Quantification of N-acetylcysteamine activated methylmalonate incorporation into polyketide biosynthesis

Article abstract of DOI:10.3762/bjoc.9.75

Polyketides are biosynthesized through consecutive decarboxylative Claisen condensations between a carboxylic acid and differently substituted malonic acid thioesters, both tethered to the giant polyketide synthase enzymes. Individual malonic acid derivatives are typically required to be activated as coenzyme A-thioesters prior to their enzyme-catalyzed transfer onto the polyketide synthase. Control over the selection of malonic acid building blocks promises great potential for the experimental alteration of polyketide structure and bioactivity. One requirement for this endeavor is the supplementation of the bacterial polyketide fermentation system with tailored synthetic thioester-activated malonates. The membrane permeable N-acetylcysteamine has been proposed as a coenzyme A-mimic for this purpose. Here, the incorporation efficiency into different polyketides of N-acetylcysteamine activated methylmalonate is studied and quantified, showing a surprisingly high and transferable activity of these polyketide synthase substrate analogues in vivo.

Full text of DOI:10.3762/bjoc.9.75

Quantification of N-acetylcysteamine activated
methylmalonate incorporation into
polyketide biosynthesis
Stephan Klopries1, Uschi Sundermann1,2 and Frank Schulz*1,2
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2013, 9, 664–674.
1Fakultät für Chemie, Technische Universität Dortmund,
Otto-Hahn-Str. 6, 44221 Dortmund, Germany and 2Abteilung für
Chemische Biologie, Max-Planck-Institut für Molekulare Physiologie,
Otto-Hahn-Str. 11, 44227 Dortmund, Germany
doi:10.3762/bjoc.9.75
Received: 10 October 2012
Accepted: 11 March 2013
Published: 05 April 2013
Email:
Frank Schulz* - frank3.schulz@tu-dortmund.de
Associate Editor: A. Kirschning
* Corresponding author
© 2013 Klopries et al; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
biosynthesis; coenzyme A; malonic acid; polyketide; polyketide
synthase
Abstract
Polyketides are biosynthesized through consecutive decarboxylative Claisen condensations between a carboxylic acid and differ-
ently substituted malonic acid thioesters, both tethered to the giant polyketide synthase enzymes. Individual malonic acid deriva-
tives are typically required to be activated as coenzyme A-thioesters prior to their enzyme-catalyzed transfer onto the polyketide
synthase. Control over the selection of malonic acid building blocks promises great potential for the experimental alteration of
polyketide structure and bioactivity. One requirement for this endeavor is the supplementation of the bacterial polyketide fermenta-
tion system with tailored synthetic thioester-activated malonates. The membrane permeable N-acetylcysteamine has been proposed
as a coenzyme A-mimic for this purpose. Here, the incorporation efficiency into different polyketides of N-acetylcysteamine acti-
vated methylmalonate is studied and quantified, showing a surprisingly high and transferable activity of these polyketide synthase
substrate analogues in vivo.
Introduction
Polyketides are ubiquitous natural products and find wide- ditional diversity is introduced by the incorporation of different
spread application in current medicine and agriculture. Polyke- carboxylic acid starter units and a range of different extender
tide synthases (PKS), giant multienzyme complexes, play a units, usually coenzyme A-activated malonic acid derivatives,
pivotal role in their biosynthesis. PKS generate molecular with varying substituents at C-2 [4,5].
complexity and diversity through a number of stepwise conden-
sations in analogy to fatty acid synthases but with optional and Current experiments to generate biosynthetic polyketide diver-
varying degrees of reduction in each step (Figure 1) [1-3]. Ad- sity focus on different aspects of the biosynthetic reaction
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Beilstein J. Org. Chem. 2013, 9, 664–674.
