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Light cut-out magnetic particle molecale label, its preparation method and aplication in multipeptide synthesis preparation

A technology of molecular labeling and magnetic particles, which is applied in the application fields of large-scale synthesis and purification of polypeptides, can solve the problems of no discovery, achieve low cost, make up for the defects and deficiencies of process methods, and save the investment in instruments and reagents

Inactive Publication Date: 2005-03-23
西安蓝晶生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There have been many reports of photocleavable molecular linkages as indirect application molecules in polypeptide synthesis, linked to resin carriers, and as safe capture linkages (Cano, et all, J.Org.chem, 2002, 67, 129-135), and there are also Photoremovable links (linker) are used as backbone molecules to connect with other carrier materials, such as with AMPS resin, CPG glass beads, etc. (products of BiosearchTechnologies Inc. in the United States). For specific connection examples, see literature reports (D.H.Rich and S.K.Gurwara, J. Am.Chem.Soc., 1975, 97, 1575; H.venkatesan and M.M.Greengurg, ibid., 1996, 61, 525; C.P.Homes and D.G..Jones ibid., 1995, 60, 2318; M.R.Carrasco, et al. Tetrahedron Lett.1997, 38, 6331.), but there is no report that such backbone molecules are connected with magnetic particles for polypeptide labeling

Method used

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  • Light cut-out magnetic particle molecale label, its preparation method and aplication in multipeptide synthesis preparation
  • Light cut-out magnetic particle molecale label, its preparation method and aplication in multipeptide synthesis preparation
  • Light cut-out magnetic particle molecale label, its preparation method and aplication in multipeptide synthesis preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Synthesis of Magnetic Particle Molecular Tags

[0042] Weigh 1 gram of Fe 3 o 4 Magnetic particles, the outer periphery of the magnetic particles has been coated with NH 2 terminal, terminal NH 2 The molar number is 1mmol / g, weigh 2mmol of photocleavable linker molecule 4-Hydroxymethyl-2-methoxy-5-nitrophenoxybutyri Acid (4-hydroxymethyl-2-methoxy-5-nitrophenoxybutyric acid) , using anhydrous HOBT, DIEA, 4-fold molar excess, in 5ml DMF solution system, activate photoremovable molecules, add the liquid activated for 5-8 minutes to the magnetic particles, and react in the dark for 2.5 hours, the reaction is complete , using magnet adsorption and precipitation to precipitate the molecular tags linked by reaction, washing with DMF, DCM, and methanol respectively and precipitating three times, drying under reduced pressure for later use, and obtaining 1.32 g of linked magnetic particle molecular tags.

Embodiment 2

[0043] Embodiment 2: synthetic preparation 0.5mmol P23 peptide

[0044] The synthetic preparation sequence is SKYTESFVAAFKRAGAGVEKAEA-NH 2 , a polypeptide with a sequence length of 23 amino acids.

[0045] Weigh 0.5 grams of Wang resin with a degree of substitution of d=1mmol / g, add it to the polypeptide reactor, and assemble and synthesize the polypeptide using the standard Fmoc coupling method. After each step of amino acid connection, use excess acetic anhydride according to the ratio of acetic anhydride:DMF=1:1 (V / V), 4-6mL blocked unreacted NH 2 end, to prevent the continued assembly and synthesis of the truncated sequence, and cut off the terminal NH after the last amino acid is synthesized 2 protection of.

[0046] Wash well to keep the peptide-resin moist, weigh 2.5 grams of the magnetic particle molecular label prepared in Example 1, and use HOBT, PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, hexafluorophosphate-1-oxyl Tripyrrolidinyl ...

Embodiment 3

[0047] Example 3 Synthetic preparation of 1 mmol P38 peptide

[0048] The synthetic preparation sequence is HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRIKNK-NH 2 A polypeptide with a sequence length of 38 amino acids

[0049] Weigh 0.5 grams of Rink Amide-AM resin with a degree of substitution of d=0.3-0.8mmol / g and add it to the polypeptide reactor, assemble and synthesize the polypeptide using the standard Fmoc coupling method, and use excess acetic anhydride to press acetic anhydride after each step of amino acid connection: DMF=1:1 (V / V), 4-6mL blocked unreacted NH 2 end, to prevent the continued assembly and synthesis of the truncated sequence, and cut off the terminal NH after the last amino acid is synthesized 2 2.5 g of the magnetic particle molecular tag prepared in Example 1 was weighed, and 4 times the molar amount of HOBT, PyBOP, and DIEA was used as a catalyst to activate the molecular tag in 5 ml of DMF to activate the 5- After 8 minutes, add the peptide-resin to the re...

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Abstract

A molecular label of magnetic particles, which can be optically excised, is prepared by linking the photosensitive molecular radial to the magnetic particle. It can be used to synthesize the high-purity complete-length target polypeptide through derivation marking to conventional solid polypeptide, cutting, magnetic separation, optically excising the molecular label, and final separation.

Description

technical field [0001] The present invention relates to a photoremovable magnetic particle molecular label and its preparation method and application, in particular to a photochemically sensitive photocleavable magnetic particle molecular label, its preparation method and its application in the large-scale synthesis and purification of polypeptides. application. Background technique [0002] Since scientists invented the solid-phase peptide method in the 1940s, thousands of peptides of various sequences have been synthesized using 9-fluorenylmethoxycarbonyl (Fmoc-) or tert-butoxycarbonyl (Boc-) , many new peptide drugs are synthesized by this method. With the rapid development of modern biotechnology, molecular biology, and proteome projects, scientists and various biomedical research and development institutions have begun to pay great attention to substances that have important physiological activities in biological systems such as polypeptides. More and more scientists h...

Claims

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Application Information

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IPC IPC(8): C07K1/13
Inventor 王亚宏
Owner 西安蓝晶生物科技有限公司
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