Biocatalysis at Work: Applications in the Development of Sagopilone

Article abstract of DOI:10.1002/cmdc.201500138

For the antitumour agent sagopilone, an epothilone analogue, a large-scale synthesis was developed to synthesise the active pharmaceutical ingredient for clinical trials, exploring enzymatic and microbial methods to produce chiral building blocks on a mul

Full text of DOI:10.1002/cmdc.201500138

DOI: 10.1002/cmdc.201500138
Full Papers
Biocatalysis at Work: Applications in the Development of
Sagopilone
Kathrin Gottfried,[b] Ulrich Klar,[a] Johannes Platzek,[b] and Ludwig Zorn*[a]
For the antitumour agent sagopilone, an epothilone analogue,
a large-scale synthesis was developed to synthesise the active
pharmaceutical ingredient for clinical trials, exploring enzymat-
ic and microbial methods to produce chiral building blocks on
a multi-kilogram scale. The three building blocks were identi-
fied as key intermediates in the synthesis and needed to be
produced with high optical purity in yields higher than those
previously published. The improved syntheses of two of these
building blocks are detailed herein. For building block A, the
chemical research synthesis was abandoned, and a novel
chemical route was developed leading to building block A via
an enzymatic hydrolysis process. For building blocks C, replace-
ment of a chemical catalytic procedure by a microbial process
meant that the development of a new starting material could
be avoided, thereby accelerating the development process. For
the clinical development process, a human metabolite of sago-
pilone was required as a reference. To accelerate the synthesis
of the metabolite, no chemical synthesis was investigated;
rather, we relied solely on oxidoreductases. The human metab-
olite of sagopilone was synthesised on a multi-gram scale in
a single-step process using genetically engineered E. coli ex-
pressing human cytochrome P450 enzyme 2C19. The integra-
tion of enzymatic and microbial processes provided tools that
enable the synthesis of highly functionalised intermediates and
metabolites.
Introduction
Searching the rich sources of nature to isolate and
analyse novel compounds as candidates for pharma-
cological treatments is well established.[1] In the area
of cancer therapy, a plethora of potent anticancer
compounds have been isolated from natural sour-
ces[2] such as plants (e.g., paclitaxel from Taxus brevi-
folia)[3] and microorganisms (e.g., epothilones from
Sorangium cellulosum, a myxobacterium strain).[4]
In addition to the exploitation of biological sys-
tems as a source of new bioactive lead compounds,
microorganisms or isolated enzymes are widely used
to perform chemical transformations as an alternative
to traditional chemical catalysts.[5] There are many ex-
amples of the use of these biological tools, either as
whole cells[6] or isolated enzymes.[7] In addition to
originating from a renewable source, biological catalysts have
the advantage of being highly selective[8] and exceptionally
beneficial for introducing chirality into functionalised mole-
cules.[9] Biotransformations have thus gained significant impor-
tance in industrial processes.[5g]
Figure 1. Epothilone B and analogues.
aimed at identifying an optimised analogue as a development
candidate was initiated in our company in 1997.[11] Although
epothilone B is derived from a natural source, our research pro-
gram was not limited to accessing new derivatives by semisyn-
thesis, but instead it took a total synthesis approach. After the
de novo synthesis of more than 350 epothilone analogues, sa-
gopilone (ZK-EPO, 2; Figure 1) was selected as the develop-
ment candidate based on its most beneficial overall pharmaco-
logical profile.[11b] The synthesis of 2 was subjected to process
optimisation and scale-up to provide the active pharmaceutical
ingredient for clinical trials.[12] In phase II clinical studies, sago-
pilone was found to be highly efficacious in first- and second-
line treatments of ovarian cancer,[13] prostate cancer,[14] melano-
ma,[15] and glioblastoma.[16]
Epothilone B (1, Figure 1) has shown strong antiproliferative
activity, but is accompanied by considerable toxicity in animal
models at therapeutic doses.[10] Therefore, a research program
[a] Dr. U. Klar, Dr. L. Zorn
Bayer Pharma AG, Global Drug Discovery
Medicinal Chemistry, 13353 Berlin (Germany)
E-mail: ludwig.zorn@bayer.com
[b] Dr. K. Gottfried, Dr. J. Platzek
Bayer Pharma AG, Global Drug Discovery
Chemical Development, 42119 Wuppertal (Germany)
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Results and Discussion
Preparation of building block A
An alternative access was re-
quired for the synthesis of build-
ing block A, as the research ap-
proach using optically active
(À)-pantolactone (5) as the ex-
chiral-pool starting material was
not applicable for a development
approach due to the use of haz-
ardous reagents in several
steps.[12d] A novel approach was
therefore developed based on
nitrile 6 (Scheme 1).[12d] This pre-
cursor was then transformed
into building block A by a se-
quence of chemical transforma-
tions.[18]
First, however, the optically
active nitrile precursor 6 had to
be synthesised. To introduce the
chirality, we relied on the enzy-
matic kinetic resolution of race-
mic acetate 11 (Table 1).[19] More
than 20 enzymes were screened,
and the lipase AY30 from Candi-
Scheme 1. Retrosynthetic analysis of sagopilone (2), with the resulting building blocks A, B, and C.
Table 1. Enzymatic kinetic resolution by hydrolysis of acetate 11 using
Candida rugosa lipase AY30 (Amano).[a]
Scheme 1 depicts the strategic bond formations and result-
ing building blocks that were used for the development pro-
cess, as reported elsewhere.[12d] Key reactions for the coupling
of building blocks involved an aldol reaction (formation of the
C6ÀC7 bond), a Wittig olefination (formation of the C12ÀC13
olefin as a precursor for the epoxide moiety), and a macrolacto-
nisation (ring closure).[12] All three building blocks A–C contain
at least one stereogenic centre. Chemical and biocatalytic ap-
proaches were investigated in parallel in order to develop
robust and economical syntheses for these building blocks. De-
tails of the biocatalytic approaches are described herein.
Incubation
time [h]
ee [%][b]
R-alcohol 12
ee [%][b]
S-acetate 13
Yield [%][b]
R-alcohol 12
5
24
48
76
89
91
88
11
16
91
98
3
10
11
23
168
In addition to implementing an efficient and scalable syn-
thetic route to sagopilone, we were also faced with the need
to provide gram quantities of its main human metabolite 3
(Figure 1). Traditionally, metabolites are synthesised via the
same synthetic sequence as their parent, except that a different
starting material or a modified intermediate with an additional
functionality would be selected.[17] If this strategy was applied
here, more than 37 steps for the synthesis of metabolite 3
would be required![12d] Clearly, such an approach would be
time-consuming and uneconomical. Therefore, we relied on
a biotransformation of sagopilone (2) to directly access its
main human metabolite 3 in a single chemical transformation.