Figure 1: The most intensively studied PKS, deoxyerythronolide B synthase (DEBS), which catalyzes the key steps in the biosynthesis of the antibi-
otic erythromycin. DEBS catalyzes the extension of a propionate starter unit with six equivalents of methylmalonyl-coenzyme A (MM-CoA). After six
rounds of decarboxylative Claisen condensations and varying degrees of reduction of the initially formed β-keto thioesters, the polyketide core of
erythromycin is released from the enzyme via a terminal esterase [6-8]. Abbreviations: AT: acyltransferase, ACP: acyl carrier protein, KS: ketosyn-
thase, KR: ketoreductase, DH: dehydratase, ER: enoylreductase, TE: thioesterase.
cascade. Mainly by genetic replacement or deletion of various for in vivo applications. Especially the bioavailability and the
fragments of PKS, alterations in chain length [9-11], redox coupled acceptance efficiency to the corresponding CoA-esters
pattern [12-14], stereochemistry [15-17], and starter unit diver- remain to be clarified. This has recently become highly rele-
sity [18-20] were in several instances introduced, giving rise to vant, as the first example for an acyltransferase domain with
a significant number of polyketide derivatives to date. However, artificially broadened substrate specificity has been constructed
the accessible extender unit diversity is currently restricted to a and introduced in the biosynthetic pathway towards
small number of different malonate units and changes in the erythromycin in place of the native AT6 domain [39]. This has
polyketide side chain pattern are highly sought after [4,17,21- led to the formation of 2-propargylerythromycin through the
26]. Different strategies can be pursued to introduce non-native incorporation of 2-propargylmalonate into the biosynthetic
extender units into the PKS machinery. They rely on the pathway, activated as SNAC-thioester and supplied to the
replacement of an acyltransferase domain of a given PKS bacterial fermentation. In this step, the exogenously supplied
module with another domain possessing different substrate synthetic building block competes with the endogenous methyl-
specificity by using various different strategies. Subsequently, malonyl-CoA (MM-CoA) for acceptance by the same enzyme
thioester activated non-native malonate derivatives can be domain; hence, the result of this experiment was the formation
synthesized by additional heterologously expressed biosyn- of the wild-type erythromycin product as a mixture with the
thetic pathways leading to the respective derivative of malonyl- new propargylated derivative. It is now of interest to judge the
CoA [17] or malonyl-ACP [27], which are supplied in vivo to efficiency of SNAC- versus CoA-activation. This can show to
the mutated PKS. Another option is the exogenous supply of the which extent the activation influences the choice of the extender
malonate derivative [28], typically activated as N-acetylcys- units in the case that a promiscuous acyltransferase domain can
teamine thioesters (malonyl-SNAC) [29-40]. Despite its catalyze the incorporation of a natural and a non-natural
apparent simplicity, the latter option is not well characterized building block.
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We here report the characterization of deuterated methyl- additionally formed dialkylation product. The subsequent
malonyl-SNAC ester incorporation into polyketides in vivo and thioesterification with N-acetylcysteamine (SNAC) smoothly
therefore in direct competition to endogenous MM-CoA, to yielded compound 3 in 82% yield by using CDI to activate the
determine the relative incorporation efficiency and the concen- malonic acid.
trations required to saturate polyketide synthases with the artifi-
cial extender unit donor. This determines the impact of SNAC- Enzymatic cleavage of the tert-butyl group was tested using the
versus CoA-activation and gives rise to rapid optimization of commercially available lipases CAL-A and CAL-B and gave
feeding experiments using exogenously supplied malonate- only small amounts of the desired product at low reaction rates
derivatives. These experiments point the way towards the with no detectable decarboxylation of the products. In contrast,
incorporation of non-native malonate derivatives into biosyn- TFA-promoted cleavage resulted in quantitative decarboxyla-
thetic pathways en route to new polyketide derivatives.
tion even at low temperatures, low acid concentrations and short
reaction times. Subsequently, Lewis-acid-mediated reactions
were examined by using either ZnBr2 or TiCl4. Thus, 2.5
equivalents of TiCl4 rapidly lead to cleavage of the tert-butyl
group, yielding compound 4 virtually byproduct free. The reac-
tion product was found to be prone to decomposition upon
concentration and storage. Work-up of the Lewis-acid-medi-
ated t-Bu-cleavage reaction by the addition of aqueous NaHCO3
buffer (pH = 8.0), followed by removal of precipitated TiO2 and
DCM led to an aqueous solution of the reaction product, which
Results and Discussion
Synthesis of D3-labeled malonyl-SNAC-
esters
The incorporation of artificially activated exogenous methyl-
malonate is most straightforwardly monitored by LC/ESI–MS.
