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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: 2013-07-24
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 in that it mainly includes the following steps:

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

[0094]

[0095] B. Synthesis of amino acid protected peptidyl resin formula (IV) on the CPG resin of formula (III), wherein X is a polypeptide sequence

[0096]

[0097] C. Conjugate formula (V) to peptidyl resin formula (IV) to form a peptide-linker resin formula (VI) with a peptide-linking group, wherein 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-link base resin, thereby forming the peptide-link base-conjugate resin formula (VII) with the peptide-link base-conjugate

[0100]

[0101...

Embodiment 1

[0179] 1) the connection of formula (I) and CPG:

[0180] Dissolve 2-8 equivalents of formula (I), HOBt and HBTU in 3 mL of DMF, add 2-8 equivalents of DIPEA, activate for 15 minutes, add to 500 mg of ammoniated CPG, and shake at room temperature for 1 hour. The reaction solution was filtered off, the resin was washed three times successively with 5 mL each of DMF, methanol and DCM, and fully dried. The completion of the reaction was detected by ninhydrin, unreacted amino groups were blocked with acetic anhydride, the resin was washed three times with 5 mL each of DMF, methanol and DCM, and fully dried. Get 1-3mg CPG, add 5mL10% acetonitrile solution of p-toluenesulfonic acid, measure the absorbance value at 507nm after constant volume and calculate as 180μmol / g (that is, the amount of formula (I) contained in every gram of resin).

[0181] 2) Synthesis of peptides on derivatized CPG

[0182] The CPG derivatized in step 1) was treated with 5% trichloroacetic acid for 3 minut...

Embodiment 2

[0192] 1) The connection of formula (I) and CPG is the same as in Example 1.

[0193] 2) The CPG derivatized in step 1) was treated with 5% trichloroacetic acid for 3 minutes to remove DMT, rinsed with methanol and DCM, and dried. From the sequence (KLLKKL), according to the Fmoc or Boc method, use the above resin to remove DMT to synthesize the target polypeptide sequence, and use an appropriate reagent to remove the protecting group on the amino group at the end of the sequence. Take the Boc method as an example:

[0194] Dissolve 2-8 equivalents of Boc-Lys(Fmoc)-OH, HOBt, and HBTU protected by side chain amino Fmoc in 4 mL of DMF, add 2-8 equivalents of DIPEA, activate for 15 minutes, and add a certain amount of 1) To obtain CPG, shake the reaction at room temperature for 1 hour. The reaction solution was filtered off, the resin was washed three times successively with 5 mL each of DMF, methanol and DCM, and fully dried. Take 1-3 mg of CPG, add an appropriate amount of 2...

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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 field of solid-phase synthesis method and application technology of polypeptides, and specifically relates to a novel peptide-linker-conjugate and a solid-phase synthesis method thereof. Background technique [0002] Improving the cell permeability of nucleic acid-based drugs such as antisense oligonucleotides, peptide nucleic acids, and promising small interfering RNA (siRNA) is one of the key issues that must be addressed if they are to be developed into useful therapeutic drugs. Many techniques have been used to improve the intracellular uptake of these compounds, such as high-pressure intravenous injection, liposomes, and nanopolymers, but these methods have side effects to varying degrees that limit their development. [0003] Cell-permeable peptides are emerging transporters developed in recent years, and have been widely used in the intracellular transport of some non-cell membrane-permeable macromolecules. For the ...

Claims

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Application Information

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