[a] Conditions: acetate 11 (1 mg), ethanol (50 mL), phosphate buffer
(66 mm, pH 7, 1 mL), 378C, 1000 rpm. [b] Determined by chiral HPLC.
da rugosa (EC 3.1.1.3, Amano) was found to be highly selective,
providing an easy route to the desired S-enantiomer as acetate
13.[20]
Even after days, only trace amounts of the S-acetate 13 were
hydrolysed; thus, a rapid reaction workup was not required,
minimising the possibility of a decreased yield of 13. Without
enzyme, acetate 11 was not hydrolysed. In the next step, the
reaction needed to be performed on a gram scale. In the de-
velopment process, higher concentrations (>1 gLÀ1) of acetate
11 were required, and additives to dissolve the acetate needed
to be minimised. It was established that, at 408C, acetate 11
was soluble in the buffer without any additional solvent re-
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quired. The enzymatic hydrolysis could be performed
at this temperature with the same excellent stereose-
lectivity and yield as before. Thus, to increase
throughput, the temperature was raised to 408C
which allowed an increase in the concentration of
racemic acetate 11 to 40 gLÀ1.[20] In bulk quantities,
the lipase was supplied as a granulated product
(lipase AYS, Amano) instead of a powder; neverthe-
less, similar results were obtained. To supply suffi-
cient material for the initial development process,
a 45-kg batch of the racemic acetate 11 in a buffer at
Scheme 4. Enzymatic kinetic resolution by hydrolysis of acetate 17 and acetylation of
alcohol 18.
pH 6 (actively controlled by the addition of 1m sodium hydrox-
ide solution) at 408C was hydrolysed to yield, after chromatog-
raphy, 22 kg (yield: 98%, theoretically achievable maximum
yield of 50%) of the S-acetate 13 with >99% ee.
As the enzymatic kinetic resolution of esters 16 was unsuc-
cessful, we turned to the enzymatic kinetic resolution of race-
mic acetate 17 and to acetylation of the corresponding alcohol
18 (Scheme 4).[23] The hydrolysis option was investigated with
more than 60 enzymes, with the best result being obtained at
pH 6 with lipase G50 from Penicillium camembertii (EC 3.1.1.3,
Amano); however, only a moderate 46% enantiomeric excess
of S-alcohol 15 was achieved. Acetylation of 18 with, for exam-
ple, vinyl acetate was not selective, and this approach was
abandoned. In the meantime, an improved chemical synthetic
route starting from Roche ester 8 had been developed,[12d] so
the search for an alternative enzymatic process was discontin-
ued.
Preparation of building block B
The chemical research synthesis of building block B also began
with an ex-chiral-pool starting material, the well-known (À)-
Roche ester 8.[12d] During the development process, novel
chemical and biocatalytic approaches to the S-enantiomer of
building block B were evaluated. Initially,
a biooxidation
screening was performed to explore the option of transform-
ing the commercially available ketone 14 directly into the de-
sired S-enantiomer of keto alcohol 15 (Scheme 2). More than
Preparation of building block C
In building block C a stereogenic centre substituted with a hy-
droxy group, protected as a silyl ether, was needed which was
initially derived from carboxylic acid precursor 10 (Scheme 1).
This precursor was first converted into the corresponding ben-
zothiazole, and then the acid functionality was transformed
into an aldehyde group. The aldehyde was subjected to an
Evans aldol reaction to access diastereomeric oxazolidinone-
based b-hydroxy amides.[12d] Isolation and purification of the
desired stereoisomer were hampered by limited stereoinduc-
tion and facile epimerisation of the oxazolidinone stereocentre
under deprotonation conditions. Therefore, a new approach
was devised which avoided the Evans aldol step and
instead used b-keto ester 19 as the key intermediate
(Scheme 5).
Scheme 2. Stereoselective hydroxylation of ketone 14.
135 strains from our corporate collection of fungi and bacteria
were screened without success. This screening was hampered
by the volatility of ketone 14.
Next, the enzymatic kinetic separation of four racemic esters
16 was explored (Scheme 3).[21] These esters were accessed by
Chemical as well as biocatalytic options were in-
vestigated for achieving the enantioselective reduc-
tion of 19 to the S-alcohol 20. Asymmetric hydroge-
nation and transfer hydrogenation were hampered
on a large scale by the fact that more than 1% of an
expensive catalytic system [Ru/(R)-Xyl-SOLPHOS] was
needed to obtain a highly enantio-enriched pro-
duct.[12d] Additionally, and even more troubling, poi-
soning of the catalyst by traces of sulfur impurities,
originating from the synthesis of the benzothiazole moiety, led
to unreproducible results and thereby placed scale-up cam-
paigns at risk.
Scheme 3. Enzymatic kinetic resolution by hydrolysis of esters 16.
oxidative ring opening of the inexpensive commercially avail-
able 2,6-dimethylcyclohexanone with, for instance, potassium
permanganate and subsequent esterification under standard
conditions.[22] A screening with 30 commercially available ester-
ases and lipases was performed, but none of the enzymes
showed a sufficient turnover rate with useful enantiomeric
ratios. Therefore, this approach was terminated.
Hence, the alternative biocatalytic approach for accessing b-
hydroxy ester 20 became the last resort. The most economical
process to introduce the required functionality is the stereose-
lective reduction of the keto group of 19, as up to 100% of
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1–4% of the starting material could still be detected in the fer-
mentation mixture.
With Pichia strains, the b-keto ester 19 was reduced nearly
completely, even at substrate concentrations as high as 2 gLÀ1.
Pichia wickerhamii (IFO 1278) was identified as a highly produc-
tive biocatalyst for the desired transformation, as 2 gLÀ1 of
substrate 19 were reduced within 24 h, and the product 20
was obtained with >99% ee.
In a next step, we studied further substrate concentration in-
creases with this yeast strain. In all samples, the b-keto ester
19 was reduced exclusively to the S-enantiomer with
>99% ee. At a concentration of 4 gLÀ1 all of the b-keto ester
19 was reduced, but at a concentration of 6 gLÀ1 and higher,
more than 20% of the b-keto ester was still detected after
72 h. Therefore, we opted to use 4 gLÀ1 in a shake-flask fer-
mentation process. In this way, b-keto ester 19 (4 g) was incu-
bated for 114 h. The culture broth was then extracted with
ethyl acetate, and the extracts were purified by column chro-
matography, which gave the S-enantiomer 20 in 78% yield
(3.14 g) with >99.5% ee.
