Novel peptide-link base-conjugate and solid phase synthesis method thereof

A conjugate and conjugation technology, applied in peptide preparation methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve the impact of oligonucleotide synthesis, affect coupling efficiency, and have fewer side chain protecting groups and other problems to achieve the effect of avoiding synthesis difficulties, simple structure, and minimizing product loss

Inactive Publication Date: 2010-12-01
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First of all, such side chain protecting groups are relatively small, and will affect the synthesis of oligonucleotides, such as affecting the coupling efficiency, the conventional I 2 Oxidation must be changed to tBuOOH oxidation
On the other hand, the same problem exists if the oligonucleotides are synthesized first
Oligonucleotides are sensitive to acidity. The Boc strategy is no longer used. Only the Fmoc strategy can be used. The protective group of the side chain can only be removed by weak acidity or catalytic reduction. Not only are there fewer protective groups, but also such modification Amino acids also need to be prepared individually, which also hinders the development of this method

Method used

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  • Novel peptide-link base-conjugate and solid phase synthesis method thereof
  • Novel peptide-link base-conjugate and solid phase synthesis method thereof
  • Novel peptide-link base-conjugate and solid phase synthesis method thereof

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preparation example Construction

[0092] According to the present invention, the solid phase synthesis method for preparing peptide-linker-conjugates is characterized by the following steps:

[0093] A. Use the structure of formula (I) and the ammoniated CPG resin of formula (II) to synthesize a CPG resin with extended link arms, formula (III), where m is 0, 1, 2, 3, 4 or 5

[0094]

[0095] B. Synthesize a peptide-based resin with protected amino acids on the CPG resin of formula (III), formula (IV), where X is the polypeptide sequence

[0096]

[0097] C. The formula (V) is conjugated to the peptide-based resin formula (IV) to form a peptide-linker resin formula (VI) with a peptide-linker group, where A is a substituted or unsubstituted benzene ring or carbon atom , N is 0, 1, 2, 3, 4 or 5

[0098]

[0099] D. Synthesize the target conjugate on the peptide-linker resin to form a peptide-linker-conjugate resin formula (VII)

[0100]

[0101] E. Remove at least one side chain protecting group from at least one amino ac...

Embodiment 1

[0179] 1) The connection between formula (I) and CPG:

[0180] Dissolve 2-8 equivalents of formula (I), HOBt and HBTU in 3mL DMF, add 2-8 equivalents of DIPEA, activate for 15 minutes, add to 500mg of ammoniated CPG, shake at room temperature for 1 hour. The reaction solution was filtered off, and the resin was washed three times with 5 mL each of DMF, methanol and DCM, and dried sufficiently. Ninhydrin detected the completion of the reaction, the unreacted amino group was blocked with acetic anhydride, and the resin was washed three times with 5 mL each of DMF, methanol and DCM, and fully dried. Take 1-3 mg of CPG, add 5 mL of 10% p-toluenesulfonic acid in acetonitrile, measure the absorbance at 507 nm after constant volume, and calculate it to be 180 μmol / g (that is, the amount of formula (I) per gram of resin).

[0181] 2) Synthesize peptides on derivatized CPG

[0182] The CPG derivatized in step 1) was treated with 5% trichloroacetic acid for 3 minutes to remove DMT, washed wi...

Embodiment 2

[0191] 1) The connection between formula (I) and CPG is the same as in Example 1.

[0192] 2) Treat the CPG derivatized in step 1) with 5% trichloroacetic acid for 3 minutes to remove DMT, rinse with methanol and DCM, and dry. From the sequence (KLLKKL), follow the Fmoc or Boc method to synthesize the target polypeptide sequence with the above-mentioned DMT-removed resin, and remove the protective group on the amino terminal of the sequence with an appropriate reagent. Take the Boc method as an example:

[0193] Dissolve 2-8 equivalents of Boc-Lys(Fmoc)-OH, HOBt and HBTU protected by the side chain amino group Fmoc in 4mL DMF, add 2-8 equivalents of DIPEA, activate for 15 minutes, add to a certain amount of 1) In the obtained CPG, the reaction was shaken at room temperature for 1 hour. The reaction solution was filtered off, and the resin was washed three times with 5 mL each of DMF, methanol and DCM, and dried sufficiently. Take 1-3 mg of CPG, add an appropriate amount of 20% p...

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Abstract

The invention discloses a novel peptide-link base-oligonucleotide compound and a solid phase gradual synthesis method thereof. The peptide-link base-oligonucleotide compound provided by the invention has the structure of a general formula (VIII), wherein X is a polypeptide structure part, A is substituted and unsubstituted benzene ring or carbon atom, and n is 0,1,2,3,4 or 5. The peptide conjugating oligonucleotide compound of the invention has the characteristics of simple structure, convenient synthesis and the like, the product has membrane permeability, can serve as a molecular biology tool for researching antisense oligonucleotide and small-interference RNA (siRNA) and has potential drug development prospect. The solid phase gradual synthesis method of the invention avoids difficult synthesis of the current biomacromolecule conjugate, simplifies synthesis operation, and is convenient to prepare the decorated oligonucleotide compound with high flux and multiple target spots.

Description

Technical field [0001] The invention belongs to the technical field of polypeptide solid-phase synthesis methods and applications, and specifically relates to a novel peptide-linker-conjugate and a solid-phase synthesis method thereof. Background technique [0002] For some nucleic acid-based drugs such as antisense oligonucleotides, peptide nucleic acids, and promising small interfering RNA (siRNA) to develop into useful therapeutic drugs, improving their cell permeability is one of the key issues that must be resolved. Many techniques have been used to increase the intracellular uptake of such compounds, such as high-pressure intravenous injection, liposomes, and nanopolymers, but these methods have different levels of side effects that limit their development. [0003] Cell-permeable peptides are emerging transport carriers developed in recent years and have been widely used for intracellular transport of some non-cell membrane-permeable macromolecules. For the intracellular tr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K1/06C07K1/04C12Q1/68
Inventor 杨振军刘洋王晓峰张礼和
Owner PEKING UNIV
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