RU2458066C1 - Method for producing peptide exenatide - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and chemical technology. What is offered is a method for producing the peptide Exenatide. The peptide Exenatide prepared by synthesis may be used for producing drugs for type 2 diabetes mellitus.

EFFECT: higher yield of the unpurified peptide (42%-60%), as well as higher purity (58%-75%) and content of the end product in a mixture (33%-42%); besides, a cheaper condensing agent (DIC) is used at all the condensation stages, while the synthesis of more than one short fragments simultaneously enables faster end peptide process.

3 cl, 2 tbl, 8 ex

Description

The invention relates to the field of medicine and chemical technology, can be used in the manufacture of medicines for the treatment of type 2 diabetes.

Diabetes mellitus is a group of metabolic diseases characterized by pathologically elevated levels of glucose in the blood, which occurs due to defects in insulin secretion, insulin action, or both defects. The number of patients with diabetes in the world at the end of 1999 was 120-140 million people, and this number is predicted to double by 2025 [1].

Treatment for diabetes is to maintain normal blood glucose levels. Various drugs are used for this purpose, for example, sulfonylureas, metformin and insulin. Exenatide is one of the drugs, the effectiveness of the treatment of which non-insulin-dependent diabetes mellitus is proven in clinical trials in humans.

Exenatide is a polypeptide, originally known as exendin-4 (exendin-4), the method of isolation of which from the venom of the lizard Heloderma suspectum and the amino acid sequence HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS-NH ₂ (1), were first published in April 1992 [2].

Exenatide has a hypoglycemic effect and is a drug against type 2 diabetes [3].

It is known [4] that a polypeptide of formula (1) can be obtained by the solid-phase method using Rink amide resins (Rink amide, Rink amide AM, Rink amide MBHA) according to the Fmoc / t-Bu strategy by sequential chain extension of one amino acid. The disadvantage of this method is the use of expensive polymer carriers, as well as a long synthesis time by sequential chain extension.

A known method for producing a polypeptide of the formula (1) [5] by the solid-phase method using Sieber amide resin according to the Fmoc / t-Bu strategy by sequential chain extension by one amino acid. The disadvantages of this method are the use of an expensive polymer carrier and a long synthesis time by sequential chain growth, as well as the use of expensive reagents (HATU and HOAt) for attaching the first amino acid.

The disadvantages of the method of producing exenatide by sequential chain growth [4, 5] is the possibility of doubling and / or elimination of one of the amino acids of the sequence, as well as side processes in the lateral functional groups, which complicates the purification of the target product, and, as a consequence, low yield [6 ]. The disadvantages of this method include the fact that with the sequential chain extension of long peptides, the formation of ordered structures [6] and / or aggregation of growing chains is possible, which complicates the synthesis of the target product.

A known method of producing a polypeptide of the formula (1) [7] by the combined method using both solid-phase synthesis according to the Fmoc / t-Bu strategy and classical in solution using pseudoproline. The patent provides a synthetic scheme (10) for preparing exenatide from a first peptide fragment (12), a second peptide fragment (14), and a third peptide fragment (16). The fourth peptide fragment (20) is obtained from the third fragment by a condensation reaction with serine amide. Then, the fourth fragment is combined with the second fragment to obtain the fifth fragment (22). Ultimately, the fifth fragment is combined with the first to obtain the target peptide. It is known [7] that the key problem in solid-phase and solution synthesis of exenatide is the presence of a sequence of three glutamic acid residues at positions 15, 16, 17. It is especially difficult to carry out chemical synthesis of peptide fragments provided that they contain several consecutive glutamic acid residues, since the sequence of repeating glutamic acid residues tends to form a regular structure. This greatly complicates the effective continuation of the construction of the fragment through the glutamine chain.

In the patent [7] it is indicated that long peptide fragments including a repeating glutamine sequence can be easily obtained if the fragments also contain one or more pseudoproline residues as a substitute for two corresponding amino acid residues. These proposed pseudoproline are dipeptides containing at least one amino acid selected from the serine-threonine group, where the side chain is protected by an oxazolidine ring.

In the patent [7] it is indicated that fragment (12) has at least one pseudoproline and is X ^{j, k} exenatide (1-m), where 1 X ^{j, k} is a pseudoproline residue, m ranges from 15-20 and C-terminal the amino acid of fragment (12) is numbered in accordance with the numbering of amino acid residues in exenatide.

The second peptide fragment (14) is exenatide (nq), where n is m + 1 (m is defined previously according to the first fragment from 15 to 20), and q has a number from 25 to 30. The third peptide fragment (16) is considered as exenatide (q + 1-38).

The disadvantage of this method is the use of pseudoproline in the synthesis of exenatide fragments, which significantly increases the cost of the target product.

The closest in technical essence and the achieved result to the proposed method is a method for producing the polypeptide (1) by the combined method using both solid-phase synthesis according to the Fmoc / t-Bu strategy and classical in solution [6]. According to this patent, the entire exenatide sequence is divided into four shorter fragments: 1-10, 11-21, 22-29 and 30-39 (the N- and C-terminal amino acids of the fragments are numbered according to the numbering of amino acid residues in the exenatide).

Known fragment 1-10 Boc-His ¹ (Trt) -Gly-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Leu ¹⁰ -OH (sequence No. 11) was obtained by solid phase synthesis according to the Fmoc / t-Bu strategy on a 2-chloro-trityl chloride resin using Fmoc-Asp (OtBu) -OPfp and condensing agents such as DIC / HOBt and PyBOP. A piperidine / DBU mixture in DMF or NMP was used to remove the temporary Fmoc-protecting group. A 3.0-0.5% solution of TFA in dichloromethane was used to cleave the obtained fragment from the polymer support. The target peptide of sequence No. 11 was obtained with a yield of 99% and a purity of 95%.

Known Fragment 11-21 Fmoc-Ser (tBu) -Lys (Boc) -Gln (Trt) -Met-Glu ¹⁵ (OtBu) -Glu (OtBu) -Glu (OtBu) -Ala-Val-Arg ²⁰ (Pbf) - Leu-OH (sequence No. 8) was obtained analogously to fragment 1-10 using TOTU as a condensing agent upon addition of Fmoc-Arg (Pbf) -OH and DIC / HOBt for addition of other amino acids. The target peptide of sequence No. 8 was obtained with a yield of 104% and a purity of 94%.

Known fragment 22-29 Fmoc-Phe-Ile-Glu (OtBu) -Trp (Boc) -Leu-Lys (Boc) -Asn (Trt) -Gly-OH (sequence No. 3) was obtained similarly to fragment 1-10 using Fmoc -Asn (Trt) -OPfp and DIC / HOBt to attach other amino acids. The target peptide of sequence No. 3 was obtained with a yield of 93% and a purity of 95%.

