C
J
) 39.7 Hz), 30.0 (m), and 27.7 (d, J
Carbonyldiimidazole (0.0855 g, 0.527 mmol) was added in one portion
to a stirred solution of [3,4-13C2]-4-oxopentanoic acid (0.051 g, 0.44
mmol) in CH2Cl2 (1 mL). The solution was stirred for a further 30
min. 4-Dimethylaminopyridine (0.016 g, 0.13 mmol) was then added,
and the mixture was stirred for another 30 min, followed by addition
of N-caprylcysteamine (0.177 g, 1.10 mmol) in one portion. The reaction
mixture was stirred overnight and then quenched by addition of H2O
(10 mL). The mixture was extracted with EtOAc (3 × 10 mL), and
the organic layers were combined, dried over MgSO4, and concentrated.
The thioester product was recovered by chromatography on silica gel
1
faint yellow oil (1.54 g, 73.3%): H NMR (250 MHz, CDCl3) δ 17.8
1
13
(1H, dd, J H- ) 6.5, 4.5 Hz), 7.3 (5H, m), 5.2 (2H, s), 3.6 (3H, s),
C
1
1
1
1
3.0 (2H, t, J H- H ) 6.8 Hz), 2.6 (2H, tt, J H- H ) 6.8 Hz) and 2.3 (3H,
dd, J H- C ) 6.1, 2.2 Hz); 13C NMR (67 MHz, CDCl3) δ 199.3, 193.9
1
13
13C-13
C
13C-13
13C-13
C
(enriched, d, J
) 63.6 Hz) 183.5 (d, J
) 70.1 Hz), 172.6,
C ) 36.2 Hz), 66.6,
135.5, 128.5, 128.4, 128.3, 72.7 (enriched, d, J
51.5, 33.6, 28.4, and 24.7.
S-[2-(Octanoylamino)ethyl] [5,6-13C2]-4,6-Dioxoheptanethioate
(15). Pd on C (5%, 1.077 g) was added to a solution of 14 (1.54 g, 5.0
mmol) in MeOH (40 mL), and the mixture was stirred under a H2
atmosphere (balloon) for 3 h. Celite was added, and the suspension
filtered. The filtrate was concentrated, and the product, methyl [5,6-
13C2]-4,6-dioxoheptanoate, was obtained as an oil (0.64 g, 74.4%) that
was carried through to the next reaction without further purification.
Aqueous LiOH solution (2.5 M, 4.5 mL) was added to a stirred solution
of methyl [5,6-13C2]-4,6-dioxoheptanoate (0.64 g, 3.7 mmol) in THF
(13.5 mL) and methanol (9 mL). The single phase solution was stirred
at rt overnight. The reaction was extracted with EtOAc (5 × 150 mL),
and the organic extracts were combined, dried, filtered, and concentrated
to give a yellow solid (0.46 g, 73%). The material was carried through
without further purification. Dicyclohexylcarbodiimide (0.47 g, 2.3
mmol) was added to a stirred solution of 4,6-dioxoheptanoic acid (0.18
g, 1.1 mmol), N-caprylcysteamine (0.36 g, 1.8 mmol), and DMAP
(0.037 g, 0.03 mmol) in CH2Cl2 (12 mL). After two days the copious
white precipitate was removed by filtration. The filtrate was concentrated
and then fractionated on silica gel, eluting with EtOAc-EtOH-hexanes
eluting with EtOAc-hexanes (6:4) as a white solid (0.126 g, 93.8%):
1
1
1
H NMR (400 MHz, CDCl3) δ 5.90 (1H, br), 3.40 (2H, q, J H- H ) 6.6
1
13
Hz), 3.00 (2H, q), 2.82 (2H, p), 2.61 (1H, q), 2.16 (3H, dd, J H-
)
C
1
13
1
1
5.86 Hz, J H- C ) 1.47 Hz), 2.10 (2H, J H- H ) 7.32 Hz), 1.58 (2H, p,
J H- H ) 7.3 Hz), 1.25 (9H, m), 0.75 (3H, t, J H- H ) 6.59 Hz); 13C
NMR (100 MHz, CDCl3) δ 206.0 (d, J C ) 38.1 Hz) 198.6, 173.3,
1
1
1
1
13C-13
13C-13
C
39.2, 38.0 (d, J
28.6, 25.6, 22.5, and 14.0.
) 38.1 Hz), 36.5, 31.6, 29.7 (m), 29.2, 28.9,
Acknowledgment. Financial support of this work from the NIH
(CA77347) is gratefully acknowledged.
Supporting Information Available: 13C NMR spectra of methyl
nonactate and methyl homononactate prepared from nonactin isolated
in isotope feeding experiments. This material is available free of charge
via the Internet at http://pubs.acs.org.
1
(38:2:60), to give the title compound (0.31 g, 78.3%): H NMR (250
References and Notes
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Y.; Masui, N.; Fuji, F.; Takaki, Y.; Inoue, A.; Horikoshi, K.
