CS271584B1 - Method of solid phase peptide preparation - Google Patents

Abstract

The solution concerns the method of preparation of solid-base peptides. The principle consists of the fact that the anchoring of carboxyterminal amino acid and gradual building of peptide strand including washing steps is performed in an ultrasonic field. A reagent with polystyrene carrier is placed into an ultrasonic bath. While being occasionally mixed, the mixture of polymeric carrier and reagents, or, eventually, of wash solutions, is treated by ultrasound. In an ultrasonic environment, the reaction is significantly accelerated.

Description

(57) The ee solution relates to a process for preparing solid phase peptides. The principle is that the anchoring of the carboxyterminal amino acid and the gradual construction of the peptide chain, including the washing steps, are carried out in an ultrasonic field. The reactor 8 with the polystyrene support is placed in an ultrasonic bath. A mixture of the polymeric carrier and the reactants and / or reactants is treated with occasional mechanical stirring. of washing solutions by ultrasound. Ultrasonication significantly accelerates the reaction.

271,584 (11).

(13) Bl (51). Cl. ⁵

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CS 271584 Bl

The invention relates to a process for preparing solid phase peptides in an ultrasonic field.

The preparation of the solid phase peptides is characterized as a sequence of repeating reactions and washing operations that take place on a polymeric support. It is characterized in that it is not possible to purify the reaction intermediates. Therefore, high demands are placed on the course of the individual reactions, including their complete conversion. The degree of conversion can be influenced by: reaction time, changing the reaction environment (e.g., from DCM to DMF), a suitable catalyst (e.g., DMAP), repeating the reaction with a re-weighing of the reagents, resp. by changing the reagents. It is often questioned that the reaction proceeds very slowly, after which the processes are lengthy, with increased consumption of reagents and / or reagents. adverse reactions (eg racemization in long-term catalysis of DMAP).

The above-mentioned disadvantages of solid phase peptide preparation are eliminated by the process according to the invention, which consists in the fact that the individual reaction steps of the successive construction of the peptide chain are carried out in an ultrasonic field.

The use of an ultrasonic field in the preparation of the solid phase peptides of the invention is accomplished by placing a polystyrene-type polymeric carrier cross-linked with divinylbenzene, preferably one percent, functionalized with suitable functional groups, preferably p-methylbenzhydryl, into the reactor. The reaction component is added to the polymeric support and the reactor is placed in an ultrasonic bath. The N-alpha-amino groups were protected with an acid-labile protecting group, preferably a Boc group, and its cleavage in the individual phases of the synthesis was carried out acidolytically, preferably with a mixture of TFA and DCM or HCl in DCM. The side functional groups of the multifunctional amino acids were protected with benzyl-type groups. The condensation of protected amino acids was carried out by suitably activated derivatives, preferably HOBt esters. The completion of the reaction was monitored by a color change of the acid-base indicator, preferably bromophenol blue, MS. No. 268230. All steps of sequential construction of the peptide chain were performed in an ultrasonic field. Upon completion of the synthesis, the peptide from the resin was cleaved and at the same time the protecting groups of the multifunctional amino acids were removed acidolytically, preferably by treatment with liquid HF. The crude product was purified by cell chromatography on supports used in peptide chemistry, preferably supports acting as molecular sieves. The peptides were characterized by amino acid analysis and their purity was verified by chromatographic and electrophoretic methods.

The process of preparing solid phase peptides in the ultrasonic field of the invention accelerates reactions and washing operations on a polymeric support, allows complete conversion in a short time, simplifies peptide preparation, and saves material (reduced reagent, solvent and catalyst consumption).

The following examples of the process for preparing solid phase peptides in the ultrasonic field of the invention are illustrative, but not limiting.