Known fragment 30-39 Fmoc-Gly-Pro-Ser (tBu) -Ser (tBu) -Gly-Ala-Pro-Pro-Pro-Ser (tBu) -NH ₂ (sequence No. 4) was obtained by two different methods.

The first method: the known fragment 30-38 Fmoc-Gly-Pro-Ser (tBu) -Ser (tBu) -Gly-Ala-Pro-Pro-Pro-OH (sequence No. 12) was obtained on a 2-chloro-trityl chloride resin using Fmoc-Pro-Pro-OH dipeptide to attach to the polymer carrier, DIC / HOBt as a condensing agent, and a solution of 25% piperidine with 2.5% HOBt in DMF to remove the temporary Fmoc-protecting group. A 3.0-0.5% solution of TFA in dichloromethane was used to cleave the obtained fragment from the polymer support. The target peptide of sequence No. 11 was obtained with a yield of 106% and a purity of 97%.

Condensation of the known fragment 30-38 with H-Ser (tBu) -NH ₂ was carried out in solution using TOTU as a condensing agent. The target peptide of sequence No. 4 was obtained with a yield of 92% and a purity of 97%.

The second method: the known fragment 30-39 Fmoc-Gly-Pro-Ser (tBu) -Ser (tBu) -Gly-Ala-Pro-Pro-Pro-Ser (tBu) -NH ₂ (sequence No. 4) was obtained by solid-phase method with using Sieber amide resin according to the Fmoc / t-Bu strategy. TCTU and Cl-HOBt were used as a condensing agent, and the Fmoc-Pro-Pro-OH dipeptide fragment was used for chain extension. The target peptide of sequence No. 4 was obtained with a yield of 60% and a purity of 78%.

Known 22-39 Fmoc-Phe-Ile-Glu (OtBu) -Trp (Boc) -Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Ser (tBu) -Gly -Ala-Pro-Pro-Pro-Ser (tBu) -NH ₂ (sequence No. 6) was obtained in NMP solution by condensation of fragments No. 3 and No. 4 in a 1: 1 molar ratio with preliminary release of the Fmoc-protective group of fragment No. 4 (yield 99%, purity 91%). As the condensing agent used TBTU. The target peptide of sequence No. 6 was obtained with a yield of 100% and a purity of 89%.

Known 11-39 Fmoc-Ser (tBu) -Lys (Boc) -Gln (Trt) -Met-Glu ¹⁵ (OtBu) -Glu (OtBu) -Glu (OtBu) -Ala-Val-Arg ²⁰ (Pbf) - Leu-Phe-Ile-Glu (OtBu) -Trp (Boc) -Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Ser (tBu) -Gly-Ala-Pro- Pro-Pro-Ser (tBu) -NH ₂ (sequence No. 9) was obtained in solution by condensation of fragments No. 8 and No. 6 in a 1: 1 molar ratio with preliminary release of the Fmoc-protecting group of fragment No. 6 (100% yield, 87% purity ) TOTU was used as a condensing agent. The target peptide of sequence No. 6 was obtained with a yield of 96% and a purity of 80%.

Known fragment 1-39 Boc-His ¹ (Trt) -Gly-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Leu ¹⁰ -Ser (tBu ) -Lys (Boc) -Gln (Trt) -Met-Glu ¹⁵ (OtBu) -Glu (OtBu) -Glu (OtBu) -Ala-Val-Arg ²⁰ (Pbf) -Leu-Phe-Ile-Glu (OtBu) -Trp (Boc) -Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Ser (tBu) -Gly-Ala-Pro-Pro-Pro-Ser (tBu) -NH ₂ (sequence No. 1) was obtained in solution by condensation of fragments No. 11 and No. 9 in a 1: 1 molar ratio with preliminary release of the Fmoc-protecting group of fragment No. 9 (yield 98%, purity 78%). PyBOP was used as a condensing agent. The target peptide of sequence No. 6 was obtained with a yield of 100% and a purity of 79%.

The release of the temporary protective Fmoc group was carried out in suitable solvents, such as CH ₂ Cl ₂ , NMP or DMF with the addition of diethylamine. The selection of fragments was carried out using DIPE.

Complete release was carried out in a TFA solution containing water, TIS, NH ₄ I and DMS. The final mixture was obtained with a total yield of 42% and a target peptide content of 33%.

The disadvantage of this method of obtaining a mixture containing a polypeptide of the formula (1) is a low yield (42%) at the last stage of synthesis and a low content (33%) of the target peptide in the product. Another disadvantage of this method is the use of various condensing agents, such as TOTU, TBTU, TCTU, PyBOP, as well as pentafluorophenyl esters of aspartic acid and asparagine, which complicate the production process and release allergens and carcinogens during the reaction [8, 9]. The use of such reagents and the dipeptide fragment of Fmoc-Pro-Pro-OH increases the cost of production of the drug. It is also undesirable to use explosive DIPE in several stages of synthesis (table 1).

Table number 1 Reagent comparison table Synthesis stage Used reagents according to the method [6] Reagents Used Getting Fragments OPfP esters, PyBOP, TOTU, DIC / HOBt Dic / hobt Fragment condensation TBTU, TOTU, RUVOR Dic / hobt Selection after release Dipe MTBE

In addition, the use of four long peptide fragments (8, 10, and 11 amino acid residues) as starting compounds for the synthesis of the entire sequence can lead to problems that exist in the method of sequential chain extension of one amino acid, namely: a long synthesis time, possible doubling and / or exclusion of one of the amino acids of the sequence, side processes in the lateral functional groups.

The technical result of the claimed invention is to increase the yield (42% -60%), purity (58% -75%) and the content of the target product in the mixture (33% -42%), the use of cheaper condensing agent (DIC) at all stages of condensation ( table 1) and process acceleration. The specified technical result is achieved by the method consisting in the condensation of peptide fragments of the compounds of formula (1) according to scheme 34 + 5, according to which the protected polypeptide of the formula (2) Boc-His ¹ (Trt) -Gly-Glu (OtBu) -Gly-Thr (tBu ) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Leu ¹⁰ -Ser (tBu) -Lys (Boc) -Gln (Trt) -Met-Glu ¹⁵ (OtBu) -Glu (OtBu) - Glu (OtBu) -Ala-Val-Arg ²⁰ (Pbf) -Leu-Phe-Ile-Glu (OtBu) -Trp (Boc) -Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Ser (tBu) -Gly-OH is condensed in solution with a pentapeptide of formula (3) H-Ala-Pro-Pro-Pro-Ser-NH ₂ obtained by the release of a protected pentapeptide of formula (4) X-Ala-Pro- Pro-Pro-Ser (tBu) -NH ₂ , where X may be an Fmoc, Boc or Z protecting group. In the condensation of polypeptide (2) and pentapeptide (3), condensation methods common in peptide synthesis, for example, the carbodiimide method, can be used.

