Linear and cyclic hymenistatin I (HS I) analogues with dipeptide segments Ile2-Pro3, Pro3-Pro4 and Val6-Pro7 replaced by their tetrazole analogues Ile-Ψ[CN4]-Ala3, Pro3-Ψ[CN4]-Ala4 and Val6-Ψ[CN4]-Alawere synthesized by the solid phase peptide synthesis method and cyclized with the TBTU and/or HATU reagent. The peptides were examined for their immunosuppressive activity in the lymphocyte proliferation test (LPT)

Kociolek, K.

Kowalski, M.L.

Silberring, J.

Smoluch, M.T.

Szkudlinska, B.

Zabrocki, J.

Zubrzak, P.

Zubrzak, Paweł

Kociołek, Karol

Smoluch, Marek

Silberring, Jerzy

Kowalski, Marek

Szkudlińska, Barbara

Zabrocki, Janusz

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Search for new synthetic immunosuppressants II. Tetrazole analogues of hymenistatin I.

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Communication
Search for new synthetic immunosuppressants II.
Tetrazole analogues of hymenistatin I
Pawe³ Zubrzak1, Karol Kocio³ek1, Marek Smoluch2, Jerzy Silberring2, Marek L.
Kowalski3, Barbara Szkudliñska3 and Janusz Zabrocki1 
1Institute of Organic Chemistry, Technical University of £ódŸ, £ódŸ, Poland; 2Faculty of
Chemistry, Jagiellonian University, Kraków, Poland; 3Department of Immunology, Medical
University of £ódŸ, £ódŸ, Poland.
Received: 19 September, 2001; accepted: 7 December, 2001
Key words: hymenistatin I, cis -amide bond, 1,5-disubstituted tetrazole ring, immunosuppression
Linear and cyclic hymenistatin I (HS I) analogues with dipeptide segments Ile
2
-Pro
3
,
Pro
3
-Pro
4
and Val
6
-Pro
7
replaced by their tetrazole analogues Ile
2
-[CN4]-Ala
3
,
Pro
3
-[CN4]-Ala
4
and Val
6
-[CN4]-Ala
7
were synthesized by the solid phase peptide
synthesis method and cyclized with the TBTU and/or HATU reagent. The peptides
were examined for their immunosuppressive activity in the lymphocyte proliferation
test (LPT).
A serious problem of transplantology, a dy-
namically expanding branch of contemporary
medicine, is survival of the transplanted or-
gan. A wider use of two existing and effective
immunosuppressants, cyclosporin A (CsA,
sandimmun®) [1] and FK-506 [2], as potent
drugs for the prevention of graft rejection is
limited by their side effects. In this situation,
the search for new immunosuppressants ex-
hibiting a similar mechanism of action but de-
Vol. 48 No. 4/2001
1151–1154
QUARTERLY

Presented at the XVI Polish Peptide Symposium, September 1–4, 2001, Jagiellonian University,
Kraków, Poland.

This work was supported by the State Committee for Scientific Research (KBN, Poland), grant 4 P05F
004 13.