This requires stable-isotope-labeled material, preferably a
D3-label.
For adding exogenous malonates to bacterial fermentations, was analyzed by mass spectrometry and TLC. Separately, the
millimolar concentrations of the SNAC-activated substrates reaction was quenched with a buffer of NaHCO3 in D2O to
were expected to be required in the fermentation media, making enable 1H NMR analysis of the reaction product. It was found
reliable and preparative-scale synthetic procedures to obtain the that the TiCl4-based t-Bu-cleavage under these conditions gives
SNAC-ester necessary. Hence, we decided to apply a modular quantitative conversion and no significant byproducts.
approach to our synthesis with an intrinsic transferability to
variously substituted malonates. After optimization, mono-t-Bu- Feeding experiments
protected malonate provided the best starting point for the syn- The frequently studied biosynthetic routes of erythromycin
thesis (Scheme 1) [34].
[6-8] and rapamycin [41] were chosen as model pathways for
the incorporation of the D3-label (Figure 2). These two polyke-
The sterically demanding protective group prevented decar- tides are produced by bacteria from two different genera and
boxylation during the subsequent alkylation and thioesterifica- possess different malonate incorporation patterns, thus
tion steps. Alkylation was achieved on a millimole-scale by providing significantly different test systems, albeit applying
using D3-iodomethane with LDA as base. After optimization, the same biosynthetic logic. By means of LC/ESI–MS analysis
the isolated yield of 2 was 37%. However, when CH3I was used of the fermentation extracts, the incorporation was to be quanti-
as electrophile for comparison, the yield reached 54% plus the fied in an erythromycin-producing strain of Saccharopolyspora
Scheme 1: Synthesis of SNAC-activated D3-methylmalonate. a: 2.1 equiv [(CH3)2CH]2NLi, 1 equiv CD3I, abs. THF, −78 °C → rt, Ar, 18 h, 37%.
b: 1.3 equiv N,N'-carbonyldiimidazole, 0.3 equiv DMAP, 1.5 equiv N-acetylcysteamine, abs. THF, 0 °C → rt, Ar, 18 h, 82%; c: 2.5 equiv TiCl4, CH2Cl2,
Ar, 0 °C → rt, 6 h, then aq NaHCO3 (pH 8.0), quant. (by TLC and 1H NMR).
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Beilstein J. Org. Chem. 2013, 9, 664–674.
Figure 2: Structures of erythromycin (left) and rapamycin (right). In this experiment both compounds were labeled in their methyl side chains with
deuterium.
erythraea (NRRL B-24071) and a rapamycin-producing strain experimental section for details on the fermentation system)
of Streptomyces hygroscopicus (NRRL 5491) through the [42]. In contrast, fermentation for the incorporation of
corresponding shift in the isotope ratio.
D3-methylmalonyl-SNAC into rapamycin biosynthesis was
carried out in 50 mL Erlenmeyer flasks, as fermentation of this
For feeding experiments, the carbonate buffer used in the syn- polyketide in deep-well plates was found to be poorly repro-
thesis of 4 was supplemented with soluble media components, ducible. Compound 4 was added in a series of concentrations to
individually prepared for the two bacterial strains (see experi- both fermentations, and the incorporation was quantified
mental section for details). This avoids a deleterious dilution of (Figure 3).
growth media during fermentation, even at high concentrations
of 4. It is important to note that 4 was synthesized in its racemic Erythromycin
form, whereas PKS exclusively accept (S)-MMCoA. This was It was observed that with the exogenous racemic malonate 4
shown for the biosynthesis of erythromycin and is assumed to supplied at a concentration of 1.25 mM, a single building block
be analogous for homologous systems [7]. Thus, the concentra- out of six endogenous methylmalonyl-CoA building blocks,
tion of (S)-4 that is available for the PKS is half of the fed was replaced in ~17% of the detected erythromycin. Addition-
concentration.
ally, at concentrations higher than 2.5 mM, a second exoge-
nously supplied building block was incorporated, and a third at
To analyze the incorporation of D3-methylmalonyl-SNAC into concentrations above 10 mM. At a concentration of 33 mM a
the erythromycin biosynthesis, fermentations were carried out triple incorporation was observed in ~20% of the detected
in triplicate in 24-well plates by using the Duetz system, erythromycin, measured after 5 days of fermentation (see
allowing for small-scale and reproducible fermentations (see Figure 3A and Figure 4 for MS spectra). A randomized distribu-
Figure 3: Relative incorporation of the D3-label into erythromycin (A) and rapamycin (B), depending on the fed concentration of rac-4. Color coding:
green: no incorporation; blue: single; red: double; magenta: triple incorporation as detected by LC/ESI–MS.
