RU2175973C1 - Method of synthesis of tetradecapeptide - Google Patents

Abstract

FIELD: protein chemistry, biochemistry. SUBSTANCE: invention describes a novel method of synthesis of tetradecapeptide of the general formula (I): H-Thr-Glu-Lys-Arg-Arg-Glu-Thr-Val-Glu-Arg-Glu-Lys-Glu-OH. Method involves condensation of pentapeptide of the formula: Z-Thr(But)-Glu(But)-Lys(Boc)-Lys(Boc)-Arg-OH with nonapeptide of the formula: Arg-Glu(But)-Thr(But)-VAl-Glu(But)-Arg-Glu(But)- Lys(Boc)-Glu(But)-OBut followed by removal of protective groups in synthesized semiproduct and preparing the end product. EFFECT: simplified process, increased yield of end product. 2 cl

Description

The invention relates to the field of chemistry of peptide compounds, as well as to the field of the pharmaceutical industry, and in particular to a new method for producing tetradecapapeptide (HEP-I) of formula (I)
H-Thr-Glu-Lys-Lys-Arg-Arg-Glu-Thr-Val-Glu-Arg-Glu-Lys-Glu-OH
which may find application in medicine.

 The peptide may find application, in particular, in the treatment of AIDS and other similar diseases. AIDS medications cannot cure AIDS patients after infection. The main problem is that the human immunodeficiency virus mutates rapidly, leading to a change in the proteins that are the target of antiviral drugs.

 Thus, the creation of a new method for producing a promising drug is important for practical medicine.

 A known method of producing tetradecapapeptide (HEP-I) of the formula (I) (Patent of the Russian Federation, RU 2127599), which consists in the fact that its synthesis is carried out by the solid-phase method by stepwise extension of the peptide chain, starting from the C-terminal glutamic acid associated with the polymer matrix, using the corresponding N-tert-butyloxycarbonylamino acids in the presence of a condensing agent, followed by treatment of the obtained protected peptidyl polymer with deblocking agents.

 The disadvantage of this method is the low yield of the target product, the impossibility of analytical control in the process of multi-stage synthesis, as well as the impossibility of a significant increase in the scale of synthesis.

 The synthesis method described above is the only one and, thus, the closest in technical essence and the achieved result to the proposed method.

 The objective of the invention is to simplify the process and increase the yield of the target product.

The proposed method that solves this problem is the condensation of the pentapeptide of the formula (II)
Z-Thr (But) -Glu (But) -Lys (Boc) -Lys (Boc) -Arg-OH
with the nonapeptide of formula (III)
Arg-Glu (But) -Thr (But) -Val-Glu (But) -Arg-Glu (But) -Lys (Boc) -Glu (But) -OBut
The obtained protected tetradecapapeptide of the formula (IV)
Z-Thr (But) -Glu (But) -Lys (Boc) -Lys (Boc) -Arg-Arg-Glu (But) -Thr (But) - Val-Glu (But) -Arg-Glu (But) - Lys (Boc) -Glu (But) -OBut
interact sequentially with
a) trifluoroacetic acid
b) hydrogen in the presence of a palladium catalyst
and the target product is purified and isolated in pure form or in the form of an acetate salt.

 Intermediates can be obtained in various ways.

 The intermediate protected pentapeptide of formula (II) used in the method can be obtained by conventional peptide synthesis methods, for example, stepwise condensation of amino acids using activated esters.

In this case, the pentapeptide of the formula (II) is prepared starting from arginine as a free base by sequential addition of N _α- carbobenzoxy-N _ε -t-butyloxycarbonyl-L-lysine, N _α- carbobenzoxy-N _ε -tert-butyloxycarbonyl-L lysine, N-carbobenzoxy-γ-tert-butyl-glutamic acid and N-carbobenzoxy-O-tert-butyl-threonine. Condensations are carried out using activated esters. Carbobenzoxy protecting groups from protected intermediate peptides are removed by catalytic hydrogenolysis. The intermediate protected nonapeptide of formula (III) used in the method can be obtained by conventional peptide synthesis methods, for example, stepwise condensation of amino acids using activated esters.