To whom correspondence should be addressed: Janusz Zabrocki, Institute of Organic Chemistry, Tech-
nical University of £ódŸ, 90-543 £ódŸ, Poland; tel. (48 42) 636 6588; fax: (48 42) 636 5530;
e-mail: zabrocki@ck-sg.p.lodz.pl
Abbreviations: ESI-MS, electrospray ionizat ion mass spectroscopy; HATU,
O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate; HS I, hymenistatin I;
LPT, lymphocyte proliferation test; PHA, phytohemagglutinin; SPPS, solid phase peptide synthe-
sis;TBTU, O-(benzotriazol-1-yl)-N,N,N,N-tetramethyluronium tetrafluoroborate.
void of toxicity, especially in the group of nat-
urally existing immunomodulatory peptides
and their analogues, has become an important
goal for medicinal chemistry.
Hymenistatin I [HS I] is a natural cyclic octa-
peptide of the sequence cyclo(Ile1-Ile2-Pro3-
Pro4-Tyr5-Val6-Pro7-Leu8-) isolated by Petit et
al., in 1990 [3]. It has been shown by Siemion
and coworkers [4] that hymenistatin I demon-
strates some immunosuppressive activity in
respect to both humoral (PFC) and cellular
(DTH) immune response. A characteristic fea-
ture of this compound is the presence of a
cis-amide bond between Pro3-Pro4 residues, a
VIa turn in the Ile2-Tyr5 region and a I or
II turn in the Val
6
-Ile
1
region in CHCl3 and
Me2SO solution, respectively [3, 5].
To evaluate the significance of the Ile
2
-Pro
3
,
Pro3-Pro4 and Val6-Pro7 peptide bonds for HS
I biological activity, we have synthesized three
linear and three cyclic hymenistatine I ana-
logues, with the dipeptide segments Ile2-Pro3,
Pro3-Pro4 and Val6-Pro7 replaced by their
tetrazole analogues Ile2-[CN4]-Ala
3, Pro3-
[CN4]-Ala
4 and Val6-[CN4]-Ala
7, respec-
tively.
Ile
1
-Ile
2
-[CN
4
]-Ala
3
-Pro
4
-Tyr
5
-Val
6
-Pro
7
-Leu
8
5
Ile
1
-Ile
2
-Pro
3
-[CN
4
]-Ala
4
-Tyr
5
-Val
6
-Pro
7
-Leu
8
6
Ile
1
-Ile
2
-Pro
3
-Pro
4
-Tyr
5
-Val
6
-[CN
4
]-Ala
7
-Leu
8
7
cyclo(Ile
1
-Ile
2
-[CN
4
]-Ala
3
-Pro
4
-Tyr
5
-Val
6
-Pro
7
-Leu
8
) 8
cyclo(Ile
1
-Ile
2
-Pro
3
-[CN
4
]-Ala
4
-Tyr
5
-Val
6
-Pro
7
-Leu
8
) 9
cyclo(Ile
1
-Ile
2
-Pro
3
-Pro
4
-Tyr
5
-Val
6
-[CN
4
]-Ala
7
-Leu
8
) 10
The 1,5-disubstituted tetrazole ring,
[CN4], has been proposed as a surrogate for
cis-amide bonds, making it a valuable tool in
the design of conformationally constrained
peptidic receptor probes [6–10]. Since native
hymenistatin I has been demonstrated to have
only weak biological activity which precludes
its use as an immunosuppressant we decided
to look for immunosuppressive activity of
hymenistatin I analogues, employing a stan-
dard test used for the assessment of immuno-
suppressive activity.
MATERIALS AND METHODS
The synthesis of desired peptides
c(Ile2-[CN4]-Ala
3)HS I 8, c(Pro3-[CN4]-
Ala4)HS I 9 and c(Val6-[CN4]-Ala
7)HS I 10
as well as the native compound was achieved
by fragment condensation on a polymeric sup-
port (Merrifield type resin) using TBTU or
HATU as a coupling reagent. The synthetic
strategy is shown for peptide 8 as an example
(Fig. 2). The tetrazole building blocks 4 was
synthesized separately in solution (Fig. 1, Ta-
ble 1) following a previously reported strategy
[8, 9] and were used in the solid phase peptide
synthesis procedure. The known proclivity of
the C-terminal -carbon of tetrazole dipep-
1152 P. Zubrzak and others 2001
Figure 1. Scheme of tetrazole
tripeptides synthesis.
tides to epimerize [7–9] on prolonged base ex-
posure prompted us to omit the neutralization
step in the solid phase synthesis of peptides
containing the tetrazole unit.
The cyclization of linear precursors was per-
formed in solution using TBTU and/or HATU
as a coupling reagent (Fig. 2). The structure of
all peptides was confirmed by ESI-MS (Ta-
ble 2).
All compounds demonstrated sufficient solu-
bility and were tested for immunosuppressive
activity by the lymphocyte proliferation test
(LPT). Lymphocytes were isolated from pe-
ripheral blood of healthy volunteers. Cells
were stimulated with phytohemagglutinin
(PHA) and incubated with different concen-
trations of the agents under study or cyclo-
