New synthesis of harzialactone A via kinetic resolution using recombinant Fusarium proliferatum lactonase

Article abstract of DOI:10.1016/j.tetasy.2010.01.021

A new synthesis of harzialactone A together with its stereoisomers was achieved starting from phenyl acetone. The key step of this new route is the kinetic bioresolution of racemic cis- and trans-isomers by recombinant Escherichia coli JM109 cells expressing Fusarium proliferatum lactonase gene (reFPL). Harzialactone A was isolated in excellent ee (>99%), while moderate to good enantioselectivities (80% to >99% ee) were obtained for its isomers.

Full text of DOI:10.1016/j.tetasy.2010.01.021

Tetrahedron: Asymmetry 21 (2010) 237–240
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
New synthesis of harzialactone A via kinetic resolution using recombinant
Fusarium proliferatum lactonase
Bing Chen a, Hai-Feng Yin b, Zhen-Sheng Wang b, Jian-He Xu a,
*
a Laboratory of Biocatalysis and Bioprocessing, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong
Road, Shanghai 200237, China
b PepTech (Shanghai) Pharmaceutical Co. Ltd, 388 Yindu Road, Shanghai 200231, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 December 2009
Accepted 18 January 2010
A new synthesis of harzialactone A together with its stereoisomers was achieved starting from phenyl
acetone. The key step of this new route is the kinetic bioresolution of racemic cis- and trans-isomers
by recombinant Escherichia coli JM109 cells expressing Fusarium proliferatum lactonase gene (reFPL). Har-
zialactone A was isolated in excellent ee (>99%), while moderate to good enantioselectivities (80% to >99%
ee) were obtained for its isomers.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
O
O
O
O
O
O
O
O
Harzialactone A (Scheme 1) is an antitumor marine metabolite
isolated from Trichoderma harianium OUPS-N115 strain and has
been reported to exhibit cytotoxic activity against the P388 lym-
phocytic leukaemia test system in cell cultures.1 Much effort has
been directed towards the asymmetric synthesis of this natural
product. Although Ito et al. established its absolute configuration
by the Mosher ester method, Mereyala et al. revised its configura-
tion and developed synthetic routes from -glucose (seven steps,
15% overall yield).2 They also prepared (ꢀD)-harzialactone A from
OH
OH
OH
OH
cis-(-)
cis-(+)
D
-xylose (eight steps, 24% yield).3 In 2007, Sudalai et al. reported
another route employing proline-catalyzed sequential
a-amino-
oxylation–HWE olefination of 3-phenylpropanal (four steps,
26.1% overall yield).4 Jian et al. also synthesized (ꢀ)-harzialactone
A in 3 steps from malic acid (yield of 40%).5
trans-(-)
trans-(+)
Previously, we have developed a method to prepare 4-substi-
tuted 2-hydroxy-4-butyrolactone using robust recombinant Fusar-
ium proliferatum lactonase and assign the relative and absolute
structure.6 Herein, we applied this method to synthesize the natu-
ral product harzialactone A and its three stereoisomers in a facile
and ‘green’ way for the first time using recombinant F. proliferatum
lactonase (reFPL) with defined relative and absolute structure.
Scheme 1. Structures of four isomers of harzialactone A.
perature overnight. Routine work-up of this reaction mixture affor-
ded compound 2. After saponification and acidification, compound
3 precipitated out as white needle crystals from water and was
submitted directly to subsequent NaBH4 reduction. After acidificat-
ion and lactonization, compound 5 was obtained (dr cis:trans/3:2)
and separated by column chromatography on silica gel. Previous
reports have demonstrated the asymmetric syn- and anti-reduction
of 1,3-diketo may be well manipulated by changing the reducing
agents and reaction temperature.7
With cis-5 and trans-5 in hand, we submitted them to enzy-
matic resolution (Scheme 2). Both racemic cis-5 and trans-5 were
readily recognized by recombinant Escherichia coli JM109 cells har-
boring plasmid pET28a carrying F. proliferatum lactonase (reFPL)
gene.8 No side reactions were observed. E. coli JM109 harboring
2. Results and discussion
Condensation (Scheme 2) of phenyl acetone and ethyl oxalate
by freshly prepared NaOEt (1.1 equiv) in ethyl ether solution was
carried out at ꢀ20 °C for 2 h, and then left to warm to room tem-
* Corresponding author. Fax: +86 21 64252250.
E-mail address: jianhexu@ecust.edu.cn (J.-H. Xu).
0957-4166/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2010.01.021

