G Model
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Fig. 1. Structures of 4-aminocyclopent-2-en-1-ol analogues.
with the same restriction enzymes. The correct linear pPIC9K-
CALB construct, obtained after digested with Sal I restriction
enzyme, was inserted into the host genome through electro-
transformation means of 1500 V and 8 ms. Integration of CALB into
the genome of P. Pastoris in AOX I or His 4 site was verified by PCR
analysis using vector- and genome-specific primers, and positive
clones were further selected by G418 resistance. With the regular
addition of methanol to activate the alcohol oxidase in P. Pastoris
under the condition of 28 8C and 220 rpm for 168 h in BMMY
medium.
Fig. 2. (a) Agarose gel electrophoresis of PCR analysis using vector- and genome-
specific primers. M, DNA marker 8000; lane 1, PCR product by using vector-specific
primers 30AOX I and
a-factor; lane 2, PCR product by using genome-specific
primers. (b) Coomassie-stained gel after SDS-PAGE analysis of supernatant proteins
expressed from Pichia Pastoris after purification by 50% ammonium sulfate
precipitation. M, protein marker; lanes 1–6, supernatant samples collected at
day 2 to day 7, respectively.
Reactions were initiated with 1 g/L of compound 3 and 0.4 mg/mL
CALB suspended in 1 mL phosphate buffer pH 7.0, then shaken at
different temperatures ranging from 20 8C to 70 8C. The results
showed that the rate of reaction was significantly improved at
50–60 8C, and then decreased as temperature continued to rise.
The optimal reaction pH was subsequently studied at 60 8C.
The reaction was carried out from pH 3.0 to pH 9.0. It was found
that the reaction was faster under weak acidic conditions, and the
optimal pH value was 5.0. Optimal enzyme concentrations and
reaction times were further investigated to reduce the spontane-
ous hydrolysis of substrates. The results showed that the optimal
enzyme concentration was about 0.4 mg/mL. However, the
optimal reaction time was different for different substrates, and
the results were listed in Table 1.
Several substrates (Fig. 1) were prepared for enzymatic
resolution. Compound
1 was prepared by four steps from
hydroxylamine hydrochloride and cyclopentadiene with the
catalysis of Mo(CO)6 according to the literature procedures [16–
18]. Compounds 2, 3 and 4 were prepared based on literature
procedure by replacing Ac2O with propionyl chloride, butyryl
chloride, and benzoyl chloride, respectively, at the last step and
were subjected to 1H NMR analysis to confirm their structures [19].
Compound 5 (Fig. 1) was prepared as a white solid by a slightly
modified method [16–20].
The hydrolysis reaction with CALB was first performed in a
closed centrifuge tube, which was initiated with 1 g/L of compound
and 0.4 mg/mL CALB suspended in 1 mL phosphate buffer pH 7.0.
After incubation at 60 8C for 4 h, the reaction solution was further
extracted with 1 mL isopropyl ether. Finally, the isopropyl ether
The reaction extract of compound 3 was assayed by HPLC. As
shown in Fig. 3, CALB performs an excellent enantioselectivity with
racemic 3, and gives the target product (1S,4R)-6 in a yield of 45.7%
and an enantiomeric excess of 99.5%.
extract (10
mL) was applied to HPLC assay [21].
The research was finally conducted under optimal conditions
as mentioned above. The results revealed that CALB showed
excellent enantioseletivity with compounds 1, 2 and 3. Conver-
sion of compounds 1, 2 and 3 gave the target product 6 in 99.3%,
98.8% and 99.5% ee, respectively. However, the spontaneous
hydrolysis of these substrates made it difficult to obtain product 6
in high yield. Those results are summarized in Table 1. Subse-
quently, as shown in Scheme 1, when the reaction time was
lengthened to 8 h, we obtained (1S,4R)-6 in >94.0% ee with the
yield up to 42.0%. At the same time, the ester 7 was produced with
approximately 99.0% ee and the yield up to 43.0% which may be
used for other applications.
3. Results and discussion
The PCR analysis result using vector- and genome-specific
primers was as follows (Fig. 2a). As expected, with the regular
addition of methanol to activate the alcohol oxidase in P. Pastoris,
the target protein was successfully expressed and accumulated
and finally precipitated at 50% ammonium sulfate saturation
(Fig. 2b).
Initially, compound 3 was chosen as model substrate to
optimize reaction conditions. As temperature can significantly
influence the solubility of the substrate and activity of the enzyme,
we first investigated the effect of temperature on the conversion.
Table 1
Biocatalysis of different substrates by CALB.a
Compound
Substrate
Time (h)
ee-6 (%)b
Yield (%)c
1
2
3
4
5
cis-N-(benzylcarbamoyl)-4-aminocyclopent-2-en-1-O-acetate
cis-N-(benzylcarbamoyl)-4-aminocyclopent-2-en-1-O-propionate
cis-N-(benzylcarbamoyl)-4-aminocyclopent-2-en-1-O-butyrate
cis-N-(benzylcarbamoyl)-4-aminocyclopent-2-en-1-O-benzoate
cis-N-acetyl-4-aminocyclopent-2-enol 1-O-acetate
4
4
99.3
98.8
99.5
0
34.5
42.8
45.7
0
2.5
24
12
20.1
11.3
a
The reactions were conducted at 60 8C and pH 5.0 with 0.4 mg/mL CALB and substrates at 1 g/L.
The analytical HPLC methods are found in references 21.
b
c
The yields are determined according to the peak area of HPLC.
Please cite this article in press as: H.-J. Wen, et al., Enantioselective synthesis of (1S,4R)-N-(benzylcarbamoyl)-4-aminocyclopent-2-en-
Wen, Hui-Jiao