Example 1

400 mg of p-methylbenzhydrylamine copoly (styrene-1% divinylbenzene) resin (amino group content 0.4 meq / g) was charged to a flow reactor (10 ml internal volume). The synthesis was carried out on a manually operated synthesizer of its own design (see the author's certificate no. 262081) by the continuous flow method according to the author. The flow reactor was placed in a beaker filled with DMF so that the entire contents of the reactor were immersed in liquid. Similarly, the β-reactor beaker was placed in an ultrasonic bath filled with water, the α-amino groups were protected with the Boc group, the side chains of the multifunctional amino acids were protected with benzyl-type groups. Arg with a p-toluenesulfone group. Protected amino acids were activated for condensation by HOBt esters. One synthetic cycle included the following steps:

CS 271584 B1 (1) cleavage of Boc protecting groups, 40% TFA in DCM, 30 min (3 min wash, 27 min at rest):

(2) washing, DCM, to a weakly acidic effluent (controlled by moistened pH paper);

(3) neutralization, 5% DIEA in chloroform, 3 min;

(4) washing, DMF, to neutral effluent;

(5) coupling: protected amino acid, 0.8 meq in DCM (or DCM: DMF, 4: 1 if necessary), add HOBt, 0.8 meq in DMF, add DCC, 0.8 meq in DCM, 30 min at 20 ° C, filter Ν, Ν-dicyclohexylurea, wash DCM, evaporate DCM under reduced pressure (t <25 ° C), dilute DMF, filter, wash DMF, make DMF to 0.2 M, add a few drops Of a 0.001 M solution of bromophenol blue in DMF (70 mg / 100 ml), do not spray into the flow reactor, allow to react until the acid-base indicator changes from blue to yellow;

(6) wash, DMF, 5 min.

The flow rate during the flushing cycles was about 20-30 ml / min, the inert gas overpressure was 20-50 kPa. The synthesis was terminated by cleavage of the Boc group from the last condensed amino acid (step (1)) of the peptidyl resin was washed with DCM, methanol and dried under a stream of nitrogen. Yield 920 mg. The crude peptide was obtained after cleavage of the peptidyl resin with liquid hydrogen fluoride. After removal of HF, the product and resins were suspended in ethyl acetate, filtered, washed with ethyl acetate, and the product extracted into 20% acetic acid and lyophilized. Yield 330 mg. The crude peptide was purified on a column (2.6 x 100 cm) containing as a stationary phase a polyamide gel (Biogel P4), a mobile phase of 20% acetic acid in water. Fractions containing homogeneous product were pooled and lyophilized. The resulting product had an amino acid analysis is consistent with the theoretical values, the purity of the product was confirmed by analytical HPLC (Rt 4.5 min, mobile phase 50% MeOH, 50% water containing 0.1% TFA), _{characterized} by thin layer chromatography (eluent: n-butanol : acetic acid: water - 4: 1: 1) and electrophoresis on paper at pH 2.5.

Yield 94 mg Gly-Glu-Arg-Asp-Arg-Aep-Arg-Ser-Ile-Arg-Leu-Val-A8n-Gly-Ssr-Leu-NH2

Example 2

Following the procedure of Example 1, peptide Ib was synthesized. A p-methylbenzyl group was used to protect the cysteine side group, the peptidyl resin yield was 820 mg, the crude product yield 262 mg. The resulting product had an amino acid analysis consistent with the theoretical values, the purity of the product was confirmed by analytical HPLC (Rt 4.5 min, mobile phase 40% MeOH, 60% water containing 0.1% TFA), thin layer chromatography (n-butanol system) acetic acid: water -4: 1: 1) and electrophoresis on paper at pH 2.5.

Yield 132.5 mg H-Cys-Val-Pro-Thr-Aep-Pro-Asn-Pro-Gln-Glu-Val-Val-HH2

Example 3 ’

The peptide Ic was synthesized according to the procedure of Example 1. A p-methylbenzyl group was used to protect the cysteine side group. The yield of peptidyl resin was 1,005 mg, the yield of crude product was 34-6 mg. The resulting product had an amino acid analysis consistent with theoretical values, the purity of the product was confirmed by analytical HPLC (Rt 6.0 min, mobile phase 45% MeOH, 55% water containing 0.1% TFA), thin layer chromatography (n-butanol system) acetic acid: water -4: 1: 1) and electrophoresis on paper at pH 2.5.

