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Continuous, solvent-free and non-enzymatic peptide synthesis by reactive extrusion

A solvent-free, reactor-based technology used in peptide synthesis to solve problems such as unproven peptide coupling stereoselectivity and no diversity allowed

Pending Publication Date: 2020-10-30
CENT NAT DE LA RECHERCHE SCI +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the polymerization is based on a single α-L-amino acid ester monomer, this method does not allow any possibility of finely controlling the diversity of the α-L-amino acid sequences of the peptides.
Also, the study did not demonstrate stereoselectivity of peptide conjugation
Furthermore, utilization of this enzyme was previously shown to be ineffective for coupling α-D-amino acid residues (Hernández, J.G. et al., Green Chem. 2017, 19, 2620-2625)

Method used

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  • Continuous, solvent-free and non-enzymatic peptide synthesis by reactive extrusion
  • Continuous, solvent-free and non-enzymatic peptide synthesis by reactive extrusion
  • Continuous, solvent-free and non-enzymatic peptide synthesis by reactive extrusion

Examples

Experimental program
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Effect test

Embodiment approach

[0193] In some embodiments of the continuous synthesis method described herein, among the monomer units contained in compound (I') or compound (II'), Rb1 or Rb3 is selected from the group consisting of hydrogen atom, methyl group, -CH2-CH2-CH2-NH-C(=NH)NH2, -CH2-C(=O)-NH2, -CH2-COOH, -CH2-SH, -(CH2)2-C(=O)-NH2 , -(CH2)2-COOH, -CH2-imidazolyl, -CH(CH3)-CH2-CH3, -CH2-CH(CH3)2, -(CH2)4-NH2, -CH2-CH2-S-CH3 , -CH2-phenyl, -CH2OH, -CH(OH)-CH3, -CH2-indolyl, -CH2-phenyl-OH and isopropyl, and the other group in Rb1 and Rb3 is a hydrogen atom . In some of these embodiments, Rb2 is a hydrogen atom. This covers embodiments in which the compound of formula (II') contains one or more amino acid residues selected from the group consisting of: glycine residues, alanine residues, arginine residues, asparagine residues Groups, aspartic acid residues, cysteine ​​residues, glutamine residues, glutamic acid residues, histidine residues, isoleucine residues, leucine residues, lysine Residues, m...

Embodiment 1

[0240] Example 1: General method for continuous and solvent-free peptide synthesis by reactive extrusion at controlled temperature

[0241] Considering the application of reactive extrusion in peptide synthesis, it was decided to study a co-rotating twin-screw extruder (MC15 micro-mixer, ) By combining Boc-Trp-OSu with HCl.H-Gly-OMe and NaHCO 3 The ability to react to generate the dipeptide Boc-Trp-Gly-OMe without solvent. However, since there is no molten phase (which prevents proper rotation of the screw), the reaction mixture cannot be extruded. Heating the extruder barrel at 100°C cannot solve this problem. In order to improve the flow of the reaction mixture in the extruder barrel, various liquid additives were screened to determine that acetone was the best liquid additive. Then, the Boc-Trp-OSu (1.0 equivalent), HCl.H-Gly-OMe (1.1 equivalent) and NaHCO 3 (1.2 equivalents) and acetone (η = 0.15mL / g) (η is defined as the ratio of the volume of liquid (in mL) divided by th...

Embodiment 2

[0247] Example 2: Synthesis of various enantiomerically pure dipeptides and tripeptides

[0248] Then these optimized reaction conditions are applied to the synthesis of Boc-Trp-Phe-OMe, Boc-Trp-Phe-OMe can be isolated with 61% yield and> Diastereomeric excess value of 99% (Table 2, entry 1). Happily, Boc-Asp(OBzl)-OSu is even more reactive than Boc-Trp-OSu, because Boc-Asp(OBzl)-Phe- can be obtained without recycling the reaction mixture in the extruder. OMe. The residence time of 1.5 minutes in the extruder barrel is sufficient to completely convert Boc-Asp(OBzl)-OSu, resulting in a 92% yield of Boc-Asp(OBzl)-Phe-OMe, de (diastereomer excess value )> 99% (Table 2, entry 2). After removing Boc by solvent-free gaseous HCl treatment, HCl.H-Trp-Gly-OMe and HCl.H-Trp-Phe-OMe were reacted with Boc-Asp(OBzl)-OSu in the extruder. The corresponding Boc-Asp(OBzl)-Trp-Gly-OMe tripeptide and Boc-Asp(OBzl)-Trp-Phe-OMe tripeptide were produced at 86% and 89% yields, with excellent purity...

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Abstract

The present disclosure relates to a continuous, solvent-free and non-enzymatic method for synthesizing a compound of formula (I): Ra-POLYPEP-Rc (I) wherein: POLYPEP is a poly-amino acid compound, Ra and Rc are as specified in the disclosure, which method comprises the steps of: a) feeding an extrusion reactor with (1) a compound of formula (II) Ra-PEPNt-Rg (II) wherein; PEPNt is a mono- or a poly-aminoacid compound, Ra and Rg are as specified in the disclosure, and (2) a compound of formula (III) H-PEPCt-Rc (III) wherein: PEPCt is a mono- or a poly-amino acid compound, and Rc is as defined inthe disclosure in the absence of any solvent, so that the compound of formula (II) and the compound of formula (III) react together for generating a compound of formula (I), and b) collecting the compound of formula (I) from the extrusion reactor.

Description

Technical field [0001] The present invention relates to the field of peptide synthesis. Background technique [0002] Generally, peptides have high usefulness in various industry fields including the food industry, the diagnostic industry, the cosmetic industry, and the therapeutic industry. Therapeutic peptides exhibit a wide range of advantageous characteristics, making them one of the most promising active pharmaceutical ingredients (K. Fosgerau, T. Hoffmann, Drug Discov. Today 2015, 20, 122; JLLau, MKDunn, Biorg.Med.Chem .2017, DOI: 10.1016 / j.bmc.2017.06.052.). In fact, they are biologically active at very low doses, with high selectivity, good tolerance and biodegradability into non-toxic amino acids. However, the industrial production of peptides is carried out in solution or by solid phase peptide synthesis (SPPS), and is plagued by the large amount of toxic organic solvents required in the synthesis and purification steps (Bray, Nature Rev. Drug Discov. 2003, 2,587 ; A...

Claims

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Application Information

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IPC IPC(8): C07K1/06B29C48/39
CPCB29C48/397B29C48/40C07K1/063B29K2089/00C07K1/061
Inventor 伊夫·耶博伊本杰明·盖拉德尼古拉斯·勒莫伊格尼弗雷德里克·拉马蒂让·马丁内斯托马斯-泽维尔·梅特罗
Owner CENT NAT DE LA RECHERCHE SCI