Scheme 5. Building block C: retrosynthetic analysis and stereoselective
reduction of the b-keto ester 19.
the desired enantiomer is possible.[24] In contrast, in the case of
enzymatic resolution of racemic hydroxy esters, for example,
only up to 50% yield can be obtained, necessitating inversion
of the undesired stereocentre to increase the overall yield.[25]
Another possibility for obtaining up to 100% yield is through
a dynamic kinetic resolution process, but special molecular fea-
tures are required.[26] Therefore, we focused on establishing
a direct biocatalytic reduction of b-keto ester 19.
For a large-scale microbial reduction process, it is not suffi-
cient to only achieve a high turnover rate and high enantio-
meric purity of the desired enantiomer. In addition, ease of
product isolation is equally important which, in whole-cell bio-
transformations, is sometimes hampered by the formation of
lipophilic byproducts. Thus, a microorganism was needed
which allowed high turnover and product isolation by simple
extraction and crystallisation steps.
After this favourable result, the fermentation process was
scaled up further. In one of the first batches, the b-keto ester
19 (109 kg) was incubated with P. wickerhamii (IFO 1278) in
a 50 m3 fermenter. b-Hydroxy ester 20 was extracted with
methyl isobutyl ketone and crystallised from diisopropyl ether
and cyclohexane, affording the S-enantiomer in 79% yield
(86.3 kg) with 99% purity and 99.8% ee (<0.1% of keto ester
19).[28]
In an alternative approach, the corresponding b-keto tert-
butyl ester 21 could be reduced with Issatchenkia orientalis
(NCYC 45) in a shake-flask fermentation process to yield the S-
enantiomer 22 in 59% yield (Scheme 6). Highly selective reduc-
Toward this end, an explorative screening of 30 yeast strains
was conducted. Pure samples of both enantiomers of the b-hy-
droxy ester product were synthesised by alternative methods
to allow direct HPLC-based analyses of the screening samples.
In all cases, b-keto ester 19 was nearly completely consumed
after 24 h, and, in most cases, the S-enantiomer 20 was detect-
ed as the main enantiomer formed; however, degradation of
product 20 was observed with certain strains if incubation was
continued for 72 h.
Scheme 6. Stereoselective reduction of the b-keto tert-butyl ester 21.
After these encouraging first results, the screening was ex-
panded, and more than 300 strains of yeasts and anaerobic
bacteria from our corporate collection of microorganisms were
screened. We began with a substrate concentration of 100 and
200 mgLÀ1 of b-keto ester 19 for yeasts and anaerobic bacte-
ria, respectively. For promising hits from this initial screening,
fermentation conditions were further optimised with a focus
on substrate concentration and the testing of various fermen-
tation media. Several subsets of strains were incubated in dif-
ferent media and with b-keto ester 19 at 200–10000 mgLÀ1.
The best results were achieved with a medium consisting of
50 gLÀ1 glucose and 20 gLÀ1 corn steep liquor.[27] Our goal was
to establish high turnover and enantioselectivity with substrate
tions of this class of b-keto esters with yeasts proved to be
a quite general process for accessing the corresponding chiral
hydroxy esters, but the process does require substrate-specific
optimisation of the exact parameters.[12c]
Biooxidation of sagopilone to access its main human
metabolite
Safety profiling of major human metabolites is a key compo-
nent in the development of each new drug candidate and is
strictly regulated.[29] In general, metabolites resulting from de-
constructive pathways (e.g., dealkylative pathways) are easily
accessible from intermediates in the synthetic routes to the
parent compounds. In contrast, metabolites resulting from
complexity-increasing pathways (e.g., hydroxylative pathways)
require the development of a new synthetic route. Depending
on the overall complexity of the drug, this may result in signifi-
concentrations >1 gLÀ1
.
Four strains showed good to excellent preference for forma-
tion of the S-enantiomer 20. Issatchenkia orientalis strains
needed more than 24 h to consume more than 1 gLÀ1 of start-
ing material 19. Even after 72 h at a concentration of 2 gLÀ1,
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cant capacity requirements. Therefore, major efforts have been
undertaken in recent years to develop methodologies for the
direct oxidation of drugs to their metabolites. Currently, all
major pharmaceutical companies use biocatalytic platforms to
generate human metabolites by the direct biooxidation of
drug candidates. In more recent years, newly developed chem-
ical CH functionalisation methodologies have started to com-
plement these biocatalytic processes. An excellent overview of
current technologies for accessing metabolites is available.[30]
As the most efficient option, we elected to investigate the
direct oxidative biotransformation of sagopilone (2). Encourag-
ingly, there were recent reports of the successful biooxidation
of epothilone B (1; 610 mg) to epothilone F (23; 126 mg, 21%
isolated yield) using Amycolata autotrophica ATCC 35203
(Scheme 7).[31] In this case, isolation of the desired product was
complicated by the formation of a second, regioisomeric hy-
CYP enzymes along with complementary P450 reductases. In
addition to CYP isoform diversity, our toolbox comprises fur-
ther diversity dimensions resulting, for example, from using dif-
ferent E. coli host strains and employing different expression
strategies (different N-terminal modifications, use of dual as
well as bicistronic CYP-P450 reductase constructs). For exam-
ple, we had 12 different engineered E. coli strains recombinant-
ly expressing CYP2C19 at hand, which were screened in a mi-
crotitre plate setup under standardised conditions. Under the
screening conditions at pH 7.4, sagopilone (2) was stable, and
two strains showed promising results with nearly 10% of the
main metabolite 3 being formed. For further optimisation we
focused on one strain, which is based on E. coli DH5a-LPSd as
host organism and contains a bicistronic plasmid with N-termi-
nally modified hCYP3A4 and the human P450 oxidoreductase
POR.
We applied a process for me-
tabolising sagopilone which is
known as ‘resting cell’ fermenta-
tion.[5g,36] In this process, the mi-
croorganism that functions as
the biocatalyst is incubated in
a shaking flask or in a fermenter
for a defined period of time and
is harvested by centrifugation.