The polypeptide of formula (2) can be obtained by conventional methods of solid-phase synthesis, for example, stepwise condensation of amino acids or fragment condensation using carbodiimides or other condensing agents. In this case, the polypeptide of formula (2) is divided into 5 shorter fragments 1-9 Fmoc-His (Trt) -Gly-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) - Asp (OtBu) -OH (formula 5), 10-14 Fmoc-Leu ¹⁰ -Ser (tBu) -Lys (Boc) -Gln (Trt) -Met-OH (formula 6), 15-20 Fmoc-Glu ¹⁵ ( OtBu) -Glu (OtBu) -Glu (OtBu) -Ala-Val-Arg ²⁰ (Pbf) -OH (formula 7), 21-24 Fmoc-Leu-Phe-Ile-Glu (OtBu) -OH (formula 8) and 25-34 Fmoc-Trp (Boc) -Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Ser (tBu) -Gly-OH (formula 9). All fragments can be obtained by known methods, for example, by solid-phase synthesis using various polymeric carriers and activating agents. In this case, the fragments were obtained on a 2-chloro-trityl chloride resin using DIC / HOBt as a condensing agent. The polypeptide of formula (2) was obtained by the convergent solid phase method by condensing the above fragments on a 2-chloro-trityl chloride resin using DIC / HOBt as a condensing agent in high yield and purity.

The small length of the peptide fragments (from 4 to 10 amino acid residues) allows the use of a polymer carrier with a high load and not the use of expensive condensing agents without loss as the resulting peptides. Thanks to the use of 6 fragments, the synthesis of which can be carried out in parallel, at comparable costs (table 2), the time of complete synthesis is reduced, the final mixture can be obtained with a higher yield and content of the polypeptide of the formula (1).

Comparison Table No. 2 The number of moles of amino acids spent on synthesis Amino acid The number of moles of AK in 1 mole of peptide (1) Spent moles for synthesis by the method [6] Moles spent on synthesis Ala 2 3-4 5 Arg one 1.5-2 2.4 Asn one 1.5-3 1.5 Asp one 2 2.4 Ile one 1.5-3 3 Gln one 1.5-2 3 Glu 5 7.5-10 14.4 Gly 5 7.1-11.1 7.2 His one 1.5-3 1.5 Leu 3 2.7-5.2 6.5 Lys 2 3-5 4.5 Met one 1.5-2 2.4 Phe 2 3-6 6 Pro four 5.2-6.2 10.5 Ser 5 7-8.5 12 Trh 2 3-6 6 Trp one 1.5-3 1.5 Val one 1.5-2 3

Qualitative reactions

Kaiser test. A small amount of resin was washed several times with ethanol. 2 drops of the following solutions were added to the resin: 5% ninhydrin in ethanol, 80% phenol in ethanol and KCN in pyridine (2 ml of 0.001 M KCN in 98 ml of pyridine). The mixture was heated to 120 ° C. Blue staining of the resin indicated the presence of a free amino group.

The completeness of the condensation in the solid-state synthesis method was also controlled using the indicator of bromphenol blue. 3 drops of a 1% solution of bromphenol blue in dimethylacetamide (DMA) were added to the reaction mixture. As the reaction progressed, the color of the solution changed from blue to yellow.

Estimation of the landing of the first amino acid on the resin

Landing was estimated by the amount of dibenzofulven released as a result of the release of the Fmoc group. For this, a solution of dibenzfulvene in N, N′-dimethylformamide, obtained by the release and washing of the polymer support, was chromatographically analyzed. The dibenzfulven concentration was calculated from its peak area using a calibration curve.

Analytical high performance liquid chromatography was performed on a KNAUER Smartline 2500 chromatograph equipped with a spectrophotometric detector at a wavelength of 214 nm. For analytical HPLC, a Waters X-Bridge C18, 5 μm, 4.5 × 10 mm column was used at an eluent flow rate of 1 ml / min.

Example 1

Solid-phase synthesis of the claimed fragment 1-9 Fmoc-His (Trt) -Gly-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -OH (formula 5) .

In a solid-phase reactor, 2 g of 2-chlorotrityl chloride resin (1.5 mmol / g capacity) in 15 ml of DCM were suspended, held for 5 minutes and the resin was filtered off. A solution of 1.42 g (3.3 mmol) of the C-terminal amino acid FmocAsp (OtBu) OH and 2 ml (12.0 mmol) of DIPEA in 10 ml of DCM was added to the resin, stirred for 2 hours at room temperature. The resin was filtered and washed with 2 × 10 ml DCM. The resin was treated with 2 × 10 ml of a mixture of DCM / methanol / DIPEA (17: 2: 1) for 10 min, washed with 2 × 10 ml of DCM and 3 × 10 ml of DMF. 10 ml of a 20% solution of diethylamine in DMF were charged into the reactor, stirred for 5 minutes and filtered; this operation was repeated again for 20 minutes, after which the resin was washed with 5 × 10 ml of DMF. (Step 4) A cooled solution of 6.0 mmol of the following protected amino acid, 0.98 g (7.2 mmol) of HOBt and 1.12 ml (7.2 mmol) of DIC in 10 ml of DMF was added to the resin, stirred at room temperature. The completeness of the reaction was monitored by changing the color of bromphenol blue. The resin was filtered, washed with 6 × 10 ml of DMF. (Operation 5) Operations 4 and 5 were repeated until the desired sequence was obtained. Then the resin was washed with 2 × 10 ml of DCM, treated with 30 × 10 ml of 1% TFA in DCM; the resulting solutions were combined in a flask containing 40 ml of a 10% solution of pyridine in methanol. The mixture was concentrated on a rotary evaporator, the product was precipitated with water, filtered off, washed with plenty of water, and dried in air. The target peptide of formula 5 was obtained with a yield (by planting the first amino acid) of 99% - 4.71 g and a purity of 99 +%.

Example 1a

The load was increased under laboratory conditions to 15 g of resin in the reactor. The addition of the first amino acid Fmoc-Asp (OtBu) -OH to a 2-chlorotrityl chloride resin was carried out in the presence of DIPEA in methylene chloride. Landing was evaluated once chromatographically by the amount of dibenzofulven generated during release. The Fmoc group was removed with a 20% solution of diethylamine in dimethylformamide. The condensations of the following Fmoc amino acids were carried out using DIC / HOBt reagents in dimethylformamide with one and a half fold excesses of activated amino acids. The fragment was cleaved from the substrate by repeated treatment with a 1% solution of TFA in methylene chloride. The target peptide of formula 5 was obtained with a yield of 99% (by planting the first amino acid) and a purity of 99 +%.

Example 2

Solid-phase synthesis of the claimed fragment 10-14 Fmoc-Leu ¹⁰ -Ser (tBu) -Lys (Boc) -Gln (Trt) -Met-OH (formula 6).