In this case, the nonapeptide of formula (III) is prepared as follows:
1. The condensation of the pentapeptide of the formula (V)
Z-Glu (But) -Thr (But) -Val-Glu (But) -Arg-OH
with a tripeptide of the formula (VI)
Glu (But) -Lys (Boc) -Glu (But) -OBut
using dicyclohexylcarbodiimide receive a protected octapeptide of the formula (VII)
Z-Glu (But) -Thr (But) -Val-Glu (But) -Arg-Glu (But) -Lys (Boc) -Glu (But) -OBut
2. The octapeptide of formula (VII) is subjected to catalytic hydrogenolysis to obtain an octapeptide of formula (VIII)
Glu (But) -Thr (But) -Val-Glu (But) -Arg-Glu (But) -Lys (Boc) -Glu (But) -OBut
3. The octapeptide of formula (VIII) is condensed with carbobenzoxy arginine using dicyclohexylcarbodiimide to give the protected nonapeptide of formula (IX)
Z-Arg-Glu (But) -Thr (But) -Val-Glu (But) -Arg-Glu (But) -Lys (Boc) -Glu (But) -OBut
4. Using catalytic hydrogenolysis, the protected nonapeptide of formula (IX) is converted to the nonapeptide of formula (III).

 In turn, the pentapeptide of formula (V) and the tripeptide of formula (VI) are obtained by sequential condensation of amino acids at one amino acid, starting from the C-terminus, with activated esters of protected amino acids, removing the N-protecting groups by catalytic hydrogenolysis.

 The final product is prepared as follows.

 The obtained protected pentapeptide of formula (II) is condensed with a nonapeptide of formula (III) using dicyclohexylcarbodiimide to give a protected tetradeapeptide of formula (IV) in high yield (more than 90%). Protective groups tert. butyl type from the protected tetradecapapeptide is removed with trifluoroacetic acid, after which carbobenzoxy protecting groups are removed by catalytic hydrogenolysis.

 Due to the use of the readily available pentapeptide of the formula (II) obtained in high yield, as well as the use of the nonapeptide of the formula (III) in the proposed method, it is possible to obtain the target product with a higher yield in comparison with the synthesis from any other intermediates.

 Example.

Abbreviations:
DMF - dimethylformamide
CDI - dicyclohexylcarbodiimide
HOBT - N-hydroxybenzotriazole
General procedure for the removal of a carbobenzoxy group by catalytic hydrogenation:
The protected peptide is dissolved in methanol at the rate of 15-50 ml of solvent per 1 g of peptide, 0.1 g of 10% palladium-carbon catalyst is added in coal, and a weak stream of hydrogen is passed with stirring until the starting product disappears. Control is carried out by thin layer chromatography. The solution was filtered to remove the catalyst and evaporated.

 Thin layer chromatography.

It is carried out on Merck-Kieselgel glass plates in the systems:
ethyl acetate-hexane 1: 1 (system 1)
Chloroform-methanol-acetic acid-water 60: 45: 7: 13 (system 2)
n-Butanol-acetic acid-water 3: 1: 1 (system 3)
Z-Lys (Boc) -Glu (But) -OBut (a)
47.7 g (100 mmol) of Z-Lys (Boc) -OSu are dissolved in 150 ml of DMF, 28.5 g (110 mmol) of H-Glu (But) -OBut are added. After 4 hours, the absence of activated ether was checked using TLC (system 1), the reaction mixture was diluted with 400 ml of ethyl acetate, washed with 200 ml of 10% acetic acid, 100 ml of water, 200 ml of 3% sodium carbonate, and evaporated. The resulting oil (71.2 g) was used without further purification. The output is quantitative. R _f 0.43 (1).

Z-Glu (But) -Lys (Boc) -Glu (But) -OBut (b)
71.2 g of crude (a) are hydrogenated in 700 ml of methanol, filtered, evaporated, the residue H-Lys (Boc) -Glu (But) -OBut is dissolved in 200 ml of DMF and 45.6 g (105 mmol) of Z-Glu (But ) -OSu. After two hours, add 3 ml of Me ₂ N (CH ₂ ) ₃ NH ₂ to remove excess activated ester. After 30 minutes, the reaction mixture was diluted with 500 ml of ethyl acetate, washed with a mixture of 300 ml of water and 150 ml of a saturated solution of NaCl, 200 ml of 2% sulfuric acid, 100 ml of water, 200 ml of 3% sodium carbonate, and evaporated. R _f 0.90 (1).