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Beilstein J. Org. Chem. 2013, 9, 664–674.
Figure 4: ESI–MS spectra of feeding experiments with an erythromycin-producing culture of S. erythraea. The mass spectra show the change in the
isotope ratio of the detected erythromycin, depending on the concentration of 4 in the fermentation medium. (A) control, 0 mM 4; (B) 1.25 mM 4; (C)
2.5 mM 4; (D) 5 mM 4; (E) 10 mM 4; (F) 15 mM 4; (G) 30 mM 4. The data are quantified by subtraction of the intensity of the mass for all relevant ions
(beginning from 734.3 in steps of three, corresponding to the D3-label) of the control from the experiments. An analogous figure (Figure S9) for the
feeding studies with a rapamycin-producing culture of S. hygroscopicus can be found in Supporting Information File 1.
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Beilstein J. Org. Chem. 2013, 9, 664–674.
tion over all six MMCoA-utilizing DEBS-modules can be incorporation of hydrolyzed and subsequently CoA-activated 4,
assumed. With this detection method, the random incorporation but clearly shows that the SNAC activation itself indeed drives
of the D3-label was measured over all six modules of the the incorporation independently from further activation in vivo.
erythromycin PKS.
This is in accordance with an observation made with 2-propar-
Rapamycin
gylmalonyl-SNAC (5), whose acceptance by a specific acyl-
In the case of rapamycin, feeding was carried out at a maximal transferase variant in the erythromycin PKS leads to the forma-
concentration of 10 mM. At this concentration no triple tion of 2-propargylerythromycin A (Scheme 2) [39]. In that
incorporation was detectable. The single incorporation was experiment, the active site of DEBS AT6 was redesigned to
observed in the same way as for the erythromycin feeding study increase acceptance of 5 for incorporation into erythromycin A.
at a concentration of 1.25 mmol of 4 in ~13% of the detected A targeted mutation was introduced into the erythromycin
rapamycin. The second incorporation was observed at a concen- producer S. erythraea. The wild-type strain showed no
tration higher than 2.5 mmol. Again, the incorporation was incorporation of 5 over the background signal. However, when
observed over the whole five days of the fermentation and it can the mutation Val295Ala was introduced into DEBS AT6, a
be assumed that the incorporation is randomly distributed over significant fraction of the complete erythromycin A production
all seven MMCoA-utilizing modules (out of 14 in total) of the was redirected towards the generation of 2-propargylery-
rapamycin PKS (RAPS). This indicates a similar efficiency of thromycin A [39].
the whole incorporation process, beginning with membrane
transfer and ending with the trans-thioesterification onto the The feeding studies for that experiment were carried out in
enzyme. Consequently, the exogenous MM-SNAC can actually fermentations of S. erythraea, thereby utilizing the full produc-
compete with MM-CoA if provided in millimolar concentra- tivity of this microorganism at the expense of overcoming its
tions. Through the relatively straightforward synthetic access to complexity. When 2-propargylmalonic acid (6a) or its dimethyl
this compound, these concentrations are easily reachable and ester 6b were fed to the culture of S. erythraea DEBS AT6*, no
can be used to overcome the expectedly weaker productivity incorporation into erythromycin was observed. Furthermore, the
inside the PKS machinery.
low catalytic promiscuity of wild type MatB malonyl-CoA
synthetases was recently described [40], pointing out the need
In order to quantify a potential in vivo replacement of SNAC for an artificial and versatile activation mechanism as described
with the native activator coenzyme A, which would blur the here, to enable what we have termed enzyme-directed mutasyn-
results, we directly compared the incorporation of D3-methyl- thesis.