Extremophiles 1999, 3, 29–34.
1
1
1
13C
J H- C ) 4.8, 164.8 Hz), 3.65 (0.5H, dd, J H- ) 6.4, 89.2 Hz), 3.4
1
1
1
1
(2H, q, J H- H ) 6.0 Hz), 3.02 (2H, t, J H- H ) 6.4 Hz), 2.87 (2H, t,
1
1
1
1
1
13
J H- H ) 6.8 Hz), 2.7 (2H, t, J H- H ) 6.8 Hz), 2.2 (1H, dd, J H-
)
C
1
13C
1.2, 6.4 Hz), 2.1 (2H, m), 2.00 (2H, dd, J H- ) 3.6, 6.4 Hz), 1.57
1
1
1
1
(2H, t, J H- H ) 6.8 Hz), 1.25 (9H, br, m), and 0.84 (3H, t, J H- H
)
6.4 Hz); 13C NMR (100 MHz, CDCl3) δ 208.3 (d, J
C ) 38.2 Hz),
13C-13
13C-13
13C-13
)
C
201.7 (d, J
62.6 Hz), 173.4, 99.7 (d, J
Hz), 43.0 (d, J
C ) 36.6 Hz), 199.6, 198.4, 194.8, 187.1 (d, J
13C-13
C
13C-13
) 36.6
C
) 64.1 Hz), 57.7 (d, J
13C-13
13C-13
)
C
C ) 39.7 Hz), 39.3, 38.3, 36.6, 33.9 (dd, J
10.7, 31.1 Hz), 31.6, 29.2, 28.9, 28.6, 25.6, 23.6, 22.5, and 14.0.
1-Benzyl 4-Ethyl [2-13C1]-([1-13C1]-2-Acetyl)succinate (17). To a
solution of 13 (0.36 g, 1.86 mmol) in benzene (10 mL) was added in
small portions 60% NaH in mineral oil (0.078 g, 1.95 mmol). The
mixture was stirred at rt for 1 h. Ethyl bromoacetate (0.25 mL, 2.23
mmol) was added via syringe. The solution was heated at 90 °C
overnight, after which the reaction was quenched by careful addition
of H2O (10 mL), followed by the addition of EtOAc (10 mL). The
mixture was shaken, and the organic layer recovered. The aqueous layer
was extracted with EtOAc (2 × 20 mL). The organic layers were
combined and dried over MgSO4, filtered, and concentrated. Purification
via silica gel chromatography, eluting with EtOAc-hexanes (40:60),
gave 1-benzyl 4-methyl [2-13C1]-([1-13C1]-2-acetyl)succinate (0.405 g,
1
77.8%): H NMR (400 MHz, CDCl3) δ 7.30 (5H, m), 5.15 (2H, d),
1
1
4.17 (0.5H, m), 4.08 (2H, q, J H- H ) 6.6 Hz), 3.85 (0.5H, m), 2.90
1
13
1
1
(2H, m), 2.30 (3H, d, J H- C ) 6.6 Hz), and 1.20 (3H, t, J H- H ) 6.6
13
13C-13
C
Hz); C NMR (100 MHz, CDCl3) δ 202.5 (keto, enriched, d, J
) 35.1 Hz), 201.7 (keto, enriched, d, J
13C-13
C ) 36.6 Hz), 175.0 (enol,
13C-13
enriched, d, J
enriched, d, J
) 76.3 Hz), 128.8, 128.7, 128.5, 94.8 (enol,
C ) 76.3 Hz), 67.7, 61.2, 59.5 (keto, enriched, d,
13C-13
C
13C-13
C
13C-13
) 36.6 Hz), 32.5
C
J
) 35.1 Hz), 54.8 (keto, enriched, d, J
(d), 30.2 (m), and 14.3.
S-[2-(Octanoylamino)ethyl] [3,4-13C2]-4-Oxopentanethioate (18).
17 (0.405 g, 1.45 mmol) was mixed with HCl (2 M, 8 mL) and heated
in an oil bath at 105 °C for 3.5 h, then stirred gently overnight at rt.
The reaction was extracted with EtOAc (3 × 20 mL). The organic
phases were combined, dried over MgSO4, and concentrated; the
product was purified by silica gel chromatography, eluting with
EtOAc-hexanes-AcOH (50:49:1), as a white solid (0.104 g, 61.1%):
(20) Rathke, M. W.; Cowan, P. J. J. Org. Chem. 1985, 50, 2624–2626.
(21) Woo, A. J.; Strohl, W. R.; Priestley, N. D. Antimicrob. Agents
Chemother. 1999, 43, 1662–1668.
(22) Nikodinovic, J.; Dinges, J. M.; Bergmeier, S. C.; McMills, M. C.;
Wright, D. L.; Priestley, N. D. Org. Lett. 2006, 8, 443–445.
(23) Bramer, C. O.; Steinbuchel, A. Microbiology 2001, 147, 2203–2214.
NP100421V