Synthesis of a cyanopeptide-analogue with trypsin activating properties

Article abstract of DOI:10.1016/S0960-894X(00)00101-3

An efficient synthesis of a peptidic analogue of cyanobacterial metabolites with proposed serine protease inhibitory activity has been developed. Surprisingly, one trypsin activating compound was obtained. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00101-3

Bioorganic & Medicinal Chemistry Letters 10 (2000) 779±781
Synthesis of a Cyanopeptide-Analogue with Trypsin Activating
Properties
Gregor Radau* and Daniel Rauh
Institute of Pharmacy, Ernst-Moritz-Arndt-University, Pharmaceutical/Medicinal Chemistry, Friedrich-Ludwig-Jahn-Str. 17,
D-17487 Greifswald, Germany
Received 26 November 1999; accepted 7 February 2000
AbstractÐAn ecient synthesis of a peptidic analogue of cyanobacterial metabolites with proposed serine protease inhibitory
activity has been developed. Surprisingly, one trypsin activating compound was obtained. # 2000 Elsevier Science Ltd. All rights
reserved.
Cyanobacteria are well-known sources of interesting
metabolites, many of which possess signi®cant biologi-
cal activity.1 Thus, cyanobacteria have been subjected to
systematical screenings. Cyanopeptides show in addi-
tion to hepato- and neurotoxic properties a broad
spectrum of biological activities, including antitumor,2
immunosuppressive,3 and antimicrobial4 e€ects as well
as angiotensin-converting enzyme inhibitory action5 and
cardioactive e€ects.6
Reversed-phase HPLC and Chiral GC analysis have
con®rmed that 1 and 2 contain no standard l-amino
acids.12 Isoleucine shows d-allo con®guration; if the
residue was a l- or a l-allo-isoleucine, the side-chain
would produce steric con¯icts with the bulky 2-carboxy-
6-hydroxy-octahydroindole (Choi) sulfate. Agmatine
constitutes the basic side-chain and seems to be essential
for inhibiting the above mentioned serine proteases. The
relative stereochemistry of the Choi-sulfate was deduced
by NMR studies; the absolute con®guration of 2 was
determined by means of X-ray crystallographic methods
of the cocrystallized 2-trypsin-complex (Brookhaven
Protein Data Bank, code: 1aq7).13 The X-ray data
provide information about atypical trypsin-inhibitor
interactions, which are caused by the non-peptidic par-
tial structure of 2 and mainly based on usual hydrogen
bonds and van der Waals interactions. Furthermore,
there is evidence for other interactions such as the sul-
fate moiety protruding into the solvent or the strong,
water-mediated interaction between the hydroxy group
of the 4-hydroxyphenyllactic acid (Hpla) and the amino
acids Cys220 and Ser146 of trypsin.
Besides the hepatotoxic cyclic peptides (microcystines),
cyanobacteria of the genera Microcystis, Oscillatoria,
and Anabaena usually produce non-toxic peptides that
inhibit serine proteases, which play a central role in the
human organism.7 10 Failures of one or more of these
enzymes may cause a state of imbalance between protease
and antiprotease (e.g. endogenous protease inhibitors)
and can lead to an excess of proteolytic activity and/or
to the development of diseases such as pancreatitis.11
Because of their biological potency, the linear cyano-
peptides aeruginosin 98-A, aeruginosin 98-B (1, 2; from
Microcystis aeruginosa)12 and aeruginosin 102-A (3,
Microcystis viridis)7 could be considered as new lead
structures for the design and synthesis of new selective
serine protease inhibitors. Each of these compounds
inhibits trypsin, thrombin, and plasmin. Compound 3 is
the strongest inhibitor of all (IC50: 0.2/0.04/0.3 mg/mL),
while 1 and 2 are equipotent (IC50: 0.6 and 0.6/7.0 and
10.0/6.0 and 7.0 mg/mL).
The conspicuous lack of any interactions of 2 with
trypsin's catalytic triade, Ser195, His57, and Asp102, is
unique. The Ser195 residue does not show close contacts
to any atoms of 2, this discovery may mean that a new
mode of inhibition di€ering from the standard mechan-
isms of serine protease inhibitors is possible.13
Both the diversity of the above mentioned interaction of
2 with trypsin and the discussion of a new inhibiting
mechanism of serine proteases justify additional inves-
tigations of structure activity relationships between
*Corresponding author. Tel.: +49-3834-864-880; fax: +49-3834-
864-874; e-mail: radau@pharmazie.uni-greifswald.de
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00101-3