Yield 201 mg H-Ile-Asn-Cys-Thr-Arg-Pro-Asn-Asn-Asn-Thr-Arg-Lye-Ser-Ile-Arg-Ile

Example 4

The peptide Id was synthesized according to the procedure of Example 1. · Formyl group was used to try the side function of tryptophan · Peptidyl-resin yield was 945 mg, crude product yield 401 mg · The resulting product had amino acid analysis equal to β theoretical, purity The product was confirmed by analytical HPLC (Rt 7.5 min, mobile phase 45% MeOH, 55% water containing 0.1% TPA), thin layer chromatography (n-butanol: acetic acid: water · 4: 1: 1 system) ) and electrophoresis on paper at pH 2.5 ·

Yield: 199 mg H-Gly-Gly-Gly-Asp-Met-Arg-Asp-Asn-Trp-Arg-Ser-Glu-Leu-Tyr-NH2

Example 5

The peptide Ιβ was synthesized according to the procedure of Example 1. · Forjyl group was used to protect the lateral function of tryptophan · peptidyl resin yield 840 mg, crude product yield 368 mg · The resulting product had amino acid analysis consistent with theoretical values, product purity was confirmed by analytical HPLC (Rt 7.5 min, mobile phase 55% MeOH, 45% water containing 0.1% TPA), thin layer chromatography (n-butanol: acetic acid: water -4: 1: 1 system) and electrophoresis on paper at pH 2.5 *

Yield 186 mg H-Val-Pro-Trp-Asn-Ala-Ser-Trp-Ser-Asn-Lys-Ser-Leu-Glu-Gln-Ile-NH2

Example 6

Chloromethylated copoly (atyrene-1% divinylbenzene) resin (2.0 g, 0.8 meq / g active chlorine content) was esterified with KP by Boc glycine (Gly content 0.76 meq / g) and placed in a reaction vessel self-built automatic synthesizer · One synthetic cycle included the following steps:

(1) Cleavage of Boc protecting groups, 40% TPA in DCM, 1x5 min, 1 x 30 min?

(2) wash, DCM, 3x2 min:

(3) neutralization, 5% DIEA in chloroform, 2x3 min:

(4) wash, DCM, 3x2 min:

(5) condensation: protected amino acid, 5 mmol in DCM (or DCM: DMP, 4: 1 if necessary), add HOBt · 5 mmol in DMP, add DCC, 5 mmol in DCM, 30 min at 20 ° C, filter N , N-dicyclohexylurea, wash with DCM, evaporate with DCM under reduced pressure.

(t <25 ° C), dilute DMP, filter, wash DMP, make DMP to 0.2 M, add a few drops of a 0.001 M solution of bromophenol blue in DMP (70 mg / 100 ml), allow to react until the acid-base indicator blue to yellow color:

(6) wash, DMP, 2x2 min:

(7) wash, DCM, 3x2 min:

The following amino acids were condensed in each cycle: Вос-Leu, BocPro, Boc-Cys (Bzl), Boc-Asn, Boc-Gln, Boc-Ile, Boc-Tyr and Boc-Cys (Bzl) · Synthesis was terminated by cleavage of the Boc group from the last condensed amino acid (step 1) and the peptidyl resin was washed with DCM, methanol and dried with a dry nitrogen stream · Yield was 3.8 g · Crude peptide was obtained after ammonolysis of peptidyl resin, protecting groups were removed with sodium in liquid ammonia removal of liquid ammonia was the product

CS 271584 B1 oxidized with 0.01 M potassium ferricyanide solution at pH 6.8 (30 min). The acidified solution (pH 3-4) was desalted on an IRA 400 column in an H + cycle. Yield 1.18 g. The desalted crude peptide was purified on a column (2.6 x 100 cm) containing as a stationary phase a polyamide gel (Biogel P4), mobile phase 20% acetic acid in water. Fractions containing a homogeneous product were pooled and lyophilized. The resulting product had an amino acid analysis consistent with theoretical values. The purity of the product was confirmed by analytical HPLC (Rt 4.5 min, mobile phase 50% MeOH, 50% water containing 0.1% TFA), thin layer chromatography (n-butanol: acetic acid: water - 4: 1 system): 1) and electrophoresis on paper at pH 2.5.