HCl • H-Glu (But) -Lys (Boc) -Glu (But) -OBut (c)
All protected tripeptide (b) is dissolved in 500 ml of methanol, hydrogenated with a palladium catalyst, the solution is filtered and a 10% solution of hydrogen chloride in dioxane is added with stirring to pH 4 (control with indicator paper). The solution was evaporated in vacuo, the residue was dried in vacuo of an oil pump with a cooled trap. The dry amorphous residue weighs 83.0 g (quantitative yield calculated on Z-Lys (Boc) -OSu). R _f 0.38 (1).

Z-Glu (But) -Arg-OH (g)
56.4 g (130 mmol) of Z-Glu (But) -OSu are dissolved in 150 ml of DMF, 21.8 g (140 mmol) of arginine are added and stirred at 30-35 ^o for a day. The clear viscous solution was diluted with 500 ml of n-butanol, 300 ml of water and 100 ml of saturated NaCl solution were added. The organic phase is washed with 200 ml of water, evaporated, the residue is triturated with 400 ml of ether, filtered and washed with ether. Yield 62.1 g. 97%. (126 mmol). R _f 0.58 (2).

Z-Val-Glu (But) -Arg-OH (d)
62.1 g (g) was dissolved in 600 methanol, hydrogenated, evaporated, the residue was dissolved in 200 ml of DMF and 150 mmol (52.2 g) of Z-Val-OSu was added. After 6 hours, 400 ml of ether and 400 ml of water are added to the reaction mixture and the mixture is kept at 0 ^° for 24 hours. The precipitate formed is washed with water, ether and dried in air. Yield 61.5 g. 87%. (104 mmol). R _f 0.61 (2).

Z-Thr (But) -Val-Glu (But) -Arg-OH (e)
56.4 g (95.2 mmol) of the protected tripeptide (g) are dissolved in 500 ml of methanol, hydrogenated, evaporated, the residue is dissolved in 150 ml of DMF and 44.6 g (95 mmol) of Z-Thr (But) -ONB is added.

After 6 hours, the solution was diluted with 500 ml of n-butanol, 300 ml of water and 100 ml of a saturated NaCl solution were added. The organic phase is washed with 200 ml of water, evaporated, the residue is triturated with 400 ml of ether, filtered and washed with ether. Yield 62.6 g. 87%. (83.5 mmol). R _f 0.69 (2).

Z-Glu (But) -Thr (But) -Val-Glu (But) -Arg-OH (g)
62.6 g (83.5 mmol) of the protected tetrapeptide (e) are dissolved in a mixture of 700 ml of methanol and 100 ml of water, hydrogenated, evaporated, the residue is dissolved in 150 ml of DMF and 36.9 g (85 mmol) of Z-Glu ( But) -OSu. After 6 hours, the solution was diluted with 500 ml of n-butanol, 300 ml of water and 100 ml of a saturated NaCl solution were added. The organic phase is washed with 200 ml of water, evaporated, the residue is triturated with 400 ml of ether, filtered and washed with ether. Yield 70.6 g. 90%. (75.6 mmol). R _f 0.45 (2).

Z-Glu (But) -Thr (But) -Val-Glu (But) -Arg-Glu (But) -Lys (Boc) - Glu (But) -OBut • HCl (h)
In a mixture of 300 ml of DMF and 300 ml of DMSO, 70.6 g (g) (75.6 mmol), 57 g (III) (80.4 mmol), 20.2 g of HOBT (150 mmol) are dissolved, several distilled off under vacuum milliliters of a solvent to remove water, cooled with ice and 21 g (100 mmol) of CDI are added with stirring.

After 3 hours, add 3 ml of N-methylmorpholine and leave overnight at room temperature. The viscous reaction mass is mixed with 600 ml of chloroform and 400 ml of n-butanol, heated to 50 ^° , 400 ml of water are added and filtered warm. The organic layer was washed with 800 ml of water heated to 50 ^° , evaporated to the state of a flowing gel, and 1.5 L of hexane was added. The resulting precipitate was filtered off, washed with ethyl acetate-hexane 1: 3, and dried in air. Yield 114.7 g. 93%. (70.5 mmol). R _f 0.71 (3).