malonate to the incorporation of 4 into the biosynthesis of
rapamycin (Supporting Information File 1, Figure S8). At 2 mM Conclusion
feeding concentration, on average 29% of the extracted Based on the single incorporation of 4 a relative incorporation
rapamycin was D3-labeled in the case of 4 but only 17% in the rate of around 40% could be observed at concentrations below
case of the non-SNAC-activated analogue. At 4 mM feeding 10 mM. When the substrate concentration is increased the
concentration, 4 gave 44% incorporation and the analogue 31%. single incorporation stagnates or decreases; instead double- and
This corresponds to a 1.5- to 1.7-fold more efficient incorpor- even triple-incorporation is observed in amounts increasing
ation upon SNAC-activation. This indicates the potential with the concentration of 4. Overall up to ~60% of
Scheme 2: Incorporation of a propargylated malonic acid derivative into erythromycin through an active-site mutation in the acyltransferase in DEBS
module 6 (DEBS AT6*) [39].
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Beilstein J. Org. Chem. 2013, 9, 664–674.
erythromycin and rapamycin is D3-labelled to a varying extent trations of (S)-4 and seems to be transferable between different
in the presence of 10 mM rac-4, corresponding to 5 mM of the species and enzymes, hence providing a good starting point for
naturally accepted (S)-enantiomer.
individual optimizations.
Experimental
Significant incorporation of SNAC-activated methylmalonate 4
into the biosynthesis of erythromycin and rapamycin requires General
no significantly different concentrations of (S)-4 between the Unless otherwise stated, materials for chemical synthesis were
two fermentations (typically ≥1 mM). This indicates a suffi- obtained from commercial suppliers (Sigma-Aldrich, Alfa
cient reactivity with PKS. However, when considering that the Aesar, Fluka, Acros) in the highest purity available and used
compound competes with endogenous MM-CoA, which is without further purification. Dry solvents were purchased from
present at micromolar concentration in actinomycete cells [43], Sigma-Aldrich, stored over molecular sieves and used as
it is apparent that analogous malonate derivatives will have to supplied. Solvents used for extraction and chromatography were
be supplied in large amounts to ideally enable saturation of purchased from Thermo Fisher Scientific. Flash chromatog-
acyltransferase variants with artificial building blocks. It also raphy was carried out using Acros silica gel 60 (35–70 µm
has to be taken into account that 4 has to migrate from the mesh). Thin-layer chromatography (TLC) was performed on
growth medium into the intracellular environment before aluminium-backed, precoated silica gel (60 F245) from Merck
reacting with the PKS; currently no statement on the intracel- with cyclohexane/EtOAc or DCM/MeOH mixtures as mobile
lular concentration of the artificial building block can be made. phases. Spots were detected by staining with KMnO4 solution
(5.0 g KMnO4, 33 g K2CO3, 10 mL 5% aqueous NaOH in
The incorporation of non-native biosynthetic building blocks 500 mL H2O) and subsequent heat treatment.
into polyketide biosynthesis is an emerging field of research
and requires either the elaborated engineered biosynthesis of NMR spectra were recorded by using a Varian Mercury 400
such a building block in the host or the straightforward supply (400 MHz, 1H; 100 MHz, 13C) spectrometer and calibrated
with an exogenous SNAC-activated malonate. However, this using residual undeuterated solvent as an internal reference.
feeding has not yet been systematized, and especially the High-resolution mass spectra were recorded at LTQ Orbitrap
competition of the non-native thioester activation with the with Accela HPLC-System (column Hypersil Gold, length
endogenous coenzyme A had yet to be quantified. We here 50 mm, inside diameter 1 mm, particle size 1.9 µm, ionization
show that N-acetylcysteamine is indeed competitive to coen- method: Electrospray Ionization). Products were characterized
zyme A in its ability to activate methylmalonate for utilization by NMR (1H, 13C) and HRMS.
by polyketide synthases. Interestingly, the efficiency of
ESI-method for quantification of D3-label
incorporation is comparable between two rather different
polyketide producers from two different bacterial genera, indi- incorporation
cating a potentially more general trend in the values measured Mass-to-charge ratios of the extracts were analyzed by HPLC
in this study.