238
B. Chen et al. / Tetrahedron: Asymmetry 21 (2010) 237–240
O
O
a
c
b
O
OH
O
O
OH
O
O
1
2
3
O
*
O
O
d
*
*
OH
*
OH OH
OH
harzialactone A
4
5
Scheme 2. The synthetic route to harzialactone A [( )-5]. Reagents and conditions: (a) 1.1 equiv NaOEt/1.1 equiv ethyl oxalate/Et2O/ꢀ20 °C/overnight; (b) (i) LiOH/THF–H2O
(1:1), (ii) Concentrated HCl; (c) 4 equiv NaBH4/CH3OH; (d) concentrated HCl/60 °C.
OH
(S)
O
OH
O
(2R,4S)
(2S,4R)
*
O
(R)
O
O
OH
OH
O
*
OH
(R)
(S)
cis-5
O
O
(S)
OH
O
(2S,4S)
(2R,4R)
(S)
*
O
OH
O
*
OH
OH
(R)
O
trans-5
OH
O
(R)
O
OH
OH
Scheme 3. Separation of cis- and trans-4-benzyl 2-hydroxy-4-butyrolactones by column chromatography combined with bioresolution catalyzed by Fusarium lactonase.
Bioresolution conditions: 30 °C; 10 ml KPB buffer (pH 6.4, 50 mM); 2% (v/v) MeCN; 10 mM substrate; 4 IU wet cells.
plasmid pET28a alone showed no hydrolytic activity, indicating
that the asymmetric hydrolysis was totally due to the activity of
reFPL.
(ꢀ)-(2S,4S)-Harzialactone A isolated in this process was con-
taminated with unreacted (+)-isomer. The enzymatic reaction
was extended to 30 min to hydrolyze completely the undesired
In a typical resolution (Scheme 3), racemic trans-harzialactone
A (0.1 mmol) in 10 ml (10 mM) of buffer (KPB, 50 mM, pH 6.4)
was treated with 30 ll of the biocatalyst (4 IU, substrate-to-cata-
Table 1
Results of resolution of harzialactone A catalyzed by recombinant Fusarium prolifer-
atum (reFPL)
lyst ratio of 20/1),9 and MeCN was used as the cosolvent (2%, v/
v). On completion of the resolution (ꢁ10 min), the mixture was
centrifuged at 10,000 g to remove the biocatalyst. The undesired
(ꢀ)-(2S,4S) harzialactone A was removed by extensive extraction
of the supernatant with ethyl acetate, and the aqueous layer was
acidified to pH 2.0 with dilute HCl and heated for 1 h at 60 °C.
The cyclized lactone was then extracted with ethyl acetate, and
the combined extract was concentrated under vacuum to afford
(+)-(2R,4R)-harzialactone A as colorless crystals.
½ ꢂ
a 2D0
Entry
Absolute configuration
ee (%)
Isolated yield (%)
1
2
3
4
(2R,4S)-5
(2S,4R)-5
(2S,4S)-5
(2R,4R)-5
80.2a
97.5a
>99b
43.8
44.8
47.2
45.9
—
ꢀ9.0
ꢀ32.5
>99b
+33.5
a
Analyzed by a chiral HPLC AD-H column.
Analyzed by a chiral HPLC OJ-H column.
b