Then, in a second fermentation
step, this cell mass is used as the
biocatalyst by incubation with
the substrate of choice. The cell
mass can be washed with aque-
ous solutions containing salts
Scheme 7. Biooxidation of epothilones to hydroxylated derivatives.
and nutrients or can be used
without washing. For the trans-
formation process, the cell mass
droxylation product. With a second, not freely accessible mi-
croorganism the isolated yield of epothilone F could be in-
creased to 33%.[31]
is suspended in the transformation medium. The biocatalysts
can be produced ready for immediate use or may be stored at
À808C in cryo buffer to prevent decomposition. For the opti-
misation of a resting cell fermentation, both steps need to be
investigated: the fermentation that produces the biocatalyst,
and also the transformation step.
Unfortunately, incubation of 2 with eight different Amycolata
strains gave disappointing results. Even after extended incuba-
tion times, the desired hydroxylation product was only formed
with less than 5% turnover.[32] With A. autotrophica ATCC
35203, sagopilone remained mostly unchanged, with trace
amounts of two hydrolysis products being formed.[33] Screen-
ing of a further set of wild-type microorganisms, which had al-
ready been used successfully for other drug biooxidations, also
failed to reveal any hits.
Initially, we incubated our E. coli strain in a 10-L steel fer-
menter to isolate cell material as resting cells which could be
used for further evaluation of the transformation step. Using
fresh cell material and 14C-labelled sagopilone to allow quan-
tification of the turnover, we were able to achieve 90% turn-
over into the desired metabolite within 22 h.
Sagopilone is metabolised in the human body by the cyto-
chrome P450 enzyme CYP2C19 which requires a complementa-
ry P450 reductase as well as NADPH for closing the catalytic
cycle. Several efforts toward designing E. coli strains recombi-
nantly expressing human CYP enzymes together with P450 re-
ductases and their use as whole-cell biooxidation catalysts
have been reported.[34] Based on these pioneering studies, a bi-
ooxidation toolbox was developed by the LINK consortium
which is still used by several pharmaceutical companies.[35]
Along the same lines, we have constructed a toolbox of
E. coli strains which recombinantly express the major human
Then, we investigated various fermentation conditions for
the production of the active biocatalyst (on a 10-L scale). The
time point for harvesting the cell mass was varied (i.e., from
144 h to 120 h) and different media were tested (see the Ex-
perimental Section below for details of the optimal medium).
As an example, in the case of cells from batch 3 (Table 2),
a medium with an altered nitrogen and carbon source was
tested: peptone from meat (2 gLÀ1) was omitted, and the glyc-
erol concentration was increased (from 4 to 5 gLÀ1), but the
amount of tryptone (12 gLÀ1) and yeast extract (24 gLÀ1) were
unchanged. Under the screening conditions with a sagopilone
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Table 2. Influence of biocatalyst quality on sagopilone turnover.[a]
Table 3. Influence of biocatalyst concentration on sagopilone turnover.[a]
Cell
material
batch no.[b]
Fermentation
time for cell
mass [h]
[Sagopilone]
Sagopilone
Metabolite
OD550
[Sagopilone]
Sagopilone
[%][b]
Metabolite
[mgLÀ1
]
(2) [%][c]
3 [%][c]
[mgLÀ1
]
3 [%][b]
3
3
3
3
5
5
5
5
10
10
10
10
25
50
75
100
25
50
75
100
25
50
75
20
30
60
77
29
33
24
97
91
80
65
100
86
88
90
1
1
1
1
2
2
2
2
3
3
3
3
144
144
144
144
120
120
120
120
120
120
120
120
25
50
75
100
25
50
75
100
25
50
75
<1
<1
15
81
56
62
46
80
74
77
72
79
75
67
56
70
40
<1
<1
9
<1
<1
<1
<1
<1
<1
12
35
<1
<1
<1
<1
100
100
26
[a] Conditions: biocatalyst production as for batch 2 in Table 2; biotrans-
[a] Conditions: 278C, 20 h, at OD550 10, 165 rpm. [b] For batches 1 and 2,
an optimal medium was used (see Experimental Section). For batch 3, an
altered medium was used: tryptone (12 gLÀ1), yeast extract (24 gLÀ1),
glycerol (5 gLÀ1). [c] Determined by HPLC.
formation conditions: 278C, 20 h, 165 rpm. [b] Determined by HPLC.
100 mm pH 7.4 potassium phosphate buffer (100 L) containing
1% glucose. Sagopilone (2; 9 g) dissolved in N,N-dimethylfor-
mamide (DMF; 367 mL) was added to the transformation
medium, which was incubated for 23 h under a partial oxygen
concentration of 50%. Then, the culture broth was harvested
and extracted twice with methyl isobutyl ketone. HPLC analysis
of the organic layers showed complete turnover of sagopilone
into the desired metabolite 3; notably, no other regioisomeric
hydroxylation products were detected. The combined organic
layer was concentrated and purified by silica gel chromatogra-
phy to afford the desired metabolite 3 (5.03 g) in 54% isolated
yield, in analytically pure form.
concentration of 25 mgLÀ1, these two changes (fermentation
time and medium) did not influence the turnover rate into the
human metabolite (79–81%); however, differences in the bio-
catalyst quality became apparent upon investigation of higher
sagopilone concentrations (Table 2).
Cells from batch 2 (harvested at an earlier time point) al-
lowed for an increase in the sagopilone concentration to
100 mgLÀ1 without the turnover being compromised (Table 2).
Older cells (batch 1) or cells from fermentations with the
changed medium (batch 3) showed significantly decreased
turnover rates at sagopilone concentrations beyond 50 mgLÀ1.
These results underscore the importance of optimising fermen-
tation time and the medium for biocatalyst production. We
now routinely use the sagopilone hydroxylation reaction as an
in-house quality-control assay for each CYP2C19 biocatalyst
batch before it is used with new substrates.
Conclusions
We have shown that parallel investigation of chemical and en-
zymatic processes can be integrated into the development
process of a complex chiral drug candidate on a large scale.
For two of the three required chiral building blocks for the syn-
thesis of sagopilone, superior processes were developed based
on biocatalytic key steps. In addition, we have described the
multi-gram-scale direct biotransformation of a highly complex
drug into its human metabolite by using a recombinant hCYP
biocatalyst. To our knowledge, this hCYP biocatalytic process is
the most complex application of such a biocatalyst, as well as
the largest scale, reported to date.
We also investigated variations in the transformation step;
for example, we determined the necessary concentration of
the biocatalyst for complete sagopilone turnover. The concen-
tration of cells, as gauged by the optical density (OD), was set
at OD 10 (determined at 550 nm) for the above studies. For
the large-scale fermentation, we wanted to decrease the
amount of biocatalyst required by decreasing the cell concen-
tration; however, as shown from the results in Table 3, an
OD550 of 10 was necessary for complete transformation of
100 mgLÀ1 of sagopilone. When the biocatalyst concentration
was decreased to OD550 5, up to 50 mgLÀ1 of sagopilone could
be transformed. This demonstrated that the process was al-
ready running at the limit of oxidation capacity of the cells,
and hence a decrease in cell concentration was not feasible.
