S. M. A. Salam et al. / Tetrahedron: Asymmetry 17 (2006) 22–29
29
enzyme solution was added to DMSO (0.4 ml) dissolv-
ing CBZ-Phe-OCam (72 mg, 0.2 mmol). The resulting
solution was rapidly shaken and shock frozen in liquid
nitrogen. After shock freezing, the frozen reaction mix-
ture was incubated in a freezer (ꢀ24 ꢀC). After 2 h, the
ice was melted at room temperature and the precipitates
collected by filtration and washed with water
(2 · 50 ml). The precipitates were dried in vacuum, and
then recrystallized from methanol–diethyl ether: yield,
74 mg (83%); mp 237–238 ꢀC (lit.:20 mp 228–228.5 ꢀC);
½aꢁD ¼ ꢀ32:4 (c 0.82, DMF). H NMR (DMSO-d6) d
2.64–3.04 (4H, m, 2 · CH2–C6H5), 4.19–4.25 (1H, m,
NH–CH), 4.44–4.49 (1H, m, NH–CH), 4.93 (2H, s, O–
CH2–C6H5), 7.11 (1H, s, CONH), 7.18–7.34 (15H, m,
3 · C6H5), 7.40 (1H, s, CONH), 7.49 (1H, d,
J = 8.5 Hz, NH–CH), 8.03 (1H, d, J = 8.3 Hz, NH–
CH). 13C NMR (DMSO-d6) d 37.3, 37.7, 53.6, 56.2,
65.2, 126.2, 127.4, 127.6, 128.0, 128.3, 129.1, 129.2,
137.0, 137.7, 138.0, 155.7, 171.1, 172.6. HRMS
(FAB+) MH+ calcd for C26H28N3O4: 446.2080; found:
446.2078.
of each component was based on the fact that only ben-
zene ring absorbs at 254 nm in proportion to its number.
The diastereomers [D–L (or L–D) and L–L] were also sep-
arated on the same column. The retention times of the
relevant diastereomers were as follows. CBZ-Phe-Phe-
NH2 and CBZ-D-Phe-Phe-NH2 (or CBZ-Phe-D-Phe-
NH2) were eluted after 18.3 and 19.3 min, respectively,
when 50% (v/v) aq CH3CN was used as eluent. CBZ-
Leu-Leu-NH2 and CBZ-D-Leu-Leu-NH2 (or CBZ-
Leu-D-Leu-NH2) were eluted after 31.1 and 36.1 min,
respectively, with an eluent of 40% (v/v) aq CH3CN.
26
1
References
1. Drauz, K.; Waldmann, H. Enzyme Catalysis in Organic
Synthesis; Wiley-VCH: Weinheim, Germany, 2002.
2. Bordusa, F. Chem. Rev. 2002, 102, 4817–4867.
3. Miyazawa, T.; Nakajo, S.; Nishikawa, M.; Hamahara, K.;
Imagawa, K.; Ensatsu, E.; Yanagihara, R.; Yamada, T.
J. Chem. Soc., Perkin Trans. 1 2001, 82–86.
4. Ha¨nsler, M.; Jakubke, H.-D. J. Pept. Sci. 1996, 2, 279–
289.
5. Schuster, M.; Aaviksaar, A.; Jakubke, H.-D. Tetrahedron
1990, 46, 8093–8102.
6. Haensler, M.; Wissmann, H.-D.; Wehofsky, N. J. Pept.
Sci. 2000, 6, 366–371.
7. Ullmann, D.; Bordusa, F.; Salchert, K.; Jakubke, H.-D.
Tetrahedron: Asymmetry 1996, 7, 2047–2054.
8. Kuhl, P.; Zacharias, U.; Burckhardt, H.; Jakubke, H.-D.
Monatsh. Chem. 1986, 117, 1195–1204.
9. Miyazawa, T.; Tanaka, K.; Ensatsu, E.; Yanagihara, R.;
Yamada, T. J. Chem. Soc., Perkin Trans. 1 2001, 87–93.
10. Miyazawa, T.; Ensatsu, E.; Yabuuchi, N.; Yanagihara, R.;
Yamada, T. J. Chem. Soc., Perkin Trans. 1 2002, 390–395.
11. Schuster, M.; Aaviksaar, A.; Haga, M.; Ullmann, U.;
Jakubke, H.-D. Biomed. Biochim. Acta 1991, 50, 84–89.
12. Salam, S. M. A.; Kagawa, K.; Kawashiro, K. Biotechnol.
Lett. 2005, 27, 1199–1203.
13. Margolin, A. L.; Tai, D.-F.; Klibanov, A. M. J. Am.
Chem. Soc. 1987, 109, 7885–7887.
14. Kawashiro, K.; Sugahara, H.; Sugiyama, S.; Hayashi, H.
Biotechnol. Bioeng. 1997, 53, 26–31.