In a solid phase reactor, 1.5 g of 2-chlorotrityl chloride resin (1.5 mmol / g) was suspended in 15 ml of DCM, kept for 5 minutes and the resin was filtered off. A solution of 0.81 g (2.3 mmol) of the C-terminal amino acid Fmoc-Met-OH and 1.5 ml (9.0 mmol) of DIPEA in 10 ml of DCM was added to the resin, stirred for 2 hours at room temperature. The resin was filtered and washed with 2 × 10 ml DCM. The resin was treated with 2 × 10 ml of a mixture of DCM / methanol / DIPEA (17: 2: 1) for 10 min, washed with 2 × 10 ml of DCM and 3 × 10 ml of DMF. 10 ml of a 20% solution of diethylamine in DMF were charged into the reactor, stirred for 5 minutes and filtered; this operation was repeated again for 20 minutes, after which the resin was washed with 5 × 10 ml of DMF. A cooled solution of 4.5 mmol of the following amino acid, 0.73 g (5.4 mmol) of HOBt and 0.85 ml (5.4 mmol) of DIC in 10 ml of DMF was added to the resin, stirred at room temperature. The completeness of the reaction was monitored by changing the color of bromphenol blue. The resin was filtered, washed with 6 × 10 ml of DMF. Operations 4 and 5 were repeated until the desired sequence was obtained. Then the resin was washed with 2 × 10 ml of DCM, treated with 20 × 10 ml of 1% TFA in DCM; the resulting solutions were combined in a flask containing 40 ml of a 10% solution of pyridine in methanol. The mixture was concentrated on a rotary evaporator, the product was precipitated with water, filtered off, washed with plenty of water, and dried in air. The target peptide of formula 6 was obtained with a yield of 2.84 g - 105% and a purity of 96%.

Example 2a

The load was increased under laboratory conditions to 15 g of resin in the reactor. A fragment of formula 6 was obtained similarly to fragment 1-9 using DIC / HOBt as a condensing agent. The target peptide of formula 6 was obtained with a yield of 106% and a purity of 95%.

Example 3

Solid-phase synthesis of the claimed fragment 15-20 Fmoc-Glu ¹⁵ (OtBu) -Glu (OtBu) -Glu (OtBu) -Ala-Val-Arg ²⁰ (Pbf) -OH (formula 7).

In a solid-phase reactor, 2 g of 2-chlorotrityl chloride resin (1.5 mmol / g) were suspended in 15 ml of DCM, kept for 5 min and the resin was filtered off. A solution of 1.80 g (3.3 mmol) of the C-terminal amino acid FmocArg (Pbf) OH and 2 ml (12.0 mmol) of DIPEA in 10 ml of DCM was added to the resin, stirred for 2 hours at room temperature. The resin was filtered and washed with 2 × 10 ml DCM. The resin was treated with 2 × 10 ml of a mixture of DCM / methanol / DIPEA (17: 2: 1) for 10 min, washed with 2 × 10 ml of DCM and 3 × 10 ml of DMF. 10 ml of a 20% solution of diethylamine in DMF were charged into the reactor, stirred for 5 minutes and filtered; this operation was repeated again for 20 minutes, after which the resin was washed with 5 × 10 ml of DMF. A cooled solution of 6.0 mmol of the following amino acid, 0.98 g (7.2 mmol) of HOBt and 1.12 ml (7.2 mmol) of DIC in 10 ml of DMF was added to the resin, stirred at room temperature. The completeness of the reaction was monitored by changing the color of bromphenol blue. The resin was filtered, washed with 6 × 10 ml of DMF. Operations 4 and 5 were repeated until the desired sequence was obtained. Then the resin was washed with 2 × 10 ml of DCM, treated with 30 × 10 ml of 1% TFA in DCM; the resulting solutions were combined in a flask containing 40 ml of a 10% solution of pyridine in methanol. The mixture was concentrated on a rotary evaporator, the product was precipitated with water, filtered off, washed with plenty of water, and dried in air. The target peptide of formula 7 was obtained with a yield of 2.13 g - 98% and a purity of 99%.

Example 3a

The load was increased under laboratory conditions to 15 g of resin in the reactor. A fragment of formula 7 was obtained similarly to fragment 1-9 using DIC / HOBt as a condensing agent. The target peptide of formula 7 was obtained with a yield of 97% and a purity of 99%.

Example 4

Solid-phase synthesis of the claimed fragment 21-24 Fmoc-Leu-Phe-Ile-Glu (OtBu) -OH (formula 8).

In a solid-phase reactor, 2 g of 2-chlorotrityl chloride resin (1.5 mmol / g) were suspended in 15 ml of DCM, kept for 5 min and the resin was filtered off. A solution of 1.92 g (4.5 mmol) of the C-terminal amino acid FmocGlu (OtBu) OH and 2 ml (12.0 mmol) of DIPEA in 10 ml of DCM was added to the resin, stirred for 2 hours at room temperature. The resin was filtered and washed with 2 × 10 ml DCM. The resin was treated with 2 × 10 ml of a mixture of DCM / methanol / DIPEA (17: 2: 1) for 10 min, washed with 2 × 10 ml of DCM and 3 × 10 ml of DMF. 10 ml of a 20% solution of diethylamine in DMF were charged into the reactor, stirred for 5 minutes and filtered; this operation was repeated again for 20 minutes, after which the resin was washed with 5 × 10 ml of DMF. A cooled solution of 6.0 mmol of the following amino acid, 0.98 g (7.2 mmol) of HOBt and 1.12 ml (7.2 mmol) of DIC in 10 ml of DMF was added to the resin, stirred at room temperature. The completeness of the reaction was monitored by changing the color of bromphenol blue. The resin was filtered, washed with 6 × 10 ml of DMF. Operations 4 and 5 were repeated until the desired sequence was obtained. Then the resin was washed with 2 × 10 ml of DCM, treated with 30 × 10 ml of a 1% solution of TFA in a mixture of DCM / THF (2: 1); the resulting solutions were combined in a flask containing 40 ml of a 10% solution of pyridine in methanol. The mixture was concentrated on a rotary evaporator, the product was precipitated with water, filtered off, washed with plenty of water, and dried in air. The target peptide of formula 8 was obtained with a yield of 2.38 g - 100% and a purity of 99 +%.

Example 4a

The load was increased under laboratory conditions to 15 g of resin in the reactor. A fragment of formula 8 was obtained similarly to fragment 1-9 using DIC / HOBt as a condensing agent. The target peptide of formula 8 was obtained with a yield of 100% and a purity of 99 +%.

Example 5

Solid-phase synthesis of the claimed fragment 25-34 H-Trp (Boc) -Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Ser (tBu) -Gly-OH (formula 9).