To verify the robustness of our optimised biotransformation
conditions, we performed a 1-L preparative-scale transforma-
tion with sagopilone (90 mg) which, after extractive workup
and chromatography, gave the desired metabolite 3 in 66%
yield (61 mg).
Experimental Section
All microorganisms were handled under a clean bench or under
sterile conditions using the corresponding equipment. All solutions
were either sterilised at 1218C or sterile-filtered. Screening proce-
dures are described in the literature: enzymatic ester hydrolysis,[37]
enzymatic esterification,[38] and microbial transformations.[39]
Finally, cryo-conserved cells (4 L; 50 L combined fermenta-
tion volume, four individual batches) were resuspended in
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residue at 408C. The mixture was cooled to 58C. The product was
isolated using a plate filter and dried at 508C, yielding the desired
S-alcohol 20 (160 kg, 88%; >99% ee): [a]2D0 =À36.4 (c=1.1 in
(3S)-1-(Benzyloxy)-4-cyano-4-methylpentan-3-yl acetate (13)
and (3R)-5-(benzyloxy)-3-hydroxy-2,2-dimethylpentanenitrile
(12) by enzymatic kinetic resolution
1
CHCl3); H NMR (400 MHz, CDCl3): d=1.27 (t, J=7.15 Hz, 3H), 2.73–
A 2000-L reactor was charged with water (1115 kg), Na2HPO4
(10.16 kg), and KH2PO4 (3.92 kg). An 81-kg solution of the racemic
acetate 11 (44.5 kg, 161.6 mol) in MTBE was added, and the solu-
tion was adjusted to pH 6 with 1m NaOH. The temperature was
set at 408C, and the MTBE was removed by distillation under re-
duced pressure. Then, lipase AYS (Amano, 22.3 kg) was added, and
the mixture was stirred at 408C for 68 h. The pH was controlled
and kept at pH 6.0 by adding 1m NaOH. The reaction mixture was
extracted with EtOAc (2”1785 L). The organic phase was filtered
over diatomaceous earth and concentrated under reduced pres-
sure. The crude material was separated by chromatography [Kro-
masil NP 10 mm (11 kg), n-hexane/MTBE gradient] yielding the S-
acetate 13 (21.87 kg, 98%; >99.5% ee) and the R-alcohol 12
(14.21 kg, 75%; >97.5% ee).
2.86 (m, 5H), 3.40 (d, J=3.51 Hz, 1H), 4.20 (q, J=7.03 Hz, 2H), 5.27
(dt, J=8.34, 3.98 Hz, 1H), 7.41 (dd, J=8.28, 1.51 Hz, 1H), 7.80 (d,
J=8.28 Hz, 1H), 7.95 ppm (d, J=1.25 Hz, 1H); MS (ESI) m/z (%):
265 (38) [M]+, 178 (100), 176 (37), 150 (86), 109 (33).
(1S,3S,7S,10R,11S,12S,16R)-10-Allyl-7,11-dihydroxy-3-[2-(hy-
droxymethyl)-1,3-benzothiazol-5-yl]-8,8,12,16-tetramethyl-
4,17-dioxabicyclo[14.1.0]heptadecane-5,9-dione (sagopilone
metabolite, 3)
Expression of the P450 enzyme CYP2C19 was performed in E. coli
DH5a-LPSd pCW_d3_2C19 (#6), a strain engineered in our laborato-
ry in 2004. The strain was stored at À808C in a 50% glycerol solu-
tion.
S-Acetate 13: tR (Chiralpak AD 20 mm, 250”4.6 mm, 308C,
1.5 mLminÀ1, hexane/EtOH 98:2, UV 204 nm)=5.91 min; 1H NMR
(400 MHz, [D6]DMSO): d=1.26 (s, 3H), 1.31 (s, 3H), 1.69–1.79 (m,
1H), 1.99–2.08 (m, 4H), 3.34–3.41 (m, 1H), 3.44–3.50 (m, 1H), 4.40
(sym m, 2H), 4.98 (dd, J=10.61, 2.02 Hz, 1H), 7.24–7.37 ppm (m,
5H); MS (ESI) m/z (%): 293 (100) [M+NH4]+, 276 (71) [M+H]+.
Oxford trace elements solution: FeCl3·6H2O (27 gLÀ1), ZnCl2
(1.31 gLÀ1), CoCl2·6H2O (2.87 gLÀ1), CuCl2·2H2O (1.27 gLÀ1), B(OH)3
(0.5 gLÀ1), CaCl2·2H2O (1.32 gLÀ1), Na2MoO4·2H2O (2.35 gLÀ1), and
37% HCl (100 mL) in water (1 L).
A 500-mL Erlenmeyer flask was charged with an aqueous solution
(100 mL) containing tryptone (16 gLÀ1), NaCl (10 gLÀ1), and yeast
extract (10 gLÀ1). The solution was adjusted to pH 7.2–7.4 with
16% NaOH and 16% H3PO4. The flask was sterilised in an autoclave
at 1218C for 20 min. An aqueous ampicillin solution was added to
the flask so that the final concentration was 50 mgmLÀ1. To the
flask was added the glycerol stock solution (50 mL) containing the
E. coli strain #6. The flask was kept in an incubator at 378C for 17 h
at 165 rpm. A 10-L fermenter was charged with tryptone (12 gLÀ1),
yeast extract (24 gLÀ1), peptone from meat (2 gLÀ1) [enzymatic
(tryptic) digested], KH2PO4 (2.2 gLÀ1), K2HPO4 (9.4 gLÀ1), and 87%
glycerol (4.6 gLÀ1). The mixture was sterilised at 1218C for 30 min.
At 378C, the following solutions were added: ampicillin (0.5 g) in
water (20 mL), riboflavin (10 mg) in water (20 mL), thiamine hydro-
chloride (3.37 g) in water (10 mL), and the Oxford trace elements
solution (2.5 mL). After 2 h, the inoculation culture (100 mL) from
the 500-mL flask was added. The fermentation mixture was stirred
at 315 rpm and aerated with 3.3 LminÀ1 of air at pH 6.7. After 3 h,
the optical density (OD550) reached 1.058. The temperature was de-
creased to 278C, and isopropyl b-d-1-thiogalactopyranoside (IPTG;
2.38 g) in water (40 mL) and 5-aminolevulinic acid (838 mg) in
water (40 mL) were added. After 6 h, the pH decreased, and feed-
ing with a sterile aqueous solution of glucose (50%) was started to
maintain the pH at 6.7. After 123 h, the culture broth was centri-
fuged, yielding cell mass (527.7 g) which was resuspended in cryo
buffer (50% glycerol in water; 500 mL). The cell mass was stored at
À808C.