coupled to a mass spectrometer. The separations were carried
out on an Accela HPLC-System (consisting of pump, autosam-
Knowledge about the concentrations of SNAC-activated pler, column oven and PDA detector) coupled online to an
building blocks required for feeding experiments under condi- Orbitrap mass spectrometer equipped with a LTQ XL linear ion
tions optimal for polyketide fermentation is critical, as unneces- trap (Thermo Electron Corporation, Dreieich, Germany) using
sary consumption of the rather expensive material should be the standard electro spray ionization source. Parallel UV
avoided, as should an underdosage, leading to false-negative absorption was detected at 210 and 254 nm. All solvents were
outcomes or low yields. The results obtained clearly show the LC–MS grade (Chromasolv, Sigma-Aldrich, Munich,
benefit of providing an activated building block, which is espe- Germany). A 5 µL amount of each sample was injected by an
cially relevant for non-native substances, but apparently also in autosampler (T = 10 °C) onto a CC12514 Nucleor C18 Gravity
the case of pseudo-native building blocks as the cellular column (3 µm particle size; Macherey-Nagel Germany) using a
machinery is not sufficiently efficient to achieve maximal flow rate of 500 µL/min.
incorporation rates.
Erythromycin: A linear gradient starting with 80% solvent A/
Overall, this study serves as a knowledge source for future 20% solvent B for one minute and increasing to 0% solvent A/
experiments aiming at the incorporation of non-native building 100% solvent B in 10 min. After that the column was washed
blocks into PKS. The efficiency of SNAC as a CoA-mimic is with 0% solvent A/100% solvent B for 5 min and re-equili-
demonstrated in a direct competition at low millimolar concen- brated to the starting conditions for an additional 5 min (solvent
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Beilstein J. Org. Chem. 2013, 9, 664–674.
A: water containing 0.1% formic acid; solvent B: acetonitrile 15 min. Then the cooling bath was removed and 433 mg
containing 0.1% formic acid).
(190 µL, 3.12 mmol) D3-MeI was added dropwise. Afterwards
the brown reaction mixture was stirred for 18 h at rt, then
Rapamycin: A linear gradient starting with 18% solvent A2/ cooled to 0 °C and quenched with 5 mL saturated NH4Cl solu-
82% solvent B2 for one minute and increasing to 0% solvent tion. The organic layer was recovered and the solvent removed
A2/100% solvent B2 in 10 min. After that the column was in vacuo. The resulting slurry was diluted with 50 mL saturated
washed with 0% solvent A2/100% solvent B for 5 min and NaHCO3 solution and washed with 50 mL of EtOAc. The
re-equilibrated to the starting conditions for an additional 5 min organic layer was washed twice with 50 mL sat. NaHCO3 solu-
(solvent A2 water containing 10 mM NH4OH, solvent B2 tion. The combined water phase was acidified to pH = 1 with
methanol containing 10 mM NH4OH and 125 mL THF/2.5 L). conc. HCl and extracted three times with 50 mL EtOAc. The
combined organic layers were dried over Na2SO4, and the crude
For mass spectrometric detection the electrospray ionization product was purified by column chromatography (cyclohexane/
was carried out in positive (erythromycin)/negative (rapamycin) EtOAc 95:5) to obtain 200 mg (37%) of 2 as a slightly yellow
ionization mode by using a source voltage of 4 kV. The capil- oil. Rf 0.5 (DCM/MeOH 9:1); HRMS (GC–MS): [M + H]+
lary voltage was set to 18 V, the capillary temperature to calcd for C8H122H3O4, 178.11532; found, 178.11512; 1H NMR
275 °C, and the tube lens voltage to 115 V. Spectra were (400 MHz, CDCl3-d1) 1.46 (s, 9H), 3.36 (s, 1H); 13C NMR
acquired in full scan centroid mode with a mass-to-charge range (101 MHz, CDCl3-d1) 21.1, 28.2, 47.0 ,82.7, 169.6, 176.6.
from 200 to 2000.