In a solid-phase reactor, 1 g of 2-chlorotrityl chloride resin (1.5 mmol / g) was suspended in 8 ml of DCM, kept for 5 minutes and the resin was filtered off. A solution of 0.51 g (2.1 mmol) of the C-terminal amino acid FmocGlyOH and 1 ml (6.0 mmol) of DIPEA in 8 ml of DCM was added to the resin, stirred for 2 hours at room temperature. The resin was filtered and washed with 2 × 10 ml DCM. The resin was treated with 2 × 10 ml of a mixture of DCM / methanol / DIPEA (17: 2: 1) for 10 min, washed with 2 × 10 ml of DCM and 3 × 10 ml of DMF. 10 ml of a 20% solution of diethylamine in DMF were charged into the reactor, stirred for 5 minutes and filtered; this operation was repeated again for 20 minutes, after which the resin was washed with 5 × 10 ml of DMF. A cooled solution of 3.0 mmol of the following amino acid, 0.49 g (3.6 mmol) of HOBt and 0.56 ml (3.6 mmol) of DIC in 10 ml of DMF was added to the resin, stirred at room temperature. The completeness of the reaction was monitored by changing the color of bromphenol blue. The resin was filtered, washed with 6 × 10 ml of DMF. Operations 4 and 5 were repeated until the desired sequence was obtained. Then, 10 ml of a 20% solution of diethylamine in DMF was loaded into the reactor, stirred for 5 min, and filtered; this operation was repeated again for 20 minutes, after which the resin was washed with 5 × 10 ml of DMF. An analytical sample of the resin containing the decapeptide of formula 9 was washed with 3 × 1 ml DCM and treated with a mixture of TFA: TIS: H ₂ O (95: 0.25: 0.25) for two hours. The resulting solution was concentrated on a rotary evaporator, the product was precipitated with MTBE, filtered off, washed with a large amount of MTBE, and dried in air. The purity of the unlocked peptide was 97%.

Example 5a

The load was increased under laboratory conditions to 15 g of resin in the reactor. A fragment of formula 9 was obtained similarly to fragment 1-9 using DIC / HOBt as a condensing agent.

Example 6

Solid-phase synthesis of the claimed fragment 1-34 Boc-His- (Trt) -Gly-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Leu-Ser ( tBu) -Lys (Boc) -Gln (Trt) -Met-Glu (OtBu) -Glu (OtBu) -Glu (OtBu) -Ala-Val-Arg (Pbf) -Leu-Phe-Ile-Glu (OtBu) - Trp (Boc) -Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Ser (tBu) -GlyOH (formula 2).

Example 6a: Sequential Chain Extension

In a solid-phase reactor, 2 g of 2-chlorotrityl chloride resin (1.5 mmol / g) were suspended in 15 ml of DCM, kept for 5 min and the resin was filtered off. A solution of 0.18 g (0.6 mmol) of the C-terminal amino acid FmocGlyOH and 2 ml (12.0 mmol) of DIPEA in 6 ml of DCM was added to the resin, stirred for 2 hours at room temperature. The resin was filtered and washed with 2 × 10 ml DCM. The resin was treated with 2 × 10 ml of a mixture of DCM / methanol / DIPEA (17: 2: 1) for 10 min, washed with 2 × 10 ml of DCM and 3 × 10 ml of DMF. 10 ml of a 20% solution of diethylamine in DMF were charged into the reactor, stirred for 5 minutes and filtered; this operation was repeated again for 20 minutes, after which the resin was washed with 5 × 10 ml of DMF. A cooled solution of 1.2 mmol of the following amino acid, 0.19 g (1.4 mmol) of HOBt and 0.22 ml (1.4 mmol) of DIC in 8 ml of DMF was added to the resin, stirred at room temperature. The completeness of the reaction was monitored by changing the color of bromphenol blue. The resin was filtered, washed with 6 × 10 ml of DMF.

Operations 4 and 5 were repeated until the desired sequence was obtained. Then the resin was washed with 2 × 10 ml of DCM, treated with 30 × 10 ml of 1% TFA in DCM; the resulting solutions were combined in a flask containing 40 ml of a 10% solution of pyridine in methanol. The mixture was concentrated on a rotary evaporator, the product was precipitated with water, filtered off, washed with plenty of water, and dried in air. The target peptide of formula 2 was obtained with a yield of 1.12 g - 51% and a purity of 79%.

Example 6b: Converged Method

To a resin containing a decapeptide of formula 9, a cooled solution of 1.8 mmol of the following fragment 21-24, 0.50 g (3.6 mmol) of HOBt and 0.55 ml (3.6 mmol) of DIC in 8 ml of DMF was added, mixed at room temperature. The completeness of the reaction was monitored by changing the color of bromphenol blue. The resin was filtered, washed with 6 × 10 ml of DMF. Then, 10 ml of a 20% solution of diethylamine in DMF was loaded into the reactor, stirred for 5 min, and filtered; this operation was repeated again for 20 minutes, after which the resin was washed with 5 × 10 ml of DMF.

Operations 4 and 5 were repeated with subsequent protected fragments 15-20, 10-14 and 1-9. Then the resin was washed with 2 × 10 ml of DCM, treated with 30 × 10 ml of 1% TFA in DCM; the resulting solutions were combined in a flask containing 40 ml of a 10% solution of pyridine in methanol. The mixture was concentrated on a rotary evaporator, the product was precipitated with water, filtered off, washed with plenty of water, and dried in air. The target protected peptide of formula 2 was obtained with a yield of 1.4 g - 62% and a purity of 95 +%.

Example 6c

The load was increased under laboratory conditions to 15 g of resin in the reactor. A fragment of formula 2 was obtained using DIC / HOBt as a condensing agent with a yield of 87% and a purity of 95%.

Example 7 Synthesis of a Fragment of H-Ala-Pro-Pro-Pro-Ser-NH ₂ , (Formula 3)

Example 7a: Solid Phase Converged Method

In a solid-phase reactor, 1 g of 2-chlorotritylamine resin (1.5 mmol / g) was suspended in 8 ml of DCM, kept for 5 minutes and the resin was filtered off. A cooled solution of 3 mmol of the first Fmoc amino acid, 3.6 mmol of HOAt and 3.6 mmol of DIC in 8 ml of DMF was added to the resin, stirred for 5 days at room temperature. The resin was filtered, washed with 10 ml of DMF (5 × 1 min). A solution of 4.5 mmol of Ac ₂ O and 4.5 mmol of DIPEA in 8 ml of DMF was added to the resin, stirred for 1 h and filtered. The resin was washed with 10 ml of DMF (5 × 1 min). 10 ml of a 20% solution of Et ₂ NH in DMF was added to the resin, stirred for 5 minutes and filtered. The operation was repeated for 20 minutes. The completion of the release reaction was checked using the Kaiser test. Washed resin D with 10 ml of DMF (5 × 1 min). The landing of the first amino acid was evaluated chromatographically by the amount of dibenzofulven formed when the Fmoc group was removed. A solution of 4.5 mmol of tert-butyloxycarbonyl-prolyl-prolyl-proline, 9 mmol of HOBt and 9 mmol of DIC in 8 ml of DMF was added to the resin, stirred for 160 min at room temperature; the resin was filtered off. The completeness of the condensation was observed by the color of the bromophenol blue indicator. The resin was washed with 10 ml of DMF (5 × 1 min) and 10 ml of DCM (3 × 1 min).

