Method for synthesizing monopegylation fixed point modification thymopoietin

A technology of PEGylation and fixed-point modification, which is applied in the field of biomedicine, can solve the problems that the yield of PEGylated thymopentin is only 2%, it is difficult to remove FMOC units, and the condensation reaction of amino acids is difficult, so as to prolong the circulation in the body Time, increase bioavailability, effect of synthetic route maturity

Inactive Publication Date: 2012-05-09
倪京满 +1
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Problems solved by technology

But, the reaction of this method will be carried out on the wang resin of the Lys (PEG) macromolecule that has condensed earlier, repeatedly condensation removes operations such as Fmoc
Since the steric hindrance between the wang resin as the carrier polymer and the PEG (5000da) polymer is too large, the removal of Fmoc is related to the steric hindrance, and the reaction conditions are limited by the steric hindrance conditions. Therefore, the protection of Fmoc removal is not only li...

Method used

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  • Method for synthesizing monopegylation fixed point modification thymopoietin
  • Method for synthesizing monopegylation fixed point modification thymopoietin
  • Method for synthesizing monopegylation fixed point modification thymopoietin

Examples

Experimental program
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Effect test

Embodiment 1

[0031] (1) Solid-phase synthesis of thymopentin with protective groups

[0032] A: Soak 1.5g of Fmoc-Tyr(tBu)-Wang resin (1%DVB, 100-200 mesh, S=0.3mmol / g) in dichloromethane (DCM) for 30 minutes, drain, and use dimethylformamide (DMF) after washing (three washes, three minutes each); add a mixed solution of piperidine and dimethylformamide (DMF) (piperidine / dimethylformamide = 1 / 4 (v / v)) Add to the washed resin, stir mechanically for 30 minutes, remove the Fmoc protecting group, wash with DMF (wash three times, each time for three minutes), and obtain the deprotected resin Tyr(tBu)-Wang. Add indene detection reagent (5% ninhydrin alcohol solution), heat in boiling water for three minutes, blue color appears, and the Fmoc group is completely removed.

[0033]B: amino acid FMOC-Val-OH 458mg (1.35mmol, 3eq)), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) 512.06mg (1.35mmol ,3eq), 1-hydroxybenzotriazole (HoBt) 182.66mg (1.35mmol, 3eq), dissolved in DMF i...

Embodiment 2

[0054] (1) Solid-phase synthesis of thymopentin with protective groups

[0055] A: Same as Example 1.

[0056] B: Change the amount of amino acid to FMOC-Val-OH: 305mg (0.9mmol, 2eq), FMOC-Asp(oBzl)-OH: 401mg (0.9mmol, 2eq), FMOC-Lys(BOC)-OH: 422mg (0.9 mmol, 2eq), Z-Arg (NO 2 )-OH: 318mg (0.9mmol, 2eq), benzotriazole-N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU): 170mg (0.45mmol, 1eq), 1- Hydroxybenzotriazole (HoBt): 61mg (0.45mmol, 1eq), the others are the same as in Example 1, and the thymopentin Z-Arg (NO 2 )-lys-Asp(oBzl)-Val-Tyr pure product 245.8mg, yield 53.83%.

[0057] (2) Liquid phase synthesis of PEGylated Thymopentin

[0058] Thymopentin Z-Arg (NO 2The amount of )-lys-Asp(oBzl)-Val-Tyr pure product is 42.7mg (3eq, 0.045mmol), the monomethoxypolyethylene glycol modification (mPEG-SPA) is 150mg (0.015mmol, 1eq), Others are identical with embodiment 1, get Z-Arg (NO 2 )-lys(mPEG)-Asp(oBzl)-Val-Tyr pure product 91.48mg, yield rate 55.7%.

[0059] ...

Embodiment 3

[0062] (1) Solid-phase synthesis of thymopentin with protective groups

[0063] A: Same as Example 1.

[0064] B: Change the amount of amino acid to FMOC-Val-OH: 611mg (1.8mmol, 4eq), FMOC-Asp(oBzl)-OH: 801mg (1.8mmol, 4eq), FMOC-Lys(BOC)-OH: 843mg (1.8 mmol, 4eq), Z-Arg (NO 2 )-OH: 635mg (1.8mmol, 4eq), benzotriazole-N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU): 341mg (0.9mmol, 2eq), 1- Hydroxybenzotriazole (HoBt): 122mg (0.9mmol, 2eq), the others are the same as in Example 1, and the thymopentin Z-Arg with a protective group is finally obtained (NO 2 )-lys-Asp(oBzl)-Val-Tyr pure product 265mg, yield 62.12%.

[0065] (2) Liquid phase synthesis of PEGylated Thymopentin

[0066] Thymopentin Z-Arg (NO 2 The amount of )-lys-Asp(oBzl)-Val-Tyr pure product is 189.6mg (0.2mmol, 20eq), the monomethoxypolyethylene glycol modification (mPEG-SPA) is 100mg (0.01mmol, 1eq), Others are identical with embodiment 1, get Z-Arg (NO 2 )-lys(mPEG)-Asp(oBzl)-Val-Tyr pure prod...

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Abstract

The invention provides a method for synthesizing monopegylation fixed piont modification thymopoietin, which belongs to the field of biomedicine. According to the method, firstly, a solid phase is adopted for synthesizing TP5 containing protecting groups, the defect that the fluorenylmethyl chloroformate (FMOC) unit removal operation of the method in the prior art is difficult to carry out is avoided, then, the modified polyethylene glycol is connected onto the lysine side chain amino at the fixed point in a liquid phase, the goal of fixed point modification is reached, simultaneously, the space steric hindrance of the solid phase wang resin is avoided through the liquid phase environment, finally, all protecting groups are removed in one step through palladium carbon catalytic hydrogenation to obtain the pegylation thymopoietin with the long-action effect, the synthesis process is simple and convenient, the cost is low, the yield is high (the total yield is higher than 20 percent), and the industrial production is convenient. The pegylation thymopoietin synthesized by the method can not be easily hydrolyzed by esoteric protease, the renal clearance can be obviously reduced because the molecular weight is heavy, and the internal circulation time is greatly prolonged, so the goals of obviously prolonging the half-life period and improving the bioavailability of organisms are reached.

Description

Technical field [0001] The present invention is the field of biomedical technology and involves a synthesis method for a single polyethylene glycol fixed -point modification. Background technique [0002] The thymopptide (TP5) is an artificial synthetic small molecular peptide. Its amino acid sequence is served as ARG-LYS-Asp-Val-Tyr. It has the same biological activity of the body's immune balance as the endogenous thymine Ⅱ.For a variety of immune dysfunction or low, TP5 can make the immune function normal through the two -way regulatory effect of promoting or suppressing.At present, it is clinically used to treat tumors and autoimmune diseases.However, after the TP5 is injected in the human body, it is quickly degraded by protease and amino peptidase. The half -life of the drug prototype is only 30 seconds.At present, only frozen dried powder injections and solution -type injections are available in clinical applications. Therefore, for patients with long -term medication, TP5...

Claims

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Application Information

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IPC IPC(8): C07K7/06C07K1/113C07K1/06C07K1/04C07K1/02
CPCY02P20/55
Inventor 倪京满王锐史敏
Owner 倪京满
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