Synthesis of tert-butyl 3-((2-acetamidoethyl)thio)-2-D3-
Synthesis
methyl-3-oxopropanoate (3): 810 mg (4.65 mmol) of com-
Synthesis of lithium diisopropylamine (LDA): Diisopropyl- pound 2 were dissolved in 10 mL abs. THF under Ar. Subse-
amine (2 mL,14.23 mmol) (freshly distilled from NaH) was quently, 981 mg (6.05 mmol) CDI and 170 mg (1.40 mmol)
mixed with 5 mL abs. THF under Ar. The solution was cooled DMAP were added at 0 °C, and the mixture was stirred for
to −78 °C. Then 9.78 mL (15.65 mmol) n-butyllithium (1.5 M 45 min at 0 °C before 1.109 g (9.30 mmol) SNAC was added
solution in hexane) was added dropwise, and the reaction mix- dropwise. The reaction mixture was stirred for another 30 min
ture was stirred for 40 min at −78 °C. After 40 min the white at 0 °C and then for 18 h at rt. The solvent was removed in
suspension was allowed to reach room temperature and the clar- vacuo and the residue was suspended in 50 mL 0.1 M HCl. The
ified solution was used immediately.
suspension was extracted three times with 50 mL of DCM. The
combined organic layers were collected, dried over Na2SO4,
Synthesis of N-acetylcysteamine (SNAC) [25]: Cysteamine and purified by column chromatography (DCM/MeOH 99:1) to
hydrochloride (11.4 g, 100 mmol), 25.2 g (300 mmol) NaHCO3 obtain 1.05 g (82%) of 3 as a slightly yellow oil. Rf 0.5 (DCM/
and 5.6 g (100 mmol) KOH were added to 500 mL of deion- MeOH 9:1); HRMS (LCMS–ESI): [M + H]+ calcd for
ized H2O. After everything was dissolved, 9.5 mL (100 mmol) C12H192H3O4NS, 279.14524; found, 279.14567; [M + Na]+
acetic anhydride was added dropwise. After stirring at room calcd for C12H192H3O4NSNa, 301.12718; found, 301.12749;
temperature for 2 h, the light rose solution was brought to 1H NMR (400 MHz, CDCl3-d1) 1.39 (s, 9H), 1.90 (s, 3H),
pH = 4 with conc. HCl and extracted three times with 100 mL 2.98–3.02 (m, 2H), 3.32–3.39 (m, 2H), 3.48 (s, 1H), 6.34 (bs,
EtOAc. The combined organic layers were dried over Na2SO4 1H); 13C NMR (101 MHz, CDCl3-d1) 23.2, 27.8, 28.8, 39.6,
and purified by column chromatography (DCM/MeOH 99:1) to 54.9, 82.4, 168.6, 170.5, 196.8.
obtain 7.56 g (61%) of the desired product as a colorless oil.
Rf 0.42 (DCM/MeOH 9:1); HRMS (GC–MS): [M + H]+ calcd Synthesis of 3-((2-acetamidoethyl)thio)-2-D3-methyl-3-
for C4H10ONS, 120.04776; found, 120.04730; 1H NMR oxopropanoic acid (4): 513 mg (1.84 mmol) of compound 3
(400 MHz, CDCl3-d1) 1.34–1.38 (t, J = 8.4 Hz, 1H), 1.97 (s, were dissolved in 50 mL abs. DCM under Ar. At 0 °C 873 mg
3H), 2.60–2.66 (m, 2H), 3.36–3.40 (m, 2H), 6.33 (bs, 1H); 13C (505 µL, 4.66 mmol) TiCl4 was added dropwise. The dark
NMR (101 MHz, CDCl3-d1) 23.1, 24.5, 42.6, 170.5.
brown reaction mixture was stirred for 30 min at 0 °C, then for
another 6 h at room temperature. After 6 h (DC-control) the
Synthesis of 3-tert-butoxy-2-D3-methyl-3-oxopropanoic acid reaction mixture was quenched with 19 mL of the individual
(2): 3-tert-Butoxy-3-oxopropanoic acid (500 mg, 3.12 mmol) NaHCO3-based feeding buffer (see below) in an ice bath to
(freshly purified by column chromatography before use) was reach a final concentration of 0.1 M of the product. DCM was
dissolved in 10 mL abs. THF under Ar. The solution was cooled evaporated at room temperature and the remaining white slurry
to −78 °C. Subsequently, 7.17 mmol of freshly prepared LDA was transferred to 50 mL polypropylene tubes. After centrifuga-
were slowly added and the resulting mixture was stirred for tion for 4 min to remove TiO2, the resulting clear solution was
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Beilstein J. Org. Chem. 2013, 9, 664–674.
