KR-20260065855-A - Synthesis of peptides, peptide nucleic acids (PNA), and oligonucleotides using membrane extraction

Abstract

The present invention relates to a system and method for producing peptides and oligonucleotides ("tides") using a reactor utilizing identification membrane technology to separate by-products and excess reagents from a reaction mixture. The identification membrane technology is based on the application of a membrane configured to act as a solid extraction membrane between the reaction chamber and the extraction chamber within the reactor by utilizing the affinity for the reagents and by-products present within the reaction chamber.

Inventors

• 오르머로드, 도미닉
• 소, 윙 호
• 그라테캣, 이자스쿤
• 카브리, 월터
• 톨로멜리, 알레산드라
• 페라짜노, 루치아

Assignees

• 비토 엔브이
• 알마마 테르 스투디오룸 유니베르시타‘ 디 볼로냐

Dates

Publication Date

20260511

Application Date

20240903

Priority Date

20230904

Claims (16)

1. A liquid system for synthesizing peptides and oligonucleotides (tides), comprising a housing (1) including a reaction chamber (2) and an extraction chamber (3) separated from each other by a membrane (4), wherein the membrane is configured to act as a solid extraction membrane between the reaction chamber and the extraction chamber, wherein the solid extraction membrane has an affinity for reagents present in the reaction chamber and optionally also by-products, has a surface polarity that matches the polarity of the reaction solution, and the solid extraction membrane has a surface polarity opposite to the polarity of the extraction solution.
2. In Article 1, A liquid phase tide synthesis system comprising a supply section (5) and an extraction section (6), wherein the supply section is configured to circulate a reaction solution (7) across a reaction chamber and the extraction section is configured to circulate an extraction solution (8) across an extraction chamber.
3. In Article 2, A liquid-phase Tide synthesis system in which the circulation of the extraction solution (8) across the extraction chamber is a closed loop.
4. In Article 2, The above membrane is a liquid phase tide system having a membrane surface polarity that causes the membrane (4) separating the reaction chamber and the extraction chamber to be saturated with the reaction solvent.
5. In Article 4, The above reaction solvent is a liquid-phase titan system selected from ethyl acetate, anisole, or DMF.
6. In Article 2, A liquid phase tide synthesis system comprising a reaction supply vessel (9) configured to supply a reaction solution into a reaction chamber through a reaction chamber inlet (10), the above supply section.
7. In Article 6, The above reaction supply vessel is a liquid phase tide synthesis system including a filling opening (12).
8. In any one of the aforementioned claims, The above film (4) is a liquid phase tide system, which is a ceramic TiO2 film with a thickness of about 1 to 5 nm.
9. Claim 9
10. In any one of paragraphs 2 through 9, The above extraction solution is a reactive extraction solution, a liquid-phase Tide system.
11. In Article 10, The above extraction solution is a liquid-phase tide system selected from an aqueous acid solution or ethanol.
12. A liquid phase synthesis method for synthesizing peptides and oligonucleotides (tides), wherein the method comprises the use of a solid phase extraction membrane (4) for separating and extracting reagents and optionally by-products from a reaction solution into an extraction solution, wherein the solid phase extraction membrane (4) has a surface polarity that matches the polarity of the reaction solution and is opposite to the polarity of the extraction solution.
13. In Article 12, A liquid phase tide synthesis method in which the above membrane has a membrane surface polarity that causes the solid extraction membrane (4), which separates the reaction solution from the extraction solution, to be saturated with the reaction solvent.
14. In Article 13, A method for synthesizing liquid-phase tides, wherein the reaction solvent is selected from ethyl acetate, anisole, or DMF.
15. In any one of paragraphs 12 to 14, The above extraction solution is a reactive extraction solution for reagents and optionally by-products from a reaction solution, and the extraction solution is selected from an aqueous acid solution or ethanol, a method for synthesizing liquid-phase tides.
16. Use of a solid extraction membrane (4) defined in any one of the aforementioned claims in a liquid phase tide synthesis method and/or system.