To the resin was added 11 ml of a solution of TFA in DCM (3: 8), stirred for 1 h at room temperature. The resin was filtered, washed with 10 ml of DCM (3 × 1 min), 10 ml of DMF (5 × 1 min), 10 ml of a 10% solution of DIPEA in DMF (3 × 2 min). The completion of the release reaction was checked using the Kaiser test. The resin was washed with 10 ml of DMF (5 × 1 min). A cooled solution of 6 mmol of the last Fmoc amino acid, 6.6 mmol of HOBt and 6.6 mmol of DIC in 8 ml of DMF was added to the resin, stirred for 160 min at room temperature; the resin was filtered off. The completeness of the condensation was observed by the color of the bromophenol blue indicator. The resin was washed with 10 ml of DMF (5 × 1 min). 10 ml of pure TFA was added to the resin, stirred for 12 hours at room temperature. The solution was filtered into a dry flask. The remaining peptide was washed from the resin with 10 ml of pure TFA (3 × 2 min). TFA was distilled off on a rotary evaporator, the residue was poured with MTBE. The precipitate was filtered off, washed with MTBE, and dried in air. The target peptide of formula 3 was obtained with a yield of 0, 30 g - 30% and a purity of 94%.

Example 7b: the Classic method in solution

Synthesis of Boc-Pro-Pro-OH

To a solution of 12.9 g (0.041 mol) of tert-butyloxycarbonyl-proline succinimide ester in 37 ml of dioxane was added with stirring a solution of 9.47 g (0.082 mol) of proline and 3.29 g (0.041 mol) of sodium hydroxide in ml of water, reaction the mixture was left overnight. The solution was evaporated by 2/3, acidified with 1 M sulfuric acid to pH ~ 4. The product was salted out with sodium chloride, extracted with ethyl acetate. An ethyl acetate solution was washed with saturated sodium chloride solution, dried with sodium sulfate. The solvent was distilled off, the residue was crystallized from 40 ml of ethyl acetate. Yield 12.3 g (96%).

Synthesis of Boc-Pro-Pro-OSu

To a solution of 8.35 g (0.027 mol) of Boc-Pro-Pro-OH and 3.41 g (0.030 mol) of N-hydroxysuccinimide in 110 ml of tetrahydrofuran, a cooled solution of 6.13 g (0.030 mol) of dicyclohexylcarbodiimide in 15 ml was added with stirring tetrahydrofuran. After 2 hours, precipitated urea was filtered off. The solution was evaporated, the residue was crystallized from isopropyl alcohol. Yield 10.1 g (91%).

Synthesis of Boc-Pro-Pro-Pro-OH

To a solution of 6.75 g (0.017 mol) of Boc-Pro-Pro-OSu in 25 ml of dioxane was added a solution of 3.83 g (0.034 mol) of proline in 17 ml of 1M sodium hydroxide; the reaction mixture was allowed to stir at room temperature overnight. The reaction mixture was washed with diethyl ether, acidified with 1M sulfuric acid to pH ~ 4. The product was salted out with sodium chloride, extracted with ethyl acetate. An ethyl acetate solution was washed with saturated sodium chloride solution, dried with sodium sulfate. The solvent was distilled off, the residue was crystallized from 40 ml of ethyl acetate. Yield 6.68 g (95%).

Synthesis of TFA * Pro-Pro-Pro-OH

3 g (0.007 mol) of Boc-Pro-Pro-Pro-OH was dissolved in 10 ml of trifluoroacetic acid, the solvent was distilled off after half an hour, 30 ml of isopropyl alcohol was added, and evaporated again. The residue was crystallized from 40 ml MTBE. Yield 3.01 g (94%).

Synthesis of Z-Ala-Pro-Pro-Pro-OH

To a solution of 17.8 g (0.042 mol) of prolyl-prolyl-proline trifluoroacetate and 15.3 ml (0.092 mol) of diisopropylethylamine in 90 ml of dioxane was added a solution of 15.5 g (0.048 mol) of N-benzyloxycarbonyl-alanine in 90 ml of dioxane, left to mix at room temperature overnight. The completeness of the reaction was checked by HPLC. The dioxane was evaporated to dryness, the residue was dissolved in 210 ml of water. The solution was washed with diethyl ether. The product was salted out, extracted with n-butanol. The butanol solution was washed with saturated aqueous sodium chloride, then with distilled water. The solution was evaporated; the residue was dried by evaporation with isopropyl alcohol, crystallized from methyl tert-butyl ether. The product was filtered off, washed with methyl tert-butyl ether, and dried in air. Yield 19.6 (91%).

Synthesis of Z-Ala-Pro-Pro-Pro-Ser (tBu) -NH ₂

To a cooled solution of 17.5 g (0.034 mol) of benzyloxycarbonyl-alanyl-prolyl-prolyl-proline and 5.8 g (0.036 mol) of O-tert-butyl-seryl amide in 100 ml of tetrahydrofuran, a cooled solution of 8.2 g ( 0.040 mol) N, N′-dicyclohexylcarbodiimide in 40 ml of tetrahydrofuran. The reaction mixture was left for two days at room temperature, the completeness of the reaction was checked by HPLC. The precipitated dicyclohexylurea was filtered off, washed with tetrahydrofuran, and the solvent was distilled off on a rotary evaporator. The residue was crystallized from methyl tert-butyl ether. The product was filtered off, washed with methyl tert-butyl ether, and dried under vacuum. Yield 20.7 g (92%).

Synthesis of H-Ala-Pro-Pro-Pro-Ser (tBu) -NH ₂

To a solution of 16.2 g (0.025 mol) of Z-Ala-Pro-Pro-Pro-Ser (tBu) -NH ₂ in 500 ml of methanol was added a catalyst - 10% palladium-carbon, hydrogen was passed with stirring. The progress of the reaction was monitored by HPLC. After filtration, the solvent was distilled off on a rotary evaporator, the residue was dried by evaporation with isopropyl alcohol. Yield 12.7 g (99%), purity 97%.

Synthesis of Z-Ala-Pro-Pro-Pro-Ser-NH ₂

To a solution of 0.66 g (0.0017 mol) of trifluoroacetate TFA * Pro-Pro-Pro-Ser-NH ₂ and 0.64 g (0.0020 mol) of Z-Ala-OSu in 20 ml of DMF was added 0.37 ml (0.0034 mol) of N-methylmorpholine was left overnight at room temperature. The completeness of the reaction was checked by HPLC. The solvent was distilled off on a rotary evaporator, the residue was poured with water, and extracted with n-butanol. The solution was evaporated, the residue was crystallized from methyl tert-butyl ether. The precipitate was filtered off, washed with methyl tert-butyl ether, and dried in air. Yield 0.92 g (92%).