A 100-L fermenter was filled with water (~90 L), and K2HPO4
(1230 g), KH2PO4 (400 g), and Synperonic (2.5 mL) were added. The
mixture was sterilised at 1218C for 30 min. EDTA solution (0.5m in
water; 100 mL) and sterile-filtered glucose solution (50% in water;
2 L) were added. The solution was regulated between pH 7.2 and
7.4 with 16% NaOH. At 278C, the fermenter was inoculated with
the cell solution in cryo buffer (4 L). The partial oxygen concentra-
tion was set at 20%. Sterile water was added to adjust to a 100 L
volume. The mixture was aerated with 33.3 LminÀ1 of air and the
partial oxygen concentration was set at 50% and maintained by
automatic regulation of the stirrer velocity. Sagopilone (2; 9 g,
16.6 mmol) in DMF (367 mL) was added. After 23 h, the culture
R-Alcohol 12: tR (Chiralpak AD 20 mm, 250”4.6 mm, 308C,
1.5 mLminÀ1, hexane/EtOH 98:2, UV 204 nm)=15.54 min; 1H NMR
(400 MHz, [D6]DMSO): d=1.22 (s, 3H), 1.25 (s, 3H), 1.48–1.57 (m,
1H), 1.83–1.92 (m, 1H), 3.46 (ddd, J=10.67, 6.25, 2.02 Hz, 1H),
3.52–3.62 (m, 2H), 4.46 (s, 2H), 5.39 (d, J=6.32 Hz, 1H), 7.24–
7.37 ppm (m, 5H); MS (ESI) m/z (%): 251 (100) [M+NH4]+, 234 (46)
[M+H]+; Anal. calcd for C14H19NO2: C 72.07, H 8.21, N 6.00, found:
C 71.85, H 8.41, N 5.87.
Ethyl (3S)-3-hydroxy-3-(2-methyl-1,3-benzothiazol-5-yl)propa-
noate (20) by reduction of b-keto ester 19
The yeast strain Pichia wickerhamii with access number IFO 1278
was purchased from the culture collection of the Institute for Fer-
mentation, Osaka, Japan. A 2-L Erlenmeyer flask was charged with
an aqueous solution (500 mL) containing glucose (50 gLÀ1) and
corn steep liquor (20 gLÀ1). The solution was adjusted to pH 6.25
with 16% NaOH. The flask was sterilised in an autoclave at 1218C
for 20 min. The culture medium was inoculated with a cryo culture
(10 mL; 50% glycerol) of P. wickerhamii IFO 1278. The culture broth
was incubated on a shaker at 308C for 24 h at 180 rpm. The culture
broth (500 mL) from the 2-L flask was added to a sterile 5000-L fer-
menter containing glucose (50 gLÀ1), corn steep liquor (20 gLÀ1),
and an antifoam agent (0.1 gLÀ1). The mixture was incubated at
288C for 38 h. The culture broth was transferred to a 50000-L fer-
menter containing an aqueous solution (50000-L) of glucose
(50 gLÀ1) and corn steep liquor (20 gLÀ1) that had been sterilised
at 1218C for 30 min. The mixture was adjusted to pH 6 with 16%
NaOH. After 0.5 h, the b-keto ester 19 (180 kg, 683.6 mol) dissolved
in DMF was added as a solution (200 gLÀ1) over 15 h. The mixture
was incubated at 288C. After 22 h, the concentration of the S-alco-
hol 20 had reached 3.5 gLÀ1, and the fermentation process was
terminated.
The culture broth was extracted with methyl isobutyl ketone (1:1).
The layers were separated, and the organic layer was partly con-
centrated. The fermentation product was filtered over charcoal,
and the filtrate was concentrated under reduced pressure. A solu-
tion of diisopropyl ether and cyclohexane (1:1.5) was added to the
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Comasseto, L. H. Andrade, M. Capelari, Q. B. Cass, A. L. M. Porto, Enzyme
Microb. Technol. 2005, 36, 937–946.
[7] a) M. O’Neill, D. Beecher, D. Mangan, A. S. Rowan, A. Monte, S. Sroka, J.
Modregger, B. Hundle, T. S. Moody, Tetrahedron: Asymmetry 2012, 23,
583–586; b) A. Hietanen, T. Saloranta, R. Leino, L. T. Kanerva, Tetrahe-
dron: Asymmetry 2012, 23, 1629–1632.
[8] a) S. Ramaswamy, B. Morgan, A. C. Oehlschlager, Tetrahedron Lett. 1990,
31, 3405–3408; b) K. A. Stein, P. L. Toogood, J. Org. Chem. 1995, 60,
8110–8112; c) J. García, S. Fernandez, M. Ferrero, Y. S. Sanghvi, V. Gotor,
J. Org. Chem. 2002, 67, 4513–4519.
[9] a) E. J. Corey, P. T. Lansbury, Jr., J. Am. Chem. Soc. 1983, 105, 4093–4094;
b) A. Romero, C.-H. Wong, J. Org. Chem. 2000, 65, 8264–8268.
[10] C. R. Harris, S. J. Danishefsky, J. Org. Chem. 1999, 64, 8434–8456.
[11] a) U. Klar, B. Roehr, F. Kuczynski, W. Schwede, M. Berger, W. Skuballa,
Synthesis 2005, 301–305; b) U. Klar, B. Buchmann, W. Schwede, W. Sku-
balla, J. Hoffmann, R. B. Lichtner, Angew. Chem. Int. Ed. 2006, 45, 7942–
7948; Angew. Chem. 2006, 118, 8110–8116.
[12] a) J. Westermann, J. Platzek, O. Petrov (Schering AG), Int. PCT Pub. No.
WO2004/108697 A1, 2004; b) J. Platzek, O. Petrov, M. Willuhn, K.-D.
Graske (Schering AG), Int. PCT Pub. No. WO2005/101950 A1, 2005; c) U.
Klar, J. Platzek, L. Zorn (Schering AG), Int. PCT Pub. No. WO2005/
064006 A1, 2005; d) U. Klar, J. Platzek, Synlett 2012, 23, 1291–1299.
[13] G. Rustin, N. Reed, G. C. Jayson, J. A. Ledermann, M. Adams, T. Perren, C.
