Y. Wu et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2331–2333
2333
Stability to trypsin in vitro
general the thrombolytic activities of 7–9 were not
changed, at least when their C-terminals were amidated
by 1. On the other hand, however, if their N-terminals
were acylated by 1 the thrombolytic activities of 7–9
were banished completely. In accordance with the con-
formational analysis using Sybyl version 6.4 it may be
clear that after the introduction of 1 into the N-terminal
of the peptides the flexibility of the conformations were
not improved at all and the stretch conformations were
formed. After introduction of 1 into the C-terminal of
the peptides the flexibility of the conformations was
obviously changed with the formation of rigid bII0 turn
in which the hydrogen bond between the NH of Ala3
residue and the C¼O of carboline resulted in the desired
conformational rigidity. In the enzyme promotion
hydrolysis experiments 25, 26 and 27 exhibited higher
stability to trypsin than 22, 23 and 24. Perhaps in the
used conditions the stretch conformation is easy to
access for trypsin; in contrast the bII0 turn is difficult to
access for trypsin. This kind of bII0 turn with free NH2
of Lys residue as the head may be responsible for the
enzymatic stability and the thrombolytic activity. Con-
sidering the synthetic difficulty of the corresponding
cyclic peptides, this special modification, such as 7–9 by
introducing 1 at their C-terminals to increase their con-
formational rigidity, may be another way of accessing
the cyclic peptide. The detailed conformational analysis
based on the NMR data is proceeding now.
10 mg of the tested compound were dissolved in 1 mL of
phosphate buffer (pH 8). To the solution 0.5 mg of
trypsin was added. The reaction mixture was kept at
37 ꢀC and the concentration of the tested compound
was monitored every 1 h using HPLC; the mobile phase
was 25% of CH3OH in water containing 0.1%
CF3COOH. The results indicated that the depletion of
22, 23, and 24 was observed after the enzyme promotion
hydrolysis had proceeded for 5, 8, and 9 h, respectively.
On the other hand, however, the concentrations of 25,
26 and 27 were not changed, even when the enzyme
promotion hydrolysis had proceeded for more than 24 h.
Conformational Analysis
Based on the calculated results using Sybyl version 6.4
the conformations of 22–27 were analyzed. The con-
formational analysis indicated that in the used condi-
tions the lowest conformational energy for 22, 23, 24,
25, 26, and 27 was 8.853, 10.787, 1.779, 10.640, 9.399,
and 1.834 kcal/mol, respectively, under which 23, 24, 25
take the stretch conformation and 26, 27, 28 exhibited
bII0 turn with intramolecular hydrogen bond consisting
of the NH of Ala3 residue and the C¼O of the carbo-
line. The exact data are listed in Table 2.
Acknowledgements
Discussion
Using the usual procedure (S)-tetrahydro-b-carboline-3-
carboxylic acid (1), peptides 7–9 and the pseudopeptides
22–27 were obtained in good yield. The bioassay of 7, 8,
9, 22, 23, 24, 25, 26 and 27 in vivo suggested that in
The author Shiqi Peng wishes to thank the Key Basic
Research Project (G1998051111) of China for financial
support.
References and Notes
Table 2. Key data for conformation analysis
1. Zhao, M.; Peng S. J. Prakt. Chem. 1999, 341, 668.
2. Zhao, M.; Zhang, Lr.; Yu, T.; Peng, S. Chinese J. Med.
Chem. 1999, 6 (2), 125 ; Chem. Abstr. 124, 290228y.
3. Lin, N.; Zhao, M.; Wang, C.; Peng, S. Bioorg. Med. Chem.
2002, 12, 585.
4. Yang, X.; Peng, S.; Jiang, T.; Zhang, Z.; Winterfeldt, E.
Chinese J. Mag. Res. 1991, 6 (3), 317; Chem. Abstr. 116,
129342f.
Compd
Distance between
˚
O and H (A)
The lowest energy
(kcal/mol)
Conformation
22
23
24
25
26
27
7.504
7.614
7.579
2.064
1.938
1.857
10.640
9.399
1.834
8.853
10.787
1.777
Stretch
Stretch
Stretch
bII0 turn
bII0 turn
bII0 turn
5. Lin, N.; Yang, J.; Wang, C.; Zhao, M.; Peng, S.; Song, D.;
Wong, J. J. Beiging Med. Univ., 2000, 32, 283.
Wu, Yanfen