Synthesis of Fmoc-Ala-Pro-Pro-Pro-Ser-NH ₂

To a solution of 0.57 g (0.0014 mol) of TFA * Pro-Pro-Pro-Ser-NH ₂ and 0.65 g (0.0016 mol) of Fmoc-Ala-OSu in 20 ml of dimethylformamide was added 0.31 ml ( 0.0028 mol) of N-methylmorpholine, left overnight at room temperature. The completeness of the reaction was checked by HPLC. The solvent was distilled off on a rotary evaporator, the residue was precipitated from water, and extracted with n-butanol. The solution was evaporated, the residue was crystallized from methyl tert-butyl ether. The precipitate was filtered off, washed with methyl tert-butyl ether, and dried in air. Yield 0.91 g (95%).

Synthesis of Boc-Ala-Pro-Pro-Pro-Ser-NH ₂

To a solution of 0.70 g (0.0018 mol) of TFA * Pro-Pro-Pro-Ser-NH ₂ and 0.56 g (0.0020 mol) of Boc-Ala-OSu in 20 ml of DMF was added 0.40 ml ( 0.0036 mol) of N-methylmorpholine, left overnight at room temperature. The completeness of the reaction was checked by HPLC. The solvent was distilled off on a rotary evaporator, the residue was precipitated from water, extracted with n-butanol, salted out from the aqueous layer and again extracted with n-butanol. The solution was evaporated, the residue was crystallized from MTBE. The precipitate was filtered off, washed with MTBE, and dried in air. Yield 0.98 g (98%).

Synthesis of H-Ala-Pro-Pro-Pro-Ser-NH ₂

0.80 g (0.0014 mol) of Boc-Ala-Pro-Pro-Pro-Ser-NH _{2 was} dissolved in a mixture of TFA: dichloromethane 1: 1; after an hour, the solution was evaporated, the residue was crystallized from MTBE, the product was dried under vacuum. Yield 0.65 g (99%), purity 98%).

Example 8

Condensation in solution of a fragment of formula (2) and H-Ala-Pro-Pro-Pro-Ser (tBu) -NH ₂

320 mg (55.2 μmol) of the fragment of formula (2) and 140 mg (221 μmol) of the H-Ala-Pro-Pro-Pro-Ser (tBu) -NH ₂ fragment were dissolved in 5 ml of N-methylpyrrolidone, 30 mg (221 μmol) HOBt and 35 μl (221 μmol) DIC. The progress of the reaction was monitored by HPLC. The reaction mixture was poured with water, the precipitate was filtered off, washed with water and dried in air. Yield 0.32 g (92%).

Example 8a

The condensation in solution of a fragment of the formula (2) and TFA * H-Ala-Pro-Pro-Pro-Ser-NH ₂ (formula 3)

9.0 g (0.00156 M) of the fragment of formula (2) and 1.97 g (0.0031 M) of the TFA * H-Ala-Pro-Pro-Pro-Ser-NH ₂ fragment were dissolved in 100 ml of N-methylpyrrolidone, 0.42 g (0.0031 M) HOBt and 1.02 ml (0.0031 M) DIC. The progress of the reaction was monitored by HPLC. The reaction mixture was poured with water, the precipitate was filtered off, washed with water and dried in air. Yield 9.98 g (99%).

Example 9. The removal of protective groups

10.58 g of the peptide was treated with 200 ml of a mixture of TFA: TIS: EDT: H ₂ O 37: 1: 1: 1 for 2 hours. The reaction mixture was filtered and evaporated on a rotary evaporator. The residue was triturated in MTBE; the solid precipitate was filtered off, dried under vacuum. The product was dissolved in 100 ml of an aqueous solution of 1% acetic acid, kept for 24 hours, lyophilized. The crude peptide of formula 1 was obtained with a yield of 10.02 g - 60% (calculated on the peptide content), a purity of 75% and a target product content of 42%.

As examples 1-9 show, as a result of the proposed synthesis, the yield of the crude peptide of formula 1 is increased (42% -60%), the purity (58% -75%) and the content of the target product in the mixture (33% -42%) also increase. In addition, a cheaper condensing agent (DIC) is used at all stages of condensation (Table 1). The simultaneous synthesis of several short fragments allows you to speed up the process of obtaining the target peptide. The peptide obtained during the synthesis can be used in the manufacture of medicines for the treatment of type 2 diabetes mellitus.

Peptide Formulas:

H-His ¹ -Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu ¹⁰ -Ser-Lys-Gln-Met-Glu ¹⁵ -Glu-Glu-Ala-Val-Arg ²⁰ -Leu-Phe- Ile-Glu-Trp ²⁵ -Leu-Lys-Asn-Gly-Gly ³⁰ -Pro-Ser-Ser-Gly-Ala ³⁵ -Pro-Pro-Pro-Ser-NH ₂ (Formula 1)

Boc-His ¹ (Trt) -Gly-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Leu ¹⁰ -Ser (tBu) -Lys (Boc ) -Gln (Trt) -Met-Glu ¹⁵ (OtBu) -Glu (OtBu) -Glu (OtBu) -Ala-Val-Arg ²⁰ (Pbf) -Leu-Phe-Ile-Glu (OtBu) -Trp (Boc) -Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Ser (tBu) -Gly-OH (formula 2)

H-Ala-Pro-Pro-Pro-Ser-NH ₂ (Formula 3)

X-Ala-Pro-Pro-Pro-Ser (tBu) -NH ₂ , (formula 4), where X = Fmoc, Boc, Z

Fmoc-His (Trt) -Gly-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -OH (formula 5)

10-14 Fmoc-Leu ¹⁰ -Ser (tBu) -Lys (Boc) -Gln (Trt) -Met-OH (formula 6)

15-20 Fmoc-Glu ¹⁵ (OtBu) -Glu (OtBu) -Glu (OtBu) -Ala-Val-Arg ²⁰ (Pbf) -OH (formula 7)

21-24 Fmoc-Leu-Phe-Ile-Glu (OtBu) -OH (Formula 8)

25-34 Fmoc-Trp (Boc) -Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Ser (tBu) -Gly-OH (formula 9)

List of abbreviations

Designation Title Structure Boc tert-butyloxycarbonyl

Bop benzotriazol-1-yl-hydroxy-tris- (dimethylamino) phosphonium hexafluorophosphate

Bzl benzyl

DBU 1,8-diazabicyclo [5.4.0] undec-7-ene

Dic N, N'-diisopropylcarbodiimide

Fmoc 9-fluorenylmethyloxycarbonyl

Hatu O- (1H-benzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium hexafluorophosphate