15. Schwyzer, R.; Iselin, B.; Rittel, W.; Sieber, P. Helv. Chim.
Acta 1956, 39, 872–883.
16. Moriniere, J. L.; Danree, B.; Lemoine, J.; Guy, A. Synth.
Commun. 1988, 18, 441–444.
17. Fulcrand, V.; Jacquier, R.; Lazaro, R.; Viallefont, P. Int.
J. Pept. Protein Res. 1991, 38, 273–277.
18. Capellas, M.; Benaiges, M. D.; Caminal, G.; Gonzalez,
26
CBZ-Phe-Ala-NH2: yield, 68%; mp 212–214 ꢀC; ½aꢁD
¼
ꢀ11:6 (c 0.53, MeOH) {lit.:20 mp 205–205.5 ꢀC;
25
1
½aꢁD ¼ ꢀ9:8 (c 1.0, MeOH)}. H NMR (DMSO-d6) d
1.23 (3H, d, J = 7.1 Hz, CH3), 2.70–3.06 (2H, m,
CH2–C6H5), 4.19–4.29 (2H, m, 2 · NH–CH), 4.94 (2H,
s, O–CH2–C6H5), 7.00 (1H, s, CONH), 7.18–7.34
(11H, m, 2 · C6H5+CONH), 7.50 (1H, d, J = 8.5 Hz,
NH–CH), 8.03 (1H, d, J = 7.6 Hz, NH–CH). 13C
NMR (DMSO-d6) d 18.5, 37.3, 48.0, 56.1, 65.2, 126.2,
127.4, 127.6, 128.0, 128.3, 129.2, 137.0, 138.1, 155.8,
171.0, 174.0. HRMS (FAB+) MH+ calcd for
C20H24N3O4: 370.1767; found: 370.1798.
CBZ-D-Phe-Phe-NH2: yield, 79%; mp 212–213 ꢀC;
26
½aꢁD ¼ ꢀ3:8 (c 0.80, MeOH). Found: C, 69.81; H,
6.20; N, 9.46. Calcd for C26H27N3O4: C, 70.09; H,
1
6.11; N, 9.43. H NMR (DMSO-d6) d 2.42–3.06 (4H,
m, 2 · CH2–C6H5), 4.20–4.25 (1H, m, NH–CH), 4.47–
4.52 (1H, m, NH–CH), 4.91 (2H, s, O–CH2–C6H5),
7.15–7.34 (16H, m, 3 · C6H5+CONH), 7.38 (1H, d,
J = 8.5 Hz, NH–CH), 7.46 (1H, s, CONH), 8.35 (1H,
d, J = 8.5 Hz, NH–CH). 13C NMR (DMSO-d6) d 37.3,
37.8, 53.6, 56.1, 65.2, 126.1, 126.2, 127.4, 127.7, 127.9,
128.3, 129.2, 136.9, 137.9, 138.0, 155.8, 171.2, 172.9.
IR (m/cmꢀ1) 3427, 3291, 3204, 1692, 1670, 1639, 1543.
HRMS (FAB+) MH+ calcd for C26H28N3O4:
446.2080; found: 446.2084.
´
´
G.; Lopez-Santın, J.; Clapes, P. Biotechnol. Bioeng. 1996,
50, 700–708.
4.7. HPLC analyses
19. Martinez, J.; Laur, J.; Castro, B. Tetrahedron Lett. 1983,
24, 5219–5222.
20. Miyazawa, T.; Hiramatsu, M.; Murashima, T.; Yamada,
T. Biocatal. Biotransform. 2003, 21, 93–100.
21. Yang, P. S.; Rising, M. M. J. Am. Chem. Soc. 1931, 53,
3183–3184.
22. Anderson, G. W.; Zimmerman, J. E.; Callahan, F. M.
J. Am. Chem. Soc. 1964, 86, 1839–1842.
23. Hill, R. R.; Birch, D.; Jeffs, G. E.; North, M. Org. Biomol.
Chem. 2003, 1, 965–972.
A JASCO instrument BIP-I equipped with a JASCO
UV-1575 monitor was used for HPLC. The amounts
of the acyl donor, peptide and the hydrolysis product
of the acyl donor were determined by reversed phase
HPLC analysis under the following conditions: column,
Inertsil ODS-80A (4.6 / · 250 mm, GL Sciences);
eluent, aq CH3CN containing a small amount of TFA
(0.1%, v/v); flow rate, 0.5 ml minꢀ1; detection, UV at
254 nm. A Shimadzu C-R6A data processor was used
for the integration of the peak areas. The determination
24. Chen, S.-T.; Chen, S.-Y.; Chen, H.-J.; Huang, H.-C.;
Wang, K.-T. Bioorg. Med. Chem. Lett. 1993, 3, 727–
733.