Poole, M. Lind, M. Persic, S. Essapen, M. Gore, H. Calvert, C. Stredder, A.
Wagner, M. Giurescu, S. Kaye, Ann. Oncol. 2011, 22, 2411–2416.
[14] J. Graff, D. C. Smith, L. Neerukonda, M. Alonso, G. R. Jones, T. M. Beer,
Jasco Rep. ASCO Annual Meeting, J. Clin. Oncol. 2008, 26, No. 15S (May
20 Suppl.), Abstr. 5141.
[15] R. C. DeConti, A. P. Algazi, S. Andrews, P. Urbas, O. Born, D. Stoeckigt, L.
Floren, J. Hwang, J. Weber, V. K. Sondak, A. I. Daud, Br. J. Cancer 2010,
103, 1548–1553.
[16] A. Silvani, P. Gaviani, A. Fiumani, V. Scaioli, E. Lamperti, M. Eoli, A. Bot-
turi, A. Salmaggi, J. Neuro-Oncol. 2009, 95, 61–64.
broth was transferred to an extraction vessel. The fermenter was
rinsed with water (50 L), which was also transferred to the extrac-
tion vessel. Methyl isobutyl ketone (50 L) was added to the reac-
tion culture mixture which was gently stirred for 23 h. The layers
were separated, and the culture was extracted again with methyl
isobutyl ketone (10 L). The combined organic layers were concen-
trated to 50 mL. Methanol (150 mL) was added, and the solution
was concentrated to dryness. The residue was subjected to silica
gel chromatography with a gradient of EtOAc and hexane, yielding
1
3 (5.03 g, 54%) as a pale-yellow foam: H NMR (600 MHz, CD3CN):
d=0.93 (s, 3H), 1.01 (d, J=6.78 Hz, 3H), 1.20 (s, 3H), 1.25 (s, 3H),
1.33–1.59 (m, 6H), 1.65–1.71 (m, 1H), 2.11–2.18 (m, 1H), 2.19–2.23
(m, 1H), 2.31–2.36 (m, 1H), 2.38–2.46 (m, 2H), 2.52–2.57 (m, 1H),
2.81–2.86 (m, 2H), 3.39 (td, J=6.78, 4.52 Hz, 1H), 3.43 (d, J=
6.78 Hz, 1H), 3.70 (td, J=6.31, 3.95 Hz, 1H), 4.11 (brs, 1H), 4.16
(ddd, J=9.22, 6.96, 4.52 Hz, 1H), 4.92 (brs, 2H), 4.96–4.99 (m, 1H),
5.00–5.04 (m, 1H), 5.76 (ddt, J=17.22, 9.98, 7.29 Hz, 1H), 6.03 (dd,
J=9.41, 2.64 Hz, 1H), 7.44–7.46 (m, 1H), 7.95–7.98 ppm (m, 2H);
13C NMR (151 MHz, CD3CN): d=18.1, 20.2, 22.0, 22.6, 23.1, 30.9,
33.1, 33.2, 36.3, 36.7, 39.9, 50.7, 53.7, 62.4, 62.6, 62.7, 72.6, 74.6,
75.1, 117.0, 120.9, 122.9, 123.9, 134.9, 137.0, 140.5, 154.4, 171.3,
176.4, 217.4 ppm; MS (ESI) m/z (%): 560 (100) [M+H]+, 367 (27).
Acknowledgements
The authors thank Petra Helfrich and Sabine Trenner for their ex-
cellent laboratory work. We thank Joerg Knaeblein and Thomas
Faupel for the E. coli strains. We also thank Ingo Hartung for
helpful discussions and suggestions.
[17] a) K. C. Nicolaou, M. R. V. Finlay, S. Ninkovic, F. Sarabia, Tetrahedron
1998, 54, 7127–7166; b) K. C. Nicolaou, S. Ninkovic, M. R. V. Finlay, F.
Sarabia, T. Li, Chem. Commun. 1997, 2343–2344.
[18] Synthesis
of
building
block A
from
S-acetate
13:
Keywords: antitumor agents · biocatalysis · enzymes · gene
expression · oxidoreductases
[1] a) K.-H. Lee, J. Nat. Prod. 2004, 67, 273–283; b) M. S. Butler, J. Nat. Prod.
2004, 67, 2141–2153; c) D. J. Newman, G. M. Cragg, K. M. Snader, J. Nat.
Prod. 2003, 66, 1022–1037; d) G. M. Cragg, P. G. Grothaus, D. J.
Newman, J. Nat. Prod. 2014, 77, 703–723; e) L. F. Tietze, H. P. Bell, S.
Chandrasekhar, Angew. Chem. Int. Ed. 2003, 42, 3996–4028; Angew.
Chem. 2003, 115, 4128–4160.
[2] a) D. G. I. Kingston, J. Nat. Prod. 2009, 72, 507–515; b) T. A. M. Gulder,
B. S. Moore, Angew. Chem. Int. Ed. 2010, 49, 9346–9367; Angew. Chem.
2010, 122, 9534–9556; c) A. K. Mukherjee, S. Basu, N. Sarkar, A. C.
Ghosh, Curr. Med. Chem. 2001, 8, 1467–1486.
[3] M. C. Wani, H. L. Taylor, M. E. Wall, P. Coggon, A. T. McPhail, J. Am. Chem.
Soc. 1971, 93, 2325–2327.
[19] a) P. Borowiecki, M. Fabisiak, Z. Ochal, Tetrahedron 2013, 69, 4597–
4602; b) E. Bellur, I. Freifeld, D. Boettcher, U. T. Bornscheuer, P. Langer,
Tetrahedron 2006, 62, 7132–7139.
[20] J. Westermann, O. Petrov, J. Platzek (Schering AG), Int. PCT Pub. No.
WO2003/014068 A1, 2003.
[4] K. Gerth, N. Bedorf, G. Hçfle, H. Irschik, H. Reichenbach, J. Antibiot.
1996, 49, 560–563.