B. Chen et al. / Tetrahedron: Asymmetry 21 (2010) 237–240
239
(2R,4R)-5. Then (ꢀ)-(2S,4S)-harzialactone A was isolated from the
4. Experimental
reaction mixture as colorless crystals (>99% ee) by simple
extraction.
4.1. Synthesis of intermediate 2
The asymmetric hydrolysis of racemic cis-harzialactone using
reFPL was also accomplished using similar procedure. Both
(2S,4R)-5 and (2R,4S)-5 were obtained in moderate to high ee val-
ues and the results are summarized in Table 1.
To a slurry of newly prepared NaOEt (0.1 mol) and Et2O solution
(50 ml), a mixture of diethyl oxalic acid (0.1 mol) and phenyl ace-
tone (0.1 mol) in dry Et2O (25 ml) solution was added carefully in
small portions at ꢀ20 °C within 2 h and then kept overnight at
room temperature. The mixture was then poured into diluted
HCl and extracted with ethyl acetate. The combined organic layers
were dried over anhydrous sodium sulfate, filtered, and concen-
trated in vacuo. Purification of the product 2 was carried out by
column chromatography on silica gel (petroleum ether/ethyl
acetate = 4:1).
We have previously reported the hydrolytic properties of reF-
PL towards various 4-aromatic 2-hydroxy-4-butyrolactone.6
Herein, when the phenyl group was replaced with a benzyl
group an increase in reFPL activity was observed. When (+)-
(2R,4R)-harzialactone A was the desired product, the bioresolu-
tion process was completed within less than 10 min in a sub-
strate-to-catalyst ratio of 20/1, which was far more efficient
(about 15-fold) than that of 4-phenyl 2-hydroxy-4-butyrolactone
reported previously. This feature of reFPL with the combination
of convenient access to racemic substrates could compensate
for the drawbacks in the theoretical yield and contribute to
the robustness of this process.
The relative configurations of cis- and trans-lactones were con-
firmed by 1H NMR. The assignment of the absolute structure was a
challenge.1–5 Ito et al. first assigned the configuration of native har-
zialactone A to be (2S,4S) (+33.3) using a Mosher ester method in
1998, after they isolated this compound. However, this conclusion
was contested by Mereyala et al. in 1999, who described the con-
4.2. Synthesis of intermediate 3
To a 250-ml round-bottom flask was added crude compound 2
(40 mmol) in 50 ml THF, followed by LiOH (60 mmol) in 50 ml
water in one portion. Saponification was terminated when com-
pound 2 was consumed completely by TLC analysis (petroleum
ether/ethyl acetate = 1:1). The mixture was concentrated in vacuo
to remove THF and the residue was extracted with ethyl acetate
(50 ml) three times. The aqueous layer was acidified with 6 N
HCl and large amount of compound 3 precipitated. This precipitate
was filtered and washed with water (100 ml) three times and dried
in vacuo. It can be used without further purification.
cise synthesis of harzialactone A from D-glucose. They assigned the
native harzialactone A to be (2R,4R), and this assignment is further
supported by later results. Our specific rotations (Table 1) were
consistent with Mereyala et al.’s reports.
4.3. Synthesis of harzialactone
We had studied the enzymatic hydrolysis of 2-hydroxy-4-
butyrolactones and found that Fusarium lactonase specifically rec-
ognizes (2R)-hydroxyl group and leaves (2S)-lactones intact.10 As
for 4-substituted 2-hydroxy-4-butyrolactone, we also have devel-
oped a CD method6 to assign the absolute structure and this meth-
od was confirmed by X-ray diffraction. Herein, we applied this
method to assign the absolute structure of harzialactone A. The
To a stirred 250-ml round-bottom flask, crude compound 3
(40 mmol) in 50 ml MeOH was added, followed by NaBH4
(190 mmol) in small portions at room temperature. The mixture
was stirred for another 3 h. Next, 50 ml water was added to
decompose residual NaBH4 and the mixture of 4 was concen-
trated in vacuo to remove the solvent. Then the mixture was
acidified to pH 1.0–2.0 and heated at 60 °C to afford racemic
harzialactone 5. Compound 5 was then extracted with ethyl ace-
tate and dried over anhydrous sodium sulfate. The separation of
the diastereomers was done by column chromatography on silica
gel (petroleum ether/ethyl acetate = 6:1–3:1). NMR data of cis-
harzialactone A: 1H NMR (300 MHz, CDCl3) d 7.21–7.32 (m,
5H), 4.49–4.60 (m, 2H), 3.46 (br, 1H), 2.91–3.17 (m, 2H), 2.56–
2.65 (m, 1H), 1.91–1.99 (m, 1H); 13C NMR (75 MHz, CDCl3): d
177.6, 135.5, 129.4, 128.7, 127.2, 77.4, 68.6, 41.3, 36.6 ppm.
NMR data of trans-harzialactone A: 1H NMR (300 MHz, CDCl3):
CD spectra of (2S,4R)-5 and (2S,4S)-5 (Figure 1) both showed posi-
*
tive C.E. curves in the n?
p
transition region at ca. 225 nm indicat-
ing an (S)-configuration of the hydroxyl group. These results are
also consistent with Mereyala et al.’s finding.
100
−− trans-(2
S,4S)
−− cis-(2
S,4R)
80
60
40
20
0
d
7.19–7.33 (m, 5H), 4.87–4.95 (m, 1H), 4.07–4.13 (dd,
J = 8.1 Hz, 8.1 Hz, 1H), 2.96–2.98 (m, 2H), 2.39–2.23 (m, 2H)
ppm. 13C NMR (75 MHz, CDCl3): d 177.7, 135.3, 129.6, 128.8,
127.3, 78.4, 67.1, 41.1, 34.5 ppm.
CD/mdeg
4.4. Expression of recombinant F. proliferatum (reFPL)
lactonase
200
250
300
FPL gene sequence was ligated to pET28a. The ligation mixture
was introduced by the transformation into E. coli JM109 and then
screening of clones containing the restriction fragment was per-
formed. The resultant plasmid was designated as pET28a-FPL.
The recombinant E. coli JM109 (DE3) containing pET28a-FPL was
cultured aerobically at 37 °C in 50 ml LB medium until OD600
reached 0.7, at which point the cultures were immediately shifted
to 23 °C. IPTG was added at a final concentration of 0.5 mM to in-
duce the lac promoter. After further cultivation, the cells were har-
vested by centrifugation and washed thoroughly with
physiological saline, and were then used for further studies.
-20
λ/nm
Figure 1. CD spectra of harzialactone A (10ꢀ4 mM in MeOH).
3. Conclusion
In conclusion, we have developed a chemo-enzymatic route to
harzialactone A for the first time with a defined configuration,
which is considered to be more eco-friendly.