780
G. Radau, D. Rauh / Bioorg. Med. Chem. Lett. 10 (2000) 779±781
serine proteases such as trypsin and its e€ectors. In this
paper, we describe the synthesis and inhibition tests of
the trypsin e€ector 10 by taking 2 into consideration as
a lead structure (Scheme 1).
transformed into the appropriate active ester by ester-
i®cation with N-hydroxysuccinimide and then subjected
to the completely unprotected l-isoleucine to give 5.
Compound 8 was obtained by DMAP/DCC-catalyzed
esteri®cation of Boc-l-proline (6) with 2-(1-piperazino)-
ethanol (7) followed by the Boc-cleavage by the means
of TFA. DCC-catalyzed coupling of 5 with 8 exclusively
lead to the formation of 10, whose structure has been
proved by NMR and MS studies.14
In order to simplify the stereochemical obstacles in 2 on
the route to useful synthetic analogues, e.g., the target
molecules 9 and 11, we substituted the chiral Hpla unit
in 2 by 3-(4-hydroxyphenyl)-propionic acid (4) and
decreased the sterically hindered Choi moiety to l-pro-
line. Through these modi®cations, we circumvent the
necessity of using the expensive d-allo-isoleucine and
were able to applicate l-isoleucine. To provide a basic
C-terminal side-chain, which is essential for the inhibi-
tion of trypsin, agmatine in 2 was exchanged for 2-(1-
piperazino)ethanol, since syntheses of the extraordinarily
expensive agmatine only succeeded in very low yields. If
this side-chain turns out to be not basic enough to inhi-
bit trypsin e€ectively, it will have to be transformed into
the appropriate guanyl derivative 11.
Surprisingly, 10 turned out to be a trypsin activator
instead of behaving as an inhibitor. The inhibition tests
were performed by using N-a-benzoyl-dl-arginyl-4-
nitroanilide (BAPNA) as the substrate of trypsin.
BAPNA was cleaved into N-a-benzoyl-dl-arginine and
4-nitroaniline, whose absorption at 405 nm was mea-
sured as an indicator of enzyme activity. Compound 10,
at a concentration between 20 and 40 mM, increases the
activity of trypsin by 70%. Whether this e€ect depends
on the concentration of BAPNA (1 mg/mL) and/or is
speci®c for BAPNA has to be clari®ed by further tests.
Thus, the desired building blocks 5 and 8 were synthe-
sized by using standard methods in peptide synthesis
(Scheme 1). 3-(4-Hydroxyphenyl)-propionic acid (4) was
In conclusion, we have developed a synthesis of a new
serine protease activator in high yields under moderate
Scheme 1. (i) HOSu/DCC/EA, 78%; (ii) NaHCO3/L-Ile-OH/EtOH, 76%; (iii) DMAP/DCC/DCM, 70%; (iv) TFA/NaHCO3, quant.; (v) DCC/EA,
56% Abbreviations: DCC: N,N-dicyclohexylcarbodiimide; DCM: dichloromethane; DMAP: N,N-dimethyl-amino-pyridine; EA: ethyl acetate;
HOSu: N-hydroxysuccinimide; TFA: tri¯uoroacetic acid.