sterile filtered and used directly for feeding experiments. × 0.5 cm agar piece was used to inoculate the preculture, 7 mL
Rf 0.16 (MeOH/DCM 1:9); HRMS: [M + H] calcd for RapV7 Seed Medium [47] supplemented with 0.16 mL 20%
C8H112H3O4NS, 223.08264; found, 223.08286; 1H NMR glucose, and cultivated for 48 h at 180 rpm and 28 °C. The main
(400 MHz, CDCl3-d1) 1.95 (s, 3H), 3.10–3.13 (t, J = 6.2 Hz, culture in 7 mL MD6 medium [47] (supplemented with
2H), 3.38–3.41 (t, J = 6.2 Hz, 2H), 3.88 (s, 1H).
0.35 mL 40% fructose and 0.1 mL L-lysine (140 mg/mL)) was
mixed with feeding buffer (containing different concentrations
of substrate) and inoculated with 0.35 mL preculture before
Preparation of the Feeding buffers
Erythromycin: 5 g glucose, 50 g Glucidex IT29 (Roquette), incubation at 26 °C and 160 rpm for six days.
83.83 g NaHCO3, and 186 mg Na2CO3 were dissolved in 1 L of
Millipore water to obtain 1 M carbonate feeding buffer at Afterwards, 10 mL methanol and glass beads were added to the
pH = 8.
culture, and the slurry was shaken for a further 3 h. The
resulting suspension was transferred into polypropylene tubes
Rapamycin: 30 g starch (corn), 1 g yeast, 30 g Toasted and centrifuged, and 1 mL of the supernatant was filtered and
Nutrisoy, 19 g Dextrin, 10 g NaCl, 83.83 g NaHCO3, and used for analysis.
186 mg Na2CO3 were dissolved in 1 L of Millipore water to
obtain the 1 M carbonate feeding buffer at pH = 8.
Supporting Information
Feeding experiments
Supporting Information File 1
All experiments were performed in triplicate. Concentrations of
4 tested for incorporation in the biosynthesis of erythromycin
and rapamycin were: 0 mM; 0.625 mM; 1.25 mM; 2.5 mM;
5.0 mM; 10.0 mM; 15.0 mM; and 33.33 mM; the latter two only
for erythromycin. A comparison of the incorporation of 4 and
D3-methylmalonic acid into the biosynthesis of rapamycin was
carried out at 2.0 mM and 4.0 mM.
NMR spectra of synthesized compounds and ESI–MS
spectra of D3-propionate incorporation.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-9-75-S1.pdf]
Acknowledgements
Generous financial support from the Beilstein Institut zur
Förderung der Chemischen Wissenschaften is acknowledged.
The authors thank the Fonds der Chemischen Industrie
for a Liebig fellowship to F. S. and a doctoral stipend to U. S.
U. S. is a fellow of the International Max Planck Research
School of Chemical Biology in Dortmund, Germany. Candida
antarctica lipases A and B were a generous donation from
c-LEcta GmbH (Leipzig, Germany), Glucidex was kindly
donated by Roquette GmbH (Frankfurt, Germany).
Erythromycin: Saccharopolyspora erythraea NRRL-B-24071
was grown on ABB13 agar [44] at 30 °C. Fermentation was
performed by using the 24 deep-well format Duetz system,
containing three glass beads per well. The Duetz system
employs a multilayer cover for deep-well plates to ensure repro-
ducible oxygen distribution over all wells in a plate [42]. We
found the 24-well Duetz system particularly suitable for actino-
mycetes in contrast to standard plate covers with air-permeable
foils. A 3 mL volume of TSB was inoculated with a 0.5 cm ×
0.5 cm agar slice of a sporulating petri-dish culture and culti-
vated for 48 h at 30 °C. The main culture consisting of 3 mL
SM3 [45] complemented with feeding-buffer (containing
different concentrations of substrate), was inoculated with
0.125 mL preculture and incubated for 5 days at 30 °C and
180 rpm.
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