HBTU O- (1H-7-azabenzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium hexafluorophosphate

HOAt 1-hydroxy-7-azabenzotriazole

Hobt 1-hydroxybenzotriazole

Pbf 2,2,4,6,7-pentamethyldihydro-benzofuran-5-sulfonyl

tBu Tert-butyl

TBTU tetrafluoroborate O- (1H-benzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium

TFA Trifluoroacetic acid CF ₃ COOH Trt Trityl

Z Benzyloxycarbonyl

MTBE Methyl tert-butyl ether

Hosu N-hydroxysuccinimide

EDT Ethandithiol

Dipa Diisopropylethylamine

DMF N, N-dimethylformamide

Information sources

1. Diabetes mellitus, WHO information, Fact Sheet No. 138, Reviewed November (1999).

2. Eng, J. et al., Isolation and Characterization of Exendin-4, an Exendin-3 Analogue, from Heloderma suspectum Venom. J. Biol. Chem., Vol. 267, Issue 11, 7402-7405, Apr, 1992.

3. US 5424286 Exendin-3 and exendin-4 polypeptides, and pharmaceutical compositions comprising the same.

4. CN 101357938A. Method for synthesizing exenatide from solid phase polypeptide.

5. CN 101538324 A. Method for preparing Exenatide.

6. WO 201100664 A2. Process for the production of Exenatide and of an Exenatide analogue (prototype).

7. US2009149628 A1. Insulinotropic peptide synthesis using solid and solution phase combination techniques.

8. H. Gausepohl, et al. in "Peptides, Chemistry and Biology, Proc. 12 ^th American Peptide Symposium."

9. A. Smith and J. E. Rivier (Eds.) ESCOM, Lieden 1992, pp. 523.

Claims (3)

1. The method of producing exenatide peptide (1)
peptide (1) has the formula (I)

characterized in that the peptide (1) is obtained by condensation of two fragments (A) and (B) by classical synthesis in solution; wherein the peptide fragment (A) is a modified part of the peptide (1); in the peptide fragment (A), the N-terminal amino acid is the amino acid position 1 in the peptide (1), the C-terminal amino acid is the amino acid position 34 in the peptide (1);
peptide fragment (A) has an N-terminal urethane-type PGA protecting group in the peptide fragment (A) side functional groups are protected;
peptide fragment (A) has the formula (II)

peptide fragment (A) was obtained by condensation of five peptide fragments (C), (D), (E), (F) and (G) by solid-phase convergent synthesis; the peptide fragment (C) is a modified part of the peptide (1); in the peptide fragment (C), the N-terminal amino acid is the amino acid occupying position 25 in the peptide (1), the C-terminal amino acid is the amino acid occupying position 34 in the peptide (1);
the peptide fragment (C) is attached to a polymer carrier,
the peptide fragment (C) does not have an N-terminal protecting group,
in the peptide fragment (C) lateral functional groups are protected; peptide fragment (C) has the formula (IX)

the peptide fragment (D) is a modified part of the peptide (1);
in the peptide fragment (D), the N-terminal amino acid is the amino acid position 21 in the peptide (1), the C-terminal amino acid is the amino acid position 24 in the peptide (1);
the peptide fragment (D) has an N-terminal urethane type PGD protecting group; side functional groups are protected in the peptide fragment (D);
the peptide fragment (D) has the formula (VIII)

the peptide fragment (E) is a modified part of the peptide (1);
in the peptide fragment (E), the N-terminal amino acid is the amino acid occupying position 15 in the peptide (1), the C-terminal amino acid is the amino acid occupying position 20 in the peptide (1);
the peptide fragment (E) has an N-terminal urethane type PGE protecting group; in the peptide fragment (E), side functional groups are protected;
peptide fragment (E) has the formula (VII)

the peptide fragment (F) is a modified part of the peptide (1);
in the peptide fragment (F), the N-terminal amino acid is the amino acid occupying position 15 in the peptide (1), the C-terminal amino acid is the amino acid occupying position 20 in the peptide (1);
the peptide fragment (F) has an N-terminal urethane type PGF protecting group; side functional groups are protected in the peptide fragment (F);
the peptide fragment (F) has the formula (VI)

the peptide fragment (G) is a modified part of the peptide (1);
in the peptide fragment (G), the N-terminal amino acid is the amino acid occupying position 15 in the peptide (1), the C-terminal amino acid is the amino acid occupying position 20 in the peptide (1);
the peptide fragment (G) has an N-terminal urethane-type PGA protecting group in the peptide fragment (G) side functional groups are protected;
peptide fragment (G) has the formula (V)

then step by step condense the claimed five peptide fragments in the following order:
step one: the peptide fragment (D) is condensed with the peptide fragment (C), resulting in a peptide fragment (DC) having an N-terminal PGD protecting group and attached to a polymer carrier;
peptide fragment (DC) has the formula (X)

step two: release the N-terminal PGD protecting group of the peptide fragment (D);
step three: the peptide fragment (E) is condensed with a peptide fragment (DC), resulting in a peptide fragment (EC) having an N-terminal PGE protecting group and attached to a polymer carrier;
the peptide fragment (EC) has the formula (XI)

fourth step: release the N-terminal protective group PGE of the peptide fragment (E);
step five: the peptide fragment (F) is condensed with the peptide fragment (EC), resulting in a peptide fragment (FC) having an N-terminal PGF protecting group and attached to a polymer carrier;
peptide fragment (FC) has the formula (XII)

step six: release the N-terminal protective group of the PGF peptide fragment (FC);
step seven: the peptide fragment (G) is condensed with the peptide fragment (FC), resulting in a peptide fragment (A) having an N-terminal PGA protecting group and attached to a polymer carrier;
after which the peptide fragment (A) is separated from the polymer carrier; and then the peptide fragment (A) is condensed with the peptide fragment (B), resulting in a peptide (1) having an N-terminal PGA protecting group;
then release the N-terminal protective group of the PGA peptide (1) and at this stage the protective groups of the side functional groups of the peptide (1) are released,
wherein the peptide fragment (B) is a modified part of the peptide (1); in the peptide fragment (B), the N-terminal amino acid is the amino acid position 35 in the peptide (1), the C-terminal amino acid is the amino acid position 39 in the peptide (1);
peptide fragment (B) has the formula (III)

the peptide fragment (B) was obtained by classical synthesis in solution, solid-phase synthesis, or a combination of solution and solid-phase syntheses. 2. The method according to claim 1, characterized in that the peptide fragment (A) or peptide fragment (B) obtained by solid-phase peptide synthesis. 3. The method according to claim 1, characterized in that the peptide fragment (A) is obtained by solid-phase peptide synthesis, and the peptide fragment (B) by classical synthesis in solution.