240
B. Chen et al. / Tetrahedron: Asymmetry 21 (2010) 237–240
4.5. Enzyme assay
Acknowledgements
The standard assay mixture comprised of 2.5% (w/v) racemic
pantolactone and appropriate amount of the cells with a final vol-
ume of 10.0 ml. The reaction was performed at 30 °C, and the pH
was automatically controlled at 6.7–6.9 with 0.1 M NaOH. The
hydrolysis rate of the lactone was calculated based on the rate of
NaOH titration. One unit of lactonase (1 IU) was defined as the
This research was financially supported by National Natural Sci-
ence Foundation of China (Grant Nos. 20773038 and 20902023),
Ministry of Science and Technology (Grant Nos. 2009CB724706
and 2009ZX09501-016), and China National Special Fund for State
Key Laboratory of Bioreactor Engineering (Grant No. 2060204).
amount of the enzyme that converted 1.0 lmol of pantolactone
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4.6. Resolution of substrates by recombinant E. coli cells
Appropriate amounts of wet cells were re-suspended in 10 ml
potassium phosphate buffer (KPB, 50 mM, pH 6.4) and the enzyme
activity of this aqueous solution was measured as 10 IU/100 ll.
General procedures were as following: To a 10 ml potassium phos-
phate buffer (KPB, 50 mM, pH 6.4) was added an appropriate vol-
ume of this aqueous solution and 0.1 mmol of cis-5 (or trans-5)
in 200 ll methanol. This mixture in 100 ml flask was kept at
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the desired product reached the appropriate enantiomeric purity.
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extracted with ethyl acetate.
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biocatalyst.
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