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Method for synthesizing thymosins

A technology of thymosin and thymus, applied in the field of biomedical chemistry

Inactive Publication Date: 2009-07-15
BCN PEPTIDES SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0040] It can be seen from the above that although the synthesis of long peptides in the academic field has been disclosed in the prior art, pseudoproline has not been applied on an industrial scale or for the synthesis of thymosin, which supports the invention of the present invention Novelty and ability and inventive step

Method used

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  • Method for synthesizing thymosins
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  • Method for synthesizing thymosins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0137] Example 1: Synthesis of Thymosin α1

[0138]The Fmoc-Asn-OH C-terminal residue was introduced on 2-chlorotrityl resin lacking amino acids to obtain 0.85 mmol / g functionalization after the introduction of the first amino acid. 0.8 g of amino acids (2.13 mmol, 0.5 eq.) were introduced on 2.5 g of resin (f = 1.6 mmol / g resin, 4 mmol). Resin and amino acid were weighed in separate containers and allowed to dry under vacuum for at least 4 hours. Dissolve the amino acid in 25 mL of dry DCM (at 4 sieve). DIEA (0.7 mL, 1.6 mmol, 1 eq.) was added and it was stirred for 5 minutes. A 1:1 solution (2.8 ml) of DIEA:DCM (1.4 mL, 3.2 mmol, 2 eq.) was prepared and added to the reaction mixture. It was stirred for another 40 minutes, then 4 mL of dry MeOH was added and allowed to react for 10 minutes, after which time the resin was filtered in a synthesis reactor equipped with a filter plate and a key, with the following washes:

[0139] Step Reagent Repeat Time

[0140] 1 DCM ...

Embodiment 2

[0154] Example 2: Synthesis of Thymosin β4

[0155] Thymosin β4 was synthesized using a solid-phase peptide synthesis strategy using Fmoc / tBu protection. For the introduction of the first amino acid on 2-chlorotrityl resin (1 g, 1.6 mmol / g), a DCM solution of Nα-Fmoc-protected amino acid (0.5 eq) and DIEA (3 eq.) was used to A functionalization of 0.85 mmol / g was achieved after amino acid. It was mechanically stirred for 40 min, then 4 mL of dry MeOH was added and allowed to react for 10 min. The resin was filtered in a synthesis reactor equipped with a filter plate and a key, and the following washes were performed with DCM and DMF. The resin was treated with 5% piperidine in DMF for 1 x 10 min and 20% piperidine in DMF for 1 x 15 min and rinsed with DMF. Once the first amino acid has been introduced, the remaining amino acids are Na-Fmoc-protected amino acids (3 eq.), HOBT (3 eq.) and DIPCDI (3 eq.) in DMF, the equivalents being calculated relative to the functionalizat...

Embodiment 3

[0163] Example 3: Synthesis of Thymosin β15

[0164] The synthesis method is similar to that used for thymosin β4. Using Fmoc-Lys(Boc)-Ser(Ψ Me，Me pro)-OH dipeptide replaces amino acids 14-15 with Fmoc-Asn(Trt)-Thr(Ψ Me，Me pro)-OH, and Fmoc-Glu(OtBu)-Thr(Ψ Me，Me pro)-OH. Coupling is only required at Lys(3).

[0165] The peptidyl resin Ser(tBu)-Asp(OtBu)-Lys(Boc)-Pro-Asp(OtBu)-Leu-Ser(tBu)-Glu(OtBu)-Val-Glu(OtBu)- Thr(tBu)-Phe-Asp(OtBu)-Lys(Boc)-Ser(Ψ M e，Mepro)-Lys(Boc)-Leu-Lys(Boc)-Lys(Boc)-Thr(tBu)-Asn(Trt)-Thr(Ψ Me，Me pro)-Glu(OtBu)-Glu(OtBu)-Lys(Boc)-Asn-Thr(tBu)-Leu-Pro-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Thr(Ψ Me，Me pro)-Ile-Gln-Gln-Glu(OtBu)-Lys(Boc)-Glu(OtBu)-Tyr(tBu)-Asn-Gln-Arg(Pbf)-Ser(tBu)-resin.

[0166] By making it with TFA:H 2 The O 95:5 mixture was reacted for 1 hour 30 minutes to cleave the peptide from the resin, precipitate it with diethyl ether and lyophilize it. The crude product was purified by semi-preparative RP-HPLC and the final product was char...

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Abstract

The invention relates to a method for obtaining thymosins and / or the pharmaceutically acceptable salts thereof by means of solid-phase synthesis on polymeric supports, which comprises the steps of linearly synthesizing the thymus-derived peptides in solid phase, incorporating at least one Thr or Ser in the sequence, in pseudoproline dipeptide form, obtaining the thymosins by treating the peptidyl resin with trifluoroacetic acid, and purifying the thymosins by RP-HPLC. The present invention also protects the isolated and / or purified compound obtained by means of said method and also the use thereof in the preparation of a medicinal product.

Description

technical field [0001] The present invention relates to the field of biomedical chemistry. In particular, the present invention relates to a new method for the chemical synthesis of thymosin in high yield and purity by means of solid-phase peptide synthesis. The method is based on the introduction of pseudoproline derivatives at key positions in the thymosin sequence. The pseudoproline oxazolidine ring prevents structuring of the growing peptide chain anchored to the resin during elongation of the peptide sequence, preventing aggregation and synthetic failure of the peptidyl resin. Background technique [0002] Thymosins are a family of biochemically and functionally distinct polypeptides with important physiological functions. They were first described in 1966 and initially isolated in the thymus, subsequently they were identified in several tissues and cells [Goldstein, A.L., Slater, F.D., White, A., Preparation and partial purification of a thymiclymphocytopoietic facto...

Claims

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Application Information

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IPC IPC(8): C07K14/575C07K17/08A61K38/22A61P29/00A61P35/00
CPCC07K1/065C07K14/57581A61K38/00A61P29/00A61P35/00A61P37/00A61P37/04A61P9/00A61K38/22C07K14/575C07K17/08
Inventor J·费尔南德斯卡尔亚多B·蓬萨提奥维奥尔斯J·克莱门特罗德里格斯R·鲁维雷斯费雷尔S·帕冯费尔南德斯
Owner BCN PEPTIDES SA
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