Z-Arg-Glu (But) -Thr (But) -Val-Glu (But) -Arg-Glu (But) -Lys (Boc) - Glu (But) -OBut • 2HClO ₄ (s)
Dissolve with heating 31.7 g (19.5 mmol) (h) in 700 ml of methanol, hydrogenate, evaporate, the residue is dissolved in 120 ml of DMF at 35-40 ^o . Separately, 7.4 g (24 mmol) of Z-Arg-OH, 24 mmol (2.7 ml) of 72% perchloric acid and 6.5 g (48 mmol) of HOBT are dissolved in 50 ml of DMF, and several milliliters of solvent are distilled off in vacuo to remove water. An octapeptide solution was added, cooled with ice, and 6.3 g (30 mmol) of CDI were added with stirring. After 2 hours, 2.2 ml of N-methylmorpholine and another 2.6 g of CDI are added. After a day, the reaction mixture was diluted with 700 ml of n-butanol, 300 ml of water and 100 ml of a saturated NaCl solution were added. The organic phase is washed twice with a mixture of 250 ml of water and 2 ml of 8M perchloric acid, then with a mixture of 200 ml of water and 100 ml of a saturated solution of sodium bicarbonate, evaporated, the residue is triturated with a mixture of 400 ml of ethyl acetate and 400 ml of hexane, filtered and washed with the same solvent. Yield 28.4 g. 81%. (15.8 mmol). R _f 0.56 (3).

Z-Lys (Boc) -Arg-OH (k)
23.3 g (48.8 mmol) of Z-Lys (Boc) -OSu are dissolved in 70 ml of DMF, 8.1 g (52 mmol) of arginine are added and stirred at 30-35 ^o for a day. A clear, viscous solution was diluted with 400 ml of n-butanol, 300 ml of water and 100 ml of a saturated solution of NaCl were added. The organic phase is washed with 200 ml of water, evaporated, the residue is triturated with 400 ml of ether, filtered and washed with ether. Yield 25.2 g. 96%. (47 mmol). R _f 0.50 (2).

Z-Lys (Boc) -Lys (Boc) -Arg-OH (L)
Dissolve 25.2 r (47 mmol) (k) in 300 ml of methanol, hydrogenate, evaporate the residue, dissolve in 150 ml of DMF and add 24.8 g (52 mmol) of Z-Lys (Boc) -OSu. After 6 hours, the solution was diluted with 500 ml of n-butanol, 300 ml of water and 100 ml of a saturated NaCl solution were added. The organic phase is washed with 200 ml of water, evaporated, the residue is triturated with 400 ml of ether, filtered off and washed with ether. Yield 32.3 g (42.2 mmol). R _f 0.72 (2).

Z-Glu (But) -Lys (Boc) -Lys (Boc) -Arg-OH (m)
Dissolve 32.3 g (42.2 mmol) of the tripeptide (L) in 600 methanol, hydrogenate, evaporate, dissolve the residue in 100 ml of DMF and add 46 mmol (20) of Z-Glu (But) -OSu. After 6 hours, 400 ml of ether and 400 ml of water are added to the reaction mixture and the mixture is kept at 0 ^° for 24 hours. The precipitate formed is washed with water, ether and dried in air. Yield 27 g. 49%. (28.4 mmol). R _f 0.77 (2).

Z-Thr (But) -Glu (But) -Lys (Boc) -Lys (Boc) -Arg-OH (n)
27 g (28.4 mmol) of the protected tetrapeptide (m) are dissolved in 500 ml of methanol, hydrogenated, evaporated, the residue is dissolved in 150 ml of DMF and 15 (32 mmol) Z-Thr (But) -ONB is added. After 6 hours, the solution was diluted with 500 ml of n-butanol, 300 ml of water and 100 ml of a saturated NaCl solution were added. The organic phase is washed with 200 ml of water, evaporated, the residue is triturated with 400 ml of ether, filtered and washed with ether. Yield 30.3 g (27.3 mmol). R _f 0.80 (2).

Z-Thr (But) -Glu (But) -Lys (Boc) -Lys (Boc) -Arg-Arg-Glu (But) -Thr (But) -Val-Glu (But) -Arg-Glu (But) - Lys (Boc) -Glu (But) -OBut • 3HClO ₄ (o)
When heated, 28.4 g (15.8 mmol) of protected nonapeptide (s) are dissolved in 800 ml of methanol, hydrogenated, the residue is dissolved in 400 ml of DMF, 17.7 g (16 mmol) of pentapeptide (n), 16 mmol 72- are added. % perchloric acid (1.8 ml), 4.7 g (35 mmol) of HOBT and a few milliliters of solvent are distilled off to remove water. Under ice cooling and stirring, add 5.25 g (25 mmol) of CDI, then after 2 hours 1 ml of N-methylmorpholine. It is stirred for 24 hours at room temperature, evaporated to 1/3 of the volume in a vacuum of an oil pump, the residue is mixed with 300 ml of hexane, 300 ml of ethyl acetate and 500 ml of water. The precipitate was filtered off, washed with water and ethyl acetate-hexane, and dried in air. Yield 42.3 g. 93%. R _f 0.51 (3).

