Pharmaceutical Chemistry Journal
Vol. 35, No. 7, 2001
IMMUNOSTIMULANT ACTIVITY OF THE COORDINATION
COMPOUNDS OF ISOLEUCINE – TRYPTOPHAN DIPEPTIDES
WITH ZINC IONS
1
1
2
3
S. D. Isupov, A. N. Shakhmatov, Z. N. Yusupov, and K. Kh. Khaidarov
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 35, No. 7, pp. 9 – 11, July, 2001.
Original article submitted February 5, 2000.
Secondary immunodeficiency states related to T-cell im-
munity disorders are treated by immunomodulants such as
tactivin (T-activin) and thymogen [1]. Tactivin is a mixture
of peptides isolated from thymus [2], while thymogen is ba-
sed on a synthetic glutamyl – tryptophan dipeptide [3]. Hy-
perimmunity and immunodeficiency states are usually trea-
ted with a composition comprising a dipeptide mixture with
the general formula X-Trp, where X is glutamic acid, gluta-
mine, leucine, and isoleucine [4]. It is known that complexa-
tion with metal (zinc, iron, platinum) ions leads to an increa-
se in the specific activity of medicinal preparations [5]. In
this context, we have studied the effect of complex formation
with zinc ions on the immunostimulant activity of an isoleu-
cine – tryptophan dipeptide.
The dipeptide was obtained by condensation of carbo-
benzoxy-L-isoleucine with L-tryptophan using dicyclohexyl-
carbodiimide as a condensing agent and 1-hydroxybenzotria-
zole as a nucleophilic additive. The carboxy group of tryp-
tophan was protected by forming a salt with triethylamine.
Upon purification, the protected peptide was unblocked by
catalytic hydrogenation in the presence of palladium (10%
Pd on activated charcoal). Preliminary purification of the
free dipeptide was performed by extraction with n-butanol,
while the final purification was effected by HPLC. The free
dipeptide yield after lyophilization amounted to 98%.
respectively. The experimental dissociation constants are as fol-
–
4
lows: isoleucine, K = 7.41 ´ 10
–
(pK = 3.13) and
1
– 2
1
10
K = 2.45 ´ 10
(pK = 9.61); tryptophan, K = 1.12 ´ 10
2
2
1
– 11
(
pK = 1.95) and K = 3.63 ´ 10
(pK = 10.44); isoleuci-
– 6
1
2
2
ne – tryptophan dipeptide, K = 7.4 ´ 10 (pK = 5.13) and
–
1
1
11
K = 1.45 ´ 10
(pK = 10.84). According to the available
2
2
published data [7], the dissociation constants of amino acids
are pK = 2.36 and pK = 9.68 for isoleucine and pK = 2.38
and pK = 9.39 for tryptophan. The difference between our
1
2
1
2
values and the published data can be explained by the fact
that our pH titration procedure was performed in aqueous
amino acid solutions without maintaining constant ionic
strength.
Solutions of the zinc-containing coordination compo-
unds were obtained by interaction of dilute aqueous solutions
of dipeptide and zinc acetate at at a reagent concentration of
–
4
3
.133 ´ 10 mole/liter, pH 6.0, and a temperature of
110 – 120°C. The reaction was conduced in the dark without
access to air in order to eliminate oxidation of the indole gro-
up of tryptophan. The formation of a target coordination
compound was confirmed by pH titration measurement, the
results of which are presented in Fig. 2. The presence of an
equivalence point in the titration curve confirms the coordi-
nation of dipeptide to zinc ion.
We also attempted to determine the composition of the
coordination compound using the method of isomolar series
Before the synthesis of coordination compounds, it was
necessary to determine the pH range in which the target di-
peptide occurs in the zwitterion form. For this purpose, we
performed the pH titration of isoleucine, tryptophan, and the
isoleucine – tryptophan dipeptide and constructed the corres-
ponding distribution diagrams using a method described in
[
(
8]. As can be seen from the results of these experiments
Figs. 3 and 4), an increase in the optical density of the iso-
molar solution with decreasing dipeptide concentration takes
place at a wavelength of 310 – 345 nm. The calculation con-
ducted for various wavelengths showed that the coordination
compounds are characterized by a zinc to dipeptide ratio of
[
6]. According to these data (Fig. 1), isoleucine, tryptophan,
and the isoleucine – tryptophan dipeptide occur in the zwitte-
rion form in the pH range 4.0 – 9.0, 3.0 – 10.0, and 6.0 – 8.0,
1
ion compositions of [ZnL (H O) ] and [ZnL ] , respecti-
: 2 and 1 : 6, which corresponds to the possible complex
2
+
2+
2
2
4
6
1
vely (here, L denotes the isoleucyl – tryptophan fragment).
The immunostimulant activity of dipeptide and the coor-
dination compounds was evaluated in vivo by determining
the titer of specific antibodies in the blood serum of calves
”
Zand” Enterprise, Dushanbe, Tajikistan.
2
3
Tajik State National University, Dushanbe, Tajikistan.
Nikitin Institute of Chemistry, Academy of Sciences of the Republic of
Tajikistan, Dushanbe, Tajikistan.
355
0
091-150X/01/3507-0355$25.00 © 2001 Plenum Publishing Corporation