G. Radau, D. Rauh / Bioorg. Med. Chem. Lett. 10 (2000) 779±781
781
conditions. Further studies of the structure activity
relationship and the mechanism of the activation have
to follow.
Wijayaratne, R.; Szewczyk, S.; Patterson; G. M. L. Tetra-
hedron 1996, 52, 395.
9. Okano, T.; Sano, T.; Kaya, K. Tetrahedron Lett. 1999, 40,
2379.
10. Murakami, M.; Sun, Q.; Ishida, K.; Matsuda, H.; Okino,
T.; Yamaguchi, K. Phytochemistry 1997, 45, 1197.
11. Nakayama, Y.; Senokuchi, K.; Nakai, H.; Obata, T.;
Kawamura, M. Drugs Fut. 1997, 22, 285.
12. Murakami, M.; Ishida, K.; Okino, T.; Okita, Y.; Matsuda,
H.; Yamaguchi, K. Tetrahedron Lett. 1995, 36, 2785.
13. Sandler, B.; Murakami, M.; Clardy, J. J. Am. Chem. Soc.
1998, 120, 595.
Acknowledgements
The authors would like to thank the Fonds der Che-
mischen Industrie and the BMBF for ®nancial support.
References and Notes
14. 5: 1H NMR (200 MHz, DMSO-d6): d (ppm)=0.80 (t, 3 H,
Ile-CH2-CH3); 0.83 (t, 3 H, Ile-CH±CH3); 1.14 (m, 1 H, Ile-
CH2); 1.31 (m, 1 H, Ile-CH2); 1.70 (m, 1 H, Ile-CH); 2.34 (t, 2
H, PhCH2±CH2±COOR); 2.68 (t, 2 H, PhCH2±CH2±COOR);
4.16 (t, 1 H, Ile-Ha); 6.64 (d, 8.0 Hz, 2 H, Harom.); 6.98 (d, 8.0 Hz,
2 H, Harom.); 7.97 (d, 8.0 Hz, 1 H, Ile-NH); 9.13 (s, 1 H, OH).
13C NMR (70 MHz, DMSO-d6): d (ppm)=11.1 (CH3CH2,
Ile); 16.3 (CH3CH-, Ile); 24.6 (CH2, Ile); 30.3 (Ph±CH2±CH2-
R); 36.2 (Ph±CH2±CH2-R); 36.9 (b-CH, Ile); 56.0 (a-CH, Ile);
114.9 (CHarom.); 129.0 (CHarom.); 131.2 (C-1arom.); 155.3 (C-
4arom.); 171.7 (CONHR); 173.1 (COOH) [a]2d0 11.5ꢀ (MeOH;
c=2); mp: 122±126 ꢀC (H2O). Compound 8 was generated in
situ from the appropriate N-Boc-proline derivative and was
subjected to 5 without further puri®cation. Compound 10:
NMR studies (300 MHz, MeOH-d4) could not distinguish
between the structures of 9 and 10. With the help of MS we
were able to assign the fragmentation scheme to the structure
of 10. MS (EI, 70 eV): m/z (intensity %)=488 (M+, 18), 360
(15), 262 (29), 234 (30), 128 (8), 127 (5), 107 (44), 98 (15), 70
(basis, 100).
1. For reviews see: Namikoshi, M; Rinehart, K. L. J. Ind.
Microbiol. 1996, 17, 373; Yasumoto, T.; Satake, M. Chimia
1998, 52, 63; Moore, R. E. J. Ind. Microbiol. 1996, 16, 134;
Sings, H. L.; Rinehart, K. L. J. Ind. Microbiol. 1996, 17, 385.
2. Trimurtulu, G.; Ohtani, I.; Patterson, G. M. L.; Moore, R.
E.; Corbett, T. H.; Valeriote, F. A.; Demchik, L. J. Am. Chem.
Soc. 1994, 116, 4729.
3. Zhang, L.-H.; Longley, R. E.; Koehn, F. E. Life Sciences
1997, 60, 751.
4. Pergament, I.; Carmeli, S. Tetrahedron Lett. 1994, 35, 8473.
5. Okino, T.; Matsuda, H.; Murakami, M.; Yamaguchi, K.
Tetrahedron Lett. 1993, 34, 501.
6. Moore, R. E.; Bornemann, V.; Niemczura, W. P.; Gregson,
J. M.; Chen, J.-L.; Norton, T. R.; Patterson, G. M. L.; Helms,
G. L. J. Am. Chem. Soc. 1989, 111, 6128.
7. Matsuda, H.; Okino, T.; Murakami, M.; Yamaguchi, K.
Tetrahedron 1996, 52, 14501.
8. Bonjouklian, R.; Smitka, T.A.; Hunt, A. H.; Occolowitz, J.
L.; Perun, T. J. Jr., Doolin, L.; Stevenson, S.; Knauss, L.;