H-Thr-Glu-Lys-Lys-Arg-Arg-Glu-Thr-Val-Glu-Arg-Glu-Lys-Glu-OH • nAcOH (p)
All protected tetradecapapeptide (o) was dissolved in 300 ml of trifluoroacetic acid, held for 1 hour at 25 ^° , evaporated, the residue was triturated with 500 ml of ether, filtered off, washed with ether and dissolved in 500 ml of water. The aqueous solution is filtered, non-polar impurities are extracted with 200 ml of n-butanol. The organic phase is discarded and the aqueous phase is mixed with 200 ml of methanol, 2 g of 10% palladium-carbon catalyst are added in coal, heated to 40 ^° and 30 ml of a saturated solution of ammonium formate are added portionwise over 3 hours. The solution was stirred at room temperature for 20 hours, filtered and applied to a column containing 1 L of SP-Sephadex in 0.2 M pyridine acetate buffer solution. Separation is carried out in a gradient of 0.2 M - 1.3 M pyridine acetate buffer; fractions containing the product are evaporated, evaporated twice with water and lyophilized from an aqueous solution. Additional purification is carried out by preparative high pressure chromatography on a reverse phase carrier. The product yield with a purity of 95-97% 13.2 g. (44%).

 Amino acid analysis: threonine 1.9 (2), valine 1.0 (1), lysine 3.1 (3), arginine 2.9 (3), glutamic acid 5.2 (5).

[α] _D -27 ^° (c = 0.5 in 95% acetic acid). The product is also characterized by a mass spectrum and a spectrum of nuclear magnetic resonance.

 HPLC retention time ≅ 8.5 min.

High performance liquid chromatography (HPLC) was carried out on a Gilson chromatograph (France) under the following conditions:
- ODS column with a size of 250 x 4.6 mm filled with a sorbent with a reversed phase with a particle size of 5 microns;
- mobile phase A: 0.2% solution of trifluoroacetic acid in 0.1 M sodium chloride solution;
- mobile phase B: acetonitrile;
- gradient mode: from 35 to 45% of phase B for 10 min;
- flow rate 1.0 ml / min;
- detection at a wavelength of 220 nm;
- sensitivity 0.1 OE.

Thin-layer chromatography is performed on Merck-Kieselgel-60 plates in systems:
(1) Chloroform-ethyl acetate-acetic acid 8: 26: 0.2
(2) Chloroform-methanol-acetic acid-water 75: 15: 5: 2
(3) Chloroform-methanol 9: 1.

Claims (2)

1. The method of producing tetradecapapeptide threonyl-glutamyl-lysyl-lysyl-arginyl-arginyl-glutamyl-threonyl-valyl-glutamyl-arginyl-glutamyl-lysyl-glutamate
H-Thr-Glu-Lys-Lys-Arg-Arg-Glu-Thr-Val-Glu-Arg-Glu-Lys-Glu-OH,
characterized in that the pentapeptide of the formula
Z-Thr (X) -Glu (X) -Lys (Y) -Lys (Y) -Arg-OH
subjected to condensation with a nonapeptide of the formula
Arg-Glu (X) -Thr (X) -Val-Glu (X) -Arg-Glu (X) -Lys (Y) -Glu (X) -OX,
the resulting protected tetradeapeptide of the formula
Z-Thr (X) -Glu (X) -Lys (Y) -Lys (Y) -Arg-Arg-Glu (X) -Thr (X) -Val-Glu (X) -Arg-Glu (X) - Lys (Y) -Glu (X) -OX,
where X, Y, Z is hydrogen or protective groups,
subjected to interaction with trifluoroacetic acid, hydrogenated in the presence of a catalyst and the final product is isolated.  2. The method according to claim 1, characterized in that X is a tert-butyl group (But), Y is a tert-butyloxycarbonyl group (Boc), and Z is benzyloxycarbonyl.