Description

Synthesis of peptides, peptide nucleic acids (PNA), and oligonucleotides using membrane extraction The present invention relates to a system and method for producing peptides and oligonucleotides ("tides") using a reactor utilizing discriminating membrane technology to separate byproducts and excess reagents from a reaction mixture using an environmentally friendly organic solvent. The identification membrane technology is based on the application of a membrane configured to act as a solid phase extraction membrane between the reaction chamber and the extraction chamber within the reactor, and this membrane limits the amount of organic solvent and water required to complete the coupling-deprotection sequence that must be repeated by utilizing its affinity for the reagents and byproducts present in the reaction chamber. Chemical synthesis of peptides, PNAs, and oligonucleotides is a repetitive technique such as solid phase peptide synthesis (SPPS) or liquid phase peptide synthesis (LPPS) (Green Chemistry 2022, 24, 975-1020). Membrane-based techniques can be applied to fluorenylmethoxycarbonyl (Fmoc) LPPS (WO2016188835A1; US8664357; Angew. Chem. Int. Ed. 2021, 60, 7786-7795) and convergent fragment-based synthesis (Org. Process Res. Dev. 2021, 25, 1628-1636) for peptides and PNAs. Liquid phase oligonucleotide synthesis (LPOS) using a membrane to remove impurities and byproducts was described by Livingston (Org. Process Res. Dev. 2016, 20, 1439-1452). The use of a separation barrier between the reaction chamber and the feed chamber to separate byproducts and excess reagents is known in the field of titan synthesis via LPPS and LPOS. In these solutions, the membrane acts as a simple physical barrier with a molecular cut-off value to retain the reaction product within the reaction chamber. The barriers used in such systems can be provided in various forms, such as porous membranes, porous particles, hollow fibers, and fritted discs. Porous barriers can be single-layer or multi-layered and can take the form of sheets, particles, hollow fibers, tubes, etc. Various configurations can be designed to retain desired components within the reaction zone and remove unwanted components from the reaction zone. By providing a source of low molecular weight reactants to the reaction zone to replenish or increase the consumption of these components during the reaction process, while simultaneously removing low molecular weight reaction products, the expression period can be significantly extended and a high molar ratio of protein or oligonucleotide products relative to the template present in the reaction medium can be provided. In its simplest configuration, this system consists of a membrane bag containing a reaction zone, while retaining a solution outside the membrane that provides the desired level of low molecular weight reaction components within the reaction zone. Thus, through exchange across the membrane, the low molecular weight products generated by the reaction are continuously dialyzed into the external solution, and the reaction components are continuously replenished within the reaction zone. A disadvantage of this system, applied to the synthesis of peptides and oligonucleotides, is that a large volume of the external solution is required to maintain an appropriate flow rate across the barrier and to keep the amount of low molecular weight products within the reaction zone to a minimum. Consequently, the process mass intensity (PMI), which is the total process mass per kg of product (kg/kg), increases sharply compared to solid phase technology, and process costs also increase. Another disadvantage is the increase in process time. Again, when compared to solid-state technology, the time required to perform this task is longer than that required to wash away impurities from growing peptides, oligonucleotides, or PNAs immobilized on solid-state resins. This is mainly because the amount of solvent required to wash away impurities from the system using Livingston’s membrane process is much greater than that required in solid-state synthesis, and therefore, unless a very large membrane surface area is used, the operation time is bound to be significantly longer. Therefore, it would be desirable to develop an improved system for tide synthesis that can operate at low trans-membrane pressure and generate less waste solution, with the goal of achieving a lower PMI and competitive process time than the corresponding SPPS. Various aspects of the present invention will now be discussed in more detail with reference to the accompanying drawings, and like reference numerals indicate like attributes. FIG. 1 is a schematic diagram of a liquid phase tide synthesis system according to the present invention. Figure 2 is a graph of Ln (1-extraction efficiency) over time for TMB-ester (solid line) and DBF-piperidine adduct (dotted line) across a Puramem selective membrane (square) and a ceramic PS- TiO2 membrane (circle), respect