1406
B. Koksch et al. / Tetrahedron: Asymmetry 15 (2004) 1401–1407
(RS)-H-(aCF2Cl)Phe-NH2 (0.212 g) was (partially) dis-
solved in H2O (2.79 mL). The amidase preparation
(29 mg being 0.13 g/g substrate) from O. anthropi
(NCIMB 40321) was then added and the mixture shaken
(160 rpm) at 37 ꢁC with the pH ꢂ 8.5. NH3 determina-
tion indicated a conversion of less than 1% after 21 h
and approximately 4% after 68 h.
4.7.2. Synthesis of (S,S)-Z-Ala-(aTfm)Ala-NH2. N-Me-
thyl-morpholine (1.0 mmol) and isobutyl chloroformate
(1.2 mmol) were added to a stirred solution of 0.5 mmol
(112 mg) Z-Ala-OH in 20 mL dry ethyl acetate at
ꢀ15 ꢁC. After 30 min a solution of 0.5 mmol (50 mg) of
H-(aTfm)Ala-NH2, which was recovered from the pre-
parative enzymatic resolution experiment (see Section
4.3), in 20 mL dry ethyl acetate was added. The reaction
mixture was stirred at ꢀ15 ꢁC for 1 h and at room
temperature over night. The reaction mixture was
washed successively with H2O, dilute citric acid, H2O,
satd NaHCO3 and H2O. The organic layer was dried
over MgSO4, evaporated in vacuo and purified by flash
chromatography (ethyl acetate:n-hexane 3:1 (v/v);
4.6. Enzymatic resolution of (RS)-H-(aCF2H)Phe-NH2
(RS)-H-(aCF2H)Phe-NH2 (0.124 g, 0.58 mmol) was
(partially) dissolved in H2O (1.24 mL). The amidase
preparation (15.8 mg, being 0.13 g/g substrate) from
O. anthropi (NCIMB 40321) was then added and the
mixture shaken (160 rpm) at 37 ꢁC with the pH ꢂ 8.5.
NH3 determination indicated that the conversion was
approximately 51% after 21 h. The enzyme was removed
by filtration and the reaction mixture analyzed by
HPLC. The conversion appeared to be 58% with the ee
values of the amide and acid being 98.7% and 67.9%,
respectively, corresponding to an E value of 25.
1
Rf ¼ 0:27). Yield: 78%. H NMR (360 MHz, CDCl3) d:
1.31 (d, 3H, J ¼ 7 Hz), 1.46 (s, 3H), 4.21 (m, 1H), 4.38
(m, 1H), 7.21–7.29 (m, 5H), 5.93 (s, 1H), 7.42 (s,
1H) ppm; 13C NMR (360 MHz, CDCl3) d: 16.90, 17.89,
62.53 (q, J ¼ 27 Hz), 53.27, 67.44, 127.70 (q,
J ¼ 286 Hz), 127.32, 128.17, 128.55, 129.08, 135.69,
171.23, 172.51 ppm; 19F NMR (235 MHz, CDCl3) d:
ꢀ0.39(s) ppm; no signal for a second diastereomer was
found.
4.7. Peptide synthesis and proof of configuration
4.7.1. Synthesis of (S,R,S)-Z-Phe-(aTfm)Ala-Ala-NH2.
1.0 mmol (157 mg) of the H-(aTfm)Ala-OH batch, which
was obtained as a product of the preparative enzymatic
resolution (see Section 4.3), was dissolved in 20 mL dry
dichloromethane. 1.1 mmol DIC and 1.0 mmol (88 mg)
H-Ala-NH2 were added. The reaction mixture was
stirred at room temperature for 12 h. After filtration of
the precipitate, the mother liquor was evaporated in
vacuo and the resulting product (mainly consisting of H-
(aTfm)Ala-Ala-NH2) used without further purification.
Acknowledgements
This work was supported by the Deutsche Forschungs-
gemeinschaft (Innovationskolleg ‘Chemisches Signal
und biologische Antwort’). The authors wish to grate-
fully acknowledge Dr. Claudia Piazza for her help in the
enzymatic resolutions and Dr. Lucien Duchateau and
Drs. E. Houben for developing and performing the
(chiral) HPLC analyses.
N-Methyl-morpholine (0.5 mmol) and isobutyl chloro-
formate (0.6 mmol) were added to a stirred solution of
0.5 mmol (150 mg) Z-Phe-OH in 20 mL absolute ethyl
acetate at ꢀ15 ꢁC. After 30 min 0.5 mmol (114 mg) H-
(aTfm)Ala-Ala-NH2 in 10 mL dry ethyl acetate were
added. The reaction mixture was stirred at ꢀ15 ꢁC for
1 h and at room temperature overnight. The reaction
mixture was washed successively with H2O, dilute citric
acid, H2O, satd NaHCO3 and H2O. The organic layer
was dried over MgSO4, evaporated in vacuo and puri-
fied by flash chromatography (ethyl acetate/n-hexane 4:1
(v/v); Rf ¼ 0:30). Yield: 61%.
References and notes
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D. T.; Goddard, W. A., III; DeGrado, W. F.; Tirrell, D.
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1
[MþH]: 509; H NMR (360 MHz, d4-methanol) d: 1.32
(d, J ¼ 8:6 Hz, 3H), 1.65 (s, 3H), 2.90 (dd, J ¼ 14:0,
8.9 Hz, 1H), 3.10 (dd, J ¼ 14:0, 6.1 Hz, 1H), 4.31 (m,
1H), 4.40 (m, 1H), 5.05 (s, 2H), 7.23–7.35 (m, 10H) ppm;
13C NMR (360 MHz, d4-methanol) d: 17.69, 19.11,
38.15, 50.69, 58.01, 63.54 (q, J ¼ 27 Hz), 67.75, 125.80
(q, J ¼ 285 Hz), 127.84, 128.63, 129.00, 129.45, 129.51,
130.33, 137.94, 138.06, 158.66, 167.86, 174.45,
177.23 ppm; 19F NMR (235 MHz, CDCl3) d:
0.85(s) ppm. This 19F NMR shift corresponds to the
(S,R,S)-diastereomer; the 19F NMR shift of the (S,S,S)-
diastereomer in CDCl3 was determined earlier3 to be
4.9 ppm.
€
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