[5] a) W. Charney, H. L. Herzog, Microbial Transformations of Steroids, Aca-
demic Press, New York, 1967; b) K. Kieslich, Microbial Transformations of
Non-Steroid Cyclic Compounds, Georg Thieme Publishers, Stuttgart,
1976; c) K. Drauz, H. Waldmann, Enzyme Catalysis in Organic Synthesis
VCH, Weinheim, 1995; d) B. L. Goodwin, Handbook of Biotransformations
of Aromatic Compounds, CRC Press, Boca Raton, 2005; e) N. M. Shaw,
K. T. Robins, A. Kiener, Adv. Synth. Catal. 2003, 345, 425–435; f) S. M.
Roberts, J. Chem. Soc. Perkin Trans. 1 2000, 611 –633; g) K. Liese, K. Seel-
bach, C. Wandrey, Industrial Biotransformations, Wiley-VCH, Weinheim,
2006; h) K. Faber, Biotransformations in Organic Chemistry, 6th ed.,
Springer, Berlin, 2011; i) K. Drauz, H. Grçger, O. May, Enzyme Catalysis in
Organic Synthesis, 3rd ed., Wiley-VCH, Weinheim, 2012; j) J. Whittall, P.
Sutton, Practical Methods for Biocatalysis and Biotransformations, Wiley,
Chichester, 2009.
[21] a) G. Cardillo, A. Tolomelli, C. Tomasini, J. Org. Chem. 1996, 61, 8651–
8654; b) G. M. Salamonczyk, K. Han, Z.-W. Guo, C. J. Sih, J. Org. Chem.
1996, 61, 6893–6900; c) N. Itaya, H. Maeda, Y. Sato (Sumitomo Chemical
Co., Ltd.), US Pat. No. US2009/0118534 A1, 2009.
[22] a) G. A. Russell, G. W. Holland, K.-Y. Chang, R. G. Keske, J. Mattox, C. S. C.
Chung, K. Stanley, K. Schmitt, R. Blankespoor, Y. Kosugi, J. Am. Chem.
Soc. 1974, 96, 7237–7248; b) H. Hocke, Y. Uozumi, Tetrahedron 2003,
59, 619–630.
[23] a) K. A. Babiak, J. S. Ng, J. H. Dygos, C. L. Weyker, Y.-F. Wang, C.-H. Wong,
J. Org. Chem. 1990, 55, 3377–3381; b) E. Santaniello, S. Casati, P. Ciuffre-
da, G. Meroni, A. Pedretti, G. Vistoli, Tetrahedron: Asymmetry 2009, 20,
1833–1836.
[24] M. G. Perrone, E. Santandrea, A. Scilimati, C. Syldatk, V. Tortorella, Tetra-
hedron: Asymmetry 2005, 16, 1473–1477.
[25] H. Danda, T. Nagatomi, A. Maehara, T. Umemura, Tetrahedron 1991, 47,
8701–8716.
[6] a) M. D. Mihovilovic, B. Mueller, M. M. Kayser, J. D. Stewart, P. Stanetty,
Synlett 2002, 0703–0706; b) A. Luna, M.-C. GuitiØrrez, R. Furstoss, V. Alp-
hand, Tetrahedron: Asymmetry 2005, 16, 2521–2524; c) L. C. Ricci, J. V.
ChemMedChem 2015, 10, 1240 – 1248
www.chemmedchem.org
1247
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
[26] a) M. Sharfuddin, A. Narumi, Y. Iwai, K. Miyazawa, S. Yamada, T. Kakuchi,
H. Kaga, Tetrahedron: Asymmetry 2003, 14, 1581–1585; b) B. Martín-
Matute, M. Edin, K. Bogµr, F. B. Kaynak, J.-E. Bäckvall, J. Am. Chem. Soc.
2005, 127, 8817–8825; c) H. Pellissier, Tetrahedron 2003, 59, 8291–
8327.
[35] a) K. Schroer, M. Kittelmann, S. Lütz, Biotechnol. Bioeng. 2010, 106, 699–
706; b) S. P. Hanlon, T. Friedberg, C. R. Wolf, O. Ghisalba, M. Kittelmann
in Modern Biooxidation: Enzymes, Reactions and Applications (Eds.: R. D.
Schmid, V. B. Urlacher), Wiley-VCH, Weinheim, 2007, pp. 233–252.
[36] a) J. Calzada, M. T. Zamarro, A. Alcon, V. E. Santos, E. Diaz, J. L. Garcia, F.
Garcia-Ochoa, Appl. Environ. Microbiol. 2009, 75, 875–877; b) H.
Hummel-Marquardt, T. Schmitz, M. Kennecke, A. Weber (Schering AG),
US Pat. No. US5700666 A, 1997; c) U. C. Banerjee, Enzyme Microb. Tech-
nol. 1993, 15, 1037–1041; d) M. Cantarella, L. Cantarella, A. Gallifuoco,
A. Spera, Enzyme Microb. Technol. 2006, 38, 126–134.
[37] J. García, S. Fernµndez, M. Ferrero, Y. S. Sanghva, V. Gotor, J. Org. Chem.
2002, 67, 4513–4519.
[38] Y.-F. Wang, J. L. Lalonde, M. Momongan, D. E. Bergbreiter, C.-H. Wong, J.
Am. Chem. Soc. 1988, 110, 7200–7205.
[39] a) S. Stahl, R. Greasham, M. Chartrain, J. Biosci. Bioeng. 2000, 89, 367–
371; b) S.-H. Hu, G. Genain, R. Azerad, Steroids 1995, 60, 337–352.
[27] In this fermentation medium,
a few strains produced increased
amounts of tyrosol, a secondary metabolite isolated from yeasts.
[28] In the course of this work, the fermentation process was improved fur-
ther, and more b-keto ester 19 could be reduced in a 50 m3 fermenter.
[29] US Department of Health and Human Services Food and Drug Adminis-
tration Center for Drug Evaluation and Research (CDER), www.fda.gov/
OHRMS/DOCKETS/98fr/FDA-2008-D-0065-GDL.pdf (accessed March
2015).
[30] K. P. Cusack, H. F. Koolman, U. E. W. Lange, H. M. Peltier, I. Piel, A. Vasu-
devan, Bioorg. Med. Chem. Lett. 2013, 23, 5471–5483.
[31] W. Li, J. A. Matson, X. Huang, K. S. Lam, G. A. McLure (Bristol-Myers
Squibb Co.), US Pat. No. US6780620 B1, 2004.
[32] Retrospective quantification based on authentic product standard.
[33] Increase in molecular weight by 18 Da; no structural assignment was
undertaken.
[34] a) J. A. R. Blake, M. Pritchard, S. Ding, G. C. M. Smith, B. Burchell, C. R.
Wolf, T. Friedberg, FEBS Lett. 1996, 397, 210–214; b) A. Parikh, E. M. J.
Gillam, F. P. Guengerich, Nat. Biotechnol. 1997, 15, 784–788.
Received: March 30, 2015
Published online on May 28, 2015
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