morpholine with stirring. 0.3 ml isobutylchloro-c◦arbonate was
added into the mixture on an ice–salt bath (-15 C). After 1–
2 min activation, Pro-OMe·HCl (0.47 g, 286 mM) was dissolved
into the mixture and reacted for 1.5 h. After removal of the
solvent in vacuo, the residue was dissolved in ethyl acetate,
washed with 1 M NaHCO3, 10% (w/v) citric acid and a
saturated NaCl solution, then dried with anhydrous MgSO4 and
evaporated. Boc-Tyr-Pro-OMe (95.6% yield, 98.3% purity) was
obtained as a yellow oil.
Boc-Tyr-Pro-OMe (0.7 g) was then dissolved in 10 ml
methanol, followed by 10 ml 1 M NaOH solution. After
stirring at room temperature for 10 h, a 20 ml saturated
NH4Cl solution was added. The pH of the solution was then
adjusted to 3 with 10% (w/v) citric acid. The mixture was
extracted with ethyl acetate and washed with a saturated NaCl
solution, dried with anhydrous MgSO4 and evaporated. Boc-
Tyr-Pro-OH (87.4% yield, 95.8% purity) was obtained as a white
powder. The synthesized Boc-Tyr-Pro-OH was confirmed by
mass spectrometry (m/z (ESI) 401.1 [M + Na]+).
in the following gradient mode: 0–5 min, 20% B; 5–30 min, 20–
100% B; 30–40 min, 100% B. The flow rate of the eluent was
1.0 ml min-1 and the oven temperature was 30 ◦C.
Purification of endomorphin-1
High speed countercurrent chromatography (TAUTO TBE-
300B, diameter of tube = 2.6 mm, total volume = 300 ml and
a 20 ml sample loop) was applied to separate the final product
(Tyr-Pro-Trp-Phe-NH2). The solvent system for the separation
was ethyl acetate–methanol–water (6 : 1 : 6, v/v). The effluent
was monitored with a UV-vis detector at 280 nm and the peak
fractions were collected separately.
Acknowledgements
Financial support for this research from the National Program
on Key Basic Research Project (2009CB724700) and the Key
Program of the National Natural Science Foundation of China
(20936002) is gratefully acknowledged.
Enzymatic synthesis of Boc-Tyr-Pro-Trp-Phe-NH2
References
Boc-Tyr-Pro-OH (0.042 g, 20 mM) and Trp-Phe-NH2 (0.056 g,
40 mM) were dissolved in 6 ml water-saturated ethyl acetate.
Then, 2 ml of crude protease PT121 (15 000 U ml-1) was added,
and incubated with shaking at 37 ◦C and 200 rpm for 10 h.
At the end of the reaction, the organic layer was separated
and washed with 1 M NaHCO3, 10% (w/v) citric acid and a
saturated NaCl solution, and then dried with anhydrous MgSO4.
Evaporation of the solvent under reduced pressure gave the
crude yellow product. The synthesized Boc-Tyr-Pro-Trp-Phe-
NH2 was confirmed by mass spectrometry (m/z (ESI) 733.9
[M + Na]+).
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Quantitative analysis of the peptide products
The peptide products were analyzed by HPLC (Dionex P680)
using a Kromasil 100-5C18 column (4.6 mm ¥ 250 mm,
Kromasil, Sweden) and a UVD170U detector at 280 nm. The
mobile phase was solvent A (water) and solvent B (methanol)
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The Royal Society of Chemistry 2011
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