Yield 940 mg

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2

Example 7

400 mg of p-methylbenzhydrylamine copoly (styrene-1% divinylbenzene) resin (content of free amino groups 0.4 meq / g) was placed in two flow reactors (10 ml internal volume). The reactors were coupled and two peptides of the same sequence were prepared according to the procedure described in Example 1, except that only in the first reactor the reactions were accelerated by ultrasound. Approximately 1/8 of the peptidyl resin was removed from both reactors and another amino acid Boc-Nle was coupled to the residue. After cleavage of the Boc groups, the crude peptides were obtained by hydrogen fluoride cleavage. 22.6 mg of Leu-Gln-Pro-Gln-Asn-Pro-Ser-Gln-Gln-Gln-Pro-Gln-NH2 from reactor 1 and 22.4 mg of Leu-Gln-Pro-Gln-Asn-Pro were obtained. -Ser-Gln-Gln-Gln-Pro-Gln-NH 2 from reactor 2; 175.2 mg of Nle-Leu-Gln-Pro-Gln-Asn-Pro-Ser-Gln-Gln-Gln-Gln-Pro-Gln-NH2 from Reactor 1 and 180.5 mg of Nle-Leu-Gln-Pro-Gln-Asn -Pro-Ser-Gln-Gln-Gln-Pro-Gln-NH2 from Reactor 2.

The products had amino acid analyzes consistent with theoretical values. Purity was determined by HPLC. The following conditions were used for the determination:

250 x 4 mm column packed with C18 reverse phase, flow rate 0.8 ml / min, solvent A 90% acetonitrile in water with 0.1% TFA addition, solvent 5% acetonitrile in water with 0.1% TFA addition «gradient profile in% AvB: 0-5 min 0.1%, 5 to 95 min 0.1 to

35.2%, 95 to 115 min 35.2 to 58.8%, 115 to 116 min. 58.8 to 99.9%, 116 to 122 min 99 »9%, 122 to 123 min 0.1%.

Analysis revealed that the peptides compared were highly pure, identical substances with the same elution profile. The same results were obtained with thin layer chromatography and electrophoresis. The savings of ultrasonic synthesis are shown in the table:

Table

Evaluation of comparative synthesis

Monitored data Synthesis with ultrasound without ultrasound Savings Condensation time 210 min 520 min 310 min Number of condensation repetitions lx 4x Consumption of chemicals: DCM 1300 ml 1 600 ml 300 ml DMP 650 ml 800 ml 150 ml Act. component 14 mmol 17 mmol 3 mmol Yield 100% 47.9% 46,8%

CS'271584 B1

Explanation of abbreviations used in the text:

HPLC high performance liquid chromatography TPA trifluoroacetic acid DCM dichloromethane DMP N, N-dimethylformamide HOBt N-hydroxybenzotriazole Boc t-butyloxycarbonyl DIEA ethyl diisopropylamine MeOH metha nol DCC N, N'-dicyclohexylcarbodiimide HP hydrogen fluoride Arg arginine Asp aspartic acid Cys cysteine Gin glutamine Gly glycine Glu glutamic acid Ile isoleucine Leu leucine Lys lysine Met methionine For proline Ser serine Thr threonine Trp tryptophan Wall valine

SUBJECT OF THE INVENTION

Claims (1)

SUBJECT OF THE INVENTION Process for the preparation of solid phase peptides, characterized in that the individual reaction steps of successive construction of the peptide chain are carried out in an ultrasonic field.