sporin A. Incorporation of [3H]thymidine,
which was added to the cultures 24 h before
cell harvesting, was determined in a liquid
scintillation counter and expressed as counts
per minute (c.p.m.).
RESULTS AND DISCUSSION
All synthesized analogues did not affect lym-
phocyte proliferation, thus are devoid of
immunosuppressive activity as assayed by the
LPT (Table 3). It was very surprising that
even native hymenistatin I did not affect lym-
phocyte proliferation in this kind of biological
test. On the other hand, our study does not ex-
clude that the examined compounds could
have an immunomodulatory effect on other
immunological functions. In fact it has previ-
ously been noted that the native compound
demonstrated some immunosuppressive ac-
tivity in animal models (mice). However, the
relevance of the biological activity of native
hymenistatin I described in animal models by
Cebrat et al. [4] to effective immunosuppres-
sion in humans is not clear and the lack of ef-
fect of the native compound on cytokine (IL-2
or IL-6) generation makes its clinical potential
use unlikely. Unfortunately, as demonstrated
Vol. 48 Hymenistatin I analogues 1153
Table 1. Analytical data for tetrazole tripeptides
Compound Yield [%]
[]D (c = 1,
MeOH)
tR Rf
c
Mr
d
calc./found
Boc-Ile-Ile-[CN4]-Ala-OBzl 69.9 –69.4 16.42
a
0.56
1
514.6/515.1
Boc-Ile-Pro-[CN4]-Ala-OBzl 79.8 –17.9 12.21
a
0.53
1
498.6/498.9
Boc-Tyr(2,6-diCl)-Val-[CN4]-Ala-Obzl 68.2 –53.7 17.5
a
0.64
1
697.6/698.2
Boc-Ile-Ile-[CN4]-Ala-OH 95.2 –54.8 5.46
b
0.64
2
424.5/424.8
Boc-Ile-Pro-[CN4]-Ala-OH 98.1 –16.2 5.62
b
0.58
2
408.4/409.1
Boc-Tyr(2,6-diCl)-Val-[CN4]-Ala-OH 96.4 –52.3 6.21
b
0.68
2
607.4/607.9
a
Analytical HPLC with linear gradient 50–70% B/25 min. (A) 0.05% trifluoroacetic acid in water, and (B) 0.038% trifluoroacetic
acid in acetonitrile/H2O (90:10, v/v);
b
gradient 40–60% B/25 min.
c
TLC systems: 1 = dichloromethane/acetone (30:1, v/v); 2 =
butanol/acetic acid/water (4:1:1, by vol.).
d
Relative molecular mass calculated/found by FAB-MS measurements.
Figure 2. Scheme of c(Ile
2
-[CN4]-Ala
3
)HS I syn-
thesis.
in our study, modification of hymenistatin I
by a 1,5-disubstituted tetrazole ring did not
improve its immunosuppressive activity in vi-
tro to any significant degree.
1154 P. Zubrzak and others 2001
Table 2. Analytical data for hymenistatin I analogues
Peptide Yield
a
(%) tR
b
(min) Mr
c
calc./found
5 Linear (Ile
2
-[CN4]-Ala
3
)HS I 83.1 6.37 910.07/909.87
6 Linear (Pro
3
-[CN4]-Ala
4
)HS I 90.4 9.22 910.07/910.01
7 Linear (Val
6
-[CN4]-Ala
7
)HS I 81.2 6.18 910.07/909.92
8 c(Ile
2
-[CN4]-Ala
3
)HS I 24.4 10.86 892.06/892.02
9 c(Pro
3
-[CN4]-Ala
4
)HS I 23.8 14.03 892.06/891.96
10 c(Val
6
-[CN4]-Ala
7
)HS I 21.3 10.14 892.06/892.08
a
Peptide content in crude product as shown by analytical HPLC with linear gradient 30–90% B/25 min. (A) 0.05% trifluoroacetic
acid in water, and (B) 0.038% trifluoroacetic acid in acetonitrile/H2O (90:10, v/v);
b
gradient as above;
c
relative molecular mass
calculated/found by ESI-MS measurements.
Table 3. Effect of the compounds studied on lymphocyte proliferation
Compound studied
Control solution
(PHA 10 g/ml)
2.5 g/ml 5 g/ml 10 g/ml 20 g/ml
Linear (Ile
2
-[CN4]-Ala
3
)HS I 172505 178933 169858 168503 165870
Linear (Pro
3
-[CN4]-Ala
4
)HS I 172505 186371 188707 179547 187384
Linear (Val
6
-[CN4]-Ala
7
)HS I 172505 177748 166299 154650 163309
c(Ile
2
-[CN4]-Ala
3
)HS I 200864 207386 199482 215386 227716
c(Pro
3
-[CN4]-Ala
4
)HS I 200864 206011 213888 207746 215366
c(Val
6
-[CN4]-Ala
7
)HS I 200864 207154 202170 212090 220303
Native HS I 200864 204662 211458 217956 224532
Results are shown in c.p.m. (counts per minute)
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Search for new synthetic immunosuppressants II. Tetrazole analogues of hymenistatin I

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Search for new synthetic immunosuppressants II. Tetrazole analogues of hymenistatin I

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